Immunomodulatory and anticancer activities of some novel 2-substituted-6-bromo-3-methylthiazolo[3,2-a]benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.200800189

Ethyl 6-bromo-3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-carboxylate 2 was prepared by the ambient temperature bromination of ethyl 3-methyl-1,3- thiazolo[3,2-a]benzimidazole-2-carboxylate 1. The acid hydrazide 4 was obtained by the reaction of ester 2 w

Full text of DOI:10.1002/ardp.200800189

230
Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
Full Paper
Immunomodulatory and Anticancer Activities of Some Novel
2-Substituted-6-bromo-3-methylthiazolo[3,2-a]benzimidazole
Derivatives
Hatem A. Abdel-Aziz1, Amira M. Gamal-Eldeen2, Nehal A. Hamdy1, and Issa M. I. Fakhr1
1 Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
2 Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, Biochemistry Department,
National Research Centre, Dokki, Cairo, Egypt
Ethyl 6-bromo-3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-carboxylate 2 was prepared by the
ambient temperature bromination of ethyl 3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-carboxy-
late 1. The acid hydrazide 4 was obtained by the reaction of ester 2 with hydrazine hydrate.
Treatment of compound 4 with benzaldehyde or 2-thiophenaldehyde yielded the corresponding
hydrazones 6a and 6b, respectively, while the reaction of acid hydrazide 4 with ethoxymethy-
lene malononitrile (7a) or with ethyl ethoxymethylene cyanoacetate (7b) in refluxing ethanol
afforded pyrazole derivatives 9a and 9b, respectively. Taken together, from the biological inves-
tigations compounds 9a and 9b were the most significant inhibitors of LPS-stimulated NO gener-
ation from Raw murine macrophage 264.7, and, as another result, compounds 2 and 4 had a
weak radical scavenging activity against DPPH radicals. Moreover, 2, 4, and 9a had a concomi-
tant strong cytotoxicity against both colon carcinoma cells (HCT-116) and hepatocellular carci-
noma cells (Hep-G2) while 9b showed specific cytotoxicity only against colon carcinoma cells.
Keywords: Anticancer / Immunomodulatory / Pyrazole / Thiazolo[3,2-a]benzimidazole /
Received: October 20, 2008; accepted: January 9, 2009
DOI 10.1002/ardp.200800189
non-competitive mGluR1 antagonist [1, 2] and 3-(4-chlor-
Introduction
ophenyl)thiazolo[3,2-a]benzimidazole-2-acetic acid or
tilomisole (WY-18,251) has been used in the treatment of
cancer [8] and rheumatoid arthritis [9] beside its benefits
as both anti-inflammatory [10] and immunomodulatory
agent [11]. In addition, several pyrazole derivatives are of
significant pharmaceutical importance as anticancer
agents [12, 13].
Enlightened by these findings and in continuation of
our interest in the chemical and biological properties of
thiazolo[3,2-a]benzimidazole derivatives [14, 15] as well
as in the course of our previous studies on the synthesis
of biologically active heterocycles [16–19], we present in
this work a novel attempt to synthesize a sequence of
compounds starting from the key precursor ethyl 6-
bromo-3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-car-
boxylate 2 and to investigate their anti-inflammatory,
immunomodulatory, and anticancer activities.
Thiazolo[3,2-a]benzimidazole derivatives have a wide
range of biological and pharmacological activities with
therapeutic potential [1, 2]. These compounds are used
for treatment of cancer [3] and bone diseases [4] and treat-
ment of metabotropic glutamate receptor type 1
(mGlu1)-related diseases (epilepsy, inhibition of nerve
cell death, Parkinson's disease, migraine headache, cere-
bral infarction, neurogenic pain, and anxiety disorder)
[5–7]. Among those derivatives, 6-amino-N-cyclohexyl-N,3-
dimethyl-thiazolo[3,2-a]-benzimidazole-2-carboxamide
(YM-298198) has been reported as a potent, selective, and
Correspondence: Hatem A. Abdel-Aziz, Applied Organic Chemistry De-
partment, National Research Centre, Dokki, Cairo 12622, Egypt.
E-mail: hatem_741@yahoo.com
Fax: +20 2 337 601 77
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Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
Thiazolo[3,2-a]benzimidazole Derivatives
231
Scheme 1. Synthesis of compounds 2 and 4
completely similar to those of 1-(6-bromo-3-methyl-1,3-
thiazolo[3,2-a]benzimidazol-2-yl)ethanone [21] this pro-
vided a firm support to the assigned structure 2 and
easily ruled out the other structure 3A.
Next, the reaction of 6-bromo-3-methylthiazolo[3,2-
a]benzimidazole-2-carboxylic acid ethyl ester 2 with
hydrazine hydrate in refluxing ethanol gave the hydra-
zide 4. Its IR spectrum revealed the appearance of three
absorption bands at 3305, 3220, and 3150 cm–1 due to
NH2 and NH functions in addition to the carbonyl absorp-
tion band at 1631 cm–1. Its mass spectrum showed a peak
corresponding to its molecular ion at m/z = 325 [M+]
(Scheme 1).
Results and discussion
Ethyl 3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-carbox-
ylate 1 was prepared according to literature methods
[20]. Ethyl 6-bromo-3-methyl-1,3-thiazolo[3,2-a]benzimi-
dazole-2-carboxylate 2, which has not been reported hith-
erto, was prepared by bromination of compound 1. Thus,
the ester 1 reacts with bromine in acetic acid at room
temperature to give one isolable product, which was ana-
lyzed correctly for C13H11BrN2O2S (Scheme 1), however
structures 3A-3C seemed also possible (Scheme 1).
The 1H-MR spectrum of the isolated product revealed
the characteristic triplet and quartet signals due to the
ethoxy function of the ester group at d = 1.33 and d = 4.34.
It also showed the singlet signal of the methyl function
in position 3 at d = 3.07 in addition to the pattern of a
1,2,4-substituted benzene in the aromatic region which
is represented as one duplet of duplet signal and two
duplets at d = 7.54 (dd, J = 8.7, 1.8 Hz, 1H, ArH), 7.64 (d, J =
8.4 Hz, 1H, ArH) and at d = 8.18 (d, J = 1.5 Hz, 1H, ArH).
These findings exclude the possibilities of bromination
at C5 or C8 (1,2,3-substituted benzene pattern is represent
as three duplets of duplet signals), which correspond to
structures 3B and 3C, respectively, but this is not enough
to exclude structure 3A where possibly bromination may
take place at C7. Recently, we synthesized and confirmed
the structure of 1-(6-bromo-3-methyl-1,3-thiazolo[3,2-
a]benzimidazol-2-yl)ethanone [21] on the basis of its sin-
gle crystal X-ray diffraction as structure analogue for
Furthermore, the treatment of compound 4 with ben-
zaldehyde 5a or 2-thiophenaldehyde 5b, in refluxing
ethanol yielded the corresponding hydrazones 6a and
6b, respectively (Scheme 2). The structures of these hydra-
zones were established on the basis of their spectral data.
For example, the IR spectrum of 6a revealed a band of NH
absorption at 3149 cm–1 in addition to a band of carbonyl
1
function at 1646 cm–1, whereas its H-NMR spectrum
revealed the appearance of a signal due to the -CH=N- pro-
ton at d = 8.11 and a D2O-exchangeable signal due to NH
function at d = 11.87.
On the other hand, the reaction of hydrazide 4 with
ethoxymethylene malononitrile 7a or with ethyl ethoxy-
methylene cyanoacetate 7b in ethanol afforded pyrazole
derivatives 9a and 9b, respectively (Scheme 2). The struc-
tures of reaction products were established on the basis
of their spectral data. For example, the IR spectrum of 9a
revealed a bands of the amino group at 3290 and
3160 cm–1 in addition to absorption bands at 2228 and
1
compound 2. However, in the H-NMR spectrum of the
reaction product the signals due to 1,2,4-substituted ben-
zene system appeared in the aromatic region, which are
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H. A. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
Scheme 2. Synthesis of compounds 6a, b and 9a, b.
1678 cm–1 corresponding to nitrile and carbonyl func-
These overall macrophage proliferative activity of
tions, respectively, while its 1H-NMR showed a singlet sig- most of the tested compounds needs to be investigated
nal of pyrazole proton at d = 8.04 in addition to D2O- whether it is specific for macrophages only or towards
exchangeable broad singlet at d = 8.17 of the NH2 group. other immune cells as well. Thus, we tested the effect of
Its mass spectrum showed a peak corresponding to its these compounds on the proliferation of T-lymphocytes
molecular ion at m/z = 401 (cf. Experimental, Section 3).
(1301 cell line). Our findings indicated that 2, 4, and 6a
Macrophages are the first line of defense in innate were strong dose-dependant inducer of T-lymphocytes
immunity against microbial infection. Phagocytes engulf growth at doses of 25–100 lg/mL, and they seem to have
and kill microorganisms and present antigens for trig- low slope of the growth curve with the increase of doses
gering adaptive immune responses [22]. During phagocy- (Fig. 1b). Compound 9a had no effect on T-lymphocytes
tosis, macrophages secrete preformed granule constitu- growth (Fig. 1b). On the other hand, 6a, and 9a were cyto-
ents and newly synthesized products that play a critical toxic against T-lymphocytes and their calculated IC50 val-
role in tissue repair [22]. Accordingly, the induction of ues were 94.2 and 88.2 lg/mL, respectively.
macrophage proliferation is crucial in the assessment of
the innate immunity.
Our results suggested that the induced macrophages
and lymphocytes proliferation is due to the skeleton of 6-
To investigate the possible effect of the tested com- bromo-3-methyl-1,3-thiazolo[3,2-a]benzimidazole itself
pounds on the growth of Raw 264.7 cells, macrophages and that the substitution with phenylmethylene in the
were incubated with different concentrations of the com- hydrazone derivative 6a and with carbonitrile in the pyr-
pounds for 24 h. The treatment with compounds azole derivative 9a led to the depression in the prolifera-
depicted in Schemes 1 and 2 (2, 4, 6a, 6b, 9a, and 9b) tion of macrophage and the cytotoxicity against lympho-
revealed a variable effect on the macrophage prolifera- cytes. On the other hand, the substitution with thienyl-
tion, as shown in Fig. 1a. Here, 6a and 9a are shown as methylene in the hydrazone derivative 6b resulted in a
insignificant growth stimulants (P>0.05), 2, 4, 6b, and 9b dramatic enhancement in the proliferation of both types
were significant dose-dependant growth stimulators of immune cells.
(P a 0.05 at doses 50 and 100 lg/mL), except for 2, with
In inflammation many mediators are involved includ-
the highest growth induction at 50 lg/mL, which then ing cytokine secretions, and inflammation mediators
was decreased with the increase of the dose. The treat- like nitric oxide (NO) [23]. NO is a highly reactive free rad-
ment with M-CSF, which was used as a reference inducer ical and it can form a number of oxidation products such
.
of macrophage proliferation, induced the macrophage as NO2, NO2 , N2O3, and S-nitrosothiols [22]. In inflamma-
proliferation to 1.78 € 0.14-fold of the control.
tory diseases, NO is produced in large quantities by the
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Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
Thiazolo[3,2-a]benzimidazole Derivatives
233
Figure 2. Anti-inflammatory activity of two doses (12.5 lg/mL as
black bars, and 25 lg/mL as white bars) of the synthesized com-
pounds on the LPS-stimulated NO generation from macro-
phages, in comparison with LPS-treated and untreated macro-
phages (grey bars).
The nitrite concentration, as an index of NO generation, was
measured by Griess assay (Mean € S.D.; n = 4).
As a mimic in-vitro model of inflammation, macro-
phages were stimulated by incubation with the bacterial
LPS, which activates the macrophage functions and the
release of inflammatory mediators including enhance-
ment of iNOS, the generation of NO, and the secretion of
the pro-inflammatory cytokines. Since most of the cellu-
larly generated NO is converted immediately into
nitrites, Raw 264.7 were stimulated with LPS and the
nitrite level was measured in cell culture supernatants
before and after treatment with different compounds
(12.5 and 25 lg/mL) to investigate their possible anti-
inflammatory activity as indicated from the inhibition of
the LPS-stimulated NO generation.
The results revealed that 9a and 9b were the most sig-
nificant inhibitors of LPS-stimulated NO generation in
both tested doses with inhibition percentage ranged
from 56.0 to 88.2% (P a 0.05) (Fig. 2), which suggest a
potential anti-inflammatory activities of those com-
pounds. Additionally, compounds 2, 4, and 6a were also
strong inhibitors of the generated NO, at the high dose
(25 lg/mL), which may be due to the skeleton of 6-bromo-
3-methyl-1,3-thiazolo[3,2-a]benzimidazole itself. Our
findings suggested that the pyrazole substitution in the
Figure 1. The effect of the treatment with different doses of the
synthesized compounds in Scheme 1 and Scheme 2 on the pro-
liferation of Raw macrophages 264.7 (a) and 1301 T-lympho-
cytes (b), as measured after 24 h of treatment by MTT assay.
The results are represented as the percentage of control cells
(Mean € S.D.; n = 4).
action of inducible NO synthase (iNOS), and cyclooxyge-
nase-2 (COX-2) [24], leading to inflammation and persis-
tent pain. Subsequently, finding of new anti-inflamma-
tory agents represents a real need to help the patients of
inflammatory diseases.
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H. A. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
derivatives (9a and 9b) of 6-bromo-3-methyl-1,3-thia-
zolo[3,2-a]benzimidazole led to moderate anti-inflamma-
tory activity as indicated from the NO inhibition, and
that substitution with carbonitrile in the pyrazole deriv-
ative 9a was more effective in the NO inhibition. The
treatment with L-NMMA (NG-monomethyl-L-arginine), as a
reference NO inhibitor, resulted in a significant inhibi-
tion of the NO production with IC50 value of 7.69 lg/mL.
The inhibition of the generated NO by the tested com-
pounds may be due to a direct scavenging capacity of NO,
an inhibition of iNOS pathway, and / or a modulation of
other factors in the NO cascade such as transcriptional
factors. To investigate the radical scavenging activity of
those compounds, we submitted them to a DPPH assay,
which revealed that all of the compounds had no radical
scavenging activity at the tested concentrations >100 lg/
mL, except for compounds 2 and 4 with calculated SC50
values of 78.2 and 88.0 lg/mL, respectively, which are rel-
atively weak antioxidant activities compared with the
reference antioxidant; ascorbic acid (SC50 = 8.6 lg/ml).
These findings suggested that the NO inhibition was not
due to direct scavenging of NO, but rather due to other
molecular pathways.
Figure 3. The IC50 values of the cytotoxic compounds against
Hep G2 cells (black bars) and HCT-116 cell (white bars), as
measured after 24 h of treatment by MTT assay.
The results are represented as mean € S.D.; n = 4.
Exploring the cytotoxic effect of the tested compounds
on cancer cell lines, Hep-G2 and HCT-116 cells were
treated with and without doses of the compounds, and
submitted to the MTT assay, a metabolic cytotoxicity
assay. The experiment showed that compounds 6a and
6b had no effect on the growth of both types of cells,
while compounds 2, 4, and 9a had a concomitant cyto-
toxic effect on both types of cells as indicated by their
IC50 values as noted in Fig. 3, in addition to compound 9b
a weak cytotoxic compound against HCT-116 cells. As
shown in Fig. 3, the most remarkable cytotoxic com-
pounds against Hep-G2 cells were 9a>4, and against HCT-
116 were 9a > 2. This suggests that 9a is a non-specific
anticancer compound but with lower cytotoxicity than
the reference anticancer agent (paclitaxel), which was
found to have an IC50 value of 710 nM against Hep-G2
cells and 825 nM against HCT-116 cells, respectively.
These findings suggested that substitution with car-
bonitrile in the pyrazole derivative 9a of 6-bromo-3-
methyl-1,3-thiazolo[3,2-a]benzimidazole led to strong
cytotoxicity against both types of solid tumor cells Hep-
G2 and HCT-116 as indicated from the low IC50 values
18.4 and 12.5 lg/mL, respectively, and that the hydrazide
derivative 4 was more cytotoxic (IC50 = 21.2 lg/mL)
cursors in the synthesis of a sequence of derivatives as
promising potent biologically active agents. Taken
together from the biological investigations, 9a and 9b
were the most significant inhibitors of LPS-stimulated
NO generation, and 2 and 4 had a weak radical scaveng-
ing activity against DPPH (1,1-diphenyl-2-picryl-hydrazyl)
radicals. Moreover, 2, 4, and 9a had a concomitant strong
cytotoxicity against colon carcinoma and hepatocellular
carcinoma cells and 9b had a specific cytotoxicity against
colon carcinoma cells only.
The authors have declared no conflict of interest.
Experimental
Melting points were measured with a Gallenkamp apparatus
(Weiss-Gallenkamp, London, UK) and are uncorrected. IR spectra
were recorded on Shimadzu FT-IR 8101 PC infrared spectropho-
tometer (Shimadzu, Tokyo, Japan). The NMR spectra were
recorded on a Varian Mercury VX-300 NMR spectrometer (Varian
Inc., Palo Alto, CA, USA). 1H-spectra were run at 300 MHz in deu-
terated dimethylsulphoxide (DMSO-d6). Chemical shifts are
quoted in d [ppm] and were related to that of the solvents. Mass
spectra were measured on a GCMS-QP1000 EX spectrometer (Shi-
madzu) at 70 eV. Elemental analyses were carried out at the
Microanalytical center of Cairo University. Follow up of the reac-
tions and checking the compound purities were made by TLC on
silica gel-precoated aluminum sheets (Type 60 F254; Merck,
Darmstadt, Germany). Ethyl 3-methyl-1,3-thiazolo[3,2-a]benzimi-
dazole-2-carboxylate 1 [20] was prepared by the reported method.
against Hep-G2 cells than the original ester 2 (IC50
53.2 lg/mL).
=
In conclusion, we have investigated the reactivity and
synthetic potency of ethyl 6-bromo-3-methyl-1,3-thia-
zolo[3,2-a]benzimidazole-2-carboxylate 2 as reactive pre-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
Thiazolo[3,2-a]benzimidazole Derivatives
235
[M+] (26.5), 412 (50.3), 292 (90.8), 223 (63.6). Anal. calcd. for
C18H13BrN4OS (413.29): C, 52.31; H, 3.17; N, 13.56; S, 7.76. Found:
C, 52.00; H, 3.35; N, 13.72; S, 7.55.
Chemistry
Ethyl 6-bromo-3-methyl-1,3-thiazolo[3,2-a]benz-
imidazole-2-carboxylate 2
A solution of ester 1 (2.6 g, 10 mmol) in glacial acetic acid
(50 mL) was stirred at room temperature while a bromine solu-
tion (1.6 g, 10 mmol) in glacial acetic acid (10 mL) was added
drop-wise over a period of 30 min. The mixture was stirred at
room temperature for further 30 min. The partially oily reaction
mixture was poured into crushed ice. The solid was filtered off,
washed with water and dried. Recrystallization from EtOH
afforded compound 2 as white needles.
6-Bromo-3-methyl-N9-[1-thien-2-ylmethylene]-1,3-
thiazolo[3,2-a]benzimidazole-2-carbohydrazide 6b
Greenish yellow crystals; yield: 68%, m.p.: >3008C; IR (KBr) mmax
/
1
cm–1: 3131 (NH), 1642 (C=O), 1547 (C=N); H-NMR (DMSO-d6) d:
3.19 (s, 3H, CH3), 7.15-7.73 (m, 5H, ArH), 8.23 (d, J = 1.5 Hz, 1H,
ArH of C5), 8.27 (s, 1H, -N=CH-), 11.99 (s, 1H, NH, D2O exchange-
able); MS m/z (%): 422 [M+ + 3] (8.4), 421 [M+ + 2] (16.1), 420 [M+ + 1]
(58.8), 419 [M+] (10.5), 309 (24.1), 295 (100), 221 (47.0), 70 (15.8).
Anal. calcd. for C16H11BrN4OS2 (419.32): C, 45.83; H, 2.64; N, 13.36;
S, 15.29. Found: C, 46.05; H, 2.65; N, 13.59; S, 15.57.
Yield: 68%; m.p.: 203-2058C; IR (KBr) mmax/cm–1: 1707 (C=O),
1597 (C=N); 1H-NMR (DMSO-d6) d: 1.33 (t, J = 7.2 Hz, 3H, CH3), 3.07
(s, 3H, CH3), 4.34 (q, J = 7.2 Hz, 2H, CH2), 7.54 (dd, J = 8.7, 1.8 Hz,
1H, ArH of C7), 7.64 (d, J = 8.4 Hz, 1H, ArH of C8), 8.18 (d, J = 1.5 Hz,
1H, ArH of C5); MS m/z (%): 342 [M+ + 3] (63.4), 341 [M+ + 2] (14.98),
340 [M+ + 1] (82.1), 339 [M+] (16.77), 338 (100), 312 (40.04), 310
(42.35). Anal. calcd. for C13H11BrN2O2S (339.21): C, 46.03; H, 3.27;
N, 8.26; S, 9.45. Found: C, 45.88; H, 3.32; N, 8.44; S, 9.37.
5-Amino-1-[(6-bromo-3-methyl-1,3-thiazolo[3,2-
a]benzimidazol-2-yl)carbonyl]-1H-pyrazole-4-carbonitrile
9a
White powder; yield: 52%, m.p.: >3008C; IR (KBr) mmax/cm–1: 3290,
3160 (NH2), 2228 (C=N), 1678 (C=O), 1626 (C=N); 1H-NMR (DMSO-
d6) d: 3.24 (s, 3H, CH3), 7.55 (dd, J = 8.4, 1.8 Hz, 1H, ArH of C7), 7.66
(d, J = 8.7 Hz, 1H, ArH of C8), 8.04 (s, 1H, pyrazole), 8.17 (br. s, 2H,
NH2, D2O exchangeable), 8.28 (d, J = 1.5 Hz, 1H, ArH of C5); MS m/z
(%): 403 [M+ + 2] (12.88), 402 [M+ + 1] (23.62), 401 [M+] (35.03), 400
(47.74), 292 (100), 232 (55.61), 220 (65.79). Anal. Calcd. for
C15H9BrN6OS (401.24): C, 44.90; H, 2.26; N, 20.95; S, 7.99. Found:
C, 44.68; H, 2.11; N, 21.17; S, 7.75.
6-Bromo-3-methyl-1,3-thiazolo[3,2-a]benzimidazole-2-
carboxylic acid hydrazide 4
A mixture of compound 2 (3.39 g, 10 mmol) and hydrazine
hydrate (0.6 mL, 99%) in 100 mL of absolute ethanol was
refluxed for 5 h. The separated white solid was filtered off and
recrystallized from EtOH / DMF to give the title compound 4.
Yield: 74%; m.p.: 304-3068C; IR (KBr) mmax/cm–1: 3305, 3220,
3150 (NH, NH2), 1631 (C=O), 1583 (C=N);1H-NMR (DMSO-d6) d: 2.98
(s, 3H, CH3), 4.61 (br. s, 1H, NH2, D2O exchangeable), 7.52 (dd, J =
8.7, 1.8 Hz, 1H, ArH of C7), 7.67 (d, J = 8.7 Hz, 1H, ArH of C8), 8.19
(d, J = 1.5 Hz, 1H, ArH of C5), 9.71 (br. s, 1H, NH, D2O exchange-
able); MS m/z (%): 328 [M+ + 3] (2.94), 327 [M+ + 2] (5.84), 326 [M+ + 1]
(100), 325 [M+] (97.4), 324 (70.09), 295 (84.49), 292 (83.09), 223
(36.33), 221 (42.75). Anal. calcd. for C11H9BrN4OS (325.18): C,
40.63; H, 2.79; N, 17.23; S, 9.86. Found: C, 40.46; H, 2.77; N, 17.04;
S, 9.68.
Ethyl 5-amino-1-[(6-bromo-3-methyl[1,3]thiazolo[3,2-
a]benzimidazol-2-yl)carbonyl]-1H-pyrazole-4-carboxylate
9b
White powder; yield: 50%, m.p.: >3008C; IR (KBr) mmax/cm–1: 3262
(NH2), 1703, 1671 (2 C=O), 1616 (C=N); 1H-NMR (DMSO-d6) d: 1.26
(t, J = 7.2 Hz, 3H, CH3), 3.09 (s, 3H, CH3), 4.15 (q, J = 7.2 Hz, 2H,
CH2), 7.58 (dd, J = 8.7, 1.8 Hz, 1H, ArH of C7), 7.66 (d, J = 8.4 Hz, 1H,
ArH of C8), 8.04 (s, 1H, pyrazole), 8.17 (br. s, 2H, NH2, D2O
exchangeable), 8.22 (d, J = 1.5 Hz, 1H, ArH of C5); MS m/z (%): 450
[M+ + 2] (54.29), 449 [M+ + 1] (43.44), 448 [M+] (35.49), 447 (86.37),
295 (100), 292 (54.5), 155 (24.7). Anal. calcd. for C17H14BrN5O3S
(448.29): C, 45.55; H, 3.15; N, 15.62; S, 7.15. Found: C, 45.81; H,
3.32; N, 15.35; S, 7.37.
Reaction of 6-bromo-3-methylthiazolo[3,2-
a]benzimidazole-2-carboxylic acid hydrazide (4) with
benzaldehyde (5a) and thiophenaldehyde (5b),
ethoxymethylene malononitrile (7a) and ethyl
ethoxymethylene cyanoacetate (7b)
Cell culture
A mixture of the hydrazide 4 (0.33 g, 1 mmol) and benzaldehyde
(5a) or 2-thiophenaldehyde (5b), and ethoxymethylene malono-
nitrile (7a) or ethyl ethoxymethylene cyanoacetate (7b) (1 mmol)
in ethanol (50 mL) was refluxed for 4 h. The formed solid prod-
uct was collected by filtration, washed with ethanol and dried.
Recrystallization from the proper solvent afforded the corre-
sponding hydrazones 6a–6b and pyrazole derivatives 9a–9b,
respectively.
Several human cell lines were used to test the anticancer activity
including: lymphoblastic leukemia (1301 cells, a generous gift
from The Training Center of DakoCytomation, Elly, UK), hepato-
cellular carcinoma (Hep-G2) and colon carcinoma (HCT-116),
and Raw murine macrophage (RAW 264.7) (ATCC, VA, USA). Cells
were routinely cultured in DMEM (Dulbeco's Modified Eagle's
Medium), except RAW 264.7 cells, which were grown in RPMI-
1640 at 378C in humidified air containing 5% CO2. Media were
supplemented with 10% fetal bovine serum (FBS), 2 mM L-gluta-
mine, containing 100 units/mL penicillin G sodium, 100 units/
mL streptomycin sulphate, and 250 ng/mL amphotericin B.
Monolayer cells were harvested by trypsin / EDTA treatment,
except for RAW 264.7 cells, which were collected by gentle scrap-
ing. The tested compounds were dissolved in dimethyl sulphox-
ide (DMSO, 99.9%, HPLC grade) and diluted 1000-fold in the
assays. In all the cellular experiments, results were compared
with DMSO-treated cells. Compound dilutions were tested
6-Bromo-3-methyl-N9-[1-phenylmethylene]-1,3-
thiazolo[3,2-a]benzimidazole-2-carbohydrazide 6a
White powder; yield: 60%, m.p.: >3008C; IR (KBr) mmax/cm–1: 3149
1
(NH), 1646 (C=O), 1548 (C=N); H-NMR (DMSO-d6) d: 3.18 (s, 3H,
CH3), 7.47-7.57 (m, 5H, ArH), 7.67 (dd, J = 8.7, 1.8 Hz, 1H, ArH of
C7), 7.79 (d, J = 7.2 Hz, 1H, ArH of C8), 8.11 (s, 1H, -N=CH-), 8.25 (d, J
= 1.2 Hz, 1H, ArH of C5), 11.87 (s, 1H, NH, D2O exchangeable); MS
m/z (%): 416 [M+ + 3] (4.9), 415 [M+ + 2] (19.6), 414 [M+ + 1] (100), 413
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H. A. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 230–237
before assays for endotoxin using Pyrogent1 Ultra gel clot assay,
and they were found endotoxin free. All experiments were
repeated four times, unless mentioned, and the data were repre-
sented as (mean € S.D.). Except mentioned, cell culture material
was obtained from Cambrex BioScience (Copenhagen, Den-
mark), and all chemicals were from Sigma (USA), unless men-
tioned otherwise.
The results were compared with the anti-proliferative activity of
paclitaxel, a reference anticancer drug.
Antioxidant assay
The antioxidant capacities of the tested compounds were stud-
ied through their scavenging activity against 1,1-diphenyl-2-
picryl-hydrazyl (DPPH) radicals [26, 31]. The bleaching of DPPH
was monitored at an absorbance of 515 nm. The percentage of
DPPH bleaching utilized for SC50 (half maximal scavenging con-
centration) was calculated as follows: 0% is the absorbance of
DPPH with solvents (ethanol) and 100% is the absorbance of
DPPH with an efficient scavenger (10 mM ascorbic acid, AA). The
results were compared with ascorbic acid activity, as a reference
antioxidant.
Proliferation of immune cells
Macrophages and lymphocytes are essential immune cells. Raw
macrophage 264.7 and 1301 cells (T-lymphocytes) were relevant
cell lines to study the effect of the synthesized compounds on
the immune cells proliferation, which was estimated by the 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) assay [25]. The yellow tetrazolium salt of MTT is reduced by
mitochondrial dehydrogenases in metabolically active cells to
form insoluble purple formazan crystals, which are solubilized
by the addition of a detergent. Cells (56104 cells/well) were incu-
bated with various concentrations of the compounds at 370C in
a FBS-free medium, before being submitted to the MTT assay.
The absorbance was measured with an ELISA reader (BioRad,
Mꢀnchen, Germany) at 570 nm. The relative cell viability was
determined by the amount of MTT converted to the insoluble
formazan salt. The data are expressed as the mean percentage of
viable cells as compared to the respective control cultures
treated with the solvent. Treatment of macrophage with 1000 U/
mL recombinant macrophage colony-stimulating factor (M-CSF,
Pierce, USA) was used as positive control.
Statistical analysis
Data were statistically analyzed using IBM computer supplied
with Statistical Package for Social Scientists (SPSS) 10.00 for win-
dows (SPSS Inc., Chicago, USA). The student's unpaired t-test as
well as the one-way analysis of variance (ANOVA) test was used to
detect the statistical significance. p value more than 0.05 was
considered insignificant.
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