Novel Biginelli dihydropyrimidines with potential anticancer activity: A parallel synthesis and CoMSIA study

Article abstract of DOI:10.1016/j.ejmech.2009.05.014

Novel Biginelli dihydropyrimidines of biological interest were prepared using p-toluene sulphonic acid as an efficient catalyst. All the thirty-two synthesised dihydropyrimidines were evaluated for their in vitro antioxidant activity using DPPH method. Only, compounds 28 and 29 exhibited reasonably good antioxidant activity. Furthermore, the synthesised Biginelli compounds were subjected for their in vitro anticancer activity against MCF-7 human breast cancer cells. The title compounds were tested at the concentration of 10 μg. Compounds exhibited activity ranging from weak to moderate and, from moderate to high in terms of percentage cytotoxicity. Among them, compounds 10 and 11 exhibited significant anticancer activity. In order to elucidate the three-dimensional structure-activity relationships (3D QSAR) towards their anticancer activity, we subjected them for comparative molecular similarity indices analysis (CoMSIA). Illustration regarding their synthesis, analysis, antioxidant activity, anticancer activity and 3D QSAR study is described.

Full text of DOI:10.1016/j.ejmech.2009.05.014

European Journal of Medicinal Chemistry 44 (2009) 4192–4198
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel Biginelli dihydropyrimidines with potential anticancer activity: A parallel
synthesis and CoMSIA study
,
a
b
b
B.R. Prashantha Kumar a , Gopu Sankar , R.B. Nasir Baig , Srinivasan Chandrashekaran
*
a Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ootacamund 643 001, Tamilnadu, India
b Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Biginelli dihydropyrimidines of biological interest were prepared using p-toluene sulphonic acid as
an efficient catalyst. All the thirty-two synthesised dihydropyrimidines were evaluated for their in vitro
antioxidant activity using DPPH method. Only, compounds 28 and 29 exhibited reasonably good anti-
oxidant activity. Furthermore, the synthesised Biginelli compounds were subjected for their in vitro
anticancer activity against MCF-7 human breast cancer cells. The title compounds were tested at the
Received 30 March 2009
Received in revised form 12 May 2009
Accepted 14 May 2009
Available online 23 May 2009
concentration of 10 mg. Compounds exhibited activity ranging from weak to moderate and, from
Keywords:
moderate to high in terms of percentage cytotoxicity. Among them, compounds 10 and 11 exhibited
significant anticancer activity. In order to elucidate the three-dimensional structure–activity relation-
ships (3D QSAR) towards their anticancer activity, we subjected them for comparative molecular simi-
larity indices analysis (CoMSIA). Illustration regarding their synthesis, analysis, antioxidant activity,
anticancer activity and 3D QSAR study is described.
Biginelli dihydropyrimidines
Anticancer activity
MCF-7 cells
CoMSIA
3D QSAR
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
a large number of multifunctionalized dihydropyrimidines of
medicinal use [9–12]. Dihydropyrimidines show a diverse range of
Drug discovery and development is a very laborious and costly
process involving synthesis and screening of diverse organic
compounds. In this regard, multicomponent reactions (MCRs) are
of increasing importance in the field of medicinal chemistry [1–5].
Currently, attention is put on speed, diversity, and efficiency in the
drug discovery process [6]. MCRs can provide products with the
diversity needed for the discovery of new lead compounds or lead
optimization employing combinatorial chemistry techniques. The
search and discovery for new MCRs on one hand [7], and the full
exploitation of already known multicomponent reactions on the
other hand, are therefore of considerable current interest.
In 1893, Pietro Biginelli has reported on the acid-catalyzed
cyclocondensation reaction of ethylacetoacetate, benzaldehyde and
urea. The reaction was carried out by simply heating a mixture of
the three components dissolved in ethanol with a catalytic amount
of HCl at reflux temperature. The product of this novel one-pot,
three-component synthesis that precipitated on cooling the reac-
tion mixture was identified correctly by Biginelli as dihydropyr-
imidin-2-one [8]. The scope of this reaction was gradually extended
by the variation of all three building blocks, allowing access to
biological activities. They are known to possess activities such as
antibacterial [13] and antiviral (nitractin) [14], antitumor [15–18],
analgesic and anti-inflammatory [19], antiplatelet aggregation [20],
and antihypertensive activity [21]. Thus development of method-
ologies for efficient lead structure identification and for pharma-
cophore variation of dihydropyrimidine motif has always attracted
the attention of pharmaceutical industry [22].
Herein, we report a simple protocol for the parallel synthesis of
some novel Biginelli dihydropyrimidines using appropriate
building blocks and acid catalyst. Also we present anticancer,
antioxidant activities of the title compounds along with 3D QSAR
study to establish the structure–activity relationships towards their
anticancer activity.
2. Results and discussion
2.1. Chemistry
Our strategy for the syntheses of title compounds was designing
the suitable and appropriate library. Hence, we identified appro-
priate building blocks, synthesised and later subjected for Biginelli
condensation reaction. o-Chloroacetoacetanilide (1) was syn-
thesised by refluxing equimolar amounts of o-chloroaniline and
* Corresponding author. Tel.: þ91 423 2443393x223; fax: þ91 423 2442937.
E-mail address: prashantha@jsscpooty.org (B.R. Prashantha Kumar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.014

B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
4193
ethylacetoacetate under solvent free conditions according to
Scheme 1 [23,24].
O
R
N
X
O
O
+
[a]
R'
N
H
NH
H2N
R CHO +
NH Y
The parallel synthesis of 1,4-dihydropyrimidines was carried out
by multicomponent-cyclisation reaction of equimolar amounts of
aryl or heteryl aldehyde, o-chloroacetoacetanilide or N-methyl-
acetoacetamide or cyanoguanidine and excess of urea or thiourea in
the presence of catalytic amounts of p-toluene sulphonic acid
(PTSA) in ethanol (Scheme 2). Some of the reaction mixtures
needed reflux for 24 h for the synthesis of 2–5, 8–11, 18–21 and 33
1,4-dihydropyrimidines. Whereas, all other 1,4-dihydropyrimidines
are obtained just by simple stirring at room temperature for 24–
30 h. In all the cases reaction went smoothly to give the corre-
sponding 1,4-dihydropyrimidines’ 2–32 (67–81%) in moderate to
good yields. The compound 33 was the only compound synthesised
using cyanoguanidine and obtained in a very poor yield of 12%. We
were unable to improve the yield of 33 even after several trials by
varying the acid catalysts. Hence, we could not extend this meth-
odology for the parallel synthesis of 1,4-dihydropyrimidines
derivatives using cyanoguanidine. The Biginelli 1,4-dihydropyr-
imidines 2–33 were synthesised relatively easy using PTSA as an
efficient catalyst and it does not demand any anhydrous conditions
unlike the other Lewis acid catalysts. The compounds 2–33 were
prepared using a simple distilled ethanol without further drying.
Alternatively, for those title compounds which required reflux were
synthesised by microwave (MW) irradiation technique. By MW
method the reaction time was reduced to 30–40 min. However, the
yields were found to be similar to that of conventional method. One
more advantage of MW method over the conventional method is
it’s comparatively easy to perform the parallel synthesis. All the
synthesised Biginelli dihydropyrimidine compounds are novel and
not reported elsewhere. All the synthesised compounds showed
satisfactory analytical results. The characteristic peak for
R'
NH
X
Y
2-33
R' = 2-Cl Ph, CH3
X = O, S, NH
Y = H, CN
Scheme 2. Reagents and conditions for the parallel synthesis of Biginelli dihy-
dropyrimidines: (a) PTSA, C2H5OH, stirred for 24–30 h at room temperature and some
of the title compounds which require heating were synthesised by refluxing reaction
mixtures for 24 h or by irradiating with microwaves for 30–40 min at 500 W power.
values for the synthesised compounds when tested at the
concentration of 10 mg are shown in Table 1. Among them,
compounds 10 and 11 showed significant activity against MCF-7
cells with cinnamoyl moiety at the fourth position of 1,4-dihy-
dropyrimidine ring system. Compounds 6, 7 and 18 also exhibited
potential anticancer activity having furan and pyridine moieties at
the same position.
2.4. CoMSIA study
In order to establish 3D structure–activity relationships towards
their anticancer activity, we have performed the CoMSIA study. The
total set of compounds was initially divided randomly into two
groups as a training and test set, with 26 compounds in the training
set and 6 compounds in 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 the sets. The % cytotoxicity was converted
logarithm to the base 10, as it would give numerically larger values
for the active compounds than those of the inactive compounds
(pACA). The training set compounds were used to develop the
CoMSIA model and the test set compounds were used to validate
the developed model.
symmetric CH proton in HNMR spectrum between 5 and 6
confirmed the formation of dihydropyrimidines.
d ppm,
2.2. In vitro antioxidant activity
For the selected or developed CoMSIA model, the cross-vali-
dated correlation coefficient (q2) value of the training set was 0.51
with four principal components. The non-cross-validated r2 value
was 0.933 with a standard error of estimation (SEE) of 0.113 and
a Fischer’s covariance ratio (F) of 43.856 (significant at the 99%
level). The field fit alignment of the dihydropyrimidines and the
correlation between the experimental cytotoxicity and the pre-
dicted cytotoxicity (pACA) are shown in Figs. 1 and 2, respectively.
The statistical results have been indicated the stability of the
selected CoMSIA model. The predictive ability of the model was
further validated using the external test set of 6 compounds. The
test set compounds were predicted closer to their experimental
values. Compound 17 was an exception to this statement because of
large difference between the predicted and experimental activities.
Possible reason could be the structurally closer compound i.e.
compound 16 in the training set has showed approximately two
folds more activity when compared to the compound 17. The results
authenticated the good prediction ability of the developed 3D-
QSAR model. In the developed CoMSIA model, the contributions of
steric, electrostatic, hydrophobic, hydrogen-bond acceptor and
hydrogen-bond donor fields were found to be 11.7%, 34.2%, 24.7%,
20.4%, and 9.1%, respectively.
The synthesised compounds were screened for their possible
antioxidant activity by DPPH (2,2-diphenyl-1-picrylhydrazyl)
method [25]. Among the thirty-two title compounds, compounds
28 and 29 with 3-nitro phenyl moiety at the fourth position of
dihydropyrimidine showed good antioxidant activity with IC50
value of 58 and 63
thirty compounds failed to exhibit the antioxidant activity when
tested, primarily, at 100 g concentrations.
mg concentrations, respectively. The remaining
m
2.3. In vitro anticancer activity
Furthermore, the compounds were subjected for in vitro anti-
cancer screening against MCF-7 breast cancer cells using trypan
blue dye exclusion tests [26,27]. The motive for us to check the
anticancer activity of the synthesised compounds was, some
reports that have claimed significant anticancer activity for some of
the 1,4-dihydropyrimidines [15–18]. The obtained % cytotoxicity
O
O
NH2
HN
The structure–activity relationships based on the above CoM-
SIA contour maps are as follows. 1,4-Dihydropyrimidine scaffold is
the basic requirement for the cytotoxicity, as all the compounds
are superimposed and aligned on that part of the substructure
(Fig. 3). The dihydropyrimidine ring with anilide portion at the
fifth position seems to be the part of pharmacophore (common
substructure) for this class of compounds to exhibit the anticancer
O
O
Cl
Cl
solvent free
+
O
reflux, 2h
64%
1
Scheme 1. Synthesis of o-chloroacetoacetanilide.

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B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
Table 1
Biological activity data and calculated values for 1,4-dihydropyrimidine derivatives.
O
R
N
H
R'
NH
N
Y
X
2-33
Cpd no
R
X
% Cytotoxicity
pACA
Exp activity
Pred activity
2
Phenyl
Phenyl
4-Methoxoxyphenyl
4-Methoxoxyphenyl
Furan-2-yl
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
25
30
47
51
76
69
49
58
71
79
13
29
45
52
18
07
72
59
35
48
57
45
61
27
41
47
39
23
41
49
64
35
1.3979
1.4771
1.6720
1.7075
1.8808
1.8388
1.6901
1.7634
1.8512
1.8976
1.1139
1.4623
1.6532
1.7160
1.25552
0.8450
1.8573
1.7708
1.5440
1.6812
1.7558
1.6532
1.7853
1.4313
1.6127
1.6720
1.5910
1.3617
1.6127
1.6901
1.8061
1.5440
1.412
1.5839
1.626
1.758
1.7291
1.873
1.632
1.812
1.809
1.7897
1.149
1.417
1.684
1.674
1.336
1.4845
1.748
1.834
1.5440
1.6812
1.7558
1.6532
1.7853
1.4313
1.6127
1.6720
1.3998
1.3617
1.6403
1.6901
1.8061
1.5440
3a
4
5
6a
7
Furan-2-yl
8
9
2-Hydroxyphenyl
2-Hydroxyphenyl
Cinnamoyl
10
11a
12
13
14
15
16
17a
18
19
20
21
22
23
24
25
26
27
28a
29
30a
31
32
33
Cinnamoyl
3-Nitrophenyl
3-Nitrophenyl
4-Hydroxy-3-methoxyphenyl
4-Hydroxy-3-methoxyphenyl
4-Bromophenyl
4-Bromophenyl
Pyridin-4-yl
Pyridin-4-yl
Phenyl
4-Methoxyphenyl
Furan-2-yl
2-Hydroxyphenyl
Furan-2-yl
Phenyl
4-Methoxyphenyl
Cinnamoyl
3-Nitrophenyl
3-Nitrophenyl
4-Hydroxy-3-methoxyphenyl
4-Hydroxy-3-methoxy-phenyl
Pyridine-4-yl
O
S
O
S
S
S
S
O
O
O
O
S
O
S
O
O
NH
Phenyl
a
Test set compounds; R0 ¼ CH3 for compounds 20–32, whereas R0 ¼ o-chloro phenyl for all the remaining compounds; Y ¼CN for compound 33, whereas Y ¼ H for
compounds 1–32.
activity. A large green contour at the fourth position of aldehyde
substitution of 1,4-dihydropyrimidine ring system, indicates that
the substitution favouring the activity. Evidence for this is
compounds 10 and 11, as they possess a lengthy cinnamoyl
substitution at that position. A small yellow contour near the
third position of same in steric contour map indicates that, there
should not be any substitution or steric extension in that region.
Evidence for this is most of the active compounds did not had
substitution at that position. The blue contour over the dihy-
dropyrimidine nucleus indicates that, electronegative atoms like
nitrogen at the first and third positions are essential for the
activity. Blue coloured contour over the aldehyde portion indi-
cates that, presence of electronegative atoms like oxygen and
nitrogen in heterocyclic ring enhances the activity (compounds 6,
7, 18, 19 and 32). In Fig. 4, the white contour near the fourth
position of aldehyde substitution indicates that, hydrophobic
groups like aromatic rings will favour the activity (compounds 10
and 11). The cyan contour after the white indicates that, further
extension with hydrophobic rings will not contribute to the
activity. The orange contour near the second position of aldehyde
substitution indicates that, presence of hydrogen-bond donors
partially contributes to the activity (compounds 8 and 9). The
purple contour at the fourth and second positions indicates that
presence of hydrogen-bond acceptors will increase the activity (5,
9, 15, 18 and 32). The compounds with bromine at fourth position
failed to show anticancer activity (compounds 16 and 17).
3. Conclusions
We have synthesised a library of novel Biginelli compounds of
biological interest. The title compounds were prepared using PTSA
as an efficient catalyst. Compounds 28 and 29 showed good anti-
oxidant activity. Compounds 10 and 11 are considered to be the
candidate compounds to investigate further, as they exhibited
significant anticancer activity against MCF-7 human breast cancer
cells. CoMSIA, being one of the important methods of 3D QSAR
elucidated the structure–activity relationships for the title
compounds towards their anticancer activity. Present 3D-QSAR
model can be used to design the new ligands of this class for their
anticancer activity.

B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
4195
2
1.8
1.6
1.4
1.2
1
1
1.2
1.4
1.6
1.8
2
Exp Activity
Fig. 2. Correlation between the observed and predicted activities of the developed
CoMSIA model.
at reflux temperature for compounds 2–5, 8–11, 18–21 and 33. The
remaining title compounds were synthesised by simple stirring at
room temperature for about 24–30 h (6, 7, 12–17, 22–32). The
reactions were monitored through TLC. After the completion of
reaction, the mixtures were allowed to cool. The solid products
formed were filtered, washed with water to remove the unreacted
urea or thiourea or cyanoguanidine and dried. Products were
further purified by recrystallisation with methanol. Alternatively,
the reactions which required reflux were driven by irradiation with
microwaves at 500 W power for about 30–40 min under similar
conditions using conical flasks with funnel placed over it. Inter-
mittent cooling was given for 5 min after every 5 min of MW
irradiation. However, the yields were found to be more or less same.
But, microwave method was found to be convenient to perform the
parallel synthesis when compared to the conventional method.
Fluorescence exhibited by most of the dihydropyrimidines was
considered for their detection on thin layer chromatograms.
Compound no 2: Pale yellowish amorphous solid (71%); mp 158–
160 ꢀC; IR (KBr, cmꢁ1): 3625 (N–H), 3026 (ArC–H), 2953 (AliC–H),
1955 (C]S),1670 (C]O),1581 (ArC]C), 749 (C–Cl); 1H NMR (DMSO-
Fig. 1. Training set molecules after alignment by field fit method.
4. Experimental
4.1. Chemistry
The synthesised compounds were characterised by MP, IR, NMR
and MASS spectral analyses. TLC was performed using 2% ethyl
acetate in chloroform as a mobile phase on aluminium plates pre-
coated with silica gel GF. The melting points of the compounds
were determined using melting apparatus by open capillary
method and are uncorrected. The IR spectra of the compounds were
recorded on FT-IR spectrometer (Perkin–Elmer Infrared-283) using
KBr pellet technique. 1H NMR spectra were recorded on AV-III
(400 MHz FT-NMR) using DMSO-d6 as solvent and TMS as internal
standard. The mass spectra were recorded using Shimadzu LCMS
2010A spectrometer under electro spray ionisation technique.
Microwave irradiation was carried out using Whirlpool domestic
microwave oven.
d6): d 2.2 (s, 3H, CH3), 5.5 (s, 1H, CH), 7.2–7.6 (m, 9H, ArH), 9.3 (s, 1H,
NH), 9.5 (s,1H, NH), 10.0 (s,1H, NH); MS (m/z): M þ 1 calculated 358;
found 358.46; mass fragments (m/z): 324, 263, 231, 146.
Compound no 3: Pale yellow crystals (79%); mp 207–209 ꢀC; IR
(KBr, cmꢁ1): 3600 (N–H), 3050 (ArC–H), 2925 (AliC–H), 1664
(C]O), 1654 (C]O), 1623 (ArC]C), 750 (C–Cl); 1H NMR (DMSO-
4.1.1. Synthesis of o-chloroacetoacetanilide (1)
d6): d 2.2 (s, 3H, CH3), 5.5 (s, 1H, CH), 7.1–7.6 (m, 9H, ArH), 7.8 (s, 1H,
Preparation of 2-chloro acetoacetanilide was carried out as per
Scheme 1. Ethylacetoacetate (0.01 M) and o-chloroaniline (0.01 M)
were mixed and refluxed for about 2 h under solvent free condi-
tions. The yellowish liquid formed was then heated on a water bath
for 30 min. The reaction mixture was allowed to cool. The crude
solid obtained was filtered and washed with ether. The product was
recrystallised from aqueous alcohol.
NH), 8.9 (s, 1H, NH), 9.5 (s, 1H, NH); MS (m/z): M þ 1 calculated 342;
found 341.87; mass fragments (m/z): 311, 279, 231, 167.
Compound no 4: Yellow crystals (80%); mp 236–238 ꢀC; IR (KBr,
cmꢁ1): 3443 (N–H), 3012 (ArC–H), 2933 (AliC–H), 1941 (C]S), 1744
(C]O), 1700 (ArC]C), 1211 (C–O), 746 (C–Cl); 1H NMR (DMSO-d6):
d
2.1 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 7.1–7.6 (m, 9H, ArH), 7.8 (s, 1H,
NH), 8.9 (s, 1H, NH), 9.5 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 386;
found 385.68; mass fragments (m/z): 345, 280, 186, 140, 115, 101.
Compound no 5: Pale yellowish amorphous solid (70%); mp
222–224 ꢀC; IR (KBr, cmꢁ1): 3262 (N–H), 3005 (ArC–H), 2828 (AliC–
H), 1677 (C]O), 1631 (C]O), 1609 (ArC]C), 1113 (C–O), 750 (C–Cl);
Compound no 1: White crystals (64%); mp 104–106 ꢀC; IR (KBr,
cmꢁ1): 3208 (N–H), 3066 (ArC–H), 2932 (AliC–H), 1704 (C]O),
1538 (C]C), 763 (C–Cl); 1H NMR (DMSO-d6):
d 2.18 (s, 3H, CH3),
3.48 (s, 2H, CH2), 7.11–7.47 (m, 4H, ArH), 9.92 (s, 1H, NH). MS (m/z):
M þ 1 calculated 212; found 211.90; mass fragments (m/z): 127, 89.
1H NMR (DMSO-d6):
d 3.5 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 5.4 (s, 1H,
CH), 7.0–7.6 (m, 8H, ArH), 7.8 (s, 1H, NH), 8.7 (s, 1H, NH), 9.5 (s, 1H,
NH); MS (m/z): M ꢁ 1 calculated 370; found 369.82; mass frag-
ments (m/z): 345, 331, 317, 164, 155, 98, 83.
4.1.2. Synthesis of 1,4-dihydropyrimidines (2–33)
Preparation of 1,4-dihydropyrimidines by one-pot multicom-
ponent reaction was carried out as per Scheme 2. The mixture of
Compound no 6: Pale brownish amorphous solid (68%); mp
178–180 ꢀC; IR (KBr, cmꢁ1): 3240 (N–H), 3005 (Ar–CH), 2972 (AliC–
H), 1946 (C]S), 1676 (C]O), 1630 (ArC]C), 1158 (C–O), 748 (C–Cl);
appropriate b-ketoester (0.005 M, 2-chloro acetoacetanilide or N-
methylacetoacetamide), urea or thiourea or cyanoguanidine
(0.0075 M), and appropriate aldehyde (0.005 M) with catalytic
amount of PTSA (0.025 g) was transferred to a round bottom flask
containing 15 ml of ethanol to serve as a solvent. The round bottom
flask was stirred to dissolve the reactants. The mixture was heated
1H NMR (DMSO-d6):
d 2.1 (s, 3H, CH3), 5.5 (s, 1H, CH), 6.3 (s, 1H, CH),
6.5 (s, 1H, CH), 7.2–7.8 (m, 4H, ArH), 9.2 (s, 1H, NH), 9.5 (s, 1H, NH),
10.1 (s, 1H, NH). M þ 1 calculated 348; found 348.32; mass frag-
ments (m/z): 315, 311, 307, 220, 156, 79.

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B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
Fig. 3. CoMSIA steric and electrostatic SD ꢂ coefficient contour plot; green contours
indicate regions where steric bulk is favorable and yellow contours indicate regions
where steric bulk is not favored. Blue contours indicate regions where electronegative
groups increase activity and red contours indicate regions where electropositive
groups increase activity.
Fig. 4. CoMSIA hydrophobic and hydrogen-bond donor SD ꢂ coefficient contour plot;
white contours indicate regions where hydrophobicity is favorable and cyan contours
indicate regions where hydrophobicity is not favored. Orange contours indicate regions
where hydrogen-bond donors increase activity and yellow contours indicate regions
where hydrogen-bond donors decrease activity. Purple contours indicate regions where
hydrogen-bond acceptors increase activity and green contours indicate regions where
hydrogen-bond donors decrease activity (not contributed in the above contour).
Compound no 7: Pale brownish amorphous solid (79%); mp
146–148 ꢀC; IR (KBr, cmꢁ1): 3290 (N–H), 3117 (ArC–H), 2910 (AliC–
H), 1663 (C]O), 1639 (C]O),1590 (ArC]C),1103 (C–O), 744 (C–Cl);
Compound no 13: White amorphous solid (80%); mp 187–
190 ꢀC; IR (KBr, cmꢁ1): 3294 (N–H), 3016 (ArC–H), 2917 (AliC–H),
1594 (ArC]C),1660 (C]O),1666 (C]O),1527 (NO2), 753 (C–Cl); 1H
1H NMR (DMSO-d6):
d
2.2 (s, 3H, –CH3), 4.3 (s, 1H, CH), 4.5 (s, 1H,
NMR (DMSO-d6): d 2.2 (s, 1H, CH3), 5.4 (s, 1H, CH), 6.9–7.1 (m, 8H,
CH), 5.6 (s, 1H, CH), 7.2–7.8 (m, 4H, ArH), 9.7 (s, 1H, NH), 9.8 (s, 1H,
NH), 10.1 (s, 1H, NH). M ꢁ 1 calculated 330; found 329.79; mass
fragments (m/z): 310, 301, 233, 117.
ArH), 8.9 (s, 1H, NH), 9.3 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated
385; found 385.60; mass fragments (m/z): 358, 263.
Compound no 14: Pale yellowish amorphous solid (80%); mp
196–199 ꢀC; IR (KBr, cmꢁ1): 3208 (N–H), 3500 (O–H), 3090 (ArC–H),
2930 (AliC–H), 1964 (C]S), 1678 (C]O), 1255 (C–O), 765 (C–Cl); 1H
Compound no 8: Yellowish amorphous solid (81%); mp 200–
203 ꢀC; IR (KBr, cmꢁ1): 3200 (N–H), 3397 (O–H), 3067 (ArC–H),
2932 (AliC–H), 1950 (C]S), 1671 (C]O), 1601 (ArC]C), 753 (C–Cl);
NMR (DMSO-d6): d 1.5 (s, 3H, CH3), 3.7 (s, 3H, OCH3), 4.8 (s, 1H, NH),
1H NMR (DMSO-d6):
d
1.7 (s, 3H, CH3), 3.5 (s, 1H, CH), 4.7 (s, 1H, CH),
8.9 (s, 1H, NH), 6.7–7.2 (m, 6H, ArH), 9.4 (s, 1H, OH), 8.9 (s, 1H, NH).
M þ 1 calculated 404; found 404.12; mass fragments (m/z): 389,
211, 123.
7.1–7.8 (m, 8H, ArH), 9.0 (s, 1H, NH), 9.4 (s, 1H, NH), 9.9 (s, 1H, NH);
MS (m/z): M ꢁ 1 calculated 372; found 371.77; mass fragments (m/
z): 328, 219, 195, 74.
Compound no 9: Pale yellowish amorphous solid (75%); mp
199–204 ꢀC; IR (KBr, cmꢁ1): 3399 (N–H), 3500 (O–H), 2920 (AliC–
H), 1676 (C]O), 1657 (C]O), 1607 (Ar–C]C), 751 (C–Cl); 1H NMR
Compound no 15: Pale yellowish amorphous solid (75%); mp
179–182 ꢀC; IR (KBr, cmꢁ1): 3190 (N–H), 3420 (O–H), 3090 (ArC–H),
2976 (AliC–H), 1676 (C]O), 1658 (C]O), 1612 (C]C), 1212 (C–O),
751 (C–Cl); 1H NMR (DMSO-d6):
d 1.4 (s, 3H, CH3), 3.7 (s, 3H, OCH3),
(DMSO-d6):
d
2.5 (s, 3H, CH3), 4.2 (s, 1H, OH), 5.3 (s, 1H, CH), 7.0–7.7
4.7 (s, 1H, NH), 6.6–7.0 (m, 6H, ArH), 8.8 (m, 1H, Ar–OH), 8.7 (s, 1H,
NH), 8.9 (s, 1H, NH), 9.3 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 386;
found 386.11; mass fragments (m/z): 273, 153, 127, 100, 83.
Compound no 16: Pale yellowish amorphous solid (74%); mp
202–205 ꢀC; IR (KBr, cmꢁ1): 3213 (N–H), 3091 (ArC–H), 3008 (AliC–
H), 1950 (C]S), 1671 (C]O), 1009 (C–Br), 748 (C–Cl); 1H NMR
(m, 8H, ArH), 9.0 (s, 1H, NH), 9.6 (s, 1H, NH), 9.8 (s, 1H, NH). M þ 1
calculated 358; found 358.19; mass fragments (m/z): 345, 287, 231,
220, 92.
Compound no 10: Yellowish crystals (70%); mp 227–230 ꢀC; IR
(KBr, cmꢁ1): 3399 (N–H), 3063 (ArC–H), 1950 (C]S), 1606 (ArC]C),
1673 (C]O), 751 (C–Cl); 1H NMR (DMSO-d6):
d
2.5 (s, 3H, CH3), 4.5
(DMSO-d6): d 2.2 (s, 3H, CH3), 5.4 (s, 1H, CH), 7.1–7.6 (m, 8H, ArH),
(s, 1H, CH), 4.6 (s, 1H, CH), 5.5 (s, 1H, CH), 7.2–7.6 (m, 9H, ArH), 7.8 (s,
1H, NH), 8.0 (s, 1H, NH), 8.6 (s, 1H, NH). M ꢁ 1 calculated 382; found
381.88; mass fragments (m/z): 357, 310, 256, 120.
9.3 (s,1H, NH), 9.6 (s,1H, NH),10.1 (s,1H, NH). M þ 1 calculated 437;
found 436.63; mass fragments (m/z): 416, 378, 254, 111.
Compound no 17: Colourless crystals (81%); mp 195–198 ꢀC; IR
(KBr, cmꢁ1): 3282 (N–H), 2962 (AliC–H), 1679 (C]O), 1647 (C]O),
Compound no 11: Pale yellowish amorphous solid (72%); mp
211–215 ꢀC; IR (KBr, cmꢁ1): 3231 (N–H), 3025 (ArC–H), 2930 (AliC–
1586 (ArC]C), 1025 (C–Br), 753 (C–Cl); 1H NMR (DMSO-d6):
d 2.1
H), 1689 (C]O), 750 (C–Cl), 1640 (C]O); 1H NMR (DMSO-d6):
d
2.5
(s, 3H, CH3), 5.3 (s,1H, CH), 7.2–7.6 (m, 8H, Ar–H), 8.8 (s,1H, NH), 9.2
(s, 1H, NH), 10.2 (s, 1H, NH). M þ 1 calculated 421; found 420.81;
mass fragments (m/z): 405, 372, 297, 103.
Compound no 18: Yellow crystals (76%); mp 139–143 ꢀC; IR (KBr,
cmꢁ1): 3600 (N–H), 3002 (ArC–H), 1604 (C]O), 1473 (C]N), 1824
(C]S), 730 (C–Cl); M ꢁ 1 calculated 357; found 346.58; mass
fragments (m/z): 331, 303, 217, 98.
(s, 3H, CH3), (s, 1H, CH), 5.5 (s, 1H, CH), 7.2–7.8 (m, 8H, ArH), 8.6 (s,
1H, NH), 10.2 (s, 1H, NH). M þ 1 calculated 368; found 368.21; mass
fragments (m/z): 348, 264, 120.
Compound no 12: Yellow crystals (77%); mp 193–196 ꢀC; IR (KBr,
cmꢁ1): 3390 (N–H), 3012 (ArC–H), 2919 (AliC–H), 1884 (C]S), 1644
(C]O), 1512 (NO2), 750 (C–Cl); 1H NMR (DMSO-d6):
d 2.5 (s, 3H,
CH3), 7.2–7.8 (m, 9H, ArH), 10.4 (s, 1H, Ar–NH). M þ 1 calculated
Compound no 19: Yellowish amorphous solid (78%); mp 137–
403; found 402.79; mass fragments (m/z): 389, 312, 219, 134.
140 ꢀC; IR (KBr, cmꢁ1): 3178 (N–H), 3032 (ArC–H), 2976 (AliC–H),

B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
4197
1728 (C]O), 1604 (ArC]C), 1681 (C]O), 1537 (C]N), 749 (C–Cl).
M þ 1 calculated 343; found 343.27; mass fragments (m/z): 327,
289, 167, 120.
8.1 (s,1H, NH), 8.8 (s,1H, NH); MS (m/z): M ꢁ 1 calculated 289; found
288.91; mass fragments (m/z): 247, 123, 100, 83.
Compound no 30: Colourless crystals (73%); mp 243–246 ꢀC; IR
(KBr, cmꢁ1): 3628 (O–H), 3388 (N–H), 3096 (ArC–H), 2968 (AliC–H),
1958 (C]S), 1666 (C]O), 1200 (C–N), 1168 (C–O); 1H NMR (DMSO-
Compound no 20: Colourless crystals (80%); mp 156–158 ꢀC; IR
(KBr, cmꢁ1): 3511 (N–H), 3021 (ArC–H), 2850 (AliC–H), 1812 (C]S),
1686 (C]O), 1245 (C–N); 1H NMR (DMSO-d6):
d
2.0 (s, 3H, CH3), 2.5
d6): d 2.0 (s, 3H, CH3), 2.5 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 5.1 (s, 1H,
(s, 3H, CH3), 5.3 (s, 1H, CH), 7.1–7.4 (m, 5H, ArH), 7.8 (s, 1H, NH), 9.3
(s, 1H, NH), 9.8 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 260; found
259.71; mass fragments (m/z): 228, 184.
CH), 6.6 (s, 1H, OH), 6.7–6.9 (m, 3H, ArH), 7.7 (s, 1H, NH), 9.0 (s, 1H,
NH), 9.2 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 306; found 306.36;
mass fragments (m/z): 255, 216.
Compound no 31: Pale yellowish amorphous solid (70%); mp
200–202 ꢀC; IR (KBr, cmꢁ1): 3261 (N–H), 3016 (ArC–H), 2932 (AliC–
H), 1699 (C]O), 1620 (C]O), 1221 (C–N), 1085 (C–O); 1H NMR
Compound no 21: Pale yellow crystals (75%); mp 201–204 ꢀC; IR
(KBr, cmꢁ1): 3380 (N–H), 3076 (ArC–H), 2910 (AliC–H), 1844 (C]S),
1714 (C]O), 1245 (C–N), 1195 (C–O); 1H NMR (DMSO-d6):
d 2.0 (s,
3H, CH3), 2.4 (s, 3H, CH3), 2.8 (s, 3H, OCH3), 5.2 (s, 1H, CH), 6.8–7.3
(m, 4H, ArH), 7.6 (s, 1H, NH), 9.2 (s, 1H, NH), 9.8 (s, 1H, NH). M þ 1
calculated 292; found 291.69; mass fragments (m/z): 250, 198, 126.
Compound no 22: Pale brownish amorphous solid (75%); mp
230–233 ꢀC; IR (KBr, cmꢁ1): 3332 (N–H), 3395 (OH), 3020 (ArC–H),
2958 (AliC–H), 1932 (C]S), 1713 (C]O), 1240 (C–N), 1195 (C–O); 1H
(DMSO-d6): d 2.0 (s, 3H, CH3), 2.5 (s, 3H, CH3), 3.7 (s, 3H, OCH3), 5.1
(s, 1H, CH), 6.7–6.9 (m, 3H, ArH), 7.5 (s, 1H, NH), 8.4 (s, 1H, –NH), 8.9
(s, 1H, –NH). M þ 1 calculated 292; found 291.84; mass fragments
(m/z): 279, 186, 101.
Compound no 32: Light greenish amorphous solid (78%); mp
146–149 ꢀC; IR (KBr, cmꢁ1): 3432 (N–H), 3050 (ArC–H), 2930 (AliC–
H), 1668 (C]O), 1238 (C–N), 1295 (C–N). M þ 1 calculated 247;
found 246.63; mass fragments (m/z): 219, 158, 120, 88.
Compound no 33: Pale yellowish amorphous solid (12%); mp
246–248 ꢀC; IR (KBr, cmꢁ1): 3421 (N–H), 3025 (ArC–H), 2913 (AliC–
H), 1722 (C]O), 1592 (ArC]C), 526 (C–Cl); 1H NMR (DMSO-d6):
NMR (DMSO-d6):
d 2.0 (s, 3H, CH3), 2.6 (s, 3H, CH3), 5.2 (s, 1H, CH),
6.1 (s, 1H, CH), 6.3 (s, 1H, CH), 7.7 (s, 1H, NH), 9.3 (s, 1H, NH), 9.9 (s,
1H, NH); MS (m/z): M ꢁ 1 calculated 250.30; found 249.85; mass
fragments (m/z): 216, 203.
Compound no 23: Pale yellowish amorphous solid (71%); mp
220–222 ꢀC; IR (KBr cmꢁ1): 3395 (O–H), 3332 (N–H), 3082 (ArC–H),
2948 (AliC–H), 1950 (C]S), 1717 (C]O), 1357 (C–N); 1H NMR
d
2.1 (s, 3H, CH3), 6.2 (s,1H, CH), 7.0–7.7 (m, 9H, ArH), 9.2 (s,1H, NH),
9.4 (s, 1H, NH), 9.7 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 364.0;
(DMSO-d6): d 1.7 (s, 3H, CH3), 2.6 (s, 3H, CH3), 4.3 (s, 1H, CH), 4.5 (s,
found 363.94; mass fragments (m/z): 328, 312, 257, 231.
1H, OH), 7.1–7.3 (m, 4H, ArH), 8.0 (s, 1H, NH), 8.9 (s, 1H, NH), 9.1 (s,
1H, NH). M þ 1 calculated 278; found 277.79; mass fragments (m/z):
216, 185, 120.
4.2. In vitro antioxidant activity
Compound no 24: Pale brownish amorphous solid (74%); mp
The DPPH free radicals formed in this assay will be reduced to
a corresponding hydrazine when it reacts with hydrogen donors.
The DPPH radical is purple in colour and upon reaction with
hydrogen donors of the antioxidant changes to yellow colour. It is
a discolouration assay, which is evaluated by the addition of the
antioxidant or test compound to a DPPH solution in ethanol and the
decrease in absorbance was measured.
253–256 ꢀC; IR (KBr, Cmꢁ1): 3451 (N–H), 3086 (ArC–H), 2980 (AliC–
H), 1726 (C]O), 1638 (C]O), 1182 (C–N); 1H NMR (DMSO-d6):
d 2.1
(s, 3H, CH3), 2.6 (s, 3H, CH3), 3.9 (s,1H, CH), 5.3 (s,1H, OH), 5.7 (s,1H,
CH), 6.8 (s, 1H, NH), 7.5 (s, 1H, NH), 8.0 (s, 1H, NH). M þ 1 calculated
236; found 235.81; mass fragments (m/z): 210, 187, 119.
Compound no 25: Pale yellowish amorphous solid (79%); mp
216–219 ꢀC; IR (KBr, cmꢁ1): 3296 (N–H), 3019 (ArC–H), 2939 (AliC–
The assay was carried out in a 96-well microtitre plate. To 200
of each of DPPH ethanolic solution, 10 l of each of the test
compound (100 g) or standard (ascorbic acid, 10 g) solution was
ml
H), 1697 (C]O),1648 (C]O); 1H NMR (DMSO-d6):
d
2.3 (s, 3H, CH3),
m
2.5 (s, 3H, CH3), 5.2 (s, 1H, CH), 7.2–7.6 (m, 5H, Ar–H), 7.8 (s, 1H, NH),
8.0 (s, 1H, NH), 8.6 (s, 1H, NH). M þ 1 calculated 246; found 245.93;
mass fragments (m/z): 221, 176, 96.
m
m
added separately to wells of the microtitre plate. The plates were
incubated at 37 ꢀC for 30 min and the absorbance of each solution
was measured at 490 nm, using ELISA reader. The IC50 values
(concentration which inhibits 50% of free radicals) for the
compounds 28 and 29 were determined by serial dilution method
Compound no 26: Yellowish crystals (76%); mp 180–184 ꢀC; IR
(KBr, cmꢁ1): 3468 (N–H), 3108 (ArC–H), 2962 (AliC–H), 1717 (C]O),
1654 (C]O), 1220 (C–N), 1245 (C–O); 1H NMR (DMSO-d6):
d 2.2 (s,
3H, CH3), 2.5 (s, 3H, CH3), 3.7 (s, 3H, OCH3), 5.3 (s, 1H, CH), 7.0–7.4
(m, 4H, Ar–H), 7.5 (s, 1H, NH), 7.9 (s, 1H, NH), 8.5 (s, 1H, NH). M þ 1
calculated 276; found 276.25; mass fragments (m/z): 250, 178, 99.
Compound no 27: Yellowish amorphous solid (72%); mp 170–
173 ꢀC; IR (KBr, cmꢁ1): 3285 (N–H), 3082 (ArC–H), 2941 (AliC–H),
1647 (C]O),1655 (C]O),1194 (C–N),1620 (C]C); 1H NMR (DMSO-
at the concentrations below 100 mg.
4.3. In vitro anticancer activity
Anticancer activity of the compounds was evaluated by deter-
mining the percentage viability of MCF-7, human breast cancer cells
using the trypan blue dye exclusion technique. MCF cells were
cultured in the peritoneal cavity of healthy albino mice weighing
25–30 g by injecting a suspension of MCF cells (1 ꢂ106 cells/ml)
intraperitoneally. The cells were aspirated aseptically from the
peritoneal cavity of the mice on day 15. The cells were washed with
Hank’s balanced salt solution (HBSS) and centrifuged for 10–15 min
in the cooling centrifuge. The pellet was resuspended with HBSS
and the process was repeated three times. Finally, the cells were
suspended in a known quantity of HBSS and the cell count was
adjusted to 1 ꢂ106 cells/ml. 0.1 ml of the diluted cell suspension
was distributed into eppendorf tubes and exposed to 0.1 ml of the
d6): d 2.1 (s, 3H, CH3), 2.5 (s, 3H, CH3), 3.5 (s, 1H, CH), 5.0 (s, 1H, CH),
7.0–7.4 (m, 5H, Ar–H), 7.5 (s, 1H, NH), 7.8 (s, 1H, NH), 8.0 (s, 1H, NH),
8.2 (s, 1H, NH); MS (m/z): M ꢁ 1 calculated 270; found 269.82; mass
fragments (m/z): 214, 203, 173, 102.
Compound no 28: Yellowish amorphous solid (80%); mp 170–
173 ꢀC; IR (KBr, cmꢁ1): 3440 (N–H), 3098 (ArC–H), 1840 (C]S),
2972 (AliC–H), 1652 (C]O), 1350 (NO2); 1H NMR (DMSO-d6):
d 2.1
(s, 3H, CH3), 2.5 (s, 3H, CH3), 5.3 (s, 1H, CH), 7.7–8.0 (m, 4H, ArH), 8.2
(s, 1H, NH), 9.5 (s, 1H, NH), 10.1 (s, 1H, NH); MS (m/z): M ꢁ 1
calculated 305; found 305.09; mass fragments (m/z): 267, 143, 115,
102.
Compound no 29: Pale yellow crystals (76%); mp 194–197 ꢀC; IR
(KBr, cmꢁ1): 3362 (N–H), 3006 (ArC–H), 2932 (AliC–H), 1711 (C]O),
test compound (10 m
g) and incubated at 37 ꢀC under 5% CO2 for 3 h.
After 3 h, a trypan blue dye exclusion test was performed to
determine the percentage viability. The pooled cells were mixed
with 0.4% yield trypan blue in a ratio of 1:1 and the number of
1675 (C]O),1220 (C–N),1344 (NO2); 1H NMR (DMSO-d6):
d
2.0 (s, 3H,
CH3), 2.6 (s, 3H, CH3), 5.3 (s, 1H, CH), 7.7 (s, 1H, NH), 7.8 (m, 4H, ArH),

4198
B.R. Prashantha Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 4192–4198
stained, non-stained and total number of cells was counted using
a haemocytometer. Cell count taken from cells grown in the
absence of the test compound was taken as 100% cell survival
(control). Percentage cytotoxicity was calculated by using the
formula for triplicate samples:
developed model was further evaluated by predicting activities of
the external test set compounds.
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