A83543 compounds, their preparation process and their uses

A83543 compound, its preparation method and its use (additional one)

<u>Field of invention</u>

The present invention relates to the use of S. spinosum (<u>Saccharopoly-spora spinosa</u>) The SPINOSYN compound produced by chemical modification. These compounds have insecticidal activity.

<u>Background of the invention</u>

U.S. Patent No. 5,362,634 confirms that the fermentation product A83543 is a family of related compounds produced by Saccharomyces spinnera, and this patent document is incorporated herein for reference. These compounds have been called A, B, C, D, E, F, G, H, J, K, L, M, N, O, P, Q, U, S, T, U, V, W Factors or components such as, Y, etc. (see also PCT WO 93/09126 and PCT WO 94/20518) and are referred to as Spinozin A, B, etc. hereinafter. These Spinozin compounds can be used for the control of spiders, nematodes and insects. The naturally occurring spinoxin compound is a 5,6,5-tricyclic ring system fused to a 12-membered macrolide, neutral sugar (rhamnose) and amine sugar (forosamine) (see Kirst et al., (1991),<u>Tetrahedron Letters</u>, 32: 4839). If the compound does not contain amine sugars, it is called A, D and other antipseudoglycan compounds. The preferred names used to refer to the pseudosaccharide adjuvant compound are Spinozin A 9-Psa, Spinozin D 9-Psa and the like. Spinozin compounds generally have the following structure:<img file="TW487559B_D0001.tif" /><img file="TW487559B_D0002.tif" /><img file="TW487559B_D0003.tif" /><img file="TW487559B_D0004.tif" />

<img file="TW487559B_D0005.tif" />

The naturally occurring Spinozin compounds can be produced by fermentation of strains NRRL 18719, 18537, 18538, 18539, 1843 and 18823. These strains have been deposited and made part of the inoculum preservation collection of the Agricultural Research Institute of the United States Department of Agriculture.

As mentioned above, Spinozin compounds are particularly effective against insects of Lepidoptera and Diptera species. Spinozin compounds are quite environmentally friendly and have interesting toxicological profiles. However, in some applications, it is ideal to have a longer residual effect than spinoxin disclosed in conventional patent cases and public cases. Spinozin analogs with longer residual effects can be used to control on fruits and nuts or to control codling moths that affect pear fruit.

<u>Summary of Invention</u>

The present invention disclosed in this article is developed by S. spinosum (<u>Saccharopoly-spora spinosa</u>) Synthetic modification of the produced products to produce intermediate products and/or insecticidal products for the agricultural and animal health markets. Many synthetic modifications are performed on rhamnose and forosamine through hydrogenation, epoxidation, reduction, halogenation, oxidation, addition of alkyl groups, addition of nitrogen groups and macrolides. The addition and removal of substituents are performed on the molecule.

<u>Detailed description of the invention</u>

The compound of the present invention is prepared directly or indirectly by modifying the compound, which is naturally produced by S. spinosum. See US Patent No. 5,362,634 issued to DowElanco, which is incorporated herein by reference. The compounds of the present invention have been shown to have activity against insects, spiders and nematodes. Therefore, the compound of the present invention can be used to produce other compounds or the compound of the present invention itself can be used to inhibit or deactivate insects. Contact the inhibitory or deactivating amount of the compound with insects or the places where they are infested. Insect's "hazard place" refers to the place where the insect or the ring culture or its eggs appear, the air around the insect or the insect, the food that the insect or the insect eats, or the objects in contact with it. Generally, these compounds are applied to the leaves of plants, which are food for insects or larvae. Unless otherwise specified herein, the following words and terms have the following meanings: The term "haloalkyl" means an alkyl group having 1 to 4 carbon atoms, in which at least one halogen is bonded to the carbon atom. The term "halogen" means Cl, F, Br, or I. The term "alkanol group" means an alkyl group having 1 to 4 carbon atoms in which at least one carbonyl group is attached to the alkyl group. The term "alkyl hydroxylamine" means a substituent of the following chemical formula wherein R¹⁰And R¹¹Each independently is an alkyl group having 1 to 4 carbon atoms, or an alkanol group having 1 to 5 carbon atoms. The term "protected hydroxyl" means substitution by methyl ester, such as but not limited to methoxymethyl, tetrahydrofuran, and ethyl ester, such as but not limited to 1-ethoxyethyl, and Benzyl ether substitution, such as but not limited to p-methoxyl group, substituted by silyl ether, such as but not limited to silyl ether having 1 to 2 carbon atoms, generally having 1 to 2 carbons Ethers of atoms, generally esters with 1 to 2 carbon atoms, more particularly formates or acetates, and carbonates are generally carbonates with 1 to 2 carbon atoms, more particularly methyl carbonate , And generally sulfonic acid esters with 1 to 2 sulfur atoms, more particularly methyl sulfonate. These protecting groups are in Green, TW Wuts, PGM<u>Protective group for organic synthesis</u>(Organic Ge Groups in Organic Syntheses, John Wiley and Sons, New York, 1991, pages 17-18) are described in more detail, which is incorporated herein by reference in its entirety. The term "protected amino group" means carbamates with 1 to 2 carbon atoms, amides with 1 to 4 carbon atoms, generally n-methanyl and n-acetyl, And, for example, the imine of the n-ylidene group. These protecting groups are further described in Greene<u>Protective group for organic synthesis</u>(Organic Groups in Organic Syntheses). The term "inhibiting insects or worms" means that the number of living insects or worms is reduced, or the number of eggs is reduced. "Deactivation amount" means the amount of compound used to cause a countable decrease in the population of insects or worms being treated. Generally, the compound used is about 1 to about 1,000 ppm (or 0.01 to 1 Kg/mu).

These compounds are generally effective against aster leafhoppers (<u>Macrostelesfascifrons</u>, aster leafhopper), beet armyworm (<u>Spodopteraexigua</u>, beet armyworm), peanut root knot nematodes (<u>Meloidogyne arenaria</u>, peanutroot knot nematode), cotton aphid (<u>Aphis gossypii</u>, cotton aphids), Cotton Leaf(<u>Tetra-anychusurticae</u>, two spotted spider mite), eleven star claw leaf beetle (<u>Diabrotica undecimpunctata howardi</u>, Southern corn rootworm), American cotton bollworm (<u>Heliothis zea</u>,cotton bollworm), Philippine corn wax cicada (<u>Peregrinus maidis</u>,corn planthopper), Tobacco leaf moth (<u>Heliothis virescens</u>, Tobbacco budworm), German cockroach (Blattella germanicus, German cockroach), European corn borer (<u>Ostrinia nubilalis,European cornborer</u>), Black-tailed Leafhopper (<u>Nephotettixcinciticeps</u>, green rice leafhopper), brown planthopper (<u>Nilaparvata lugens</u>, brown planthopper), and Chilo suppressalis, rice stem borer (Chilo suppressalis, rice stem borer), etc.

Synthetic compounds are generally made by modifying rhamnose, modified amino sugar, or starting from 9- or 17-pseudoglycan compounds and then substituting rhamnose or amino sugar for non-sugars or different sugars. Synthetic compounds are also modified by 5,6,5-tricyclic and/or 12-membered macrocyclic pseudoglycan compounds of naturally occurring compounds as described in the Examples herein. The compounds claimed herein include all isomers of the compound and any acid addition salts and isomers thereof.

There are several diastereomers of the compounds requested in this article. Because of the majority of stereogenic centers, the diastereomers can be expected to have insecticidal activity. Although some diastereomers may be more effective than other diastereomers, the present invention treats all diastereomers equally.

In Part A of Example 17, the rhamnose at positions R5', R6', and R7' of Formula I are added by adding a long-chain alkyl group, an aryl group and/or a long-chain alkyl group with halogen substituents, Addition modification of aryl groups. Esters can also be formed at the R5', R6' and R7' positions. Additional sugars can also be substituted at these positions. Another modification method is to add substituents containing atoms such as nitrogen, sulfur, phosphorus and silicon at these positions. In addition to the modification on rhamnose, the double bond between the C5 and C6 positions in Formula I is modified by epoxidation or hydrogenation.

In Part B of Example 17, the amino sugar is substituted by non-sugar substituents via ester linkages. Esters generally have a nitrogen heterocyclic group, halogen, or amine group. In Part C of Example 17, the amino sugar is changed from H or methyl to long-chain alkyl, aryl, quaternary ammonium salt or N-oxide by changing the substituent on the nitrogen. In Part D of Example 17, the double bond positions of Cl3 and Cl4 are modified by hydrogenation, epoxidation, reduction and addition such as alkyl and nitrogen groups of hydroxylamine and cyanide compounds. In Part E of Example 17, the Cl7 substituent of the macrolide was removed to obtain a double bond and/or the C5 and C6 positions were modified with halogens, epoxides and alkoxides. In part F of Example 17, the positions of C5 and C6 were modified by hydrogenation, epoxidation, halogenation, oxidation, and addition of heteroatom substituents and esters.

In part G of Example 17, other sugars such as rhamnose derivatives were substituted for amine sugars. In Part H of Example 17, the Spinozin starting material was alkylated or N-demethylated. The resulting material is best used as a starting material for the manufacture of other compounds. In Part I of Example 17, deoxidized rhamnose analogs were prepared and further modified with ester or heteroatom substitution. In Part J of Example 17, rhamnose is replaced with other sugars and non-sugars such as esters and ethers. In the K part of Example 17, the C9 position of the 5,6,5-tricyclic moiety in the molecule was modified by oxidizing the hydroxyl group to a ketone and then adding an alkyl group to the C-0 double bond. Furthermore, the hydroxyl group at the C9 position is deoxidized or substituted with a nitrogen-containing group.

The compounds disclosed in this article can not only be used in the production of agricultural products, but the acid addition salts of these compounds are also ideal products. Acid addition salts can be prepared from the compounds disclosed in Formula I, II, IV, VI, VIII, and XV. The salt of the compound uses standard techniques for preparing salts, which are well-known to those who are familiar with the art. Salts can be neutralized to form acid addition salts. Particularly useful acid addition salts include, but are not limited to, those formed by standard reactions of organic and inorganic acids, such as sulfuric acid, hydrogen nitric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, and maleic acid. Acid, fumaric acid, cholic acid, dihydroxy acid, galactaric acid, glutamic acid, camphor acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid , Salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, etc.

The steps and formulas used to prepare the composition are traditional techniques in the field of conventional agriculture or pest control technology. The composition can be concentrated or dispersed in water, or can be used in powder, bait or granular formulations. The dispersion is generally an aqueous suspension or emulsion prepared from a concentrated formula of the compound. Water-soluble or water-dispersible or emulsifiable formulations are solid, wettable powder, or liquid, known as emulsifiable concentrates or aqueous dispersions. The wettable powder can be coagulated or pressed to form water-dispersible particles. These particles contain a mixture of compounds, inert carriers and surfactants. The concentration of the compound is generally between about 0.1% to about 90% by weight. Inert carriers are generally attapulgite, montmorillonite and diatomaceous earth or purified silicate.

The surfactant contains about 0.5% to about 10% wettable powder, and the surfactant is generally sulfonated lignin, concentrated sulfonate, sulfonate, alkyl sulfonate, alkyl sulfonate, Or non-ionic defined active agents, such as alkylphenol ethylene oxide adducts or mixtures thereof.

The emulsifiable concentrate of the compound of the present invention generally contains about 50 to about 500 grams of compound per liter of liquid, which is equivalent to about 10 to about 50% dissolved in an inert carrier, which is a mixture of solvents or emulsifiers that are not miscible with water. . Organic solvents include organics, such as xylene and petroleum fractions, such as the high boiling point of petroleum and olefins, including heavy naphtha and aromatic naphtha. Other available organics include ene-based solvent rosin derivatives, such as cyclohexanone and complex aliphatic ketones and complex alcohols. All emulsifiers of emulsifiable concentrates are generally mixed ionic or non-ionic surfactants, such as those mentioned above and their equivalents.

Aqueous suspensions containing water-insoluble substances can be prepared in which the compound is dispersed in the aqueous vehicle at a concentration of generally about 5 wt% to about 50 wt%. The suspension is preferably prepared by finely grinding the compound and vigorously mixing it with the water, surfactant, and dispersant vehicles described herein. Inert ingredients, such as inorganic salts and synthetic or natural gums, can also be used as needed to increase the density and/or viscosity of the aqueous vehicle.

The precipitated flowable substance can be prepared by dissolving active molecules in a solvent and surfactant or interface active polymer that can be mixed with water. When these formulations are mixed with water, the active compound precipitates and surfactants control the size of the resulting microcrystalline precipitates. The size of the crystals can be controlled by selecting a specific polymer and surfactant mixture.

The compound of the present invention can also be applied as a granular composition applied to the soil. The granular composition generally contains from about 0.5% to about 10% by weight of the compound. The compound is dispersed in an inert carrier, which is generally clay or an equivalent substance. Generally speaking, a granular composition is prepared by dissolving the compound in a suitable solvent and applying it to a preformed granular carrier of the desired particle size. The particle size is generally about 0.5 mm to 3 mm. The granular composition can also be formed by forming a mass or paste of the carrier and compound, drying the combined compound, and pulverizing the mass or paste to form the desired particle size.

The compound can also be combined with a suitable organic solvent. Organic solvents are generally mild petroleum lubricants widely used in agriculture. These compositions are generally applied as a spray. More particularly, the compound is applied as a dispersion in a liquid carrier, and the liquid carrier is water. The compound can also be applied in the form of an aerosol composition. The compound is dissolved in an inert carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container, where the mixture is dispersed through an atomizing valve. The propellant mixture contains a low boiling point halocarbon, which can be mixed with an organic solvent or an aqueous dispersion pressurized with an inert gas or a gaseous halocarbon.

The amount of compound applied to insects and infestations is not critical and can be easily determined by the skilled person. Generally, the concentration that provides ideal control is about 10 ppm to about 5,000 ppm. For crops such as soybeans and cotton, the application rate is about 0.01 to about 1 kg/ha, and the compound is applied in a spray formulation of 5 to 50 gal/A. The compound can also be applied to any insects or places where they live. These locations are generally cotton, soybean or vegetable crops, fruits and hazel trees, grape vines, houses or decorative plants. The compounds of the present invention can also be used to treat animals to control arthropods, namely insects and spiders, which are pests on animals. These arachnid pests generally invade the host's in vitro ("ecto") surface; the agents used to control such pests are preferably "topical parasiticides".

All animals are vulnerable to such pests, although vertebrate hosts have the most serious problems. Humans are the latent hosts of many parasites, and in tropical areas and in areas lacking sanitary facilities, parasite infestation is a common medical problem. In addition, many domestic animals are highly vulnerable to parasites, such as cattle, sheep, pigs, goats, buffaloes, deer, piglets, chickens, turkeys, ducks, geese, ostriches, etc. Horses and other recreational animals are susceptible to parasites, as are minks and other animals that use fur for feeding purposes, as are rats, rats, and other experimental and research animals. Companion-type animals, such as dogs and cats, are highly susceptible to parasites, and because of their close relationship with humans, such parasitic diseases can also occur in humans who accompany these animals. Fish, crustaceans and other aquatic species are also vulnerable to parasites. In short, parasitic diseases involve hosts that are mainly the entire animal range.

The economic cost of the spread of ectoparasites is considerable. In the field of livestock breeding, animals suffer from problems such as feed efficiency and reduced growth rate. The production of milk and wool is harmed and endangered the production of wool, hides and furs. Animals become susceptible to secondary microbial infections and parasites. Even when ectoparasites do not harm health and production, they can also cause discomfort that cannot be ignored.

Although many parasiticides are available, they have encountered various problems, including limited types of action, environmental toxicity, repeated treatment requirements, and in many cases, ectoparasite resistance. Therefore, there is still a need for new topical parasiticides.

The compound of the present invention provides a new tool in medical equipment for controlling ectoparasites. In a specific example of the present invention, the present invention focuses on a method for inhibiting or killing arthropod parasites on host objects, which comprises contacting the parasites with an effective amount of the compound of the present invention.

The compounds of the present invention can be used to control a wide variety of arthropod parasites. Representative parasites that can be controlled by the compounds of the present invention are as follows:

American flower tick, Amblyomma spotted tick, Persian tick, Boophilus miniature, Oxtail itch, Bovine worm, Dog worm, Brachycephalosporum angifera, American big tick, Gallus gallus, Ixodes sheep, chicken Feather, knee scabies, ear beak tick, horse itch, sheep itch, Rhipicephalus sanguineus, scabies, Aedes, Anopheles, Culex pipiens, common mosquitoes, feather lice, spiral cone flies, spotted mosquitoes, Temperate bed bugs, spiral cone flies, dog cetaceans, cat cetaceans, C. midges, midges, midges, sheep feather lice, skin flies, red-tailed stomach flies, horse stomach flies, horse nasal stomach flies, tsetse flies, harassment Horn flies, donkey blind lice, cattle blind lice, body blind lice, pig blind lice,<u>Hydrotaea irritans</u>, Cowhide flies, striated skin flies, long jaw lice,<u>Linognathus pedalis</u>, Long jaw louse, Lucilia sericata, sheep louse, housefly, sheep nose fly, louse, midges, black flower fly, scurvy mosquito, pubic louse, hunting, black mosquito, tube louse, buffalo blind louse, fly , Horsefly, mealworm, cone hunting.

The in vitro parasiticidal activity of the compound of the present invention can be achieved when it comes into contact with parasites. The contact can be eggs, larvae, adults, or other life stages.

"Contact" includes the parasite ingesting the compound of the invention.

The delivery technology of external parasiticides is well-known to those who are familiar with the technology. Generally speaking, the compound of the present invention is applied to the surface of the animal body, whereby the compound of the present invention can come into contact with the parasites already on the host and the parasites that will attack the host within the effective period. Generally speaking, the compound is formulated as a liquid formula, which can be sprayed on the surface of the animal or poured on the surface of the animal. Another traditional treatment method is "immersion", in which the treated cattle are roughly immersed in a dilution of insecticide. For some hosts and parasites, the formula can be a powder that can be spread on the host, or shampoo or lotion for animal bathing. The collars of cats or dogs can also be used as a way of delivering external parasiticides, whereby the external parasiticides can be directly delivered to the surface of the animal.

In other techniques, the external parasiticides are applied to places where animals often haunt. Therefore, the compounds can be brought into contact with parasites as if they were applied directly to the host. As far as livestock are concerned, dusting bags are also well-known. They adopt a positioning method, in which cows will inevitably rub against the bag so that the compound can come into contact with parasites.

In another embodiment, the compound of the present invention can be used to control insects and arachnids, which are parasites in the excrement of cattle and other animals. In an embodiment of the present invention, the compound of the present invention can pass through the intestinal tract and be embedded in excrement through oral administration. The control of parasites in excrement can indirectly protect animals from parasites.

The compound of the present invention is formulated as a parasiticide that can be used in methods known to those skilled in the art. In general, the formula may include the compound of the present invention and one or more physiologically acceptable adjuvants. The formulation contains a concentrate, wherein the active agent of the present invention is present in a concentration of 0.001 to 98.0 percent, and the other contents can be physiologically acceptable carriers. In such formulations, especially those with a content of the compounds of the present invention below 50%, sometimes they can be used directly, but these formulations can also be diluted with other physiologically acceptable carriers to form a relatively dilute therapeutic formulation. The latter formulation may contain a lower concentration of active agent of 0.001 to 0.1 percent.

In another embodiment, the compound of the present invention can be effectively combined with other parasiticides or repellents, the latter being a conventional internal parasiticide ("endo"=internal, which controls internal parasites, the Parasites are generally flat worms and nematodes). Representative internal parasiticides include the following:

Abamectin, Endothiamidazole, Avermectin, Butadiene, Mya venom, Dichlorvos, Doramectin, Epsiprantel, Febantel, Fenthiodazole, Flubendazole, Ivenmectin, Levotetraimidazole, Mebendazole, Mitroscanate, Dichlorvos, Moxidectin , Netobimin, Niclosamide, Nitroisothiocyanidin, Sulazolamide, Oxbendazole, Hexahydro, one, Closulon, Closantel, Diacetaminophen, Nitroxide , amoxicillin, chlorfensulfamide, triclofenidazole.

Representative internal parasiticides include:

Abamectin, Alphmethrin, Amitraz, Avermectin, Mya venom, Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyromazine, Deltamethrin, Diamon, Diflubenzufon, Dioxin, Doramectin, Amphos, Flumethhalinate, Flumethhalerate Hexaflumuron, Ivermectin, Benzene hexachloride, Lufenuron, Malathion, Methoprene, Metriphonate, Moxidectin, Permethrin, Phosme, Pirimiphos, Properamphos, Proxacarb, Rotodione, Thiomethicone, Methoxycarb, Trichlorfon, Zetacypermethrin, Bt biotoxin and boric acid.

<u>Example</u>

<u>General experiment part</u>

Unless otherwise specified, the reagents and solvents used were purchased directly from commercial suppliers and all reactions were carried out at room temperature (20-22°C) using constant-speed magnetic stirring. All reactions involving organometallic reagents, humidity sensitive reagents, or metal hydride reagents are carried out in commercially available anhydrous solvents and under dry nitrogen. Utilize NaCl, NaHCO₃, NH₄Cl and other salts for distribution, extraction, or washing refer to saturated aqueous solutions of these salts. The reaction is generally "gradually completed" by using an organic solution of the above-mentioned salt solution to extract the product;₂CO₃, Na₂SO₄, Or MgSO₄The organic layer was dried, filtered and evaporated under reduced pressure. Reverse phase thin layer chromatography (RPTLC) was performed on a glass bottom plate combined with octadecylsilane (0.2 mm, taken from Whatman). Chromatography refers to flash chromatography and is performed on E. Merck silica gel 60 (20-400 mesh). Reverse phase high performance liquid chromatography (RPHPLC) was performed on silica gel (Rain Dynamax 60A, 8μm) combined with Cl8. All melting points are measured in an open capillary and uncorrected. Measured at 300 to 400 MHz and 75 or 101 MHz respectively¹HNMR and¹³C NMR spectrum. Mass spectrometry data was measured by electrospray ionization (ESI). Elemental analysis is provided by the analysis laboratory of Dowlanco or Midwest Microlabs.

<u>Example 1 Synthesis of Spinozin A Glycoside</u>

A sample of Spinozin A (8.50 g, 11.61 mmol) was dissolved in EtOH (100 mL). Add water (100 mL), and while stirring, add 4NH₂SO₄(aq) Solution (200 mL) was added to the solution. Under a nitrogen atmosphere, the resulting mixture was heated to reflux temperature for 4.5 hours. Cool to room temperature, dilute with toluene (250 mL) and ethyl acetate (300 mL), and add brine (300 mL). After extraction, the phases were separated. The aqueous layer was extracted again with ethyl acetate (100 mL). Continuous use of diluted brine (3X) and 5% NaHCO₃(2X) Wash the combined organic layer and then use anhydrous MgSO₄Dry and concentrate under reduced pressure. Make the residue flash SiO₂Purified on a column (250 g; ethyl acetate). After concentrating the purified part by chromatography to dryness, a glassy solid (4.08 g, 87%) can be obtained, [a]₅₈₉=-145.6°(MeOH), [a]₅₈₉=-131.7°(CHCl₃)。

<u>Example 2 Synthesis of Spinozin A17-Psa</u>

Dissolve Spinozin A sample (20.0 gms, 27.4 mmol) in 300mL1N H₂SO₄Neutralize and heat to 80°C for 2 hours with magnetic stirring. The reaction was then cooled to room temperature. Use suction to filter the sediment, and use freshly prepared 1N H₂SO₄washing. Then the solid product was dissolved in dichloromethane, washed with brine, and K₂CO₃Dry and evaporate under reduced pressure. Purified by silica gel chromatography using 70% EtOAc in hexane to obtain Spinozin 17-Psa (14.46 gms; 89%) as a colorless glass.

<u>Example 3 Synthesis of Spinozin A9-Psa</u>

A suspension of N-chlorosuccinimide (1.20 gm, 9.00 mmol) in dichloromethane (45 ml) was cooled to -78°C under nitrogen. Pure diethyl sulfide (1.07 ml, 9.90 mmol) was added to the suspension for 5 minutes, and the mixture was stirred at -78°C for 0.5 hour. Spinozin J (2.15 gm, 3.00 mmol) dissolved in 8 mL of dichloromethane was slowly added to the mixture for 15 minutes while keeping the reaction temperature below -60°C. When the addition was complete, the solution was stirred at -78°C for 6 hours. Then triethylamine (1.25 mL, 9.00 mmol) was added dropwise and the solution was allowed to warm to room temperature. The reaction was diluted with 40 mL of dichloromethane and poured into 20 mL of 1N sodium bisulfate and 30 mL of water. The layers were separated and the organic layer was extracted with 30 mL saturated sodium bicarbonate and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was dissolved in MeOH (100 ml). Add anhydrous K₂CO₃(4.15 gm, 30.00 mmol) and stir the suspension at room temperature. After stirring for 2 hours, the reactant was cooled to 0°C and water was evaporated to 1/5 of the reactant volume in a rotary evaporator. Add dichloromethane (100 mL) and water (100 mL) and separate the layers. The aqueous layer was extracted with 3 x 50 mL of dichloromethane and combined with the organic extracts and washed with water and then brine solution. The solution was dried with sodium sulfate, filtered and concentrated in a rotary evaporator to obtain a viscous yellow oil. The product was purified by flash chromatography (250 gm silica gel, 0.5% concentrated sulfuric acid in 5% methanol in dichloromethane) to obtain a white foamy Spinozin A9-Psa (1.52 gm; 93 %)¹H NMR(CDCl₃)δ78(brs,1, H-13), 4.63(m, 1, H-21), 4.43(m, 2, H-1", H-9), 2.23(s, 6, N(CH₃)₂)。

Example 4<u>Preparation of Spinozin using Saccharopolysporaspinosa NRRL 18395 culture</u>

<u>Part AOscillating flask fermentation</u>

Inoculate a freeze-dried pellet or a culture of Spinocystis NRRL 18395 in the form of a suspension maintained in liquid nitrogen into a nutrient medium containing composition A and composition B (medium B is more suitable for large-scale production) , Where the nutrient medium A contains (content expressed in %) tryptic soy medium*3.0%, yeast extract 0.3%, MgSO₄7H₂O 0.2%, glucose 0.5%, maltose 0.4%, deionized water qs 1 liter, unadjusted pH (*purchased from Baltimore Biolabs) and nutrient medium B contains enzyme hydrolyzed casein** 3.0%, yeast extract 0.3%, MgSO₄7H₂O0.2%, glucose 1.0%, deionized water qs1 liter, use sodium hydroxide to adjust the pH from 6.2 to 6.5, (**purchased from NZ Amine A, Sheffield Products, PO Box638, Norwich, NY 13815). Prepare slant medium or plate medium by adding 2.5% agar-agar to vegetative seed culture medium A or B. The inoculated slant medium is cultured at 30°C for about 10 to 14 days. The heated slant culture medium is scraped with a sterile instrument to loosen the spores and remove and soak the mycelium clusters. About 1/4 of the obtained loosened spores and culture growth are inoculated to the first stage vegetative seed culture medium. In addition, liquid nitrogen ampoules can be used to inoculate the first-stage medium. When the culture is kept in liquid nitrogen, the ampoule is prepared using an equal volume of the nutrient culture (cultured for 48-72 hours, 30°C) and a suspension medium. The suspension medium contains lactose (100 g), glycerol (200 mL) and deionized water (qs to 1 liter). Inoculate the liquid nitrogen ampoule into a 500-mL Erlenmeyer flask containing 100 mL of nutrient medium (or a 250-mL Erlenmeyer flask containing 50 mL of medium). The culture was incubated on a shaker at a speed of 250 rpm around a 2 inch (5.08 cm) circle at 30°C for 48 hours. Inoculate the inoculated culture (5%v/v inoculum) into 100 mL of a productive medium with the following composition: (content expressed in %) glucose 4.0%, partial enzyme hydrolyzed* vegetable protein 1.5-3.0 %, cottonseed powder** 1.0%, CaCO₃(Reagent grade or industrial grade) 0.3%, soybean oil 1.0%, tap water qs1 liter (pre-sterilized and adjusted to pH 7.0 with NaOH), *Sheftone H, Sheffield Products, **Proflo, Traders Protein, PO BOX 8407, Memphis , TN 38108. The inoculated productive culture medium is placed in a 500-mL Erlenmeyer flask at 28-30°C, on a shaker at a speed of 250 rpm, around a 2-inch circle, and cultured for 6 to 8 days.

<u>B. Stirred bioreactor fermentation</u>

In order to provide a large amount of inoculum, 10 mL of the first-stage medium prepared as described in Part A was inoculated into 400 mL of the second-stage medium with the same composition as the first-stage medium. The second-stage medium was placed in a 2-L wide-mouthed Erlenmeyer flask at 30°C on a shaker at a speed of 250 rpm around a 2-inch circle, and cultured for 48 hours. Inoculate the prepared second-stage nutrient medium into 80-115 liters of sterile productive medium prepared as described in section A. If necessary, additional soybean oil can be added to control foaming.

Ferment the inoculated productive medium in a 165-L stirred bioreactor at a temperature of 28°C for 5 to 8 days. The air flow in the stirring tank and the speed of the stirrer are controlled by a computer to keep the dissolved oxygen content at 50% or higher of the air saturation.

<u>Example 5 Separation of Spinozin A, B, C and D</u>

The fermentation medium (225 L) prepared as described in Example 4 was filtered with a filter aid (1% Hyflo), and the separated biomass was washed with water (~50 L). The biomass was then stirred with methanol (~100 L) for about 1 hour and filtered. The methanol filtrate was concentrated to a volume of about 1 liter. The concentrate was extracted 3 times with ether (1 L each time). Concentrate the combined ether extracts to a volume of about 200 mL. The concentrate (8 mL) was partially chromatographed in a silica gel column (RP-8 Lobar, size B, EM Science, EM Industries Division, 30 Inc.). Repeat this step a total of 12 times (cycles). The instrument settings and implementation steps for preparative chromatography are in the "Autoprep" mode as follows:

A complete "Autoprep" HPLX system includes three RaininRabbit HPX pumps, a pressure component, a Gilson Model 20 1BHPLC fractional collector, an Isco-v4 absorbance detector and an Apple Macintosh Plus computer. The complete system is configured according to the Dynamax HPLC Method Administrator's Manual from Rainin Instruments. The "Autoprep" HPLC structure has the advantage of system automation to allow repeated preparative separation under virtually identical conditions to have practically identical results. Collecting and merging corresponding parts obtained from multiple operations can provide chromatographic analyzable volume without the need for large-scale tubing. The two solvent mixtures (A) and (B) were used in the equal power mode (ISOCRATIC) with a flow rate of 0.8 mL/min. Solvent system A contains 95 mL CH₃OH, 95 mLCH₃CN, 10 mLH₂O, and solvent system B contains 100 mL CH₃OH, 100 mLCH₃CN, 0 mL H₂O. The power equalization mixture used contains 60% solvent B.

The duration of each dissolution cycle is 28.0 minutes. Discard the precipitate 16 minutes before each cycle. The following precipitates are eluted in 6 times, 2 minutes per tube (16 mL per tube). The 6-tube final fraction (chromatographic fraction) obtained from the automatically combined fractions of each cycle (12 times in total). The activity of each final lysate against mosquito larvae was analyzed and the presence of active Spinozine compounds was also determined by analytical HPLC. Then the active fractions were combined according to the activity and HPLC profiles and further purified. The purification system uses the same Autoprep" HPLC and solvent system, but with high resolution, and 21.4-mm×25- pre-filled with 8μ C-18 reverse phase silica gel. cm preparative column (Rainin Dynamax) to obtain Spinozin A, B, C and D. Spinozin A and D can be obtained from CH₃OH/H₂O crystallized out.

<u>Example 6 Purification of Spinozin A and D</u>

The fermentation medium (10 L) was prepared according to the method described in Part A of Example 4, but 1) 200 mL productive medium was used in a 1-L flask; 2) soybean oil was removed from the production medium; and 3) at 30°C Cultivate for 4-6 days. Filter the medium. Discard the filtrate containing 4 mcg Spinozin A/mL and non-detectable amounts of Spinozin B, C, or D/mL. The biomass was washed with water and extracted with methanol for 1 hour. The extract (7 L) contains 72 mcg Spinozin A/mL and 7 mcg Spinozin D/mL. The methanol extract was concentrated to a volume of 5 L, and added to HP-20 resin (150 mL, Mitsubishi Chemical Industry Co., Ltd., Japan) in water (2 L). The mixture was stirred for 1 hour. Then the HP-20 resin mixture was placed in the glass column. Use methanol: water (1:1, 1L), the initial effluent and precipitate are not active. Using methanol: water (7: 3, 1 L), the second precipitate contains a trace amount of Spinozin A. The subsequent eluate using methanol (1 L) contains Spinozin A and Spinozin D activity. The methanol precipitate is concentrated and combined with 2 similar eluates obtained from other elution operations and concentrated to dryness. The residue was dissolved in 75 mL methanol:THF (4:1) and precipitated by adding to 10 volumes of acetone. The mixture was filtered, and the filtrate was concentrated to dryness. The residue was dissolved in methanol (25 mL) and placed in a 5.5×90-cm column of LH-20Sephadex (Pharmacia LKB Biotechnology Company, USA). The column was prepared with methanol. The HPLC method described in Example 1 was used to collect and analyze 125 25- mL of eluted fractions.

The eluted fractions containing the desired compound are combined and concentrated. The residue was dissolved in methanol (10 mL) and placed into a 41.1-mm×25-cm preparative column filled with 8μ C-18 reverse phase silica gel (Rainin Dynamax). The column is adjusted under the conditions of methanol: acetone: water (37.5:37.5:25). After placing the sample, develop the sample with a 180-min linear gradient of the following solvents: Solvent System A is 37.5 mL CH₃OH, 37.5 mL CH₃N, 25 mLH₂O, and solvent system B is 45 mL CH₃OH, 45 mL CH₃CN, 0 mLH₂O. The eluted fractions containing Spinozin A were combined, dried, dissolved in t-BuOH (5 mL) and freeze-dried to obtain 778 mg of pure Spinozin A. The eluted fractions containing Spinozin D were combined and concentrated and chromatographed as described above with the D eluted fractions of the similar separation operation of 6 using the same column, but with different solvents. The column is adjusted under the conditions of methanol: acetone: water (40:40:20). The solvent system used to expand the sample in the column with a 180-min linear gradient operation is: solvent system A contains 40 mL CH₃OH, 40 mL CH₃N, 20 mLH₂O, and solvent system B contains 95 mL CH₃OH, 95 mL CH₃CN, 10 mL H₂O. Combine and concentrate the fractions containing D. The residue was dissolved in t-BuOH (5 mL) and freeze-dried to obtain 212 mg Spinozin D.

<u>Example 7 Ingredients Spinozin E, F, G, H and J and Spinozin</u>

<u>A 17-Psa separation</u>

The fermentation medium (8 L) prepared by the procedure similar to that described in Example 4 was processed according to the method described in Example 4. Combine the fractions containing the desired compound obtained from the LH-20 Sephadex column with the corresponding fractions obtained from other similar fermentations. Because the production of pseudoglycan compounds of ingredients E, F, G, H, J and A is very small, it must be subjected to multiple fermentations to provide sufficient amounts for further purification.

The secondary factor collection part prepared in this way and containing approximately 1.6 g of solid 15 substances was placed into the HPLC column (Rainin Dynamx) pre-packed with 8 micron C-18 reverse phase silica gel (ODS) as described in Example 4 . String to CH₃OH: CH₃CN: H₂O (75:75:50) adjustment, and the gradient is based on the following solvent system from 100% solvent (A) to 50% (B): solvent system A 75%CH₃OH, 75%CH₃CN, 50%H₂O and solvent system B 95%CH₃OH, 75%CH₃CN, 50%H₂O, collected in 25-ml fractions. Collect

<img file="TW487559B_D0006.tif" />

The collected fraction 5 (100 mL) was concentrated to a residue, dissolved in methanol (1 mL) and placed on a 21.4-mm×250-mm HPLC column (Rainin Dynamx) as described in Example 5. Use the following solvent system (A) to adjust the column: solvent system A30: 30: 40 CH₃OH/CH₃CN/H₂O(1N NH₄OAc, pH 5), and solvent B 95:95:10 CH₃OH/CH₃CN/H₂O(1NNH₄OAc, pH 5), using a 120-minute linear gradient from 100% solvent (A) to 50% solvent (B) to collect 15-mL eluate at 7.5 mL/min. Continue to collect the precipitate at 50% (B) for an additional 60 minutes. Collect the following melting points:

<img file="TW487559B_D0007.tif" />

These collected fractions are combined with the collected fractions obtained from the chromatographic process with the starting materials of other use types. As in the method described in this article, the combined collected fraction is further purified by column chromatography; desalted in HP-20 resin using standard techniques; and concentrated and freeze-dried to obtain the following components:

<img file="TW487559B_D0008.tif" />

*By mass spectrometry

<u>Example 8 Preparation of Spinozin K, Spinozin O and Spinozin Y using culture NRRL 18743</u>

<u>Part A. Shaking flask fermentation</u>

A culture of Spinoza NRRL 18743 in the form of freeze-dried pellets or a suspension maintained in liquid nitrogen was inoculated into a nutrient medium having the following composition:

<img file="TW487559B_D0009.tif" />

*Baltimore Biological Laboratory, Cockeysville, MD

The slant medium or plate medium can be prepared by adding 2.5% agar-agar to the nutrient medium. The inoculated slant medium is cultured at 30°C for about 10 to about 14 days. The mature slant culture medium is scraped with a sterile instrument to loosen the spores and remove and macerate the mycelium cluster. About 1/4 of the pine spores and cultures thus obtained were inoculated into 50 mL of the first-stage nutritive inoculum culture medium. In addition, liquid nitrogen ampoules can be used to inoculate the first-stage medium.

Dilute it with a homogenous nutrient medium, a sterile suspension of glycerol: lactose: water (2:1:7) at 1:1 (volume: volume), and distribute it into a sterile tube (1.5 ml/tube) to prepare a liquid -Nitrogen-the original inoculum. The diluted culture is then stored under liquid nitrogen and in a suitable storage container and used as the original inoculum for the cultivation of shake-flask cultures and fermentation tank cultures.

Quickly thaw the liquid nitrogen ampoule and inoculate 0.5 mL into a 250-mL Erlenmeyer flask containing 50mL nutrient medium. The culture was incubated at 32°C in a shaker at a rate of 250 rpm around a 2-inch circle for 48 hours.

Inoculate the cultured culture (5% v/v inoculum) into 25 mL of productive medium with the following composition:<img file="TW487559B_D0010.tif" /><img file="TW487559B_D0011.tif" />

*Chemical milk nutrients, Sheffield Products, Norwich, NY

**Proflo, graders Protein, Memphis TN

The inoculated productive medium was cultured in a 250-mL wide-mouthed Erlenmeyer flask, and it was cultured in a 2-inch circle at a rate of 250 rpm at 30°C for 7 days.

<u>B. Stirred bioreactor fermentation</u>

In order to provide a large amount of inoculum, 10 mL of the first-stage nutrient medium prepared as described above was inoculated into 400 mL of the second-stage nutrient medium with the same composition as the first-stage nutrient medium. The purity of the nutrient medium in the second stage is &gt;98%. The collected fraction containing Spinozin K separated by the first preparative HPLC and the repurified Spinozin O were combined, concentrated to 200 ml, and desalted in the same manner as Spinozin O. The eluted fractions containing ingredient K with a purity of &gt;98% were collected, concentrated to dryness, and freeze-dried with t-BuOH to obtain Spinozin K (11.1 g; &gt;99% purity).

<u>Example 9 Isolation of Spinozin K, Spinozin O and Spinozin Y from strain NRRL 18743</u>

The fermentation medium (260-L) was prepared roughly according to the method described in Part B of the above Example. After adjusting the pH to 3.0 with 5N HCl, acetone (260-L) was added to the entire medium. The resulting mixture was filtered through a ceramic filter to obtain a filtrate (480-L), and the filtrate was kept under refrigeration every other week. The medium/acetone filtrate was adjusted to pH 12 with 25% NaOH and the filler was filled to a steel tube column (10-L, 10 cm×122cm) containing HP-20ss resin (Mitsubishi Chemical Industries, Ltd., Japan) with a flow rate of 0.5 L/min ) Filter 2 times through ceramic filter before. Take CH₃CN-CH₃OH-0.1%aq.NH₄OAc (Using NH₄OH adjusted to pH 8.1) (25:25:50:20-L). Use CH₃CH-CH₃OH-0.1%aq.NH₄OAc (Using NH₄OH was adjusted to pH 8.1) (95:95:10:30-L) Spinozin K, O and Y were precipitated at a flow rate of 1-L/min. Concentrated liquid (30-L) re-dissolved in CH₃OH, then concentrate to dryness, re-dissolve in CH₃OH (100 ml), followed by CH₃Precipitation in CS (2-L). Remove the resulting precipitate by filtration, with CH₃CN cleans the precipitate, and discards the sediment: Concentrate the combined filtrate and cleaning solution (3-L) to dryness. The obtained residue was redissolved in dichloromethane (50 ml) and placed in a silica gel (EM grade 62.60-200 mesh) column (7.5 cm×50 cm) equilibrated in acetone. String to CH₃CN is the eluent, followed by CH₃CN-CH₃OH(9:1:20-L), then CH₃CN-CH₃OH (8:2:10-L), collect 1-L eluted fractions. Collect eluted fractions 11-30 and concentrate to dryness. Dissolve the resulting residue in CH₃OH (50 mL) and put (10 dissolution operations) preparative reverse phase HPLC column (Rainin Dynamax-60A, 8μm Cl8, 41.4 mm ID×25 cm and 41.4 mm×5 cm protective components), the column Is balanced to H₂O-CH₃OH-CH₃CH; (50:175:175, including 0.1% NH₄OAc). The column is operated at a rate of 40 ml/min and a 60-minute drive H₂O CH₃OH-CH₃CN to (50:175:175; containing 0.1; NH₄OAc) to H₂O-CH₃OH CH₃CH(10:45:45; contains 0.1; NH₄OAc) linear gradient precipitation. A variable wavelength UV detector adjusted to 250 nm is used to monitor the separation process. Collection corresponding to a small amount of Spinozin Y (collection part 1, 1-L), Spinozin K (collection part 2, 8-L) and Spinozin O (collection part 3, 4-L) The first 3 peaks of the elution solution (collect the results of 10 elution operations). Spinozin K was concentrated to a small volume, and then desalted by re-chromatography in the same column without elution with buffer. The effluent corresponding to the absorption peak was concentrated to dryness, dissolved in t-BuOH, and freeze-dried to obtain pure Spinozin K (7.3 g). Spinozine O was desalted and freeze-dried in a similar manner to obtain Spinozine O (1.4 g). Desalting Spinozin Y by similar chromatography (Rainin Dynamax-60A 8 pmCl8 column, 21.4 mmID×25 cm, with 21.4 mm×5 cm protective components) and freeze-drying in a similar manner to obtain pure Spinozin Nuoxin Y (46 mg).

<u>Example 10 Preparation of Spinozin J, Spinozin L, Spinozin M, and Spinozin N using NRRL 18719</u>

Utilize Spinner's yeast (<u>Saccharopolyspora spinosa</u>) NRRL18719 culture to obtain Spinozin J, Spinozin L, Spinozin M, and Spinozin N.

<u>Part A: Shaking flask fermentation</u>

A culture of Spinoza NRRL 18743 in the form of freeze-dried pellets or a suspension maintained in liquid nitrogen was inoculated into a nutrient medium having the following composition:<img file="TW487559B_D0012.tif" /><img file="TW487559B_D0013.tif" />

The slant medium or plate medium can be prepared by adding 2.5% agar-agar to the nutrient medium. The inoculated slant medium is cultured at 30°C for about 10 to about 14 days. The heated slant medium culture was scraped with a sterile instrument to loosen the spores and remove and macerate the mycelium clusters. Approximately 1/4 of the pine spores and cultures thus obtained were inoculated into 50 mL of the first-stage vegetative inoculum culture medium. In addition, liquid nitrogen ampoules can be used to inoculate the first-stage medium.

When the culture is kept in liquid nitrogen, use a homogenous-nutrient medium (cultivate at 30°C for 48-72 hours), use a sterile suspension to dilute 1:1 (volume:volume), and distribute to a sterile tube (1.5 ml/tube) prepare ampoules. The suspension contains lactose (100 g), glycerin (200 ml), and deionized water (qs to 1 liter).

Inoculate the liquid nitrogen ampoule into a 500-mL Erlenmeyer flask (or a 250-ml flask containing 50 ml of nutrient medium) containing 100 ml mL of nutrient medium. The culture was incubated at 30°C in a shaker at a speed of 260 rpm around a 2-inch (5.08 cm) circle for 48 hours.

The cultured culture (10% v/v inoculum) liquid nitrogen ampoule, depending on the size of the flask, is inoculated into 50 ml or 100 ml productive medium with the following composition:

<img file="TW487559B_D0014.tif" />

Adjust the pH to pH 7.0 with 1N NaOH and sterilize at 120°C for 40 min.

*Chemical milk nutrients, Sheffield Products, Norwich, NY13815

**Proflo, graders Protein, Memphis TN 38018

***The amount of methyl oleate is 30 ml.

The inoculated productive medium was cultured in a 250-ml or 500-ml flask, and it was cultured for 7 to 10 days at 37° C. in a shaker at a speed of 260 rpm around a 2-inch (5.08 cm) circle.

<u>B. Stirred reactor fermentation</u>

In order to provide a large amount of inoculum, 10 ml of the first-stage medium prepared as described in section A above was inoculated to 400 ml of the second-stage nutrient medium with the same composition as the first-stage medium. The second-stage medium was cultured in a 2-L wide-mouth flask, and cultured for 48 hours at 30°C in a shaker at a speed of 260 rpm around a 2-inch circle. The second-stage nutrient medium (2L) of the culture was inoculated into 80 to 115 liters of sterile productive medium prepared as described in section A above.

Ferment the inoculated productive medium in a 165-L stirred bioreactor at a temperature of 30°C for 7 to 10 days. The air flow in the stirring tank and the speed of the stirrer are controlled by a computer to maintain the dissolved oxygen concentration at or above 60% to about 80% air saturation.

The following table shows the amount of Spinozin J, Spinozin L, Spinozin M, Spinozin M, and Spinozin N prepared from the NRRL 18719 culture.<img file="TW487559B_D0015.tif" /><img file="TW487559B_D0016.tif" />

<u>Example 11 Preparation of Spinozin J, Spinozin L, Spinozin M, and Spinozin N using culture NRRL 18719</u>

The fermentation medium (105-L) was prepared by the above method, and the pH was adjusted to 10 (initial pH 6.8) by adding 5N NaOH. The resulting mixture was filtered through a ceramic filter. The filtrate was discarded, a mixture of acetone and water (1:1, 50L) was added to the mycelial solid, and the resulting mixture was filtered. A second mixture of acetone and water (1:1, 50 L) was added to the mycelial solids, and the resulting mixture was adjusted to pH 3.0 with 25% sulfuric acid. The resulting mixture was filtered, and a third mixture of acetone and water (1:1 50 L) was added to the mycelial solid. The resulting mixture was filtered and the acidic filtrates were combined.

The combined filtrates were extracted with hexane (10 L). Separate the phases and add a second portion of hexane (10 L) to the water phase. The pH of the resulting mixture was adjusted to pH 10 using 5N NaOH. Dilute the resulting emulsion with 50 L of water. The phases were separated and the hexane was extracted with a third portion of hexane (10 L). The phases are separated and the second and third hexane extracts are combined and concentrated to a volume of about 4 liters. When standing, the concentrate is divided into three phases, an aqueous phase, an emulsified liquid phase, and an organic phase. The organic phase was freeze-dried to obtain 15.29 g of crude product.

The crude product was dissolved in methanol (500 mL), filtered, and concentrated to dryness under reduced pressure. Dissolve the residue in methanol (20 ml) in the second part and put it into LH-20SEPHADEX (Pharmacia LKB Biotechnology, Inc. Piscataway, NJ, 7.5 cm×46 cm), elute with methanol and collect 25 ml of eluent share. Using an HPLC system, the eluted fractions were analyzed to determine the eluted fractions containing Spinozin compound. Combine the eluted fractions 18-50 and concentrate to dryness.

The residue was dissolved in a mixture of methanol, acetone and water (5:5:1), and a 1 ml portion was transferred to a preparative reverse phase HPLC column (Rainin Dynamax-60A, Cl8, 41.4 mm×300 mm, 8 mm particles, 60 holes, Woburn, MA) medium chromatography. The column is eluted with a mixture of methanol, acetone and water (87.5:87.5:25) added to a final concentration of 0.1% (pH 7.6) with ammonium acetate. The eluted fractions were analyzed by HPLC system, and similar eluted fractions were combined and concentrated to obtain three semi-pure concentrates A, B, and C.

The semi-pure concentrate C was re-chromatographed on the system described in the preceding paragraph, and re-chromatography was performed 10 times, with 200 ml filling for each re-chromatography operation. The elution of each operation is combined and concentrated to obtain the products Cl and C2. Preparation C2 was subjected to a third chromatography; however, water was used to replace 0.1% ammonium acetate (desalting step). The eluted fractions containing Spinozin L with an HPLC purity of at least 99.5% were combined and concentrated. The residue was crystallized from ethanol/water (1:1) to obtain 2.4 g Spinozin L.

Combine preparation C2 and semi-pure concentrate B and desalinate as described in the previous paragraph (12×200 mL operation); however, the desired compound is a combination of methanol, acetone and water (11:11:3) The mixture dissolves. The eluted fractions containing Spinozin J with an HPLC purity of at least 99.5% are combined and concentrated. The residue was dissolved in hot t-butanol and freeze-dried to obtain 4.3 g Spinozin J.

The semi-pure concentrate A was chromatographed according to the above method, but the desired compound was eluted with a mixture of methanol, acetone and water (37.5:37.5:25) added with ammonium acetate to a final concentration of 0.1%. The eluted fractions of each operation (4 times in total) were combined and concentrated to obtain preparations A1, A2, and A3.

The aforementioned column chromatography preparation A1 was used; however, the column was eluted with a mixture of methanol, acetone and water (2:2:1). The eluted fractions containing Spinozin M with an HPLC purity of at least 99.5% are combined and concentrated. The residue was dissolved in hot t-butanol and freeze-dried to obtain 136 mg Spinozine M.

Preparation A2 was chromatographed and processed according to the method described in the preceding paragraph to obtain 71 mg Spinozin N.

<u>Example 12 Preparation of Spinozin Q using culture NRRL 18823</u>

<u>Part A. Shaking flask fermentation</u>

A culture of Spinoza NRRL 18823 in the form of freeze-dried pellets or a suspension maintained in liquid nitrogen was inoculated into a nutrient medium having the following composition:

<img file="TW487559B_D0017.tif" />

Can be inoculated into the first-stage medium by liquid nitrogen ampoules. This ampoule is prepared by homogeneous culture (48-72 hours incubation, 30°C), diluted 1:1 (volume:volume) with a sterile suspension, and distributed into sterile tubes (1.5ml/tube) . The suspension contains pore sugar (100 g), glycerin (200 ml), and deionized water (qs to 1 L). Inoculate the liquid nitrogen ampoule into a 250-ml jar containing 50 ml nutrient medium. The culture was cultured for 48 hours in a shaker at a speed of 250 rpm around a 2-inch (5.08 cm) circle at 32°C.

The cultured culture (5% v/v inoculum) was inoculated into a 50 mL Erlenmeyer flask containing a productive medium with the following composition:<img file="TW487559B_D0018.tif" /><img file="TW487559B_D0019.tif" />

The inoculated productive medium was cultured in a 250-mL Erlenmeyer flask, and it was cultured in a 2-inch circle at a rate of 250 rpm at 30°C for 7 days.

<u>B. Stirred bioreactor fermentation</u>

In order to provide a large amount of inoculum, inoculate 10 mL of the 10 mL first-stage nutrient medium prepared in part A above to 400 mL of the second-stage nutrient medium with the same composition as the first-stage nutrient medium. The second-stage nutrient medium was cultured in a 2 L wide-mouthed Erlenmeyer flask at 32°C at a rate of 260 rpm around a 2-inch circle for 48 hours. The prepared second-stage nutrient medium (2 L) was used to inoculate 115 liters of sterile productive medium as described in section A above.

The inoculated productive medium was fermented in a 165-L stirred bioreactor at a temperature of 30°C for 7 days. The air flow in the stirring tank and the speed of the stirrer are controlled by a computer to maintain the dissolved oxygen concentration at or above 60% to about 80% air saturation.

<u>Example 13 Separation of Spinozin Q, Spinozin R, Spinozin S and Spinozin T from the culture NRRL 18823</u>

The fermentation medium (100 L; the titer of Spinozin H obtained was 303 pg/ml, and the titer of Spinozin Q was 50 pg/ml) was prepared according to the method described in the above examples, and it was refrigerated for 2 days before treatment. After adjusting the pH to 3.0 with 5N HCl, acetone (100 L) was added to the entire medium. The resulting mixture was filtered through a ceramic filter to obtain a filtrate (170 L), which was kept under refrigeration until every other week. The culture medium/acetone filtrate was adjusted to pH 13 and filtered through a ceramic filter before being filled into a steel tube (10 L; 10×122 cm). The column contained HP-20SS resin (Mitsubishi Chemical Industries, Ltd., Japan). ) And the flow rate is 1 L/min. This column is at a flow rate of 1 L/min, using a solvent "A" (0.1%aq.NH₄OAc, use NH₄OH adjusted to pH 8.1) and solvent "B" (CH₃CN CH₃OH1: 1) Gradient of mixing, collect 4 L of eluted fractions. The pump system is programmed to generate a gradient from 0 to 50% B in 1 minute, then a gradient from 50 to 100% B in 90 minutes, and then 100% B power is delivered equally for 15 minutes. HPLc analysis indicated that the fraction 17 (4 L) contained mostly Spinozin R and other more polar substances and a small amount of Spinozin T and H; fraction 18-22 contained mostly Spinozin Spinozin H and a small amount of Spinozin R and Q, and a small amount of Spinozin S and more polar substances; the eluted fraction 23-24 contains Spinozin H and Q. The HPLC analysis of the collected part suggests the following total amount: Spinozin H 23.0 g; Spinozin Q 3.4 g; Spinozin R2.0 g; Spinozin S 0.2 g; Spinozin T 0.2 g.

<u>Example 14 Recovery of Spinozin Q, Spinozin R, Spinozin S, and Spinozin T from Spinozin-producing strains</u>

Fermentation medium (85 L; Spinozin H obtained with a titer of 302 pg/ml, and Spinozin Q with a titer of 44 pg/ml) prepared from the Q-producing strain, and refrigerated overnight before treatment. After adjusting the pH to 3.0 with 5N HCl, acetone (90 L) was added to the entire medium. The resulting mixture was filtered through a ceramic filter to obtain a filtrate (176 L), which was kept under refrigeration until every other week. The medium/acetone filtrate was adjusted to pH 13 with 50% NaOH and filtered through a ceramic filter (140L filtrate) before filling the steel pipe (10 L; 10×122 cm). The column contained HP-20SS resin ( Mitsubishi Chemical Industries, Ltd., Japan) and the flow rate is 1 L/min. This column is tied to a flow rate of 1 L/min, using the solvent "A" (0.1%aq.NH₄OAc, use NH₄OH adjusted to pH 8.1) and solvent "B" (CH₃CNCH₃OH 1:1) Gradient of mixing, collect 4 L (approximate value) of eluted fractions. The pump system is programmed to generate a gradient from 0 to 50% B in 1 minute, then a gradient from 50 to 100% B in 90 minutes, and then 100% B power is delivered equally for 15 minutes. HPLC analysis indicated that collection part 1 (dissolved fractions 16-21; 24.5L) contained Spinozin H (12.32g) and Q (0.34g); collection part 2 (dissolved fractions 22-25; 16 L) Contains Spinozin H (4.66 g), Q (2.06 g), R, S and T.

<u>A. Separation of pure ingredient Q</u>

Concentrate the collection part 2 to dryness, redissolve it in dichloromethane (50 ml), and fill it with a glass column (5.5 cm) containing silica gel (EM grade 62, 60-200 mesh) and equilibrated in dichloromethane ×30 cm). The column was washed with dichloromethane (2 L), and then the eluent was developed with dichloromethane-methanol (95:5) to collect 250 ml of eluent. The fractions 3 obtained through 15 operations were combined and concentrated to a residue, then dissolved in ethanol/water (400 ml) and allowed to maintain at room temperature every other week. The obtained crystals were washed with cold ethanol/water (1:1) and dried to obtain 6.1 g of dry crystals, which were found to contain 68.7% Spinozin H and 31.2% Spinozin Q by HPLC analysis. The dry crystalline substance 2 was dissolved in tetrahydrofuran/methanol (1:1) and placed in a preparative reverse phase HPLC column (Rainin Dynamax60A 8pm Cl8, 41.4mmID×25 cm, with 41.4mm×5 cm) in 12 operations. Protective components). The column is eluted at a flow rate of 50 ml/min, using the solvent "A" (H₂O-CH₃CN-CH₃OH; 30: 35: 35, containing 0.1% NH₄OAc) and solvent "B" (H₂O-CH₃CN-CH₃OH 10:45:45, containing 0.1% NH₄OAc) Gradient of mixing. The pump system is programmed to generate a gradient from 50 to 100% B within 60 minutes. The separation process is monitored by a variable wavelength UV detector adjusted at 250 nm.

Peak 1 contains Spinozine H (99%; 6 L) that precipitates first, followed by Spinozine Q. Combine peak 2 of all operations (12 times) (containing Spinozin H 20%, ingredient Q 80%; 8 L) and concentrate to 500 ml, then put it in the same column under the same mobile phase conditions Operate 5 times. Collect part 2 (2 L) and balance it with H₂O-CH₃OH-CH₃The same column of CN (20:40:40) to desalt with 99% pure Spinozin Q. The string is H₂O-CH₃OH-CH₃CN (10:45:45) was eluted, and 10 eluted parts were collected (one part every 3 minutes). Combine fractions 2 to 7, concentrate to a residue, and dissolve in hot ethanol (80 ml). Add equal amount of H₂O and allow the solution to cool overnight. Collect the resulting crystals in a filter and cool the EtOH-H₂O (1:1) washing, and drying to obtain 1.5 g of pure Spinozin Q.

<u>Example 15 Insecticide composition</u>

<u>A. Aqueous suspension</u>

<img file="TW487559B_D0020.tif" /><img file="TW487559B_D0021.tif" />

<img file="TW487559B_D0022.tif" />

<u>Example 17 Synthetic modification</u>

<u>Part A rhamnose-modified derivatives</u>

<u>Example A1-(Compound 9-O-(5,6-dihydro derivative of 2,3,4-tri-O-ethyl-α-L-rhamnosyl) Spinozin A 9-Psa</u>

The compound 9-O-(2,3,4-tri-O-ethyl-α-L-rhamnosyl) Spinozin A 9-Psa (106 mg, 0.137 mmol) was dissolved in 5 mL of toluene. Add \-triphenylphosphine rhodium(I) chloride (11.9 mg, 0.0128 mmol). The head space of the flask was evacuated and nitrogen was introduced 3 times. Vacuum and introduce hydrogen 3 times. Use a spherical flask to maintain the flask in hydrogen and heat to 110-120°C. After 3.5 hours, the flask was cooled to room temperature and the hydrogen was evacuated and replaced with nitrogen. The toluene solvent was evaporated and replaced with 20 mL of ether. The ether solution was extracted with 3×10 mL 1H HCl. Combine the acid extracts and neutralize with 10 ml 4N NaOH. The neutralized suspension was extracted with 3×10 mL ether, and the ether extracts were combined, washed with 20 mL brine, and washed with K₂CO₃Dry and evaporate under reduced pressure. Available compound 9-O-(2,3,4-tri-O-ethyl-α-L-rhamnosyl 5,6-dihydro derivative) Spinozin A 9-Psa compound 5 ,6-Dihydro derivative, 39.4 mg, 36.8% part¹H-NMR δ 6.84 (1H, bs), 1.01 (1H, m), 0.68 (1H, m).

<u>Example A2-2'-O-Dichloroacetyl Spinozin Q</u>

Dissolve the compound Spinozin Q (302 mg, 0.412 mmol) in dry CH₂Cl₂(8 ml). The solution was stirred under nitrogen and DMAP (diisopentyl methyl phosphonate, 269 mg, 2.20 mmol) was added. Then dichloroacetic anhydride (492 mg, 315 μl, 2.05 mmol) was introduced. After 45 minutes, pyridine (1.5 ml) was added, followed by EtOAc (50 ml) and toluene (50 ml). Take 5%aq.NaHCO₃(3×) Extract solution. Take anh.Na₂SO₄The organic phase is dried and dried under reduced pressure. The residue is flashing SiO₂Purified on a column (30 g/EtOAc followed by 5% EtOH in EtOAc). The compound 2'-O-dichloroacetylspinoxine Q obtained from the purely eluted fractions;

<img file="TW487559B_D0023.tif" />

<u>Example A3-2'-O-Trifluoroacetyl Spinozin Q</u>

Dissolve the compound Spinozin Q (330 mg, 0.451 mmol) in dry CH₂Cl₂(10 ml). The solution was stirred under nitrogen and DMAP (diisopentyl methyl phosphonate, 306 mg, 2.50 mmol) was added. Then trifluoroacetic anhydride (473 mg, 320 μl, 2.25 mmol) was introduced. After 14 hours, pyridine (1.5 ml) was added, followed by EtOAc (50 ml) and toluene (50 ml). Take 5%aq.NaHCO₃(3×) Extract solution. Take anh.Na₂SO₄The organic phase is dried and dried under reduced pressure. The residue is flashing SiO₂Purified on a column (100 g/EtOAc followed by 5% EtOH in EtOAc). The compound 2'-O-Trifluoroacetosin Q; 199 mg, 54%¹H NMR(CDCl₃)δ6.69 (1H, bs), 5.43 (1H, bs), 5.26 (1H, dd: 2.9, 1.6 Hz), 1.66 (3H, CH₃, Bs);¹³C-NMR(CDCl₃) δ 156.7 (d: 32.5 Hz), 114.0 (q: 286 Hz) ppm.

<u>Example A4-2'-O-(p-trifluoromethyl)benzyl spinoxin Q</u>

Dissolve the compound Spinozin Q (430 mg, 0.587 mmol) in dry CH₂Cl₂(10ml). DMAP (diisopentyl methyl phosphonate, 367 mg, 3.0 mmol) was added, followed by trifluoromethyl benzyl chloride (613 mg, 440 ml, 2.94 mmol). After stirring for 2 hours at room temperature, the reaction mixture was diluted with EtOAc (100μl) and toluene (30 ml), and the solution was continuously filled with brine and 5% aq.NaHCO₃(3×)Cleaning. Take anh.Na₂SO₄The organic layer is dried and dried under reduced pressure. The residue is flashing SiO₂Separate on a column (100 g/EtOAc followed by 5% EtOH in EtOAc) to obtain the compound 2'-O-p-trifluoromethyl)benzylspinoxine Q; 473 mg, 89%¹H NMR(CDCl₃)δ8.13 (1H, bd: 8.1 Hz), 7.68 (1H, bd: 8.2 Hz), 6.73 (1H, bs), 5.45 (1H, bs), 5.42 (1H, dd: 3.3, 1.8 Hz), 1.69 (3H, CH₃, Bs): Elemental analysis: for C₄₉H₆₈NO₁₁F₃Estimated value: C 65.10, H 7.58, N 1.55: Actual value: C 64.89, H 7.55, N 1.56.

<u>Example A5-(5S,6R)-epoxy-2'-O-(p-trifluoromethyl)benzyl spinoxin Q</u>

The compound (5S, 6R)-epoxy-spinoxine Q (406 mg, 0.543 mmol) was dissolved in dry CH₂Cl₂(10 ml). DMAP (diisopentyl methyl phosphonate, 369 mg, 3.0 mmol) was added, followed by trifluoromethyl benzyl chloride (623 mg, 445 μl, 2.98 mmol). After stirring for 14 hours at room temperature, triethylamine (2 ml) was added and stirring was continued for 30 minutes. The reaction mixture was diluted with EtOAc (100 ml) and toluene (30 ml), and the solution was continuously filled with brine, 5% aq. NaHCO₃(2×) and water (1×) cleaning. The organic layer was concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to provide the compound (5S,6R)-epoxy-2'-O-(p-trifluoromethyl)benzylspinoxine Q; 391 mg, 78% IR v 1728, 1664, 1324, 1272 cm^-1；¹H NMR(CDCl₃)δ8.1²(1H, bd: 8.² Hz), 7.6⁷(1H, bd: 8.2 Hz), 6.54 (1H, bs), 5.42 (1H, dd: 3.2, 1.8 Hz), 1.31 (3H, CH₃, Bs).

<u>Example A6-2'-O-Dichloroacetyl Spinozin H</u>

The compound Spinozin H (170 mg, 0.237 mmol) was dissolved in dry pyridine (3 ml). DMAP (diisopentyl methyl phosphonate, 30 mg, 2.46 mmol) was added. Stir at room temperature and add dichloroacetic anhydride (0.077 ml, 120 mg, 0.50 mmol). After 30 minutes, toluene (50 ml) and EtOAc (50 ml) were added. Then rinse the solution with water, then 5%aq.NaHCO₃(3×)Cleaning. Take Na₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (15 g/EtOAc) to provide the compound 2'-O-dichloroacetylspinoxine H; 155 mg, 79% CI MS m/z 830(M+3), 828(M+1 ):¹H NMR(CDCl₃) δ 6.70 (1H, bs), 5.99 (1H, s), 5.18 (1H, s), 5.18 (1H, dd: 3.0, 2.0 Hz).

<u>Example A7-(2'R)-(diethyl)phosphite Spinozin H</u>

The compound Spinozin H (351 mg, 0.489 mmol) was dissolved in dry pyridine (5 ml). DMAP (diisopentyl methyl phosphonate, 62 mg, 0.51 mmol) was added and stirring was continued for 5 minutes at room temperature under nitrogen. Add diethyl chlorophosphite (157 mg, 0.145 ml, 1.0 mmol). After 30 minutes, toluene (50 ml) and EtOAc (50 ml) were added. Continuously use salt water (2×) and 5%aq.NaHCO₃Wash the solution and concentrate. The residue is flashing SiO₂Separate on a column (100g/EtOAc) to obtain compound 2'R-(diethyl)phosphite Spinozin H; 306 mg, 75% CI MS m/z 838(M+1):¹H NMR(CDCl₃)δ6.69(1H, bs), 4.40(1H, m), 3.83(4H, 2×CH₂, M), 1.18 (6H, 2×CH₃, M); elemental analysis: for C₄₄H₇₂NO₁₂Estimated value of P: C 63.06, H 8.66, N 1.67; actual value: C 62.94, H 8.88, N 1.71.

<u>Example A8-(2'R)-(Diethyl) phosphorothioate Spinozin H</u>

The compound Spinozin H (261 mg, 0.364 mmol) was dissolved in dry pyridine (3 ml). DMAP (diisopentyl methyl phosphonate, 60 mg) was added, followed by diethyl chlorothiophosphate (152 mg, 125 μl, 0.80 mmol). The reaction mixture was stirred at room temperature under nitrogen. After 12 hours, toluene (50 ml) and EtOAc (50 ml) were added. First with salt water and then with 5%aq.NaHCO₃Extraction solution. Take Na₂SO₄The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (30g/EtOAc) to obtain compound 2'R-(diethyl)thiophosphate Spinozin H; 84 mg, 27% CI MS m/z 878(M+1);¹H-NMR(CDCl₃)δ6.74(1H, bs), 4.80(1H, m), 4.22(4H, 2×CH₂, M), 1.30 (6H, 2×CH₃, M).

<u>Example A9-2'-O-(Ethyl) ethanedioxin H</u>

The compound Spinozin H (720 mg, 1.003 mmol) was dissolved in dry dichloroethane (15 ml). DMAP (diisopentyl methyl phosphonate, 80 mg) was added, followed by dry triethylamine (2 ml). The reaction mixture was stirred at room temperature under nitrogen. Add ethyl ethylene diacyl chloride (1 ml). After stirring for 1 hour, the reaction mixture was diluted with toluene (50 ml) and EtOAc (100 ml). The solution is continuously used in saline, 5%aq.NaHCO₃(3×), and water (1×) cleaning. Take Na₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified on a column (110 g/EtOAc) to obtain the 2'-O-(ethyl) ethanedioxin H of the compound Spinozin H; 668mg, 81%CI MS m/z818(M+1 );¹H-NMR(CDCl₃)δ6.69 (1H, bs), 5.22 (1H, dd: 3.0, 1.8 Hz), 4.27 (2H, q: 7.2 Hz), 1.29 (3H, t: 7.2 Hz); elemental analysis: for C₄₄H₆₇NO₁₃Estimated value: C 64.60, H8.26, N 1.71: Actual value: C64.63, H 8.41, N 1.80.

<u>Example A10-2'-O-Trichloroacetyl Spinozin H</u>

The compound Spinozin H (295 mg, 0.411 mmol) was dissolved in dry pyridine (5 ml). Trichloroacetoxychloride compound (1 ml, excess) was added and the reaction mixture was stirred at room temperature under nitrogen for 14 hours. Toluene (50 ml) and EtOAc (50 ml) were added. Take 5%aq.NaHCO₃Extraction solution. Take Na₂SO₄The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified on a column (60g/EtOAc) to obtain the compound 2'-O-Trichloroacetylspinoxine H; 249 mg, 70% CI MS m/z 866(M+5), 862(M+1) ;¹H-NMR(CDCl₃)δ6.70(1H, bs), 5.18(1H, /dd: 2.2, 1.8Hz), 2.16(6H, 2×CH₃, S).

<u>Example A11-2'-O-Chloroacetyl Spinozin H</u>

The compound Spinozin H (306 mg, 0.43 mmol) was dissolved in dry pyridine (5 ml). Chloroacetin (0.50 ml) was added dropwise. The reaction mixture was stirred at room temperature under nitrogen. After 3 hours, toluene (50 ml) and EtOAc (50 ml) were added. Continuously use salt water, 5%aq.NaHCO₃And water cleaning solution. Take anh.Na₂SO₄The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80g/EtOAc) to provide compound 2'-O-Chloroacetylspinoxine H; 71 mg, 21%¹H-NMR(CDCl₃)δ 6.72 (1H, bs), 5.16 (1H, dd: 2.2, 1.8 Hz), 3.69 (2H, bs);¹³C-NMR(CDCl₃)d 169.9(C=0), 41.4(-CH₂Cl) ppm.

<u>Example A12-(3'R)-2'-keto-3'-methoxy-3'-(thiomethyl)methylspinoxine H</u>

The compound Spinozin H (3.10 g, 4.32 mmol) was dissolved in dry DMSO (8 ml). Stir the mixture at room temperature under nitrogen and add triethylamine (3 g, 7 equivalents), and then add Py-SO₃Complex (2.06 g, 12.9 mmol). The reaction mixture was stirred at room temperature for 14 hours. Toluene (100ml) and EtOAc (100ml) were added. Continuously use salt water, 5%aq.NaHCO₃And water extraction solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Column (SiO₂/EtOAc) and then separated by HPLC on a reverse phase C-18 column with 10% water in methanol as the mobile phase. Evaporation of the pure fraction obtained can provide compound (3'R)-2'-keto-3'-methoxy-3'-(thiomethyl)methylspinoxine H; 697 mg, 39%IR v1738, 1721, 1661, 1038 cm^-1; EI MS m/z 776(M+);¹H-NMR(CDCl₃)δ6.71(1H, bs), 3.99(1H, m: W_h/2=18Hz), 3.53(3H, CH₃, S), 3.36 (3H, CH₃, S), 2.19 (6H, 2×CH₃, Bs), 2.08 (3H, CH₃, S).

<u>Example A13-2'-O-Acetyl-2'-episospinoxine H and 2'-episospinoxine H</u>

Dissolve the compound 2'-ketospinoxine H (2.95 g, 4.12 mmol) in dry Et₂O (60 ml). Cool the solution to 0°C under nitrogen and add Li(T-BuO)₃AlH (97%, 1.62 g, 6.18 mmol, 1.5 equivalents). Stirring was continued for 20 minutes at 0°C. The reaction mixture was carefully quenched with brine. Ether (50 ml) was added and the phases were separated. Take 5%aq.NaHCO₃(4×) Clean the organic layer to anh.K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Column (SiO₂/EtOAc). Therefore, it is possible to obtain 2'-epimer alcohols that are not separated on silica or by HPLC on C-18/acetone-MeOH-water (60-30-10). Dissolve 2'-alcohols (1.84 g, 2.56 mmol) in dry CH₂Cl₂(10 ml). Add pyridine (5 ml) and DMAP (80 mg), then add Ac₂O (3 ml, excess). Stir at room temperature under nitrogen for 8 hours. The reaction mixture was diluted with EtOAc (50 ml) and benzene (50 ml). The solution is continuous with saline (2×) and 5%aq.NaHCO₃(2×)Cleaning. Take Na₂SO₄The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Chromatography in a column (230-400 m, 120 g/EtOAc) to obtain pure compound a) 2'-o-acetyl-2'-epispinoxine H; 1.176 g, 60%,¹H-NMR(CDCl₃)δ6.67 (1H, bs), 4.83 (1H, d: 3.8 Hz), 4.52 (1H, dd: 10.0, 3.8 Hz), 3.47 (6H, 2×CH₃, S), 2.14(6H, 2×CH₃, S), 2.01 (3H, CH₃, S) ppm. Add the compound 2'-O-acetyl-2'-epi-spinoxine h (280 mg, 0.368 mmol) dissolved in EtOH (5ml), and then add 10% aq.K₂CO₃Solution (3 ml). The reaction mixture was stirred at room temperature under nitrogen for 15 minutes. Brine (10 ml) was added, followed by EtOAc (50 ml) nhe PhH (50 ml). Separate the phases. Wash the organic layer with water (3×), and use K₂CO₃Dry and concentrate under reduced pressure. Residue flash SiO₂Purified in a column (80 g/EtOAc) to obtain compound b) 2'-epi-spinoxine H; 228 mg, 86%¹H-NMR(CDCl₃)δ6.67(1H, bs), 4.68(1H, 3.8Hz), 3.55(3H, CH₃, S), 3.46 (3H, CH₃, S), 2.14 (6H, CH₃, S) ppm;¹³C-NMR(CDCl₃)δ203.2, 172.9, 147.9. 144.7, 144.5, 129.7, 129.3, 103.9, 97.6, 86.1, 84.9, 81.1, 77.3, 77.1, 74.1, 73.0, 67.6, 65.4, 61.3, 61.1, 49.8, 48.2, 48.1, 46.5 , 42.0, 41.7, 41.2 (2×C), 37.9, 37.0, 34.8, 34.6, 31.4, 30.6, 28.9, 22.2, 19.5, 18.9, 18.2, 16.6, 9.9ppm.

<u>Example A14-2'-O-Trimethylsilyl Spinozin H</u>

Dissolve the compound Spinozin H (1.59 g, 2.21 mmol) in CH₂Cl₂(10 ml) and DMAP (0.80 g) was added. The mixture was stirred at room temperature under nitrogen. TMS trifluoromethanesulfonate (0.90 ml, excess) was slowly introduced into the mixture via an injection syringe and stirring was continued for 1 hour. EtOAc (50 ml) and PhH (50 ml) were added. Take 5%aq.NaHCO₃(3×) Cleaning solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (100 g/EtOAc) to obtain the compound 2'-O-dimethylsilyl Spinozin H; 1.615 g, 92% mp=163°C(Et₂O): EIMS 791(M+);¹H-NMR(CDCl₃)δ6.69(1H, bs), 3.81(1H, dd: 2.6, 2.0Hz), 3.46(3H, CH₃, S), 3.38 (3H, CH₃, S), 2.16(6H, 2×CH₃, Bs) ppm.

<u>Example A15-2'-O-Ethyl Spinozin H</u>

Dissolve the compound Spinozin H (2.21 g, 3.08 mmol) in CH₂Cl₂(60 ml), add 10% NaOH aqueous solution (25 ml) and K₂CO₃Solid (5g), followed by diethyl sulfate (8ml, excess). The reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Join H₂O (50 ml) and CH₂Cl₂(50ml), the phases are separated, the organic layer is separated by H₂O(2×) cleaning, with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Separated by column chromatography (230-400m, 250 g/EtOAc). The pure fraction can provide the compound 2'-O-ethyl Spinozin H; 635 mg, 28% CI MS m/z 746(M+1);¹H-NMR(CDCl₃)δ6.71(1H, bs), 3.61(2H, CH₂, M), 3.49 (3H, CH₃, S), 3.42 (3H, CH₃, S), 2.17 (6H, 2×CH₃, S), 1.18 (3H, CH₃, T: 7.0 Hz) ppm;¹³C-NMR(CDCl₃)δ67.0(-CH₂-), 16.0(-CH₃)ppm; C₄₂H₆₇NO₁₀Elemental analysis: estimated value: C 67.61, H 9.07, N 1.88; actual value: C 67.80, H 1.87, N 9.20.

<u>Example A16-(2'R)-(ethyl) carbonate of Spinozin H</u>

Triphenylphosphine (6.5 g, 24.8 mmol) was dissolved in dry THF (30 ml). Diethyl azodicarboxylate (4.8 ml. 5.32 g, 30.5 mmol) was added and the mixture was stirred at room temperature for 5 minutes. Add pure ethanol (1.43 ml1.14 g, 24.8 mmol). After stirring for another 5 minutes, the compound (1.20 g, 1.67 mmol) was introduced. Stirring was continued for 24 hours at room temperature under nitrogen. Add ethyl acetate (50 ml) and benzene (100 ml). With salt water, 5%aqNaHCO₃(2×) and water (1×) continuous cleaning solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Separated in a column (120 g/EtOAc) to obtain Spinozin H (2'R)-(ethyl) carbonate compound; 233 mg, 18% IR v 1747, 1720, 1645, 1263 cm^-1；¹H-NMR(CDCl₃)δ 6.74 (1H, bs), 4.95 (1H, dd: 3.3, 1.8 Hz), 4.19 (2H, q: 7.1 Hz) ppm; C₄₃H₆₇NO₁₂Elemental analysis: estimated value: C 65.37, H 8.55, N 1.97; actual value: C 65.44, H8.49, N 2.05.

<u>Example A17-2'-O-Trifluoromethanesulfonyl Spinozin H</u>

Spinozin H compound (2.12 g, 2.95 mmol) was dissolved in dry 1,2-dichloroethane (3 ml). Add pyridine (3 ml). The solution was cooled to 0°C under nitrogen. Add trifluoromethanesulfonic anhydride (1.20 ml, 7.0 mmol). After stirring for 1 hour, PhH (50 ml) and EtOAc (100 ml) were added. Solution with 5%aqNaHCO₃(3×)Cleaning. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Separate on a column (160 g/EtOAc) to obtain 2'-O-trifluoromethanesulfonyl spinoxin H; 1.77 g, 71% IR v 1415, 1212, 1068 and 918 cm^-1；¹H-NMR(CDCl₃) δ 6.72 (1H, bs), 4.93 (1H, dd: 2.8, 1.9 Hz) ppm.

<u>Example A18-2'-On- propan Jishibinnuo embodiment oct H</u>

Dissolve the compound Spinozin H (1.22 g, 1.70 mmol) in CH₂Cl₂(20 ml) and add 25% NaOH aqueous solution (15 ml), then add K₂CO₃(2 g), tetrabutylammonium chloride (1.2 g) and yltriethylammonium chloride (1.5 g). 1-iodopropane (10 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Join CH₂Cl₂(20 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Separated by column chromatography (120 g/EtOAc). The concentrate of the pure eluted fraction can provide the compound 2'-On-Propyl Spinozin H; 535 mg, 41%)¹H-NMR(CDCl₃)δ6.67(1H, bs), 2.13(6H, 2×CH₃, X), 0.81 (3H, CH₃, T: 7.4 Hz) ppm; C₄₃H₆₉NO₁₀Elemental analysis: estimated value: C 67.95, H 9.15, N 1.84; actual value: C 67.74, H9.37, N 1.84.

<u>Example A19-2'-On-Pentyl Spinozin H</u>

Dissolve the compound Spinozin H (1.02 g, 1.42 mmol) in CH₂Cl₂(20 ml) and add 25% NaOH aqueous solution (15 ml), then add K₂CO₃(3.5 g), tetrabutylammonium chloride (0.8 g), and triethylammonium chloride (1.3 g). 1-iodopropane (15 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(20 ml) and separate the phases. Wash the organic layer with water (3×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Separated by column chromatography (200 g/EtOAc). The concentrate of pure eluted fractions can provide the compound 2'-On-Pentyl Spinozin

<img file="TW487559B_D0024.tif" />

<u>Example A20-2'-O-kispinoxine H</u>

Dissolve the compound Spinozin H (1.48 g, 2.06 mmol) in CH₂Cl₂(20 ml) and add 25% NaOH aqueous solution (15 ml), then add K₂CO₃(3 g), Tetrabutylammonium Chloride (0.85 g) and Menthyl Triethylammonium Chloride (1.2 g). Phosphorus chloride (10 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(20 ml) and separate the phases. Wash the organic layer with water (3×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Separated by column chromatography (200 g/EtOAc). The concentrate of the pure eluted fraction can provide the compound 2'-O--based Spinozin H;

<img file="TW487559B_D0025.tif" />

<u>Example A21-2'-O-Acetyl Spinozin-Q</u>

Dissolve the compound Spinozin Q (11.0 mg, 0.015 mmol) in dry CH₂Cl₂(3 ml). Add triethylamine (5 ml). Stir the solution at room temperature under nitrogen and add Ac₂0 (0.70 ml, excess). After 20 hours, EtOAc (50 ml) and PhH (20 ml) were added. With salt water, 5%aqNaHCO₃(4×) and water (1×) continuous cleaning solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (5 g/EtOAc) to obtain compound 2'-O-acetylspinoxine Q;

<img file="TW487559B_D0026.tif" />

<u>Example A22-3'-On-Propyl Spinozin J</u>

The compound Spinozin J (331 mg, 0.433 mmol) was dissolved in CH₂Cl₂(10 ml) and add 15% NaOH aqueous solution (5 ml), then add K₂CO₃(0.70 g), tetrabutylammonium chloride (0.3 g), and menthyltriethylammonium chloride (0.83 g). 1-iodopropane (4.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Join CH₂Cl₂(50 ml) and water (50 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (80 g/EtOAc). Concentrate of pure eluted fractions can provide compound 2'-On-propyl history

<img file="TW487559B_D0027.tif" />

<u>Example A23-3'-O-Ethyl Spinozin J</u>

Dissolve the compound Spinozin J (747 mg, 1.04 mmol) in CH₂Cl₂(20 ml) and add 15% NaOH aqueous solution (10 ml), then add K₂CO₃(1.10 g) and yltriethylammonium chloride (1.2 g). 1-Iodoethane (5.5 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Join CH₂Cl₂(50 ml) and water (50 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified in a column (100 g/EtOAc). The concentrate of the pure eluted fractions can provide a) the compound Spinozin J (192 mg) and b) the compound 3'-O-ethyl Spinozin J;

<img file="TW487559B_D0028.tif" />

<u>Example A24-3'-O-Ethyl Spinozin K</u>

The compound Spinozin K (30.6 mg, 0.043 mmol) was dissolved in CH₂Cl₂(5 ml). Add 15% NaOH aqueous solution (5 ml), then add K₂CO₃(0.300 g) and menthyl triethylammonium chloride (0.41 g). 1-Iodoethane (2.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(50 ml) and water (50 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (10 g/EtOAc). The concentrate of the pure eluate can provide a) compound (15.1 mg) and b) compound 3'-O-ethyl spinoxin

<img file="TW487559B_D0029.tif" />

<u>Example A25-3'-O-Ethyl Spinozin L</u>

The compound Spinozin L (330 mg, 0.451 mmol) was dissolved in CH₂Cl₂(15 ml). Add 20% NaOH aqueous solution (10 ml), then add K₂CO₃(1.30 g) and yltriethylammonium chloride (1.1 g). 1-Iodoethane (5.0 ml) was added and the reaction mixture was vigorously stirred at room temperature under nitrogen for 96 hours. Join CH₂Cl₂(50 ml) and water (50 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (85 g/EtOAc). The concentrate of the pure eluted fraction can provide the compound 3'-O-ethyl Spinozin L;

<img file="TW487559B_D0030.tif" />

<u>Example A26-3'-O-Allyl Spinozin J</u>

Dissolve the compound Spinozin J (370 mg, 0.515 mmol) in CH₂Cl₂(15 ml). Add 15% NaOH aqueous solution (10 ml), then add K₂CO₃(1.40 g) and menthyl triethylammonium chloride (0.98 g). Allyl bromide (3.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 28 hours. Join CH₂Cl₂(50 ml) and water (50 ml) and separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue was dissolved in toluene (10 ml) and heated to reflux temperature for 10 minutes. The solution was cooled to room temperature and concentrated under reduced pressure. The residue is flashing SiO₂Purified on a column (80 g/EtOAc). The concentrate of the pure eluted fraction can provide the compound 3'-O-allyl Spinozin J; 121 mg, 31%¹H-NMR(CDCl₃)δ6.67(1H,

<img file="TW487559B_D0031.tif" />

<u>Example A27-3'-O-propargyl Spinozin J</u>

The compound Spinozin J (392 mg, 0.546 mmol) was dissolved in CH₂Cl₂(15 ml). Add 15% NaOH aqueous solution (10 ml), then add K₂CO₃(1.50 g) and menthyl triethylammonium chloride (1.10 g). The propargyl bromide (80% solution in toluene; 4.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 40 hours. Join CH₂Cl₂(50 ml) and water (50 ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue was dissolved in toluene (10 ml) and heated to reflux temperature for 20 minutes. The solution was cooled to room temperature and concentrated under reduced pressure. The residue is flashing SiO₂Purified on a column (80 g/EtOAc). The concentrate of the pure eluted fraction can provide the compound 3'-O-propargyl Spinozin J;

<img file="TW487559B_D0032.tif" />

<u>Example A28-3'-O-(cyclopropyl)methyl spinoxine J</u>

Dissolve the compound Spinozin J (470 mg, 0.655 mmol) in CH₂Cl₂(10 ml). Add 15% NaOH aqueous solution (15 ml), then add K₂CO₃(1.50 g) and menthyl triethylammonium chloride (1.10 g). The (cyclopropyl)methyl bromide compound (.09 ml) was added and the reaction mixture was vigorously stirred at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(50 ml) and water (50 ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (90 g/EtOAc). The concentrate of the pure eluted fractions can provide a) compound Spinozin J (216 mg) and b) compound 3'-O-(cyclopropyl)methyl spinozin J; 25.1 mg, 9%

<img file="TW487559B_D0033.tif" />

<u>Example A29-3'-O-(chloro)methyl spinoxine J</u>

The compound Spinozin J (510 mg, 0.71 mmol) was dissolved in CH₂Cl₂(5.0 ml). Join K₂CO₃(1.10 g). The reaction flask was immersed in a water bath (10°C) and 10% NaOH aqueous solution (10 ml) was added with vigorous stirring, followed by yltriethylammonium chloride (1.20 g). Chloroiodomethane (5.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (70 g/EtOAc). The concentrate of the pure elution fraction can provide a) compound (111 mg) and b) compound 3'-O-(chloro)methylspinoxine J; 120 mg, 28%)¹NMR(CDCl₃)δ

<img file="TW487559B_D0034.tif" />

<u>Example A30-3'-O-(pentafluorophenyl)-thiocarbonyl carbonate of Spinozin L</u>

The compound Spinozin L (733 mg, 1.0 mmol) was dissolved in dry pyridine (5.0 ml). Stir under nitrogen at room temperature (water bath). Within 5 minutes, (pentafluorophenyl) chlorothiocarbonyl carbonate (1.5 ml, excess) was added dropwise. After 10 minutes, PhH (50 ml) and EtOAc (50 ml) were added. The solution is 5%aq.NaHCO₃(3×). The organic layer is anh.Na₂SO₄Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain 3'-O-(pentafluorophenyl)-thiocarbonyl carbonate of Spinozin L; 712 mg, 74%,¹N-

<img file="TW487559B_D0035.tif" />

<u>Example A31-3'-Deoxy-spinoxine L</u>

The 3'-O-(pentafluorophenyl)-thiocarbonyl carbonate (690 mg, 0.72 mmol) of the compound Spinozin L was dissolved in dry toluene (10 ml). Add tri-n-butyltin hydride (0.50 ml, 1.86 mmol) at room temperature under nitrogen. Then AIBN (20 mg) was added. The reaction mixture was heated to reflux temperature for 15 minutes. After cooling to room temperature, the solution was concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (75 g/EtOAc) to obtain the compound 3'-deoxybinoxine

<img file="TW487559B_D0036.tif" />

<u>Example A32-3'-O-(isopropyl) carbonate of Spinozin J</u>

The compound Spinozin J (330 mg, 0.46 mmol) was dissolved in dry HMPA (3.5 ml). Silver carbonate (0.66 g) was added. The reaction mixture was stirred at room temperature under nitrogen for 5 minutes. Introduce 2-iodopropane (1.5 ml). The reaction mixture was stirred for 18 hours. EtOAc (20 ml) and PhH (100 ml) were added. Wash the organic layer with water (3×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Separate in a column (20 g/EtOAc) to obtain a) compound (98 mg) and b) compound Spinozin J 3'-O-(isopropyl) carbonate

<img file="TW487559B_D0037.tif" />

<u>Example A33-3'-O-Dichloroacetyl Spinozin L</u>

The compound Spinozin L (244 mg, 0.33 mmol) was dissolved in dry pyridine (4 ml). Dichloroacetic anhydride (0.50 ml, excess) was added and stirring was continued for 16 hours at room temperature under nitrogen. EtOAc (50 ml) and PhH (100 ml) were added. The solution is 5%aqNaHCO₃(3×)Cleaning. Take anh.K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain the compound 3'-O-dichloroacetylspinoxine L;

<img file="TW487559B_D0038.tif" />

<u>Example A34-3'-O-Dichloroacetyl Spinozin J</u>

The compound Spinozin J (302 mg, 0.42 mmol) was dissolved in dry pyridine (5 ml). Dichloroacetic anhydride (0.50 ml, excess) was added and stirring was continued for 16 hours at room temperature under nitrogen. EtOAc (50 ml) and PhH (100 ml) were added. The solution is 5%aqNaHCO₃(3×)Cleaning. Take anh.K₂CO₃The dried organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80g/EtOAc) to obtain the compound 3'-O-dichloroacetylspinoxine J; 293 mg, 84%¹H-NMR

<img file="TW487559B_D0039.tif" />

<u>Example A35-3'-On-Butyl Spinozin J</u>

The compound Spinozin J (296 mg, 0.41 mmol) was dissolved in CH₂Cl₂(5.0 ml). Join K₂CO₃(1.10 g). The reaction flask was immersed in a water bath (10° C.) and 15% NaOH aqueous solution (10 ml) was added with vigorous stirring, followed by yltriethylammonium chloride (0.88 g). 1-Iodobutane (3.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 40 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (60 g/EtOAc). The concentrate of pure elution fraction can provide a) compound (211 mg) and b) compound 3'-On-butyl history

<img file="TW487559B_D0040.tif" />

<u>Example A36-3'-O-Isobutyryl Spinozin J</u>

The compound Spinozin J (359 mg, 0.50 mmol) was dissolved in dry pyridine (4 ml). Add N,N-dimethylamine pyridine (66 mg). The reaction was cooled to 10°C under nitrogen (water bath). Isobutyryl chloride (2.0 ml, excess) was added and stirring was continued for 70 hours at room temperature under nitrogen. EtOAc (50 ml) and PhH (100 ml) were added. Organic layer with 5%aq.NaHCO₃(3×)Cleaning. Take anh.K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain 3'-O-isobutyryl Spinozin J;

<img file="TW487559B_D0041.tif" />

<u>Example A37-3'-O-Tepentyl Spinozin J</u>

The compound Spinozin J (426 mg, 0.59 mmol) was dissolved in dry pyridine (4 ml). Add N,N-dimethylamine pyridine (116 mg). The reaction was cooled to 10°C under nitrogen (water bath). Add pivaloyl chloride (2.0 ml, excess) and continue stirring for 70 hours at room temperature under nitrogen. EtOAc (50 ml) and PhH (100 ml) were added. Organic layer with 5%aq.NaHCO₃(3×)Cleaning. Take anh.K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain 3'-O-Pentyl Spinozin J;

<img file="TW487559B_D0042.tif" />

<u>Example A38-5,6-Dihydro-3'-O-ethyl Spinozin J</u>

The compound 5,6-dihydro-spinoxine J (399 mg, 0.554 mmol) was dissolved in CH₂Cl₂(5.0 ml). Join K₂CO₃(1.10 g). The reaction flask was immersed in a water bath (10°C) and 15% NaOH aqueous solution (10 ml) was added with vigorous stirring, followed by tetrabutylammonium hydrogen sulfate (0.90 g). 1-Iodoethane (3.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Water (2×) for organic layer, K₂CO₃Drying and CH₂Cl₂(100 ml) and water (50ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified in a column (100 g/EtOAc). The concentrate of pure elution fraction can provide 5,6-dihydro-3'-O-ethyl history

<img file="TW487559B_D0043.tif" />

<u>Example A39-5,6-Dihydro-3'-On-Propyl Spinozin J</u>

The compound 5,6-dihydro-spinoxine J (8 mg, 0.119 mmol) was dissolved in CH₂Cl₂(3.0 ml). Join K₂CO₃(0.68 g). The reaction flask was immersed in a water bath (10°C) and a 20% aqueous NaOH solution (6 ml) was added with vigorous stirring, followed by tetrabutylammonium hydrogen sulfate (0.81 g). 1-iodopropane (1.20 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 20 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Water (2×) for organic layer, K₂CO₃Drying and CH₂Cl₂(100 ml) and water (50ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (25 g/EtOAc). The concentrate of pure eluted fractions can provide 5,6-dihydro-3'-O-propyl Spinozin J;

<u>Example A40-2'-Table-2'-O-Ethyl Spinozin H</u>

The compound 2'-O-acetyl-2'-spinoxine H (502 mg, 0.664 mmol) was dissolved in CH₂Cl₂(4.0 ml). Join K₂CO₃(1.10 g). The reaction flask was immersed in a water bath (10°C) and a 20% aqueous NaOH solution (10 ml) was added with vigorous stirring, followed by tetrabutylammonium hydrogen sulfate (0.82 g). 1-Iodoethane (1.20 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 22 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Water (2×) for organic layer, K₂CO₃Drying and CH₂Cl₂(100ml) and water (50ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (80 g/EtOAc). The concentrate of pure eluted fraction can provide 2'-table-2'-O-ethyl spinoxin H; 132 mg, 27%¹H-NMR(CDCl₃)δ6.66

<img file="TW487559B_D0044.tif" />

<u>Example A41-5,6-Dihydro-3'-On-Butyl Spinozin J</u>

The compound 5,6-dihydro-spinoxine J (93 mg, 0.129 mmol) was dissolved in CH₂Cl₂(3.0 ml). Join K₂CO₃(0.70 g). The reaction flask was immersed in a water bath (10°C) and a 20% aqueous NaOH solution (10 ml) was added with vigorous stirring, followed by tetrabutylammonium hydrogen sulfate (0.66 g). 1-Iodobutane (2.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 20 hours. Join CH₂Cl₂(100 ml) and water (50 ml). Separate the phases. Water (2×) for organic layer, K₂CO₃Drying and CH₂Cl₃(100 ml) and water (50ml). Separate the phases. Wash the organic layer with water (2×) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified on a column (25 g/EtOAc). Concentrates of pure leaching fractions can provide 5,6-dihydro-3'-O-butyl Spinnol

<img file="TW487559B_D0045.tif" />

<u>Example A42-2'-O-(S-Methyl-N-ylcarbimidylthio) Spinozin H</u>

In a cold (0°C), suitably stirred sodium hydride (50% dispersion in mineral oil, 12.0 mg, 0.14 mmol) in anhydrous THF (1.5 mL) suspension, add dropwise within 2-3 minutes A solution of Spinozin H (0.1 g, 0.14 mmol) in THF (1.5 mL). The mixture was stirred at 0°C for 30 minutes, and then a portion of N--based isothiocyanate (25 μl, 0.18 mmol) was added by means of a syringe. The cooling bath was removed and the mixture was stirred at room temperature for 4.5 hr. Then a portion of methyl iodide (30 μl, 0.48 mmol) was added and the mixture was stirred for 30 minutes. Then the solvent was removed under reduced pressure and the residue was dissolved in anhydrous ether (20 mL). Filter through diatomaceous earth to remove any insoluble matter and rinse with ethanol. The filtrates were combined and washed to leave 0.13 g of crude 2'-O-(S-methyl-N-ylcarbimidothio) spinoxin H as a colorless oil:

<img file="TW487559B_D0046.tif" />

<u>Example A43-2'-O-Trichloroacetimido Spinozin H X504447</u>

In a cold (0°C), suitably stirred sodium hydride (50% dispersion in mineral oil, 12.0 mg, 0.26 mmol) in anhydrous THF (2.0 mL) suspension, add dropwise within 2-3 minutes A solution of Spinozin H (0.1 g, 0.14 mmol) in THF (2.0 mL). The mixture was stirred at 0°C for 20 minutes, followed by adding a portion of trichloroacetate (20 μL, 0.2 mmol) and stirring at 0°C for 2 hours. Then a portion of glacial acetic acid (16 μL, 0.27 mmol) was added and the reaction mixture was stirred for 10 minutes under cooling. Then the solvent was removed under reduced pressure and the crude 2'-O-trichloroacetimidospinoxine H was purified by flash chromatography on silica (40 mL), using 3% methanol in dichloromethane as the solvent. Leaching. Get pure 2'-O-trichloroacetimido Spinol in the form of white foam

<img file="TW487559B_D0047.tif" />

<u>Example A44-3'-O-Trifluoromethanesulfonyl Spinozin J</u>

Spinozin J (0.2 g, 0.28 mmol) and dry CH in cold (-10°C), properly stirred₂Cl₂(5 mL) of dry pyridine (0.1 mL, 1.26 mmol) solution, add a part of triflic acid (60 μL, 0.35 mmol) by means of a syringe. Stir the resulting orange-brown solution at -10°C for 1.5 h, then add CH₂Cl₂(20 mL) and dilute with water (5 mL), saturated Na₂CO₃(4 mL), then washed with water (5 mL), and finally dried with (MgSO₄). The concentrate leaves 216 mg (91%) 3'-O-Trifluoromethanesulfonyl Spinozin J, which is pure enough for use without further purification:¹NMR(CDCl₃) δ 4.98 (dd, 1, H-3').

<u>Example A45-9-O (2,3,4-tris-O-ethyl-L-α-rhamnosyl) Spinozin A 9-Psa: 9-O-(2,3,4- Tri-O-ethyl-β-LL-rhamnosyl) Spinozin A 9-Psa 3:1 mixture</u>

Dry CH of Spinozin A 9-Psa (2.1 g, 3.86 mmol) and pyridine p-toluenesulfonate (1.3 g, 5.17 mmol) in cold (0°C), properly stirred₂Cl₂(200 ml) solution containing powdered 4A molecular sieves (2.6 g), within 20 minutes, add O-(2,3,4-tri-O-ethyl-α-L-rat Plumose (pyranosyl) trichloroimine acetate (5.0 g, 12.75 mmol) in CH₂Cl₂(20 mL) solution. After 3 hours at 0°C, add another part of imide ester (2.0 g) of CH₂Cl₂(6 mL) solution. Then the cooling bath was removed and the reaction mixture was stirred at room temperature for 16 hours. Then more imine ester (0.8 g) was added. After another 7 hours, the reaction mixture was filtered through diatomaceous earth and filtered with CH₂Cl₂(25 mL) Wash diatomaceous earth and combine the filtrate with saturated Na₂CO₃(2×80 mL) Wash and dry (MgSO₄). The concentrate leaves a residue of 11.5 g, which is subjected to flash chromatography on silica (700 mL) using 3% MeOH in CH₂Cl₂The solution is an eluent to produce 2.0 g (67%) of compound a) 9-O-(2,3,4-tris-O-ethyl-α-L-rhamnosyl) Spinozin A 9 -Psa: 9-O-(2,3,4-tris-O-ethyl-β-LL-rhamnosyl) Spinozin A 9-Psa 3:1 mixture. The mixture was passed through a 21.4 mm(id)×20 cm(1) Rainin reverse phase Cl8 column by HPLC, using 4% H₂O (contains 0.01% NH₄The methanol solution of OH) is the eluent separated into 10 parts of about 200 mg. The β-mutaisomer precipitates first. Compound b) 9-O-(2,3,4-tri-O-ethyl-β-LL-rhamnosyl) Spinozin A 9-Psa: 0.35 g; colorless foam;¹NMR(CDCl₃)δ 4.36 (s, 1, H-1'), 4.45 (m, 2, H-1", H-9). Compound c) 9-O-(2,3,4-Tri-O-B -Α-L-rhamnosyl) Spinozin A 9-Psa; 1.2 g, colorless foam;¹NMR(CDCl₃) δ 4.43 (bd, 1, H-1"), 4.47 (s, 1, H-1').

<u>Example A46-3'-keto Spinozin J</u>

A suspension of N-chlorobutadiamide (104.7 mg, 0.78 mmol) in dichloromethane (2.6 ml) was cooled to -78°C under nitrogen. Isopropyl sulfide (125 μl, 0.86 mmol) was added to the suspension and the mixture was stirred at -78°C for 0.5 hr. Then spinoxin J (184.6 mg, 0.26 mmol) in dichloromethane (1 ml) was added slowly. When the addition was complete, the solution was stirred at -78°C for 6.25 hr, and then triethylamine (109 μL, 0.78 mmol) was added, and the solution was added to room temperature to make it red. After heating, add Et₂O (6 mL) 5% MeOH in dichloromethane was purified by eluent to obtain a colorless semi-solid 3'-ketospinoxine J (151.2 mg, 82%).

<img file="TW487559B_D0048.tif" />

<u>Example A47-(4'R)-4'-Demethoxy-4'-amino Spinozin A</u>

To a solution of 4'-keto Spinozin K (115.3 mg, 0.16 mmol) in methanol (2 ml), ammonium acetate (149.3 mg, 1.9 mmol) was added, followed by cyanoborohydride (10 mg, 0.16 mmol) . The reaction mixture was stirred at room temperature for 7 hours. Then the mixture was diluted with 1N HCl and washed with ether. Then it was extracted with fresh ether. Take MgSO₄Dry and evaporate at room temperature under reduced pressure. The crude product was separated on silica by chromatography and eluted with 10% methanol in dichloromethane and then with 50% methanol in dichloromethane in a single step. (4'R)-4'-Demethoxy-4'-amino Spinozin A (23.3 mg; 20% yield) is a colorless glass, which is the most polar substance after separation. FDMS, m/e (relative intensity) 717 (60): 716 (M⁺，100)，142(35)，114(30)。

<u>Example A48-2'-oxy-3'-alkenyl-desmethoxy-spinoxine H</u>

The reaction was carried out according to the method described in Example 3 to prepare Spinozin A 9-Psa, using Spinozin H and Spinozin J (5.02 gm, 7.0 mmol), N-chlorobutadiamide ( 35 of 2.68 gm, 20 mmol), dichloromethane (90 mL), dimethyl sulfide (3.1 mL, 42.0 mmol), triethylamine (2.8 mL, 21.0 mmol) and potassium carbonate (12.8 gm, 92.2 mmol): 65 mixture. Available Spinozin A 9-Psa (2.32 gm; 94% yield, based on Spinozin J) and 2'-oxy-3'-alkenyl 4'-demethoxy-Spinno Xin H (290 mg; 55% yield, based on Spinozin H), FDMS, m/e (relative intensity) 684 (M⁺, 30), 683 (100), is a white solid.

<u>Example A49-2'-(aL-2,3,4-tri-O-methylrhamnosyl) Spinozin H 3'-(aL-2,3,4-tri-O-methyl Rhamnosyl) Spinozin J</u>

To a solution of Spinozin H and Spinozin J (35:65, 1.0 gm, 1.44 mmol) in anhydrous dichloromethane, add 1-bromo-2,3,4-tri-O-ethyl Rhamnose¹(1.26 g, 3.57 mmol), followed by diisopropylamine (301 ml, 1.73 mmol) and then silver triflate (456.8 mg, 1.73 mmol). The reaction mixture was stirred at room temperature in a dark room for 2 days. The mixture was then diluted with dichloromethane and washed with water. Filter dichloromethane to remove insoluble materials, and use MgSO₄Dry and concentrate at room temperature under reduced pressure. The crude product was separated from the unreacted starting material by chromatography on silica, with 5% MeOH in CH₂Cl₂The solution is eluent. The crude product mixture was purified by preparative HPLC on a Cl8 column with ethyl acetate: methanol: 0.1% NH₄OAc (42.5:42.5:12.5 to 45:45:10 with a linear gradient of 90 minutes) was used as the eluent. The resulting compound a) 2'-(α-L-2,3,4-tri-O-methylrhamnosyl) Spinozin H (47 mg), FDMS, m/s (relative intensity) 990 ( M⁺, 100) and compound b) 3'-(aL-2,3,4-tri-O-methylrhamnosyl) Spinozin J (16 mg), FDMS, m/e (relative intensity) 990 (M⁺, 25), 989(70), 988(70), are white solids.

¹Fisher, E.; Bergmann, M.; A. Chem. Ber. 1920, 53, 2363.

<u>Example A50-2'-(N-hydroxy)imino Spinozin H</u>

To crude 2'-keto Spinozin H (238.3 mg, 0.33 mmol) and hydroxylamine hydrochloride (24.9 mg, 0.36 mmol) in pure ethanol (10 ml), add anhydrous sodium acetate (60.0 mg, 0.73 mmol) ). The reaction mixture was stirred at room temperature for 3 hours. Then the mixture was diluted with dichloromethane, washed with water, brine, and Na₂SO₄Dry and evaporate at room temperature under reduced pressure. The product was purified by silica chromatography using 5% methanol in dichloromethane as the eluent. The obtained 2'-(N-hydroxy) imino Spinozin H (73.9 mg) is a white solid, FDMS, m/e (relative intensity) 731 (M⁺，50)，73(100)。

<u>Example A51-4'-keto Spinozin K</u>

The reaction was carried out using Spinozin K (1.49 gm, 2.07 mmol) according to the method described in Example A46. Available 4'-keto Spinozin K (1.48 gm; about 100% yield), a viscous white solid, FDMS, m/e (relative strength) 715 (M⁺，100)。

<u>Example A52-2'-(N-hydroxy)imino-4'-desmethoxy-3'4'-dehydrospinoxine H</u>

Using 2'-oxy-3'-alkenyl-4'-desmethoxy Spinozin H (579.8 mg, 0.85 mmol) as the starting material, the reaction was carried out according to the method described in Example A50. Available 2'-(N-hydroxy)imino-4'-desmethoxy-33,4'-dehydrospinoxine H (223 mg, 42% yield, based on the recovered starting material Benchmark) slightly white solid. FDMS, m/e (relative intensity) 699 (M⁺，70)，698(100)。

<u>Bao Shi example A53-9-O-2,3,4-tri-O-ethyl-α-L-rhamnosyl spinoxine A 9-Psa hemiglutarate</u>

The compound 9-O-2,3,4-tri-O-ethyl-α-L-rhamnosyl) Spinozin A 9-Psa (99.6 mg, 0.128 mmol) was dissolved in 2 mL of acetone and added A solution of glutaric acid (8.5 mg, 0.064 mmol) in 2 mL acetone. After the solution was stirred for 3 hours, the solvent was then removed under reduced pressure. The compound 9-O-2,3,4-tri-O-ethyl-α-L-rhamnosylspinoxine A 9-Psa hemiglutarate is available, 105.7 mg, the estimated value of analysis, to C₄₄H₇₁N₁₀1/2(C₅H₈O₄): C 66.48, H 8.99, N 1.67; actual value C 66.30, H 8.58, N 1.71; mp. 76-82°C.

<u>Example A54-3'-O-Ethyl Spinozin J Hemiglutarate</u>

Compound 3'-O-Ethyl Spinozin J (130.4 mg, 0.174 mmol) was dissolved in 2 mL of acetone and glutaric acid (11.5 mg, 0.087 mmol) in 2 mL of acetone was added. The solution was stirred overnight. Evaporate the solvent to obtain 3'-O-Ethyl Spinozin J Hemiglutarate, 131 mg. Analyze the estimated value, for C₄₂H₆₇NO₁₀1/2(C₅H₈O₄): C 65.82, H 8.81, N 1.72; actual value C 65.65, H 8.46, N 1.72; mp. 75-82°C.

<u>Example A55-3'-O-pentadeuteroethyl Spinozin J Hemiglutarate</u>

The compound 3'-O-pentadeuteroethyl Spinozin J (97.4 mg, 0.129 mmol) was dissolved in 3 mL of acetone and glutaric acid (8.6 mg, 0.065 mmol) in 2 mL of acetone was added. The solution was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to obtain the compound 3'-O-pentadeuteroethyl spinoxin J hemiglutarate, 102 mg. Analyze the estimated value, for C₄₂H₆₂NO₁₀D₅1/2(C₅H₈O₄): C 65.02, H 9.27, N 1.70; actual value C65.12, H 9.01, N 1.85; mp. 104-114°C.

<u>Example A56-5,6-Dihydro-3'-On-Propyl Spinozin J Hemiglutarate</u>

The compound 5,6-dihydro-3'-On-propyl Spinozin J (88.3 mg, 0.115 mmol) was dissolved in 3 mL acetone and glutaric acid (7.5 mg, 0.056 mmol) was added to a 2 mL acetone solution. The solution was stirred at room temperature overnight. The solvent was evaporated under reduced pressure to obtain compound 5,6-dihydro-3'-On-propylspinoxine J hemiglutarate, 86.1 mg. Analyze the estimated value, for C₄₃H₇₁NO₁₀1/2(C₅H₈O₄): C 65.99, H 9.12, N 1.69; actual value C65.30, H 10.00, N 1.77; mp. 74-84°C.

<u>Example A57-3'-O-Ethyl Spinozin L Hemiglutarate</u>

The compound 3'-O-ethyl Spinozin L (84.2 mg, 0.110 mmol) was dissolved in 3 mL of acetone and glutaric acid (7.5 mg, 0.056 mmol) was added to a 3 mL acetone solution. The solution was stirred at room temperature overnight. The solvent was evaporated under reduced pressure to obtain compound 3'-O-ethylspinoxine L hemiglutarate, 91.0 mg. Analyze the estimated value, for C₄₃H₆₉NO₁₀1/2(C₅H₈O₄): C66.16, H 8.90, N 1.69; actual value C65.43, H 9.16, N 1.69; mp. 77-82°C.

<u>Example A58-(3'S)-4'-nor-3'-deoxy-3'-[(R)-(α-methoxy, α-propanoxy)methyl]spinoxine J and (3'S)-4'-Nor-3'-Deoxy-3'-[(S)-(α-Methoxy, α-Propyloxy)methyl] Spinozin J</u>

The compound 3'-O-Trifluoromethanesulfonyl Spinozin J (488 mg, 0.574 mmol) was dissolved in dry DMF (20 mL). Cesium propionate compound and propionic acid (1.09 g, excess) were added. The reaction mixture was cooled to room temperature while ultrasonic shaking (55% amplitude) at 660W for 15 minutes. PhH (70 ml) and EtOAc (70 ml) were added. Take 2%aq.NaHCO₃Solution (2×) and saturated aq.NaHCO₃(1×) Cleaning solution. Take the organic phase to K₂CO₃Dry and concentrate under reduced pressure. Residue by SiO₂Purification by flash tube column chromatography to obtain a mixture (275 mg). This sample was then repeated by RF C-18 HPLC (MeOH-H₂O: 90-10) to obtain compound a) (3'S)-4'-nor-3'-[(R)-(α-methoxy, α-propionyloxy)methyl]spinoxine J ; 62 mg, 23%):¹NMR(CDCl₃)δ 5.96 (1H, d: 9.2 Hz) 4.99 (1H, s) ppm and compound b) (3'S)-4'-nor-3'-deoxy-3'-[(S)-(α- Methoxy, α-Propyloxy)methyl] Spinozin J; 84 mg, 31%:¹NMR(CDCl₃) δ 5.89 (1H, dd: 9.4, 1.2 Hz), 4.99 (1H, bs). The stereoisomeric chemistry of compound C(3') can be confirmed by NOEDS and molecular model results.

<u>Example A59-(3'S)-4'-nor-3'-deoxy[(RS)-a-chloro-a-methoxy)methyl]spinoxine J</u>

The compound 3'-O-Trifluoromethanesulfonyl Spinozin J (496 mg, 0.583 mmol) was dissolved in dry DMF (14 mL). Lithium chloride (1.80 g, excess) was added. The reaction mixture was cooled to room temperature while being ultrasonically shaken (660 W, 52% amplitude) for 7 minutes. The solution is washed with diluted brine (2×) and water (2×). Organic layer with K₂CO₃Dry, filter and concentrate under reduced pressure. Put the residue in short SiO₂/EtOAc column purification and then repeatedly by RP C-18 HPLC (MeOH-H₂O: 92-8) Purification. Purification record produced epimer (3'S)-4'-nor-3'-deoxy [R/Sa-chloro-a-methoxy)methyl] Spinozin J; 45 mg, 11%: 5.02 (0.5H, s), 5.00 (0.5 H, s), 4.57 (0.5 H, d: 9.5 Hz), 4.55 (0.5 H, d: 4.5 Hz), 2.16 (6 H, 2×CH)₃, S).

<u>Example A60-(3'S)-4'-nor-3'-deoxy[(R)-(α-fluoro,α-methoxy)methyl]spinoxine J(3'S)-4'- Demethyl-3'-deoxy[(S)-(α-fluoro, α-methoxy)methyl] Spinozin J</u>

The compound Spinozin J (697 mg, 0.97 mmol) was dissolved in dry toluene (10 mL). Under nitrogen, pyridine (1 ml) and diethylamine sulfur trifluoro compound (DAST: 0.70 mmol, 0.85 g, 5.2 mmol) were added with stirring. The reaction mixture was immediately warmed to boiling point. After 10 minutes, the solution was allowed to cool to room temperature. Benzene (50 ml) and EtOAc (75 ml) were added. Take 5%aq.NaHCO₃Aqueous cleaning solution. Organic layer with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂The column (80 g/EtOAc) was separated to obtain a white solid (626 mg). A part of this sample (400 mg) was further used RF C-18 HPLC (MeOH-H₂O: 90-10) Separation to obtain compound a) (3'S)-4'-nor-3'-deoxy[(R)-(α-fluoro, α-methoxy)methyl]spinoxine J; 82 mg, 21%:¹NMR(CDCl₃)δ5.13 (dd: 66.3, 5.4Hz) ppm and compound b) (3'S)-4'-nor-3'-deoxy[(S)-(α-fluoro,α-methoxy)methyl ] Spinozin J; 292 mg, 64%):¹NMR(CDCl₃) δ 5.26 (dd: 65.1, 8.6 Hz) ppm.

<u>Example A61-(1'S, 2'S)-[1'-(2'S)]methyl-2'-deoxy-fluorospinoxine H</u>

The compound Spinozin H (916 mg, 1.28 mmol) was dissolved in dry toluene (15 mL). Add pyridine (1.8 ml). The solution was heated to reflux temperature under nitrogen. At the beginning of the heating process, DAST (0.62 ml, 4.6 mmol) was added. After boiling for 10 minutes, the reaction mixture was cooled to room temperature and carefully reduced to 5% aq. NaHCO₃Sudden cold. Benzene (50 ml) and EtOAc (75 ml) were added. Take 5%aq.NaHCO₃(3×) Cleaning solution. Organic layer with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Purification by column (100 g/EtOAc), collecting the eluted fractions to obtain the compound (1'S, 2'S)-[1'-(2'S)]methyl-2'-deoxy-fluorospinoxine H; 992 mg, 72%:¹NMR(CDC1₃) δ 4.99 (dd: 52.8, 7.0 Hz) ppm.

<u>Example A62-3'(E)-(Nn-butyl)imino Spinozin J</u>

The compound 3'-O-Trifluoromethanesulfonyl Spinozin J (591 mg, 0.695 mmol) was dissolved in dry DMF (20 mL). Add 15%TBAF/Al₂O₃(2.7 g, excess). The reaction flask was placed in a water cooling bath and ultrasonically shaken (55% amplitude) at 660W for 10 minutes. EtOAc (50 ml) and PhH (70 ml) were added. Filter the solution, extract with water (3×) and extract with K₂CO₃dry. The organic layer was extracted under reduced pressure. The residue was passed through a flash chromatography column (SiO₂, 75 g/EtOAc) to provide the compound (3'((E)-(Nn-butyl)imino Spinow

<img file="TW487559B_D0049.tif" />

<u>Example A63-3'-O-pentadeuteroethyl Spinozin J</u>

The compound Spinozin J (882 mg, 1.22 mmol) was dissolved in dichloromethane (5 ml). Join K₂CO₃(1.42 g). Place the reaction flask in a water cooling bath (ca. 10°C). While stirring, add 15% aq.NaOH (15 ml), then add TEBA-Cl (0.92 g) and NBu₄HSO₄(0.30 g). Then add C₂D₅I (5 g: 31 mmol) and the reaction mixture was stirred vigorously for 48 hours at room temperature. Then dichloromethane (100 ml) and water (100 ml) were added. After extraction, the phases are separated. The organic layer was washed with water (2×) and concentrated under reduced pressure. The residue is flashing SiO₂Purified in a column (120 g/EtOAc) to obtain the compound 3'-O-pentadeuteroethyl Spinozin J; 815 mg, 88%:

<img file="TW487559B_D0050.tif" />

<u>Example A64-3'-Deoxy-3'-azospinoxine J (1:1 3'and R3'S mixture) and (3'S)-4'-nor-3'-deoxy-3'- [(RS)-(a-azo, α-methoxy)methyl] Spinozin J</u>

The compound 3'-O-trifluoromethanesulfonyl (440 mg, 0.518 mmol) was dissolved in dry DMF (20 ml). Sodium azide (1.44 g, excess) was added. At room temperature, the mixture was ultrasonically oscillated (660 W, 55% amplitude) for 10 minutes. EtOAc (50 ml) and PhH (100 ml) were added. At 5%aq.KHCO₃Solution (3×). The organic layer is anh.K₂CO₃Dry, filter and concentrate under reduced pressure. The residue was chromatographed with SiO₂(50 g/EtOAc) and by RP C-18 HPLC (MeOH-H₂O: 88-12) to obtain compound a) 3'-deoxy-3'-azospinoxine J ((1:1 3'R and 3'S mixture); 40 mg, 10%

<img file="TW487559B_D0051.tif" />

<u>Example A65-(3'S)-4'-nor-3'-deoxy-3'-[(RS)-α-chloro,α-methoxy)methyl]spinoxine J</u>

The compound 3'-O-Trifluoromethanesulfonyl Spinozin J (448 mg, 0.574 mmol) was dissolved in dry DMF (20 ml). Lithium chloride (1.80 g, excess) was added. The mixture was cooled to room temperature while ultrasonically oscillating the mixture (660 W, 52% amplitude) for 7 minutes. PhH (70 ml) and EtOAc (50 ml) were added. The solution is washed with diluted brine (2×) and water (2×). Organic layer with K₂CO₃Dry, filter and concentrate under reduced pressure. Put the residue in short SiO₂/EtOAc column purification and then repeatedly by RP C-18 HPLC (MeOH-H₂O: 92-8) Purification. Purification record of the difference of (3'S)-4'-nor-3'-deoxy-3'-[(RS)-α-chloro,α-methoxy)methyl]spinoxine J Isomers<img file="TW487559B_D0052.tif" /><img file="TW487559B_D0053.tif" />

<u>Example A66-5,6-Dihydro-2'-O-Ethyl Spinozin H</u>

The compound 5,6-dihydrospinoxine H (462 mg, 0.64 mmol) was dissolved in dichloromethane (6 ml). Add potassium carbonate powder (1.0 g). Place the reaction flask in a water cooling bath (ca. 10°C). While stirring, add 10% aq.NaOH (15 ml), then add NBu₄HSO₄(0.30 g) solid. Then iodoethane (4.0 ml) was added. The reaction mixture was stirred vigorously at room temperature under nitrogen for 46 hours. Add dichloromethane (100 ml) and water (100 ml). After extraction, the phases are separated. The organic layer is anh.K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain compound 5,6-dihydro-2'-O-ethylspinoxine H; 336 mg, 70%:

<img file="TW487559B_D0054.tif" />

<u>Example A67-5,6-Dihydro-2'-On-Propyl Spinozin H</u>

The compound 5,6-dihydrospinoxine H (488 mg, 0.68 mmol) was dissolved in dichloromethane (8 ml). Add potassium carbonate powder (1.1 g). Place the reaction flask in a water cooling bath (ca. 10°C). While stirring, add 10% aq.NaOH (20 ml), then add NBu₄HSO₄(0.95 g) solid. Then iodo n-propane (4.0 ml) was added. The reaction mixture was stirred vigorously for 46 hours at room temperature under nitrogen. Add dichloromethane (100 ml) and water (100 ml). After extraction, the phases are separated. The organic layer is anh.K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Purified in a column (80 g/EtOAc) to obtain compound 5,6-dihydro-2'-On-propylspinoxine H; 355 mg, 69%:

<img file="TW487559B_D0055.tif" />

<u>Example A68-3'-Epispinoxine J</u>

The compound 3'-keto-spinoxine J (3.61 g, 5.04 mmol)) was dissolved in ether (100 ml). The solution was cooled to 0°C under nitrogen. Add a portion of lithium tri-t-butoxyaluminum hydride (1.45 g; 97%, 5.53 mmol). Stirring was continued for 15 minutes at 0°C, and then the remaining LTBAH (105 g, 4.00 mmol) was introduced. Continue stirring for another 35 minutes. Add benzene (60 ml). Slowly decompose excess hydride with saturated brine (20 ml) at 0°C. Separate the phases. The organic layer is continuously treated with brine-H₂O(8:1), wash with 10% aq.NaOH solution, and with water. The organic phase is anh.K₂CO₃Dry, filter and concentrate under reduced pressure. The obtained product (3.215 g, 89%) was obtained by flash tube column chromatography (160 g, SiO₂/EtOAc) to obtain 95% pure compound 3'-epispinoxine J; 2.715 g, 75%:

<img file="TW487559B_D0056.tif" />

<u>Example A69-3'-Table-3'-O-Trifluoromethanesulfonyl Spinozin J</u>

Compound 3'-Epispinoxine J (1.53 g, 2.13 mmol) was dissolved in dry dichloromethane (20 ml). Add pyridine (5.0 ml, dry). The solution was cooled to 0°C under nitrogen. Slowly introduce trifluoromethanesulfonic anhydride (1.0 ml, excess) by means of a syringe. Stirring was continued for 16 hours at 0°C. EtOAc (50 ml) and PhH (100 ml) were added. With diluted brine (2×) and 5%aq.KHCO₃Solution (2×) Extract the solution. The organic layer is anh.Na₂SO₄, Filter and concentrate under reduced pressure. The residue is flashing SiO₂Column (100 g/EtOAc) to obtain compound 3'-table-3'-O-trifluoromethanesulfonyl Spinozin J; 1.36

<img file="TW487559B_D0057.tif" />

<u>Example A70-3'-O-(α,α,β-trifluoro-γ-butene)-α-spinoxine J</u>

The compound Spinozin J (790 mg, 1.10 mmol) was dissolved in dry dichloromethane (5 ml). Add potassium carbonate (1.1 g), followed by 15% aq. sodium hydroxide solution (15 ml), Bu₄NHSO₄(0.80 g) and 4-bromo-1,1,2-trifluorobutene (3.0 g, excess). DMSO (4.0 ml) was added. Stirring was continued for 16 hours at room temperature under nitrogen. Add dichloromethane (100 ml) and water (100 ml). After extraction, the phases are separated. Organic phase with K₂CO₃Dry, filter and concentrate under reduced pressure. The residue is flashing SiO₂Column (100 g/EtOAc) to obtain the compound 3'-O-(α,α,β-trifluoro-γ-butene)-α-kispinnol

<img file="TW487559B_D0058.tif" />

And (b) Recover the compound Spinozin J (520 mg, 75%).

<u>Example A71-2'-Epi-3'-keto 0 Spinozin J</u>

3'-keto Spinozin J (755 mg, 1.05 mmol) was dissolved in dry THF (10 ml). Add tetraethylsilane (1.0 ml), then add 15% Bu₄NF/Al₂O₃(0.55 g, excess). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. Benzene (70 ml) and EtOAc (30 ml) were added. At 5%aq.KHCO₃Solution (2×). Take K₂CO₃The organic layer was dried, filtered, and concentrated under reduced pressure. The residue is flashing SiO₂Column (120 g/EtOAc) to obtain (a) 3'-keto Spinozin J (290 mg, 38%) and (b) compound 2'-table-3'-

<img file="TW487559B_D0059.tif" />

<u>Example A72-2'-table-3'-table Spinozin J</u>

Compound 2'-epi-3'-keto Spinozin J (305 mg, 0.426 mmol) was dissolved in dry THF (10 ml) and dry ether (10 ml) was added. Cool the solution to 0°C and stir under nitrogen while adding a portion of Li(t-Buo)₃AlH (200 mg, 0.76 mmol). Stirring is continued for 30 minutes at this temperature. Then it was carefully quenched with salt water. Make the mixture in Et₂Partition between O and water containing 5% NaOH. The organic layer is continuous with water containing 5% NaOH and 5% aq.KHCO₃(2×)Cleaning, with K₂CO₃Dry, filter and concentrate under reduced pressure to obtain a white foam containing only 2'-epi-3'-epispinoxine J; 303 mg, 99% ¹H-

<img file="TW487559B_D0060.tif" />

<u>Example A73-2'-table-3'-table-3'-O-ethyl Spinozin J</u>

Compound 2'-epi-3'-epispinoxine J (250 mg, 0.348 mmol) was dissolved in dichloromethane (5 ml). Potassium carbonate (1.0 g) was added. Place the flask in a cooling bath (10°C), add 20% NaOH (20 ml), and then add Bu₄NHSO₄(0.71 g), iodoethane (2.0 ml) and DMSO (5 ml). The reaction mixture was stirred vigorously under nitrogen for 60 hours. Add dichloromethane (100 ml) and water (100 ml). After extraction, the phases are separated. Organic layer with K₂CO₃Dry, filter and concentrate under reduced pressure. The residue is flashing SiO₂Column (30 g/EtOAc) to provide 2'-table-3'-table-3'-O-ethyl Spinozin J, 43mg

<img file="TW487559B_D0061.tif" />

<u>Example A74-3'-Deoxy-3'-fluorospinoxine J (1:1 mixture of 3'isomers)</u>

3'-Epispinoxine J (570 mg, 0.794 mmol) was dissolved in dry dichloromethane (10 ml). The solution was cooled to -25°C under nitrogen. Diethylamine sulfur trifluoride (315 ml, 2.38 mmol) was introduced via a syringe. Stirring was continued for 3 hours at -25°C under nitrogen, and then the temperature was increased within 30 minutes. Add pyridine (0.5 ml) and continue stirring for another 15 minutes at room temperature. Make the mixture in CH₂Cl₂(100 ml) and 5%aq.KHCO₃(150 ml) is divided into two parts. The organic layer is continuously 5%aq.KHCO₃Clean with K₂CO₃Dry, filter and concentrate under reduced pressure. The residue is flashing SiO₂Column (100 g/EtOAc) and then RP C-18 HPLC (10% water in methanol) to obtain the compound 3'-deoxy-3'-fluorospinoxine J, 3'aF: 3' of bF

<img file="TW487559B_D0062.tif" />

<u>Example A75-3'-O-phenyl Spinozin J</u>

3'-Epispinoxine J (148 mg, 0.206 mmol) was dissolved in dry xylene (3 ml). The solution was stirred at room temperature under nitrogen and diphenylphosphine (118 mg, 0.45 mmol) was added, followed by phenol (42.5 mg, 0.45 mmol). After stirring for 15 minutes, diethyl azodicarboxylate (DEAD, 75 ml (95%), 0.45 mmol) was added to the mixture. Stir at room temperature for 16 hours, then dilute with PhH (70 ml) and EtOAc (50 ml), and use 5% aq.KHCO₃Wash and dry with potassium carbonate. The residue is flashing SiO₂Column purification and then separation on RP C-18 HPLC column (12% water in methanol) to obtain (a) recovered 3'-epispinoxine J (101 mg: 68%) and (b) Compound 3'-O-phenyl Spinozin J; 3.1 mg, 1.9%¹H-

<img file="TW487559B_D0063.tif" />

<u>Example A76-3'-O-(S-phenyl) disulfide carbonate of Spinozin J</u>

Spinozin J (1.20 mg, 1.67 mmol) was dissolved in dry dichloromethane (10 ml). Add pyridine (dry, 2 ml), followed by DMAP (70 mg). The reaction mixture was stirred at room temperature under nitrogen and cooled in water. Slowly add phenylchlorodithioformate (1.80 ml, excess) via syringe. The mixture was then stirred at room temperature under nitrogen for 16 hours. PhH (70ml) and EtOAc (70ml) were added. Organic layer with 5% KHCO₃(4×)Cleaning, with K₂CO₃Dry and concentrate under reduced pressure. Flash SiO₂Column (120 g/EtOAc) separation to provide compound 3'-O-(S-phenyl) disulfide carbonate; 780 mg, 54%¹H NMR

<img file="TW487559B_D0064.tif" />

<u>Example A77-3'-O-(pentafluorophenyl)thiocarbonate of Spinozin J</u>

Spinozin J (1.52 mg, 2.12 mmol) was dissolved in dry acetone (15 ml). Add DMAP (0.84 g, 6.88 mmol) and dissolve. The solution was cooled to 0°C under nitrogen. Slowly add pentafluorophenyl chlorothiocarbamate (1.60 ml, 10.0 mmol) dropwise. The reaction mixture was stirred at room temperature for 16 hours. PhH (70 ml) and EtOAc (50 ml) were added. At 5% KHCO₃(3×) Extract the solution and use K₂CO₃dry. The organic layer was concentrated under reduced pressure. Flash SiO₂The column (120 g/EtOAc) was separated to provide the compound 3'-O-(pentafluorophenyl)thiocarbonate; 1.77 g, 88%,¹N-NMR

<img file="TW487559B_D0065.tif" />

<u>Example A78-3'-O-[(3'R)-3'-Deoxyspinoxine J]-3'-kispinoxine J, (3'R) and (3'S)3'-ene A 1:1 mixture of propyl-3'deoxyspinoxine J, and (3'S)-3'-allyl-3'-deoxyspinoxine J</u>

The 3'-O-(pentafluorophenyl)thiocarbonate (1.65 g, 1.75 mmol) (1.52 mg, 2.12 mmol) of the compound Spinozin J was dissolved in dry toluene (25 ml). Allyl tributyltin (1.70 ml, 5.32 mmol) was added, followed by AIBN (180 mg). The mixture was heated to reflux temperature under nitrogen for 20 hours. The mixture is cooled, concentrated and directly put into the flash SiO₂Column (220 g/EtOAc). The purified fraction was further subjected to RP C-18 HPLC (10% water in methanol) to provide compound a) 3'-O-[(3'R)-3'-deoxyspinoxine J] -3'-Kispenoxin J; 373 mg

<img file="TW487559B_D0066.tif" />

2.24, and compound b) 1:1 mixture of (3'R) and (3'S)3'-allyl-3'-deoxyspinoxine J; 144 mg, 11%. The mixture was further repeated by RP C-18 HPLC on a 5 micron Rainin column to obtain compound c) (3'S)-3'-allyl-3'-deoxyspinoxine J;

<u>Example A79-(14R)-13,14-dihydro-3'(E)-(methoxycarbonyl)methylene-3'-deoxyspinoxine J</u>

The compound 3'(E)-(methoxycarbonyl)methylene-3'-spinoxine J (308 mg, 0.39 mmol) was dissolved in Et₂O (10 ml). Add glacial acetic acid (5 ml). Stir the mixture at room temperature and add NaBH₃CN (180 mg, excess). Stirring was continued for 16 hours at room temperature under nitrogen. PhH (100 ml) and EtOAc (50 ml) were added. With diluted brine (2×), followed by 5%aq.NaHCO₃extraction. The organic layer is anh.K₂CO₃Dry, filter and concentrate under reduced pressure. Flash SiO₂Column (55 g, EtOAc) was purified to provide compound (14R)-13,14β-dihydro-3'(E)-(methoxycarbonyl)methylene-3'-deoxyspinnol

<img file="TW487559B_D0067.tif" />

<u>Example A80-3'(E)-(hydroxymethyl)methylene-3'-deoxyspinoxine J, (14S,21S)-1,21-deoxy-13,14-dihydro- 1,21-Dihydroxy-3'(E)-(hydroxymethyl)methylene-3'-deoxy-1,21-spinoxine J</u>

The compound 3'(E)-(methoxycarbonyl)methylene-3'-deoxyspinoxine J (340 mg, 0.44 mmol) was dissolved in dry dichloromethane (5 ml). Dry THF (1.5 ml) was added. The solution was cooled to 0°C under nitrogen and DIBALH (1.95 ml, 1.0 M cyclohexane solution, 1.95 mmol) was added. Continue stirring for 45 minutes. Add saturated NH dropwise at 0°C₄OH-NH₄Cl (1:1) solution. Add PhH (100 ml) and EtOAc (70 ml), and add saturated NH₄OH-NH₄Cl(1:1) solution, then 2N aq.NaOH, and finally 5%aq.KHCO₃The solution (2×) was extracted. Organic layer with K₂CO₃Dry, filter and concentrate under reduced pressure. Flash SiO₂Column (30 g/EtOAc) was separated to provide compound a) 3'(E)-(hydroxymethyl)methylene-3'-deoxyspinoxine J

<img file="TW487559B_D0068.tif" />

<u>Example A81-4'-On-Propyl Spinozin K</u>

Spinozin K (322 mg, 0.448 mmol) was dissolved in dry dichloromethane (4ml). Potassium carbonate (1.1 g) is added. Place the flask in a water bath (ca. +10°C). Add 15% NaOH aqueous solution (25 ml), then add Bu₄NHSO₄(1.2 g), iodo n-propane (3.0 ml) and DMSO (4 ml). After 16 hours, import additional Bu₄NHSO₄(0.70 g). Stir vigorously at room temperature under nitrogen for a total of 60 hours. Water (100 ml) and dichloromethane (100 ml) were added. After extraction, the phases are separated. The organic layer is anh.K₂CO₃Dry, filter and dry under reduced pressure. The residue is flashing SiO₂Separate in a column (100 g/EtOAc) to provide the compound 4'-On-Propyl Spinozin K; 225 mg, 66%¹H-

<img file="TW487559B_D0069.tif" />

<u>Example A82-Methyl-2,3,4-tri-O-ethyl-L-rhamnoseranose</u>

Methyl L-rhamnose and pyranose (Fischer, E. Chem. Ber., 1895, 28, 1158) into α and β isomers (21.0 g, 0.118 mole) were added to the 50 with proper stirring. % (w/w) a mixture of aqueous sodium hydroxide, DMSO (100 ml) and tetrabutylammonium hydrosulfate (20.0 g, 0.059 mmol). Iodoethane (75.0 mL) was added to the mixture. A slightly exothermic reaction can be seen within 20-30 minutes before the reaction (external cooling, ice bath is used to maintain the reaction temperature close to 25°C). After reacting for 3 hours, add more than 75 ml of ethyl iodide. After another 3 hours, add more than 75 ml of ethyl iodide and more than 50% NaOH solution (200 ml). After another 3 hours, add more than 70 ml of ethyl iodide and more than 50% NaOH solution (100 ml), and stir the mixture overnight. The total reaction time is 24 hours. Dilute the mixture with water (200 ml) and extract as much as possible with dichloromethane. The dichloromethane extract was washed with brine (200 ml) and dried (MgSO₄) And evaporation. The amorphous residue was triturated with hexane (300 ml) and filtered. The collected solids were combined with hexane (100 mL) and the filtrates and concentrated, yielding 42.0 g of crude product. Perform flash chromatography with 900 ml of silica, using 10% ethyl acetate in hexane as the eluent. After pre-dissolving 800 ml, collect 200 ml of eluate. The clean methyl 2,3,4-tri-O-ethyl-L-rhamnose pyran (25.5 g,

<img file="TW487559B_D0070.tif" />

<u>Example A83-2,3,4-Tri-O-ethyl-L-rhamnose</u>

Mix methyl 2,3,4-tris-O-ethyl-L-rhamnose pyranose (11.0 g, 0.042 mol) in 110 ml of 4:1 (v/v) trifluoroacetic acid: water The solution was stirred at room temperature for 24 hours. The solution was concentrated under reduced pressure (~1.0 mm, 30-35°C) to near dryness, and the residue was diluted with dichloromethane (200 mL). Separate the aqueous phase and the organic phase and wash the organic phase with saturated sodium bicarbonate solution (40 mL), brine (40 mL) and dry, then dry with magnesium sulfate, and concentrate, leaving 11.0 g of crude product. Flash chromatography with 950 mL of silica, using 25% ethyl acetate in hexane as the eluent, to obtain 9.5 g (91%) of pure triethylrhamnus, which are α and β epimers Mixture and nearly colorless

<img file="TW487559B_D0071.tif" />

<u>Example A84-O-(2,3,4-tris-O-ethyl-α-L-rhamnosyl)-trichloroacetate imitate</u>

Add a portion of the freshly distilled trichloroethane (12.0 mL, 0.12 mmol) to cold (0-5°C), properly stirred triethylrhamnose (2.2 g, 8.86 mmol) in dry dichloromethane (80 mL) solution. Partial addition method: 60% sodium hydride in mineral oil (0.35 g, 8.87 mmol is 100%) solution is added to the solution within 1-2 minutes. (Note: Foaming and hydrogen release). After 20 minutes of reaction, the cooling bath was removed and the mixture was stirred at room temperature for 5.5 hours. Then cool the mixture to 0-5°C and add a portion of silicone gel (6.0 g). After stirring for 10 minutes, the mixture was filtered and the collected solids were washed with dichloromethane (25 mL). Under reduced pressure (~25 mm, 30-35°C), the combined filtrate and washing liquid were evaporated to dryness and the residue was treated with hexane (60 mL) to separate some flocculent solids. After keeping for 30 minutes, the solid was filtered (celite) and washed with hexane (25 mL). Then, the combined filtrate and washing solution were concentrated to dryness under reduced pressure (~25 mm, 30-35°C), leaving 2.9 g of crude imidate as pale yellow oil, by¹HNMR spectrum analysis shows that it only contains epimers and is pure enough to be used directly without further purification 1 (Note: imidate cannot be subjected to silica chromatography and will decompose in short-circuit distillation under reduced pressure (~0.1 mm)):¹HNMR(CDCl₃)δ8.54(s,

<img file="TW487559B_D0072.tif" />

<u>Example A85-3'(E)-(Methoxycarbonyl)methylene-3'-deoxyspinoxine J(3'(Z)-(Methoxycarbonyl)methylene-3'-deoxy Spinozing J</u>

The compound 3'-keto Spinozin J (609 mg, 0.851 mmol) was dissolved in dry toluene (25 ml). Add methoxycarbonylmethylenetriphenylphosphorane (2.2 g, excess). The reaction mixture was heated to reflux temperature under nitrogen for 2 hours. Cool to room temperature and flash SiO directly₂Chromatography on a column (220 g/EtOAc). The fractions with the expected polarity were combined and concentrated under reduced pressure to obtain a crude product (584 g). The sample was repeated by RP C-18 HPLC (12%H₂O in methanol solution) was separated to obtain: recovery of the starting 3'-ketospinoxine J (49mg), compound a) 3'(Z)-(methoxycarbonyl)methylene-3'-deoxyspine Nosin J

<img file="TW487559B_D0073.tif" />

And compound b), 3'(E)-(methoxycarbonyl)methylene-3'-deoxyspinoxine

<img file="TW487559B_D0074.tif" />

<u>Example A86- Spinozin A 9-Psa</u>

To a solution of 3'-keto Spinozin J (1.89 gm, 2.64 mmol) in methanol (100 ml), potassium carbonate (anhydrous; 1.82 gm: 13.2 mmol) was added and the mixture was stirred at room temperature for 2 hours. Then diethyl ether (100 ml) was added and the mixture was filtered. The filtrate was evaporated at room temperature to obtain a yellow solid. The yellow solid was dissolved in dichloromethane and washed with water followed by brine and dried over magnesium sulfate. The dichloromethane was then evaporated under reduced pressure to obtain a colorless semi-solid (1.53 gm). The semi-solid was purified by flash chromatography with a single step gradient of 5% methanol in dichloromethane-10% methanol in dichloromethane to obtain Spinozin A 9-Psa (1.09 gm, 76% yield) Rate), it is a glass with white deviation.

<u>Example A87-3'-keto-spinoxine D</u>

The reaction was carried out according to the method described in Example A46, with Spinozin L (997.4 mg, 1.36 mmol) as the starting material, and 3'-keto Spinozin D (850 mg) was obtained as a colorless semi-solid. NMR showed that the product contained impurities of diisopropyl sulfide, but the product could be used without further purification.

<u>Example A88- Spinozin D 9-Psa</u>

The reaction was carried out according to the method described in Example A86, with 3'-keto Spinozin D (770 mg, 1.06 mmol) as the starting material, and a colorless glassy Spinozin D 9-Psa (246 mg, 1.06 mmol) was obtained. 42% yield).

<u>Example A89-2'-keto-spinoxine H</u>

Diethyl sulfide (1.8 mL, 16.74 mmol) was added dropwise over 2-3 minutes to a cold (-78°C), nitrogen blanketed suspension of N-chlorobutadiamide (1.7 g, 12.73 mmol). The resulting solution was stirred at -78°C for 30 minutes. While maintaining a reaction temperature of &lt;-65°C, spinoxin H (3.0 g, 4.18 mmol) in dichloromethane (25 mL) was added dropwise within 10 minutes . After maintaining at -78°C for 3 hours, while maintaining a reaction temperature of &lt;-65°C, diethylamine (4.1 ml, 29.47 mmol) was added dropwise within 5 minutes. The cooling bath was then removed and the reaction was allowed to warm to room temperature during 20-30 minutes. Add dichloromethane (80 ml) and wash with 0.2N HCl (150 mL) and brine (100 mL) and dry with magnesium sulfate. The remaining concentrate was 4.2 g semi-solid. After flash chromatography on silica (325 mL), 3% methanol in dichloromethane was used as the eluent to obtain a colorless foam of Spinozin H 2'-ketone (2.8 g, 93%) :¹HNMR(CDC1₃) δ 4.68 (S, 1, H-1'), 4.12 (d, 1, H-3'), 3.97 (m, 1, H-5').

<u>Implement A90-Spinozing A 9-Psa</u>

Combine 2'-keto Spinozin H (1.0 g, 1.39 mmol), p-toluenesulfonate (0.32 g, 1.75 mmol) and tripropylamine (0.33 ml, 1.75 mmol) in 1,4-dioxane ( The 40 ml) solution was heated to reflux temperature under nitrogen with stirring for 30 hours. Then the solvent was removed under reduced pressure and the residue was chromatographed on silica (100 mL), using 5% methanol in dichloromethane as the eluent to obtain a colorless foam of Spinozin A 9-Psa ( 0.39 g

<img file="TW487559B_D0075.tif" />

<u>Example A91- Spinozin B 9-Psa</u>

The reaction was carried out according to the method described in Example 3, with Spinozin M (199.3 mg, 0.28 mmol) as the starting material. After preliminary purification by chromatography on silica, in one step, eluted with 7% methanol in dichloromethane solution followed by 10% methanol in dichloromethane solution. Separated by preparative HPLC on a C18 column with ethyl acetate: methanol: 0.1% ammonium acetate (60 minutes linear gradient from 30:30:40 to 32.5:32.5:35). The result is Spinozin B 9-Psa (49 mg, 33% yield), a white solid, FDMS, m/e (relative intensity) 530 (M+, 60), 529 (100).

<u>Example A92-(5R,6S)-5,6-Ethoxy-3'-On-PropylSpain L and (5R,6S)-5,6-Ethoxy-3'-On-PropylSpane Nosin L</u>

In a nitrogen environment, dissolve compound 3'-On-propyl Spinozin L (1.07 g, 1.38 mmol) in dichloromethane (25 ml) and add m-CBPA (50%, 1.88 g, 5.44 mmol) and The reaction was stirred for 24 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and extracted with 10% sodium bisulfate solution (3), followed by extraction with saturated sodium bicarbonate solution (3×25 ml). The organic layer was washed with brine (25 ml) and Anhydrous potassium carbonate dried product to obtain a yellow solid (0.80 g). The crude product was purified by reverse phase HPLC (methanol: 0.1% aq. ammonium hydroxide, 90: 10) to obtain (5R,6S)-5,6-ethane Oxygen-3'-On-Propyl Spinoxine L (26.6 mg, 2.4%): Partial¹HNMRδ6.68

<img file="TW487559B_D0076.tif" />

<u>Example A93-3'-O,N-bis-(trideuteromethyl) Spinozin M</u>

Spinozin J (1.34 g, 1.87 mmol) was dissolved in dichloromethane (8ml). Potassium carbonate (1.0 g) was added, followed by 20% sodium hydroxide solution (20 ml), tetrabutylammonium hydrogen sulfate (0.77 g), and dideuteromethyl iodide (5.0 g). The reaction mixture was stirred vigorously for 20 hours. Upon gradual completion, the crude mixture was suspended in m-xylene (25 ml) and heated to reflux temperature for 1 hour. Then the mixture was cooled and chromatographed on silica gel (ethyl acetate) to provide 3'-O,N-bis(trideuteromethyl) Spinozin M (0.883 g, 64%) as a white solid: MS m/z 737. C₄₁H₅₉D₆NO₁₀Estimated value of the analysis: C, 66.73; H, 8.06; N, 1.90. Actual value: C, 66.92; H, 7.84; N2.13.

<u>Example A94-3'-(Z)-(carboxy)methylene spinoxin J</u>

(Methoxycarbonyl)methylene Spinozin J (80 mg, 0.104 mmol) was dissolved in THF (5 ml). Add methanol (3 ml) and saturated lithium hydroxide solution (3 ml). The mixture was stirred under nitrogen for 6 hours and ethanol (2 ml) was added, then the mixture was concentrated. The residue was dissolved in ethyl acetate (50 ml) and washed with brine 3 times. The organic layer was dried with sodium sulfate, concentrated and subjected to HPLC (80:20, methanol/water) to obtain a white off solid 3'-(Z)-(carboxy)methylenespine

<img file="TW487559B_D0077.tif" />

<u>Example A95-4'-Epispinoxine K</u>

The compound 4'-ketospinoxine K (235 mg, 0.328 mmol) was dissolved in dry THF (10 ml). Add dry ether (10 ml) and cool the solution under nitrogen at 0°C. While stirring vigorously, lithium tri-tert-butoxide aluminum hydride (194 mg, 0.74 mmol) was added. After 30 minutes, saturated brine was carefully added, followed by ether (100 ml). The mixture was washed 3 times with 2N sodium hydroxide/saline solution, followed by washing with 5% sodium bicarbonate solution. The organic layer was dried with potassium carbonate, filtered and concentrated under reduced pressure to obtain 4'-episospinokine K

<img file="TW487559B_D0078.tif" />

<u>Example A96-4'-Epispinoxine A</u>

The compound 4'-keto Spinozin K (149 mg, 0.207 mmol) was treated with methyl iodide, and then heated to reflux temperature in m-xylene. According to the synthetic compound 3'-O,N-bis(trideuteromethyl) Base) Spinozin M, using uranium carbonate (1.2 g), 20% aqueous sodium hydroxide solution (25 ml), methyl iodide (5.0 ml) and tetrabutylammonium hydrogen sulfate (0.80 g). The crude pyrolysis product was purified by HPLC on silica gel (ethyl acetate) and additionally purified by HPLC (86:14, methanol/water). The obtained compound is a white solid 4'-epibinosine A (74 mg,

<img file="TW487559B_D0079.tif" />

<u>Example A97-4'-trifluoromethyl-4'-episospin K</u>

The compound 4'-ketospinoxine K (170 mg, 0.237 mmol) was dissolved in dry THF (10 ml). Stir under nitrogen at 0°C and add trifluoromethyl(trimethyl)silane (241 mg, 1.69 mmol). Stirring was continued for 6 hours at 0°C. After gradual completion, the crude product was dissolved in dichloromethane (5 ml). Add water (1 ml), triethylammonium chloride (100 mg) and KHF₂(300 mg) and the mixture was stirred for 2 hours. The crude product provided is generally gradually completed to be purified on silica gel (ethyl acetate) and additionally purified by HPLC (88:12, methanol/water) to provide 4'-trifluoromethyl-4'-episobene Nosin K (65 mg, 35%)

<img file="TW487559B_D0080.tif" />

<u>Example A98-(5R,6S)-3'-dehydro-5,6-ethoxy-3'-methylene Shishinosin J and (5S,6R)-3'-deoxy-5,6- Ethoxy-3'-Methylene Spinozin J</u>

The compound 3'-deoxy-3'-methylenespinoxine J (640 mg, 0.896 mmol) was dissolved in dichloromethane (100 ml). Cool the solution to 0°C and add m-CPBA (1.06 g, ca. 3 mmol) part by part. Stirring was continued for 1 hour at 0°C. A 10% sodium bisulfite solution (100 ml) was added, followed by dichloromethane (150 ml). After extraction, the phases are separated, and the organic phase is gradually reacted in the usual manner. Purified by silica gel column and then separated by HPLC (88:12, methanol/water) to obtain a) compound (5R,6S)-3'-deoxy-5,6-ethoxy-3'-methylene Spinozin J (20 mg, 3

<img file="TW487559B_D0081.tif" />

And (5S,6R)-3'-deoxy-5,6-ethoxy-3'-methylenespinoxine J (170

<img file="TW487559B_D0082.tif" />

<u>Example A99-3'''-(4'''(((Spinozin J-3'-O-)yl)methyl)phenyl 3'''-trifluoromethyl)-3'' 'H-diazirine</u>

3-(4-(Bromomethyl)phenyl-3-difluoromethyl-3H-diazirine (6.0 g, 21.5 mmol) was dissolved in dichloromethane (6 ml). Spinozin J (1.75 g , 2.4 mmol) in dichloromethane (5 ml) was added to this solution, followed by tetrabutylammonium hydrogen sulfate (1.1 g) and 25% aqueous sodium hydroxide solution. The reaction mixture was stirred vigorously for 20 hours. Gradually completed and Chromatography on silica gel (ethyl acetate) to provide the compound 3'''-(4'''(((spinozin J-3'-O-)yl)methyl)phenyl 3'''- Trifluoromethyl)-3'''H-diazirine (1.393 g

<img file="TW487559B_D0083.tif" />

<u>Example A100-3'-O-isopropenyl Spinozin J</u>

The 3'-O-acetate of Spinozin J (3.74 g, 4.92 mmol) was dissolved in dry THF (50 ml). Dry pyridine (10 ml) was added and the solution was cooled to -78°C under nitrogen. Slowly add 0.5 M Tebbe reagent in toluene solution (25ml, 12.5mmol). After stirring at -78°C for 30 minutes, the temperature was increased to +25°C and stirring was continued for 1 hour. The mixture was cooled to 0°C and a 15% aqueous sodium hydroxide solution (20 ml) was added very slowly. After that, ether (150 ml) was added. Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to provide the compound 3'-O-isopropenyl Spinozin J (1.99 g, 53%), which is

<img file="TW487559B_D0084.tif" />

<u>Example A101-3'-O-isopropyl Spinozin J</u>

3'-O-isopropenyl Spinozin J (0.828 g, 1.09 mmol) was dissolved in dry toluene (12 ml). Add triethylsilane (338 g, 0.9 mmol). The mixture was cooled to +10°C under nitrogen and TFA (912 mg, 8 mmol) was added within 1 minute. Stirring is continued for 5 minutes, then triethylamine (1.5 ml) is added. Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to obtain the product, which is purified by HPLC (90:10, methanol/water) to provide 3'-O-isopropylspinoxine J (218 mg, 26%), as a white solid

<img file="TW487559B_D0085.tif" />

<u>Example A102-Mixture of Compound-3'-On-Propyl Spinozin J (60%-85%) and Compound 3'-On-Propyl Spinozin L (40-15%)</u>

The technical mixture of Spinozin J (60%-85%) and Spinozin L (40-15%) was dissolved in dichloromethane (50 ml). Potassium carbonate (12 g) and 20% aqueous sodium hydroxide solution (100 ml) were added, followed by n-propyl iodide (35 ml), tetrabutylammonium hydrogen sulfate (4.6 g) and DMSO (50 ml). The mixture was stirred vigorously for 72 hours. Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to provide the compound 3'-On-propyl spinoxin J (60%-85%) and the compound 3'-On-propyl spinoxin L ( 40-15%) mixture (8.87g) as a white solid:¹HNMRδ 6.65 (s, 1H), 5.37 (s, ca. 0.2H), 0.85 (t, J=7.4, 3H).

<u>Example A103-Mixture of Compound 3'-On-Propyl Spinozin J (60%-85%) and Compound 3'-On-Propyl Spinozin L (40-15%) Citrate</u>

A mixture (205 mg, 0.275 mmol) of compound 3'-On-propyl Spinozin J (60%-85%) and compound 3'-On-propyl Spinozin L (40-15%) Acetone (8 ml) was added to a solution of citric acid monohydrate (57.9 mg, 0.275 mmol) in acetone (3 ml) and the solution was stirred for 2 hours. The solvent was removed by a rotary evaporator to obtain the compound 3'-On-Propyl Spinoxin J (60%-85%) and the compound 3'-On-Propyl Spinoxin L (40-15%) citric acid Salt mixture (254 mg).

<u>Example A104-Compound 5,6-Dihydro-3'-On-Propyl Spinozin J (60%-85%) and Compound 3'-On-Propyl Spinozin L (40-15%) mixture</u>

The mixture (8.35 g) of compound 3'-On-propyl Spinozin J (60%-85%) and compound 3'-On-propyl Spinozin L (40-15%) was dissolved in ethanol (250 ml). Add cyclohexane (60 ml). The mixture was cooled to 0°C under nitrogen and 10% Pd/C catalyst (7.0 g) was added. The reaction mixture was gently heated to reflux temperature for 3 hours. Then cool to 0°C and add pyridine (2ml), filter the catalyst, concentrate the filtrate, and then dissolve in ether (300ml). Generally completed gradually, the compound 5,6-dihydro-3'-On-Propyl Spinozin J (60%-85%) and the compound 3'-On-Prop Spinoxin L (40-15%) can be provided The mixture (7.40 g) is a white solid:

<img file="TW487559B_D0086.tif" />

<u>Example A105-Compound 5,6-Dihydro-3'-On-Propyl Spinozin J (60%-85%) and Compound 3'-On-Propyl Spinozin L (40-15%) Lemon Acid mixture</u>

The mixture of compound 5,6-dihydro-3'-On-propyl Spinozin J (60%-85%) and compound 3'-On-propyl Spinozin L (40-15%) (205 mg, 0.269 mmol) was dissolved in acetone (8 ml) and a solution of citric acid monohydrate (56.7 mg, 0.269 mmol) in acetone (3 ml) was added and the solution was stirred for 2 hours. The solvent was removed by a rotary evaporator to obtain the compound 3'-On-Propyl Spinozin J (60%-85%) and the compound 3'-On-Prop Spinoxin L (40-15%) citric acid Salt mixture (211 mg).

<u>Example A106-Compound 5,6-Dihydro-3'-On-isopropyl Spinozin J</u>

3'-O-isopropenyl Spinozin J (280 mg, 0.369 mmol) was reacted with triethylsilane (110 mg, 0.94 mmol) and TFA (340 mg, 3.0 mmol) within 20 minutes, according to compound 3' -O-Isopropyl Spinozin J (Example A101) steps. After separating the crude product by HPLC (90:10, methanol/water), 5,6-dihydro-3'-On-isopropylspinoxine J (47 mg, 17%) can be obtained as a white solid: ESI MS m/z 763 (M+1).

<u>Example A107-3'-O-ethyl-N-formyl-spinoxine M and compound 3'-O-ethylspinoxine M</u>

3'-O-Ethyl Spinozin J (2.20 g, 2.95 mmol) was dissolved in ether (25 ml). The solution was then added to a solution of sodium acetate (10.3 g) in ethanol (50 ml) and water (20 ml) pre-refluxed for 30 minutes under nitrogen. Iodine (4.8 g) was then added, and after 5 minutes, 0.2N aqueous sodium hydroxide solution was added. Stirring was continued for 10 minutes at 50°C. The reaction mixture was combined with saturated sodium bisulfite solution (200 ml) and the reaction was gradually carried out in the usual manner. Flash chromatography (ethyl acetate) on silica gel followed by a 30% ethanol in ethyl acetate solution to provide a) 3'-O-ethyl-N-methanyl-spinoxine M (0.380g , 17%) is a yellow glassy solid: ESI MS m/z 760 (M+1); and compound b) 3'-0-ethyl Spinozin M (0.799 g, 37%), a white off-white solid: ESI MS m/z 732 (M+1).

<u>Example A108-N-[2'''-(Methoxycarbonyl)vinyl]-3'-O-ethyl-spinoxine M</u>

3'-O-Ethyl Spinozin M (455 mg, 0.622 mmol) was dissolved in dry dichloromethane (20 ml). Methyl propiolate (2.20 ml, excess) was added and the mixture was stirred at room temperature under nitrogen. After 18 hours, the reaction was gradually carried out and purified on a silica gel column (ethyl acetate) to obtain N-[2'''-(methoxycarbonyl)vinyl]-3'-O-ethyl-Spinnor Xin M (454 mg, 89%) as a white solid: ESI MS m/z 816 (M+1).

<u>Example A109-3``'-(4'''(((Spinozin H-2'-O-)yl)methyl)phenyl 3'''-trifluoromethyl)-3'' 'H-diazirine</u>

Spinoxin H (1.40 g, 1.95 mmol) and 3-(4-(bromoethyl) phenyl-3-difluoromethyl-3H-diazirine (2.70 g, 9.7 mmol) according to 3'''- (4 '' '(((Shibinnuoxin J-3'-O-) yl) methyl) phenyl 3' '' - trifluoromethylphenyl) -3 '' 'H-diazirine method step of quenching the reaction .Can obtain 3'''-(4'''(((Spinozin H-2'-O-)yl)methyl)phenyl 3'''-trifluoromethyl)-3''' H-diazirine (251 mg, 14%) is a white solid: ESI MS m/z 916 (M+1).

<u>Example A110-3'''-(4'''(((3-O-Ethyl Spinozin MN-) yl)methyl-)phenyl-3'''-trifluoromethyl))- 3'''H-diazirine</u>

3'-O-Ethyl Spinozin M (79 mg, 0.108 mmol) was dissolved in dry DMF (5 ml). Add triethylamine (0.50 ml), followed by 3-(4-(bromomethyl)phenyl-3-trifluoromethyl-H-diazirine (1.0 g, 3.6 mmol) in dry dichloromethane (4 ml ). The mixture was stirred at room temperature in a dark room, and chromatographed on silica gel (ethyl acetate) to obtain 3"'-(4"'(((3'-O-ethylspinoxine MN-) Yl)methyl-)phenyl-3'''-trifluoromethyl)-3'''H-diazirine (90 mg, 90%), as a yellow glassy solid: ESI MS m/z 930(M+ 1).

<u>Example A111-3'-allyl-3'-epispinoxine J</u>

3'-keto Spinozin J (3.40 g, 4.799 mmol) was dissolved in ether (40 ml). The solution was stirred vigorously under nitrogen at +10°C and allyl tributyltin (4.0 ml, excess) was added. After 5 minutes, lithium perchloride (20.0 g) was slowly introduced. Maintain vigorous stirring for 20 hours. More ethyl acetate (100 ml) was added and the reactants were allowed to react gradually. The crude product was chromatographed on silica gel (ethyl acetate) to obtain 3'-allyl-3'-epispinoxine J (2.32

<img file="TW487559B_D0087.tif" />

<u>Example A112-(14R/S-3'-allyl-13,14-dihydro-3'-epispinoxine J</u>

3'-allyl-3'-epispinoxine J (1.14 g, 1.50 mmol) was dissolved in dry dichloromethane (12 ml). The solution was cooled to -78°C under nitrogen and a part of diphenylselenyl chloride (650 mg, 3.32 mmol) was scabed. After 10 minutes, the mixture was heated to 0°C. After another 10 minutes, add triethylamine (0.50 ml) and continue stirring at 0°C for 10 minutes. The reaction was gradually carried out and chromatographed on silica gel (ethyl acetate) to obtain a white solid (1.028 g). This material was dissolved in dry toluene (20 ml). Add tri-n-butyltin hydride (1.80 ml, excess) and AIBN (110 mg). The mixture was heated to reflux temperature under nitrogen for 15 minutes. After cooling to room temperature, the mixture was concentrated under reduced pressure and purified by silica gel (ethyl acetate) chromatography. Then separated by HPLC (92:8, methanol/water) to provide (14R/S-3'-allyl-13,14-dihydro-3'-epispinoxine J (188 mg, 16%) ), is a white solid:¹HNMRδ5.45-5.75 (m, 3H), 5.04 (m, 2H). ESI MS m/z 760 (M+1).

<u>Example A113-Compound 5,6-dihydro-3'-epi-N-3'-propyl spinoxin J and compound 5,6-dihydro-3'-epi-3'-propyl spinoxine J</u>

The compound 3'-allyl-3'-epispinoxine J (218 mg, 0.287 mmol) was dissolved in ethanol (30 ml). Cool the solution to 0°C under nitrogen. Add 10% Pd/C (911 mg), followed by cyclohexane (10 ml, not purified before use). The mixture was heated to reflux temperature under nitrogen for 3 hours. Add triethylamine (1 ml). After gradual reaction and chromatography on silica gel, the crude product was separated on HPLC (94:6, methanol/water). A) compound 5,6-dihydro-3'-epi-N-3'-propyl spinoxine J (28 mg, 12%) can be obtained, which is a yellow glass

<img file="TW487559B_D0088.tif" />

(M+1) and compound b) 5,6-dihydro-3'-table-3'-propyl Spinozin J (94mg, 43%) is a white solid:

<img file="TW487559B_D0089.tif" />

<u>Example A114-3'-O-vinyl Spinozin J</u>

Spinozin J (1.2 g, 1.67 mmol) was dissolved in ethyl vinyl ether (50 ml, excess). Mercury acetate (3.5 g) was added. The mixture was heated to reflux temperature under nitrogen for 4 hours. After gradual reaction and chromatography on silica gel (ethyl acetate), 3'-O-vinyl Spinozin J (163 mg, 13%) can be obtained as a white solid: ESI MS m/z 744(M+ 1).

<u>Example A115-3'-O-(N-imidazole) sulfonyl spinoxine J</u>

Spinozin J (2.40 g, 3.34 mmol) was dissolved in dimethylformamide (15 ml). The solution was cooled to 0°C under nitrogen and imidazole (6.8 g, excess) was added. The mixture was stirred for 10 minutes. After that, thiochloride (1.90 ml) was slowly introduced via a syringe, and the mixture was warmed to room temperature and stirring was continued for 16 hours. Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to provide 3'-O-(N-imidazole) sulfonyl spinoxin J (1.52 g, 54%) as a white solid: ESI MS m/z 848 (M+1).

<u>Example A116-4'-Nor-3'-(E)(Methoxy)methylene Spinozin J</u>

3'-O-(N-imidazole) Spinozin J (1.21 g, 1.43 mmol) was dissolved in dry toluene (20 ml). Add triethylamine chloride (5.0 g, excess) and add the solution to reflux temperature under nitrogen. After 5 hours, the mixture was concentrated under reduced pressure. The crude product was purified by chromatography on silica gel (ethyl acetate) and then separated by HPLC (90:1, methanol/water) to obtain 4'-nor-3'-(E)(methoxy) Spinozin methyl J (61 mg, 6%), white

<img file="TW487559B_D0090.tif" />

<u>Example A117-5,6-Dihydro-3'-O-vinyl Spinozin J</u>

5,6-Dihydro-Spinozine J (1.85 g, 2.57 mmol) was dissolved in vinyl ether (50 ml, filtered). Mercury acetate (4.2 g, excess) was added. The reaction mixture was heated to reflux temperature for 4 hours. Then sodium carbonate solid (10 g) was added. Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to provide 5,6-dihydro-3'-O-vinylspinoxine J (1.02 g, 53%):

<img file="TW487559B_D0091.tif" />

<u>Example A118-2'-O-vinyl Spinozin H</u>

Spinozin H (2.03 g, 2.83 mmol) was reacted with butyl vinyl ether and separated from the reaction mixture according to the procedure described in 5,6-dihydro-3'-O-ethylene Spinozin J. The obtained 2'-O-vinylspinoxine H (1.09 g, 52%) was a white solid: ESI MS m/z 744 (M+1).

<u>Example A119-3'-O-(dimethyl) phosphate of Spinozin J</u>

Spinozin J (0.903 g, 1.26 mmol) was dissolved in dry pyridine (2.5ml). The solution was cooled to 0°C under nitrogen and carbon tetrabromide (0.91 g, 2.75 mmol) was added. After stirring for 5 minutes, slowly introduce trimethylphosphorus P(OMe) via a syringe₃(0.38 ml, 3.12 mmol). The cooling bath was removed and the mixture was stirred at room temperature for 3 hours. Generally, the reaction is gradually carried out and chromatographed on silica gel (15% ethanol in ethyl acetate) to provide Spinozin J's 3'-O-(dimethyl) phosphate (0.910 g, 88%), which is white Solid: ESI ME m/z 826 (M+1).

<u>Example A120-2'-O-(dimethyl) phosphate of Spinozin H</u>

Combine Spinozin H (0.65 g, 0.905 mmol) with carbon tetrabromide (0.63 g, 1.90 mmol) and trimethylphosphorus (0.30 ml, 2.46 mmol), 3'-O- (Dimethyl) phosphate is the reaction described in the step. This provides 2'-O-(dimethyl) phosphate of Spinozin H (0.230 g, 31%) as a white solid: ESI MS m/z 826 (M+1).

<u>Example A121-(14S)-13,14-dihydro-3'-O-ethyl-spinoxine M</u>

3'-O-Ethyl Spinozin M (810 mg, 1.106 mmol) was dissolved in dry ether (80 ml). A portion of lithium tri-tertiary butoxyaluminum hydride (97% powder), 1.48 g, 5.6 mmol) was added. The mixture was stirred under nitrogen at room temperature for 5 hours. Then the solution was cooled to 0°C and slowly quenched with saturated saline (5 ml). More diethyl ether (100 ml) was added and the mixture was extracted with 2N aqueous sodium hydroxide solution (2 times) and saturated sodium bicarbonate solution (1 time). The organic layer was dried with anhydrous potassium carbonate, filtered and concentrated. The crude product was chromatographed on silica gel (20% ethanol in ethyl acetate) to obtain (14S)-13,14-dihydro-3'-O-ethyl-spinoxine M (766 mg, 94%) , Is a white solid;

<img file="TW487559B_D0092.tif" />

<u>Example A 122-(14S)-13,14-dihydro-3'-O-ethyl-spinoxine M</u>

(14S)-13,14-Dihydro-3'-O-ethyl Spinozin M (326 mg, 0.444 mmol) was dissolved in dry dichloromethane (10 ml). Add triethylamine (3.0ml) and iodoethane (2.0ml, excess). The mixture was stirred under nitrogen at room temperature for 4 hours. Generally, the reaction is gradually carried out and chromatographed on silica gel (15% ethanol in ethyl acetate) to provide (14S)-13,14-dihydro-3'-O-ethyl-spinoxine M (337 mg, 99%), as a white solid: ESI MS m/z 762 (M+1).

<u>Example A 123-(14S)-13,14-dihydro-3'-On-propyl Spinozin J, compound (1R/1S,15R,21S)-15-deoxy-1,15-oxy-3 '-On-Propyl-1,21-off-spinoxine J1-hemiacetal and compound (15R)-15-deoxy-15-hydroxy 3'-On-propyl spinoxin J</u>

Within 16 hours, according to the procedure of (14S)-13,14-dihydro-3'-O-ethyl Spinozin M, 3'-On-propyl Spinozin J (1.87 g, 2.46 mmol) was reacted with a solution of lithium tri-tert-butoxide aluminum hydride (97% powder, 2.02 g, 7.71 mmol) in ether (100 ml). Generally, the reaction is gradually carried out and chromatographed on silica gel (ethyl acetate) to provide a) (14S)-13,14-dihydro-3'-On-propyl Spinozin J (1.19 g, 63%), which is White solid:

<img file="TW487559B_D0093.tif" />

And b) a mixture of compound (0.66 g), which was further analyzed by HPLC (88:12, methanol/water) to obtain c) (1R/1S,15R,21S)-15-deoxy-1,15-oxygen -3'-On-Propyl-1,21-off-spinoxine J 1-hemiacetal (77 m

<img file="TW487559B_D0094.tif" />

; And d) (15R)-15-deoxy-15-hydroxy 3'-On-propyl Spinozin J (44 mg, 2.3%), as a white solid:

<img file="TW487559B_D0095.tif" />

<u>Example A124-(14S)-5,6,13,14-tetrahydro-3'-On-propyl Spinozin J</u>

In a period of 3 hours, make 5,6-dihydro-3'-On-propyl Spinnol according to the procedure described in (14S)-13,14-dihydro-3'-O-ethylspinoxin M Xin J (1.88 g, 2.47 mmol) was reacted with a solution of lithium tri-tert-butoxide aluminum hydride (97% powder, 2.40 g, 9.16 mmol) in ether (100 ml). In the gradual reaction and chromatography on silica gel (ethyl acetate) can provide (14S)-5,6,13,14-tetrahydro-3'-On-propylspinoxine J (1.440 g, 76% ), for white

<img file="TW487559B_D0096.tif" />

<u>Example A125-3'-O-(2-Methoxyethyl)-Spinozin J</u>

Spinozin J (718 mg, 1.00 mmol) was dissolved in DMSO (1.4 ml) and dichloromethane (1.4 ml). Add potassium carbonate (0.90 g, 6.5 mmol), 20% sodium hydroxide aqueous solution (5.2 ml), tetra-n-butylammonium hydrogen sulfate (339 mg, 1.00 mmol) and 2-methoxyethyl bromide (1.00 ml, 10.6 mmol). The mixture was partitioned between ether and water. The organic layer was extracted with ether. The combined organic layer was washed with water (3 times) and saturated sodium bicarbonate, dried with anhydrous potassium carbonate and evaporated to produce a yellow oil. This oil was chromatographed in a reverse phase column (Kromasil'Cl8, silica ODS, 100, 10m, spherical, 25cm×20 mm), using 88% methanol and 12% water (containing 0.1% v/vconc. Hydrogen Aqueous ammonium oxide) to produce a white amorphous solid (249 mg, 32%).¹HNMR d 3.78 (m, 2H), 3.38 (s, 3H).

<u>Example A126-3'-Methoxymethyl-Spinozine J</u>

Spinozin J (359 mg, 0.500 mmol) was dissolved in dichloromethane (2ml) and the resulting solution was cooled to 0°C. Add diisopropylethylamine (105 ml, 0.600 mmol) in a single portion and then add bromomethyl methyl ether (90%, 50 ml, 0.5 mmol) in divided portions. The precipitate formed immediately can be reconstituted in a few minutes. The mixture was stirred at 0°C for 2.5 hours and at room temperature for another 2.5 hours. The mixture was cooled to 0°C and additional amine (0.32 ml, 1.8 mmol) and bromide (0.15 ml, 1.7 mmol) were added. The mixture was stirred for another hour. A saturated aqueous sodium hydrogen carbonate solution (3 ml) was added and the mixture was stirred at room temperature for 17 hours. The mixture was diluted with dichloromethane and washed with sodium bicarbonate solution, water (2 times), saturated sodium bicarbonate solution, and brine (2 times). The mixture was dried with anhydrous potassium carbonate and evaporated to produce a yellow oil. This oil was chromatographed in a reverse phase column (Kromasil'Cl8, silica ODS, 100, 10m, spherical, 25cm×20 mm), using 85% methanol and 15% water (containing 0.1% v/v conc. Aqueous ammonium hydroxide) to produce a white glassy solid (87 mg, 3%).¹HNMR d 3.45 (s, 3H); MS m/z 762.6 (M+H estimated value is 762.5).

<u>Example A127-5,6-Dihydro-3'-methoxymethyl-spinoxine J</u>

Place 3'-methoxymethyl-spinoxine J (243 mg, 0.319 mmol) and palladium/activated carbon (10%, 200 mg) in a 25-ml round bottom flask. Ethanol (7.25 ml) and cyclohexane (1.75 ml, 17.3 mmol) were added and the resulting solution was heated to reflux temperature for 3 hours. The mixture was continuously filtered through diatomaceous earth and PTFE membrane (Gelman, Acrodisc 13CR, 0.2mm). The mixture was concentrated under reduced pressure and the residue was chromatographed on a reverse phase column (Kromasil'Cl8, silica ODS, 100, 10m, spherical, 25cm×20 mm), using 85% methanol and 15% water (containing 0.1% v/v conc. ammonium hydroxide aqueous solution) to produce an amorphous solid (68 mg, 28%). MS m/z 764.7 (the estimated value of M+H is 764.5).

<u>Example A128-3'-O-cyanomethyl-spinoxine L and 3'-O-trimethylsilyl-spinoxine L</u>

Spinozin L (366 mg, 0.500 mmol) was dissolved in DMF (1 ml) and the solution was cooled to -15°C. 1.0 M photoresist adhesion promoter (0.53 ml, 0.53 mmol) was added dropwise to produce a very dark solution, which allowed to maintain at 0°C for 65 hours. The reaction mixture was partitioned between ethyl acetate and saturated sodium bicarbonate. The organic layer was diluted with sodium bicarbonate aqueous solution, washed with sodium thiosulfate dilution and brine. The mixture was dried with anhydrous magnesium sulfate and the solvent was removed under reduced pressure to produce a dark oil (380 mg). The oil is chromatographed in a reverse phase column (Kromasil'Cl8, silica ODS, 100, 10m, spherical, 25cm×20 mm), using 90% methanol and 10% water (containing 0.1% v/v conc. Ammonium hydroxide aqueous solution) to produce two products, 3'-O-cyanomethyl-spinoxine L (48 mg, 12%),¹HNMR d 4.25, 4.44 (abq, J=14.9, 2 H); MS m/z 771.6 (the estimated value of M+H is 771.5); and 3'-O-trimethylsilyl-spinoxine L( 241 mg, 60%),¹HNMR d 0.19 (s, 9H): MS m/z 804.7 (M+H estimated value is 804.5).

<u>Example A129-3'-O-carbon-t-butoxymethyl-spinoxine J</u>

Spinozin J (718 mg, 1.00 mmol) and tetra-n-butylammonium hydrosulfate (34 mg, 0.10 mmol) were dissolved in dichloromethane (3.5 ml). T-Butyl bromoacetate (2.95 ml, 20.0 mmol) and powdered potassium hydroxide (1.0 g, 15 mmol) were continuously added in a single portion and the reaction mixture was stirred at room temperature. After 40 minutes, tetra-n-butylammonium hydrogen sulfate (64 mg, 0.2 mmol) was added. After another 60 minutes, the mixture was diluted with water and extracted with dichloromethane (1 time) and ethyl acetate (1 time). The combined organic layer was washed with water (2 times), saturated sodium bicarbonate and brine, dried with potassium carbonate and magnesium sulfate and evaporated to produce a yellow oil (4.13 g). This oil was placed under reduced pressure (0.9 Torr) for 2 hours to produce oil (2.3 g). The oil was chromatographed on silica gel (165 g) with ethyl acetate and then 10% methanol in ethyl acetate to produce

<img file="TW487559B_D0097.tif" />

<u>Example A130-4''-N-desmethyl-4''N-(2-fluoroethyl)5,6-dihydro 3'-O-propyl Spinozin J</u>

According to the method of Example CZ, 1-bromo-2-fluoroethane (0.20 ml, 0.34g, 2.7 mmol), sodium iodide (0.04 g, 0.3 mmol), (i-Pt)₂NEt (0.40 ml, 0.30 g, 2.3 mmol), 4"-N-desmethyl-5,6-dihydro 3'-O-propyl Spinozin J (0.300 g, 0.401 mmol), and DMF to prepare the compound .MPLC (25:7 to 50:50 ethyl acetate/cyclohexane) to obtain 4"-N-desmethyl-4"N-(2-fluoroethyl)5,6-dihydro as a white powder -3'-O-Propyl Spinozin J.

<u>Example A131-4"-N-[3'-O-(9-fluorenylmethoxycarbonyl)β-propylamino]spinoxin J and 3'-O-(β-propylamino)spin Nosin J</u>

Add trimethylamine (0.25 ml, 0.18 g, 1.8 mmol) and HC=C(Me)OCOCl (0.10 ml, 0.11g, 0.92 mmol) to Fmoc-N(H)-b-Ala-COOH (0.26 g , 0.84 mmol) 0°C, Spinozin J (0.502 g, 0.699 mmol), and DMAP (0.02 g, 0.2 mmol) in dichloromethane (4 ml) at 0°C. After 2 hours, the mixture was heated to room temperature for 20 hours. The mixture was evaporated. MPLC(SiO₂, 0: 100 to 20: 80 methanol/dichloromethane) to obtain 0.17 g (24%) of 4"-N-[3'-O-(9-fluorenylmethoxycarbonyl)β-propylamine sulfonyl] Spinozin J is a white powder: MS(m+H⁺) Expected value: 1011.6. Actual value: 1011.6 and 3'-O-(β-propylamino) Spinozin J, 0.21 g, (38%), white powder: MS(m+H⁺ &amp; m+2H⁺) Expected value: 789.5 &amp; 395.2. Actual value: 395.5. The Fmoc group is easily deprotected under the reaction conditions and can be regarded as the product of the present invention.

<u>Example A132-4"-N-desmethyl-5,6-dihydro-3'-O-propyl Spinozin J</u>

Add F-TEDA (0.98 g, 2.8 mmol) to the CH of 5,6-dihydro-3'-On-propyl Spinozin J (0.94 g, 1.23 mmol)₃CN solution (10m). After 10 minutes, the organic layer was partitioned between ethyl acetate and 0.1 M hydrochloric acid. The organic layer was washed sequentially with sodium bicarbonate and brine. The organic layer was dried with magnesium sulfate, filtered, and evaporated. MPLC(SiO₂, 5:95 to 10:90 methanol/dichloromethane) to obtain 0.64 g (70%) of 4"-N-desmethyl-5,6-dihydro-3'-O-propyl Spinozin J , It is a white powder and can be recycled (5,6-Dihydro-3'-On-Propyl Spinozin J0.25 g (27%)).

<u>Example A133-1'-epi-spinozin K</u>

Within 15 minutes, O-(2,3-di-O-methyl-4-O-benzyl-α-L-rhamnosyl-ranosyl) trichloroacetimidate (5.5 g, 11.5 mmol) in dichloromethane (60 ml) was added drop by drop to Spinozin A 9-Psa (1.25 g, 2.30 mmol) and pyridine-p-toluenesulfonate (0.815 g, 3.24 mmol) of dry dichloromethane (120 ml) in a solution containing powdered 4A molecular sieves (2.0 g). After stirring for 4 days at room temperature, the mixture was filtered with diatomaceous earth, the diatomaceous earth was washed with dichloromethane (100 ml) and the combined filtrate was washed with saturated sodium carbonate (2×60 ml) and brine (75 ml) and Lotion. After concentration, 8.2 g of residue remained, which was flash chromatographed with silica (650ml) using 3% methanol in dichloromethane to produce 1.1g (58%) of the coupling product, colorless foam 3α: 1β epimer. This material was dissolved in methanol (30 ml) and anhydrous calcium carbonate was added to the solution (0.98 g, 7.1 mmol). The solution was stirred at room temperature for 6 hours, acidified by the dropwise addition of 2N hydrochloric acid (6.8 ml) and then concentrated under reduced pressure to near dryness. The residue was partitioned between water (40 ml) and dichloromethane (150 ml). The organic extract is then saturated with sodium bicarbonate (40 ml) and brine (40 ml) and dried with magnesium sulfate. After concentration, 0.72 g of crude debenzylated product remained. Purified by flash chromatography of silica (80 ml), using 4% methanol in dichloromethane to produce 0.5 g of clean products, which are Spinozin K and 1'-epi-Spinozin K The 3:1 mixture. By HPLC with a 41.4 mm×25 cm (1) Rainin reverse phase Cl8 (8μm) column, using 15% water (containing 0.01% ammonium hydroxide) in methanol as the eluent, the product was separated into 3 pieces of about 180 The part of mg. β-epimer, 1'-epi-spinozin K precipitated first: 110 mg;

<img file="TW487559B_D0098.tif" />

<u>Example A134-4'-O-Propyl Spinozin K</u>

Add a portion of propionic anhydride (0.1 ml, 0.77 mmol) to a properly stirred solution of Spinozin K (72 mg, 0.1 mmol) and DMAP (~2mg) in dry pyridine (1.0 ml) at room temperature, And the mixture was stirred for 20 hours. The pyridine was removed under reduced pressure, the residue was dissolved in dichloromethane (25 ml) and the dichloromethane solution was washed with water (5 ml), saturated sodium carbonate solution (25 ml), brine (5 ml) and Dry over magnesium sulfate. After filtering off the dry reagents, the dichloromethane solution was concentrated to about 6 ml and stirred with polyvinylpyridine (0.3 g) for 15 minutes to neutralize any residual propionate in the product. After filtration of the resin, the dichloromethane was evaporated and 85 mg of crude product remained. Purified by flash chromatography of silica (30ml), using 3% methanol in dichloromethane to produce 71 mg of 4'-O-propanospinoxine K as a colorless foam:¹HNMRδ 6.76

<img file="TW487559B_D0099.tif" />

<u>Example A135-(9-O-(2,3,4-tris-O-methyl-D-rhamnosyl) analogue of Spinozin A</u>

Compound 9-O-(2,3,4-tri-O-methyl-D-rhamnosylpyranosyl) Spinozin A, 3.5α:1β epimer at position 1' The mixture was prepared by the same procedure as in Example A45, with a yield of 82%, consisting of 0.54 g (1.0 mmol) Spinozin A 9-Psa, 0.35 g (1.4 mmol) pyridine p-toluene sulfuric acid and 2.0 g (5.7 mmol) O-(2,3,4-methyl-α-D-rhamnosyl)-trichloroacetimidate was prepared. The α and β epimers were performed by reverse phase HPLC with Cl8 bonded silica gel, using 10% water (containing 0.1% ammonium hydroxide) in methanol. The beta epimer is the first to precipitate out. β epimer: 85 mg; colorless foam;¹HNMR(CDCl₃)δ6.78<img file="TW487559B_D0100.tif" /><img file="TW487559B_D0101.tif" />

<u>Example A136-3'-O-(2,2,2-trifluoroethyl) Spinozin J</u>

Add a part of 60% sodium hydride mineral oil (224 mg, 5.6 mmol as 100%) suspension to cold (0-5°C), suitably stirred Spinozin J (500 mg, 0.7 mmol) anhydrous THF (20 ml) solution. When H₂When the release stopped (after about 10 minutes), trifluoroethyl triflate (J. Org. Chem. 1973, 38, 3673: Tetrahedron 1965, 21, 1) (0.9 g, 3.2 mmol) was added dropwise. The cooling bath was removed after 1 hour, and the reaction was still stirred at room temperature for 4 hours. The mixture was then cooled to 0-5°C, and water (20 ml) was added dropwise over 15 minutes. The mixture was extracted with ether (2×50 ml). The organic extract was washed with brine (25 ml) and dried with magnesium sulfate. Purify by flash chromatography of silica (120 ml), using 3% methanol in dichloromethane as the eluent. The partially purified product (150 mg) was purified by 41.4 mm (id) × 25 mm (1) Rainin reverse phase C8 (8 μm) column, using 10% water (containing 0.1 ammonium hydroxide) in methanol as the eluent , To produce 58mg of clean 3'-O-(2,2,2-trifluoroethyl) Spinozin J product, which is

<img file="TW487559B_D0102.tif" />

<u>Example A137-3'-O-(2,3,4-tris-O-ethyl)-α-rhamnose (pyranosyl) Spinozin J and 3'-O-(2,3, 4-tri-O-ethyl)-β-rhamnose (pyranosyl) Spinozin J</u>

The 9:1 epimer mixture of α:β at position 1''' (0.33 g, 62% yield) was obtained from Spinozin J (0.4 g, 0.56 mmol), pyridine P-toluenesulfonate (0.18 g, 0.72 mmol) and O-(2,3,4-tris-O-ethyl α-L-rhamnosyl pyranosyl (0.18 g, 0.72 mmol) and O- (2,3,4-Tri-O-ethyl-α-L-rhamnosylpyranyl) trichloroacetimidate (1.6 g, 4.08 mmol) was reacted by the same procedure as in Example A45 The mixture was prepared by reverse phase HPLC with Cl8 bonded silica (8 mm) column (41.1 mm(id)×25 cm(1)), using 6% water (containing 0.15% ammonium hydroxide) in methanol as the column Eluent. β-epimer precipitates first. β-epimer: 5 mg; colorless foam;¹HNMR(CDCl₃)δ6.76(s,

<img file="TW487559B_D0103.tif" />

<u>Example A138-4'-deoxy-3'-dehydro-3'-alkenyl-spinozin J</u>

Add a part of 2,2,2-trifluoroethanol (15μl, 0.21 mmol) to a suspension of cold (0°C) 60% sodium hydride mineral oil 7.5 mg, 0.188 mmol is 100%) in dry DMF (2.5 ml) and Stir under refrigeration for 20 minutes. A portion of 3'-O-trifluoromethanesulfonyl-3'-epi-spinoxine J (100 mg, 0.118 mmol) was added to the current clean solution, and the mixture was stirred at room temperature for 18 hours. Add silica gel (200 mg), stir the mixture for 10 minutes, then filter and wash the collected silica with dichloromethane. The filtrate and washings were combined and concentrated to dryness under reduced pressure and the residue was dissolved in ethyl acetate (20 ml). Then the ethyl acetate solution was washed with brine (10 ml) and dried with magnesium sulfate. Evaporate the solvent to leave 76 mg of residue, which was flash chromatographed on silica (10 ml) using 3% methanol in dichloromethane as the eluent to obtain 43 mg of 4'-dehydrogen-3'-de Oxygen-3'-alkenyl-spinozin J, a colorless foam:

<img file="TW487559B_D0104.tif" />

<u>Example A139-3'-O-propyl Spinozin L</u>

Add powdered potassium hydroxide (1g) to Spinozin L (0.732g, 1.0 mmol), tetrabutyllithium bromide (0.1 mmol) and n-propyl bromide (2.7g, 22.0 mmol). A solution of methyl chloride (3 ml) was stirred under nitrogen at 25°C for 1.5 hours. Water (10 ml) was added and the layers were separated. The aqueous layer was extracted with dichloromethane (3×20 ml) and the combined extracts were dried over sodium sulfate, filtered, and concentrated. Triturated with pentane to obtain 3'-O-propyl Spinozin L (0.72 g, 93%) as a colorless glass. EMI MS, m/z 774.7(M⁺)。

<u>Example A140-3'-O-propyl Spinozin J&amp;L</u>

Powdered potassium hydroxide (1g) was added to Spinozin J&amp;L (0.732g, 1.0 mmol), tetrabutyllithium bromide (0.032 g, 0.1 mmol) in bromopropane (5 ml)) solution and under nitrogen and Stir at 25°C for 3 hours. Add ether (20 ml) and water (10 ml) and separate the phases. The aqueous layer was extracted with ether (2×20 ml) and dried over magnesium sulfate. The combined ether extracts were filtered and concentrated. Triturated with pentane to obtain 3'-O-propyl Spinozin J &amp; L (0.71 g, 92%) as a colorless glass. EMI MS of 3'-O-propyl Spinozin J, m/z760.2(M⁺). EMI MS of 3'-O-propyl Spinozin L, m/z 774.3(M⁺)。

<u>Example A141-3'-O-Ethyl Spinozin L</u>

Add powdered potassium hydroxide (1g) to Spinozin L (0.732g, 1.0 mmol), tetrabutyllithium bromide (0.032 g, 0.1 mmol) in bromoethane (5 ml)) and place it under nitrogen And stirring at 25°C for 0.5 hours. Add ether (20 ml) and water (10 ml) and separate the phases. The aqueous layer was extracted with ether (2×20 ml) and dried over magnesium sulfate. The combined ether extracts were filtered and concentrated. Triturated with pentane to obtain 3'-O-ethyl Spinozin L (0.72 g, 94%) as a colorless glass. EMI MS of 3'-O-propyl Spinozin J, m/z760.3(M⁺)。

<u>Example A141-3'-O-propyl Spinozin Q</u>

Spinozin Q (732 mg, 1 mmol), tetrabutyl lithium bromide (34 mg, 0.1 mmol), 1-bromopropane (4 ml, 24 mmol), and dichloromethane (1 ml) were placed A 50 ml round bottom flask equipped with magnet stirring. Stir the solution in a water bath at room temperature and add a part of sodium hydroxide (500 mg, 7.5 mmol, 85% pure, mechanically pulverized before use). The resulting mixture was stirred for 2 hours, and then the reaction was gradually completed by partitioning between 25 ml ethyl acetate and 5 ml water. The phases were separated and the ether phase was washed with 1×5 ml water and 1×5 ml saturated sodium chloride , Dry with magnesium sulfate, and remove the solvent with a rotary evaporator. The residue was chromatographed with 30 g of silica gel, and ethanol/ethyl acetate/hexane (2:50:50) was used as the eluent to obtain compound 3'-O-propyl Spinozin Q, which was white and brittle High-quality foam (610 mg, purity 97.5%, analyzed by HPLC).

<u>Part B modified pseudoglycan compounds by substituting non-sugar derivatives for amino sugars</u>

<u>Example B1-17-O-Acetyl-Spinozin A 17-Psa</u>

A solution of Spinozin A (2.00 gm, 2.73 mmol) in glacial acetic acid (50 ml) was heated to reflux temperature for 5 hours and then stirred at room temperature for 12 hours. The reaction mixture was evaporated to a small volume, and then poured into saturated sodium bicarbonate solution. The aqueous mixture was extracted with ether. The ether was washed with brine, dried with potassium carbonate and evaporated at room temperature and reduced pressure to obtain a yellow glass (1.48 gm). The product was separated by chromatography on silica, and a 40% ethyl acetate solution in methyl hexane was used as the eluent. The isolated 17-O-acetyl-spinoxin A 17-Psa (478.9 mg, yield 28%) is a white solid, FDMS, m/e (relative intensity) 632 (M⁺-H, 100), and the separated Spinozin A17-Psa (846.2 mg, yield 52%) are colorless glass.

<u>Example B2-17-O-(3-Dimethylaminopropyl) Spinozin A 17-Psa</u>

Compound 17-O-(Propyl) Spinozin A 17-Psa (206.3 mg, 0.32 mmol) was dissolved in dimethylamine (5 ml) in an ice bath and the reaction mixture was capped and kept at -5°C Stir for 2 hours. Then when the dimethylamine is distilled through the acid scrubber, the reaction mixture is allowed to warm to room temperature. The residue was dissolved in aqueous HCl and washed with ether. The aqueous solution was basified with 5N sodium hydroxide and extracted with fresh ether. The ether obtained from the alkaline extract was washed with brine, dried with sodium carbonate and dried at room temperature under reduced pressure. The obtained 17-O-(3-dimethylaminopropyl) Spinozin A 17-Psa (177.1 mg, 80% yield) is a colorless glass, FDMS, m/e (relative strength) 689(M⁺，100)。

<u>Example B3-17-O-(3-Propanoyl) Spinozin A 17-Psa</u>

To Spinozin A 17-Psa (364.3 mg, 0.61 mmol) in chloroform (10 ml), add propyl chloride (75 ml, 0.92 mmol) and then add diisopropylethylamine (160 ml, 0.92 mmol). The reaction mixture was heated to reflux temperature for 6 hours, then propyl chloride (75 ml, 0.92 mmol) and diisopropylethylamine (160 ml, 0.92 mmol) were added and the mixture was refluxed for another 12 hours. The reaction mixture was then cooled to room temperature and diluted with dichloromethane. The dichloromethane was washed with saturated sodium bicarbonate, dried with potassium carbonate and evaporated at room temperature under reduced pressure. The crude material was chromatographed on silica and eluted with 20% ethyl acetate in dichloromethane. The obtained 17-O-(3-propanyl) Spinozin A 17-Psa (322 mg, 82% yield) was a colorless glass, FDMS, m/e (relative strength) 643 (M⁺-H, 100).

<u>Example B4-17-O-(Chloroacetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B3, using Spinozin A 17-Psa (205.4 mg, 0.35 mmol) as the starting material, and chloroacetyl chloride, and using 40% ethyl acetate in hexane as the starting material Chromatography eluent. The obtained 17-O-(chloroacetyl) Spinozin A 17-Psa (194.7 mg, 83% yield) is a white deviating glass, FDMS, m/e (relative strength) 666 (M⁺-H, 100), 190 (20).

<u>Example B5-17-O-(4-Chlorobutanol) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B3, using Spinozin A 17-Psa (206.4 mg, 0.35 mmol) as the starting material, and chlorobutyryl chloride, and using 35% ethyl acetate in hexane as the chromatography Eluent. The obtained 17-O-(chlorobutanyl) Spinozin A 17-Psa (224.3 mg, 92% yield) was a white deviating glass, FDMS, m/e (relative strength) 696 (M⁺，100)，189(20)。

<u>Example B6-17-O-(Dimethylamine Acetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B2, with 17-O-(chloroacetyl)spinoxine A 17-Psa (99.4 mg, 0.15 mmol) as the starting material. The obtained 17-O-(dimethylamine acetyl) Spinozin A17-Psa (79.1 mg, 78% yield) is a beige glass, FDMS, m/e (relative strength) 675 (M⁺-H, 100).

<u>Example B7-17-O-(4-iodobutanol) Spinozin A 17-Psa</u>

To a solution of 17-0-(4-chlorobutanol) Spinozin A 17-Psa (91.9 mg, 0.13 mmol) in acetone (3 ml), sodium iodide (198 mg, 1.3 mmol) was added. When a precipitate formed, the reaction mixture was heated to reflux temperature for 18 hours. The mixture was cooled to room temperature, diluted with water and extracted with dichloromethane. The dichloromethane was dried with potassium carbonate and evaporated at room temperature under reduced pressure. 17-O-(4-iodobutanol) Spinozin A 17-Psa (94.7 mg, 93% yield) can be obtained as a beige glass, FDMS, m/e (relative strength) 832 ( 10), 786(M⁺-H, 100), 704(20), 377(30).

<u>Example B8-17-O-(4-Dimethylbutanol) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B2, starting with 17-O-(4-iodobutanol)) Spinozin A 17-Psa (216.9 mg, 0.28 mmol). The obtained 17-O-(4-dimethylaminobutanol) Spinozin A 17-Psa (155.7 mg, 79% yield) is a beige glass, FDMS, m/e (relative strength) 704 (M⁺，100)。

<u>Example B9-17-O-(N'-Methyl-N-yl acetate) Spinozin A 17-Psa</u>

To a solution of 17-O-(4-chloroacetyl) Spinozin A 17-Psa (150.5 mg, 0.23 mmol) in chloroform (7 ml), add diisopropylamine (59 μl, 0.34 mmol), and then add 1-Methylphosphonium (38 μl, 0.34 mmol). The reaction mixture was allowed to stir at room temperature for 1 hour, and then heated to reflux temperature for 2.5 hours. The mixture was cooled to room temperature for 3 days. Then add 1-methyl (0.5 ml) and heat the mixture to reflux temperature for 4 hours. The reaction mixture was then cooled to room temperature and poured into saturated sodium bicarbonate solution, and extracted with ether. The ether portion was washed with brine, dried over magnesium sulfate and evaporated at room temperature and reduced pressure. The crude product was purified by chromatography on silica and precipitated with 7% methanol in dichloromethane and then with 20% methanol in dichloromethane (single step). The obtained 17-O-(N'-methyl-N-acetic acid) Spinozin A 17-Psa (63.1 mg, 38% yield) was a colorless glass, FDMS, m/e (relative Strength) 730 (M⁺，100)，731(80)。

<u>Example B10-17-O-(N-morphoyl acetate) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B9, with 17-O-(chloroacetyl)spinoxine A 17-Psa (152.3 mg, 0.23 mmol) and moxan (0.5 ml at a time) as starting materials. Reflux for 14 hours and extract with dichloromethane. The obtained 17-O-(N-morphoyl acetate) Spinozin A 17-Psa (141.3mg; 86% yield) is a white off-glassy substance , FDMS, m/e (relative intensity) 717 (M⁺，100)。

<u>Example B11-17-O-(2-(1-imidazolyl)acetyl) Spinozin A 17-Psa</u>

To a solution of sodium hydride (50% suspension in mineral oil; 15.8 mg, 0.33 mmol) in THF (5 ml), imidazolyl (23.1 mg, 0.34 mmol) was added. 17-O-Bromoacetylspinoxine A 17-Psa (201.1 mg, 0.28 mmol) was added to the mixture, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane and washed with water. Then the dichloromethane part was washed with salt, dried with potassium carbonate, and evaporated at room temperature under reduced pressure. The crude product was purified by chromatography on silica and eluted with 5% ethanol in ethyl acetate. The obtained 17-O-(2-(1-imidazolyl)acetyl) Spinozin A 17-Psa (117 mg, 60% yield) is a colorless glass, FDMS, m/e (relative strength )699(M⁺，100)，698(40)，189(40)，101(70)。

<u>Example B12-17-O-(2-(4-(2-pyrimidinyl)-1-hexahydroyl)acetinyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B11, using 17-O-(bromoacetyl)spinoxine A 17-Psa (209.6 mg, 0.29 mmol), and 2-(1-hexahydroyl)pyrimidine as The starting material and dichloromethane using 5% methanol were the chromatographic eluent. The obtained 17-O-(2-(4-(2-pyrimidinyl)-1-hexahydroyl)acetyl)spinoxine A 17-Psa (233.6 mg, 97% yield), It is white deviating from glass, FDMS, m/e (relative intensity) 717 (M⁺，60)，794(100)。

<u>Example B13-17-O-(2-(4-Methylamino-1-pyridinyl)acetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B11, with 17-O-(bromoacetyl)spinoxine A 17-Psa (201.8 mg, 0.28 mmol) and 4-dimethylamine pyridine as starting materials, Stir at room temperature for 3 days, and use 10% methanol in dichloromethane and then 100% methanol (single step) as the eluent. The obtained 17-O-(2-(4-methylamino-1-pyridinyl) ethanoyl) Spinozin A 17-Psa (104.2 mg, 49% yield) was a white solid, FDMS ,M/e(relative intensity)759(M⁺，100)。

<u>Example B14-17-O-(4-pyridinecarbonyl) Spinozin A 17-Psa</u>

To a suspension of isonicotinic acid (50.7 mg, 0.41 mmol) in dichloromethane (10 ml), add DMAP (18.5 mg, 0.4 mmol) and Spinozin A17-Psa (202.1 mg, 0.34 mmol), and Then DCC (107.8 mg, 0.52 mmol) was added. The reaction mixture was stirred at room temperature for 2.5 days, and then diluted with ether and filtered. The filtrate was evaporated at room temperature under reduced pressure. The residue was purified by chromatography on silica and eluted with 50% ethyl acetate in hexane. The obtained 17-O-(4-pyridinecarbonyl) Spinozin A 17-PSa (116.1 mg, 74% yield) was a colorless glass, FDMS, m/e (relative strength) 696 (M⁺，100)，694(40)。

<u>Example B15-17-O-(hexahydroacetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B11, with 17-O-(bromoacetoxy)spinoxine A 17-Psa (206.4 mg, 0.29 mmol), and hexahydro as starting materials. The obtained 17-O-(hexahydroacetyl) Spinozin A 17-Psa (127.1 mg, 61% yield) was a beige solid, FDMS, m/e (relative intensity) 791(40) , 718(40), 717(M⁺，65)。

<u>Example B16-17-O-(N-Methyl-L-proline) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B14, with Spinozin A 17-Psa (207.7 mg, 0.36 mmol) and N-methylproline (63.7 mg, 0.43 mmol) as starting materials. Obtained 17-O-(N-methyl-L-proline) Spinozin A 17-Psa (65.9 mg, 43% yield, based on recovered Spinozin A17-Psa), FDMS , M/e (relative intensity) 703(95), 702(M⁺，100)。

<u>Example B17-17-O-[N-(2-hexahydropyridineethyl)]amine acetylspinoxine A 17-Psa</u>

The reaction was carried out as described in Example B14, starting with 17-O-bromoacetylspinoxine A 17-Psa (205.1 mg, 0.29 mmol) and 1-(2-aminoethyl)-hexahydropyridine substance. After chromatography on silica, the eluent was dissolved in 10% methanol in dichloromethane, and the product was further subjected to reverse phase HPLC on a Cl8 column with ethyl acetate: methanol: 0.1% ammonium acetate (35:35: 30 to 45:45:10, linear gradient within 60 minutes) to obtain 17-O-[N-(2-hexahydropyridinethyl)]amine acetylspinoxine A 17-Psa (11 mg , 5% yield), white solid, FDMS, m/e (relative intensity) 760 (90), 759 (M⁺，100)。

<u>Example B19-17-O-(Ethoxycarbonylacetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B3, with Spinozin A 17-Psa (207.7 mg, 0.35 mmol) and diethyl chlorinated malonate (484 ml, 3.78 mmol; only added once). The obtained 17-O-(ethoxycarbonyl acetyl) Spinozin A 17-Psa (153.6 mg, 62% yield) was a colorless solid, FDMS, m/e (relative intensity) 704 (M⁺，100)，189(51)。

<u>Example B19-17-O-(Bromoacetyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example B3, using Spinozin A 17-Psa (4.06 g, 6.9 mmol) and bromoacetyl bromide as starting materials, and using 10% ethyl acetate in dichloromethane solution as Chromatography eluent. The resulting 17-O-(bromoacetyl) Spinozin A 17-Psa (2.92 g, 59% yield) was a white off-solid, FDMS, m/e (relative intensity) 712 (M⁺，50)，713(85)。

<u>Example B20-17-O-(p-aminophenacetin) Spinozin A 17-Psa</u>

Compound 17-O-(p-nitrophenylacetoxy) Spinozin A17-Psa (101.6 mg, 0.134 mmol) was dissolved in ethanol (5 ml) and tin(II) chloride (142.6 mg, 0.75 mmol) was added ). The yellow color of the solution quickly disappeared and replaced with a white suspension. The reaction was heated to reflux temperature for 3.5 hours, cooled to room temperature, and allowed to remain overnight. After gradual reaction to form a residue, the residue was purified by chromatography on silica gel (ethyl acetate/hexane) to provide 17-O-(p-aminophenethyl)spinoxine A 17-Psa (45.3 mg, 46% yield), a white solid, FDMS m/z 723. C₄₁H₅₇NO₁₀Estimated value of analysis: C, 68.03; H, 7.94; N, 19.3. Actual value: C, 67.53; H, 7.91; N, 2.41.

<u>Example B21-17-O-(O-Chlorobenzyl) Spinozin A 17-Psa</u>

The compound was prepared as described in Example B46, using o-chlorobenzoic acid (51 mg, 0.32 mmol), DMAP (47 mg, 0.38 mmol), Spinozin A17-Psa (135 mg, 0.228 mmol), and DCC (49 mg, 0.23 mmol). The obtained 17-O-(o-chlorobenzyl) Spinozin A 17-Psa (69 mg, 41%) was a white solid, FDMS m/z 729.

<u>Example B22-17-O-(m-Chlorophenethyl) Spinozin A 17-Psa</u>

The compound was prepared as described in Example B46, using m-chlorophenylacetic acid (42.3 mg, 0.24 mmol), DMAP (45.2 mg, 0.37 mmol), Spinozin A 17-Psa (134.4 mg, 0.22 mmol), and DCC (51.2 mg, 0.24 mmol). The obtained 17-O-(m-chlorophenethyl) Spinozin A 17-Psa (101.5 mg, 60%) was a white solid: FDMS m/z 742.

<u>Example B23-17-O-Formyl Spinozin A 17-Psa</u>

The compound Spinozin A17-Psa (104 mg, 0.18 mmol) was dissolved in benzene (5 ml) and dimethylformamide dimethyl acetal (0.10 g, 0.84 mmol) was added. The solution was heated to reflux temperature for 2 hours, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on silica gel (ethyl acetate/hexane gradient, 20:80 to 50:50) to provide 17-O-formylspinoxine A17-Psa (35.8 mg, 32.8%) , Is a white solid: FDMS m/z 618.

<u>Example B24-17-O-(ortho-benzyl)benzyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2-benzoic acid (83 mg, 0.36 mmol), DMAP (38 mg, 0.31 mmol), Spinozin A17-Psa (150 mg, 0.25 mmol), and DCC (58 mg, 0.28 mmol). The obtained 17-O-(ortho-benzyl)benzyl) Spinozin A 17-Psa (37.6 mg, 18.5%) was a white solid: FDMS m/z 799.

<u>Example B25-17-O-(O-Chlorophenylacetoxyspinoxine A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2-chlorophenylacetic acid (69 mg, 0.40 mmol), DMAP (38 mg, 0.31 mmol), Spinozin A 17-Psa (148 mg, 0.25 mmol), and DCC (64 mg, 0.31 mmol). The obtained 17-O-(o-chlorophenylacetoxyspinoxine A 17-Psa (92mg, 49%) was a white solid: FDMS m/z 743, 745. C₄₁H₅₅O₁₀Estimated value of Cl analysis: C, 66.25; H, 7.46. Actual value: C, 66.23; H, 7.36.

<u>Example B26-17-O-(O-Phenylbenzyl Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2-phenylbenzoic acid (78 mg, 0.39 mmol), DMAP (37 mg, 0.30 mmol), Spinozin A17-Psa (142 mg, 0.24 mmol), and DCC (60 mg, 0.29 mmol). The obtained 17-O-(ortho-phenylbenzylspinoxine A 17-Psa (70 mg, 38%) was a white solid: FDMS m/z 770.

<u>Example B27-17-O-(ortho,p-dichlorophenethyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2,4-dichlorophenylacetic acid (66 mg, 0.32 mmol), DMAP (52 mg, 0.26 mmol), Spinozin A17-Psa (142 mg, 0.24 mmol) , And DCC (55 mg, 0.26 mmol). The resulting O-(ortho,p-dichlorophenethyl) Spinozin A17-Psa (108.5 mg, 57.9%) was a white solid: FDMS m/z 777.

<u>Example B28-17-O-(O-isopropylbenzyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2-isopropylbenzoic acid (72 mg, 0.43 mmol), DMAP (43 mg, 0.35 mmol), Spinozin A17-Psa (171 mg, 0.28 mmol), And DCC (72 mg, 0.35 mmol). The obtained 17-O-(o-isopropylbenzyl) Spinozin A 17-Psa (76.7 mg, 36.0%) was a white solid: FDMS m/z 737. . C₄₃H₆₀O₁₀Estimated value of analysis: C, 70.08; H, 8.21. Actual value: C, 70.33; H, 8.28.

<u>Example B29-17-O-(ortho, o-dichlorophenethyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2,6-dichlorobenzoic acid (61 mg, 0.30 mmol), DMAP (40.6 mg, 0.332 mmol), Spinozin A17-Psa (131 mg, 0.22 mmol) , And DCC (59 mg, 0.28 mmol). The obtained 17-O-(ortho, o-dichlorophenacetin) Spinozin A17-Psa (101 mg, 58.5%) was a white solid: FDMS m/z 777

<u>Example B30-17-O-spinoxine A 17-Psa</u>

The compound Spinozin A 17-Psa (120 mg, 0.20 mmol) was dissolved in 5 ml of dimethylformamide and silver oxide (50 mg, 0.21 mmol) and bromide group (120 mg, 0.70 mmol) were added. ). The reaction was stirred for 1 hour and during this period additional bromide groups (200 mg, 1.4 mmol) and silver oxide (40 mg, 0.42 mmol) were added. The reaction was stirred for another 12 hours, after which the reaction was heated to 80-90°C and bromide base (100 mg) and silver oxide (20 mg) were added every hour over 3 hours. The reaction was cooled to room temperature, and the filtrate was washed with ether (50 ml), filtered with deionized water, and then with brine. The ether solution was dried with magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (ethyl acetate/hexane gradient, 20:80 to 50:50) to provide 17-O-ylspinoxine A 17-Psa (6.3 mg, 4.5%) , Is a colorless glass: FDMS m/z 618: partly

<img file="TW487559B_D0105.tif" />

<u>Example B31-17-O-(p, m(dichlorophenylacetoxy) spinoxine A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 3,4-dichlorophenylacetic acid (136 mg, 0.66 mmol), DMAP (70 mg, 0.57 mmol), Spinozin A17-Psa (170 mg, 0.29 mmol) , And DCC (70 mg, 0.34 mmol). The obtained 17-O-(p, m(dichlorophenylacetoxy) Spinozin A 17-Psa (171.2 mg, 76.5%) is a white solid: FDMS m/z 776, 777, 778, 779, 780. C₄₁H₅₂O₁₀Cl₂Estimated value from analysis: C, 63.32; H, 7.00; Cl, 9.12. Actual value: C, 63.37; H6.91; Cl, 9.31.

<u>Example B32-17-O-(p-(N,N-dimethylamine)benzyl)penoxine A17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 2,6-dimethylaminobenzoic acid (30 mg, 0.18 mmol), DMAP (22 mg, 0.18 mmol), Spinozin A17-Psa (98 mg, 0.16 mmol), and DCC (40 mg, 0.19 mmol). The obtained 17-O-(p-(N,N-dimethylamine)benzyl)penoxine A17-Psa (39.5 mg, 32%) was a white solid: FDMS m/z777. C₄₂H₅₉NO₁₀Estimated value from analysis: C, 68.36; H, 8.06; N, 1.90. Actual value: C, 68.20; H, 8.28; N, 2.12.

<u>Example B33-17-O-(p-methoxybenzyl)benoxine A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 4-methoxybenzoic acid (44 mg, 0.29 mmol), DMAP (41 mg, 0.33 mmol), Spinozin A17-Psa (108 mg, 0.18 mmol), And DCC (44 mg, 0.21 mmol). The obtained 17-O-(p-methoxybenzyl)benoxine A 17-Psa (101.7 mg, 76.4%) is a white solid: FDMS m/z 724,725.

<u>Example B34-17-O-(Benzoyl)benoxine A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using benzoic acid (33 mg, 0.27 mmol), DMAP (28 mg, 0.23 mmol), Spinozin A 17-Psa (98 mg, 0.17 mmol), and DCC (41 mg , 0.20 mmol). The obtained 17-O-(benzyl)benoxine A 17-Psa (71.0 mg, 61.7%) is a white solid: FDMS m/z695. C₄₀H₅₄O₁₀Estimated value from analysis: C, 69.14; H, 7.83. Actual value: C, 69.35; H, 7.69.

<u>Example B35-17-O-(p-isopropylbenzyl) pentoxin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 4-isopropylbenzoic acid (58 mg, 0.35 mmol), DMAP (51 mg, 0.41 mmol), Spinozin A 17-Psa (129 mg, 0.21 mmol) , And DCC (80 mg, 0.38 mmol). The obtained 17-O-(p-isopropylbenzyl) pentoxin A17-Psa) (33.1 mg, 20.5%) was a white solid: FDMS m/z 736.

<u>Example B36-17-O-Phenyl Acetyl Binoxine A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using phenylacetic acid (30 mg, 0.22 mmol), DMAP (4 mg, 0.34 mmol), Spinozin A 17-Psa (121 mg, 0.21 mmol), and DCC ( 47 mg, 0.22 mmol). The obtained 17-O-phenylacetylbinoxine A 17-Psa (86.0 mg, 59.3%) was a white solid: FDMS m/z 709. C₄₁H₅₆O₁₀Estimated value from analysis: C, 69.47; H, 7.96. Actual value: C, 69.28; H, 7.94.

<u>Example B37-17-O-(p-methoxyphenacetinyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 4-methoxyphenylacetic acid (39 mg, 0.23 mmol), DMAP (45 mg, 0.36 mmol), Spinozin A 17-Psa (123 mg, 0.20 mmol) ), and DCC (67 mg, 0.32 mmol). The obtained 17-O-(p-methoxyphenethyl) Spinozin A17-Psa (97.8 mg, 63.5%) was a white solid: FDMS m/z 739.

<u>Example B38-17-O-(p-Nitrophenylacetoxy) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 4-nitrophenylacetic acid (86 mg, 0.47 mmol), DMAP (66 mg, 0.54 mmol), Spinozin A 17-Psa (255 mg, 0.43 mmol) , And DCC (100 mg, 0.48 mmol). The obtained 17-O-(p-nitrophenethyl) Spinozin A17-Psa (194.1.59, 6%) was a white solid: FDMS m/z 753. C₄₁H₅₅NO₁₂Estimated value from analysis: C, 65.32; H, 7.35; N, 1.86. Actual value: C, 65.58; H, 7.54; N, 2.05.

<u>Example B39-17-O-(m-nitrophenylacetoxy) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 3-nitrophenylacetic acid (86 mg, 0.37 mmol), DMAP (62 mg, 0.50 mmol), Spinozin A 17-Psa (185 mg, 0.31 mmol) , And DCC (83 mg, 0.40 mmol). The resulting 17-O-(m-nitrophenethyl) Spinozin A17-Psa (166.1, 70.3%) is a white solid: C₄₁H₅₅NO₁₂Estimates from analysis: C, 65.32; H, 7.35; N, 1.86. Actual value: C, 65.13; H, 7.49; N, 1.92.

<u>Example B40-17-O-(m-benzotrifluoride acetyl) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 3-Trifluorotolylacetic acid (101 mg, 0.49 mmol), DMAP (64 mg, 0.52 mmol), Spinozin A 17-Psa (274 mg, 0.46 mmol) , And DCC (109 mg, 0.52 mmol). The resulting O-(m-benzotrifluoride acetyl) Spinozin A17-Psa (302.2 mg, 84.6%) was a white solid: C₄₂H₅₅O₁₀F₃Estimated value from analysis: C, 64.93; H, 7.14. Actual value, C, 65.07; H, 7.39.

<u>Example B41-17-Epi-O-Methyl Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B47, using 17-epi-spinoxine A 17-Psa (20 mg, 0.033 mmol), Proton Sponge^(5 mg, 0.03 mmol) and trimethyloxonium tetrafluoroborate (14 mg, 0.065 mmol). The obtained 17-epi-O-methylspinoxine A 17-Psa (7.1 mg, 35%) is a white solid: part

<img file="TW487559B_D0106.tif" />

<u>Example B42-17-O-(m-Methoxyphenacetin) Spinozin A 17-Psa</u>

The compound was prepared according to the procedure described in Example B46, using 3-methoxyphenylacetic acid (98 mg, 0.59 mmol), DMAP (71 mg, 0.58 mmol), Spinozin A 17-Psa (290 mg, 0.49 mmol) , And DCC (112 mg, 0.54 mmol). The obtained 17-O-(m-methoxyphenethyl) Spinozin A 17-Psa (308.7 mg, 85.1%) was a white solid: C₄₂H₅₈O₁₁Estimated value from analysis: C, 68.27; H, 7.91. Actual value: C, 68.25; H, 7.92.

<u>Example B43-17-O-(Tetrahydropyran-2-yl) Spinozin A C-17-Psa</u>

Combine Spinozin A C-17-Psa (0.50 g, 0.85 mmol), 3,4-dihydro-2H-pyran (0.45 mL, 0.41 g, 4.9 mmol) and TsOHHCl (0.01 g, 0.05 mmol) The solution of) in dichloromethane (4 ml) was stirred for 24 hours. The mixture was partitioned between ether and sodium bicarbonate. The organic layer was washed with brine, dried over magnesium sulfate, filtered and evaporated. MPLC (silica, 50:50, ether/hexane) can yield 0.35 g (61%) of 17-O-(tetrahydropyran-2-yl)spinoxine A C-17-Psa, which is 1:1 A mixture of non-mirror isomers and a clean colorless glass.

<u>Example B44-17-O-(4-nitrobenzyl)-17-epi-spinoxine A 17-Psa</u>

A benzene solution of dimethyl azocarboxylate (0.4 ml, 2.49 mmol) is added to properly stirred Spinozin A 17-Psa (0.3 g, 0.51 mmol) at room temperature within 2-3 minutes , A benzene solution (12 ml) of triphenylphosphine (0.65 g, 2.49 mmol) and p-nitrobenzoic acid (0.37 g, 2.20 mmol). The resulting solution was stirred at room temperature for 48 hours. The solvent was removed and the residue was flash chromatographed on silica (150ml), using 2.5% acetone in dichloromethane as the eluent to obtain 110 mg of 17-O-(4-nitrobenzyl )-17-Table-Spinozin A 17-Psa. The sample was recrystallized from 2:1 acetone/water, and the sample was colorless needles. mp 159-161°C;¹H NMR(CDCl₃)δ7.32

<img file="TW487559B_D0107.tif" />

<u>Example B45-17-O-(4-Nitrophenylacetoxy)-17-epi-spinoxine A 17-Psa</u>

A portion of 1,3-dicyclohexylcarbodiimide (21 mg, 0.11 mmol) was added to a properly stirred 4-nitro-phenyllactic acid (20 mg, 0.11 mmol) at room temperature, 4-bis A solution of methylaminopyridine (3 mg, 0.024 mmol) and 17-epi-spinoxine A-17-Psa (59 mg, 0.1 mmol) in dichloromethane (2 ml). The resulting mixture was stirred at room temperature for 2 hours, then diluted with more dichloromethane (2.0 ml) and filtered to remove insoluble materials. The filtrate was evaporated and the residue was flash chromatographed on silica (25 ml), using 25% ethyl acetate in methane as the eluent to obtain 17-O-(4-nitrophenethyl acetonitrile)- 17-table-spinozin A 17-Psa (70 mg), a colorless foam:

<img file="TW487559B_D0108.tif" />

<u>Example B46-17-O-(p-Chlorophenethyl) Spinozin A 17-Psa</u>

The compound p-chlorophenylacetic acid (50 mg, 0.29 mmol) and dimethylamine pyridine (DMAP, 50 mg, 0.40 mmol) were dissolved in dichloromethane (8 ml) and spinoxin was added to the solution A 17-Psa (164.7 mg, 0.279 mmol), and then dicyclohexylcarbodiimide (DCC, 66 mg, 0.32 mmol) was added. The reaction was stirred overnight. The reaction was filtered to remove solids and chromatographed on silica gel (ethyl acetate/hexane gradient, 20:80 to 50:50) to provide 17-O-(p-chlorophenylacetoxy)spinoxine A 17 -Psa (128.4 mg, 61.8%) as a white solid: FDMS m/z 743. C₄₁H₅₅O₁₀Estimated value of Cl analysis: C, 66.25; H, 7.46. Actual value: C, 65.98; H, 7.31.

<u>Example B47-17-O-Methyl Spinozin A 17-Psa</u>

The compound Spinozin A 17-Psa (267 mg, 0.452 mmol) was dissolved in dichloromethane (5 ml) and Proton Sponge was added^(107 mg, 0.50 mmol) and trimethyloxonium tetrafluoroborate (104 mg, 0.702 mmol). The mixture was stirred for 3 hours and gradually reacted to form a white fluffy solid (0.20 g). The solid was purified by reverse phase chromatography (methanol/water 90:1) to provide 17-O-methylspinoxine A 17-Psa (135.8 mg, 49.6%) as a white solid: IR

<img file="TW487559B_D0109.tif" />

<u>Part C: Modifications in the substituents of the amino sugar</u>

<u>Example Cl-N-Acetyl Spinozin B</u>

To Spinozin B (200 mg, 0.28 mmol) in chloroform (5 ml), add diisopropylethylamine (72.7 μl, 0.42 mmol) and then add acetyl chloride (30 μl, 0.42 mmol) . The reaction mixture was stirred at room temperature for 0.5 hours. The mixture was then diluted with dichloromethane and saturated sodium bicarbonate. The dichloromethane portion was dried with potassium carbonate and evaporated at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 5% methanol in dichloromethane. N-Acetyl Spinozin B (239 mg, about 100% yield) is available as a colorless glass body, FDMS, m/e, (relative strength) 759 (M⁺-H, 10), 714 (60), 189 (50), 170 (50), 101 (100).

<u>Example C2-N-Benzyl Spinozin B</u>

The reaction was carried out according to the steps described in Example C1, with Spinozin B (200 mg, 0.28 mmol) and benzyl chloride as starting materials. The obtained N-benzyl Spinozin B (227.4 mg, about 100% yield) is a light yellow glass-like body, FDMS, m/e (relative strength) 821 (M⁺-H, 5), 776(20), 232(30), 189(35), 103(100).

<u>Example C3-N-Allyl Spinozin B</u>

The reaction was carried out according to the steps described in Example C1, with Spinozin B (200 mg, 0.28 mmol) and allyl bromide as starting materials. The reaction was stirred at room temperature for 4 days, and then evaporated at room temperature under reduced pressure, and a 40% ethyl acetate solution in hexane was used as the chromatographic eluent. The obtained allyl spinoxine B (148.9 mg, 70% yield) is a colorless glass, FDMS, m/e (relative strength) 757 (M⁺-H, 100).

<u>Example C4-N-Benzyl Spanacine B</u>

The reaction was carried out according to the steps described in Example C1, starting with Spinozin B (200 mg, 0.28 mmol) and bromide. The obtained N-benzyl Spinozin B (216.6 mg, 96% yield) is a colorless glass, FDMS, m/e (relative strength) 807 (M⁺-H, 100).

<u>Example C5-N-Methyl Spinozin A Iodide</u>

To Spinozin A (111.6 mg, 0.15 mmol) in chloroform (5 ml), add methyl iodide (200 μl). The reaction mixture was stirred at room temperature for 24 hours followed by the addition of methyl iodide (200 μl), and the reaction mixture was stirred at room temperature for another 24 hours. Then the solvent was evaporated under reduced pressure at room temperature and the residue was pressed with ether. The obtained N-methylspinoxine A iodide (101 mg, 76% yield) was a white off-white solid, FDMS, m/e (relative intensity) 747 (M⁺，100)。

<u>Example C6-N-oxyspinoxine A</u>

To an ice-cooled solution of 81.2% pure Spinozin A (2.07 gm, 2.83 mmol) in dichloromethane (100 ml), m-chloroperoxybenzoic acid (450 mg, 2.57 mmol) was added. The reaction mixture was allowed to warm to room temperature for 1 hour, and then the reaction mixture was stirred at room temperature for 5 hours. The reaction was quenched by adding sodium bicarbonate solution. The organic phase was separated, dried over magnesium sulfate, and evaporated at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 10% methanol in dichloromethane. The resulting N-oxy Spinozin A (1.59 gm, 92% yield), as a light yellow glass, FDMS, m/e (relative strength) 762.9 (20), 731.8 (M⁺-O, 40), 686.8 (100), 401.7 (25).

<u>Example C7-4"-hydroxyspinoxine A</u>

The reaction was carried out as described in Example Cl3, with N-oxyspinoxine A (509.8 mg, 0.68 mmol) as the starting material. A white precipitate formed during the reaction and was separated by filtration. The precipitate was then pressed and ground with ether. The ether was evaporated under reduced pressure at room temperature and the residue was dissolved in methanol (20 ml) and sodium borohydride (258 mg, 6.82 mmol) was added and the reaction mixture was stirred at room temperature for 7 hours. The mixture was then evaporated to a small volume at room temperature under reduced pressure and diluted with ether. The ether portion was washed with water and brine, dried with potassium carbonate, and evaporated at room temperature and reduced pressure. The product was separated by chromatography on silica and eluted with 70% ethyl acetate in hexane. The obtained 4"-hydroxyspinoxine A (92.1 mg, 19% yield) was a colorless glass and was a 3:1 isomeric mixture, FDMS, m/e (relative strength) 704 (M⁺，80)，591(30)，190(70)，115(100)。

<u>Example C8-3",4"-dihydro-4"-deamine Spinozin C</u>

N-oxy Spinozin A (505.9 mg, 0.68 mmol) was heated to 120°C under nitrogen. The gas began to release at 115°C, and the room continued to be heated until the gas released (approximately 15 minutes). Then the mixture was slowly cooled to room temperature. The product was separated by chromatography on silica and eluted with 5% methanol in dichloromethane. The obtained 3",4"-dihydro-4"-deamine Spinozin C (91 mg, 19% yield), FDMS, m/e (relative intensity) 696 (M⁺, 80), 589(20), 189(50), 101(100), N-formyl Spinozin B (43.3 mg; 9% yield), Spinozin A (102.1 mg; 21% yield) Rate), and Spinozin B (114.5 mg; 23% yield), all are colorless glass.

<u>Example C9-N-(N'-yl-3-methyl) Spinozin C</u>

To Spinozin C (213.6 mg, 0.3 mmol) in methanol (2 ml) was added N-yl-3- carboxyaldehyde (83.7 mg, 0.36 mmol). The reaction mixture was stirred at room temperature for 20 hours, and sodium cyanoborohydride (43.5 mg, 0.7 mmol) was added. Stir for another 2 hours at room temperature. TLC (eluted with 10% methanol in dichloromethane) showed that the reaction was incomplete, however, the solvent was evaporated to a small volume under reduced pressure at room temperature. The mixture was then diluted with dichloromethane. The dichloromethane part was washed with water and brine, dried with potassium carbonate and distilled at room temperature and reduced pressure. The product was separated by chromatography on silica and eluted with 70% ethyl acetate in hexane. The obtained N-(N'-yl-3-methyl) Spinozin C (83.8 mg, 30% yield) is a light yellow glass, FDMS, m/e (relative strength) 1140 (15), 923(M⁺，100)。

<u>Example Cl0-N-Demethyl-N-formyl Spinozin J</u>

To Spinozin J (990.4 mg, 1.38 mmol) in anhydrous dichloromethane (25 ml), add pyridine dichromate (622.6 mg, 1.65 mmol). The reaction mixture was stirred at room temperature for 3.75 days and then filtered through Celite. Wash the diatomaceous earth with fresh dichloromethane. Combine the dichloromethane fractions and evaporate the dichloromethane at room temperature under reduced pressure. The product was separated by chromatography on silica and eluted with ethyl acetate. The obtained N-desmethyl-N-formyl spinoxin J (360 mg, 36% yield) was a white solid, EIMS, m/e (relative intensity) 732 (M⁺，40)，715(100)，175(20)，101(25)。

<u>Example Cl1-N-yl Spinozin A Bromide</u>

The reaction was carried out as described in Example C5, starting with Spinozin A (100 mg, 0.14 mmol) and bromide group, and refluxing for 6 days. The obtained N-yl Spinozin A bromide salt (86.6 mg, 69% yield) is a white off-white solid, FDMS, m/e (relative intensity) 976(10), 822(M⁺，100)，189(60)，142(100)。

<u>Example Cl2-4"-Demethyl-(dimethylamine)-4"-azacyclopropenyl Spinozin A</u>

To Spinozin C (205.2 mg, 0.29 mmol) in acetone (2 ml), add monochloroacetaldehyde (39.1 mg, 0.62 mmol). The reaction mixture was stirred at room temperature for 2 days. Reverse phase HPLC (eluted with 44% ethyl acetate, 44% methanol and 12% (0.5% ammonium acetate solution) showed that the reaction was incomplete. The mixture was then diluted with saturated sodium bicarbonate solution and extracted with dichloromethane. Dichloromethane part Then washed with brine, dried with potassium carbonate and evaporated at room temperature and reduced pressure to obtain a colorless glass (199 mg). The product was separated on a Cl8 column by preparative HPLC, and ethyl acetate: methanol: 0.1 % Ammonium acetate (90 minutes linear gradient elution from 35:35:30 to 45:45:10). The resulting 4"-desmethyl-(dimethylamine)-4"-azacyclopropenyl spinnol Xin A (30 mg, 14% yield), white solid, EIMS, m/e (relative intensity) 729 (M⁺，100)。

<u>Example Cl3-4"-Demethyl-(dimethylamine)-4"-oxy Spinozin A</u>

To a solution of N-oxy Spinozin A (203.8 mg, 0.27 mmol) in benzene (10 ml), add acetic anhydride (500 ml). The reaction mixture was stirred at room temperature for 5 days. The mixture was then evaporated at room temperature under reduced pressure. The residue (after standing at room temperature for 6 days) was separated by chromatography on silica and eluted with 2.5% methanol in dichloromethane. The obtained a) 4"-desmethyl-(dimethylamine)-4"-oxyspinoxine A (59.9 mg, 32% yield) is an unstable colorless glass, FDMS, m/e (Relative strength) 704 (MH⁺, 10), 592(90), 190(95), 101(100), and compound b) N-acetylspinoxine B (69.2 mg, 34% yield), are colorless glass.

<u>Example Cl4-N-(N'-Methylaminomethyl) Spinozin B</u>

Spinozin B compound (200 mg, 0.278 mmol) and methyl isocyanate (0.30 g, 5.26 mmol) were combined in 10 ml of toluene and the mixture was stirred at room temperature for 3 days. The reaction mixture was partitioned between ether/water and separated into layers. The extracted aqueous layer was extracted with 3×25 ml of ether and the ether extracts were combined. The ether extract was washed with 5 ml of saline solution, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica column (230-400 m, dichloromethane: methanol, 95:5, v/v) of medium pressure to obtain N-(N'-methylaminomethanyl)spin Nosin B (0.14 g, 66%). C₄₄H₆₆N₂O₁₁Elemental analysis: estimated value C, 65.09; H, 8.31; N3.31; actual value C, 64.80; H, 8.31; N, 3.34; FAB MS (m/z) 775 (M+1).

<u>Example Cl5-N-Methanesulfonyl Spinozin B</u>

Dissolve Spinozin B compound (200 mg, 0.279 mmol) in 5 ml of dichloromethane, and add methanesulfonyl chloride (0.11 g, 0.96 mmol) and diisopropylethylamine (0.13 g, 1.00 mmol) . Stir at room temperature for 2 hours, then pour 10 ml each of ether and water. Layers occurred and the aqueous layer was extracted with 2×10 ml water. The ether extracts were combined, washed with 10 ml saline solution, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting residue was chromatographed on a silica column (230-400 m, dichloromethane: methanol, 95:5, v/v) of moderate pressure to obtain compound N-methanesulfonyl spinoxin B (0.14 g , 63%). C₄₁H₆₅NO₁₂Elemental analysis of S: estimated value C, 61.86; H, 8.23<u>；</u>N 1.76; actual value C, 61.73; H, 7.83; N, 1.65.

<u>Example Cl6-N-ethoxycarbonyl Spinozin B</u>

Spinozin B compound (214 mg, 0.298 mmol) was dissolved in 5 ml of dichloromethane and ethyl chloroformate (0.15 g, 0.14 mmol) and diisopropylethylamine (0.12 g, 0.93 mmol) were added. The reaction was stirred at room temperature for 2 hours. The reaction was poured into 10 ml water/10 ml ether and the layers were separated. The aqueous layer was extracted with 2×10 ml diethyl ether. Combine the ether extracts and wash with 10 ml saturated sodium bicarbonate and then with 10 ml brine. The ether solution was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. Chromatography on a silica column (230-400 m, dichloromethane: methanol, 95:5, v/v) of medium pressure to obtain N-ethoxycarbonyl spinoxin B (0.19 g, 80%) . C₄₃H₆₇NO₁₂Elemental analysis: estimated value C, 65.38; H, 8.55; N1.77; actual value C, 64.80; H, 8.53; N, 1.24; FAB MS (m/z) 790 (M+1).

<u>Example Cl7-N-Trifluoromethyl Acetyl Spinozin B</u>

Spinozin B compound (118.9 mg, 0.165 mmol) was dissolved in 5 ml of dichloromethane and diisopropylethylamine (0.10 g, 0.77 mmol) and trifluoroacetic anhydride (0.10 g, 0.48 mmol) were added. The reaction was stirred at room temperature for 20 hours. The reaction was poured into ether/saturated sodium carbonate solution and the layers were separated. The aqueous layer was extracted with 2×10 ml ether. The ether extracts were combined and the extracts were washed with 30 ml of saline solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Chromatography was performed on a silica column (230-400 m, dichloromethane/hexane, 40:60, v/v) of medium pressure to obtain the compound N-trifluoromethyl acetylspinoxine B. C₄₂H₆₂NO₁₁F₃Elemental analysis: estimated value C, 61.97; H, 7.52; N 1.31; actual value C, 60.88; H, 7.60; N, 1.46; FAB MS (m/z) 813 (M+1).

<u>Example Cl8-N-Formyl Spinozin B</u>

Spinozin B compound (107.3 mg, 0.150 mmol) was added to 10.0 ml ethyl formate and the mixture was heated to reflux temperature. After 1 hour at reflux temperature, the reaction was cooled to room temperature and the solvent was removed under reduced pressure. Chromatography was performed on a silica column (230-400 m, dichloromethane/methanol 95:5, v/v) of medium pressure to obtain compound N-formylspinoxine B; 103.1 mg, 92.3%. C₄₁H₆₃NO₁₁Elemental analysis: estimated value C, 66.02; H, 8.51; N 1.88; actual value C, 64.67; H, 8.75; N, 2.03; FAB MS (m/z) 745 (M+1).

<u>Example Cl9-N-aminomethanospinoxine C</u>

Silver cyanate (3.7 g, 24 mmol) was suspended in 10 ml of benzene and silicon tetrachloride (1 g, 5.8 mmol) was added. The reaction immediately changed color, from light gray to dark purple suspension. The reaction was heated to reflux temperature for 1 hour, cooled to room temperature and filtered to remove any solids. The filtrate was concentrated under reduced pressure to obtain silicon tetracyanate as a colorless oil. Dissolve all the silicon tetrachlorate in 10 ml of benzene and add the compound Spinozin C (93 mg, 0.132 mmol) in 2 ml of benzene. The resulting solution was heated to reflux temperature for 30 minutes. The reaction was cooled to room temperature and concentrated under reduced pressure. 20 ml of 90% isopropanol/water was added to the residue and the reaction was heated to reflux temperature for 30 minutes. Cool to room temperature and concentrate under reduced pressure. The residue was suspended in dichloromethane and filtered to remove solids. The residue was chromatographed on a gravity silica column (230-400 m, dichloromethane/methanol 95:5, v/v) to obtain compound N-aminomethylspinoxine C (28.2 mg, 28.6%) . C₄₀H₆₂N₂O₁₁Elemental analysis: estimated value C, 64.32; H, 8.37; N3.75; actual value C, 64.04; H, 8.12; N, 4.04; FAB MS (m/z) 746 (M+1).

<u>Example C20-N-Trifluoromethanesulfonyl Spinoxine B</u>

Spinozin B compound (136 mg, 0.189 mmol) was dissolved in 5 ml of dichloromethane and diisopropylethylamine (0.10 g, 0.77 mmol) and difluoromethanesulfonic anhydride (0.10 g, 0.35 mmol) were added. The reaction was stirred at room temperature for 1 hour. The reactant was poured into a solution of 20 ml each of ether/saturated sodium carbonate. The layers were separated and the aqueous layer was extracted with 2×10 ml ether. The ether extracts were combined and the extracts were washed with 25 ml of saline solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Chromatography was performed on a silica column (230-400 m, dichloromethane/hexane, 50:50, v/v) at a medium pressure to obtain the compound N-trifluoromethanesulfonylspinoxine B. C₄₁H₆₂NO₁₂SF₃Elemental analysis: estimated value C, 57.94; H, 7.35; N 1.65; actual value C, 58.07; H, 7.52; N, 1.70; FAB MS (m/z) 849 (M+1).

<u>Example C21- Spinozin A N-[(E)-Prop-1'''-enoic acid]-3'''-ammonium salt, and ethyl [Spinozin B N-2''' -Methyl(Z)-Propyl-1'''-enoic acid]-3'''-ammonium salt</u>

Spinozin A compound (0.68 g, 0.929 mmol) was dissolved in dichloromethane (3 ml) and water (50 ml) was added. The mixture was stirred at 0°C and a portion of ethyl propionate (Aldrich, 2.20 g, 22.4 mmol) was added. The cooling bath was removed and the reaction mixture was stirred for 30 minutes, then it was ultrasonically shaken (in a 50 W Fishers ultrasonic cleaner) for 12 minutes. After that, dioxane (400 ml) was added and the solvent was removed under reduced pressure. The residue was purified by flash chromatography in silica gel (230-400 m, 80 g/THF, followed by methanol, followed by 15% water in methanol). The pure fractions obtained by chromatography can be obtained a) ethyl [spinoxine B N-2'''-methyl(Z)-prop-1'''-enoic acid]-3'''-ammonium Salt (296 mg,<img file="TW487559B_D0110.tif" /><img file="TW487559B_D0111.tif" />

<u>Example C22- Spinozin B N-(2'''-Methyl-(Z)-Prop-1'''-enoic acid)-3'''-ester</u>

Spinozin B compound (192 mg, 0.267 mmol) was dissolved in dry dichloromethane (25 ml). A portion of methyl propionate (Aldrich 1.80 g, 21.4 mmol) was added. The reaction mixture was stirred at room temperature under nitrogen for 5 hours. The solvent and excess ethyl propionate were removed under reduced pressure. The residue was purified on a flash silica gel column (230-400 m, 50 g/THF). The pure elution fraction can obtain Spinozin B N-(2'''-methyl-(Z)-prop-1'''-enoic acid)-3'''-ester (205 mg,

<img file="TW487559B_D0112.tif" />

<u>Example C23-N-(cyano)methyl spinoxine B</u>

Spinozin B compound (263 mg, 0.370 mmol) was dissolved in dry dichloromethane (7 ml). The solution was stirred at room temperature under nitrogen. Triethylamine (1.0 ml) was added, followed by bromoacetate (1.2 ml, excess). The reaction mixture was stirred at room temperature for 3 days. Add ethyl acetate (50 ml) and PhH (50 ml), and the solution was continuously washed with water (3×) and 5% sodium bicarbonate (2×). The organic layer was dried with anhydrous potassium carbonate and concentrated under reduced pressure. The residue was then dried at 0.005 Torr for 3 hours to obtain the compound N-(cyano)methylspinoxine B (260 mg, 94%):¹³C-NHR(CDC1₃)d 117.8(CN) and 43.8(N-CH₂-CN) ppm.

<u>Example C24-4"-N-Ethyl Spinozin B</u>

Alkylation step A: Stir a solution of Spinozin B compound (0.50 g, 0.70 mmol) in DMF (1.5 ml), and sequentially dilute the solution with iodoethane (0.11 ml, 0.21 g, 1.4 mmol) and (i-Pr)₂NEt (0.36 mL, 0.27 g, 2.1 mmol) treatment. After 20 hours, the mixture was treated with ca. 1 mL of 1 M sodium thiosulfate solution to decompose any residual iodine. The resulting mixture was partitioned between ether and saturated brine. The organic layer was washed with brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to yield 0.61 g. The residue was purified by MPLC (silica, 5:95 methanol/ethyl acetate) to obtain 0.47 g (90%) of 4"-N-ethyl Spinozin B as a white powder. C₄₂H₆₇NO₁₀Analysis estimates: C, 67.62; H, 9.05; N, 1.88. Actual value: C, 67, 11; H, 9.73; N, 1.92 and C, 67.17; H, 9.54; N, 1.98.

<u>Example C25-4"-N-(1-propyl) Spinozin B</u>

The compound was prepared according to the alkylation step A of Example 24, using Spinozin B (0.50 g, 0.70 mmol), DMF (1.5 mL), 1-iodopropane (0.14 mL, 0.24 g, 1.4 mmol), and (i-Pr)₂NEt (0.36 ml, 0.27 g, 2.1 mmol). Purified by MPLC (silica, 2:98 methanol/ethyl acetate) to obtain 0.41 g (77%) of 4"-N-(1-propyl) Spinozin B as a white powder. C₄₃H₆₉NO₁₀Analysis estimates: C, 67.96; H, 9.15; N, 1.84. Actual value: C, 68.06; H, 9.25; N, 1.97.

<u>Example C26-4"-N-(2-Methyl-prop-1-yl) Spinozin B</u>

The compound was prepared according to the alkylation step A of Example 24, using Spinozin B (0.95 g, 1.3 mmol), DMF (2.8 mL), 1-iodo-2-methylpropane (0.32 mL, 0.51 g, 2.8 mmol), and (i-Pr)₂NEt (0.73 ml, 0.54 g, 4.2 mmol). Purified by MPLC (silica, 50:50 methanol/ethyl acetate) to obtain 0.07 g (7%) of 4"-N-(1-propyl) Spinozin B as a white powder. C₄₄H₇₁NO₁₀Analytical estimates: C, 68.28; H, 9.25; N, 1.81. Actual value: C, 68.33; H, 9.26; N, 1.88.

<u>Example C27-4"-N-(2-Methyl-prop-1-yl) Spinozin B</u>

The compound was prepared according to the alkylation step A of Example 24, using Spinozin B (0.95 g, 1.3 mmol), DMF (2.8 mL), cyclopropane methyl bromide (0.27 mL, 0.38 g, 2.8 mmol) , Sodium iodide (0.05 g, 0.3 mmol) and (i-Pr)₂NEt (0.73 ml, 0.54 g, 4.2 mmol). Purify by MPLC (silica, 2:98 methanol/ethyl acetate) to obtain 0.34 g (33%) of 4"-N-(2-methyl-prop-1-yl)spinoxine B, which is White powder. C₄₄H₆₉NO₁₀Analysis estimates: C, 68.45; H, 9.01; N, 1.81. Actual value: C, 68.36; H, 9.22; N, 1.76.

<u>Example C28-N-(N'-(1,1-Dimethylethoxycarbonyl)glycamido) Spinozin B</u>

Coupling step A: A solution of Boc-Gly-OH (0.16 g, 0.91 mmol) and BOP-Cl (0.23 g, 0.90 mmol) in dichloromethane (7ml) stirred at -15°C under nitrogen Gifmoxine (0.12 ml., 0.11 g, 1.1 mmol) treatment. After 1.5 hours, the mixture was sequentially treated with Spinozin B (0.50 g, 0.70 mmol) and N-methylformosine (0.12 ml, 0.11 g, 11.1 mmol). The temperature was maintained at -15°C for 4 hours, and then the material was gradually warmed to room temperature for 10 hours. The mixture was evaporated under reduced pressure, and the residue was purified by MPLC (silica, 50:50 to 100:0 ethyl acetate/hexane) to provide 0.61 g (99%) of N-(N'-(1, 1-Dimethylethoxycarbonyl)glycamido)spinoxine B, white powder. C₄₇H₇₄N₂O₁₃Analytical estimates: C, 64.51; H, 8.52; N, 3.20. Actual value: C, 63.76; H, 8.62; N, 3.29, and C, 63.78; H, 8.60; N, 3.43.

<u>Example C29-N-(N'-(1,1-Dimethylethoxycarbonyl)-β-propylamino) Spinozin B</u>

This compound was prepared according to the coupling part described in Example 28, using Boc-β-Ala-OH (0.17 g, 0.90 mmol) and BOP-Cl (0.23 g, 0.90 mmol) in dichloromethane (7ml ) The solution was filled with N-methylformolanium (2×0.12 ml, 0.22 g, 2.2 mmol) and Spinozin B (0.50 g, 0.70 mmol) in a nitrogen atmosphere. The residue was purified by MPLC (silica, 70:30 to 100:0 ethyl acetate/hexane) to provide 0.62g (99%) of N-(N'-(1,1-dimethylethoxy) Carbonyl)-β-propylamino) Spinozin B is a white powder. C₄₈H₇₆N₂O₁₃Analysis estimates: C, 64.84; H, 8.62; N, 3.15. Actual value: C, 64.38; H, 8.67; N, 3.22.

<u>Example C30-N-(N'-(Phenylmethoxycarbonyl)-L-propylamino) Spinozin B</u>

This compound was prepared according to the coupling part described in Example 28, using Cbz-L-Ala-OH (0.21g, 0.94 mmol) and BOP-Cl (0.234g, 0.94 mmol), dichloromethane (7ml ), N-methyl formoxine (2×0.12 ml, 0.22 g, 2.2 mmol) and Spinozin B (0.50 g, 0.70 mmol). The residue was purified by MPLC (silica, 75:25 ethyl acetate/hexane) to provide 0.68 g (99%) of N-(N'-(phenylmethoxycarbonyl)-L-propylamino) Spinozin B is a white powder. C₅₁H₇₄N₂O₁₃Analysis estimates: C, 66.36; H, 8.08; N, 3.03. Actual value: C, 2 rotamers:¹H-NMR(CDCl₃)7.3-7.4(M, 5H), [5.06+5.10 y5.04(5+2d, J=12.23 y12.3, Σ=2H, 2.87 y2.76(25, Σ=3H).

<u>Example C31-N-(N',N'-Dimethylglycamidospinoxine B</u>

This compound is prepared according to the coupling part described in Example 28, wherein N(Me₂) Gly-OH (0.09 g, 0.9 mmol) and BOP-Cl (0.254g, 0.98 mmol), dichloromethane (7ml), N-methyl formaldehyde (2×0.12 ml, 0.22g, 2.2 mmol) and Spinozin B (0.50 g, 0.70 mmol). The residue was purified by MPLC (silica, 5:95 methanol/dichloromethane) to provide 0.23 g (41%) of N-(N',N'-dimethylglycaminospinoxine B as White powder. C₄₄H₇₀N₂I₁₁Analytical estimates: C, 65.81; H, 8, 79; N, 3, 49. Actual value: C, M+H+ estimated value 803.5 actual value 803.6.

<u>Example C32-N-(N'-Methylthioaminomethyl) Spinozin B</u>

Spinozin B (203 mg, 0.28 mmol) was dissolved in 10 ml of toluene and methyl isocyanate (100 mg, 1.36 mmol) was added, and the solution was stirred for 1 hour. After the gradual reaction, the crude mixture was chromatographed on silica gel (1:1, ethyl acetate/hexane) to provide N-(N'-methylthioaminomethanyl) Spinozin B (0.14 g, 64%), is a white solid, MS m/z 791.)-L-propylamine) Spinozin B is a white powder. C₄₂H₆₆N₂O₁₀Analytical estimates of S: C, 63.77; H, 8.41; N, 3.54. Actual value: C, 63.57, H, 8.42N, 3.63.

<u>Example C33-N-(N',N'-Dimethylmethylmethymidine) Spinozin B</u>

Spinozin C (102 mg, 0.14 mmol) was dissolved in dimethylformamide dimethyl acetal (5 ml) and the solution was heated to reflux temperature for 30 minutes. The reaction was cooled to room temperature and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (95:5 methanol/ethyl acetate) to provide N-(N',N'-dimethylmethylmethyl)spinoxine B (56.2 mg, 51% ), is a yellow-brown solid: FDMS m/z 758.

<u>Example C34-N-(N',N'-dimethylaminomethanyl) Spinozin B</u>

Spinozin B (0.10 g, 0.14 mmol) was dissolved in dimethylaminomethanyl chloride (0.1 g, 0.93 mmol), and N,N-diisopropylethylamine (0.10 g, 0.77 mole) Dissolve in dichloromethane (10 ml) and stir for 24 hours. The solvent was evaporated under reduced pressure and the residue was purified by chromatography on silica gel (95:5 ethyl acetate/methanol). N-(N',N'-dimethylaminomethanyl) Spinozin B (76.8 mg, 70%) is available as a white solid: FDMS m/z 788.

<u>Example C35-N-Cinyl Spinozin B</u>

Spinozin B (98 mg, 0.14 mmol) was dissolved in dichloromethane (5 ml) and N,N-diisopropylethylamine (26 mg, 0.2 mmol) and octyl chloride (30 mg, 0.18 mmol) was added ) And the reaction was stirred for 20 hours. After gradual completion of the reaction, the residue was purified by chromatography on silica gel (95:5 methanol/ethyl acetate) to provide N-octanylspinoxine B (61.9 mg, 53.8%): FDMS m/z 844 .C₄₈H₇₇NO₁₁: C, 68.29; H, 9.19; N, 1.65. Actual value: C, 68.03; H, 9.29; N, 1.84.

<u>Example C36-N-nitrospinoxine B</u>

Spinozin B (1.01 mg, 1.40 mmol) was suspended in water (10 ml) and cooled to 0°C in an ice bath. Add 1N HCl (10 ml) to this cold suspension and stir the reaction until all solids are dissolved. (Note: A little heating is necessary to dissolve all solids). The solution was cooled down to 0°C and sodium nitrate (500 mg, 7.2 mmol) was added and the reaction was stirred at 0°C. During the reaction, a white precipitate formed during the reaction. After 2 hours, additional sodium nitrite (500 mg, 7.2 mmol) was added and the reaction was stirred for another 1 hour. The solid was collected by vacuum filtration and washed with cold water (2×). The solid is air-dried to obtain N-nitroso Spinozin B (1.00 g, 95%) as a white solid: FDMS m/z 748. Analysis C₄₀H₆₂N₂O₁₁Estimated value: C, 64.32; H, 8.37; N, 3.75. Actual value: C, 64.52; H, 8.26; N, 3.45.

<u>Example C37-N-Benzyl Spinozin B</u>

The compound was prepared according to the method described in Example C78, using Spinozin B (109 mg, 0.152 mmol), N,N-diisopropyldiethylamine (0.1 g, 0.8 mmol), and benzenesulfonyl chloride (26.3 mg, 0.15 mmol). N-Benzyl Spinozin B (78.6 mg, 60.5%) can be obtained as a white solid: FDMS m/z 857.

<u>Example C38-N-benzylidene Spinozin C</u>

The compounds Spinozin C (106 mg, 0.15 mmol), benzaldehyde (0.1g, 0.9 mmol) and camphorsulfonic acid (5 mg) were dissolved in benzene (15 ml) and the reaction was heated to reflux temperature for 1 hour. The solvent was evaporated under reduced pressure and the residue was purified by chromatography on silica gel (ethyl acetate/hexane gradient 20:80 to 40:60) to provide N-benzylidene spinoxin C (65.5 mg, 55.0%), as a white solid: FDMS m/z 746, 791.

<u>Example C39-N,N-Diacetylspinoxine C</u>

The compound was prepared according to the method described in Example C78, using Spinozin C (102 mg, 0.144 mmol), N,N-diisopropyldiethylamine (0.1 g, 0.8 mmol), and acetyl chloride ( 0.1, 1 mmol). N,N-Diacetyl-kispenoxin C can be obtained as a white solid: FDMS m/z 787. Analysis C₄₃H₆₅N₁O₁₂Estimated value: C, 65.54; H, 8.31. Actual value: C, 64.64; H, 8.08.

<u>Example C40-N,N-Diacetylspinoxine C</u>

The compound was prepared according to the method described in Example C79, using Spinozin C (81 mg, 0.11 mmol), methyl isocyanate (0.5 g, 0.9 mmol). The compound N,N-diacetylspinoxine C (61.8 mg, 71.0%) can be obtained as a white solid: FDMS m/z 760. Analysis C₄₁H₆₄N₂O₁₁Estimated value: C, 64, 71; H, 8.48; N3.68. Actual value: C, 64.48; H, 8.70; N3.59.

<u>Example C41-4"-Demethyl-(dimethylamino)-4"-(N-(E)-(2-hydroxy, 3,5-di-t-coylphenyl) imino group history Benoxine A and 4"-desmethyl-(dimethylamino)-4"-(N-(Z)-(2-hydroxy, 3,5-di-t-coylphenyl) imine Kispinosine A</u>

The compound Spinozin C (0.55g, 0.78mmol) was dissolved in methanol/tetrahydrofuran (6:1) and 1,3-di-t-butyl-1,2-benzene (228 mg, 1.03 mmol) and the reaction was stirred for 24 hours. During this reaction, the reactants changed from dark red to light yellow-green. The solvent was removed by a rotary evaporator and the crude green solid was purified by chromatography on silica gel (ethyl acetate/hexane, 1:1) to provide compound 4"-desmethyl-(dimethylamino)4 "-(N-(E)-(2-hydroxy, 3,5-di-t-coylphenyl) imino Spinozin A (228 mg, 32.2%):

<img file="TW487559B_D0113.tif" />

<u>Example C42-4"-Demethyl-(dimethylamino)-4"-oxy Spinozin A</u>

The compound 4"-desmethyl-(dimethylamino)-4"-[N-(E)-(2-hydroxy, 3,5-di-t-butylphenyl) imino Spino Octane A (204 mg, 0.225 mole) was suspended in methanol/tetrahydrofuran/water, 6:1:1 (8 mL), and the compound was dissolved by adding tetrahydrofuran (6 mL). To this solution was added oxalic acid dihydrate (60 mg, 0.47 mmol) and the reaction was stirred for 20 hours, followed by heating at 50-60°C for 4 hours. After the gradual reaction was completed, the residue was purified by cyclotron chromatography on silica gel (ethyl acetate/hexane, 1:1) to extract the compound 4"-desmethyl-(dimethylamino)-4 "-Oxy Spinozin A (43.2 mg, 27.3%), as a white solid: partly¹H-NMR δ6.75

<img file="TW487559B_D0114.tif" />

<u>Example C43-(4"S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A</u>

Compound 4"-desmethyl-(dimethylamino)-4"-oxy Spinozin A (78 mg, 0.11 mmole) was dissolved in ether (10 mL) and cooled to 0°C in an ice bath. To this cold solution was added lithium tri-tert-butoxide (48 mg, 0.19 mmol) and the reaction was stirred for 20 minutes. The reaction was quenched with saturated sodium chloride (3ml), the reaction was gradually carried out, and the residue was purified by cyclotron chromatography on silica gel (ethyl acetate/hexane, 1:1) to provide compound (4"S) -4"-Demethyl-(dimethylamino)-4"-hydroxyspinoxine A (46.6 mg,

<img file="TW487559B_D0115.tif" />

<u>Example C44-(4"S)-4"-Demethyl-(dimethylamino)-4"-Acetyloxy Spinozin A</u>

The compound (4"S)-4"-desmethyl(dimethylamino)-4"-hydroxyspinoxine A (10 mg, 0.14 mmol) was dissolved in dichloromethane and acetic anhydride (0.05 g , 0.5 mmol) and pyridine (0.1 g, 1 mmol) and stir the reactants for 2 hours. Gradually the reaction is completed to obtain the compound (4"S)-4"-desmethyl-(dimethylamino)-4" -Acetyloxy Spinozin A (5.7 mg, 57%), no

<img file="TW487559B_D0116.tif" />

<u>Example C45-N-desmethyl-N-formyl-spinoxine D</u>

The compound N-desmethyl-N-formyl-spinoxine D (1.08 g; 1.47 mmol) was dissolved in ethyl formate (25 ml) and the solution was heated at reflux temperature for 14 hours. The solvent was evaporated by a rotary evaporator and by reverse phase HPLC (methanol/0.1%aq. ammonium hydroxide, 85:15) to provide N-desmethyl-N-methanyl-spinoxin D (645.6 mg, 57.6%), white foam: partly

<img file="TW487559B_D0117.tif" />

<u>Example C46-4"-N-[(2,4-Difluorophenyl)aminocarbonyl] Spinozin B</u>

According to the method of Example C80, the compound was prepared from 2,4-difluorophenyl isocyanate (78 ml, 98 mg, 0.63 mmol) and Spinozin B (0.30 g, 0.42 mmol). MPLC(SiO₂, 50:50, EtOAc/hexane) to obtain 0.36 g (99%) 4"-N-[(2,4-difluorophenyl)aminocarbonyl]spinoxine B as a white powder: MS ( m+H⁺) Expected value: 873.5. Actual value: 873.8.

<u>Example C47-4"-N-[(3,4-Difluorophenyl)aminocarbonyl] Spinozin B</u>

According to the method of Example C80, the compound was prepared from 3,4-difluorophenyl isocyanate (0.12 g, 0.64 mmol) and Spinozin B (0.30 g, 0.42 mmol). MPLC(SiO₂, 64:40 to 100:0, EtOAc/hexane) to obtain 0.38 g (99%) 4"-N-[(3,4-difluorophenyl)aminocarbonyl] Spinozin B, white Powder: MS(m+H⁺) Expected value: 905.4. Actual value: 905.6.

<u>Example C48-4"-N-[(4-methoxyphenyl)aminocarbonyl] Spinozin B</u>

According to the method of Example C80, the compound was prepared from 4-methoxyphenyl isocyanate (81 ml, 93 mg, 0.63 mmol) and Spinozin B (0.30 g, 0.42 mmol). MPLC(SiO₂, 64:40, EtOAc/hexane) to obtain 0.36 g (99%) 4"-N-[(4-methoxyphenyl)aminocarbonyl] Spinozin B as a white powder: MS (m +H⁺) Expected value: 867.5. Actual value: 867.8.

<u>Example C49-4"-N-(1-propyl) Spinozin B</u>

According to the method of Example C81, 1-iodopropane (0.14 ml, 0.24g, .1.4 mmol), (i-Pr)₂Compounds were prepared with NEt (0.36 ml, 0.27 g, 2.1 mmol), Spinozin B (0.50 g, 0.70 mmol), and DMF (1.5 ml). MPLC(SiO₂, 50:50, EtOAc/hexane) to obtain 0.41 g (77%) 4"-N-(1-propyl) Spinozin B as a white powder. C₄₃H₆₆NO₁₀Analysis: Estimated value: C, 67.96; H, 9.15; N, 1.84. Actual value: C, 68.06; H, 9.25; N, 1.97.

<u>Example C50-4"-N-(2-methyl-1-propyl) Spinozin B</u>

According to the method of Example C81, 1-iodo-2-methylpropane (0.32 ml0.51 g, 2.8 mmol), (i-Pr)₂Compounds were prepared with NEt (0.73 ml, 0.54 g, 4.2 mmol), Spinozin B (0.95 g, 1.3 mmol), and DMF (2.8 ml). MPLC(SiO₂, 50:50, EtOAc/hexane) to obtain 0.07 g (7%) 4"-N-(2-methyl-1-propyl) Spinozin B as a white powder (Spinoxine can also be recovered Xin B, 0.71 g (75%)): MS (m+H⁺) Expected value: 774.5. Actual value: 774.8. C₄₄H₇₁NO₁₀Analysis: Estimated value: C, 68.28; H, 9.25; N, 1.81. Actual value: C, 68.33; H, 9.25; N, 1.88.

<u>Example C51-4"-N-[(Cyclopropyl)methyl] Spinozin B</u>

According to the method of implementing C81, from (bromomethyl) cyclopropane (0.27 ml, 0.38 g, 2.8 mmol), NaI (0.05 g, 0.3 mmol), (i-Pr)₂-NEt (0.73 mL, 0.54 g, 4.2 mmol), Spinozin B (0.95 g, 1.3 mmol), and DMF (2.8 mL) to prepare the compound. MPLC (50:50, EtOAc/hexane) to obtain 0.34 g (33%) 4"-N-[(cyclopropyl)methyl] Spinozin B as a white powder (Spinozine can also be recovered B, 0.40 g (42%) Spinozin B). C₄₄H₆₉NO₁₀Analysis: Estimated value: C, 68.45; H, 9.01; N, 1.81. Actual value: C, 68.36; H, 9.22; N, 1.76.

<u>Example C52-4"-N-ethyl-5,6-dihydrospinoxine B</u>

According to the method of Example C81, from bromoethane (60 ml, 0.12 g, 0.75 mmol), (i-Pr)₂-NEt (0.20 mL, 0.15 g, 1.1 mmol), 5,6-dihydrospinoxine B (0.27 g, 38 mmol), and DMF (2 mL) to prepare the compound. MPLC (40:60, EtOAc/hexane) to obtain 0.22 g (79%) of 4"-N-ethyl-5,6-dihydrospinoxine B as a white powder. C₄₂H₆₉NO₁₀Analysis: Estimated value: C, 67.44; H, 9.30; N, 1.87. Actual value: C, 67.52; H, 9.07; N, 1.78.

<u>Example C53-5,6-Dihydro-4"-N-(2-methyl-1-propyl) Spinozin B</u>

According to the method of Example C81, 1-iodo-2-methylpropane (0.10 ml, 0.16 g, 0.87 mmol), (i-Pr)₂-NEt (0.23 mL, 0.17 g, 1.3 mmol), 5,6-dihydrospinoxine B (0.31 g, 0.43 mmol), and DMF (2 mL) to prepare the compound. MPLC(SiO₂, 40:60, EtOAc/hexane) to obtain 0.16 g (48%) of 5,6-dihydro-4"-N-(2-methyl-1-propyl)spinoxine B as a white powder .C₄₄H₇₃NO₁₀Analysis: Estimated value: C, 68.10; H, 9.48; N, 1.80. Actual value: C, 68.10; H, 9.37; N, 1.87.

<u>Example C54-4"-N-(2-fluoroethyl) Spinozin B</u>

According to the method of Example C81, 1-bromo-2-fluoroethane (0.10 ml, 0.17 g, 0.3 mmol), NaI (0.04 g, 0.67 mmol), (i-Pr)₂Compounds were prepared with NEt (0.36 mmol, 0.27 g, 2.1 mmol), Spinozin B (0.48 g, 0.67 mmol), and DMF (2 mL). MPLC (30:70 to 40:60, EtOAc/hexane) to obtain 0.30 g (59%) of 4"-N-(2-fluoroethyl) Spinozin B as a white powder. C₄₂H₆₆NO₁₀Analysis: Estimated value: C, 66.03; H, 8.71; N, 1.83. Actual value: C, 65.89; H, 9.11; N, 1.92.

<u>Example C55-4"-N-(2,2,2-trifluoroethyl) Spinozin B</u>

According to the method of Example C81, 2,2,2-trifluoroethyl trifluoromethanesulfonate (0.34 g, 1.5 mmol), (i-Pr)₂Compounds were prepared with NEt (0.36 mmol, 0.27 g, 2.1 mmol), Spinozin B (0.38 g, 0.53 mmol), and DMF (2 mL). MPLC (30:70, EtOAc/hexane) to obtain 0.32 g (76%) of 4"-N-(2,2,2-trifluoroethyl)spinoxine B as a white powder. C₄₂H₆₄NO₁₀Analysis: Estimated value: C, 63.06; H, 8.06; N, 1.75. Actual value: C, 62.78; H, 7.97; N, 1.66.

<u>Example C56-4"-N-[2-(N',N'-dimethylamino)ethyl] Spinozin B</u>

According to the method of Example C81, Me₂(CH₂)₂ClHCl(0.30 g, 2.1 mmol), (i-Pt)₂Compounds were prepared with NEt (0.50 mL, 0.37 g, 2.9 mmol), Spinozin B (0.50 g, 0.70 mmol), and DMF (2 mL). MPLC(20:80, MeOH/CH₂Cl₂) To obtain 0.04 g (7%) 4"-N-[2-(N',N'-dimethylamino)ethyl] Spinozin B as a white powder (0.44 g (88 %) Spinozin B). MS (m+H⁺) Expected value: 789.5. Actual value: 789.8.

<u>Example C57-4"-N-(2-propyl) Spinozin B</u>

According to the method of Example C81, 2-iodopropane (0.11 ml, 0.19g, 1.1 mmol), (i-Pt)₂Compounds were prepared with NEt (0.30 mL, 0.22 g, 1.7 mmol), Spinozin B (0.40 g, 0.56 mmol), and DMF (2 mL). MPLC(SiO₂, 50:50, EtOAc/hexane) to obtain 0.09 g (21%) 4"-N-(2-propyl) Spinozin B as a white powder: MS (m+H⁺) Expected value: 760.5. Actual value: 760.6. C₄₃H₆₉NO₁₀Analysis: Estimated value: C, 67.96; H, 9.15; N, 1.84. Actual value: C, 68.04; H, 9.32; N, 1.85.

<u>Example C58-4"-N-(1-butyl) Spinozin B</u>

According to the method of Example C81, 1-iodobutane (0.13 ml, 0.21g, 1.1 mmol), (i-Pr)₂Compounds were prepared with NEt (0.30 mL, 0.22 g, 1.7 mmol), Spinozin B (0.40 g, 0.56 mmol) and DMF (2 ml). MPLC(SiO₂, 50:50 EtOAc/hexane) to obtain 0.41 g (95%) 4"-N-(1-butyl) Spinozin B as a white powder. MS (m+H⁺) Expected value: 774.5. Actual value: 774.7.

<u>Example C59-4"-N.4"-N-(Butane-1.4-diyl) Spinozin B iodide salt</u>

According to the method of Example C81, 1,4-diiodobutane (0.11 ml, 0.26 g, 0.83 mmol), (i-Pr)₂Compounds were prepared with NEt (0.30 mL, 0.22 g, 1.7 mmol), Spinozin B (0.30 g, 0.42 mmol) and DMF (1 ml). MPLC(SiO₂, 25:75 methanol/dichloromethane) to obtain 0.18 g (47%) 4"-N,4"-n-(but-1,4-diyl)spinoxine B iodide salt, which is a yellow-brown powder .

<u>Example C60-4"-N,4"-N-(pentyl-1,2-diyl)spinoxine B iodide salt</u>

According to the method of Example C81, 1,5-diiodobutane (0.12 ml, 0.26 g, 0.81 mmol), (i-Pr)₂Compounds were prepared with NEt (0.30 mL, 0.22 g, 1.7 mmol), Spinozin B (0.30 g, 0.42 mmol) and DMF (1 ml). MPLC(SiO₂, 25:75 methanol/dichloromethane) to obtain 0.10 g (26%) 4"-N,4"-N-(penta-1,2-diyl)spinoxine B iodide salt, which is a yellow-brown powder .

<u>Example C61-4"-N,4"-N-(Butane-1,4-diyl) Spinozin C</u>

According to the method of Example C81, 1,4-diiodobutane (0.22 ml, 0.52 g, 1.7 mmol), (i-Pr)₂Compounds were prepared with NEt (0.44 mL, 0.33 g, 2.5 mmol), Spinozin C (0.60 g, 0.57 mmol) and DMF (4 ml). MPLC(SiO₂, 5:95 methanol/dichloromethane) to obtain 0.10 g (23%) 4"-N,4"-N-(butan-1,4-diyl)spinoxine C as a white powder. MS(m+H⁺) Expected value: 758.5. Actual value: 758.6.

<u>Example C62-4"-N,4"-N-(penta-1,5-diyl) Spinozin C</u>

According to the method of Example C81, 1,5-diiodopentane (0.25 ml, 0.54 g, 1.7 mmol), (i-Pr)₂Compounds were prepared with NEt (0.44 mL, 0.33 g, 2.5 mmol), Spinozin C (0.60 g, 0.57 mmol) and DMF (4 ml). MPLC(SiO₂, 50:50 ethyl acetate/hexane) to obtain 0.31 g (70%) of 4"-N,4"-N-(penta-1,4-diyl)spinoxine C as a white powder. MS(m+H⁺) Expected value: 772.5. Actual value: 772.7.

<u>Example C63-4"-N,4"-N-Diethyl Spinozin C</u>

According to the method of Example C81, from ethyl iodide (0.23 ml, 0.45 g, 2.9 mmol), (i-Pr)₂Compounds were prepared with NEt (0.60 mL, 0.45 g, 3.4 mmol), Spinozin C (0.40 g, 0.57 mmol) and DMF (2 ml). MPLC(SiO₂, 50:50 ethyl acetate/hexane) to obtain 0.38 g (88%) 4"-N,4"-N-diethyl Spinozin C as a white powder: MS (m+H⁺) Expected value: 760.5. Actual value: 760.7. C₄₃H₆₉NO₁₀Analysis value: C, 67.96; H, 9.15; N, 1.84. Actual value: C, 68.01; H, 9.47; N, 1.83.

<u>Example C64-4"-N,4"-N-(2-butene-1,4-diyl) Spinozin C and 4"-N,4"-N-(butadiene-1, 4-diyl) Spinozin C</u>

According to the method of Example C81, 1,4-dichlorobut-2-ene (0.10 ml, 0.12 g, 0.95 mmol), NaI (0.02 g, 0.1 mmol), (i-Pr)₂These compounds were prepared with NEt (0.22 mL, 0.16 g, 1.3 mmol), Spinozin C (0.29 g, 0.41 mmol) and DMF (1.5 ml). MPLC(SiO₂, 20:80 ethyl acetate/dichloromethane) to obtain 0.11 g (35%) 4"-N,4"-N-(2-butene-1,4-diyl) Spinozin C, which is White powder: MS(m+H⁺) Expected value: 765.5. Actual value: 756, 6, and 4"-N,4"-N-(butadiene-1,4-diyl) Spinozin C: 0.11 g (35%), white powder: MS (m+ H⁺) Expected value: 754.5. Actual value: 75.8. The mild room temperature oxidation has shown the formation of this product.

<u>Example C65-4"-N-[(6-Chloro-3-ridinyl)methyl]spinoxine B</u>

According to the method of Example C81, (6-chloro-3-oxidinyl) chloromethane (0.14 g, 0.8 6 mmol), (i-Pr)₂Compounds were prepared with NEt (0.22 mL, 0.16 g, 1.3 mmol), Spinozin B (0.30 g, 0.42 mmol) and DMF (1 ml). MPLC(SiO₂, 25:75 to 75:25 ethyl acetate/hexane) to obtain 0.30 g (86%) 4"-N-[(6-chloro-3-ridinyl)methyl]spinoxine B, which is White brown powder.

<u>Example C66-4"-N-[N'-(2,2-Dimethylethoxycarbonyl)-β-propylamino] Spinozin B</u>

According to the method of Example C77, NMM (2x0.12 ml, 0.22 g, 2.2 mmol), Boc-N(H)-β-Ala-CO₂H (0.17 g, 0.90 mmol), BOP-Cl (0.23 g, 0.90 mmol), Spinozin B (0.50 g, 0.70 mool) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 70:30 to 100:0 ethyl acetate/hexane) to obtain 0.62 g (99%) 4"-N-[N'-(2,2-dimethylethoxycarbonyl)-β-propylamine Base] Spinozin B, white brown powder, MS(m+H⁺) Expected value: 889.5. Actual value: 889.7. C₄₈H₇₆N₂O₁₃Analysis: Estimated value: C, 64.84; H, 8.62; N, 3.15. Actual value: C, 64.38; H, 8.87; N, 3.22.

<u>Example C67-4"-N-[N'-(Phenylmethoxycarbonyl)-L-lactamino] Spinozin B</u>

According to the method of Example C77, NMM (2x0.12 ml, 0.22 g, 2.2 mmol), Boc-N(H)-L-Ala-CO₂H (0.21 g, 0.94 mmol), BOP-Cl (0.24 g, 0.94 mmol), Spinozin B (0.50 g, 0.70 mmol) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 75:25, ethyl acetate/hexane) to obtain 0.68 g (99%) of 4"-N-[N'-(phenylmethoxycarbonyl)-L-propylamino]spinoxine B, White powder: MS(m+H⁺) Expected value: 923.5. Actual value: 923.7.

<u>Example C68-4"-N-[N',N'-dimethylglycamido)] Spinozin B</u>

According to the method of Example C77, NMM (2x0.12 ml, 0.22 g, 2.2 mmol), N(Me)₂-Gly-CO₂H (0.09 g, 0.9 mmol), BOP-Cl (0.25 g, 0.98 mmol), Spinozin B (0.50 g, 0.70 mmol) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 5: 95, MeOH/CH₂Cl₂) To obtain 0.43 g (77%) 4"-N-[N',N'-dimethylglycinyl)] Spinozin B as a white powder: MS(m+H⁺) Expected value: 803.5. Actual value: 803.6.

<u>Example C69-4"-N-[N',N'-(pentan-1,5-diyl)-β-propylamino]spinoxine B</u>

According to the method of Example C77, NMM (2x0.12 ml, 0.22 g, 2.2 mmol), N, N (CH₂)₅-β-Ala-CO₂H (0.14 g, 0.89 mmol), BOP-Cl (0.26 g, 1.0 mmol), Spinozin B (0.50 g, 0.70 mmol) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 5:95 to 10:90, MeOH/CH₂Cl₂) To obtain 0.54 g (90%) 4"-N-[N',N'-(pentyl-1,5-diyl)-β-propylamino]spinoxine B as a white powder: MS( m+H⁺) Expected value: 857.6. Actual value: 857.8.

<u>Example C70-4"-N-[N',N'-Diethyl-β-propylamine] Spinozin B</u>

According to the method of Example C77, NMM (0.24 ml, 0.22 g, 2.2 mmol &amp; 0.12 ml, 0.11 g, 1.1 mmol), N, N (Et)₂-β-Ala-CO₂HHCl (0.16 g, 0.88 mmol), BOP-Cl (0.26g, 1.0 mmol), Spinozin B (0.50 g, 0.70 mmol) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 5:95 to 10:90, MeOH/CH₂Cl₂) To obtain 0.46 g (78%) 4"-N-[N',N'-diethyl-β-propylamino]spinoxine B as a white powder: MS(m+H⁺) Expected value: 845.6. Actual value: 845.8.

<u>Example C71-4"-N-[N'-(9-fluorenylmethoxycarbonyl)-β-propylamino]spinoxine B and 4"-N-(β-propylamino)spin Nosin B</u>

According to the method of Example C77, NMM (2x0.12 ml, 0.22 g, 2.2 mmol &amp; 0.12 ml, 0.11 g, 1.1 mmol), Fmoc-N(H)-β-Ala-CO₂H (0.26 g, 0.84 mmol), BOP-Cl (0.23 g, 0.90 mmol), Spinozin B (0.502 g, 0.699 mmol) and CH₂Cl₂(7 ml) The compound was prepared. MPLC(SiO₂, 0: 100 to 20: 80, MeOH/CH₂Cl₂) To obtain 0.03 g (4%) 4"-N-[N'-(9-fluorenylmethoxycarbonyl)-β-propylamine phenyl]spinoxine B as a white powder: MS(m+H⁺) Expected value: 1011.6. 4"-N-(β-propylamino) Spinozin B 0.27 g (49%), white powder: MS(m+H⁺) Expected value: 789.5. Actual value: 789.7. The mild deprotection of the Fmoc group under the reaction conditions has indicated the formation of this product.

<u>Example C72-4"-N-(diethoxyphosphatidyl) Spinozin B</u>

Will (EtO)₂P(O)Cl (91 ml, 0.11 g, 0.63 mmol) and (i-Pt)₂NEt (0.22 ml, 0.16 g, 1.3 mmol) was sequentially added to Spinozin B (0.30 g, 0.42 mmol) in CH₂Cl₂(4 ml) solution. After 2 days, the mixture was in CH₂Cl₂With NaHCO₃Between balance. Wash the organic layer with brine, and use (Na₂SO₄) Drying and filtering and evaporation. MPLC(SiO₂, EtOAc) to obtain 0.27 g (75%) of 4"-N-(diethoxyphosphinyl) Spinozin B as a white powder.

<u>Example C73-N-Trifluoroacetyl-N-desmethylspinoxine D</u>

Add a portion of trifluoroacetic anhydride (0.16 ml, 1.1 mmol) to the cold (0°C), properly stirred N-desmethyl spinoxin D (0.4 g, 0.54 mmol) and triethylamine (0.2 ml , 1.5 mmol) in ethyl acetate. The cooling bath was removed and the solution was stirred at room temperature for 40 minutes, then poured into ice water (40 ml). The product was extracted in ethyl acetate (3x15 ml) and saturated NaHCO₃(6 ml) and brine (6 ml) to wash the organic extracts, and use (MgSO₄)dry. Evaporate the solvent to leave 0.4 g of clean N-trifluoroacetyl-N-desmethylspinoxine D, which is a colorless foam:¹H-NMR(CDCl₃)δ

<img file="TW487559B_D0118.tif" />

<u>Example C74-N-(2,2,2-Trichloroethoxy)carbonyl-N-desmethylspinoxine A</u>

Anhydrous K₂CO₃(0.12 g, 0.87 mmole) to dry benzene added to properly stirred Spinozin A (1.5 g, 2.0 mmol) and 2,2,2-trichloroethyl chloroformate (0.8 ml, 5.5 mmol) (20 ml) solution. The resulting mixture was heated at reflux temperature for 48 hours, then cooled to room temperature and poured into ice water (100 ml). The product was extracted in ethyl acetate (3x40 ml) and the organic extract was washed with brine (40 ml) and washed with (MgSO₄)dry. The residue (2.7 g) after evaporation of the solvent was subjected to flash chromatography with silica (170 ml), using 2:1 hexane/ethyl acetate as the eluent to obtain N-(2,2,2-tri Chloroethoxy) carbonyl-N-desmethylspinoxine A (1.3 g) as a colorless foam

<img file="TW487559B_D0119.tif" />

<u>Example C75-N-(2,2,2-Trichloroethoxy)carbonyl-N-desmethylspinoxine D</u>

In 2-3 minutes, add 2,2,2-trichlorochloroformate (0.07 ml, 0.51 mmol) in CH₂Cl₂(2 ml) The solution was added dropwise to the stirred N-desmethylspinoxine D (0.15 g, 0.20 mmol) and pyridine (0.3 ml) in CH₂Cl₂(4 ml) solution. The solution was stirred at room temperature for 6 hours, then diluted with water (5 ml). Separate the organic layer and continuously use 1N HCl (5 ml), saturated NaHCO₃(5 ml) and saline (5 ml), and washed with (MgSO₄)dry. The remaining residue (0.42 g) was concentrated, and the residue was flash chromatographed with silica (90 ml) using 2:1 hexane/ethyl acetate as the eluent to obtain 0.15 g (80%) The N-(2,2,2-trichloroethoxy)carbonyl-N-desmethylspinoxine D is a colorless foam:¹H-NMR(CDCl₃)δ6.76(s,

<img file="TW487559B_D0120.tif" />

<u>Example C76-N-(2,2,2-Trichloroethoxy)carbonyl-N-desmethyl-7-hydroxyspinoxine D</u>

5% SeO on silica (0.21 g)₂And tertiary butyl hydroxyperoxide (90%, 0.07 mmol) in CH₂Cl₂(3 ml) The suspension was stirred at room temperature for 15 minutes. In this mixture, add N-tri-chloroethoxycarbonyl-N-desmethylspinoxine D (0.15 g, 0.16 mmol) in CH dropwise within 2-3 minutes₂Cl₂(2 ml) solution. The mixture was stirred at room temperature for 1.5 hours and then filtered. The collected solid is CH₂Cl₂(10 ml) Wash and combine the filtrate and wash with water (5 ml) and brine (5 ml) and wash with (MgSO₄)dry. The remaining 0.11 g residue was concentrated. The residue was flash chromatographed with silica (90 ml) using 4% methanol in CH₂Cl₂To obtain 63 mg of N-(2,2,2-trichloroethoxy)carbonyl-N-desmethyl-7-hydroxyspinoxine D as a white foam

<img file="TW487559B_D0121.tif" />

<u>Example C77-4"-N-[N'-(2,2-Dimethylethoxycarbonyl)glycamido] Spinozin B</u>

Add 4-methylmorphine (NMM) (0.12 ml, 0.11 g, 1.1 mmol) to Boc-N(H)-Gly-CO of -15c₂CH of H (0.16 g, 0.91 mmol) and BOP-Cl (0.23 g, 0.90 mmol)₂Cl₂(7 ml) in solution. After 1.5 hours, the volume was reduced by about half under reduced pressure. Spinozin B (0.50 g, 0.70 mmol) and NMM (0.12 ml, 0.11 g, 1.1 mmol) were added sequentially. The mixture was heated to room temperature for 20 hours; the mixture was evaporated. MPLC(SiO₂, 50:50 to 100:0, EtOAc/hexane) to obtain 0.61 g (99%) of 4"-N-[N'-(2,2-dimethylethoxycarbonyl)glycamido] history Binoxin B, white powder: MS(m+H⁺) Expected value: 875.5. Actual value: 875.9.

<u>Example C78-N-Chloroacetyl Spinozin B</u>

Spinozin B (104.4 mg, 0.145 mmol) was dissolved in dichloromethane (5 ml) and N,N-diisopropylethylamine (0.1 g, 0.8 mmol) and dichloromethane f (0.1 g, 0.9 mmol) and stir the reaction for 1 hour. After the residue gradually reacted to form, it was purified by silica gel (ethyl acetate/hexane, 50:50) chromatography to provide N-chloroacetylspinoxine B (79.1 mg, 68%) as a colorless glass Material: FDMS m/z 794. C₄₂H₆₄NO₁₁Analysis of Cl: Estimated value: C, 63.50, H, 8.12; N, 1.76. Actual value: C, 63.32; H, 8.04; N, 1.58.

<u>Example C79-N-(N'-Ethylthioaminomethyl) Spinozin B</u>

The compound Spinozin B (97.8 mg, 0.136 mmol) was dissolved in toluene (4 ml) and ethyl isothiocyanate (0.05 g, 0.6 mmol) was added and the reaction was heated at reflux temperature for 2 hours. The toluene solvent was removed under reduced pressure and the residue was purified by chromatography on silica gel (ethyl acetate/hexane, gradient 20:80 to 50:50) to provide the compound N-(N'-ethylthioaminomethyl) (Ki) Spinozin B, a colorless glass: FDMS m/z 804,805.

<u>Example C80-4"-N-[(4-(trifluoromethoxy)phenyl]aminocarbonyl Spinozin B</u>

Add 4 (trifluoromethoxy) phenyl isocyanate (94 ml, 0.13 g, 0.63 mmol) and DMF (3 ml, 3 mg, 0.04 mmol) to Spinozin B (0.30 g, 0.42 mmol) in ether (4 ml). The mixture was heated to room temperature for 15 minutes. The excess isocyanate was decomposed via the addition of methanol (1 ml) and the mixture was evaporated. MPLC(SiO₂, 50:50, ethyl acetate/hexane) to obtain 0.37 g (97%) of 4"-N-[(4-(trifluoromethoxy)phenyl]aminocarbonyl Spinozin B, white Powder: MS(m+H⁺) Expected value: 921.5. Actual value: 921.6.

<u>Example C81-4"-N-Ethyl Spinozin B</u>

Iodoethane (0.11 ml, 0.21 g, 1.4 mmol) and (i-Pt)₂NEt (0.36 ml, 0.27 g, 2.1 mmol) was sequentially added to Spinozin B (0.05 g, 0.70 mmol) in DMF (1.5 ml). By TLC (SiO₂) And HPLC (C₁₈) Monitor the reaction process. After showing full conversion (1-5 days), the reaction was evaporated. The residue was partitioned between ethyl acetate and sodium bicarbonate. The organic layer was washed with brine, with (MgSO₄) Dry, filter and evaporate. MPLC(SiO₂, 50:50 ethyl acetate/hexane) to obtain 0.55 g (89%) 4"-N-ethyl Spinozin B as a white powder: MS (m+H⁺) Expected value: 746.5. Actual value: 746.7.

<u>Example C82-(4"S)-4"-Demethyl-(dimethylamino)-4"-methoxy Spinozin A</u>

The compound (4"S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (2.21 g; 3.08 mmol) was dissolved in CH₂Cl₂(60 ml). Add water (20ml), 10% aq.NaOH (25 ml) and K₂CO₃Solid (5 g), followed by dimethyl sulfate (8 ml, excess). The reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Add water (50 ml) and CH₂Cl₂(50 ml) to separate the organic layer with H₂O(2x) cleaning, with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Column chromatography (230-400 m, 250g/ethyl acetate) separation.

<u>Example C83-4"-(S)-4"-desmethyl-(dimethylamino)-4"-ethoxy Spinozin A</u>

The compound (4"S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (2.21 g; 3.08 mmol) was dissolved in CH₂Cl₂(60 ml). Add water (20ml), 10% aq.NaOH (25 ml) and K₂CO₃The solid (5 g), followed by the addition of diethyl sulfate (8 ml, excess). The reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Add water (50 ml) and CH₂Cl₂(50 ml) to separate the organic layer with H₂O(2x) cleaning, with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Column chromatography (230-400 m, 250g/ethyl acetate) separation.

<u>Example C84-4"-(S)-4"-desmethyl-(dimethylamino)-4"-propoxy Spinozin A</u>

The compound 4"-(S)-4"-desmethyl-(dimethylamino; -4"-hydroxyspinoxine A (1.22 g, 1.70 mmol) was dissolved in CH₂Cl₂(20 ml). Add water (20 ml), 10% NaOH aqueous solution (25 ml), then add K₂CO₃(2 g), tetrabutylammonium chloride (1.2 g) and mensyltriethylammonium chloride (1.5 g). 1-iodopropane (10 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 48 hours. Dichloromethane (20 ml) was added and the phases were separated. Wash the organic layer with water (2x) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Purified by column chromatography (120 g/ethyl acetate).

<u>Example C85-4"-(S)-4"desmethyl-(dimethylamino)-4"-chloromethyl spinoxine A</u>

The compound 4"-(S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (510 mg, 0.71 mmol) was dissolved in CH₂Cl₂(5.0 ml) and add K₂CO₃(1.10 g). The reaction flask was immersed in a water bath (10° C.) and 15% NaOH aqueous solution (10 ml) was added with vigorous stirring, followed by the addition of menthyl triethylammonium chloride (1.20 g). Chloroiodomethane (5.0 ml) was added and the reaction mixture was stirred vigorously at room temperature under nitrogen for 72 hours. Add dichloromethane (100 ml) and water (50 ml). Separate the phases. Wash the organic layer with water (2x) and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Purified by column chromatography (70 g/ethyl acetate).

<u>Example C86-4"-(S)-4"-desmethyl-(dimethylamino)-4"-trimethylsilanoxy Spinozin A</u>

The compound 4"-(S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (510 mg, 0.71 mmol) was dissolved in CH₂Cl₂(10.0 ml). Add dimethylamine pyridine (0.80 g). The mixture was stirred at room temperature under nitrogen. Slowly introduce trimethylsilyl trifluoromethanesulfonate (0.90 ml, excess) via a syringe and continue stirring for 1 hour. Add ethyl acetate (50 ml) and benzene (50 ml). With 5% aq, NaHCO₃Solution (3x) cleaning solution. Organic layer with K₂CO₃Drying and drying under reduced pressure. The residue is flashed by SiO₂Purified by column chromatography (100 g/ethyl acetate).

<u>Example C87-(4"S)-4"-Demethyl-(dimethylamino)-4"-Acetyloxy Spinozin A</u>

The compound (4"S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (10 mg, 0.14 mmol) was dissolved in dichloromethane and acetic anhydride (0.05 g, 0.5 mmol) and pyridine (0.1 g, 1 mmol), and the reactant was stirred for 2 hours. The reaction was gradually reacted to obtain compound (4"S)-4"-desmethyl-(dimethylamino)-4 "-Acetyloxy Spinozin A.

<u>Example C88-4"-(S)-4"-desmethyl-(dimethylamino)-4"-chloroacetoxy Spinoxine A</u>

The compound 4"-(S)-4"-desmethyl-(dimethylamino)-4"-hydroxyspinoxine A (306 mg, 0.43 mmol) was dissolved in dry pyridine (5 ml). Chloroacetic acid chloride (0.50 ml) was added dropwise. The reaction mixture was stirred at room temperature under nitrogen. After 3 hours, toluene (50 ml) and ethyl acetate (50 ml) were added. The solution was continuously heated with brine, 5% NaHCO₃(2x) and water cleaning. Anhydrous Na₂SO₄The organic layer is dried and dried under reduced pressure. The residue is flashed by SiO₂Purified by column chromatography (80 g/ethyl acetate).

<u>Example C89-Compound 3",4"-dihydro-4"-deamino-5,6-dihydrospinoxine A</u>

5,6-Dihydrospinoxine A N-oxide (1.26 g, 1.68 mmol) was dissolved in dry THF (10 ml). The solution was cooled to -78°C and pyridine (5ml) was added. While stirring, trifluoroacetic anhydride (1.8 ml, 2.67 g, 12.7 mmol) was added. After 16 hours, ether (120 ml) was added, and the mixture was extracted with dilute brine (2x), followed by 10% KHCO₃(2X) Extraction, and use K₂CO₃Dry and concentrate under reduced pressure. The residue is flashed by SiO₂Column chromatography (ether) was separated to obtain compound 3",4"-dihydro-4"-deamino-5,6-dihydrospinoxine A (122 mg, 10.5%) as a white solid:¹H-NMR δ 6.82(

<img file="TW487559B_D0122.tif" />

<u>Example C90-Compound 5,6-dihydro-N-methanoyl-spinoxine B and compound 4"-deamino-5,6-dihydro-4"-ketospinoxine C</u>

5,6-Dihydrospinoxine A (1.43 g, 1.95 mmol) was dissolved in dichloromethane (25 ml) and pyridine (6.0 ml) was added. The flask was cooled to +10°C. Bromine (3.0 ml) was added with vigorous stirring under nitrogen. Water (2.0 ml) was added and stirring was continued for 1 hour. Ether (200 ml) was added, and the mixture was continuously added with water (10% NaHSO₃) Solution, water and 10% KHCO₃Aqueous solution extraction, with anhydrous K₂CO₃Dry, filter and concentrate. The crude product was divided into 2 equal parts. Part of it by flash chromatography (SiO₂/Et₂O) Purification and repeated RP (C-18) HPLC (92:8, MeOH/H₂O) Isolated to obtain compound 5,6-dihydro-N-formyl-spinoxine B (230 mg, 31%) as a white solid:¹H-NMR δ.7.95(b

<img file="TW487559B_D0123.tif" />

-Deamino-5,6-dihydro-4"-keto Spinozin C (168 mg, about 12%). The above steps were repeated on a 5-fold larger scale, which resulted in the production of pure 4"-de Amino-5,6-dihydro-4"-keto Spinozin C (212 mg, 3.1%), white

<img file="TW487559B_D0124.tif" />

<u>Example C91-Compound 4"-deamino-5,6-dihydro-4"(S)-hydroxyspinoxine C</u>

4"-Deamino-5,6-dihydro-4"-keto Spinozin C (192 mg, 0.273 mmol) was dissolved in dry ether (50 ml). The solution was cooled to 0°C and a portion of lithium tri-t-butylaluminum hydride (97%, 145 mg, 0.55 mmol) was added. Stir continuously for 15 minutes at the same temperature. The reaction mixture was then carefully quenched with brine. Add ether (100 ml) and extract the mixture with 2N NaOH (2x), followed by 10% KHCO₃Aqueous solution extraction, with K₂CO₃Dry and concentrate. Crude product is flashing SiO₂Column (EtOAc) chromatographic purification and RP (C-18) HPLC (92:8, MeOH/H₂O) Isolated to obtain compound 4"-deamino-5,6-dihydro-4"(S)-hydroxyspinoxine C (91 mg, 47%) as a white solid:

<img file="TW487559B_D0125.tif" />

<u>Example C92-Compound 4"-Deamino-5,6-dihydro-4"(S)-Methoxy Spinozin C</u>

4"-Deamino-5,6-dihydro-4"(S)-hydroxyspinoxine C (66 mg, 0.093 mmol) was dissolved in dichloromethane (1.5 ml). Iodomethane (2.0 ml) was added, followed by 40% NaOH aqueous solution (3.0 ml) and tetrabutylphosphonium bromide (320 mg). Stir the reaction mixture vigorously at room temperature under nitrogen and add powdered KOH (10 ml) and Et₂O (100 ml), wash the mixture with water, and then with 10% KHCO₃Wash with aqueous solution (2x). Anhydrous K₂CO₃The organic layer was dried, filtered, and dried under reduced pressure. The residue is flashing SiO₂String (CH₂Cl₂-Et₂O) Purification to provide compound 4"-deamino-5,6-dihydro-4"(S)-methoxyspinoxine C as a white solid:¹H-NMR δ6.82(bs, 1H),

<img file="TW487559B_D0126.tif" />

<u>Part D Modification examples of the 13' and 14' positions on the tricyclic part of the compound of formula I D1-(14R)-13,14-dihydro-spinoxine A</u>

In a nitrogen atmosphere, in a pure ethanol (20 ml) solution of Spinozin A (1.0 gms, 1.37 mmol), sodium borohydride (520 mg, 13.7 mmol) was added partly within 0.5 hours. The reaction mixture was stirred at room temperature for 45 minutes and then quenched by the addition of saturated aqueous ammonium chloride solution. Then it was diluted with water and extracted with dichloromethane. Take K₂CO₃The dichloromethane portion was dried and evaporated at room temperature under reduced pressure. The product was purified by chromatography on silica and diluted with 5% methanol in chloroform. Because the separation effect obtained was not good, the chromatography was repeated, and after the product was recovered, it was diluted with ethyl acetate. The obtained (14R)-13,14-dihydro-spinnosine A is a colorless glass, FDMS, m/e (relative strength) 734 (M⁺，100)，733(60)。

<u>Example D2-(14S)-5,6,13,14-tetrahydro-spinoxine A</u>

The reaction was carried out as described in Example D5, with Spinozin A (106.8 mg, 0.15 mmol) as the starting material. The obtained (14S)-5,6,13,14-tetrahydro-spinoxine A (85.5 mg; yield 80%) is a colorless, slightly viscous solid, FDMS, m/e (relative strength) 736 (M⁺，100)。

<u>Example D3-(14R)-5,6,13,14-tetrahydro-spinoxine A</u>

To (14R)-13,14-dihydrospinoxine A (210.3 mg, 0.29mmol) in benzene (9 ml) solution, add R(triphenylphosphine) rhodium(I) chloride (47.2 mg, 0.05) mmol) and place the reaction mixture under a hydrogen atmosphere. The mixture was stirred at room temperature for 4 days, and then the solvent was evaporated under reduced pressure at room temperature. The product was purified by chromatography on silica, diluted with 5% methanol in dichloromethane. The product was further purified by chromatography on silica, diluted with 70% ethyl acetate. The obtained (14R)-5,6,13,14-tetrahydro-spinozin A is a colorless glass, FDMS, m/e (relative strength) 735 (M⁺，100)。

<u>Example D4-(13R,14S)-13,14-epoxy-spinoxine A</u>

To Spinozin A (200.7 mg, 0.27 mmol) in methanol (3 ml), add 30% hydrogen peroxide (41 ml, 1.35 mmol) solution and then add sodium hydroxide (19 mg, 0.33 mmol) ). The reaction mixture was stirred at 0°C for 1 hour, and then heated to room temperature. After stirring for 3 hours at room temperature, the mixture was diluted with dichloromethane. Then wash dichloromethane with water, wash with 5% sodium thiosulfate, and wash with K₂CO₃Dry and evaporate at room temperature under reduced pressure. The obtained (13R,14S)-13,14-epoxy-spinoxine A (200.2mg, 99%) is a colorless glass, FDMS, m/e (relative strength) 747 (M⁺，100)。

<u>Example D5-(14S)-5,6,13,14-tetrahydro-deoxyspinoxine J</u>

To a solution of 3'-deoxy Spinozin J (108.8 mg, 0.16 mmol) in ethyl acetate (50 ml), alumina/palladium (100 mg) was added. The reaction mixture was placed under 60 psi of hydrogen and stirred at room temperature for 8 hours. After filtering the catalyst, the solvent was evaporated under reduced pressure at room temperature. The residue was purified by chromatography on silica and diluted with 2.5% ethanol in ethyl acetate. The obtained (14S)-5,6,13,14-tetrahydro-deoxy Spinozin J is a white solid, FDMS, m/e (relative intensity) 707 (65), 706 (MH⁺，100)。

<u>Example D6-(14S)-13,14-dihydro-spinoxine A</u>

Spinozin A (454 mg, 0.620 mmol) was dissolved in ether (50 ml). Under nitrogen and room temperature, lithium tri-t-butoxyaluminum hydride (475 mg, 97%, 1.81 mmol) was added to this solution with vigorous stirring. Stirring is continued for 18 hours. It was then diluted with ethyl acetate (70 ml) and toluene (50 ml) and carefully quenched with brine. The resulting mixture was washed with brine (2x) and then with 5% aqueous sodium bicarbonate (2x). Anhydrous K₂CO₃Dry and concentrate under reduced pressure. By flashing SiO₂The residue was purified by column (30 g, 0.05% pyridine in ethyl acetate) to obtain compound (14S)-13,14-dihydro-spinoxine A (421 mg, 92%) needles (acetic acid Ethyl ester-hexane). mp150-

<img file="TW487559B_D0127.tif" />

<u>Example D7-13-(N-hydroxy)amino-13,14-alkenyl spinoxine A</u>

Spinozin A (3.54 g, 4.71 mmol) was dissolved in ethanol (80 ml), a 10% aqueous sodium hydroxide solution (15 ml) was added, and then hydroxylammonium hydrochloride (2.0 g, excess) was added immediately. The reaction mixture was stirred for 45 minutes. Then ethyl acetate (50 ml) and toluene (100 ml) were added. Extract with brine followed by 5% sodium bicarbonate (3x). Take K₂CO₃The organic layer is dried and dried under reduced pressure. Let the residue be completely dried under reduced pressure and dissolve CH₂Cl₂(50 ml). Add triethylamine (5 ml) followed by methanesulfonyl chloride (2.5 ml). After stirring for 30 minutes, toluene (70 ml) and ethyl acetate (50 ml) were added. Wash the solution with 5% sodium bicarbonate (3x). Organic layer with K₂CO₃Dry and concentrate under reduced pressure. The residue was dissolved in dry toluene (30 ml), DBU (4.0 ml) was added and the reaction mixture was heated under reflux (under nitrogen) for 30 minutes. The solution was cooled, toluene (50 ml) was added and the solution was extracted with water (4x). Take K₂CO₃Dry and concentrate under reduced pressure. By flash column chromatography (SiO₂/EtOAc) separation and then by preparative HPLC (C-18 coated with silica, 10% methanol solution in water as the mobile phase) to obtain the compound 13-(N-hydroxy)amino-13,14-alkenyl history Binoxin A

<img file="TW487559B_D0128.tif" />

<u>Example D8-(13R)-13-cyano-14,15(E)-alkenyl-15-O-trimethylsilyl Spinoxine A</u>

The compound Spinozin A (5.68 g, 7.76 mmol) was dissolved in DMSO (40 ml). The solution was stirred at room temperature (water bath) under nitrogen. Add trimethylsilyl cyanide (3.0 ml). After stirring for 5 minutes, LiCN (1.1 g, excess) was added part by part. Stirring is continued for 30 minutes, and ethyl acetate (50 ml) and PhH (100 ml) are added. At 10%aq.K₂CO₃(3x) Solution extraction. Organic layer with anhydrous K₂CO₃Dry and concentrate under reduced pressure. Flash SiO₂Purification by column (220 g/EtOAc). Evaporate the pure fraction to provide compound (13R)-13-cyano-14,15(E)-alkenyl-15-O-trimethylsilyl Spinoxine A; 5.33 g, 82%)¹H-NMR(CDCl₃，300

<img file="TW487559B_D0129.tif" />

<u>Example D9-(13R,14R)-13-cyano-17-deoxy-13,14-dihydro-16,17(E)-alkenylspinoxine A X509471 and (13R,14R)-13 -Cyano-13,14-Dihydro Spinol A X509488</u>

The compound (13R)-13-cyano-14,15(E)-alkenyl-15-O-dimethylsilyl Spinoxine H (557 mg, 0.67 mmol) was dissolved in CH₂Cl₂(30ml). Add water (50 ml) and KHF₂(5.0 g), followed by tetrabutylammonium chloride (1.36 g). The mixture was stirred at room temperature under nitrogen for 1 hour. Add dichloromethane (70 ml) and water (50 ml). Separate the phases and wash the organic layer with water (2x), with K₂CO₃Dry and concentrate under reduced pressure. The residue is flashing SiO₂Column (230-400 m, 100 g/EtOAc) was separated to obtain compound a)(13R,14R)-13-cyano-17-deoxy-13,14-dihydro-16,17(E)- Ene

<img file="TW487559B_D0130.tif" />

<u>Example D10-(13R)-13-cyano-14,15(E)-alkenyl-2'-O-difluoromethanesulfonyl-15-O-trimethylsilyl-spinoxine H X507995</u>

The compound 2'-O-Trifluoromethanesulfonyl Spinozin H (663 mg, 0.78 mmol) was dissolved in dry DMSO (7 ml). The solution was stirred at room temperature under nitrogen and trimethylsilane cyanide (1.0 ml, excess) was added. At this time, it is necessary to cool the reaction mixture to room temperature. After stirring for 5 minutes, a portion of anhydrous lithium cyanide powder (310 mg, 9.4 mmol) was added. Continue to vigorously stir for another 30 minutes. Add ethyl acetate (50 ml) and PhH (100 ml). Continuously extract the solution with brine, with brine and 5%aq.NaHCO₃(2X) Dilution. Flash SiO₂The residue was purified by column (100 g/EtOAc) to obtain compound (13R)-13-cyano-14,15(E)-alkenyl-2'-O-difluoromethanesulfonyl-15-O- Trimethylsilyl-spinoxine H, 507 mg, 69% IR v 2238,

<img file="TW487559B_D0131.tif" />

<u>Example D11(13R,14R)-13,14-dihydro-13-(hydroxy)amino Spinozin A</u>

The compound Spinozin A (1.0 g, 1.3 mmol) was dissolved in 10 ml methanol and hydroxylammonium hydrochloride (0.23 g, 3.3 mmol) was added. Potassium hydroxide (0.15 g, 2.6 mmol) was added to the solution and the reaction was stirred at room temperature. Within a few minutes, the colorless solution then turned into a turbid white precipitate. The reaction was stirred at room temperature overnight. After stirring for 18 hours at room temperature, the white suspension was poured into a separatory funnel containing 30 ml of saturated sodium bicarbonate solution and 30 ml of ether. The layers were separated and the aqueous layer was extracted with 2x50 ml ether. The ether extracts were combined, washed with 30 ml brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The obtained compound is (13R,14R)-13,14-dihydro-13-(hydroxy)amino Spinozin A; 0.92 g, 88%, is a white solid. C₄₁H₆₈N₂O₁₁Analysis:

<img file="TW487559B_D0132.tif" />

<u>Example D12-(13R,14R)-13,14-dihydro-13-(N-methyl-N-hydroxy)amino Spinozin A</u>

The compound Spinozin A (1.01 g, 1.36 mmol) was dissolved in 5 ml methanol and N-methylhydroxyammonium hydrochloride (0.23 g, 3.3 mmol) was added. Potassium hydroxide (148 mg, 2.63 mmol) was added to the solution and the reaction was stirred at room temperature. After 1 minute, the reaction became cloudy. The reaction was stirred at room temperature for 18 hours, and the ingredients were poured into a separatory funnel containing 30 ml of saturated sodium bicarbonate solution and 30 ml of dichloromethane. The layers were separated and the dichloromethane layer was extracted with 2x50 ml dichloromethane. The dichloromethane extracts were combined, washed with 30 ml brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The obtained compound is (13R,14R)-13,14-dihydro-13-(N-methyl-N-hydroxy)amino Spinozin A; 0.89 g, 83%, white solid. C₄₂H₇₀N₂O₁₁The analysis: estimated value: C 64.76, H 9.06, N 3.60; actual value: C 64.88, H 9.03, N 3.78: MS m/z(FD)779(M+1).

<u>Example D13-(13R,14R)-13,14-dihydro-13-(hydroxy)amino Spinozin A 17-Psa</u>

Following the steps described in Example D11, Spinozin A 17-Psa (207 mg, 0.35 mmol), hydroxylammonium hydrochloride (96.0 mg, 1.38 mmol), potassium hydroxide (74.6 mg, 1.32 mmol), and 5 ml methanol. The resulting compound (13R,14R)-13,14-dihydro-13-(hydroxy)amino Spinozin A 17-

<img file="TW487559B_D0133.tif" />

<u>Example D14-(13R,14R)-13,14-dihydro-13-(N-ylidene-N-oxo)aminospinoxine A</u>

The compound (13R, 14R)-13,14-dihydro-13-(hydroxy)amino Spinozin A (139 mg, 0.181 mmol) was dissolved in 5 ml toluene and benzaldehyde (44 mg, 0.41 mmol) was added. The reaction was heated to reflux temperature for 1 hour, cooled to room temperature, and concentrated under reduced pressure. By using SiO under moderate pressure₂String (CH₂Cl₂: CH₃The residue was purified by OH, 97: 3 (V/V)) to obtain compound (13R, 14R)-13,14-dihydro-13-(N-ylidene-N-oxo)aminospine

<img file="TW487559B_D0134.tif" />

<u>Example D15-(13R,14R)-13,14-dihydro-13-(N-methyl-N-hydroxy)amino Spinozin D</u>

The compound Spinozin D (0.53 g, 0.71 mmol) was suspended in 10 ml of methanol and N-methylhydroxylamine (103 mg, 1.24 mmol) was added. To this suspension was added potassium hydroxide (103 mg, 1.83 mmol) and stirred at room temperature. After stirring for 1 minute, the reaction became more turbid from visible. The reaction was stirred at room temperature for 48 hours. The reactant was poured into a separatory funnel containing 25 ml of dichloromethane and 30 ml of saturated sodium bicarbonate, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. By HPLC (Cl8, CH₃OH: H₂O, 95: 5 (V/V)) The residue was purified. The obtained compound (13R, 14R)-13,14-dihydro-13-(N-methyl-N-hydroxy)aminospinoxine D; 180 mg, 32%. Note: During chromatographic purification, the compound (13R,14R)-13,14-dihydro-13-(N-methyl-N-hydroxy)amino Spinozin D will be partially decomposed into the compound Spinozin

<img file="TW487559B_D0135.tif" />

<u>Example D16-(14S)-13,14-dihydro-spinoxine A 17-Psa</u>

Suspend compound (14S)-13,14-dihydrospinoxine A (380 mg, 0.518 mmol) in 10 ml water and add 2 ml 1N H₂SO₄. No change can be observed when the acid is added to the solution. The reactants are heated to 95-100°C, during which the solids dissolve to obtain a colorless solution. After heating for 1 hour, a white precipitate formed and the reaction was cooled to room temperature. The solid was collected by filtration under reduced pressure, washed with 3x10 ml of cold water and air-dried. The resulting compound (14S)-13,14

<img file="TW487559B_D0136.tif" />

<u>Example D17-(14R)-13,14-dihydro-spinoxine A 17-Psa</u>

Suspend compound (14R)-13,14-dihydrospinoxine A (397 mg, 0.541 mmol) in 10 ml water and add 2 ml 1N H₂SO₄. No change can be observed when the acid is added to the solution. The reactants are heated to 95-100°C, during which the solids dissolve to obtain a colorless solution. After heating for 20 minutes, a white precipitate began to form, which reconstituted in 1 hour. The reaction was cooled to room temperature and formed a glassy solid. The glassy solid was extracted with 2x25 ml diethyl ether, washed with 20 ml brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting compound (14R)-13,14-dihydro-spinoxine A 17-Psa,

<img file="TW487559B_D0137.tif" />

<u>Example D18-(13R,14S)-13,14-dihydro-13-methylspinoxine A</u>

Cuprous iodide (1.34 g, 7.0 mmol) was suspended in 20 ml of anhydrous ether under nitrogen. The reaction was cooled to 0°C in an ice bath and 1.4 M methyl lithium in ether (9.0 ml, 12.6 mmol) solution was slowly added within 3 minutes. A 5 ml ether solution of the compound Spinozin A (2.04 g, 2.80 mmol) was slowly added to the reaction within 10 minutes. A bright yellow solution is available, which will gradually lose its color and get a precipitate. After 30 minutes, the reaction was poured into a separatory funnel containing 25 ml of saturated sodium bicarbonate solution and 25 ml of ether. The layers were separated and the aqueous layer was extracted with 2x50 ml ether. The ether was filtered to remove suspended solids and washed with 25 ml brine, dried over magnesium sulfate and concentrated under reduced pressure. The obtained residue was subjected to HPLC (Cl8, CH₃OH: H₂O, 95: 5, V/V) to obtain the compound (13R, 14S)-13,14-dihydro-13-methylspinnol

<img file="TW487559B_D0138.tif" />

<u>Example D19-(13R,14S)-13,14-dihydro-13-n-butylspinoxine A</u>

Cuprous iodide (1.0 g, 5.2 mmol) was suspended in 20 ml of anhydrous ether under nitrogen. The reaction was cooled to 0°C in an ice bath and a 1.4 M solution of n-butyllithium in hexane (7.0 ml, 11 mmol) was slowly added within 5 minutes. Stir at 0°C for 30 minutes to get a dark brown solution. A 5 ml ether solution of the compound Spinozin A (2.13 g, 2.9 mmol) was slowly added to the reaction within 5 minutes. After stirring for 15 minutes, the reaction was poured into a separatory funnel containing 50 ml of 50% concentrated ammonium hydroxide aqueous solution and extracted with 3x30 ml of ether. Combine the ether extracts and wash with 50 ml concentrated ammonium hydroxide and 30 ml saturated aqueous sodium bicarbonate solution. The ether solution was dried with magnesium sulfate and concentrated under reduced pressure. The resulting compound: (13R,14S)-13,14-dihydro-13-n-butylspine

<img file="TW487559B_D0139.tif" />

<u>Example D20-(14S,15S)-13,14-dihydro-15-hydroxyspinoxine A</u>

The compound Spinozin A (600 mg, 0.820 mmol) was dissolved in dry ether (100 ml). Add lithium tri-t-butoxide aluminum hydride (296 mg, 97%, 1.13 mmol). After stirring for 5 minutes under nitrogen, the reaction mixture was cooled to 78°C and a portion of LiAlH was added₄(29 mg, 0.763 mmol). Stirring was continued for 25 minutes at -78°C/nitrogen. Then the cooling bath was changed to 0°C and stirring was continued for 2 hours. Then slowly introduce brine (10 ml) and add ether (70 ml). After separating the extract phase, the organic layer was washed with brine (3x) and washed with K₂CO₃Dry, filter and concentrate under reduced pressure. Flash SiO₂Purification by column (20 g/ethyl acetate) and then separation with chromatotron (1.0 mm, 10% EtOH/EtOAc). These steps provide the compound (14S,15S)-13,14-dihydro-15-hydroxyspinoxine A, 78 mg, 13%:¹H-NMR

<img file="TW487559B_D0140.tif" />

<u>Example D21-(14S,15S,17S,21S)-1,21-deoxy-13,14-dihydro-1,21-off-1,15,21-trihydroxyspinoxine A and [ (15S)-Hydroxy-Spinoxine A</u>

Dissolve Spinozin A (446 mg, 0.610 mmol) in dry Et₂O (60ml). At room temperature under nitrogen, lithium tri-t-butoxyaluminum hydride (148 mg, 0.583 mmol) was added with stirring. After 10 minutes, add LiAlH₄(19 mg, 0.50 mmol) and at RT/N₂Continue stirring for 50 minutes. The mixture was cooled to 0°C and brine was added dropwise to quench the remaining hydride. Join Et₂O (70 ml), after separating the extract layer, wash the organic layer with brine (3x), and wash the organic layer with K₂CO₃Dry, filter and concentrate under reduced pressure. In flash SiO₂The residue was separated on a column (25 g/5% EtOH in EtOAc) to obtain compound a) (14S, 15S, 17S, 21S)-1,21-deoxy-13,14-dihydro-1,21 -Broken-1,15,21-Trihydroxy Spinozin A, 71 mg, 15%:¹H-NMR(CDCl₃，

<img file="TW487559B_D0141.tif" />

xCH₃, s) ppm, and a mixture of compound b) product (313 mg), which was further separated (1.0mm, 15% EtOH/EtOAc) on a Chromatotron rotating disk. These separations can provide compound b)[(15S)-hydroxyspinnolometer

<img file="TW487559B_D0142.tif" />

<u>Example D22-(14R)-13,14-dihydro-15-hydroxyspinoxine A</u>

Spinozin A (2.17 g, 2.96 mmol) was dissolved in ethanol (75 ml) and sodium borohydride (1.1 g, 29 mmol) was added carefully within 5 minutes. The reaction mixture was stirred for 3 hours and carefully quenched with 1 N HCl (15 ml). After the gradual reaction was completed, the crude material (1.68 g) was chromatographed on silica gel (dichloromethane/methanol, 95:5) to provide (14R)-13,14-dihydrospinoxine A (635 mg, 29.2%) and (14R)-13,14-dihydro-15-hydroxyspinoxine A (317 mg, 14.6%), are white solids. Compound (14R)-13,14-dihydro-15-hydroxyspinoxine A:¹³C-NMR d 174.75, 131.52,

<img file="TW487559B_D0143.tif" />

<u>Example D23-(13R,14S,15R/S)-15-hydroxy-13,14-epoxy Spinozin A</u>

The compound (13R,14S)-13,14-epoxy Spinozin A (0.53 g, 0.71 mmol) was dissolved in ethanol (10 ml) and sodium borohydride (36 mg, 0.96 mmol) was added and the reactant was stirred 3. Hour. Additional sodium borohydride (100 mg, 2.64 mmol) was added and the reaction was stirred for 72 hours. The reaction was quenched with acetone (5 ml), then 1N HCl (1 ml) was added and the reaction was gradually completed to obtain a residue, which was subjected to reverse phase HPLC (methanol/0.1% aq. ammonium hydroxide, 90: 10) Purification. The resulting compound (13R,145,15S)-15-hydroxy-13,14-epoxy Spinozin A (78.9 mg, 14.8%): part¹H-NMR

<img file="TW487559B_D0144.tif" />

<u>Example D24-(14S)-13,14-dihydrospinoxine D</u>

The compound Spinozin D (1.00 g, 1.34 mmol) was dissolved in anhydrous tetrahydrofuran (25 ml) under nitrogen, and lithium tri-t-butoxyaluminum hydride (1.01 g, 3.97 mmol) was added and the reaction was stirred. Objects for 24 hours. The reaction was quenched with ethyl acetate (40 ml) and brine (10 ml) and the phases were separated. The aqueous layer was extracted with ethyl acetate (25 ml) and the layers were separated after filtering through filter paper. The ethyl acetate extracts were combined and washed with brine (25 ml), dried over anhydrous potassium carbonate and concentrated under reduced pressure. The residue (360 mg) was purified by reverse phase HPLC (methanol/0.1%aq. ammonium hydroxide, 95:5) to obtain (14S)-13,14-dihydrospinoxine D (189 mg, 18.9%) ), as a white solid: MS m/z 748.7C₄₂H₆₉NO₁₀Analysis: Estimated value: C, 67.44; H, 9.30; N, 1.87. Actual value: C, 67.21; H, 9.20; H, 1.69.

<u>Example D25-1,21-Deoxy-1,21-Cross-1,15,21-Trihydroxyspin A, (15R/S)-15-hydroxyspinoxine A, (14R,16, 17Z)-17-deoxy-16,17-dehydro-13,14-dihydrospinoxine A17-Psa, and (14R)-13,14-dihydrospinoxine A</u>

The compound Spinozin A (5.12 g, 7.00 mmol) was dissolved in anhydrous ether (75 ml) and cooled to 0°C. Methyl ether (15.0 ml, 7.5 mmol) was added to the ethylene glycol solution of lithium aluminum hydride (0.5 M) and quenched immediately by the addition of water (0.30 ml), followed by the addition of 15% aqueous sodium hydroxide and water (1.0 ml) ). The suspended solids were filtered off and the filtrate was rotary evaporated under reduced pressure to provide a white solid (4.62 g). Reverse phase HPLC (methanol/0.1% aq. sodium hydroxide, 90:10) was used to provide the compound 1,21-deoxy-1,21-broken-1,15,21-trihydroxy<img file="TW487559B_D0145.tif" /><img file="TW487559B_D0146.tif" />

<u>Example D26-(15R,14S)-15-deoxy-15-hydroxy-5,6,13,14-tetrahydrospinoxine A and compound (1R/S,14S,15R,21S)-15 -Deoxy-1,15-oxa-1,2-clo-5,6,13,14-tetrahydrospinoxine A 1-hemiacetal</u>

Dissolve (14S)-5,6,13,14-tetrahydrospinoxine A (2.60 g, 3.53 mmol) in dry Et₂O (90 ml). Cool the solution to 0°C under nitrogen and add LiAlH₄(95% powder, 268 mg, 6.8 mmol). The mixture was stirred at 0°C for 10 minutes and then carefully saturated with NH₄10% aqueous ammonium hydroxide solution (10 ml) of Cl was quenched. Generally, the reaction is gradually completed and chromatographed on silica gel (ethyl acetate, then 15% ethanol in ethyl acetate) to provide a)(15R,14S)-15-deoxy-15-hydroxy-5,6,13, 14-tetrahydrospinoxine A (1.261 g,

<img file="TW487559B_D0147.tif" />

<u>Example D27-(14S)-15-Deoxy-15,16(E)-alkenyl-5,6,13,14-tetrahydrospinoxine A, (14S,15R/S)-15- Fluoro-5,6,13,14-tetrahydrospinoxine A, and (14S)-15-deoxy-15,16(E)-alkenyl-5,6,13,14-tetrahydro Spinozing A</u>

(15R,14S)-15-Deoxy-15-hydroxy-5,6,13,,14-tetrahydrospinoxine A (0.484 g, 0.65 mmol) was dissolved in dichloromethane (4 ml). The solution was cooled to 0°C under nitrogen and mooxansulfur trifluoride (0.50 ml, 4.1 mmol) was added slowly. Remove the cooling bath and continue stirring at room temperature for 4 hours. Generally, the reaction is gradually completed and chromatographed on silica gel (ethyl acetate) to provide a product mixture, which is further subjected to HPLC (90:10, MeOH/H₂O) Separation. Obtained: a) (14S)-15-deoxy-15,16(E)-alkenyl-5,6,13,14-tetrahydrospinoxine A (48 mg, 10%), white solid:¹H-NMR

<img file="TW487559B_D0148.tif" />

<u>Example D28-13, 14-a-methanol-spinoxine A</u>

Sodium hydride (60% distributed in oil, 52 mg, 1.3 mmol) was weighed into the flask and placed in a nitrogen atmosphere. The suspension is mainly stirred with pentane (2 ml) and the pentane is removed by a pipette. Residual pentane was removed by evaporation under a stream of nitrogen. Suspend the dried NaH in DMSO (4 ml), cool the resulting slurry to 15° C. and add iodinated dimethyl oxide (286 mg, 1.3 mmol) in a single portion (attention-vigorous gas generation initially). The reaction mixture was stirred at this temperature for 40 minutes while the mixture was a clear solution. A solution of Spinozin A (827 mg, 1.13 mmol) in dry toluene (1 ml) and DMSO (1 ml) was added dropwise within 5 minutes. The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0°C, and Et₂Dilute with O and quench with saturated sodium bicarbonate solution and water. Make the mixture in Et₂Partition between O and saturated sodium bicarbonate solution. Take Et₂O extract the aqueous layer and combine the ether phases and wash with water (3 times), and dilute with sodium bicarbonate and salt. The mixture was dried with anhydrous sodium carbonate and the solvent was removed under dry nitrogen to give a white solid (812 mg). The solid was chromatographed on silica gel (81 g) using EtOAc followed by 2% MeOH in EtOAc as the eluent to give a white solid (785 mg, 93%)¹H-NMR δ loss 6.85 (br s, 1H); MS m/z 746.5 (estimated value of M+H, 746.5).

<u>Example D29-13,14-epoxy-15-hydroxyspinoxine A</u>

Compound 13,14-epoxy Spinozin A (250 mg, 0.3 mmol) dissolved in Et₂O (20 ml). The mixture was stirred vigorously under nitrogen at room temperature and lithium tri-t-butoxyaluminum hydride (153 mg, 0.6 mmol) was added. Stirring is continued for 18 hours. The reaction mixture was quenched with brine and extracted with ether (3×20 ml). Combine the ether extracts with anhydrous Na₂SO₄Dry and concentrate under reduced pressure. The residue was purified on preparative TLC (EtOAc) to provide compound 13,14-epoxy-15-hydroxyspinoxine A (140 mg, 56%) as a white solid.

<img file="TW487559B_D0149.tif" />

<u>Example D30-13,14-dihydro-2-phenylspinoxine A</u>

Under nitrogen, add n-butyllithium (4.8 ml, 7.7 mmol) and HMPA (1.0 ml) to a solution of diisopropanol (1.01 ml, 7.7 mmol) in THF (20 ml) at -30°C and cool to- 40°C. A solution of Spinozin A (1.0 g, 1.4 mmol) in THF (10 ml) was added to the reaction mixture over a period of 10 minutes and stirred for 1 hour. The reaction mixture was quenched with ammonium chloride solution (10 ml) and extracted with ether (3x40 ml). Combine the ether extracts and add anhydrous Na₂SO₄Dry and concentrate under reduced pressure. The residue was chromatographed on a preparative HPLC Cl8 column, with 10% water (0.1% NH₄OH) was diluted with methanol solution to obtain compound 13,14-dihydro-2-phenylselenium Spinozin A (0.24 g, 19%),

<img file="TW487559B_D0150.tif" />

<u>Example D31-3,14-Dihydrospinoxine A</u>

The compound (2Z)-2,3-dihydrospinoxine A (20 mg, 0.03 mmol) was dissolved in Et₂O (5 mL). The mixture was stirred vigorously under nitrogen at room temperature and lithium tri-t-butoxyaluminum hydride (7.6 mg, 0.03 mmol) was added. After stirring for 18 hours, lithium tri-t-butoxyaluminum hydride (7.6 mg, 0.03 mmol) was added. Continue stirring for 1 hour. The reaction mixture was diluted with brine and extracted with ether (3x15 ml). Combine the ether extracts with anhydrous Na₂SO₄Dry and concentrate under reduced pressure. The residue was purified on a C-18 column of preparative HPLC with 10% water (containing 0.1% NH₄OH) was eluted in methanol solution to obtain compound 3,14-dihydrospinoxine A (11 mg, 50%), which was a white off-flaky solid.¹H-

<img file="TW487559B_D0151.tif" />

<u>Part E Modification of the pseudoglycan at the C-17 position of the tricyclic part of formula I</u>

<u>Example E1-17-Deoxy-17-amino Spinozin A 17-Psa</u>

To 17-keto Spinozin A 17-Psa (318.7 mg, 0.54 mmol) in methanol (6 ml) was added ammonium acetate (417.1 mg, 5.4 mmol) and then sodium cyanoborohydride (45 mg, 0.76 mmol). The reaction mixture was stirred at room temperature for 5 days. The solvent was evaporated under reduced pressure at room temperature. The residue was dissolved in 1N HCl and washed with ether. The aqueous layer was then basified with 5N NaOH and saturated with sodium chloride. The aqueous layer was then extracted with fresh ether. Wash the ether part with salt water, use K₂CO₃Dry and evaporate at room temperature under reduced pressure. The resulting 17-deoxy-17-amino Spinozin A 17-Psa (68.2 mg; yield 21%) is a colorless glass, a 7:1 mixture of isomers, FDMS, m/e( Relative strength)744(5),591(20),590(MH⁺，40)，112(100)。

<u>Example E2-(16,17Z)-17-Dehydro-16,17-Dehydrospinoxine A 17-Psa</u>

To a suspension of N-methylspinoxine A iodide (203.7 mg, 0.23 mmol) in THF (9 ml), sodium hydride (50% suspension in mineral oil, 42.9 mg, 0.89 mmol) was added. The reaction mixture was heated to reflux temperature for 1 hour, and then extracted with ether. Wash the ether part with salt water, use K₂CO₃Dry and concentrate at room temperature under reduced pressure. The crude product was chromatographed on silica, eluted with hexane and then with a 40% ethyl acetate in hexane solution in a one-step gradient. The obtained (16,17Z)-17-deoxy-16,17-dehydrospinoxine A 17-Psa (89.5 mg, yield 67%) was a white glass, FDMS, m/e( Relative strength) 1144 (20), 573 (M⁺，60)，101(100)。

<u>Example E3-(16,17E)-17-deoxy-16,17-dehydrospinoxine A 17-Psa, (17,18E)-17-deoxy-17,18-deoxy Penoxine 17-Psa and (16,17Z)-17-deoxy-16,17-dehydrospinoxine A 17-Psa</u>

The reaction was carried out as described in Example E2, using Spinozin A (5.1 gm, 6.97 mmol) as the starting material. By preparative HPLC in C₁₈Separate the product on the column with ethyl acetate: methanol: 0.1% NH₄OAc (20:40:40) was eluted. The obtained (16,17E)-17-deoxy-16,17-dehydrospinoxine A 17-Psa (119 mg; yield 3%), FDMS, m/e (relative intensity) 572 (M⁺, 100), (17,18E)-17-deoxy-17,18-dehydrospinoxine 17-Psa (634mg; yield 16%), FDMS, m/e (relative intensity) 572 (M⁺, 100), and (16,17Z)-17-deoxy-16,17-dehydrospinoxine A 17-Psa (689 mg; yield 17%) are all white solids.

<u>Example E4-(5R,6R)-5-(2-hydroxyethoxy)-6-bromospinosine A 17-Psa</u>

To a suspension of Spinozin A (154.1 mg, 0.21 mmol) in ethylene glycol (5 ml), N-bromosuccinimide (79.8 mg, 0.45 mmol) was added. The reaction mixture was heated to 80°C for 1.5 hours, and then cooled to room temperature. The mixture was diluted with dichloromethane and washed with water. Then wash the dichloromethane part with brine, and use K₂CO₃Dry and evaporate at room temperature. The product was separated by chromatography on silica and eluted with 70% ethyl acetate in hexane and then 5% methanol in dichloromethane. The obtained compound a) Spinozin A17-Psa (41.7 mg; yield 34%) was a colorless glass. (5R,6R)-5-(2-hydroxyethoxy)-6-bromospinoxine A 17-Psa was further purified by chromatography on silica, dissolved in 70% ethyl acetate in hexane Analysis. The obtained compound b) (5R, 6R)-5-(2-hydroxyethoxy)-6-bromospinoxine A17-Psa (50.2 mg, yield 33%), as a colorless glass, FDMS, m /e (relative intensity) 734 (~5), 732 (MH⁺，60)，189(100)。

<u>Example E5-(5R,6R)-5,6-epoxy-17-O-dimethylsilyl Spinoxine A 17-Psa</u>

To (5S,6R)-5,6-epoxy Spinozin A 17-Psa (503.7 mg, 0.83 mmol) in ether (20 ml), add chlorotrimethylsilane (211 ml, 1.66 mmol) And diisopropylethylamine (289 ml, 1.66 mmol). The reaction mixture was stirred at room temperature for 23 hours, then additional chlorotrimethylsilane (105 ml, 0.83 mmol) and diisopropylethylamine (144 ml, 0.83 mmol) were added. The reaction mixture was stirred for another 18 hours at room temperature. Decant the ether and triturate the remaining precipitate with fresh ether. Combine the ether parts, wash with saturated NaHCO3 aqueous solution, and wash with K₂CO₃Dry and evaporate at room temperature under reduced pressure. The crude product was purified by chromatography on silica and eluted with 50% ethyl acetate in hexane. The obtained (5R, 6R)-5,6-epoxy-17-O-dimethylsilyl Spinozin A 17-Psa (521.1 mg; yield 92%) was a colorless glass, FDMS, m /e (relative intensity) 679 (75), 678 (M⁺，100)，190(30)，100(35)。

<u>Example E6-(5R, 6R)-5,6-epoxy Spinozin A 17-Psa</u>

To a solution of Spinozin A 17-Psa (1.0 gm, 1.71 mmol) in dichloromethane (45 ml), m-chloroperoxybenzoic acid (591.7 mg, 3.34 mmol) was added. The reaction mixture was stirred at room temperature for 23 hours. Dilute the mixture with dichloromethane and add saturated NaHCO₃Clean with K₂CO₃Dry and evaporate at room temperature under reduced pressure. The crude product was purified by chromatography on silica and eluted with 15% acetone in dichloromethane. The obtained (5R, 6R)-5,6-epoxy Spinozin A 17-Psa (1.04 gm; yield is about 100%) is a colorless glass, FDMS, m/e (relative strength) 607 ( M⁺，70)，190(100)，101(99)。

<u>Example E7-17-keto-spinoxine A 17-Psa</u>

To a suspension of N-chlorobutadiamide (108.6 mg, 0.81 mmol) in dichloromethane (2.6 ml) at -78°C, isopropyl sulfide (125 ml, 0.86 mmol) was added. The reaction mixture was stirred at -78°C for 30 minutes, and then Penspinoxine A 17-Psa (150.1 mg, 0.25 mmol) in dichloromethane (1 ml) was slowly added. The reaction mixture was stirred at -78°C for 3 hours, then triethylamine (109 ml, 0.78 mmol) was added and when the color turned red, the reaction mixture was heated to room temperature. Then the mixture was diluted with dichloromethane, washed with 0.1N HCl, brine, and washed with MgSO₄Dry and evaporate at room temperature under reduced pressure. The crude product was purified by chromatography on silica and eluted with 40% ethyl acetate in hexane. The obtained 17-keto-spinoxine A 17-Psa (84.7 mg; yield 58%) is a colorless glass, FDMS, m/e (relative strength) 588 (M⁺，100)。

<u>Example E8-17-Deoxy-16,17-Dehydrospinoxine A Ag</u>

The reaction was carried out as described in Example 3, using Spinozin J (15.02 gm, 21 mmol) as the starting material. Will K₂CO₃After treatment with the methanol solution, the reaction mixture was filtered, and then evaporated at room temperature under reduced pressure. The residue was kept at room temperature for 24 hours without further purification, and then triturated with dichloromethane. Then dichloromethane was evaporated under reduced pressure at room temperature. The crude product was purified by chromatography on silica and eluted with 5% methanol in dichloromethane. The resulting crude 17-deoxy-16,17-dehydrospinoxine A Ag is a yellow semi-solid and Spinoxine A 9-Psa (1.02 gm; yield 9%) is a colorless glass . The crude 17-deoxy-16,17-dehydrospinoxine A Ag was further purified by preparative HPLC on a Cl8 column, with ethyl acetate: methanol: 0.1% NH₄OAc (35:35:30) eluted. The obtained pure 17-deoxy-16,17-dehydrospinnosine A Ag (1.81 gm; 224 yield) is a white glass, FDMS, m/e (relative strength) 460(10), 385( M⁺，55)，384(100)。

<u>Example E9-17-Deoxy-17-cyano-13,14-dihydro-13-cyanospinoxine A 17-Psa</u>

The compound Spinozin A (97 mg, 0.13 mmol) was dissolved in 5 mL methanol and potassium cyanide (59.8 mg, 0.92 mmol) was added and the reaction was stirred overnight. After the gradual reaction was completed, the crude product (0.05 g) was chromatographed on silica gel (ethyl acetate/hexane 20:80 to 50:50 gradient) to provide compound 17-deoxy-17-cyano- 13,14-Dihydro-13-cyanospinoxine A 17-

<img file="TW487559B_D0152.tif" />

<u>Example E10-17-Deoxy-3'-O-[2'''-(dimethylamino)ethyl]-16,17-(Z)-alkenylspinoxine J 17-Psa</u>

Spinozin J (1.20 g, 1.67 mmol) was reacted with 1-chloro-2-dimethylamine ethane (its hydrochloride salt was formed in situ), followed by 3'-O,N-bis(trideuterium) The steps described in (methyl) Spinozin M are carried out. Chromatography and HPLC separation on silica gel (ethyl acetate) (88:12, MeOH/H₂O) to provide 17-deoxy-3'-O-[2'''-(dimethylamino)ethyl]-16,17-(Z)-alkenylspinoxine J 17-Psa( 109 mg, 10%), as a white solid:¹H-NMR δ 6.39 (s, 1H), 5.63 (m, 1H), 2.18 (s, 6H), 1.84 (s, 3H).

<u>Example E11-(13R,14S)-13,14-epoxy Spinozin A, compound (13R,14S)-16,17-dehydro-17-epoxy-16,17-(Z)- Alkenyl-13,14-epoxy Spinozin A 17-Psa, and the compound (13R,14S,16S,17R)-17-deoxy-13,14-cyclohydrogen-16,17-epoxy Kispinosine A 17-Psa</u>

Spinozin A (5.06 g, 6.91 mmol) was dissolved in ethanol (35 mL). Then tetrahydrofuran (15 mL) was added, followed by 10% aqueous NaOH (15 mL) and 30% aqueous hydrogen peroxide (6 mL, excess). Stir at room temperature for 4 hours. Generally, it is gradually formed by reaction and chromatographed on silica gel (ethyl acetate) to obtain: a) (13R,14S)-13,14-epoxy Spinozin A (3.18 g, 61.5%) as a white solid: ESI MSm/z 748(M+1); and b) a mixture of less polar compounds, which was subjected to flash silica gel column chromatography (7%Et₂O in dichloromethane) to obtain: c) (13R,14S)-16,17-dehydro-17-deoxy-16,17(Z)-alkenyl-13,14-epoxy Spinnol Sim A 17-

<img file="TW487559B_D0153.tif" />

<u>Example E12-(2R/S)-16,17-dihydro-17-deoxy-16,17-(E/Z)-alkenyl-2-ethylspinoxine F 17-Psa</u>

Diisopropylamine (0.658 mL, 5.0 mmol) was dissolved in dry THF (10 mL). The solution was cooled to -23°C under nitrogen and n-BuLi (2.5M hexane solution 2.0 mL, 5.0 mmol) was added dropwise. Stir at -23°C for 30 minutes and cool the mixture to -78°C. Spinozin F (1.03 g, 1.434 mmol) in dry THF (3 mL) was added dropwise to the reaction mixture. After 5 minutes, the reaction temperature rose to -23°C. After 48 minutes, iodoethane (1.25 mL, 15.6 mmol) was introduced. Using stirring, allow the temperature to rise to room temperature within 2.5 hours. Generally, the reaction is gradually formed and chromatographed on silica gel (ethyl acetate) to obtain the product (441 mg), which is chromatographed on silica gel (ethyl acetate) repeatedly. The supplied (2R/S)-16,17-dihydro-17-deoxy-16,17(E/Z)-alkenyl-2-ethylspinoxine F 17-Psa (152 mg, 18 %), is white

<img file="TW487559B_D0154.tif" />

<u>Example E13-17-Table-Spinozine A 17-Psa</u>

Add a portion of sodium borohydride (14.2 mg, 0.37 mmol) to cold (-20°C) 17-keto Spinozin A 17-psa (220 mg, 0.37 mmol) and cerium(III) chloride heptahydrate ( 140 mg, 0.37 mmol) in methanol (6 mL) solution (note: foaming and H₂Released). The mixture was stirred at -20°C for 20 minutes, and then quenched at this temperature by adding 1N HCl (0.5 mL) dropwise over 2-3 minutes. Then the mixture was diluted with ether (40 mL) and the organic layer was washed with brine (2x5 mL) and washed with MgSO₄dry. After concentration, 290 mg of solid residue remained, which was flash chromatographed on silica (40 ml) using 3% MeOH in CH₂Cl₂It is the eluent to obtain 220 mg of white powder, which is a 4:1 mixture of 17-epi-Spinozin A 17-Psa and Spinozin A 17-Psa. Separated by reverse phase HPLC with Cl8 bonded silica column (41.4 mm(id)x25 cm(1)), using 15%H₂MeOH of O is a solution. Spinozin A 17-Psa precipitates first. 17-Watch-Spinozing A 17-

<img file="TW487559B_D0155.tif" />

<u>Modification of the C-5 and C-6 positions of the tricyclic moiety of Spinozin A in Part F compound-</u>

<u>Example F1-(5R,6R)-5-(2-hydroxyethoxy)-6-bromospinoxine A</u>

To spinoxin (1.0 gml 1.37 mmol) and N-bromosuccinimide (289.5 mg, 1.63 mmol) in ethylene glycol (20 ml) suspension, add 5 N HCl (295 ul, 1.47 mmol) ). The reaction mixture turned yellow and was stirred at room temperature for 1.5 hours, during which time the color faded to clear. Pour the mixture into saturated NaHCO₃The aqueous solution was extracted with ether. Take MgSO₄The ether was dried and evaporated at room temperature under reduced pressure. Crude (5R, 6R)-5-(2-hydroxyethoxy-6-bromospinoxine A (896.8 mg, yield 75%) was obtained. The compound was purified by chromatography on silica to obtain 5 % Methanol in dichloromethane solution was eluted to obtain (5R, 6R)-5-(2-hydroxyethoxy)-6-bromospinoxine A as a white glass, FDMS, m/e( Relative strength) 873 (100), 871 (M⁺，80，).

<u>Example F2-(5S,6R)-5,6-dihydroxy-5,6-dioxospinoxine A</u>

In Spinozin A (487.8 mg, 0.67 mmol) and trimethylamine-N-oxide dihydrate (108 mg, 0.97 mmol) in an aqueous (0.5 ml) t-butanol (3 ml) suspension, Add pyridine (60 ml, 0.74 mmol) followed by iron tetroxide (0.1 M: 40 ml, 0.004 mmol). The reaction mixture was heated to reflux temperature under a nitrogen atmosphere for 19 hours. Then cool the dark mixture to room temperature and pour 20% NaHSO₃The aqueous solution was extracted with ether. Wash the ether part with brine, and MgSO₄Dry and evaporate at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 7% methanol in dichloromethane. The obtained (5S,6R)-5,6-dihydroxy-5,6-dihydrospinoxine A (282.1 mg; yield 55%) is a beige solid, FDMS, m/e (relative intensity) 766 (M+, 60), 765 (100).

<u>Example F3-(5R)-5-(2-hydroxyethoxy)-6,7-deoxyspinoxine A</u>

In (5R, 6R)-5-(2-hydroxyethoxy)-6-bromospinoxine A (299.9 mg, 0.34 mmol) in anhydrous THF (10 ml), add sodium sulfide (50% of Mineral oil dispersion; 69.3 mg, 1.44 mmol) and release gas. The reaction mixture was stirred at room temperature for 3.5 hours, and then poured into water. The water portion was extracted with ether. Wash the ether part with salt water, use K₂CO₃Dry and evaporate at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 5% methanol in dichloromethane. The obtained (5R)-5-(2-hydroxyethoxy)-6,7-dehydrospinoxine A (123.9 mg; yield 50%, based on the recovered starting material) is light Yellow glass, FDMS, m/e (relative intensity) 791.8 (M⁺，100)，419.5(30)。

<u>Example F4-(5S,6R)-spinoxine A carbonate</u>

In (5S,6R)-5,6-dihydrospinoxine A (85 mg, 0.11 mmol) and ethylene carbonate (109.2 mg, 1.2 mmol) in benzene (3 ml), add anhydrous K₂CO₃(40.2 mg, 0.29 mmol) and the mixture was heated to reflux temperature. After 2.5 hours, the mixture was cooled to room temperature and diluted with dichloromethane. Wash the dichloromethane part with water and brine, and use K₂CO₃Dry and evaporate at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 7% methanol in dichloromethane. The obtained (5S, 6R)-spinoxine A carbonate (65.1 mg; yield 75%) was a white solid, FDMS, m/e (relative intensity) 792 (M⁺，70)，791(100)。

<u>Example F 5-(5S,6S)-5-acetoxy-6-bromospinoxine A and (5R,6R)-5-acetoxy-6-bromo-5,6-dihydro Binoxin A</u>

To Spinozin A (511.7 mg, 0.7 mmol) in glacial acetic acid (5ml), add N-bromosuccinimide (152.9 mg, 0.86 mmol). The reaction was stirred at room temperature for 24 hours and slowly Pour in 5Np NaOH (35ml). The aqueous mixture was then saturated with NaCl and extracted with ether. Extract the ether part with brine and use K₂CO₃Dry and evaporate at room temperature under reduced pressure. The crude mixture (544.2 mg) is available. The mixture was purified by chromatography on silica, eluted with 7% methanol in dichloromethane, and the isomers were separated by preparative HPLC on a Cl8 column. Acetate: methanol: 0.1% NH₄OAc (41:41:18 to 42:42:16, linear gradient over 60 minutes) was eluted. The obtained (5S, 6S)-5-acetoxy-6-bromospinoxine A, FDMS, m/e (relative intensity) 874(50), 873(98), 872(48), 871(M⁺, 100) and (5R, 6R)-5-acetoxy-6-bromo-5,6-dihydrospinoxine A, FDMS, m/e (relative intensity) 874(60), 873(90 ), 872(45), 871(M⁺, 100), is a white solid.

<u>Example F6-5,6-Dibromospinoxine A 17-Psa</u>

To a solution of Spinozin A (257.2 mg, 0.35 mmol) in carbon tetrachloride (10 ml), bromine (18 ml, 0.35 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours, during which time the color of the reaction mixture faded to pale yellow. The solvent was evaporated under reduced pressure at room temperature, however, TLC (eluted with 10% methanol in dichloromethane) showed that the reaction was incomplete. The residue was redissolved in carbon tetrachloride (10 ml) and bromine (18 ul, 0.35 mmol) was added. The reaction mixture was stirred at room temperature for 3 days, during which time the color of the reaction mass faded. Then dilute with dichloromethane and wash with 5% sodium thiosulfate. Take MgSO₄The dichloromethane portion was dried and evaporated at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 3% methanol in dichloromethane. The obtained 5,6-dibromospinosine A 17-Psa (142.9 mg; yield 54%) is a white solid, FDMS, m/e (relative intensity) 750 (M⁺，10)，189(30)，101(100)。

<u>Example F7-5,6-Dihydrospinoxine A (246088)</u>

Spinozin A (504 mg; 0.69 mmol) in dry benzene (30 ml), add tris(triphenylphosphine) rubidium chloride (50.6 mg; 0.055 mmol) and place the mixture in an atmospheric generator under hydrogen. After stirring with a magnet (without monitoring the temperature) for 6 hours and then keeping it under a nitrogen atmosphere for 19 hours, a small amount of NMR showed that the reaction was about 2/3 complete. Add additional rubidium chloride (triphenylphosphine) (50 mg; 0.055 mmol) and replace the mixture to a hydrogen atmosphere. It was then magnetized for 24 hours and evaporated at room temperature under reduced pressure. The residue was initially subjected to flash chromatography on silica using 5% MeOH in dichloromethane. The resulting product (489.1 mg) was a yellow glass 9-like substance. The product was further purified by delta-preparative reverse phase HPLC with CH₃CN[38%]: MeOH[38%]: 0.05%NM₄OAC[24%] to CH₃CN[45%]: MeOH[45%]: 0.05%NH₄Gradient dissolution of OAc[10%]. The obtained 5,6-dihydrospinoxine A (246088) (275.4 mg; yield 54%) was a pale yellowish white glass. part

<img file="TW487559B_D0156.tif" />

<u>Example F8-(5S,6R)-5,6-Epoxy Spinozin A and (5R,6S)-Epoxy Spinozin A</u>

Under nitrogen, (5S,6R) and (5R,6S)-epoxy Spinozin A,N-oxide (2.6 gms; 3.4 mmol) in 300 ml chloroform solution 6:1 mixture solution, add three Phenylborane (823 mg, 3.4 mmol) and stirred at room temperature for 6 hours. The reaction was diluted with dichloromethane and washed with 1N NaOH. Take K₂CO₃The dichloromethane portion was dried, and evaporated under reduced pressure to obtain a white off-glass (2.57 gms). The crude material was purified by delta preparative reverse phase HPLC with MeOH[42%]: CH3CN[42%]: 0.25% NH₄OAc[16%] dissolve for 22 minutes and then use MeOH[45%]: CH₃CN[45%]: 0.25%NH₄OAc[10%] eluted for 14 minutes. The obtained (5S,6R)-5,6-epoxy Spinozin A (0.26 gms, 11%), part of¹H-NMR(CDCl₃)d<img file="TW487559B_D0157.tif" />All are white solids.

<u>Example F9-(5S,6R)-Epoxy Spinozin D and (5R,6S)-Epoxy Spinozin D</u>

The reaction was carried out as described in Example F9, using a 6:1 mixture of (5S,6R) and (5R,6S)-epoxyspinoxine D,N-oxide (450 mg, 0.58 mmol) as the starting material . The resulting (5S, 6R)-epoxy Spinozin D (16.4<img file="TW487559B_D0158.tif" />body. The low yield is due to the loss on the reverse phase HPLC column.

<u>Example F10-(5S,6R) and (5R,6S)-epoxy Spinozin A, N-oxide mixture</u>

To Spinozin A (212.2 mg, 0.238 mmol; 82.1% purity) in 10 ml dichloromethane solution, m-chloroperoxybenzoic acid (135 mg, 0.771 mmol) was added and stirred at room temperature for 3 hours. In saturated NaHCO₃Partition between and dichloromethane. The phases were separated and the aqueous layer was extracted with fresh dichloromethane. Combine the organic layers with MgSO₄Dry and evaporate under reduced pressure to give a white solid (246.8 mg). Chromatography on silica to purify the crude product, using 10% MeOH in dichloromethane to 20% MeOH in dichloromethane in one step gradient elution. The obtained 6:1 mixture of (5S, 6R) and (5R, 6S)-epoxy Spinozin A, N-oxide is a white solid (181.2 mg; about 100%); part

<img file="TW487559B_D0159.tif" />

<u>Example F11-(5S,6R) and (5R,6S)-epoxy spinoxin D,N-oxide</u>

The reaction was carried out as described in Example F10, with Spinozin D (517.9 mg, 0.70 mmol) as the starting material. The obtained 6:1 mixture of (5S, 6R) and (5R, 6S)-epoxy Spinozin D, N-oxide (450.4 mg; yield 83%) was a yellow glass. part¹H-NMR(CDCl₃) δ 6.71 and 6.60 (1H, s), 1.40 (3H, s).

<u>Example F12-(5R,6R)-5,6-Dibromo-4"-keto Spinozin A and (5R,6R)-5,6-Dibromo Spinozin B</u>

The compound Spinozin A (1.98 g, 2.70 mmol) is dissolved in CH₂Cl₃(90 ml). Triethylamine (10 ml) was added and the reaction mixture was cooled to 0°C. Bromine (5.0 g, 27.8 mmol) was added with vigorous stirring. After 15 minutes, the temperature was raised to room temperature and stirring was continued for another 30 minutes. Take CH₂Cl₂(100ml) Dilute the mixture and continuously add water (2x) and 10% NaHSO₃The aqueous solution is serially diluted. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. The residue is flashing SiO₂Column (230-400 m, 120 g/EtOAC, followed by EtOH) to obtain: a) compound (5R, 6R)-5,6-dibromo-4"-ketospinoxine A (342

<img file="TW487559B_D0160.tif" />

<u>Example F13-5-Chlorospinosine A and 6-chlorospinnosine A</u>

Dissolve the compound Spinozin A (2.62 g, 3.58 mmol) in dry CH₂Cl₂(30 ml). The solution was cooled to 0°C under nitrogen. A portion of phenylselenylene chloride (Aldrich, 98%; 0.909 g, 4.65 mmol) was added with vigorous stirring. After 7 minutes, a portion of m-CPBA (Aldrich, 50%; 4.46 g, 12.9 mmol) was introduced. Join CH₂Cl₂(50 ml) and keep stirring at 0°C for 15 min. After that, join Et₃N (5.0 ml). After stirring for another 10 minutes, further add CH₂Cl₂(100 ml) The reaction mixture was diluted. Take 10%NaHSO₃(2x) Washing solution. Continuous water (1x), 5% NaHCO₃(3x), 5%K₂CO₃(1x) and water (1x) to clean the organic phase, then use anhydrous K₂CO₃Dry, filter and concentrate under reduced pressure. Flash SiO₂The residue was purified by a column (230-400 m, 120 g; EtOAC is the eluent). The reduced pressure concentrate of the homogeneous elution fraction was subjected to TLC to provide a white solid (2.23 g). Then this material was subjected to preparative reverse phase HPLC [two Millipore RCM40 mm cartridges, Cl8-coated 6m silica; eluent: 10% water (containing 0.05v/v% NH₄OH) in MeOH] Purification. The collected eluted fractions can obtain 5-chlorospinoxine A, which is a white glassy solid (1.188 g, 43%).¹H-NMR and H, H-COSY spectrum analysis showed that this substance was a mixture containing about 15% 6-chlorospinoxine A6. Use Rainin reverse phase column (with spherical 5m, 10OA, C-18 coating filling) and 15% water (containing 0.05 v/v% NH₄The methanol solution of OH) was used as the mobile phase to complete the separation of the 235 mg sample of the mixture. The obtained compound is as follows: compound a) 5-chlorospinoxine A

<img file="TW487559B_D0161.tif" />

<u>Example F14-5,6-cis-dihydroxy-5,6-dihydrospinoxine B (2:1 mixture of (5S, 6R) and (5R, 6S) isomers respectively)</u>

The compound Spinozin A (3.26 g, 4.45 mmol) was dissolved in acetone (70 ml). Water (30 ml) was added, followed by N-methylmorphine-N-oxide (645 mg, 5.5 mmol). After the N-oxide is dissolved, add 0s0₄(2.5% t-BuOH solution 2.5%, 0.0015 mmol). The reaction mixture was stirred at room temperature. After 24 hours, EtOAc (100 ml) and benzene (100 ml) were added. Continuously with 10% NaHSO₃Aqueous solution (1x), water (1x) and 5% NaHCO₃(1x) Wash the mixture. Take K₂CO₃The organic layer is dried and dried under reduced pressure. Flash SiO₂The residue was separated by column (EtOAc, then 10% MeOH in EtOAc) to obtain 5,6-cis-dihydroxy-5,6-dihydrospinoxine A mixture ((5S,6R) and (5R, 6S) 2:1 mixture of isomers, 2.236 g, 66%) and 5,6-cis-dihydroxy-5,6-dihydrospinoxine B ((5S,6R) and (5R,6S) Heterogeneous

<img file="TW487559B_D0162.tif" />

<u>Example F15-(5s,6R)-5,6-Dihydroxy Spinozin A dichloroketene acetal and (5s,6R)-5,6-bis(trichloro-acetoxy) Spinozing A</u>

Dissolve (5s,6R)-5,6-dihydroxy-5,6-dihydrospinoxine A (376mg, 0.491 mmol) in dry CH₂Cl₂(10 ml). Add pyridine (3 ml) followed by N,N-dimethylamino pyridine (600 mg). The reaction mixture was stirred at room temperature under nitrogen and trichloroacetoxychloride (1.00 ml, excess) was added. After 1 hr, the mixture was diluted with EtOAc (100 ml) and PhH (50 ml). Continuously use salt water (1x) and 5% NaHCO₃Aqueous solution (2x) to clean the solution, and use Na₂SO₄dry. The organic layer was concentrated under reduced pressure. Flash SiO₂The residue was separated by column (60 g/EtOAc) to obtain: compound a) (5s,6R)-5,6-dihydroxyspinoxine A dichloroketene acetal (104 mg<img file="TW487559B_D0163.tif" /><img file="TW487559B_D0164.tif" />

<u>Example F16-5-keto-6,7-alkenyl-6-hydroxyspinoxine A</u>

A 2:1 mixture of (5S,6R)and(5R,6S)5,6-cis-dihydroxy-5,6-dihydrospinoxine A (1.51 g, 1.97 mmol) was dissolved in dry CH₂Cl₂(5 ml). Separately, N-chlorobutadiamide (0.793 g, 5.91 mmol) was dissolved in dry CH₂Cl₂(15 ml). At -78°C, ethyl sulfide (0.83 ml, 7.68 mmol) was added to the NCS solution and stirred at -78°C for 30 minutes. Then the glycol solution was introduced and stirring was continued for 1.5 hours at -78°C. Then triethylamine (2 ml, dry) was added and the solution was slowly brought to room temperature under nitrogen within 2 hours. Add EtOAC (50 ml) and PhH (100 ml). Continuously with 5% NaHCO₃(3x) and water (1x) cleaning solution, followed by K₂CO₃Dry and concentrate under reduced pressure. Flash SiO₂The residue was purified by a column (230-400 m, 120 g, EtOAC, followed by 10% EtOH in EtOAc) to obtain 1.20 g with a slight inclusion, which can be easily obtained from Et₂O crystallized to obtain 5-keto-6,7-alkenyl-6-hydroxyspinoxine A (1.01 g, 674) IR (CHCl₃)

<img file="TW487559B_D0165.tif" />

<u>Example F17-(5R,6R)-5,6-dibromospinoxine A</u>

Dissolve the compound Spinozin A (1.59 g, 2.17 mmol) in dry CH₂Cl₂(3 ml). The solution was stirred at room temperature. A portion of phenylselenylene bromide (98%, 1.05 g, 4.34 mmol) was added. After stirring for 30 minutes, the solution was cooled to 0°C. MCPBA (2.5 g, about 7 mmol) was added and stirring was continued for 30 minutes at 0°C. Then triethylamine (3 ml) was added and stirred for 10 minutes, and the mixture was diluted with benzene (150 ml) and EtoAc (50 ml). Continuously with 10% NaHSO₃(2x) Aqueous solution, brine (1x) and 5% NaHCO₃(2x) Washing solution. Take K₂CO₃The organic phase was dried and concentrated under reduced pressure. Flash column chromatography (SiO₂/EtOAC) to purify the residue and then by HPLC (C-18 coating, 6m, 100 A filling; 5%H₂The MeOH solution of O is the mobile phase). The pure fraction can provide a) unreacted compound Spinozin A and b) compound (5R)

<img file="TW487559B_D0166.tif" />

<u>Example F18-5-keto-6,7-alkenyl-6-(p-chloro)hydrospinoxine A</u>

The compound 5-keto-6,7-alkenyl-6-hydroxyspinoxine A (556 mg, 0.73 mmol) was dissolved in dry CH₂Cl₂(10 ml). Add pyridine (5 ml) and DMAP (300 mg). The solution was stirred at room temperature under nitrogen and p-chlorobenzyl chloride (1.0 ml, excess) was added. After 15 minutes, the reaction mixture was diluted with EtOAc (50 ml) and PhH (100 ml). The solution is continuously mixed with brine (2x), water (1x) and 5% NaHCO₃(2x) The aqueous solution is diluted. Take Na₂SO₄The organic phase was dried and concentrated under reduced pressure. By flash column chromatography (SiO₂/EtOAc) to obtain the compound 5-keto-6,7-alkenyl-6-(p-chloro)oxy Spinozin A (634 mg, 96%)¹H-NMR(CDCl₃)d: 8.03

<img file="TW487559B_D0167.tif" />

s) ppm.

<u>Example F19-5,6-bis(trimethylsilyloxy)-5,7(8)-diene Spinozin A</u>

The compound 5-keto-6,7-alkenyl-6-hydroxyspinoxine A (91.40g, 1.84 mmol) was dissolved in dry CH₂Cl₂(15 ml). The solution was stirred at room temperature under nitrogen and triethylamine (dry, 3 ml) was added. After 15 minutes, trimethylsilyl triflate (1.5 ml) was introduced and the reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with PhH (50 ml) and EtOAc (100 ml). Continuously use salt water (1x), 5% NaHCO₃Aqueous solution (3x) and H₂O (1x) extraction solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. In flash SiO₂The residue was separated on a column (120g/EtoAc+0.2% Py) to obtain the compound 5,6-bis(trimethylsilyloxy)-5,7(8)-diene Spinozin A (

<img file="TW487559B_D0168.tif" />

<u>Example F20-5-keto-6,7-alkenyl-6-t-butylbis-methylsilanoxy Spinoxine A</u>

The compound 5-keto-6,7-alkenyl-6-hydroxyspinoxine A (1.025g, 1.345 mmol) was dissolved in CH₂Cl₂(Dry, 20 ml). Add pyridine (2ml). The solution was stirred at room temperature under nitrogen and t-BDMS triflate (98%, 471 ml, 2.01 mmol) was added. Stirring is continued for 14 hours. The mixture was diluted with EtoAc (80 ml) and PhH (50 ml). With 5%NaHCO₃(2x) Washing solution. Take K₂CO₃The organic layer was dried and concentrated under reduced pressure. By flash tube column chromatography (230-400m SiO₂, 100 g, EtOAc) to purify the residue to obtain compound 5-keto-6,7-enyl-6-t-butyldi-methylsilanoxy Spinozin A (1.030 g, 874)¹H-NMR(CDCl3)d:

<img file="TW487559B_D0169.tif" />

<u>Example F21-(5S)-5,6-Dihydro-5-hydroxyspinoxine A(6s)-5,6-dihydro-6-hydroxyspinoxine A(6R)-5,6- Dihydro-6-hydroxyspinoxine A</u>

Mercury trifluoroacetate (1.12 g, 2.62 mmol) was suspended in 30 ml of THF:water, 2:1 (V/V). Spinozin A (0.98 g, 1.34 mmol) was added to this yellow suspension. The reaction was then stirred at room temperature for 30 minutes. The reaction was treated with 7 mL 1N NaOH and then 2.5 mL 0.5M NaBH₄Of3M NaOH solution treatment. The reaction turned black and supported the precipitate. The solid was allowed to settle to the bottom of the flask and the liquid was poured into a separatory funnel containing 50 ml of ether. The layers were separated and the aqueous layer was extracted with 2x25 mL diethyl ether. Combine the ether extracts and add 30 mL saturated NaHCO₃Aqueous solution cleaning, with K₂CO₃Dry and concentrate under reduced pressure. By medium pressure liquid chromatography (230-400 m, SiO₂, CH₂Cl₂: MeOH, 95: 5 (V/V)) The residue was purified to obtain compound a) (5S)-5,6-dihydro-5-hydroxyspinoxine A (349.7 mg, 30.4%)<img file="TW487559B_D0170.tif" />Mixture of 6-hydroxyspinoxine A and (6R)-5,6-dihydro-6-hydroxyspinoxine A, (6s)-5,6-dihydro-6-hydroxyspinoxine A And (6R)-5,6-dihydro-6-hydroxyspinoxine A can be applied by HPLC (C-18 coating, 6m, 100 A filling; 10%H₂MeOH solution of O as the mobile phase) to provide compound b) (6R)-5,6-dihydro-6-hydroxyspinoxine A (7.1 mg, 1%) part¹H-

<img file="TW487559B_D0171.tif" />

<u>Example F22-(5R,6R)-5-acetoxy-6-thiomethoxy Spinozin A</u>

Compound Spinozin A (2.64 g, 3.61 mmol) solution CH₃CN (dry, containing 1% ethyl sulfide; 30 ml). This solution was maintained at room temperature and dimethyl(methylthio)tetrafluoroborate (1.10 g, 5.61 mmol) was added and the mixture was ultrasonically shaken (50 W ultrasonic cleaner) for 10 minutes. Add anhydrous sodium acetate powder (2.5 g, excess) and ultrasonic shake for 1 hour. Dilute the solution with EtoAc (50 ml) and PhH (100 ml) and add 5% K₂CO₃, 5%NaHCO₃Aqueous solution and H₂O cleaning solution. Take Na₂SO₄The organic layer was dried and concentrated under reduced pressure. By SiO₂The above flash tube column chromatography was used to purify the residue and preparative HPLC (C-18 coating, 6m, 100 A filling; 8%H₂The MeOH solution of O is the mobile phase). The pure fraction can provide the compound (5R, 6R)-5-acetoxy-6-thiomethoxyspinoxine A (2.07 g, 68%)¹H-NMR<img file="TW487559B_D0172.tif" /><img file="TW487559B_D0173.tif" />

<u>Example F23-(5R,6R)-5-acetoxy-6-methylsulfonyl spinoxine A</u>

The compound (5R, 6R)-5-acetoxy-6-thiomethoxy Spinozin A (1.05 g, 1.25 mmol) was dissolved in CH₂Cl₂(100 ml). The solution was cooled to 0°C and while stirring, meta-CPBA (50%, 1.95 g, 5.6 mmol) was added part by part. After 40 minutes, use CH₂Cl₂(50 ml) The reaction mixture was diluted. Continuously with 10% NaHSO₃Aqueous solution (2x), water (1x) and 5% NaHCO₃Aqueous (2x) cleaning solution. Take Na₂SO₄The organic layer was dried and concentrated under reduced pressure. By flash column chromatography (230-400 m SiO₂/EtOAc, followed by 10% EtOH in EtOAc) to purify the residue to obtain compound (5R, 6R)-5-acetoxy-6-methanesulfonylspinoxine A (966 mg, 89%)

<img file="TW487559B_D0174.tif" />

<u>Example F24-(5R,6R)-6-bromo-5-hydroxyspinoxine J</u>

The compound Spinozin A (1.33 g, 1.32 mmol) was dissolved in DMSO (15 ml). Water (5 ml) was added, causing precipitation. Introduce concentrated H while stirring₂SO₄(1.8 mmol). Dissolve the precipitate immediately. The solution was cooled to 0°C and N-bromosuccinimide (322 mg, 1.8 mmol) was added. After stirring for 15 minutes at 0°C, the reaction mixture was poured into 50 ml saturated NaHCO₃In aqueous solution. Take Et₂O extract the mixture. The combined organic extracts were washed with brine and washed with K₂CO₃Dry and concentrate under reduced pressure. The residue was purified by reverse phase HPLC (C-18Dynamax column, 20% water in MeOH solution as the mobile phase) to obtain compound (5R, 6R)-6-bromo-5-hydroxyspinoxine J (1.30 g ,

<img file="TW487559B_D0175.tif" />

<u>Example F25-5,6-Dihydrospinoxine A 9-Psa</u>

The compound Spinozin A 9-Psa (46.5 mg, 0.0856 mmol) was dissolved in 5 mL of toluene. Add \-triphenylphosphine rhodium(I) chloride (8.5 mg, 0.0092 mmol) to this solution. The top area of the flask was evacuated and nitrogen was introduced 3 times. Vacuum and introduce hydrogen 3 times. The flask with the spherical shape is maintained under hydrogen and heated to 110-120°C. After 3.5 hours, the flask was cooled to room temperature and hydrogen was evacuated and replaced with nitrogen. The toluene was evaporated and replaced with 20 ml. The ether solution was extracted with 3x10mL 1N HCl. The acid extracts were combined and neutralized with 10 ml 4N NaOH. Extract the neutralized suspension with 3x10 mL of ether, combine the ether extracts, wash with 20 mL of brine, and use K₂CO₃Dry and concentrate under reduced pressure. The obtained compound 5,6-dihydrospinoxine A 9-Psa (29.5 mg, 63%) part¹H-NMR(CDCl₃) d6.84 (1H, bs,), 1.01 (1H, m), 0.68 (1H, m).

<u>Example F26-5,6-Dihydrospinoxine A 17-Psa</u>

According to the steps described in Example F25 above, the compound Spinozin A 17-Psa (270.8 mg, 0.458 mmol) was added to 32.8 mg [(Ph)₃P]₃RhCl treatment. After evaporating toluene, by medium pressure liquid chromatography (230-400 mSiO₂, CH₂Cl₂: MeOH, 95:5 (V/V) to purify the compound. The obtained compound 5,6-dihydrospinoxine A 17-Psa (188.1 mg, 69.2%) part

<img file="TW487559B_D0176.tif" />

<u>Example F27-5,6-Dihydrospinoxine J</u>

Following the procedure described in Example F25 above, the compound Spinozin J (1.00 g, 1.39 mmol) was treated with 91.3 mg [(Ph)3P]3RhCl in 20 mL toluene. The obtained compound 5,6-dihydrospinoxine J (0.70 g, 70%) part¹H-NMR(CDCl₃) d6.84 (1H, bs) 3.82 (1H, dt), 1.01 (1M, m), 0.69 (1H, m).

<u>Example F28-5,6-Dihydrospinoxine H</u>

The compound Spinozin H (2.57 g, 3.58 mmol) was dissolved in a round bottom flask containing 20 ml of toluene. [Ph3P] 3RhCl (169.2 mg, 0.183 mmol) in 5 mL toluene was added to the colorless solution. The flask was equipped with a reflux condenser and the top area of the flask was evacuated 3 times and nitrogen was introduced. The nitrogen was evacuated 3 times and hydrogen was introduced. The flask with the spherical shape was maintained under 1 atm of hydrogen and heated to 100-110°C in an oil bath. After 10 hours, the heat source was removed and the flask was evacuated 3 times, and nitrogen was introduced after each evacuation. The contents of the flask were hot filtered through a 3" celite column under nitrogen. The celite was washed with 100 mL of ether to dissolve the remaining compounds. The filtrate was extracted with 3x50 mL of 1N HCl. The acid extracts were combined and reversed with 25 mL of ether. Extract and basify with 50 mL 4N NaOH. The precipitated white solid is extracted into ether (2x25 mL) and the acetone extract is washed with 25 mL saline solution. The ether solution is filtered through a plug of celite and concentrated under reduced pressure The obtained compound 5,6-dihydrospinoxine H (2.24 g, 87.14). C₄₀H₆₅NO₁₀The analysis value: estimated value: C 66.73, H 9.10, N 1.95; actual value: C 66.31, H 9.01, N 2.02. Cl MS (m/z) 721 (M+1).

<u>Example F29-5,6-Dihydrospinoxine A hemiglutarate</u>

The compound 5,6-dihydrospinoxine A (232.2 mg, 0.316 mmol) was dissolved in 3 mL of acetone. Add a solution of glutaric acid (20.8 mg, 0.157 mmol) in 3 mL acetone and stir the solution spacer at room temperature. The solvent was removed under reduced pressure to obtain compound 5,6-dihydrospinoxine A hemiglutarate (250.8 mg). C₄₁H₆₇NO₁₀1/2(C₅H₈O₄Analysis of ): estimated value: C 65.31, H 8.94, N 1.75; actual value: C 65.14, H 8.78, N 1.87; mp.83-92°C.

<u>Example F30-5,6-Dihydrospinoxine B</u>

Hydrogenation step A: Combine Spinozin B (1.10 g, 1.53 mmol) and (Ph₃P)₃A PhMe (15 mL) solution of RhCl (0.05 g, 0.09 mmol) was placed in a Parr device and kept in a hydrogen environment (45-50 psi) at 104-107°C for 2 hours. After cooling to room temperature, the mixture was evaporated under reduced pressure and the residue was decolorized (Et₂O/charcoal) and filter. The filtrate was evaporated under reduced pressure and passed through MPLC (SiO₂, 5: 95 to 10: 90 MeOH/CH₂Cl₂) Purification to obtain 0.90 g (82%) of 5,6-dihydrospinoxine B as a white powder. C₄₀H₆₅NO₁₀Analysis: Estimated value: C, 66.73; H, 9.10; N, 1.95. Actual value: C, 66.53; H, 9.14; N, 2.15.

<u>Example F31-Synthesis of compound (5S, 6R)-epoxy-spinoxine Q</u>

Spinozin Q (970 mg, 1.32 mmol) was dissolved in dichloromethane (150150 ml). The solution was cooled to 0°C and a portion of m-CPBA (1.19 g about 50% reagent, about 3.45 mmol) was added. After the m-CPBA was dissolved, immediately put the reaction mixture in the refrigerator. After 40 hours, use 10% NaHSO₃(2x), with water (1x) and with 5% NaHCO₃(2x) The reaction mixture was extracted with an aqueous solution. Organic layer with anhydrous K₂CO₃Dry, filter and concentrate under reduced pressure. The residue is flashing SiO₂The column (230-400 m, 70 g/EtOAc) was separated. The chromatographic homogeneous eluate was concentrated under reduced pressure to obtain a white solid (694 mg). The white solid (88 mg) precipitated from this substance was dried with Et₂O performs precipitation. Ether solution, after evaporation, compound (5S, 6R)-epoxy-spinoxine Q can be obtained,

<img file="TW487559B_D0177.tif" />

<u>Example F32-(5S,6R)-5,6-dihydro-5,6-dihydroxyspinoxine (65%) and (5R,6S)-5,6-dihydro-5,6-two Mixture of hydroxyspinoxine A (35%)</u>

Spinozin A (3.26 g, 4.45 mmol) was dissolved in acetone (70 mL). Add water (30 mL), followed by N-methylmorphonium N-oxide (645 mg, 5.5 mmol), and osmium tetroxide (2.5% in 2-methyl-2-propanol; 160 mg, 0.015 mmol). After 1 hour, another part of OsO4 (300 mg) was added. After 24 hours, mix the mixture with 10% NaHSO₃(200 mL) The aqueous solutions were combined and reacted gradually. The crude product was purified on a short flash column with silica gel (10% MeOH in EtOAc) to provide unisolated (5S,6R)-5,6-dihydro-5,6-dihydroxyspinoxine ( 65%) and (5R,6S)-5,6-dihydro-5,6-dihydroxy Spinozin A (35%) mixture (35%) (2.24 g, 66%), white solid:¹H-NMRd 6.71 (s, 1H), 3.93 (m, 0.65 H), 3.88 (m, 0.35 H), 2.11 (s, 6H).

<u>Example F33-5,6-Dihydrospinoxine B</u>

Combine Spinozin B (1.10 g, 1.53 mmol) and (Ph₃P)₃PhMe (15 mL) solution of RhCl (0.08 g, 0.09 mmol) was placed on the Parr device and placed in H₂Gas environment (45-50 psi) and 104-107°C. After 2 hours, the reaction was filtered and evaporated. The residue solution Et₂0 and pulped with charcoal. The slurry was filtered and evaporated. MPLC(5: 95MeOH/CH₂Cl₂) 0.90 g (82%) of 5,6-dihydrospinoxine B can be obtained as a white powder. C₄₀H₆₅NO₁Analysis: Estimated value: C, 66.73; H, 9.10; N, 1.95, actual value: C, 66.53; H, 9.14; N, 2.15.

<u>Example F34-5,6-Dihydrospinoxine D</u>

In a 100 ml Parr container, add 10% Pd/C and cyclohexane to a pure ethanol (60 mL) solution of Spinozin D (2.5 g, 3.4 mmol). The suspension was heated at 120°C for 48 hours. After cooling, the reaction mixture was filtered through a pad of Celite and concentrated under reduced pressure to obtain 2.4 g of a 2:1 mixture of 5,6-dihydrospinoxine D and spinoxin D. The residue (200 mg) was dissolved in dichloromethane (20 mL) and cooled to 0°C. MCPBA (95 mg, 0.45 mmol) was added to the reaction mixture and stirred at 25°C for 16 hours. The reaction mixture was quenched with sodium bicarbonate solution and the layers were separated. The aqueous layer was extracted with dichloromethane (2x20 mL). Combine the combined extract with 10% NaHCO₃Stir for 3 hours, wash with brine, and use anhydrous Na₂SO₄Dry and concentrate under reduced pressure. The residue was purified on preparative HPLC using Cl8 column, 10% water (0.1% NH₄OH) was eluted in methanol to obtain compound 5,6-dihydrospinoxine D (10 mg). MS

<img file="TW487559B_D0178.tif" />

<u>Part G is modified by replacing amino sugars with different sugars</u>

<u>Example G1-17-O-(2-Iodo-2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl)] Spinozin A 17-Psa</u>

To Spinozin A17-Psa (105.4 mg, 0.18 mmol) in acetone (0.86 ml) solution, add 3,4-di-O-acetyl-6-deoxy-L-hexenose ( Aldrich, 52 ul, 0.35 mmol) and then N-iodosuccinimide (80.5 mg, 0.36 mmol) was added. The reaction mixture was stirred at room temperature for 19 hours. Then dilute the dark mixture with dichloromethane and add NaHSO₃Wash with an aqueous solution and then wash with 5% sodium thiosulfate. Take MgSO₄The dichloromethane portion was dried and evaporated at room temperature under reduced pressure. The product was purified by preparative HPLC at 45:45:10/acetate: methanol: 0.05% NH₄The OAc aqueous solution is eluted. The resulting 17-O-(2-iodo-2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl)) Spinozin A 17-Psa (49 mg ; Yield 29%), colorless glass, FDMS, m/e (relative strength) 930 (M⁺-H, 20), 929 (30), 190 (100).

<u>Example G2-17-O-(2-Deoxy-α-L-rhamnosyl) Spinozin A 17-Psa</u>

The reaction was carried out as described in Example G3 below, with 17-O-(2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl) Spinozin A 17 -Psa (271.2 mg, 0.34 mmol) is the starting material. The obtained 17-O-(2-deoxy-α-L-rhamnosyl) Spinozin A 17-Psa (187 mg; 77% yield) was a white glass, FDMS, m/e( Relative strength) 1432 (20), 752 (30), 720 (M⁺，100)，206(50)。

<u>Example G3-17-O-(2-Iodo-2-deoxy-α-L-rhamnosyl) Spinozin A 17-Psa</u>

In 17-O-(2-iodo-2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl) Spinozin A 17-Psa (107.8 mg, 0.12 Add a methanol (9 ml) solution of methanol (2 ml) to a saturated ammonia solution of methanol (2 ml). The reaction mixture was capped and stirred at room temperature for 19 hours. The mixture was evaporated at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 5% methanol in dichloromethane. The obtained 17-O-(2-iodo-2-deoxy-α-L-rhamnosyl) Spinozin A 17-Psa (74 mg; yield 75%) is a colorless glass, FDMS ,M/e(relative intensity)848(MH⁺，20)，591(100)，189(65)，101(90)。

<u>Example G4-17-O-(2-Deoxy-3,4-di-O-acetyl-α-L-rhamnosyl) Spinozin A 17-Psa</u>

In 17-O-(2-iodo-2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl) Spinozin A 17-Psa (515.3 mg, 0.55 mmol) in toluene (85 ml), add tri-n-butylsilane (780 ul, 2.85 mmol) followed by a trace of AIBN. The reaction mixture was heated to reflux temperature for 30 minutes, and then cooled to room temperature for 1.5 hours. The mixture was evaporated at room temperature under reduced pressure. The mixture was evaporated by chromatography on silica and eluted with 40% ethyl acetate in hexane. The obtained 17-O-(2-deoxy-3,4-di-O-acetyl-α-L-rhamnosyl) Spinozin A 17-Psa((321.5mg; yield 73% ), white deviating glass, FDMS, m/e (relative intensity) 804 (M⁺-H, 100), 190 (55), 101 (70).

<u>Example G5-3'-Demethoxy Spinozin C</u>

The reaction was carried out as described in Example H1 below, with 3'-desmethoxy Spinozine B (240 mg, 0.35 mmol) as the starting material. The obtained 3'-demethoxy Spinozin C (126.1 mg; yield 54%) was a pale yellow solid, FDMS, m/e (relative intensity) 676(40), 675(100), 674(M⁺，80)，673(35)。

<u>Example G6-3'-Demethoxy-17-keto Spinozin A 17-Psa</u>

The reaction was carried out according to the method described in Example E7 above, with 3'-desmethoxy Spinozine A 17-Psa (809.8 mg, 1.4 mmol). The obtained 3'-demethoxy-17-keto Spinozin A 17-Psa (501.4 mg; yield 64%) was a colorless glass, FDMS, m/e (relative strength) 559 ( M⁺，45)，558(100)，159(10)。

<u>Example G7-17-O-(2-Acetamido-2-dehydro-3,4,6-tetra-O-acetyl-β-D-glucopyranosyl Spinozin A 17 -Psa</u>

Spinozin A 17-psa (1.0 g, 1.70 mmol), 2-methyl--(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D -Pyran type glucoyl)-[2,1-d]-2-oxazol (Nakabayashi, S.; Warren, CD; Jeanloz, RW Carbohydr. Res. 1986, 150, C7) (0.56 g, 1.70 mmol) And pyridine -p-toluene acid salt (75 mg, 0.30 mmol) in 1,2-dichloromethane (65 mL) solution was heated at reflux temperature and stirred for 48 hours, and then cooled to room temperature. Then add saturated Na₂CO₃(2 mL) and stir the mixture at room temperature for 5 minutes and then add water (15 mL) and CH₂Cl₂(30 mL) Dilute. Separate the water layer and add CH₂Cl₂(30 mL) Wash and combine the washing solution with the organic layer. Then the combined organic layer was washed with brine (25 mL) and dried (MgSO₄), and concentrate. The residue (1.4 g) was flash chromatographed with silica (160 mL), using 3% MeOH in CH₂Cl₂It is the eluent to obtain 0.6 g of crude glycosylation product. It was purified by reverse phase HPLC with a Rainin micro-absorption Cl8 column (41.4 mm(id)x25 cm(1)) in three parts of 200 mg, using 10% water in MeoH eluent to obtain 151 mg 17-O- (2-Acetylamino-2-deoxy-3,4,6-tetra-O-acetyl-β-D-glucopyranosylspinoxine A 17-Psa. From 1:2 EtOAc /The sample recrystallized from hexane is colorless needles, mp 199-200°C;¹H-NMR(CDCl3)

<img file="TW487559B_D0179.tif" />

<u>Example G8-17-O-(2'-acetamido-2-deoxy-β-D-glucopyranosyl) Spinozin A 17-Psa</u>

In 17-O-(2-acetamido-2-deoxy-bD-glucopyranosyl) Spinozin A17-psa (35 mg, 0.038 mmol) in MeOH (5 mL) solution, add A part of 60% NaH (2 mg) mineral oil dispersion oil. The resulting solution was stirred at room temperature for 25 minutes, and then neutralized with glacial acetic acid. Evaporate the solvent and flash the chromatographic residue with silica (25 mL) using 12% MeOH in CH₂Cl₂The solution is an eluent to obtain 17-O-(2'-acetamido-2-deoxy-β-D-glucopyranosyl) Spinozin A 17-Psa (27 mg), which is Colorless foam:¹H-NMR(CDCl₃)δ6.83(s, 1H, H-13), 4.83<img file="TW487559B_D0180.tif" /><img file="TW487559B_D0181.tif" />

<u>Example G9-2-O-Acetyl-α-D-Deglycamino bromide hydrobromide</u>

Diacetyl glycosamine hydrochloride (Flynn, EH; Sigal, MV, Jr.; Wiley, PF; Gerzon, KJAm. Chem. Soc. 1954, 76, 3120) (0.9 g, 3.05 mmol) in Add to a properly stirred solution prepared from acetic anhydride (0.75 mL) and 30% HBr in acetic acid (3.75 mL) at room temperature. The solution was stirred at room temperature for 1 hour, and then over 10 minutes, diethyl ether (45 ml) was partially added in 4-5 ml portions to precipitate the hydrobromide product; initially the oil was allowed to flow out but was scraped off and Continue to stir to solidify. The hygroscopic hydrobromide was filtered under nitrogen, washed with ether (3x40 mL), and then dried in a rotary evaporator at 30°C and reduced pressure (-30 mm) to produce 2-O-acetate -Α-D-Deglycosamine bromide hydrobromide (1.1 g), off-white powder. The powder can be used directly

<img file="TW487559B_D0182.tif" />

<u>Example G10-17-O-(β-D-Glycosamino) Spinozin A 17-Psa</u>

A portion of 2-O-acetyl-α-D-deglycosamine bromide hydrobromide (1.1 g, 3.0 mmol) was added to suitably stirred Spinozin A 17-Psa (0.5 g, 0.85 mmol) ) And dimethylpyridine (0.26 mL, 2.2 mmol) in 1,2-dichloromethane solution. The solution was heated to 60-65°C and kept at this temperature for 24 hours, cooled to 25°C and treated with 0.5 N HCl (5.2 mL). The mixture was stirred at 25°C for 10 minutes and then added to CH₂Cl₂(20 mL) Dilute. Separate the organic layer and add water (5 mL), saturated NaHCO₃(6 mL) and brine (5 mL) washed and dried (MgSO₄). Flash chromatography on silica (150 mL) to leave 0.6 g of concentrate, using 3% MeOH in CH₂Cl₂The solution is an eluent to obtain 0.25 g of 17-O-(β-D-deglycosinyl) Spinozin A17-Psa, which is a mixture of 4β:1α and a white foam. Dissolve it in MeoH (7 mL) and add a 0.1M NaOMe solution in MeOH (1.5 mL). The solution was stirred at room temperature for 6 hours, then acetic acid (10 mL) was added and the solution was stirred for 10 minutes. The solvent was evaporated and 200 mg of 17-O-(β-D-deglycamino) Spinozin A 17-Psa remained, which was a 4:1 β/α mutamer mixture. β-mutamel (80 mg, white foam) was separated by reverse phase HPLC in 3 parts on Rainin C-18 column (41.4 mm(id)x25cm(1)), using 45:45:10 CH₃CN/MeOH/2%NH₄OAc is soluble

<img file="TW487559B_D0183.tif" />

<u>Example G11-α-D-aminoglycosyl bromide hydrobromide</u>

D-amino sugar (Boeck, LD; Chio, H.; Eaton, TE; Godfrey, OW, Jr.; Michel, KH; Nakatsukasa, WM; Yao, RC-F. European Patent, 0 375 316 A1 (1990) ) (0.1 g, 0.63 mmol) was added in one portion to 1 mL of a 30% HBr acetic acid: acetic anhydride mixture that has been suitably stirred and refrigerated (about 10°C) 5:1 (v/v). The resulting solution was stirred at cooling temperature for 10 minutes, and then at room temperature for 1 hour. Then diethyl ether (7 mL) was added partially in 1.0 mL portions over 5 minutes. The oil in the hydrobromide product was initially allowed to flow out but solidified by scraping and continuous stirring. The hygroscopic hydrobromide was filtered under nitrogen, washed with ether (4×10 mL), and then dried in a rotary evaporator at about 30° C. under reduced pressure. It can be used for glycosylation immediately. Produce (ID-amino sugar bromine compound hydrobromide: 0.17 g (89%), white off-white powder:¹H-NMR(

<img file="TW487559B_D0184.tif" />

<u>Example G12-1"-Table-Spinozin A</u>

Add HgBr2 (0.1 g, 0.28 mmol) in CH₂Cl₂(10 mL) The suspension was stirred rapidly at room temperature for 10 minutes. (Dissolve about 2/3HgBr during this period₂). Use 4A molecular sieve (0.15 g) to pulverize and add Spinozin A 17-PSa (0.11 g, 0.18 mmol) and stir the mixture for 10 minutes. Then, within 1 hour, add α-D-aminosugar hydrogen bromide dropwise Bromate (0.17g, 0.56 mmol) in CH₂Cl₂(4.0 mL) solution. Stir for another 3 hours, add saturated Na₂CO₃(4 mL) and stir the mixture for 10 minutes. By filtering the mixture through diatomaceous earth and distilling the collected mixture into CH₂Cl₂(15 mL) and water (5 mL) to wash. Combine the organic layers of the filtrate and separate the washing liquid and add CH₂Cl₂(15mL) Wash the water layer. Combine the combined organic extracts with 10% KI aqueous solution (2x4 mL), 10% NaHCO₃(5 mL) and brine (10 mL) washed and dried (MgSO₄). After evaporation, a residue of 0.15 g remains, which is flash chromatographed with silica (40 mL) using 4% MeOM in CH₂Cl₂To obtain a clean glycosylation product (49 mg) as an eluent, it is a 3:1 mixture of 1"-epi-Spinozin A and Spinozin A. This mixture is dissolved in CH₃CN (1.5 mL) and 4.6 mm (id) x 250 mm (1) Apex phenyl column in two consecutive arrays analyzed by HPLC to separate mutagenic isomers with 40:40:20CH₃CN/NeOH/2%NH₄OAc is the eluent and the flow rate is 1.5 mL/min. About 30 injections of 40-45uL, each containing about 1.5 mg of the prepared mixture. The obtained 1"-table Spinozin A is a colorless foam:¹H-NMR(CDCl₃)δ6.75(s,

<img file="TW487559B_D0185.tif" />

<u>Example G13-17-O-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl) Spinozin A 17-Psa</u>

Spinozin A 17-PSa (0.3 g, 0.5 mmol) and HgBr₂(90 mg, 0.25 mmol) in 1,2-dichloromethane (20 mL) solution, heat the powder containing 4A molecular sieve and heat to reflux temperature and collect 3 mL solvent by distillation. In this refluxing mixture, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (0.48 g, 1.17 mmol) bis was added dropwise within 10 minutes A solution of methyl chloride (5 mL). Collect more solvent (3 mL) and continue to reflux for 20 hours. Then add more bromide (0.15 g) and HgBr₂(90 mg) and reflux for 5 hours. The reaction mixture was then cooled to room temperature and filtered through Celite. The collected solid is CH₂Cl₂(20mL) Wash and combine the filtrate and washing liquid and continuously wash with 10% KI (2x10 mL), water (15 mL) and brine (15 mL) and then dry (MgSO₄). Evaporation leaves a residue of 0.75 g, which is flash chromatographed on silica (90 mL) using 3% MeOH in CH₂Cl₂To obtain 0.2 g of 17-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)spinoxine A 17-Psa, which is no

<img file="TW487559B_D0186.tif" />

<u>Production of H-part distribution glycosyls, pseudosaccharides, glycosyls and other alkylated spinoxins</u>

<u>Example H1- Synthesis of Spinozin C from Spinozin B</u>

A solution of Spinozin B (1.00 gms, 1.4 mmol) in methanol (45 ml) was cooled to 3°C under nitrogen. A freshly prepared 1M methanol solution of sodium methoxide (7.1 ml, 7.1 mmol) was added and then a portion of iodine solid (1.8 gms, 7.1 mmol) was added. The reaction was stirred at 3°C for 4.5 hours. (HPLC showed that the reaction was 80% complete) and then poured into 5% sodium thiosulfate/diluted ammonium hydroxide solution and extracted with ether. Wash the ether extract with brine and dry (K₂CO₃) And concentrated under reduced pressure at room temperature. The crude product was purified by reverse phase HPLC on a Cl8 column, and eluted with methanol:acetate:0.05% ammonium acetate (45:45:10) to obtain Spinozin C (361 mg). Of the product (MS, and¹H-NMR) is the same as the naturally produced Spinozin C.

<u>Example H2- Spinozin D Ag</u>

To Spinozin D 9-Psa (132 mg, 0.24 mmol) in water (5 ml), add 1N H dropwise₂SO₄To pH 1.7 and homogenize the mixture. The solution was heated to 80°C for 3.75 hours, during which time the oil was separated from the solution. The mixture was cooled to room temperature and dichloromethane was added to dissolve the oil. The aqueous phase was separated and extracted with fresh dichloromethane. Combine the dichloromethane parts, quickly add 1N H₂SO₄Clean with K₂CO₃Dry and evaporate to room temperature to obtain a pale yellow glass (82.9 mg). The product was purified by flash chromatography with 5% MeOH in dichloromethane to obtain Spinozin D Ag (63.6 mg, yield 63%) as a colorless glass.

<u>Example H3-Synthesis of Spinozin B from Spinozin A</u>

A 80% methanol/water (125 ml) solution of Spinozin A (5.0 gms, 5.13 mmol) and sodium acetate trihydrate (4.68 gms, 34.4 mmol) was heated to 47°C under nitrogen. After adding a portion of iodine in solid form (1.75 gms, 68.0 mmol), the pH value is reduced from 10 to 8, and a brown color can be obtained. The pH is maintained at 8-9 by adding 1N sodium hydroxide at a fixed time. The reaction was heated for 2.5 hours (during this time the color faded to light yellow), and then cooled to room temperature. The solution was poured into a solution of water (250 ml) and ammonium hydroxide (50 ml) and extracted with ether. Wash the ether extract with brine and dry (K₂CO₃), and concentrated at room temperature under reduced pressure. The crude product was purified on a Cl8 column by reverse phase HPLC, and eluted with methanol:acetate:0.054 ammonium acetate (45:45:10) to obtain Spinozin B (2.52 gms), which (MS,¹H-NMR, 13C NMR, IR, and OR) are the same as naturally produced Spinozin B.

<u>Example H4-N-Demethylspinoxine J</u>

The reaction was carried out as described in Example H3, with Spinozin J (105.4 mg, 0.15 mmol) as the starting material; however, the extraction operation was carried out with dichloromethane instead of ether. The obtained N-desmethylspinoxine J (57.3 mg; 54%) was a pale yellow glass.

<u>Example H5-N-Demethylspinoxine L</u>

The reaction was carried out as described in Example H4, with Spinozin L (102.5 mg, 0.14 mmol) as the starting material. The obtained N-desmethylspinoxine L (66.5 mg; 664) was a white glass.

<u>Example H6-N-Demethyl Spinozin K</u>

The reaction was carried out as described in Example H4, with Spinozin K (101.5 mg, 0.14 mmol) as the starting material. The obtained N-desmethylspinoxine K (79.3 mg; 81%) was a white glass.

<u>Example H7-N-Demethylspinoxine D</u>

A suspension of Spinozin D (5.10 gms, 6.8% mmol) and sodium acetate (10.3 gm, 125 mmol) in 80% methanol/water (500 ml) was heated to 50°C, and nitrogen gas was bubbled through the liquid 15 Minutes (pH=8.96). When adding a portion of iodine in solid form (3.15 gms, 12.4 mmol), the pH drops to pH 8, and a brown color can be obtained. The pH is maintained at 8-9 by adding 1N sodium hydroxide at a fixed time. The reaction was heated for 1.5 hours (during this time the color faded to light yellow), and then cooled to room temperature. Add 10% sodium bisulfite (100 mL) to neutralize unreacted iodine. The reaction was concentrated in a rotary evaporator to a total volume of 150 mL and the solution was extracted with 3x100 mL ethyl acetate. The ethyl acetate extracts were combined and washed with brine solution (100 mL). The ethyl acetate solution was dried with potassium carbonate and concentrated in a rotary evaporator. You can get 5.0 gm yellow oil. The crude product was subjected to flash chromatography (500 mL SiO₂, CH₂Cl₂: CH₃OH, 95:05) was purified to obtain N-desmethylspinoxine D (3.79 gms, 75.8

<img file="TW487559B_D0187.tif" />

<u>Example H8-N,N-Didesmethyl Spinozin K</u>

The reaction was carried out as described in Example H1, using N-desmethylspinoxine K (891 mg, 1.27 mmol) as the starting material; however, the extraction operation was carried out with ethyl acetate instead of ether. The obtained N,N-didesmethylspinoxine K (463.6 mg) was a colorless glass.

<u>Example H9- Spinozin F 17-Psa</u>

The compound was prepared from 35 mg (0.049 mmol) Spinozin F as described in Spinozin E 17-Psa (Example H10). Spinozin F

<img file="TW487559B_D0188.tif" />

<u>Example H10- Spinozin E 17-Pseudoglycan compound</u>

Add a portion of 1N H to a properly stirred 35 mg Spinozin E in water (0.7 mL) solution₂SO₄(0.1 mL) solution. The resulting solution was heated to 90-100°C and kept at this temperature for 24 hours. During this period, the pseudoglycan compound obtained was separated. Then the mixture was cooled to room temperature and the crude product was extracted into dichloromethane. Then wash the organic extract with saturated NaCl and dry (MgSO₄). After evaporation, the crude 17-pseudoglycan compound is left, which is purified by silica gel chromatography using 4% methanol in CH₂Cl₂The solution is an eluent to obtain 26 mg (92%) Spinozin E 17-pseudoglycone compound,

<u>Part I Deoxyrhamnose Derivatives</u>

<u>Example I1-N-Demethyl-2'-deoxy Spinozin Q</u>

Compound 2'-Deoxy Spinozin Q (940 mg, 1.31 mmol) was dissolved in hot 70% MeOH/30% pH 9 buffer (40 ml). Sodium acetate trihydrate (1.3 gms, 9.6 mmol) was added, and the suspension was heated to 47°C. Then add iodine in solid form (438 mg, 1.7 mmol). After stirring at 47°C for 4 hours, additional iodine (150.8 mg, 0.59 mmol) was added. The mixture was stirred at 47°C for another 4 hours, and then cooled to room temperature and stirred for another 12 hours. The solution was poured into 5% sodium thiosulfate and extracted with Et20. Wash the Et20 part with salt water and dry it (K₂CO₃), and steamed at room temperature. The residue is in a silica gel column (5%MeOH/CH₂Cl₂) Was chromatographed and provided N-desmethyl-2'-deoxyspinoxine Q (462.7 mg; yield 56%, based on the recovered starting material) as a pale pink solid; FDMS, m /z (relative intensity) 702 (100), 159 (10).

<u>Example I2-N-Demethyl-3'-deoxy Spinozin J</u>

The reaction was carried out as described in Example 11, using 3'-deoxyspinoxine J (1.51 gms, 2.16 mmol) as the starting material. The resulting N-desmethyl-3'-deoxy Spinozin J (937.6 mg; yield 63%) was a white solid; FDMS, m/e (relative intensity) 687(100)159(10).

<u>Example I3-3'-Deoxy Spinozin J 17-Psa</u>

Suspend 3'-Deoxy Spinozin J (257.2 mg, 0.37 mmol) in 1N H₂SO₄(5 ml) solution and heated to reflux temperature for 2.25 hours. Then the mixture was cooled to room temperature, with CH₂Cl₂Dilute and wash with water. Then wash CH with salt water₂Cl₂Part, dry (K₂CO₃) And evaporate at room temperature. The residue was chromatographed on a silica gel column (50% EtOAc/hexane) to provide 3'-deoxy Spinozin J 17-Psa (112 mg; yield 54%); FDMS.m/e, (relative Intensity) 560 (100), 159/16).

<u>Example I4-9-O (1-tetrahydropyranyl) Spinozin A 9-Psa (a mixture of α and β isomers)</u>

The compound Spinozin A 9-Psa (106.6 mg, 0.20 mmol) was dissolved in benzene (10 ml)<u>Merge</u>Add dihydropyran (21 ml, 0.23 mmol) followed by a catalytic amount of p-toluenesulfonic acid. The mixture was heated to reflux temperature for 16 hours. Since no reaction was observed, more dihydropyran (200ml) was added. The mixture was refluxed in a Dean/Stark trap for 6 hours and then cooled to room temperature. Take CH₂Cl₂The mixture was diluted and washed with 1N NaOH. Wash CH with salt water₂Cl₂Part, dry (K₂CO₃) And concentrated at room temperature. The residue (105 mg, mostly Spinozin A 9-Psa) was dissolved in benzene (10 ml), and dihydropyran (200 ml, 2.2 mmol) was added, followed by p-toluenesulfonic acid (42.9 mg , 0.23 mmol). The mixture was stirred at room temperature for 1 hour, and then diluted with Et20. With saturated NaHCO₃, Salt water cleaning Et₂Part O, dry (K₂CO₃) And evaporate at room temperature. The residue was chromatographed on a silica gel plate (100% EtOAc, then 10% EtOH/EtOAc, then 100% MeOH, in 2 steps) to provide 9-O-(1-tetrahydropyranyl)spinoxine A 9-Psa (a mixture of α and β isomers) (116.8 mg; yield 93%); FDMS m/e (relative intensity) 628(100), 142(5).

<u>Example I5-3'-O Acetyl Spinozin J</u>

The compound Spinozin J (207.9 mg, 0.29 mmol) was dissolved in pyridine (2 ml) and acetic anhydride (140 ml, 1.5 mmol) was added. The mixture was stirred at room temperature for 24 hours, and then evaporated at room temperature. Put the residue on a silica gel column (7% MeOH/CH₂Cl₂) Chromatography to provide 3'-O-acetylspinoxine J (148.8 mg, yield 68%) as a colorless glass. FDMS, m/e (relative intensity) 759 (100), 283 (4), 142 (7).

<u>Example I6-2'-O-Acetyl Spinozin H</u>

The reaction was carried out as described in Example I5, with Spinozin H (211.9 mg, 0.29 mmol) as the starting material. The obtained 2'-O-acetylspinoxine H (175.2 mg, yield 80%) was a colorless glass. FDMS, m/e (relative intensity) 759 (100), 142 (6).

<u>Example I7-4'-O-Acetyl Spinozin K</u>

The reaction was carried out as described in Example I5, with Spinozin K (207.0 mg, 0.29 mmol) as the starting material. The obtained 4'-O-Acetyl Spinozin K (204.9 mg, yield 93%) was a white glass. FDMS, m/e (relative intensity) 759 (100).

<u>Example I8-4'-O-[(S-methyl)dithiocarbonyl] Spinozin K</u>

Spinozin K (201.0 mg, 0.28 mmol) and imidazole (catalytic amount) were dissolved in anhydrous THF (2 ml), and stirred at room temperature under nitrogen. Sodium hydride (50% mineral oil solution; 25 mg, 0.52 mmol) was added to the solution, followed by carbon disulfide (90 ml, 1.5 mmol) and then methyl iodide (90 ml, 1.44 mmol). After stirring for 1 hour at room temperature, acetic acid (90ml) was added, and the mixture was poured into saturated NaHCO₃middle. Followed by CH₂Cl₂Extract the water layer. Wash CH with salt water₂Cl₂Part, dry (MgSO₄) And evaporated at room temperature to provide 4'-O-[(S-methyl)dithiocarbonyl]spinoxine K (218.1 mg, yield 97%) as a yellow glass. FDMS, m/e (relative intensity) 808 (100).

<u>Example I9-3'-O-[(S-methyl)dithiocarbonyl)] Spinozin J</u>

The reaction was carried out as described in Example I8, with Spinozin J (207.1 mg, 0.29 mmol) as the starting material. The obtained 3'-O-[(S-methyl)dithiocarbonyl)] Spinozin J (224.0 mg, yield 96%) was a yellow glass. FDMS, m/z (relative intensity) 808 (100).

<u>Example I10-2'-O-[(S-methyl)dithiocarbonyl] Spinozin H</u>

The reaction was carried out as described in Example I8, with Spinozin H (204.0 mg, 0.28 mmol) as the starting material. The obtained 2'-O-[(S-methyl)dithiocarbonyl]spinoxine H (218.8 mg, yield 97%) was a yellow glass. FDMS, m/z (relative intensity) 808 (100).

<u>Example I11-2'-O-[(S-methyl)dicarbonyl]spinoxine Q</u>

The reaction was carried out as described in Example I8, with Spinozin Q (1.00 gms, 1.4 mmol) as the starting material. The obtained 2'-O-[(S-methyl)dicarbonyl]spinoxine Q (1.13 gms, yield 98%) was a yellow glass. FDMS, m/z, (relative intensity) 822 (100).

<u>Example I12-3'-O-(S-methyl)dithiocarbonyl) Spinozin L</u>

Spinozin L (1.65 gms, 2.2 mmol) was dissolved in anhydrous THF (50ml) and cooled in an ice bath under nitrogen. Add imidazole (21.2 mg, 0.3 mmol) to the solution, then add sodium hydride (60% mineral oil solution; 105.0 mg, 2.6 mmol) followed by carbon disulfide (700 ml, 11.64 mmol) and methyl iodide (700 ml, 11.1 mmol). The mixture was stirred at room temperature for 1 hour, then a small amount of NaH was added and stirring was continued for 1 hour. Pour the mixture into saturated ammonium chloride solution and add CH₂Cl₂extraction. Dry CH₂Cl₂Part (K₂CO₃) And evaporated at room temperature to provide 3'-O-(S-methyl)dithiocarbonyl)spinoxine L (1.86 mg, yield 100%) as a yellow solid. FDMS, m/e (relative intensity) 821 (100), 160 (4).

<u>Example I13-4'-Deoxy-Spinozin K</u>

The compound 4'-O-[(S-methyl)dithiocarbonyl] Spinozin K (182.5 mg, 0.23 mmol) was dissolved in toluene (10 ml). Tributyltin hydride (93 ml, 0.35 mmol) and AIBN (catalytic amount) were added and the solution was refluxed. After another 2 hours, more tributyltin hydride (100 ml) was added. The mixture was refluxed for another 12 hours and stirred at room temperature for 5 hours. Then the solvent was evaporated to a small volume and then chromatographed on a silica gel column (80% EtOAc/hexane) to provide 4'-deoxy Spinozin K (59.9 mg, yield 37%) as a colorless glass Things. FDMS, m/e (relative intensity) 702 (100).

<u>Example I14-3'-Deoxy Spinozin J</u>

The compound 3'-O-[(S-methyl)dithiocarbonyl] Spinozin J (849.1 mg, 1.05 mmol) was dissolved in anhydrous toluene (50 ml). Fresh tributyltin hydride (500 ml, 1.6 mmol) and AIBN (10.2 mg) were added and the mixture was refluxed. After 8 hours, AIBN (32.8 mg) was added, and reflux was continued for 2 hours. After standing at 5°C for 48 hours, the solvent was evaporated to a small volume. The residue was chromatographed on a silica gel column (3.5% MeOH/CH₂Cl₂) To provide 3'-deoxyspinoxine J (557.0 mg, yield 76%) as a colorless glass. FDMS, m/z (relative intensity) 701 (100).

<u>Example I15-2'-Deoxy Spinozin H</u>

The compound 2'-O-[(S-methyl)dithiocarbonyl] Spinozin H (1.15 gms, 1.4 mmol) was dissolved in anhydrous toluene (50 ml). Fresh tributyltin hydride (750 ml, 2.8 mmol) and AIBN (30 mg) were added and the mixture was refluxed. After 2.5 hours, AIBN (20.5 mg) was added and reflux continued for 5 hours. After stirring for 11.5 hours at room temperature, the solvent was evaporated to a small volume and the residue was chromatographed on a silica gel column (3.5% MeOH/CH₂Cl₂) To provide 2'-deoxyspinoxine H (821.5 mg, yield 84%) as a colorless glass. FDMS, m/z (relative intensity) 701 (100).

<u>Example I16-2'-Deoxy Spinozin Q</u>

The compound 2'-O-[(S-methyl)dithiocarbonyl] Spinozin Q (1.08 gms, 1.3 mmol) was dissolved in dry toluene (50 ml). Fresh tributyltin hydride (750 ml, 2.8 mmol) and AIBN (47.5 mg) were added and the mixture was refluxed for 2.5 hours and then cooled to room temperature. After 20 hours, the solvent was evaporated and the residue was applied to a silica gel column (3.54 MeOH/CH₂Cl₂) Upper chromatography to provide 2'-deoxyspinoxine Q (912.9 mg, yield 98%) as a colorless glass. FDMS, m/z (relative intensity) 716 (100).

<u>Example I17-3'-Deoxy Spinozin L</u>

The reaction was carried out as described in Example I16, with 3'-O-(3-methylthiocarbonyl)spinoxine L (1.85 gms, 2.25 mmol). 3'-Deoxy Spinozin L (1.16 gms, 74% yield), a colorless glass. FDMS, m/e (relative intensity) 716 (100).

<u>Example I18-9-O-(α-L-3,4-Di-O-acetylrhamnosyl) Spinozin A 9-Psa</u>

Combine Spinozin A 9-Psa (1.56 gms, 2.9 mmol) and 1-bromo-2,3,4-tri-O-acetylrhamnose¹(1.52 gms, 4.3 mmol) dissolved in CH₂Cl₂(75 ml; anhydrous). Tetramethoxine (1.0 ml, 8.4 mmol) was added, followed by silver triflate (823.8 mg, 3.2 mmol), and the reaction flask was covered with aluminum foil. After stirring for 24 hours, the reaction mixture was filtered through diatomaceous earth at room temperature and in a dark room, and filtered with CH₂Cl₂Clean the diatomaceous earth. Collect the filtrate and add saturated NaHCO₃Washing and drying (MgSO₄) And evaporate at room temperature. The residue was placed under high vacuum to produce a viscous yellow oil 3.29 gms. Put the substance in a silica gel column (5%EtOH/CH₂Cl₂, And then wash the column with 100% EtOH) to provide the product 9-O-(α-L-3,4-di-O-acetylrhamnosyl) Spinozin A 9-Psa( 259.6 mg, 15% based on the recovered Spinozin A 9-Psa, it is a colorless glass). IR(KBr, cm^-1) 3480.99 (OH), 2939.92, 1747.73, 1661.89, 1457.41, 1373.49, 1232.67, 1164.19, 1125.61, 1042.66, 988.64, 902.80. FDMS (m/z, relative intensity) 774 (100). (C₄₂H₆₃NO₁₂Analysis of ): Estimated value: C: 65.18, H: 8.20, N: 1.81; actual value: C: 64.94, H: 8.28, N: 1.97.

<u>Example I19-3,4-Di-O-ethyl-6-deoxy-L-glucenose</u>

Combine iodoethane (32 mL, 0.40 mol), 3,4-di-O-acetyl-6-deoxy-L-glucenose (4.28 g, 0.20 mol), 50% NaOH aqueous solution (43 mL , 0.81 mol) and DMSO (11.4. mL, 0.16 mol) were sequentially added to a 300-mL round bottom three-necked flask equipped with a top stirrer. Start stirring and add tetrabutylammonium hydrosulfate (2.04 g, 0.006 mol) in a single portion. Stirring is continued for 66 hours. Mixture in water and Et₂Balance between O. Take Et₂O (20 mL) extract the water layer twice and the last time with 1:l Et₂O-CH₂Cl₂(20 mL) extraction. The organic layers were combined, washed successively with water, diluted sodium thiosulfate aqueous solution, water and brine, dried with sodium sulfate and potassium carbonate, and concentrated under reduced pressure. A white solid precipitated during evaporation. The mixture was diluted with pentane and the solids were removed by filtration. The solvent was removed from the filtrate under reduced pressure to produce a light yellow oil (3.2 g, 87%), which was sufficiently homogeneous for subsequent reactions without additional purification.¹H-NMR d 6.32(dd, J=6.1,

<img file="TW487559B_D0189.tif" />

<u>Example I20-3,4-Di-On-propyl-6-deoxy-L-glucenose</u>

Combine iodine n-propane (39 mL, 0.40 mol), 3,4-di-O-acetyl-6-deoxy-L-glucenose (4.28g, 0.20 mol), 50% NaOH aqueous solution (43 mL, 0.81 mol) and DMSO (11.4 mL, 0.16 mol) were sequentially added to a 300-mL round bottom three-necked flask equipped with a top stirrer. Start stirring and add tetrabutylammonium hydrosulfate (2.04 g, 0.006 mol) in one portion. The mixture was stirred at room temperature for 17 hours. The mixture is equilibrated between water and pentane. The aqueous layer was extracted with additional pentane. The organic layers were combined and washed successively with water (3 times), diluted sodium thiosulfate, diluted sodium bicarbonate and brine. The solvent was removed under reduced pressure (first 20 torr and then 0.1 torr) to obtain a light yellow oil (4.0 g, 94%), which was ready for use without further purification.¹H-NMR d<img file="TW487559B_D0190.tif" /><img file="TW487559B_D0191.tif" />

<u>Example I21-3,4-Di-Oi-propyl-6-deoxy-L-glucenose</u>

Iodine i-propane (40 mL, 0.40 mol), 3,4-di-O-acetyl-6-deoxy-L-glucenose (4.28 g, 0.20 mol), 50% NaOH aqueous solution (43 mL, 0.81 mol) and DMSO (11.4 mL, 0.16 mol) were added to a 300-mL round bottom three-necked flask equipped with a top stirrer. Start stirring and add tetrabutylammonium hydrosulfate (2.04 g, 0.006 mol) in a single portion. The mixture was stirred at room temperature for 67 hours and at reflux temperature for 24 hours. The mixture was cooled to room temperature and equilibrated between water and pentane. The aqueous layer was extracted with pentane. The organic layers were combined and washed with water (4 times), diluted sodium thiosulfate, water and saturated sodium bicarbonate. Remove the solvent under reduced pressure (first 20 Torr and then 0.1 Torr to produce a light yellow oil (1.2 g, 28%), which is ready for use and

<img file="TW487559B_D0192.tif" />

<u>Example I22-9-(2-Deoxy-3-O,4-O-di-n-propyl-1-a-rhamnosyl)-spinoxine A 9-pseudoglycan Compounds and 9-(2-deoxy-3O,4-O-di-n-propyl-1-β-rhamnosyl)-spinoxine A 9-pseudoglycone compound</u>

Using the same steps and reactants as described in Example I29, the amount used is as follows: Spinozin A 9-pseudoglycone compound (544 mg, 1.00 mmol), CH₂Cl₂Ligand compound (4 mL), 3,4-Di-On-propyl-6-deoxy-L-glucenose (429 mg, 2.00 mmol) and camphorsulfonic acid (279 mg, 1.20 mmol). The crude product was chromatographed on silica gel (80 g) with 3% MeOH in CHCl₃Solution dissolution and inverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25 cm × 20 mm), with 90% MeOH and 10% water (containing 0.1% v/v concentrated NH₄OH aqueous solution) in order to provide the mutagenic isomers which are all white foams; α-mutagenic isomers (307 mg,

<img file="TW487559B_D0193.tif" />

<u>Example I23-9-(2-Deoxy-3-O,4-O-di-i-propyl-1-a-rhamnosyl)-spinoxine A 9-pseudoglycan Compound</u>

Using the same steps and reactants as in Example I29, the usage amount is as follows: Spinozin A 9-pseudoglycone compound (543 mg, 1.00 mmol), CH₂Cl₂(4 mL), 3,4-Di-On-propyl-6-deoxy-L-glucose (428 mg, 2.00 mmol) and camphorsulfonic acid (279 mg, 1.20 mmol). The crude product was chromatographed on silica gel (100 g) with EtoAc and on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25cm×20 mm) with 90% MeOH and 10% water ( Contains 0.1% v/v concentrated NH₄OH aqueous solution) to produce a white powder (306 mg, 40%).¹H-NMR d 4.84 (br d, J=2.9, 1H), 1.40-1.13 (m, 25H).

<u>Example I24-1-(O,O-Diethyldithiophosphoryl)-2-deoxy-3,4-di-O-acetyl-rhamnose</u>

3,4-Di-O-acetyl-6-deoxy-L-glucenose (2.14 g, 10.0 mmol) was dissolved in benzene (20 mL). Within 5 minutes, a solution of O,O-diethylsulfophosphoric acid (1.96 g, 10.5 mmol) in benzene (10 mL) was added and the resulting mixture was stirred at room temperature for 18 hours. Additional dithiophosphoric acid (0.10 g, 0.54 mmol) was added and the mixture was stirred for another 20 hours. The mixture was extracted with diluted sodium bicarbonate aqueous solution (2 times), water and brine. The mixture Na₂SO₄And MgSO₄dry. The solvent was removed under reduced pressure to give a yellow oil (4.03 g, 101%).

<u>Example I25-9-(2-deoxy-3-O,4-O-diethyl-1-α-rhamnosyl)-spinoxine A 9-pseudoglycone compound</u>

The 4A molecular sieve powder (500 mg) and AgF (685 mg, 5.40 mmol) were suspended in acetone (2 mL). Add a single part of 1-(O,O-diethyl dithiophosphoryl)-2-deoxy-3,4-di-O-acetyl-rhamnose (400mg, 1.00 mmol) ethyl Solution. The resulting dark green-brown mixture was stirred at room temperature for 6 days. The mixture was filtered through Celite to remove the black precipitate, the solvent was evaporated and the residue was dissolved in EtOAc-Et₂O to 1:1 mixture (20 mL). The mixture was washed successively with NaOH (2 times), water (2 times) and brine. Evaporate the solvent and put the residue on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25 cm×20 mm), using 90% MeoH and water (containing 0.1% v/v concentrated NH₄OH aqueous solution) chromatography to give an amorphous white solid (36 mg, 9%).¹H-NMR d 4.85 (br d, J2.9, 1H), 2.08 (s, 3H), 2.01 (s, 3H).

<u>Example I26-9-(2H-5-R-acetoxy)-5,6-dihydro-6-S-methyl-2-a-pyranyl)-spinoxine A-9- Pseudoglycan compounds</u>

Spinoxin A 9-pseudoglycan compound (272 mg, 0.500 mmol) and 3,4-di-O-acetyl-6-deoxy-L-glucenose (214 mg, 1.00 mmol) dissolved in CH₂Cl₂(3 mL). The substance that passed through the molecular sieve (4A, 40 mg) and camphorsulfonic acid (128 mg, 0.550 mmol) were added. After 18 hours, additional camphorsulfonic acid (20 mg, 0.086 mmol) was added. The mixture was stirred for 3 days. Take CH₂Cl₂(10 mL) The mixture was diluted and diluted with a 1:1:1 mixture of water-saturated sodium bicarbonate aqueous solution-1.0N NaOH. Take CH₂Cl₂(2 times) The aqueous layer was extracted and the combined organic layer was washed with water, dried with anhydrous sodium sulfate and evaporated to obtain a dark oil. Put the residue on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25 cmX20 mm), using 90% MeoH and 10% water (containing 0.1% v/v concentrated NH₄OH aqueous solution) chromatography to produce a white foam (44 mg, 11%).¹H-NMR d5.92-5.73 (m, 4H), 5.01 (br s, 1H), 2.09 (s, 3H).

<u>Example I27-5,6-dihydro-9-(2-deoxy-3-O,4-O-diethyl-1-α-rhamnosyl)-spinoxine A 9-pseudo Glycogen compounds</u>

The 9-(2-deoxy-3-O,4-O-diethyl-1-a-rhamnosyl)-spinoxine A 9-pseudoglycone compound (200 mg, 273 mmol) and cyclohexane (0.40 mL, 3.95 mmol) were dissolved in pure ethanol (4 mL). Palladium/charcoal (10%, 20 mg: 19 mmol) was added carefully and the resulting mixture was heated at reflux temperature for 3 hours. The mixture was stirred at room temperature for another 17 hours. The mixture was filtered through Celite and evaporated under a stream of nitrogen. Put the residue on a reverse phase column (Kromasil'Cl8, silica ODS, 100A, 10 m, spherical, 25 cmX20 mm), using 90% MeoH and 10% water (containing 0.1% v/v concentrated NH₄OH aqueous solution) chromatographed to give a glassy solid (45 mg, 22%).¹H-NMR d 6.83 (br s, 1H), lack of 2H-multiplex between 5.9-5.7.

<u>Example I28-5,6-Dihydro-9-(2-deoxy-3-O,4-O-di-n-propyl-1-α-rhamnosyl)-spinoxine A 9-pseudoglycan compound</u>

Add cyclohexane (3.0 mL, 28.6 mmel) and palladium/charcoal (10%, 43 mg) to pure ethanol (3 mL). Add 9-(2-deoxy-3-O,4-O-di-n-propyl-1-a-rhamnosyl)-spinoxine A 9-pseudoglycone compound (188 mg, 0.248 mmol) in ethanol (2 mL) and the mixture was heated at reflux temperature for 5 hours. At 1 hour and 4.5 hours, 38 mg and 67 mg of palladium/charcoal were added to the reaction mixture, respectively. The mixture is cooled, filtered through diatomaceous earth and concentrated into a thick oil. Dissolve the residue in Et₂O and wash with concentrated sodium bicarbonate and brine, dry with anhydrous potassium carbonate and evaporate to produce light gray foam. The substance was placed on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25 cmX20 mm), using 92% MeoH and 8 water (containing 0.1% v/v concentrated NH₄OH aqueous solution) chromatography to produce an amorphous powder (80 mg, 42%).¹H-NMR d 6.73 (br s, 1H), lack of 2H-multiplex between 5.9-5.7.

<u>Example I29-9-(2-Deoxy-3-O,4-O-diethyl-1-α-rhamnosyl)-spinoxine A 9-pseudoglycone compound</u>

Dissolve Spinozin A 9-pseudoglycone compound (136 mg, 0.250 mmol) in CH₂Cl₂(1 mL). A single portion of 3,4-di-O-ethyl-6-deoxy-L-glucenose (93 mg, 0.50 mmol) and camphorsulfonic acid (70 mg, 0.30 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 hour. Saturated aqueous sodium bicarbonate solution was added and the mixture was diluted with EtOAc (5 mL). The mixture was equilibrated between saturated sodium bicarbonate (10 mL) and a mixture of 1.0 N NaOH (2 mL) and EtOAc (10 mL). The aqueous layer was extracted with additional EtOAc. The combined organic layer was successively washed with saturated sodium bicarbonate and brine and washed with anhydrous Na₂SO₄dry. Remove the solvent under reduced pressure and place the residue on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10 m, spherical, 25 cmX20 mm), using 90% MeoH and 10% water (containing 0.1% v /vConcentrate NH₄OH aqueous solution) chromatography to give an amorphous white solid (74 mg, 40%).

<img file="TW487559B_D0194.tif" />

<u>Part J is modified by the substitution of rhamnose to modify the pseudoglycan compound A2 Example J1-9-O-[(1'S,2'S,3'S)-2-methyl-4-(1',2'-bis- Acetyloxy-3'-hydroxybutyl)-5-hydroxy-1,3-dioxolane-2-yl]spinoxine A 9-Psa in the mixture of dioxolane positions 2 and 5</u>

To Spinozin A 9-Psa (100.8 mg, 0.19 mmol) in dichloromethane, add 1-bromo-2,3,4-tri-O-acetyl-rhamnose¹(67.9 mg, 0.19 mmol), followed by silver triflate (52.3 mg, 0.2 mmol) and then diisopropylethylamine (35 mg, 0.2 mmol). The mixture was stirred for 3.5 hours at room temperature in a dark room. Then the mixture was diluted with dichloromethane and washed with water. Then wash the dichloromethane part with brine and use MgSO₄Dry and concentrate at room temperature under reduced pressure. The product is separated from the unreacted starting material by chromatography on silica, and eluted with 5% methanol in dichloromethane. The resulting 9-O-[(1'S,2'S,3'S)-2-methyl-4-(1',2'-bis-acetoxy-3'-hydroxybutyl)-5-hydroxy-1, 3-Dioxolane-2-yl] Spinozin A 9-Psa in a mixture of dioxolane positions 2 and 5 (75.2 mg; yield 49%), FDMS, m/e (relative intensity) 816(M⁺，70)，815(100)，585(10)。

<u>Example J2-9-O-[(1'S,2'S,3'S)-2-methyl-4-(1',2'-3'-trihydroxybutyl)-5-hydroxy-1,3-dioxide Pentan-2-yl] Spinozin A 9-Psa is a mixture of positions 2 and 5 of the dioxolane ring</u>

In 9-O-[(1's,2'S,3'S)-2-methyl-4-(1',2'-bis-acetoxy-3'-hydroxybutyl)-5-hydroxy-1,3 -Dioxolane-2-yl] Spinozin A9-Psa at the positions 2 and 5 of the dioxolane ring (104.2 mg, 0.12mmol) in methanol (4 ml) solution, add a small amount of sodium hydride ( 60% mineral oil dispersion) and generate gas. The reaction mixture was stirred at room temperature for 15 minutes. The mixture was then diluted with dichloromethane and washed with saturated aqueous ammonium chloride solution. Wash the dichloromethane part with brine and use MgSO₄Dry and concentrate at room temperature under reduced pressure. The resulting 9-O-[(1'S,2'S,3'S)-2-methyl-4-(1',2',3'-trihydroxybutyl)-5-hydroxy-1,3-dioxolane- 2-base] Spinozin A9-Psa is a mixture of positions 2 and 5 of dioxolane (44.2 mg; yield 50%), as a beige solid, FDMS, m/e (relative intensity) 733 (95 ), 732 (M⁺，100)，543(10)。

<u>Example J3-9-O-(2-Methoxyethoxymethyl) Spinozin A 9-Psa</u>

To Spinozin A 9-Psa (474.3 mg, 0.87 mmol) in dichloromethane (20 ml), add diisopropylethylamine (225 μl, 1.3 mmol) and 2-methoxyethoxy Methyl chloride (100 ul, 0.87 mmol). The reaction mixture was heated to reflux temperature for 24 hours and then stirred at room temperature for 2 days. Then dilute the mixture with dichloromethane, and with saturated NaHCO₃Water solution cleaning. Take K₂CO₃The dichloromethane portion was dried and concentrated under reduced pressure at room temperature. The product was separated from the unreacted material by chromatography on silica, and was eluted with a 4% methanol solution in dichloromethane, followed by a 10% methanol solution in dichloromethane. The resulting 9-O-(2-methoxyethoxymethyl)spinoxine A9-Psa (226.5 mg; yield 56%, based on the recovered starting material) was a colorless glass. FDMS, m/e (relative intensity) 632 (M⁺，40)，631(100)。

<u>Example J4-9-O-Methyl Spinozin A 9-Psa</u>

To Spinozin A 9-Psa (200.1 mg, 0.37 mmol) in THF (2ml), sodium hydride (50% mineral oil dispersion; 34.6 mg, 0.72 mmol) was added, followed by methyl iodide (90 ul , 1.4 mmol). The reaction mixture was stirred at room temperature for 3.5 hours with slow evaporation. Dilute the mixture with ether and wash with water. Then wash the ether part with salt water, with K₂CO₃Dry and concentrate at room temperature under reduced pressure. The product was separated from unreacted substances by chromatography on silica, and eluted with 7% methanol in dichloromethane. The obtained 9-O-methylspinoxine A 9-Psa (75.6 mg; yield 37%) is a white glass, FDNS, m/e (relative strength) 558 (M⁺，60)，557(100)。

<u>Example J5-9-O[(2R,3S,4S,5R,9R)-2-methyl-3-acetoxy-4-dimethylamino-7-methyl-1,6,8 -Trioxo[4.3.0]bicyclononane-7-yl]spinoxine A 9-Psa</u>

Spinozin A 9-Psa (564.5 mg, 1.04 mmol) and 1-bromo-2,4-di-O-acetylmycaminobromide²(487.4 mg, 1.15 mmol) in anhydrous dichloromethane (30 ml) solution, add tetramethylammonium (358 ml, 3.0 mmol) and silver triflate (298.4 mg, 1.18 mmol). The reaction mixture was stirred at room temperature in a dark room for 20 hours. The reaction mixture was then diluted with dichloromethane and washed with 1N NaOH. Then wash the dichloromethane part with brine and use K₂CO₃Dry and evaporate at room temperature under reduced pressure. The product was separated from unreacted substances by chromatography on silica, and eluted with 5% methanol in dichloromethane. The resulting 9-O-[(2R,3S,4S,5R,9R)-2-methyl-3-acetoxy-4-dimethylamino-7-methyl-1,6,8- Trioxo[4.3.0]bicyclononane-7-yl]spinoxine A 9-Psa (101.9 mg; yield 13%, based on the recovered starting material), a mixture of isomers . FDMS, m/e (relative intensity) 828 (20), 800 (M⁺-H, 100), 759 (20), 543 (30).

<u>Example J6-9-O-Acetyl Spinozin A 9-Psa</u>

To Spinozin A 9-Psa (167.3 mg, 0.31 mmol) in chloroform (5ml), add diisopropylethylamine (220 uI, 1.26 mmol) and then add acetyl chloride (220 ml, 3.1 mmol). The reaction mixture was stirred at room temperature for 2 hours. Then the mixture was diluted with ether and washed with 1N NaOH, with K₂CO₃Dry and evaporate at room temperature under reduced pressure. The product was purified by chromatography on silica and eluted with 50% ethyl acetate in hexane. The obtained 9-O-Acetyl Spinozin A 9-Psa (110.6 mg; yield 61%) is a white deviating glass, FDMS, m/e (relative intensity) 586(50), 585(M⁺，100)。

<u>Example J7-9-O-Carboethoxy Acetyl Spinozin A 9-Psa</u>

The reaction was carried out as described in Example J6, starting with Spinozin A 9-Psa (177 mg, 0.33 mmol) and ethylmalonyl chloride. The 9-O-Carboethoxyacetylspinoxine A 9-Psa (124.4 mg; yield 57%) obtained was a white deviating glass, FDMS, m/e (relative strength) 658 (MH⁺，100)。

<u>Example J8-9-O, N-Dimethyl Spinozin A 9-Psa Iodide</u>

To Spinozin A 9-Psa (101.1 mg, 0.19 mmol) in DMF (0.5 ml) solution, silver(II) oxide (81.9 mg, 0.35 mmol) and methyl iodide (35 ul, 0.56 mmol) were added. The reaction mixture was stirred in a dark room at room temperature for 7 days. The mixture was then diluted with ether and filtered. The precipitate was dissolved in water and washed with fresh ether. The water layer was partially freeze-dried and the residue was triturated with ether and dried under a stream of nitrogen. The obtained 9-O,N-dimethylspinoxine A 9-Psa iodide (13.5 mg; yield 10%) was a yellow-brown solid, FDMS, m/e (relative intensity) 761(10), 573 (30), 572(M⁺，100)，188(25)。

<u>Example J9-9-O-[(S-methyl)dithiocarbonyl) Spinozin A 9-Psa</u>

To Spinozin A 9-Psa (205.5 mg, 0.38 mmol) and imidazole crystals in anhydrous THF (2 ml) solution, add sodium hydride (50% mineral oil dispersion; 33.8 mg, 0.7 mmol), then add carbon disulfide (90 ul, 1.5 mmol) followed by methyl iodide (90 ul, 1.4 mmol). The reaction mixture was stirred at room temperature and then glacial acetic acid (90 ul) was added. Then pour the reactant into saturated NaHCO₃The aqueous solution was extracted with dichloromethane. Wash the dichloromethane part with brine and use MgSO₄Dry and evaporate at room temperature under reduced pressure. The obtained 9-O-[(S-methyl)dithiocarbonyl)spinoxine A 9-Psa (230 mg; yield 95%) is a yellow glass, FDMS,^-m/e (relative intensity) 634 (M⁺，60)，633(100)，142(50)。

<u>Example J10-9-O-[(2R,3S,4S,5R,9R)-2-methyl-3-hydroxy-4-dimethylamino-7-methyl-1,6,8-tri Oxo[4.3-0]bicyclononane-7-yl]spinoxine A 9-Psa</u>

The reaction was carried out as described in Example J2, with 9-O-[(2R,3S,4S,5R,9R)-2-methyl-3-acetoxy-4-dimethylamino-7-methyl Base-1,6,8-trioxo[4.3.0]bicyclononane-7-yl]spinoxine A 9-Psa (40 mg, 0.05 mmol) was used as the starting material. The resulting 9-O-[(2R,3S,4S,5R,9R)-2-methyl-3-hydroxy-4-dimethylamino-7-methyl-1,6,8-trioxo [4.3-O]Bicyclononane-7-yl] Spinozin A 9-Psa (15.2 mg; yield 40%), FDMS, m/e (relative intensity) 759 (M⁺，100)，543(35)。

<u>Example J11-9-Om-Methoxyoxy-3-spinoxine A 9-pseudoglycone compound</u>

Same procedure as described in Example J18, but immediately add pyrrolidine and pyridine and Et in the reactants₂O replaces EtOAc. Use the same chromatographic conditions to produce an amorphous white solid (110 mg, 54%),¹H-NMR d

<img file="TW487559B_D0195.tif" />

<u>Example J12-9-O-p-Methoxyoxy Spinozin A 9-pseudoglycone compound</u>

Follow the steps described in Example J18, but immediately add pyrrolidine and pyridine and use Et in the reactants₂O replaces EtOAc. Use the same chromatographic conditions to produce an amorphous white solid (100 mg, 49%),¹H-NMR d 7.98

<img file="TW487559B_D0196.tif" />

<u>Example J13-9-table-9-O-phenoxyacetyl-spinoxine A 9-pseudoglycone compound</u>

Spinozin A 9-pseudoglycone compound (544 mg, 1.00 mmol) and triphenylphosphine (525 mg, 2.00 mmol) were dissolved in benzene (5 mL) and the solution was cooled to 4°C. Diethyl azocarboxylate (0.31 mL, 2.00 mmol) was added dropwise at a rate to maintain the temperature below 5°C and the color of the reagent disappeared between each drop. While maintaining 5°C-11°C, the mixture was stirred for another 20 minutes and without cooling for 60 minutes. The reaction was quenched with water and diluted with aqueous sodium bicarbonate solution and partitioned between EtOAC and the diluted aqueous sodium bicarbonate solution. The aqueous layer was extracted with EtOAc solution (2 times). The combined organic layer was washed successively with diluted sodium bicarbonate (2 times) and brine. The mixture was dried with anhydrous sodium sulfate and evaporated to produce an oily solid. Dissolve the solid in Et₂O and extract with diluted HCl (3 times). Combine the milky water layer and add Et₂O extraction. Adjust the pH of the water layer to close to 10 with 1.0 N NaOH. The combined EtOAc fractions were washed with brine (2 times) and dried over anhydrous sodium sulfate. The solvent was evaporated and the residue was chromatographed on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10m, spherical, 25 cm X 20 mm) with 90% MeOH and 10% water (containing 0.1% v/ v thick NH₄OH aqueous solution) to produce a white amorphous solid (0.54 g<img file="TW487559B_D0197.tif" /><img file="TW487559B_D0198.tif" />

<u>Example J14-9-O(2,3,5-Tri-O-methyl)-α-L-Ribanosylspinoxine A 9-Psa and 9-O-(2,3,5-Tri -O-methyl-β-L-ribopyranospinoxine A 9-Psa</u>

Obtained a yield of 84% α: β9-O-(2,3,5-tri-O-methyl)-α-L-(ribopyranosyl) Spinozin A 9-Psa 9:1 The mixture was obtained from Spinozin A 9-psa (0.33 g, 0.63 mmol), pyridine p-toluenesulfonate (0.2 g, 0.8 mmol) and O-(2,3,5-tri-O-methyl) The reaction of β-β-L-ribopyranosyl)trichloroacetimidate (1.6 g, 4.75 mmol) was carried out in the same procedure as in Example J18. The α and β isomers were separated by HPLC with a 41.4 mm (id) x 25 cm (1) reverse phase Cl8 column. The α-mutaisomer precipitates first. Alpha-mutant: 100 mg;

<img file="TW487559B_D0199.tif" />

<u>Example J15-9-O-(2,3,4,6-tetra-O-methyl)-α-L-mannosylspinoxine A 9-Psa and 9-O-(2,3 ,4,6-Tetra-O-methyl-β-L-mannosyl) Spinozin A 9-Psa</u>

α and β9-O-(2,3,4,6-Tetra-O-methyl)-α-L-mannanosyl) Spinozin A 9-Psa 1.9:1 mutagenic isomer The mixture (0.4 g, yield 71%) was obtained from Spinozin A 9-psa (0.4 g, 0.74 mmol), pyridinium p-toluenesulfonate (0.25 g, 1.0 mmol) and O-(2 ,3,4,6-Tetra-O-methyl-α-L-mannanosyl) tris-chloroacetimidate (1.7 g, 4.49 mmol) reaction, the reaction is based on the example Perform the same steps as described in J18. The mutagenic isomers were separated by reverse phase HPLC, using 2 parts of Cl8 bonded silica (8μm) column (41.4 mm(id)x 25 cm(1)), with 10% H₂O (containing 0.1% NH₄OH) MeOH solution is the eluent. Beta mutagenic isomers are precipitated first. Beta mutamer: 18 mg

<img file="TW487559B_D0200.tif" />

<u>Example J16-9-O-(N-desmethyl-N(2,2,2-trichloroethoxycarbonyl)-α-D-aminoglycosyl) Spinozin A 9-Psa and 9- O-(N-desmethyl-N-2,2,2-trichloroethoxycarbonyl)-β-D-aminoglycosyl) Spinozin A 9-Psa</u>

Add a portion of trimethylsilyl trifluoromethanesulfonate (0.7 mL, 3.6 mmol) to the cold (-40°C), properly stirred N-desmethyl-N-(2,2,2-trichloro Ethoxy)carbonyl-1-O-(4-nitrooxy)-D-amino sugar (about 6α:1β mutarotonic mixture) (0.7 g, 1.49 mmol) and 4A molecular sieve (Beads, 8-12 mesh, 2.0 g) of CH₂Cl₂(30 mL) and Et₂O (30 mL) in the suspension of the mixture. The mixture was heated to -10°C for 20 minutes, and while maintaining the temperature of the reactants at -10°C, spinoxin A 9-Psa (0.5 g, 0.92 mmol) of CH was added dropwise within 20 minutes₂Cl₂(30 mL) solution. After stirring for 4 hours at 10°C, the whole mixture was poured into saturated NaHCO with proper stirring₃(240 mL) and ice (about 100 g). Separate the organic layer and add CH₂Cl₂(2 x 75 mL) The aqueous layer was extracted. With saturated NaHCO₃(75 mL) The combined organic layer was washed, then washed with brine (75 mL) and dried (MgSO₄). 1.1 g residue remained in the concentrate, and the residue was flash chromatographed with silica (175 mL) using 3% MeOH in CH₂Cl₂For eluent, to obtain 0.6 g (77%) of clean glycosylated 9-O-(N-desmethyl-N-(2,2,2-trichloroethoxycarbonyl-α-D-amine) Spinozin A 9-Psa, a mixture of 3:1 mutamers from α to β, is a colorless foam. The mixture is separated by reverse phase HPLC and a silica column bonded with Cl8 [41.4 mm(id)x 25 cm(1)), using 5%H₂O (containing 0.15% NH₄The solution of OH) in MeOH is the eluent to obtain individual pure metamorphic foreign matter. The β-mutaisomer precipitates first. β-mutamel: 140 mg; none

<img file="TW487559B_D0201.tif" />

<u>Example J17-9-O-(N-desmethyl-β-D-aminoglycosyl) Spinozin A 9-Psa</u>

The compound was prepared by the procedure used in Example J20. Obtain 37 mg of 9 from 100 mgof9-O-(N-desmethyl-N-2,2,2-trichloroethoxycarbonyl)-β-D-aminoglycosyl) Spinozin A 9-Psa -O-(N-desmethyl-β-D-aminoglycosyl) Spinozin A 9-Psa, white foam:¹H-NMR(

<img file="TW487559B_D0202.tif" />

<u>Example J 18-9-OO-Methylhydroxyloxyspinoxine A 9-pseudoglycone compound</u>

Dissolve Spinozin A 9-pseudoglycone compound (163 mg, 0.300 mmol) in CH₂Cl₂(0.6 mL). Add O-Methoxyoxychloride (56 ul, 0.38 mmol) and triethylamine (53 ul, 0.38 mmol). After 3 hours, 4-oxalidinoxidine (3 mg, 0.02 mmol) was added and stirred for 15 hours. The reaction was partitioned between EtOAc (5 mL) and 0.5N aqueous NaOH (5 mL). Continuously wash the organic layer with 0.5N NaOH aqueous solution and brine, and use anhydrous MgSO₄Dry and concentrate under reduced pressure. The residue was chromatographed on a reverse phase column (Kromasil' Cl8, oxosilica ODS, 100A, 10 m, spherical, 25cm X 20 mm), using 90% MeOH and 10% H₂O (contains 0.14v/v concentrated NH₄OH aqueous solution) to produce an amorphous white solid (110 mg, 54%)

<img file="TW487559B_D0203.tif" />

<u>Example J19-9-O-(2,3,4-Tri-O-methyl-L-lyxolanosyl) Spinozin A 9-Psa</u>

Dry CH containing powdered 4A molecular sieve (0.8 g) in properly stirred Spinozin A 9-Psa (0.4 g, 0.74 mmol) and pyridine p-toluenesulfonate (0.25 g, 1.0 mmol)₂Cl₂(50 mL) in the solution, add O-(2,3,4-tris-O-methyl-aL-lysopyranyl) tris-chloroacetimidate (2.3 g, 6.8 mmol) CH₂Cl₂(10 mL) solution. The reaction mixture was stirred for 72 hours, and then filtered through Celite, with CH₂Cl₂(25 mL) Wash the collected solids and combine the filtrate with saturated Na₂CO₃(2x20 ml) and saline (20 mL) washing, and drying (MgSO₄). After concentration, 2.8 g of residue was left. The residue was flash chromatographed with silica (240 mL), using 3% MeOH in CH₂Cl₂The solution is eluted to produce 0.45 g (85%) of a clean coupling product, which is β: αg-O-(2,3,4-tri-O-methyl-L-lysopyranosyl) Spinner Sin A 9-Psa 1.5:1 mixture of mutagenic isomers. The mixture was separated by HPLC and used in 41.4mm(id)x 25 cm(1) reverse phase Cl8 column in 3 approximately 150 mg fractions, using 10% H₂O (contains 0.15% NH₄OH) MeOH solution is the eluent. The β-mutaisomer precipitates first. β-mutamel: 102 mg; colorless foam;¹H-NMR<img file="TW487559B_D0204.tif" /><img file="TW487559B_D0205.tif" />

<u>Example J20-9-O-(N-desmethyl-aD-aminoglycosyl) Spinozin A 9-Psa</u>

Add activated Zn powder (0.4g) to 9-O-(N-desmethyl-N-(2,2,2-trichloroethoxycarbonyl)-aD-aminoglycosyl) spain that has been properly stirred A solution of Nosin A9-Psa 0.1 g, 0.12 mmol) in THF (3 mL), MeOH (0.5 mL) and 1M NH40Ac were added, and the mixture was stirred at room temperature for 3 days. The solid was removed by filtration and washed with THF (5 mL). Combine the filtrate and washing liquid and evaporate to dryness and add CH₂Cl₂(30 mL) Collect the residue. This CH₂Cl₂The solution is saturated with Na₂CO₃(5 mL) and brine washing and drying (MgSO₄). After concentration, 66 mg of residue was left. The residue was chromatographed with silica (20 mL) using 8% MeOH in CH₂Cl₂Solution to obtain 42 mg of 9-O-(N-desmethyl-aD-aminoglycosyl) Spinozin A 9-Psa as a white foam,¹H-NMR

<img file="TW487559B_D0206.tif" />

<u>K Part A83543 Tricyclic Pseudoglycan Compound A2 Modification on C9</u>

<u>Example K1-9-Epispinoxine A 9-Psa</u>

To a solution of 9-keto Spinozin A 9-Psa (207.9 mg, 0.38 mmol) in anhydrous methanol (10 ml), sodium borohydride (23 mg, 0.61 mmol) was added. The reaction mixture was stirred at room temperature for 40 minutes, then diluted with dichloromethane and washed with water. Wash the dichloromethane part with brine, use K₂CO₃Dry, and dry at room temperature and under reduced pressure. Separate the product on Cl8 column by preparative HPLC with ethyl acetate: methanol: 0.1% NH₄OAc (35:35:30 to 40:40:20, linear gradient within 60 minutes) dissolution. Obtained 9-Epispinoxine A 9-Pea (57mg; yield 28%), FDMS, m/e (relative intensity) 1084/(15), 544 (M⁺, 45), 543 (100) and Spinozin A 9-Psa (42 mg; yield 20%).

<u>Example K2-(9S) and (9R)-9-methylspinoxine A 9-Psa</u>

To a solution of 9-ketospinoxine A 9-Psa (336.8 mg, 0.62 mmol) in anhydrous THF (7 ml), methyl magnesium chloride (3.0 M; 250ul, 0.75 mmol) was added. During the addition of Grignard reagent, the reaction mixture was heated to reflux temperature, and then stirred at room temperature for 1 hour. Additional methylmagnesium chloride (500 u1, 1.5 mmol) was added and the mixture was stirred at room temperature for another 1 hour. The mixture was diluted with ether and washed with water. The ether part was filtered through celite to remove magnesium salt, washed with brine, and washed with K₂CO₃Dry and concentrate at room temperature under reduced pressure. Separate the product on Cl8 column by preparative HPLC with ethyl acetate: methanol: 0.1% NH₄OAc (30:30:40 to 45:45:10, linear gradient over 60 minutes) dissolution. The obtained (9S)-9-methylspinoxine A 9-Psa (93 mg; yield 27%), FDMS, m/e (relative intensity) 558 (M⁺, 30), 557(100) and (9R)-9-methylspinoxine A 9-Psa (55' mg; yield 16%), FDMS, m/e (relative intensity) 558 (M⁺，40)，557(100)。

<u>Example K3-9-deoxy-spinoxine A 9-Psa and 9-deoxy-8,9-dehydrospinoxine A 9-Psa</u>

To 9-O-[(S-methyl)dithiocarbonyl] Spinozin A 9-Psa (219.7mg; 0.35 mmol) in toluene (10 ml), add tributyltin hydride (250 ml, 1.0 mmol) and a trace amount of AIBN. The reaction mixture was heated to reflux temperature for 20 hours, and there was no obvious reaction. Add AIBN (micro) and continue heating for 28 hours. The mixture was then stirred at room temperature for 4 days and the solvent was removed at room temperature under reduced pressure. The product was initially purified by chromatography on silica, eluted with 5% methanol in dichloromethane, and then separated from the unreacted starting material by preparative HPLC on a Cl8 column. Ethylene: methanol: 0.14%NH₄OAc (40:40:20 to 45:45:10, linear gradient over 90 minutes). The obtained 9-deoxy-8,9-dehydrospinoxine A 9-Psa (8 mg; yield 4%), FDMS, m/e (relative intensity) 526 (M⁺, 65), 525 (100) and 9-deoxy-spinoxine A 9-Psa (34mg; 18% yield), FDMS, m/e (relative intensity) 528 (M⁺, 55), 527 (100), all are white solids.

<u>Example K4-9-O-Methyl Spinozin A Ag</u>

The reaction was carried out as described in Example 2, using 9-O-methylspinoxine A 9-Psa (188.8 mg, 0.34 mmol) as the starting material. The obtained 9-O-methyl Spinozin A Ag (126.9 mg; yield 90%) was a white solid, FDMS, m/e (relative intensity) 417 (M⁺，40)，416(100)。

<u>Example K5-9-Deoxy-9-N-morphoyl) Spinozin A 9-Psa</u>

In anhydrous methanol (7 ml) solution of 9-ketospinoxine A 9-Psa (154.4 mg, 0.28 mmol), moxan (244 u1, 2.8 mmol) was added. The reaction mixture was stirred at room temperature for 45 minutes, then sodium cyanoborohydride (84.5 mg, 1.3 mmol) was added. Stirring was continued for 7 hours at room temperature, and then diluted with ether. Wash the ether part with water, then wash with brine, and wash with MgSO₄Dry and concentrate at room temperature under reduced pressure. The product is separated from unknown impurities by chromatography on silica, and eluted with 5% methanol in dichloromethane. The obtained 9-deoxy-9-N-morphonyl) Spinozin A 9-Psa (48.5mg; yield 28%) is a mixture of isomers, FDMS, m/e (relative intensity) 614 (30), 613 (M+, 50), 612 (100).

<u>Implement Pour K6-9-Deoxy Spinozin A Ag</u>

The reaction was carried out as described in Example 2, using 9-deoxyspinoxine A 9-Psa (335 mg, 0.63 mmol) as the starting material. The obtained 9-deoxy Spinozin A Ag (129.5 mg; yield 53%) was a white solid, FDMS, m/e (relative intensity) (M⁺，45)，386(100)。

<u>Example K7-9-ketospinoxine A 9-Psa</u>

Suspend N-chlorobutadiamide (1.4 g, 10.5 mmal) in CH₂Cl₂(35 ml) and cool to -70°C under nitrogen. After adding diethyl sulfide (825 ul, 11.2 mmol) and stirring at -70°C for 30 minutes, slowly add Spinozin A 9-Psa (2.0 g, 3.7 mmol) in CH₂Cl₂(13 ml) solution. After stirring for 2 hours at -70°C, triethylamine (1.4 ml, 10 mmol) was added and the reaction was allowed to warm to room temperature. After stirring at room temperature for 20 hours, and standing at 0°C for 24 hours, with CH₂Cl₂Dilute the solution and use H₂O cleaning. Then wash CH with salt water₂Cl₂Part, dry (MgSO₄), and concentrated at room temperature under reduced pressure. The residue is in a silica gel column (7%MeOH/CH₂Cl₂) Upper chromatography to provide 9-ketospinoxine A 9-Psa (1.83 g, 91%) as a white glass. FDMS, m/e (relative intensity) 541 (100).

<u>Example K8-1"-α/β-9-O phenoxyacetyl-spinoxine A 9-pseudoglycone compound</u>

Spinozin A 9-pseudoglycone compound (163 mg, 0.300 mmol) and 4-oxrolidinoxidine (6 mg, 0.04 mmol) were dissolved in CH₂Cl₂(1 mL). Phenoxyacetyl chloride (55 mL, 0.40 mmol) was added in a single portion. The mixture was stirred at room temperature for 18 hours. The mixture was quenched with solid sodium bicarbonate and partitioned between EtOAc and aqueous sodium bicarbonate. The organic layer was washed with water and brine and dried with anhydrous magnesium sulfate. The solvent was evaporated and the residue was chromatographed on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10m, spherical, 25 cm X 20 mm), using 90% MeOH and water (containing 0.1% v/v) Concentrated NH₄OH aqueous solution) to obtain a white solid (108 mg,

<img file="TW487559B_D0207.tif" />

<u>Example K9-9-Epi-Spinozin A 9-Pseudoglycan compound</u>

Suspend the 9-table-9-O-phenoxyacetyl-spinoxine A 9-pseudoglycone compound (0.44 g, 0.65 mmol) in a mixture of MeOH (9 mL) and water (1 mL) middle. Potassium carbonate (135 mg, 0.97 mmol) was added to the resulting slurry. Add 3 ml of 9:1 MeOH-water along the wall of the beaker. After 4 hours, the homogeneous solution was equilibrated between EtOAc and 1:1 water-saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted with EtOAc and the combined organic phase was washed successively with diluted sodium bicarbonate (2 times) and brine. Dry with anhydrous sodium sulfate and remove the solvent under reduced pressure to produce a glassy solid (366 mg, 104%)¹H-NMR d 4.50 (br q, J=6.0, 1H).

<u>Example K10-9-table-9-(2-O,3-O,4-O-triethyl-1-α-rhamnosyl)-spinoxine A 9-pseudoglycone compound And 9-table-9-(2-O,3-O,4-O-triethyl-1-β-rhamnosyl)-spinoxine A 9-pseudoglycone compound</u>

The 9-epi-spinoxine A 9-pseudoglycan compound (340 mg, 0.625 mmol) was dissolved in CH₂Cl₂(6 mL). To this solution was added molecular sieves (4A; 420 mg) and pyridine tosylate (210 mg, 0.837 mmol). This mixture was cooled to 0°C and maintained at this temperature, while adding 1-(α/β)-2-O,3-O,4-O-triethyl-rhamnose dropwise within 10 minutes CH₂Cl₂(5mL) solution. The mixture was stirred at 0°C for 40 minutes and at room temperature for 18 hours. Reaction mixture in CH₂Cl₂And sodium bicarbonate aqueous solution. The organic layer was washed with diluted sodium bicarbonate aqueous solution, water (2 times) and brine. Remove the solvent under reduced pressure and place the residue on silica gel (60 g) with 3% MeOH in CH₂Cl₂Solution chromatography. The fractions containing the product were collected and the solvent was removed under reduced pressure. The residue was chromatographed on a reverse phase column (Kromasil' Cl8, silica ODS, 100A, 10m, spherical, 25 cm X 20 mm) with 92% MeOH and 8% water (containing 0.1% v/v concentrated NH₄OH aqueous solution) was eluted to obtain two kinds of mutagenic isomers of amorphous white solid, α-(142 mg,

<img file="TW487559B_D0208.tif" />

<u>Part L is modified by the formation of Spinozin dimer</u>

<u>Example L1-Bis[(Spinnosine J-3'-O-) base]-methane[formaldehyde bis(Spinnosine J-3'-o-) acetal</u>

According to the procedure described in compound 3'-O,N-bis(trideuteromethyl) Spinozin M, potassium carbonate (3.0 g), tetrabutylammonium hydrogen sulfate (1.80 g), 15% NaOH aqueous solution ( 50 mL) and CH₂Br₂(4.5 mL) Make Spinozin J (0.93g, 1.29 mmol) and CH₂Br₂reaction. After the gradual reaction was completed, the crude product was purified by chromatography on silica gel and then by HPLC (88:12, MeOH/H₂O) separation to provide formaldehyde bis(spinoxine J-3'-O-) acetal (47 mg, 54) as a white solid: ESI MS m/z 1448 (M+1).

<u>Example L2[S]R/S-Bis[(Spinozin J-3'-O-yl]sulfide</u>

Spinozin J (2.70 g, 3.76 mmol) was dissolved in dry pyridine (10 mL). Add dry dichloromethane (4 mL). The mixture was stirred under nitrogen and cooled to -78°C. Slowly add Thionyl Chloride (0.90 mL, 12.3 mmol). The mixture was heated to room temperature and stirred for 20 hours. Generally, the reaction is gradually completed and chromatographed on silica gel (ethyl acetate, then 10% EtOH in EtOAc) to provide [S]R/S-bis[(spinoxin J-3'-O-) group] Sulfide (1180 mg, 42%) as a white solid: ESI MS m/z 1482 (M+1).

<u>Example L3-4"-N-Pentamethylene bridged Spinoxin B dimer and 4"-N,4"-N-(pent-1,5-diyl) Spinoxin B bromide salt</u>

According to the method of Example C24, 1,5-dibromopentane (38 mL, 64 mg, 0.28 mmol), (i-pr)₂Compounds were prepared with NEt (0.30 mL, 0.22 g, 1.7 mmol), Spinozin B (0.40 g, 0.56 mmol), and DHF (1.5 mL). MPLC (0: 100 to 20: 80 MeOH/CH₂Cl₂) Obtain 0.07 g (17%) of 4"-N-Pentamethylene bridged Spinozin B dimer as a white powder (Spinozine B can also be recycled, 0.16 g (40%)): MS(m⁺H+ &amp; m⁺2H+/2) Expected value: 1504.0 &amp; 752.5. Actual value: 1504.1 &amp; 753.1 and 4"-N,4"-N-(Pent-1,5-diyl) Spinozin B bromide, 0.17 g(35%): MS(m-Br-) Expected value: 786.5. Actual value: 786.7.

<u>Part M Modification of position 2 on the macrocyclic part of formula I</u>

<u>Example M1-2-Ethyl Spinozin A</u>

Following the procedure for the 2-methyl analog of Spinozine A as described in Example M23, ethane iodide (1.0 g, 6.4 mmol) was substituted for methyl iodide. Therefore, 1 gram of Spinozin A can produce 0.35 grams of pure isomeric 2-ethyl Spinozine A by preparative reverse phase chromatography. C₄₃H₆₉NO₁₀The estimated value of the analysis is: C, 67.96; M, 9.15; N, 1.84. The actual value is C, 67.65; H, 9.01: N, 1.96.

<u>Example M2-2-Ethoxycarbonyl Spinozin A</u>

According to the preparation process of the 2-methyl analog of Spinozin A as described in Example M23, cyanoformate (0.69 g, 6.98 mmol) was substituted for the electrophile. Preparative reverse phase chromatography (C-18, washing with a gradient of 85-98% methanol 0.1% NH4OH/water) can produce 0.14 g of 2-ethoxyl Spinozin A. C₄₄H₆₉NO₂The analytical estimate of the value is: C, 65.72; M, 8.65; N, 1.74. The actual value is: C, 65.77; H, 8.38; N, 1.79.

<u>Example M3-2-Methylthio Spinolyl A</u>

According to the preparation process of the 2-methyl analog of Spinozin A as described in Example M23, dimethyl sulfide (0.66 g, 6.98 mmol) was substituted for the electrophile. Preparative reverse phase chromatography (C-18, washing with a gradient of 85-98% methanol: 0.1% NH4OH/water) can produce 0.20 g of 2-methylthiospinoxine A.¹H-NMR (300 MHz, CDCl₃)d 2.2(s, 3H, cis CH₃). C₄₄H₆₉NO₂The analytical estimates of is: C, 65.72; M, 8.65; N, 1.74. The actual value is: C, 65.77; H, 8.38; N, 1.79. C₄₂H₆₇NO₁₀The analytical estimates of S are: C, 64.83; H, 8.68; N, 1.80; S, 4.1. The actual value is: C, 65.18; H, 10.43; N, 1.76: S, 3.66.

<u>Example M4-2-Bromo Spinozin A</u>

According to the preparation process of the 2-methyl analog of Spinozin A as described in Example M23, 1,2-dibromotetrafluoroethane (0.9 g, 3.47 mmol) was substituted for electrophilic at -70°C Reagents. Preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH/water gradient washing) can produce 0.5 g of 2-bromo spinoxine A, C₄₁H₆₄NO₁₀The analytical estimates of Br are: C, 60.73; H, 7.95; N, 1.72. The actual value is C, 62.14; H, 7.93; N, 1.79.

<u>Example M5-2-(2'-Hydroxy) R and S isomers of Ethyl Spinozin A</u>

According to the preparation process of the 2-methyl analog of Spinozine A as described in Example M23, 1,2-dibromotetrafluoroethane (0.9 g, 3.47 mmol) was substituted for electrophilic at -70°C Reagents. Preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH/water gradient washing) can produce two R and S isomers of 2-(2'-hydroxy)ethyl Spinozin A (0.25g each) isomer 1:C₄₃H₆₉NO₁₁The analytical estimates are: C, 66.55; H, 8.96; N, 1.80. Actual value: C, 66.48; H, 10.6; N, 1.84, isomer 2: C₄₃H₆₉NO₁₁The analytical estimates of is: C, 66.55; H, 8, 96; N, 1.80. The actual value is: C, 65.82; H, 10.07; N, 1.72.

<u>Example M6-2-Phenylthio Spinozine A</u>

According to the preparation process of the 2-methyl analog of Spinozin A as described in Example M23, dimethyl sulfide (1.52 g, 6.98 mmol) was substituted for the electrophile. Using preparative reverse phase chromatography (C-18, with 85-98% methanol:

0.1 of NH₄OH/water gradient washing) can produce 0.71 g of 2-phenylthiospinoxine A. C₄₇H₆₉NO₁₀The analytical estimates of S are: C, 67.19; H, 8.28; N, 1.67; S, 3.81. The actual value is: C, 67.12: H, 8.51; N, 1.65; S, 3.34.

<u>Example M7-2-Formyl Spinozin A</u>

This compound was prepared similarly to Example M23, except that ethyl formate (0.56 mL, 6.98 mmol) was substituted as the electrophile at -70°C. Preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH/water gradient washing) can produce 0.1 g of 2-formylspinoxine A. C₄₂H₆₆NO₁₁The analytical estimates are: C, 66.37; H, 8.75; N, 1.84.7; S, 3.81. The actual value is: C, 63.77; H, 8.46; N, 1.88.

<u>Example M8-2-Methylsulfinyl Spinoxine A, N-oxide</u>

The stirred 2-methylthio Spinozin A solution (1.0 g, 1.3 mmol) was cooled to 0°C in 10 ml of anhydrous methanol and m-chloroperbenzoic acid (50%, 0.67 g) was added in batches. , 1.9 mmol). Observe that it exotherms to 10°C. After stirring for 1 hour, the reaction mixture was poured into 10% HCl and washed twice with ether. The aqueous layer was basified with NaHCO3 and concentrated under reduced pressure to a solid, and then triturated with 25 ml of ether three times and 25 ml of EtoAc twice to form a powder. The organic layer material is combined and concentrated. Preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH/water gradient washing) can produce 0.36 grams of light yellow, 2-methanesulfinyl spinoxin A,-oxide solid foam. C₄₂H₆₇NO₁₂The analytical estimates of S are: C, 62.27; H, 8.34; N, 1.73; S, 3.95. The actual value is: C, 58.63; H, 8.07; N, 1.64; S, 4.12.

<u>Example M9-2-Methylsulfinyl Spinoxine A</u>

The stirred 2-methylthiospinoxine A solution (0.5 g, 0.65 mmol) in 10 ml of anhydrous methanol solution was cooled to -30°C and 5.5 M m-chloroperbenzoic acid in methanol was added via a syringe Acid (50%, 0.67 g, 1.9 mmol). Concentrate on a rotary evaporator, then dissolve the residue in 10 ml of 10% HCl, and wash twice with 30 ml of Et20. NaHCO₃It was basified and extracted 3 times with ether/EtOAc, dried, and concentrated on a rotary evaporator to give 0.45 g of oil. Preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH/water gradient washing) can produce 0.36 g of 2-methanesulfinyl spinoxine A. C₄₂H₆₇NO₁₁The analytical estimates of S are: C, 63.53; H, 8.50; N, 1.76; S, 4.02. The actual values are: C, 63.17; H, 9.50; N, 1.77; S, 4.02.

<u>Example M10-2-hydroxymethylspinoxine A and 2-N-pyridylmethylspinoxine A</u>

Pyrimidine (0.06 g, 0.7 mmol) was added to a solution of 2-methanylmethyl spinoxine A (0.44 g, 0.6 mmol) in 25 mL of MeOH at 20°C. After 15 minutes, the solution was cooled to 0°C and sodium cyanoboride (0.5 g, 0.9 mmol) was added in portions to maintain its temperature below 10°C. After 20 minutes, add 10% HCl to this reactant and add Et₂O washed twice. NaHCO₃It was basified and extracted 3 times with EtOAc. The combined organic layer was dried and concentrated to an oily substance via a rotary evaporator. Preparative reverse phase chromatography (C-18, washed with a gradient of 85-98% methanol: 0.1% NH4OH) can produce 0.1 g of 2-hydroxymethylspinoxine A and 2-n- Pyridinyl methyl spinoxine A versus 2-hydroxymethyl spinoxine A. C₄₂H₆₇NO₁₁The analytical estimates of is: C, 66.19; H, 8.86; N, 1.84. The actual value is: C, 64.95; H, 8.65; N, 1.83. p-2-n-pyridylmethylspinoxine A. C₄₇H₇₆N₂O₁₀The analytical estimates of is: C, 68.08; H, 9.24; N, 3.38. The actual value is: C, 67.89; H, 9.31; N, 3.13.

<u>Example M11-2-(1-Methoxy-1-hydroxymethyl) Spinozin A</u>

The enol compound (4.1 mmol) of Spinozin A can be prepared according to the general procedure described in Example M23. Ethyl formate (1.5g, 20 mmoI) was added dropwise for more than 10 minutes, where it was cooled (-70°C), and then allowed to slowly warm to -30°C. Then pour this solution into one which has been stirred and cooled (0°C) over saturated NH₄Cl (75 mL) and ether (75 mL) in water. The organic layer was separated and washed with brine, dried, and concentrated to an oil. HPLC reverse phase chromatography (85-98% methanol: 0.1% NH₄OH) can produce 1.5 grams of pure hemiacetal. C₄₃H₆₉NO₁₂The analytical estimates of is: C, 65.12; H, 8.78; N, 1.77. The actual value is: C, 65.16; H, 8.65; N, 1.83.

<u>Example M12-2-Phenylselenoyl Spinozin A</u>

Following the procedure for the 2-methyl analog of Spinozin A as described in Example M23, methylselenochloride (0.26 g, 1.37 mmol) was substituted for methyl iodide. So by preparative reverse phase chromatography (C-18, 85-98% methanol: 0.1% NH₄OH gradient washing) 1 g of Spinozin A can produce two isomers of 2-phenylselenoyl Spinozin A (R and S) (0.19 g and

<img file="TW487559B_D0209.tif" />

<u>Example M13-(R and S)2-ethoxycarbonylmethylspinoxine A</u>

Spinozin A enol (1.37 mmol) can be prepared according to the general procedure described in Example M23. Add ethyl bromoacetate (0.76 g, 4.5 mmol) at -60°C, and then allow it to slowly warm to 0°C. Then pour this solution into saturated NH₄Cl aqueous solution. And Et₂Extract twice with 50 ml of O, and extract the combined organics with 50 ml of 0.1N HCl, and then use saturated NaHCO to produce the acidic aqueous layer₃Alkalize, then use Et₂O 50 ml extraction twice. After drying and concentrating the combined organic layer to produce one gram of oil, it was subjected to reverse phase chromatography. To extract gradient 85-98% methanol: 0.1% NH₄OH can produce two 2-ethoxycarbonylmethyl spinoxin A isomers (0.17 g, 17% and 0.13 g, 13%). Spinozin A (0.2g) can also be recovered.

<u>Example-M14-2-Phenylselenooxy Spinozin A and 2,3-Dihydro Spinozin A</u>

A solution of 2-phenylselenooxy Spinozin A (main isomer; 0.20 g, 0.22 mmol) in 10 ml of anhydrous MeOH, cool to -60°C under N2 and magnet stirring, and add three parts of MCPBA at the same time (0.06 g, approximately 0.25 mmol of 57-85% pure MCPBA) over 15 minutes. The solution can be heated to room temperature for more than 1 hour, and then 50 ml of Et₂O diluted, saturated NaHCO₃Solution cleaning. The organic layer was extracted with 30 mL of 1N HCl. The acidic water layer is saturated with NaHCO₃The solution alkalized it (pH 9) and added 25 ml of Et₂O extract twice. Organic layer with MgSO₄Dried and concentrated to an oily substance. By chromatography (C-18, using a gradient of 85-90% methanol: 0.1% NH₄OH) produces two substances. The first type (0.06 g) is 2-phenylselenooxy Spinozine A. The second product (0.02 g) is 2,3-dehydrospinoxine A. Selenium oxide (0.4g, 0.44mmol) was added to 10ml of toluene and heated under reflux for 30 minutes. After cooling, it was diluted with ether and washed with 25ml of 1N HCl. Separate the water layer, the pH value is NaHCO₃The aqueous solution is adjusted to 9, and then 50 ml of Et₂O extraction twice, the combined organic layer was dried, concentrated and chromatographed (C-18, using an extraction gradient of 85-90% methanol: 0.1% NH₄OH) produces 0.10 g of 2,3-dehydrogen

<img file="TW487559B_D0210.tif" />

<u>Example-M15-2-Phenylselenooxy Spinozine A and 2,3-Dehydrospinoxine A</u>

A solution of 2-phenylselenooxy Spinozin A (subisomer; 0.20 g, 0.25 mmol) in 10 ml of anhydrous MeOH was cooled to -60°C under N2 and magnet stirring, and three parts of MCPBA were added at the same time (0.065 g, approximately 0.25 mmol of 57-85% pure MCPBA) over 15 minutes. The solution can be heated to room temperature for more than 1 hour, and then 50 ml of Et₂O diluted, saturated NaHCO₃Solution cleaning. The organic layer was extracted with 30 mL of 1N HCl. The acidic water layer is saturated with NaHCO₃The solution is alkalized (pH 9) and then 25 ml of Et₂O extract twice. Organic layer with MgSO₄Dried and concentrated to an oily substance. By chromatography (C-18, using a gradient of 85-98% methanol: 0.1% NH₄OH) Purify a part of this selenide (0.2 g). C₄₇H₆₉NO₁₁Analytical estimates of Se: C, 62.51; H, 7.70; N, 1.55. The actual value is: C, 62.13; H, 1.64; N, 7.73. The remaining crude selenide (0.2 g) was heated in 10 ml of benzene for 45 minutes, and after cooling, it was concentrated and chromatographed (C-18, using an extraction gradient of 85-98% methanol: 0.1% NH₄OH). 0.07 g of 2,3-dehydrospinoxine A yellow foam was produced. This substance decomposes at room temperature.¹H-NMR (300 MHz, CDCl₃)

<img file="TW487559B_D0211.tif" />

<u>Example M16- Spinozin A, t-butyldimethylsilyl ketal</u>

Add 2 ml of HMPA and 1 g of t-butyldimethylsilyl chloride (6.9 mmol) to a magnetically stirred and cooled (-40°C) solution of LDA (6.8 mmol) in 20 ml of THP. Then Spinozin A (1.0 g, 1.36 mmol) in 2 ml of THF was added for more than 2 min, and the solution was allowed to slowly warm to 0°C. The resulting solution was poured into 50 ml of water and 50 ml of ether. After the layers were separated, the organic phase was washed with brine, dried and concentrated. Chromatography (85-98%MeOH/H₂O elution gradient) produced 0.55 g of pure Spinozin A, t-butyldimethylsilyl ketal.¹H-NMR (300 MHz,

<img file="TW487559B_D0212.tif" />

<u>Example M17-2-Chlorospinosine A</u>

The TBS ketal (0.1g, 0.12mmol) of Benoxine A in 5ml of anhydrous THF was stirred with a magnet and cooled to -78°C, and then a portion of N-chlorosuccinimide (15 mg, 0.125 mmol. After allowing the solution to slowly warm to room temperature, it was separated between 30 ml of ether and 30 ml of saline solution. The organic layer was dried and concentrated to an oil, and then chromatographed (reversely Phase, 85-98%MeOH/H₂The extraction gradient) to obtain 40 mg of 2-(R)-chlorospinoxine¹H-NMR (300 MHz, CDCl₃)d 4.68(s, 1H; C2-H). MS M+H 766:

<u>Example M18-2-fluorospinoxine A</u>

The TBS ketal (0.05g, 0.06mmol) of Spinozin A in 5ml of anhydrous THF was stirred by a magnet and cooled to -78°C, and then a portion of XeF2 (15 mg, 0.09 mmol) was added. After allowing the solution to slowly warm up to room temperature, it was distinguished between 30 ml of ether and 30 ml of saline solution. The organic layer was dried and concentrated to an oil, and then chromatographed (reverse phase, 85-98% MeOH/H₂The extraction gradient) to obtain 18 mg of 2-fluorospinoxine A.¹H-NMR (300 MHz, CDCl₃)d 5.12 (d, J=50Hz, 1H; C2-H). MS M+H 750.

<u>Example M19-2-(Dimethylamino)iminomethyl Spinozine A</u>

To a solution of 2-spinoxine (1-methoxy-1-hydroxymethyl) (0.1 g, 0.13 mmol) in 3 ml of MeOH, add 11 mg (0.18 mmol) of N,N-dimethylhydrazine . Then it was heated in a steam bath for 2 hours, then cooled and concentrated. The residue was chromatographed (reverse phase, 85-98% MeOH/H₂O's extraction gradient) produces 3 mg of .¹H-NMR (300 MHz, CDCl₃)d 6.65(d, J=7 Hz, 1H; C2-CH=NNMe₂); 4.1 (dd, J=7.4 Mz, 1H; C2-CH); 2.78 (s, 6H). MS M+H 802.8.

<u>Example M20-2-Hydroxyiminomethyl Spinozine A</u>

To a solution of 2-spinoxine (1-methoxy-1-hydroxymethyl) (0.06 g, 0.08 mmol) in 3 ml of MeOH, 10 mg (0.14 mmol) of hydroxylamine hydrochloride was added. Then it was heated in a steam bath for 2 hours, then cooled and concentrated. Et₂O fill up and wash with NaHCO3 solution. After drying and concentrating the organic layer, 0.05 g of cis and trans isomers of were produced.<img file="TW487559B_D0213.tif" /><img file="TW487559B_D0214.tif" />

<u>Example M21-2-Cyanospinoxine A</u>

To a solution of 2-(1-methoxy 1-hydroxymethyl) Spinozin A (0.4 g, 0.5 mmol) in 5 ml of MeOH, add N, N-dimethylhydrazine (0.05 g, 0.8 mmol), It was heated at reflux for 1 hour, then cooled and concentrated in vacuo. The residue was covered in 1 ml of MeOH and stirred, and a solution of magnesium monoperphthalate (MMPP, 85%; 0.5 g, 0.8 mmol) in 3 ml of MeOH was cooled (-40° C.). The solution can be warmed at room temperature and stirred overnight, after which it is poured into saturated 10 ml NaHCO₃In aqueous solution, then Et₂O 25 ml extraction twice. After drying and concentrating the organic solution, it was chromatographed (reverse phase, 85-98% MeOH/H₂O extraction gradient) to produce 85 mg of 2-cyanospinozine AM+H 757.5.¹H-NMR

<img file="TW487559B_D0215.tif" />

<u>Example M22-2-cyano-2-fluorospinoxine A</u>

Under N2, add butyl lithium (1.6 M, 0.1 mL, 0.16 mmol) and diisopropylamine (30 ul, 0.2 mmol). After 30 minutes, a solution of 2-cyanospinoxine A (0.105 g, 0.14 mmol) in 0.5 mL of anhydrous THF was added. This solution can be stirred for another 0.5 hour, then add N-fluorobenzenesulfoximine (NFSi; 50 mg, 0.16 mmol), stir at -40°C for 0.5 hour, then allow it to slowly warm to 0°C, then pour Enter Et₂O inside, and wash with saline solution. Then it was dried and concentrated to obtain a light yellow oil, which was chromatographed (reverse phase, 85-98% MeOH/H₂O elution gradient) produces 35 mg of 2-cyano-2-fluorospinoxine A. MS M+H 775.6.

<u>Example M232-Methyl Spinozin A isomer</u>

Set the N2 inlet device in a 50 ml 3-neck round bottom flask, add a funnel and a thermometer, and pour 15 ml of anhydrous THF. The solution was stirred magnetically and cooled to -40°C. Add more butyl lithium (1.6M in hexane, 4.5 mL, 7.2 mmol) followed by diisopropylamine (1.0 mL, 7.2 mmol) dropwise over 2 minutes. After the addition was complete, the solution was cooled to -70°C, and HMPA (1.2 mL) and Spinozin A (1.0 g, 1.37 mmol) in 3 mL of anhydrous THF were added. The solution can be slowly warmed to -40°C for more than 1 hour, then cooled to -70°C, and iodomethane (1 g, 7 mmol) in 2 ml of THF is added dropwise, maintaining the temperature at-during the addition. Below 60°C. Remove the cold bath and allow the solution to slowly warm up to -10°C (estimated 45 minutes). The solution was poured into ice water and extracted with 50 ml of diethyl ether twice to obtain an organic product. Combine the organic layer and clean with saline solution, MgSO₄After drying, it is concentrated into a pale yellow jelly. By the prepared reverse phase chromatography (Cl8, the gradient of 85-98% methanol: 0.1% NH₄OH) two 2-methyl spinoxine A analogs (162.9 mg and 69.8 mg) isomers were obtained. The remaining material from the separation is spinoxine A which does not react.

Main isomers:

<img file="TW487559B_D0216.tif" />

Subisomers:

<img file="TW487559B_D0217.tif" />

<u>Part N Modification of the tricyclic part of the compound of formula I</u>

<u>Example N1-5-Hydroxyspinoxine D, 7-methanospinoxine D, 7,8-dehydrospinoxine D and 7,11-dehydrospinoxine D</u>

Spinozin D (7.5 g, 10 mmol) was dissolved in 20 ml of dioxane (dioxane) and 40 ml of formic acid (90%), cooled to -10 °C and magnetically stirred, while adding a part of the two Selenium oxide (2.3 g, 2.1 mmol) maintains this solution at this temperature for 4 hours, and then concentrates under vacuum while maintaining the temperature at 0°C or below. Then the reddish residue was overlaid with ether (250 ml) and saturated NaHCO was added₃ 100 ml. The layer fluid is carefully shaken until the end of the gas generation, and then filtered with diatomaceous earth and separated into layers. The organic layer was washed with brine, dried and concentrated to become a semi-solid substance. Recrystallization from MeOH yielded 4.5 g of a mixture of 5- and 7-hydroxyspinoxine D formate esters. Recrystallization from MeOH yields pure 7-formylspinoxine D, mp. 184°C. MS M+H 790.8. The residue (estimated 2.5 g) was covered with 30 ml of THF and added to a solution of 0.5 g of LiOOH in 10 ml of water. This solution may be allowed to stir magnetically for 3 hours at room temperature, and then be separated between EtOAc and water. The organic layer is dried (MgSO₄) And concentrated, the residue was chromatographed (reverse phase, 80-98% MeOH/H₂O extraction gradient) The first major slope peak obtained is a mixture of 5- and 7-hydroxyspinoxine D. Recrystallization with MeOH removed (a small amount) of the 7-isomer and yielded pure 1.4 g of pure 5-hydroxyspinoxine D, mp. 115°C. M.SM+H 762.4. The second major slope peak is 7,8-dehydro Spinozin D, mp. 150°C MSM+H 744.4. C₄₂H₆₅NO₁₀The analytical estimated value of is: C, 67.81; H, 8.81; N, 1.88, the actual value is: C, 67.60; H, 8.81; N, 1.88. A small slope peak immediately before the 7,8-dihydro isomer was eluted and separated, and it was determined to be 7,11-dihydrospinoxine D, mp. 160°C. MS 744.7. C₄₂H₆₅NO₁₀The analytical estimates of is: C, 67.81; H, 8.81; N, 1.88. The actual value is: C, 67.68; H, 9.05; N, 1.73.

<u>Example N2-7,11,12-5-bis-dehydro (X543351) Spinozin D</u>

Spinozin D (3.5 g, 9 mmol) was dissolved in 10 ml of dioxane and 20 ml of formic acid (90%), cooled to -10°C and stirred magnetically, while adding a portion of SeO2 (2.3 g , 2.1 mmol) maintain this solution at this temperature for 4 hours, and then allow it to warm to room temperature. Stirring was continued overnight, after which the reddish residue was overlaid with ether (250 ml) and saturated NaHCO was added₃ 100 ml. The layer fluid is carefully shaken until the end of the gas generation, and then filtered with diatomaceous earth and separated into layers. The organic layer was washed with brine, dried and concentrated. The residue was chromatographed (reverse phase, gradient from 85 to 98% in 0.1% NH₄MeOH in OH aqueous solution can collect 7,11,12,5-bis-dehydrospinoxine D (30mg) at 320 nm_maxThe trace fraction of elution. MS M+H 742.7.

<u>Example N3-5-keto-6,7-dehydrospinoxine D</u>

At room temperature, magnetically stir in 5 ml CH₂Cl₂A mixture of 5- and 7-hydroxyspinoxine D isomers (0.60 g, 0.79 mmol) in the mixture, and PDC (1.6 mmol) was added. After 1 hour, the solution was partitioned between water and EtOAc, the organic layer was separated and washed with brine, then dried and concentrated. Extract the residue first by Et₂O can remove the chromium salt through the silica gel column, and then through the reverse phase column, (80-98%MeOH/H₂O gradient) can obtain 60 mg of the title compound, mp 148°C. .MSH+H760.7.

<u>Example N4-5-Fluoro-6,7-dehydrospinoxine D and 7-fluorospinoxine D</u>

Magnet stirs 8 ml CH of 5- and 7-hydroxy Spinozin D isomer (1.0 g, 1.34 mmol)₂Cl₂The mixture was cooled to 0°C under nitrogen. Then DAST (0.32 g, 1.5 eq) was added dropwise over 5 minutes. After 30 minutes, pour it into 25 ml of bicarbonate aqueous solution, and then add 30 ml of CH₂Cl₂After extraction, the organic layer is dried and concentrated, then reverse phase column (80-98%MeOH/H₂O elution gradient) produces 4 distillates. The first product isolated was recrystallized with MeOH to obtain 0.16 g of 5-fluoro-6,7-dehydrospinoxine D, mp. 165°C. The MS M+H peak is 764.7. The second part of the distillate obtained by the extraction is 7-fluorospinnosine D, and the MSM+H peak is at 760.8. The remaining distillates are mainly unsaturated (7,8- and 7,11-dehydrogen )substance.

<u>Example N5-5,6-dihydro-6,7-dehydrospinoxine D and 5,6-dihydro-7,11-dehydrospinoxine D</u>

To 0.2 g (0.27 mmol) 7,8-dihydrospinoxine D in 5 ml ethanol, add 0.5 ml cyclohexane and 50 mg (cat) wetted Pd(OH)₂/C. The solution was heated at reflux temperature for 2 hours, then it was cooled, filtered and concentrated to an oil. Chromatography (reverse phase column, 80-98% methanol/water gradient solution) provides two new products. The first (40 mg) is 5,6-dihydro-7,11-to

<img file="TW487559B_D0218.tif" />

<u>Example 18-Utilization of insecticides and pesticides</u>

The compound of the present invention shows activity against various insects and worms. More particularly, the compounds of the present invention show activity against watermelon aphids, which are members of the order Homoptera. Other members of the order Homoptera include leafhoppers, planthoppers, pear yellow big lice, apple suckers, scales, whiteflies, foam cicadas, and many other host-specific aphids. The activity on thrips Wen's thrips can also be observed, which is a member of the Thysanoptera order. The compound of the present invention can also be resistant to subtropical armyworm, which is a member of Lepidoptera. Other typical members of this order include the codling moth, cutworm, cloth moth, Indian corn borer, leaf roller, American cotton bollworm, European corn borer, cabbage borer, ganache looper, American cotton bollworm, bag moth, European larva caterpillar, sodwebworm, grass armyworm.

The compound of the present invention can be used to reduce insects and serogroups, and can be used to inhibit insects or serogroups, which comprises applying the compounds of the present invention as described in the above examples to insects in an effective insect or inactive amount Or the place of haunt. The results are reported in the following table, in which the following are the abbreviations used in the table:

ALH means purple-wand leafhopper

BAW means beet leaf moth

CA means cotton aphid

NEM means peanut root knot nematodes

SCRW means Eleven Star Melon Leaf Beetle

TBW means Tobacco leaf moth

TSSM means cotton leaf

GECR means German cockroach

When evaluating the pesticidal activity, each test compound was prepared into a 400 ppm solution, and the solution was then diluted with water to a lower concentration. The 400ppm solution was prepared by mixing 19.2 ml of 0.05% Tween 20 (polyoxyethylene (20) sorbitan monolaurate) in water and 8 mg of the compound of the present invention in 8 ml of acetone/ethanol (9/1) solution. The determination of the activity against the purple leafhopper is as follows. This test is performed using concentrations of 400 ppm and 50 ppm. A 1 oz plastic cup contains a cotton wick sprayed with 0.4 ml of formulated material using a fan-shaped mist cone nozzle. Allow excess water to evaporate. Put 5 to 10 adult cicadas anesthetized by carbon dioxide into each cup. Cover the cup and keep it at room temperature for 24 hours. The mortality rate can be calculated.

The activity against beet leaf moth was evaluated as follows. This test is carried out using concentrations of 400 ppm and 50 ppm. The general purpose Lepidoptera artificial food is diluted to half the strength with 5% non-nutritive agar gum. Take 8 ml of food material and distribute it in a 1-ounce feeding cup. One hour before the treatment, 35-40 eggs are distributed on the surface of the foodstuff. Then the cup is sprayed with the formulated substance through the fan-shaped mist cone nozzle. The treated cup was allowed to air dry before being sealed with a plastic lid, and the cup was left at room temperature for 6 days. The activity is determined by the total number of live and dead larvae and the size of the live larvae.

The activity against cotton aphid and cotton leaf moth was evaluated as follows. The Golden crookneck squash plant line grows to extend the cotyledon stage (approximately 6 to 8 days). Before applying the test substance, the plants were infected with cotton aphid and cotton leaf moth by cutting the transfer sheet of the pest group from the preserved strain for 16-24 hours. Before spraying the test substance, the transfer sheet was quickly removed from the pumpkin plant. The test is carried out at concentrations of 400 ppm and 50 ppm. The plants were sprayed with the test substance using an atomizing sprayer at 17 psi. The surface of the leaf and the back of the leaf should be coated until it is flowable, and then allowed to dry. The activity of each compound was measured 3 days after treatment. The activity is based on the number of cicadas or cicadas that are only sprayed with solvents.

The activity against peanut root knot nematodes was evaluated as follows. Place 5 untreated cucumber seeds on the bottom of a clean 1-ounce cup, add 20 g of clean white sand, and rotate the bottom while spraying the cup so that 1.0 ml of 400 ppm solution can be placed on the sand. Add 2.5-3.0ml of deionized water containing 300 to 500 nematodes to each bad seed. Place the cup in the environmental growth chamber for 10 to 12 days at a temperature of 76 to 856°F and an atmospheric humidity of 50 to 60%. After 10 to 12 days, evaluate by inverting the cup and observing the mortality of nematodes and the damage to cucumber plants.

The activity of anti-eleven star cucurbita leaf beetle was evaluated by adding 1 ml of a test solution containing the determined concentration to 16 g of poor soil containing corn kernels. The resulting concentration in the soil is 24 ppm. After drying for 1.5 to 2 hours, put 5 fourth-instar larvae of Eleven star melon leaf beetle into each cup. After 3 to 4 days, check the number of live insects in the soil by emptying the cup on a plate.

The evaluation of the activity against the tobacco leaf moth is as follows. Dilute the general purpose Lepidoptera artificial food with 5% non-nutritive agar gum to half the strength. Take 8 ml of food material and distribute it in a 1-ounce feeding cup. 1 hour before the treatment, distribute 35-40 eggs on the surface of the foodstuff. Then the cup is sprayed with the formulated substance through the fan-shaped mist cone nozzle. The test system uses 400 ppm and 50 ppm. The treated cup was allowed to air dry before being sealed with a plastic lid, and the cup was left at room temperature for 6 days. The activity is determined by the total number of live and dead larvae and the size of the live larvae.

The anti-German cockroach activity is evaluated as follows. Put 8ml of alfalfa, which is mainly green pest food, into a 1-ounce feeding cup. Then, the cup is sprayed with the modulated substance through the fan-shaped mist cone nozzle. The test is carried out using concentrations of 400 ppm and 50 ppm. The test cup was allowed to air dry for 24 hours and infected with German cockroaches that were five and four instars later than the third instar. Cover the cup and keep it in an environmental growth chamber for 10 days at a temperature of 76-85°C. The activity is evaluated based on the total number of surviving and dead insects.

<u>Utilization of killing agent</u>

The method of the present invention is implemented according to standard techniques for the application of disinfectants. Generally speaking, good killing activity can be expected at the level of 1-10 1bs/acre. The compounds can be formulated into the forms described herein. When formulated as a dispersion, the disinfectant is generally applied in the form of an infusion around the growing plants or enhanced use through stimulation systems. When used in granular form, the pesticide can be mixed with the soil before planting, or applied in a strip form to the surface of the seed row, the sowing area and then mixed with the soil, or it can be applied as a side coat to the grown crop.

The following activities are found by the following compounds:<img file="TW487559B_D0219.tif" /><img file="TW487559B_D0220.tif" /><img file="TW487559B_D0221.tif" /><img file="TW487559B_D0222.tif" /><img file="TW487559B_D0223.tif" /><img file="TW487559B_D0224.tif" /><img file="TW487559B_D0225.tif" /><img file="TW487559B_D0226.tif" />

<img file="TW487559B_D0227.tif" />

Example 19 Spinozin Derivatives of Example 17

<u>Control of Sting Flies and Blow Flies</u>

The test steps are as follows:

The compound to be evaluated is dissolved in 1 part of acetone and 1 part of ethanol to provide a compound stock solution with a concentration of 5000 ppm. The solution was shaken in ultrasonic shaking for 15 minutes. Put the stock solution part in a 15 ml test tube and add part of bovine serum to provide the desired compound dilution. Place the dental floss core in each test tube so that the serum can saturate the core.

For the test of blowfly, about 20 blowfly larvae are placed in the center of the top of the saturated floss core, and the test tube is tightly plugged with cotton and cultured at 27°C and 70% humidity for 24 to 48 hours. Count the mortality of the larvae caused by the control medium and adjust the mortality to determine the percentage of efficiency against blowfly.

For the test of adult sting flies, the saturated core is placed on the filter paper of the petri dish, and about 10 cold surviving hungry sting flies are placed in the center of the bottom of the dish. Cover the petri dish and incubate at 27°C and 60% relative humidity for 48 hours. Mortality readings were recorded at 24 and 48 hours, respectively, and any mortality was adjusted with the control vehicle to determine the effectiveness of anti-sting fly cover adults.

The results are reported as follows:<img file="TW487559B_D0228.tif" /><img file="TW487559B_D0229.tif" /><img file="TW487559B_D0230.tif" /><img file="TW487559B_D0231.tif" /><img file="TW487559B_D0232.tif" /><img file="TW487559B_D0233.tif" />