Synthesis and evaluation of novel triazoles and mannich bases functionalized 1,4-dihydropyridine as angiotensin converting enzyme (ACE) inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.09.027

A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory activity. Screening of above synthesized compounds fo

Full text of DOI:10.1016/j.bmc.2014.09.027

Bioorganic & Medicinal Chemistry 22 (2014) 5824–5830
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel triazoles and mannich bases
functionalized 1,4-dihydropyridine as angiotensin converting
enzyme (ACE) inhibitors
Ravindra M. Kumbhare , Umesh B. Kosurkar , Pankaj K. Bagul b,c, Abhinav Kanwal b,c, K. Appalanaidu a,
a,
⇑
a
a
b,c,
⇑
Tulshiram L. Dadmal , Sanjay Kumar Banerjee
a
Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500607, India
Present Address: Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon 122016, India
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2014
Revised 9 September 2014
Accepted 11 September 2014
Available online 19 September 2014
A series of novel diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate embedded triazole and
mannich bases were synthesized, and evaluated for their angiotensin converting enzyme (ACE) inhibitory
activity. Screening of above synthesized compounds for ACE inhibition showed that triazoles functional-
ized compounds have better ACE inhibitory activity compared to that of mannich bases analogues.
Among all triazoles we found 6h, 6i and 6j to have good ACE inhibition activity with IC50 values
0
.713
l
M, 0.409
l
M and 0.653
l
M, respectively. Among mannich bases series compounds, only 7c
M.
Keywords:
Angiotensin converting enzyme
Inhibitors
resulted as most active ACE inhibitor with IC50 value of 0.928
l
Ó 2014 Published by Elsevier Ltd.
1
,4-Dihydropyridine
Triazole
Mannich bases
1
. Introduction
like skin rash, loss of taste and dry cough are reported with capto-
pril. All those side effects of captopril are thought to be because of
5
Hypertension is the leading cause of cardiovascular disease
the presence of mercapto group which provides strong chelating
action. After captopril, research has been aimed in finding potent,
1
6
affecting one in three adults worldwide. It is the primary cause of
stroke, coronary artery disease and sudden cardiac death. Many fac-
tors are believed to be involved in the pathogenesis of hypertension,
its progression and complications. Renin angiotensin aldosterone
system (RAAS) plays an important role in controlling blood volume
selective ACE inhibitors that would not contain a mercapto (SH)
functional group and would have a weaker chelating function. Enal-
april, lisinopril, ramipril and benzapril are the newly synthesized
peptide based molecules available in market. However, all of them
2
,3
7
and hypertension. Angiotensin converting enzyme (ACE) is a key
enzyme regulating the rate limiting step in the RAAS pathway. ACE
converts angiotensin I to angiotensin II which is the main active
component for hypertension. Angiotensin II causes vasoconstriction
still carry the same set of side effects with less severity. So there
is a need to synthesis non-peptide based analogue for ACE inhibition
with fewer side effects.
A survey of literature revealed that 1,4-dihydropyridine deriva-
tives have received much attention during recent years due to its
4
along with increased blood volume and water retention. To inhibit
8
AngII mediated hypertension and associated complication, several
drugs have been synthesized and tested as ACE inhibitors. Captopril
was the first peptide-based ACE inhibitor which developed in 1975
and was considered a breakthrough. However, several side effects
versatile biological activities such as antidepressant, antihyper-
9
10
11
12
tensive, antithrombotic, anticonvulsant, cardiotonic, anti-
1
3
14
15
bacterial, antiHIV, and calcium channel blockers. However,
little attention has been directed towards the BACE-1 blocking
1
6
activity of these compounds. DHP can be converted to the qua-
ternary amines leading to their retention in the brain.17 Another
characteristic feature of DHP scaffold is the possibility of structural
modifications via introducing various chemical substituents in dif-
ferent positions of the DHP ring and hence providing remarkable
⇑
⇑
Corresponding authors. Present Address: Drug Discovery Research Center
DDRC), Translational Health Science and Technology Institute (THSTI), Gurgaon
(
1
2
22016, India. Tel.: +91 11 26741358 (S.K.B.), tel.: +91 40 27191776; fax: +91 40
7193185 (R.M.K.).
E-mail addresses: kumbhare@iict.res.in (R.M. Kumbhare), skbanerjee@thsti.res.
18
changes in pharmacological profile. Dihydropyridine drugs, such
in (S.K. Banerjee).
http://dx.doi.org/10.1016/j.bmc.2014.09.027
0
968-0896/Ó 2014 Published by Elsevier Ltd.

R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
5825
N
N
O
O
O N
2
O
O
O
O
O
O
O
O
O
N
O
O
O
O
N
N
H
N
H
N
H
Lacidipine
Manidipine
Darodipine
2
3
1
Figure 1. Drugs containing 1,4-dihydropyridine framework.
as nifedipine, nicardipine, amlodipine and other drugs must be
used with caution in patients with coronary heart disease because
of the increased mortality observed in patients treated with these
drugs. One of the most discussed undesirable effects is the reflex
tachycardia produced by dihydropyridines.
logical activity a new series of triazole and mannich base functional-
ized 1,4-dihydropyridine have been synthesized.
2. Results and discussion
2.1. Synthesis
1
9
Darodipine 1 (Fig. 1) are dihydropyridine derivatives calcium
antagonists that easily pass into the brain, showing high affinity
for cerebral L-type voltage-sensitive calcium channel (VSCC). One
of the most vascular selective of the dihydropyridines derivatives
Lacidipine, 2 is a calcium channel blocker developed for oral admin-
istration for use in mild to moderate hypertension,20 and is widely
used in therapy since the early 1990s. It exhibits anti-atherosclerotic
Substituted salicylaldehyde is the starting material in the synthe-
sis of 1,4-dihydropyridine derivatives(6 and 7). It was propargylated
with propargyl bromide to yield 4 in excellent yield. Aldehyde group
from 4 reacted with ethylacetoacetate and ammonium acetate
under reflux condition in ethanol solvent to give 1,4-dihydropyri-
dine 5 in very good yield. Now, to obtain the triazole compounds
(6a–j) by click reaction, 5 was reacted with various aliphatic and aryl
azides in dry THF using 5 mol % CuI to provide 1,4-disubstituted
triazole products. All the compounds obtained in very good yields
21
and antioxidant effects. Manidipine 3 is a lipophilic and highly
2
2
vasoselective agents and has long lasting activity for the treat-
ments of hypertension. Manidipine is a dihydropyridine (DHP) cal-
cium channel antagonist, which effectively reduced blood pressure
in patients which mild to moderate hypertension and effectively
maintains reduced blood pressure levels throughout the dosing
periods of 24 h. Hence in continuous of our efforts for the structural
modificationon heterocyclic moiety23 and thus to improve their bio-
1
(78–85%) were confirmed by IR, H and mass (ESI and HRMS) spec-
tral data. Also, mannich bases (7a–k) of compound 5 were synthe-
2
4
1
sized by reported method and products confirmed by IR, H and
mass (ESI and HR-MS) spectral data (See Schemes 1-3).
Compound
R
R1
R
R N
2
Compound
6a
5Br
7a
5Br
HN
O
N N
S
HN
N
N
6
b
c
5Br
7b
5Br
N
N
CF3
OH
7c
3,5-dibromo HN
6
3
,5-dibromo
COOH
OCF3
HN
7
d
e
3,5-dibromo
6d
3,5-dibromo
7
3,5-dibromo HN
N
F
6
e
3,5-dibromo
3,5-dibromo
SCF3
3,5-dibromo
HN
N
7
f
N
6
f
HN
7
g
h
3 OCH3
3 OCH3
O
6
g
h
3,5-dibromo
3 OCH3
OH
7
HN
HN
O
F
N
S
6
7i
3 OCH3
3 OCH3
N
N N
HN
N
6
i
3 OCH3
7
j
CF3
S
6j
3 OCH3
C8F
17
7k
3 OCH3 HN
COOC H5
2

5
826
R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
CHO
OH
and histidyl-leucine (HL). The extent HA released is directly pro-
Br
K2CO3 , Acetone
Reflux, 2 h
CHO
O
R
R
portional to the ACE activity. In this screening method, the released
hippuric acid from the substrate hippurryl-histyl-leucine (HHL)
was mixed with pyridine and benzene sulfonyl chloride. The
resulting yellow color (410 nm) is directly proportional to
the released hippuric acid and thus with the ACE activity. All the
new compounds 6a–j and 7a–k tested in the desired concentra-
tions did not show any significant absorbance at 410 nm under
control conditions. Being simple, sensitive and rapid nature, the
colorimetric method is used for evaluating ACE inhibitory activity
of new analogues 6a–j and 7a–k. Lisonipril, a known ACE inhibitor
drug has been used as standard for comparison with the inhibitory
activity of synthesized new analogues.
R = 5 Br, 3,5-dibromo, 3OCH3
4
Scheme 1.
C2H5OOC
R
NH
CHO
O
O O
Ethanol
R
NH4COOCH3
H C
3
O C H
Reflux,3h
COO2CH5
2
5
O
5
The evaluation results of all 21 new compounds for ACE
inhibitory activity are presented in Figure 2. The experiments car-
ried out for test compounds at five different concentrations
Scheme 2.
(
100 nm to 2 lM) revealed that 6h, 6i, 6j and 7c showed dose
dependent inhibition of ACE activity (Fig. 3). The mechanism for
obtaining different ACE inhibition activity for different compounds
need to be explored further. However, the increased chelating
activity of those active compounds with zinc ions of the enzyme
cannot be ruled out. The IC50 value of these active compounds
are given in Table 1.The results obtained here will be explored
further study the binding nature and to generate lead molecules
for drug discovery.
5
a
b
C H OOC
C H OOC
2 5
2
5
NH
NH
The toxicity evaluation of four most potent ACE inhibitors 6h,
6
i, 6j and 7c was assessed by (3-5-dimethylthiazol-2-yl)-2,5-
1
0
R
R
diphenyltetrazolium bromide (MTT) assay against A549 (adeno
COOC2H5
N
COOC2H5
O
O
O
N
NR2
N
7a-k
6a-j
HN R1
Scheme 3. Reagents and conditions: (a) R
1 2 3
NHCOCH N , dry THF, CuI, 24 h, rt; (b)
Neutral Al , CuI, THF, HCHO, HNR , 12 h, rt.
2
O
3
2
2
.2. Pharmacology
The in vitro angiotensin converting enzyme (ACE) inhibitory
activity of new compounds 6a–j and 7a–k were measured using
recent colorimetric high-throughput screening method developed
by Jimsheena and Gowda.25 Most of the antihypertensive peptides
have been characterized by the rabbit lung ACE inhibitor assay,
based on the hydrolysis of the synthetic peptide hippurylhistidyl-
leucine (HHL). HHL is hydrolyzed by ACE to hippuric acid (HA)
Figure 3. Dose-dependent angiotensin converting enzyme (ACE) inhibition activity
of test compounds and standard compound.
Figure 2. In vitro angiotensin converting enzyme (ACE) inhibition of new analogues and standard drug.

R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
5827
Table 1
than 200 mesh was used for column chromatography. Appropriate
names for all the new compounds were given with the help of
Chem Bio Office 2012.
IC50 values of active compounds selected from preliminary screening
S. No.
Compound code
IC50 (lM)
1
2
3
4
5
6h
6i
6j
7c
Std.
0.713
0.409
0.653
0.928
0.281
4
.1. General procedure for the preparation of compound (4)
Salicylaldehyde (1 mmol) was dissolved in acetone (10 mL),
2 3
K CO (1.3 mmol) was added. The propargyl bromide (1 mmol)
was slowly added drop wise to the above mixture over a period
of 15 min at room temperature. Reaction mixture was refluxed
for 2 h. After completion of reaction, solvent was removed under
reduced pressure. The residue was washed with n-hexane repeat-
edly to remove excess propargyl bromide, treated with distilled
water to remove excess potassium carbonate and solid product
separated was filtered and dried to get product 4.
Table 2
MTT assay inhibitory activities (IC50) in
lM
S. No.
Compound code
A549-cells
HEK-cells
1
2
3
4
5
6h
6i
6j
7c
>1000
>1000
>1000
>1000
2.6
>1000
>1000
>1000
>1000
0.69
4.2. General procedure for the preparation of compound (5)
Doxorubicin
An appropriate propargylated salicylaldehyde (10 mmol), ethyl
acetoacetate (20 mmol) and ammonium acetate (22 mmol) were
dissolved in ethanol and refluxed for 1 h. After completion of the
reaction, reaction mixture was cooled to room temperature and
product was extracted with ethyl acetate, washed with water
and brine solution. Column chromatography was performed using
silica gel (60–120 mesh), eluted with ethyl acetate and hexane
carcinomic human alveolar basal epithelial) and HEK 293 (normal
human embryonic kidney) cell lines. The inhibitory activities (IC50
in M are summarized in Table 2. The compounds 6h, 6i, 6j and 7c
)
l
exhibited their 50% inhibitory concentrations (IC50) >1000
lM and
are considered nontoxic.
(
20%) to get product 5.
3
. Conclusion
In conclusion, we have described an efficient synthesis and
4
.3. General procedure for the preparation of compound (6)
To a solution of dipolarophile 5 (1 mmol) in dry THF (5 mL), CuI
evaluation of ACE inhibition activity of novel 1,4-dihydropyridine
derivatives functionalized with triazoles and mannich bases. Com-
pounds 6h, 6i, 6j and 7c exhibit promising ACE inhibition property
comparable to Lisinopril drug. Cytotoxicity study of these com-
pounds showed that all four compounds are nontoxic as assessed
by measuring IC50 value in A549 and HEK cell lines, respectively.
Screening all the new compounds using in vitro ACE inhibition
assay resulted triazole as more potent ACE inhibitors than the
respective mannich base analogues by thousand folds. Among
the series of triazole 6a–j, three compounds 6h, 6i, 6j were resulted
(
5 mol %) was added. Then aryl/aliphatic azide (1 mmol) was added
slowly at room temperature under nitrogen atmosphere and
continued stirring for 24 h. Solvent was removed under reduced
pressure, the residue was diluted with water and extracted with
ethyl acetate. The combined organic layers were washed with
2 4
water, brine, dried over Na SO , filtered and concentrated under
reduced pressure. This was further purified by column chromatog-
raphy using (ethyl acetate/n-hexane, 40:60) as eluent to obtained
the pure product 6.
as most active ACE inhibitors with IC50 values of 0.713
.409 M and 0.653 M, respectively. Compared with ACE inhibi-
tory activity (IC50) of various triazole, mannich base 7c showed
relatively higher IC50 value (0.928 M). The results reveal that
binding of heteroaryl triazole at ACE binding pocket is more
pronounced through zinc ion chelation than the triazole of natural
origin. The information generated here could be of use to generate
lead molecules for drug discovery.
lM,
4
.3.1. Diethyl4-(5-bromo-2-((1-(2-(cyclohexylamino)-2-
0
l
l
oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl-
1
,4-dihydropyridine-3,5-dicarboxylate 6a
Pale yellow solid, mp = 84 °C, IR (KBr)
l
m
max: 3350, 2933, 1688,
À1
1
1
217, 1038 cm
CH
s, 6H, Me), 3.68 (s, 1H), 3.83–4.09 (m, 4H), 4.86 (s, 1H, pyridine
CH), 4.99 (s, 2H, NCH ), 5.16 (s, 2H, OCH ), 6.83–6.89 (m, 1H, Ar-
;
H NMR (DMSO, 500 MHz): d = 1.07 (t, 6H,
J = 6.9 Hz, CO
2
2
Me), 1.13–1.44 (m, 5H), 1.54–1.83 (m, 5H), 2.21
(
2
2
H), 7.16–7.23 (m, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 7.77 (s, 1H, pyridine
4
. Experimental section
NH), 7.92 (s, 1H, CONH), 8.31 (s, 1H, triazole H). MS (ESI) m/z 644
+
+
[
M+H] ; HR-MS (ESI) calcd for C30
38 6 5
H O N Br [M+H] : 644.2078.
Melting points were measured with a Fischer–Johns melting
Found: 644.2082.
point apparatus and are uncorrected. IR spectra were recorded as
À1
neat liquids or KBr pellets and absorptions are reported in cm
.
1
H NMR spectra were recorded on 300 (Bruker) and 500 MHz (Var-
ian) spectrometers in appropriate solvents using TMS as internal
standard or the solvent signals as secondary standards and the
chemical shifts are shown in d scales. Coupling constants J are
expressed in Hertz. High-resolution mass spectra were obtained
by using ESI-QTOF mass spectrometry. Reagents and all solvents
were analytically pure and were used without further purification.
All the experiments were monitored by analytical thin layer chro-
matography (TLC) performed on silica gel GF254 pre-coated plates.
After elution, plate was visualized under UV illumination at
4.3.2. Diethyl 4-(5-bromo-2-((1-(2-oxo-2-(5-(trifluoromethyl)-
1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth
oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate 6b
White solid, mp = 160 °C, IR (KBr)
m
max: 3349, 2935, 1690, 1216,
H NMR (DMSO, 300 MHz): d = 1.11 (t, 6H, J = 6.9 Hz,
Me), 2.28 (s, 6H, Me), 3.88–4.15 (m, 4H), 4.89 (s, 1H, pyri-
dine CH), 5.09 (s, 2H, OCH ), 5.28 (s, 2H, NCH ), 6.86–6.93 (m,
À1
1
1039 cm
;
CO CH
2
2
2
2
1H, Ar-H), 7.18–7.26 (m, 1H, Ar-H), 7.58–7.63 (m, 1H, Ar-H), 7.81
(s, 1H, pyridine NH), 7.98 (s, 1H, CONH), 8.39 (s, 1H, triazole H).
+
2
54 nm for UV active materials. Further visualization was achieved
MS (ESI) m/z 715 [M+H] ; HR-MS (ESI) calcd for C26
27
H O
6
N
8
BrF
3
S
+
by staining with PMA and charring on a hot plate. Silica gel finer
[M+H] : 714.0826. Found: 714.0784.

5
828
R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
4
.3.3. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4-
4.3.8. Diethyl 4-(2-((1-(2-(6-fluorobenzo[d]thiazol-2-ylamino)-
2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-methoxyphenyl)-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6h
dihydropyridin-4-yl)-4,6-dibromophenoxy)methyl)-1H-1,2,3-
triazol-1-yl)acetamido)benzoic acid 6c
White solid, mp = 158 °C, IR (KBr)
m
max: 3316, 2979, 1684, 1208,
H NMR (DMSO, 300 MHz): d = 1.08 (t, 6H, J = 6.9 Hz,
Me), 2.25 (s, 6H, Me), 3.81–4.10 (m, 4H), 4.85 (s, 1H, pyri-
dine CH), 5.11 (s, 2H, OCH ), 5.32 (s, 2H, NCH ), 6.91–6.98 (m,
H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.79 (s, 1H, pyridine NH),
.97 (s, 1H, CONH), 8.43 (s, 1H, Triazole H), 10.48 (s, 1H, COOH).
Pale green solid, mp = 100 °C, IR (KBr)
m
max: 3334, 2936, 1701,
H NMR (DMSO, 500 MHz): d = 1.26 (t, 6H,
Me), 2.31 (s, 6H,Me), 3.28 (s, 3H, OCH ),
3.62–4.18 (m, 4H), 4.91 (s, 1H, pyridine CH), 5.22 (s, 2H,
OCH ), 5.45 (s, 2H, NCH ), 6.57–7.47 (m, 3H, Ar-H), 7.51–7.83
À1
1
À1
1
1
122 cm
;
1231, 1078 cm
;
CO CH
2
2
J = 6.9 Hz, CO CH
2
2
3
2
2
4
7
2
2
(m, 3H, Ar-H), 8.0 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H),
10.24 (s, 1H, CONH). MS (ESI) m/z 665 [M+H] ; HR-MS
+
+
MS (ESI) m/z 762 [M+H] ; HR-MS (ESI) calcd for C31
H31Br
2
N
5
O
8
+
+
[
M+H] : 761.9935. Found: 761.9915.
(ESI) calcd for
65.2193.
C
32
34
H O
7
N
6
FS [M+H] : 665.2188. Found:
6
4
.3.4. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluorometh-
oxy)benzylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
4.3.9. Diethyl4-(3-methoxy-2-((1-(2-oxo-2-(5-(trifluoromethyl)-
1,3,4-thiadiazol-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)meth oxy)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6i
2
,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6d
Pale yellow solid, mp = 100 °C, IR (KBr) max: 3320, 2975, 1685,
H NMR (DMSO, 500 MHz): d = 1.08 (t, 6H,
Me), 2.19 (s, 6H, Me), 3.88–4.19 (m, 4H), 4.42
d, 2H, J = 5.9 Hz, benzyl H), 4.81 (s, 1H, pyridine CH), 5.13 (s, 2H,
m
À1
1
1
213, 1021 cm
CH
;
White solid, mp = 132 °C, IR (KBr)
m
max: 3351, 2933, 1689, 1215,
H NMR (DMSO, 500 MHz): d = 1.14 (t, 6H, J = 6.9 Hz,
Me), 2.15 (s, 6H, Me), 3.82 (s, 3H, OCH ), 3.89–4.13 (m,
4H), 5.0 (s, 1H, pyridine CH), 5.17 (s, 2H, OCH ), 5.53 (s, 2H,
NCH ), 6.65–7.17 (m, 3H, Ar-H), 7.43 (s, 1H, pyridine NH), 8.03
(s, 1H, triazole H), 10.92 (s, 1H, CONH). MS (ESI) m/z 666 [M+H] ;
À1
1
J = 6.9 Hz, CO
(
2
2
1039 cm
;
CO CH
2
2
3
OCH
2
), 5.23 (s, 2H, NCH
2
), 7.16–7.24 (m, 2H, Ar-H), 7.32–7.42 (m,
2
2
8
H, Ar-H), 7.52 (s, 1H, pyridine NH), 7.72–7.80 (m, 2H, Ar-H),
2
+
.05 (s, 1H, triazole H), 8.86 (t, 1H, J = 5.9 Hz, CONH). MS (ESI) m/
+
+
+
z 815 [M+H] ; HR-MS (ESI) calcd for C32
H
33
O
N
7 2
Br
2
F
4
[M+H] :
HR-MS (ESI) calcd for C28
664.1802.
29 7 7 3
H O N F S [M+H] : 664.1795. Found:
8
14.0483. Found: 814.0525.
4
.3.5. Diethyl4-(3,5-dibromo-2-((1-(2-oxo-2-(4-(trifluoromethyl
4.3.10. Diethyl 4-(2-((1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-
heptadecafluorodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-
methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate 6j
thio)phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
2
,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 6e
White solid, mp = 134 °C, IR (KBr) max: 3334, 2968, 1681, 1209,
H NMR (DMSO, 300 MHz): d = 1.24 (t, 6H, J = 6.9 Hz,
Me), 2.18 (s, 6H, Me), 3.91–4.16 (m, 4H), 4.85 (s, 1H, pyri-
dine CH) 5.17 (s, 2H, OCH ), 5.38 (s, 2H, NCH ), 7.49–7.79 (m, 6H,
Ar-H), 7.85 (s, 1H, pyridine NH), 8.13 (s, 1H, triazole H), 10.66 (s,
m
À1
1
1
CO
026 cm
CH
;
Yellow viscous liquid, IR (neat)
m
max: 3353, 2939, 1679, 1228,
H NMR (DMSO, 300 MHz): d = 1.15 (t, 6H, J = 6.9 Hz,
Me), 2.26 (s, 6H, Me), 2.85 (t, 2H, J = 7.6 Hz, CF CH ), 3.84
(s, 3H, OCH ), 3.94–4.26 (m, 4H), 4.72 (t, 2H, J = 7.6 Hz, NCH ),
5.08 (s, 1H, pyridine CH), 5.20 (s, 2H, OCH ), 6.66–7.12 (m, 3H,
Ar-H), 7.27 (s, 1H, pyridine NH), 7.91 (s, 1H, triazole H). MS (ESI)
À1
1
1054 cm
CO CH
;
2
2
2
2
2
2
2
2
3
2
+
1
H, CONH). MS (ESI) m/z 818 [M+H] ; HR-MS (ESI) calcd for
2
+
C
31
30 7 4 2 3
H O N Br F S [M+H] : 818.0284. Found: 818.0226.
+
+
m/z 903 [M+H] ; HR-MS (ESI) calcd for C33
01.1888. Found: 901.1871.
30 6 4
H O N F17 [M+H] :
9
4
.3.6. Diethyl 4-(3,5-dibromo-2-((1-(2-(cyclopropylamino)-2-
oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2,6-dimethyl-
1
,4-dihydropyridine-3,5-dicarboxylate 6f
Yellow solid, mp = 76 °C, IR (KBr) max: 3321, 2944, 1678, 1211,
, 500 MHz): d = 0.43–0.60 (m, 2H, cyclo-
), 0.70–0.91 (m, 2H, cyclopropyl CH ), 1.04 (t, 6H,
J = 6.9 Hz, CO CH Me), 2.63 (s, 6H, Me), 2.72 (m, 1H, cyclopropyl
CH), 3.88–4.21 (m, 4H), 4.88 (s, 1H, pyridine CH), 5.18 (s, 2H,
OCH ), 5.40 (s, 2H, NCH ), 7.35–7.42 (m, 2H, Ar-H), 7.83 (s, 1H, pyr-
4.4. General procedure for the preparation of compound (7)
m
À1
1
1
029 cm ; H NMR (CDCl
3
To a solution of terminal acetylene (2 mmol) in THF was added
secondary amine (2.1 mmol), 37% formaldehyde (2 mmol), CuI
(0.2 mmol) and 3 g neutral alumina. This reaction mixture was stir-
red at room temperature for 12 h. The progress of reaction was
monitored by TLC. Solvent was removed under reduced pressure
and extracted with ethyl acetate, washed with water, brine and
propyl CH
2
2
2
2
2
2
idine NH), 8.11 (s, 1H, triazole H), 10.63 (s, 1H, CONH). MS (ESI) m/z
6
+
+
82 [M+H] ; HR-MS (ESI) calcd for
C
27
H31Br
2
N
5
O
6
[M+H] :
2 4
dried over Na SO . Product was purified by column chromatogra-
6
81.0122. Found: 681.0132.
phy using (ethyl acetate/n-hexane, 50:50) as eluent to afford the
pure product 7.
4
.3.7. 3-(2-(4-((2-(3,5-Bis(ethoxycarbonyl)-2,6-dimethyl-1,4-
dihydropyridin-4-yl)-4,6-dibromo phenoxy)methyl)-1H-1,2,3-
4.4.1. Diethyl4-(5-bromo-2-(4-morpholinobut-2-ynyloxy)
triazol-1-yl)acetamido)propanoic acid 6g
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7a
Yellow solid, mp = 78 °C, IR (KBr)
m
max: 3332, 2932, 1727, 1234,
H NMR (DMSO, 500 MHz): d = 1.21 (t, 6H, J = 6.9 Hz,
Me), 2.24 (s, 6H, Me), 2.42 (t, 2H, J = 7.2 Hz), 3.48–3.58
m, 2H), 3.89–4.18 (m, 4H), 4.89 (s, 1H, pyridine CH), 5.20 (s, 2H,
OCH ), 5.42 (s, 2H, NCH CO), 7.52–7.68 (m, 2H, Ar-H), 7.89 (s,
Brown solid, mp = 73 °C, IR (KBr)
m
max: 3324, 2935, 1692,
500 MHz): d = 1.31 (t, 6H,
Me), 2.39 (s, 6H, Me), 2.61 (m, 4H), 3.38
), 3.82 (m, 4H, morpholine), 4.14 (m, 4H, morpho-
line), 4.73 (s, 2H, OCH ), 5.21 (s, 1H, pyridine CH), 5.94 (s, 1H,
À1
1
À1
1
1
105 cm
;
1213 cm
J = 6.9 Hz, CO
(s, 2H, NCH
;
H
CH
2 2
3
NMR (CDCl ,
CO CH
2
2
(
2
2
2
2
1
1
H, pyridine NH), 8.15 (s, 1H, triazole H), 10.58 (s, 1H, CONH),
pyridine NH), 7.20–7.57 (m, 3H, Ar-H). MS (ESI) m/z 562
+
+
+
3.20 (s, 1H, COOH). MS (ESI) m/z 714 [M+H] ; HR-MS (ESI) calcd
[M+H] ; HR-MS (ESI) calcd for C27
H
33
O
6
N
2
Br [M+H] : 561.1595.
+
for C27
H31Br
2
N
O
5 8
[M+H] : 713.0154. Found: 713.0143.
Found: 561.1578.

R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
5829
4
.4.2. Diethyl 4-(5-bromo-2-(4-(4-(pyridin-2-yl)piperazin-1-
2
4.60 (s, 2H, OCH ), 5.09 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine
yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
NH), 6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS
+
+
3
,5-dicarboxylate 7b
(ESI) m/z 497 [M+H] ; HR-MS (ESI) calcd for C28
H
37
O
6
N
2
[M+H] :
Brown solid, mp = 76 °C, IR(KBr)
m
max: 3329, 2929, 1691, 1437,
497.2646. Found: 497.2628.
À1
1
1
CO
3
2
6
212 cm
CH Me), 2.29 (s, 6H, Me), 2.64 (m, 4H), 3.02 (s, 2H, NCH
.55 (m, 4H, piperazine H), 4.04 (m, 4H, piperazine H), 4.62 (s,
H, OCH ), 5.11 (s, 1H, pyridine CH), 5.94 (s, 1H, pyridine NH),
.43–6.86 (m, 3H, Ar-H), 7.07–7.69 (m, 3H, Ar-H), 8.19 (m, 1H,
;
H NMR (DMSO, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz,
2
2
2
),
4.4.8. Diethyl 4-(3-methoxy-2-(4-morpholinobut-2-ynyloxy)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7h
2
Yellow solid, mp = 80 °C, IR (KBr)
m
max: 3324, 2934, 1692,
, 300 MHz): d = 1.20 (t, 6H, J = 6.9 Hz,
Me), 2.29 (s, 6H, Me), 2.50 (m, 4H), 3.30 (s, 2H, NCH ),
3.72 (t, 4H, J = 4.5 Hz), 3.81 (s, 3H, OCH ), 3.98–4.10 (m, 4H), 4.65
s, 2H, OCH ), 5.12 (s, 1H, pyridine CH), 6.17 (s, 1H, pyridine NH),
À1
1
1215 cm
; H NMR (CDCl
3
Ar-H). MS (ESI) m/z 638 [M+H]+; HR-MS (ESI) calcd for
CO CH
2
2
2
+
C
37
H
26
O
3
N
7
[M+H] : 637.1989. Found: 637.2004.
3
(
2
4
.4.3. Diethyl4-(3,5-dibromo-2-(4-(4-(2-hydroxyethyl)piperazin
6.70 (dd, 1H, J = 2.5 Hz, Ar-H), 6.86–6.93 (m, 2H, Ar-H). MS (ESI)
+
+
-1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
m/z 513 [M+H] ; HR-MS (ESI) calcd for C28
H
37
O
7
N
2
[M+H] :
3
,5-dicarboxylate 7c
513.2595. Found: 513.2586.
Brown solid, mp = 74 °C, IR (KBr)
m
max: 3412, 3327, 2932, 1694,
, 500 MHz): d = 1.05 (t, 6H,
Me), 2.28 (s, 6H, Me), 2.62 (s, 8H, piperazine
H), 2.69 (t, 2H, J = 5.9 Hz), 3.41 (s, 2H, NCH ), 3.48 (m, 2H), 3.66
s, 1H), 4.10 (m, 4H), 4.36 (s, 2H, OCH ), 4.76 (s, 1H, pyridine
CH), 5.13 (s, 1H, pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50
À1
1
1
441, 1215 cm
;
H NMR (CDCl
3
4.4.9. Diethyl 4-(3-methoxy-2-(4-(4-methylpiperazin-1-yl)but-
2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicar-
boxylate 7i
J = 6.9 Hz, CO
2
CH
2
2
(
2
Yellow solid, mp = 118 °C, IR (KBr)
m
max: 3328, 2930, 1695,
, 300 MHz): d = 1.29 (t, 6H,
Me), 2.29 (s, 9H), 2.37 (s, 8H), 3.37 (s, 2H,
), 3.96–4.08 (m, 4H), 4.57 (s, 2H, OCH ),
.08 (s, 1H, pyridine CH), 6.49 (s, 1H, pyridine NH), 6.70 (dd, 1H,
À1
1
1439, 1214 cm
J = 6.9 Hz, CO CH
NCH
;
H NMR (CDCl
3
+
(
d, 1H, J = 2.5 Hz, Ar-H). MS (ESI) m/z 682 [M+H] ; HR-MS (ESI)
2
2
+
calcd for C32
H
28
O
7
N
4
Br [M+H] : 682.1034. Found: 682.0975.
2
), 3.80 (s, 3H, OCH
3
2
5
4
2
.4.4. Diethyl4-(3,5-dibromo-2-(4-(4-methylpiperazin-1-yl)but-
-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
J = 2.5 Hz, Ar-H), 6.83–6.95 (m, 2H, Ar-H). MS (ESI) m/z 526
+
+
[M+H] ; HR-MS (ESI) calcd for C29
H
40
O
6
N
3
[M+H] : 526.2912.
dicarboxylate 7d
Found: 526.2904.
Brown solid, mp = 81 °C, IR (KBr) mmax: 3328, 2931, 1696, 1439,
À1
1
1
214 cm
CO CH Me), 2.31 (s, 9H), 2.36 (s, 8H), 3.39 (s, 2H, NCH
H), 4.59 (s, 2H, OCH ), 5.08 (s, 1H, pyridine CH), 6.98 (s, 1H,
pyridine NH), 7.36 (d, 1H, J = 2.3 Hz, Ar-H), 7.50 (d, 1H, J = 2.5 Hz,
;
H NMR (CDCl
3
, 300 MHz): d = 1.21 (t, 6H, J = 6.9 Hz,
4.4.10. Diethyl 4-(3-methoxy-2-(4-(4-phenylpiperazin-1-yl)but-
2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicar-
boxylate 7j
2
2
2
), 4.05 (m,
4
2
Brown solid, mp = 82 °C, IR (KBr)
m
max: 3335, 2937, 1692, 1438,
, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz,
Me), 2.28 (s, 6H, Me), 2.45–2.87 (m, 4H), 2.95–3.30 (m,
H), 3.38 (s, 2H, NCH ), 3.79 (s, 3H, OCH ), 3.94–4.12 (m, 4H),
4.65 (s, 2H, OCH ), 5.12 (s, 1H, pyridine CH), 6.32 (s, 1H, pyridine
+
À1
1
Ar-H). MS (ESI) m/z 654 [M+H] ; HR-MS (ESI) calcd for C28
H
38
5
O N
3
1216 cm
; H NMR (CDCl
3
+
Br
2
[M+H] : 654.1173. Found: 654.1006.
CO CH
2
2
4
2
3
4
.4.5. Diethyl 4-(3,5-dibromo-2-(4-(4-(4-fluorophenyl)piperazin
2
-
3
1-yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
,5-dicarboxylate 7e
NH), 6.65–7.05 (m, 5H, Ar-H), 7.18–7.40 (m, 3H, Ar-H). MS (ESI)
+
+
m/z 588 [M+H] ; HR-MS (ESI) calcd for C34
H
42
O
6
N
3
[M+H] :
Brown solid, mp = 70 °C, IR (KBr)
m
max: 3331, 2930, 1693, 1440,
588.3068. Found: 588.3069.
À1
1
1
CO
4
5
4
215 cm
CH Me), 2.31 (s, 6H, Me), 2.60–2.82 (m, 4H), 2.99–3.26 (m,
H), 3.41 (s, 2H, NCH ), 3.92–4.20 (m, 4H), 4.69 (s, 2H, OCH ),
3
; H NMR (CDCl , 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz,
2
2
4.4.11. Diethyl 4-(2-(4-(4-(ethoxycarbonyl)piperidin-1-yl)but-2-
ynyloxy)-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxylate 7k
2
2
.11 (s, 1H, pyridine CH), 6.37 (s, 1H, pyridine NH), 6.75–7.08 (m,
H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, Ar-H), 7.49 (d, 1H, J = 2.5 Hz, Ar-
Brown solid, mp = 84 °C, IR (KBr)
m
max: 3339, 2936, 1693, 1436,
, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz,
Me), 1.26 (t, 3H, J = 7.6 Hz), 1.63–2.01 (m, 4H), 2.30 (s,
H, Me), 2.34–2.51 (m, 1H), 2.59–2.97 (m, 4H), 3.30 (s, 2H,
NCH ), 3.80 (s, 3H, OCH ), 4.03 (q, 2H, J = 6.8 Hz), 4.08–4.22 (m,
4H), 4.62 (s, 2H, OCH ), 5.10 (s, 1H, pyridine CH), 6.36 (s, 1H, pyr-
idine NH), 6.70 (dd, 1H, J = 3.0 Hz, Ar-H), 6.85–6.93 (m, 2H, Ar-H).
+
À1
1
H). MS (ESI) m/z 734 [M+H] ; HR-MS (ESI) calcd for C33
H
37
O
6
N
Br
2 2
F
1218 cm
; H NMR (CDCl
3
+
[
M+H] : 734.0997. Found: 734.1058.
CO CH
2
2
6
4
.4.6. Diethyl 4-(3,5-dibromo-2-(4-(4-(pyridin-2-yl)piperazin-1-
yl)but-2-ynyloxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-
2
3
2
3
,5-dicarboxylate 7f
Yellow solid, mp = 75 °C, IR (KBr)
+
m
max: 3329, 2928, 1692, 1437,
MS (ESI) m/z 583 [M+H] ; HR-MS (ESI) calcd for C32
H
43
O
8
N
2
À1
1
+
1
CO
3
(
211 cm
CH Me), 2.28 (s, 6H, Me), 2.55–2.76 (m, 4H, piperazine H),
.40 (s, 2H, NCH ), 3.48–3.62 (m, 4H, piperazine H), 3.93–4.17
m, 4H), 4.65 (s, 2H, OCH ), 5.09 (s, 1H, pyridine CH), 6.23 (s, 1H,
;
H NMR (CDCl
3
, 300 MHz): d = 1.22 (t, 6H, J = 6.9 Hz,
[M+H] : 583.3014. Found: 583.3017.
2
2
5. ACE inhibition assay
2
2
pyridine NH), 6.54–6.75 (m, 3H, Ar-H), 7.34 (d, 1H, J = 2.3 Hz, Ar-
ACE inhibition assay was performed using the method
2
5
H), 7.49 (d, 1H, J = 2.5 Hz, Ar-H), 8.15–8.22 (m, 1H, Ar-H). MS
described by Jimsheena and Gowda. Rabbit lung acetone powder
(Sigma Aldrich, USA) was used as a source of ACE enzyme. 1 gm of
rabbit lung acetone powder was incubated with 10 mL of 0.05 M
sodium borate buffer pH 8.2 containing 0.3 M NaCl and 0.5% Triton
X-100 at 4 °C for 24 h followed by centrifugation at 4 °C,
12,000 rpm for 30 min. The supernatant was then collected and
stored in aliquots and was used as a source of ACE enzyme. ACE
activity was assayed by monitoring the release of Hippuric acid
(HA) from the hydrolysis of hippuryl-histidyl-leucine (HHL)
(Sigma–Aldrich, USA). ACE solution was preincubated with test
+
(
[
ESI) m/z 717 [M+H] ; HR-MS (ESI) calcd for C33
39
H O
6
N
2
Br
2
+
M+H] : 717.1169. Found: 717.1118.
4
.4.7. Diethyl 4-(3-methoxy-2-(4-(pyrrolidin-1-yl)but-2-ynyloxy)
phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 7g
Brown solid, mp = 122 °C, IR (KBr) max: 3334, 2938, 1693,
, 300 MHz): d = 1.19 (t, 6H, J = 6.9 Hz,
Me), 1.62–1.93 (m, 4H), 2.30 (s, 6H, Me), 2.46–2.74 (m,
H), 3.41 (s, 2H, NCH ), 3.79 (s, 3H, OCH ), 3.93–4.13 (m, 4H),
m
À1
1
1
210 cm
; H NMR (CDCl
3
CO CH
2
2
4
2
3

5
830
R.M. Kumbhare et al. / Bioorg. Med. Chem. 22 (2014) 5824–5830
and standard drug solution for 10 min at 37 °C. The enzyme reac-
tion was started by adding 0.05 M sodium borate buffer (pH 8.2)
containing 0.3 M NaCl and 5 mM substrate (HHL) followed by
incubation at 37 °C for 30 min. The reaction was arrested by the
for the award of a fellowship and R.M.K thanks S.E.R.C., Depart-
ment of Science & Technology, Government of India for financial
assistance under the Fast Track Scheme for young scientists
(SR/FTP/CS-031/2010). SKB is thankful to Department of
Biotechnology (DBT) for providing Ramalingaswami Fellowship.
addition of 100
00 L of pyridine (SD Fine chemical, India) was added followed
by 100 L of benzyl sulfonyl chloride (BSC) (SD Fine chemical,
lL of 0.1 M HCl. After stopping the reaction,
2
l
l
References and notes
India). The solution was mixed by inversion for 1 min and cooled
on ice. The yellow color developed was measured at 410 nm. The
decreased concentration of HA in the test reaction compared with
the control reaction was expressed as percentage inhibition and
calculated from the equation: Inhibition% = 100 À [T/C] Â 100,
where T = absorbance of test reaction and C = absorbance of control
reaction. The therapeutic drug Lisinopril was used as reference ACE
inhibitor.
1
2
3
.
.
.
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6
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. Cytotoxicity assay in cell lines
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Schleifer, K. J. J. Med. Chem. 1999, 42, 2204; (c) Visentin, S.; Amiel, P.; Frittero,
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A549-cells (Human Lung Adenocarcinoma Epithelial Cell Line)
10. Carlos Sunkel, E.; Fau de Casa Juana, Miguel; Cillero, Francisco Javier; Priego,
and HEK293 cells (Human Embryonic Kidney Cell Line) were pur-
chased from National Centre for Cell Science (NCCS), Pune. Both
cell lines were maintained in Dulbecco’s modified Eagle’s medium
Jaime G.; Pilar Ortega, M. J. Med. Chem. 1988, 31, 1886.
1
1. (a) Surendra Kumar, R.; Idhayadhulla, A.; Jamal Abdul Nasser, A.; Kavimani, S.;
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Swain, S. P.; Sarangi, P. Acta Pol. Pharm. 2009, 66, 147.
(
(
DMEM, Sigma, USA) with 10% heat-inactivated fetal bovine serum
FBS, Lonza), 100 U/mL penicillin, 100 U/mL streptomycin, and
12. Beyer, T.; Gansohr, N.; Gjörstrup, P.; Ravens, U. Naunyn Schmiedebergs Arch
Pharmacol. 1986, 334, 488.
1
1
3. Refat, Hala M.; Fadda, A. A. Eur. J. Med. Chem. 2013, 70, 419.
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Schinazi, R. F. J. Med. Chem. 1990, 33, 2188.
2
mM
CO . Cells were passaged every 2–3 days to maintain exponential
growth.
L-glutamine at 37 °C in a humidified atmosphere of 5%
2
1
5. (a) Miri, R.; Javidnia, K.; Mirkhani, H.; Kazemi, F.; Hemmateenejad, B.; Edraki,
N.; Mehdipour, A. R. DARU 2008, 16, 263; (b) Kumar, R. S.; Idhayadhulla, A.;
Abdul Nasser, A. J.; Selvin, J. Eur. J. Med. Chem. 2011, 46, 804.
7
. In vitro cytotoxicity assay
16. (a) Choi, S. J.; Cho, J. H.; Im, I.; Lee, S. D.; Jang, J. Y.; Oh, Y. M.; Jung, Y. K.; Jeon, E.
S.; Kim, Y. C. Eur. J. Med. Chem. 2010, 45, 2578; (b) Al-Nadaf, A.; Abu Sheikha, G.;
Taha, M. O. Bioorg. Med. Chem. 2010, 1, 9.
The cytotoxicity of the different compounds (Table 1) was
1
1
1
2
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MTT
bromide.
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
DMEM,
1
0
4
1 Â 10 cells/well were seeded in 100
lL
supplemented with 10% FBS in each well of 96-well micro culture
plates and incubated for 24 h at 37 °C in a CO
desired concentrations (10 M–1000 M) of the compounds were
made and added to the wells with respective vehicle control. After
4 h of incubation, 10 L MTT (5 mg/mL) was added to each well
2
incubator. The
l
l
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2
l
and the plates were further incubated for 4 h. Then the supernatant
from each well was carefully removed, formazan crystals were dis-
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solved in 100
l
L of DMSO, and absorbance was recorded at 540-nm
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wavelength. Cell survival rate was determined by comparing the
absorbance value of treated cells with that in the control cells.
5
0% effective concentration (EC50) values were decided by probit
analysis.
4772.
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We are thankful to the Director of IICT for providing facilities.
U.B.K., P.K.B., A.K. and K. A. thank for Council of Scientific and
Industrial research (CSIR) and T.L.D. thank for UGC, New Delhi,
K.; Gowda, L. R. Peptides 2010, 31, 1165.