Synthesis and in vitro anticancer and antitubercular activity of diarylpyrazole ligated dihydropyrimidines possessing lipophilic carbamoyl group

Article abstract of DOI:10.1016/j.bmcl.2012.02.101

A series of dihydropyrimidine derivatives were synthesized by utilizing Biginelli reaction and evaluated for their in vitro anticancer activity against MCF-7 human breast cancer (HBC) cell line using sulforhodamine B (SRB) assay and antitubercular activity against Mycobacterium tuberculosis (MTB) H ₃₇Rv using Microplate Alamar Blue Assay (MABA). Compounds 13p, 13t were exhibited 70.6% and 63.7% of HBC cell growth inhibition at 10 μM concentration. Interestingly compound 13p was also found to be the most potent in the series against MTB H₃₇Rv with MIC value of 0.125 μg/mL.

Full text of DOI:10.1016/j.bmcl.2012.02.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2708–2711
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro anticancer and antitubercular activity
of diarylpyrazole ligated dihydropyrimidines possessing lipophilic
carbamoyl group
Rama Krishna Yadlapalli a, O.P. Chourasia a, Kiranmayi Vemuri b, Manjula Sritharan b,
Ramu Sridhar Perali c,
⇑
a Department of Chemistry, Dr. Harisingh Gour University (A Central University), Sagar 470 003, MP, India
b Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
c School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydropyrimidine derivatives were synthesized by utilizing Biginelli reaction and evaluated
for their in vitro anticancer activity against MCF-7 human breast cancer (HBC) cell line using sulforhoda-
mine B (SRB) assay and antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv using
Microplate Alamar Blue Assay (MABA). Compounds 13p, 13t were exhibited 70.6% and 63.7% of HBC cell
Received 21 January 2012
Revised 27 February 2012
Accepted 29 February 2012
Available online 7 March 2012
growth inhibition at 10
l
M concentration. Interestingly compound 13p was also found to be the most
g/mL.
Ó 2012 Published by Elsevier Ltd.
potent in the series against MTB H37Rv with MIC value of 0.125
l
Keywords:
Dihydropyrimidines
1,3-Diarylpyrazoles
Multicomponent Reaction
Anticancer
Antitubercular
Substituted 1,3-diarylpyrazoles1 and 4-aryl-3,4-dihydro-2(1H)-
pyrimidone esters (DHPMs)2 are found to be interesting molecular
scaffolds for de-novo drug design towards the identification of
novel non-steroidal anti-inflammatory drug (NSAID)s, antiviral
and anticancer agents. Diverse analogs of 1,5-diarylpyrazoles also
reported to exhibit impressive cyclooxygenase-2 (COX-2) inhibi-
tory potency.3 Due to the elevated levels of COX-2 enzyme in
breast cancer cells, COX-2 is one of the targets to be selectively
inhibited to suppress osteolytic bone metastasis as well as angio-
genesis in tumor cells. In this direction several NSAIDs were syn-
thesized and their COX-2 inhibitory activities were evaluated. As
a result, celecoxib (CelebrexÒ) 1, a selective COX-2 inhibitor is in
the market for the treatment of both osteoarthritis and adult rheu-
matoid arthritis and monastrol 2, a mitotic kenensin EG5 inhibitor4
were identified (Fig 1). A similar kind of molecular skeleton is pres-
ent in nitractin 3, an antiviral agent. 1,3-Diarylpyrazole derivative,
lonazolac 4 (IrritrenÒ), is also in the market for the treatment of
inflammatory diseases. DHPMs are also important structural
motifs present in potent calcium channel blockers, antihyperten-
sive agents,5 adrenergic antagonists6 and neuropeptide Y (NPY)
antagonists,7 fatty acid transporter FATP4 inhibitors.8 Very
recently DHPMs with potential anticancer activity against MCF-7
human breast cancer cells9 was reported.
On the other hand pyrimidine derivatives were also evaluated
as selective inhibitors of dihydrofolate reductase (DHFR), a key en-
zyme in the folate cycle,10 from Mycobacterium tuberculosis
(MTB).11 Diarylpyrazole based dihydropyrimidine derivatives were
found to be the most active compounds in vitro with MIC values
upto 0.02 lg/mL against MTB and were more potent than isonia-
zid.12 Several DHPM derivatives bearing a lipophilic group (car-
bamoyl moiety) were also shown to be potential antitubercular
agents.13 These compounds could act as precursors which after
penetration of the compound into the cell wall of MTB would be
converted to the carboxylate anions by enzymatic hydrolysis.14
The above observations prompted us to evaluate the bioactivity
of 1,3-diarylpyrazole ligated DHPMs possessing lipophilic carbam-
oyl moiety. Toward the synthesis of these double pharmacophore
containing molecules, we adopted a general and diversity oriented
multi component reaction (MCR) involving a one pot condensation
of an activated 1,3-dicarbonyl system, 1,3-diarylpyrazole carbalde-
hyde and urea or thio-urea.
Differentially substituted acetoacetanilide derivatives possess-
ing electron releasing groups (ERG) and electron withdrawing
groups (EWG) on the anilide were selected for the MCR reaction.
These acetoacetanilides (8a–8f) were prepared by refluxing
⇑
Corresponding author.
E-mail address: prssc@uohyd.ernet.in (R.S. Perali).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2012.02.101

R. K. Yadlapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2708–2711
2709
SO2NH2
A two step protocol was adopted for the synthesis of 1,3-diary-
lpyrazole-4-carbaldehyde derivatives. Accordingly, reaction of ace-
tophenones (9a and 9b) with phenylhydrazine in acetic acid at
25 °C provided the corresponding hydrazones 10a and 10b. These
hydrazones upon reaction with excess Vilsmeier reagent, with
the in situ generated halomethyleneiminium salt, resulted the for-
mation of 1,3-diarylpyrazole-4-carbaldehyde derivatives 11a and
11b in excellent yield (Scheme 2). The expected target dihydropy-
rimidine derivatives (13a–13w) were prepared by refluxing
equimolar amounts of b-ketoester (ethylacetoacetate, 5)/acetoace-
tanilides (8a–8f), pyrazole-carbaldehydes (11a and 11b) and ex-
cess amount of urea/thiourea (1.5 equiv) with catalytic amount
of p-toluenesulfonic acid (40 mol %) in ethanol (Scheme 3).
Interestingly better yields were obtained using thiourea com-
pared to urea. The formation of DHPMs was confirmed by observ-
ing the characteristic peaks of the three components that were
used for MCR in the product NMR. All the DHPMs showed the
asymmetric CH proton in 1H NMR spectrum between d 5.30–
5.80 ppm, as well as a singlet at d 2.00–2.40 ppm equivalent to
the methyl group on the dihydropyrimidine ring. Further in 13C
NMR spectra showed chemical shifts at d 173.0–175.0 ppm corre-
sponding to C@S and d 152.0–154.0 ppm assigning to C@O on the
dihydropyrimidine ring. The 13C NMR peaks of carbonyl present
in ester and carbamoyl group were found to be in between d
164.0–167.0 ppm. The electron rich quaternary carbon of DHPM
which is adjacent to the asymmetric center was observed in be-
tween d 99.0–109.0 ppm as expected. All the final products were
also confirmed by taking LC–MS and observing the molecular ion
peak. Finally the elemental analysis results were within 0.4% of
the theoretical values assuring the purity of the final products for
bioactivity studies.
OH
O
O
NH
N
N
CH3
N
H
S
F3C
Celecoxib(®Celebrex) 1
Monastrol 2
(COX-2 inhibitor)
(mitotic kinesin EG5 inhibitor)
COOH
O2N
O
O
N
N
O
NH
Cl
N
H
O
Lonazolac (®Irritren) 4
Nitractin 3
(Antiviral)
(Anti-inflammatory agent)
Figure 1. Dihydropyrimidine and diarylpyrazole structures with biological
importance.
NH2
O
O
O
O
Neat reaction
R
+
reflux, 80 oC
R
N
H
O
24 h
5
6a-6f
8a-8f (31-42%)
only in case of 6f
Con., HCl,
6a R = H
6b
reflux , 80 oC, 24 h
0 o
C
R = 4-Me
6c R = 4-OMe
6d
R = 4-Cl
6e R = 2-Cl
NO2
O2N
6f R = 4-NO2
O
NH
However, when R1 is H, X is O and R2 is either 4-CH3OC6H4NH or
4-ClC6H4NH (13g and 13i) the products were obtained as diastereo-
meric mixtures in the ratio of 71:29 and 83:17, respectively.16 Due
to this reason these two compounds were not further evaluated for
bioactivity studies. Moreover, this observation also confirms that
substituent’s at R1 and on phenyl ring of the carbamoyl group play
an important role in the product confirmation.
Out of 23 compounds (13a–13w) that were synthesized 21
were subjected for in vitro anticancer screening against MCF-7
human breast cancer cells using SRB assay protocol.17 Each com-
pound was tested at four dose levels (1 Â 10À7 M, 1 Â 10À6 M,
1 Â 10À5 M and 1 Â 10À4 M). Appropriate positive controls were
run in each experiment and each experiment was repeated thrice.
As shown in the Table 1 six compounds, 13b, 13j, 13m, 13n, 13p
and 13t, were found to inhibit growth of MCF-7 cells at GI50 (con-
centration of compound causing 50% inhibition of cell growth) of
N
H
Croton derivative
7
Scheme 1. Synthesis of acetoacetanilide derivatives.
O
OHC
H
PhNHNH2
DMF
N
N
Ph
R1
POCl3, 2 h,
CH3COOH
N
R1
N
25 oC, 15 min
90 o
C
Ph
R1
9a R1 = H
9b
10a
10b
11a
11b
(87%)
(83%)
R
1 = Cl
Scheme 2. Synthesis of 1,3-diaryl-1H-pyrazole-4-carbaldehydes.
less than 50.0
lM. Out of all, compounds 13p (entry 16), 13t (entry
equimolar amounts of corresponding substituted anilines and
ethylacetoacetate under solvent free conditions (Scheme 1).15
20) exhibited excellent growth inhibition of MCF-7 HBC cell lines
with 70.6% and 63.7%, respectively at 10 lM concentration.
Ph
N
N
Ph
H2N
O
O
N
N
p-TsOH
O
R1
+
+
X
R2
EtOH
R2
H2N
NH
R1
7 h, reflux
CHO
X
N
H
12a
X = O
5, 8a 8f
11a
-
13a 13n
-
(59-93%)
12b X = S
12a
12b
5, 8a-8c
11b
11b
13o 13v
-
(65-91%)
23 compounds
8d
12a
13w (46%)
Scheme 3. One-pot synthesis of 1,3-diarylpyrazole ligated dihydropyrimidine derivatives.

2710
R. K. Yadlapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2708–2711
Table 1
Inhibitory effect of the synthesized compounds upon MCF-7 cells and Mycobacterium tuberculosis H37Rv
Ph
Breast Cancer Cell Line MCF-7
% Growth inhibition
MTB H37Rv
Visual MABA MICsd
N
N
Growth inhibition
O
R1
R2
NH
X
N
H
13a-13w
Entry
R1
R2
X
Products
at 10À5 M (10
l
M)
at 10À4 M (100
lM)
GI50(
l
M)c
(lg/mL)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OC2H5
OC2H5
C6H5NH
C6H5NH
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
13a
13b
13c
13d
13e
13f
<10
30.4
<10
<10
11.9
19.7
—
86
60.8
42.0
80.6
88.2
71.7
56.1
—
16
32
32
32
32
16
—
8
—
8
32
16
16
32
0.25
0.125
2
32
32
2
À12.0b
63.2
57.4
68.6
84.3
—
4-CH3C6H4NH
4-CH3C6H4NH
4-CH3OC6H4NH
4-CH3OC6H4NH
4-ClC6H4NH
4-ClC6H4NH
2-ClC6H4NH
2-ClC6H4NH
4-NO2C6H4NH
4-NO2C6H4NH
OC2H5
OC2H5
C6H5NH
C6H5NH
4-CH3C6H4NH
4-CH3C6H4NH
4-CH3OC6H4NH
4-CH3OC6H4NH
4-ClC6H4NH
13ga
13h
13ia
13j
<10
—
63.9
—
77.3
—
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
48.6
13.9
16.5
27.3
48.4
17.1
70.6
19.6
24.2
13.6
63.7
<10
<10
<10
À12.8b
72.2
68.5
À17.7b
99.3
70.3
99.2
71.7
74.7
70.4
84.9
72.1
72.0
88.7
34.7
66.6
68.8
35.5
41.4
68.8
33.2
65.5
61.5
69.7
43.5
69.7
70.0
56.9
13k
13l
13m
13n
13o
13p
13q
13r
13s
13t
13u
13v
13w
1
16
32
GI50 = Concentration of compound causing 50% inhibition of cell growth. The bold fonts indicate the best activity values obtained in bioactivity studies, out of all the
compounds that were screened.
a
Obtained as a diastereomeric mixture so that the activity tests were not conducted.
b
% Control growth, negative numbers indicate the cell kill.
c
Each compound is tested at four dose levels 10À7 M, 10À6 M, 10À5 M, 10À4 M, and each experiment is repeated thrice.
d
The experiment was done in duplicate each time and was done twice to confirm the MIC values and confirmed in the BacT/ALERTÒMP Mycobacteria detection system
(bioMérieux, France).
Compound 13p showed maximum inhibition of cell growth with
GI50 of 33.2 lM. In addition 13p also exhibited good dose–re-
sponse ( Fig. 2). The above structure–activity relationship (SAR)
studies revealed that compounds containing X = S were more po-
tent than the X = O counter parts. Based on this observation we
may conclude that presence of thio-urea functional group in
DHPMs enhances the anticancer activity of these type of scaffolds.
Due to the presence of 1,3-diarylpyrazole along with lipophilic
carbamoyl moiety all the 21 compounds were also tested against
MTB H37Rv. The minimum inhibitory concentration (MIC) values
of compounds (13a–13w) were obtained by the Microplate Alamar
Blue Assay18 (MABA) after 5 days of incubation at 37 °C (Table 1,
column 9). The experiment was done in duplicate each time and
was repeated twice to check the reproducibility of the MIC values
and also confirmed in the BacT/ALERTÒMP Mycobacteria detection
system (bioMérieux, France). More interestingly, compounds 13p
and 13o, with a MIC value of 0.125 and 0.25
to be the most potent in the series. Compounds 13q, 13t and 13u
were also expressed good MIC values of 2.0, 2.0 and 1.0 g/mL,
lg/mL, were found
Figure 2. Dose response curve for in vitro anticancer activity of 13p at 0.1
M 12%, 10 M 70.6% and 100 M 99.2%.
lM 4.1%,
l
1
l
l
l
respectively. These compounds could be derivatized further to-
wards the discovery of more potent antimicrobial agents. The
above SAR studies revealed that compounds with chlorine (Cl) at
R1 in the series, were more potent (Compounds 13o, 13p, 13q,
13t and 13u) when compared to compounds in which R1 = H. These
results also suggest that replacing the alkyl ester moiety (R2) with
lipophilic carbamoyl does influence the GI50 values against MCF-7
breast cancer cells as well as MIC values against MTB H37Rv.
In conclusion, a Biginelli reaction was implemented towards the
synthesis of diarylpyrazole with DHPM possessing lipophilic car-
bamoyl groups. These novel scaffolds showed moderate anticancer
activity against MCF-7 breast cancer cell lines as well as good to
excellent antitubercular activity against MTB H37Rv. Out of all,
compound 13p was found to be the most effective and potent
molecule against both anticancer and antitubercular activity. The

R. K. Yadlapalli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2708–2711
2711
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.101.
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