CHEM. RES.

CHINESE UNIVERSITIES 2009, 25(6), 841845

*Corresponding author. E-mail: huliming@bjut.edu.cn
Received November 28, 2008; accepted March 10, 2009.
Supported by the National Basic Research Program of China(No.2009CB930200), Beijing City Education Committee, China
(No.KM20061000 5029) and Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdic-
tion of Beijing Municipality, China.

Synthesis and Biological Activities of Quinoline
Derivatives as HIV-1 Integrase Inhibitors
LUO Zai-gang
1
, ZENG Cheng-chu
1
, WANG Fang
2
, HE Hong-qiu
1
, WANG Cun-xin
1
,
DU Hong-guang
2
and HU Li-ming
1*

1. College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, P. R. China;
2. College of Science, Beijing University of Chemical Technology, Beijing 100029, P. R. China

Abstract Based on the structure of the integrase core domain and pharmacophore perception, the authors picked out
the hit quinolone derivative 1 as the lead compound via virtual screen in ACD, MDDR, NCI and Chinese Herb
three-dimensional database with the aid of DOCK4.0 program and synthesized a series of analogues of compound 1.
Their primary anti-HIV properties against integrase reveal that 6-position methyl group on the benzene ring of
quinolone plays a more important role than chlorine, 7-position methyl group or no substituted group. But the
title compounds exhibit little difference when the substituted group was phenyl or thienyl on the pyridine ring of
quinoline.
Keywords Quinolone; Integrase; Anti-HIV activity
Article ID 1005-9040(2009)-06-841-05

1 Introduction
HIV-1 integrase(IN), along with HIV-1 reverse
transcriptase and HIV-1 protease, is an essential en-
zyme for retroviral replication and represents an im-
portant target for interrupting the viral replication
cycle
[1]
.
IN is an attractive target because it has no coun-
terpart in mammalian cells, therefore, IN inhibitors
should have high selectivity and low toxicity
[2]
. As a
kind of HIV-1 inhibitors, quinolone derivatives have
aroused great interest due to their extremely versatile
small molecules, easily synthesized at low cost on a
large scale and well-known biochemical properties
that make them very suitable pharmacophore
structures
[3,4]
. The first quinolone-based structure
(GS-9137)
[5]
with very strong antiretroviral properties
owns its anti-HIV activity exclusively to the inhibition
of the viral enzyme integrase.
Pharmacophore perception is one of the widely
used analogue-based drug design methods where
structural information of known lead compounds is
utilized to identify novel and more potent com-
pounds
[6]
. The quinolone-based structure was also as-
sumed to be the new pharmacophore for designing
new generation HIV-1 IN inhibitors
[7]
. We envisage
that pharmacophore perception is an ideal approach
for discovering a new generation IN inhibitors. A three
dimensional database was virtually screened to iden-
tify novel integrase inhibitors. Based on the structure
of the integrase core domain, a preliminary search of
the compounds in ACD, MDDR, NCI and Chinese
Herb Database were carried out via DOCK4.0 pro-
gram
[8]
.
Clustering and drug-likeness predicting were in-
vestigated to filter the docking collection and pick out
the hit compounds. Finally, compound 1(Scheme 1)
was selected as one of the quinolone derivatives, with
lower binding energies and good drug-likeness. In this
study, we synthesized the title compounds 18
(Scheme 2) and the potential activities of these com-
pounds as HIV-1 integrase inhibitors were also eva-
luated.





Scheme 1 Chemical structure of compound 1

842 CHEM. RES. CHINESE UNIVERSITIES Vol.25






















Reagents and conditions: a. Na
2
SO
4
/H
2
O, trichloroacetaldehyde hydrate, hydroxylamine hydrochloride, appropriate anilines, 6070 C, 5 h, 75%91%;
b. 98% H
2
SO
4
, 90 C, 30 min, 43%81%; c. 2-acetylthiophene or acetophenone, NaOH/H
2
O, reflux 5 h or KOH/EtOH, reflux 2472 h, 56%77%;
d. SOCl
2
, CHCl
3
, reflux; e. 8% KOH/EtOH, r. t., 3 h, 94%; f. 1,3-dibrompropane, DMF, 40 C, 1.5 h, 83%; g. piperazine, K
2
CO
3
, KI, DMF/CH
3
CN,
reflux, 4 h, 55%; h. 95% EtOH, 50% hydrazine hydrate, reflux 30 min, 65%; i. NaHCO
3
, THF, reflux 2 h, 20%50%.
Scheme 2 Synthesis of the title compounds 18
2 Chemical Properties
Our general synthetic route was applied to the
new inhibitors quinolone derivatives 18 with sym-
metry structures as shown in Scheme 2. The substi-
tuted quinoline-4-carbonyl chlorides 2532, the one
key intermediate, were prepared in four steps. The
synthesis of compounds 912 from commercially
available anilines, trichloroacetaldehyde hydrate and
hydroxylamine hydrochloride were performed in the
presence of Na
2
SO
4
in water at 6070 C for 5 h. But
the cyclization in the next step to obtain isatins
1316 could be occurred at two positions when subs-
tituted group R
2
was chlorine or methyl. Based on the
literature
[9]
,

the main products of isatins 15 and 16
could be achieved by controlling the pH below 2 of
the filtrate in the workup. Isatins were prepared under
standard conditions with 2-acetylthiophene in the
presence of NaOH in water refluxing for 5 h or ace-
tophenone in the presence of KOH in ethanol reflux-
ing over 24 h, these two methods delivered
2-substituted quinolone acids 1724, and then qui-
nolone acids reacted with thionyl chloride to yield
acyl chlorides 2532. We noted that the other key
intermediate, compound 36, could be synthesized via
Gabriel reaction. Potassium phthalimide 33 was
treated with 1,3-dibromopropane in DMF at 40 C for
1.5 h to give compound 34. By reaction with pipera-
zine, compound 34 was converted to compound 35 in
the presence of K
2
CO
3
and catalyst KI in the mixed
solvents of DMF and CH
3
CN. The intermediate com-
pound 36 could be obtained after hydrazinolysis of
compound 35. Finally, a series of compounds 18
were achieved by compound 36 reacting with com-
pounds 2532 and the structures were confirmed by
1
H NMR,
13
C NMR, IR and ESI-MS.
3 Experimental
All the solvents were of commercial quality and
were dried and purified by conventional methods.
Melting points were measured on XT4A electrother-
mal with an uncorrected apparatus. Infrared(IR)
spectra were recorded as thin films on KBr plates with
a Bruker vertex 70 IR Spectrophotometer.
1
H and
13
C NMR spectra were recorded on a Bruker AV400
spectrometer in solvent(CDCl
3
or DMSO-d
6
) with
TMS as internal reference. MS spectra(ESI) were
recorded on a Bruker ESQUIRE 6000 mass
No.6 LUO Zai-gang et al. 843

spectrometer. The intermediate compounds 912
[10]
,
1316
[10,11]
, 1724
[12,13]
, 2532
[14]
, 34
[15,16]
, 35
[17,18]

and 36
[19]
were prepared according to the correspond-
ing literature methods.
3.1 General Synthetic Procedure of Compounds
18
Compound 36(0.45 g, 2.25 mmol) was dissolved
in dried THF(5 mL) and this solution was added
dropwise to a mixture of each of compounds 2532
(4.50 mmol), NaHCO
3
(0.95 g, 11.25 mmol) and dried
THF(20 mL) under vigorous stirring. The resulting
mixture was kept pH 89 with NaHCO
3
and refluxed.
After 2 h, TLC analysis indicated the reactant of each
of compounds 2532 was disappeared. The mixture
was then concentrated in vacuo, diluted with water
(30 mL) and extracted with CHCl
3
(15 mL3). The
combined organic layer was dried over Na
2
SO
4
,
filtered, and concentrated. The residue was purified by
flash chromatography(chloroform/ethanol as eluent)
to afford the title compounds 18 as white crystal.


3.1.1 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
[2-(2-thienyl) -4-quinolinecarboxamide](1)
White crystal, yield 22%, m. p. 227228 C.
1
H NMR(CDCl
3
, 400 MHz), : 1.57(m, 4H, CH
2
),
1.82.3(m, 12H, CH
2
N), 3.57(q, J=5.6 Hz, 4H,
CH
2
CH
2
NH), 7.17(t, 2H, ArH), 7.51(m, 4H,
ArH), 7.73(m, 4H, ArH), 7.82(s, 2H, ArH),
8.09(d, J=8.0 Hz, 2H, ArH), 8.18(d, J=8.4 Hz, 2H,
ArH), 8.47(t, 2H, NH).
13
C NMR(DMSO-d
6
, 100
MHz), : 26.47, 38.34, 53.32, 56.07, 115.85, 123.82,
125.82, 127.26, 128.13, 129.07, 129.29, 130.50,
130.82, 144.02, 144.75, 148.10, 152.12, 166.78.
ESI-MS, m/z: 675.2[M+H]
+
.
3.1.2 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
(2-phenyl -4-quinolinecarboxamide)(2)
White crystal, yield 48%, m. p. 234235 C.
1
H NMR(CDCl
3
400 MHz), : 1.53(m, 4H, CH
2
),
1.553.1(m, 12H, CH
2
N), 3.55(q, J=5.6 Hz, 4H,
CH
2
CH
2
NH)7.397.65(m, 8H, ArH), 7.75(t, 2H,
ArH), 7.88(s, 2H ArH), 8.028.18(m, 4H,
ArH), 8.19(d, J=8.0 Hz, 2H, ArH), 8.27(d, J=
8.2 Hz, 2H, ArH), 8.46(t, 2H, NH).
13
C NMR
(DMSO-d
6
, 100 MHz), : 26.50, 38.35, 53.32, 56.07,
117.01, 123.88, 125.80, 127.54, 127.76, 129.36,
130.00, 130.32, 130.62, 138.71, 143.83, 148.40,
156.26, 166.97. ESI-MS, m/z: 663.2[M+H]
+
, 685.2
[M+Na]
+
.
3.1.3 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
[6-methyl-2-(2-thienyl)-4-(quinolinecarboxamide)](3)
White crystal, yield 48%, m. p. 231233 C.
1
H NMR(CDCl
3
, 400 MHz), : 1. 57(m, 4H,
CH
2
CH
2
CH
2
), 1.622.42(m, 12H, NCH
2
), 2.53
(s, 6H, CH
3
), 3.57(q, 4H, CH
2
CH
2
NH), 7.147.16
(m, 2H, ArH), 7.457.47(m, 2H, ArH), 7.55(d,
J=8.4 Hz, 2H, ArH), 7.70(m, 2H, ArH), 7.77(s,
2H, ArH), 7.91(t, 4H, ArH), 8.45(b, 2H, NH). IR
(KBr), /cm
1
: 1642.6(
C=O
).
13
C NMR(DMSO-d
6
,
100 MHz), : 21.84, 26.44, 38.28, 53.27, 56.08,
115.83, 123.77, 124.55, 127.70, 129.00, 129.09,
130.12, 132.87, 136.81, 143.35, 144.86, 146.68,
151.25, 166.89. ESI-MS, m/z: 703.2[M+H]
+
, 725.1
[M+Na]
+
.
3.1.4 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
(6-methyl-2-phenyl-4-quinolinecarboxamide)(4)
White crystal, yield 50%, m. p. 218219 C.
1
H NMR(CDCl
3
, 400 MHz), : 1.53(m, 4H, CH
2
),
1.562.48(m, 12H, CH
2
N), 2.55(s, 6H, CH
3
),
3.56(q, 4H, CH
2
CH
2
NH), 7.507.68(m, 8H, ArH),
7.83(s, 2H, ArH), 8.028.20(m, 8H, ArH),
8.45(b, 2H, NH).
13
C NMR(CDCl
3
, 100 MHz), :
21.91, 23.57, 40.92, 52.61, 57.57, 116.51, 123.54,
124.07, 127.38, 128.90, 129.51, 129.76, 132.43,
137.32, 139.08, 142.67, 147.50, 155.75, 167.38.
IR(KBr), /cm
1
: 1637(
C=O
). ESI-MS, m/z: 691.3
[M+H]
+
, 713[M+Na]
+
.
3.1.5 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
[6-chloro-2-(2-thienyl)-4-quinolinecarboxamide](5)
White crystal, yield 20%, m. p. 247248 C.
1
H NMR(DMSO-d
6
, 400 MHz), : 1.72(m, 4H,
CH
2
), 2.232.44(m, 12H, CH
2
N), 3.42(q, 4H,
CH
2
CH
2
NH), 7.26(t, 2H, ArH), 7.80(m, 4H,
ArH), 8.04(d, 2H, ArH), 8.12(m, 4H, ArH),
8.20(s, 2H, ArH), 8.95(t, 2H, NH).
13
C NMR
(DMSO-d
6
, 100 MHz), : 23.81, 36.95, 52.30, 56.05,
117.59, 124.46, 124.77, 129.07, 129.23, 131.07,
131.27, 131.35, 131.81, 141.81, 144.00, 146.55,
152.59, 166.54. ESI-MS, m/z: 743.1[M+H]
+
, 765
[M+Na]
+
.
3.1.6 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
(6-choro-2-phenyl-4-quinolinecarboxamide)(6)
White crystal, yield 31%, m. p. 239241 C.
1
H NMR(DMSO-d
6
, 400 MHz), : 1.56(m, 4H,
CH
2
CH
2
CH
2
), 1.652.15(m, 12H, CH
2
N), 3.52(q,
4H, CH
2
CH
2
NH)), 7.507.91(m, 10H, ArH),
8.128.32(m, 8H, ArH), 8.52(t, 2H, NH).

844 CHEM. RES. CHINESE UNIVERSITIES Vol.25

13
C NMR(DMSO-d
6
, 100 MHz), : 23.79, 37.01,
53.37, 56.12, 118.91, 124.61, 124.79, 128.11, 129.44,
130.84, 131.43, 131.58, 132.34, 137.75, 142.17,
146.35, 156.75, 166.59. ESI-MS, m/z: 731.2[M+H]
+
,
753.1[M+Na]
+
.
3.1.7 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
[7-methyl-2-(2-thienyl)-4-quinolinecarboxamide](7)
White crystal, yield 25%, m. p. 223224 C.
1
H NMR(DMSO-d
6
, 400 MHz), : 1.561.73(m,
4H, CH
2
), 2.122.48(m, 12H, CH
2
N), 2.50(s,
6H, CH
3
), 3.52(q, 4H, CH
2
CH
2
NH), 7.227.25(m,
2H, ArH), 7.407.46(m, 2H, ArH), 7.75(d,
J=8.4 Hz, 2H, ArH), 7.82(t, 2H, ArH), 7.96(t, 2H,
ArH), 8.018.19(m, 4H, ArH), 8.84(t, 2H, NH).
13
C NMR(CDCl
3
, 100 MHz), : 21.71, 29.70, 40.72,
52.36, 57.31, 114.54, 121.55, 124.91, 126.06, 128.09,
128.63, 128.84, 129.20, 134.45, 140.69, 144.86,
148.87, 151.76, 167.08. ESI-MS, m/z: 703.2[M+H]
+
,
725.1[M+Na]
+
.
3.1.8 N,N'-(1,4-Piperazinediyldi-3,1-propanediyl)bis
(7-methyl-2-phenyl -4-quinolinecarboxamide)(8)
White crystal, yield 43%, m. p. 229230 C.
1
H NMR(CDCl
3
, 400 MHz), : 1.491.54(m,
4H, CH
2
), 1.942.05(m, 12H, CH
2
N), 2.56(s,
6H, CH
3
), 3.52(q, 4H, CH
2
CH
2
NH), 7.39(d, J=5.0
Hz, 2H, ArH ), 7.457.52(m, 6H, ArH), 7.80
(s, 2H, ArH), 7.95(s, 2H, ArH), 8.108.16
(m, 6H, ArH), 8.43(b, 2H, NH).
13
C NMR(CDCl
3
,
100 MHz), : 21.78, 23.65, 40.79, 52.56, 57.43,
115.76, 121.59, 124.93, 127.44, 128.88, 129.04,
129.45, 129.58, 139.13, 140.50, 143.09, 149.12,
156.66, 167.31. ESI-MS, m/z: 691.3[M+H]
+
, 713.3
[M+Na]
+
.
3.2 Bioassay Conditions
(1) Donor DNA: D01: 5P-ACCCTTTTAGTCA-
GTGTGGAAAATCTCTAGCAGT-3(P=phosphated);
D02: 3-GAAAATCAGTCACACCTTTTAGAGATC-
GTCA-5. TargetDNA: T01: 5-TGACCAAGGGCT-
AATTCACT-3-biotin; T02: biotin-3-ACTGGTTCC-
CGATTAAGTGA-5. D01+D02 and T01+T02 were
mixed in a ratio of 1:1 at a concentration of 100
mol/L in purified water, heated to 95 C for 5 min,
slowly cooled down to room temperature, and stored
at 20 C until use. (2) Plate coating was performed as
described by Hazuda et al.
[20]
. (3) The plate wells
were washed three times with phosphate buffered
saline(PBS) and once with reaction buffer(25 mmol/L
piperazine-N,N-bis-2-ethanesulfonic acid, pH 7.0, 10
mmol/L -mercapto-ethanol, 5% glycerol, 0.1 mg/mL
BSA, and 10 mmol/L MnCl
2
). HIV-IN(integrase)(2.5
g in 3 L) was added and preincubated in the wells
in reaction buffer for 10 min, test compounds were
added in DMSO and preincubated with IN for 30 min
followed by the addition of 3 L target DNA(27
mol/L), and the reaction mixture was incubated at
37 C for 2 h. (4) Afterwards, the wells were washed
three times with PBS containing 0.1% Tween 20, and
100 L of 1:1000 diluted alkaline phosphatase conju-
gate streptavidin was added to the wells and incubated
for 45 min at 37 C. Finally, wells were washed three
times with PBST and 100 L of p-NPP substrate(0.1
mol/L Na
2
CO
3
, pH 9.5, 6.7 mmol/L p-NPP, 2.0
mmol/L MgCl
2
) was added to them. Plates were read
at 405 nm with a 1420 Multilabel counter VICTOR
reader.
4 Biological Assay
The title compounds 18 were tested in vitro for
strand-transfer inhibition in the presence of MnCl
2

according to a previously method
[20]
. However, the
primary results of anti-HIV properties against inte-
grase did not show good inhibitory effect as our ex-
pectation(Table 1).
Table 1 HIV IN inhibition activity of compounds
18(100 mol/L)
The data reveal that all the compounds possess
inhibition activities to a certain extent at a concentra-
tion of 100 mol/L with Baicalein as the contrast.
Worthwhile, the inhibitory activities of compounds 3
and 4 are better than those of the others, which indi-
cates that the 6-position methyl group on the benzene
ring of quinolone acts plays a more important role
than the chlorine or no substituted group. When com-
pounds 7 and 8 have 7-position methyl group on the
benzene ring of quinolone, a loss of activity in the
strand-transfer inhibition assay was observed as well.
Moreover, the data also reveal the title compounds
exhibit little difference when R
3
was phenyl or thienyl
on the pyridine ring.

Compound Inhibitory rate(%) Compound Inhibitory rate(%)
1 4.64 6 7.26
2 5.01 7 18.01
3 35.25 8 14.32
4 37.16 Baicalen 100
5 5.36 Control 0
No.6 LUO Zai-gang et al. 845


5 Conclusions
In summary, based on the structure of the inte-
grase core domain and the pharmacophore perception,
the authors picked out the hit compound 1 as the lead
compound through virtually screen in ACD, MDDR,
NCI and Chinese Herb three-dimensional database via
DOCK4.0 program and synthesized quinolone deriva-
tives 18. Their primary anti-HIV properties against
integrase were different with the substituted groups R
1

or R
2
on the benzene ring of quinoline. But the title
compounds exhibit little difference when R
3
was
phenyl or thienyl on the pyridine ring of quinoline.
Under the computational molecular docking, further
studies directing toward novel HIV-1 integrase inhibi-
tor analogues of quinoline derivatives made the au-
thors find one of them with promising bioactivity.

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