Design, synthesis, and evaluation of the anticancer properties of a novel series of carboxamides, sulfonamides, ureas, and thioureas derived from 1,2,4-oxadiazol-3-ylmethyl-piperazin-1-yl substituted with pyrazolo[1,5-a]pyrimidine derivatives

Article abstract of DOI:10.1007/s00706-016-1723-9

Abstract: A series of novel carboxamides, sulfonamides, ureas, and thioureas derived from 1,2,4-oxadiazol-3-ylmethyl-piperazin-1-yl substituted with pyrazolo[1,5-a]pyrimidine analog were designed and synthesized. The newly synthesized compounds were characterized by ¹H NMR, ¹³C NMR, ESI–MS, and IR and were tested for their in vitro antiproliferative activity by MTT assay. Out of these twenty derivatives, five compounds showed good anticancer activity against HeLa cell line. These are superior with less than 10?μg/cm³ of IC₅₀ when compared to the marketed anticancer drug paclitaxel with 30?μg/cm³ of IC₅₀ against Hela cell line. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1723-9

Monatsh Chem
DOI 10.1007/s00706-016-1723-9
ORIGINAL PAPER
Design, synthesis, and evaluation of the anticancer properties
of a novel series of carboxamides, sulfonamides, ureas,
and thioureas derived from 1,2,4-oxadiazol-3-ylmethyl-piperazin-
1
-yl substituted with pyrazolo[1,5-a]pyrimidine derivatives
1
1
2
1
3
A. K. Ajeesh Kumar
•
Kanya B. Nair
•
Yadav D. Bodke
•
Ganesh Sambasivam
•
Kishore G. Bhat
Received: 22 December 2015 / Accepted: 28 February 2016
Ó Springer-Verlag Wien 2016
Abstract A series of novel carboxamides, sulfonamides,
ureas, and thioureas derived from 1,2,4-oxadiazol-3-yl-
methyl-piperazin-1-yl substituted with pyrazolo[1,5-
a]pyrimidine analog were designed and synthesized. The
Graphical abstract
1
newly synthesized compounds were characterized by H
1
3
NMR, C NMR, ESI–MS, and IR and were tested for their
in vitro antiproliferative activity by MTT assay. Out of
these twenty derivatives, five compounds showed good
anticancer activity against HeLa cell line. These are
3
superior with less than 10 lg/cm of IC50 when compared
3
to the marketed anticancer drug paclitaxel with 30 lg/cm
of IC50 against Hela cell line.
Keywords Carboxamide Á Sulfonamide Á Urea Á
Thiourea Á Pyrazolo[1,5-a]pyrimidine Á 1,2,4-Oxadiazole Á
Piperazine Á Anticancer activity
Introduction
Pyrazole represents one of the most active classes of hete-
rocyclic compounds, since many of them possesses a wide
range spectrum of biological activities like potential inhi-
bitors of HIV-1 [1], analgesic drugs [2, 3], antihypertensive
agents [4], anticancer activity [5, 6]. Also, compounds
containing substituted pyrimidine derivatives are of sig-
nificant biological importance and used as antibacterial [7],
antifungal [8], antitumor [9], antiviral [10], anti-inflamma-
tory [11], and antihypertensive [12] agents.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1723-9) contains supplementary
material, which is available to authorized users.
&
A. K. Ajeesh Kumar
ajuedat@gmail.com
1
2
Anthem Biosciences Pvt. Ltd., # 49, Bommasandra Industrial
Area, Bommasandra, Bangalore 560 099, Karnataka, India
In general, pyrazole derivatives are utilized for the
synthesis of other fused heterocyclic systems. Among
these, pyrazolopyrimidines which mimic structurally with
biogenic purines [13, 14] have considerable chemical and
pharmacological importance and are bioisosters to tria-
zolothienopyrimidines [15], imidazoquinazolines [16], and
pyrimidoquinazolines [17]. Many analogs of pyrazolo[1,5-
Department of PG Studies and Research in Industrial
Chemistry, School of Chemical Sciences, Kuvempu
University, Jnana Sahyadri, Shankaraghatta, Shimoga
5
77 451, Karnataka, India
3
Department of Microbiology, Maratha Mandal’s NGH
Institute of Dental Sciences and Research Centre, Belgaum
5
90 010, Karnataka, India
123

A. K. Ajeesh Kumar et al.
a]pyrimidine are associated with diverse pharmacological
activities [18–22] like tuberculostatic [23], antimicrobial
activities [24], neuroleptic [25], CNS depressant [26], and
antihypertensive [27]. Moreover, pyrazolopyrimidines
have useful properties as antimetabolite in purine bio-
chemical reactions.
Derivatives of 1,2,4-oxadiazole constitute an important
family of heterocyclic compounds [28]. Since many of them
exhibit a remarkable biological activity [29, 30] and find
wide usage as dyes, photosensitive electrical materials,
polymer precursors, and stabilizers, the synthesis and
transformations have received great attention for a long time.
In particular, 2-aryl-5-substituted 1,2,4-oxadiazoles
have been reported to show antibacterial [31], antifungal
Fig. 1 Chemical structure of compound 12l which showed good
antiproliferative activity
ylmethyl-piperazin-1-yl substituted with pyrazolo[1,5-a]-
pyrimidine derivative involves four consecutive schemes.
First part involves the synthesis of 5,7-dimethylpyra-
zolo[1,5-a]pyrimidine-3-carboxylic acid as shown in
Scheme 1. This involves the synthesis of ethyl 5-amino-
1H-pyrazole-4-carboxylate (2) as the initial counterpart
and in general, it was carried out by two steps protocol [55,
56]. But we have carried out the synthesis by a single pot
reaction from the commercially available ethyl cyanoac-
etate (1), dimethylformamide dimethylacetal, and
hydrazine hydrate in acetic acid medium under mild
heating with dimethylformamide as solvent. The ethyl
5-amino-1H-pyrazole-4-carboxylate (2) was converted
to the ethyl 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-car-
boxylate (3) with acetyl acetone in acetic acid medium at
82 °C in good yield [57]. After having synthesized the
known compound 3, the 5,7-dimethylpyrazolo[1,5-a]-
pyrimidine-3-carboxylic acid (4) was synthesized by
hydrolyzing the ester derivative 3 with sodium hydroxide
in ethanol and water medium under reflux condition.
[
32], analgesic, anti-inflammatory [33], and hypoglycemic
activities. Also 1,2,4-oxadiazoles are well-known com-
pounds with promising physiological activities [34, 35].
1
,2,4-Oxadiazole rings occur widely in biologically active
synthetic compounds and are often used in drug discovery
as good bioisosters of amides and esters [28, 36, 37].
Furthermore, they have been reported to have agonist for
cortical muscarinic receptors [38–41], benzodiazepine [42,
4
3], 5-HT1D (5-hydroxytryptamine) receptors [29], and as
antagonists for 5-HT [30] or histamine H receptors [44].
3
3
They showed activity against several breast and colorectal
cancer cell lines [45–47].
A number of piperazine derivatives have been shown to
possess a variety of pharmacological properties like anti-
histamanic [48], analgesic [49], antiinflammatory [50],
anti-HIV [51], antimalarial [52], antitubercular [53], and
antimicrobial activity [54]. Hence, piperazine is found to
be an important structural feature in some synthetic drugs
such as prazosin, lidoflazine, and urapidil.
0
Second part involves the synthesis of tert-butyl 4-(N -
Prompted by these observations and as a continuation of
our ongoing research program in the synthesis of biologi-
cally active molecules, we envisaged the synthesis of novel
molecules based on the three lead pharmacophores realized
viz., pyrazolo[1,5-a]pyrimidine, 1,2,4-oxadiazole, and
piperazine. In this communication, we disclose the syn-
thesis of a novel series of carboxamides, sulfonamides,
ureas, and thioureas derived from 1,2,4-oxadiazol-3-
ylmethyl-piperazin-1-ylsubstituted with pyrazolo[1,5-a]-
pyrimidine analog with representative example, i.e., com-
pound 12l as shown in Fig. 1, showing good growth
inhibition against HeLa cell line.
hydroxycarbamimidoyl)piperazine-1-carboxylate (8) in
three steps as shown in Scheme 2. The piperazine-1-car-
boxylic acid tert-butyl ester (6) was prepared by heating
piperazine (5) with BOC-anhydride in methanol at 50 °C
for 2 h [58]. The tert-butyl 4-(cyanomethyl)piperazine-1-
carboxylate (7) was synthesized by reaction with
piperazine-1-carboxylic acid tert-butyl ester and
chloroacetonitrile with potassium carbonate in acetonitrile
at 60 °C for 5 h. After having synthesized the nitrile
0
derivative, tert-butyl 4-(N -hydroxycarbamimidoyl)piper-
azine-1-carboxylate (8) was prepared by reacting tert-butyl
4-(cyanomethyl)piperazine-1-carboxylate (7) with hydrox-
ylamine hydrochloride in the presence of sodium carbonate
at reflux temperature with methanol for 5 h.
Results and discussion
Chemistry
The third part involves the synthesis of scaffold,
5
,7-dimethyl-3-(3-piperazin-1-ylmethyl-1,2,4-oxadiazol-5-
yl)pyrazolo[1,5-a]pyrimidine (11) as shown in Scheme 3.
This involves the synthesis of 4-[2-(5,7-dimethylpyra-
zolo[1,5-a]pyrimidine-3-carboxamido)-2-(hydroxyimino)-
ethyl]piperazine-1-carboxylic acid tert-butyl ester (9)
Synthesis of a series of novel carboxamides, sulfonamides,
ureas, and thioureas derived from 1,2,4-oxadiazol-3-
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
Scheme 1
Scheme 2
Scheme 3
derived from 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-
0
corresponding free amine, 5,7-dimethyl-3-(3-piperazin-1-
carboxylic acid (4) and tert-butyl 4-(N -hydr oxycar-
ylmethyl-1,2,4-oxadiazol-5-yl)pyrazolo[1,5-a]pyrimidine (11)
by deprotecting BOC group of piperazine ring from the
cyclized 1,2,4-oxadiazole moiety. This step was carried out
using trifluoroacetic acid as the reagent in dichloromethane
solvent at room temperature. The trifluoroacetate salt
obtained was made basic with saturated potassium
carbonate solution and extracted with dichlor-
omethane:methanol (100:5). The crude product obtained
after evaporation was recrystallised from ethyl
acetate:hexane (1:8) medium at room temperature to get
the scaffold as off white solid.
bamimidoyl)piperazine-1-carboxylate (8) using EDCI•HCl
and HOBt with N-methylmorpholine in dimethylfor-
mamide solvent at room temperature for 6 h. Cyclization
of this amide derivative was initially tried by heating with
0
N,N -dicyclohexylcarbodiimide (DCC) in ethanol at 80 °C.
But the isolation involved the purification by column
chromatography due to the formation of dicyclohexylurea
(
DCU) from the coupling agent owing to lower yields. We
then employed 1,8-diazabicycloundec-7-ene (DBU) as the
dehydrating agent. So, the amide intermediate was further
cyclised to the corresponding 1,2,4-oxadiazole, 4-[5-(5,7-
dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-3-
yl)methyl]piperazine-1-carboxylic acid tert-butyl ester (10)
by heating at 90 °C with DBU in N,N-dimethylformamide
The fourth part involves the synthesis of target mole-
cules 12a–12t as shown in Scheme 4. The free amine
obtained after BOC cleavage was derivatized into four
series of final target molecules. First series involves the
synthesis of urea derivatives 12a–12e obtained by treating
amine and the corresponding isocyanates in THF medium
(
DMF) solvent for 2 h. In order to synthesize novel com-
pounds from above intermediate, we have prepared the
123

A. K. Ajeesh Kumar et al.
Scheme 4
1 13
Compound 10 was elucidated by its IR, H and C NMR,
at room temperature. The second series involves the syn-
thesis of thiourea derivatives 12f–12j obtained by reacting
amine and the corresponding isothiocyanates in THF
medium at room temperature. The third series involves the
synthesis of carboxamide derivatives 12k–12o obtained by
the reacting amine and corresponding acid chlorides with
N-methylmorpholine as base in dimethylacetamide as sol-
vent at room temperature. The fourth series involves the
synthesis of sulfonamide derivatives 12p–12t obtained by
reacting amine and the corresponding sulfonyl chlorides
with N-methylmorpholine as base in dimethylacetamide as
solvent at room temperature.
1
and LC–MS analyses. The H NMR of compound 10 did
not show the peak for –NH group of carboximidamide,
2
1
3
thereby confirming the oxadiazole ring formation. The
C
NMR showed a signal at 170.87 ppm confirming the
presence of oxadiazole ring in the molecule. Compound 11
1
was elucidated by its IR, H and C NMR, and LC–MS
13
1
analyses. The H NMR of free amine scaffold did not show
the peak for BOC group of piperazine, thereby confirming
the free piperazine ring in the molecule. The IR spectrum
-
1
showed peak at 3327 cm which attributed to the –N–H
stretch vibration of the amine from the piperazine ring.
The results of all the newly synthesized compounds
The structures for all the above compounds were con-
firmed by spectral studies. The structure of the amide
compound, 4-[2-(5,7-dimethylpyrazolo[1,5-a]pyrimidine-
1
13
12a–12t were confirmed by H NMR, C NMR, LC–MS,
and IR analyses and given in the ‘‘Experimental’’ sec-
tion. The structures of urea derivatives 12a–12e were
confirmed by the following spectral studies. In general, the
FT-IR spectrum of 12a contained a broad band at
3
-carboxamido)-2-(hydroxyimino)ethyl]piperazine-1-car-
boxylic acid tert-butyl ester (9) was elucidated by its IR,
1
13
H and C NMR, and LC–MS analyses. The H NMR
1
-
1
spectrum of 9 showed singlet at d = 10.69 ppm, for one
proton which corresponds to the amide NH proton.
1631 cm which was attributed to the carbonyl stretching
1
vibration of the corresponding urea derivative. The H
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
NMR spectrum of 12a contained a broad singlet with a
chemical shift of 6.37 ppm which was consistent with the
Anticancer evaluation
1
3
proton of the –NH from the urea link. C NMR spectrum
of 12a contained a signal at 158.98 ppm which was con-
sistent with the carbonyl group from the urea link. The
structure of compound 12a was further confirmed by mass
spectrometry which gave a molecular ion peak with an m/z
value of 433.4 for [M?H] which was consistent the
molecular formula C H N O . The structures of thiourea
All compounds were screened for their in vitro anti-cancer
activity against representative human cervical cancer cell
line called HeLa. Paclitaxel was used as a reference stan-
dard. The data generated from this study Table 1 showed
that some of the target compounds exhibit good potency in
inhibiting the growth of HeLa cell line. Compounds 12b,
12e, 12k, 12l, 12m, and 12r were showed good anti pro-
liferative activity against HeLa cell line. Interestingly,
among these compounds, the in vitro anticancer activity of
compounds 12b, 12e, 12k, 12l, and 12r are superior to the
marketed anti-cancer drug paclitaxel. However, some of
the synthesized compounds are less potent when compared
to paclitaxel.
2
2 24 8 2
derivatives 12f–12j were confirmed by the following
spectral studies. In general, the FT-IR spectrum of 12i
-
1
contained a broad band at 1629 cm which was attributed
to the C=S stretching vibration of the corresponding
1
thiourea derivative. The H NMR spectrum of 12i con-
tained a broad singlet with a chemical shift of 6.72 ppm
which was consistent with the proton of the –NH from the
The anticancer activity of these novel compounds 12a–
12t suggested that introduction of the urea derivatives,
carboxamides, and sulfonamides were increased the
antiproliferative activity when compared to the thiourea
derivatives. In addition to these observations, we have
concluded that the optimum anticancer activity observed
for compounds containing one or more halogen
derivatives.
1
3
thiourea link. The C NMR spectrum of 12i contained a
signal at 181.63 ppm which was consistent with the thio-
carbonyl group from the thiourea link. The structure of
compound 12i was further confirmed by mass spectrometry
which gave a molecular ion peak with an m/z value of
4
29.4 for [M?H] which was consistent the molecular
formula C H N OS.
20 28 8
The structures of carboxamide derivatives 12k–12o
were confirmed by the following spectral studies. In gen-
eral, the FT-IR spectrum of 12o contained a broad band at
Conclusion
-
1
1
654 cm which was attributed to the carbonyl stretching
vibration of the corresponding carboxamide derivative. The
1
A series of novel carboxamides, sulfonamides, ureas, and
thioureas derived from pyrazolo[1,5-a]pyrimidine scaffold
were designed and successfully synthesized. The synthesis
involves the preparation of both 5,7-dimethylpyrazolo[1,5-
a]pyrimidine-3-carboxylic acid and 4-(N-hydroxycar-
bamimidoylmethyl)piperazine-1-carboxylic acid tert-butyl
ester. The coupling reaction between both leads to get the
corresponding amide 4-{2-[(5,7-dimethyl-1,3-dihydropy-
razolo[1,5-a]pyrimidine-3-carbonyl)amino}-2-(hydroxy-
imino)ethyl]piperazine-1-carboxylic acid tert-butyl ester.
This amide was further cyclized to the corresponding
heterocyclic 4-[5-(5,7-dimethylpyrazolo[1,5-a]pyrimidin-
3-yl)-1,2,4-oxadiazol-3-ylmethyl]piperazine-1-carboxylic
acid tert-butyl ester with DBU in DMF heating condition.
The BOC deprotection was carried out with trifluoroacetic
acid in dichloromethane medium to get the scaffold 5,7-
dimethyl-3-(3-piperazin-1-ylmethyl-1,2,4-oxadiazol-5-yl)-
pyrazolo[1,5-a]pyrimidine. The synthesized compounds
H NMR spectrum of 12o contained a sharp singlet with a
chemical shift of 7.78 ppm which was consistent with the
protons of the 2,4,6-trichlorobenzene ring from the car-
1
3
boxamide link. The C NMR spectrum of 12o contained a
signal at 162.20 ppm which was consistent with the car-
bonyl group from the carboxamide group. The structure of
compound 12o was further confirmed by mass spectrom-
etry which gave a molecular ion peak with an m/z value of
5
22.1 for [M?H], which was consistent the molecular
formula C H Cl N O . Finally, the structures of sulfon-
2
2
20
3 7 2
amide derivatives 12p–12t were confirmed by the
following spectral studies. In general, the FT-IR spectrum
-
of 12p contained a broad band at 1346 cm , which was
1
attributed to the sulfonyl antisymmetric stretching vibra-
1
3
tion of the corresponding sulfonamide derivative. The
NMR spectrum of 12p contained two signals at 155.88 and
59.22 ppm with a coupling constant of J = 250.50 Hz
C
1
1
13
which were consistent with the fluorine group from the
sulfonamide derivative. The structure of compound 12p
was further confirmed by mass spectrometry which gave a
molecular ion peak with an m/z value of 506.4 for [M?H]
which was consistent the molecular formula C H
were characterized by H NMR, C NMR, ESI–MS, and
IR. The new compounds were tested for their in vitro
antiproliferative activity by MTT assay. Out of these
twenty derivatives, compounds 12b, 12e, 12k, 12l, and 12r
showed good anticancer activity against HeLa cell line.
2
1 21-
3
ClFN O S. The spectral data generated in the current study
7
These are superior with less than 10 lg/cm of IC50 when
compared to the marketed anticancer drug paclitaxel with
3
were in good agreement with the assigned structures of all
the novel molecules synthesized in this series.
3
30 lg/cm of IC50 against Hela cell line.
123

A. K. Ajeesh Kumar et al.
Table 1 Anticancer activity of compounds 12a–12t
Compound Concentration/ OD % of cell lysis IC50
Table 1 continued
Compound Concentration/ OD
lG
% of cell lysis IC50
at 492 nm as observed
lG
at 492 nm as observed
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10
0.632
0.510
0.495
0.680
0.646
0.630
0.690
0.682
0.640
0.362
0.340
0.311
0.802
0.790
0.786
0.540
0.486
0.465
0.281
0.262
0.211
0.721
0.740
0.789
0.521
0.428
0.410
0.781
0.721
0.681
0.516
0.415
0.411
0.562
0.428
0.416
0.281
0.224
0.211
0.320
0.316
0.312
0.408
0.388
0.341
No lysis
25
30 lg
12n
12o
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
10
0.712
0.682
0.620
0.745
0.660
0.412
0.490
0.432
0.420
0.632
0.510
0.496
0.281
0.261
0.200
0.802
0.790
0.786
0.782
0.626
0.424
0.862
0.512
0.484
0.401
No lysis
25
[30 lg
30 lg
2
3
0
0
50
50
2
10
No lysis
25
30 lg
No lysis
25
2
3
0
0
50
50
2a
2b
2c
2d
2e
2f
2g
2h
2i
10
25
[30 lg
[10 lg
No activity
30 lg
12p
12q
12r
50
20 lg
2
3
0
0
25
50
50
[50
No lysis
25
10
50
30 lg
2
3
0
0
75
100
50
10
No lysis
No lysis
No lysis
25
75
\10 lg
No activity
30 lg
2
3
0
0
[75
100
10
12s
No lysis
No lysis
No lysis
No lysis
25
2
3
0
0
[25
50
10
100
\10 lg
[30 lg
20 lg
12t
2
3
0
0
100
100
50
10
No lysis
25
Control
No lysis
No lysis
25
2
3
0
0
Paclitaxel 10
30 lg
[25
25
20
30
10
50
2
3
0
0
50
[50
No lysis
No lysis
No lysis
25
Experimental
10
No activity
20 lg
2
3
0
0
Chemicals were obtained from Sigma-Aldrich Co. Final
purifications were carried out using Merck silica gel
10
60–120 mesh size. TLC experiments were performed on
2
3
0
0
50
alumina-backed silica gel 40 F254 plates (Merck, Darm-
stadt, Germany). The plates were illuminated under UV
[50
[25
50
2j
2k
2l
10
20 lg
(
254 nm) and KMnO . Melting points were determined
4
1 13
using B u¨ chi B-540. All H and C NMR spectra were
2
3
0
0
50
recorded on Bruker AM-300 and Bruker AM-400 (300 and
13
1
400 MHz for H NMR and 75 and 100 MHz for
10
75
\10 lg
\10 lg
10 lg
C
2
3
0
0
100
NMR), Bruker BioSpin Corp., Germany. Molecular
weights of unknown compounds were checked by LC MS
6200 series Agilent Technology. Chemical shifts are
reported in ppm (d) with reference to internal standard
TMS. The signals are designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; brs, broad singlet. IR for all
the synthesized compounds was recorded using a Bruker
Alpha FT-IR spectrometer using a diamond ATR single
reflectance module (24 scans).
100
10
75
2
3
0
0
[75
[75
50
2m
10
2
3
0
0
75
[75
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
5
-Amino-1H-pyrazole-4-carboxylic acid ethyl ester
reduced pressure to remove methanol. The crude material
obtained was purified by column chromatography on silica
gel eluted with chloroform/methanol (100:4, v/v) to afford
compound 6 as off white solid (35.6 g, 55 %). M.p.:
(
2, C H N O )
6
9 3 2
To a solution of 25 g of ethyl cyanoacetate (1, 0.221 mol)
3
3
in 25 cm dimethylformamide was added 37.5 cm glacial
acetic acid (0.077 mol) followed by 47.40 g of N,N-
dimethylformamide dimethylacetal (0.397 mol) drop wise
at room temperature and stirred for 1 h. To the resultant
1
45.5–46.8 °C; TLC: R = 0.35 (CHCl –MeOH 9:1); H
f
3
NMR (300 MHz, CDCl ): d = 1.38 (9H, s, CH ),
3
3
2.29–2.39 (1H, m, NH), 2.60 (4H, t, J = 4.95 Hz,
13
3
pale yellow color solution was added 25 cm hydrazine
N(CH ) , 3.20 (t, 4H, J = 5.4 Hz) ppm;
C NMR
2
2
hydrate (0.42 mol) drop wise at 0 °C and the reaction
medium was stirred for 2 h 50 °C. After completion of the
reaction, the reaction medium was diluted with 2.5 dm of
(100 MHz, DMSO-d ): d = 28.5, 45.9 (two peaks), 78.9,
6
?
154.4 ppm; MS: m/z = 187.3 [M?H] .
3
4-(Cyanomethyl)piperazine-2-carboxylic acid tert-butyl
3
water and extracted with 2 9 700 cm of ethyl acetate. The
ester (7)
This compound was prepared following the protocol
mentioned in the literature [60] and obtained as an off
combined organic layer was dried over sodium sulfate,
filtered, and concentrated under reduced pressure to obtain
the crude product as pale brown liquid. The crude product
was further purified by column chromatography over silica
gel, eluted with chloroform/methanol (95:5, v/v) to afford
the compound 2 as a white solid (29 g, 85 %). M.p.: 106.7–
1
white solid (32 g, 88 %). M.p.: 91.5–93.7 °C; H NMR
(
300 MHz, DMSO-d ): d = 1.40 (9H, s, CH ), 2.41 [4H, t,
6
3
J = 5.1 Hz, N(CH ) ], 3.35 [t, 4H, J = 5.4 Hz, N(CH ) ],
2
2
2 2
3.76 (2H, s, CH ) ppm; and values were in correlation with
2
1
08.9 °C; TLC: R = 0.22 (CHCl –MeOH 8:2); IR (ATR):
f
3
reported literature.
vꢀ = 3193 (–NH), 2972 (=C–H), 1662 (ester –C=O), 1551
-
1 1
–C–C), 1496 (ester –C–O), 1337 (C–N) cm ; H NMR
(
(
(
(
(
4-(N-Hydroxycarbamimidoylmethyl)piperazine-1-car-
boxylic acid tert-butyl ester (8)
400 MHz, CDCl ): d = 1.23 (3H, t, J = 8 Hz, CH ), 4.15
2H, q, J = 8 Hz, OCH CH ), 5.98 (2H, bs, ArNH ), 7.45
3
1
1H, bs, ArH), 12.04 (1H, bs, pyrazole NH) ppm; C NMR
3
3
This compound was prepared following the protocol
mentioned in the literature [61] and obtained as off white
2
2
3
1
100 MHz, DMSO-d ): d = 14.9, 59.1, 94.0, 139.9, 151.8,
solid (20 g, 70 %). M.p.: 236.6–238.6 °C; H NMR
6
?
64.2 ppm; LC–MS: m/z = 156.7 [M?H] .
1
(300 MHz, DMSO-d ): d = 1.39 (9H, s, CH ), 2.30 [4H,
6
3
t, J = 4.8 Hz, N(CH ) ], 2.83 (2H, s, CH ), 3.38 [4H, t,
2
2
2
Ethyl 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxy-
late (3)
This compound was prepared as per the reported literature
J = 8.1 Hz, N(CH ) ], 5.24 (2H, s, NH ), 8.89 (1H, s, OH)
2
2
2
ppm and values were in correlation with reported literature.
[
59] and obtained as a pale yellow solid (34 g, 98 %).
1
4-[2-(5,7-Dimethylpyrazolo[1,5-a]pyrimidine-3-carboxam-
ido)-2-(hydroxyimino)ethyl]piperazine-1-carboxylic acid
tert-butyl ester (9, C H N O )
M.p.: 102.5–105.2 °C; H NMR (400 MHz, CDCl3):
d = 1.30 (3H, t, J = 8 Hz, CH ), 2.58 (3H, s, ArCH ),
3
3
20 29 7 4
2
.71 (3H, s, ArCH ), 4.27 (2H, q, J = 8 Hz, OCH CH ),
3
To a solution of 13 g of compound 4 (0.067 mol) and
2
3
3
18.44 g of compound 8 (0.071 mol) in 65 cm of
dimethylformamide was added 17.98 g of 1-(3-dimethy-
7
.12 (1H, s, ArH), 8.53 (1H, s, ArH) ppm.
5
,7-Dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid
laminopropyl)-3-ethylcarbodiimide
0.094 mol) followed by 14.39 g of 1-hydroxybenzotria-
zole (0.094 mol) and 4 g of N-methylmorpholine
0.033 mol) at room temperature under nitrogen atmo-
hydrochloride
(
4)
This compound was prepared as per the reported literature
57, 59] and obtained as off white solid (25 g, 96 %). M.p.:
(
[
(
1
1
76.8–179.9 °C; H NMR (400 MHz, CDCl ): d = 2.56
3
sphere. The resultant reaction mixture was stirred for 6 h at
room temperature. After completion of the reaction,
reaction mixture was quenched with drop-wise addition
(
3H, s, ArCH ), 2.70 (3H, s, ArCH ), 7.09 (1H, s, ArH),
3 3
8
.49 (1H, s, ArH) ppm, 12.24(1H, bs, acid) which is
consistent with literature values.
3
of 650 cm of ice-cold water and stirred for further 30 min
Piperazine-1-carboxylic acid tert-butyl ester
at room temperature. The solid precipitated out was
3
filtered, washed with 100 cm of cold water and dried
(
6, C H N O )
18 2 2
9
To a solution of 30 g of piperazine (5, 0.348 mol) in
1
under vacuum at 50 °C for 1 h to afford compound 9 as an
off white solid (22 g, 75 %). M.p.: 164.3–167.1 °C; TLC:
R = 0.28 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3490
3
20 cm of methanol was added a solution of 83.54 g di-
3
tert-butyl carbonate (0.382 mol) in 90 cm of methanol
drop wise over a period of 45–60 min at 0 °C under
nitrogen atmosphere. The reaction mixture was heated to
f
3
(amide –NH), 2981 (=C–H), 2812 (–OH), 1712 (ester –
C=O), 1685 (C=N), 1630 (amide –C=O), 1550 (–C–C),
-1 1
1476 (ester –C–O), 1475 (amide –C–O) cm ; H NMR
50 °C for 2 h. After completion of the reaction, the
reaction mixture was concentrated completely under
(300 MHz, DMSO-d ): d = 1.39 (9H, s, CH ), 2.38–2.40
6
3
123

A. K. Ajeesh Kumar et al.
[
4H, m, N(CH ) ], 2.60 (3H, s, CH ), 2.73 (3H, s, CH ),
3
(400 MHz, DMSO-d ): d = 2.42–2.49 [4H, m, N(CH ) ],
2
2
3
6
2 2
3
.06 (2H, s, CH ), 3.34–3.36 [4H, m, N(CH ) ], 6.52 (1H,
2 2 2
2.63 (3H, s, CH ), 2.67–2.69 [4H, m, N(CH ) ], 2.76 (3H,
3 2 2
s, NH), 6.91 (1H, s, OH), 7.15 (1H, s, pyrimidine H), 8.71
s, CH ), 3.63 (2H, s, CH ), 7.21 (1H, s, pyrimidine H), 8.84
3 2
1
3
1H, s, pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
13
(
(1H, s, pyrazole H) ppm; C NMR (75 MHz, DMSO-d6):
d = 16.9, 24.9, 45.8 (two peaks), 53.2, 54.1 (two peaks),
95.2, 111.5, 145.1, 146.4, 147.4, 163.3, 167.4, 170.6 ppm;
d = 17.0, 25.0, 28.5 (three peaks), 43.0, 52.7 (two peaks),
5
1
7.0, 79.2, 101.0, 111.0, 146.6, 147.4, 147.5, 154.2, 156.9,
?
59.6, 163.0 ppm; LC–MS: m/z = 432.0 [M?H] .
?
LC–MS: m/z = 314.3 [M?H] .
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
Synthesis of substituted urea derivatives 12a–12e (general
procedure)
3
To a solution of compound 11 (1.00 mmol) in 4 cm of
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid
tert-butyl ester (10, C H N O )
3
2
0
27
7
To a solution of 20 g of compound 9 (0.046 mol) in
1
tetrahydrofuran, corresponding isocyanates (1.05 mmol)
were added drop wise at 0 °C under nitrogen atmosphere.
The reaction medium was stirred at the same temperature
for 30 min and then allowed to stir at room temperature for
3
00 cm of dimethyl formamide, 8.3 cm of DBU
3
(
0.055 mol) was added drop wise at room temperature
under nitrogen atmosphere. The resultant reaction mixture
was stirred at 90 °C for 2 h. After completion of reaction,
the reaction mixture was quenched with drop-wise addition
1
h under nitrogen atmosphere. After completion of
reaction, the reaction mixtures were concentrated under
reduced pressure and purified on silica gel column
chromatography with chloroform/methanol (100:2, v/v) to
afford corresponding urea derivatives 12a–12e.
3
of 1 dm of ice-cold water and stirred for further 30 min at
room temperature. The solid precipitated out was filtered,
3
washed with 200 cm of cold water and dried under
vacuum at 50 °C for 1 h to afford compound 10 as off
white solid (15.9 g, 83 %). M.p.: 166.0–168.3 °C; TLC:
R = 0.35 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 2969 (=C–
4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid pheny-
f
3
H), 1678 (ester –C=O), 1556 (–C–C), 1427 (ester –C–O),
-
lamide (12a, C22H N O )
24 8 2
1
1
1
323 (–C–N) cm
;
H NMR (300 MHz, DMSO-d6):
From 0.100 g of compound 11 (0.319 mmol) and 0.040 g
of phenyl isocyanate (0.335 mmol), compound 12a was
obtained as an off white solid (0.12 g, 85 %) after
chromatography on a silica gel column with chloroform/
methanol (100:2, v/v). M.p.: 189.2–192.2 °C; TLC:
d = 1.39 (9H, s, CH ), 2.51–2.59 [4H, m, N(CH ) ], 2.64
3
2 2
(
3H, s, CH ), 2.74 (3H, s, CH ), 3.07 (2H, s, CH ),
3 3 2
3
8
.34–3.36 [4H, m, N(CH ) ], 7.22 (1H, s, pyrimidine H),
2 2
1
.84 (1H, s, pyrazole H) ppm; C NMR (100 MHz,
3
DMSO-d ): d = 17.0, 24.9, 28.5 (three peaks), 52.5 (two
R
f
= 0.43 (CHCl
3
–MeOH 9:1); IR (ATR): vꢀ = 3301 (–
6
peaks), 52.6 (two peaks), 79.2, 95.2, 111.6, 145.2, 146.5,
NH), 2920 (=CH), 1631 (amide –C=O), 1541 (–C–C), 1437
-
1 1
1
47.5, 154.2, 163.5, 167.4, 170.8 ppm; LC–MS:
(amide –C–O), 1363 (Ar–C–N) cm ; H NMR (400 MHz,
CDCl ): d = 2.74 (3H, s, ArCH ), 2.76–2.83 [4H, m,
N(CH ) ], 2.84 (3H, s, ArCH ), 3.59–3.69 [4H, m,
?
m/z = 414.4 [M?H] .
3
3
2
2
3
5
,7-Dimethyl-3-(3-piperazin-1-ylmethyl-1,2,4-oxadiazol-5-
N(CH ) ], 3.90 (2H, s, –CH –), 6.37 (1H, bs, urea NH),
2
2
2
yl)pyrazolo[1,5-a]pyrimidine (11, C H N O)
1
5 19 7
6
7
8
.84 (1H, s, pyrimidine H), 7.04 (1H, t, J = 7.2 Hz, ArH),
To a solution of 15 g of compound 10 (0.036 mol) in
1
.26–7.34 (2H, m, ArH), 7.34 (2H, d, J = 8.4 Hz, ArH),
13
.72 (1H, s, pyrazole H) ppm; C NMR (75 MHz, DMSO-
3
3
50 cm of dichloromethane was added 45 cm of triflu-
oroacetic acid drop wise at 0 °C under nitrogen
atmosphere. The reaction mixture was slowly allowed to
raise the temperature to 25 °C and stirred for 6 h. After
completion of reaction, the reaction mixture was concen-
trated under reduced pressure and the residue was dissolved
d6): d = 16.9, 24.9, 44.1, 52.4, 52.7, 95.2, 111.6, 121.0,
1
21.5, 140.3, 143.0, 145.1, 146.5, 147.4, 155.2, 163.4,
?
67.3, 170.8 ppm; LC–MS: m/z = 433.4 [M?H] .
1
4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid [4-(tri-
3
in water, basified with 75 cm of saturated potassium
3
carbonate solution and extracted with 2 9 150 cm of
fluoromethoxy)phenyl]amide (12b, C23H F N O )
23 3 8 3
dichloromethane/methanol (100:5, v/v). The combined
organic extracts were dried over sodium sulfate, filtered,
and concentrated completely to obtain the crude product as
pale yellow solid. The crude material was further purified
by recrystallization with ethyl acetate/hexane (1:8, v/v) at
room temperature to afford compound 11 as an off white
From 0.100 g of compound 11 (0.319 mmol) and 0.068 g
of 4-(trifluoromethoxy)phenyl isocyanate (0.335 mmol),
compound 12b was obtained as an off white solid (0.14 g,
87 %) after chromatography on a silica gel column with
chloroform/methanol (100:2, v/v). M.p.: 179.6–182.4 °C;
TLC: R = 0.38 (CHCl
–MeOH 9:1); IR (ATR): vꢀ = 3333
f 3
solid (8.5 g, 75 %). M.p.: 147.8–150.9 °C; TLC: R = 0.11
(–NH), 2921 (=CH), 1634 (amide –C=O), 1535 (–C–C),
1463 (amide –C–O), 1359 (Ar–C–N), 1281 (C–O–C)
-1 1
cm ; H NMR (400 MHz, CDCl ): d = 2.65–2.71 [4H,
3
f
(
CHCl –MeOH 9:1); IR (ATR): vꢀ = 3327 (–NH), 2958
3
-
1
1
(
=C–H), 1556 (–C–C), 1323 (–C–N) cm
;
H NMR
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
m, N(CH ) ], 2.73 (3H, s, ArCH ), 2.83 (3H, s, ArCH ),
2
137.6, 140.7, 145.0, 146.3, 147.3, 155.2, 163.2, 167.2,
?
170.7 ppm; LC–MS: m/z = 447.3 [M?H] .
2
3
3
3
.61–3.55 [4H, m, N(CH ) ], 3.86 (2H, s, –CH –), 6.43
2
2
2
(
1H, bs, urea NH), 6.84 (1H, s, pyrimidine H), 7.13 (2H, d,
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid
3,4-dichlorophenyl)amide (12e, C H Cl N O )
J = 8.4 Hz, ArH), 7.35–7.39 (2H, m, ArH), 8.72 (1H, s,
3
pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
1
(
2
2
22
2 8 2
d = 15.9, 23.9, 43.0 (two peaks), 51.4, 51.6 (two peaks),
9
1
From 0.100 g compound 11 (0.319 mmol) and 0.063 g of
3,4-dichlorophenyl isocyanate (0.335 mmol), compound
4.2, 110.5, 115.8, 118.3, 120.9,123.4 (q,
JC-
=
253.6 Hz), 118.4, 120.6, 139.3, 141.9, 144.1, 145.5,
F
1
2e was obtained as off white solid (0.15 g, 92 %) after
1
46.4, 154.1, 162.4, 166.3, 169.8 ppm; LC–MS: m/
?
chromatography on a silica gel column with chloroform/
methanol (100:2, v/v). M.p.: 196.7–198.7 °C; TLC:
R = 0.38 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 337 (–
z = 517.3 [M?H] .
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid
cyclopentylamide (12c, C H N O )
4
f
3
NH), 3065 (=CH), 1624 (amide –C=O), 1550 (–C–C), 1477
-
(amide –C–O), 1378 (Ar–C–N), 818 (–C–Cl) cm ; H
1
1
2
8
21
8
2
From 0.100 g compound 11 (0.319 mmol) and 0.037 g of
cyclopentyl isocyanate (0.335 mmol), compound 12c was
obtained as an off white solid (0.12 g, 89 %) after
chromatography on a silica gel column with chloroform/
methanol (100:2, v/v). M.p.: 115.5–118.2 °C; TLC:
R = 0.40 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3446
NMR (400 MHz, DMSO-d ): d = 2.56–2.60 [4H, m,
6
N(CH ) ], 2.63 (3H, s, ArCH ), 2.77 (3H, s, ArCH ),
2
2
3
3
3.41–3.51 [4H, m, N(CH ) ], 3.76 (2H, s, –CH –), 7.21
2 2 2
(
(
1H, s, pyrimidine H), 7.45 (2H, d, J = 12 Hz, ArH), 7.82
1H, s, ArH), 8.80 (1H, s, urea NH), 8.83 (1H, s, pyrazole
1
3
f
3
H) ppm; C NMR (100 MHz, DMSO-d ): d = 17.0, 24.9,
6
(
NH), 2967 (=CH), 1643 (amide –C=O), 1551 (–C–C),
41.4, 46.0, 52.1, 52.2, 52.81, 95.2, 111.6, 129.1, 131.8,
-
442 (amide –C–O), 1336 (Ar–C–N) cm ; H NMR
1
1
1
1
34.2, 135.1, 144.2, 145.0, 146.0, 146.6, 147.4, 159.1,
?
163.4, 167.2, 170.8 ppm; LC–MS: m/z = 502.9 [M?H] .
(
400 MHz, DMSO-d ): d = 1.31–1.46 (4H, m, cyclopen-
6
tane CH ), 1.58–1.60 (2H, m, cyclopentane CH ),
2
2
Synthesis of substituted thiourea derivatives 12f–12j
1
.72–1.77 (2H, m, cyclopentane CH ), 2.45–2.48 [4H, m,
2
(
general procedure)
To a solution of compound 11 (1.00 mmol) in 4 cm
of tetrahydrofuran, corresponding isothiocyanates
1.05 mmol) were added drop wise at 0 °C under nitrogen
N(CH ) ], 2.64 (3H, s, ArCH3), 2.76 (3H, s, ArCH ),
3
2
2
3
3
.28–3.30 [4H, m, N(CH ) ], 3.71 (2H, s, –CH –),
2 2 2
3
.85–3.89 (1H, m, cyclopentyl CH), 6.21 (1H, d,
(
J = 6.8 Hz, urea NH), 7.22 (1H, s, pyrimidine H), 8.34
1
3
atmosphere. The reaction medium was stirred at the same
temperature for 30 min and then allowed to stir at room
temperature for 1 h under nitrogen atmosphere. After
completion of reaction, the reaction mixture was concen-
trated under reduced pressure and purified on silica gel
column chromatography with chloroform/methanol (100:3,
v/v) to afford corresponding urea derivatives 12f–12j.
(
1H, s, pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
d = 16.5, 25.2 (two peaks), 28.7, 32.9 (two peaks), 43.7
two peaks), 52.3, 52.5, 52.7 (two peaks), 95.2, 111.5,
45.1, 146.4, 147.4, 157.6, 163.4, 167.4, 170.8 ppm; LC–
(
1
?
MS: m/z = 425.4 [M?H] .
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carboxylic acid
m-tolylamide (12d, C H N O )
3 26 8 2
4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carbothioic acid
2
From 0.100 g compound 11 (0.319 mmol) and 0.045 g of 3-
methylphenyl isocyanate (0.335 mmol), compound 12d was
obtained as an off white solid (0.13 g, 90 %) after chro-
matography on a silica gel column with chloroform/methanol
phenylamide (12f, C22H N OS)
24 8
From 0.100 g compound 11 (0.319 mmol) and 0.045 g of
phenyl isothiocyanate (0.335 mmol), compound 12f was
obtained as an off white solid (0.12 g, 82 %) after
chromatography on a silica gel column with chloroform/
methanol (100:3, v/v). M.p.: 199.2–201.7 °C; TLC:
(
100:2, v/v). M.p.: 164.9–167.4 °C; TLC: R = 0.42
f
(
CHCl –MeOH 9:1); IR (ATR): vꢀ = 3301 (–NH), 3064 (–
3
CH), 2921 (=CH), 1632 (amide –C=O), 1546 (–C–C), 1428
-1 1
(
amide –C–O), 1318 (Ar–C–N) cm ; H NMR (400 MHz,
R
f
= 0.44 (CHCl
3
–MeOH 9:1); IR (ATR): vꢀ = 3296 (–
DMSO-d ): d = 2.24 (3H, s, ArCH ), 2.56 [4H, t,
NH), 2921 (=CH), 1631 (thiourea –C=S), 1533 (–C–C),
6
3
-
1
1
J = 4.4 Hz, N(CH ) ], 2.64 (3H, s, ArCH ), 2.77 (3H, s,
2
1441 (thiourea –C–S), 1320 (Ar–C–N) cm ; H NMR
(300 MHz, DMSO-d ): d = 2.52–2.52 [4H, m, N(CH ],
2.64 (3H, s, ArCH ), 2.77 (3H, s, ArCH ), 3.79 (2H, s, –
CH –), 3.89–3.99 [4H, m, N(CH ], 7.09–7.11 (1H, m,
2
3
ArCH ), 3.46 [4H, t, J = 4.4 Hz, N(CH ) ], 3.76 (2H, s, –
2 2
6
)
2 2
3
CH –), 6.74 (1H, d, J = 7.6 Hz, urea NH), 7.09 (1H, t,
2
3
3
J = 7.6 Hz, ArH), 7.22–7.24 (2H, m, ArH), 7.27 (1H, s,
2
)
2 2
ArH), 8.43 (1H, s, pyrimidine H), 8.85 (1H, s, pyrazole H)
3
ArH), 6.23 (1H, s, thiourea NH), 7.28 (4H, d, J = 3.6 Hz,
1
ppm; C NMR (75 MHz, DMSO-d ): d = 16.8, 21.5, 22.4,
ArH), 8.85 (1H, s, pyrimidine H), 9.32 (1H, s, pyrazole H)
1
6
3
4
4.0, 52.3, 52.6, 95.1, 111.4, 117.0, 120.5, 122.7, 128.4,
ppm; C NMR (100 MHz, DMSO-d ): d = 16.9, 24.7,
6
123

A. K. Ajeesh Kumar et al.
4
1
1
6.0, 53.4, 54.0, 95.3, 111.3, 123.8, 128.9, 129.7, 130.8,
32.0, 135.0, 145.1, 146.4, 147.4, 163.3, 167.4, 171.1,
?
79.8 ppm; LC–MS: m/z = 449.3 [M?H] .
chromatography on a silica gel column with chloroform/
methanol (100:3, v/v). M.p.: 144.3–145.9 °C; TLC:
R = 0.45 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3393 (–
f
3
NH), 3069 (–CH), 2920 (–CH ), 2852 (=CH), 1629 (thiourea
3
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
–
C=S), 1534 (–C–C), 1463 (thiourea –C–S), 1334 (Ar–C–N)
oxadiazol-3-ylmethyl]piperazine-1-carbothioic acid iso-
-1 1
cm ; H NMR (300 MHz, DMSO-d ): d = 1.44 [9H, s, –
6
propylamide (12g, C H N OS)
9 26 8
1
C(CH ) ], 2.51–2.59 [4H, m, N(CH ) ], 2.63 (3H, s, ArCH ),
3
3
2 2
3
From 0.100 g compound 11 (0.319 mmol) and 0.034 g of
isopropyl isothiocyanate (0.335 mmol), compound 12g was
obtained as a white solid (0.11 g, 84 %) after chromatogra-
phy on a silica gel column with chloroform/methanol (100:3,
v/v). M.p.: 175.0–177.5 °C; TLC: R = 0.41 (CHCl –
2
6
.76 (3H, s, ArCH ), 3.69–3.85 [6H, m, –CH –, N(CH ) ],
3 2 2 2
.72 (1H, s, thiourea NH), 7.21 (1H, s, pyrimidine H), 8.82
1
3
1H, s, pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
(
d = 14.4, 24.9, 29.4 (three peaks), 47.6, 52.1, 52.6 (two
peaks), 53.6, 95.2, 111.5, 145.1, 146.4, 147.4, 163.4, 167.2,
f
3
MeOH 9:1); IR (ATR): vꢀ = 3335 (–NH), 3010 (–CH),
?
70.8, 181.6 ppm; LC–MS: m/z = 429.4 [M?H] .
1
2
966 (–CH ), 2866 (=CH), 1631 (thiourea –C=S), 1531 (–C–
3
-1 1
C), 1447 (thiourea –C–S), 1346 (Ar–C–N) cm ; H NMR
4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carbothioic acid
propylamide (12j, C H N OS)
(
300 MHz, DMSO-d ): d = 1.12 (6H, d, J = 6.3 Hz,
6
RCH ), 2.51–2.59 [4H, m, N(CH ) ], 2.64 (3H, s, ArCH ),
3
3
2 2
19 26 8
2
.77 (3H, s, ArCH ), 3.75 (2H, s, –CH –), 3.79–3.88 [4H, m,
3
From 0.100 g compound 11 (0.319 mmol) and 0.034 g of n-
propyl isothiocyanate (0.335 mmol), compound 12j was
obtained as an off white solid (0.12 g, 87 %) after chro-
matography on a silica gel column with chloroform/methanol
2
N(CH ) ], 4.48–4.54 (1H, s, R CH), 7.22 (1H, s, pyrimidine
2
2
2
H), 7.31 (1H, d, J = 7.8 Hz, thiourea NH), 8.84 (1H, s,
1
pyrazole H) ppm;
3
C NMR (75 MHz, DMSO-d6):
d = 16.8, 22.3 (two peaks), 24.8, 47.3, 47.4, 52.1, 52.3,
(100:3, v/v). M.p.: 154.5–156.4 °C; TLC: R = 0.46
f
9
1
5.1, 111.4, 145.0, 146.3, 147.3, 163.2, 167.1, 170.6,
?
80.6 ppm; LC–MS: m/z = 415.4 [M?H] .
(CHCl –MeOH 9:1); IR (ATR): vꢀ = 3348 (–NH), 3002 (–
3
C–H), 2926 (=CH), 1605 (thiourea –C=S), 1533 (–C–C),
-
1
1
440 (thiourea –C–S), 1326 (Ar–C–N) cm ; H NMR
1
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
(
400 MHz, DMSO-d ): d = 0.84 (3H, t, J = 8 Hz, –CH ),
6
3
oxadiazol-3-ylmethyl]piperazine-1-carbothioic acid cyclo-
1
2
.49–1.58 (2H, m, –CH –), 2.50–2.52 [4H, m, N(CH ) ],
2 2 2
hexylamide (12h, C H N OS)
2 30 8
2
.63 (3H, s, ArCH ), 2.76 (3H, s, ArCH ), 3.40 (2H, t,
3
3
From 0.100 g compound 11 (0.319 mmol) and 0.047 g of
cyclohexyl isothiocyanate (0.335 mmol), compound 12h
was obtained as an off white solid (0.13 g, 88 %) after
chromatography on a silica gel column with chloroform/
methanol (100:3, v/v). M.p.: 186.1–188.5 °C; TLC:
R = 0.39 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3309
J = 10 Hz, –CH –), 3.74 (2H, s, –CH –), 3.78–3.82 [4H, m,
2
2
N(CH ) ], 7.21 (1H, s, pyrimidine H), 7.69 (1H, t, J = 3 Hz,
2
2
1
3
thiourea NH), 8.82 (1H, s, pyrazole H) ppm; C NMR
(
75 MHz, DMSO-d ): d = 11.6, 16.8, 22.2, 24.8, 47.3, 47.4,
6
5
1
2.1, 52.3, 95.1, 111.4, 145.0, 146.3, 147.3, 163.3, 167.1,
?
70.7, 181.6 ppm; LC–MS: m/z = 416.2 [M?H] .
f
3
(
–NH), 3080 (–CH), 2924 (=CH), 1632 (thiourea –C=S),
-1
1
1
525 (–C–C), 1443 (thiourea –C–S), 1350 (Ar–C–N) cm ;
Synthesis of substituted carboxamides 12k–12o (general
procedure)
3
To a solution of the compound 11 (1.00 mmol) in 3 cm of
H NMR (400 MHz, DMSO-d ): d = 1.00–1.12 (2H, m,
6
cyclohexane CH ), 2.25–3.00 (4H, m, cyclohexane CH ),
2
2
2
.62–2.71 (2H, m, cyclohexane CH ), 2.81–2.89 (2H, m,
2
dimethylacetamide was added N-methylmorpholine
(5.00 mmol) followed by drop wise addition of corre-
sponding acid chlorides (1.30 mmol) at 0 °C under
nitrogen atmosphere. The reaction medium was stirred at
the same temperature for 10 min and then allowed to stir at
room temperature for 1 h under nitrogen atmosphere. After
completion of reaction, water was added to the reaction
mixture and stirred for 10 min. The solid precipitated out
was filtered, washed with water and dried under vacuum to
afford the corresponding carboxamides 12k–12o.
cyclohexane CH ), 2.52–2.59 [4H, m, N(CH ) ], 2.64 (3H, s,
2 2
2
ArCH ), 2.73 (3H, s, ArCH ), 3.71 (2H, s, –CH –), 3.75–3.83
3
3
2
[
(
4H, m, N(CH ) ], 4.11–4.20 (1H, m, cyclohexyl CH), 7.19
2 2
1H, s, pyrimidine H), 7.23 (1H, d, J = 7.2 Hz, thiourea
1
3
NH), 8.80 (1H, s, pyrazole H) ppm; C NMR (75 MHz,
DMSO-d ): d = 16.9, 24.9, 25.5, 25.7, 32.5, 47.4, 52.1, 52.4,
6
5
4.9, 95.2, 111.5, 145.1, 146.5, 147.4, 163.4, 167.1, 170.8,
?
80.7 ppm; LC–MS: m/z = 455.4 [M?H] .
1
4
-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazine-1-carbothioic acid
(3,4-Dichlorophenyl){4-[5-(5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)-1,2,4-oxadiazol-3-ylmethyl]piperazin-1-
yl}methanone (12k, C H Cl N O )
tert-butylamide (12i, C H N OS)
2
0 28 8
From 0.100 g compound 11 (0.319 mmol) and 0.039 g of
tert-butyl isothiocyanate (0.335 mmol), compound 12i was
obtained as an off white solid (0.12 g, 86 %) after
2
2
21
2 7 2
From 0.100 g compound 11 (0.319 mmol) and 0.087 g of
3,4-dichlorobenzoyl chloride (0.414 mmol), compound
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
3
3
1
2k was obtained as off white solid (0.14 g, 89 %) after
= 270.75 Hz), 124.0 (q, J = 7.5 Hz), 126.6 (q, J
C-F C-
F
F
2
= 6.8 Hz), 129.5 (q, JC-F = 75.8 Hz), 129.9, 131.3,
recrystallization with water. M.p.: 224.4–226.9 °C; TLC:
R = 0.38 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 2911
137.3, 145.1, 146.5, 147.4, 163.4, 167.2, 167.8, 170.8 ppm;
?
LC–MS: m/z = 486.3 [M?H] .
f
3
(
(
=CH), 1628 (–C=O), 1547 (–C–C), 1440 (–C–O), 1379
-
Ar–C–N), 827 (–C–Cl) cm
1
1
;
H NMR (400 MHz,
(
3,5-Dimethylphenyl){4-[5-(5,7-dimethylpyrazolo[1,5-
DMSO-d ): d = 2.55–2.65 [4H, m, N(CH ) ], 2.62 (3H,
6
2 2
a]pyrimidin-3-yl)-1,2,4-oxadiazol-3-ylmethyl]piperazin-1-
s, ArCH ), 2.77 (3H, s, ArCH ), 3.59–3.69 [4H, m,
3
3
yl}methanone (12n, C H N O )
2
4 27 7 2
N(CH ) ], 3.75 (2H, s, –CH –), 7.21 (1H, s, pyrimidine
2
2
2
From 0.100 g compound 11 (0.319 mmol) and 0.070 g of
3,5-dimethylbenzoyl chloride (0.414 mmol), compound
H), 7.38 (1H, dd, J = 4 Hz, 8 Hz, ArH), 7.76–7.70 (2H, m,
1
ArH), 8.83 (1H, s, pyrazole H) ppm; C NMR (100 MHz,
3
1
2n was obtained as off white solid (0.12 g, 85 %) after
DMSO-d ): d = 17.0, 24.8, 41.8, 46.1, 52.5, 52.7, 95.0,
6
recrystallization with water. M.p.: 176.6–179.4 °C; TLC:
R = 0.39 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3077 (–
CH), 2915 (=CH), 1627 (–C=O), 1537 (–C–C), 1463 (–C–
1
11.4, 128.6, 129.0, 131.7, 134.0, 144.8, 145.0, 146.5,
f
3
1
46.9, 147.4, 158.6, 162.8, 166.7, 171.0 ppm; LC–MS: m/
?
z = 486.4 [M?H] .
-
1 1
O), 1377 (Ar–C–N) cm ; H NMR (300 MHz, DMSO-
(
3,5-Difluorophenyl){4-[5-(5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)-1,2,4-oxadiazol-3-ylmethyl]piperazin-1-
yl}methanone (12l, C H F N O )
d ): d = 2.28 (6H, s, ArCH ), 2.59–2.48 [4H, m, N(CH ) ],
6
3
2 2
2.63 (3H, s, ArCH ), 2.76 (3H, s, ArCH ), 3.51–3.65 [4H,
3 3
2
2
21
2
7
2
m, N(CH ) ], 3.75 (2H, s, –CH –), 6.96 (2H, s, ArH), 7.06
2 2 2
From 0.100 g compound 11 (0.319 mmol) and 0.073 g of
,5-difluorobenzoyl chloride (0.414 mmol), compound 12l
was obtained as a white solid (0.13 g, 87 %) after
(
1H, s, ArH), 7.21 (1H, s, pyrimidine H), 8.83 (1H, s,
1
pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
3
3
d = 17.0, 21.2 (two peaks), 24.9, 47.5, 52.4, 52.6, 95.2,
1
recrystallization with water. M.p.: 198.9–201.2 °C; TLC:
11.6, 124.9, 131.1, 136.3, 138.0, 145.1, 146.5, 147.4,
R = 0.37 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 2927
f
3
163.4, 167.3, 169.6, 170.8 ppm; LC–MS: m/z = 446.4
?
M?H] .
(
(
=CH), 1627 (–C=O), 1542 (–C–C), 1434 (–C–O), 1378
-
[
1
1
Ar–C–N), 1124 (–C–F) cm
;
H NMR (300 MHz,
DMSO-d ): d = 2.48–2.53 [4H, m, N(CH ) ], 2.63 (3H,
[4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
oxadiazol-3-ylmethyl]piperazin-1-yl](2,4,6-tri-
6
2 2
s, ArCH ), 2.76 (3H, s, ArCH ), 3.51–3.65 [4H, m,
3
3
N(CH ) ], 3.76 (2H, s, –CH –), 7.15 (2H, d, J = 4.8 Hz,
2
chlorophenyl)methanone (12o, C22H20Cl N O )
3 7 2
2
2
ArH), 7.21 (1H, s, pyrimidine H), 7.34 (1H, t, J = 9 Hz,
From 0.100 g compound 11 (0.319 mmol) and 0.101 g of
2,4,6-trichlorobenzoyl chloride (0.414 mmol), compound
12o was obtained as a white solid (0.15 g, 90 %) after
recrystallization with water. M.p.: 187.2–189.3 °C; TLC:
1
3
ArH), 8.82 (1H, s, pyrazole H) ppm; C NMR (100 MHz,
DMSO-d ): d = 17.0, 24.9, 41.9, 47.3, 52.3, 52.7, 95.2,
6
2
2
1
05.4 (d, J = 26 Hz), 105.6 (d, J = 26 Hz), 110.8
C-F C-F
2
d, J = 26 Hz), 111.6, 139.8 (d, J = 8 Hz), 145.2,
3
(
R
f
= 0.44 (CHCl
3
–MeOH 9:1); IR (ATR): vꢀ = 2956
C-F
C-F
1
1
1
46.5, 147.5, 162. 6 (d, J = 249 Hz), 163.5, 164.1 (d,
C-F
(=CH), 1654 (–C=O), 1545 (–C–C), 1441 (–C–O), 1326
-1
1
JC-F = 249 Hz), 166.7, 167.3, 170.8 ppm; LC–MS:
?
m/z = 454.4 [M?H] .
(Ar–C–N), 857 (–C–Cl) cm
DMSO-d ): d = 2.53–2.54 (2H, m, NCH
2H, m, NCH –), 2.63 (3H, s, ArCH ), 2.76 (3H, s,
;
H NMR (400 MHz,
–), 2.60–2.62
6
2
(
2
3
{4-[5-(5,7-Dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-
ArCH ), 3.19 (2H, t, J = 5 Hz, NCH –), 3.67 (2H, t,
2
3
oxadiazol-3-ylmethyl]piperazin-1-yl}[3-(trifluoro-
J = 5 Hz, NCH –), 3.77 (2H, s, –CH –), 7.21 (1H, s,
2
2
methyl)phenyl]methanone (12m, C H F N O )
2
3 22 3 7 2
pyrimidine H), 7.78 (2H, s, ArH), 8.83 (1H, s, pyrazole H)
13
From 0.100 g compound 11 (0.319 mmol) and 0.086 g of
-(trifluoromethyl)benzoyl chloride (0.414 mmol), com-
ppm; C NMR (100 MHz, DMSO-d ): d = 17.0, 24.9,
6
3
4
1
1
1.5, 46.1, 52.2, 52.2, 52.8, 95.2, 111.6, 128.7 (two peaks),
pound 12m was obtained as off white solid (0.14 g, 88 %)
after recrystallization with water. M.p.: 185.4–187.6 °C;
TLC: R = 0.40 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3085
31.9 (two peaks), 134.1, 135.0, 145.1, 146.5, 147.5, 162.2,
?
63.5, 167.2, 170.8 ppm; LC–MS: m/z = 522.1 [M?H] .
f
3
(
(
–ArH), 1627 (–C=O), 1542 (–C–C), 1441 (–C–O), 1335
1
Synthesis of substituted sulfonamides 12p–12t (general
procedure)
3
To a solution of compound 11 (1.00 mmol) in 3 cm of
-
1
Ar–C–N), 1023 (–C–F) cm
;
H NMR (300 MHz,
DMSO–d ): d = 2.52–2.48 [4H, m, N(CH ) ], 2.62 (3H,
6
2 2
s, ArCH ), 2.76 (3H, s, ArCH ), 3.55–3.69 [4H, m,
3
dimethylacetamide was added N-methylmorpholine
(5.00 mmol) followed by drop wise addition of corre-
sponding sulfonyl chlorides (1.30 mmol) at 0 °C under
nitrogen atmosphere. The reaction medium was stirred at
the same temperature for 10 min and then allowed to stir at
room temperature for 1 h under nitrogen atmosphere. After
3
N(CH ) ], 3.76 (2H, s, –CH –), 7.21 (1H, s, pyrimidine
2
2
2
H), 7.68 (2H, d, J = 6.3 Hz, ArH), 7.73 (1H, s, ArH), 7.87
1H, t, J = 5.7 Hz, ArH), 8.82 (1H, s, pyrazole H) ppm;
(
1
3
C NMR (75 MHz, DMSO-d ): d = 16.9, 24.8, 41.9 (two
6
1
peaks), 47.3 (two peaks), 52.2, 95.2, 111.5, 124.3 (q, J
C-
123

A. K. Ajeesh Kumar et al.
completion of reaction, water was added to the reaction
mixture and stirred for 30 min at room temperature. The
solid precipitated out was filtered, washed with water and
dried under vacuum at 50 °C for 1 h to afford the
corresponding sulfonamides 12p–12t.
12r was obtained as an off white solid (0.13 g, 86 %) after
recrystallization with water. M.p.: 199.4–201.3 °C; TLC:
R = 0.44 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 2832
f
3
(=CH), 1631 (sulfonyl –S=O), 1541 (–C–C), 1439 (sul-
fonyl –C–O), 1352 (Ar–C–N), 1323 (–SO ), 937 (–C–F)
2
-
1
1
cm
; H NMR (400 MHz, DMSO-d ): d = 2.52–2.60
6
3
-{3-[4-(4-Chloro-3-fluorobenzenesulfonyl)piperazin-1-yl-
[
4H, m, N(CH ) ], 2.61 (3H, s, ArCH ), 2.74 (3H, s,
2
2
3
methyl]-1,2,4-oxadiazol-5-yl}-5,7-dimethylpyrazolo[1,5-
ArCH ), 3.02–3.12 [4H, m, N(CH ) ], 3.72 (2H, s, –CH –),
2 2
3
2
a]pyrimidine (12p, C H ClFN O S)
2
1
21
7 3
7.20 (1H, s, pyrimidine H), 7.40–7.50 (2H, m,
From 0.100 g compound 11 (0.319 mmol) and 0.095 g of
-chloro-3-fluorobenzenesulfonyl chloride (0.414 mmol),
compound 12p was obtained as an off white solid (0.13 g,
ArH),7.72–7.78 (2H, m, ArH), 8.79 (1H, s, pyrazole H)
13
4
ppm; C NMR (100 MHz, DMSO-d ): d = 17.0, 24.9,
6
2
5.9, 51.8, 52.0, 95.1, 111.6, 118.0 (d, J = 22 Hz),
4
C-F
8
2
5 %) after recrystallization with water. M.p.: 202.0–
2
24.2 (d, J = 14 Hz), 125.6 (d, J = 3 Hz), 131.2
4
1
C-F
C-F
04.1 °C; TLC: R = 0.46 (CHCl –MeOH 9:1); IR (ATR):
3
d, J = 8 Hz), 131.3, 136.5 (d, J = 8 Hz), 145.1,
3
f
3
(
C-F C-F
vꢀ = 3087 (=CH), 1548 (–C–C), 1346 (sulfonyl –S=O),
1
46.5, 147.5, 158. 7 (d, J = 253 Hz), 163.4, 167.2,
1
1
C-F
-
1
1
1
328 (Ar–C–N), 1055 (–C–F), 817 (–C–Cl) cm
;
H
?
70.8 ppm; LC–MS: m/z = 472.3 [M?H] .
NMR (400 MHz, DMSO-d ): d = 2.52–2.60 [4H, m,
6
5,7-Dimethyl-3-{3-[4-(naphthalene-1-sulfonyl)piperazin-1-
N(CH ) ], 2.61 (3H, s, ArCH ), 2.74 (3H, s, ArCH ),
3
2
2
3
ylmethyl]-1,2,4-oxadiazol-5-yl}pyrazolo[1,5-a]pyrimidine
12s, C H N O S)
2
.92–3.01 [4H, m, N(CH ) ], 3.71 (2H, s, –H –), 7.20 (1H,
2 2 2
(
s, pyrimidine H), 7.58 (1H, d, J = 8 Hz, ArH), 7.78 (1H, d,
25 25 7 3
From 0.100 g compound 11 (0.319 mmol) and 0.094 g of 1-
naphthylsulfonyl chloride (0.414 mmol), compound 12s was
obtained as an off white solid (0.13 g, 81 %) after recrys-
tallization with water. M.p.: 210.6–212.9 °C; TLC:
J = 8 Hz, ArH), 7.87 (1H, t, J = 16 Hz, ArH), 8.78 (1H, s,
1
3
pyrazole H) ppm;
C NMR (75 MHz, DMSO-d6):
d = 16.9, 24.8, 46.1 (two peaks), 51.5 (two peaks), 51.8,
2
3
9
5.1, 111.5, 116.5 (d, J = 23.25 Hz), 125.1 (d, J
C-F C-
2
3.75 Hz), 125.5 (d, J = 18 Hz), 132.3, 136.2 (d,
R = 0.45 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3081 (–
=
f
3
F
3
C-F
1
CH), 2922 (=CH), 1631 (sulfonyl –S=O), 1540 (–C–C), 1437
-1 1
sulfonyl –C–O), 1381 (Ar–C–N), 1322 (–SO ) cm ; H
JC-F = 6 Hz), 145.1, 146.4, 147.4, 157.5 (d, JC-
250.50 Hz), 163.4, 167.2, 170.8 ppm; LC–MS:
(
=
2
F
?
m/z = 506.4 [M?H] .
NMR (400 MHz, DMSO-d ): d = 2.54–2.60 [4H, m,
6
N(CH ) ], 2.66 (3H, s, ArCH ), 2.74 (3H, s, ArCH ),
2
2
3
3
3
1
-{3-[4-(Cyclopropanesulfonyl)piperazin-1-ylmethyl]-
,2,4-oxadiazol-5-yl}-5,7-dimethylpyrazolo[1,5-a]pyrim-
3.05–3.15 [4H, m, N(CH ) ], 3.67 (2H, s, –CH –), 7.19 (1H,
2 2 2
s, pyrimidine H), 7.62–7.73 (3H, m, ArH), 8.08 (1H, d,
J = 8 Hz, ArH), 8.13 (1H, d, J = 4 Hz, ArH), 8.27 (1H, d,
J = 8 Hz, ArH), 8.65 (1H, d, J = 12 Hz, ArH), 8.75 (1H, s,
idine (12q, C H N O S)
8 23 7 3
1
From 0.100 g compound 11 (0.319 mmol) and 0.058 g of
cyclopropylsulfonyl chloride (0.414 mmol), compound
1
3
pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
d = 17.0, 24.9, 45.8, 51.9, 52.0, 95.2, 111.6, 125.1, 127.4,
128.6, 129.5, 130.7, 132.4, 134.4, 135.1, 145.2, 146.5, 147.5,
1
2q was obtained as a white solid (0.11 g, 83 %) after
recrystallization with water. M.p.: 236.5–238.2 °C; TLC:
R = 0.48 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3094
?
f
3
163.5, 167.2, 170.8 ppm; LC–MS: m/z = 504.2 [M?H] .
(
–CH), 2975 (=CH), 1629 (sulfonyl –S=O), 1540 (–C–C),
-
1
3-{3-[4-(4-Chlorobenzenesulfonyl)piperazin-1-ylmethyl]-
1,2,4-oxadiazol-5-yl}-5,7-dimethylpyrazolo[1,5-a]pyrim-
idine (12t, C H ClN O S)
1
1
443 (sulfonyl –C–O), 1374 (Ar–C–N), 1329 (–SO ) cm ;
2
H NMR (300 MHz, DMSO-d ): d = 0.91–0.99 (4H, m,
6
2
1
22
7 3
cyclopropane CH ), 2.57–2.62 [4H, m, N(CH ) ], 2.63
2 2 2
From 0.100 g compound 11 (0.319 mmol) and 0.087 g of
-chlorobenzenesulfonyl chloride (0.414 mmol), com-
(
(
3H, s, ArCH ), 2.68–2.71 (1H, m, cyclopropyl CH), 2.76
3
4
3H, s, ArCH ), 3.15–3.22 [4H, m, N(CH ) ], 3.77 (2H, s, –
3 2 2
pound 12t was obtained as a white solid (0.14 g, 87 %)
after recrystallization with water. M.p.: 207.6–210.6 °C;
TLC: R = 0.46 (CHCl –MeOH 9:1); IR (ATR): vꢀ = 3083
CH –), 7.22 (1H, s, pyrimidine H), 8.83 (1H, s, pyrazole H)
2
1
3
ppm; C NMR (100 MHz, DMSO-d ): d = 12.4, 16.9,
2
6
f
3
4.7, 45.7, 48.8, 52.2, 53.8, 95.5, 111.4, 144.9, 146.2,
(
ArH), 1548 (–C–C), 1443 (sulfonyl –S–O), 1348 (sulfonyl
1
47.29, 158.9, 163.0, 167.2, 170.6 ppm; LC–MS:
-
1 1
–S=O), 835 (–C–Cl) cm ; H NMR (400 MHz, DMSO-
?
m/z = 418.3 [M?H] .
d ): d = 2.56–2.60 [4H, m, N(CH ) ], 2.61 (3H, s, ArCH ),
6
2 2
3
3
1
-{3-[4-(2-Fluorobenzenesulfonyl)piperazin-1-ylmethyl]-
,2,4-oxadiazol-5-yl}-5,7-dimethylpyrazolo[1,5-a]pyrim-
2.74 (3H, s, ArCH ), 2.90–3.03 [4H, m, N(CH ) ], 3.71
3 2 2
(2H, s, –CH –), 7.19 (1H, s, pyrimidine H), 7.70 (2H, d,
2
idine (12r, C H FN O S)
2
J = 8.8 Hz, ArH), 7.73 (2H, d, J = 8.4 Hz, ArH), 8.79
1
1
22
7 3
3
From 0.100 g compound 11 (0.319 mmol) and 0.081 g of
-fluorobenzenesulfonyl chloride (0.414 mmol), compound
(1H, s, pyrazole H) ppm; C NMR (100 MHz, DMSO-d6):
d = 17.0, 24.9, 46.2, 51.6, 51.9, 95.2, 111.6, 129.9, 130.0,
2
1
23

Design, synthesis, and evaluation of the anticancer properties of a novel series of…
1
34.2, 138.7, 145.2, 146.5, 147.5, 163.5, 167.2, 170.8 ppm;
Acknowledgments We would like to thank the Management Team
at Anthem Biosciences Pvt. Ltd., Bangalore, India, for their valuable
support and allocation of proper resources for the completion of this
work. We would also like to thank the Analytical Chemistry Team
at the Department of Analytical chemistry, Anthem Biosciences,
Bangalore, India, for carrying out all the analytical work described
in this study. Also, we would like to thank the Molecular Biology
Team at the Maratha Mandal Institute of Technology, Belagavi,
Karnataka, India, for conducting the anti-proliferative studies by
MTT assay.
?
LC–MS: m/z = 489.1 [M?H] .
Anticancer activity
All compounds were screened for their in vitro anti-cancer
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-dimethythia-
zol-2-yl)-2,5-diphenyltetrazolium 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, isopropanol) and then
released solubilized formazan reagent is measured spec-
trophotometrically. Since reduction of MTT can only occur
in metabolically active cells, the level of activity is a
measure of the viability of these cells.
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