New Benzimidazoles and Their Antitumor Effects with Aurora A Kinase and KSP Inhibitory Activities

Article abstract of DOI:10.1002/ardp.201400441

A newly synthesized series of anticancer compounds comprising thiazolo[3,2-a]pyrimidine derivatives 6a-q bearing a benzimidazole moiety was produced via a one-pot reaction of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-cyanoacetamide 5 with 2-aminothiazole and an appropriate aromatic aldehyde. Compound 7 was obtained via the reaction of 4-(1H-benzo[d]imidazol-2yl)benzenamide 1 with carbon disulphide and methyl iodide in the presence of concentrated aqueous solution of NaOH, then treated with o-phenylenediamine to give N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-1H-benzo[d]imidazol-2-amine 8. The structures of the newly synthesized compounds were confirmed by analytical and spectroscopic measurements (IR, MS, and ¹H NMR). The synthesized products were screened and studied for their in vitro antitumor activity against three human cancer cell lines (namely colorectal cancer cell line HCT116, human liver cancer cell line HepG2, and human ovarian cancer cell line A2780) and their Aurora A kinase and KSP inhibitory activities. All newly synthesized compounds revealed marked results comparable with the standard drug CK0106023. The compounds 6e and 6k of the thiazolopyrimidine derivatives were the most active compounds when tested against the three cell lines in comparison with the standard drug CK0106023, and showed potent dual KSP and Aurora A kinase inhibition.

Full text of DOI:10.1002/ardp.201400441

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
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Full Paper
New Benzimidazoles and Their Antitumor Effects with Aurora A
Kinase and KSP Inhibitory Activities
Amira S. Abd El-All1, Asmaa A. Magd-El-Din1, Fatma A. F. Ragab2, Mahmoud ElHefnawi3,
Mohamed M. Abdalla4, Shadia A. Galal1, and Ahmed A. El-Rashedy1
1
Department of Natural and Microbial Product, Research Division of Pharmaceutical and Drug Industries,
National Research Centre, Dokki, Cairo, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Giza, Egypt
Biomedical Informatics and Chemoinformatics Group, Center of Excellence for Advanced Sciences,
2
3
National Research Centre, Dokki, Cairo, Egypt
Research Unit, Saco Pharm. Co., 6 October City, Egypt
4
A newly synthesized series of anticancer compounds comprising thiazolo[3,2-a]pyrimidine derivatives 6a–
q bearing a benzimidazole moiety was produced via a one-pot reaction of N-(4-(1H-benzo[d]imidazol-2-
yl)phenyl)-2-cyanoacetamide 5 with 2-aminothiazole and an appropriate aromatic aldehyde. Compound
7 was obtained via the reaction of 4-(1H-benzo[d]imidazol-2yl)benzenamide 1 with carbon disulphide
and methyl iodide in the presence of concentrated aqueous solution of NaOH, then treated with
o-phenylenediamine to give N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-1H-benzo[d]imidazol-2-amine 8.
The structures of the newly synthesized compounds were confirmed by analytical and spectroscopic
1
measurements (IR, MS, and H NMR). The synthesized products were screened and studied for their
in vitro antitumor activity against three human cancer cell lines (namely colorectal cancer cell line HCT116,
human liver cancer cell line HepG2, and human ovarian cancer cell line A2780) and their Aurora A kinase
and KSP inhibitory activities. All newly synthesized compounds revealed marked results comparable
with the standard drug CK0106023. The compounds 6e and 6k of the thiazolopyrimidine derivatives
were the most active compounds when tested against the three cell lines in comparison with the
standard drug CK0106023, and showed potent dual KSP and Aurora A kinase inhibition.
Keywords: Aurora A kinase inhibition / Benzimidazole / Human cancer cells / KSP inhibition /
Thiazolo[3,2-a]pyrimidine
Received: November 25, 2014; Revised: November 25, 2014; Accepted: March 20, 2015
DOI 10.1002/ardp.201400441
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
among the most extensively studied classes of heterocyclic
compounds due to their chemotherapeutic application such
Introduction
Benzimidazoles of both natural and synthetic source are the
key components of many active compounds [1], since they are
as antitumor [2], antiviral [3–5], antimicrobial [6–8], antihy-
pertensive, anti-inflammatory [9, 10], and 5-HT3 receptor
antagonistic [11]. Although benzimidazole is one of the
oldest and chemically well-studied nitrogen heterocycles as
multi-component nucleus, it was longly neglected till 1950
when it was discovered that this moiety was an integral part
of the structure of vitamin B12. Since then, it has been
recognized as the versatile nucleus for building molecules
possessing broad spectrum of biological activity. Benzimid-
azole has a poly functional ability to carry out various organic
Correspondence: Dr. Amira S. Abd El-All, Department of Natural
and Microbial Product, Research Division of Pharmaceutical and
Drug Industries, National Research Centre, Dokki, Cairo 12622,
Egypt.
E-mail: amira19661@hotmail.com
Fax: þ20 233370931.
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reactions together with disclosing fascinating aspects which
led to the synthesis of a large variety of benzimidazole
derivatives of which many have been demonstrated to exhibit
powerful pharmacological activity [12].
These facts together with the powerful biological activity
of pyrimidine moiety which is widely used as a key building
block for pharmacological agent led us to bind these two
interesting moieties and specifically its derivative thiazolo-
pyrimidine hoping to build significant places in medicinal
chemistry research [13].
Pyrimidine and fused pyrimidine derivatives are one of the
most prominent structures found in nucleic acid including
uracil, thymine, cytosine, adenine, and guanine and are the
fundamental building blocks for DNA and RNA. They also play
an essential role in several biological processes, found in
nucleoside antibiotics, antibacterial, and cardiovascular as
well as considerable chemical reactions [14]. The key role of
pyrimidine’s action is playing in cellular processes which
makes it valuable for drug discovery.
Thiazolo[4,5-d]pyrimidine derivatives have acquired a grow-
ing importance in the field of medicinal chemistry and are
considered as thia-analogs of the natural purine bases such as
adenine and guanine, because of their biological potential
while some thiazolo[3,2-a]pyrimidines have been demon-
strated to be associated with potent immune modulating
properties [38]. Thiazolo[3,2-a]pyrimidines are also of phar-
macological interest due to their ant-inflammatory [39, 40],
psychopharmacological [41], bactericidal [42], and antiviral
activity as inhibitors of HIV-1 reverse transcriptase [43].
The main target of this article is to build a new compound
bearing the two interesting moieties of benzimidazole and
thiazolopyrimidine hoping to achieve a pronouncing progress
in the field of drug discovery against several antitumor cell
lines.
Fu et al. [44] synthesized four series of dihydropyrazolo-
[3,4-b]pyridines 2a,b and benzo[4,5]imidazo[1,2-a]pyrimi-
dines 3a,b as dual KSP and Aurora A kinase inhibition.
Despite showing moderate KSP and Aurora
A kinase
Thiazolo nucleus possesses different biological activities
such as antihypertensive [15], anti-inflammatory [16], anti-
schizophrenic [17], antibacterial [18], anti-HIV [19], hypnotic
[20], antiallergic [21], and more recently analgesic [22],
fibrinogen receptor antagonists with antithrombotic activity
[23], inhibitors of bacterial DNA gyrase B [24], and antitumor
and cytotoxic activities.
Thiazolo[4,5-d]pyrimidine have been of interest due to their
ability to inhibit2-methylerythritol-2,4-cylodiphosphate syntheses
[25]. They have been also used as analgesic and antiparkinsonian
agent [26], modulators of Transient Receptor Potential Vanilloid-
receptor 1 (TRPV1) [27], anticancer agent [28–30], pesticides [31],
phosphate inhibitor [32, 33], acetylcholinesterase inhibitor [34]
and antimicrobial substances [35–37].
inhibition, compound 3a,b displayed significant cytotoxic
activity in micromolar range, especially against HCT116 and
HepG2 cell lines (Fig. 1).
Motivated by the above findings, the present investigation
deals with the synthesis of a new series of benzimidazole
derivatives N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-7-amino-
8,8a-dihydro-(5-substiuted benzylidine)-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide 6a–q and studies its KSP and
Aurora A kinase inhibitions.
Serial derivatives of thiazolo[3,2-a]pyrimidine-6-carboxa-
mide bearing benzimidazole moiety 6a–q were synthesized
via a one-pot reaction. Bis-benzimidazole moiety 8 was
formed via formation of compound 7, and its reaction with
o-phenylenediamine. New design approach highly potent
Figure 1. Compounds 2a,b and 3a,b showing KSP and Aurora A kinase inhibition.
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NH2
NH2
N
reflux
NH2
poly-phosphoric acid
or o-phosphoric acid
NH
HOOC
NH2
Scheme 1. Preparation of 4-(1H-benzo[d]imidazol-2-yl)benzenamine.
human antitumor cell lines namely colorectal cancer cell line
HCT116, human liver cancer cell line HepG2, and human
ovarian cancer cell line A2780 and Aurora A kinase and KSP
inhibitory activities were described.
phosphoric acid [48] as dehydrating agent instead of
polyphosphoric acid (Scheme 1). The structure of compound
1 was confirmed by different spectroscopic tool. Its IR
spectrum (KBr/cmꢀ1) showed an absorption band at 3350
and 3217 which corresponds to (NH, NH2), a band at 1629 due
to C N. The 1H NMR spectrum of compound 1 was revealed
–
–
proton at d 5.58 and 12.46 ppm which was assigned to the
appearance of NH2 and NH group exchangeable with D2O,
two doublet protons at d 6.63 and 7.79 ppm characterized to
AB system for aniline ring and multiple protons at 7.08 and
7.46 ppm related to phenyl ring.
Results and discussion
Chemistry
4-(1H-Benzo[d]imidazol-2-yl)benzenamine (1) was prepared
as previously reported by condensation of o-phenylenedi-
amine with p-aminobenzoic acid in the presence of poly-
phosphoric acid [45–47]. This method yielded phosphorous
It has been now found that reaction of compound 1 with
ethyl cyanoacetate in refluxing ethanol afforded the corre-
complex that was difficult to get rid of. In the present work,
this compound 1 was prepared with higher purity using o-
sponding
N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-cyano-
acetamide 5 through the mechanism illustrated in Scheme 2.
NH2
O
−C2H5OH
N
OEt
N
Ar-CHO
NH
S
N
CN
NH2
O
N
H
N
H
CN
5
1
H2N
O
NH
H
N
S
NH
N
N
Ar
6a–q
c = C6H4-Cl (m)
g = C6H4-OCH3 (p)
Ar a =
f =
d = C6H4-Cl (p)
i = C6H4-OH (p)
b = C6H4-Cl (o)
e = C6H4-OCH3 (o)
j = C6H4-NO2 (o)
C6H5
C6H4-OCH3 (m)
C6H4-NO2 (p)
h = C6H4-OH (o)
k =
o = C H -2,5-OCH
3
n = C6H3-2,4-OCH3
l = C6H4-CH3 (p) m = C6H4-N(CH3)2 (p)
6
3
q = C H -3,4,5-OCH
p = C6H3-3-OCH3-4OH
6
3
3
Scheme 2. Preparation of 5H-thiazolo[3,2-a]pyrimidine-6-carboxamide derivatives 6a–q.
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benzimidazoleandNH amide, a bandat2370 duetoC
ꢀNanda
ꢀ
ꢀ
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The structures of compounds 5 were confirmed on the basis of
synthesized 5H-thiazolo[3,2-a]pyrimidine-6-carboxamide 6a–
their analytical and spectral data. Its IR spectrum (KBr/cmꢀ1
)
q showed disappearance of the band corresponding to CꢀꢀN
ꢀꢀ
ꢀꢀ
showed two bands at 3334 and 3055 corresponding to NH of
at 2370 cmꢀ1 and appearance of band at 3315–3345 cmꢀ1
corresponding to NH2 groups in addition to characteristic
bands for each compound (see Experimental section).
Structure 6c was supported by its mass spectrum (m/z 501)
(Mþ), which agrees with its molecular formula C26H21ClN6OS.
Its 1H NMR spectrum (DMSO-d6, d ppm) showed six signals
at 2.46, 4.30, 6.09, 10.14, 10.55, and 12.18 due to H5 of
pyrimidine, NH2, H8 of pyrimidine, NH amide, NH pyrimidine,
and NH benzimidazole which was exchangeable, multiple
protons (14H) at 7.13–7.53, 7.14–77.70, and 8.07 due to
aromatic, thiazole, and benzimidazole rings.
band at 1673 due to C O. Its 1H NMR spectrum (DMSO-d6,
–
–
d ppm) showed three signals at 3.78, 10.59, and 12.04 due to
CH2, NH amide which was exchangeable, NH benzimidazole
which was exchangeable. The 10H protons for aromatic and
benzimidazole ring appeared as a multiplet at 6.63–7.68, 7.78,
and at 8.11. Moreover, the structure was also supported by
its mass spectrum (m/z 275.90) (M–), which agrees with its
molecular formula C16H12N4O.
The present investigation deals with the synthesis of a new
series of benzimidazole derivatives of N-(4-(1H-benzo[d]-
imidazol-2-yl)phenyl)-7-amino-8,8a-dihydro-(5-substituted
benzylidine)-5H-thiazolo[3,2-a]pyrimidine-6-carboxamide
6a–q. Compounds 6a–q were synthesized via a one-pot
reaction of 5 with appropriate aromatic aldehyde with
different functional groups in different position and 2-
amino thiazole in refluxing acetic acid (Scheme 2). The
traditional approach for preparation of thiazolopyrimidine
derivatives has been made from either of the two directions.
Either the pyrimidine ring is constructed first and thiazole
ring formed in the terminal step of the synthetic se-
quence [40, 49], or the thiazole ring is prepared initially
and pyrimidine ring formed in the terminal stage [50, 51].
The suggested mechanism for preparation of 5H-thiazolo-
[3,2-a]pyrimidine-6-carboxamide derivatives 6a–q is illustrat-
ed in Scheme 3.
Dimethyl-(4-(1H-benzo[d]imidazol-2-yl)phenyl)carbonimido-
dithioate 7 (carbonates) has been synthesized by the reaction
of compound 1 with carbon disulphide in the presence of
10 M sodium hydroxide and methyl iodide in ice cold solution
(Scheme 4). Further N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-
1H-benzo[d]imidazol-2-amine
8 was synthesized by the
reaction of compound 7 with o-phenylenediamine in the
presence of N,N-dimethylformamide in refluxing (Scheme 4).
The structure of compound 8 was established on the basis of
spectroscopic and analytical data. Structure 8 was supported
by its mass spectrum (m/z 326) (Mþ), which agrees with its
molecular formula C20H15N5. Its IR spectrum (KBr/cmꢀ1
)
showed three bands at 3107, 3175, and 3232 which
corresponds to 3NH group and a band at 1608 related to
1
–
C N. Its H NMR spectrum (DMSO-d , d ppm) showed multiple
–
6
peaks of 12H proton at 6.96–8.90 due to aromatic ring, six
signals at 10.43, 12.43, and 12.50 due to 3NH which was
exchangeable.
Novel synthesized compounds 6a–q structure was con-
firmed by different spectroscopic tools. The IR spectra of the
O
S
O
–2H2O
N
H2N
N
NH
N
N
H
Ar
CH3COOH
O
NH2
NH
O
NH2
NH
H
N
N
N
cyclization
NH
NH
N
H
N
H
Ar
N
S
Ar
S
6a–q
Scheme 3. Mechanism of preparation of compounds 6a–q.
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H
N
N
SCH3
SCH3
10 M NaOH
CH3I
CS2
NH2
N
N
H
1
N
7
NH2
NH2
H
N
N
H
N
DMF
N
N
H
–2CH3-SH
8
Scheme 4. Preparation of N-(4-1H-benzo[d]imidazol-2-yl)phenyl)-1H-benzo[d]imidazol-2-amine.
Biological screening
line after the specified time. The concentration required for
50% inhibition of cell viability (IC50) is calculated and the
results are given in Table 1. The results were compared
to the antiproliferative effects of the reference control N-[1-
(3-benzyl-7-chloro-4-oxoquinazolin-2-yl)propyl]-4-bromo-N-
[3-(dimethylamino)propyl]benzamide CK0106023 [53]. The
results are presented in Table 1.
In vitro cytotoxicity
In vitro cytotoxic activity screening of the newly synthesized
compounds was carried out utilizing three different human
tumor cell lines namely colorectal cancer cell line HCT116,
human liver cancer cell line HepG2, and human ovarian cancer
cell line A2780 obtained from the American Type Culture
Collection (Rockville, MD, USA). The tumor cells are main-
tained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat inactivated fetal calf serum
(GIBCO), penicillin (100 U/mL), and streptomycin (100 mg/mL)
at 37°C in humidified atmosphere containing 5% CO2. Cells at
a concentration of 0.50 ꢁ 106 are grown in a 25 cm2 flask in
5 mL of complete culture medium.
The antiproliferative activity is measured in vitro using the
sulforhodamine b stain (SRB) assay according to the previous
reported standard procedure [52]. Cells are inoculated in
96-well micro titer plate (104 cells/well) for 24 h before
treating with the tested compounds to allow attachment of
cell to the wall of the plate. Tested compounds are dissolved
in DMSO at 1 mg/mL immediately before use and diluted to
the appropriate volume just before addition to the cell
culture. Different concentrations of the compounds under
test were added to the cells. Triplicate wells are prepared for
each individual dose.
The HCTI16 cell line was more sensitive to the tested
compounds than the other two tested cell lines. The tested
compounds showed IC50 in the range of 1.20–7.34 mM. Six
compounds 6b, 6c, 6e, 6f, 6k, and 6q showed IC50 < 2 mM,
the most active derivatives against the cell line was 6b N-(4-
(1H-benzo[d]imidazol-2-yl)phenyl)-7-amino-8,8a-dihydro-(2-
chlorophenyl)-5H-thiazolo[3,2-a]pyrimidine-6-carboamide.
Concerning the three chlorophenyl derivatives 6b, 6c, and 6d,
it is obvious that the 2-chlorophenyl derivative 6b was the
most active (IC50 ¼ 1.60 mM) while the 4-chlorophenyl isomer
6d was the least active (IC50 ¼ 7.34 mM). Similarly, concerning
the three methoxy derivatives 6e, 6f, and 6g, the most active
isomer was the 2-methoxyphenyl 6e (IC50 ¼ 1.20 mM) while
the least active was the 4-methoxyphenyl 6g (IC50 ¼ 3.11 mM).
The trimethoxyphenyl derivatives 6q showed higher cytotoxic
activity (IC50 ¼ 1.76 mM) than both 2,4-dimethoxyphenyl deriv-
ative 6n (IC50 ¼ 3.77 mM) and 2,5-dimethoxyphenyl derivative
6o (IC50 ¼ 2.83 mM). Conversion of the 2-methoxyphenyl
substituent 6e to 2-hydroxyphenyl 6h markedly decreased
the activity (IC50 ¼ 1.20 mM and 5.51 mm, respectively). On
contrast, the 4-hydroxyphenyl derivative 6i was more active
than the 4-methoxyphenyl derivatives 6g (IC50 ¼ 2.57 and
3.11 mM, respectively). With respect to the two nitrophenyl
derivatives 6j and 6k, the 4-nitrophenyl isomer was much
more active than the 2-nitrophenyl isomer (IC50 ¼ 1.32 and
4.14 mM, respectively).
Monolayer cells were incubated with the compounds for
48 h at 37°C and in atmosphere of 5% CO2. After 48 h, cells are
fixed, washed, and stained for 30 min with 0.4% w/v SRB
dissolved in 1% acetic acid. Unbound dye is removed by four
washes with 1% acetic acid, and attached stain was recovered
with Tris-EDTA buffer. Color intensity is measured in an ELISA
reader. The relation between surviving fraction and drug
concentration is plotted to get the survival curve for each cell
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Table 1. IC50 (mM) of the newly synthesized compounds;
IC50 values defined as the concentration corresponding to
50% growth inhibition.
respectively). In addition, the 2-nitrophenyl derivative 6j
was four times less active than the 4-nitro phenyl derivative
6k (IC50 ¼ 8.28 and 2.34 mM, respectively). Again, the
trimethoxy derivative 6g showed higher activity than
the two dimethoxy derivatives 6n and 6o (IC50 ¼ 3.21,
7.45, and 5.44 mM, respectively). The 4-methylphenyl
derivative 6l showed good activity against this cell line
(IC50 ¼ 4.89 mM). The 4-dimethylamino derivative showed
enhanced activity (IC50 ¼ 3.97 mM). Finally, the two com-
pounds 7 and 8 showed cytotoxic activity with IC50 ¼ 4.56
and 6.67 mM, respectively, against HCT116 cell line and
less potent against A2780 with IC50 ¼ 17.16 and 27.98 mM,
respectively, while they showed better activity against
HepG2 with IC50 ¼ 9.20 and 14.01 mM, respectively.
IC50 (mM)
Compounds
HCTI16
A2780
HepG2
1
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
60
5.01
2.34
1.60
1.93
7.34
1.20
1.45
3.11
5.51
2.57
4.14
1.32
6.07
2.13
3.77
2.83
3.42
1.76
4.56
6.67
1.00
19.39
7.29
4.47
5.71
31.61.
3.10
10.22
4.41
2.89.
3.57
15.56
2.11
2.60
6.04
11.35
4.89
8.28
2.34
12.61
3.97
7.45
5.44
6.71
3.21
9.20
14.01
2.15
3.96
10.52
21.91
8.24
15.18
3.50
24.76
6.45
13.44
9.31
11.89
5.05
17.16
27.98
3. 02
KSP inhibitory activity
Kinesin spindle protein, known as KSP or Hs Eg5, has recently
emerged as a key target for selective antimitotic cancer
therapy. KSP inhibitory activity was assessed by measuring the
release of inorganic phosphate from ATP hydrolysis through
absorbance detection of
a malachite green-phosphate
6p
6q
7
8
complex [54]. The IC50 values of the target compounds
against KSP were determined by measuring the activities of
microtubule-activated ATPase activity. Monastrol, CPUYL064,
and CK0106023 were used as positive controls.
The correlation between KSP inhibition and Aurora A
kinase inhibition and suppression of tumor growth is
validated as an important cancer target. We have been able
to design, synthesize, and evaluate a series of benzimidazoles
carrying a thiazolopyrimidine moiety as dual inhibitors of KSP
and Aurora A kinase. All the compounds identified through
this new design approach were highly potent KSP and Aurora
A inhibitory.
CK0106023
Regarding the A2870 cell line, the tested compounds showed
IC50 in the range of 3.10–24.76 mM. Three compounds 6e, 6f,
and 6k showed cytotoxic activity comparable to the standard
drug (IC50 ¼ 3.10, 3.96, and 3.50 mM respectively). Concerning
the three chlorophenyl derivatives 6b, 6c, and 6d, the most
active isomer was the 2-chlorophenyl 6b, while the least
active one was the 4-chlorophenyl 6d (IC50 ¼ 4.47 and
31.61 mM, respectively). Conversion of the 2-methoxyphenyl
6e to 2-hydroxyphenyl 6h decreased the cytotoxic activity
(IC50 ¼ 3.10 and 21.91 mM, respectively), while conversion of
the 4-methoxyphenyl derivatives 6g to 4-hydroxy increased
the activity (IC50 ¼ 10.52 and 8.24 mM, respectively). The
4-nitrophenyl derivative 6k also showed increased activity
than the 2-nitrophenyl 6j (IC50 ¼ 3.50 and 15.18 mM, respec-
tively). The trimethoxyphenyl derivatives 6q showed better
activity (IC50 ¼ 1.76 mM) than 2,4-dimethoxyphenyl derivatives
6n (IC50 ¼ 5.05 mM) and 2,5-dimethoxyphenyl derivatives 6o
(IC50 ¼ 2.83 mM).
The two derivatives 6e and 6k can serve as lead compounds
for further development as they showed potent dual KSP
and Aurora A kinase inhibition in addition to their in vitro
cytotoxic activity against the three tested cell lines.
Aurora kinase inhibitory activity
Aurora kinases (Auroras A, B, and C) are vital component
playing a critical role in regulating many of the processes that
are pivotal to mitosis, and they are required for healthy cell
growth and proliferation. It was reported that Aurora A
kinase is overexpressed in a broad range of tumor cell lines
and human primary tumors; thus, the inhibition of Aurora A
kinase, by small molecules, is becoming an area of major
current interest in oncology drug discovery [55]. In order to
obtain more potent compounds with specific inhibitory effect
against Aurora A kinase, we used the Aurora A kinase activity
assay kit developed by Cell Signaling.
The results are presented in Table 2 showing that all the
synthesized compounds exhibited significant inhibiting activ-
ities toward kinesin spindle protein KSP with the IC50 values in
the range of 0.0009–0.0055 mM and also toward Aurora A
kinase with the IC50 values in the range of 1.22–10.51 mM.
Compound 6a with a phenyl substituent showed remarkable
Tested against the HepG2 cell line, the synthesized
compounds showed IC50 in the range of 2.11–15.56 mM. Six
compounds 6b, 6c, 6e, 6f, 6k, and 6n showed good activity
with respect to the standard drug. Concerning the three
chlorophenyl derivatives 6b, 6c, and 6d, the most active
isomer was the 2-chlorophenyl derivative and the least active
one was the 4-chlorophenyl (IC50 ¼ 2.89 and 15.56 mM,
respectively). Similarly, the 2-methoxy phenyl derivative 6e
was the most active and the least active one was the 4-
methoxy phenyl derivative 6g (IC50 ¼ 2.11 and 6.04 mM,
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Benzimidazole Derivatives as Antitumor Agents
Archiv der Pharmazie
Table 2. KSP and Aurora A kinase inhibitory activities of
the newly synthesized compounds.
Conclusion
The most active compounds from the thiazolopyrimidine
derivative against the three tested cell lines were 6e and 6k
comparable to the standard drug. The correlation between
KSP inhibition and Aurora A kinase inhibition and suppression
of tumor growth is validated as an important cancer target.
We have been able to design, synthesize, and evaluate a series
of benzimidazoles carrying a thiazolopyrimidine moiety as
dual inhibitors of KSP and Aurora A kinase. All the compounds
identified through this new design approach were highly
potent KSP and Aurora A inhibitory. The two derivatives 6e
and 6k can serve as lead compounds for further development,
as they showed potent dual KSP and Aurora A kinase
inhibition in addition to their in vitro cytotoxic activity
against the three tested cell lines.
Aurora A kinase
Compounds
KSP IC50 (mM)
IC50 (mM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
0.0018
0.0012
0.0014
0.0055
0.0009
0.0011
0.0023
0.0041
0.0019
0.0034
0.0010
0.0045
0.0016
0.0031
0.0021
0.0028
0.0013
5.47
2.7
1.71
2.15
10.51
1.22
1.53
3.79
7.48
3.02
5.96
1.37
8.38
2.41
5.32
3.38
4.75
1.92
>50
40.34
>50
Experimental
6q
Chemistry
Monastrol
CPUYL064
CK0106023
All melting points were measured on a Gallenkamp melting
point apparatus and were uncorrected. Microanalyses of the
compounds were performed in the Micro-analytical Labora-
tory Center (Faculty of Science, Cairo University, Cairo, Egypt).
The IR spectra (4000–400 cmꢀ1) were recorded using KBr
pellets on a Perkin Elmer 1650 FT-IR instrument. The 1H NMR
(500 MHz) spectra were recorded on a Varian spectrometer
using DMSO-d6 as a solvent and TMS as an internal standard;
chemical shift was reported in ppm. The mass spectroscopy
was carried out using Finnigan mat SSQ 7000 (Thermo
Instrument Systems, Inc., USA) at 70 and 20 eV.
0.18
0.029
The inhibitory activities against KSP were determined by
measuring the MT-activated ATPase activity.
activity toward KSP and Aurora A kinase (IC50 ¼ 0.0018 and
2.7 mM, respectively). Concerning the chlorophenyl derivatives
6b, 6c, and 6d, the highest activity against KSP and Aurora A
kinase was observed in compound 6b which has a 2-
chlorophenyl substituent (IC50 ¼ 0.0012 and 1.71mM, respec-
tively). On the other hand, the isomer 6d with a 4-chlorophenyl
substituentpossessedmarkeddiminishedactivity(IC50 ¼ 0.0055
and 10.51 mM, respectively). Similarly, the 2-methoxyphenyl
derivative 6e was the most active (IC50 ¼ 0.0009 and 1.22 mM,
respectively), andthe leastactiveonewas the 4-methoxyphenyl
derivative 6g (IC50 ¼ 0.0023 and 3.79 mM, respectively). The
trimethoxyphenyl derivative 6q showed good activity
Preparation of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-2-
cyanoacetamide (5)
A mixture of 4-(1H-benzo[d]imidazol-2-yl)benzenamine 1
(0.001 mol) and excess ethyl cyanoacetate in absolute ethanol
(20 mL) was boiled under reflux for 3 h. The product separated
upon storing the reaction mixture to cool at room tempera-
ture. Then product was collected and crystallized from
ethanol.
toward KSP and Aurora
A
kinase (IC50 ¼ 0.0013 and
1.92 mM, respectively) more than 2,4-dimethoxyphenyl
derivatives 6n (IC50 ¼ 0.0031 and 5.32 mM, respectively) and
2,5-dimethoxyphenyl derivatives 6o (IC50 ¼ 0.0021 and
3.38 mM, respectively). The 4-nitrophenyl derivative 6k also
showed increased activity toward KSP and Aurora A kinase
(IC50 ¼ 0.001 and 1.37 mM, respectively) than the 2-nitrophenyl
6j (IC50 ¼ 0.0034 and 5.96mM, respectively). Similarly, the
4-hydroxyphenyl derivatives 6i also showed increased activity
toward KSP and Aurora A kinase (IC50 ¼ 0.0019 and 3.02 mM,
respectively) than the 2-hydroxyphenyl 6j (IC50 ¼ 0.0041 and
7.48 mM, respectively). Finally, the 4-dimethylaminophenyl
derivative 6m showed good activity against the KSP and
Aurora A kinase (IC50 ¼ 0.0016 and 2.41 mM, respectively).
The 4-methylphenyl derivative 6l showed diminished activity
(IC50 ¼ 0.0045 and 8.38 mM, respectively).
Yield 85%, m.p. 265–267°C. 1H NMR (500 MHz, DMSO-d6):
3.78 (s, 2H, CH2), 6.63–7.68 (m, 4H, aromatic), 7.78 (m, 2H, H4,
and H7 benzimidazole), 8.11 (m, 2H, H5, and H6 benzimid-
azole), 10.59 (s, 1H, NH amide exchangeable), 12.04 (s, 1H, NH
benzimidazole, exchangeable). MS: m/z 275.90 (M, 20.53%).
IR (KBr, n/cmꢀ1): n (NH benzimidazole) 3334, n (NH amide, str.)
ꢀꢀ
–
–
3055, n (CꢀꢀN) 2370, n (C O) 1673, n (C N benzimidazole ring)
–
–
ꢀꢀ
1605. Anal. calcd. for C16H12N4O (%) M.W. 276: C, 69.55; H,
4.38; N, 20.28. Found: C, 69.70; H, 4.17; N, 20.09.
Preparation of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-7-
amino-5-(substituted benzylidine)-8,8a-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxamide (6a–q)
An equimolar amount (0.01 mol) mixture of cyanoacetamide
compound 5 with the appropriate aldehyde and 2-amino
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7

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
A. S. Abd El-All et al.
Archiv der Pharmazie
thiazole in glacial acetic acid (50 mL) was heated under reflux
for appropriate time. The reaction mixture cooled at room
temperature for 24 h. Then product was filtered off and
crystallized from methanol.
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
chlorophenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxamide (6d)
Yield 81%, m.p. 290–292°C. 1H NMR (500 MHz, DMSO-d6): 2.49
(s, 1H, H5 pyrimidine ring); 4.28 (br, 2H, NH2, exchangeable
D2O); 5.89 (s, 1H, H8a pyrimidine ring); 7.11–7.53 (m, 10H,
thiazole, and aromatic rings), 7.72 (m, 2H, H4, and H7,
benzimidazole); 8.06 (m, 2H, H5, H6, benzimidazole); 10.16
(s, 1H, NH amid exchangeable D2O); 10.55 (s, 1H, NH pyrimidine
exchangeable D2O); 12.02 (s, 1H, NH benzimidazole exchange-
able D2O). MS: m/z 501 (Mþ, 13.39%). IR (KBr, n/cmꢀ1): n (NH2)
3351, 3295, n (NH amid) 3261, n (NH pyrimidine) 3215, n (NH
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-
phenyl-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-
carboxamide (6a)
Yield 83%, m.p. 297–299°C. 1H NMR (500 MHz, DMSO-d6): 2.47
(s, 1H, H5 pyrimidine); 4.30 (br, 2H, NH2 exchangeable D2O);
6.20 (s, 1H, H8a pyrimidine); 6.70–7.50 (m, 11H, aromatic, and
thiazole); 7.70 (m, 2H, H4, H7 benzimidazole), 8.06 (m, 2H, H5,
H6 benzimidazole); 10.18 (s, 2H, NH amid exchangeable D2O);
10.55 (s, 1H, NH pyrimidine exchangeable D2O); 12.06 (br, 1H,
NH benzimidazole exchangeable D2O). MS: m/z 466 (Mþ), IR
(KBr, n/cmꢀ1): n (NH2) 3320, 3295, n (NH amid) 3270, n (NH
–
–
benzimidazole) 3128, n (C O) 1673, n (C N benzimidazole ring)
–
–
1603, n (C–S benzimidazole ring) 1370; n (C–Cl) 749. Anal. calcd.
for C26H21ClN6OS (%) M.W. 501: C, 62.33; H, 4.22; N, 16.77; Cl,
7.08; S, 6.40. Found: C, 62.30; H, 4.21; N, 16.79; Cl, 7.01; S, 6.05.
–
pyrimidine) 3196, n (NH benzimidazole) 3127, n (C O) 1677, n
–
–
(C N benzimidazole ring) 1605, n (C–S benzimidazole ring)
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2-
methoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6e)
–
1375, 3196. Anal. calcd. for C26H22N6OS (%) M.W. 466: C,
66.93; H, 4.75; N, 18.01; S, 6.87. Found: C, 66.99; H, 4.76; N,
18.04; S, 6.49.
Yield 80%, m.p. 297–298°C. 1H NMR (500 MHz, DMSO-d6): 2.47
(s, 1H, H5 pyrimidine ring); 3.85 (s, 3H, OCH3); 4.40 (br, 2H,
NH2, exchangeable D2O); 5.96 (s, 1H, H8a pyrimidine ring);
7.00–7.53 (m, 10H, thiazole, and aromatic rings), 7.72 (m, 2H,
H4, and H7, benzimidazole); 8.07 (m, 2H, H5, H6, benzimid-
azole); 10.17 (s, 1H, NH amid exchangeable D2O); 10.45 (s, 1H,
NH pyrimidine exchangeable D2O); 12.01 (s, 1H, NH benz-
imidazole exchangeable D2O). MS: m/z 497 (Mþ2, 33.20%). IR
(KBr, n/cmꢀ1): n (NH2) 3321, 3290, n (NH amid) 3272, n (NH
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2-
chlorophenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxamide (6b)
Yield 73%, m.p. 255–257°C. 1H NMR (500 MHz, DMSO-d6): 2.42
(s, 1H, H5 pyrimidine); 4.32 (br, 2H, NH2 exchangeable D2O);
6.19 (s, 1H, H8a pyrimidine); 7.09–7.54 (m, 10H, aromatic, and
thiazole); 7.71 (m, 2H, H4, H7 benzimidazole), 8.06 (m, 2H, H5,
H6 benzimidazole); 10.14 (s, 2H, NH amid exchangeable D2O);
10.55 (s, 1H, NH pyrimidine exchangeable D2O); 12.14 (br, 1H,
NH benzimidazole exchangeable D2O). MS: m/z 501, 503 (M,
Mþ2). IR (KBr, n/cmꢀ1): n (NH2) 3320, 3290, n (NH amid) 3262, n
–
pyrimidine) 3220, n (NH benzimidazole) 3127, n (C O) 1678, n
–
–
(C N benzimidazole ring) 1604, n (C–S benzimidazole ring)
–
1369. Anal. calcd. for C27H24N6O2S(%) M.W. 497: C, 65.30; H,
4.87; N, 16.92; S, 6.46. Found: C, 65.36; H, 4.88; N, 16.95; S, 6.10.
–
(NH pyrimidine) 3210, n (NH benzimidazole) 3128, n (C O)
–
–
1677, n (C N benzimidazole ring) 1605, n (C–S benzimidazole
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(3-
methoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6f)
–
ring) 1369; n (C–Cl) 750. Anal. calcd. for C26H21ClN6OS (%)
M.W. 501: C, 62.33; H, 4.22; N, 16.77; Cl, 7.08; S, 6.40. Found: C,
62.39; H, 4.23; N, 16.82; Cl, 7.05; S, 6.04.
Yield 82%, m.p. 295°C. 1H NMR (500 MHz, DMSO-d6): 2.47 (s,
1H, H5 pyrimidine ring); 3.80 (s, 3H, OCH3); 4.40 (br, 2H, NH2,
exchangeable D2O); 5.96 (s, 1H, H8a pyrimidine ring); 7.14–
7.54 (m, 10H, thiazole, and aromatic rings), 7.72 (m, 2H, H4,
and H7, benzimidazole); 8.07 (m, 2H, H5, H6, benzimidazole);
10.19 (s, 1H, NH amid exchangeable D2O); 10.50 (s, 1H, NH
pyrimidine exchangeable D2O); 12.10 (s, 1H, NH benzimid-
azole exchangeable D2O). MS: m/z 496 (Mþ, 52.32%). IR (KBr,
n/cmꢀ1): n (NH2) 3320, 3295, n (NH amid) 3260, n (NH
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(3-
chlorophenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxamide (6c)
Yield 65%, m.p. 252–254°C. 1H NMR (500 MHz, DMSO-d6): 2.46
(s, 1H, H5 pyrimidine ring); 4.30 (br, 2H, NH2, exchangeable
D2O); 6.09 (s, 1H, H8a pyrimidine ring); 7.13–7.53 (m, 10H,
thiazole, and aromatic rings), 7.14–77.70 (m, 2H, H4, and H7,
benzimidazole); 8.07 (m, 2H, H5, H6, benzimidazole); 10.19 (s,
1H, NH amid exchangeable D2O); 10.51 (s, 1H, NH pyrimidine
exchangeable D2O); 12.02 (s, 1H, NH benzimidazole ex-
changeable D2O). MS: m/z 501 (Mþ). IR (KBr, n/cmꢀ1): n (NH2)
3350, 3290, n (NH amid) 3262, n (NH pyrimidine) 3200, n (NH
–
pyrimidine) 3200, n (NH benzimidazole) 3124, n (C O) 1673,
–
–
n (C N benzimidazole ring) 1605, n (C–S benzimidazole ring)
–
1370. Anal. calcd. for C27H24N6O2S (%) M.W. 497: C, 65.30; H,
4.87; N, 16.92; S, 6.46. Found: C, 65.31; H, 4.82; N, 16.91; S, 6.13.
–
–
benzimidazole) 3127, n (C O) 1673, n (C N benzimidazole
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
methoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6g)
–
–
ring) 1604, n (C–S benzimidazole ring) 1377; n (C–Cl) 749. Anal.
calcd. for C26H21ClN6OS (%) M.W. 501: C, 62.33; H, 4.22; N,
16.77; Cl, 7.08; S, 6.40. Found: C, 62.37; H, 4.24; N, 16.82; Cl,
7.09; S, 6.09.
Yield 75%, m.p. 275–277°C. 1H NMR (500 MHz, DMSO-d6): 2.47
(s, 1H, H5 pyrimidine ring); 3.84 (s, 3H, OCH3); 4.30 (br, 2H,
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8

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Benzimidazole Derivatives as Antitumor Agents
Archiv der Pharmazie
NH2, exchangeable D2O); 5.92 (s, 1H, H8a pyrimidine ring);
7.15–7.54 (m, 10H, thiazole, and aromatic rings), 7.71 (m, 2H,
H4, and H7, benzimidazole); 8.05 (m, 2H, H5, H6, benzimid-
azole); 10.17 (s, 1H, NH amid exchangeable D2O); 10.52 (s,
1H, NH pyrimidine exchangeable D2O); 12.112 (s, 1H, NH
benzimidazole exchangeable D2O). MS: m/z 498 (Mþ,
52.32%). IR (KBr, n/cmꢀ1): n (NH2) 3350, 3269, n (NH amid)
3260, n (NH pyrimidine) 3207, n (NH benzimidazole) 3122, n
benzimidazole); 8.79 (m, 2H, H5, H6, benzimidazole); 10.14 (s,
1H, NH amid exchangeable D2O); 10.64 (s, 1H, NH pyrimidine
exchangeable D2O); 12.10 (s, 1H, NH benzimidazole ex-
changeable D2O); MS: m/z 511 (M, 28%), 172 (m1, 100%).
IR (KBr, n/cmꢀ1): n (NH2) 3320, 3263, n (NH amid) 3201, n (NH
–
pyrimidine) 3160, n (NH benzimidazole) 3051, n (C O) 1670,
–
–
n (C N benzimidazole ring) 1605, n (C–S benzimidazole ring)
–
1369. Anal. calcd. for C26H21N7O3S (%) M.W. 511: C, 61.04; H,
4.14; N, 19.17; S, 6.27. Found: C, 61.10; H, 4.15; N, 19.20; S, 6.09.
–
–
(C O) 1673,
n (C N benzimidazole ring) 1603, n (C–S
–
–
benzimidazole ring) 1370. Anal. calcd. for C27H24N6O2S (%)
M.W. 497: C, 65.30; H, 4.87; N, 16.92; S, 6.46. Found: C, 65.31;
H, 4.82; N, 16.91; S, 6.13.
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
nitrophenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxamide (6k)
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2-
hydroxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6h)
Yield 96%, m.p. 275–277°C. 1H NMR (500 MHz, DMSO-d6): 2.48
(s, 1H, H5 pyrimidine ring); 4.29 (br, 2H, NH2, exchangeable
D2O); 5.78 (s, 1H, H8a pyrimidine ring); 7.08–7.13 (m, 10H,
thiazole, and aromatic rings), 7.72 (m, 2H, H4, and H7,
benzimidazole); 8.06 (m, 2H, H5, H6, benzimidazole); 10.16 (s,
1H, NH amid exchangeable D2O); 10.57 (s, 1H, NH pyrimidine
exchangeable D2O); 12.08 (s, 1H, NH benzimidazole ex-
changeable D2O); MS: m/z 511 (M, 34%). IR (KBr, n/cmꢀ1): n
(NH2) 3319, 3260, n (NH amid) 3192, n (NH pyrimidine) 3163, n
Yield 91%, m.p. 220–222°C. 1H NMR (500 MHz, DMSO-d6): 2.46
(s, 1H, H5 pyrimidine ring); 4.80 (br, 2H, NH2, exchangeable
D2O); 6.02 (s, 1H, H8a pyrimidine ring); 6.80–7.53 (m, 10H,
thiazole, and aromatic rings), 7.71 (m, 2H, H4, and H7,
benzimidazole); 8.07 (m, 2H, H5, H6, benzimidazole); 10.20 (s,
1H, NH amid exchangeable D2O); 10.40 (s, 1H, NH pyrimidine
exchangeable D2O); 10.80 (s, 1H, NH benzimidazole ex-
changeable D2O); 12.00 (br, 1H, OH exchangeable D2O); MS:
m/z 481 (Mꢀ1, 9.08%). IR (KBr, n/cmꢀ1): n (OH) 3520, n (NH2)
3319, 3270, n (NH amid) 3200, n (NH pyrimidine) 3167, n (NH
–
–
(NH benzimidazole) 3052, n (C O) 1670, n (C N benzimidazole
–
–
ring) 1605, n (C–S benzimidazole ring) 1373. Anal. calcd. for
26H21N7O3S (%) M.W. 511: C, 61.04; H, 4.14; N, 19.17; S, 6.27.
C
Found: C, 61.08; H, 4.10; N, 19.18; S, 6.11.
–
–
benzimidazole) 3052, n (C O) 1677, n (C N benzimidazole
–
–
ring) 1604, n (C–S benzimidazole ring) 1374. Anal. calcd. for
26H22N6O2S (%) M.W. 482: C, 64.71; H, 4.60; N, 17.42; S, 6.64.
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
methylphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
C
Found: C, 64.88; H, 4.62; N, 17.45; S, 6.24.
pyrimidine-6-carboxamide (6l)
Yield 63%, m.p. >300°C. 1H NMR (500 MHz, DMSO-d6): 2.06 (s,
3H, CH3); 2.46(s, 1H, H5 pyrimidine ring); 4.30 (br, 2H, NH2,
exchangeable D2O); 5.84 (s, 1H, H8a pyrimidine ring); 6.81–
7.95 (m, 10H, thiazole, and aromatic rings), 7.78 (m, 2H, H4,
and H7, benzimidazole); 8.06 (m, 2H, H5, H6, benzimidazole);
10.17 (s, 1H, NH amid exchangeable D2O); 10.60 (s, 1H, NH
pyrimidine exchangeable D2O); 12.09 (s, 1H, NH benzimid-
azole exchangeable D2O). MS: m/z (480Mþ1). IR (KBr, n/cmꢀ1):
n (NH2) 3352, 3266, n (NH amid) 3205, n (NH pyrimidine) 3172, n
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
hydroxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6i)
Yield 90%, m.p. 229–230°C. 1H NMR (500 MHz, DMSO-d6): 2.46
(s, 1H, H5 pyrimidine ring); 4.27 (br, 2H, NH2, exchangeable
D2O); 6.90 (s, 1H, H8a pyrimidine ring); 7.14–7.53 (m, 10H,
thiazole, and aromatic rings), 7.72 (m, 2H, H4, and H7,
benzimidazole); 8.08 (m, 2H, H5, H6, benzimidazole); 10.16 (s,
1H, NH amid exchangeable D2O); 10.40 (s, 1H, NH pyrimidine
exchangeable D2O); 10.60 (s, 1H, NH benzimidazole ex-
changeable D2O); 12.09 (br, 1H, OH exchangeable D2O); MS:
m/z 480 (Mꢀ2, 13.32%). IR (KBr, n/cmꢀ1): n (OH) 3580, n (NH2)
3322, 3250, n (NH amid) 32197, n (NH pyrimidine) 3163, n (NH
–
–
–
(NH benzimidazole) 3050, n (C O) 1698, n (C N benzimidazole
–
ring) 1610, n (C–S benzimidazole ring) 1371. Anal. calcd. for
28H27N6OS (%) M.W. 480: C, 67.48; H, 5.03; N, 17.49; S, 6.69.
C
Found: C, 67.54; H, 5.05; N, 17.52; S, 6.30.
–
–
benzimidazole) 3051, n (C O) 1677, n (C N benzimidazole
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
(dimethylamino)phenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6m)
–
–
ring) 1605, n (C–S benzimidazole ring) 1374. Anal. calcd. for
26H22N6O2S (%) M.W. 482: C, 64.71; H, 4.60; N, 17.42; S, 6.64.
C
Found: C, 64.77; H, 4.61; N, 17.45; S, 6.27.
Yield 71%, m.p. 295–297°C. 1H NMR (500 MHz, DMSO-d6): 2.06
(s, 6H, 2CH3); 2.46 (s, 1H, H5 pyrimidine ring); 4.30 (br, 2H, NH2,
exchangeable D2O); 5.84 (s, 1H, H8a pyrimidine ring); 6.81–
7.59 (m, 10H, thiazole, and aromatic rings), 7.78 (m, 2H, H4,
and H7, benzimidazole); 8.06 (m, 2H, H5, H6, benzimidazole);
10.17 (s, 1H, NH amid exchangeable D2O); 10.60 (s, 1H, NH
pyrimidine exchangeable D2O); 12.09 (s, 1H, NH benzimid-
azole exchangeable D2O). MS: m/z 511 (Mþ2, 38.46%). IR
(KBr, n/cmꢀ1): n (NH2) 3327, 3258, n (NH amid) 3210, n (NH
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2-
nitrophenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]pyrimidine-
6-carboxamide (6j)
Yield 86%, m.p. 215–217°C. 1H NMR (500 MHz, DMSO-d6): 2.46
(s, 1H, H5 pyrimidine ring); 4.31 (br, 2H, NH2, exchangeable
D2O); 5.85 (s, 1H, H8a pyrimidine ring); 7.15–7.93 (m, 10H,
thiazole, and aromatic rings), 8.26 (m, 2H, H4, and H7,
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A. S. Abd El-All et al.
Archiv der Pharmazie
–
pyrimidine) 3167, n (NH benzimidazole) 3051, n (C O) 1673, n
–
–
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(3,4,5-
trimethoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6q)
–
(C N benzimidazole ring) 1607, n (C–S) 1375. Anal. calcd. for
28H27N7OS (%) M.W. 509: C, 65.99; H, 5.34; N, 19.24; S, 6.29.
C
Found: C, 66.05; H, 5.36; N, 19.27; S, 6.52.
Yield 69%, m.p. 265–267°C. 1H NMR (500 MHz, DMSO-d6): 2.47
(s, 1H, H5 pyrimidine ring); 3.61(s, 3H, 3OCH3); 3.68 (s, 3H,
3OCH3); 3.80 (s, 3H, 3OCH3); 4.16 (br, 2H, NH2 exchangeable
D2O); 5.83 (s, 1H, H8a pyrimidine ring); 6.55–7.55 (m, 8H,
thiazole, and aromatic rings); 7.72 (m, 2H, H4, and H7,
benzimidazole); 8.08 (m, 2H, H5, H6, benzimidazole); 10.20 (s,
1H, NH amid exchangeable D2O); 10.53 (s, 1H, NH pyrimidine
exchangeable D2O); 12.07 (s, 1H, NH benzimidazole ex-
changeable D2O). MS: m/z 554 (Mꢀ2, 21.66%). IR (KBr, n/cmꢀ1):
n (NH2) 3315, 3272, n (NH amid) 3203, n (NH pyrimidine) 3174, n
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2,4-
dimethoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6n)
Yield84%,m.p.268–270°C. 1HNMR(500MHz,DMSO-d6):2.46(s,
1H, H5 pyrimidine ring); 3.72 (s, 3H, 3OCH3); 3. 75 (s, 3H, 2OCH3);
4. 26 (br, 2H, NH2, exchangeable D2O); 5.96 (s, 1H, H8a
pyrimidine ring); 7.53–7.59 (m, 9H, thiazole, and aromatic
rings), 7.71 (m, 2H, H4, and H7, benzimidazole); 8.06 (m, 2H, H5,
H6, benzimidazole); 10.74 (s, 1H, NH amid exchangeable D2O);
11.96 (s, 1H, NH pyrimidine exchangeable D2O); 12.74 (s, 1H, NH
–
–
(NH benzimidazole) 3046, n (C O) 1678, n (C N benzimidazole
–
–
ring) 1602, n (C–S) 1371. Anal. calcd. for C29H28N6O4S (%) M.W.
556: C, C, 62.57; H, 5.07; N, 15.10; S, 5.76. Found: C, 63.63; H,
5.09; N, 15.13; S, 5.44.
benzimidazoleexchangeableD2O). MS:m/z 526(Mþ),465(Mþ2
,
–2OCH3, 30.77%). IR (KBr, n/cmꢀ1): n (NH2) 3323, 3250, n (NH
amid) 3203, n (NH pyrimidine) 3170, n (NH benzimidazole) 3048,
–
–
n (C O) 1677, n (C N benzimidazole ring) 1606, n (C–S) 1370.
Preparation of dimethyl-4-(1H-benzo[d]imidazol-2-yl)-
phenylcarbonimidodithioate (7)
–
–
Anal. calcd. for C28H26N6O3S (%) M.W. 526: C, 63.86; H, 4.98; N,
15.96; S, 6.09. Found: C, 64.90; H, 4.96; N, 16.00; S, 5.78.
To a well-stirred ice cold solution of 1 (0.025 mol, 5.25 g) in
dimethyl formamide (10 mL) was added 10 M NaOH aqueous
solution (4 mL). Carbon disulphide (0.05 mol, 3.01 g) was
added with further stirring for 30 min. Finally, methyl iodide
(0.025 mol,1.55 g) was added and the mixture was stirred for
3 h, poured in ice cold water, and the resulting solid was
washed with water and recrystallized from aq. ethanol. Yield
81%, m.p. 189–190°C. 1H NMR (500 MHz, HDMS-d6): 3.93 (s,
6H, 2SCH3); 8.01–6.8 (m, 8H, aromatic); 11.82 (br, 1H, NH
exchangeable). MS m/z: 313 (Mþ). IR (KBr, n/cmꢀ1): n (NH)
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(2,5-
dimethoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-a]-
pyrimidine-6-carboxamide (6o)
Yield 93%, m.p. 210–212°C. 1H NMR (500 MHz, DMSO-d6): 2.46
(s, 1H, H5 pyrimidine ring); 3.05 (s, 3H, 3OCH3); 3.65 (s, 3H,
2OCH3); 4.26 (br, 2H, NH2, exchangeable D2O); 6.20 (s, 1H, H8a
pyrimidine ring); 6.82–7.53 (m, 9H, thiazole, and aromatic
rings), 7.71 (m, 2H, H4, and H7, benzimidazole); 8.07 (m, 2H, H5,
H6, benzimidazole); 10.15 (s, 1H, NH amid exchangeable D2O);
10.50 (s, 1H, NH pyrimidine exchangeable D2O); 12.00 (s, 1H, NH
–
–
3434; n (C N) 1632; n (C N benzimidazole) 1608; n (2C–S) 1263,
–
–
1210. Anal. calcd. for C16H15N3S2 (M.W.: 313) Calc: C, 61.31; H,
4.82; N, 13.41; S, 20.46; Found C 61.53, H 4.77, N 13.40; S, 19.29.
benzimidazoleexchangeableD2O).MS:m/z526(Mþ),465(Mþ2
,
–2OCH3, 30.77%). IR (KBr, n/cmꢀ1): n (NH2) 3325, 3261, n (NH
amid)3205, n (NH pyrimidine) 3174, n (NH benzimidazole) 3050,
Preparation of N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-
1H-benzo[d]imidazol-2-amine (8)
–
–
–
n (C O) 1670, n (C N benzimidazole ring) 1603, n (C–S) 1372.
–
Anal. calcd. for C28H26N6O3S (%) M.W. 526: C, 63.86; H, 4.98;
N, 15.96; S, 6.09. Found: C, 64.92; H, 4.99; N, 15.99; S, 5.75.
To a solution of 7 (1.5 g, 0.005mol) in N,N-dimethylformamide
(20 mL) was added a solution of o-phenylenediamine (0.43 g,
0.004mol) in DMF (15mL) with stirring at room temperature.
The reaction mixture was refluxed for 8 h. The mixture was
cooledthenpouredoncrushedice.Theresultingsolidwasdried
and recrystallized from ethanol. Yield 93%, m.p. 269–271°C.
1H NMR (500MHz, HDMS-d6): 8.9–7.16 (m, 12H, aromatic);
10.43, 12.43, 12.50 (s, 3H, 3NH exchangeable). MS m/z: 326
N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-7-amino-5-(4-
hydroxy-3-methoxyphenyl)-8,8a-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxamide (6p)
Yield 97%, m.p. >300°C. 1H NMR (500 MHz, DMSO-d6): 2.46 (s,
1H, H5 pyrimidine ring); 3.75(s, 3H, OCH3); 4.26 (br, 2H, NH2
exchangeable D2O); 6.00 (s, 1H, H8a pyrimidine ring); 7.14–
7.52 (m, 9H, thiazole, and aromatic rings), 7.71 (m, 2H, H4, and
H7, benzimidazole); 8.04 (m, 2H, H5, H6, benzimidazole);
10.15 (s, 1H, NH amid exchangeable D2O); 10.30 (s, 1H, NH
pyrimidine exchangeable D2O); 10.90 (s, 1H, NH benzimid-
azole exchangeable D2O); 12.50 (br, 1H, OH exchangeable
D2O). MS: m/z 511 (M, 38.46%). IR (KBr, n/cmꢀ1): n (OH) 3590, n
(NH2) 3345, 3261, n (NH amid) 3205, n (NH pyrimidine) 3170, n
(Mþ1). IR (KBr, n/cmꢀ1): n (3NH) 3312, 3232, 3107; n (C N
–
–
benzimidazole) 1608. Anal. calcd. for C20H15N5 (M.W: 325) Calc:
C, 73.83; H, 4.65; N, 21.52; Found C 73.89, H 4.57, N 21.55.
Pharmacology
All the newly synthesized compounds were evaluated in
Research Unit (Saco Pharm. Co., 6 October City, Egypt).
–
–
(NH benzimidazole) 3056, n (C O) 1673, n (C N benzimidazole
Preparation of Eg5
–
–
ring) 1603, n (C–S) 1369. Anal. calcd. for C27H24N6O3S (%) M.W.
512: C, 63.40; H, 4.72; N, 16.43; S, 6.26. Found: C, 63.33; H, 4.73;
N, 16.23; S, 6.41.
Coding regions were PCR amplified from a template
containing full-length human Eg5. The primers used were
forward 50-TATAGG GCG AAT TCC GCC ATG GCG TCG CAG
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Benzimidazole Derivatives as Antitumor Agents
Archiv der Pharmazie
CCA-30 and reverse 50-ACG GGC TGC AGC AAG CTC GAG TTT
TAAACG TTC TAT-30. The region encoding residues 2–386 was
subcloned into pET28a (NOVAGEN). Protein expression in
Escherichia coli cells was induced with 0.5 mM isopropyl b-D-I-
thiogalactopyranoside IPTG. Cells were harvested after 20 h of
growth at 20 °C and then disrupted by sonication. The soluble
lysate was clarified by centrifugation and applied to a SP-
Sepharose column (Amersham Pharmacia Biotech) in a buffer
A (20 mM 1,4-piperazineddiethanesulfonic acid sodium salt
Na-PIPES, pH 6.3; 20 mM NaCl; 1 mM MgCl2; 1 mM ethylene
glycol-bis(b-aminoethyl ether)-N,N,N0,N0-tetraactic acid tetra-
sodium salt Na-EGTA). Protein was eluted with a linear
gradient of 20–1000 mM NaCl. Eg5 was identified by SDS–
PAGE, and then applied to Mono-Q columns (Amersham
Pharmacia Biotech) in a buffer B (20 mM Tris–HCl, pH 8.8;
1 mM MgCl2; 1 mM Na-EGTA). A gradient from 0 to 1000 mM
NaCl was used to elute Eg5. Fractions were analyzed by SDS–
PAGE. The most concentrated fraction was dialyzed against
ATPase buffer (20 mM Na-PIPES, pH 7.5; 1 mM MgCl2; 1 mM
Na-EGTA) and then aliquoted, frozen in liquid nitrogen, and
stored at ꢀ80 °C.
control experiment was done under the same conditions
without the tested compounds. The reaction was stopped by
addition of 50 mmol/L ethylene diamine tetra acetic acid
EDTA (pH 8). Then, 30 mL reaction mixtures were transferred
to a streptavidin-coated 96-well plate (PerkinElmer, Inc.) and
incubated at room temperature for 60 min. After washing
thrice with PBS/T, the phosphor PLK (Ser10) antibody (Cell
Signaling Technology) was added to the plate for further
incubation at 37°C for 60 min. After washing, HRP-labeled
secondary antibody was added. After incubation at room
temperature for 30 min, the plate was finally washed five
times and the fluorescence signal was determined with BMG
Polar star Galaxy (Germany) at 450 nm. The data were
analyzed using Microsoft Excel to obtain the IC50 of the
tested compounds.
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the final reaction of the assay contained 20 mM PIPES, pH 7.5,
5.0 mM MgCl2, 1 mM EGTA, 10 mM paclitaxel, 0.6 mM tubulin
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