Synthesis of quinolin-2-one alkaloid derivatives and their inhibitory activities against HIV-1 reverse transcriptase

Article abstract of DOI:10.3390/molecules16097649

Based on an established common pharmacophore of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNTTIs), a series of quinolin-2-one derivatives were synthesized and assayed for their in vitro activities against HIV-1 reverse transcriptase (RT) for the first time. Some of the tested compounds were active against HIV-1 RT. Compounds 4a2 and 4d2 showed inhibitory activities with IC50 values of 0.21 and 0.15 μM, respectively, with a mode of interaction with RT residues of the allosteric pocket similar to that of efavirenz.

Full text of DOI:10.3390/molecules16097649

Molecules 2011, 16, 7649-7661; doi:10.3390/molecules16097649
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Quinolin-2-one Alkaloid Derivatives and Their
Inhibitory Activities against HIV-1 Reverse Transcriptase
Pi Cheng 1,3,*, Qiong Gu 2, Wei Liu 1, Jian-Feng Zou 1, Yang-Yong Ou 3, Zhong-Yong Luo 3 and
Jian-Guo Zeng 1,3,
*
1
National Research Center of Engineering Technology For Utilization of Functional Ingredients
From Botanicals, College of Horticulture and Landscape Architecture, Hunan Agricultural
University, Changsha 410128, Hunan, China; E-Mails: lwhncs618@163.com (W.L.);
zoujf2005@163.com (J.-F.Z.)
2
3
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, Guangdong,
China; E-Mail: guqiong@sysu.edu.cn (Q.G.)
Hunan Engineering Research Center of Botanical Extract, Changsha410128, Hunan, China;
E-Mails: oyy713@163.com (Y.-Y.O.); zhongyongluo@hotmail.com (Z.-Y.L.)
* Authors to whom correspondence should be addressed; E-Mails: picheng55@126.com (P.C.);
ginkgo@world-way.net (J.-G.Z.); Tel.: +86-731-8468-6560; Fax: +86-731-8468-6560.
Received: 16 June 2011; in revised form: 4 August 2011 / Accepted: 20 August 2011 /
Published: 7 September 2011
Abstract: Based on an established common pharmacophore of HIV-1 non-nucleoside
reverse transcriptase inhibitors (NNTTIs), a series of quinolin-2-one derivatives were
synthesized and assayed for their in vitro activities against HIV-1 reverse transcriptase (RT)
for the first time. Some of the tested compounds were active against HIV-1 RT.
Compounds 4a2 and 4d2 showed inhibitory activities with IC50 values of 0.21 and 0.15
μM, respectively, with a mode of interaction with RT residues of the allosteric pocket
similar to that of efavirenz.
Keywords: quinolin-2-one; alkaloid; synthesis; HIV-1 RT; activity

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1. Introduction
AIDS (Aquired Immune Deficiency Syndrome), caused by human immunodeficiency virus (HIV),
a RNA dependent retrovirus, remains one of the major causes of death in the World. HIV-1 reverse
transcriptase (RT) is one of the enzymes crucial for the HIV virus replication cycle. Upon entering a
host cell, the single stranded viral RNA is converted to double-stranded DNA catalyzed by RT and
then the virus DNA inserts into the genome of the host cell. Inhibition of reverse transcriptase (RT),
the HIV-encoded polymerase which directs both RNA and DNA synthesis, has been proven to be one
of the most effective ways to block viral multiplication [1-5].
Based on the ready availability of X-ray crystal structures of inhibitor-HIV RT complexes, Freeman
and colleagues have identified the common pharmacophore N-(4-chlorophenyl)acetamide (1) from an
array of known non-nucleoside reverse transcriptase inhibitors [6] such as efavirenz, DPC961,
HBY097 and the newly reported benzoimidazol-2-one [7] and dihydroquinoxalin-2-one [8] NNTRIs
(Figure 1).
Figure 1. NNRTI pharmacophore N-(4-chlorophenyl)acetamide (1) and related anti-HIV-1
RT active compounds.
Me
O
O
Me
Me
S
F3C
F3C
MeO
N
Cl
Cl
NH
O
N
H
S
N
H
O
N
H
O
HBY097
Efavirenz
DPC961
Cl
SO2
Cl
Cl
Cl
N
Cl
N
O
N
O
N
H
O
N
H
H
1
N-(4-chlorophenyl)acetamide
benzoimidazol-2-one
dihydroquinoxalin-2-one
A series of substituted 2-quinolones (2, Figure 2) were synthesized and evaluated as HIV-1
inhibitors in Freeman’s research. As to natural products, a similar quinolin-2-one alkaloid 3 isolated
from Euodia roxburghiana reported by McCormick also showed inhibitory activity with an IC50 of 8
μM in an HIV-1 reverse transcriptase (RT) assay [9].
Figure 2. quinolin-2-one NNRTIs and newly designed derivatives.
R
R
O
O
Y
F
R
O
X
Cl
N
H
N
H
O
N
H
O
N
H
O
X = H or Cl
Y =S, SO, SO2 or O
5
3
2
4

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Based on the pharmacophore established by Freeman, we designed and synthesized a series of
4-substituted quinolin-2-one alkaloids 4 and 5, as shown in Figure 2. We envisaged that replacement of
the cyclopropylethynyloxy moiety with a hydrophobic aromatic ring would retain the inhibitory
activity against HIV-1 RT [4,10-13]. In compounds 4, the group Y could be oxygen, sulfur, sulfinyl or
sulfonyl units, which were flexible linkers to connect the hydrophobic phenyl group with the quinolin-
2-one scaffold. To identify the importance of the linker, compounds 5 were synthesized with an
aromatic ring directly connected to quinoline-2-one scaffold. In this paper, we report the synthesis of
compounds 4 and 5 and the evaluation of the synthesized compounds for their in vitro inhibitory
activities against HIV-1 RT for the first time.
2. Results and Discussion
2.1. Chemistry
The syntheses of compounds 4 is summarized in Schemes 1 and 2, respectively. 3-Hydroxy-
quinolin-2-one (8, Scheme 1) has been the subject of intensive synthetic studies for a long time.
Among the synthetic strategies used is the Friedel-Crafts reaction [6]. Freeman reported that a
treatment with aniline or 4-chloroaniline 6 and excess of diethyl malonate in diphenyl ether at 250 °C
for 24 h afforded cyclized compound 8 directly. But in our study, target compound 8 was not obtained
under these conditions and only some amide intermediates could be detected by HPLC-ESIMS. We thus
irradiated a mixture of aniline and diethylmalonate (2:1 molar ratio) under microwaves and prepared
corresponding dimalonamide 7 in good yield. Heating N,N’-di(4-chlorophenyl)malonamide in
polyphosphoric acid (PPA) at 140–150 °C afforded the 4-hydroxy-2-quinolone 8 (Scheme 1) [14].
Refluxing compound 8 in POCl3 provided 2,4,6-trichloroquinoline 9 in good yield. A phenylthio group
was then installed on the quinoline ring to afforded compounds 10, which were converted to target
compounds 4a through a microwave assisted hydrolysis in mixture of TFA and HCl [15]. Compounds
4a were oxidized to target compounds 4b as racemates, and these were then converted to compounds
4c by reaction with another 1.1 equiv. of 3-chlorobenzoperoxoic acid (m-CPBA).
Scheme 1. Synthesis of target compounds 4a–4c.
Reagents and conditions: (a) diethylmalonate, MW (320 W), 10 min; (b) PPA, 140–150 °C, 6 h;
(c) POCl3, reflux, 3 h; (d) benzenethiol, 1.0 eq, Et3N, DMF, 0 °C→rt, overnight; (e) TFA, HCl,
MW (320 W), 20 min; (f) m-CPBA, 1.1equiv., CH2Cl2, MeOH, rt; (g) m-CPBA, 1.1equiv., CH2Cl2,
MeOH, rt.

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The synthesis of compounds 4d shared the same synthetic precursor 2,4,6-trichloroquinoline 9
(Scheme 2). Through treatment of compound 9a with various phenols followed by hydrolysis in acid
mixtures, compounds 4d were synthesized in moderate yields using the conditions described above
without need for further optimization.
Scheme 2. Synthesis of target compounds 4d.
Reagents and conditions: (h) phenol, 1.0 eq. Et3N, DMF, 0 °C→rt, overnight; (i) TFA, HCl, MW
(320 W), 20 min.
The synthetic route of target compounds 5 was summarized in Scheme 3. The synthesis of 5 by
various methods has been reviewed. The majority of compounds 13 were prepared by the reaction
of aryl esters with ortho-lithiated Boc-protected 4-chloroaniline via the formation of the dianion
species with t-BuLi (2.2 equiv) followed by deprotection of the Boc group (Scheme 3) [16,17]. A
tandem amidation/Knoevengel condensation of compounds 13 with ethyl acetate gave compounds 5 in
good yields.
Scheme 3. Synthesis of target compounds 5.
Reagents and conditions: (j) (Boc)2O, DIEA, DCM; (k) −78 °C, t-BuLi, then add R1-PhCOOMe at
−40 °C and warmed up to 0 °C, then 3 N HCl, EtOH, reflux, in three steps; (l) 0 °C, LiHMDS,
EtOAc.
2.2. Enzymatic Activities
Compounds 4a–4d were tested in RT inhibition assays and proved to be active as inhibitors of
HIV-1 RT (Table 1). The enzymatic data highlighted that derivative 4a2 (IC50 = 0.21 μM) and 4d2
(IC50 = 0.15 μM) turned out to be more active than 4a3 (IC50 = 13 μM), respectively, and suggested
that the presence of a chlorine substituent at C-6 may lead to an increase of potency and influence the
affinity to the enzyme. This finding is in agreement with the N-(4-chlorophenyl)acetamide
pharmacophore established by Freeman, and new type of anti-HIV-1 RT compounds could be
designed based on the pharmacophore model.

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Table 1. HIV-1 RT inhibitory activities of compounds 4–5. a
Compounds
4a1
X
Y
S
S
R
H
IC50 (μM) b,c
5.6
Cl
Cl
H
4a2
4a3
4b1
4b2
4c1
3,5-CH3
3,5-CH3
H
3,5-CH3
H
3,5-CH3
H
3,5-CH3
H
0.21
18
8.9
2.6
49
10
3.0
0.15
>100
>100
>100
S
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
SO
SO
SO2
SO2
O
O
/
/
/
4c2
4d1
4d2
5a
5b
5c
2-F
2-Cl
a All values are the mean of two independent experiments; b The inhibition of recombinant
HIV-1 RT activity was performed with a commercially available ELISA kit (Roche)
according to the instructions of the manufacturer. IC50 = effective concentration that
c
inhibits 50% of HIV-1 RT; Efavirenz: an HIV-1 RT inhibitor used as positive control.
IC50 = 6 nM.
In addition, the type of linker which connected the aromatic ring with the quinoline scaffold
might be crucial for potent anti-HIV-1 RT activity in the title compounds. Comparing compounds 4a2
(IC50 = 0.21 μM), 4b2 (IC50 = 2.6 μM) and 4c2 (IC50 = 10 μM), a decrease of activity against RT could
be observed as the degree of oxidation of the sulfur atom increased. The role of the substituents of the
phenyl ring on the biological activities was also investigated, as shown in Table 1. In compounds 4a1
(IC50 = 5.6 μM) and 4a2 (IC50 = 0.21 μM), two hydrophobic methyl group on the phenyl ring led to
potent enzyme activity, and may contribute to the ability to bind with the hydrophobic site of RT. In
contrast, compounds 5a–5c exhibited obviously decreased activities towards the enzyme. We envisagd
that hydrophobic interaction ability was influenced by the rigid conjugation system between the phenyl
ring and the quinolin-2-one scaffold.
2.3. Docking Studies
To investigate the binding mode of the synthesized quinolin-2-ones, computational modeling was
performed using CDOCK. The CHARMM force field was used for the energy minimizations in the
docking process. The coordinates of the RT-wfavirenz complex (PDB code: 1FK9) [12,18] were
downloaded, and the molecular structure of the most potent compounds 4a2 and 4d2 were docked into
its active site. The best docked conformation of compounds 4a2 and 4d2 were shown in Figure 3,
together with the experimental position of efavirenz and the 3D common-feature pharmacophore
model. Compounds 4a2 and 4d2 interact with RT residues of the allosteric pocket in a fashion similar
to known NNRTIs. The p-chloroaniline moiety has a hydrophobic contact with the surrounding
residues such as Tyr318, Pro236, Leu234 and Val106. The carboxamide group of the molecule
interacts with the main-chain of Lys101 by hydrogen bond interactions. The 3,5-dimethylphenyl
fragment occupies a hydrophobic pocket consisting of Pro95, Tyr181, Tyr188 and Trp229, which is

Molecules 2011, 16
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occupied by the cyclopropyl-propynyl of efavirenz in the complex. One of the two methyl groups on
the benzene ring adopts a similar orientation to the cyclopropyl group of efavirenz.
Figure 3. CDOCK-modeled binding mode of 4a2 (left) and 4d2 (right) in comparison with
the crystal structure (1FK9 in PDB) of efavirenz (orange colored carbon atoms). The key
hydrogen bond is illustrated with green lines.
3. Experimental
3.1. Chemistry
3.1.1. Instruments and Materials
Column chromatography silica gel (200-300 mesh) and TLC plate (Qingdao Meijin Chemical Inc.;
1
Qingdao; China); MS data were obtained on an AutoSpec-3000 or Agilent HPLC-ESIMS; H-NMR
spectra were recorded on Bruker 300M or DRX-500 spectrometers and chemical shifts were given in δ
with TMS as an internal reference. All the reagents are commercial available. Tetrahydrofuran (THF)
was refluxed over Na before use.
3.1.2. General Procedure for Preparation of Compounds 4
3.1.2.1. Synthesis of Intermediates 7
Aniline or 4-chloroaniline (100 mmol), and diethyl malonate (50 mmol) were properly mixed in a
250 mL beaker. The obtained mixture was irradiated in a microwave oven at the power of 320 W for
5 min. After irradiation, the crude product was recrystallised in ethanol to afford the desired product.
bis(4-Chlorophenyl)malonamide (7a): yield 95%, white solid, ESIMS: m/z: 323 (M+H)+
Diphenylmalonamide (7b): yield 91%, white solid, ESIMS: m/z: 255 (M+H)+

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3.1.2.2. Synthesis of Intermediates 8
A mixture of 7a or 7b (2.0 mmol) with 5–6 times by weight polyphosphoric acid were stirred in an
oil bath at 140–150 °C for 6 h. Then the mixture was cooled, diluted with water and the resultant gum
solidified by standing over night. The solid was filtered and dried in air. It was recrystallised from
ethanol to afford corresponding hydroxyquinolones as creamy crystals.
6-Chloro-4-hydroxyquinolin-2(1H)-one (8a): white solid, yield: 82%, 1H-NMR (300 MHz,
DMSO-d6, δ ppm): 5.80 (s, 1H), 7.68 (s, 1H), 7.27 (dd, 1H, J = 8.6, 4.8 Hz), 7.53 (d, 1H, J = 8.6 Hz),
11.35 (brs, 1H, NH).
1
4-Hydroxyquinolin-2(1H)-one (8b): white solid, yield: 85%, H-NMR (300 MHz, DMSO-d6,
δ ppm): 5.83 (s, 1H), 7.51–7.68 (m, 3H), 7.23 (d, 1H, J = 8.0 Hz), 11.33 (brs, 1H, NH).
3.1.2.3. Synthesis of Intermediates 9
A mixture of 8 (84.47 mmol) in POCl3 (150 mL) was heated under reflux for 3h. After cooling
down, the mixture was slowly added to crushed ice while shaking. The precipitate was collected by
filtration and washed several times with water. The product was dried under vacuum overnight to give
the products as white solid which are pure enough for further use.
1
2,4,6-Trichloroquinoline (9a): white solid, yield 70%, H-NMR (500 MHz, CDCl3, δ ppm): 8.00
(s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.56 (s, 1H); ESIMS: m/z: 232 (M+H)+.
1
2,4-Dichloroquinoline (9b): white solid, yield 66%, H-NMR (500 MHz, CDCl3, δ ppm): 8.16
(d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.47
(s, 1H); ESIMS: m/z: 198 (M+H)+.
3.1.2.4. Synthesis of Intermediates 10
To a solution of 9a or 9b (10.0 mmol) in DMF (10 mL) was added Et3N (2.78 mL, 20 mmol) and a
solution of thiol (10 mmol) in DMF (10 mL) dropwise at 0 °C. The reaction was warmed up after 10 min
and stirred overnight at rt. 200 mL of EtOAc was added to the mixture and washed with water and
brine successively. The organic layer was separated and dried over Na2SO4. The product was purified
on a column after removal of the solvent under vacuum. In most of cases, less than 7% of
dithio-substituted quinoline was formed.
1
2,6-Dichloro-4-(phenylthio)quinoline (10a): amorphous powder, yield: 69%. H-NMR (300 MHz,
CDCl3, δ ppm): 8.11 (s, 1H), 7.88–7.50 (m, 2H), 7.18–7.09 (m, 5H), 6.72 (s, 1H); ESIMS: m/z:
306 (M+H)+.

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1
2,6-Dichloro-4-(3,5-dimethylphenylthio)quinoline (10b): amorphous powder, yield: 69%. H-NMR
(300 MHz, CDCl3, δ ppm): 8.13 (s, 1H), 7.88–7.50 (m, 2H), 7. 24 (s, 2H), 7.19 (s, 1H), 6.62 (s, 1H),
2.34 (s, 6H); ESIMS: m/z: 334 (M+H)+.
2-chloro-4-(3,5-dimethylphenylthio)quinoline (10c): amorphous powder, yield: 59%. 1H-NMR
(300 MHz, CDCl3, δ ppm): 8.23 (d, J = 8.0 Hz, 1H), 7.99–7.57 (m, 3H), 7.23 (s, 1H), 7.21 (s, 1H),
7.16 (s, 1H), 6.63 (s, IH), 2.33 (s, 6H); ESIMS: m/z: 282 (M+H)+.
3.1.2.5. Synthesis of Target Compounds 4a
A solution of compound 10 (0.50 mmol) in TFA (2 mL) and HCl (6 N, 2 mL) was heated under
microwave radiation at 120 °C for 20 min. After cooling down, water (20 mL) was added. The
precipitate was collected by filtration and washed with water several times. The product was air dried
(71–85%) and was pure enough without further purification. The solubility of the products in organic
solvents is poor.
6-Chloro-4-(phenylthio)quinolin-2(1H)-one (4a1): amorphous powder, yield: 71%. 1H-NMR (300 MHz,
DMSO-d6, δ ppm): 11.13 (bs, 1H), 7.81 (s, 1H), 7.68–7.30 (m, 2H), 7.08–6.89 (m, 5H), 5.72 (s, 1H);
ESIMS: m/z: 288 (M+H)+; HRESIMS: calc for C15H11ClNOS [M+H]+ 288.0250, found 288.0261.
6-Chloro-4-(3,5-dimethylphenylthio)quinolin-2(1H)-one (4a2): amorphous powder, yield: 77%.
1H-NMR (300 MHz, DMSO-d6, δ ppm): 11.38 (bs, 1H)7.76 (s, 1H), 7.68–7.30 (m, 2H), 7. 22 (s, 2H),
7.18 (s, 1H), 5.82 (s, 1H), 2.34 (s, 6H); ESIMS: m/z: 316 (M+H)+; HRESIMS: calc for C17H15ClNOS
316.0563, found 316.0547.
4-(3,5-dimethylphenylthio)quinolin-2(1H)-one (4a3): amorphous powder, yield: 85%. 1H-NMR
(300 MHz, DMSO-d6, δ ppm): 11.66 (bs, 1H), 7.88 (d, J = 8.0 Hz, IH), 7.89–7.43 (m, 3H), 7.251
(s, 2H), 7.13 (s, 1H), 5.88 (s, IH), 2.32 (s, 6H); ESIMS: m/z: 282 (M+H)+; HRESIMS: calc for
C17H16NOS [M+H]+ 282.0953, found 282.0950.
3.1.2.6. Synthesis of Target Compounds 4b
To a solution of compound 4a (0.25 mmol) in a mixture of CH2Cl2 and MeOH (50 mL) was added
1.1 equiv. of 3-chlorobenzoperoxoic acid. After stirring overnight, water (50 mL) was added. The
mixture was extracted with CH2Cl2 and evaporated to give a residue which were further purified on
silica gel column to give compounds 4b (66–75%).
6-Chloro-4-(phenylsulfinyl)quinolin-2(1H)-one (4b1): amorphous powder, yield: 66%. 1H-NMR
(300 MHz, DMSO-d6, δ ppm): 11.85 (bs, 1H), 7.91 (s, 1H), 7.88–7.47 (m, 7H), 5.92 (s, 1H), 2.39 (s, 6H);
ESIMS: m/z: 304 (M+H)+; HRESIMS: calc for C15H11ClNO2S [M+H]+ 304.0199, found 304.0208.
6-Chloro-4-(3,5-dimethylphenylsulfinyl)quinolin-2(1H)-one (4b2): amorphous powder, yield: 75%.
1H-NMR (300 MHz, DMSO-d6, δ ppm): 11.40 (bs, 1H), 7.96 (s, 1H), 7.88–7.55 (m, 2H), 7. 47 (s, 1H),

Molecules 2011, 16
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7. 42 (s, 1H), 7.40 (s, 1H), 6.01 (s, 1H), 2.32 (s, 6H); ESIMS: m/z: 332 (M+H)+; HRESIMS: calc for
C17H15ClNO2S [M+H]+ 332.0512, found 332.0509.
3.1.2.7. Synthesis of Target Compounds 4c
To a solution of compound 4b (0.2 mmol) in a mixture of CH2Cl2 and MeOH (50 mL) was added
1.1 equiv. 3-chlorobenzoperoxoic acid. After stirring overnight, 50 mL of water was added. The
mixture was extracted with CH2Cl2 and evaporated to give a residue which were further purified on
silica gel column to give compound 4b (60–66%).
1
6-Chloro-4-(phenylsulfonyl)quinolin-2(1H)-one (4c1): amorphous powder, yield: 64%. H-NMR (300
MHz, CDCl3): 11.51 (bs, IH), 8.10 (s, 1H), 7.91–7.49 (m, 5H), 7.39 (d, J = 8.0 Hz, 1H), 7.29
(d, J = 8.0 Hz, 1H), 6.12 (s, 1H); ESIMS: m/z: 320 (M+H)+; HRESIMS: calc for C15H11ClNO3S
[M+H]+ 330.0148, found 320.0154.
6-Chloro-4-(3,5-dimethylphenylsulfonyl)quinolin-2(1H)-one (4c2): amorphous powder, yield: 60%.
1H-NMR (300 MHz, CDCl3, δ ppm): 11.86 (bs, IH), 8.00 (s, 1H), 7.98–7.69 (m, 2H), 7.57 (s, 2H),
7.39 (s, 1H), 5.95 (s, 1H), 2.35 (s, 6H); ESIMS: m/z: 348 (M+H)+; HRESIMS: calc for C17H15ClNO3S
[M+H]+ 348.0461, found 348.0470.
3.1.2.8. Synthesis of Target Compounds 4d
To a solution of 9a (10.0 mmol) in DMF (10 mL) was added Et3N (2.78 mL, 20 mmol) and a
solution of phenol (10 mmol) in DMF (10 mL) dropwise at 0 °C. The reaction was warmed up after 10
min and stirred for 24h at rt. 200 mL of EtOAc was added to the mixture and washed with water and
brine successively. The organic layer was separated and dried over Na2SO4. The product 11 was
purified on column after removal of solvent under vacuum with yield of 75–86%. A solution of
compound 11 (0.50 mmol) in TFA (2 mL) and HCl (6 N, 2 mL) was then heated under microwave
radiation at 120 °C for 20 min. After cooling down, 20 mL of water was added. The precipitate was
collected by filtration and washed with water for several times. The product was air dried (71–85%)
and was pure enough without further purification.
1
6-Chloro-4-phenoxyquinolin-2(1H)-one (4d1): amorphous powder, yield: 71%. H-NMR (300 MHz,
DMSO-d6, δ ppm): 11.94 (bs, 1H), 7.71 (s, 1H), 7.60–7.10 (m, 2H), 7.01–6.67 (m, 5H), 5.70 (s, 1H);
ESIMS: m/z: 272 (M+H)+; HRESIMS: calc for C15H11ClNO2 [M+H]+ 272.0478, found 272.0470.
1
6-Chloro-4-(3,5-dimethylphenoxy)quinolin-2(1H)-one (4d2): amorphous powder, yield: 77%. H-NMR
(300 MHz, DMSO-d6, δ ppm ): 11.64 (bs, 1H)7.70 (s, 1H), 7.65–7.31 (m, 2H), 6.89 (s, 2H), 6.93
(s, 1H), 5.72 (s, 1H), 2.33 (s, 6H); ESIMS: m/z: 300 (M+H)+; HRESIMS: calc for C17H15ClNO2
[M+H]+ 300.0791, found 300.0775.

Molecules 2011, 16
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3.1.3. General Procedure for Preparation of Compounds 5
3.1.3.1. Synthesis of Intermediate 12
4-Chloroaniline (19.2 g, 0.1 mol), DIEA (0.12 mol) and toluene (40 mL) were placed in a 100 mL
flask and stirred at rt, then Boc2O (26 g, 0.12 mol) was added, and the solution was stirred at rt until
the completion of the reaction monitored by TCL. Then the solution was poured into water and
extracted with DCM and then the organic layer was washed by water and dried over anhydrous
Na2SO4. Then the organic solvent was removed under vacuum to give crude product which was further
purified on Si gel column chromatography and eluted with petroleum ether (PET)-EtOAc (6:1) to give
1
the compound tert-butyl 4-chlorophenylcarbamate (12) 21.3 g, white solid, yield 94%. H-NMR
(CDCl3, δ ppm): 7.31–7.22 (m, 4H), 6.54 (brs, 1H, NH), 1.51 (s, 9H, CH3); positive FABMS m/z (%):
227 [M]+ (60), 172 (83), 57 (100).
3.1.3.2. Synthesis of Compound 13
Compound 12 was taken as an example. To a 100 mL flask, Boc2O (11.4 g, 50 mmol) and
anhydrous THF (30 mL) were added under an atmosphere of N2. The solution was stirred and cooled to
−78 °C, then t-BuLi solution in THF (110 mmol) was added dropwise under N2. The solution was kept
at −78 °C under stirring for 1 h and then warmed up to −40°C. Methyl benzoate (60 mmol, in 10 mL
THF) was added dropwise under N2 in 15 min. The solution was kept in −40 °C for another 1 h and
warmed up to rt. The solution was poured into ice cooled NH4Cl solution and extracted with EtOAc.
The combined organic layer was washed with brine and dried over anhydrous Na2SO4 and then
evaporated to give crude N-Boc protected 13. Without further purification, the crude product was
dissolved in 30 mL EtOH, and then 3 mol/L HCl solution (10 mL) was added. The mixture was
refluxed until the completion of the reaction. Then the solution was adjusted to pH 8 using saturated
NaHCO3 solution and extracted with DCM successively. The organic layer was washed with brine and
dried over anhydrous Na2SO4 and then evaporated to give a residue which was further purified on
Si CC to give compound 13 (3.46 g, yield: 30%).
(2-Amino-5-chlorophenyl)(phenyl)methanone (13a): 1H-NMR (DMSO-d6, δ ppm): 7.64–7.33
(m, 7H), 6.99 (d, J = 8.8 Hz, 1H), 6.08 (brs, 2H, NH2); positive FABMS m/z (%): 232 [M+1]+ (100).
(2-Amino-5-chlorophenyl)(2-fluorophenyl)methanone (13b): yellow amorphous powder, yield 27%,
1H-NMR (DMSO-d6, δ ppm): 7.50–6.15 (m, 6H), 6.67 (d, J = 8.8 Hz, 1H), 6.38 (brs, 2H, NH2);
positive FABMS m/z (%): 250 [M+1]+ (100). 249 [M]+ (100).
(2-Amino-5-chlorophenyl)(2-chlorophenyl)methanone (13c): yellow amorphous powder, yield 31%,
1H-NMR (DMSO-d6, δ ppm): 7.46–7.11 (m, 6H), 6.68–6.65 (m, 1H), 6.47 (brs, 2H, NH2); positive
FABMS m/z (%): 266 [M+1]+ (100).

Molecules 2011, 16
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3.1.3.3. Synthesis of Compounds 5
Compound 5a was taken as an example. A 30 mL, 2-necked flask was charged with compound 13a
(1 mmol, 231 mg) and THF (5 mL). The resulting solution was cooled to 0 °C and LiHMDS (6 mL,
1M in THF) was added over 5 min. The internal temperature was controlled <5 °C for 10 min, then
EtOAc (144 mg, 2 mmol) was added over 1 min. The reaction solution was allowed to warm up to rt
and stirred at rt for 2h. Then water (5 mL) was added, and the reaction mixture was stirred at rt for
another 24 h. The solution was poured into 50 mL ice cooled water, and a water suspension formed.
The suspension was filtered and the solid was further purified on Si CC eluted with CHCl3/CH3OH
(97:3) to give white amorphous powder 5a (212 mg, yield: 83%).
1
6-Chloro-4-phenylquinolin-2(1H)-one (5a): white amorphous powder, yield:70% H-NMR (DMSO-d6,
δ ppm): 12.02 (s, 1H, NH), 7.60–7.39 (m, 7H), 7.26 (s, 1H), 6.45 (s, 1H); positive FABMS m/z (%):
256 [M+1]+ (50), 73 (100). HRESIMS: calc for C15H11ClNO [M+H]+ 256.0529, found 256.0548.
1
6-Chloro-4-(2-fluorophenyl)quinolin-2(1H)-one (5b): white amorphous powder, yield: 77%, H-NMR
(CDCl3-CD3OD, δ ppm): 8.16–8.10 (m, 3H), 8.01–7.96 (m, 3H), 7.89–7.87 (m, 2H); EI-MS m/z (%):
275 [M+2]+ (6), 274 [M+1]+ (4), 273 [M]+ (18), 246 (17), 210 (65), 131 (100), 117 (50), 105 (21), 91
(84): HRESIMS: calc for C15H10ClFNO [M+H]+ 274.0435, found 274.0448.
6-Chloro-4-(2-chlorophenyl)quinolin-2(1H)-one (5c): white amorphous powder, yield: 80%, 1H-NMR
(DMSO-d6, δ ppm): 12.11 (s, 1H, NH), 7.67–7.40 (m, 4H), 6.84 (d, J = 2.2 Hz, 1H), 6.47 (s, 1H);
EI-MS m/z (%): 293 [M+4]+ (4), 293 [M+3]+ (3), 291 [M+2]+ (10), 289 [M]+ (20), 167 (30), 149 (100);
HRESIMS: calc for C15H10Cl2NO [M+H]+ 290.0139, found 290.0151.
3.2. General Procedure for HIV-1 RT Inhibitory Assay
HIV-1 reverse transcriptase (RT) activity was measured using a commercially available ELISA RT
kit (Roche) according to the instructions of the manufacturer. The compounds were incubated with
DIG-labeled reaction mixture at 37 °C for 2 h, then anti-DIG-POD solution was added, followed by
substrate ABTS. Efaviren was used as a positive control. The absorbency at 405 nm/490 nm (A405/490)
was read on Bio-Tek ELx 800 ELISA reader.
3.3. General Procedures for Docking Study
CDOCK was used for the docking study. The CHARMM force field was used for the energy
minimizations in the docking process. The X-ray crystal structure of reverse transcriptase complexed
with efavirenz was retrieved from PDB (PDB code: 1FK9). The active site was defined as all residues
within 6.5 Å radius of the cocrystallized efavirenz. Starting from the ligand configuration, a set of
10 different orientations was randomly generated and ranked according to their binding free energy.
The docking mode was chosen on the basis of binding affinity rank.

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4. Conclusions
In summary, a group of quinoneline-2-one analogues with activity against HIV-1 RT were
synthesized based on the SAR established by Freeman and known crystal structures. The SAR and
docking studies of the titled compounds were analyzed. Analogues 4a2 and 4d2 demonstrated the
most activity among these analogues with a similar mode of interaction with RT residues of the
allosteric pocket as known NNRTIs. Further structure modifications and cell-based in vitro anti-HIV-1
assay studies of these quinolin-2-one derivatives will be reported in due course.
Acknowledgments
The authors are grateful to Qiong Gu’s research group for the docking study performed at the
School of Pharmaceutical Sciences, Sun Yat-sen University. This work was partly supported by the
Talent Introduction Project in Hunan Agricultural University (11YJ04).
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Sample Availability: Samples of the all compounds are available from the authors.
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