Design, synthesis and pharmacological evaluation of pyrimidobenzothiazole-3-carboxylate derivatives as selective L-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmc.2015.09.009

L-type voltage gated calcium channels play essential role in contraction of various skeletal and vascular smooth muscles, thereby plays important role in regulating blood pressure. Dihydropyridine receptors have been targeted for development of newer antihypertensive agents, one of the structurally analogs nucleus dihydropyrimidines have been reported earlier by us as a potential agent toward development of calcium channel modulator. A pre-synthetic QSAR was run and on the basis of structure activity relationship a series of twenty three molecules was synthesized and studied by myosin light chain kinase assay (MLCK), Angiotensin Converting Enzyme (ACE) colorimetric assay, non-invasive blood pressure (NIBP) and invasive blood pressure (IBP) methods. Molecules with significant efficacy were studied for their single crystal X-ray diffraction, molecular docking, molecular dynamics and post-synthetic QSAR. The NIBP and IBP methods screened molecules with better percentage inhibition versus time compared to standard drug Nifedipine. The lead compound ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido [2,1-b] [1,3] benzothiazole-3-carboxylate (26) presented a triclinic structure with polymeric chain packing in lattice. 26 exhibited IC₅₀on MLCK assay of 2.1 ± 1.7 μM with selectivity of L-type calcium channels and comparative to Nifedipine. It offered satisfactory physicochemical properties with partition coefficient of (C log P) 4.64. Its pharmacokinetic profile is also good with C_max at 0.40 μg/ml by oral route with T_max reaching in 0.5 h which means in 30 min. 26 also exhibits superior t_1/2 of 5.4 h and oral bioavailability of (F) 56.75% with an AUC_∞ of 0.84 μg h/ml. Molecular docking studies indicates toward the interaction of lead compound via hydrogen bonds with Lys144, Glu181 and Asp183, it forms the Van der Walls interactions with Ser18, Asp20, Asn187, Pro185, Glu180, Glu181 and Arg10 with Glide score and Glide energy to be -3.602 and -47.098, respectively. Post-synthetic QSAR of newly synthesized molecules indicates toward improvement with respect to steric descriptor which contributed negatively in former series.

Full text of DOI:10.1016/j.bmc.2015.09.009

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacological evaluation of
pyrimidobenzothiazole-3-carboxylate derivatives as selective L-type
calcium channel blockers
b
a
c
d
Rupesh Chikhale a, , Sonali Thorat , Amit Pant , Ankush Jadhav , Krishna Chary Thatipamula ,
⇑
Ratnadeep Bansode e, G. Bhargavi d, Nazira Karodia e, M. V. Rajasekharan d, Anant Paradkar e,
Pramod Khedekar a,
⇑
a Computer Aided Drug Design Laboratory, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Mahatma Jyotiba Fuley Shaikshanik Parisar,
Amravati Road, Nagpur 440033, MS, India
b Analytical Chemistry Laboratory, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440033, MS, India
c Centre for Bioinformatics, Pondicherry University, Pondicherry 605014, India
d School of Chemistry, University of Hyderabad, C. R. Rao Road, Hyderabad 500 046, India
e Centre for Pharmaceutical Engineering Science, Faculty of Life Sciences, University of Bradford, Bradford BD7 1DP, West Yorkshire, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
L-type voltage gated calcium channels play essential role in contraction of various skeletal and vascular
smooth muscles, thereby plays important role in regulating blood pressure. Dihydropyridine receptors
have been targeted for development of newer antihypertensive agents, one of the structurally analogs
nucleus dihydropyrimidines have been reported earlier by us as a potential agent toward development
of calcium channel modulator. A pre-synthetic QSAR was run and on the basis of structure activity rela-
tionship a series of twenty three molecules was synthesized and studied by myosin light chain kinase
assay (MLCK), Angiotensin Converting Enzyme (ACE) colorimetric assay, non-invasive blood pressure
(NIBP) and invasive blood pressure (IBP) methods. Molecules with significant efficacy were studied for
their single crystal X-ray diffraction, molecular docking, molecular dynamics and post-synthetic QSAR.
The NIBP and IBP methods screened molecules with better percentage inhibition versus time compared
to standard drug Nifedipine. The lead compound ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido [2,1-b]
[1,3] benzothiazole-3-carboxylate (26) presented a triclinic structure with polymeric chain packing in
Received 20 July 2015
Revised 3 September 2015
Accepted 4 September 2015
Available online xxxx
Keywords:
Benzothiazole
Hypertension
3D-QSAR
Myosin light chain kinase
ACE
lattice. 26 exhibited IC50on MLCK assay of 2.1 1.7
comparative to Nifedipine. It offered satisfactory physicochemical properties with partition coefficient
of (ClogP) 4.64. Its pharmacokinetic profile is also good with Cmax at 0.40 g/ml by oral route with
Tmax reaching in 0.5 h which means in 30 min. 26 also exhibits superior t1/2 of 5.4 h and oral bioavailabil-
ity of (F) 56.75% with an AUC0–1 of 0.84 g h/ml. Molecular docking studies indicates toward the inter-
lM with selectivity of L-type calcium channels and
l
l
action of lead compound via hydrogen bonds with Lys144, Glu181 and Asp183, it forms the Van der Walls
interactions with Ser18, Asp20, Asn187, Pro185, Glu180, Glu181 and Arg10 with Glide score and Glide
energy to be ꢀ3.602 and ꢀ47.098, respectively. Post-synthetic QSAR of newly synthesized molecules
indicates toward improvement with respect to steric descriptor which contributed negatively in former
series.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
skeletal and smooth muscle cells. These are also responsible for
the regulation of neurohormones, gene expression, neuronal sur-
L-type calcium channels are the voltage-gated calcium channels
essential for coupling excitation to cause contraction in cardiac,
vival, synaptic efficacy and regulation of transmitters by expres-
sion in neurons and endocrine cells.1 Kanngiesser et al., reported
the first isolation of L-type calcium channel or the dihydropyridine
receptor along with their cloning.2 L-type is a unique calcium
channel that specifically binds to dihydropyridine modulators. Cal-
cium channel blockers (CCBs), antagonist and modulators are
⇑
Corresponding authors at present address: Advanced Centre for Treatment,
Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi
Mumbai 410 210, India. Tel.: +91 8983387871 (R.C.).
E-mail address: rupeshchikhale7@gmail.com (R. Chikhale).
http://dx.doi.org/10.1016/j.bmc.2015.09.009
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chikhale, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.009

2
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
myosin leading to muscle contraction. Amplification of signal is
achieved by calcium-induced calcium release from the sarcoplas-
mic reticulum through ryanodine receptors.
Nifedipine causes initial influx of calcium that inhibits the con-
tractile processes of smooth muscle cells. It causes dilation of the
coronary and systemic arteries, increase in oxygen delivery to the
myocardial tissue, decrease in total peripheral resistance and
decrease in the systemic blood pressure resulting into decreased
afterload, this vasodilatory effect results in an overall decrease in
blood pressure. 1,4-DHPs causes systemic vasodilating and vascu-
lar pressure lowering effect which may lead to tachycardia and
positive inotropy and hence not used to treat angina. These classes
of compounds also face another limitation of short plasma half life
related to the high rate of metabolic oxidation of pyridine nucleus
leading to high frequency of dosing. These effects has proved to be
limitation toward the 1,4-DHPs use in therapy as calcium channel
blocker. 1,4 Dihydropyrimidines (1,4-DHPMs) are the structurally
analogous compounds that have been widely studied as substitute
for the 1,4-DHPs (Fig. 1 types I and II). Pyrimidine nucleus occurs
naturally as a fundamental part of the nucleic acids, is present in
biologically important compounds with antiviral,5 antitumor6
and cardiovascular agent.7 Recently research interest has focused
on the development of 1,4-DHPMs (Fig. 1. types II and IV) the
aza analogs of 1,4-DHPs.8–15
In our previous effort 1,4-DHPM derivatives exhibited compar-
ative antihypertensive activity to nifedipine in Deoxycorticos-
terone acetate salt (DOCA) rat model by non-invasive blood
pressure (NIBP) and invasive blood pressure method (IBP), the
report consisted of N-3 substitution with various phenacyl bro-
mides (Scheme 1).16 The synthetic scheme consisted of simple
two step protocol leading to final fifteen derivatives. o-Methyli-
sourea (1) reacted with ethyl acetoacetate (2) and substituted ary-
laldehydes (3) under the influence of catalytic amount of
hydrochloric acid to yield the corresponding 1,4-DHPMs (4), these
were further substituted with suitable phenacyl bromides (5) to
give N-3 substituted 1,4-DHPMs (6). However, the duration of
Figure 1. Structural analogy of 1,4-DHPs and 1,4-DHPMs.
diverse group of compounds that regulate activity of voltage gated
L-type calcium channels. They cause vasodilation, negative ino-
tropy, negative chronotropy and negative dromotropy specifically
at the atrioventricular node. Benzothiazepines, 1,4-dihydropyridi-
nes (1,4-DHPs), phenylalkylamines are three classes of compounds
that act as calcium channel blockers.1 These classes differ in their
basic structure as well as in their selectivity toward L-type calcium
channels. Nifedipine is a representative 1,4-DHP widely used in
therapy, it acts by decreasing arterial smooth muscle contractil-
ity.3,4 Subsequently vasoconstriction is caused by inhibiting the
influx of calcium ions through L-type calcium channels. Calcium
ions entering through these channels bind to calmodulin then
these calcium-bound calmodulin binds to and activates myosin
light chain kinase (MLCK). Activated myosin light chain kinase
mediates phosphorylation of the regulatory light chain subunit of
R
R
NH
O
DMF
O
H
H3C
O
+
+
NaHCO3
H2N
O
H3C
O
N
H3C
O
CH3
O
H
CH3
1
H3C
N
H
O
2
3
4 (a-c)
O
R1
Pyridine
Br
R
5 (a-c)
R1
O
H
N
H3C
O
O
H3C
N
H
O
CH3
6 (a-o)
Comp. 6a 6b
6c
6d 6e
6f
Cl
6g
6h
6i
6j
6k
6l
6m
6n
6o
R
H
H
H
H
Cl Cl
OH OH
Cl
OH
CH3 CH3
Cl
CH3
OCH3 OCH3 OCH3
Cl OCH3
R1
Cl OCH3
H
Cl OCH3
H
OCH3
H
OCH3
H
Scheme 1. Synthetic route for preparation of compounds 6(a–o), this is the previously reported scheme form our lab.16
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
S
N
NH2
NH
S
H3C
CH3
S
NH
N
S
Δ
0-5 °C
33
SMIT
NH
HN
N
NH2
NH2
Phenylthiourea
NH4SCN
N
Bromine
NH2
EtOH:H2O (1:1), Reflux
N
Xylene
Reflux
CH3
Aniline
7
30
H3C
34
H
O
O
O
O
R1
R
R1
TMOA
H3C
O
H3C
O
2, 8
O
R
H
2, 8
9
S
N
NH
N
O
N
R1
R1
N
H3C
O
S
H3C
N
H3C
10-29
31 R1 = C2H5
32 R1 = CH3
Sr. No. Compd.
R
R1
Sr. No. Compd.
R
R1
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
10
11
12
13
14
15
16
17
18
19
2-Cl
4-Cl
4-F
CH3 11.
CH3 12.
CH3 13.
CH3 14.
CH3 15.
CH3 16.
CH3 17.
CH3 18.
CH3 19.
20
21
22
23
24
25
26
27
28
29
2-Cl
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
4-Cl
4-F
2-OH
4-OH
2-OH
4-OH
4-OCH3
3-NO2
4-NO2
(CH3)2N
4-OCH3
3-NO2
4-NO2
(CH3)2N
4-OH-3-OCH3 CH3 20.
4-OH-3-OCH3 C2H5
Scheme 2. Synthetic route for preparation of target compounds 10–34. Reagents and conditions 10–29: HCl, CH3OH, reflux, 8–12 h. 30, 31 and 32: S-methyl isothiourea,
C2H5OH/H2O (1:1), reflux, 4 h; trimethyl ortho acetate (TMOA), dry reaction, 120 °C, 40–60 min. 34: xylene, reflux, 8 h, azeotropic distillation of H2O.
action or the half-life (T1/2) needed to be improved in order to
obtain compounds with longer duration of action compared to
nifedipine.
depending upon the 1,2 diketones (2, 8) involved in reaction. Com-
pound 30 was further treated with pentane 2,4 dione (33) in pres-
ence of xylene and removal of water under the conditions of
azeotropic distillation provides with N-(4,6-dimethylpyrimidin-2-
yl)-1,3-benzothiazol-2-amine (34). The synthesised compounds
were evaluated for their potency, selectivity and duration of action
by means of animal models and structure activity relationship was
essential on the basis of computational studies.
In continuation to our efforts for the development of 1,4-
DHPMs with better antihypertensive activity and greater T1/2 we
designed some new 1,4-DHPMs based on work reported by Atwal
et al. and Krylsky et al..15,17,18 In this article we report effects of
modification on the 1,4-DHPM’s pyrimidine ring and further its
QSAR analysis. Initially we started with the modification of the Big-
inelli’s classical one pot reaction, 2-aminobenzothiazole (7) reacted
with methyl acetoacetate (8) or ethyl acetoacetate (2) and various
substituted arylaldehydes (3) leading to ethyl or methyl 4-(substi-
tuted phenyl)-2-methyl-4H-pyrimido[2,1-b][1,3]benzothiazole-3-
carboxylates (10–29) in range of 60–80% synthetic yields
(Scheme 2). 2-Aminobenzothiazole was further treated with S-
methyl isothiourea (SMIT) to obtain benzothiazolyl guanidine
(30). Benzothiazolyl guanidine is a highly functionalised molecule;
it was further treated with 2 and 8 in presence of access of tri-
methyl orthoacetate (TMOA) under presence of inert conditions
of nitrogen to provide with 1-[2-(1,3-benzothiazol-2-ylamino)-4-
ethyl-6-methylpyrimidin-5-yl]ethanone (31) and 1-[2-(1,3-ben-
zothiazol-2-ylamino)-4,6-dimethylpyrimidin-5-yl]ethanone (32)
2. Experimental section
2.1. Chemicals, reagents and instruments
Chemicals were obtained from Sigma Aldrich, Germany and Alfa
Aeser, United Kingdom. Melting points (mp) were detected with
open capillaries using Thermonik Precision Melting point cum Boil-
ing point apparatus (C-PMB-2, Mumbai, India) and are uncor-
rected. Chromatography of the synthesized intermediates and
title compounds were performed on silica gel pre-coated plates
(Merck: 100–200 mesh). Thin layer chromatography (TLC) was
used to monitor the progress and/or completion of the reactions.
IR spectra (KBr) were recorded on FTIR-8400s spectrophotometer
Please cite this article in press as: Chikhale, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.009

4
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(Shimadzu, Japan). 1H and 13C NMR was obtained using a Bruker
Advance-II 400 Spectrometer on 400 MHz using tetramethylsilane
(TMS) as internal standard. All chemical shift values were recorded
as d (ppm), coupling constant value J is measured in hertz, the
peaks are presented as s (singlet), d (doublet), t (triplet), br s (broad
singlet), dd (double doublet), m (multiplet). The purity of com-
pounds was controlled by thin layer chromatography (Merck, silica
gel, HF254-361, type 60, 0.25 mm, Darmstadt, Germany). X-ray
data was collected on a BRUKER-AXS SMART APEX CCD X-ray
(100 MHz, DMSO-d6): d 17.58, 51.68, 57.01, 99.49, 103.80,
114.50, 115.72, 115.94, 124.17, 124.55, 127.96, 129.88, 129.96,
135.94, 160.86, 161.31, 163.31, 164.52, 200.86; IR (KBr, cmꢀ1):
3090, 1720, 1687, 1475, 1310; MS: (ESI) m/z [M+H]+ calcd for
[C19H15FN2O2S]+: 354.08; found: 355.19.
2.2.5. Methyl 4-(2-hydroxyphenyl)-2-methyl-4H-pyrimido
[2,1-b][1,3]benzothiazole-3-carboxylate (13)
% yield = 70%, mp 153–155 °C; 1H NMR (400 MHz, DMSO-d6) d
2.58 (s, 3H), 3.73 (s, 3H), 4.7 (s, 1H), 5.3 (s, 1H, OH), 6.85 (t, 2H,
J = 8.66 Hz, 8.73 Hz), 7.30 (d, 1H, J = 7.82 Hz), 7.54 (d, 1H,
J = 1.06 Hz), 7.95 (t, 2H, J = 8.11 Hz, 8.72 Hz), 8.40 (t, 2H,
diffractometer, using graphite-monochromatic Mo K
a radiation
(=0.71073 Å). Mass spectra (ESI-MS) were recorded at ESI-MS spec-
trometer (DPX-400, Bruker, USA). Cammag High Performance Thin
Layer Chromatography (HPTLC) system with Linomat IV sample
J = 7.5 Hz, 8.70 Hz); 13C NMR (100 MHz, DMSO-d6):
d 25.51,
applicator, Hamilton Bonaduz Schweiz 100
l
l syringe, scanner III
53.37, 58.33, 111.14, 118.21, 122.53, 125.35, 126.18, 127.45,
127.81, 128.50, 129.53, 131.04, 134.46, 140.92, 143.70, 153.35,
160.58, 163.40; IR (KBr, cmꢀ1): 3380, 3090, 1718, 1690, 1475,
1310; MS: (ESI) m/z [M+H]+ calcd for [C19H16N2O3S]+: 352.08;
found: 352.81.
with winCATS software version 1.4.1., along with pre-coated silica
Gel 60F254 TLC plates. Shimadzu High Performance Liquid Chro-
matography (HPLC) system with SPD-20A prominence Photodiode
Array (PDA) detector and LC-20AB prominence LC solution soft-
ware were used in this study.
2.2.6. Methyl 4-(4-hydroxyphenyl)-2-methyl-4H-pyrimido[2,1-
b][1,3]benzothiazole-3-carboxylate (14)
2.2. Chemistry
% yield = 70%, mp 179–181 °C; 1H NMR (400 MHz, DMSO-d6): d
1.23 (s, 3H), 2.31 (s, 3H), 6.36 (s, 1H), 6.65 (t, 2H, J = 8.68 Hz,
8.72 Hz), 7.18 (d, 1H, J = 7.96 Hz), 7.21 (d, 1H, J = 1.04 Hz), 7.40
(m, 3H), 7.72 (d, 1H, J = 8.16 Hz), 9.49 (s, 1H, OH); 13C NMR
2.2.1. General procedure for synthesis of 2-methyl-4-
substituted phenyl-4H-pyrimido[2,1-b] [1,3]benzothiazole-3-
carboxylate derivatives (10–29)
A mixture of the ethyl or methyl acetoacetate (1 mmol), substi-
tuted benzaldehyde (1 mmol), and 2-aminobenzothiazole
(1 mmol) in presence of methanol was refluxed for 6–12 h, few
drops of hydrochloric acid was added to this mixture as catalyst.
Completion of the reaction was monitored by TLC. Excess of sol-
vent was removed under distillation, then the mixture was allowed
to cool at room temperature, crude product solidifies overnight.
Residue obtained was washed with petroleum ether followed by
diethyl ether for two or three times. This was further purified by
re-crystallization from a mixture of DMF/ethanol (1:1) to yield
60–80% of pure product.
(100 MHz, DMSO-d6):
d 23.18, 50.84, 56.02, 102.90, 112.36,
115.17, 122.81, 123.90, 126.50, 128.25, 132.14, 137.58, 153.76,
157.29, 162.45, 166.10; IR (KBr, cmꢀ1): 3380, 3082, 1718, 1690,
1468, 1309; MS: (ESI) m/z [M+H]+ calcd for [C19H16N2O3S]+:
352.08; found: 352.70.
2.2.7. Methyl 4-(4-methoxyphenyl)-2-methyl-4H-pyrimido
[2,1-b][1,3]benzothiazole-3-carboxylate (15)
% yield = 74%, mp 231–233 °C; 1H NMR (400 MHz, DMSO-d6): d
2.32 (s, 3H), 3.34 (s, 3H), 3.56 (s, 3H, OCH3), 6.43 (s, 1H), 6.82 (t, 2H,
J = 8.64 Hz, 8.76 Hz), 7.00 (d, 1H, J = 7.96 Hz), 7.19 (d, 1H,
J = 1.14 Hz), 7.34 (m, 2H), 7.46 (d, 1H, J = 8.18 Hz), 7.75 (d, 1H,
J = 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): d 23.20, 50.87, 54.94,
55.90, 102.79, 112.36, 113.88, 117.68, 120.82, 122.81, 126.76,
128.22, 130.88, 133.74, 137.52, 152.79, 153.97, 158.97, 162.55,
166.05, 166.37; IR (KBr, cmꢀ1): 3082, 1718, 1690, 1468, 1309,
1267; MS: (ESI) m/z [M+H]+ calcd for [C20H18N2O3S]+: 366.10;
found: 367.10.
2.2.2. Methyl 4-(2-chlorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (10)
% yield = 62%, mp 159–161 °C; 1H NMR (400 MHz, DMSO-d6): d
2.38 (s, 3H), 3.58 (s, 3H), 7.32 (d, 1H, J = 8.11 Hz), 7.45 (t, 2H,
J = 8.65 Hz, 8.74 Hz), 7.76 (d, 1H, J = 7.83 Hz), 7.98 (d, 1H,
J = 1.06 Hz), 8.00 (m, 2H), 8.59 (s, 1H), 8.91(s, 1H); 13C NMR
(100 MHz, DMSO-d6):
d 33.98, 77.50, 84.25, 120.72, 121.78,
123.76, 126.20, 131.52, 148.33, 156.17, 160.53, 191.91; IR (KBr,
cmꢀ1): 3090, 1718, 1690, 1475, 1310; MS: (ESI) m/z [M+H]+ calcd
for [C19H15ClN2O2S]+: 370.05; found: 370.98.
2.2.8. Methyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (16)
% yield = 80%, mp 243–245 °C; 1H NMR (400 MHz, DMSO-d6): d
2.34 (s, 3H), 3.63 (s, 3H), 6.72 (s, 1H), 7.21 (t, 2H, J = 8.60 Hz,
8.78 Hz), 7.61 (d, 1H, J = 7.92 Hz), 7.78 (d, 1H, J = 1.15 Hz), 7.87
(m, 2H), 8.31 (d, 1H, J = 8.18 Hz), 8.32 (d, 1H, J = 7.5 Hz); 13C NMR
2.2.3. Methyl 4-(4-chlorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (11)
% yield = 60%, mp 163–165 °C; 1H NMR (400 MHz, DMSO-d6): d
2.31 (s, 3H), 3.50 (s, 3H), 7.19 (s, 1H), 7.32 (t, 2H, J = 8.66 Hz,
8.74 Hz), 7.45 (d, 1H, J = 7.83 Hz), 7.75 (d, 1H, J = 1.06 Hz), 8.00 (t,
2H, J = 8.11 Hz, 8.72 Hz), 8.59 (s, 2H); 13C NMR (100 MHz, DMSO-
d6): d 33.92, 53.14, 58.81, 115.25, 118.50, 120.72, 121.79, 123.77,
124.57, 126.21, 126.72, 127.81, 131.50, 134.15, 140.10, 143.60,
148.33, 156.15, 160.56; IR (KBr, cmꢀ1): 3095, 1715, 1690, 1480,
1405; MS: (ESI) m/z [M+H]+ calcd for [C19H15ClN2O2S]+: 370.05;
found: 370.28.
(100 MHz, DMSO-d6):
d 23.38, 51.03, 55.74, 101.87, 112.37,
121.50, 122.80, 123.02, 123.33, 124.34, 127.01, 130.43, 133.34,
137.15, 143.43, 147.82, 155.20, 163.11, 165.75; IR (KBr, cmꢀ1):
3082, 1718, 1690, 1510, 1468, 1309, 1140; MS: (ESI) m/z [M+H]+
calcd for [C19H15N3O4S]+: 381.07; found: 382.00.
2.2.9. Methyl 2-methyl-4-(4-nitrophenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (17)
% yield = 74%, mp 232–234 °C; 1H NMR (400 MHz, DMSO-d6): d
2.34 (s, 3H), 2.51 (s, 3H), 6.68 (s, 1H), 7.21 (t, 2H, J = 8.35 Hz,
8.80 Hz), 7.31 (d, 1H, J = 7.92 Hz), 7.46 (d, 1H, J = 1.15 Hz), 7.74
(m, 2H), 8.15 (d, 1H, J = 8.18 Hz), 8.17 (d, 1H, J = 7.5 Hz); 13C NMR
2.2.4. Methyl 4-(4-fluorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (12)
% yield = 67%, mp 176–178 °C; 1H NMR (400 MHz, DMSO-d6): d
2.50 (s, 3H), 3.73 (s, 3H), 6.81 (s, 1H), 7.24 (t, 2H, J = 8.68 Hz,
8.72 Hz), 7.26 (d, 1H, J = 7.96 Hz), 7.51 (d, 1H, J = 1.04 Hz), 7.60
(m, 2H), 7.68 (d, 1H, J = 8.16 Hz), 8.10(d, 1H, J = 7.8 Hz); 13C NMR
(100 MHz, DMSO-d6):
d 23.37, 51.01, 55.87, 101.69, 112.24,
122.80, 123.95, 126.96, 128.28, 137.16, 147.20, 148.14, 155.16,
163.07, 165.71; IR (KBr, cmꢀ1): 3088, 1720, 1700, 1495, 1468,
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
1300, 1143; MS: (ESI) m/z [M+H]+ calcd for [C19H15N3O4S]+:
381.07; found: 382.06.
2.2.15. Ethyl 4-(2-hydroxyphenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (23)
% yield = 65%, mp 159–161 °C; 1H NMR (400 MHz, DMSO-d6) d
1.93 (t, 3H, J = 8.32 Hz, 7.45 Hz), 2.89 (s, 3H), 4.3 (q, 2H,
J = 2.49 Hz, 2.01 Hz), 5.3 (s, 1H, OH), 6.85 (t, 2H, J = 8.66 Hz,
8.73 Hz), 7.30 (d, 1H, J = 7.82 Hz), 7.54 (d, 1H, J = 1.06 Hz), 7.95 (t,
2H, J = 8.11 Hz, 8.72 Hz), 8.40(t, 2H, J = 7.5 Hz, 8.70 Hz); 13C NMR
2.2.10. Methyl 4-[4-(dimethylamino)phenyl]-2-methyl-4H-
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (18)
% yield = 59%, mp 203–205 °C; 1H NMR (400 MHz, DMSO-d6): d
1.90 (s, 3H), 2.8 (s, 6HN(CH3)2), 3.5 (s, 3H), 4.4 (s, 1H), 7.36 (t, 2H,
J = 8.38 Hz, 8.70 Hz), 7.48 (d, 1H, J = 7.90 Hz), 7.54 (d, 1H,
J = 1.15 Hz), 7.7 (m, 2H), 7.9 (d, 1H, J = 8.13 Hz), 8.4 (d, 1H,
J = 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): d 22.43, 27.12, 39.24,
58.54, 58.16, 114.36, 118.65, 121.28, 124.55, 125.23, 127.17,
127.36, 130.68, 131.83, 132.52, 135.19, 134.63, 147.14, 155.66,
162.07, 164.50; IR (KBr, cmꢀ1): 3080, 1720, 1700, 1475, 1305,
1140; MS: (ESI) m/z [M+H]+ calcd for [C21H21N3O2S]+: 379.13;
found: 380.22.
(100 MHz, DMSO-d6):
d 25.59, 53.77, 58.32, 69.75, 111.44,
118.25, 122.57, 125.52, 126.88, 127.59, 127.80, 128.53, 129.55,
131.42, 134.67, 140.22, 143.76, 153.52, 160.88, 163.14; IR (KBr,
cmꢀ1): 3380, 3090, 1717, 1690, 1475, 1310; MS: (ESI) m/z [M
+H]+ calcd for [C20H18N2O3S]+: 366.10; found: 367.07.
2.2.16. Ethyl 4-(4-hydroxyphenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (24)
% yield = 67%, mp 172–174 °C; 1H NMR (400 MHz, DMSO-d6) d
1.19 (t, 3H, J = 7.46 Hz, 7.49 Hz), 2.31(s, 3H), 3.34 (d, 1H,
J = 7.8 Hz), 4.05 (q, 2H, J = 2.53 Hz, 2.73 Hz), 6.34 (s, 1H), 6.65 (t,
2H, J = 8.68 Hz, 8.72 Hz), 7.24 (d, 1H, J = 7.96 Hz), 7.40 (d, 1H,
J = 1.04 Hz), 7.42 (m, 2H), 7.73 (d, 1H, J = 8.16 Hz), 9.48 (s, 1H,
OH); 13C NMR (100 MHz, DMSO-d6): d 14.08, 23.15, 56.13, 59.38,
103.05, 112.34, 115.09, 122.80, 124.20, 122.80, 128.41, 132.17,
137.61, 153.56, 157.27, 162.33, 165.61; IR (KBr, cmꢀ1): 3365,
3080, 1718, 1695, 1470, 1310; MS: (ESI) m/z [M+H]+ calcd for
[C20H18N2O3S]+: 366.10; found: 367.10.
2.2.11. Methyl 4-(4-hydroxy-3-methoxyphenyl)-2-methyl-4H-
pyrimido[2,1-b][1,3] benzothiazol-3-carboxylate (19)
% yield = 68%, mp 167–169 °C; 1H NMR (400 MHz, DMSO-d6) d
2.50 (s, 3H), 3.35 (s, 3H), 3.71 (s, 3H, OCH3), 6.37 (s, 1H), 6.65 (t,
2H, J = 8.38 Hz, 8.82 Hz), 6.75 (d, 1H, J = 7.90 Hz), 7.03 (d, 1H,
J = 1.16 Hz), 7.19 (m, 2H), 7.73 (d, 1H, J = 7.7 Hz), 9.08 (s, 1H,
OH); 13C NMR (100 MHz, DMSO-d6): d 23.13, 50.82, 55.58, 56.29,
102.84, 111.41, 112.50, 115.50, 119.31, 122.80, 123.93, 126.74,
132.70, 137.65, 146.49, 147.14, 153.70, 162.48, 166.12; IR (KBr,
cmꢀ1): 2990, 1717, 1690, 1480, 1310, 1130; MS: (ESI) m/z [M
+H]+ calcd for [C20H18N2O4S]+: 382.09; found: 383.02.
2.2.17. Ethyl 4-(4-methoxyphenyl)-2-methyl-4H-pyrimido
[2,1-b][1,3]benzothiazole-3-carboxylate (25)
2.2.12. Ethyl 4-(2-chlorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (20)
% yield = 79%, mp 191–192 °C; 1H NMR (400 MHz, DMSO-d6): d
1.43 (t, 3H, J = 8.36 Hz, 7.53 Hz), 1.71 (s, 3H), 3.56 (s, 3H, OCH3),
4.59 (q, 2H, J = 2.53 Hz, 2.73 Hz), 6.34 (s, 1H), 7.17 (t, 2H,
J = 8.64 Hz, 8.76 Hz), 7.40 (d, 1H, J = 7.96 Hz), 7.54 (d, 1H,
J = 1.14 Hz), 7.65 (m, 2H), 7.85 (d, 1H, J = 8.18 Hz), 8.3 (d, 1H,
J = 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): d 26.55, 53.64, 55.79,
58.29, 69.15, 116.55, 117.85, 123.46, 125.48, 126.38, 127.17,
128.47, 129.16, 129.59, 131.43, 134.18, 139.59, 146.14, 155.57,
161.13, 164.50; IR (KBr, cmꢀ1): 3082, 1718, 1690, 1468, 1309,
1267; MS: (ESI) m/z [M+H]+ calcd for [C21H20N2O3S]+: 380.11;
found: 380.98.
% yield = 63%, mp 234–236 °C; 1H NMR (400 MHz, DMSO-d6): d
1.32 (t, 3H, J = 6.65 Hz, 8.01 Hz), 3.03 (s, 3H), 3.9 (q, 2H, J = 1.20 Hz,
2.48 Hz), 6.41 (s, 1H), 6.90 (t, 2H, J = 8.65 Hz, 8.74 Hz), 7.32 (d, 1H,
J = 7.83 Hz), 7.52 (d, 1H, J = 1.06 Hz), 7.66 (m, 2H), 7.92 (d, 1H,
J = 8.11 Hz), 8.26(d, 1H, J = 7.5 Hz); 13C NMR (100 MHz, DMSO-
d6): d 23.14, 52.34, 56.18, 68.44, 114.52, 117.15, 122.09, 124.91,
126.37, 127.81, 127.39, 128.74, 128.71, 131.20, 132.51, 139.51,
142.36, 152.72, 159.47, 164.09; IR (KBr, cmꢀ1): 3095, 1720, 1690,
1475, 1315; MS: (ESI) m/z [M+H]+ calcd for [C20H17ClN2O2S]+:
384.06; found: 384.98.
2.2.18. Ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (26)
2.2.13. Ethyl 4-(4-chlorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (21)
% yield = 83%, mp 254–256 °C; 1H NMR (400 MHz, DMSO-d6): d
1.22 (t, 3H, J = 8.12 Hz, 7.42 Hz), 2.50(s, 3H), 4.07 (q, 2H, J = 2.26 Hz,
2.41 Hz), 6.70 (s, 1H), 7.21 (t, 2H, J = 8.60 Hz, 8.78 Hz), 7.32 (d, 1H,
J = 7.92 Hz), 7.61 (d, 1H, J = 1.15 Hz), 7.77 (m, 2H), 7.79 (d, 1H,
J = 8.18 Hz), 8.36 (d, 1H, J = 7.5 Hz); 13C NMR (100 MHz, DMSO-
d6): d 13.97, 23.31, 55.83, 59.70, 101.94, 112.42, 121.78, 122.77,
123.00, 123.31, 124.34, 126.99, 130.47, 133.40, 137.19, 143.53,
147.66, 155.10, 163.03, 165.19; IR (KBr, cmꢀ1): 3082, 1720, 1690,
1512, 1468, 1319, 1145; MS: (ESI) m/z [M+H]+ calcd for
[C20H17N3O4S]+: 395.09; found: 396.02.
% yield = 64%, mp 227–229 °C; 1H NMR (400 MHz, DMSO-d6): d
1.38 (t, 3H, J = 6.70 Hz, 8.20 Hz), 2.87 (s, 3H), 3.8 (q, 2H, J = 1.49 Hz,
2.54 Hz), 6.58 (s, 1H), 6.90 (t, 2H, J = 8.66 Hz, 8.74 Hz), 7.32 (d, 1H,
J = 7.83 Hz), 7.52 (d, 1H, J = 1.06 Hz), 7.90 (t, 2H, J = 8.11 Hz,
8.72 Hz), 8.35(t, 2H, J = 7.5 Hz, 8.76 Hz); 13C NMR (100 MHz,
DMSO-d6): d 25.44, 53.47, 58.85, 68.19, 111.20, 118.54, 122.09,
124.45, 126.91, 127.34, 127.85, 128.47, 129.78, 131.32, 134.65,
140.16, 143.63, 153.77, 160.73, 163.90; IR (KBr, cmꢀ1): 3091,
1715, 1694, 1480, 1401; MS: (ESI) m/z [M+H]+ calcd for
[C20H17ClN2O2S]+: 384.06; found: 384.86.
2.2.19. Ethyl 2-methyl-4-(4-nitrophenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (27)
2.2.14. Ethyl 4-(4-fluorophenyl)-2-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (22)
% yield = 79%, mp 234–236 °C; 1H NMR (400 MHz, DMSO-d6): d
1.56 (t, 3H, J = 8.34 Hz, 7.82 Hz), 2.01 (s, 3H), 4.35 (q, 2H,
J = 2.16 Hz, 2.45 Hz), 6.15 (s, 1H), 7.29 (t, 2H, J = 8.35 Hz, 8.80 Hz),
7.46 (d, 1H, J = 7.92 Hz), 7.58 (d, 1H, J = 1.15 Hz), 7.7 (m, 2H), 7.9
(d, 1H, J = 8.18 Hz), 8.3 (d, 1H, J = 7.5 Hz); 13C NMR (100 MHz,
DMSO-d6): d 27.16, 54.44, 58.26, 70.55, 114.31, 118.34, 122.18,
124.45, 126.23, 127.76, 128.37, 130.74, 130.38, 132.83, 137.93,
138.65, 147.63, 155.63, 162.24, 164.75; IR (KBr, cmꢀ1): 3085,
1723, 1691, 1515, 1470, 1315, 1135; MS: (ESI) m/z [M+H]+ calcd
for [C20H17N3O4S]+: 395.09; found: 396.01.
% yield = 64%, mp 201–203 °C; 1H NMR (400 MHz, DMSO-d6): d
2.03 (t, 3H, J = 7.50 Hz, 8.65 Hz), 3.65 (s, 3H), 4.1 (q, 2H, J = 2.49 Hz,
2.01 Hz), 6.84 (s, 1H), 7.10 (t, 2H, J = 8.68 Hz, 8.72 Hz), 7.47 (d, 1H,
J = 7.96 Hz), 7.51 (d, 1H, J = 1.04 Hz), 7.60 (m, 2H), 7.83 (d, 1H,
J = 8.16 Hz), 8.11(d, 1H, J = 7.8 Hz); 13C NMR (100 MHz, DMSO-
d6): d 27.51, 52.44, 56.39, 65.70, 113.43, 118.25, 123.06, 125.27,
126.82, 127.13, 128.63, 128.98, 129.58, 131.12, 134.75, 139.71,
147.60, 154.67, 161.28, 162.59; IR (KBr, cmꢀ1): 3090, 1720, 1687,
1475, 1310; MS: (ESI) m/z [M+H]+ calcd for [C20H17FN2O2S]+:
368.09; found: 368.89.
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
2.2.20. Ethyl 4-[4-(dimethylamino)phenyl]-2-methyl-4H-
pyrimido[2,1-b][1,3]benzothiazole-3-carboxylate (28)
% yield = 62%, mp 198–200 °C; 1H NMR (400 MHz, DMSO-d6):
d1.29 (t, 3H, J = 8.12 Hz, 7.72 Hz), 2.92 (s, 3H), 3.26 (s, 6H, N
(CH3)2), 4.67 (q, 2H, J = 2.65 Hz, 2.21 Hz), 6.15 (s, 1H), 7.36 (t, 2H,
J = 8.38 Hz, 8.70 Hz), 7.48 (d, 1H, J = 7.90 Hz), 7.54 (d, 1H,
J = 1.15 Hz), 7.7 (m, 2H), 7.9 (d, 1H, J = 8.13 Hz), 8.4 (d, 1H,
J = 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): d 22.54, 27.14, 39.46,
58.64, 58.76, 69.56, 114.26, 118.56, 121.87, 124.35, 125.28,
127.27, 127.36, 130.76, 131.78, 132.52, 135.39, 134.68, 147.49,
155.36, 162.57, 164.15; IR (KBr, cmꢀ1): 3097, 1715, 1700, 1449,
1308, 1142; MS: (ESI) m/z [M+H]+ calcd for [C22H23N3O2S]+:
393.15; found: 394.01.
of solvent was removed under distillation, and then the mixture
was allowed to cool at room temperature. The precipitate formed
was filtered off, recrystallized from DMF, and dried to give 55%
yield.
% yield = 62%, mp 272–274 °C; 1H NMR (400 MHz, DMSO-d6): d
1.32 (t, 3H, J = 1.66 Hz, 1.71 Hz,CH3), 2.25(s, 3H, Ar-CH3), 2.76 (s,
3H, Ar-CH3), 4.12(s, 1H, NH), 7.25 (t, 2H, J = 8.38 Hz, 8.70 Hz),
7.43 (d, 1H, J = 7.90 Hz), 7.55 (d, 1H, J = 1.15 Hz); 13C NMR
(100 MHz, DMSO-d6,) d 20.84, 27.84, 120.61, 122.48, 127.88,
147.53, 150.42, 164.54, 168.17, 179.73; IR (KBr, cmꢀ1): 1694,
1720, 3087, 1470; MS: (ESI) m/z [M+H]+ calcd for [C15H14N4OS]+:
298.08; found: 299.06.
2.2.25. N-(4,6-Dimethylpyrimidin-2-yl)-1,3-benzothiazol-2-
amine (34)
2.2.21. Ethyl 4-(4-hydroxy-3-methoxyphenyl)-2-methyl-4H-
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (29)
% yield = 67%, mp 232–234 °C; 1H NMR (400 MHz, DMSO-d6) d
1.22 (t, 3H, J = 8.12 Hz, 7.72 Hz), 2.50 (s, 3H), 3.34 (s, 3H, OCH3),
3.84 (s, 3H), 4.07 (s, 1H), 6.66 (t, 2H, J = 8.38 Hz, 8.82 Hz), 6.76 (d,
1H, J = 1.16 Hz), 7.21 (m, 2H), 7.73 (d, 1H, J = 8.14 Hz), 9.07 (s,
1H, OH); 13C NMR (100 MHz, DMSO-d6): d 14.13, 23.08, 55.57,
56.36, 59.41, 102.97, 111.62, 112.52, 115.51, 119.49, 122.80,
126.71, 132.76, 137.67, 146.48, 147.05, 153.50, 162.36, 165.64;
MS: (ESI) m/z [M+H]+ calcd for [C21H20N2O4S]+: 396.11; found:
397.11.
Benzothiazolyl guanidine 30 (5 mmol) was refluxed with acetyl
acetone 33 (5 mmol) in presence of xylene (25 ml) for 9 h. Comple-
tion of the reaction was monitored by TLC. Excess of solvent was
removed under azeotropic conditions of distillation, and allowed
to cool at room temperature. The precipitate formed was filtered
off, recrystallized from DMF, and dried to give 62% yield.
% yield = 73%, mp 159–161 °C; 1H NMR (400 MHz, DMSO-d6): d
2.23 (s, 6H, CH3), 3.78 (s, 1H, NH), 6.30 (s, 1H, Pyrimidine), 7.31 (m,
1H), 7.74 (m, 1H), 7.99 (m, 2H); 13C NMR (100 MHz, DMSO-d6,) d
30.31, 51.01, 106.41, 118.78, 120.56, 121.71, 123.58, 126.12,
131.35, 148.41, 157.59, 166.22; IR (KBr, cmꢀ1): 3431.2, 3342.2,
1634.7, 1621.3, 1495.4, 1462.5; MS: (ESI) m/z [M+H]+ calcd for
[C13H12N4S]+: 256.07; found: 257.12.
2.2.22. 1-(1,3-Benzothiazol-2-yl)guanidine (30)
2-Aminobenzothiazole (1 mmol) and S-methylisothiourea
hemisulfate salt (1 mmol) was refluxed for sixteen hours in a mix-
ture of ethanol/water (1:1), after which the solvent was removed
under pressure to obtain the flaky solid. The crude product was
boiled in water and treated with 0.5 M NaOH to bring pH of solu-
tion toward neutral, then allowed to cool gradually. White crude
solid was obtained, it was recrystallized from ethanol to yield
white crystalline needle shaped crystals.
2.3. In vitro studies
All synthesised compounds were subjected to biological studies
initially for determination of mode of action by myosin light chain
kinase assay method followed by the angiotensin-I converting
enzyme colorimetric assay.
% yield = 72%, mp 214–216 °C; 1H NMR (400 MHz, DMSO-d6): d
3.34 (s, 2H, NH2), 7.0 (m, 1H, Ar), 7.20 (m, 1H, Ar), 7.31 (t, 1H,
J = 7.52 Hz, Ar), 7.46 (s, 2H, NH2), 7.63 (dd, 1H, J = 6.96 Hz, Ar).
13C NMR (100 MHz, DMSO-d6,) d 78.81, 117.55, 120.61, 120.75,
125.24, 130.73, 152.64, 166.25; IR (KBr, cmꢀ1): 3424, 3339, 1655,
1601, 1491, 1460, 1618; MS: (ESI) m/z [M+H]+ calcd for
[C8H8N4S]+: 192.04; found: 193.14.
2.3.1. Myosin light chain kinase assay (MLCK)
MLCK inhibitory activity screens were performed by the
method reported by Gao et al.. Initially all the reagents were pre-
pared; kinase assay buffer, kinase dilution buffer, kinase solution,
ATP stock solution,
c-
32P-ATP assay combination, 1% phosphoric
acid solution and substrate solution.19,20
The active MLCK was thawed along with kinase assay buffer,
substrate solution and kinase dilution buffer on ice, whereas the
2.2.23. 1-[2-(1,3-Benzothiazol-2-ylamino)-4-ethyl-6-
methylpyrimidin-5-yl]ethanone (31)
c-
32P-ATP Assay combination was thawed at room temperature.
Benzothiazolyl guanidine 30 (5 mmol), ethyl acetoacetate 2
(5 mmol) in the trimethyl orthoacetate (5 ml) was refluxed for
60 min. Completion of the reaction was monitored by TLC. Excess
of solvent was removed under distillation, and then the mixture
was allowed to cool at room temperature. The precipitate formed
was filtered off, recrystallized from DMF, and dried to give 50%
yield.
Two parallel pre-cooled microcentrifuge tubes were prepared con-
taining sample and blank control. In the first tube 10 mL of kinase
solution, 5 mL of substrate, 2.5 mL of cold water and 5 mM CaCl2
solution containing 0.75 mg Calmodulin was added to make up
the volume upto 20 mL. In case of blank control, instead of sub-
strate 5 mL of cold water was used. The reaction was initiated by
adding c-
32P-ATP combination leading the final volume to 25 mL
% yield = 57%, mp 285–287 °C; 1H NMR (400 MHz, DMSO-d6): d
1.28 (t, 3H, J = 1.64 Hz, 1.68 Hz,CH3), 2.37(s, 3H, CH3), 2.59 (m, 5H,
Ar-CH3), 4.32(s, 1H, NH), 7.36 (t, 2H, J = 8.38 Hz, 8.70 Hz), 7.48 (d,
1H, J = 7.90 Hz), 7.54 (d, 1H, J = 1.15 Hz); 13C NMR (100 MHz,
DMSO-d6,) d 19.45, 29.82, 121.21, 122.86, 127.38, 147.57, 149.32,
164.58, 168.74, 199.39; IR (KBr, cmꢀ1): 1690, 1718, 3090, 1475;
MS: (ESI) m/z [M+H]+ calcd for [C16H16N4OS]+: 312.10; found:
313.10.
then it was incubated in a water bath for 15 min at 30 °C. The reac-
tion was stopped and about 20 mL of reaction mixture spotted on a
strip of phosphocellulose P81 paper then dried. This strip was
washed with 1% phosphoric acid solution thrice for about ten min-
utes. In the same manner control was also treated but washing of
control strip was avoided, both the strips were dried and then
radioactivity was calculated on the scintillation counter in pres-
ence of scintillation fluid. The corrected cpm was determined by
subtracting the count of blank control value, the values were calcu-
lated by following formula;
2.2.24. 1-[2-(1,3-Benzothiazol-2-ylamino)-4,6-
dimethylpyrimidin-5-yl]ethanone (32)
Specific Radioactivity (SR) of ATP (cpm/nmol)
Benzothiazolyl guanidine 30 (5 mmol), methyl acetoacetate 8
(5 mmol) in the trimethyl orthoacetate (5 ml) was refluxed for
60 min. Completion of the reaction was monitored by TLC. Excess
cpm of 5ml of g ꢀ 32P-ATP assay combination
SR ¼
nmol of ATP
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
2.5. Single crystal X-ray studies
cpm = value from control
nmol of ATP = 1.25 nmol (5 ml of 250 mM ATP Assay Cocktail)
X-ray data for compounds 12 and 26 was collected on a BRU-
KER-AXS SMART APEX CCD X-ray diffractometer, using graphite-
Specific Kinase Activity (SA) (nmol/min/mg).
monochromatic Mo K
a radiation (=0.71073 Å). Data was reduced
D
cpm ꢁ ð25=20Þ
SR ꢁ E ꢁ T
using SAINTPLUS,30 and a multi-scan absorption correction using
SADABS31 was performed. The structure was solved using
SHELXS-97 and full matrix least-squares refinements against F2
were carried out using SHELXL-97.32 All ring hydrogen atoms were
assigned on the basis of geometrical considerations and were
allowed to ride upon the respective carbon atoms. All hydrogen
atoms were assigned fixed Uiso values, equal to 1.2Ueq of the parent
atom for ring and 1.5Ueq for methyl hydrogen. Crystallographic
data and structure refinement parameters are presented in Tables
8–10 for compounds 12 and 26, the CCDC deposition number for
12 is CCDC 1059194 and for 26 is CCDC 1059195.
nmol=min=mg ¼
SR = specific radioactivity of the ATP (cpm/nmol ATP)
D
cpm = cpm of the sample ꢀ cpm of the blank
25 = total reaction volume
20 = spot volume
T = reaction time (minutes)
E = amount of enzyme (mg)
2.3.2. Angiotensin-I converting enzyme colorimetric assay
The Angiotensin Converting Enzyme (ACE) inhibitor screening
assay was done based on colorimetric, high-throughput method
developed by Jimsheena and Gowda.21 Earlier ACE activity estima-
tion was carried out by reversed-phase High-Performance Liquid
Chromatography (RP-HPLC)22 or capillary electrophoresis,23 these
were highly expensive and time consuming methods requiring
expensive instrumentation as well. Most assays are based on the
hydrolysis of hippuryl-histidyl-leucine (HHL) to hippuric acid
(HA) and histidylleucine (HL) and the extent of hippuric acid
released is considered directly proportional to ACE activity. In this
colorimetric method released HA was complexed with pyridine
and benzene sulfonyl chloride (BSC) leading to formation of a yel-
low color, which was measured at 410 nm. The porcine kidney ACE
powder and the hippuryl-histidyl-leucine (HHL) was obtained
from Sigma–Aldrich Chemicals, the HHL substrate was used for
assay directly.
To study the inhibitory activity of synthesized compounds and
to determine their IC50 values a high throughput assay was per-
formed. In a 96-wellquartz microtiter plate a total volume of
0.25 mL each well was maintained. The assay mixture contained
0.125 mL of 1 M sodium borate buffer pH 8.2 containing 0.3 M
NaCl, 0.05 mL of 5 mM HHL, and 0.025 mL of ACE enzyme. The
consecutive two rows were used to assay ACE at different dilu-
tions in a total reaction volume of 0.0375 mL. The intermediate
and last rows were used to assay the inhibitory activity of the
compounds 11–29, 31, 32 and 34. This mixture was incubated
at 37 °C for 30 min and then stopped by addition of 0.2 mL of
1 M HCl. To this mixture 0.4 mL of pyridine was added followed
by 0.2 mL of benzene sulfonyl chloride (BSC), it was mixed prop-
erly by inverting the container for one minute and then cooled
on ice; yellow color developed was measured at 410 nm. The
IC50 value is defined as the concentration of inhibitor required
to decrease ACE activity by 50%. A minimum of 3–5replicates
were taken for data analysis the percent inhibition curves were
2.6. In silico studies
2.6.1. Structure prediction and validation
The full length experimentally solved structure for calcium
channel protein is not available till date in Protein structure
databases. The sequence of protein was then retrieved from NCBI
genome database.33 Three dimensional structure of calcium
channel protein was generated using ab initio method via
Rosetta structure prediction tool.34,35 Rosetta use Metropolis
Monte Carlo method coupled with energy functions, most of
these molecular properties can be derived from structural infor-
mation datasets such as Protein Data Bank (PDB).36 The pre-
dicted structure was evaluated using SAVE server tool which
provide information on quality of modeled structure. SAVEs pro-
vides Ramachandran Plot, analyzes the residue-by-residue geom-
etry of protein and plot the graph of overall stereo-chemical
quality of structure. Verify-3D tool analyzes the amino acid
sequence (1D) with generated atomic model (3D) compatibility
of structure and determine statistical error value of non-bonded
interactions between different atom types compared by highly
refined structures was plot by ERRAT score.37–41
2.6.2. Molecular docking and dynamics study
To confirm best inhibitor of the series or the interaction
between selected compound and modeled calcium channel pro-
tein, molecular docking studies was carried out with help of Glide
docking module of Schrodinger. The CASTP server (Computed Atlas
of Surface Topography of Proteins) was employed and active site
cavity was selected.42,43 The active site cavity is a space for better
binding of ligand to fit inside the protein structure which inhibits
the functions of protein by blocking the binding affinity toward
other molecules. The lead compound library was generated for
maximum number of possible conformations using LigPrep mod-
ule of Schrodinger and docking with protein was carried out using
Glide docking module.44 The molecular dynamics studies were car-
ried out to evaluate the binding affinity, stability and conforma-
tional changes of protein–ligand complexes. The molecular
dynamics studies were carried out using Gromacs 5.0.4 package
for time period of 10 ns.45
plotted using
a minimum of five determinations for each
inhibitor concentration, and IC50 values computed from these
logarithmic plots.
2.4. Pharmacological evaluation
The method for induction of hypertension by DOCA salt hyper-
tension method,24,25 non-invasive (tail cuff) and invasive (carotid
artery cannulation) method,26 Ulcerogenic studies27 were devel-
oped and reported earlier form our lab.16 PK/PD studies were car-
ried out according to standard protocols and reported articles
earlier form our lab.28,29 The detailed protocols are mentioned in
Supplementary material.
2.7. Three dimensional quantitative structure activity
relationship studies using k-Nearest Neighbor Molecular Field
Analysis (kNN-MFA 3D QSAR) method
Three-dimensional structures were drawn for each molecule in
ChemDraw Ultra 8.0 molecular design suite and the geometries
were optimized using Monte Carlo conformational analysis, Merck
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8
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Molecular Force Fields (MMFF) and other charges in the Vlife MDS
engine 4.3.2.24 The Vlife MDS engine consist of the 2D and 3D stud-
ies, we selected the 3D studies to explain QSAR for the newly syn-
thesized compounds. The molecules were aligned by the template
based techniques using the basic nucleus. 3D descriptors such as
Tripos force field, electrostatic, steric and hydrophobic field types,
with cut-off values of 10.0 and 30.0 kcal/mol respectively with
Gasteiger and Marsilli charge type was selected for building the
model, about 5100 field descriptors were studied and analyzed.
As the model is based on grid based method a rectangular grid
was formed initially around the molecules.
similar to Mannich condensation. In the next step the iminium
intermediate generated undergoes a nucleophilic addition on the
ketoesters, this intermediate undergoes condensation with the sec-
ondary amine group of 2-aminobenzothiazoleto yield the cyclized
product. In this case the 2-aminobenzothiazole plays role of
thiourea as in regular Biginelli reaction. Earlier to our report Heravi
et al. has presented synthesis of several derivatives of pyrimi-
dobenzothazolyl by means of novel N-sulfonicacid modified poly
(styrene-maleic anhydride) catalyst46 however, our synthetic pro-
tocol provided with better % yields, simple procedure and low cost
of synthesis.
The set of molecules was dived into training and test set based
on the sphere exclusion method leading to a training set/test set of
10:5 molecules for the series 6a–6o and training set/test set of
14:6 molecules for the series 10–29. In pursuit of better model
we studied all the three possible methods based on the kNN-
MFA namely k-Nearest Neighbor-Stepwise Variable Selection, k-
Nearest Neighbor-Genetic Algorithm, k-Nearest Neighbor-Simu-
lated Annealing. The descriptors were obtained in form of field
points and correlation plots for all the molecules.
Spectroscopic studies establish the structures of this series,
the IR spectrum showed characteristic absorption band for
(C@O) belonging to the esters at about 1690, 1718 cmꢀ1, (C–H)
stretching around 3090 cmꢀ1, (C–C) stretching at 1475 cmꢀ1, in
compounds with nitro derivatives absorptions were found
around 1468 cmꢀ1, compounds with hydroxyl group substitu-
tions also exhibited characteristic absorption band around
3380 cmꢀ1 1H NMR studies of this series exhibited characteristic
.
peaks for nucleus and substituted groups. The pyrimidobenzoth-
iazolyl nucleus was confirmed by presence of methyl group at
the sixth position of ring nucleus corresponding to 2 ppm as tri-
plet, aromatic protons exhibited in the range of 6.3–8.5 ppm for
the conjugated benzene ring of benzothiazole. The proton on
fourth carbon peculiar to dihydropyrimidine nature of Bigenilli
derivatives was observed around 5.3–6.7 ppm. The protons cor-
responding to methyl and ethyl groups of the carboxylate chain
were exhibited around 2.4–3.6 ppm as singlet or triplet. The
ethylene protons were exhibited at about 4.0 ppm with multiplet
peak behavior. 13C NMR spectra exhibited the aliphatic and aro-
matic regions significantly with the seven carbons in aliphatic
region between 13 and 78 ppm and remaining carbon nuclei
around 100–165 ppm justifying the aromatic nature of com-
pounds. The structure for all derivatives were finally confirmed
on the basis of HRMS studies and were found to be correct based
on the molecular ion peak analysis and the M+H and MꢀH peaks
on the spectra.
2.8. High Performance Liquid Chromatography (HPLC) and High
Performance Thin Layer Chromatography (HPTLC) studies
The HPLC studies were carried out on Shimadzu prominence
system with PDA detector, samples were analyzed with a station-
ary phase column Zorbax SB-C18 by Agilent and stationary phase
was methanol 100% v/v. The sample was injected at a volume of
20 ll using a rhynodyne injector with a flow rate maintained at
1.0 ml/min at 25 °C. The detection wavelength was set at 343 nm
for compound 12 and at 258 nm for compound 26.48,49
The HPTLC studies were carried out for compounds 12 and 26
under optimized conditions for chromatography. Stationary phase
was the pre-coated silica gel 60 F254 plates with a thickness of
200
lm. Sample was applied in form of band with a width of
5 mm and volume 5
l
l. After several trials mobile phase was set
at toluene/acetonitrile (9:1). Ascending separation technique was
employed with a pre-saturated chamber for 15 min prior to run
up to 90 mm. Scanning of plate was carried out under scanning
mode of absorbance/reflectance at wavelength of 343 nm for com-
pound 12 and at 258 nm for compound 26 with a scanning speed of
20 nm/s.
3.1.2. Synthesis of compounds 30–34
Synthesis of benzothiazolyl guanidine (30) was accomplished
by simple condensation reaction between 7 and S-methyl isoth-
iourea hemisulfate salt (SMIT), the reaction proceeded under the
condition of reflux in a mixture of water/ethanol (1:1) yielding
the product as white crystalline material after neutralising with
dilute sodium hydroxide. This step in synthesis is very useful for
generating guanidine at the lab scale. It was characterized with
IR spectroscopy which exhibited the prominent peak of primary
amine at 3424.1 and 3339.2 cmꢀ1. The 1H NMR spectra exhibited
protons belonging to amines around 3.63 ppm and the aromatic
protons for benzothiazole at about 6.9–7.6 ppm. 13C NMR reflects
the single aliphatic carbon at 78.54 ppm in 30 and the remaining
carbon nuclei are found in aromatic region of above 100 ppm
accounting for seven nucleus.
3. Results
3.1. Chemistry
The synthetic route and method of synthesis for obtaining the
designed compounds is outlined in Schemes 2, Scheme 1 is already
reported in our previous article and hence its synthetic data is not
mentioned here.
3.1.1. Synthesis of compounds 10–29
Compound 30 was reacted with ethyl acetoacetate 2 or methyl
acetoacetate 8 in presence of trimethyl orthoacetate (TMOA) under
the conditions of reflux to obtain compounds 31 and 32 respec-
tively by methods reported by Potapov et al.17,18 The compounds
were characterized by IR, it exhibited the characteristic absorption
band for (C@O) at about 1690, 1718 cmꢀ1, (C–H) stretching around
3090 cmꢀ1, (C–C) stretching at 1475 cmꢀ1. The 1H NMR spectra
exhibited the aromatic nature with aromatic protons around 7.5–
8.5 ppm, the secondary amine proton formed a broad singlet at
4.2 ppm in both the derivatives. The differentiating point was
occurrence of a multiplet at about 2.5 in case of 31and disappear-
ance of the same to form a singlet with three protons in 32. Further
these compounds were confirmed by 13C NMR and Mass spectra.
2-Aminobenzothiazole 7 was obtained by methods earlier
reported so we have not mentioned its synthesis.16 Compound 7
in presence of ethyl or methyl ester 2, 8 and various substituted
benzaldehydes 9 under the influence of catalytic amount of
hydrochloric acid yields compounds 10–29 with methanol as
solvent. All compounds were obtained in satisfactory yield of
60–80% in about 14 h based on the TLC monitoring of reaction pro-
gress, they were recrystallized from solvent system of dimethyl
formamide/ethanol (1:1). This synthesis is based on the principles
of one pot multicomponent reaction (MCR) of Biginelli type.
Reaction mechanism can be explained in two steps, initially the
aldehyde and 2-aminobenzothiazole undergoes condensation
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Table 1
Data of the in vitro studies for MLCK assay, ACE assay and ClogP by the Shake-Flask method
R
R4
N
R1
R3
N
R2
ClogPc ClogPd
a
b
Sr. No. Compd
R
R1
R2
R3
R4
IC50 for MLCK IC50 for
assay ( M) ACE assay
M)
l
(
l
O
1
2
6a
6b
–OCH3
–OCH3
–CH3
–CH3
–COOC2H5 23.3 1.4
–COOC2H5 33.3 8.3
>100
>100
4.82
5.61
NT
NT
O
Cl
O
3
4
5
6c
6d
6e
–OCH3
–OCH3
–OCH3
–CH3
–CH3
–CH3
–COOC2H5 13.3 8.4
–COOC2H5 31.3 8.3
–COOC2H5 41.3 8.6
>100
>100
>100
5.04
5.53
6.32
NT
NT
NT
OCH3
O
Cl
Cl
O
Cl
O
6
7
8
6f
–OCH3
–OCH3
–OCH3
–CH3
–CH3
–CH3
–COOC2H5 18.2 8.4
–COOC2H5 10.2 8.4
–COOC2H5 15.2 1.4
>100
>100
>100
5.52
4.15
4.94
NT
NT
NT
OCH3
Cl
O
O
6g
6h
OH
OH
Cl
O
9
10
11
6i
–OCH3
–OCH3
–OCH3
–CH3
–CH3
–CH3
–COOC2H5 9.8 8.4
–COOC2H5 19.2 8.4
–COOC2H5 29.2 4.1
>100
>100
>100
4.37
5.32
6.10
NT
NT
NT
OH
OCH3
O
6j
CH3
O
6k
CH3
Cl
O
12
13
6l
–OCH3
–OCH3
–CH3
–CH3
–COOC2H5 5.1
6
>100
>100
5.54
4.74
NT
NT
CH3
OCH3
O
6m
–COOC2H5 7.4 1.4
OCH3
(continued on next page)
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10
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1 (continued)
a
b
Sr. No. Compd
R
R1
R2
R3
R4
IC50 for MLCK IC50 for
assay ( M) ACE assay
M)
ClogPc ClogPd
l
(
l
O
14
15
6n
6o
–OCH3
–OCH3
–CH3
–CH3
–COOC2H5 30.3 8.6
–COOC2H5 4.1 1.1
>100
>100
5.52
4.96
NT
NT
OCH3
Cl
O
OCH3
OCH3
Cl
N
16
17
18
19
20
21
22
23
24
25
26
27
28
10
11
12
13
14
15
16
17
18
19
20
21
22
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
–COOCH3
9.8 8.4
9.1 1.6
7.3 2.3
4.3 1.5
4.21 1.3
2.14 1.1
2.04 1.7
2.04 1.7
4.37 1.4
3.67 3.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.49
5.49
4.92
4.06
4.11
4.69
4.52
4.52
4.94
3.95
6.01
6.01
5.44
NT
S
N
5.44
4.85
NT
Cl
F
S
N
S
N
HO
S
N
NT
OH
S
N
4.64
4.45
4.49
NT
OCH3
S
N
NO2
S
N
NO2
S
N
CH3
N
CH3
S
N
OCH3
3.89
NT
OH
S
N
Cl
–COOC2H5 10.8 8.6
–COOC2H5 11.1 1.6
–COOC2H5 13.3 2.3
S
N
NT
Cl
F
S
N
5.32
S
N
HO
29
30
23
24
–CH3
–CH3
–COOC2H5 4.7 1.5
–COOC2H5 5.9 1.3
>100
>100
4.58
4.63
NT
NT
S
N
S
OH
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
Table 1 (continued)
a
b
Sr. No. Compd
R
R1
R2
R3
R4
IC50 for MLCK IC50 for
assay ( M) ACE assay
M)
ClogPc ClogPd
l
(
l
N
S
31
25
–CH3
–COOC2H5 2.17 1.2
>100
5.22
5.14
OCH3
NO2
N
S
32
33
34
26
27
28
–CH3
–CH3
–CH3
–COOC2H5 2.1 1.7
–COOC2H5 3.04 1.4
–COOC2H5 7.37 1.4
>100
>100
>100
4.64
4.64
5.47
4.57
4.70
NT
N
NO2
S
N
CH3
N
CH3
S
OCH3
N
S
35
29
–CH3
–COOC2H5 3.67 3.7
>100
4.48
4.37
OH
N
H2
H2
H2
N
36
37
31
32
–CH3
–CH3
—
—
–C2H5 –COCH3
>100
>100
>100
>100
NT
NT
NT
NT
S
N
N
–CH3
–CH3
S
N
N
38
39
34
–CH3
—
—
—
–CH3
—
–CH3
—
>100
>100
NT
NT
S
Nifedipine*
—
2.02 1.1
2.34 3.1
3.57
3.86
NT—Not Tested.
a
IC50 determined from the MLCK assay (
l
M).
b
c
IC50 determined from the ACE assay (
lM).
Calculated by Cambridgesoft corporation’s ChemDraw Ultra 11.0.
Determined by the Shake-Flask method with HPLC.
Nifedipine (standard drug).
d
*
Figure 2. IC50 values for MLCK assay for compound series 6a–6o and 10–34. The results for 6a–6o are found to be insignificant as compared to the standard drug Nifedipine
whereas compounds 15, 16, 17, 25 and 26 exhibits comparable inhibitory action.
Compound 34was obtained on reaction between 30 and acetyl ace-
tone 33 under the reflux condition n presence of xylene as solvent.
Its structure was confirmed on the basis of above mentioned tech-
niques 1H NMR spectra of 34 exhibited characteristic peaks at
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12
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Antihypertensive activity obtained by non-invasive tail-cuff method at a dose of 10 mg/kg
Compd
Average blood pressure (mm Hg) at time (h)
0
1
2
3
4
5
6
7
8
9
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
32
34
Control
226
226
225
224
228
225
226
226
226
222
226
230
220
225
225
226
225
225
225
226
225
226
225
225
225
4
8
1
3
3
4
2
2
4
3
4
3
5
3
4
8
3
4
6
3
8
4
8
2
1
222
220
214
216
220
222
224
226
222
220
222
220
210
220
222
224
214
222
223
224
224
224
224
224
214
8
5
2
6
6
8
3
3
8
6
8
6
6
5
8
5
2
8
5
6
8
3
5
1
2
220
200
201
205
210
221
210
215
220
210
220
210
200
210
221
210
202
220
210
210
225
225
225
225
201
9
5
1
3
3
9
8
8
9
3
9
3
5
3
9
5
1
9
6
3
2
3
4
1
1
193
193
193
180
183
195
190
195
193
193
193
190
185
190
195
193
193
190
195
193
224
224
224
224
193
2
4
3
5
5
2
4
4
2
5
2
5
1
5
2
4
3
2
8
5
8
6
4
3
3
185
170
182
170
170
180
172
170
185
178
185
175
175
175
180
178
182
178
180
178
222
221
222
224
182
4
5
2
7
7
4
5
5
4
7
4
7
7
7
4
5
2
4
5
7
4
5
8
4
2
175
161
173
165
161
170
160
165
175
161
175
160
160
160
175
161
173
172
160
161
211
214
213
224
173
8
4
1
2
2
8
4
4
8
2
8
2
2
4
8
4
1
8
8
2
1
8
4
1
1
170
161
161
150
150
168
142
140
170
150
170
140
140
145
162
155
163
167
145
142
208
210
209
225
151
6
4
2
5
5
6
4
4
6
5
6
5
5
6
6
4
6
6
3
5
5
4
2
1
2
165
155
141
142
141
155
135
135
165
141
165
125
135
137
155
155
140
160
140
138
207
209
206
224
131
4
6
2
6
6
4
6
6
4
6
4
6
5
7
4
6
6
4
6
6
8
3
4
4
2
161
150
138
130
138
145
130
130
161
138
161
120
135
131
150
150
131
158
135
134
207
209
206
225
118
6
7
1
7
7
6
7
7
6
7
6
7
5
7
6
7
4
6
7
7
4
2
4
2
1
155
140
128
126
135
135
128
120
155
133
155
115
125
131
150
145
119
152
128
130
206
208
205
224
108
3
5
2
5
5
3
5
5
3
5
3
5
5
5
3
5
1
3
5
5
8
5
6
3
2
150
135
113
120
132
115
118
120
150
131
150
120
124
130
140
135
110
145
128
124
205
207
205
225
103
3
8
3
8
8
3
8
8
3
8
3
8
7
8
3
8
5
3
7
8
7
5
1
2
3
Nifedipine (standard)
Table 3
Comparative study of inhibition (%) for antihypertensive activity by NIBP method
Compd
Average blood pressure (mm Hg) at time (h)
0
1
2
3
4
5
6
7
8
9
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
32
34
Control
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.77
2.65
4.89
3.57
3.51
1.33
0.88
0.00
1.77
0.90
1.77
4.35
4.55
2.22
1.33
0.88
4.89
1.33
0.89
0.88
0.44
0.88
0.44
0.44
4.89
2.65
14.60
14.60
14.22
19.64
19.74
13.33
15.93
13.72
14.60
13.06
14.60
17.39
15.91
15.56
13.33
14.60
14.22
15.56
13.33
14.60
0.44
18.14
24.78
19.11
24.11
25.44
20.00
23.89
24.78
18.14
19.82
18.14
23.91
20.45
22.22
20.00
21.24
19.11
20.89
20.00
21.24
1.33
22.57
28.76
23.11
26.34
29.39
24.44
29.20
26.99
22.57
27.48
22.57
30.43
27.27
28.89
22.22
28.76
23.11
23.56
28.89
28.76
6.22
24.78
28.76
28.44
33.04
34.21
25.33
37.17
38.05
24.78
32.43
24.78
39.13
36.36
35.56
28.00
31.42
27.56
25.78
35.56
37.17
7.56
26.99
31.42
37.33
36.61
38.16
31.11
40.27
40.27
26.99
36.49
26.99
45.65
38.64
39.11
31.11
31.42
37.78
28.89
37.78
38.94
8.00
28.76
33.63
38.67
41.96
39.47
35.56
42.48
42.48
28.76
37.84
28.76
47.83
38.64
41.78
33.33
33.63
41.78
29.78
40.00
40.71
8.00
31.42
33.63
40.27
49.78
46.43
42.11
48.89
47.79
46.90
33.63
40.99
33.63
47.83
43.64
42.22
37.78
40.27
51.11
35.56
43.11
45.13
8.89
11.50
10.67
8.48
7.89
1.78
7.08
4.87
2.65
5.41
2.65
8.70
9.09
6.67
1.78
7.08
10.22
2.22
6.67
7.08
0.00
0.44
0.00
0.00
10.67
38.05
43.11
43.75
40.79
40.00
43.36
46.90
31.42
40.09
31.42
50.00
43.18
41.78
33.33
35.84
47.11
32.44
43.11
42.48
8.44
0.88
0.44
0.44
14.22
2.21
1.33
0.44
19.11
5.31
5.33
0.44
23.11
7.08
7.11
0.00
32.89
7.52
8.44
0.44
41.78
7.52
8.44
0.00
47.56
7.96
8.89
0.44
52.00
8.41
8.89
0.00
54.22
Nifedipine (standard)
2.35 ppm contributing to six protons of methyl groups on the
pyrimidine ring and the singlet of one proton of pyrimidine at
6.28 ppm.
ing as it is known that benzazoles act by both mechanisms of CCBs
and ACE inhibitors.
3.2.1. MLCK studies
3.2. In vitro studies
Myosin light chain kinase assay (MLCK) was carried out to
determine the mode of action of newly synthesized compounds.
Compounds of the series 6a–6o and 10–34 were subjected to assay
and IC50 was determined on the basis of specific kinase activity
(SA). It was observed that from the series 6a–6o, compounds 6l,
Myosin light chain kinase assay and Angiotensin-I converting
enzyme colorimetric assay were carried out to determine the mode
of action for compounds under screening. The distinction between
modes of action helped select the animal model and in vivo screen-
6m and 6o were found to posses IC50 in range of 4–8 lM whereas
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
13
induced hypertension in rat model. The non-invasive blood pres-
sure method enumerates systolic blood pressure (SBP), whereas
invasive or the carotid artery cannulation method helps determine
diastolic blood pressure (DBP) along with mean arterial blood pres-
sure (MAP). The compounds which exhibited promising results
were further evaluated for their severity index to determine
ulcerogenic nature of compounds as treatment of hypertension is
very long and subject has to be on therapy for life time. On same
grounds selected compounds were subjected to physicochemical
and pharmacokinetic studies.
3.3.1. Non invasive blood pressure method (NIBP)
This method provided with systolic blood pressure (SBP), obser-
vations made from recording of variations in blood pressure by
tail-cuff method is summarized in Table 2. We selected the deriva-
tives for NIBP measurements on basis of substitution in final mole-
cule so that structure activity relationship can be established.
Likewise compounds with electron donors, withdrawers and halo-
gens as substitutions were selected for this study. Para-methoxy
and meta-nitro derivatives 15, 16, 25 and 26 exhibited remarkable
antihypertensive activity, Para-nitro derivatives 17 and 24 exhib-
ited good antihypertensive effect at 10 mg/kg. Compounds 13,
14, 23 and 24 had were found to be moderately potent compared
to 10, 20, 21 and 22 which have shown poor response compared
to the standard drug. The % inhibition with respect to dose of
10 mg/kg is detailed in Table 3 and Figure 3. Data is presented as
means SEM and a repeated measure for analysis of variance
was used to determine the statistical significance between and
within groups. The differences were considered statistically signif-
icant at a P level lower than 0.0001 and F value for all the com-
pounds are F: 28.67 0.05. From the Analysis of Variance
(ANOVA) and % inhibition studies it was found that compounds
11, 12, 15, 16, 17, 19, 22, 25, 26, 27 and 29 exhibited significant
to good antihypertensive activity and were further tested at lower
dose levels of 5 and 2.5 mg/kg.
Figure 3. Graph showing decrease in systolic blood pressure (SBP) as % inhibition
with respect to time on hourly basis by the non-invasive blood pressure method.
6b, 6d, 6e, 6k and 6n gave very insignificant results in assay test
(Table 1). These findings are in consent with previously deter-
mined in vivo studies on these molecules; however the difference
between biochemical assay and pharmacological screening is
based on the concentration of substance being administered. To
verify the MLCK activity of series 10–34, same protocol was fol-
lowed and the specific kinase studies revealed that 15, 16, 17, 25
and 26 exhibits IC50 in range of 2–3.7 lM which is close to the con-
centration of standard drug Nifedipine at 2.02 lM (Fig. 2).
3.2.2. ACE colorimetric assay
Angiotensin Converting Enzyme (ACE) inhibitor assay was car-
ried out following the MLCK assay for differentiating mode of
action of compounds under study. Method applied involves a col-
orimetric determination for inhibitory activity of compounds
under study and compare it with standard drug Nifedipine. It
3.3.2. Invasive blood pressure method (IBP)
This method provides with diastolic blood pressure and mean
arterial blood pressure since the cannulation is done on the carotid
artery. Compounds 11, 12, 15, 16, 17, 19, 22, 25, 26, 27 and 29
exhibited promising results by NIBP method; these were subjected
to IBP technique to evaluate effect on diastolic blood pressure. The
results for diastolic activity or the IBP is given in Table 4, the per-
cent inhibition exhibited by selected compounds with respect to
standard drug Nifedipine is given in Table 5. It was found that com-
pounds 12 and 26 exhibited promising results with respect to ini-
tiation of inhibition and length of time. The data is presented as
means SEM, repeated measure analysis of variance was used to
was found that all compounds showed an IC50 above 100 lM com-
pared to Nifedipine which exhibited IC50 of 2.34 3.1 (Table 1).
This result indicates that compounds under study act by modulat-
ing the Calcium channels rather than ACE mechanism.
3.3. Pharmacological evaluation
Compounds from the series 10–34 were subjected to antihyper-
tensive activity by Deoxycorticosterone Acetate salt (DOCA-salt)
Table 4
Antihypertensive activity obtained by direct cannulation of carotid method (10 mg/kg dose)
Compd
Average blood pressure (mm Hg) at time (H)
0
0.15
0.30
1.0
2.0
3.0
4.0
11
12
15
16
17
19
22
25
26
27
29
Control
226
225
226
225
226
226
224
226
225
226
226
225
225
2
1
2
1
2
2
3
3
6
2
2
2
1
221
217
216
214
221
216
220
218
216
221
216
224
211
3
2
3
2
3
3
6
6
2
3
3
1
2
216
199
210
190
216
210
203
210
180
216
210
225
194
8
1
8
1
8
8
3
3
1
8
8
1
1
190
190
190
180
190
190
180
193
176
190
190
224
189
4
3
4
3
4
4
5
5
3
4
4
3
3
170
168
165
161
170
165
165
168
160
170
165
224
165
5
2
5
2
5
5
7
7
2
5
5
4
2
156
148
156
131
156
156
150
156
130
156
156
224
141
4
1
4
1
4
4
2
2
1
4
4
1
1
114
124
130
110
116
130
125
132
107
116
130
225
104
8
8
5
8
8
5
8
8
8
8
5
7
8
Nifedipine (standard)
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14
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 6
Table 5
Ulcerogenic activity of compounds from Scheme 2
Comparative study of Percent (%) inhibition for antihypertensive activity by direct
cannulation of carotid artery method (10 mg/kg dose)
Compound (50 mg/kg)
Ulcerogenic activity (severity index)
Compd
Average blood pressure (mm Hg) at time (H)
0.15 0.30 1.0 2.0 3.0 4.0
2.21 4.42 15.93 24.78 30.97 49.56
3.56 11.56 15.56 25.33 34.22 44.89
4.42 7.08 15.93 26.99 30.97 42.48
4.89 15.56 20.00 28.44 41.78 51.11
11
12
15
16
17
19
22
25
0.36
0.35
0.54
0.25
0.20
0.20
0.25
0.36
0.20
0.35
0.25
0.00
0.54
1.97
0
11
12
15
16
17
19
22
25
26
27
29
Control
0
0
0
0
0
0
0
0
0
0
0
0
0
2.21
4.42
1.79
3.54
4.00
2.21
4.42
0.44
4.42 15.93 24.78 30.97 48.67
7.08 15.93 26.99 30.97 42.48
9.38 19.64 26.34 33.04 44.20
7.08 14.60 25.66 30.97 41.59
26
27
29
20.00
21.78 28.89 42.22 52.44
Control
Nifedipine
Ibuprofen
4.42 15.93 24.78 30.97 48.67
7.08 15.93 26.99 30.97 42.48
0.00
0.44
0.44
0.44
0.00
Nifedipine (standard)
6.22 13.78 16.00
26.67 37.33 53.78
Table 1 along with theoretical values generated by computational
algorithm. It was observed that experimental and theoretical val-
ues of logP are in good understanding with each other exhibiting
the coefficient of partition around 5 which is in agreement to the
Lipinski scale of 5 for drug likeness of a new molecule under study.
3.3.5. Pharmacokinetic studies
In this study Nifedipine was used as standard drug for evaluat-
ing pharmacological and pharmacodynamic-kinetic studies of
newly synthesised compounds. Nifedipine is known to exhibit
bioavailability of 56–77% by oral route and gains a mean plasma
concentration in range of about 0.16 lg/ml in 30–60 min. Four
compounds 12, 16, 22 and 26 were subjected to pharmacokinetic
studies based on the substitutions and their performance as anti-
hypertensive activity. It was observed that 12 exhibited a Cmax of
1.80 and 0.59 lg/ml by intravenous and oral route respectively
with a Tmax of 0.5 and bioavailability of 44.37% which is quite good
for compound with halogen as substitution. 16 and 22 also gave
good results with bioavailability of 56.37 and 53.00% respectively.
Figure 4. Graph showing decrease in diastolic blood pressure (DBP) as % inhibition
with respect to time on hourly basis by the carotid artery cannulation method.
26 exhibited highest availability of 56.75% with a Cmax of 0.41
lg/
ml by oral route and a t1/2 of 5.4 h and AUC of 0.78
lg h/ml (see
Table 7).
obtain the statistical significance within and between the groups.
The ANOVA results provide with P level lower that 0.0001 and F
value for all compounds are F: 10.60 0.05, the results are shown
in Figure 4.
3.4. Single crystal X-ray studies
Two compounds from this series viz., methyl 4-(4-fluo-
rophenyl)-2-methyl-4H-pyrimido[2,1b] [1,3] benzothiazole-3-car-
boxylate 12and ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido
[2,1-b] [1,3] benzothiazole-3-carboxylate 26 were selected for sin-
gle crystal X-ray studies to determine their atomic and molecular
organization and geometric orientation in crystal lattice. The
details regarding crystallographic data and structure refinement
is presented in Tables 8–10. The compound 12 crystallised into
monoclinic system with space group P21/c, a = 12.629(3) Å,
3.3.3. Ulcerogenic studies
Compounds exhibiting significant antihypertensive properties
were further screened for their ulcerogenic activity. The test com-
pounds exhibited ulcerogenic activity ranging from 0.20 to 0.56 on
the severity index whereas nifedipine as a standard drug displayed
similar reading at the 0.54 whereas Ibuprofen an NSAID exhibited
severity of 1.97. The results are given in Table 6, structurally com-
pounds with nitro or methoxy group substitution like in 17, 19 and
29 displayed less severity of about 20–25 whereas compounds
with halogens as substitution displayed a little higher severity of
about 36. However, it is observed that these derivatives are quit
safe compared to NSAIDs like Ibuprofen.
b = 6.7018(16) Å, c = 22.365(5) Å,
a = 90°, b = 104.819(4)°, c = 90°,
Z = 4 and V = 1830.0(8) Å3. The unit cell consist of two molecules
of 12 in a dimeric interaction via a chlorine atom, proton on first
nitrogen and sulfur in the benzothiazole portion of molecule are
involved in this dimeric formation with a prominent Cl–H bond
length of 2.082(2) Å. The ORTEP and dimeric view of 12 is pre-
sented in Figures 5 and 6. The compound 26 crystallised into tri-
3.3.4. Physicochemical studies
ꢀ
clinic system with space group P1; a = 8.3412(15) Å, b = 10.5799
Compounds found to be exhibiting good antihypertensive activ-
ity by both methods were subjected to determination of their par-
tition coefficient (logP) by shake-flask method. Compounds were
treated in a binary system of Water: Octanol and subjected to
UV-HPLC analysis for estimation of concentration of compounds
in each layer after set interval of time. Results are reported in
(19) Å, c = 11.2033(19) Å,
a = 96.871(3)°, b = 101.345(3)°,
c
= 101.897(3)°, Z = 2 and V = 935.2(3) Å3. The unit cell consists of
four molecules forming a polymeric chain of compound 26 with
S1ꢂ ꢂ ꢂO1 (3.104) Å contacts along the c-axis. ORTEP and 1D view
of 26 is presented in Figures 7 and 8, respectively.
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
15
Table 7
Study on selected compounds for pharmacokinetic and bioavailability studies
Table 9
Selected bond distances (Å) for compound 12
Sr.
No.
Compd Route Dose
(mg/kg)
Cmax
Tmax t1/2 AUC0–1
Fc (%)
F(1)–C(14)
O(1)–C(17)
N(1)–C(1)
N(2)–C(1)
C(2)–C(11)
C(4)–C(19)
C(6)–C(7)
C(9)–C(10)
1.360(3) S(1)–C(1)
1.196(3) O(2)–C(17)
1.324(3) N(1)–C(6)
1.331(3) N(2)–C(4)
1.522(3) C(3)–C(4)
1.500(3) C(5)–C(6)
1.385(3) C(7)–C(8)
1.724(2) S(1)–C(5)
1.330(3) O(2)–C(18)
1.408(3) N(1)–C(2)
1.393(3) C(2)–C(3)
1.345(3) C(3)–C(17)
1.382(3) C(5)–C(10)
1.377(4) C(8)–C(9)
1.759(2)
1.452(3)
1.475(3)
1.519(3)
1.476(3)
1.385(3)
1.379(4)
(lg/ml) (h)
(h)
(lg h/ml)
1
2
3
4
12
16
22
26
i.v.a
10
p.o.b 20
1.81
0.56
—
0.5
2.6
5.5
1.51
0.67
—
44.37
i.v.
p.o.
10
20
1.51
0.33
—
0.6
2.8
5.8
1.49
0.84
—
56.37
1.378(4) C(11)–C(12) 1.376(3) C(11)–C(16) 1.384(3)
i.v.
p.o.
10
20
1.74
0.48
—
0.6
2.6
6.0
1.62
0.86
—
53.00
C(12)–C(13) 1.385(4) C(13)–C(14) 1.359(5) C(14)–C(15) 1.350(5)
C(15)–C(16) 1.382(4) N2–H2A 0.880(2) Cl–H2A 2.082(2)
i.v.
10
20
1.63
0.40
—
0.5
2.5
5.4
1.48
0.84
—
56.75
p.o.
a
b
c
Intra Venous route.
Per Oral route.
Oral bioavailability.
We engaged the dynamic modeling program Rosetta for our pur-
pose of generating the channel structure. The predicted structure
was approx 600 Amino acid (aa) length, which has three domains;
(1) Transport protein domain (1–416 aa) consisting three alpha-
helix and four beta-sheets, (2) RNA binding like domain (417–
544 aa) consisting of five helix and five sheets and (3) the helical
Chaperone domain (545–604 aa) (Fig. 9). The structure was vali-
dated using SAVE server for stereo-chemical qualities which pro-
vides the Ramachandran plot of 98.5% in favoured region
(Fig. 10), 1.1% in allowed region with overall quality factor of
85.420 predicted by ERRAT and 86.92% of Verfy-3D value average
against the 3D-1D score (Supplementary material). These observa-
tions from SAVEs concluded that the modeled structure is reliable
for further analysis.
3.5. In silico studies
3.5.1. 3D-struture prediction and validation
Functions of any protein is dependent on its 3 dimensional
geometry, that is, 3D structure of that protein which is hard to
obtain from the liner sequence. Computer generated 3D structures
of proteins is widely used to predict the structures as the gap
between experimentally obtained structures and protein sequence
availability is increasing by days. The two main methods viz.,
Homology modeling (Template-based modeling) and ab initio
modeling are widely accepted and used for the prediction of 3D
structure. If the experimental work provides similar protein struc-
ture then the framework can be copied for target protein to con-
struct the structure, but in absence of template (experimentally
solved structure) ab initio modeling (de novo modeling) is prefered
to construct the structure of protein.43 In present article we have
used ab initio modeling method to construct the structure because
experimentally solved structures were insufficient to provide
homology based modeling. Protein Data Bank (PDB) contains struc-
ture for calcium channel with approximate 200 amino acid lengths.
3.5.2. Docking and dynamics studies
Molecular docking studies were carried out for the selection of
good inhibitory mechanism of calcium channel protein from iden-
tified derivative compounds with help of Glide tool of
Schrodinger.47 The investigations suggested that compounds 12
and 26 shown good connectivity with first domain of protein via
hydrogen bonds and other interactions like Van der Walls interac-
tions. 12 forms hydrogen bonds with Lys144 and Glu181, it forms
Van der Waals interactions with Ser18, Asp20, Asn187, Pro185,
Table 8
Crystallographic data and structure refinement for compound 12 and 26
Compound
Methyl 4-(4-fluorophenyl)-2-methyl-4H-
pyrimido[2,1b] [1,3] benzothiazole-3-
carboxylate (12)
Ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido
[2,1-b] [1,3] benzothiazole-3-carboxylate (26)
CCDC deposition number
Formula
Formula weight
Crystal system
Space group
a (Å)
CCDC 1059194
19H16ClFN2O2S
390.85
Monoclinic
P21/c
12.629(3)
6.7018(16)
22.365(5)
90
104.819(4)
90
1830.0(8)
4
CCDC 1059195
C20H17N3O4S
395.44
C
Triclinic
ꢀ
P 1
8.3412(15)
10.5799(19)
11.2033(19)
96.871(3)
101.345(3)
101.897(3)
935.2(3)
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
2
T (K)
298(2)
298(2)
k (Å)
0.71073
1.419
0.348
0.71073
1.404
0.206
Dcalc (g cmꢀ3
)
l
(mmꢀ1
)
F (000)
808
412
Crystal size (mm)
h range (°)
h/k/l indices
Reflection collected
Unique reflections [Rint
Goodness-of-fit
0.32 ꢁ 0.30 ꢁ 0.28
1.668–26.080
ꢀ15, 15/ꢀ8, 8/ꢀ27, 27
18132
3617 [R(int) = 0.0323]
1.095
0.20 ꢁ 0.19 ꢁ 0.16
1.880–26.085
ꢀ10, 10/ꢀ13, 13/ꢀ13, 13
9812
3666 [R(int) = 0.0308]
1.033
]
R1[I > 2r(I)]
0.0497
0.0634
wR2 (all data)
0.1214
0.1700
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16
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 10
analysis of structure activity relationship study by Stepwise Vari-
Selected bond distances (Å) for compound 26
able Selection (SWV), Genetic Algorithm (GA) and Simulated
Annealing (SA) methods. SWV method selects variables form a
original pool of set and thus optimizes nearest neighbor (k). The
process of natural selection and survival of the fittest is the princi-
pal behind GA method. The SA method is based on process of
annealing involving distortion and reformation of confirmation is
observed. Both the set of compounds were subjected to QSAR anal-
ysis by applying all the three methods of MFA.24
S(1)–C(5)
O(2)–C(17)
O(4)–N(3)
N(1)–C(2)
N(3)–C(13)
C(3)–C(4)
C(5)–C(10)
C(7)–C(8)
1.739(3) S(1)–C(1)
1.334(4) O(2)–C(18)
1.208(3) N(1)–C(1)
1.472(3) N(2)–C(1)
1.469(4) C(2)–C(3)
1.358(4) C(3)–C(17)
1.383(4) C(5)–C(6)
1.375(4) C(8)–C(9)
1.741(3) O(1)–C(17)
1.446(4) O(3)–N(3)
1.363(3) N(1)–C(6)
1.289(4) N(2)–C(4)
1.513(4) C(2)–C(11)
1.464(4) C(4)–C(20)
1.395(4) C(6)–C(7)
1.387(5) C(9)–C(10)
1.208(3)
1.210(3)
1.394(3)
1.387(4)
1.523(3)
1.506(4)
1.384(4)
1.373(5)
C(11)–C(12) 1.379(3) C(11)–C(16) 1.385(4) C(12)–C(13) 1.383(4)
C(13)–C(14) 1.375(4) C(14)–C(15) 1.365(4) C(15)–C(16) 1.378(4)
C(18)–C(19) 1.227(7)
It was observed that in both series Model C obtained from SA-
kNN MFA explained 3D QSAR in a satisfactory manner. Observed
and predicted pIC50 values for compounds for 6a–6o and 10–29
is presented in Table 11. The contour map for compounds 6a–6o
exhibited comparative statistical coefficients by SA-kNN MFA
model like q2 (0.71), q2_se (0.12), perd_r2 (0.15) and perd_r2se
(0.42). The contributing descriptors for this model are enlisted in
Table 12 and Figure 13, these are electrostatic, hydrophobic and
steric in nature. The hydrophobic descriptor H_233 (0.2438–
0.2952) in positive range and toward carboxylate arm suggests
that an increase of hydrophobic character in this region will con-
tribute toward activity. Descriptors H_4 (0.1599–0.1885), H_370
(0.2665–0.3338) and H_352 (0.1959–0.2373) are in the region
around methyl substitution at sixth position of pyrimidine ring
suggesting for enhancing hydrophobic character for better activity
of molecules. H_339 (0.2588–0.3072) is toward the substituted
phenyl region and have a positive range suggesting for possibility
of bulkier substitution in this region. H_715 (0.2039–0.2665) and
H_809 (0.2900–0.5174) lie in the region of N-3 substitution of phe-
nyl moiety and suggest for hydrophobic groups in this region.
The electrostatic descriptors E_131 [(ꢀ0.1676)(ꢀ0.0620)] and
E_140 [(ꢀ0.0831)(ꢀ0.0056)] are in the negative range of scale indi-
cating that presence of several substitutions on the substituted
phenyl ring contributes negatively toward the activity. A single
steric descriptor S_778 [(ꢀ0.0050)(ꢀ0.0044)] was observed on
the contour plot that significantly suggest for removal of steric
effect to enhance the activity of designed molecules around the
pyrimidine ring position of 1–3, this descriptor has directed us to
design molecules with lower steric effect around the N-1, C-2
and N-3 positions of the nucleus.
Glu180, Glu181 and Arg10. The docking energy score, that is, Glide
score and Glide energy for 12 was observed as ꢀ3.682 and ꢀ41.318
respectively proving it of having good binding affinity. Compound
26 forms hydrogen bonds with Lys144, Glu181 and Asp183, it
forms the Van der Walls interactions with Ser18, Asp20, Asn187,
Pro185, Glu180, Glu181 and Arg10 (Fig. 11). The docking energy
score, that is, Glide score and Glide energy for 26 was found to
be ꢀ3.602 and ꢀ47.098, respectively proving that it has good bind-
ing affinity.
The molecular dynamics studies were carried out for 10 ns to
explore the structural stability, conformational changes and bind-
ing interactions. The stability of protein-lead compound complexes
was defined by Potential energy graphs and RMSD graphs. The
potential energy graph shows the average energy of 1.88 E-09
and 1.72 E-09 KJ/mol for compound 12 and 26, respectively which
suggest that these compounds were energetically stable
(Fig. 12A and B). Whereas the root mean square deviation (RMSD)
graph shows that complexes were maintains their stability during
the period of simulation. The stable deviations were recorded
between 2 ns and 8 ns period (Fig. 12C and D). The overall atomic
fluctuations of amino acids, that is, RMSF graph indicate that
amino acid of binding region is more flexible during interactions
while other amino acids maintaining the protein conformation
(Fig. 12E and F).
3.5.3. 3D QSAR model generated by kNN-MFA method
The contour map for compounds 10–29 exhibited comparative
statistical coefficients by SA-kNN MFA model like q2 (0.8788),
q2_se (0.1008), perd_r2 (ꢀ0.8432) and perd_r2se (0.3493). The con-
tributing descriptors for this model are enlisted in Table 13 and
Figure 14, these are electrostatic, hydrophobic and steric in nature.
The electrostatic descriptor E_484 [(ꢀ1.8374)(ꢀ1.8055)], E_332
[(ꢀ5.6892)(1.3061)] and E_620 [(ꢀ5.0924)(3.0039)] lie in the
region of phenyl ring with substitutions it is found that all of these
are exhibiting negative values symbolizing that compounds with
halogen substituted suffers from low activity due to the
electrostatic factor. E_510 [(ꢀ3.2476)(0.0972)] and E_88
[(0.7295)(1.8011)] in the bulkier region of pyrimidobenzothiazole
The 3D QSAR models were generated for both series 6a–6o and
10–29 separately since the nucleus of both sets of molecules under
study varies structurally due to conjugation of five remembered
ring of benzothiazole with the pyrimidine in the later series.
k-Nearest Neighbor Molecular Field Analysis method allows for
Figure 5. ORTEP view of the compound 12. Hydrogen atoms are omitted for clarity
Figure 6. Dimeric view of compound 12, it makes a hydrogen bonded dimer
and the thermal ellipsoids are represented at the 30% probability level.
through sulfur, chloride ion and pyrimidinum hydrogen in the lattice.
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
17
removal or modification in groups on that position. H_654
[(0.3548)(0.4459)] is the only hydrophobic descriptor in the con-
tour map indicating the positive contribution made by the car-
boxylate arm of the molecule.
3.6. High Performance Liquid Chromatography (HPLC) and High
Performance Thin Layer Chromatography (HPTLC) studies
Compounds 12 and 26 were studied for their analytical charac-
teristic and were found to exhibit satisfactory results. In HPTLC
analysis, the Rf for 12 was found at 4.2 and for 26 it was found to
be 4.2 as well. These compounds were found to be pure on the
HPTLC study. HPLC studies were carried out on compound 12
and 26, it was found that synthesized compounds exhibit peak
purity of 99.99% at retention time of 3.045 min and peak purity
of 95.08% at common retention time of 3.045 min respectively
(Fig. 15).
Figure 7. ORTEP view of the compound 26. Hydrogen atoms are omitted for clarity
and the thermal ellipsoids are represented at the 30% probability level.
4. Discussion
A global WHO report says that hypertension and other cardio-
vascular diseases are accountable to approximately 17 million
deaths a year. Of these, hypertension is responsible for 9.4 million
deaths due to elevated blood pressure related issues alone. Burden
of this disease is not only of developed or developing nations but
also on underdeveloped nations, an overall estimated expense of
3.76 trillion US$ was estimated for low and middle income
moiety contributes positively toward the activity exhibited by
these molecules. S_585 [(30.0000)(30.0000)] and S_229 [(2.8396)
(30.0000)] are the steric descriptors and indicated the positive con-
tribution of pyrimidobenzothiazole ring. S_626 [(ꢀ0.0536)
(ꢀ0.0243)] and S_272 [(ꢀ0.1355)(ꢀ0.0579)] indicates the negative
contribution made by substitution on the phenyl ring suggesting of
Figure 8. View of 1D–polymeric chain of compound 26 along the c-axis.
Figure 9. Modeled 3D structure of calcium channel protein obtained from modeling program Rosetta exhibiting Domains 1, 2 and 3 along with N and C-terminal.
Please cite this article in press as: Chikhale, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.009

18
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
year. Since last five years only one drug Perindopril,50 an endothe-
lial receptor antagonist has been approved by US FDA.51 It is lead-
ing to situation with need for new drug with longer acting
potential and with affordable cost of treatment calls for discovery
of new molecules to tackle this situation.
In an attempt to develop newer, better antihypertensive agent
we had employed dihydropyrimidine nucleus for synthesis of
structurally analogous derivatives for Nifedipine. We successfully
synthesized fifteen compounds and evaluated these molecules
for their antihypertensive activity. However, these molecules had
one major flaw toward their bioavailability and duration of action
as it was not found to be satisfactory. To solve this problem we car-
ried out pre-synthetic 3D QSAR studies prior to synthesis on this
series of molecules (Fig. 13), it was found that two descriptors
one hydrophobic (H_715) and one steric (S_778) contributed pos-
itively and negatively to the activity respectively. This region of
the contour plot refers to 1st and 2nd positions of the dihydropy-
rimidine nucleus, which are constant irrespective of substitutions
in the proposed series of molecules. The substitution on other posi-
tions varies according to aromatic aldehyde or phenacyl bromide
employed in the synthesis. Keeping in mind we designed molecule
with bulky nature at 1st and 2nd positions of the dihydropyrim-
idine nucleus, this was achieved by introducing 2-aminoben-
zothaizole instead of methyl urea in the three component
reaction. The reactions were carried out to yield twenty three
new molecules with conjugated or non-conjugated 2-aminoben-
zothiazole. Initial screening was carried out by the MLCK assay
and ACE colorimetric assay to confirm the mechanism of action
of these compounds, compounds 10–29 responded well to the
MLCK assay but 31, 32 and 34 did not show any inhibition in this
Figure 10. Ramchandran Plot for the calcium channel protein.
countries in the year 2013 for treatment of cardiovascular diseases.
America also suffers from the agony of this disease with about 70
million adults suffering from hypertension making this figure as 1
of every 3 adults and a cost of approximately 46 billion US$ each
study and exhibited IC50 above 100
lM. Compounds 15, 16, 17, 25
and 26 exhibited a IC50in the range of 2–3.7
l
M comparable to the
Figure 11. Docking poses of protein and lead compounds 12 and 26. (A) and (C) Protein interacted with compound 12, (B) and (D): protein interacted with compound 26.
Hydrogen bonds are shown in Black color (dot line), amino acids who involved in hydrogen bond with lead compound are labeled and amino acids involved in other
interaction are shown in Stick formation.
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
19
Figure 12. Graphical representation of molecular dynamic studies at 10 ns (10000 ps). (A) and (B) are Potential Energy graphs for compounds 12 and 26, respectively. (C) and
(D) are RMS Deviation (RMSD) graphs for compounds 12 and 26 resp. E and F are RMS Fluctuation graphs for compounds 12 and 26, respectively.
standard drug Nifedipine with 2.02
l
M. These compounds did not
subjected to non-invasive method of tail cuff measurements of the
rats with DOCA salt induced hypertension. It was observed that
compounds with methoxy and nitro substitutions on the phenyl
ring at positions 3 and 4 provided with moderate to highest activ-
ity which is observed in compounds 15, 16, 17, 24, 25 and 26. This
led to the development of a model which represents the SAR for
dihydropyrimidine derivatives and extends to studies described
earlier by Atwal et al. (Fig. 16). It was found that the compounds
which have shown comparative activity to Nifedipine gave same
results in the invasive method of antihypertensive model. Com-
pounds 15, 16, 17, 19, 22, 25, 26, 27 and 29 have exhibited good
results in the IBP method with P lower level of 0.0001 and F value
show any inhibitory action on the ACE colorimetric assay explain-
ing the mode of action of the derivatives to be via calcium channel
modulating action.
In order to screen the compounds for antihypertensive activity
we employed two methods of evaluation, first one was needed to
determine the effect on systolic blood pressure and second model
to determine the diastolic blood pressure. Rationally it should be
that only compounds screened from MLCK assay should have been
studied by in vitro studies, however, the compounds needed to be
studied as well in the live organism rather than only by biochem-
ical assay method. All the compounds 10–34 from this series were
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20
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 11
3D QSAR data by the k-Nearest Neighbor (kNN) method for 6a–6o and 10–29
R
R1
R
S
O
H
O
H3C
O
H
N
N
R1
O
H3C
N
H
O
H3C
CH3
N
10-29
6a-6o
Sr. No. Compd
R
R1
Model A
Model B
Model C
3D QSAR by SWV-kNN MFAa
3D QSAR by GA-kNN MFAb
3D QSAR by SA-kNN MFAc
d
Obs. Act
Pre. Acte
Resf
0.05897
0.184112 ꢀ2.52244 ꢀ2.397447
Obs. Act
Pre. Act
Res
Obs. Act
Pre. Act
Res
0.118276
0.149983
1
2
3
4
5
6a
6b
6c
6d
6e
6f
H
H
H
Cl
Cl
Cl
OH
OH
H
Cl
ꢀ2.36735
ꢀ2.52244
ꢀ2.30833
ꢀ2.36735 ꢀ2.067167
0.300183 ꢀ2.36735 ꢀ2.249074
0.124993 ꢀ2.52244 ꢀ2.372457
ꢀ2.338288
OCH3 ꢀ2.12385
ꢀ2.343606 ꢀ0.219756 ꢀ2.12385 ꢀ2.214093 ꢀ0.09024
ꢀ2.12385 ꢀ2.267553 ꢀ0.1437
H
Cl
ꢀ2.49554
ꢀ2.61595
ꢀ2.453467
ꢀ2.339126
ꢀ2.3785
0.042073 ꢀ2.49554 ꢀ2.145026
0.276824 ꢀ2.61595 ꢀ2.352551
0.350514 ꢀ2.49554 ꢀ2.343539
0.263399 ꢀ2.61595 ꢀ2.401738
0.152001
0.214212
6
OCH3 ꢀ2.26007
ꢀ0.11843
ꢀ2.26007 ꢀ2.42473
ꢀ0.16466
ꢀ2.26007 ꢀ2.418315 ꢀ0.15825
7
8
9
6g
6h
6i
H
Cl
ꢀ2.00860
ꢀ2.18184
ꢀ2.013902 ꢀ0.0053
ꢀ2.00860 ꢀ2.134277 ꢀ0.12568
0.102696 ꢀ2.18184 ꢀ2.437514 ꢀ0.25567
ꢀ2.019535 ꢀ0.028315 ꢀ1.99122 ꢀ2.136976 ꢀ0.14576
ꢀ2.00860 ꢀ2.008675 ꢀ7.5E-05
ꢀ2.079144
ꢀ2.18184 ꢀ2.26261
ꢀ0.08077
OH
OCH3 ꢀ1.99122
ꢀ1.99122 ꢀ2.018131 ꢀ0.02691
0.199192 ꢀ2.28330 ꢀ2.288739 ꢀ0.00544
0.044374
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
6j
6k
6l
CH3
CH3
CH3
OCH3
OCH3
OCH3
2-Cl
4-Cl
4-F
2-OH
4-OH
4-OCH3
3-NO2
4-NO2
(CH3)2N
4-OH-3-OCH3 CH3
2-Cl
4-Cl
4-F
2-OH
4-OH
4-OCH3
3-NO2
4-NO2
(CH3)2N
H
Cl
ꢀ2.28330
ꢀ2.46538
ꢀ2.402065 ꢀ0.118765 ꢀ2.28330 ꢀ2.084108
ꢀ2.387886
ꢀ2.34026
ꢀ2.06117
ꢀ2.303452
0.077494 ꢀ2.46538 ꢀ2.382306
0.083074 ꢀ2.46538 ꢀ2.421006
OCH3 ꢀ1.70757
ꢀ0.63269
ꢀ0.19194
ꢀ1.70757 ꢀ2.08289
ꢀ1.86923 ꢀ2.16448
ꢀ0.37532
ꢀ1.70757 ꢀ2.323888 ꢀ0.61632
6m
6n
6o
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
Cl
ꢀ1.86923
ꢀ2.48144
ꢀ0.29525
ꢀ1.86923 ꢀ2.057553 ꢀ0.18832
0.177988 ꢀ2.48144 ꢀ2.378382
0.103058 ꢀ2.48144 ꢀ2.337509
0.143931
OCH3 ꢀ1.61278
ꢀ2.144176 ꢀ0.531396 ꢀ1.61278 ꢀ2.075296 ꢀ0.46252
ꢀ1.61278 ꢀ2.136396 ꢀ0.52362
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
ꢀ1.99122
ꢀ1.95904
ꢀ1.86332
ꢀ1.63346
ꢀ1.62428
ꢀ1.33041
ꢀ1.30963
ꢀ1.30960
ꢀ1.64048
ꢀ1.56466
ꢀ2.03342
ꢀ2.04532
ꢀ2.12385
ꢀ1.67209
ꢀ1.77085
ꢀ1.33645
ꢀ1.32221
ꢀ1.48287
ꢀ1.86746
ꢀ1.56466
ꢀ1.75232
ꢀ1.98176
ꢀ1.74308
ꢀ1.74213
ꢀ1.62624
ꢀ1.31823
ꢀ1.32533
ꢀ1.32545
ꢀ1.31673
ꢀ1.73175
ꢀ1.99927
ꢀ1.95382
ꢀ1.92507
ꢀ1.72946
ꢀ1.84967
ꢀ1.5037
0.2389
ꢀ0.02272
0.12024
ꢀ0.10867
ꢀ0.00196
0.01218
ꢀ0.0157
ꢀ0.01585
0.32375
ꢀ0.16709
0.03415
ꢀ1.99122 ꢀ1.81807
ꢀ1.95904 ꢀ1.83263
ꢀ1.86332 ꢀ1.44496
ꢀ1.63346 ꢀ1.66728
ꢀ1.62428 ꢀ1.59642
ꢀ1.33041 ꢀ1.32144
ꢀ1.30963 ꢀ1.32317
ꢀ1.30960 ꢀ1.44714
ꢀ1.64048 ꢀ1.44099
ꢀ1.56466 ꢀ1.70138
ꢀ2.03342 ꢀ2.07807
ꢀ2.04532 ꢀ2.04193
ꢀ2.12385 ꢀ1.81428
ꢀ1.67209 ꢀ1.94813
ꢀ1.77085 ꢀ1.65288
ꢀ1.33645 ꢀ1.43128
ꢀ1.32221 ꢀ1.55728
ꢀ1.48287 ꢀ1.59043
ꢀ1.86746 ꢀ1.55723
ꢀ1.56466 ꢀ1.59198
0.17315
0.12641
0.41836
ꢀ1.99122 ꢀ1.99122
ꢀ1.95904 ꢀ1.921
0
0.03804
ꢀ0.18158
ꢀ0.03863
ꢀ0.1911
0.0208
ꢀ1.86332 ꢀ2.0449
ꢀ1.63346 ꢀ1.67209
ꢀ1.62428 ꢀ1.81538
ꢀ1.33041 ꢀ1.30961
ꢀ1.30963 ꢀ1.31072
ꢀ1.30960 ꢀ1.31885
ꢀ1.64048 ꢀ1.53263
ꢀ1.56466 ꢀ1.60719
ꢀ2.03342 ꢀ1.73073
ꢀ2.04532 ꢀ2.06947
ꢀ2.12385 ꢀ2.00522
ꢀ1.67209 ꢀ1.63346
ꢀ1.77085 ꢀ1.85423
ꢀ1.33645 ꢀ1.56779
ꢀ1.32221 ꢀ1.95652
ꢀ1.48287 ꢀ1.67013
ꢀ1.86746 ꢀ1.68631
ꢀ1.56466 ꢀ1.61462
ꢀ0.03382
0.02786
0.00897
ꢀ0.01354
ꢀ0.13754
0.19949
ꢀ0.13672
ꢀ0.04465
0.00339
ꢀ0.00109
ꢀ0.00925
0.10785
ꢀ0.04253
0.30269
ꢀ0.02415
0.11863
0.03863
ꢀ0.08338
ꢀ0.23134
ꢀ0.63431
ꢀ0.18726
0.18115
ꢀ0.04996
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
0.0915
0.19878
ꢀ0.05737
ꢀ0.07882
ꢀ0.16725
ꢀ0.33482
0.16617
0.36464
ꢀ0.10417
0.30957
ꢀ0.27604
0.11797
ꢀ0.09483
ꢀ0.23507
ꢀ0.10756
0.31023
ꢀ1.65703
ꢀ1.3167
ꢀ1.50282
ꢀ1.66883
4-OH-3-OCH3 C2H5
ꢀ0.02732
Test Set molecules.
a
k-Nearest Neighbor-Stepwise Variable Selection.
k-Nearest Neighbor-Genetic Algorithm.
k-Nearest Neighbor-Simulated Annealing.
Minimum Inhibitory Concentration.
Predicted activity.
b
c
d
e
f
Residual.
for these compounds at 10.60 0.05, signifying that the inter and
intra correlation between compounds is statistically significant.
Our goal was to enhance the duration of action and improve phar-
macokinetic properties of the dihydropyrimidines in order to
achieve this goal we further screened the compounds on basis of
their ulcerogenic character, since the drugs for hypertension need
to be consumed at least daily. Compounds with methoxy and nitro
substitutions exhibited moderate to less severity of 2.20–0.56
which was safe similar to Nifedipine compared to NSAIDs like
Ibuprofen with severity of 1.97.
Nifedipine at 3.86. The values obtained by shake flask method were
very close to results obtained from simulation algorithm generated
values. Several compounds based on their pharmacological profile
were subjected to pharmacokinetic studies to determine their
bioavailability by oral and intra venous routes. It was found that
compound 12 had less oral bioavailability compared to 16, 22
and 26. These compounds exhibited more than half the quantity
of drug available in blood for activity with a Tmax of around 0.5
and Cmax above 0.41 lg/ml with AUC of 0.78 lg h/ml. These obser-
vations indicate toward success in obtaining compounds with bet-
On the basis of ulcerogenic studies compounds were subjected
to determination of their partition coefficient (logP) compounds
12, 16, 19 and 26 exhibited values of 4.85, 4.45, 3.89 and 4.57,
respectively. However, these values are little higher than that of
ter bioavailability and half life than Nifedipine.
Physical properties and packing characters of compounds in
their crystal lattice is of utmost importance for further develop-
ment of compounds into drug formulation. These properties will
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
21
Table 12
The selected k-Nearest Neighbor Simulated Annealing (SA-kNN MFA) QSAR equation along with statistical parameters employed for selecting the model for 6a–6o
Model no.
C
Selected descriptors
k
N
Descriptor range
q2
q2_se
Pred r2
Pred_r2se
Degree of freedom
H_233
H_4
3
10
H_233 0.2438 to 0.2952
H_4 0.1599 to 0.1885
H_715 0.2039 to 0.2665
H_809 0.2900 to 0.5174
H_370 0.2665 to 0.3338
H_339 0.2588 to 0.3072
E_131 0.1676 to 0.0620
S_778 0.0050 to 0.0044
H_352 0.1959 to 0.2373
E_140 0.0831 to 0.0056
0.7129
0.1292
0.1572
0.4243
ꢀ1
H_715
H_809
H_370
H_339
E_131
S_778
H_352
E_140
K = Nearest number.
N = Molecules in the test set.
2 = Cross-validated correlation coefficient.
q2_se = Standard error of cross-validated correlation coefficient.
q
r
2 = Squared correlation coefficient.
Pred_r2 = r2 for external test set.
Pred_r2se = Standard error of predicted squared regression.
Figure 13. Field points exhibiting contributing descriptors for 3D-QSAR model C by SA-kNN MFA for series 6a–6o.
Table 13
The selected k-Nearest Neighbor Simulated Annealing (SA-kNN MFA) QSAR equation along with statistical parameters employed for selecting the model for 10–29
Model no.
C
Selected descriptors
k
N
Descriptor range
q2
q2_se
Pred r2
Pred_r2se
0.3493
Degree of freedom
3
S_585
S_229
E_484
E_332
H_654
E_510
E_88
S_626
S_272
E_620
5
14
S_585 30.0000 to 30.0000
S_229 2.8396 to 30.0000
E_484 1.8374 to 1.8055
E_332 5.6892 to 1.3061
H_654 0.3548 to 0.4459
E_510 3.2476 to 0.0972
E_88 0.7295 to 1.8011
S_626 0.0536 to 0.0243
S_272 0.1355 to 0.0579
E_620 5.0924 to 3.0039
0.8788
0.1008
ꢀ0.8432
K = Nearest number.
N = Molecules in the test set.
q
2 = Cross-validated correlation coefficient.
q2_se = Standard error of cross-validated correlation coefficient.
r
2 = Squared correlation coefficient.
Pred_r2 = r2 for external test set.
Pred_r2se = Standard error of predicted squared regression.
Please cite this article in press as: Chikhale, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.009

22
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 14. Field points exhibiting contributing descriptors for 3D-QSAR model C by SA-kNN MFA for series 10–29.
determine their stability, inter and intra molecular interactions, co-
crystallization, compatibility with other materials specially adju-
vant, we studied the single crystal X-ray diffraction characteristics
of two selected compounds 12 and 26 from our series of deriva-
tives. Compound 12 was found to be monoclinic in structure with
faint yellow color; its crystal structure exhibits the interaction of
proton on nitrogen with the chloride ion for formation of the crys-
tal lattice. It is found that the packing of crystal lattice need the
interaction between two molecules forming a dimer in connection
with the chloride ion entrapped between two molecules. In case of
compound 26 the crystal packing exhibited a packing of four mole-
cules making polymeric chain within the lattice, interaction
between sulfur of one unit with oxygen of next unit forming a
chain. These compounds were found to be stable and satisfy most
of the requirements for further development as per the need of for-
mulations of solid and liquid forms.
The single crystal X-ray diffraction study provided with bond
angles, bond length and orientation of molecules in crystal
lattice, these parameters were used further for studying the
molecular docking of these molecules. To justify and establish
the interaction of synthesized compounds as antihypertensive
agents earlier we determined their specificity on the basis of
MLCK assay and the ACE assay. It was found that these com-
pounds were specific toward voltage gated calcium channels in
the MLCK assay and displayed satisfactory results by in vivo
and in vitro eva0luation studies. To carry out the molecular
docking studies we needed the crystal structure of receptor and
a 3D model which is not available for the calcium channels.
The most appropriate channel structure available on the Protein
Data Bank is PDB: 1T0J. This was not a complete structure and
needed to be modeled. Rosetta server was used to develop a
complete 3D model for calcium channel by ab intio or the de novo
modeling, this is a first report on 3D model for calcium channel
and docking thereon. The generated model was validated on
the basis of Ramchandran Plot and found to be highly accurate
with 98.5% of favoured region and the overall quality was further
verified and found to be good by ERRAT and Verify-3D
score. Docking was tried on various portions of the channel but
satisfactory results were not obtained, then we selected the
first domain (Fig. 9) as our docking target and it provided with
satisfactory results. A dock score of ꢀ3.682 and ꢀ3.602 was
exhibited by compounds 12 and 26, respectively which are good
for compounds with less number of rotatable bonds as in pre-
sented series of molecules. To validate our findings from molec-
ular docking molecular dynamics studies were carried out.
Selected compounds exhibited stability with the protein in terms
of average energy, this was determined on basis of structural
stability, conformational changes and binding interactions. RMSD
calculated was measured between 2 and 8 ns and overall fluctu-
ations indicated that the region of binding is quit flexible during
interaction with docked molecules in complex along with other
amino acids maintaining their conformation during this process.
The results obtained from in vitro and in vivo studies goes par-
allel and it was found that compounds with less IC50 values gave
good results at constant doses in NIBP and IBP evaluation. To
understand the improvement from 6a–o series to 10–34 series of
compounds we used the MLCK results as an important parameter
to carryout 3D-QSAR analysis. A repeat 3D-QSAR analysis was car-
ried out and contour plot analyzed, it was found that the region
under our study, that is, positions 1 and 2 of the pyrimidobenzoth-
iazole nucleus successfully yielded results, previously negatively
contributing steric descriptor has now yielded a positively con-
tributing steric descriptor (S_229) suggesting that this modifica-
tion has positively contributed to the activity (Fig. 14),
a
electrostatic descriptor (E_88) also suggest for positive contribu-
tion of the benzothiazole conjugation. These results clearly sug-
gests regarding the SAR of dihydropyrimidines with few
directions like presence of methyl, ethyl and propyl substitutions
on the left arm of dihydropyrimidine ring improves the activity
by 10 to 60 folds and hence these are very important for biological
activity, whereas presence of propyl, ethyl methyl improves the
activity merely by 4.7 to 3.8 folds only (Fig. 16). It signifies the
need for presence of such substitutions on the molecule, substitu-
tions on the aromatic phenyl ring contributes toward the activity
positively with a series of sequence as 3-NO2 > 3-Cl > 3-CF3. Substi-
tution on the right portion of dihydropyrimidine molecule leading
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R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
23
Figure 15. HPTLC densitogram and HPLC chromatogram for compounds 12 and 26.
to cyclic ring conjugated or fused has always contributed positively
to the pharmacological profile of the nucleus.
These molecules 12 and 26 were further studied for their
chromatographic properties on the basis of HPLC and HPTLC
analysis and it was found that synthesized compounds exhibit
peak purity of 99.99% at retention time of 3.045 min and peak
purity of 95.08% at common retention time of 3.045 min, respec-
tively. The methods were developed to determine purity of com-
pounds and the process related impurities in molecules, it was
found to be in the acceptable range for newly synthesised com-
pounds and provides with possibility to obtain compounds with
high purity.
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24
R. Chikhale et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
6. Crystallographic data
The crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the Cambridge
Crystallographic Data Centre with deposition no. CCDC 1059194,
CCDC 1059195. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
(fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.Uk).
Acknowledgements
RVC is thankful to Council of Scientific and Industrial Research
(CSIR) New Delhi, India for providing financial support in form of
SRF: 09/128/0080/2011/EMR-I. PBK acknowledges financial sup-
port by University Grants Commission (UGC) New Delhi, India in
form of Major Research Project 41-744/2012 (SR). P.B.K. also
thanks the Commonwealth Scholarship Commission (CSC), UK for
Commonwealth Academic Fellowship INCF-2014-107 for the year
2014–15.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.009.
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