Design, synthesis, and biological evaluation of 1-(thiophen-2-yl)-9H-pyrido[3,4-b]indole derivatives as anti-HIV-1 agents

Article abstract of DOI:10.1111/cbdd.12456

A novel series of 1-(thiophen-2-yl)-9H-pyrido [3,4-b]indole derivatives were synthesized using DL-tryptophan as starting material. All the compounds were characterized by spectral analysis such as ¹H NMR, Mass, IR, elemental analysis and evalua

Full text of DOI:10.1111/cbdd.12456

Chem Biol Drug Des 2014
Research Article
Design, Synthesis, and Biological Evaluation of
1-(thiophen-2-yl)-9H-pyrido[3,4-b]indole Derivatives
as Anti-HIV-1 Agents
Penta Ashok1, Cui-Lin Lu2,3, Subhash Chander1,
therapy, HAART) AIDS-related deaths are decreasing in
the recent years with 1.6 million deaths reported in 2012
compared to 2.3 million in 2005 (1). HAART is often
called as drug-cocktail, is the combination of 3 or 4 dif-
Yong-Tang Zheng2,* and Sankarnarayanan
Murugesan1,*
1Medicinal Chemistry Research Laboratory, Department of
ferent antiretroviral drugs. WHO recommends the use of
Pharmacy, Birla Institute of Technology and Science,
Pilani, Rajasthan 333031, India
combination therapy for the treatment of HIV infection to
decrease emergence of drug resistance and to reduce
side-effects (2). HAART effectively reduce the viral repli-
cation rate and decreases plasma viral load below the
detection level which resulted in significant delay in dis-
ease progression (3), but it is unable to eradicate the
virus completely from the infected patients. Hence, long-
term or permanent treatment is required because of the
chronic nature of infection which results in emergence of
resistance, side-effects, and toxicity (4). Thus, develop-
ment of new anti-HIV agents acting on novel drug tar-
gets is recommended to overcome lifelong treatment,
resistance, and adverse effects.
2Key laboratory of Animal Models and Human Disease
Mechanisms of Chinese Academy of Sciences & Yunnan
Province, Kunming Institute of Zoology, Chinese Academy
of Sciences, Kunming, Yunnan 650223, China
3Kunming College of Life Science, University of Chinese
Academy of Sciences, Kunming, Yunnan 650204, China
*Corresponding authors: Yong-Tang Zheng,
zhengyt@mail.kiz.ac.cn; and Sankarnarayanan Murugesan,
murugesan@pilani.bits-pilani.ac.in
A novel series of 1-(thiophen-2-yl)-9H-pyrido [3,4-b]
indole derivatives were synthesized using DL-trypto-
phan as starting material. All the compounds were
characterized by spectral analysis such as 1H NMR,
Mass, IR, elemental analysis and evaluated for inhibi-
tory potency against HIV-1 replication. Among the
reported analogues, compound 7g exhibited signifi-
cant anti-HIV activity with EC50 0.53 lM and selectiv-
ity index 483; compounds 7e, 7i, and 7o displayed
moderate activity with EC50 3.8, 3.8, and 2.8 lM and
selectivity index >105, >105, and 3.85, respectively.
Interestingly, compound 7g inhibited p24 antigen
expression in acute HIV-1IIIB infected cell line C8166
with EC50 1.1 lM. In this study, we also reported the
Lipinski rule of 5 parameters, predicted toxicity pro-
file, drug-likeness, and drug score of the synthesized
analogues.
b-carboline represents a tricyclic pyrido[3,4-b]indole ring
system present in large number of natural products iso-
lated from different sources like marine sponges (5), ter-
ritorial plants (6), and fast food (7). The b-carboline
skeleton has privileged position in medicinal chemistry,
as compounds containing b-carboline scaffold displayed
diverse biological activities such as antimalarial, antileish-
manial, antibacterial, anticancer, antithrombotic, and
antiviral activity (8). Harman is the first b-carboline alka-
loid reported for anti-HIV activity and few of its synthetic
derivatives displayed potent anti-HIV activity (6). b-carbo-
line alkaloid flazin showed weak anti-HIV-1 activity,
where as its amido derivative flazinamide exhibited
potent activity through interfering with early stage of
infection by inhibiting the cell to cell transmission (9).
Along with these natural alkaloids, synthetic b-carboline
derivatives with different substitutions mainly on position
1 and 3 were reported for anti-HIV activity, displayed
significant activity through multiple mechanisms such as,
inhibiting cell to cell transmission and blocking the Tat–
TAR interaction (10–12) (Figure 1). With our ongoing pro-
ject and our continuous interest to develop b-carboline
derivatives as anti-HIV-1 agents, in this study, we have
reported design, synthesis, and biological evaluation of
1-(thiophen-2-yl)-b-carboline derivatives against HIV-1 cell
line and HIV-1 p24 antigen production.
Key words: AIDS, C8166 cells, HAART, HIV-1, indole, p24
antigen, b-carboline
Received 21 July 2014, revised 2 September 2014 and
accepted for publication 15 October 2014
According to UNAIDS-2013 report, it is estimated that,
2.3 million new infections were reported, and 35.3 mil-
lions are living with human immunodeficiency virus (HIV)
infection in the year 2012. With the availability of
effective treatment regimen (highly active anti-retroviral
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12456
1

Ashok et al.
Figure 1: Structure of reported b-carboline
derivatives and their anti-HIV activity.
the reaction, as monitored by TLC, the solvent was evapo-
rated under reduced pressure. The reaction mixture was
extracted with ethyl acetate (2 9 20 mL), combined
organic layers were dried over Na2SO4, and concentrated
under vacuum to afford (7a–p).
Methods and Materials
Chemistry
General methods
All solvents and reagents purchased from Sigma or Merck
companies are used as received without further purifica-
tion. Solvent system used throughout experimental work
for running thin layer chromatography (TLC) was ethyl
acetate and hexane mixture (5:5) to monitor the reaction.
Column chromatography was performed using silica gel
(100–200 mesh, SRL, Mumbai, India) as stationary phase,
and ethyl acetate and hexane mixture as mobile phase.
Melting points are uncorrected and were determined in
open capillary tubes on a Precision Buchi B530 (Flawil,
Switzerland) melting point apparatus containing silicon oil.
IR spectra of the synthesized compounds were recorded
using FTIR spectrophotometer (Shimadzu IR Prestige 21,
Mumbai, India). 1H NMR spectra were recorded on a Bru-
ker DPX-400 spectrometer (Bruker India Scientific Pvt.
Ltd., Mumbai, India) using TMS as an internal standard
(chemical shifts d in ppm). Elemental analysis was per-
formed on Vario EL III M/s Elementar C, H, N, and S ana-
lyzer (Elementar Analysensysteme GmbH, Hanau,
Germany). The ESMS were recorded on MICROMASS
Quattro-II LCMS system (Waters Corporation, Milford, MA,
USA).
(4-phenylpiperazin-1-yl)(1-(thiophen-2-yl)-9H-pyrido
[3,4-b]indol-3-yl)methanone (7a). White solid; %Yield
84; mp 152–154 °C; IR mmax/cmÀ1 (KBr): 3250, 1622, 1556,
1494, 1433, 744; 1H NMR (400 MHz, CDCl3): d 10.71
(s, 1H), 8.40 (s, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.94
(d, J = 3.4 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.49
(t, J = 7.5 Hz, 1H), 7.42 (d, J = 5.0 Hz, 1H), 7.24–7.15
(m, 2H), 6.91 (d, J = 8.1 Hz, 2H), 6.87–6.74 (m, 3H), 4.09–
3.96 (m, 4H), 3.35–3.25 (m, 4H); MS (ESI) m/z 439 (M + 1+);
Anal. Calcd for C26H22N4OS: C, 71.21; H, 5.06; N, 12.78;
S, 7.31, Found: C, 71.18; H, 5.10; N, 12.82; S, 7.29.
(4-(4-methoxyphenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7b). White solid;
%Yield 86; mp 182–184 °C; IR mmax/cmÀ1 (KBr): 3232,
1626, 1554, 1506, 1431, 1238, 723; 1H NMR (400 MHz,
CDCl3): d 10.98 (s, 1H), 8.22 (s, 1H), 7.93–7.77 (m, 2H),
7.50 (d, J = 8.0 Hz, 1H), 7.36–7.26 (m, 2H), 7.12–6.95
(m, 2H), 6.73 (d, J = 9.0 Hz, 2H), 6.63 (d, J = 9.0 Hz,
2H), 3.91–3.79 (m, 4H), 3.54 (s, 3H), 3.01–2.89 (m, 4H);
MS (ESI) m/z 468.9 (M + 1+); Anal. Calcd for
C
27H24N4O2S: C, 69.21; H, 5.16; N, 11.96; S, 6.84,
Found: C, 69.24; H, 5.18; N, 11.92; S, 6.82.
General procedure for the preparation of 7
(1-(thiophen-2-yl)-9H-pyrido[3,4-b]indol-3-yl)(4-p-
To a stirred solution of (5) (0.29 g, 0.001 mol) in dry THF,
HOBt (0.16 g, 0.001 mol) and EDC. HCl (0.23 g,
0.001 mol) were added, and the mixture was stirred for
30 min. To the reaction mixture, substituted phenylpiper-
azine (6a–p) (0.001 mol) was added at ice-cold tempera-
ture and the reaction mixture was subsequently further
stirred at room temperature for 6 h. After completion of
tolylpiperazin-1-yl)methanone (7c). White solid;
%
Yield 80; mp 232–-234 °C; IR mmax/cmÀ1 (KBr): 3296,
1616, 1556, 1433, 709; 1H NMR (400 MHz, CDCl3): d
8.76 (s, 1H), 8.51 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.79
(d, J = 2.0 Hz, 1H), 7.65–7.525 (m, 3H), 7.41–7.35 (m,
1H), 7.29–7.28 (m, 1H), 7.14 (d, J = 7.4 Hz, 2H), 6.94 (d,
2
Chem Biol Drug Des 2014

Anti-HIV Activity of b-Carboline Derivatives
J = 7.4 Hz, 2H), 4.25–4.15 (m, 4H), 3.40–3.25 (m, 4H),
2.32 (s, 3H); MS (ESI) m/z 453 (M + 1+); Anal. Calcd for
C27H24N4OS: C, 71.65; H, 5.35; N, 12.38; S, 7.09, Found:
C, 71.67; H, 5.38; N, 12.35; S, 7.05.
%Yield 76; mp 160–162 °C; IR mmax/cmÀ1 (KBr): 3326,
1634, 1556, 1487, 744; H NMR (400 MHz, CDCl3): d 8.98
1
(s, 1H), 8.48 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.80 (d,
J = 3.1 Hz, 1H), 7.65–7.58 (m, 2H), 7.54 (d, J = 5.0 Hz,
1H), 7.38–7.34 (m, 1H), 7.28–7.26 (m, 1H), 7.23 (t,
J = 8.1 Hz, 1H), 6.96 (t, J = 2.1 Hz, 1H), 6.90–6.84 (m,
2H), 4.20–4.02 (m, 4H), 3.43–3.37 (m, 4H); Anal. Calcd for
C26H21ClN4OS: C, 66.02; H, 4.48; N, 11.85; S, 6.78,
Found: C, 66.05; H, 4.52; N, 11.82; S, 6.82.
(4-(4-chlorophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7d). White solid;
%Yield 78; mp >250 °C; IR mmax/cmÀ1 (KBr): 3192, 1642,
1556, 1492, 1433, 748; 1H NMR (400 MHz, CDCl3): d
8.98 (s, 1H), 8.48 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.80
(d, J = 3.1 Hz, 1H), 7.66–7.58 (m, 2H), 7.54 (d,
J = 5.0 Hz, 1H), 7.38–7.63 (m, 1H), 7.28–7.26 (m, 1H),
7.23 (t, J = 8.1 Hz, 1H), 6.96 (t, J = 2.1 Hz, 1H), 6.91–
6.83 (m, 2H), 4.21–4.01 (m, 4H), 3.45–3.35 (m, 4H); MS
(ESI) m/z 473 (M + 1+, 100%), 475 (M + 3+, 33%); Anal.
Calcd for C26H21ClN4OS: C, 66.02; H, 4.48; N, 11.85; S,
6.78, Found: C, 66.00; H, 4.49; N, 11.88; S, 6.81.
(4-(2-methoxyphenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7i). White solid;
%Yield 70; mp 148–150 °C; IR mmax/cmÀ1 (KBr): 3356,
1638, 1526, 1427, 1246, 740; 1H NMR (400 MHz,
CDCl3):
d
8.88 (s, 1H), 8.47 (s, 1H), 8.14 (d,
J = 7.9 Hz, 1H), 7.80 (dd, J = 3.7, 0.9 Hz, 1H), 7.65–
7.59 (m, 2H), 7.54 (dd, J = 5.1, 1.0 Hz, 1H), 7.38–7.34
(m, 1H), 7.28–7.26 (m, 1H), 7.10–6.97 (m, 3H), 6.93
(dd, J = 8.0, 1.2 Hz, 1H), 4.22–4.08 (m, 4H), 3.92 (s,
3H), 3.32–3.22 (m, 4H); MS (ESI) m/z 468.9 (M + 1+);
Anal. Calcd for C27H24N4O2S: C, 69.21; H, 5.16; N,
11.96; S, 6.84, Found: C, 69.22; H, 5.20; N, 11.98; S,
6.79.
(4-(4-nitrophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (7e). White solid;
%Yield 76; mp >250 °C; IR
m
max/cmÀ1 (KBr): 3226,
1648, 1502, 1492, 1330, 744; 1H NMR (400 MHz,
CDCl3): d 8.77 (s, 1H), 8.52 (s, 1H), 8.25–8.07 (m, 3H),
7.71–7.47 (m, 4H), 7.42–7.25 (m, 1H), 7.14–7.02 (m,
1H), 6.90–6.78 (m, 2H), 4.35–3.82 (m, 4H), 3.65–3.55
(m, 4H); Anal. Calcd for C26H21N5O3S: C, 64.58; H,
4.38; N, 14.48; S, 6.63, Found: C, 64.59; H, 4.35; N,
14.51; S, 6.67.
(1-(thiophen-2-yl)-9H-pyrido[3,4-b]indol-3-yl)(4-o-
tolylpiperazin-1-yl)methanone (7j). White solid; %Yield
78; mp 186–188 °C; IR mmax/cmÀ1 (KBr): 3398, 1612,
1556, 1427, 740; 1H NMR (400 MHz, CDCl3): d 8.83 (s,
1H), 8.49 (s, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.80 (d,
J = 3.5 Hz, 1H), 7.62 (d, J = 3.7 Hz, 2H), 7.54 (d,
J = 4.4 Hz, 1H), 7.37 (dt, J = 8.0, 4.1 Hz, 1H), 7.30 (s,
1H), 7.23 (t, J = 7.6 Hz, 2H), 7.11 (d, J = 7.7 Hz, 1H),
7.05 (t, J = 7.4 Hz, 1H), 4.10 (d, J = 20.0 Hz, 4H), 3.11
(d, J = 3.2 Hz, 4H), 2.39 (s, 3H); Anal. Calcd for
C27H24N4OS: C, 71.65; H, 5.35; N, 12.38; S, 7.09, Found:
C, 71.63; H, 5.32; N, 12.39; S, 7.12.
(4-(4-fluorophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7f). White solid;
%Yield 78; mp >250 °C; IR
m
max/cmÀ1 (KBr): 3207,
1624, 1554, 1433, 748; 1H NMR (400 MHz, CDCl3):
d 8.77 (s, 1H), 8.52 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H),
7.80 (d, J = 2.8 Hz, 1H), 7.63 (d, J = 3.6 Hz, 2H), 7.58–
7.53 (m, 1H), 7.42–7.36 (m, 1H), 7.32–7.29 (m, 1H),
7.06–6.94(m, 4H), 4.22–4.04 (m, 4H), 3.34–3.28 (m,
4H); Anal. Calcd for C26H21FN4OS: C, 68.40; H, 4.64;
N, 12.27; S, 7.02, Found: C, 68.43; H, 4.62; N, 12.30;
S, 7.06.
(4-(2-chlorophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone
(7k). White
solid; %Yield 72; mp 148–150 °C; IR mmax/cmÀ1 (KBr):
3306, 1624, 1562, 1446, 752; 1H NMR (400 MHz,
CDCl3): d 8.80 (s, 1H), 8.50 (s, 1H), 8.16 (d, J = 7.9 Hz,
1H), 7.79 (dd, J = 3.7, 1.0 Hz, 1H), 7.63 (d, J = 3.7 Hz,
2H), 7.55 (dd, J = 5.1, 1.0 Hz, 1H), 7.45–7.34 (m, 2H),
7.31–7.26 (m, 3H), 7.13 (dd, J = 8.0, 1.5 Hz, 1H), 7.04
(td, J = 7.8, 1.5 Hz, 1H), 4.22–4.06 (m, 4H), 3.32–3.21
(m, 4H); Anal. Calcd for C26H21ClN4OS: C, 66.02; H,
4.48; N, 11.85; S, 6.78, Found: C, 66.06; H, 4.52; N,
11.82; S, 6.76.
(4-(3-methoxyphenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7g). White solid;
%Yield 82; mp 170–172 °C; IR mmax/cmÀ1 (KBr): 3387,
1602, 1554, 1485, 1433, 1249, 742; 1H NMR (400 MHz,
CDCl3): d 8.78 (s, 1H), 8.52 (d, J = 0.5 Hz, 1H), 8.17 (d,
J = 7.8 Hz, 1H), 7.79 (dd, J = 3.7, 1.0 Hz, 1H), 7.63 (dd,
J = 4.5, 1.0 Hz, 2H), 7.56 (dd, J = 5.1, 1.0 Hz, 1H),
7.41–7.35 (m, 1H), 7.32-7.29 (m, 1H), 7.24 (t,
J = 8.2 Hz, 1H), 6.63 (dd, J = 8.2, 1.8 Hz, 1H), 6.55 (t,
J = 2.3 Hz, 1H), 6.49 (dd, J = 7.9, 2.1 Hz, 1H), 4.22–
4.14 (m, 2H), 4.13-4.02 (m, 2H), 3.84 (s, 3H), 3.44–3.33
(m, 4H); Anal. Calcd for C27H24N4O2S: C, 69.21; H,
5.16; N, 11.96; S, 6.84, Found: C, 69.19; H, 5.12; N,
11.93; S, 6.80.
(4-(2-fluorophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone
(7l). White
solid; %Yield 68; mp 154–156 °C; IR mmax/cmÀ1 (KBr):
1
3271, 1634, 1502, 1433, 707; H NMR (400 MHz, CDCl3):
d 10.16 (s, 1H), 8.45 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H),
7.94–7.88 (m, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.58–7.53
(m, 1H), 7.51–7.45 (m, 1H), 7.32–7.28 (m, 1H), 7.24–7.22
(m, 1H), 7.13–6.93 (m, 4H), 4.16–4.05 (m, 4H), 3.30–3.22
(4-(3-chlorophenyl)piperazin-1-yl)(1-(thiophen-2-yl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (7h). White solid;
Chem Biol Drug Des 2014
3

Ashok et al.
(m, 4H); Anal. Calcd for C26H21FN4OS: C, 68.40; H, 4.64;
N, 12.27; S, 7.02, Found: C, 68.42; H, 4.68; N, 12.25; S,
7.01.
maintained at 37 °C in 5% CO2 in RPMI-1640 medium
supplemented with 10% heat-inactivating FBS (Gibco).
HIV-1IIIB was prepared from the supernatants of H9/HIV-
1IIIB cells. The 50% HIV-1 tissue culture infectious dose
(TCID50) in C8166 cells was determined and calculated by
Reed and Muench method. Virus stocks were stored in
small aliquots at À70 °C. The titre of virus stock was
1.0 9 108 TCID50/mL.
(4-(2,3-dichlorophenyl)piperazin-1-yl)(1-(thiophen-2-
yl)-9H-pyrido[3,4-b]indol-3-yl)methanone (7m). White
solid; %Yield 70; mp 188–190 °C; IR mmax/cmÀ1 (KBr):
1
3274, 1662, 1542, 1436, 765; H NMR (400 MHz, CDCl3):
d 8.88 (s, 1H), 8.49 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.79
(d, J = 3.6 Hz, 1H), 7.63–7.62 (m, 2H), 7.54 (dd, J = 5.1,
0.8 Hz, 1H), 7.39–7.35 (m, 1H), 7.30–7.26 (m, 1H), 7.25–
7.18 (m, 2H), 7.04 (dd, J = 6.9, 2.7 Hz, 1H), 4.17–4.11
(m, 4H), 3.28–3.20 (m, 4H); Anal. Calcd for
C26H20Cl2N4OS: C, 61.54; H, 3.97; N, 11.04; S, 6.32,
Found: C, 61.57; H, 3.95; N, 11.08; S, 6.36.
Cytotoxicity assay
The cellular toxicity of compounds on C8166 cells was
assessed by MTT colorimetric assay. Briefly, 100 lL of
4 9 105 cells were plated into 96-well plates, 100 lL of
various concentrations of compounds was added and
incubated at 37 °C in a humidified atmosphere of 5% CO2
for 72 h. Initially, 100 lL of supernatant was discarded,
MTT reagent was added and incubated for 4 h, 100 lL
50% DMF-15% SDS was added. After the formazan was
dissolved completely, the plates were read on a Bio-Tek
ELx 800 ELISA reader at 570 nm/630 nm. 50% cytotoxic-
ity concentration (CC50) was calculated (9).
(4-(pyridin-4-yl)piperazin-1-yl)(1-(thiophen-2-yl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (7n). White solid;
%Yield 68; mp 140–142 °C; IR mmax/cmÀ1 (KBr): 3207,
1641, 1554, 1415, 740; 1H NMR (400 MHz, CDCl3): d
9.12 (s, 1H), 8.55 (s, 1H), 8.32 (d, J = 6.5 Hz, 2H), 8.17
(d, J = 7.9 Hz, 1H), 7.82 (dd, J = 3.6, 0.8 Hz, 1H), 7.64–
7.62 (m, 2H), 7.55 (dd, J = 5.1, 0.8 Hz, 1H), 7.40–7.36
(m, 1H), 7.28–7.26 (m, 1H), 6.74 (d, J = 6.6 Hz, 2H),
4.23–4.07 (m, 4H), 3.65–3.55 (m, 4H); Anal. Calcd for
C25H21N5OS: C, 68.32; H, 4.82; N, 15.93; S, 7.30, Found:
C, 68.35; H, 4.83; N, 15.89; S, 7.32.
Inhibition of syncytia formation
In the presence or absence of various concentrations of
samples, 4 9 104 C8166 cells were infected with HIV-1
at a multiplicity of infection (MOI) of 0.04, and cultured
IIIB
(4-(pyridin-2-yl)piperazin-1-yl)(1-(thiophen-2-yl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (7o). White solid;
%Yield 64; mp 128–130 °C; IR mmax/cmÀ1 (KBr): 3383,
in 96-well plates at 37 °C in 5% CO2 for 3 days. Zidovu-
dine was used as a positive control. At 3 days post-infec-
tion, the cytopathic effect (CPE) was measured by
counting the number of syncytia in each well of 96-well
plates under an inverted microscope (1009). The inhibi-
tory percentage of syncytia formation was calculated by
the percentage of syncytia number in sample-treated cul-
ture compared to that in infected control culture. 50%
effective concentrations (EC50) and selectivity index (SI,
CC50/EC50) were calculated (9).
1
1624, 1577, 1485, 1436, 738; H NMR (400 MHz, CDCl3):
d 8.77 (s, 1H), 8.54 (s, 1H), 8.25 (d, J = 3.8 Hz, 1H), 8.19
(d, J = 7.8 Hz, 1H), 7.80 (d, J = 3.6 Hz, 1H), 7.63 (d,
J = 3.8 Hz, 2H), 7.58–7.51 (m, 2H), 7.43–7.34 (m, 1H),
7.32–7.29 (m, 1H), 6.74–6.66 (m, 2H), 4.18–4.02 (m, 4H),
3.86–3.75 (m, 4H); Anal. Calcd for C25H21N5OS: C, 68.32;
H, 4.82; N, 15.93; S, 7.30, Found: C, 68.29; H, 4.79; N,
15.96; S, 7.26.
(4-benzylpiperazin-1-yl)(1-(thiophen-2-yl)-9H-pyrido
[3,4-b]indol-3-yl)methanone (7p). White solid; %Yield
76; mp 160–162 °C; IR mmax/cmÀ1 (KBr): 3388, 1602,
1554, 1485, 1425, 700; 1H NMR (400 MHz, CDCl3): d
8.90 (s, 1H), 8.42 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.82–
7.75 (m, 1H), 7.70–7.46 (m, 3H), 7.43–7.18 (m, 7H), 3.98–
3.94 (m, 4H), 3.62 (s, 2H), 2.68–2.30 (m, 4H); MS (ESI) m/
z 453 (M + 1+); Anal. Calcd for C27H24N4OS: C, 71.65; H,
5.35; N, 12.38; S, 7.09, Found: C, 71.69; H, 5.32; N,
12.41; S, 7.12.
Inhibition of HIV-1 p24 antigen production in acute
infection
The inhibitory effect of compound on HIV-1 replication
in-vitro was examined by quantification of p24 expression
using capture ELISA. Briefly, C8166 cells were inoculated
with HIV-1IIIB (M.O.I. = 0.03) in the absence or presence of
various concentrations of compound at 37 °C for 2 h to
allow for viral absorption. It was then washed three times
with PBS. The cells were plated at 3 9 104 cells/well with
or without various concentrations of compound and incu-
bated at 37 °C in a humidified atmosphere of 5% CO2 for
4 days. HIV-1 p24 expression in cell-free supernatants
was assayed by ELISA. The inhibitory percentage of p24
antigen production was calculated by the OD490/630 value
of sample-treated culture compared to that in infected
control culture. 50% effective concentrations (EC50) and
selectivity index (SI, CC50/EC50) were calculated (9).
Biological evaluation
Cells and virus
C8166 cells and HIV-1IIIB were kindly donated by Medical
Research Council, AIDS Regent Project. The cells were
4
Chem Biol Drug Des 2014

Anti-HIV Activity of b-Carboline Derivatives
COOH
CO2Et
CO2Et
CO2Et
a
c
b
NH2
NH
NH2
N
N
H
N
H
N
H
N
H
S
S
2
3
4
1
d
O
COOH
N
e
N
R
N
N
N
H
HN
N
H
N
R
S
S
7a–p
6a–p
5
Scheme 1: Reagents and conditions: (a) thionyl chloride, ethanol, reflux, 30 min, 76%; (b) thiophene-2-carbaldehyde, trifluoroacetic acid,
DCM, rt, 3 h, 78%; (c) KMnO4, THF, rt, 24 h, 72%; (d) 50% aq. NaOH, reflux, 30 min, 68%; (e) EDCI, HOBt, THF, piperazines, 0 °C-rt
6 h, 64–86%.
Molecular properties
afford the intermediate ethyl 1-(thiophen-2-yl)-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indole-3-carboxylate (3). Upon
Good pharmacokinetic properties are essential for any
new chemical entity to complete drug discovery process
successfully. In recent time, in-silico prediction of molecu-
lar properties of new chemical entity is getting more inten-
sion in medicinal chemistry. Molecular parameters, such
as ClogP (≤5), Molecular weight (≤500), number of hydro-
gen bond acceptors (≤5) and number of hydrogen bond
donors (≤10), and aqueous solubility have much impact on
pharmacokinetic properties of ligands. Drug-likeness is a
qualitative concept used in drug design for how ‘drug-like’
a substance is with respect to factors like bioavailability.
Around 80% of the marketed drugs have positive drug-
likeness value. The drug score is a calculated value to
judge the compound’s overall potential to qualify as drug.
Drug score (ds) of a molecule depends up on cLogP,
molecular weight, logS, drug-likeness, and toxicity risks of
the molecule. By taking the importance of these molecular
properties into consideration, in this study, we predicted,
Lipinski rule of five parameters, toxicity profile, drug-like-
ness, and drug score values of these new chemical
entities using molinspiration cheminformatics (http://www.
molinspiration.com) and osiris property explorer (http://
www.organic-chemistry.org/prog/peo/) (13).
oxidation with potassium permanganate, ethyl 1-(thiophen-
2-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (4) was formed,
followed by alkaline ester hydrolysis to obtain 1-(thiophen-
2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (5) as key
intermediate. The carboxylic acid group-containing key
intermediate (5) was treated with appropriate amines
(substituted aryl-piperazines 6a–p) in the presence of 1-hy-
droxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (EDCI) to get the
desired products (7a–p) in good to excellent yields (14,15).
Synthesized compounds were characterized by spectral
data such as IR, 1H NMR, Mass, and elemental analysis.
In general, the IR spectra of the synthesized compounds
showed N-H stretching at around 3398–3207 cmÀ1
,
C=O absorption around 1648–1602 cmÀ1
absorption (methoxy) band at around 1249–1238 cmÀ1
,
C–O–C
,
and C–Cl absorption band at around 752–744 cmÀ1. The
1H NMR spectrum of the reported compounds showed
characteristic singlet around d 8.52–8.22 due to position 4
proton of the b-carboline ring, indole NH proton value was
varied with d value range from 10.71 to 8.76, remaining
aromatic protons were observed from the range of d 8.32
to 6.66 with their respective pattern of splitting, sometimes
as multiplets due to overlapping of the peaks. Eight piper-
azine protons appeared as two multiplets around d value
4.35–3.79 and 3.65-3.05. In analogues 7b, 7g, and 7i
methoxy protons appeared as singlet at d 3.54, 3.84, and
3.92, respectively, aromatic methyl protons as singlet at
2.39 and 2.32 and in benzyl piperazine derivative benzyl
protons appeared as singlet at d 3.62. In mass spectral
analysis, M + 1 peak appeared as parent peak. Elemental
analysis indicated that the calculated and observed values
were within the acceptable limits (Æ 0.05%).
Results and Discussion
Chemistry
Designed analogues were synthesized using the synthetic
protocol illustrated in Scheme 1. The compounds were
synthesized from the starting material, DL-Tryptophan (1)
in a sequence of reactions. Initially, ethyl ester of trypto-
phan (2) was synthesized from DL-Tryptophan (1) using
thionylchloride in ethanol and followed by Pictet-Spengler
reaction in the presence of trifluoroacetic acid (TFA) to
Chem Biol Drug Des 2014
5

Ashok et al.
Table 1: Biological activity of the synthesized compounds
Inhibition of p24 antigen
production
Inhibition of syncytia formation
Comp. Code
R
CC50 (lM)a
EC50 (lM)b
SIc
EC50 (lM)b
SIc
7a
7b
7c
7d
7e
7f
Phenyl
>400
>400
>400
>400
>400
>400
256
>400
>400
>400
>400
>400
>400
>400
10.6
102.1
>200
>200
>200
3.8
118.8
0.53
17.8
3.8
38.7
>200
18.8
95.8
60.8
2.8
>3.9
–
–
NTd
NTd
NTd
NTd
7.6
–
–
–
–
4-Methoxyphenyl
4-Methylphenyl
4-Chlorophenyl
4-Nitrophenyl
4-Fluorophenyl
3-Methoxyphenyl
3-Chlorophenyl
2-Methoxyphenyl
2-Methylphenyl
2-Chlorophenyl
2-Fluorophenyl
2,3-Dichlorophenyl
4-Pyridyl
–
>105
>3.4
483
>22.5
>105
>10.3
–
>21.3
>4.2
>6.6
3.85
>32
>52.6
–
232.7
>14.3
>71.4
–
–
>11.4
–
–
–
>19.6
1178800.0
NTd
1.1
7g
7h
7i
27.9
5.6
7j
NTd
NTd
35.2
NTd
NTd
NTd
20.4
0.004
7k
7l
7m
7n
7o
7p
2-Pyridyl
Benzyl
Zidovudine
>400
4715.2
12.5
0.002
2357600.0
aCC50: concentration required to reduce the viability of C8166 cells by 50%, as determined by MTT method.
bEC50: concentration of compound required to achieve 50% protection of C8166 cell cultures against HIV-induced cytotoxicity.
cSI: selectivity index (CC50/EC50).
dNot tested.
Biological evaluation
phenyl ring with heterocyclic ring pyridine led to increased
cytotoxicity, and replacement with benzyl ring resulted in
considerable increase in anti-HIV-1 activity without any
effect on cytotoxicity.
All the synthesized compounds of analytical purity were
used for biological evaluation such as inhibition of HIV rep-
lication and cytotoxicity (Table 1). Compounds were evalu-
ated for HIV-1 replication inhibition potency by measuring
suppression of syncytia formation. Among the reported
analogues, compound 7g exhibited significant inhibition of
syncytia formation with EC50 0.53 lM. Compounds 7e, 7i,
and 7o displayed moderate inhibition of HIV-1 replication
with EC50 3.8, 3.8, and 2.8 lM respectively. Compounds
7h, 7j, 7m, 7n, and 7p showed weak anti-HIV-1 activity.
All the synthesized analogues were evaluated for cytotoxic-
ity using MTT assay method against C8166 cell lines.
Among these analogues, two compounds 7o and 7g dis-
played cytotoxicity against C8166 cell lines, CC50 values
were found to be 10.6 and 256 lM and rest of the com-
pounds not showed any toxicity at 400 lM.
Compounds exhibited inhibition of HIV-1 syncytia forma-
tion were also evaluated for inhibition potency against
HIV-1IIIB p24 antigen production. Compound 7g showed
significant inhibition of p24 production in acute HIV-1
infected C8166 cell lines with EC50 value of 1.1 lM.
Molecular Properties
Predicted in-silico molecular properties of the reported 1-
(thiophen-2-yl)-b-carboline derivatives were shown in
Table S1. From this study, we found that compounds dis-
played significant in-vitro anti-HIV activity were following the
Lipinski rule of five parameters with single violation, which
indicating that these compounds may not have any bioavail-
ability problem (16). Reported b-carboline derivatives
showed medium risk of mutagenic and low risk of tumori-
genic, irritant, reproductive effects. Compounds also showed
good drug-likeness (positive values) and drug score values
except compound 7e showed less drug-likeness and drug
score values, due to the presence of nitro group.
Structure activity relationship study
In the present study, we explored the phenyl ring attached
to piperazine moiety with different substitutions and
replacements. In this series, proto compound with phenyl
substitution displayed weak inhibition of HIV-1 replication.
Analogues with ortho–para directing group substitutions
on position 4 of phenyl ring, were not showed any anti-
HIV-1 activity even at maximum tested concentration,
whereas meta directing nitro group substitution resulted in
significant increase in potency. Ortho–para directing
groups on position 2 and 3 favours the anti-HIV-1 activity,
and potency was increased many folds. Replacement of
Conclusion
In the present study, we reported synthesis and anti-HIV-1
activity of novel series of 1-(thiophen-2-yl)-9H-pyrido[3,4-b]
6
Chem Biol Drug Des 2014

Anti-HIV Activity of b-Carboline Derivatives
indole derivatives. Among the reported analogues, com-
pounds 7g, 7e, 7i, and 7o exhibited significant to moder-
ate anti-HIV-1 activity with EC50 values 0.53, 3.8, 3.8, and
2.8 lM with good selectivity index 483, >105, >105, and
3.85, respectively. Interestingly, compound 7g also dis-
played significant suppression of p24 antigen production
with EC50 value of 1.1 lM. SAR studies stated that substi-
tution of electron donating ortho–para directing groups on
position 2 and 3 as well as electron withdrawing group
substitution on position 4 favours the anti-HIV-1 activity.
In-silico molecular predication studies revealed these com-
pounds following Lipinski rule of five parameters and have
good pharmacokinetic profile.
7. Adachi J., Mizoi Y., Naito T., Ogawa Y., Uetani Y., Nin-
omiya I. (1991) Identification of tetrahydro-b-carboline-
3-carboxylic acid in foodstuffs, human urine and
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Acknowledgments
The authors are grateful to SAIF, Panjab University,
Chandigarh, India for providing NMR spectra. One of the
authors Ashok Penta, acknowledges Council of Scientific
and Industrial Research, New Delhi, India for financial sup-
port in the form of Senior Research Fellowship.
11. Yu X., Lin W., Pang R., Yang M. (2005) Design, syn-
thesis and bioactivities of TAR RNA targeting b-carbo-
line derivatives based on Tat–TAR interaction. Eur J
Med Chem;40:831–839.
12. Brahmbhatt K.G., Ahmed N., Sabde S., Mitra D.,
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Conflict of Interest
immunodeficiency
virus.
Bioorg
Med
Chem
Lett;20:4416–4419.
None.
13. Penta A., Chander S., Ganguly S., Murugesan S.
(2014) De novo design and in-silico studies of novel
1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
carboxylic acid derivatives as HIV-1 reverse transcrip-
tase inhibitors. Med Chem Res;23:3662–3670.
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Chem Biol Drug Des 2014
7