Cell cycle disruption and apoptotic activity of 3-aminothiazolo[3,2-a]benzimidazole-2-carbonitrile and its homologues

Article abstract of DOI:10.1016/j.ejmech.2010.02.025

3-Aminothiazolo[3,2-a]benzimidazole-2-carbonitrile (2) was prepared and upon hydrolysis using concentrated sulfuric acid or phosphoric acid resulted in the corresponding 3-aminothiazolo[3,2-a]benzimidazole-2-carboxamide derivative (3). Cyclization of the 2 using acetic anhydride or formic acid gave the corresponding pyrimido[4′,5′:4,5]thiazolo[3,2-a]benzimidazol-4(3H)-one (5) in good yields. Acetylation of 2 with acetic anhydride in pyridine afforded N-acetylaminothiazolo[3,2-a]benzimidazole-2-carbonitrile (6). In vitro antiproliferative activities of synthesized compounds were investigated at The National Cancer Institute (NCI), USA, according to their applied protocol. Compound 6 revealed significant antiproliferative activity, however, weak activity was shown by the other derivatives. Cell cycle disruption and apoptotic activity of 6 were studied, interestingly, 6 has the ability to arrest G2/M phase and it can induce apoptosis in time dependant manner.

Full text of DOI:10.1016/j.ejmech.2010.02.025

European Journal of Medicinal Chemistry 45 (2010) 2689e2694
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Cell cycle disruption and apoptotic activity of 3-aminothiazolo[3,2-a]
benzimidazole-2-carbonitrile and its homologues
,
,d
Abdelwareth A.O. Sarhan a, Abdullah Al-Dhfyan b, Maha A. Al-Mozaini b c, Chaker N. Adra b
,
,f,
*
Tarek Aboul-Fadl e
a Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt
b Stem Cell Therapy Program, King Faisal Specialized Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia
c Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
d Transplantation Research Center (TRC), Brigham & Women's Hospital and Children's Hospital Boston, Harvard Medical School, Boston, MA, USA
e Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
f Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Aminothiazolo[3,2-a]benzimidazole-2-carbonitrile (2) was prepared and upon hydrolysis using
concentrated sulfuric acid or phosphoric acid resulted in the corresponding 3-aminothiazolo[3,2-a]
benzimidazole-2-carboxamide derivative (3). Cyclization of the 2 using acetic anhydride or formic acid
gave the corresponding pyrimido[40,50:4,5]thiazolo[3,2-a]benzimidazol-4(3H)-one (5) in good yields.
Acetylation of 2 with acetic anhydride in pyridine afforded N-acetylaminothiazolo[3,2-a]benzimidazole-
2-carbonitrile (6). In vitro antiproliferative activities of synthesized compounds were investigated at The
National Cancer Institute (NCI), USA, according to their applied protocol. Compound 6 revealed signifi-
cant antiproliferative activity, however, weak activity was shown by the other derivatives. Cell cycle
disruption and apoptotic activity of 6 were studied, interestingly, 6 has the ability to arrest G2/M phase
and it can induce apoptosis in time dependant manner.
Received 8 September 2009
Received in revised form
26 January 2010
Accepted 9 February 2010
Available online 16 February 2010
Keywords:
3-Aminothiazolo[3,2-a]benzimidazole-2-
carbonitrile
Cytotoxic activity
Cell cycle disruption
G2/M phase
Ó 2010 Elsevier Masson SAS. All rights reserved.
Apoptosis
1. Introduction
cycle G2 checkpoint abrogation strategy attractive against cancer
[18,19]. From a medicinal chemistry point of view, synthetic small
Unlimited and uncontrolled cell proliferation is characteristic of
tumor cells [1,2]. Disruption of cell cycle has a crucial role in cancer
progression [3], as a result of this carcinogenesis can be controlled
by agent which have an effect of cell proliferation. Thus various
natural and synthetic agents are gaining widespread attention due
to their cell cycle regulation and modulation activity [4e17]. In fact,
all the suspected contributory factors for oncogenesis and muta-
gens, such as viruses and inherited predisposing factors e have
been shown to impair G1 checkpoint function. Consequently, more
than half of all human cancer cells with impaired G1 checkpoint
function rely on the G2 checkpoint to survive against the DNA
damage which most cytotoxic cancer treatments cause. The G2 cell
cycle checkpoint is rarely used by normal cells, which makes a cell
molecule modulators of the G2/M checkpoint are of particular
interest. Several clinically important anticancer compounds, such
as vinca alkaloids and taxanes, which act as prominent inhibitors of
G2/M transition, can be an effective for controlling some cancer
types. Nonetheless, all of these compounds disrupt tubulin directly
and they have complex chemical structures that restrict chemical
modification. In addition, several prominent disrupters of tubulin,
such as nocodazole and colchicine, lack antitumor efficacy. There-
fore, novel chemical structures that block G2/M phase transition
are valuable as pharmacological probes and as lead structures for
future therapeutic agents [20]. The antitumor activity of thiazolo
[3,4-a]benzimidazoles on human T-lymphoblastic CEM leukemia
cells has been reported [21e24]. According to these reports thia-
zolobenzoimidazole derivatives exert their antitumor effect by
activating the programmed cell death pathway (apoptosis). Thia-
zolo[3,4-a]benzimidazoles were also shown to effect the P-glyco-
protein mechanism in the HL60R cells, thus suggesting that they
are not suitable substrates for the multidrug transporter. In
* Corresponding author. Current address: Department of Pharmaceutical Chem-
istry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi
Arabia. Tel.: þ966 146 77341; fax: þ966 146 76220.
E-mail addresses: fadl@aun.edu.eg, fadl@ksu.edu.sa (T. Aboul-Fadl).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.025

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A.A.O. Sarhan et al. / European Journal of Medicinal Chemistry 45 (2010) 2689e2694
NH2
addition, they caused more apoptosis in drug resistant cells [25].
The mechanisms of this selective activity in multidrug resistant
(MDR) cells are not yet understood; however, this property may be
useful in the treatment of MDR malignancies to selectively kill Pgp-
expressing neoplastic cells only while preserving Pgp-negative
normal cells [26].
N
CN
a
N
NH
O
N
S
N
N
S
2
5
This work presents the synthesis of some thiazolo[3,2-a]benz-
imidazole derivatives, in vitro apoptotic effect, cell cycle regulation
and modulation activities.
b
O
2. Results and discussion
HN
S
N
2.1. Chemistry
N
CN
N
N
The synthesis and reactions of the 3-aminothiazolo[3,2-a]
benzimidazole-2-carbonitrile (2) was achieved as previously
reported by Sarhan et al. [27,28] from 2-mercaptobenzimidazole
(1). Applying the same procedures but instead of bromomalono-
nitrile, the ethyl bromocyanoacetate was reacted with 2-mercap-
tobenzimidazole (1) in ethanol in the presence of potassium
hydroxide. This step was followed by refluxing the resulting
precipitate in absolute ethanol and anhydrous sodium acetate to
give the corresponding 3-aminothiazolo[3,2-a]benzimidazole-2-
ethoxycarbonyl (4) as colorless crystals in 41% yield. Upon hydro-
lysis of 2 using concentrated sulfuric acid at room temperature or
heating with phosphoric acid at 70 ꢀC, the 3-aminothiazolo[3,2-a]
benzimidazole-2-carboxamide (3) was obtained as colorless crys-
tals in 50 and 56% yield, respectively [29], Scheme 1.
Refluxing of 3-aminothiazolo[3,2-a]benzimidazole-2-carbon-
itrile (2) in a mixture of formic acid, formamide and DMF (1:1:1)
afforded the corresponding pyrimido[40,50:4,5]-benzimidazole-4
(3H)-one (5) as colorless crystals in 38% yield [30,31]. Compound 5
could be also prepared upon refluxing of compound 2 in formic acid
only in 31% yield but along with the desired product, an undesired
benzimidazolyl-2-thioacetic acid was obtained in 36% yield.
Treatment of 2 with acetic anhydride in pyridine (1:1) at 50e60 ꢀC
afforded 3-N-acetylaminothiazolo[3,2-a]benzimidazole-2-carbon-
itrile (6) as white crystals in 55% yield [32], as shown in Scheme 2.
Structures of these synthesized compounds were verified using
spectral and elemental analyses and were consistent with the
assigned structures. The new compound 4 was identified and
characterized in a similar manner.
N
S
OH
N
5
6
Scheme 2. Synthesis of compounds 5 and 6. Reagents and conditions (a) HCONH2/
HCO2H/DMF, reflux, 3 h, 100 ꢀC and (b) Ac2O, pyridine, 50e60 ꢀC, 2 h.
protocol, each cell line is inoculated and preincubated on a micro-
titer plate. Test agents are then added at a single concentration
(10ꢁ4 M) and the culture is incubated for 48 h. Results for each test
agent are reported as the percent of growth of the treated cells
when compared to the untreated control cells [33,34]. According to
our results, among the synthesized compounds, only compound 6
revealed significant antiproliferative activity, however, week
activities were shown by other derivatives. The mode of action of
the antiproliferative activity of 6, such as the cell cycle disruption
and apoptotic activity of this compound were investigated.
To gain insight into the mechanism of antiproliferation, the
effect on cell cycle distribution was investigated by fluorescence-
activated cell sorting (FACS) analysis. MDA-MB-231 cells were
exposed to 100 mM of compound 6 for 24, 48 and 72 h which
resulted in the accumulation of G2/M phase, from 25.71 to 42, 46.22
and 45.79, respectively. This was also accompanied by compensa-
tory decrease in G1 phase cells (Fig. 1A and B). Compound 6 caused
a time dependant G2/M phase arrest, since the majority of accu-
mulation effect was detected in the first 24 h exposure time.
Compound 6 only had minor effect on S phase. These results sug-
gested that compound 6 inhibited the cellular proliferation via G2/
M phase arrest in a time dependant manner.
2.2. In vitro antiproliferative activity
2.3. Apoptotic effect of compound 6
In vitro antiproliferative activity of the synthesized compounds
was investigated according to the NCI recommendations. In this
The apoptotic effect of compound 6 was evaluated using
annexin V staining and a time dependent relationship was found
(Fig. 2A and B). The apoptotic population increased from 9.54% in
the control group to 23.77%, 54.59% and 80% increased after 24, 48
and 72 h exposure time using 100 mM of compound 6 for incuba-
tion. The cytotoxic effect of compound 6 is not correlated to the
NH2
N
CN
1) a
2) b
N
SH
S
N
H
N
2
increase of necrotic cell population.
1
1) d
2) e
1) c
2) b
3. Conclusions
NH2
NH2
S
O
O
Synthesis of 3-aminothiazolo[3,2-a]benzimidazole-2-carbon-
itrile and its derivatives starting from 2-mercaptobenzimidazole
are reported in this manuscript. In vitro antiproliferative activity of
N
N
OEt
NH2
S
N
4
N
these compounds demonstrated that compound
6 possesses
3
notable activity in G2/M phase arrest and induction of apoptosis in
a time dependant manner. These results suggest that compound 6
has the potential to be developed into a useful cell cycle specific
blocking agent. For this reason, further investigations are in
Scheme 1. Reported synthesis of compounds 2e4. Reagents and conditions (a) BrCH
(CN)2/KOH, EtOH; (b) AcONa/EtOH, reflux; (c) BrCH(CN)CO2Et/KOH, EtOH; (d) H2SO4,
RT and (e) H3PO4, 70 ꢀC.

A.A.O. Sarhan et al. / European Journal of Medicinal Chemistry 45 (2010) 2689e2694
2691
Fig. 1. (A) Time dependant effect of compound 6 on cell cycle distribution. MDA-MB-231 cells were treated with 100 mM of compound 6 and analyzed at, 24 (a), 48 (b) and 72 h (c)
by DNA flow cytometry. (B) Histograms show the number of cells per channel (vertical axis) vs DNA content (horizontal axis). The values indicate the percentage of cells in the
relevant phases of the cell cycle. The data shown was representative of three independent experiments with similar findings.

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A.A.O. Sarhan et al. / European Journal of Medicinal Chemistry 45 (2010) 2689e2694
Fig. 2. (A) Time dependant apoptotic response of MDA-MB-231 cells. MDA-MB-231 cells were treated with 100 mM of compound 6 and analyzed at, 24 (a), 48 (b) and 72 h (c) by
flow cytometry. Cells were harvested and incubated with annexin V-FITC and PI as described in Section 4.8. 10,000 Cells were analyzed per determination. Dot plots show annexin
V-FITC binding on the X axis and PI staining on the Y axis. Dots represent cells as follows: lower left quadrant, normal cells (FITCꢁ/PIꢁ); lower right quadrant, apoptotic cells (FITCþ/
PIꢁ); upper right quadrant, late apoptotic cells (FITCþ/PIþ), upper left quadrant (FITCꢁ/PIþ) necrotic cells. The results shown here are representative of three experiments.
(B) Histograms show time dependant apoptotic response of MDA-MB-231 cells treated by compound 6.
progress for the cell cycle and apoptotic activity of compound 6 in
addition to structural modulation of the 3-aminothiazolo[3,2-a]
benzimidazole-2-carbonitrile moiety.
University, Egypt). Chemical shifts are given in d ppm values, rela-
tive to the tetramethylsilane (TMS) as internal standard, J values are
given in Hz. CDCl3 was used as a deuterated solvent unless other-
wise stated. MS Spectra were obtained using a JEOL JMS-600 mass
spectrometer at ionization energy of 70 eV. Elemental analyses
were carried out at the Micro analytical laboratory of the Chemistry
Department (Assiut University, Egypt).
4. Experimental section
4.1. General methods
Cell culture MDA-MB-231 cells were cultured in RPMI 1640
medium supplemented with 100 ml/l fetal bovine serum with
addition of 100 U/ml penicillin, 100 U/ml streptomycin. Cells in
All melting points were determined using a kofler hot e stage. IR
Spectra were measured on a Perkin-Elmer FT spectrometer 1710 by
KBr techniques. 1H NMR Spectra were recorded at room tempera-
ture on a Varian EM-390, 90 MHz Spectrometer or on a Jeol LA
400 MHz FT-NMR spectrometer (Central Lab. Spectral Unit, Assiut
suspension (500 mL) were added to each 60 mm culture dishes and
incubate for 24 h at 37 ꢀC in a humidified atmosphere of 50 ml/l
CO2 in air. Cells were treated with the most active five members of

A.A.O. Sarhan et al. / European Journal of Medicinal Chemistry 45 (2010) 2689e2694
2693
the NCI antitumor screening program. Compound 6 is the only
active member which induced apoptosis by disruption of cell cycle.
4.5. Synthesis of pyrimido[40,50:4,5]thiazolo[3,2-a]benzimidazol-4
(3H)-one (5)
Thizolo[3,2-a]benzimidazole derivative 2 (2.14 g, 0.1 mol) was
dissolved and refluxed in formic acid, formamide and DMF (30 ml)
10 ml of each at 100 ꢀC for 12 h. The hot reaction mixture was then
cooled, diluted with water and the formed precipitate was collected
by filtration. The product was further purified by crystallization
from acetic acid to give the corresponding pyrimidone as white
crystals in 38% yield. Analytical data of compounds 5 was in satis-
factory agreement with the previously published data in the liter-
ature [30].
4.2. Synthesis of 3-aminothiazolo[3,2-a]benzimidazole-2-
carbnitrile (2)
A mixture of 2-mercaptobenzimidazole (1, 1.5 g, 1.0 mmol) and
KOH (1.1 mmol) was stirred in ethanol (25 ml) at room temperature
until complete solubility occurred. The solution is then filtered off
and stirred in an ice bath for further 10 min. A solution of freshly
prepared bromomalononitrile (1.1 mM) in ethanol was added drop
wise in an ice bath and stirring was continued for further 15 min.
The formed precipitate was filtered, washed with water (3ꢂ) fol-
lowed by dilute ethanol wash and finally dried. The precipitate was
then refluxed in absolute ethanol in the presence of fused sodium
acetate for 3 h. The resulting precipitate was collected by filtration,
washed with water (3ꢂ) followed by crystallization from pyridine
or dilute acetic acid to give the corresponding 3-aminothiazolo[3,2-
a]benzimidazole-2-carbnitrile (2) as colorless crystals in 50% yield.
Analytical data of compound 2 was in satisfactory agreement with
the previously published literature [31,32].
4.6. Synthesis of N-acetyl aminothiazolo[3,2-a]benzimidazole-2-
carbonitrile (6)
Compound 2 (1.0 g, 4.6 mM) was stirred in a mixture of Ac2O/
pyridine 1:1 (20 ml) at 50e60 ꢀC for 2 h. The reaction was worked
up and crystallized from ethanol following the method that
described in the literature to give 6 as white crystals in 55% yield,
mp. 247e249 ꢀC [29].
IR (KBr),
n
¼ 3220 m, 2220s, 1686s, 1617s, 1594s, 1489s, 1451s,
1217s, 754s, 741s cmꢁ1. 1H NMR (90 MHz, TFA)
aromatic-H), 3.0 (s, 3H, CH3).
d
¼ 7.8e8.8 (m, 4H,
4.3. Synthesis of ethyl 3-aminothiazolo[3,2-a]benzimidazole-2-
carboxylate (4)
EI-MS m/z (%) ¼ 256 (Mþ,18), 240 (1), 214 (100),187 (11),165 (1),
160 (2), 149 (3), 134 (4), 118 (5), 95 (2), 90 (60), 77 (3), 70 (5), 63 (3),
55 (4), 45 (2).
Anal. calcd. for C12H8N4OS: C 56.2, H 3.1, N 21.9, S 12.5, found: C
56.4, H 3.2, N 21.8, S 12.3.
4 Was prepared in a similar manner as the synthesis of
compound 2. Instead of bromomalononitrile a solution of ethyl
bromocyanoacetate in absolute ethanol was used. After the reac-
tion was complete, the resulting precipitate was collected and
crystallized from ethanol to give the expected ester 4 as colorless
needles crystals in 41% yield, mp. 228e230 ꢀC.
4.7. Flow cytometric analysis of cellular DNA content
IR (KBr)
n
¼ 3380s, 3292s, 3216s, 3148, 1672s, 1632s, 1608s,
1540s, 1496s, 1440s, 1300s, 752s cmꢁ1
.
2 ꢂ 106 Cells were fixed in 1 ml ethanol (70%) for 60 min at room
1H NMR(400 MHz, CDCl3þ trifluoroacetic acid(TFA)): 8(s, 2H,
NH2), 7.7(m, 1H, aromatic-H), 7.4e7.1 (m, 3H, aromatic-H), 4.3(q,
J ¼ 7.5, 14.5, 2H, CH3CH2), 1.4(t, J ¼ 12.5, 3H, CH3CH2).
temperature. Harvested cells were resuspended in 1 ml Na citrate
(50 mM) containing 250
60 min. Next, cells were resuspended in the same buffer containing
g propidium iodine (PI) and incubated for 30 min before being
analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA).
The percentage of cells in various cell cycle phases was determined
by using Cell Quest Pro software (Becton Dickinson).
m
g RNase A and incubated at 50 ꢀC for
13C NMR (75 MHz, CDCl3 þ TFA): 170.28 (CO), 161.69 (C-9a),
152.21 (C-3), 133.99 (C-4a, C-8a), 125.63 (C-6, C-7), 122.66 (C-5, C-
8), 117.82, 113.93 (C-2, CN), 62.48 (CH2), 14.20 (CH3).
4
m
MS (EI 70 eV) m/z (%): 261 (Mþ, 100), 215 (79), 187 (9), 161 (17),
145 (25), 134 (11), 118 (10), 102 (10), 90 (19), 76 (5), 63 (7).
4.4. Synthesis of 3-aminothiazolo[3,2-a]benzimidazole-2-
carboxamide (3)
4.8. Measurement of annexin V binding by flow cytometry
It has been shown that loss of phospholipids asymmetry of the
plasma membrane is an early event of apoptosis [35,36]. Annexin V
binds to negatively charged phospholipids, like phosphatidylserine.
During apoptosis, the cells react with annexin V once chromatin
condenses but before the plasma membrane loses its ability to
exclude propidium iodide (PI). Hence, by staining cells with
a combination of fluorescein isothiocyanate (FITC) annexin V and PI
it is possible to detect non-apoptotic live cells, early apoptotic cells
and late apoptotic or necrotic cells [37,38]. Annexin-V staining was
performed using the Vybrant Apoptosis Assay Kit# 2 (Molecular
Probe) following the manufacturer's recommendations. Annexin-V
stained cells were analyzed by flow cytometry, measuring the
fluorescence emission at 530 and less than 575 nm.
Thizolo[3,2-a]benzimidazole derivative 2 (2.14 g, 0.1 mol) was
dissolved in phosphoric acid 85% (20 ml) and stirred at 60e70 ꢀC
for 30 min. The reaction mixture was diluted with ice/cold water
and neutralized with ammonium hydroxide. The resulting precip-
itate was collected by filtration, dried and recrystallized from
ethanol to give the corresponding amide derivative 3 as silvery
crystals, yield 1.3 g (56%), mp. 249e25 ꢀC.
IR (KBr)
n
¼ 3448s, 3363s, 3196s, 1656s, 1604s, 1543s, 1483s,
1449s, 1206s, 750s cmꢁ1
.
1H NMR (400 MHz, DMSO-d6): 8.2e8.1 (m, 1H, aromatic-H), 7.5
(m, 3H, 1H, aromatic-H and 2H, CONH2), 7.4e7.1 (m, 4H, 2H,
aromatic-H and 2H, NH2).
13C NMR (75 MHz, CDCl3 þ TFA): 167.35 (CO), 150.43 (C-9a),
145.30 (C-3), 135.61 (C-4a, C-8a), 126.71, 126.73 (C-5, C-8), 129.54
(C-6, C-7), 115.43, 112.72 (C-2, CN).
Acknowledgment
EI-MS m/z (%) ¼ 232 (Mþ, 100), 215 (52), 187 (2), 161 (13), 145
(27), 134 (7), 118 (11), 102 (5), 90 (11), 76 (6), 63 (5).
Anal. calcd. for C10H8N4OS: C 51.7, H 3.5, N 24.1, S 13.8, found: C
51.5, H 3.4, N 24.0, S 13.7.
Assiut University, Egypt is deeply acknowledged for supporting
this work. National Cancer Institute (NCI), USA is also appreciated
for antiproliferative screening of our compounds.

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