Synthesis of new 1,2,3-triazole linked benzimidazole molecules as anti-proliferative agents

Article abstract of DOI:10.1080/00397911.2017.1289412

One pot click chemistry is used to link triazole and benzimidazole pharmacophore to get N-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)aniline and its derivatives. Flexible linkages in the form of –CH₂–R or –O–R/–N–R were designed during synthesis. All the newly synthesized compounds were characterized by FT-IR and NMR spectroscopy as well as high-resolution mass spectrometry. Selected compounds were screened for in vitro anti-proliferative activity using National Cancer Institute (NCI)-60 human tumor cell line screening program. The most potent structure N-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-chloroaniline 7e showed 40% growth inhibition in renal cancer cell line (UO-31) at 10 μM concentration.

Full text of DOI:10.1080/00397911.2017.1289412

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Synthesis of New 1,2,3-triazole Linked
Benzimidazole Molecules as Anti-Proliferative
Agents
Ishani I. Sahay & Prasanna S. Ghalsasi
To cite this article: Ishani I. Sahay & Prasanna S. Ghalsasi (2017): Synthesis of New 1,2,3-triazole
Linked Benzimidazole Molecules as Anti-Proliferative Agents, Synthetic Communications, DOI:
10.1080/00397911.2017.1289412
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Date: 10 February 2017, At: 09:46

Synthesis of New 1,2,3-triazole Linked Benzimidazole Molecules as Anti-
Proliferative Agents
Ishani I. Sahay1, Prasanna S. Ghalsasi1
1Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of
Baroda, Vadodara, Gujarat, India
Corresponding Authors: Prasanna S. Ghalsasi. Email: prasanna.ghalsasi@gmail.com
Abstract
One pot Click chemistry is employed to link trizole and benzimidazole pharmacophore to
get N-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)aniline and
its derivatives. Flexible linkages in the form of –CH2-R or –O-R/ -N-R were designed
during synthesis. All the newly synthesized compounds were characterized by FT-IR and
NMR spectroscopy as well as high resolution mass spectrometry. 1H NMR study showed
interesting fluxional rotation in one of the molecules due to intramolecular hydrogen
bonding. Selected compounds were screened for in vitro anti-proliferative activity using
NCI (National Cancer Institute)-60-human-tumor-cell line-screening program. The most
potent
structure
N-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-chloroaniline7e showed 40% growth inhibition in renal cancer cell line
(UO-31) at 10µM concentration.
Graphical abstract
KEYWORDS: Benzimidazole, Click Reaction, 1,2,3-triazole, anti-proliferative.
1

INTRODUCTION
Pharmacophore driven synthesis for achieving biological activity is well known in
literature. But designing efficient, high yielding regio-specific synthesis with more than
one pharmacophore in a single molecule still remained challenge.
The broad and the potent activity of triazoles and benzimidazoles have established them
as pharmacologically significant scaffolds[1-4]in an array of drug categories such as anti-
microbial, anti-inflammatory, analgesic, anti-peptic, anti-viral, anti-neoplastic, anti-
tubercular, anti parkinsons, anti-diabetic, anti-depressant (Figure 1).
Over the last decade 1,2,3-triazoles[5-9] have received increasing attention in medicinal
chemistry[10-14] and this was made possible because of the discovery of the highly useful
and widely applicable 1,3-dipolar cycloaddition reaction between azide and alkynes
(click chemistry) [15]catalysed by copper salts and ruthenium complexes[16]. The most
striking features of considering 1,2,3-triazoles as drug candidate[17-20], schematically
shown in Figure 2, are 1) formation of hydrogen bonds for improving solubility and
ability to interact with biomolecular targets (hydrogen bond acceptor sites- N2 and
N3centers, and hydrogen bond donor site - C5 center); 2) can act as intercalating agent via
π–π stacking interactions; 3) can participate in C-H hydrogen bonding interactions; 4) can
substitute for an amide linkage without altering the binding pose; 5) are highly stable to
metabolic degradation as compared to other compounds containing three adjacent
nitrogen (N) atoms can be metabolically inert (1,2,4-triazoles) [21-23]
.
2

On the other hand, benzimidazole, a heterocyclic aromatic organic compound, is an
important pharmacophore[24-25] and a privileged structure in medicinal chemistry,
especially for anti-cancer activity[26-30]. It is observed that, hydrogen bond donor and
acceptor site i.e N1 and N3 in the benzimidazole molecule (see Figure 3), which exhibits
tautomerism,[31] play a critical role in binding to the biological targets.
RESULTS AND DISCUSSION
Our efforts were focused on synthesizing new triazole linked benzimidazole molecules
and checking its anti proliferative activity, keeping PAINS (Pan Assay Interference
Compounds)[32] structure in mind. Most PAINS function as reactive chemicals rather than
discriminating drugs. The designing of compounds, see Figure 4, was driven by three
basic principles: 1) inserting flexible linker between benzimidazole and triazole
pharmacophore: use of methylene bridge; 2). Derivatizing triazole at C4 position with
flexible group: CH2-O/ CH2-N; and 3). increasing solubility and/or bioavailability by
derivatizing benzimidazole pharmacophore: N-ethylation. Interestingly, this design
strategy forced us to use three different synthetic routes, for synthesizing eleven new
compounds, as discussed below.
Strategy 1: 2-Chloro benzimidazole,2, was synthesized by reacting o-phenylenediamine
with two different reagents 1) 2-chloro acetyl chloride and 2) 2-chloro acetic acid[33-
35](Scheme 1). Both these reactions gave comparable yield. 2 was transformed into 2-
(azidomethyl)-1H-benzo[d]imidazole, 3, using sodium azide in dry DMSO[33]. The
reaction condition, especially solvent selection, was optimized to get the best yields. In
3

accordance with theory, DMSO,aprotic polar solvent proved choice of solvent, since it
favors conversion of 2 to 3, SN2 reaction, as well as 1,3-dipolar cycloaddition. Without
isolating compound 3, in situ, alkyne derivative was added to obtain final compound 4.
Fluorine is known for its pharmacophorical activity therefore its addition on triazole
nucleus was planned and carried out similar to Scheme 1.
Strategy 2: Our aim in this strategy is to derivatise triazole ring at C4 position using the
N-(prop-2-yn-1-yl) aniline/phenolic groups. Substituted phenol and aniline derivatives
were allowed to react with propargyl bromide in the presence of K2CO3base and DMF as
solvent[36-37](Scheme 2).Terminal alkynes is known to provide an efficient method for the
synthesis of triazole[38]. Krim and co-workers[39] reported (Prop-2-ynyloxy) benzene as an
intermediate, for the synthesis of triazole via Huigen dipolar cycloaddition method using
click chemistry, which is followed in the present study.
To extend our study, we thought of using paracetamol as an additional pharmacophore.
We synthesized, 7d, where phenolic group of paracetomol was attached at C4 position of
triazole.
Strategy 3: Bioavailability and solubility of benzimidazole can be increased by inserting
ethyl or methyl groups at N3 position. Therefore, we carried out direct
methlylation/ethylation of compound 2. But all the attempts resulted in difficult to
characterize polymeric product. Hence, N-ethylation is carried out initially; on o-nitro
aniline (Scheme 3). o-Nitro aniline was converted to N-ethyl derivative using ethyl iodide
4

as per the reported procedure[40]. Further, compound 9 was reduced to compound 10
using iron powder[41]. All the further steps were followed according to Scheme 1.
All column purified eleven new molecules (Table 1) with triazole linked benzimidazole
pharamacophore, were characterized by standard spectroscopic techniques. Literature
showed hydrogen bonding ability of C3 proton in triazole molecule [21]. Interestingly, we
observed dramatic upfield shift of 0.63 ppm for the same proton in 1H NMR spectra for
13 and not in 4b, as shown in Figure 5. This implies that intramolecular hydrogen
bonding in 13 forces rotation of benzimidazole molecule, a fluxional rotation, for fixing
geometry. The NMR result for 13 is paving our path to understand the molecular aspect
of this compound (role of ethyl group). Constant efforts are being put in this direction to
understand this phenomenon which will be published later.
All the molecules were initially screened for anti-proliferative activity in silico by
National Cancer Institute (NCI), USA (Experimental details are provided in supporting
information). Compound 4b, 7d, 7e and 13 were further selected for actual screening
anti-proliferative activity at 10µM concentration. Figure 6 shows comparative study of
compound 4b, 7d, 7e and 13 on selected cell lines SR(Leukamia), SNB-75(CNS),
A498(kidney), UO-31(kidney). 7e shows 40% growth inhibition in UO-31 renal cancer
cell line (mean growth inhibition) at 10µM concentration. Although this activity is
moderate, the structure of 7e can help in developing or designing novel drug candidates.
EXPERIMENTAL
5

This includes synthesis and characterization of one of the new molecules. Experimental
procedures for the intermediates and rest of the derivatives are presented in supporting
information.
MATERIAL AND METHODS
All the compounds were purified using column chromatography (2000- 400 mesh silica)
before characterization. TLC analysis was done using pre-coated silica on aluminium
sheets. Melting points were recorded in Thiele’s tube using paraffin oil and are
uncorrected. FT-IR (KBr pellets) spectra were recorded in the 4000-400 cm–1 range using
a Perkin-Elmer FT-IR spectrometer. The NMR spectra were obtained on a Bruker AV-III
400 MHz spectrometer using TMS as an internal standard. The chemical shifts were
reported in parts per million (ppm), coupling constants (J) were expressed in hertz (Hz)
and signals were described as singlet (s), doublet(d), triplet(t), broad (b) as well as
multiplet (m). High resolution mass spectrometry was performed on Micromass Q-TOF
Micro instrument. All chemicals and solvents were of commercial grade and were used
without further purification.
General Method For Synthesis Of 2-((4-Phenyl-1H-1,2,3-Triazol-1-Yl)Methyl)-1H-
Benzo[D]Imidazole 4a
Synthesis of compound 4a[42] was done in single step. A mixture of compound 2 (0.500g,
3mmol) and sodium azide (0.195g, 3mmol) was charged in DMSO (5mL) at room
temperature for 15-16hrs [22]. Reaction completion was monitored using thin layer
chromatography (TLC, EtOAc:PET, 7:3). After in situ formation of compound 3, the
6

respective alkyne derivative (0.321g, 3.14mmol) was added and mixture of CuSO4.5H2O
(0.112g, 0.4mmol) and sodium ascorbate (0.267g, 1.35mmol) in water (0.5mL) was
added as shown in Scheme 1[25-26]. The reaction was stirred at room temperature and its
completion was monitored by TLC. Compound 4a was then isolated and further purified
by column chromatography using various concentrations of ethyl acetate and petroleum
ether to afford the desired compound 4a as a white solid. FT-IR (KBr) γ: 690.52(Mono
substitution), 738.74(o-substitution), 1089.78(C-N stretch), 1276.88(C-N stretch),
1442.75(N=N), 1587.42(C=N), 3095.75(N-H stretch) cm-1. 1H NMR(400MHz,DMSO-
d6) δ: 5.96( 2H, s, ), 7.19( 2H, t, J=9.6Hz) , 7.35( 1H, m ), 7.45( 2H, t, J=8Hz), 7.50 (1H,
13
d, J=7.6Hz), 7.61(1H, d, J=7.6Hz), 7.89(2H, d, J=8Hz), 8.71 (1H, s), 12.69(1H, s); C
NMR (100MHz, DMSO-d6) δ: 52.7, 127.4, 130.4, 133.3, 134.2, 135.7, 151.8, 153.5
ppm; HR-MS (ESI+) [C16H13N5+Na] : Calculated-298.1069, Observed-298.1064
1-Ethyl-2-((4-(4-Fluorophenyl)-1H-1,2,3-Triazol-1-Yl)Methyl)-1H-
Benzo[D]Imidazole 13:
Title compound was synthesized following the same procedure as reported for compound
4a and isolated as white solid. FT-IR (KBr) γ: 734.88 (o-substitution), 829.39(p-
substitution), 1228.66(C-N stretch), 1458.18(N=N), 1496.76(-C=N of benzimidazole),
2972.31(-C-H); 1H NMR (400 MHz, DMSO-d6) δ: 1.21(3H, t, J=7.2Hz), 4.40( 2H, q,
J=7.2Hz), 6.08(2H, s), 7.22(1H, d, J=7.2Hz), 7.28(3H, t, J=8.8Hz), 7.62(2H, t, J=6.8Hz),
7.93(2H, t, J=5.2Hz), 8.08( 1H, s); 13C NMR (100MHz, DMSO-d6) δ: 15.3, 38.8, 46.6,
111, 116.2, 116.4, 119.7, 122.4, 122.6, 123.2, 127.5, 127.5, 127.7, 127.8, 135.2, 142.5,
7

146.3, 148.3, 161.1, 163.5 ppm ; HR-MS (ESI+) [C18H16FN5+Na] : Calculated-
344.1287, Observed-344.1284
CONCLUSION
In conclusion, click reaction can conjoin triazole pharmacophore with benzimidazole in a
faster and efficient method. The reaction proceeds without isolation of azide intermediate
with out compromising in good yields. Commercially available o-phenylenediamine was
used during the synthesis of series of N-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-
1,2,3-triazol-4-yl)methyl)aniline compounds. During this investigation, 1H NMR study
clearly showed interesting fluxional rotation, for 13, due to intramolecular C-H…N
hydrogen bonding. The in-vitro anticancer activity of selected compounds using NCI-60
human cell line screening program revealed that most of the title compounds showed
moderate bioactivity at 10µM concentration. Flexible structured molecule, 7e, N-((1-
((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-chloroaniline
showed a 40% growth inhibition in human renal cancer cell line (UO-31) which needs
further investigation.
ACKNOWLEDGEMENT:
PSG thank Department of Science and Technology, New Delhi (SR/S1/IC-43/2009) for
financial support. IIS thanks JRF and SRF for UGC-BSR fellowship. The authors thank
the National Cancer Institute for 60 human cancer cell line screenings in the NCI's
Developmental Therapeutics Program (DTP) in vitro cell line screening.
8

CONFLICT OF INTEREST
The authors declare no conflict of interest.
SUPPLEMENTARY DATA
Full experimental details and analytical data for new molecules can be found via the
Supplementary Content section of this article’s webpage.
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Table 1. New benzimidazole linked triazole compounds
No
Terminal Alkyne
Product
Yield*(
%)
Melting
Point
(°C)
1
2
98
95
185-187
135-139
105-107
3
85
4
5
95
96
135-140
130-135
13

6
7
94
96
155-160
210-215
8
85
175-180
9
92
95
180-185
105-110
10
11
95
175-180
*Yields are given for the final step
14

Scheme 1. Synthesis of 2-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-1H-
benzo[d]imidazole (4a and 4b)
15

Scheme 2. Synthesis ofN-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)aniline and its derivatives
16

Scheme 3. Synthesis of 1-ethyl-2-((4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)-1H-
benzo[d]imidazole
17

Figure 1. List of FDA approved drugs with benzimidazole or triazole in their core
structure.
18

Figure 2. 1,2,3-triazoles as potential binding interactions
19

Figure 3. Benzimidazole binding interaction sites
20

Figure 4. Designing of new triazole linked benzimdazole compounds
21

Figure 5. Plausible reason for the shift of peak in the 1H NMR spectra
22

Figure 6.% Growth Inhibition on selected cell lines at 10µM concentration
23