Biological evaluation and structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole derivatives as anti-leishmanial agents

Article abstract of DOI:10.1016/j.bioorg.2018.11.037

A series of piperazinyl-β-carboline-3-carboxamide derivatives were designed through a molecular hybridization approach. Designed analogues were synthesized, characterized and evaluated for anti-leishmanial activity against Leishmania infantum and Leishmania donovani. In L. infantum inhibition assay, compounds 7d, 7g and 7c displayed potent inhibition of promastigotes (EC₅₀ 1.59, 1.47 and 3.73 μM respectively) and amastigotes (EC₅₀ 1.4, 1.9 and 2.6 μM respectively). SAR studies revealed that, para substitution of methoxy, chloro groups and methyl group on ortho position favored anti-leishmanial activity against L. infantum. Among these analogues 7d, 7h, 7n and 7g exhibited potent inhibition against L. donovani promastigotes (EC₅₀ 0.91, 4.0, 4.57 and 5.02 μM respectively), axenic amastigotes (EC₅₀ 0.9, 3.5, 2.2 and 3.8 μM respectively) and intracellular amastigotes (EC₅₀ 1.3, 7.8, 5.6 and 6.3 μM respectively). SAR studies suggested that, para substitution of methoxy group, para and meta substitution of chloro groups and benzyl replacement recommended for significant anti-leishmanial against L. donovani.

Full text of DOI:10.1016/j.bioorg.2018.11.037

Accepted Manuscript
Biological Evaluation and Structure activity relationship of 9-methyl-1-phe-
nyl-9H-pyrido[3,4-b]indole derivatives as anti-leishmanial agents
Penta Ashok, Subhash Chander, Terry K. Smith, Rajnish Prakash Singh, Prabhat
Nath Jha, Murugesan Sankaranarayanan
PII:
S0045-2068(18)30494-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.037
YBIOO 2645
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 May 2018
14 October 2018
21 November 2018
Please cite this article as: P. Ashok, S. Chander, T.K. Smith, R. Prakash Singh, P. Nath Jha, M. Sankaranarayanan,
Biological Evaluation and Structure activity relationship of 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole derivatives
as anti-leishmanial agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biological Evaluation and Structure activity relationship of 9-methyl-1-phenyl-9H-
pyrido[3,4-b]indole derivatives as anti-leishmanial agents
Penta Ashoka, Subhash Chandera,b, Terry K. Smithc,Rajnish Prakash Singhd, Prabhat Nath Jhad,
Murugesan Sankaranarayanana*
a,*Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science
Pilani, Pilani Campus, Pilani-333031, Rajasthan. India. E. mail: penta.ashok@gmail.com, murugesan@pilani.bits-
pilani.ac.in
bSchool of Pharmacy, Maharaja Agrasen University, Atal ShikshaKunj, Solan, Himachal Pradesh 174103. E. mail:
subhashsaininiper@gmail.com
cSchools of Biology & Chemistry, BSRC, The University, St. Andrews, Fife Scotland. KY16 9ST, UK. E.
mail: tks1@st-andrews.ac.uk
dDepartment of Biological Sciences, Birla Institute of Technology & Science Pilani, Pilani Campus, Pilani 333031,
Rajasthan, India. E. mail: prabhatjha@pilani.bits-pilani.ac.in
ABSTRACT
A series of piperazinyl-β-carboline-3-carboxamide derivatives were designed through a
molecular hybridization approach. Designed analogues were synthesized, characterized and
evaluated for anti-leishmanial activity against Leishmania infantum and Leishmania donovani.
In L. infantum inhibition assay, compounds 7d, 7g and 7c displayed potent inhibition of
promastigotes (EC50 1.59, 1.47 and 3.73 µM respectively) and amastigotes (EC50 1.4, 1.9 and 2.6
µM respectively). SAR studies revealed that, para substitution of methoxy, chloro groups and
methyl group on ortho position favored anti-leishmanial activity against L. infantum. Among
these analogues 7d, 7h, 7n and 7g exhibited potent inhibition against L. donovani promastigotes
(EC50 0.91, 4.0, 4.57 and 5.02 µM respectively), axenic amastigotes (EC50 0.9, 3.5, 2.2 and 3.8
µM respectively) and intracellular amastigotes (EC50 1.3, 7.8, 5.6 and 6.3 µM respectively). SAR
studies suggested that, para substitution of methoxy group, para and meta substitution of chloro
groups and benzyl replacement recommended for significant anti-leishmanial against L.
donovani.
Keywords: Molecular hybridization; Leishmaniasis; Leishmania infantum; Leishmania
donovani; Promastigotes; Amastigotes

1. Introduction
Leishmaniasis is caused by intracellular protozoan Leishmania spp parasites and is considered as
one of the most neglected tropical diseases. Among various clinical forms of leishmaniasis,
visceral leishmaniasis is the most severe, affecting internal organs like bone marrow, liver and
spleen. VL is also known as kala-azar and is lethal if untreated in over 95% of cases. VL caused
by Leishmania donovani and Leishmania infantum[1]. Leishmaniasis control is majorly
dependent upon chemotherapy that includes decade old drugs, due to no effective vaccines.
Antimonial drugs such as sodium stibogluconate and meglumineantimoniate have been used for
the last 70 years, however increasing incidence of resistance has been reported [2]. Anti-
leishmanial drugs such as amphotericin B, diamidine, pentamidine and paromomycin has been
restricted by cost, toxicity and resistance [3, 4]. Miltefosine is originally developed as anti-cancer
agent, has been approved by FDA for leishmaniasis treatment [5]. Miltefosine has restricted use
in pregnancy and has high risk of resistance due to long half-life (150 h). Clinical efficacy of
miltefosine has been decreased in countries like India, where it is used extensively [6]. Based
upon these collective facts, there is an increased need of new anti-leishmanial agents with good
therapeutic profiles.
Nature and natural products remain an important source for the development of therapeutic
agents. Natural products especially alkaloids have been known for anti-infective activity. β-
carboline represents a tricyclic pyrido[3,4-b]indole ring system has reported for wide spectrum
of biological activities. β-carboline derivatives displayed different biological activities such as
anti-microbial [7], anti-cancer [8], anti-leishmanial [9, 10], anti-malarial [11, 12], anti-tubercular
[13], anti-viral [14] and anti-thrombotic activities [15]. β-carboline alkaloids such as Harmine,
annomontine and manzamines (Fig. 1) exhibited potent anti-leishmanial activity [9, 16, 17].
Manzamine is a group of β-carboline alkaloids, possess complex structure of -carboline moiety
attached to a pentacyclic diamine ring having both eight and thirteen membered rings on a
pyrrolo[2,3-i] isoquinoline framework. Large numbers of manzamine alkaloids are isolated from
different marine sponge species and exhibited potent anti-leishmanial activity. Beside these
natural β-carboline alkaloids, several synthetic β-carboline derivatives having different
substitutions on 1, 2, 3 and 9 positions have exhibited significant anti-leishmanial activity [17,
2

18]. Among these synthetic β-carboline derivatives, β-carboline derivatives with N-
alkylcarboxamide group on 3 position displayed potent anti-leishmanial activity and attracted us
to design new β-carboline derivatives [19-22]. The piperazine moiety has privileged position in
medicinal chemistry with diverse biological activities [23]. Bisarylpiperazine derivatives
exhibited activities such as anti-leishmanial [24], anti-microbial [23, 25, 26], anti-protozoal [27,
28], anti-cancer [29], anti-tubercular [29, 30] and anti-viral [31](Fig. 1). Based upon literature
reports, in the present study we designed β-carboline and piperazine hybrid molecules (Fig. 1)
using molecular hybridization approach.
Fig. 1. Structure of reported and designed β-carboline-piperazine hybrid molecules
2. Results and Discussion
2.1. Chemistry
3

The synthetic scheme for β-carbolinepiperazine derivatives was illustrated in scheme 1. The
titled compounds were synthesized using DL-Tryptophan (1) as starting material. Initial
esterification of DL-Tryptophan (1) using thionylchloride to get an ethyl ester of tryptophan (2),
was followed by pictet-spengler reaction in presence of trifluoroacetic acid to obtain tricyclic
ethyl 2,3,4,9-tetrahydro-1-phenyl-1H-pyrido[3,4-b]indole-3-carboxylate (3). Upon oxidation
with potassium permanganate, ethyl-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (4) was
obtained, and is continued by 9-N methylation with methyl iodide in presence of potassium
hydroxide to acquire ethyl-9-methyl-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (5). Further
alkaline ester hydrolysis of compound (5) produced 9-methyl-1-phenyl-9H-pyrido[3,4-b]indole-
3-carboxylic acid (6) as key intermediate. The carboxylic acid key intermediate (6) was treated
with appropriate amines (aryl-substitutedpiperazines) in presence of coupling agent 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and hydroxybenzotriazole (HOBt) to
obtain the desired products (7a-p) in good yields [32-34].
Scheme 1: Reagents and conditions: i) thionylchloride, ethanol, reflux, 30 min, 76 %; ii) benzaldehyde,
trifluoroacetic acid, DCM, rt, 3 h, 82 %; iii) KMnO4, THF, rt, 24 h, 68 %; iv) methyliodide, KOH, DMSO, rt, 30
min, 72 %; v) 50 % aq. NaOH, reflux, 30 min, 78 %; vi) EDCl, HOBt, THF, piperazines, 0 ºC-rt 6 h, 62-82 %.
2.2. Biological evaluation
2.2.1. Cytotoxicity evaluation
Initially all these reported β-carboline derivatives were evaluated for cytotoxicity against HeLa
cell lines at 500 μM by an Alamar blue assay, which showed most of these compounds were
non-toxic to HeLa cells at tested concentration. Compounds which showed cytotoxicity against
HeLa cells were further evaluated to determine cytotoxic concentration (CC50) value. Among
these reported β-carboline derivatives, compound 7o and 7p showed cytotoxicity (CC50 29.7 and
27.3 μM, respectively) against HeLa cells.
2.2.2. Anti-leishmanial screening
Anti-leishmanial activity of these analogues was determined by evaluating their inhibition
activity against both promastigote, amastigote forms of Leishmania infantum and Leishmania
donovani strains. Compounds were screened against promastigote forms of the Leishmania
strains to determine their effective concentration (EC50) values. Finally, compounds which
exhibited potent inhibition of promastigote (EC50 <20 µM) were further evaluated against
4

amastigote forms of respective strain using established axenic and intracellular amastigote assay
methodologies. Anti-leishmanial drugs miltefosine and pentamidine were used as standards for
comparison purpose.
2.2.2.1. Leishmaniainfantum
2.2.2.1.1. Anti-promastigote activity. Anti-promastigote activity of these reported β-carboline
derivatives was determined by Alamar blue assay method. In in-vitro evaluation against
promastigotes of L. infantum, compounds 7g, 7d, and 7c displayed potent anti-promastigote
activity (EC50 1.47, 1.59 and 3.73 µM respectively) than standard drugs pentamidine and
miltefosine(EC50 8.31 and 12.36 µM respectively). Compounds 7a, 7f, 7j and 7k displayed
significant inhibition of L. infantum promastigotes (EC50 9.45, 13.8, 19.7 and 17.2 µM
respectively) and inhibition potency was comparable with standard drugs.Analogues 7n, 7b and
7h exhibited moderate activity (EC50 20.4, 24.3 and 21.7 µM respectively), while 7d, 7h, 7l, 7m,
7o and 7p showed weak anti-promastigote activity (EC50 72.4, 50.9, 64.9, 93.0, 29.7 and 45.7
µM respectively) against L. infantum. Especially, compounds 7d and 7g exhibited most
promising inhibition of L. infantum and are five times more potent than the standard drugs.
Structure activity relationship studies suggested that, para substitution with ortho-para directing
groups (methoxy and chloro) enhanced promastigotes inhibition activity significantly. Ortho
substitution with methyl group has increased the anti-promastigote activity significantly while
substitution of other ortho-para directing and meta directing groups (methoxy, chloro)
substitution on phenyl ring has decreased the activity drastically. Replacement of phenyl ring
with benzyl and pyridyl ring was not favorable for anti-promastigote activity.
2.2.2.1.2. Anti-amastigote activity. Compounds exhibited significant inhibition of L. infantum
promastigotes (EC50<20 μM) were further evaluated against axenic amastigote forms of L.
infantum.Alamar blue assay method was used to determine inhibition potential of these β-
carboline derivatives against (human life stage forms) axenic amastigotes. In L. infantum axenic
amastigote inhibition assay, compounds 7d, 7g and 7c displayed potent activity (EC50 1.4, 1.9
and 2.6 µM respectively) better than standard drugs pentamidine and miltefosine(EC50 2.7 and
4.8 µM). Compound 7a showed axenic anti-amastigote activity (EC50 4.5 µM) against L.
infantum and potency was comparable with standard drugs. Compounds, 7j, 7k and 7f exhibited
significant inhibition of L. infantum axenic amastigotes (EC50 14.6, 15.0 and 15.3). In-vitro anti-
5

amastigote activity results (table 1) suggested that these β-carboline derivatives were more potent
against axenic amastigote forms of L. infantum.
Table 1 Anti-leishmanial activity against L. infantum
aSI = CC50/IC50, ND – Not Determined
Structure Activity Relationship (SAR). Among these piperzinyl-β-carboline derivatives, un-
substituted proto compound displayed significant anti-leishmanial activity against both
promastigote and amastigotes of L. infantum. A series of β-carboline derivatives with different
ortho-para and meta directing groups on various positions of phenyl were studied to determine
their effect on anti-leishmanial activity. Substitution of ortho-para directing methyl group on
ortho position of phenyl ring has increased anti-leishmanial activity significantly against both
forms, whereas methyl substitution on para position has reduced the activity. Ortho-para
directing groups such as methoxy and chloro on para substitution strongly enhanced the anti-
leishmanial activity, while ortho substitution lessen the activity and meta substitution drastically
decreased the activity. Meta directing nitro and fluoro group substitution on para position
decreased the activity considerably and fluoro substitution on ortho position severely decreased
the activity. Dual substitution on ortho and meta position with chloro declined the activity.
Moreover, phenyl ring replacement with pyridine and benzyl ring has adversely affected the anti-
leishmanial activity. SAR studies suggested that, meta position of phenyl ring is highly
susceptible, substitution with any group drastically reduced the activity. Ortho-para directing
methoxy, chloro groups on para position and methyl group on ortho position favored the activity.
Phenyl ring replacement with heterocyclic rings is not recommended for anti-leishmanial activity
against L. infantum.
2.2.2.2. Leishmaniadonovani
2.2.2.2.1. Anti-promastigote activity. L. donovani promastigote inhibition potency of these
reported β-carboline derivatives was determined by Alamar blue assay method. Among these
analogues, compound 7d exhibited potent inhibition (EC50 0.91 µM) of L. donovani
promastigotes than standard drugs pentamidine and miltefosine (EC50 6.4 and 3.12 µM). Anti-
promastigote activity of analogues 7h, 7n and 7g (EC50 4.0, 4.57, and 5.02 µM respectively) is
equipotent as that of standard drugs. Compounds 7f, 7p, 7c, 7a, 7k, 7j, 7b, 7i, and
6

7odisplayedsignificant inhibition (EC50 8.42, 8.5, 9.45, 11.9, 12.4, 13.1, 14.2, 15.2 and 19.5 µM
respectively) of promastigote and inhibition potency is comparable with standard
drugs.Compound 7e showed moderate (EC50 28.5 µM) anti-promastigote activity and 7l and 7m
displayed weak inhibition (EC50 85.9 and 117.5 µM) of L. donovani promastigotes.
2.2.2.2.2. Axenic amastigote screening. Compounds exhibited significant anti-promastigote
activity (EC50<20 µM), were further screened against axenic amastigote (human life cycle
parasite form) forms. Among these derivatives, Analogues 7d, 7h, 7n and 7g showed potent
inhibition (EC50 0.9, 2.2 3.5 and 3.8 µM respectively) of axenic amastigotes. Especially,
compound 7d exhibited two to three times more potent anti-amastigote activity than pentamidine
and miltefosine (EC50 1.6 and 2.8 µM). Compounds 7c, 7b, 7f, 7j, 7k and 7a exhibited
significant inhibition (EC50 5.3, 5.8, 6.4, 7.5, 9.3 and 10.0 µM respectively) of L. donovani
axenic amastigotes. Compounds 7p, 7o and 7i displayed moderate anti-amastigote activity (EC50
12.3, 13.9 and 19.4 µM respectively). Axenic amastigote assay results revealed that these β-
carboline derivatives exhibited potent inhibition of axenic amastigotes of L. donovani.
2.2.2.2.3. Intracellular amastigote screening. Although, axenic amastigotes assay most
commonly used to screen new leishmanial agents are different from intracellular amastigotes in
terms of protein expression and drug susceptibility [40, 41]. Hence in our present study,
compounds displayed potent inhibition of promastigote and axenic amastigotes were further
evaluated against intracellular amastigotes to ensure their activity against clinical relevant forms
of parasite. Intracellular amastigote assay results suggested that, most of these analogues
displayed potent inhibition of L. donovanimacrophagic amastigotesand the potency was
comparable with miltefosine and many folds higher than pentamidine (table 2). Especially,
compound 7d ((4-(4-methoxyphenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-
3-yl)methan-one) exhibited five times potent inhibition (EC50 1.3 µM) of intracellular
amastigotes compared to miltefosine (EC50 6.4 µM). Compounds 7n and 7g were displayed
potent anti-amastigote activity (EC50 5.6 and 6.3 µM) than standard drugs miltefosine and
pentamidine with better selectivity index. Anti-amastigote potency of 7b, 7c, 7h, 7o and 7p
(EC50 7.2, 7.8, 7.9, 7.9 and 9.4 µM) is comparable with miltefosine and better than pentamidine.
Compounds 7k, 7f, 7a, 7j and 7i exhibited good anti-amastigote activity (EC50 10.1, 11.8, 13.5,
15.4 and 22.0 µM) with better selectivity.
7

Table 2 Anti-leishmanial activity against L. donovani
aSI = CC50/IC50, ND – Not Determined
Structure Activity Relationship (SAR). Structure activity relationship study suggests that,
substitution or replacement of phenyl ring has significant effect on anti-leishmanial activity as
well as cytotoxicity of these analogues. Among these analogues, un-substituted phenyl derivative
displayed significant inhibition of promastigotes, axenic amastigote and intracellular amastigotes
of L. donovani. Ortho-para directing methyl group substitution on ortho and para position
enhanced the potency against axenic amastigotes and intracellular amastigotes of L. donovani
significantly, while on para substitution anti-promastigote activity altered marginally. Methoxy,
chloro group substitution had strong impact on anti-leishmanial activity of these analogues,
position of substitution is vital in their inhibition activity. Ortho substitution of methoxy group
showed excellent increase in inhibition potency against promastigotes and both amastigote forms
of L. donovani. While methoxy substitution on ortho position slightly increased the anti-
leishmanial activity, meta substitution resulted in drastic decline in activity. Chloro substitution
on para and meta position has significantly increased the inhibition potency against tested forms,
while on ortho substitution has marginally affected the activity. Meta directing nitro and fluoro
substitution on para position haven’t showed any considerable effect on inhibition potency of
these analogues. However, fluoro substitution on ortho position and chloro di-substitution (ortho
and meta) significantly decreased the anti-leishmanial potency. Replacement of phenyl ring with
benzyl ring has significantly enhanced the activity. Even though pyridyl replacement resulted in
increased anti-leishmanial activity, it also enhanced the cytotoxicity of these analogues. SAR
studies suggested that, ortho-para directing methoxy group on para position, chloro group on
para and meta position and benzyl replacement was recommended for anti-leishmanial activity of
these analogues against L. donovani.
Through the present study, we have identified new lead molecules with potent anti-leishmaninal
activity against L.infantumand L.donovani. Structure activity relationship studies would be
utilised to optimize the lead molecules to generate clinically effective against both drug sensitive
and resistance strains of L.infantumand L.donovani. Exact mechanism of action of these
molecules for thier potential activity is needs to be evaluated. We hypothesize based upon
8

literature reports, that these molecules may exhibit the observed potent activity through
interaction with nucleic acids.
3. Conclusion
In summary, we have successfully applied molecular hybridization technique to design
piperazinyl-β-carboline-3-carboxamide derivatives as anti-leishmanial agents. Designed
molecules were synthesized and evaluated for anti-leishmanial activity against L. infantum and L.
donovani. Analogues displayed significant inhibition of promastigote forms (EC50< 20 μM) were
further evaluated against amastigote forms of respective species. Most of these reported
analogues displayed potent anti-leishmanial activity (promastigote and amastigote forms) against
the both tested strains with better selectivity index. Compounds such as, 7d,7g and 7c
wereexhibited potent inhibition of promastigote (EC50 1.59, 1.47 and 3.73 µM respectively) and
amastigote (EC50 1.4, 1.9 and 2.6 µM respectively) of L. infantum than standard drugs
miltefosine, pentamidine. SAR studied suggested that, ortho-para directing methoxy, chloro
groups substitution on para position as well as methyl group on ortho position favored the anti-
leishmanial activity against L. infantum.
More interestingly, most of these compounds showed potent anti-leishmanial activity against L.
donovani. Especially, compound 7d displayed potent inhibition of L. donovani promastigotes,
axenic amastigotes and intracellular amastigotes with EC50 0.91, 0.9 and 1.3 µM respectively.
Analogues 7n, 7g and 7h were exhibited potent inhibition activity against all tested forms of L.
donovani. SAR studies suggested that, ortho-para directing methoxy group on para position,
chloro group on para and meta position were recommended for anti-leishmanial activity against
L. donovani. Although, pyridyl replacement has increased the inhibition activity as well
cytotoxicity and benzyl replacement has enhanced the anti-leishmanial activity with better
selectivity.
4. Experimental protocols
4.1. Chemistry
All solvents and reagents purchased from Sigma or Merck companies are used as received
without further purification. Solvent system used throughout the experimental work for running
Thin Layer Chromatography (TLC) was ethyl acetate and hexane mixture (6:4) in order to
monitor the reaction. Column chromatography was performed using silica gel (100-200 mesh,
9

SRL, India) as stationary phase and mixture of ethyl acetate and hexane 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 FT-IR spectrophotometer (Shimadzu IR Prestige
1
21, India). H NMR spectra were recorded on a Bruker DPX-400 spectrometer (Bruker India
Scientific Pvt. Ltd., Mumbai) using TMS as an internal standard (chemical shifts in d, ppm).
Elemental analysis was performed on Vario EL III M/s Elementar C, H, N and S analyzer
(ElementarAnalysensysteme GmbH, Germany). The ESMS were recorded on MICROMASS
Quattro-II LCMS system (Waters Corporation, Milford, USA).
4.2. General procedure for the preparation of 7
To a stirred solution of (6) (0.29 g, 0.001 mol) in dry THF, HOBt (0.16 g, 0.012 mol) and EDCI
HCl (0.23 g, 0.012 mol) were added and continued stirring for 30 min. To the reaction mixture,
substituted phenylpiperazine (0.001 mol) was added under ice cold temperature and the reaction
mixture was further stirred at room temperature for 6 h. After completion of reaction as
monitored by TLC, solvent was evaporated under vacuum. Reaction mixture was extracted with
ethyl acetate (2 x 20 mL), collected organic layer was dried over Na2SO4 and concentrated under
vacuum to get (7a-p).
4.2.1.
(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-phenylpiperazin-1-yl)methanone
(7a)White solid; Yield 76 %; mp 232-234 °C; Rf 0.45 (Hexane : EtOAc = 6:4); IR νmax/cm-
1(KBr): 3043, 1636, 1564, 1468; 1H NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 8.21 (d, J = 7.8 Hz,
1H), 7.70-7.61 (m, 3H), 7.58-7.44 (m, 4H), 7.36 (t, J = 7.2 Hz, 1H), 7.30-7.28 (m, 2H), 6.96-6.90
(m, 3H), 4.04 (brs, 4H), 3.52 (s, 3H), 3.33-3.23 (m, 4H); ESI-MS: m/z 447 ((M+1), 100 %);
Anal. Calcdfor C29H26N4O: C, 78.00; H, 5.87; N, 12.55, Found: C, 78.02; H, 5.85; N, 12.51.
4.2.3. (9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-p-tolylpiperazin-1-yl)methanone (7b)
White solid; Yield 72 %; mp 188-190 °C; Rf 0.40 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3059, 1652, 1558, 1448; H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H),
7.68-7.64 (m, 3H), 7.58-7.53 (m, 3H), 7.48 (d, J = 8.3 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.11 (d,
J = 8.2 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 4.06-4.04 (m, 4H), 3.54 (s, 3H), 3.29-3.18 (m, 4H),
10

2.30 (s, 3H); ESI-MS m/z 461 ((M+1), 100 %); Anal. Calcdfor C30H28N4O: C, 78.23; H, 6.13; N,
12.16, Found: C, 78.22; H, 6.16; N, 12.14.
4.2.4. (9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-o-tolylpiperazin-1-yl)methanone (7c)
White solid; Yield 64 %; mp 148-150 °C; Rf 0.43 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3053, 1636, 1552, 1419; H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 8.21 (d, J = 7.7 Hz, 1H),
7.65-7.62 (m, 3H), 7.55-7.50 (m, 3H), 7.45 (d, J = 8.2 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.20-
7.15 (m, 2H), 7.01-6.99 (m, 2H), 4.02-4.00 (m, 4H), 3.51 (s, 3H), 3.06-2.94 (m, 4H), 2.35 (s,
3H); ESI-MS m/z 461 ((M+1), 100 %); Anal. Calcdfor C30H28N4O: C, 78.23; H, 6.13; N, 12.16,
Found: C, 78.20; H, 6.12; N, 12.18.
4.2.5.(4-(4-methoxyphenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7d)
White solid; Yield 82 %; mp 170-172 °C; Rf 0.30 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
3064, 1651, 1516, 1454, 1255; 1H NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 8.21 (d, J = 7.8 Hz,
1H), 7.66-7.62 (m, 4H), 7.54-7.50 (m, 4H), 7.45 (d, J = 8.3 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H),
6.86 (d, J = 8.8 Hz, 2H), 4.10 (brs, 4H), 3.77 (s, 3H), 3.51 (s, 3H), 3.25-3.17(m, 4H); ESI-MS
m/z 477 ((M+1), 100 %); Anal. Calcdfor C30H28N4O2: C, 75.61; H, 5.92; N, 11.76, Found: C,
75.65; H, 5.88; N, 11.71.
4.2.6.(4-(3-methoxyphenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7e)
White solid; Yield 74 %; mp 196-198 °C; Rf 0.35 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3057, 1625, 1556, 1454, 1435, 1253; H NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 8.21 (d, J =
7.8 Hz, 1H), 7.66-7.62 (m, 3H), 7.56-7.50 (m, 3H), 7.46 (d, J = 8.3 Hz, 1H), 7.36 (t, J = 7.5 Hz,
1H), 7.20 (t, J = 8.0 Hz, 1H), 6.62-6.48 (m, 3H), 4.13- 4.07 (m, 4H), 3.80 (s, 3H), 3.52 (s, 3H),
3.36-3.26 (m, 4H); ESI-MS m/z 477 ((M+1), 100 %); Anal. Calcdfor C30H28N4O2: C, 75.61; H,
5.92; N, 11.76, Found: C, 75.58; H, 5.94; N, 11.79.
4.2.7.(4-(2-methoxyphenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7f)
11

White solid; Yield 62 %; mp 168-170 °C; Rf 0.35 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
3053, 1643, 1556, 1496, 1240; 1H NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 8.23 (d, J = 7.8 Hz,
1H), 7.68-7.64 (m, 3H), 7.58-7.52 (m, 3H), 7.48 (d, J = 8.3 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H),
7.06-7.04 (m, 1H), 6.96-6.90 (m, 3H), 4.08 (brs, 4H), 3.90 (s, 3H), 3.54 (s, 3H), 3.22-3.12 (m,
4H); ESI-MS m/z 477 ((M+1), 100 %); Anal. Calcdfor C30H28N4O2: C, 75.61; H, 5.92; N, 11.76,
Found: C, 75.63; H, 5.97; N, 11.73.
4.2.8.(4-(4-chlorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7g)
White solid; Yield 76 %; mp 160-162 °C; Rf 0.40 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3055, 1633, 1556, 1471, 756; H NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 8.24 (d, J = 7.8 Hz,
1H), 7.68-7.64 (m, 3H), 7.56-7.53 (m, 3H), 7.50-7.48 (m, 1H), 7.41-7.37 (m, 1H), 7.26 (d, J =
8.9 Hz, 2H), 7.02-6.95 (m, 2H), 4.10 (brs, 4H), 3.54 (s, 3H), 3.34-3.25 (m, 4H); ESI-MS m/z 481
((M+1), 100 %), 483 ((M+3), 33 %); Anal. Calcdfor C29H25ClN4O: C, 72.42; H, 5.24; N, 11.65,
Found: C, 72.39; H, 5.26; N, 11.63.
4.2.9.(4-(3-chlorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7h)
White solid; Yield 70 %; mp 130-132 °C; Rf 0.42 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3069, 1614, 1556, 1487, 736; H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 8.19 (d, J = 7.9 Hz,
1H), 8.01-7.99 (m, 2H), 7.66-7.53 (m, 5H), 7.39-7.35 (m, 1H), 7.21 (t, J = 8.1 Hz, 1H), 6.93 (t, J
= 2.1 Hz, 1H), 6.88-6.82 (m, 2H), 4.12-4.06 (m, 4H), 3.54 (s, 3H), 3.38-3.31 (m, 4H); ESI-MS
m/z 481 ((M+1), 100 %), 483 ((M+3), 33 %); Anal. Calcdfor C29H25ClN4O: C, 72.42; H, 5.24;
N, 11.65, Found: C, 72.46; H, 5.27; N, 11.68.
4.2.10.(4-(2-chlorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7i)
White solid; Yield 66 %; mp 152-154 °C; Rf 0.40 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3043, 1629, 1556, 1435, 734; H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 8.21 (d, J = 7.8 Hz,
1H), 7.65-7.62 (m, 3H), 7.56-7.44 (m, 4H), 7.46-7.44 (m, 2H), 7.24-7.20 (m, 1H), 7.05-6.97 (m,
2H), 4.04 (brs, 4H), 3.51 (s, 3H), 3.19-3.08 (m, 4H);ESI-MS m/z 481 (((M+1), 100 %), 483
12

((M+3), 33 %); Anal. Calcdfor C29H25ClN4O: C, 72.42; H, 5.24; N, 11.65, Found: C, 72.40; H,
5.20; N, 11.64.
4.2.11.(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-(4-nitrophenyl)piperazin-1-
yl)methanone (7j)
Yellow solid; Yield 70 %; mp 200-202 °C; Rf 0.30 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3053, 1633, 1597, 1471, 1303, 754; H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 8.19-8.14 (m,
3H), 7.97 (d, J = 7.1 Hz, 2H), 7.64-7.51 (m, 5H), 7.38-7.34 (m, 1H), 6.84 (d, J = 9.4 Hz, 2H),
4.19-4.06 (m, 4H), 3.51 (s, 3H), 3.64-3.56 (m, 4H);ESI-MS m/z 492 ((M+1),100 %); Anal.
Calcdfor C29H25N5O3: C, 70.86; H, 5.13; N, 14.25, Found: C, 70.82; H, 5.12; N, 14.28.
4.2.12.(4-(4-fluorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7k)
White solid; Yield 76 %; mp 188-190 °C; Rf 0.40 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
1
3057, 1614, 1556, 1487, 736; H NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 8.23 (d, J = 7.7 Hz,
1H), 7.68-7.65 (m, 3H), 7.58-7.53 (m, 3H), 7.48 (d, J = 8.4 Hz, 1H), 7.38 (td, J = 7.6, 0.8 Hz,
1H), 7.06-6.96 (m, 2H), 6.96-6.88 (m, 2H), 4.06-4.05 (m, 4H), 3.54 (s, 3H), 3.26-3.16 (m,
4H);ESI-MS m/z 465 ((M+1),100 %); Anal. Calcdfor C29H25FN4O: C, 74.98; H, 5.42; N, 12.06,
Found: C, 74.94; H, 5.45; N, 12.03.
4.2.13.(4-(2-fluorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7l)
White solid; Yield 68 %; mp 168-170 °C; Rf 0.45 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
3055, 1633, 1556, 1471, 1435, 756; 1H NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 8.21 (d, J = 7.8
Hz, 1H), 7.65-7.62 (m, 3H), 7.55-7.50 (m, 3H), 7.45 (d, J = 8.3 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H),
7.07-6.93 (m, 4H), 4.05-4.04 (m, 4H), 3.51 (s, 3H), 3.23-3.13 (m, 4H); ESI-MS m/z 465
((M+1),100 %); Anal. Calcdfor C29H25FN4O: C, 74.98; H, 5.42; N, 12.06, Found: C, 74.96; H,
5.39; N, 12.10.
4.2.14.(4-(2,3-dichlorophenyl)piperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (7m)
13

White solid; Yield 74 %; mp 166-168 °C; Rf 0.32 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
3059, 1614, 1556, 1438, 790, 736; 1H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 8.21 (d, J = 7.8
Hz, 1H), 7.67-7.62 (m, 3H), 7.58-7.48 (m, 3H), 7.46 (d, J = 8.3 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H),
7.20-7.11 (m, 2H), 6.95 (dd, J = 7.6, 1.9 Hz, 1H), 4.04 (brs, 4H), 3.51 (s, 3H), 3.18-3.08 (m,
4H);ESI-MS m/z 516 (((M+1), 100 %), 518 ((M+3), 66 %), 518 ((M+5), 10 %)); Anal. Calcdfor
C29H24Cl2N4O: C, 67.58; H, 4.69; N, 10.87, Found: C,67.62; H, 4.64; N, 10.91.
4.2.2. (4-benzylpiperazin-1-yl)(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)methanone (7n)
White solid; Yield 78 %; mp 158-160 °C; Rf 0.40 (Hexane : EtOAc = 6:4); IR νmax/cm-1(KBr):
3055, 1614, 1552, 1438, 1409; 1H NMR (400 MHz, CDCl3): δ 8.50 (s, 1H), 8.22 (d, J = 7.7 Hz,
1H), 7.66-7.62 (m, 3H), 7.56-7.52 (m, 3H), 7.47 (d, J = 8.3 Hz, 1H), 7.39-7.34 (m, 5H), 7.30-
7.28 (m, 1H), 3.91-3.87 (m, 4H), 3.57 (s, 2H), 3.52 (s, 3H), 2.62-2.48 (m, 4H); ESI-MS m/z 461
((M+1), 100 %); Anal. Calcdfor C30H28N4O: C, 78.23; H, 6.13; N, 12.16, Found: C, 78.26; H,
6.11; N, 12.19.
4.2.15.(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-(pyridin-4-yl)piperazin-1-
yl)methanone (7o)
Yellowish white solid; Yield 68 %; mp 198-200 °C; Rf 0.35 (Hexane : EtOAc = 2:8); IR
νmax/cm-1 (KBr): 3053, 1620, 1558, 1456; 1H NMR (400 MHz, CDCl3): δ 8.59 (s, 1H), 8.31 (d,
J = 5.4 Hz, 2H), 8.24 (d, J = 7.8 Hz, 1H), 7.68-7.62 (m, 3H), 7.58-7.55 (m, 3H), 7.50 (d, J = 8.4
Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 6.68 (d, J = 6.0 Hz, 2H), 4.10-4.03 (m, 4H), 3.51 (s, 3H), 3.53-
3.42 (m, 4H);ESI-MS m/z 448 ((M+1), 100 %); Anal. Calcdfor C28H25N5O: C, 75.15; H, 5.63;
N, 15.65, Found: C, 75.19; H, 5.68; N, 15.68.
4.2.16.(9-methyl-1-phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-(pyridin-2-yl)piperazin-1-
yl)methanone (7p)
Yellowish white solid; Yield 62 %; mp 174-176 °C; Rf 0.30 (Hexane : EtOAc = 2:8); IR
1
νmax/cm-1 (KBr): 3053, 1614, 1556, 1487, 1471; H NMR (400 MHz, CDCl3): δ 8.57 (s, 1H),
8.24-8.21 (m, 2H), 7.69-7.65 (m, 3H), 7.58-7.47 (m, 5H), 7.38 (t, J = 7.5 Hz, 1H), 6.69-6.66 (m,
2H), 4.02 (brs, 4H), 3.71-3.69 (m, 4H), 3.55 (s, 3H); ESI-MS m/z 448 ((M+1), 100 %); Anal.
Calcdfor C28H25N5O: C, 75.15; H, 5.63; N, 15.65, Found: C, 75.12; H, 5.61; N, 15.62.
14

4.3. Biological evaluation
4.3.1. Cytotoxicity assay
HeLa cell cytotoxicity studies were carried out as described previously. Briefly, the cells were
cultured in DMEM supplemented with 10% fetal calf serum and 2 mM L-glutamine.Cells were
plated at initial cell concentration of 2 × 104 cells/well and incubated with the compounds for
∼65 h prior to addition of Alamar Blue solution for further 5 h [35].
4.3.2. Promastigote assay
L. donovanipromastigotes were cultured at 37°C in M199 medium supplemented with 10 %
heat-inactivated fetal calf serum.L. infantum JPCM5 MCAN/ES/98/LLM-87 promastigotes were
cultured at 26°C in modified Eagle’s medium (HOMEM) [36]. Parasites were incubated with
serial dilutions of compounds for 72 h, followed by Alamar blue-based assay as previously
described [37].
4.3.3. Axenic amastigote assay
L. donovaniLdBOB axenic amastigotes or L. infantum JPCM5 MCAN/ES/98/LLM-87 were
cultured at 37°C in modified Eagle’s medium (HOMEM; L. infantum) and axenic amastigotes
were incubated for 72 h with compounds, followed by Alamar blue-based assay as previously
described [37].
4.3.4. Intra macrophage L. donovani (amastigote) assay
THP-1 (human monocytic leukemia) cells were a kind gift from Dr Susan Wylie (Dundee) and
maintained in minimal essential medium plus 10% (vol/vol) FBS. An intracellular Leishmania
assay using LdBOB amastigotes were performed utilizing PMA differentiated THP-1 cells
infected overnight with axenic amastigotes, prior to compounds being added and incubated for
further 72 h, subsequent microscopy-based readout was used to determine EC50 values [37].
Briefly, THP-1 cells (20,000 per well, 200 µl) were plated into 96 well plates in presence of 10
nM PMA and incubated at 37oC in 5% CO2 atmosphere for 75 h. The adhered cells were then
washed with phosphate buffered saline supplemented with 1 mM CaCl2, 0.5 mM MgCl2, 0.1%
(w/v) bovine serum albumin and using LdBOB amastigotes added to all wells at a multiplicity of
infection of 5 (100,000 amastigotes per well) and incubated for 18 h. Any remaining
extracellular amastigotes were removed and the adhered cells were washed with the same
15

supplemented PBS solution as above. Pre-aliquoted compounds in a serial dilution were added
from another plate, such that the total well volume was 200 μL and incubated for further 72 h.
Cells were fixed with 100% methanol, stained with Giemsa and examined microscopically.
Number of intracellular amastigotes (~200 macrophages per well) were determined and the
percentage infection was established compared to an untreated control (100%) allowing EC50
values to be calculated [37].
Conflicts of interest
There are no conflicts of interest declared by the authors.
Acknowledgements
We are thankful to SAIF, Panjab University, Chandigarh and Central Instrumentation
Laboratory, Guru Jambheshwar University of Science and Technology, Hisar, India for
providing us NMR spectra. We are grateful to SAIF, CSIR-CDRI, Lucknow, India for Mass
spectral data. One of the authors, Ashok Penta, acknowledges Council of Scientific and
Industrial Research, New Delhi, India for financial support in the form of Senior Research
Fellowship. This work was also partially supported by TKS’s Welcome Trust project grant
093228and the European Community’s Seventh Framework Program under grant agreement
No.602773 (Project KINDRED).
References
[1] S.N. Suryawanshi, S. Kumar, R. Shivahare, S. Pandey, A. Tiwari, S. Gupta, Design, synthesis
and biological evaluation of aryl pyrimidine derivatives as potential leishmanicidal agents,
Bioorg. Med. Chem. Lett. 23 (2013) 5235-5238.
[1] A. Chattopadhyay, M. Jafurulla, A novel mechanism for an old drug: Amphotericin B in the
treatment of visceral leishmaniasis, Biochem. Biophys. Res. Comm. 416 (2011) 7-12.
[2] L. Kedzierski, Leishmaniasis Vaccine: Where are we today?, J. Glob. Infect. Dis. 2 (2010)
177-185.
16

[3] P.J. Guerin, P. Olliaro, S. Sundar, M. Boelaert, S.L. Croft, P. Desjeux, M.K. Wasunna,
A.D.M. Bryceson, Visceral leishmaniasis: Current status of control, diagnosis, treatment, a
proposed research and development agenda, Lancet Infect. Dis. 2 (2002) 494-501.
[4] K.M. Wasan, E.K. Wasan, P. Gershkovich, X. Zhu, R.R. Tidwell, K.A. Werbovetz, J.G.
Clement, S.J. Thornton, Highly effective oral amphotericin-B formulation against Murine
Visceral Leishmaniasis, J. Infect. Dis. 200 (2009) 357-360.
[5] S.L. Croft, J. Engel, Miltefosine-discovery of the antileishmanial activity of phospholipid
derivatives, Trans. Royal Soci. Trop. Med. Hyg. 100 (2006) S4-S8.
[6] J. Mishra, S. Singh, Miltefosine resistance in Leishmaniadonovani involves suppression of
oxidative stress-induced programmed cell death, Exper. Parasitol. 135 (2013) 397-406.
[7] G. Nenaah, Antibacterial and antifungal activities of (beta)-carboline alkaloids of
Peganumharmala (L) seeds and their combination effects, Fitoterapia. 81 (2010) 779-782.
[8] H. Huang, Y. Yao, Z. He, T. Yang, J. Ma, X. Tian, Y. Li, C. Huang, X. Chen, W. Li, S.
Zhang, C. Zhang, J. Ju, Antimalarial β-carboline and Indolactam alkaloids from
Marinactinosporathermotolerans, a Deep Sea Isolate, J. Nat. Prod. 74 (2011) 2122-2127.
[9] P. Ashok, H. Lathiya, S. Murugesan, Manzamine alkaloids as antileishmanial agents: A
review, Eur. J. Med. Chem. 97 (2015) 928-936.
[10] P. Ashok, S. Chander, T.K. Smith, M. Sankaranarayanan, Design, synthesis and biological
evaluation of piperazinyl-β-carbolinederivatives as anti-leishmanial agents, Eur. J. Med. Chem.
150 (2018) 559-566.
[11] P. Ashok, S. Ganguly, S. Murugesan, Manzamine alkaloids: isolation, cytotoxicity,
antimalarial activity and SAR studies, Drug Discov. Today. 19 (2014) 1781-1791.
[12] P. Ashok, S. Ganguly, S. Murugesan, Review on in-vitro anti-malarial activity of natural β-
carboline alkaloids, Mini. Rev. Med. Chem. 13 (2013) 1778-1791.
[13] A. Penta, S. Franzblau, B. Wan, S. Murugesan, Design, synthesis and evaluation of
diarylpiperazine derivatives as potent anti-tubercular agents, Eur. J. Med. Chem. 105 (2015) 238-
244.
17

[14] P. Ashok, S. Chander, J. Balzarini, C. Pannecouque, S. Murugesan, Design, synthesis of
new β-carboline derivatives and their selective anti-HIV-2 activity, Bioorg. Med. Chem. Lett. 25
(2015) 1232-1235.
[15] J. Yasuhara-Bell, Y. Lu, Marine compounds and their antiviral activities, Antivir. Res. 86
(2010) 231-240.
[16] C. Di Giorgio, F. Delmas, E. Ollivier, R. Elias, G. Balansard, P. Timon-David, In vitro
activity of the β-carboline alkaloids harmane, harmine and harmaline toward parasites of the
species Leishmaniainfantum, Exp. Parasitol. 106 (2004) 67-74.
[17] T.S. Kam, K.M. Sim, T. Koyano, K. Komiyama, Leishmanicidal alkaloids from
Kopsiagriffithii, Phytochemistry. 50 (1999) 75-79.
[18] R. Kumar, S. Khan, A. Verma, S. Srivastava, P. Viswakarma, S. Gupta, S. Meena, N. Singh,
J. Sarkar, P.M.S. Chauhan, Synthesis of 2-(pyrimidin-2-yl)-1-phenyl-2,3,4,9-tetrahydro-1H-β-
carbolines as antileishmanial agents, Eur. J. Med. Chem. 45 (2010) 3274-3280.
[19] D.C. Reis, M.C. Pinto, E.M. Souza-Fagundes, L.F. Rocha, V.R. Pereira, C.M. Melo, H.
Beraldo, Investigation on the pharmacological profile of antimony (III) complexes with
hydroxyquinoline derivatives: anti-trypanosomal activity and cytotoxicity against human
leukemia cell lines, Biometals. 24 (2011) 595-601.
[20] C.M.B. Leite Silva, F.P. Garcia, J.H.d.S. Rodrigues, C.V. Nakamura, T. Ueda-Nakamura, E.
Meyer, A.L.T.G. Ruiz, M.A. Foglio, J.E. de Carvalho, W.F. da Costa, Synthesis, antitumor,
antitrypanosomal and antileishmanial activities of benzo [4,5] canthin-6-ones bearing the N′-
(substituted benzylidene)-carbohydrazide and N-alkylcarboxamide groups at C-2, Chem. Pharm.
Bull. 60 (2012) 1372-1379.
[21] R. Pedroso, L. Tonin, T. Ueda-Nakamura, B.D. Filho, M. Sarragiotto, C. Nakamura, Beta-
carboline-3-carboxamide derivatives as promising antileishmanial agents, Ann. Trop. Med.
Parasitol. 105 (2011) 549-557.
[22] H. Volpato, lito, V. Desoti, N. Cristina, J. Cogo, M.R. Panice, M.H. Sarragiotto, S.d.O.
Silva, T. Ueda-Nakamura, C.V. Nakamura, The effects of N-butyl-1-(4-dimethylamino)phenyl-
18

1,2,3,4-tetrahydro-β-carboline-3-carboxamide against Leishmaniaamazonensis are mediated by
mitochondrial dysfunction, Evid. Complement. Alternat. Med. 2013 (2013) 7.
http://dx.doi.org/10.1155/2013/874367
[23] R.D. Taylor, M. MacCoss, A.D.G. Lawson, Rings in Drugs, J. Med. Chem. 57 (2014) 5845-
5859.
[24] A. Mayence, J.J. VandenEynde, L. LeCour Jr, L.A. Walker, B.L. Tekwani, T.L. Huang,
Piperazine-linked bisbenzamidines: a novel class of antileishmanial agents, Eur. J. Med. Chem.
39 (2004) 547-553.
[25] S. Wang, X. D. Jia, M.L. Liu, Y. Lu, H. Y. Guo, Synthesis, antimycobacterial and
antibacterial activity of ciprofloxacin derivatives containing a N-substituted benzyl moiety,
Bioorg. Med. Chem. Lett. 22 (2012) 5971-5975.
[26] R.V. Patel, P. Kumari, D.P. Rajani, K.H. Chikhalia, Synthesis, characterization and
pharmacological
activities
of
2-[4-cyano-(3-trifluoromethyl)phenyl
amino)]-4-(4-
quinoline/coumarin-4-yloxy)-6-(fluoropiperazinyl)-s-triazines, J. Fluorine Chem. 132 (2011)
617-627.
[27] A. Mayence, J.J. VandenEynde, F.M. Krogstad, D.J. Krogstad, M.T. Cushion, T.L. Huang,
Parallel solution-phase synthesis of conformationally restricted congeners of pentamidine and
evaluation of their antiplasmodial activities, J. Med. Chem. 47 (2004) 2700-2705.
[28] C. Fytas, G. Zoidis, N. Tzoutzas, M.C. Taylor, G. Fytas, J.M. Kelly, Novel lipophilic
acetohydroxamic acid derivatives based on conformationally constrained spiro carbocyclic 2,6-
diketopiperazine scaffolds with potent trypanocidal activity, J. Med. Chem. 54 (2011) 5250-
5254.
[29] B. Chetan, M. Bunha, M. Jagrat, B.N. Sinha, P. Saiko, G. Graser, T. Szekeres, G. Raman, P.
Rajendran, D. Moorthy, A. Basu, V. Jayaprakash, Design, synthesis and anticancer activity of
piperazinehydroxamates and their histone deacetylase (HDAC) inhibitory activity, Bioorg. Med.
Chem. Lett. 20 (2010) 3906-3910.
[30] Y. Wang, J. Ai, Y. Wang, Y. Chen, L. Wang, G. Liu, M. Geng, A. Zhang, Synthesis and c-
Met kinase inhibition of 3,5-disubstituted and 3,5,7-trisubstituted Quinolines: Identification of 3-
19

(4-acetylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7-(trifluoromethyl)quinoline
anticancer agent, J. Med. Chem. 54 (2011) 2127-2142.
as
a
novel
[31] D.L. Romero, R.A. Morge, M.J. Genin, C. Biles, M. Busso, L. Resnick, I.W. Althaus, F.
Reusser, R.C. Thomas, W.G. Tarpley, Bis(heteroaryl)piperazine (BHAP) reverse transcriptase
inhibitors: structure-activity relationships of novel substituted indole analogs and the
identification
of
1-[(5-methanesulfonamido-1H-indol-2-yl)carbonyl]-4-[3-[(1-
methylethyl)amino]pyridinyl]piperazinemono methane sulfonate (U-90152S), a second-
generation clinical candidate, J. Med. Chem. 36 (1993) 1505-1508.
[32] S. Hutait, M. Nayak, A. Penta, S. Batra, Convenient synthesis of a library of lactam-fused β-
carbolines via the Ugi Reaction, Synthesis. 03 (2011) 419-430.
[33] R. Mahesh, T. Devadoss, D.K. Pandey, S. Bhatt, Discovery of new anti-depressants from
structurally novel 5-HT3 receptor antagonists: Design, synthesis and pharmacological evaluation
of 3-ethoxyquinoxalin-2-carboxamides, Bioorg. Med. Chem. Lett. 21 (2011) 1253-1256.
[34] V. Singh, S. Hutait, S. Biswas, S. Batra, Versatility of substituted 1-formyl-9H-β-carbolines
for the synthesis of new fused β-carbolines via intramolecular 1,3-dipolar cycloaddition, Eur. J.
Org. Chem. 2010 (2010) 531-539.
[35] L. Zhou, G. Stewart, E. Rideau, N.J. Westwood, T.K. Smith, A class of 5-nitro-2-
furancarboxylamides with potent trypanocidal activity against Trypanosoma bruceiin vitro, J.
Med. Chem. 56 (2013) 796-806.
[36] S.O. Oyola, K.J. Evans, T.K. Smith, B.A. Smith, J.D. Hilley, J.C. Mottram, P.M. Kaye, D.F.
Smith, Functional analysis of Leishmania cyclopropane fatty acid synthetase, PloS one. 7 (2012)
51300.
[37] M. De Rycker, I. Hallyburton, J. Thomas, L. Campbell, S. Wyllie, D. Joshi, S. Cameron,
I.H. Gilbert, P.G. Wyatt, J.A. Frearson, Comparison of a high-throughput high-content
intracellular Leishmaniadonovani assay with an axenic amastigote assay, Antimicrob. Agents
Chemother. 57 (2013) 2913-2922.
20

Scheme 1
21

Table 1 Anti-leishmanial activity of the titled compounds against L. infantum
L. infantum
promastigote
EC50 (µM)
L. infantum
Comp. Code
R
CC50 (µM)
S.Ia
S.Ia
Axenic
amastigote
EC50 (µM)
4.5±0.3
-C6H5
-4CH3C6H4
-2CH3C6H4
-4OCH3C6H4
-3OCH3C6H4
-2OCH3C6H4
-4ClC6H4
>500
>500
9.45 ± 0.82
24.3 ± 1.3
3.73 ± 0.28
1.59 ± 0.11
72.4 ± 6.7
13.8 ± 0.9
1.47 ± 0.32
50.9 ± 4.8
21.7 ± 0.6
19.7 ± 1.7
17.2 ± 0.82
64.9 ± 5.7
93.0 ± 7.5
20.4 ± 1.4
61.1 ± 5.4
45.7 ± 3.3
8.31 ± 0.18
12.6±1.1
>52.9
>20.5
>134.0
>314.5
>6.9
>111.1
7a
ND
2.6±0.2
1.4±0.1
ND
-
7b
>500
>192.3
7c
>500
>357.1
7d
>500
-
7e
>500
>36.2
>340.1
>9.8
15.3±1.1
1.9±0.2
ND
>32.7
7f
>500
>263.2
7g
-3ClC6H4
>500
-
7h
-2ClC6H4
>500
>23.0
>25.3
>29.1
>7.7
ND
-
7i
-4NO2C6H4
-4FC6H4
>500
14.6±2.9
15.0±1.3
ND
>34.2
7j
>500
>33.3
7k
-2FC6H4
>500
-
-
-
-
-
7l
-2,3-diClC6H5
-CH2C6H5
-4C5H4N
>500
>5.3
ND
7m
7n
>500
>24.5
0.48
ND
29.7 ± 1.5
273 ± 21.6
ND
7o
-2C5H4N
5.97
ND
7p
Pentamidine
Miltefosine
2.7±0.4
4.8±0.8
22

Table 2 Anti-leishmanial activity of the titled compounds against L. donovani
L. donovani
Axenic
L. donovani
L. donovani
Promastigote
EC50 (µM)
Compound
Code
CC50
(µM)
intracellular
amastigote
EC50 (µM)
S.Ia
S.Ia
S.Ia
amastigote
EC50 (µM)
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
29.7 ±
1.5
11.9 ± 1.2
14.2 ± 1.2
9.45 ± 0.72
0.91 ± 0.09
28.5 ± 1.8
8.42 ± 0.45
5.02 ± 0.36
4.0 ± 0.25
15.2 ± 0.85
13.1 ± 1.5
12.4 ± 1.4
85.9 ± 7.3
117.5 ± 6.4
4.57 ± 0.24
>42.0
>35.2
>52.9
>549.5
>17.5
>59.4
>99.6
>125
10.0±0.3
5.8±0.3
5.3±0.4
0.9±0.1
ND
50.0
86.2
94.3
555.6
-
13.5±2.0
7.9±0.4
9.4±1.3
1.3±0.1
ND
37.0
63.3
53.2
384.6
-
7a
7b
7c
7d
7e
7f
6.4±0.2
3.8±0.5
2.2±0.1
19.4±1.2
7.5±0.4
9.3±0.5
ND
78.1
131.6
227.3
25.8
66.7
53.8
-
11.8±0.7
6.3±0.3
7.8±1.3
22.0±2.1
15.4±0.9
10.1±1.1
ND
42.4
79.4
64.1
22.7
32.5
49.5
-
7g
7h
7i
>32.9
>38.2
>40.3
>5.8
7j
7k
7l
>4.3
ND
-
ND
-
7m
7n
>109.4
3.5±0.2
142.9
5.6±0.9
89.3
19.5 ± 2.8
8.5 ± 0.75
1.52
13.9±0.4
12.3±0.6
2.1
7.2±0.5
7.9±0.3
4.1
7o
7p
273 ±
21.6
45.73
22.2
34.6
Pentamidine
Miltefosine
6.40 ± 0.11
3.12±0.16
1.6±0.1
2.8±0.4
23.7±1.8
6.4±0.3
aSI = CC50/IC50, ND – Not Determined
23

Biological Evaluation and Structure activity relationship of 9-methyl-1-phenyl-9H-
pyrido[3,4-b]indole derivatives as anti-leishmanial agents
Penta Ashoka, Subhash Chandera,b, Terry K. Smithc,Rajnish Prakash Singhd, Prabhat Nath Jhad,
Murugesan Sankaranarayanana*
a,*Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science
Pilani, Pilani Campus, Pilani-333031, Rajasthan. India. E. mail: penta.ashok@gmail.com, murugesan@pilani.bits-
pilani.ac.in
bSchool of Pharmacy, Maharaja Agrasen University, Atal ShikshaKunj, Solan, Himachal Pradesh 174103. E. mail:
subhashsaininiper@gmail.com
cSchools of Biology & Chemistry, BSRC, The University, St. Andrews, Fife Scotland. KY16 9ST, UK. E.
mail: tks1@st-andrews.ac.uk
dDepartment of Biological Sciences, Birla Institute of Technology & Science Pilani, Pilani Campus, Pilani 333031,
Rajasthan, India. E. mail: prabhatjha@pilani.bits-pilani.ac.in
Highlights
 New anti-leishmanial agents designed through molecular hybridization approach.
 7d showed potent anti-leishmanial activity against both L. infantum & L. donovani.
 7d EC50 against L. infantum promastigotes, axenic amastigotes 1.59 & 1.4 µM.
 7d EC50 against L. donovani promastigotes, axenic & intracellular amastigotes 0.91 &
0.91 & 1.4 µM.
24

Biological Evaluation and Structure activity relationship of 9-methyl-1-phenyl-9H-
pyrido[3,4-b]indole derivatives as anti-leishmanial agents
Penta Ashoka, Subhash Chandera,b, Terry K. Smithc, Rajnish Prakash Singhd, Prabhat Nath Jhad,
Murugesan Sankaranarayanana*
a,*Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science
Pilani, Pilani Campus, Pilani-333031, Rajasthan. India. E. mail: penta.ashok@gmail.com, murugesan@pilani.bits-
pilani.ac.in
bSchool of Pharmacy, Maharaja Agrasen University, Atal ShikshaKunj, Solan, Himachal Pradesh 174103. E. mail:
subhashsaininiper@gmail.com
cSchools of Biology & Chemistry, BSRC, The University, St. Andrews, Fife Scotland. KY16 9ST, UK. E. mail:
tks1@st-andrews.ac.uk
dDepartment of Biological Sciences, Birla Institute of Technology & Science Pilani, Pilani Campus, Pilani 333031,
Rajasthan, India. E. mail: prabhatjha@pilani.bits-pilani.ac.in
Anti-leishmanial activity EC50 (µM)
L. infantum: promastigotes 1.59 and axenic amastigotes 1.4.
L.donovani: promastigotes 0.9, axenic amastigotes 1.4 and intracellular amstigotes 1.3
25