DE4320570C2 - Process for the production of C-nucleosides and C-nucleoside analogs, new C-nucleosides and C-nucleoside analogues and their use - Google Patents

Description

The invention relates to a method for producing C- Nucleosides and C-nucleoside analogues, new C-nucleosides and C- Nucleoside analogues and their use.

The new C-nucleosides and C-nucleoside analogs have so far been able to cannot be synthesized.

6- (D-Ribofuranosylmethyl) pyrimidines are known from J. Org. Chem. 43, 2925 (1978), 47, 5115 (1982), 51, 1058 (1986), Liebigs Annalen 6, 957 (1986), J. Chem. Soc. Perkin I 201 (1983).

For the therapy of viral and cancer diseases is one Series of nucleosides have been synthesized and tested. Several of these are used today in medical practice, u. a. Acyclovir (Schaeffer et al, Nature 1978, 272, 583-85) and 3'- Azidothymidine (Broder et al, Proc. Natl. Acad. Sci., USA, 1985,82,7096). Others are used as therapeutic agents just before, u. a. for 5- (2-bromovinyl) -2'-deoxyuridine and 3'- Fluorothymidine.

A disadvantage of all of these connections is theirs Instability. The N-C bond between the heterocycle and the Sugar part is relatively easily hydrolytically or enzymatically ge cleave, resulting in ineffective degradation products (York, J .; J org. Chem. 1981 (46), 2171; Mansuri, M., J. Med. Chem. 1989 (32), 461). With acyclovir, the patient must e.g. B. 10 times Amount of the therapeutically necessary dose can be administered. With 5- (2-bromovinyl) -2'-deoxyuridine after 6 hours Body of the patient only about 1% of the amount used available. 3'-azido and 3'-fluorothymidine are highly effective, but too toxic for long-term treatment of a patient.

The 2 ', 3'-dideoxyadenosine derived from adenine occur moreover deamination and nucleoside cleavages what the use of the highly effective connection is crucial limited (Nucleosides & Nucleotides 8 (56), 659-71 (89) and  J. Med. Chem. 1988, 31, 2040-48). An attempt was made to replace it of the adenosine residue by inosine bring about an improvement because there is little conversion into adenosine in the body he follows. But it was also a decisive breakthrough unavailable.

The invention aims to be therapeutic, in particular Virostatic and cancerostatic compounds for ver to provide, which one with the known substances comparable effectiveness and high stability. she are said to be potential therapeutic agents with low stress for be suitable for the patient. Another object of the invention consists of building blocks for novel oligonucleotides provide.

The aim of the invention is achieved with the new C-nucleosides of the general formula I according to claims 4 to 11.

These compounds are significantly more stable than their analogs without -C bridge between nucleobase and sugar residue. Since the Substituent on the methyl group at C6 of the pyrimidine ring can be varied as desired, the invention large number of compounds I provided that a selective inhibition of enzymes which are responsible for the Virus multiplication is significant. Are particularly stable the compounds I according to the invention 11th

The compounds I are virostatic and / or cancerostatic. Their effectiveness has been tested on bone marrow stem cells. The compounds show an inhibition in the bone marrow stem cell proliferation test that is comparable to the known substances and in some cases even higher. It lies z. B. 6- (β-D-2 ', 3'-didehydro-2', 3'-didesoxyribofuranos-1-yl) methyl-4-aminopyrimidine under 10 ^-4 molar.

Another very important application of the  Compounds I according to the invention consists in their use as Building blocks for synthetic oligonucleotides. Under synthetic Oligonucleotides become RNA and DNA oligonucleotides, including especially ribozymes (also ribozymes from DNA and RNA building blocks) and antisense oligonucleotides. Preferably be the compounds mentioned in claim 4 are used for this.

Such connections are produced according to the known Patterns, some typical ways are shown below using the example of Thymidins are described. To build DNA Oligonucleotides is first thymidine to 5'-dimethoxy-trityl Derivative tritylated, then implemented via Cyanoethyl (diisopropyl) phosphordiamidit. The resulting 5'-Di methoxy-trityl-3'-cyanoethyl (diisopropyl) phosphamidite is added closing in a manner known per se in the oligonucleotide Synthesizer implemented further.

To build RNA oligonucleotides after tritylation protection of the 2'-OH group z. B. done by the silyl radical. The construction of ribozymes and antisense oligonucleotides takes place in basically the same way.

One possibility lies in the possibility of building up novel ribozymes great advantage of the invention. A problem of the previously known Ribozymes are that they are not sufficiently stable. you has tried to increase stability, u. a.

• - by substitution of the 2'-OH group, e.g. B. by F or by NH ₂ ,
• - by replacing the phosphate residues with thiophosphates and
• - by replacing the phosphate residues with phosphonates.

In no case, however, was the stability of the ribozymes for the intended uses are sufficient.

With the compounds according to the invention, the ribozyme structure in a new way depending on the intended Intended purpose. Thus, a suitable substitution on  C5 of the pyrimidine ring of compounds I the polarity influence differently (e.g. a 5-fluorine substitution to lower the pK value, which is a higher Acidity means).

Compounds which are particularly suitable for the construction of the ribozyme are 6- (β-D-ribofuranos-1-yl) methyl-4-aminopyrimidine and 6- (β-D-ribofuranos-1-yl) methyl-2-amino-4-hydroxy-pyrimidine and their derivatives substituted in the 2'-position by F, NH ₂ , H or ara-OH. These compounds can be used as phosphate esters, thiophosphate esters or as phosphonates.

Are special for building antisense oligonucleotides suitable 6- (β-D-2'-deoxyribofuranos-1-yl) methyl-4-aminopyrimidine and 6- (β-D-2'-Desoxyribofuranos-1-yl) methyl-2-amino-4-hydroxy- pyrimidine and its ribose analogues.

These compounds can also be used as phosphate esters, Thiophosphate ester or be used as phosphonates. The The novel antisense oligonucleotides produced show a increased specific activity and increased binding ability on the desired carrier, which results in improved applications opportunities.

The compounds I are prepared according to claim 1 by reacting an esterified or etherified 1-halogenose of the general formula II with C-metalated pyrimidines or their mono-, di- or trimethylsilyloxy ether or alkyl ether of the general formula III, the latter optionally in 2 - and / or 4-position NH ₂ groups are protected, preferably by a dimethylaminomethylene group, at temperatures from -70 ° C to + 100 ° C, preferably at -70 ° C to -50 ° C.

The detailed procedure is as follows.

The Halogenose II is in an inert solvent such as Alka dissolved or aromatics, cooled to -70 ° C to 0 ° C and  added dropwise with the C-metalated pyrimidine III. These metal compounds III are by implementing the corresponding persilylated or otherwise etherified pyrimidine Derivatives with organometallic compounds such. B. Butyllithium or sodium amide. Then that will Reaction mixture preferably brought to room temperature and if necessary, heated briefly to complete the implementation. After completion of the implementation, recognizable by unusual Metal salt is obtained after the usual chemical operations the desired product.

Another variant of the implementation according to the invention is preferred when using etherified halogenoses, is that one the halogenose II in an inert solvent and the C-metallated pyrimidine III is added with constant stirring. Of is essential for the success of the invention absolute freedom from water.

The invention is intended to be explained in more detail below by means of exemplary embodiments are explained.

example 1 6- (β-D-2'-Desoxyribofuranos-1-yl) methyl-uracil

1.42 g (5 mmol) of 2,4-bis (trimethylsiloxy) -6-methyl-pyrimidine are abs. In 10 ml. Dissolved hexane, cooled to -70 ° C and added dropwise with 4 ml of 1.5 molar butyllithium solution in hexane under argon. The solution is allowed to warm to 0 ° C. and is added dropwise to a solution of 1.95 g (5 mmol) of 2-deoxy- (3.5-) which is initially cooled to -70 ° C. and later warms to 0 ° C. di-toluyl) -ribosyl chloride in 20 ml abs. Toluene. If the reaction solution is neutral, it is evaporated in a rotary evaporator and deacylated with sodium methylate and separated on silica gel (Merck 60) with chloroform-methanol (9/1) into α, β anomers.
F: 125 °, M = 242.2 = C ₁₀ H ₁₄ N ₂ O ₅ according to MS Varian CH ₇ 300 MHz
NMR: H '= 6.4146 ppm = β.

The β-anomer can be converted into cytosine,  Isocytosine and. a. Derivatives are implemented that the Purine derivatives adenine, inosine, xanthine, guanine and the like. a. correspond.

Example 2 6- (β-D-2-Desoxyribofuranos-1-yl) methyl-4-hydroxy-pyrimidine

5.5 g (~ 30 mmol) of 4- (trimethylsilyoxy) -6-methyl-pyrimidine (TK 196 ° C) in 30 ml abs. Dissolved toluene and added dropwise at 0 ° C with 20 ml of 1.6 M butyllithium in hexane (protective gas). The lithium salt solution is at 0 ° C in a stirred solution of 10 g (~ 30 mmol) of 2-deoxy- (3,5-di-toluyl) - ribosyl chloride in 100 ml abs. Toluene dropped. The reaction is then briefly warmed to complete the reaction. The toluene solution is extracted with cold water, the organic phase is dried (MgSO 4), the solvent is evaporated and the residue is deacylated with 100 ml of 1/20 m NaOCH solution. The solution is then neutralized with Dowex H + ion exchanger, the glycoside and unreacted pyrimidine are subsequently detached from the ion exchanger and separated into a and β anomers via a silica gel column.
Syrup, M = 226.23 = C ₁₀ H ₁₄ N ₂ O ₄ mass peak CH ₇ (Varian)

Example 3 6- (β-D-2 ', 3'-didehydro-2', 3'-didesoxyribofuranos-1-yl) methyl-4- hydroxy-pyrimidine

226 mg (1 mmol) 6- (β-D-2-deoxyribofuranos-1-yl) methyl-4-hydroxy pyrimidine are abs in a known manner in 20 ml. Pyridine with 300 mg trityl chloride and after a further 20 hours at 0 ° C with 0.25 ml Mesyl chloride added. After hydrolysis of the excess Chlorides in water ice, the residue in chloroform taken up, successively in cold dilute sulfuric acid, water and extracted bicarbonate solution.

The chloroform solution is spun in, the residue in Acetic anhydride warmed until the pyrimidine ring was completely acylated is. A partial detritylation disturbs the subsequent one Elimination not. The one obtained after spinning in again  The residue is taken up in 5 ml of DMF and with 5 ml of ethyl boiled diisopropylamine under reflux until the Starting compound no longer in the thin layer chromatogram is detectable. After rotating in again, the Protective groups removed in a known manner and the remaining Residue on silica gel 60 (Merck) with chloroform / methanol 9/1 Cut.

The title compound is characterized by that typical for ene compounds Bromine discoloration demonstrated.

Example 4 6- (β-D-2 ', 3'-didehydro-2', 3'-didesoxyribofuranos-1-yl) methyl-4- amino-pyrimidine

50 mg of the compound prepared in Example 3 are in 2 ml Hexamethyldisilazane and 1 drop of DMF slowly warmed until the Substance goes into solution. Excess hexamethyldisilazane removed in vacuo, and the residue is mixed with 2 ml liquefied ammonia and catalytic amounts of ammonium chloride in the Bomb tube heated at 160 ° C overnight. After evaporating the Ammoniaks the residue is taken up in methanol and on Silica gel 60 (Merck) separated with chloroform / methanol 9/1.

Example 5 6- (hydroxyethyloxyethyl) -2,4-dimethoxypyrimidine

74 mg of 1,3-dioxolane are dissolved in 1 ml of absolute hexane and a solution of 0.13 ml (1 mmol) of trimethylbromosilane in 1 ml of absolute hexane is added. After 24 hours at 20 ° C., the dioxolane is split to bromomethoxy-ethoxy-trimethylsilane, so that it is at -70 ° C. to a solution of 154 mg (1 mmol) of 2,4-dimethoxy-6-methylpyrimidine in 10 ml of THF and 0 , 8 ml of a 1.5 m Bu-Li solution is added dropwise. After a reaction time of 6 hours at -70 ° C and subsequent heating to 20 ° C, the reaction mixture is concentrated and, if necessary, chromatographed on silica gel 60 with chloroform. Yield. 60-80% of a colorless oil,
MS: 229.234 = M + 1 corresponds to C ₁₀ H ₁₆ N ₂ O ₄
UV: (pH 7) max 258 nm
NMR: 5H 6.40; 2,4-dimethoxy 4.00; 2'H 3.7; 5'H 4.3 ppm

Example 6 6- (5-hydroxypentyl) -2,4-dimethoxypyrimidine

1 ml of absolute THF is mixed with 0.13 ml (1 mmol) of trimethylbromosilane and after 24 hours at 20 ° C. the THF is cleaved to 1-bromo-5- (trimethylsilyloxy) butane. This solution is added dropwise at -70 ° C. to a solution of 154 mg (1 mmol) of 2,4-dimethoxy-6-methylpirimidine in 10 ml of THF and 0.8 ml of a 1.5 m Bu-Li solution. After a reaction time of 10 hours at -70 ° C., the temperature is brought to 20 ° C. and the mixture is concentrated. Yield: 70% colorless oil,
MS: 226.26 = M = C ₁₁ H ₁₈ N ₂ O ₃
UV: (pH 7) max 258 nm
NMR: 5H 6.19; 2,4-dimethoxy 3.97; 3.93 5'H 3.64; 1'H 2.59 ppm

Claims (19)

1. Process for the preparation of C-nucleosides and C-nucleoside analogs of the general formula I
in which
R ₁ = H, Hal, OH, SH, NH ₂ , N ₃ , CN,
R ₂ = H, Hal, OH, SH, NH ₂ , N ₃ , CN, NH-NH ₂
R ₃ = H, Hal, OH, NH ₂ , N ₃ , alkyl, aryl, NO, NO ₂ NH-NH ₂ , NH-aryl, NH-alkyl, CH ₂ -Hal, vinyl, bromovinyl,
R ₄ = H, Hal, OH, alkyl, NH ₂ , NH alkyl, NH aryl, CN,
R ₅ = H, Hal, OH, alkyl, NH ₂ , NH alkyl, NH aryl, CN,
R ₄ and R ₅ together = O, S, N-aryl, N-alkyl, N-NH-aryl, N-NH-alkyl,
R ₆ =
or an alkyl, aryl, aralkyl, allyl, vinyl, alkoxyalkyl, aroxyalkyl, acyloxyalkyl, hydroxyalkyl, thioalkyl, aminoalkyl radical
and
R ₇ = H, Hal, OH,
R ₈ = H, Hal, OH,
R ₉ = H, Hal, OH, N ₃
where if R ₇ = OH, R ₈ cannot be OH and vice versa and
R ₁₀ = OH, PO ₄ , CH ₂ -PO ₃ , or salts,
characterized in that an esterified or etherified 1-halogenose of the general formula II
where R ₁₁ = H, O-acyl, O-alkyl, O-aryl, O-silyl,
or a compound from the group consisting of alkyl hal, aryl hal, aralkyl hal, allyl hal, vinyl hal, alkoxyalkyl hal, aroxyalkyl hal, acyloxyalkyl hal, acylthioalkyl hal, acylaminoalkyl hal, trialkylsilyl hal
with C-metalated pyrimidines or their mono- or bis-, trimethylsilyloxy ethers or alkyl ethers of the general formula III, the NH ₂ groups which are optionally present in the 2- and / or 4-position are protected, preferably by a dimethyl aminomethylene group,
in which R ₁ to R _{5 has} the abovementioned meaning and
Me = Li, Na is,
implemented at temperatures from -70 ° C to + 100 ° C, preferably at -70 ° C to -50 ° C, and subsequently, if necessary, further implemented with operations customary in nucleoside chemistry. 2. The method according to claim 1, characterized in that the halogenose II at -70 ° C to 0 ° C in an inert solvent, preferably in alkanes, Ethers or aromatics, dissolved and added dropwise with the C-metallized Pyrimidine III is added in the same solvent, the reaction mixture subsequently brought to room temperature and optionally to Completion of the implementation is warmed up briefly. 3. The method according to claim 1 and 2, characterized in that the corresponding persilylated pyrimidine derivatives with organometallic Reacts compounds such as butyllithium or sodium amide and the formed Reacts compound III in the reaction mixture directly with the halogenose II. 4. 6-methylene substituted nucleosides of
4-hydroxypyrimidine,
4-aminopyrimidine,
2-amino-4-hydroxypyrimidine
and the
2,4-diaminopyrimidine
and their 5-halogen and 5-amino derivatives with the following carbohydrate components:
2 ', 3'-dideoxy-ribose
3'-azido-2 ', 3'-dideoxy-ribose
3'-fluoro-2 ', 3'-dideoxy-ribose
2'-fluoro-ara-2 ', 3'-dideoxy-ribose
2 ', 3'-didehydro-2', 3'-dideoxy-ribose. 5. 6- (β-D-2 ', 3'-dideoxyribofuranos-1'-yl) methyl-4-hydroxy pyrimidine. 6. 6- (β-D-2 ', 3'-dideoxyribofuranos-1'-yl) methyl-4-amino pyrimidine. 7. 6- (β-D-2 ', 3'-didehydro-2', 3'-dideoxy-ribofuranos-1'-yl) methyl-4-hydroxypyrimidine.   8. 6- (β-D-2 ', 3'-didehydro-2', 3'-dideoxy-ribofuranos-1'-yl) methyl-4-aminopyrimidine. 9. 6- (β-D-2 ', 3'-dideoxy-3'-fluoro-ribofuranos-1'-yl) methyl- 4-hydroxypyrimidine. 10. 6- (β-D-2 ', 3'-dideoxy-3'-fluoro-ribofuranos-1'-yl) methyl 4-aminopyrimidine. 11. 6- (β-D-2 ', 3'-dideoxy-3'-azido-ribofuranos-1'-yl) methyl 4-hydroxypyrimidine. 12. Use of the compounds according to claim 4 as a virostatic. 13. Use of the compounds according to claim 4 as Cancer therapeutic. 14. Use of the compounds according to claim 4 as building blocks for synthetic oligonucleotides. 15. Use according to claim 14 as building blocks for synthetic RNA oligonucleotides. 16. Use according to claim 14 as building blocks for synthetic DNA nucleotides. 17. Use according to claim 14 and 15 for the synthesis of novel ribozymes. 18. Use according to claim 14 and 15 for the synthesis of novel antisense oligonucleotides. 19. Use according to claim 14 and 16 for the synthesis of novel antisense oligonucleotides.