Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity

Article abstract of DOI:10.1016/j.ejmech.2019.111699

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of no

Full text of DOI:10.1016/j.ejmech.2019.111699

European Journal of Medicinal Chemistry 183 (2019) 111699
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock
protein 90 (HSP90) inhibitors with significant anti-HIV activity
Jay Trivedi a, Afsana Parveen b, Farhana Rozy b, Alapani Mitra a, Chandralata Bal b,
,
c
,
, *
Debashis Mitra a **, Ashoke Sharon b
a National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune, 411007, India
b Department of Chemistry, Birla Institute of Technology, Mesra, Ranchi, 835215, India
c Centre for DNA Fingerprinting and Diagnostics, Hyderabad, 500039, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2019
Received in revised form
11 September 2019
Accepted 12 September 2019
Available online 16 September 2019
The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emer-
gence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In
continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, syn-
thesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their
anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory
potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV
activity of these molecules suggests that 2l has ~3.5 fold better therapeutic index than AUY922, the
second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load
independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse
Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free
in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven
gene expression. Taken together, structural rationale, modeling studies and characterization of biological
activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and
thus opens a new horizon to develop novel anti-HIV therapeutic candidates.
Keywords:
HSP90
anti-HIV compound
Isoxazole
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
of infected individuals, the fact remains that we are still far from
eradicating the disease from human population [6]. With limited
Since its discovery in 1983 as the causative agent of Acquired
immunodeficiency syndrome (AIDS) [1e3], Human immunodefi-
ciency virus (HIV) have claimed more than 38 million lives till date
[4]. HIV-1 primarily infects vital cells of the immune system that
includes CD4þ T-cells, monocytes and macrophages, which results
in the death of infected as well as bystander cells by multiple
mechanisms [5]. Eventually, severely compromised immunity in-
vites opportunistic infections and cancer to thrive, ultimately
resulting in the death of infected individuals. With currently
practiced combination antiretroviral therapy (cART), although we
have been able to control AIDS to a great extent and prolong the life
resources, HIV-1 is not able to complete its life cycle within the host
on its own [7]. HIV-1 exploits the host machinery not only to
produce new functional viral progeny but also to evade from host
immune defense system [8]. Several studies in the past decade have
identified over 900 different host cellular factors and HIV de-
pendency factors (HDFs), modulation of many of which inhibits
viral replication in the host without modulating the host physi-
ology [9e15]. These studies suggest that targeting host cellular
factors involved in viral life cycle along with viral targets may help
us to combat the limitations of current arsenal of anti-HIV medi-
cations more efficiently. Heat shock protein 90 (HSP90) not only
shapes the consequence and fate of genetic variations in humans
but also plays a dynamic role in the life cycle of a wide range of
virus species [16e20]. In case of HIV-1 infection, HSP90 is a crucial
factor for regulation of LTR driven gene expression through cellular
* Corresponding author.
** Corresponding author.
E-mail addresses: dmitra@nccs.res.in, dmitra@cdfd.org.in (D. Mitra), asharon@
bitmesra.ac.in (A. Sharon).
NF-kB pathway and reactivation of latent viral reservoirs [21e23].
The vital role of HSP90 in virus life cycle was confirmed when
https://doi.org/10.1016/j.ejmech.2019.111699
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
several HSP90 inhibitors, including Geldanamycin (the first gener-
ation HSP90 inhibitor) and its analogues, were reported to block
virus propagation in the host cells at non-toxic concentrations
[24e28].
The second generation HSP90 inhibitor, AUY922 (Fig. 1) was
found to prevent reactivation of latent HIV-1 reservoirs in CD4þ T-
cells in recent studies [29,30].A pivotal role of HSP90 in HIV-1
transcription and potential of HSP90 inhibitors to inhibit HIV-1
LTR driven gene expression opens new scope to obtain novel as
well as more specific HSP90 inhibitors to eliminate HIV from the
host.
The activity of second generation HSP90 inhibitor AUY922 to
control HIV-1 LTR driven gene expression and reactivation of latent
reservoirs induced us to consider it in designing of our new scaffold.
Here, we report a new route for the synthesis of fifteen novel 2-
isoxazol-3-yl-acetamide analogues (I) and discovery of potential
anti-HIV lead candidate targeting HSP90.
Scheme 1. A proposed reaction mechanism for one-step scaffold transformation from
pyranone carboxamide (1a-o) to isoxazole acetamide scaffold (2a-o) through intra-
molecular ring transformation.
2. Results and discussion
Table 1
An anti-HIV activity of synthesized compounds 2a-o including reference compound
AUY922 at Highest Non-Cytotoxic Concentration.
2.1. Chemistry
A novel ring contraction strategy implemented as the one-step
transformation of 2H-pyranone to isoxazole through intra-
molecular nucleophilic ring opening by hydroxyamino functional
group (-NHOH) followed by decarboxylation. For this purpose, the
4-hydroxyamino-N-substituted-2-oxo-6-aryl-2H-pyran-3-
carboxamides (1a-o) were synthesized starting from aryl aceto-
phenones in three steps following the reported procedure by our
group [31]. Then appropriate 1a-o was stirred in dioxane using
NaHCO3 as a base at 80 ꢀC for 24 h.
We propose that hydroxyl group of eNHOH made a nucleophilic
attack on the C-6 of 2H-pyranone ring to initiate ring opening
(Scheme 1). Aromatization followed by decarboxylation yields the
2-isoxazol-3-yl-acetamide derivatives (2a-o). All the synthesized
compounds (Table 1) were characterized by physical and spectro-
scopic analysis.
Compd.
R1
R2
HNCa (nM)
%
Inhibitionb
AUY922
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
e
e
1000
1000
1000
1000
100
88.35 1.64
89.13 2.86
98.41 0.44
23.5 2.57
33.33 1.1
85.37 3.49
49.04 2.02
13.41 1.53
59.58 0.71
27.7 2.11
94.79 3.25
60.88 1.47
85.03 1.63
95.45 1.37
53.06 1.71
56.85 1.83
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-Me-Ph
2-Me-Ph
3-Me-Ph
4-F-Ph
4-Cl-Ph
4-OH-Ph
4-OMe-Ph
4-COOMe-Ph
2-OH-Ph
n-Pr
500
100
1000
1000
100
1000
100
1000
1000
1000
100
2.2. Biology
i-Pr
Fifteen novel derivatives (2a-o) were first evaluated for their
Highest non-cytotoxic concentration (HNC) in TZM-bl cells using
MTT based cell proliferation assay [32]. Further, the anti-HIV ac-
tivities at their respective HNCs were analyzed by luciferase-based
reporter assay in TZM-bl cells infected with 0.5 MOI of HIV-1NL4-3
and the results are reported in Table 1. The first compound (2a)
with two unsubstituted phenyl groups significantly inhibited
replication of virus up to 89.0% at its HNC of 1000 nM. When we
introduced a methyl group at 4-position on R2 (2b), there was no
change in HNC, however, the inhibition potential was enhanced to
98.0%. Then 2c and 2d were synthesized with a methyl group at 2-
2m
2n
2
4-OMe-Ph
furan-2-yl
thien-2-yl
3-Me-Ph
4-OH-Ph
4-Me-Ph
a
Highest non-cytotoxic concentration (HNC) in nM.
% Inhibition of virus transcription at HNC.
b
and 3-position respectively. Though 2c was like 2a-b in terms of
cytotoxicity, we observed a nearly 3-fold drop in its anti-HIV ac-
tivity. However, just shifting the methyl group to 3-position (2d), a
major drop in HNC, as well as the ability to inhibit replication of HIV
was observed.
Therefore, we focused to do alteration only at the 4-position of
R2. The cytotoxicity of halo analogues (2e-f) was increased (drop in
HNC) as well as the inhibitory potential was reduced. Introduction
of a hydrophilic hydroxyl group (2g) made the compound inactive
(with <15% inhibition). When we masked the polar hydroxy group
to increase the hydrophobic character in 2h, the result was increase
in inhibitory potential. In addition, 2i with a masked carboxyl group
(methyl ester) was tested and found to be less active than 2h, along
with enhanced cytotoxicity. These results indicate that a hydro-
phobic group (e.g. methyl) is preferred over polar groups (halo/
hydroxy) at 4-position on R2 for better inhibition of HIV-1. We had
observed that a non-polar group at 2-position on R2 (2c) was not
good for anti-HIV activity. Therefore, we changed it to a polar
Fig. 1. The chemical structure of second generation HSP90 inhibitor AUY922 and novel
scaffold I for synthesis and anti-HIV activity evaluation.

J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
3
Table 3
hydroxy group (2j) and to our surprise the compound was highly
active with no change in cytotoxicity.
Therapeutic Index of 2l in different cell types.
Further, the potential of R2 for an aliphatic group (2k-l), various
combinations of R1 and R2 groups (2m-o) were tested. Interesting
to note that the isopropyl group at R2 (2l) showed significant in-
hibition. The replacement of the phenyl ring at R1 with 4-
methoxyphenyl (2m) enhanced inhibition but replacement with
thiophene or furan (2q-r) didn't yield any significant result. Thus,
structure-activity relationship (SAR) studies provided the positive
role of a hydrophobic group at R2 position. From the above anti-HIV
and cytotoxicity studies, six molecules 2a-b, 2e, 2j and 2l-m with
inhibitory potentials >85% were selected for detailed biological
studies.
The therapeutic index [TI ¼ CC50/IC50] of selected compounds
2a-b, 2e, 2j, and 2l-m were determined in human CD4þ T-cell line
CEM-GFP [33]. For this, we first determined CC50 (Cytotoxic Con-
centration 50%, Table 2) values by MTT based cell proliferation
assay. We then determined the IC50 (Inhibitory concentration 50%,
Table 2) values in CEM-GFP cell line infected with 0.5 MOI of HIV-
Cell types.
CC50
(
m
M)
IC50 (nM)
Therapeutic Index
Jurkat J6
U937
hPBMCs
60.27 1.23
56.65 1.0
67.88 1.14
52.67 3.32
52.99 2.68
56.2 4.58
1144.3
1069.1
1207.83
isolates of HIV. We tested the anti-HIV potential of 2l at its HNC on
various isolates of HIV-1 like HIVe1IIIB (X4 tropic, subtype-B), HIV-
89.6 (R5X4 tropic, subtype-B), HIV-1ADA (R5 tropic, subtype-B) and
subtype C isolate HIV-1IndieC in both T-cell line, CEM-GFP and
hPBMCs. Our results indicate that 2l significantly inhibits replica-
tion of all HIV-1 isolates tested in both cell lines as well as in pri-
mary cells as compared to their respective untreated control
(Fig. 2aeb).
Further, we tested the anti-HIV activity of 2l at its HNC in CEM-
GFP and hPBMCs infected with a different multiplicity of infections
(MOI) (0.01, 0.1 and 1 MOI). The results suggest that 2l efficiently
inhibits the replication of HIV-1 at low (0.01 MOI) as well as high
viral load (1 MOI) as compared to untreated controls (Fig. 2ced).
To analyze if 2l has any effect on viral target enzymes, we tested
its effect on Reverse Transcriptase (RT), Integrase (IN) and Protease
(PR) in a cell-free in vitro assay system at HNC. Our results indicate
that 2l does not significantly modulate these viral enzymes as
compared to their known inhibitors (Fig. 3aec). These results
strengthen the probability of inhibition of HSP90 by 2l to show its
anti-HIV activity. Molecular modelling and docking studies
including drug-like property estimation further support the po-
tential of 2l as promising anti-HIV lead candidate, which demon-
strate binding into the ATP binding pocket of HSP90.
1
1
NL4-3. Determination of TI indicates that 2l has approximately 3.5-
folds better therapeutic index than AUY922 and approximately 1.5-
folds better than AZT.
Further, we then focused on 2l as it has the best therapeutic
index among the tested molecules and analyzed its potential to
inhibit HIV-1 replication in different cell types as well as its po-
tential to inhibit HIV-1 replication in various viral isolates and viral
load. In addition, we studied the effect of 2l on viral enzymes and its
potential to inhibit HIV-1 LTR driven gene expression in HSP90
dependent manner.
The replicative behavior of HIV-1 greatly varies in the T-cells as
well as monocytes and macrophages. T-cells mainly act as the
producers of progeny virions, whereas monocytes and macro-
phages behave as the reservoirs and govern the cell-mediated
transmission of HIV-1 [34e36]. Because of this differential
behavior, many drugs fail to counter virus in certain cell types,
especially in monocytes and macrophages. Hence, to elucidate the
behavior of 2l in different cell types, we determined its TI in
another human T-cell line (Jurkat J6) and a monocytic cell line
(U937). We finally validated the cell line based results in primary
cells (human PBMCs) isolated from healthy seronegative donors.
Our results indicate that 2l shows a cell type independent response
and it significantly inhibits HIV-1NL4-3 replication in all the cell
types tested (TI ¼ >1000, Table 3).
2.3. Modelling studies
The computational design process was initiated utilizing the
binding profile of AUY922 (reported to control HIV-1 reactivation)
with HSP90 [29]. The side chains of AUY922 lacks flexibility and we
Reverse transcription is an extremely fast process with lack of
30-50 exonuclease proofreading activity which allows the virus to
develop progeny virions with extensive mutations [37,38]. These
extensive hypermutations, homologous recombination in hosts and
superinfection results in the development of various genetically
distinct isolates of HIV-1 in a single host. In certain cases, in-
dividuals are reported to have developed resistant mutations to the
current medications even before exposure to cART [39,40]. Hence,
it is very important to study the potential of antivirals on different
Table 2
Therapeutic Index (TI) of selected molecules with reference to AUY922 in cell-based
assays.
Compd.
CC50
(m
M)
IC50 (nM)
Therapeutic Index (TI)
AZT
AUY922
2a
2b
2e
2j
2l
2m
13.88 0.29
31.11 2.44
22.97 2.025
44.01 0.84
11.85 1.39
42.89 0.87
65.56 2.48
15.67 0.17
12.74 0.54
66.25 1.38
287.92 3.98
86.83 2.62
87.05 0.38
252.92 5.13
39.48 2.36
284.18 9.61
1089.48
469.58
79.78
506.85
136.13
169.58
1660.58
55.14
Fig. 2. HIV-1 replication inhibition potential of 2l in various virus isolates and viral
load in CEM-GFP and hPBMCs cell line. CEM-GFP (a) and hPBMCs (b) were infected
with 0.5 MOI of different isolates of HIV-1 in the absence or presence of 2l (at HNC).
72-hours post infection supernatant was collected and virus concentration was
determined by p24 antigen capture ELISA (n ¼ 3). CEM-GFP (c) and hPBMCs (d) were
infected with different MOI of HIV-1NL4-3(0.01, 0.1 and 1). The Supernatant was
collected at different time points post-infection and virus production was determined
by p24 antigen capture ELISA (n ¼ 3).

4
J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
Fig. 4. Binding mode of 2l with the active site of HSP90 showing hydrophilic in-
teractions (hydrogen bonding as yellow dash line) of isoxazole ring and C]O of the
amide with Ser52, and Thr184 through water bridge. The isopropyl group favorably
interacts with Val150, Phe138 and Leu107 (green mesh surface) in the hydrophobic
cavity of HSP90 active site. In addition, Lys58 and Gly97 interact through water and
form a cavity for proper binding of 2l within the active site of HSP90. (For interpre-
tation of the references to colour in this figure legend, the reader is referred to the Web
version of this article.)
Fig. 3. Test of 2l on RT, IN and PR. In a cell-free assay system, 2l was tested for its
effect on viral enzymes: RT (a), IN (b) and PR (c). Nevirapine (NVP), Raltegravir (RLT)
and Indinavir (IND) was used as positive control inhibitors respectively in these assays
(n ¼ 3).
nucleotide entry, which results in disruption of chaperone complex
and ultimately ends in proteasomal degradation of HSP90 as well as
the client proteins [43e45]. Also, as mentioned earlier, HSP90 plays
a crucial role in HIV-1 LTR mediated transcription and gene
expression [23,24,46e48].
intended to introduce flexibility with isoxazole moiety and to
enhance drug-like properties to avoid possible toxicity. Therefore,
one of the aromatic moieties on AUY922 was shifted on to the
acetamido functionality (R2 on NH) on I. In addition to the struc-
tural rigidity, the hydroxy group (green) in AUY922 may be further
reducing its flexibility due to H-bonding with oxygen of isoxazole
moiety which is avoided in I (R1 ¼ aryl, het-aryl). Accordingly,
fifteen new 2-isoxazol-3-yl-acetamide analogues (2a-2o) were
selected for synthesis. The modeling studies of 2a-2o and AUY922
(reference molecule) were conducted on HSP90 [41]. The docking
study followed by binding mode analysis suggests the probability
of newly designed scaffold (I) to act as an HSP90 inhibitor. The N-
terminal domain of HSP90 is the ATP binding domain, which uti-
lizes the energy of ATP-binding or its hydrolysis to drive various
functions within the cell. Therefore, the structure of HSP90 boun-
ded to an inhibitor at N-terminal domain (PDB: 4LWH) [42] was
obtained from protein data bank and utilized for docking and
binding mode analysis after appropriate structural correction in
term of bond order, water treatment, minimization, etc. In-silico
ADMET profiles of 2a-o and AUY922 were calculated. The calcu-
lated LogP (octanol/water) value for AUY922 is 2.46, however the
logP values of 2a-o were in the range of 3.5 which indicates their
better lipophilic profile. The apparent MDCK permeability of
AUY922 showed very poorly (23 nm/s), however, 2l showed great
MDCK permeability of 821 nm/s, which confirms its superior
ADMET profile.
2.4. Mechanistic study of inhibition of HIV-1 replication
Hence, to further elucidate the mechanistic details of 2l medi-
ated inhibition of HIV-1 replication, we explored its effect on LTR
driven gene expression. For this, we first studied the role of HSP90
on LTR driven gene expression by silencing and overexpression
studies (Fig. 5aeb). These studies indicate that LTR mediated
transcription is significantly compromised upon silencing of HSP90
whereas the increased LTR activity was observed upon over-
expression of the same. Further, the immunoblotting analysis
suggests that the treatment of 2l results in significantly reduced
HSP90 protein levels (Fig. 5ced). Finally, we studied the LTR
mediated gene expression upon 2l treatment. These results suggest
that upon 2l treatment the Tat-mediated LTR driven gene expres-
sion is significantly abolished as compared to the cells transfected
with LTR-Luc and Tat alone. These results collectively confirm that
2l inhibits HSP90 which has an inhibitory effect on HIV-1 Tat-
mediated transactivation of LTR.
3. Conclusions
Currently, more than 28 US FDA approved drugs and their for-
mulations, as part of combination therapy have been helping us to
manage AIDS-related complication to a significant extent. But the
fact remains that we are still far from eradicating the disease from
the human population. One of the major hurdles is the emergence
of drug-resistant viral strains and the inability to detect and destroy
latent viral reservoirs [49]. During its replication within the host,
HIV extensively exploits cellular machinery at various stages of its
life cycle and thus relies on a large number of HDFs for successful
propagation. Modulation of these HDFs may help us to inhibit virus
replication without affecting the host [50,51]. HSP90 plays a critical
role in HIV-1 LTR driven transcription and inhibition of HSP90 by
small molecule inhibitors were reported to inhibit HIV-1 tran-
The X-ray crystal structure and docking pose reveal the binding
mode of AUY922 with the active site of HSP90 and the active site
water mediates bridge interaction between AUY922 with Ser52 and
Asp102, further Asp93 and Gly97. It is observed that 2l occupies the
same binding site using hydrophilic interaction with Ser52 and
Thr184 including hydrogen bonding (yellow dash line, Fig. 4)
through water bridge. Lys58 interacts with Gly97 through water
forming a cavity for proper binding of 2l within the active site of
HSP90. In addition, the major hydrophobic interactions of the iso-
propyl group on 2l with Val150, Phe138 and Leu107 (green mesh
surface) further favors the binding. Considering the superior
ADMET profile over AUY922 and favorable binding mode, 2l can be
a potential lead candidate for HSP90. Mechanistically, binding of
HSP90 inhibitors in the ATP binding pocket results in blockage of
scription at HNC [46]. In case of HIV, HSP90 also regulates NF-
kB
pathway by a positive feedback mechanism which is required for

J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
5
Taken together, we report the synthesis of fifteen new 2-
isoxazol-3-yl-acetamide analogues (2a-2o). Further, the molecu-
lar modeling and biological studies confirm the potential of 2l as a
potential drug candidate for anti-HIV therapeutics acting through
host protein (HSP90) modulation.
4. Experimental
4.1. General
All reactions were carried out in oven-dried glassware under
nitrogen atmosphere. The chemicals and solvents were purchased
from Spectrochem, Across or Sigma-Aldrich. Reactions were
monitored by thin layer chromatography (TLC) (Kieselgel 60 F 254,
Merck) and visualized under UV light (254 nm). Crude was purified
on silica gel (100e200 mesh). 1H and 13C NMR were done on
JEOL400 MHz spectrophotometers in CDCl3 or DMSO‑d6. Chemical
shifts are reported as ppm (d) relative to TMS (d 0.0) as internal
standard. In 1H NMR data multiplicity is defined as: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet).
Mass spectra were recorded on 6430 Triple quardrupole mass
spectrometer. IR spectra were recorded on a Perkin-Elmer 1600
series FT-IR spectrophotometer. UV spectra were recorded on a
Perkin Elmer, USA, Lambda 25 UVeVisible spectrophotometer.
Melting points were determined on a Veego melting point appa-
ratus and are uncorrected.
Fig. 5. Inhibition of HIV-1 LTR driven gene expression in HSP90 dependent manner
by 2l. (a) HEK-293T cells were transfected with HSP90 siRNA (20 nM) using RNAi Max.
24-hours post-transfection, cells were co-transfected with pLTR-Luc and pNL4-3 using
PEI. (b) HEK-293T cells were co-transfected with pLTR-Luc, pNL4-3 and HSP90 clone
using PEI. 48-hours post-transfection, cells were harvested in both cases and Lumi-
nescence and Fluorescence were measured using Molecular Devices M5 microplate
reader. (c) CEM-GFP cells were infected with 0.5 MOI of HIV-1NL4-3 in presence or
absence of 2l. DMSO treated cells were considered as vehicle controls. 72-hours post
infection, HSP90 protein expression was measured by immunoblotting. (d) Densito-
metric analysis of immunoblots. (e) HEK-293T cells were co-transfected with p-LTR-
Luc and Tat in the presence or absence of 2l using PEI. 48-hours post-transfection, cells
were harvested and Luminescence and Fluorescence were measured. All the experi-
ments were performed in three biological replicates (p < 0.05 ¼ *, p < 0.01 ¼ **,
p < 0.001 ¼ ***) (n ¼ 3).
4.2. Procedure for the synthesis of substituted 2-isoxazol-3-yl-
acetamide (2a-o)
To the solution of appropriate 4-(hydroxyamino)-N-substituted-
2-oxo-6-phenyl-2H-pyran-3-carboxamide (1a-o, 0.31 mmol) in
dioxane, NaHCO3 (6.2 mmol) was added and stirred for 24 h at
80 ꢀC. The reaction was monitored by TLC, the solvent was removed
under vacuum and ice-cold water was added. The resulted pre-
cipitate was filtered, dried and was purified on silica gel (100e200
mesh) column with ethyl acetate and n-hexane (10% EtOAc:n-
hexane) as eluents to obtain 2a-o.
latency reversal [23,24]. Hence, targeting HSP90 may be advanta-
geous not only to block virus transcription but also in shock and kill
approach for latency reversal. We synthesized fifteen new 2-
isoxazol-3-yl-acetamide analogues (2a-2o) and studied their anti-
HIV potential in HeLa based reporter cell line TZM-bl at HNC
which indicates that 2a-b, 2e, 2j, and 2l-m inhibit HIV-1 replication
significantly (>85%). We further characterized the anti-HIV poten-
tial of these selected analogues by determining their therapeutic
index in CEM-GFP cell line. Our results indicate that 2l has
approximately 3.5-fold better therapeutic index than AUY922.
Molecular docking indicates that 2l binds an N-terminal ATP
binding domain of HSP90 utilizing hydrophobic interaction with
Leu107, Phe138 and Val150 for favorable Vander Waal's contact
along with hydrophilic interaction with Ser52 and Thr184. The
combination of hydrophilic as well as hydrophobic interactions and
superior ADMET profile makes 2l as a significant drug candidate for
HIV. The replicative behavior of HIV-1 is different in various cell
types and due to which, some drugs might fail to block virus
replication in certain cell types. Hence, we have tested 2l in various
cell types including T-cell line and monocytic cell line and further
validated results in primary cells. Our results suggest that 2l in-
hibits HIV-1 replication significantly (TI > 1000) in all the cell types
tested. Further, we tested the potential of 2l on different isolates of
HIV-1 and found that it significantly inhibits all the isolates tested
both in CEM-GFP cells as well as in hPBMCs. Our results also indi-
cate that 2l potentially inhibits HIV-1 replication at higher viral
load (up to 1 MOI) as well both in cell line and primary cells.
Interestingly, 2l does not significantly modulate viral enzymes like
RT, IN and PR as compared to their known inhibitors in a cell-free
in vitro assay system at its HNC. Further, we found that 2l has a
significant inhibitory effect on Tat-mediated transactivation and
LTR driven gene expression.
4.2.1. N-phenyl-2-(5-phenylisoxazol-3-yl)acetamide (2a)
White powder; Yield: 62%; mp: 151e152 ꢀC; UV (MeOH)
l
¼ 254 nm; IR: (KBr) (
n
, cmꢁ1): 1662 (C]O), 3059 (OH), 3279
max
(NH); MS-ESI (m/z): 279.4[Mþþ1]; 1H NMR (400 MHz, DMSO‑d6):
d
3.83 (s, 2H, CH2), 7.01 (s,1H, Isoxazole), 7.06e7.07 (t, J ¼ 6.4 Hz,1H,
ArH), 7.29e7.32 (t, J ¼ 6.4 Hz, 2H, ArH), 7.50e7.52 (m, 3H, ArH),
7.59e7.61 (m, 2H, ArH), 7.85e7.87 (m, 2H, ArH), 10.38 (s, 1H, NH
amide), 13C NMR (400 MHz, DMSO‑d6):
d 33.9, 101.1, 119.2, 123.6,
125.5, 126.6, 128.2, 128.8, 129.1, 130.4, 138.9, 159.6, 166.4, 168.8,
HRMS: m/z ([MþH]þ) Calcd for C17H14ClN3O: 279.1128; Found:
279.1129.
4.2.2. 2-(5-phenylisoxazol-3-yl)-N-p-tolylacetamide (2b)
White crystalline powder; Yield: 46%; mp: 177e178 ꢀC; UV
(MeOH)
l
¼ 255 nm; IR: (KBr) (
n
, cmꢁ1): 1658 (C]O), 3043
max
(OH), 3290 (NH); MS-ESI (m/z): 293.1 [Mþþ1]; 1H NMR (400 MHz,
DMSO‑d6)
d 2.28 (s, 3H, Me), 3.83 (s, 2H, CH2), 6.86e6.87 (bs, 2H,
ArH), 7.07 (s,1H, Isoxazole), 7.18 (d, J ¼ 8 Hz, 2H, ArH), 7.38e7.46 (m,
3H, ArH), 7.81 (m, 2H, ArH), 10.26 (s, 1H, NH amide), 13C NMR
(400 MHz, DMSO‑d6):d 21.7, 34.5, 55.9, 101.1, 115.2, 116.9, 120.2,
124.7, 127.8, 129.2, 138.5, 139.4, 160.1, 161.3166.9, 169.4; HRMS: m/z
([MþH]þ) Calcd for C18H16N2O2: 293.1285; Found: 293.1289.
4.2.3. 2-(5-phenylisoxazol-3-yl)-N-o-tolylacetamide (2c)
Off white powder; Yield: 74%; mp: 175e176 ꢀC; UV (MeOH)
¼ 257 nm; IR: (KBr) (
l
n
, cmꢁ1): 1658 (C]O), 3047 (OH), 3271
max

6
J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
(NH); MS-ESI (m/z): 293.4 [Mþþ1], 294.4 [Mþþ2]; 1H NMR
101.1, 113.4, 120.8, 125.5, 126.6, 126.9, 129.3, 130.4, 132.0, 155.4,
159.7, 165.8, 168.8; HRMS: m/z ([MþH]þ) Calcd for C18H16N2O3:
309.1234; Found: 309.1235.
(400 MHz, DMSO‑d6):
d 2.21 (s, 3H, Me), 3.86 (s, 2H, CH2), 7.01 (s,
1H, Isoxazole), 7.06e7.21 (m, 3H, ArH), 7.39 (m, 1H, ArH), 7.48e7.55
(m, 3H, ArH), 7.85e7.87 (d, J ¼ 8 Hz, 2H, ArH), 9.71 (s, 1H, NH
amide), 13C NMR (400 MHz, DMSO‑d6):
d 18.4, 33.9, 101.6, 125.6,
4.2.9. Methyl 4-(2-(5-phenylisoxazol-3-yl) acetamido) benzoate
126.1, 126.5, 127.4, 130.0, 130.9, 132.4, 136.5, 160.4, 167.0, 169.3;
HRMS: m/z ([MþH]þ) Calcd for C18H16N2O2: 293.1285; Found:
293.1289.
(2i)
Light yellow powder; Yield: 34%; mp: 168e169 ꢀC; UV (MeOH)
¼ 254 nm; IR: (KBr) (
l
n
, cmꢁ1): 1678 (C]O), 3125 (OH), 3325
max
(NH), 1701 (C]O of COOMe); MS-ESI (m/z): 337.1 [Mþþ1]; 1H NMR
4.2.4. 2-(5-phenylisoxazol-3-yl)-N-m-tolylacetamide (2d)
(400 MHz, DMSO‑d6) d 3.82 (s, 3H, OMe), 3.89 (m, 2H, CH2), 7.03 (s,
White powder; Yield: 71%; mp: 138e139 ꢀC; UV (MeOH)
l
1H, Isoxazole), 7.51e7.55 (m, 3H, ArH), 7.74e7.76 (d, J ¼ 8 Hz, 2H,
¼ 255 nm; IR: (KBr) (
n
, cmꢁ1): 1658 (C]O), 3062 (OH), 3275
max
ArH), 7.86e7.88 (d, J ¼ 8 Hz, 2H, ArH), 7.92e7.94 (d, J ¼ 8 Hz, 2H,
(NH); MS-ESI (m/z): 293.4 [Mþþ1], 294.4 [Mþþ2]; 1H NMR
ArH), 10.68 (s, 1H, NH amide), 13C NMR (400 MHz, DMSO‑d6):
d 34.0,
(400 MHz, DMSO‑d6) d 2.26 (s, 3H, Me), 3.81 (s, 2H, CH2), 6.86e6.88
51.9, 101.1, 118.6, 124.2, 125.5, 126.8, 129.2, 130.3, 143.2, 159.3, 165.8,
167.0, 168.8; HRMS: m/z ([MþH]þ) Calcd for C19H16N2O4: 337.1183;
Found: 337.1184.
(m,1H, ArH), 7.0 (s,1H, Isoxazole), 7.16e7.20 (m,1H, ArH), 7.37e7.52
(m, 5H, ArH), 7.85e7.87 (d, J ¼ 6.4 Hz, 2H, ArH), 10.27 (s, 1H, NH
amide), 13C NMR (400 MHz, DMSO‑d6):
d 21.7, 34.5, 101.6, 117.0,
120.3, 124.8, 126.1, 127.3, 129.2, 129.8, 130.9, 138.5, 139.4, 160.2,
166.8,169.3; HRMS: m/z ([MþH]þ) Calcd for C18H16N2O2: 293.1285;
Found: 293.1291.
4.2.10. N-(2-hydroxyphenyl)-2-(5-phenylisoxazol-3-yl) acetamide
(2j)
Light yellow powder; Yield: 64%; mp: 149e150 ꢀC; UV (MeOH)
¼ 253 nm; IR: (KBr) (
l
n
, cmꢁ1): 1662 (C]O), 3136 (OH), 3383
max
4.2.5. N-(4-fluorophenyl)-2-(5-phenylisoxazol-3-yl) acetamide
(2e)
(NH); MS-ESI (m/z): 295.1[Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
White crystalline powder; Yield: 63%; mp: 173e174 ꢀC; UV
d
3.94 (s, 2H, CH2), 6.74e6.78 (m, 1H, Isoxazole), 6.86e6.96 (m, 3H,
n
, cmꢁ1): 1658 (C]O), 3086
ArH), 7.48e7.55 (m, 3H, ArH), 7.80e7.88 (m, 3H, ArH), 9.53 (s, 1H,
(MeOH)
l
¼ 256 nm; IR: (KBr) (
max
NH amide), 9.81 (s, 1H, OH), 13C NMR (400 MHz, DMSO‑d6):
d
33.6,
(OH), 3259 (NH); MS-ESI (m/z): 297.4[Mþþ1]; 1H NMR (400 MHz,
101.1,115.3,118.9,122.3,124.7,125.5,126.0,126.9,129.2,130.3,147.8,
159.8, 166.6, 168.7; HRMS: m/z ([MþH]þ) Calcd for C17H14N2O3:
295.1077; Found: 295.1079.
DMSO‑d6):
d 3.83 (s, 2H, CH2), 7.03 (s, 1H Isoxazole), 7.12e7.19 (m,
2H, ArH), 7.52 (m, 3H, ArH), 7.62 (bs, 2H, ArH), 7.84e7.92 (m, 2H,
ArH), 10.41 (s, 1H, NH amide), 13C NMR (400 MHz, DMSO‑d6):
d
34.4,
101.6, 115.8, 116.0, 121.5, 126.1, 127.4, 129.8, 130.9, 135.7, 160.1, 166.8,
169.4; HRMS: m/z ([MþH]þ) Calcd for C17H13FN2O2: 297.1034;
Found: 297.1039.
4.2.11. 2-(5-phenylisoxazol-3-yl)-N-propylacetamide (2k)
White powder; Yield: 63%; mp: 115e116 ꢀC; UV (MeOH)
l
¼ 256 nm; IR: (KBr) (
n
, cmꢁ1): 1643 (C]O), 3121 (OH), 3294
max
(NH); MS-ESI (m/z): 245.2[Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
4.2.6. N-(4-chlorophenyl)-2-(5-phenylisoxazol-3-yl) acetamide (2f)
Yellow powder; Yield: 68%; mp: 166e167 ꢀC; UV (MeOH)
l
d
0.83e0.87 (m, 3H, Me), 1.40e1.46 (m, 2H, CH2), 3.02e3.07 (m, 2H,
¼ 254 nm; IR: (KBr) (
n
, cmꢁ1): 1685 (C]O), 3067 (OH), 3263,
max
CH2), 3.56 (s, 2H, CH2), 6.92 (s, 1H, Isoxazole), 7.49e7.55 (m, 3H,
3298 (NH); MS-ESI (m/z): 313.1 [Mþþ1], 315.1 [Mþþ2]; 1H NMR
ArH), 7.84e7.86 (m, 2H, ArH), 8.18 (s, 1H, NH amide), 13C NMR
(400 MHz, DMSO‑d6)
d 3.85 (s, 2H, CH2), 7.02 (s, 1H, Isoxazole),
(400 MHz, DMSO‑d6):d 11.4, 22.3, 32.9, 40.6, 101.0, 125.5, 126.5,
7.37e7.40 (m, 2H, ArH), 7.49e7.55 (m, 3H, ArH), 7.64e7.66 (m, 2H,
126.9, 129.3, 130.3, 160.0, 167.3, 168.6; HRMS: m/z ([MþH]þ) Calcd
ArH), 7.86e7.88 (d, J ¼ 8 Hz, 2H, ArH), 10.54 (s, 1H, NH amide), 13
C
for C14H16N2O2: 245.1285; Found: 245.1285.
NMR (400 MHz, DMSO‑d6):d 33.9, 101.1, 120.8, 125.6, 126.6, 126.8,
127.1, 128.7, 129.1, 129.3, 137.9, 159.5, 166.6, 168.8; HRMS: m/z
([MþH]þ) Calcd for C17H13ClN2O2: 313.0738; Found: 313.0734.
4.2.12. N-isopropyl-2-(5-phenylisoxazol-3-yl)acetamide (2l)
White powder; Yield: 59%; mp: 153e154 ꢀC; UV (MeOH)
l
¼ 255 nm; IR: (KBr) (
n
, cmꢁ1): 1647 (C]O), 3063 (OH), 3298
max
4.2.7. N-(4-hydroxyphenyl)-2-(5-phenylisoxazol-3-yl) acetamide
(NH); MS-ESI (m/z): 245.2 [Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
(2g)
d
1.07e1.09 (m, 6H, Me), 3.53 (s, 2H, CH2), 3.81e3.87 (m, 1H, CH),
Off white powder; Yield: 57%; mp: 214e215 ꢀC; UV (MeOH)
l
6.92 (s, 1H, Isoxazole), 7.49e7.55 (m, 3H, ArH), 7.84e7.86 (m, 2H,
¼ 256 nm; IR: (KBr) (
n
, cmꢁ1): 1662 (C]O), 3121 (OH), 3267
max
ArH), 8.11 (s, 1H, NH amide), 13C NMR (400 MHz, DMSO‑d6):
d
22.3,
(NH); MS-ESI (m/z): 295.1[Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
32.9, 33.8, 40.7, 101.1, 125.5, 126.9, 129.3, 130.3, 160.0, 166.4, 168.6;
HRMS: m/z ([MþH]þ) Calcd for C14H16N2O2: 245.1285; Found:
245.1278.
d
3.77 (s, 2H, CH2), 6.69e6.71 (d, J ¼ 8.4 Hz, 2H, ArH), 7.00 (s, 1H,
Isoxazole), 7.24e7.39 (d, J ¼ 8.4 Hz, 2H, ArH), 7.49e7.55 (m, 3H,
ArH), 7.86e7.89 (d, J ¼ 8.4 Hz, 2H, ArH), 9.20 (s, 1H, NH amide),
10.06 (s, 1H, OH), 13C NMR (400 MHz, DMSO‑d6):
d 33.8, 101.1, 115.1,
121.0, 125.5, 126.6, 126.9, 129.3, 130.4, 130.6, 153.5, 159.8, 165.6,
168.8; HRMS: m/z ([MþH]þ) Calcd for C17H14N2O3: 295.1077;
Found: 295.1079.
4.2.13. 2-(5-(4-methoxyphenyl)isoxazol-3-yl)-N-m-tolyl acetamide
(2m)
Pure white powder; Yield: 29%; mp: 167e168 ꢀC; UV (MeOH)
l
¼ 258 nm; IR: (KBr) (
n
, cmꢁ1): 1659 (C]O), 3059, 3132 (OH),
max
4.2.8. N-(4-methoxyphenyl)-2-(5-phenylisoxazol-3-yl) acetamide
3252 (NH); MS-ESI (m/z): 323.2 [Mþþ1]; 1H NMR (400 MHz,
(2h)
DMSO‑d6) d 2.27 (s, 3H, Me), 3.78 (s, 3H, OMe), 3.82 (s, 2H, CH2),
Pure white powder; Yield: 61%; mp: 148e149 ꢀC; UV (MeOH)
l
6.86 (s, 1H Isoxazole), 6.87e6.89 (m, 1H, ArH), 7.06e7.08 (m, 1H,
ArH), 7.17e7.19 (m, 2H, ArH), 7.37e7.39 (m, 1H, ArH), 7.45 (s, 1H,
¼ 249 nm; IR: (KBr) (
n
, cmꢁ1): 1651 (C]O), 3090 (OH), 3290
max
(NH); MS-ESI (m/z): 309.1 [Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
ArH), 7.80e7.82 (d, J ¼ 8 Hz, 2H, ArH), 10.23 (s, 1H, NH amide), 13
C
d
3.71 (s, 3H, OMe), 3.79 (m, 2H, CH2), 6.88e6.90 (m, 2H, ArH), 6.91
NMR (400 MHz, DMSO‑d6):d 20.4, 33.8, 101.1, 119.2, 125.5, 126.8,
(s, 1H, Isoxazole), 7.50e7.55 (m, 5H, ArH), 7.86e7.88 (m, 2H, ArH),
129.1, 129.2, 130.3, 132.4, 136.4, 159.6, 166.0, 168.7; HRMS: m/z
([MþH]þ) Calcd for C19H18N2O3: 323.1390; Found: 323.1382.
10.18 (s, 1H, NH amide), 13C NMR (400 MHz, DMSO‑d6):
d
33.8, 55.2,

J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
7
4.2.14. 2-(5-(furan-2-yl)isoxazol-3-yl)-N-(4-hydroxyphenyl)
4.3.4. Virus isolates, generation of viral stocks and determination of
virus infectivity
acetamide (2n)
Yellow powder; Yield: 41%; mp: 170e171 ꢀC; UV (MeOH)
l
Molecular clones for HIV-1NL4-3, HIVe1IIIB, HIV-1ADA, HIV-189.6
virus isolates were obtained from NIH, AIDS Reagent Program, NIH,
USA [52]. HIV-1IndieC, which is a full length infectious molecular
clone of subtype-C HIV-1 prevalent in India was a kind gift of Dr. M.
Tatsumi, Japan [53]. Virus stock was prepared by transfecting these
molecular clones in HEK-293T cells using CalPhos Mammalian
Transfection Kit (Clontech, Takara, USA). Briefly, 2.5 ꢂ 106 cells were
seeded in 90 mm petri-dish and incubated at 37 ꢀC for adherence.
12-hours post seeding; cells were transfected with various molec-
ular clones independently according to manufacturer's protocol.
36-hours post transfection, supernatant was collected and virus
was pelleted at 28,000 rpm at 4 ꢀC for two and a half hours using
Beckman ultracentrifuge in SW28 rotor. The pellet was suspended
in serum free RPMI and used for infection. For infectivity of the
¼ 252 nm; IR: (KBr) (
n
, cmꢁ1): 1647 (C]O), 3067, 3136 (OH),
max
3267 (NH); MS-ESI (m/z): 285.1 [Mþþ1]; 1H NMR (400 MHz,
DMSO‑d6)
d 3.76 (s, 2H, CH2), 6.68 (s, 1H Isoxazole), 6.70e6.75 (m,
3H, ArH), 7.13 (s, 1H, ArH), 7.35e7.37 (m, 2H, ArH), 7.93 (s, 1H, ArH),
9.20 (s, 1H, OH), 10.04 (s, 1H, NH amide), 13C NMR (400 MHz,
DMSO‑d6): d 33.9, 100.8, 111.6, 112.8, 115.6, 121.5, 131.0, 145.9, 154.0,
160.0, 160.9, 165.9.
4.2.15. 2-(5-(thiophen-2-yl)isoxazol-3-yl)-N-p-tolyl acetamide
(2o)
White powder; Yield: 46%; mp: 160e161 ꢀC; UV (MeOH)
l
¼ 250 nm; IR: (KBr) (
n
, cmꢁ1): 1655 (C]O), 3094 (OH), 3290
max
(NH); MS-ESI (m/z): 299.4 [Mþþ1]; 1H NMR (400 MHz, DMSO‑d6)
prepared virus stocks,
b-galactosidase staining assay was per-
d
2.20 (s, 3H, CH3), 3.75 (s, 2H, CH2), 6.81 (s, 1H, Isoxazole),
formed as described earlier [47]. Briefly, TZM-bl cells were infected
with different concentrations of the virus prepared and incubated
at 37 ꢀC in humidified CO2 incubator. 36-hours post incubation,
cells were washed with 1X PBS and fixed with 0.5% Glutaraldehyde.
Staining solution with X-gal was added post fixation and blue
stained cells were counted within 12 h of staining.
7.06e7.08 (d, J ¼ 8.4 Hz, 2H, ArH), 7.18e7.20 (m, 1H, ArH) 7.43e7.45
(d, J ¼ 8.8 Hz, 2H, ArH), 7.67e7.68 (m, 1H, ArH), 7.76e7.77 (m, 1H,
ArH),10.20 (s, 1H, NH amide), 13C NMR (400 MHz, DMSO‑d6):
d 20.9,
34.2, 100.9, 119.7, 128.3, 128.8, 129.1, 129.6, 129.8, 132.9, 136.9, 160.2,
164.5, 166.5.
4.3.5. HIV-1 infection and anti-HIV activity assay
4.3. Biological assay protocols
TZM-bl cells were seeded at the density of 0.15*10^6 cells/well in
24-well plate. 12-hours post seeding cells were infected with 0.5
MOI of HIV-1NL4.3 and treated with molecules at HNC. 48-hours
post infection, cell were lysed and mixed with Steady-Glo sub-
strate in equal 1:1 ratio. Luminescence was then measured using
Molecular Devices M5 microplate reader. CEM-GFP, Jurkat J6, U937
and hPBMCs were infected with different isolates of HIV-1 as
described previously [32]. Briefly, cells were incubated with 0.5
4.3.1. Cell culture
Jurkat J6 and U937 cell lines were obtained from NCCS Cell Re-
pository, NCCS, Pune, India. Both TZM-bl and CEM-GFP cell lines
were obtained from NIH AIDS Reagent Program, Division of AIDS,
NIH, USA. CEM-GFP, Jurkat J6 and U937 cell lines were grown in
RPMI-1640 medium (Invitrogen, USA) and TZM-bl cells were grown
in DMEM medium, both containing 10% fetal bovine serum (FBS) in
MOI of different isolates of HIV-1 in presence of polybrene (1 mg/
presence of 100 U/ml of penicillin (Invitrogen, USA) and 100
streptomycin (Invitrogen, USA) to avoid bacterial contamination.
500 g/ml G418 (Invitrogen, USA) was added to the media con-
mg/ml
mL) at 37 ꢀC in humidified CO2 incubator with intermittent mixing.
Four hours post incubation cells were washed with serum free
RPMI-1640 and plated in complete RPMI-1640. Cells were then
treated with molecules at different concentrations. Untreated cells
were taken as negative control and DMSO treated cells were taken
as vehicle control. 72-hours post infection, supernatant was
collected and virus production was determined using p24 antigen
capture ELISA kit (Advanced Bioscience Laboratories, USA) ac-
m
taining CEM-GFP cells as a selective antibiotic.
4.3.2. Isolation of human peripheral blood mononuclear cells
(hPBMCs)
Blood of normal seronegative donors was obtained from Indian
Serological Institute (ISI) blood bank, Navi Peth, Pune. hPBMCs
were isolated from the blood using Histopaque-1077 (Sigma, USA)
by gradient centrifugation as described previously [47]. Briefly,
blood was diluted using serum free RPMI-1640. Diluted blood was
then carefully layered on top of Histopaque-1077 without dis-
turbing the gradient. PBMCs were separated by centrifugation at
1800 rpm for 25 min. PBMCs were then collected and washed twice
with serum free RPMI and seeded at the density of 106 cells/mL of
media and activated with PHA and used for further experiments.
cording to manufacturer's protocol. Briefly, 100 mL of viral particle
containing supernatant was loaded in to the p24 primary antibody
coated wells in presence of lysis buffer and incubated at 37 ꢀC. 1-
hour post incubation, wells were washed using ELISA washer
(Biorad, USA) and HRP-conjugate solution was added to the well
and incubated at 37 ꢀC for 1-h. After incubation, wells were again
washed and substrate solution was added and incubated at room
temperature for 30 min to allow to blue colour to develop. The
reaction was stopped using stop solution and absorbance was read
at 450 nm using Molecular Devices M5 microplate reader. Per-
centage Inhibition of virus production was calculated using the
following formula.
4.3.3. MTT cell proliferation assay
Cytotoxicity of synthesized molecules was determined by MTT
cell proliferation assay kit, (Roche, Germany) as described earlier
[32]. Briefly, cells were seeded at the density of 25,000 cells/well in
ðp24 Conc:of untreated ꢁ p24 Conc:of treatedÞ
% Inhibition ¼
ðp24 Conc:of untreatedÞ
96 well plate containing 100 mL of complete media. Molecules at
ꢂ 100
different concentration was added to the media and incubated in
humidified CO2 incubator. Untreated cells were taken as negative
control and DMSO treated cells were taken as vehicle control. 48-
hours post incubation, 10
m
L of MTT (5 mg/mL) was added in each
4.3.6. RNA dependent DNA polymerase activity assay of HIV-1 RT
(RDDP assay)
Effect of 2l on RNA dependent DNA polymerase activity of HIV-1
RT enzyme was tested with HIV-1 reverse transcriptase
well in dark and incubated at 37 ꢀC for the formation of formazan
crystals. 4-hours post incubation, the crystals were dissolved using
100 mL isopropanol and the absorbance was read at 570 nm.

8
J. Trivedi et al. / European Journal of Medicinal Chemistry 183 (2019) 111699
colorimetric assay kit (Roche, Germany) according to manufac-
turer's protocol. Nevirapine treated wells were taken as positive
control whereas untreated wells were taken as negative control
and DMSO treated wells were taken as vehicle control.
Abbreviations used
cART
HSP
Combination antiretroviral therapy
Heat Shock Proteins
ELISA
Enzyme Linked Immunosorbent Assay
4.3.7. HIV-1 integrase activity assay
hPBMCs Human Peripheral Blood Mononuclear Cells
2l was tested for its effect, if any, on HIV-1 Integrase enzyme
using HIV-1 integrase assay kit (XpressBio, USA) according to
manufacturer's protocol. Raltegravir treated wells were taken as
positive control whereas untreated wells were taken as negative
control and DMSO treated wells were taken as vehicle control.
IND
HNC
LTR
Indinavir
Highest Non-cytotoxic Concentration
Long Terminal Repeats
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
MTT
NVP
Nevirapine
4.3.8. HIV-1 protease activity assay
NF-
RLT
MDCK
ADMET
k
B
Nuclear Factor kappa B
Raltegravir
Madin-Darby Canine Kidney
absorption, distribution, metabolism, excretion, and
toxicology
Effect of 2l on HIV-1 Protease enzyme was determined using
HIV-1 Fluorometric Protease Activity Assay Kit, (Biovision, USA)
according to manufacturer's protocol. Briefly, in a single step re-
action, HIV-1 protease enzyme was diluted in reaction buffer and
10 nmol/well was loaded in 96 well plate in presence or absence of
2l. Indinavir treated wells were taken as positive control whereas
untreated wells were taken as negative control and DMSO treated
wells were taken as vehicle control. 15-minutes post addition,
Fluorescence was measured using Molecular Devices M5 micro-
plate reader at Ex/Em ¼ 330/450 nm.
TLC
PEI
thin layer chromatography
Polyethylenimine.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111699.
4.4. Molecular modeling protocols
References
The structure of HSP90 inhibitor bound FJ5 ligand (PDB: 4LWH)
[42], N terminal domain was obtained from protein data bank. The
modeling studies and ADMET calculation were performed using
software from Schrodinger [41] and the receptor were prepared
using Protein Preparation Wizard for docking studies. The pdb file
(4LWH) were pre-processed and bond orders were assigned,
hydrogen were removed and added, zero order bonds to metals
were created, the selenomethionine were converted to methio-
nines and waters beyond 5 Å of ligand were removed. H-bonds
were assigned at neutral pH. Heavy atoms were converged to 0.30 Å
RMSD (Root Mean Square Distance). The protein was minimized
with OPLS2005 force field. The structures of compounds to be
docked were drawn on ChemDraw, converted to 3D structure using
ChemBio 3D. Structures were minimized using MacroModel and
the most stable bioactive conformation was selected using ConfGen
module, after which the ligands were used for docking studies.
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static and H bond cut-offs were 7.0, 12.0 and 4.0 Å respectively.
5000 iterations of the Polak-Ribier Conjugate Gradient (PRCG)
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This work was supported by Science and Engineering Research
Board (SERB), India (EMR/2017/003331) grant to DM and AS and
intramural funding from NCCS. JT is thankful to DST for INSPIRE
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