2
A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O-
+N
N
N
NHBn
S
N
HN
N
N
HN
N
N
HN
N
OH
N
HN
N
HO
N
Et
N
OH
N
3
1
OH
HO
2
C6H5
N
O
H
N
O
S
O
O
N
N
N
N
CF3
N
R1
R2
N
NH
HO
O
N
N
N
HN
R
CF3
R3
OH
4
6a-x
5
Figure 1. Chemical structures of pyrazolopyrimidines (1–4), E7010 (5), 2-anilinonicotiyl-pyrazolo[1,5-a]pyrimidine conjugates (6a–x).
remarkable selectivity against different types of cancers.25 As
described here, pyrazolopyrimidines and 2-anilinonicotinyl scaf-
folds are of considerable interest in the development of anticancer
compounds. Modifications on the pyrazolo[1,5-a]pyrimidine
nucleus and 2-anilinonicotic acids have resulted in a large number
of compounds that exhibit significant anticancer activities.
In continuation to our efforts on the development of useful syn-
thetic protocols suitable for applications in the field of combinato-
rial chemistry and chemistry-driven drug discovery.26–28
Additionally, the medicinal importance of these moieties with
impressive anticancer profile and their effect on various molecular
targets as potential cancer chemotherapeutic agents an attempt
has been made in the present study to link the pyrazolo[1,5-a]
pyrimidine moiety to the 2-anilinopyridyl structural component
of E7010. The conformationally restricted cyclic piperazine amide
has been employed as a linker for this purpose resulting in a new
class of compounds. These newly synthesized chemical entities
have been evaluated for their antiproliferative potential and cell
cycle perturbations. Moreover detailed studies have been carried
out to understand their mechanistic aspects of regulating the cell
proliferation in MCF7 cell line via estrogen signaling.
The synthesis of the target conjugates have been performed in a
convenient manner as outlined in Schemes 1 and 2. The synthetic
route employed starts with the synthesis of b-diketoesters (8a, 8b,
8c, 8d and 8e) with some modification to the one reported in the
literature.29 For this purpose, different substituted acetophenones
(7a–e) on oxylation by dimethyl oxalate in the presence of
sodium methoxide provides the b-diketoesters (8a–e). Different
acetophenones with 4-chloro, 4-fluoro, 3-methoxy, 4-methoxy
and 3,4,5-trimethoxy substitutions have been employed. A conve-
nient protocol was employed for the synthesis of fused pyrazolo
[1,5-a]pyrimidines compounds. The b-diketoester on cyclization
with an aza-heterocyclic amine, i.e., 3-amino-5-phenyl pyrazolo
in the presence of catalytic amount of cerric ammonium nitrate
(CAN, 5 mol %) in ethanol at room temperature affords various sub-
The synthesis of these new conjugates (6a–x) has been accom-
plished by the synthetic route depicted in Scheme 2. The ethyl
ester of 2-chloronicotinic acid 11 and different substituted anilines
12a–e are refluxed in ethylene glycol to give the coupled product
of 2-anilino nicotinic acid esters 13a–e. The anilines used are with
different substitutions such as 4-fluoro, 4-methoxy, 3,4-dimethoxy
and 3,4,5-trimethoxysubstitutions with a view to bring diversity in
the conjugates for a better understanding of their SAR. The esters
13a–e on base hydrolysis on treating with 2 N sodium hydroxide
solution in ethanol affords 2-anilinonicotinic acids (14a–e) in
quantitative yields. These acids are converted to amides by cou-
pling them with commercially available Boc-protected piperazine
in presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
and hydroxyl benzotriazole (EDCl/HOBt) in dry dimethylfor-
mamide at 0 °C. To effect the coupling of 2-anilinonicotinyl piper-
azineamide
(15a–e)
and
pyrazolo[1,5-a]pyrimidine
acid
intermediates (10a–e), the Boc-protection on the piperazine moi-
ety of 15a–e is removed by treatment with trifluoroacetic acid in
dichloromethane. This was followed by the crucial coupling
assisted by EDCl/HOBt methodology in dry DMF to yield the
desired 2-anilinonicotinyl linked pyrazolo[1,5-a]pyrimidine conju-
gates (6a–x) as shown in Scheme 2 (Supplementary information).
By employing this efficient and convenient synthetic route, we
have synthesized twenty four new 2-anilinonicotinyl linked pyra-
zolo[1,5-a]pyrimidine conjugates that are joined through confor-
mationally restricted piperazineamide spacers.
These conjugates have been evaluated for their biological prop-
erties such as cytotoxic activity, cell viability and effects on pro-
teins such as estrogen receptor, Bcl-2, survivin and cyclin D1 in
MCF breast cancer cell lines in order to understand the mechanism
involved during cell division arrest of cancer cells.
In order to study the possible cytotoxic effect by these series of
conjugates (6a–x), MTT assay was conducted. Here SiHa, HeLa (cer-
vical cancer cell line), MCF-7 (breast cancer), IMR-32 (neuroblas-
toma) were treated at concentration ranging from 0.25 to 8 lM
stituted
2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
of conjugates for 48 h. It was observed that conjugates 6a, 6c, 6j,
6m, 6o, 6q, 6r, 6s, 6t and 6w were effective against all the cells.
Conjugates 6a, 6c were found to be most effective among the series
against the cell lines tested (Fig. 2 and Table1). From cytotoxic data
it is also evident that these molecules are specific to MCF-7, HeLa
esters (9a–e), which upon base hydrolysis with sodium hydroxide
provides the corresponding carboxylic acids (10a–e).Then with
pyrazolo[1,5-a]pyrimidine acid intermediates (10a–e) in hand,
we proceeded to synthesize the target conjugates.
Kamal, Ahmed
