Synthesis of novel Hantzsch dihydropyridines and Biginelli dihydropyrimidines of biological interest: A 3D-QSAR study on their cytotoxicity

Article abstract of DOI:10.1007/s00044-009-9195-7

We report a library consisting of some novel Hantzsch dihydropyridines and Biginelli dihydropyrimidines of biological interest as well as their synthesis and analysis. The important steps in the synthetic part were found to be Hantzsch and Biginelli multicomponent reactions. The synthesized compounds were screened for their in vitro antibacterial activity against two gram-positive bacteria: Staphylococcus aureus and Bacillus subtilis. The title compounds did not exhibit potential antibacterial activity. Furthermore, compounds were subjected to in vitro cytotoxicity against Vero cells. Compounds exhibited weak, moderate, or high cytotoxicity. Compounds 4a, 4b, 4c, 4f, 4g, 4h, 4i, 7i, 7l, 7m, and 7r exhibited potential cytotoxicity. CoMFA study has resulted in the identification of structural features contributing toward their cytotoxicity.

Full text of DOI:10.1007/s00044-009-9195-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:344–363
DOI 10.1007/s00044-009-9195-7
ORIGINAL RESEARCH
Synthesis of novel Hantzsch dihydropyridines
and Biginelli dihydropyrimidines of biological interest:
a 3D-QSAR study on their cytotoxicity
B. R. Prashantha Kumar Æ Pankaj Masih Æ
E. Karthikeyan Æ Ankur Bansal Æ Suja Æ
Pottekad Vijayan
Received: 15 November 2008 / Accepted: 17 March 2009 / Published online: 23 April 2009
¨
Ó Birkhauser Boston 2009
Abstract We report a library consisting of some novel Hantzsch dihydropyridines
and Biginelli dihydropyrimidines of biological interest as well as their synthesis and
analysis. The important steps in the synthetic part were found to be Hantzsch and
Biginelli multicomponent reactions. The synthesized compounds were screened for
their in vitro antibacterial activity against two gram-positive bacteria: Staphylo-
coccus aureus and Bacillus subtilis. The title compounds did not exhibit potential
antibacterial activity. Furthermore, compounds were subjected to in vitro cytotox-
icity against Vero cells. Compounds exhibited weak, moderate, or high cytotoxicity.
Compounds 4a, 4b, 4c, 4f, 4g, 4h, 4i, 7i, 7l, 7m, and 7r exhibited potential cyto-
toxicity. CoMFA study has resulted in the identification of structural features
contributing toward their cytotoxicity.
Keywords Dihydropyrimidines ꢀ Dihydropyridines ꢀ CoMFA ꢀ Cytotoxicity
Introduction
Dihydropyridines and dihydropyrimidines (Eisner and Kuthan, 1972; Stout and
Meyers, 1982; Kappe, 1993, 2003) represent important and extensively studied
compounds belonging to the class of calcium channel blockers. Many researchers
have attempted to determine the synthetic routes and various biological activities of
these compounds. These developments led to the preparation and pharmacological
evaluation of 1,4-dihydropyridines (DHP) and 3,4-dihydropyrimidines (DHPM) of
B. R. Prashantha Kumar (&) ꢀ P. Masih ꢀ E. Karthikeyan
Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ootacamund, TN 643 001,
India
e-mail: brprashant@yahoo.com
A. Bansal ꢀ Suja ꢀ P. Vijayan
Department of Biotechnology, JSS College of Pharmacy, Ootacamund, TN 643 001, India

Med Chem Res (2010) 19:344–363
345
biological interest (Loev et al., 1972, 1974; Meyer et al., 1981; Mager et al., 1992;
Kappe, 2000a, b; Dallinger et al., 2004).
The discovery during the 1930s that a dihydropyridine (dihydronicotinamide
derivative, NADH), ‘‘hydrogen transferring coenzyme’’ consequently became
important in biological system, has generated numerous studies on the biochemical
properties of dihydropyridines and their bioisosteres dihydropyrimidines (Bruce and
Bencovic, 1966). The dihydropyrimidine-5-carboxylate core has been found in
several marine natural products (Heys et al., 2000; Aron and Overman, 2004).
DHPM derivatives are regarded as privileged structures in drug research (Horton
et al., 2003; Kappe, 2003; Eynde and Watte, 2003; Shanumugam et al., 2003).
The credit for the first synthesis of dihydropyridines is attributed to Arthur
Hantzsch for work performed a century ago (Hantzsch, 1882). The Hantzsch 1,4-
dihydropyridines synthesis involves the reaction of a 1,3-dicarbonyl compound with
aldehyde and ammonia (Jones, 1984). In 1893, P. Biginelli reported the first
synthesis of dihydropyrimidine by simple one pot condensation reaction of 1,3-
dicarbonyl compound, aldehyde, and urea (Biginelli, 1893). During the past
decades, the scope of the original cyclocondensation reaction was gradually
extended by variation of all three building blocks, allowing access to a large number
of structurally diverse multifunctionalized DHPs and DHPMs.
The present interest for Hantzsch 1,4-dihydropyridines and Biginelli 1,4-
dihydropyrimidines is mainly due to their close structural relationship to similar
drugs and compounds reported in the literature for their antitubercular (Desai et al.,
2001; Prashantha Kumar et al., 2008), antimicrobial (Hooper et al., 1982; Snider
and Shi, 1993; Aron and Overman, 2004), and anticancer activities (Hattori et al.,
2003; Tsuji et al., 2004).
Results and discussion
Chemistry
Synthesis of Hantzsch 1,4-dihydropyridines derivatives was achieved by adopting
steps as outlined in the Scheme 1. Refluxing ethyl acetoacetate 1 and p-
chloroaniline 2 under neat conditions for 2 h gave p-chloro acetoacetanilide 3 at
30% yield. The p-chloro acetoacetanilide 3 was subjected to Hantzsch method of
1,4-dihydropyridines synthesis. Hantzsch one pot multicomponent reaction involved
0.01 mole of p-chloro acetoacetanilide 3, 0.005 moles of aryl or heteroaryl
aldehydes, and excess of 25% aqueous NH3 using ethanol as a solvent under reflux
condition for 12–18 h gave a corresponding 1,4-dihydropyridines 4a–k. After
recrystallization, yields were found to be between 30 and 36% (Table 1).
Synthesis of 1,4-dihydropyrimidines by adopting Biginelli synthetic protocol
involving one pot multicomponent reaction was performed by following steps as
outlined in Scheme 2 (Kappe and Stadler, 2004). In the first step, ethyl acetoacetate
1 and p-anisidine 5 reacted under neat conditions resulting in the formation of p-
methoxy acetoacetanilide 6 with the yield of 40%. p-Methoxy acetoacetanilide 6
was further taken for the Biginelli condensation reaction by reacting it with urea or

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O
O
a
O
O
H2N
Cl
+
Cl
N
+
N
Cl
O
H
H
O
O
b
2
1
3
RCHO
O
R
O
Cl
N
H
N
H
Cl
N
H
4a-4k
Scheme 1 Reagents and conditions for the synthesis of DHPs: (a) neat, reflux, 2 h and (b) ethanol, 25–
30% aqueous ammonia solution, reflux for 12–18 h
thiourea and aryl or heteroaryl aldehyde in the presence of catalytic amount of p-
toluenesulfonic acid (PTSA). The reaction times were found to be between 22 and
26 h. Nineteen 7a–s, various substituted-1,4-dihydropyrimidines, were synthesized
with the yield ranging from 18 to 51% (Table 1).
Biginelli 1,4-dihydropyrimidines, 7a–s, were synthesized relatively easily by
using PTSA as an efficient catalyst compared with anhydrous AlCl3 or HCl. The
present protocol best describes the synthesis of Biginelli dihydropyrimidines. All
the reported Biginelli dihydropyrimidines compounds were found to be novel and
not reported elsewhere. Most of the reported Hantzsch dihydropyridines were found
to be novel.
Antibacterial activity
Dihydropyridines and dihydropyrimidines are known to posses’ antitubercular
activity. All the synthesized compounds were screened for their potential
antibacterial activity by cup plate method against two gram-positive bacteria, such
as Staphylococcus aureus and Bacillus subtilis, because gram-positive bacteria
share some common structural features with the Mycobacterium tuberculosis.
However, none of the titled compounds exhibited significant antibacterial activity
when tested in triplicate at 50 lg concentration, as preliminary antibacterial study.
Possibly, it seems that the p-chloroanilide and p-methoxyanilide portion of
structures would not have contributed toward their antibacterial activity to come
out as candidate compounds to investigate further for the same.
In vitro cytotoxicity assay
The synthesized compounds were subjected to in vitro cytotoxicity assay against
Vero cells. The motive for us to check the cytotoxicity for the synthesized

Med Chem Res (2010) 19:344–363
347
Table 1 Structures of the synthesized 1,4-dihydropyridines and 1,4-dihydropyrimidines, their cytotox-
icity on Vero cells, and their predicted cytotoxicity by CoMFA
Compound
no.
R
X
Reaction CTC50
time (h)
pCTC50
(lM)
Predicted
pCTC50
(lg/ml)
4a
4ba
4c
4d
4e
4f
phenyl
–
12
14
14
16
15
13
12
18
12
14
12
23
25
22
25
22
24
26
24
23
23
23
25
24
23
22
23
24
26
25
15
20
1.516
1.420
1.302
0.333
0.800
1.315
1.642
1.392
1.017
0.913
0.538
0.176
1.371
0.367
0.408
0.192
0.173
0.343
0.176
1.383
0.776
0.697
1.680
1.698
0.164
0.157
0.207
-0.101
1.529
0.116
1.687
1.411
1.510
0.162
0.805
1.452
1.263
1.452
0.951
0.898
0.579
0.351
1.310
0.540
0.344
0.320
0.107
0.326
0.161
1.494
0.748
0.586
1.725
1.563
0.282
0.084
0.294
0.100
0.947
0.363
4-methoxyphenyl
2-furyl
–
–
24
4-hydroxy-3-methoxyphenyl
4-pyridyl
–
250
78
–
3-nitrophenyl
3-chlorophenyl
cinnamyl
–
26
4g
4h
4i
–
12
–
21
H
–
40
4ja
4k
7a
7ba
7c
7d
7ea
7f
2-hydroxyphenyl
4-[N,N-dimethyl]-phenyl
phenyl
–
62
–
155
225
15
O
S
O
S
O
S
O
S
O
O
S
O
S
O
S
O
S
O
O
phenyl
4-methoxyphenyl
4-methoxyphenyl
2-furyl
158
150
210
230
160
246
15
2-furyl
7g
7ha
7i
2-hydroxyphenyl
2-hydroxyphenyl
cinnamyl
7j
3-nitrophenyl
3-nitrophenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
3-chlorophenyl
3-chlorophenyl
4-[N,N-dimethyl]phenyl
4-[N,N-dimethyl]phenyl
4-pyridyl
64
7k
7l
80
8
7m
7n
7o
7pa
7q
7r
7s
8
254
270
236
500
10
H
200
a
Test set compounds
compounds was that some reports in the past have claimed significant anticancer
activity for similar kind of substructures (Kawase et al., 2002). The assay was
performed by the sulforhodamine B (SRB) method (Philip et al., 1990). Almost all
of the titled compounds exhibited weak, moderate, or high cytotoxicity. Com-
pounds, 4a, 4b, 4c, 4f, 4g, 4h, 4i, 7i, 7l, 7m, and 7r exhibited significant cytotoxic
activity with lesser CTC50 values (Table 1).

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O
O
O
a
+
H2N
O
O
N
O
H
O
1
5
6
NH2
b
H2N
RCHO
,
X
R
O
NH
NH
X
O
NH
X= O or S
R= Aryl or Heteroaryl
7a-7s
Scheme 2 Reagents and conditions for the synthesis of DHPMs: (a) reflux for 9 h and (b) PTSA,
C2H5OH, stirring for 22–26 h at rt
3D-QSAR study
To establish the structure activity relationship of the title compounds toward their
cytotoxic activity, 3D-QSAR study was performed by using comparative molecular
field analysis (CoMFA) method (Kubinyi and Wolff, 1995; Cramer et al., 1988a).
Of 30 title compounds, 24 compounds constituted the training set and remaining 6
compounds, which are congeneric to the training set compounds, constituted the test
set. Test and training set compounds were chosen manually such that low-,
moderate-, and high-activity compounds were present in approximately equal
proportions in both sets. The training set compounds were used to develop the
CoMFA model and the test set compounds were used to validate the developed
model. The structures of compounds were sketched, energy minimized, and
analyzed for their conformations according to the protocol implemented in the
SYBYL 6.7. Both the test set and training set molecules were aligned separately by
field fit method. The aligned training set molecules were subjected to the CoMFA
analysis.
CoMFA model was developed considering all the necessary statistical param-
eters. For the developed CoMFA model, the partial least square (PLS) analyses
yielded cross-validated q2 value by leave one out method (LOO) of 0.568 with six
principle components. No outlier was removed because the obtained q2 value was
good enough to retain all the compounds in the training set to build the model.
Statistical parameters for the developed CoMFA model are given in Table 2. Steric
and electrostatic contributions surrounding the aligned training set molecules were
found to be 51.5 and 48.5%, respectively. The developed model was further
validated by predicting the activities of the test set compounds. Plot depicting
correlation between the experimental activity and the calculated activity is shown in
Fig. 1.
Contour plot depicting the steric and electrostatic fields of the compounds
generated based on the contributions toward the PLS model is shown in Fig. 2. The

Med Chem Res (2010) 19:344–363
349
Table 2 PLS data obtained for a developed CoMFA model consisting of 24 compounds
Parameter
Value
q2 value (LOO)
0.568
6
No. of components
Standard error of prediction (cross-validated)
r2 (non-cross-validated)
Standard error of estimate (non-cross-validated)
F value
0.212
0.898
0.113
81.782
0
P value
Steric contribution
0.515
0.485
Electrostatic contribution
Fig. 1 Plot of experimental vs. predicted activities for the training set compounds
green (sterically favorable) and yellow (sterically unfavorable) and blue (electro-
statically favorable for electronegative atoms) and red (electrostatically unfavorable
for electronegative atoms) regions in the contour represent 80 and 20% level
contributions, respectively.
The structure activity relationships based on the above CoMFA contour map are
as follows. 1,4-Dihydropyridine and 1,4-dihydropyrimidine motifs being the
bioisosteric pharmacophores are required for the cytotoxicity. The pharmacophore
part in the above contour map is clearly visible as exactly superimposed part of the
aligned molecules (common substructure). Anilide portion of dihydropyrimidine
and dihydropyridine also seems to be the part of pharmacophore because we can see
some compounds with significant activity from both the classes. Large, green
contours at fourth position of 1,4-dihydropyridine and 1,4-dihydropyrimidine ring

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Med Chem Res (2010) 19:344–363
Fig. 2 1,4-Dihydropyridines and 1,4-dihydropyrimidines CoMFA steric SD 9 coefficient contour plot
system indicate the substitution favoring the activity. Evidence for this is that all the
dihydropyridines and dihydropyrimidines possessed the different substitution at that
position. The small yellow contour on the nitrogen atoms of the basic bioisosteric
scaffolds indicates that there should not be any substitution or steric extension over
them or in that region. The small green contour map at the one side of anilide
portion indicates that 1,4-dihydropyridines are relatively more potent having anilide
portions on either side of the scaffold (4a and 4g). In contrast to this,
dihydropyrimidines had anilide portion on one side only and still exhibited the
cytotoxic activity (7q exhibited least activity amongst all, whereas 7l and 7r showed
significant activity). The yellow and blue contours at the para position of phenyl
ring indicated that substitution with bulky (methyl groups) and electropositive
substitutions will not favor toward cytotoxic activity (compounds 7q and 4k
containing N,N-dimethyl groups).
Conclusions
A series of novel Hantzsch dihydropyridines and Biginelli dihydropyrimidines of
biological interest were synthesized and analyzed for their structures. The Biginelli
compounds were prepared by using PTSA as an efficient catalyst. Compounds, such
as 4h, 7r, and 7m, exhibited potential cytotoxicity and are considered the candidates
to investigate further for the same. CoMFA, a ligand-based approach, is used in
rational drug design to build activity model, which provides important information
on possible improvements in ligand structure to increase the cytotoxicity. The
developed CoMFA model is the most significant and reliable model and indicates
the importance of substitution at the fourth position of 1,4-dihydropyridines and 1,4-
dihydropyrimidines toward the cytotoxicity.

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351
Experimental
General
The melting points of the synthesized compounds were determined in open
capillaries using Veego VMP-1 apparatus and are expressed in °C and are
uncorrected. The IR spectra of compounds were recorded on Shimadzu FT-IR
1
spectrometer using KBR pellet technique and are expressed in cm-1. H NMR
spectra was recorded on Bruker DRX-300 (300 MHz FT-NMR) using DMSO-d6 as
solvent and TMS as internal standard. The chemical shifts were reported in d ppm.
Mass spectra were obtained using Shimadzu LCMS 2010A under ESI ionization
technique.
Procedure for the preparation of p-chloro acetoacetanilide (3)
1 (0.01 M) and 2 (0.01 M) were mixed and refluxed for approximately 2 h. The
light-yellowish liquid formed was then heated on a water bath to remove the alcohol
formed during the reaction. After allowing the reaction mixture to cool, crude
crystals were obtained. Purification was performed by stirring crude crystals with
cold diethyl ether for approximately 20 min using a mechanical stirrer. After
allowing it to stand for 30 min, followed by filtration, gave 3 in a pure form.
p-Chloro acetoacetanilide (3)
White crystalline solid, mp 145–152°C, Yield 30%, IR (KBr, cm-1): 3249 (N–H),
3070 (ArC–H), 2980 (AliC–H), 1714 (C=O, ketone), 1658 (C=O, amide), 1552
(C=C), 1342 (C–N), 669 (C–Cl), 1H-NMR (DMSO-d6) d: 2.20 (s, 3H, CH3), 3.55 (s,
2H, CH2), 7.33 (d, 2H, ArH), 7.62 (d, 2H, ArH), 10.22 (s, 1H, NH), MS (m/z): M?
calculated 211, found, 210.95, Mass fragments (m/z): 168.02, 155.05.
General procedure for the preparation of 1,4-dihydropyridines by one
pot-multicomponent, Hantzsch method of synthesis (4a–k)
Preparation of 4a–k by one pot multicomponent reaction was performed according
to Scheme 1. The mixture of 3 (0.01 M), appropriate aldehyde (0.005 M), and 3 ml
of 25–30% aqueous ammonia solution were transferred to a round bottom flask
containing 15 ml of ethanol. The reaction mixture was refluxed for 12–18 h. One
milliliter of 25% aqueous ammonia solution was added for every 3 h during the
reflux. The reactions were monitored through TLC using 20% ethyl acetate in pet
ether as solvent system. Soon after the reaction was completed, the reaction mixture
was allowed to cool. The solid product formed was filtered and washed with cold
methanol to get Biginelli compounds. Biginelli condensation reactions can be
performed in a parallel synthetic way with ease.

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N,N-Bis(4-chlorophenyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-
dicarboxamide (4a)
Colorless amorphous solid, mp 195–200°C, yield 30%, IR (KBr, cm-1): 3290 (N–
H), 3074 (ArC–H), 3000 (AliC–H), 1652 (C=O, amide), 1490 (ArC=C), 1332 (C–
1
N), 703 (C–Cl), H-NMR (DMSO-d6) d: 2.07 (s, 6H, (CH3)2), 5.07 (s, 1H, CH),
6.13 (bs, 1H, NH), 7.20 (d, 4H, ArH), 7.27 (d, 4H, ArH), 7.45 (m, 1H, CH), 7.59 (m,
4H, ArH), 9.26 (s, 2H, (NH)2). MS (m/z): M? calculated 492, found 491.55, Mass
fragments (m/z): 474.55, 461.55, 414.65, 322.80.
N,N-Bis(4-chlorophenyl)-4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxamide (4b)
White amorphous solid, mp 225–229°C, yield 35%, IR (KBr, cm-1): 3338 (N–H),
3001 (ArC–H), 2908 (AliC–H), 1668 (C=O, amide), 1508 (ArC=C), 1303 (C–N),
1240 (C–O), 675 (C–Cl), 1H-NMR (DMSO-d6) d: 2.07 (s, 6H, (CH3)2), 3.63 (s, 3H,
OCH3), 5.07 (s, 1H, CH), 5.79 (bs, 1H, NH), 6.74 (d, 2H, ArH), 7.12 (d, 2H, ArH),
7.29 (d, 4H, ArH), 7.60 (d, 4H, ArH), 9.41 (s, 2 H, (NH)2). MS (m/z): M? calculated
522, found 521.86, Mass fragments (m/z): 506.3, 478.71, 412.35, 225.12.
N,N-Bis(4-chlorophenyl)-4-(furan-2-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxamide (4c)
Colorless amorphous solid, mp 198–202°C, yield 29%, IR (KBr, cm-1): 3299 (N–
H), 3126 (ArC–H), 3066 (AliC–H), 1666 (C=O, amide), 1537 (ArC=C), 1249 (C–
N), 1163 (C–O), 690 (C–Cl). 1H-NMR (DMSO-d6) d: 2.10 (s, 6H, (CH3)2), 5.13 (s,
1H, CH), 6.13 (bs, 1H, NH),7.23 (d, 4H, ArH), 7.46 (d, 4H, ArH), 7.59 (t, 1H, CH),
7.84 (d, 1H, ArH), 7.91(d, 1H, ArH), 9.15 (s, 2H, (NH)2). MS (m/z): M? calculated
482, found 481.55, Mass fragments (m/z): 327.80.
N,N-Bis(4-chlorophenyl)-4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxamide (4d)
Bluish amorphous solid, mp 280–284°C, yield 33%, IR (KBr, cm-1): 3380 (O–H),
3361 (N–H), 3126 (ArC–H), 3020 (AliC–H), 1660 (C=O, amide), 1515 (ArC=C),
1307 (C–N), 1249(C–O), 688 (C–Cl). 1H-NMR (DMSO-d6) d : 2.50 (s, 6H, (CH3)2),
3.54 (s, 3H, OCH3), 5.20 (s, 1H, CH), 5.79 (bs, 1H, NH), 6.82–6.98 (m, 3H, ArH),
7.01(s, 1H, ArH), 7.32 (d, 4H, ArH), 7.47 (d, 4H, ArH), 9.07 (s, 1H, OH), 10.45 (s,
2H, (NH)2). MS (m/z): M? calculated 538, found 538.20, Mass fragments (m/z):
522.50, 502.50, 370.70, 352.75.
N,N-Bis(4-chlorophenyl)-4-(4-pyridyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxamide (4e)
Colorless amorphous solid, mp 275–281°C, yield 36%, IR (KBr, cm-1): 3286 (N–
H), 3110 (ArC–H), 2999 (AliC–H), 1679 (C=O, amide), 1631 (C=C), 1595(C=N),

Med Chem Res (2010) 19:344–363
353
1321 (C–N), 1242 (C–O), 690 (C–Cl), 1H-NMR (DMSO-d6) d: 2.09 (s, 6H, (CH3)2),
5.06 (s, 1H, CH), 6.19 (bs, 1H, NH), 7.17 (d, 2H, ArH), 7.30 (d, 4H, ArH), 7.60 (d,
4H, ArH), 8.03 (d, 2H, ArH), 9.59(d, 2H, (NH)2), MS(m/z): M? calculated 493,
found 493.50, Mass fragments (m/z): 382.70.
N,N-Bis(4-chlorophenyl)-4-(3-nitrophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxamide (4f)
1
Yellowish amorphous solid, mp 201–202°C, yield 32%, H-NMR (DMSO-d6) d:
2.18 (s, 6H, (CH3)2), 5.31 (s, 1H, CH), 6.35 (bs, 1H, NH), 7.28 (d, 4H, ArH), 7.52 (t,
1H, CH), 7.55 (d, 4H, ArH), 7.64 (d, 1H, CH), 7.96 (d, 1H, ArH), 8.05 (s, 1H, ArH),
9.60 (s, 2H, (NH)2), MS (m/z): M? calculated 537, found 537.45, Mass fragments
(m/z): 522.50, 507.50, 494.50, 479.55, 425.65.
4-(3-Chlorophenyl)-N,N-bis(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxamide (4g)
Brownish semisolid, mp 65–69°C, yield 33%, 1H-NMR (DMSO-d6) d: 2.02 (s, 6H,
(CH3)2), 5.74 (s, 1H, CH), 6.21 (bs, 1H, NH), 7.12 (d, 1H, CH), 7.22 (t, 1H, CH),
7.30 (d, 4H, ArH), 7.40 (d, 2H, ArH), 7.49 (s, 1H, ArH), 7.59 (d, 4H, ArH), 9.54 (s,
2H, (NH)2), MS (m/z): M? calculated 526, found 526.50, Mass fragments (m/z):
491.55, 475.55, 460.60.
(E)-N,N-Bis(4-chlorophenyl)-2,6-dimethyl-4-styryl-1,4-dihydropyridine-3,5-
dicarboxamide (4h)
1
Colorless amorphous solid, mp 180–183°C, yield 36%, H-NMR (DMSO-d6) d:
2.20 (s, 6H, (CH3)2), 5.29 (s, 1H, CH), 6.02 (s, 1H, NH), 6.21 (d, 1H, CH), 6.64 (d,
1H, CH), 7.14 (m, 5H, ArH), 7.46 (d, 4H, ArH), 7.65 (d, 4H, ArH), 9.49 (s, 2H,
(NH)2). MS (m/z): M? calculated 518, found 518.85, Mass fragments (m/z): 413.60,
390.70, 357.45, 341.35.
N,N-Bis(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxamide (4i)
1
Yellowish amorphous solid, mp 118–123°C, yield 35%, H-NMR (DMSO-d6) d:
2.00 (s, 6H, (CH3)2), 3.33 (s, 2H, CH2), 7.32 (d, 4H, ArH), 7.65 (d, 4H, ArH), 7.88
(s, 1H, NH), 9.21 (s, 2H, (NH)2), MS (m/z): M? calculated 416, found 416.65, Mass
fragments (m/z): 401.65, 388.70, 303.85.
N,N-Bis(4-chlorophenyl)-4-(2-hydroxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-
3,5-dicarboxamide (4j)
Brownish amorphous solid, mp 170–174°C, yield 31%, IR (KBr, cm-1): 3390 (O–
H), 3361 (N–H), 3066 (ArC–H), 2988 (AliC–H), 1658 (C=O, amide), 1525
1
(ArC=C), 1311 (C–N), 1230 (C–O), 661 (C–Cl), H-NMR (DMSO-d6) d: 2.19 (s,
6H, (CH3)2), 5.26 (s, 1H, CH), 5.80 (bs, 1H, NH), 6.68 (d, 1H, ArH), 6.78 (t, 1H,

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Med Chem Res (2010) 19:344–363
ArH), 6.93 (t, 1H, CH), 7.20 (d, 1H, CH), 7.31 (d, 4H, ArH), 7.61 (d, 4H, ArH), 9.33
(s, 2H, (NH)2), 10.16 (s, 1H, OH). MS (m/z): M? calculated 508, found 50812, Mass
fragments (m/z): 479.31, 432.22, 398.10.
N,N-Bis(4-chlorophenyl)-4-(4-(dimethylamino)phenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxamide (4k)
Yellowish amorphous solid, mp 150–155°C, yield 30%, IR (KBr, cm-1): 3296 (N–
H), 3040 (ArC–H), 2932 (AliC–H), 1631 (C=O, amide), 1560 (ArC=C), 1352 (C–
N), 1236 (C–O), 638 (C–Cl). 1H-NMR (DMSO-d6) d: 2.13 (s, 3H, (CH3)2), 3.01 (s,
6H, 2 9 NCH3), 3.70 (s, 3H, OCH3), 5.79 (s, 1H, CH), 6.03 (bs, 1H, NH), 6.92 (d,
4H, ArH), 7.10 (m, 4H, ArH), 7.34 (d, 4H, ArH), 9.21 (s, 2H, NH). MS (m/z): M?
calculated 535, found 535.45, Mass fragments (m/z): 517.50, 501.50, 491.55,
461.55, 455.60, 429.65, 422.65, 407.65.
Procedure for the preparation of p-methoxy acetoacetanilide (6)
1 (0.01 M) and 5 (0.01 M) were mixed and refluxed for approximately 9 h; the
blackish liquid formed was then heated on a water bath to remove the alcohol
formed during the reaction. After allowing the reaction mixture to cool, crude
crystals were obtained, filtered, and the residue was washed with cold diethyl ether
to get light blue-colored crystals of p-methoxy acetoacetanilide.
p-Methoxy acetoacetanilide (6)
Light-blue crystalline solid, mp 183–185°C, yield 35%, IR (KBr, cm-1): 3573 (N–
H, broad), 3002 (ArC–H), 2960 (AliC–H), 1716 (C=O, ketone), 1654 (C=O, amide),
1
1467 (C=C), 1249 (O–C), H-NMR (DMSO-d6) d: 2.18 (s, 3H, CH3), 3.48 (s, 2H,
CH2), 3.70 (s, 3H, OCH3), 6.85–6.87 (d, 2H, ArH), 7.45–7.47 (d, 2H, ArH), 9.92 (s,
1H, NH). MS (m/z): M? calculated 207, found 207.95, Mass fragments (m/z):
205.95, 188.0.
General procedure for the preparation of 1,4-dihydropyrimidines by one pot-
multicomponent, Biginelli synthesis (7a–s)
The mixture of 6 (0.005 M), urea/thiourea (0.0075 M), and appropriate aldehyde
(0.005 M) with catalytic amount of PTSA in 15 ml of ethanol was stirred for 22–
26 h. The reactions were monitored through TLC using 30% ethyl acetate in pet
ether as solvent system. After the reaction was complete, the reaction mixture was
cooled in a refrigerator and filtered. The precipitate obtained was washed
thoroughly with water to remove unreacted urea/thiourea and dried. The crude
solid product was recrystallized with ethanol to give the pure compounds (7a–s).
This reaction can be performed in a parallel synthetic way by taking above building
blocks and varying the aldehydes for each reaction mixture.

Med Chem Res (2010) 19:344–363
355
6-Ethyl-1,2,3,4-tetrahydro-N-(4-methoxyphenyl)-2-oxo-4-phenylpyrimidine-5-
carboxamide (7a)
Ash-colored solid, mp 240–242°C, yield 51%, IR (KBr, cm-1): 3278 (N–H), 3114
(ArC–H), 2912 (AliC–H), 1681 (C=O, amide), 1519 (C=C), 1176 (O–C), H-NMR
1
(DMSO-d6) d: 2.01 (s, 3H, CH3), 3.67 (s, 3H, OCH3), 5.37 (s, 1H, CH), 6.79–6.81
(d, 2H, ArH), 7.20–7.35 (m, 5H, ArH), 7.41 (d, 2H, ArH), 7.54 (s, 1H, NH), 8.66 (s,
1H, NH), 9.41 (s, 1H, NH). MS (m/z): M? calculated 337, found 336.78, Mass
fragments (m/z): 317.16, 252.35.
N-(4-Methoxyphenyl)-6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (7b)
1
Light-greenish solid, mp 209–212°C, yield 41%, H-NMR (DMSO-d6) d: 2.04 (s,
3H, CH3), 3.68 (s, 3H, OCH3), 5.38 (s, 1H, CH), 6.81 (d, 2H, ArH), 7.23–7.44 (m,
5H, ArH), 7.48 (d, 2H, ArH), 9.40 (s, 1H, NH), 9.59 (s, 1H, NH), 9.95 (s, 1H, NH),
MS (m/z): M? calculated 353, found 353.75. Mass fragments (m/z): 341.80, 329.80,
315.85, 277.90, 272.90.
N-Bis(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (7c)
Light-bluish colored solid, mp 238–240°C, yield 40%, IR (KBr, cm-1): 3328 (N–
H), 3067 (ArC–H), 2954 (AliC–H), 1681 (C=O, amide), 1514 (C=C), 1284 (O–C),
1H-NMR (DMSO-d6) d: 2.01 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 5.32 (s, 1H, CH),
6.79 (d, 2H, ArH), 6.85–7.16 (m, 4H, ArH), 7.41 (d, 2H, ArH), 7.47 (s, 1H, NH),
8.62 (s, 1H, NH), 9.32 (s, 1H, NH). MS (m/z): M? calculated 367, found 366.75,
Mass fragments (m/z): 353.0, 348.75, 330.80, 305.85.
N-Bis(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (7d)
1
Light-greenish solid, mp 218–220°C, yield 41%, H-NMR (DMSO-d6): d 2.04 (s,
3H, CH3), 3.70 (s, 3H, OCH3), 5.31 (s, 1H, CH), 6.80 (d, 2H, ArH), 6.88 (d, 2H,
ArH), 7.14 (d, 2H, ArH), 7.44 (d, 2H, ArH), 9.35 (s, 1H, NH), 9.55 (s, 1H, NH), 9.91
(s, 1H, NH). MS (m/z): M? calculated 383, found 382.80, Mass fragments (m/z):
321.60, 252.35, 237.45, 211.40.
4-(Furan-2-yl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-
5-carboxamide (7e)
Dark-brownish solid, mp 234–236°C, yield 40%, IR (KBr, cm-1): 3292 (N–H),
3012 (ArC–H), 2918 (AliC–H), 1666 (C=O amide), 1519 (C=C), 1244 (C–O), H-
1
NMR (DMSO-d6): d 2.05 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 5.8 (s, 1H, CH), 6.18
(d, 2H, ArH), 7.41 (m, 3H, ArH), 7.62 (d, 2H, ArH), 8.73 (s, 1H, NH), 9.41 (s, 1H,

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Med Chem Res (2010) 19:344–363
NH), 9.98 (s, 1H, NH). MS (m/z): M? calculated 327, found 327.13, Mass fragments
(m/z): 311.40, 252.35, 227.35, 211.40.
4-(Furan-2-yl)-N-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7f)
Ash-colored solid, mp 208–211°C, yield 37%, 1H-NMR (DMSO-d6): d 2.06 (s, 3H,
CH3), 3.72 (s, 3H, OCH3), 5.41 (s, 1H, CH), 6.21 (d, 2H, ArH), 6.82 (d, 2H, ArH),
7.42–7.60 (m, 3H, ArH), 9.45 (s, 1H, NH), 9.58 (s, 1H, NH), 10.3 (s, 1H, NH), MS
(m/z): M? calculated 343, found 343.80, Mass fragments (m/z): 319.80, 306.85,
287.9, 268.90.
4-(2-Hydroxyphenyl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7g)
1
Light-greenish solid, mp 226–227°C, yield 38%, H-NMR (DMSO-d6): d 2.04 (s,
3H, CH3), 3.70 (s, 3H, OCH3), 5.70 (s, 1H, CH), 6.72 (d, 2H, ArH), 7.50–7.58 (m,
4H, ArH), 8.70 (s, 1H, NH), 9.30 (s, 1H, NH), 9.80 (s, 1H, OH), 10.00 (s, 1H, NH).
MS (m/z): M? calculated 353, found 352.40, Mass fragments (m/z): 307.7, 232.85,
207.4, 121.80.
4-(2-Hydroxyphenyl)-N-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7h)
1
Light-ash-colored solid, mp 235–237°C, yield 34%, H-NMR (DMSO-d6): d 1.75
(s, 3H, CH3), 3.70 (s, 3H, OCH3), 4.61 (s, 1H, CH), 6.83–7.41 (m, 8H, ArH), 9.0 (s,
1H, NH), 9.02 (s, 1H, NH), 9.05 (s, 1H, OH), 10.02 (s, 1H, NH). MS (m/z): M?
calculated 369, found 369.10, Mass fragments (m/z): 349.91, 212.32, 120.76.
(E)-N-(4-Methoxyphenyl)-6-methyl-2-oxo-4-styryl-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (7i)
Light-greenish-colored solid, mp 209–214°C, yield 29%, IR (KBr, cm-1): 3346 (N–
H), 3056 (ArC–H), 2967 (AliC–H), 1668 (C=O, amide), 1512(ArC=C),
1
1618(AliC=C), H-NMR (DMSO-d6): d 2.04 (s, 3H, CH3), 3.73 (s, 3H, OCH3),
5.80 (s, 1H, CH), 6.19–6.51 (m, 4H, ArH), 7.20–7.59 (m, 4H, ArH), 9.93 (s, 1H,
NH), 10.0 (s, 1H, NH), 10.13 (s, 1H, NH). MS (m/z): M ? 1 calculated 364, found
364.75, Mass fragments (m/z): 323.80, 304.85, 292.85, 276.90.
N-(4-Methoxyphenyl)-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7j)
Light-bluish solid, mp 257–259°C, yield 39%, 1H-NMR (DMSO-d6): d 2.04 (s, 3H,
CH3), 3.67 (s, 3H, OCH3), 5.48 (s, 1H, CH), 6.80 (d, 2H, ArH), 7.38 (d, 2H, ArH),
7.62–7.73 (m, 4H, ArH), 8.11 (s, 1H, NH), 8.85 (s, 1H, NH), 9.48 (s, 1H, NH). MS

Med Chem Res (2010) 19:344–363
357
(m/z): M? calculated 382, found 382.70, Mass fragments (m/z): 357.75, 335.80,
306.85, 277.90.
N-(4-Methoxyphenyl)-6-methyl-4-(3-nitrophenyl)-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7k)
Light-greenish solid, mp 246–248°C, yield 35%, IR (KBr, cm-1): 3346 (N–H),
3010(ArC–H), 2979(AliC–H), 1890 (C=S), 1674 (C=O, amide), 1558 (C=C), 1521
(NO2), 1350 (NO2), 1203 (C–O). 1H-NMR (DMSO-d6) d: 2.04 (s, 3H, CH3), 3.68 (s,
3H, OCH3), 5.49 (s, 1H, CH), 7.39 (d, 2H, ArH), 7.68–8.15 (m, 6H, ArH), 9.56 (s,
1H, NH), 9.65 (s, 1H, NH), 10.15 (s, 1H, NH). MS (m/z): M? calculated 398, found
397.79. Mass fragments (m/z): 315.21, 311.12, 278.29, 139.23.
4-(4-Hydroxy-3-methoxyphenyl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7l)
1
Light-greenish solid, mp 185–186°C, yield 47%, H-NMR (DMSO-d6) d: 2.03 (s,
3H, CH3), 3.67 (s, 3H, OCH3), 5.28 (s, 1H, CH), 6.66–6.83 (m, 5H, ArH), 7.42 (d,
2H, ArH), 8.99 (s, 1H, NH), 9.29 (s, 1H, NH), 9.54 (s, 1H, OH), 9.86 (s, 1H, NH).
MS (m/z): M? calculated 383, found 383.70, Mass fragments (m/z): 369.70, 355.75,
340.80, 303.85.
4-(4-Hydroxy-3-methoxyphenyl)-N-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7m)
Pale-yellowish solid, mp 202–206°C, yield 37%, IR (KBr, cm-1): 3388 (O–H),
3258 (N–H), 3058 (ArC–H), 2993 (AliC–H), 1656 (C=O, amide), 1964 (C=S), 1510
1
(ArC=C), 1176 (C–O), H-NMR (DMSO-d6): d 2.01 (s, 3H, CH3), 3.15 (s, 3H,
OCH3), 4.82 (s, 1H, CH), 6.61–6.85 (m, 5H, ArH), 7.29 (d, 2H, ArH), 8.86 (s, 1H,
NH), 8.89 (s, 1H, NH), 9.36 (s, 1H, OH), 9.38 (s, 1H, NH). MS (m/z): M? calculated
399, found 399.41, Mass fragments (m/z): 358.1, 301.23.
4-(3-Chlorophenyl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7n)
Light-bluish solid, mp 212–214°C, yield 38%, 1H-NMR (DMSO-d6) d: 2.02 (s, 3H,
CH3), 3.67 (s, 3H, OCH3), 5.35 (s, 1H, CH), 6.81 (t, 3H, ArH), 7.27–7.41 (m, 5H,
ArH), 8.74 (s, 1H, NH), 9.46 (s, 1H, NH), 9.9 (s, 1H, NH). MS (m/z): M? calculated
371, found 371.70, Mass fragments (m/z): 357.75, 344.75, 330.80, 308.85.
4-(3-Chlorophenyl)-N-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7o)
Light-greenish solid, mp 205–207°C, yield 41%, IR (KBr, cm-1): 3394 (N–H),
3050 (ArC–H), 2954 (AliC–H), 1942 (C=S), 1685 (C=O, amide), 1544 (C=C), 1224
(C–O), 734 (C–Cl). 1H-NMR (DMSO-d6) d: 2.05 (s, 3H, CH3), 3.72 (s, 3H, OCH3),

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5.36 (s, 1H, CH), 6.82 (d, 2H, ArH), 7.18 (d, 2H, ArH), 7.25–7.43 (m, 4H, ArH),
9.44 (s, 1H, NH), 9.62 (s, 1H, NH), 10.05 (s, 1H, NH). MS (m/z): M? calculated
387, found 387.39, Mass fragments (m/z): 372.0, 359.01.
4-(4-(Dimethylamino)phenyl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7p)
1
Light-greenish solid, mp 224–225°C, yield 42%, H-NMR (DMSO-d6) d: 2.23 (s,
3H, CH3), 3.03 (s, 3H, N–CH3), 3.70 (s, 3H, OCH3), 5.39 (s, 1H, CH), 5.42 (d, 2H,
ArH), 6.52 (d, 2H, ArH), 7.10 (m, 4H, ArH), 9.83 (s, 1H, NH), 9.91 (s, 1H, NH),
9.93 (s, 1H, NH). MS (m/z): M? calculated 380, found 380.55. Mass fragments (m/
z): 367.50, 350.0, 332.55, 324.90, 316.50.
4-(4-(Dimethylamino)phenyl)-N-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7q)
Light-greenish solid, mp 198–200°C, yield 39%, IR (KBr, cm-1): 3341 (NH), 3043
(ArC–H), 2972 (AliC–H), 1942, (C=S), 1658 (C=O amide), 1512 (ArC=C),1033
(C–O), 1H-NMR (DMSO-d6): d 2.07 (s, 3H, CH3), 2.48 (s, 3H, N–CH3), 3.67 (s, 3H,
OCH3), 5.39 (s, 1H, CH), 6.66 (d, 2H, ArH), 6.80 (d, 2H, ArH),7.04 (d, 2H, ArH),
7.42 (d, 2H, ArH), 9.28 (s, 1H, NH), 9.50 (s, 1H, NH), 9.84 (s, 1H, NH). MS (m/z):
M? calculated 396, found 396.10, Mass fragments (m/z): 381.12, 352.0.
N-(4-Methoxyphenyl)-6-methyl-2-oxo-4-(pyridin-4-yl)-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7r)
Light-bluish solid, mp 196–198°C, yield 34%, IR (KBr, cm-1): 3288 (NH), 3068
(ArC–H), 2959 (AliC–H), 1670 (C=O amide), 1539 (ArC=C), 1292 (C–O). H-
1
NMR (DMSO-d6) d: 2.02 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 5.37 (s, 1H, CH), 7.22–
7.78 (m, 8H, ArH), 8.99 (s, 1H, NH), 9.29 (s, 1H, NH), 9.86 (s, 1H, NH). MS (m/z):
M? calculated 338, found 338.29, Mass fragments (m/z): 323.18, 232.85, 121.80.
N-(4-Methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide
(7s)
Colorless solid, mp 224–226°C, yield 18%, 1H-NMR (DMSO-d6): d 2.16 (s, 3H, CH3),
3.70 (s, 3H, OCH3), 3.58 (d, 2H, CH2), 6.88 (d, 2H, ArH), 7.14 (d, 2H, ArH). MS (m/z):
M? calculated 261, found 261.21, Mass fragments (m/z): 246.03, 217.19, 151.0.
Antibacterial activity
All of the synthesized compounds were screened for their antibacterial activity
against two gram-positive bacteria: B. subtilis and S. aureus (Frankel et al., 1970).
The primary screening was performed by using the agar disc-diffusion method using
Muller-Hinton agar medium. The compounds were tested at the concentration of
50 lg per well. Sterile nutrient agar plates were prepared in petri dishes under

Med Chem Res (2010) 19:344–363
359
aseptic conditions; 0.1 ml of each standardized test organism was spread onto agar
plates and cavity was done by using a sterile borer of diameter 6 mm. Then, 50 lg
per well of compounds or standard drug solution of streptomycin (10 lg) and
DMSO solvent were placed in each cavity, separately. The plates were maintained
at ?4°C for 1 h to allow diffusion of solution into the medium; then the plates were
incubated at 37°C for 24 h and observed for zone of inhibition.
In vitro cytotoxicity
Short-term in vitro cytotoxicity assay was performed using Vero cells according to
the standard procedure (Moldeus et al., 1978). SRB is a bright-pink aminoxanthene
dye with two sulfonic groups. Under mild acidic conditions, SRB binds to protein
basic amino acid residues in trichloroacetic acid fixed cells to provide a sensitive
index of cellular protein content that is linear over a cell density range of at least
two orders of magnitude. After incubation, the solutions in the wells were flicked off
and 100 ll of different concentrations (2–500 lg) of compounds were added to the
cells and incubated at 37°C for 3 days in 5% CO2 atmosphere. The microscopic
examinations were performed and observations were recorded every 24 h. After,
72 h, 50% trichloroacetic acid (25 ll) was added to each well and the plates were
incubated for 1 h at 4°C. The supernatant was then removed, and the cells were
washed with water, air-dried, and stained, each well with SRB for 30 min. The
unbound dye was removed by washing with 1% acetic acid and the plates were air-
dried. Tris base (10 mM, 100 ll) was added to wells to solubilize the dye. The
plates were vigorously shaken for 5 min, and the absorbance was measured using
microtiter plate reader at 540 nm. The mean absorbance of triplicate was recorded.
Mean absorbance taken from cells grown in the absence of the test compound was
taken as 100% cell survival (control). The percentage growth inhibition was
calculated using the following formula:
Growth inhibition % ¼ 100 ꢁ ½sample absorbance/control absorbanceꢂ ꢃ 100
The percentage growth inhibition was plotted against concentration and the CTC50
(concentration required to reduce viability by 50%) value was calculated.
QSAR study
Biological data, designing training, and test sets
The 30 substituted 1,4-dihydropyrimidines (19 compounds) and 1,4-dihydropyri-
dines (11 compounds) were subjected to CoMFA analysis. The cytotoxicity was
converted into micromolar cytotoxicity by dividing the original values by their
respective molecular weights and taking the negative logarithm to the base 10
(pMCTC50), because it gives numerically larger value for the active compound than
that of the inactive compound. The training set was designed to account for all of
the structural and activity variations among the compounds. The rest of the
compounds constituted the test set to validate the model generated by the CoMFA

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Med Chem Res (2010) 19:344–363
study. Training and test set compounds, their structures, and their cytotoxicity are
shown in Table 1.
Computational details
Molecular-modeling software, SYBYL 6.7 installed (supplied by TRIPOS, USA) on
a silicon graphics workstation with IRIX 6.5 operating system, was used for the
three-dimensional structure generation and molecular-modeling studies. All of the
compounds were built from fragments available in the SYBYL database. Each
structure was fully geometry-optimized using the standard Tripos force field with a
distance-dependent dielectric function until a root mean square (rms) deviation of
˚
0.001 kcal/mol A was achieved (Clark et al., 1989). In our experience, Tripos force
field was found to be better for geometry optimization of 1,4-dihydropyridines and
1,4-dihydropyrimidines class of compounds compared with the other tools available
in SYBYL. The partial atomic charges required for the electrostatic interactions
were computed using the Gasteiger-Marsili method as implemented in the SYBYL
(Gasteiger and Marsili, 1980).
Conformational searching
The conformational search was performed using systematic search protocol. The
rotatable bonds in all molecules were searched from 0 to 360° in 10° increments.
The minimum energy conformation obtained was subsequently used in the analyses.
The obtained conformations for the title compounds visibly were found to be similar
to that of the X-ray crystallographic conformations (Andreas et al., 1999).
Alignment of the training and test sets
The important requirement for CoMFA is that the 3D structures of the molecules to
be analyzed are aligned according to a suitable conformation template, which is
assumed to adopt a ‘‘bioactive conformation’’ (Cramer et al., 1988b). Molecules in
the database were aligned by using the ‘‘Database Align’’ routine available in the
SYBYL. The compounds were fitted to the common substructure of 1,4-dihydro-
pyridines and 1,4-dihydropyrimidines. The aligned training set molecules are shown
in Fig. 2. Field fit method was used to optimize the alignment of molecules to a
previously calculated steric and electrostatic field (Clark et al., 1990).
CoMFA models
CoMFA steric and electrostatic interaction fields were calculated at each 3D cubic
˚
lattice intersection point of a regularly spaced grid 2.0 A in x, y, and z directions to
encompass the aligned molecules. The steric term (Lennard-Jones 6–12 potential)
and electrostatic (Coulomb potential) field interactions were calculated using the sp3
˚
carbon probe atom (1.52 A Van der Waals radius) carrying ?1 charge, with a
distance dependant dielectric at each lattice point. The charges were determined
using the Gasteiger–Marsili method. The energy calculation was performed for all

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361
grid points such that all energies were constrained to be between -30 and 30 kcal/
mol.
A Partial Least Squares (PLS) analysis was performed to derive the 3D-QSAR
models, utilizing the CoMFA standard scaling for the molecular fields. The activity
data, pMCTC50, was used as the dependant variable and the predictive value of the
model, represented by the q2, was evaluated using the leave-one-out (LOO) cross-
validation method. To speed up calculations and to reduce the noise between the
calculated columns, column filtering was used to exclude the columns with a
variance less than 2.0 units. The optimum number of components was determined
based on the standard error of prediction and q2 values. A final robust model was
generated by excluding four outliers. The conventional r2 values also were
calculated. After the 3D-QSAR models were established for our structures,
graphical examination of the model depicted which areas around the molecules
should contain a greater or lesser amount of electronegative, electropositive groups
and whether sterics were an important factor for affinity toward cytotoxic activity.
To further validate or to ensure the developed CoMFA model, the activities of the
training set compounds were predicted. The actual and predicted values of the
compounds are listed in Table 1.
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