Synthesis and biological evaluation of N4-(hetero)arylsulfonylquinoxalinones as HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.02.039

A series of novel N⁴-(hetero)arylsulfonylquinoxalinone derivatives were prepared in a straight and efficient way. Of all the synthesized compounds, five compounds exhibited potent anti-HIV-1 replication activities with IC₅₀ value at

Full text of DOI:10.1016/j.bmc.2009.02.039

Bioorganic & Medicinal Chemistry 17 (2009) 2767–2774
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of N4-(hetero)arylsulfonylquinoxalinones
as HIV-1 reverse transcriptase inhibitors
*
Bailing Xu , Yan Sun, Ying Guo, Yingli Cao, Tao Yu
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
A series of novel N4-(hetero)arylsulfonylquinoxalinone derivatives were prepared in a straight and effi-
cient way. Of all the synthesized compounds, five compounds exhibited potent anti-HIV-1 replication
activities with IC50 value at the level of 10À7 mol/L. Preliminary structure–activity relationships were
studied in details and that will shed light on the discovery of more potent non-nucleoside reverse-trans-
criptase inhibitors.
Article history:
Received 8 December 2008
Revised 17 February 2009
Accepted 19 February 2009
Available online 25 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Quinoxalinone
HIV-1 Reverse transcriptase inhibitor
Structure–activity relationship
1. Introduction
the lead compound 1, a wide array of N4-(hetero)arylsulfonylqui-
noxalinone analogs were designed and synthesized, and their anti-
Since the human immunodeficiency virus 1 (HIV-1) was first
confirmed as the causative agent of acquired immunodeficiency
syndrome (AIDS), there had been an intensive investigation aimed
at developing anti-HIV drugs. Up to now, more than 20 anti-HIV
drugs, classified into seven groups, respectively, had been ap-
proved for clinical treatment.1 Among these clinic drugs, non-
nucleoside reverse-transcriptase inhibitors (NNRTIs), which inter-
act with a specific allosteric non-substrate binding site on HIV-1
reverse transcriptase, have proved to be effective anti-HIV drugs
because of their high potency, low toxicity and improved pharma-
cokinetics.2,3 To date, three NNRTIs, including Nevirapine, efavi-
renz and delavirdine, have been approved for clinical treatment
of HIV infection (Fig. 1). Efavirenz acted as an essential component
of the highly active anti-retroviral therapy (HAART).4,5 Neverthe-
less, HIV has a low genetic barrier to generate resistance to NNRTIs,
which accounts for the rapid emergence of drug resistance.6 There-
fore, an enormous effort is continued to develop novel chemical
entities with improved sensitivity and resistance profiles.
viral activities were evaluated. Herein, the detailed synthetic
procedure, anti-HIV-1 activity and preliminary structure–activity
relationships (SAR) studies of these new quinoxalinone chemical
entities are described as follows.
2. Chemistry
The compound 1 and its analogs 5–22 were synthesized
straightforward as shown in Scheme 1. In the presence of bases
(DIEA or K2CO3), the aromatic nucleophilic substitution reaction
of the structurally diverse o-halo nitrobenzene 2a–c with natural
or unnatural amino acids or their esters provided the key interme-
diates 3a–i in the yield of 20–80%. The quinoxalinone scaffold 4
was achieved by the reductive cyclization of intermediates 3
in situ in moderate to high yield (37–98%). In the course of opti-
mizing reaction condition, a wide range of reducing reagents such
as Na2S2O4/K2CO3, Pd–C/H2, Pd–C/HCOONH4 or Fe/HOAc has been
attempted. As Pd–C/HCOONH4 was employed in the cyclization
reaction, compounds 4a, 4e and 4i were generated in high yield
(88–96%); while moderate to good yield (37–98%) was obtained
when Na2S2O4/K2CO3 was chosen as the reducing reagent (e.g.,
4b, 4d, 4f and 4g). Compound 4c was afforded in 66% yield via
the catalytic hydrogenation of compound 3c in the presence of
10% Pd–C. After attempting several reducing reagents, Fe/HOAc
was used to reduce compound 3h to give compound 4h in 49%
yield smoothly. Finally, sulfonylation of quinoxalinones 4 with var-
ious aryl or heteroaryl sulfonyl chloride in the presence of pyridine
provided the desired quinoxalinone derivatives 1, 5–22.
In our ongoing study to search for new chemical entities of
NNRTIs by screening our in-house library, 6-fluoro-N4-(quinoline-
8-sulfonyl)-3,4-dihydroquinoxalin-2(1H)-one
1 was identified
with anti-HIV activity at micromolar level (Fig. 1). The lead com-
pound 1 is a quinoxaline-based chemical entity, and this subunit
also was presented in the potent NNRTIs such as GW420867x7,8
and HBY097.9 With a purpose to improve the anti-HIV activity of
* Corresponding author. Tel./fax: +86 10 63166764.
E-mail address: xubl@imm.ac.cn (B. Xu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.039

2768
B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
O
HN
N
HN
F3C
HN
S
NH
N
Cl
O
O
N
N
N
N
O
N
O
O
O
H
Nevirapine
Efavirenz
Delavirdine
N
O
O
O
O
O
S
O
F
N
F
N
O
N
S
N
H
N
O
N
H
S
H
1
IC50 = 0.98 µM
HBY097
GW420867x
Figure 1. Chemical structures of FDA-approved NNRTIs and quinoxaline-based NNRTIs.
Ar
S
R3
O
O
H
N
H
N
R1
R2
X
R1
R2
R1
R2
R3
O
R1
R2
N
R3
O
a
COOR4
c
b
NO2
NO2
N
H
N
H
3
4
2
1, 5-22
2a: R1 = F, R2 = H, X = F
3a: R1 = F, R2 = H, R3 = H, R4 = C2H5
3b: R1 = F, R2 = H, R3 = CH3, R4 = H
3c: R1 = F, R2 = H, R3 = C2H5, R4 = H
3d: R1 = F, R2 = H, R3 = CH2CH2SCH3, R4 = CH3
3e: R1 = F, R2 = H, R3 = CH2COOCH3, R4 = CH3
3f: R1 = Cl, R2 = H, R3 = H, R4 = C2H5
3g: R1 = Cl, R2 = H, R3 = CH3, R4 = H
3h: R1 = Cl, R2 = H, R3 = C2H5, R4 = H
3i: R1 = H, R2 = CF3, R3 = H, R4 = C2H5
4a: R1 = F, R2 = H, R3 = H
4b: R1 = F, R2 = H, R3 = CH3
4c: R1 = F, R2 = H, R3 = C2H5
4d: R1 = F, R2 = H, R3 = CH2CH2SCH3
4e: R1 = F, R2 = H, R3 = CH2COOCH3
4f: R1 = Cl, R2 = H, R3 = H
4g: R1 = Cl, R2 = H, R3 = CH3
4h: R1 = Cl, R2 = H, R3 = C2H5
4i: R1 = H, R2 = CF3, R3 = H
2b: R1 = Cl, R2 = H, X = Cl
2c: R1 = H, R2 = CF3, X = Cl
Scheme 1. Reagents and conditions: (a) amino acid/K2CO3, ethanol/water or amino acid ester/DIEA, CH3CN; (b) Na2S2O4/K2CO3/ethanol/water or H2/Pd–C/ethanol; Pd–C/
HCOONH4; Fe/HOAc; (c) ArSO2Cl or heteroaryl sulfonyl chloride, pyridine, CH2Cl2.
tively; while compound 7 showed sevenfold less anti-HIV-1 activ-
ity than that of lead compound 1, probably due to the more bulky
size of the quinoline-8-sulfonyl group.
3. Biological results and discussion
The anti-HIV-1 activities of all target compounds 5–22 were
evaluated by a cell-based HIV-1 replication pharmacological model
which was set up by HIV-1(pNL4-3) core packed with vesicular
stomatitis virus glycoprotein. The level of HIV-1 replication was
presented by a reporter gene expression (i.e., luciferase activity)
in infected cells. The results are expressed as IC50 values. Azidothy-
midine (AZT) and Efavirenz were used as reference molecules
(Tables 1–3).
Table 1
The chemical structures and anti-HIV replication activities of compounds 1, 5–11a
Ar
O
S
O
The quinoxaline derivatives GW420867x and HBY097 were re-
ported with potent anti-HIV activities.7–9 Both GW420867x and
HBY097 have a common iso-propyloxy carbonyl group on N-4 po-
sition; while within our lead compound 1, a heteroaryl sulfonyl
functionality is installed on N-4 instead of an iso-propyloxy car-
bonyl group. Hence, our initial investigation was performed on
the modification of the substituents on N-4 position. As shown in
Table 1, compound 6 exhibited a comparable anti-HIV-1 replica-
tion activity to lead compound 1. Meanwhile, compounds 7–11
were designed and synthesized as shown in Scheme 1 on the basis
of the following rationales: (1) the known SAR studies on quinox-
alines8 showed that the advantageous effect of small alkyl group
on C-3 to the anti-HIV activity; (2) the installment of methyl group
on C-3 could be easily completed using alanine as nucleophilic re-
agent. As expected, compounds 9 and 10 exhibited increased anti-
HIV-1 activity in comparison with compounds 5 and 6, respec-
F
N
R3
O
N
H
b
Compound
R3
ArSO2
Quinoline-8-sulfonyl
3-Cyanobenzene-1-sulfonyl
2-Methyloxycarbonylthiophene-3-sulfonyl
Quinoline-8-sulfonyl
4-Nitrobenzene-1-sulfonyl
3-Cyanobenzene-1-sulfonyl
2-Methyloxycarbonylthiophene-3-sulfonyl
2,5-Dichlorothiophene-3-sulfonyl
IC50 (lM)
1
5
6
7
8
9
10
11
H
H
H
CH3
CH3
CH3
CH3
CH3
0.98
>100
2.02
7.0
>100
55
0.2
>100
a
AZT and Efavirenz were used as reference molecules; IC50 for AZT was 48 nM;
IC50 for Efavirenz was 1.3 nM.
b
Compound dose (lM) required to inhibit the HIV activity by 50%.

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
2769
Table 2
The chemical structures and anti-HIV-1 replication activities of compounds 6, 10 and
12–14a
S
O
O
O
S O
F
N
R3
N
H
O
R3
H
CH3
C2H5
CH2CH2SCH3
CH2COOCH3
IC50 (lM)
b
Compound
6
10
12
13
14
2.02
0.2
5.0
>100
>100
Figure 2. FlexX-modeled binding mode of 10 (carbon atoms colored cyan) in
comparison with the crystal structure (1FK9 in PDB) of Efavirenz (carbon atoms
colored orange). The key hydrogen bond is illustrated with yellow line. Molecular
image was generated with UCSF Chimera.13
a
AZT and Efavirenz were used as reference molecules; IC50 for AZT was 48 nM;
IC50 for Efavirenz was 1.3 nM.
b
Compound dose (lM) required to inhibit the HIV activity by 50%.
1 vs 19 and 7 vs 20). However, the introduction of CF3 group on
C-7 position is detrimental to the anti-HIV-1 activity (15 vs 18,
19 vs 22). The most potent compounds 10 and 16 were discovered
Studies of a series of known crystal structures of the complexes
of NNRTIs and HIV-1 RT indicated that a small hydrophobic group
incorporated in NNRTIs played a key role for triggering the confor-
mational switch of Tyr181 and lead to the increased binding affin-
ity.8,10 As shown in Table 1, compound 10 exhibited the most
with IC50 value of 0.2
on C-6 position.
lM with either fluoro or chloro substituent
To investigate the binding mode of the synthesized arylsulfo-
nylquinoxalinones, computational modeling was performed using
FlexX algorithm implemented in SYBYL 6.9.11 The coordinates of
the RT–Efavirenz complex (pdb code: 1FK9)12 was downloaded,
and the molecular structure of the most potent compound 10
was docked into its active site. As shown in Figure 2, the orienta-
tion and position of compound 10 is quite similar to that of Efavi-
renz. The quinoxalinone ring has a hydrophobic contact with the
surrounding residues such as Leu100, Val106, Leu234, Pro236
and Tyr318. The crucial hydrogen bonding interaction, which is
also frequently used for some other typical NNRTIs to bind to RT,
was formed between the carbonyl oxygen of Lys101 and NH of
the quinoxalinone compound 10. The 2-methoxylcarbonyl-3-sulfo-
nylthiophene fragment lies in a hydrophobic pocket consisting of
Pro95, Tyr181, Tyr188 and Trp229, which is occupied by the cyclo-
propyl-propynyl of Efavirenz in the complex. As expected, the
methyl substituent of compound 10 takes a similar orientation to
the trifluoromethyl group of Efavirenz. In consistent with the
above SAR studies, the larger C-3 substituents resulted in de-
creased activity probably due to their steric interference with the
residues Val106, Val179 and Gly190 in the binding site.
potent inhibitory activity with an IC50 value of 0.2 lM, so we pre-
served the 2-methoxylcarbonyl-3-sulfonylthiophene and further
explored the effect of various 3-substitutent. As depicted in Table
2, the bulky group on C-3 position has a significant impact on
the anti-HIV potency of the synthesized compounds. As R3 group
increase in bulky size, the activity of the corresponding compound
tends to decrease. For example, the larger R3 group such as
CH2CH2SCH3 and CH2COOCH3 lead to a complete loss of the anti-
HIV activity (e.g., compounds 13 and 14); In contrast, the smaller
alkyl group such as methyl and ethyl are tolerated.
The role of the substitutes of the phenyl ring on the biological
activities was also investigated as shown in Table 3. Compounds
(I) and (II) were chosen as template due to their potent anti-HIV-
1 activity. The replacement of 6-fluoro with chloro give rise to an
advantageous effect on the activity in moderate degree (6 vs 15,
Table 3
The chemical structures and anti-HIV-1 replication activities of compounds 15–22a
S
O
4. Conclusions
N
O
O
S
O
O
S
O
R1
R2
N
R3
In summary, a wide array of novel heteroarylsulfonylquinoxa-
line-based chemical entities was synthesized as potent NNRTIs
with IC50 at submicromolar level. The SAR and molecular modeling
studies of the titled compounds were analyzed, and that will serve
as guidance to further development of more potent quioxalines
NNRTIs.
R1
R2
N
R3
O
N
H
O
N
H
(I)
(II)
b
Compound
Structure
R1
R2
R3
IC50 (lM)
15
16
17
18
19
20
21
22
I
I
I
I
II
II
II
II
Cl
Cl
Cl
H
Cl
Cl
Cl
H
H
H
H
CF3
H
H
H
CF3
H
CH3
C2H5
H
H
CH3
C2H5
H
0.83
0.2
6.3
>100
0.33
0.65
>100
>100
5. Experimetal
5.1. Chemistry
5.1.1. General
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. 1H NMR (300 MHz) on a Varian
Mercury 300 spectrometer was recorded in DMSO-d6 or CDCl3.
Chemical shifts are reported in d (ppm) units relative to the inter-
a
AZT and Efavirenz were used as reference molecules; IC50 for AZT was 48 nM;
IC50 for Efavirenz was 1.3 nM.
b
Compound dose (lM) required to inhibit the HIV replication activity by 50%.

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B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
nal standard tetramethylsilane (TMS). High resolution mass spec-
tra (HRMS) were obtained on an Agilent Technologies LC/MSD
TOF spectrometer. All chemicals and solvents used were of reagent
grade without purified or dried before use. All the reactions were
monitored by thin-layer chromatography (TLC) on pre-coated sil-
ica gel G plates at 254 nm under a UV lamp. Column chromatogra-
phy separations were performed with silica gel (200–300 mesh).
J2 = 5.1 Hz, 1H), 4.34 (s,2H); HRMS (ESI): m/z, calcd for
C15H11N3O3FS [M+H+]: 332.0505, found 332.0504.
5.1.4. 6-Fluoro-4-(2-methyoxycarbonylthiophene-3-sulfonyl)-
3,4-dihydroquinoxalin-2-(1H)-one (6)
Following the preparation protocol of Section 5.1.2.3, the mix-
ture of compound 4a (0.083 g, 0.5 mmol) and 2-methyoxycarbon-
ylthiophene-3-sulfonyl chloride (0.14 g, 0.6 mmol) in pyridine
was stirred at room temperature for 48 h to provide the title com-
pound as light yellow solid (0.04 g, 22% yield); mp 182–184 °C. 1H
NMR (300 MHz, DMSO-d6, d ppm) d 10.53 (s, 1H), 7.95 (d,
J = 5.4 Hz, 1H), 7.27 (d, J = 5.1 Hz,1H), 7.24 (dd, J1 = 9.6 Hz,
J2 = 2.7 Hz, 1H), 7.09 (td, J1 = 8.7 Hz, J2 = 2.7 Hz, 1H), 6.86 (dd,
J1 = 8.7 Hz, J2 = 5.4 Hz, 1H), 4.41 (s,2H), 3.72 (s, 3H); HRMS (ESI):
m/z, calcd for C14H12N2O5FS2 [M+H+]: 371.0166, found 371.0166.
5.1.2. 6-Fluoro-4-(quinoline-8-sulfonyl)-3,4-dihydroqui-
noxalin-2-(1H)-one (1)
5.1.2.1. Ethyl 2-(5-fluoro-2-nitrophenyl)-acetate (3a). To a stir-
red solution of 2,4-difluoronitro-benzene 2a (6.36 g, 40 mmol) in
CH3CN (20 mL) was added glycine ethyl ester hydrochloride
(6.70 g, 48 mmol) followed by addition of a solution of DIEA
(12.41 g, 96 mmol) in CH3CN (10 mL). The reaction mixture was stir-
red at room temperature for 24 h. The solvent was evaporated to af-
ford the crude product which was purified by recrystalization in 95%
ethanol. Compound 3a was obtained as yellow needle crystal
(7.67 g,79% yield): mp 101–104 °C; 1H NMR (300 MHz, CDCl3, d
ppm) d 8.55 (br s, 1H), 8.26 (dd, J1 = 9.3 Hz, J2 = 6.3 Hz, 1H), 6.44
(ddd, J1 = 9.6 Hz, J2 = 7.2 Hz, J3 = 2.1 Hz, 1H), 6.34 (dd, J1 = 11.1 Hz,
J2 = 2.4 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 4.04 (d, J = 4.8 Hz, 2H), 1.33
(t, J = 6.9 Hz, 3H).
5.1.5. 6-Fluoro-3-methyl-4-(quinoline-8-sulfonyl)-3,4-dihydro-
quinoxalin-2-(1H)-one (7)
5.1.5.1. 2-(5-Fluoro-2-nitrophenylamino)propanoic acid (3b).
To a stirred solution of K2CO3 (5.53 g, 40 mmol) and alanine
(1.78 g, 20 mmol) in water (50 mL) was added dropwise 2,4-difluo-
ronitrobenzene 2a (3.82 g, 24 mmol) in ethanol (30 mL). The reac-
tion mixture was stirred at room temperature for 12 h. The solvent
was evaporated in vacuo. The residue was dissolved in water
(100 mL) and washed with ether (30 mL Â 3). The aqueous phase
was adjusted to pH 2 with 2 N HCl aqueous solution, and then ex-
tracted with EtOAc (50 mL Â 4). The organic layer was dried over
anhydrous MgSO4, concentrated and purified with column chroma-
tography on silica gel to afford compound 3b as yellow solid (3.0 g,
66% yield); mp 118–120 °C. 1H NMR (300 MHz, CDCl3, d ppm) d
8.44 (d, J = 6.3 Hz, 1H), 8.26 (dd, J1 = 9.3 Hz, J2 = 6.0 Hz, 1H), 6.49–
6.37 (m, 2H), 4.27 (m, J = 6.9 Hz, 1H), 1.68 (d, J = 7.2 Hz, 3H).
5.1.2.2. 6-Fluoro-3,4-dihydroquinoxalin-2-(1H)-one (4a). The
reaction mixture of compound 3a (3.91 g, 16.14 mmol), HCOONH4
(15.26 g, 242.15 mmol) and 10% Pd–C (6.85 g, 3.23 mmol) in eth-
anol (300 mL) was stirred for 20 h. The solid was removed by fil-
tration and the filtrate was concentrated to afford the yellow solid
which was purified by column chromatography on silica gel with
petroleum ether–EtOAc (v/v = 2:1). Compound 4a (2.57 g) was
obtained as yellow solid (2.57 g, 96% yield); mp 165–169 °C. 1H
NMR (400 MHz, DMSO-d6, d ppm) d 10.22 (s, 1H), 6.66 (dd,
J1 = 8.4 Hz, J2 = 5.6 Hz, 1H), 6.43 (dd, J1 = 10.4 Hz, J2 = 2.8 Hz, 1H),
6.34 (td, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 6.20 (s, 1H), 3.72 (s, 2H).
5.1.5.2. 6-Fluoro-3-methyl-3,4-dihydroquinoxalin-2-(1H)-one
(4b). To a stirred solution of K2CO3 (2.76 g, 20 mmol) and Na2S2O4
(10.69 g, 60 mmol) in water (60 mL) was added dropwise com-
pound 3b (2.28 g, 10 mmol) in ethanol (80 mL). The reaction mix-
ture was stirred at room temperature until TLC indicated the
reaction completed. The solvent was evaporated in vacuo. The res-
idue was dissolved in water (100 mL) and extracted with EtOAc
(50 mL Â 3). The organic layer was dried over anhydrous MgSO4,
concentrated and purified with column chromatography on silica
gel to afford compound 4b as yellow solid (1.3 g, 74% yield); mp
111–113 °C. 1H NMR (300 MHz, CDCl3, d ppm) d 9.20 (s, 1H), 6.70
(dd, J1 = 8.7 Hz, J2 = 5.1 Hz, 1H), 6.38–6.47 (m, 2H), 4.02 (q,
J = 6.6 Hz, 1H), 3.96 (br s, 1H), 1.46 (d, J = 6.9 Hz, 3H).
5.1.2.3. 6-Fluoro-4-(quinoline-8-sulfonyl)-3,4-dihydroquinoxa-
lin-2-(1H)-one (1). To a well stirred solution of compound 4a
(0.12 g, 0.70 mmol) in pyridine (10 mL) was added quinoline-8-
sulfonyl chloride (0.19 g, 0.84 mmol). The reaction mixture was
stirred at room temperature for 12 h and then heated at 50 °C for
24 h. The saturated CuSO4 aqueous solution was added to quench
the reaction. The reaction mixture was extracted with EtOAc
(30 mL Â 3) and the organic layer was washed with saturated Cu-
SO4(aq) (30 mL Â 2), dried over anhydrous MgSO4, concentrated to
give the crude product which was purified by column chromatog-
raphy on silica gel to afford compound 1 as white solid (0.12 g,
48%); mp 195–196 °C. 1H NMR (300 MHz, CDCl3, d ppm) d 8.92
(dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H), 8.46 (dd, J1 = 7.5 Hz, J2 = 1.2 Hz,
1H), 8.18 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 8.14 (br s, 1H), 8.00 (dd,
J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.66 (dd, J1 = 10.2 Hz, J2 = 2.7 Hz, 1H),
7.57 (t, J = 7.8 Hz, 3H), 7.44 (dd, J1 = 8.4 Hz, J2 = 4.2 Hz, 1H), 6.70
(td, J1 = 8.4 Hz, J2 = 2.7 Hz, 1H), 6.51 (dd, J1 = 8.7 Hz, J2 = 5.1 Hz,
1H), 4.89 (s, 2H); HRMS (ESI): m/z, calcd for C18H12N2O4F2
[M+H+]: 358.0759, found 358.0793.
5.1.5.3. 6-Fluoro-3-methyl-4-(quinoline-8-sulfonyl)-3,4-dihy-
droquinoxalin-2-(1H)-one (7). Following the preparation proto-
col of Section 5.1.2.3, the mixture of compound 4b (0.13 g,
0.70 mmol) and quinoline-8-sulfonyl chloride (0.19 g, 0.84 mmol)
in pyridine was stirred to provide the title compound as yellow oil
(0.094 g, 36% yield); 1H NMR (300 MHz, DMSO-d6, d ppm) d 10.20
(s, 1H), 8.83 (dd, J1 = 4.2 Hz, J2 = 1.5 Hz, 1H), 8.48 (dd, J1 = 8.4 Hz,
J2 = 1.5 Hz, 1H), 8.39 (d, J = 7.2 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.68
(m, 2H), 7.49 (dd, J1 = 10.2 Hz, J2 = 2.7 Hz, 1H), 6.85 (td, J1 = 8.4 Hz,
J2 = 2.7 Hz, 1H), 6.63 (dd, J1 = 8.7 Hz, J2 = 5.7 Hz, 1H), 5.35 (q,
J = 7.5 Hz, 1H), 1.29 (d, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C18H15N3O3FS [M+H+]: 372.0813, found 372.0826.
5.1.3. 6-Fluoro-4-(3-cyanobenzene-1-sulfonyl)-3,4-dihydroqui-
noxalin-2-(1H)-one (5)
Following the preparation protocol of Section 5.1.2.3, the mix-
ture of compound 4a (0.083 g, 0.5 mmol) and 3-cyanobenzene-1-
sulfonyl chloride (0.12 g, 0.6 mmol) in pyridine was stirred at room
temperature to provide the title compound as light yellow solid
(0.08 g, 48% yield); mp 258–262 °C. 1H NMR (300 MHz, DMSO-d6,
d ppm) d 10.32 (s, 1H), 8.20 (dt, J1 = 7.2 Hz, J2 = 1.5 Hz 1H), 7.93
(s, 1H), 7.66–7.75 (m, 2H), 7.37 (dd, J1 = 9.6 Hz, J2 = 2.7 Hz, 1H),
7.20 (td, J1 = 8.4 Hz, J2 = 2.7 Hz, 1H), 6.79 (dd, J1 = 9.0 Hz,
5.1.6. 6-Fluoro-3-methyl-4-(4-nitrobenzene-1-sulfonyl)-3,4-
dihydroquinoxalin-2-(1H)-one (8)
Following the preparation protocol of Section 5.1.2.3, the mix-
ture of compound 4b (0.25 g, 1.40 mmol) and 4-nitrobenzene-1-
sulfonyl chloride (0.37 g, 1.68 mmol) in pyridine was stirred to
provide the title compound as yellow solid (0.34 g, 67% yield);

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
2771
mp 240–241 °C. 1H NMR (300 MHz, DMSO-d6, d ppm) d 10.19(s,
1H), 8.32(d, J = 8.7 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.25–7.54(m,
2H), 6.66 (dd, J1 = 8.7 Hz, J2 = 5.7 Hz, 1H), 6.46 (dd, J1 = 10.2 Hz,
J2 = 2.7 Hz, 1H), 6.36 (td, J1 = 8.7 Hz, J2 = 2.7 Hz, 1H), 3.78(q,
J1 = 6.6 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H); HRMS (ESI): m/z, calcd for
C15H13N3O5FS [M+H+]: 366.0554, found 366.0573.
C (0.127 g, 0.06 mmol) in ethanol (20 mL) was subjected to hydroge-
nation under the pressure of 60 psi for 30 min. The reaction mixture
was filtered and the filtrate was concentrated and purified with col-
umn chromatography to afford the compound 4c as brown solid
(38 mg, 66% yield); mp 133–135 °C. 1H NMR (300 MHz, CDCl3, d
ppm) d 8.46 (s, 1H), 6.63 (dd, J1 = 9.6 Hz, J2 = 6.9 Hz, 1H), 6.43 (d,
J = 7.8 Hz, 1H), 6.40 (d, J = 9.6 Hz, 1H), 4.02 (s, 1H), 3.87 (t,
J1 = 6.0 Hz, 1H), 1.84 (m, 2H), 1.03 (t, J = 7.2 Hz,, 3H).
5.1.7. 6-Fluoro-3-methyl-4-(3-cyanobenzene-1-sulfonyl)-3,4-
dihydroquinoxalin-2-(1H)-one (9)
The mixture of compound 4b (0.05 g, 0.3 mmol) in CH2Cl2
(6 mL) and 3-cyanobenzene-1-sulfonyl chloride (0.18 g, 0.9 mmol)
was added pyridine (0.12 g, 1.5 mmol) in CH2Cl2 (4 mL). The reac-
tion mixture was stirred at room temperature to provide the title
compound as yellow solid (0.09 g, 88% yield); mp 213–215 °C. 1H
NMR (300 MHz, DMSO-d6, d ppm) d 10.38 (s, 1H), 8.19 (dt,
J1 = 7.8 Hz, J2 = 1.5 Hz, 1H), 7.92 (s, 1H), 7.63–7.74 (m, 2H), 7.40
(dd, J1 = 9.3 Hz, J2 = 2.7 Hz, 1H), 7.22 (td, J1 = 8.7 Hz, J2 = 3.0 Hz,
1H), 6.81(dd, J1 = 9.0 Hz, J2 = 5.4 Hz, 1H), 4.62 (q, J = 6.6 Hz, 1H),
1.15 (d, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C16H13N3O3FS
[M+H+]: 346.0662, found 346.0688.
5.1.10.3. 6-Fluoro-3-ethyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one (12). Following the
preparation protocol ofSection 5.1.7, the mixture of compound 4c
(19 mg, 0.1 mmol), 2-methyoxycarbonylthiophene-3-sulfonyl
chloride (48 mg, 0.2 mmol) and pyridine (40 mg, 0.5 mmol) in
anhydrous CH2Cl2 (2 mL) was stirred to provide the title compound
as light yellow oil (15 mg, 38% yield); 1H NMR (300 MHz, acetone-
d6, d ppm) d 9.43 (s, 1H), 7.80 (d, J = 5.4 Hz, 1H), 7.48 (dd,
J1 = 9.9 Hz, J2 = 2.7 Hz, 1H), 7.33 (d, J = 5.1 Hz, 1H), 6.96–7.02 (m,
2H), 4.77 (dd, J1 = 10.2 Hz, J2 = 5.1 Hz, 1H), 3.76 (s, 3H), 1.68 (m,
1H), 1.44 (m, 1H), 1.00 (t, J1 = 7.5 Hz, 3H); HRMS (ESI): m/z, calcd
for C17H15NO4F2S2 [M+H+]: 399.0410, found 399.0417.
5.1.8. 6-Fluoro-3-methyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxa-lin-2-(1H)-one (10)
5.1.11. 6-Fluoro-3-((2-methylthio)-ethyl)-4-(2-methyoxycar-
bonylthiophene-3-sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one
(13)
5.1.11.1. Methyl 2-(5-fluoro-2-nitrophenylamino)-4-methylthio-
butanoate (3d). Following the preparation protocol of Section
5.1.2.1, the mixture of 2,4-difluoronitrobenzene 2a (0.79 g,
5.0 mmol), methionine methyl ester hydrochloride (1.20 g,
6.0 mmol) and DIEA (1.55 g, 12 mmol) in CH3CN was stirred at room
temperature for 12 h and then refluxed to afford compound 3d
(1.44 g) as yellow oil in yield 95%.
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4b (0.05 g, 0.3 mmol), 2-methyoxycarbonylthioph-
ene-3-sulfonyl chloride (0.22 g, 0.9 mmol) and pyridine (0.12 g,
1.5 mmol) in CH2Cl2 was stirred to provide the title compound as
yellow solid (0.05 g, 44% yield); mp 184–186 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm) d 10.58 (s, 1H), 7.95 (d, J = 5.1 Hz, 1H), 7.31 (dd,
J1 = 7.5 Hz, J2 = 2.7 Hz, 1H), 7.29 (d, J = 5.1 Hz, 1H), 7.10 (td, J1 = 8.7 Hz,
J2 = 2.7 Hz, 1H), 6.88 (dd, J1 = 9.0 Hz, J2 = 5.7 Hz, 1H), 4.80 (q, J = 7.5 Hz,
1H), 3.69 (s, 3H), 1.18 (d, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C15H14N2O5FS2 [M+H+]: 385.0322, found 385.0313.
5.1.11.2. 6-Fluoro-3-((2-methylthio)-ethyl)-3,4-dihydroquinox-
alin-2-(1H)-one (4d). Following the preparation protocol of Sec-
tion 5.1.5.2, a solution of K2CO3 (553 mg g, 4.0 mmol), Na2S2O4
(2.09 g, 12 mmol) and compound 3d (605 mg, 2.0 mmol) in the
mixture of water and ethanol was stirred at room temperature
for 30 min to afford compound 4d (475 mg) as light yellow oil in
yield 99%. 1H NMR (300 MHz, CDCl3, d ppm) d 8.69 (s, 1H), 6.66
(dd, J1 = 7.5 Hz, J2 = 5.1 Hz, 1H), 6.44 (t, J = 8.4 Hz, 1H), 6.43 (d,
J = 8.7 Hz, 1H), 4.38 (s, 1H), 4.09 (m, 1H), 2.74–2.62 (m, 2H),
2.22–2.16 (m, 1H), 2.13 (s, 3H), 2.08–1.99 (m, 1H).
5.1.9. 6-Fluoro-3-methyl-4-(2,5-dichlorothiophene-3-sulfonyl)-
3,4-dihydroquinoxalin-2-(1H)-one (11)
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4b (0.045 g, 0.25 mmol), 2,5-dichlorothiophene-3-
sulfonyl chloride (0.19 g, 0.75 mmol) and pyridine (0.10 g,
1.25 mmol) in CH2Cl2 was stirred to provide the title compound
as yellow solid (0.075 g, 76%); mp 203–206 °C. 1H NMR
(400 MHz, CDCl3, d ppm) d 8.24 (s, 1H), 7.49 (dd, J1 = 9.2 Hz,
J2 = 2.8 Hz, 1H), 7.01 (td, J1 = 8.4 Hz, J2 = 2.8 Hz, 1H), 6.83 (s, 1H),
6.77 (dd, J1 = 8.8 Hz, J2 = 4.8 Hz, 1H), 4.92 (q, J = 7.2 Hz, 1H), 1.37
(d, J1 = 7.2 Hz, 1H); HRMS (ESI): m/z, calcd for C13H10N2O3FS2Cl2
[M+H+]: 394.9494, found 394.9531.
5.1.11.3. 6-Fluoro-3-((2-methylthio)-ethyl)-4-(2-methyoxycar-
bonylthiophene-3-sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one
(13). Following the preparation protocol of Section 5.1.7, the mix-
ture of compound 4d (48 mg, 0.2 mmol), 2-methyoxycarbonylthi-
ophene-3-sulfonyl chloride (144 mg, 0.6 mmol) and pyridine
(79 mg, 1.0 mmol) in anhydrous CH2Cl2 was stirred to provide
the title compound (20 mg) as yellow oil in yield 22%. 1H NMR
(300 MHz, CDCl3, d ppm) d 9.05 (s, 1H), 7.56 (d, J = 9.3 Hz, 1H),
7.41 (d, J = 5.1 Hz, 1H), 7.34 (d, J = 5.1 Hz, 1H), 6.89 (t, J = 7.8 Hz,
1H), 6.78 (m, 1H), 5.15 (dd, J1 = 9.9 Hz, J2 = 3.9 Hz, 1H), 3.76 (s,
3H), 2.64 (m, 2H), 2.06 (s, 3H), 1.96 (m, 2H); HRMS (ESI): m/z, calcd
for C17H17N2O5FS3Na [M+Na+]: 467.0181, found 467.0180.
5.1.10. 6-Fluoro-3-ethyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxa-lin-2-(1H)-one (12)
5.1.10.1. 2-(5-fluoro-2-nitrophenylamino)butanoic acid (3c). The
reaction mixture of K2CO3 (0.28 g, 2.0 mmol) and 2-aminobutanoic
acid (0.10 g, 1.0 mmol) in water (2 mL) and 2,4-difluoronitrobenzene
2a (0.19 g, 1.2 mmol) in ethanol (0.5 mL) was heated by microwave
(power 50 W, temperature 120 °C) for 30 min. The reaction mixture
was concentrated in vacuo. The residue was dissolved in water
(40 mL) and washed with ether (10 mL Â 3). The aqueous phase was
adjusted to pH 2 with 5% HCl aqueous solution, and then extracted with
EtOAc (20 mL Â 2). The organic layer was dried over anhydrous MgSO4,
concentrated and purified with column chromatography on silica gel
to afford compound 3c as yellow oil (0.075 g, yield 31.0%); 1H NMR
(300 MHz, CDCl3, d ppm) d 8.47 (d, J = 6.6 Hz, 1H), 8.26 (dd,
J1 = 9.0 Hz, J2 = 6.3 Hz, 1H), 6.44 (m, 2H), 4.16 (dd, J1 = 12.0 Hz,
J2 = 5.1 Hz, 1H), 2.05 (m, 2H), 1.10 (t, J = 7.2 Hz,, 3H).
5.1.12. 6-Fluoro-3-((2-methoxycarbonyl)-methyl)-4-(2-methy-
oxycarbonylthiophene-3-sulfonyl)-3,4-dihydroquinoxalin-2-
(1H)-one (14)
5.1.12.1. Dimethyl 2-(5-fluoro-2-nitrophenylamino)-succinate
(3e). Following the preparation protocol of Section 5.1.2.1, the
mixture of 2,4-difluoronitrobenzene 2a (0.79 g, 5.0 mmol), glu-
tamic acid dimethyl hydrochloride (1.19 g, 6.0 mmol) and DIEA
(1.55 g, 12 mmol) in CH3CN was stirred at room temperature and
then refluxed for 24 h to afford compound 3e (735 mg) as yellow
5.1.10.2. 6-Fluoro-3-ethyl-3,4-dihydroquinoxalin-2-(1H)-one
(4c). The mixture of compound 3c (0.073 g, 0.3 mmol) and 10% Pd–

2772
B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
oil in yield 49%. 1H NMR (300 MHz, CDCl3, d ppm) d 9.26 (br s, 1H),
8.25 (dd, J1 = 9.3 Hz, J2 = 6.0 Hz, 1H), 6.42–6.53 (m, 2H), 4.57 (m,
1H), 3.81 (s, 3H), 3.74 (s, 3H), 2.98 (d, J = 6.0 Hz, 2H).
in water and 2,4-dichloronitrobenzene 2b (1.15 g, 6.0 mmol) in
ethanol was heated by microwave (power 50 W, temperature
150 °C) for 30 min. The reaction mixture was concentrated in va-
cuo. The residue was dissolved in water (60 mL) and washed with
ether (10 mL Â 3). The aqueous phase was adjusted to pH 2 with
5% HCl aqueous solution, and the solid (380 mg) was precipitated
in yield 31%, mp 138–141 °C. 1H NMR (300 MHz, DMSO-d6, d
ppm) d 8.42 (d, J = 7.2 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H), 7.09 (d,
J = 2.1 Hz, 1H), 6.76 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 4.56 (m,
J1 = 6.9 Hz, 1H), 1.47 (d, J = 6.9 Hz, 3H).
5.1.12.2. 6-Fluoro-3-((2-methoxycarbonyl)-methyl)-3,4-dihy-
droquinoxalin-2-(1H)-one (4e). Following the preparation proto-
col of Section 5.1.2.2, a reaction mixture of compound 3e (735 mg,
2.45 mmol), HCOONH4 (2.32 g, 36.75 mmol) and 10% Pd–C (1.04 g,
0.49 mmol) in ethanol (40 mL) was stirred to afford the yellow solid
0.56 g, yield 95%, mp 165–169 °C. 1H NMR (300 MHz, CDCl3, d ppm) d
8.68 (d, J = 7.8 Hz, 1H), 8.25 (dd, J1 = 9.3 Hz, J2 = 6.0 Hz, 1H), 6.42–6.53
(m, 2H), 4.57 (m, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 2.98 (d, J = 6.0 Hz, 2H).
5.1.14.2. 6-Chloro-3-methyl-3,4-dihydroquinoxalin-2-(1H)-one
(4g). The preparation method is same as that of compound 3b. A
solution of K2CO3 (276 mg, 2.0 mmol), Na2S2O4 (1.04 g, 6.0 mmol)
and compound 3g (245 mg, 1.0 mmol) in the mixture of water
and ethanol was stirred at room temperature to afford compound
4g (73 mg) as light red solid in yield 37%, mp 119–121 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm) d 10.28 (s, 1H), 6.58–6.69 (m, 3H),
6.28 (s, 1H), 3.80 (q, J =6.6 Hz, 1H), 1.23 (d, J = 6.6 Hz, 3H).
5.1.12.3. 6-Fluoro-3-((2-methoxycarbonyl)-methyl)-4-(2-
methyoxycarbonylthiophene-3-sulfonyl)-3,4-dihydroquinoxa-
lin-2-(1H)-one (14). Following the preparation protocol of Section
5.1.2.3, the mixture of compound 4e(48 mg, 0.2 mmol) and 2-methy-
oxycarbonylthiophene-3-sulfonyl chloride (144 mg, 0.6 mmol) in
pyridine (5 mL) was stirred to provide the title compound (70 mg)
as yellow oil in yield 80%. 1H NMR (300 MHz, CDCl3, d ppm) d 8.52
(br s, 1H), 7.55 (dd, J1 = 9.6 Hz, J2 = 2.7 Hz, 1H), 7.41 (d, J = 5.4 Hz,
1H), 7.38 (d, J = 5.4 Hz, 1H), 6.87 (dd, J1 = 7.8 Hz, J2 = 2.7 Hz, 1H),
6.73 (dd, J1 = 8.7 Hz, J2 = 5.1 Hz, 1H), 5.60 (dd, J1 = 8.4 Hz, J2 = 5.1 Hz,
1H), 3.81 (s, 3H), 3.66 (s, 3H), 2.71 (m, 2H); HRMS (ESI): m/z, calcd
for C17H16N2O7FS2 [M+H+]: 443.0377, found 443.0380.
5.1.14.3. 6-Chloro-3-methyl-4-(2-methyoxycarbonylthiophene-
3-sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one (16). Following
the preparation protocol of Section 5.1.7, the mixture of compound
4g (39 mg, 0.20 mmol), 2-methyoxycarbonylthiophene-3-sulfonyl
chloride (24 mg, 0.1 mmol) and pyridine (40 mg, 0.5 mmol) in
anhydrous CH2Cl2 (2 mL) was stirred to provide the title compound
(22 mg) in yield 55%, light yellow solid, mp 193–194 °C. 1H NMR
(300 MHz, acetone-d6, d ppm) d 9.51 (s, 1H), 7.83 (d, J = 5.1 Hz,
1H), 7.63 (d, J = 2.4 Hz, 1H), 7.32 (d, J = 5.4 Hz, 1H), 7.25 (dd,
5.1.13. 6-Chloro-4-(2-methyoxycarbonylthiophene-3-sulfonyl)-
3,4-dihydroquinoxalin-2-(1H)-one (15)
5.1.13.1. Ethyl 2-(5-chloro-2-nitrophenyl)-acetate (3f). Follow-
ing the preparation protocol of Section 5.1.2.1, a solution of 2,4-
dichloronitrobenzene 2b (15.36 g, 80 mmol), glycine ethyl ester
hydrochloride (13.40 g, 96 mmol) and DIEA (24.81 g, 192 mmol)
in CH3CN (60 mL) was stirred at room temperature to afford com-
pound 3f (4.85 g) as yellow solid, yield 23%, mp 96–99 °C. 1H NMR
(300 MHz, CDCl3, d ppm) d 8.47 (br s, 1H), 8.16 (d, J = 9.6 Hz, 1H),
6.71 (m, 2H), 4.30 (q, J = 7.2 Hz, 2H), 4.06 (d, J = 5.4 Hz, 2H), 1.33
(t, J = 6.9 Hz, 3H).
J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 6.99 (d,
J =8.7 Hz, 1H), 5.00 (q,
J = 6.9 Hz, 1H), 3.76 (s, 3H), 1.29 (d, J = 7.2 Hz, 3H); HRMS (ESI):
m/z, calcd for C15H14N2O5S2Cl [M+H+]: 401.0027, found 401.0012.
5.1.15. 6-Chloro-3-ethyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one (17)
5.1.15. 1. 2-(5-Chloro-2-nitrophenylamino)butanoic acid (3h). Fol-
lowing the preparation protocol of Section 5.1.10.1, a reaction mixture
of K2CO3 (553 mg, 4.0 mmol) and 2-aminobutanoic acid (206 mg,
2.0 mmol) in water and 2,4-dichloronitrobenzene (461 mg, 2.4 mmol)
in ethanol was heated by microwave (power 50 W, temperature
180 °C) for 30 min. Compound 3h (108 mg) was obtained as brown
oil in yield 21%. 1H NMR (300 MHz, DMSO-d6, d ppm) d 8.41 (d,
J = 7.5 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.12 (d, J = 2.1 Hz,1H), 6.76 (dd,
J1 = 9.3 Hz, J2 = 2.1 Hz, 1H), 4.58 (m, 1H), 1.89 (m, 2H), 0.87 (t,
J = 7.5 Hz, 3H).
5.1.13.2. 6-Chloro-3,4-dihydroquinoxalin-2-(1H)-one (4f). Fol-
lowing the preparation protocol of Section 5.1.5.2, a solution of
K2CO3 (5.19 g, 37.52 mmol), Na2S2O4 (19.60 g, 112.56 mmol) and
compound 3f (4.85 g, 18.76 mmol) in the mixture of water and eth-
anol was stirred at room temperature to afford compound 4f
(1.92 g) as light red solid in yield 56%, mp 179–182 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm) d 10.32 (s, 1H), 6.67 (d, J = 8.4 Hz, 1H),
6.65 (d, J = 2.1 Hz, 1H), 6.56 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H), 6.20 (s,
1H), 3.73 (s, 2H).
5.1.15.2. 6-Chloro-3-ethyl-3,4-dihydroquinoxalin-2-(1H)-one
(4h). Fe power (247 mg, 4.2 mmol) was added to a solution of
compound 3h (108 mg, 0.42 mmol) in acetic acid (2 mL). The reac-
tion mixtur was refluxed for 1 h and cooled to room temperature.
Fe powder was filtered off and the filtrate was concentrated. The
residue was dissolved in EtOAc (20 mL) and washed with saturated
NaHCO3 (20 mL Â 2), dried over anhydrous MgSO4 and concen-
trated to give the crude product which was purified by column
chromatography. Compound 4h (43 mg) was obtained as light
brown solid in yield 49%. 1H NMR (300 MHz, DMSO-d6, d ppm) d
10.29 (s, 1H), 6.62 (m, 3H), 6.29 (s, 1H), 3.70 (m, 1H), 1.61 (m,
2H), 0.90 (t, J = 7.5 Hz, 3H).
5.1.13.3. 6-Chloro-4-(2-methyoxycarbonylthiophene-3-sulfo-
nyl)-3,4-dihydroquinoxalin-2-(1H)-one (15). Following the
preparation protocol of Section 5.1.7. the mixture of compound
4f (55 mg, 0.30 mmol), 2-methyoxycarbonylthiophene-3-sulfonyl
chloride (24 mg, 0.1 mmol) and pyridine (40 mg, 0.5 mmol) in
anhydrous CH2Cl2 (2 mL) was stirred to provide the title compound
(23 mg) in yield 59%, white solid, mp 158–159 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm) d 10.62 (s, 1H), 7.96 (d, J = 5.4 Hz,
1H), 7.42 (d, J = 2.4 Hz, 1H), 7.28 (dd, J1 = 9.6 Hz, J2 = 2.4 Hz, 1H),
7.25 (d, J = 5.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 4.41 (s, 2H), 3.71
(s, 3H); HRMS (ESI): m/z, calcd for C13H10N2O6NaS3 [M+Na+]:
408.9599, found 408.9607.
5.1.15.3. 6-Chloro-3-ethyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one (17). Following the
preparation protocol of Section 5.1.7, the mixture of compound 4h
(42 mg, 0.20 mmol), 2-methyoxycarbonylthiophene-3-sulfonyl
chloride (24 mg, 0.1 mmol) and pyridine (40 mg, 0.5 mmol) in
anhydrous CH2Cl2 (2 mL) was stirred to provide the title compound
(21 mg) in yield 51%, light yellow solid, mp 93–95 °C. 1H NMR
5.1.14. 6-Chloro-3-methyl-4-(2-methyoxycarbonylthiophene-
3-sulfonyl)-3,4-dihydroquinoxa-lin-2-(1H)-one (16)
5.1.14.1. 2-(5-Chloro-2-nitrophenylamino)propanoic acid (3g). Fol-
lowing the preparation protocol of Section 5.1.10.1, a reaction mix-
ture of K2CO3 (1.38 g, 10.0 mmol) and alanine (445 mg, 5.0 mmol)

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
2773
(300 MHz, acetone-d6, d ppm) d 9.48 (br s, 1H), 7.82 (d, 1H,
J = 5.1 Hz), 7.71 (d, 1H, J = 2.4 Hz), 7.31 (d, 1H, J = 5.4 Hz), 7.24
(dd, J1 = 8.7 Hz, J2 = 2.4 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.76 (dd,
J1 = 10.2 Hz, J2 = 4.8 Hz, 1H), 3.87 (s, 3H), 1.67 (m, 1H), 1.45 (m,
3H); HRMS (ESI): m/z, calcd for C18H15N3O3SCl [M+H+]: 388.0517,
found 388.0518.
5.1.19. 6-Chloro-3-ethyl-4-(quinoline-8-sulfonyl)-3,4-
1H), 1.01 (t, J = 7.5 Hz, 3H)
;
HRMS (ESI): m/z, calcd for
dihydroquinoxalin-2-(1H)-one (21)
C16H16N2O5S2Cl [M+H+]: 415.0183, found 415.0183.
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4h (42 mg, 0.20 mmol), quinoline-8-sulfonyl chloride
(137 mg, 0.6 mmol) and pyridine (79 mg, 1.0 mmol) in anhydrous
CH2Cl2 (2 mL) was stirred to provide the title compound (31 mg)
in yield 39%, white solid, mp 107–108 °C. 1H NMR (300 MHz, ace-
tone-d6, d ppm) d 9.16 (s, 1H), 8.88 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H),
8.41–8.48 (m, 2H), 8.22 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.90 (d,
J = 2.4 Hz, 1H), 7.70 (dd, J1 = 8.4 Hz, J2 = 7.5 Hz, 1H), 7.63 (dd,
J1 = 8.4 Hz, J2 = 4.2 Hz, 1H), 6.97 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H),
6.69 (d, J = 8.4 Hz, 1H), 5.32 (dd, J1 = 10.8 Hz, J2 = 4.8 Hz, 1H), 1.83
(m, 1H), 1.53 (m, 1H), 1.11 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd
for C19H17N3O3SCl [M+H+]: 402.0673, found 402.0675.
5.1.16. 7-Trifluoromethyl-4-(2-methyoxycarbonylthiophene-3-
sulfonyl)-3,4-dihydroquinoxa-lin-2-(1H)-one (18)
5.1.16.1. Ethyl2-(4-trifluoromethyl-2-nitrophenyl)-acetate(3i). Fol-
lowing the preparation protocol of Section 5.1.2.1, a solution of
2-chloro-5-trifluoromethylnitrobenzene 2c (9.02 g, 40 mmol),
glycine ethyl ester hydrochloride (6.70 g, 48 mmol) and DIEA
(12.4 g, 96 mmol) in CH3CN (60 mL) was stirred at room temper-
ature for 24 h and then refluxed for 48 h to afford compound 3i
(8.81 g) as yellow needle crystal, yield 75%, mp 109–111 °C.
5.1.16.2. 7-Trifluoromethyl-3,4-dihydroquinoxalin-2-(1H)-one
(4i). Following the preparation protocol of Section 5.1.2.2, a reac-
tion mixture of compound 3j (10.69 g, 36.58 mmol), HCOONH4
(34.60 g, 548.75 mmol) and 10% Pd–C (15.51 g, 7.32 mmol) in eth-
anol (600 mL) was stirred to afford the yellow solid 6.94 g, yield
88%, mp 150–152 °C. 1H NMR (400 MHz, DMSO-d6, d ppm) d
10.43 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.96 (s, 1H), 6.72 (d,
J = 8.4 Hz, 1H), 6.61 (s, 1H), 3.84 (s, 2H).
5.1.20. 7-Trifluoromethyl-4-(quinoline-8-sulfonyl)-3,4-
dihydroquinoxalin-2-(1H)-one (22)
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4i (32 mg, 0.15 mmol), quinoline-8-sulfonyl chloride
(102 mg, 0.45 mmol) and pyridine (59 mg, 0.75 mmol) in anhy-
drous CH2Cl2 (5 mL) was stirred to provide the title compound
(60 mg) in yield 97%, light yellow solid, mp 205–206 °C. 1H NMR
(300 MHz, CDCl3, d ppm) d 8.92 (dd, J1 = 4.2 Hz, J2 = 1.5 Hz, 1H),
8.45 (dd, J1 = 7.5 Hz, J2 = 1.2 Hz, 1H), 8.19 (dd, J1 = 8.1 Hz,
J2 = 1.8 Hz, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.64 (br s, 1H), 7.54 (t,
J = 7.5 Hz, 1H), 7.49 (dd, J1 = 8.4 Hz, J2 = 4.2 Hz, 1H), 7.21 (d,
J = 8.7 Hz, 1H), 6.76 (s, 1H), 4.94 (s, 2H); HRMS (ESI): m/z, calcd
for C18H13N3O3F3S [M+H+]: 408.0624, found 408.0604.
5.1.16.3. 7-Trifluoromethyl-4-(2-methyoxycarbonylthiophene-
3-sulfonyl)-3,4-dihydroquinoxalin-2-(1H)-one (18). Following
the preparation protocol of Section 5.1.7, the mixture of compound
4i (65 mg, 0.30 mmol), 2-methyoxycarbonylthiophene-3-sulfonyl
chloride (24 mg, 0.1 mmol) and pyridine (40 mg, 0.5 mmol) in
anhydrous CH2Cl2 (2 mL) was stirred to provide the title compound
(28 mg) in yield 66%, light yellow solid, mp 161–162 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm) d 10.77 (s, 1H), 7.98 (d, J = 5.4 Hz,
1H), 7.61 (d, J = 5.4 Hz,1H), 7.38 (dd, J1 = 8.7 Hz, J2 = 1.2 Hz, 1H),
7.33 (d, J = 5.1 Hz, 1H), 7.16 (d, J = 1.5 Hz, 1H), 4.48 (s, 2H), 3.69
(s, 3H); HRMS (ESI): m/z, calcd for C16H11NO4F4S2 [M+H+]:
421.0060, found 421.0062.
5.2. Anti-HIV activity assay
Vesicular stomatitis virus glycoprotein (VSV-G) plasmid was co-
transfected with env-deficient HIV-1 vector, pNL4-3.luc.R-E-,14,15
into 293 cells by using modified Ca3(PO4)2 method.16 Briefly, 293
cells (100 mm plate) were co-transfected with 8
lg HIV-1 vector
and 3 g VSVG DNA. After 16 h, plates were washed by PBS, and
l
5.1.17. 6-Chloro-4-(quinoline-8-sulfonyl)-3,4-dihydro-
quinoxalin-2-(1H)-one (19)
fresh media DMEM with 10% FBS was added into the plates. Forty
eight hours post-transfection, supernatant was harvested and fil-
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4f (37 mg, 0.20 mmol), quinoline-8-sulfonyl chloride
(137 mg, 0.6 mmol) and pyridine (79 mg, 1.0 mmol) in anhydrous
CH2Cl2 (2 mL) was stirred to provide the title compound (31 mg)
in yield 41%, light yellow solid, mp 220–221 °C. 1H NMR
(300 MHz, CDCl3, d ppm) d 8.94 (dd, J1 = 4.2 Hz, J2 = 1.5 Hz, 1H),
8.46 (dd, J1 = 8.1 Hz, J2 = 1.2 Hz, 1H), 8.18 (dd, J1 = 8.1 Hz,
J2 = 1.8 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 2.4 Hz, 1H),
7.57 (t, J1 = 8.1 Hz, 1H), 7.49 (dd, J1 = 12.9 Hz, J2 = 8.7 Hz, 1H),
7.46 (s, 1H), 6.93 (m, 1H), 6.43 (d, J = 8.4 Hz, 1H), 4.89 (s, 2H);
HRMS (ESI): m/z, calcd for C17H13N3O3SCl [M+H+]: 374.0366, found
374.0383.
tered through a 0.45 lm filter. The supernatant contained VSVG/
HIV-1 pseudotyped virions which were quantified by p24 concen-
trations which were detected by ELISA (ZeptoMetrix,
Cat.:0801111). VSVG/HIV-1 viral solution was diluted to 0.2 ng
p24/mL and can be used directly or stored at À80 °C.
One day prior to infection, 293ET cells were plated on 24-well
plates at the density of 6 Â 104 cells per well. Compound was incu-
bated with target cells for 15 min prior to adding VSVG/HIV-
1(0.5 mL/well) for infection. The same amount of solvent alone
was used as control. After post-infection for 48 h, cells were lysed
in 50 lL cell lysis reagent (Promega). Luciferase activity of the cell
lysate was measured by a FB15 luminometer (Berthold Detection
System) according to the manufacture’s instructions.
5.1.18. 6-Chloro-3-methyl-4-(quinoline-8-sulfonyl)-3,4-
dihydroquinoxalin-2-(1H)-one (20)
5.3. Computational studies
Following the preparation protocol of Section 5.1.7, the mixture
of compound 4g (20 mg, 0.10 mmol), quinoline-8-sulfonyl chloride
(68 mg, 0.3 mmol) and pyridine (40 mg, 0.5 mmol) in anhydrous
CH2Cl2 (2 mL) was stirred to provide the title compound (32 mg)
in yield 82%, white solid, mp 214–215 °C. 1H NMR (300 MHz, ace-
tone-d6, d ppm) d 9.18 (s, 1H), 8.88 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H),
8.45 (m, 2H), 8.23 (dd, J1 = 8.1 Hz, J2 = 1.5 Hz, 1H), 7.88 (d,
J = 2.1 Hz, 1H), 7,70 (dd, J1 = 8.4 Hz, J2 = 7.5 Hz, 1H), 7.63 (dd,
J1 = 8.4 Hz, J2 = 4.2 Hz, 1H), 6.99 (dd, J1 = 8.7 Hz, J2 = 2.1 Hz, 1H),
6.72 (d, J = 8.4 Hz, 1H), 5.57 (q, J = 7.2 Hz, 1H), 1.40 (d, J = 7.2 Hz,
All calculations and manipulations were performed using FlexX
software package integrated in SYBYL 6.911, running on SGI Fuel
workstation. The X-ray crystal structure of reverse transcriptase
complexed with efavirenz was retrieved from PDB (PDB code:
1FK9).12 In FlexX docking, the Receptor Description File (RDF) de-
scribed the active site environment. It contains the information
about the protein, its amino acids, the active site, non-amino acid
residues, and specific torsion angles. The active site was defined
as all residues within 6.5 Å radius of the cocrystallized efavirenz.

2774
B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 2767–2774
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Acknowledgments
This work is supported by the drug discovery and development
foundation of the Institute of Materia Medica, Chinese Academy of
Medical Science. We are grateful to Professor Yanshen Guo for his
helpful discussion on molecular modeling.
References and notes
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