Synthesis and HIV-1 integrase inhibition activity of some N-arylindoles

Article abstract of DOI:10.1248/cpb.56.720

Eight simple N-arylindoles were designed, synthesized and evaluated as human immunodeficiency virus (HIV)-1 integrase inhibitors in vitro for the first time. Among these compounds, 3b, 3e and 3g demonstrated significant anti-HIV-1 integrase activity. Especially 3b showed the highest anti-HIV-1 integrase activity with EC₅₀ value of 7.88 μg/ml and TI value of 24.61. Meantime, some structure-activity relationships were also observed and will provide a new lead for design and discovery of more potent N-arylindoles as HIV-1 integrase inhibitors.

Full text of DOI:10.1248/cpb.56.720

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Notes
Chem. Pharm. Bull. 56(5) 720—722 (2008)
Vol. 56, No. 5
Synthesis and HIV-1 Integrase Inhibition Activity of some N-Arylindoles
Hui XU, ,a Wu-Qing LIU,b Ling-Ling FAN,a Yang CHEN,a Liu-Meng YANG,b Lei LV,a and
*
,b
*
Yong-Tang ZHENG
a Laboratory of Pharmaceutical Synthesis, College of Sciences, Northwest A&F University; Yangling 712100, China: and
b Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Diseases Mechanisms,
Kunming Institute of Zoology, Chinese Academy of Sciences; Kunming 650223, China.
Received December 2, 2007; accepted February 13, 2008; published online February 22, 2008
Eight simple N-arylindoles were designed, synthesized and evaluated as human immunodeficiency virus
(HIV)-1 integrase inhibitors in vitro for the first time. Among these compounds, 3b, 3e and 3g demonstrated sig-
nificant anti-HIV-1 integrase activity. Especially 3b showed the highest anti-HIV-1 integrase activity with EC50
value of 7.88 mg/ml and TI value of 24.61. Meantime, some structure–activity relationships were also observed
and will provide a new lead for design and discovery of more potent N-arylindoles as HIV-1 integrase inhibitors.
Key words N-arylindole; anti-human immunodeficiency virus type 1; integrase inhibitor; synthesis
In the past two decades, a worldwide search has been and cytotoxicity (CC50) in cell-based assays against HIV-1
made for new chemotherapeutic agents targeting the human integrase replication in acutely infected C8166 cells and
immunodeficiency virus (HIV). However, many drugs have C8166 cells, respectively. In addition, the therapeutic index
only limited or transient clinical benefit due to their side ef- (TI) was also calculated as shown in Table 1. 3ꢀ-Azido-3ꢀ-de-
fects and the development of virus–drug resistance.1) There- oxythymidine (AZT) was used as a positive control.
fore, the development of new, selective and safe HIV-1 inte-
As indicated in Table 1, among these compounds, 3b, 3e
grase inhibitors still remains a high priority for medical re- and 3g showed the more potent anti-HIV-1 integrase activity
search. N-Arylindoles are central structural scaffolds in many with EC50 values of 7.88, 11.24 and 19.22 mg/ml, and TI val-
pharmacologically important and bioactive molecules, which ues of 24.61, 9.48 and 8.26, respectively. Especially 3b ex-
display antiestrogen,2) analgesic,3) neuroleptic,4) antiallergy,5) hibited the most potent and promising anti-HIV-1 integrase
5-HT6 receptor antagonists,6) and FTase inhibitors (FTIs) ac- activity (TIꢁ24.61). On the contrary, compounds 3d and 3h
tivity.7) Although Merino et al. reported that a set of pyrim- showed lower TI values (1.90 for 3d, and 1.28 for 3h) and
ido[5,4-b]indole derivatives possess anti-HIV-1 activity,8) to higher cytotoxicity (5.40 mg/ml for 3d, and 14.41 mg/ml for
the best of our knowledge, little attention has been paid to 3h) when compared with the others.
the anti-HIV-1 integrase activity of the single N-arylindoles,
From the comparative study, it is possible to draw some
with low-molecular weight. As part of our program aimed at structure–activity relationships as shown in Table 1. The
the discovery and development of bioactive molecules,9—11) cytotoxicity (CC50), anti-HIV-1 integrase activity (EC50) and
herein we report the synthesis and anti-HIV-1 integrase activ- TI values of 3a and 3b were 91.44/191.9 mg/ml, 35.82/7.88
ity of some single N-arylindoles with various functional
groups.
Results and Discussion
Eight simple N-arylindoles 3a—h (Fig. 1) were prepared
successfully by cross-coupling various indoles with activated
fluoroarenes via nucleophilic aromatic substitution (SNAr) re-
1
actions as shown in Chart 1, and were characterized by H-
NMR (400 MHz), HR-MS or elemental analysis, EI-MS and
melting point. Subsequently, the N-arylindoles 3a—h were
tested in vitro for their anti-HIV-1 integrase activity (EC50)
Chart 1. The Synthetic Route of N-Arylindoles 3a—h
Table 1. Anti-HIV-1 Integrase Activity of N-Arylindoles (3a—h) in Vitro
Compounds
CC50a) (mg/ml)
EC50b) (mg/ml)
TIc)
3a
3b
3c
91.44
191.9
ꢂ200
5.40
35.82
7.88
82.28
2.93
2.55
24.61
ꢂ2.66
1.90
3d
3e
3f
3g
99.61
11.24
37.76
19.22
12.09
0.007
9.48
ꢂ6.63
8.26
ꢂ200
157.14
14.41
3h
1.28
184034.28
AZTd)
1288.24
a) CC50 (50% cytotoxic concentration), concentration of drug that causes 50% reduc-
tion in total C8166 cell number, Drugs with CC50 values ꢂ200 mg/ml cannot be tested
at higher concentrations for a more exact CC50 value due to the effect of the solvent
DMSO; b) EC50 (50% effective concentration), concentration of drug that reduces syn-
cytia formation by 50%; c) therapeutic index (TI) is a ratio of the CC50 value/EC50
value; d) AZT was used as a positive control.
Fig. 1. Structures of Different N-Arylindoles 3a—h
∗ To whom correspondence should be addressed. e-mail: orgxuhui@nwsuaf.edu.cn; zhengyt@mail.kiz.ac.cn
© 2008 Pharmaceutical Society of Japan

May 2008
721
using TMS as internal standard and CDCl3 as solvent. HR-MS and EI-MS
were carried out with APEX II Bruker 4.7T AS and Thermo DSQ GC/MS
instruments, respectively. Elemental analysis was executed on Carlo-Erba
1106 CHN microanalyzer.
mg/ml, and 2.55/24.61, respectively. Obviously, the TI value
of 3b was almost 10 times of that of 3a, while the cytotoxic-
ity of 3b was decreased 2 times compared with 3a. Mean-
while, the CC50, EC50 and TI values of 3d and 3e were
General Procedure for the Synthesis of N-Arylindoles 3a—h The
5.40/99.61 mg/ml, 2.93/11.24 mg/ml, and 1.90/9.48, respec- mixture of the appropriate activated fluoroarenes (1, 1.0 mmol), the indoles
(2, 1.2 mmol), and anhydrous Cs2CO3 (2.0 mmol) in DMSO (2 ml) in 25 ml
rockered flask was stirred at 40 °C in an air atmosphere until complete con-
sumption of the starting material checked by TLC. Then ice water (40 ml)
was added to the above mixture, and the latter was extracted by EtOAc
tively. Accordingly, the TI value of 3e was almost 6 times of
that of 3d, while the cytotoxicity of 3e was significantly de-
creased 19 times compared with 3d. That is, introducing
ortho-nitro group on the N-phenyl ring of indoles, would
lead to give compounds possessing more potent anti-HIV-1
integrase activity than those having para-nitro group on the
N-phenyl ring of indoles; Moreover, the cytotoxicity of the
compounds having ortho-nitro group on the N-phenyl ring
of indoles, were significantly decreased when compared
with those having para-nitro group on the N-phenyl ring of
indoles (3b vs. 3a, 3e vs. 3d). However, when introducing
para-nitro group on the N-phenyl ring of 3-methylindole, the
corresponding compound showed the more potent anti-HIV-1
integrase activity than the one having ortho-nitro group on
the N-phenyl ring of 3-methylindole (3g vs. 3h). For exam-
ple, the CC50, EC50 and TI values of 3g and 3h were 157.14/
14.41 mg/ml, 19.22/12.09 mg/ml, and 8.26/1.28, respectively.
Consequently, the TI value of 3g was more than 6 times of
(60 mlꢃ3). Subsequently, the combined organic phase was washed by brine
(40 ml), dried over anhydrous Na2SO4, concentrated in vacuo and purified by
preparative TLC to give the pure N-arylation indoles, which were character-
ized by 1H-NMR (400 MHz), HR-MS or elemental analysis, EI-MS and mp.
3a: Yield: 86%, yellow solid, mp 109—109.5 °C; 1H-NMR (400 MHz,
CDCl3) d: 6.77 (1H, d, Jꢁ3.2 Hz), 7.21 (2H, m), 7.37 (1H, d, Jꢁ3.6 Hz),
7.64 (4H, m), 8.39 (2H, d, Jꢁ8.8 Hz); EI-MS m/z: 238 (Mꢄ, 100); HR-MS
m/z: 239.0818 [MꢄH]ꢄ, Calcd 239.0815.
3b: Yield: 94%, orange solid, mp 69—70 °C; 1H-NMR (400 MHz, CDCl3)
d: 6.72 (1H, d, Jꢁ3.2 Hz), 7.11 (4H, m), 7.53 (2H, m), 7.68 (2H, m), 8.01
(1H, d, Jꢁ8.4 Hz); EI-MS m/z: 238 (Mꢄ, 100); HR-MS m/z: 239.0818
[MꢄH]ꢄ, Calcd 239.0815.
3c: Yield: 76%, white solid, mp 96—96.5 °C; 1H-NMR (400 MHz,
CDCl3) d: 6.76 (1H, d, Jꢁ3.6 Hz), 7.18 (2H, m), 7.33 (1H, d, Jꢁ8.4 Hz),
7.40 (1H, d, Jꢁ3.2 Hz), 7.46 (1H, m), 7.60 (1H, d, Jꢁ8.4 Hz), 7.69 (2H, m),
7.83 (1H, d, Jꢁ7.6 Hz); EI-MS m/z: 218 (Mꢄ, 100); HR-MS m/z: 219.0919
[MꢄH]ꢄ, Calcd 219.0917.
3d: Yield: 67%, yellow solid, mp 220—221 °C; 1H-NMR (400 MHz,
that of 3h, while the cytotoxicity of 3g was almost decreased CDCl3) d: 6.95 (1H, d, Jꢁ3.6 Hz), 7.53 (1H, d, Jꢁ3.2 Hz), 7.61 (1H, d,
Jꢁ8.8 Hz), 7.70 (2H, d, Jꢁ8.4 Hz), 8.18 (1H, dd, Jꢁ8.8 Hz, 2.0 Hz), 8.46
11 times compared with 3h. In addition, the EC50 and TI
values of 3b, 3e, 3f and 3h were 7.88, 11.24, 37.76 and
12.09 mg/ml, and 24.61, 9.48, ꢂ6.63 and 1.28, respectively;
Therefore, whether introducing electron-withdrawing (nitro
group) or electron-donating group (methyl group) on the in-
dole’s ring of N-(2-nitrophenyl)indole (3b) will give less ac-
tive compounds than 3b.
Interestingly, once the nitro group of N-(2-nitrophenyl)in-
dole (3b) was substituted by cyano group to give N-(2-
cyanophenyl)indole (3c), the anti-HIV-1 integrase activity of
which was decreased sharply. For example, the EC50 and TI
values of 3b and 3c were 7.88/82.28 mg/ml, and 24.61/
ꢂ2.66, respectively. The anti-HIV-1 integrase activity of 3b
was nearly 10 times of that of 3c. Based upon the above in-
vestigation, the nitro group certainly is an important func-
tional group for 3b being good HIV-1 integrase inhibitory
activity. Furthermore, efforts to explain the reason why 3b
showed the most potent anti-HIV-1 integrase activity are on-
going in our laboratory.
(2H, d, Jꢁ8.8 Hz), 8.66 (1H, d, Jꢁ2.0 Hz); EI-MS m/z: 283 (Mꢄ, 28).
3e: Yield: 91%, orange solid, mp 104.5—106 °C; 1H-NMR (400 MHz,
CDCl3) d: 6.90 (1H, d, Jꢁ3.2 Hz), 7.10 (1H, d, Jꢁ9.2 Hz), 7.32 (1H, d,
Jꢁ3.2 Hz), 7.59 (1H, dd, Jꢁ8.0 Hz, 0.8 Hz), 7.68 (1H, m), 7.81 (1H, m),
8.08 (2H, m), 8.63 (1H, d, Jꢁ1.6 Hz); EI-MS m/z: 283 (Mꢄ, 100); HR-MS
m/z: 284.0592 [MꢄH]ꢄ, Calcd 284.0588.
3f: Yield: 37%, orange solid, mp 96.5—97 °C; 1H-NMR (400 MHz,
CDCl3) d: 1.94 (3H, s), 6.67 (1H, d, Jꢁ3.2 Hz), 6.92 (1H, d, Jꢁ6.8 Hz),
7.05 (2H, m), 7.49 (2H, m), 7.66 (2H, m), 7.97 (1H, dd, Jꢁ8.0 Hz, 1.2 Hz);
EI-MS m/z: 252 (Mꢄ, 95); HR-MS m/z: 253.0973 [MꢄH]ꢄ, Calcd
253.0972.
3g: Yield: 90%, yellow solid, mp 137—139 °C; 1H-NMR (400 MHz,
CDCl3) d: 2.39 (3H, s), 7.18 (1H, s), 7.24 (2H, m), 7.63 (2H, d, Jꢁ8.4 Hz),
7.64 (2H, d, Jꢁ8.8 Hz), 8.36 (2H, d, Jꢁ8.8 Hz); EI-MS m/z: 252 (Mꢄ, 100);
Anal. Calcd for C15H12N2O2: C, 71.42; H, 4.76; N, 11.11. Found: C, 71.54;
H, 4.52; N, 10.98.
1
3h: Yield: 98%, red liquid; H-NMR (400 MHz, CDCl3) d: 2.35 (3H, s),
6.90 (1H, s), 7.11 (3H, m), 7.43 (2H, m), 7.61 (2H, m), 7.94 (1H, dd,
Jꢁ8.0 Hz, 1.2 Hz); EI-MS m/z: 252 (Mꢄ, 80); HR-MS m/z: 253.0971
[MꢄH]ꢄ, Calcd 253.0972.
Anti-HIV-1 Integrase Activity Assay. Cells and Virus Cell line
(C8166) and the laboratory-derived virus (HIV-1IIIB) were obtained from
MRC, AIDS Reagent Project, UK. 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 ꢅ70 °C.12)
MTT-Based Cytotoxicity Assay Cellular toxicity of compounds 3a—h
on C8166 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) method as described previously.13) Briefly, cells
were seeded on 96-well microtiter plate in the absence or presence of vari-
ous concentrations of compounds in triplicate and incubated at 37 °C in a
humid atmosphere of 5% CO2 for 3 d. The supernatants were discarded and
MTT reagent (5 mg/ml in PBS) was added to each well, then incubated for
4 h, 100 ml of 50% N,N-dimethylformanide (DMF)–20% sodiumdodecyl
sulfate (SDS) was added. After the formazan was dissolved completely, the
plates were read on a Bio-Tek Elx 800 ELISA reader at 595/630 nm. The cy-
totoxic concentration that caused the reduction of viable C8166 cells by 50%
(CC50) was determined from dose–response curve.
Conclusion
In conclusion, eight simple N-arylindoles were designed,
synthesized and evaluated as HIV-1 integrase inhibitors in
vitro. Three compounds 3b, 3e and 3g demonstrated signifi-
cant anti-HIV-1 integrase activity as displayed in Table 1. Es-
pecially 3b showed the most promising and best activity
against HIV-1 integrase. In order to decrease cytotoxicity and
increase anti-HIV-1 integrase activity, further structural mod-
ifications of N-arylindoles will be conducted in our research
group.
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.). Melting points were
Syncytia Assay In the presence of 100 ml various concentrations of
compounds, C8166 cells (4ꢃ105/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
1
determined on a digital melting-point apparatus and were uncorrected. H-
NMR spectra were recorded on a Bruker Avance DMX 400 MHz instrument

722
Vol. 56, No. 5
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)ꢁCC50/EC50.14)
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Acknowledgments This work has been supported by the program for
New Century Excellent University Talents (NCET-06-0868), State Educa-
tion Ministry of China, and the Science & Technology Research Plan in
Shaanxi Province of China (No. 2006K01-G31-04). We also would like to
acknowledge Key Scientific and Technological Projects of Yunnan province
(2004NG12), National 973 project of China (2006CB504200), The Knowl-
edge Innovation Program of CAS (KSCX1-YW-R-24).
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