HU205089B - Process for producing new thymine derivatives and pharmaceutical compositions comprising same - Google Patents

Abstract

The present invention relates to novel thimine derivatives of formula (I), the epimers and anomers thereof, wherein A represents a substituted 1-hexopyranosyl ring, which carries a substituent being not suitable for forming phosphate esters instead of at least one of the hydroxy groups being in position 2, 3 and 6, and at least one of the hydroxy group is protected with a protecting group. The compounds of formula (I) which are free of protecting groups, are useful against retrovirus (HIV) therefore they can be used for the treatment of AIDS.

Description

The present invention relates to novel thymine (5-methyluracil) derivatives, epimers and anomes, and to pharmaceutical compositions containing them as active ingredients. The novel compounds of the present invention are useful in the treatment of AIDS by their anti-retroviral (HIV) activity.

The novel thymine derivatives of the invention are represented by formula (I)

R ¹ and R ² is hydrogen,>

One of R ³ and R ⁴ is an azido group and the other is hydrogen,

One of R ^{s and} R ⁶ being one hydrogen atom and the other being a hydroxy group,

R ⁷ is methyl

The compounds of formula I according to the invention are prepared by reacting a compound of formula Ia in which

R ¹ , R ² , R ³ , R ⁴ and R ⁷ are as defined above, R ⁵ 'and R ⁶ ' are different, represent a hydrogen atom or an acyloxy protecting group, remove the acyloxy protecting group and optionally separate the epimers.

Compounds of formula (Ia) are prepared by reacting thymine with a compound of formula (II):

R ² , R ³ , R ⁴ and R ⁷ are as defined for formula (I),

R ³ 'and R ⁶ ' are different hydrogen or acyloxy protecting groups,

R is C 1-4 alkyl.

The spread of acquired immune deficiency disease (AIDS) is increasing worldwide, and researchers are stepping up efforts to search for pathogenic anti-retroviral (HIV) drugs.

It is known that retroviral DNA polymerase (reverse transcriptase) plays an exceptional and essential role in the life cycle of HIV and may also be a target for the fight against YMS (J. Med. Chem. 30, 862, 1987). It is also well known that the most effective drugs for treating DNA and RNA viral infections are the nucleosides. In the context of anti-AIDS studies, a long-known compound, 3'-azido-3'-deoxythymidine (AZT), marketed as Retrovir (Zidovudine) by Wellcome (J (Org. Chem. 29, 2076, 1964, J. Med. Chem., 21, 109, 1978).

The anti-AIDS effect of AZT is due to its ability to inhibit the formation of viral DNA through competitive inhibition or chain termination. AZT is first activated in the cell by phosphorylation in the form of AZT triphosphate, an analogue of thymidine triphosphate, one of the building blocks of DNA. During competitive inhibition, AZT triphosphate binds to the point of reverse transcriptase to which nucleoside triphosphates otherwise attach in physiological processes.

In the case of chain termination, the tear transcriptase "makes a mistake" and incorporates AZT triphosphate into the growing DNA chain instead of thymidine triphosphate. Since AZT contains an azido group at the C-3 'position unlike thymidine, which is no longer capable of forming a phosphate ester, the chain growth is terminated and the inactive form of the provir is formed.

In recent years, a number of modified analogs of thymidine have been produced in both the agonic and β-D-ribofuranosyl moieties. Since the dideoxynucleosides were particularly promising in their anti-hatás activity, the sugar moiety of thymidine, whose C-3 'hydroxyl group is not only azido but also nitrile (Tetrahedron Lett. 29,941 1988) and hydrogen (Proc. Natl. Acad. Sci. USA 83 1911 1986) and fluorine atom (Tetrahedron Lett. 44 625 1988). The logical sequence is terminated by the β-D-glycero-pentenofuranosyl derivative having C-2 'and C-3'. a double bond between their atoms (Biochem. Pharmacol. 36, 311, 1987).

The derivatives listed above, including AZT, are pyrimidine nucleosides, the sugar component of which is in the βD-pentofuranosyl ring form and none of the molecules at the C-3 'position is capable of forming the phosphate ester necessary for the growth of the viral DNA chain. However, of these, only high activity against ΗΓΥ was observed in AZT. Experience with invitro T cell culture has shown that AZT is effective against HIV at concentrations of 1-5 / μΜ and atoxic levels below 2050 / μΜ. However, the acyclic derivative obtained by opening the pyranoside ring is completely ineffective (J. Med. Chem. 32, 73-76 1989).

Therapeutic efficacy also depends on the ease with which the compound enters the cell and the degree of phosphorylation of the molecules. Studies have shown that phosphorylation of different derivatives is effected with different efficiencies. Presumably, the AZT C-3 'azo group plays a very important role in this regard. This is further supported by the fact that, for example, the 2 ', 3'-dideoxy analogue (which has a hydrogen atom at the C-3' position), which has a much less potent activity against HVV, has only a much smaller amount of phosphorylation required for activation.

In our studies, we have sought to produce types of molecules that are capable of binding to reverse transcriptase and can be incorporated into the viral DNA chain, while also providing sufficient phosphorylation to produce the activated form of the molecules.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel novel thymine derivative of formula (I) which is more potent, less toxic and therefore more useful than the molecules known to date.

Surprisingly, it has been found that, unlike the results obtained so far in nucleoside analogs, the anti-HIV activity is not exclusively linked to a nucleoside-type molecular structure containing a five-membered sugar moiety.

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Thus, the present invention is based on the discovery that by replacing the five-membered sugar moiety of thymidine with thermodynamically more stable six-membered monosaccharide units having a pyranosyl structure, we obtain polydioxy-pyranosyl ring molecules having the following formula (I): Its anti-HIV (HIV) activity is significantly better than the derivatives known so far, but their toxicity is lower.

Presumably, the polydeoxy nature of the six-membered sugar component plays a major role in the development of a more favorable antiviral activity, since the phosphate ester, which is essential for the construction of the viral DNA chain, is not required at all possible points of attachment.

The thimine derivatives of formula (Ia) may be prepared by the reaction of thymine with a hexopyranoside of formula (II).

The hexopyranosyl monosaccharide of formula (II) is preferably in the L-configuration or optionally in the D-configuration.

Of particular importance for the biological activity of thymine derivatives of formula I are the substituents R ³ and R ⁴ for azido and hydrogen, and the substituents R ⁵ and R ⁶ for hydroxyl and hydrogen. According to the formula (I), (Ia) and (II), wherein R ³ and R ⁴ and R ⁵ and R ⁶ may be assigned to axial or equatorial positions, the hexopyranosyl sugar moiety is a ribo-, xylo-, arabino- or indicates a lixo configuration.

For the preparation of the compounds of formula (Ia) according to the invention, it is preferred that the aglycone (thymine) and the glycoside of the formula (II) in which R ⁵ 'and R ⁶ ' are interchangeably acyl-preferably -nitrobenzoyloxy and hydrogen, in the presence of excess silylating agent, preferably hexamethyldisilazane and trimethylchlorosilane, at a temperature of 100-150 ° C until homogeneous.

Glycosylation can be carried out immediately after removal of the excess silylating agent in vacuo, in a suitable inert solvent, preferably dry acetonitrile, preferably using a promoter, preferably in the presence of trifluoromethanesulfonic acid trimethylsilyl ester, at full boiling point of the solvent.

A further advantage in carrying out the reaction is that the reaction mixture is maintained at 90-95 ° C in the presence of a powdered molecular sieve and in portions of the promoter agent.

Particularly advantageous for the recovery of the compound of formula (Ia) thus obtained is that one of R ⁵ 'or R ⁶ ' represents a p-nitrobenzoyloxy group, since the β-anomers of these products are crystallized in good yield, and can be filtered out of the α-glycoside remaining in the solution.

In a preferred embodiment of the invention, compounds of formula I can be prepared from compounds of formula Ia by reacting a compound of formula Ia wherein R ⁵ 'or R ⁶ ' is p-nitrobenzoyloxy wherein R ¹ , R ² , R ³ , R ⁴ and R ⁷ are as defined above, R ⁵ 'and R ⁶ ' are different, hydrogen or p-nitrobenzoyloxy protecting group on silica gel 60, benzene methanol, the β-anomer obtained in crystalline form is dissolved in anhydrous methyl alcohol and the C-4 'acyloxy group is removed in the presence of a catalytic amount of sodium methylate. The target product may be purified by column chromatography if necessary.

Optionally, it is easier to carry out the deoxygenated compound of formula (Ia) obtained after glycosylation wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ ', R ⁶ ' and R ⁷ 'are as defined above The C-4 'acyloxy group is saponified with anhydrous methanolic ammonia solution or sodium methylate and the resulting α- and β-anomeric mixture is separated by column chromatography as described above.

The removal of the acyloxy group is carried out at 20-25 ° C for 2-6 hours in both process variants and the pH of the reaction mixture is adjusted to pH 5-6 with dilute mineral acid, acetic acid or a hydrogen cation exchange resin. The solution was evaporated to dryness in vacuo. The residue was dried and purified by column chromatography as described above.

The present invention also provides pharmaceutical compositions comprising the compounds of formula (I) as the active ingredient, which may be formulated for oral or parenteral administration.

Oral formulations may contain 150-250 mg of the active ingredient in a single dose, homogenized with excipients and excipients commonly used in pharmaceutical formulations, and compressed into tablets or capsules.

An injectable preparation for intramuscular administration can be prepared by dissolving the active compound in known pharmaceutical diluents, preferably in the form of a 0.3-0.5% solution in sterile ampoules, due to the favorable water solubility of the compounds of the formula I and the appropriate stability of their aqueous solution.

For intravenous use, the active ingredient can be infused into any commonly used intravenous fluid (eg, saline, 5% dextrose solution).

Comparative studies with AZT show that 1- (3-azido-2,3,6-trideoxy-4-L-ribo-hexapyranosyl) -thymine at 25 μ a was infected with HTLV-IIIB (HIV) infected with H9 (continuous T4). lymphocyte line) inhibited the proliferation of HIV and the spread of infection in cell culture. Microscopic examination of the cultures (morphology, refraction) showed that the substance was not toxic at 100 μΜ.

The invention is illustrated by the following examples, without limiting our claims.

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Example 1 Isolation of 1- (3-azido-2,3,6-trideoxy-4-L-ribo-hexopyranosyl) thymine

0.3 mmol of thymine and 0.15 mmol of methyl 3-azido-4-O-pnitrobenzoyl-2,3,6-trideoxy-PL-ribo-hexopyran-5-oxide in 1.5 ml of hexamethyldisilazane and 0.15 ml of trimethylchloro silane was treated at 120-130 ° C for 3 hours while the reaction mixture became homogeneous. The reaction mixture was then evaporated to dryness by addition of anhydrous toluene. The residue was dissolved in 3 ml of anhydrous acetonitrile and reacted with 0.15 ml of trifluoromethanesulfonic acid trimethylsilyl ester at 90 ° C for 3 hours. A further 0.15 mL of trifluoromethanesulfonic acid trimethylsilyl ester was added and the reaction continued for a further 2 hours. By this time, the layer chromatography showed that the glycosylation reaction was virtually complete. The reaction mixture was then degassed by addition of benzene in vacuo and the residue evaporated on a silica gel 60 (30 x 3 cm) column with benzene / methanol (9:

1) Chromatography with a mixture of solvent (3-4 ml / 30 min per fraction). Fractions 24-32 were combined and concentrated in vacuo to give a crystalline product which was isolated by filtration.

Yield: 85%

Mp 166-169 ° C 25

TLC: _Rf = 0.54 (96: 4 chloroform-methanol)

Formula: CjgHjgNgOy

Molecular weight: 430.37

Analysis calculated found N 19.52 N = 19.34 IR (KBr): 2100 ^cm-1 (azido) 30

1600-1720 cm- ¹ (thyminecarbonyl and estercarbonyl)

1605 cnr ^l (C = C)

1522.1345 cm ^-1 (nitro group)

945 cm &^lt; ^-1 >

1 H NMR and COSY (200 MHz, MeOD) δ ppm:

8.41 to 8.23 (4H, m, ₂ phenyl PNO)

7.54 (1H, q, -CH =) 4 JCH, CH ₃ =

1.2 Hz 40

6.02 (1H, dd, H-1) 3 J, 2ax = 11.0 Hz

3J1, 2eq = 2.7 Hz

5.11 (1H, dd, H-4) 313.4 = 3.2 Hz 3 J4.5 = 9.8 Hz 45 4.54 (1H, ddd, H-3) 4.33 (1H, dq, H-5) 2.35 (LH, ddd, H 3Jax, 3 = 3.2 2AX) Hz 50

2Jax, 2eq = 13.9 Hz

2.12 (1H, ddd, H2eq)

1.90 (3H, d, thymine-55)

CH ₃ )

1.28 (3H, d, CH3-5 ') 3JMe, M-5 =

6.1 Hz

0.80 mmol of the previously isolated and characterized p-nitrobenzoyl derivative was dissolved in 50 ml of anhydrous 60 methanol, 1.5 ml of 0.1 N methanolic sodium methoxide solution was added and allowed to stand at room temperature for 2 hours. The solution was then adjusted to pH 6 with acetic acid, evaporated to dryness in vacuo, and the residue dried in a vacuum desiccator over potassium hydroxide and calcined calcium chloride. The white crystals thus obtained were washed with a little anhydrous ether and chromatographed on silica gel 60 columns with benzene / methanol (9: 1) as before.

Yield: 72%

M.p. 199-201 ° C [α] [] -24.5 ° (c = 0.51, methanol)

TLC: R _f = 0.20 (9: 1 Benzene-methanol) Water solubility: 5 mg / ml Molecular formula: C _U H ₁₅ N ₅ O ₄ .C ₆ H ₆ Molecular weight: 359.40 Elemental analysis:

Calculated for C, 56.80; H, 5.88; N, 19.49

Found: 54.82; 54.32 H Found: 5.73; 5.79 N Found: 19.79; 19.82

IR (KBr): 3400-3370 cm ^-1 (OH)

3320

2982

2930 ^cm-1 (NH) ^cm-1 (CH ₃₎ ^cm-1 (-CON)

1680-1610 cm ^-1 (C = O; C = C)

940 cm ' ¹ (tympanic vibration)

1 H NMR (200 MHz, MeOD) δ ppm: 7.48 (1H, q, CH =) 4 J H, CH ₃ = 1.2 Hz

5.86 (1H, dd, H-1) 3 J, 2ax -12.0 Hz

- 3J1 * 2eq = 2.5 Hz 4.12 (1H, ddd, H-3 313, 4 = 3.2 Hz 3J3.2eq = 3.0 3J3, 2ax - Hz 3.0 Hz 3.51 (1H, dd, H-4) 34.5 = 9.5 Hz 2.13 (1H, ddd, H- 2J2ax, 2eq = 12 2AX) Hz 1.97 (1H, ddd, H- 1.87 2eq) (3H, d, timin 1.27 CH ₃ ) (3H, d, CH ₃ -5 ')

Example 2 Isolation of 1- (3-azido-2,3,6-trideoxy-α- and -β-L-ribohexopyranosyl) thymine

The glycosylation reaction was carried out as described in Example 1, and the resulting crude product solution in benzene-methanol (9: 1) was filtered through a pad of silica gel (60 mL) without suction and then de-solvented in vacuo. The resulting α- and β-anomeric mixture was isolated together, the p-nitrobenzoyl group of which was removed in anhydrous methanolic medium as in Example 1 with sodium methylate. The saponified product was then chromatographed on a silica gel 60 (40 x 2 cm) column in benzene / methanol (9: 1) (1 mL / 30 min). The amount of α-anomer formed in Akis was removed from the column in fractions 77-105.

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Yield: 15% [α] 25 D + 21.80 ° (c = 0.83, MeOH)

The β-L-isomer was obtained by vacuum evaporation after fractionation of 119-137 fractions.

Yield: 65%

M.p .: 199-202 ° C [α] D 20 -21.6 ° (c = 0.50; MeOH)

TLC: _Rf = 0.27 (9: 1 benzene-MeOH)

Claims (5)

PATENT CLAIMS R ⁵ 'and R ⁶ ' as defined in claim 1 are hydrogen or p-nitrobenzoyloxy. A process for the preparation of novel thymine compounds of the formula I, their anomers and epimers, wherein R ¹ and R ² are hydrogen, One of R ^{3 and} R ⁴ is azido and the other is hydrogen, R ^s or R ⁶ are a hydrogen atom, the other is hydroxy, R ⁷ is a methyl group comprising reacting thymine with a compound of formula II wherein R ² , R ³ , R ⁴ and R ⁷ are as defined for formula I, R ⁵ 'and R ⁶ ' are different hydrogen or acyloxy protecting groups, R is C 1 -C 4 alkyl, followed by a compound of formula (Ia) thus obtained R 1, R ² , R ³ , R ⁴ and R ⁷ are as defined in the scope, R ⁵ 'and R ⁶ ' are different, represent a hydrogen atom or an acyloxy protecting group, the acyloxy protecting group is removed and, if desired, the epimers are separated. 2. A process according to claim 1, wherein R ¹ , R ¹ , R ² , R ³ , R ⁴ , R ⁷ are as defined above. 3. The process of claim 1 wherein the compound of formula II is methyl 3-azido-4-O-p-nitrobenzoyl-2,3,6-trideoxy-β-L-ribohexopyridanoside. 4. A process for the preparation of a pharmaceutical composition comprising as active ingredient a thymine derivative represented by the formula (I) according to claim 1, wherein R 1 to R ⁷ are as defined in claim 1, together with one or more pharmaceutically acceptable carriers and / or excipients. and mixed into a pharmaceutical composition. 5. A process according to claim 4 wherein the active ingredient is 1- (3-azido-2,3,6-trideoxy-β-L-ribohexopyranosyl) thymine.