Anticancer potential of some imidazole and fused imidazole derivatives: Exploring the mechanism: Via epidermal growth factor receptor (EGFR) inhibition

Article abstract of DOI:10.1039/d0md00146e

Imidazole-based epidermal growth factor receptor (EGFR) inhibitors were computationally designed and synthesized. All the compounds were assessed for their anti-proliferative activity against five cancer cell lines, viz., MDA-MB-231 (breast), T47D (breast) and MCF-7 (breast), A549 (lung) and HT-29 (colorectal). Compounds 2c and 2d emerged as better anticancer molecules with no toxicity towards normal cells. 2c and 2d inhibited EGFR enzymatic activity in vitro with IC50 values of 617.33 ± 0.04 nM and 710 ± 0.05 nM, respectively. In order to further improve the potency, we explored an unoccupied area of the ATP binding domain of EGFR and analysed an in silico interaction model of 2c and 2d-EGFR complexes that guided and allowed substitution of the 4-fluorophenyl ring (2c and 2d) with 4-(4-methylpiperazinyl)-3-nitrophenyl at the N-9 position, resulting in compound 3c with a better binding score and potent EGFR inhibitory activity (IC50: 236.38 ± 0.04 nM), which was comparable to the positive control erlotinib (239.91 ± 0.05 nM). 3c exhibited a great improvement in anticancer potency with inhibition of cell growth of all cancer cell lines at very low micromolar concentrations (IC50 = 1.98 to 4.07 μM). Further investigation revealed that 3c also induced an increase in ROS levels in cancer cells in a mitochondrial-independent manner and halted the cell cycle at the sub-G1 phase.

Full text of DOI:10.1039/d0md00146e

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RESEARCH ARTICLE
Anticancer potential of some imidazole and fused
imidazole derivatives: exploring the mechanism
Cite this: DOI: 10.1039/d0md00146e
via epidermal growth factor receptor (EGFR)
inhibition†
b
Sourav Kalra,a Gaurav Joshi,b Manvendra Kumar,b Sahil Arora,
Harsimrat Kaur,c
a
a
b
*
*
*
Sandeep Singh, Anjana Munshi and Raj Kumar
Imidazole-based epidermal growth factor receptor (EGFR) inhibitors were computationally designed and
synthesized. All the compounds were assessed for their anti-proliferative activity against five cancer cell
lines, viz., MDA-MB-231 (breast), T47D (breast) and MCF-7 (breast), A549 (lung) and HT-29 (colorectal).
Compounds 2c and 2d emerged as better anticancer molecules with no toxicity towards normal cells. 2c
and 2d inhibited EGFR enzymatic activity in vitro with IC50 values of 617.33 0.04 nM and 710 0.05 nM,
respectively. In order to further improve the potency, we explored an unoccupied area of the ATP binding
domain of EGFR and analysed an in silico interaction model of 2c and 2d–EGFR complexes that guided
and allowed substitution of the 4-fluorophenyl ring (2c and 2d) with 4-(4-methylpiperazinyl)-3-nitrophenyl
at the N-9 position, resulting in compound 3c with a better binding score and potent EGFR inhibitory
activity (IC50: 236.38 0.04 nM), which was comparable to the positive control erlotinib (239.91 0.05
nM). 3c exhibited a great improvement in anticancer potency with inhibition of cell growth of all cancer cell
lines at very low micromolar concentrations (IC50 = 1.98 to 4.07 μM). Further investigation revealed that 3c
also induced an increase in ROS levels in cancer cells in a mitochondrial-independent manner and halted
the cell cycle at the sub-G1 phase.
Received 4th May 2020,
Accepted 13th June 2020
DOI: 10.1039/d0md00146e
rsc.li/medchem
overexpression and elevation in multiple cancer subtypes
including non-small cell lung cancer (NSCLC), breast cancer,
Introduction
Cancer accounts for a number of deaths worldwide and is the
second leading cause of death after cardiovascular disorders.1
Cancer cells possess inherent hallmarks that include tissue
invasion, metastasis, evasion of apoptosis, angiogenesis,
inflammation and immortality2 that make them insensitive to
conventional anticancer agents. Considering this scenario,
the development of anticancer drugs targeting the major
hallmarks was put forth.3 Amongst the various anticancer
drug targets known, protein kinases are the most studied as
druggable targets.4 The epidermal growth factor receptor
(EGFR) belongs to the ErbB family of receptor tyrosine
kinases5 and is one of the effective drug targets owing to its
and ovarian cancer.6 The EGFR is considered to be the driver
of cell proliferation, migration, adhesion and cell survival
which consequently lead to tumorigenesis via auto-
phosphorylation by activating an intracellular signalling
cascade.
Considering the centralized role of kinases, there had
been a revolution in the development of EGFR inhibitors7
(Fig. 1). First-generation EGFR inhibitors such as erlotinib
and gefitinib (quinazoline scaffold) were approved by the
USFDA in 2002 and 2004, respectively, for the treatment of
NSCLC.8 Eventually, the first-generation EGFR inhibitors
were ineffective due to acquired point mutations, T790M and
L858R, that hinder their binding at the ATP kinase domain
of EGFR. This further gave way to the development of
second-generation EGFR inhibitors (canertinib, dacomitinib
and afatinib) which were able to interact with CYS797 via
covalent binding and hinder the ATP binding at the active
site.9 The clinical trials were promising initially but toxicity
a Department of Human Genetics and Molecular Medicine, School of Health
Sciences, Central University of Punjab, Bathinda, 151001, India.
E-mail: anjanadurani@yahoo.co.in; sandeepsingh82@gmail.com
b Department of Pharmaceutical Sciences and Natural Products, School of Basic
and Applied Sciences, Central University of Punjab, Bathinda, 151001, India.
E-mail: raj.khunger@cup.edu.in, raj.khunger@gmail.com
c Desh Bhagat Dental College and Hospital, Mandi Gobindgarh, India
† Electronic supplementary information (ESI) available: Copies of spectra and
docking results. See DOI: 10.1039/d0md00146e
(skin
and
gastrointestinal)
halted
their
further
development.10 Thereafter, pyrimidine-based irreversible,
third-generation EGFR inhibitors (WZ4002 and osimertinib)
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A trisubstituted imidazole (11g) based on a p38α MAPK
inhibitor template, recently reported by the Juchum group
against mutated EGFR L858R/T790M/C797S, had
a
reasonable orientation in the active site of mutant EGFR
kinase domain.15
We exercised target hopping of 11g along with molecular
docking to design new compounds (Fig. 2), 1 and 2, based on
the considerations that (a) the p-fluorophenyl residue at the
N-1 position of imidazole (1) or the N-9 position of fused
imidazole (2) is favoured and compatible not only with the
gatekeeper residue (Thr766) but also with the mutated one
(MET) in the binding pocket, thus making it suitable for
clinically challenging EGFR in addition to its metabolic
stability; (b) hydrogen bond interactions of designed
compounds with MET769 are conserved; (c) another phenyl
ring of 1 (substituted at N-5; amino group) or 2 (substituted
at the C-2 position) orients towards the DFG motif (ASP831,
PHE832 and GLY833) which also interacts with the
p-hydroxyphenyl residue of 11g; (d) –CN (1) or –CONH2 (2)
interacts with PRO770; and (e) they are easy to synthesize.
Further, the designed compounds do not contain pyridinyl
imidazoles in their framework which can potentially inhibit
cytochrome p450 and lead to toxic side effects.16 In the
present work, the rational design of 1 and 2 followed by their
synthesis along with detailed in vitro biological evaluation
including cancer cell based antiproliferative effects,
enzymatic inhibition, alteration of ROS levels and cell cycle
analysis are reported. Molecular docking at the EGFR ATP
kinase domain further helped in achieving high EGFR
potency in the nM range.
Fig. 1 Chemical structures of some EGFR inhibitors.
were developed; they possessed a much better safety profile
than the second generation due to their lower affinity and
binding for wild-type EGFR.11 Irreversible inhibition of
tyrosine kinase activity results in continuous suppression of
EGFR signalling as compared to reversible inhibition.12 The
phase trials suggested that the prolonged use of third-
generation inhibitors was associated with off-target side
effects followed by C797S, BRAF and MEK mutations.13 Very
recently, fourth-generation EGFR inhibitors (e.g. EAI045) have
been disclosed to overcome the concomitant EGFR T790M
and C797S mutations, but their safety profile in the clinic is
yet to be established.14
Results and discussion
Synthesis procedures
For the synthesis of target compounds 1 (1a–1r) and 2
(2a–2d), a synthesis plan was designed (Scheme 1) that
utilized the starting materials 6, 8 and 9 which in turn can
be synthesized from a common precursor, ethyl N-((Z)-2-
amino-1,2-dicyanovinyl)formimidate (4). The condensation of
Fig. 2 Design of target compounds 1 and 2 based on target hopping of 11g. 1c, 2c1 (as a representative compound) and 11g were docked at the
ATP binding domain of EGFR (PDB ID: 1M17).
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Scheme 1 Reagents and conditions: (i) CHIJOEt)3, 1,4-dioxane, 80 °C, 6 h. (ii) Aniline hydrochloride, EtOH, rt, 3 h. (iii) KOH (1 M), rt, 6 h. (iv)
p-TsOH, MeOH, rt, 2 h. (v) DBU, EtOH, rt, 2 h. (vi) H2SO4, MeOH, rt, 1 h.
commercially available diaminomaleonitrile (3) (1.1 equiv.)
with triethyl orthoformate (1.1 equiv.) yielded a formimidate
ester, 4.17 4, when treated with p-fluororaniline and
p-fluorobenzylamine, underwent a nucleophilic substitution
reaction under the catalytic influence of aniline
hydrochloride, yielding 5 and 7, respectively, which under
strong basic conditions afforded ring cyclised products, 6
and 8, respectively (Scheme 1), while 5 when kept under mild
basic conditions underwent ring cyclisation to form 9.
conditions. In the case of the reaction with 3,4-dimethoxy-
and 3,4,5-trimethoxybenzaldehydes, 2a and 2c, respectively,
were obtained as partially oxidized products, whereas in the
case of 4-methoxy- and 4-isopropyl benzaldehydes fully
aromatized products, 2b and 2d, respectively, were formed.
All the final compounds were fully characterized by mp, IR,
NMR and HRMS techniques.
Biological evaluation
Cytotoxic potential of target compounds. To evaluate the
cytotoxic potential of the target compounds (1a–1r and 2a–2d),
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)-based antiproliferative assay was performed against
five cancer cell lines, viz., MDA-MB-231 (breast), T47D (breast)
and MCF-7 (breast),19 A549 (lung) and HT-29 (colorectal). The
respective cancer cells were treated with the target compounds
at varying concentrations of 1, 5 and 25 μM and incubated for
48 h. Erlotinib was taken as a positive control. The results
suggested that nine compounds 1i, 1k, 1m, 1o, 1p, and 2a–2d
exhibited good antiproliferative activity in at least two cancer
cell lines employed (Table 1).
Synthesis of target compounds 1a–1r
The synthesis of target compounds 1a–1j (n = 0) and 1k–1r (n
= 1) was successfully achieved by reacting the corresponding
aryl aldehydes (1 equiv.) with 6 and 8, respectively, in the
presence of a catalytic amount of p-TsOH (10 mol%).18
Synthesis of target compounds 2a–2d
9 (1 equiv.) underwent cyclocondensation with aryl aldehydes
(1 equiv.) under acidic conditions (1 drop of H2SO4) to afford
the target compounds 2a–2d in good yields (>60%). Two
types of products were observed while using the same
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Table 1 The antiproliferative potential of the target compounds (1 and 2) against various cancer cell lines
Inhibitory potential (IC50 (μM) SD)a of target compounds of growth of cancer cells
Code
MDA-MB-231
T47D
MCF-7
A549
HT-29
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
2a
2b
2c
18.74 0.23
12.34 0.55
13.36 0.14
13.6 0.38
16.28 0.52
17.09 0.43
17.17 0.2
21.45 0.16
4.27 0.32
20.45 0.25
2.87 0.28
17.58 0.68
3.44 0.31
16.05 0.34
2.87 0.43
2.67 0.14
10.54 0.74
>25
13.74 0.17
16.32 0.44
15.86 0.62
20.10 0.21
20.39 0.26
19.43 0.47
15.26 0.54
19.95 0.63
7.23 0.22
>25
17.5 0.76
22.42 0.22
5.72 0.62
21.48 0.56
9.19 0.78
6.14 0.24
8.2 0.21
8.32 0.32
7.09 0.26
9.1 0.44
9.88 0.52
9.28 0.31
18.12 0.16
13.6 0.23
16.28 0.55
17.09 0.22
17.17 0.41
>25
2.96 0.17
14.66 0.32
9.71 0.23
13.13 0.17
2.02 0.26
10.87 0.42
4.62 0.30
1.32 0.17
9.13 0.14
24.34 0.47
2.29 0.22
10.94 0.27
2.29 0.23
10.6 0.41
1.04 0.15
13.74 0.17
16.32 0.31
15.86 0.16
20.10 0.55
20.39 0.18
19.43 0.49
15.26 0.32
19.95 0.23
7.23 0.18
>25
16.5 0.37
22.42 0.41
5.72 0.16
21.48 0.27
9.19 0.19
6.14 0.42
13.78 0.37
>25
9.99 0.52
11.45 0.29
9.33 0.31
9.99 0.16
7.85 0.28
14.65 0.42
9.87 0.19
9.04 0.26
10.89 0.24
10.64 0.28
11.35 0.36
3.87 0.35
4.53 0.27
11.09 0.22
12.57 0.18
7.46 0.49
3.99 0.32
5.71 0.21
3.46 0.16
>30.00 (ref. 25)
>25
8.67 0.15
1.22 0.29
6.76 0.32
2.29 0.35
5.46 0.19
10.68 0.72
5.87 0.36
9.96 0.31
5.35 0.22
9.80 (ref. 24)
2.37 0.18
11.03 0.39
3.29 0.19
11.02 0.38
4.63 0.32
2d
Erlotinib
a
Data are expressed as mean values SD of three independent experiments.
The lead compounds 1i, 1k, 1m, 1o, 1p, and 2a–2d were
further investigated for their toxicity toward human
peripheral blood mononuclear cells (HPBMCs) using the
MTT assay at 10 μM concentration. The results showed that
compounds 1m and 1i possessed extreme toxicity, whereas
compounds 2a and 1k were found to possess mild toxicity
(Fig. 3A). However, compounds 1o, 1p, 2b, 2c and 2d did not
induce any toxicity in HPBMCs and were further assessed for
Fig. 3 (A) MTT assay on HPBMCs using selected compounds 1i, 1k, 1m, 1o, 1p and 2a–2d; compounds 1i, 1k, 1m, and 2a showed toxicity towards
the HPBMCs. The graph depicts the percentage survival of the cells after treatment. (B) MTT assay on normal breast cells (HBL-100) using
compounds 1o, 1p, 2b, 2c, and 2d. The assay was conducted at a concentration of 10 μM and incubated for 48 h. The graph depicts the
percentage survival of cells after treatment. (C) Disruption of HBL-100 cell morphology by 1o, 1p and 2b suggesting toxicity, whereas compounds
2c and 2d did not produce any morphological alteration towards HBL-100 cells, suggesting their non-cytotoxic potential.
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their cytotoxicity (if any) on normal breast cells (HBL-100; as
control in the experiment). The results suggested that 2c and
2d were non-cytotoxic to HBL-100 (Fig. 3B and C) at 10 μM
concentration.
(in the case of hypoxic tumours);22 further it could be utilized
upon conversion to NH2 and subsequent alkylation as a
handle to design irreversible inhibitors of EGFR by making a
covalent bond with remote cysteine residues.23
2c and 2d, being the most promising anticancer molecules
of the series, were chosen further to establish the mechanism
of action as proposed by evaluating their ability to inhibit
ATP-dependent phosphorylation of EGFR. The results
suggested that 2c and 2d inhibited in vitro enzymatic activity
of EGFR with IC50 of 617.33 0.04 nM and 710 0.05 nM,
respectively, in comparison to the positive control, erlotinib
(IC50 = 239.91 0.05 nM) (Fig. 4).20
Since 3c emerged to be the best fit, it was synthesized
(Scheme 2) and assessed for anticancer evaluation in vitro.
3c was characterized by mp, IR, NMR and HRMS
techniques. The 1H NMR spectrum showed characteristic
amide NH2 protons as a singlet at δ 8.10. The protons of the
N-methyl piperazine ring appeared as two singlets, each
corresponding to 4 protons at δ 3.12 and δ 2.85, whereas
N-methyl protons appeared at δ 2.21. The 13C NMR signal for
carbonyl carbon of amide was observed at δ 165.04. The
HRMS (TOF-ESI) spectrum confirmed the mass of the
compound to be 519.2094 [M + H]+.
Compound 3c was anticancerous, induced cancer cell
death by promoting oxidative stress in a mitochondrial
independent manner, and was anti-EGFR. When tested
against the same cancer cell lines, 3c inhibited their growth
at a much lower concentration (IC50 = 1.98 to 4.07 μM;
Table 2) as compared to its parent compound 2c (compare
Table 1 with Table 2) and erlotinib, indicating its broad-
spectrum anticancer activity. 3c was further assessed for its
cytotoxicity against normal cells, i.e. HPBMCs and HBL-100
cells, and no noteworthy toxicity (Fig. 6A and B) was
observed in 3c.26 3c was also able to elevate the ROS
concentration inside MDA-MB-231 cancer cells when
incubated for 48 h at a sub-IC50 concentration using the
H2DCFDA assay (Fig. 6C and D). The alteration in ROS
was measured and analysed using flow cytometer on FL2-
In order to achieve a low nM anti-EGFR IC50 value
comparable to that of erlotinib, we utilised a molecular
modelling tool to explore the unoccupied area of the ATP
binding domain of EGFR and analysed an in silico interaction
model of 2c and 2d–EGFR complexes that guided and allowed
chain lengthening substitution of the 4-fluorophenyl ring (2c
and 2d) with the 4-(4-methylpiperazinyl)-3-nitrophenyl) moiety
at the N-9 position, which yielded compounds 3c and 3d,
respectively (Fig. 5). As expected, the binding dock scores of
3c and 3d were higher than those of their corresponding
parent compounds (2c; R isomer and 2d), and 3c was
energetically more favoured in the EGFR binding site (dock
score = −7.545) than 3d (dock score = −6.99). 3c maintained
important and conserved interactions such as hydrogen bond
formation (CONH2) with MET769 (1.8 Å), while a similar
interaction in the case of 3d was weak and observed at a
longer distance (2.4 Å). The N-methyl piperazinyl ring of 3c
was found to be near the DFG motif (ASP831, PHE832 and
GLY833), whereas the nitro group was observed to interact
with ASP831. In addition to improvement in the binding
affinity of the inhibitor, the N-methyl piperazinyl moiety
could also potentiate the water solubility and bioavailability
of 3c.21 Likewise, the –NO2 group upon bioreduction could
selectively target hypoxic tumours by intercalating with DNA
H
channel. Next, the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethyl-imidacarbocyanine iodide)-based mitochondrial
membrane permeability assay indicated (Fig. 6E and F)
that 3c caused an increase in the red/green ratio
(membrane depolarization), suggesting the progression of
cell death in a mitochondrial-independent pathway.27 Next,
Fig. 4 (A) Dose–response curves showing percentage inhibition of phosphorylation in EGFR as induced by investigational compounds 2c and 2d
in comparison with the positive control erlotinib. (B) The enzymatic inhibitory concentration (IC50) obtained as a consequence of inhibition in
phosphorylation.
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Fig. 5 Design of compounds based on chain lengthening of 2c and 2d. Designed compounds 3c and 3d were evaluated on the basis of the
docking score and the important interactions; MET769 (H-bond) interaction was observed in 3c (1.8 Å) and 3d (2.4 Å).
Scheme 2 Synthesis of best fit compound 3c and reagents and conditions: (i) 4-fluoro-3-nitroaniline, aniline hydrochloride, EtOH, rt, 3 h, 82%, (ii)
DBU, EtOH, rt, 2 h, 67%, (iii) 3,4-dimethoxybenzaldehyde, H2SO4, 1 drop MeOH, (iv) N-methyl piperazine, DMF, 50 °C, rt, 4 h, K2CO3.
Table 2 Anti-proliferative effect of 3c on cancer cells
Inhibitory potential (IC50 (μM) SD)a of 3c
Breast cancer cells
Code
Lung cancer cells
MCF-7
MDAMB-231
T47D
A549
H1299
Entry
1
2
3c
3.26 0.11
5.46 0.19
4.07 0.29
9.8 (ref. 24)
4.05 0.22
>30.00 (ref. 25)
1.98 0.19
1.04 0.15
2.09 0.24
3.05 0.23
Erlotinib
a
Data are expressed as mean values SD of three independent experiments.
the effect of compound 3c on cell cycle progression using
propidium iodide-based cell cycle analysis suggested that
3c induced cell cycle arrest at the sub-G1 phase
(Fig. 6G and H). Furthermore, 3c inhibited in vitro the
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Fig. 6 MTT assay of 3c against HPBMC (A) and HBL100 cells (B). (C) and (D) depict the relative fluorescence altered by 3c under the influence of
ROS generated in MDA-MB-231 cells as confirmed by the H2DCFDA-based assay and interpreted using a flow cytometer. (E) Quantification ratio of
J-aggregates vs. J-monomers depicting mitochondrial membrane permeability as detected by JC-1 dye and analyzed via a flow cytometer. (F) The
relative ratio of red/green (J-aggregates vs. monomers) altered by treatment with 3c incubated for 48 h (G) Graph depicting the effect of 3c on cell
cycle progression in MDA-MB-231 cells after 48 h of treatment at sub-IC50 concentration. The result signifies sub-G1 arrest as identified using PI
dye and analyzed using a flow cytometer. (H) Histogram shift defines the overlapping and similar pattern of arrest by 3c at the sub-G1 phase.
enzymatic activity of EGFR at a much lower concentration
(Fig. 7A; compare with 2c) with an IC50 of 236.38 0.04
nM which was comparable to that of the positive control,
erlotinib (IC50 = 239.91 0.05 nM) (Fig. 7B).
In order to explore the alternative anticancer
mechanismIJs) of 3c in addition to EGFR inhibition, we
performed in silico binding studies of 3c with proteins
which are mostly over-expressed in cancer such as Her-2,
ERα, PR, etc. (Table S1 and Fig. S1: see the ESI†). However,
the results were not promising and did not encourage us
to carry out further in vitro binding studies. Cell cycle
arrest at sub-G1 by 3c suggested that it could also be
provoked by the inhibition of other mitogens such as
CDK2, 4 or 6.28
However, experimental results suggested that 3c was not a
strong inhibitor of CDKs (IC50 >500 nM with all CDKs, i.e. 2,
4, and 6), further endorsing EGFR inhibition as one of the
major anticancer mechanisms.
Fig. 7 (A) Dose–response curves showing the percentage inhibition of phosphorylation in EGFR induced by 3c and erlotinib. (B) The enzymatic
inhibitory concentration (IC50) obtained as a consequence of inhibition in phosphorylation.
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Experimental section
5-Amino-1-(4-fluorophenyl)-1H-imidazole-4-carbonitrile (6).
General
Reagents of AR/GR quality were purchased from Sigma-
Aldrich, S.D. Fine chemicals, Loba-Chemie Pvt. Ltd., and
Sisco Research Laboratory and were used without further
purification. A Mettler Toledo (for quantity measurements
<10 mg) and a Sartorius analytical balance (BSA224S-CW)
(for quantity measurements >10 mg) were used for weighing
purposes. An SGW heating mantle, Tarson Spinot Digital,
ILMVAC rotary digital evaporator, digital hot plate and NSW
oven/vacuum oven were used during the course of the
reaction. The progress of the reaction was monitored by TLC,
using petroleum ether/ethyl acetate and chloroform/methanol
as the mobile phase on pre-coated Merck TLC (TLC silica gel
60, F254) plates in a JSGW UV/fluorescence analysis cabinet
and/or iodine chamber. Melting points were recorded on
Stuart melting point apparatus (SMP-30) with open glass
capillary tubes and were uncorrected. Infrared (IR) spectra of
compounds were recorded with KBr on a Bruker FT-IR
spectrophotometer. 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded at Panjab University,
Chandigarh, Indian Institute of Technology, Ropar, and the
Central University of Rajasthan in CDCl3/DMSO-d6 on a
Bruker Advance II (400 MHz)/or JEOL NMR spectrometer
using TMS (δ = 0) as an internal standard. Mass spectra were
recorded on a Shimadzu GCMS-QP2010 and DST-FIST funded
GCMS instruments with EI mode at the Central
Instrumentation Laboratory (CIL) and DPSN, the Central
University of Punjab, and HRMS analysis was performed at
IIT, Ropar (Punjab, India). The CDK assay was outsourced at
Life Technologies SelectScreen™ Biochemical Profiling Lab.
To a suspension of 4 (3 g, 21.42 mmol) in methanol (1.5
mL) were added p-fluoroaniline (1.85 ml, 17.14 mmol) and
aniline hydrochloride (2.7 mg, 0.021 mmol). The reaction
mixture was stirred for 3 h at rt (TLC). The precipitate so
obtained (5) was filtered, washed with diethyl ether, dried
and used for the next step without further purification. A
suspension of 5 (1 g, 4.42 mmol) was stirred in 1 M KOH
(15 mL) at rt for
6 h (TLC) to obtain 5-amino-1-(4-
fluorophenyl)-1H-imidazole-4-carbonitrile (6). The crude
product was extracted with EtOAc (3 × 20 mL) and further
recrystallized from EtOAc to obtain the pure compound (6).
Yield: 89%; color: brown solid; mp: 192–196 °C. IR (KBr,
cm−1): 3334 and 3295 (NH2 stretch), 2211 (CN stretch), 1577
(NH2 bend), 1248 (C–F). 1H NMR (400 MHz, DMSO-d6) δ:
7.53–7.50 (2H, m), 7.42–7.38 (3H, m), 6.18 (2H, s, D2O
exchangeable NH2). HRMS (TOF-ESI) calcd for C10H7FN4:
202.0655 [M]+; observed: 203.0719 [M + H]+.
Representative procedure for the synthesis of compounds
1a–1j
A
mixture
of
6
(0.5
g,
2.48
mmol),
3,4,5-
Chemistry
trimethoxybenzaldehyde (0.49 g, 2.48 mmol) and p-TsOH
(0.042 g, 10 mol%) in methanol (10 mL) was stirred at rt for
2 h. The progress of the reaction was monitored by TLC,
using a petroleum ether and ethyl acetate solvent system (4 :
1). The precipitate so obtained was filtered and washed with
an excess of methanol. The compound was dried and further
recrystallized from methanol.
N-(2-Amino-1,2-dicyano-vinyl)-formimidic acid ethyl ester
(4).17
(E)-1-(4-Fluorophenyl)-5-((3,4,5-trimethoxybenzylidene)-
amino)-1H-imidazole-4 carbonitrile (1a).
To a suspension of 2,3-diaminomaleonitrile (3, 3 g, 27.75
mmol) in 1,4-dioxane (25 ml) was added triethyl orthoformate
(4.5 mL). The reaction mixture was refluxed at 80 °C for 6 h
(TLC). The dark brown mass formed after the evaporation of
dioxane was extracted with diethyl ether (3 × 50 mL) and
filtered. The yellow needle-shaped crystals 4 formed were
used for the next step without any further purification. Yield:
82%; color: yellow; mp: 135 °C (reported 134–136 °C). IR
(KBr, cm−1): 3418, 3390 (NH2 stretch), 2244, 2258 (CN
Yield: 82%; color: light yellow; mp: 120–122 °C. IR (KBr,
cm−1): 2222 (CN), 1654 (CN), 1236 (C–O), 1164 (C–F). 1H
NMR (CDCl3, 400 MHz): 9.01 (1H, s), 7.67 (1H, s), 7.45–7.42
(2H, m), 7.27–7.21 (2H, m), 7.08 (2H, s), 3.93 (3H, s), 3.89
1
stretch). H NMR (400 MHz, CDCl3, TMS = 0) δ: 1.37 (3H, t, J
= 7.2 Hz), 4.29 (2H, q, J = 7.3 Hz), 7.99 (2H, s, D2O
exchangeable NH2), 7.97 (1H, s).
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(6H, s). 13C NMR (100 MHz, CDCl3): 163.79, 161.35, 153.61,
146.94, 142.71, 136.41, 130.48, 130.34, 127.43, 127.34, 116.57,
116.34, 115.59, 106.57, 100.49, 61.17, 56.30. HRMS (TOF-ESI)
calcd for C20H17FN4O3: 380.1285 [M]+; observed: 381.1366 [M
+ H]+.
The mentioned procedure was further utilized for the rest
of the compounds in the series. The physical data of the
compounds are as follows:
(E)-5-((2,5-Dimethoxybenzylidene)amino)-1-(4-
fluorophenyl)-1H-imidazole-4-carbonitrile (1b).
Yield: 87% colour: dark yellow, mp: 105–107 °C. IR (KBr,
cm−1): 3453 (OH), 2219 (CN), 1654 (CN), 1215 (C–O), 1173
(C–F). 1H NMR (CDCl3, 400 MHz): 8.96 (1H, s), 7.61 (1H, s),
7.42–7.38 (3H, m), 7.22–7.18 (3H, m), 6.97 (1H, m), 6.08 (1H,
s, D2O exchangeable OH), 3.89 (3H, s). 13C NMR (100 MHz,
CDCl3): 164.04, 163.38, 160.88, 152.20, 148.14, 147.73, 136.05,
130.38, 127.13, 127.07, 126.82, 125.81, 116.26, 116.03, 115.97,
115.48, 110.29, 55.71. HRMS (TOF-ESI) calcd for C18H13FN4O2:
336.1023 [M]+; observed: 337.1081 [M + H]+.
(E)-1-(4-Fluorophenyl)-5-((4-hydroxybenzylidene)amino)-
1H-imidazole-4-carbonitrile (1e).
Yield: 86%; color: bright yellow; mp: 125–127 °C. IR (KBr,
cm−1): 2224 (CN), 1659 (CN), 1221 (C–O), 1172 (C–F). 1H
NMR (CDCl3, 400 MHz): 9.48 (1H, s), 7.66 (1H, s), 7.42–7.38
(3H, m), 7.22–7.16 (2H, m), 7.06–7.03 (1H, m), 6.89 (1H, m),
3.49 (3H, s), 3.12 (3H, s). 13C NMR (100 MHz, DMSO-d6):
189.59, 156.72, 153.57, 148.04, 133.19, 128.69, 128.58, 125.99,
124.76, 123.70, 117.52, 117.34, 117.11, 114.96, 110.82, 56.84.
HRMS (TOF-ESI) calcd for C19H15FN4O2: 350.1179 [M]+;
observed: 351.1250 [M + H]+.
Yield: 78%; color: pale yellow; mp: 130–132 °C. IR (KBr,
cm−1): 3449 (OH), 2224 (CN), 1666 (CN), 1235 (C–O), 1166
(C–F). 1H NMR (CDCl3, 400 MHz): 9.66 (1H, s), 8.60 (1H, s),
7.38–7.35 (3H, m), 7.26–7.23 (2H, m), 7.41–7.38 (2H, m),
6.58–6.53 (2H, m). HRMS (TOF-ESI) calcd for C17H11FN4O:
306.0917 [M]+; observed: 307.0989 [M + H]+.
(E)-1-(4-Fluorophenyl)-5-((4-nitrobenzylidene)amino)-1H-
imidazole-4-carbonitrile (1c).
(E)-1-(4-Fluorophenyl)-5-((4-methoxybenzylidene)amino)-
1H-imidazole-4-carbonitrile (1f).
Yield: 82%; color: yellow solid; mp: 112–114 °C. IR (KBr,
cm−1): 2227 (CN), 1603 (CN), 1519 (NO), 1348 (N–O),
1116 (C–F).1H NMR (CDCl3, 400 MHz): 9.22 (1H, s), 8.29
(2H, d, J = 8 Hz), 7.96 (2H, d, J = 8 Hz), 7.71 (1H, s),
7.41–7.38 (2H, m), 7.29–7.24 (2H, m). HRMS (TOF-ESI)
calcd for C17H10FN5O2: 335.0819 [M]+; observed: 336.0881
[M + H]+.
Yield: 85%, colour: yellow, mp: 134–136 °C. IR (KBr, cm−1):
1654 (CN), 1247 (C–O). 1H NMR (CDCl3, 400 MHz): 9.00
(1H, s), 7.76 (2H, d, J = 8 Hz), 7.61 (1H, s), 7.42–7.38 (2H, m),
7.22–7.18 (2H, m), 6.94 (2H, d, J = 8 Hz), 3.86 (3H, s). 13C
NMR (100 MHz, CDCl3): 164.41, 162.45, 161.25, 158.19,
153.70, 146.78, 145.72, 129.68, 129.17, 125.09, 125.00, 116.36,
116.13, 113.43, 54.93. MS (EI): m/z 319 [M − 1]+.
( E ) - 1 - ( 4 - F l u o r o p h e n y l ) - 5 - ( ( 4 - h y d r o x y - 3 -
methoxybenzylidene)amino)-1H-imidazole-4-carbonitrile (1d).
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(E)-1-(4-Fluorophenyl)-5-((2-hydroxy-5-nitrobenzylidene)-
amino)-1H-imidazole-4-carbonitrile (1g).
MHz): 9.00 (1H, s), 7.63 (1H, s), 7.44–7.39 (3H, m), 7.38–7.36
(1H, m), 7.22–7.18 (2H, m), 6.94 (1H, d, J = 8 Hz), 3.99 (3H,
s), 3.87 (3H, s). 13C NMR (100 MHz, DMSO-d6): 56.03, 56.39,
91.37, 110.04, 111.83, 117.07, 117.29, 117.56, 126.61, 128.12,
128.53, 128.63, 130.18, 133.14, 148.04, 149.72, 154.74, 163.63,
165.52. HRMS (TOF-ESI) calcd for C19H15FN4O: 350.1179 [M]+;
observed: 351.1260 [M + H]+.
(E)-1-(4-Fluorophenyl)-5-((4-isopropylbenzylidene)amino)-
1H-imidazole-4-carbonitrile (1j).
Yield: 84%, colour: creamish, mp: 128–130 °C. IR (KBr,
cm−1): 2224 (CN), 1590 (CN), 1451 (NO), 1272 (N–O),
1070 (C–F). 1H NMR ((CDCl3 + DMSO-d6), 400 MHz): 10.77
(1H, s), 9.49 (1H, s), 7.97 (1H, d, J = 8 Hz), 7.83 (1H, s), 7.44–
7.40 (2H, m), 7.26 (1H, d, J = 3 Hz), 7.21–7.15 (2H, m), 6.95–
6.92 (1H, dd, J12 = 2.4 Hz, J13 = 8 Hz). HRMS (TOF-ESI) calcd
for C17H10FN5O3: 351.0768 [M]+; observed: 352.0833 [M + H]+.
( E ) - 1 - ( 4 - F l u o r o p h e n y l ) - 5 - ( ( 3 - h y d r o x y - 4 -
methoxybenzylidene)amino)-1H-imidazole-4-carbonitrile (1h).
Yield: 82%, colour: pale yellow, mp: 140–142 °C. IR (KBr, cm−1):
2223 (CN), 1653 (CN), 1166 (C–F). 1H NMR (CDCl3, 400
MHz): 9.06 (1H, s), 7.75 (2H, d, J = 8 Hz), 7.64 (1H, s), 7.43–7.39
(2H, m), 7.32 (2H, d, J = 8 Hz), 7.24–7.19 (2H, m), 2.99–2.93
(1H, sep), 1.27 (3H, s), 1.25 (3H, s). MS (EI): m/z 332 [M]+.
Synthesis of 5-amino-1-(4-fluorobenzyl)-1H-imidazole-4-
carbonitrile (8)
Yield: 82%, colour: off-white, mp: 110–112 °C. IR (KBr cm−1):
3452 (OH), 2222 (CN), 1654 (CN), 1224 (C–O), 1021 (C–F).
1H NMR (CDCl3, 400 MHz): 8.98 (1H, s), 7.62 (1H, s), 7.44–
7.39 (3H, m), 7.33–7.30 (1H, dd, J12 = 1.6 Hz, J13 = 8.5 Hz),
7.22–7.20 (2H, m), 6.91 (1H, d, J = 8 Hz), 5.72 (1H, s, D2O
exchangeable OH), 3.99 (3H, s). 13C NMR (100 MHz, CDCl3):
164.09, 163.8, 160.74, 152.48, 147.58, 146.94, 136.17, 130.33,
128.04, 127.14, 127.05, 124.17, 116.34, 116.11, 113.88, 110.88,
99.32, 55.83. HRMS (TOF-ESI) calcd for C18H13FN4O2:
336.1023 [M]+; observed: 337.1094 [M + H]+.
To a suspension of 4 (3 g, 21.42 mmol) in methanol (1.5 mL)
were added p-fluorobenzylamine (1.85 ml, 17.14 mmol) and
aniline hydrochloride (2.7 mg, 0.021 mmol). The reaction
mixture was stirred at rt for 3 h. The precipitate so obtained
was filtered, washed with diethyl ether and dried to afford 7
and used without any further purification. Yield: 78%. A
suspension of 7 (1 g, 3.88 mmol) was stirred in 1 M KOH
solution (15 mL) to obtain 5-amino-1-(4-fluorobenzyl)-1H-
imidazole-4-carbonitrile (8). The reaction was monitored
using TLC. The crude product was extracted with ethyl
acetate (3 × 20 mL) and further recrystallized from ethyl
acetate to obtain the pure compound (8). Yield: 68%; color:
coffee brown solid; mp: 201–203 °C. IR (KBr, cm−1): 3395,
3321 (NH2 stretch), 2243 (CN stretch), 1561 (NH2 bend), 1124
(E)-5-((3,4-Dimethoxybenzylidene)amino)-1-(4-
fluorophenyl)-1H-imidazole-4-carbonitrile (1i).
1
(C–F). H NMR (400 MHz, CDCl3 + DMSO-d6) δ: 7.1–7.10 (2H,
m), 6.97–6.93 (3H, m), 5.43 (2H, s, D2O exchangeable NH2),
4.93 (2H, s). 13C NMR (CDCl3 + DMSO-d6, 100 MHz): 163.27,
161.30, 147.23, 132.2, 132.22, 131.07, 129.27, 129.20, 116.54,
115.89, 115.72, 46.51. MS (EI): m/z 216 [M]+.
Yield: 82%, colour: cream, mp: 120–122 °C; IR (KBr, cm−1):
2222 (CN), 1659 (CN), 1237 (C–O). 1H NMR (CDCl3, 400
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Representative procedure for the synthesis of compounds
1i–1r
To a suspension of 8 (0.1 g, 0.38 mmol, 1 equiv.) in methanol
(10 ml), 3,4,5-trimethoxybenzaldehyde (0.074 g, 0.38 mmol, 1
equiv.) was added followed by addition of p-TsOH (0.006 g,
10 mol%). The reaction mixture was stirred for 2 h at rt. The
completion of the reaction was confirmed by TLC, using a
petroleum ether and ethyl acetate elution system (4 : 1). The
precipitate so obtained was filtered and washed with an
excess of methanol. The compound was dried and further
recrystallized from methanol (1k).
(E)-1-(4-Fluorobenzyl)-5-((3,4,5-trimethoxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (1k).
Yield: 88%; color: orange color solid; mp: 110–112 °C; IR (KBr,
cm−1): 2224 (CN), 1660 (CN), 1517 (NO), 1344 (N–O), 1228 (C–
F). 1H NMR (CDCl3, 400 MHz): 9.03 (1H, s), 8.19 (2H, d, J = 8 Hz),
7.95–7.94 (2H, m), 7.49–7.46 (1H, m), 7.12–7.10 (2H, m), 6.90–6.87
(2H, m), 5.13 (2H, s). 13C NMR (CDCl3, 100 MHz): 160.26, 149.90,
145.08, 140.19, 137.58, 129.84, 129.55, 124.08, 116.11, 115.96,
115.56, 100.75, 89.73, 47.45. HRMS (TOF-ESI) calcd for
C18H12FN5O2: 349.0975 [M]+; observed: 350.0986 [M + H]+.
(E)-1-(4-Fluorobenzyl)-5-((4-hydroxy-3-methoxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (1n).
Yield: 84%; color: yellow color solid; mp: 159–161 °C. IR
(KBr, cm−1): 2216 (CN), 1650 (CN), 1238 (C–O), 1136 (C–F).
1H NMR (CDCl3, 400 MHz): 8.99 (1H, s), 7.46 (1H, s), 7.26–
7.24 (2H, m), 7.16 (1H, s), 7.07–7.03 (3H, m), 5.22 (2H, s),
3.95 (3H, s), 3.94 (6H, s). HRMS (TOF-ESI) calcd for
C21H19FN4O3: 394.1441 [M]+; observed: 395.1426 [M + H]+.
Using the above-mentioned procedure, the following
compounds were synthesized. The physical data of the
compounds are as follows:
Yield: 79%; color: light grey; mp: 161–163 °C. IR (KBr, cm−1):
3451 (OH), 2218 (CN), 1654 (CN), 1214 (C–O), 1158 (CF). 1H
NMR (CDCl3, 400 MHz): 8.97 (1H, s), 7.44–7.43 (2H, m), 7.26–7.23
(4H, m), 7.06–7.02 (2H, m), 6.15 (1H, s), 5.21 (2H, s), 3.98 (3H, s).
13C NMR (100 MHz, DMSO-d6): 163.34, 152.21, 147.00, 146.91,
136.13, 11.46, 129.78, 129.71, 128.37, 124.19, 116.19, 115.96,
115.79, 113.88, 110.83, 55.98, 47.10. MS (EI): m/z 350 [M]+.
(E)-1-(4-Fluorobenzyl)-5-((4-methoxybenzylidene)amino)-
1H-imidazole-4-carbonitrile (1o).
(E)-5-((2,5-Dimethoxybenzylidene)amino)-1-(4-
fluorobenzyl)-1H-imidazole-4-carbonitrile (1l).
Yield: 81%; color: bright golden solid; mp: 178–180 °C. IR (KBr,
cm−1): 2221 (CN), 1654 (CN), 1224 (C–O), 1168 (C–F). 1H NMR
(CDCl3, 400 MHz): 9.47 (1H, s), 7.57 (1H, s), 7.42 (1H, s), 7.24–
7.21 (2H, m), 7.04–7.02 (3H, m), 6.92–6.89 (1H, m), 5.18 (2H, s)
3.88 (3H, s), 3.80 (3H, s). HRMS (TOF-ESI) calcd for
C20H17FN4O2: 364.1336 [M]+; observed: 365.1409 [M + H]+.
(E)-1-(4-Fluorobenzyl)-5-((4-nitrobenzylidene)amino)-1H-
imidazole-4-carbonitrile (1m).
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Yield – 83%; color: dark yellow solid; mp: 125–127 °C. IR
(KBr, cm−1): 2217 (CN), 1654 (CN), 1221 (C–O), 1166 (C–F).
1H NMR (400 MHz, DMSO-d6) δ: 9.02 (1H, s), 7.90–7.88 (2H,
d, J = 8 Hz), 7.43 (1H, s), 7.26–7.23 (2H, m), 7.06–7.00 (4H,
m), 5.21 (2H, s), 3.90 (3H, s). 13C NMR (100 MHz, DMSO-d6)
δ: 163.78, 163.55, 163.09, 161.68, 147, 136.04, 131.32, 129.69,
129.69, 128.05, 116.20, 116.03, 114.57, 99.36, 55.61, 47.28.
HRMS (TOF-ESI) calcd for C19H15FN4O: 334.1229 [M]+;
observed: 335.1236 [M + H]+.
(E)-1-(4-Fluorobenzyl)-5-((3-hydroxy-4-methoxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (1p).
Yield: 85%; yellow color solid; mp: 158–160 °C; IR (KBr,
cm−1): 2213 (CN), 1653 (CN), 1217 (C–O), 1168 (C–F). 1H
NMR (400 MHz, DMSO-d6): 9.35 (1H, s), 7.86 (1H, d, J = 7.2
Hz), 7.42 (1H, s), 7.26–7.22 (2H, m), 7.04 (2H, m), 6.80–6.87
(1H, m), 5.19 (2H, s), 4.03 (3H, s), 3.96 (3H, s), 3.90 (3H, s).
13C NMR (DMSO-d6, 100 MHz, δ with TMS = 0): 160.03,
158.26, 155.87, 147.92, 135.96, 129.67, 129.60, 122.88, 121.93,
116.19, 116.02, 107.93, 99.50, 62.50, 61.04, 56.26, 47.27. MS
(EI): m/z 394 [M]+.
Synthesis of 5-amino-1-(4-fluorophenyl)-1H-imidazole-4-
carbimidoyl cyanide (9)
Yield: 83%; color: yellow solid; mp: 167–169 °C; IR (KBr,
cm−1): 3450 (OH), 2220 (CN), 1654 (CN), 1275 (C–O). 1H
NMR (CDCl3, 400 MHz): 8.76 (1H, s), 8.23 (1H, brs), 7.42 (1H,
s), 7.35 (1H, s), 7.28 (1H, s), 7.21–7.19 (2H, m), 7.15–7.12 (3H,
m), 5.07 (2H, s), 3.83 (3H, s). 13C NMR (100 MHz, CDCl3):
163.34, 152.21, 147.00, 146.91, 136.13, 11.46, 129.78, 129.71,
128.37, 124.19, 116.19, 115.96, 115.79, 113.88, 110.83, 55.98,
47.10. HRMS (TOF-ESI) calcd for C19H15FN4O2: 350.1179 [M]+;
observed: 351.1173 [M + H]+.
(E)-5-((3,4-Dimethoxybenzylidene)amino)-1-(4-
fluorobenzyl)-1H-imidazole-4-carbonitrile (1q).
To a solution of 5 (1 g, 4.78 mmol) were added 1–2 drops of
DBU to obtain 5-amino-1-(4-fluorophenyl)-1H-imidazole-4-
carbimidoyl cyanide (9). The reaction mixture was stirred at rt
for 2 h (TLC). The precipitate so obtained was filtered,
washed with diethyl ether and dried to afford 9. Yield: 83%;
color: apple green; mp: 120–122 °C. IR (KBr, cm−1): 2221 (CN
1
stretch), 3421, 3321 (N–H), 1632 (CN), 1220 (C–F). H NMR
(400 MHz, DMSO-d6) δ: 11.08 (1H, s, NH), 7.52–7.50 (2H, m),
7.41–7.39 (3H, m), 6.67 (2H, s, NH2). 13C NMR (100 MHz,
DMSO-d6) δ: 165.81, 163.53, 161.09, 148, 144.61, 143.76,
143.58, 132.69, 130.64, 128.16, 128.07, 117.38, 117.28. HRMS
(TOF-ESI) calcd for C11H8FN5: 229.0764 [M]+; observed:
230.0837 [M + H]+.
Yield: 84%; color: light yellow solid; mp: 172–174 °C; IR
(KBr, cm−1): 2217 (CN), 1654 (CN), 1223 (C–O), 1167 (C–F).
1H NMR (CDCl3, 400 MHz): 8.97 (1H, s), 7.47–7.42 (3H, m),
7.24–7.23 (2H, m), 7.04 (2H, m), 6.95 (1H, d, J = 8 Hz), 5.19
(2H, s), 3.96 (3H, s), 3.94 (3H, s). 13C NMR (100 MHz,
CDCl3): 163.78, 161.80, 149.15, 148.81, 145.53, 133.19,
129.81, 129.52, 129.49, 128.73, 128.66, 120.04, 117.00,
116.64, 116.47, 111.19, 111.13, 95.52, 55.94, 55.91, 49.30.
HRMS (TOF-ESI) calcd for C20H17FN4O2: 364.1336 [M]+;
observed: 365.1419 [M + H]+.
Representative procedure for the synthesis of compounds
2a–2d
To a mixture of 9 (0.1 g, 0.44 mmol, 1 equiv.) in methanol (5
ml) was added 3,4,5-trimethoxybenzaldehyde (1 equiv.)
followed by addition of 1 drop of diluted sulfuric acid. The
reaction mixture was stirred for 1 h at rt. The progress of the
reaction was monitored by TLC, using a petroleum ether and
ethyl acetate solvent system (2 : 1) as eluent. The precipitate
so obtained was filtered and washed with excess methanol.
(E)-1-(4-Fluorobenzyl)-5-((2,3,4-trimethoxybenzylidene)-
amino)-1H-imidazole-4-carbonitrile (1r).
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The compound was dried and further recrystallized from
methanol to afford pure 2a.
9-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)-3,9-dihydro-
2H-purine-6-carboxamide (2a).
Yield: 62%; color: dark orange solid; mp: 133–135 °C. IR
(KBr, cm−1): 3346, 3280 (NH2), 1651 (CO), 1232 (C–O), 1171
1
(C–F). H NMR (400 MHz, DMSO-d6) δ: 8.14 (2H, s), 8.04 (1H,
s), 7.93 (2H, s), 7.32 (2H, m), 7.03 (1H, s), 6.85 (2H, s), 6.12
(1H, s), 3.72 (3H, s), 3.68 (3H, s). 13C NMR (100 MHz, DMSO-
d6) δ: 162.55, 148.96, 148.93, 145.02, 119.94, 118.77, 116.66,
116.54, 116.43, 111.77, 110.31, 110.26, 56.05, 55.92. HRMS
(TOF-ESI) calcd for C20H18FN5O3: 395.1393 [M]+; observed:
396.1531 [M + H]+; observed: 418.1341 [M + Na]+.
Yield: 76%; color: dark yellow; mp: 145–147 °C. IR (KBr,
cm−1): 3355 and 3299 (NH2), 1643 (CO), 1588 (CN), 1228
(C–O), 1165 (C–F). H NMR (400 MHz, DMSO-d6) δ: 8.18 (1H,
1
s), 8.15 (1H, s), 8.08 (1H, s), 7.91–7.88 (2H, m), 7.40–7.37 (1H,
m), 7.32 (2H, m), 6.71 (2H, s), 6.1 (1H, s), 3.66 (6H, s), 3.58
(3H, s). 13C NMR (100 MHz, DMSO-d6) δ: 162.4, 161.45,
159.04, 157.91, 153.56, 153.04, 145.22, 139.74, 137.61, 133.80,
123.39, 123.31, 117.20, 116.98, 116.92, 116.64, 116.41, 103.86,
60.47, 56.56, 56.28. HRMS (TOF-ESI) calcd for C21H20FN5O4:
9-(4-Fluorophenyl)-2-(4-isopropylphenyl)-9H-purine-6-
carboxamide (2d).
425.1499 [M]+; observed: 426.1589 [M
448.1589 [M + Na]+.
+
H]+; observed:
Using the above-mentioned protocol, the remaining
compounds were synthesized. The physical data of the
compounds are as follows:
9-(4-Fluorophenyl)-2-(4-methoxyphenyl)-9H-purine-6-
carboxamide (2b).
Yield: 68%; color: off white solid; mp: 156–158 °C. IR
(KBr, cm−1): 3313, 3299 (NH2), 1650 (CO), 1548 (CN),
1132 (C–F). 1H NMR (400 MHz, DMSO-d6) δ: 9.05 (1H, s),
8.47 (1H, s, CONH), 8.40 (2H, d, J = 8 Hz), 8.02 (1H, s,
CONH), 8.01 (2H, m), 7.54–7.51 (2H, m), 7.36 (2H, d, J =
8 Hz), 2.95–2.91 (1H, sep), 1.20 (6H, d). 13C NMR (100
MHz, DMSO-d6) δ: 165.09, 163.08, 160.63, 158.44,
154.36, 151.74, 148.83, 147.56, 135.22, 130.49, 128.79,
127.12, 126.43, 126.35, 117.20, 116.97, 32.21, 24.22.
HRMS (TOF-ESI) calcd for C21H18FN5O: 375.1495 [M]+;
observed: 376.1566 [M + H]+.
Yield: 65%; color: off white solid; mp: 115–117 °C. IR
(KBr, cm−1): 3442, 3392 (NH2), 1658 (CO), 1578
(CN), 1245 (C–O), 1171 (C–F). 1H NMR (400 MHz,
DMSO-d6) δ: 9.02 (1H, s), 8.48 (1H, s, CONH), 8.45–8.42
(2H, d, J = 8 Hz), 8.05 (1H, s, CONH), 8.03–7.98 (2H, m),
7.53–7.49 (2H, m), 7.05–7.03 (2H, m), 5.21 (2H, s), 3.80
(3H, s). 13C NMR (100 MHz, DMSO-d6) δ: 165.13, 163.05,
161.93, 160.6, 158.29, 154.38, 148.78, 147.30, 130.34,
129.69, 129.96, 126.42, 126.34, 117.19, 116.96, 114.52,
55.61. HRMS (TOF-ESI) calcd for C19H14FN5O2: 363.1131
[M]+; observed: 364.1280 [M + H]+; observed: 386.1087
[M + Na]+.
Synthesis of 5-amino-1-(4-fluoro-3-nitrophenyl)-1H-imidazole-
4-carbimidoylcyanide (11)
2-(3,4-Dimethoxyphenyl)-9-(4-fluorophenyl)-3,9-dihydro-
2H-purine-6-carboxamide (2c).
To a suspension of 4 (3 g, 21.42 mmol) in methanol (10 mL)
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were added p-fluoro-3 nitroaniline (2.7 g, 17.30 mmol) and
aniline hydrochloride (2.7 mg, 0.021 mmol). The reaction
mixture was stirred for 3 h at rt. The precipitate so obtained
was filtered, washed with diethyl ether and dried to afford 10
and was used without any further purification. Yield: 82%.
To a solution of 10 (1 g, 3.64 mmol) were added 1–2 drops of
DBU to obtain 5-amino-1-(4-fluorophenyl)-1H-imidazole-4-
carbimidoyl cyanide (11). The reaction mixture was stirred for
2 h at rt. The precipitate so obtained was filtered, washed
with diethyl ether and dried to afford 11, which was used for
the next step without further purification. Yield: 67%; color:
green color; mp: 120–122 °C. IR (KBr, cm−1): 3399 (N–H),
2280 (CN stretch), 1654 (CN), 1538 (NO), 1342 (N–O),
1261 (C–F). MS (EI): m/z 274 [M]+.
the crude mixture was extracted with ethyl acetate (10 mL ×
3) and water. The organic layer was evaporated under vacuum
using
a rotary evaporator to afford an orange colored
compound which was recrystallized from methanol to afford
pure 3c. Yield: 65%; color: orange solid; mp: 181–183 °C. IR
(KBr, cm−1): 3320, 3291 (NH2), 1680 (CO), 1620 (CN),
1548 (NO), 1372 (N–O). 1H NMR (400 MHz, DMSO-d6) δ:
9.15 (1H, s), 8.76 (1H, s), 8.53 (1H, s), 8.22–8.16 (2H, m), 8.10
(2H, s), 7.58 (1H, d, J = 8 Hz), 7.11 (1H, d, J = 8 Hz), 3.91 (3H,
s), 3.85 (3H, s), 3.12 (4H, s), 2.85 (4H, s), 2.25 (3H, s). 13C
NMR (100 MHz, DMSO-d6) δ: 165.04, 158.14, 154.05, 151.69,
149.20, 148.83, 146.69, 144.83, 141.94, 130.25, 129.83, 128.29,
127.80, 122.74, 122.04, 120.71, 111.92, 111.38, 56.06, 55.92,
54.90, 51.29, 46.17. HRMS (TOF-ESI) calcd for C25H26N8O5:
518.2026 [M]+; observed: 519.2094 [M + H]+.
Synthesis of 2-(3,4-dimethoxyphenyl)-9-(4-fluoro-3-
nitrophenyl)-9H-purine-6-carboxamide (12)
Biological evaluation
Cell culture. Cancer and normal cells were purchased
from the National Cell Repository situated at NCCS, Pune.
The cells were grown in DMEM containing 10% fetal bovine
serum (FBS) and antibiotic solution (1× Penstrip, Invitrogen).
The maintenance of cell growth was attained under standard
conditions inside a CO2 incubator with 5% CO2 and 95%
humidity at 37 °C. Cells were seeded at a density of between
1 and 3 × 104 cells per cm2 and sub-cultured using trypsin
(trypsinisation) when 70–80% confluent.
2,3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl
tetrazolium
To a suspension of 11 (0.4 g, 1.45 mmol, 1 equiv.) in
methanol (5 ml), 3,4-dimethoxybenzaldehyde (0.3 g, 1 equiv.)
was added and the mixture was stirred at rt after the addition
of 1 drop of H2SO4 for 1 h. The completion of the reaction
was confirmed by TLC, and the product (12) was filtered and
washed with methanol and was used for the next step
without further purification. Yield: 85%; dark yellow color
solid; mp: 172–174 °C; IR (KBr, cm−1): 2218 (CN), 1655
(CO), 1512 (NO), 1323 (N–O), 1216 (C–F). MS (EI): m/z
438 [M]+.
bromide (MTT) assay. The MTT assay was performed under
normoxic conditions. The cells were counted using an
automated cell counter and approximately 8 × 103 cells in
100 μl of DMEM were seeded in each well of 96 well plates.29
For human peripheral blood mononuclear cells (HPBMCs),
RPMI medium was used instead of DMEM. Cells were further
treated with the investigational compounds that were
subjected to serial dilution (prior) in biological grade DMSO.
The treatment was made at varying concentrations and for 48
h under standard conditions. After a specific time period the
medium was discarded and MTT was added (10 μl MTT dye
per well (5 mg ml−1 in PBS stock)). The cells were further
incubated to allow for the formation of formazan; after the
stipulated time of 4 h, the crystal was dissolved in DMSO and
absorbance was read using a microplate reader at 570 nm.
The results are presented as mean SD. The IC50 values of
these interesting compounds were calculated. HPBMCs were
cultured and used as per the protocol no. CUPB/cc/14/IEC/
4483 approved by the Institutional Ethics Committee of the
Central University of Punjab, Bathinda, according to
guidelines issued by the Indian Council of Medical Research
(ICMR), Govt. of India.
Synthesis of 2-(3,4-dimethoxyphenyl)-9-(4-(4-methylpiperazin-
1-yl)-3-nitrophenyl)-9H-purine-6-carboxamide (3c)
Reactive oxygen species (ROS) and mitochondrial membrane
integrative assay
1-Methyl piperazine (0.42 mL, 4.378 mmol) and K2CO3 (2
equiv., 0.803 g) were added to 12 (200 mg, 2.91 mmol)
dissolved in DMF (0.5 mL). The reaction mixture was stirred
for 4 h (TLC) at 50 °C. After the completion of the reaction,
To perform ROS and mitochondrial permeability assay, H2-
DCFDA and JC-1 dyes were employed. Cells were cultured
and treatment was given using the above-mentioned
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procedures. After the stipulated incubation time of 48 h, each
dye was added separately and the fluorescence intensity was
measured using a BD Accuri C6 flow cytometer.
For CDK4/cyclin D1 inhibitory assessment, a 2X CDK4/cyclin
D1/Rb substrate mixture was prepared in 50 mM HEPES pH
7.5, 0.01% BRIJ-35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT,
and 0.02% NaN3. The final 10 μL kinase reaction mixture
consisted of 7.5–30 ng CDK4/cyclin D1 and 1 μM Rb substrate
in 32.5 mM HEPES pH 7.5, 0.005% BRIJ-35, 5 mM MnCl2, 0.5
mM EGTA, 1 mM DTT, and 0.01% NaN3. After the 1 hour
kinase reaction incubation, 5 μL of detection mix was added. A
similar protocol was followed for the CDK4/cyclin D3.
For CDK6/cyclin D1, a 2X CDK6/cyclin D1/Rb substrate
mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-
35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT, and 0.02% NaN3.
The final 10 μL kinase reaction mixture consisted of 1.75–7
ng CDK6/cyclin D1 and 1 μM Rb substrate in 32.5 mM
HEPES pH 7.5, 0.005% BRIJ-35, 5 mM MnCl2, 0.5 mM EGTA,
1 mM DTT, and 0.01% NaN3. After the 1 hour kinase reaction
incubation, 5 μL of detection mix was added.
Cell cycle analysis
Cells were cultured in 30 mm dishes and treated with the
selected investigational compounds using the above-
mentioned protocols. After the desired time period of 48 h,
the media was removed and the cells were washed and
trypsinized. Further, the cells were fixed using 70% ethanol
to allow the passage of propidium iodide (PI) inside the cells.
After fixing for 4 h at −20 °C, the cells were centrifuged (2200
rpm) and washed subsequently with 1X PBS. After this,
RNAse was added and the cells were kept aside for 15 min;
then PI was added to each vial and the cells were incubated
for 30 min in the dark before analysing them in a flow
cytometer.
Molecular modelling studies. Molecular docking studies
were carried out using the Maestro 11.4 molecular docking
suite (Glide). All the designed ligands were imported in the
Maestro-scratch project in a 2D format which were drawn
using ChemDraw Professional. Ligands were prepared using
Ligprep to convert in 3D format. The force field used was
OPLS3 and all ionization states were generated at pH 7.0
2.0. Tautomers were generated for each neutralised or
ionized molecule. In the computation, specified chiralities
were retained and generated, at the most 10 per ligand. The
3D structure of the EGFR was imported from the RCSB
Protein Data Bank, PDB ID 1M17, using Protein Preparation
Wizard in which the workspace structure was pre-processed
where bond orders were assigned, the CCD database was
used, hydrogens were added, and zero-order bonds to metal
and disulphide bonds were created. Water beyond 5.00 Å was
deleted and pre-processed. After pre-processing, the
workspace was analysed and an active chain was selected in
which prepared ligands were to be docked. Later on, the
protein was refined which included optimization, removal of
waters and finally restrained minimization. After the
completion of ligand and protein preparation, receptor grid
generation was done in which a grid is formed around an
active site and a receptor-grid file is generated. Molecular
docking was carried out by docking the prepared ligand
molecule into the active ATP binding site of the minimized
protein using the receptor grid file. The flexible docking was
done using the extra precision mode and RMSD was
computed for the input ligandIJs).
Epidermal growth factor receptor (EGFR) inhibitory activity
The EGFR inhibitory activity of the investigational
compounds was determined using a commercially available
assay kit (z-Lyte kinase assay kit-tyr4 peptide (catalogue no.
PV3193)). The assay was performed in accordance with the
manufacturer's protocol and as per our previously published
reports.20a,c The percentage inhibition of kinase was
measured at Ex/Em 400, 445 and 520 nm using a microplate
reader. The calculation to deduce emission ratio was done by
dividing the coumarin emission (445 nm) by the fluorescein
emission (520 nm).
The extent of phosphorylation was calculated using the
given formula:
% Phosphorylation
ðEmission ratio × F100%Þ − C100%
¼ 1 −
ðC0% − C100%Þ þ ½Emission ratio ðF100% − F0%Þꢀ
where F100% = average fluorescein emission signal of the
100% Phos. Control; C100% = average coumarin emission
signal of the 0% Phos. Control; C0% = average coumarin
emission signal of the 100% Phos. Control; F0% = average
fluorescein emission signal of the 0% Phos. Control.
Cyclin-dependent kinase assays. The CDK assay was
outsourced at the Life Technologies SelectScreen™
Biochemical Profiling Lab, USA. The brief protocol is as
follows:
For CDK2/cyclin A inhibitory assessment, a 2X CDK2/
cyclin A/Ser/Thr 12 mixture was prepared in 50 mM
HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM
EGTA. The final 10 μL kinase reaction mixture consisted
of 1.22–10.3 ng CDK2/cyclin A and 2 μM Ser/Thr 12 in 50
mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1
mM EGTA. After the 1-hour kinase reaction incubation, 5
μL of a 1 : 4096 dilution of Development Reagent A was
added.
Conclusion
In the current work, we emphasized target hopping of 11g
along with molecular docking to design new fused imidazole
derivatives. The rational design led to the synthesis of target
molecules that were assessed for in vitro biological
evaluation. Among all the imidazole-bearing compounds, two
compounds 2c and 2d were found to possess selective
anticancer potential against five cancer cell lines; they were
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devoid of toxicity towards normal cells and inhibited EGFR at
the nM level. We further tuned the EGFR binding ability of
the compounds by exploring a 3D interaction model of 2c
and 2d–EGFR complexes and extending the chain length by
substitution of the 4-fluorophenyl ring (2c and 2d) with
4-(4-methylpiperazinyl)-3-nitrophenyl) at the N-9 position,
resulting in a best fit compound 3c with maximum binding
interactions with residual amino acids. 3c was synthesized
and found to possess more potent anti-EGFR and anticancer
activities than the parent compound 2c and the positive
control in addition to induction of oxidative stress in cancer
cells and cell cycle arrest at the sub-G1 phase. Further, our
efforts (in silico and in vitro) to explore the possibility of
inhibition of other overexpressed proteins by 3c also
suggested EGFR inhibition as one of the major anticancer
mechanisms. We will be interested to determine the effect of
3c on mutated EGFR (T790M and L858R) and its efficacy in
hypoxic lung cancer models (in vitro and in vivo) in the future
and the results will be published in due course.
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Author contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version
of the manuscript.
9 (a) M. Hossam, D. S. Lasheen and K. A. Abouzid, Covalent
EGFR
inhibitors:
binding
mechanisms,
synthetic
Conflicts of interest
approaches, and clinical profiles, Arch. Pharm., 2016, 349(8),
573–593; (b) X. Lu, L. Yu, Z. Zhang, X. Ren, J. B. Smaill and
K. Ding, Targeting EGFRL858R/T790M and EGFRL858R/
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There are no conflicts to declare.
Acknowledgements
We are thankful to the Vice-Chancellor and Dean of Academic
Affairs for providing the funds to support the present work.
The authors thank CIL, CUPB, for data analysis. GJ thanks
CSIR, New Delhi (Grant no. 05/1051IJ0011)/2018-EMR-I), MK
thanks DST-SERB, New Delhi (Grant no. EMR/2017/002702/
HS) and SA thanks Bristol Myers Squibb, USA (Grant
No.53803645), for providing research fellowships. GC-MS
analysis service using GC-MS equipment under DST-FIST
support to the Department of Pharmaceutical Sciences and
Natural Products is also duly acknowledged.
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