The synthesis and immunotropic activity of a new azathioprine analog - 2-Amino-6-(1-ethyl-2-methyl-4-nitroimidazolyl-5-mercapto)purine

Full text of DOI:10.1023/A:1010383903327

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-ETHYL-
2
-METHYL-4-NITROIMIDAZOLYL-5-MERCAPTO)PURINE
1
2
3
3
P. M. Kochergin, E. V. Aleksandrova, L. S. Tolvinskaya, I. B. Zhukova,
5
3
4
1
V. G. Pukhal’skaya, L. Yu. Telegin, L. A. Pevnitskii, and V. S. Korsunskii
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 11, pp. 9 – 11, November, 2000.
Original article submitted June 29, 2000.
O
N
S
Azathioprine (Az) is among the currently available effec-
tive immunodepressants which is widely used in the treat-
ment of autoimmune and rheumatic diseases [1, 2], idio-
pathic 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.
P2S
5
HN
N
HN
H N
N
C H N
2
5
H N
N
N
I
N
H
2
2
H
C H NH
2
2
5
(
COOC H )
(CONHC H )
2 5 2
2
5
2
C H OH
2 5
II
HNO
C
H
Cl
C H
2 5
Previous studies [7, 10] have described the immunode-
pressive properties of the thioguanine analog of Az, 2-ami-
no-6-(1-methyl-4-nitroimidazolyl-5-mercapto)purine (tia-
miprine), 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 thiogua-
nine, the strongest imunodepressant of which was 2-amino-
2
5
PCl5 Cl
N
N
3
CH3
CH3
POCl3
H SO
2
4
N
N
O N
III
2
IV
C H
2
5
S
N
N
CH3
NaOH
N
N
I + IV
6
-(1-ethyl-2-methyl-4-nitroimidazolyl-5-mercapto)purine
C-87) [12].
The present report provides a more detailed description
H O
2
NO2
H N
N
N
H
2
(
· H O
2
C-87
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:
EXPERIMENTAL CHEMICAL PART
Thioguanine (I). Thioguanine was prepared from gua-
nine [13] and P S as described in [14]. The yield of pure
2
5
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 pre-
pared from compound II and PCl in POCl as described in
1
Center for Drug Chemistry, All-Russia Research Institute of Pharmaceuti-
cal Chemistry, Moscow.
2
Zaporozhskii State Medical University, Ukraine.
Russian State Medical University, Moscow.
3
5
3
4
Center for Theoretical Studies of Physicochemical Pharmacology, Rus-
sian Academy of Sciences, Moscow.
[
16]. The yield was 60 – 64% and the boiling temperature
20
was 100 – 101°C (12 mm), nD 1.4980.
5
Medical-Genetic Scientific Center, Russian Academy of Medical Sci-
ences, Moscow.
Corresponding author: V. S. Korsunskii
1-Ethyl-2-methyl-4-nitro-5-chlorimidazole (IV). This
was prepared by nitration of compound III with a mixture of
579
0
091-150X/00/3411-0579$25.00 © 2001 Plenum Publishing Corporation

5
80
P. M. Kochergin et al.
AFC, % of control
AFC, % of control
2
Àz
Ñ-87
2
1
0
1
Regression line
1
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
(
LD ): 36 mg/kg for compound C-87 and 90 mg/kg for Az.
50
1) Compound C-87; 2) Az; 3) control.
–
0
50
100
150
200
mg/kg
Estimation of acute toxicity was performed as described
Fig. 1. Relationship between the suppression of the immune re-
sponse 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.
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.
concentrated HNO and H SO as described in [17]. The
3
2
4
yield was 81 – 82% and the melting temperature was
8 – 90°C.
-Amino-6-(1-ethyl-2-methyl-4-nitroimidazolyl-5-mer-
8
Studies of the suppression of the primary immune re-
sponse were based on the use of sheep RBC as antigen, at a
2
capto)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%
8
dose of 5 ´ 10 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 in-
dexes (TI) were calculated as TI = LD /ED . Preliminary
aqueous NaOH was stirred for 3 – 3.5 h at 70°C. CH COOH
3
(
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 filtra-
tion, washed with water, and recrystallized from water (with
charcoal). The yield was 39.6 g (82%) and the melting tem-
perature was 225 – 226°C (with degradation). The Rf
5
0
50
experiments [12] were conducted identify the optimum dos-
age times for C-87 and Az in relation to the antigenic stimu-
lus: 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].
(
C H OH : H O : CH COOH, 5 : 4 : 1) was 0.45.
4 9 2 3
–
IR spectral data (n, cm ): 1340, 1550 (NO ); 3150,
1
2
3
210, 3320, 3420 (NH and NH). The melting temperature
2
from [11] was 225 – 226°C.
After identification of the optimal dosage time for com-
pound C-87, the suppression of immune responses to RBC
by different doses was compared with that of Az.
EXPERIMENTAL PHARMACOLOGICAL PART
As shown from the data presented in Fig. 1, compound
C-87 produced significantly greater suppression of the ani-
mals’ 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
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.

The Synthesis and Immunotropic Activity of a New Azathioprine Analog
581
AFC, % of control
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).
2
1
0
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) im-
mune responses were studied in parallel. As in the preceding
part of the study, compound C-87 gave more effective sup-
pression 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.
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2
3
Fig. 3. The effects of compound C-87 and azathioprine on immune
responses to Vi antigen. Substances were given at equitoxic doses
1
2
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