Effective synthesis of benzimidazoles-imidazo[1,2-a]pyrazine conjugates: A comparative study of mono-and bis-benzimidazoles for antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2019.07.042

A novel series of 6-substituted-8-(1-cyclohexyl-1H-benzo[d]imidazole-6-yl)imidazo[1,2-a]pyrazine and 6-substituted-8-(1-benzyl-1H-benzo[d]imidazole-6-yl)imidazo[1,2-a]pyrazine is first time synthesized and screen in vitro biological activity for 60 human cancer cell lines representing nine different cancer types. Derivatives 10 and 36 show antitumor activity for all tested cell lines, display comparable full panel mean-graph midpoint growth inhibition (MG_MID GI₅₀) values of 2.10 and 2.23 μM, respectively. Furthermore, these derivatives show strong binding interactions with DNA and bovine serum albumin (BSA), studied through absorption, emission, and circular dichroism techniques. These spectroscopic studies reveal that imidazo[1,2-a]pyrazine-benzimidazoles 10 and 36, intercalate with ct-DNA as a leading interaction for fundamental biologically significant effects, with monobenzimidazole show better activity than bisbenzimidazole. These experiments have confirmed that the imidazo[1,2-a]pyrazine and benzimidazole moieties are efficient pharmacophores to trigger binding to DNA. These compounds have also interacted with bovine serum albumin protein that demonstrating high values of binding constant.

Full text of DOI:10.1016/j.ejmech.2019.07.042

Accepted Manuscript
Effective synthesis of benzimidazoles-imidazo[1,2-a]pyrazine conjugates: A
comparative study of mono-and bis-benzimidazoles for antitumor activity
Iqubal Singh, Vijay Luxami, Kamaldeep Paul
PII:
S0223-5234(19)30663-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.042
EJMECH 11539
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2019
Revised Date: 2 July 2019
Accepted Date: 12 July 2019
Please cite this article as: I. Singh, V. Luxami, K. Paul, Effective synthesis of benzimidazoles-
imidazo[1,2-a]pyrazine conjugates: A comparative study of mono-and bis-benzimidazoles for
antitumor activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.07.042.
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ACCEPTED MANUSCRIPT
Effective synthesis of benzimidazoles-imidazo[1,2-a]pyrazine conjugates: A
comparative study of mono-and bis-benzimidazoles for antitumor activity
Iqubal Singh, Vijay Luxami and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology,
Patiala-147001, India
A novel series of imidazo[1,2-a]pyrazine-benzimidazole is first time synthesized and screen in
vitro biological activity for 60 human cancer cell lines representing nine different cancer types.

ACCEPTED MANUSCRIPT
Effective synthesis of benzimidazoles-imidazo[1,2-a]pyrazine conjugates: A
comparative study of mono-and bis-benzimidazoles for antitumor activity
Iqubal Singh, Vijay Luxami and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology, Patiala-
147001, India
Email: kpaul@thapar.edu
ABSTRACT
A novel series of 6-substituted-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine
and 6-substituted-8-(1-benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine is first time
synthesized and screen in vitro biological activity for 60 human cancer cell lines representing nine
different cancer types. Derivatives 10 and 36 show antitumor activity for all tested cell lines, display
comparable full panel mean-graph midpoint growth inhibition (MG_MID GI50) values of 2.10 and
2.23 ꢀM, respectively. Furthermore, these derivatives show strong binding interactions with DNA and
bovine serum albumin (BSA), studied through absorption, emission, and circular dichroism
techniques. These spectroscopic studies reveal that imidazo[1,2-a]pyrazine- benzimidazoles 10 and
36, intercalate with ct-DNA as a leading interaction for fundamental biologically significant effects,
with monobenzimidazole show better activity than bisbenzimidazole. These experiments have
confirmed that the imidazo[1,2-a]pyrazine and benzimidazole moieties are efficient pharmacophores
to trigger binding to DNA. These compounds have also interacted with bovine serum albumin protein
that demonstrating high values of binding constant.
Keywords: Imidazo[1,2-a]pyrazine; benzimidazole; anticancer activity; DNA interaction; bovine
serum albumin
INTRODUCTION
DNA is one of the key targets for cytotoxic antiproliferative drugs. Generally, these antitumor agents
damage DNA or block DNA synthesis, thus, responsible for inhibition of nucleic acid precursor
biosynthesis, or disrupt hormonal stimulation of cell growth [1]. So, more efficient, less toxic, and
target-specific non-covalently DNA binding anticancer drug needs to be developed. Extensive efforts
have been currently centred on the development of new anticancer drugs based on combination of two
active pharmacophores, particularly, bicyclic moieties, that are effective for binding and cleavage of
DNA [2] under physiological conditions. The structures of benzimidazole and imidazo[1,2-a]pyrazine
facilitate the compound bind strongly with DNA and hence, affect the critical metabolic routes. A
large number of derivatives based on these moieties has been designed and evaluated for antitumor
activity. Amongst these compounds, nocodazole], a 2-thienyl carbonyl benzimidazole, carbendazim
[4], a benzimidazole carbamate and Veliparib [5], a pyrrolidine-2-yl benzimidazole are used in the
clinic while mebendazole is currently undergoing clinical trials (Figure-1) [6]. The anticancer activity
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of benzimidazoles appear to derive from their ability to form strong complexes with nucleic acids and
thus induce DNA damage, and exert related effects such as topoisomerase poisoning, telomerase
inhibition and inhibition of gene transcription [7-9]. Consequently, extensive studies on the mode and
mechanism of interaction of benzimidazoles for binding to DNA are also reported from our
laboratory. To regulate the interaction properties of the drugs, different substituents as well as new
rings (quinazoline, triazine, purine, naphthalimide etc) were introduced with benzimidazole moiety
(Figure 1, A-D) [10-13]. With these derivatives, pharmacological data showed that the maximum
cytotoxicity was obtained with compounds substituted at position -5 or -6 of benzimidazole, and in
particularly with 2-chlorophenyl, 2-methoxyphenyl and pyrrolidine analogues. Additionally, it turned
out that some of these compounds were evaluated towards cell lines and DNA, displayed positive
activity on tumor and strong binding interactions with DNA. We anticipated that the proposed
molecules with 6-substituted benzimidazole would also bind with DNA, and favour the interaction
with nucleophilic sites of nucleic acid bases. Elucidation of the DNA binding activity of these
analogues might provide a new array of seeking other pharmacological activities.
To check these binding interactions and antitumor activity, we studied more closely the chemical
reactivity of imidazo[1,2–a]pyrazines-benzimidazole conjugates with variation of substitution at 6-
position of imidazo[1,2-a]pyrazine. We have also prepared symmetrical and unsymmetrical
benzimidazole substituted-imidazo[1,2-a]pyrazines, whereby compounds showed pronounced
antitumor activity. It has been observed that the most promising compound inhibited tumor growth,
which was related to their high DNA binding capacity, and the imidazo[1,2-a]pyrazine and
benzimidazole substituted derivatives showed the prominent activity [14,15]. From all of the above-
mentioned reflections, it has prompted us to prepare novel derivatives of 6-substituted-8-(1-
cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine and 6-substituted-8-(1-benzyl-1H-
benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (Figure 2), detailed study of their interactions with
DNA, and antitumor activity on the panel of cancer cell lines.
At the same time, proteins have also attracted enormous research interest as a prime molecular
target. Amongst the serum albumins, bovine serum albumin (BSA) is the most important protein
present in plasma, carries several exo- and endogenous compounds [16]. Attention has now been
centred on the protein required to drive and transportation of the drug to the target site for antitumor
activity [17]. Thus, it is essential to explore drug-protein interaction, as most of the drugs bound to
serum albumin are usually transported as a protein complex.
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Figure 1. Structures of benzimidazole derivative
Figure 2. Derivatives of 6-substituted-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-
a]pyrazine and 6-substituted-8-(1-benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine
RESULTS AND DISCUSSION
Chemistry: All compounds (9-27 and 34-36) shown in Figure 2 were prepared according to the
Schemes 1-2 starting from 1,3-dibromobenzene (Figures S1-S56). The commercial available 1,3-
dibromobenzene 1 was nitrated in the presence of sulphuric acid and nitric acid to afford 2 in 95%
yield which was regioselectively substituted with cyclohexylamine and benzylamine in DMF,
afforded 3a and 3b, respectively. Formation of 3a was confirmed using NMR spectral analysis, with
the multiplets of eleven protons corresponding to cyclohexyl ring in the range of δ 3.46-1.22 ppm.
Boronation of 3a and 3b using bis(pinacolato)diboron in the presence of Pd(PPh3)2Cl2 and KOAc
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provided 4a and 4b, respectively. Characteristic signals of twelve protons of four methyl groups of
boronate at δ 1.35 ppm, confirmed the formation of 4a. Suzuki-Miyaura cross-coupling of 4a-b with
dibromo-imidazo[1,2-a]pyrazine 5 [18] and Pd(PPh3)4, afforded compound 6a-b alongwith traces of
disubstituted products. Disappearance of singlet of methyl groups at δ 1.35 ppm of boronate and
1
appearance of aromatic protons of imidazo[1,2-a]pyrazine in the range of δ 8.69-7.77 ppm in H
NMR spectrum confirmed the formation of 6a. Reduction of derivatives with sodium dithionite in
ammonia provided amines 7a-b followed by cyclization with triethylorthoformate in acetic acid to
obtain intermediates 8a and 8b in 90% and 80% yields, respectively. Appearance of one additional
proton at aromatic region confirmed the formation of compounds 8a and 8b. Derivatives 3b-7b have
not been separated from column chromatography and used further without purification. Suzuki
reactions of intermediates with unsubstituted and substituted phenyl, thienyl and naphthyl boronates
were carried out in CH3CN:H2O using Pd(PPh3)4 and K2CO3 afforded 9-27 in 75-85% yields (Scheme
1, Figure S1). Appearance of five protons of phenyl ring, three protons of imidazopyrazine and four
protons of benzimidazole at aromatic region confirmed the formation of compound 9.
Scheme 1. Synthesis of 6-substituted-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-
a]pyrazine a
aReagents and conditions: (a) H2SO4, HNO3, DCM, 0 °C, 30 min., 95%; (b) Cyclohexyl amine/benzyl amine,
K2CO3, DMF, 100 °C, 18 h; (c) Bis(pinacolato)diboron, Pd(PPh3)2Cl2, KOAc, 1,4-dioxane, reflux, 10 h; (d)
Pd(PPh3)4, K2CO3, CH3CN : water (9:1), N2, reflux, 10-12 h; (e) Na2S2O4, aq. NH3, THF : water, rt, 1 h; (f)
Triethylorthoformate, AcOH, rt, 10 min., 80-90%; (g) ArB(OH)2, Pd(PPh3)4, K2CO3, CH3CN : water (9:1), N2,
reflux, 12-15 h, 75-85%.
Interestingly, the scope of another benzimidazole at C-6 position of imidazo[1,2-a]pyrazine for
Suzuki reaction was also explored with different synthetic route (Scheme 2). Nitration of the
commercial available 1,3- and 1,4-dibromobenzene (1 and 28) to yield 2 and 29, respectively, which
were substituted with cyclohexylamine in DMF, afforded 3a and 30. Formation of 30 was confirmed
using NMR spectral analysis, by the multiplet of eleven protons, corresponding to cyclohexyl ring in
the range of δ 3.52-1.25 ppm and broad singlet at δ 8.12 ppm of NH. Reductions of 3a and 30 were
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carried out with sodium dithionite in the presence of ammonia to afford 31a and 31b, respectively,
followed by cyclization with triethylorthoformate in acetic acid, produced respective benzimidazoles
32a and 32b. Boronation of derivatives using bis(pinacolato)diboron in the presence of Pd(PPh3)2Cl2
and KOAc afforded 33a and 33b. Suzuki-Miyaura cross-couplings of benzimidazole boronates 33a-b
with intermediate 8a-b have been performed using Pd(PPh3)4 and K2CO3 to afford 34-36 in 75-81%
yields. Appearance of three protons of imidazopyrazine and eight protons of benzimidazoles at
aromatic region, and twenty two protons of two cyclohexyl rings at δ 3.42-1.31 ppm confirmed the
formation of compound 34.
Scheme 2. Synthesis of 6/8-(1-benzyl/cyclohexyl-1H-benzo[d]imidazol-5/6-yl)-6/8-(1-cyclohexyl-
1H-benzo[d]imidazol-5/6-yl)imidazo[1,2-a]pyrazine b
NO2
NO2
NH2
N
a
b
d
c
Br
Br
Br
Br
Br
N
Br
Br
NH
NH
1, 28
2, 29
32a-b
3a, 30
31a-b
Br
28 =
1 =
Br
e
Br
Br
N
N
N
N
N
O
B
f
N
N
N
N
N
Ar2 =
O
Ar1 =
33a-b
Ar2
N
Ar1
34-36
(35) 75%
(34) 78%
N
N
N
Ar1 =
Ar2 =
N
(36) 81%
bReagents and conditions: (a) H2SO4, HNO3, DCM, 0 °C, 30 min., 93-95%; (b) Cyclohexyl amine, K2CO3,
DMF, 100 °C, 18 h; (c) Na2S2O4, aq. NH3, THF : water (3:2), rt, 1 h; (d) Triethylorthoformate, AcOH, rt, 10
min.; (e) Bis(pinacolato)diboron, Pd(PPh3)2Cl2, KOAc, 1,4-dioxane, reflux, 12 h; (f) 8a-b, Pd(PPh3)4, K2CO3,
CH3CN : water (9:1), N2, reflux, 10-12 h, 75-81%.
X-ray crystal structure. The molecular structure and the assignment of 9 were unambiguously
confirmed by single-crystal X-ray diffraction study (Figure 3, S57).
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Figure 3. X-ray crystal structure of compound 9 (CCDC No. 1828636)
Compound 9 consists of two planar fragments, benzimidazole and imidazo[1,2-a]pyrazine which
are in conjugation with phenyl ring present at C6-position of imidazo[1,2-a]pyrazine. Compound 9
crystallizes with Z = 4 in the space group P2/n (Tables S2-S9). Torsion angles along C-N and C-C of
imidazo[1,2-a]pyrazine are 174.66 (13) Å and 5.0 (2) Å, respectively, while torsion angle along C-C
bond of benzimidazole is 6.3 (2) Å. Moreover, the C-C bond length between imidazo[1,2-a]pyrazine
and benzimidazole is shifted to double bond length (1.48 Å). The six membered cyclohexyl ring
present at benzimidazole ring is deviated from planarity that exist in chair conformation. Atom
systems C2-C1-C14 and C3-C4-C15 having angle strain, slightly deviated by 1.8º and 1.5º,
respectively from the ideal tetrahedral value. The bond length of N5-C25 (1.31 Å) of benzimidazole
ring is shorter having double bond character than longer bond length of N1-C25 (1.36 Å), indicating
the presence of cyclohexyl group at N1 position and imidzao[1,2-a]pyrazine attached at the 6-position
of benzimidazole (C7). Similarly, phenyl ring present at 6-position of imidazo[1,2-a]pyrazine having
the bond length of 1.48 Å, indicating conjugation of these two moieties. Indeed, C-C bond length of
imidazo[1,2-a]pyrazine with benzimidazole and phenyl ring agrees well with the standard double
bond length of sp2 hybridized carbon atoms in the rings. There is thus strong conjugation between
these different planar moieties constitute the skeleton of molecule.
Molecular properties and drug-likeness. Good bioavailability can be achieved with appropriate
balance between solubility and partitioning properties. We have subjected a series of imidazol[1,2-
a]pyrazine-benzimidazole derivatives (9-27 and 34-36) for the prediction of lipophilicity and
solubility with Lipinski’s “Rule of Five” [19]. The results from the calculations revealed that all the
derivatives fulfilled the Lipinski’s rule except compounds 34-36, in which violation was due to high
molecular mass (Table S10). Experimental log P was determined by the octanol/water partition
coefficient using UV-visible spectroscopy. All of the compounds showed log P values less than 5
indicating good lipophilicity to penetrate into the cell. Compounds possessed less number of rotatable
bonds (3-5) and therefore, exhibited less conformational flexibility. These compounds showed
Topological Polar Surface Area (TPSA) value of < 70 Å2 that is also supported the drug absorption in
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digestive tract as well as blood-brain barrier penetration [20,21]. Calculation of percentage oral
absorption (%ABS = 109 - 0.345 TPSA) concluded that all derivatives were likely to be absorbed
well, having calculated oral absorption in the range of 86-92%.
Biological studies. Antiproliferative activity. National Cancer Institute (NCI) has obtained
samples from the NCI’s panel of 60 human tumor cell lines and measured a variety of targets in these
cell lines including gene mutations, mRNA levels, protein levels and enzyme activities. The
compound might inhibit these targets, or, the compound interacts with a related target (maybe a
component of the same metabolic pathway). The compound might cause some sort or damage that is
repaired by the targets. The target might regulate genes whose products are inhibited by the
compound. The compound might interact with a cellular component and trigger a checkpoint that
requires the target, thus might kill the cell. In a preliminary test, compounds were assayed at single
dose concentration (10-5 M) in the full panel of NCI 60 cancer cell lines [22]. The tested compounds
showed diverse but strong antiproliferative effect on the evaluated panel of cell lines and most of the
compounds exhibited more than 50% inhibition of tumor growth at micromolar concentration. As
revealed from results of 21 tested compounds, six compounds with wide range of growth percentage
displayed strong growth inhibitory activity (-88.03 to 99.61) at 10 µM concentration (Table 1). The
monobenzimidazole derivative 10 demonstrated superior activity than bisbenzimidazole with
cytostatic effects towards 42 cell lines while later showed cytotoxic effect with 43 cancer cell lines (in
case of compound 36). Thus, amongst the monobenzimidazoles, 4-methoxyphenyl at C6 position of
imidazo[1,2-a]pyrazine 10 indicated better cytostatic activity than phenyl 9, 4-ethylphenyl 24 and 4-
formylphenyl 26 derivatives in the series of compounds. Such findings denote that bisbenzimidazole
moiety is more favorable for cytotoxic and mono benzimidazole for cytostatic activity. This
superiority in the activity might be attributed to the corresponding increase in binding interactions
through π bonding and therefore, permeability and penetration into cancer cells. On referring to the
total number of sensitive cell lines for tested compounds, it has been found that most of the target
compounds exhibited broad spectrum anti-tumor activity covering different cancer subpanels.
Amongst these sensitive cell lines to tumor, colon cancer cell line HCC-2998 was found to be highly
sensitive with negative growth percentage value (lethal effect) for derivative 36. Central nervous
system (SF-539) cancer cell lines were noticed to be the most susceptible cell lines for derivative 10.
Moreover, T-47D (breast cancer) was also proved to be the most responsible cells to compounds 9, 24
and 26. Compounds 10 and 36 attributed pronounced antitumor activity over the majority of tested
cancer cell lines. The detailed anti-tumor activities on growth inhibition of NCI-60 human cancer cell
lines at the single dose of 10 µM, are shown in Tables S11-S13.
Table 1. Overview of the preliminary anticancer assay at single dose concentration of 10 µM
Compd.
Mean
Range of growth
inhibition
The most
sensitive
Positive
Positive No. of sensitive
growth
cytostatic cytotoxic cell lines/Total
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percent
59.72
20.32
65.98
71.82
54.67
-26.50
cell lines
T-47D
effectb
17/57
42/57
14/59
9/59
effectc
1/57
cell lines
18/57
51/57
14/59
9/59
-11.09a to 92.04
-43.35a to 98.64
0.55 to 94.82
9
SF-539
9/57
10
24
26
34
36
T-47D
0/59
0.53 to 98.01
T-47D
0/59
7.04 to 89.11
-88.03a to 99.61
RPMI-8226
HCC-2998
24/59
16/59
0/59
24/59
59/59
43/59
aNegative (-) indicates the cell killed. bThe ratio between number of cell lines with percent growth from 0 to 50
c
and total number of cell lines. The ratio between number of cell lines with percent growth of <0 and total
number of cell lines.
Compounds 10 and 36 have been shown interesting growth inhibitory activity in the preliminary
single dose screen and were evaluated for the advance 5-dose (10-4-10-8 M) testing mode against the
full panel. In five dose assay, the monobenzimidazole derivative seemed to contribute better activity
toward tumor growth which is evident from full panel mean-graph midpoint growth inhibition
(MIG_MID GI50) of compounds 10 (2.10 µM) and 36 (2.23 µM) (Table-2) as these values disguise
significant cell selectivity. The mean GI50 graph of 4-methoxyphenyl (10) showed potent activity in
the leukemia, non-small cell lung, colon, CNS, renal, prostate and breast cancer subpanels. Compound
10 showed GI50 of 422 nM for K-562 and 890 nM for SR of Leukemia cancer, GI50 of 622 nM for
NCI-H460 and 424 nM for NCI-H522 of non-small cell lung cancer, GI50 of 399 nM for HCT-116,
and 383 nM for HT29 cells of colon cancer, 361 nM for SF-295 of CNS cancer and 312 nM for
MDA-MB-435 cell lines of melanoma. Compound 10 also showed selectivity to other panel of cell
lines as shown in Table S14. Compound 36 showed GI50 of 799 nM for 786-0 cancer cell lines
corresponding to renal (Table S15). Higher LC50 values of compounds (usually > 100 µM) to most of
the cell panels indicated their low toxicity profile.
Table 2. Median growth inhibitory (GI50, µM), total growth inhibitory (TGI, µM) and median lethal
concentrations (LC50, µM) of in vitro evaluation of compounds 10 and 36 against subpanel of human
tumor cancer cell lines.
Compd Activity
I
II
III
IV
V
VI
VII
VIII IX
MIG_
MIDa
(µM)
GI50
TGI
LC50
GI50
TGI
LC50
1.60 1.65 1.24 1.18 6.95 3.04 1.30 0.84 1.11
2.10
10
36
b
b
5.05 6.15 9.23 2.49 3.20 7.64
19.23
7.57
b
b
b
b
b
b
b
b
b
b
1.91 2.18 1.97 2.11 2.00 2.46 3.01 2.50 1.95
5.57 4.56 3.74 2.82 4.04 5.92 3.62 4.82 3.6
2.23
4.30
4.76
b
b
b
b
b
b
b
3.41 6.12
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I, leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII,
a
b
prostate cancer; IX, breast cancer. Full panel mean-graph midpoint (MIG_MID) (µM). Compounds showed values > 100 µM.
Cytotoxicity against human normal cell line. To evaluate the safety, cytotoxicity effect of
the two most potent compounds (10 and 36) was determined by means of colorimetric assay
(MTT assay) against human normal cell line (Hek293) at 10-4, 10-5, 10-6, 10-7 and 10-8 M
concentrations. It has been observed that derivative 10 showed only 19.05%, 16.51%,
15.07%, 14.79% and 13.96% cytotoxicity to Hek293 cells whereas compound 36 exhibited
18.05%, 14.53%, 14.11%, 13.60% and 12.08% cytotoxicity to Hek293 cells at above said
concentrations (Figure 4). Cytotoxicity data shown that compounds 10 and 36 have low
cytotoxicity against mammalian cells, indicating the compounds were able to selectively kill
the cancer cells.
Figure 4. Effect of cytotoxicity of compounds 10 and 36 on human normal cell line Hek293
DNA interaction studies. The mode of anticancer activity of imidazo[1,2-a]pyrazine-
benzimidazole conjugates might be grounded on DNA damage. Two most potent compounds 10 and
36 were studied with ct-DNA to investigate the effect of these compounds on antiproliferative
activity. X-ray crystal structure analysis of these derivatives showed the planarity of compounds and
previous studies performed with planar heterocycles had demonstrated for occurrence of an
intercalative binding process upon complexation with DNA. So, these compounds might bound with
DNA via inercalation. Here, we have studied the capacity of binding of imidazo[1,2-a]pyrazine-
benzimidazole derivatives to DNA macromolecule with different spectroscopic methods like UV-
visible, fluorescence and circular dichroism.
UV-visible spectroscopy. The absorption spectra of compounds 10 and 36 (20 µM) showed intense
bands at 290 nm and 295 nm, respectively. On addition of ct-DNA (0-15 ꢀM), hypochromic shifts
were observed in phosphate buffer at pH 7.4. The absorbance of compounds 10 and 36 was decreased
by 20.83% and 15.27%, respectively (Figure 5a and S58). During the titration with DNA, an
isobestic point (10: 280 nm and 36: 290 nm) was observed which suggested a single mode of binding
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with compounds. Hypochromic effect observed in absorption spectra of compounds in the presence of
ct-DNA is characteristic of an intercalative binding [23]. On the basis of interaction of compounds
with DNA, the binding constants (Kb) for compound–DNA complexes have been determined from
Benesi-Hildebrand equation (equation 1) [24] which were found to be 3.34 ± 0.13 × 104 M-1 (95CI =
3.193 to 3.487 x 104) for 10 and 3.18 ± 0.01 × 104 M-1 (95CI = 3.169 to 3.191 x 104) for 36 (Figure
S59). These binding constants concluded that compound 10 has more affinity to bind with ct-DNA
than 36.
Any compound interacts with DNA, generally, interfere in the replication of DNA and
stop the division of the cancer cells. So, strong DNA binding activity corresponds to the
strong anticancer activity. In the present study, compound 10 interacted with DNA more
strongly than compound 36. Similarly, compound 10 (GI50 = 2.10 µM for full panel mean-
graph midpoint) showed more anticancer activity than compound 36 (GI50 = 2.23 µM for full
panel mean-graph midpoint) in 60 human cancer cell line studies, indicating the relationship
of DNA-binding and anticancer activity.
Figure 5. Effect of incremental addition of ct-DNA on (a) absorption and (b) emission spectra of
compound 10 in phosphate buffer at pH 7.4.
Thermal denaturation studies. Thermal denaturation experiments were performed for further
confirmation of interaction of compounds with DNA. The ability of the drug to protect ct-DNA
against thermal denaturation was used as an indication of their capacity to bind DNA and to stabilize
the double strand [25]. ꢀTm values for 10 and 36 on complexation with ct-DNA have been found to be
22 ºC and 12 ºC, respectively. ꢀTm values indicated that compound 10 has a strong affinity to bind
with DNA due to less steric hindrance and thus stabilized DNA against heat denaturation.
Fluorescence spectroscopy. Emission studies provide additional information regarding the
interaction mode of compounds with DNA. The binding of compounds with DNA was studied by
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maintaining the concentration of compounds 10 and 36 at 5 µM while varying the concentration of ct-
DNA (10: 0-110 µM; 36: 0-40 µM) in phosphate buffer (pH 7.4) at 298 K. On excitation of
compounds 10 at 290 nm and 36 at 295 nm, showed intense emission bands at 470 and 450 nm,
respectively. On increasing the concentration of ct-DNA to compounds, gradual quenching of the
fluorescence intensities by 75% and 35%, for compounds 10 and 36, respectively, were observed
without any significant change in emission maxima (Figures 5b and S60). Stern Volmer equation
(equation-2) [26] was performed to know more about the quenching process and to differentiate the
probable quenching mechanism. KSV was calculated from ratio of slope to intercept of Stern-Volmer
plot (Figure S61a) and found to be 4.65 ± 0.51 ×104 M-1 (95CI = 4.073 to 5.227 x 104) and 1.19 ±
0.15 ×104 M-1 (95CI = 1.020 to 1.359 x 104) for 10 and 36, respectively at 298 K (Table 3).
Compound 10 had the more KSV value, suggesting that compound 10 bound more strongly to ct-DNA
than 36.
A linear Stern-Volmer plot was obtained with compounds 10 and 36, suggesting the possibility of
single sort of binding process, either static or dynamic binding, which can be distinguished by
bimolecular binding/quenching constant (Kq). The value of Kq was calculated using KSV in equation 2
where the average fluorescence lifetime (τo) [27] is 10−8 s in the absence of DNA (analyte). The values
of Kq were observed to be 4.65 × 1012 M-1 s−1 and 1.19 × 1012 M-1 s−1 for respective compounds 10
and 36 (Table 3) which are much greater than typically observed for dynamic enhancement (~1 × 1010
M−1 s−1) [28]. The values of Kq indicated that interaction probably involved the static quenching with
formation of complex at ground state.
Table 3. Quenching and binding parameters of compounds upon interaction with ct-DNA
Compds. T (K)
K
SV (×104) (M-1) Kq (×1012) (M-1s-1)
aR
Kb (×104 M-1)
n
bR
298
308
318
298
4.65
1.68
0.73
1.19
4.65
1.68
0.73
1.19
0.9567
0.9445
0.9669
0.9921
36.52
1.29
1.08
0.58
1.21
0.9850
0.9614
0.9645
0.9916
10
4.57
0.02
9.58
36
aR (KSV and Kq) and bR (Kb and n) are the correlation coefficients
Modified Stern-Volmer equation (equation-3) [29] is generally used to calculate the binding
constant (Kb) and the number of binding sites (n) and in the present study, were found to be 36.52 ±
0.22 × 104 M-1 (95CI = 36.271 to 36.769 x 104) and 1.29 for compound 10 and 9.58 ± 1.3 × 104 M-1
(95CI = 8.109 to 11.051 x 104) and 1.21 for compound 36 at 298 K (Figure S61b). Compound 10
showed 1.5 times more binding interaction than 36 suggested that 10 bound strongly to ct-DNA. To
determine the effect of temperature on DNA binding with compounds, emission spectra of compound
10 (better binder with DNA) were recorded at 308 K and 318 K. On increasing the concentration of
ct-DNA (308 K: 0-115 µM.; 318 K: 0-95 µM) to compound 10 (5 µM), there is gradual quenching of
the fluorescence intensities by 65% and 60% at 308 K and 318 K, respectively, without any
11

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significant change of wavelength in emission maxima (Figure S62). The values of the Stern-Volmer
quenching constant [KSV; 1.68 ± 0.32 × 104 M-1 (95CI = 1.318 to 2.042 x 104) and 0.73 ± 0.08 × 104
M-1) (95CI = 0.515 to 0.945 x 104)], bimolecular quenching constant (Kq; 1.68 ×1012 M-1 s−1 and 0.7
×1012 M-1 s−1), the binding constant [Kb; 4.57 ± 0.16 × 104 M-1 (95CI = 4.389 to 4.751 x 104) and 0.02
± 0.005 × 104 M-1 (95CI = 0.014 to 0.026 x 104)] and the average number of binding site (n; 1.08 and
0.58) were calculated at 308 K and 318 K (Table 3, Figure S63). A decrease in binding constant is
observed with increasing temperature, indicating a reduction in the stability of the DNA-compound
adduct at higher temperature with exothermic nature of the binding process.
Thermodynamic parameters were calculated for compound 10 according to the van't Hoff
equation (equation 4) where enthalpy change (ꢀH) and entropy change (ꢀS) have been found to be -
69.28 Kcal M-1 and -205.94 cal M-1 K-1, respectively (Table 4, Figure S64). The negative value of
ꢀH showed that binding process is exothermic. Accordingly, the negative values of ꢀH and ꢀS
indicated that the hydrogen bonding and van der Waals contact played key role in the binding of
compound to DNA [30]. Moreover, the negative values of ꢀG obtained at different temperatures
revealed the favourable spontaneous nature of the binding process. It is noteworthy that increase
observed in ꢀG value with temperature indicating the influence of temperature for interaction of
compound with DNA.
Table 4. Thermodynamic parameters for the interaction of DNA with compound 10 at three different
temperatures.
T (K) ∆H (Kcal M-1) ∆S (cal M-1 K-1) ∆G (Kcal M-1)
aR
298
308
318
-69.28
-205.94
-7.91
-5.85
-3.79
0.9305
aR is the correlation coefficient
Ethidium bromide (EB) displacement. To evaluate the intercalation properties of compounds 10
and 36 with DNA, ethidium bromide (EB) displacement assay was performed. EB, a fluorescence
probe, has a planar structure that simply binds with DNA by an intercalative binding mode [31]. The
emission spectra of ethidium bromide-ct DNA complex (3 µM : 30 µM) at excitation of 520 nm,
showed an intense band at 606 nm. The fluorescence intensity of EB-DNA complex at 606 nm
decreased markedly with increasing the concentration of compounds (0-85 µM), indicated that some
of the EB molecules, which were intercalated into DNA base pairs, have been replaced by compounds
and released into the aqueous medium (Figures 6a and S65). The values of the Stern-Volmer
quenching constant [KSV; 0.82 ± 0.01 × 104 M-1 (95CI = 0.809 to 0.831 x 104) for compound 10 and
0.74 ± 0.01 × 104 M-1 (95CI = 0.729 to 0.751 x 104) for compound 36] and bimolecular quenching
constant (Kq; 0.82 ×1012 M-1 s−1 for compound 10 and 0.74 ×1012 M-1 s−1 for compound 36) were
calculated (Figure S66).
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Figure 6. (a) Effect of incremental addition of compound 10 on emission spectrum of complex of EB
(3 µM) and ct-DNA (30 µM); (b) CD spectra of free ct-DNA (40 µM) (blue line), 10/ct-DNA
complex (red line) and 36/ct-DNA complex (purple line) at ratio r[compound/ct-DNA] = 0.025.
Circular Dichroism (CD). In order to get insight into the conformation of ct-DNA on interaction
with compounds, circular dichroism technique was used. The ct-DNA can eventually acquire induced
CD spectrum (ICD) upon binding to molecules, from which mutual orientation of the ct-DNA could
be derived, consequently giving useful information about mode of interaction. It was obvious that
DNA had the characteristic CD signal in the UV-region, one negative band at 246 nm because of the
right handed B-form helicity, and one positive band at 278 nm because of the base stacking [32] was
observed. Addition of compounds 10 and 36 resulted in decrease in intensity of the ct-DNA, CD
spectrum at (λ) 246 nm and increase of CD band at (λ) 278 nm (Figure 6b). These compounds
yielded weak negative induced CD (ICD) band between (λ) 350 and 400 nm, thus excluding binding
into the minor groove of ct-DNA as a dominant interaction. The results obtained for compounds 10
and 36 together with weak negative ICD band points toward intercalative binding mode of interaction
with ct-DNA [33,34].
Bovine Serum Albumin (BSA) interactions. Serum albumin (SA) is the most abundant protein
in plasma which involves in the transport of drugs within body. Binding to these proteins may lead to
loss or enhancement of biological properties of the original drug. The possible binding interactions of
compounds 10 and 36 with BSA, structural homology of human serum albumin (HSA), have been
investigated by absorption and emission experiments.
UV-Visible spectroscopy. Absorption spectrum is a simple and appropriate technique to discover
the structural changes of protein and to investigate the protein-ligand complex formation. Absorption
spectra of BSA (10 µM) in phosphate buffer (pH 7.4) have been explored with incremental addition
of compounds (10 and 36) at 0-8 µM. The absorption band at 280 nm has been observed due to
aromatic amino acids of BSA (Trp, Tyr, and Phe) which can be influenced by species that interact
with this protein [35]. The intensity of the absorption peak of BSA has been increased markedly with
increasing concentration of the compounds (Figures 7a and S67). These variations originated from
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changes in the conformation and the polarity of the microenvironment around the aromatic residues of
BSA. These, in turn, resulted from penetration of compounds into the structure of BSA.
The values of binding constant (Kb) for the interaction of compounds with BSA were used to
compare the affinity of each derivative towards serum albumin. The binding constants (Kb) were
calculated using Benesi-Hildebrand equation (equation 1) [24] by plot of 1/(A-Ao) versus
1/[compound] (Figure S68) from the ratio of the intercept to the slope and found to be 1.95 ± 0.27 ×
105 M-1 (95CI = 1.644 to 2.256 x 105) for 10 and 2.65 ± 0.22 ×104 M-1 (95CI = 2.401 to 2.899 x 104)
for 36. Since the value of Kb is directly related to the extent of BSA binding and provide relative
affinities, in the present study, indicating that monobenzimidazole derivatives (10) have more ability
to penetrate into the cell than bisbenzimidazole moieties (36).
Figure 7. Effect of incremental addition of compound 10 on (a) absorption and (b) emission spectra
of BSA (10 µM, phosphate buffer, pH 7.4).
Fluorescence spectroscopy. The effects of 10 and 36 on the emission of BSA have also been
evaluated to gain more information on the interaction between the compounds and protein. Upon
excitation at 280 nm, BSA exhibited an intense fluorescent emission band at around 350 nm mainly
due to the presence of tryptophan residues. On increasing the concentration of compounds 10 (0-10
µM) and 36 (0-10 µM) to BSA (10 µM), quenching of the emission bands (75% for 36) at 350 nm
were observed, however, 10 showed ratiometric response toward BSA (Figures 7b and S69). The
emission intensity ratio at 470/350 nm has been found to be changed from 0.08 to 1.08, clearly
depicted the ~12-fold ratiometric response indicating strong interaction of compound 10 with BSA.
A comparison has also been made for the binding affinity with the help of Stern-Volmer equation
(equation-2) [26] that showed the relation between the quenching extent for each compound and the
strength of their interactions with BSA. The values of Stern-Volmer constant (KSV) and the apparent
bimolecular quenching constant (Kq) were calculated to evaluate the efficiency of quenching and the
accessibility of the fluorophores to the quenchers (compounds) (Figure S70a). A linear Stern-Volmer
plot was obtained with derivatives suggesting that single sort of binding process occurs either static or
14

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dynamic that can be distinguished by calculating bimolecular quenching constant (Kq) from equation-
2 using the lifetime of the fluorophore (τo) [27] 10-8 s in the absence of the quencher (compound).
The values of KSV for 10 and 36 were found to be 1.16 ± 0.034 × 105 M-1 (95CI = 1.121 to 1.198 x
105) and 1.14 ± 0.035 × 105 M-1 (95CI = 1.101 to 1.179 x 105), respectively. Compound 10 showed
slightly higher value of KSV than 36, indicating that compound 10 has better affinity to bound with
BSA. As shown in Table 5, the values of Kq for 10 and 36 have been found to be 1.16 and 1.14 × 1013
M-1s-1, which are significantly larger than the diffusion-controlled limit (~1 ×1010 M-1 s-1) [28],
indicating binding of the compounds to BSA probably involves the static quenching with formation of
complex at ground state.
The binding constants (Kb) and average number of binding sites (n) of imidazo[1,2-a]pyrazine-
benzimidazole conjugates have been calculated using the modified Stern-Volmer equation (equation-
3) [29] from plot of log (F₀-F)/F versus log [compound] (Figure S70b) and were found to be 4.68 ±
0.017 × 105 M-1 (95CI = 4.661 to 4.699 x 105) and 1.50 for compound 10, and 1.08 ± 0.021 × 105 M-1
(95CI = 1.056 to 1.104 x 105) and 1.01 for compound 36. Compound 10 showed the higher binding
constant than 36, indicating strong affinity with BSA. These binding parameters for the interactions
suggested that serum albumins might act as carrier proteins for these compounds and their metabolites
in delivering them to target tissues.
Table 5. Quenching and binding parameters for the interaction of BSA with compounds 10 and 36
Kb (×105 M-1)
Compd
K
SV (×105)(M-1) Kq (×1013) (M-1s-1)
aR
n
bR
1.16
1.14
1.16
1.14
0.9475
0.9578
4.68
1.08
1.50 0.9700
1.01 0.9771
10
36
aR (KSV and Kq) and bR (Kb and n) are the correlation coefficients
Molecular docking studies. Molecular docking is an important tool for researchers to speculate
the interaction between potential drug (ligand) and biomolecules. In the present study, we have
carried out docking using AutoDock suite (vina) [36] to confirm the mode of binding between
compounds 10 and 36 with DNA (pdb 1BNA) [37]. The optimized cluster was ranked by energy
levels in the best conformation of the ligand-DNA modelled structures, and the minimum binding
energies of the DNA with compounds 10 and 36 have been found to be -10.7 and -9.3 Kcal/mol,
respectively (Table S16). Methoxy group in case of 10 and N of benzimidazole in case of 36 are also
involved in the H-bonding with purine moiety of the guanidine group. Hydrogen bond has been
formed between the oxygen linked methoxy of 10 and H-22 hydrogen atom (d = 2.5 Å) association
with N-2 of guanine (DG14 of the B chain). Similarly, compound 36 formed two hydrogen bonds
with guanine. These hydrogen bonds were formed by the N of benzimidazole with the H-3 hydrogen
atom (d = 2.6 Å) associated with N-3 of guanine (DG16 of B chain) and H-21 hydrogen atom (d = 2.5
Å) associated with N-2 of guanine (DG10 of A chain) (Figure 8). Hence, because of the strong π -π
stacking interaction and the hydrogen bonding between compounds and the nucleic acid base, the
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torsional motion of compounds reduces substantially in the DNA solution, leading to change in its
emission yield.
(a)
(b)
Figure 8. Molecular docking of DNA (1BNA) with compounds (a) 10 and (b) 36, obtained from
Discovery Studio.
CONCLUSION
Derivatives of new conjugated ring systems 6-substituted-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-
yl)imidazo[1,2-a]pyrazine and 6-substituted-8-(1-benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-
a]pyrazine obtained in good to moderate yields from the key intermediate 6,8-dibromo-imidazo[1,2-
a]pyrazine, exhibiting excellent cytotoxicity activity against about 60 human tumor cell lines with
GI50 values reaching nanomolar concentrations in some of the cell lines. A particular efficacy was
observed with monobenzimidazole derivative 10 against leukemia, colon, CNS and melanoma
subpanels (GI50 = 0.31-0.39 ꢀM). The results with DNA interactions suggested that the fluorescence
of compounds 10 and 36 were quenched significantly and the probable quenching mechanism was a
static quenching process. The binding mode of these compounds to DNA was an intercalation
binding, which was supported by the results from ethidium bromide assay, circular dichroism and
DNA-melting measurements. Interestingly, a satisfactory correlation was observed between the extent
of DNA interaction and the antiproliferative activity. Moreover, the interaction of compounds 10 and
36 with bovine serum albumin proteins were studied through fluorescence emission spectroscopy that
revealing the binding to BSA with relatively high stability constants (Kb = 1.08-4.68 × 105 M-1) which
rely between optimum range to suggest binding, transfer and release upon arrival at their targets.
Thus, novel imidazo[1,2-a]pyrazine-benzimidazole conjugates with superior bioactivity profile and
their mode of interactions with DNA have been described, and the objective is thus fully reached.
EXPERIMENTAL SECTION
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Chemistry. General Methods. All commercially available compounds (Aldrich, Merck,
Spectrochem etc.) were used without further purification. All the recorded melting points were
1
13
uncorrected and measured in open capillaries. All H and C NMR characterization were performed
on Jeol ECS 400 NMR spectrometer, which was operated at 400 MHz for 1H nuclei and 100 MHz for
13C nuclei, taking CDCl3 as solvent. Chemical shifts are reported in parts per million (ppm) and TMS
was used as an internal reference. Coupling constants (J) were reported in hertz (Hz). The synthesized
compounds were characterized by mass spectra using Water Micromass-Q-T of Micro. Elemental
analysis has been done with Thermo Scientific (Flash 2000) analyzer. Purification of synthesized
compounds through column chromatography was done with the help of silica gel having mesh size of
60-120/100-200 using hexane/ethyl acetate and chloroform/methanol in various polarity system. UV-
Vis studies were carried out on a Shimadzu UV-2600 machine using slit width of 1.0 nm and matched
quartz cells. Emission spectra were determined on a Varian Cary Eclipse fluorescence spectrometer.
CD spectra were carried out on Applied Photophysics CD spectrophotometer. Absorption and
fluorescence scans were saved as ACS II files and further processed in Excel™ to produce all graphs
shown. Anticancer activities were evaluated at 9 panels of cancer cells including 60 different cancer
cell lines at NCI, USA. These cancer cell lines are: panel (cell lines); Leukemia (CCRF-CEM, HL-
60(TB), K-562, MOLT-4, RPMI-8226, SR), Non-Small Cell Lung Cancer (A549/ATCC, EXVX,
HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-522), Colon Cancer (COLO
205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539,
SNB-19, SNB-75, U251), Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2,
SK-MEL-28, SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1, OVCAR-3, OVCAR-
4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal Cancer (786-0, A498, ACHN, CAKI-1,
RXF 393, SN12C, TK-10, UO-31), Prostate Cancer (PC-3, DU-145), Breast Cancer (MCF7, MDA-
MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468).
2,4-dibromo-1-nitrobenzene (2) [38]: 1,3-Dibromobenzene (1) (5 gm, 21.18 mmol) in
dichloromethane (15 ml) and sulphuric acid (10 ml) was treated with dropwise addition of cooled
mixture of HNO3 and H2SO4 (4:1) at 0˚C and stirred for 30 min. Then, reaction was allowed to stir at
room temperature. The reaction mixture was quenched by pouring into ice. The precipitated product
was filtered and thoroughly washed with water. Air dried the crude to obtained the desired product (2)
5.53 gm, 95% yield; Rf 0.3 (hexane); mp 68-71 oC.
5-Bromo-N-cyclohexyl/benzyl-2-nitroaniline (3a-b): 2,4-Dibromo-1-nitrobenzene (2) (3 gm,
10.71 mmol), cyclohexyl amine / benzyl amine (1.27 gm / 1.37 gm, 12.85 mmol) and potassium
carbonate (1.47 gm, 10.71 mmol) were taken in dry dimethylformamide (20 ml) in 100 ml dried round
bottom flask and stirred the reaction mixture for 18 h at 100 ˚C. On completion of the reaction, 100
mL water was added to the reaction mixture at room temperature and extracted with ethyl acetate (3 ×
50 mL). Extract was dried over anhydrous Na2SO4, filtered and evaporation of solvent to get crude
17

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product. Purification of the crude was done by column chromatography using hexane/ethyl acetate
(20:1) as solvent system, to yield the desired product 3a-b.
5-Bromo-N-cyclohexyl-2-nitroaniline (3a): Yellow solid; 2.36 gm, 74% yield; Rf 0.3
(hexane); mp 90-93 oC; 1H NMR (CDCl3, 400 MHz): δ (ppm) 8.10 (d, J = 7.00 Hz, 1H, NH), 7.97 (d,
2
3
J = 9.12 Hz, 1H, ArH), 6.97 (d, J = 1.92 Hz, 1H, ArH), 6.65 (dd, J = 9.16 Hz, J = 2.04 Hz, 1H,
ArH), 3.46-3.39 (m, 1H, cyclohex-CH), 2.01-1.98 (m, 2H, cyclohex-CH2), 1.78-1.74 (m, 2H,
cyclohex-CH2), 1.64-1.59 (m, 1H, cyclohex-CH2), 1.45-1.22 (m, 5H, cyclohex-CH2); 13C NMR
(CDCl3, 100 MHz): δ (ppm) 145.0, 131.6, 130.5, 128.6, 128.3, 118.2, 116.6 (ArC), 51.1 (CH), 32.6
(CH2), 25.5 (CH2), 24.5 (CH2).
N-Cyclohexyl/benzyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (4a-b):
5-Bromo-N-cyclohexyl/benzyl-2-nitroaniline (3a-b) (2 gm, 6.68 mmol), bis(pinacolato)diboron (2.03
gm / 1.99 gm, 8.01 mmol), potassium acetate (0.98 gm / 1.35 gm, 10.02 mmol),
palladium(II)bis(triphenylphosphine)dichloride (1.0 mol%) and 1,4-dioxane (20 mL) were charged in
an oven dried RBF. The reaction mixture was stirred at reflux condition for 10 h until the aryl halide
was completely consumed as determined by thin layer chromatography. Solvent was evaporated under
reduced pressure followed by addition of 100 ml of water into it. The crude product was extracted
using chloroform (3 × 50 mL). Extract was completely dried using anhydrous Na2SO4 and filtered to
obtain the crude product. The product was purified via column chromatography using ethyl acetate
and hexane as eluents to obtain desired product (4a-b).
N-Cyclohexyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (4a): Reddish
o
1
yellow solid; 1.80 gm, 78% yield; Rf 0.5 (10% ethylacetate in hexane); mp 120-122 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 8.10 (d, J = 8.56 Hz, 2H, NH & ArH), 7.26 (s, 1H, ArH), 6.94 (d, J =
8.72 Hz, 1H, ArH), 3.69-3.61 (m, 1H, cyclohex-CH), 2.03-2.00 (m, 2H, cyclohex-CH2), 1.80-1.75 (m,
2H, cyclohex-CH2), 1.65-1.61 (m, 1H, cyclohex-CH2), 1.50-1.26 (m, 17H, cyclohex-CH2 & boronate-
CH3); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 144.0, 135.7, 132.8, 125.8, 120.9, 120.1, (ArC),
84.5 (C), 50.4 (CH), 32.9 (CH2), 25.0 (CH2), 24.9 (CH3), 24.4 (CH2). MS (ESI): m/z 346.2 (M++1);
Anal Calcd for C18H27BN2O4: C, 62.44; H, 7.86; N, 8.09; found C, 62.40; H, 7.80; N, 8.13.
5-(6-Bromoimidazo[1,2-a]pyrazin-8-yl)-N-cyclohexyl/benzyl-2-nitroaniline
(6a-b):
3,6-
Dibromoimidazo[1,2-a]pyrazine (5) (2 g, 7.22 mmol) in acetonitrile:water (9:1),
N-
cyclohexyl/benzyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (4a-b) (2.49 gm /
2.58 gm, 7.22 mmol) and K2CO3 (1.0 g, 7.22 mmol) were added under inert atmosphere. Then,
Pd(PPh3)4 (5 mol%) was added with continued nitrogen purging and refluxed the reaction for 10-12 h.
The solvent was evaporated under reduced pressure followed by 50 ml water was added and extracted
with chloroform. Chloroform layer was dried over sodium sulphate. Purification of the crude product
was obtained by column chromatography by adopting hexane:ethylacetate (9:1) as eluents (6a-b).
18

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5-(6-Bromoimidazo[1,2-a]pyrazin-8-yl)-N-cyclohexyl-2-nitroaniline (6a): Reddish solid; 2.09
o
1
gm, 70% yield; Rf 0.3 (10% ethylacetate in hexane); mp 152-154 C; H NMR (CDCl3, 400 MHz): δ
(ppm) 8.67 (d, J = 1.40 Hz, 1H, ArH), 8.30 (s, 1H, ArH), 8.28 (d, J = 9.08 Hz, 1H, ArH), 8.20 (d, J =
2
3
7.08 Hz, 1H, NH), 7.87 (d, J = 0.64 Hz, 1H, ArH), 7.80 (dd, J = 9.08 Hz, J = 1.72 Hz, 1H, ArH),
7.75 (d, J = 0.68 Hz, 1H, ArH), 3.79-3.69 (m, 1H, cyclohex-CH), 2.21-2.15 (m, 2H, cyclohex-CH2),
2.04-1.99 (m, 2H, cyclohex-CH2), 1.83-1.76 (m, 3H, cyclohex-CH2), 1.69-1.61 (m, 2H, cyclohex-CH2
), 1.54-1.43 (m, 1H, cyclohex-CH2 ); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 147.3, 144.5, 144.0,
141.2, 136.6, 126.9, 125.9, 120.9, 120.1, 119.2, 117.4, 115.2, 114.1 (ArC), 51.4 (CH), 31.5 (CH2),
25.6 (CH2), 24.9 (CH2).
5-(6-Bromoimidazo[1,2-a]pyrazin-8-yl)-N1-cyclohexyl/benzylbenzene-1,2-diamine
(7a-b):
Round bottom flask was charged with 5-(6-bromoimidazo[1,2-a]pyrazin-8-yl)-N-cyclohexyl/benzyl-
2-nitroaniline (6a-b) (2 gm, 4.80 mmol) and sodium dithionite (4.18 gm / 4.10 gm, 24.03 mmol) in
THF:Water (3:2) mixture. Ammonia solution (5 ml) was added to stirred reaction and further stirred
at room temperature for 1h. The reaction mixture was extracted with ethyl acetate. Ethyl acetate layer
was dried over sodium sulphate. Crude brown product obtained was directly used for further reaction
without purification.
6-Bromo-8-(1-cyclohexyl/benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (8a-b): 5-
(6-Bromoimidazo[1,2-a]pyrazin-8-yl)-N1-cyclohexyl/benzylbenzene-1,2-diamine (7a-b) (7) (1 gm,
2.59 mmol) was stirred with triethylorthoformate (0.385 gm / 0.375 gm, 2.59 mmol) in acetic acid at
room temperature for 10 min. Reaction was quenched in water and treated with NaHCO3 to basify the
reaction and extracted using chloroform. Sodium sulphate was used to dry the chloroform layer.
Purification of crude product was done through column chromatography using ethyl acetate and
hexane (3:7) as eluents.
6-Bromo-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (8a): Light
o
1
brown solid; 0.91 gm, 90% yield; Rf 0.5 (40% ethylacetate in hexane); mp 175-178 C; H NMR
2
3
(CDCl3, 400 MHz): δ (ppm) 9.00 (d, J = 1.52, 1H, ArH), 8.72 (dd, J = 8.68 Hz, J = 1.72 Hz, 1H,
ArH), 8.23 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.93 (d, J = 8.52 Hz, 1H, ArH), 7.88 (d, J = 1.16 Hz, 1H,
ArH), 7.72 (d, J = 1.20 Hz, 1H, ArH), 4.44-4.36 (m, 1H, cyclohex-CH), 2.30 (d, J = 11.44 Hz, 2H,
cyclohex-CH2), 2.01 (d, J = 13.80 Hz, 2H, cyclohex-CH2), 1.88-1.78 (m, 3H, cyclohex-CH2), 1.64-
1.53 (m, 2H, cyclohex-CH2), 1.43-1.28 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ
(ppm) 149.6, 145.8, 142.3, 138.7, 136.0, 133.5, 129.5, 124.2, 122.6, 120.0, 117.7, 114.0, 112.6 (ArC),
55.3 (CH), 33.6 (CH2), 25.7 (CH2), 25.4 (CH2). MS (ESI): m/z 395.0 (M++1); Anal Calcd for
C19H18BrN5: C, 57.59; H, 4.58; N, 17.67; found C, 57.48; H, 4.70; N, 17.93.
8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)-6-bromoimidazo[1,2-a]pyrazine (8b): Light brown
o
1
solid; 0.77 gm, 80% yield; Rf 0.4 (40% ethylacetate in hexane); mp 182-185 C; H NMR (CDCl3,
3
400 MHz): δ (ppm) 8.99 (d, J = 1.40 Hz, 1H, ArH), 8.71 (dd, 2J = 8.68 Hz, J = 1.36 Hz,1H, ArH),
19

ACCEPTED MANUSCRIPT
8.20 (s, 1H, ArH), 8.00 (s, 1H, ArH), 7.95 (d, J = 8.72 Hz, 1H, ArH), 7.85 (d, J = 0.92 Hz, 1H, ArH),
7.70 (d, J = 0.92 Hz, 1H, ArH), 7.37-7.28 (m, 5H, ArH), 5.48 (s, 2H, CH2-benzyl); 13C{1H} NMR
(CDCl3, 100 MHz): δ (ppm) 149.4, 145.9, 144.9, 138.6, 135.9, 135.2, 134.0, 129.9, 129.0, 128.3,
127.5, 124.2, 122.5, 120.1, 117.5, 113.8, 112.5 (ArC), 48.9 (CH2-benzyl). MS (ESI): m/z 403.0
(M++1); Anal Calcd for C20H14BrN5: C, 59.42; H, 3.49; N, 17.32; found C, 59.48; H, 3.60; N, 17.13.
6-Aryl-8-(1-cyclohexyl/benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (9-27):
6-Bromo-8-(1-cyclohexyl/benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (8a-b)
(150 mg, 0.378 mmol), arylboronic acid (0.378 mmol), K2CO3 (52.27 mg, 0.378 mmol) and Pd(PPh3)4
(5 mol%) were taken in a mixture of acetonitrile : water (9:1). Reaction mixture was refluxed for 12-
15 h under nitrogen until the completion of the reaction (checked by TLC). Solvents were evaporated
under reduced pressure. Water (50 ml) was added to the mixture and extracted with chloroform.
Chloroform layer was dried over sodium sulphate to get the crude product. Crude was further purified
by column chromatography using hexane:ethylacetate as eluents.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-phenylimidazo[1,2-a]pyrazine (9): Light green
o
1
solid; 111.64 mg, 85% yield; Rf 0.5 (40% ethylacetate in hexane); mp 194-196 C; H NMR (CDCl3,
2
3
400 MHz): δ (ppm) 9.18 (s, 1H, ArH), 8.79 (dd, J = 8.60 Hz, J = 1.48 Hz, 1H, ArH), 8.37 (s, 1H,
ArH), 8.10 (s, 1H, ArH), 8.05 (d, J = 7.44 Hz, 2H, ArH), 7.95 (d, J = 8.60 Hz, 1H, ArH), 7.83 (s, 1H,
ArH), 7.73 (s, 1H, ArH), 7.51 (t, J = 7.36 Hz,2H, ArH), 7.42 (t, J = 7.12 Hz, 1H, ArH), 4.42-4.34 (m,
1H, cyclohex-CH), 2.28 (d, J = 12.04 Hz, 2H, cyclohex-CH2), 1.98 (d, J = 11.36 Hz, 2H, cyclohex-
CH2), 1.87-1.77 (m, 3H, cyclohex-CH2), 1.60-1.51 (m, 2H, cyclohex-CH2), 1.38-1.27 (m, 1H,
cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 148.8, 145.4, 142.0, 138.9, 138.7, 136.7,
135.1, 133.4, 130.9, 128.9, 128.6, 126.2, 123.8, 119.8, 114.4, 113.7, 112.7 (ArC), 55.4 (CH), 33.5
(CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 394.3 (M++1); Anal Calcd for C25H23N5: C, 76.31; H,
5.89; N, 17.80; found C, 76.42; H, 5.87; N, 17.94.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazine
o
(10): Light green solid; 121.76 mg, 76% yield; Rf 0.6 (40% ethylacetate in hexane); mp 196-198 C;
2
3
1H NMR (CDCl3, 400 MHz): δ (ppm) 9.17 (d, J = 1.04, 1H, ArH), 8.76 (dd, J = 8.56 Hz, J = 1.40
Hz, 1H, ArH), 8.33 (s, 1H, ArH), 8.10 (s, 1H, ArH), 8.01 (d, J = 8.84 Hz, 2H, ArH), 7.94 (d, J = 8.64
Hz, 1H, ArH), 7.83 (d, J = 0.68 Hz, 1H, ArH), 7.73 (d, J = 0.64 Hz, 1H, ArH), 7.03 (d, J = 8.84 Hz,
2H, ArH), 4.43-4.35 (m, 1H, cyclohex-CH), 3.86 (s, 3H, CH3), 2.30 (d, J = 11.60 Hz, 2H, cyclohex-
CH2), 1.99 (d, J = 13.60 Hz, 2H, cyclohex-CH2), 1.88-1.78 (m, 3H, cyclohex-CH2), 1.62-1.52 (m, 2H,
cyclohex-CH2), 1.40-1.33 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 160.2,
148.9, 141.9, 138.9, 138.7, 135.1, 133.5, 131.0, 129.4, 127.6, 123.8, 119.9, 114.3, 114.2, 112.6, 112.6
(ArC), 55.5 (CH), 55.4 (OCH3), 33.6 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 424.3 (M++1);
Anal Calcd for C26H25N5O: C, 73.74; H, 5.95; N, 16.54; found C, 73.67; H, 5.78; N, 16.48.
20

ACCEPTED MANUSCRIPT
4-(8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazin-6-yl)benzaldehyde (11):
o
1
Light green solid; 130.75 mg, 82% yield; Rf 0.4 (40% ethylacetate in hexane); mp 198-201 C; H
NMR (CDCl3, 400 MHz): δ (ppm) 10.07 (s, 1H, CHO), 9.21 (s, 1H, ArH), 8.77 (dd, 2J = 8.52 Hz, 3J =
1.36 Hz, 1H, ArH), 8.54 (s, 1H, ArH), 8.26 (d, J = 8.24 Hz, 2H, ArH), 8.10 (s, 1H, ArH), 8.01 (d, J =
8.32 Hz, 2H, ArH), 7.95 (d, J = 8.64 Hz, 1H, ArH), 7.89 (d, J = 0.80 Hz, 1H, ArH), 7.81 (d, J = 0.64
Hz, 1H, ArH), 4.43-4.35 (m, 1H, cyclohex-CH), 2.31 (d, J = 11.20 Hz, 2H, cyclohex-CH2), 2.01-1.97
(m, 2H, cyclohex-CH2), 1.90-1.79 (m, 3H, cyclohex-CH2), 1.63-1.52 (m, 2H, cyclohex-CH2), 1.40-
1.29 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 191.9 (CO), 149.4, 142.6,
139.0, 137.3, 136.2, 135.6, 130.5, 130.4, 126.7, 123.7, 120.1, 114.9, 114.7, 112.8 (ArC), 55.5 (CH),
33.6 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 422.5 (M++1); Anal Calcd for C26H23N5O: C,
74.09; H, 5.50; N, 16.62; found C, 74.42; H, 5.25; N, 16.57.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(naphthalen-1-yl)imidazo[1,2-a]pyrazine (12):
o
1
Light green solid; 137.59 mg, 82% yield; Rf 0.4 (40% ethylacetate in hexane); mp 200-203 C; H
2
3
NMR (CDCl3, 400 MHz): δ (ppm) 9.07 (d, J = 1.12 Hz, 1H, ArH), 8.80 (dd, J = 8.60 Hz, J = 1.40
Hz, 1H, ArH), 8.29-8.27 (m, 2H, ArH), 8.08 (s, 1H, ArH), 7.96 (d, J = 7.08 Hz, 1H, ArH), 7.92 (s,
1H, ArH), 7.90 (d, J = 8.64 Hz, 1H, ArH), 7.80 (d, J = 0.52 Hz, 1H, ArH), 7.70 (dd, 2J = 6.96 Hz, 3J
= 0.84 Hz,1H, ArH), 7.58-7.53 (m, 2H, ArH), 7.53-7.47 (m, 2H, ArH), 4.38-4.30 (m, 1H, cyclohex-
CH), 2.27 (d, J = 11.44 Hz, 2H, cyclohex-CH2), 1.96 (d, J = 13.64 Hz, 2H, cyclohex-CH2), 1.85-1.75
(m, 3H, cyclohex-CH2), 1.55-1.46 (m, 2H, cyclohex-CH2), 1.34-1.25 (m, 1H, cyclohex-CH2); 13C{1H}
NMR (CDCl3, 100 MHz): δ (ppm) 148.9, 142.0, 140.3, 138.7, 135.3, 135.2, 134.1, 133.5, 131.8,
130.9, 129.4, 128.5, 127.7, 126.5, 126.1, 125.9, 125.4, 124.2, 119.9, 117.2, 114.2, 112.5 (ArC), 55.4
(CH), 33.5 (CH2), 25.7 (CH2), 25.4 (CH2); MS (ESI): m/z 444.5 (M++1); Anal Calcd for C29H25N5: C,
78.53; H, 5.68; N, 15.79; found C, 78.23; H, 5.89; N, 15.49.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(4-fluorophenyl)imidazo[1,2-a]pyrazine (13):
o
1
Light green solid; 121.42, 75% yield; Rf 0.5 (40% ethylacetate in hexane); mp 186-188 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 9.18 (d, J = 1.20 Hz, 1H, ArH), 8.74 (dd, 2J = 8.56 Hz, 3J = 1.48 Hz, 1H,
ArH), 8.35 (s, 1H, ArH), 8.09 (s, 1H, ArH), 8.04 (d, J = 5.36 Hz, 1H, ArH), 8.02 (d, J = 5.32 Hz, 1H,
ArH), 7.94 (d, J = 8.64 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.20 (t, J = 8.68 Hz, 2H,
ArH), 4.42-4.34 (m, 1H, cyclohex-CH), 2.29 (d, J = 11.56 Hz, 2H, cyclohex-CH2), 1.99 (d, J = 13.64
Hz, 2H, cyclohex-CH2), 1.88-1.78 (m, 3H, cyclohex-CH2), 1.62-1.50 (m, 2H, cyclohex-CH2), 1.40-
13
1.27 (m, 1H, cyclohex-CH2); C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 164.5, 162.0, 149.1, 145.6,
142.1, 138.9, 138.0, 135.3, 133.5, 132.9, 130.7, 128.1, 128.0, 123.7, 120.0, 116.0, 115.8, 114.4, 113.3,
112.7 (ArC), 55.4 (CH), 33.6 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 412.3 (M++1); Anal
Calcd for C25H22FN5: C, 72.97; H, 5.39; N, 17.02; found C, 72.72; H, 5.20; N, 16.85.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(thiophen-3-yl)imidazo[1,2-a]pyrazine
(14):
o
1
Light green solid; 125.43 mg, 83% yield; Rf 0.4 (40% ethylacetate in hexane); mp 180-183 C; H
21

ACCEPTED MANUSCRIPT
2
3
NMR (CDCl3, 400 MHz): δ (ppm) 9.13 (d, J = 0.96 Hz, 1H, ArH), 8.73 (dd, J = 8.68 Hz, J = 1.52
Hz, 1H, ArH), 8.32 (s, 1H, ArH), 8.08 (s, 1H, ArH), 8.02 (m, 1H, ArH), 7.94 (d, J = 8.60 Hz, 1H,
2
ArH), 7.83 (d, J = 0.72 Hz, 1H, ArH), 7.73 (d, J = 0.64 Hz, 1H, ArH), 7.60 (dd, J = 5.04 Hz,3J =
1.20 Hz, 1H, ArH), 7.45 (dd, 2J = 5.00 Hz,3J = 3.08 Hz,1H, ArH), 4.42-4.35 (m, 1H, cyclohex-CH),
2.30 (d, J = 12.08 Hz, 2H, cyclohex-CH2), 1.99 (d, J = 10.96 Hz, 2H, cyclohex-CH2), 1.88-1.78 (m,
3H, cyclohex-CH2), 1.61-1.52 (m, 2H, cyclohex-CH2), 1.39-1.28 (m, 1H, cyclohex-CH2); 13C{1H}
NMR (CDCl3, 100 MHz): δ (ppm) 149.3, 145.6, 142.0, 139.0, 138.9, 135.6, 135.1, 133.5, 130.7,
126.8, 125.1, 123.8, 123.0, 120.0, 114.3, 113.1, 112.6 (ArC), 55.4 (CH), 33.6 (CH2), 25.7 (CH2), 25.5
(CH2); MS (ESI): m/z 400.2 (M++1); Anal Calcd for C23H21N5S: C, 69.15; H, 5.30; N, 17.53; S, 8.02;
found C, 69.47; H, 5.59; N, 17.65; S, 8.40.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(m-tolyl)imidazo[1,2-a]pyrazine (15): Light
o
1
green solid; 121.78 mg, 79% yield; Rf 0.6 (40% ethylacetate in hexane); mp 182-185 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 9.16 (d, J = 1.04 Hz, 1H, ArH), 8.79 (dd, 2J = 8.64 Hz, 3J = 1.52 Hz, 1H,
ArH), 8.41 (s, 1H, ArH), 8.08 (s, 1H, ArH), 7.95 (d, J = 8.60 Hz, 1H, ArH), 7.91 (s, 1H, ArH), 7.85
(s, 1H, ArH), 7.83 (s, 1H, ArH), 7.76 (s, 1H, ArH), 7.41 (t, J = 7.60 Hz, 1H, ArH), 7.22 (s, 1H, ArH)
4.43-4.35 (m, 1H, cyclohex-CH), 2.46 (s, 3H, CH3), 2.31 (d, J = 11.44 Hz, 2H, cyclohex-CH2), 2.00
(d, J = 13.56 Hz, 2H, cyclohex-CH2), 1.89-1.79 (m, 3H, cyclohex-CH2), 1.62-1.51 (m, 2H, cyclohex-
CH2), 1.42-1.31 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 149.0, 145.6,
142.0, 139.0, 139.0, 138.6, 136.7, 135.2, 133.5, 130.9, 129.5, 128.9, 127.1, 123.9, 123.4, 120.0, 114.3,
113.6, 112.6 (ArC), 55.4 (CH), 33.5 (CH2), 25.7 (CH2), 25.5 (CH2), 21.6 (CH3); MS (ESI): m/z 408.6
(M++1); Anal Calcd for: C26H25N5: C, 76.63; H, 6.18; N, 17.19; found C, 76.53; H, 6.14; N, 17.45.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(4-ethylphenyl)imidazo[1,2-a]pyrazine (16):
o
1
Light green solid; 130.75 mg, 82% yield; Rf 0.6 (40% ethylacetate in hexane); mp 184-186 C; H
2
3
NMR (CDCl3, 400 MHz): δ (ppm) 9.16 (d, J = 0.76 Hz, 1H, ArH), 8.79 (dd, J = 8.60 Hz, J = 1.40
Hz, 1H, ArH), 8.39 (s, 1H, ArH), 8.08 (s, 1H, ArH), 8.00 (d, J = 8.16 Hz, 2H, ArH), 7.94 (d, J = 8.64
Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.35 (d, J = 8.12 Hz, 2H, ArH), 4.43-4.35 (m,
1H, cyclohex-CH), 2.75 (q, J = 7.60 Hz, 2H, CH2), 2.31 (d, J = 11.60 Hz, 2H, cyclohex-CH2), 2.00 (d,
J = 13.56 Hz, 2H, cyclohex-CH2), 1.89-1.78 (m, 3H, cyclohex-CH2), 1.63-1.52 (m, 2H, cyclohex-
CH2), 1.40-1.30 (m, 1H, cyclohex-CH2), 1.30 (t, J = 7.60 Hz, 3H, -CH3 ); 13C{1H} NMR (CDCl3, 100
MHz): δ (ppm) 149.0, 145.5, 145.0, 141.9, 139.0, 135.1, 134.3, 133.5, 131.0, 128.5, 126.3, 123.9,
120.0, 114.2, 113.2, 112.6 (ArC), 55.4 (CH), 33.6 (CH2), 28.7 (CH2), 25.7 (CH2), 25.5 (CH2), 15.6
(CH3); MS (ESI): m/z 422.5 (M++1); Anal Calcd for C27H27N5: C, 76.93; H, 6.46; N, 16.61; found C,
76.82; H, 6.31; N, 16.75.
1-(4-(8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazin-6-yl)phenyl)ethan-1-
one (17): Light green solid; 131.81 mg, 80% yield; Rf 0.6 (40% ethylacetate in hexane); mp 201-204
oC; 1H NMR (CDCl3, 400 MHz): δ (ppm) 9.21 (s, 1H, ArH), 8.78 (dd, 2J = 8.60 Hz, 3J = 1.16 Hz, 1H,
22

ACCEPTED MANUSCRIPT
ArH), 8.53 (s, 1H, ArH), 8.20 (d, J = 8.40 Hz, 2H, ArH), 8.13 (s, 1H, ArH), 8.10 (d, J = 8.40 Hz, 2H,
ArH), 7.96 (d, J = 8.64 Hz, 1H, ArH), 7.89 (s, 1H, ArH), 7.81 (s, 1H, ArH), 4.44-4.37 (m, 1H,
cyclohex-CH), 2.66 (s, 3H, CH3), 2.32 (d, J = 12.32 Hz, 2H, cyclohex-CH2), 2.01 (d, J = 13.80 Hz,
2H, cyclohex-CH2), 1.90-1.79 (m, 3H, cyclohex-CH2), 1.63-1.53 (m, 2H, cyclohex-CH2), 1.40-1.30
(m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 197.8 (C=O), 141.2, 139.0,
137.5, 136.9, 135.5, 130.9, 129.1, 126.3, 124.1, 119.7, 114.7, 112.9 (ArC), 55.6 (CH), 33.5 (CH2),
26.8 (CH3), 25.7 (CH2), 25.4 (CH2); MS (ESI): m/z 436.4 (M++1); Anal Calcd for C27H25N5O: C,
74.46; H, 5.79; N, 16.08; found C, 74.36; H, 5.89; N, 16.40.
6-(4-Chlorophenyl)-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (18):
o
1
Light green solid; 126.15 mg, 78% yield; Rf 0.6 (40% ethylacetate in hexane); mp 189-193 C; H
NMR (CDCl3, 400 MHz): δ (ppm) 9.18 (s, 1H, ArH), 8.74 (dd, 2J = 8.64 Hz, 3J = 1.36 Hz, 1H, ArH),
8.40 (s, 1H, ArH), 8.11 (s, 1H, ArH), 8.01 (d, J = 8.56 Hz, 2H, ArH), 7.94 (d, J = 8.64 Hz, 1H, ArH),
7.86 (d, J = 0.60 Hz, 1H, ArH), 7.76 (d, J = 0.60 Hz, 1H, ArH), 7.47 (s, 1H, ArH), 7.45 (s, 1H, ArH),
4.43-4.35 (m, 1H, cyclohex-CH), 2.30 (d, J = 12.12 Hz, 2H, cyclohex-CH2), 2.00 (d, J = 13.76 Hz,
2H, cyclohex-CH2), 1.88-1.76 (m, 3H, cyclohex-CH2), 1.62-1.52 (m, 2H, Cyclohex-CH2), 1.39-1.31
(m, 1H, Cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 149.1, 145.4, 142.0, 138.9,
137.7, 135.4, 135.3, 134.7, 133.4, 130.7, 129.1, 127.5, 123.8, 119.9, 114.5, 113.6, 112.7 (ArC), 55.5
(CH), 33.6 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 428.4 (M++1); Anal Calcd for C25H22ClN5:
C, 70.17; H, 5.18; N, 16.37; found C, 70.25; H, 5.14; N, 16.32.
6-(4-Bromophenyl)-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (19):
o
1
Light green solid; 139.44 mg, 78% yield; Rf 0.6 (40% ethylacetate in hexane); mp 197-200 C; H
NMR (CDCl3, 400 MHz): δ (ppm) 9.18 (s, 1H, ArH), 8.74 (dd, 2J = 8.56 Hz, 3J = 1.40 Hz, 1H, ArH),
8.39 (s, 1H, ArH), 8.12 (s, 1H, ArH), 7.94 (d, J = 8.56 Hz, 3H, ArH), 7.85 (s, 1H, ArH), 7.75 (s, 1H,
ArH), 7.62 (d, J = 8.52 Hz, 2H, ArH), 4.42-4.35 (m, 1H, cyclohex-CH), 2.30 (d, J = 12.60 Hz, 2H,
cyclohex-CH2), 2.00 (d, J = 13.68 Hz, 2H, cyclohex-CH2), 1.88-1.79 (m, 3H, cyclohex-CH2), 1.61-
1.51 (m, 2H, cyclohex-CH2), 1.40-1.30 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ
(ppm) 149.1, 138.9, 137.7, 135.7, 135.3, 132.0, 130.7, 128.6, 127.8, 127.4, 126.8, 123.8, 122.9, 119.9,
114.5, 113.6, 112.8 (ArC), 55.5 (CH), 33.5 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 472.2
(M++1); Anal Calcd for C25H22BrN5: C, 63.57; H, 4.69; N, 14.83; found C, 63.27; H, 4.34; N, 14.95.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(2-methoxyphenyl)imidazo[1,2-a]pyrazine
o
(20): Light green solid; 128.17 mg, 80% yield; Rf 0.5 (40% ethylacetate in hexane); mp 191-194 C;
1H NMR (CDCl3, 400 MHz): δ (ppm) 9.14 (s, 1H, ArH), 8.86 (s, 1H, ArH), 8.76 (dd, 2J = 8.64 Hz, 3J
2
= 1.40 Hz, 1H, ArH), 8.38 (dd, J = 7.76 Hz,3J = 1.76 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.94 (d, J =
8.60 Hz, 1H, ArH), 7.85 (d, J = 1.04 Hz, 1H, ArH), 7.76 (d, J = 0.84 Hz, 1H, ArH), 7.40-7.36 (m, 1H,
ArH), 7.20-7.15 (m, 1H, ArH), 7.06 (d, J = 8.24 Hz, 1H, ArH), 4.44-4.36 (m, 1H, cyclohex-CH), 3.97
(s, 3H, CH3), 2.31 (d, J = 12.36 Hz, 2H, cyclohex-CH2), 2.00 (d, J = 13.76 Hz, 2H, cyclohex-CH2),
23

ACCEPTED MANUSCRIPT
1.88-1.78 (m, 3H, cyclohex-CH2), 1.64-1.52 (m, 2H, cyclohex-CH2), 1.40-1.32 (m, 1H, cyclohex-
CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 156.8, 141.8, 138.8, 135.2, 135.0, 131.2, 131.0,
129.6, 125.3, 123.9, 121.3, 119.8, 118.4, 114.3, 112.4, 111.4 (ArC), 55.8 (CH), 55.4 (OCH3), 33.6
(CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 424.3 (M++1); Anal Calcd for C26H25N5O: C, 73.74; H,
5.95; N, 16.54; found C, 73.77; H, 5.72; N, 16.36.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(2-fluorophenyl)imidazo[1,2-a]pyrazine (21):
o
1
Light green solid; 127.64 mg, 82% yield; Rf 0.4 (40% ethylacetate in hexane); mp 192-194 C; H
2
3
NMR (CDCl3, 400 MHz): δ (ppm) 9.20 (d, J = 0.92 Hz, 1H, ArH), 8.74 (dd, J = 8.56 Hz, J = 1.40
Hz, 1H, ArH), 8.67 (s, 1H, ArH), 8.46-8.41 (m, 1H, ArH), 8.13 (s, 1H, ArH), 7.95 (d, J = 8.64 Hz,
1H, ArH), 7.87 (s, 1H, ArH), 7.78 (s, 1H, ArH), 7.67-7.62 (m, 1H, ArH), 7.35-7.33 (m, 1H, ArH),
7.21-7.16 (m, 1H, ArH), 4.43-4.37 (m, 1H, cyclohex-CH), 2.31 (d, J = 12.12 Hz, 2H, cyclohex-CH2),
2.00 (d, J= 13.80 Hz, 2H, cyclohex-CH2), 1.89-1.79 (m, 3H, cyclohex-CH2), 1.66-1.52 (m, 2H,
cyclohex-CH2), 1.43-1.32 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 149.1,
141.9, 138.8, 135.3, 133.4, 132.2, 132.1, 130.8, 130.0, 129.9, 128.6, 128.5, 124.8, 123.8, 119.9, 117.9,
117.8, 116.3, 116.1, 114.6, 112.8 (ArC), 55.5 (CH), 33.6 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI):
m/z 412.3 (M++1); Anal Calcd for C25H22FN5: C, 72.97; H, 5.39; N, 17.02; found C, 72.73; H, 5.42;
N, 17.42.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(thiophen-2-yl)imidazo[1,2-a]pyrazine
(22):
o
1
Light green solid; 123.92 mg, 82% yield; Rf 0.4 (40% ethylacetate in hexane); mp 188-191 C; H
NMR (CDCl3, 400 MHz): δ (ppm) 9.16 (s, 1H, ArH), 8.79 (dd, 2J = 8.64 Hz, 3J = 1.28 Hz, 1H, ArH),
8.34 (s, 1H, ArH), 8.10 (s, 1H, ArH), 7.93 (d, J = 8.64 Hz, 1H, ArH), 7.82 (s, 1H, ArH), 7.71 (s, 1H,
ArH), 7.55 (d, J = 3.40 Hz, 1H, ArH), 7.38 (d, J = 5.00 Hz, 1H, ArH), 7.12 (q, J = 3.76 Hz, 1H, ArH),
4.41-4.34 (m, 1H, cyclohex-CH), 2.30 (d, J = 12.12 Hz, 2H, cyclohex-CH2), 1.99 (d, J = 13.72 Hz,
2H, cyclohex-CH2), 1.89-1.79 (m, 3H, cyclohex-CH2), 1.66-1.47 (m, 2H, cyclohex-CH2), 1.39-1.29
(m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 148.9, 145.4, 142.1, 141.9, 138.8,
135.3, 134.8, 133.4, 130.5, 128.1, 126.6, 124.0, 123.1, 119.8, 114.4, 112.7, 111.8 (ArC), 55.5 (CH),
33.5 (CH2), 25.7 (CH2), 25.5 (CH2); MS (ESI): m/z 400.2 (M++1); Anal Calcd for C23H21N5S: C,
69.15; H, 5.30; N, 17.53; S, 8.02; found C, 69.14; H, 5.33; N, 17.32; S, 8.24.
8-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-6-(3-(trifluoromethyl)phenyl)imidazo[1,2-
a]pyrazine (23): Light green solid; 148.42 mg, 85% yield; Rf 0.6 (40% ethylacetate in hexane); mp
198-201 oC; 1H NMR (CDCl3, 400 MHz): δ (ppm) 9.17 (s, 1H, ArH), 8.83 (dd, 2J = 8.64 Hz, 3J = 1.36
Hz, 1H, ArH), 8.50 (s, 1H, ArH), 8.37 (s, 1H, ArH), 8.26 (d, J = 7.60 Hz, 1H, ArH), 8.11 (s, 1H,
ArH), 7.96 (d, J = 8.60 Hz, 1H, ArH), 7.89 (s, 1H, ArH), 7.80 (s, 1H, ArH), 7.70-7.61 (m, 2H, ArH),
4.43-4.35 (m, 1H, cyclohex-CH), 2.31 (d, J = 12.24 Hz, 2H, cyclohex-CH2), 2.01 (d, J = 13.76 Hz,
2H, cyclohex-CH2), 1.91-1.81 (m, 3H, cyclohex-CH2), 1.65-1.62 (m, 2H, cyclohex-CH2), 1.40-1.33
(m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 149.1, 142.1, 138.9, 137.6, 137.2,
24

ACCEPTED MANUSCRIPT
135.5, 133.4, 131.2, 130.7, 129.4, 129.3, 125.2, 124.5, 124.1, 123.1, 119.7, 114.7, 114.1, 112.7 (ArC),
55.7 (CH), 33.5 (CH2), 25.7 (CH2), 25.4 (CH2); MS (ESI): m/z 462.4 (M++1); Anal Calcd for
C26H22F3N5: C, 67.67; H, 4.81; N, 15.18; found C, 67.82; H, 4.99; N, 15.00.
8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)-6-(4-ethylphenyl)imidazo[1,2-a]pyrazine (24): Light
o
1
brown solid; 132.20 mg, 83% yield; Rf 0.3 (40% ethylacetate in hexane); mp 179-181 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 9.08 (s, 1H, ArH), 8.83 (dd, 2J = 8.52 Hz, 3J = 1.44 Hz, 1H, ArH), 8.37
(s, 1H, ArH), 8.00 (s, 1H, ArH), 7.97-7.94 (m, 3H, ArH), 7.81 (s, 1H, ArH), 7.73 (s, 1H, ArH), 7.37-
7.28 (m, 7H, ArH), 5.47 (s, 2H, benzyl-CH2), 2.75 (q, J = 7.60 Hz, 2H, ethyl-CH2), 1.31 (t, J = 7.64
Hz, 3H, ethyl-CH3); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 148.7, 145.6, 145.0, 144.6, 138.9,
135.5, 135.0, 134.1, 131.5, 129.1, 128.5, 128.4, 127.7, 126.2, 124.1, 120.0, 114.3, 113.2, 112.4 (ArC),
49.1 (benzyl-CH2), 28.7 (ethyl-CH2), 15.7 (ethyl-CH3); MS (ESI): m/z 430.6 (M++1); Anal Calcd for
C28H23N5: C, 78.30; H, 5.40; N, 16.31; found C, 78.38; H, 5.28; N, 16.10.
8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)-6-(m-tolyl)imidazo[1,2-a]pyrazine (25): Light brown
o
1
solid; 124.80 mg, 81% yield; Rf 0.3 (40% ethylacetate in hexane); mp 184-187 C; H NMR (CDCl3,
2
3
400 MHz): δ (ppm) 9.08 (d, J = 1.12 Hz, 1H, ArH), 8.82 (dd, J = 8.64 Hz, J = 1.44 Hz, 1H, ArH),
8.40 (s, 1H, ArH), 8.01 (s, 1H, ArH), 7.98 (d, J = 8.60 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.83 (s, 1H,
ArH), 7.74 (s, 1H, ArH), 7.41-7.28 (m, 7H, ArH), 7.25 (d, J = 7.56 Hz, 1H, ArH), 5.49 (s, 2H, benzyl-
CH2), 2.47 (s, 3H, CH3); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 148.8, 145.7, 144.6, 138.9,
138.6, 136.7, 135.5, 135.1, 134.1, 131.4, 129.5, 129.1, 128.8, 128.4, 127.6, 127.0, 124.1, 123.4, 120.1,
114.3, 113.7, 112.4 (ArC), 49.1 (benzyl-CH2), 21.7 (CH3); MS (ESI): m/z 416.5 (M++1); Anal Calcd
for: C27H21N5: C, 78.05; H, 5.09; N, 16.86; found C, 77.95; H, 5.02; N, 17.04.
4-(8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazin-6-yl)benzaldehyde (26): Light
o
1
brown solid; 125.83 mg, 79% yield; Rf 0.3 (40% ethylacetate in hexane); mp 182-185 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 10.09 (s, 1H, CHO), 9.11 (s, 1H, ArH), 8.83 (d, J = 8.48 Hz, 1H, ArH),
8.53 (s, 1H, ArH), 8.23 (d, J = 8.12 Hz, 2H, ArH), 8.04-7.95 (m, 4H, ArH), 7.87 (s, 1H, ArH), 7.79 (s,
1H, ArH), 7.38-7.30 (m, 5H, ArH), 5.50 (s, 2H, benzyl-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ
(ppm) 191.9 (CO), 144.8, 142.5, 138.9, 137.2, 136.2, 135.6, 135.4, 131.0, 130.3, 129.1, 128.4, 127.6,
126.6, 124.1, 120.2, 114.9, 114.7, 112.7 (ArC), 49.2 (benzyl-CH2); MS (ESI): m/z 430.6 (M++1);
Anal Calcd for C27H19N5O: C, 75.51; H, 4.46; N, 16.31; found C, 75.58; H, 4.50; N, 16.30.
8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)-6-(naphthalen-1-yl)imidazo[1,2-a]pyrazine (27): Light
o
1
brown solid; 137.30 mg, 82% yield; Rf 0.3 (40% ethylacetate in hexane); mp 183-186 C; H NMR
(CDCl3, 400 MHz): δ (ppm) 9.04 (d, J = 1.04 Hz, 1H, ArH), 8.81 (dd, 2J = 8.68 Hz, 3J = 1.56 Hz, 1H,
ArH), 8.26 (s, 1H, ArH), 8.23 (d, J = 8.48 Hz, 1H, ArH), 7.98-7.90 (m, 6H, ArH), 7.78 (s, 1H, ArH),
7.67 (d, J = 6.92 Hz, 1H, ArH), 7.58-7.54 (m, 2H, ArH), 7.52-7.45 (m, 2H, ArH), 7.31-7.27 (m, 2H,
ArH), 7.25-7.23 (m, 1H, ArH), 5.43 (s, 2H, benzyl-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm)
148.8, 145.6, 144.7, 140.3, 138.7, 135.4, 135.2, 135.2, 134.1, 134.1, 131.8, 131.3, 129.4, 129.1, 128.5,
25

ACCEPTED MANUSCRIPT
128.3, 127.7, 127.6, 126.6, 126.1, 125.8, 125.4, 124.3, 120.1, 117.3, 114.2, 112.4 (ArC), 49.1 (benzyl-
CH2); MS (ESI): m/z 452.5 (M++1); Anal Calcd for C30H21N5: C, 79.80; H, 4.69; N, 15.51; found C,
79.65; H, 4.55; N, 15.76.
1,4-Dibromo-2-nitrobenzene (29): Procedure for synthesis of 1,4-dibromo-2-nitrobenzene
(29) is similar as that for the synthesis of 2,4-dibromo-1-nitrobenzene (2) using 1,4-dibromobenzene
(28).
4-Bromo-N-cyclohexyl-2-nitroaniline (30): Procedure for synthesis of 4-bromo-N-cyclohexyl-
2-nitroaniline (30) is similar as that for the synthesis of 5-bromo-N-cyclohexyl-2-nitroaniline (3a)
using 1,4-dibromo-2-nitrobenzene (29). Yellow solid; 2.40 gm, 75% yield; Rf 0.3 (hexane); mp 93-96
1
oC; H NMR (CDCl3, 400 MHz): δ (ppm) 8.27 (d, J = 2.40 Hz, 1H, ArH), 8.12 (d, J = 7.08 Hz, 1H,
2
3
NH), 7.44 (dd, J = 9.20 Hz, J = 2.32 Hz, 1H, ArH), 6.79 (d, J = 9.28 Hz, 1H, ArH), 3.52-3.44 (m,
1H, cyclohex-CH), 2.05-2.02 (m, 2H, cyclohex-CH2), 1.81-1.74 (m, 2H, cyclohex-CH2), 1.68-1.64
(m, 1H, cyclohex-CH2), 1.47-1.25 (m, 5H, cyclohex-CH2);13C{1H} NMR (CDCl3, 100 MHz): δ (ppm)
143.7, 138.8, 136.1, 131.8, 129.0, 116.0, 114.8, 114.2, 105.8 (ArC), 51.2 (CH), 32.6 (CH2), 25.5
(CH2), 24.5 (CH2).
5-bromo-N1-cyclohexylbenzene-1,2-diamine (31a): 5-Bromo-N-cyclohexyl-2-nitroaniline
(3a) (1 g, 3.34 mmol) and sodium dithionite (2.9 g, 16.72 mmol) were charged in THF:water (3:2)
mixture. Ammonia solution (3 ml) was added to the reaction mixture at room temperature and further
stirred for 1h. The reaction mixture was extracted with ethyl acetate. Ethyl acetate was dried over
Na2SO4. Crude brown product was directly used for further reaction without purification.
4-bromo-N1-cyclohexylbenzene-1,2-diamine (31b): Similar synthesis route has been adopted
using 4-bromo-N-cyclohexyl-2-nitroaniline (30).
6-bromo-1-cyclohexyl-1H-benzo[d]imidazole (32a): 5-Bromo-N1-cyclohexylbenzene-1,2-
diamine (31a) (1 g, 3.71 mmol) was stirred with triethylorthoformate (0.550 g, 3.71 mmol) in acetic
acid at room temperature for 10 min. Reaction mixture was quenched in water and further treated with
NaHCO3 to basify the reaction and extracted using chloroform. Sodium sulphate was used to dry the
chloroform layer. Column chromatography was performed to purify the crude product with the help of
hexane and ethyl acetate (3:7) as eluent.
5-bromo-1-cyclohexyl-1H-benzo[d]imidazole (32b): Similar synthesis route has been adopted
using 4-bromo-N1-cyclohexylbenzene-1,2-diamine (31b).
1-cyclohexyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (33a): 6-
bromo-1-cyclohexyl-1H-benzo[d]imidazole (32a) (2 gm, 7.16 mmol), bis(pinacolato)diboron (2.19
gm, 8.60 mmol), potassium acetate (1.05 gm, 10.75 mmol) and palladium(II)bis(triphenylphosphine)
dichloride (5.0 mol%) were added in 1,4-dioxane (20 mL). The reaction mixture was refluxed for the
period of 12 h until aryl halide got completely consumed as determined by thin layer chromatography.
Solvent of reaction was evaporated under reduced pressure followed by addition of 100 ml water to
the reaction mixture. The crude product was extracted using chloroform (3 × 50 mL). Extract was
26

ACCEPTED MANUSCRIPT
dried over anhydrous Na2SO4, filtered to obtain the crude product. Crude material was purified via
column chromatography using ethyl acetate and hexane as solvent system to obtain desired reddish
yellow product.
1-cyclohexyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (33b):
Similar synthesis route has been adopted using 5-bromo-1-cyclohexyl-1H-benzo[d]imidazole (32b).
6,8-(Bisbenzimidazole)imidazo[1,2-a]pyrazine (34-36): 1-Cyclohexyl-6-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (33a) or 1-cyclohexyl-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-benzo[d]imidazole (33b) (1.0 eq), 8a-b (1.0 eq., 150 mg), K2CO3 (1.0 eq.) and
Pd(PPh3)4 (5 mol%) were taken in a mixture of acetonitrile : water (9:1). Reaction mixture was
refluxed for 12-15 h under nitrogen until the completion of the reaction (checked by TLC). Solvents
were evaporated under reduced pressure. Water (50 ml) was added to the mixture and extracted with
chloroform. Chloroform layer was dried over sodium sulphate to get the crude product. Crude was
further purified by column chromatography using hexane:ethylacetate as eluents.
6,8-Bis(1-yclohexyl-1H-benzo[d]imidazol-6-yl)imidazo[1,2-a]pyrazine (34): Light brown
o
solid; 152.15 mg, 78% yield; Rf 0.5 (40% ethylacetate in hexane); mp 225-228 C; 1H NMR (CDCl3,
2
3
400 MHz): δ (ppm) 9.17 (d, J = 0.84 Hz, 1H, ArH), 8.75 (dd, J = 8.64 Hz, J = 1.36 Hz, 1H, ArH),
8.48 (s, 1H, ArH), 8.24 (s, 1H, ArH), 8.11 (s, 1H, ArH), 8.07 (s, 1H, ArH), 7.97 (d, J = 8.60 Hz, 1H,
ArH), 7.91-7.84 (m, 3H, ArH), 7.80 (d, J = 0.72 Hz, 1H, ArH), 4.44-4.28 (m, 2H, 2 x cyclohex-CH),
2.27 (s (b), 4H, 2 x cyclohex-CH2), 2.00 (d, J = 9.92 Hz, 4H, 2 x cyclohex-CH2), 1.86-1.80 (m, 6H, 2
x cyclohex-CH2), 1.61-1.51 (m, 4H, 2 x cyclohex-CH2), 1.39-1.29 (m, 2H, 2 x cyclohex-CH2);
13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 149.2, 149.2, 145.5, 144.2, 142.0, 141.5, 141.5, 139.5,
139.0, 135.3, 134.1, 133.5, 131.8, 130.9, 123.8, 120.6, 120.5, 120.0, 114.3, 113.7, 112.7, 108.7 (ArC),
55.5 (CH), 55.4 (CH), 33.6 (CH2), 25.7 (CH2), 25.7 (CH2), 25.5 (CH2), 25.4 (CH2); MS (ESI): m/z
516.6 (M++1); Anal Calcd for C32H33N7: C, 74.54; H, 6.45; N, 19.01; found C, 74.65; H, 6.71; N,
19.32.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-8-(1-cyclohexyl-1H-benzo[d]imidazol-6-
yl)imidazo[1,2-a]pyrazine (35): Light brown solid; 146.30 mg, 75% yield; Rf 0.5 (80% ethylacetate in
hexane); mp 204-207 oC; 1H NMR (CDCl3, 400 MHz): δ (ppm) 9.17 (s, 1H, ArH), 8.80 (dd, 2J = 8.68
3
Hz, J = 1.48 Hz, 1H, ArH), 8.47 (s, 1H, ArH), 8.45 (d, J = 1.00 Hz, 1H, ArH), 8.11-8.06 (m, 3H,
ArH), 7.95 (d, J = 8.40 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.79 (d, J = 0.64 Hz, 1H, ArH), 7.56 (d, J =
8.56 Hz, 1H, ArH), 4.44-4.34 (m, 1H, cyclohex-CH), 4.33-4.20 (m, 1H, cyclohex-CH), 2.31 (t, J =
12.80 Hz, 4H, 2 x cyclohex-CH2), 2.00-1.97 (m, 4H, 2 x cyclohex-CH2), 1.91-1.74 (m, 6H, 2 x
cyclohex-CH2), 1.65-1.48 (m, 4H, 2 x cyclohex-CH2), 1.41-1.31 (m, 2H, 2 x cyclohex-CH2); 13C{1H}
NMR (CDCl3, 100 MHz): δ (ppm) 149.05, 145.45, 141.82, 141.46, 139.62, 139.02, 135.15, 133.93,
133.55, 131.47, 131.08, 124.00, 121.76, 119.97, 118.14, 114.39, 114.32, 113.39, 112.59, 110.58,
108.72, 77.42, 77.10, 76.79, 55.78, 55.34, 33.67, 33.42, 25.78, 25.74, 25.54, 25.48. (ArC), 55.7 (CH),
27

ACCEPTED MANUSCRIPT
55.3 (CH), 33.6 (CH2), 33.4 (CH2), 25.7 (CH2), 25.7 (CH2), 25.5 (CH2), 25.4 (CH2); MS (ESI): m/z
516.6 (M++1); Anal Calcd for C32H33N7: C, 74.54; H, 6.45; N, 19.01; found C, 74.39; H, 6.38; N,
18.71.
8-(1-Benzyl-1H-benzo[d]imidazol-6-yl)-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-
yl)imidazo[1,2-a]pyrazine (36): Light green solid; 157.28 mg, 81% yield; Rf 0.5 (90% ethylacetate in
hexane); mp 131-134 oC; 1H NMR (CDCl3, 400 MHz): δ (ppm) 9.13 (d, J = 1.12 Hz, 1H, ArH), 8.87
2
2
(dd, J = 8.64 Hz,3J = 1.48 Hz, 1H, ArH), 8.45 (s, 2H, ArH), 8.07 (s, 1H, ArH), 8.06 (dd, J = 8.60
Hz,3J = 1.60 Hz, 1H, ArH), 8.00 (s, 1H, ArH), 7.99 (d, J = 8.64 Hz, 1H, ArH), 7.83 (d, J = 0.84 Hz,
1H, ArH), 7.77 (s, 1H, ArH), 7.55 (d, J = 8.56 Hz,1H, ArH), 7.38-7.28 (m, 5H, ArH), 5.50 (s, 2H,
benzyl-CH2), 4.28-4.20 (m, 1H, cyclohex-CH), 2.28 (d, J = 11.16 Hz, 2H, cyclohex-CH2), 2.02 (d, J =
13.64 Hz, 2H, cyclohex-CH2), 1.87-1.78 (m, 3H, cyclohex-CH2), 1.59-1.49 (m, 2H, cyclohex-CH2),
1.40-1.29 (m, 1H, cyclohex-CH2); 13C{1H} NMR (CDCl3, 100 MHz): δ (ppm) 148.6, 145.5, 144.6,
144.3, 141.4, 139.4, 138.9, 135.5, 135.0, 134.2, 133.9, 131.5, 131.3, 129.1, 128.4, 127.7, 124.2, 121.6,
120.0, 118.1, 114.3, 113.3, 112.4, 110.5 (ArC), 55.7 (CH), 49.0 (benzyl-CH2), 33.4 (CH2), 25.7
(CH2), 25.4 (CH2); MS (ESI): m/z 524.6 (M++1); Anal Calcd for C33H29N7: C, 75.69; H, 5.58; N,
18.72; found C, 75.63; H, 5.63; N, 18.75.
Procedure for in vitro anticancer screening
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. Cells are inoculated into 96 well microtiter
plates in 100 ml at plating densities ranging from 5,000 to 40,000 cells/well depending on the
doubling time of individual cell lines. The microtiter plates are then incubated at 37 °C, 5% CO2, 95%
air and 100% relative humidity for 24 h.
After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of
the cell population for each cell line. Experimental drugs are solubilized in DMSO at 400-fold the
desired final maximum test concentration and stored frozen prior to use. At the time of drug addition,
an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 µg/ml gentamicin. Additional four, 10-fold or ½
log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of
100 µL of these different drug dilutions are added to the appropriate microtiter wells, resulting in the
required final drug concentrations. Following drug addition, the plates are incubated for an additional
°
48 h at 37 C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is
terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold
°
50% (w/v) TCA and incubated for 60 min at 4 C. The supernatant is discarded, and the plates are
washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4%
(w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 min at room
temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and
the plates are air dried and then subsequently solubilized with 10 mM trizma base, and the absorbance
28