S. Chander et al.
BioorganicChemistry79(2018)212–222
Scheme 1. Synthetic route for the titled compounds (3a–5j).
hydrogen significantly reduced its activity (27.26 µM) while substitu-
tion with 2-methoxy (5c) markedly increased the potency (0.27 µM),
moreover compound 5c was found to be the most potent among the all
tested compounds. In contemporary to this, methoxy at meta position
(5d) drastically reduced the activity. Among halogen substituted com-
pounds, chloro at ortho position (5e) favored the RT inhibition activity
(0.76 µM) while, chloro as well as bromo at meta position (5f and 5 g)
was found unfavorable. Compounds with stronger electron with-
drawing nitro group at meta position (5h) and compounds having di-
substitution with methoxy and chloro (5i and 5j) also showed weak
Table 1
Optimization of the reaction conditions to obtain hit compound 3a.
S. No Solvent Base
Reaction
condition
Inference
a1
a2
DMF
ACN
K2CO3, (2 Eq.) rt, 4 h
Multiple spots (not isolated)
30% yield (after column
purification)
88% yield, single spot
reaction
Et3N, (2 Eq.)
heating, 80 °C,
3 h
rt, 6 h
a3
EtOH
Et2NH,
(1.2 Eq.)
NMR, 13C NMR, FTIR, ESI-MS and elemental analysis. Both enantiomers
of the compound 5c were separated by using chiral preparative HPLC.
Overall, SAR studies revealed that halogens like bromo and chloro
at 5th position of oxindole ring is crucial for RT inhibition activity,
further, methoxy or chloro groups at ortho position of phenyl ring sig-
nificantly favored their RT inhibition activity (Fig. 2).
2.2. Biological activity
2.2.1. In-vitro HIV-1 RT inhibition assay
2.2.2. Evaluation of anti-HIV-1 activity and cytotoxicity
Initially, synthesized virtual hit (3a) was tested for wild HIV-1 RT
inhibitory activity using RT assay kit (Roche). In the study, marketed
drug efavirenz was used as a reference positive control while 5% DMSO
was taken as negative control [36,37]. Virtual hit compound 3a dis-
played moderate inhibitory potential against HIV-1 RT with IC50 of
15.54 µM. In the next phase, keeping 5-bromo intact at oxindole system,
compounds without substitution (3b) as well as different substitutions
at phenyl ring (3c–f) were tested. Interestingly, compound without
substitution at phenyl ring 3b showed better potency (5.22 µM) than
the virtual hit 3a. Further, electron donating methyl and methoxy
groups at meta (3c) and ortho (3d) position enhanced the potency
(IC50 = 1.38 and 0.82 µM, respectively). Furthermore, halogen chloro
group at ortho position (3e) also favored the inhibition potency
(2.03 µM), while bromo at meta position (3f) markedly decreased the
RT inhibition potency (32.37 µM). Further, to investigate the role of 5-
bromo at the oxindole ring on its RT inhibitory potential, compounds
4a–f were synthesized which possessed hydrogen instead of bromo and
subsequently tested against HIV-1 RT. Unfortunately, replacement of
bromo with hydrogen at oxindole was not found favorable for RT in-
hibition potency and except 4d, other compounds (4a, 4b, 4c, 4e and
4f) exhibited weak to least potency.
All the synthesized compounds 3a–5j were evaluated for anti-HIV-1
activity against wild strain HIV-1IIIB in T lymphocyte cells by syncytia
formation assay. The assay is based upon the cytopathic effect (CPE) of
HIV-1 and measured the viability of HIV-1 infected T lymphocyte cells
[38]. The cytotoxicity of the synthesized compounds on T cell lines was
also assessed by MTT colorimetric assay method [39].
Among the compounds having bromo substitution at oxindole ring
(3a–f), virtual hit 3a and compound 3f showed weak potency while
four compounds (3b, 3c, 3d and 3e) showed superior potency with
EC50 < 11 µM. It is worthy to note that all four compounds (3b, 3c, 3d
and 3e) also showed low micromolar potency against HIV-1 RT.
Moreover, compound 3d exhibited sub-micromolar potency against
HIV-1 (EC50 = 0.19 µM) as well as HIV-1 RT (IC50 = 0.82 µM) with
selectivity index > 2105. Among the compounds 4a–f, none of the
compounds showed superior activity against HIV-1 RT, in similar pat-
tern all the compounds showed weak or least activity against HIV-1
also. Compounds bearing 5-chloro at oxindole ring (5a–5j), displayed
weak to potent activity against HIV-1. Three compounds 5b, 5c and 5e
displayed low micromolar potency, particularly compound 5c having
methoxy substitution at ortho position exhibited nano molar potency
(0.090 µM) against HIV-1 with selectivity index > 4444. Further,
compound 5f having meta chloro substitution exhibited moderate anti-
HIV-1 activity (14.51 µM), while rest of the compounds (5a, 5d, 5g, 5h,
5i and 5j) showed weak to least activity. Overall, with few exceptions,
most of the compounds showed good to excellent co-relation between
RT inhibition and anti-HIV-1 activity (see Table 2).
Next, compounds having 5-chloro at the oxindole ring and different
substitution at the phenyl ring (5a–j) were synthesized and evaluated
for RT inhibition activity. Compound 5a is a bioisoster of virtual hit 3a,
but in term of RT inhibition potential, it showed better activity
(IC50 = 5.92 µM) than 3a. Further, replacement of para fluoro with
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