Synthesis of terphenyl benzimidazoles as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.01.004

A series of new terphenyl benzimidazoles (3a-z and 3aa-ad) were synthesized and evaluated for their anticancer activity. All the 30 compounds have shown moderate to good anticancer potency, however some of the compounds (3j, 3m-t and 3aa-ad) exhibited prominent anticancer potency with GI₅₀ values ranging from <0.1 to 9.72 μM. These compounds exhibit G2/M phase arrest and the analysis of tubulin by Western blot experiments in case of 3t and 3ad shows the disturbances that are caused in the ratio of soluble versus polymerized tubulin in cells. Compounds 3t and 3ad are the most promising candidates amongst the series and has the potential to be taken up for further detailed studies either alone or in combination with the existing therapies.

Full text of DOI:10.1016/j.ejmech.2012.01.004

European Journal of Medicinal Chemistry 50 (2012) 9e17
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of terphenyl benzimidazoles as tubulin polymerization inhibitors
,
c
,
a
b
a
a
a
Ahmed Kamal a , M. Kashi Reddy , Thokhir B. Shaik , Rajender , Y.V.V. Srikanth , V. Santhosh Reddy ,
*
G. Bharath Kumar a, Shasi V. Kalivendi b
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500607, India
b Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad 500607, India
c Catalytic Chemistry Chair, Chemistry Department, College of Science, King Saud University, 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:
A series of new terphenyl benzimidazoles (3aez and 3aaead) were synthesized and evaluated for their
anticancer activity. All the 30 compounds have shown moderate to good anticancer potency, however
some of the compounds (3j, 3mꢀt and 3aaꢀad) exhibited prominent anticancer potency with GI50
Received 19 July 2011
Received in revised form
3 January 2012
Accepted 5 January 2012
Available online 12 January 2012
values ranging from <0.1 to 9.72 mM. These compounds exhibit G2/M phase arrest and the analysis of
tubulin by Western blot experiments in case of 3t and 3ad shows the disturbances that are caused in the
ratio of soluble versus polymerized tubulin in cells. Compounds 3t and 3ad are the most promising
candidates amongst the series and has the potential to be taken up for further detailed studies either
alone or in combination with the existing therapies.
Keywords:
Terphenyls
Suzuki coupling
Benzimidazoles
Anticancer agents
Tubulin polymerization
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
inhibitors is that they bind to the tubulin binding sites thereby
stabilizing (taxol, 1c) or destabilizing (nocodazole, 1b) microtubule
Cancer is a major worldwide health problem which is the
second leading cause of mortality in developed countries [1] and
most frightening disease in humans. Improvements in treatment
and prevention have led to a decrease in cancer deaths, but the
number of new diagnoses continues to rise. Treatment of cancer
cells with agents that interfere with microtubule assembly causes
mitotic arrest and eventually cell death. Microtubules are among
the most successful targets for anticancer therapy [2]. Composed
of dynamic polymers of tubulin, microtubules are elements of the
cell cytoskeleton that play key roles in cell shape [3]. During
mitosis, tightly regulated microtubule dynamics is essential for
spindle formation and chromosomal separation. Therefore, rapidly
dividing cells are more susceptible to inhibitors of tubulin poly-
merization than non-dividing cells and impair microtubule
dynamics and consequently arrest cells during mitosis [4]. The
hallmark of microtubule-interfering drugs is mitotic arrest that
eventually leads to apoptosis [5]. The mode of action of tubulin
assembly. Disruption of microtubule leads to cell cycle arrest at
G2/M phase followed by apoptotic cell death [6]. Various naturally
occurring compounds which act as tubulin targeting agents are
currently in clinical trials, which stabilize or destabilize microtu-
bule by specifically binding to anyone of the three reported
binding sites [7]. Current microtubule inhibitory agents used in
the clinic have severe side effects, and development of resistance
is frequent.
Over the past few years there is a considerable interest in the
development and pharmacology of heteroaromatic organic
compounds, such as benzimidazoles [8,9], due to their wide range
of biological activities such as antitumor [10ꢀ12], antibacterial
[13,14], antihypertensive [15], anti-inflammatory [16], vasodilator
[17] and antiviral [18ꢀ20] agents. Benzimidazole substituted
derivatives are also known inhibitors of cyclin dependent kinase or
tyrosine kinase and are useful for inhibiting cell proliferation for
the treatment of cancer [21ꢀ23]. The fact that benzimidazole
residue is a constituent of vitamin B12 supports their potential use
as therapeutics. Due to the structural similarity of benzimidazole
nuclei with some naturally occurring compounds such as purine,
they can easily interact with biomolecules of the living systems.
Bisbenzimidazole derivatives such as Hoechst 33258 (1a) has
undergone Phase I clinical evaluation as an anticancer agent and
* Corresponding author. Division of Organic Chemistry, Indian Institute of
Chemical Technology, Hyderabad 500607, India. Tel.: þ91 40 27193157; fax: þ91 40
27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.004

10
A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
the inhibition of topoisomerase and DNA helicase has been
proposed as its mechanism of action [24,25]. Moreover, terphenyls
are aromatic hydrocarbons comprising of a chain of three benzene
rings. These compounds can be structurally classified into three
types in which the terminal rings are o-, m- and p-substitutions of
the central aromatic ring, most natural terphenyls are para-ter-
phenyl derivatives. A number of terphenyl containing molecules
have been synthesized as useful therapeutic agents for various
diseases over the past few years including potent immunosup-
pressant [26], neuroprotective [27] antioxidant [28] antithrombotic
[29], anticoagulant [30], antibacterial [31] and anticancer activity
(2) [32ꢀ35]. Thus, due to the diverse range of the pharmacological
activities of terphenyl and its derivatives, it was considered of
interest to explore different substituted terphenyl derivatives as
a pharmacophore in the design of benzimidazole compounds.
In our continuing search for anticancer agents, we synthesized
a number of pharmacologically important heterocyclic compounds
[36ꢀ38]. Our strategy, as outlined in this paper is to discover new
microtubule inhibitors from a small molecule library of compounds
that contain the benzimidazole core structures (Fig.1). Thus a thirty
compound library of a new class of terphenyl benzimidazoles was
designed, synthesized and evaluated for their anticancer activity
against different human cancer cell lines apart from their effect on
tubulin polymerization.
3. Results and discussion
3.1. Evaluation of biological activity
3.1.1. Anticancer activity
These terphenyl benzimidazole compounds (3aꢀz and 3aaꢀad)
were evaluated for their anticancer activity in selected human
cancer cell lines of breast, cervix, colon, lung, oral, ovarian and
prostate by using sulforhodamine B (SRB) method. The compounds
that exhibit GI50 ꢁ 10ꢀ5 M are considered to be active on the
respective cancer cell lines. All the 30 compounds showed
moderate to good anticancer potency with GI50 values ranging from
<0.1 to 100 mM. Interestingly, all these new compounds showed
significant activity in the breast (MCF7), lung (A549), and oral (KB)
cancer cell lines. From the structure activity relationship (SAR)
studies, some interesting trends have been observed in these
compounds. With respect to substitution on the terphenyl moiety
of benzimidazole derivatives, all the 3,4-dimethoxy (3h, 3s and
3ac) and 3,4,5-trimethoxy (3i, 3t and 3ad) containing compounds
showed better anticancer activity against most of the cancer cell
lines compared to other compounds. Substitution on the o-phe-
nylenediamine, hydroxy group containing compounds (3kꢀt)
enhanced anticancer activity compared to without substitution
(3aꢀj) and methyl (3uꢀad) group containing benzimidazole
compounds. Among all the compounds synthesized, compounds 3j,
3mꢀt and 3aaꢀad, exhibit significant anticancer activity with GI50
2. Chemistry
values ranging from <0.1 to 9.72
mM, while the positive control
adriamycin demonstrated the GI50 in the range of 0.1e7.25
against the panel of eleven human cancer cell lines and the
results are summarized in Table 2.
m
M
The preparation of substituted terphenyl benzimidazoles is
depicted in Scheme 1. The structures of target compounds are listed
in Table 1. Synthesis of biphenyl systems utilizing the chemo-
selective Pd-catalyzed Suzuki cross-coupling reaction between 1-
bromo-4-iodobenzene (4) and 4-formyl phenyl boronic acid (5) in
toluene/ethanol as a solvent in the presence of catalytic amount of
tetrakis (triphenylphosphine) palladium (0) using 2 M sodium
carbonate solution provides biphenyl aldehyde (6), which
undergoes Suzuki cross-coupling reaction with different boronic
acids (7aꢀj) by employing the above mentioned procedure affords
terphenyl aldehydes (8aꢀj). The oxidative cyclization of o-phenyl-
enediamines (9aꢀc) and terphenyl aldehydes (8aꢀj) with sodium
metabisulphite in ethanol/water solvent system provides the ter-
phenyl benzimidazole compounds 3aꢀz and 3aaꢀad as shown in
Scheme 1.
3.1.2. Effect on cell cycle arrest
To investigate the mechanism underlying the antiproliferative
effect of these terphenyl benzimidazole compounds, we analyzed
the cell cycle distribution by treating A549 cells at 5 mM concen-
tration for 48 h. The cell cycle dependent DNA content was deter-
mined by flow cytometry using propidium iodide staining
according to the standard protocol [39]. The effect of these
compounds (3n, 3o, 3s, 3t and 3abꢀad) on cell cycle along with
nocodazole (employed as a standard) was examined. Amongst
these, 3t, 3o and 3ad showed significant increase in the G2/M phase
in comparison with control and the standard as seen in Fig. 2.
Fig. 1. Chemical structures of Hoechst 33258 (1a), nocodazole (1b), taxol (1c) 3,4,5-trimethoxy terphenyl carboxylic acid (2) and terphenyl benzimidazoles (3aꢀz and 3aaꢀad).

A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
11
Scheme 1. Synthesis of terphenyl benzimidazoles (3aꢀz and 3aaꢀad): Reagents and conditions: (i) Pd(PPh3)4, 2M Na2CO3, toluene/EtOH, 2 h, reflux, 68%; (ii) Pd(PPh3)4, 2M Na2CO3,
toluene/EtOH, 2ꢀ3 h, reflux, 72ꢀ79%; (iii) Na2S2O5, EtOH/H2O, 4ꢀ6 h, 70ꢀ80 ꢂC, 51ꢀ70%.
3.1.3. Effect on tubulin polymerization
have shown considerable inhibition of tubulin polymerization i.e.,
98.3% and 48.8% respectively, while the compound 3o showed
36.3% inhibition (in the order of 3t > 3ad > 3o) in comparison with
Tubulin polymerization dynamics within the cells are critical for
completion of mitosis and are frequently targeted by agents that
induce mitotic arrest which interfere with the mitotic spindle
formation [40]. If the function of microtubules is blocked, cell
differentiation will arrest which leads to the accumulation of cells
at G2/M phase in cell cycle analysis and it eventually leads to cell
death [41]. Therefore, the three potent compounds namely 3t, 3o
and 3ad which induced cell cycle arrest at G2/M phase were eval-
uated for their inhibitory effects on tubulin polymerization along
with positive control, nocodazole (1b). The compounds 3t, 3ad
nocodazole which showed 58.9% inhibition at 3
as shown in Fig. 3.
mM concentration
3.1.4. Analysis of free versus polymerized tubulin by Western
blotting
The microtubules constantly undergo polymerization and depo-
lymerization cycles which are mainly mediated by a- and b-tubulin,
and altering this dynamic balance is considered as a potential target
for drug development towards cancer [42,43] (e.g- taxol or colchi-
cines). In order to extend the in vitro effects of the compounds on
tubulin polymerization to the cellular effects i.e., ratio of free vs
Table 1
Structures of compounds 3aꢀz and 3aaꢀad, melting points and their yields.
polymerized tubulin, Western blot analysis of
was performed by treating the cells with nocodazole (1
(1 M), 3o, 3t and 3ad (5 M) for 24 h as described in the “Materials
a-tubulin in A549 cells
mM), taxol
m
m
and Methods”. Results indicate that while the ratio of free to poly-
merized tubulin in controls is 1 (normalized value), following treat-
ment with compounds 3o, 3t and 3ad the values increased to 1.1, 1.4
and 1.3 respectively, indicating the tubulin inhibitory effects of these
compounds even under intracellular conditions. Under similar
experimental conditions nocodozole (1b), a known microtubule
disrupting agent and taxol (1c), microtubule stabilizer altered the
ratio of free to polymerized tubulin was found to be 1.5 and 0.5 (50%
decrease) as shown in Fig. 4a and b.
.
Compound R1
R2
R3
R4 R5
Melting point ꢂ
C
Yield % (mg)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
H
H
H
F
CF3
OCF3
OH
OCH3
N(CH3)2
H
H
H
H
H
H
H
H
Cl
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
162ꢀ164
227ꢀ229
238ꢀ240
260ꢀ262
234ꢀ236
139ꢀ141
156ꢀ158
197ꢀ199
242ꢀ244
134ꢀ136
196ꢀ198
290ꢀ292
291ꢀ293
279ꢀ281
278ꢀ280
152ꢀ154
165ꢀ167
250ꢀ252
201ꢀ203
205ꢀ207
53% (182)
51% (186)
58% (240)
55% (236)
54% (195)
53% (199)
50% (194)
51% (193)
51% (231)
55% (239)
65% (235)
62% (235)
70% (301)
68% (303)
59% (223)
53% (207)
51% (205)
70% (277)
63% (265)
68% (307)
58% (208)
65% (245)
68% (291)
60% (266)
52% (195)
60% (234)
66% (265)
70% (275)
55% (231)
59% (265)
4. Conclusion
In conclusion, a new class of terphenyl benzimidazoles were
synthesized and tested for their in vitro anticancer activities against
eleven human cancer cell lines. All the compounds showed moderate
togoodanticancerpotencyagainstmostof thetestedcancercelllines,
however some of the compounds (3j, 3mꢀt and 3aaꢀad) exhibited
significant anticancer potency GI50 values in the range of
OCH3
H
H
3j
OCH3 OCH3
3k
3l
H
H
H
H
H
H
H
H
H
H
F
CF3
OCF3
OH
OCH3
N(CH3)2
H
OH
OH
OH
OH
OH
OH
OH
OH
3m
3n
3o
3p
3q
3r
<0.1e9.72 mM. The FACS analysis showed more population in G2/M
phase, indicating that these compounds (3o, 3t and 3ad) could
inhibit tubulin polymerization, further biological assays like
analysis of free versus polymerized tubulin by Western blotting was
carried out. It was observed from the results that the supernatant
fraction has increased free tubulin content upon treatment with
these compounds (3o, 3t and 3ad) with a concomitant decrease in
the polymerized tubulin. This investigation reveals that synthesis of
Cl
3s
OCH3
OCH3 OH
OCH3 OH
H
H
H
H
H
H
H
3t
OCH3 OCH3
3u
3v
3w
3x
3y
3z
3aa
3ab
3ac
3ad
H
H
H
H
H
H
H
H
H
H
F
CF3
OCF3
OH
OCH3
N(CH3)2
H
H
H
H
H
H
H
H
H
H
H
CH3 179ꢀ181
CH3 282ꢀ284
CH3 249ꢀ251
CH3 290ꢀ292
CH3 248ꢀ250
CH3 147ꢀ149
CH3 139ꢀ141
CH3 215ꢀ217
CH3 171ꢀ173
CH3 143ꢀ145
benzimidazoles incorporating
a substituted terphenyl moiety
enhances the anticancer activity. Based on the results from the
biological studies it is evident that compounds 3t and 3ad have the
potential to be taken up for further detailed studies either alone or
in combination with existing therapies as potential tubulin
polymerization inhibitors.
Cl
OCH3
OCH3
OCH3
OCH3 OCH3

12
A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
Table 2
GI50 values (in m
M)a for compounds 3aez and 3aaead in selected human cancer cell lines.
Compound
A549b
Guravb
j
Hop62b
MCF-7c
ZR-75-1c
A2780d
32.12
DWDe
j
KBe
Colo205f
PC3g
SiHah
j
j
j
j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
41.21
33.21
36.63
38.02
33.11
38.12
30.06
31.11
7.60
2.11
8.32
0.17
3.61
0.19
0.17
7.69
7.10
2.40
1.31
<0.1
3.63
6.03
3.52
3.56
3.52
6.33
3.51
2.80
2.10
<0.1
7.25
39.23
36.21
41.35
38.42
30.51
33.28
41.23
36.31
7.60
6.38
2.20
3.01
2.31
2.21
0.19
3.11
2.31
3.42
0.19
<0.1
31.62
30.21
31.04
31.02
12.07
3.11
3.13
1.21
0.19
0.17
39.71
41.34
47.22
39.10
41.01
47.20
39.30
32.32
9.25
2.98
4.01
6.92
4.32
3.31
3.19
4.90
4.20
2.28
2.71
2.11
32.11
24.91
41.23
41.21
21.20
28.26
6.24
2.26
2.19
2.11
0.17
41.23
41.20
j
j
j
39.34
48.23
35.63
29.68
j
j
j
j
39.13
48.31
33.90
45.23
j
j
j
j
j
j
j
38.43
33.28
j
j
j
47.21
36.02
46.30
47.01
j
j
j
j
38.11
35.11
45.20
2.76
j
j
j
j
j
j
j
j
j
j
j
j
2.19
9.23
7.10
1.11
2.86
3.19
3.60
0.19
6.63
8.21
2.31
1.11
0.17
3.31
16.52
12.01
11.03
11.26
9.27
6.24
2.12
1.31
0.17
0.17
41.22
39.73
9.71
35.61
6.32
5.11
3.80
2.65
8.20
4.10
2.41
2.11
0.19
27.41
35.03
2.41
2.41
18.21
41.13
7.20
3.45
3.10
0.19
1.79
31.12
21.60
9.21
41.01
41.22
6.10
2.90
2.92
8.64
3.01
2.80
2.71
0.17
41.22
24.61
12.26
12.28
31.22
2.11
33.71
47.11
9.72
1.24
15.31
2.26
7.41
0.19
6.61
8.42
2.56
1.24
4.20
9.43
9.04
8.31
3.20
2.27
2.41
3.21
8.25
2.26
2.21
0.19
39.31
33.52
24.23
24.21
41.22
36.27
4.23
2.74
2.12
2.37
0.14
42.10
8.01
9.68
22.21
3.18
3.10
2.70
6.40
9.21
2.10
1.40
0.19
28.31
33.12
38.18
41.12
41.21
8.32
1.21
1.22
3.41
8.32
2.60
1.63
6.32
8.60
1.32
0.19
6.20
4.98
9.71
9.10
3.72
8.41
2.41
4.80
6.24
8.40
1.11
0.17
35.24
41.02
33.24
28.26
33.53
21.21
7.11
3.60
3.51
0.17
0.14
3s
3t
0.17
j
3u
3v
3w
3x
3y
2.56
j
j
30.62
2.01
2.01
30.61
j
21.21
30.01
j
j
j
3z
3aa
3ab
3ac
3ad
ADRi
2.21
2.80
2.13
0.19
3.23
2.51
1.72
0.17
0.10
9.21
2.78
2.32
0.19
0.14
8.12
2.67
2.56
0.18
1.81
0.17
0.16
a
50% growth inhibition and the values are mean of three determinations.
b
c
d
e
f
Lung cancer cell line.
Breast cancer cell line.
Ovarian cancer cell line.
Oral cancer cell line.
Colon cancer cell line.
Prostate cancer cell line.
Cervix cancer cell line.
Adriamycin.
g
h
i
j
100.
5. Experimental protocols
indicator. Column chromatography was performed with Merck
60e120 mesh silica gel. 1H NMR and 13C NMR spectra were
recorded on Gemini Varian-VXR-unity (200 MHz) or Bruker
UXNMR/XWIN-NMR (300 M Hz) instruments. Chemical shifts are
5.1. General
All chemicals were purchased from Aldrich (SigmaeAldrich, St.
Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company,
Ward Hill, MA, USA) or Spectrochem Pvt. Ltd (Mumbai, India) and
were used without further purification. Reactions were monitored
by TLC, performed on silica gel glass plates containing 60GF-254,
and visualizat ion on TLC was achieved by UV light or iodine
reported in parts per million (d in ppm) relative to the peak for
tetramethylsilane (TMS) as an intrernal standard, coupling
constants are reported in hertz (Hz). Elemental analysis was ꢃ0.4%
Fig. 3. Effect on tubulin polymerization: tubulin polymerization assay was carried out
in a reaction mixture that contained PEM buffer and GTP (1 mM) in the presence or
Fig. 2. Effect on cell cycle: A549 lung cancer cells in 6 well plates were treated with
absence of test compounds (3o, 3t and 3ad) at 3 mM concentration. The reaction was
compounds 3n, 3o, 3s, 3t, 3abꢀad (5
m
M), and nocodozole (1b, 1
m
M) for 48 h. Cells
initiated by the addition of GTP to all the wells. Tubulin polymerization was monitored
by the increase in fluorescence at 420 nm (excitation wavelength is 360 nm) was
measured for 1 h at 1 min interval in a multimode plate reader (Tecan) at 37 ꢂC.
Nocodazole (1b) was used as the positive control.
were trypsinized, washed with PBS and were fixed in 70% ethanol. Before analysis, cells
were finely dispersed and stained with propidium iodide. Stained cells were analyzed
by flow cytometry.

A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
13
chromatography using ethyl acetate and hexane to afford the pure
terphenylaldehydes (8).
5.1.2.1. [1,10;40,100]Terphenyl-400-carbaldehyde (8a). White solid (yield
190 mg, 74%): Rf ¼ 0.3 (10% ethyl acetate/hexane); mp: 150e152 ꢂC;
1H NMR (CDCl3, 300 MHz):
d 10.07 (s, 1H, eCHO), 7.98 (d, 2H,
J ¼ 8.3 Hz, ArH), 7.81 (d, 2H, J ¼ 8.3 Hz, ArH), 7.73 (s, 4H, ArH), 7.65
(d, 2H, J ¼ 7.3 Hz, ArH), 7.48 (t, 2H, J ¼ 7.7 Hz, ArH), 7.40 (s, 1H, ArH);
MS (ESI): m/z 259 (M þ 1)þ.
5.1.2.2. 4-Fluoro[1,10;40,100]Terphenyl-400-carbaldehyde (8b). White
solid (yield 210 mg, 76%): Rf ¼ 0.3 (10% ethyl acetate/hexane); mp:
134e136 ꢂC; 1H NMR (CDCl3, 300 MHz):
d 10.06 (s, 1H, eCHO), 7.98
(d, 2H, J ¼ 8.3 Hz, ArH), 7.83 (d, 2H, J ¼ 8.1 Hz, ArH), 7.75 (d, 2H,
J ¼ 8.4 Hz, ArH), 7.68 (d, 2H, J ¼ 8.4 Hz, ArH), 7.61e7.66 (m, 2H,
ArH), 7.17 (t, 2H, J ¼ 8.6 Hz, ArH); MS (ESI): m/z 277 (M þ 1)þ.
5.1.2.3. 4-Trifluoromethyl[1,10;40,100]Terphenyl-400-carbaldehyde
(8c). White solid (yield 241 mg, 74%): Rf ¼ 0.3 (10% ethyl acetate/
hexane); mp: 148e150 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.08 (s,
1H, eCHO), 7.99 (d, 2H, J ¼ 8.3 Hz, ArH), 7.81 (d, 2H, J ¼ 8.3 Hz, ArH),
7.67 (q, 4H, J ¼ 3.7, 8.3 Hz, ArH), 7.59 (d, 2H, J ¼ 9.0 Hz, ArH), 7.02 (d,
2H, J ¼ 9.0 Hz, ArH); ESIꢀMS: m/z 326 (M þ 1)þ.
Fig. 4. a. Western blot analysis of
A549 cells treated with compounds 3t, 3o and 3ad at 5
(1b) and taxol (1c) were used at 1 M for 24 H. b. Ratio of soluble versus polymerized
a
-tubulin in the soluble and polymerized fractions of
mM concentration, nocodazole
m
tubulin. Tubulin band intensity from soluble and polymerized fractions was obtained
by densitometry and the values are represented as the ratio of soluble to polymerized
tubulin.
5.1.2.4. 4-Trifluoromethoxy[1,10;40,100]Terphenyl-400-carbaldehyde
(8d). White solid (yield 246 mg, 72%): Rf ¼ 0.3 (12% ethyl acetate/
hexane); mp: 155e157 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.09 (s,1H,
eCHO), 7.99 (d, 2H, J ¼ 8.2 Hz, ArH), 7.81 (d, 2H, J ¼ 8.2 Hz), 7.68 (q,
4H, J ¼ 3.7, 8.2 Hz, ArH), 7.60 (d, 2H, J ¼ 9.0 Hz, ArH), 7.03 (d, 2H,
J ¼ 9.0 Hz, ArH); MS (ESI): m/z 342 (M þ 1)þ.
of the theritical values. ESI spectra were recorded on Micro mass,
Quattro LC using ESIþ software with capillary voltage 3.98 kV and
ESI mode positive ion trap detector. Melting points were deter-
mined with an Electro thermal melting point apparatus, and are
uncorrected.
5.1.2.5. 4-Hydroxy[1,10;40,100]Terphenyl-400-carbaldehyde (8e). White
solid (yield 209 mg, 76%): Rf ¼ 0.3 (20% ethyl acetate/hexane); mp:
151e153 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.06 (s,1H, eCHO), 7.98
5.1.1. Synthesis of 40-bromobiphenyl-4-carbaldehyde (6)
(d, 2H, J ¼ 8.7 Hz, ArH), 7.83 (d, 2H, J ¼ 8.0 Hz, ArH), 7.69 (q, 4H,
J ¼ 5.09, 8.0 Hz, ArH), 7.50 (d, 2H, J ¼ 8.7 Hz, ArH), 6.92 (d, 2H,
J ¼ 8.7 Hz, ArH); MS (ESI): m/z 275 (M þ 1)þ.
To a solution of 1-bromo-4-iodobenzene (4, 282 mg, 1 mmol) in
3 mL of toluene was added a catalytic amount (0.4% mol) of
tetrakis-triphenylphosphine palladium (0) and 0.5 mL of aqueous
2 M Na2CO3. A solution of 4-formyl phenylboronic acid (5, 150 mg
1 mmol) in 1 mL of ethanol was added, stirred at room temperature
under a nitrogen atmosphere. After 10 min, the solution was heated
to reflux for 2 h in an argon atmosphere. TLC showed disappearance
of starting materials, this reaction mixture is cooled to room
temperature and extracted with ethyl acetate (3 ꢄ 30 mL) and the
organic phase was washed with water and brine, dried over
anhydrous Na2SO4 and the solvent was removed under reduced
pressure to get the crude product. This residue was further purified
by column chromatography using ethyl acetate and hexane to
afford the pure bromobiphenylaldehyde (6) as a white solid, mp:
5.1.2.6. 4-Methoxy[1,10;40,100]Terphenyl-400-carbaldehyde (8f). White
solid (yield 225 mg, 78%): Rf ¼ 0.3 (12% ethyl acetate/hexane); mp:
170e172 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.07 (s,1H, eCHO), 7.97
(d, 2H, J ¼ 8.3 Hz, ArH), 7.80 (d, 2H, J ¼ 8.3 Hz, ArH), 7.66 (q, 4H,
J ¼ 3.77, 8.3 Hz, ArH), 7.59 (d, 2H, J ¼ 9.0 Hz, ArH), 7.01 (d, 2H,
J ¼ 9.0 Hz, ArH), 3.87 (s, 3H, eOCH3); MS (ESI): m/z 289 (M þ 1)þ.
5.1.2.7. 4-N,N-Dimethylamino[1,10;40,100]Terphenyl-400-carbaldehyde
(8g). White solid (yield 237 mg, 79%): Rf ¼ 0.3 (12% ethyl acetate/
hexane); mp: 141e143 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.05 (s,1H,
eCHO), 7.96 (d, 2H, J ¼ 8.7 Hz, ArH), 7.81 (d, 2H, J ¼ 8.0 Hz, ArH),
7.67 (q, 4H, J ¼ 5.0, 8.0 Hz, ArH), 7.48 (d, 2H, J ¼ 8.7 Hz, ArH), 6.90 (d,
2H, J ¼ 8.7 Hz, ArH), 2.44 (s, 6H, ꢀN(CH3)2); MS (ESI): m/z 302
(M þ 1)þ.
138e140 ꢂC: Yield 176 mg, 68%: 1H NMR (CDCl3, 300 MHz):
d 10.06
(s, 1H, eCHO), 7.96 (d, 2H, J ¼ 8.6 Hz, ArH), 7.72 (d, 2H, J ¼ 8.6 Hz,
ArH), 7.61 (d, 2H, J ¼ 9.2 Hz, ArH), 7.50 (d, 2H, J ¼ 9.2 Hz, ArH); MS
(ESI): m/z 260 (M þ 1)þ.
5.1.2.8. 3-Chloro[1,10;40,100]Terphenyl-400-carbaldehyde (8h). White
solid (yield 219 mg, 75%): Rf ¼ 0.3 (10% ethyl acetate/hexane); mp:
5.1.2. General procedure for terphenyl aldehydes (8aꢀj)
131e133 ꢂC; 1H NMR (CDCl3, 300 MHz):
d 10.05 (s, 1H, eCHO),
To a solution of bromobiphenylaldehyde (6, 260 mg, 1 mmol)
and various phenyl boronic acids (7aej, 1 mmol) in 2 M aqueous
sodium carbonate (2 mL) and toluene/ethanol (9:3 mL) is added
a catalytic amount (0.4% mol) of tetrakis-triphenylphosphine
palladium, and the mixture was reflux under argon atmosphere
for 2e3 h. After completion of reaction, the suspension is cooled
and extracted with ethyl acetate (3 ꢄ 30 mL) and the organic phase
was washed with water and brine, dried over anhydrous Na2SO4
and the solvent was removed under reduced pressure to get the
crude product. This residue was further purified by column
7.92e7.99 (m, 2H, ArH), 7.78 (d, 2H, J ¼ 8.8 Hz, ArH), 7.66e7.74 (m,
4H, ArH), 7.58e7.64 (m, 2H, ArH), 7.45e7.52 (m, 1H, ArH), 7.39 (t,
1H, J ¼ 7.3 Hz, ArH); MS (ESI): m/z 293 (M þ 1)þ.
5.1.2.9. 3,4-Dimethoxy[1,10;40,100]Terphenyl-400-carbaldehyde (8i). White
solid (yield 248 mg, 78%): Rf ¼ 0.3 (15% ethyl acetate/hexane); mp:
154e156 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.04 (s,1H, eCHO), 7.94
(d, 2H, J ¼ 8.3 Hz, ArH), 7.76 (d, 2H, J ¼ 8.3 Hz, ArH), 7.64 (q, 4H,
J ¼ 4.5, 8.3 Hz, ArH), 7.08e7.16 (m, 2H, ArH), 6.85e6.93 (d, 1H,

14
A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
J ¼ 9.0 Hz, ArH), 3.96 (s, 1H, eOCH3), 3.92 (s, 3H, eOCH3); MS (ESI):
m/z 319 (M þ 1)þ.
ArH), 7.67 (d, 2H, J ¼ 7.5 Hz, ArH), 7.45e7.60 (m, 2H, ArH), 7.34 (d,
3H, J ¼ 8.3 Hz, ArH), 7.01e7.14 (m, 1H, ArH), 6.85 (d, 3H, J ¼ 7.55 Hz,
ArH), 6.70 (d, 1H, J ¼ 7.5 Hz, ArH); IR (KBr) (nmax/cmꢀ1): 3448ꢀ3612
(NH and ꢀOH), 2932, 2892, 1351, 1240, 813; MS (ESI): m/z 363
(M þ 1)þ; Anal. Calcd for C25H18N2O: C, 82.88; H, 5.03; N, 7.73,
found: C, 82.81; H, 4.96; N, 7.68.
5.1.2.10. 3,4,5-Trimethoxy[1,10;40,100]Terphenyl-400-carbaldehyde
(8j). White solid (yield 265 mg, 76%): Rf ¼ 0.3 (18% ethyl acetate/
hexane); mp: 122e124 ꢂC; 1H NMR (CDCl3, 200 MHz):
d 10.06 (s,1H,
eCHO), 7.96 (d, 2H, J ¼ 8.0 Hz, ArH), 7.78 (d, 2H, J ¼ 8.0 Hz, ArH),
7.66 (q, 4H, J ¼ 3.6, 8.0 Hz, ArH), 6.79 (s, 2H, ArH), 3.95 (s, 6H,
2 ꢄ eOCH3), 3.89 (s, 3H, eOCH3); MS (ESI): m/z 349 (M þ 1)þ.
5.1.3.6. 2-[4-Methoxy(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3f). Brown solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H NMR
(CDCl3þDMSO-d6 300 MHz):
d
12.86 (brs, 1H, ꢀNH), 8.29 (d, 2H,
5.1.3. General procedure for the preparation benzimidazole
derivatives (3aez and 3aaead)
J ¼ 7.5 Hz, ArH), 7.71e7.82 (m, 4H, ArH), 7.66 (d, 2H, J ¼ 7.9 Hz, ArH),
7.61 (d, 2H, J ¼ 8.3 Hz, ArH), 6.97e7.15 (m, 5H, ArH), 6.68 (d, 1H,
J ¼ 7.5 Hz, ArH), 3.88 (s, 3H, eOCH3); IR (KBr) (nmax/cmꢀ1): 3431 (NH),
2941, 2888,1438,1230, 838; MS (ESI): m/z 377 (M þ 1)þ; Anal. Calcd for
C26H20N2O: C, 82.96; H, 5.35; N, 7.44, found: C, 82.91; H, 5.29; N, 7.38.
To a stirred solution of terphenylaldehydes (8aej, 1 mmol) in
ethanol/H2O (10:1 mL), a catalytic amount (0.02 mmol) of Na2S2O5
and O-phenylenediamines (9aec, 1 mmol) was added. The result-
ing mixture was stirred at 70e80 ꢂC for 4e6 h. After completion of
the reaction as indicated by TLC, this reaction mixture cooled to
room temperature and extracted with ethyl acetate (3 ꢄ 30 mL).
The combined layers were washed with water, brine solution, dried
over anhydrous Na2SO4. Evaporation of the solvent under vacuum
produced the crude product that was further purified by column
chromatography using ethyl acetate/hexane to afford the pure
benzimidazole compounds (3aez and 3aaead).
5.1.3.7. 2-[4-N,N-Dimethylamino(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3g). Brown solid; Rf ¼ 0.3 (40% ethyl acetate/hexane);
1H NMR (CDCl3þDMSO-d6 300 MHz):
12.85 (brs, 1H, ꢀNH), 8.28
d
(d, 2H, J ¼ 7.7 Hz, ArH), 7.71 (d, 2H, J ¼ 8.1 Hz, ArH), 7.64 (d, 2H,
J ¼ 7.7 Hz, ArH), 7.39e7.55 (m, 2H, ArH), 7.33 (d, 3H, J ¼ 8.2 Hz,
ArH), 7.00e7.12 (m, 1H, ArH), 6.90 (d, 3H, J ¼ 7.5 Hz, ArH), 6.71 (d,
1H, J ¼ 7.6 Hz, ArH), 2.43 (s, 6H, ꢀN(CH3)2); IR (KBr) (nmax/cmꢀ1):
3428 (NH), 2938, 2869, 1404, 1229, 840; MS (ESI): m/z 390
(M þ 1)þ; Anal. Calcd for C27H23N3: C, 82.26; H, 5.94; N, 10.81,
found: C, 82.19; H, 5.81; N, 10.66.
5.1.3.1. 2-[(1,10;40,100)Terphenyl]-1H-benzo[d]imidazole (3a). Brown
solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H NMR (CDCl3,
200 MHz):
d
12.80 (brs, 1H, ꢀNH), 8.29 (d, 2H, J ¼ 8.2 Hz, ArH),
7.70e7.82 (m, 6H, ArH), 7.66 (d, 2H, J ¼ 8.2 Hz, ArH), 7.31e7.51 (m,
4H, ArH), 7.00e7.15 (m, 2H, ArH), 6.70 (t, 1H, J ¼ 7.2 Hz, ArH); IR
(KBr) (nmax/cmꢀ1): 3412 (NH), 2918, 2831,1302, 1250, 810; MS (ESI):
m/z 347 (M þ 1)þ; Anal. Calcd for C25H18N2: C, 86.68; H, 5.24; N,
8.09, found: C, 86.61; H, 5.18; N, 8.04.
5.1.3.8. 2-[3-Chloro(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3h). Brown solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H NMR
(CDCl3þDMSO-d6 200 MHz):
d
12.86 (brs, 1H, ꢀNH), 8.29 (d, 2H,
J ¼ 7.5 Hz, ArH), 7.72e7.81 (m, 4H, ArH), 7.60e7.70 (m, 4H, ArH),
7.30e7.52 (m, 4H, ArH), 7.22e7.27 (m, 2H, ArH); 13C NMR
(CDCl3þDMSO-d6 50 MHz):
d 150.9, 141.7, 138.6, 133.9, 129.7, 129.1,
128.3, 127.2, 126.7, 126.3, 124.6, 121.9, 119.1, 114.5; IR (KBr) (nmax/
5.1.3.2. 2-[4-Fluoro(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3b). Brown solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H NMR
cmꢀ1): 3429 (NH), 2922, 2852, 1313, 1277, 744; MS (ESI): m/z 380
(M þ 1)þ; Anal. Calcd for C25H17ClN2: C, 78.84; H, 4.53; N, 7.38,
found: C, 78.77; H, 4.47; N, 7.33.
(CDCl3, 300 MHz):
d
12.88 (brs,1H, ꢀNH), 8.30 (d, 2H, J ¼ 7.7 Hz, ArH),
7.71e7.81 (m, 4H, ArH), 7.82 (d, 4H, J ¼ 8.3 Hz, ArH), 7.37e7.60 (m, 4H,
ArH), 7.13 (d, 2H, J ¼ 8.3 Hz, ArH); IR (KBr) (nmax/cmꢀ1): 3442 (NH),
2930, 2821,1362,1230, 790; MS (ESI): m/z 365 (M þ 1)þ;Anal. Calcdfor
C25H17FN2: C, 82.41; H, 4.72; N, 7.68, found: C, 82.35; H, 4.63; N, 7.62.
5.1.3.9. 2-[3,4-Dimethoxy(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3i). Pale yellow solid; Rf ¼ 0.3 (45% ethyl acetate/hexane); 1H NMR
(CDCl3, 200 MHz):
d
12.88 (brs, 1H, ꢀNH), 8.31 (d, 2H, J ¼ 8.0 Hz,
5.1.3.3. 2-[4-Trifluoromethyl(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3c). Pale yellow solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H NMR
ArH), 7.71 (d, 2H, J ¼ 8.0 Hz, ArH), 7.64 (s, 4H, ArH), 7.55 (d, 2H,
J ¼ 8.0 Hz, ArH), 7.38 (s, 1H, ArH), 7.12e7.22 (m, 2H, ArH), 7.08
(d, 1H, J ¼ 7.5 Hz, ArH), 6.99 (d, 1H, J ¼ 8.3 Hz, ArH), 3.96 (s, 3H,
eOCH3), 3.93 (s, 3H, eOCH3); 13C NMR (CDCl3þDMSO-d6 50 MHz):
(CDCl3þDMSO-d6 300 MHz):
d
12.86 (brs, 1H, ꢀNH), 8.30 (d, 2H,
J ¼ 8.3 Hz, ArH), 7.74 (q, 4H, J ¼ 6.0, 8.3 Hz, ArH), 7.60e7.68 (m, 4H,
ArH), 7.29e7.38 (m, 4H, ArH), 7.24 (d, 2H, J ¼ 8.3 Hz, ArH); IR (KBr)
d
150.9, 149.0, 148.6, 140.7, 139.4, 137.4, 132.1, 129.0, 127.0, 126.8,
(
nmax/cmꢀ1): 3432 (NH), 2961, 2830, 1332, 1206, 741; MS (ESI): m/z
122.1, 118.7, 112.1, 110.2, 55.5; IR (KBr) (nmax/cmꢀ1): 3426 (NH),
2925, 2852,1431,1248, 832; MS (ESI): m/z 406 (M þ 1)þ; Anal. Calcd
for C27H22N2O2: C, 79.78; H, 5.46; N, 6.89, found: C, 79.73; H, 5.41;
N, 6.85.
415 (M þ 1)þ; Anal. Calcd for C26H17F3N2: C, 75.35; H, 4.16; N, 6.73,
found: C, 75.27; H, 4.08; N, 6.68.
5.1.3.4. 2-[4Trifluoromethoxy(1,10;40,100)terphenyl]-1H-benzo[d]imid-
azole (3d). Brown solid; Rf ¼ 0.4 (45% ethyl acetate/hexane); 1H
5.1.3.10. 2-[3,4,5-Trimethoxy(1,10;40,100)terphenyl]-1H-benzo[d]imid-
NMR (CDCl3þDMSO-d6 300 MHz):
d
12.85 (brs, 1H, ꢀNH), 8.31 (d,
azole (3j). Pale yellow solid; Rf ¼ 0.3 (48% ethyl acetate/hexane); 1H
2H, J ¼ 8.3 Hz, ArH), 7.76 (q, 4H, J ¼ 6.0, 8.3 Hz, ArH), 7.62e7.70 (m,
NMR (CDCl3þDMSO-d6 200 MHz):
d
12.89 (brs, 1H, ꢀNH), 8.25 (d,
4H, ArH), 7.27e7.36 (m, 4H, ArH), 7.24 (d, 2H, J ¼ 8.2 Hz, ArH); 13C
2H, J ¼ 8.5 Hz, ArH), 7.66e7.79 (m, 2H, ArH), 7.59 (s, 2H, ArH),
7.33e7.53 (m, 4H, ArH), 7.03e7.17 (m, 1H, ArH), 6.87 (d, 2H,
J ¼ 8.5 Hz, ArH), 6.70 (d, 1H, J ¼ 8.7 Hz, ArH), 3.93 (s, 6H,
2 ꢄ eOCH3), 3.88 (s, 3H, eOCH3); IR (KBr) (nmax/cmꢀ1): 3421 (NH),
2913, 2863, 1449, 1292, 830; MS (ESI): m/z 436 (M þ 1)þ; Anal.
Calcd for C28H24N2O3: C, 77.08; H, 5.54; N, 6.43, found: C, 77.01; H,
5.48; N, 6.38.
NMR (CDCl3þDMSO-d6 50 MHz):
d 157.3, 150.9, 148.0, 140.9, 135.0,
129.2, 127.5, 126.9, 125.9, 121.8, 120.7, 119.1, 114.5, 110.1; IR (KBr)
(
nmax/cmꢀ1): 3413 (NH), 2938, 2829, 1340, 1290, 808; MS (ESI): m/z
431 (M þ 1)þ; Anal. Calcd for C26H17F3N2O: C, 72.52; H, 4.13; N, 6.51,
found: C, 72.41; H, 4.09; N, 6.44.
5.1.3.5. 2-[4-Hydroxy(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(3e). Brown solid; Rf ¼ 0.3 (40% ethyl acetate/hexane); 1H NMR
5.1.3.11. 4-Hydroxy-2-[(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
(CDCl3þDMSO-d6 300 MHz):
d
12.87 (brs, 1H, ꢀNH), 10.03 (s,
(3k). Brown solid; Rf ¼ 0.4 (50% ethyl acetate/hexane); 1H NMR
1H, ꢀOH, ArH), 8.30 (d, 2H, J ¼ 7.5 Hz, ArH), 7.74 (d, 2H, J ¼ 8.3 Hz,
(CDCl3, 200 MHz):
d
12.87 (brs, 1H, ꢀNH), 10.01 (s, 1H, ꢀOH), 8.30

A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
15
(d, 2H, J ¼ 8.2 Hz, ArH), 7.71e7.82 (m, 6H, ArH), 7.67 (d, 2H,
J ¼ 8.2 Hz, ArH), 7.33e7.52 (m, 4H, ArH), 7.02e7.16 (m, 2H, ArH),
6.71 (t, 1H, J ¼ 7.9 Hz, ArH); 13C NMR (CDCl3þDMSO-d6 50 MHz):
5.1.3.17. 4-Hydroxy-2-[4-N,N-Dimethylamino(1,10;40,100)terphenyl]-1H-
benzo[d] imidazole (3q). Pale yellow solid; Rf ¼ 0.3 (50% ethyl
acetate/hexane); 1H NMR (CDCl3þDMSO-d6 300 MHz):
d 12.86 (brs,
d
149.2, 146.2, 141.0, 139.7, 139.5, 138.1, 128.5, 127.9, 127.1, 126.9,
1H, ꢀNH), 10.01 (brs, 1H, ꢀOH), 8.30 (d, 2H, J ¼ 7.7 Hz, ArH),
7.61e7.75 (m, 4H, ArH), 7.46 (t, 1H, J ¼ 7.0 Hz, ArH), 7.31 (d, 4H,
J ¼ 8.3 Hz, ArH), 7.00e7.12 (m, 1H, ArH), 6.88 (d, 2H, J ¼ 7.7 Hz, ArH),
126.6, 126.3, 123.3, 107.2, 104.8; IR (KBr) (nmax/cmꢀ1): 3419ꢀ3652
(NH and ꢀOH), 2968, 2839, 1343, 1238, 861; MS (ESI): m/z 363
(M þ 1)þ; Anal. Calcd for C25H18N2O: C, 82.88; H, 5.01; N, 7.73,
found: C, 82.82; H, 4.97; N, 7.68.
6.72 (d, 1H, J ¼ 7.7 Hz, ArH), 2.44 (s, 6H, ꢀN(CH3)2); IR (KBr) (nmax
/
cmꢀ1): 3418ꢀ3604 (NH and ꢀOH), 2938, 2849, 1362, 1238, 828; MS
(ESI): m/z 406 (M þ 1)þ; Anal. Calcd for C27H23N3O: C, 80.01; H,
5.75; N, 10.31; found: C, 79.92; H, 5.68; N, 10.25.
5.1.3.12. 4-Hydroxy-2-[4-fluoro(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3l). Brown solid; Rf ¼ 0.3 (50% ethyl acetate/hexane); 1H
NMR (CDCl3, 300 MHz):
d
12.84 (brs, 1H, ꢀNH), 10.50 (s, 1H, ꢀOH),
5.1.3.18. 4-Hydroxy-2-[3-chloro(1,10;40,100)terphenyl]-1H-benzo[d]
8.29 (d, 2H, J ¼ 8.1 Hz, ArH), 7.77 (t, 4H, J ¼ 7.9 Hz, ArH), 7.61e7.69
(m, 4H, ArH), 7.12e7.20 (m, 2H, ArH), 7.06 (t, 1H, J ¼ 7.9 Hz, ArH),
6.70 (d, 2H, 7.9 Hz, ArH); 13C NMR (CDCl3þDMSO-d6 50 MHz):
imidazole (3r). Brown solid; Rf ¼ 0.4 (45% ethyl acetate/hexane);
1H NMR (CDCl3, 300 MHz):
d
12.85 (brs, 1H, ꢀNH), 10.61 (s,
1H, ꢀOH), 8.28 (d, 2H, J ¼ 8.0 Hz, ArH), 7.74e7.79 (m, 3H, ArH),
7.64e7.72 (m, 3H, ArH), 7.56 (d, 1H, J ¼ 7.5 Hz, ArH), 7.48 (t, 1H,
J ¼ 7.5 Hz, ArH), 7.33e7.52 (m, 2H, ArH), 7.16 (d, 1H, J ¼ 8.0 Hz, ArH),
7.07 (t, 1H, J ¼ 8.0 Hz, ArH), 6.71 (d, 1H, J ¼ 7.5 Hz, ArH); IR (KBr)
d
159.3, 149.5, 140.6, 138.6, 138.3, 135.9, 129.6, 128.8, 128.1, 127.9,
126.8, 126.5, 122.8, 115.4, 114.9, 106.8; IR (KBr) (nmax/cmꢀ1):
3405ꢀ3633 (NH and ꢀOH), 2924, 2853, 1264, 815; MS (ESI): m/z
381 (M þ 1)þ; Anal. Calcd for C25H17FN2O: C, 78.93; H, 4.48; N, 7.31,
found: C, 78.93; H, 4.48; N, 7.31.
(
nmax/cmꢀ1): 3412ꢀ3639 (NH and ꢀOH), 2959, 2830, 1302, 1223,
831; MS (ESI): m/z 397 (M þ 1)þ; Anal. Calcd for C25H17N2ClO2: C,
75.66; H, 4.33; N, 7.06, found: C, 75.61; H, 4.26; N, 7.09.
5.1.3.13. 4-Hydroxy-2-[4-trifluoromethyl(1,10;40,100)terphenyl]-1H-
benzo[d]imidazole (3m). Brown solid; Rf ¼ 0.5 (50% ethyl acetate/
5.1.3.19. 4-Hydroxy-2-[3,4-dimethoxy(1,10;40,100)terphenyl]-1H-benzo
hexane); 1H NMR (CDCl3, 300 MHz):
d
12.83 (brs, 1H, ꢀNH), 10.61(s,
[d]imidazole (3s). White solid; Rf ¼ 0.3 (55% ethyl acetate/hexane);
1H, ꢀOH), 8.28 (d, 2H, J ¼ 7.6 Hz, ArH), 7.63e7.81 (m, 8H, ArH), 7.31
(d, 2H, J ¼ 7.6 Hz, ArH), 7.03e7.20 (m, 2H, ArH), 6.74 (t, 1H,
J ¼ 7.6 Hz, ArH); IR (KBr) (nmax/cmꢀ1): 3402ꢀ3609 (NH and ꢀOH),
2952, 2862,1308,1240, 801; MS (ESI): m/z 431 (M þ 1)þ; Anal. Calcd
for C26H17F3N2O: C, 72.58; H, 4.12; N, 6.49, found: C, 72.52; H, 4.08;
N, 6.42.
1H NMR (CDCl3, 200 MHz):
d
12.90 (brs,1H, ꢀNH),10.05 (s,1H, ꢀOH),
8.32 (d, 2H, J ¼ 8.3 Hz, ArH), 7.70 (d, 2H, J ¼ 8.3 Hz, ArH), 7.65 (s, 4H,
ArH), 7.54 (d, 1H, J ¼ 8.3 Hz, ArH), 7.39 (s, 1H, ArH), 7.13e7.21 (m, 2H,
ArH), 7.07 (d,1H, J ¼ 7.5 Hz, ArH), 6.97 (d,1H, J ¼ 8.3 Hz, ArH), 3.95 (s,
3H, ArH, eOCH3), 3.91 (s, 3H, eOCH3); 13C NMR (CDCl3þDMSO-d6
75 MHz):
d 158.7, 149.6, 148.8, 148.4, 140.8, 139.6, 137.8, 132.6, 131.5,
128.7,126.8,126.6,122.9,118.8,111.6,109.9,106.9, 55.6; IR (KBr) (nmax
/
5.1.3.14. 4-Hydroxy-2-[4-trifluoromethoxy(1,10;40,100)terphenyl]-1H-
benzo[d]imidazole (3n). Brown solid; Rf ¼ 0.4 (50% ethyl acetate/
cmꢀ1): 3414ꢀ3629 (NH and ꢀOH), 2926, 2881, 1408, 1249, 752; MS
(ESI): m/z 423 (M þ 1)þ; Anal. Calcd for C27H22N2O3: C, 76.72; H, 5.24;
N, 6.63, found: C, 76.53; H, 5.18; N, 6.58.
hexane); 1H NMR (CDCl3, 300 MHz):
d
12.81 (brs, 1H, ꢀNH), 10.63 (s,
1H, ꢀOH), 8.28 (d, 2H, J ¼ 7.5 Hz, ArH), 7.64e7.82 (m, 8H, ArH), 7.32 (d,
2H, J¼ 7.5 Hz, ArH), 7.04e7.20 (m, 2H, ArH), 6.73(t,1H, J¼ 7.5 Hz, ArH);
IR (KBr) (nmax/cmꢀ1): 3412ꢀ3599 (NH and ꢀOH), 2958, 2831, 1318,
1223, 789; MS (ESI): m/z 447 (M þ 1)þ; Anal. Calcd for C26H17F3N2O2:
C, 69.92; H, 3.85; N, 6.31, found: C, 69.87; H, 3.81; N, 6.24.
5.1.3.20. 4-Hydroxy-2-[3,4,5-trimethoxy(1,10;40,100)terphenyl]-1H-
benzo[d]imidazole (3t). Pale yellow solid; Rf ¼ 0.3 (55% ethyl
acetate/hexane); 1H NMR (CDCl3þDMSO-d6 200 MHz):
d 12.91 (brs,
1H, ꢀNH),10.07 (s,1H, ꢀOH), 8.26 (d, 2H, J ¼ 8.5 Hz, ArH), 7.64e7.78
(m, 2H, ArH), 7.58 (s, 2H, ArH), 7.31e7.52 (m, 3H, ArH), 7.02e7.15
(m, 1H, ArH), 6.86 (d, 2H, J ¼ 8.5 Hz, ArH), 6.69 (d, 1H, J ¼ 8.7 Hz,
5.1.3.15. 4-Hydroxy-2-[4-hydroxy(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3o). Brown solid; Rf ¼ 0.3 (50% ethyl acetate/hexane);
ArH), 3.93 (s, 6H, 2 ꢄ eOCH3), 3.88 (s, 3H, eOCH3); IR (KBr) (nmax
/
1H NMR (CDCl3þDMSO-d6 300 MHz):
d
12.87 (brs, 1H, ꢀNH), 10.06
cmꢀ1): 3412ꢀ3671 (NH and ꢀOH), 2969, 2833, 1398, 1223, 813; MS
(ESI): m/z 453 (M þ 1)þ; Anal. Calcd for C28H24N2O4: C, 74.32; H,
5.32; N, 6.16, found: C, 74.25; H, 5.22; N, 6.09.
(brs, 2H, ꢀOH), 8.29 (d, 2H, J ¼ 7.5 Hz, ArH), 7.63e7.76 (m, 4H, ArH),
7.47 (t, 1H, J ¼ 6.9 Hz, ArH), 7.33 (d, 4H, J ¼ 8.3 Hz, ArH), 7.02e7.14
(m, 1H, ArH), 6.86 (d, 2H, J ¼ 7.5 Hz, ArH), 6.70 (d, 1H, J ¼ 7.5 Hz,
ArH); 13C NMR (CDCl3þDMSO-d6 75 MHz):
d
155.8, 149.4, 146.2,
5.1.3.21. 5-Methyl-2-[(1,10;40,100)terphenyl]-1H-benzo[d]imidazole
141.6, 139.4, 131.5, 128.5, 128.2, 127.4, 126.8, 126.4, 123.1, 122.0,
115.3, 114.7, 107.1; IR (KBr) (nmax/cmꢀ1): 3415ꢀ3651 (NH and ꢀOH),
2961, 2860,1344,1238, 831; MS (ESI): m/z 379 (M þ 1)þ; Anal. Calcd
for C25H18N2O2: C, 79.35; H, 4.77; N, 7.41, found: C, 79.26; H, 4.71;
N, 7.33.
(3u). Light yellow solid; Rf ¼ 0.4 (40% ethyl acetate/hexane); 1H
NMR (CDCl3, 300 MHz):
d
12.88 (brs, 1H, ꢀNH), 8.31 (d, 2H,
J ¼ 8.1 Hz, ArH), 7.70e7.80 (m, 5H, ArH), 7.68 (d, 4H, J ¼ 8.3 Hz, ArH),
7.35e7.59 (m, 4H, ArH), 6.70 (d, 1H, J ¼ 7.3 Hz, ArH), 2.45 (s, 3H,
eCH3); IR (KBr) (nmax/cmꢀ1): 3436 (NH), 2939, 2869, 1452, 1332,
813; MS (ESI): m/z 361 (M þ 1)þ; Anal. Calcd for C26H20N2: C, 86.65;
H, 5.56; N, 7.76, found: C, 86.58; H, 5.51; N, 7.72.
5.1.3.16. 4-Hydroxy-2-[4-methoxy(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3p). Coffee solid; Rf ¼ 0.3 (45% ethyl acetate/hexane);
1H NMR (CDCl3þDMSO-d6 300 MHz):
d
12.89 (brs, 1H, ꢀNH), 10.04
5.1.3.22. 5-Methyl-2-[4-fluoro(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3v). Brown solid; Rf ¼ 0.4 (40% ethyl acetate/hexane);
(s, 1H, ꢀOH), 8.30 (d, 2H, J ¼ 7.5 Hz, ArH), 7.70e7.81 (m, 3H, ArH),
7.67 (d, 2H, J ¼ 7.1 Hz, ArH), 7.60 (d, 2H, J ¼ 8.0 Hz, ArH), 6.98e7.14
1H NMR (CDCl3, 300 MHz):
d
12.88 (brs, 1H, ꢀNH), 8.31(d, 2H,
(m, 5H, ArH), 6.69 (d, 1H, J ¼ 7.5 Hz, ArH), 3.86 (s, 3H, eOCH3); 13
C
J ¼ 8.3 Hz, ArH), 7.70e7.80 (m, 4H, ArH), 7.83 (d, 4H, J ¼ 8.3 Hz, ArH),
7.35e7.59 (m, 3H, ArH), 7.10 (d, 2H, J ¼ 7.7 Hz, ArH), 2.49 (s, 3H,
NMR (CDCl3þDMSO-d6 75 MHz):
d 158.9, 149.6, 146.5, 140.8, 139.3,
137.6, 132.0, 129.6, 129.4, 128.7, 127.4, 126.9, 126.4, 122.9, 114.0,
106.9, 54.9; IR (KBr) (nmax/cmꢀ1): 3421ꢀ3650 (NH and ꢀOH),
2924, 2853, 1402, 1258, 817; MS (ESI): m/z 393 (M þ 1)þ; Anal.
Calcd for C26H20N2O2: C, 79.56; H, 5.14; N, 7.12, found: C, 79.48; H,
5.08; N, 7.17.
eCH3); 13C NMR (CDCl3þDMSO-d6 50 MHz):
d 160.3, 150.1, 141.0,
140.4,139.2,138.3,135.9,129.0,128.5,127.8,127.0,126.8,123.3,115.7,
110.8, 30.6; IR (KBr) (nmax/cmꢀ1): 3424 (NH), 3030, 2921, 2855,1440,
1302, 813; MS (ESI): m/z 379 (M þ 1)þ; Anal. Calcd for C26H19FN2: C,
82.55; H, 5.05; N, 7.42, found: C, 82.47; H, 4.48; N, 7.36.

16
A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
5.1.3.23. 5-Methyl-2-[4-trifluoromethyl(1,10;40,100)terphenyl]-1H-ben
zo[d]imidazole (3w). Brown solid; Rf ¼ 0.4 (40% ethyl acetate/
5.1.3.29. 5-Methyl-2-[3,4-dimethoxy(1,10;40,100)terphenyl]-1H-benzo
[d]imidazole (3ac). Pale yellow solid; Rf ¼ 0.3 (45% ethyl acetate/
hexane); 1H NMR (CDCl3, 300 MHz):
d
12.78 (brs, 1H, ꢀNH), 8.28 (d,
hexane); 1H NMR (CDCl3, 200 MHz):
d
12.89 (brs, 1H, ꢀNH),
d 8.13
2H, J ¼ 8.9 Hz, ArH), 7.86 (s, 1H, ArH), 7.79e7.83 (m, 4H, ArH), 7.74
(q, 4H, J ¼ 2.8, 8.9 Hz, ArH), 7.58 (d, 1H, J ¼ 7.7 Hz, ArH), 7.35 (d, 2H,
J ¼ 8.9 Hz, ArH), 7.04 (d, 1H, J ¼ 7.7 Hz, ArH), 2.46 (s, 3H, eCH3); IR
(KBr) (nmax/cmꢀ1): 3436 (NH), 2952, 2861, 1402, 1362, 833; MS
(ESI): m/z 429 (M þ 1)þ; Anal. Calcd for C27H19F3N2: C, 75.71; H,
4.51; N, 6.54, found: C, 75.66; H, 4.47; N, 6.51.
(d, 2H, J ¼ 8.1 Hz, ArH), 7.68 (d, 2H, J ¼ 8.0 Hz, ArH), 7.63 (s, 4H, ArH),
7.53 (d, 1H, J ¼ 8.2 Hz, ArH), 7.38 (s, 1H, ArH), 7.12e7.20 (m, 2H,
ArH), 7.06 (d, 1H, J ¼ 7.5 Hz, ArH), 6.95 (d, 1H, J ¼ 8.2 Hz, ArH), 3.96
(s, 3H, eOCH3), 3.93 (s, 3H, eOCH3), 2.44 (s, 3H, eCH3); IR (KBr)
(
nmax/cmꢀ1): 3429 (NH), 2930, 2836, 1452, 1368, 843; MS (ESI): m/z
421 (M þ 1)þ; Anal. Calcd for C28H24N2O2: C, 79.98; H, 5.74; N, 6.66,
found: C, 79.92; H, 5.69; N, 6.61.
5.1.3.24. 5-Methyl-2-[4trifluoromethoxy(1,10;40,100)terphenyl]-1H-ben
zo[d]imidazole (3x). Brown solid; Rf ¼ 0.4 (45% ethyl acetate/
5.1.3.30. 5-Methyl-2-[3,4,5-trimethoxy(1,10;40,100)terphenyl]-1H-benzo
hexane); 1H NMR (CDCl3, 300 MHz):
d
12.54e12.63 (brs, 1H, ꢀNH),
[d]imidazole (3ad). Brown solid; Rf ¼ 0.3 (45% ethyl acetate/
8.29 (d, 2H, J ¼ 8.5 Hz, ArH), 7.85 (s, 1H, ArH), 7.78e7.82 (m, 4H,
ArH), 7.73 (q, 4H, J ¼ 2.8, 8.5 Hz, ArH), 7.59 (d, 1H, J ¼ 7.6 Hz, ArH),
7.35 (d, 2H, J ¼ 8.5 Hz, ArH), 7.04 (d, 1H, J ¼ 7.6 Hz, ArH), 2.48 (s, 3H,
eCH3); IR (KBr) (nmax/cmꢀ1): 3436 (NH), 2961, 2848, 1432, 1382,
hexane); 1H NMR (CDCl3þDMSO-d6 400 MHz):
d 12.90 (brs,
1H, ꢀNH), 8.28 (d, 2H, J ¼ 8.5 Hz, ArH), 7.73e7.82 (m, 4H, ArH), 7.67
(d, 2H, J ¼ 8.5 Hz, ArH), 7.55 (d,1H, J ¼ 8.5 Hz, ArH), 7.44 (s,1H, ArH),
7.07 (d, 1H, J ¼ 8.5 Hz, ArH), 6.85 (s, 2H, ArH), 3.96 (s, 6H,
814; 13C NMR (CDCl3þDMSO-d6 50 MHz):
d
157.3,150.8,148.1, 142.4,
2 ꢄ eOCH3), 3.92 (s, 3H, eOCH3), 2.50 (s, 3H, eCH3); IR (KBr) (nmax
/
141.1, 135.2, 129.1, 127.5, 126.9, 125.8, 121.8, 120.7, 119.1, 114.5, 110.1,
30.2; MS (ESI): m/z 445 (M þ 1)þ; Anal. Calcd for C27H19F3N2O:
C, 72.95; H, 4.32; N, 6.34, found: C, 72.91; H, 4.25; N, 6.23.
cmꢀ1): 3428 (NH), 2932, 2829, 1456, 1340, 808; MS (ESI): m/z 451
(M þ 1)þ; Anal. Calcd for C29H26N2O3: C, 77.35; H, 5.82; N, 6.21,
found: C, 77.28; H, 5.77; N, 6.17.
5.1.3.25. 5-Methyl-2-[4-hydroxy(1,10;40,100)terphenyl]-1H-benzo[d]
5.1.4. Cell cycle analysis
Human lung cancer cell lines (A549) in 6 well plates were
incubated for 48 h in the presence or absence of test compounds
imidazole (3y). Brown solid; Rf ¼ 0.3 (40% ethyl acetate/hexane); 1H
NMR (CDCl3, 300 MHz):
d
12.88 (brs, 1H, ꢀNH), 10.03 (s, 1H, ꢀOH),
8.31 (d, 2H, J ¼ 7.7 Hz, ArH), 7.80 (m, 2H, ArH), 7.63e7.76 (m, 4H, ArH),
7.35e7.59 (d, 3H, J ¼ 8.0 Hz, ArH), 7.02e7.14 (m,1H, ArH), 6.85 (d, 2.H,
J ¼ 7.5 Hz, ArH), 6.69 (d, 1H, J ¼ 7.5 Hz, ArH), 2.44 (s, 3H, eCH3); IR
(KBr) (nmax/cmꢀ1): 3429ꢀ3635 (NH and ꢀOH), 2953, 2832, 1408,
1326, 819; MS (ESI): m/z 377 (M þ 1)þ; Anal. Calcd for C26H20N2O: C,
82.95; H, 5.35; N, 7.44, found: C, 82.88; H, 5.28; N, 7.39.
(5 mM) and nocodazole (1b, 1 mM). Cells were harvested with
Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 ꢂC for 30 min,
ethanol was removed by centrifugation and cells were stained with
250 mL of DNA staining solution [10 mg of Propidium Iodide (PI),
0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dis-
solved in 100 mL of sterile water] at room temperature for 30 min
in the dark. The DNA contents of 20,000 events were measured by
flow cytometry (DAKO CYTOMATION, Beckman Coulter, Brea, CA).
Histograms were analyzed using Summit Software.
5.1.3.26. 5-Methyl-2-[4-methoxy(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3z). Pale yellow solid; Rf ¼ 0.3 (40% ethyl acetate/
hexane); 1H NMR (CDCl3þDMSO-d6 300 MHz):
d 12.86 (brs,
1H, ꢀNH), 10.05 (s, 1H, ArH), 8.29 (d, 2H, J ¼ 7.5 Hz, ArH), 7.71e7.82
(m, 3H, ArH), 7.66 (d, 2H, J ¼ 7.1 Hz, ArH), 7.61 (d, 2H, J ¼ 8.1 Hz, ArH),
6.97e7.15 (m, 5H, ArH), 6.68 (d, 1H, J ¼ 7.5 Hz, ArH), 3.88 (s, 3H,
eOCH3), 2.44 (s, 3H, eCH3); IR (KBr) (nmax/cmꢀ1): 3433 (NH), 2981,
2831, 1401, 1323, 818; MS (ESI): m/z 391 (M þ 1)þ; Anal. Calcd for
C27H22N2O: C, 83.09; H, 5.68; N, 7.17, found: C, 83.01; H, 5.63; N, 7.14.
5.1.5. Tubulin polymerization assay
An in vitro assay for monitoring the time dependent polymeri-
zation of tubulin to microtubules was performed employing
a fluorescence-based tubulin polymerization assay kit (BK011,
Cytoskeleton, Inc.) according to the manufacturer’s protocol
[44,45]. The reaction mixture in a final volume of 10 mL in PEM
buffer (80MM PIPES, 0.5 mm EGTA, 2MM MgCl2, pH 6.9) contained,
2 mg/mL bovine brain tubulin, fluorescent reporter, 1 mM GTP in
5.1.3.27. 5-Methyl-2-[4-N,N-Dimethylamino(1,10;40,100)terphenyl]-1H-
benzo[d] imidazole (3aa). Brown solid; Rf ¼ 0.3 (40% ethyl acetate/
the presence or absence of test compounds 3o, 3t and 3ad (3 mM) at
hexane); 1H NMR (CDCl3, 300 MHz):
d
12.03 (brs, 1H, ꢀNH), 8.33 (d,
37 ꢂC. Tubulin polymerization was followed by monitoring the
fluorescence-enhancement due to the incorporation of a fluores-
cence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm)
was measured for 1 h at 1 min interval in a multimode plate reader
(Tecan). Nocodazole (1b) was used as a positive control under
similar experimental conditions.
2H, J ¼ 7.7 Hz, ArH), 7.81 (m, 2H, ArH), 7.61e7.73 (m, 4H, ArH),
7.33e7.62 (d, 3H, J ¼ 7.8 Hz, ArH), 7.00e7.15 (m, 1H, ArH), 6.86 (d,
2.H, J ¼ 7.7 Hz, ArH), 6.70 (d, 1H, J ¼ 7.7 Hz, ArH), 2.45 (s,
6H, ꢀN(CH3)2), 2.41 (s, 3H, eCH3); IR (KBr) (nmax/cmꢀ1): 3438 (NH),
2933, 2844, 1432, 1313, 871; MS (ESI): m/z 404 (M þ 1)þ; Anal. Calcd
for C28H25N3: C, 82.92; H, 6.38; N, 10.39, found: C, 82.81; H, 6.24;
N, 10.26.
5.1.6. Analysis of soluble versus polymerized tubulin in cells
5.1.3.28. 5-Methyl-2-[3-chloro(1,10;40,100)terphenyl]-1H-benzo[d]
imidazole (3ab). Brown solid (66% yield): Rf ¼ 0.4 (40% ethyl
acetate/hexane); mp: 215e217 ꢂC; 1H NMR (CDCl3, 300 MHz):
Cells were plated in 24-well dishes, grown to 60e80% con-
fluency, and treated with either no drug or 5
mM concentration of
3t, 3o and 3ad drugs alone, nocodozole (1b) and taxol (1c) (at 1
m
M
d
12.84 (brs, 1H, ꢀNH), 8.29 (d, 2H, J ¼ 8.3 Hz, ArH), 7.75e7.81 (m,
concentration) were used as positive and negative controls. Cells
were incubated with drugs for 24 h, later the media was removed,
cells were rinsed in 1X PBS at 22 ꢂC, harvested at the same
temperature in lysis buffer containing 0.1 M Pipes, 1 mM EGTA,
1 mM MgSO4, 30% glycerol, 5% DMSO, 5 mM GTP, 0.125% NP-40, and
protease inhibitors, including aprotinin [200 units/mL], pH 6.9 and
then centrifuged at 15000g at 22 ꢂC for 30 min in an Sorvall Leg-
endmicro 21R model temperature controlled centrifuge (Thermo
scientific), to separate polymerized (P) from soluble (S) tubulin.
4H, ArH), 7.63e7.71 (m, 3H, ArH), 7.50e7.58 (m, 2H, ArH), 7.40e7.47
(m, 2H, ArH), 7.34 (d, 1H, J ¼ 8.6 Hz, ArH), 7.05 (d 1H, J ¼ 8.6 Hz,
ArH), 2.49 (s, 3H, eCH3); 13C NMR (CDCl3þDMSO-d6 50 MHz):
d
150.4, 141.5, 140.3, 138.8, 137.9, 133.8, 131.2, 130.0, 129.1, 128.5,
126.9, 126.6, 124.7, 123.7, 114.7, 29.9; IR (KBr) (nmax/cmꢀ1): 3418
(NH), 2922, 2854, 1466, 1387, 823; MS (ESI): m/z 395 (M þ 1)þ; Anal.
Calcd for C26H19ClN2: C, 79.08; H, 4.85; N, 7.19, found: C, 79.02; H,
4.81; N, 7.12.

A. Kamal et al. / European Journal of Medicinal Chemistry 50 (2012) 9e17
17
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Y. Ikenishi, Y. Terui, J. Antibiot. 51 (1998) 445e450;
Pellets of polymerized “P” tubulin were resuspended in a volume of
lysis buffer equal to the soluble “S” fraction, and resolved in 7% SDS/
PAGE as described earlier [37]. After transfer to NC membrane
immunoblotting was performed with mouse anti a-tubulin anti-
body [DMIA, Sigma, St. Louis, MO], followed by an FITC-conjugated
secondary antibody (Sigma). The blot was imaged using Phosphor
imager (Fugifilm, Japan). Quantitative analysis of the soluble and
polymer fractions was done by densitometry using Gene-box
(Syngene).
(b) K. Kawada, A. Arimura, T. Tsuri, M. Fuji, T. Komurasaki, S. Yonezawa,
A. Kugimiya, N. Haga, S. Mitsumori, M. Inagaki, T. Nakatani, Y. Tamura,
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[28] (a) I.K. Lee, B.S. Yun, S.M. Cho, W.G. Kim, J.P. Kim, I.J. Ryoo, H. Koshino, I.D. Yoo,
J. Nat. Prod. 59 (1996) 1090e1092;
Acknowledgements
(b) B.S. Yun, I.K. Lee, J.P. Kim, I.D. Yoo, J. Antibiot. 53 (2000) 114e122;
(c) D.N. Quang, T. Hashimoto, M. Nukada, I. Yamamoto, M. Tanaka, Y. Asakawa,
Planta Med. 69 (2003) 1063e1066;
(d) D.N. Quang, T. Hashimoto, M. Nukada, I. Yamamoto, M. Tanaka,
Y. Asakawa, Chem. Pharm. Bull. 51 (2003) 1064e1067.
The authors M. K. R.; V. S. R and G. B. K are thankful to CSIR, Y. V. V
thankful to UGC New Delhi, for the award of research fellowships.
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