Discovery of novel antileishmanial agents in an attempt to synthesize pentamidine - Aplysinopsin hybrid molecule

Article abstract of DOI:10.1021/jm900564x

In an attempt to synthesize pentamidine-aplysinopsin hybrid molecule 25, a lead molecule 8 (containing Z-configured aplysinopsin moiety) was identified for antileishmanial activity. Optimization of lead 8 provided 24 (containing E-configured aplysinopsin)

Full text of DOI:10.1021/jm900564x

J. Med. Chem. 2009, 52, 5793–5802 5793
DOI: 10.1021/jm900564x
Discovery of Novel Antileishmanial Agents in an Attempt to Synthesize Pentamidine-Aplysinopsin
Hybrid Molecule
Sharad Porwal,† Shikha S. Chauhan,† Prem M. S. Chauhan,*,† Nishi Shakya,‡ Aditya Verma,‡ and Suman Gupta‡
†Division of Medicinal & Process Chemistry, Central Drug Research Institute, Lucknow 226001, India, and ‡Division of Parasitology,
Central Drug Research Institute, Lucknow 226001, India
Received March 27, 2009
In an attempt to synthesize pentamidine-aplysinopsin hybrid molecule 25, a lead molecule 8
(containing Z-configured aplysinopsin moiety) was identified for antileishmanial activity. Optimization
of lead 8 provided 24 (containing E-configured aplysinopsin) possessing 10 times more activity and
401-fold less toxicity than the drug pentamidine in cell based assays. Synthesis of 24 was possible,
surprisingly, because of two innate reactivities of indole-3-carbaldehyde which provided it in diastereo-
and regio-selectively pure form without recourse to the long reaction pathway.
Introduction
B is a highly effective option; however, this drug formulation
is very expensive (US $2800 per treatment), limiting its use in
most endemic regions. Although recent clinical trials with
injectable paromomycin have shown encouraging results, an
expanded catalog of new drugs for the VL causing parasite
L. donovani is required to tackle the problem of resistance.1
In spite of some side effects of pentamidine, the broad range
of its (dicationic class of molecules in general) biological
activities,5 relatively low propensity toward the development
of resistance (resistance to pentamidine itself has never been a
significant problem in the field, despite its widespread use as a
prophylactic),6 and our previous work on this class of mole-
cules7 prompted us to develop some novel dicationic class of
molecules as potential antileishmanial agents with improved
efficacy and selectivity than pentamidine. From a literature
search, we found that aplysinopsins (a class of natural pro-
ducts possessing cyclic guanidine function)8 act on similar
biological targets (plasmepsin II and serotonin receptors) as a
dicationic class of molecules.9 Taking inspiration from a
fragment based drug discovery (FBDD) approach,10,11 we
designed a hybrid molecule 25 (Figure 1), where one amidi-
nophenoxy function of pentamidine has been replaced with
aplysinopsin.
Because of their synthesis in biological settings, small
molecule natural products have in built selectivity and pha-
macokinetic profile (necessary for a drug molecule).12 Hence,
incorporating a drug fragment with natural product may
provide molecules with better activity and less toxicity.13,14
Herein, we describe our efforts for the synthesis of hybrid
molecule 25 and its analogues and the discovery of a new class
of antileishmanial agents in this endeavor.
Chemistry. To synthesize compound 25 and its analogues,
we adopted the route depicted in Scheme 1. Direct synthesis
of aplysinposin and its subsequent coupling with the penta-
midine fragment was not feasible because of poor yield in
the synthesis of aplysinopsin and a lack of chemo selectivity
in its subsequent coupling with the petamidine fragment
[mainly p-(hydroxypentyl)benzamidine]. As we proceeded
according to Scheme 1, we encountered a problem in direct
Leishmaniasis is a vector born parasitic disease of the
tropics and subtropics which is manifested in four major
clinical forms (cutaneous leishmaniasis, mucocutaneous leish-
maniasis, visceral leishmaniasis, and post kala-azar dermal
leishmaniasis or PKDLa) depending on the causative species
of the protozoan Leishmania. In all of the above forms of
leishmaniases, visceral leishmaniasis (VL) is lethal, if left
untreated. There are ≈70,000 deaths, and 1.5 million new
cases emerge per year due to VL. Majority of VL cases
(g90%) occur in just six countries: India, Nepal, Bangladesh,
Sudan, Ethiopia, and Brazil. The situation has become com-
plicated because of the emergence of PKDL, which appears in
0-6 months after the successful curing of VL.1 The WHO has
declared VL a neglected and emerging disease.2
Antimonials are the first line of treatment options for VL,
which were discovered almost 70 years ago. These suffer from
major side effects including cardiac arrhythmia and pancrea-
titis. Besides their toxicity, treatment failure with antimonials
use has increased; some times, as high as 62% in some of the
regions.1 Second line treatment options for VL are pentami-
dine, miltefosin, and amphotericin B. However, all of these
drugs suffer from moderate to severe side effects. Pentami-
dine, an aromatic diamidine, is not active orally and can lead
to renal, pancreatic, and hepatic toxicity along with hypoten-
sion and dysglycemia.3 Miltefosine, an alkylphosphocholine,
has a long half-life (100-200 h) in humans and a low
therapeutic ratio, the characteristics that could encourage
the development of resistance. It is not suitable for use during
pregnancy because of tetratogenecity and also cause mild to
severe gastrointestinal side effects.4 Liposomal amphotericin
*To whom correspondence should be addressed. Phone: 091 522
2612411. Fax: 091 522 2623405. E-mail: prem_chauhan_2000@yahoo.
com.
a Abbreviations: TBHP, tert-Butylhyrdroperoxide; DIPEA, diiso-
propylethylamine; TBAB, tetrabutylammonium bromide; VL, visceral
leishmaniasis; PKDL, post kala-azar dermal leishmaniasis; FBDD,
fragment based drug discovery; SE: standard error.
r
2009 American Chemical Society
Published on Web 09/10/2009
pubs.acs.org/jmc

5794 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Porwal et al.
Figure 1. Hybrid of pentamidine and aplysinopsin.
Scheme 1. Compounds Formed (1-9) in an Initial Attempt to Synthesize Molecule 25a
a Reagents and conditions: (a) DMF, POCl3, 0 °C-rt. (b) p-cyanophenoxypentylbromide, toluene, NaOH (aq.), TBAB, rt. (c) ethanolamine (1 equiv.),
ethanol (absolute), 60 °C. (d) MeOH (dry), HCl (g). (e) MeOH, MeONa. (f) TBHP/ NH3 (aq.), MeOH, steel vessel. bSee Supporting Information.
condensation of 2-imino-1-methyl-5H-imizazole-4-one (creati-
nine), which occurred in poor yield and provided a mixture
of diastereomers (E- and Z-). Therefore, we first condensed
2-thio-4-imidazolidinon (2-thiohydantoin) with intermediate 9
to provide compounds 8, 13, and 14 in their Z- geometry.
Conversion of the cyano function of compound 8 to amidine
(Pinner synthesis) again proved difficult because of its high
insolubility in MeOH and the presence of many NH centers
(Scheme 1).
We tried a novel approach for the conversion of the cyano
function of 8 to amidine by first complexing it with Zn2þ
(using anhydrous ZnCl2) and then using neat amine to form
the desired amidine. But instead of getting amidine, we
observed the guanylation of amine with substrate 8 to form
compounds 1-6 (Scheme 2). Guanylation of amine was
known using HgCl2, Bi(III) salts, and Mukaiyama’s re-
agent.15 This is the first example of the use of anhydrous
ZnCl2 in guanylation of amines. In the absence of anhydrous
ZnCl2, guanylation occurred in very poor yield with many
side products.
In the second phase of our endeavor, we synthesized
various analogues of compound 8, which was found active
in antileishmanial screening. To synthesize intermediates 26
and 27 (Scheme 3) containing compounds other than the
p-cyanophenoxy moiety, we have to change the sequence of
coupling by first replacing one bromo group of 1,5-dibro-
mopentane with indole-3-carbaldehyde and then replacing
the other with piperazine and phenol, respectively, instead of
doing the opposite, which provided only dimer. Similarly,
intermediate 28 (Scheme 4) was synthesized to finally obtain
compound 18. S-methylated derivative 15 was synthesized
under controlled methylation conditions (Scheme 3).
In the third phase (Schemes 4, 5, and 6), we attempted to
synthesize other analogues of lead molecule 8 with a focus on

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5795
the modification of its cyano functional group, i.e., to incor-
porate amidine functionality in 8. Compound 21 was ob-
tained from compound 8 by oxidative amination of thiocar-
bonyl function (Scheme 1). For the synthesis of compound
19, direct reaction of hydroxylamine hydrochloride with 8
yielded an inseparable mixture of unknown compounds,
which was characterized neither by Z- nor E-isomers.
Therefore, we prepared intermediate 29 for the syn-
thesis of 19 and 24 by coupling of p-amidoximophenol with
1-bromopentylindole-3-carbaldehyde. Two regio-isomers were
formed in the reaction, but desired isomer 29 (phenolic OH-
substituted isomer) precipitated out in pure form after initial
purification by column chromatography (Scheme 4). In con-
trast to that described above, simple n-alkylation of p-amidox-
imophenol provided an inseparable mixture of two regio-
isomers. Compounds 17 and 24 were formed in exclusive
E-configuration because of the innate reactivity of indole-3-
carbaldehyde (Schemes 1 and 4, respectively).16
Amide analogue 20was synthesized(Scheme 5) by first con-
verting the cyano function of p-cyanophenoxypentyl bromide
to amide and then proceeding as described for compound 8.
Synthesis of compound 23 was started from alkylation of
NH of azaindole to form 31 followed by Vilsmeir formyla-
tion. Reverse of the sequence for the synthesis of 23 was not
feasible because of poor yield in Vilsmeir formylation of
azaindole (Scheme 6).
Scheme 2. Guanylation of Amine in an Attempt to Convert the
Cyano Function of 8 to Amidinea
Indole-3-carbaldehyde derivatives 29 and 30 (Schemes 4
and 5, respectively) behave unusually in Knoevenagel con-
densation with 2-thiohydantoin. These derivatives instead of
providing the usual Z- configured molecules, yielded almost
1:1 mixtures of E- and Z-isomers. The behavior of these
indole-3-carbaldehydes derivatives where indole NH was
substituted with p-amido- and p-amidoximo-phenoxypentyl
was surprising in comparison to that of p-cyanophenoxy-
pentyl substituted indole derivative 9, which provided only
the Z-isomer. The change of the cyano function of p-cyano-
phenoxypentylindole-3-carbaldehyde (9) to amidoxim or
amide function may have disturbed the geometry of Knoe-
venagel condensation of CH acid (2-thiohydantoin) by
interference in the transition state (TS) of the reaction.
a Reagents and conditions: (a) ZnCl2 (anhy.), R2NH2 (aliphatic
amines or anilines), neat, 80-90 °C.
Scheme 3. Synthesis of Compounds (11, 12, 15, and 16)a for Optimization of 8b
a See Scheme 1 for 10, 13, and 14. b Reagents and conditions: (a) 1,5-dibromopentane (5 equiv.), K2CO3, acetone, 60 °C. (b) DIPEA, AcCN, 80 °C.
(c) Ethanolamine (1 equiv.), ethanol (absolute), 60 °C. (d) MeI, K2CO3, acetone, 0 °C. (e) MeI, K2CO3, DMF, 70 °C.

5796 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Porwal et al.
Scheme 4. Synthesis of Compounds (18, 19, 24)a in a Further Attempt at Optimizing 8b
a See Scheme 1 for 21 and 22, Scheme 5 for 20, and Scheme 6 for 23. b Reagents and conditions: (a) K2CO3, acetone, 60 °C. (b) AcCN, K2CO3, 60 °C.
(c) 2-Thiohydantoin or 1-Me-2-thiohydantoin, ethanolamine (1 equiv.), ethanol (absolute), 60 °C.
Scheme 5. Synthesis of Compound 20 in a Further Attempt at Optimizing 8a
a Reagents and conditions: (a) NaOH (aq.), H2O2, MeOH. (b) indole-3-carbaldehyde, K2CO3, acetone, 60 °C. (c) 2-Thiohydantoin or
1-Me-2-thiohydantoin, ethanolamine (1 equiv.), ethanol (absolute), 60 °C.
Results and Discussion
luciferase activity of the drug treated parasites with that
of the untreated control (see Experimental Section). Using
the above screening protocols, we observed that compounds
2-4 have shown >90% inhibition of the promastigote stage
of parasites, but none has shown activity against the amasti-
gote stage of L. donovani. Later, compounds 5 and 6, in which
the p-cyanophenoxypentyl chain has been removed, were
screened. This change has even diminished the compounds’
anti promastigote activity. Finally, the screening of precursor
compounds 7-9 provided a hit molecule, 8, which showed
62% growth inhibition of amastigotes in infected macro-
phages at 12.5 μg/mL without showing any toxicity (Table 1).
To find new antileishmanial agents, we screened initially
formed guanylated derivatives 1-4 against the promastigote
stage of L. donovani (using firefly luciferase gene transfected
promastigotes). The structures werefirst screenedat 10 μg/mL
concentration. If more than 90% inhibition was observed,
the compounds were further screened against the intra-
cellular amastigote stage of the parasite. For the screening
at the amastigote stage of parasite, mouse macrophages were
infected with firefly luciferase expressing promastigotes,
which transformed to amastigotes in macrophages. The inhi-
bition of amastigote growth was determined by comparison of

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5797
Scheme 6. Synthesis of Compound 23 in a Further Attempt at Optimizing 8a
a Reagents and conditions: (a) DMF, POCl3, 0 °C-rt. (b) NaH, DMF, rt, under N2. (c) 2-Thiohydantoin, ethanolamine (1 equiv.), ethanol
(absolute), 60 °C.
Table 1. Preliminary Antileishmanial Screening of Initially Formed Compounds 1-9
a Percent inhibition measured at 10 μg/mL (for Prom.) and at 12.5 μg/mL (for Amat.). b Prom.: promastigote. c Amat.: intracellular amastigote. d NI:
no inhibition. e Toxicity IC50 against J774 cell line >100 μg/mL. f IC50 values are the average of two independent assays expressed as average ( standard
error.
Surprisingly, the same compound 8 was also found active in
vivo. Compound 8 showed on average 62.1 ( 9.8 (SE)%
parasite reduction in four treated hamsters at a dosage of 4 ꢀ
50 mg/kg given through the intraperitonial route. Since in
vitro parasite reduction (Table 1) and in vivo reduction (see
above) are the same, it may appear that compound 8 has
complete bioavailabity. Also, no adverse reactions were ob-
served during and post-treatment in treated hamsters, which
are in accordance with no in vitro toxicity of compound 8
(Table 1 footnote e).
From the above promising data about compound 8, wenext
proceeded to optimize this lead molecule. A series of com-
pounds 10-17 were synthesized with variation in chain length
(13 and 14), thiocarbonyl function (10, 15, and 16), cyano
(12) and phenolic moieties (11), and change in geometry (17).
These were screened for antileishmanial activity. As was

5798 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Table 2. Antileishmanial Activity of Compounds 10-17
Porwal et al.
a Percent inhibition measured at 10 μg/mL (for Prom.) and at 12.5 μg/mL (for Amat.). b Prom.: promastigote. c Amat.: intracellular amastigote. d NI: no
inhibition. e Cytotoxicity for the J774 cell line. f NT: not toxic, i.e., IC50 > 100 μg/mL. g ND: not determined (because of poor activity compared to that of
8 in the Prom. assay).
evident from Table 2, none of these compounds was found to
be as promising as compound 8. Either these showed signifi-
cantly less activity than 8 in the promastigote assay (<90% at
10 μg/mL) or were toxic compared to 8 (compounds 13 and
17). Hence, it became evident that p-cyanophenoxypentyl and
2-thiohydantoin moieties were essential for the optimum
activity of compound 8.
To enhance the activity of 8, we next focused on modifying
its cyano functionality by converting it to amide, amidoxim,
etc. We also envisaged synthesizing the 2-imino analogue of 8
toseetheeffect on activity. A seriesof compounds18-24were
synthesizedin the third phase and directly screenedagainst the
amastigote internalized in macrophages (which is more rele-
vant for drug discovery) because of the high probability of
their being active.
In this endeavor, we found that the introduction of 2-
aminopyridine, which mimics amidine function, in place of
the p-cyanophenoxy moiety in compound 8 proved to be
almost ineffective (compound 18, Table 3). This may be due
to the one carbon shortage compared to the p-amidinophe-
noxypentyl fragment of pentamidine. Conversion of the
cyano function of compound 8 to amidoxim proved to be
totally ineffective (compound 19), and conversion to amide
also did not provide any significant activity (compound 20).
Reduction in the active diastereomer may be the reason for
that since both compounds were formed as an ≈1:1 mixture of
E- and Z-isomers. There is also a possibility that structures
19 and 20 may be inherently inactive (also see below).
Surprisingly, compound 24 with pure E-configuration
proved to be the most promising compound of the series with
IC50 = 2.0 μM and SI = 53.35 for the reduction of parasite of
infected macrophages. In the intracellular amastigote assay,
compound 24 was found to be 10 times more active than the
drug pentamidine, while it showed 401 times less toxicity for
human macrophages than the above drug. There are many
examples of natural product based drug molecules which pos-
sess good bioavailability and selectivity profile (e.g., rapamy-
cin and mevastatin).12,19 Incorporation of the 2-thio analogue
of aplysinopsin (a privileged structure, which acts on similar
targets) may have provided the requisite selectivity to 24.
Replacing the indole ring of compound 8 with azaindole
(compound 23) has only improved the activity of compound
8, in all synthesized compounds with Z- configuration. There
were two other interesting findings.
First, the conversion of the 2-thio function of compound 8
to 2-imino or substituted imino (which is equivalent to
cyclic guanidine function, compounds 21 and 1-4), totally
diminished the activity. Lipophilicity of the cationic center of

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5799
Table 3. Antileishmanial Activity of Compounds 18-24
a Intracellular amastigote. b Percent inhibition at 10 μg/mL. c Cytotoxicity for KB cell line. d ND: not determined. e N/A: not applicable. f Control:
pentamidine isethionate salt. g G. Iso.: geometric isomer. h IC50 values are the average of two independent assays expressed as average ( standard error.
i Mix.: mixture of E- and Z-isomers.
diamidines has already been reported as the enhancer of
biological activity against similar species of protozoa;17 this
may be the reason for the activity enhancement of compound
8 compared to that of 21.
counterpart of 8 but formed as almost a 50:50 mixture of
E- and Z-isomers, may be inherently inactive, though further
work is necessary to confirm this.
Conclusions
Second, amidoxim containing compound 24 (possessing
E-configuration) has shown a better activity profile than its
cyano counterpart 17, which is in the same E-configuration
(88.2% versus 46.4% inhibition of the promastigote stage of
parasite at 10 μg/mL for 24 and 17, respectively). p-Amidino-
phenoxy functionality is already known as the recognition
motif for various transporters involved in pentamidine up-
take.18 The selective enhancement of parasite cell permeability
may be the reason for the better activity of 24 over 17.
Furthermore, molecule 8, which is in Z-configuration, is
active, while 17, which is almost similar in functionality
(except a Me group) but has opposite geometry, is inactive
in the promastigote assay (Tables 1 and 2). These findings
suggest that for a given geometry (E- or Z-), a particular set of
functionality is essential for bioactivity. An active molecule in
a given geometry may not be as active in the opposite
geometry. Hence, compound 19, which is the amidoxime
In conclusion, in an effort to optimize the activity of lead
molecule 8 based on the aplysinopsin pentamidine hybrid
scaffold, we found active molecules in both E- (compound 24)
and Z (compound 23)-subseries of compounds. Incorpora-
tion of pentamidine substructure to the 2-thio analogue of the
natural product aplysinopsin (compound 24) increased both
the selectivity and activity of the parent drug pentamidine.
The concept may also be useful in improving the bioactivity
profile of privileged natural product structure for other
therapeutic areas. Future studies will be directed for a more
detailed understanding of SAR (by finding suitable chemistry
to synthesize 19 as a pure Z-isomer, by variation of the
cationic and linker part of compound 24 in the E-subseries
and by replacing the benzo part of the indole ring by the
bioisostere of pyridine) to optimize the in vivo efficacy of this
class of molecules.

5800 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Porwal et al.
Experimental Section
In Vivo Assay. The in vivo leishmanicidal activity was deter-
mined in golden hamsters (Mesocricetus auretus) infected with
MHOM/IN/80/Dd8 strain of L. donovani obtained through the
courtesy of PCC Garnham, Imperial College, London (U.K.).
The method of Beveridge,22 as modified by Bhatnagar et al.,23
was used for in vivo screening. Golden hamsters (of either sex)
weighing 40-45 g were infected intracardially with 1 ꢀ 107
amastigotes per animal. The infection was well adapted to the
hamster model and establishes itself in 15-20 days. Meanwhile,
hamsters gain weight (85-95 g) and can be subjected to repeated
spleen biopsies. Pretreatment spleen biopsy in all of the animals
was carried out to assess the degree of infection. The animals
with þ1 infection (5-15 amastigotes/100 spleen cell nuclei) were
included in the chemotherapeutic trials. The infected animals
were randomized into several groups on the basis of their
parasitic burdens. Five to six animals were used for each test
sample. Drug treatment by i.p. route was initiated after 2 days of
biopsy and continued for 5 consecutive days. Post-treatment
biopsies were done on day 7 of the last drug administration, and
amastigote counts were assessed by Giemsa staining. Intensity
of infection in both treated and untreated animals, and also the
initial count in treated animals were compared, and the efficacy
was expressed in terms of percentage inhibition (PI) using the
following formula:
Promastigote Viability Assay. The L. donovani promastigotes
(MHOM/IN/Dd8; originally obtained from Imperial college,
London) were transfected with the firefly luciferase gene, and
the transfectants were maintained in medium 199 (Sigma
Chemical Co., USA) supplemented with 10% fetal calf serum
(GIBCO) and 1% penicillin (50 U/mL) and streptomycin
(50 μg/mL) solution (Sigma) under pressure of G418 (Sigma).
The in vitro effect of the compounds on the growth of promas-
tigotes was assessed by monitoring the luciferase activity of
viable cells after treatment. The transgenic promastigotes of late
log phase were seeded at 6 ꢀ 104/100 μL medium 199/well in 96-
well flat-bottomed microtiter (MT) plates (CELLSTAR) and
incubated for 72 h in medium alone or in the presence of test
dilutions of drugs in DMSO.20 Parallel dilution of DMSO was
used as controls. After incubation, an aliquot (50 μL) of
promastigote suspension was aspirated from each well of a 96-
well plate and mixed with an equal volume of Steady Glo reagent
(Promega), and luminescence was measured by a luminometer.
The values were expressed as relative luminescence unit (RLU).
The inhibition of parasitic growth is determined by comparison
of the luciferase activity of drug treated parasites with that of
untreated controls by the following general formula:
PI ¼ 100 -½ANAT ꢀ 100=ðINAT ꢀ TIUCÞꢁ
percentage inhibition ¼ ðN - n ꢀ 100Þ=N
where PI is percent inhibition of amastigote multiplication,
ANAT is actual number of amastigotes in treated animals,
INAT is initial number of amastigotes in treated animals, and
TIUC is times increase of parasites in untreated control animals.
Synthetic Chemistry. Nuclear magnetic spectra were recorded
on 200 MHz, 300 MHz, and 400 MHz spectrometers. In the case
of multiplets, the signals are reported as intervals. Signals were
abbreviated as s, singlet; d, doublet; t, triplet; m, multiplet. The
electron spray mass spectra were recorded on a triple quadru-
pole mass spectrometer. The samples (dissolved in suitable
solvents such as methanol/acetonitrile/water) were introduced
into the ESI source through a syringe pump at the rate of
5 μL/min. The ESI capillary was set at 3.5 kV, and the cone
voltage was 40 V. The spectra were collected in 6 s scans, and the
printouts are averaged spectra of 6-8 scans. Infrared spectra
were recorded using a Perkin-Elmer RX-1 spectrometer; the
values were reported in cm-1. The purity of all tested com-
pounds was ascertained on the basis of their elemental analysis
and was g95%.
Compounds Listed in Table 1. Synthesis of 1-(p-Cyanophe-
noxypentyl)indole-3-carbaldehyde (9). To a stirred solution of
indole-3-carbaldehyde (1.0 g, 1.0 equiv) and, tetrabutylammo-
nium bromide (0.1 equiv) and (p-cyanophenoxy)pentylbromide
(1.2 equiv) in toluene (5.0 mL) was added 1.0 mL 20% w/v
aqueous NaOH solution. The reaction mixture was stirred for
1.5 h until the reaction was complete. The product separated as a
semisolid mass, which was decanted off. Crude solid was recrys-
tallized from 1% MeOH in chloroform solution. Yield: 71%. M.
P. >250 °C. 1H NMR (200 MHz, DMSO-d6): δ 1.48-1.62 (m,
2H), 1.77-1.91 (m, 2H), 1.95-2.04 (m, 2H), 3.97 (t, 2H, J 6.2
Hz), 4.22 (t, 2H, J 7.0 Hz), 6.88 (d, 2H, J 8.8 Hz) 7.29-4.40 (m,
3H), 7.56 (d, 2H, J 8.8 Hz), 7.72 (s, 1H), 8.29-8.33 (m, 1H), 10.00
(s, 1H). 13C NMR (50 MHz, DMSO-d6): δ 23.3 (CH2), 26.7
(CH2), 27.0 (CH2), 47.3 (CH2), 67.9 (CH2), 110.9 (CH), 115.6 (2
CH), 122.5 (CH), 123.3 (CH), 124.6 (CH), 134.5 (CH), 138.8
(CH), 184.5 (CH), 119.6 (C), 125.8 (C), 137.5 (C), 104.5 (C), 118.6
(C), 162.3 (C). FAB Mass [M þ H]þ 333. IR 1656.4, 1600.1. Anal.
Calcd for C21H20N2O2: C, 75.88; H, 6.06; N, 8.43; O, 9.63.
Found: C, 75.57; H, 6.03; N, 8.47.
where N is the average relative luminescence unit (RLU) of
control wells, and n is the average RLU of treated wells.
Screening on Infected Macrophages. For assessing the activity
of compounds against the amastigote stage of the parasite,
mouse macrophage cell line (J-774A.1) infected with promasti-
gotes expressing the luciferase firefly reporter gene was used.
Cells were seeded in a 96-well plate (1.5 ꢀ 104cell/100 μL/well) in
RPMI-1640 containing 10% fetal calf serum, and the plates
were incubated at 37 °C in a CO2 incubator. After 24 h, the
medium was replaced with fresh medium containing stationary-
phase promastigotes (4 ꢀ 103/100 μL/well). Promastigotes in-
vade the macrophage and are transformed into amastigotes. The
test material in appropriate concentrations (0.195-12.5 μg/mL)
in complete medium was added after replacing the previous
medium, and the plates were incubated at 37 °C in a CO2
incubator for 72 h. After incubation, the drug containing the
medium was decanted, and 50 μL of PBS was added to each well
and mixed with an equal volume of Steady Glo reagent. After
gentle shaking for 1-2 min, the reading was taken with a
luminometer. The inhibition of parasitic growth is determined
by comparison of the luciferase activity of drug treated parasites
with that of untreated controls as described above.
Cytotoxicity Assay. The cell viability was determined using
the MTT assay. J774.A-1 cell line or KB cell line was maintained
in RPMI medium (Sigma), supplemented with 10% fetal calf
serum and 40 mg/mL gentamycin. Exponentially growing cells
(1 ꢀ 104cells/100 μL/well) were incubated with different drug
concentrations for 72 h and were incubated at 37 °C in a
humidified mixture of CO2 and 95% air in an incubator. Stock
solutions of compounds were initially dissolved in DMSO and
further diluted with fresh complete medium. After incubation,
25 μL of MTT reagent (5 mg/mL) in PBS medium, followed by
syringe filtration, was added to each well and incubated at 37 °C
for 2 h. At the end of the incubation period, the supernatant was
removed by tilting the plate completely without disturbing the
cell layer, and 150 μL of pure DMSO was added to each well.
After 15 min of shaking, the readings were recorded as absor-
bance at 544 nm on a microplate reader. The cytotoxic effect was
expressed as 50% lethal dose, i.e., as the concentration of a
compound which provoked a 50% reduction in cell viability
compared to that of a cell in culture medium alone. IC50 values
were estimated through the preformed template as described by
Huber and Koella.21
Representative Procedure for the Synthesis Compound 8. 1-(p-
Cyanophenoxypentyl)indole-3-carbaldehyde (9) (0.245 g, 1.0
equiv) and 2-thiohydantoin (1.0 equiv) were stirred in the
presence of ethanolamine (1.5 equiv) in absolute ethanol at
60 °C for 2-3 h. Compounds were precipitated out, filtered, and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5801
crystallized from methanol or acetone. Yield: 82%. M.P. 240 °C.
1H NMR (200 MHz, CDCl3): δ 1.45-1.65 (m, 2H), 1.78-1.91
(m, 2H), 1.91-2.09 (m, 2H), 4.06 (t, 2H, J 6.2 Hz), 4.30 (t, 2H, J
7.0 Hz), 6.84 (s, 1H), 7.07 (d, 2H, J 8.8 Hz), 7.23-738 (m, 2H),
7.63 (d, 1H, J 7.1 Hz), 7.74 (d, 2H, J 8.8 Hz), 7.87 (d, 1H, J 7.3
Hz). 13C NMR (50 MHz, DMSO-d6): δ 23.0 (CH2), 28.5 (CH2),
29.9 (CH2), 46.3 (CH2), 68.1 (CH2), 108.6 (CH), 111.2 (CH),
118.3 (CH), 115.7 (2 CH), 120.5 (CH), 122.9 (CH), 131.7 (CH),
135.1 (2 CH), 112.9 (C), 128.2 (C), 136.5 (C), 104.6 (C), 118.6
(C), 162.8 (C), 122.4 (C), 165.2 (C), 177.3 (C). Anal. Calcd for
C24H22N4O2S: C, 66.96; H, 5.15; N, 13.01. Found: C, 66.72; H,
5.14; N, 12.98.
2H), 1.74-1.78 (m, 2H), 1.89-1.94 (m, 2H), 3.57 (s, 3H), 3.89 (t,
2H, J 6.3 Hz), 4.22 (t, 2H, J 6.9 Hz), 5.40 (s, broad, 2H), 6.77 (d,
2H, J 8.7 Hz), 6.85 (s, 1H), 7.17-7.22 (m, 2H), 7.42 (d, 1H, J 7.8
Hz), 7.52 (d, 2H, J 8.7 Hz), 7.84 (d, 1H, J 7.4 Hz), 9.03 (s, 1H).
13C NMR (50 MHz, DMSO-d6): δ 23.7 (CH2), 29.0 (CH2), 30.1
(CH2), 30.5 (CH2), 47.1 (CH2), 68.1 (CH2), 111.4 (CH), 112.7
(CH), 114.7 (2 CH), 119.4 (CH), 121.6 (CH), 123.4 (CH), 127.5
(2 CH), 133.9 (CH), 109.0 (C), 120.6 (C), 126.3 (C), 129.3 (C),
136.6 (C), 151.8 (C), 160.1 (C), 163.9 (C), 173.8 (C). ESMS [M þ
H]þ 478. Anal. Calcd for C25H27N5O3S: C, 62.87; H, 5.70; N,
14.66. Found: C, 62.62; H, 5.67; N, 14.62.
Representative Procedure for the Synthesis of Compound 1.
Compound 8 and anhydrous ZnCl2 were stirred in dry metha-
nol at 60 °C for 0.5 h. Methanol was removed under vacuum,
and neat butyl amine (dried over anhydrous KOH pellet) was
added to the remaining solid residue just to cover it (2-3 mL).
The reaction mixture was stirred at 80 °C for 7-8 h until
the reaction was complete. It was cooled and added to 8 mL,
1% aqueous hydrochloric acid solution. The reaction mixture
was extracted by ethylacetate (3 ꢀ 4 mL) and chromatographed
over 100-200 mesh silica gel eluted using methanol in chloro-
form solvent system with increasing concentration of methanol
from 0.1% to 2.0%. Yield: 64%. Semi solid. 1H NMR (200 MHz,
acetone-d6): δ 0.84 (t, 3H, 7.2 Hz), 1.31-1.84 (m, 10H), 3.42 (t, 2H,
J 7.2 Hz), 3.89 (t, 2H, J 6.2 Hz), 4.20 (t, 2H, J 7.0 Hz), 6.82 (s, 1H),
6.82 (d, 2H, J 8.8 Hz), 7.06-7.14 (m, 2H), 7.39 (d, 1H, J 7.4 Hz),
7.53 (d, 2H, J 8.8 Hz), 7.84 (d, 1H, J 7.2 Hz), 8.20 (s, 1H). 13C
NMR (50 MHz, acetone-d6):δ14.5 (CH3), 21.1 (CH2), 24.4 (CH2),
32.9 (CH2), 47.8 (CH2), 47.5 (CH2), 29.1-30.6 (2 CH2 merged with
acetone-d6 peaks), 69.3 (CH2), 104.7 (CH), 111.3 (CH), 116.7 (2
CH), 120.2 (CH), 121.4 (CH), 123.5 (CH), 135.2 (2 CH), 136.5
(CH), 107.9 (C), 129.3 (C), 132.5 (C), 104.5 (C), 118.2 (C), 162.4
(C), 121.9 (C), 163.7 (C), 173.7 (C). FAB Mass [M þ H]þ 471.
Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found:
C, 71.37; H, 6.62; N, 14.86.
Acknowledgment. We thank Sophisticated Analytical
Instruments Facility, CDRI for providing analytical data,
Dr. Neena Goyal, Biochemistry Division, for providing
transgenic L. donovani promastigotes, and CSIR, New Delhi,
for providing financial assistance to S.P. and N.S. CDRI
Communication No: 7738.
Supporting Information Available: Synthesis procedures and
analytical data of all remaining compounds and intermediates.
This material is available free of charge via Internet at http://
pubs.acs.org.
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1
M.P. 220 °C (decomposed). H NMR (200 MHz, DMSO-d6):
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1
Compound 18. Yield: 73%. M.P. >240 °C. H NMR (200
MHz, DMSO-d6): δ 1.41-1.46 (m, 2H), 1.71-1.77 (m, 2H),
1.88-1.93 (m, 2H), 3.86 (t, 2H, J 6.0 Hz), 4.18 (t, 2H, J 6.8 Hz),
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