Anthranilamide-Pyrazolo[1,5-a]pyrimidine Conjugates as p53 Activators in Cervical Cancer Cells

Article abstract of DOI:10.1002/cmdc.201200205

A library of new anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates were designed, synthesized, and evaluated for their anticancer activity in cervical cancer cells such as HeLa and SiHa that possess low levels of p53. All 24 conjugates showed antiproliferative activity, while some of them exhibit significant cytotoxicity. In assays related to cell-cycle distribution, these conjugates induced G₂/M arrest in HeLa cells and G₁ cell-cycle arrest in SiHa cells. Immunocytochemistry assays revealed that these compounds cause nuclear translocation of p53, thereby indicating the activation of p53. In cervical cancer cells, the p53 protein is degraded by E6 oncoprotein. Immunoblot and RT-PCR analyses proved the presence of mitochondria-mediated apoptosis with involvement p53 target genes such as BAX, Bcl2, and p21 (CDKI). Moreover, these compounds increased the phosphorylated forms of p53 and provide signals for apoptosis induction. Interestingly, one of the conjugates, (2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)methanone, is the most promising candidate in this series and has the potential to be taken up for further detailed studies.

Full text of DOI:10.1002/cmdc.201200205

MED
DOI: 10.1002/cmdc.201200205
Anthranilamide–Pyrazolo[1,5-a]pyrimidine Conjugates as
p53 Activators in Cervical Cancer Cells
Ahmed Kamal,*[a] Jaki R. Tamboli,[a] M. Janaki Ramaiah,[b] S. F. Adil,[c] G. Koteswara Rao,[b]
A. Viswanath,[a] Adla Mallareddy,[a] S. N. C. V. L. Pushpavalli,[b] and Manika Pal-Bhadra*[b]
A library of new anthranilamide–pyrazolo[1,5-a]pyrimidine con-
jugates were designed, synthesized, and evaluated for their an-
ticancer activity in cervical cancer cells such as HeLa and SiHa
that possess low levels of p53. All 24 conjugates showed anti-
proliferative activity, while some of them exhibit significant cy-
totoxicity. In assays related to cell-cycle distribution, these con-
jugates induced G2/M arrest in HeLa cells and G1 cell-cycle
arrest in SiHa cells. Immunocytochemistry assays revealed that
these compounds cause nuclear translocation of p53, thereby
indicating the activation of p53. In cervical cancer cells, the
p53 protein is degraded by E6 oncoprotein. Immunoblot and
RT-PCR analyses proved the presence of mitochondria-mediat-
ed apoptosis with involvement p53 target genes such as BAX,
Bcl2, and p21 (CDKI). Moreover, these compounds increased
the phosphorylated forms of p53 and provide signals for apop-
tosis induction. Interestingly, one of the conjugates, (2-phenyl-
7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-
(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)methanone,
is the most promising candidate in this series and has the po-
tential to be taken up for further detailed studies.
Introduction
Cervical cancer is the second most prevalent cancer in women
worldwide, both in terms of incidence and mortality rates.[1]
The occurrence of cervical cancer is higher than that
of breast cancer, especially in India.[2] High-risk
human papillomavirus (HPV) types 16 and 18 are re-
sponsible for more than 70% of cancer cases. Pro-
gression of cells from one phase to another is regu-
lated by cyclin-dependent kinases (CDKs) and cy-
clins.[3] Therefore, various therapeutic strategies that
target the major players in cell-cycle regulation have
been developed; these include compounds that
cause reactivation or overexpression of cellular CDK
inhibitors such as p21. Increased expression of CDK
inhibitors causes cytostasis and senescence.[4] Al-
though there is no mutation in the p53 gene in
human HeLa S3 cervical cancer cells, p53 function
and its production are impaired due to the expres-
(2),[18] and BMS 387032 (3),[19] as shown in Figure 1. However,
there are opportunities to identify and develop additional
sion of the E6 oncoprotein of HPV, which promotes
the degradation of p53.[5,6] Abrogation of functional
p53 is responsible for malignant transformation.[7]
Figure 1. Structures of flavopiridol (1), roscovitine (2), BMS 387032 (3), anthranilamide
AAL993 (4), CI-1040 (5), and anthranilamide–pyrazolo[1,5-a]pyrimidine conjugates 6a–x.
Therefore, any drug that enhances p53 production apart from
cell-cycle control is likely to be of great value in the control
and eradication of cervical cancer.[8]
[a] Dr. A. Kamal, J. R. Tamboli, A. Viswanath, A. Mallareddy
Division of Organic Chemistry
CSIR-Indian Institute of Chemical Technology, Hyderabad 500007 (India)
E-mail: ahmedkamal@iict.res.in
Pyrazolo[1,5-a]pyrimidine derivatives have been reported as
inhibitors of CDKs that are involved in mediating the transmis-
sion of mitogenic signals and numerous other cellular
events[9–15] including cell proliferation, migration, differentia-
tion, metabolism, and immune responses. It has also been ob-
served that many of these derivatives may block the prolifera-
tion of various cancer cell lines.[16] A number of selective CDK
inhibitors have been described, and some of them currently
undergoing clinical trials are flavopiridol (1),[17] roscovitine
[b] Dr. M. J. Ramaiah, G. Koteswara Rao, Dr. S. N. C. V. L. Pushpavalli,
Dr. M. Pal-Bhadra
Chemical Biology Laboratory
CSIR-Indian Institute of Chemical Technology, Hyderabad 500007 (India)
E-mail: manika@iict.res.in
[c] Dr. S. F. Adil
Chemistry Department, College of Science
King Saud University, Riyadh (Saudi Arabia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200205.
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novel CDK inhibitors that possess superior biological profiles
than those of the presently known candidates. Hence, a consid-
erable challenge in this area is the identification of new conju-
gates composed of pharmacophores of known antitumor
agents that enhance selectivity and antitumor activity. An an-
thranilamide moiety is present in agents such as AAL993 (4)
and PD 184352 (CI-1040; 5),[20,21] which is considered responsi-
ble for their potent antitumor properties[22,23] (Figure 1). Simi-
larly, over the last decade, a number of piperazine derivatives
have been synthesized to exploit their chemotherapeutic po-
tential.[24–26] Symmetrical bifunctional agents equipped with
a piperazine moiety in the linker have been reported as
a promising class of antitumor compounds with remarkable se-
lectivity against colon cancers.[27] Recently, trans-diamine di-
chloroplatinum(II) complexes with piperazine ligands were re-
ported that exhibit significant cytotoxicity.[28] In continuation of
these efforts, the present work describes the design and syn-
thesis of a series of anthranilamide–pyrazolo[1,5-a]pyrimidine
conjugates (6a–x) to explore their potential as a new class of
anticancer agents. All these compounds were evaluated for
their anticancer activity against a panel of human cancer cell
lines, and the most promising compounds among them (6 f, 6l
and 6r) were evaluated for cytotoxicity, cell-cycle effects, in-
duction of apoptosis, and effects on tumor suppressor genes
(p53 and p21). Interestingly, this new class of conjugates has
a unique mode of action that differs from that of other known
antitumor compounds.
sodium methoxide to provide the b-diketo esters 8a–f. Hetero-
cycle formation via dehydration coupling with an aza-heterocy-
clic amine, i.e., 3-amino-5-phenylpyrazole, afforded various
substituted 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
esters[30] (9a–f), which, upon base hydrolysis with sodium hy-
droxide, provided the corresponding carboxylic acids 10a–f.
One of the precursors 14c was synthesized as reported[31] by
us previously; however, the other precursors 14a, 14b, and
14d were prepared by treatment of isatoic anhydride (11) with
N-Boc piperazine at a molar ratio of 1:1.2, and then held at
reflux in 1,4-dioxane to give the 4-(2-aminobenzoyl)-N-Boc pi-
perazine 12 in nearly quantitative yield. Upon reductive amina-
tion of this intermediate with various heteroaryl and aromatic
aldehydes using sodium cyanoborohydride in methanol with
catalytic amounts of acetic acid afforded compounds 13a–d.
These, upon deprotection with triflouroacetic acid, gave the re-
quired precursors 14a–d as shown in Scheme 2. Synthesis of
Results and Discussion
Scheme 2. Reagents and conditions: a) N-Boc piperazine, 1,4-dioxane, reflux,
4 h; b) R4-CHO, NaCNBH3, MeOH, CH3COOH, RT, 22 h; c) TFA, CH2Cl2, RT, 5 h.
Chemistry
Synthesis of the target conjugates was performed in a conven-
ient manner as outlined in Schemes 1, 2 and 3. As shown in
Scheme 1, the b-diketo esters 8a, 8b, 8d, and 8e were synthe-
sized as reported earlier.[29] The various substituted acetophe-
nones were oxylated by dimethyl oxalate in the presence of
anthranilamide–pyrazolo[1,5-a]pyrimidine conjugates 6a–x was
carried out by using these precursors (14a–f). The target con-
jugates were synthesized by coupling 14a–d to different sub-
stituted
2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
acids (10a–f) in presence of EDCI and HOBt to afford the corre-
sponding anthranilamide–pyrazolo[1,5-a]pyrimidine conjugates
6a–x (Scheme 3). All the synthesized compounds were charac-
1
terized by H and 13C NMR spectroscopy, as well as mass spec-
trometry. Conjugates 6a–x were evaluated for their anticancer
activity in selected human cancer cell lines.
Biology
Anticancer activity
Some representative anthranilamide–pyrazolo[1,5-a]pyrimidine
conjugates were tested against 60 human cancer cell lines de-
rived from nine types of cancer (lung, leukemia, colon, melano-
ma, ovarian, renal, prostate, breast, and CNS) at a single dose
at the National Cancer Institute (NCI; Bethesda, MD, USA).
Three anthranilamide–pyrazolo[1,5-a]pyrimidine conjugates—
6 f, 6l, and 6r—that exhibited considerable activity in the pri-
Scheme 1. Reagents and conditions: a) dimethyl oxalate, NaOMe, THF, RT,
8 h; b) HCl (cat.), EtOH, reflux, 2 h; c) 2n NaOH, MeOH, reflux, 2 h.
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mary screen were further evalu-
ated against the panel of 60
human cancer cell lines at five
concentrations, and the results
are listed in Table 1. These three
compounds exhibit
a
broad
spectrum of activity against vari-
ous cancer cell lines with GI50
values in the range of 0.58–
53 mm. Specifically, conjugate 6r
exhibits
significant
activity
against most of the cancer cell
lines, with GI50 values ranging
from 0.58–6.8 mm.
Cell viability (MTT) assay
Scheme 3. Reagents and conditions: a) EDCI/HOBt, CH2Cl2, 08C!RT, 8 h.
In view of the encouraging GI50
Table 1. Anticancer activity of anthranilamide–pyrazolo[1,5-a]pyrimidine
conjugates in human cancer cell lines.
values, we next evaluated the
cytotoxicity of these compounds by MTT assay. Compounds
such as 6 f, 6l, and 6r, which exhibited efficient antiprolifera-
tive activity in the preliminary screen, were evaluated for their
cytotoxicity against two representative human cervical cancer
cell lines: HeLa and SiHa; MTT assays were performed by treat-
ing cells for this compound series.[32] Determination of prolifer-
ation by MTT assay clearly showed that all three compounds
are highly cytotoxic, more so than roscovitine, a positive con-
trol used in the study. Concentrations <1 mm were found to
have negligible effects on cytotoxicity. Dose–response analyses
were carried out at conjugate concentrations of 1, 2, and 4 mm,
and from these data, IC50 values were deduced. IC50 values for
these conjugates were in the ranges of 1.46–2.72 and 1.56–
2.70 mm for the HeLa and SiHa cell lines, respectively. From the
biological data (inhibition of proliferation) for all the conju-
gates, it is apparent that compounds 6 f, 6l, and 6r are the
most effective in both cervical cancer cell lines (Table 2). Inter-
estingly, the substitution pattern (R1, R2, and R3) on the 7-
phenyl ring of the pyrazolo[1,5-a]pyrimidine subunit considera-
bly affects the anticancer activity of these conjugates. Trisubsti-
tuted compounds such as 3,4,5-trimethoxy analogues display
higher potency than those of other substitution patterns on
the pyrazolo[1,5-a]pyrimidine moiety phenyl ring. In contrast,
heteroaryl and aromatic aldehyde substitutions on the phenyl
ring of the anthranilamide subunit do not exhibit a significant
effect on the potency of such analogues. These aspects reveal
that 3,4,5-trimethoxy substitution on the 7-phenyl ring of the
pyrazolo[1,5-a]pyrimidine subunit is essential for maintaining
antitumor activity.
GI50 [mm]
GI50 [mm]
Cancer panel
Cell lines
6 f[b] 6l[c] 6r[d] Cancer panel
6 f[b] 6l[c] 6r[d]
Cell lines
Leukemia
HL-60(TB)
K-562
MOLT-4
SR
Ovarian
3.7 6.1 1.98 IGROV1
1.9 1.38 1.5 OVCAR-3
4.9 43 3.3 OVCAR-4
NT[e] 3.3 0.58 OVCAR-5
3.5 NT[e] NT[e] OVCAR-8
4.0 NT[e] NT[e] NCI/ADR-RES
SK-OV-3
5.58 13.9 7.7
2.5 3.0 2.69
6.83 5.5 4.37
12 15 10
4.47 30 4.3
1.2 2.6 1.9
2.67 5.5 4.4
CCRF-CEM
RPMI-8226
Non-small-cell lung
A549/ATCC
EKVX
Renal
2.8 5.6 3.5 786-0
6.0 53 4.9 A498
3.8 11 6.0 ACHN
NT[e] 2.41 NT[e] CAKI-1
8.4 6.39 4.44 RXF 393
4.3 15.7 4.96 SN12C
5.3 NT[e] 5.6 TK-10
2.8 4.26 3.3 UO-31
1.0 1.67 2.2
4.6 11.4 4.49
2.99 8.4 2.1
7.3 4.9 4.2
1.9 2.78 1.39
2.0 2.45 2.3
5.0 29 17
6.0 20 6.8
4.87 6.99 1.5
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
Prostate
1.3 4.0 4.1 PC-3
7.3 14.3 5.8 DU-145
3.6 4.49 NT[e]
3.6 4.43 4.2
1.1 3.78 3.5
2.2 4.18 2.3
3.27 5.0 3.96
1.2 3.16 2.78
2.98 4.7 3.67
Breast
5.2 5.9 4.89 MCF7
2.3 3.78 2.18 MDA-MB-231/ATCC 3.8 8.67 7.0
1.5 2.5 3.0
2.7 9.85 2.5 HS 578T
5.0 8.9 6.7 BT-549
1.56 3.3 1.98 T-47D
3.6 5.76 3.37 MDA-MB-468
Melanoma
3.79 6.0 4.8 SK-MEL-28
7.3 NT[e] NT[e] SK-MEL-5
3.8 5.9 4.0 UACC-257
0.3 1.16 0.66 UACC-62
3.2 3.7 4.37
2.66 36 3.8
5.3 6.26 3.27
3.2 8.38 NT[e]
2.46 2.71 2.1
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
Effect on cell cycle
12.9 9.6 5.2
1.46 2.85 1.58
3.9 NT[e] 4.97
2.3 2.8 2.37
Previous studies have demonstrated that roscovitine, a CDK in-
hibitor, controls cell growth by cell-cycle arrest.[33] Therefore,
we considered it important to understand the role of these
conjugates (6a–x) on cell-cycle progression in cells where p53,
the main cell-cycle checkpoint, is expressed at negligible
levels.[34,35] Treatment of HeLa cells with these compounds at
[a] Compound concentration required to decrease cell growth to half
that of untreated cells. [b] 6 f (NSC755290). [c] 6l (NSC755289). [d] 6r
(NSC755288). [e] Not tested.
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2 mm (the IC50 concentration) caused an increase in the per-
centage of cells in the G2/M phase and a decrease of those in
the G1 phase (i.e., G2/M arrest). However, treatment of SiHa
cells with the same compounds caused G1 arrest (Figure 2; see
also Supporting Information tables 3 and 4).
BrdU incorporation assay
Interestingly, compounds 6 f, 6l, and 6r showed enhanced cy-
totoxicity apart from this significant effect on cell cycle, there-
by prompting us to take up a detailed investigation on these
conjugates. The cell-cycle arrest caused by 6 f, 6l, 6r, and ro-
scovitine (ros) was confirmed by a 5-bromo-2-deoxyuridine
(BrdU) cell proliferation assay (Figure 3). HeLa cells treated with
conjugates showed no change in BrdU incorporation relative
to control. This confirms G2/M arrest for HeLa, whereas SiHa
cells treated with conjugates showed a decrease in BrdU incor-
poration; these data strongly confirm G1 phase cell-cycle
arrest. It is plausible that different cells originated from the
same cancer phenotype respond differently to the same conju-
gates.
Table 2. In vitro cytotoxicity of compounds.
IC50 [mm][a]
Compound
SiHa
HeLa
6a
6b
6c
6d
6e
6 f
6g
6h
6i
6j
6k
2.08ꢀ0.09
2.71ꢀ0.12
2.24ꢀ0.22
2.23ꢀ0.18
2.07ꢀ0.44
1.91ꢀ0.17
1.93ꢀ0.32
2.33ꢀ0.39
2.16ꢀ0.11
2.19ꢀ0.50
1.83ꢀ0.49
1.61ꢀ0.30
2.06ꢀ0.23
2.04ꢀ0.48
2.35ꢀ0.05
2.34ꢀ0.38
1.81ꢀ0.22
1.56ꢀ0.30
2.14ꢀ0.42
1.99ꢀ0.17
2.71ꢀ0.39
2.40ꢀ0.16
2.40ꢀ0.58
2.31ꢀ0.54
2.69ꢀ0.35
2.05ꢀ0.05
2.42ꢀ0.44
1.81ꢀ2.24
1.89ꢀ0.13
2.46ꢀ0.24
1.51ꢀ0.20
2.72ꢀ0.02
2.02ꢀ0.38
2.02ꢀ0.16
1.73ꢀ0.18
1.56ꢀ0.12
1.54ꢀ0.18
2.07ꢀ0.36
2.38ꢀ0.30
2.35ꢀ0.05
2.12ꢀ0.15
1.99ꢀ0.17
1.46ꢀ0.15
1.95ꢀ0.19
1.99ꢀ0.64
2.13ꢀ0.05
1.95ꢀ0.42
2.35ꢀ0.27
1.76ꢀ0.24
2.20ꢀ0.12
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
Roscovitine[b]
Figure 3. BrdU assay: HeLa and SiHa cells were treated with compounds
(ros, 6 f, 6l, and 6r) at a final concentration of 2 mm and incubated for 24 h.
BrdU incorporation was monitored to confirm G1 cell-cycle arrest by test
compounds in SiHa cells. Statistical significance of effects observed in com-
pound-treated cells relative to control (ctrl): ***p<0.001, **p<0.01.
[a] Compound concentration required to inhibit cancer cell proliferation
by 50%; data are expressed as the mean ꢀSD from the dose–response
curves of at least three independent experiments. [b] Positive control ref-
erence compound.
Figure 2. Cell-cycle distribution: HeLa (top row) and SiHa (bottom row) cells treated with roscovitine, 6 f, 6l, and 6r were studied immediately following an in-
cubation period of 24 h. HeLa cells show an increased percentage of cells arrested in the G2/M phase relative to control cells, whereas SiHa cells exhibit G1
cell-cycle arrest.
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Effect on p53 and dependent genes
is indispensible for DNA fragmentation, chromatin condensa-
tion, and apoptotic body formation.[40] Because an increase in
the levels of p53 protein cause its nuclear translocation, we
were interested in investigating downstream signaling events
such as induction of apoptosis, which is caused by caspases.
Therefore, caspase-3 activity was assayed to understand the in-
duction of apoptosis by these compounds. Interestingly, com-
pound-treated cells showed a near twofold increase in cas-
pase-3 activation relative to control (untreated) cells; the
known CDK inhibitor roscovitine was used as a positive control
(Figure 5). Thus, this study revealed that the caspase-mediated
apoptotic pathway is responsible for the antiproliferative activi-
ty of these compounds (Figure 6).
p53 is a tumor suppressor that plays a crucial role in cell-cycle
arrest at G1 and G2/M and in apoptotic processes.[36,37] In gener-
al, the activation of p53 occurs by phosphorylation at serine
residues 46 and 15; it then translocates to the nucleus upon
receipt of stress signals induced by anticancer agents.[38] Imme-
diately after activation, p53 further controls genes such as p21,
Bcl2, and BAX,[38] each of which contain p53 binding sites in
their promoters. Thus HeLa cells were treated with compounds
6 f, 6l, and 6r at 2 mm (Figure 4A), and after incubation for
24 h, gene expression patterns were studied by RT-PCR analy-
sis. Interestingly, p53 and p21 transcript levels increased in the
compound-treated cells, wherein GAPDH serves as an internal
control. To further confirm p53 activation, immunofluorescence
experiments were carried out with 6r-treated HeLa cells, with
roscovitine as control. We observed complete nuclear translo-
cation in compound-treated cells, whereas control-untreated
cells showed p53 protein localization in both the nucleus and
cytoplasm. This strongly suggests the p53-activating nature of
these compounds[38] (Figure 4B). We also observed a decrease
in the levels of Bcl2 proteins and increased expression of phos-
phorylated forms of p53 as well as BAX protein levels in com-
pound-treated cells (Figure 4C). These results support the acti-
vation of p53 by these conjugates.
Conclusions
In the present study, a series of anthranilamide–pyrazolo[1,5-
a]pyrimidine conjugates were synthesized and evaluated for
their anticancer activity. Cervical cancer cell lines that express
negligible p53 protein levels were selected in this study. These
compounds exhibited significant anticancer activity, with IC50
values in the ranges of 1.46–2.72 and 1.56–2.7 mm in HeLa and
SiHa cells, respectively. Compounds 6 f, 6l, and 6r were found
to be much more cytotoxic than the other compounds in this
series. Studies also showed that 6 f, 6l, and 6r caused the
highest G2/M cell-cycle arrest in HeLa cells and G1 phase arrest
in SiHa cells. They not only regulate cell cycle, but also induce
expression of p53 as well as p21 transcript levels. These com-
pounds also induce the BAX protein and decrease the Bcl2
protein levels, which are dependent on p53 and cause apopto-
sis in a caspase-3 dependent manner. Interestingly, molecules
Effect on caspase-3 activation
Caspases are cysteine proteases that are known to be the
main executors in the process of apoptosis.[39] Among all cas-
pases, caspase-3 is a frequently activated death protease and
Figure 4. Purine conjugates activate p53 and induce mitochondria-mediated apoptosis: A) RT-PCR analysis was conducted to examine the change in gene ex-
pression levels of p53 and its target gene p21 in HeLa cells treated with roscovitine, 6 f, 6l, and 6r for 24 h. B) p53 localization was examined in cells treated
with conjugate 6r; roscovitine was used as positive control. C) The activated (i.e., phosphorylated) forms of p53 as well as genes that are tightly associated
with apoptosis, such as BAX and Bcl2, were examined by Western blot analysis; b-actin was used as loading control.
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60 GF254, and visualization was carried out by UV light or iodine in-
dicator. Column chromatography was performed with Merck 60–
120 mesh silica gel. 1H NMR spectra were recorded on Bruker
UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR Unity (400,
500 MHz) instruments. Chemical shifts (d) are reported in ppm
downfield from an internal TMS standard. ESIMS data were record-
ed on a Micromass Quattro LC using ESI+ software at a capillary
voltage of 3.98 kV and ESI mode positive ion trap detector. High-
resolution MS (HRMS) data were recorded on a QSTAR XL Hybrid
MS–MS mass spectrometer. Melting points were determined with
an Electro thermal melting point apparatus, and are uncorrected.
High-performance liquid chromatography (HPLC) analyses for de-
termining the purity of synthesized compounds were performed
on a Shimadzu SPD-10A (UV/Vis detector) LC-10AT instrument
[column: Luna 5 mm C18(2) 250ꢁ4.60 mm Mightysil RP-18 GP 250ꢁ
4.6 mm (5 mm); mobile phase: 80% A (CH3CN) and 20% B (H2O) in
15 min; flow rate: 1 mLminꢁ1; injected sample: 10 mL; column tem-
perature: 278C; wavelength: 254 nm]. The purity of all compounds
was ꢂ95% based on analytical HPLC. See the Supporting Informa-
tion for data for compounds 8c–14d.
Figure 5. Effect of purine conjugates on caspase-3 activity in HeLa cells: The
increased activity of caspase-3 during apoptosis after treatment with conju-
gates 6 f, 6l and 6r at 2 mm was measured by fluorimetry. The cleavage of
the peptide substrate by active caspase-3 releases the fluorophore AFC,
which was quantified at an excitation wavelength of 400 nm and an emis-
sion wavelength of 505 nm (DEVD-CHO is the indicated inhibitor of caspase-
3 in the rightmost column). Roscovitine (ros) was used as positive control.
Statistical significance of effects observed in compound-treated cells relative
to control (ctrl): ***p<0.001, **p<0.01.
(2,7-Diphenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(3,4,5-trime-
thoxybenzylamino)benzoyl)piperazin-1-yl)methanone (6a): Con-
jugate 6a was prepared by amide bond formation between piper-
azin-1-yl-(2-(3,4,5-trimethoxybenzylamino)phenyl)methanone (14a,
160 mg, 0.41 mmol) and 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-
carboxylic acid (7a, 154 mg, 1.2 mmol) in dry CH2Cl2. The coupling
reagents EDCI (1.2 mmol) and HOBt (1.2 mmol) were added, and
the reaction mixture was stirred at room temperature for 10 h.
After completion of reaction as indicated by TLC, the reaction mix-
ture was quenched with NaHCO3 and extracted in EtOAc (4ꢁ
25 mL) from the ice-cold aqueous layer and dried over anhydrous
Na2SO4. The resulting product 6a was purified by column chroma-
tography to afford a yellow solid: 223 mg, 80% yield. Rf =0.29
~
(EtOAc/hexane, 7:3); mp: 111–1128C; IR (KBr) n=3373, 2932, 1757,
1627, 1550, 1234, 841, 760 cmꢁ1 1H NMR (300 MHz, CDCl3): d=
;
3.78–3.82 (m, 4H), 3.84 (s, 9H), 3.87–3.96 (m, 4H), 4.29 (s, 2H), 5.6
(brs, 1H), 6.58 (d, 2H, J=9 Hz), 6.68–6.74 (m, 2H), 7.07–7.15 (m,
2H), 7.34 (d, 1H, J=5.2 Hz), 7.41–7.53 (m, 5H), 7.61 (t, 2H, J=
2.2 Hz), 8.01 (dd, 2H, J=1.5, 8.3 Hz), 8.20–8.25 ppm (m, 2H);
13C NMR (300 MHz, CDCl3): d=42.9, 46.9, 47.9, 56, 60.7, 94.2, 94.6,
103.8, 107.1, 112.2, 116.1, 116.2, 118.5, 126.4, 126.5, 127.1, 127.8,
128.5, 128.9, 129.2, 129.6, 130.7, 131.5, 132.4, 134.5, 147.2, 149,
151.1, 156.8, 165.8, 170.7 ppm; MS (ESI): 683 [M+H]+; HRMS (ESI)
calcd for C40H39N6O5 [M+H]+ 683.2740, found: 683.2732; anal.
calcd for C40H38N6O5: C 70.36, H 5.61, N 12.31, O 11.72, found: C
70.32, H 5.55, N 12.28, O 11.70; purity 95.10% (HPLC).
Figure 6. Schematic diagram representing the action of analogues on p53
activation by phosphorylation: Activated p53 translocates to the nucleus
and regulates Mdm2 levels and further acts on mitochondria, where a de-
crease in Bcl2 oncoprotein and an increase BAX protein levels causes cas-
pase-mediated apoptosis.
(7-(4-Nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-
(3,4,5-trimethoxybenzylamino)benzoyl)piperazin-1-yl)metha-
none (6b): Compound 6b was prepared by following the method
described for the preparation of compound 6a, employing pipera-
zin-1-yl-(2-(3,4,5-trimethoxybenzylamino)phenyl)methanone (14a,
160 mg, 0.41 mmol) and 7-(4-nitrophenyl)-2-phenylpyrazolo[1,5-
a]pyrimidine-5-carboxylic acid (7b, 176 mg, 0.49 mmol) to afford
pure compound 6b as a yellow solid in 244 mg, 82% yield. Rf =
that can induce p53, particularly in cervical cancer cell lines
(HeLa and SiHa), are of great value and are likely to be useful
in the treatment of cancer, either alone or in combination with
other anticancer agents.
~
0.31 (EtOAc/hexane, 7:3); mp: 113–1148C; IR (KBr) n=3380, 3065,
Experimental Section
2925, 1629, 1604, 1549, 1257, 831, 755 cmꢁ1 1H NMR (300 MHz,
;
Chemistry
CDCl3): d=3.69–3.79 (m, 4H), 3.80 (s, 3H), 3.83 (s, 6H), 3.84–3.91
(m, 2H), 3.92–4.0 (m, 2H), 4.27 (s, 2H), 5.69 (brs, 1H), 6.55 (s, 2H),
6.66 (t, 2H, J=8.3 Hz), 7.03 (s, 1H), 7.09 (d, 1H, J=6.0 Hz), 7.17–
7.24 (m, 2H), 7.34 (s, 1H), 7.36–7.48 (m, 3H), 7.59 (t, 2H, J=3.7 Hz),
7.97 (d, 2H, J=7.5 Hz), 8.19–8.25 ppm (m, 1H); 13C NMR (300 MHz,
CDCl3): d=42.8, 47.5, 47.9, 56.4, 60.9, 94.4, 103.1, 106.9, 112, 114,
All chemicals and reagents were obtained from Aldrich (Sigma–Al-
drich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai,
India), and were used without further purification. Reactions were
monitored by TLC performed on glass plates containing silica gel
&6
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ChemMedChem 0000, 00, 1 – 13
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These are not the final page numbers!

Anthranilamide–Pyrazolo[1,5-a]pyrimidine Conjugates
MED
116.2, 118.2, 126.3, 126.9, 127.4, 128.3, 128.7, 128.9, 129.3, 129.8,
130.5, 131.7, 132.6, 134.7, 148, 149.2, 151.3, 152.4, 156.9, 162.2,
165.5, 170.8 ppm; MS (ESI): 728 [M+H]+; HRMS (ESI) calcd for
C40H38N7O7 [M+H]+ 728.2432, found: 728.2413; anal. calcd for
C40H37N7O7: C 66.01, H 5.12, N 13.47, O 15.39, found: C 65.98, H
5.09, N 13.46, O 15.37; purity 95.30% (HPLC).
6.71 (t, 2H, J=7.9 Hz), 7.05–7.16 (m, 3H), 7.24 (d, 1H, J=8.3 Hz),
7.35 (s, 1H), 7.41–7.52 (m, 3H), 7.89 (dd, 1H, J=2.2, 8.3 Hz), 7.98–
8.06 ppm (m, 3H); 13C NMR (300 MHz, CDCl3): d=42.9, 47.4, 48, 56,
60.8, 94.4, 104, 106.2, 110.8, 112.2, 112.3, 116.2, 118.2, 122.7, 123.2,
126.4, 127.9, 128.7, 129.2, 131.4, 132.5, 134.5, 136.9, 146.8, 147,
148.5, 149.2, 151, 151.6, 153.3, 156.6, 166, 170.7 ppm; MS (ESI): 742
[M+]; purity 98.20% (HPLC).
(2-Phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(3,4,5-tri-
methoxybenzylamino)benzoyl)piperazin-1-yl)methanone
(6c):
(2-Phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)(4-(2-(3,4,5-trimethoxybenzylamino)benzoyl)piperazin-1-yl)-
methanone (6 f): Compound 6 f was prepared by following the
method described for the preparation of compound 6a, employing
piperazin-1-yl-(2-(3,4,5-trimethoxybenzylamino)phenyl)methanone
Compound 6c was prepared by following the method described
for the preparation of compound 6a, employing piperazin-1-yl-(2-
(3,4,5-trimethoxybenzylamino)phenyl)methanone (14a, 160 mg,
0.41 mmol) and 2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidine-5-car-
boxylic acid (7c, 161 mg, 0.49 mmol) to afford pure compound 6c
as a yellow solid in 214 mg, 75% yield. Rf =0.33 (EtOAc/hexane,
(14a,
160 mg,
0.41 mmol)
and
2-phenyl-7-(3,4,5-
trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7 f,
198 mg, 0.49 mmol) to afford pure compound 6 f as a yellow solid
in 221 mg, 70% yield. Rf =0.23 (EtOAc/hexane, 7:3); mp: 114–
~
7:3); mp: 116–1178C; IR (KBr) n=3371, 2927, 2835, 1631, 1605,
1550, 1239, 838, 755 cmꢁ1 1H NMR (300 MHz, CDCl3): d=2.49 (s,
;
~
3H), 3.75–3.83 (m, 4H), 3.85–3.91 (m, 4H), 3.92 (s, 3H), 3.93 (s, 6H),
4.36 (s, 2H), 5.47 (brs, 1H), 6.74 (t, 1H, J=4.4 Hz), 7.06–7.14 (m,
3H), 7.19 (t, 1H, J=8.8 Hz), 7.27–7.29 (m, 2H), 7.31 (s, 1H), 7.38–
7.42 (m, 3H), 7.46 (t, 2H, J=7.7 Hz), 8.01 (d, 2H, J=6.6 Hz),
8.15 ppm (d, 2H, J=7.7 Hz); 13C NMR (300 MHz, CDCl3): d=21.6,
42.9, 43.1, 47.5, 56.1, 56.2, 61, 94.5, 105.9, 106.7, 116.9, 117.6, 118.7,
118.9, 126.6, 127.6, 127.9, 128.7, 128.9, 129.3, 129.4, 130.3, 130.4,
131, 132.6, 142, 145.8, 147.3, 149.2, 151.2, 153.5, 156.8, 161.2,
166.0 ppm; MS (ESI): 697 [M+H]+; HRMS (ESI) calcd for C41H41N6O5
[M+H]+ 697.3109, found: 697.3118; anal. calcd for C41H40N6O5: C
70.67, H 5.79, N 12.06, O 11.48, found: C 70.62, H 5.74, N 12.04, O
11.46; purity 95.10% (HPLC).
115 8C; IR (KBr) n=3384, 2934, 2834, 1627, 1581, 1551, 1239, 832,
1
761 cmꢁ1; H NMR (300 MHz, CDCl3): d=3.72–3.82 (m, 4H), 3.84 (s,
9H), 3.86–3.95 (m, 4H), 3.97 (s, 6H), 3.99 (s, 3H), 4.29 (s, 2H), 5.59
(brs, 1H), 6.59 (d, 2H, J=4.4 Hz), 6.69–6.74 (m, 2H), 7.09–7.15 (m,
2H), 7.36 (s, 1H), 7.40–7.52 (m, 4H), 7.57 (s, 2H), 8.0 ppm (d, 2H,
J=7.7 Hz); 13C NMR (300 MHz, CDCl3): d=42.9, 44.2, 47.9, 56, 56.3,
60.7, 61, 94.6, 104.1, 106.7, 107, 107.3, 112.3, 116.2, 118.2, 125.3,
126.4, 127.8, 127.9, 128.8, 129.2, 131.5, 132.8, 134.5, 137, 140.8,
146.8, 147.4, 149.2, 151, 153.3, 155.8, 165.8, 170.7 ppm; MS (ESI):
773 [M+H]+; HRMS (ESI) calcd for C43H45N6O8 [M+H]+ 773.3293,
found: 773.3289; anal. calcd for C43H44N6O8: C, 66.83; H, 5.74; N,
10.87; O, 16.56 found: C, 66.85, H 5.70, N 10.84, O 16.54; purity
98.40% (HPLC).
(7-(4-Methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-
(2-(3,4,5-trimethoxybenzylamino)benzoyl)piperazin-1-yl)metha-
none (6d): Compound 6d was prepared by following the method
described for the preparation of compound 6a, employing pipera-
zin-1-yl-(2-(3,4,5-trimethoxybenzylamino)phenyl)methanone (14a,
160 mg, 0.41 mmol) and 7-(4-methoxyphenyl)-2-phenylpyrazolo-
[1,5-a]pyrimidine-5-carboxylic acid (7d, 169 mg, 0.49 mmol) to
afford pure compound 6d as a yellow solid in 221 mg, 76% yield.
(2,7-Diphenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(furan-2-ylme-
thylamino)benzoyl)piperazine-1-yl)methanone (6g): Compound
6g was prepared by following the method described for the prep-
aration of compound 6a, employing (2-(furan-2-ylmethylamino)-
phenyl)(piperazin-1-yl)methanone (14b, 160 mg, 0.56 mmol) and
2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
acid
(7a,
211 mg, 0.67 mmol) to afford pure compound 6g as a yellow solid
in 231 mg, 71% yield. Rf =0.56 (EtOAc/hexane, 7:3); mp: 110–
~
Rf =0.28 (EtOAc/hexane, 7:3); mp: 104–1058C; IR (KBr) n=3392,
2931, 2837, 1630, 1601, 1549, 1256, 831 cmꢁ1 1H NMR (300 MHz,
;
111 8C; IR (KBr) n=3334, 3062, 2922, 1654, 1611, 1581, 1224, 847,
~
1
CDCl3): d=3.64–3.73 (m, 4H), 3.74 (s, 3H), 3.77 (s, 6H), 3.78–3.84
(m, 2H), 3.86 (s, 3H), 3.87–3.93 (m, 2H), 3.78–3.84 (m, 2H), 4.20 (s,
2H), 5.62 (brs, 1H), 6.49 (s, 2H), 6.55–6.63 (m, 2H), 6.94 (s, 1H),
6.99–7.05 (m, 3H), 7.09–7.21 (m, 2H), 7.28–7.41 (m, 3H), 7.92 (d,
2H, J=6.9 Hz), 8.21 ppm (d, 2H, J=8.8 Hz); 13C NMR (300 MHz,
CDCl3): d=42.9, 47.5, 48, 55.5, 56.1, 60.8, 94.4, 104.1, 106.2, 112.3,
114, 116.3, 122.7, 126.6, 127.9, 128.8, 129.2, 131.3, 131.4, 131.5,
132.6, 134.6, 146.9, 147.1, 149.3, 151.1, 152, 153.4, 156.8, 162.2,
166.1, 170.8 ppm; MS (ESI): 713 [M+H]+; HRMS (ESI) calcd for
C41H41N6O6 [M+H]+ 713.3087, found: 713.3120; anal. calcd for
C41H40N6O6: C, 69.09; H, 5.66; N, 11.79; O, 13.47, found: C 69.03, H
5.69, N 11.80, O 13.44; purity 99.50% (HPLC).
767 cmꢁ1; H NMR (300 MHz, CDCl3): d=3.70–3.99 (m, 8H), 4.33 (s,
2H), 5.5 (brs, 1H), 6.21 (d, 1H, J=2.2 Hz), 6.29 (s, 1H), 6.64–6.77
(m, 2H), 7.01–7.11 (m, 2H), 7.17–7.24 (m, 1H), 7.34 (s, 2H), 7.36–
7.48 (m, 3H), 7.59 (d, 3H, J=2.8 Hz), 7.97 (d, 2H, J=7.1 Hz),
8.22 ppm (d, 2H, J=3.0 Hz); 13C NMR (300 MHz, CDCl3): d=40.8,
42.9, 47.4, 94.6, 107.1, 110.3, 112, 116.5, 118.8, 126.6, 127.1, 127.9,
128.5, 128.7, 129, 129.2, 129.4, 130.5, 131.2, 131.3, 132.4, 141.9,
146.4, 147.2, 149, 151.2, 152.2, 156.8, 165.8, 170.5 ppm, MS (ESI):
583 [M+H]+; HRMS (ESI) calcd for C35H31N6O3 [M+H]+ 583.2890,
found: 583.2884; anal. calcd for C35H30N6O3: C 72.15, H 5.19, N
14.42, O 8.24, found: C 72.06, H 5.22, N 14.44, O 8.22; purity
98.85% (HPLC).
(7-(3,4-Dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-
yl)(4-(2-(3,4,5-trimethoxybenzylamino)benzoyl)piperazin-1-yl)-
methanone (6e): Compound 6e was prepared by following the
method described for the preparation of compound 6a, employing
piperazin-1-yl-(2-(3,4,5-trimethoxybenzylamino)phenyl)methanone
(14a, 160 mg, 0.41 mmol) and 7-(3,4-dimethoxyphenyl)-2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7e, 183 mg,
0.49 mmol) to afford pure compound 6e as a yellow solid in
222 mg, 73% yield. Rf =0.27 (EtOAc/hexane, 7:3); mp: 115–1168ꢁC1;
~
4-(2-(Furan-2-ylmethylamino)benzoyl)piperazin-1-yl)(7-(4-nitro-
phenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)methanone (6h):
Compound 6h was prepared by following the method described
for the preparation of compound 6a, employing (2-(furan-2-ylme-
thylamino)phenyl)(piperazin-1-yl)methanone
(14b,
160 mg,
0.56 mmol) and 7-(4-nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimi-
dine-5-carboxylic acid (7b, 241 mg, 0.67 mmol) to afford pure com-
pound 6h as a yellow solid in 245 mg, 70% yield. Rf =0.5 (EtOAc/
~
hexane, 7:3); mp: 107–1088C; IR (KBr) n=3383, 3012, 2962, 1625,
IR (KBr) n=3393, 2836, 1626, 1597, 1552, 1237, 813, 761 cm
;
1585, 1556, 1238, 841, 760 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d=
1H NMR (300 MHz, CDCl3): d=3.78–3.83 (m, 4H), 3.85 (s, 9H), 3.87–
3.73–3.81 (m, 4H), 3.84–3.88 (m, 2H), 3.89–3.93 (m, 2H), 4.33 (s,
3.97 (m, 4H), 4.02 (s, 6H), 4.29 (s, 2H), 5.61 (brs, 1H), 6.60 (s, 2H),
2H), 5.45 (brs, 1H), 6.23 (s, 1H), 6.31 (s, 1H), 6.72 (t, 1H, J=6.9 Hz),
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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ÞÞ
These are not the final page numbers!

A. Kamal, M. Pal-Bhadra, et al.
MED
~
6.78 (d, 1H, J=8.9 Hz), 7.07–7.15 (m, 2H), 7.27–7.29 (m, 1H), 7.32
(s, 1H), 7.36 (s, 1H), 7.41 (d, 1H, J=5.9 Hz), 7.46 (t, 2H, J=6.9,
7.9 Hz), 7.6 (t, 2H, J=2.0, 2.9 Hz), 8.0 (d, 2H, J=7.9 Hz), 8.22 ppm
(t, 2H, J=2.9, 3.9 Hz); 13C NMR (300 MHz, CDCl3): d=40.9, 42.9,
47.4, 94.6, 107.1, 110.3, 112, 116.6, 118.8, 119.1, 126.6, 127.9, 128.5,
128.7, 129.2, 129.4, 130.5, 131.1, 131.3, 132.4, 141.9, 146.4, 147.2,
149.1, 151.2, 152.2, 156.9, 165.8, 170.5 ppm; MS (ESI): 728 [M+H]+;
HRMS (ESI) calcd for C35H30N7O5 [M+H]+ 628.2289, found:
628.2283; anal. calcd for C35H29N7O5: C 66.98, H 4.66, N 15.62, O
12.75 found: C 66.91, H 4.62, N 15.64, O 12.73; purity 95.13%
(HPLC).
IR (KBr) n=3373, 3000, 2927, 1627, 1577, 1550, 1262, 852,
1
808 cmꢁ1; H NMR (300 MHz, CDCl3): d=3.58–3.95 (m, 8H), 3.99 (s,
6H), 4.33, (s, 2H), 5.61 (brs, 1H), 6.24 (d, 2H, J=2.9 Hz), 6.60–6.78
(m, 2H), 6.99–7.11 (m, 3H), 7.25 (s, 1H), 7.31–7.48 (m, 5H), 7.84 (d,
1H, J=8.8 Hz), 7.98 ppm (d, 3H, J=6.2 Hz); 13C NMR (300 MHz,
CDCl3): d=42.7, 42.9, 47.5, 56, 56.1, 94.4, 106.3, 107.1, 110.7, 110.8,
112.5, 112.8, 116.6, 118.7, 118.9, 122.8, 122.9, 123.2, 126.3, 126.5,
127.9, 128.8, 129.2, 131.4, 132.6, 142, 146.4, 146.8, 148.6, 151.7,
152.2, 156.7, 166.1, 170.6 ppm; MS (ESI): 643 [M+H]+; HRMS (ESI)
calcd for C37H35N6O5 [M+H]+ 643.2668, found: 643.2685; anal.
calcd for C37H34N6O5: C 69.14, H 5.33, N 13.08, O 12.45, found: C
69.17, H 5.30, N 13.06, O 12.42; purity 95.40% (HPLC).
(4-(2-(Furan-2-ylmethylamino)benzoyl)piperazin-1-yl)(2-phenyl-
7-p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)methanone (6i): Com-
pound 6i was prepared by following the method described for the
preparation of compound 6a, employing (2-(furan-2-ylmethylami-
no)phenyl)(piperazin-1-yl)methanone (14b, 160 mg, 0.56 mmol)
and 2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid
(7c, 220 mg, 0.67 mmol) to afford pure compound 6i as a yellow
solid in 226 mg, 68% yield. Rf =0.57 (EtOAc/hexane, 7:3); mp: 116–
(4-(2-(Furan-2-ylmethylamino)benzoyl)piperazin-1-yl)(2-phenyl-
7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)metha-
none (6l): Compound 6l was prepared by following the method
described for the preparation of compound 6a, employing (2-
(furan-2-ylmethylamino)phenyl)(piperazin-1-yl)methanone
(14b,
160 mg, 0.56 mmol) and 2-phenyl-7-(3,4,5-
trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7 f,
271 mg, 0.67 mmol) to afford pure compound 6l as a yellow solid
in 278 mg, 74% yield. Rf =0.3 (EtOAc/hexane, 7:3); mp: 107–1088C;
~
117 8C; IR (KBr) n=3374, 2927, 1742, 1612, 1577, 1254, 856,
1
764 cmꢁ1; H NMR (500 MHz, CDCl3): d=2.48 (s, 3H), 3.69–3.96 (m,
~
8H), 4.36 (s, 2H), 5.66 (brs, 1H), 6.59 (s, 1H), 6.74 (d, 2H, J=
8.1 Hz), 7.05–7.15 (m, 2H), 7.20 (t, 1H, J=7.5 Hz), 7.29 (d, 2H, J=
8.5 Hz), 7.35–7.53 (m, 6H), 8.02 (d, 2H, J=6.9 Hz), 8.15 ppm (d, 2H,
J=7.9 Hz); 13C NMR (300 MHz, CDCl3): d=21.5, 42.8, 42.9, 47.7,
94.4, 106.4, 112, 116.7, 119.1, 124.6, 125, 126.7, 127, 127.6, 127.9,
128.7, 129.4, 129.5, 129.8, 132.4, 140.1, 142.3, 142.7, 146.2, 147.5,
148.9, 151.6, 156.9, 166.4, 170.7 ppm; MS (ESI): 597 [M+H]+; HRMS
(ESI) calcd for C36H33N6O3 [M+H]+ 597.2461, found: 597.2469; anal.
calcd for C36H32N6O3: C 72.47, H 5.41, N 14.08, O 8.04 found: C
72.51, H 5.38, N 14.06, O 8.02; purity 98.63%, (HPLC).
IR (KBr) n=3375, 3054, 2932, 1627, 1578, 1550, 1245, 837,
762 cmꢁ1 1H NMR (300 MHz, CDCl3): d=3.73–3.83 (m, 4H), 3.85–
;
3.95 (m, 4H), 3.97 (s, 6H), 3.98 (s, 3H), 4.34 (s, 2H), 5.51 (brs, 1H),
6.24 (d, 1H, J=3.0 Hz), 6.33 (dd, 1H, J=2.2, 3.7 Hz), 6.7–6.82 (m,
2H), 7.11 (s, 1H), 7.25–7.32 (m, 2H), 7.37 (d, 2H, J=6.0 Hz), 7.41–
7.51 (m, 3H), 7.56 (s, 2H), 8.02 ppm (dd, 2H, J=1.5, 8.3 Hz);
13C NMR (300 MHz, CDCl3): d=40.9, 42.9, 47.5, 56.4, 61, 94.6, 106.7,
107, 107.1, 110.3, 112, 116.6, 118.8, 125.4, 126.4, 127.9, 128.8, 129.3,
130.5, 131.3, 132.5, 140.8, 141.9, 146.4, 146.8, 149.3, 151.2, 152.2,
153.1, 156.8, 165.9, 170.5 ppm; MS (ESI): 673 [M+H]+; HRMS (ESI)
calcd for C38H37N6O6 [M+H]+ 673.2774, found: 673.2777; anal.
calcd for C38H36N6O6: C 67.84, H 5.39, N 12.49, O 14.27, found: C
67.86,H 5.33, N 12.50, O 14.25; purity 98.26% (HPLC).
(4-(2-(Furan-2-ylmethylamino)benzoyl)piperazin-1-yl)(7-(4-me-
thoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)methanone
(6j): Compound 6j was prepared by following the method de-
scribed for the preparation of compound 6a, employing (2-(furan-
2-ylmethylamino)phenyl)(piperazin-1-yl)methanone (14b, 160 mg,
0.56 mmol) and 7-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyri-
midine-5-carboxylic acid (7d, 231 mg, 0.67 mmol) to afford pure
compound 6j as a yellow solid in 250 mg, 73% yield. Rf =0.36
(2,7-Diphenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(thiophen-2-yl-
methylamino)benzoyl)piperazin-1-yl)methanone (6m): Com-
pound 6m was prepared by following the method described for
the preparation of compound 6a, employing piperazin-1-yl-(2-(thi-
ophen-2-ylmethylamino)phenyl)methanone
(14c,
160 mg,
~
(EtOAc/hexane, 7:3); mp: 110–1118C; IR (KBr) n=3377, 2923, 1624,
0.53 mmol) and 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
acid (7a, 198 mg, 0.63 mmol) to afford pure compound 6m as
a yellow solid in 250 mg, 79% yield. Rf =0.50 (EtOAc/hexane, 7:3);
1604, 1549, 1257, 832, 760 cmꢁ1 1H NMR (300 MHz, CDCl3): d=
;
3.73–3.80 (m, 4H), 3.82–3.88 (m, 4H), 3.92 (s, 3H), 4.32 (s, 2H), 5.58
(brs, 1H), 6.20 (d, 1H, J=2.2 Hz), 6.27 (d, 1H, J=3.3 Hz), 6.67 (t,
1H, J=7.7 Hz), 6.74 (d, 1H, J=8.8 Hz), 7.0 (s, 1H), 7.05–7.10 (m,
3H), 7.20–7.25 (m, 1H), 7.30 (s, 1H), 7.33–7.39 (m, 2H), 7.43 (t, 2H,
J=7.7 Hz), 7.97 (d, 2H, J=7.7 Hz), 8.27 ppm (d, 2H, J=8.8);
13C NMR (300 MHz, CDCl3): d=40.8, 42.8, 47.4, 55.4, 94.4, 106.1,
107.1, 110.3, 112.0, 114.0, 116.6, 118.8, 122.6, 126.6, 127.9, 128.7,
129.1, 131.3, 131.7, 132.6, 141.9, 146.4, 146.9, 148.9, 149.2, 151.1,
155, 156.7, 162, 166, 170.5 ppm; MS (ESI): 613 [M+H]+; HRMS (ESI)
calcd for C36H33N6O4 [M+H]+ 613.2563, found: 613.2580; anal.
calcd for C36H32N6O4: C 70.57, H 5.26, N 13.72, O 10.45, found: C
70.52, H 5.28, N 13.75, O 10.42; purity 95.70% (HPLC).
~
mp: 117–1188C; IR (KBr) n=3012, 2931, 1757, 1684, 1631, 1588,
1234, 831, 767 cmꢁ1 1H NMR (300 MHz, CDCl3): d=3.75–3.84 (m,
;
4H), 3.85–3.96 (m, 4H), 4.54 (s, 2H), 5.49 (brs, 1H), 6.71–6.81 (m,
2H), 6.95–6.99 (m, 1H), 7.0–7.03 (m, 1H), 7.19 (d, 1H, J=2.4 Hz),
7.13 (dd, 1H, J=7.5, 7.7 Hz), 7.21 (dd, 1H, J=5.0, 4.9) Hz, 7.24–7.28
(m, 1H), 7.33 (d, 1H, J=2.2 Hz), 7.40–7.51 (m, 3H), 7.58–7.63 (m,
3H), 7.99–8.04 (m, 2H), 8.20–8.25 ppm (m, 2H); 13C NMR (300 MHz,
CDCl3): d=42.8, 42.9, 47.5, 94.6, 107.1, 112.1, 116.8, 119.2, 124.4,
124.8, 126.6, 126.8, 127.1, 127.7, 128.5, 128.7, 128.9, 129.2, 129.4,
130.5, 131.2, 131.3, 132.4, 142.6, 146.1, 147.2, 149, 151.2, 156.8,
165.8, 170.5 ppm; MS (ESI): 599 [M+H]+; HRMS (ESI) calcd for
C35H31N6O2S [M+H]+ 599.2228, found: 599.2223; anal. calcd for
C35H30N6O2S: C 70.21, H 5.05, N 14.04, O 5.34, S 5.36, found: C
70.18, H5.08, N 14.01, O 5.32, S 5.38; purity 95.80% (HPLC).
(7-(3,4-Dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-
yl)(4-(2-(furan-2-ylmethylamino)benzoyl)piperazin-1-yl)metha-
none (6k): Compound 6k was prepared by following the method
described for the preparation of compound 6a, employing (2-
(7-(4-Nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-
(furan-2-ylmethylamino)phenyl)(piperazin-1-yl)methanone
(14b,
(thiophen-2-ylmethyl)benzoyl)piperazin-1-yl)methanone
(6n):
160 mg, 0.56 mmol) and 7-(3,4-dimethoxyphenyl)-2-
Compound 6n was prepared by following the method described
for the preparation of compound 6a, employing piperazin-1-yl-(2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7e, 251 mg,
0.67 mmol) to afford pure compound 6k as a yellow solid in
251 mg, 70% yield. Rf =0.35 (EtOAc/hexane, 7:3); mp: 102–1038C;
(thiophen-2-ylmethylamino)phenyl)methanone
(14c,
160 mg,
0.53 mmol) and 7-(4-nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimi-
&8
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ChemMedChem 0000, 00, 1 – 13
ÝÝ
These are not the final page numbers!

Anthranilamide–Pyrazolo[1,5-a]pyrimidine Conjugates
MED
dine-5-carboxylic acid (7b, 226 mg, 0.63 mmol) to afford pure com-
pound 6n as a yellow solid in 272 mg, 80% yield. Rf =0.48 (EtOAc/
(7-(3,4-Dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-
yl)(4-(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)me-
thanone (6q): Compound 6q was prepared by following the
method described for the preparation of compound 6a, employing
piperazin-1-yl-(2-(thiophen-2-ylmethylamino)phenyl)methanone
(14c, 160 mg, 0.53 mmol) and 7-(3,4-dimethoxyphenyl)-2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7e, 236 mg,
0.63 mmol) to afford pure compound 6q as a yellow solid in
265 mg, 76% yield. Rf =0.32 (EtOAc/hexane, 7:3); mp: 96–978C; IR
~
hexane, 7:3); mp: 109–1108C; IR (KBr) n=3378, 2937, 2887, 1627,
1
1607, 1584, 841, 752 cmꢁ1; H NMR (300 MHz, CDCl3): d=3.74–4.03
(m, 8H), 4.53 (s, 2H), 5.64 (brs, 1H), 6.65–6.75 (m, 2H), 6.91–6.95
(m, 1H), 6.98 (d, 1H, J=3.0 Hz), 7.04 (s, 1H), 7.09 (d, 1H, J=7.5 Hz),
7.14–7.27 (m, 2H), 7.34 (s, 1H), 7.36–7.48 (m, 3H), 7.58 (t, 2H, J=
3.0 Hz), 7.97 (d, 2H, J=8.3 Hz), 8.22 (dd, 1H, J=3.0, 6.7 Hz),
8.3 ppm (d, 1H, J=8.3 Hz); 13C NMR (300 MHz, CDCl3): d=42.8,
47.4, 94.6, 106.7, 107.1, 112.1, 116.7, 119.1, 124.4, 124.8, 126.5,
126.8, 127.3, 127.7, 128.5, 128.7, 129.1, 129.4, 130.5, 131.2, 131.3,
132.3, 142.6, 146, 147.2, 149, 156.8, 165.8, 170.4 ppm; MS (ESI): 644
[M+H]+; HRMS (ESI) calcd for C35H30N7O4S [M+H]+ 644.2049,
found: 644.2068; anal. calcd for C35H29N7O4S: C, 65.30; H, 4.54; N,
15.23; O, 9.94; S, 4.98, found: C 65.25, H 4.57, N 15.25, O 9.91, S
4.96; purity 95.50%, (HPLC).
ꢁ1
(KBr) n=3372, 2925, 1626, 1576, 1551, 1261, 805 cm
;
1H NMR
~
(300 MHz, CDCl3): d=3.60–3.77 (m, 4H), 3.78–3.88 (m, 4H), 3.94 (s,
6H), 4.46, (s, 2H), 5.44 (brs, 1H), 6.59–6.77 (m, 2H), 6.86–7.10 (m,
5H), 7.11–7.24 (m, 2H), 7.28 (s, 1H), 7.32–7.46 (m, 3H), 7.81 (d, 1H,
J=6.7 Hz), 7.95 ppm (t, 3H, J=6.7 Hz); 13C NMR (300 MHz, CDCl3):
d=42.8, 42.9, 47.4, 56, 56.1, 94.4, 106.2, 110.8, 112.1, 112.5, 116.7,
119.2, 122.8, 123.2, 124.4, 124.8, 126.4, 126.8, 127.7, 128.7, 129.1,
131.1, 132.5, 142.6, 146.1, 146.7, 148.6, 149.3, 151.1, 151.7, 156.6,
165.9, 170.4 ppm; MS (ESI): 659 [M+H]+; HRMS (ESI) calcd for
C37H35N6O4S [M+H]+ 659.2440, found: 659.2432; anal. calcd for
C37H34N6O4S: C, 67.46; H, 5.20; N, 12.76; O, 9.71; S, 4.87, found: C
67.49, H 5.18, N 12.73, O 9.73, S 4.85. purity 96.80% (HPLC).
(2-Phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)(4-(2-(thiophen-
2-ylmethylamino)benzoyl)piperazin-1-yl)methanone (6o): Com-
pound 6o was prepared by following the method described for
the preparation of compound 6a, employing piperazin-1-yl-(2-(thi-
ophen-2-ylmethylamino)phenyl)methanone
(14c,
160 mg,
0.53 mmol) and 2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidine-5-car-
boxylic acid (7c, 207 mg, 0.63 mmol) to afford pure compound 6o
as a yellow solid in 253 mg, 78% yield. Rf =0.52 (EtOAc/hexane,
(2-Phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)(4-(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)me-
thanone (6r): Compound 6r was prepared by following the
method described for the preparation of compound 6a, employing
piperazin-1-yl-(2-(thiophen-2-ylmethylamino)phenyl)methanone
~
7:3); mp: 112–1138C; IR (KBr) n=3375, 2941, 1684, 1628, 1580, 883,
1
803 cmꢁ1; H NMR (300 MHz, CDCl3): d=2.51 (s, 3H), 3.75–3.82 (m,
4H), 3.84–3.90 (m, 2H), 3.92–3.99 (m, 2H), 4.52 (s, 2H), 5.63 (brs,
1H), 6.65–6.75 (m, 2H), 6.91–6.95 (m, 1H), 6.97–7.00 (m, 1H), 7.03
(s, 1H), 7.09 (d, 1H, J=7.5 Hz), 7.15–7.23 (m, 2H), 7.32 (s, 1H),
7.36–7.44 (m, 5H), 7.95–8.0 (m, 2H), 8.14 ppm (d, 2H, J=8.3 Hz);
13C NMR (300 MHz, CDCl3): d=21.6, 42.9, 43, 47.5, 94.5, 106.7,
112.3, 116.8, 119.2, 124.5, 124.9, 126.6 126.9, 127.7, 127.8, 128.7,
129.2, 129.3, 129.5, 131.3, 132.6, 140.7, 142, 142.6, 146.1, 147.3,
149.2, 151.2, 156.8, 166, 170.5 ppm; MS (ESI): 613 [M+H]+; HRMS
(ESI) calcd for C36H33N6O2S [M+H]+ 613.2382, found: 613.2374;
anal. calcd for C36H32N6O2S: C 70.57, H 5.26, N 13.72, O 5.22, S 5.23,
found: C 70.61, H 5.23, N 13.69, O 5.24, S 5.21; purity 98.80%
(HPLC).
(14c,
160 mg,
0.53 mmol)
and
2-phenyl-7-(3,4,5-
trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7 f,
255 mg, 0.63 mmol) to afford pure compound 6r as a yellow solid
in 270 mg, 74% yield. Rf =0.3 (EtOAc/hexane, 7:3); mp: 120–1218C;
IR (KBr) n=3378, 3056, 2935, 1632, 1587, 1565, 1263, 843,
761 cmꢁ1
3.95 (m, 4H), 3.97 (s, 6H), 3.98 (s, 3H), 4.54 (d, 2H, J=3.0 Hz), 5.50
(brs, 1H), 6.70–6.81 (m, 2H), 6.96 (dd, 1H, J=3.7, 5.2 Hz), 7.0–7.03
(m, 1H), 7.10–7.15 (m, 2H), 7.19–7.24 (m, 1H), 7.28 (s, 1H), 7.36 (s,
1H), 7.39–7.52 (m, 3H), 7.56 (s, 2H), 8.02 ppm (dd, 2H, J=1.5,
8.3 Hz); 13C NMR (300 MHz, CDCl3): d=42.8, 42.9, 47.5, 56.3, 60.9,
68.2, 94.6, 106.7, 107, 107.2, 112.1, 116.8, 119.1, 120.2, 124.4, 124.8,
125.3, 126.2, 126.4, 126.8, 127.7, 128.6, 128.8, 129.2, 131.2, 132.4,
152.8, 153, 156.7, 165.8, 170.4 ppm; MS (ESI): 689 [M+H]+; HRMS
(ESI) calcd for C38H37N6O5S [M+H]+ 689.2542, found: 689.2547;
anal. calcd for C38H36N6O5S: C 66.26, H, 5.27, N 12.20, O 11.61, S
4.66, found: C 66.28, H 5.29, N 12.17, O 11.59, S 4.64; purity
97.90% (HPLC).
~
;
1H NMR (300 MHz, CDCl3): d=3.75–3.85 (m, 4H), 3.86–
(7-(4-Methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)(4-
(2-(thiophen-2-ylmethylamino)benzoyl)piperazin-1-yl)metha-
none (6p): Compound 6p was prepared by following the method
described for the preparation of compound 6a, employing pipera-
zin-1-yl-(2-(thiophen-2-ylmethylamino)phenyl)methanone
(14c,
160 mg, 0.53 mmol) and 7-(4-methoxyphenyl)-2-phenylpyrazolo-
[1,5-a]pyrimidine-5-carboxylic acid (7d, 217 mg, 0.63 mmol) to
afford pure compound 6p as a yellow solid in 269 mg, 81% yield.
(4-(2-(Benzo[d][1,3]dioxol-4-ylmethylamino)benzoyl)piperazin-1-
yl)(2,7-diphenylpyrazolo[1,5-a]pyrimidin-5-yl)methanone
(6s):
~
Rf =0.40 (EtOAc/hexane, 7:3); mp: 98–998C; IR (KBr) n=3376, 3065,
Compound 6s was prepared by following the method described
for the preparation of compound 6a, employing (2-(benzo[d]-
[1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-1-yl)methanone
(14d, 160 mg, 0.47 mmol) and 2,7-diphenylpyrazolo[1,5-a]pyrimi-
dine-5-carboxylic acid (7a, 176 mg, 0.56 mmol) to afford pure com-
pound 6s as a yellow solid in 233 mg, 78% yield. Rf =0.56 (EtOAc/
1
2997, 2933, 1757, 1684, 1625, 1577, 1245, 893, 840 cmꢁ1; H NMR
(300 MHz, CDCl3): d=3.72–3.83 (m, 4H), 3.84–3.91 (m, 4H), 3.92 (s,
3H), 4.53 (s, 2H), 5.48 (brs, 1H), 6.72 (t, 1H, J=7.4 Hz), 6.77 (d, 1H,
J=7.4 Hz), 6.94–7.02 (m, 2H), 7.05–7.13 (m, 4H), 7.19 (d, 1H, J=
5.3 Hz), 7.24 (d, 1H, J=9.5 Hz), 7.30 (s, 1H), 7.38–7.43 (m, 1H), 7.47
(t, 2H, J=7.4 Hz), 8.02 (d, 2H, J=7.4 Hz), 8.27 ppm (d, 2H, J=
8.4 Hz); 13C NMR (300 MHz, CDCl3): d=42.8, 42.9, 47.5, 55.5, 94.4,
106.2, 112.2, 114, 116.8, 119.2, 122.7, 123.5, 124.5, 124.9, 126.6,
126.9, 127.8, 128.8, 129.2, 131.2, 131.3, 132.6, 146.1, 146.9, 149.3,
151.2, 156.7, 162.1, 166.1, 170.5 ppm; MS (ESI): 629 [M+H]+; HRMS
(ESI) calcd for C36H33N6O3S [M+H]+ 629.2381, found: 629.2362;
anal. calcd for C36H32N6O3S: C 68.77, H 5.13, N 13.37, O 7.63, S 5.10,
found: C 68.74, H 5.15, N 13.39, O 7.61, S 5.11; purity 97.40%
(HPLC).
~
hexane, 7:3); mp: 118–1198C; IR (KBr) n=3376, 2930, 2834, 1667,
1607, 1560, 1244, 841, 760 cmꢁ1 1H NMR (500 MHz, CDCl3): d=
;
3.76–3.84 (m, 4H), 3.85–3.96 (m, 4H), 4.26 (s, 2H), 5.56 (brs, 1H),
5.94 (s, 2H), 6.65–6.86 (m, 5H), 7.12 (d, 2H, J=9.63), 7.19–7.25 (m,
1H), 7.33 (s, 1H), 7.40–7.51 (m, 3H), 7.57–7.65 (m, 3H), 8.02 (d, 2H,
J=6.7 Hz), 8.23 ppm (dd, 2H, J=2.0, 6.9 Hz); 13C NMR (300 MHz,
CDCl3): d=42.5, 42.9, 47.5, 94.7, 107.7, 108.3, 112.3, 116.1, 117.9,
120.3, 124.2, 126.51, 126.6, 127.9, 128.4, 128.6, 128.9, 129, 129.3,
129.5, 130.7, 131.5, 132.6, 132.7, 146.7, 146.9, 147.2, 147.9, 149.8,
151.2, 156.95, 165.9, 170.8 ppm; MS (ESI): 637 [M+H]+ HRMS (ESI)
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
9
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ÞÞ
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A. Kamal, M. Pal-Bhadra, et al.
MED
calcd for C38H33N6O4 [M+H]+ 637.2556, found: 637.2547; anal.
calcd for C38H32N6O4: C, 71.68, H 5.07, N 13.20, O 10.05, found: C
71.71, H 5.05, N 13.17, O 10.02; purity 95.30%, (HPLC).
[M+H]+ 667.2663, found: 667.2657; anal. calcd for C39H34N6O5: C
70.26, H 5.14, N, 12.60, O, 12.00, found: C 70.29, H 5.12, N 12.57, O
11.98; purity 95% (HPLC).
(4-(2-(Benzo[d][1,3]dioxol-5-ylmethylamino)benzoyl)piperazin-1-
yl)(7-(4-nitrophenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl)me-
thanone (6t): Compound 6t was prepared by following the
method described for the preparation of compound 6a, employing
(2-(benzo[d][1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-1-yl)me-
thanone (14d, 160 mg, 0.47 mmol) and 7-(4-nitrophenyl)-2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7b, 201 mg,
0.56 mmol) to afford pure compound 6t as a yellow solid in
256 mg, 80% yield. Rf =0.54 (EtOAc/hexane, 7:3); mp: 108–1098ꢁC1;
1H NMR (300 MHz, CDCl3): d=3.74–3.98 (m, 8H), 4.26 (s, 2H), 5.57
(brs, 1H), 5.94 (s, 2H), 6.64–6.88 (m, 5H), 7.08–7.18 (m, 1H), 7.33 (s,
2H), 7.4–7.51 (m, 4H), 7.59–7.65 (m, 2H), 8.02 (dd, 2H, J=1.7,
6.9 Hz), 8.23 ppm (dd, 2H, J=2.2, 6.0 Hz); 13C NMR (300 MHz,
CDCl3): d=42.7, 42.9, 47.5, 94.7, 101, 106.7, 107.2, 107.8, 108.3,
112.2, 116.2, 118.3, 119.1, 120.4, 123.6, 126.6, 128, 128.6, 128.8,
129.2, 129.3, 129.5, 130.6, 131.2, 131.4, 132.5, 132.7, 146.8, 147.1,
151.1, 156.9, 165.9, 170.7 ppm; MS (ESI): 682 [M+H]+; anal. calcd
for C38H31N7O6: C 66.95, H 4.58, N 14.38, O 14.08, found: C 66.91, H
4.62, N 14.36, O 14.11; purity 95.50% (HPLC).
(4-(2-(Benzo[d][1,3]dioxol-5-ylmethylamino)benzoyl)piperazin-1-
yl)(7-(3,4-dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-
yl)methanone (6w): Compound 6w was prepared by following
the method described for the preparation of compound 6a, em-
ploying (2-(benzo[d][1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-
1-yl)methanone (14d, 160 mg, 0.47 mmol) and 7-(3,4-dimethoxy-
phenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7e,
210 mg, 0.56 mmol) to afford pure compound 6w as a yellow solid
in 222 mg, 68% yield. Rf =0.35 (EtOAc/hexane, 7:3); mp: 115–
~
~
IR (KBr) n=3380, 2927, 1634, 1587, 1556, 1243, 884, 803 cm
;
116 8C; IR (KBr) n=3395, 2927, 1627, 1550, 1505, 1256, 853,
808 cmꢁ1 1H NMR (300 MHz, CDCl3): d=3.76–3.84 (m, 4H), 3.84–
;
3.98 (m, 4H), 3.99 (s, 6H), 4.24 (s, 2H), 5.69 (brs, 1H), 5.92 (s, 2H),
6.59–6.82 (m, 4H), 7.0–7.10 (m, 3H), 7.18 (t, 1H, J=7.5 Hz), 7.25 (s,
1H), 7.34 (s, 1H), 7.36–7.48 (m, 3H), 7.85 (d, 1H, J=8.3 Hz),
7.98 ppm (t, 3H, J=2.2 Hz); 13C NMR (300 MHz, CDCl3): d=42.8,
47.3, 47.4, 55.9, 56, 94.4, 100.8, 106.2, 107.6, 108.2, 110.7, 112.3,
112.7, 116.1, 118.2, 120.2, 122.7, 123.1, 126.1, 126.4, 127.8, 128.7,
129.1, 131.3, 132.4, 132.6, 146.6, 146.7, 146.8, 146.9, 147.8, 148.5,
149.2, 151, 156.6, 165.9, 170.7 ppm; MS (ESI): 697 [M+H]+; HRMS
(ESI) calcd for C40H37N6O6 [M+H]+ 697.2770, found: 697.2781; anal.
calcd for C40H36N6O6: C 68.95, H, 5.21, N 12.06, O 13.78, found: C
68.93, H 5.23, N 12.09, O 13.80; purity 97.60% (HPLC).
(4-(2-(Benzo[d][1,3]dioxol-5-ylmethylamino)benzoyl)piperazin-1-
yl)(2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)methanone
(6u): Compound 6u was prepared by following the method de-
scribed for the preparation of compound 6a, employing (2-
(benzo[d][1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-1-yl)metha-
none (14d, 160 mg, 0.47 mmol) and 2-phenyl-7-p-tolylpyrazolo[1,5-
a]pyrimidine-5-carboxylic acid (7c, 184 mg, 0.56 mmol) to afford
pure compound 6u as a yellow solid in 214 mg, 70% yield. Rf =
(4-(2-(Benzo[d][1,3]dioxol-5-ylmethylamino)benzoyl)piperazin-1-
yl)(2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-
5-yl)methanone (6x): Compound 6x was prepared by following
the method described for the preparation of compound 6a, em-
ploying (2-(benzo[d][1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-
1-yl)methanone (14d, 160 mg, 0.47 mmol) and 2-phenyl-7-(3,4,5-
trimethoxyphenyl)-pyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7 f,
226 mg, 0.56 mmol) to afford pure compound 6x as a yellow solid
in 235 mg, 69% yield. Rf =0.29 (EtOAc/hexane, 7:3); mp: 113–
~
0.57 (EtOAc/hexane, 7:3); mp: 109–1108C; IR (KBr) n=3379, 2937,
1628, 1603, 1565, 1234, 851, 760 cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d=2.49 (s, 3H), 3.75–3.84 (m, 4H), 3.86–3.94 (m, 4H), 4.26 (s, 2H),
5.56 (brs, 1H), 5.93 (d, 2H, J=6.0 Hz), 6.69 (t, 1H, J=7.5 Hz), 6.75–
6.84 (m, 3H), 7.04–7.14 (m, 2H), 7.19–7.25 (m, 1H), 7.32 (t, 2H, J=
4.5 Hz), 7.38–7.51 (m, 5H), 8.0 (dd, 2H, J=3.7, 12 Hz), 8.16 ppm (d,
2H, J=8.3 Hz); 13C NMR (300 MHz, CDCl3): d=21.7, 42.7, 42.9, 47.8,
94.6, 100.7, 106.6, 107.0, 112.1, 116.4, 117.2, 118.0, 119.8, 123.5,
125.8, 126.3, 127.5, 128.2, 128.5, 129.0, 129.4, 129.7, 130.2, 130.7,
131.3, 132.4, 146.2, 147.4, 149.6, 151.1, 156.4, 166.0, 170.6 ppm; MS
(ESI): 651 [M+H]+; purity 97.50% (HPLC).
~
114 8C; IR (KBr) n=3389, 2934, 1627, 1549, 1508, 1242, 831,
803 cmꢁ1 1H NMR (300 MHz, CDCl3): d=3.75–3.84 (m, 4H), 3.86–
;
3.94 (m, 4H), 3.97 (s, 6H), 3.98 (s, 3H), 4.26 (s, 2H), 5.55 (brs, 1H),
5.93 (s, 2H), 6.69 (t, 2H, J=6.9 Hz), 6.75–6.85 (m, 2H), 7.09–7.13 (m,
2H), 7.23 (t, 1H, J=6.9 Hz), 7.36 (s, 1H), 7.39–7.50 (m, 4H), 7.57 (s,
2H), 8.02 ppm (d, 2H, J=7.9 Hz); 13C NMR (300 MHz, CDCl3): d=
42.9, 47.4, 47.5, 56.3, 61, 94.6, 100.9, 106.7, 107, 107.7, 108.3, 112.2,
116.1, 118.2, 120.3, 125.4, 126.4, 127.9, 128.6, 128.8, 129.3, 130.3,
131.4, 132, 132.4, 132.7, 146.8, 146.9, 149.3, 151.1, 153.1, 156.8,
165.9, 170.7 ppm; MS (ESI): 727 [M+H]+ HRMS (ESI) calcd for
C41H39N6O7 [M+H]+ 727.2864, found: 727.2869; anal. calcd for
C41H38N6O7: C 67.76, H 5.27, N 11.56, O 15.42, found: C 67.78,H 5.24,
N 11.53, O 15.40; purity 95.30%, (HPLC).
(4-(2-(Benzo[d][1,3]dioxol-5-ylmethylamino)benzoyl)piperazin-1-
yl)(7-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl-
methanone (6v): Compound 6v was prepared by following the
method described for the preparation of compound 6a, employing
(2-(benzo[d][1,3]dioxol-5-ylmethylamino)phenyl)(piperazin-1-yl)me-
thanone (14d, 160 mg, 0.47 mmol) and 7-(4-methoxyphenyl)-2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid (7d, 193 mg,
0.56 mmol) to afford pure compound 6v as a yellow solid in
Biology
Cell culture: Human cervical epithelial cancer cell lines HeLa and
SiHa, purchased from the American Type Culture Collection (ATCC),
were maintained in RPMI and Dulbecco’s modified Eagle’s medium
(DMEM) (Invitrogen), respectively, and were supplemented with
2 mm Glutamax (Invitrogen), 10% fetal calf serum, and 100 UmLꢁ1
penicillin and 100 mgmLꢁ1 streptomycin sulfate (Sigma). Cell cul-
tures were maintained at 378C under a humidified atmosphere
containing 5% CO2.
256 mg, 82% yield. Rf =0.36 (EtOAc/hexane, 7:3); mp: 122–1238C;
ꢁ1
~
IR (KBr) n=3380, 2897, 1628, 1603, 1549, 1253, 929, 832 cm
;
1H NMR (500 MHz, CDCl3): d=3.75–3.82 (m, 4H), 3.84–3.89 (m, 2H),
3.93 (s, 3H), 3.94–3.97 (m, 2H), 4.24 (s, 2H), 5.68 (brs, 1H), 5.93 (s,
2H), 6.62–6.67 (m, 2H), 6.79 (d, 2H, J=12.7 Hz), 7.08 (d, 3H, J=
7.2 Hz), 7.18 (t, 1H, J=8.2 Hz), 7.25 (s, 2H), 7.31 (s, 1H), 7.38 (d, 1H,
J=7.2 Hz), 7.44 (t, 2H, J=7.2 Hz), 7.98 (d, 2H, J=7.2 Hz), 8.28 ppm
(d, 2H, J=8.2 Hz); 13C NMR (300 MHz, CDCl3): d=42.9, 47.4, 55.3,
94.6, 100.8, 106.1, 107.6, 108.2, 112.1, 113.9, 116.1, 118.3, 120.2,
122.6, 124.2, 126.5, 127.8, 128.2, 128.6, 129, 129.7, 131.2, 132.5,
132.7, 146.6, 146.8, 147.8, 149.2, 151.1, 156.6, 162, 165.9,
170.6 ppm; MS (ESI): 667 [M+H]+; HRMS (ESI) calcd for C39H35N6O5
MTT assay: Cell viability was assessed by MTT assay, which assays
mitochondrial function based on the ability of viable cells to
reduce MTT to insoluble formazan crystals by mitochondrial dehy-
drogenase activity. HeLa and SiHa cells were seeded in a 96-well
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Anthranilamide–Pyrazolo[1,5-a]pyrimidine Conjugates
MED
plate at a density of 1ꢁ104 cells per well. After overnight incuba-
tion, cells were treated with compounds roscovitine (ros) and 6a–
x at concentrations of 1, 2, 4, and 8 mm and incubated for 24 h.
The cell culture medium was then discarded and replaced with
10 mL MTT dye. Plates were incubated at 378C for 2 h. The result-
ing formazan crystals were solubilized in 100 mL extraction buffer.
The optical density (OD) was read at l 570 nm with a microplate
reader (Multi-mode Varioskan instrument, Themo Scientific).
of Quantity One software (version 4.1.1) using the Bio-Rad image
analysis system (CA, USA).
Caspase-3 assay: A caspase-3 fluorescence assay kit (Clonetech, CA,
USA) was used to evaluate caspase-3 activity, following procedures
provided by the manufacturer. HeLa cells were treated with com-
pounds ros, 6 f, 6l, and 6r at 2 mm as obtained from FACS analysis.
Cell lysates were added to the 2ꢁ reaction buffer containing DTT
and caspase substrate. Incubation was carried out at 378C for 1 h.
Readings were taken at an excitation wavelength of 400 nm and
an emission wavelength of 505 nm.
Cell cycle analysis: HeLa and SiHa cells (5ꢁ105) were seeded in
60 mm dishes and allowed to grow for 24 h. Compounds (6a–x)
were added at a final concentration of 2 mm to the culture media,
and cells were incubated for an additional 24 h. Cells were harvest-
ed with Trypsin–EDTA, fixed with ice-cold 70% EtOH at 48C for
30 min, washed with phosphate-buffered saline (PBS) and incubat-
ed with 1 mgmLꢁ1 RNase A solution (Sigma) at 378C for 30 min.
Cells were collected by centrifugation at 2000 rpm (313 g, Sorvall
swinging-bucket rotor, model #75002000, Heraeus Multifuge 1S-R,
Thermo Scientific) for 5 min and further stained with 250 mL DNA
staining solution [10 mg propidium iodide (PI), 0.1 mg trisodium
citrate, and 0.03 mL Triton X-100 dissolved in 100 mL sterile Milli-Q-
purified water at room temperature for 30 min in the dark]. The
DNA contents of 20000 events were measured by flow cytometry
(Dako Cytomation, Beckman Coulter, Brea, CA, USA). Histograms
were analyzed using Summit Software V4.3.02.
Immunoflouroscence microscopy to study p53 localization: HeLa cells
were seeded on glass cover slips, incubated for 24 h in the pres-
ence or absence of test compounds ros and 6l at 2 mm. At the
end of incubation, cells were fixed with 4% paraformaldehyde,
0.02% glutaraldehyde in PBS, and permeabilized by dipping the
cells in 100% methanol (ꢁ208C). Cover slips were then blocked
with 1% BSA in PBS for 1 h followed by incubation with a primary
antibody against p53 followed by secondary antibody. At the end
of experiments, cells were washed and fixed for confocal microsco-
py (Olympus).
Protein extraction and Western blot analysis: HeLa cells (5ꢁ105)
were seeded in a 60 mm dish and allowed to grow for 24 h. Com-
pounds ros, 6 f, 6l, and 6r were added to the culture media at
2 mm, and the cells were incubated for an additional 24 h. Total
cell lysates were obtained by lysing the cells in ice-cold RIPA buffer
(1ꢁPBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS)
containing 100 mgmLꢁ1 PMSF, 5 mgmLꢁ1 aprotinin, 5 mgmLꢁ1 leu-
peptin, 5 mgmLꢁ1 pepstatin, and 100 mgmLꢁ1 NaF. After centrifu-
gation at 12000 rpm (6349 g, fixed-angle rotor, model #75003348,
Heraeus Multifuge 1S-R, Thermo Scientific) for 10 min, the protein
in supernatant was quantified by the Bradford method (Bio-Rad)
using a Multimode Varioskan instrument (Thermo-Fischer Scientif-
ic). Protein (50 mg per lane) was applied to a 12% SDS–polyacryl-
amide gel. After electrophoresis, the protein was transferred to
a poly(vinylidine difluoride) (PVDF) membrane (Amersham Biosci-
ences). The membrane was blocked at room temperature for 2 h in
1ꢁTBS+0.1% Tween 20 (TBST) containing 5% blocking powder
(Santa Cruz Biotechnology). The membrane was washed with TBST
for 5 min, and primary antibody was added and incubated at 48C
overnight. p53 Ser46 and p53 Ser15 were purchased from Cell Sig-
naling, and b-actin, Bcl2, and BAX were purchased from Imgenex.
Membranes were washed with TBST three times for 15 min, and
the blots were visualized with chemiluminescence reagent
(Thermo-Fischer Scientific). X-ray films were developed and fixed
with reagents purchased from Kodak.
BrdU cell proliferation assay: This assay was carried out by using
the 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kit (Milli-
pore) to assess the effect of compounds such as 6 f, 6l, and 6r on
the proliferation of HeLa and SiHa cells. Roscovitine was used as
positive control. Cells (1ꢁ104) were seeded and allowed to grow
for 24 h. BrdU was added and allowed to incorporate for 5 h fol-
lowed by the addition of test compounds (ros, 6 f, 6l, and 6r) at
a concentration of 2 mm for 24 h. Fixation was done for 30 min at
room temperature. The cells were then washed, anti-BrdU antibody
was added; this binds BrdU that was incorporated in the cell. After
incubation for 1 h, anti-BrdU goat anti-mouse horseradish perox-
idase (HRP)-conjugated secondary antibody (100 mL, 1:2000) was
added and incubated for 30 min. Washing procedures were fol-
lowed according to the manufacturer’s instructions. TMB substrate
(100 mL) was added and incubated for another 30 min at room
temperature. A decrease in OD450 indicates that the cells are arrest-
ed in the G1 phase.
Semi-quantitative reverse transcription PCR (RT-PCR): Total RNA was
extracted using an RNeasy mini kit (Qiagen, USA) and reverse tran-
scribed into cDNA using superscript II reverse transcriptase (Invitro-
gen Life Technologies). PCR was carried out with specific primers
(Table 3) in a PCR thermocycler (Takara Bioscience). The products
were separated by agarose gel (1%) electrophoresis followed by
staining with ethidium bromide and visualization under UV light.
The signal intensity of respective bands was measured by means
Statistical analysis: Statistical analyses were performed using
GraphPad software to evaluate significant differences between
control and test samples. All variables were tested in three inde-
pendent experiments. Results are reported as the mean ꢀSD; *p<
0.05, **p<0.01, ***p<0.001.
Acknowledgements
Table 3. Primers used in RT-PCR.
Primer
Sequence
Product
size [bp]
J.R.T. is thankful to DST (India), and A.V. and A.M.R. thank CSIR
(India) for the award of research fellowships.
GAPDH forward
GAPDH reverse
p21 gene forward
p21 gene reverse
p53 gene forward
p53 gene reverse
5’-GGG AAG GTG AAG GTC GGA GT-3’
5’-TTG AGG TCA ATG AAG GGG TCA-3’
5’-CAC CGA GAC ACC ACT GGA GG-3’
5’-GAG AAG ATC AGC CGG CGT TT-3’
5’-AGG TTG GCT CTG ACT GT-3’
110
260
220
Keywords: antitumor agents · apoptosis · caspase-3 · cell
cycle · roscovitine
5’-TTG ACG TGG TGA GGC TC-3’
ChemMedChem 0000, 00, 1 – 13
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A. Kamal, M. Pal-Bhadra, et al.
MED
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Revised: May 18, 2012
Published online on && &&, 0000
&12
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These are not the final page numbers!

FULL PAPERS
Joining forces: A series of anthranila-
mide–pyrimidine conjugates were syn-
thesized and evaluated for anticancer
activity. Not only do these compounds
regulate the cell cycle, they also induce
p53 expression and p21 transcription
levels. The conjugates also induce BAX
protein and decrease Bcl2 protein
A. Kamal,* J. R. Tamboli, M. J. Ramaiah,
S. F. Adil, G. Koteswara Rao, A. Viswanath,
A. Mallareddy, S. N. C. V. L. Pushpavalli,
M. Pal-Bhadra*
&& – &&
Anthranilamide–Pyrazolo[1,5-
a]pyrimidine Conjugates as p53
Activators in Cervical Cancer Cells
levels, effecting caspase-3-mediated
apoptosis. Compounds that can induce
p53 are of great value and are likely to
be useful in the treatment of cancer.
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&13
&
ÞÞ
These are not the final page numbers!