RU2104282C1 - Nucleoside phospholipid derivatives and pharmaceutical composition - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: product: compounds of the formula (I): $$$ where $$$ and $$$ - linear or branched $$$-alkyl chain; n = 0, 1 or 2; B means nucleotide residue. Invention relates to method of their synthesis and antiviral medicinal agents containing these compounds. EFFECT: improved method of synthesis. 5 cl, 9 tbl

Description

,
где0 R₁ - неразветвленная или разветвленная, насыщенная или ненасыщенная алкильная цепь с 8-15 углеродными атомами, которая может быть однократно или многократно замещена фенильными группами, галогеном, C₁-C₆-алкокси-, C₁-C₆-алкилфенильными группами, галогеном, C₁-C₆-алкокси-, C₁-C₆-алкилмеркапто-, C₁-C₆-алкоксикарбонил-, C₁-C₆-алкилсульфинил- или C₁-C₆-алкилсульфонил-группами,
R₂ - неразветвленная или разветвленная алкильная цепь с 8-15 углеродными атомами, которая может быть замещена при необходимости однократно или многократно фенильными группами, галогеном, C₁-C₆-алкокси-, C₁-C₆-алкилмеркапто-, C₁-C₆-алкоксикарбонил или C₁-C₆-алкилсульфонил-группами,
R₃ - водород или гидрокси-группа,
R₄, R₅ - соответственно водород или из остатков R₄ и R₅ галоген, гидрокси-, циано- или азидо-группа и кроме того,
R₃ и R₄ могут представлять собой еще одну связь между C-2' и C-3',
n - 1 или 2, а
B - одно из следующих соединений:
1.

,
причем R₆ может быть водород, алкильная цепь с 1-4 углеродными атомами или галоген,
2.

,
при этом R₇ - это может быть водород, алкильная цепь с 1 - 4 углеродными атомами или галоген,
3.

,
при этом R₈ может быть водород, алкильная цепь с 1 - 4 углеродными атомами, галоген, или гидрокси- или амино-группа,
4.

,
причем R₉ может быть водород или амино-группа, и
R₁₀ означает водород, галоген, C₁-C₆-алкокси, C₁-C₆-алкилмеркапто, или амино-группу, которая может быть моно- или ди- замещена посредством C₁-C₆-алкил, C₁-C₆-алкокси-, гидрокси-, C₂-C₆-алкил- и/или C₃-C₆-циклоалкил-, арил-, гетарил-, аралкил- или гетарилалкил-групп, которые при желании в арильном или в гетарильном остатке могут быть еще замещены одной или несколькими гидрокси-, метокси-, или алкильными группами, или галогеном, или аллилом, который при необходимости может быть замещен моно- или диалкил- или алкокси-группами,
их таутомеры и их физиологически совместимые соли неорганических и органических кислот или оснований, а также способ их получения и лекарственных средств, содержащих эти соединения.The aim of the invention are new phospholipid derivatives of nucleosides of General formula 1,

,
where 0 R ₁ is an unbranched or branched, saturated or unsaturated alkyl chain with 8-15 carbon atoms, which may be substituted once or repeatedly by phenyl groups, halogen, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylphenyl groups, halogen, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkyl mercapto, C ₁ -C ₆ alkoxycarbonyl, C ₁ -C ₆ alkylsulfinyl or C ₁ -C ₆ alkylsulfonyl groups,
R ₂ is an unbranched or branched alkyl chain with 8-15 carbon atoms, which may be optionally substituted once or repeatedly by phenyl groups, halogen, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkyl mercapto, C ₁ - C ₆ alkoxycarbonyl or C ₁ -C ₆ alkylsulfonyl groups,
R ₃ is hydrogen or a hydroxy group,
R ₄ , R ₅ respectively hydrogen or from the residues R ₄ and R ₅ halogen, hydroxy, cyano or azido group and in addition,
R ₃ and R ₄ may be another bond between C-2 'and C-3',
n is 1 or 2, and
B is one of the following compounds:
1.

,
and R ₆ may be hydrogen, an alkyl chain with 1-4 carbon atoms or halogen,
2.

,
while R ₇ - it can be hydrogen, an alkyl chain with 1 to 4 carbon atoms or halogen,
3.

,
wherein R ₈ may be hydrogen, an alkyl chain with 1 to 4 carbon atoms, a halogen, or a hydroxy or amino group,
4.

,
wherein R ₉ may be hydrogen or an amino group, and
R ₁₀ means hydrogen, halogen, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkyl mercapto, or an amino group which may be mono or di-substituted by C ₁ -C ₆ alkyl, C ₁ -C _6- alkoxy-, hydroxy-, C ₂ -C ₆ -alkyl- and / or C ₃ -C ₆ -cycloalkyl-, aryl-, hetaryl-, aralkyl- or hetarylalkyl groups which, if desired, are in the aryl or hetaryl radical may also be substituted with one or more hydroxy, methoxy, or alkyl groups, or halogen, or allyl, which may optionally be substituted with mono or dialkyl or alkoxy groups,
their tautomers and their physiologically compatible salts of inorganic and organic acids or bases, as well as the method for their preparation and medicines containing these compounds.

 Since the compounds of general formula I contain asymmetric carbon atoms, all optically active forms and racemic mixtures of these compounds are also the subject of the invention.

 In J. Biol. Chem. 265, 6112/1990 / describes a method for the preparation and use of liponucleotides as antiviral drugs. However, only those associated with known nucleosides, such as AZT and ddC, dimyristoylphosphatidyl and dipalmitoylphosphatidyl residues with their structure of fatty acid esters were investigated and synthesized here.

 In J. Med. Chem. 33, 1380/1990 / describes nucleoside conjugates of thioether lipids with cytidine diphosphate, which have an antitumor effect and which could find application in oncology.

 Chem. Pharm. Bull. 36, 209/1988 / describes 5 '- / 3-SN-phosphatidyl / nucleosides with antileukemic activity, as well as their enzymatic synthesis from the corresponding nucleosides and phosphocholines in the presence of phospholipase D with transferase activity.

 Enzymatic synthesis of liponucleotides is also described in Tetrahedron Lett. 28, 199/1987 / and Chem. Pharm. Bull. 36, 5020/1988 /.

 The compounds of this invention also have valuable pharmacological properties. In particular, they are suitable for the treatment and prevention of infections caused by DNA viruses, such as Herpes - Simplex virus / simple lichen /, cytomegaly virus, Papova virus, herpes zoster virus - chicken pox or Epstein-Barr virus or RNA viruses like Toga viruses or especially retro viruses like onco viruses HTLV-I and II, as well as Visna lentiviruses and the human immune weakness virus HIV-1 and 2.

 Particularly useful are compounds of formula I for the treatment of clinical manifestations of retroviral HIV infections in humans, such as in prolonged generalized lymphadenopathy / PJL /, the progressive stage of the related AIDS complex / ARC / and the clinical picture of AIDS.

 Unexpectedly, it has now been found that the compounds of general formula I inhibit the propagation of DNA and / or RNA viruses at a stage specific for the DNA or RNA transcription virus. These substances, through the inhibition of the Reverse Transkriptase enzyme, can affect the reproduction of retroviruses / cm. Proc. Nati. Acad Sci. USA 83, 1911, 1986 or Nature 325, 773 1987 /. Of particular therapeutic interest is the inhibitory effect on HIV virus, the culprit of the disease of immune weakness - AIDS. For the treatment of AIDS, only 3'-acido-3 'deoxythymidine / DE-A-3608606 / is currently allowed in AIDS patients. However, the toxic side effects of 3'-acido-3'-deoxythymidine on the bone marrow in about 50% of treated patients necessitate a blood transfusion. The compounds of general formula I do not have this disadvantage. They have an antiviral effect, not manifesting themselves cytotoxically in pharmacologically significant doses.

 The compounds of this invention and their pharmaceutical preparations can also be used in combination with other drugs for the treatment and prevention of these infections. Examples of other drugs contain agents that can be used to treat and prevent HIV infections or diseases associated with this disease, such as 3'-acido-3'-deoxythymidine, 2 ', 3'-dideoxynucleosides, such as 2', 3 '-dideoxycytidine, 2', 3'-dideoxyadenosine and 2 ', 3'-dideoxyinosine, acyclic nucleosides / e.g. acyclovir /, interferon, such as A-interferon, renal excretion inhibitors, such as probenicide, nucleoside transport inhibitors, such as dipyridamole, as well as immunomodulators, such as measures, interleukin II or stimulation factors, such as, for example, granulocyte factor - Markofag - colonies. The compounds of this invention and another drug can be dispensed, respectively, individually, simultaneously in one single preparation or in two separate preparations, or at different time periods, so that a synergistic effect is achieved.

 As possible salts of compounds of the general formula I, it is possible to consider primarily alkaline salts, alkaline-earth and ammonium salts of the phosphate group. As alkaline salts, lithium, sodium and potassium salts are preferred. As alkaline earth salts, in particular, magnesium and calcium salts are considered. Ammonium salts according to the invention are understood to mean salts containing an ammonium ion, which can be substituted up to four times with alkyl radicals with 1-4 carbon atoms and / or aralkyl radicals, preferably benzyl radicals. The substituents may be the same or different.

 Compounds of the general formula I may contain basic groups, in particular amino groups, which, with the aid of the corresponding acids, can be converted into salts formed by the addition of acids. How acids can be considered in this case, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, fumaric, succinic, tartaric, citric, lactic, maleic or methanesulfonic acid.

In the general formula, IR ₁ is a predominantly unbranched C ₁₀ -C ₁₄ alkyl group which may still be substituted with a C ₁ -C ₆ alkoxy or C ₁ -C ₆ alkyl mercapto group. R ₁ represents, in particular, a decyl, undecyl, dodecyl, tridecyl, or tetradecyl group. As the C ₁ -C ₆ alkoxy substituent, R ₁ are preferably considered methoxy, ethoxy, butoxy and hexyloxy groups. If R _{1 is} substituted with a C ₁ -C ₆ alkyl mercapto residue, then this should be understood, in particular, methyl mercapto, ethyl mercapto, propyl mercapto, butyl mercapto and hexyl mercapto residue, and n is one of the numbers 0 , 1 or 2.

R ₂ means a substantially unbranched C ₁₀ -C ₁₄ alkyl group which may still be substituted with a C ₁ -C ₆ alkoxy group or a C ₁ -C ₆ alkyl mercapto group. R ₂ represents, in particular, a decyl, undecyl, dodecyl, tridecyl, or tetradecyl group. As C ₁ -C ₆ alkoxy substituents, R ₂ are preferably considered methoxy, ethoxy, propoxy, butoxy and hexyloxy groups.

If R _{2 is} substituted with a C ₁ -C ₆ alkyl mercapto residue, then this is understood in particular as a methyl mercapto residue, ethyl mercapto, butyl mercapto and hexyl mercapto radical.

R ₄ and R ₅ preferably mean respectively hydrogen or one of the two residues, preferably a cyano or acid group, or a halogen atom like fluorine, chlorine, bromine or iodine.

Particularly preferred are compounds in which R ₃ and R ₄ represent a hydrogen atom, R ₅ cyano, acido or fluoro; R ₅ means hydrogen, and R ₃ / R ₄ represent another bond between C-2 'and C-3'.

In bases B of general formula I, the radicals R ₆ or R ₇ preferably represent a hydrogen atom, a methyl, ethyl, propyl or butyl radical, or a halogen atom such as fluorine, chlorine, bromine or iodine. Especially preferred for R ₆ or R _{7 is} a hydrogen atom, a methyl or ethyl residue and a chlorine or bromine atom.

The R ₈ residue is predominantly a hydrogen atom, a methyl, ethyl, propyl or butyl residue, an amino group or a halogen atom such as fluorine, chlorine, bromine or iodine, preferably chlorine or bromine.

R ₁₀ means mainly a hydrogen atom, fluorine, chlorine or a bromine atom, a C ₁ -C ₆ alkoxy group, in particular a methoxy, ethoxy, propoxy, butoxy or hexyloxy group, a C ₁ -C ₆ alkyl mercapto group, in particular methyl mercapto, ethyl mercapto, butyl mercapto or hexyl mercapto group, or an amino group, which may be mono- or disubstituted on a C ₁ -C ₆ alkyl group, such as, for example, methyl, ethyl, butyl or hexyl a group on a hydroxy, C ₂ -C ₆ alkyl group, such as, for example, a hydroxyethyl, hydroxypropyl, hydroxybutyl, or hydroxyhexyl group, on a C ₃ -C ₆ y cycle an alkyl radical, such as, for example, a cyclopropyl, cyclopentyl or cyclohexyl radical, on an aryl, preferably phenyl, on an aralkyl radical, such as benzyl, which in this case can be substituted by one or more hydroxy or methoxy groups, C ₁ -C ₆ -alkyl groups, such as, for example, methyl, ethyl, propyl, butyl or hexyl groups or on halogen atoms such as fluorine, chlorine or bromine. The amino group may also be substituted with a heterarylalkyl or hetaryl residue, such as, in particular, a thienyl, furyl or pyridyl residue. By a heterarylalkyl radical is preferably meant a thienylmethyl radical, a furylmethyl radical or a pyridylmethyl radical.

Preferably, the linked nucleosides in the claimed liponucleotides of the general formula I are as follows:
-2 ', 3'-dideoxy-3'-azidouridine
-2 ', 3'-dideoxyinosine
-2 ', 3'-dideoxyguanosine
-2 ', 3'-dideoxycytidine
-2 '3'-dideoxyadenosine
-3'-deoxythymidine
-2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / O-methylbenzyl / adenosine
-2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / 2-methylpropyl / adenosine
-2 ', 3'-dideoxy-3'-acidoguanosine-3'-deoxy-3'-acidothymidine
-2 ', 2'-dideoxy-3'-fluoro-5-chlorururidine
-3'-deoxy-3'-fluorothymidine
-2 ', 3'-dideoxy-3'-fluoroadenosine-2', 3'-dideoxy-3'-fluoro-2,6-diaminopurinriboside
-2 ', 3-dideoxy-2', 3'-didehydrocytidine
-3'-deoxy-2 ', 3'-didehydrothymidine
-3'-deoxy-2 ', 3'-didehydrothymidine
-3'-deoxy-3'-acidothymidine.

,
где R₁, R₂ и n имеют указанные значения, подвергают взаимодействию с соединением общей формулы III,

,
где R₃ представляет собой водород или гидрокси-группу, защищенную посредством обычной для специалиста кислородной защитной группой,

- соответственно водород, галоген, ацидо-, циано-остаток, или один из остатков

гидрокси-группы, защищенной посредством обычной для специалиста кислородной защитной группой, или

- еще одну связь, и
B имеет указанные значения,
в присутствии фосфорокситрихлорида и сложного эфира фосфорной кислоты и третичного азотного основания, например пиридина или триэтиламина, в инертном растворителе, как, например, толуол, и после осуществленного гидролиза согласно обычному в химии нуклеозида способу отщепляют кислородные защитные группы, или
соединение общей формулы IV:

,
в которой
R₁, R₂ и n имеют вышеназванные значения, подвергают взаимодействию с соединением общей формулы III, в которой

и B имеют указанные значения, в присутствии фосфолипазы в инертном растворителе, как, например, хлороформ, в присутствии соответствующего буфера, и после завершения реакции при необходимости согласно обычному в химии нуклеозидов способу отщепляют кислородную защитную группу.Compounds of general formula I can be prepared in that
1. The compound of General formula II,

,
where R ₁ , R ₂ and n have the indicated meanings, are reacted with a compound of the general formula III,

,
where R ₃ represents hydrogen or a hydroxy group protected by a conventional specialist oxygen protective group,

- respectively, hydrogen, halogen, acido, cyano residue, or one of the residues

a hydroxy group protected by a specialist’s oxygen protecting group, or

- another connection, and
B has the indicated meanings,
in the presence of phosphoroxytrichloride and an ester of phosphoric acid and a tertiary nitrogen base, for example pyridine or triethylamine, in an inert solvent, such as toluene, and after the hydrolysis is carried out according to the usual method of nucleoside chemistry, the oxygen protecting groups are cleaved, or
the compound of General formula IV:

,
wherein
R ₁ , R ₂ and n have the above meanings, are reacted with a compound of general formula III, in which

and B have the indicated meanings, in the presence of a phospholipase in an inert solvent, such as chloroform, in the presence of an appropriate buffer, and after completion of the reaction, if necessary, the oxygen protecting group is cleaved according to the usual method of nucleoside chemistry.

 The preparation of compounds of general formulas II and IV are described in Lipids 22, 947 (1987) and in DE-A-3039629.

 The preparation of compounds of formula III are described, for example, in European patent EP-A-0286028 and WO 90/08147.

 Compounds similar to general formula I are described in EP-A-0350287. There, however, only a 1,2-complex glycerol diester is described.

 In US patent N 4734232, CL. A 61 K 31/70, 1988, discloses pharmaceutical compositions having inhibitory activity against viral reverse transcriptase based on nucleotide phosphates and containing pharmaceutically acceptable carriers.

 The drug-containing compounds of formula I for the treatment of viral infections can be administered enteral or parenteral in liquid or solid form. In this case, the usual forms of application are considered, such as tablets, capsules, dragees, syrup, solutions or suspensions. As the injection medium, water is used predominantly containing additives conventional in injection solutions, such as stabilizing agents, dissolution agents and buffers. Such additives are, for example, tartrate and citrate buffers, ethanol, a complexing agent, such as ethylenediaminetetraacetic acid and its non-toxic salts, high molecular weight polymers, such as liquid polyethylene oxide for regulating viscosity. Liquid fillers are, for example, starch, lactose, mannitol, methyl cellulose, talc, finely divided silicic acids, high molecular weight fatty acids like stearic acid, gelatin, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats, solid high molecular weight polymers, like polyethylene glycols, etc. Formulations suitable for oral administration may, if desired, have flavorings or sweeteners.

 Dosage may depend on various factors, such as method of application, type of application, age or individual condition. The proposed compounds are usually used in amounts of 0.1-100 mg, mainly 0.2-80 mg per day and per kg of body weight. It is preferable that the daily dosage can be divided into 2-5 applications, and for any application, 1-2 tablets are dispensed with a biologically active substance content of 0.5-500 mg. These tablets can have such a delayed effect, as a result of which the number of applications per day is reduced to 1-3. The content of the biologically active substance of tablets with delayed action can deliver 2-1000 mg. A biologically active substance can also be given as a result of prolonged infusions, with amounts of 5-1000 mg per day usually being sufficient.

In the sense of the proposed invention, in addition to the compounds named in the examples and all the meanings of the substituents mentioned in the claims, the following compounds of the formula I are considered:
1. (3-Dodecyl mercapto-2-decyloxy) propyl ester of 2 ', 3'-dideoxy-3'-fluoro-5-chlorururidine) -5'-phosphoric acid,
2. / 3-dodecyl-sulfinyl-2-decyloxy / propyl ether / 3'-deoxy-3'-azido-thymidine / -5'-phosphoric acid,
3. / 3-dodecyl-sulfonyl-2-decyloxy / propyl ether / 3'-deoxy-3'-acidido-thymidine / -5'-phosphoric acid,
4. / 3-dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxycytidine / -5'-phosphoric acid,
5. / 3-dodecyl mercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxyinosine / -5'-phosphoric acid,
6. / 3-dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3-dideoxyguanosine / -5'-phosphoric acid,
7. / 3-dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxyadenosine / -5'-phosphoric acid,
8. / 3-dodecylmercapto-2-decyloxy / propyl ether / 3'-deoxythymidine / -5'-phosphoric acid,
9. / 3-dodecylmercapto-2-decyloxy / propyl ether / 3'-deoxy-2 ', 3'-didehydrothymidine / -5'-phosphoric acid,
10. / 3-dodecylmercapto-2-decyloxy / propyl ether / 3'-deoxy-3'-fluorothymidine / -5'-phosphoric acid,
11. / 3-dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxy-3'-acidoguanosine / -5'-phosphoric acid,
12. / 3-dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxy-3'-fluoro-2,6-diaminopurinriboside / -5'-phosphoric acid,
13. / 3-dodecylmercapto-2-decyloxy / propyl ether [2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / 2-methylpropyl / adenosine] -5'-phosphoric acid,
14. / 3-dodecylmercapto-2-decyloxy / propyl ether [2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / o-methylbenzyl / - adenosine] -5'-phosphoric acid,
15. / 3-decylmercapto-2-dodecyclooxy / propyl ether / 2 ', 3'-dideoxy-2', 3'-didehydrocytidine / -5'-phosphoric acid,
16. / 3-undecylmercapto-2-dodecyloxy / propyl ether / 2 ', 3'-dideoxy-3'-fluoroadenosine / -5'-phosphoric acid,
17. / 3-decylsulfonyl-2-dodecyloxy / propyl ether / 2 ', 3'-dideoxy-3'-acidouridine / -5'-phosphoric acid,
18. / 3-decylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxycytidine / -5'-phosphoric acid,
19. / 3-dodecylmercapto-2-dodecyloxy / propyl ether / 2 ', 3'-dideoxyinosine / -5'-phosphoric acid,
20. / 3-tetradecylmercapto-2-decyloxy / propyl ether / 3'-deoxy-3'-acidothymidine / -5'-phosphoric acid,
21. / 3-pentadecylmercapto-2-decyloxy / propyl ether / 3'-deoxy-3'-acidothymidine / -5'-phosphoric acid,
22. / 3-tridecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxyinosine / -5'-phosphoric acid,
23. / 3-dodecylmercapto-2-octyloxy / propyl ether / 2 ', 3'-dideoxyinosine / -5'-phosphoric acid.

 Example 1a (3-Dodecyl mercapto-2-decyloxy) propyl ester / 3'-deoxy-3'-azido'-thymidine / -5'-phosphoric acid.

To a solution of 1.25 g / 3 mmol / 3-dodecyl mercapto-2-decyloxy-1-propanol and 1.2 ml / 8.6 mmol / triethylamine in 40 ml abs. ether, instilled in a nitrogen atmosphere at 0 ^° C. 0.42 ml / 4.5 mmol / POCl ₃ and stirred for 45 minutes. Then it was warmed to room temperature, a solution of 800 mg / 3 mmol / 3'-deoxy-3'-azido-thymidine / AZT / in a mixture of 15 ml abs was instilled. ether and 20 ml abs. toluene and stirred for 6 hours under reverse flow / thin layer chromatography - control /.

After cooling, 50 ml of water was added, the mixture was stirred vigorously for 2 hours, then the organic phase was separated, dried over Na ₂ SO ₄ and concentrated in a rotary evaporator. The residue was purified by preparative column chromatography on silica gel 60 with a dichloromethane / methanol ratio 9: 1 as solvent. The yield was 540 mg / 24% of theoretical value /.

Melting point 187 ^o C, sintering, decomposition temperature 220-223 ^o C with the appearance of brown staining.

³¹ p - NMR = 0.59 ppm.

 Example 1b (3-Dodecyl mercapto-2-decyloxy) propyl ester / 3'-deoxy-3'-azido-thymidine / -5'-phosphoric acid.

Similar to the recipe in Chem. Pharm. Bull. 36, 5020 (1988), suspended 2 mmol. AZT and 5000 units. phospholipase D in 4 ml of sodium acetate / CaCl ₂ buffer was diluted with a solution of 6 mmol. 3-dodecyl mercapto-2-decyloxypropyl-1-phosphoric acid monocholine ester in 160 ml of chloroform and heated for 8 hours to 45 ^° C.

It was then dried over Na ₂ SO ₄ and the solvent was removed in vacuo. The residue was purified as described in Example 1 by column chromatography. The yield was 51%. The product turned out to be identical to the product from example 1 a / melting point, DC, ¹ H - and ³¹ p - NMR /.

Example 2. / 3-Undecylmercapto-2-undecyloxy / propyl ether / 3'-deoxy-3'-azido-thymidine / -5'-phosphoric acid was obtained analogously to example 1 a. Yield 27%, melting point 218-222 ^o C / decomposition /.

Example 3. / 3-Dodecylmercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / o-methylbenzyl / adenosine-5'-phosphoric acid.

680 mg / 1.37 mmol / 3-dodecylmercapto-2-decyloxy / phosphoric acid propyl ester in 20 ml abs. pyridine was diluted with 337 mg / 1 mmol / 2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - / o-methylbenzyl / adenosine, and after adding 1.37 g / 6.7 mmol / DCC stirred for 24 hours at room temperature / thin layer chromatography - control /. Pyridine was then removed in vacuo, the residue was suspended in ether and filtered off from undissolved urea. After evaporation of the solvent, the filtrate was purified by column chromatography on silica gel 60 with a dichloromethane / methanol ratio 95/5 as solvent. Yield 220 mg / 26% of theoretical value /.

R _f = 0.68 / CH ₂ Cl ₂ / CH ₃ OH / H ₂ O 13/5 / 0.8 /.

 Example 4. / 3-Dodecyl mercapto-2-decyloxy / propyl ether / 3'-deoxy-3'-azidothymidine / -5'-phosphoric acid.

 Analogously to example 3 of 13.5 / 3-dodecyl mercapto-2-decyloxy / propyl ester of phosphoric acid, 5.5 g of AZT and 27 g of DCC in 350 ml of abs. pyridine, by 30-hour stirring at room temperature and purification as described above, the corresponding liponucleotide was obtained in 62% yield / analytical data were identical to those specified in example 1 /.

 Example 5. / 3-Dodecyl mercapto-2-decyloxy / propyl ether / 3'-deoxythymidine / -5'-phosphoric acid.

 Analogously to example 3 of 1.3 g / 3-dodecyl mercapto-2-decyloxy / propyl ester of phosphoric acid, 500 mg of 3'-deoxythymidine and 2.6 g of DCC in 40 ml of abs. pyridine, as a result of stirring for 24 h at room temperature and chromatographic purification, the corresponding liponucleotide was obtained in 51% yield.

R _f = 0.45 / CH ₂ Cl ₂ / CH ₃ OH / H ₂ O 12/5 / 0.8 /.

 Example 6. / 3-Dodecyl mercapto-2-decyloxy / propyl ether / 2 ', 3'-dideoxyinosine / -5'-phosphoric acid.

 Analogously to example 3 of 1.3 g / 3-dodecyl mercapto-2-decyloxy / propyl ester of phosphoric acid, 500 mg of 2 ', 3'-dideoxyinosine and 2.6 g of DCC in 40 ml of abs. pyridine as a result of 40-hour stirring at room temperature and chromatographic purification obtained named liponucleotide, with a yield of 61%.

R _f = 0.38 / CH ₂ Cl ₂ / CH ₃ OH / H ₂ O 13/5 / 0.8 /.

 Physical data for the preparation of compounds are given in table 1.

 Examples of the preparation of salts.

 Example 7. Obtaining the crude (3-dodecylthio-2-decyloxy) propyl ester of the product (3-deoxy-3-azido-thymidine) -5 phosphoric acid.

509 g (0.95 mol) of 3-dodecylthio-2-decyloxy-propanol-1-monophosphate was dissolved in 2.84 L of dry pyridine at room temperature. 443 g (1,467 mol) of triisopropyl benzenesulfonyl chloride are added to the solution, then 229 g (0.857 mol) of 3-deoxy-3-azido-thymidine (AZT) are dissolved after 10 minutes with stirring at room temperature. The brown solution was stirred overnight at room temperature. After adding water, the mixture becomes cloudy, but with subsequent stirring, it again becomes transparent. The solution was concentrated in vacuo at a bath temperature of 60 ^° C. in a rotary evaporator and the residue was distilled twice with 2.8 L of toluene until pyridine was removed. After evaporation, a semi-solid residue is poured into 5.7 L of diisopropyl ether and stirred for about an hour in an ice bath. The pyridine salt of triisopropylbenzenesulfonic acid is filtered off with suction and washed with 700 ml of diisopropyl ether in portions. The salt was dried at 50 ^° C (615 g) and recrystallized. The ether filtrate is shaken three times with 1.8 L of 2N. HCl. If good phase separation does not occur, the emulsion layer is carefully aspirated on the fibrin layer. The organic phase is dried with sodium sulfate and concentrated to constant weight. Obtain 677 g of crude phosphoric acid diester (106% of theory, counting 13 tons) by liquid chromatography: about 76% surface.

 Example 8. Purification of a crude phosphoric acid diester by formation of a Ca salt. 283 g (0.38 mol) of the crude product of Example 1 are dissolved in 1.73 L of methanol and 850 ml of water with stirring at room temperature; dropwise a solution of 60.1 g of acetate hydrate in 400 ml of water with stirring at room temperature for 45 minutes. A greasy precipitate forms, which is stirred for a long time (for example, overnight). In this case, the precipitate becomes powdery. The calcium salt is filtered off and the wet salt is intensively mixed with 1 liter of acetone at room temperature for about 30 minutes. Filter and dry. Exit 192 g.

 The calcium salt is suspended in 1.2 L of methyl tert-butyl ether and 276 ml of 2N. HCl and mix vigorously until the salt decomposes and a slightly turbid aqueous phase forms. After phase separation, the methyl tert-butyl phase is washed twice with 250 ml of a saturated NaCl solution, dried over sodium sulfate and concentrated.

 The residue after evaporation; 179.1 g (63.2% of the administered amount); liquid chromatography 90.41% by weight.

 Example 9. Purification of the crude product by preparative liquid chromatography.

Preparative system:
stationary phase: Merck Lichroprep RP, 15-25 microns, mobile phase: 0.02 molar solution of NaH ₂ PO ₄ 12.5% + 87.5% methanol.

125 g of the crude product of Example 2 are dissolved at approximately 35 ^° C. in 750 ml of methanol. 1.5 L of the mobile phase is added and the pH is set to 5 with concentrated NaOH. The solution is filtered and the filtrate is supplemented with a mobile phase of up to 2.5 l. This solution is chromatographed in 500 ml portions.

A maximum of the main product is fractionated and a 1.2 L fraction is separated. The combined purified fractions from five separations (6 L) are concentrated in vacuo on a rotary evaporator at a bath temperature of 40 ^° C. to a viscous residue. The residue is washed out of the apparatus in portions with water for a total of 500 ml; establish a semi-concentrated hydrochloric acid pH = 2.

 Extracted with methyl tert-butyl ether (1 • 2 L; 2 • 1 L); the phases are clearly separated and may be slightly cloudy. In the third extraction, both phases are usually transparent.

The combined methyl tert-butyl extracts for dehydration were shaken twice and 250 ml of a saturated NaCl solution. The organic phase is dried over Na ₂ SO ₄ and concentrated, liquid chromatography about 99.5 pov.%.

 Example 10. Obtaining Na-salt.

 278.3 g (0.327 mol) of the chromatographic purified product of Example 3 are dissolved in 4.8 L of methanol at room temperature, mixed with 760 ml of water (it becomes milky).

Set using 1n. NaOH (about 300 ml) pH = 5.8 with respect to the reference electrode and a cloudy solution is filtered. The clear filtrate is concentrated in vacuo on a rotary evaporator. The evaporation residue is distilled four times with toluene. The dry residue after evaporation is poured into 5.8 L of acetone and stirred for about 1 hour at room temperature. It is filtered off, washed with a small amount of acetone and dried at 50 ^o C.

 Yield 278 g (97% of theory, counting on purified free acid), liquid chromatography 99.45% by weight.

 Example 11. Obtaining Na-salt by precipitation from a solution of toluene.

3 g (0.0978 mol) of the purified free acid of Example 3 are dissolved in 500 ml of toluene. A slightly cloudy solution is filtered and mixed with the amount of 30% sodium methylate necessary for salt formation with stirring. A clear, slightly yellow solution is slowly pinned under stirring in 3 L of acetone. If necessary, stirred overnight, a powdery residue precipitated. It is filtered off, washed with acetone and a slightly sticky product is dried at 50 ^° C. Yield 66.2 g (88.1% of theory).

The following are the compounds obtained analogously to Example 7 as a crude product, enriched through calcium salt as described in Example 8, purified as described in Example 9 by preparative liquid chromatography and converted to sodium salt as described in Example 11:
1. The sodium salt of (3-undecyl mercapto-2-undecyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, so pl. 218-222 ^o C with decomp., R _f = 0.55 (isopropanol: butyl acetate: water = 5: 3: 2);
2. The sodium salt of (3-dodecyloxy-2-decyloxy) propyl ether (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, so pl. 205-211 ^o C with decomp., R _f = 0.60 (isopropanol: butyl acetate: water = 5: 3: 2);
3. sodium salt of (3-decylmerconato-2-dodecyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, mp> 206 ^° C, decomp .; R _f = 0.24 (methanol: water = 8: 2);
4. The sodium salt of (3-decylmercanto-2-decyloxy) propyl ester of (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, mp> 207 ^° C with dec, R _f = 0.45 (isopropanol: butyl acetate: water = 5: 3: 2);
5. the sodium salt of hexadecyl ether (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, so pl. 227-230 ^o C with dec, R _f = 0.55 (isopropanol: butyl acetate: water = 5: 3: 2);
6. The sodium salt of (3-tetradicylmerconto-2-decyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, so pl. 155 ^o C, R _f = 0.30 (acetic ether: methyl = 3: 1;
7. The sodium salt of (3-dodecylmercanto) propyl ester of (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid, mp> 200 ^° C, decomp .; R _f = 0.20 (ethyl acetate: methanol = 3: 1);
8. the sodium salt of (3-dodecylmercanto-2-dodecyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid,
mp> 170 ^° C with dec, R _f = 0.25 (ethyl acetate: methanol = 3: 1);
9. The sodium salt of (3-decylmercanto-2-hexadecyloxy) propyl ester (3'-deoxy-3'-azidomidine) -5'-phosphoric acid, so pl. 135 ^o C, R _f = 0.35 (acetic ether: methanol = 3: 1);
10. The sodium salt of (2-dodecyloxy) tetradecyl ether (3 '- deoxy-3'-azidothymidine) -5'-phosphoric acid, so pl. 83 ^o C, R _f = 0.35 (acetic ether: methanol = 3: 1);
11. The sodium salt of (3-tridecylmercanto-2-decyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid; mp> 190 ^° C with dec, R _f = 0.20 (ethyl acetate: methanol = 3: 1);
12. The sodium salt of (3-dodecyl mercanto-2-octyloxy) propyl ester (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid; mp> 200 ^o C with dec, R _f = 0.60 (methylene chloride: methanol: water = 65:25: 4) ^* ;
13. The sodium salt of (3-undecylmercanto-2-decyloxy) propyl ester of (3'-deoxy-3'-azidothymidine) -5'-phosphoric acid; mp> 200 ^° C with dec, R _f = 0.25 (ethyl acetate: methanol = 3: 1);
14. sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ester (3'-deoxy-thymidine) -5'-phosphoric acid, so pl. > 175 ^° C with dec, R _f = 0.45 (isopropanol: butyl acetate: water = 5: 3: 2);
15. sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ester of thymidine-5'-phosphoric acid, so pl. 251-254 ^o C with decomp., R _f = 0.45 (n-propanol: water = 9: 1);
16. sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ether (6-mercantopurin-9-β-D-ribofuranoside) -5'-phosphoric acid, mp> 200 ^° C decomp., R _f = 0, 45 (isopropanol: butyl acetate: conc. Ammonia: water = 50: 30: 5: 15);
17. sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ester (5-fluorouredine) -5'-phosphoric acid, mp> 210 ^° C decomp., R _f = 0.55 (methylene chloride: methanol: water = 65: 25: 4);
18. sodium salt of (3-Dedocylmercanto-2-decyloxy) propyl ester (2 ', 3'-dideoxy-cytidine) -5'-phosphoric acid, so pl. 202-205 ^o C decomp., R _f = 0.50 (isopropanol: butyl acetate: water = 5: 3: 2);
19. sodium, salt of (3-undecylmercanto-2-undecyloxy) propyl ester (3'-deoxy-3'-fluorothymidine) -5'-phosphoric acid, mp> 200 ^° C with decomposition, R _f = 0, 15 (dichloromethane: methanol = 8: 2);
20. The sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ester of 2 '- (9- {/ (1-hydroxymethyl) ethoxy / methyl} guanidine) -phosphoric acid, so pl. > 170 ^° C with dec, R _f = 0.22 (isopropanol: butyl acetate: water: conc. Ammonia = 50:30:15: 5);
21. sodium salt of (3-dodecylmercanto-2-decyloxy) propyl ester 2 '- (9 {/ (1-hydroxymethyl) ethoxy / methyl} guanine) -phosphoric acid, R _f = 0.73 (water: methanol 0.5 : 9.5 on RP-8), R _f = 0.30 (dichloromethane: methanol: water = 6.5: 2.5: 0.4 on silica gel;
22. sodium salt of (3-dodecyl mercapto-2-decyloxy) propyl ester 2 '- / 9- (ethoxymethyl) guanine / -phosphoric acid, R _f = 0.77 (water: methanol = 0.5: 9.5 per RP -8), R _f = 0.35 (dichloromethane: methanol: water = 6.5: 2.5: 0.4 on silica gel).

 The recipe.

 BM 21 1290 a (the compound of example 1a), finely ground, mixed in a turbo mixer with the specified in table. 2 with the amount of lactose and fill hard gelatin capsules with a size of 0.

The efficacy and tolerability of AZT and the compounds of Example 1a of this application (p. 1a) in a Friend virus (BF) model of leukemia
1) Materials and methods.

 Female mice aged 6-8 weeks were infected on day 0 with 0.2 ml of virus-containing spleen cell extract. Animals were injected daily from 0 day (start: 1 hour after infection) to 13 days 3'-deoxy-3'-azidothymidine (AZT) and (3-dodecyl-thio-2-decyloxy) -propyl ether (3'-deoxy- 3'-azidothymidine-5'-phosphoric acid (s.r. 1a) at the doses indicated in Tables 3 and 4. Before starting therapy and on day 13, body weight and blood count were determined, as well as on day 14 after killing - determined the weight of the spleen as a parameter of viremia.

 2) Results.

 Tab. 3 and 4 show the dose-dependent effect of AZT and c. pr. 1a for enlarged spleen caused by viruses (Table 3) or for restoration of virus-dependent suppression of saturation with bone marrow cells, respectively (Table 3).

 The data table. 3 and 4 indicate that the compound of example 1a is more effective than AZT.

 The following results represent the relationship between the activity and molecular structure of the claimed esters of nucleotide and lipid.

 These results were obtained with various combinations in two series on a model of leukemia called Friend virus in vitro. These results were compared with the results obtained for AZT or in control, respectively.

 1. In this document, test compounds are indicated by the internal number of the applicant. From the table. 5 it is clear which compounds these numbers belong to.

 2. Series 1: Relationship between the activity and molecular structure of the ester lipid (EL) nucleoside in a model of leukemia caused by Friend virus in vitro.

a) experimental design:
female BaIb / c mice (age 7–9 weeks, from Iffa Credo, Belgium) were infected on day 0 of the experiment with 0.2 ml of diluted 1:10 (FSB) VG and from +3 to +13 days daily were injected with them intraperitoneally substances listed in table. 6 (dose: 50 mg / kg / day) or FSB (phosphate buffered saline) (control I and II). On day 14, all animals were killed. The number of animals in each experiment was 10 (n = 10).

 b) Research.

 On day 13, i.e., when the therapy was completed, a small blood picture consisting of eight different hematological parameters was examined using standard techniques. After killing the animals on day +14, the weight of the spleen was determined.

 c) Results (see tab. 6).

 ca) In the table. Figure 4 shows the median values for spleen weight and platelet count. The weight of the spleen at +14 days after infection with Friend virus correlates with viremia and is a disease parameter that can be easily measured. According to the results of all studies, we can conclude that the number of platelets in the peripheral blood, which is also very easy to calculate, is also a parameter of viremia, which correlates with it and with the weight of the spleen, respectively.

cb) As can be seen from the table. 4, the claimed compounds containing AZT as a nucleoside are active against viremia. The results also indicate that there is a relationship between the activity and the molecular structure of the tested in vitro EL nucleoside conjugates. The following trends are determined (arrows indicate a decrease in activity):
cba) BM 21.1291 (C ₁₁ / C ₁₁ ) _ → BM 21.1290 (C ₁₂ / C ₁₀ ) BM 21.1388 (C ₁₀ / C ₁₂ )
cbb) BM 21.1290 (S) _ → BM 21.1380 (SO) _ → BM 21.1384 (SO ₂ )
coc) BM 21.1290 (AZT) _ → BM 21.1365 (ddI)
As shown by (cbc), the claimed compounds containing ddI as a nucleoside are less active than compounds containing AZT, however, the tested compound (BM 21.1365) is as active as AZT (spleen weight is greater, platelet count is greater), in while for this compound, toxic side effects on bone marrow observed for AZT are not expected.

 Comparison: the spleen weight of a healthy, uninfected and untreated BaIb / c mouse is normal 0.1–0.2 g. The platelet count is normally about 600–800 • 10 μl.

 3. Series 2: the effect of AZT, BM 21.1290 and some other EL-AZT conjugates (including R / S - BM 21.1290) on the course of Friend virus leukemia in BaIb / c mice (a partial repetition of previous experiments).

 a) Setting up experiments (see 2.a).

b) Measured parameters:
ba) body weight at 0 and +13 days,
bb) RT (reverse transcriptase) on days 0 and +14 (RT on day 0 only as a test, 1 animal from the group) (after killing the animals, the serum was separated individually and frozen at -70 ^° C to determine RT),
bc) small blood count at 0 and +13 days (blood picture at day 0 only for verification, 1 animal from the group),
bd) spleen weight at +14 day.

 c) The results are presented in table. 7-9.

 Each table provides the necessary explanations, including significant differences found between the tested groups.

Claims (5)

1. Phospholipid derivatives of nucleosides of the general formula I

where R ₁ linear or branched alkyl C ₈ - C _{1 5} ;
R ₂ linear or branched alkyl C ₈ - C _{1 5} ;
n 0, 1 or 2,
Group

is -2 ', 3'-dideoxyinosine, -2,3'-dideoxycytidine, -3'-deoxythymidine, -2', 3'-dideoxy-2 ', 3'-didehydro-N ⁶ - (O-methylbenzyl) - adenosine, -3'-deoxy-3'-fluorothymidine or -3'-deoxy-3'-azidothymidine,
or their tautomers, or physiologically acceptable salts of inorganic or organic acids or bases. 2. The compounds according to claim 1, where R ₁ C _{1 0} - C _{1 4} -alkyl. 3. Compounds according to claims 1 or 2, where R ₂ C ₈ is C _{1 2} -alkyl. 4. Phospholipid derivatives of nucleosides of the formula I according to claims 1 to 3, where the group

is a residue selected from -2 ', 3'-dideoxyinosine, -2', 3'-dideoxycytidine, -3'-deoxythymidine, -2 ', 3'-dideoxy-2', 3'-didehydro-N ⁶ - ( O-methylbenzyl) adenosine, -3'-deoxy-3'-fluorothymidine, 3'-deoxy-3'-azidothymidine.  5. A pharmaceutical composition inhibiting viral reverse transcriptase containing, as an active principle, phospholipid derivatives of nucleosides and a pharmaceutically acceptable carrier and adjuvant, characterized in that the compounds according to claim 1 are used as phospholipid derivatives of nucleosides in an effective amount.

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