S. Chander et al. / Bioorganic Chemistry 67 (2016) 75–83
81
The progress of reaction was monitored by thin layer chromatogra-
phy (TLC) using ethyl acetate and hexane (in suitable proportion)
as mobile phase. Melting points were uncorrected and determined
in open capillary tubes on a Precision Buchi B530 (Flawil, Switzer-
land) melting point apparatus containing silicon oil. The IR spectra
of the synthesized compounds were recorded using FTIR spec-
trophotometer (Shimadzu IR Prestige 21, Shimadzu, Mumbai,
India). 1H NMR spectra were recorded on Bruker DPX-400 spec-
trometer (Bruker India Scientific Pvt. Ltd., Mumbai, India) using
TMS as an internal standard (chemical shifts in d). Elemental anal-
ysis was performed on Vario EL III M/s Elementar C, H, N, and S
analyzer (Elementar Analysensysteme GmbH, Hanau, Germany).
ESI-MS were recorded on MICROMASS Quattro-II LCMS system
(Waters Corporation, Milford, USA).
further stirred at room temperature for 6–8 h. After completion
of reaction as per TLC, reaction mass was taken in a separating fun-
nel, to this 25 ml more DCM was added, and organic layer was
washed with saturated sodium bicarbonate solution (50 ml).
Organic layer was then washed with distilled water (50 ml), DCM
layer was separated and dried over anhydrous sodium sulphate
and finally evaporated to afford the final compounds 6a-o [34].
3.3. In-vitro HIV-1 RT screening
In-vitro HIV-1 RT inhibitory activity of the synthesized com-
pounds was evaluated using Roche diagnostics kit in accordance
with the kit protocol [35]. This is based upon colorimetric assay
method in which marketed drug efavirenz was used as reference
compound. Procedure followed is briefly described here; the reac-
tion mixture was set with template primer complex, RT enzyme
and dNTPs in a lysis buffer with or without inhibitors. The reaction
mixture was incubated at 37 °C for 1 h and then transferred to
streptavidin-coated microtitre plate (MTP). The biotin-labeled
dNTPs that were incorporated in the template due to activity of
RT, bound to streptavidin. The unbound dNTPs were washed using
wash buffer and anti-DIG-POD was added to the MTP. The DIG-
labeled dNTPs incorporated in the template were bound to an
anti-DIG-POD antibody. The unbound anti-DIG-POD was washed
again with washing buffer and the peroxide substrate (ABST) was
added to the MTP. A colored reaction product was produced during
the cleavage of the substrate catalyzed by a peroxide enzyme. The
absorbance of the sample was determined as an optical density
(OD) at 405 nm using a micro titer plate ELISA reader. The final
value of OD taken is average of duplicate results and % inhibition
of HIV-1 RT was calculated using the below mentioned formula.
3.2.1. Synthesis of 6-methoxy-1,2,3,4-tetrahydroquinoline (2)
To the stirred solution of 6-methoxyquinoline
1 (5 g,
31.44 mmol) in ethanol, Ni-Al alloy (2.5 g) was added and stirring
was continued for 30 min. Further, reaction mass was cooled to 0–
4 °C and then aqueous sodium hydroxide solution (10% w/v, 50 ml)
was added slowly. After the complete addition, reaction mixture
was further stirred at room temperature for 6 h. After completion
of reaction as per TLC, reaction mass was passed through a tight
celite bed, further ethanol (2 ꢂ 75 ml) was passed through the
celite bed. Combined filtrate was evaporated on rotary evaporator
till its volume remained one-fourth to its original volume. Further,
reaction mass was acidified with 2 N HCl, until its pH became neu-
tral. Further, reaction mass was taken in separating funnel and
extracted twice with ethyl acetate (2 ꢂ 150 ml). Combined ethyl
acetate layer was first washed with water (300 ml) and then with
brine (300 ml). Ethyl acetate layer was dried over anhydrous
sodium sulphate and finally evaporated on rotary evaporator to
afford the compound 2 as crude oil. Crude 2 was purified by col-
umn chromatography using 5% ethyl acetate in hexane as mobile
phase and silica (mesh size 100–200) as stationary phase, which
afforded the pure compound 2, as light yellow oil with 87% yield
[33].
Initially, compounds were screened at 100
lM concentration,
finally IC50 was calculated using double dilution method.
ꢀ
ꢁ
OD at 405 nm with inhitor
OD at 405 nm without inhitor
% Inhibition ¼ 100 ꢀ
ꢂ 100
3.4. Cytotoxicity and anti-HIV activity
3.2.2. Synthesis of 3-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)
propanoic acid (4)
Synthesized compounds 6a-o were evaluated for cytotoxicity
upon the CD4+ line of T cells (C8166) by MTT colorimetric assay
[34]. anti-HIV-1 activity (HIV-1IIIB strain) of all compounds were
determined by the cytopathic effect (CPE) method, which mea-
sured the viability of HIV-1IIIB infected C8166 cells [36,37]. In this
study, marketed drug zidovudine was used as positive control,
details of experimental work and procedures followed for cytotox-
icity and anti-HIV-1 assay studies are given in the supplementary
part of this manuscript (Section 3.4).
3-Bromopropanoic acid 3 (2.67 g, 21.4 mmol) was added por-
tion wise to the stirring solution of 6-methoxy-1,2,3,4-tetrahydro
quinoline 2 (3.5 g, 21.4 mmol) in acetonitrile, containing triethyl
amine (5.40 g, 53.5 mmol) as base and catalytic amount of potas-
sium iodide. Reaction mixture was refluxed and progress of the
reaction was monitored using TLC. After completion of reaction,
as per TLC (after 5 h), acetonitrile was evaporated on rotary evap-
orator, 100 ml of water was added to reaction mixture and
extracted with hexane (150 ml). Further, aqueous layer was sepa-
rated and neutralized with 6 N HCl, and taken in a separating fun-
nel, and twice extracted with ethyl acetate (2 ꢂ 250 ml). Combined
ethyl acetate layer was first washed with water (250 ml) and then
subsequently with brine (250 ml). Ethyl acetate layer was dried
over anhydrous sodium sulphate and finally evaporated on rotary
evaporator to afford compound 4 as light yellow semisolid with
73% yield.
3.5. Docking studies
Docking studies of four significantly active compounds (6a, 6b,
6j and 6o) along with reference compound rilpivirine were per-
formed using Glide 5.9 [38] (Extra Precision) running on maestro
version 9.4, in order to investigate their in-silico inhibitory poten-
tial against three different HIV-1 RT strains. Enzyme used for the
docking study were wild HIV-1 RT (Pdb ID: 3MEE), and mutant
strains involved K103N (Pdb ID: 3TAM) and K103N/Y181C (Pdb
ID: 4I2Q). All three selected proteins were retrieved from RCSB Pro-
tein Data Bank in complex with their co-crystallized ligands. Pro-
tein preparation wizard of Schrödinger suite was used for the
preparation of selected proteins. Proteins were pre-processed sep-
arately by deleting the substrate co-factor as well as the crystallo-
graphically observed water molecules (water without H bonds),
followed by optimization of hydrogen bonds. After assigning
3.2.3. General procedure for the synthesis of 3-(6-methoxy-3,4-
dihydroquinolin-1(2H)-yl)-1-(4-phenylpiperazin-1-yl)propan-1-one
derivatives (6a-o)
To the stirred solution of 3-(6-methoxy-3,4-dihydroquinolin-1
(2H)-yl) propanoic acid 4 (0.235 g, 1 mmol) in dry DCM, HOBt
(0.16 g, 1.2 mmol) and EDCI. HCl (0.23 g, 1.2 mmol), triethyl amine
(0.253 g, 2.5 mmol) were added and stirring was continued for
30 min. at room temperature. To the reaction mixture, correspond-
ing piperazines (5a-o) were added and the reaction mixture was