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

The present invention relates to a class of novel L-carnitine and acyl L-carnitine esters, their use as cationic lipids suitable for the beneficial intracellular delivery of pharmacologically active compounds that facilitate transmembrane transport or enhance their interaction with specific sites of the cell membrane (receptors).

The term "intracellular delivery" as used herein refers to the transfection of a cell with naturally derived or modified polynucleotides or plasmids (gene delivery), or to introduce drugs or immunogenic peptides into the cell.

Generally, many pharmacologically active substances, such as e.g. polypeptides, proteins or drugs, must penetrate into the cell in order to have an effect on cellular function, at the cellular or molecular level. For these molecules, the cell membrane creates a selectively impermeable barrier. The cell membrane is actually protective, preventing the entry of potentially toxic substances, but also the passage of compounds with therapeutic activity. The complex composition of the cell membrane includes phospholipids, glycolipids and proteins; their function is influenced by cytoplasmic components such as Ca⁺⁺ and other ions, ATP, microfilaments, microtubules, enzymes and proteins that bind Ca⁺⁺. The interaction between the structural and cytoplasmic components of cells and the response to external signals are mechanisms responsible for the selectivity among different cell types. The membrane barrier effect can be overcome by complexing the substances with lipid preparations that duplicate the structure of naturally occurring membrane lipids. These lipids are able to fuse with the membrane and release substances connected with them into the cell. The lipid complexes are not only capable of facilitating intracellular transport through membrane fusion, but can also reduce the repulsive force between the membrane and the molecule to enter the cell. Amphipathic lipids such as membrane phospholipids form lipid vesicles or liposomes in aqueous systems.

Liposomes are vesicles in which a given volume of water is completely enclosed by one or more membranes composed of lipid molecules, usually phospholipids. Phospholipids, which consist of a hydrophilic head and a pair of carbon chains (hydrophobic tails), are the main components of biological membranes. In an aqueous solution, the hydrophobic tails join together to get rid of water molecules, while the hydrophilic heads interact with the medium, spontaneously creating populations of bubbles of varying diameters. Lipids are generally bipolar, neutral, or anionic. These vesicles can be used as carriers for drugs, small molecules, proteins, nucleotides, and plasmids.

Over the past years, cationic liposomes, a class of positively charged vesicles made from synthetic lipids, have found wide application in the transfer of genetic material into the cell. The negative DNA charge can interact with the positive cationic charge of the lipids to form a stable DNA-liposome complex. The simplicity and universality of this technology made liposomes important carriers in gene transport in human gene therapy. Today, most vectors used in gene therapy and approved by the NIH Recombinant Advisory Committee include viral and synthetic systems.

Viral infection employs a series of mechanisms to attack a specific cell and transfer the DNA to the nucleus. The rationale for the use of viral vectors in gene therapy is based on the possibility of replacing viral genes with genes that code for therapeutic functions, without eliminating the ability of the viral particle to infect the cell. The limitations of viral therapy relate to viral components that may be immunogenic, cytopathogenic, and recombinogenic.

Great hopes are placed on the use of cationic lipids in gene therapy. These vectors have great potential as compared to vectors of biological origin, because they are much safer, less toxic and are also capable of switching on genes with a large dimension. However, compared to biological-type vectors, these vectors have a low efficiency of intracellular gene transcription. However, it should be remembered that the use of such transfection systems is in the initial phase of research. Cationic lipids play a very important role in DNA-lipid complex formation, cell-complex interaction, membrane fusion, intracellular release of DNA, and in transcription.

PL 204 418 B1

There are important examples of in vivo use of cationic liposomes. The first clinical trial of gene therapy for the treatment of melanoma was performed by introducing an expression vector containing a human liposome linked to the HLA-B7 gene. Another important application relates to the treatment of pulmonary cystic fibrosis by administration via the pulmonary route or via intranasal spray of the liposome associated with the expression vector SV-4OC-FTR. Other clinical trials involving the use of liposomes in cancer gene therapy are ongoing.

In general, four components are identified in the structure of cationic lipids: a positively charged cationic head, a spacer group, an anchor lipid, and a binding link.

The cationic head is responsible for the interactions between cationic liposomes and DNA, between the DNA-liposome complex and the cell membrane, and other cell components. It consists of mono- or polycationic groups which (depending on the number of charges) can be substituted in various ways.

The spacer group is the part of the molecule that separates the cationic head from the hydrophobic tail and is involved in ensuring optimal contact between the cationic head and the negative charges of DNA phosphates.

The anchor lipid is the nonpolar hydrocarbon part of the molecule and determines the physical properties of the lipid bilayer such as its stiffness and the amount of exchange with membrane lipids.

By "connecting bond" is meant the bond between the hydrocarbon chains and the rest of the molecule. This bond determines the chemical stability and biodegradability of the cationic lipids.

In recent years, the use of liposomes has consistently increased in the cosmetics sector. The success of liposomes in this field is due to the fact that these compounds are very well tolerated by the skin. They are used both as carriers for active ingredients and as compounds enhancing their absorption.

The scientific and patent literature contains many references to the preparation and use of liposomes; however, there is very little literature describing the use of carnitine derivatives useful for gene transport, while no literature is available on drug transport dealing with techniques for producing compounds distantly resembling those with which this invention is concerned.

EP patent application No. 0 279 887 describes the use of a carnitine derivative, i.e. phosphatidyl carnitine, optionally in mixture with other phospholipids and lipids (cholesterol, phosphatidylcholine, phosphatidylserine), for the preparation of liposomes.

In the given example of liposome production, phosphatidyl carnitine liposomes are obtained which include propranolol, a drug known to have antihypertensive, anti-anginal and anti-arrhythmic activity. The carnitine derivative is used herein based on the established pathway of carnitine towards the heart muscle. Rather, this tropism avoids metabolizing the liposomes in the liver before they achieve their desired end.

The presence of phosphatidyl carnitine also allows the oral use of liposomes as they are resistant to intestinal lipases.

In J. Med. Chem., Jun. 18, 1998; 41 (13): 2207-15, numerous L-carnitine esters useful for gene delivery have been described, but have not been described or proposed as useful agents for drug delivery.

WO 96/399193 describes novel target drug agents that target the mitochondria via the translocation of the carnitine-acylcarnitine system, but do not suggest that they are useful for liposome production.

EP 559 625 B1 describes numerous L-carnitine and acyl L-carnitine esters endowed with selective gastrointestinal muscle relaxant activity.

In recent years, molecular biologists have identified numerous defects at the chromosomal level that cause hereditary diseases in humans.

An important branch of modern medicine concerns the treatment of genetic diseases with the use of gene therapy methods.

As already mentioned, cationic liposomes are widely used for the intracellular delivery of pharmacologically active compounds, facilitating transmembrane transport or enhancing their interaction with specific sites of the cell membrane (receptors).

These vectors have great potential compared to vectors of biological origin because they are safer, less toxic, and are also capable of switching on genes with large

In dimensions. However, compared to biological-type vectors, they have a low efficiency of intracellular gene transcription.

In addition, gene transport mediated by common cationic lipids requires that plasmid DNA and cationic lipids be kept separate and their mixing is performed immediately prior to gene transfer.

Attempts to stabilize the polynucleotide complexes have so far been unsuccessful; in fact, the complexes only remain stable for a short period.

There is therefore a strongly recognized need in the field of gene therapy or gene transfer and drug delivery to obtain stable, reproducible, site-specific systems that are also active after an appropriate period of time.

It has now been found that a class of cationic lipids potently active in enhancing the intracellular delivery of pharmacologically active compounds includes novel L-carnitine and acyl L-carnitine esters.

These new compounds are stable and highly selective because they are site specific in reaching the target organ.

These distinctive features make them particularly useful in transporting active compounds directly to a site where they can exert their pharmacological activity.

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

<img file="PL204418B1_D0001.tif" />

wherein:

n is an integer from 1 to 3;

R is hydrogen or a straight-chain or branched alkanoyl with 2-6 carbon atoms; R1 and R2, which may be the same or different, represent a saturated or unsaturated straight acyl chain with 3-20 carbon atoms; and X^- is the anion of a pharmacologically acceptable acid.

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

Examples of R1 and R2 are hexanoyl, undecanoyl, myristoyl, palmitoyl or oleoyl.

Preferred examples of compounds according to the invention are:

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

- acetyl L-carnitine bromide ester with 2-hydroxyacetyl-13-dipalmitoyl glycerol (ST 771);

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

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

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

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

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

- ester of propionyl L-carnitine bromide with 1,3-dihexanoyl-2-hydroxyacetylglycerol (ST 808). By an anion of a pharmacologically acceptable acid we mean any anion of an acid that does not increase undesirable toxic effects or other side effects.

These acids are well known to pharmacologists and those skilled in pharmaceutical technology. Examples of anions, but not limited to those mentioned, are: chloride; bromide;

iodide; aspartate; hydro aspartate; citrate; hydrogen citrate; tartrate; bitartrate; phosphate;

hydrogen phosphate; fumarate; hydrogen fumarate; glycerophosphate; glucosophosphate; lactate; maleate; hydrogen maleate; mucan; orothonate; oxalate; hydrogen oxalate; sulfate; bisulfate; trichloroacetate; trifluoroacetate; methanesulfonate; palmitate and hydropalmitate.

PL 204 418 B1

The subject compounds are used for the production of liposomes. Therefore, the invention also relates to liposomes containing such compounds. The liposome of the invention also contains accessory lipids that are selected from the group consisting of cholesterol, 1-palmitoyl-2-oleoylphosphatidylcholine, or dioleylphosphatidylcholine.

The invention also includes the use of the subject liposome for the preparation of a composition useful for the transport of pharmacologically active compounds.

The pharmacologically active compound contained in the liposome is a naturally occurring or modified plasmid or polynucleotide.

A plasmid or polynucleotide is useful in gene therapy, it carries coded information for a peptide or protein useful as a vaccine.

For use in the liposome of the invention, an active compound is used which is a drug selected from the group consisting of an anti-cancer, anti-angiogenic, anti-viral, anti-bacterial, anti-fungal, anti-protozoan, cardiovascular active compound, or immunogenic peptides.

Preferably, the drug is an anti-cancer or anti-angiogenic agent.

The anticancer agent is preferably selected from the group consisting of taxol or a camptothecin derivative.

Said camptothecin derivative is selected from the group consisting of:

- 7-carbonitrile camptothecin;

- 7-benzyloxyiminomethylcamptothecin and

- 7-butoxyiminomethylcamptothecin.

The invention also includes the use of the subject liposome for the preparation of a composition useful for the transport of pharmacologically active compounds.

Compounds of formula (I), in the form of liposomes, are agents useful for either delivering naturally occurring or modified plasmids or nucleotides useful in gene therapy, or are those which encode a peptide or protein useful as a vaccine and for general drug delivery such as e.g. . anticancer drugs, antiviral agents, antibacterial, antifungal, antiprotozoal agents, drugs useful in the treatment of diseases of the cardiovascular system or immunogenic peptides and other drugs useful in therapy.

Liposomes containing a compound of formula (I) are prepared by conventional techniques well known to those having basic skills in the art; see, e.g., Alien ™ Drugs 56, 747-56 (1998). Liposomes in accordance with the present invention can also be prepared using other ingredients well known in the art of liposome technology. In one embodiment of the present invention, the liposomes may contain auxiliary lipids, which term is well understood in the art.

Examples of accessory lipids are cholesterol, 1-palmitoyl-2-oleoylphosphatidylcholine, or dioleylphosphatidylcholine.

The liposomes according to the invention exist as suitable compositions which are also the subject of the present application. The compositions are pharmaceutical in nature and optionally contain acceptable carriers and / or excipients.

The compounds of formula (I), in the form of liposomes, may also be useful in the preparation of cosmetic compositions, both containing the liposome per se as the cosmetically active substance and in the delivery of substances with cosmetic activity, such as e.g. moisturizers, nutrients, for cleansing the face, anti-wrinkle, anti-cellulite and anti-stretch marks.

Liposome compositions containing compounds of formula (I) may be administered orally or parenterally, intravenously, intramuscularly, subcutaneously, transdermally or by nasal or oral spray.

The procedure for the preparation of compounds of formula (I) according to the invention is shown in the following reaction diagram, which is intended to be a diagram that applies to the entire general formula (I). One skilled in the art can readily obtain all groups designated R, R1 and R2, and this is because all the necessary reagents are commercially available or disclosed in the literature and the reaction conditions are generally appropriate throughout the scope of the present invention and any modification, if necessary, is normally is based on generally available knowledge.

PL 204 418 B1

<img file="PL204418B1_D0002.tif" />

With reference to the above reaction diagram 1, the preparation of the compounds of formula (I) according to the invention is illustrated below.

Example 1. Preparation of propionyl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 772)

a) Preparation of 1,3-dihydroxypropan-2-one 1,3-dipalmitate (1)

Dihydroxyacetone (7 g; 0.078 mol) was dissolved in 300 ml of anhydrous chloroform at 0 ° C (external temperature) under an anhydrous nitrogen atmosphere.

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

The resulting mixture, which was raised to ambient temperature, was stirred for 24 hours.

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

The separated organic phase was dried over anhydrous sodium sulfate, filtered on a cellulose filter and concentrated to dryness to obtain the crude product (1).

Pure product (1) was obtained by crystallization from 500 ml of ethyl alcohol.

30.4 g of product (1) are obtained. Yield 73%. Melting point = 80-81 ° C

H.¹NMR (CDCl3): 0.9 (6H, t, CH3CH2-); 1.3 (48H, m, (CH2) n = 24); 1.55 (4H, m, -OCOCH2CH2-);

2.4 (4H, t, -OCOCH2-); 4.7 (4H, s, -OCCH2-).

b) Preparation of 1,2,3-trihydroxypropane 1,3-dipalmitate (2)

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

The temperature of the milky white suspension obtained was raised to 5 ° C (external temperature) and sodium borohydride (500 mg; 13 mmol) was added in small portions. The suspension was stirred for 30 minutes at 5 ° C.

Glacial acetic acid was then added slowly until the turbulence of the fluid due to decomposition of the excess sodium borohydride ceased, finally obtaining a solution.

Chloroform (100 ml) was added to the solution, which was accompanied by the formation of a two-phase system.

PL 204 418 B1

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

The organic solution containing compound (2) was dried with sodium sulfate, filtered and concentrated to dryness to give a wax-like product.

The product (2) was obtained by crystallization of the crude product from wax-like acetone.

4.8 g of product (2) are obtained. Yield 94%. Melting point = 71-72 ° C

H.¹NMR (CDCl3): 0.9 (6H, t, CH3CH2-); 1.3 (48H, m, (CH2) n = 24); 1.55 (4H, m, -OCOCH2CH2-);

2.4 (4H, t, -OCOCH2-); 4.2 (5H, m, -CHCH2O-).

c) Preparation of 1,3-dipalmitoyl-2-bromoacetylglycerol (3)

The product (2) (2.5 g; 4.4 mmol) was dissolved in dry chloroform (50 ml) with stirring at 0 ° C (external temperature).

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

The reaction mixture was held for 30 minutes at 0 ° C (external temperature) and for 30 minutes at ambient temperature.

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

After the reaction mixture was dried (sodium sulfate) and concentrated to dryness, the product (3) was purified by crystallization from acetone.

2.5 g of product (3) are obtained. Yield 89%. Melting point = 46-47 ° C

H.¹NMR (CDCl3): 0.9 (6H, t, CH3CH2-); 1.3 (48H, m, (CH2) n = 24); 1.55 (4H, m, -OCOCH2CH2-);

2.4 (4H, t, -OCOCH2-); 3.9 (2H, s, -OCH2COO-); 4.2-4.4 (5H, m, -CHCH2O-); 5.25 (1H, m, CHCH2O-).

d) Preparation of L-propionyl carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoylglycerol (4)

The inner salt of L-propionyl carnitine (0.95 g, 4.4 mmol), previously dried under high vacuum at 40 ° C, was suspended in anhydrous dimethylformamide (approx. 20 ml).

Product (3) (3 g, 4.7 mmol) was added to the suspension in small portions. The slurry was slowly heated to 38 ° C and kept under these conditions until a solution was obtained.

After 10 minutes, the solution was cooled to 0 ° C over 30 minutes. The resulting precipitate was filtered, washed with diethyl ether and dissolved in chloroform (100 ml). The resulting opalescent solution (30 ml) was filtered through celite and concentrated. To the latter solution was added hexane (100 ml) and the resulting precipitate of the product (4) was filtered and dried under high vacuum at 35 ° C. 3.19 g of the title compound were obtained.

Yield: 80%. Melting point = 127-128 ° C.

[and]²⁵D = 3.9 (C = 1% chloroform).

Elemental analysis: C47H88BrNO10

<td></td><td>C%</td><td>H%</td><td>N%</td><td>Br%</td>

<td>Calculated</td><td> 62,23</td><td> 9,78</td><td> 1,54</td><td> 8,81</td>

<td>Found</td><td> 62,73</td><td> 10,15</td><td> 0,79</td><td> 8,77</td>

H.¹NMR (CDCl3): 0.9-0.95 (6H, t, CH3CH2CH2-); 1.1-1.2 (3H, t, CH 3 CH 2 CO); 1.2-1.4 (24H, m, (CH2) n = 24); 1.5-1.6 (4H, m, -OCCH 2 CH 2 -); 2.3-2.4 (4H, t, -OCCH 2 CH 2 -); 2.4-2.45 (4H, dd, -CHCH2O-); 2.95 (2H, d, -CH2COOCH2COO-); 3.5 (9H, s, N (CH3) 3); 4.2 (4H, m, CH2OCOCH2); 4.35 (2H, m, -CH2N-); 4.65 (2H, d, d, -OCH2CO-); 5.25 (1H, m, -OCH2CHCH2O-); 5.75 (1H, m, -CHCH2N-).

Examples 2-7. The following compounds were prepared according to the previous example:

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

- acetyl L-carnitine bromide ester with 2-hydroxyacetyl-1,3-dipalmitoyl glycerol (ST 111);

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

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

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

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

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

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

One advantageous embodiment of the invention described herein is the preparation of liposomes with anti-cancer drugs, and particularly liposomes which act as carriers for the camptote8.

Tin compounds, for example those disclosed in WO 97/31003. In a more preferred embodiment, the described invention provides liposomes for the transfer of camptothecins of general formula (IV):

<img file="PL204418B1_D0003.tif" />

wherein:

R7 is the group -C (R11) = NO (n) R10, wherein R10 is hydrogen or a C1-C5 alkyl or C1-C5 alkenyl group, straight or branched chain, or a C3-C10 cycloalkyl group, or a straight chain or branched group ( C3-C10) cycloalkyl (C1-C5) alkyl, or C6-C14 aryl, or straight or branched chain (C5-C14) aryl- (C1-C5) alkyl, or heterocycle or straight or branched heterocycle (C1-C5 ) alkyl, the above-mentioned heterocyclic group containing at least one heteroatom selected from nitrogen atoms, optionally substituted with (C1-C5) alkyl, and / or oxygen and / or sulfur; the above-mentioned groups as alkyl, alkenyl, cycloalkyl, aryl, arylalkyl, heterocyclic or heterocycloalkyl substituted optionally by other groups selected from: halogen, hydroxy, C1-C5 alkyl, C1-C5 alkoxy, phenyl, cyano, nitro, -NR12R13 wherein R12 and R13 may be the same or different and represent a hydrogen atom, a straight or branched (C1-C5) alkyl, a -COOH group, or one of its pharmaceutically acceptable esters; or the group - CONR14R15, in which R14 and R15 may be the same or different and are hydrogen, straight or branched (C1-C5) alkyl; or

R10 is an aroyl group C6-C10 optionally substituted with one or more groups selected from: halogen, hydroxy, C1-C5 straight or branched alkyl, C1-C5 straight or branched alkoxy, phenyl, cyano, nitro, -NR16R17, wherein R16 and R17 may be the same or different, and represent hydrogen, C1-C5 straight or branched alkyl;

R10 is polyaminoalkyl; or

R10 is a glycosyl group; n is 0 or 1;

R11 is hydrogen, C1-C5 straight or branched alkyl, C1-C5 straight or branched alkenyl, C3-C10 cycloalkyl, straight or branched (C3-C10) cycloalkyl (C1-C5) alkyl, C6-C14 aryl, straight or branched chain (C6-C14) aryl (C1-C5) alkyl;

R8 and R9, which may be the same or different, are hydrogen, hydroxy, C1-C5 straight or branched alkoxy;

their N1-oxides, single isomers, especially the syn and anti isomers of the group -C (R11) = NO (n) R10; their possible enantiomers, diastereoisomers and related mixtures; their pharmaceutically acceptable salts and their active metabolites.

Compounds of formula (IV) are described in European Patent Application No. 99,830,124.6, filed on March 9, 1999.

Regarding the compounds of formula (IV) in which n is 1 and R10 is as defined above, except aroyl, they can be prepared starting from 7-aldehyde camptothecin (formula IVa, R11 hydrogen) or 7-ketocamptothecin (formula IVa, R11 is other than hydrogen).

PL 204 418 B1

<img file="PL204418B1_D0004.tif" />

wherein R7 is the group -C (R11) = O and R11 is as defined in formula (IV), R8 and R9 are as defined in formula (IV). The compound of formula (IVa) is reacted with a compound of formula (Va) R10O-NH2 in which R10 is as described above to give compounds of formula (I) in which R7 is a group -C (R11) = NO- R10, R10 is as defined in formula (IV), except aroyl.

The reaction can be carried out by conventional methods known to those skilled in the art, in a process normally leading to the formation of oximes. The preferred molar ratio of the 7-aldehyde or 7-ketocamptothecin to hydroxylamine should be in the range from 1: 3 to 3: 1. The corresponding hydroxylamine salt can also be used. The reaction is performed in the presence of a base, e.g. an inorganic base such as potassium carbonate, or an organic base such as triethylamine or diazabicyclononane, using polar solvents, preferably methanol or ethanol, and reacting at temperatures ranging from ambient temperature to the boiling point of the solvent, optionally in the presence of dehydrating agents, e.g. sodium sulfate or magnesium, molecular sieves. If required, the reaction can also be carried out in the presence of a catalyst, e.g. a Lewis acid.

Alternatively, the above-mentioned compounds can be prepared from 7-aldehyde camptothecin oxime (prepared as described by Sawada in Chem. Pharm. Bull. 39, 2574 (1991)) or 7-ketonocamptothecin, or from the corresponding 7-acylcamptothecin by reaction with the halide R10-X wherein X is preferably iodine, in a polar solvent, e.g. tetrahydrofuran or an alcohol and in the presence of a base, e.g. sodium hydride or potassium carbonate.

Regarding the compounds of formula (IV) in which n is 1 and R10 is aroyl as defined in formula (IV), they can be prepared starting from camptothecin 7-oxime, the preparation of which is described in the previous paragraph, by reaction with chlorides acyl R10-COCl in polar solvents and in the presence of a base, preferably pyridine or directly in pyridine as described by Cho in J. Org. Chem. 62, 2230 (1997).

Regarding the compounds of formula (IV) in which n is 0 and R10 is as defined above, except aroyl, they can be prepared starting from 7-aldehyde camptothecin (formula IVa, R11 is hydrogen) or 7-ketocamptothecin (formula IVa, R11 is other than hydrogen).

<img file="PL204418B1_D0005.tif" />

wherein R7 is the group -C (R11) = O and R11 is as defined in formula (IV), R8 and R9 are as defined in formula (IV). The compound of formula (IVa) is reacted with the compound of formula 10

Of (Vb) R10NH2, in which R10 is as defined above, to give compounds of formula (IV) in which R7 is a group -C (R11) = N-R10, R10 is defined as in formula (IV) ), except for aroyl. The reaction can be carried out using conventional methods known to those skilled in the art of pharmaceutical technology, the process being the normal formation of imines. The preferred molar ratio of the 7-keto or 7-aldehyde camptothecin to the imine should be in the range from 1: 3 to 3: 1. The corresponding amine salt can also be used. The reaction is carried out in the presence of a base, e.g. an inorganic base such as potassium carbonate, or an organic base such as triethylamine or diazabicyclononane, using polar solvents, preferably methanol or ethanol, and carrying out the reaction at temperatures ranging from ambient temperature to the boiling point of the solvent, optionally in the presence of dehydrating agents, e.g. sodium or magnesium sulfate, molecular sieves. If required, the reaction may be carried out in the presence of a catalyst, e.g. a Lewis acid as described by Moretti and Torre in Synthesis, 1970, 141; or Kobayashi et al., Synlett, 1977, 115).

7-aldehyde camptothecin and camptothecin 7-oxime are described in European patent application EP 0 056 692 and in the above-cited article by Sawada, Chem. Pharm. Bull. 39, 2574 (1991).

The N1-oxides of compounds of formula (IV) are prepared by known methods of heteroaromatic nitrogen oxidation, preferably by oxidation with acetic or trifluoroacetic acid and hydrogen peroxide or by reaction with organic peracids (A. Albini and S. Pietra, Heterocyclic N-oxides, CRC, 1991).

Regarding the varying meaning of R10 found in the various formulas of the V reagents, these reagents are commercially available or can be prepared by methods known in the literature which those skilled in the art may use to supplement their own knowledge of the subject.

Pharmaceutically acceptable salts are obtained by conventional methods described in the literature and need not be further described.

Preferred camptothecin derivatives, in addition to 7-carbonitrile camptothecin (CPT 83), are also 7-benzyloxyiminomethyl camptothecin (CPT 172) and 7-butoxyiminomethyl camptothecin (CPT 184).

Example 8. 7-Benzyloxyiminomethylcamptothecin (CPT 172)

500 mg (1.33 mmol) of 7-formylcamptothecin is dissolved in 100 ml of ethanol. 15 ml of pyridine and 638 mg (4 mmol) of O-benzylhydroxylamine hydrochloride are added. The solution is heated to reflux for 5 hours. The solvent is evaporated off in vacuo and the residue thus obtained is purified by flash chromatography on silica gel using a hexane / ethyl acetate 4: 6 mixture as eluant. Yield 65%. Melting point: 200-205 ° C with decomposition.

The product obtained is an approximately 8: 2 mixture of the two syn and anti isomers (isomer A - Rf 0.32; isomer B - Rf 0.19, on Merck 60 F254 silica gel; eluant: hexane: ethyl acetate 3: 7) ).

HPLC: the analyzes were performed on a device equipped with a four-phase pump (HP 1050) with a Rheodyne injector (20 µΐ loop), and the diode detector (HP 1050) was operated by the HPLC-ChemStation program. Spectra were read in the range from 200 to 600 nm and chromatograms were recorded at 360 and 400 nm.

Reverse phase column chromatography (C18 column, Rainin CIS; 25 x 0.4 cm, Varian) was performed with a RP18 precolumn. The analysis was performed using a linear elution gradient starting from an acetonitrile: water ratio of 30:70 to 100% acetonitrile over 20 minutes, with a flow rate of 1 ml / minute. The retention time was: 12.51 minutes for the B isomer and 14.48 minutes for the A isomer.

¹H-NMR (300 MHz; DMSO-d6): δ 0.88 (t, H3 - 18A + H3-18B), 1.87 (8m, 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 - 17A + H2 - 17B ), 6.53 (s, -OHA + -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, H9A), 9.38 (s, CH = NA).

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

Example 9. 7-Butoxyiminomethylcamptothecin (CPT 184)

400 mg (1.06 mmol) of 7-formylcamptothecin is dissolved in 80 ml of ethanol. 12 ml of pyridine and 400 mg (3.18 mmol) of ot-butylhydroxylamine hydrochloride are added. The solution is heated to reflux for 4 hours. The solvent was evaporated in vacuo and the residue obtained was purified by flash chromatography on silica gel using hexane / ethyl acetate 4: 6 as eluant. 322 mg (0.72 mmol) of a yellow solid are obtained. Yield 68%. Melting point: 250 ° C with decomposition.

PL 204 418 B1

The product obtained is an approximately 8: 2 mixture of the two syn and anti isomers (isomer A - Rf 0.31; isomer B - Rf 0.24, on Merck 60 F254 silica gel; eluant: hexane: ethyl acetate 3: 7) ).

HPLC: Analyzes were performed on a device equipped with (HP 1050) with a Rheodyne injector (20 µl loop) and the diode detector (HP 1050) was operated by the HPLC-ChemStation program. Spectra were read in the range from 200 to 600 nm and chromatograms were recorded at 360 and 400 nm.

A C18 column (Rainin C18; 25 x 0.4 cm, Varian) was used with a RP18 precolumn. The analysis was performed using a linear elution gradient starting from an acetonitrile: water ratio of 30:70 to 100% acetonitrile over 20 minutes, with a flow rate of 1 ml / min. The retention time was: 12.92 minutes for the B isomer and 14.61 minutes for the A isomer.

¹H-NMR (300 MHz; DMSO-d6): δ: 0.88 (t, H3 - 18A + H3 - 18B), 1.30 (s, t-but. B), 1.47 (s, t- cylinder A), 1.87 (m, H2 - 19A + H2 - 19B), 5.18 (s, H2 - 5 B), 5.37 (H2 - 5 A), 5.42 (s, H2 - 17A + H2 - 17B), 6.54 (s, OH A + -OH B), 7.35 (s H -14A), 7.36 (s, H -14B) 7.69-7.83 (m , H11A + 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 - 12A), 9.31 (s, CH A).

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

Preparation of liposomes

The compounds of the invention can be used to prepare multilayer liposomes (MLV) and monolayer liposomes (SUVs), both as dry powders and as suspensions in solutions.

Compounds of the invention, prepared as described in Examples 1-7, are used to prepare liposomes according to the following procedure. The appropriate amount of the compound is dissolved in chloroform; this solution is concentrated to dryness under high vacuum with a rotary evaporator until a lipid film is obtained. The lipid film is dried under high vacuum until the remaining traces of solvent are removed and then dissolved in tert-butyl alcohol or water. The thus obtained solution is lyophilized to obtain a soft, dry powder.

The powders are hydrated with a suitable amount of aqueous solution to obtain a liposome of the compound used, which is then complexed with the polynucleotide or with the required drug.

Another method of producing liposomes consists in adsorbing the lipid film consisting of a compound of the invention in a solvent on a suitable inert carrier such as sorbitol, mannitol or other pharmacologically acceptable carbohydrates. The mixture is dried under high vacuum to give a solid that can be hydrated easily and very quickly before use.

Dry powder formulations have the advantage of being stable over a long period of time and being easy to handle.

In addition, the compounds of the invention can be used to prepare liposomes linked to DNA or to the required drug in the form of a dry powder according to the following procedure. The compound of the invention is dissolved in tert-butyl alcohol or in water; the solution thus obtained is mixed with the DNA or the drug of interest, and the mixture is lyophilized to obtain a complex which may be referred to as proliposome-DNA or proliposome-drug complex, in the form of a soft, dry powder.

The powders (proliposomes) so obtained can be used for the preparation of pharmaceutical compositions which can be administered in the form of an aerosol, or alternatively parenterally or orally when reconstituted with water or a suitable buffer solution.

Liposomes complexed with DNA or with solid drugs can also be obtained by adsorption of the lipid membrane onto an inert support such as sorbitol, mannitol, or other carbohydrates prepared by the method described above.

Study of liposome formation

Liposome formation was investigated by a colorimetric method using a water-soluble dye according to the following procedure. An aqueous solution of the water-soluble dye Arsenazo III was obtained (MW = 776.37; 2.3 mg / ml).

This solution was used instead of water to hydrate the lipid membranes prepared as described above. An aliquot of the suspension containing the dye-coating liposome was diluted 100-fold with water.

2 ml of the liposome suspension was used to obtain the first optical density reading at 660 nm; the reading was obtained with the same sample designated as blank. 200 μΐ size12

The amount of CaCb (15 mg / ml; 100 mM) was added to the first sample and the optical density was measured at 660 nm against a blank to which was added 200 μl of water. The absorbance value obtained was determined as reading 2. 100 µl of Triton X-100 solution (5% by volume; 0.26% final concentration) was added to the sample and 200 µl of water was added to the blank; reading the optical density at 660 nm provided information about the optical density value defined as reading 3. To calculate the percentage of dye contained in liposomes, the following formula was used:

% dye contained in liposomes = reading 3 - reading 2 reading 3 • 100

The percentage of coated dye is a measure of liposome formation and is on average about 40%: the determination of the liposome dimensions was carried out using the laser light scattering method with positive results.

Example 10. Preparation 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 in 20 ml of chloroform. The solution was concentrated until a lipid film was obtained on the surface of the glass of the flask.

After removing the last traces of chloroform using a high vacuum pump, 20 ml of tert-butyl alcohol was added to the lipid membrane. In order to obtain a clear solution it had to be heated to 60 ° C. The solution was immediately frozen at -70 ° C with liquid nitrogen and lyophilized for 24 hours. To obtain the final suspension of the SUV liposomes, the lyophilized product, hydrated with PBS solution (20 ml), was sonicated for 20 minutes at 0 ° C. Filtration was then performed on a 400 nm filter to remove traces of titanium released by the ultrasonic probe.

Testing the physical stability of the preparation

The physical stability of the formulation was examined by turbidimetry by recording a TDC time curve at 800 nm, at 20 ° C for 6 hours. The steady trend of turbidity indicated the stability of the formulation, no precipitation was observed.

HeLa cells were transfected with plasmid DNA with ST 772 liposome. Figures 1 and 2 show the efficiency of transfection of plasmid DNA with ST 772 liposome into HeLa cells. For this purpose, densitometric analysis of DNA extracted from ST 272 transfected cells and with DOTAP as the relevant cationic lipid was used. The results of the blot analysis reveal amounts of plasmid DNA of the same order that were obtained with DOTAP.