The Synthesis and Immunotropic Activity of a New ... - Springer Link

Pharmaceutical Chemistry Journal

Vol. 34, No. 11, 2000

SEARCH FOR NEW DRUGS THE SYNTHESIS AND IMMUNOTROPIC ACTIVITY OF A NEW AZATHIOPRINE ANALOG – 2-AMINO-6-(1-ETHYL2-METHYL-4-NITROIMIDAZOLYL-5-MERCAPTO)PURINE P. M. Kochergin,1 E. V. Aleksandrova,2 L. S. Tolvinskaya,3 I. B. Zhukova,3 V. G. Pukhal’skaya,3 L. Yu. Telegin,4 L. A. Pevnitskii,5 and V. S. Korsunskii1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 11, pp. 9 – 11, November, 2000. Original article submitted June 29, 2000.

Azathioprine (Az) is among the currently available effective immunodepressants which is widely used in the treatment of autoimmune and rheumatic diseases [1, 2], idiopathic pulmonary fibrosis [3], clinical transplantation [4 – 6], etc. However, this agent has a narrow therapeutic ratio [7] and produces serious side effects [8, 9]. Thus, the search for new immunodepressants remains important in contemporary theoretical and practical medicine. Previous studies [7, 10] have described the immunodepressive properties of the thioguanine analog of Az, 2-amino-6-(1-methyl-4-nitroimidazolyl-5-mercapto)purine (tiamiprine), though this compound has relatively high toxicity and has therefore not found widespread clinical application. Another report [11] described the synthesis of a series of new 6-nitroimidazolyl-substituted derivatives of thioguanine, the strongest imunodepressant of which was 2-amino6-(1-ethyl-2-methyl-4-nitroimidazolyl-5-mercapto)purine (C-87) [12]. The present report provides a more detailed description of the results of a comparative study of the acute toxicity and immunotropic actions of compounds C-87 and Az. C-87 was synthesized according to the scheme:

O

S

HN H2N

P2S5

N N

C2H5N

N H

C2H5NH2

(COOC2H5)2

C2H5OH

HN

N N

H2N

N H

I (CONHC2H5)2 II

PCl5

Cl

N

POCl3

C2H5

N

HNO3

CH3

O2N

S NaOH H2O

N

N H2N

N

H2SO4

III

I + IV

Cl

N N

N H

N

C2H5 CH3

IV

C2H5

N

CH3

NO2 · H2O

C-87

EXPERIMENTAL CHEMICAL PART Thioguanine (I). Thioguanine was prepared from guanine [13] and P2S5 as described in [14]. The yield of pure product was 26 – 27% and the melting point was > 350°C. N,N¢-Diethyloxamide (II). This was prepared from diethyloxalate and ethylamine as described in [15]. The yield was 85 – 86% and the melting point was 177 – 178°C. 1-Ethyl-2-methyl-5-chlorimidazole (III). This was prepared from compound II and PCl5 in POCl3 as described in [16]. The yield was 60 – 64% and the boiling temperature was 100 – 101°C (12 mm), n D20 1.4980. 1-Ethyl-2-methyl-4-nitro-5-chlorimidazole (IV). This was prepared by nitration of compound III with a mixture of

1

Center for Drug Chemistry, All-Russia Research Institute of Pharmaceutical Chemistry, Moscow. 2 Zaporozhskii State Medical University, Ukraine. 3 Russian State Medical University, Moscow. 4 Center for Theoretical Studies of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow. 5 Medical-Genetic Scientific Center, Russian Academy of Medical Sciences, Moscow. Corresponding author: V. S. Korsunskii

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0091-150X/00/3411-0579$25.00 © 2001 Plenum Publishing Corporation

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P. M. Kochergin et al.

AFC, % of control

AFC, % of control Àz Ñ-87 Regression line

2

2

1

1

0

0

1

2

3

Fig. 2. The effects of compound C-87 and azathioprine on immune responses to sheep RBC. Substances were given in equitoxic doses (LD50): 36 mg/kg for compound C-87 and 90 mg/kg for Az. 1) Compound C-87; 2) Az; 3) control. –1 0

50

100

150

200 mg/kg

Fig. 1. Relationship between the suppression of the immune response to RBC and the doses of compound C-87 and azathioprine. Here and in Figs. 2 and 3, mean geometric AFC counts per spleen are presented with 95% confidence intervals.

concentrated HNO3 and H2SO4 as described in [17]. The yield was 81 – 82% and the melting temperature was 88 – 90°C. 2-Amino-6-(1-ethyl-2-methyl-4-nitroimidazolyl-5-mercapto)purine Hydrate (C-87). A mixture of 23.8 g (0.143 mole) of thioguanine (I), 27.0 g (0.143 mole) of nitrochlorimidazole IV, and 14.5 g (0.145 mole) of 40% aqueous NaOH was stirred for 3 – 3.5 h at 70°C. CH3COOH (4 – 5 ml) was added to the boiling reaction mixture until the aqueous layer gave an acidic reaction, when the mixture was cooled to 15 – 20°C, the precipitate was collected by filtration, washed with water, and recrystallized from water (with charcoal). The yield was 39.6 g (82%) and the melting temperature was 225 – 226°C (with degradation). The Rf (C4H9OH : H2O : CH3COOH, 5 : 4 : 1) was 0.45. IR spectral data (n, cm – 1): 1340, 1550 (NO2); 3150, 3210, 3320, 3420 (NH2 and NH). The melting temperature from [11] was 225 – 226°C. EXPERIMENTAL PHARMACOLOGICAL PART Studies of the toxic and immunodepressant actions of compound C-87 and Az were performed on male CBA/CaLacSto mice weighing 18 – 20 g. Both substances were used as suspensions in 1% starch gel and were given i.p.

Estimation of acute toxicity was performed as described by Litchfield and Wilcoxon [18]. The external appearance was monitored over a period of 14 days, along with behavior and deaths. The LD50 of compound C-87 was 80 mg/kg (62.2 – 97.5 mg/kg), while that of Az was 350 mg/kg (286 – 428 mg/kg). The Az toxicity curve was smother than that of C-87, showing that the two substances have different toxicity dynamics. Studies of the suppression of the primary immune response were based on the use of sheep RBC as antigen, at a dose of 5 ´ 108 cells, and polysaccharide Vi antigen (Vi-Ag), at a dose of 5 mg, both administered to mice i.v. in sterile physiological saline. Control mice received the same volume of i.p. starch gel. Immune responses in experimental and control animals were assessed four days after immunization in terms of the number of antibody-forming cells (AFC) per spleen by the local hemolysis method [19]. Therapeutic indexes (TI) were calculated as TI = LD50/ED50. Preliminary experiments [12] were conducted identify the optimum dosage times for C-87 and Az in relation to the antigenic stimulus: the immune responses mice to RBC and Vi-Ag were maximally suppressed by both agents when given one day after antigens (“day +1”). The time-effect curve for Az was consistent with other published data [7]. After identification of the optimal dosage time for compound C-87, the suppression of immune responses to RBC by different doses was compared with that of Az. As shown from the data presented in Fig. 1, compound C-87 produced significantly greater suppression of the animals’ immune responses than Az. The strongest inhibition of immunogenesis by compound C-87 occurred over the dose range 30 – 50 mg/kg (decreasing the number of AFC per

The Synthesis and Immunotropic Activity of a New Azathioprine Analog spleen to less than 10% of control). The TI of compound C-87 was significantly greater than that of Az (13.3 and 8.0 respectively). At the final stages of the study, the actions of equitoxic doses of Az and compound C-87 on thymus-dependent (RBC, Fig. 2) and thymus-independent (Vi-Ag, Fig. 3) immune responses were studied in parallel. As in the preceding part of the study, compound C-87 gave more effective suppression of the immune responses to both antigens than Az. Neither C-87 nor Az showed any preferential suppression of T-dependent or T-independent immune responses. Thus, compound C-87 is of interest in terms of further studies as a potential immunodepressant. REFERENCES 1. E. Berger and L. Rumbach, Rev. Med. Int., 20, 3465 – 3505 (1999). 2. T. Munster and D. E. Furst, Clin. Exp. Rheumatol., 17(6), 529 – 536 (1999). 3. R. P. Baughman and E. E. Lower, Clin. Immunother., 6(6), 431 – 442 (1996). 4. C. G. Groth, J. M. Backman, J. M. Morales, et al., Transplantation, 67(7), 1036 – 1042 (1999). 5. N. Perico and G. Remuzzi, Drugs, 54(4), 533 – 570 (1999). 6. G. N. Pershin, Zh. VKhO im. D. I. Mendeleeva, 15(2), 216 – 222 (1970). 7. L. N. Filitis, Yu. A. Sorkina, G. N. Pershin, et al., Farmakol. Toksikol., 34(6), 708 – 713 (1971). 8. J. N. Stolk, A. M. Boerbooms, R. A. De Abreu, et al., Arthritis Rheum., 41(10), 1858 – 1866 (1998). 9. M. F. Neurath, R. Wanitschke, M. Peters, et al., Gut, 44(5), 625 – 628 (1999). 10. R. V. Petrov and V. M. Man’ko, Immunodepressors [in Russian], Meditsina, Moscow (1971), p. 31. 11. P. M. Kochergin, E. V. Aleksandrova, V. S. Korsunskii, and V. S. Shlikhunova, Khim. Geterotsik. Soed., No. 2, 221 – 224 (2000).

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AFC, % of control 2

1

0

1

2

3

Fig. 3. The effects of compound C-87 and azathioprine on immune responses to Vi antigen. Substances were given at equitoxic doses (LD50). 1) Compound C-87; 2) Az; 3) control.

12. P. M. Kochergin, L. S. Tolvinskaya, I. B. Zhukova, et al., in: Proceedings of the V Russian Scientific Conference, “Humans and Drugs” [in Russian], Moscow (1998), pp. 578 – 579. 13. P. M. Kochergin, L. V. Persanova, E. V. Aleksandrova, et al., Khim. Geterotsik. Soed., No. 3, 388 – 390 (1995). 14. D. Daves, W. Noell, R. Robins, et al., J. Am. Chem. Soc., 82, 263 (1960). 15. P. M. Kochergin and K. S. Bushueva, Zh. Priklad. Khimii, 35, 2108 – 2109 (1962). 16. P. M. Kochergin, Zh. Obshch. Khimii, 34, 2735 – 2739 (1964). 17. V. S. Korsunskii, P. M. Kochergin, and V. S. Shlikhunova, Khim. Farm. Zh., 23(2), 249 – 250 (1989). 18. M. L. Belen’kii, Elements of the Quantitative Estimation of Pharmacological Effects [in Russian], Leningrad (1963), pp. 81 – 106. 19. N. K. Jerne and A. A. Nordin, Science, 140(3565), 405 (1963).