Design, synthesis and in-vitro evaluation of novel tetrahydroquinoline carbamates as HIV-1 RT inhibitor and their antifungal activity

Article abstract of DOI:10.1016/j.bioorg.2015.12.005

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are vital class of drugs in treating HIV-1 infection, but drug resistance and toxicity drive the need for effective new inhibitors with potent antiviral activity, less toxicity and improved physicoc

Full text of DOI:10.1016/j.bioorg.2015.12.005

Accepted Manuscript
Design, synthesis and in-vitro evaluation of novel tetrahydroquinoline carba-
mates as HIV-1 RT inhibitor and their antifungal activity
Subhash Chander, Penta Ashok, Yong-Tang Zheng, Ping Wang, Krishnamohan
S. Raja, Akash Taneja, Sankaranarayanan Murugesan
PII:
S0045-2068(15)30043-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.12.005
YBIOO 1866
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 November 2015
16 December 2015
16 December 2015
Please cite this article as: S. Chander, P. Ashok, Y-T. Zheng, P. Wang, K.S. Raja, A. Taneja, S. Murugesan, Design,
synthesis and in-vitro evaluation of novel tetrahydroquinoline carbamates as HIV-1 RT inhibitor and their antifungal
activity, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.12.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and in-vitro evaluation of novel tetrahydroquinoline carbamates as
HIV-1 RT inhibitor and their antifungal activity
Subhash Chandera, Penta Ashoka, Yong-Tang Zhengb, Ping Wangb, Krishnamohan S. Rajaa, Akash Tanejaa, and
Sankaranarayanan Murugesana,*
a,*Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology &
Science, Pilani-333031. Rajasthan. India. E. mail: murugesan@pilani.bits-pilani.ac.in
b Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Disease
Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan 650223. P.R. China. E. mail: zhengyt@mail.kiz.ac.cn
Abstract: Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are vital class of drugs in
treating HIV-1 infection, but drug resistance and toxicity drive the need for effective new inhibitors
with potent antiviral activity, less toxicity and improved physicochemical properties. In the present
study, twelve novel 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl)ethyl phenylcarbamate
derivatives were designed as inhibitor of HIV-1 RT using the ligand based drug design approach and
in-silico evaluated for drug-likeness properties. Designed compounds were synthesized, characterized
and in-vitro evaluated for RT inhibitory activity against wild HIV-1 RT. Among these, four
compounds (6b, 6i, 6j and 6l) exhibited significant inhibition of HIV-1 RT (IC50 ≤ 20 µM). Among
four compounds, most active compounds 6b and 6j inhibited the RT activity with IC50 8.12 and 5.42
µM respectively. Docking studies of compounds 6b and 6j were performed against wild HIV-1 RT in
order to predict their putative binding mode with selected target. Further, cytotoxicity and anti-HIV
activity of compounds 6b and 6j were evaluated on T lymphocytes (C8166 cells). All the synthesized
compounds were also evaluated for antifungal activity against Candida albicans and Aspergillus niger
fungal strains.
Keywords: Tetrahydroquinoline, Antiretroviral, Reverse Transcriptase, Drug-likeness, Cytotoxicity,
Docking, Antifungal
1. INTRODUCTION
Reverse Transcriptase (RT) is one among the various HIV targets which has been deeply
characterized, its central role in viral replication makes it attractive target for drug design. Non-
Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) is an important class of drugs, playing vital
role in drug combination therapy called ‘‘Highly Active Antiretroviral Therapy’’ (HAART) which is
currently used for HIV treatment [1]. HAART includes combination of three or more anti-HIV drugs
in order to reduce the emergence of drug resistance. NNRTIs are the key component of HAART due
to their high potency, low toxicity, high selectivity and specificity [2,3]. Although, after the advent of
HAART, the mortality rate due to AIDS reduced up to 60–80%, which made the AIDS a chronic
manageable disease from its fatal nature. However, management of HIV/AIDS is still worrisome and
complex due to certain problem associated with current therapy, like development of drug resistance,
poor drug tolerability, monitoring of therapy and efficacy. So, all these factors drive the need for

effective new inhibitors with potent antiviral activity, less toxicity and improved physicochemical
properties [4,5].
1,2,3,4-Tetrahydroquinoline (THQ) scaffold containing natural products as well as synthetic
derivatives have privileged position in the medicinal chemistry and reported for diverse biological
activities like antimicrobial, antiviral, antifungal, antimalarial, antileishmanial, antitubercular,
antidiabetic, antithrombotic and anticancer [6-12]. Exploring the anti-HIV activity of
tetrahydroquinoline nucleus, Su and team reported a series of tetrahydroquinolines which exhibited
potent inhibitory activity against HIV-1 RT by blocking its allosteric site (Fig. 1). Most potent
compound of the series A1 (Fig. 1) exhibited inhibitory activity against wild, as well as two mutant
strains (K103N and Y181C) of HIV-1 RT with IC50 value of 1, 1 and 4 nM respectively. Another
bioisosters of the same compound A2 also inhibited the activity of three selected RT strains with IC50
value of 18, 18 and 99 nM respectively [13]. In another study, a novel series of 5-alkyl-2-arylthio-6-
((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated for
HIV-1 RT inhibitory activity, in which compounds B1 and B2 exhibited potency comparable to
nevirapine [14]. In one more study, derivatives of (S)-2-((2S,4R)-2-(3-aminophenyl)-1,2,3,4-
tetrahydroquinolin-4-yl)cyclopentanone; C1 and C2 exhibited moderate anti-HIV activity in
recombinant virus assay (RVA) method [15].
Fig. 1 Tetrahydroquinoline analogs as anti-HIV agents
The availability of crystal structures of HIV-1 RT along with potent inhibitors helped in identifying
the factors which can be taken as basis for designing novel and potent inhibitors against HIV-1 RT.
Although, NNRTIs are structurally diverse compounds, still they contain numerous ubiquitous
fragments in their structures and possess a common pharmacophoric model. This model includes an
aromatic ring able to participate in <pi>-<pi> interactions, amide or thio-amide moieties capable of
hydrogen bonding and one or more hydrocarbon-rich domain that participate in hydrophobic
interactions with RT enzyme [16].
In the present study, we took structural features from molecules which inhibited the activity of HIV-1
RT at low nanomolar concentration (D, E and Efavirenz Fig. 2) [17] and constructed a prototype
compound 6. Designed prototype compound 6 constitute two hydrophobic parts; tetrahydroquinoline
along with chloro phenyl ring at one terminal and phenyl ring at the other terminal, both connected by

the hydrophilic open carbamate linker. Presence of open carbamate linker and tetrahydroquinoline
moiety provides extra flexibility to the designed compound in order to optimize the interactions at the
receptor site. Further, series of twelve compounds was generated by making the different substitution
at the prototype compound (6a-l).
Fig. 2 Literature reported potential HIV-1 RT inhibitors and the designed prototype
Physicochemical and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicological)
properties play very crucial role in discovery and development of novel drugs at the earlier stage.
Currently available drugs in the market possess a balance of desirable ADMET properties and
intrinsic potency. However, determination of ADMET of all novel molecules using iv-vivo models is
very challenging and costly task. So considering these facts, we in-silico predicted these properties of
the designed compounds using Qik-prop module of Schrödinger [18] as well as online tools;
admetSAR [19] and FAF-Drugs [20]. Once after assurance that, in-silico predicted ADMET
properties of the designed compounds possessed likeness properties, designed compounds were
synthesized, characterized and in-vitro evaluated for RT inhibitory activity against wild strains of
HIV-1 RT. Docking studies were performed for the two significantly active compounds against wild
HIV-1 RT in order to predict their putative binding mode with the selected target. Further, these two
compounds were also evaluated for cytotoxicity as well as their cytoprotective efficacy against HIV-1
in C8166 cells.
Generally people living with HIV/AIDS face major health threats due to their weak immune system,
which make them susceptible to infections called “opportunistic” infections (OIs), which are the most
common cause of death among HIV/AIDS patients [21, 22]. Among the various “opportunistic”
infections, drug resistance fungal infections are also responsible for the major health threats, which
further drive the need for the search of new potential antifungal agents. Many studies revealed the
antifungal potential of compounds containing THQ nucleus [23-25], which provoked us for the
antifungal evaluation of the synthesized compounds. All synthesized compounds were evaluated for
antifungal activity against fungal strains of Candida albicans and Aspergillus niger.

2. MATERIALS AND METHOD
2.1 In-silico prediction of physicochemical and ADMET parameters
In order to predict the drug likeness behavior of the designed compounds, their physicochemical and
ADMET properties were in-silico predicted using different tools like Qikprop module of Schrödinger,
admetSAR and FAF-Drugs3. Different parameters predicted were; molecular weight, total solvent
accessible surface area, number of hydrogen bond donor, number of hydrogen bond acceptor,
octanol/water partition coefficient, aqueous solubility, apparent caco-2 cell permeability, brain/blood
partition coefficient, number of rotatable bonds, rat acute toxicity and mutagenicity.
2.2 Chemistry
All solvents and reagents purchased from Sigma or SD fine companies were used as received without
further purification. The progress of reaction was monitored by thin layer chromatography (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, Switzerland)
melting point apparatus containing silicon oil. The IR spectra of the synthesized compounds were
1
recorded using FTIR spectrophotometer (Shimadzu IR Prestige 21, Shimadzu, Mumbai, India). H
NMR spectra were recorded on Bruker DPX-400 spectrometer (Bruker India Scientific Pvt. Ltd.,
Mumbai, India) using TMS as an internal standard (chemical shifts in δ). ESI-MS were recorded on
MICROMASS Quattro-II LCMS system (Waters Corporation, Milford, USA).
2.3 In-vitro HIV-1 RT screening
In-vitro HIV-1 RT inhibitory activity of the synthesized compounds was evaluated using Roche
diagnostics kit in accordance with the kit protocol [26]. This is based upon colorimetric assay method
in which marketed drug efavirenz was used as reference compound. Procedure followed is briefly
described here; the reaction 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 streptavidine-coated microtitre plate (MTP). The biotin-labeled dNTPs that were
incorporated in the template due to activity of RT, bound to streptavidine. 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 formula. Initially compounds were screened
at 100 µM concentration; finally IC50 was calculated using the double dilutions methods.

2.4 Docking studies
Docking studies of the two most active compounds (6b and 6j) were performed using Glide 5.9 [27]
running on maestro version 9.4, to investigate their putative binding mode in the NNIBP (Non-
Nucleoside Inhibitory Binding Pocket) of HIV-1 RT [28]. Enzyme used for the docking study was
wild HIV-1 RT, retrieved from RCSB Protein Data Bank (PDB ID: 3MEE) in complex with
rilpivirine. Protein preparation wizard of chr dinger suite was used for preparation of selected
protein. Protein was pre-processed separately by deleting the substrate co-factor as well as the
crystallographically observed water molecules (water without H bonds), followed by optimization of
hydrogen bonds. After assigning charge and protonation state, finally energy was minimized with
Root Mean Square Deviation (RMSD) value of 0.30 Å using Optimized Potentials for Liquid
Simulations-2005 (OPLS-2005) force field [29]. Finally energy minimized protein and co-crystallized
ligand was employed to build energy grids using the default value of protein atom scaling (1.0 Å)
within a cubic box of dimensions, centered on the centroid of the X-ray ligand pose. The structures of
the 6b and 6j were drawn using ChemSketch and converted to 3D structure with the help of 3D
optimization tool. By using LigPrep 2.6 [30] module, the drawn ligands were geometry optimized;
partial atomic charges were computed using OPLS-2005 force field. Finally, 32 poses were included
with different tautomeric and steric features for docking studies. Finally, prepared ligands were
docked with prepared protein using Glide 5.9 module, in extra precision mode (XP). The best docked
pose (with lowest Glide score value) obtained from Glide was analyzed. RMSD value was calculated
between the experimental binding mode of rilpivirine as in X-ray co-crystallized protein-ligand
complex and re-docked pose to ensure accuracy and reliability of the docking procedure.
2.5 Evaluation of cytotoxicity and anti-HIV activity
Compounds 6b and 6j were further selected for cytotoxicity evaluation on T cell lines (C8166) by
MTT colorimetric assay [31]. Anti-HIV-1 activity (HIV-1IIIB strain) of both these two compounds was
determined by the cytopathic effect (CPE) method, which measured the viability of HIV-1IIIB infected
C8166 cells [32]. During this study, marketed drug zidovudine was used as positive control, detailed
methodology and procedure of cytotoxicity and anti-HIV-1 assay is given in the supplementary part
of the manuscript.
2.6 Antifungal activity
All the synthesized compounds were also screened for in-vitro antifungal activity against two fungal
strains; Candida albicans (MTCC 3958) and Aspergillus niger (MTCC 9933). Antifungal activity of
the test compounds was expressed in terms of ZOI (Zone of Inhibition) and MIC (Minimum
Inhibitory Concentration). Fluconazole was used as reference drug for the study and DMSO was used
as a negative control. Procedure followed is described briefly; autoclaved suitable growth medium
(Malt Yeast Agar for Candida albicans and Czapek Yeast Extract Agar-CYA for Aspergillus niger) in
double strength were poured into autoclaved petri plates. Further, the agar plates were swabbed with

100µl inocula of each test organism under aseptic condition. After the adsorption, wells of 6 mm
diameter were made by the sterile metallic borer and the solution of working compound was poured
into the wells. The plates were incubated at 2 C for 48 h and ZOI was calculated [33]. Similarly,
MIC values were calculated using broth double dilution method for each compound, taking 100µl
inocula of each fungal culture after incubation at 2 C for 48 h.
3. RESULTS AND DISCUSSION
3.1 In-silico prediction of physicochemical and ADMET parameters
Physicochemical and ADMET parameters of the designed compounds were in-silico predicted and are
shown in Table 1, (except Mol. wt. which is shown in Table 3). The optimum range of drug likeness
parameters (followed by 95% known drug molecules) along with their abbreviations are shown in
Table 2 (34). The results of the predicted drug-likeness parameters revealed that, the value of
descriptors like mol. wt, HBD, HBA and logP lied within the optimum range as described in Table 2.
Although, compounds 6d and 6l, exhibited the low value of logS (highlighted by bold font) than the
optimum range (–6.5 to 0.5), but predicted logS deviate by a very small margin from the optimum
range, which may not change the ADMET properties much significantly, so, both these compounds
were also selected for further study. Other descriptors like SASA, logBB, Rot were also found within
the optimum range. Apart from these descriptors, all tested compounds showed good predicted oral
bioavailability (% human oral absorption >75). Predicted rat acute toxicity of all compounds laid in
“class III” on toxicity scale generally class III is considered as suitable for the druggable point of
view. Further, none of the compounds possessed mutagenicity (predicted in qualitative terms). So,
overall considering all the predicted parameters, majority of the compounds possessed the drug-
likeness properties.
Table 1 In-silico predicted physicochemical and ADMET parameters
Comp.
code
Rat acu.
tox.g
SASAa
HBDb
HBAc
ClogP logS
PCacod
logBBe
Rotf
Mut.h
666.92
695.19
1
4
5.41
5.81
-5.96 4364.71 0.08
-6.36 5415.96 0.17
-6.43 4388.60 0.07
5
2.85
2.77
None
None
6a
6b
1
4
5
698.84
698.55
670.72
675.51
675.50
690.12
704.16
704.31
753.13
794.67
1
1
1
1
1
1
1
1
1
1
4
4
4
4
4
4
7
7
6
6
5.72
5.72
5.60
5.66
5.66
5.90
5.10
5.10
6.10
5.54
5
5
5
5
5
5
6
6
7
7
2.83
2.81
2.78
2.89
2.91
2.85
2.69
2.67
2.60
2.66
None
None
None
None
None
None
None
None
None
None
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
4385.09 0.07
-6.62
-6.15 4731.37 0.19
-6.21 4386.54 0.19
-6.21 4387.41 0.20
-6.38 4383.63 0.25
-6.08 524.68
-6.08 526.00
-0.95
-0.95
-6.37 2422.50 -0.33
1313.13 -0.68
-6.73
a Solvent accessible surface area, b No. of hydrogen bond donors, c No. of hydrogen bond acceptors, d Predicted
apparent Caco-2 cell permeability in nm/sec, Predicted brain/blood partition coefficient, No. of rotatable
bonds, g Rat acute toxicity, h Mutagenicity
e
f

Table 2 Optimum range of physicochemical and ADMET parameters
S. No.
Molecular descriptor
Molecular weight (Mol wt)
Optimum range
130-725
300-1000
0-6
2-20
-2-6.5
–6.5- 0.5
<25 poor, > 500 great
–3.0-1.2
1
2
3
4
5
6.
7
8
9
Total solvent accessible surface area in square angstroms (SASA)
No. of hydrogen bond donor groups (HBD)
No. of hydrogen bond acceptor groups (HBA)
Octanol/water partition coefficient (logP)
Aqueous solubility, in mol dm–3 (logS)
Apparent Caco-2 cell permeability in nm/sec (PCaco)
Brain/blood partition coefficient (logBB)
No. of rotatable bonds (Rot)
0 to 15
3.2 Chemistry
Designed compounds 6a-l were synthesized in three steps, details of the optimum reaction conditions
used for the synthesis of target compounds are given below (scheme 1). First step involved reaction of
tetrahydroquinoline (1) with 4-chlorophenacyl Bromide 2, which afforded compound 3 as
intermediates. In second step, keto group of intermediate 3 reduced to hydroxyl using sodium
borohydride as reducing agent. Further, reaction of intermediate 4 with different substituted phenyl
isocyanates (5a-l) in the presence of sodium hydride as base afforded the final compounds (6a-l) in
good to excellent yield.
Scheme 1 Reagents and conditions: (a) DMF, Et3N, 0 ˚C- rt, 12 h (b) NaBH4 dry MeOH 0 ˚C- rt, 6 h
(c) NaH, dry THF, 0 ˚C-rt, 5-8 h
Synthesis of 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl)ethanone (3)
2-bromo-1-(4-chlorophenyl) ethanone 2 (9.28 g, 40 mmol) was added portion wise to the stirred ice
cold solution of 1,2,3,4-tetrahydroquinoline 1 (5.32 g, 40 mmol) in DMF, containing triethyl amine
(14.42 ml, 100 mmol) as base. Reaction was further stirred at rt and progress of the reaction was
monitored using TLC. After completion of reaction, as per TLC (after 12 h) 350 ml water was added
to the reaction mixture, and then twice extracted with ethyl acetate using separating funnel (2x250
ml). Combined ethyl acetate layer was twice washed with ice cold water (2x500 ml), and then washed
with brine water (500 ml). Ethyl acetate layer was dried over anhydrous sodium sulphate and finally
evaporated on rotary evaporator to afford the crude compound 3. Crude compound 3 was further
purified by column chromatography using ethyl acetate and hexane as solvent system (3% ethyl

acetate in hexane) and silica gel (mess size 100-200) as stationary phase which afforded the
compound 3 as light yellow solid with 68% yield.
Synthesis of 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl) ethanol (4)
To the ice cooled stirring solution of intermediate 3 (7.0 g, 24.40 mmol) in dry methanol, sodium
borohydride (2.70 g, 73.20 mmol) was added portion wise, after the complete addition of sodium
borohydride, reaction mixture was stirred at rt and progress of the reaction was monitored by TLC.
After completion of reaction as per TLC (after 6 h), methanol was evaporated using rotary evaporator
and 200 ml of ice water was slowly added to the reaction mass. Further, reaction mixture was twice
extracted with ethyl acetate using separating funnel (2x250 ml). Combined ethyl acetate layer was
first washed with ice cold water (500 ml), and then washed with brine water (500 ml). Ethyl acetate
layer was dried over anhydrous sodium sulphate and finally evaporated on rotary evaporator to afford
the compound 4, as white solid with 92% yield [35].
General procedure for the synthesis of 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl)ethyl
phenylcarbamate derivatives (6a-l)
Sodium hydride (hexane washed, 0.052 g, 2.2 mmol) was added portion wise to the ice cooled stirred
solution of intermediate 4 (0.574 g, 2 mmol) in dry THF. Further, corresponding isocyanate (5a-l) was
slowly added under ice cooled condition. After that, reaction mixture was stirred at rt for next 5-8 h;
progress of the reaction was monitored by TLC. After completion of reaction, reaction mixture was
quenched with 0.1 N HCl (1 ml) under ice cooled condition. Further, THF was evaporated from the
reaction mass, ethyl acetate (20 ml) was added. Ethyl acetate layer was twice washed with distilled
water (2x20 ml), and then with brine water (20 ml). Organic layer was dried over anhydrous sodium
sulphate and finally evaporated on rotary evaporator to afford the final corresponding derivatives 6a-l
[36]. Preliminary characteristics data of the synthesized compounds 6a-l are shown in Table 3.
Table 3 Preliminary characteristics data of the synthesized compounds
Comp.
Code
Melting point
R
Mol. formula
Mol. Wt
% Yield
(˚C)
H
C24H23ClN2O2
C25H25ClN2O2
C25H25ClN2O2
C25H25ClN2O2
C24H22ClFN2O2
C24H22ClFN2O2
C24H22ClFN2O2
C24H22Cl2N2O2
C24H22ClN3O4
C24H22ClN3O4
C27H27ClN2O4
C27H27ClN2O4
406.14
420.16
420.16
420.16
424.14
424.14
424.14
440.11
451.13
451.13
478.17
478.17
106-108
88
82
85
83
78
79
81
87
73
75
78
76
6a
2-Me
3-Me
4-Me
2-F
3-F
4-F
76-78
6b
6c
6d
6e
6f
6g
6h
6i
120-122
138-140
88-90
106-108
124-126
166-168
164-166
166-168
76-78
4-Cl
3-NO2
4-NO2
2-COOEt
4-COOEt
6j
6k
6l
132-134

Synthesized compounds were purified using suitable purification techniques like column
chromatography and re-crystallization techniques as per requirement. Purified compounds were
characterized by non-spectral methods (melting point and thin layer chromatography) as well as
1
spectral analysis (IR, H NMR and Mass). IR spectra of the synthesized compounds showed the
expected absorption bands, for example all compounds possessed hydrogen of carbamate bond
(−OCONH-), a corresponding stretching peak (sharp, strong) appeared in IR spectra of compounds at
the region 3200–3350 cm-1. Another characteristic peak (sharp, strong), appeared in the region 1700-
1750 cm-1 corresponding to the stretching of carbonyl group (-OC=ONH-). Proton NMR signals and
proton counting of the title compounds were also observed in their expected region. NMR spectrum of
the unsubstituted compound 6a is explained below in order to gain an idea about the peak pattern of
the whole series. Two protons at 3rd carbon of THQ ring displayed multiplets with δ values 1. 9 –
1.73. Further two benzylic proton at the forth position of THQ ring appeared as dd at δ 2.71.
Interestingly, two protons at 2nd position of THQ appeared in discrete peaks (ddd) at δ 3.02 and 3.21.
Two protons adjacent to nitrogen, outside the ring also appeared in two discrete peaks (dd) at δ 3.40
and 3.87. Further, single proton at benzylic carbon connected with highly electronegative oxygen
atom is highly deshielded, which appeared (dd) at δ 6.02. Further, all aromatic protons appeared at
their expected region, moreover nitrogen proton of carbamate (NH) due to strong deshielding
appeared (dd) in the aromatic region near δ 6.7 . ESI-MS of the synthesized compounds showed the
corresponding M+1 peak.
3.3 HIV-1 RT inhibitory activity
Purified compounds were screened for the HIV-1 RT inhibitory assay using assay kit (Roche),
efavirenz was used as reference drug during the studies. The results of RT in-vitro assay revealed that,
around four compounds (6b, 6i, 6j and 6l) exhibited significant potency (highlighted in bold font in
Table 4) with IC50 less than 20 µM. Compound 6a (un-substituted phenyl analogues) of series
exhibited moderate inhibition of RT activity (IC50 36.98 µM). Further, substitution of phenyl with
methyl group at ortho position markedly increased the potency against HIV-1 RT (IC50 8.12 µM),
while methyl substitution at meta and para position decreased the potency (IC50 68.26 and 83.60
respectively). Among the compounds containing fluoro substitution at phenyl ring (6e, 6f and 6g),
potency against HIV-1 RT varied in the order ortho < meta < para (IC50 90.30, 72.14 and 48.57 µM
respectively). Substitution with electron withdrawing chloro group at the para position of phenyl (6h)
resulted enhancement in the RT inhibition activity (IC50 23.76 µM) compared to the unsubstituted
compound 6a. Further, substitution with nitro group at the para position of phenyl ring (6i) markedly
increased the potency (IC50 5.42 µM), while nitro substitution at meta position slightly decrease its
potency (IC50 14.87 µM). Compound 6k substituted with ethyl carboxylate group at the ortho position
of phenyl ring retained the potency (IC50 38.97), but interestingly substitution with same group at para
position (6l) markedly enhanced the potency (IC50 12.34) against HIV-1 RT. Standard drug efavirenz

inhibited the activity of HIV-1 RT with IC50 0.025 µM). So, SAR studies revealed that, electron
donating group at ortho position and electron withdrawing group of moderate size at para position of
phenyl ring resulted enhancement in potency against HIV-1 RT.
Table 4 Results of in- vitro HIV-1 RT inhibition studies
Comp.
Code
aRT inhibition
IC50 (µM)
R
36.98
8.12
H
6a
6b
6c
6d
6e
6f
6g
6h
6i
2-Me
3-Me
4-Me
2-F
3-F
4-F
68.26
83.60
90.30
72.14
48.57
23.76
14.87
5.42
38.97
12.34
0.025
4-Cl
3-NO2
4-NO2
2-COOEt
4-COOEt
6j
6k
6l
Efavirenz
avalue is mean of duplicate experiment,
each value exhibited SD less than ± 10%,
from mean
3.4 Docking studies
In order to predict the exact binding mode of two most active compounds (6j and 6b) within NNIBP
of HIV-1 RT, docking studies were carried out. The value of RMSD obtained between experimental
binding mode as in X-ray and re-docked pose (Fig. 3) for rilpivirine (Co-crystallized ligand in target
protein) was 0.6, which suggested that, docking procedure could be relied on to predict the binding
mode of the selected compounds. The analysis of best docked pose of compound 6j (Fig. 4a )
revealed that, first hydrophobic wing (contains tetrahydroquinoline and chloro phenyl ring) part of 6j,
forms hydrophobic interaction with amino acids Pro-95, Trp-229, Tyr-181, Leu-100, Val-106, Tyr-
318, while second aromatic wing (p-nitro phenyl) exhibited hydrophobic interactions with Val-179.
The keto group of the central carbamate linker showed dual hydrogen bonding interaction with Lys-
101 and Lys-103. While, in best docked pose of compound 6b, its first hydrophobic wing displayed
hydrophobic interaction with amino acids Leu-100, Val- 179, Glu-138, Pro-95, Tyr-181, Trp-220,
Tyr-188, while second aromatic wing (methyl phenyl) showed similar interaction with Val-106, Tyr-
318 (Fig. 4b).
So these hydrophobic and hydrogen bonding interactions of 6j with HIV-1 RT may be responsible for
its enhanced in-vitro activity. Although, compound 6b not displayed any hydrogen bonding
interaction but its prominent hydrophobic interaction with receptor may be responsible to overcome
the deficiency of former interactions and retention of significant RT inhibitory activity during in-vitro
studies.

Fig. 3 Superimposed view of best scoring pose of rilpivirine (blue) with its X-ray pose (green) in 3MEE.
Fig. 4a
Fig. 4b
Fig. 4 Docked pose of compound 6j (Fig. 4a) and 6b (Fig. 4b) inside the NNIBP of 3MEE, showing
hydrophobic (green dotted lines) and hydrogen bonding interactions (pink dotted lines)
3.5 Cytotoxicity and anti-HIV-1 activity
Anti-HIV activity of compounds 6b and 6j was determined by measuring the inhibitory percentage of
syncytia (multinucleated giant cell) formation in sample-treated culture compared to that in infected
control culture. Compounds 6b and 6j exhibited the EC50 values of 11.10 and 2.76 µg/ ml respectively
against HIV-1 mediated syncytia formation. Further, compounds 6b and 6j exhibited CC50 values of
108.73 and 18.95 µg/ml upon C8166 cell. So, overall compounds 6b and 6j displayed safety index of
9.8 and 6.9 respectively. In this study, reference drug zidovudine exhibited most potent anti-HIV
activity as well as least cytotoxicity with EC50 and CC50 values of 0.0019 µg/ ml and 1389.95 µg/ ml
respectively.

3.6 Antifungal activity
Among the compounds (6a-l), screened for antifungal activity, seven compound (6e, 6f, 6g, 6h, 6i,
6k, and 6l) showed significant, three compounds (6a, 6d and 6j) exhibited moderate, while other
compounds possessed weak to least inhibitory potential against the growth of Candida albicans
(Table 5). Against Aspergillus niger, six compounds (6d, 6e, 6f, 6g, 6h and 6l) exhibited significant,
two compounds (6a and 6j) showed moderate, while other compounds displayed weak to least
inhibitory potential. Interestingly, five compounds (6e, 6f, 6g, 6h and 6l) significantly inhibited the
growth of both fungal strains. Moreover, compounds 6g, 6h and 6l showed promising antifungal
activity against Candida albicans, in which compound 6h displayed antifungal activity almost
comparable to reference drug Fluconazole with ZOI 20 mm and MIC 8 µg/ml. Overall, Candida
albicans was found to be more sensitive compared to Aspergillus niger against the tested compounds.
Table 5 Antifungal evaluation results of the synthesized compounds 6a-l
Candida albicans
Aspergillus niger
ZOIa
MIC
ZOIa
MIC
Comp.
Code
(mm)
(µg/ml)
(mm)
(µg/ml)
13
10
-
64
128
-
13
-
128
-
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
10
16
18
16
18
17
12
14
-
128
64
64
64
32
64
128
64
-
14
16
18
19
20
19
14
16
20
21
64
64
32
16
8
32
128
64
16
8
18
22
64
8
Fluconazole
aZOI ≥15; significantly active (bold font), ZOI 13-14; moderately
active, ZOI 10-12; weakly active, ZOI≤10 least active MIC was
not calculated for least active compounds.
4. CONCLUSIONS
Twelve novel 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl)ethyl phenylcarbamate derivatives
were designed as inhibitor of HIV-1 RT using ligand based drug design approach. Further, basis upon
the in-silico predicted physicochemical and ADMET parameters, designed compounds were found to
be Drug-like. Designed compounds were synthesized, purified, characterized and in-vitro evaluated
for RT inhibitory activity against the wild HIV-1 RT strain, in which four compounds exhibited
significant inhibition of HIV-1 RT (IC50 ≤ 20 µM) in which most active compounds 6b and 6j
inhibited the RT activity with IC50 8.12 and 5.42 µM respectively. SAR studies of compounds 6a-l
revealed that, electron donating group at ortho position and electron withdrawing group of moderate size at

para position of prototype compound 6, favoured the RT inhibitory potency. In the docking studies against
wild HIV-1 RT, compound 6b showed very prominent hydrophobic interaction, while compound 6j
exhibited strong hydrogen as well as hydrophobic bonding interactions with selected RT. These
prominent interactions of both compounds with target protein may be responsible for their significant
RT inhibitory potency during the in-vitro studies. Further, compounds 6b and 6j also displayed anti-
HIV activity (in CPE method on C8166 cell) with EC50 11.10 and 2.76 µg/ml. In cytotoxicity studies,
compounds 6b and 6j exhibited CC50 108.73 and 18.95 µg/ml respectively performed on C8166 cell,
and showed overall safety index of 9.8 and 6.9 respectively. So, overall studies can be helpful for
further lead optimization or designing of novel potent anti-HIV agents. The result of antifungal
screening of all the synthesized compounds revealed that, five compounds (6e, 6f, 6g, 6h and 6l)
significantly inhibited the growth of both tested fungal strains. Moreover, compound 6h showed
promising antifungal activity against Candida albicans almost comparable to reference drug
Fluconazole.
5. ACKNOWLEDGMENTS
Authors gratefully acknowledge BITS-Pilani for providing the necessary facilities to do this work.
This work was carried out under a grant from Science and Engineering Research Board of Department
of Science and Technology, New Delhi. (Ref. No: SR/FT/LS-58/2011), the STS Program of CAS
(KFJ-EW-STS-026) and Collaborative Innovation Center for Natural Products and Biological Drugs
of Yunnan
6. CONFLICT OF INTEREST
The authors declared that there is no conflict of interest.
7. REFERENCES
[1] J.R. Brechtl, W. Breitbart, M. Galietta, S. Krivo, B. Rosenfeld, The use of highly active anti
retroviral therapy (HAART) in patients with advanced HIV infection: impact on medical,
palliative care and quality of life outcomes. J. Pain Symptom Manag. 21 (2001) 41–51.
[2] M.J. Kozal, Drug-resistant human immunodeficiency virus. Clin. Microbiol. Infect. 15 (2009)
69–73.
[3] B. Crabtree-Ramirez, A. Villasis-Keever, A. Galindo-Fraga, C. del Rio, J. Sierra-Madero,
Effectiveness of Highly Active Antiretroviral Therapy (HAART) among HIV-infected patients
in Mexico. AIDS Res. Hum. Retroviruses 26 (2010) 373-378.

[4] L. Palombi, M. Dorrucci, I. Zimba, Immunologic response to Highly Active Antiretroviral
Therapy and mortality reduction in a cohort of Human Immunodeficiency Virus–positive
persons in Mozambique. Am. J. Trop. Med. Hyg. 83 (2010) 1128-1132.
[5] P. Chong, P. Sebahar, M. Youngman, Rational design of potent non-nucleoside Inhibitors of
HIV-1 reverse transcriptase. J. Med. Chem. 55 (2012) 10601-10609.
[6] A. Mitchinson, A. Nadin, Saturated nitrogen heterocycles. J Chem. Soc. Perkin. Trans.1 18
(1999) 2553-2582.
[7] J.P. Michael, Quinoline, quinazoline and acridone alkaloids. Nat. Prod. Rep. 25 (2008) 166-
187.
[8] S. Kumar, S. Bawa, H. Gupta, Biological activities of quinoline derivatives. Mini. Rev. Med.
Chem. 9 (2009) 1648-1654.
[9] V. Sridharan, P.A. Suryavanshi, J.C. Menendez, Advances in the chemistry of
tetrahydroquinolines. Chem. Rev. 111 (2011) 7157-7259.
[10] A.A. Rashad, A.J. Jones, V.M. Avery, J. Baell, P.A. Keller, Facile synthesis and preliminary
structure-activity analysis of new sulfonamides against Trypanosoma brucei. ACS Med. Chem.
Lett. 5 (2014) 496-500.
[11] G.A. Jacoby, M.A. Corcoran, D.C. Hooper, Protective effect of Qnr on agents other than
quinolones that target DNA gyrase. Antimicrob. Agents Chemother. 59 (2015) 6689-6695.
[12] S.B. Wang, X.F. Wang, B. Qin, E. Ohkoshi, K.Y. Hsieh, E. Hamel, M.T. Cui, D.Q. Zhu, M.
Goto, S.L. Morris-Natschke, K.H. Lee, L. Xie, Optimization of N-aryl-6-methoxy-1,2,3,4-
tetrahydroquinolines as tubulin polymerization inhibitors. Bioorg. Med. Chem. 23 (2015) 5740-
5747.
[13] D.S. Su, J.J. Lim, E. Tinney, B.L. Wan, M.B. Young, K.D. Anderson, Substituted
tetrahydroquinolines as potent allosteric inhibitors of reverse transcriptase and its key mutants.
Bioorg. Med. Chem. Lett. 19 (2009) 119-5123.
[14] J. Zhang, P. Zhan, J. Wu, Z. Li, Y. Jiang, W. Ge, C. Pannecouque, E. De Clercq, X. Liu,
Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-
yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase
inhibitors. Bioorg. Med. Chem. 19 (2011) 4366-76.
[15] L.M. Bedoya, Quinoline-based compounds as modulators of HIV transcription through NF-
kappa B and Sp1 inhibition. Antiviral Res. 87 (2010) 338-344.
[16] S. Yang, C. Pannecouque, D. Daelemans, Molecular design, synthesis and biological evaluation
of BP-O-DAPY and O-DAPY derivatives as non-nucleoside HIV-1 reverse transcriptase
inhibitors. Eur. J. Med. Chem. 65 (2013) 134–43.
[17] O.S. Pedersen, E.B. Pedersen, Non-nucleoside reverse transcriptase inhibitors: the NNRTI
boom; Antivir. Chem. Chemother. 10 (1999) 285-314.
[18] Qikprop. New York, Schrödinger, LLC, (2013) Version 3.8

[19] F. Cheng, W. Li, Y. Zhou, J. Shen, Z. Wu, G. Liu, admetSAR: a comprehensive source and
free tool for assessment of chemical ADMET properties. J. Chem. Inf. Model. 52 (2012) 3099-
3105.
[20] D. Lagorce, O. Sperandio, J.B. Baell, M.A. Miteva, B.O. Villoutreix, FAF-Drugs: a web server
for compound property calculation and chemical library design. Nucl. Acids Res. 43 (2015)
W200-W207.
[21] M. Ghate, S. Deshpande, S. Tripathy, M. Nene, P. Gedam, S. Godbole, M. Thakar, A. Risbud,
R. Bollinger, S. Mehendale, Incidence of common opportunistic infections in HIV-infected
individuals in Pune, India: analysis by stages of immunosuppression represented by CD4
counts. Int. J. Infect. Dis. 13 (2009) e1-e8.
[22] S.D. Patel, D.M. Kinariwala, T.B. Javadekar, Clinico-microbiological study of opportunistic
infection in HIV seropositive patients. Indian J. Sexual. Transm. Dis. 32 (2011) 90-93.
[23] J.M. Urbina, J.C. Cortes, A. Palma, S.N. Lopez, S.A. Zacchino, R.D. Enriz, Inhibitors of the
fungal cell wall: synthesis of 4-aryl-4-N-arylamine-1-butenes and related compounds with
inhibitory activities on beta(1-3)glucan and chitin synthases. Bioorg. Med. Chem. 8 (2000) 691-
698.
[24] P. Bendale, S. Olepu, P.K. Suryadevara, V. Bulbule, K. Rivas, L. Nallan, Second generation
tetrahydroquinoline-based protein farnesyltransferase inhibitors as antimalarials. J. Med. Chem.
50 (2007) 4585-4605.
[25] M. Gutierrez, U. Carmona, G. Vallejos, L. Astudillo, Antifungal activity of
tetrahydroquinolines against some phytopathogenic fungi. Zeitschrift fur Naturforschung C. 67
(2012) 551-556.
[26] J. Eberle, C.W. Knopf, Nonisotopic assays of viral polymerases and related proteins. Methods
Enzymol. 275 (1996) 257–276.
[27] Glide, Schrödinger, LLC, New York, (2013) version 5.9.
[28] R.A. Friesner, R.B. Murphy, M.P. Repasky, Extra precision glide: docking and scoring
incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem.
49 (2006) 6177-6196.
[29] W.L. Jorgensen, D.S. Maxwell, R.J. Tirado, Monte Carlo investigations of solvent effects on
the chorismate to prephenate rearrangement. J. Am. Chem. Soc. 118 (1996) 11225-11236.
[30] Lig-Prep, Schrödinger, LLC, New York, 2013, version 2.6.
[31] G. H Zhang, Q. Wang, J.J. Chen, X.M. Zhang, S.C. Tam, Y.T. Zheng. Anti-HIV-1 effect of
scutellarin. Biochem. Biophys. Res. Commun. 334.(2005) 812-816.
[32] R.R. Wang, Q. Gu, Y.H. Wang, X.M. Zhang, L.M. Yang, J. Zhou, J.J. Chen, Y.T. Zheng. Anti-
HIV-1 activities of compounds isolated from the medicinal plant Rhus chinensis. J.
Ethnopharmacol. 117 (2008) 249-256.

[33] S. Magaldi, S. Mata-Essayag, C. Hartung de Capriles, C. Perez, M.T. Colella, C. Olaizola, Y.
Ontiveros, Well diffusion for antifungal susceptibility testing. Int. J. Infect. Dis. 8 (2004) 39-45.
[34] Qikprop, User Manual, Schrödinger, LLC, Chapter-1, Version 3.8, (2013), 2-5.
[35] D, Kupfer, Altered selectivity of reduction of steroidal carbonyls. Tetrahedron 15 (1961) 193-
196.
[36] K.K Roy, S. Tota, T. Tripathi, Lead optimization studies towards the discovery of novel
carbamates as potent AChE inhibitors for the potential treatment of Alzheimer’s disease.
Bioorg. Med. Chem. 20 (2012) 6313-6320.

Graphical abstract

Highlights
 Novel Tetrahydroquinoline carbamates were designed as HIV-1 RT inhibitor using
ligand based drug design approach and in-silico evaluated for drug likeness properties
 Designed compounds were synthesized and in-vitro evaluated for HIV-1 RT
inhibitory activity against wild strain
 Two most active RT inhibitors were selected for further studies like docking, anti-
HIV activity and cytotoxicity
 All synthesized compounds were evaluated for antifungal activity against Candida
albicans and Aspergillus niger fungal strains