Identification of a diverse indole-2-carboxamides as a potent antileishmanial chemotypes

Article abstract of DOI:10.1016/j.ejmech.2016.01.028

A novel series of highly diverse indole-2-carboxamides was synthesized utilizing the isocyanide based multicomponent reaction (IMCR)-post modification approach and were identified as potential antileishmanial chemotype. Among the synthesized 18 analogues, 12 analogues exhibited better antileishmanial activity against intracellular amastigotes form of Leishmania donovani (IC₅₀values of 0.6-7.5 μM) as compared to standard drugs miltefosine and sodium stibogluconate. The compounds were also non-toxic towards Vero cells. Compounds 2b, 2m and 2p with significant in vitro activity were then evaluated for their in vivo efficacy following intraperitoneal route. These three compounds at a concentration of 50 mg/kg/day for 5 consecutive days showed 70.0, 63.5 and 63.4% inhibition of Leishmania amastigotes, respectively at day 7 post treatment in hamster model of visceral leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2016.01.028

Accepted Manuscript
Identification of a diverse Indole-2-carboxamides as a potent antileishmanial
chemotypes
Shashi Pandey, Shikha S. Chauhan, Rahul Shivahare, Abhisheak Sharma, Swati
Jaiswal, Suman Gupta, Jawahar Lal, Prem M.S. Chauhan
PII:
S0223-5234(16)30029-0
10.1016/j.ejmech.2016.01.028
EJMECH 8318
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 April 2015
Revised Date: 15 January 2016
Accepted Date: 16 January 2016
Please cite this article as: S. Pandey, S.S. Chauhan, R. Shivahare, A. Sharma, S. Jaiswal, S. Gupta,
J. Lal, P.M.S. Chauhan, Identification of a diverse Indole-2-carboxamides as a potent antileishmanial
chemotypes, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.028.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Identification of a diverse Indole-2-carboxamides as a potent antileishmanial
chemotypes
Shashi Pandey,a Shikha S. Chauhan,a Rahul Shivahare,b Abhisheak Sharma,c, d Swati Jaiswal,c, d
Suman Gupta,b Jawahar Lalc, d and Prem M. S. Chauhana*
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
bDivision of Parasitology, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
c
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
d
Academy of Scientific and Innovative Research, New Delhi, India

ACCEPTED MANUSCRIPT
Identification of a diverse Indole-2-carboxamides as a potent antileishmanial
chemotypes
Shashi Pandey,a Shikha S. Chauhan,a Rahul Shivahare,b Abhisheak Sharma,c, d Swati Jaiswal,c, d
Suman Gupta,b Jawahar Lalc, d and Prem M. S. Chauhana*
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
bDivision of Parasitology, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
c
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
d
Academy of Scientific and Innovative Research, New Delhi, India
Abstract: A novel series of highly diverse indole-2-carboxamides was synthesized utilizing the
isocyanide based multicomponent reaction (IMCR)-post modification approach and were
identified as potential antileishmanial chemotype. Among the synthesized 18 analogues, 12
analogues exhibited better antileishmanial activity against intracellular amastigotes form of
Leishmania donovani (IC50 values of 0.6 - 7.5 ꢀM) as compared to standard drugs miltefosine
and sodium stibogluconate. The compounds were also non-toxic towards Vero cells. Compounds
2b, 2m and 2p with significant in vitro activity were then evaluated for their in vivo efficacy
following intraperitoneal route. These three compounds at a concentration of 50 mg/kg/day for 5
consecutive days showed 70.0, 63.5 and 63.4 % inhibition of Leishmania amastigotes,
respectively at day 7 post treatment in hamster model of visceral leishmaniasis.
Keywords: Leishmania donovani, Hamster model, Indole-2-Carboxamide, Multicomponent
reaction.
*Corresponding Author Tel.: 91 522 2612411; fax: +91 522 2623405, E-mail addresses:
prem_chauhan_2000@yahoo.com; premsc58@hotmail.com.
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1. Introduction
Leishmaniasis is one of the most devastating neglected tropical diseases representing the serious
public health problems in developing countries [1]. Leishmaniasis is second only to malaria in
terms of the worldwide burden inflicted by protozoan parasitic diseases [2]. According to WHO
estimations, there are almost 1.3 million new cases of leishmaniasis and 20,000 to 30,000 deaths
annually [3]. This disease is caused by the protozoan parasites of the genus Leishmania and
transmitted to the humans by the bite of female phlebotomine sandfly. It may manifest in three
clinical forms, cutaneous, muco-cutaneous and visceral leishmaniasis (VL). Among all, VL (also
known as Kala-azar) caused by Leishmania donovani (India and Near East) and L. infantum
(around the Mediterranean Sea), is the most severe form of the disease in which vital organs of
the body viz. liver, spleen and bone marrow are affected [4]. Currently, no effective vaccines are
available [5], therefore the current treatment of leishmaniasis still relies on handful number of
drugs such as, antimony based drugs, meglumine antimoniate (Glucantime), sodium
stibogluconate (Pentostam), paromomycin, amphotericin B, and miltefosine [6]. Since its
discovery, pentavalent antimonials have been used extensively as first line drug for the treatment
of VL. However, requirement of high dose of parenteral administration, resistance issues, long-
term treatment and severe side effects including cardiac arrhythmia and pancreatitis restricted the
use of first line therapy in affected regions [7]. Furthermore, the second line drugs, pentamidine
and amphotericin-B also suffer from moderate to severe side effects [8]. In the past decades,
antileishmanial chemotherapy has been noticeably improved by the introduction of new
liposomal formulation of amphotericin B, paromomycin, and serendipitous invention of
miltefosine.
Although, single dose liposomal amphotericin B is a highly effective alternative with
almost no side effect but its high cost makes this formulation unaffordable for poor people of
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developing nations [9-10]. Moreover, miltefosine, a phosphocholine analogue, is introduced as
first orally active drug for the treatment of VL. Despite the fact that this drug is effective, it has a
long half-life (150-200 h), consequently prone to resistance development and has teratogenic
effect in the women of child-bearing age [11]. The aforementioned limitations of the existing
drugs along with the development of drug resistance especially in HIV−Leishmania co-infected
patients [12] as well as the dearth of new antileishmanial drug candidates clearly reflects toward
the efforts to hunt for novel drugs based on new molecular scaffolds capable of meeting the
unmet needs in antileishmanial chemotherapy. In this regard, there is a need to develop new
medicinal chemistry approaches to find out novel lead compounds that might populate a pipeline
of new therapeutics.
Additionally, medicinal chemistry deliberately depends on robust synthetic methods
capable of creating chemical diversity to satisfy the demand for the number and the quality of
compounds for drug discovery. In this regard, multicomponent reactions (MCR) especially
IMCRs provide versatile synthetic platforms for the preparation of libraries of diverse and
complex ‘drug-like’ heterocyclic compounds. Among IMCRs, Ugi reaction has achieved
profound attention for the synthesis and discovery of new lead structures and different types of
biologically active scaffolds [13-15].
Indoles are omnipresent in natural products as well as in numerous biologically active
compounds [16-17]. Literature search revealed that various indole containing natural products
have shown potent antileishmanial activity [18-19]. Also, various indole containing scaffolds
like
[1,2,4]triazinoindole-[1,3,5]triazines
[20],
[1,2,4]triazinoindole-pyrimidine
[20],
pentamidine-aplysinopsin hybrids [21], triazino indole-quinolines [22] have been previously
synthesized in our lab that have shown potential antileishmanial activity. Moreover, we have also
synthesized analogues of coscinamide (indole containing natural products) i.e. 8,9-
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Dihydrocoscinamide B [23], indolyl glyoxylamides [24] and explored their antileishmanial
activity. While the naturally occurring coscinamides have not been reported to show any
antileishmanial activity, the synthesized analogues showed potent antileishmanial activity [23-
24].
Recently, we have developed an efficient and facile multicomponent based methodology
which involves Ugi-post modification for the preparation of diverse 1H-indole-2-carboxamide
and 1H-pyrrole-2-carboxamide scaffold [25]. Furthermore, 1H-indole-2-carboxamide and 1H-
pyrrole-2-carboxamide templates are present in natural products [26] as well as in numerous
biologically active synthetic compounds. These scaffolds are also associated with broad range of
biological activities [27-29]. However, to the best of our knowledge; the antileishmanial activity
of these bioactive pharmacophores has not been reported to date.
Inspired by these encouraging facts and as a part of our ongoing interest toward the
design and synthesis of novel heterocycles as antileishmanial agent, [19-24, 30-31] we have
synthesized a series of diverse indole-2-carboxamide derivatives and screened them for
antileishmanial activity.
2. Results and discussion
2.1. Chemistry
The highly functionalized indole-2-carboxamides and pyrrole-2-carboxamides were synthesized
using our previously developed [25] two-step procedure as outlined in Scheme 1. In first step,
triethylamine was added to a solution of amino ester hydrochloride in methanol and the resulting
solution was stirred at room temperature for 10 min. To the stirred solution, corresponding
aldehyde, 1H-indole-2-carboxylic acid and isocyanide were successively added that produced
corresponding indole-fused diketopiperazines 1(a-f) in good to excellent yields. In the next step,
compound 1 was refluxed with various amines in ethanol to afford highly functionalized
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carboxamides 2(a-r) in good to excellent yields. It is noteworthy here that the final products
were obtained by subsequent regioselective ring opening of diketopiperazine unit 1 via an
intermolecular transamidation reaction under mild condition. Moreover, our synthetic strategy is
amenable for the quick and easy synthesis of a library of highly diverse analogues.
2.2. Biological assay
2.2.1. In vitro antileishmanial activity and cytotoxicity
In the endeavor to identify the activity of indole-2-carboxamides, we have synthesized a series of
18 compounds and screened them against intracellular amastigote form of L. donovani and their
cytotoxicity was assessed on mammalian kidney fibroblast cells (Vero cell line). The results are
summarized in Table 1. We have also tested the compounds for their cytotoxicity on murine
macrophages (J-774A.1 cell line) and results are depicted in supplementary Table S1. Most of
the synthesized compounds displayed excellent antiamastigote activity that was manifold better
than the activity of standard drug, miltefosine. Initially, we synthesized derivatives 2(a-d) with
R1 as p-chloro and 2(e-g) with R1 as 3, 4, 5-trimethoxy and different substitutions at R2. Among
these two groups of synthesized compounds, compound 2b with p-chloro substituent at R1 was
found to be most active. Encouraged with the good activity profile of compound 2b, we
undertook the synthesis of a variety of structural analogues with p-chloro group retained at R1
and variation at R and R2 in the hope to improve the activity profile. The structure-activity
relationship studies of these compounds suggested that the activity of these compounds were
greatly influenced by the substituent at R2. In case of aromatic substitution at R2, the activity of
compounds strongly depended on the substitution of benzyl ring at R2. Analogue 2a with
unsubstituted benzyl ring and analogues 2c and 2d, with p-chloro and 3,4 dichloro substituted
benzyl ring showed moderate antiamastigote activity, while analogue 2b with an p-
methoxybenzyl group showed potent activity with IC50 = 1.2 µM. In case of aliphatic substituent,
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the introduction of ethanol group at R2 in compound 2i resulted in complete loss in
antileishmanial activity, whereas analogue 2j having 3-propyl morpholine at R2 exhibited potent
antileishmanial activity against amastigotes with IC50 = 2.1 µM which was 4 fold more active
than standard drug miltefosine. Moreover, both the compounds (2i and 2j) were non-toxic
towards Vero cells (CC50 = 225.5 and 211.8 µM, respectively). These results suggested that
presence of free polar group (here hydroxyl) in 2i had detrimental effect on the in vitro
antiamastigote activity. Introduction of 4-methylpyridine as heterocyclic aromatic moiety at R2
in analogue 2k demonstrated a slight decreased antileishmanial activity. Further, we replaced the
4-methoxy group in 2b by 3-methoxy group in 2l and we found that the compound 2l also
showed good activity with IC50 = 1.9 µM. Our SAR studies indicated that the introduction of
methoxy substituent at benzyl ring of R2 plays a crucial role in the antiamastigote activity.
Instigated with the potent activity profile and selectivity index of halogen containing
analogues having p-chloro group at position R1, we synthesized analogues 2m and 2n with p-
bromo group at position R1. Both these synthesized compounds also showed excellent
antiamastigote activity with IC50 values of 1.0 and 1.5 ꢀM, respectively as compared to standard
drug miltefosine and were non-toxic towards Vero cells (CC50 > 400 ꢀM). Furthermore, these
compounds also showed very high selective index (SI = >400 and >266, respectively) that was
66 and 44 fold more than standard drug miltefosine. Next, replacement of tertiary butyl group of
2b with 1,1,3,3-tetramethyl butyl group at position R in 2o resulted in slight decrease in
antileishmanial activity. In view of the fact, that the compounds 2b, 2n and 2o having 4-methoxy
benzyl group at R2 showed potent activity with high selectivity, we designed compounds 2p-2r
with 4-methoxy intact at position R1 and variations at R2. Interestingly, among all the
synthesized analogues in present series, compound 2p with R2 as 4-methoxybenzyl group, was
found to be the most potent with IC50 value of 0.6 µM which is 14-fold more active than
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miltefosine (IC50 = 8.4 µM). Additionally, compound 2p was non-toxic to Vero cells (CC50
=
>400 ꢀM) and has high selectivity (SI >666). Compound 2q with 4-chlorobenzyl group exhibited
moderate antiamastigote activity (IC50 = 7.50 ꢀM) while compound 2r with pyridine-4-ylamine
group showed potent antiamastigote activity (IC50 = 1.4 ꢀM).
2.2.2. In vivo antileishmanial activity in hamster model
Encouraged with the results of these initial set of analogous, we selected the best three
compounds (2b, 2m and 2p) on the basis of their in vitro potency for further evaluation
accordingly; in vivo tests were performed in the L. donovani/golden hamster model. The aqueous
solutions of test compounds were administered for five consecutive days at 50 mg/kg/day by
intraperitoneal (ip) route. The post-treatment (p.t.) splenic biopsies were done on day 7 of the last
dose administration and amastigote counts were assessed by Giemsa staining. All the test
compounds 2b, 2m and 2p showed good percentage inhibition of 70.0 ± 13.3%, 63.5 ± 10.3 and
63.4 ± 8.2 %, respectively in Leishmania parasite multiplication (Figure 1). Furthermore, we
have not observed any abnormal behavior of animals at the time of dosing.
2.2.3. Cell death analysis
In order to confirm the mode of killing of Leishmania parasite by the action of best active
compound 2b, we have performed cell death analysis experiment using annexin V- FITC
(stained apoptotic cells) and propidium iodide (stained necrotic cells). In our results, we have
found that this derivative has potential to kill the parasite by the apoptotic way of death.
Leishmania promastigotes were incubated with compound 2b for various time points (12-72h),
labeled with annexin V- FITC and propidium iodide and analyzed by flow cytometry.
Flow cytometric analysis revealed that after 12, 24 and 48 h incubation with compound 2b (at
IC50 concentration 0.9 ꢀM against promastigotes) [32], 49.10, 56.17 and 73.30% of
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promastigotes, respectively, stained positive for annexin V as shown in lower right quadrant
(Figure 2B-D) as compared to untreated promastigotes, where cells were unstained with both the
dyes (Figure 2A). The maximum phosphatidylserine exposure was observed at 72 h incubation
(Figure 2E), where 79.07% promastigotes were in early apoptotic phase. Almost negligible cells
were stained with propidium iodide, indicating that killing mode of promastigotes by compound
2b via apoptosis.
2.2.4. Preliminary pharmacokinetic studies
The compelling in vitro and in vivo antileishmanial activity exhibited by compound 2b
encouraged us to perform in vivo pharmacokinetic studies of compound 2b. Pharmacokinetic
studies revealed that the animals tolerated the treatment, as no peculiarities in the behavior of
animals were observed. Calculations of the pharmacokinetic parameters were based on the mean
serum concentrations and were performed by use of the noncompartmental approaches using
Phoenix WinNonlin, version 6.3 (Certara Inc, Missouri, USA). Following per oral (po, 50
mg/kg) and intravenous (5 mg/kg) administration, the compound was detected in serum upto 24
h. After po dose, it was rapidly absorbed (tmax1, 1 h) and slowly eliminated (MRT, 8.5 h) with
multiple peak phenomenon (Figure 3 and Table 2). It may be due to the delayed gastric
emptying, enterohepatic recirculation and variability of absorption in different regions of
gastrointestinal tract [33]. The compound possesses adequate oral bioavailability (34.9 %) with
an average Cmax 2852.6 ng/ml. The volume of distribution (4.4 L/kg) is higher than the total
blood volume (0.064 L/kg; Nagarjun and Lal) of the rat and systemic clearance (0.6 L/h/Kg) is
lower than the total hepatic blood flow in rats (2.9 L/h/kg) [34] indicating extravascular
distribution with negligible extrahepatic elimination. Hence, the pharmacokinetic parameters
support the good oral exposure of the compound to exhibit antileishmanial activity.
3. Conclusion
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In conclusion, we have synthesized highly functionalized indole-2-carboxamide via Ugi-post
transformation approach. Biological evaluation of these new chemical entities revealed that most
of the synthesized compounds exhibited better potency against the intracellular amastigote form
of L. donovani than the standard drug miltefosine and SSG in the in vitro system and were not
found to be cytotoxic. The lead compound 2b was found to be 7-fold more active than
miltefosine against the amastigotes and eradicated the parasites via apoptotic mode of killing.
Significant inhibition of parasites in hamster model of VL and pharmacokinetic parameters also
supports the candidature of compound 2b as a potential antileishmanial lead. Our findings
underscore the importance of the indole-2-carboxamides as an important lead scaffold in the
design and synthesis of antileishmanial agents.
4. Experimental Methods
4.1. General information: All commercially available starting materials and solvents were
reagent grade, and used without further purification. Reactions were carrying out under dry
glassware with magnetic stirring. IR spectra were recorded on a FTIR spectrophotometer
Shimadzu 8201 PC and are reported in terms of frequency of absorption (cm-1). 1H NMR and 13C
NMR spectra were recorded on Bruker Supercon Magnet Avance 400 and 300 spectrometers
using TMS as an internal reference and the samples were dissolved in suitable deuterated
solvents (Chemical shifts (δ) are given in ppm relative to TMS and coupling constants (J) in Hz).
HR-DART MS were recorded on JEOL, JMS T100LC Accu TOF. Melting points were
determined in open capillary tubes on an electrically heated block and were uncorrected. Thin-
layer chromatography (TLC) was carried out with silica gel plates (silica gel 60 F254), that were
visualized by exposure to ultraviolet light. Purity of final compounds was determined by
analytical HPLC, which was carried out on a Waters HPLC system (model pump: 515, detector
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PDA-2998). HPLC analysis conditions: Thermo ODS-2 Hypersil C18 (5.0µM), 4.6x250 mm
column flow rate 1.0 mL/min. All evaluated compounds are ≥ 95% pure.
4.1.1. General Procedure for the Preparation of Compound 1(a-f)
To a solution of amino ester hydrochloride (1 mmol) in methanol (3 mL) was added
triethylamine (1.1 mmol) and the resulting solution was stirred at room temperature for 10 min.
To the stirred solution were successively added corresponding benzaldehdye (1 mmol), 1H-
indole-2-carboxylic acid 4 (1 mmol) and isocyanide (1 mmol). The mixture was stirred at room
temperature for 30 min. The progress of the reaction was monitored by TLC. After completion of
the reaction, solvent was evaporated at reduced pressure and purified through column
chromatography (eluent: CHCl3/MeOH) using 100−200 mesh silicagel to afford the desired
product [25].
4.1.2. General procedure for the preparation of compound 2(a-r)
To a stirred solution of required fused diketopiperazine (1 mmol) in ethanol (5mL) corresponding
amine (1.2 mmol) was added and the reaction mixture was heated at reflux for 4-8 h. The
progress of the reaction was monitored by TLC. After completion of the reaction, solvent was
evaporated at reduced pressure and purified through column chromatography (eluent: CHCl3/
MeOH) using 100−200 mesh silica gel to afford the desired product 2(a-r). Characterization of
Compounds 2(a-h) [25].
4.2. Characterization of Compounds 2(i-r)
4.2.1. N-(2-(tert-butylamino)-1-(4-chlorophenyl)-2-oxoethyl)-N-(2-((2-hydroxyethyl)amino)-2-
oxoethyl)-1H-indole-2-carboxamide (2i)
White solid; Yield 84%; Mp: 112-113 °C; IR (KBr): 3421, 3019, 1657, 1521 cm-1; HPLC–PDA:
tr =5.04 min (% area = 99.10%); 1H NMR (400 MHz, CDCl3) δ = 9.63(br s, 2H), 7.66 (d, 1H, J =
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7.9 Hz), 7.40-7.36(m, 4H), 7.31-7.27(m, 1H), 7.13-7.10 (m, 1H), 6.90 (s, 1H), 6.09-5.52(m, 2H),
13
4.34-3.25(m, 8H), 1.27 (s, 9H); C NMR (100 MHz, CDCl3) δ = 170.2, 169.9, 164.4, 136.1,
135.9, 131.4, 131.2, 129.7, 127.8, 127.6, 125.3, 122.5, 120.8, 111.7, 107.0, 66.3, 61.7, 52.2, 51.1,
42.7, 28.3: HRMS (ESI) calcd for [C25H29ClN4O4 + H+] 485.1950; found 485.1950
4.2.2.
N-(2-(tert-butylamino)-1-(4-chlorophenyl)-2-oxoethyl)-N-(2-((3-
morpholinopropyl)amino)-2-oxoethyl)-1H-indole-2-carboxamide (2j)
White solid; Yield 81%; Mp: 98-100 °C; IR (KBr): 3441, 3030, 1647, 1516 cm-1; ; HPLC–PDA:
tr =5.64 min (% area = 95.01%); 1H NMR (400 MHz, CDCl3) δ = 9.66(s, 1H), 9.44 (br s, 1H),
7.66 (d, 1H, J = 8.0 Hz), 7.41-7.26 (m, 5H), 7.13-7.09 (m, 1H), 6.92 (s, 1H), 5.93(s, 1H), 5.49(s,
1H), 4.40-4.29 (m, 1H), 3.90-3.82(m, 1H), 3.59(s, 4H), 3.36(s, 2H), 2.28(m, 5H), 1.93(s, 2H),
13
1.68-1.65(m, 2H), 1.31 (s, 9H); C NMR (100 MHz, CDCl3) δ = 170.1, 169.7, 164.0, 153.9,
138.9, 138.2, 135.9, 128.3, 128.1, 128.0, 127.9, 127.0, 125.2, 122.8, 120.6, 111.5, 107.1, 107.0,
66.8, 60.8, 56.0, 51.9, 50.3, 43.4, 28.3; HRMS (ESI) calcd for [C30H38ClN5O4 + H+] 568.2685;
found 568.2684.
4.2.3. N-(2-(tert-butylamino)-1-(4-chlorophenyl)-2-oxoethyl)-N-(2-oxo-2-((pyridin-4-ylmethyl)
amino)ethyl)-1H-indole-2-carboxamide (2k)
White solid; Yield 74%; Mp:132-134 °C; IR (KBr): 3434, 2921, 1647, 1516 cm-1; ; HPLC–PDA:
tr =6.01 min (% area = 96.33%); 1H NMR (400 MHz, CDCl3) δ = 10.35(br s, 1H), 9.55(s, 1H),
8.37(s, 2H), 7.64 (d, 1H, J = 8.0 Hz), 7.42(d, 1H, J = 8.3 Hz), 7.35-7.29(m, 3H), 7.21(d, 2H, J =
8.0 Hz), 7.15-7.11(m, 3H), 6.88(s, 1H), 5.81(s, 1H), 5.53(s, 1H), 4.55-4.39(m, 3H), 3.80(br s,
1H), 1.27 (s, 9H); 13C NMR (100 MHz, CDCl3) δ = 169.8, 163.9, 149.6, 147.1, 136.0, 135.9,
131.1, 129.8, 127.8, 127.7, 125.5, 122.8, 122.7, 120.9, 111.6, 107.1, 66.4, 52.2, 50.6, 42.3, 28.3:
HRMS (ESI) calcd for [C29H30ClN5O3 + H+] 532.2110 ; found 532.2110.
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4.2.4. N-(2-(tert-butylamino)-1-(4-chlorophenyl)-2-oxoethyl)-N-(2-((3-methoxybenzyl)amino)-
2-oxoethyl)-1H-indole-2-carboxamide (2l)
White solid; Yield 80%; Mp: 156-158 °C; IR (KBr) : 3414, 1656, 1591, 1397 cm-1; ; HPLC–
PDA: tr = 5.38 min (% area = 96.26%); 1H NMR (400 MHz, CDCl3) δ = 9.96(br s, 1H), 9.46(s,
1H), 7.64(d, 1H, J = 7.9 Hz), 7.40(d, 1H, J = 8.2 Hz), 7.31-7.28(m, 3H), 7.17-7.10(m, 4H), 6.87-
13
6.76(m, 4H), 5.69(s, 1H), 5.49(s, 1H), 4.60-4.36(m, 3H), 3.76-3.70 (m, 4H), 1.25 (s, 9H); C
NMR (100 MHz, CDCl3) δ = 169.5, 169.3, 164.1, 135.9, 131.3, 129.7, 129.4, 127.8, 125.3,
122.7, 120.7, 113.7, 112.9, 111.6, 107.2, 66.23, 55.05, 52.0, 50.8, 43.5, 28.3; HRMS (ESI) calcd
for [C31H33ClN4O4 + H+] 561.2263; found 561.2261.
4.2.5.
N-(1-(4-bromophenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(2-((3-
morpholinopropyl)amino)-2-oxoethyl)-1H-indole-2-carboxamide (2m)
White solid; Yield 82%; Mp:134-136 °C; IR (KBr): 3425, 3020, 1661, 1398 cm-1; HPLC–PDA:
1
tr = 6.72 min (% area = 99.16 %); H NMR (400 MHz, CDCl3) δ = 9.66(s, 1H), 9.43(br s, 1H),
7.66 (d, 1H, J = 8.0 Hz), 7.56(d, 2H, J = 7.3 Hz), 7.41(d, 1H, J = 8.2 Hz), 7.30-7.27(m, 2H),
7.13-7.09 (m, 1H), 6.92 (s, 1H), 5.93(s, 1H), 5.48(s, 1H), 4.30-4.26 (m, 1H), 3.85-3.83(m, 1H),
3.59(s, 4H), 3.36(s, 2H), 2.28(s, 5H), 1.88(s, 2H), 1.70-1.65(m, 2H), 1.31 (s, 9H); 13C NMR (100
MHz, CDCl3) δ = 169.3, 164.1, 135.9, 132.57, 131.5, 127.9, 125.3, 124.0, 122.7, 120.7, 111.6,
107.0, 66.9, 66.6, 56.4, 53.5, 52.1, 51.1, 38.0, 28.4, 25.6; HRMS (ESI) calcd for [C30H38BrN5O4
+ H+] 612.2180 ; found 612.2181.
4.2.6. N-(1-(4-bromophenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(2-((4-methoxybenzyl)amino)-
2-oxoethyl)-1H-indole-2-carboxamide (2n)
White solid; Yield 88%; Mp:128-130 °C; IR (KBr): 3427, 3020, 1660, 1516 cm-1; HPLC–PDA:
tr = 5.49 min (% area = 99.34%) 1H NMR (300 MHz, CDCl3) δ = 9.82 (br s, 1H), 9.55 (s, 1H),
12

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7.63 (d, 1H, J = 7.8 Hz), 7.43-7.38(m, 3H), 7.31-7.27(m, 1H), 7.22(d, 2H, J = 6.7 Hz), 7.14-7.06
(m, 3H), 6.85 (br s, 1H), 6.78 (d, 2H, J = 8.4 Hz), 5.78 (s, 1H), 5.49 (s, 1H), 4.53-4.29 (m, 3H),
3.75-3.73 (m, 4H), 1.24 (s, 9H); 13C NMR (100 MHz, CDCl3) δ = 169.4, 169.1, 164.4, 158.7,
136.0, 132.5, 131.6, 130.3, 129.6, 127.8, 127.6, 125.1, 123.9, 122.5, 120.6, 113.7, 111.7, 107.0,
65.8, 55.1, 51.8, 50.8, 42.9, 28.2; HRMS (ESI) calcd for [C31H33BrN4O4 + H+] 605.1758; found
605.1757.
4.2.7.
N-(1-(4-chlorophenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-N-(2-((4-
methoxybenzyl)amino)-2-oxoethyl)-1H-indole-2-carboxamide (2o)
White solid; Yield 78%; Mp: 205-207 °C; IR (KBr): 3422, 3019, 1662, 1384 cm-1; HPLC–PDA:
tr = 5.49 min (% area = 97.16%); 1H NMR (400 MHz, CDCl3) δ = 9.89 (br s, 1H), 9.32 (s, 1H),
7.64 (d, 1H, J = 8.0 Hz), 7.39(d, 1H, J = 8.2 Hz), 7.32-7.28 (m, 3H), 7.25-7.19 (m, 4H), 7.14-
7.11 (m, 1H), 6.85 (s, 1H), 6.79(d, 1H, J = 8.3 Hz), 5.53(s, 1H), 5.31 (br s, 1H), 4.54-4.32 (m,
3H), 3.89-3.76 (m, 4H), 1.71(d, 1H, J = 13.4 Hz), 1.53(d, 1H, J = 14.7 Hz), 1.39(d, 6H, J = 14.3
13
Hz), 0.89(s, 9H); C NMR (100 MHz, CDCl3) δ = 168.9, 168.6, 163.8, 158.8, 135.8, 131.3,
130.5, 129.7, 127.9, 125.2, 122.7, 120.7, 113.8, 111.5, 107.1, 67.0, 56.3, 52.7, 51.4, 42.9, 31.4,
31.3, 28.6, 27.9; HRMS (ESI) calcd for [C35H41ClN4O4 + H+] 617.2889; found 617. 2888.
4.2.8.
N-(2-(tert-butylamino)-1-(4-methoxyphenyl)-2-oxoethyl)-N-(2-((4-
methoxybenzyl)amino)-2-oxoethyl)-1H-indole-2-carboxamide (2p)
White solid; Yield 92%; Mp: 198-200 °C; IR (KBr): 3422, 3019, 1654, 1513 cm-1; HPLC–PDA:
1
tr = 6.71 min (% area = 100.00%). H NMR (400 MHz, CDCl3) δ = 10.1(br s, 1H), 9.53 (s, 1H),
7.63 (d, 1H, J = 7.9 Hz), 7.40 (d, 1H, J = 8.2 Hz), 7.30(d, 1H, J = 7.5 Hz), 7.24(d, 2H, J = 7.9
Hz), 7.13-7.09 (m, 3H), 6.85 (br s, 1H), 6.81-6.74(m, 4H), 5.68 (s, 1H), 5.54 (s, 1H), 4.59-4.54
(m, 1H), 4.40-4.29 (m, 2H), 3.79-3.74(m, 7H), 1.24(s, 9H); 13C NMR (100 MHz, CDCl3) δ =
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170.3, 169.4, 164.2, 160.3, 158.6, 135.9, 131.3, 130.6, 129.6, 128.1, 127.8, 124.9, 124.5, 122.6,
120.5, 114.8, 113.6, 111.6, 106.9, 66.0, 55.1, 51.7, 50.4, 42.8, 28.2; HRMS (ESI) calcd for
[C32H36N4O5 + H+] 557.2758; found 557.2758.
4.2.9. N-(2-(tert-butylamino)-1-(4-methoxyphenyl)-2-oxoethyl)-N-(2-((4-chlorobenzyl)amino)-
2-oxoethyl)-1H-indole-2-carboxamide (2q)
White solid; Yield 89 %; Mp: 130-132 °C; IR (KBr): 3420, 3020, 1658, 1610, 1516 cm-1;
HPLC–PDA: tr = 5.47 min (% area =96.69 %); 1H NMR (400 MHz, CDCl3) δ = 10.35 (br s, 1H),
9.53 (s, 1H), 7.63 (d, 1H, J = 8.0 Hz), 7.42 (d, 1H, J = 8.3 Hz), 7.31-7.27 (m, 1H), 7.24-7.22 (m,
2H), 7.18-7.09 (m, 5H), 6.84 (s, 1H), 6.81 (d, 2H, J = 8.3 Hz), 5.69 (s, 1H), 5.56(br s, 1H), 4.61-
4.56 (m, 1H), 4.42-4.29 (m, 2H), 3.80-3.67(m, 4H), 1.24(s, 9H); 13C NMR (75 MHz, CDCl3) δ =
170.4, 169.7, 164.1, 160.4, 136.9, 135.9, 132.8, 131.2, 129.7, 128.3, 128.0, 127.8, 125.1, 124.3,
122.6, 120.6, 114.8, 111.6, 106.9, 66.1, 55.2, 51.8, 50.4, 42.7, 28.2; HRMS (ESI) calcd for
[C31H33ClN4O4 + H+] 561.2263; found 561.2263.
4.2.10.
N-(2-(tert-butylamino)-1-(4-methoxyphenyl)-2-oxoethyl)-N-(2-oxo-2-((pyridin-4-
ylmethyl) amino)ethyl)-1H-indole-2-carboxamide (2r)
White solid; Yield 76 %; Mp: 212-213 °C; IR (KBr): 3421, 3019, 1650, 1513 cm-1; HPLC–PDA:
tr = 7.45 min (% area = 95.05 %); 1H NMR (400 MHz, DMSO-d6) δ = 11.69(br s, 1H), 9.20-
8.77(m, 1H), 8.46-8.25(m, 2H), 7.58-7.46(m, 2H), 7.25-7.21(m, 3H), 7.08-6.70(m, 5H), 6.08-
5.85(m, 1H), 4.37-4.19(m, 2H), 3.96-3.97(m, 1H), 3.78(s, 3H), 1.28(s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ = 169.3, 163.2, 158.7, 148.8, 147.2, 135.5, 130.9, 130.1 129.2, 127.8, 126.4, 126.1,
123.3, 121.7, 121.1, 119.4, 113.5, 111.7, 103.6, 62.9, 54.6, 49.9, 49.2, 40.7, 27.8; HRMS (ESI)
calcd for [C30H33N5O4 + H+] 528.2605; found 528.2605.
4.3. Biological assay
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4.3.1. In vitro antiamastigote assay
For assessing the activity of compounds against the amastigote stage of parasite, mouse
macrophage cell line (J-774A.1) infected with WHO reference strain (MHOM/IN/80/Dd8) of
promastigote (expressing luciferase firefly reporter gene) was used. Macrophages were seeded in
a 96 well plate (4 x 104/mL/100µL/well) in RPMI-1640 containing 10% Foetal calf serum and
the plates were incubated at 37 ºC in a 5% CO2 incubator. After 24 h, the medium was replaced
with fresh medium containing stationary phase promastigotes (4 x 105/mL/100µL/well).
Promastigotes invade the macrophage and are transformed into amastigotes. Each well of the
plate was washed with plain RPMI medium after 24 h of incubation to remove the un-
internalized promastigotes. The test compounds were added at two fold dilutions up to 7 points
in complete medium starting from 40µM concentration after replacing the previous medium and
the plates were incubated at 37ºC in a CO2 incubator for 72 h. After incubation, the drug
containing medium was aspirated and 50 µL PBS was added in each well and mixed with an
equal volume of Steady Glo reagent. After gentle shaking for 1–2 min, the reading was taken in
a luminometer[32]. The values are expressed as relative luminescence units (RLU). Data were
transformed into a graphic program (Excel). IC50 of antileishmanial activity was calculated by
nonlinear regression analysis of the concentration response curve using the four parameter Hill
equations.
4.3.2. Cytotoxicity assay
The cell viability was determined using the MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay [35]. As described earlier, mammalian fibroblast cells (Vero cell
line) and murine macrophage cells (J-774A.1) (1 x 105 cells/mL/100µL/well) were incubated
with test compounds at 7 concentrations starting from 400µM. After 72 h of incubation, 25 ꢀL
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of MTT reagent (5mg/mL) in PBS medium was added to each well and incubated at 37°C for 2
h. At the end of the incubation, the supernatant were removed and 150 µL of pure DMSO was
added to each well. After 15 min of incubation, the readings were recorded as absorbance at 544
nm on a micro plate reader. Fifty percent cytotoxic concentration (CC50) values were estimated
as described by Huber & Koella (1993) [36]. The selectivity index (SI) for each compound was
calculated as ratio between, cytotoxicity on Vero cells (CC50) and activity (IC50) against
Leishmania amastigotes.
4.3.3. In vivo trial in L. donovani / hamster model
The in vivo antileishmanial activity was determined in golden hamsters (Mesocricetus auratus)
infected with MHOM/IN/80/Dd8 strain of Leishmania donovani [37]. Golden hamsters (Inbred
strain) of either sex weighing 40–45g were infected intracardiacally with 1x 107 amastigotes per
animal. After establishment of infection in 15 days, pre-treatment spleen biopsy in all the
animals was carried out to assess the degree of infection. The animals with +1 infection (5–10
amastigotes/ 100 spleen cell nuclei) were included in the studies. Five to six infected animals
were randomized into several groups used for each test compound. Drug treatment by
intraperitoneal or oral route was initiated after two days of biopsy and continued for five
consecutive days. SSG and miltefosine are used as reference drugs. Post-treatment biopsies were
done on day 7 of the last dose administration and amastigote counts were assessed by Giemsa
staining method [36]. Intensity of infection in both, treated and untreated animals, and also the
initial count in treated animals was compared and the efficacy was expressed in terms of
percentage inhibition (PI) using the following formula:-
PI = 100 – [(ANAT x 100)/ (INAT x TIUC)]
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Where PI is Per cent Inhibition of amastigotes multiplication, ANAT is Actual Number of
Amastigotes in Treated animals, INAT is Initial Number of Amastigotes in Treated animals and
TIUC is Time Increase of parasites in Untreated Control animals.
4.3.4. Double staining with annexin V-FITC and propidium iodide (Death cell analysis)
For the detection of mode of killing by annexin V-FITC and propidium iodide (PI) double
staining in Leishmania promastigotes incubated with compound, the apoptosis detection kit
(Sigma Aldrich) was used as per manufacturer’s instructions. Briefly, log phase promastigotes (1
x 106/ mL / well) were incubated with test compound for 12, 24, 48 and 72 h time points. After
incubation, washed and centrifuged promastigotes were incubated with 5 µL of annexin V- FITC
and 10 µL of propidium iodide for 30 min at room temperature. Fluorescence of cells was
determined with Cell Quest FACSCalibur (Becton Dickinson). At least 10,000 cells were
analyzed for each sample. Cells stained with the dyes, annexin V-FITC and propidium iodide
were analysed by flow cytometry using 488 nm and 536 nm excitation wavelengths and 530 nm
and 617 nm emission wavelengths on FL1 and FL2 channel, respectively [32].
4.2.5. Pharmacokinetics of 2b in rats
The pharmacokinetic studies of the compound 2b was carried out in young and healthy male
Sprague-Dawley rats weighing 250 ± 25 g. The rats were obtained from Laboratory Animal
Division of the Institute and were housed in plastic cages under standard laboratory conditions
with a regular 12 h day-night cycle. Standard pelleted laboratory chow (Goldmohar Laboratory
Animal Feed, Lipton India Ltd, Chandigarh, India) and water were allowed ad libitum. The rats
were acclimatized to this environment for at least two days before conducting the experiments.
The oral dose was administered after overnight fasting (12-16 h). The study was conducted in
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three rats per time point. In all the experiments, euthanasia and disposal of carcasses were carried
out as per the guidelines of Local Ethics Committee for animal experimentation.
Suspension formulation of the compound was prepared by triturating the compound in 2% (v/v)
tween 80 in water in a pestle with mortar. A single 50 mg/kg dose was given to conscious rats by
oral gavage in a volume of approximately 1 mL/250 g rat and the time of dosing was recorded.
Blood samples were collected at 0.5, 1, 2, 4, 6, 8, 10, 18, 24 and 48 h post dose. Solution
formulation containing 20 mg compound was prepared by dissolving the compound in dimethyl
sulfoxide, ethanol, tween 80, propylene glycol (10:20:2:48, % v/v) and normal saline (q.s.) to
obtain 10 mg/ml of 2b. The formulation was administered intravenously to conscious rats (via
the femoral vein) at a dose of 5 mg/kg. Blood was collected at 0.083, 0.33, 0.5,1, 2, 4, 6, 8, 10,
24 and 48 h post dose from each rat in microtubes (Axygen, CA, USA).
Two blood samples were withdrawn from each animal. An initial 0.5 mL blood sample
was drawn by cardiac puncture under light anesthesia followed by a sample drawn from the
inferior vena cava (terminal sample) from the dosed rats using a 24G needle and a syringe in a
clean and dry test tube. The total volume of blood collected from each rat for the first sample
was not more than 10% of the total body volume. The blood was allowed to clot, by keeping the
tube on a slant for approximately 45 min. Then it was centrifugated at 3000 rpm for 10 min at
4°C and the serum was separated into clean and neatly labeled tubes. All samples were stored at -
80°C until analysis.
Chromatographic separations and quantification of the compound was achieved by a
reverse phase HPLC method on a Discovery HS C-18 column (5 ꢀm, 150 x 4.6 mm id) preceded
with a guard column (5 ꢀm, 20 x 4.0 mm, id) packed with the same material under isocratic
condition at a flow rate of 1 ml/min. The HPLC system used in this study consisted of a pump
(LC-10AT VP with FCV-10AL VP), degasser (DGU-14A) and auto-injector (SIL-HTc, fixed
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with a 100 ꢀl loop) (Shimadzu, Japan). Eluents were monitored at 295 nm with UV-Vis multiple
wavelength detector (Shimadzu, Japan) and chromatograms were integrated using Class-VP
(version 6.12 SP5) software. The mobile phase composition was aqueous ammonium acetate
buffer (0.01M) and acetonitrile (35:65, %v/v) and it was degassed by ultrasonication for 10 min
before use. The HPLC system was equilibrated for approximately 30 min before commencement
of analysis and chromatography was carried out at ambient temperature. The lower limit of
quantification for the analytical method was 25 ng/ml of test analyte in serum. The mean and
SEM of the serum concentrations of the compound at each time point was calculated using
Microsoft Excel for Windows. Due to multiple peak phenomenons, all pharmacokinetic
parameters were calculated by noncompartmental model using WinNonlin program, version 6.3
(Certara Inc, Missouri, USA).
Acknowledgements
SP and SSC are thankful to University Grant Commission and Council of Scientific and
Industrial Research, New Delhi, India, for financial support in the form of fellowship. We are
also thankful to R. K. Purshottam and Dayanand Vishwakarma for providing the HPLC data and
the SAIF Division of CDRI, Lucknow, for providing the spectroscopic data.
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Captions
Table 1. In vitro antileishmanial activity against L. donovani amastigotes and cytotoxicity of
indole-2-carboxamides 2(a-r).
Table 2. Pharmacokinetic parameters of 2b in male Sprague Dawley rats.
Figure 1. In vivo antileishmanial efficacy in L. donovani / golden hamster model.
Figure 2. Cell death analysis using annexin V-FITC and propidium iodide (PI) double staining
in Leishmania promastigotes incubated with compound 2b.
Figure 3. Plasma concentration-time profile of 2b after single oral (50 mg/kg) and intravenous
dose (5 mg/kg) in male Sprague Dawley rats (n=3). Bar represents SEM.
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Table 1. In vitro antileishmanial activity against L. donovani amastigotes and cytotoxicity of
indole-2-carboxamides 2(a-r)
Compound
Antiamastigote
activity
IC50a (in µM)
Cytotoxicityb on Vero Selectivity Indexc
cells
SI
CC50 (in µM)
2a
2b
2c
2d
2e
2f
8.8 ± 1.0
248.4 ± 9.6
145.6 ± 9.1
>400
28
121
>36
7
1.2 ± 0.3
10.9 ± 1.3
7.5 ± 1.1
>40
52.2 ± 6.1
176.1 ± 8.4
70.3 ± 8.1
72.9 ± 5.9
73.0 ± 6.8
225.5 ± 10.3
211.8 ± 5.2
>400
<4
9.2 ± 1.2
6.3 ± 0.9
14.5 ± 1.4
>40
7
2g
2h
2i
11
5
<5
2j
2.1 ± 0.4
6.4±1.0
1.9 ± 0.6
1.0 ± 0.3
101
>62
>210
>400
2k
2l
>400
2m
>400
2n
2o
1.5 ± 0.4
3.1 ± 0.8
>400
>400
>266
>129
2p
2q
0.6 ± 0.2
7.5 ± 0.9
1.4 ± 0.6
56.1 ± 3.2
>400
399.6 ± 0.5
109.1 ± 6.7
>400
>666
53
2r
78
SSGd
>7
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MFe
8.4 ± 1.2
53.1 ± 4.5
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b
aIC50 (50 % maximum inhibitory concentration) and CC50 (50 % maximum cytotoxic concentration)
values are the average (mean ± standard deviation) of two independent assays done in duplicates,
cSelectivity Index (SI) is defined as the ratio of CC50 on vero cells to IC50 against L. donovani intracellular
amastigotes. dSSG (sodium stibo-gluconate) and eMF (miltefosine) were used as reference drugs.
Table 2. Pharmacokinetic parameters of 2b in male Sprague Dawley ratsa
Parameters
Oral
Intravenous
1
2
3
1
2
3
1589.5 ± 146.6
2280 ± 23.2
Cmax (ng/ml)
1633.1 ± 138.8
1390 ± 42.4
2852.6 ± 424.4
-
0.083
0.5
-
1
6
tmax (h)
10
AUClast (ng h/ml)
MRT (h)
27190
8.5
7786
7.4
4.4
0.6
-
Vss (L/kg)
12.8
1.5
Clearance (L/h/kg)
Bioavailability (%)
34.9
aEach value represents the average of three rats dosed orally (50 mg/kg) and intravenously (5 mg/kg);
Values of Cmax are mean ± SEM; AUClast = area under the serum concentration-time curve up to last
observation, Cmax = serum peak concentration, tmax = time to Cmax, MRT = mean residence time, Vss =
volume of distribution at steady-state
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Figure 1: In vivo antileishmanial efficacy in L. donovani / golden hamster model.
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Figure 2. Cell death analysis using annexin V-FITC and propidium iodide (PI) double staining in
Leishmania promastigotes incubated with compound 2b. (A) Untreated promastigotes, (B) 12 h
incubation, (C) 24 h incubation, (D) 48 h incubation and (E) 72 h incubation with compound 2b. The
lower left quadrant shows unstained living cells (Annexin-V and PI negative), the upper left shows
necrotic cells (Annexin-V negative and PI positive), the upper right indicates late apoptotic cells (both
Annexin-V and PI positive), and the lower right shows early apoptotic cells (Annexin-V positive and PI
negative). The data are representative of two independent experiments done in duplicates
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Figure 3. Plasma concentration-time profile of 2b after single oral (50 mg/kg) and intravenous dose (5
mg/kg) in male Sprague Dawley rats (n=3). Bar represents SEM.
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