WO1995018816A1

WO1995018816A1 - Novel nucleoside-lipid addition compounds, their production and their pharmaceutical use

Info

Publication number: WO1995018816A1
Application number: PCT/DE1995/000041
Authority: WO
Inventors: Hans Brachwitz; Peter Langen; Iduna Fichtner; Wolfgang E. Berdel; Matthias Baeseler; Uwe Lachmann; Karin Dressler; Yvonne Thomas
Original assignee: Max-Delbrück-Centrum für Molekulare Medizin
Priority date: 1994-01-07
Filing date: 1995-01-04
Publication date: 1995-07-13
Also published as: DE4400310A1; EP0738274A1

Abstract

The invention relates to novel nucleoside-lipid addition compounds of general formula (I) including all enantiomeric forms and all pharmaceutically usable salts of said compounds. The field of use is in the chemical and pharmaceutical industry. The compounds (I) with a diphosphate or phosphono-phosphate grouping are produced by condensing nucleoside-5'-phosphonophosphoamidates with the corresponding lipid phosphates or phosphonates or by condensing the lipid phosphoamidates or lipid phosphonoamidates with the corresponding 5'-nucleoside monophosphates. The compounds (I) containing a triphosphate or a phosphonodiphosphate group are obtained by condensing a nucleoside diphosphoamidate with an alkyl phosphate or alkyl phosphonate or by reacting a lipid phosphoamidate or lipid phosphonoamidate with a 5'-nucleoside phosphate or by condensing a lipid diphosphate or a lipid phosphonophosphate with a 5'-nucleoside monophosphoamidate or by reacting a lipid phosphoamidate or a lipid phosphonophosphoamidate with a 5'-nucleoside monophosphate. The compounds of formula (I) have a cytostatic effect.

Description

New nucleoside-lipid adducts, their production and their pharmaceutical use

The invention relates to new nucleoside-lipid adducts of the general formula I including all enantiomeric forms and all pharmaceutically usable salts of these compounds. The invention further relates to processes for the preparation of the compounds I and their pharmaceutical use. The field of application of the invention is thus the chemical and pharmaceutical industries.

The naturally occurring cytidine phosphate diacylglycerols (CDP-DAG), which are an important intermediate in cellular phosphatidylinositol synthesis, have no cytostatic activity. However, it is known that structure-modified CDP-DAG analogs inhibit the growth of tumor cells, but have little effect on the growth of homopoetic stem cells (Brachwitz et al., J. Cancer Res. (1 990) 1 1 6 (Suppl.ll), 993; Langen et al., Anticancer Res. J_2 (1 992) 2109). The aim of the invention was now to synthesize new cytostatically active nucleoside-lipid adducts which are introduced into the cell in analogy to the naturally occurring CDP-DAG and which, owing to their structure, have a high level of biostability and therefore a longer residence time in the organism and / or inhibit the proliferation of tumor cells by blocking natural pathways of degradation or synthesis of metabolically modified cleavage product and which are therefore suitable for pharmaceutical use.

The goal is solved according to the claims.

The invention relates to new nucleoside-lipid adducts of the general formula I

wherein

a) R is a straight-chain or branched, saturated or a mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted hydrocarbon radical with 1-22 carbon atoms means or

b) R CH - A CE A 1 1

CH - B CH t 1

CH ₂ ^- CH ₂ B

is what

A is saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano (C5-C30) alkoxy or alkenoxy,

B has one of the meanings given for A or denotes hydrogen, hydroxyl, halogen, cyano, acyloxy (C2-C22) or a group of the general formula 0- (CH2) _χ -CF3, where x is 0-3. c) Nk is a nucleoside, preferably cytidine, 2'-deoxycytidine, thymidine, adenosine and ara-cytidine residue, d) n is 0 or 1 and m is 1 or 2.

Preferred connections are:

Cytidine 5'-octylphosphonophosphate (Formula I: R = CgH ^, n = 0, m = 1, Nk = cytidine

Rest),

Cytidine 5'-dodecylphosphonophosphate (Formula I: = - 2 ^ 2 ^J ö ' ^{n =} ^' ^m = 1 ι Nk = Cytidin-

Rest),

Cytidine 5'-tetradecylphosphonophosphate (Formula I: R

n = 0, m = 1,

Nk = cytidine residue),

Cytidine 5'-hexadecylphosphonophosphate (Formula I: R = C- | gH33, n = 0, m = 1,

Nk = cytidine residue),

Cytidine 5'-octadecylphosphonophosphate (Formula I: R = C. | gH37, n = 0, m = 1,

Nk = cytidine residue

Cytidine 5 '- (3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R = C-] gH3 -0-

(CH ₂ > 3, n = 0, m = 1, Nk = cytidine residue),

Cytidine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R = C-j gH33-

0-CH ₂ -CHCI-CH ₂ , n = 0, m = 1, Nk = cytidine residue) _i Cytidine 5'-hexadecylphosphonodiphosphate (Formula I,

n = 0, m = 2, Nk = cytidine residue) _x

Cytidine 5'-hexadecyl triphosphate (Formula I, R = C | H33, n = 0, m = 2, Nk = cytidine residue), 2'-deoxycytidine 5'-dodecylphosphonophosphate (Formula I: = C- | 2H25. n = 0, m = 1, Nk = 2'-deoxycytidine residue),

2'-deoxycytidine-5'-hexadecylphosphonophosphate (Formula I:

n = 0, m = 1, Nk = 2-deoxycytidine residue),

2'-deoxycytidine-5'-dodecyldiphosphate (formula I: R = C-j2H25 n = 1, m = 1, Nk = 2'-deoxycytidine residue),

2'-deoxycytidine-5'-hexadecyldiphosphate (formula I: ^ = ^ - \ Q ^ 23 'n = 1, m = 1, Nk = 2'-deoxycytidine residue),

Thymidine-5'-dodecylphosphonophosphate (formula I: R = C | 2H25. N = 0, m = 1, Nk = thymidine residue),

Thymidine-5'-hexadecylphosphonophosphate (formula I: = C-ι H33, n = 0, m = 1, Nk = thymidine residue),

Thymidine-5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (formula I; R = C ₁₆ H ₃₃ -0-CH2-CHCI-CH2-, n = 0, m = 1, Nk = thymidine Residue), thymidine 5'-dodecyl diphosphate (formula I: R = Ci2 ^H 25 'n = 1, m = 1, Nk = thymidine), thymidine 5'-hexadecyl diphosphate (formula I:

n = 1, m = 1, Nk = thymidine residue),

Thymidine-5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate (formula I,: R = C ₁ H33-0-CH2_ CHCI-CH ₂ , n = 1, m = 1, NK = thymidine residue) ,

Adenosine 5'-dodecylphosphonophosphate (formula I: R = Ci2H25 'n = 0, m = 1, Nk = adenosine residue),

Adenosine 5'-hexadecylphosphonophosphate (formula I: R = C ₁ H3, n = 0, m = 1, Nk = adenosine residue),

Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R = C ₁₆ H33-0-CH2-CHCI-CH ₂ , n = 0, m = 1, Nk-adenosine Residue), adenosine 5'-dodecyl diphosphate (formula I: R = C ^ 2 * ^ 25 ' ^{n =} ^' ^{m =} 1. Nk = adenosine residue),

Adenosine 5'-hexadecyl diphosphate (formula I: R = C | gH 3, n = 1, m = 1, Nk = adenosine residue),

Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate (Formula I: R- = C ₁₆ H33-0-CH ₂ -CHCI-CH2, n = 1, m = 1, Nk-adenosine residue ), Ara-cytidine-5-hexadecylphosphonophosphate (formula I: = C- | gH33, n = 0, m = 1, Nk = ara-cytidine residue),

Ara-cytidine 5'-octadecylphosphonophosphate (Formula I: R

n = 0, m = 1, Nk = ara-cytidine residue), Ara-cytidine 5'-hexadecyl diphosphate (formula I: R = C _j gH3 * - ₇ , n = 1, m = 1, Nk = ara-cytidine residue),

Ara-cytidine 5'-octadecyl diphosphate (formula I: R = C ^ gH3, n = 1, m = 1, Nk = ara-cytidine residue),

The nucleoside-lipid adducts of the general formula I, in which a diphosphate (n = 1, m = 1) or a phosphonophosphate group (n = 0, m = 1) is present, are obtained either by condensation of the nucleoside monophosphoamidates with the corresponding alkyl phosphates or alkyl phosphonates. Another variant of the method consists in the implementation of an activated lipid phosphate or phosphonate component, e.g. B. in the form of an amidate with the corresponding nucleoside monophosphate. The nucleoside-lipid adducts of the formula I which have a triphosphate (n = 1, m = 2) or a phosphonodiphosphate grouping (n = 0, m = 2) can be correspondingly prepared by reacting nucleoside monophosphoamidates with alkyl diphosphates or Obtained alkyl phosphonates. These compounds can also be synthesized by condensation of the nucleoside diphosphoamidates with the corresponding alkyl phosphates or alkyl phosphonates.

Further synthesis variants consist in the reaction of a nucleoside diphosphate with an alkylphosphoamidate or alkylphosphonoamidate and in the reaction of a nucleoside monophosphate with an alkyldiphosphoamidate or alkylphosphonophosphoamidate. These reactions generally take place in a polar water-free solvent, preferably pyridine, at 20-50 ° C. The products are isolated and, if necessary, purified by chromatography.

The compounds of general formula I and their salts are biologically active and have a pronounced antitumor activity, which u. a. on human immoralized breast epithelial cells H 184 A 1 and on human breast tumor cells (Matu) in vitro. The selectivity of the antiproliferative effect is shown by a significantly lower inhibition of growth of bone marrow cells in vitro by the compounds according to the invention.

Tab. 1 shows the inhibition of cell growth of human breast epithelial cells H 184 1 and human breast tumor cells of selected compounds of the general formula I: Tab. 1 IC ₅₀ - selected (50% inhibition of cell growth in μM) selected substances of formula I.

Substance (Formula I, R, n, m, Nk =) H 184 A 1 ^a 'Matu ^b)

C ₁₄ H ₂₉ , 0, 1, cyt 30 55

^C 16 ^H 33 '° <- ¹ - cyt 15 31 C ₁₈ H ₃₇ , 0, 1, Cyt 23 31 C ₁₆ H ₃ 3θ (CH ₂ ) 3, 0, 1, Cyt 50 40 ^C 16 ^H 33 ^0CH 2 ^C HC1CH ₂ , 0, 1, Cyt 60 58 ^C 16H ₃ 3 '** - <2, Cyt 20 21 ^C 16 ^H 33' ° <- * -. ^τh y 52 22 C ₁₆ H330CH ₂ CHC1CH2, 0, 1, Thy 57 53 C ₁₆ H ₃ 3, 1, 1, Thy 44 58 C ₁₆ H330CH ₂ CHC1CH2, 1, 1, Thy 43 45

^C 16 ^H 33 <° <L ^Ad 18 21

^C 16 ^H 33 <! ' ¹ > ^Ad 42 33

^C 16 ^H 33 '°' ^x > ara-cyt 2.1 0.65

^C 16 ^H 33 * - * - '' ara-cyt 2.3 0.65

^a 'Immortalized human breast epithelial cells ^b ' Human breast tumor cells

Cyt = cytidine residue, Thy = thymidine residue, Ad = adenosine residue, ara-cyt = ara-cytidine residue

In the case of the compounds according to the invention, indications of a selective antitumor effect in vitro can also be found, H. the growth of normal cells in the comparable concentration range is inhibited far less than that of tumor cells, so that a distributed use in human medicine for the treatment and prophylaxis of tumor diseases is possible.

The compounds of general formula I and their salts can be used as cytostatic agents, preferably in a 3-5% mixture with pharmaceutically customary carriers and additives. ■

The invention will be explained in more detail below by means of exemplary embodiments.

Execution examples

example 1

Cytidine 5'-octylphosphonophosphate (Formula I: R = CoH ^, n = 0, m = 1, Nk = cytidine

Rest)

305 mg (1.57 mmol) octylphosphonic acid were treated with 1.5 g (2.1 mmol) cytidine 5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexyl carboxamidine salt) in 40 ml dry pyridine at 38 ° C 72 Hours stirred. The mixture was then evaporated to dryness in vacuo, codistilled three times with dry toluene and the residue was dried in vacuo, then taken up in CHC / methanol 1: 1 and the solution was adjusted to pH 3 by dropwise addition of 0.5 M HCl. The phases were separated and the aqueous phase extracted twice with CHCl3. After the combined CHCIg phases were washed again with a little water, drying was carried out over Na2SC-4. The solution was concentrated, the residue was dissolved in about 15 ml of CHCl3. After adding 30 ml of methanol / H 2: 1, the solution was adjusted to pH 9 with methanolic ammonia. After the phase separation, the CHCIg phase was extracted three times with 20 ml of methanol / H2O 2: 1. The combined aqueous phases were evaporated to dryness in vacuo and codistilled three times with toluene. The crude product was crystallized out by adding cold acetone. The resulting crude product contains small amounts of unreacted phosphonate. The further purification was carried out on a CM-52 cellulose column (50 g). It was washed with CHCI3 / CH3OH 8: 2 until no phosphate was detectable in the 1 50 ml fractions, which were concentrated to about 4 ml. Then the CH3OH content was increased to 50% and the clean substance was eluted. The eluate was evaporated to dryness and crystallized with acetone. 513 mg of clean substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, CHCI3 / CH3OH / H2O / CH3COOH = 50: 30: 8: 4) R _f = 0.37. Example 2

Cytidine 5'-dodecylphosphonophosphate (Formula I: R = C- _] 2 * ^ 25 'n = 0, m = 1, Nk = cytidine

Rest)

360 mg (1.45 mmol) of dodecylphosphonate were mixed with 1.32 g (1.9 mmol) of cytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) in 30 ml of dry pyridine with stirring at 35 ° C implemented in 72 hours. The batch was worked up and the crude product was purified according to Example 1 on a CM-52 cellulose column (25 g). 90 mg of pure product were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck finished plates, CHCl3 / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.29.

Example 3

Cytidine 5'-tetradecylphosphonophosphate (Formula I: R = C- _] 4H25, n = 0, m = 1,

Nk = cytidine residue)

500 mg (1.8 mmol) of tetradecylphonate were treated with 900 mg (1.3 mmol) of cytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) in 40 ml of dry pyridine at 35 ° C Stirring implemented in 5 days. The mixture was worked up and the crude substance was purified on a CM-52 cellulose column (25 g) as described in Example 1. 273 mg of clean substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck finished plates, CHCl3 / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.3.

Example 4

Cytidine-5'-hexadecylphosphonophosphate (Formula I: f ^ C- _j ßH, n = 0, m = 1,

Nk = cytidine residue)

326 mg (1.06 mmol) hexadecylphosphonate were mixed with 700 mg (1.01 mmol) cytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) in 30 ml dry pyridine at 30 ° C. with stirring 70 hours implemented. The mixture was worked up and the crude product was purified on a CM-52 cellulose column (25 g) as described in Example 1. 185 mg of clean substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, CHCI3 / CH3OH / H2O / CH3COOH 50: 30: 8: 4) R _f = 0.3. Example 5

Cytidine 5'-octadecylphosphonophosphate (Formula I: R - ⁼ Ci 8 '"* 37' n = 0, m = 1,

Nk = cytidine residue)

500 mg (1.5 mmol) of octadecylphosphonate were mixed with 1.2 g (1.72 mmol) of cytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) in 40 ml of dry pyridine at 38 ° C Stirring implemented in 4 days. The mixture was worked up and purified on a CM-52 cellulose column (25 g) in accordance with Example 1. 200 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, CHCI3 / CH3OH / H2O / CH3COOH = 50: 30: 8: 4) R _f = 0.37.

Example 6

Cytidine 5 '- (3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R - C-j ßH33-0-

(CH ₂ ) ₃ , n = 0, m = 1, Nk = cytidine residue)

According to Example 1, 799.2 mg (1, 15 mmol) of cytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) with 364 mg (1 mmol) of 3-hexadecyloxypropylphosphonic acid at 35 ° C implemented. The batch was worked up as described in Example 1. 1 87.5 mg of pure substance were obtained in the form of the disodium salt (dihydrate).

TLC (silica gel 60, Merck finished plates, CH ₃ CI / CH3θH / H ₂ 0 / CH ₃ COOH = 50: 30: 8: 4) R _f = 0.45.

Example 7

Cytidine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R = Ci ßH3 -

0-CH ₂ -CHCI-CH ₂ . n = 0, m = 1, Nk = cytidine residue)

According to Example 1, 793.2 mg (1, 15 mmol) of cytidine-5-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt) with 400 mg (1 mmol)

2-chloro-3-hexadecyloxypropyl-1-phosphonic acid reacted at 35 ° C. The batch was worked up as described in Example 1. 266 mg of pure substance were obtained in the form of the disodium salt (dihydrate).

TLC (silica gel 60, prefabricated Merck plates, CH3CI / CH3OH / H2O / CH3COOH 50: 30: 8: 4) R _f =

0.31. Example 8

Cytidine 5'-hexadecylphosphonodiphosphate (Formula I, R = C- _] gH33, n = 0, m = 2,

Nk = cytidine residue)

909.2 mg (1.15 mmol) of cytidine-5'-diphosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol of water) were dried three times with 30 ml of dry toluene on a rotary evaporator to dryness brought and then taken up with 25 ml of dry DMSO. After the addition of 306 mg (1 mmol) of hexadecylphosphonic acid, the mixture was stirred at 25 ° C. with the exclusion of moisture for 60 hours. The conversion was monitored by thin layer chromatography (silica gel 60, Merck finished plates, n-propanol / water / conc. NH3 60:20:10).

To isolate the product mixture from the DMSO solution, five times the molar amount of Nal was dissolved in acetone (25 ml) and this solution was added to the DMSO reaction solution with stirring. The precipitate obtained was dissolved in 20% methanol, placed on a column filled with RP 18 material and eluted with the same solvent to remove by-products. The cytidine hexadecylphosphonophosphate was desorbed by gradually increasing the methanol content of the eluent. 50 mg of pure substance in the form of the trisodium salt (dihydrate) were obtained.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / water / conc. NH3 60:20: 10) R _f = 0.4.

Example 9

Cytidine 5'-hexadecyl triphosphate (formula I, R = C ^ gH3, n = 0, m = 2, Nk = cytidine residue)

According to Example 8 from cytidine-5'-diphosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol water) and hexadecylphosphoric acid at 35-50 ° C. The substance is obtained as a disodium salt (dihydrate).

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NHg / water 60: 10:20) R _f = 0.4.

Example 10

2'-deoxycytidine-5'-dodecylphosphonophosphate (formula I: R = C- _| 2H25 n = 0, m = 1,

Nk = 2'-deoxycytidine residue)

A mixture of 250 mg (1 mmol) of dodecylphosphonate and 770.3 mg (1, 15 mmol) of 2'-deoxycytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexyl-carboxamidine salt with 1.5 mol Water) was vacuumed twice with absolute toluene evaporated, the residue dried over P2O5 was absolute in 40-50 ml. Dissolved pyridine and the solution stirred for 70 h at room temperature. The processing took place as in

Example 1 described. For the chromatographic purification of the substance a

CM-52 cellulose column used. 131 mg of product were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NHg / water 60: 10:20)

R _f = 0.53.

Example 1 1

2'-deoxycytidine-5'-hexadecylphosphonophosphate (formula I: R = C _j gH33, n = 0, m = 1,

Nk = 2-deoxycytidine residue)

As described above, 306.4 mg (1 mmol) of hexadecylphosphonate were mixed with 770.3 mg (1, 15 mmol) of 2'-deoxycytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1 , 5 mol water) implemented. The batch was worked up in accordance with Example 1. 140 mg of product were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NHg / water 66:10:20) R _f = 0.54.

Example 12

2'-deoxycytidine-5'-dodecyldiphosphate (formula I: R = C ^ 2H25- n = 1, m = 1, Nk = 2'-

Deoxycytidine residue)

287.6 mg (1.08 mmol dodecylphosphate were mixed with 832.7 mg (1.24 mmol) 2'-deoxycytdine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol Water) according to Example 1. The batch was worked up as described there, 207 mg in the form of the disodium salt (dihydrate) were obtained.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NHg / water 66: 10:20)

R _f = 0.53.

Example 13

2'-deoxycytidine-5'-hexadecyl diphosphate (formula I: R = Cι gH3 ₃ , n = 1, m = 1, Nk = 2'-

Deoxycytidine residue)

According to Example 10, 322.4 mg (1.0 mmol) of hexadecyl phosphate with 770.3 mg (1.5 mmol) of 2'-deoxycytidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'- dicyclohexylcarboxamidinsalz with 1, 5 mol water) implemented. The processing and

Cleaning was carried out according to Example 1. 210 mg of pure product in the form of

Obtained disodium salt.

TLC (silica gel 60 Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66: 10:20), R _f =

0.54.

Example 14

Thymidine-5'-dodecylphosphonophosphate (formula I: R = C ^ 2H25 'n = 0, m = 1,

Nk = thymidine residue)

225.7 mg (0.90 mmol) of dodecylphosphonate and 710.1 mg (1.04 mmol) of thymidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol of water) were prepared in accordance with the example 1 implemented. The crude product was cleaned using a preparative TLC plate (Kieselgel 60, Merck finished plates, F254). 80 mg of the substance were applied 1: 1 to the starting line in 1 - 2 ml CHCI3 / CH3OH. After development twice in CHCI3 / CH3OH / H2O / CH3COOH 50: 30: 8: 4, the UV-visible band was extracted with approx. 30 ml CHCI3 / CH3OH 2: 1. The residue obtained after evaporation was crystallized by treatment with acetone. 18 mg of pure substance in the form of the disodium salt were obtained from a TLC plate.

TLC (silica gel 60, Merck prefabricated plates, (n-propanol / conc. NHg / water 66: 10:20) R _f = 0.54.

Example 1 5

Thymidine-5'-hexadecylphosphonophosphate (formula I: = C- | gH 3, n = 0, m = 1,

Nk = thymidine residue)

According to the instructions in Example 1, 200 mg (0.65 mmol) of hexadecylphosphonate with 514 mg (0.75 mmol) of thymidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol of water) ) implemented. The substance was purified as described in Example 14. With 60 mg of substance applied, 14 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60 Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66: 10:20), R _f = 0.55. Example 16

Thymidine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I,

R = C _{1 6} H ₃ 3-0-CH2-CHCI-CH2-, n = 0, m = 1, Nk = thymidine residue)

According to Example 1, 200 mg (0.5 mmol) of 2-chloro-3-hexadecyloxypropyl-1-phosphonic acid with 400 mg (0.48 mmol) of thymidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 moles of water). The substance was isolated and purified as indicated in Example 1. 56 mg of pure substance in the form of the disodium salt (dihydrate) could be obtained. TLC (silica gel 60 Merck prefabricated plates, propanol / conc. NHg / water 66: 10:20) R _f = 0.56.

Example 17

Thymidine 5'-dodecyl diphosphate (Formula I: R = C. _| 2 ^ -25 'n = 1, m = 1, Nk = thymidine)

234 mg (0.88 mmol) dodecyl phosphate were reacted with 692 mg (1.01 mmol) thymidine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol water). The batch was worked up in accordance with Example 1. The substance was isolated and purified as described in Example 14. With 70 mg of substance applied, 22 mg of pure substance in the form of the disodium salt (monohydrate) could be obtained.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66:10:20) R _f = 0.54.

Example 1 8

Thymidine δ'-hexadecyl diphosphate (formula I: R - = C _| gH33, n = 1, m = 1, Nk = thymidine

Rest)

244 mg (0.508 mmol) hexadecyl phosphate were mixed with 400 mg (0.58 mmol) thymidine 5-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol water) in approx. 35 ml abs. Pyridine implemented according to the instructions in Example 1. The substance was isolated and purified by means of preparative TLC according to Example 14. With 70 mg of substance applied, 18 mg of pure substance in the form of the disodium salt (dihydrate) could be obtained.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66: 10:20) R _f = 0.55. Example 1 9

Thymidine-5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate (Formula I,: R = C- | gH33-0-CH2_

CHCI-CH ₂ , n = 1, m = 1, NK = thymidine residue)

According to Example 1, 288.4 mg (0.7 mmol) of 2-chloro-3-hexadecyloxypropyl-1-phosphate with 548 mg (0.8 mmol) of thymidine-5'-monophosphomorpholidate (as 4-

Morpholine-N, N'-dicyclohexylcarboxamidinsalz with 1, 5 mol water) reacted and worked up. The resulting substance is purified as described in Example 1.

1 23 mg of pure substance in the form of the disodium salt (dihydrate) could be obtained.

TLC (silica gel 60, Merck prefabricated plates, propanol / conc. NH ₃ / water 66: 10:20) R _f = 0.56.

Example 20

Adenosine 5'-dodecylphosphonophosphate (Formula I: ^ ⁼ ^ - \ 2 ^ 2 ' ^{n =} 0' nr. = 1,

Nk = adenosine residue)

A mixture of 250 mg (1 mmol) dodecylphosphonate and 81 6.3 mg (1, 15 mmol) adenosine-δ'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol water) was twice with abs. Rotated in toluene. The mixture obtained was dried in vacuo over P2O5, in 40-50 ml of absolute. Dissolved pyridine and stirred at room temperature. The reaction was followed by TLC (CHCl3 / CH3OH / H2O / - CH3COOH 50: 30: 8: 4). After 70 h, the pyridine was distilled off and the product was taken up in 1.5 ml of water, 30 ml of CH3OH and 25 ml of CHCl3 and adjusted to pH 4 with 1 N formic acid. The upper phase was extracted twice with chloroform. The organic phases were combined, dried, brought to pH 9 with methanolic NH3 and evaporated to dryness. 178 mg were obtained.

The substance obtained was purified by means of HPLC on an RP1 8 column; it is obtained as the free acid with 2.5 mol of water.

TLC (silica gel 60 Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66: 10:20) R _f = 0.52.

Example 21

Adenosine-5'-hexadecylphosphonophosphate (Formula I: R = C ^ gH33, n = 0, m = 1,

Nk = adenosine residue)

According to Example 20, 300 mg (0.98 mmol) of hexadecylphosphonate with 798.7 mg (1.13 mmol) of adenosine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclo- hexylcarboxamidine salt reacted with 1.5 mol water). 340 mg of crude product were obtained. The substance was isolated and purified as described in Example 20.

TLC (silica gel 60 Merck prefabricated plates, n-propanol / conc. NHg / water 66: 1 0:20)

R _f = 0.51.

Example 22

Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I:

^{R = C} 1 6 ^H 33 ^" ° ^{" CH} 2 ^{"CHCI" CH} 2 ' ^{n = 0} ' ^{m = 1} 'Nk adenosine residue)

According to Example 1, 200 mg (0.5 mmol) of 2-chloro-3-hexadecyloxypropyl-1-phosphonic acid 409.2 mg (0.58 mmol) of adenosine-5'-monophosphomorpholidate (as 4-morpholine-N, N'- dicyclohexylcarboxamidinsalz with 1, 5 mol water) implemented. The mixture was worked up and the end product was purified as indicated in Example 1. 94 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60 Merck prefabricated plates, CH ₃ CI / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.38.

Example 23

Adenosine 5'-dodecyl diphosphate (formula I: R = Cj ₂ H ₂ g, n = 1, m = 1, Nk = adenosine residue)

According to Example 20, 300 mg (1.3 mol) of dodecyl phosphate with 91 9.57 mg (1.3 mmol) of adenosine 5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol Water) implemented. 290 mg of crude product were obtained. The reaction mixture was worked up and the substance was purified as indicated in Example 20.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NHg / water 66: 10:20) R _f = 0.52.

Example 24

Adenosine 5'-hexadecyl diphosphate (formula I: R = C- | gH 3, n = 1, m = 1, Nk = adenosine residue)

In accordance with Example 20, 300 mg (0.92 mmol) of hexadecyl phosphate were reacted with 750 mg (1.07 mmol) of adenosine-5'-monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol of water) . There were 1 60 mg of crude product receive. The reaction mixture was worked up and the substance was purified as described in Example 20.

TLC (silica gel 60, Merck prefabricated plates, n-propanol / conc. NH ₃ / water 66:10:20) R _f = 0.51.

Example 25

Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate

(Formula I: R = C _{1 6} H33-0-CH ₂ -CHCI-CH2, n = 1, m = 1, Nk adenosine residue)

According to Example 1, 223 mg (0.54 mmol) of 2-chloro-3-hexadecyloxypropyl-1-phosphate with 438.8 mg (0.62 mmol) of adenosine 5'-monophosphomorpholidate (as 4-morpholine-N, N ' -dicyclohexylcarboxamidinsalz with 1, 5 mol water) implemented. The mixture was worked up and the end product was purified on a CM-52 cellulose column as shown in Example 1. 123 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck prefabricated plates, CH ₃ CI / CH3θH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.38.

Example 26

Ara-cytidine-5-hexadecylphosphonophosphate (Formula I: • ^P - ⁼ ^ - \ Q ^ 33 'n = 0, m = 1,

Nk = ara-cytidine residue)

A mixture of 310 mg (0.97 mmol) hexadecylphosphonate and 758 mg 1,09 mmol) ara-cytidine monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol water) was spun in several times with dry toluene P ₂ Og dried in a desiccator, dissolved in 40 ml of absolute pyridine and stirred for 16 days at room temperature. After the reaction had ended, the mixture was concentrated in vacuo and redistilled twice with toluene. The residue obtained was dissolved in 60 ml of CHCl3 / - CH3OH / H2O 2: 3: 1 and, after the addition of 6 ml of formic acid, stirred for 30 minutes at room temperature. The aqueous phase was then extracted twice with CHCl 3. The combined CHC ^ phases were dried over sodium sulfate and evaporated to dryness. The crude product obtained was purified by column chromatography on 25 g of CM-52 cellulose. To remove by-products, the elution was first carried out with CHCl 3, CHCl 4 / methanol mixtures 9: 1, 8: 2. Then the percentage of ethanol was gradually increased to a ratio of 1: 1. The pure substance obtained from these fractions is crystallized by treatment with acetone. 130 mg of pure substance were obtained in the form of the disodium salt. TLC (silica gel 60, Merck finished plates, CH ₃ CI / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.46.

Example 27

Macaw cytidine 5'-octadecylphosphonophosphate (Formula I: R = Cι gH3 ₇ , n = 0, m = 1,

Nk = ara-cytidine residue)

300 mg (0.95 mmol) of octadecylphosphonate and 750 mg (1.05 mmol) of ara-cytidine monophosphomorpholidate (as 4-morpholine-N, N'-dicyclohexylcarboxamidine salt with 1.5 mol of water) were reacted at 35 ° C. in accordance with Example 26. The reaction mixture was worked up and the product was isolated as shown in Example 26 using a CM-52 column. 130 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck finished plates, CH ₃ CI / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.45.

Example 28

Ara-cytidine 5'-hexadecyl diphosphate (formula I: R - = C- _| gH3 ** ₇ , n = 1, m = 1, Nk = ara-cytidine residue)

According to Example 26, 310 mg (0.97 mmol) of hexadecyl phosphate were reacted with 758 mg (1.1 mmol) of ara-cytidine monophosphomorpholidate at room temperature. The reaction mixture was worked up and the product was isolated as shown in Example 26 using a CM-52 column. 100 mg of pure substance were obtained in the form of the disodium salt.

TLC (silica gel 60, Merck finished plates, CH ₃ CI / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.46.

Example 29

Ara-cytidine 5'-octadecyl diphosphate (Formula I: = C- _| gH3 ₇ , n = 1, m = 1, Nk = ara-cytidine residue)

In accordance with Example 26 from 278 mg (0.97 mmol) octadecyl phosphate and 750 mg (1.05 mmol) ara-cytidine monophosphomorpholidate at room temperature. The reaction mixture was worked up and the product was isolated as shown in Example 26 using a CM-52 column. 178 mg of pure substance were obtained in the form of the disodium salt. TLC (silica gel 60, Merck finished plates, CH ₃ CI / CH ₃ OH / H ₂ 0 / CH ₃ COOH 50: 30: 8: 4) R _f = 0.46.

Claims

claims

1 . New nucleoside-lipid adducts of the general formula I, including all enantiomeric forms and all pharmaceutically acceptable salts of these compounds,

wherein

a) R is a straight-chain or branched, saturated or a mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted hydrocarbon radical having 1-22 carbon atoms, or

b) R CH - A CH - A

I I

CH - B or CH -

I l

is what

A saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano test C5-C3Q) - alkoxy or -alkenoxy

means

B has one of the meanings given for A or hydrogen, hydroxyl, halogen, cyano, acyloxy (C ₂ -C ₂₂ ) or a group of the general formula 0- (CH ₂ ) _χ CF ₃ , where x is 0-3, means

c) Nk is a nucleoside, preferably cytidine, 2'-deoxycytidine, thymidine, adenosine and ara-cytidine residue,

d) n is 0 or 1 and m is 1 or 2.

2. Cytidine-5'-alkylphosphonophosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 1 and Nk is a cytidine residue, and their salts.

3. Cytidine-5'-alkylphosphonodiphosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 2 and Nk is a cytidine residue, and their salts.

4. Cytidine-5'-alkyl triphosphates of the formula I, in which R has the meanings already mentioned, n = 1, m = 2 and Nk is a cytidine radical, and their salts

5. 2'-deoxycytidine-5'-alkylphosphonophosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 1 and Nk is a 2'-deoxycytidine radical, and their salts.

6. 2'-deoxycytidine-5'-alkyldiphosphates of the formula I, in which R has the meanings already mentioned, n = 1, m = 1 and Nk is a 2'-deoxycytidine radical, and their salts.

7. thymidine-5'-alkylphosphonophosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 1 and Nk is a thymidine radical, and their salts.

8. Thymidine-5'-alkyldiphosphates of the formula I, in which R has the meanings mentioned, n = 1, m = 1 and Nk is a thymidine radical, and their salts.

9. Adenosine-5'-alkylphosphonophosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 1 and Nk is an adenosine radical, and their salts.

10. Adenosine-5'-alkyldiphosphates of the formula I, in which R has the meanings mentioned, n = 1, m = 1 and Nk is an adenosine radical, and their salts.

1 1. Ara-cytidine-5'-alkylphosphonophosphates of the formula I, in which R has the meanings already mentioned, n = 0, m = 1 and Nk is an ara-cytidine radical, and their salts.

12. Ara-cytidine-5'-alkyldiphosphates of the formula I, in which R has the meanings already mentioned, n = 1, m = 1 and Nk is an ara-cytidine radical, and their salts.

13. Ara-cytidine-δ'-alkylphosphonodiphosphates, of formula I, in which R has the meanings already mentioned, n = 0, m = 2 and Nk is an ara-cytidine radical, and their salts.

14. Ara-cytidine-5'-alkyl triphosphates of the formula I, in which R has the meanings already mentioned, n = 1, m = 2 and Nk is an ara-cytidine radical, and their salts.

15. Cytidine 5'-octylphosphonophosphate (Formula I: R = CgH- _j , n = 0, m = 1, Nk = cytidine residue)

16. Cytidine 5'-dodecylphosphonophosphate (Formula I: R = C _j 2H25 'n = 0, m = 1, Nk = cytidine residue)

17. Cytidine 5'-tetradecylphosphonophosphate (Formula I: R = C ^ 4H25 _< n = 0, m = 1, Nk = cytidine residue)

18. Cytidine-5'-hexadecylphosphonophosphate (Formula I: R - * ⁼ C _| gH33, n = 0, m = 1, Nk = cytidine residue)

19. Cytidine 5'-octadecylphosphonophosphate (Formula I: R - = C- gH3 * 7, n = 0, m = 1, Nk = cytidine residue)

20. Cytidine 5 '- (3-hexadecyloxypropyl-1-phosphono) phosphate (formula I: = C- _| gH33-0- (CH ₂ ) ₃ , n = 0, m = 1, Nk = cytidine residue)

21. Cytidine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R ^{= C} 16 ^H 33 "° ^{" CH} 2 ^{"CHCI" CH} 2' ^{n = 0} ' ^{m = 1} ' Nk = cytidine residue)

22. cytidine-5'-hexadecylphosphonodiphosphate (formula I, = C | H33, n = 0, m = 2, Nk = cytidine residue)

23. Cytidine-5'-hexadecyl triphosphate (formula I, R =

n = 0, m = 2, Nk = cytidine residue)

24.2'-deoxycytidine-5'-dodecylphosphonophosphate (formula I: = C. | 2H25. N = 0, m = 1, Nk = 2'-deoxycytidine residue)

25.2'-deoxycytidine-5'-hexadecylphosphonophosphate (Formula I:

n = 0, m = 1, Nk = 2-deoxycytidine residue)

26.2'-deoxycytidine-5'-dodecyl diphosphate (formula I:

n = 1, m = 1, Nk = 2'-deoxycytidine residue)

27.2'-deoxycytidine-5'-hexadecyldiphosphate (formula I: R = C- | gH33, n = 1, m = 1, Nk = 2'-deoxycytidine residue)

28. Thymidine-5'-dodecylphosphonophosphate (Formula 1: R =

n = 0, m = 1, Nk = thymidine residue)

29. Thymidine-5'-hexadecylphosphonophosphate (Formula I: =

n = 0, m = 1, Nk = thymidine residue)

30. Thymidine-5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (formula I, R = C ₁₆ H ₃₃ -0-CH2-CHCI-CH2-, n = 0, m = 1, Nk = Thymidine residue)

31. thymidine-5'-dodecyl diphosphate (formula I: R = C | 2H25 'n = 1, m = 1, Nk = thymidine)

32. thymidine-5'-hexadecyldiphosphate (formula I: R = C-ιgH 3, n = 1, m = 1, Nk = thymidine residue)

33. Thymidine-5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate (Formula I: R

CH ₂ .CHCI-CH ₂ , n = 1, m = 1, NK = thymidine residue)

34. adenosine 5'-dodecylphosphonophosphate (formula I: = Ci2l ~ -25 ' ^{n =} 0 / m = 1, Nk = adenosine residue)

35. adenosine-5'-hexadecylphosphonophosphate (formula I: R = CιgH33, n = 0, m = 1, Nk = adenosine residue)

36. Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1-phosphono) phosphate (Formula I: R = C ₁₆ H33-0-CH2-CHCI-CH ₂ , n = 0, m = 1, Nk- Adenosine residue)

37. adenosine 5'-dodecyl diphosphate (formula I: R = C- * 2H25- n = 1, m = 1, Nk = adenosine residue)

38. adenosine 5'-hexadecyl diphosphate (formula I: R = Ci6 * "- 33 'n = 1, m = 1, Nk = adenosine residue)

39. Adenosine 5 '- (2-chloro-3-hexadecyloxypropyl-1) diphosphate (Formula I:

CH ₂ -CHCI-CH ₂ , n = 1, m = 1, Nk-adenosine residue)

40. Ara-cytidine-5-hexadecylphosphonophosphate (formula I: R = C _| gH33, n = 0, m = 1, Nk = ara-cytidine residue)

41. Ara cytidine 5'-octadecylphosphonophosphate (Formula I:

n = 0, m = 1, Nk = ara-cytidine residue)

42. Ara cytidine 5'-hexadecyl diphosphate (formula I: R = C ₁ gH3, n = 1, m = 1, Nk = ara cytidine residue)

43. Ara cytidine 5'-octadecyl diphosphate (formula I: = C ₁ gH3 * ₇ , n = 1, m = 1, Nk = ara cytidine residue)

44. Process for the preparation of the compounds of the general formula I, characterized in that a compound of the general formula II,

0 I)

(O) n P - OH II

OH in which R and n has the meaning given for the compound I, is condensed with a nucleoside 5'-phosphoamidate, preferably nucleoside 5'-monophosphomorpholidate or an amidate derived from the phosphate or phosphonate II with a nucleoside 5 ' -monophosphate is condensed.

45. A process for the preparation of compounds of the general formula I in which m = 2 and R, n and Nk have the meanings already mentioned, characterized in that a compound of the general formula II in which R and n are for formula I has the meanings mentioned, is reacted with a nuleoside 5'-diphosphoamidate or an amidate derived from the phosphate or phosphonate of the general formula II is reacted with a nucleoside 5'-diphosphate or a compound of the general formula III,

0 0 II II

R - (0) _n - P - O - P - OH III

1 I

OH OH

in which R and n have the meanings already mentioned, are reacted with a nucleoside 5'-monophospoamidate or an amidate derived from the diphosphate or phosphonophosphate of the general formula III is reacted with a nucleoside 5'-monophosphate.

46. cytostatic agent which contains one or more compounds of the general formula I as active ingredient.

47. Cytostatic, which contains 3-5% active ingredient of formula I in addition to the pharmaceutically customary carriers and additives.