Molecular Docking Studies and Synthesis of Amino-oxy-diarylquinoline Derivatives as Potent Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1055/a-0968-1150

In this study, amino-oxy-diarylquinolines were designed using structure-guided molecular hybridization strategy and fusing of the pharmacophore templates of nevirapine (NVP), efavirenz (EFV), etravirine (ETV, TMC125) and rilpivirine (RPV, TMC278). The anti-HIV-1 reverse transcriptase (RT) activity was evaluated using standard ELISA method, and the cytotoxic activity was performed using MTT and XTT assays. The primary bioassay results indicated that 2-amino-4-oxy-diarylquinolines possess moderate inhibitory properties against HIV-1 RT. Molecular docking results showed that 2-amino-4-oxy-diarylquinolines 8(a-d) interacted with the Lys101 and His235 residue though hydrogen bonding and interacted with Tyr318 residue though π-π stacking in HIV-1 RT. Furthermore, 8a and 8d were the most potent anti-HIV agents among the designed and synthesized compounds, and their inhibition rates were 34.0% and 39.7% at 1 μM concentration. Interestingly, 8a was highly cytotoxicity against MOLT-3 (acute lymphoblastic leukemia), with an IC ₅₀ of 4.63±0.62 μg/mL, which was similar with that in EFV and TMC278 (IC ₅₀ 7.76±0.37 and 1.57±0.20 μg/ml, respectively). Therefore, these analogs of the synthesized compounds can serve as excellent bases for the development of new anti-HIV-1 agents in the near future.

Full text of DOI:10.1055/a-0968-1150

Published online: 07.11.2019
Original Article
Thieme
Makarasen Arthit et al. Molecular docking studies and… Drug
Res 2019; 00: 00–00
Molecular Docking Studies and Synthesis of Amino-oxy-
diarylquinoline Derivatives as Potent Non-nucleoside HIV-1
Reverse Transcriptase Inhibitors
Authors
1
3
3
1
1
1
Arthit Makarasen , Mayuso Kuno , Suwicha Patnin , Nanthawan Reukngam , Panita Khlaychan , Sirinya Deeyohe ,
Pakamas Intachote , Busakorn Saimanee , Suchada Sengsai , Pornthip Boonsri , Apinya Chaivisuthangkura ,
Wandee Sirithana , Supanna Techasakul
2
2
2
3
3
4
1
Affiliations
Supporting Information for this article is available
1
Department of Chemistry, Laboratory of Organic
online at http://www.thieme-connect.de/products.
Synthesis, Chulabhorn Research Institute, Laksi, Bangkok,
Thailand
ABSTRACT
2
Biological Activity Test and Screening Unit, Central
Facilities, Chulabhorn Research Institute, Laksi, Bangkok,
Thailand
In this study, amino-oxy-diarylquinolines were designed using
structure-guided molecular hybridization strategy and fusing
of the pharmacophore templates of nevirapine (NVP), efavirenz
3
4
Department of Chemistry, Faculty of Science,
Srinakharinwirot University, Wattana, Bangkok, Thailand
Department of Chemical Technology, Faculty of Science
and Technology, Suan Dusit University, Bangplat,
Bangkok
(
EFV), etravirine (ETV, TMC125) and rilpivirine (RPV, TMC278).
The anti-HIV-1 reverse transcriptase (RT) activity was evalu-
ated using standard ELISA method, and the cytotoxic activity
was performed using MTT and XTT assays. The primary bioas-
say results indicated that 2-amino-4-oxy-diarylquinolines pos-
sess moderate inhibitory properties against HIV-1 RT. Molecu-
lar docking results showed that 2-amino-4-oxy-diarylquinolines
Keywords
synthesis, quinoline, molecular docking, hybrid, anti HIV-1
activity
8
(a-d) interacted with the Lys101 and His235 residue though
hydrogen bonding and interacted with Tyr318 residue though
π-π stacking in HIV-1 RT. Furthermore, 8a and 8d were the most
potent anti-HIV agents among the designed and synthesized
compounds, and their inhibition rates were 34.0% and 39.7 %
at 1 µM concentration. Interestingly, 8a was highly cytotoxic-
ity against MOLT-3 (acute lymphoblastic leukemia), with an IC50
of 4.63 ± 0.62 µg/mL, which was similar with that in EFV and
TMC278 (IC50 7.76±0.37 and 1.57±0.20µg/ml, respectively).
Therefore, these analogs of the synthesized compounds can
serve as excellent bases for the development of new anti-HIV-1
agents in the near future.
received
accepted
08.03.2019
02.07.2019
Bibliography
DOI https://doi.org/10.1055/a-0968-1150
Published online: 7.11.2019
Drug Res 2019; 69: 671–682
©
Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Dr. Supanna Techasakul
Department of Chemistry
Laboratory of Organic Synthesis, Chulabhorn Research
Institute
Laksi
1
0210 Bangkok
Thailand
Tel.: + 66/255/38 555, Fax: + 66/255/38 549
supanna@cri.or.th
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
6
71

Original Article
Thieme
Introduction
Material and Methods
Acquired immune deficiency syndrome (AIDS) is caused by human
immunodeficiency virus (HIV). The research on this disease has
been extensive for over 40 years, but no cure has been found. Mul-
ti-drug therapy against HIV, known as highly active antiretroviral
therapy (HAART), has become the most standard and efficient
treatment regimen for HIV infection. HAART generally involves in-
hibitors targeting reverse transcriptase (RT) and protease enzymes
The starting materials and other reagents for synthesis were pur-
chased from Aldrich Company and Tokyo Chemical Industry and
were used as received without further purification unless otherwise
indicated. The melting points were measured using an SMP3 Stu-
artTM digital melting point apparatus from Bibby Sterlin, Ltd. To
confirm the structure, we analyzed the product by using nuclear
magnetic resonance (NMR) and mass spectrometry. Proton and
carbon NMR spectra were obtained using the Bruker AvanceII-300
spectrometer at 300 and 75 MHz, respectively. High-resolution
mass spectra were measured with an ESI-TOF, i. e., MicroTOF mass
spectrometer (Bruker Daltonics, Germany). HepG2(hepato-
carcinoma), MOLT-3 (acute lymphoblastic leukemia), HuCCA-1
(cholangiocarcinoma), A549 (lung carcinoma), and MRC-5 (normal
embryonic lung cell) cell lines were purchased from Hyclone Labo-
ratories. Doxorubicin (purity ≥ 98 %) and etoposide (purity 98 %)
were purchased from Aldrich Company.
[
1–3]. Among the currently available drugs in HAART, RT inhibitors
are the mainstay of most of the frontline HIV combination thera-
pies. The RT inhibitors can be divided into two classes of drugs,
namely, those belonging to the nucleoside reverse transcriptase
(
NRTIs) and those belonging to the non- nucleoside reverse tran-
scriptase (NNRTIs) [4]. NRTIs inhibit the RT through the active site,
whereas the NNRTIs bind to a site far from the active site [5–7]. NN-
RTIs are important components of the HAART regimen, owing to
their unique antiviral activity, high specificity and low cytotoxicity.
The five structurally diverse NNRTIs, namely, nevirapine (NVP), de-
lavirdine (DLV), efavirenz (EFV) [8,9], etravirine (ETV, TMC125) and
rilpivirine (RPV, TMC278) [10, 11], were approved by FDA for clini-
cal use. Searching for new anti-HIV-1 RT agent with either higher
potency or less toxicity is necessary, and researchers focus on find-
ing solution to this problem. Molecular hybridization approach is
used to design and develop new molecules. This approach depends
on the combination of physiochemical and pharmacological activ-
ities to produce newly hybridized compounds with improved affin-
ity and efficiency compared with drugs containing two or more
known compounds. In the present study, we studied the molecu-
lar hybridization of NVP, EFV, and TMC278 to generate a series of
disubstituted quinoline derivatives and to explore the new anti-HIV
agents.
Quinoline is a heterocyclic aromatic nitrogen compound wide-
ly used as a parental compound to synthesize molecules with med-
ical benefits. Quinoline derivatives are an important class of hete-
rocycles, being the chief components of many naturally occurring
products. The quinoline derivative possesses various pharmaco-
logical properties, such as anticancer, antimycobacterial, antimi-
crobial, anticonvulsant, anti-inflammatory and cardiovascular ac-
tivities [12–16]. The applications of quinoline derivatives utilizes
in almost every branch of medicinal chemistry. In recent years,
large numbers of quinoline derivatives, including mono-, di-, tri-,
tetra- and heterocyclic substituents have been synthesized and
their cytotoxic activity reported [17]. Our research program aimed
at the discovery of HIV-1 RT using the quinoline as a core structure
to maintain the characteristic of the original base structure. The
molecular hybridizations are characterized by a core structure of
quinoline and the substituent for two positions in the core struc-
ture. Similar side chains of TMC125 and TMC278 were used to con-
firm the potential of this core structure in the NNRTI activity. Here-
in, we reported the synthesis and antiviral activity of amino-oxy-
diarylquinoline derivatives together with some measured and
calculated physicochemical properties, such as binding energy,
number of ligand conformations and interaction. The possible
structures and the structure-activity relationships are discussed in
detail to obtain further insights into these analogs.
Molecular docking studies
The geometry of quinoline derivatives, namely, 5(a-d) and 8(a-d)
was fully optimized using the density functional theory (DFT) at
B3LYP/6–31 G (d, p) level implemented in Gaussian 03. The crystal
structure of HIV-1(4G1Q) was obtained from the Protein Data Bank.
4G1Q is a crystal structure of HIV-1 RT in a complex with TMC278,
which is an NNRTI. Before docking, the protein was prepared by re-
moving water molecules, the ligand, and other unnecessary small
molecules from the crystal structure of the ligand-HIV-1 RT com-
plex (PDB code: 4G1Q). The binding interactions of quinoline de-
rivatives with HIV-1 were simulated by molecular docking using the
Autodock 4.2. Autodock default settings were used for parameters
such as the population size for the Lamarckian Genetic Algorithm
(LGA) (set to 150 individuals) and the number of genetic algorithm
(set to 200). The maximum number of evaluations was performed
for 2500000 and the maximum number of generations was select-
ed for 27 000. The grid box size used was 80 × 80 × 80 A° and the
center applied was as follows: x = 49.082, y = − 28.29, z = 37.541;
the spacing was 0.375 A°. The lowest energy conformation was
used to visualize the docked conformation using the Discovery stu-
dio 4.0 software. The identification of ligand binding modes was
done by iteratively evaluating a number of ligand conformations
and estimating energy of their interactions with the target. Exper-
iment were performed in triplicate and repeated three times with
similar results.
Synthesis
6
-Nitroquinolin-4-ol (2)
A mixture of concentrated nitric acid (2.5 mL) and concentrated
sulfuric acid (2.5 mL) was slowly added to a solution of 4-hydrox-
yquinoline (1) (2.0 g, 13.8 mmol) in concentrated sulfuric acid
(2.5mL) at −15°C. The mixture was stirred for 2h, and the temper-
ature of the reaction mixture was slowly raised from − 15 to 0 °C.
The mixture was quenched with ice-water (1:1). The precipitated
crude product, that is, 2, was collected by filtration and dried under
high vacuum (2.2g, 84%), yellow solid, and mp. 217.5°C–218.4°C.
1
The H-NMR (300 MHz, dimethyl sulfoxide-d6 [DMSO-d6]) condi-
tions were as follows: δ ppm 6.20 (d, 1 H, J = 7.5 Hz), 7.73 (d, 1 H,
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
6
72

+
J=9.1Hz), 8.06 (d, 1H, J=7.5Hz), 8.42 (dd, 1H, J=9.2, 2.7Hz), 8.84
d, 1 H, J = 2.7 Hz), and 12.33 (bs, 1 H). The 13C-NMR (75 MHz, DM-
SO-d6) conditions were as follows: δ ppm 110.2, 110.5, 120.2,
160.7. Finally, the HRMS ( + ESI) was C18H14N O [M + H] ; it re-
3 3
(
quires 320.1035, but has 320.1020.
4
-(4′-formylphenoxy)-6-nitroquinoline (4c)
1
21.6, 124.6, 125.9, 140.9, 142.6, 143.7, and 176.3. Finally, the
+
HRMS (+ESI) was C H N O [M+H] ; it requires 191.0457, but has
With 90 % yield, the synthesis started with 100 mg (0.48 mmol) of
9
7
2
3
1
91.0448.
3 to obtain 127 mg of 4c, which consisted of white solid and mp.
1
2
03.8°C–204.3°C. The H-NMR (300MHz, CDCl ) conditions were
3
4
-Chloro-6-nitroquinoline (3)
as follows: δ ppm 6.78 (d, 1 H, J = 5.2 Hz), 7.40 (dd, 2 H, J = 6.8,
1.7 Hz), 8.06 (dt, 2H, J = 8.6, 1.9 Hz), 8.26 (d, 1H, J = 9.3 Hz), 8.55
(dd, 1 H, J = 9.3, 2.6 Hz), 8.90 (d, 1 H, J = 5.2 Hz), 9.30 (d, 1 H,
A catalytic amount of DMF was added to the mixture of 2 (1.0 g,
.3mmol) and POCl (20mL) in a sealed tube. The sealed tube was
5
3
flushed with argon, and the reaction mixture was heated at 100°C
J=2.5Hz), and 10.07 (s, 1H). The 13C-NMR (75MHz, CDCl ) condi-
3
for 5h. The excess of POCl was removed from the reaction mixture
tions were as follows: δ ppm 106.1, 119.3, 120.6, 121.3, 123.9,
3
under high vacuum. Furthermore, 10% aqueous NH OH was added
131.2, 132.3, 134.3, 151.8, 154.6, 158.5, 162.0, and 190.4. Final-
4
+
to the residue and extracted thrice with ethyl acetate. The com-
bined organic layers were washed with saturated NaCl and dried
over Na SO . The crude product was purified on silica gel column
ly, the HRMS (+ESI) was C16H10N O [M+H] ; it requires 294.0641,
2
4
but has 295.0714.
2
4
4
-(4′-cyanophenoxy)-6-nitroquinoline (4d)
(
eluent: hexane/ethyl acetate 1:1) to obtain 3 (1.0 g, 91 %), white
1
solid and mp. 140.8 °C–141.5 °C. The H-NMR (300 MHz, CDCl )
With 99 % yield, the synthesis started with 100 mg (0.48 mmol) of
3
conditions were as follows: δ ppm 7.67 (d, 1H, J = 3.5 Hz), 8.23 (d,
3 to obtain 138 mg of 4d, which consisted of white solid, mp.
1
1
H, J = 6.9 Hz), 8.55 (dd, 1 H, J = 6.9, 1.2 Hz), 8.97 (dd, 1 H,
212.4°C–213.3°C. The H-NMR (300MHz, CDCl ) conditions were
3
J=3.5Hz), and 9.20 (bs, 1H). The 13C-NMR (75MHz, CDCl ) condi-
tions were as follows: δ ppm 121.4, 122.9, 123.9, 125.9, 132.0,
as follows: δ ppm 6.77 (d, 1 H, J = 3.9 Hz), 7.36 (d, 2 H, J = 6.5 Hz),
7.84 (d, 2 H, J = 6.45 Hz), 8.26 (d, 1 H, J = 6.7 Hz), 8.55 (dd, 1 H,
J = 6.9, 1.8 Hz), 8.91 (d, 1 H, J = 3.8 Hz), and 9.27 (d, 1 H, J = 1.9 Hz).
3
1
44.6, 151.0, and 153.3. Finally, the HRMS (+ESI) was C H ClN O
9 6 2 2
+
The 13C-NMR (75 MHz, CDCl ) conditions were as follows: δ ppm
[
M + H] ; it requires 209.0118, but has 209.0112.
3
1
1
06.1, 110.2, 117.8, 119.1, 120.5, 121.7, 124.0, 131.3, 134.8,
45.6, 151.8, 154.5, 157.2, and 161.7. Finally, the HRMS ( + ESI)
General procedure for the preparation of 4-phenoxy-
-nitroquinoline 4(a-d)
6
+
was C16H10N O [M + H] ; it requires 292.0722, but has 292.0709.
3
3
A mixture of 3 (0.48 mmol) and hydroxyl benzene (0.57 mmol) in
DMF (4mL) with anhydrous potassium carbonate (0.96mmol) was
heated in a sealed tube and stirred at 120 °C for 5 h. After cooling,
the mixture was poured into ice-water, and the mixture was ex-
tracted thrice with ethyl acetate. The combined organic layers were
washed with saturated NaCl and dried over Na SO . The crude prod-
General procedure for the preparation of 4-oxyaryl-
-(4'-cyanophenyl)-aminoquinoline 5(a-d)
6
A mixture of 4(a-d) (0.10mmol) and SnCl ·2H O (0.50mmol) in ab-
2
2
solute ethanol (3mL) was sonicated in a water bath at 30°C for 2h.
When the whole starting material was completely consumed in the
reaction (identified by the TLC), the mixture was cooled to room tem-
perature and the solvent was evaporated. The residue was dissolved
with ethyl acetate and filtered through a short silica gel column. The
crude product was used for further reaction without purification. A
mixture of 6-(4-phenoxy)-quinolinamine (0.10 mmol), 4-iodoben-
2
4
uct was purified on silica gel column (eluent: hexane/ethyl acetate)
to obtain 4-phenoxy-6-nitroquinoline 4(a-d)
4
-(2′,6′-Dimethyl-4′-formylphenoxy)-6-nitroquinoline (4a)
With 97 % yield, the synthesis started with 100 mg (0.48 mmol) of
3
to obtain 150 mg of 4a, which consisted of white solid, and mp.
zonitrile (0.12 mmol), Pd(OAc) (0.01 mmol), SPhos (0.01 mmol)
2
1
1
83.0 °C–183.7°C. The H-NMR (300MHz, CDCl ) conditions were
and Cs CO (0.15 mmol) in DMF (5 mL) was stirred and heated at
3
2
3
as follows: δ ppm 2.25 (s, 6H), 6.41 (d, 1H, J=5.2Hz), 7.77 (s, 2H),
.27 (d, 1 H, J = 9.3 Hz), 8.56 (dd, 1 H, J = 9.3, 2.3 Hz), 8.82 (d, 1 H,
J = 5.2 Hz), 9.42 (d, 1 H, J = 2.5 Hz), and 10.03 (s, 1 H). The 13C-NMR
75 MHz, CDCl ) conditions were as follows: δ ppm 103.4, 119.1,
100°C for 10h. After cooling, the corresponding solution was evap-
orated in vacuo. The residue was purified on silica gel column
(eluent: hexane/ethyl acetate) to obtain 5(a-d).
8
(
3
4
-(2′,6′-Dimethyl-4′-formylphenoxy)-6-(4′′-cyanophenyl)-
1
1
19.6, 123.8, 130.8, 131.0, 131.1, 132.0, 134.5, 145.4, 151.7,
aminoquinoline (5a)
54.2, 154.7, and 160.9. Finally, the HRMS (+ESI) was C18H15N O4
2
+
[
M + H] ; it requires 323.1032, but has 323.1024.
With 38 % yield (2 steps), the synthesis started with 32.4 mg
(
0.10 mmol) of 4a to obtain 15.2 mg of 5a, which consisted of yel-
4
-(2′,6′-Dimethyl-4′-cyanophenoxy)-6-nitroquinoline (4b)
low solid and mp. 276.5 °C–277.2 °C. 5a was obtained in 28.2 %
overall yield (5 steps from 1). The H-NMR (300 MHz, CDCl ) con-
1
With 90 % yield, the synthesis started with 100 mg (0.48 mmol) of
3
3
to obtain 138 mg of 4b, which consisted of white solid, and mp.
ditions were as follows: δ ppm 2.22 (s, 6 H), 6.24 (d, 1H, J=5.2Hz),
6.57 (s, 1H), 7.15–7.22 (m, 2H), 7.54–7.60 (m, 2H), 7.64 (dd, 1H,
J = 8.0, 2.6 Hz), 7.73 (s, 2 H), 8.10–8.18 (m, 2 H), 8.55 (d, 1 H,
1
2
26.0°C–226.7°C. The H-NMR (300MHz, CDCl ) conditions were
3
as follows: δ ppm 2.20 (s, 6H), 6.36 (d, 1H, J=3.9Hz), 7.55 (s, 2H),
8
3
.27 (d, 1H, J=7.0Hz), 8.57 (dd, 1H, J=7.0, 1.7Hz), 8.82 (d, 1H, J =
.9 Hz), and 8.39 (d, 1H, J = 1.8 Hz). The 13C-NMR (75 MHz, CDCl3)
J=5.2Hz), and 10.00 (s, 1H). The 13C-NMR (75MHz, CDCl ) condi-
tions were as follows: δ ppm 14.1, 14.1, 102.6, 109.0, 115.9, 121.4,
3
conditions were as follows: δ ppm 16.0, 103.2, 110.7, 118.1, 119.0,
19.5, 123.9, 131.2, 132.7, 133.3, 145.5, 151.8, 153.0, 154.7, and
125.2, 130.4, 130.9, 132.2, 133.9, 134.3, 138.8, 146.9, 149.3,
1
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
6
73

Original Article
Thieme
1
54.9, 159.0, and 191.2. Finally, the HRMS (+ESI) was C25H20N O2
dried over Na SO . The crude product was purified on a silica gel
column (eluent: hexane/ethyl acetate) to obtain 7(a-d).
3
2
4
+
[
M + H] ; it requires 394.1550, but has 394.1549.
4
-(2′,6′-Dimethyl-4′-cyanophenoxy)-6-(4′′-cyanophenyl)-
4-(2′,6′-Dimethyl-4′-formylphenoxy)-2-chloroquinoline
aminoquinoline (5b)
(7a)
With 59 % yield (2 steps), the synthesis started with 20.0 mg
With 69 % yield, the synthesis started with 1.0g (5.05 mmol) of 6
to obtain 1.1 g of 7a, which consisted of white solid and mp.
(
0.06 mmol) of 4b to obtain 14.5 mg of 5b, which consisted of yel-
1
low solid and mp. 289.5 °C–290.1 °C. 5b was obtained in 40.6 %
154.6°C–156.9°C. The H-NMR (300MHz, CDCl ) conditions were
3
1
overall yield (5 steps from 1). The H-NMR (300 MHz, CDCl ) con-
as follows: 2.23 (s, 6 H), 6.20 (s, 1 H), 7.65 (td, 1 H, J = 7.5, 1.2 Hz),
3
ditions were as follows: δ ppm 2.15 (s, 6 H), 6.20 (d, 1H, J=4.9Hz),
7.75 (s, 2 H), 7.82 (td, 1 H, 7.5, 1.2 Hz), 8.04 (dd, 1 H, 8.3, 0.9 Hz),
6
.61 (bs, 1 H), 7.18 (d, 2 H, J = 8.7 Hz), 7.56 (bs, 2 H), 7.57 (d, 2 H,
J = 2.4 Hz), 7.63 (dd, 1 H, J = 9.0, 2.4 Hz), 8.07–8.15 (m, 2 H), and
.55 (d, 1 H, J = 5.1 Hz). The 13C-NMR (75 MHz, CDCl ) conditions
and 8.4 (dd, 1 H, 8.3, 0.9 Hz). The 13C-NMR (75 MHz, CDCl ) condi-
3
tions were as follows: 16.1, 102.9, 119.5, 121.7, 126.7, 128.5,
131.5, 132.1, 134.4, 148.8, 151.3, 154.6, 161.0, and 191.2. Final-
ly, the HRMS ( + ESI) was C18H15ClNO2 [M + H] + ; it requires
312.0791, but has 312.0791.
8
3
were as follows: δ ppm 16.0, 102.5, 102.5, 108.9, 110.1, 115.8,
1
1
18.3, 119.5, 121.3, 125.1, 130.9, 132.9, 133.2, 133.9, 138.7,
46.7, 147.0, 149.5, 153.7, and 158.5. Finally, the HRMS ( + ESI)
+
4-(2′,6′-Dimethyl-4′-cyanophenoxy)-2-chloroquinoline
was C25H19N O [M + H] ; it requires 391.1553, but has 391.1550.
4
(
7b)
4
(
-(4′-formylphenoxy)-6-(4′′-cyanophenyl)-aminoquinoline
5c)
With 52 % yield (2 steps), the synthesis started with 20.9 mg
0.07 mmol) of 4c to obtain 13.4 mg of 5a, which consisted of yel-
With 62 % yield, the synthesis started with 0.30g (1.51 mmol) of 6
to obtain 0.29 g of 7b, which consisted of white solid and mp.
1
184.3°C–185.0°C. The H-NMR (300MHz, CDCl ) conditions were
3
(
as follows: 2.20 (s, 6 H), 6.2 (s, 1 H), 7.53 (s, 2 H), 7.65 (td, 1 H,
J = 9.2, 1.5 Hz), 7.83 (td, 1 H, J = 9.2, 1.5 Hz), 8.04 (dd, 1 H, J = 8.3,
1.2 Hz), and 8.38 (dd, 1 H, J = 8.3, 1.2 Hz). The 13C-NMR (75 MHz,
low solid and mp. 116.0 °C–117.0 °C. 5c was obtained in 44.2 %
overall yield (5 steps from 1). The H-NMR (400 MHz, CDCl ) con-
1
3
ditions were as follows: δ ppm 6.40 (bs, 1H), 6.75 (d, 1H, J=5.1Hz),
CDCl ) conditions were as follows: 15.9, 102.7, 110.4, 118.1, 119.3,
3
7
2
8
1
.12 (bd, 2 H, J = 8.8 Hz), 7.29 (bd, 2 H, J = 8.6 Hz), 7.51–7.57 (m,
H), 7.59 (dd, 1 H, J = 9.0, 2.6 Hz), 7.91 (d, 1 H, J = 2.5 Hz), 7.96–
.02 (m, 2H), 8.13 (d, 1H, J = 9.0 Hz), 8.69 (d, 1H, J = 5.1 Hz), and
121.6, 126.8, 128.4, 131.5, 132.7, 133.3, 148.8, 151.1, 153.2, and
+
160.7. Finally, the HRMS ( + ESI): C18H14ClN O [M + H] ; it requires
2
309.0795, but has 309.0783.
0.0 (s, 1H). The 13C-NMR (100MHz, CDCl ) conditions were as fol-
3
4
-(4′-formylphenoxy)-2-chloroquinoline (7c)
lows: δ ppm 103.2, 107.2, 108.7, 115.9, 119.4, 120.3, 122.6,
1
1
25.2, 130.9, 132.2, 133.4, 133.9, 138.8, 146.7, 149.4, 159.2,
With 67 % yield, the synthesis started with 0.30g (1.51 mmol) of 6
to obtain 0.29 g of 7c, which consisted of white solid and mp.
60.0, and 190.5. Finally, the HRMS ( + ESI) was C23H16N O2
3
+
1
[
M + H] ; it requires 366.1237, but has 366.1232.
113.9°C–114.2°C. The H-NMR (300MHz, CDCl ) conditions were
3
as follows: 6.64 (s, 1H), 7.35 (d, 2H, J=8.7Hz), 7.61 (td, 1H, J=7.8,
4
(
-(4′-cyanophenoxy)-6-(4′′-cyanophenyl)-aminoquinoline
5d)
With 63 % yield (2 steps), the synthesis started with 20.9 mg
0.07mmol) of 4d to obtain 16.4mg of 5d, which consisted of yellow
1
1
.2 Hz), 7.81 (td, 1 H, J = 7.8, 1.2 Hz), 8.04 (m, 3 H), and 8.26 (dd,
H, J = 8.4, 0.9 Hz). The 13C-NMR (75 MHz, CDCl ) conditions were
3
as follows: 106.5, 120.4, 121.0, 121.8, 126.9, 128.5, 131.6, 132.3,
134.0, 149.0, 151.0, 159.1, 161.9, and 190.5. Finally, the HRMS
(
+
solid and mp. 214.0°C–214.6°C. 5d was obtained in 47.7% overall
yield (5 steps from 1). The H-NMR (300MHz, CDCl ) conditions were
( + ESI) was C16H11ClNO [M + H] ; it requires 284.0478, but has
2
1
284.0465.
3
as follows: δ ppm 6.40 (bs, 1H), 6.73 (d, 1H, J=5.0Hz), 7.11 (bd, 2H,
J=8.64, 7.24 (bd, 2 H, J=8.70), 7.50–7.56 (m, 2 H), 7.58 (dd, 1H,
J=9.2, 2.5Hz), 7.75 (bd, 2H, J=8.7Hz), 7.87 (d, 1H, J=2.4Hz) , 8.12
4
-(4′-cyanophenoxy)-2-chloroquinoline (7d)
With 60 % yield, the synthesis started with 0.30g (1.51 mmol) of 6
to obtain 0.26 mg of 7d, which consisted of white solid and mp.
214.1°C–215.0°C. The H-NMR (300MHz, CDCl ) conditions were
(
d, 1H, J=9Hz), and 8.69 (d, 1H, J=4.98Hz). The 13C-NMR (75MHz,
1
CDCl ) conditions were as follows: δ ppm 103.3, 107.3, 108.5, 108.9,
3
3
116.0, 118.1, 119.4, 120.6, 122.6, 124.0, 125.2, 131.1, 133.9, 134.6,
as follows: 6.63 (s, 1H), 7.32 (d, 2H), 7.60 (t, 1H, J = 7.2 Hz), 7.78–
1
38.9, 146.7, 149.4, and 158.7. Finally, the HRMS ( + ESI) was
7.83 (m, 3 H), 8.02 (d, 1H, J = 8.4 Hz), and 8.22 (d, 1H, J = 8.4 Hz).
+
The 13C-NMR (75 MHz, CDCl ) conditions were as follows: 106.4,
C23H15N O [M+H] ; it requires 363.1240, but has 363.1246.
4
3
1
1
09.6, 117.9, 119.6, 120.2, 121.2, 121.6, 126.9, 128.4, 131.5,
34.4, 134.7, 148.8, 150.8, 157.6, 161.5. Finally, the HRMS (+ESI)
General procedure for the preparation of 2-chloro-4-
phenoxyquinoline 7(a-d)
+
was C16H10ClN O [M+H] ; it requires 281.0482, but has 281.0470
2
A mixture of 2,4-dichloroquinoline (6) (5.05 mmol) and hydroxyl
benzene (5.56 mmol) in DMF (15 mL) with anhydrous cesium car-
bonate (10.10 mmol) was heated in a sealed tube and stirred at
General procedure for the preparation of 2-amino-4-
oxydiarylquinoline 8(a-d)
8
0 °C for 16 h. After cooling, the mixture was poured into ice-wa-
A mixture of 2-chloro-4-phenoxyquinoline 7(a-d) (0.13 mmol),
ter, and the mixture was extracted thrice with ethyl acetate. The
combined organic layers were washed with saturated NaCl and
4-iodobenzonitrile (0.17 mmol), Pd(OAc) (0.01 mmol), SPhos
2
(0.01mmol) and Cs CO (0.20mmol) in DMF (7mL) was stirred and
2
3
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6
74

heated at 100 °C for 5 h. After cooling, the corresponding solution
was evaporated in vacuo. The residue was purified on a silica gel
column (eluent: hexane/ethyl acetate) to obtain 8(a-d).
4
-(2′,6′-Dimethyl-4′-formylphenoxy)-2-(4′′-cyanophenyl)-
aminoquinoline (8a)
With 69% yield, the synthesis started with 40.0 mg (0.13mmol) of
7
a to obtain 34.8mg of 8a, which consisted of white solid and mp.
2
91.3 °C–292.2 °C. 8a was obtained in 47.6 % overall yield (2 steps
1
from 6). The H-NMR (300MHz, CDCl ) conditions were as follows:
3
2
1
7
1
1
1
.26 (s, 6 H), 5.73 (s, 1 H), 6.95 (bs, 1 H), 7.46 (td, 1 H, J = 7.6,
.0 Hz), 7.56 (m, 2 H), 7.70 (s, 2 H), 7.74 (m, 1 H), 7.87 (m, 2 H),
.91 (bd, 1 H, J = 8.6 Hz), 8.30 (dd, 1 H, J = 8.2, 1.1 Hz), and 9.96 (s,
H). The 13C-NMR (75 MHz, CDCl ) conditions were as follows:
3
6.1, 93.1, 104.0, 117.6, 118.2, 119.5, 121.5, 123.9, 127.3, 130.9,
32.5, 133.2, 134.1, 144.6, 148.7, 153.5, 155.2, 160.7, and 191.4.
+
Finally, the HRMS ( + ESI) was C25H20N O [M + H] ; it requires
3
2
3
94.1550, but has 394.1559.
4
-(2′,6′-Dimethyl-4′-cyanophenoxy)-2-(4′′-cyanophenyl)-
aminoquinoline (8b)
With 60% yield, the synthesis started with 70.0 mg (0.23mmol) of
7
b to obtain 53.0mg of 8b, which consisted of white solid and mp.
2
56.5 °C–257.0 °C. 8b was obtained in 37.2 % overall yield (2 steps
▶
Fig. 1 Overlay of the conformations of nevirapine (NVP) (black),
1
from 6). The H-NMR (300MHz, CDCl ) conditions were as follows:
2
3
efavirenz (EFV) (dark grey), and rilpivirine (TMC278) (light grey) in
the binding pocket of HIV-1 RT.
.23 (s, 6 H), 5.81 (s, 1 H), 7.41–7.46 (m, 1 H), 7.47 (s, 2 H), 7.56
(
dd, 2H, J=7.1, 1.8Hz), 7.71 (m, 1H), 7.87–7.99 (m, 3H), and 8.27
▶
Fig. 2 Design of a disubstituted quinoline based on the structure guide using the molecular hybridization of NVP, EFV and TMC278 as the basic structure.
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
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75

Original Article
Thieme
(
dd, 1 H, J = 8.2, 1.1 Hz). The 13C-NMR (75 MHz, CDCl ) conditions
6.42 (s, 1H), 7.43 (t, 1H, J=7.1Hz), 7.50–7.59 (m, 2 H), 7.66–7.77
(m, 3 H), 7.77–7.88 (m, 1 H), 8.04–8.15 (m, 4 H), 9.80 (s, 1 H), and
10.1 (s, 1 H). The 13C-NMR (75 MHz, DMSO-d6) conditions were as
follows: 97.8, 102.0, 117.6, 117.9, 119.6, 121.2, 123.6, 126.7,
3
were as follows: 16.0, 93.3, 103.6, 109.4, 117.4, 118.2, 119.6,
1
1
21.3, 123.8, 127.2, 130.9, 133.0, 133.1, 133.2, 144.8,148.6,
53.8, 154.0, and 160.2. Finally, the HRMS ( + ESI) was C25H19N O
4
+
[
M + H] ; it requires 391.1553, but has 391.1548.
130.7, 132.1, 133.1, 133.5, 145.4, 148.1, 154.1, 159.1, 160.4 and
+
1
91.9. Finally, the HRMS ( + ESI) was C23H16N O [M + H] ; it re-
3
2
4
(
-(4′-formylphenoxy)-2-(4′′-cyanophenyl)-aminoquinoline
8c)
With 62% yield, the synthesis started with 70.0 mg (0.25mmol) of
quires 366.1237, but has 366.1229.
4
-(4′-cyanophenoxy)-2-(4′′-cyanophenyl)-aminoquinoline
(
8d)
7
c to obtain 55.9 mg of 8c, which consisted of white solid and mp.
2
67.1 °C–268.0 °C. 8c was obtained in 41.5 % overall yield (2 steps
With 59% yield, the synthesis started with 70.0 mg (0.25mmol) of
7d to obtain 53.3mg of 8d, which consisted of white solid and mp.
1
from 6). The H-NMR (300MHz, CDCl ) conditions were as follows:
3
2
78.5 °C–278.7 °C. 8d was obtained in 35.4 % overall yield (2 steps
1
from 6). The H-NMR (300MHz, CDCl ) conditions were as follows:
3
6
.59 (s, 1 H), 7.18–7.24 (m, 2 H), 7.25–7.31 (m, 2 H), 7.47–7.54
(
m, 1 H), 7.60–7.65 (m, 3 H), 7.66–7.80 (m, 3 H), and 8.08 (d, 1 H,
J = 7.9 Hz). The 13C-NMR (75 MHz, CDCl ) conditions were as fol-
3
lows: 103.9, 108.5, 118.1, 119.8, 119.9, 121.6, 125.9, 126.9,
1
28.0, 131.1, 133.2, 134.4, 147.8, 148.7, 156.0, 158.7, and 160.2.
+
Finally, the HRMS ( + ESI) was C23H15N O [M + H] ; it requires
4
3
63.1240, but has 363.1232.
HIV-1 RT inhibition assay
The inhibition assay of HIV-1 RT (wt) was performed by utilizing the
template/primer hybrid poly(A)×oligo (dT)15, digoxigenin- and bi-
otin-labeled nucleotides, an antibody to digoxigenin that was con-
jugated to peroxidase (anti-DIG-POD), and the peroxidase substrate
ABTS. The incorporation quantities of the digoxigenin- and biotin-
labeled dUTP into DNA represented the activity of HIV-1 RT activ-
ity. The HIV-RT inhibition assay was implemented using an RT assay
kit (Roche), and the procedures for assaying RT inhibition were per-
formed as described in the kit protocol [18–20]. The tested com-
pound and the three control drugs, namely, NVP, EFV, and TMC278,
were used at 1 µM concentration to determine the percentage in-
hibitory values. The reaction mixture consisted of template/prim-
er complex, 2′-deoxy-nucleotide-5′–triphosphates (dNTPs), and
RT enzyme in the lysis buffer with or without an inhibitor. After in-
cubation for 1 h at 37 °C, the reaction mixture was transferred to
streptavidin-coated microtiter plate (MTP). The biotin-labeled
dNTPs that were incorporated in the template because of the pres-
ence of RT were bound to strepavidine. The unbound dNTPs were
washed using a wash buffer, and then, antidigoxigenin-peroxidase
▶
4
Fig. 3 Conformations of rilpivirine (TMC278) (black) and
-(4′-formylphenoxy)-6-(4′′-cyanophenyl)-aminoquinoline (grey) in
the binding pocket of HIV-1 RT.
▶
Fig. 4 Synthesis of 6-amino-4-oxydiaylquinoline derivatives Reagents and conditions: a HNO3/H2SO4, −15°C to 0°C; b POCl3, cat. DMF, 100°C
sealed tube; c hydroxyl benzene (Ar OH), K CO , DMF, 100°C sealed tube; d SnCl , EtOH, sonicated at 30°C; e 4-iodobenzonitrile, Pd(OAc) , SPhos,
1
2
3
2
2
Cs CO , DMF, 100°C.
2
3
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
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76

▶
Table 1 Inhibitory activity (%) of the compounds 5(a-d), NVP, EFV, and
(anti-DIG-POD) was added to the MTP. The DIG-labeled dNTPs in-
corporated in the template was bound to the anti-DIG-POD anti-
body. The unbound anti-DIG-POD was also washed elaborately for
a
TMC278 at 1µM against HIV-1 RT.
Com-
pounds
Ar1
inhibition (%)
against HIV-1
RTb
5
times by using a wash buffer. Finally, the peroxide substrate
(ABST) was added to the MTP. A colored reaction product emerged
during the cleavage of the substrate catalyzed by peroxide enzyme.
The absorbance of the sample was measured at OD405 (reference
wavelength: approx. 490nm) by using the MTP (ELISA) reader. The
percentage inhibitory activity of RT inhibitors was calculated by
comparing with that of a sample without an inhibitor. The result-
ing color intensity was directly proportional to the RT activity. The
percentage inhibitory values were calculated by the following for-
mula: Inhibition % = {1−([O.D. value with RT and inhibitor−O.D.
value without RT and inhibitors] / [O.D. value without inhibitors
5
5
5
5
a
b
c
2,6-dimethyl-4-formylphenoxy
2,6-dimethyl-4-cyanophenoxy
4-formylphenoxy
28.73 ± 2.01
25.46 ± 1.75
34.91 ± 1.84
32.43 ± 1.50
53.39 ± 1.70
96.69 ± 0.85
97.06 ± 0.98
d
4-cyanophenoxy
NVP
EFV
TMC278
a
The RT kit was commercially available and supplied by Roche, and the
b
data were obtained by standard ELISA. Results were expressed as
(
with RT)−O.D. value without RT and inhibitors])}× 100.
mean ± standard error of Inhibitory activity ( %).
Cytotoxic activity
▶
Table 2 The binding energy and the interaction between each of the
Cell line were seeded in a 96-well microplate (Costar No. 3599),
6-amino-4-oxydiarylquinoline derivatives 5(a-d), NVP, EFV and TMC278
3
4
1
00μL/well, at a density of 5 × 10 to 2×10 cells/well. Background
and the HIV-1 RT from molecular docking.
control wells contained the same volume as the complete culture
medium. The microplate was incubated for 24 h at 37 °C, with 5 %
Compound
Binding energy (kcal/mol)a
NOCb
17 ± 3
5
5
5
5
a
b
c
− 12.01 ± 0.02
− 12.16 ± 0.06
− 11.80 ± 0.01
− 12.21 ± 0.01
− 7.91 ± 0.01
− 9.25 ± 0.01
− 12.53 ± 0.01
CO , and 95 % humidity (Shellab). Samples at various concentra-
2
20 ± 5
106 ± 6
110 ± 3
125 ± 3
130 ± 5
133 ± 5
tions were added to the microplate, which was incubated for an-
other 48 h. Cell viability was determined by staining with the MTT
assay [3(4,5-dimethylthiazol-2-yl)-2–5-diphenyl tetrazolium bro-
mide (Sigma-Aldrich) [21–24]. The reagent was dissolved in phos-
phate buffered saline at 5 mg/mL, and filtered to sterilize and re-
move a small amount of insoluble residue present in some batches
of MTT. MTT solution (10µL/100µL medium) was added to all wells
d
NVP
EFV
TMC278
a
Results were expressed as mean ± standard error of binding energy
of each assay, and plates were incubated at 37° C, 5% CO , and 95%
b
2
(
kcal/mol). Results were expressed as mean ± standard error of
humidity for 2–4 h. Subsequently, DMSO (100 µL, Merck, Germa-
ny) was added to dissolve the resulting formazan by sonication. The
number of ligand conformations
▶
Fig. 5 Binding interactions between 6-amino-4-oxydiaylquinoline derivatives 5(a-d) and HIV-1 RT surrounding the designed compounds (dis-
tance in°A).
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Original Article
Thieme
Results and Discussion
plates were read on a microplate reader (Molecular Devices, CA,
USA) by using a test wavelength of 550nm and a reference wave-
length of 650 nm. The XTT assay for suspension cells was acute
lymphoblastic leukemia (MOLT-3) [23]. Plates were incubated for
Molecular docking was used to predict the binding site, the bind-
ing energy and the interaction between assigned compounds and
the HIV-1 RT to propose the potential of the assigned molecules in
evaluating the new anti-HIV-1 RT agent. In our molecular docking
approach, the new target molecule was obtained from the molec-
ular hybridization for NVP, EFV and TMC278 with HIV-1 RT to observe
the conformation and interaction between these compounds and
the HIV-1 RT binding site. The combination and the overlaying of
NVP, EFV and TMC278 in the binding site of HIV-1 RT allowed the
development of a new hybrid structure, which was based on the sub-
units for each of the NVP, EFV, and TMC278, as shown in ▶Fig. 1.
Resulting from the molecular hybridization, 4,6-disubstituted qui-
noline was designed as a core structure that corresponded with the
square frame as in ▶Fig. 2. The 4,6-disubstituted quinoline may
maintain the characteristics of the original base structure, espe-
cially in the EFV structure. The substituents in the assigned mole-
cule were selected from those of TMC125 and TMC278 as an amino
aryl and oxy aryl groups, respectively.
4
0
h after the addition of 50 µL of mixture of 1 mg/mL (5 mL) and
.383mg/mL (100µL) of phenazine methosulfate. The absorbance
of the formed orange formazan compounds was measured at the
wavelengths of 492 and 690 nm. IC50 values were determined as
the drug and sample concentrations at 50 % inhibition of the cell
growth.
The molecular docking was applied to stimulate the conforma-
tions of the assigned molecules, that is, 4-(4'-formylphenoxy)-6-(4"-
cyanophenyl)-aminoquinoline. The optimized molecule was ar-
ranged in a similar pattern with the TMC278 at the binding site of
HIV-1 RT (▶Fig. 3). Therefore, 6-amino-2-oxydiarylquinoline deriv-
atives were selected and synthesized to observe the anti-RT activity.
6
-Amino-4-oxydiarylquinoline 5(a-d) was synthesized as shown
in ▶Fig. 4. The 6-nitroquinolin-4-ol (2) was obtained from the ni-
tration of 4-hydroxy-6-nitroquinoline (1) using nitric and sulfuric
acids. The 6-nitroquinolin-4-ol (2) was subjected with phosphorus
oxychloride to afford the 4-chloro-6-nitroquinoline (3). The 6-ami-
no-4-oxydiarylquinoline 5(a-d) was obtained from 4-chloro-6-ni-
troquinoline (3) by replacing the chlorine with hydroxyl benzene
under basic conditions. 4-Oxyaryl-6-nitroquinoline (4) was treated
with a reducing agent to generate aryl amine, followed by the cross
▶
Fig. 6 Conformations of 6-amino-4-oxydiaylquinoline derivatives
5
(a-b) (black) and 5(c-d) (grey) in the binding pocket of HIV-1 RT.
▶
Fig. 7 Binding interactions between 2-amino-4-oxydiaylquinoline derivatives 8(a-d) and HIV-1 RT surrounding the designed compounds
(
distance in°A).
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
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78

coupling reaction with 4-iodobenzonitrile using Pd(OAc) to obtain
centration for each of the synthesized compounds by using the RT
assay and colorimetric method. The result was demonstrated in
▶Table 1. The inhibitory activity (%) for each compound was lower
than that of NVP, EFV, and TMC278, which were used as anti-RT
drugs. The molecular docking was used to expand and support the
experimental results; for instance, the anti-RT activity considered
the designed molecules. The binding energy, the number of ligand
conformation and the interaction for 6-amino-4-oxydiarylquino-
line derivatives 5(a-d) in the HIV-1 RT was calculated, as shown in
2
6
-amino-4-oxydiarylquinoline 5(a-d).
The 6-amino-4-oxydiarylquinoline derivatives 5(a-d) were test-
ed for percentage inhibitory activity against HIV-1 RT at 1 µM con-
▶
Table 2 and ▶Fig. 6.
The binding energies of 5(a-d) are relatively similar and lower
than those of anti-RT drugs, thereby possibly causing a higher
stability for the interaction. In the molecular docking, we found
that 5(a-d) were bound to the same location in the HIV-1 RT. From
▶
Fig. 5, the aromatic ring in the core structure and in the substit-
uent at 6-position of quinoline 5(a-b) exhibited the hydrophobic
interactions with the indole ring of Trp229 and with the aromatic
ring of Tyr188 and Tyr181 by using π-π stacking interaction in the
HIV-1 RT. Moreover, the formyl group and the cyano group in the
substituent at 4-position of quinoline 5(a, b) displayed the hydro-
gen bond interactions with the hydroxyl group of Try318 and with
the amino group of Lys103 in the HIV-1 RT, respectively. Whereas,
the NH-amino group and the cyano group in the substituent at
6
-position of quinoline 5(c-d) demonstrated the hydrogen bond-
ing interactions with the carboxyl group on Lys101 and with the
carboxylic acid group on His235. The hydrogen bonding interac-
tion between 5(c-d) and HIV-1 RT displayed shorter, and greater
than that of 5(a-b), moreover, the number of ligand conformation
of 5(c-d) exhibited five times of 5(a-b). Thus, the different confor-
mations and interaction for each of the compounds had various ef-
fects on the anti-RT activity. Additionally, 5(c-d) exhibited an anti-
RT activity that was higher than that of 5(a-b), thereby correspond-
ing to the interaction information from the molecular docking.
According to many studies, the hydrogen bonding interaction be-
tween the active compounds and the Lys101 residue in the HIV-1
RT is often found in NNRTIs [25]. From those results, some com-
pounds demonstrated different conformations and interaction, as
shown in ▶Fig. 5 and 6. The 6-amino-4-oxydiarylquinoline deriva-
tives may not be suitable for the interaction and binding with HIV-1
RT because the conformations were not arranged in the same
pattern, resulting in difficulty in studying and designing the target
molecules. Therefore, 2-amino-4-oxydiarylquinoline derivatives
▶
Fig. 8 Conformations of 2-amino-4-oxydiaylquinoline derivatives
8
(a-d) in the binding pocket of HIV-1 RT.
▶
Table 3 Binding energy and the interaction between 2-amino-4-oxydia-
rylquinoline derivatives 8(a-d) and HIV-1 RT from molecular docking.
Binding energy (kcal/mol)a
NOCb
Compound
8
8
8
8
a
a
b
c
− 13.18 ± 0.01
− 13.69 ± 0.02
− 12.06 ± 0.01
− 12.56 ± 0.01
77 ± 8
89 ± 6
66 ± 4
92 ± 4
d
8(a-d) were assigned and docked instead of the 6-amino-4-oxydiar-
Results were expressed as mean ± standard error of binding energy
b
ylquinoline 5(a-d) to observe the potent HIV-1 NNRTIs.
(
kcal/mol). Results were expressed as mean ± standard error of
number of ligand conformations
Molecular docking results of 2-amino-4-oxydiarylquinoline qui-
noline derivatives 8(a-d) showed that all compounds bound to
▶
Fig. 9 Synthesis of 2-amino-4-oxydiaylquinoline derivatives Reagents and conditions: a hydroxyl benzene (Ar1OH), Cs2CO3, DMF, 80°C sealed
tube; b 4-iodobenzonitrile, Pd(OAc) , SPhos, Cs CO , DMF, 100°C.
2
2
3
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
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79

Original Article
Thieme
HIV-1 RT with an identical location using 3 hydrogen bonding. First
bonding structured from the nitrogen atom in the quinoline core-
structure with the hydrogen atom of the amino group on Lys101.
Second bonding created from the hydrogen atom in the amino sub-
stituent at 2-position of quinoline with the oxygen atom of the car-
boxyl group on Lys101. Third bonding displayed from the nitrogen
atom in the cyano group at the side chain of substituent in 2-posi-
tion of quinoline with the hydrogen atom of the carboxylic acid
group on His235 as shown in ▶Fig. 7 and 8. The aldehyde group
on 8a and 8c and the cyano group on 8b and 8d indicated different
binding energies, as demonstrated in ▶Table 3. Comparing the
substituent effect between the cyano group (8c) and the aldehyde
group (8d) on the binding site with HIV-1 RT, we found that 8d,
which was the derivative with the cyano group, indicated consid-
erably greater binding energy values than 8c, which was the deriv-
ative with the aldehyde group. Meanwhile, the addition of methyl
group to the aromatic cyano derivative (8b) led to an improved
binding corresponded with the binding energy obtained from the
addition of methyl group to the aromatic aldehyde derivative (8a).
Thus, 2,4-disubstitued quinoline as 2-amino-4-oxydiarylquinoline
derivatives 8(a-d) may provide a stronger inhibiting activity for
HIV-1 RT than 6-amino-4-oxydiarylquinoline derivatives 5(a-d).
The 2-amino-4-oxydiarylquinoline derivatives 8(a-d) were syn-
thesized from 2,4-dichloroquinoline (6) by using nucleophilic sub-
stitution reaction under basic conditions, followed by cross-cou-
pling reaction as shown in the ▶Fig. 9. The 2-amino-4-oxydiar-
ylquinoline 8(a-d) was subjected to testing the percentage
inhibitory activity against HIV-1 RT, with the RT assay using the col-
orimetric method; the result is presented in ▶Table 4.
The 2-amino-4-oxydiarylquinoline 8(a) demonstrated a higher
inhibitory activity against HIV-1 RT than 8(c). The result was cor-
responded with the information from the molecular docking and
the interaction with the HIV-1 RT. The binding energy of 8(a) was
exhibited lower than 8(c), and, the NOC value of 8(a) was showed
higher than 8(c). Additionally, the hydrophobic interaction be-
tween the aromatic substituent at 4-position of quinoline and the
aromatic ring of Tyr181 was found only in 8(a). Moreover, the
2-amino-4-oxydiarylquinoline 8(d) exhibited a stronger inhibitory
activity against HIV-1 RT than 8(b), thereby corresponding to the
information from the interaction with the HIV-1 RT. 8(d) displayed
the hydrophobic interaction between the aromatic substituent at
4-position of quinoline and the indole ring of Trp229, whereas 8(b)
was not. The molecular docking results of 2-amino-4-oxydiarylqui-
noline derivatives 8(a-d) showed that 8(d) provided the best value
of the number of ligand conformation. Those results implied that
8(d) have a good inhibitory activity compared with other designed
compounds. However, the activity may not be corresponded to the
molecular docking in some cases, because the molecular docking
was used to predict the binding side, binding energy, and the in-
teraction between designed compounds and the receptors in the
computer, thereby possibly causing a different result in the biolog-
ical testing. Besides the inhibition of HIV-1 RT activity, 2-amino-
4
-oxydiaylquinoline derivatives 8(a-d) and three common drugs
▶
Table 4 Inhibitory activity (%) of the compounds 8(a-d) against HIV-1 RT.a
(NVP, EFV, and TMC278) were evaluated for cytotoxicity against
various cancer cell lines, as shown in ▶Table 5. In the literatures,
the NNRTIs, namely, NVP, EFV, and TMC278, have been reportedly
toxic against a wide range of cancer cells in vitro [26–33] and only
have been reported a minor toxicity against normal tissue cells [26].
The toxicity of NNRTIs against cancer cells promoted the idea to
use these drugs in patients infected with HIV-1 to prevent or even
treat cancer. In a cytotoxic assay, 8b, 8d and NVP demonstrated a
weak activity against the evaluated cancer cell lines and normal cell
lines (MRC-5). 8a, 8c, EFV, and TMC278 exhibited a strong activity
against acute lymphoblastic leukemia (MOLT-3) cells (IC50 values
Compounds
Ar1
inhibition (%)
against HIV-1 RTb
8
8
8
8
a
a
b
c
2,6-dimethyl-4-formylphenoxy
2,6-dimethyl-4-cyanophenoxy
4-formylphenoxy
34.02 ± 2.37
27.85 ± 1.64
19.40 ± 1.62
39.71 ± 1.67
d
4-cyanophenoxy
The RT kit was commercially available and supplied by Roche, and
b
data were obtained by standard ELISA. Results were expressed as
mean ± standard error of Inhibitory activity ( %).
▶
Table 5 In vitro cytotoxic activity of the compounds 8(a-d) against human cancer cell lines and normal cell line.
a
b
Compounds
Cell lines [IC50 (µg/mL)]
HepG2
MOLT-3
HuCCA-1
A549
MRC-5
8
8
8
8
a
b
c
25.95 ± 2.40
%C = 38.25c
26.76 ± 2.80
%C = 44.80c
%C = 15.37c
27.33 ± 1.23
32.02 ± 4.19
0.23 ± 0.02
21.14 ± 0.70
4.63 ± 0.62
19.04 ± 16.02
5.71 ± 1.33
%C = 6.00c
%C = 29.00c
7.76 ± 0.37
1.57 ± 0.20
–
11.58 ± 0.768
%C = 3.00c
10.84 ± 0.735
%C = 23.00c
%C = 11.00c
18.83 ± 0.756
22.46 ± 0.621
0.89 ± 0.035
–
20.13 ± 1.195
%C = 21.00c
44.46 ± 1.647
%C = 26.00c
%C = 18.00c
19.00 ± 0.565
%C = 42.00c
0.19 ± 0.007
–
27.46 ± 1.68
%C = 7.36c
39.41 ± 1.73
%C = 12.03c
%C = 1.02c
30.17 ± 1.90
%C = 14.42c
1.42 ± 0.04
–
d
NVP
EFV
TMC278
Doxorubicin
Etoposide
a
0.048 ± 0.007
HepG2 (Hepatocarcinoma), MOLT-3 (Acute lymphoblastic leukemia), HuCCA-1 (Cholangiocarcinoma), A549 (Lung carcinoma), MRC-5 (normal
b
embryonic lung cell). Results were expressed as mean ± standard error of inhibition perceptual for all cell lines. Doxorubicin and etoposide were used
as the positive control. Experiments were performed in triplicate. Inactive (IC50 ≥ 50 µg/mL); reported in percentage cytotoxicity at the substance
c
concentration of 50µg/mL.
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
6
80

▶
Fig. 10 Binding interactions between nevirapine (NVP), efavirenz (EFV), and rilpivirine (TMC278) and HIV-1 RT surrounding the designed com-
pounds (distance in°A).
of 4.63 ± 0.62, 5.71 ± 1.33, 7.76 ± 0.37 and 1.57 ± 0.20 µg/mL, re-
spectively). 8a, 8c, EFV, and TMC278 had substantial cytotoxicity
in the evaluated cancer cell line whereas TMC278 had moderate
cytotoxicity against normal embryonic lung (MRC-5) cells. Howev-
er, 8d had high percentage inhibitory activity against HIV-1 RT com-
pared with other synthesized compounds and had very low cyto-
toxic against normal embryonic lung (MRC-5) cells compared with
EFV and TMC278.
energy. It means that the physicochemical properties and the phar-
macological properties of 2-amino-4-oxydiarylquinoline deriva-
tives may be improved using the Lewis base as nitrogen atom to
increase the interaction such as the number of hydrogen bonding
resulting to strong binding and good solubility. Further improve
the structure and the activity for HIV-1 RT inhibitor and also the ef-
ficiency synthetic method of these 2-amino-4-oxydiarylquinoline
derivatives are underway in our laboratory and will be reported in
the near future.
Finally, in this study, we also reported the binding energies,
number of conformations, interactions and characterization of new
HIV-1 RT inhibitors that evaluated the possibility to use quinoline
as a structure basis. The molecular docking results of efavirenz,
nevirapine and rilpivirine demonstrated that all compounds bound
to HIV-1 RT on Lys101 using hydrogen bonding except for the efa-
virenz, that also bound on Lys103 using hydrogen bonding. π-π
stacking between HIV-1 RT in the amino acid residue named of
Trp229 and Tyr181 and rilpivirine were also found in the interac-
tion information from the molecular docking calculation, as shown
in ▶Fig. 10. The interactions between rilpivirine or the designed
compound 8a and 8d and HIV-1 RT were similar to those of hydro-
gen bonding and π-π stacking, with similar distance for each of
amino acid residue. However, 8a and 8d exhibited more hydrogen
bonding interaction which was displayed between His235, amino
acid residue in HIV-1 RT, with the cyano group, side chain of sub-
stituent in 2-position of quinoline. Compound 8a and 8d have pos-
sessed percentage inhibitory against HIV-1 RT at 34.0 and 39.7%,
respectively. Those values are less than that of the commercial
drugs which was used for the molecular hybridization such as NVP,
EFV and TMC278. Based on the structure of 8a, we found that add-
ing the nitrogen atom into the quinoline core-structure such as
Conclusions
2-amino-4-oxydiarylquinoline derivatives could inhibit HIV-1 RT at
a stronger manner than 6-amino-4-oxydiarylquinoline, except for
the 4-formylphenoxy substituent. According to the molecular dock-
ing results, the 2-amino-4-oxydiarylquinoline was more suitable
for binding with the HIV-1 RT and more possible to study the sub-
stituent effect than the 6-amino-4-oxydiarylquinoline, because the
former bound with the HIV-1 RT with an identical location. The per-
centage inhibitory activity against HIV-1 RT was also influenced by
the substituent in the aromatic side chain of oxy-amino-aryl-quin-
oline. Furthermore, 4-(2′,6′-Dimethyl-4′-formylphenoxy)-2-(4′′-
cyanophenyl)-aminoquinoline (8a) and 4-(4′-cyanophenoxy)-2-
(4′′-cyanophenyl)-aminoquinoline (8d) could inhibit HIV-1 RT at
the level similar with that of NVP. In addition, 8a exhibited a strong
cytotoxic activity against MOLT-3 cells, similar with those of EFV
and TMC278. 8d demonstrated very low cytotoxicity against nor-
mal embryonic lung (MRC-5) cells. Therefore, 2-amino-4-oxydiar-
ylquinoline derivatives can be further improved for the develop-
ment of HIV-1 RT inhibitors, thereby providing the opportunity to
treat HIV and cancer with one drug.
1
,6-naphthyridine, together with the aromatic substituent at 2-po-
sition of quinoline demonstrated high NOC value and low biding
Makarasen A et al. Anti-HIV of Amino-oxy-Diarylquinoline … Drug Res 2019; 69: 671–682
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81

Original Article
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The authors are grateful for the financial support given by Chulab-
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GmbH, Roche, Applied Science, Sandhofer Strasse. 116: D-68305
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