Esters of l-carnitine or alkanoyl l-carnitines useful as cationic lipids for the intracellular delivery of pharmacologically active compounds.

Description

The invention described herein refers to the class of novel esters of L-carnitine and acyl L-carnitine and their use as cationic lipids which are suitable for supporting intracellular transport of pharmaceutically active compounds, by their transfer through membrane, or to promote their interaction with certain cell sites (receptors).

The invention described herein also relates to other known esters of L-carnitine and acyl L-carnitine, which are useful for the same purpose as the novel compounds mentioned above.

What is meant by the term "intracellular transport" is the cellular gene or plasmid of natural origin or alteration, administered by therapeutic activity (gene transfer) or the introduction of drugs or immunogenic peptides into the cells.

Many pharmaceutical drugs, such as, for example, polypeptides and proteins or drugs generally need to enter the cells to apply their function to the subcellular molecular level. For these molecules the cell membrane consists of a selective bracket. Frumuhimnan, in fact, performs a protective workforce that prevents the entry of potentially toxic substances, but also leads to compounds possessing behavioral activity. The complex combination of cell membranes comprises phospholipid, glycolipid and protein; hips affect her activity with factors like Ca⁺⁺ and ions of ions, ATP, microparticles, microbes, enzymes and proteins that bind Ca⁺⁺. The interaction between the cellular and peripheral factors of the cell and the vidbragdid of external signals is responsible for the selectivity shown by and medal of many different cellular organisms. The hemispheric effect of the membranes can be overcome by combining substances in complexes with lipid formulations that reconstruct the composition of membrane membranes that occur in nature. These lipids have the ability to merge at the membranes and release the substances that are combined with them inside the cells. The lipid complexes do not only treat intracellular interactions by combining the membranes, but also can minimize load loss between the molecules that penetrate into the cell. Doubable lipids, such as the phospholipid membrane, form lipid blisters of lipids in the aqueous systems.

Liposomes are a bleeding pair whose water volume is completely closed with one more more membrane consisting of lipid molecules, usually phospholipids. Phospholipid, which consists of hydrophobic head and a pair of carbon chains (hydrophobic halves), are the main components of organic membranes. In the aqueous solution, the hydrophobic halves adhere to the exclusion of water, while the hydrophobic headers interact with the waist, which suddenly form a number of leaves with different diameters. Lipidis are usually bi-ionic, neutral and anionic. These platelets can be used as ferries of drugs, small molecules, proteins, nuclei and plasmids.

For the past few years, cationic liposomes, which are a category of positively charged leaves made from ready-made lipids, are very important for the transfer of inherited substances inside the cells. Negative loading DNA can interact with positive cations of the cationic lipids, forming the DNA-lipid complexion. Simplicity and versatility of this technology has made liposomes an important ferry for the distribution of genes to genetic behaviors in human subjects. Today, the majority of peacemakers reach as genetic conductors, and approvals of Radiology Advisory Committee NIH, to virus systems and ready-made systems.

Viral infection involves a series of complex processes in order to assault a particular cell and carry the DNA into the nucleus. The basic prerequisite for the use of viral viruses is based on the possibility of replacing the viruses with gene coding for the treatment mode without removing the virus's ability to infect cells. Viral therapy limits contain those viral factors that can be immunogenic, cytokine-dependent and cause rearrangement.

High hopes are attached to the use of cationic lipids in gene therapy. These genetic features possess great potential compared to those of biological origin, as they are much safer, less toxic and also have the ability to implement high-level genes.

However, as compared to biotype genotypes, they still have low yield on intracellular gene recoding. It should be borne in mind, however, that the use of such a retrieval system is at the initial stage of research. Cationic lipids play a very important role in the formation of the DNA lipid complex, in cellular interaction, in merging with the membrane, in the release of DNA within the cell and in transcription.

There are important examples of the use of cationic lipids in living bodies. The first clinical trial of genetic therapy was performed by implementing an expression vector that contained the human liposome-complex HLA-B7 gene for the treatment of melanoma. Another important use refers to the treatment of pulmonary fibrosis in the lungs through a respiratory tract or as a nasal spray on the liposome-complexed expression vector SV-40C-FTR. Other clinical studies involving the use of liposomes in cancer treatment are currently underway.

Four basic elements are usually identified in the construction of cationic lipids: the positively charged cationic head, the spacer, the festulipid and the linkage.

The cationic head is responsible for the interaction between cationic lipids and DNA, in between

DNA liposome complex and cell membrane and other parts of the cell. It consists of single or multicandionic groups (which depends on the number of loads) that can be varied.

The spacecraft is part of the molecule that separates the cationic head from the hydrophobic tail and contributes to ensuring the maximum connection between the cationic haze and the negative charge of the DNA phosphates.

The festulipid is the nonpolar hydrocarbon component and determines the physical properties of the double lipid layer, such as the rigidity and rate of interchange of membrane glycol. What is meant by a "linker" is the connection between the hydrocarbon chain and the leaving of the molecule. This interface determines chemical stability and the biological degradability of cationic lipids.

In recent years, the use of liposomes has increased steadily in the cosmetics industry. The success of the liposomes in this field is due to the fact that these compounds are well tolerated by the skin. They are used both as ferries for active ingredients and as compounds that support later absorption.

Published science and patent materials are full of references to the proliferation and use of liposomes; However, there are very few references describing the use of carnitine derivatives useful in gene distribution, as there are no documents dealing with known methods of production of compounds similar to microorganisms consistent with the invention described. is here.

Patent Application EP 0 279 887 describes the use of a derivative of carnitine, i.e. phosphatidyl carnitine, optionally in combination with other phospholipids and lipids (cholesterol, phosphatidylcholine, phosphatidylserine), in the production of liposomes.

In the examples of liposomes, lipoproteins of phosphatidyl carnitine are produced as introduced propranolol, a medicine known to be effective as an anti-cardiovascular and anti-arrhythmic agent. The carnivorous skin is noted here for the apparent cardiac fatigue of carnitine. This tendency makes it possible to prevent the fractures from breaking through the nest rather than reaching the desired destination.

Continuous phosphatidyl carnitine also allows for oral administration of liposomes, as they are resistant to liposomal intestines.

In J. Med. Chem. 1998 Jun 18; 41 (13): 2207-15, there are described a number of esters of L-carnitine that are used for gene distribution, but they are not described as suggested by drug users for drug distribution.

WO 96/39193 discloses innovative targeting agents which are directed into incubation through carnitine-acyl carnitine traslocase kerfid, but do not indicate those used in the preparation of lipids.

EP 559 625 B1 discloses a number of esters of L-carnitine and acyl carnitine which possess the selective vasoconstrictor activity on the digestive tract.

In recent years, molecular interventions have identified a number of defects at chromosome levels that cause inherited diseases in human subjects.

An important field of modern medicine is concerned with the treatment of these inherited diseases by using behavioral information for genetic behavior.

As has been observed, cationic lipids are widely used in intracellular distribution of drug compounds, by transferring through membrane studies to their interaction at a particular cell membrane pathway (transient).

These genera have great potential midad with those of biological origin, as they are much safer, less poisonous, and also have the ability to incorporate a high staerd gene. As compared to genotypes of biological gerd, they have a low yield on intracellular transcription.

Furthermore, requiring gene retention of the medium of its conventional cationic lipid through plasmid DNA and cationic lipid is kept separate, and upon their preparation, it is performed immediately prior to gene transfer.

Attempts to support these multiculturalists have yet to be fought by giving encouraging support, in fact, they are only supportive of a matter of time.

As a result of genetic resuscitation and drug distribution, therefore, there is a strong need for stable, repeatable seat-specific systems that are also active after a suitable long period of time.

It has been found that a cationic lipid group which is strongly active in promoting intracellular distribution of drug-containing compounds consisting of new esters of L-carnitine and acyl L-carnitines.

These new compounds are stable and highly selective because they are local-5 selected to reach the target organ.

This feature makes them particularly useful for transferring active compounds directly to the place where they can apply their drug activity.

The compounds of the invention as general formula (I):

described here are compounds with

<img file="IS2476B_D0001.tif" />

| \

R1

R2 (I) wherein:

n is an integer from 1 to 3;

R is hydrogen or alkanoyl, straight or branched, with 2-6 carbon atoms;

Ri and R₂, which may be the same or different, represent saturated or unsaturated straight-chain alkyl, with 3-20 carbon atoms; and X is the anion of pharmaceutically acceptable acid.

Examples of R are acetyl, propionyl, butyryl, valeryl and isovaleryl.

Examples of Ri and R₂ are hexanoyl, octaneoyl, myristoyl, palmitoyl or oleoyl.

Preferred examples of compounds according to the invention are:

- ester of L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 770);

- ester of acetyl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 771);

- ester of propionyl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 772);

- ester of isobutyryl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 773);

- ester of isovaleryl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 774);

- ester of L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 810);

- ester of acetyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 809).

ester of propionyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 808).

What is meant by anion of pharmaceutically acceptable acid is any anion an acid that does not cause undesirable toxicity or side effects.

These acids are well-known pharmacists and experts in the field of pharmaceutical technology.

Examples of these anions, but not only those listed herein, are: chloride; bromide; iodide; aspartate; sýruaspartat; citrate; acid citrate; acid tartrate; phosphate; acid phosphate; fumarate; glycerophosphate; glycerophosphate; glucose phosphate; lactate; maleate; acid maleate; mucate; orotate; oxalate; sýruoxalat; sulfate; trichloroacetate; trichloroacetate; methanesulfonate; pamate and syrupamate.

Compounds of formula (I), in the form of liposomes, are agents useful both for natural or altered plasmid distribution or nuclei useful in gene therapy, or as a code for peptides or proteins useful as a vaccine, and for general distribution drugs, such as, for example, anticancer agents, antiviral agents, bactericidal agents, fungicides, antidiabetics, drugs useful in treating cardiovascular diseases, or immunogenic peptides and other medicinal products useful in therapy.

Lipomycin containing the compound of formula (I) are prepared using conventional methods, a well-known person of ordinary skill in the art; see, for example, Allen TM Drugs 56, 747-56 (1998). Lipomycin according to the present invention may also be prepared by using other factors well known in the art of lipoprotein technology. In one embodiment of the present invention, liposomes may contain a helpful lipid, a term well known in the art. Examples of helper lipids are cholesterol, 1-palmitoyl-2-oleoyl phosphatidylcholine or dioleyl phosphatidylcholine.

Liposomes of the present invention are appropriately presented as combinations. In the manifestation of the distribution of pharmaceutical compounds, the compositions are separated as pharmaceutically, which may comprise pharmaceutically acceptable ferries and / or excipients.

Compounds of formula (I), in the form of liposomes, may also be useful in the preparation of excipient compositions comprising both the liposomes per se as active excipients and for dispersion of cosmetic agents, such as, for example, moisturizing agents, nutrients, anti-corrosion agents, anti-wrinkle agents, anti-dermatologic agents and agents that inhibit skin throat ulceration.

Liposomes composed of compounds of formula (I) may be administered by mouth or via the gastrointestinal tract, intravenously, intramuscularly, subcutaneously, subcutaneously, or in the form of nasal or oral contraceptives.

The process for the preparation of compounds of formula (I) according to the invention is illustrated by the following reaction catalytic converters, which are intended to include this general formula (I). A skilled person can easily get all the groups assigned to R, Ri and R₂, where all necessary reagents are available on the market or published in published materials and reaction conditions are generally applicable to the overall inventions of the present invention and any change, if necessary usually achieved within generally accepted knowledge.

CH.OH

I 'c = o

I

CH.OH

CICO- (CH.) "CH.

CH.OCO (CH,) "CH,

C-0

I

CH.OCO (CK,) "CK,

In ») a)

CH_;OCO (CH_:), "CK,

C-0

I

CH_OCO &lt;CH_;) ,, C - :.

N * BH,

CK.OCO {Cri)._MCn.

I ''

Choe

I

CtLOCO (CH)._MCK, (2) a,

CK.OCO &lt;CK.) ,. CH,

CH-OH

I

CKOCO (CH.) "CH, coccct. Br

CK.OCO (CH)_MCH

CH-OCOCH.Br

I '

CKOCO &lt;CH.), CH, (3) c&gt;

<img file="IS2476B_D0002.tif" />

CH.OCCKCH _) .. CH_;

CH -OCOCH.Br &lt;L.oco &lt;ch.) "Ch₎ ru no-ntru \ rv

<img file="IS2476B_D0003.tif" />

CH.OCO &lt;CK.&gt; "CH₎ Z

With reference to reaction catalyst 1 above, the production of compounds of formula (I) is shown below.

DÆM11

Progression of esters of propionyl L-carnitine bromide with 2-hydroxysetyl 1,3-dipalmitoylglycerol (ST 772)

a) Preparation of 1,3-dihydroxyprdpan-2-dn 1,3-dipalmititate (11

Dihydroxyacetone (7g, 0.078 mol) was dissolved in 300 mL of anhydrous silica at 0 ° C (external temperature) under anhydrous nitrogen flux.

In the solution thus obtained, palmitoyl chloride (44 g, 0.16 mol) and anhydrous pyridine (15 mL) were added dropwise.

The temperature of the blonde that was photographed, which had come to room temperature, was maintained by stirring for 24 hours.

The mixture was then extracted in the following order: with 300 mL of 0.5% hydrochloric acid solution, 300 mL of 5% sodium bicarbonate aqueous solution and finally with 300 mL of water.

The separated organic phase was dehydrated with anhydrous sodium sulfate, filtered with cellulosic acid and concentrated to dryness to give crude product (1).

The pure product (1) was obtained by crystallization from 500 mL of ethyl alcohol.

30.4 g of product (1) were obtained.

Yield: 73%

bm = 80-81 ° C

H¹NMR (CDCl3)₃): 0.9 (6H, t, CH₃CH, -k 1.3 (48H, m, (CH₂)_{n = 24}); 1.55 (4H, m, -OCOCH, CH₂-): 2.4 (4H, t, -OCQCH9-): 4.7 (4H, s) -OCH3

b) Preparation of 1,2,3-trihydroxyprop-1,3-dip palmitate (2)

Water (7.5 mL) was slowly added to product (1) (5 g, 9 mmol), dissolved in tetrahydrofuran (125 mL) and toluene (25 mL) upon stirring.

The temperature of the milk white suspension obtained was brought to 5 ° C (external temperature) and sodium borohydride (500 mg, 13 mmol) was added in small portions. The suspension was kept for 30 minutes at 5 ° C.

Glacial acetic acid was then added slowly until the agitation caused by the degradation of excess sodium borohydride, eventually yielding a solution.

Chloroform (100 mL) was added to the solution, forming a biphasic system.

The lower organic phase consisting of CHCl₃ was separated, extracted in the following order: with water (25 ml), sodium bicarbonate (25 ml of 10% aqueous solution) and water (25 ml).

The organic solution containing (2) was dehydrated with sodium sulfate, filtered and concentrated to dryness, yielding a waxy product.

Product (2) was obtained by crystallization of the waxy raw product in acetone.

4.8 g of product (2) were obtained.

Yield: 94%. bm = 71-72 ° C

H¹NMR (CDCl 3): 0.9 (6H, t, CH 2 CH 3 -): 1.3 (48H, m,₂)_{n = 24}); 1.55 (4H, m, -QCOCH9CH9-): 2.4 (4H, t, -COC H9-): 4.2 (5H, m, --CHCH2O-)

c) Preparation of 1,3-dipalmityl-2-bromoacetyl qlycerol (3)

Product (2) (2.5 g, 4.4 mmol) was dissolved in anhydrous chloroform (50 mL) with stirring and at 0 ° C (external temperature).

In the solution thus obtained was slowly added pyridinium (0.42 ml) and dropwise 3 ml of chloroform solution containing bromoacetyl chloride (0.43 ml, 5.2 mmol).

The reaction mixture was kept for 30 minutes at 0 ° C (outside temperature) and for 30 minutes at room temperature.

The reaction mixture was then treated in the following order with: 1% hydrochloric acid aqueous solution (approximately 50 mL), 5% sodium bicarbonate aqueous solution (approximately 50 mL) and water.

Product (3) was purified by acetone crystallization, after dehydration of the reaction mixture (with sodium sulfate) and strengthening to dryness.

2.5 g of product (3) were obtained.

Yield: 89%. bm = 46-47 ° C

H¹NMR (CDCl3): 0.9 (6H, t, CH_aCH3): 1.3 (48H, m, (CH₂)_{n = 24}); 1.55 (4H, m, -OCOCH 2 CH 2 -): 2.4 (4H, t, -OCOCH 3 -); 3.9 (2H, s, -OCH 4.2-4.4 (5H, m, - CHCH, O-): 5.25 (1H, m, CHCH2O-).

d) Preparation of esters of L-propionyl carnitine bromide with 2-hydroxyazetyl-1,3-dipalmitylalphserol (4)

L-propionyl carnitine inner salt (0.95 g, 4.4 mmol) previously dried in vacuo at 40 ° C was suspended in anhydrous dimethylformamide (approximately 20 mL).

Product (3) (3 g, 4.7 mmol) was added to the suspension in small portions. The suspension was warmed slowly to 38 ° C and maintained under these conditions until dissolution was obtained.

After 10 minutes, the solution was brought to 0 ° C for 30 minutes. A precipitate was obtained, which was filtered and washed with ethyl ether and dissolved in chloroform (100 mL). The opalescent solution obtained (30 mL) was filtered on celite and concentrated. In this latter solution was added hexane (100 mL), and the resulting precipitate of product (4) was filtered and dried under vacuum at 35 ° C.

3.19 g of the title compound were obtained.

Yield: 80%. bm = 127-128 ° C [α]²⁵d = -3.9 (C = 1% chloroform)

<td colspan="5">Preliminary analysis of C₄₇H88BrNO₁₀</td>

<td></td><td>C%</td><td>H%</td><td>n%</td><td>br%</td>

<td>calculated Recommended</td><td>62.23 62.73</td><td>9.78 10.15</td><td>1.54 0.79</td><td>8.81 8.77</td>

H¹ NMR (CDCl3): 0.9-0.95 (6H, t, CH₃CH₂CH₂-); 1.1-1.2 (3H, t, CH₂CH, CO): 1.2-1.4 (24H, m, CH₂n = 24); 1.5-1.6 (4H, m, -OCCH, CH_r): 2.3-2.4 (4H, t, -OCCH 2 CH 2 -); 2.4-2.45 (4H, dd,

-CHCHgQ-k 2.95 (2H, d, -CH₂COOCH, COO-1: 3.5 (9H, s, N (CH₃)₃); 4.2 (4H, m, -CH 2 COOCH 2): 4.35 (2H, m, -CH₂N-1; 4.65 (2H, dd, -OCH 3 CO-k 5.25 (1H, m, -OCH_PCHCH, O-): 5.75 (1H, m, -CHCH₂N-).

EXAMPLES 2-7

The following compounds were prepared in the same manner as in the above example:

- ester of L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 770);

- ester of acetyl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 771);

- ester of isobutyryl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 773);

- ester of isovaleryl L-carnitine bromide with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (ST 774);

ester of L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 810);

- ester of acetyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 809).

- Propionyl L-carnitine bromide ester with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 808),

One preferred embodiment of the invention described herein consists of the proliferation of liposomes with a cancer drug, and especially a fetal system acting as a ferret for camptothecin, for example those disclosed in WO 97/31003. In addition to a preferred embodiment, the invention described herein provides lipotransmitter distribution of the general formula (IV):

<img file="IS2476B_D0004.tif" />

where: r₇ is -C (R₁₁) = N-0_(n) Rio hfipur, pair of R₁₀ is a hydrogen or a C1 -C6 alkyl or C1-C5 alkenyl group, a direct compound of claim C₃-C₁₀ cycloalkyl group, direct chemical article (C₃-C₁₀) cycloalkyl-C1-C6) alkyl group, efia (C₆-C₁₄) aryl, a direct chemical branch (C₆-C₁₄) aryl- (C1-C5) alkyl group, a heterocyclic straight or branched heterocyclic C1-C8 alkyl group, said heterocyclic optionally containing at least one substituent selected from the atoms of nitrogen, optionally substituted by a (C1 -C6) alkyl group, and / or oxygen and / or sulfur dioxide; said alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, heterocyclic or heterocycloalkyl groups optionally substituted with five or more substituents selected from: halogen, hydroxy, C 1 -C 6 alkyl, C 1 -C 8 alkoxy, phenyl, cyano, nitro, -NR₁₂R₁₃, pair as r₁₂ and r₁₃, which can be verified, same or different, are hydrogen, straight-chain branched (C 1 -C 6)₅) alkyl, -COOH group, one of its pharmaceutically acceptable esters; efia -CONR₁₄R₁₅ The chip, pair of R₁₄ and r₁₅, which can be verified as such, are hydrogen, straight-chain branched (C 1 -C 6) alkyl; or alkaline

R₁₀ see C₆-C₁₀ aroyl residues optionally substituted by one or more additional substituents selected from: halogen, hydroxy, straight-chain branched C1-C8 alkyl, straight-chain branched C1-C8 alkoxy, phenyl, cyano, nitro, -NR₁₆R₁₇, pair as r₁₆ and r₁₇, can be verified as diverse, hydrogen, straight-chain branched C1-C5 alkyl;

R₁₀ is a polyaminoalkyl residue; or

R₁₀ is a glycosyl residue; n is the number 0 or 1;

Is hydrogen, straight-chain branched C 1 -C 6 alkyl, straight or branched C 1 -C 8 alkenyl, C₃-C₁₀Cycloalkyl, straight or branched (C₃-C₁₀) cycloalkyl - (CrC₅) alkyl, C₆-C₁₄ aryl, straight or branched (C₆-C₄) aryl - (CrC₅) alkyl;

R₈ and Rg, which may be the same or different, are hydrogen, hydroxyl, straight or branched C1 -C8 alkoxy;

Their noxides, single disappears, especially syn and anti disappears -C (Rn) = N-O (_n) Group of Rio, 10 of their possible enantiomers, diastereomeric and related mixtures, pharmaceutically acceptable salts thereof and their active metabolites.

Compounds of formula (IV) are described in European Patent Application No. 99830124.6, filed March 9, 1999.

In the case of compounds of formula (IV) wherein n is 1 and R₁₀ as defined above, with the exception of aroyl, these compounds may be prepared starting with camptothecin-7-aldehyde (formula IVa, Rn hydrogen) or camptothecin-7-keto (formula IVa, R_n other than hydrogen).

<img file="IS2476B_D0005.tif" />

(IVa) wherein R₇ is -O2,) = O group, and R_n is as defined by formula (IV), R₈ and r₉ are as defined by formula (IV). Formula (IVa) compound is reacted with formula (Va) compound R1₀O-NH₂, where R₁₀ is like force above, to give a compound of formula (I), wherein R₇ is -C (R₁i) = NOR₁₀ the group, r₁₀ is defined as in formula (IV) except for aroyl.

The reaction can be carried out by the skilled worker on the stage, the process consisting of an oxima-based synthesis. Preferably, the molar ratio of camptothecin-7-aldehyde should increase 7-keto versus hydroxylamine in the range of 1: 3 to 3: 1. Fluid-containing hydroxyl salts can also be used. The reaction is carried out in a vapor deposition base, for example an inorganic base such as potassium carbonate, promoting an organic base such as triethylamine enhancing diazabicyclononium, using the use of a polar solvent, preferably methane sulfonate, and performing the reaction at a temperature ranging from room temperature to the boiling point of the solvent, optionally in the presence of dehydration agents, for example sodium or magnesium sulfate, molecular sieves. If necessary, the reaction can also be carried out in the presence of catalysts, for example, Lewis acid.

Alternatively, the compounds mentioned above may be prepared from oxime camptothecin-7-aldehyde (obtained as described in Sawada et al., Chem. Pharm, Bull. 39, 2574 (1991)), or 7- ketone or from the corresponding 7-acyl-cocotothecin by reaction with R₁₀-X halide, wherein X is preferably iodine, in a polar solvent, for example tetrahydrofuran or alcohols, and in the presence of a base, for example, sodium hydride or potassium carbonate.

In the case of compounds of formula (IV) wherein n is 1 and R_1o is aroyl, as defined in formula (IV), these compounds may be prepared starting with camptothecin-7-oxime, wherein the prodrug thereof was described in the last paragraph, with R₁₀-COCI acyl chloride, in polar solvents, and in the presence of a base, preferably pyridine, directly in pyridine, as described by Cho et al., J. Org. Chem. 62, 2230 (1997).

The derivative of a compound of formula (IV) wherein n is 0 and R₁₀ as defined above, with the exception of aroyl, the compounds may be prepared starting with camptothecin-7-aldehyde (formula IVa, Rn hydrogen) or camptothecin-7-ketone (formula IVa, but hydrogen).

<img file="IS2476B_D0006.tif" />

where R₇ is -C (Rn) = O group, and Rn is as defined by formula (IV), R₈ and r_g are as defined by formula (IV). Formula (IVa) Compound is reacted with Formula (Vb) Compound R₁₀NH₂, pair as r₁₀ is as defined above, to give a compound of formula (IV), wherein R₇ is -C (Rn) = NR₁₀ the group, r₁₀ is defined as in formula (IV) except for aroyl. The reaction may be carried out by competent methods of well-known pharmacologists, the process consisting of the traditional synthesis of imine. Preferably, the molar ratio of camptothecin-7-aldehyde or 7-ketone versus hydroxylamine should be in the range of 1: 3 to 3: 1. Vifieigandi is in salt can also verify the use. The reaction is carried out in a concentrated base for the purpose of an inorganic base such as potassium carbonate, an organic base such as triethylamine or diazabicyclonon, using a liquid solvent, preferably the methane of the ethylene, and effecting a reaction temperature between the room temperature and the supernatant temperature of the solvent, optionally Vapor disturbances, such as sodium magnesium sulphate, molecular sieves. If necessary, can also be reactivated by induction of catalysts, for example, Lewis acid, as described, by Daemis, by Moretti and Torre, Synthesis, 1970,141; Efia by Kobayashi et al., Synlett, 1977, 115).

Combothosin-7-aldehyde and camptothecin-7-oxide are described in European Patent Application EP

0056692 and in the article referred to above by Sawada et al., Chem. Pharm. Bull. 39, 2574 (1991).

The hydroxides of the compounds of formula (IV) are prepared in accordance with known oxidation methods of heteroaromatic nitrogen, preferably oxidation with acetic or trifluoroacetic acid and hydrogen peroxide, or by reaction with organic peroxyacids (A. Albini and S. Pietra, Heterocyclic N-Oxides, CRC , 1991).

In terms of changing the importance of Rio present in various reagents of Formula V, these reagents are available on the market, or may be prepared in accordance with methods known from light materials which the expert in the field can take as a addition to his or her knowledge of the subject matter.

Pharmaceutically acceptable salts are obtained by conventional methods described in the literature, which do not require further descriptions.

EXAMPLE 8 15

7-benzyloxyiminomethylcometothecin (CPT172)

500 mg (1.33 mmol) of formyl-camptothecin are dissolved in 100 ml of ethanol. 15 ml of pyridine and 638 mg (4 mmol) of O-benzylhydroxylamine hydrochloride are added. The solution is refluxed for 5 hours. The solvent is evaporated in vacuo and the residue obtained is purified by flash chromatography on silica gel using 4: 6 bps hexane / ethyl acetate as a phase phase.

Yield: 65%

bm: 200-205 ° C degrees.

The resulting product consists of approximately 8: 2 parts of the syn and anti neighborhoods (disappearing A: rt 0.32, disappearing B, rt 0.19, Merck 60 F254 silica gel; Phosphate: hexane / ethyl acetate 3: 7).

HPLC: The analyzes were performed on a four-day basis (HP 1050) with Rheodyne Insertion Lock (20 μl loop) and diddufylkis except (HP 1050) run by the HPLC-ChemStation program. Rdfasdfnun was performed in the range of 200 to 600 nm and lithritin was recorded at 360 and 400 nm.

C18 column with a phase (Rainin C18; 25x0.4 cm, Varian) was used together with an RP18 compound. The analysis was carried out at a linear elution stage starting from acetone nitrate 30:70 with 100% acetonitrile over 20 minutes, with a flow rate of 1 ml / min. The racing times were: 12.51 min for turbine B and 14.48 min for turbine A.

¹1 H-NMR (300 MHz; DMSO-d₆): δ: 0.88 (t, H₃-18A + H₃-18B), 1.878m, (H₂-19A + H₂-19B), 5.18 (s,

H₂-5B), 5.21 (8s, H₂- Ph B), 5.30 (H₂-Ph A), 5.40 (s, H₂-5A), 5.45 (s, H₂-17 + H₂-17B), 6.53 (s,

-OH A + -OH B), 7.3-7.6 (m, Ar A + Ar B + H-14A + H-14B), 7.75 (m, H-11A + H-11B), 7.85 -7.95 (m, H-10A + H-10B), 7.98 (dd, H-12B), 8.18-8.27 (m, H-12A + H9-B), 8.45 s, CH = NB), 8.59 (dd, H-9A), 9.38 (s, CH = NA).

Mass m / z 481 (M⁺ 100) 374 (30) 330 (70) 300 (30) 273 (20) 243 (20) 91 (34).

DÆIUII9

7-butoxyiminomethyl combocothecin (CPT 184)

400 mg (1.06 mmol) of 7-formylkamotothecin are dissolved in 80 ml of ethanol. 12 ml of pyridine and 400 mg (3.18 mmol) of Ot-butylhydroxylamine hydrochloride are charged. The solution is refluxed for 4 hours. The solvent is evaporated at ambient temperature and the resulting residue is purified by flash chromatography on silica gel using a 4: 6 mixture of hexane / ethyl acetate as a mobile phase.

322 mg (0.72 mmol) were obtained from a yellow solid.

Yield: 68% bm: 250 ° C degrees.

The product obtained consists of approximately 8: 2 mixture of syn and anti-two (disappearing A: rt 0.31, disappearing B, rt 0.24, Merck 60 F254 silica gel, travel phase: hexane / ethyl acetate 3: 7) .

HPLC: The analyzes were performed on a four-day basis (HP 1050) with a Rheodyne Insertion Lock (20 μl loop) and a diode fillet (HP 1050) run by the HPLC-ChemStation program. Batch collection was carried out in the range of 200 to 600 nm and chromatography recorded at 360 and 400 nm.

C18 secondary phase column (Rainin C18; 25x0.4 cm, Varian) was used in combination with an RP18 compound. The analysis was carried out at a linear elution stage starting from acetonitrile 30:70 to 100% acetonitrile for 20 minutes at a flow rate of 1 ml / min. The racing times were: 12.92 min for turbine B and 14.61 min for turbine.

¹1 H-NMR (300 MHz; DMSO-d₆): δ: 0.88 (t, H₃-18A + H₃-18B), 1.30 (s, t-butyl), 1.47 (s, t-butyl), 1.87 (m, H₂-19A + H₂-19B), 5.18 (s, H₂-5B), 5.37 (H₂-5A), 5.42 (s, H₂-17 + H₂-17B), 6.54 (s, -OH A + -OH B), 7.35 (s H-14A), 7.36 (s, H-14B), 7.69-7.83 (m, H-11A + H -11B), 7.85-7.98 (m, H-10A + H-10B), 8.07 (dd, H-9B), 8.16-8.27 (m, H-9A + H- 12B), 8.40 (s, CH B), 8.62 (dd, H-9A), 9.31 (s, CH A).

Mass m / z 448 (M⁺ 28) 391 (40) 374 (100) 362 (40) 330 (34) 57 (17).

PRODUCTION ON FITUKORNUM

The compounds according to the invention can be used to prepare multifaceted nitrous oxide (MLV) and single-phase nitrous oxide (SUV), both in the form of dry powder and as aqueous suspension.

The compounds according to the invention, prepared as described in Examples 1-7, are used to prepare liposomes according to the following method. The dissolved amount of the compound is dissolved in chloroform, the solution is concentrated under vacuum to dryness in a rotary evaporator until a lipid film is obtained. The lipid film is dried at an accelerator until the last residue of solvent has been removed and then dissolved in tert-butyl alcohol or with water. The solution thus obtained is a freeze-drying, which gives a soft dry powder.

The powder is filled with a suitable amount of an aqueous solution, which gives a liposome of the compound used, which is then complexed with the polynucleotide or with the desired drug.

Another method of manufacturing liposomes consists of collecting a lipid film comprising a compound of the invention in a solvent, or a suitable inert excipient such as sorbitol, mannitol or other pharmaceutically acceptable carbohydrates. The mixture is dried in vacuo, which gives a solid which can easily and accurately verify water for use.

Compositions in the form of dry dust show the advantages of power being stable over a long period of time, and are easy to use.

Further, the compounds according to the invention can be used for the purpose of preparing a lipid comprising a DNA comprising the desired drug, in the form of dry powder, according to the following processes. A compound of the invention is dissolved in tert-butyl alcohol undergoes water; The solution thus obtained is the DNA molecule that promotes a proliferative drug and the compound is lyophilized, thus providing complexes that can be defined as anti-corneal DNA enhancing anti-corneal drugs, in the form of a soft dry powder.

Pancreatic cancer can verify the ability to produce drug preparations that can be verified. Through aerosols, promote when strengthening the benefits, re-solubilised with water, promote a high level of buffer solution, they can be verified by oral lymph node.

Lipids that contain DNA-enhancing drugs, can also be obtained by the use of a proliferation of lipid aggregation lipids, such as sorbitol, mannitol, boosting other carbohydrates, using power, as described above.

Test for the formation of lipids

The formation of the liposomes was carried out by means of photoconductivity, using the force of water-soluble pigments, according to the following processes. Aqueous solution of water-soluble Arsenazo III was obtained (m.p. = 776.37: 2.3 mg / mL).

This solution was used in a stack of water for the production of lipid layers that came from the survival mentioned above.

A dose of the suspension containing the fatty alcohols containing the intestinal pigment was diluted

10O-fold with water.

Two mL of liposomal suspension were power consumption to obtain first reading light intensity of 660 nm; the reading was obtained according to a comparable sample, which was defined as a zero sample. 200 μl of CaCl₂solution (15 mg / mL; 100 mM) was charged to the first sample, and the light density measured against the zero sample, which was added 200 μl of water. Gleypnigildifl was the color of the readings. 2. Vifl continued with the force of baeta in the sample 100 μl of Triton X-100 solution (5% v / v, 0.26% final concentration) and in the zero sample 200 μl of water; The light density flux of 660 nm gave the light density defined as reading 3. For the calculation of the percentage of integral coloring, the following formula was used:

. ,. , Reading 3 - Reading 2x 100% of color that is internal reading = Reading 3

The percentage of ink-colored pigments gives a measure of the formation of the liposomes, and is approximately 40% on average: the size of the liposomes was performed using a positive-release laser distribution.

EXAMPLE OF PRODUCTION ON FITUKORNUM

EXAMPLE 8

Production of taxol-ST 772 SUV liposomes (1:70) mg, 0.0234 mmol of taxol and 1485 mg, 1.638 mmol of ST 772 were dissolved chloroform.

The solution was a reinforced pair of lipid film obtained on the surface of the glass bottle.

After removal of the final residues of chloroform with a ventricular pump, 20 mL of tert-butyl alcohol was added to the lipid film. In order to obtain a clear solution, it had to be heated to 60 ° C. The solution was simultaneously frozen at -70 ° C with liquid nitrogen and lyophilized for 24 hours.

To close the SUV liposomal suspension, the freeze-dried product was watered with PBS solution (20 mL), boiled for 20 minutes at 0 ° C.

Filtration was then carried out on a 400 nm filter to exclude residues of titanium released to the silencer.

Test of the physical status of the production

The physical stability of the production was tested by using a quantum measurement by recording on TDC (Hourly Curve) at 800 nm at 20 ° C for 6 hours.

Fixed moresty, indicating production stability, was recorded, without any challenge.

Gene Delivery

Production of liposome DNA fleece

Lipids and plasmid DNA were adequately diluted separately in PBS. The DNA was added to the lipid cells, and the lipid-DNA complex was left for about 30 minutes at 4 ° C to facilitate the formation of a continuous liposome DNA interaction.

In experimental experiments, 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) was used as a reference for cationic lipid; 2.5 μg of plasmid DNA were used per 2x10⁵ HeLa cells, and the concentration of lipids was 9 μm.

In experimental experiments, both DOTAP and [2,3- (dioleoyl) propyl] trimethylammonium (DOTMA) were used as criteria for cationic lipids.

The mole ratios defined in the results show in nmol concentrations of the relevant cationic lipids per mg of DNA.

In the induction experiments in the living body, 25 μg of plasmid DNA was used in each animal. Plasmid pCMVIuc data in these experiments contained the cDNA luciferase gene for the transcriptional control of the control region of the cytomegalovirus.

Quantitative determination of the activity of lupiferases

The protein activity of luciferase in cells and tissues was determined by using Boehringer

Mannheim set (item number 1666 893).

The cells were purged 3 times in PBS and then removed from the plate by dissolving buffer (100 mM potassium phosphate pH 7.8, 1 mM dipotreitol - DTT) and passed through three continuous circles of freezing and piling. After centrifugation in 1.5 ml of eppendorf glands, the flotifl of lubrication measurements were no more than 5 hours after the extraction of the proteins.

Emission irradiation measurements were made using a light sensor at 562 nm. After initial freezing in liquid N₂ and paired with fineness, the tissues were redissolved in a decomposition buffer and incubated for 10-15 minutes on ice.

The samples were then separated into 2 ml eppendorf gels and flotifl was tested for luciferase activity.

Spot analysis

Cell DNA was extracted according to the basic demarcation method described by Sanbrook, Fritsch and Maniatis in Molecular Cloning, 1989.

The amount of DNA extracted from the cells was preceded by nylon filters (Boehringer) using the Biorad stain test device. The filters were then premixed for 4 hours at a 65 ° C vial solution containing 0.5 M sodium pyrophosphate (NaPi), 1 mM EDTA, 7% SDS. The clue that was marked with³²P (alaa) was prepared by force using plasmid pCMVIuc DNA as a format and randomized Amersham kit. The filter was mixed in the same premix buffer for 12 hours at 42 ° C using power using 1x10⁶ CPM / ml. The filter was placed for 3 10 minutes washing

65 ° C in a buffer containing 40 mM NaPi, 1% SDS. The radiation analysis was performed with the help of phosphoricides using phosphorous layers activated by beta-radiation which are read and quantified by using light-emitting system in conjunction with the image analysis program. The density measurement performed on the spot test was performed using an IP-LabGel image analysis program.

Plasmid DNA induction test

A number of plasmid DNA induction tests were performed both in the experimental and in the living body.

Both DOTAP and DOTMA were used as criterion for cationic lipids, the induction rate, which has been adequately identified by Abkenet et al., Proc. Natl. Acad. Sd. USA 1993, 90, 6518. In the experiments in the living body, several different mole ratios of cationic lipids to plasmid DNA were identified for the purpose of determining the activity of cationic lipids and the most effective concentration for gene transfer. The infusion density of the different lipids was determined both in vitro and in living cells using the ferrous luciferase agent present in the pCMVIuc plasmid, as its efficacy in respect of relative luminous units (previously described) facilitated the quantification.

Alternatively, gene transduction efficiency on the number of cationic lipids was measured by concentration measurement (phosphoric) on samples of DNA extracted from genetic cells pre-adsorbed on nitrocellulose sebum (spot test) and mixed with ³²P-labeled plasmid DNA tagged, as previously described.

Resuscitation test in a living body

Genetic resonance resuscitation resemblance to ST 983 liposomes: DNA mole ratios in this experiment is the understanding of liver, lung and cardiac dysfunction on the ST 983 liposome: plasmid DNA mole ratios were evaluated. The following nmoles of the lipids on pg of DNA were tested: 12: 1.24: 1.36: 1 and 48: 1.

Groups of 6 Balb / c mice weighing approximately 20 g were treated with the above-mentioned levels of liposome DNA fragments in a 200 μL volume of PBS intravenously and sacrificed 24 hours after administration of the complex.

Luciferase activity extracted from lung, cardiovascular and hepatic tissues revealed the overwhelming distribution of luciferase to lung at all molar ratios detected. In fact, about 99% of total luciferase extracted from these three tissues was located in the lungs. Liposomes: DNA molar ratio 12: 1 proved the best.

Plasmid DNA introduction into HeLa cells with ST 772 liposomes

Figures 1 and 2 show the induction activity of ST 772 liposome on plasmid DNA in HeLa cells. For this purpose, a concentration analysis was performed on a DNA insert extracted from the cells introduced by ST 272 and with DOTAP as a reference cationic lipid. The results of the spot test analysis show the amount of plasmid DNA of the same magnitude obtained with DOTAP.