Synthesis and evaluation of β-carboline derivatives as inhibitors of human immunodeficiency virus

Article abstract of DOI:10.1016/j.bmcl.2010.06.052

A series of β-carboline derivatives were synthesized by utilizing aromatization and chemoselective alkylation method recently reported from our laboratory. Synthesized derivatives were evaluated for anti-HIV activity in human CD4+ T cell line (CEM-GFP) infected with HIV-1 NL_4.3 virus. 1-Formyl-β-carboline-3-carbxylic acid methyl ester (15) showed inhibition of human immunodeficiency virus at IC₅₀ = 2.9 μM.

Full text of DOI:10.1016/j.bmcl.2010.06.052

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4416–4419
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of b-carboline derivatives as inhibitors of human
immunodeficiency virus
a
Keyur G. Brahmbhatt a, Nafees Ahmed a, Sudeep Sabde b, Debashis Mitra b, , Inder Pal Singh ,
*
Kamlesh K. Bhutani a,
*
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India
b National Center for Cell Science (NCCS), Pune University Campus, Ganeshkhind, Pune, Maharashtra 411 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of b-carboline derivatives were synthesized by utilizing aromatization and chemoselective alkyl-
ation method recently reported from our laboratory. Synthesized derivatives were evaluated for anti-HIV
activity in human CD4+ T cell line (CEM-GFP) infected with HIV-1 NL4.3 virus. 1-Formyl-b-carboline-3-
Received 27 April 2010
Revised 8 June 2010
Accepted 9 June 2010
Available online 12 June 2010
carbxylic acid methyl ester (15) showed inhibition of human immunodeficiency virus at IC50 = 2.9 lM.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anti-HIV
b-Carboline
1-Formyl-b-carboline
CEM-GFP cells
An urgent need for the discovery of newer agents for the treat-
ment of AIDS demands that all approaches to drug discovery
should be exploited aggressively. Among the possible approaches,
the one from natural products has made unique and vital contribu-
tions to drug discovery in many disease areas. Natural products are
rich source for biologically active compounds. The conversion of
these biologically active compounds into derivatives better suited
for medicinal use has become a useful strategy for many modern
drug discovery programs. Many natural products have shown
promising anti-HIV activities which have been elaborately re-
viewed by our group.1 Several natural products have been used
as lead molecules for further modifications to improve their anti-
HIV activities.2–5
tial interaction of regulatory protein Tat to trans-activation
response region (TAR).13,14 Furthermore, C-1 substituted alkaloid
flazine (2) and its amide derivative (3) have also been reported
for their anti-HIV activity.15
This led us to study various b-carboline analogues as anti-HIV
agents as a part of our ongoing work. In this Letter, we report syn-
thesis and evaluation of several b-carboline derivatives for anti-
HIV activity using human CD4+ reporter T cell line (CEM-GFP) in-
fected with HIV-1 NL4.3
.
b-Carboline derivatives, obtained from various natural and syn-
thetic sources, show variety of important biological activities;6–11
including recent reports on their anti-HIV potential (Fig. 1). N9-
butylharmine (1), a semi-synthetic derivative of widely occurring
alkaloid harmine, has been found to inhibit HIV replication in H9
lymphocytes.12 Presence of alkyl group at N9 and methyl group
at C-1 are essential for better anti-HIV activity. 1-Methyl-b-carbo-
line-3-carboxamide derivative (4) bearing guanidinium group has
been known to inhibit replication of HIV-1 by hindering the essen-
* Corresponding authors. Tel./fax: +91 172 2232208 (K.K.B); tel.: +91 202
5708151; fax: +91 202 5692259 (D.M).
E-mail addresses: dmitra@nccs.res.in (D. Mitra), kkbhutani@niper.ac.in (K.K.
Bhutani).
Figure 1. Anti-HIV b-carboline derivatives.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.052

K. G. Brahmbhatt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4416–4419
4417
Scheme 1. Synthesis of b-carboline derivatives: Reagents and conditions: (i) dicyclohexylcarbodiimide (DCC), CH2Cl2, 20 °C, 3 h then CF3COOH, 50 °C, 1 h, 85%; (ii) NaH,
anhydrous DMF, 28 °C (7 62% 4 h; 13 80% 30 min); (iii) MeSO2Cl, Et3N, CH2Cl2, 0 °C, 2 h then amine, 20 °C, 6 h (8a 70%; 8b 68%; 8c 72%); (iv) NaH, anhydrous DMF, 0 °C, 30 min
then, RX (X = Br or I), 28 °C, 3 h (9a 82%; 9b 80%; 9c 83%; 9d 78%; 9e 75%; 14a 85%; 14b 78%; 14c 80%; 14d 75%; 14e 85%); (v) SOCl2, MeOH, 4 h, 30 °C, 96%; (vi) SeO2, dioxane,
reflux, 67%; (vii) Ac2O, pyridine, reflux, 3 h, 60%; (viii) inverse addition: n-BuI, anhydrous DMF, degassing by freeze-pump–thaw then NaH, 28 °C, oxygen-free argon
atmosphere, 30 min (11 77%; 12 13%).
The overall strategy followed for the synthesis of b-carboline
derivatives is shown in Scheme 1. N-acetyl tryptophan (5),
obtained by the Schotten–Baumann method from commercially
available dl-tryptophan, was cyclized by a modified Bischler–
Napieralski condensation to afford 1-methyl-3,4-dihydro-b-carb-
oline-3-carboxylic acid (6) in 85% yield.16 Methyl ester derivative
(10) was synthesized by reacting 6 with thionyl chloride in
methanol.17 Compounds 6 and 10 were aromatized by employ-
ing sodium hydride in anhydrous DMF to yield 7 and 13 in
62% and 80%, respectively.18 Compound 7 was reacted with
methanesulfonyl chloride in dichloromethane in the presence
of triethyl amine to obtain reactive mesyl ester which was re-
acted in situ with piperidine, morpholine or thiomorpholine to
generate 8a–c in 70%, 68% and 72% yield, respectively.19 1-
Methyl-b-carboline-3-carboxylic acid methyl ester (13) and thi-
omorpholine derivative (8c) were alkylated to afford N9-alkyl
derivatives 9a–e20 and 14a–e,18 respectively. Synthesis of 11a
was attempted by alkylation of 10, however, these attempts re-
sulted in formation of C-3 alkylated derivative (11) in 77% yield
along with 12 as a result of chemoselective alkylation in oxygen
free conditions.18 Mixture of 11 and 12 was separated by dry
column vacuum chromatography (DCVC).
Table 1
Anti-HIV activity of synthesized compounds
Entry Compound Non-cytotoxic
% Inhibition p24 ELISA at non-
cytotoxic concentration
Entry Compound Non-cytotoxic
% Inhibition p24 ELISA at non-
cytotoxic concentration
code
concentratione
(lM)
code
concentratione
(lM)
1
2
3
4
5
6
7
8
9
6
17.54
1.71
27.12
1.61
16.48
NIa
NI
16.9
NI
NI
NI
NI
NI
17.26
12.17
12
13
14
15
16
17
18
19
20
21
12
13
14.12
4.17
19.66
3.73
3.54
1.69
15.13
9.83
3.59
4.98
NI
21.3
NI
NI
NI
8a
8b
8c
9a
9b
9c
9d
9e
10
11
14a
14b
14c
14d
14e
15
6.22
19.76
23.84
21.46
1.36
18.59
8.39
NI
NI
78.17b,c
NI
16
AZT
10
11
89.75b,d
a
NI = no inhibition.
b
IC50 = concentration of compound required to achieve 50% protection of CEM-GFP cells (n = 3) from virus, as determined by p24 ELISA assay, CC50 = concentration of
compound required to reduce proliferation of CEM-GFP cells (n = 3) by 50%, as determined by MTT assay and T.I.(therapeutic index) = CC50/IC50
.
c
IC50 = 2.9
l
M, CC50 = 20.3
M, CC50 = 23.04
Non-cytotoxic concentration = highest concentration where the cell viability is more than 95%.
lM and TI = 7.
d
e
IC50 = 1.04
l
l
M and TI = 23.07.

4418
K. G. Brahmbhatt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4416–4419
17. Lippke, K. P.; Schunack, W. G.; Wenning, W.; Muller, W. E. J. Med. Chem. 1983,
Compound 13 underwent oxidation upon treatment with sele-
26, 499.
nium dioxide to yield 1-formyl derivative (15) in 67% which was re-
fluxed with acetic anhydride and pyridine (10:1) to obtain canthin-
6-one-3-carboxylic acid methyl ester (16) in 60% yield.21
18. Brahmbhatt, K. G.; Ahmed, N.; Singh, I. P.; Bhutani, K. K. Tetrahedron Lett. 2009,
50, 5501.
19. General procedure for preparation of amide derivatives (8a–c): To a mixture of 7
(1.60 g, 7.08 mmol) and triethyl amine (2.86 g, 28.3 mmol) in anhydrous
dichloromethane (10 ml), methanesulfonyl chloride (0.97 g, 8.5 mmol) was
added dropwise at 0 °C under positive argon atmosphere. The reaction mixture
was allowed to stir at 28 °C for 2 h followed by addition of amine [a: piperidine
(0.66 g, 7.79 mmol), b: morpholine (0.68 g, 7.79 mmol) or c: thiomorpholine
(0.80 g, 7.79 mmol)]. The resulting mixture was allowed to stir for another 6 h
at 28 °C. The reaction mixture was poured into cold water, and extracted with
chloroform (3 Â 100 ml). The combined organic phase was washed with water
(3 Â 200 ml) and with brine (3 Â 200 ml), then dried over anhydrous sodium
sulfate and evaporated in vacuo. The crude product obtained was purified by
column chromatography (Silica Gel #60–120, hexane/EtOAc-gradient) to
obtain compound 8a, 8b or 8c in 70%, 68% and 72% yield, respectively
depending on the amine used.
All synthesized analogues were first evaluated for their cytotox-
icity in MTT based cell viability assay22 in CEM-GFP cells. Based on
the results of MTT assay, non-cytotoxic concentration of each ana-
logue was used for determination of in vitro anti-HIV activity in
CEM-GFP cells23–26 as shown in Table 1. Only the active com-
pounds showing more than 60% inhibition at non-cytotoxic con-
centration were further evaluated to determine their CC50 and
IC50 values. The results are shown in Table 1.
In our preliminary screenings, compounds 6, 8c, 10 and 13 were
found to show inhibition of HIV-1 NL4.3 (Table 1, entries 1, 4, 10
and 13). N-Alkylated derivatives of harmine have been reported
to show better activity than harmine.12 Based on this observation,
8c and 13 were alkylated to afford 9a–e and 14a–e, respectively,
whereas, alkylation of 10 resulted in formation of 11 and 12. It is
noteworthy that N-alkylation of 8c and 13 was detrimental and re-
sulted in loss of activity (Table 1, entries 5–9 and 14–18). This
observation suggested a different mode of action and/or a different
binding site for b-carboline-3-carboxylic acid derivatives as com-
pared to harmine (7-methoxy-b-carboline) derivatives. Interest-
ingly, 1-formyl-b-carboline-3-carboxylic acid methyl ester
(1-Methyl-b-carboline-3-yl)(piperidin-1-yl)methanone (8a): 1H NMR (400 MHz,
CDCl3 ppm): d 9.41 (br s, 1H), 7.99 (s, 1H), 7.92 (d, 1H, J = 7.84 Hz), 7.45–7.51 (m,
2H), 7.20–7.23 (m, 1H), 3.81 (br s, 2H), 3.54 (br s, 2H), 2.69 (s, 3H), 2.74 (br s, 2H),
2.69 (s, 3H), 1.70 (br s, 4H), 1.56 (br s, 2H). 13C NMR (100 MHz, CDCl3, ppm): d
168.5, 142.2, 139.9, 139.7, 133.7, 127.3, 127.2, 120.9, 120.7, 119.1, 111.8, 110.9,
47.7, 42.7, 25.5, 24.6, 23.7, 19.2. MS (APCI): m/z 294.3 [M+1]+.
(1-Methyl-b-carboline-3-yl)(morpholino)methanone (8b): 1H NMR (300 MHz,
DMSO-d6, ppm): d 8.08 (d, 2H, J = 9.38), 7.34–7.45 (m, 2H), 7.07 (t, 1H, J = 7.30),
3.43 (br s, 8H), 2.58 (s, 3H). 13C NMR (75.5 MHz, DMSO-d6s, ppm): d 168.4, 142.2,
141.1, 141.0, 134.9, 128.7, 127.6, 122.4, 121.5, 120.1, 113.9, 112.5, 48.0, 42.7,
20.7. MS (APCI): m/z 296.1 [M+1]+.
(1-Methyl-b-carboline-3-yl)(thiomorpholino)methanone(8c): 1H NMR (400 MHz,
CDCl3, ppm): d 9.48 (br s, 1H), 8.09 (s, 1H), 7.92 (d, 1H, J = 7.60 Hz), 7.49–7.45 (m,
2H), 7.24–7.20 (m, 1H), 4.12 (br s, 2H), 3.92 (br s, 2H), 2.85 (br s, 2H), 2.74 (br s,
2H), 2.69 (s, 3H). 13C NMR (100 MHz, CDCl3, ppm): d 169.8; 142.4, 140.8, 140.6,
134.9, 128.4, 128.3, 121.8, 121.7, 120.3, 113.7, 111.9, 50.3, 45.3, 28.1, 27.4, 20.3.
MS (APCI): m/z 312.2 [M+1]+.
derivative (15) showed inhibition of HIV at IC50 of 2.9
CC50 of 20.3 M. Anti-HIV potential of 15 is encouraging as there
are no reports of 1-formyl derivative of b-carboline showing anti-
lM and
l
20. General procedure for alkylation of (1-methyl-b-carboline-3-yl)(thiomorpho
lino)methanone (8c): Compound 8c (250 mg, 0.80 mmol) was stirred in
anhydrous DMF (10 ml) at room temperature in argon atmosphere. It was
cooled to 0–5 °C and then sodium hydride (60%) (38.6 mg, 0.96 mmol) was
added. The reaction mixture was allowed to stir for 30 min at 28 °C. Alkyl halide
(3 mmol) was added dropwise and the reaction mixture was stirred for 3 h at
28 °C. The resulting mixture was then poured into cold water, and extracted with
ethyl acetate (3 Â 50 ml). The organic phase was washed with water (2 Â 25 ml)
and brine (2 Â 25 ml), then dried over anhydrous sodium sulfate and evaporated
in vacuo. The crude product was purified by column chromatography (Silica Gel
#60–120, hexane/EtOAc-gradient) to obtain compound 9a–e in 82%, 80%, 83%,
78% and 75%, respectively.
HIV activity in the literature.
In conclusion, we have described synthesis and biological eval-
uation of b-carboline derivatives for anti-HIV activity. Out of vari-
ous compound tested, 1-formyl-b-carboline-3-carboxylic acid
methyl ester derivative (15) was found to be active against HIV.
Further modification of the selected lead is under progress, and re-
sults will be published in a due course of time.
(9-Benzyl-1-methyl-b-carboline-3-yl)(thiomorpholino)methanone(9a): 1H NMR
(400 MHz, DMSO-d6, ppm): d 8.51 (s, 1H), 8.43 (d, 1H, J = 7.24), 7.74 (d, 1H,
J = 7.78), 7.67 (d, 1H, J = 6.60), 7.38 (d, 1H, J = 6.68), 7.25–7.30 (m, 3H), 6.99(d, 2H,
Acknowledgment
Authors thank the Department of Biotechnology, Ministry of
Science and Technology, Government of India for the financial sup-
port to this project (grant no. BT/PR7020/Med/14/930/2005 Dt. 23/
05/2006).
J = 6.16), 5.98 (s, 2H), 3.96 (br s, 2H), 3.76 (br s, 2H), 2.87 (s, 3H), 2.71(br s, 4H). 13
C
NMR (100 MHz, DMSO-d6, ppm): d 166.5, 142.5, 140.6, 140.0, 138.3, 134.5, 129.6,
129.4, 128.9, 127.3, 125.3, 122.3, 120.8, 120.4, 113.8, 110.8, 49.6, 47.4, 44.4, 27.1,
26.6, 21.6. MS (APCI): m/z 402.2 [M+1]+.
(9-Prenyl-1-methyl-b-carboline-3-yl)(thiomorpholino)methanone (9b): 1H NMR
(400 MHz, CDCl3, ppm): d 8.29 (s, 1H), 8.14 (d, 1H, J = 7.80 Hz), 7.60 (dt, 1H,
J1 = 8.28, J2 = 1.00 Hz), 7.43 (d, 1H, J = 8.36 Hz), 7.31 (dt, 1H, J1 = 7.72,
J2 = 0.44 Hz), 5.18–5.22 (m, 3H), 4.12 (br s, 2H), 4.01 (br s, 2H), 3.03 (s, 3H, –
CH3), 2.81 (br s, 2H), 2.76 (br s, 2H), 1.91 (s, 3H), 1.74 (s, 3H). 13C NMR (100 MHz,
CDCl3, ppm): d 169.0, 141.7, 139.8, 135.2, 134.7, 129.3, 128.4, 121.5, 121.3, 120.8,
120.1, 114.2, 109.8, 63.5, 50.2, 45.2, 43.3, 27.8, 27.1, 24.9, 23.0, 18.1. MS (APCI):
m/z 380.3 [M+1]+.
References and notes
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doi:10.1002/jhet.364, in press.
9-(4-Fluorobenzyl)-1-methyl-b-carboline-3-yl(thiomorpholino)methanone(9c):
1H NMR (400 MHz, CDCl3, ppm): d 8.33 (s, 1H), 8.18 (d, 1H, J = 7.88 Hz), 7.59 (dt,
1H, J1 = 7.24, J2 = 1.00), 7.38 (m, 2H, J = 8.38. Hz), 6.97 (m, 4H), 5.81 (s, 2H,), 4.11
(br s, 2H), 3.97 (br s, 2H), 2.93 (s, 3H, –CH3), 2.83 (br s, 2H), 2.75 (br s, 2H). 13CNMR
(100 MHz, CDCl3, ppm): d 163.1, 160.6, 142.2, 141.2, 139.7, 135.1, 133.0, 129.8,
129.1, 126.7, 126.6, 121.5, 120.9, 120.6, 115.7, 115.5, 114.0, 109.4, 63.3, 50.1,
45.1, 28.4, 27.5, 24.6. MS (APCI): m/z 420.3 [M+1]+.
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1H
NMR (400 MHz, CDCl3, ppm): d 8.28 (s, 1H), 8.12 (d, 1H, J = 7.80 Hz), 7.65 (dt,
1H, J1 = 7.16, J2 = 1.16 Hz), 7.54 (d, 1H, J = 8.36 Hz), 7.35 (dt, 1H, J1 = 7.88,
J2 = 0.80 Hz), 5.30 (d, 2H, J = 2.44), 4.12 (br s, 2H), 3.99 (br s, 2H), 3.14 (s, 3H, –
CH3), 2.82 (br s, 2H), 2.76 (br s, 2H), 2.38 (t, 1H, J = 2.44). 13C NMR (100 MHz,
CDCl3, ppm): d 168.8, 143.1, 141.4, 139.9, 135.0, 130.0, 128.9, 121.8, 121.8, 121.0,
114.4, 109.4, 78.3, 73.7, 50.4, 45.4, 34.7, 28.1, 27.4, 23.2. MS (APCI): m/z 350.3
[M+1]+.
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(9-Butyl-1-methyl-b-carboline-3-yl)(thiomorpholino)methanone (9e): 1H NMR
(400 MHz, CDCl3, ppm): d 8.29 (s, 1H), 8.14 (d, 1H, J = 7.76 Hz), 7.61 (dt, 1H,
J
1 = 7.32, J2 = 1.12 Hz), 7.49 (d, 1H, J = 8.36 Hz), 7.31 (dt, 1H, J1 = 7.76,
J2 = 0.60 Hz), 4.56 (t, 2H, J = 7.84), 4.12 (br s, 2H), 4.02 (br s, 2H), 3.05 (s, 3H),
2.81 (br s, 2H), 2.76 (br s, 2H), 1.81–1.89 (m, 2H), 1.47 (m, 2H), 1.00 (t, 3H,
J = 7.36). 13CNMR (100 MHz, CDCl3, ppm): d 168.8, 141.6, 141.4, 139.3, 134.9,
129.2, 128.3, 121.3, 121.0, 120.0, 114.0, 109.6, 63.2, 63.0, 44.4, 32.6, 24.7, 24.5,
22.9, 19.8, 13.4. MS (APCI): m/z 368.2 [M+1]+.

K. G. Brahmbhatt et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4416–4419
4419
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infection. GFP expression is due to trans-activation by Tat protein of stably
integrated long terminal repeat regulated GFP gene. This cell line is used
widely for determination of anti-HIV activity due to easy visualization of
infected cells.23 CEM-GFP cells were infected with HIV-1 NL4.3 virus at a
multiplicity of infection (MOI) of 0.05 using the standard protocol previously
published.24,25 The cells were then incubated with samples for up to 8 days
post infection. Virus production was assayed in the culture supernatant on
day-8 post infection by p24 antigen capture ELISA (Perkin–Elmer, USA).
26. General procedure for evaluation of anti-HIV activity: CEM-GFP is a human CD4+
reporter T cell line which expresses green fluorescent protein (GFP) upon HIV