Purine-benzimidazole hybrids: Synthesis, single crystal determination and in vitro evaluation of antitumor activities

Article abstract of DOI:10.1016/j.ejmech.2015.02.036

In an effort to identify novel compounds for the treatment of cancer, a diverse array of potential bioactive hybrid, purine-benzimidazole was synthesized in good yields through nucleophilic substitution at C6 position of purine ring with versatile cyclic amines at C2 position. The structures of newly prepared compounds were confirmed by IR, ¹H, ¹³C NMR, mass spectroscopy and, in case of 19, by single crystal Xray diffraction analysis. The newly synthesized compounds were evaluated against 60 human tumour cell lines at one dose concentration level. Compound 6 exhibited significant growth inhibition and was evaluated as 60 cell panel at five dose concentration levels. Compound 6 proved to be 1.25 fold more active than the positive control 5-FU, with GI50 value of 18.12 μM (MG-MID). Interaction of the compounds with Aurora-A enzyme involved in the process of propagation of cancer, has also been investigated. Compound 6 showed selectivity towards Aurora-A kinase inhibition with IC50 value of 0.0l mM. Molecular docking studies in the active binding site provided theoretical support for the experimental biological data acquired.

Full text of DOI:10.1016/j.ejmech.2015.02.036

Accepted Manuscript
Purine-benzimidazole hybrids: Synthesis, single crystal determination and in vitro
evaluation of antitumor activities
Alka Sharma, Vijay Luxami, Kamaldeep Paul
PII:
S0223-5234(15)00132-4
10.1016/j.ejmech.2015.02.036
EJMECH 7718
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 September 2014
Revised Date: 23 December 2014
Accepted Date: 19 February 2015
Please cite this article as: A. Sharma, V. Luxami, K. Paul, Purine-benzimidazole hybrids: Synthesis,
single crystal determination and in vitro evaluation of antitumor activities, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.036.
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ACCEPTED MANUSCRIPT
Purine-benzimidazole hybrids: Synthesis, single crystal determination and in vitro
evaluation of antitumor activities
Alka Sharma, Vijay Luxami, Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147004 INDIA
∗
To whom corresponding should be addressed. Tel.: +91-94656-70595; Fax: +91-175-236-
498;
4
E-mail address: kpaul@thapar.edu (K.Paul)
N
CH3
N
HN
N
N
N
H
N
N
6
0 Human Cancer Cell Lines
Aurora-A Kinase Inhibitor
IC50 = 0.01 µM
GI = 18.12 µM
50

ACCEPTED MANUSCRIPT
Purine-benzimidazole hybrids: Synthesis, single crystal determination and in vitro
evaluation of antitumor activities
Alka Sharma, Vijay Luxami, Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147004 INDIA
∗
To whom corresponding should be addressed. Tel.: +91-94656-70595; Fax: +91-175-236-
498;
4
E-mail address: kpaul@thapar.edu (K. Paul)
Abstract
In an effort to identify novel compounds for the treatment of cancer, a diverse array of
potential bioactive hybrid, purine-benzimidazole was synthesized in good yields through
nucleophilic substitution at C6 position of purine ring with versatile cyclic amines at C2
1
13
position. The structures of newly prepared compounds were confirmed by IR, H, C NMR,
mass spectroscopy and, in case of 19, by single crystal X-ray diffraction analysis. The newly
synthesized compounds were evaluated against 60 human tumour cell lines at one dose
concentration level. Compound 6 exhibited significant growth inhibition and was evaluated
as 60 cell panel at five dose concentration levels. Compound 6 proved to be 1.25 fold more
active than the positive control 5-FU, with GI value of 18.12 µM (MG-MID). Interaction of
5
0
the compounds with Aurora-A enzyme involved in the process of propagation of cancer, has
also been investigated. Compound 6 showed selectivity towards Aurora-A kinase inhibition
with IC value of 0.0l µM. Molecular docking studies in the active binding site provided
5
0
theoretical support for the experimental biological data acquired.
Keywords: Purine, Benzimidazole, X-ray structure, Antitumor activity, Aurora kinase
inhibitor, Molecular modeling
1
. Introduction
Cancer is becoming a major health problem in developing and undeveloped countries
1,2]. Although major advances have been made in the chemotherapeutic management of
[
some patients, the continued commitment to the laborious task of discovering new
anticancer agents remains critically important. In the course of identifying various
chemical substances which may serve as leads for designing novel antitumor agents, we
are particularly interested in the present work of molecular hybridizations with purine and
1

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benzimidazole derivatives which have been identified as a new class of cancer
chemotherapeutic agents with significant therapeutic efficacy against solid tumors [3]. It
is well known that purine derivatives are potent inhibitors of Aurora kinase [4], cyclin
dependent kinase (CDK) [5], epidermal growth factor receptor (EGFR) and vascular
endothelial growth factor receptor (VEGFR) [6]. Consequently, various approaches have
been adopted to enhance the potency and selectivity of these inhibitors. These efforts led
to discovery of many drugs such as Olomoucine, Roscovitine, R-CR8 and Dinaciclib
(Figure 1) that have also maintained selectivity towards various kinases. Olomoucine and
Roscovitine are first discovered CDK inhibitors while the optically pure R-enantiomer of
Roscovitine (Seliciclib) is currently being evaluated as an oncology drug candidate in
patient diagnosed with non small cell lung cancer and nasopharyngeal cancer [7] or other
malignancies [8]. The anti-HIV/HBV drugs abacavir and penciclovir are some of the
purine drugs, also available presently in the market [9]. Similarly, benzimidazole and its
derivatives are categorized as the important pharmacophores and privileged sub-structures
in medicinal chemistry owing to their involvement as a key component for various
biological activities [10]. Benzimidazoles are among the important heterocyclic
compounds that found in natural and non-natural products such as vitamin B12, marine
alkaloid kealiiquinone [11] etc. Some of their derivatives are marketed as anti-fungal
agent such as Carbendazim [12], anti-helmintic agents such as Mebendazole and
Thiabendazole [13] and anti-psychotic drug such as Pimozide [14].
Figure 1
In view of the previous rationale and in continuation of an ongoing program aiming at
finding new structure leads with potential chemotherapeutic activities using molecular
hybridization [15], new series of molecular hybrids using purine and benzimidazole have
been synthesized and screened for in vitro antitumor activity as well as Aurora A kinase
inhibitors. These series comprises the derived 2,6-disubstituted purine pharmacophore that
are structurally related to Olomoucine and Roscovitine. In the present study, the
substitution pattern at the 2,6-disubstituted purine and benzimidazole pharmacophore
were selected so as to confer different electronic environment that would affect the
activity of target molecules. SAR, QSAR and molecular modelling studies were used to
identify the structural features required for the antitumor properties of these new hybrid
series.
2

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2
. Results and Discussion
.1. Chemistry
2
The synthetic strategy to prepare the purine-benzimidazole hybrids (3a-d and 4-19) has
been depicted in scheme 1. The target compounds were achieved in three steps using 3,4-
dihydro-1H-purin-2,6-(5H,9H)-dione as starting material. Refluxing of 3,4-dihydro-1H-
purin-2,6-(5H,9H)-dione with phosphorus oxychloride in the presence of triethylamine for
5
h afforded 2,6-dichloropurine (1). Treatment of compound 1 with 2a [15] in the
presence of isopropyl alcohol (IPA) at room temperature for 24 h gave 3a in 79% yield.
Similarly, compound 1 was also treated with 2b-d under the same reaction conditions to
give compounds 3b-d with 47-94% yields. Refluxing of compounds 3a-d with different
secondary amines in ethanol for 48 h and after purification with column chromatography
gave pure compounds 4-19 with moderate to excellent yields (Table-1). All the
1
13
synthesized compounds were well characterized by IR, H, C NMR, mass spectroscopy
supporting information) and in case of compound 19, by single crystal X-ray diffraction
analysis (Figure 2).
(
Scheme 1
Table 1
Figure 2
2
.2. In vitro anticancer screening
All the synthesized compounds were submitted to National Cancer Institute (NCI) disease-
oriented human cell lines for in-vitro evaluation as antitumor activities (Table S1). Fourteen
compounds (3a-d, 4, 6-11, 16, 18-19) were evaluated against 60 cell lines at a single dose of
1
0 µM concentration [16-19] and their outputs were reported as a mean graph of the percent
growth of treated cells, and presented as percentage growth inhibition (GI %). Compound 6
exhibited significant growth inhibition and was evaluated for further 60 cell panel at five dose
concentration levels.
Preliminary in vitro antitumor screening was revealed that only compounds belonging
to the series 4-11 showed significant inhibition for most of cancer cell lines. The percentage
of inhibition for cancer cells were more than 50% in a number of the tested derivatives. On
the contrary, compounds 18 and 19 showed weak activities compared with other compounds
and percentage of inhibition did not reach 30% except one or two cell lines. These variations
3

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could be correlated to the difference in positions of allyl or butyl group on the benzimidazole
moiety in which the distance between the core purine moiety and alkyl chains of
benzimidazole are important factor for affecting antitumor activity. Compound 6 proved to be
active towards lung cell NCI-H60 with GI value of 99.63%; colon cells HT29 and KM12
with GI values of 87.58% and 86.91%; melanoma cell MDA-MB-435 with GI value of
9
0.84%; renal cells A498, ACHN and TK-10 with GI values of 98.65%, 82.19% and 88.17%
respectively. Meanwhile, compound 9 showed selectivity towards leukemia cell MOLT-4
with GI value of 90.95%; CNS cell SNB-75 with GI value of 88.56%; ovarian cell OVCAR-4
with GI value of 80.98% and breast cells BT-549 and T-47D with GI values of 82.04% and
9
3.22% respectively (Supporting Information).
From the above data, it is clear that compound 6 is the most active member of the series.
Consequently, this active compound was carried over and tested against a panel of different
tumor cell lines at five dose concentration level (Table S2). Compound 6 showed specificity
towards colon, CNS and ovarian cancer cell lines with GI values of 3.16, 2.00 and 1.34 µM
5
0
respectively. MG-MID revealed that compound 6 is 1.25 fold more active than 5-fluorouracil
(
5-FU), with GI value of 18.12 µM (Table-2).
50
Table 2
.3. Aurora-A kinase inhibition of compounds
2
Based on the anti-cancer activities of the compounds, it was desirable to investigate some of
the compounds for probable cellular targets. Interactions of compounds with Aurora-A
enzyme involved in the process of propagation of cancer, has been investigated with the help
of enzyme immunoassay using Aurora-A kinase inhibitor screening kit [20]. It has been
shown that compounds 3a and 5 displayed least activity towards Aurora A with IC values
5
0
of 8.00 and 8.50 µM respectively. Amines substitution at C2 position of purine with
morpholine and pyrrolidine resulted compounds 4 and 6 respectively that showed excellent
inhibitory activity towards Aurora-A with IC50 values of 0.02 and 0.01 µM (Table-3).
Substitution of 3a with piperidine (compound 5) decreased the inhibitory activity while
substitution with 4-methylpiperazine increased the Aurora A inhibitory activity with IC50
value of 0.072 µM. Replacement of N-allyl group with N-butyl group (compound 3b) of
benzimidazole resulted in increase in potency with IC value of 0.075 µM. Substitution of
5
0
3
b at C2 position of purine with pyrrolidine resulted in almost 10 fold decrease in inhibitory
activity. Therefore, it seems that the compound 6 under present investigation probably target
4

ACCEPTED MANUSCRIPT
Aurora-A for exhibiting best anti-cancer activity. Ligand efficiency (LE) has also been
determined for these compounds that indicated the higher efficiency of compound 6 with LE
=
0.39 for binding to the enzyme. The partition coefficient of the molecules was also studied
in octanol/water systems for the determination of log P values by shake-flask method [21]
Supporting Information). It has been indicated that compound 6 showed higher log P value
(
that supported the dependency of lipophilicity with higher activity of this compound towards
Aurora A inhibition and cancer cell lines.
Table 3
2
.4. Quantitative structure-activity relationship (QSAR)
IC values of purine-benzimidazole hybrid compounds were first calculated by Aurora-A
5
0
kinase assay kit. Geometries of these compounds were refined by means of semi empirical
2
2
method PM3 using QSARINS Software [22]. We have calculated q , r , RMSE , RMSE and
cv
tr
F using log P, heat of formation, molar refractivity and steric energy to get equation 1. With
the addition of each parameter, there is improvement of the correlation coefficient. So on
collectively using these four parameters into a multiple linear regression analysis, we
obtained much improved in the correlation coefficient (eq. 1)
log (IC ) = 25.4623 +14.2235 log P + 0.1660 heat of formation – 0.3082 molar refractivity +
5
0
1
.1124 steric energy
2
2
n = 7 q
= 0.9185, r = 0.994,
...........eq.(1)
LOO
RMSE = 1.0444, RMSE = 0.2825, F = 83.3054
cv
tr
2
2
Here, n - number of samples, r - correlation coefficient, q - cross validated, obtained by
leave one out method and F - fischer statistics.
Equation 1 appears to be the best QSAR model obtained by the multiple linear regression
analysis, considered significant for the activity (Figure 3). Eq (1) showed the positive
contribution of log P and steric energy indicating the importance of lipophilicity and steric
hindrance of purine and benzimidazole rings for the activity of hybrid.
Figure 3
2
.5. Structure–activity relationship (SAR)
Structure-activity correlation, based on the number of cancer cell lines and Aurora A kinase
inhibitors revealed that the nature of the substituents at C2- and C6-positions of purine
affected the biological functions. Regarding 60 human tumor cell line studies, compounds 4,
5

ACCEPTED MANUSCRIPT
6
-11 showed comparatively higher activity than their isomers 16, 18-19 suggested that there
is much difference in antitumor activity with orientations of alkyl chain of benzimidazole. In
the first series of compounds, when the allyl group is replaced with butyl group, a
comparable affinity is found (compare 3a and 3b), but when chloro of 3a is constrained in a
cyclic morpholine system (compound 4), some activity is increased although other rings are
well tolerated as illustrated by pyrrolidine (6) and 4-methylpiperazine (7) analogues and
former showed higher activity than later. Similarly, substitution at C2-position of 3b with
piperidine leading to compound 9 that has increased the activity while decrease in activity
was observed with morpholine, pyrrolidine and 4-methylpiperazine (8, 10-11). Another series
of compounds 16, 18-19, substitution of 3d with morpholine (16) increases the activity while
pyrrolidine (18) and 4-methylpiperazine (19) decreases the activity. SAR study of Aurora A
kinase inhibitory activity was also demonstrated that substitution of 3a with morpholine and
pyrrolidine showed almost 400-fold and 800-fold increase in activity while replacement of
these cyclic substituent with 4-methylpiperazine decreases the affinity slightly (compound 7).
These studies indicated that substitution of the purine heterocycle with various cyclic
secondary amines leads to highly potency towards 60 human cancer cell line activities as well
as Aurora A kinase inhibitions.
2
.6. Molecular docking studies
Molecular docking studies were also carried out for compound 6, which has been proved to
be most active compound. Although the cellular targets were not defined in the experimental
anticancer investigation of these molecules, to look into the possible interactions at the
enzymatic level, we have carried out the docking studies [23] of compound 6 in the active
sites of Aurora-A kinase (PDB ID 2WTV and 2XNE) using builder tool kit of software
package ArgusLab 4.0.1 (www.arguslab.com) [24].
Docking of compound 6 in the active site of Aurora-A (2WTV) showed hydrogen
bonding interaction of N atom of five membered purine moiety in the active site of His644
(d = 2.48 Ǻ, d = 2.09 Ǻ and d = 2.79 Ǻ) amino acid residue. Linker NH group of purine and
benzimidazole showed H-bonding interaction with Asp622 (d = 2.39 Ǻ) amino acid residue
and nitrogen atom of benzimidazole moiety showed H-bonding interaction with Ser625 (d =
2
.59 Ǻ) amino acid residue. Pyrrolidine moiety also showed hydrogen bonding of N atom
with active site of Arg626 (d = 1.87 Ǻ) amino acid residue (Figure 4). Therefore, docking of
compound 6 in the active site of these enzymes indicated the probable mode of action for
anticancer activities.
6

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The binding mode of purine-benzimidazole-based kinase inhibitors were also elucidated by
co-crystallisation of Aurora-A (2XNE) and compared with isoform Aurora B/C. The N9 of
purine in compound 6 showed hydrogen bonding with carbonyl group of Leu215 (d = 2.60
Å), amino groups of Leu 264 (d = 2.81 Å) and Gly216 (d = 1.91 Å) in the hinge region of the
kinase. Purine N7 is hydrogen bonded to the amino group of Gly216 (d = 2.36 Å) and the
nitrogen atom of pyrrolidine to the carbonyl and amino groups of Thr217 (d = 2.39 Å and d =
2
.32 Å) as shown in figure 5. N1 atom of purine also showed hydrogen bonding with
carbonyl group Thr217 (d = 2.66 Å). Linker NH group of benzimidazole and purine showed
hydrogen bonding interaction with amino group of Thr217 (d = 2.91 Å). The benzimidazole
with allyl chain of the inhibitor sits in a hydrophobic pocket formed the gatekeeper residues
Val147, Glu211, Lys162 and Ala160. Importantly, the pyrrolidine moiety substituted at C2
position of purine resides in close proximity to Val218 of Aurora-A (3.3 Å closest contact),
whereas the equivalent residue in Aurora-B/C is a leucine. This is one of the three active site
sequence differences between Aurora-A and Aurora-B/C. The Arg220 side chain in Aurora-A
(lysine in Aurora-B/C) points away from the active site. We exploited this observation in the
design of compounds with substantially enhanced selectivity in inhibiting Aurora-A over
isoforms -B and -C. It was envisaged that isoform selectivity for Aurora-A could be achieved
by the introduction of a C2 purine and pyrrolidine moieties bearing an electron rich
substituent capable of interacting with Leu215, Thr217 and Arg220 in Aurora-A, which
would sterically clash with the equivalent residue in Aurora-B/C. It should be noted that
Bavetsias et. al. and Coumar et al. recently reported respective imidazo[1,2-a]pyrazine and
pyrazole-based Aurora-A selective inhibitor, and rationalised the selectivity for inhibition of
Aurora-A over Aurora-B/C by proposing a similar argument, that is, hydrogen bonding
interaction with the backbone NH of Thr217 in Aurora-A and steric clash with the equivalent
residue in Aurora B/C [25].
Figure 4
Figure 5
3
. Conclusion
The present work has led to the development of novel hybrids of purine-benzimidazole
molecules and some of which shown promising antitumor activities. Compound 6 showed
-5
more active in most of the cancer cell lines at 10 M concentration and showed sensitivity
towards colon cancer, CNS cancer and ovarian cancer with GI values of 3.16, 2.00 and 1.36
5
0
7

ACCEPTED MANUSCRIPT
µM respectively at five dose concentration level. As per the results of enzyme immunoassays,
compound 6 exhibited significant activity for inhibition of Aurora-A enzyme with IC value
5
0
of 0.01 µM. Subsequently, QSAR model for the affinity of this new series of Aurora A kinase
inhibitors with physicochemical descriptors were developed with good predictive ability.
Molecular modelling studies indicating considerable interactions of these compounds in the
active site of amino acids of Aurora-A over Aurora B/C kinase enzyme that also favor the
enzyme immunoassay results. The experimental (60 human cancer cell lines and Aurora A
kinase enzyme) as well as theoretical studies (QSAR and molecular modelling) clearly
predicted activity of purine-benzimidazole hybrids. These preliminary encouraging results of
biological screening could offer an excellent framework in this field and may lead to
discovery of potent antitumour agents.
4
. Experimental Section
4
.1. Chemistry
All chemicals and solvents of commercial grade were used without further purification
and were supplied by loba, spectrochemicals and Sigma-Aldrich. Melting points were
1
13
determined in open capillaries and are uncorrected. H and C NMR spectra were recorded
1
13
on Jeol ECS 400 ( H, 400 MHz; C, 100 MHz) NMR spectrometer respectively, using
CDCl and DMSO-d as solvents. The chemical shifts were expressed in parts per million
3
6
with TMS as internal reference and J values are given in hertz. Mass Spectra of the
synthesized compounds were recorded at Waters Micromass Q-Tof Micro. The crystal
structure was collected on Bruker AXS KAPPA APEX II CCD diffractometer. Reactions
were monitored by thin layer chromatography (TLC) with silica plate coated with silica gel
HF-254 and column chromatography was performed with silica gel 60-120 mesh.
Ethylacetate: hexane and chloroform: methanol were adopted solvent systems.
4
5
1
.2. General procedure for the preparation of 1/3-butyl-2-methyl-1H/3H-benzo[d]imidazole-
-ylamine (2b, 2d)
/3-Butyl-2-methyl-5-nitro-1H/3H-benzimidazole was synthesized by previous reported
method by the alkylation of 2-methyl-5-nitro-benzimidazole (0.05 mol) with butyl bromide
0.075 mol) in the presence of NaH (0.126 mol) in THF at room temperature for 8h. To a
suspension of SnCl .2H O (135 mmol) in 2N HCl (95.2 ml), 1/3-butyl-2-methyl-5-nitro-
(
2
2
°
1
H/3H-benzimidazole (36.4 mmol) was heated at 110 C for 7 h. After the completion of the
reaction (monitored by TLC), the suspension was neutralized with 2N NaOH and diluted with
8

ACCEPTED MANUSCRIPT
ethanol. Filtered the solid product and extracted the filtrate with chloroform, dried over
Na SO , filtered and concentrated to get mixture of products which were separated through
2
4
column chromatography using ethylacetate:methanol (9.5:0.5) to get pure solid compounds
2
b and 2d.
4
.2.1. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-ylamine (2b)
°
-1
Brownish powder (450 mg, 52% yield) m.p. 138-140 C. IR υmax (KBr, cm ); 3314,
1
3
184, 2955, 2921, 1624, 1465, 1401, 1211. H NMR (CDCl ); δ 7.46 (d, J = 8.72 Hz, 1H,
3
CH), 7.08 (d, J = 8.24 Hz, 1H, ArH), 6.67-6.61 (m, 1H, ArH), 4.04-3.96 (m, 2H, N-CH2),
.55 (s, 3H, CH ), 1.77-1.72 (m, 2H, CH ), 1.39-1.34 (m, 2H, CH ), 0.97 (t, J = 3.66 Hz, 3H,
2
3
2
2
+
CH ). EIMS, m/z; 203.4 (M + 1).
3
4
.2.2. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-ylamine (2d)
°
-1
Brownish powder (750 mg, 86% yield) m.p. 138-140 C. IR υmax (KBr, cm ); 3349,
1
3
198, 2956, 2926, 1624, 1454, 1406, 1213. H NMR (CDCl ); δ 7.44 (d, J = 8.24 Hz, 1H,
3
2
3
CH), 6.62 (dd, J = 8.64 Hz, J = 2.28 Hz, 1H, ArH), 6.56 (d, J = 2.28 Hz, 1H, ArH), 3.98 (t,
J = 7.32 Hz, 2H, N-CH ), 2.52 (s, 3H, CH ), 1.76-1.69 (m, 2H, CH ), 1.41-1.32 (m, 2H,
2
3
2
+
CH ), 0.96 (t, J = 7.36 Hz, 3H, CH ). EIMS, m/z; 203.4 (M + 1).
2
3
4
.3. General procedure for the preparation of 3a-3d
To a solution of 2a-d (5.34 mmol) and isopropyl alcohol (25 ml), 2,6-dichoropurine (5.29
mmol) was added and stirred at room temperature for 24 h. After washing the crude solid
with IPA, dried to obtain pure white solid of 3a-d.
4
.3.1. N-(1-allyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-chloro-9H-purin-6-amine (3a)
°
-1
White powder (1.4 g, 79% yield) m.p. 270 C (d). IR υmax (KBr, cm ); 3354, 3305,
1
3
199, 1637, 1596, 1496, 1302, 1250, 1166. H NMR (DMSO-d ); δ 13.36 (bs, 1H, NH),
6
1
0.58 (bs, 1H, NH), 8.66 (s, 1H, CH), 8.60 (s, 1H, CH), 7.97 (d, J = 9.16 Hz, 1H, ArH), 7.80
(
d, J = 8.68 Hz, 1H, ArH), 6.08-6.04 (m, 1H, CH), 5.36 (d, J = 10.52 Hz, 1H, CH ), 5.29 (d, J
2
1
3
=
16.96 Hz, 1H, CH ), 5.11 (d, J = 5.04 Hz, 2H, N-CH ), 2.56 (s, 3H, CH ). C NMR
2 2 3
(
DMSO-d ); δ 152.5, 151.5, 151.2, 137.6, 132.1, 131.2, 128.3, 119.0, 112.6, 46.9, 12.2.
6
+
EIMS, m/z; 340.1 (M + 1).
4
.3.2. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-chloro-9H-purin-6-amine (3b)
9

ACCEPTED MANUSCRIPT
°
-1
White powder (0.9 g, 47% yield) m.p. 260 C (d). IR υ
(KBr, cm ); 3380, 2958, 1648,
max
1
1
498, 1433, 1232, 1109. H NMR (DMSO-d ); δ 10.62 (bs, 1H, NH), 8.72 (bs, 1H, NH), 8.59
6
(s, 1H, CH), 8.32 (s, 1H, ArH), 7.87 (d, J = 1.46 Hz, 1H, ArH), 7.75 (d, J = 9.16 Hz, 1H,
ArH), 4.36 (t, J = 7.32 Hz, 2H, N-CH ), 2.82 (s, 3H, CH ), 1.92-1.85 (m, 2H, CH ), 1.51-
2
3
2
1
3
1
1
3
.41 (m, 2H, CH ), 1.01 (t, J = 7.36 Hz, 3H, CH ). C NMR (DMSO-d ); δ 152.6, 151.1,
2 3 6
50.3, 137.4, 136.7, 132.9, 117.3, 115.2, 101.0, 46.8, 25.1, 19.5, 13.6, 12.2. EIMS, m/z;
+
56.1 (M + 1).
4
.3.3. N-(3-allyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-chloro-9H-purin-6-amine (3c)
°
-1
White powder (1.7 g, 94% yield) m.p. 285 C (d). IR υmax (KBr, cm ); 3341, 3122,
1
2
1
9
964, 1646, 1559, 1422, 1230, 1128. H NMR (DMSO-d ); δ 10.54 (bs, 1H, NH), 8.69 (bs,
6
H, NH), 8.48 (s, 1H, CH), 8.27 (d, J = 11.92 Hz, 1H, ArH), 7.82 (s, 1H, ArH), 7.71 (d, J =
.16 Hz, 1H, ArH), 6.05-5.98 (m, 1H, CH), 5.40 (t, J = 16.52 Hz, 2H, CH ), 4.97 (d, J = 6.40
2
1
3
Hz, 2H, N-CH ), 2.77 (s, 3H, CH ). C NMR (DMSO-d ); δ 152.4, 151.4, 136.6, 130.8,
2
3
6
+
1
4
1
19.9, 119.8, 119.4, 115.1, 114.9, 103.8, 46.9, 12.0. EIMS, m/z; 340.1 (M + 1).
.3.4. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-chloro-9H-purin-6-amine (3d)
°
-1
White powder (1.6 g, 86% yield) m.p. 280 C (d). IR υmax (KBr, cm ); 3350, 2959, 1649,
1
500, 1432, 1235, 1112. H NMR (DMSO-d ); δ 10.65 (bs, 1H, NH), 8.78 (bs, 1H, NH), 8.53
6
(s, 1H, CH), 8.35 (s, 1H, ArH), 7.82 (d, J = 8.72 Hz, 1H, ArH), 7.75 (d, J = 8.68 Hz, 1H,
ArH), 4.34 (t, J = 7.36 Hz, 2H, N-CH ), 2.79 (s, 3H, CH ), 1.89-1.80 (m, 2H, CH ), 1.46-
2
3
2
1
3
1
1
.37 (m, 2H, CH ), 0.96 (d, J = 7.32 Hz, 3H, CH ). C NMR (TFA+CDCl ); δ 156.5, 150.6,
2 3 3
49.3, 147.8, 139.5, 135.2, 131.7, 127.1, 121.5, 109.3, 105.4, 45.5, 30.4, 19.6, 12.4, 10.7.
+
EIMS, m/z; 356.4 (M + 1).
4
.4. General procedure for the preparation of compounds 4-19
(1/3-allyl/butyl-2-methyl-1H/3H-benzo[d]imidazol-5-yl)-2-chloro-9H-purin-6-amine
(0.29 mmol) was refluxed with amines (0.73 mmol) in ethanol (20 ml) for 2-3 days. Reaction
was monitored by TLC, crude solid obtained with evaporation of the solvent under vacuum
that was purified by column chromatography using chloroform : methanol as eluents to give
pure compounds 4-19.
4
.4.1. N-(1-allyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-morpholino-9H-purin-6-amine (4)
°
-1
White powder (65 mg, 75% yield) m.p. 239-240 C. IR υmax (KBr, cm ); 3322, 3066,
1
2
975, 1626, 1588, 1478, 1424, 1293, 1168, 1110. H NMR (DMSO-d ); δ 10.03 (bs, 1H,
6
1
0

ACCEPTED MANUSCRIPT
NH), 8.20 (s, 1H, CH), 7.83 (s, 1H, ArH), 7.66 (s, 1H, ArH), 7.46 (d, J = 8.28 Hz, 1H, ArH),
5
.96-5.93 (m, 1H, CH), 5.24 (d, J = 10.56 Hz, 1H, CH ), 4.99 (d, J = 17.44 Hz, 1H, CH ),
2 2
1
3
4
.72 (d, J = 2.76 Hz, 2H, N-CH ), 3.80 (s, 8H, CH ), 2.59 (s, 3H, CH ). C NMR (DMSO-
2
mor
2
3
d6); δ 159.2, 151.9, 142.5, 134.1, 133.4, 131.7, 131.1, 116.9, 115.9, 110.1, 108.9, 66.7, 45.6,
+
4
5.1, 29.4, 13.6. EIMS, m/z; 391.0 (M + 1).
4
.4.2. N-(1-allyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(piperidin-1-yl)-9H-purin-6-amine
(5)
°
-1
White powder (60 mg, 53% yield) m.p. 242-244 C. IR υmax (KBr, cm ); 3312, 2929,
1
1
1
604, 1581, 1475, 1302, 1255, 1174. H NMR (CDCl ); δ 10.10 (bs, 1H, NH), 8.22 (d, J =
3
2
3
.96 Hz, 1H, CH), 7.97 (s, 1H, ArH), 7.53 (dd, J = 8.68 Hz, J = 2.28 Hz, 1H, ArH), 7.22
(
d, J = 8.68 Hz, 1H, ArH), 5.98-5.91 (m, 1H, CH), 5.24 (d, J = 10.52 Hz, 1H, CH ), 4.99 (d, J
2
=
16.92 Hz, 1H, CH ), 4.72 (d, J = 5.04 Hz, 2H, N-CH ), 3.80 (s, 4H, CH ), 2.58 (s, 3H,
2 2 pip 2
1
3
CH ), 1.82 (s, 2H, CH ), 1.67 (s, 4H, CH ). C NMR (CDCl ); δ 159.4, 152.2, 152.1,
3
pip
2
pip
2
3
1
1
42.8, 135.0, 134.2, 131.7, 131.5, 117.4, 116.1, 110.6, 109.0, 50.9, 46.0, 45.9, 25.9, 25.0,
+
3.8. EIMS, m/z; 389.0 (M + 1).
4
.4.3. N-(1-allyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(pyrrolidin-1-yl)-9H-purin-6-amine
(6)
°
-1
White powder (66 mg, 60% yield) m.p. 260 C (d). IR υmax (KBr, cm ); 3388, 2969,
1
1
1
642, 1589, 1478, 1344, 1282, 1172. H NMR (DMSO-d ); δ 10.21 (bs, 1H, NH), 9.05 (s,
6
H, CH), 8.47 (d, J = 1.84 Hz, 1H, ArH), 7.66 (t, J = 4.60 Hz, 1H, ArH), 7.28 (d, J = 8.68
Hz, 1H, ArH), 6.02-5.95 (m, 1H, CH), 5.21 (d, J = 11.00 Hz, 1H, CH ), 4.95 (d, J = 16.96
2
Hz, 1H, CH ), 4.79 (d, J = 4.84 Hz, 2H, N-CH ), 3.61 (s, 4H, N- CH ), 2.55 (s, 3H, CH ),
2
2
pyr
2
3
1
3
2
1
4
6
.00 (t, J = 6.40 Hz, 4H, pyrCH ). C NMR (DMSO-d ); δ 157.9, 152.0, 142.5, 135.4, 132.6,
2 6
+
30.8, 116.9, 115.7, 109.6, 109.3, 47.1, 45.8, 25.7, 13.7. EIMS, m/z; 375.0 (M +1).
.4.4. N-(1-allyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-(4-methylpiperazin-1-yl)-9H-purin-
-amine (7)
°
-1
White powder (44 mg, 74% yield) m.p. 70-72 C. IR υmax (KBr, cm ); 3412, 2971, 1638,
1
1
598, 1461, 1307, 1263, 1101. H NMR (CDCl ); δ 10.01 (bs, 1H, NH), 8.16 (d, J = 1.84 Hz,
3
2
3
1
H, CH), 7.78 (s, 1H, ArH), 7.65 (s, 1H, ArH), 7.50 (dd, J = 8.72 Hz, J =1.84 Hz, 1H,
ArH), 5.95-5.92 (m, 1H, CH), 5.25 (d, J = 11.00 Hz, 1H, N-CH ), 5.00 (d, J = 16.96 Hz, 1H,
2
CH ), 4.73 (d, J = 4.60 Hz, 2H, N-CH ), 3.90 (s,
CH 4H), 2.59 (s, 3H, CH ), 2.38 (s,
piperazine 2, 3
2
2
1
3
3
1
H, CH ), 1.25 (s,
CH , 4H). C NMR (CDCl ); δ 153.7, 152.2, 134.9, 131.3, 117.4,
piperazine 2 3
3
+
16.3, 116.2, 110.7, 108.9, 54.8, 46.6, 45.8, 29.6, 13.7. EIMS, m/z; 404.3 (M + 1).
1
1

ACCEPTED MANUSCRIPT
4
.4.5. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2-morpholino-9H-purin-6-amine (8)
°
-1
White powder (72 mg, 63% yield) m.p. 228-232 C. IR υmax (KBr, cm ); 3317, 3060,
1
2
962, 1589, 1480, 1428, 1292, 1261, 1170, 1107. H NMR (DMSO-d ); δ 12.23 (bs, 1H,
6
NH), 8.76 (s, 1H, NH), 8.17 (s, 1H, CH), 7.83 (d, J = 2.72 Hz, 1H, ArH), 7.69 (s, 1H, ArH),
7
.62 (d, J = 7.36 Hz, 1H, ArH), 4.17 (t, J = 7.32 Hz, 2H, N-CH ), 3.90 (t, J = 4.82 Hz, 4H,
2
CH ), 2.62 (s, 3H, CH ), 2.61 (t, J = 1.84 Hz, 4H, CH ), 1.85-1.77 (m, 2H, CH ), 1.45-
mor
2
3
mor
2
2
13
1
.37 (m, 2H, CH ), 1.01 (t, J = 7.32 Hz, 3H, CH ). C NMR (DMSO-d ); δ 158.7, 151.2,
2 3 6
1
41.7, 133.9, 130.5, 115.6, 109.5, 108.6, 66.2, 63.3, 48.9, 44.7, 43.1, 42.9, 31.2, 19.5, 13.3.
+
EIMS, m/z; 407.5 (M + 1).
4
.4.6. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2(piperidin-1-yl)-9H-purin-6-amine
(9)
°
-1
White powder (82 mg, 72% yield) m.p. 218-220 C. IR υmax (KBr, cm ); 3206, 2979,
1
1
643, 1558, 1500, 1421, 1230, 1113. H NMR (DMSO-d ); δ 12.00 (bs, 1H, NH), 8.11 (s,
6
1
H, CH), 7.59 (d, J = 11.92 Hz, 2H, ArH), 7.25 (d, J = 8.72 Hz, 1H, ArH), 4.11 (t, J = 7.32
Hz, 2H, N-CH ), 3.78 (s, 4H, CH ), 2.58 (s, 3H, CH ), 1.80-1.73 (m, 2H, CH ), 1.61 (s, 6H,
2
pip
2
3
2
1
3
CH ), 1.40-1.35 (m, 2H, CH ), 0.96 (t, J = 7.32 Hz, 3H, CH ). C NMR (DMSO-d ); δ
pip
2
2
3
6
1
1
58.7, 151.2, 142.1, 133.8, 130.6, 115.2, 109.4, 108.4, 45.3, 43.1, 31.2, 25.1, 24.4, 19.5, 13.4,
+
3.2. EIMS, m/z; 405.5 (M + 1).
4
.4.7. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2(pyrrolidin-1-yl)-9H-purin-6-amine
(10)
°
-1
White powder (87 mg, 80% yield) m.p. 280 C (d). IR υmax (KBr, cm ); 3340, 2957,
1
1
1
626, 1580, 1450, 1339, 1277, 1112. H NMR (DMSO-d ); δ 12.42 (bs, 1H, NH), 9.32 (s,
6
H, NH), 8.70 (s, 1H, CH), 8.24 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.50 (d, J = 8.72 Hz, 1H,
ArH), 4.12 (t, J = 7.32 Hz, 2H, N-CH ), 3.61 (s, 4H, CH ), 2.56 (s, 3H, CH ), 2.01 (t, J =
2
pyr
2
3
6
3
1
1
.44 Hz, 4H, pyrCH ), 1.77-1.73 (m, 2H, CH ), 1.41-1.35 (m, 2H, CH ), 0.97 (t, J = 7.32 Hz,
2 2 2
1
3
H, CH ). C NMR (TFA+CDCl ); δ 151.1, 150.9, 149.6, 149.4, 140.6, 138.3, 135.0, 134.7,
3
3
+
31.7, 127.9, 122.9, 105.8, 49.6, 47.4, 30.8, 19.6, 13.1, 12.4, 11.5. EIMS, m/z; 391.2 (M +
).
4
6
.4.8. N-(1-butyl-2-methyl-1H-benzo[d]imidazol-5-yl)-2(4-methylpiperazine-1-yl)-9H-purin-
-amine (11)
1
2

ACCEPTED MANUSCRIPT
°
-1
White powder (65 mg, 55% yield) m.p. 225-228 C. IR υmax (KBr, cm ); 3350, 2935,
1
1
1
583, 1470, 1426, 1366, 1253, 1147. H NMR (DMSO-d ); δ 12.45 (bs, 1H, NH), 9.47 (bs,
6
H, NH), 8.21 (s, 1H, CH), 8.05 (s, 1H, ArH), 7.83 (s, 1H, ArH), 7.43 (d, J = 8.72 Hz, 1H,
ArH), 4.08-4.02 (m, 2H, N-CH ), 3.68 (s, 4H,
CH ), 2.47 (s, 3H, CH ), 2.45 (s, 4H,
piperazine 2 3
2
CH ), 2.36 (s, 3H, CH ), 1.69-1.63 (m, 2H, CH ), 1.28-1.22 (m, 2H, CH ), 0.87 (t, J =
piperazine
2
3
2
2
1
3
7
1
3
.36 Hz, 3H, CH ). C NMR (DMSO-d ); δ 158.7, 152.4, 152.3, 151.9, 151.7, 151.3, 138.8,
3 6
37.8, 137.0, 134.9, 133.2, 117.9, 116.2, 115.4, 113.5, 103.2, 101.2, 54.5, 45.9, 44.3, 43.1,
+
1.3, 19.8, 13.6, 13.6. EIMS, m/z; 420.5 (M + 1).
4
.4.9. N-(3-allyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-morpholino-9H-purin-6-amine (12)
°
-1
White powder (120 mg, 96% yield) m.p. 260 C (d). IR υmax (KBr, cm ); 3394, 2958,
1
1
626, 1583, 1458, 1309, 1101. H NMR (DMSO-d ); δ 12.29 (bs, 1H, NH), 9.19 (bs, 1H,
6
NH), 8.28 (s, 1H, CH), 8.01 (s, 1H, ArH), 7.69 (s, 1H, ArH), 7.49 (d, J = 8.72 Hz, 1H, ArH),
6
4
.03-5.97 (m, 1H, CH), 5.20 (d, J = 10.52 Hz, 1H, CH ), 4.86 (d, J = 16.96 Hz, 1H, CH ),
2 2
1
3
.75 (s, 2H, N-CH ), 3.73 (s, 4H, CH ), 2.57 (s, 4H, CH ), 2.53 (s, 3H, CH ). C NMR
2
mor
2
mor
2
3
(
DMSO-d ); δ 158.6, 151.7, 151.2, 150.8, 137.5, 135.9, 134.7, 134.5, 131.4, 117.8, 116.0,
6
+
1
14.9, 113.4, 100.6, 66.2, 44.9, 44.7, 30.4, 13.1. EIMS, m/z; 391.4 (M + 1).
4
.4.10. N-(3-allyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(piperidin-1-yl)-9H-purin-6-amine
(13)
°
-1
White powder (98 mg, 85% yield) m.p. 243-245 C. IR υ (KBr, cm ); 3275, 2925,
max
1
1
623, 1583, 1437, 1425, 1305, 1255, 1191. H NMR (DMSO-d ); δ 12.21 (bs, 1H, NH), 9.02
6
(bs, 1H, NH), 8.40 (s, 1H, CH), 7.93 (s, 1H, ArH), 7.64 (s, 1H, ArH), 7.49 (d, J = 8.72 Hz,
1
1
2
1
3
H, ArH), 6.01-5.97 (m, 1H, CH), 5.20 (d, J = 10.52 Hz, 1H, CH ), 4.88 (d, J = 16.96 Hz,
2
H, CH ), 4.74 (s, 2H, N-CH ), 3.80 (t, J = 5.52 Hz, 4H, CH ), 2.54 (s, 3H, CH ), 1.67 (s,
2
2
pip
2
3
13
H, pipCH ), 1.61 (s, 4H, CH ). C NMR (DMSO-d ); δ 158.5, 150.9, 150.7, 137.2, 135.8,
2
pip
2
6
34.8, 134.7, 131.4, 117.7, 116.0, 114.7, 100.2, 45.2, 44.9, 25.2, 24.4, 13.2. EIMS, m/z;
+
89.5 (M + 1).
4
.4.11. N-(3-allyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(pyrrolidin-1-yl)-9H-purin-6-amine
(14)
°
-1
White powder (60 mg, 55% yield) m.p. 240 C (d). IR υ
(KBr, cm ); 3320, 2964,
max
1
1
633, 1578, 1460, 1337, 1275, 1204, 1113. H NMR (DMSO-d ); δ 12.36 (bs, 1H, NH), 9.26
6
(bs, 1H, NH), 8.63 (s, 1H, CH), 8.15 (s, 1H, ArH), 7.68 (s, 1H, ArH), 7.47 (d, J = 5.28 Hz,
1
3

ACCEPTED MANUSCRIPT
1
H, ArH), 6.04-5.96 (m, 1H, CH), 5.16 (d, J = 10.08 Hz, 1H, CH ), 4.82 (s, 1H, CH ), 4.78
2
2
(
d, J = 9.16 Hz, 2H, N-CH ), 3.57 (s, 4H, N- CH ), 2.52 (s, 3H, CH ), 1.99 (t, J = 6.44 Hz,
2 pyr 2 3
1
3
4
H, pyrCH ). C NMR (DMSO-d ); δ 156.8, 150.8, 150.4, 136.2, 135.1, 134.3, 131.8, 131.7,
2 6
+
1
17.1, 116.9, 115.4, 114.4, 102.2, 100.2, 46.3, 44.6, 24.7, 12.7. EIMS, m/z; 375.5 (M + 1).
4
6
.4.12. N-(3-allyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(4-methylpiperazin-1-yl)-9H-purin-
-amine (15)
°
-1
White powder (70 mg, 62% yield) m.p. 159-162 C. IR υmax (KBr, cm ); 3313, 2936,
1
1
629, 1601, 1583, 1476, 1440, 1364, 1257, 1139. H NMR (DMSO-d ); δ 12.38 (bs, 1H,
6
NH), 9.38 (bs, 1H, NH), 8.30 (s, 1H, CH), 8.14 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.45 (d, J =
.68 Hz, 1H, ArH), 6.05-5.98 (m, 1H, CH), 5.17 (d, J = 10.52 Hz, 1H, CH ), 4.80 (s, 1H,
8
2
CH ), 4.75 (s, 2H, N-CH ), 3.73 (s, 4H,
CH ), 2.52 (s, 3H, CH ), 2.49 (s, 3H, CH ),
piperazine 2 3 3
2
2
1
3
2
1
1
.27 (s, 4H, piperazineCH ). C NMR (DMSO-d ); δ 158.5, 152.0, 151.4, 150.9, 137.5, 136.3,
2 6
+
34.4, 132.2, 117.7, 115.8, 115.1, 100.9, 54.5, 45.8, 45.0, 44.2, 13.2. EIMS, m/z; 404.5 (M +
).
4
.4.13. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-morpholino-9H-purin-6-amine (16)
°
-1
White powder (74 mg, 65% yield) m.p. 218 C (d). IR υmax (KBr, cm ); 3353, 2931,
1
1
600, 1585, 1471, 1431, 1382, 1254, 1124. H NMR (DMSO-d ); δ 12.46 (s, 1H, NH), 9.42
6
(s, 1H, NH), 8.25 (s, 1H, CH), 7.80 (s, 1H, ArH), 7.50 (d, J = 8.72 Hz, 1H, ArH), 7.43 (d, J =
9
.04 Hz, 1H, ArH), 4.12 (t, J = 7.32 Hz, 2H, N-CH ), 3.73 (s, 4H, CH ), 2.54 (d, J = 2.28
2
mor
2
Hz, 4H, morCH ), 2.53 (s, 3H, CH ), 1.77-1.72 (m, 2H, CH ), 1.39-1.32 (m, 2H, CH ), 0.99 (t,
2
3
2
2
1
3
J = 7.32 Hz, 3H, CH ). C NMR (DMSO-d ); δ 158.6, 137.7, 136.5, 134.7, 134.6, 117.6,
3
6
+
1
15.2, 101.0, 99.4, 66.1, 48.6, 44.9, 31.2, 30.6, 25.3, 19.6, 13.6. EIMS, m/z; 407.5 (M + 1).
4
.4.14. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(piperidin-1-yl)-9H-purin-6-amine
(17)
°
-1
Light brown (78 mg, 72% yield) m.p. 278 C (d). IR υ
(KBr, cm ); 3328, 2924,
max
1
1
627, 1603, 1583, 1480, 1307, 1252, 1126. H NMR (DMSO-d ); δ 12.37 (bs, 1H, NH), 9.38
6
(bs, 1H, NH), 8.27 (s, 1H, CH), 7.80 (s, 1H, ArH), 7.40 (d, J = 8.72 Hz, 1H, ArH), 7.36 (d, J
=
8.24 Hz, 1H, ArH), 4.04 (t, J = 7.36 Hz, 2H, N-CH ), 3.71 (t, J = 5.04 Hz, 4H, pipCH2),
2
2
1
.45 (s, 3H, CH ), 1.69-1.61 (m, 2H, CH ), 1.56 (s, 2H, CH ), 1.49 (s, 4H, CH ), 1.29-
3 2 pip 2 pip 2
13
.21 (m, 2H, CH ), 0.89 (t, J = 7.36 Hz, 3H, CH ). C NMR (DMSO-d ); δ 158.5, 152.4,
2
3
6
1
4

ACCEPTED MANUSCRIPT
1
1
51.6, 150.9, 137.8, 136.5, 134.8, 117.8, 115.2, 113.1, 100.9, 45.2, 42.9, 31.3, 25.3, 24.5,
+
9.6, 13.7, 13.6. EIMS, m/z; 405.5 (M + 1).
4
.4.15. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(pyrrolidin-1-yl)-9H-purin-6-amine
(18)
°
-1
White powder (77 mg, 70% yield) m.p. 260 C (d). IR υmax (KBr, cm ); 3321, 2956,
1
1
581, 1521, 1467, 1382, 1274, 1198, 1115. H NMR (DMSO-d ); δ 12.39 (bs, 1H, NH), 9.27
6
(bs, 1H, NH), 8.67 (s, 1H, CH), 8.22 (s, 1H, ArH), 7.48 (d, J = 7.80 Hz 1H, ArH), 7.37 (d, J
=
8.68 Hz 1H, ArH), 4.09 (t, J = 10.08 Hz, 2H, N-CH ), 3.59 (s, 4H, pyrCH ), 2.52 (s, 3H,
2
2
CH ), 1.98 (t, J = 5.96 Hz, 4H, CH ), 1.78-1.68 (m, 2H, CH ), 1.40-1.31 (m, 2H, CH ),
3
pyr
2
2
2
1
3
0
1
1
.94 (t, J = 7.32 Hz, 3H, CH ). C NMR (DMSO-d ); δ 157.2, 152.2, 150.3, 137.2, 135.3,
3 6
34.7, 131.6, 121.9, 119.0, 117.5, 114.5, 100.1, 62.0, 48.6, 46.6, 43.0, 31.4, 25.3, 19.6, 13.6,
+
3.4. EIMS, m/z; 391.5 (M + 1).
4
6
.4.16. N-(3-butyl-2-methyl-3H-benzo[d]imidazol-5-yl)-2-(4-methylpiperazin-1-yl)-9H-purin-
-amine (19)
°
-1
White powder (96 mg, 82% yield) m.p. 238-240 C. IR υmax (KBr, cm ); 3393, 2955,
1
1
1
7
2
624, 1583, 1442, 1355, 1252, 1136. H NMR (DMSO-d ); δ 12.29 (bs, 1H, NH), 9.08 (bs,
6
H, NH), 8.30 (s, 1H, CH), 7.96 (d, J = 3.20 Hz, 1H, ArH), 7.48 (d, J = 5.88 Hz, 1H, ArH),
.46 (s, 1H, ArH), 4.12-4.08 (m, 2H, N-CH ), 3.82 (s, 4H,
CH ), 2.58 (s, 3H, CH ),
piperazine 2 3
2
.49 (t, J = 4.6 Hz, 4H, piperazineCH ), 2.32 (s, 3H, CH ), 1.85-1.75 (m, 2H, CH ), 1.40-1.36
2
3
2
1
3
(
m, 2H, CH ), 0.96 (t, J = 6.84 Hz, 3H, CH ). C NMR (DMSO-d ); δ 158.5, 150.5, 137.5,
2 3 6
1
34.6, 134.5, 117.7, 114.9, 100.5, 56.3, 54.4, 45.7, 44.1, 43.0, 31.2, 25.0, 19.6, 18.1, 13.4.
+
EIMS, m/z; 420.5 (M + 1).
4
.5. 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% CO , 95% air and 100% relative humidity for 24 h.
2
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
1
5

ACCEPTED MANUSCRIPT
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% CO , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated
2
by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold
°
5
0% (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
0 min at room temperature. After staining, unbound dye is removed by washing five times
(
1
with 1% acetic acid and the plates are air dried and then subsequently solubilized with 10
mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of
5
15 nm. Using the seven absorbance measurements [time zero (T ), control growth (C), and
z
test growth in the presence of drug at the five concentration levels (T )], the percentage
i
growth is calculated at each of the drug concentration levels. Percentage growth inhibition is
calculated as:
[
(Ti -Tz)/(C - Tz)] × 100 for concentrations for which T ≥ Tz; [(Ti -Tz)/Tz] × 100 for
i
concentrations for which Ti < Tz.
Three dose response parameters are calculated for each experimental agent. Growth
inhibition of 50% (GI ) is calculated from [(Ti -Tz)/(C - Tz)] × 100 = 50. The drug
5
0
concentration resulting in total growth inhibition (TGI) is calculated from T = Tz. The LC
i
50
is calculated from [(Ti-Tz)/Tz]×100 = 50 [16,19, 26].
Acknowledgment
We wish to thank to UGC, New Delhi (41-322/2012, SR) and CSIR, New Delhi
02(0034)/11/EMR-II) for providing funds. Sincere thank to Prof. Sanjay Saxena and Mahiti
(
Gupta, department of biotechnology for helping Aurora kinase activities. NCI, USA is
gratefully acknowledged for investigating antitumor activities.
References
1
6

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[
1] R.V. Kumara, S. Bhasker, J. Cancer Policy 2 (2014) 63. [2] M.N. Noolvi, H.M. Patel,
V. Bhardwaj, A. Chauhan, Eur. J. Med. Chem. 46 (2011) 2327.[3] W.B. Parker, Chem.
Rev. 109 (2009) 2880.
[
[
[
[
4] A.M. D’Alise, G. Amabile, M. Iovino, F.P.D. Giorgio, M. Bartiromo, F. Sessa, F. Villa,
A. Musacchio, R. Cortese, Mol. Cancer Ther. 7 (2008) 1140.
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L. Meijer, J. Endicott, J. Med. Chem. 43 (2000) 1282.
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7] W.S. Hsieh, R. Soo, B.K. Peh, T. Loh, D. Dong, D. Soh, L.S. Wong, S. Green, J.
Chiao, C.Y. Cui, Y.F. Lai, S.C. Lee, B. Mow, R. Soong, M. Salto-Tellez, B.C. Goh,
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[
8] C.L. Tourneau, S. Faivre, V. Laurence, C. Delbaldo, K. Vera, V. Girre, J. Chiao, S.
Armour, S. Frame, S.R. Green, A. Gianella-Borradori, V. Dieras, E. Raymond, Eur. J.
Cancer 46 (2010) 3243.
[
[
9] M. Ferrero, V. Gotor, Chem. Rev. 100 (2000) 4319.
10] (a) G. Singh, M. Kaur, C. Mohan, Int. J. Pharm. 4 (2013) 82. (b) Y. Bansal, O. Silakari,
Bioorg. Med. Chem. 20 (2012) 6208. (c) P. Singla, V. Luxami, K. Paul RSC Adv. 4
(2014) 12422.
[
[
[
11] S. Nakamura, N. Tsuno, M. Yamashita, I. Kawasaki, S. Ohta, Y. Ohishi, J. Chem. Soc.
Perkin Trans.1 (2001) 429.
12] E.S. Park, H.J. Lee, H. Park, M.N. Kim, K.H. Chung, J.S. Yoon, J. Appl. Poly. Sci.
8
0 (2001) 728.
13] (a) J.C. Hazelton, B. Iddon, H. Suschitzky, L.H. Woolley, Tetrahedron 51 (1995) 10771.
b) C.S. Labaw, R.L. Webb, U.S 4285878, 1981; Chem. Abstr. 95 (1981) 168837. (c) P.
(
Kohler, Int. J. Parasitol. 31 (2001) 336.
[
14] P. Meisel, H.J. Heidrich, H.J. Jaensch, E. Kretzschmar, S. Henker, G. Laban, DD
243284, 1987; Chem. Abstr. 107 (1987) 217629. (b) D. Kyle, R.R. Goehring, B. Shao,
WO 001,039775; Chem. Abstr. 135 (2001) 33477.
[
15] (a) A. Sharma, V. Luxami, K. Paul, Bioorg. Med. Chem. Lett. 23 (2013) 3288. (b) K.
Paul, A. Sharma, V. Luxami, Bioorg. Med. Chem. Lett. 24 (2014) 624.
16] M.R. Grever, S.A. Sehepartz, B.A. Chabners, Semin. Oncol. 19 (1992) 622.
[
1
7

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[
17] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J.
Langley, P. Cronise, A. Vaigro-Wolff, M. Gray- Goodrich, H. Campbell, J. Mayo, M.
Boyd, J. Natl. Cancer Inst. 83 (1991) 757.
[
[
18] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91.
19] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H.
Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990) 1107.
[
[
20] CycLex Aurora-A kinase Assay/Inhibitor Screening kit Cat# CY-1165.
21] A.I. Caco, L.C. Tome, R. Dohrn, I.M. Marrucho, J. Chem. Eng. Data 55 (2010) 3160
and references therein.
[
22] QSARINS (QSAR-INSubria): QSARINS, software for QSAR MLR model
Development and validation, version 2.1- 2014, QSAR Research Unit in Environmental
Chemistry and Ecotoxicology, Department of theoretical and applied sciences (DiSTA),
University of Insubria, Via J.H. Dunant 3, Varese, Italy. http://www.qsar.it.
23] M.M. Neaz, M. Muddassar, F.A. Pasha, S.J. Cho, Acta Pharmacol. Sin. 31 (2010) 244.
24] Compounds were constructed and docked with builder toolkit of the software package
ArgusLab 4.0.1 (www.arguslab.com).
[
[
[
25] (a) N. Bouloc, J. M. Large, M. Kosmopoulou, C. Sun, A. Faisal, M. Matteucci, J.
Reynisson, N. Brown, B. Atrash, J. Blagg, E. McDonald, S. Linardopoulos, R. Bayliss,
V. Bavetsias, Bioorg. Med. Chem. Lett. 20 (2010) 5988, (b) M. S. Coumar, J.-S. Leou,
P. Shukla, J.-S. Wu, A. K. Dixit, W.-H. Lin, C.-Y. Chang, T.-W. Lien, U.-K. Tan, C.-H.
Chen, J. T.-A. Hsu, Y.-S. Chao, S.-Y. Wu, H.-P. Hsieh, J. Med. Chem. 52 (2009) 1050.
26] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D.L. Fine, B.J.
Abbott, J.G. Mayo, R.H. Shoemaker, M.R. Boyd, Cancer Res. 48 (1988) 589.
[
Figures Captions
Figure 1. Purine related drugs
Figure 2. ORTEP diagram of compound 19 (CCDC No. 1022534)
Figure 3. Plot of observed vs predicted Aurora A kinase inhibitory activity (expressed as log
(
IC ) values for compounds 3a-3b, 4-7 and 10 using eq 1). Predicted values were obtained
50
with a leave-one-out cross-validation procedure.
Figure 4. Compound 6 docked in active site of Aurora-A (2WTV). H-bonds of compound 6
with different amino acids residues are visible. Carbon atoms are given in green colour.
1
8

ACCEPTED MANUSCRIPT
Figure 5. Compound 6 docked in active site of Aurora-A (2XNE). H-bonds of compound 6
with different amino acids residues are visible. Carbon atoms are given in green colour.
Scheme 1. Synthetic route for the preparation of target compounds 4-19
Table 1. Physicochemical properties of the newly synthesized compounds 3a-d and 4-19
Table 2. Compound 6 and 5-FU median growth inhibitory (GI , µM), total growth
5
0
inhibitory (TGI, µM) and median lethal concentrations (LC , µM) of in vitro subpanel
5
0
tumour cell lines.
Table 3. IC ligand efficiency and experimental log P values of tested compounds
5
0,
NH
N
NH
N
NH
N
N
N
N
N
N
N
N
N
N
N
HN
HN
N
HO
H
CH3 OH
CH3 OH
Roscovitine
R-CR8
Olomoucine
-
HO
+
O
N
NH
N
HN
N
N
N
N
N
HN
N
N
OH
OH
H
N
N
N
N
2
H N
N
2
OH
OH
Penciclovir
Abacavir
Dinaciclib
Figure 1. Purine related drugs
Figure 2. ORTEP diagram of compound 19 (CCDC No. 1022534)
1
9

ACCEPTED MANUSCRIPT
Figure 3 Plot of observed vs predicted Aurora A kinase inhibitory activity (expressed as log
(
IC ) values for compounds 3a-3b, 4-7 and 10 using eq 1). Predicted values were obtained
50
with a leave-one-out cross-validation procedure.
Figure 4. Compound 6 docked in active site of Aurora-A (2WTV). H-bonds of compound 6
with different amino acids residues are visible. Carbon atoms are in given green colour.
2
0

ACCEPTED MANUSCRIPT
Figure 5. Compound 6 docked in active site of Aurora-A (2XNE). H-bonds of compound 6
with different amino acids residues are visible. Carbon atoms are given in green colour.
R
R
N
N
R
CH3
CH3
N
N
NH
NH
N
N
CH3
N
N
N
HNR1R
N
N
2
H N
2
Ethanol, Reflux
NH
NH
N
3a,3b
Cl
R R N
2 1
2
a, 2b
O
Cl
4-11
N
N
N
N
N
HN
POCl / Base
Reflux
N
3
CH3
CH3
N
N
H
N
NH
O
N
H
Cl
N
1
NH
H
R
N
N
R
N
N
HNR R2
N
CH3
1
N
N
H
Ethanol, Reflux
N
H
H2N
Cl
N
N
R R N
R
2 1
3
c, 3d
12-19
2
c, 2d
R; 2a = 2c = Allyl: R; 2b = 2d = butyl
Scheme 1 Synthetic route for the preparation of target compounds 4-19
Table 1. Physicochemical properties of the newly synthesized compounds 3a-d and 4-19
o
Compds
Starting
Material
NHR R
%
yields
79
47
94
86
75
53
60
M.pt. ( C)
Molecular
formulae
1
2
3
a
2a
2b
2c
2d
3a
3a
3a
3a
--
--
--
--
270 (d)
260 (d)
285(d)
280 (d)
239-240
242-244
260(d)
70-72
C
C
C
C
16
17
16
17
H
14
H
18
H
14
H
18
N
7
N
7
N
7
N
7
Cl
Cl
Cl
Cl
3
b
3c
3
d
4
morpholin-4-yl
piperidin-1-yl
pyrrolidin-1-yl
C
20
H
22
N
8
O
5
6
7
C
C
C
21
H
24
N
8
N
8
N
9
H
H
20
21
22
4-methylpiperazin-1-yl
74
25
2
1

ACCEPTED MANUSCRIPT
8
9
3b
3b
3b
3b
3c
3c
3c
3c
3d
3d
3d
3d
morpholin-4-yl
piperidin-1-yl
pyrrolidin-1-yl
63
72
80
55
96
85
55
62
65
72
70
82
228-232
218-220
280 (d)
225-228
260(d)
C
C
C
C
C
C
C
C
21
H
26
N
8
O
8
H
H
H
N
N
N
22
21
22
28
26
29
1
0
1
2
3
4
5
6
7
8
9
8
1
1
1
1
1
1
1
1
1
4-methylpiperazin-1-yl
morpholin-4-yl
piperidin-1-yl
pyrrolidin-1-yl
4-methylpiperazin-1-yl
morpholin-4-yl
piperidin-1-yl
pyrrolidin-1-yl
4-methylpiperazin-1-yl
9
H
22
N
8
O
8
20
243-245
240(d)
H
H
H
N
N
N
21
20
21
24
22
25
8
159-162
218 (d)
278 (d)
260 (d)
238-240
9
C
H
26
N
8
O
21
C
C
C
H
H
H
N
N
N
22
21
22
28
26
29
8
8
9
Table 2. Compound 6 and 5-FU median growth inhibitory (GI , µM), total growth
5
0
inhibitory (TGI, µM) and median lethal concentrations (LC , µM) of in vitro subpanel
5
0
tumour cell lines.
Compds Activity
I
II
III
IV
V
VI
VII VIII IX
MG-
MID
a
b
b
b
b
b
b
b
b
b
b
6
GI50
TGI
LC50
GI50
TGI
LC50
54.5
3.16 2.00 29.6 1.34
18.12
b
b
b
b
b
b
b
b
b
b
b
b
b
b
5
-FU
15.1
b
8.4 72.1 70.6 61.4 45.6 22.7 76.4
22.60
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
I, Leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian
cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer.
a
Full panel mean-graph midpoint (µM).
Compounds showed values >100 µM.
b
Table 3. IC ligand efficiency and experimental log P values of tested compounds
5
0,
a
Compounds
IC (µM)
Ligand efficiency Experimental log P
5
0
3
a
8.00
0.07
0.02
8.50
0.01
0.07
0.70
0.29
0.38
0.24
0.35
0.39
0.29
0.33
2.48
1.28
1.59
1.55
2.53
1.25
1.36
3
b
4
5
6
7
1
0
a
Calculated using the formula: LE = [ -1.4× log 10 (IC (M))]/(number of non hydrogen atoms).
50
2
2

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
A new series of regioisomeric purine-benzimidazole hybrids.
Single X-ray determination of compound 19
In vitro evaluation of synthesized compounds for their antitumor activity.
In vitro evaluation of Aurora kinase inhibitors
Molecular modeling in active sites of Aurora A kinase.

ACCEPTED MANUSCRIPT
Supporting Information
Purine-benzimidazole hybrids: Synthesis, single crystal determination and in vitro
evaluation of antitumor activities
Alka Sharma, Vijay Luxami, Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147004, India.
Tel.: +91-94656-70595; fax: +91-175-236-4498;
E-mail: kpaul@thapar.edu
Table of Contents
S. No.
Contents
H and C NMR spectra of compounds
Page No.
S2-S22
S23-S24
S25
1
13
1
2
3
4
60 human tumor cell line (one dose results)
Five dose results of compound 6
Aurora A kinase studies
S25-S26
5
Shake Flask Method for lipophilicity
determination
S26-S27
6
7
X-ray crystallographic data of compound 19
Docking of compound 6
S27-S36
S36
S1

ACCEPTED MANUSCRIPT
H N
2
N
CH3
N
1
Figure S1. H NMR spectrum of compound 2b in CDCl
3
N
CH3
N
H2N
1
Figure S2. H NMR spectrum of compound 2d in CDCl .
3
S2

ACCEPTED MANUSCRIPT
N
N
CH3
HN
N
N
N
H
Cl
N
1
Figure S3. H NMR spectrum of compound 3a in DMSO-d .
6
N
CH3
N
HN
N
N
N
Cl
N
H
1
3
Figure S4. C NMR spectrum of compound 3a in DMSO-d .
6
S3

ACCEPTED MANUSCRIPT
N
CH3
N
HN
N
N
N
N
Cl
N
CH3
H
N
HN
N
N
N
H
Cl
N
1
Figure S5. H NMR spectrum of compound 3b in DMSO-d .
6
1
3
Figure S6. C NMR spectrum of compound 3b in DMSO-d .
6
S4

ACCEPTED MANUSCRIPT
N
CH3
N
HN
N
N
N
Cl
N
H
1
Figure S7. H NMR spectrum of compound 3c in DMSO-d .
6
N
CH3
N
HN
N
N
N
Cl
N
H
1
3
Figure S8. C NMR spectrum of compound 3c in DMSO-d .
6
S5