Synthesis and anti-leishmanial evaluation of 1-phenyl-2,3,4,9-tetrahydro-1H-β-carboline derivatives against Leishmania infantum

Article abstract of DOI:10.1016/j.ejmech.2016.08.014

In the present study, antileishmanial activity of sixteen novel series of tetrahydro-β-carboline derivatives against transgenic infrared fluorescent Leishmania infantum strain has been reported. Among these reported analogues, most of the compounds exhibited potent inhibition against both promastigote (IC₅₀from 1.99?±?1.40 to 20.69?±?0.95?μM) and amastigote (IC₅₀from 0.67?±?0.05 to 4.16?±?0.008?μM) forms of L.?infantum. Moreover, compound 7l, displayed most potent and selective inhibition of parasite amastigote form with IC₅₀0.67?±?0.05?μM, selectivity index >298.5 and was comparable with standard drug amphotericin B. From this study, a new class of tetrahydro-β-carboline derivatives with potent antileishmanial activity was identified and it needs further extensive study to optimize the lead molecules to win the battle against severe and neglected disease leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2016.08.014

European Journal of Medicinal Chemistry 123 (2016) 814e821
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and anti-leishmanial evaluation of 1-phenyl-2,3,4,9-
tetrahydro-1H-b-carboline derivatives against Leishmania infantum
a
a
b
b
Penta Ashok , Subhash Chander , Ana Tejería , Laura García-Calvo ,
b
a, *
Rafael Bala n~ a-Fouce , Sankaranarayanan Murugesan
Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science, Pilani 333031, India
a
b
Departmento de Ciencias Biomedicas, Facultad de Veterinaria, Universidad de Leon, Leon 24071, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2016
Received in revised form
In the present study, antileishmanial activity of sixteen novel series of tetrahydro-b-carboline derivatives
against transgenic infrared fluorescent Leishmania infantum strain has been reported. Among these re-
ported analogues, most of the compounds exhibited potent inhibition against both promastigote (IC50
8
August 2016
from 1.99 ± 1.40 to 20.69 ± 0.95
L. infantum. Moreover, compound 7l, displayed most potent and selective inhibition of parasite amasti-
gote form with IC50 0.67 ± 0.05 M, selectivity index >298.5 and was comparable with standard drug
amphotericin B. From this study, a new class of tetrahydro- -carboline derivatives with potent anti-
mM) and amastigote (IC50 from 0.67 ± 0.05 to 4.16 ± 0.008 mM) forms of
Accepted 9 August 2016
Available online 10 August 2016
m
b
Keywords:
Leishmaniasis
Neglected disease
leishmanial activity was identified and it needs further extensive study to optimize the lead molecules to
win the battle against severe and neglected disease leishmaniasis.
© 2016 Elsevier Masson SAS. All rights reserved.
Tetrahydro-b-carboline
Promastigote
Amastigote
1. Introduction
last five decades; unfortunately, 60% of VL cases in India (Bihar)
become un-responsive to antimonials due to developed resistance.
Leishmaniasis is one of the major neglected diseases and is
In spite of its adverse effects and cost, polyene antifungal drug
amphotericin B is the drug of choice where resistance to antimo-
nials is developed [5]. Usefulness of second line drugs such as
pentamidine and paromomycin has been restricted [6,7]. Miltefo-
sine originally developed as anticancer agent, is the only oral drug
used for the treatment of VL and usage is limited with toxic effects
like nephrotoxicity, hepatotoxicity and teratogenicity. Moreover,
recently low sensitivity to miltefosine is observed in countries like
India where it has been used extensively [8]. In addition, enhanced
prevalence in immuno compromised patient and lack of effective,
safe and availability of antileishmanial drugs increases the need to
develop novel antileishmanial agents with good selectivity index
(SI).
prevalent in 98 countries throughout the world. According to WHO
reports, 310 millions are living at risk places, 1.3 million new in-
fections and 30,000 deaths take place annually [1]. Leishmaniasis
has been classified as three different clinical forms (i. e) Cutaneous
Leishmaniasis (CL), Mucocutaneous Leishmaniasis (MCL) and
Visceral Leishmaniasis (VL) depending on parasite species [2].
Visceral Leishmaniasis (also known as kala azar) is the most severe
form of leishmaniasis, caused by L. donovani in Bangladesh,
Ethiopia, India, Nepal, South Sudan and Sudan. L. infantum is the
etiological agent of VL in countries at both shores of the Mediter-
ranean Basin, whereas L. chagasi is the predominant species in
South America [3]. VL often affects internal organs such as liver,
spleen, bone marrow and is usually fatal if left untreated [4]. Even
though, leishmaniasis control mainly depends on chemotherapy,
the first-line therapy for leishmaniasis is limited with decade old
drugs. Pentavalent antimonials (sodium stibogluconate and
meglumine antimoniate) have been used as first line drugs for the
Nature remains as potential source to produce antiinfective
agents and large number of natural products especially alkaloids
displayed potent antileishmanial activity. Among the various group
of natural alkaloids,
b-carbolines stay as one of the important
alkaloidal group as it present in large number of natural products
isolated from different sources like territorial plants [9], marine
sponge [10], fast food [11] and humans [12]. b-Carboline skeleton
has privileged position in medicinal chemistry with various bio-
logical activities such as anticancer [13], antithrombotic [14],
*
Corresponding author.
E-mail address: murugesan@pilani.bits-pilani.ac.in (S. Murugesan).
http://dx.doi.org/10.1016/j.ejmech.2016.08.014
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
815
antimicrobial [15], antimalarial [16,17], antileishmanial [18e23],
antitubercular [24,25] and antiviral activity [26,27]. Natural -car-
boline alkaloids such as harman, harmaline, harmine, buchtienine,
annomontine and manzamine-A (Fig. 1) displayed moderate to
potent antileishmanial activity [28e30]. Manzamines are a group of
trifluoroacetic acid via Pictet-Spengler reaction. Other in-
termediates 2-Chloro-N-(substituted)phenylacetamides (6a-p)
were synthesized from substituted anilines (4a-p) and chloroacetyl
chloride (5) by using triethylamine as base and dichloromethane as
solvent with good to excellent yields. To obtain desired compounds
(7a-p),1-Phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (3) was
treated with 2-Chloro-N-(substituted)phenylacetamides (6a-p) in
b
b
-carboline alkaloids possess complex structure of
moiety attached to a pentacyclic ring both eight and thirteen
membered rings on pyrrolo[2,3-i]isoquinoline framework.
b-carboline
a
presence of K
2 3
CO in dimethylformamide as solvent in yields of
Several manzamine alkaloids (Fig. 1) displayed potent anti-
leishmanial activity and we recently reviewed the antileishmanial
activity of manzamines [31]. With the optimistic antileishmanial
68e90% [34e36].
Synthesized 1-Phenyl-2-(1-phenyl-3,4-dihydro-1H-pyrido[3,4-
b]indol-2(9H)-yl)ethanone derivatives were characterized by
1
13
potency of these natural alkaloids, several synthetic
b
-carboline
spectral analysis such as, IR, H NMR, C NMR, Mass and elemental
derivatives were also evaluated for antileishmanial activity and
displayed potent inhibition against leishmania parasites
analysis. IR spectra of the synthesized compounds showed N-H
ꢀ1
stretching at 3394 to 3282 cm , C]O absorption at 1698 to
ꢀ
1
[
18,19,21e23]. Based on the above facts and our continuous interest
to develop novel -carboline derivatives as antileishmanial agents,
in the present work, synthesis and antileishmanial evaluation of
sixteen novel tetrahydro- -carboline derivatives has been reported.
1682 cm , C-C aromatic stretching around 1481 to 1456, C-O ab-
ꢀ
1
b
sorption (methoxy) band at 1249 to 1246 cm , C-Cl absorption
ꢀ1
band at 1062 to 1044 cm and in nitro derivative N-O asymmetric
ꢀ1 1
b
stretching was observed around 1519 cm . H NMR spectrum of
the compounds showed singlet peak of one hydrogen at 5.14 to
4.83 due to position-1 methine proton of the -carboline ring, two
protons of phenacyl methylene group appeared as two doublets,
each of one proton at 4.14 to 4.03 and 3.92 to 3.71 with high
To the best of knowledge, these compounds have never been re-
ported for antileishmanial activity elsewhere. In-silico prediction of
molecular properties of new chemical entities has gained interest
in recent medicinal chemistry. Molecular properties play crucial
role in absorption, distribution, metabolism, excretion and toxicity
d
b
d
coupling constant values of ~17 Hz, due to geminal hydrogen
coupling. Four aliphatic hydrogens of two methylene groups of
(
ADMET) properties of ligands. Compounds follow Lipinski rule of
five parameters may show good oral bio-availability and are able to
develop as oral drug candidates [32,33]. In the present study,
tetrahydro-
3.35e3.27, 3.23e3.11, 3.03e2.97 and 2.92e2.89 respectively,
methoxy protons appeared as singlet at ~3.80 and methyl protons
as singlet at ~2.4. Indole NH proton position was varied with
b-carboline were appeared as four multiplets around
d
calculated Lipinski parameters of these novel
b
-carboline de-
d
rivatives using Molinspiration Cheminformatics and Osiris property
explorer was also reported.
d
d
value 7.96 to 7.30. In mass spectral analysis, Mþ1 peak appeared
as parent ion peak. Elemental analysis data showed that, the
calculated and observed values are within the acceptable limits
2
. Results and discussion
(±0.05%).
2.1. Chemistry: synthesis of tetrahydro-b-carboline derivatives
2.2. Molecular properties
The designed tetrahydro-
sized as per the illustrated protocol in Scheme 1. Key intermediate
-Phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (3) was syn-
b-carboline derivatives were synthe-
Good pharmacokinetic properties and toxicity profiles are
essential for any new chemical entities to complete drug discovery
1
thesized from tryptamine (1) and benzaldehyde (2) in presence of
process and to become successful drugs. Hence, in-silico prediction
Fig. 1. Structure of natural antileishmanial
b
-carboline alkaloids with their IC50 values.

816
P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
ꢂ
2 3
Scheme 1. Synthetic protocol for the designed analogues. Reagents and conditions: (a) Trifluoroacetic acid, DCM, rt, 24 h; (b) Triethylamine, DCM, 0 C, 30 min; (c) K CO , DMF, 3 h.
of molecular properties of new chemical entity became very much
useful nowadays. Compounds, which follow Lipinski parameters
such as, ClogP (ꢁ5), Molecular weight (ꢁ500), number of hydrogen
bond acceptors (ꢁ5) and number of hydrogen bond donors (ꢁ10)
may not have the bioavailability problems. Molecules violating
more than one time from these rules may have some undesirable
effect on their pharmacokinetic properties [37]. By taking the
importance of these molecular properties into consideration, in the
present study, Lipinski parameters of these new chemical entities
respectively) and comparable with amphotericin
B
(IC50
0.32 ± 0.05 M). Surprisingly, compound 7k showed decreased
m
activity against amastigote forms. More interestingly, few com-
pounds are inactive against promastigotes but they exhibited
potent inhibition of amastigotes especially compound 7j displayed
IC50 at sub-micromolar concentration. Potent antiamastigote ac-
tivity is mainly depends on lipophilicity of these analogues, it may
play important role in compound transportation across macro-
phage membrane. Most of these analogues have not shown any
using
Molinspiration
Cheminformatics
(http://www.
cytotoxicity to splenocytes at 200 mM, as toxicity is one of the major
molinspiration.com) and Osiris property explorer (http://www.
organic-chemistry.org/prog/peo) was predicted. From the results,
it was found that, compounds 4d, 4i, 4n, 4 and 4p showed violation
from one Lipinski parameter i.e., ClogP, predicted that these com-
pounds may have logP value higher than 5. It is well known that,
single violation from any of these Lipinski parameters will not have
significant effect on pharmacokinetic properties of ligand [33,37].
Hence, from the study of in-silico prediction of molecular proper-
ties, it was concluded that, the new chemical entities might not
have any bioavailability problems. Predicted in-silico molecular
properties are shown in Table 1.
concerns with antileishmanial drugs.
In the present study, the effect of phenyl ring attached to amide
nitrogen with different electron donating and withdrawing sub-
stituents on antileishmanial activity was explored. Among these
reported analogues, the prototype compound with un-substituted
phenyl ring displayed potent antiamastigotes activity with IC50
value of 1.68 mM. Substitutions on para position of phenyl ring with
electron donating and withdrawing substituents resulted in mar-
ginal decrease in activity. Upon meta substitution with ortho-para
directing groups had increased the potency faintly while meta
directing nitro group produced considerable increase in potency
and trifluoromethyl group had minimal effect on inhibition po-
tency. Substitutions on ortho position have variable effect on anti-
leishmanial potency, methoxy group weakly decreased, whereas
methyl group produced the most potent analogue of the series with
significant increase in potency and chloro substitution caused little
increase in potency. Steric groups like, dimethyl substitution and
replacement of phenyl ring with naphthyl ring reduced the activity
vaguely. Even though, it is very tough to conclude structure activity
relationship studies with limited number of analogues, but
maximum efforts has been made to summarize the same, substi-
tution on meta position is favorable for activity especially with
electron withdrawing groups followed by donating groups, ortho
substitution with electron donating substituents was advantageous
for antileishmanial activity, followed by electron withdrawing
groups and steric group substitution was not desirable. Replace-
ment with bulkier naphthyl ring retained the potency with mar-
ginal decrease in activity and para substitutions on phenyl ring
were not recommended for potent antileishmanial activity.
2.3. Antileishmanial activity and cytotoxicity
All the synthesized sixteen novel tetrahydro-b-carboline de-
rivatives were initially evaluated for antipromastigotes activity at
three concentrations 1, 10, 100 M respectively. Compounds dis-
played potent inhibition at 100 M were further evaluated to
m
m
determine IC50 against promastigotes. Among the reported ana-
logues, most of the compounds exhibited potent inhibition against
promastigotes of L. infantum strain with IC50 ranges from 1.99 to
2
0.69
mM. Antileishmanial drug, amphotericin
B
(IC50
0
.77 ± 0.15
mM) was used as standard to compare the potency of the
reported analogues. In these reported compounds, compound 7k
showed potent inhibition of promastigotes with IC50
1.99 ± 1.40
mM.
With optimistic results of these tetrahydro-
b-carboline de-
rivatives against promastigotes, all the sixteen analogues were
evaluated against amastigote form using splenocytes from experi-
mentally infected BALB/c mice with infrared L. infantum strains.
Interestingly, intracellular amastigotes were more sensitive to most
3. Conclusion
of these tetrahydro-
exhibited potent inhibition with IC50 value ranges from 0.67 to
.46 M. Among these reported analogues, compounds 7l and 7j
displayed most potent activity with IC50 values (0.67 and 0.87
b-carboline derivatives and these analogues
In summary, antileishmanial activity of novel sixteen tetrahy-
6
m
dro-b-carboline derivatives has been reported against both pro-
mM
mastigote and amastigote forms of L. infantum. Most of these

P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
Table 1 (continued )
817
Table 1
Predicted In-silico molecular properties of the synthesized compounds.
Comp. code Compound structure
Mol.Wt ClogP HBAa HBDb
Comp. code Compound structure
Mol.Wt ClogP HBAa HBDb
7n
449
5.37
2
4
7a
7b
7c
381
411
395
4.61
4.51
4.93
2
2
2
4
5
4
7
o
409
5.84
2
4
7p
431
5.79
2
4
7
7
7
d
e
f
415
426
399
5.23
4.48
4.67
2
2
2
4
7
4
Values in bold indicates ClogP is greater than 5.
a
Hydrogen Bond Acceptor.
Hydrogen Bond Donor.
b
analogues exhibited potent antileishmanial activity with good SI.
Especially, two analogues 7l and 7j exhibited selective and potent
inhibition of amastigotes with IC50 values 0.67 and 0.87
mM
respectively and potency was comparable with amphotericin B.
Structure activity relationship study suggested that, substitution on
meta, ortho positions showed favorable effect, while replacement
with bulkier group had minimal effect on activity and para sub-
stitution was not desirable. Moreover, we need further exhaustive
studies in order to optimize the identified lead molecules and to
find out exact molecular mechanism of action for their potent
antileishmanial activity.
7
7
g
411
395
4.51
4.93
2
2
5
4
h
4. Experimental section
4.1. Chemistry
7i
415
5.23
2
4
All solvents and reagents purchased from Sigma or Merck
companies are used as received without further purification. Sol-
vent system used throughout 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
7
7
j
426
411
4.48
4.51
2
2
7
5
performed using silica gel (100e200 mesh, SRL, India) as stationary
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 spectropho-
tometer (Shimadzu IR Prestige 21, India). H NMR and C NMR
spectra were recorded on a Bruker DPX-400 spectrometer (Bruker
India Scientific Pvt. Ltd., Mumbai) using TMS as an internal stan-
k
1
13
dard (chemical shifts in d). Elemental analysis was performed on
7
7
l
395
415
4.93
4.23
2
2
4
4
Vario EL III M/s Elementar C, H, N and S analyzer (Elementar Ana-
lysensysteme GmbH, Germany). The ESMS were recorded on
MICROMASS Quattro-II LCMS system (Waters Corporation, Milford,
USA).
m
4.1.1. General procedure for the synthesis of 2-(3,4-dihydro-1-
phenyl-1H-pyrido [3,4-b] indol-2(9H)-yl)-N-phenylacetamide 7a-p
(exemplified as 7a)
To a stirred solution of 3 (0.47 g, 2 mmol) in dry DMF (3 mL),
K CO (0.83 g, 6 mmol) was added and stirred the reaction mixture
2 3
at room temperature for 30 min. Thereafter, 2-Chloro-N-phenyl-
acetamide 6a (0.34 g, 2 mmol) was added and the reaction mixture
was stirred for additional 3 h at room temperature. On completion
of the reaction as monitored by TLC, the contents were poured into

818
P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
ice water (50 mL) whilst stirring with a glass rod. Thereafter, the
aqueous layer was extracted with EtOAc (3 ꢃ 20 mL). The organic
layers were combined and washed with brine (40 mL), dried with
anhydrous Na SO and concentrated to yield the product (0.59 g
2 4
from 0.47 g, 78%). For analytical grade, the compound was passed
4.1.6. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
N-(4-nitrophenyl)acetamide (7e)
Compound 7e was prepared in 70% yield as white solid from 3
and 2-Chloro-N-(4-nitrophenyl)acetamide (4e) according to the
above mentioned general procedure. The compound was purified
through short silica gel (100e200 mesh) column chromatography
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.28).
9.62 (brs, 1H),
.25e8.19 (m, 2H), 7.72e7.66 (m, 2H), 7.59 (dd, J ¼ 6.4, 1.9 Hz, 1H),
.39e7.36 (m, 6H), 7.26e7.24 (m, 1H), 7.22e7.15 (m, 2H), 4.82 (s,
ꢂ
1
(
ethylacetate: hexane ¼ 6:4, R
f
0.40) to get 7a as a white solid.
mp 210e212 C. H NMR (400 MHz, CDCl ): d
3
8
7
4
.1.2. 2-(3,4-Dihydro-1-phenyl-1H-pyrido[3,4-b]indol-2(9H)-yl)-
N-phenylacetamide (7a)
White solid. mp 110e112 C. H NMR (400 MHz, CDCl
brs, 1H), 7.60e7.58 (m, 1H), 7.54 (d, J ¼ 7.6 Hz, 2H), 7.40e7.33 (m,
H), 7.25e7.23 (m, 1H), 7.20e7.11 (m, 3H), 4.80 (s, 1H), 3.44 (d,
J ¼ 16.6 Hz, 1H), 3.36e3.32 (m, 1H), 3.21 (d, J ¼ 16.6 Hz, 1H),
1
3
H), 3.48 (d, J ¼ 16.9 Hz, 1H), 3.36e3.27 (m, 2H), 3.16e3.11 (m, 1H),
ꢀ1
.09e2.98 (m, 2H). IR (KBr, cm ):
n 3288, 1681, 1537, 1506, 1342,
ꢂ
1
):
d
9.26
þ
3
1300. MS (ESI) m/z 427.1 (M þ H ). Anal. Calcd for C25
22 4 3
H N O : C,
(
8
70.41; H, 5.20; N, 13.14. Found: C, 70.38; H, 5.24; N, 13.12.
4.1.7. N-(4-Fluorophenyl)-2-(3,4-dihydro-1-phenyl-1H-pyrido[3,4-
ꢀ1
3
1
.14e3.08 (m, 1H), 3.01e2.96 (m, 2H). IR (KBr, cm ):
n
3302, 1672,
519, 1448. MS (ESI) m/z 382.4 (M þ H ). C NMR (100 MHz,
DMSO) 169.46, 141.52, 138.98, 136.84, 134.54, 129.58, 129.19,
28.73, 128.14, 126.93, 123.89, 121.20, 119.82, 118.82, 118.20, 111.59,
07.69, 62.39, 57.75, 47.93, 20.76. Anal. Calcd for C25 O: C,
b]indol-2(9H)-yl)acetamide (7f)
þ
13
Compound 7f was prepared in 72% yield as white solid from 3
and 2-Chloro-N-(4-fluorophenyl)acetamide (4f) according to the
above mentioned general procedure. The compound was purified
d
1
1
23 3
H N
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.32).
9.23 (brs, 1H),
.60e7.58 (m, 1H), 7.51e7.47 (m, 2H), 7.39e7.36 (m, 6H), 7.25e7.22
m, 1H), 7.21e7.14 (m, 2H), 7.07e7.01 (m, 2H), 4.79 (s, 1H), 3.43 (d,
7
8.71; H, 6.08; N, 11.02. Found: C, 78.72; H, 6.05; N, 11.04.
ꢂ
1
3
mp 102e104 C. H NMR (400 MHz, CDCl ): d
7
(
4.1.3. 2-(3,4-Dihydro-1-phenyl-1H-pyrido[3,4-b]indol-2(9H)-yl)-
J ¼ 16.7 Hz, 1H), 3.36e3.31 (m, 1H), 3.21 (d, J ¼ 16.7 Hz, 1H),
N-(4-methoxyphenyl)acetamide (7b)
ꢀ
1
3
1
7
.16e3.08 (m, 1H), 3.02e2.95 (m, 2H). IR (KBr, cm ):
n
3290, 1693,
Compound 7b was prepared in 90% yield as white solid from 3
and 2-Chloro-N-(4-methoxyphenyl)acetamide (4b) according to
the above mentioned general procedure. The compound was pu-
þ
514. MS (ESI) m/z 400.2 (M þ H ). Anal. Calcd for C25
H22FN O: C,
3
2.51; H, 8.11; N, 10.57. Found: C, 72.53; H, 8.15; N, 10.61.
rified by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
4
.1.8. 2-(3,4-Dihydro-1-phenyl-1H-pyrido[3,4-b]indol-2(9H)-yl)-
ꢂ
1
0
.32). mp 98e100 C. H NMR (400 MHz, CDCl
3
):
d
9.12 (brs, 1H),
N-(3-methoxyphenyl)acetamide (7g)
7.58e7.56 (m, 1H), 7.43e7.34 (m, 8H), 7.19e7.12 (m, 3H), 6.86e6.84
Compound 7g was prepared in 80% yield as white solid from 3
and 2-Chloro-N-(3-methoxyphenyl)acetamide (4g) according to
the above mentioned general procedure. The compound was pu-
(
2
1
7
m, 2H), 4.75 (s, 1H), 3.78 (s, 3H), 3.41e3.29 (m, 2H), 3.18e3.05 (m,
ꢀ1
H), 2.96e2.92 (m, 2H). IR (KBr, cm ):
246. MS (ESI) m/z 412.5 (M þ H ). Anal. Calcd for C26
n
3298, 1668, 1517, 1452,
: C,
þ
25 3 2
H N O
rified by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
5.89; H, 6.12; N, 10.21. Found: C, 75.92; H, 6.13; N, 10.20.
ꢂ
1
0
.34). mp 138e140 C. H NMR (400 MHz, CDCl
3
)
d
9.26 (s, 1H),
7
7
(
1
.65e7.54 (m, 1H), 7.41e7.34 (m, 6H), 7.32 (t, J ¼ 2.0 Hz, 1H),
.27e7.21 (m, 2H), 7.21e7.14 (m, 2H), 7.00 (d, J ¼ 7.9 Hz, 1H), 6.69
dd, J ¼ 8.2, 2.0 Hz, 1H), 4.79 (s, 1H), 3.83 (s, 3H), 3.43 (d, J ¼ 16.7 Hz,
4.1.4. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
N-p-tolylacetamide (7c)
H), 3.38e3.29 (m, 1H), 3.24e3.06 (m, 2H), 3.04e2.93 (m, 2H). IR
Compound 7c was prepared in 86% yield as white solid from 3
and 2-Chloro-N-p-tolylacetamide (4c) according to the above
mentioned general procedure The compound was purified by col-
ꢀ
1
þ
(KBr, cm ):
n
3284, 1666, 1537, 1248. MS (ESI) m/z 412.2 (M þ H ).
25 3 2
Anal. Calcd for C26H N O : C, 75.89; H, 6.12; N, 10.21. Found: C,
7
5.87; H, 6.15; N, 10.18.
umn chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.36). mp
ꢂ
3
02e104 C. H NMR (400 MHz, CDCl ): d 9.17 (brs, 1H), 7.56 (dd,
1
1
4.1.9. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
J ¼ 6.3, 2.6 Hz, 1H), 7.41e7.35 (m, 8H), 7.22e7.20 (m, 1H), 7.15e7.11
N-m-tolylacetamide (7h)
(
m, 4H), 4.77 (s, 1H), 3.42e3.38 (m, 1H), 3.32e3.30 (m, 1H),
ꢀ
1
Compound 7h was prepared in 72% yield as white solid from 3
and 2-Chloro-N-m-tolylacetamide (4h) according to the above
mentioned general procedure. The compound was purified by
3
3
C
.19e3.06 (m, 2H), 2.97e2.94 (m, 2H), 2.31 (s, 3H). IR (KBr, cm ):
307, 1668, 1523, 1486. MS (ESI) m/z 396.3 (M þ H ). Anal. Calcd for
n
þ
26 25 3
H N O: C, 78.96; H, 6.37; N, 10.62. Found: C, 78.94; H, 6.39; N,
column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.36). mp
ꢂ
3
24e126 C. H NMR (400 MHz, CDCl ) d 9.23 (s, 1H), 7.59 (dd,
10.60.
1
1
J ¼ 6.1, 2.5 Hz, 1H), 7.45e7.30 (m, 8H), 7.27e7.12 (m, 4H), 6.95 (d,
4
.1.5. N-(4-Chlorophenyl)-2-(3,4-dihydro-1-phenyl-1H-pyrido
J ¼ 7.5 Hz, 1H), 4.79 (s, 1H), 3.43 (d, J ¼ 16.6 Hz, 1H), 3.34 (m, 1H),
ꢀ
1
[3,4-b]indol-2(9H)-yl)acetamide (7d)
3.24e3.08 (m, 2H), 3.04e2.93 (m, 2H), 2.37 (s, 3H). IR (KBr, cm ):
n
þ
Compound 7d was prepared in 76% yield as white solid from 3
3267, 1666, 1537, 1282. MS (ESI) m/z 396.2 (M þ H ). Anal. Calcd for
and 2-Chloro-N-(4-chlorophenyl)acetamide (4d) according to the
above mentioned general procedure. The compound was purified
26 25 3
C H N O: C, 78.96; H, 6.37; N, 10.62. Found: C, 78.97; H, 6.36; N,
10.64.
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.38).
9.28 (brs, 1H), 7.59
dd, J ¼ 6.2, 2.1 Hz, 1H), 7.51e7.47 (m, 2H), 7.39e7.35 (m, 6H),
ꢂ
1
mp 130e132 C. H NMR (400 MHz, CDCl
3
):
d
4.1.10. N-(3-Chlorophenyl)-2-(3,4-dihydro-1-phenyl-1H-pyrido
[3,4-b]indol-2(9H)-yl)acetamide (7i)
Compound 7i was prepared in 70% yield as white solid from 3
and 2-Chloro-N-(3-chlorophenyl)acetamide (4i) according to the
above mentioned general procedure. The compound was purified
(
7
3
3
.32e7.29 (m, 2H), 7.25e7.22 (m,1H), 7.19e7.16 (m, 2H), 4.79 (s,1H),
.43 (d, J ¼ 16.7 Hz, 1H), 3.35e3.31 (m, 1H), 3.22 (d, J ¼ 16.7 Hz, 1H),
ꢀ1
.16e3.08 (m, 1H), 3.02e2.97 (m, 2H). IR (KBr, cm ):
n
3323, 1678,
þ
3þ
1519, 1490, 1062. MS (ESI) m/z 416.1 (M þ H ), 418.1 (M þ H ).
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.38).
9.30 (brs, 1H), 7.64 (t,
J ¼ 1.9 Hz, 1H), 7.59 (dd, J ¼ 6.2, 2.2 Hz, 1H), 7.41e7.352 (m, 7H),
ꢂ
1
Anal. Calcd for C25
H22ClN
3
O: C, 72.19; H, 5.33; N, 10.10. Found:
mp 120e122 C. H NMR (400 MHz, CDCl ): d
3
72.22; H, 5.29; N, 10.14.

P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
819
Table 2
Results of in-vitro anti-leishmanial activity of the synthesized compounds.
Comp. code
CC50a
IC50a
Selectivity Index(SI)b
IC50a
Selectivity index(SI)b
Promastigotes
Amastigotes
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
>200
>200
>200
145.91 ± 20.83
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>20
12.37 ± 1.37
>100
>16.17
na
>10.39
9.64
1.68 ± 0.04
6.46 ± 0.4 7
6.31 ± 1.28
3.75 ± 0.63
5.29 ± 0.25
4.16 ± 0.08
1.23 ± 0.08
1.48 ± 0.03
1.51 ± 0.50
0.87 ± 0.09
5.35 ± 0.17
0.67 ± 0.05
1.05 ± 0.03
2.02 ± 0.31
2.64 ± 0.33
2.25 ± 0.28
0.32 ± 0.05
>119
>30.9
>31.7
38.9
19.25 ± 2.29
15.13 ± 0.58
8.10 ± 0.43
15.10 ± 1.16
14.58 ± 1.56
7.63 ± 0.41
6.68 ± 0.93
>100
1.99 ± 1.40
10.43 ± 0.44
20.69 ± 0.95
45.18 ± 8.48
16.95 ± 1.52
>100
>24.69
>13.25
>13.72
>26.21
>29.94
na
>100.5
>19.18
>9.67
>4.43
>11.8
na
>37.8
>48.1
>162.6
>135.1
>132.4
>229.8
>37.4
>298.5
>190.5
>99.0
>75.75
>88.88
>62.5
Amphotericin B
0.77 ± 0.15
>259.74
na-not applicable.
a
Values in
Selectivity Index ¼ CC50/IC50
m
M.
b
.
7
.26e7.23 (m, 2H), 7.19e7.16 (m, 2H), 7.10 (dd, J ¼ 8.0, 1.0 Hz, 1H),
mentioned general procedure. The compound was purified by
column chromatography (ethylacetate: hexane ¼ 6:4, R 0.38). mp
ꢂ
3
170e172 C. H NMR (400 MHz, CDCl ): d 9.23 (brs, 1H), 8.15 (dd,
4
.79 (s, 1H), 3.43 (d, J ¼ 16.7 Hz, 1H), 3.33e3.30 (m, 1H), 3.22 (d,
f
1
J ¼ 16.7 Hz, 1H), 3.17e3.08 (m, 1H), 3.02e2.96 (m, 2H). IR (KBr,
ꢀ
1
þ
cm ):
n
3251, 1672, 1523, 1044. MS (ESI) m/z 416.2 (M þ H ), 418.2
J ¼ 8.1, 0.8 Hz, 1H), 7.59 (dd, J ¼ 6.1, 2.9 Hz, 1H), 7.42e7.38 (m, 5H),
7.30 (brs, 1H), 7.25e7.15 (m, 5H), 7.05 (td, J ¼ 7.5, 1.1 Hz, 1H), 4.77 (s,
1H), 3.52 (d, J ¼ 16.5 Hz, 1H), 3.42e3.37 (m, 1H), 3.21e3.10 (m, 2H),
3þ
(
M þ H ). Anal. Calcd for C25
3
H22ClN O: C, 72.19; H, 5.33; N, 10.10.
Found: C, 72.15; H, 5.36; N, 10.07.
ꢀ
1
3
.01e2.94 (m, 2H), 2.29 (s, 3H). IR (KBr, cm ): 3286, 1664, 1531,
þ
4.1.11. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
1456. MS (ESI) m/z 396.3 (M þ H ). Anal. Calcd for C26
25 3
H N O: C,
N-(3-nitrophenyl)acetamide (7j)
78.96; H, 6.37; N, 10.62. Found: C, 78.92; H, 6.40; N, 10.60.
Compound 7j was prepared in 68% yield as white solid from 3
and 2-Chloro-N-(3-nitrophenyl)acetamide (4j) according to the
above mentioned general procedure. The compound was purified
4.1.14. N-(2-Chlorophenyl)-2-(3,4-dihydro-1-phenyl-1H-pyrido
[3,4-b]indol-2(9H)-yl)acetamide (7m)
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.28).
Compound 7m was prepared in 70% yield as white solid from 3
and 2-Chloro-N-(2-chlorophenyl)acetamide (4m) according to the
above mentioned general procedure. The compound was purified
ꢂ
1
mp 156e158 C. H NMR (400 MHz, CDCl ): d 9.49 (brs, 1H), 8.34 (t,
3
J ¼ 2.1 Hz, 1H), 7.98e7.92 (m, 2H), 7.6e7.59 (m, 1H), 7.50 (t,
J ¼ 8.2 Hz, 1H), 7.41e7.37 (m, 6H), 7.26e7.24 (m, 1H), 7.20e7.17 (m,
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.32).
ꢂ
3
mp 156e158 C. H NMR (400 MHz, CDCl ): d 9.99 (brs, 1H), 8.48
1
2
3
H), 4.82 (s, 1H), 3.48 (d, J ¼ 16.8 Hz, 1H), 3.37e3.27 (m, 2H),
ꢀ
1
.17e3.12 (m, 1H), 3.06e2.91 (m, 3H). IR (KBr, cm ):
n
3288, 1672,
(dd, J ¼ 8.3, 1.5 Hz, 1H), 7.60e7.58 (m, 1H), 7.48e7.46 (m, 2H),
7.41e7.37 (m, 4H), 7.30e7.28 (m, 2H), 7.24e7.22 (m, 1H), 7.18e7.15
(m, 2H), 7.07e7.03 (m, 1H), 4.78 (s, 1H), 3.51 (d, J ¼ 16.6 Hz, 1H),
3.89e3.34 (m, 1H), 3.30e3.16 (m, 2H), 3.01e2.93 (m, 2H). IR (KBr,
þ
1
535, 1454, 1350. MS (ESI) m/z 427.3 (M þ H ). Anal. Calcd for
25 22 4 3
C H N O : C, 70.41; H, 5.20; N, 13.14. Found: C, 70.44; H, 5.18; N,
13.10.
ꢀ
1
þ
cm ):
n
3234, 1672, 1519, 1052. MS (ESI) m/z 416.1 (M þ H ), 418.1
3
þ
4.1.12. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
(M þ H ). Anal. Calcd for C25
3
H22ClN O: C, 72.19; H, 5.33; N, 10.10.
N-(2-methoxyphenyl)acetamide (7k)
Found: C, 72.21; H, 5.30; N, 10.09.
Compound 7k was prepared in 68% yield as white solid from 3
and 2-Chloro-N-(2-methoxyphenyl)acetamide (4k) according to
the above mentioned general procedure. The compound was pu-
4.1.15. N-(3-(Trifluoromethyl)phenyl)-2-(3,4-dihydro-1-phenyl-
1H-pyrido [3,4-b]indol-2(9H)-yl) acetamide (7n)
rified by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
Compound 7n was prepared in 72% yield as white solid from 3
and 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide (4n) ac-
cording to the above mentioned general procedure. The compound
was purified by column chromatography (ethylacetate:
ꢂ
1
0
8
.36). mp 106e108 C. H NMR (400 MHz, CDCl
3
):
d
9.91 (s, 1H),
.37 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.60 (dd, J ¼ 5.8, 3.0 Hz, 1H), 7.44 (dd,
J ¼ 7.5, 2.0 Hz, 2H), 7.37e7.34 (m, 4H), 7.25e7.23 (m, 1H), 7.20e7.14
ꢂ
1
(
(
1
(
m, 2H), 7.08 (td, J ¼ 7.8, 1.6 Hz, 1H), 6.98 (td, J ¼ 7.8, 1.1 Hz, 1H), 6.92
dd, J ¼ 8.1, 1.1 Hz, 1H), 4.78 (s, 1H), 3.91 (s, 3H), 3.46 (d, J ¼ 16.6 Hz,
hexane ¼ 6:4, R
f
0.38). mp 160e162 C. H NMR (400 MHz, CDCl ):
7.56e7.53 (m, 1H), 7.35e7.30 (m, 5H), 7.27e7.19 (m, 8H), 7.16e7.13
3
d
H), 3.37e3.33 (m, 1H), 3.19e3.12 (m, 2H), 2.97e2.92 (m, 2H). IR
(m, 2H), 4.61 (s, 1H), 3.24 (d, J ¼ 16.5 Hz, 1H), 3.09e3.06 (m, 1H),
ꢀ1
þ
KBr, cm ):
n
3305, 1666, 1537, 1282. MS (ESI) m/z 412.3 (M þ H ).
2.99 (d, J ¼ 16.5 Hz, 1H), 2.86e2.82 (m, 1H), 2.79e2.73 (m, 2H). IR
ꢀ
þ
1
Anal. Calcd for C26
25
H N
3
O
2
: C, 75.89; H, 6.12; N, 10.21. Found: C,
(KBr, cm ):
n
3342, 1658, 1519, 1454, 972. MS (ESI) m/z 450.1
7
5.88; H, 6.10; N, 10.22.
(M þ H ). Anal. Calcd for C29
25 3
H N O: C, 69.48; H, 4.93; N, 9.35.
Found: C, 69.52; H, 4.88; N, 9.32.
4.1.13. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
N-o-tolylacetamide (7l)
4.1.16. N-(2,4-dimethylphenyl)-2-(1-phenyl-3,4-dihydro-1H-pyrido
Compound 7l was prepared in 74% yield as white solid from 3
and 2-Chloro-N-o-tolylacetamide (4l) according to the above
[3,4-b]indol-2(9H)-yl)acetamide (7o)
ꢂ
Compound 7 was prepared in 70% yield as white solid from 3

820
P. Ashok et al. / European Journal of Medicinal Chemistry 123 (2016) 814e821
ꢂ
and 2-Chloro-N-(2,4-dimethylphenyl)acetamide (4 ) according to
added at different concentrations for 72 h and the fluorescence
emission signal was recorded at 700 nm channel in an infrared
imager Odyssey (LI-COR) [39].
the above mentioned general procedure. The compound was pu-
rified by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
ꢂ
1
0
.34). mp 88e90 C. H NMR (400 MHz, CDCl
3
):
d
9.15 (brs, 1H), 7.96
(
7
d, J ¼ 8.1 Hz, 1H), 7.58 (dd, J ¼ 6.1, 2.9 Hz, 1H), 7.41e7.37 (m, 5H),
4.2.3. Cytotoxicity
.31 (brs, 1H), 7.24e7.22 (m, 1H), 7.18e7.14 (m, 2H), 7.04e7.00 (m,
To determine the cytotoxicity of the reported analogues in
mammalian cells, the alamar blue staining method was used on
drug-exposed splenocytes derived from uninfected mice, according
to manufacturer's recommendations. The animal research
described in this manuscript was complied with Spanish (Ley 32/
2007) and European Union Legislation (2010/63/UE). The used
protocols were approved by the Animal Care Committee of Uni-
versidad de Le oꢀ n (Spain).
2
H), 4.77 (s, 1H), 3.50 (d, J ¼ 16.5 Hz, 1H), 3.40e3.36 (m, 1H),
3
.19e3.10 (m, 2H), 3.00e2.91 (m, 2H), 2.30 (s, 3H), 2.25 (s, 3H). IR
ꢀ1
(
KBr, cm ):
n
3302, 1687, 1519, 1454, 742. MS (ESI) m/z 410.5
O: C, 79.19; H, 6.65; N, 10.26; O,
þ
(
3
M þ H ). Anal. Calcd for C27
27 3
H N
.91. Found: C, 79.19; H, 6.65; N, 10.26; O, 3.91.
4.1.17. 2-(3,4-Dihydro-1-phenyl-1H-pyrido [3,4-b]indol-2(9H)-yl)-
N-(naphthalen-1-yl)acetamide (7p)
Compound 7p was prepared in 80% yield as white solid from 3
and 2-Chloro-N-(naphthalen-4-yl)acetamide (4p) according to the
above mentioned general procedure. The compound was purified
Acknowledgements
The authors are thankful to SAIF, Panjab University, Chandigarh
and Central Instrumentation Laboratory, Guru Jambheshwar Uni-
versity of Science and Technology, Hisar, India for providing NMR
spectra in this study. The authors are also grateful to SAIF, CSIR-
CDRI, Lucknow, India for Mass spectral data. One of the author,
Ashok Penta, acknowledges Council of Scientific and Industrial
Research, New Delhi, India for financial support in the form of Se-
nior Research Fellowship. This research was supported by Minis-
terio de Economía y CompetitividadMinisterio de Economía y
Competitividad (MINECO; AGL2010-16078/GAN and CYTED
214RT0482).
by column chromatography (ethylacetate: hexane ¼ 6:4, R
f
0.38).
9.91 (brs, 1H), 8.25
dd, J ¼ 7.6, 0.8 Hz, 1H), 7.91e7.89 (m, 1H), 7.83e7.80 (m, 1H), 7.68
d, J ¼ 8.2 Hz, 1H), 7.62 (dd, J ¼ 6.2, 2.7 Hz, 1H), 7.55e7.52 (m, 2H),
.50e7.47 (m, 3H), 7.42e7.39 (m, 4H), 7.27e7.25 (m, 1H), 7.22e7.18
m, 2H), 4.85 (s, 1H), 3.63 (d, J ¼ 16.6 Hz, 1H), 3.52e3.48 (m, 1H),
ꢂ
1
3
mp 160e162 C. H NMR (400 MHz, CDCl ): d
(
(
7
(
3
.32 (d, J ¼ 16.6 Hz, 1H), 3.26e3.21 (m, 1H), 3.09e3.00 (m, 2H). IR
ꢀ
1
þ
(
KBr, cm ):
n
3291, 1672, 1531, 1494. MS (ESI) m/z 432.5 (M þ H ).
25 3
Anal. Calcd for C29H N O: C, 80.72; H, 5.84; N, 9.74. Found: C,
8
4
4
0.70; H, 5.82; N, 9.76.
.2. Biological activity
Appendix A. Supplementary data
.2.1. Antipromastigote activity
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.014.
All the synthesized analytical pure compounds were used to
measure the inhibitory potency against promastigotes of infrared
fluorescent protein 1.4 (IFP1.4)-L. infantum (BCN150) strain. The
assay has been carried out using promastigotes, which were
cultured in a 96-well plate in the presence of graded concentrations
Conflict of interest
The authors declared that, conflict of interest is none.
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(
1, 10 and 100
m
M) of the test compounds, 200
m
L of a promastigote
6
culture 1 ꢃ 10 and 2
mL of the drugs per well. Plates were incu-
ꢂ
bated for 48 h at 26 C. After 48 h, growth of promastigotes was
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