Synthesis and exploration of QSAR model of 2-methyl-3-[2-(2-methylprop-1- en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one as potential antibacterial agents

Article abstract of DOI:10.3109/14756366.2011.587814

Present communication deals with the synthesis of novel 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a] benzimidazol-4(3H)-one derivatives under phase transfer catalysis (PTC) conditions using benzyl triethyl ammonium chloride (BTEAC) as PTC. It also elicits the studies on in vitro antimicrobial evaluation of synthesized compounds against a representative genera of gram-negative and gram-positive bacteria i.e., Bacillus subtilis, Staphylococcus aureus, Pseudomonas diminuta and Escherichia coli. All the compounds have been found to manifest profound antimicrobial activity. Moreover, extensive quantitative structure-activity relationship (QSAR) studies have been performed to deduce a correlation between molecular descriptors under consideration and the elicited biological activity. A tri-parametric QSAR model has been generated upon rigorous statistical treatment.

Full text of DOI:10.3109/14756366.2011.587814

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 294–301
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.587814
RESEARCH ARTICLE
Synthesis and exploration of QSAR model of 2-methyl-3-[2-(2-
methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]
benzimidazol-4(3H)-one as potential antibacterial agents
Pratibha Sharma1, Ashok Kumar1, Manisha Sharma1, Jitendra Singh1, Prabal Bandyopadhyay1,
Manisha Sathe2, and M.P. Kaushik2
1School of Chemical Sciences, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore, India and 2Defence
Research and Development Establishment, Jhansi Road, Gwalior, India
Abstract
Present communication deals with the synthesis of novel 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-
1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one derivatives under phase transfer catalysis (PTC) conditions using
benzyl triethyl ammonium chloride (BTEAC) as PTC. It also elicits the studies on in vitro antimicrobial evaluation of
synthesized compounds against a representative genera of gram-negative and gram-positive bacteria i.e., Bacillus
subtilis, Staphylococcus aureus, Pseudomonas diminuta and Escherichia coli. All the compounds have been found to
manifest profound antimicrobial activity. Moreover, extensive quantitative structure-activity relationship (QSAR)
studies have been performed to deduce a correlation between molecular descriptors under consideration and the
elicited biological activity. A tri-parametric QSAR model has been generated upon rigorous statistical treatment.
Keywords: Phase transfer catalysis, benzimidazole, quantitative structure-activity relationship (QSAR),
antibacterial activity
Introduction
astemizole, esomeprazole, albendazole, mebendazole,
e fight against bacterial infection is one of the great
success challenges of medicinal chemistry. Multiple
drug-resistant micro-organisms1,2 are becoming com-
mon causes of infections in the acute and long-term
care. e emergence of these resistant microorganisms
has created a major concern and there exists an urgent
need of antibacterial agents in structural classes distinct
from known antibacterial agents. A systematic perusal
of literature reveals that benzimidazole skeleton has
gained a reputation as a drug of privileged sub-structure
fenbendazole, oxfendazole, oxibendazole, albenda-
zolesulfonamide, thiabendazole, thiophanate, feb-
antel, nelotrimin, and triclabendazole, carbendazim,
fuberidazole7–12. Nowadays, in view of the affinity dis-
played by them towards a variety of enzymes and pro-
tein receptors, compounds containing benzimidazole
sub-structure are being recognized as a drug of choice in
the current drug design scenario.
e advent of high throughput screening technolo-
gies has impacted significantly on the methodologies
that are used for the synthesis of a number of medicinal
compounds. e implementation in the laboratory of
these synthetic technologies to increase the number of
molecules generated by chemists is now a pre-requisite
to competitive advantage in the field. Several publica-
tions describing synthesis of benzimidazole in solu-
tion phase and on solid supports have appeared in the
owing to its extensive use in medicinal chemistry3–6
.
Interest in benzimidazole containing structures stems
from their widespread occurrence in molecules that
display a plethora of useful biological properties. ey
are characterized by a broad spectrum of activity against
roundworms (nematodes), an ovicidal effect, and a
wide safety margin. Current clinical example includes
Address for Correspondence: Dr. Pratibha Sharma, PhD, School of Chemical Sciences, Devi Ahilya University, Takshashila Campus,
Khandwa Road, Indore-452001 (M.P.), India. Tel.: +91-731-2460208; Fax: +91-731-2470352. E-mail: drpratibhasharma@yahoo.com
(Received 13 January 2011; revised 08 May 2011; accepted 10 May 2011)
294

Synthesis and exploration of QSAR model 295
literature13–17. us, keeping in view the ever-increasing
demand of new antibiotic agents, it was thought of
our interest to design the synthesis of a novel series of
benzimidazoles i.e., 2-methyl-3-[2-(2-methylprop-1-
en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]ben-
zimidazol-4(3H)-one (Figure 1) under phase transfer
catalysis (PTC) conditions.
Moreover, in order to explore the medicinal potential
of these synthesized compounds, comprehensive QSAR
studies have been performed to establish a correlation
between the various physicochemical parameters and
the elicited antibacterial behaviour.
ethyl 3-oxobutanoate as the substrates. In this strategy,
1H-benzimidazol-2-amine (1), obtained by the reaction
of 1,2-phenylenediamine dihydrochloride with cyano-
gen bromide, was treated with substituted azo-coupled
products i.e., ethyl 3-oxo-2-(phenyldiazenyl)butano-
ate (2a–r). Furthermore, the obtained precursor com-
pound 2-methyl-3-(phenyldiazenyl) pyrimido[1,2-a]
benzimidazol-4(3H)-one (3a–r) was then treated with
3-methylbut-1,2-dien-1-ylidene, generated in situ as per
Scheme 1, under PTC conditions to obtain 2-methyl-
3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1
-yl]pyrimido[1,2-a]benzimidazole-4(3H)-one
(4a–r)
Systematic perusal of literature reveals that a num-
ber of methods have been reported for the synthesis
of different benzimidazole derivatives under variable
experimental conditions18–22. However, most of the
existing methods to design benzimidazole skeleton
requires the insertion of a carbon into a precursor with
ortho heteroatoms on a benzene ring. Moreover, most
of the methods have not been found to be quite acces-
sible from the viewpoints of both yield and economics
of the reaction. us, in order to cater the needs asso-
ciated with synthetic aspects, herein, we would like to
present unique approach to synthesize benzimidazole
derivatives under PTC conditions considering inser-
tion-cyclisation sequence onto azo functionality of the
precursor.
(Figure 1) as the final product (Scheme 2).
From the viewpoint of mechanistic consider-
ations, the formation of 4a–r could be rationalized
by an initial trap of dimethylvinylidene carbene by
2-methyl-3-(phenyldiazenyl)pyrimido[1,2-a] benzim-
idazol-4(3H)-one at−N=N- to form three membered
ring, which is thermally labile compared with the car-
bocyclic analogue because of much smaller bond dis-
sociation energy23 of N–N bond than C–C bond. Hence,
it is rearranged to 2-methyl-3-[2-(2-methylprop-1-en-1
-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimida-
zole-4(3H)-one (4a–r).
Materials and methods
us an important preparative methodology
for the synthesis of 2-methyl-3-[2-(2-methylprop-
1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]
benzimidazol-4(3H)-one (4a–r) has been delineated
incorporating substituted aniline derivatives, urea and
All the chemicals and other reagents used were of AR
grade purity. All the synthesized compounds were
characterized by IR, 1H-NMR, 13C NMR, FAB-Mass
spectroscopy and elemental analyses. Melting points
were determined on an electro-thermal apparatus by
open capillary method and are uncorrected. e NMR
spectra were recorded on Bruker DRX300 instrument
(300 MHz) in CDCl3 using TMS as an internal standard.
Chemical shifts are reported in δ (ppm) values. All IR
spectra were run on Shimadzu 460 spectrophotometer
in KBr Discs; frequencies are reported in cm−1. FAB mass
spectra (FAB-MS) were recorded on a JEOL SX 102 Mass
Spectrometer. To ascertain the purity of all the synthe-
sized compounds, analytical thin layer chromatography
was performed on (E Merck silica gel G0.50mm plate no.
5700) using acetonitrile, methanol, and water (50:30:20,
v/v) as eluting system. Elemental analyses were per-
formed on Elementar Vario EL-III Carlo-Erba-1108
equipment.
Synthesis of 1H-benzimidazol-2-amine (124)
A solution of 1,2-phenylenediamine dihydrochloride
(0.45 g, 2.5 mmol) in 5 mL of water was cooled to 0°C and
Figure 1. Structure of synthesized 2-methyl-3-[2-(2-methylprop-
1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-
4(3H)-one (4a–r).
Scheme 1. Generation of 3-methylbuta-1,2-dien-1-ylidene (dimethylvinylidene carbene) intermediate.
© 2012 Informa UK, Ltd.

296 P. Sharma et al.
Scheme 2. An overview of synthesis of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-
4(3H)-one (4a–r).
treated with a solution of cyanogen bromide (0.60 mL,
5 M in acetonitrile, 3.0 mmol) and solid NaHCO3 (0.41 mg,
4.9 mmol). e solution was stirred at ambient tempera-
ture for 40–45 h. e mixture was made basic with 1 M
aqueous Na2CO3 and the solution was concentrated
under reduced pressure. e residue was triturated
with hot ethanol, and the ethanolic solution was filtered
and concentrated under reduced pressure to obtain the
compound 1 in appreciable yield.
1H-benzimidazol-2-amine (1): Yield 85%; mp 135–
136°C; Anal. Calcd. for C7H7N3 (R=H): C, 63.14; H, 5.30; N,
31.56%; Found: C, 63.10; H, 5.28; N, 31.53%; IR (υ cm−1):
3045 (C-H, sp2), 3210 (NH, bonded), 3175 (NH, free),
1654 (C=N), 1626, 1586, 1444 (C…C, ring str) 958, 859,
1
742 (sub. phenyl); H-NMR (300 MHz, CDCl3) δ (ppm):
4.0 (s, 2H, NH2), 5.0 (s, NH), 7.6–7.9 (m, 4H, Ar-H); 13C
NMR (CDCl3) δ (ppm): 117.41, 124.34, 136.66, 158.62;
FAB-MS: 134 (M+H)+.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and exploration of QSAR model 297
50% of aqueous potassium hydroxide (15 mL), benzyl-
triethylammonium chloride (BTEAC) (0.57 g, 2.5 mmol)
and benzene (5 mL) was taken and stirred thoroughly for
30 min. To this, a pertinent 2-methyl-3-(phenyldiazenyl)
pyrimido[1,2-a] benzimidazol-4(3H)-one (3a–r) (2.5
mmol) was added slowly and stirred for further 5–7h
under nitrogen atmosphere. While stirring was going
on, 3-chloro-3-methyl-1-butyne (25 mmol) (Scheme 1)
in benzene (5 mL) was added slowly to the mixture. e
contents were diluted with water (120mL), followed
by extraction with ether (120 mL) to afford crude prod-
uct. It was purified on an alumina column (benzene as
eluent) so as to finally obtain 2-methyl-3-[2-(2-methyl-
prop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]
benzimidazol-4(3H)-one (4a–r). eir purity was further
ascertained upon performing TLC resolution on E Merck
silica gel-G plates using acetonitrile, methanol, and water
(50:30:20, v/v) as eluting system. An overview of all the
synthetic steps is depicted in Scheme 2.
General procedure for synthesis of ethyl 3-oxo-2-[(E)-
phenyldiazenyl]butanoate (2a-r25)
Different substituted coupled products were synthesized
as the precursors taking ethyl 3-oxobutanoate as the
active methylene compound. Pertinent aniline deriva-
tives (0.01 M) were dissolved in 4 mL HCl and 4 mL dis-
tilled water and kept at freezing temperature. To this, an
aqueoussolutionofsodiumnitrite(0.69 g, 0.01 M)in5 mL
of distilled water was added dropwise with continuous
stirring, keeping the temperature in the vicinity of 0–5°C.
Meanwhile, in another beaker ethyl 3-oxobutanoate
(1 mL), sodium acetate (7.0 g) and ethyl alcohol (25 mL)
were taken and cooled in an ice bath. Now the diazotized
solution (Scheme 2) was added to this solution dropwise
with thorough stirring under temperature-controlled
conditions. e reaction mixture was kept for overnight
period, filtered through suction, washed well with water
and dried in vacuum. Fine yellow crystals of ethyl 3-oxo-
2-[(E)-phenyldiazenyl]butanoate (2a–r) were obtained.
Ethyl 3-oxo-2-[(E)-phenyldiazenyl]butanoate (2a):
Yield 82%; mp 145–146°C; Anal. Calcd. for C12H14N2O3:
C, 62.11; H, 5.76; N, 19.06%; Found: C, 62.08; H, 5.71;
N, 19.01%; IR (υ cm−1): 3050 (C-H, sp2), 2945 (C-H, sp3),
2010 (N=N), 1750 (C=O, ester), 1618, 1580, 1449 (C…C,
ring str), 1118 (C-O), 958, 859, 742 (sub. phenyl); 1H-NMR
(300 MHz, CDCl3) δ (ppm): 1.1 (t, 3H, CH2CH3, J= 6.5
Hz), 2.0 (s, 3H, CH3), 5.4 (q, 2H, CH2CH3, J= 6.5 Hz), 6.1
(s, CH), 7.2-7.6 (m, 5H, Ar-H); 13C NMR (CDCl3) δ (ppm):
21.24, 27.42, 86.14, 122.62, 125.10, 129.25, 150.20, 172.02;
FAB-MS: 235 (M+H)+.
2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benz-
imidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one
(4a): Yield 76%; mp 122–123°C; Anal. Calcd. for C22H19N5O:
C, 71.53; H, 5.18; N, 18.96%; Found: C, 71.50; H, 5.15; N,
18.96%; IR (υ cm−1): 3085 (C-H, sp2), 2952 (C-H, sp3),
1720 (C=O), 1631 (C=C/C=N), 1620, 1522, 1440 (C…C,
1
ring str), 952, 853, 741 (sub. phenyl); H-NMR (300 MHz,
CDCl3) δ (ppm): 1.85 (s, 6H, 2 × CH3, isopropenyl), 2.02 (s,
3H, CH3), 4.54 (s, CH, pyrimidine), 5.13 (s, CH, methine),
7.27 (m, 4H, H11, H12, H21, H22), 7.66 (m, 4H, H10, H13,
H20, H23); 13C NMR (CDCl3) δ (ppm): 17.44 (C24), 20.25
(C28), 24.27 (C27), 65.82 (C3), 110.02, 114.12, 115.28,
123.02, 123.26, 130.74, 134.28, 138.91, 139.24 (aromatic
ring), 141.64 (C6, C16), 144.66 (C26), 165.16 (C4), 169.57
(C2); FAB-MS: 370 (M+H)+.
General procedure for synthesis of 2-methyl-3-
(phenyldiazenyl)pyrimido[1,2-a]benzimidazol-4(3H)-
one (3a–r)
2-methyl-3-[4-Chloro-2-(2-methylprop-1-en-1-yl)-1-
H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4-
(3H)-one (4f): Yield 79%; mp 126–127°C; Anal. Calcd.
for C22H18N5 ClO: C, 65.43; H, 4.49; N, 17.34%; Found: C,
65.40; H, 4.46; N, 17.31%; IR (υ cm−1): 3052 (C-H, sp2),
2950 (C-H, sp3), 1725 (C=O), 1621 (C=C/C=N), 1605,
1550, 1440 (C—C, ring str), 952, 840, 755 (sub.phenyl),
An equimolar (0.01 M) quantity of 1H-benzimidazol-
2-amine (1) and ethyl 3-oxo-2-[(E)-phenyldiazenyl]
butanoate (2a–r) was taken in a 250 cc RB flask and 10 mL
freshly prepared sodium ethoxide was added to this. e
mixture was heated under reflux condition for 30 min.
e solid product thus formed was cooled, dehydrated
by anhydrous sodium sulphate, filtered and recrystal-
lized from ethanol. e obtained yield was 70–86%.
2-methyl-3-(phenyldiazenyl) pyrimido[1,2-a] benzim-
idazol-4(3H)-one (3a): Yield 78%; mp 163–164°C; Anal.
Calcd. for C17H13N5O: C, 67.32; H, 4.32; N, 32.09%; Found:
C, 67.26; H, 4.28; N, 23.05%; IR (υ cm−1): 3055 (C-H, sp2),
2940 (C-H, sp3), 2051 (N=N), 1621 (C=C/C=N), 1620, 1532,
1445 (C…C, ring str), 955, 850, 745 (sub. phenyl); 1H-NMR
(300MHz, CDCl3) δ (ppm): 2.4 (s, 3H, CH3), 5.6 (s, CH),
7.30 -7.61 (m, 9H, Ar-H); 13C NMR (CDCl3) δ (ppm): 20.20,
70.62,114.20,115.24,122.64,123.25,125.20,130.18,137.02,
139.56, 141.54, 159.22, 165.50; FAB-MS: 304 (M+H)+.
1
6.3 (C-Cl); H-NMR (300 MHz, CDCl3) δ (ppm): 1.82 (s,
6H, 2 × CH3, isopropenyl), 1.99 (s, 3H, CH3), 4.62 (s, CH,
pyrimidine), 5.82 (s, CH, methine), 7.18 (d, 1H, H21),
7.24 (t, 1H, H22), 7.24 (d, 2H, H11, H12), 7.62 (d, 2H, H10,
H13), 7.48 (d, 1H, H23); 13C NMR (CDCl3) δ (ppm): 17.46
(C24), 19.22 (C28), 25.29 (C27), 65.34 (C3), 113.35, 114.13,
115.28, 120.74, 123.07, 124.12, 124.49, 130.75, 138.32,
138.90, 140.37 (aromatic ring), 141.50 (C6, C16), 143.52
(C26), 164.74 (C4), 167.88 (C2); FAB-MS: 405 (M+H)+.
2-methyl-3-[4-methyl-2-(2-methylprop-1-en-1-yl)-1-
H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4-
(3H)-one (4i): Yield 84%; mp 122–123°C; Anal. Calcd.
for C23H21N5O: C, 72.04; H, 5.52; N, 18.26%; Found: C,
72.00; H, 5.49; N, 18.22%; IR (υ cm−1): 3062 (C-H, sp2),
2971 (C-H, sp3), 1726 (C=O), 1629 (C=C/C=N), 1612,
1525, 1437 (C…C, ring str), 954, 849, 743 (sub. phenyl);
1H-NMR (300 MHz, CDCl3) δ (ppm): 1.80 (s, 6H, 2 × CH3,
General procedure for synthesis of 2-methyl-3-[2-
(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]
pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r26)
In a 100 mL three-necked bolt-head flask, fitted with a
dropping funnel and a mechanical stirrer, a mixture of
© 2012 Informa UK, Ltd.

298 P. Sharma et al.
isopropenyl), 1.95 (s, 3H, CH3), 2.12 (s, 3H, ArCH3), 4.52 (s,
CH, pyrimidine), 5.75 (s, CH, methine), 7.17 (t, 1H, H22),
7.44 (d, 1H, H23), 7.56 (d, 1H, H21), 7.24 (d, 2H, H11,
H12), 7.68 (d, 2H, H10, H13); 13C NMR (CDCl3) δ (ppm):
17.02 (Ar-CH3), 18.06 (C24), 20.12 (C28), 24.89 (C27),
66.14 (C3), 112.25, 114.13, 115.22, 122.95, 123.04, 124.18,
126.24, 130.77, 138.82, 139.08, 140.37 (aromatic ring),
142.33 (C6, C16), 144.11 (C26), 165.64 (C4), 168.28(C2);
FAB-MS, 384 (M+H)+.
In vitro antibacterial assay
Broth micro-dilution assay
e newly synthesized compounds were screened
for their antibacterial activity against Pseudomonas
diminuta, Escherichia coli, Bacillus subtilis and
Staphylococcus aureus using broth micro-dilution
method. Approximately 109 cells of respective bacterial
cells were inoculated in 10 mL Mueller-Hinton broth.
Each synthesized compound (200 µg) was added and
mixed well by shaking at 200rpm and the plates are incu-
bated at 37°C for 48–72 h. After every 6-h interval, 1 mL of
cell culture were withdrawn and subjected to serial dilu-
tion (10−1–10−6) and spread on nutrient agar plates and
the plates were incubated at 37°C for 24 h. e bacterial
colonies were counted by colony form unit (CFU) in each
dilution to find out the antibacterial activity of the tested
compounds.
e MIC (µg/mL) of a compound was determined
according to the lowest concentration that inhibited vis-
ible growth of bacteria after incubation at 37°C for 24 h.
IC50 (µg/mL) represents that inhibitory concentration of
the compound, at which the 50% inhibition of the growth
of bacteria occurred in vitro. IC50 is also converted to the
pIC50 scale (−log IC50), in which higher values indicate
exponentially greater potency. Mueller-Hinton broth was
used as the test medium. Each experiment in the anti-
bacterial assays was replicated twice in order to define
the MIC and IC50 values. Ampicillin was used as standard
antibacterial agent.
2-methyl-3-[4-hydroxy-2-(2-methylprop-1-en-1-yl)-
1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4-
(3H)-one (4j): Yield 83%; mp 120–121°C; Anal. Calcd. for
C22H19N5O2: C, 68.56; H, 4.97; N, 18.17%; Found: C, 68.53;
H, 4.95; N, 18.15%; IR (υ cm−1): 3345 (O-H), 3056 (C-H,
sp2), 2925 (C-H, sp3), 1718 (C=O), 1620 (C=C/ C=N), 1595,
1521, 1450 (C…C, ring str), 950, 852, 744 (sub. phenyl);
1H-NMR (300 MHz, CDCl3) δ (ppm): 1.85 (s, 6H, 2 × CH3,
isopropenyl), 1.97 (s, 3H, CH3), 4.67 (s, CH, pyrimidine),
5.78 (s, CH, methine), 6.25 (s, 1H, OH), 6.95(d,1H, H21),
7.08 (t, 1H, H22), 7.15 (d, 1H, H23), 7.29 (d, 2H, H11, H12),
7.68 (d, 2H, H10, H13). 13C NMR (CDCl3) δ (ppm): 17.48
(C24), 19.22 (C28), 25.25 (C27), 66.18 (C3), 105.62, 107.84,
121.82, 123.05, 123.18, 114.15, 115.28, 130.74, 138.91,
140.39, 145.08 (aromatic ring), 141.58 (C6, C16), 143.53
(C26), 164.70 (C4), 168.22 (C2); FAB-MS: 386 (M+H)+.
2-methyl-3-[6-methyl-2-(2-methylprop-1-en-1-yl)-1-
H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4-
(3H)-one (4k): Yield 76%; mp 121–22°C; Anal. Calcd. for
C23H21N5O: C, 72.04; H, 5.52; N, 18.26%; Found: C, 72.02;
H, 5.49; N, 18.25%; IR (υ cm−1): 3068 (C-H, sp2), 2979
(C-H, sp3), 1721 (C=O), 1625 (C=C/C=N), 1615, 1528,
1441 (C…C, ring str), 952, 847, 746 (sub. phenyl); 1H-NMR
(300 MHz, CDCl3) δ (ppm): 1.85 (s, 6H, 2 × CH3, isopro-
penyl), 1.98 (s, 3H, CH3), 2.32 (s, 3H, ArCH3), 4.64 (s, CH,
pyrimidine), 5.83 (s, CH, methine), 7.24 (d, 2H, H11,
H12), 7.43 (s, 1H, H23), 7.49 (d, 1H, H21), 7.58 (d, 1H,
H21), 7.68 (d, 2H, H10, H13); 13C NMR (CDCl3) δ (ppm):
18.06 (C24), 21.42 (Ar-CH3), 20.24 (C28), 24.96 (C27),
66.92 (C3), 114.13, 115.12, 115.34, 123.04, 126.04, 130.74,
131.22, 132.84, 138.82, 138.88, 140.37 (aromatic ring),
141.84 (C6, C16), 143.78 (C26), 165.25 (C4), 169.28(C2);
FAB-MS: 384 (M+H)+.
2-methyl-3-[4-nitro-2-(2-methylprop-1-en-1-yl)-1-
H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4-
(3H)-one (4m): Yield 73%; mp 124–25°C; Anal. Calcd. for
C22H19N6O3: C, 63.76; H, 4.38; N, 20.28%; Found: C, 63.73;
H, 4.35; N, 20.25 %; IR (υ cm−1): 3080 (C-H, sp2), 2959
(C-H, sp3), 1725 (C=O), 1625 (C=C/C=N), 1612, 1520, 1440
(C…C, ring str), 1331 (NO2) 952, 855, 745 (sub. phenyl);
1H-NMR (300 MHz, CDCl3) δ (ppm): 1.80(s, 6H, 2 × CH3,
isopropenyl), 1.94 (s, 3H, CH3), 4.64 (s, CH, pyrimidine),
5.23 (s, CH, methine), 7.23 (d, 2H, H11, H12), 7.45 (t, 1H,
H22), 7.62 (d, 2H, H10,H13), 7.95 (d, 1H, H23), 8.18(d,1H,
H21); 13C NMR (CDCl3) δ (ppm): 17.43 (C24), 19.28 (C28),
25.27 (C27), 64.88 (C3), 114.23, 115.26, 118.65, 121.32,
123.02, 123.95, 130.75, 133.08, 137.06, 138.90, 139.87
(aromatic ring), 141.53 (C6, C16), 143.53 (C26), 164.66
(C4), 166.28 (C2); FAB-MS: 415 (M+H)+.
Results and discussion
In order to screen these synthesized benzimidazoles
as more potent lead compounds, QSAR study was per-
formed on substituted 2-methyl-3-[2-(2-methylprop-
1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]
benzimidazol-4(3H)-one (4a–r) derivatives. e struc-
ture of more active member of the series is shown in
Figure 2. Fifty percent inhibitory concentration [IC50]
of the substituted 2-methyl-3-[2-(2-methylprop-1-en-
1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimi-
dazol-4(3H)-one (4a–r) was calculated and converted
to the negative logarithmic scale (pIC50) (Table 1). A
number of physicochemical parameters viz., Connolly
accessible area (CAA), Connolly molecular area (CMA),
molar refractivity (MR), dipole-dipole energy (DDENE),
non-1,4 VDW energy (NVDW), VDW 1,4 energy (VDW)
were calculated by the Chem office 6.0 using Chem.
3D Pro package27. Geometry optimization was done by
PM3 method incorporated in MOPAC28 server of the
ChemOffice 6.0 program. Single point energies calcula-
tion for the lead compounds was done by RHF (Restricted
Hartree-Fock: closed shell) wave function. HyperChem.
Release 8.0.3 Molecular Modeling System29 was used
for the calculation of van der Waal surface volume
(WVOL), van der Waal surface area (WSA), surface area
grid (SAG), hydrophobicity (logP), molecular refractiv-
ity (REF), molecular polarizability (P), and molecular
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and exploration of QSAR model 299
orbital energies (HOMO and LUMO). Initial energy min-
imizations were done by employing molecular mechan-
ics methods. ese initially optimized geometries were
optimized by PM3 method employing Fletcher-Reeves
(conjugate gradient) algorithm with a termination RMS
gradient of 0.0001 kcal/(Å mol).
All the statistical analyses were carried out by the com-
puter program Valstat30. Correlation analysis between
molecular descriptors (calculated by ChemOffice and
HyperChem packages), and biological activity was
performed. e dataset were divided into test set and
training set containing three and fifteen compounds,
respectively. All possible combinations of parameters
were considered for the QSAR study. Multiple regres-
sion analysis was carried out using in vitro antimicro-
bial inhibition activities as the dependent variable and
all molecular descriptors as independent variables in
all possible combinations. e statistical quality of the
regression equations was justified on the basis of signifi-
cant statistical parameters viz., percentage of explained
variance (% EV), variance ratio (F), and standard error
of estimate (SEE). All the final equations have regression
coefficients, intercepts and variance ratio (F) significant
to more than 95% confidence level. Use of more than
one variable in the multivariate equation was justified
by an autocorrelation study. e predictive powers of
the equations were validated by the leave one out (LOO)
cross validation method. Predicted residual sum of
square (PRESS), total sum of squares (SSY) and cross-
validated r2 (r2CV) were considered for the validation of
the models.
Tri-parametric QSAR model for activity against
P. diminuta
pIC50 = −27.711 (–1.99)+2.592 (–0.300) MR −
(1)
0.013 (–0.008) CMA −0.124 (–0.060) DDENE
n= 18, F = 273.089, PRESS = 0.311, SSY = 5.406, PRESS/
Figure 2. PM3 optimized geometry of more active 2-methyl-3-[2-
(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]
benzimidazol-4(3H)-one.
SSY = 0.058, r2CV = 0.942,
% EV = 93.805, SEE = 0.144,
r2bsp = 0.989, Bootstrapping std = 0.006, Chance < 0.001,
Q2 = 0.967, SPRESS = 0.121, SDEP = 0.104
Table 1. e in vitro antimicrobial inhibition activities of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]
pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r).
pIC50
S. No.
1
Entry
4a
4b
4c
4d
4e
4f
R
P. diminuta
−3.0934
−1.1761
−1.5563
−2.1303
−2.2304
−2.4232
−3.1761
−2.4624
−2.5315
−3.1303
−2.5740
−2.3324
−1.9542
−1.3424
−1.7634
−2.6021
−2.5575
−2.3617
−3.2175
B. subtilis
−3.2574
−1.3617
−1.7243
−2.2900
−2.3802
−2.5843
−3.3426
−2.6021
−2.6928
−3.3118
−2.7380
−2.4843
−2.1139
−1.5441
−1.9294
−2.7076
−2.6857
−2.4698
−3.3802
E. Coli
−3.1746
−1.2553
−1.6532
−2.2041
−2.3222
−2.5119
−3.2565
−2.5441
−2.6021
−3.2041
−2.6628
−2.4393
−2.0607
−1.4472
−1.8751
−2.6284
−2.5911
−2.3802
−3.2742
S. aureus
−3.3222
−1.4472
−1.7634
−2.3617
−2.4698
−2.6532
−3.4108
−2.6902
−2.7559
−3.3598
−2.8162
−2.5563
−2.1903
−1.6232
−2.0253
−2.8062
−2.7324
−2.5441
−3.4150
-H
2
4-OC2H5
6-C2H5
6-NO2
4-OCH3
4-Cl
3
4
5
6
7
4g
4h
4i
6-OH
4-COOH
4-CH3
4-OH
6-CH3
6-OCH3
4-NO2
4-C2H5
4-Br
8
9
10
11
12
13
14
15
16
17
18
19
4j
4k
4l
4m
4n
4o
4p
4q
4r
5-CH3
5-Cl
5-OCH3
--
A
A, Ampicilline.
© 2012 Informa UK, Ltd.

300 P. Sharma et al.
Contribution of parameters to model is MR: CMA:
DDENE:: 85.306: 12.958: 1.000. In the above equation
% EV, F, SEE, PRESS, SSY, r2CV, r2bsp, SPRESS are percent-
age of explained variance, ratio between the variances
of observed and calculated activities, standard error of
estimate, predicted residual sum of squares, total sum of
squares, cross validated r2, Bootstrapping r2 and standard
deviation error of prediction, respectively.
Equation (1) explains up to 93.805% of the variation
of the activities. However, MR is the key factor to control
biological activity, which can be attributed to steric inter-
action occurring in polar species. It is related to molar
volume (V) and the refractivity index (η) according to the
relation:
Contribution of parameters to model is MR: CMA:
DDENE:: 85.306: 12.958: 1.000.
pIC50 = −27.493 (–1.917) +2.498 (–0.289) MR −
(4)
0.011 (–0.007) CMA −0.114 (–0.058) DDENE
n= 18, F = 282.885, PRESS = 0.054, SSY = 4.630, PRESS/
SSY = 0.012, r2CV = 0.988,
% EV = 98.736, SEE = 0.060,
r2bsp = 0.990, Bootstrapping std = 0.006, Chance < 0.001,
Q2 = 0.964, SPRESS = 0.123, SDEP = 0.106
Contribution of parameters to model is MR: CMA:
DDENE:: 89.055: 12.034: 1.000.
In all the above models, MR term has significant con-
tribution as supported by statistics of the analysis. us,
it is revealed from all the proposed models deduced
through equations 1-4 that the MR is the main governing
factor. Moreover, increase in the percentage contribu-
tion of CMA also signifies the importance of Connolly
molecular area (CMA).
MR[( 21)/( 22)]V
which reveals that MR is a measure of bulk due to its
relation with molar volume. However, it also contains
a polarizability component η. Hence, the positive coef-
ficient of MR corresponds to higher activity of these
compounds. On the other hand, negative coefficient of
the CMA signifies the importance of Connolly molecular
area in the activity. Lower value of this index corresponds
to higher activity of these compounds.
Conclusion
A variety of benzimidazole derivatives have been suc-
cessfully synthesized in appreciable yields and screened
in vitro for their antimicrobial activities against both
strains of gram-positive and gram-negative bacteria.
Moreover, quantitative structure-activity relationship
studies have been performed to explore their inher-
ent antimicrobial potential. All the synthesized com-
pounds have shown excellent antimicrobial activities.
It is evident from the developed QSAR model that MR,
CMA, and DDENE parameters were the main governing
physicochemical factors for the displayed antimicrobial
activities. Such a QSAR evaluation would open up future
perspectives to use these compounds as new lead com-
pounds in drug discovery processes.
Further, PRESS is a cross-validation parameter whose
value less than SSY (sum of the squares of response
value) points out that the model predicts better than
Chance and can be considered statistically important.
To be a reasonable QSAR model, PRESS/SSY ratio
should be smaller than 0.4 and its value smaller than
0.11 indicates an excellent model. e data indicate
that for all the proposed models, this ratio is < 0.1 sug-
gesting all of them to be excellent models. Furthermore,
the value of cross-validated correlation coefficient (r2
)
and Bootstrapping r2 are further supporting the predCicV-
tive power of these explaining models.
Moreover, the compounds have also been screened
against E. coli, B. subtilis and S. aureus bacteria and the
tri-parametric QSAR models are presented in the equa-
tion 2, 3, and 4, respectively.
Declaration of interest
We are thankful to the Defence Research Development
Organization (DRDO), New Delhi (India), and Central
Drugs Research Institute (CDRI), Lucknow (India), for
providing financial assistance and spectro-analytical
facilities, respectively.
pIC50 = −27.440 (–1.885)+2.531 (–0.285) MR −
(2)
0.012 (–0.007) CMA −0.124 (–0.057) DDENE
n= 18, F = 295.238, PRESS = 0.057, SSY = 4.670, PRESS/
SSY = 0.012, r2CV = 0.988,
% EV = 98.679, SEE = 0.062,
r2bsp = 0.989, Bootstrapping std = 0.007, Chance < 0.001,
Q2 = 0.971, SPRESS = 0.111, SDEP = 0.095
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