Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of α-(Benzoylamino)-β-substituted Acrylic Amide Derivatives of Pyrazolo[1,5-a]pyrimidine

Article abstract of DOI:10.1002/jhet.3029

A novel series of α-(benzoylamino)-β-substituted acrylic amide derivatives of pyrazolo[1,5-a]pyrimidine has been synthesized using a convergent multistep synthesis. The synthesized compounds were characterized by ¹H NMR, ¹³C NMR, ESI-MS, and IR analyses. Those new compounds were screened for their in vitro antiproliferative activity using an MTT assay analysis. Out of nine derivatives synthesized in the current study, compounds 13g, 13d, 13h, and 13i exhibited the greatest anticancer activities in HeLa and HepG₂ cell lines. The in vitro anticancer activity of compound 13g against HeLa, HepG₂, and MCF-7 cell lines is superior to the marketed drugs paclitaxel and SAHA.

Full text of DOI:10.1002/jhet.3029

Month 2017 Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel
Series of α-(Benzoylamino)-β-substituted Acrylic Amide Derivatives of
Pyrazolo[1,5-a]pyrimidine
b
a
a
A. Sasikumar,a,b V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
*
aAnthem Biosciences Pvt. Ltd., # 49, Bommasandra Industrial Area, Bommasandra, Bangalore 560 099, Karnataka, India
bDepartment of Biomedical Sciences, School of Bio Sciences and Technology, VIT University, Tamilnadu 632014, India
*
E-mail: v.mohan@vit.ac.in
Received April 19, 2017
DOI 10.1002/jhet.3029
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
A novel series of α-(benzoylamino)-β-substituted acrylic amide derivatives of pyrazolo[1,5-a]pyrimidine
has been synthesized using a convergent multistep synthesis. The synthesized compounds were character-
1
ized by H NMR, 13C NMR, ESI-MS, and IR analyses. Those new compounds were screened for their in
vitro antiproliferative activity using an MTT assay analysis. Out of nine derivatives synthesized in the current
study, compounds 13g, 13d, 13h, and 13i exhibited the greatest anticancer activities in HeLa and HepG2 cell
lines. The in vitro anticancer activity of compound 13g against HeLa, HepG2, and MCF-7 cell lines is supe-
rior to the marketed drugs paclitaxel and SAHA.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
interest because of their wide range of biological
activities. Several compounds of this class show
interesting antitrypanosomal [20] and antischistosomal
activities [21]. They are used as HMG-CoA reductase
inhibitors [22], COX-2 selective inhibitors [23], 30:50-
cyclic-AMP phosphodiesterase inhibitors [24], CRF1
antagonists [25], selective peripheral benzodiazepine
receptor ligands [26], potassium channel openers (KCO’s)
[27], and histamine-3 receptor ligands [28].
Oxazolones are important synthon for the synthesis of
several class compounds such as amino acids, dyes,
drugs, and biological active compounds. Literature survey
reveals that compounds containing oxazolone core exhibit
important biological activities such as analgesic [29],
anticancer [30], antiinflammatory [31], neuroleptic [32],
sedative [33], antidiabetic [34], and antiobesity activities
[35]. Substituted oxazolones on reaction with various
Pyrazole and its derivatives, a class of well-known
heterocycle, hold important position in medicinal and
pesticide chemistry for their diverse biological activities
[1,2]. Natural products containing pyrazole nucleus are
very scarce, but many synthetic pyrazoles are known to be
biologically active [3]. Examples of leading drugs
containing pyrazole rings are celebrex [4], sildenafil
(Viagra) [5], zaleplon [6], and fipronil [7]. Substituted
pyrazole derivatives are known for their biological and
pharmacological activities [8] not only as antiinflammatory
[9], antibacterial [10], antifungal [11], and anticancer [12]
but also important useful starting materials for the
synthesis of other fused heterocyclic systems.
Pyrimidines are the most important six-member
heterocyclic containing nitrogen atoms on 1,3 positions.
Compounds containing substituted pyrimidine derivatives
are known to possess biological activities such as
antibacterial [13], antifungal [14], antiinflammatory [15],
anti-HIV [16], and anticancer activities [17]. In addition
to these, they are known to possess potential central
nervous system depressant properties [18] and can act as
calcium channel blockers [19].
heterocyclic
amines,
thiosemicarbazide
and
sulphonamides, in the presence of dry pyridine, acetic
acid, or acetic anhydride gave three different derivatives
such as imidazolinone, α-acylamino acrylic acid, or
triazinone derivatives.
Among three derivatives mentioned previously,
biological activity of both imidazolinone and triazinone
derivatives were well established in the literature,
whereas the biological importance of α-acylamino acrylic
Fused derivative of pyrazole and pyrimidine viz.
pyrazolo[1,5-a]pyrimidine has attracted considerable
© 2017 Wiley Periodicals, Inc.

A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
amide derivatives was not studied in detail. A detailed
literature survey reveals that synthesis and biological
importance of acrylic amides containing pyrazolo[1,5-a]
pyrimidine were not reported yet.
Therefore, considering the aforementioned data
described, we present here our recent work on design and
synthesis of novel α-(benzoylamino)-β-substituted acrylic
amide derivatives from novel oxazolone analogues of 5,7-
dimethyl-pyrazolo[1,5-a]pyrimidine using substituted
amines with representative example, that is, compound
13g as shown in Figure 1, showing good growth inhibition
against HeLa cell line, HepG2, and MCF-7.
aromatic primary amines with substituted oxazolone
derivatives. Oxazolone derivatives containing pyrazolo[1,5-
a]pyrimidine prepared from the corresponding 5,7-
dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4).
Synthesis of 5,7-dimethyl-pyrazolo[1,5-a]pyrimidine-3-
carboxylic acid (4) as shown in Scheme 1 was explained
well in detail in the literature [41].
In order to synthesize the respective oxazolones from the
aforementioned acid derivative (4), we require the
respective hippuric acid derivative (6) as the initial
counterpart. In general, the hippuric acid derivatives were
obtained by treating the corresponding acid chlorides
with glycine in the presence of sodium hydroxide
solution at room temperature [42]. Based on the literature
methods, our initial attempts to form the corresponding
acid chloride of acid (4) by using oxalyl chloride and
phosphorous chloride mediated reactions were not
successful. Alternatively, it was acheived through two-
step conversion as shown in Scheme 2. The first step
involves the preparation of 7-dimethyl-pyrazolo[1,5-a]
pyrimidine-3-carbonyl-amino-acetic acid methyl ester (5),
which was achieved by coupling acid derivative (4) with
glycine methyl ester hydrochloride by means of acid-
amine coupling agents. Further, ester (5) was saponified
to 5,7-dimethyl-pyrazolo[1,5-a]pyrimidine-3-carbonyl)-
amino]-acetic acid (6) using 10% sodium hydroxide
solution at 100°C for 1 h.
Oxazolones (8a–8e) were synthesized by treating 5,7-
dimethyl-pyrazolo[1,5-a]pyrimidine-3-carbonyl)-amino]-
acetic acid (6) with respective aldehydes (7a–7e) in the
presence of acetic anhydride and sodium acetate at
80°C, based on the reported Erlenmeyer–Plöchl reaction
[43] as shown in Scheme 3.
Amines (12a–12c) were synthesized by the following
reaction method as shown in the Scheme 4. 4-
Nitrobenzoyl chloride (9) was treated with substituted
amines (10a–10c) under basic condition medium to obtain
the corresponding nitro-substituted amides (11a–11c). The
aforementioned nitro amides were further reduced under
hydrogenation pathway to synthesize corresponding
amines (12a–12c) in good yield.
RESULTS AND DISCUSSION
Chemistry. The aim of present study was to investigate
the reactivity of substituted primary aliphatic and aromatic
amines with novel oxazolones derived from pyrazolo[1,5-
a]pyrimidine analogue. Three types of reaction can occur
when aromatic amines treated with oxazolones
derivatives in the presence of acid or base under heating
conditions. The first type involves the insertion of amine
into the oxazolone ring to obtain imidazolones [36]. The
second type involves the ring expansion to obtain
triazinones [37]. The third one involves the ring opening
of oxazolones to obtain α-(benzoylamino)-β-substituted
acrylic amides [38–40].
The method presented in this paper represents the
synthesis of acrylic amide by reaction of substituted
O
N
H
O
N
H
O
Cl
N
H
N
N
N
Our attempts as per the reported literature [42], coupling
of oxazolones (8a–8e) with substituted anilines (12a–12c)
in ethanol medium under reflux condition was not
successful. Also, the reaction carried at elevated
13g
Figure 1. Chemical structure of compound 13g, which showed good
antiproliferative activity.
Scheme 1. Synthesis of compounds 4.
O
O
OH
O
O
DMF-DMA, AcOH
NH2-NH2.H2O
CN
Acetylacetone
NaOH
O
N
O
N
N
N
N
N
N
NH2
EtOH, H2O
720C,4h
O
N
DMF, 500C, 2h
AcOH, 820C,8h
H
4
1
2
3
Journal of Heterocyclic Chemistry
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Month 2017
Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of
α-(Benzoylamino)-β-substitutedAcrylicAmideDerivativesofPyrazolo[1,5-a]pyrimidine
Scheme 2. Synthesis of compounds 6.
O
O
O
O
O
N
OH
N
EDCI.HCl, HOBt,
NMM, DMF
O
OH
O
10% NaOH
100 0C, 2h
H
H
+
N
N
N
H2N
O
N
N
N
N
N
N
R.T, 5h
HCl
4
5
6
Scheme 3. Synthesis of compounds 8a–8e.
O
O
X
O
O
O
N
H
NaOAc, Ac2O
OH
N
H
+
N
N
N
N
N
N
Reflux, 4h
X
8a - 8e
6
7a-e
8a, X = 4-chloro
8b, X = 2-bromo-5-fluoro
8c, X = 3,4-dimethoxy
8d, X = 2-chloro
8e, X = 3-chloro
temperatures with high boiling solvents like
dimethylformamide, toluene, and dimethyl sulfoxide was
also unsuccessful. But when we performed the reaction
with acetic acid under reflux condition, we observed the
formation of product exclusively the ring opened
oxazolones to get α-(benzoylamino)-β-substituted acrylic
amides as shown in Scheme 5. The presence of three
amide –NH protons in the 1H NMR of the isolated
product confirms formation of α-(benzoylamino)-β-
substituted acrylic amides.
3298 cmÀ1, which was attributed to the –NH stretching
vibration of the amide. Two other peaks were observed
at 1747 and 1650 cmÀ1 for the C=O stretch of carbonyl
groups for both amide carbonyl and ester carbonyl,
respectively. The 1H NMR spectrum of compound 5
contained a singlet with a chemical shift of 3.67 ppm,
which was consistent with the proton of the methyl
ester, as well as a triplet with a chemical shift of
8.42 ppm, which was consistent with the proton of the
amide group. The 13C NMR spectrum of compound 5
contained a signal at 171.10 ppm, which was consistent
with the carbonyl carbon of amide group. LC-MS
analysis of compound 5 gave a molecular ion peak with
an m/z value of 263.1 for [M + H], which was
consistent with the molecular formula C12H14N4O3. The
Synthesis and structural elucidation part for compound
4 was explained in detail in our earlier publication [41].
1
The structure of compound 5 was confirmed by IR, H
NMR, 13C NMR, and LC-MS analyses. The FTIR
spectrum of compound 5 contained a broadband at
Scheme 4. Synthesis of compounds 12a–12c.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
Scheme 5. Synthesis of compounds 13a–13i.
1
structure of compound 6 was confirmed by IR, H NMR,
The structure of compounds 11a–c and 12a–c was
confirmed by IR, 1H NMR, 13C NMR, and LC-MS
analyses. The FTIR spectrum of 11b contained a
broadband at 3323 cmÀ1, which was attributed to the
–NH stretching vibration of the amide. Two other
peaks were observed at 1627 and 1338 cmÀ1, which
represents the C=O stretch of carbonyl group of the
amide and –NO2 stretch for the nitro group,
13C NMR, and LC-MS analyses. The FTIR spectrum of
compound 6 contained a broadband at 3283 cmÀ1
,
which was attributed to the –NH stretching vibration of
the amide. Two other peaks were observed at 1728 and
1643 cmÀ1 for the C=O stretch of carbonyl groups for
both amide carbonyl and acid, respectively. The 1H
NMR spectrum of compound 6 contained a triplet with
a chemical shift of 8.36–8.39 ppm range, which was
consistent with the proton of the amide –NH, as well as
a broad singlet with a chemical shift of 12.71 ppm,
which was consistent with the proton of the acid group.
The 13C NMR spectrum of 6 contained a signal at
171.96 ppm, which was consistent with the carbonyl
carbon of acid functional group. LC-MS analysis of
compound 6 gave a molecular ion peak with an m/z
value of 249.3 for [M + H], which was consistent with
the molecular formula C11H12N4O3.
The structures of oxazolone derivatives 8a–8e were
confirmed by 1H NMR, 13C NMR, LC-MS, and IR
analyses, and detailed explanation was given in the
Experimental section. The structure of compound 8a was
confirmed by IR, 1H NMR, 13C NMR, and LC-MS
analyses. The FTIR spectrum of compound 8a contained a
broadband at 3423 cmÀ1, which was attributed to the exo-
olefinic –CH stretching vibration of the oxazolone. In
addition to that, a peak was observed at 1775 cmÀ1 for the
C=O stretch of carbonyl group from the oxazolone. The
1H NMR spectrum of compound 8a contained a singlet
with a chemical shift of 7.10 ppm, which was consistent
with the proton of the benzylidene as well as two singlets
with chemical shift of 7.44 and 8.17 ppm, which was
consistent with the protons from the 4-chloro-phenyl
group. The 13C NMR spectrum of compound 8a contained
a signal at 167.5 ppm, which was consistent with the
carbonyl carbon of oxazolone group. LC-MS analysis of
compound 8a gave a molecular ion peak with an m/z value
of 353.3 for [M + H], which was consistent with the
compound 8a molecular formula C18H13ClN4O2.
1
respectively. The H NMR spectrum of compound 11b
contained a broad singlet with a chemical shift of
6.08 ppm range, which was consistent with the proton
of the amide –NH, as well as a broad singlet with a
chemical shift of 4.18 ppm, which was consistent with
the junction proton of the cycloheptyl group. The 13C
NMR spectrum of 11b contained
a
signal at
166.6 ppm, which was consistent with the carbonyl
carbon of amide group. LC-MS analysis of compound
11b gave a molecular ion peak with an m/z value of
263.2 for [M + H], which was consistent with the
molecular formula C14H18N2O3. In the same way, the
analytical data for the remaining nitro compounds are
in agreement with the molecular formulae. The
structure of compound 12b was confirmed by IR, 1H
NMR, 13C NMR, and LC-MS analyses. The FTIR
spectrum of compound 12b contained two broadbands
at 3344 and 3303 cmÀ1, which were attributed to the
–NH stretching vibration for both amine group and
amide group, respectively. The peak at 1604 cmÀ1
represents for the C=O stretch of carbonyl group of
the amide. The 1H NMR spectrum of compound 12b
contained a doublet with a chemical shift of 7.71 ppm
range, which was consistent with the proton of the
amide –NH, as well as a broad singlet with a chemical
shift of 5.54 ppm, which was consistent with the
protons of the amine group. The 13C NMR spectrum
of compound 12b contained a signal at 165.5 ppm,
which was consistent with the carbonyl carbon of
amide group. LC-MS analysis of compound 12b gave
a molecular ion peak with an m/z value of 233.3 for
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of
α-(Benzoylamino)-β-substitutedAcrylicAmideDerivativesofPyrazolo[1,5-a]pyrimidine
Table 1
[M + H], which was consistent with the molecular
formula C14H20N2O. The analytical data for the
remaining amine compounds are in agreement with the
Anticancer activity of compounds 13a–13i.
Percentage growth inhibition in different cell lines
Concn.
molecular formulae.
The structure of compounds 13a–13i was confirmed by
1
IR, H NMR, 13C NMR, and LC-MS analyses. The FTIR
Compound
(μM)
HeLa
HepG2
MCF-7
spectrum of compound 13a contained three broadband
signals at 3383, 3304, and 2920 cmÀ1, respectively,
which were attributed to the –NH stretching vibration of
the three amide groups. Three other peaks were observed
at 1652, 1627, and 1602 cmÀ1, respectively, for the C=O
stretching vibration of carbonyl groups for three amide
groups. The 1H NMR spectrum of compound 13a
contained a triplet with a chemical shift of 9.03 ppm
range, which was consistent with the proton of the 4-
trifluoromethylbenzylamide group. In addition to this, a
singlet with a chemical shift of 9.95 ppm was consistent
with the proton of the acryl amide group. Also, we have
observed another singlet with a chemical shift of
10.35 ppm, consistent with the proton of the
pyrazolo[1,5-a]pyrimidyl amide group. The 13C NMR
spectrum of compound 13a contained three signals from
three different carbonyl groups of the amide group. In
addition to that, a quartet signal was observed at 120.55,
123.42, 126.13, and 129.00 ppm range with a coupling
constant of 271 Hz, which was consistent with the
trifluoromethyl carbon from the molecule. LC-MS
analysis of compound 13a gave a molecular ion peak
with an m/z value of 673.6 for [M + H], which was
consistent with the molecular formula C35H31F3N6O5.
The spectral data generated in the current study were in
good agreement with the assigned structures of all the
novel molecules synthesized in this series.
Anticancer evaluation. All the compounds synthesized
in the present study were screened for their in vitro
anticancer activity against HeLa cells, which were used as
a representative human cervical cancer cell line and
HepG2 cells, which were used as human liver cancer line
and MCF-7 cells, which were used as a representative
human breast carcinoma cancer cell line. These cell lines
were obtained from American Type Culture Collection.
Paclitaxel and SAHA were used as reference standards.
The anticancer activity of the compounds is listed in
Table 1. These data showed that some of the compounds
exhibited good inhibitory activity towards the growth of
HeLa, HePG2, and MCF-7 cell lines.
13a
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
0.77821
10.60291
13.15136 12.45136
26.07004 23.31691
33.47193 25.69444
6.732348
11.45833
2.818991
2.117264
À2.46914
1.335312
À3.4202
À6.17284
13.79822
7.81759
À1.23457
25.66766
22.3127
22.92769
34.56973
34.69055
36.33157
22.7003
16.77524
À0.17637
39.02077
36.64495
34.39153
58.01187
51.79153
49.02998
22.55193
19.21824
21.86949
34.1584
13b
28.0397
26.07004
13c
45.33074 68.3087
45.53015 63.54167
47.64268 66.73152
72.76265 59.77011
73.18087 60.9375
72.45658 58.17121
39.88327 35.79639
39.50104 29.6875
32.75434 32.87938
13d
13e
13f
68.8716
43.34975
69.85447 40.79861
67.74194 46.88716
86.38132 76.68309
81.49688 77.25694
78.90819 77.62646
13g
13h
38.0531
36.78161
32.20339 35.41667
34.21053 39.68872
55.25292 72.08539
56.34096 73.95833
57.81638 72.76265
40.6032
42.586
52.879
13i
Paclitaxel
SAHA
32.6531
37.3856
51.8135
21.0459
29.1503
40.6736
39.3258
53.3502
21.2871
34.7066
23.6659
35.2313
45.5934
48.799
HeLa, cervical carcinoma; HepG2, liver carcinoma; MCF-7, breast
carcinoma.
anticancer drugs paclitaxel and SAHA. Compounds 13a
and 13b were not shown any anti-proliferative activities
in all the three cell lines screened. Overall, the compound
13g exhibited superior anticancer activity than the
marketed anticancer drugs paclitaxel and SAHA in all the
three cell lines.
Considering the SAR of these novel compounds (13a–
13i) synthesized indicates that the introduction of chloro
functional group at the position ortho, meta, and para to
the benzylidine ring led to an increase in the anticancer
activity irrespective of amide functional group such as n-
butyl, cycloheptyl, and trifluoro methylbenzyl than chloro
functional group at meta position. It can be concluded
that anticancer activity increases when chloro substitution
in the benzylidine ring follows the order
ortho > para > meta irrespective of amide functional
group. In contrast, dimethoxy substitution to the
benzylidine ring did not show any inhibition toward all
three cell lines screened, which indicates deactivation
In particular, compounds 13g, 13d, and 13f showed
good antiproliferative activities against HeLa cells.
Compounds 13g, 13i, and 13c showed good anti-
proliferative activities against HepG2 cells. Compounds
13h, 13g, and 13e showed good anti-proliferative
activities against MCF-7 cells. Furthermore, above seven
compounds 13c, 13d, 13e, 13f, 13h, 13g, and 13i
exhibited superior anticancer activity than the marketed
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A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
property as shown from values of compounds 13a and 13b.
In addition to the presence of –F, ÀBr substitution in the
benzylidine ring showed moderate activity in all three
cell lines screened.
All together, the results of this study revealed that
compounds 13g, 13d, 13h, and 13i exhibited the greatest
anticancer activities in both the cell lines such as HeLa
cells and HepG2 cells. However, the in vitro anticancer
activity of compound 13g against HeLa, HepG2, and
MCF-7 cell lines is superior to the marketed drugs
paclitaxel and SAHA. It is noteworthy that compound
13g contained a chloro group at the ortho position of the
benzylidine ring.
Molecular weights for all the synthesized compounds were
checked by LC-MS 6200 series Agilent Technology. The
chemical shifts are reported in ppm (δ) with reference to
TMS as an internal standard. The signals are designed as fol-
lows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad
singlet. IR spectra for all the compounds were recorded
using a Bruker Alpha FTIR spectrometer using a diamond
ATR single reflectance module (24 scans).
Chemistry.
The synthesis part of 5,7-dimethyl-
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
4
was
reported in the publication [41].
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carbonyl-
amino-acetic acid methyl ester (5, C12H14N4O3).
To the
of 5,7-dimethyl-pyrazolo[1,5-a]
solution of 45
g
pyrimidine-3-carboxylic acid 4 (235.2 mmol), 44.31 g of
glycine methyl ester hydrochloride (352.8 mmol),
47.67 g of HOBT (352.8 mmol) in 600 cm3 of DMF,
142.74 g of N-methyl morpholine (588.1 mmol) was
added dropwise at room temperature and stirred for
10 min at the same temperature under argon atmosphere.
A total of 67.62 g of EDC.HCl (352.8 mmol) was added
lotwise under argon atmosphere, and the resulted reaction
medium was stirred for 5 h at room temperature.
Completion of the reaction was monitored by TLC. After
completion of the reaction, the reaction mass was
quenched with 6 dm3 of ice under stirring, and the
stirring was continued for 30 min. The precipitate was
collected by filtration, washed with water, and dried
under vacuum to afford 5,7-dimethyl-pyrazolo[1,5-a]
pyrimidine-3-carbonyl-amino-acetic acid methyl ester 5
as off white solid (54 g, 88%). MP: 199.3–201.7°C;
TLC: Rf = 0.45 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3298 (À NH), 1747 (–C=O), 1650 (ÀC=O), 1553 (ÀNH),
CONCLUSIONS
A novel series of α-(benzoylamino)-β-substituted acrylic
amide derivatives of pyrazolo[1,5-a] pyrimidine has been
designed and synthesized using a convergent approach,
and the structures of these compounds were confirmed by
1H NMR, 13C NMR, ESI-MS, and IR analyses. These
new compounds were tested for their in vitro anti-
proliferative activity using an MTT assay. Out of the nine
derivatives prepared in the current study, compounds 13g,
13d, 13h, and 13i exhibited good anticancer properties
tested against HeLa cells and HepG2 cells. However, the
in vitro anticancer activity of compound 13g against
HeLa, HepG2, and MCF-7 cell lines is superior to the
marketed drugs paclitaxel and SAHA.
The results of our in vitro anticancer studies revealed
that the majority of these derivatives showed high levels
of potency against HeLa cells, HepG2, and MCF-7 cell
lines. Further structure–activity relationship studies on
these compounds are therefore currently in progress in
our laboratory.
1
1222 (C–O–C) cmÀ1; H NMR (400 MHz, DMSO-d6):
δ = 2.62 (3H, s, ArCH3), 2.72 (3H, s, ArCH3), 3.67 (3H,
s, ÀOCH3), 4.17–4.18 (2H, d, glycine-CH2), 7.12 (1H, s,
ArH), 8.42 (1H, t, amide-NH), 8.49 (1H, s, ArH) ppm;
13C NMR (100 MHz, DMSO-d6): δ = 16.92, 24.79, 52.26,
104.06, 110.62, 145.50, 145.85, 147.56, 162.07, 162.11,
171.10 ppm; LC-MS: 263.1 (M + H); analysis calculated
for C12H14N4O3: C, 54.96, H, 5.38, N, 21.36%; found: C,
54.84, H, 5.47, N, 21.53%.
EXPERIMENTAL
General information. All chemicals were of analytical
grade and obtained from Sigma-Aldrich Co. The
purification of all the intermediates and final compounds
was carried out by column chromatography using Merck
silica gel with 230–400 mesh size. The purity of the
compounds was determined by thin layer chromatography
experiments, performed on alumina-backed silica gel
40 F254 plates (Merck, Darmstadt, Germany). The TLC ob-
servation of these plates were carried out by illuminating un-
der UV (254 nm) lamp and KMnO4 stain solution. Melting
points were determined using Buchi B-540 instrument and
are uncorrected. All 1H and 13C NMR spectra were recorded
on Bruker AM-300 (300 MHz for 1H NMR and 75 MHz for
13C NMR) and Bruker AM-400 (400 MHz for 1H NMR and
100 MHz for 13C NMR), Bruker BioSpin Corp., Germany.
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carbonyl)-
amino]-acetic acid (6, C11H12N4O3). A total of 520 cm3 of
sodium hydroxide solution (10%) was added to 52 g of
5,7-dimethyl-pyrazolo[1,5-a]pyrimidine-3-carbonyl-amino
acetic acid methyl ester 5 (198.2 mmol), and the resulted
suspension was refluxed at 100°C for 2 h. The reaction
medium became a clear brownish solution during this
period. Completion of the reaction was monitored by
TLC. After completion of the reaction, the reaction mass
was cooled to room temperature. The reaction medium
was diluted with 300 cm3 of ice and acidified to pH 3
with 40 cm3 of hydrochloric acid (12 N) under stirring,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of
α-(Benzoylamino)-β-substitutedAcrylicAmideDerivativesofPyrazolo[1,5-a]pyrimidine
and the stirring was continued for further 30 min. The
precipitate was collected by filtration, washed with water,
and dried under vacuum to afford 5,7-dimethyl-
pyrazolo[1,5-a]pyrimidine-3-carbonyl-amino-acetic acid 6
as off white solid (41 g, 85%). MP: 247.5–248.9°C;
TLC: Rf = 0.25 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3283 (ÀNH), 1728 (ÀC = O), 1643 (ÀC = O), 1556
cmÀ1) υ: 2997 (=CH), 1775 (ÀC=O), 1651 (ÀC=N), 1581
(ÀNH), 1189 (C–O–C), 1156 (C–F) cmÀ1
;
1H NMR
(400 MHz, CDCl3): δ = 2.66 (3H, s, ÀAr-Me), 2.80 (3H,
s, ÀAr-Me), 6.85 (1H, d, J = 0.4 Hz, ArH), 7.08 (1H, s),
7.44 (2H, d, J = 8.8 Hz), 8.17 (2H, d, J = 8.4 Hz), 8.67
(1H, s) ppm; 13C NMR (75 MHz, CDCl3): δ = 16.88,
24.90, 96.79, 110.98, 118.1 (2JCÀF = 23.1 Hz), 119.4
(2JCÀF = 25.2 Hz), 120.7, 124.1, 133.7 (3JCÀF = 7.8 Hz),
134.7 (3JCÀF = 9.0 Hz), 135.1, 145.6, 146.8, 147.3, 160.1,
161.3 (1JCÀF = 244.87 Hz), 163.5, 166.5 ppm; LC-MS:
414.2 (M–H); analysis calculated for C18H12BrFN4O2: C,
52.07, H, 2.91, N, 13.49%; found: C, 51.93, H, 2.99, N,
(ÀNH), 1222 (C–O–C) cmÀ1
;
1H NMR (400 MHz,
DMSO-d6): δ = 2.59 (3H, s, ArCH3), 2.71 (3H, s,
ArCH3), 4.08–4.09 (2H, d, J = 6.4 Hz, glycine-CH2),
7.12 (1H, s, ArH), 8.38 (1H, t, amide-NH), 8.50 (1H, s,
ArH), 12.67 (1H, bs, COOH) ppm; 13C NMR (100 MHz,
DMSO-d6): δ = 16.90, 24.80, 41.25, 104.19, 110.51,
145.45, 145.75, 147.46, 161.93, 161.98, 171.96 ppm;
13.65%.
4-[1-(3,4-Dimethoxy-phenyl)-meth-(E)-ylidene]-2-(5,7-
dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl)-4H-oxazol-5-one
(8c). From 5 g of compound 6 (20.14 mmol) and 3.84 g
LC-MS: 249.3 (M
+ H); analysis calculated for
C11H12N4O3: C, 53.22, H, 4.87, N, 22.57%; found: C,
53.11, H, 4.96, N, 22.73%.
of 3,4-dimethoxybenzaldehyde 7c (23.16 mmol),
compound 8c was obtained as pale yellow solid (6.2 g,
82%) after recrystallization from cold ethanol. MP:
246.8–248.4°C; TLC: Rf = 0.42 (CHCl3–MeOH 9:1); IR
(ATR, cmÀ1) υ: 3327 (=CH), 1785 (ÀC=O), 1656
(ÀC=N), 1550 (ÀNH), 1192 (C–O–C), 1161 (C–O–C)
Synthesis of substituted oxazolone derivatives 8a–8e
(general procedure).
A solution of aldehyde 7a–7e
(1.15 mmol), 5,7-dimethyl-pyrazolo[1,5-a]pyrimidine-3-
carbonyl)-amino]-acetic acid (6) (1.00 mmol), and
sodium acetate (1.00 mmol) in acetic anhydride (10 cm3)
was mixed well and heated to reflux for 4 h under argon
atmosphere. The reaction mass was cooled to room
temperature and quenched into ice cold water and then
stirred for 30 min at the same temperature. The
precipitated solid was filtered, washed with cold water,
and dried under vacuum. This crude material was further
recrystallized with cold ethanol to obtain the required
oxazolones 8a–8e.
cmÀ1 1H NMR (300 MHz, CDCl3): δ = 2.59 (3H, s,
;
ÀArMe), 2.75 (3H, s, ÀArMe), 3.85 (3H, s, ÀAr-OMe),
3.94 (3H, s, ÀAr-OMe), 7.21 (1H, s, ArH), 7.08 (2H, m,
ArH), 7.60 (1H, t, ArH), 8.42 (1H, bs), 8.76 (1H, s, ArH)
ppm; 13C NMR (75 MHz, CDCl3): δ = 17.22, 25.09,
55.88, 55.99, 97.66, 110.8, 113.64, 127.24, 129.31,
131.09, 145.62, 147.00, 149.10, 151.47, 157.83, 162.84,
167.86 ppm; LC-MS: 379.1 (M + H); analysis calculated
for C20H18N4O4: C, 63.49, H, 4.79, N, 14.81%; found:
4-[1-(4-Chloro-phenyl)-meth-(E)-ylidene]-2-(5,7-dimethyl-
C, 63.39, H, 4.90, N, 14.97%.
pyrazolo[1,5-a]pyrimidin-3-yl)-4H-oxazol-5-one (8a).
From
4-[1-(2-Chloro-phenyl)-meth-(E)-ylidene]-2-(5,7-dimethyl-
5 g of compound 6 (20.14 mmol) and 3.25 g of 4-
chlorobenzaldehyde 7a (23.16 mmol), compound 8a was
obtained as pale yellow solid (5.82 g, 82%) after
recrystallization from cold ethanol. MP: 264.9–266.1°C;
TLC: Rf = 0.36 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3298 (=CH), 17847 (ÀC=O), 1650 (ÀC=N), 1553
pyrazolo[1,5-a]pyrimidin-3-yl)-4H-oxazol-5-one (8d).
From
5 g of compound 6 (20.14 mmol) and 3.25 g of 2-
chlorobenzaldehyde 7d (23.16 mmol), compound 8d was
obtained as pale yellow solid (5.89 g, 83%) after
recrystallization from cold ethanol. MP: 248.9–250.8°C;
TLC: Rf = 0.38 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3049 (=CH), 1782 (ÀC=O), 1652 (ÀC=N), 1550 (ÀNH),
(ÀNH), 1222 (C–O–C), 985 (C–Cl) cmÀ1
;
1H NMR
1
1193 (C–O–C), 939 (C–Cl) cmÀ1; H NMR (400 MHz,
(400 MHz, CDCl3): δ = 2.77 (3H, s), 2.84 (3H, s), 6.86
(1H, s), 7.10 (1H, s), 7.44 (2H, d, J = 8.7 Hz), 8.17 (2H, d,
J = 8.7 Hz), 8.68 (1H, s) ppm; 13C NMR (100 MHz,
CDCl3): δ = 17.27, 25.35, 97.39, 111.16, 127.20, 129.13,
132.51, 133.2, 133.4, 136.48, 145.92, 147.14, 147.4,
159.39, 163.50, 167.52 ppm; LC-MS: 353.3 (M + H);
analysis calculated for C18H13ClN4O2: C, 61.28, H, 3.71,
N, 15.88%; found: C, 61.18, H, 3.80, N, 16.03%.
4-[1-(2-Bromo-5-fluorophenyl)-meth-(E)-ylidene]-2-(5,7-
dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl)-4H-oxazol-5-one
(8b). From 5 g of compound 6 (20.14 mmol) and 4.70 g of
2-bromo-5-fluorobenzaldehyde 7b (23.16 mmol),
compound 8b was obtained as pale yellow solid (6.69 g,
80%) after recrystallization from cold ethanol. MP: 248.5–
250.2°C; TLC: Rf = 0.41 (CHCl3–MeOH 9:1); IR (ATR,
CDCl3): δ = 2.76 (3H, s, ÀArMe), 2.84 (3H, s, ÀArMe),
6.86 (1H, s, ÀArH), 7.31–7.35 (1H, m, ÀArH), 7.41–
7.47 (2H, m, ÀArH), 7.66 (1H, s, ÀArH), 8.70 (1H, s,
ÀArH), 9.00 (1H, t, J = 6.4 Hz, ÀArH) ppm; 13C NMR
(100 MHz, CDCl3): δ = 17.28, 25.34, 29.73, 97.41,
111.20, 123.55, 127.17, 129.85, 131.20, 131.91, 133.17,
134.37, 136.21, 146.04, 147.16, 147.46, 160.03, 163.57,
167.39 ppm; LC-MS: 353.3 (M + H); analysis calculated
for C18H13ClN4O2: C, 61.28, H, 3.71, N, 15.88%; found:
C, 61.17, H, 3.80, N, 16.03%.
4-[1-(3-Chloro-phenyl)-meth-(E)-ylidene]-2-(5,7-dimethyl-
pyrazolo[1,5-a]pyrimidin-3-yl)-4H-oxazol-5-one (8e).
5 g of compound 6 (20.14 mmol) and 3.25 g of 3-
chlorobenzaldehyde 7e (23.16 mmol), compound 8e was
From
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
obtained as pale yellow solid (5.68 g, 80%) after
recrystallization from cold ethanol. MP: 256.3–258.1°C;
TLC: Rf = 0.37 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3073 (=CH), 1793 (ÀC=O), 1658 (ÀC=N), 1584 (ÀNH),
cmÀ1 1H NMR (300 MHz, CDCl3): δ = 1.57–1.70
;
(10H, m, cycloheptyl), 2.03–2.07 (2H, m, cycloheptyl),
4.18 (1H, bs, ÀCH-cycloheptyl), 6.08 (1H, bs, ÀNH–CO),
7.91 (1H, d, J = 7.2 Hz, ArH), 7.91 (1H, d, J = 7.2 Hz,
ArH) ppm; 13C NMR (100 MHz, DMSO-d6): δ = 14.22,
20.17, 32.01, 113.00, 122.04, 129.08, 151.87, 166.64 ppm;
1
1193 (C–O–C), 856 (C–Cl) cmÀ1; H NMR (400 MHz,
CDCl3): δ = 2.79 (3H, s, ÀArMe), 2.85 (3H, s, ÀArMe),
6.87 (1H, s, ÀArH), 7.07 (1H, s, ÀArH), 7.37–7.42 (2H, m,
ÀArH), 7.89–7.93 (1H, m, ÀArH), 8.53 (1H, s, ÀArH),
8.67 (1H, s, ÀArH) ppm; LC-MS: 353.3 (M + H); analysis
calculated for C18H13ClN4O2: C, 61.28, H, 3.71, N,
15.88%; found: C, 61.16, H, 3.79, N, 16.04%.
LC-MS: 263.2 (M
+ H); analysis calculated for
C14H18N2O3: C, 64.11, H, 6.92, N, 10.68%; found: C,
64.01, H, 7.01, N, 10.83%.
4-Nitro-N-butyl-benzamide (11c).
From 2 g of n-
butylamine 10c (17.6 mmol) and 5.58 g of 4-nitrobezoyl
chloride 9 (30.0 mmol), compound 11c was obtained as
off white solid (4.86 g. 80%) after recrystallization from
methanol. MP: 169.3–170.8°C; TLC: Rf = 0.44 (CHCl3–
MeOH 9:1); IR (ATR, cmÀ1) υ: 3300 (ÀNH), 1633
Synthesis of substituted nitro derivatives 11a–11c (general
procedure). To a solution of substituted amine 10a–10c
(1.00 mmol) in dichloromethane (10 cm3) and triethyl
amine (2.2 mmol) was added dropwise at 0°C under
nitrogen atmosphere. The resultant mixture was stirred
(ÀC=O), 1509 (ÀNH), 1342 (ÀNO2) cmÀ1
;
1H NMR
(300 MHz, CDCl3): δ = 1.01 (3H, t, J = 7.6 Hz, ÀCH3),
1.41–1.48 (2H, m, ÀCH2–CH2–), 1.60–1.67 (2H, m,
ÀCH2–CH2–), 3.5 (2H, q, J1 = 3.3, J2 = 6.9 Hz, ÀCH2),
6.17 (1H, bs, ÀNH–CO), 7.93 (1H, d, J = 8.7 Hz, ArH),
8.29 (1H, d, J = 8.7 Hz, ArH) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 13.12, 19.10, 30.50, 122.91,
128.08, 139.78, 148.32, 163.87 ppm; LC-MS: 223.1
(M + H); analysis calculated for C11H14N2O3: C, 59.45,
H, 6.35, N, 12.60%; found: C, 59.36, H, 6.46, N, 12.74%.
for
5
min followed by dropwise addition of
acid chloride (1.1 mmol) in
corresponding
dichloromethane (10 cm3) at 0°C. The reaction mixture
was slowly warmed to room temperature and stirred for
2 h under nitrogen atmosphere. The progress of the
reaction was monitored by TLC. The reaction mixture
was quenched with water (25 cm3) and extracted with
dichloromethane (2 × 20 cm3). The combined organic
layer was dried over anhydrous sodium sulfate, filtered,
and concentrated under vacuum to afford crude
compound. The crude was purified by recrystallization
from methanol (8 V) at room temperature to afford
corresponding nitro derivatives 11a–11c.
Synthesis of substituted amino derivatives 12a–12c (general
procedure).
To a solution of corresponding nitro
derivative 11a–11c (1 mmol) in 20 cm3 of methanol
(10 V) was added 10% Pd/C (10% loading) under nitrogen
atmosphere. The reaction mixture was charged to a Parr
shaker apparatus and evacuated and filled with hydrogen
gas three times at room temperature and then
hydrogenated at 3 kg/cm2 for 2 h. Completion of the
reaction was monitored by TLC. After completion of the
reaction, the reaction mixture was filtered through celite
and washed with methanol (10 V). The filtrate was
completely concentrated under high vacuum to get the
crude amine product. The crude product obtained was
further purified by stirring with diethylether (10 V) at
room temperature for 30 min. The precipitated solid was
filtered, washed with diethylether, and dried under vacuum
to afford corresponding amine derivatives 12a–12c.
4-Nitro-N-(4-trifluoromethyl-benzyl)-benzamide (11a).
From
2
g
of 3-(trifluoromethyl)benzylamine 10a
(11.4 mmol) and 2.33 g of 4-nitrobezoyl chloride 9
(12.5 mmol), compound 11a was obtained as off white
solid (3.14 g, 85%) after recrystallization from methanol.
MP: 166.2–167.8°C; TLC: Rf = 0.46 (CHCl3–MeOH 9:1);
IR (ATR, cmÀ1) υ: 3298 (ÀNH), 1631 (ÀC=O), 1507
(ÀNH), 1340 (ÀNO2) cmÀ1
;
1H NMR (400 MHz,
DMSO-d6): δ = 4.59 (2H, d, J = 6.0 Hz, ÀCH2), 7.56–
7.69 (4H, m, ArH), 8.12 (2H, dt, J1 = 2.0, J2 = 4.8 Hz,
ArH), 8.34 (2H, dt, J1 = 2.0, J2 = 4.8 Hz, ArH), 9.46 (1H,
t, J = 5.6 Hz, ÀNH–CO) ppm; 13C NMR (100 MHz,
3
DMSO-d6): δ = 42.9, 124.1 (q, JCÀF = 4 Hz), 124.3,
124.4 (q, 3JCÀF = 4 Hz), 124.7 (q, 1JCÀF = 271 Hz), 129.1,
4-Amino-N-(3-trifluoromethyl-benzyl)-benzamide (12a).
2
From
2
g
of 4-nitro-N-(3-trifluoromethyl-benzyl)-
129.7 (q, JCÀF = 32 Hz), 129.8, 131.9, 140.1, 141.1,
benzamide 11a (6.10 mmol) was hydrogenated with
0.200 g of palladium on carbon in methanol, compound
12a obtained as off white solid (1.6 g, 90%) after
recrystallization from diethylether. MP: 164.1–166.0°C;
TLC: Rf = 0.30 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3328 (ÀNH), 1648 (ÀC=O), 1534 (ÀNH), 1339 (ÀNO2)
149.5, 165.2 ppm; LC-MS: 322.7 (M À H); analysis
calculated for C15H11F3N2O3: C, 55.56, H, 3.42, N,
8.64%; found: C, 55.46, H, 3.50, N, 8.82%.
4-Nitro-N-cycloheptyl-benzamide (11b).
From 2 g of
cycloheptylamine 10b (17.6 mmol) and 3.60 g of 4-
nitrobezoyl chloride 9 (19.4 mmol), compound 11b was
obtained as off white solid (3.84 g, 83%) after
recrystallization from methanol. MP: 176.1–178.0°C;
TLC: Rf = 0.42 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3322 (ÀNH), 1627 (ÀC=O), 1516 (ÀNH), 1338 (ÀNO2)
1
cmÀ1; H NMR (400 MHz, DMSO-d6): δ = 4.49 (2H, d,
J = 6.0 Hz, ÀCH2), 5.64 (2H, bs, ÀNH2), 6.55 (2H, d,
J = 8.8 Hz, ArH), 7.53–7.59 (4H, m, ArH), 7.61 (2H, d,
J = 8.8 Hz, ArH), 8.66 (1H, t, J = 6.0 Hz, ÀNHCO)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of
α-(Benzoylamino)-β-substitutedAcrylicAmideDerivativesofPyrazolo[1,5-a]pyrimidine
ppm; 13C NMR (100 MHz, DMSO-d6): δ = 42.54, 113.0,
crude material, which was purified by column
chromatography, eluted with chloroform and methanol
mixture to afford the corresponding acrylamide
3
3
124.7 (q, JCÀF = 4 Hz), 124.0 (q, JCÀF = 4 Hz), 124.6
1
(q, JCÀF
2JCÀF = 31 Hz), 129.7, 131.8, 142.3, 152.3, 166.8 ppm;
LC-MS: 295.1 (M H); analysis calculated for
= 270 Hz), 126.1, 128.9, 129.4 (q,
derivatives 13a -13i.
5,7-Dimethyl-pyrazolo[1,5-a] pyrimidine-3-carboxylic acid
{(E)-2-(3,4-dimethoxy-phenyl)-1-[4-(3-trifluoromethyl-
benzylcarbamoyl)-phenylcarbamoyl]-vinyl}-amide (13a).
+
C15H13F3N2O: C, 61.22, H, 4.45, N, 9.52%; found: C,
61.13, H, 4.57, N, 9.66%.
From 0.250 g of compound 8c (0.66 mmol) and 0.194 g
of 4-amino-N-(4-trifluoromethyl-benzyl)-benzamide 12a
(0.66 mmol), compound 13a was obtained as off white
solid (0.35 g, 81%) after column chromatography on
silica gel column eluted with chloroform/methanol
(100:2, v/v). MP: 247.9–249.8°C; TLC: Rf = 0.35
(CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ: 3383 (ÀNH),
3304 (ÀNH), 2920 (ÀNH), 2847 (=CH), 1652 (ÀC=O),
1627 (ÀC=O), 1602 (ÀC=O), 1538 (ÀC=N), 1501
(ÀNH), 1191 (C–O–C), 1152 (C–O–C), 1116 (C–O–C)
4-Amino-N-cycloheptyl-benzamide (12b). From 2 g of 4-
nitro-N-cycloheptyl-benzamide 11b (7.60 mmol) was
hydrogenated with 0.200 g of palladium on carbon in
methanol, compound 12b obtained as off white solid
(1.5 g, 88%) after recrystallization from diethyl ether. MP:
164.4–166.2°C; TLC: Rf = 0.29 (CHCl3–MeOH 9:1); IR
(ATR, cmÀ1) υ: 3303 (ÀNH), 1604 (ÀC=O), 1503
;
(ÀNH), 1296 (ÀNO2) cmÀ1 1H NMR (400 MHz,
DMSO-d6): δ = 1.38–1.59 (10H, m, cycloheptyl), 1.60–
1.81 (2H, m, cycloheptyl), 3.88 (1H, bs, ÀCH-
cycloheptyl), 5.54 (1H, bs, ÀNH2), 6.50 (1H, d,
J = 4.2 Hz, ArH), 7.54 (1H, d, J = 4.8 Hz, ArH), 7.71 (1H,
d, J = 8.0 Hz, ÀNH–CO) ppm; 13C NMR (100 MHz,
DMSO-d6): δ = 24.47, 28.33, 34.99, 50.53, 112.89,
122.15, 129.21, 151.82, 165.57 ppm; LC-MS: 233.3
(M + H); analysis calculated for C14H20N2O: C, 72.38, H,
8.68, N, 12.06%; found: C, 72.29, H, 8.75, N, 12.23%.
1
cmÀ1; H NMR (400 MHz, DMSO-d6): δ = 2.49 (3H,
s), 2.78 (3H, s), 3.54 (3H, s), 3.77 (3H, s), 4.55 (2H, d,
J = 6 Hz), 6.84 (1H, s), 7.00 (1H, d, J = 8.4 Hz), 7.20
(1H, s), 7.30 (1H, s), 7.33 (1H, d, J = 2 Hz), 7.55–7.67
(4H, m), 7.80 (2H, d, J = 9.2 Hz), 7.87 (2H, d,
J = 8.8 Hz), 8.59 (1H, s), 9.03 (1H, t, J = 6.4 Hz), 9.95
(1H, s), 10.35 (1H, s) ppm; 13C NMR (100 MHz,
DMSO-d6): δ = 16.99, 24.57, 42.72, 55.56, 55.98,
104.01, 110.97, 112.25, 112.91, 119.33, 120.71, 123.42,
4-Amino-N-butyl-benzamide (12c).
From 2 g of 4-
nitro-N-butyl-benzamide 11c (8.90 mmol) was
hydrogenated with 0.200 g of palladium on carbon in
methanol, compound 12c obtained as off white solid
(1.5 g. 91%) after recrystallization from diethylether. MP:
159.6–161.4°C; TLC: Rf = 0.26 (CHCl3–MeOH 9:1);
IR (ATR, cmÀ1) υ: 3441 (ÀNH), 3341 (ÀNH), 1600
1
126.13, 128.84 (q, JC–F = 271 Hz), 123.03, 123.93,
3
123.96, 123.99, 124.02 (q, JC–F = 3.0 Hz), 124.21,
3
124.24, 124.27, 124.30 (q, JCÀF = 3.0 Hz), 124.51,
126.91 (d, 3JCÀF
=
22.65 Hz), 128.4 (d,
4
3JCÀF = 22.65 Hz), 128.8 (d, JCÀF = 22.65 Hz), 129.3,
4
129.5, 129.6, 129.8 (d, JCÀF = 22.65 Hz), 131.9, 141.8,
(À C = O), 1500 (ÀNH), 1350 (ÀNO2) cmÀ1
;
1H
142.7, 145.8, 146.1, 148.1, 149.0, 149.7, 160.3, 162.4,
165.0, 166.3 ppm; LC-MS: 673.1 (M + H); analysis
calculated for C35H31F3N6O5: C, 62.50, H, 4.65, N,
NMR (400 MHz, DMSO-d6):
δ = 0.90 (3H, t,
J = 7.6 Hz, ÀCH3), 1.25–1.34 (2H, m, ÀCH2–CH2–),
1.42–1.49 (2H, m, ÀCH2–CH2–), 3.19 (2H, q, J1 = 3.2,
J2 = 9.2 Hz, ÀCH2–CH2–), 5.55 (1H, bs, ÀNH2), 6.52
(2H, d, J = 8.4 Hz), 7.56 (2H, d, J = 8.0 Hz), 7.94
(1H, bs, ÀNH–CO) ppm; 13C NMR (100 MHz,
DMSO-d6): δ = 23.34, 27.23, 33.60, 50.19, 122.77,
128.20, 140.04, 148.23, 162.86 ppm; LC-MS: 193.2
(M + H); analysis calculated for C11H16N2O: C, 68.72, H,
8.39, N, 14.57%; found: C, 68.64, H, 8.48, N, 14.73%.
12.49%; found: C, 62.39, H, 4.76, N, 12.61%.
5,7-Dimethyl-pyrazolo [1,5-a]pyrimidine-3-carboxylic acid
[(E)-1-(4-butylcarbamoyl-phenylcarbamoyl)-2-(3,
4-dimethoxy-phenyl)-vinyl]-amide (13b). From 0.250 g of
compound 8c (0.66 mmol) and 0.127 g of 4-amino-N-
butyl-benzamide 12c (0.66 mmol), 13b was obtained as
off white solid (0.31 g, 83%) after column
chromatography on silica gel column eluted with
chloroform/methanol (100:2, v/v). MP: 261.3–262.9°C;
TLC: Rf = 0.38 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3242 (ÀNH), 3180 (ÀNH), 2919 (ÀNH), 2848 (=CH),
1656 (ÀC=O), 1634 (ÀC=O), 1598 (ÀC=O), 1538
(ÀC=N), 1501 (ÀNH), 1251 (C–O–C), 1188 (C–O–C),
Synthesis of acrylamide derivatives 13a–13i (general
procedure).
To a suspension of oxazolone 8a–8e
(1 mmol) in acetic acid (4 V), the corresponding amine
12a–12c (1 mmol) was added lotwise at room
temperature under nitrogen atmosphere and heated to
110°C for 6 h. The reaction mixture was poured into
water (10 V) and stirred at room temperature for 30 min.
The resulting precipitate was filtered off, washed with
water, and dried under vacuum. The aforementioned solid
was dissolved in dichloromethane and dried over
1
1145 (C–O–C) cmÀ1; H NMR (400 MHz, DMSO-d6):
δ = 0.85 (3H, t, J = 7.8 Hz, ÀCH3), 1.46–1.48 (2H, m,
ÀCH2–), 1.50–1.53 (2H, m, ÀCH2–), 2.49 (3H, s,
ÀArMe), 2.78 (3H, s, ArMe), 3.24 (2H, q, J = 6.8 Hz,
ÀCH2), 3.54 (3H, s, ÀOMe), 3.77 (3H, s, ÀOMe), 6.83
(1H, s, ArH), 7.00 (1H, d, J = 8.4 Hz, ArH), 7.20 (1H, s,
ArH), 7.30 (1H, s, ArH), 7.33 (1H, d, J = 2 Hz, ArH),
anhydrous
sodium
sulfate.
Removal
of
the
dichloromethane gave corresponding acryl amide as a
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
7.76–7.82 (4H, m, ArH), 8.32 (1H,t, J = 5.6 Hz, ÀNH–
CO), 8.59 (1H,s, ArH), 9.95 (1H,s, ÀNH–CO), 10.33
(1H,s, ÀNH–CO) ppm; 13C NMR (75 MHz, DMSO-d6):
δ = 14.12, 16.90, 20.09, 24.48, 31.75, 40.78, 55.53,
55.94, 103.97, 110.88, 112.25, 112.93, 119.20, 122.97,
124.47, 126.88, 128.12, 129.48, 129.54, 142.28, 145.72,
146.09, 148.07, 148.97, 149.66, 160.31, 162.36, 164.88,
165.98 ppm; LC-MS: 571.1 (M + H); analysis calculated
for C31H34N6O5: C, 65.25, H, 6.01, N, 14.73%; found:
C, 65.15, H, 6.11, N, 14.86%.
J = 8.0 Hz, ÀNHCO–), 8.56 (1H, s, ArH), 10.05 (1H, s,
ÀNHCO–), 10.44 (1H, s, ÀNHCO–) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 16.94, 24.47, 28.31, 29.48,
34.87, 50.86, 103.68, 110.95, 117.69, 119.30, 127.95,
128.41, 130.05, 130.28, 130.37, 130.51, 132.54, 133.47,
134.11, 142.04, 145.71, 146.13, 148.11, 160.06, 162.31,
164.18, 165.01 ppm; LC-MS: 583.3 (M À 2H); analysis
calculated for C32H33ClN6O3: C, 65.69, H, 5.68, N,
14.36%; found: C, 65.59, H, 5.77, N, 14.51%.
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
[(E)-2-(3-chloro-phenyl)-1-(4-cycloheptylcarbamoyl-
acid
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
[(E)-2-(4-chloro-phenyl)-1-(4-cycloheptylcarbamoyl-
acid
phenylcarbamoyl)-vinyl]-amide (13e).
From 0.250 g of
phenylcarbamoyl)-vinyl]-amide (13c).
From 0.250 g of
compound 8e (0.70 mmol) and 0.164 g of 4-amino-N-
cycloheptyl-benzamide 12b (0.70 mmol), compound 13e
was obtained as off white solid (0.33 g, 81%) after
column chromatography on silica gel eluted with
chloroform/methanol (100:2, v/v). MP: 259.6–261.1°C;
TLC: Rf = 0.35 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3460 (ÀNH), 3219 (ÀNH), 2921 (ÀNH), 2859 (=CH),
1689 (ÀC=O), 1641 (ÀC=O), 1589 (ÀC=O), 1561
(ÀC=N), 1529 (ÀNH), 1489 (ÀNH), 1190 (C–O–C),
compound 8a (0.70 mmol) and 0.164 g of 4-amino-N-
cycloheptyl-benzamide 12b (0.70 mmol), compound 13c
was obtained as off white solid (0.33 g, 80%) after column
chromatography
on
silica
gel
eluted
with
chloroform/methanol (100:2, v/v). MP: 258.3–260.4°C;
TLC: Rf = 0.39 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3276 (ÀNH), 3110 (ÀNH), 2919 (ÀNH), 2856 (=CH),
1668 (ÀC=O), 1625 (ÀC=O), 1591 (ÀC=O), 1550
(ÀC=N), 1525 (ÀNH), 1495 (ÀNH), 1187 (C–O–C),
;
901(C–Cl) cmÀ1 1H NMR (400 MHz, DMSO-d6):
1096 (C–Cl) cmÀ1
;
1H NMR (400 MHz, DMSO-d6):
δ = 1.44–1.65 (10H, m, cycloheptyl), 1.83–1.84 (2H, m,
cycloheptyl), 2.49 (3H, s, ÀArMe), 2.77 (3H, s, ÀArMe),
3.84–3.98 (1H,m, ÀCH, cycloheptyl), 6.78 (1H,s, ArH),
7.20 (1H, s, ArH), 7.42–7.45 (2H, m, ArH), 7.66 (1H, d,
J = 7.6 Hz, ArH), 7.75–7.83 (5H, m, ArH), 8.12 (1H, d,
J = 7.6 Hz, ÀNHCO–), 8.59 (1H,s, ArH), 10.15 (1H, s,
ÀNHCO–), 10.43 (1H, s, ÀNHCO–) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 16.97, 24.47, 24.55, 28.30,
34.87, 50.88, 103.72, 111.01, 119.15, 121.24, 128.20,
128.36, 128.61, 128.90, 129.95, 131.08, 132.85, 133.97,
136.59, 142.10, 145.73, 146.20, 148.21, 160.19, 162.54,
164.53, 165.01 ppm; LC-MS: 583.3 (M À 2H); analysis
calculated for C32H33ClN6O3: C, 65.69, H, 5.68, N,
14.36%; found: C, 65.60, H, 5.76, N, 14.52%.
δ = 1.41–1.67 (10H, m, cycloheptyl), 1.81–1.86 (2H, m,
cycloheptyl), 2.47 (3H,s, ÀArMe), 2.77 (3H,s, ÀArMe),
3.86–3.98 (1H, m, ÀCH, cycloheptyl), 6.79 (1H,s, ArH),
7.20 (1H,s, ArH), 7.47 (2H, d, J = 8.4 Hz, ArH), 7.73 (2H,
s, ArH), 7.75 (2H, s, ArH), 7.77 (2H, s, ArH), 7.82 (2H, d,
J = 8.8 Hz, ArH), 8.12 (1H, d, J = 8.0 Hz, ÀNHCO–),
8.58 (1H, s, ArH), 10.10 (1H, s, ÀNHCO–), 10.44 (1H, s,
ÀNHCO–) ppm; 13C NMR (75 MHz, DMSO-d6):
δ = 17.10, 24.60, 28.44, 35.00, 50.99, 103.94, 111.18,
119.35, 121.69, 128.52, 129.42, 130.12, 131.38, 132.37,
133.40, 133.56, 142.28, 145.88, 146.36, 148.29, 160.28,
162.63, 164.71, 165.14 ppm; LC-MS: 585.1 (M + H);
analysis calculated for C32H33ClN6O3: C, 65.69, H, 5.68,
N, 14.36%; found: C, 65.60, H, 5.77, N, 14.52%.
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
[(E)-1-(4-butylcarbamoyl-phenylcarbamoyl)-2-(2-bromo-5-
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
[(E)-2-(2-chloro-phenyl)-1-(4-cycloheptylcarbamoyl-
acid
fluorophenyl I)-vinyl]-amide (13f).
From 0.250 g of
phenylcarbamoyl)-vinyl]-amide (13d).
From 0.250 g of
compound 8b (0.60 mmol) and 0.121 g of 4-amino-N-
butyl-benzamide 12c (0.60 mmol), 13f was obtained as
off white solid (0.29 g, 80%) after column
compound 8d (0.70 mmol) and 0.164 g of 4-amino-N-
cycloheptyl-benzamide 12b (0.70 mmol), compound 13d
was obtained as off white solid (0.32 g, 78%) after
column chromatography on silica gel eluted with
chloroform/methanol (100:2, v/v). MP: 263.7–265.8°C;
TLC: Rf = 0.36 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3272 (ÀNH), 3065 (ÀNH), 2920 (ÀNH), 2854 (=CH),
1658 (ÀC=O), 1621 (ÀC=O), 1590 (ÀC=O), 1547
(ÀC=N), 1533 (ÀNH), 1503 (ÀNH), 1188 (C–O–C),
chromatography
on
silica
gel
eluted
with
chloroform/methanol (100:2, v/v). MP: 265.8–267.2°C;
TLC: Rf = 0.35 (CHCl3–MeOH 9:1); IR (ATR, cmÀ1) υ:
3246 (ÀNH), 3055 (ÀNH), 2922 (ÀNH), 2855 (=CH),
1717 (ÀC=O), 1657 (ÀC=O), 1627 (ÀC=O), 1547
(ÀC=N), 1504 (ÀNH), 1447 (ÀNH), 1248 (C–O–C),
1185 (C–F) cmÀ1 1H NMR (400 MHz, DMSO-d6):
;
1042 (C–Cl) cmÀ1
;
1H NMR (400 MHz, DMSO-d6):
δ = 0.89 (3H, t, J = 7.8 Hz, ÀCH3), 1.31–1.35 (3H, m,
ÀCH2–), 1.48–1.49 (2H, m, ÀCH2–), 1.50–1.52 (2H, m,
ÀCH2–), 2.49 (3H,s, ÀArMe), 2.75 (3H,s, ÀArMe), 3.25
(2H, q, J = 6.8 Hz, ÀCH2), 6.67 (1H, s, ArH), 7.17 (1H,
s, ArH), 7.23 (1H, t, J = 8.0 Hz, ArH), 7.60 (1H, d,
J = 9.6 Hz, ArH), 7.66–7.70 (1H, m, ArH), 7.78–7.82
δ = 1.43–1.67 (10H, m, cycloheptyl), 1.83–1.84 (2H, m,
cycloheptyl), 2.32 (3H, s, ÀArMe), 2.74 (3H, s, ÀArMe),
3.85–3.97 (1H, m, ÀCH, cycloheptyl), 6.76 (1H, s, ArH),
7.14 (1H, s, ArH), 7.34–7.39 (2H, m, ArH), 7.59 (1H, d,
J = 7.2 Hz, ArH), 7.74–7.84 (5H, m, ArH), 8.13 (1H, d,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Design, Synthesis, and Evaluation of the Anticancer Properties of a Novel Series of
α-(Benzoylamino)-β-substitutedAcrylicAmideDerivativesofPyrazolo[1,5-a]pyrimidine
(4H, m, ArH), 7.83 (2H, d, J = 6.4 Hz, ArH), 8.35 (1H, t,
J = 8.4 Hz, ÀNH–CO), 8.58 (1H, s, ArH), 10.08 (1H, s,
ÀNH–CO), 10.46 (1H,s, ÀNH–CO) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 14.22, 16.99, 20.17, 24.31,
calculated for C29H29ClN6O3: C, 63.91, H, 5.36, N,
15.42%; found: C, 63.82, H, 5.47, N, 15.55%.
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
[(E)-1-(4-butylcarbamoyl-phenylcarbamoyl)-2-(4-chloro-
acid
2
phenyl)-vinyl]-amide (13i). From 0.250 g of compound 8a
(0.70 mmol) and 0.136 g of 4-amino-N-butyl-benzamide
12c (0.70 mmol), 13i was obtained as off white solid
(0.30 g, 80%) after column chromatography on silica gel
eluted with chloroform/methanol (100:2, v/v). MP:
255.3–257.2°C; TLC: Rf = 0.34 (CHCl3–MeOH 9:1);
IR(ATR, cmÀ1) υ: 3355 (ÀNH), 3276 (ÀNH), 3115
(ÀNH), 2962 (=CH), 1671 (ÀC=O), 1632 (ÀC=O), 1593
(ÀC=O), 1552 (C=N), 1528 (ÀNH), 1499 (ÀNH), 1187
31.82, 103.66, 110.98, 117.20 (d, JCÀF = 23.8 Hz),
2
117.7 (d, JCÀF
= 22.6 Hz), 118.8, 118.99 (d,
4JCÀF = 2.7 Hz), 119.42, 128.30, 129.94, 134.60, 135.14
3
3
(d, JCÀF = 8.4 Hz), 136.22 (d, JCÀF = 8.4 Hz), 141.98,
145.76,
146.15,
148.25,
159.94,
161.66
(d,
1JCÀF = 243.3 Hz), 162.3, 163.9, 165.9 ppm; LC-MS:
609.1 (M + 2H); analysis calculated for C29H28BrFN6O3:
C, 57.34, H, 4.65, N, 13.83%; found: C, 57.24, H, 4.76,
N, 13.98%.
;
(C–O–C), 829 (C–Cl) cmÀ1 1H NMR (400 MHz,
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid
[(E)-1-(4-butylcarbamoyl-phenylcarbamoyl)-2-(2-chloro-
DMSO-d6): δ = 0.89 (3H,t, J = 7.2 Hz, ÀCH3), 1.37–
1.42 (2H, m, ÀCH2–), 1.46–1.54 (2H, m, ÀCH2–), 2.47
(3H, s, ArMe), 2.77 (3H, s, ArMe), 3.25 (2H,q,
J = 6.8 Hz, ÀCH2), 6.79 (1H, s, ArH), 7.20 (1H, s, ArH),
7.47 (2H, d, J = 8.4 Hz, ArH), 7.71 (2H, d, J = 8.4 Hz,
ArH), 7.76–7.83 (4H, m, ArH), 8.32 (1H, t, J = 5.6 Hz,
ÀNH–CO), 8.58 (1H, s, ArH), 10.10 (1H, s, ÀNH–CO),
10.43 (1H, s, ÀNH–CO) ppm; LC-MS: 545.1 (M + H);
analysis calculated for C29H29ClN6O3: C, 63.91, H, 5.36,
N, 15.42%; found: C, 63.80, H, 5.47, N, 15.54%.
phenyl)-vinyl]-amide (13g). From 0.250 g of compound 8d
(0.70 mmol) and 0.136 g of 4-amino-N-butyl-benzamide
12c (0.70 mmol), 13g was obtained as off white solid
(0.30 g, 79%) after column chromatography on silica gel
eluted with chloroform/methanol (100:2, v/v). MP: 262.9–
264.7°C; TLC: Rf = 0.36 (CHCl3–MeOH 9:1); IR (ATR,
cmÀ1) υ: 3261 (ÀNH), 3062 (ÀNH), 2920 (ÀNH), 2852
(=CH), 1658 (ÀC=O), 1624 (ÀC=O), 1608 (ÀC=O),
1547 (ÀC=N), 1505 (ÀNH), 1187 (C–O–C), 853 (C–Cl)
1
cmÀ1; H NMR (400 MHz, DMSO-d6): δ = 0.89 (3H,t,
Anticancer activity. All compounds were screened for
their in vitro anticancer activity against representative human
cancer cell line (HeLa cell line) by MTT assay. This is
a colorimetric assay that measures the reduction of
yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) by mitochondrial succinate dehydrogenase.
The MTT enters the cells and passes into the mitochondria
where it is reduced to an insoluble, colored (dark purple)
formazan product. The cells are then solubilized with an
organic solvent (e.g., dimethylsulfoxide and isopropanol)
and then the released formazan reagent is measured
spectrophotometrically. Because reduction of MTT can only
occur in metabolically active cells, the level of activity is a
measure of the viability of these cells.
The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) was made a solution in such a way that
10 mg was dissolved in 10 mL of Hank’s balanced
solution. The cell lines were maintained in 96 wells
microtiter plate containing MEM media supplemented with
10% heat inactivated fetal calf serum, containing 5% of
mixture of gentamicin, penicillin (100 units/mL), and
streptomycin (100 μg/mL) in presence of 5% CO2 at 37°C
for 3–4 days. After which, the obtained supernatant was
removed and replace MEM media with Hank’s balanced
solution, and the cells were incubated overnight. The
in vitro growth inhibitions of test compounds were assessed
by calorimetric or spectrophotometric method. This helps to
determine the conversion of MTT into formazan blue by
living cells. Remove the supernatant from the plate, add
fresh Hank’s balanced salt solution, and treated with
J = 7.2 Hz, ÀCH3), 1.31–1.35 (2H, m, ÀCH2–), 1.48–
1.49 (2H, m, ÀCH2–), 1.50–1.52 (2H, m, ÀCH2–), 2.49
(3H, s, ÀArMe), 2.74 (3H, s, ÀArMe), 3.25 (2H,q,
J = 6.8 Hz, ÀCH2), 6.75 (1H,s, ArH), 7.14 (1H, s, ArH),
7.34–7.59 (2H, m, ArH), 7.58 (1H, d, J = 4.8 Hz, ArH),
7.60–7.84 (5H, m, ArH), 8.34 (1H, t, J = 8.4 Hz, ÀNH–
CO), 8.57 (1H, s, ArH), 10.06 (1H,s, ÀNH–CO), 10.46
(1H,s, ÀNH–CO) ppm; LC-MS: 545.1 (M + H); analysis
calculated for C29H29ClN6O3: C, 63.91, H, 5.36, N,
15.42%; found: C, 63.80, H, 5.47, N, 15.54%.
5,7-Dimethyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic
[(E)-1-(4-butylcarbamoyl-phenylcarbamoyl)-2-(3-chloro-
acid
phenyl)-vinyl]-amide (13h). From 0.250 g of compound 8e
(0.70 mmol) and 0.136 g of 4-amino-N-butyl-benzamide
12c (0.70 mmol), compound 13h was obtained as off
white solid (0.31 g, 82%) after column chromatography on
silica gel eluted with chloroform/methanol (100:2, v/v).
MP: 258.8–260.2°C; TLC: Rf = 0.33 (CHCl3–MeOH 9:1);
IR (ATR, cmÀ1) υ: 3233 (ÀNH), 3103 (ÀNH), 2957
(ÀNH), 2926 (ÀNH), 2866 (=CH), 1690 (ÀC=O), 1639
(ÀC=O), 1597 (ÀC=O), 1557 (ÀC=N), 1530 (ÀNH),
1499 (ÀNH), 1187 (C–O–C), 901 (C–Cl) cmÀ1; 1H NMR
(400 MHz, DMSO-d6): δ = 0.90 (3H,t, J = 7.2 Hz, ÀCH3),
1.30–1.35 (2H, m, ÀCH2–), 1.46–1.52 (2H, m, ÀCH2–),
2.48 (3H, s, ÀArMe), 2.77 (3H, s, ÀArMe), 3.25 (2H,q,
J = 6.8 Hz, ÀCH), 6.78 (1H,s, ArH), 7.20 (1H,s, ArH),
7.40–7.47 (2H, m, ArH), 7.71 (1H, d, J = 7.6 Hz, ArH),
7.76–7.83 (5H, m, ArH), 8.32 (1H, t, J = 7.2 Hz), 8.59
(1H, s, ArH), 10.16 (1H, s, ÀNH–CO), 10.44 (1H, s,
ÀNH–CO) ppm; LC-MS: 545.1 (M + H); analysis
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Sasikumar, V. Mohanasrinivasan, A. K. Ajeesh Kumar, and D. Krishnaswamy
Vol 000
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different concentrations of compound (approximately diluted
with DMSO). The marketed anticancer drug paclitaxel was
tested as a reference compound in the assay. The control
group contains only DMSO. After 24 h of incubation at
37°C in a humidified atmosphere of 5% CO2, the medium
was replaced with MTT solution (100 μL, 5 mg/mL in
MEM medium) for further 4 h. The supernatant was
carefully aspirated, and the precipitated crystals of
formazan blue were solubilized by adding DMSO
(200 μL), and optical density was measured at wavelength
of 570 nm using LISA microplate reader. The results were
represented out in triplicates for each concentration.
Concentration at which the optical density (O.D) of treated
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2241.
Acknowledgments. We are thankful to the management, Anthem
Biosciences, Bangalore, India, for their invaluable support and al-
location of resources for this work. We would like to thank analyt-
ical chemistry team, Department of Analytical Chemistry,
Anthem Biosciences, Bangalore, India, for carrying out all the an-
alytical work. Also, we would like to thank molecular biology
team, Department of Molecular Biology, Maratha Mandal’s
NGH Institute of Dental Sciences and Research Centre, Belagavi,
Karnataka, for helping to carry out the biological studies of the
compounds reported.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet