Synthesis and biological evaluation of HQCAs with aryl or benzyl substituents on N-1 position as potential HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.07.037

A series of new 5-hydroxylquinolone-3-carboxylic acids (HQCAs) with various aryl or benzyl substituents on N-1 position were synthesized and evaluated for their anti-HIV activity in C8166 cell culture. Most of the target compounds displayed activity against wide-type HIV-1 in the low micromolar range in infected C8166 cells. The most active compound 5g exhibited activity against wild-type HIV-1 and HIV-1 mutant virus A17 with an EC₅₀ value of 3.17 and 17.88 μM, respectively. The biological results and the docking study revealed that the substitution pattern on N-1 position of the quinolone core might contribute to physicochemical properties of HQCAs and resulted in great influence on their antiviral potency.

Full text of DOI:10.1016/j.bmc.2011.07.037

Bioorganic & Medicinal Chemistry 19 (2011) 5553–5558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of HQCAs with aryl or benzyl substituents
on N-1 position as potential HIV-1 integrase inhibitors
Qiu-Qin He a, Xuan Zhang b, Hai-Qiu Wu a, Shuang-Xi Gu a, Xiao-Dong Ma a, Liu-Meng Yang b,
Yong-Tang Zheng b, Fen-Er Chen a,c,
⇑
a Department of Chemistry, Fudan University, Shanghai 200433, PR China
b Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, PR China
c Institute of Biomedical Science, Fudan University, Shanghai 200433, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 5-hydroxylquinolone-3-carboxylic acids (HQCAs) with various aryl or benzyl substituents
on N-1 position were synthesized and evaluated for their anti-HIV activity in C8166 cell culture. Most of
the target compounds displayed activity against wide-type HIV-1 in the low micromolar range in infected
C8166 cells. The most active compound 5g exhibited activity against wild-type HIV-1 and HIV-1 mutant
Received 19 June 2011
Revised 18 July 2011
Accepted 20 July 2011
Available online 27 July 2011
virus A17 with an EC50 value of 3.17 and 17.88 lM, respectively. The biological results and the docking
study revealed that the substitution pattern on N-1 position of the quinolone core might contribute to
physicochemical properties of HQCAs and resulted in great influence on their antiviral potency.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
HIV
Integrase inhibitor
Quinolone-3-carboxylic acids
Two-metal chelating mechanism
Structure–activity relationships
1. Introduction
2. Chemistry
As a promising therapeutic HIV-1 integrase (IN) inhibitor,
Elvitegravir1a,b (1, Fig. 1) has drawn a lot of attention due to its
favorable pharmacokinetic property and clinical profiles. A variety
of 1 analogs such as 4-oxoquinoline 3-carboxylic acids (OQCAs),2a,b
4-oxonaphthyridine 3-carboxylic acids (ONCAs)3 and 5-hydrox-
ylquinolone-3-carboxylic acids (HQCAs)4 (2–4, Fig. 1) have been
developed in an effort to obtain more potent and selective IN inhib-
itors. Although considerable linear, branched and cyclic alkyl
groups have been introduced on N-1 of these analogs, the effect
of aryl or benzyl group at N-1 on biological activity have not yet
been studied.
Two series of HQCAs (N-aryl-substituted compounds 5a–e and
N-benzyl-substituted compounds 5f–m) were synthesized as
depicted in Scheme 1. The key intermediate acrylates 9a–b were
prepared from methyl 2,6-difluoro-3-iodobenzoate similar to our
previously reported protocol.4 Substitution of 9a–b with appropri-
ate aryl amines or benzyl amines led to acrylates 10a–m, which
were then cyclized using DBU as a base to furnish quinolone esters
11a–m. The target compounds 5a–e were obtained from 11a–e by
ester hydrolysis and subsequent 5-OH incorporation under basic
conditions. The target compounds 5f–m was synthesized from
11f–m by methoxyl substitution with sodium methoxide and then
demethylation with BBr3.
HQCAs,4 recently reported by us, have shown anti-HIV-1 activ-
ity with low micromolar to submicromolar EC50 values. Further
docking study revealed that the anti-HIV activity of these com-
pounds might involve a two-metal chelating mechanism. With
the aim of contributing to a better understanding of the struc-
ture–activity relationships (SAR) of HQCAs, a series of new HQCAs
with various aryl or benzyl substituents on N-1 (5a–m, Fig. 1) were
synthesized and evaluated as potential IN inhibitors.
3. Results and discussion
3.1. Biological evaluation
The synthesized compounds 5a–m were evaluated by
3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide
(MTT) assay5 for cytotoxicity and antiviral activity in C8166 cells
infected with the wild-type HIV-1 (LAI strain IIIB), and the com-
pounds 5b–m were also assayed for their biological activity against
HIV-1 mutant virus (A17). Compound 4k4 ðR100 ¼ 2S-1-hydroxy-3-
methylbutan-2-ylÞ and Elvitegravir were included as reference
⇑
Corresponding author. Tel.: +86 21 65643809; fax: +86 21 65643811.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.037

5554
Q.-Q. He et al. / Bioorg. Med. Chem. 19 (2011) 5553–5558
F
O
N
OH
O
OH
O
OH
Cl
O
R3
R2
R3'
R2'
O
O
O
N
N
N
R1
R1'
OH
OQCAs
2
ONCAs
3
Elvitegravir
1
OH
O
N
OH
O
F
OH
O
OH
Cl
O
R
N
R1''
n
R'
n=0, 1
HQCAs
4
5a-m
Figure 1. Chemical structures of HIV-1 IN inhibitors.
compounds. The cytotoxicity and antiviral activities of these com-
pounds are listed in Table 1.
HIV-1 IN. The substituted benzyl group and quinolone ring exhibit
stacking interaction with C16 and A17, respectively. No inter-
p–p
First we compared the antiviral activity of the compounds with
N-substituted benzene ring 5a and N-substituted benzyl group 5g
against wild-type HIV-1, the biological results showed that 5a dis-
played much weak active than that of 5g.
action could be detected between the substituent on N-1 and IN,
suggesting that this part of the molecule might just contribute to
favorable physicochemical properties of 5c and 5g.
Next we examined the effect of introducing substituents into
the benzene ring and benzyl group of 5a and 5g, respectively.
Interestingly, the introduction of methyl group or bromide atom
at the meta or para position of the benzene ring (5b–e) led to sig-
nificantly increased activity against wild-type HIV-1, however, the
introduction of methyl group or fluoro atom at the ortho, meta or
para position of the benzene ring (5h–m) caused decreased antivi-
ral activity. And compound 5c incorporated a methyl group at the
para position of 5a was approximately 80 times more potent than
that of 5a and showed EC50 value similar to that of 5g. The intro-
duction of a second methyl group (5d) at the meta position of 5c
caused a decrease of antiviral activity against wild-type HIV-1
but enhanced activity against HIV-1 mutant virus A17.
To confirm the effect of 2-fluoro and 3-chloro substituents on
the C-6 benzyl group, we replaced the chloro substituent at 3-posi-
tion of 5g and 5j with a hydrogen atom (5f and 5i). 2-Fluoro-3-
chlorobenzyl compounds 5g and 5j appeared to be more active
against wild-type HIV-1 than the corresponding 2-fluorobenzyl
compounds 5f and 5i, respectively. And the compound 5j also dis-
played slightly stronger activity against A17 than that of 5i.
4. Conclusion
In conclusion, we designed and synthesized a series of new
HQCAs with various aryl or benzyl substituents on N-1 position
of a quinolone. All the target compounds exhibited activity against
wide-type HIV-1 in the low micromolar range in infected C8166
cells. The most active compound 5g exhibited activity against
wild-type HIV-1 and HIV-1 mutant virus A17 with an EC50 value
of 3.17 and 17.88 lM, respectively. Although in all cases, the mea-
sured activities against wild-type HIV-1 were lower than that of 4k
and Elvitegravir. The biological results showed that variation in
substitution at N-1 position of the quinolone core had great influ-
ence on antiviral potency of HQCAs. Interestingly, no interaction
between the substituent on N-1 and IN could be detected from
the docking study. These findings suggested that the substitution
pattern on N-1 position of the quinolone core might contribute
to physicochemical properties of HQCAs and strongly affect their
antiviral activity, which is consistent with the observations previ-
ously reported by us4 and Dayam et al.6
5. Experimental
3.2. Molecular modeling calculations
5.1. General procedures
With the aim to investigate the binding model of our newly
synthesized compounds with IN, molecular docking study was
performed.
Melting points were measured on a WRS-1 digital melting point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra on a
Brucker AV 400 MHz spectrometer were recorded in CDCl3. Chem-
ical shifts are reported in d (ppm) units relative to the internal
standard tetramethylsilane (TMS). Mass spectra were obtained
on an Agilent MS/5975 mass spectrometer. All chemicals and sol-
vents used were of reagent grade and were purified and dried by
standard methods before use. All air-sensitive reactions were run
under a nitrogen atmosphere. All the reactions were monitored
by TLC on pre-coated silica gel G plates at 254 nm under a UV lamp
using ethyl acetate/hexane as eluents. Flash chromatography sep-
arations were obtained on silica gel (300–400 mesh).
Compounds 5c and 5g, which displayed the most activity
against wild-type HIV-1 of N-aryl-substituted HQCAs and N-ben-
zyl-substituted HQCAs, were docked into our previously con-
structed model of the HIV-1 IN catalytic core domain (CCD)/viral
DNA complex4 using SURFLEX-DOCK SYBYLX 1.1. The docking re-
sults showed that both 5c and 5g positioned a similar orientation
in the active site of the homology model of HIV-1 IN (Fig. 2). As
shown in the previous theoretical binding model of compound
4k with IN,4 the C-5 hydroxyl, 4-ketone and 3-carboxylate in
compounds 5c and 5g also could form a two-Mg2+ chelation with

Q.-Q. He et al. / Bioorg. Med. Chem. 19 (2011) 5553–5558
5555
F
O
F
F
O
F
F
O
F
I
b
a
O
OH
O
R
R
F
6a-b
7a-b
F
O
F
O
O
O
N
c
e
d
O
O
R
R
F
8a-b
9a-b
F
O
O
F
O
N
O
O
f
O
R
R
F
NH
n
n
R'
R'
10a-m
n=0, 1
11a-m
O
O
N
O
OH
O
N
O
h
OH
OH
R
R
g
n=0
R'
R'
12a-e
5a-e
11a-m
i
n=1
F
O
O
OH
O
N
O
j
OH
OH
R
R
N
R'
R'
12f-m
5f-m
Scheme 1. Reagents and conditions: (a) (1) 3-chloro-2-flurobenzyl bromide, Zn, 1,2-dibromoethane, chlorotrimethylsilane, THF, 60 °C, 1 h, (2) Pd(PPh3)4, toluene, reflux,
overnight; (b) satd aq LiOH, THF, 50 °C, 3 h; (c) (1) CDI, THF, rt, 2 h; (2) potassium methylmalonate, MgCl2, 60 °C, overnight; (d) DMFDMA, THF, 50 °C, 3 h; (e) aryl amines or
benzyl amines, THF, 60 °C, 3–8 h; (f) DBU, DMF, 90 °C, overnight; (g) MeONa, toluene, DMF, reflux, 5–10 h; (h) BBr3, CH2Cl2, ꢁ40 °C to rt, overnight; (i) satd aq LiOH, dioxane,
50 °C, 3 h; (j) 12.5 M NaOH, dioxane, 80 °C, 1–2 days.
5.2. General procedure for the synthesis of 6a–b
residue was purified by column chromatography (silica gel, petro-
leum ether/ethyl acetate 40/1 to 25/1, v/v) to give the desired
compound.
Under N2, zinc powder (156 mg, 2.4 mmol) was suspended in
THF (5 mL), then 1,2-dibromoethane (catalytic amount) and
trimethylsilyl chloride (catalytic amount) were added at 60 °C.
After being stirred for 30 min, a solution of 3-chloro-2-fluoroben-
zyl bromide (488 mg, 2.2 mmol) or 2-fluorobenzyl bromide
(414 mg, 2.2 mmol) in THF (3 mL) was added dropwise at 60 °C.
The mixture was stirred for further 1 h to give the solution of
benzylzinc bromide. To a mixture of methyl 2,6-difluoro-3-iodo-
benzoate (596 mg, 2.0 mmol) and Pd(PPh3)4 (13 mg, 0.011 mmol)
in toluene (10 mL) was added the above solution of benzylzinc
bromide dropwise at 60 °C under N2. After completion of the
addition, the mixture was heated under reflux overnight. After
allowing the mixture to cool, toluene (15 mL) and 20% aqueous
NH4Cl solution (10 mL) were added to the reaction solution, and
the mixture was stirred and partitioned. The organic layer was
washed twice with 20% aqueous NH4Cl solution (5 mL) and twice
with satd NaHCO3 (5 mL) and then dried over MgSO4. After filtra-
tion, the filtrate was concentrated under reduced pressure. The
Compound 6a4 (R = 2-F, 3-Cl, 290 mg, 46%): colorless oil; 1H
NMR (CDCl3): d 3.95 (s, 3H, CH3), 4.00 (s, 2H, CH2), 6.87–6.92 (td,
1H, J = 8.8, J0 = 1.6 Hz, ArH), 6.99–7.03 (m, 1H, ArH), 7.05–7.09 (m,
1H, ArH), 7.24–7.30 (m, 2H, ArH).
Compound 6b (R = 2-F, 280 mg, 50%): 1H NMR (CDCl3): colorless
oil; 1H NMR (CDCl3): d 3.97 (s, 3H, CH3), 4.01 (s, 2H, CH2), 6.87–6.92
(td, 1H, J = 8.8, J0 = 1.2 Hz, ArH), 7.04–7.11 (m, 2H, ArH), 7.17–7.29
(m, 3H, ArH).
5.3. General procedure for the synthesis of 7a–b
To a solution of 6a–b (1.0 mmol) in THF (8 mL) was added satd
aq LiOH (5 mL). After being stirred at 50 °C for 3 h, the mixture was
cooled, poured into ice-water and acidified with 4 M HCl to pH ꢀ 2.
The precipitate was filtered off, washed by water, and dried to af-
ford 7 as a white solid. This crude was used directly for the next
step without further purification.

5556
Q.-Q. He et al. / Bioorg. Med. Chem. 19 (2011) 5553–5558
Table 1
CH3), 3.95 (s, 2H, CH2), 4.03 (s, 2H, CH2), 6.91–6.96 (m, 1H, ArH),
7.02–7.12 (m, 2H, ArH), 728–7.34 (m, 2H, ArH); Enol form 1H
NMR (CDCl3) d 3.83 (s, 1.3H, CH3), 4.03 (s, 0.9H, CH2), 5.44 (s,
0.4H, CH), 6.89–6.96 (m, 0.4H, ArH), 7.02–7.12 (m, 0.9H, ArH),
7.21–7.34 (m, 0.9H, ArH), 12.31 (s, 0.4H, OH).
Compound 8b (R = 2-F, 335 mg, 52%): colorless oil; Keto form/
Enol form = 2/1; Keto form 1H NMR (CDCl3) d 3.74 (s, 3H, CH3),
3.95 (s, 2H, CH2), 4.02 (s, 2H, CH2), 6.89–6.93 (m, 1H, ArH), 7.00–
7.12 (m, 2H, ArH), 7.18–7.29 (m, 3H, ArH); Enol form 1H NMR
(CDCl3) d 3.83 (s, 1.5H, CH3), 4.02 (s, 1H, CH2), 5.44 (s, 0.5H, CH),
6.87–6.91 (m, 0.5H, ArH), 7.00–7.12 (m, 1H, ArH), 7.18–7.29 (m,
1.5H, ArH), 12.31 (s, 0.5H, OH).
Anti-HIV-1 activity and cytotoxicity of compounds in C8166 cellsa
b
c
Compd
R
R0
EC50
(
l
M)
CC50
M)
SId
(
l
WT (IIIB)
A17
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
4k
1
2-F, 3-Cl
2-F, 3-Cl
2-F, 3-Cl
2-F, 3-Cl
2-F, 3-Cl
2-F
2-F, 3-Cl
2-F, 3-Cl
2-F
2-F, 3-Cl
2-F, 3-Cl
2-F, 3-Cl
2-F, 3-Cl
H
307.57
20.48
3.58
24.93
7.12
20.87
3.17
5.55
6.46
3.27
4.93
9.34
NDe
388.14
71.55
193.88
315.73
84.97
156.28
20.34
255.51
67.14
44.91
69.04
1.26
3.49
54.16
12.66
11.93
7.49
3-CH3
4-CH3
3,4-2CH3
4-Br
H
H
4-CH3
2-F
2-F
3-F
4-F
3,4-2F
43.68
67.88
32.81
33.24
51.19
17.88
29.25
27.22
24.23
19.08
22.52
24.25
0.085
6.42
46.04
10.39
13.73
14.00
7.04
5.5. General procedure for the synthesis of 9a–b
65.79
71.35
125.03
5.74
0.13
12.43
961.77
A
mixture of 8a–b (0.9 mmol) and DMFMDA (129 mg,
0.00021 0.0010
17.20 81,904.76
1.1 mmol) in THF (10 mL) was heated at 50 °C for 3 h. The reaction
mixture was cooled to room temperature and then concentrated
under reduced pressure. The residue was used directly for the next
step without further purification.
a
All data represent mean values for at least two separate experiments.
Compound concentration required to protect the cell against viral cytopath-
b
ogenicity by 50% in C8166 cells.
c
Compound concentration that decreases the normal uninfected C8166 cell
viability by 50%.
5.6. General procedure for the preparation of 10a–m
d
Selectivity index; ratio CC50/EC50
Not detected.
.
e
A mixture of 9a–b (1.5 mmol) and aryl amines or benzyl amines
(1.8 mmol) in THF (15 mL) was stirred at 60 °C for 3–8 h and then
concentrated under reduced pressure. The resulting residue 10a–m
was used directly for the next step without further purification.
5.7. General procedure for the preparation of 11a–m
A mixture of 10a–m (1.5 mmol) and DBU (2.3 mmol) in DMF
(10 mL) was stirred at 90 °C overnight, then cooled down and
poured into ice-water. The mixture was extracted by dichloro-
methane (5 mL ꢂ 3). The combined organic solution was washed
with brine (5 mL ꢂ 2), dried over MgSO4 and concentrated under
reduced pressure. The residue was recrystallized from ethyl ace-
tate to give the desired compound 11a–m.
5.8. General procedure for the preparation of 12a–e
To a solution of MeONa (270 mg, 5 mmol) in toluene (15 mL)
was added 11a–e (1.0 mmol) and one drop of DMF. After being
stirred at reflux for 5–10 h, the mixture was cooled, poured into
ice-water and acidified with 4 M HCl to pH ꢀ 2, extracted by ethyl
acetate (10 mL ꢂ 3) and concentrated to give the desired
compound 12a–e.
Figure 2. Binding model of compounds 5c (cyan) and 5g (orange) in the active site
of new homoly model of HIV-1 IN.
5.9. General procedure for the preparation of 12f–m
5.4. General procedure for the synthesis of 8a–b
To a solution of satd aq LiOH (5 mL) in dioxane (8 mL) was
added 11f–m (1.2 mmol). After being stirred at 50 °C for 3 h, the
mixture was cooled, poured into ice-water and acidified with
4 M HCl to pH ꢀ 2. The resulting precipitate was collected by filtra-
tion, washed by water, and then dried to give the crude desired
compound 12f–m. This crude was used directly for the next step
without further purification.
To a suspension of CDI (389 mg, 2.4 mmol) in THF (5 mL) was
added a solution of 7a–b (2.0 mmol) in THF (5 mL) dropwise. The
resulting mixture was stirred at room temperature for 2 h, and
then potassium 3-methoxy-3-oxopropanoate (374 mg, 2.4 mmol)
and MgCl2 (228 mg, 2.4 mmol) were added portion-wise. The reac-
tion mixture was stirred at 60 °C overnight. The resulting precipi-
tate was removed by filtration, and the filtrate was diluted with
water (5 mL) and acidified with 2 M HCl to pH 6–7. The mixture
was extracted with ethyl acetate (15 mL) and washed with water
(5 mL), satd aq NaHCO3 (5 mL), and brine (5 mL), then dried over
MgSO4. After filtration, the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography (sil-
ica gel, petroleum ether/ethyl acetate 20/1 to 15/1, v/v) to give the
desired compound.
5.10. General procedure for the preparation of 5a–e
To a solution of 12a–e (1 mmol) in dichloromethane (10 mL)
was added BBr3 (2 M, 10 mmol) dropwise at ꢁ40 °C. After being
stirred for 1 h, the mixture was allowed to warm to rt and stirred
overnight. Ice-water (8 mL) was added dropwise to the reaction
solution. The resulting mixture was extracted by dichloromethane
(10 mL ꢂ 3), washed with 5% aqueous NaHCO3 solution (5 mL),
dried and concentrated to give the desired compound 5a–e.
Compound 8a4 (R = 2-F, 3-Cl, 320 mg, 45%): colorless oil; Keto
form/Enol form = 2.3/1; Keto form 1H NMR (CDCl3) d 3.75 (s, 3H,

Q.-Q. He et al. / Bioorg. Med. Chem. 19 (2011) 5553–5558
5557
5.10.1. 6-(3-Chloro-2-fluorobenzyl)-5-hydroxy-4-oxo-1-phenyl-
1,4-dihydroquinoline-3-carboxylic acid (5a)
2H, ArH), 7.13–7.22 (m, 3H, ArH), 7.27–7.29 (m, 1H, ArH), 7.36–
7.37 (m, 3H, ArH), 7.40–7.42 (d, J = 8.8 Hz, 1H, ArH), 8.81 (s, 1H,
CH), 13.26 (s, 1H, OH), 13.74 ppm (s, 1H, COOH). MS (ESI) m/z
426 [M+Na]+; Anal. Calcd for C24H18FNO4: C, 71.46; H, 4.50; N,
3.47. Found: C, 71.14; H, 4.71; N, 3.77.
Yield 82%. mp 218–220 °C; 1H NMR (CDCl3): d 4.09 (s, 2H, CH2),
6.44–6.46 (d, J = 8.8 Hz, 1H, ArH), 6.98–7.01 (t, J = 8.0 Hz, 1H, ArH),
7.18–7.25 (m, 2H, ArH), 7.37–7.39 (m, 2H, ArH), 7.40–7.42 (d,
J = 8.8 Hz, 1H, ArH), 7.62–7.64 (m, 3H, ArH), 8.72 (s, 1H, CH),
13.15 (s, 1H, OH), 13.62 ppm (s, 1H, COOH). MS (ESI) m/z 446
[M+Na]+; Anal. Calcd for C23H15ClFNO4: C, 65.18; H, 3.57; N, 3.30.
Found: C, 64.92; H, 3.89; N, 3.59.
5.11.2. 6-(3-Chloro-2-fluorobenzyl)-1-benzyl-5-hydroxy-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (5g)
Yield 60%. mp 216–218 °C; 1H NMR (CDCl3): d 4.05 (s, 2H, CH2),
5.43 (s, 2H, CH2), 6.86–6.88 (d, J = 7.6 Hz, 1H, ArH), 6.97–6.98 (m,
1H), 7.15–7.26 (m, 5H), 7.36–7.37 (m, 2H), 7.43–7.45 (d, J = 7.2 Hz,
1H, ArH), 8.82 (s, 1H, CH), 13.26 (s, 1H, OH), 13.68 ppm (s, 1H,
COOH). MS (ESI) m/z 460 [M+Na]+; Anal. Calcd for C24H17ClFNO4:
C, 65.83; H, 3.91; N, 3.20. Found: C, 65.61; H, 4.16; N, 3.44.
5.10.2. 6-(3-Chloro-2-fluorobenzyl)-5-hydroxy-4-oxo-1-m-tolyl-
1,4-dihydroquinoline-3-carboxylic acid (5b)
Yield 80%. mp 206–208 °C; 1H NMR (CDCl3): d 2.48 (s, 3H, CH3),
4.10 (s, 2H, CH2), 6.50–6.52 (d, J = 8.8 Hz, 1H, ArH), 6.99–7.03 (t,
J = 8.0 Hz, 1H, ArH), 7.19–7.20 (m, 2H, ArH), 7.22–7.26 (m, 2H,
ArH), 7.42–7.46 (m, 2H, ArH), 7.49–7.53 (m, 1H, ArH), 8.72 (s, 1H,
CH), 13.16 (s, 1H, OH), 13.72 ppm (s, 1H, COOH). MS (ESI) m/z
460 [M+Na]+; Anal. Calcd for C24H17ClFNO4: C, 65.83; H, 3.91; N,
3.20. Found: C, 66.14; H, 3.59; N, 3.44.
5.11.3. 6-(3-Chloro-2-fluorobenzyl)-1-(4-methylbenzyl)-5-hydr-
oxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5h)
Yield 62%. mp 238–240 °C; 1H NMR (CDCl3): d 2.34 (s, 3H, CH3),
4.06 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.87–6.90 (d, J = 8.8 Hz, 1H,
ArH), 6.97–7.01 (t, J = 8.0 Hz, 1H, ArH), 7.03–7.05 (d, J = 8.0 Hz,
2H, ArH), 7.16–7.18 (d, J = 8.0 Hz, 2H, ArH), 7.20–7.24 (m, 2H,
ArH), 7.44–7.46 (d, J = 8.8 Hz, 1H, ArH), 8.80 (s, 1H, CH), 13.28 (s,
1H, OH), 13.71 ppm (s, 1H, COOH). MS (ESI) m/z 474 [M+Na]+; Anal.
Calcd for C25H19ClFNO4: C, 66.45; H, 4.24; N, 3.10. Found: C, 66.73;
H, 4.02; N, 3.42.
5.10.3. 6-(3-Chloro-2-fluorobenzyl)-5-hydroxy-4-oxo-1-p-tolyl-
1,4-dihydroquinoline-3-carboxylic acid (5c)
Yield 78%. mp 234–236 °C; 1H NMR (CDCl3): d 2.51 (s, 3H, CH3),
4.11 (s, 2H, CH2), 6.48–6.50 (d, J = 8.8 Hz, 1H, ArH), 6.99–7.04 (t,
J = 8.0 Hz, 1H, ArH), 7.22–7.28 (m, 5H, ArH), 7.41–7.43 (d,
J = 8.0 Hz, 2H, ArH), 8.73 (s, 1H, CH), 13.18 (s, 1H, OH),
13.76 ppm (s, 1H, COOH). MS (ESI) m/z 460 [M+Na]+; Anal. Calcd
for C24H17ClFNO4: C, 65.83; H, 3.91; N, 3.20. Found: C, 65.63; H,
3.53; N, 3.40.
5.11.4. 6-(2-Fluorobenzyl)-1-(2-fluorobenzyl)-5-hydroxy-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (5i)
Yield 48%. mp 225–226 °C; 1H NMR (CDCl3): d 4.08 (s, 2H, CH2),
5.47 (s, 2H, CH2), 6.89–6.91 (d, J = 8.8 Hz, 1H, ArH), 7.02–7.23 (m,
6H, ArH), 7.30–7.32 (m, 1H, ArH), 7.36–7.41 (m, 1H, ArH),
7.47–7.49 (d, J = 8.8 Hz, 1H, ArH), 8.85 (s, 1H, CH), 13.28 (s, 1H,
OH), 13.71 ppm (s, 1H, COOH). MS (ESI) m/z 444 [M+Na]+; Anal.
Calcd for C24H17F2NO4: C, 68.41; H, 4.07; N, 3.32. Found: C,
68.71; H, 4.32; N, 3.07.
5.10.4. 6-(3-Chloro-2-fluorobenzyl)-1-(3,4-dimethylphenyl)-5-
hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5d)
Yield 81%. mp 210–212 °C; 1H NMR (CDCl3): d 2.36 (s, 3H, CH3),
2.40 (s, 3H, CH3), 4.10 (s, 2H, CH2), 6.51–6.54 (d, J = 8.8 Hz, 1H,
ArH), 6.99–7.03 (t, J = 8.0 Hz, 1H, ArH), 7.10–7.14 (m, 2H, ArH),
7.22–7.26 (m, 2H, ArH), 7.35–7.37 (d, J = 8.0 Hz, 1H, ArH), 7.41–
7.43 (d, J = 8.8 Hz, 1H, ArH), 8.72 (s, 1H, CH), 13.18 (s, 1H, OH),
13.76 ppm (s, 1H, COOH). MS (ESI) m/z 474 [M+Na]+; Anal. Calcd
for C25H19ClFNO4: C, 66.45; H, 4.24; N, 3.10. Found: C, 66.17; H,
3.92; N, 3.36.
5.11.5. 6-(3-Chloro-2-fluorobenzyl)-1-(2-fluorobenzyl)-5-hydr-
oxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5j)
Yield 52%. mp 215–217 °C; 1H NMR (CDCl3): d 4.07 (s, 2H, CH2),
5.45 (s, 2H, CH2), 6.89–6.91 (d, J = 8.8 Hz, 1H, ArH), 7.00–7.01 (m,
2H, ArH), 7.11–7.23 (m, 4H, ArH), 7.35–7.40 (m, 1H, ArH),
7.48–7.50 (d, J = 8.8 Hz, 1H, ArH), 8.83 (s, 1H, CH), 13.27 (s, 1H,
OH), 13.65 ppm (s, 1H, COOH). MS (ESI) m/z 478 [M+Na]+; Anal.
Calcd for C24H16ClF2NO4: C, 63.24; H, 3.54; N, 3.07. Found: C,
63.02; H, 3.84; N, 2.85.
5.10.5. 6-(3-Chloro-2-fluorobenzyl)-1-(4-bromophenyl)-5-
hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5e)
Yield 76%. mp 207–209 °C; 1H NMR (CDCl3): d 4.10 (s, 2H, CH2),
6.44–6.46 (d, J = 8.8 Hz, 1H, ArH), 7.00–7.03 (t, J = 8.0 Hz, 1H, ArH),
7.21–7.25 (m, 2H, ArH), 7.29–7.31 (d, J = 8.8 Hz, 2H, ArH), 7.44–
7.46 (d, J = 8.8 Hz, 1H, ArH), 7.78–7.80 (d, J = 8.8 Hz, 2H, ArH),
8.69 (s, 1H, CH), 13.11 (s, 1H, OH), 13.59 ppm (s, 1H, COOH). MS
(ESI) m/z 524 [M+Na]+; Anal. Calcd for C23H14BrClFNO4: C, 54.95;
H, 2.81; N, 2.79. Found: C, 54.74; H, 2.64; N, 3.09.
5.11.6. 6-(3-Chloro-2-fluorobenzyl)-1-(3-fluorobenzyl)-5-
hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5k)
Yield 53%. mp 214–215 °C; 1H NMR (CDCl3): d 4.06 (s, 2H, CH2),
5.42 (s, 2H, CH2), 6.79–6.81 (d, J = 8.8 Hz, 1H, ArH), 6.83–6.85 (d,
J = 8.8 Hz, 1H, ArH), 6.91–6.93 (d, J = 8.0 Hz, 1H, ArH), 6.97–7.01
(t, J = 8.0 Hz, 1H, ArH), 7.03–7.07 (m, 1H, ArH), 7.19–7.25 (m, 2H,
ArH), 7.33–7.38 (m, 1H, ArH), 7.45–7.47 (d, J = 8.8 Hz, 1H, ArH),
8.12 (s, 1H, CH), 13.24 (s, 1H, OH), 13.64 ppm (s, 1H, COOH). MS
(ESI) m/z 478 [M+Na]+; Anal. Calcd for C24H16ClF2NO4: C, 63.24;
H, 3.54; N, 3.07. Found: C, 62.93; H, 3.38; N, 3.27.
5.11. General procedure for the preparation of 5f–m
To a solution of 12.5 N NaOH (5 mL) in dioxane (8 mL) was
added 12f–m (1.5 mmol). After being stirred at 80 °C for 1–2 days,
the mixture was cooled, poured into water and acidified with 2 M
HCl to pH ꢀ 2, then extracted by dichloromethane (5 mL ꢂ 3). The
combined organic solution was washed with water (5 mL ꢂ 2),
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by column chromatography (silica gel, dichlo-
romethane) to afford the desired compound 5f–m.
5.11.7. 6-(3-Chloro-2-fluorobenzyl)-1-(4-fluorobenzyl)-5-
hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5l)
Yield 48%. mp 236–238 °C; 1H NMR (CDCl3): d 4.06 (s, 2H, CH2),
5.39 (s, 2H, CH2), 6.82–6.84 (d, J = 8.8 Hz, 1H, ArH), 6.97–7.01 (t,
J = 8.0 Hz, 1H, ArH), 7.05–7.10 (m, 2H, ArH), 7.13–7.16 (m, 2H,
ArH), 7.19–7.26 (m, 2H, ArH), 7.45–7.47 (d, J = 8.8 Hz, 1H, ArH),
8.80 (s, 1H, CH), 13.26 (s, 1H, OH), 13.65 ppm (s, 1H, COOH). MS
(ESI) m/z 478 [M+Na]+; Anal. Calcd for C24H16ClF2NO4: C, 63.24;
H, 3.54; N, 3.07. Found: C, 63.55; H, 3.36; N, 3.25.
5.11.1. 6-(2-Fluorobenzyl)-1-benzyl-5-hydroxy-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (5f)
Yield 58%. mp 208–210 °C; 1H NMR (CDCl3): d 4.05 (s, 2H, CH2),
5.41 (s, 2H, CH2), 6.83–6.85 (d, J = 8.8 Hz, 1H, ArH), 6.99–7.07 (m,

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5.11.8. 1-(3,4-Difluorobenzyl)-6-(3-chloro-2-fluorobenzyl)-5-
hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5m)
Yield 30%. mp 222–224 °C; 1H NMR (CDCl3): d 4.07 (s, 2H, CH2),
5.38 (s, 2H, CH2), 6.75–6.78 (d, J = 8.8 Hz, 1H, ArH), 6.87–6.90 (m,
1H, ArH), 6.96–7.02 (m, 2H, ArH), 7.16–7.23 (m, 3H, ArH),
7.46–7.48 (d, J = 8.8 Hz, 1H, ArH), 8.79 (s, 1H, CH), 13.24 (s, 1H,
OH), 13.61 ppm (s, 1H, COOH). MS (ESI) m/z 496 [M+Na]+; Anal.
Calcd for C24H15ClF3NO4: C, 60.84; H, 3.19; N, 2.96. Found: C,
61.10; H, 3.37; N, 2.68.
References and notes
1. (a) Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.; Ito, Y.;
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Larhed, M.; Witvrouw, M.; Michiels, M.; Christ, F.; Debyser, Z.; Corelli, F. J. Med.
Chem. 2008, 51, 5125; (b) Sato, M.; Kawakami, H.; Motomura, T.; Aramaki, H.;
Matsuda, T.; Yamashita, M.; Ito, Y.; Matsuzaki, Y.; Yamataka, K.; Ikeda, S.;
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3. Nagasawa, J. Y.; Song, J.; Chen, H.; Kim, H. W.; Blazel, J.; Ouk, S.; Groschel, B.;
Borges, V.; Ong, V.; Yeh, L. T.; Girardet, J. L.; Vernier, J. M.; Raney, A. K.; Pinkerton,
A. B. Bioorg. Med. Chem. Lett. 2011, 21, 760.
Acknowledgments
4. He, Q. Q.; Gu, S. X.; Wu, H. Q.; Zhang, X.; Yang, L. M.; Zheng, Y. T.; Chen, F. E.
Bioorg. Med. Chem., in press (BMC Reference No. 9282). doi:10.1016/
j.bmc.2011.06.020.
5. (a) Zheng, Y. T.; Zhang, W. F.; Ben, K. L.; Wang, J. H. Immunopharmacol.
Immunotoxicol. 1995, 17, 69; (b) Pannecouque, C.; Daelemans, D.; De Clercq, E.
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This project was funded by China Postdoctoral Science Founda-
tion(20100470624). The work was also supported in part by grants
from the National Natural Science Foundation of China(20872018),
from Chinese Academy of Sciences (KSCX2-YW-R-185), from the
Eleventh Five-Year Key Scientific and Technological Program of
China (2009ZX09501-029), and from National Basic Research
Program of China (2009CB5223006).
6. Dayam, R.; Al-Mawsawi, L. Q.; Zawahir, Z.; Witvrouw, M.; Debyser, Z.; Neamati,
N. J. Med. Chem. 2008, 51, 1136.