R. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2430–2433
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1,2-diaminobenzene to give benzimidazolyl phenylnitr-
iles 3a–c, 4a–c, and 5a–c. Subsequent hydrolysis and
esterification converted the phenylnitriles to correspond-
ing ethyl benzoates. One exception is intermediate 8a,
which was prepared by N-methylation of 6a with
CH3I in the presence of KOH in anhydrous acetone.
Target compounds 9a–c, 10a–c, and 11a–c were
obtained by treatment of the corresponding ethyl benzo-
ates with excessive guanidine in absolute isopropanol,
followed by hydrochlorination with gaseous HCl. The
salts thus formed were further purified by silica gel col-
umn chromatography (CH2Cl2/CH3OH, 7:1).
respectively, making them 45 and 15 times more potent
than cariporide, the IC50 of which was 65 nM in the
same test.
In the in vivo study, 15 of the 16 tested compounds (ex-
cept for 31a) had cardioprotective activity against IR in-
jury at various degree (p < 0.05), among which the
infarct size and the CK level of 9a, 10a, 10c, 11a, 32a–
b, 34, 35 were significantly lower than those of the con-
trol group (p < 0.01). The infarct size of 11a, 32b, and
35, the CK level of 32b and 35 were comparable with
those of cariporide. Both the parameters of 10a and 34
consistently implied a more favorable activity than
cariporide.
Synthesis of compounds 31a–b, 32a–b, and 33–35 is
illustrated in Scheme 2. Introduction of a chlorosulfo-
nyl or bromo group into the 3-position of 4-methyl
benzoic acid resulted in 12 and 19. Further alteration
of the chlorosulfonyl group of 12 obtained 13, 15, and
17. Then the acids 13, 17, and 19 were esterified to
give their ethyl esters 14, 18, and 20, while 16 was ob-
tained by CH3I methylation of both the carboxyl and
the sulfinic acid group of 15. These esters were brom-
ized with NBS to afford corresponding benzyl bro-
mines 23–26, which were subsequently treated with
2-mercapto-(5-nitro)-1H-benzimidazole to give inter-
mediates 27a–b and 28–30. Intermediates 22a–b came
from the nucleophilic substitution of p-carbo-
xylbenzylbromide with 2-mercapto- (5-nitro)-1H-benz-
imidazole followed by esterification. The synthesis of
2-mercapto-(5-nitro)-1H-benzimidazoles referred to lit-
erature method.6 The final benzoylguanidine products
were obtained similarly with the above compounds
9a–c, 10a–c, and 11a–c.
In concern with the structure–activity relationship, it is
obvious that the para-benzimidazolyl substituted ben-
zoylguanidines were more potent than the ortho- and
meta-substituted ones, the order being para > ortho > -
meta. As mentioned earlier, that is the reason we chose
the para-substituted compounds for further chemical
investigation. Also mentioned earlier is that 5-nitro
benzimidazolyl benzoylguanidines were more active
than their non-substituted counterparts. This is recon-
firmed by compound 31a versus 31b and 32a versus
32b. Among compounds 31a–b, 32a–b, and 33–35, the
3-methylsulfonyl (34), 3-morpholinylsulfonyl (35), and
3-aminosulfonyl (32a–b) benzoylguanidines were better
than non-substituted ones (31a–b), whereas the 3-bromo
substituted analog (33) represented even less activity. By
comparing compound 9a versus 31a, 10a versus 31b, the
prolongation of the hinge seemed to diminish the activ-
ity. Thus, we may infer that the remarkably good results
of compound 34 and other compounds possessing a sul-
fonyl group may come mainly from the various sulfonyl
substituents.
NHE1 inhibitory activity of 16 target compounds and
cariporide was evaluated in rat platelet swelling assay
(PSA), in which the swelling of rat platelets was induced
by a propionate buffer (pH 6.7). The experiment was
performed as described by Rosskopf et al.,7 with minor
modifications. The half-maximal inhibitory concentra-
tion (IC50) value of the tested compounds was obtained
from the linear part of the relationship between the log
concentration and NHE activity using linear regression
analysis.
Acknowledgments
We appreciate the support from Department of Pharma-
cology, and Center for Instrumental Analysis, China
Pharmaceutical University, for their contribution in
the pharmacological test and structural confirmation.
All the target compounds were also tested for the protec-
tion against myocardial ischemic-reperfusion injury in
SD rat hearts. The pharmacological model was created
by ligating the left anterior descending coronary artery
(LAD) for 1 h and then releasing the ligature for 2 h, fol-
lowed by immediate euthanization and heart-excision.
The hearts were stained by Evans blue injection and
TTC immersion respectively to measure the area at risk
and infarct area. Infarct size was expressed as the ratio
of infarct area to area at risk. Besides, blood sample was
taken and blood serum was prepared to undergo creatine
kinase (CK) activity determination.
References and notes
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The PSA results showed that most of the tested com-
pounds inhibited rat platelet NHE-1 in a concentra-
tion-dependent manner. Compounds 10a–c, 32b, 34,
35 were superior to cariporide in NHE inhibition. The
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Zhang, Rui
