Anti Human Immunodeficiency Virus type 1 (HIV-1) agents 4. Discovery of 5,5′-(p-Phenylenebisazo)-8-hydroxyquinoline sulfonates as new HIV-1 inhibitors in vitro

Article abstract of DOI:10.1248/cpb.58.976

To search for compounds with superior anti-human immunodeficiency virus type 1 (HIV-1) activity, ten 5,5′-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates (4a-j) were synthesized and preliminarily evaluated as HIV-1 inhibitors in vitro for the first time

Full text of DOI:10.1248/cpb.58.976

976
Note
Chem. Pharm. Bull. 58(7) 976—979 (2010)
Vol. 58, No. 7
Anti Human Immunodeficiency Virus Type 1 (HIV-1) Agents 4. Discovery
of 5,5؅-(p-Phenylenebisazo)-8-hydroxyquinoline Sulfonates as New HIV-1
Inhibitors in Vitro
Xi-Wen ZENG,a,# Ning HUANG,b,# Hui XU,*,a Wen-Bin YANG,a Liu-Meng YANG,b Huan QU,a and
Yong-Tang ZHENG*,b
a Laboratory of Pharmaceutical Design & Synthesis, College of Sciences, Northwest A&F University; Yangling 712100,
China: and b Key Laboratory of Animal Models and Human Diseases Mechanisms of Chinese Academy of Sciences and
Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences; Kunming 650223, China.
Received January 30, 2010; accepted April 12, 2010; published online April 15, 2010
To search for compounds with superior anti-human immunodeficiency virus type 1 (HIV-1) activity, ten
5,5؅-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates (4a—j) were synthesized and preliminarily evaluated as
HIV-1 inhibitors in vitro for the first time. Some compounds demonstrated anti-HIV-1 activity, especially 5,5؅-
(p-phenylenebisazo)-8-hydroxyquinoline p-ethylbenzenesulfonate (4g) and 5,5؅-(p-phenylenebisazo)-8-hydrox-
yquinoline p-chlorobenzenesulfonate (4i) showed the more potent anti-HIV-1 activity with 50% effective concen-
tration (EC50) values of 2.59 and 4.01 mg/ml, and therapeutic index (TI) values of 31.77 and 24.51, respectively.
Key words 8-hydroxyquinoline; sulfonate; acquired immune-deficiency syndrome; human immunodeficiency virus-1; inhibitor
Since the first case of acquired immunodeficiency syn- sulfonyl chlorides in the presence of triethylamine. The
1
drome (AIDS) was reported in 1981, the human immunodefi- structures of the compounds were well characterized by H-
ciency virus (HIV)/AIDS has always been a global health NMR, MS, IR, and mp.
threat and the leading cause of deaths.1) In the past two
Subsequently, all the target compounds 4a—j and 3 were
decades, twenty-five drugs, including nucleoside/nucleotide evaluated for inhibitory activity against HIV-1 replication in
viral reverse transcriptase (RT) inhibitors (NRTIs), non-nu- acutely infected C8166 cells in vitro and 3Ј-azido-3Ј-de-
cleoside RT inhibitors (NNRTIs), protease inhibitors (PIs), oxythymidine (AZT) was used as a positive control. The
integrase inhibitors (INIs) and fusion (or entry) inhibitors assay results of compounds 4a—j and 3 were presented in
(FIs), were approved for clinical use in the world.2) However, Table 1. Among these compounds, 3, 4f, 4g, and 4i demon-
these drugs have only limited or transient clinical benefit due strated significant anti-HIV-1 activity with 50% effective
to their severe side effects and the emergence of viral vari- concentration (EC50) values of 0.45, 3.51, 2.59, and 4.01
ants resistant to HIV-1 inhibitors.3—5) Currently, the design mg/ml, and therapeutic index (TI) values of 18.91, 17.55,
and development of new, selective and safe HIV-1 inhibitors 31.77, and 24.51, respectively. Especially compound 4g
is therefore highly desirable in the world. Recently, some 8- exhibited the most potent and promising anti-HIV-1 activity
hydroxyquinolinyl azo derivatives have been used as corro- (TIϭ31.77). However, 4a, 4e, 4h, and 4j showed the lower
sion inhibitors for mild steel,6) ligands,7) and probes for mon- TI values less than 5.
itoring of enzymatic activity and heavy metal ions.8,9) To the
Meanwhile, some preliminary structure–activity relation-
best of our knowledge, however, little attention has been paid ships (SAR) of 4a—j and 3 were also observed. Once the
to the anti-HIV-1 activity of the single 8-hydroxyquinolinyl methylsulfonyl or benzenesulfonyl group was introduced on
azo derivatives. In our previous paper, some single N-arylsul- the 8-position of 3 (TIϭ18.91), the corresponding TI values
fonylindoles,10) benzyl phenyl ethers11) and dibenzofurans12) of 4a and 4e were sharply reduced to 3.25 and 2.21, respec-
were found to demonstrate the significant anti-HIV-1 activity tively. But when the ethyl group or the chloro atom was
in vitro. As a consequence, these encouraging results prompted introduced at 4Ј-position on the phenyl ring of 4a, the corre-
us to further study the anti-HIV-1 activity of other single sponding compounds exhibited the more potent anti-HIV-1
compounds, and in continuation of our program aimed at the activity (4g and 4i vs. 4a). For example, the TI values of 4g
discovery and development of bioactive molecules, herein we and 4i were 31.77 and 24.51, respectively, and the EC50 values
report the synthesis and anti-HIV-1 activity of some 5,5Ј-(p- of 4g and 4i were 2.59 and 4.01 mg/ml, respectively; while
phenylenebisazo)-8-hydroxyquinoline sulfonates.
the EC50 and TI values of 4a were 14.36 mg/ml and 3.25,
respectively. That is, the TI value of 4g was more than 9
times of that of 4a, while the EC50 value of 4g was signifi-
Results and Discussion
A series of novel 5,5Ј-(p-phenylenebisazo)-8-hydroxy- cantly decreased more than 5 times compared with 4a. Inter-
quinoline sulfonates 4a—j (Fig. 1) were synthesized as estingly, when the p-methyl group of 4c was substituted by
shown in Chart 1. Firstly, 1,4-benzenediamine (1) reacted the p-ethyl group to give 4g, the EC50 value of 4g was
with NaNO2 and HCl at 0—5 °C to give benzidine diazonium reduced from 13.40 (4c) to 2.59 mg/ml, while the TI value of
chloride (2), which then reacted with 8-quinolinol to yield 4g was increased from 6.24 (4c) to 31.77, i.e., the TI value of
5,5Ј-(p-phenylenebisazo)-8-hydroxyquinoline (3). Finally, ten 4g was nearly 5 times of that of 4c. When the p-bromo atom
5,5Ј-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates (4a— of 4d was substituted by the p-chloro atom to give 4i, the
j) were obtained by reaction of 3 with the corresponding EC50 value of 4i was reduced from 17.11 (4d) to 4.01 mg/ml,
∗ To whom correspondence should be addressed. e-mail: orgxuhui@nwsuaf.edu.cn; zhengyt@mail.kiz.ac.cn
© 2010 Pharmaceutical Society of Japan
#
These authors contributed equally to this work.

July 2010
977
Fig. 1. Chemical Structures of 3 and 4a—j
while the TI value of 4i was increased from 5.96 (4d) to Conclusion
24.51. Moreover, when the nitro group was introduced at the
In summary, ten novel 5,5Ј-(p-phenylenebisazo)-8-hydroxy-
meta position on the phenyl ring of 4i to give 4h, the TI quinoline sulfonates (4a—j) were synthesized and prelimi-
value of 4h was decreased from 24.51 (4i) to 4.88, and the narily evaluated as HIV-1 inhibitors in vitro for the first time.
50% cytotoxic concentration (CC50) values of 4h was sharply Some compounds demonstrated anti-HIV-1 activity, espe-
reduced from 98.31 (4i) to 13.41, i.e., the cytotoxicity of 4h cially 5,5Ј-(p-phenylenebisazo)-8-hydroxyquinoline p-ethyl-
against C8166 cells was more than 7 times of that of 4i.
benzenesulfonate (4g) and 5,5Ј-(p-phenylenebisazo)-8-hydroxy-
quinoline p-chlorobenzenesulfonate (4i) exhibited the more

978
Vol. 58, No. 7
3 as a brown solid in a 40.6% yield, mp 195—197 °C (lit.,13) 200 °C). Finally,
to a suspended solution of 3 (0.5 mmol) in dichloromethane (15 ml) at 0 °C,
sulfonyl chlorides (1.2 mmol) and triethylamine (1.5 mmol) were added in
sequence. After adding, the above mixture was stirred for 36 h at room tem-
perature, and the solvent was evaporated under reduced pressure to give the
residue, which was seperated by preparative thin-layer chromatography
(PTLC) and purified by recrystallization from cyclohexane to give the pure
products 4a—j.
4a: Yield: 21.6%, red solid, mp 123—125 °C; IR (KBr) cmϪ1: 3060,
1
1586, 1493, 1449, 1357; H-NMR (300 MHz, CDCl3) d: 9.23 (d, Jϭ8.4 Hz,
2H, H-2), 8.88 (d, Jϭ3.0 Hz, 2H, H-4), 7.95—8.03 (m, 6H, H-3, H-2Ј, H-
6Ј), 7.90 (d, Jϭ8.4 Hz, 2H, H-6), 7.72 (d, Jϭ8.4 Hz, 2H, H-7), 7.55-7.64 (m,
6H, H-4Ј, Ar-H), 7.48 (t, Jϭ8.0 Hz, 4H, H-3Ј, H-5Ј); MS (ESI-TRAP) m/z:
390 [(MϩDMSOϩ2H)ϩ/2, 100].
Chart 1. The Synthetic Route of 5,5Ј-(p-Phenylenebisazo)-8-hydroxy-
quinoline Sulfonates 4a—j
4b: Yield: 19.6%, salmon solid, mp 177—178 °C; IR (KBr) cmϪ1: 3044,
1
3015, 1609, 1497, 1473, 1369; H-NMR (300 MHz, CDCl3) d: 9.22 (d, Jϭ
Table 1. Anti-HIV-1 Activity of 5,5Ј-(p-Phenylenebisazo)-8-hydroxy-
quinoline Sulfonates (4a—j) in Vitroa)
8.4 Hz, 2H, H-2), 9.11 (s, 2H, H-2Ј), 8.81 (s, 2H, H-4), 8.46 (d, Jϭ8.0 Hz,
2H, H-4Ј), 8.37 (d, Jϭ8.0 Hz, 2H, H-6Ј), 8.02 (d, Jϭ7.2 Hz, 2H, H-3), 7.93
(d, Jϭ8.4 Hz, 2H, H-6), 7.83 (d, Jϭ8.4 Hz, 2H, H-7), 7.70 (t, Jϭ8.0 Hz, 2H,
H-5Ј), 7.55 (s, 4H, Ar-H); MS (ESI-TRAP) m/z: 435 [(MϩDMSOϩ2H)ϩ/2,
100].
Compounds CC50b) (mg/ml)
EC50c) (mg/ml)
TId)
4c: Yield: 17.2%, salmon solid, mp 142—144 °C; IR (KBr) cmϪ1: 3052,
3
4a
4b
4c
4d
4e
8.51
47.56
40.36
83.66
102.02
7.55
0.45
14.63
4.03
13.40
17.11
3.41
18.91
1
3.25
10.02
6.24
5.96
2.21
2917, 2844, 1595, 1496, 1375; H-NMR (300 MHz, CDCl3) d: 9.22 (d, Jϭ
8.4 Hz, 2H, H-2), 8.92 (s, 2H, H-4), 8.02 (d, Jϭ6.0 Hz, 2H, H-3), 7.87—
7.91 (m, 6H, H-6, H-2Ј, H-6Ј), 7.69 (d, Jϭ8.4 Hz, 2H, H-7), 7.55 (s, 4H, Ar-
H), 7.28 (d, Jϭ7.8 Hz, 4H, H-3Ј, H-5Ј), 2.41 (s, 6H, CH3); MS (ESI-TRAP)
m/z: 404 [(MϩDMSOϩ2H)ϩ/2, 100].
4d: Yield: 15.6%, salmon solid, mp 145—147 °C; IR (KBr) cmϪ1: 3083,
4f
4g
4h
4i
61.62
82.29
13.14
98.31
37.69
1139.47
3.51
2.59
2.69
4.01
19.73
0.00324
17.55
31.77
4.88
24.51
1.91
1
2924, 2844, 1573, 1496, 1379; H-NMR (300 MHz, CDCl3) d: 9.24 (d, Jϭ
7.8 Hz, 2H, H-2), 8.88 (s, 2H, H-4), 8.04 (s, 2H, H-3), 7.87—7.92 (m, 6H,
H-6, H-2Ј, H-6Ј), 7.76 (d, Jϭ8.4 Hz, 2H, H-7), 7.56—7.63 (8H, Ar-H, H-3Ј,
H-5Ј); MS (ESI-TRAP) m/z: 469 [(MϩDMSOϩ2H)ϩ/2, 100].
4j
AZTe)
352688.27
4e: Yield: 24.5%, red solid, mp 170—172 °C; IR (KBr) cmϪ1: 2925, 2844,
1
1628, 1543, 1367; H-NMR (300 MHz, CDCl3) d: 9.32 (d, Jϭ8.4 Hz, 2H,
a) Values are means of two separate experiments; b) CC50 (50% cytotoxic concentra-
tion), concentration of drug that causes 50% reduction in total C8166 cell number; c)
EC50 (50% effective concentration), concentration of drug that reduces syncytia forma-
tion by 50%; d) therapeutic index (TI) is a ratio of the CC50 value/EC50 value; e) AZT
was used as a positive control.
H-2), 9.07 (s, 2H, H-4), 8.04 (d, Jϭ6.3 Hz, 2H, H-3), 7.95 (d, Jϭ8.4 Hz, 2H,
H-6), 7.82 (d, Jϭ8.4 Hz, 2H, H-7), 7.57 (s, 4H, Ar-H), 3.52 (s, 6H, CH3);
MS (ESI-TRAP) m/z: 328 [(MϩDMSOϩ2H)ϩ/2, 100].
4f: Yield: 18.3%, red solid, mp 116—118 °C; IR (KBr) cmϪ1: 2917, 2842,
1
1594, 1496, 1376; H-NMR (300 MHz, CDCl3) d: 9.24 (d, Jϭ8.4 Hz, 2H,
H-2), 8.93 (s, 2H, H-4), 8.02 (d, Jϭ6.0 Hz, 2H, H-3), 7.88—7.96 (m, 6H, H-
6, H-2Ј, H-6Ј), 7.73 (d, Jϭ8.4 Hz, 2H, H-7), 7.57 (s, 4H, Ar-H) ,6.92 (d, Jϭ
7.2 Hz, 4H, H-3Ј, H-5Ј), 3.85 (s, 6H, OCH3); MS (ESI-TRAP) m/z: 420
[(MϩDMSOϩ2H)ϩ/2, 100].
potent anti-HIV-1 activity with EC50 values of 2.59 and 4.01
mg/ml, and TI values of 31.77 and 24.51, respectively. More-
over, the preliminary SAR showed that the ethyl group or the
chloro atom at 4Ј-position on the phenyl ring of 5,5Ј-(p-
phenylenebisazo)-8-hydroxyquinoline benzenesulfonate was
very important for possessing potent anti-HIV-1 activity. It
will pave the way for further optimal structural modifications
of the 8-hydroxyquinolinyl azo derivatives as HIV-1 inhibitors.
4g: Yield: 16.1%, red solid, mp 117—119 °C; IR (KBr) cmϪ1: 3055,
1
2969, 2925, 2849, 1593, 1493, 1469, 1372; H-NMR (300 MHz, CDCl3) d:
9.24 (d, Jϭ8.4 Hz, 2H, H-2), 8.91 (s, 2H, H-4), 8.02 (d, Jϭ7.5 Hz, 2H, H-3),
7.88—7.93 (m, 6H, H-6, H-2Ј, H-6Ј), 7.72 (d, Jϭ8.4 Hz, 2H, H-7), 7.56 (s,
4H, Ar-H), 7.29 (d, Jϭ7.8 Hz, 4H, H-3Ј, H-5Ј), 2.70 (m, 4H, CH2CH3), 1.23
(m, 6H, CH2CH3); MS (ESI-TRAP) m/z: 418 [(MϩDMSOϩ2H)ϩ/2, 100].
4h: Yield: 20.5%, red solid, mp 112—115 °C; IR (KBr) cmϪ1: 3095,
1
1593, 1536, 1497, 1347; H-NMR (300 MHz, CDCl3) d: 9.26 (d, Jϭ8.4 Hz,
2H, H-2), 8.86 (s, 2H, H-4), 8.75 (s, 2H, H-2Ј), 8.15 (d, Jϭ8.4 Hz, 2H, H-3),
8.03 (d, Jϭ7.8 Hz, 2H, H-6Ј), 7.95 (d, Jϭ8.4 Hz, 2H, H-6), 7.86 (d, Jϭ8.4
Hz, 2H, H-7), 7.67 (d, Jϭ8.4 Hz, 2H, H-5Ј), 7.58 (s, 4H, Ar-H); MS (ESI-
TRAP) m/z: 469 [(MϩDMSOϩ2H)ϩ/2, 100].
Experimental
All the solvents were of analytical grade and the reagents were used as
purchased. Thin-layer chromatography (TLC) and preparative thin-layer
chromatography (PTLC) were performed with silica gel plates using silica
gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., China). Melting points
were determined on a digital melting-point apparatus and were uncorrected.
Infrared spectra (IR) were recorded on a Thermo Nicolet Nexus FTIR-8700
spectrometer (Thermo Nicolet, U.S.A.). 1H-NMR spectra were recorded on a
Bruker Avance DMX 300 MHz instrument (Bruker, Swiss) using TMS as
internal standard and CDCl3 as solvent. Electrospray iontrap mass spectra
(electrospray ionization (ESI)-TRAP-MS) were recorded on a Bruker ESI-
TRAP Esquire 3000 (Bruker, Swiss) plus mass spectrometry instrument.
General Procedure for the Synthesis of 5,5؅-(p-Phenylenebisazo)-8-hy-
droxyquinoline Sulfonates 4a—j To a solution of 1,4-benzenediamine (1,
30 mmol) in distilled water (15 ml) at 0 °C, HCl (12 mol/l, 15 ml) was added
dropwise, followed by a solution of NaNO2 (65.9 mmol) in distilled water
(15 ml), and the temperature of the reaction mixture was kept at 0—5 °C.
After stirring for 20 min, a solution of benzidine diazonium chloride (2) was
prepared. Subsequently, the solution of 2 was added dropwise to a mixture
of 8-hydroxyquinoline (60 mmol), NaOH (60 mmol) and ethanol (30 ml) at
0—5 °C, and the mixture was stirred until a lot of brown precipitate was pro-
duced, which was filtered, washed with distilled water, and purified by
recrystallization to give 5.12 g 5,5Ј-(p-phenylenebisazo)-8-hydroxyquinoline
4i: Yield: 16.2%, salmon solid, mp 118—120 °C; IR (KBr) cmϪ1: 3095,
1
1586, 1496, 1379; H-NMR (300 MHz, CDCl3) d: 9.23 (d, Jϭ8.0 Hz, 2H,
H-2), 8.86 (s, 2H, H-4), 8.04 (s, 2H, H-3), 7.89—7.95 (m, 6H, H-6, H-2Ј, H-
6Ј), 7.75 (d, Jϭ8.4 Hz, 2H, H-7), 7.56 (s, 4H, Ar-H), 7.45 (d, Jϭ6.3 Hz, 4H,
H-3Ј, H-5Ј); MS (ESI-TRAP) m/z: 424 [(MϩDMSOϩ2H)ϩ/2, 100].
4j: Yield: 17.8%, salmon solid, mp 205—207 °C; IR (KBr) cmϪ1: 3104,
1
2917, 2844, 1678, 1591, 1497, 1376; H-NMR (300 MHz, CDCl3) d: 9.26
(d, Jϭ8.4 Hz, 2H, H-2), 8.91 (s, 2H, H-4), 8.02 (d, Jϭ6.3 Hz, 2H, H-3),
7.89—7.92 (m, 6H, H-6, H-2Ј, H-6Ј), 7.75—7.81 (m, 4H, H-7, NH), 7.64 (d,
Jϭ7.5 Hz, 4H, H-3Ј, H-5Ј), 7.56 (s, 4H, Ar-H), 2.20 (s, 6H, CH3); MS (ESI-
TRAP) m/z: 447 [(MϩDMSOϩ2H)ϩ/2, 100].
Anti-HIV-1 Activity Assay.10) Cells and Virus Cell line (C8166) and
the laboratory-derived virus (HIV-1IIIB) were obtained from MRC, AIDS
Reagent Project, U.K. C8166 was maintained in RPMI-1640 supplemented
with 10% heat-inactivated newborn calf serum (Gibco). The cells used in all
experiments were in log-phase growth. The 50% HIV-1IIIB tissue culture
infectious dose (TCID50) in C8166 cells was determined and calculated by
the Reed and Muench method. Virus stocks were stored in small aliquots at
Ϫ70 °C.

July 2010
MTT-Based Cytotoxicity Assay Cellular toxicity of compounds 3 and
979
Yunnan (2007BC006), 863 Program (2006AA020602), 973 Program
4a—j on C8166 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- (2009CB522306), the CAS (KSCX1-YW-R-24, KSCX2-YW-R-185), and
diphenyltetrazolium bromide (MTT) method as described previously. the MRC AIDS Research Project and the NIH AIDS Research and Refer-
Briefly, cells were seeded on 96-well microtiter plate in the absence or pres- ence Reagent Program for providing cell lines and viruses.
ence of various concentrations of compounds in triplicate and incubated at
37 °C in a humid atmosphere of 5% CO2 for 3 d. The supernatants were dis-
carded and MTT reagent (5 mg/ml in PBS) was added to each wells, then
incubated for 4 h, 100 ml of 50% N,N-dimethylformamide (DMF)–20%
sodium dodecyl sulfate (SDS) was added. After the formazan was dissolved
completely, the plates were read on a Bio-Tek Elx 800 enzyme-linked im-
munosorbent assay (ELISA) reader at 595/630 nm. The cytotoxic concentra-
tion that caused the reduction of viable C8166 cells by 50% (CC50) was
determined from dose-response curve.
Syncytia Assay In the presence of 100 ml various concentrations of
compounds, C8166 cells (4ϫ105/ml) were infected with virus HIV-1IIIB at a
multiplicity of infection (M.O.I.) of 0.06. The final volume per well was
200 ml. Control assays were performed without the testing compounds in
HIV-1IIIB infected and uninfected cultures. After 3 d of culture, the cyto-
pathic effect (CPE) was measured by counting the number of syncytia. Per-
centage inhibition of syncytia formation was calculated and 50% effective
concentration (EC50) was calculated. AZT (Sigma) was used as a positive
control. Therapeutic index (TI)ϭCC50/EC50.
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