Design, synthesis, and molecular docking studies of novel pyrazolyl 2-aminopyrimidine derivatives as HSP90 inhibitors

Article abstract of DOI:10.1002/ardp.201900063

A series of novel pyrazolyl 2-aminopyrimidine derivatives (7a-t) were designed based on scaffold hopping techniques, synthesized and biologically evaluated for their HSP90 inhibition and anticancer activity. Several compounds exhibited potent HSP90 inhibition with IC₅₀ values less than that of the reference standard 17-AAG (1.25 μM). The most potent compound 7t displayed excellent HSP90 inhibition with an IC₅₀ of 20 nM and in vitro antiproliferative potential against three cancer cell lines (IC₅₀ < 5 μM). 7t also induced dose dependent degradation of client proteins (pHER2 and pERK1/2) in Western blot analysis. Several structural features of 7p-t oriented the molecules to retain all the essential binding interactions with HSP90, as observed by rationalized docking studies. Therefore, the para-nitrophenyl ring on the central pyrazole ring along with the 2-amino group on the pyrimidine ring are the crucial features in the development of novel HSP90 inhibitors based on this scaffold for targeted anticancer therapy.

Full text of DOI:10.1002/ardp.201900063

Received: 2 March 2019
Revised: 6 July 2019
Accepted: 9 July 2019
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DOI: 10.1002/ardp.201900063
F U L L P A P E R
Design, synthesis, and molecular docking studies of novel
pyrazolyl 2‐aminopyrimidine derivatives as HSP90 inhibitors
Akhila Mettu1
Naikal James Prameela Subhashini3
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Venu Talla2
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Deepti Madanlal Bajaj2
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1Department of Pharmaceutical Chemistry,
Abstract
Gokaraju Rangaraju College of Pharmacy,
Kukatpally, Hyderabad, India
A series of novel pyrazolyl 2‐aminopyrimidine derivatives (7a‒t) were designed based
on scaffold hopping techniques, synthesized and biologically evaluated for their
HSP90 inhibition and anticancer activity. Several compounds exhibited potent HSP90
inhibition with IC50 values less than that of the reference standard 17‐AAG (1.25 µM).
The most potent compound 7t displayed excellent HSP90 inhibition with an IC50 of
20 nM and in vitro antiproliferative potential against three cancer cell lines
(IC50 < 5 µM). 7t also induced dose dependent degradation of client proteins (pHER2
and pERK1/2) in Western blot analysis. Several structural features of 7p‒t oriented
the molecules to retain all the essential binding interactions with HSP90, as observed
by rationalized docking studies. Therefore, the para‐nitrophenyl ring on the central
pyrazole ring along with the 2‐amino group on the pyrimidine ring are the crucial
features in the development of novel HSP90 inhibitors based on this scaffold for
targeted anticancer therapy.
2Department of Pharmacology, National
Institute of Pharmaceutical Education and
Research, Balanagar, Hyderabad, India
3Department of Chemistry, University College
of Science, Osmania University, Hyderabad,
India
Correspondence
Subhashini Naikal James Prameela,
Department of Science, University College of
Science, Osmania University, Hyderabad
500007, India.
Email: njsubhashini@yahoo.co.in
Funding information
Science and Engineering Research Board,
Department of Science and Technology,
Grant/Award Number: SR/WOSA/CS‐1003/
2014
K E Y W O R D S
active site, cancer, client proteins, HSP90 inhibitors, pyrazolyl pyrimidines
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1
INTRODUCTION
(CCT018159, G3130), oxazole (AUY922), triazole (STA‐9090),
indazole (SNX2112, SNX5422), 2‐aminopyrimidine (SNX6833,
Cancer is a disorder of multiple mechanisms, progressive with
severity and challenging life at various stages. It involves an
imbalance in cellular growth with alteration of various mechanisms
involved, leading to suppression of p53 gene and thereby evading
apoptosis.[1] Heat shock protein 90 (HSP90) is a multichaperone
complex recognized as a potential target for cancer.[2] It is reported
that the levels of HSP90 constitute 5–10% in a cancer cell compared
to 1% in normal cell.[3] Geldanamycin (GA) and radicicol (RA) are the
first identified HSP90 inhibitors with limitations in their pharmaco-
kinetic parameters. 17‐N‐Allyl amino‐17‐demethoxygeldanamycin
(17‐AAG), 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin
(17‐DMAG) are the semisynthetic analogs of GA with lesser toxicity,
however the side effects overlay their clinical use.[4,5] Later, various
small molecules with diverse structures were proposed as N‐terminal
ATP competitive antagonists possessing purine (PU3), pyrazole
BIIB021, NVPHSP990) rings and most of them are still in clinical
trials.[6–11] X‐ray crystallographic studies and molecular docking
studies of various reported inhibitors with HSP90 have shown
hydrogen bonding with Asp93, Thr184, Asp54, Asn51, Lys58 amino
acids, thereby proving their binding region as N‐terminal ATP binding
site.[12] Mostly, HER2 driven breast cancer and MAPK driven
melanomas are most sensitive to HSP90 inhibitors.[13–15] Although
the research on HSP90 inhibitors initiated decades back, still there
exists a crave for new entities, due to pivotal role of HSP90 in cancer.
Many purine analogs were exploited as HSP90 inhibitors and
several of them are in clinical trials (Figure 1). The SAR studies of these
analogs have depicted the 2‐aminopyrimidine scaffold as an essential
moiety for activity.[16,17] Therefore, we have retained the same in our
study. Further construction of our basic disubstituted pyrazolyl
pyrimidine was based on BIIB021,
wileyonlinelibrary.com/journal/ardp
a
purine based conventional
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Arch Pharm Chem Life Sci. 2019;e1900063.
© 2019 Deutsche Pharmazeutische Gesellschaft
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METTU ET AL.
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FIGURE 1 Aminopyrimidine based
potent HSP90 inhibitors
ring. A series of novel compounds with the proposed scaffold (7a‒t)
bearing various substituents were synthesized and evaluated for
anticancer activity and HSP90 inhibition.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Scheme 1 depicts the synthesis of disubstituted pyrazolyl pyrimidin‐2‐
amines (7a‒t). First step involved condensation of commercially
available substituted phenyl hydrazine (1a‒c) with substituted acet-
ophenone (2a,b) to form hydrazones 3a‒d. The obtained product upon
double Vilsmeier Haack reaction in the presence of two moles of
phosphorous oxychloride and DMF yielded 1,3‐disubstituted phenyl
pyrazole‐4‐carbaldehydes 4a‒d.[18] The free aldehyde group when
treated with substituted acetophenone (5) in the presence of strong
base under reflux conditions yielded chalcones 6a‒t.[19] The chalcone
moiety was then treated with guanidine hydrochloride using potassium
FIGURE 2 Design of the novel pyrazolyl pyrimidine derivatives
7a‒t
HSP90 inhibitor. As depicted in Figure 2, the substituted phenyl ring is
supposed to occupy the chloro position of BIIB021, substituted phenyl
pyrazole is presumed to be pyrazolo part of purine ring and 2,4‐
dichloro/4‐nitro phenyl ring is theorized to replace 2,4‐dichloro pyridine
SCHEME 1 Synthesis of disubstituted pyrazolyl pyrimidin‐2‐amines 7a‒t. Reagents and conditions: (i) gla. AcOH (cat), methanol, 60‒70°C,
6‒7 hr, 80‒90%. (ii) DMF/POCl3, 80‒90°C, 9‒10 hr, 70‒75%. (iii) 50% KOH, methanol, reflux, 12‒16 hr, 40‒55%. (iv) Guanidine‐HCl, potassium
tert‐butoxide, DMF, reflux, 10‒12 hr, 70‒80%

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TABLE 1 Structural details of synthesized compounds with results of the MTT and FP assay
MTT assay (IC50 SDa in µM)
#
R1
Cl
Cl
Cl
Cl
Cl
H
R2
Cl
R3
R4
R5
R6
MCF‐7
2.8 0.3
5.0 1.8
6.6 0.7
> 20
MDA‐MB‐231
1.6 0.1
4.4 0.8
3.5 0.8
> 20
HCT116
4.7 0.3
4.6 0.2
5.4 0.1
> 20
FP assay (IC50 SDa in µM)
4.08 1.2
6.63 1.3
3.08 1.0
n/a
7a
7b
7c
Br
H
H
OCH3
F
Cl
Br
H
H
Cl
Br
H
OCH3
OCH3
OCH3
Br
7d
7e
7f
Cl
Br
OCH3
H
Cl
Br
H
H
> 20
> 20
> 20
n/a
NO2
NO2
NO2
NO2
NO2
Cl
Br
H
H
OCH3
F
8.9 1.9
2.5 0.2
1.7 0.5
3.7 0.8
1.7 0.5
> 20
3.6 0.8
5.4 0.6
4.2 0.7
2.9 0.7
7.6 0.9
> 20
4.9 0.1
6.2 0.3
4.4 0.1
8.9 1.0
> 20
n/a
7g
7h
7i
H
Br
H
H
2.04 0.7
0.54 0.05
n/a
H
Br
H
OCH3
OCH3
OCH3
Br
H
Br
OCH3
H
7j
H
Br
H
H
0.85 0.04
n/a
7k
7l
Cl
Cl
Cl
Cl
Cl
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
H
OCH3
F
> 20
Cl
H
H
> 20
> 20
> 20
n/a
7m
7n
7o
7p
7q
7r
Cl
H
OCH3
OCH3
OCH3
Br
7.7 3.2
> 20
16.4
> 20
5.2
1
> 20
5.42 1.24
n/a
Cl
OCH3
H
15.5 1.7
> 20
Cl
H
H
9.6 0.8
> 20
2
8.54 1.62
1.08 0.08
0.08 0.004
1.06 0.02
0.05 0.006
0.02 0.003
1.26 0.03
Cl
H
H
OCH3
F
1.9 0.8
0.7 0.6
0.8 0.3
6.0 0.2
4.3 0.08
8.5 0.7
4.5 0.5
4.8 0.5
0.2 0.08
H
Cl
H
H
3.8 0.1
> 20
H
Cl
H
OCH3
H
OCH3
OCH3
Br
7s
H
Cl
OCH3
H
5.0 0.5
2.4 0.1
0.25 0.4
2
0.4
0.8 0.4
0.5
7t
H
Cl
H
17‐AAG
2
Abbreviations: FP, flourescence polarization; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; SD, standard deviation.
aAll results are mean of at least three independent determinations performed in duplicate. n/a, not available.
tertiary butoxide as base to furnish the final compounds 7a‒t. All the
synthesized compounds were purified by column chromatography using
ethyl acetate/petroleum ether solvent system.
and in vitro antiproliferative activities by the 3‐(4,5‐dimethylthiazol‐2‐
yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay on two human breast
carcinomas (MCF‐7 and MDA‐MB‐231) and colon carcinoma (HCT116).
The results are depicted in Table 1. Based on the substitutions of rings A
and B on pyrazole, all the test compounds are grouped into four types
(Figure 3). Type 4 (7p‒t) with para‐chloro group on ring A and para‐nitro
group on ring B were most active in both the assays performed with
IC50 valves for MTT assay in the range of 2.4–20 μM, 0.7–2 μM and
4.3–8.5 μM on MCF‐7, MDA‐MB‐231, and HCT‐116 cell lines,
respectively, and 0.02–1.08 μM in FP assay for HSP90 inhibition; type
2 (7f‒j) with para‐nitro group on ring A and para‐bromo group on ring B
have good anti‐proliferative activities with IC50 valves in the range of
1.7–8.9 μM, 2.9–7.6 μM, 4.4–20 μM on MCF‐7, MDA‐MB‐231, and
HCT116 cell lines, respectively, with optimum HSP90 inhibition (IC50 at
0.85–2.04 μM); type 1 series (7a‒e) with 2,4‐dichloro substitution on
ring A and para‐bromo substitution on ring B have average to good
antiproliferative activities with IC50 valves in the range of 2.8–20 μM,
1.6–20 μM, 4.6–20 μM on MCF‐7, MDA‐MB‐231, and HCT116 cell
lines, respectively, and exhibit moderate HSP90 inhibition (IC50 at
3.08–6.63 μM); type 3 (7k‒o) with 2,4‐dichloro substitution on ring A
and para‐nitro group on ring B were least active in both the tests
performed with IC50 values in the range of 7.7–20 μM, 5.2–20 μM,
15.5–20 μM on MCF‐7, MDA‐MB‐231, and HCT116 cell lines,
The synthesized compounds 7a‒t were structurally confirmed
by IR, 1H‐NMR, 13C‐NMR, ESI‐MS techniques. In general, the IR
spectrum of all the compounds exhibited characteristic peaks of free
amino group (NH2) at 3,450–3,400 cm−1 and 1,630–1,580 cm−1 for
aromatic rings (C═C). In the 1H‐NMR spectra, the methoxy groups of
compounds 7a, 7c, 7d, 7 f, 7h, 7i, 7k, 7m, 7n, 7p, 7r and 7s appeared
in the range of δ 3.95–3.50. The amino group of all the compounds
was in the range of δ 5.85–5.10 and its protons were exchangeable
with D2O, all the aromatic protons were observed in the range of δ
8.80–6.84. The 13C‐NMR spectra represent the signal for methoxy
carbons of compounds 7a, 7c, 7d, 7f, 7h, 7i, 7k, 7m, 7n, 7p, 7r, and 7s
at δ 56.1−55.2, the aromatic carbons of all the compounds between δ
165−101 ppm. All the compounds were analyzed by ESI‐MS analysis
to give peaks [M+H]+ or [M+Na]+ corresponding to their molecular
weights.
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2.2
HSP90 inhibition and antiproliferative assays
All the test compounds (7a‒t) and the reference compound 17‐AAG
were evaluated for HSP90 inhibition by fluorescence polarization assay

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FIGURE 3 Disubstituted pyrazolyl pyrimidin‐2‐amine scaffold
illustrating the four variants of the compounds
FIGURE 5 Receptor interactions of compound 7t (orange) at the
active site of HSP90. A yellow line represents hydrogen bonding with
the respective amino acid
respectively and exhibit moderate HSP90 inhibition (IC50 at
5.42–8.54 μM). Compounds 7q, 7s, and 7t displayed significantly good
results in all the three cell lines (MTT assay) with IC50 values ≤5 µM and
potent HSP90 inhibition at 80, 50, and 20 nM, respectively, than
standard 17‐AAG (1.25 µM). Compound 7t exhibited the highest HSP90
inhibition as well as potent in vitro antiproliferative efficacy against the
three cancer cell lines (IC50 values of 2.4, 0.8, 4.8 for MCF‐7, MDA‐MB‐
231, HCT‐116, respectively). Overall, mono‐substitution on ring A with
nitro group on ring B and amino group of pyrimidine are the essential
features of this scaffold for HSP90 inhibition and therefore could be the
reason for the higher activity profiles observed with type 4 series.
toward acidic hydrophilic pocket and hydrogen bonding with Asn51 and
Asp54 as depicted in Figure 5 for compound 7t. The substituents on the
phenyl ring C occupies the basic pocket, hydrogen bonding methoxy
group with Lys58. Surrounding these hydrophilic sites are the
hydrophobic interactions with Ala55, Ile96, Met98, Leu107, Ile110,
Ala111, Gly135, Phe138, Tyr139, Trp162, and Val186 amino acids
(Figure 6). Although 7k–o also possess para‐nitro group on phenyl ring
B, the presence of disubstitution on phenyl ring A oriented the
compound in a different mode thereby hindering the key interaction
with AsH93, which could be the reason for lesser activity profiles.
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2.3
Docking
All the synthesized compounds with 17‐AAG were docked at N‐terminal
ATP binding site HSP90α using Glide program embedded in Maestro
9.1 on Schrodinger engine. 5LRZ was downloaded from Protein Data
Bank due to the structural similarity of its ligand with our synthesized
compounds and binding affinities were analyzed along with essential
hydrogen bonding interactions with AsH93 (protonated form of Asp93),
Thr184, Asn51, Asp54, Lys58.[20,21] Compounds 7f‒j, 7p‒t being
monosubstituted on rings A and B oriented perfectly into the pockets
of active site. The nitro group on phenyl ring B in 7p‒t established the
crucial interaction with AsH93 and therefore could be the reason for
the good binding affinities observed with this series. The pyrimidine ring
occupies the center of active site with its free amino group orienting
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2.4
Structure‒activity relationship
Scaffold: Disubstituted pyrazolyl pyrimidin‐2‐amine pharmacophore
was the crucial moiety of all compounds (7a‒t). The amino group
retained essential interactions with HSP90 protein and the
FIGURE 4 Effects of the test compounds on HSP90 client
proteins in MCF‐7 cells
FIGURE 6 Receptor interactions of compounds 7a‒t at the active
site of HSP90

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disubstituted pyrazolyl pyrimidine scaffold oriented perfectly into
the active site of protein as observed by docking studies.
Ring A: Compounds with mono‐substitution at para position on
ring A displayed good HSP90 inhibition (7f‒j, 7p‒t) compared to
disubstituted compounds (7a‒e, 7k‒o). 4‐Chloro substituted com-
pounds (7p‒t) were more active than 4‐nitro substituted ones (7f‒j).
This could be due to the increased hydrophobic interactions seen
with chloro over nitro group.
4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
All the melting points were determined using BÜCHI Melting Point
B‐450 apparatus (Büchi Labortechnik, Switzerland). IR spectra were
recorded on Shimadzu IR Affinity‐1, FT‐IR spectrophotometer.
Nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded with Avance 300 spectrophotometer with chemical shifts
in parts per million (ppm, δ) downfield from TMS as an internal
standard using CDCl3 or DMSO solvent. High resolution mass
spectra were recorded with Agilent QTOF mass spectrometer.
Commercially available reagents and solvents were procured from
Sigma Aldrich/Merck/TCI and used without any purification unless
stated. All the synthesized compounds were purified on Buchi Flash
Chromatography using Merck silica gel 200–400#.
Ring B: Para‐nitro substitution (7a‒j) was more predominate than
para‐bromo as observed in compounds 7k‒t. Also, the nitro group
retained the essential hydrogen bonding with protonated Asp93 in
docking studies (Figure 6).
Ring C: Substituents on ring C (R4–R6) determined the various
activity profiles of all the compounds. Halo substituted compounds
(7j, 7q, 7t) were more active than methoxy substituted ones (7h, 7p,
7r, 7s). The bromo substitution (7t, 7j) was predominate than fluoro
substitution (7q, 7g). This could be due to increased hydrophobic
interactions seen with bromo over fluoro counterpart. Dimethoxy
substituted compounds (7s, 7r, 7c) were more active than mono‐
methoxy substituted ones (7p, 7a). No particular trend was observed
with position of disubstitution.
The spectral data of the investigated compounds are provided
as Supporting Information. Also, their InChI codes together with
biological evaluation data are also provided as Supporting Information.
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4.1.2
Synthetic procedures of compounds 3a‒d,
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2.5
Western blot analysis
4a‒d, 6a‒t
Effect of HSP90 inhibition was observed by the degradation of its
client proteins. pHER2 expressing breast cancers and altered MAPK
signaling induced cancers are the most prevalent ones and therefore
were selected in our study. Two compounds (7q, 7t) and reference
compound 17‐AAG were tested for their effect on the two client
proteins (pHER2 and pERK1/2) on MCF‐7 cell line. Figure 4 displays
dose dependent degradation of pHER2 and pErk1/2 with increasing
concentrations of compounds (2.5, 3.5, 4.5 µM). β‐Actin was the
loading control and 17‐AAG was taken at 0.05, 0.1, and 0.2 µM
concentrations.
Synthesis of substituted acetophenone phenyl hydrazones (3a‒d)
involves condensation of respective acetophenones 1a‒c with
substituted phenyl hydrazine 2a/2b and then synthesis of 1,3‐di‐
(substituted)phenyl‐1H‐pyrazole‐4‐carboxaldehyde (4a‒d) using the
Vilsmeier‐Haack reagent (POCl3/DMF); employing a similar proce-
dure followed by Kira et al.[18] Their analytical and spectroscopic data
agreed with reported literature. Pyrazolyl chalcones (6a‒t) were
synthesized by Claisen‐Schmidt condensation of intermediates 4a‒d
(0.01 mol) with appropriate aldehydes 5a‒e (0.01 mol) in methanol
(30 ml) and refluxed in the presence of base (40% potassium
hydroxide) for 12–14 hr. The reaction mixture was then poured into
crushed ice followed by neutralization with HCl. The solid separated
was filtered, dried and crystallized from ethanol. The purity of the
chalcones was checked by TLC. The IR spectra showed typical
absorption bands at 1,660–1,627, 1,518–1,605, 1,332–1,507 cm−1
corresponding to C═O, C═C, and NO2 functionalities, respectively. In
the 1H‐NMR spectra, doublet at 7.67–7.87 and 7.60–7.69 ppm
represents C═C bond formation as reported by Insuasty et al.[19]
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3
CONCLUSION
The present work has designed a novel scaffold with disubstituted
pyrazolyl pyrimidin‐2‐amine moiety based on scaffold hopping and
developed a series of compounds 7a‒t as novel anticancer agents
with HSP90 inhibition. Among these, compound 7t displayed
excellent HSP90 inhibition at nanomolar scale (20 nM) and in
vitro antiproliferative potential against three cancer cell lines
(IC50 < 5 µM). HSP90 inhibition was also evidenced via inhibition of
client proteins (pHER2 and pERK1/2) by compounds 7t and 7q.
Molecular docking studies well appreciated compounds 7p‒t as
excellent HSP90 inhibitors. The presence of mono substitution at
para position on rings A and B of pyrazole especially with p‐nitro
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4.1.3
General synthetic procedure for
disubstituted pyrazolyl pyrimidin‐2‐amines (7a‒t)
To the solution of intermediates 6a‒t (10 mmol) in DMF (10 ml),
guanidine hydrochloride (10 mmol) was added followed by potassium
tert‐butoxide (1 mmol) and refluxed for 6–8 hr. Then, the reaction
mixture was quenched with dil. HCl and extracted into ethyl acetate
(50 ml × 3). The organic layer was collected and the obtained crude
solid was purified by column chromatography (EtOAc/Hex = 1:2) to
obtain compounds 7a‒t.
group on ring
B and 2‐amino group of pyrimidine are the
pharmacophoric essentials of this series at active site of HSP90.
Therefore, the higher potency of these drugs with targeted action
make them superior over conventional HSP90 inhibitors.

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4‐(3‐(4‐Bromophenyl)‐1‐(2,4‐dichlorophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐
methoxyphenyl)pyrimidin‐2‐amine (7a)
(dd, 2H, ArH), 7.55–7.71 (m, 7H, ArH), 7.84–7.97 (m, 2H, ArH), 8.46
(s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.1, 163.7, 162.5, 146.7,
142.3, 133.8, 132.8, 133.2, 132.7, 132.3, 131.5, 130.0, 128.7, 122.5,
123.6, 103.7, 101.7. ESI‐MS: m/z 616 (M+H+).
Yield 65%; m.p. 145°C; FT‐IR (KBr) ν/cm−1: 3,437 (NH2), 2,748
(CH aliphatic), 1,600 (C═C aromatic). 1H‐NMR (300 MHz, CHCl3) δ
(ppm): 3.85 (s, 3H, OCH3), 5.20 (s, 2H, NH2) (D2O exchangeable),
6.84–6.99 (m, 4H, ArH), 7.39–7.42 (d, 1H, ArH, J = 9), 7.57–7.66 (m,
5H, ArH), 7.74–7.77 (d, 2H, ArH, J = 9), 8.41 (s, 1H, ArH). 13C‐NMR
(75 MHz, CDCl3): δ 164.8, 163.6, 160.0, 159.7, 147.8, 143.7, 134.5,
133.3, 132.6, 132.5, 131.8, 131.6, 128.7, 128.3, 128.1, 125.5, 123.1,
122.7, 117.8, 104.5, 111.3, 55.8. ESI‐MS: m/z 567 (M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐meth-
oxyphenyl)pyrimidin‐2‐amine (7f)
Yield 75%; m.p. 240°C; FT‐IR (KBr) ν/cm−1: 3,404 (NH2), 2,723
(CH aliphatic), 1,550 (C═C aromatic), 1,334 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.76 (s, 3H, OCH3), 5.69 (s, 2H, NH2)
(D2O exchangeable), 6.83–6.86 (d, 2H, ArH, J = 9.3), 6.88 (d, 1H, ArH),
7.47–7.59 (m, 4H, ArH), 7.74–7.77 (d, 2H, ArH, J = 9.5), 8.03–8.06 (d,
2H, ArH, J = 9.2), 8.26–8.29 (d, 2H, ArH, J = 9.3), 8.83 (s, 1H, ArH).
13C‐NMR (75 MHz, CDCl3): 165.2, 163.4, 161.8, 159.3, 145.8, 143.6,
131.7, 131.3, 130.6, 129.0, 128.4, 125.4, 124.3, 123.3, 118.7, 114.1,
105.1, 101.4, 55.8. ESI‐MS: m/z 565 (M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(2,4‐dichlorophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐
fluorophenyl)pyrimidin‐2‐amine (7b)
Yield 70%; m.p. 220°C; FT‐IR (KBr) ν/cm−1: 3,433 (NH2), 1,629
(C═C aromatic), 1,226 (C–F). 1H‐NMR (300 MHz, CHCl3) δ (ppm):
5.15 (s, 2H, NH2) (D2O exchangeable), 6.88 (s, 1H, ArH), 7.11–7.14
(d, 2H, ArH, J = 9), 7.38–7.42 (dd, 1H, ArH, J = 6), 7.57–7.59 (m, 5H,
ArH), 7.63 (s, 1H, ArH), 7.77–7.82 (q, 2H, ArH), 8.43 (s, 1H, ArH).
13C‐NMR (75 MHz, CDCl3): 164.8, 163.2, 158.9, 149.7, 145.6,
136.9, 135.6, 132.3, 132.1, 130.8, 130.2, 128.9, 128.4, 127.1,
123.0, 121.8, 115.9, 113.6, 105.5, 101.9, 101.3. ESI‐MS: m/z 556
(M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐fluoro-
phenyl)pyrimidin‐2‐amine (7g)
Yield 75%; m.p. 240°C; FT‐IR (KBr) ν/cm−1: 3,404 (NH2), 1,550
(C═C aromatic), 1338 (NO2), 1,224 (C–F). 1H‐NMR (300 MHz,
CHCl3) δ (ppm): 5.80 (s, 2H, NH2) (D2O exchangeable), 6.93 (s, 1H,
ArH), 7.02–7.07 (d, 2 H, ArH), 7.48–7.59 (m, 4H, ArH), 7.83 (d, 2H,
ArH), 8.05–8.07 (d, 2H, ArH, J = 6.4), 8.27–8.30 (d, 2H, ArH, J = 9.4),
8.89 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.7, 164.8, 162.5,
162.0, 146.1, 145.4, 145.0, 132.4, 132.0, 131.8, 131.5, 130.5, 128.7,
123.5, 123.1, 118.9, 115.0, 103.9. ESI‐MS: m/z 553 (M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(2,4‐dichlorophenyl)‐1H‐pyrazol‐4‐yl)‐6‐
(3,4‐dimethoxyphenyl)pyrimidin‐2‐amine (7c)
Yield 70%; m.p. 160°C; FT‐IR (KBr) ν/cm−1: 3,437 (NH2), 2,852
(CH aliphatic), 1,583 (C═C aromatic). 1H‐NMR (300 MHz, CHCl3) δ
(ppm): 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 5.10 (s, 2H, NH2) (D2O
exchangeable), 6.88 (s, 1H, ArH), 6.91 (s, 1H, ArH), 7.32–7.34 (d, 1H,
ArH, J = 6.4), 7.37–7.42 (m, 2H, ArH), 7.59 (m, 5H, ArH), 7.63–7.66 (d,
1H, ArH, J = 9.2), 8.45 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.2,
163.4, 159.8, 151.2, 149.0, 147.5, 136.2, 134.8, 133.4, 131.6, 130.8,
130.5, 129.7, 128.9, 128.4, 128.1, 122.9, 120.8, 120.0, 110.9, 109.4,
105.2, 55.9. ESI‐MS: m/z 598 (M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(3,4‐di-
methoxyphenyl)pyrimidin‐2‐amine (7h)
Yield 70%; m.p. 260°C; FT‐IR (KBr) ν/cm−1: 3,423 (NH2), 2,746
(CH aliphatic), 1,629 (C═C aromatic), 1,332 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.80 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
5.62 (s, 2H, NH2) (D2O exchangeable), 6.82–6.84 (d, 2H, ArH), 7.29 (s,
1H, ArH), 7.37–7.40 (d, 1H, ArH, J = 9), 7.50–7.59 (m, 4H, ArH),
8.03–8.06 (d, 2H, ArH), 8.27–8.30 (d, 2 H, ArH, J = 9), 8.83 (s, 1H,
ArH). 13C‐NMR (75 MHz, CDCl3): 165.1, 162.6, 161.1, 153.1, 149.6,
146.7, 146.3, 145.7, 131.9, 131.5, 130.6, 127.8, 123.9, 123.3, 117.6,
115.9, 111.4, 107.8, 104.6, 101.1, 56.5. ESI‐MS: m/z 595 (M+H+).
4‐(3‐(4‐Bromophenyl)‐1‐(2,4‐dichlorophenyl)‐1H‐pyrazol‐4‐yl)‐6‐
(2,4‐dimethoxyphenyl)pyrimidin‐2‐amine (7d)
Yield 70%; m.p. 140°C; FT‐IR (KBr) ν/cm−1: 3,444 (NH2), 2,752
(CH aliphatic), 1583 (C═C aromatic). 1H‐NMR (300 MHz, CHCl3) δ
(ppm): 3.85 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 5.25 (s, 2H, NH2) (D2O
exchangeable), 6.85–6.87 (d, 1H, ArH), 6.90 (s, 1H, ArH), 7.38–7.39
(d, 1H, ArH, J = 3.1), 7.40–7.42 (d, 1H, ArH, J = 6.3), 7.44 (s, 1H, ArH),
7.56–7.57 (m, 2H, ArH), 7.63–7.66 (d, 2H, ArH, J = 9.5), 8.25–8.27 (m,
2H, ArH); 8.42 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.7, 163.3,
162.7, 160.6, 159.6, 146.9, 142.6, 134.7, 133.5, 133.6, 132.6, 131.8,
131.6, 131.2, 129.4, 127.6, 123.5, 122.4, 110.2, 107.5, 104.9, 101.8,
98.6, 56.2, 55.4. ESI‐MS: m/z 619 (M+Na+).
4‐(3‐(4‐Bromophenyl)‐1‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(2,4‐di-
methoxyphenyl)pyrimidin‐2‐amine (7i)
Yield 78%; m.p. 200°C; FT‐IR (KBr) ν/cm−1: 3,446 (NH2), 2,796
(CH aliphatic), 1,581 (C═C aromatic), 1,336 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.91 (s, 3H, OCH3), 3.93 (s, 3H, OCH3),
5.20 (s, 2H, NH2) (D2O exchangeable), 6.86–6.89 (d, 2H, ArH),
7.31 (s, 1H, ArH), 7.36–7.38 (d, 1H, ArH), 7.55–7.66 (m, 4H, ArH),
7.99–8.02 (d, 2H, ArH, J = 6.5), 8.36–8.39 (d, 2H, ArH, J = 9.0),
8.66 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.1, 163.7, 160.7,
159.8, 159.4, 147.6, 145.1, 143.6, 132.7, 132.2, 131.8, 129.5,
127.7, 123.5, 118.7, 109.6, 107.2, 103.7, 101.9, 98.6, 56.3, 55.4.
ESI‐MS: m/z 595 (M+H+).
4‐(4‐Bromophenyl)‐6‐(3‐(4‐bromophenyl)‐1‐(2,4‐dichlorophenyl)‐1H‐
pyrazol‐4‐yl)pyrimidin‐2‐amine (7e)
Yield 45%; m.p. 230°C; FT‐IR (KBr) ν/cm−1: 3,448 (NH2), 1,629
(C═C aromatic), 669 (C–Br). 1H‐NMR (300 MHz, CHCl3) δ (ppm):
5.30 (s, 2H, NH2) (D2O exchangeable), 6.85 (s, 1H, ArH), 7.32–7.45

METTU ET AL.
7 of 9
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4‐(4‐Bromophenyl)‐6‐(3‐(4‐bromophenyl)‐1‐(4‐nitrophenyl)‐1H‐pyra-
5.39 (s, 2H, NH2) (D2O exchangeable), 6.36 (s, 1H, ArH), 6.45–6.47 (d,
1H, ArH), 7.36–7.39 (d, 1H, ArH), 7.50–7.61 (m, 3H, ArH), 7.87–7.90
(d, 3H, ArH, J = 9.4), 8.15–8.17 (d, 2H, ArH, J = 7.3), 8.35 (s, 1H, ArH).
13C‐NMR (75 MHz, CDCl3): 165.2, 163.6, 162.0, 161.6, 158.3, 147.9,
145.8, 141.7, 139.1, 134.9, 133.2, 132.5, 131.6, 131.0, 128.5, 126.1,
123.5, 121.8, 110.2, 103.5, 101.3, 98.8, 56.7, 55.8. ESI‐MS: m/z 562
(M+H+).
zol‐4‐yl)pyrimidin‐2‐amine (7j)
Yield 72%; m.p. 260°C; FT‐IR (KBr) ν/cm−1: 3,411 (NH2), 1,579
(C═C aromatic), 1,334 (NO2), 601 (C–Br). 1H‐NMR (300 MHz, CHCl3)
δ (ppm): 5.11 (s, 2H, NH2) (D2O exchangeable), 6.93 (d, 1H, ArH),
7.51–7.67 (m, 6H, ArH), 7.84–7.87 (d, 2H, ArH, J = 8.5), 8.00–8.03 (d,
2H, ArH, J = 9), 8.30–8.33 (d, 1H, ArH, J = 9.4), 8.38–8.41 (d, 1H, ArH,
J = 9.2), 8.64 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.7, 163.3,
162.0, 145.8, 145.5, 143.2, 135.7, 132.6, 132.0, 131.9, 128.4, 124.6,
123.7, 119.4, 103.7, 101.3. ESI‐MS: m/z 554 (M+H+).
4‐(4‐Bromophenyl)‐6‐(1‐(2,4‐dichlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐
pyrazol‐4‐yl)pyrimidin‐2‐amine (7o)
Yield 68%; m.p. 240°C; FT‐IR (KBr) ν/cm−1: 3,416 (NH2), 1,602
(C═C aromatic), 1,342 (NO2), 561 (C–Br). 1H‐NMR (300 MHz, CHCl3)
δ (ppm): 5.10 (s, 2H, NH2) (D2O exchangeable), 6.97 (s, 1H, ArH),
7.41–7.45 (dd, 1H, ArH), 7.55–7.66 (m, 4H, ArH), 7.72–7.75 (d, 2H,
ArH, J = 8.4), 7.89–7.91 (d, 2H, ArH, J = 9.2), 8.26–8.29 (d, 2H, ArH),
8.40 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.7, 164.8, 161.5,
148.9, 145.6, 140.7, 139.5, 134.6, 132.5, 131.2, 129.1, 123.7, 123.6,
103.7, 101.6. ESI‐MS: m/z 604 (M+Na+).
4‐(1‐(2,4‐Dichlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐
methoxyphenyl)pyrimidin‐2‐amine (7k)
Yield 74%; m.p. 180°C; FT‐IR (KBr) ν/cm−1: 3,421 (NH2), 2,794
(CH aliphatic), 1,631 (C═C aromatic), 1,344 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.85 (s, 3H, OCH3), 5.07 (s, 2H, NH2)
(D2O exchangeable), 6.92–6.95 (d, 3H, ArH), 7.41–7.44 (dd, 1 H, ArH,
J = 6), 7.60–7.66 (dd, 2H, ArH), 7.81–7.84 (d, 2H, ArH, J = 9),
7.90–7.93 (d, 2H, ArH, J = 9.4), 8.26–8.29 (d, 2H, ArH, J = 8.6), 8.39
(s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.1, 164.6, 163.5, 160.6,
147.8, 146.9, 140.8, 139.1, 134.6, 133.5, 132.7, 130.0, 129.5, 128.7,
127.4, 127.0, 124.4, 122.7, 112.7, 103.7, 101.8, 55.7. ESI‐MS: m/z 554
(M+H+).
4‐(1‐(4‐Chlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐meth-
oxyphenyl)pyrimidin‐2‐amine (7p)
Yield 80%; m.p. 230°C; FT‐IR (KBr) ν/cm−1: 3,441 (NH2), 2,796
(CH aliphatic), 1,629 (C═C aromatic), 1,332 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.89 (s, 3H, OCH3), 5.18 (s, 2H, NH2)
(D2O exchangeable), 6.86–6.91 (m, 3H, ArH), 7.29–7.32 (d, 2H,
ArH, J = 7.8), 7.48–7.52 (m, 2H, ArH), 7.75–7.78 (d, 2H, ArH,
J = 8.2), 7.92–7.94 (d, 2H, ArH, J = 6.2), 8.28–8.30 (d, 2H, ArH,
J = 6.5), 8.48 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.0,
163.5, 162.7, 160.8, 147.5, 145.6, 139.6, 137.6, 134.7, 132.6,
130.5, 129.5, 128.6, 128.1, 126.5, 118.6, 115.6, 107.9, 101.6,
55.4. ESI‐MS: m/z 520 (M+Na+).
4‐(1‐(2,4‐Dichlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐
fluorophenyl)pyrimidin‐2‐amine (7l)
Yield 70%; m.p. 220°C; FT‐IR (KBr) ν/cm−1: 3,429 (NH2), 1,604
(C═C aromatic), 1,344 (NO2), 1,228 (C–F). 1H‐NMR (300 MHz,
CHCl3) δ (ppm): 5.75 (s, 2H, NH2) (D2O exchangeable), 6.95 (s, 1H,
ArH), 7.03–7.06 (d, 2H, ArH, J = 9.2), 7.55–7.62 (m, 3H, ArH),
7.83–7.91 (m, 4H, ArH), 8.15–8.18 (d, 2H, ArH, J = 9.3), 8.42 (s, 1H,
ArH). 13C‐NMR (75 MHz, CDCl3): 165.3, 163.1, 161.5, 160.0, 148.8,
146.7, 137.1, 138.9, 136.2, 131.2, 131.8, 130.5, 129.4, 127.5, 123.1,
117.6, 103.7, 101.5. ESI‐MS: m/z 521 (M+H+).
4‐(1‐(4‐Chlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(4‐fluoro-
phenyl)pyrimidin‐2‐amine (7q)
Yield 65%; m.p. 280°C; FT‐IR (KBr) ν/cm−1: 3,448 (NH2), 1,597
(C═C aromatic), 1,334 (NO2), 1,228 (C–F). 1H‐NMR (300 MHz,
CHCl3) δ (ppm): 5.71 (s, 2H, NH2) (D2O exchangeable), 7.05–7.07
(d, 2H, ArH), 7.39–7.42 (d, 2H, ArH, J = 8.7), 7.79–7.82 (d, 2H, ArH,
J = 8.7), 7.87–7.96 (m, 5H, ArH), 8.16–8.19 (d, 2H, ArH, J = 8.7), 8.73
(s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.6, 163.4, 161.4, 160.4,
146.7, 145.5, 135.6, 131.5, 131.2, 131.0, 129.7, 128.5, 124.6, 119.6,
116.7, 103.7, 101.7. ESI‐MS: m/z 508 (M+Na+).
4‐(1‐(2,4‐Dichlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(3,4‐
dimethoxyphenyl)pyrimidin‐2‐amine (7m)
Yield 78%; m.p. 140°C; FT‐IR (KBr) ν/cm−1: 3,414 (NH2), 2,725
(CH aliphatic), 1,602 (C═C aromatic), 1,338 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3),
5.28 (s, 2H, NH2) (D2O exchangeable), 6.86 (s, 1H, ArH), 6.89–6.92 (d,
1H, ArH, J = 9.2), 7.29–7.33 (dd, 1H, ArH), 7.41–7.45 (dd, 1H, ArH),
7.49 (s, 1H, ArH), 7.60–7.70 (m, 2H, ArH), 7.90–7.93 (d, 2H, ArH),
8.30–8.32 (d, 2H, ArH, J = 4.6), 8.41 (s, 1H, ArH). 13C‐NMR (75 MHz,
CDCl3): 165.4, 163.5, 162.7, 149.2, 149.6, 147.5, 145.1, 141.6, 139.6,
134.6, 134.1, 131.5, 130.0, 129.7, 129.3, 126.1, 123.4, 121.6, 120.9,
111.6, 109.3, 102.9, 100.6, 56.6. ESI‐MS: m/z 562 (M+H+).
4‐(1‐(4‐Chlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(3,4‐di-
methoxyphenyl)pyrimidin‐2‐amine (7r)
Yield 72%; m.p. 230°C; FT‐IR (KBr) ν/cm−1: 3,444 (NH2), 2,725
(CH aliphatic), 1,631 (C═C aromatic), 1,332 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.91 (s, 3H, OCH3), 3.92 (s, 3H, OCH3),
5.17 (s, 2H, NH2) (D2O exchangeable), 6.86–6.89 (d, 1H, ArH, J = 8.4),
6.92 (s, 1H, ArH), 7.29–7.32 (dd, 1H, ArH), 7.47–7.50 (d, 3H, ArH,
J = 8.8), 7.76–7.79 (d, 2H, ArH, J = 9), 7.92–7.95 (d, 2H, ArH, J = 8.7),
8.28–8.31 (d, 2H, ArH, J = 9.2), 8.49 (s, 1H, ArH). 13C‐NMR (75 MHz,
CDCl3): 165.7, 164.7, 161.6, 150.8, 149.6, 147.6, 145.6, 139.7, 135.6,
4‐(1‐(2,4‐Dichlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(2,4‐
dimethoxyphenyl)pyrimidin‐2‐amine (7n)
Yield 70%; m.p. 180°C; FT‐IR (KBr) ν/cm−1: 3,433 (NH2), 2,723
(CH aliphatic), 1,579 (C═C aromatic), 1,348 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.53 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),

8 of 9
METTU ET AL.
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131.9, 131.6, 130.7, 129.2, 124.5, 120.6, 119.7, 110.0, 107.4, 103.9,
56.1. ESI‐MS: m/z 529 (M+Na+).
compounds to inhibit HSP90α. The N‐terminal domain assay kit was
procured from BPS Biosciences, San Diego, CA (#50293) and the
assay protocol was followed accordingly.[24] The master mixture was
prepared with: N wells × (15 μl of Hsp90 assay buffer + 5 μl of 40 mM
DTT + 5 μl of 2 mg/ml BSA + 40 of μl H2O). Then, 65 μl of master
mixture was added to all wells. A total of 5 μl of diluted FITC‐labeled
geldanamycin (100 nM) was added to each well labeled “positive
control”, “negative control”, and “test”. A total of 10 μl of test
compounds (7a‒c, 7g, 7h, 7j, 7m, 7o, 7p‒t) were added to each well
labeled “Test”. A total of 25 μl of assay buffer was added to the wells
labeled “Blank”. Reaction was initiated by adding 20 μl of diluted
Hsp90α (17 ng/μl), to each well designated “positive control” and
“test” and incubated at room temperature for 2–3 hr with slow
shaking. FP values were measured at wavelengths of 485 nm
(excitation) and 530 nm (emission) using Biotek Synergy H1 hybrid
reader. The fraction of tracer bound to Hsp90 was correlated to the
mP value and plotted against values of competitor concentrations.
The inhibitor concentration at which 50% of bound GM was
displaced was obtained by fitting the data. All experimental data
were analyzed using SOFTmax Pro 4.3.1 and plotted using Prism 4.0
(GraphOad Software Inc., San Diego, CA).
4‐(1‐(4‐Chlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyrazol‐4‐yl)‐6‐(2,4‐di-
methoxyphenyl)pyrimidin‐2‐amine (7s)
Yield 81%; m.p. 210°C; FT‐IR (KBr) ν/cm−1: 3,441 (NH2), 2,748
(CH aliphatic), 1,610 (C═C aromatic), 1,350 (NO2). 1H‐NMR
(300 MHz, CHCl3) δ (ppm): 3.58 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
5.04 (s, 2H, NH2) (D2O exchangeable), 6.43 (d, 1H, ArH), 6.57–6.60
(dd, 1H, ArH,), 7.23 (s, 1H, ArH), 7.46–7.49 (d, 2H, ArH, J = 8.7),
7.75–7.77 (d, 2H, ArH, J = 8.7), 7.92–7.97 (m, 3H, ArH), 8.26–8.29 (d,
2H, ArH, J = 8.7), 8.43 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 164.1,
163.6, 162.1, 159.3, 158.8, 149.3, 147.6, 139.9, 137.8, 132.9, 131.8,
129.8, 129.7, 129.1, 123.5, 122.6, 120.4, 119.0, 110.3, 105.3, 101.3,
98.7, 55.2, 55.4. ESI‐MS: m/z 529 (M+Na+).
4‐(4‐Bromophenyl)‐6‐(1‐(4‐chlorophenyl)‐3‐(4‐nitrophenyl)‐1H‐pyra-
zol‐4‐yl)pyrimidin‐2‐amine (7t)
Yield 71%; m.p. 290°C; FT‐IR (KBr) ν/cm−1: 3,435 (NH2), 1,599
(C═C aromatic), 1,344 (NO2), 567 (C–Br). 1H‐NMR (300 MHz, CHCl3)
δ (ppm): 5.78 (s, 2H, NH2) (D2O exchangeable), 7.08 (s, 1H, ArH),
7.40–7.59 (m, 4H, ArH), 7.64 (s, 1H, ArH), 7.75–7.90 (m, 2H, ArH),
7.93 (d, 2H, ArH, J = 9.3), 8.16–8.19 (d, 2H, ArH, J = 8.7), 8.25–8.28 (d,
1H, ArH), 8.76 (s, 1H, ArH). 13C‐NMR (75 MHz, CDCl3): 165.5, 163.3,
162.1, 147.8, 145.5, 132.6, 131.9, 131.2, 129.5, 128.9, 128.7, 124.2,
123.7, 120.7, 103.8, 101.5. ESI‐MS: m/z 548 (M+Na+).
|
4.2.3
Western blot analysis
MCF‐7 cells were seeded at a density of 1 × 106 in 100 cm2 petri
plates. After 24 hr culture, the cells were treated with three different
concentrations (0.5, 1, and 2 fold of IC50) of compounds 17‐AAG, 7q,
7t, or with vehicle (DMSO 0.5%), for 24 hr. After completion, cells
were trypsinised and washed with phosphate buffered saline and
then with lysis solution (50 mM Tris‐HCl pH 7.4, 150 mM NaCl, 2 mM
ethylenediaminetetraacetic acid, 1% NP‐40 (v/v), 0.1% sodium
dodecyl sulfate (SDS; w/v), 5 mM dithiothreitol, and protease
inhibitor). Lysate protein concentration was determined by the
bicinchoninic acid protein assay (Thermo Scientific) and equal
amount of protein from each lysate was loaded on SDS polyacryla-
mide gel electrophoresis and blotted onto polyvinylidene fluoride
membrane (Millipore), conjugated with specific primary antibodies
and then probed with goat anti‐rabbit antibody coupled to
peroxidase antibody. The bands were visualized on a chemilumines-
cence plate reader. Expression of pHER2 and pErk1/2 proteins was
determined by probing with specific pHER2 and pERK1/2 antibodies,
(#6942 and #9101, Cell Signaling Technology, Danvers, MA). Here, β‐
actin was taken as loading control.[25]
|
4.2
In vitro anticancer activity
|
4.2.1
3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide assay
Human breast carcinoma MDA‐MB‐231 and MCF‐7, colon carcinoma
HCT116 were cultured in RPMI medium. All the cell cultures were
augmented with 2 µM glutamine, 10% fetal bovine serum, 100 units
per ml penicillin, and 100 µg/ml streptomycin and incubated at 37°C
under 5% CO2. In vitro cell growth inhibition was assessed with the
MTT assay. Cells were seeded into 24‐well culture plates at a density
of 3,700 cells per ml for MCF‐7, 4,000 cells per ml for MDA‐MB‐231,
3,500 cells per ml for HCT116 and incubated for 24 hr. Then, the
cells were incubated with various concentrations of synthesized
compounds (7a–t) along with standard drug (17‐AAG) for 48 hr. The
media was removed and MTT reagent (5 mg/10 ml) was added and
incubated for 4 hr. Further, the obtained formazan was solubilized by
lysis buffer (10% SDS in 0.1 N HCl), DMSO (100 µl/cell), and
absorbance was recorded at 570 nm with multi detection reader
Spectramax M4, Molecular Devices USA. Control wells contained the
equivalent volume of vehicle DMSO (1%). The values shown are the
means and standard deviation of at least three independent
experiments performed in duplicate.[22,23]
|
4.3
Docking studies
The PDB entry 5LRZ (a co‐crystal of A003643501 in complex with
HSP90α) was downloaded from Protein Data Bank due to its
structural resemblance with test series and were docked at
N‐terminal ATP binding site HSP90α using Glide program embedded
in Maestro 9.1 on Schrodinger engine. The downloaded PDB was
further processed for energy minimization and then grid was
generated at active site. The ligands were subjected to ligprep
|
4.2.2
Fluorescence polarization assay
The ability of test compounds to compete for N‐terminal ATP binding
site with FITC‐labeled geldanamycin determines the potency of

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simulations to generate energy minimized 3D structures and were
docked flexibly in the protein grids using Glide‐extra precision (XP)
simulations. The final docking score (g score) was based on
interactions such as lipophilic pair term, hydrophobic enclosure
reward, hydrogen bonding and electrostatic rewards. The performed
docking was validated by superimposing the docked A003643501
with cocrystal ligand and the RMSD was less than 0.01.
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ACKNOWLEDGMENTS
[16] J. Shi, R. Van de Water, K. Hong, R. B. Lamer, K. W. Weichert, C. M.
Sandoval, S. R. Kasibhatla, M. F. Boehm, J. Chao, K. Lundgren, N.
Timple, R. Lough, G. Ibanez, C. Boykin, F. J. Burrows, M. R. Kehry, T.
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This research was supported by Women Scientist Scheme (WOS‐A),
Department of Science and Technology (DST), New Delhi, India
(Grant no. SR/WOS‐A/CS‐1003/2014).
CONFLICT OF INTEREST
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1969, 10, 109.
The authors declare that there is no conflict of interest.
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ORCID
[21] S. Roughley, L. Wright, P. Brough, A. Massey, R. E. Hubbard, Top.
Curr. Chem. 2012, 317, 61.
Naikal James Prameela Subhashini
http://orcid.org/0000-0003-
3852-6043
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