Phosphonate compounds

<u style="single">Field of invention</u> The present invention is for the treatment of novel phosphonate compounds, compositions containing them, methods of their production, and various medical disorders such as, for example, osteoporosis and other bone metabolic disorders, cancer, viral infections, etc. Regarding their uses.

<u style="single">Background of the invention</u> Phosphonate compounds have long been known to provide a variety of therapeutic benefits. A particular type of phosphonate compound with therapeutic benefit is a bisphosphonate, a pyrophosphonate analog in which the central oxygen atom of the pyrophosphate bond is replaced by carbon. Various substituents can be attached to this central carbon atom to produce bisphosphonate compound derivatives with different degrees of medicinal properties. These derivatives have the following general structure:<chemistry num="3"><img id="000002" he="68" wi="153" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>In the formula, R_aAnd R_{b b}Can independently be selected from hydroxyl, amino, sulfhydryl, halogen, or various alkyl or aryl groups, or combinations of such groups, which can be further substituted. As an example, etidronate (in the formula, R_aIs CH₃And R_{b b}Is OH); Clodronic acid, Dichloromethylene bisphosphonic acid (Cl)₂MDP) (in formula, R_aAnd R_{b b}Is Cl); pamidronate, 3-amino-1-hydroxypropyridenebisphosphonic acid (in the formula, R)_aIs ethylamino and R_{b b}Is a hydroxyl); alendronate, 4-amino-1-hydroxybutylidenebisphosphonic acid (in the formula, R)_aIs propylamino, and R_{b b}Is a hydroxyl); Olpadronate, 3-dimethylamino-1-hydroxypropyridenebisphosphonic acid (in the formula, R)_aIs dimethylaminoethyl and R_{b b}Is a hydroxyl group); as well as amino-olpadronate (IG-9402), 3- (N, N-dimethylamino) -1-aminopropyridene bisphosphonate (in the formula, R)_aIs N, N-dimethylaminoethyl, and R_{b b}Is NH₂Is).

Bisphosphonates and their substituted derivatives have the unique property of inhibiting bone resorption in vivo. Bisphosphonates also act by inhibiting apoptosis (programmed cell death) in osteoblasts. Therefore, the indications for their use are treatment and prevention of osteoporosis, Paget's disease, metastatic bone cancer, hyperparathyroidism, rheumatoid arthritis, painful dystrophy, sterno-costoclavicular. Includes treatment of hyperostosis), Gaucher's disease, Engelmann's disease, and certain non-skeletal muscle disorders (Papapoulos, SE,<u style="single">Osteoporosis</u>, R. Marcus, D. Feldman and J. Kelsey, Academic Press, San Diego, 1996, p. 1210, Table 1 (Non-Patent Document 1).

Despite the therapeutic benefits of bisphosphonates, they have pharmaceutical drawbacks as orally administered substances. One drawback is poor oral availability: small doses, such as 0.7% to 5% of the oral dose, are absorbed from the gastrointestinal tract. Oral absorption is further reduced when co-ingested with food. In addition, some currently available bisphosphonates, such as FOSAMAX (Merck; sodium alendronate), SKELID (Sanofi, Childronate) and ACTONE (Procter and Gamble, lysedronate), are available. It is known to have local toxicity and cause esophageal hypersensitivity and ulcers. Other bisphosphonates, such as amino-olpadronate, do not have an anti-resorptive effect (Van Beek, E. et al., J. et al. Bone Miner Res., 11 (10): 1492-1497 (1996) (Non-Patent Document 2)), which can inhibit osteocytic apoptosis and stimulate net bone formation (Plotkin, L. et al., J. Clin. Invest., 104 (10): 1363-1374 (1999) (Non-Patent Document 3) and US Pat. No. 5,885,973 (Patent Document 1)). Therefore, it may be useful to develop chemically modified bisphosphonate derivatives that maintain or enhance the pharmacological activity of the parent compound while eliminating or alleviating their unwanted side effects.

In addition to bisphosphonates, monophosphonates are also known to provide therapeutic benefits. One class of monophosphonates with therapeutic benefit is antiviral nucleotide phosphonates such as, for example, cidofovir, cyclic cidofovir, adefovir, tenofovir, as well as 5'-phosphonates and methylenephosphonates such as azidothymidine, ganciclovir, acyclovir. In this type of compound, the 5'-hydroxyl of the sugar moiety, or their equivalents in acyclic nucleosides (ganciclovir, penciclovir, acyclovir) that do not contain the complete sugar moiety, are replaced by phosphorus-carbon bonds. In the case of methylenephosphonate, the methylene group is replaced with 5'-hydroxyl or its equivalent, and its carbon atom is then covalently attached to the phosphonate. Various AZT structures are shown below, including compounds devised for use in the practice of the present invention. AZT itself is shown on the left. Compound A is AZT-monophosphonate, which has the usual phosphodiester bond between sugar and phosphate. In contrast, compound B (ALT 5'-phosphonate) and compound C (AZT) In 5'-methylenephosphonate), the 5'-hydroxyl of 3'-azido 2', 3'-zideoxyribose is absent and methylene linked by a phosphorus-carbon bond (AZT phosphonate) or phosphorus-carbon bond. It has been replaced by one of (AZT methylene phosphonate). Compound B and Compound C are examples of compounds useful in the practice of the present invention.<chemistry num="4"><img id="000003" he="146" wi="153" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="5"><img id="000004" he="174" wi="153" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="6"><img id="000005" he="182" wi="153" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="7"><img id="000006" he="162" wi="153" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

This type of compound may have activity as an antiproliferative or antiviral nucleotide. In cell metabolism, two additional phosphorylations occur to form the nucleotide phosphonate diphosphate, which represents the equivalent of nucleoside triphosphate. Antiviral nucleotide phosphonate diphosphate is a selective inhibitor of viral RNA or DNA polymerase or reverse transcriptase. That is, their inhibitory effect on viral polymerases is far greater than their inhibition of mammalian cell DNA polymerases α, β and γ or mammalian RNA polymerases. Conversely, antiproliferative nucleotide phosphonate diphosphates can inhibit cancer cell DNA and RNA polymerase and show very low selectivity for normal cell DNA and RNA polymerase. Nucleotide phosphonates are poorly absorbed from the gastrointestinal tract, so they require frequent parenteral administration (eg, cidofovir). In addition, the negatively charged phosphonate moiety interferes with cell invasion, resulting in reduced antiviral or antiproliferative activity. Surprisingly, the compounds of the present invention overcome the shortcomings of this type of substance.

Pharmaceutically active substances of antiviral phosphonates are known; the following US patent discloses studies on nucleotide phosphonate analogs: No. 5,672,697 (Patent Document 2) (nucleoside-5'-methylene). Phosphonate), No. 5,922,695 (Patent Document 3) (Anti-viral phosphonomethoxynucleotide analog), No. 5,977,089 (Patent Document 4) (Anti-viral phosphonomethoxynucleotide analog), No. 6,043,230 (Patent Document 5) ) (Antiviral phosphonomethoxynucleotide analog), No. 6,069,249 (Patent Document 6). So far, the preparation and use of alkylglycerol phosphates covalently attached to phosphonate-free drugs with amino, carboxyl, hydroxyl or sulfhydryl functional groups has been demonstrated. These prodrugs selectively contain a linker group or one or two additional phosphate esters between the drug and alkylglycerol phosphate (US Pat. No. 5,411,947 (Patent Document 7) and US Patent Application). No. 08 / 487,081 (Patent Document 8)). Incomplete esters of chloromethanediphosphonic acid (partial esters) are known (US Pat. No. 5,376,649 (Patent Document 9)) and dianhydrides of clodronate have been reported (Ahlmark et al., J. Med. Chem). ., 42: 1473-1476 (1999) (Non-Patent Document 4)). However, these incomplete esters are known not to release active bisphosphonates by chemical or biochemical conversion (Niemi, R. et al., J. Chrom. B., 701: 97-102 (1997). ) (Non-Patent Document 5)). A prodrug containing an alkylglycerol phosphate residue attached to an antiviral nucleoside (US Pat. No. 5,223,263 (Patent Document 10)) or a prodrug containing a phosphonocarboxylate (US Pat. No. 5,463,092). Patent Document 11)) is also disclosed.

Therefore, less toxic and more effective agents are still needed to treat various diseases, such as those caused by viral infections and inappropriate cell proliferation (eg, cancer). .. Therefore, an object of the present invention is to develop chemically modified phosphonate derivatives of pharmaceutically active substances, such as antiviral and antineoplastic agents. These modified derivatives enhance the potency of the parent compound while minimizing adverse side effects when administered to subjects in need of it.

<p num="0009"><patcit num="1"><text>U.S. Pat. No. 5,885,973</text></patcit><patcit num="2"><text>U.S. Pat. No. 5,672,697</text></patcit><patcit num="3"><text>U.S. Pat. No. 5,922,695</text></patcit><patcit num="4"><text>U.S. Pat. No. 5,977,089</text></patcit><patcit num="5"><text>U.S. Pat. No. 6,043,230</text></patcit><patcit num="6"><text>U.S. Pat. No. 6,069,249</text></patcit><patcit num="7"><text>U.S. Pat. No. 5,411,947</text></patcit><patcit num="8"><text>U.S. Patent Application No. 08 / 487,081</text></patcit><patcit num="9"><text>U.S. Pat. No. 5,376,649</text></patcit><patcit num="10"><text>U.S. Pat. No. 5,223,263</text></patcit><patcit num="11"><text>U.S. Pat. No. 5,463,092</text></patcit></p>

<p num="0010"><nplcit num="1"><text>Edited by Papapoulos, SE, Osteoporosis, R. Marcus, D. Feldman and J. Kelsey, Academic Press, San Diego, 1996, p. 1210, Table 1</text></nplcit><nplcit num="2"><text>Van Beek, E. et al., J. Bone Miner Res., 11 (10): 1492-1497 (1996)</text></nplcit><nplcit num="3"><text>Plotkin, L. et al., J. Clin. Invest., 104 (10): 1363-1374 (1999)</text></nplcit><nplcit num="4"><text>Ahlmark et al., J. Med. Chem., 42: 1473-1476 (1999)</text></nplcit><nplcit num="5"><text>Niemi, R. et al., J. Chrom. B., 701: 97-102 (1997)</text></nplcit></p>

<u style="single">Brief description of the invention</u> The present invention provides analogs of phosphonate compounds. Phosphonate compounds devised for use in accordance with the present invention are intended for the treatment of cancer, various viral infections, etc., in addition to those that reduce bone resorption or inhibit osteoblast or osteoblast apoptosis. Includes those that improve the bioactivity, selectivity, or bioavailability of nucleotide phosphonate analogs as useful. Compounds of the invention include substituted or unsubstituted alkyl glycerol, alkyl propanediols, alkyl ethanediols, or phosphonates covalently attached to related moieties (directly or indirectly via a linker molecule). In another aspect of the invention, pharmaceutical formulations containing analogs of the phosphonate compounds described herein are provided.

In another aspect of the invention, various therapies, such as methods for treating or preventing bone resorption in mammals, methods for increasing bone formation by preventing osteoblasts and osteoblast apoptosis, bone mass and strength. There are provided methods of increasing the amount of bone, treating viral infections, eg, treating disorders caused by improper cell proliferation such as cancer.

<figref num="1">The effects of the compound 1-O-hexadecyloxypropane alendronate according to the present invention on dexamethasone-induced apoptosis of MLO-Y4 osteoocytes are summarized. The line segment represents the average ± SD of the three individual measurements. White bars indicate the absence of dexamethasone, black bars are 10.^-4Indicates the presence of M dexamethasone.</figref><figref num="2">The effects of the compound 1-O-hexadecyloxypropane alendronate according to the present invention on dexamethasone-induced apoptosis of calvaria cells are summarized. The line segment represents the average ± SD of the three individual measurements. The gray bar indicates the absence of dexamethasone, and the black bar is 10.^-4Indicates the presence of M dexamethasone.</figref>

<u style="single">Detailed description of the invention</u> The phosphonate compound of the present invention has the following structure:<chemistry num="8"><img id="000007" he="58" wi="159" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>During the ceremony R₁And R₁'Is independently -H, optionally replaced -O (C₁-C₂₄) Alkyl, -O (C₁-C₂₄) Alkenyl, -O (C₁-C₂₄) Acyl, -S (C)₁-C₂₄) Alkyl, -S (C)₁-C₂₄) Alkenyl, or -S (C)₁-C₂₄) Acyl, but at least one R₁And R₁'Is not H and the alkenyl or acyl moiety optionally has 1-6 double bonds; R₂And R₂'Is independently -H, optionally replaced -O (C₁-C₇) Alkyl, -O (C₁-C₇) Alkenyl, -S (C₁-C₇) Alkyl, -S (C)₁-C₇) Alkenyl, -O (C₁-C₇) Acyl, -S (C)₁-C₇) Acyl, -N (C)₁-C₇) Acyl, -NH (C)₁-C₇) Alkyl, -N ((C₁-C₇) Alkylation))₂, Oxo, halogen, -NH₂, -OH, or -SH; R₃Is a functional group or C on any linker L^αA phosphonate derivative of a pharmaceutically active phosphonate, bisphosphonate or pharmaceutically active compound linked to the available oxygen atom above; X, if present<chemistry num="9"><img id="000008" he="36" wi="155" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>Is; L is a valence bond or formula -J- (CR)₂)_tIt is a bifunctional linking molecule of -G- (in the formula, t is an integer from 1 to 24, and J and G are independently -0-, -S-, -C (O) O-, or. -NH- and R is -H, substituted or unsubstituted alkyl, or alkenyl); m is an integer from 0 to 6; and n is 0 or 1.

In a preferred embodiment, m = 0, 1 or 2. In these preferred embodiments, R₂And R₂'Is preferably -H, so that the prodrug is a therapeutic phosphonate ethanediol, propanediol or butanediol derivative. Preferred ethanediol phosphonate species have the following formula:<chemistry num="10"><img id="000009" he="54" wi="156" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>In the formula, R₁, R₁', R₃, L and n are those defined above.

Preferred propanediol species have the following formula:<chemistry num="11"><img id="000010" he="56" wi="159" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>In the equation, m = 1 and R₁, R₁', R₃, L and n are defined in the above general formula.

Preferred glycerol species have the following formula:<chemistry num="12"><img id="000011" he="59" wi="156" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>In the formula, m = 1, R₂= H, R₂'= OH, as well as C^αR above₂And R₂'Both are -H. Glycerol is an optically active molecule. Using the stereospecific numbering convention of glycerol, the sn-3 position is the position phosphorylated by glycerol kinase. In the compound of the present invention having a glycerol residue,-(L)_n-R₃The moiety can be attached to either the sn-3 or sn-1 position of glycerol.

In all species of pharmaceutically active substances of the invention, R₁Is preferably the expression -O- (CH₂)_t-CH₃It is an alkoxy group having (t is 0 to 24 in the formula). More preferably, t is 11-19. Most preferably, t is 15 or 17.

Preferred R₃The group contains bisphosphonates known to be clinically useful, including, for example, the following compounds:<u style="single">Etidronate</u>: 1-Hydroxyethylylidene bisphosphonic acid (EDHP);<u style="single">Clodronic acid</u>: Dichloromethylene bisphosphonic acid (Cl₂MDP);<u style="single">Children</u>: Chloro-4-phenylthiomethylenebisphosphonic acid;<u style="single">Pamidronate</u>: 3-Amino-1-hydroxypropyridene bisphosphonic acid (ADP);<u style="single">Arendronate</u>: 4-Amino-1-hydroxybutylidenebisphosphonic acid;<u style="single">Orpadronate</u>: 3-Dimethylamino-1-hydroxypropyridene bisphosphonic acid (dimethyl-APD);<u style="single">Ibandronate</u>: 3-Methylpentylamino-1-hydroxypropyridenebisphosphonic acid (BM21.0955);<u style="single">EB-1053</u>: 3- (1-pyrrolidinyl) -1-hydroxypropylidenebisphosphonic acid;<u style="single">Risedronate</u>: 2- (3-Pyridinyl) -1-hydroxy-ethylidene bisphosphonic acid;<u style="single">Amino-olpadronate</u>: 3- (N, N-dimethylamino-1-aminopropyridene) bisphosphonate (IG9402) etc.

R₃Can also be selected from a variety of phosphonate-containing nucleotides (or nucleosides that can be derived to their corresponding phosphonates), which are also devised to be used herein. Preferred nucleosides are useful in the treatment of disorders caused by inappropriate cell proliferation, such as 2-chloro-deoxyadenosine, 1-β-D-arabinofuranosyl-citidine (cytarabine, ara-C), fluoro. Uridine, fluorodeoxyuridine (floxuridine), gemcitibine, cladribine, fludalabine, pentostatin (2'-deoxycoformycin), 6-mercaptopurine, 6-thioguanine, and substituted or unsubstituted 1-β-D- Includes arabinofuranosyl-guanine (ara-G), 1-β-D-arabinofuranosyl-adenosine (ara-A), 1-β-D-arabinofuranosyl-uridine (ara-U), etc. ..

Nucleosides, which are useful in treating viral infections, are also R₃It can be converted to their corresponding 5'-phosphonates for use as a group. Such phosphonate analogs typically have a 5'-hydroxyl substituted phosphonic acid group (-P0) of the antiviral nucleoside.₃H₂) Or methylene phosphonic acid group (-CH₂P0₃H₂) Is included. 5'-Hydroxy-P0₃H₂Below are some examples of substitution-induced antiviral phosphonates:<chemistry num="13"><img id="000012" he="42" wi="139" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="14"><img id="000013" he="32" wi="130" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="15"><img id="000014" he="34" wi="140" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="16"><img id="000015" he="46" wi="139" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

5'-Hydroxy-CH₂-P0₃H₂Below are some examples of substitution-induced antiviral phosphonates:<chemistry num="17"><img id="000016" he="34" wi="133" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="18"><img id="000017" he="33" wi="144" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="19"><img id="000018" he="27" wi="139" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><chemistry num="20"><img id="000019" he="32" wi="138" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>Other preferred antiviral nucleotide phosphonates devised for use in the practice of the present invention are similarly ddA, ddI, ddG, L-FMAU, DXG, DAPD, L-dA, L-dI, L-( d) Derived from antiviral nucleosides including T, L-dC, L-dG, FTC, penciclovir, etc.

In addition, antiviral phosphonates such as cidofovir, cyclic cidofovir, adefovir, tenofovir are the R of the present invention.₃It can be used as a base.

Certain compounds of the invention have one or more chiral centers, such as in sugar moieties, and can therefore be present in optically active form. Similarly, if these compounds contain alkenyl groups or unsaturated alkyl or acyl moieties, they may be present in the cis- and trans-isomers of the compounds. Additional asymmetric carbon atoms can be present in the substituents, such as alkyl groups. R isomers, S isomers, and mixtures thereof, including racemic mixtures as well as cis- and trans-isomer mixtures, are considered by the present invention. All such isomers and mixtures thereof are intended to be included in the present invention. Where a particular steric isomer is desired, by a method known in the art, or as an alternative, by using a stereospecific reaction of the starting material that contains an asymmetric center and is already partitioned. , A mixture of stereoisomers and methods that lead to division by known methods.

There are many phosphonate compounds that can be derived according to the present invention to pharmaceutically modify activity or increase oral absorption, such as the compounds disclosed in the patents below, each of which is in its entirety. Incorporated herein by reference: US Pat. Nos. 3,468,935 (etidronate), 4,327,039 (palmidronate), 4,705,651 (alendronate), 4,870,063 (bisphosphonate derivative), 4,927,814 (diphosphonate). Phosphonate), No. 5,043,437 (Phosphosphonate of azidodideoxynucleoside), No. 5,047,533 (Acyclic purine phosphonate nucleotide analog), No. 5,142,051 (N-phosphonylmethoxyalkyl derivative of pyrimidine and purine base), No. 5,183,815 ( Bone activator), No. 5,196,409 (bisphosphonate), No. 5,247,085 (antiviral purine compound), No. 5,300,671 (Gem-di)<u style="single">Phosphonate</u>), No. 5,300,687 (trifluoromethylbenzylphosphonate), No. 5,312,954 (bis- and tetrakis-phosphonate), No. 5,395,826 (guanidine alkyl-1,1-bisphosphonate derivative), No. 5,428,181 (bisphosphonate derivative), No. 5,442,101 No. 5,532,226 (trifluoromethylbenzylphosphonate), No. 5,656,745 (nucleotide analog), No. 5,672,697 (nucleoside-5'-methylenephosphonate), No. 5,717,095 (nucleotide analog), Nos. 5,760,013 (thymidylate analogs), Nos. 5,798,340 (nucleotide analogs), Nos. 5,840,716 (phosphonate nucleotide compounds), Nos. 5,856,314 (thio-substituted nitrogen-containing heterocyclic phosphonate compounds), Nos. 5,885,973 (Orpadronate), Nos. 5,886,179 (nucleotide analogs), Nos. 5,877,166 (mirror isomerically pure 2-aminopurine phosphonate nucleotide analogs), Nos. 5,922,695 (antiviral phosphonomethoxynucleotide analogs), No. Nos. 5,922,696 (ethylene and allen phosphonate derivatives of purines), Nos. 5,977,089 (antiviral phosphonomethoxynucleotide analogs), Nos. 6,043,230 (antiviral phosphonomethoxynucleotide analogs), Nos. 6,069,249 (antiviruses) Sex phosphonomethoxynucleotide analogs); Belgian Patent No. 672205 (clodronate); European Patent No. 753523 (amino-substituted bisphosphonates); European Patent Application No. 186405 (geminal diphosphonate); etc.

Certain bisphosphonate compounds have the ability to inhibit squalene synthase and lower serum cholesterol levels in mammals, including humans. Examples of these bisphosphonates are disclosed, for example, in US Pat. Nos. 5,441,946 and 5,563,128 of Pauls et al., "Phosphonate derivatives of lipophilic amines," both of which are in their entirety. Incorporated as a reference in the specification. Analogs of these squalene synthase inhibitory compounds according to the present invention, and their use in the treatment of lipid disorders in humans, are within the scope of the present invention. The ability of the bisphosphonates of the invention to be used orally or topically to prevent or treat periodontal disease is disclosed in US Pat. No. 5,270,365, which is incorporated herein by reference in its entirety. Be done.

As used herein, the term "alkyl" is a monovalent linear or branched chain or cyclic radical of 1 to 24 carbon atoms, methyl, ethyl, n-propyl, isopropyl, n. -Contains butyl, isobutyl, tert-butyl, n-hexyl, etc.

The term "substituted alkyl" as used herein further refers to hydroxy, alkoxy (lower alkyl group), mercapto (lower alkyl group), cycloalkyl, substituted cycloalkyl, heterocyclic, substituted. Heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy, halogen, trifluoromethyl, cyano, nitro, nitron, amino, amide, -C ( O) It means an alkyl group having one or more substituents selected from H, acyl, oxyacyl, carboxyl, carbamate, sulfonyl, sulfonamide, sulfryl and the like.

As used herein, the term "alkenyl" is a linear or branched hydrocarbyl group having one or more carbon-carbon double bonds and having from about 2 to up to 24 carbon atoms. And "substituted alkenyl" means an alkenyl group further having one or more of the above-mentioned substituents.

As used herein, the term "aryl" means an aromatic group having a range of 6 to up to 14 carbon atoms, and "substituted aryl" is one or more of the aforementioned. Means an aryl group further having a substituent of.

As used herein, the term "heteroaryl" comprises one or more heteroatoms (eg, N, 0, S, etc.) as part of the ring structure and ranges from 3 to a maximum of 14. It means an aromatic group having a carbon atom of, and the "substituted heteroaryl" means a heteroaryl group further having one or more of the above-mentioned substituents.

As used herein, the term "bond" or "valence bond" refers to the connection between atoms consisting of electron pairs.

As used herein, the term "pharmaceutically acceptable salt" means an addition salt of an acid and a base.

As used herein, the term "prodrug" refers to a compound that has a chemically or metabolically cleaving group and is pharmaceutically active by solvolysis or under physiological conditions in vivo. Means a derivative of a pharmaceutically active compound.

Phosphonate analogs are therapeutically effective phosphonates (or therapeutically) covalently attached to 1-O-alkylglycerols, 3-O-alkylglycerols, 1-S-alkylthioglycerols, or alkoxy-alkanols by hydroxyl groups. It contains a phosphonate derivative of a compound that is effective in its ability to be absorbed in the gastrointestinal tract more efficiently than its parent compound. Oral doses of this analog are taken directly from the mammalian gastrointestinal tract and the active drug is released in vivo by the action of endogenous enzymes. Phosphonate analogs of the invention may have a higher degree of biological activity than the corresponding underived compounds.

The compounds of the present invention have been described in prior art because the phosphonate-containing moiety directly links to an alkyl-glycerol or alkoxy-alkanol moiety and the presence of a phosphonic acid bond interferes with the enzymatic conversion to a free drug. It is an improvement over the alkylglycerol phosphate prodrug. Other linkers between these groups can be present in the improved analogs. For example, the expression -O- (CH₂)_nA bifunctional linker with -C (O) O- (where n is 1 to 24 in the formula) can attach the phosphonate to the hydroxyl group of the alkoxy-alkanol or alkyl glycerol moiety.

As mentioned above, the phosphonates of the present invention can be more highly orally absorbed. Furthermore, it is believed that cellular enzymes, rather than plasma or gastrointestinal enzymes, convert this complex to free phosphonates. A further advantage of alkoxy-alkanolphosphonates is that co-administered foods tend to reduce or eliminate phosphonate absorption, which is significantly reduced or eliminated, resulting in higher plasma levels and better patient compliance. That is.

The compounds of the present invention are mediated through the skin by application such as tablets, capsules, liquids, emulsions or suspensions, inhaled liquids or solid particles, microencapsulated particles such as sprays, transdermal patches. , Or transrectally, eg, in the form of suppositories. The lipophilic prodrug derivative of the present invention is particularly well suited for transdermal administration and delivery systems and may also be used as a toothpaste. Administration can also be administered parenterally in the form of an injectable solution.

The compositions can be prepared in their usual form, such as capsules, tablets, aerosols, solutions, suspensions, or with carriers for topical application. A pharmaceutical preparation containing the compound of the present invention can be prepared according to a conventional method as described in, for example, "Remington's Pharmaceutical Science" (1985).

The pharmaceutical carrier or diluent used can be a conventional solid or liquid carrier. Examples of solid carriers include lower alkyl ethers of lactose, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, or cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, or water. The carrier or diluent can include any of the lasting substances known in the art, such as glyceryl monostearate or distearate, alone or in admixture with wax.

If the solid support is used for oral administration, the preparation can be locked or placed in hard gelatin capsules in the form of powder or pellets. The dose of solid support varies widely, but is usually from about 25 mg to about 1 g. If a liquid carrier is used, the preparation can be a syrup, an emulsion, a soft gelatin capsule, or a sterile injectable solution, such as an aqueous or non-aqueous liquid suspension or solution. ..

Tablets are prepared by mixing the active ingredient (ie, one or more compounds of the invention) with a pharmaceutically inert inorganic or organic carrier, diluent, and / or excipient. Examples of such excipients that can be used for tablets are lactose, cornstarch or derivatives thereof, talc, stearic acid, or salts thereof. Examples of excipients suitable for gelatin capsules are vegetable oils, waxes, fats, semi-solids, and liquid polyols. These bisphosphonate prodrugs can also be produced in microencapsulated form.

For nasal drops, the preparation can contain a compound of the invention dissolved or suspended in a liquid carrier for aerosol application, particularly an aqueous carrier. The carrier may include a solubilizer such as propylene glycol, a surfactant, an absorption enhancer such as lecithin or cyclodextrin, or a preservative.

The pharmaceutical compositions of the present invention for parenteral injection can be added to pharmaceutically acceptable sterilized aqueous or non-aqueous liquids, dispersions, suspensions, or emulsions, plus sterilized injectable solutions just prior to use. Alternatively, it can contain a sterile powder for reconstitution in the dispersion.

Suitable excipients for the preparation of solutions and syrups are water, polyols, sucrose, invert sugar, glucose and the like. Suitable excipients for the preparation of injectable solutions include water, alcohols, polyols, glycerol, vegetable oils and the like.

Pharmaceuticals are further added with various additions such as preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, buffers, coating agents, antioxidants, diluents, etc. Can contain ingredients.

Optionally, the pharmaceutical compositions of the present invention follow a general formulation combined with one or more compounds exhibiting varying activities, such as antibiotics or other pharmaceutically active substances. It may contain a compound. Such combinations are also within the scope of the present invention.

The present invention provides methods for treating mammalian diseases associated with bone metabolism, viral infections, inappropriate cell proliferation and the like. These methods specifically include the step of administering therapeutically effective amounts of the prodrugs of the invention to humans or other mammals in need of them. Suitable indications for such treatment are senile osteoporosis, postmenopausal or steroid-induced osteoporosis, Paget's disease, metastatic bone cancer, hyperparathyroidism, rheumatic arthritis, painful dystrophy, thoracic-costal bones. Clavicle hyperplasia, Gaucher's disease, Engelmann's disease, certain non-skeletal muscle disorders and periodontal disease, human immunodeficiency virus (HIV), influenza, simple herpesvirus (HSV), human herpesvirus 6, cytomegalovirus Includes (CMV), hepatitis B virus, Epstein-Barr virus (EBV), varicella herpesvirus, lymphoma, hematological disorders such as leukemia.

In certain aspects of the invention, methods of preventing or treating bone loss in mammals, especially humans, are provided, the method comprising administering to a human or mammal a therapeutically effective amount of a compound of the invention. The bisphosphonate prodrug that inhibits bone resorption of the present invention is to counter osteoclast-mediated bone resorption or bone loss in a state in which a prodrug is prepared from bisphosphonate and is known to be effective. , Useful in treatment. Suitable indications for such treatment include osteoporosis, especially osteoporosis in postmenopausal women, osteoporosis associated with long-term glucocorticoid therapy, and Paget's disease of bone. Similarly, the bisphosphonate compound clodronic acid (Ostac, Boehringer-Mannheim, Mannheim, Germany) is known to reduce metastases to bone in addition to internal organs in breast cancer patients at high risk of distal metastases (Diel, IJ). Et al., New Engl. J. Med., 339 (60 357-363) (1998)). The efficacy of the bisphosphonate prodrug of the present invention can be evaluated according to the same method as for the parent compound. These are comparative measurements of bone mineral density in the lumbar spine, femoral neck, trochanteric, forearm and whole body; measurement of vertebral deformity and height in vertebral fractures, osteoporosis, bone scanning or radiography of bone lesions in metastatic disease Including combinations with and.

In another aspect of the invention, promoting osteoclasting of the invention, which inhibits osteoblast and bone cell apoptosis, resulting in a higher net bone formation rate while substantially not altering osteoclast function. The step of administering the sex compound provides a method of increasing bone mass and strength in mammals, especially humans (Plotkin et al., J. Clin. Invest., 104: 1363-1374 (1999) and Van Beek et al., J. et al. . Bone Min. Res., 11: 1492 (1996)).

Yet another aspect of the invention provides a method of treating a disease caused by a viral infection. Suitable indications for such treatment are human immunodeficiency virus (HIV), influenza, smallpox virus (HSV), human herpesvirus 6, cytomegalovirus (CMV), hepatitis B and C viruses, Epstein- Diseases caused by barvirus (EBV), varicella herpes virus, and orthopox virus (eg, largepox and smallpox, vaccinia, smallpox, bovine pox, camel pox, monkey pox, etc.), Ebola virus, papilloma Includes easily infectious viruses such as viruses.

In yet another aspect of the invention, diseases caused by inappropriate cell proliferation, such as black species, lung cancer, pancreatic cancer, gastric cancer, colon and rectal cancer, prostate and breast cancer, leukemia and lymphoma. A method of treating the disease is provided. Anti-cancer agents that can be converted to their nucleotide phosphonates for use as compounds of the invention include cytarabine (ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine, cladribine, fludarabine, pentostatin 2'-deoxycoformycin), 6-mercaptopurine and 6-thioguanine, as well as substituted or unsubstituted ara-adenosine (ara-A), ara-guanosine (ara-G), and arauridine (ara-U) Included, but not limited to. The anti-cancer compounds of the present invention may be used alone or in combination with other types of anti-cancer agents such as other metabolic antagonists or alkaloids, topoisomerase inhibitors, alkylating agents, anti-neoplastic antibiotics and the like. it can.

The prodrugs of the invention can be administered in appropriate unit doses, if desired, via oral, parenteral, topical, transrectal, and other routes.

As used herein, the term "parenteral" means subcutaneous, intravenous, intraarterial, intramuscular, or intravitreal injection, or instillation.

The term "topical" includes transrectal and inhalation spray administration, as well as the more common oral and nasal skin and mucosal pathways, and during toothpaste.

The term "effective amount", when used in the phosphonate prodrugs of the present invention, is a dose that prevents or reverses the aforementioned diseases. Effective amounts, especially for diseases related to bone metabolism, are doses that prevent, attenuate, or reverse bone resorption, such as those that occur in abnormal or excessive bone resorption, or aging, especially in postmenopausal women, or to bone in breast cancer. It is a dose that prevents or counters metastasis and metastasis to internal organs.

The "effective amount" for a disease associated with viral infection or inappropriate cell proliferation (eg, cancer) is determined with reference to the recommended amount of antiviral or anticancer parent compound. The dose selected will vary depending on the activity of the selected compound, the route of administration, the severity of the condition being treated, and the condition and history of the patient being treated. However, it is within the art to start with a dose of the compound at a level lower than the dose required to achieve the desired therapeutic effect and to taper the dose until the desired effect is achieved. Is. If desired, the daily effective dose can be divided into multiple doses intended for administration, such as 2-4 doses daily. However, specific dose levels for any particular patient are determined by a variety of factors, including body weight, general health, diet, time, route of administration, concomitant medications, and the severity of the disease being treated. It seems to be understood.

Generally, the compounds of the present invention are dispensed into unit dosage forms containing 1% to 100% of the active ingredient. The therapeutic dose range is about 0.01 to about 1,000 mg / kg / day, preferably about 0.10 mg / kg / day to 100 mg / kg / day when administered as a drug to a patient, eg, a human. The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can vary for administration the dose of the active compound that is effective in achieving the desired therapeutic response for a particular patient.

Many animal studies have demonstrated the efficacy of bisphosphonates in the prevention of bone loss under experimental conditions designed to mimic the associated clinical disease. Based on these tests, several small animal model systems are available to evaluate the effects of bisphosphonates. These tests are also useful in measuring efficacy for comparison of the bisphosphonate prodrugs of the invention. Evaluation of bisphosphonate treatment typically requires measurement of thigh ash mass and bone mass, such as as measured as trabecular volume, between treated and untreated fauna. .. Thompson, D. et al., J. Bone and Mineral Res., 5 (3): 279-286 (1990) reveal the use of such a method to assess inhibition of bone loss in fixed rats treated with aminohydroxybutanebisphosphonates. There is. Yamamoto, M. et al., Calcif Tissue Int., 53: 278-282 (1993) caused hyperthyroidism in rats, resulting in bone changes similar to those in human hyperthyroidism, and thus bisphosphonate. Histomorphological analysis of treated and untreated groups biochemically based on osteocalcin measurements and including differences in cancellous bone volume, and bone, osteoclast and osteoblast surfaces in bone sections Was compared by histological comparison. Seedor, JG et al., J. Bone and Mineral Res., 6 (4): 339-346 (1991), Allen counters bone loss in ovariectomized rats by histomorphological analysis of thigh ash mass and tibial trabecular volume. A test of the action of dronate is described. The Schink assay, which involves a histological examination of the epiphysis of growing rats, can also be used as a screening assay. Examples of screening tests for evaluating the anti-bone resorption effect of the compounds of the present invention in experimental rats with osteopenia by various strategies are shown in Example 14.

The compounds of the present invention can generally be prepared in a variety of ways, as shown in Schemes I-VI. The general phosphonate esterification methods described below are provided for purposes of illustration only and are not configured to limit the invention in any way. In fact, several methods have been developed for the direct condensation of phosphonic acids with alcohol (see, eg, RC Larock, "Comprehensive Organic Transformations", VCH, New York, 1989, p. 966 and references therein. thing). Isolation and purification of the compounds and intermediates described in these examples, if desired, such as filtration, extraction, crystallization, flash column chromatography, thin layer chromatography, distillation or a combination of these techniques. It can be carried out by any suitable separation method or purification method. A detailed description of the appropriate separation and isolation methods is given in the Examples below. Of course, other equivalent separation and isolation methods can also be used.

Scheme I outlines the synthesis of bisphosphonate prodrugs containing primary amino groups, such as pamidronate or alendronate. Example 1 provides conditions for the synthesis of 1-O-hexadecyloxypropyl-arendronate (HDP-alendronate) or 1-O-hexadecyloxypropyl-pamidronate (HDP-pamidronate). In this method, a mixture of dimethyl4-phthalimide butanoylphosphonate (1b, prepared as disclosed in US Pat. No. 5,039,819) and hexadecyloxypropylmethylphosphite (2) in a pyridine solution is treated with triethylamine. , Bisphosphonate tetraester 3b is obtained and purified by silica gel chromatography. Intermediate 2 is Kers, A., Kers, J., Stawinski, I., Sobkowski, M., Kraszewski, A., Synthesis, April 1995, pp. 427-430, obtained by ester transfer of diphenyl phosphite. Thus, diphenyl phosphite in pyridine solution is first treated with hexadecyloxypropan-1-ol and then with methanol to provide compound 2.

Scheme I<chemistry num="21"><img id="000020" he="85" wi="137" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

An important aspect of this method is that other long chain alcohols can be used in place of hexadecylpropan-1-ol to produce a variety of compounds of the invention. Treatment of Intermediate 3b with acetonitrile in acetonitrile with bromotrimethylsilane selectively cleaves the methyl ester to give the monoester 4b. Treatment with hydrazine in a mixed solvent system of compound 4b (20% methanol / 80% 1,4-dioxane) results in the removal of phthalimide protecting groups as shown. The desired alendronate prodrug is collected by filtration and converted to a triammonium salt by treatment with methanolic ammonia.

Scheme II shows the synthesis of bisphosphonate analogs lacking the primary amino group. In this case, the treatment step is similar to Scheme I, except that protection with a phthalimide group followed by deprotection with hydrazine degradation is not required.

Scheme II<chemistry num="22"><img id="000021" he="67" wi="134" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Bisphosphonates with a 1-amino group, such as amino-olpadronate, can be converted to analogs of the prodrugs of the invention using the method shown in Slightly Modified Scheme III.

Scheme III<chemistry num="23"><img id="000022" he="72" wi="135" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

A mixture of compounds 2 and 3- (dimethylamino) propionitrile is treated with anhydrous HCl followed by the addition of dimethyl phosphite to give tetraester 3. After demethylation with bromotrimethylsilane, hexadecyloxypropyl-aminoolpadronate occurs.

Scheme IV illustrates the synthesis of bisphosphonate analogs in which the lipid group is attached to the primary amino group of the parent compound rather than the phosphonate ester.

Scheme IV<chemistry num="24"><img id="000023" he="97" wi="136" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Scheme V illustrates the general synthesis of alkylglycerol or alkylpropanediol analogs of cidofovir, cyclic cidofovir, and other phosphonate esters. Treatment of 2,3-isopropyridene glycerol 1 with NaH in dimethylformamide followed by reaction with alkyl methanesulfonate yields alkyl ether 2. Removal of the isopropylidene group by acetic acid treatment followed by reaction with trityl chloride in pyridine produces Intermediate 3. Alkylation of Intermediate 3 with an alkyl halide yields Compound 4. Removal of the trityl group with an 80% aqueous acetic acid solution gives O, O-dialkylglycerol 5. Bromination of compound 5 followed by reaction with a sodium salt of cyclic cidofovir or other phosphonate-containing nucleotides produces the desired phosphonate adduct 7. Ring-opening of the cyclic adduct is achieved by reaction with aqueous sodium hydroxide solution. Preferred propanediol species can be synthesized by substituting 1-O-alkylpropane-3-ol for Compound 5 in Scheme V. Tenofovir and adefovir analogs can be synthesized by substituting cCDV with these nucleotide phosphonates in Scheme V's reaction (f). Similarly, other nucleotide phosphonates of the invention can be produced in this way.

Scheme V<chemistry num="25"><img id="000024" he="51" wi="135" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>Reagents: a) NaH, R₁OSO₂Me, DMF; b) 80% aqueous acetic acid solution; c) trityl chloride, pyridine; d) NaH, R₂-Br, DMF; e) CBr₄, Triphenylphosphine, THF; f) Cyclic cidofovir (DCMC salt), DMF; g) 0.5N NaOH

Scheme VI illustrates a general method of synthesizing nucleotide phosphonates of the invention, using 1-O-hexadecyloxypropyl-adefovir as an example. Nucleotide phosphonate (5 mmol) is suspended in anhydrous pyridine and alkoxy alcohol or alkyl glycerol derivative (6 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 10 mmol) are added. The mixture is heated to reflux and vigorously stirred until the condensation reaction is complete, monitored by thin layer chromatography. The mixture is then cooled and filtered. The filtrate is concentrated under reduced pressure, the residue is adsorbed on silica gel and purified by flash column chromatography (elution with about 9: 1 dichloromethane / methanol) to give the corresponding phosphonate monoester.

Scheme VI<chemistry num="26"><img id="000025" he="41" wi="138" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

The present invention will be described in more detail with reference to examples, but these are not intended to limit the present invention.

<p num="0076">Example 1 Synthesis of 1-O-hexadecylpropanediol-3-alendronate A. <u style="single">Hexadecyloxypropylmethylphosphite (b)</u> Hexadecyloxypropylmethylphosphite, Kers, A., Kers, I., Stawinski, J., Sobkowski, M., Kraszewski, A., Synthesis, April 1995, pp. 427-430. Was prepared using. A pyridine (25 mL) solution of hexadecyloxypropan-1-ol (6.0 g, 20 mmol) was slowly added to a pyridine (50 mL) solution of diphenyl phosphite (14 g, 60 mmol) kept at 0 ° C. After stirring the mixture for 1 hour, anhydrous methanol (10 mL) was added. After further stirring for 1 hour, the residue was adsorbed on silica gel and chromatographed using a gradient elution method (20% ethyl acetate / 80% hexane from hexanes) to evaporate the solvent to give pure compound 2. It was obtained as a waxy low melting point solid (4.5 g, yield 60%).<img id="000026" he="20" wi="133" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0077">B. <u style="single">Hexadecyloxypropyltrimethyl 4-phthalimide butanoylphosphonate (3b)</u> Dimethyl 4-phthalimide butanoylphosphonate (1b, 3.0 g, 7.9 mmol, prepared according to US Pat. No. 5,039,819) and hexadecyloxypropylmethylphosphite (2, 2.9 g, 9 mmol) in a pyridine (50 mL) solution. Triethylamine (0.2 g, 2 mmol) was added. The mixture was stirred at room temperature for 5 hours and then the solvent was removed under reduced pressure. The residue was adsorbed on silica gel and chromatographed (ethyl acetate) to give compound 3b (3.5 g, 63%) as a viscous oil.<img id="000027" he="34" wi="132" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0078">C. <u style="single">Hexadecyloxypropyl 4-phthalimide butanoylphosphonate (4b)</u> Compound 3b (3.0 g, 4.3 mmol) obtained above was dissolved in dry acetonitrile (50 mL) and cooled to 0 ° C. A solution of bromotrimethylsilane (3.9 g, 25.5 mmol) in acetonitrile (25 mL) was added slowly and stirred for an additional 2 hours. The mixture was slowly poured into cracked ice. The resulting precipitate was collected by vacuum filtration and dried under reduced pressure to give 1.2 g of 4b (42% yield).<img id="000028" he="27" wi="134" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0079">D. <u style="single">1-O-hexadecylpropanediol-3-alendronate (5b)</u> Compound 4b (300 mg, 0.45 mmol) was dissolved in a mixture of 1,4-dioxane (20 mL) and methanol (5 mL). Anhydrous hydrazine was added and the mixture was stirred at room temperature for 4 hours. The separated precipitate was collected by vacuum filtration and washed with 1,4-dioxane. The solid was suspended in ethanol and methanol ammonia (3 mL) was added. After stirring for 10 minutes, the resulting solid was collected by filtration, washed with ethanol and dried under reduced pressure to give 220 mg HDP-alendronate (5b) as a triammonium salt. Analysis by FT-IR showed that the phthalimide protecting group had been removed. Electrospray MS m / e 532 (MH)⁺), 530 (MH^-) </p><p num="0080">Example 2 Synthesis of 1-O-hexadecylpropanediol-3-pamidronate (5a) 1-O-Hexadecylpropanodiol-3-pamidronate is prepared by a similar method (for Scheme 1), except that dimethyl 3-phthalimide propanoylphosphonate (1a) is prepared with 3-phthalimide propanoic acid. Will be done. Compound 1a is concentrated with compound 2 to give trimethylbisphosphonate 3a. Deprotection in steps C and D above to give HDP-pamidronate as shown.</p><p num="0081">Example 3 Synthesis of 1-O-octadecyl-2-O-methyl sn-glycero-3-alendronate Prodrac having a lipophilic group other than hexadecyloxypropyl is prepared by replacing hexadecyloxypropan-1-ol with various long-chain alcohols in step A of Example 1. For example, 1-O-octadecyl-2-O-methyl-sn-glycerol and diphenyl phosphite are reacted in pyridine and then treated with methanol to produce 1-O-octadecyl-2-O-methyl-sn-glycerylmethyl. Get Phosphite. The dialkylphosphite and phosphonate 1b are concentrated and then deprotected in steps C and D to give 1-O-octadecyl-2-O-methyl-sn-glycero-3-alendronate. Scheme 2 represents the synthesis of other bisphosphonate complexes that do not have a primary amino group in the side chain. In this case, protection with phthalimide groups and hydrazine degradation are not required.</p><p num="0082">Example 4 Synthesis of HDP-amino-olpadronate Scheme 3 shows the synthesis of the 1-aminobisphosphonate complex. The procedure described in Compound 2, 1- (dimethylamino) propionitrile of Example 1, and Orlovskii, VV; Vovsi, BA, J. Gen Chem. USSR (Engl. Transl.) 1976, 46: 294-296. To prepare the bisphosphonate trimethyl ester 3 using. Demethylate with bromotrimethylsilane to give HDP-amino-olpadronate as described in Step C of Example 1.</p><p num="0083">Example 5 Synthesis of 1-O-hexadecylpropanediol-3-succinyl-alendronate Scheme 4 shows the synthesis of a bisphosphonate complex in which the lipid group of the parent compound is attached to a primary amino group. Tetramethyl- (4-phthalimide-1-hydrobutylidene) bisphosphonate (2.0 g, 4.4 mmol) was dissolved in 0.2 M methanol hydrazine (100 mL) and stirred at room temperature for 3 days. After concentrating the mixture in half, the solids began to separate. The solid was filtered off and the filtrate was concentrated to dryness. Proton NMR showed that this compound was a tetramethyl- (4-amino-1-hydroxybutylidene) bisphosphonate. It was dried at 50 ° C. overnight with phosphorus pentoxide. 1.2 g of compound was suspended in a mixture of pyridine (25 mL) and N, N-dimethylformamide (25 mL) and 3-succinyl-1-hexadecylpropane (1.76 g, 4.4 mmol). ) Was added. Dicyclohexylcarbodiimide (2.52 g, 12.21 mmol) was added and the mixture was stirred at room temperature for 2 days. The mixture was filtered, the filtrate was adsorbed on silica gel and flash chromatographed with dichloromethane (0% -20%) on an increasing gradient of methanol to give succinylated compounds. This was deprotected with trimethylsilyl bromide in acetonitrile to crystallize it from methanol to give the title compound purified.</p><p num="0084">Example 6 Synthesis of adefovir hexadecyloxypropyl ester and 1-O-octadecyl-sn-glyceryl ester Adefovir (1.36 g, 5 mmol) and 3-hexadecyloxy-1-propanol (1.8 g, 6 mmol) were mixed in dry pyridine and DCC (2.06 g, 10 mmol) was added. The mixture was heated to reflux, stirred for 18 hours, then cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was poured onto a short column of silica gel. The column was eluted with 9: 1 dichloromethane / methanol to give hexadecyloxypropyl-adefovir (HDP-ADV) as a white powder.</p><p num="0085"> Adefovir (1.36 g, 5 mmol) and 1-O-octadecyl-sn-glycerol (2.08 g, 6 mmol) were mixed in dry pyridine (30 mL) and DCC (2.06 g, 10 mmol) was added. The mixture was heated to reflux, stirred for 18 hours, then cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was poured onto a silica gel column. The column was eluted with 9: 1 dichloromethane / methanol to give 1-O-octadecyl-sn-glyceryl-3-adefovir.</p><p num="0086">Example 7 Synthesis of AZT-phosphonate hexadecyloxypropyl ester Phosphonate analogs of AZT (3'-azido-3'-5'-dideoxythymidine-5'-phosphonic acid) have been published in the published procedure (Hakimelahi, GH; Moosavi-Movahedi, AA; Sadeghi, MM; Tsay, SC. .; Hwu, JR Journal of Medicinal Chemistry, 1995 38, 4648-4659).</p><p num="0087"> After suspending AZT phosphonate (1.65 g, 5 mmol) in dry pyridine (30 mL), add 3-hexadecyloxy-1-propanol (1.8 g, 6 mmol) and DCC (2.06 g, 10 mmol) and heat the mixture. After refluxing and stirring for 6 hours, the mixture was cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was poured onto a silica gel column. Elute the column with 9: 1 dichloromethane / methanol and 3'-azido-3'-5'-dideoxythymidine-5'-<u style="single">Phosphonate</u>, Hexadecyloxypropyl ester was obtained.</p><p num="0088">Example 8 Synthesis of hexadecyloxypropyl, octadecyloxypropyl, octadecyloxyethyl, and hexadecyl esters in cyclic cidofovir N, N-dicyclohexyl-4-morpholincarboxymidine (DCMC, 1.0 g, 3.5 mmol) was added to a stirred N, N-DMF (25 mL) suspension of cidofovir (1.0 g, 3.17 mmol). The mixture was stirred overnight to dissolve cidofovir. The clear solution was filled in another funnel and 1,3-diclohexyl carbodiimide (1.64 g, 7.9 mmol) was added slowly (in 30 minutes) to a stirred hot pyrimidine solution (25 mL, 60 ° C). The reaction mixture was stirred at 100 ° C. for 16 hours, cooled to room temperature, and the solvent was removed under reduced pressure. Gradient elution method (CH) by adsorbing the residue on silica gel₂Cl₂Purified by flash column chromatography using + MeOH). The UV active product is finally 5: 5: 1 CH₂Cl₂/ MeOH / H₂Elution with O allowed the solvent to evaporate to give 860 mg of a white solid.¹H and³¹A P NMR spectrum showed that this was a DCMC salt of cyclic cidofovir (yield = 44%).</p><p num="0089"> Add 1-bromo-3-hexadecyloxypropane (1.45 g, 4 mmol) to a dry DMF (35 mL) solution of cyclic cidofovir (DCMC salt) (0.5 g, 0.8 mmol) and mix 6 at 80 ° C. It was heated for hours and stirred. The solution is concentrated under reduced pressure, the residue is adsorbed on silica gel, and the gradient elution method (CH)₂Cl₂Purified by flash column chromatography using + EtOH). Alkylation product 90:10 CH₂Cl₂Elution with / EtOH. The fraction containing the purified product was evaporated to give 260 mg of HDP-cyclic cidofovir (55% yield).</p><p num="0090"> Add 1-bromo-3-octadecyloxypropane (2.82 g, 7.2 mmol) to a dry DMF (35 mL) solution of cyclic cidofovir (DCMC salt) (1.0 g, 3.7 mmol) and heat the mixture at 85 ° C for 5 hours. And stirred. The solution is concentrated under reduced pressure, the residue is adsorbed on silica gel, and the gradient elution method (CH)₂Cl₂Purified by flash column chromatography using + MeOH). Alkylation product 9: 1 CH₂Cl₂Eluted with / MeOH. The fraction containing the purified product was evaporated to give 450 mg of ODP-cyclic cidofovir.</p><p num="0091"> Add 1-bromo-3-octadecyloxyethane (3.0 g, 7.9 mmol) to a dry DMF (35 mL) solution of cCDV (DCMC salt) (1.0 g, 3.7 mmol) and heat the mixture at 80 ° C for 4 hours. And stirred. The solution is concentrated under reduced pressure, the residue is adsorbed on silica gel, and the gradient elution method (CH)₂Cl₂Purified by flash column chromatography using + MeOH). Alkylation product 9: 1 CH₂Cl₂Eluted with / MeOH. The fraction containing the purified product was evaporated to give 320 mg of octadecyloxyethyl-cCDV.</p><p num="0092"> 1-Bromohexadecane (1.2 g, 4 mmol) was added to a dry DMF (35 mL) solution of cyclic cidofovir (DCMC salt) (0.5 g, 0.8 mmol) and the mixture was heated at 80 ° C for 6 hours and stirred. .. The solution is concentrated under reduced pressure, the residue is adsorbed on silica gel, and the gradient elution method (CH)₂Cl₂Purified by flash column chromatography using + MeOH). A The alkylated products 9: 1 CH₂Cl₂Eluted with / MeOH. The fraction containing the purified product was evaporated to give 160 mg of hexadecyl-cCDV.</p><p num="0093">Example 9 Synthesis of hexadecyloxypropyl, octadecyloxypropyl, octadecyloxyethyl, and hexadecyl esters in cidofovir The above hexadecyloxypropyl-cyclic CDV was dissolved in 0.5M NaOH and stirred at room temperature for 1.5 hours. The pH was adjusted to about 9 by dropping 50% acetic acid water. The precipitated HDP-CDV was isolated by filtration, washed with water, dried and recrystallized from (3: 1 p-dioxane / water) to give HDP-CDV.</p><p num="0094"> Similarly, octadecyloxypropyl-, octadecyloxyethyl-, and hexadecyl-cCDV esters were hydrolyzed and purified with 0.5M NaOH to give the corresponding sidophodiesters.</p><p num="0095">Example 10 Cyclic-Synthesis of ganciclovir phosphonate hexadecyloxypropyl ester Published Procedures for Cyclic Phosphonate Analogs of Ganciclovir (Reist, EJ; Sturm, PA; Pong, RY; Tanga, MJ and Sidwell, RW, "Synthesis of Acyclonucleoside Phosphonate for Evaluation of Antiviral Agents" of acyclonucleoside phosphonates for evaluation as antiviral agents) , pp. 17-34, J, C. Prepared using Martin (ed.), Nucleotide Analogues as Antiviral Agents, American Chemical Society, Washington, DC). The cGCV phosphonate was converted to DCMC salt in DMF, treated with 1-bromo-3-hexadecyloxypropane and the mixture was heated at 80 ° C. for 6 hours. The alkylated product was isolated by flash chromatography to give HDP-cyclic-GCV phosphonate.</p><p num="0096">Example 11 Synthesis of ganciclovir phosphonate hexadecyloxypropyl ester The HDP-cyclic-GCV phosphonate obtained above was dissolved in 0.5M NaOH and stirred at room temperature to convert to an acyclic diester. The solution was neutralized with 50% acetic acid water to precipitate the product recrystallized with 3: 1 p-dioxane / water.</p><p num="0097">Example 12 1-O-hexadecyloxypropane alendronate inhibits dexamethasone-induced apoptosis of MLO-Y4 osteoocytes MLO-Y4 osteoocytes were pretreated with indicated concentrations of 1-O-hexadecyloxypropane alendronate (HDP-alendronate) for 1 hour, then dexamethasone (final concentration 10).^-4M) Cells were incubated for 6 hours in the presence and absence. The dead cell rate was determined by the uptake of trypan blue (Plotkin et al., J Clin Invest 104: 1363-1374, 1999). The results are shown in Fig. 1. The line segment represents the mean ± SD of three independent measurements. Data were analyzed by one-way ANOVA (Student-Keuls-Newman test).^*p &lt;0.05. HDP-Alendronate 10 Dexametason-Induced Apoptosis^-8From 10^-5Inhibit with M.</p><p num="0098">Example 13 1-O-hexadecyloxypropane alendronate inhibits dexamethasone-induced apoptosis of calvaria cells Calvaria cells were taken from neonatal C57 BL / 6J mice and transferred to tissue culture. Cells are pretreated with the indicated concentration of HDP-alendronate, then 10^-4Cells were incubated for 6 hours in the presence and absence of M dexamethasone. The dead cell rate was determined by the uptake of trypan blue (Plotkin et al., J Clin Invest 104: 1363-1374, 1999). The results are shown in Fig. 2. The line segment represents the mean ± SD of the three independent measurements. The data were analyzed by one-way ANOVA (Student-Cruise-Newman test).^*p &lt;0.05. HDP-Alendronate is 10^-8At M or higher concentrations, it inhibited the increase in dexamethasone-induced death cell rate (p = &lt;0.05). Exposure of cells to 0.05 μM DEVD (a peptide inhibitor of apoptosis) did not show an increase in mortality, indicating that DEVD inhibits dexamethasone-induced apoptosis.</p><p num="0099">Example 14 Inhibition of bone resorption in ovariectomized rats by 1-O-hexadecyloxypropane alendronate 4-Amino-1-hydroxybutylidene-1,1-bisphosphonate disodium salt or 1-O-hexadecylpropanediol-3- in Sprague-Dawley female rats (250 gm to 280 gm) who underwent surgery to remove both ovaries. Alendronate is injected subcutaneously at doses of 0 mg / kg / day to 8 mg / kg / day for 4-12 weeks. At 12 weeks, the rats included in the control group are sacrificed and the femur ash is collected in each animal. Alternatively, the method of administration may be oral. The ash mass of each individual femur is measured and the value of each group is compared to an index of bone mass to measure the relative inhibition of bone loss during the treatment protocol. Animals treated with 1-O-hexadecylpropane alendronate show that bone loss is inhibited compared to control ovariectomized animals.</p><p num="0100">Example 15 Inhibition of bone resorption in human osteoporosis by 1-O-octadecyloxypropyl-alendronate Two groups of postmenopausal women are treated with placebo or 1-O-octadecyloxypropyl-alendronate at oral doses of 0.1 mg / kg / day to 100 mg / kg / day for 2-3 years, respectively. .. Each of the treatment groups is continuously observed for bone mineral density, spinal fracture incidence, radiographic test progression of spinal deformity, and height loss over the treatment period. The measurements are compared in the various treatment groups to determine their effectiveness in the form of alendronate treatment between the treatment groups. The group treated with 1-O-octadecyloxypropyl alendronate appears to have fewer fractures and a lower rate of bone mineral density loss than the placebo group.</p><p num="0101">Example 16 Stimulation of bone formation in steroid-induced human osteoporosis with 1-O-octadecyloxypropyl-amino-olpadronate A group of patients with steroid-induced osteoporosis treated with 1-O-octadecyloxypropyl-amino-olpadronate or placebo at an oral dose of 0.1 mg / kg / day to 100 mg / kg / day for 1 month to 1 year. Process the period. Each of the treatment groups is continuously observed for bone mineral density, spinal fracture incidence, radiographic test progression of spinal deformity, and height loss over this treatment period. The measurements are compared in the various treatment groups to determine the effectiveness of 1-O-octadecyloxypropyl-amino-olpadronate treatment between the treatment groups. Compared to placebo treatment, patients treated with 1-O-octadecyloxypropyl-amino-olpadronate had increased bone mineral density and reduced fractures.</p><p num="0102">Example 17 Antiviral activity and selectivity of phosphonate nucleotide analogs against human cytomegalovirus (HCMV) HCMV Antiviral Assays: Antiviral assays for HCMV DNA include Dankner, WM, Scholl, D., Stanat, SC, Martin, M., Souke, RL and Spector, SA, J. Virol. This was done as described in Methods, 21: 293-298 (1990). Briefly, subdense MRC-5 cells in 24-well culture dishes at various concentrations in Eagle's Minimal Essential Medium (E-MEM) containing 2% FBS and antibiotics. Pretreated for 24 hours. The medium was removed and the HCMV strain was added in drug-free wells within 5 days at a dilution that produced a 3-4+ cytopathic effect (CPE). The virus was adsorbed at 37 ° C for 1 hour, aspirated and the drug dilution was replaced. After incubation for 5 days, HCMV DNA was subjected to the CMV Antivirus Susceptibility Test from Diagnostic Hybrids (Athens, OH). Nucleic acid hybridization using Kit) was used to quantify in triads. The medium was removed and the cells were lysed according to the manufacturer's instructions. The solution was adsorbed on a Hybriwix filter and then hybridized overnight at 60 ° C. Hybriwix was washed at 73 ° C for 30 minutes and measured with a gamma counter. The result, EC₅₀Shown as (50% inhibitory concentration).</p><p num="0103"> Preliminary experiments were performed on cidofovir and adefovir 1-O-hexadecylpropanediol (HDP) derivatives and are shown in Table 1.</p><p num="0104">(table 1)<img id="000029" he="41" wi="144" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0105"> The results in Table 1 show that 1-O-hexadecylpropanediol-3-cyclic CDV (HDPcCDV) is&gt; 900 times more active than CDV or cyclic CDV. The selectivity index for HCMV in more cytotoxic but rapidly dividing cells was&gt; 59,000, compared to 1,900 to&gt; 2,100 for uninduced CDV. Based on these promising preliminary results, additional experiments were performed with additional compounds of the invention. These additional experiments are shown below.</p><p num="0106"> Cytotoxicity of test compounds in vitro: Subcongested human lung fibroblasts (MRC-5, American Type Culture Collection, Rockvill, MD) in 24-well plates, 2 % Treated with diluted drug in E-MEM (Gibco BRL, Grand Island, NY) supplemented with fetal bovine serum and antibiotics. After incubation at 37 ° C for 5 days, the cell monolayer was visually inspected under a microscope to estimate the drug concentration that resulted in a 50% reduction in cell number.</p><p num="0107"> The data obtained from these experiments are shown in Table 2.</p><p num="0108">(Table 2) Inhibition of human CMV replication in MRC-5 human lung fibroblasts assayed by DNA depletion<img id="000030" he="74" wi="151" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" />EC₅₀-50% effective concentration; CC₅₀ -50% cytotoxic concentration; selectivity index-CC₅₀/ EC₅₀.. EC₅₀The result is the average of 3 to 6 measurements, except that ADV is repeated once in pairs.</p><p num="0109"> As shown in the results shown in Table 2, the compounds of the present invention are uniformly more active and selective than underived cidofovir, cyclic cidofovir and adefovir.</p><p num="0110"><u style="single">Example 18</u><u style="single">In vitro effects of HDP-cCDV on poxvirus replication</u> The activity of cidofovir (CDV), cyclic cidofovir (cCDV), and 1-O-hexadecylpropanediol-3-cCDV (HDP-cCDV) in antiviral cells infected with vaccinia virus or bovine hemorrhoid virus. Activity was tested by measuring a dose-dependent reduction in cytopathic effect (CPE). Preliminary vaccinia and cowpox EC₅₀Values were determined in a CPE reduction assay for human foreskin fibroblasts (HFF). The data thus obtained is shown in Table 3.</p><p num="0111">(Table 3)<img id="000031" he="41" wi="146" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0112"> As shown in Table 3, HDP-cCDV is highly active against vaccinia virus and IC₅₀The value was 0.11 μM, whereas that of cCDV and CDV was 0.97 and 1.8 μM, respectively. HDP-cCDV is very effective in cowpox-infected cells, IC₅₀The values were &lt;0.03 μM, whereas those for cCDV and CDV were 0.72 and 2.1, respectively. Based on these promising preliminary results, the effects of the cidofovir analogs of the present invention on other orthopoxviruses were investigated.</p><p num="0113"> Poxvirus antiviral cytopathic effect (CPE) assay: For each drug concentration, 3 wells containing Vero cells were infected with 1000pfu / well of orthopoxvirus, and the other 3 remained uninfected. And the toxicity was determined. Plates were tested and stained after viral infection and untreated cells showed 4 + CPE. Neutral red was added to these media and CPE was assayed for neutral red uptake at 540 nm. 50% inhibition (EC₅₀) And cytotoxic concentration (CC₅₀) Was determined from the dose-response plot. The results are shown in Table 4.</p><p num="0114">(Table 4)<img id="000032" he="44" wi="150" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" />EC₅₀-50% effective concentration; CC₅₀ --50% cytotoxic concentration in Vero cells; selectivity index-CC₅₀/ EC₅₀.. Same abbreviation as Table 2. The result is the average of 3 measurements.</p><p num="0115"> As shown in Table 4, the compounds of the invention were substantially more active against vaccinia, cowpox, and various smallpox strains than uninduced CDV or cCDV.</p><p num="0116"><u style="single">Example 19</u><u style="single">In vivo effects of 1-O-hexadecylpropanediol-3-adefovir (HDP-ADV) on HIV-1 replication</u> Preliminary experiments on the inhibition of HIV-1 replication by the compounds of the present invention were performed as follows. The drug assay was performed as described above in Larder et al., Antimicrobial Agents &amp; Chemotherapy, 34: 436-441 (1990). HIV-1_LAIInfected HT4-6C cells were exposed to the indicated drug and incubated at 37 ° C for 3 days. These cells were fixed with crystal violet to visualize plaques. Antiviral activity was assessed as a percentage of control plaque (without drug) measured in drug-treated samples. EC₅₀Is a μM concentration that reduces the number of plaques by 50%. The activity of adefovir in HIV-1-infected HT4-6C cells was compared to AZT (sidovudine) and 1-O-hexadecylpropanediol-3-adefovir (HDPADV). The results are shown in Table 5.</p><p num="0117">(Table 5)<img id="000033" he="31" wi="144" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0118"> Adefovir is EC₅₀Was 16 μM, which was a moderate activity. AZT was highly active as expected (EC)₅₀Is 0.007 μM), but HDP-ADV is the most active of these three compounds, EC₅₀Was 0.0001 μM, which was more than 5 log times more active than adefovir itself. Based on these promising preliminary results, additional experiments were performed as follows.</p><p num="0119"> HIV-1 antiviral assay: The effect of antiviral compounds on HIV replication in CD4-expressing HeLa HT4-6C cells was measured by plaque reduction assay (Larder, BA, Chesebro, B. and Richman, DD, Antimirob. Agents Chemother., 34: 436-441 (1990)). Briefly, monolayers of HT4-6C cells were infected with 100-300 plaque forming units (PFU) virus per well on 24-well microdiluted plates. As mentioned above, various concentrations of drug were added to culture medium, Dulbecco's modified Eagle's medium containing 5% FBS and antibiotics. After 3 days at 37 ° C, the monolayer was fixed in a 10% formaldehyde solution with phosphate buffered saline (PBS) as a solvent and stained with 0.25% crystal violet to make the viral plaque visible. .. Antiviral activity was evaluated as a percentage of control plaque measured in drug-treated samples. Cytotoxicity was assayed by the method of Hostetler et al. (Antivirus Research, 31: 59-67 (1996)). The results are shown in Table 6.</p><p num="0120">(Table 6) Inhibition of HIV replication in HT4-6C cells in plaque reduction<img id="000034" he="30" wi="141" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" />EC₅₀-50% effective concentration; CC₅₀ --50% cytotoxic concentration in Vero cells; selectivity index-CC₅₀/ EC₅₀.. EC₅₀Values are the average of 4 experiments.</p><p num="0121"> As readily shown by the results in Table 6, the compound 1-O-hexadecylpropanediol-3-ADV of the present invention has more activity and selectivity than adefovir.</p><p num="0122"><u style="single">Example 21</u><u style="single">Effect of cidofovir analogs on herpes virus replication</u> HSV-1 antiviral assay: Subdense MRC-5 cells in 24-well culture dishes, media removed and HSV-1 virus-free wells in 20-24 hours 3-4 + Inoculated by diluting and adding to give CPE results. It was absorbed at 37 ° C for 1 hour, aspirated and replaced with various concentrations of drug in E-MEM containing 2% FBS and antibiotics. After incubation for approximately 24 hours, HSV DNA was quantified in triplets by nucleic acid hybridization using the HSV Antivirus Susceptibility Test Kit from Diagnostic Hybrids (Athens, OH). The medium was removed and the cells were lysed according to the manufacturer's instructions. After adsorbing the lysate on a Hybriwix filter, it hybridized overnight at 60 ° C. Hybriwix was washed at 73 ° C for 30 minutes and measured with a gamma counter. Cytotoxicity was evaluated as described in Example 17. EC obtained in this way₅₀And CC₅₀Is shown in Table 7.</p><p num="0123">(Table 7) Inhibition of human HSV replication in MRC-5 human lung fibroblasts assayed by DNA depletion<img id="000035" he="64" wi="149" file="JP5963894B2_D0001.tif" img-format="tif" img-content="drawing" />Same abbreviation as Table 2: EC₅₀-50% effective concentration; CC₅₀ -50% cytotoxic concentration; selectivity index-CC₅₀/ EC₅₀.. EC₅₀Values are the average of two experiments, excluding HDP-CDV measured once in pairs.</p><p num="0124"> As shown in Table 7, all compounds of the invention are more active against HSV-1 than underived nucleotide phosphonates, cidofovirs, or cyclic cidofovirs.</p><p num="0125"> Although the invention described above has been described in detail with illustrations and examples for clarity and understanding, those skilled in the art will deviate from the spirit or scope of the claims in view of the content of the invention. Unless otherwise, it seems clear that certain modifications and modifications can be made.</p>