Synthesis and PASS-assisted evaluation of coumarin–benzimidazole derivatives as potential anti-inflammatory and anthelmintic agents

Article abstract of DOI:10.1007/s00044-017-2036-1

Two series of novel derivatives have been designed by coupling medicinally important coumarin and benzimidazole nuclei through different linkers. These compounds have been predicted to be potent anti-inflammatory and anthelmintic by in silico studies using PASS (prediction of activity spectra for substances) software. The compounds are synthesized and evaluated for the predicted activities as well as for their in vitro antioxidant potential. Compounds of first series (4a–4f) are found good to moderate anti-inflammatory agents. Among these, compounds 4b and 4f exhibited maximum anti-inflammatory activity (45% inhibition), which is equivalent to the activity of indomethacin (48% inhibition) after 3 h (peak inflammatory response time). Compounds of second series (5a–5f) exhibit anthelmintic activity. Amongst these, compound 5f has mortality activity marginally higher than albendazole (10–11 s). Compound 5e is found to be the most potent antioxidant with remarkable EC₅₀ value (0.08 μM/mL), which is though a little less than that of ascorbic acid (0.03 μM/mL). In addition, a comparative analysis of calculated Lipinski’s parameters reveals that all test compounds have the propensity to be orally bioavailable. Based on these findings, compounds 4b, 4f, 5e, and 5f are identified as new leads to develop potent anti-inflammatory, anthelmintic, and antioxidant compounds.

Full text of DOI:10.1007/s00044-017-2036-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2036-1
ORIGINAL RESEARCH
Synthesis and PASS-assisted evaluation of
coumarin–benzimidazole derivatives as potential
anti-inflammatory and anthelmintic agents
Purva Sethi1 Yogita Bansal1 Gulshan Bansal1
●
●
Received: 2 October 2015 / Accepted: 16 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Two series of novel derivatives have been
designed by coupling medicinally important coumarin and
benzimidazole nuclei through different linkers. These
compounds have been predicted to be potent anti-
inflammatory and anthelmintic by in silico studies using
PASS (prediction of activity spectra for substances) soft-
ware. The compounds are synthesized and evaluated for the
predicted activities as well as for their in vitro antioxidant
potential. Compounds of first series (4a–4f) are found good
to moderate anti-inflammatory agents. Among these, com-
pounds 4b and 4f exhibited maximum anti-inflammatory
activity (45% inhibition), which is equivalent to the activity
of indomethacin (48% inhibition) after 3 h (peak inflam-
matory response time). Compounds of second series (5a–5f)
exhibit anthelmintic activity. Amongst these, compound 5f
has mortality activity marginally higher than albendazole
(10–11 s). Compound 5e is found to be the most potent
antioxidant with remarkable EC50 value (0.08 µM/mL),
which is though a little less than that of ascorbic acid (0.03
µM/mL). In addition, a comparative analysis of calculated
Lipinski’s parameters reveals that all test compounds have
the propensity to be orally bioavailable. Based on these
findings, compounds 4b, 4f, 5e, and 5f are identified as new
leads to develop potent anti-inflammatory, anthelmintic, and
antioxidant compounds.
Abbreviations
PASS
OPD
Prediction of activity spectrum of substances
o-Phenylenediamine
DCC
Dicyclohexylcarbodiimide
Dichloromethane
4-Dimethylaminopyridine
Dicyclohexylurea
DCM
DMAP
DCU
NCEs
SCMC
DPPH
BHT
Novel chemical entities
Sodium carboxy methyl cellulose
2,2-Diphenyl-1-picrylhydrazyl
Butylhydroxytoluene
Introduction
Benzimidazole nucleus is an integral structural component
of many drugs belonging to different therapeutic categories
such as anthelmintics (albendazole, mebendazole, and
thiabendazole), proton pump inhibitors (omeprazole, lan-
soprazole, and pantoprazole), antihistaminic (astemizole),
antiviral (enviradine), and antihypertensive (candesarten
and telmisartan). An amino group at 2-position of benzi-
midazole generates a rigid form of guanidine, which is an
indispensable pharmacophore for diverse therapeutic activ-
ities (Bansal and Silakari 2012). In addition, an aromatic
moiety such as naphthyl, heteroaryl, p-nitrophenyl, or pyr-
idyl at the same position is known to produce potent
anthelmintics (Tsukamoto et al. 1980). Coumarin is another
important heterocyclic system, which has been exploited to
develop numerous therapeutic agents such as warfarin,
phenprocoumon, and acenocoumarol as anticoagulant;
armillarisin A as antibiotic; hymecromone as choleretic and
●
●
●
Keywords Coumarin Benzimidazole Anti-inflammatory
●
Anthelmintic Antioxidant
* Yogita Bansal
yogitabansalp@rediffmail.com
1
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, India

Med Chem Res
Scheme 1 Synthesis of test
compounds of series 4 and 5
antispasmodic; and cromakalim as antihypertensive (Wu
et al. 2009). It is also explored for the development of
anticancer (Riveiro et al. 2008), antimicrobial (Gormley
et al. 1996; Manvar et al. 2011), anti-Alzheimer disease
(Anand et al. 2012), antiviral (Curini et al. 2003), anti-
hyperlipidemic (Madhavan et al. 2003), and antioxidant and
anti-inflammatory (Bansal et al. 2013) agents. Hybridization
of coumarin nucleus with other varied heterocyclics is being
exploited to develop novel hybrid molecules that possess
multiple pharmacological activities (Sandhu et al. 2014).
Coumarin–benzimidazole hybrids are reported as potential
antiviral and antitumour agents (Paul et al. 2013; Tsay et al.
2013). With an aim to further explore the potential of
coumarin–benzimidazole hybrids as privileged medicinal
scaffold, we have coupled variedly substituted coumarin
nucleus with benzimidazole through a metabolizable or
non-metabolizable linker to obtain two series of test com-
pounds. The pharmacological activities spectrum of the test
compounds was predicted by in silico method using pre-
diction of activity spectrum of substances (PASS) software,
and the compounds were evaluated for most likely phar-
macological activities.
Results and discussion
Chemistry
The test compounds were synthesized by a four step syn-
thetic scheme (Scheme 1). In first step, a substituted phenol
(1a–1f) was condensed with citric acid to produce the
corresponding coumarin-4-acetic acid derivative (2a–2f) as
an
intermediate.
The
other
intermediate,
2-
aminobenzimidazole (3), was synthesized by reactions
between o-phenylendiamine and cyanogen bromide
(CNBr). For synthesis of first series of test compounds (4a–
4f), each of the intermediates 2a–2f was refluxed with o-
phenylendiamine for 3–6 h in the presence of orthopho-
sphoric acid (OPA) as catalyst. For the second series of test
compounds, 5a–5f, each of the intermediates 2a–2f was
coupled with 3 under anhydrous conditions using dicyclo-
hexylcarbodiimide (DCC) as coupling agent. All inter-
mediates and test compounds in the scheme were purified
by recrystallization from aqueous ethanol, and their struc-
tures were ascertained by infrared (IR), 1H-nuclear magnetic
resonance (NMR), 13C-NMR, and high resolution mass
spectral (HRMS) analyzes. Disappearance of stretching

Med Chem Res
Table 1 PASS predicted
activity of test compounds
Compounds
Probability of a compound being active (Pa) or inactive (Pi)
Anthelmintic
Anti-
Antineoplastic
Anti-ischemic
Kidney
inflammtory
function
stimulant
Pa
Pi
Pa
Pi
Pa
Pi
Pa
Pi
Pa
Pi
4a
4b
4c
4d
4e
4f
0.547
0.475
0.440
0.482
–
0.013
0.027
0.039
0.025
–
0.688
0.677
0.695
0.694
0.621
0.720
–
0.051
0.057
0.048
0.048
0.091
0.036
–
0.712
0.702
0.721
0.681
0.660
0.734
0.707
0.697
0.715
0.677
0.657
0.727
0.020
0.022
0.019
0.027
0.032
0.017
0.021
0.023
0.020
0.028
0.033
0.018
0.624
0.043
0.732
0.707
0.728
0.726
0.637
0.721
0.686
0.657
0.681
0.679
0.633
0.674
0.016
0.025
0.017
0.018
0.070
0.019
0.036
0.054
0.039
0.040
0.073
0.043
0.605
0.049
0.601
0.050
0.605
0.049
–
–
–
–
0.634
0.040
5a
5b
5c
5d
5e
5f
0.797
0.752
0.757
0.725
0.654
0.747
0.003
0.003
0.003
0.004
0.005
0.004
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
bands due to –COOH in IR spectra of compounds 4a–4f,
which were otherwise noted in IR spectra of coumarin
carboxylic acids (2a–2f) ascertained that the latter is
Molecular properties calculations
Magnitude of all calculated Lipinski’s parameters were
within the acceptable range (Table 2), and hence as per
Lipinski’s rule of five, the compounds were suggested to be
orally bioavailable. Total polar surface area (TPSA) is
another parameter for optimizing ability of a drug to
permeate cells. Molecules with polar surface area not
greater than 140 Å sq can easily permeate cells, and all test
compounds have TPSA less than 140. Therefore the com-
pounds are predicted to have good cell membrane
permeability.
condensed
with
o-phenylenediamine
to
form
benzimidazole–coumarin hybrids (4). Formation of com-
pounds of series 5 was ascertained by the presence of a band
in the range of 1630–1680 cm−1 corresponding to –CONH–
linkage. In 1H-NMR spectra, the –NH– protons were
detected at δ values ranging from 4.5 to 5.0, which were
confirmed by deuterium exchange experiments. Carbon
atoms of benzene ring of coumarin moiety were detected as
downfield or upfield signals depending upon the type of
substituent present in the moiety. In HRMS analyzes, mass
of parent ion [MH+] of each compound was found to cor-
respond to its theoretical mass.
Biological assay
Anti-inflammatory activity
In-silico studies
Based on PASS prediction data, compounds 4a–4f were
evaluated for anti-inflammatory activity. All compounds
exhibited moderate to good activity (35–45% inhibition of
rat paw edema) compared to indomethacin (47% inhibition)
(Table 3). The peak anti-inflammatory effect was observed
after 3 h of carageenan injection. Compounds 4b and 4f
were maximally active (45% inhibition), and equipotent to
indomethacin (48% inhibition). Comparative evaluation of
activity data of all six compounds in the series suggested
that the activity is independent of the electronic effects of
the substituents present on coumarin nucleus.
PASS predicted activity spectrum of test compounds
PASS predicted spectrum of activity of the test compounds
(Table 1) showed that compounds 4a–4f possess good
probability of exhibiting anti-inflammatory activity (Pa
values 0.621–0.720) whereas compounds 5a–5f have pro-
pensity to act as good anthelmintics (Pa values
0.654–0.797). In addition, compounds of series 4 were also
predicted to exhibit anti-ischemic activity (0.601–0.634)
whereas compounds of series 5 were predicted to be
anticancer (0.657–0.734) and kidney function stimulant
(0.633–0.732).

Med Chem Res
Table 2 Calculated Lipinski’s parameters and TPSA of test
compounds
Compounds Log Pa TPSAb MWc
nONd nOHNHe n viol.f
4a
4b
4c
4d
4e
4f
3.00
3.92
3.92
3.90
3.41
3.50
2.14
3.07
3.07
3.07
2.64
2.65
79.12
58.89
58.89
58.89
292.30
290.32
290.32
290.32
5
4
4
4
7
4
7
6
6
6
9
6
2
1
1
1
1
1
3
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
104.71 321.29
58.89 276.30
108.22 321.29
5a
5b
5c
5d
18
5f
87.99
87.99
87.99
319.32
319.32
319.32
133.81 364.31
87.99 305.29
a
Calculated lipophilicity
Total polar surface area
Molecular weight
b
c
d
e
f
Number of hydrogen bond acceptor
Number of hydrogen bond donors
Number of violations from Lipinski’s rule of five
Anthelmintic activity
Earthworms are used for evaluation of anthelmintic activity
as they resemble, both anatomically and physiologically,
with the intestinal roundworm parasites in human beings.
All compounds of series 5 showed paralytic activity and
mortality against the helminths in a dose dependent manner
(Figs. 1 and 2, respectively). Compound 5f was found to be
the most potent paralytic agent (time for paralysis 14 min)
from the series, and equipotent to albendazole (time for
paralysis 15 min) at a concentration of 0.1%. Also, its
mortality activity was marginally greater than the activity of
albendazole at all concentrations. The activity was in con-
sonant with their PASS predicted scores that support the
applicability of PASS in reliable prediction of activity
spectrum of organic molecules.
Antioxidant activity
In vitro antioxidant activity of all compounds was evaluated
as radical scavenging potential using DPPH method. A
compound possessing antioxidant potential react with
DPPH radical and decreases the color intensity due to the
radical. The color intensity is measured spectro-
photometrically and related to the radical scavenging
activity. Kinetics of the reaction between test compound
and DPPH revealed that absorbance plateau was attained at
30 min. Hence, 30 min was taken as optimum incubation

Med Chem Res
time for evaluating antioxidant activity of the compounds.
All test compounds decreased the color intensity of DPPH
solution, which suggested that the compounds possess
radical scavenging ability. Compounds 5a–5f were found
better radical scavenger than compounds 4a–4f (Table 4).
Compound 5e was more potent (EC50 0.08 µM/mL) than
BHT (EC50 23.4 µM/mL), and less potent than ascorbic acid
(EC50 0.03 µM/mL). The results revealed that an electron
withdrawing group increases the antioxidant potential,
probably be increasing electrophilicity of the amidic nitro-
gen atom.
were also evaluated for in vitro antioxidant activity. Com-
pounds 4b and 4f emerged as the most potent anti-
inflammatory compounds, and equipotent to indometha-
cin. Compound 5f was found to exhibit paralytic activity
equivalent to albendazole and mortality marginally greater
than albendazole. All compounds also exhibited moderate
to excellent free radical scavenging activity. These findings
support the hypothesis that coupling of two different phar-
macophores can produce a single hybrid molecule that
exhibits dual/multiple pharmacological activities. The most
active compounds from these series (4b, 4f, 5e, and 5f) can
be taken as lead for design and development of novel,
potent and safe anti-inflammatory and anthelmintic medic-
inal agents.
Conclusions
Two series of coumarin–benzimidazole hybrids (4 and 5)
were designed and screened for pharmacological activities
using PASS software, and structure activity relation (SAR)
is proposed as shown in Fig. 3. The compounds were
synthesized through a four-step reaction scheme. Based on
the in silico studies, compounds of series 4 were evaluated
for anti-inflammatory activity whereas those of series 5
were evaluated for anthelmintic activity. All compounds
Materials and methods
The reactions were monitored by thin layer chromatography
using silica-gel precoated aluminum plates (Merck, Ger-
many) visualized in ultraviolet (UV) chamber at short and
long wavelengths. Melting points were recorded with open
capillary method, and were uncorrected. 1H-NMR were
Fig. 2 Anthelmintic activity of compounds 5a–5f as time of death.
Values are expressed as mean SD (n = 6)
Fig. 1 Anthelmintic activity of compounds 5a–5f as time of paralysis.
Values are expressed as mean SD (n = 6)
Fig. 3 Proposed SAR of the
coumarin–benzimidazole
coupled derivatives for varied
activities

Med Chem Res
Table 4 Antioxidant activity of the test compounds
Method A A mixture of citric acid (0.02 mol) and sulfuric
acid (0.03 mol) was stirred at room temperature for 30 min.
The mixture was kept in boiling water bath to remove
carbon monoxide (CAUTION: Fume Cupboard). As soon
as the evolution of CO gas was slackened, the flask was
removed from the bath, kept aside for 15 min or till the
reaction mixture was free from CO bubbles. The contents
were cooled to 10 °C, and (un)substituted phenol (0.02 mol)
cooled to 10 °C was added drop wise. The reaction mixture
was stirred at room temperature for 48 h, and poured over
crushed ice. The precipitates were filtered and dissolved in
saturated sodium bicarbonate solution. The solution was
acidified to afford the intermediates 2a–2f.
Compounds
EC50 (µM/mL)
4a
1.75
4.42
3.98
4.13
0.17
2.24
1.02
3.01
2.80
2.42
0.08
1.80
23.4
0.03
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
Method B A mixture of citric acid (0.02 mol), sulphuric
acid (0.03 mol) and (un)substituted phenol (0.02 mol) was
heated in a microwave oven at 10% power for 4 min. The
reaction mixture was poured over crushed ice and processed
similarly as in method A to obtain the desired product.
5f
BHT
Ascorbic acid
recorded in dimethyl sulfoxide (DMSO)-d6 on Bruker
Avance II, 400 MHz NMR spectrophotometer. For 13C-
NMR spectra, the instrument was operated at 100 MHz.
Chemical shifts were reported as δ using tetramethylsilane
as internal standard with multiplicities mentioned (br,
broad; s, singlet; d, doublet; m, multiplet; dd, double
doublet), and number of protons. Coupling constants (J)
were expressed in Hz. Infrared spectra were recorded using
KBr disks on a Perkin Elmer Fx IR Spectrophotometer.
High resolution mass spectral analysis was performed on
microTOF-Q11 mass spectrometer (Bruker Daltonics
GmbH, Germany).
Albino rats (150–200 g) of either sex were used for
evaluation of anti-inflammatory activity. The animals were
housed in cages at room temperature 27 3 °C with a 12 h
light/dark cycle, and were allowed food and water ad libi-
tum. These were randomly allocated into control, standard,
and test groups. The study was approved by institutional
animal ethical committee under CPCSEA guidelines. Red
earthworms Pheretima posthuma were procured from a
vermipost, located in village Jagatpur (Chandigarh, India),
and were got authenticated from the Department of Zool-
ogy, Punjabi University, Patiala.
7-Hydroxycoumarin-4-acetic acid (2a) Yield 67%
(method A), 78% (method B); m.p. 170 °C; IR (FT-IR) vmax
(cm−1): 3210 (Ar C–H), 2924 (Al C–H), 1710 (C=O), 1701
(C=O lactone), 1458 (C=C), 1130 (C–O), 1290 (C–OH);
1H-NMR (DMSO-d6, 400 MHz): δ 11.08 (1H, s, COOH),
7.41–6.98 (2H, m, H−5, and H-6), 6.69–6.23 (2H, m H-8,
and H-3), 4.93 (1H, dd, OH), 3.02–2.89 (2H, m, CH2); 13C-
NMR (DMSO-d6, 100 MHz): δ 171.5 (C=O), 160.2 (C-2),
112.5 (C-3), 152.4 (C-4), 150.5 (C-8a), 135.2 (C-6), 124.8
(C-5), 122.9 (C-7), 120.1 (C-4a), 104.1 (C-8), 37.0 (–CH2).
HRMS (+ESI) [M + H]+: 221.0445 (theoretical), 221.0464
(found).
7-Methylcoumarin-4-acetic acid (2b) Yield 72% (method
A), 88% (method B); m.p. 190 °C; IR (FT-IR) vmax (cm−1):
3210 (Ar C–H), 2916 (Al C–H), 1710 (C=O), 1693 (C=O
lactone), 1530 (C=C), 1120 (C–O), 1280 (C–OH); 1H-
NMR (DMSO-d6, 400 MHz): δ 7.42–7.18 (3H, m, H-5, H-
6, and H-8), 6.59–6.51 (1H, m, H-3), 2.98 (2H, s, CH2),
2.44–2.41 (3H, m, CH3); 13C-NMR (DMSO-d6, 100 MHz):
δ 171.5 (C=O), 160.8 (C-2), 154.6 (8a), 143.1 (C-7), 125.7
(C-6), 125.6 (C-5), 112.5 (C-3), 118 (C-4), 117.4 (C-8), 115
(C-4), 37.0 (–CH2), 21.3 (CH3). HRMS (+ESI) [M + H]+:
219.0652 (theoretical), 219.0681 (found).
Chemistry
6-Methylcoumarin-4-acetic acid (2c) Yield 64% (method
A), 74% (method B); m.p. 192 °C; IR (FT-IR) vmax (cm−1):
3172 (Ar C–H), 2932 (Al C–H), 1705 (C=O), 1688 (C=O
lactone), 1527 (C=C), 1155 (C–O), 1230 (C–OH); 1H-
NMR (DMSO-d6, 400 MHz): δ 11.25 (1H, s, COOH),
7.46–7.22 (2H, m, H-5, and H-7), 6.92–6.21 (2H, m, H-8,
and H-3), 3.02–2.88 (2H, m, CH2), 2.39–2.34 (3H, m,
CH3); 13C-NMR (DMSO-d6, 100 MHz): δ 171.2 (C=O),
Synthesis of coumarin-4-acetic acid analogs (2a–2f)
Coumarin-4-acetic acid is a known compound (Manwar
et al. 2008). The reaction conditions reported for this
compound were modified and optimized (method A) to
synthesis the analogs 2a–2f. These analogs were also syn-
thesized by microwave assisted method (method B).

Med Chem Res
160.8 (C-2), 155.4 (C-4), 150.5 (C-8a), 127.8 (C-7), 127.6
(C-5), 125.1 (C-6), 120.9 (C-4a), 116.9 (C-8), 112.5 (C-3),
37.0 (–CH2), 21.7 (CH3). HRMS (+ESI) [M + H]+:
219.0652 (theoretical), 219.0676 (found).
solution was cooled to room temperature and made alkaline
with aqueous ammonia. Compound 3 was separated as
precipitates, which were recrystallized as beige colored
crystals from ethanol-water mixture. Yield: 88%; m.p. 234 °
C; IR (FT-IR) vmax (cm−1): 3434 (N–H), 3116 (Ar C–H),
1636 (C=N), 1483 (N–H), 1451 (C=C), 1239 (C–N), 846
(N–H), 719 (C–H); 1H-NMR (DMSO-d6, 400 MHz): δ 7.22
(2H, dd, H-7, and H-4), 6.9 (2H, dd, H-6, and H-5), 5.4
(1H, br, NH), 4.8 (2H, br, NH2); 13C-NMR (DMSO-d6, 100
MHz): δ 164.3 (C-2), 143.6 (3a′), 115.5 (C-4), 124.8 (C-5),
123.6 (C-6), 110.6 (C-7), 148.4 (C-7a′); HRMS (+ESI) [M
+ H]+: 134.0713 (theoretical), 134.0728 (found).
8-Methylcoumarin-4-acetic acid (2d) Yield 52% (method
A), 64% (method B); m.p. 182 °C; IR (FT-IR) vmax (cm−1):
3261 (Ar C–H), 2944 (Al C–H), 1712 (C=O), 1700 (C=O
lactone), 1554 (C=C), 1182 (C–O), 1224 (C–OH); 1H-
NMR (DMSO-d6, 400 MHz): δ 11.23 (1H, m, COOH),
7.53–7.42 (2H, m, H−5, and H-7), 7.26–7.09 (2H, m, H-6,
and H-3), 2.91–2.87 (2H, s, CH2), 2.42–2.31 (3H, s, CH3);
13C-NMR (DMSO-d6, 100 MHz): δ 171.5 (C=O), 160.8
(C-2), 155.0 (C-4), 150.4 (C-8a), 132.8 (C-7), 126.3 (C-8),
125.6 (C-5), 125.3 (C-6), 120.9 (C-4a), 112.5 (C-3), 37.0
(–CH2), 15.7 (CH3). HRMS (+ESI) [M + H]+: 219.0652
(theoretical), 219.0679 (found).
Synthesis of test compounds 4a–4f
A synthetic method reported by Li et al (2006) was taken as
lead to synthesize these compounds. In general, each of the
intermediates 2a–2f (0.002 mol) was refluxed with o-phe-
nylenediamine (0.002 mol) at 200–250 °C for 3–6 h in the
presence of OPA (5 mL). After completion of reaction, the
mixture was poured in to 100–150 mL of cold water, and
the compound was separated by addition of ammonia
solution, which was recrystallized from hot aqueous ethanol
mixture.
8-Nitrocoumarin-4-acetic acid (2e) Yield 72% (method
A), 81% (method B); m.p. 212 °C; IR (FT-IR) vmax (cm−1):
3215 (Ar C–H), 2921 (Al C–H), 1709 (C=O), 1689 (C=O
lactone), 1516 (C=C), 1146 (C–O), 1229 (C–OH), 1509
1
(N=O); H-NMR (DMSO-d6, 400 MHz): δ 10.86 (1H, s,
COOH), 7.92–8.40 (2H, m, H-5, and H-7), 6.35–7.52 (2H,
m, H-6, and H-3), 2.90–2.92 (2H, dd, CH2); 13C-NMR
(DMSO-d6, 100 MHz): δ 160.8 (C-2), 112.5 (C-3), 155.0
(C-4), 121.9 (C-4a), 134.7 (C-5), 126.3 (C-6), 126.5 (C-7),
142.6 (C-8), 145.9 (C-8a), 37.0 (–CH2), 171.5 (C=O);
HRMS (+ESI) [M + H]+: 250.0346 (theoretical), 250.0371
(found).
4-[(Benzimidazol-2-yl)methyl]-7-hydroxy coumarin (4a)
Yield 59%; m.p. 233 °C; IR (FT-IR) vmax (cm−1): 3192 (Ar
C–H), 2911 (Al C–H), 1705 (C=O), 1608 (C=N), 1455 (Ar
C=C), 1390 (C–OH), 1138 (C–O), 1270 (C–N), 744
1
(C–H); H-NMR (DMSO-d6, 400 MHz): δ 7.73–7.74 (2H,
m, H-4′, and H-7′), 7.42–7.36 (2H, m, H-5, and H-6),
7.06–7.04 (2H, M, H-5′, and H-6′), 7.03–6.92 (2H, m, H-2,
and H-8), 4.68–4.81 (2H, m, OH, and H-1′), (2H, s, CH2),
3.28–3.21; 13C-NMR (DMSO-d6, 100 MHz): δ 159.9 (C-2),
159.1 (C-7), 155.6 (C-4), 154.2 (C-8a), 151.9 (C-2′), 151.7
(C-7a′), 141.5 (C-3a′), 125.8 (C-5), 124.8 (C-5′), 123.6 (C-
6′), 119.1 (C-4′), 116.1 (C-4a), 115.3 (C-6), 113.4 (C-3),
110.9 (C-7′), 104.0 (C-8), 40.4 (CH2); HRMS (+ESI) [M
+ H]+: 293.0921 (theoretical), 293.0946 (found).
Coumarin-4-acetic acid (2f) Yield 63% (method A), 68%
(method B); m.p. 182 °C; IR (FT-IR) vmax (cm−1): 3155 (Ar
C–H), 2924 (Al C–H), 1710 (C=O), 1688 (C=O lactone),
1499 (C=C), 1187 (C–O), 1250 (C–OH); 1H-NMR
(DMSO-d6, 400 MHz):
δ 11.60 (1H, m, COOH),
7.65–7.49 (2H, m, H-5, and H-7), 7.32–6.39 (3H, m, H-3,
H-6, and H-8), 3.10 (2H, s, CH2); 13C-NMR (DMSO-d6,
100 MHz): δ 170.9 (C=O), 161.0 (C-2), 155.4 (C-4), 150.0
(C-8a), 137.7 (C-6), 128.5 (C-7), 128.2 (C-5), 128.1 (C-4a),
118.1 (C-8), 111.8 (C-3), 37.0 (–CH2); HRMS (+ESI) [M
+ H]+: 205.0496 (theoretical), 205.0513 (found).
4-[(Benzimidazol-2-yl) methyl]-7-methyl coumarin (4b)
Yield 64%; m.p. 229 °C; IR (FT-IR) vmax (cm−1): 3079 (Ar
C–H), 2937 (Al C–H), 1726 (C=O), 1603 (Ar C=N), 1477
(Ar C=C), 1475 (C–O), 1272 (C–N), 1567 (N–H), 736 (Ar
1
Synthesis of 2-aminobenzimidazole 3
C–H); H-NMR (DMSO-d6, 400 MHz): δ 8.14–8.19 (m,
2H, H-4′, and H-7′), 7.81–7.84 (m, 2H, H-5, and H-7),
7.51–7.62 (m, 2H, H-5′, and H-6′), 7.21–6.79 (m, 2H, H-3,
and H-8), 4.62 (s, 1H, NH), 3.34 (m, 2H, CH2), 2.26–2.29
(m, 3H, CH3); 13C-NMR (DMSO-d6, 100 MHz): δ 161.3
(C-2), 111.4 (C-3), 155.6 (C-4), 119.9 (C-4a), 125.8 (C-5),
125.3 (C-6), 141.7 (C-7), 117.0 (C-8), 152.9 (C-8a), 21.0
(CH3), 39.4 (CH2), 152.4 (C-2′), 141.9 (C-3a′) 119.1 (C-4′),
124.6 (C-5′), 123.6 (C-6′), 111.0 (C-7′), 150.0 (7a′); HRMS
The intermediate 3 was synthesized by using the method as
reported by Leonard et al (1947). Briefly, solutions of
cyanogen bromide (0.03 mol) and o-phenylenediamine
(0.02 mol), prepared separately in 25 mL of 50% aqueous
methanol, were mixed in a 250 mL conical flask and stirred
at room temperature for 24 h. Thereafter, methanol was
recovered under vacuum on water bath. The remaining

Med Chem Res
1
(+ESI) [M + H]+: 291.1128 (theoretical), 291.1139
1510 (C–O),1503 (N–H), 1278 (C–N), 810 (Ar C–H); H-
(found).
NMR (DMSO-d6, 400 MHz): δ 7.79–7.76 (2H, m, H-4′, and
H-7′), 7.64–7.43 (2H, m, H-5, and H-7), 7.27–7.26 (2H, m,
H-5′, and H-6′), 7.24–7.10 (2H, m, H-6, and H-8), 5.10 (1H,
s, NH), 3.16 (2H, s, CH2); 13C-NMR (DMSO-d6, 100
MHz): δ 160.8 (C-2), 155.4 (C-4), 153.2 (C-8a), 152.1 (C-
2′), 151.2 (C-7a′), 140.9 (C-3a′), 128.1 (C-5), 128.1 (C-7),
126.4 (C-5′), 126.3 (C-6′), 125.3 (C-6), 121.1 (C-4a), 116.1
(C-4′), 115.0 (C-8), 112.1 (C-3), 109.5 (C-7′), 39.8 (CH2);
HRMS (+ESI) [M + H]+: 277.0972 (theoretical), 277.0995
(found).
4-[(Benzimidazol-2-yl) methyl]-6-methyl coumarin (4c)
Yield 69%; m.p. 231 °C; IR (FT-IR) vmax (cm−1): 3079 (Ar
C–H), 2937 (Al C–H), 1726 (C=O), 1603 (Ar C=N), 1477
(Ar C=C), 1475 (C–O), 1272 (C–N), 1567 (N–H), 736 (Ar
1
C–H); H-NMR (DMSO-d6, 400 MHz): δ 8.19–8.14 (2H,
m, H-4′, and H-7′), 7.84–7.81 (2H, m, H-5, and H-7),
7.62–7.51 (2H, m, H-5′, and H-6′), 7.21–6.79 (2H, m, H-3,
and H-8), 4.62 (1H, s, NH), 3.34 (2H, m, CH2), 2.29–2.26
(3H, m, CH3); 13C-NMR (DMSO-d6, 100 MHz): δ 160.8
(C-2), 155.0 (C-4), 152.4 (C-2′), 150.5 (C-8a), 150.1 (C-7a
′), 140.9 (C-3a′), 135.3 (C-6), 131.7 (C-7), 126.8 (C-5),
124.2 (C-5′), 123.6 (C-6′), 121.4 (C-4a), 119.0 (C-4′), 116.0
(C-8), 112.5 (C-3), 110.3 (C-7′), 21.3 (CH3); HRMS
(+ESI) [M + H]+: 291.1128 (theoretical), 291.1147
(found).
Synthesis of test compounds 5a–5f
The reaction conditions reported by Montalbetti et al.
(2005) to synthesize similar kind of compounds were
modified and optimized to synthesize each of these test
compounds using DCC as a coupling agent. In general, a
suspension of each of the intermediate 2a–2f (0.01 mol) and
DCC (0.01 mol) in 100 mL of dried dichloromethane
(DCM) was vigorously stirred under nitrogen for 30 min. A
solution of 3 (0.01 mol) in dried DCM (30 mL) and freshly
4-[(Benzimidazol-2-yl) methyl]-8-methyl coumarin (4d)
Yield 47%; m.p. 236 °C; IR (FT-IR) vmax (cm−1): 3054 (Ar
C–H), 2918 (Al C–H), 1720 (C=O), 1621 (Ar C=N), 1452
(Ar C=C), 1488 (C–O), 1274 (C–N), 1537 (N–H), 743 (Ar
1
C–H); H-NMR (DMSO-d6, 400 MHz): δ 8.19–8.14 (2H,
distilled
pyridine
(50 mL)
along
with
4-
m, H-4′, and H-7′), 7.84–7.81 (2H, m, H-5, and H-7),
7.62–7.51 (2H, m, H-5′, and H-7′), 7.21–6.79 (2H, m, H-3,
and H-6), 4.71–4.62 (H, s, NH), 3.42–3.34 (2H, s, CH2),
2.29–2.26 (3H, s, CH3); 13C-NMR (DMSO-d6, 100 MHz):
δ 160.8 (C-2), 155.0 (C-4), 150.4 (C-8a), 139.9 (C-7a′),
141.5 (C-2′), 138.9 (C-3a′), 132.8 (C-7), 126.3 (C-8), 125.6
(C-5), 125.3 (C-6), 123.0 (C-5′), 123.0 (C-6′), 120.9 (C-4a),
115.2 (C-4′), 115.2 (C-7′), 112.5 (C-3), 40.0 (CH2), 15.7
(CH3); HRMS (+ESI) [M + H]+: 291.1128 (theoretical),
291.1142 (found).
dimethylaminopyridine (DMAP) (4 × 10–4 mol) was
added to the stirred reaction mixture. The contents were
maintained at 0 °C for 15 min, than stirred at 0 °C for 2 h
followed by stirring at room temperature for 12 h. Dicy-
clohexylurea (DCU) was filtered, and the filtrate was dried
in vacuum to yield solid residue. The latter was mixed and
stirred with dried ethyl acetate with heating on water bath.
The ethyl acetate soluble portion was filtered, washed with
distilled water, dried over magnesium sulfate, and evapo-
rated under vacuum. The resulting crude solid was recrys-
tallized from methanol to yield the test compound.
4-[(Benzimidazol-2-yl) methyl]-8-nitro coumarin (4e)
Yield 76%; m.p. 239 °C; IR (FT-IR) vmax (cm−1): 2997 (Ar
C–H), 2885 (Al C–H), 1761 (C=O), 1640 (Ar C=N), 1508
(C–O), 1509 (C–N=O), 1450 (Ar C=C), 1296 (C–N), 812
4-[(Benzimidazol-2-yl) amino carbonyl methyl] 7-hydroxy
coumarin (5a) Yield 77%; m.p. 219 °C; IR (FT-IR) vmax
(cm−1): 3326 (N–H), 3036 (Ar C–H), 2929 (Al C–H), 1650
(C=O), 1462 (C–O), 1436 (Ar C=C), 1572 (Ar C–N=O),
1
(Ar C–H); H-NMR (DMSO-d6, 400 MHz): δ 8.44–8.03
(2H, m, H-4′, and H-7′); 7.98–7.75 (2H, m, H-5, and H-7),
7.56–7.01 (2H, m, H-5′, and H-6′), 7.02–6.78 (2H, m, H-3,
and H-6), 3.42 (2H, m, CH2); 13C-NMR (DMSO-d6, 100
MHz): δ 160.8 (C-2), 155.0 (C-4), 145.9 (C-8a), 142.6 (C-
8), 141.5 (C-2′), 138.9 (C-7a′), 138.9 (C-3a′), 134.7 (C-5),
126.5 (C-7), 126.3 (C-6), 123.0 (C-5′), 123.0 (C-6′), 121.9
(C-4a), 115.2 (C-4′), 115.2 (C-7′), 112.5 (C-3), 40.0 (CH2);
HRMS (+ESI) [M + H]+: 322.0822 (theoretical), 322.0849
(found).
1
1650 (CO–NH), 1219 (C–OH), 740 (Ar C–H); H-NMR
(DMSO-d6, 400 MHz): δ 7.44–7.42 (2H, m, H-4′, and H-
7′), 7.32–7.28 (2H, m, H-5, and H-6), 7.10–7.09 (2H, m, H-
5′, and H-6′), 7.09–7.00 (2H, m, H-3, and H-8), 4.84 (2H, s,
NH, and OH), 2.82–2.95 (2H, m, CH2); 13C-NMR (DMSO-
d6, 100 MHz): δ 164.0 (C=O), 160.8 (C-2), 158.1 (C-7),
155.0 (C-4), 154.7 (C-8a), 146.7 (C-2′), 136.6 (C-3a′),
126.2 (C-5), 123.0 (C-5′), 113.6 (C-4a), 112.6 (C-6), 112.5
(C-3), 115.2 (C-4′), 115.2 (C-6′), 102.0 (C-8), 43.1 (CH2);
HRMS (+ESI) [M + H]+: 336.0979 (theoretical), 336.0994
(found).
4-[(Benzimidazol-2-yl) methyl] coumarin (4f) Yield 77%;
m.p. 219 °C; IR (FT-IR) vmax (cm−1): 2991 (Ar C–H), 2845
(Al C–H), 1766 (C=O), 1642 (Ar C=N), 1458 (Ar C=C),

Med Chem Res
4-[(Benzimidazol-2-yl) amino carbonyl methyl]-7-methyl
coumarin (5b) Yield 55%; m.p. 287 °C; IR (FT-IR) vmax
(cm−1): 3327 (N–H), 3056 (Ar C–H), 2853 (Al C–H), 1726
(C=O), 1625 (CO–NH), 1462 (C–O), 1448 (Ar C=C), 787
d6, 400 MHz): δ 8.20–7.85 (2H, m, H-5, and H-7),
7.65–7.40 (2H, m, H-4′, and H-7′), 7.34–7.12 (3H, m, H-6,
H-6′ and H-5′), 6.99–6.89 (1H, m, H-3), 5.13 (1H, s, NH),
2.01 (2H, m, CH2); 13C-NMR (DMSO-d6, 100 MHz): δ
164.0 (C=O), 160.8 (C-2), 155 (C-4), 146.7 (C-2′), 145.9
(C-8a), 142.6 (C-8), 136.6 (C-3a′), 136.6 (C-7a′), 134.7 (C-
5), 126.5 (C-7), 126.3 (C-6), 123.4 (C-5′), 123.3 (C-6′),
121.9 (C-4a), 115.2 (C-4′), 115.2 (C-7′), 112.5 (C-3), 43.1
(CH2); HRMS (+ESI) [M + H]+: 365.0881 (theoretical),
365.0902 (found).
1
(Ar C–H); H-NMR (DMSO-d6, 400 MHz): δ 7.94–7.89
(2H, m, H-7′, and H-4′), 7.79–7.76 (2H, m, H-6′, and H-5′),
7.09–7.00 (2H, m, H-3, and H-8), 7.07–7.05 (2H, m, H-5,
and H-6), 4.69–4.57 (1H, m, NH), 2.89 (2H, m, CH2), 2.24
(3H, m, CH3); 13C-NMR (DMSO-d6, 100 MHz): δ 165.4
(C=O), 160.6 (C-2), 154.9 (C-4), 153.8 (C-8a), 152.0 (C-
2′), 149.0 (C-3a′), 143.8 (C-7a′), 143.8 (C-7), 112.7 (C-3),
118.1 (C-4a), 125.3 (C-6), 125.1 (C-5), 124.4 (C-6′), 122.4
(C-5′), 117.2 (C-8), 115.6 (C-7′), 110.5 (C-4′), 42.9 (CH2),
21.3 (CH3); HRMS (+ESI) [M + H]+: 334.1186 (theore-
tical), 334.1203 (found).
4-[(Benzimidazol-2-yl) amino carbonyl methyl] coumarin
(5f) Yield 66%; m.p. 288 °C; IR (FT-IR) vmax (cm−1):
3300–3500 (N–H), 3038 (Ar C–H), 2933 (Al C–H),1682
(C=O), 1655 (CO–NH),1457 (Ar C=C), 843 (Ar C–H);
1H-NMR (DMSO-d6, 400 MHz): δ 8.12 (1H, s, NH),
7.88–7.78 (2H, m, H-4′, and H-7′), 7.55–7.45 (2H, m, H-5,
and H-7), 7.30–7.28 (2H, m, H-6, and H-8), 7.18–7.14 (2H,
m, H-5′, and H-6′), 2.95–2.81 (2H, s, CH2); 13C-NMR
(DMSO-d6, 100 MHz): δ 164.0 (C=O), 160.8 (C-2), 155
(C-4), 153.5 (C-8a), 146.7 (C-2′), 136.6 (C-3a′), 136.6 (C-
7a′), 128.6 (C-5), 128.3 (C-7), 125.4 (C-6), 123 (C-5′), 123
(C-6′), 121 (C-4a), 116.3 (C-8), 115.2 (C-4′), 115.2 (C-7′),
112.5 (C-3), 43.1 (CH2); HRMS (+ESI) [M + H]+:
320.1030 (theoretical), 320.1052 (found).
4-[(Benzimidazol-2-yl) amino carbonyl methyl]-6-methyl
coumarin (5c) Yield 64%; m.p. 290 °C; IR (FT-IR) vmax
(cm−1): 3327 (N–H), 3056 (Ar C–H), 2853 (Al C–H), 1726
(C=O), 1462 (C–O), 1448 (Ar C=C), 1672 (CO–NH), 742
1
(Ar C–H); H-NMR (DMSO-d6, 400 MHz): δ 7.84 (1H, s,
NH), 7.56–7.49 (2H, m, H-7′, and H-4′), 7.47–7.32 (2H, m,
H-5′, and H-6′), 7.19–7.10 (2H, m, H-5, and H-7),
7.05–6.95 (2H, m, H-3, and H-8), 4.96 (1H, s, NH), 2.89
(2H, s, CH2) 2.39–2.34 (3H, m, CH3); 13C-NMR (DMSO-
d6, 100 MHz): δ 165.1 (C=O), 161.0 (C-2), 154.9 (C-4),
153.8 (C-8a), 152.1 (C-2′), 148.9 (C-3a′), 143.2 (C-7a′),
135.3 (C-6), 131.8 (C-7), 127.1 (C-5), 124.3 (C-6′), 123.4
(C-5′), 120.1 (C-4a), 116.0 (C-8), 115.5 (C-7′), 112.1 (C-3),
110.1 (C-4′), 42.8 (CH2), 21.9 (CH3); HRMS (+ESI) [M +
H]+: 334.1186 (theoretical), 334.1211 (found).
In-silico studies
PASS predicted activity spectrum of test compounds
4-[(Benzimidazol-2-yl) amino carbonyl methyl]-6-methyl
coumarin (5d) Yield 39%; m.p. 282 °C; IR (FT-IR) vmax
(cm−1): 3300–3500 (N–H), 3038 (Ar C–H), 2933 (Al
C–H),1682 (C=O), 1655 (CO–NH), 1457 (Ar C=C), 843
The biological activity of each test compound was predicted
by the computer software PASS, which predicts the activity
spectrum of a compound as probable activity (Pa) and
probable inactivity (Pi) with accuracy of prediction as high
as 85%. The activities having Pa of 0.5–0.7 for a compound
are considered probable activities of the compound (Por-
oikov et al. 2000).
1
(Ar C–H); H-NMR (DMSO-d6, 400 MHz): δ 7.84–7.70
(m, 2H, H-4′, and H-7′), 7.51–7.43 (m, 2H, H-5′, and H-6′),
7.41–7.23 (m, 2H, H-5, and H-7), 7.11–6.93 (m, 2H, H-3,
and H-6), 4.73 (s, 1H, NH), 2.50 (s, 2H, CH2), 2.36–2.20
(m, 3H, CH3); 13C-NMR (DMSO-d6, 100 MHz): δ 164
(C=O), 161.0 (C-2), 155 (C-4), 146.7 (C-2′), 145.9 (C-8a),
142.6 (C-8), 136.6 (C-7a′), 136.6 (C-3a′), 134.7 (C-5),
126.5 (C-7), 126.3 (C-6), 123 (C-5′), 123 (C-6′), 121.9 (C-
4a), 115.2 (C-4′), 115.2 (C-7′), 112.1 (C-3), 43.1 (CH2);
HRMS (+ESI) [M + H]+: 334.1186 (theoretical), 334.1211
(found).
Molecular property calculations
Molecular properties such as lipophilicity (Log P), total
polar surface area (TPSA), molecular weight (MW), hHy-
drogen bond acceptors (nON), hydrogen bond donors
(nOHNH), number of violations (nviol), and number of
rotatable bonds (nrotb) were calculated using molinspiration
calculations software. These properties helped in prediction
of intestinal absorption, blood brain barrier permeability,
and oral bioavailability according to Lipinski’s rule of 5
(Lipinski et al. 1997).
4-[(Benzimidazol-2-yl) amino carbonyl methyl]-8-nitro-
coumarin (5e) Yield 47%; m.p. 285 °C; IR (FT-IR) vmax
(cm−1): 3300–3500 (N–H), 3060 (Ar C–H), 2922 (Al
C–H), 1713 (C=O), 1654 (CO–NH), 1512 (N=O), 1490
1
(C–O), 1419 (Ar C=C), 743 (Ar C–H); H-NMR (DMSO-

Med Chem Res
Biological assays
[(AbsControl−AbsTest)/AbsControl] × 100. Different con-
centrations of each compound and standard were used in
order to obtain calibration curves. Antioxidant activity of
each compound and standard drug was evaluated in tripli-
cate and EC50 values were reported as mean SD.
Anti-inflammatory activity
Anti-inflammatory activity of test compounds of series 4
was evaluated by carrageenan-induced rat paw edema
method as reported by Winter et al. (1963). Edema was
induced by sub-plantar injection of 0.1 mL of 1% carra-
geenan solution. The test compounds were administered i.p.
as suspension in 0.5% sodium carboxy methyl cellulose
(SCMC) 30 min prior to injection of carrageenan, at a dose
equimolar to the standard drug. Indomethacin at a dose of
20 mg/kg was used as standard. SCMC (0.5%) was used as
control. Volume of the paw was measured at 0, 0.5, 1, 2, 3,
4, and 6 h intervals (Fereidoni et al. 2000). Anti-
inflammatory activity was calculated as percent inhibition
of carrageenan induced paw edema using the following
formula (Chu and Kovacs 1977).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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It was evaluated for each test compound of series 5 using
the method as described by Ajaiyeoba et al. (2001). The
worms were divided into different groups with six earth-
worms in each group, and washed with normal saline. The
target compounds were evaluated at different concentrations
(0.1, 0.2, 0.3, and 0.5% w/v) taking albendazole as standard
drug. The helminthes were placed in a solution of each
compound at each concentration in Petri plates. The time
taken for worms to become motionless was noted as
paralysis time, whereas the time taken for death of the worm
was noted as lethal time. To ascertain death, each worm was
frequently subjected to external stimuli that stimulate and
induce movement in the worms, if alive. The mean paralysis
time and mean lethal time were calculated for each com-
pound at each concentration.
Antioxidant activity
It was evaluated as hydrogen donating or radical scavenging
ability of the compound using DPPH method taking BHT
and ascorbic acid as standard drugs (Locatelli et al. 2009).
Briefly, a 700 µl solution of each test compound in metha-
nol was mixed with the same volume of DPPH solution
(100 µM in methanol). Mixture was shaken vigorously,
allowed to stand in dark at room temperature for 30 min and
absorbance was noted at 515 nm. For control reading, the
test compound solution was replaced with methanol, and for
standard reading, it was replaced with solutions of BHT or
ascorbic acid. Antiradical activity was expressed as percent
inhibition (I%) and calculated using the following equation:
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