NL8901258A - New 5-halo-2'-3'-di:deoxy-cytidine derivs. - useful as anti-retro-viral agents, esp. for treating HIV infections - Google Patents

Abstract

5-Halo-3'-azido -2',3'-dideroxycytidines (I), 5-halo-3'-fluoro-2',3' dideoxycytidines (II) and 5-halo-2',3' -didehydro-2',3'-dideoxycytidines (III) are new. The 5-chloro cpds. (Ia)-(IIIa) are specifically claimed. (Ia)-(IIIa) have ED50 values of 9, 14 and 15 micromolar respectively against the cytopathogenicity of HIV-1 in MT-4 cells. (Ia) and (IIa) have a better therapeutic index than 2',3'-dideoxycytidine. (Ia)-(IIIa) do not inhibit transformation of murine C3H/3T3 fibroblasts induced by Moloney murine sarcoma virus at concns. up to 1mM.

Description

5-Halogeno-2 ', 3'-dideoxycytidine derivatives in medicaments for the treatment of retrovirus infections.

The invention relates to new dideoxycytidine derivatives and their use in a therapeutic agent for treating retrovirus infections such as AIDS and AIDS-related diseases.

AIDS, or acquired immunodeficiency syndrome, is a pandemic immunosuppressive disease that results from depletion of helper T lymphocyte cells in the human body. The causative agent has been identified as a retrovirus and is referred to as "human immunodeficiency virus" or HIV. Currently, two different types (HIV-1 and HIV-2) of that retrovirus have been described; both types can induce AIDS or AIDS-related diseases, although HIV-1 is more widespread than HIV-2.

Many attempts to find suitable anti-HIV agents have already been made and many substances and compounds have been reported to inhibit the replication of HIV (usually type 1) in vitro. For review, see E. De Clercq, Anticancer Res., 1, 1023-1038 (1987).

Among the proposed anti-HIV compounds, 3'-azido-2 ', 3'-dideoxythymidine (azidothymidine or AZT) is currently the only compound that has been shown to be clinically useful in treating AIDS patients, compare Pischl et al, New Engl. J. Med. 317, 185-191 (1987).

In vitro tests, in addition to 3'-azido-21,31-dide-oxythymidine, also 3'-fluoro-2 '- dideoxythymidine, have been shown to be a potent inhibitor of HlV replication, compare Balzarini et al, Biochemical Pharmacology, 37, 2847-2856 (1988). Furthermore, a potent anti-HIV activity is found in 5-chloro-3'-azido and 5-chloro-3'-fluoro derivatives of 2 ', 3'-dideoxyuridine, compare Balzarini et al, Biochemical Pharm., 38, 869-894 (1989).

In the cytidine series, 2 ', 3'-dideoxycytidine has been shown to be a potent anti-HIV inhibitor, as is its 2', 3'-didehydro derivative, while its 3'-azido and 3'-fluoro derivatives are less potent , compare Balzarini et al, lc, 1988.

Further research has now found that 5-halo-3'-azido, 5-halo-3'-fluoro and 5-halo-2 ', 3 · didehydro derivatives of 2 *, 3'-dideoxycytidine have a potent and have selective anti-HIV activity comparable to that of 2 ', 3'-dideoxycytidine. This means that said compounds can be used advantageously in pharmaceutical preparations against AIDS and AIDS-related diseases, and generally in pharmaceutical preparations against retrovirus infections including hepatitis B.

The 5-halo-3'-azido, 5-halo-3'-fluoro and 5-halo-2 ', 3'-didehydro derivatives of 2', 3'-dideoxycytidine are new substances that can be taken by any usual route for nucleoside analogs can be synthesized. Preferably, a corresponding 2 ', 3'-dideoxyuridin derivative is first made, which is then converted into a 2', 3'-dideoxycytidine derivative by a known method. The halogen atom at the 5-position is preferably introduced only if the substituent at the 3 'position is already present.

It is noted that "halogeno" includes chloro, bromo, iodo and fluoro. Chloro is currently preferred.

Some examples are now given for the synthesis of the substances according to the invention.

Synthesis Example 1 5-chloro-31-azido-21,31-dideoxycytidine.

2.6 g (4.95 mmol) of 5, O-monomethoxytrityl-3, -azido-2 ', 3, -dide-oxyuridine were reacted with 1.0 g (7.5 mmol) of N-chlorosuccinimide in 100 ml of pyridine. Work-up and chromatographic purification gave 2.52 g (4.5 mmol, 91%) of a light brown foam, which was evaporated together with anhydrous pyridine and dissolved in 50 ml of dichloroethane pyridine (5: 1).

20 ml of a 10% solution of trifluoromethanesulfonic anhydride in 1,2-dichloroethane were added dropwise to the cooled solution (0 ° C) over 20 minutes. After three hours at room temperature, the starting material was completely converted according to TLC (CHCl3-MeOH 95: 5). The mixture was poured into 200 ml of ammonia saturated methanol. The solution was stirred overnight at room temperature, where, according to TLC, a new product was present in substantially equal amounts in addition to the starting material. After concentration, the residue was dissolved in ethyl acetate and washed with water and brine.

The organic layer was dried, evaporated to dryness and purified to yield 1.00 g (1.78 mmol, 40%) of recovered 5-chloro-S'-O-monomethoxytrityl-3'-azido-2 ', 3'-dideoxyuridine and 1, 26 g (2.25 mmol, 50%) of S-chloro-S'-O-monomethoxytrityl-S'-azido-2 ', 3'-dideoxycytidine as foam were obtained. UV (MeOH) Λ max288 nm. hit foam was treated with 100 ml of 80% acetic acid at 60 ° C for 30 minutes. After adsorption on silica gel, the mixture was purified (CHCl3 to CHCl3-MeOH 94: 6) to give 380 mg (1.32 mmol) of a light yellow foam which crystallized from MeOH diethyl ether. Yield 221 mg (0.77 mmol, 34%), mp: 173-175 ° C (dec).

UV (MeOH) λ 289 ftir. (€ = 7300), λ 264 nm. max min MS, m / z 286 (S, M), 146 (46, E- + 2H;, 145 (! 00, B + h), 142 (9, S), 110 (27.

B + H-Cl).

NMR (DMSO-d) <5: 2.34 (2, H-2 \ H-2 "), 3.67 (a, H-5 \ H-5"), 3.86 (n.

D

H-3 '), 4.37 (c, J - 5.5Hz, H-4'), 5.32 (br, 5 * -OH), 6.03 (t, .1 - 6Hz, Η — 1 *), 7.20 (br) and 7.83 (br) (NH4, 8.20 (s, H-6) ppm.

13 C NMR (DMSO-d) $: 37.5 (C-2 '), 59.5 (C-3f), 60.4 (C-5'>, 84.5 and 85.?

O

(C-Γ, C-4 *), 99.2 (0-5), 139.2 (C-6), 153.6 (C-2), 161.5 (C-4) ppm.

Anal. (CH ClNfiO 3) C, H.N.

Synthesis Example 2 5-chloro-3'-fluoro-2 ', 3'-dideoxycytidine 760 mg (2.47 mmol) 5-chloro-5'-O-acetyl-3'-fluoro-2', 3'-dide oxyuridine was dissolved in 24 ml of dichloroethane-pyridine (5: 1) and cooled in an ice-salt bath. 10 ml of a 10% solution of trifluoromethanesulfonic anhydride in dichloroethane was added dropwise over 10 minutes and the mixture was stirred at ambient temperature for 3 hours. TLC (CHCI3-MeOH 95: 5) shows that the starting material was completely converted. The contents were poured into 100 ml of ammonia-saturated methanol and stirred for 15 hours. Then, according to TLC (CHCl3-MeOH 9: 1), two nucleosidic products were present, the fastest moving product of which migrated together with the deacylated starting material. Flash chromatographic purification (CHCl3-MeOH 97: 3 to 9: 1) recovered 286 mg (1.08 mmol, 43%) S-chloro-S'-fluoro ^ ', 3'-dideoxyuridine and 600 mg crude brown foam obtained. After intensive purification, 148 mg (0.46 mmol, 22%) of the title compound was isolated as a white foam which crystallized from MeOH acetone. Mp: 179-180 ° C.

UV CMeOH '> na (ε * 67Cöj, λ 265 am.

max min MS * Jtfz 26a 115, M>, I4t * «3 $, E + 2H), 145 (10O. B + h), 119 (13, S), 210 (3G, B + H-Cl).

51X8 'DMS'i-d.' 4; i.?; '· 2, f.' · H-2 1, H-2 ", parclfilly pray b>, ó '3.63 (d. J * 3.5 Hz, H-5 *, H-s”), 4.20 v'lt, J = J.5 Hz, p = 27.3 Hz,> .2: (br, v'-CH), 5.29 <dd, 3 = 5.7 and 8.5 Hz, Hl *>, 7.26 (br; and 7.90 (brXM ^), 8.U (s, H-6) ppft.

NMR (PMSu-d ^ J 4; 35.3 (J = 20 Hz, C-2 '), 60.9 (J * 11 Hz, 5 "), 85.2 (j = 23.2 Hz, 33.5 (C-Γ), 95.0 (J 173.3 Hz, C-3, 99.2 (05), 139.1 (C-6), 153.5 (C-2), 161.4 (04) ppa.

Anal. (CHHCIFSjOj) C.H.N.

Synthesis Example 3 5-chloro-21,31-didehydro-21,31-dideoxycytidine.

1.46 g (5.46 mmol) 5'-O-propionyl-2 ', 3'-didehydro-2', 3'-dideoxyuridine was converted to 5'-0-propionyl-2 'in 81% yield. , 3'-didehydro-2 ', 3'-dideoxycytidine using triazole and 0-chlorophenyl dichlorophosphate, according to Sung's method of converting uridine to cytidine derivatives. Compare W.L. Sung, J. Org. Chem. 47, 3623-3628 (1982).

UV (MeOH) Xmax271 and 237 nm.

Reaction with benzoic anhydride in anhydrous pyridine gave the N-benzoylated product in 93% yield. UV (MeOH) -λjjigx 261 and 304 nm.

The protected nucleoside analog was treated with 1.5 equivalents of N-chloro-succinimide in pyridine at 100 ° C for 30 minutes. Intensive work-up provided 38% of the 5-chlorine product in addition to 21% recovered material.

UV (MeOH) Amax 261 and 331 nm.

Deprotection with methanol, saturated with ammonia, finally gave 62% of the title product which crystallized from methanol-diethyl ether. Mp: 144-145 ° C.

UV (MeOH) λ 288 rm * (ε · ^ 6 760), λ. 267 nm.

max min MS, m / z 24 3 (? »K;, Hi (97, B - K), MO (100, BrH-Cl), 58 (48, S-H).

NMR (DMSC-d) 6: 3.6) (d, J «2.6 Hz after exchatige vith 00, K-5 ', oi H-5r'), 4.81 Ca, H-4 ')> 5.C7 (t, J - 5Hs, 5'-OH), 5.91 (dm, = 5.7

Hz, K-3 *), 6.34 (dm.y - 5.7 Hz, H-2 '), 6.84 (in, Η-Γ), 7.21 (br) and? .Ó4 (' οτ) (ΝΗ?), S , l * (s, H-6) pp®.

13C NMfc (DMSO-d.) 6-61.9 (C-5 '), 87.4 (04'), 90.2 (C-1 '), 99.0 (C-5), fl 126.7 (05'), 134.4 (02 140.1 (06), 153.9 (02), 161.5 (C-4) ppm.

Anal. (C9Hi0C1N3O3) C.H.N.

In experiments leading to the invention, it was found that the compounds of Synthesis Examples 1, 2 and 3 are capable of inhibiting the cytopathogenicity of HIV-1 in MT-4 cells at a 50% effective dose (ED50) of resp. 9μΜ, 14μΜ and 15μΜ. They are nearly as active against the replication of HIV-2. Moreover, the compounds of Synthesis Examples 1 and 2 are hardly toxic to the MT-4 cells, so that they have a high selectivity index, as good or even better than that of 2 *, 3 ' -dideoxycytidine.

In investigating the ability to counteract the Moloney murine sarcoma virus (MSV) induced transformation of murine C3H / 3T3 fibroblasts, none of the derivatives of the synthesis examples showed any antiviral activity at concentrations up to 100 µM. This is in contrast to azidothymidine which is an effective inhibitor of the MSV (ED5Q = 0.027yM) and to 2 ', 3'-dideoxycytidine which has an ED50 of 10yM. Nevertheless, it is believed that said derivatives have sufficient antiviral activity against human retroviruses infecting human cells.

On the basis of this data, said compounds are useful in anti-AIDS and AIDS-related diseases and generally in anti-retroviral medicines including hepatitis B.

Therapeutic compositions containing the compounds of the invention as an active ingredient for treating retroviral infections such as AIDS or AIDS-related diseases in human practice may take the form of powders, suspensions, solutions, sprays, emulsions, ointments or creams used for topical administration for intranasal, rectal, vaginal and also for oral or parenteral (intravenous, intradermal, intramuscular, intrathecal, etc.) administration. Such formulations can be prepared by combining the active compounds (e.g. by mixing, dissolving, etc.) with pharmaceutically acceptable excipients of a neutral nature (such as aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents, additives) and further with color if desired. - and fragrances. The concentration of the active ingredient in the therapeutic composition can vary widely between 0.1% and 100% depending on the mode of administration. Furthermore, the dose of the active ingredient to be administered may vary between 0.1 mg and 100 mg per kilogram body weight.

The anti-HIV properties of the 5-halogeno-2 ', 3'-dideoxycytidine derivatives of the invention are documented by the following examples which should not be read in a limiting sense. Therein, 23'-dideoxycytidine and 3'azido-2 ', 3'-dideoxythymidine are used for comparison.

The viruses used in the examples were HIV-1 and HIV-2, respectively. obtained from the broth of persistently HIV-1 infected H9 cells and from the broth of persistently HIV-2 infected CEM cells. Furthermore, use was made of Moloney murine sarcoma virus (MSV) prepared from tumors elicited by infection in vivo from three day old NMRI mice (compare De Clercq et al., Proc. Soc. Exp. Biol. Med., 137, 590-594, 1971).

The cells used in the examples were MT-4 cells as described by I. Miyoshi et al, Gann Monogr. 28, 219-218 (1982). These cells were grown in a culture medium consisting of RPMI-1640 medium supplemented with 20 mM Hepes buffer, 10% (v / v) inactivated fetal calf serum and 2 mM glutamine. This RPMI-1640 medium is a standard medium containing inorganic salts such as NaCl, NaHCOs, Na2HPC> 4 etc., as well as glucose, various amino acids and various vitamins.

Five different substances were used as test compounds, namely:

AzddClCyd: 5-chloro-3'-azido-2 ', 3'-dideoxycytidine,

FddClCyd: 5-chloro-3'-fluoro-2 ', 3'-dideoxycytidine, D4C1C: 5-chloro-2', 3'-didehydro-2 ', 3'-dideoxycytidine, ddCyd: 2 *, 3'-dideoxycytidine ,

AzddThd: 3'-azido-2 ', 3'-dideoxythymidine.

Example 1

Inhibition of the cytopathogenic activity of HIV-1.

The test compounds were valued for their inhibitory effect on the cytopathogenic activity of HIV-1 in MT-4 cells.

In a first series of tests, MT-4 cells (5x105 cells / ml) were suspended in a fresh culture medium consisting of RPMI-1640 medium with, in addition to the aforementioned additions, 0.075% (w / v) NaHCO3, 2.5 µg / ml Fungizone ( Squibb NV). The suspension was infected with 200 CCID50 of HIV-1 per mm of cell suspension (1 CCID50 is the infectious dose for 50% of the cell culture). Immediately after infection, 100 μΐ aliquots of the cell suspension in the wells of a test plate were pooled with 100 μΐ aliquots of appropriate dilutions of the test compounds. Thus, each 200 µl well contained 20 CCID5Q of HIV and 5x10 4 MT-4 cells. After incubating for five days at 37 ° C in a humid atmosphere controlling the CC> 2 content, the number of viable cells was counted.

A second series of tests, parallel to the first, was performed on uninfected cell cultures incubated in the presence of different concentrations of the test compounds. Here too, the number of viable cells was counted afterwards. From the values found, the 50% effective dose (ED50) and the 50% cytotoxic dose {CD50) were calculated, i.e. the concentrations of test compound required to measure the number of viable cells in the virus-infected, respectively. reduce the non-infected cell cultures by 50%.

The results are shown in Table 1.

connection ED50 CD50

AzddClCyd 9 923

FddClCyd 14 1000 D4C1C 15 170 ddCyd 0.27 39

AzddThd 0.002 5.2

The table shows that the three compounds from Synthesis Examples 1-3 have a value for ED50 of respectively. Have 9yM, 14yM and 15yM. In addition, two of the three compounds have extremely low toxicity (high value of CD50) so that their therapeutic index will be the same as that of ddCyd or even better.

Experiments with HIV-2 showed a similar picture.

Example 2

Effect of various additives on the anti-HIV effect.

The experiments of Example 1 were repeated in the presence of various additives, after which the effect of these additives was determined.

In a first series of tests, MT-4 cells (106 cells per ml) were suspended in a fresh culture medium as mentioned in example 1. The suspension was infected with 200 CCID50 of HIV-1 per ml of cell suspension. Then, 50μ1 portions of the infected cell suspension in the wells of a test plate were combined with ΙΟΟμΙ portions of an appropriate dilution of a test compound and 50μ1 portions of a medium containing certain additives. After five days of incubation at 37 ° C in a humid atmosphere with control of the CO2 content, the number of viable cells was counted.

A second series of tests, parallel to the first, was performed on uninfected cell cultures, again counting the number of viable cells afterwards. From the values found, the values of ED50 and CD50 were calculated in the same manner as in Example 1.

The additives used were: dCyd 2'-deoxycytidine dThd 2'-deoxythymidine THU tetrahydrouridine dTHU 2'-deoxytetrahydrouridine

The results, along with those of Example 1, are reported in Table 2.

Table 2 shows that the addition of 1000 µM dCyd resulted in a marked decrease in the anti-Hlv activity of the compounds from Synthesis Examples 1-3. A similar decline also occurred with the addition of THU + dTHU and of dCyd + THU-dTHU.

In this regard, the compounds of the Synthesis Examples were similar to ddCyd or 2 ', 3'-dideoxy-cytidine. In contrast, the results are not comparable to those of azidothymidine, where an addition of dCyd or of dCyd + dThd results in a decrease in anti-HIV activity, but an addition of THU + dTHU, or of THU + dTHU + dCyd has no effect .

Furthermore, it appears that the additives significantly decrease the cytostatic activity of the compounds of Synthesis Examples 1 and 2, as well as at 21,3'-dideoxycytidine, while the compound of Synthesis Example 3 exhibits anomalous behavior here.

Example 3

Inhibition of Moloney murine sarcoma virus (MSV).

The test compounds were valued for their inhibitory effect on the transformation of C3H murine embryofibroblasts by Moloney murine sarcoma virus (MSV).

C3H cells were introduced into the wells of a 48-well test plate at a dose of 20000 cells per ml. After 24 hours, the cell cultures were infected with 80 focus-forming units of MSV and 120 minutes later, the culture medium was replaced with 1 ml of fresh medium containing varying concentrations of test compounds. After six days, transformation of the cell cultures was observed microscopically.

The tests showed that none of the compounds of Synthesis Examples 1-3 showed any antiviral activity at concentrations up to 1000 µM. In contrast, the transformation by AzddThd was effectively inhibited (ED 50 = 0.027 µM) while ddCyd exhibited an ED 50 of 10 µM.

Claims (14)

1. A compound selected from the group of 5-halo-3'-azido-2 ', 3'-dideoxycytidine, 5-halo-3'-fluoro-2', 3 '-dideoxycytidine and 5-halo-2', 3 "-didehydro-2", 3'-dideoxycytidine. 2.5-chloro-3'-azido-2 ', 3'-dideoxycytidine. 3.5-chloro-3'-fluoro-2 ', 3'-dideoxycytidine. 4.5-chloro-2 ', 3'-didehydro-2', 3'-dideoxycytidine. A pharmaceutical composition for use in the treatment of retrovirus infections, which composition comprises a compound from the group of 5-halogeno-31-azido-2 ', 3'-dideoxycytidine, 5-halogeno-3'-fluoro-2', 3 ' -dideoxycytidine and 5-halo-2 ', 3'-didehydro-2', 3'-dideoxycytidine as active ingredient. Pharmaceutical composition according to claim 5, which composition contains the active ingredient in a concentration between about 0.1 and about 100% by weight. Pharmaceutical preparation according to claim 5, which preparation is in the form of a powder, suspension, solution, spray, emulsion, ointment or cream. A pharmaceutical composition for use in treating AIDS or AIDS-related diseases, which composition comprises a compound from the group of 5-halogeno-31-azido-2 ', 3'-dideoxycytidine, 5-halogeno-3'-fluoro-2 'r3'-dideoxycytidine and 5-halo-2', 31-didehydro-21,3'-dideoxycytidine as active ingredient. A pharmaceutical composition according to claim 8, which composition contains the active ingredient in a concentration between about 0.1 and about 100% by weight. Pharmaceutical preparation according to claim 8, which preparation is in the form of a powder, suspension, solution, spray, emulsion, ointment or cream. A method of treating retroviral infections, which consists in obtaining a compound from the group of 5-halogeno-3'-azido-2 ', 3'-dideoxycytidine, 5-halo-geno-3'-fluoro-2' , 3'-dideoxycytidine and 5-halo-2 ', 3'-didehydro-2', 3 '-dideoxycytidine, administers to a patient suffering from a retroviral infection. A method of treating AIDS or an AIDS-related disease, which consists in comprising a compound from the group of 5-halogeno-3'-azido-2 ', 3'-dideoxycytidine, 5-halogeno-3 '-fluoro-2', 3'-dideoxycytidine and 5-halo-2 ', 3'-didehydro-2', 3'-dideoxycytidine administers to a patient suffering from AIDS or an AIDS related disease. 13. Use of a compound selected from the group of 5-halo-3'-azido-2 ', 3'-dideoxycytidine, 5-halo-geno-3'-fluoro-2', 3'-dideoxycytidine and 5-halo -2 ', 3'-didehydro-2', 3'-dideoxycytidine for the preparation of a pharmaceutical composition against retroviral infections and hepatitis B. Use of a compound selected from the group of 5-halo-3'-azido-2 ', 3'-dideoxycytidine, 5-halo-geno-3'-fluoro-2', 3'-dideoxycytidine and 5- halogeno-2 ', 3'-didehydro-2', 3'-dideoxycytidine for the manufacture of a pharmaceutical composition against AIDS and AIDS-related diseases.