Design, synthesis and biological evaluation of 1,3,6-trisubstituted β-carboline derivatives for cytotoxic and anti-leishmanial potential

Article abstract of DOI:10.1016/j.bmcl.2015.12.095

In the present study, 23 derivatives of 1,3,6-trisubstituted β-carboline were synthesized and evaluated for cytotoxic potential against four human cancer cells, namely A-549, HeLa, Hep G2 and MCF-7 as well as anti-leishmanial activity against Leishmania d

Full text of DOI:10.1016/j.bmcl.2015.12.095

Accepted Manuscript
Design, synthesis and biological evaluation of 1,3,6-trisubstituted β-carboline
derivatives for cytotoxic and anti-leishmanial potential
Nitin A. Lunagariya, Vikrantsinh M. Gohil, Varun Kushwah, Soumya Neelagiri,
Sanyog Jain, Sushma Singh, Kamlesh K. Bhutani
PII:
S0960-894X(15)30408-X
http://dx.doi.org/10.1016/j.bmcl.2015.12.095
BMCL 23456
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 July 2015
19 November 2015
29 December 2015
Please cite this article as: Lunagariya, N.A., Gohil, V.M., Kushwah, V., Neelagiri, S., Jain, S., Singh, S., Bhutani,
K.K., Design, synthesis and biological evaluation of 1,3,6-trisubstituted β-carboline derivatives for cytotoxic and
anti-leishmanial potential, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2
015.12.095
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Design, synthesis and biological evaluation of 1,3,6-trisubstituted β-carboline derivatives
for cytotoxic and anti-leishmanial potential
a
Nitin A. Lunagariya , Vikrantsinh M. Gohil , Varun Kushwah , Soumya Neelagiri , Sanyog
a
b
c
b
Jain , Sushma Singh , Kamlesh K. Bhutani *
c
a,
a
Department of Natural Products, National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S.A.S. Nagar, Punjab-160062, INDIA
b
Department of Pharmaceutics, National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S.A.S. Nagar, Punjab-160062, INDIA
c
Department of Biotechnology, National Institute of Pharmaceutical Education and Research
(NIPER), Sector 67, S.A.S. Nagar, Punjab-160062, INDIA
*
Corresponding author: K. K. Bhutani; E-mail: kkbhutani@niper.ac.in

Abstract
In the present study, 23 derivatives of 1,3,6-trisubstituted β-carboline were synthesized and
evaluated for cytotoxic potential against four human cancer cells, namely A-549, HeLa, Hep
G2 and MCF-7 as well as anti-leishmanial activity against Leishmania donovani
(
MHOM/80/IN/Dd8) promastigotes. Among the studied compounds, compounds 13c and
3q showed potent cytotoxic activity better than the parent compound 10. For instance,
1
compound 13c was found to be the most cytotoxic with IC50 of 4.72, 3.59, 3.65 and 4.17 µM
against A-549, HeLa, Hep G2 and MCF-7 respectively, while for compound 13q, IC50 were
1
5.47, 5.30, 6.15 and 13.39 µM against the same cancer cells respectively. Further, these two
compounds were found to be apoptotic in A-549 and MCF-7 cells when observed using
Annexin V/ propidium iodide staining under confocal microscope. All the compounds were
also tested for anti-leishmanial potential. In which, compounds 13u and 13c were found to
show moderate inhibition with IC50 of 23.5±9.0 and 68.0±0.0 µM respectively, while
compound 10 was the most active with IC50 of 9.0±2.8 µM, suggesting the modification at C-
6
detrimental for anti-leishmanial activity. Interestingly, amongst all, compound 13c was
found to be the most active for cytotoxic and moderately active for anti-leishmanial activity
which can be further developed as a lead for these disease areas.

Keywords
,3,6-trisubstituted β-carboline; Cytotoxic; Apoptosis; Cancer; Anti-leishmanial
1

Naturally occurring and synthetically derived β-carbolines have been studied extensively for
anti-cancer, anti-protozoal against Leishmania and Trypanosoma, anti-viral activity against
human immunodeficiency virus (HIV) and herpes simplex virus (HSV), anti-plasmodial, anti-
bacterial, anti-fungal and anti-thrombotic potential, apart from their neuropharmacological
1
effects. These biological effects have been linked with diverse modes of action and targets
2
such as intercalation of DNA , inhibition of the various enzymes such as monoamine
3
oxidase , topoisomerase I and II , kinesin spindle protein and various kinases including
4
5,6
7
cyclin-dependent kinases , IKK , B-Raf kinase , mitogen-activated protein kinase-activated
8
9
1
0
11
protein kinase 2 (MK-2) and polo-like kinase 1 (PLK1) . Simple β-carbolines such as
harmaline (1) and harmine (2) from Peganum harmala (Syrian Rue) (Fig 1) have shown good
in vitro and in vivo anti-cancer and anti-leishmanial activity and provide an excellent scaffold
1
2-15
for the structural modifications.
Figure 1 Biologically active β-and γ-carbolines
Literature search revealed that most of the structure-activity relationship (SAR) studies of β-
carboline derivatives have been reported around the modifications at C-1, C-3, N-2 and N-9
on ring-B and -C of β-carboline with the improved cytotoxic potential towards cancer
1
,16-18,20
cells.
It has been reported that appropriate substitutions at C-1, C-3 and/or N-9
1
6-18,20
enhance antitumor activity
, while substitutions at C-3 and N-9 reduce acute toxicity and

1
6,17
neurotoxicity
. On the other hand, substitution at N-2 enhances both cytotoxicity and acute
1
7
toxicity. However, substitutions at C-6, C-7 and/or C-8 on ring-A of β-carboline have not
been studied in detail. Recently, C-6, C-7 and/or C-8 substituted β-carbolines have been
1
9,20
8,9,24
reported to possess cytotoxic potential
Fig 1). One of the reported JAK2 inhibitor, 1-cyclohexylamino-7-(1-methyl-1H-pyrazol-4-
yl)-5H-pyrido[4,3-b]indole-4-carboxamide (3), containing γ-carboline nucleus, was able to
as well as studied for the inhibition of kinases
(
2
1
make classic hydrogen bond interactions with kinase hinge backbone residues. Based on
2
2
this lead and other two clinically available B-Raf kinase inhibitors vemurafenib and
2
3
sorafenib , β-carboline derivatives with 1-carboxamide and 6-sulfonamide functionality had
been designed and reported as B-Raf kinase inhibitors. Out of which, 1-carboxamide-6-(N-(3-
(
dimethylamino)propyl)-sulfamoyl)-β-carboline (4) had shown potent inhibition of wild type
9
B-Raf kinase with IC50 of 1.62 µM. In another study, 5-bromo-6-methoxy-β-carboline (5)
had been identified as non-specific inhibitor of IKK with IC50 of 1.0 µM. Further,
modifications on C-6, C-7 and C-8 had been shown the way to novel class of selective IKK
inhibitors. Out of those, 6,8-dichloro-β-carboline compound (6) with cycloalkylether moiety
on C-7, was reported with more selectivity and potency as IKK inhibitor with IC50 of 0.08
8
µM. Similarly, 7,8-dichloro-1-oxo-β-carbolines (7) had been reported as a versatile scaffold
24
with potent and selective inhibition of various kinases with unusual binding modes. Based
on these studies, combination of appropriate substitutions on ring-A, -B and -C of β-
carbolines, as shown in Figure 1, can be taken as an approach to develop potent anti-cancer
compounds with less associated toxicity. Considering these literature reports, we have
synthesized 1,3,6-trisubstituted β-carboline compounds with modification on ring -A and -C
for the evaluation of in vitro cytotoxic activity against human cancer cells.
In continuation for the search of potent anti-leishmanial compounds, our group has earlier
2
5
shown the anti-leishmanial activity of substituted 1-aryl-β-carboline derivatives. Effect of

substitution/s on ring-A of β-carboline was not studied well for anti-leishmanial potential.
This encouraged us to evaluate the synthesized 1,3,6-trisubstituted β-carbolines for any
possible anti-leishmanial activity.
Scheme 1 Synthesis of 1,3,6-trisubstituted β-carboline derivatives; Reagents and condition:
(
a) SOCl
2
, MeOH, reflux, 6 h; (b) CH
3
CHO, H SO (0.5 mM), rt, 18 h; (c) KMnO , acetone,
2
4
4
rt, 24 h; (d) conc. HNO
3
, conc. H SO
2
4
, 0-2°C, 30 min; (e) Pd/C, NaBH , MeOH, rt, 3 h; (f)
4
RCOCl, anhydrous dichloromethane, rt, 6-10 h (Collective yield of step-e and f =27-83%).
For the design of 1,3,6-trisubstituted β-carboline derivatives, the overall strategy is depicted
in Scheme 1. Synthesis of methyl 1-methyl-9H-pyrido[3,4-b]indole-3-carboxylate 10 was
2
6
done by reported methodology , with some modifications. In brief, methyl 1-methyl-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (9) was synthesized from tryptophan
methyl ester and acetaldehyde via Pictet-Spengler iminium ion cyclization in the presence of
dilute sulfuric acid (0.5 mM). Aromatization of compound 9 was carried out using potassium
permanganate in acetone to yield compound 10. Compound 10 was subsequently nitrated
using nitrating mixture of conc. nitric acid and sulfuric acid to provide methyl 1-methyl-6-
nitro-9H-pyrido[3,4-b]indole-3-carboxylate (11). Reduction of compound 11 was carried out
2
7
using reported methodology by sodium borohydride in presence of catalytic amount of Pd/C
to amino compound 12. As compound 12 was prone to oxidation, crude reaction product was
directly subjected to acylation. For acylation, amino compound 12 in anhydrous

dichloromethane was reacted with various acyl chlorides to yield compound 13a-u (Fig 2).
Collective yields of isolated compound 13a-u after reduction and acylation were found to be
2
8
2
7-83%. All the compounds and intermediates were confirmed by spectral analysis.
Figure 2 Synthesized 1,3,6-trisubstituted β-carboline derivatives 13a-u
2
Using in vitro MTT assay , all the synthesized compounds were also screened for their
9
cytotoxic potential against four human cancer cells namely A-549, HeLa, Hep G2 and MCF-
7
at the concentration of 10µM. Docetaxel was kept as positive control throughout the study.
Percentage viability of the cells is shown in Figure 3. Compounds 13c and 13q were found to
be the most active against all four cancer cell lines. The remaining compounds showed
similar or less cytotoxicity than parent compound 10. Compounds 13c and 13q were studied
further for the concentration dependant cytotoxic potential in the range of 0.1 to 50 µM (Fig
4
). Determined IC50 values of the compounds 10, 13c and 13q against human cancer cells and
normal human embryonic lung cells (L-132) are listed in the Table 1. These results indicate
that compound 13c was approximately 8-, 10-, 11- and 11-folds more cytotoxic as compared
to compound 10 against A-549, HeLa, Hep G2 and MCF-7 cells, respectively. While the

same increase in cytotoxicity for compound 13q was approximately 2-, 7-, 6- and 3-folds
against A-549, HeLa, Hep G2 and MCF-7 respectively. For the apoptosis detection, A-549
and MCF-7 cells, treated with compounds 10, 13c and 13q at concentration of 10 µM as per
3
0
manufacturer’s protocol , were visualized under Olympus FV1000 confocal microscope
using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life technologies, Oregon
USA), as shown in Figure 5. Cell cultures treated with compounds 13c and 13q showed more
apoptotic cells than compound 10. However, compounds 10, 13c and 13q were also showed
the cytotoxicity on the normal cells indicating that the compounds lack the selectivity
towards the cancer cells in inducing apoptosis. These results suggest that bulky substitutions
such as 4-tert-butyl and 3,5-bis(trifluoromethyl) containing the benzamido substitution are
favorable for the activity compared to small functional groups such as methyl, methoxy,
bromo, and trifluromethyl containing benzamido derivatives. Compounds containing alkyl
chains and small 5-membered heterocyclic ring such as furan, thiophene and 2,5-
dimethylfuran did not show any improvement in the cytotoxic activity. Inactivity of
cinnamoyl derivative and alkoxy as well as phenoxyacetamide derivatives suggest that
aromatic ring of the substituent must be directly attached to carbonyl group (e.g. benzamido),
for the better activity.
Figure 3 Cytotoxic activity of synthesized compounds 10-12 and 13a-u against A-549,
HeLa, Hep G2 and MCF-7 cells at a concentration of 10 µM. DTX= Docetaxel.

A
B
A-549
HeLa
1
20
00
120
100
80
60
40
20
0
1
8
6
4
2
0
0
0
0
0
0.1
1
10
50 100
0.1
1
10
50 100
Concentration (µM)
Concentration (µM)
Hep G2
MCF-7
C
D
1
20
00
120
100
80
60
40
20
0
1
8
6
4
2
0
0
0
0
0
0.1
1
10
50 100
0.1
1
10
50 100
Concentration (µM)
Concentration (µM)
Figure 4 Concentration dependent cell growth inhibition by compounds 10 (▬), 13c (▬),
3q (▬) and Docetaxel (▬) against human cancer cell lines (A) A-549, (B) HeLa, (C) Hep
1
G2, and (D) MCF-7.
Table 1 Cytotoxic activity of compounds 10, 13c and 13q against human cancer and normal
cell lines
IC50 (µM)*
Compound
R
A-549
HeLa
Hep G2
MCF-7
L-132
10
-H
37.84
35.61
39.59
44.97
39.28
1
3c
4.72
3.59
3.65
4.17
3.26
13q
15.47
3.41
5.30
3.23
6.15
2.86
13.39
3.60
11.22
1.69
Docetaxel
*
Values are expressed as median effect dose in µM at which 50% cells are inhibited,
calculated by CalcuSyn software 2.1 (Biosoft, Ferguson, MO).

Figure 5 Induction of apoptosis by compounds 10, 13c and 13q in A-549 and MCF-7 cells;
Live cells: no fluorescence; Apoptotic cells: green fluorescence due to Annexin V binding
only; Dead cells: both green and red fluorescence because of Annexin V and PI respectively,
collectively visible yellow-green in overlay. Scale bar = 50µm; DTX= Docetaxel.
The synthesized compounds were screened for the anti-leishmanial potential against
3
1
Leishmania donovani (MHOM/80/IN/Dd8) promastigotes, using in vitro MTT assay.
Miltefosine was kept as positive control. Among the evaluated compounds, parent β-
carboline 10 has shown the IC50 of 9.0±2.8 µM with selectivity index (S.I.) greater than 11.11
which is comparable to that of miltefosine (IC50=11.9±2.7 µM, S.I.=2.64), while 13u and 13c
derivative have shown the IC50 of 23.5±9.0 with S.I. of 3.27 and 68.0±0.0 µM with S.I. of
0
.47, respectively. (Table 2). The remaining compounds did not show any promising result.
However, based on IC50 of compounds 10, 13c and 13u, it can be concluded that substitution
on C-6 of β-carboline is inappropriate for the anti-leishmanial activity since compounds 13u
and 13c were less active than the parent compound 10.

Table 2 In vitro anti-leishmanial activity of 1,3,6-trisubstituted β-carbolines
Selectivity index
IC50 (Mean ± S.D.) (µM)
(
SI)
THP-1 differentiated
macrophages
L. donovani
Promastigotes
11.9±2.70
9.0±2.80
>100
Compound
Miltefosine
31.5 ± 2.10
2.64
10
11
12
>100
> 11.11
-
-
>100
-
-
1
3a
3b
3c
3d
3e
3f
3g
>100
-
-
1
>100
-
-
1
68.0±0.00
NS
32 ± 5.60
0.47
1
-
-
1
>100
-
-
1
NS
-
-
1
>100
-
-
13h
NS
-
-
1
3i
>100
-
-
1
3j
NS
-
-
1
3k
3l
3m
3n
NS
-
-
1
NS
-
-
1
>100
-
-
1
>100
-
-
1
3o
3p
3q
NS
-
-
1
100
-
-
1
NS
-
-
13r
>100
-
-
1
3s
3t
NS
-
-
-
1
NS
-
13u
23.5±9.00
77 ± 11.30
3.27
NS‒Not soluble in 20% DMSO
In gist, two compounds 13c and 13q showed stronger cytotoxic and apoptotic potential while
two compounds 13u and 13c showed moderate anti-leishmanial activity than the parent
compound 10. These findings suggest that combination of the substitutions on the ring -A and
-
C of β-carboline is a good starting point for improving the cytotoxic potential. The
compound 13c needs to be further optimized via synthetic modifications to get more potent
leads for the studied disease areas. On the other hand, compounds 10 and 13u, showing good
IC50 with high S.I., can be taken as lead for the further development for the search of newer
antileishmanial agents.

Acknowledgements
Authors are thankful to the Director, NIPER, for providing financial support to carry out this
work.

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8. Compound 13c: Brown solid; yield = 50%; mp: decomposed over 200°C (without
1
melting); H-NMR (400 MHz, CD
3
OD): δ 1.14 (s, 9H), 2.79 (s, 3H), 3.83 (s, 3H), 7.34
(
d, 2H, J=8.28 Hz), 7.40 (d, 1H, J=8.92 Hz), 7.65-7.74 (m, 3H), 8.37 (s, 1H), 8.56 (s,
1
3
1
1
1
3
H); C-NMR (100 MHz, CD OD): δ 16.81, 31.58, 35.89, 54.01, 114.13, 115.11,
18.32, 122.15, 126.66, 127.55, 128.64, 130.41, 131.58, 132.99, 134.94, 137.22, 141.71,
+
[M+H] 416.1974,
42.75, 156.88, 163.15, 168.77; HRMS (ESI) calcd for C25
H
26
N
3
O
3
found m/z 416.1967.
9. Cell Culture & MTT assay protocol for cytotoxicity: Four human cancer cell lines A-
49, HeLa, Hep G2 and MCF-7, and normal human embryonic lung cell line (L-132),
2
5
were procured from National Centre for Cell Sciences, Pune, India. The cells were
maintained in a suitable medium, as recommended by ATCC, containing 10% FBS
(
PAA, Austria), and antibiotics (antibiotic and anti-mycotic solution; PAA, Austria). For
5
MTT assay, cells (1 x 10 cells/well) were seeded in 96-well plates. The plates were
incubated overnight to allow the attachment of cells. The cells were then incubated with
compounds for 24 h. After treatment, the cells were incubated with MTT (0.5 mg/mL; 0.2
mL) for 3 h, and the formazan concentration was determined spectrophotometrically at
550 nm (µQuant, Bio-Tek Instruments).
3
0. Detection of apoptosis using Annexin V/PI staining: Detection of apoptosis was done
using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life technologies, Oregon

USA) as per the manufacturer’s protocol. In brief, cells were incubated with the
compounds at concentration of 10 µM. Untreated cells were kept as control. After the
incubation of 6 h, cells were washed with cold phosphate buffer solution (PBS). Then
cells were incubated with the 10 µL annexin V and PI (1 µg/mL) in 1X annexin-binding
buffer at room temperature for 15 minutes. The cells were washed with 1X annexin-
binding buffer and observed under Olympus FV1000 confocal microscope.
3
1. Protocol for anti-leishmanial activity: To investigate the drug sensitivity on Leishmania
32
donovani (MHOM/80/IN/Dd8) promastigotes, MTT assay was performed .
5
Approximately, 2 x 10 L. donovani promastigotes from logarithmic phase were seeded in
9
6-well plates and incubated for 48 h, followed by treatment with different concentrations
of drug or compounds ranging from 10 µM to 100 µM for 48 h at 24°C. Finally, 20 µL of
mg/mL MTT solution was added to each well and incubated in the dark for 4 h at 37°C.
5
The cells were centrifuged at 3000g for 10 min. The purple formazan crystals were
dissolved in 100 µL of DMSO and absorbance was measured at 540 nm in a Tecan
microplate reader. The net absorbance obtained in untreated promastigotes was
considered as 100% viability and relatively the percentage viability of promastigotes was
calculated for treated promastigotes. Miltefosine was taken as the standard drug in each
experiment. For selectivity indices, cytotoxicity assay on the THP-1 differentiated
5
macrophages were performed. In brief, THP-1 monocytes (2 x 10 cells/well) were
seeded in a 96-well plate and differentiated with 5 nM of phorbolmyristate acetate (PMA,
Sigma) for 48 h. Differentiated macrophages were treated with different concentrations
(
10-100 µM) of compounds and miltefosine for 48 h, followed by the detection of the cell
viability by MTT assay, as described above. Based on the results, IC50 and subsequently
selectivity indices were calculated.
3
2. Mosmann, T. J. Immunol. Methods. 1983, 65, 55.