Discovery of 3,3′-diindolylmethanes as potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2013.02.024

An efficient protocol for synthesis of 3,3′-diindolylmethanes using recyclable Fe-pillared interlayered clay (Fe-PILC) catalyst under aqueous medium has been developed. All synthesized 3,3′-diindolylmethanes showed promising antileishmanial activity again

Full text of DOI:10.1016/j.ejmech.2013.02.024

European Journal of Medicinal Chemistry 63 (2013) 435e443
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of 3,30-diindolylmethanes as potent antileishmanial agentsq
,
a
b
b
Sandip B. Bharate a , Jaideep B. Bharate , Shabana I. Khan , Babu L. Tekwani ,
*
Melissa R. Jacob b, Ramesh Mudududdla a, Rammohan R. Yadav a, Baljinder Singh c,
P.R. Sharma d, Sudip Maity e, Baldev Singh c , Ikhlas A. Khan , Ram A. Vishwakarma
,
b
a,c,
**
*
a Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
b National Centre for Natural Products Research (NCNPR), School of Pharmacy, University of Mississippi, MS 38677, USA
c Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
d Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
e Central Institute of Mining and Fuel Research (CIMFR), Dhanbad, Jharkhand 826001, India
a r t i c l e i n f o
a b s t r a c t
An efficient protocol for synthesis of 3,30-diindolylmethanes using recyclable Fe-pillared interlayered clay
(Fe-PILC) catalyst under aqueous medium has been developed. All synthesized 3,30-diindolylmethanes
showed promising antileishmanial activity against Leishmania donovani promastigotes as well as axenic
amastigotes. Structureeactivity relationship analysis revealed that nitroaryl substituted diindolyl-
methanes showed potent antileishmanial activity. The 4-nitrophenyl linked 3,30-diindolylmethane 8g
Article history:
Received 26 December 2012
Received in revised form
20 February 2013
Accepted 22 February 2013
Available online 28 February 2013
was found to be the most potent antileishmanial analog showing IC50 values of 7.88 and 8.37 mM against
both L. donovani promastigotes and amastigotes, respectively. Further, a pharmacophore based QSAR
model was established to understand the crucial molecular features of 3,30-diindolylmethanes essential
for potent antileishmanial activity. These compounds also exhibited promising antifungal activity against
Cryptococcus neoformans, wherein fluorophenyl substituted 3,30-diindolylmethanes were found to be
most potent antifungal agents. Developed synthetic protocol will be useful for economical and eco-
friendly synthesis of potent antileishmanial and antifungal 3,30-diindolylmethane class of compounds.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
3,30-Diindolylmethanes
Antileishmanial
Antimalarial
Leishmania donovani
Antifungal
Fe-pillared interlayered clay
1. Introduction
amphotericin B are available, which are costly and have serious
side-effects, and are getting resistant to pathogens after treatment
Parasitic diseases such as malaria, leishmaniasis, trypanosomi-
asis affect millions of people worldwide and pose a major health
problem in developing countries [1,2]. Amongst these malaria and
leishmaniasis have affected major population with increasing
number of new cases each year. Leishmaniasis is caused by proto-
zoan parasites that belong to the genus Leishmania and is trans-
mitted by the bite of certain species of sand fly (subfamily
Phlebotominae). Most of the current drugs used to treat parasitic
diseases are decades old and have many limitations, including the
emergence of drug resistance. For leishmaniasis, either the first-
line pentavalent antimonials or second-line drugs such as
for several weeks, and hence there is a need for new anti-
leishmanial agents with improved efficacy and less side-effects for
both visceral and cutaneous leishmaniasis [3,4].
Bisindole class of compounds are known to possess diverse
range of pharmacological activities such as anticancer [5e7], anti-
microbial [8e11] etc. Hamacanthin A (1), a bisindole alkaloid iso-
lated from the sponge Hamacantha sp [11] and Spongosorites sp [8]
showed potent antibacterial activity against Staphylococcus aureus
and MRS with MIC of 6.45
mM and antifungal activity against Ba-
cillus subtilis with MIC of 3.22
mM [8]. Furthermore, bisindoles have
also been reported as fluorescent molecular probes [12] of biolog-
ical interest. Amongst various antileishmanial scaffolds reported,
indole alkaloids [13e18] such as 2e3 [18] showed promising ac-
tivity against Leishmania parasite. Munoz et al. [18] observed that
dimeric indole alkaloids showed better antibacterial and anti-
leishmanial activities compared to monomeric indole alkaloids.
Dimeric alkaloids conodurine 2 and N-demethylconodurine 3
showed strong activity against the intracellular amastigote form of
Leishmania. Furthermore, 3,30-diindolylmethane (DIM, 4) is a
q
IIIM communication number. IIIM/1532/2013.
* Corresponding authors. Tel.: þ91 191 2569111; fax: þ91 191 2569333.
** Corresponding author. Medicinal Chemistry Division, Indian Institute of Inte-
grative Medicine (CSIR), Canal Road, Jammu 180001, India. Tel.: þ91 191 2569111;
fax: þ91 191 2569333.
E-mail addresses: sbharate@iiim.ac.in (S.B. Bharate), baldevsingh@iiim.ac.in
(B. Singh), ram@iiim.ac.in (R.A. Vishwakarma).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.024

436
S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
N
CH3
NH
N
H
N
MeO
H
N
H
H3C
COOMe
Br
N
Br
NH
N
H
O
N
HN
R
COOMe
1
2: R = CH3
4
3: R = H
Fig. 1. Structures of biologically active bisindole alkaloids 1e4.
cancer chemopreventive agent present in cruciferous vegetables
such as broccoli, brussels sprouts, cabbage and kale [5]. Structures
of bisindole alkaloids 1e4 are shown in Fig. 1.
results clearly indicate the significant enhancement of catalytic
activity of clay after pillaring it with iron.
Next, we studied scope of the reaction for various substituted
indoles and different aldehydes. Treatment of indole (5a) with
various substituted aryl and heteroaryl aldehydes in water in
presence of 15 mol% of Fe-PILC B produced corresponding 3,30-
diindolylmethanes 7ae7o in excellent yields as shown in Fig. 3.
Indoles substituted with electron withdrawing group (e.g. Iodo,
Fig. 4) as well as electron donating group (e.g. OMe, Fig. 5) on
treatment with various aryl and heteroaryl aldehydes produced
corresponding 3,30-diindolylmethanes in excellent yields as shown
in Figs. 4 and 5. Similarly aldehydes with both electron donating as
well as electron withdrawing groups participated well in this re-
action. Recyclability of the Fe-PILC B catalyst was checked to prove
the heterogeneous nature and its repeated use. Treatment of 1H-
indole (5a) and 4-hydroxy benzaldehyde (6a) in water in presence
of 15 mol% Fe-PILC B led to formation of 3,30-diindolylmethane 7a
in 85, 82, 78, 68% yield over four cycles respectively.
Due to promising pharmacological activities [7,19,20] of bisin-
doles or diindolylmethanes, plenty of synthetic methods are avail-
able in literature for their synthesis; however mostof these methods
involves treatment of indoles with aldehydes in presence of ho-
mogeneous acid catalysts or Lewis acids [21e24] and only few re-
ports on the use of heterogeneous catalysts such as inorganic-
supported polyoxometalates [25], polyanilineebismoclite compos-
ite [26] and zeolites [27,28]. Despite these reports, development of a
hazard-free, waste-free, and energy-efficient synthetic protocol will
be of great use for economical synthesis of this class of compounds.
In continuation to our efforts toward development of efficient syn-
thetic methodologies for preparation of biologically important
scaffolds [29e31], and our efforts in the area of indole chemistry
[32e35], herein we have developed an efficient protocol (Fig. 2) for
preparation of potent antileishmanial 3,30-diindolylmethanes.
In order to understand the efficiency and catalytic activity as a
function of catalyst composition, physical nature and surface
acidity, the Fe-PILC B catalyst was characterized for specific surface
area, temperature programmed reduction (TPR), temperature
programmed desorption (TPD) and scanning electron microscopy
(SEM) analysis. Surface area results evidenced that catalyst B is
composed of fine aggregates, which was also supported by SEM
analysis. Surface area for natural montmorillonite clay K10 and Fe-
2. Results and discussion
2.1. Synthesis of 3,30-diindolylmethanes
Keeping in mind earlier findings that requirement of acidity is
essential for the preparation of 3,30-diindolylmethanes, we
explored Fe-PILC catalysts A and B varying in their surface acidity.
Reaction between 1H-indole (5a) and 4-hydroxy benzaldehyde (6a)
was used as a model reaction (Table 1). Solvent optimization
studies revealed that methylene chloride and water were best
solvents producing higher yields. Catalyst loading as low as 15 mol%
was found to be efficient producing excellent yields of products.
Further, catalyst B was found to be better as compared to catalyst A.
Thus catalyst B and water as reaction medium at room temperature
were chosen for further scope exploration studies. Furthermore, we
compared the reactivity of Fe-PILC B with plane montmorillonite
clay K10 (entries 9 and 10). Product 7a was not formed using clay
K10 after 6 h of reaction time (entry 9), however w30% product was
formed when reaction was continued for 16 h (entry 10). These
PILC B was found to be 52 m2 gꢀ1 and 102 m2 ꢀ1, indicating in-
g
crease in the surface area in Fe-pillared clays. SEM analysis of Fe-
PILC B showed that metal is randomly distributed over the sup-
port surface with flake formation, which was also supported by
dispersion analysis (upto 99.4%). The average crystallite size of the
metal was 11.60 Å. Further, SEM analysis also showed that Fe par-
ticles were present in the form of aggregates and not as a contin-
uous film. Highly dispersed metal on the support is able to adsorb
substrate and/or reagent to the high extent. TPD analysis showed
that the Fe-PILC B contained enormously high surface acidity
compared to the original clay montmorillonite K10. Total acidity of
the catalyst was found to be 7.91 mmol NH3/g catalyst, which was
H+
R'
R'
Fe
R'
R = substituted
aryls, heteroaryls
NH
N
H
15 mol% Fe-PILC
HN
Water, 6-10 h
Indoles
R' = H, I, OMe
60-78%
+
R
3,3'-Diindolyl methanes
R CHO
Aldehydes
Antileishmanial activity against Leishmania donovani
Antifungal activity against Cryptococcus neoformans
Fig. 2. Synthesis of 3,30-diindolylmethanes using Fe-PILC catalyst.

S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
437
Table 1
I
I
Optimization of reaction conditions for synthesis of 3,30-diindolylmethanes.
R
I
R
15 mol% Fe-PILC B
+
CHO
Water
N
H
HN
NH
N
HN
8a-j
H
5b
NH
6
5a
Catalyst
Solvent
R =
+
Cl
Cl
HO
CHO
OH
7a
F
F
Br
OMe
6a
Br
8d
F
8c
OH
8b
OH
8a
8e
8f
(6 h, 68%)
(6 h, 75%) (6 h, 72%)
(6 h, 70%)
(10 h, 60%) (6 h, 71%)
Entry
Catalysta
mol%
Solvent
Temp.
Time (h)
Yieldb (%)
1.
2.
3.
4.
5.
6.
7.
8.
Fe-PILC A
Fe-PILC A
Fe-PILC A
Fe-PILC B
Fe-PILC B
Fe-PILC B
Fe-PILC B
Fe-PILC B
Clay K10
Clay K10
None
50
50
50
50
50
50
30
15
50
50
e
CH2Cl2
Water
Water
CH2Cl2
Water
Water
Water
Water
Water
Water
CH2Cl2
Water
rt
rt
60 ꢁC
rt
rt
60 ꢁC
rt
rt
rt
4
4
6
4
4
6
6
6
6
58
54
58
75
70
76
76
74
0
O
O2N
NO2
8h
8g
8i
8j
(8 h, 63%)
(6 h, 68%) (8 h, 62%)
(6 h, 70%)
9
Fig. 4. Synthesis of 5,50-diiodo-3,30-diindolylmethanes 8aej.
10
11.
12.
rt
rt
rt
16
10
10
30
0
0
None
e
further gets reduced to FeO and then consequently to Fe at 470 ꢁC
(Figure S1 of supplementary information).
a
Fe-PILC A and B vary in their surface acidity. Fe-PILC B is more acidic than Fe-
PILC A.
b
Isolated yield after silica gel column chromatography.
2.2. Biological evaluation
distributed on the surface of the clay support as 47% (3.75 mmol
NH3/g catalyst), 18% (1.40 mmol NH3/g catalyst) and 35%
(2.76 mmol NH3/g catalyst) of weak, medium and strong acidic
sites. Further, a H2-temperature-programmed reduction (TPR)
profile of the Fe-PILC B indicated that iron oxides gets reduced to Fe
in three steps. Initially Fe2O3 gets reduced to Fe3O4 at 415 ꢁC, which
All synthesized 3,30-diindolylmethanes were evaluated for
antileishmanial, antimalarial, antibacterial and antifungal, activ-
ities. The in vitro antileishmanial activity of 3,30-diindolylmethanes
was tested against a culture of both Leishmania donovani promas-
tigotes and axenic amastigotes. Several compounds showed
promising antileishmanial activity as depicted in Table 2. Amongst
all tested analogs, 5-nitrofuryl substituted 5,50-diiodo 3,30-diindo-
lylmethane 8j was found to be most promising compound against
L. donovani promastigotes showing IC50 and IC90 values of 3.02 and
R
R
15 mol% Fe-PILC
Water
B
+
CHO
N
H
6.22
mM which was comparable to control drug pentamidine.
HN
NH
However, the 4-nitrophenyl substituted 5,50-diiodo 3,30-diindolyl-
5a
6
7a-o
methane 8g showed potent activity against both promastigotes as
well as amastigotes with IC50 values of 7.88 and 8.37 mM, respec-
R =
tively. Analysis of structureeactivity relationship revealed that 3,30-
diindolylmethanes substituted with nitro-substituted aromatic
(e.g. 7l, 8g, 9g) or heteroaromatic moiety (e.g. 7o, 8j) showed potent
antileishmanial activity against L. donovani promastigotes and
amastigotes.
OH
OH
Br
OMe
OMe
7d
OMe
7e
OH
7b
OH
7a
OMe
7c
7f
All compounds were also evaluated for in vitro antimalarial ac-
tivity against chloroquine-sensitive (D6) and resistant (W2) clones
of Plasmodium falciparum via determination of plasmodial LDH
activity [36]. 2,6-Dichlorophenyl linked 3,30-diindolylmethane 7j
showed antimalarial activity against D6 clone of P. falciparum with
(6 h, 78%) (6 h, 55%) (8 h, 75%)
(8 h, 70%) (8 h, 65%) (10 h, 60%)
Cl
Br
Cl
IC50 value of 12.1
cytotoxicity towards mammalian kidney fibroblasts (vero) cells at
concentration upto 4.6 g/mL.
m
M. None of the 3,30-diindolylmethane showed
Br
F
F
Cl
F
NO2
7l
7g
7h
7i
7j
7k
m
(6h, 65%) (6 h, 70%) (6 h, 68%) (10 h, 69%) (8 h, 70%) (6 h, 72%)
The antibacterial activity was tested against S. aureus,
methicillin-resistant S. aureus, Mycobacterium intracellulare,
Escherichia coli and Pseudomonas aeruginosa. Susceptibility of these
microorganisms (except M. intracellulare) to test compounds was
determined according to a modified version of CLSI methods [37e
39]. Susceptibility of M. intracellulare was done using the modified
Alamar Blue procedure [40]. Ciprofloxacin was used as positive
control for comparison. Results of antibacterial activity are shown
in Table 3. All tested diindolylmethanes were active only against
O
S
O2N
7n
7m
7o
(6 h, 74%)
(6, 72%)
(8 h, 60%)
Fig. 3. Synthesis of 3,30-diindolylmethanes 7aeo.

438
S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
MeO
15 mol% Fe-PILC B
diindolylmethanes 7g and 7k showed potent antifungal activity
OMe
MeO
R
R
9a-k
Cl
against C. neoformans with MIC values of 2.99 and 3.49 mM
respectively. Hydroxyphenyl substituted 5,50-diiodo-diindolyl-
methanes 8a which showed potent antibacterial activity, also
exhibited promising antifungal activity against C. neoformans with
+
CHO
Water
N
H
HN
NH
5a
6
MIC of 4.24
m
M. Antifungal activity of 3,30-diindolylmethanes
R =
against C. neoformans is shown in Table 4.
Cl
2.3. Pharmacophore model for antileishmanial 3,30-
diindolylmethanes
F
F
OMe
Br
Br
9d
OH
9b
F
9c
OH
9a
A pharmacophore based QSAR model [41,42] was established for
antileishmanial 3,30-diindolylmethanes in order to understand the
key molecular features essential for antileishmanial activity against
L. donovani promastigotes. As depicted in Fig. 6, the selected
pharmacophore model comprises two H-bond donors and four
aromatic ring features. Developed model showed excellent statis-
tical parameters, thus guaranties its utility to design new promising
antileishmanial 3,30-diindolylmethanes.
9e
9f
(6 h, 66%) (6 h, 70%) (6 h, 68%) (10 h, 62%) (6 h, 70%)
(6 h, 70%)
O
S
O2N
NO2
9g
9h
9i
9j
9k
(8 h, 70%)
(8 h, 69%)
(6 h, 70%)
(6 h, 68%)
(6 h, 67%)
3. Conclusion
Fig. 5. Synthesis of 5,50-dimethoxy-3,30-diindolylmethanes 9aek.
In summary, we have developed simple and efficient green
protocol for preparation of structurally diverse 3,30-diindolyl-
methanes. Nitroaryl substituted 3,30-diindolylmethanes were
potent antileishmanial agents possessing activity comparable to
pentamidine. 3,30-Diindolylmethanes also showed promising
antibacterial and antifungal activities. Fluorophenyl substituted
3,30-diindolylmethanes were found to be most potent antifungal
compounds against C. neoformans. Thus, herein we have shown that
developed synthetic protocol is highly efficient for economical
synthesis of potent antifungal and antileishmanial diindolyl-
methanes. Furthermore, a developed statistically significant phar-
macophore model can be used for designing potent antileishmanial
3,30-diindolylmethanes.
S. aureus and methicillin-resistant S. aureus. Amongst tested com-
pounds, hydroxyphenyl substituted 5,50-diiodo-diindolylmethanes
8a and 8b showed most potent antibacterial activity against
S. aureus and MRS with MIC values in the range of 2.0e4.3 mM. 3,5-
Difluorophenyl substituted 3,30-diindolylmethane 7k also showed
potent antibacterial activity against S. aureus and MRS with MIC
values of 3.49 and 6.98
methanes 7o and 9c showed activity against M. intracellulare with
IC50 values of 52.9 and 39.6 M respectively.
m
M respectively. Only two 3,30-diindolyl-
m
Antifungal activities were evaluated against a panel of patho-
genic fungi (Candida albicans, Cryptococcus neoformans, Aspergillus
fumigatus) associated with opportunistic infections. Amphoterecin
B was used as positive control for comparison. Several compounds
showed promising activity against C. neoformans, however no ac-
tivity was found against other fungi. Fluorophenyl substituted 3,30-
4. Experimental section
4.1. General
All chemicals were obtained from SigmaeAldrich Company and
used as received. 1H, 13C and DEPT NMR spectra were recorded on
Bruker-Avance DPX FT-NMR 500 and 400 MHz instruments.
Chemical data for protons are reported in parts per million (ppm)
downfield from tetramethylsilane and are referenced to the resid-
ual proton in the NMR solvent (CDCl3, 7.26 ppm). Carbon nuclear
magnetic resonance spectra (13C NMR) were recorded at 125 MHz
or 100 MHz: chemical data for carbons are reported in parts per
Table 2
Antileishmanial activity of 3,30-diindolylmethanes against Leishmania donovani
promastigotes and amastigotes.
a,b
a,b
Entry L. donovani (IC50/IC90
)
Entry
L. donovani (IC50/IC90
)
Promastigotes Amastigotes
Promastigotes Amastigotes
7a
7b
7c
7d
7e
7f
7g
7h
7i
92.84/106.4
85.95/98.86
89.03/105.5
84.97/95.63
82.86/na
41.15/75.74
17.63/22.94
26.66/39.04
27.45/59.38
80.72/90.79
8.35/19.78
15.94/54.33
51.33/88.88 8e
na/na
na/na
75.00/na
na/na
8f
8g
24.25/41.25
7.88/10.16
na/na
23.18/44.49
8.37/38.37
na/na
42.17/79.46 8h
million (ppm, d scale) downfield from referenced to the carbon
na/na
95.86/na
8i
8j
na/na
na/na
resonance of the solvent (CDCl3, 77 ppm). ESI-MS and HRMS
spectra were recorded on Agilent 1100 LC-Q-TOF and HRMS-6540-
UHD machines. IR spectra were recorded on PerkineElmer IR
spectrophotometer. Melting points were recorded on digital
melting point apparatus.
3.02/6.22
81.06/90.98
na/na
15.75/46.55
32.70/58.46
na/na
26.79/65.31
12.08/18.81
66.55/82.85
34.65/64.92
38.07/65.85
37.27/59.98
3.29/6.21
25.32/28.93
42.89/76.31
na/na
47.62/66.82
na/na
47.54/74.52 9a
41.97/76.97 9b
35.85/75.58 9c
48.41/81.54 9d
67.40/95.61 9e
40.79/72.26 9f
7j
7k
7l
na/na
49.64/78.71
14.73/64.22
33.19/na
39.83/na
59.02/84.82
na/na
4.2. General procedure for synthesis of 3,30-diindolylmethanes
using Fe-PILC B catalyst
7m 82.04/91.90
53.44/na
9g
7n
7o
8a
8b
8c
8d
100.4/na
70.21/101.28 9h
31.54/41.82 9i
39.76/52.46 9j
31.58/47.24 9k
18.25/31.43 Pentamidine
13.14/46.58 Amphotericin B 0.17/0.35
6.61/12.18
19.07/53.20
20.77/37.94
9.84/18.22
4.49/8.47
To the solution of indole 5aec and aldehyde 6 in water was
added 15 mol% Fe-PILC B and reaction mixture was stirred at room
temperature for 6e10 h. Completion of the reaction was monitored
by TLC. After completion of reaction, reaction mixture was filtered
and filtrate was concentrated on vacuo rotavapor to get crude
product. Crude product was purified by silica gel (#100e200) col-
umn chromatography to get 3,30-diindolylmethanes 7aek, 8aej
2.9/14.3
0.13/0.37
na, not active.
a
Values are in mM.
b
IC50 and IC90 are sample concentrations that inhibit 50% and 90% growth of
L. donovani parasites compared to solvent controls.

S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
439
Table 3
Antibacterial activity of 3,30-diindolylmethanes.
Entry
IC50/MIC/MBCa
S. aureus
MRS
M. intracellulare
7a
7c
7d
7e
7f
7g
7h
7i
7j
7k
7n
7o
8a
8b
8j
9c
10.47/29.59/na
9.06/na/na
7.47/13.59/na
7.84/na/na
17.81/59.2/na
na/na/na
12.3/27.2/na
31.5/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
na/na/na
52.9/na/na
na/na/na
na/na/na
na/na/na
39.6/41.8/na
na/na/na
na/na/na
na/na/na
1.0/1.51/3.02
17.19/59.2/na
3.76/5.98/na
3.79/7.02/na
3.75/12.5/na
2.36/6.41/51.3
2.32/3.49/na
9.15/15.2/na
12.0/28.0/28.0
2.10/4.24/33.9
1.89/4.03/na
7.52/16.4/16.4
3.45/10.5/na
7.51/na/na
36.2/na/na
9.23/23.9/na
6.12/14.0/56.2
6.30/12.5/na
na/na/na
2.93/6.98/na
13.45/na/na
8.54/14.0/na
1.15/2.12/10
2.11/4.03/na
11.28/32.8/na
4.67/na/na
Fig. 6. Pharmacophore model for antileishmanial 3,30-diindolylmethanes. (a) The best
phase hypothesis DDRRRR.116 superimposed on one of the most active compound 8g.
In the figure a, ‘D’ denotes H-bond donor and ‘R’ denotes aromatic ring. (b) Correlation
graph between experimental and PHASE predicted antileishmanial activity (# of PLS
factors ¼ 3; r2 ¼ 0.885; SD ¼ 0.158; F ¼ 43.8; p ¼ 3.3 ꢂ 10ꢀ8; stability ¼ 0.529;
RMSE ¼ 0.2658; Q2 ¼ 0.592; pearson r ¼ 0.836). Training: Test ¼ 65: 35. Test set
included 7a, 7c, 7i, 7m, 8a, 8d, 9c, 9g, 9h and 9i. All remaining analogs were part of
training set.
9e
9f
9h
17.16/na/na
6.89/11.7/na
na/na/na
4.05/11.71/na
39.7/na/na
0.39/1.51/1.51
Ciprofloxacin
0.39/1.51/1.51
a
Values are in mM.
120.73, 119.69, 119.48, 118.92, 116.17, 115.97, 111.16, 102.65, 39.30; IR
(CHCl3): nmax 3401, 1574, 1384, 1044 cmꢀ1; ESI-MS: m/z 419.0376
[Mþ1]þ, 457.0693 [M þ K]þ; HRMS: m/z 417.03937 and 419.03801
calcd for C23H16BrFN2 ꢀ Hþ (417.03972 and 419.03794).
and 9aek in 60e78% yields. Compounds 7a [26,43], 7b [26e28], 7c
[27,28,43e45], 7d [46], 7f [47], 7h [22,25e28,44,48], 7j [47], 7k
[49], 7l [22,25,26,43e45], 7m [46,50], 7n [43], 7o [43], 8f [49], 9d
[51], 9f [49] and 9g [52] were characterized by comparison of their
spectral data with literature values. Spectral data for new com-
pounds is provided below.
4.2.3. 3,30-(3-Bromophenyl-methanediyl)-bisindole (7i)
Black solid; m.p. 84e86 ꢁC; 1H NMR (CDCl3, 400 MHz):
d 7.87
4.2.1. 3,30-(4-Methoxy-3-bromophenyl-methanediyl)-bisindole (7e)
(brs, 2H), 7.43 (s, 1H), 7.38e7.34 (m, 4H), 7.30e7.24 (m, 2H), 7.20e
7.11 (m, 3H), 7.07 (t, J ¼ 7.5 Hz, 2H), 6.58 (s, 2H), 5.78 (s, 1H); 13C
Black solid; m.p. 104e106 ꢁC; 1H NMR (CDCl3, 400 MHz):
d 7.86
NMR (CDCl3, 125 MHz):
d 146.50, 136.61, 131.68, 129.80, 129.30,
(s, 2H), 7.46 (d, J ¼ 12 Hz, 1H), 7.35e7.30 (m, 4H), 7.16e7.11 (m, 3H),
127.39, 126.78, 123.69, 122.38, 122.05, 119.77, 119.40, 118.88, 110.84,
39.89; IR (CHCl3): nmax 2912, 1612, 1509, 1455, 1217, 1039 cmꢀ1; ESI-
MS: m/z 401 [Mþ1]þ; HRMS: m/z 401.0457 calcd for
C23H17BrN2þHþ (401.0474).
6.96 (t, J ¼ 7.2 Hz, 2H), 6.76 (d, J ¼ 8.4 Hz, 1H), 6.58 (s, 2H), 5.74 (s,
1H), 3.73 (s, 3H); 13C NMR (CDCl3,125 MHz):
d 154.11,137.92,136.71,
133.33, 128.44, 126.85, 123.60, 121.90, 119.76, 119.39, 111.84, 111.69,
111.04, 56.28, 38.88; IR (CHCl3): nmax 3411, 2929, 1604, 1509, 1456,
1300, 1216 cmꢀ1; ESI-MS: m/z 431 [Mþ1]þ; HRMS: m/z 429.0595
calcd for C24H19BrN2O ꢀ Hþ (429.0597).
4.2.4. 5,50-Diiodo-3,30-(4-hydroxyphenyl-methanediyl)-bisindole
(8a)
Dark maroon solid; m.p. 93e95 ꢁC; 1H NMR (CDCl3, 400 MHz):
4.2.2. 3,30-(4-Fluoro-3-bromo-phenylmethanediyl)-bisindole (7g)
Maroon colored solid; m.p. 102e104 ꢁC; 1H NMR (CDCl3,
d
7.97 (s, 2H), 7.67 (d, J ¼ 1.2 Hz, 2H), 7.43e7.40 (q, J ¼ 1.6, 8.4 Hz,
2H), 7.15e7.12 (q, J ¼ 5.6, 8.4 Hz, 4H), 6.77 (d, J ¼ 6.8 Hz, 2H), 6.58 (d,
400 MHz):
d
7.90 (s, 2H), 7.50e7.45 (dd, J ¼ 2.4, 6.8 Hz, 1H), 7.37 (t,
J ¼ 1.6 Hz, 2H), 5.67 (s, 1H), 4.73 (brs, 1H, OH); 13C NMR (CDCl3,
J ¼ 7.9 Hz, 4H), 7.25e7.17 (m, 3H), 6.97e6.90 (m, 3H), 6.59 (d,
J ¼ 1.6 Hz, 2H), 5.78 (s, 1H); 13C NMR (CDCl3, 100 MHz):
d
158.52,
125 MHz):
d 153.67, 135.42, 134.22, 130.70, 129.31, 129.01, 128.44,
124.11, 119.08, 115.23, 112.95, 82.46, 38.62; IR (CHCl3): nmax 3422,
2927, 1612, 1596, 1510, 1454, 1215, 1094 cmꢀ1; ESI-MS: m/z 588.8
[M ꢀ 1]þ; HRMS: m/z 588.9251 calcd for C23H16I2N2O ꢀ Hþ
(588.9268).
156.38,141.53,136.70,133.44,129.09,129.03,126.75,123.59,122.20,
Table 4
Antifungal activity of 3,30-diindolylmethanes against C. neoformans.
Entry
IC50
MICa
MFCa
a
4.2.5. 5,50-Diiodo-3,30-(4-hydroxy-3-methoxyphenyl-
methanediyl)-bisindole (8b)
7a
7c
7d
7f
7g
7h
7i
7k
7n
7o
8a
8b
8c
8d
9f
18.91
35.45
16.88
14.64
1.94
5.56
5.33
2.32
18.93
29.64
1.80
3.92
16.78
5.58
29.59
na
27.17
59.17
2.99
14.04
12.50
3.49
30.49
na
59.17
na
27.17
na
5.98
14.04
12.50
3.49
30.49
na
4.24
na
29.85
30.67
na
1H NMR (CDCl3, 400 MHz):
d
8.00 (s, 2H), 7.69 (d, J ¼ 1.2 Hz, 2H),
7.42e7.39 (q, J ¼ 1.6, 8.4 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz, 2H), 6.83 (t,
J ¼ 8.4 Hz, 2H), 6.72 (q, J ¼ 1.6, 8.0 Hz, 1H), 6.56 (d, J ¼ 1.6 Hz, 2H),
5.67 (s, 1H), 3.76 (s, 3H); IR (CHCl3): nmax 3422, 2927, 1612, 1596,
1510, 1454, 1315,1094 cmꢀ1; ESI-MS: m/z 618.8 [M ꢀ 1]þ; HRMS: m/
z 618.9362 calcd for C24H18I2N2O2 ꢀ Hþ (618.9374).
4.24
4.2.6. 5,50-Diiodo-3,30-(4-fluoro-3-bromophenyl-methanediyl)-
bisindole (8c)
32.26
29.85
30.67
47.85
1.35
Brown crystalline solid; m.p. 145e147 ꢁC; 1H NMR (CDCl3,
18.35
0.85
400 MHz):
6H), 6.75 (s, 2H), 5.71 (s, 1H); 13C NMR (CDCl3, 100 MHz):
156.82, 140.58, 140.55, 135.75, 133.17, 130.17, 129.10, 128.85, 127.65,
d
8.02 (s, 2H), 7.87 (s, 1H), 7.53 (m, 2H), 7.21e7.02 (m,
Amphotericin B
1.35
d
158.78,
a
Values are in mM.

440
S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
124.67, 117.97, 116.40, 116.22, 113.49, 113.25, 109.10, 108.93, 83.15,
38.80; IR (CHCl3): nmax 3428, 2924, 1595, 1490, 1455, 1243,
1045 cmꢀ1; ESI-MS: m/z 668.8 [M ꢀ 1]þ; HRMS: m/z 670.8299 calcd
for C23H14BrFI2N2 þ Hþ (670.8312).
516 cmꢀ1; ESI-MS: m/z 608 [M ꢀ H]þ; HRMS: m/z 607.8965 calcd for
C
21H13I2N3O3 ꢀ Hþ (607.8963).
4.2.13. 5,50-Dimethoxy-3,30-(4-hydroxyphenyl-methanediyl)-
bisindole (9a)
4.2.7. 5,50-Diiodo-3,30-(4-bromophenyl-methanediyl)-bisindole
Dark maroon solid; m.p.140e142 ꢁC; 1H NMR (CDCl3, 400 MHz):
(8d)
d 7.76 (s, 2H), 7.36e7.17 (m, 4H), 6.85e6.79 (m, 4H), 6.76 (d,
Brown solid; m.p. 122e123 ꢁC; 1H NMR (CDCl3, 400 MHz):
d
7.94
J ¼ 7.6 Hz, 2H), 6.65 (d, J ¼ 1.6 Hz, 2H), 5.70 (s, 1H), 4.70 (brs, 1H,
OH), 3.69 (s, 6H); IR (CHCl3): nmax 3409, 2927, 1581, 1482, 1439,
1204, 1038 cmꢀ1; ESI-MS: m/z 397.2 [M ꢀ H]þ; HRMS: m/z 397.1541
calcd for C25H22N2O3 ꢀ Hþ (397.1547).
(s, 2H), 7.59 (d, J ¼ 1.2 MHz, 2H), 7.38e7.34 (m, 4H), 7.10e7.07 (m,
4H) 6.52 (d, J ¼ 1.6 Hz, 2H), 5.53 (s, 1H); 13C NMR (CDCl3, 125 MHz):
d
142.13, 135.75, 131.53, 130.62, 130.26, 129.24, 128.36, 124.37,
120.34, 118.18, 113.19, 83.06, 39.23; IR (CHCl3): nmax 3421, 2922,
1596, 1457, 1413, 1021 cmꢀ1; ESI-MS: m/z 650.8 [M ꢀ H]þ; HRMS:
m/z 650.8407 calcd for C23H15BrI2N2 ꢀ Hþ (650.8424).
4.2.14. 5,50-Dimethoxy-3,30-(4-hydroxy-3-methoxyphenyl-
methanediyl)-bisindole (9b)
Dark violet solid; m.p. 88e90 ꢁC; 1H NMR (CDCl3, 400 MHz):
4.2.8. 5,50-Diiodo-3,30-(2,6-dichlorophenyl-methanediyl)-bisindole
(8e)
d
7.83 (s, 2H), 7.25 (m, 2H), 6.87 (m, 7H), 6.65 (d, J ¼ 1.6 Hz, 2H), 5.69
(s, 1H), 3.75 (s, 3H), 3.70 (s, 6H); IR (CHCl3): nmax 3401, 2957, 2927,
2856, 1736, 1584, 1459, 1267, 1171, 1030 cmꢀ1; ESI-MS: m/z 427
[M ꢀ 1]þ; HRMS: m/z 427.1654 calcd for C26H24N2O4 ꢀ Hþ
(427.1652).
Maroon colored solid; m.p. 247e249 ꢁC; 1H NMR (CDCl3,
400 MHz):
(m, 4H), 6.74 (s, 2H), 6.59 (s, 1H); 13C NMR (CDCl3, 125 MHz):
137.68, 136.45, 135.41, 130.38, 129.55, 128.11, 128.31, 125.17, 114.66,
d 7.98 (s, 2H), 7.57 (s, 2H), 7.38e7.33 (m, 3H), 7.14e7.08
d
113.01, 83.12, 36.69; IR (CHCl3): nmax 3412, 2926, 1623, 1582, 1483,
1453, 1210 cmꢀ1; ESI-MS: m/z 640.8 [M ꢀ H]þ; HRMS: m/z
650.8526 calcd for C23H14Cl2I2N2 ꢀ Hþ (650.8540).
4.2.15. 5,50-Dimethoxy-3,30-(4-fluoro-3-bromophenyl-
methanediyl)-bisindole (9c)
Maroon solid; m.p. 93e95 ꢁC; 1H NMR (CDCl3, 400 MHz):
d 7.78
(brs, 2H), 7.46 (dd, J ¼ 2.0, 6.8 Hz, 1H), 7.27e7.23 (m, 3H), 6.98 (t,
4.2.9. 5,50-Diiodo-3,30-(4-nitrophenyl-methanediyl)-bisindole (8g)
Orange crystalline solid; m.p. 144e146 ꢁC; 1H NMR (CDCl3,
J ¼ 8.4 Hz, 1H), 6.79 (d, J ¼ 2.4 Hz, 2H), 6.70 (s, 2H), 6.58 (s, 2H), 5.64
(s, 1H), 3.64 (s, 6H); 13C NMR (CDCl3, 100 MHz):
d 158.80, 156.37,
400 MHz):
2H), 7.49e7.43 (m, 4H), 7.22 (d, J ¼ 8.4 Hz, 2H), 6.63 (d, J ¼ 1.6 Hz,
2H), 5.88 (s, 1H); 13C NMR (CDCl3, 125 MHz):
150.80, 146.79,
135.77, 130.91, 129.37, 128.17, 124.40, 123.83, 117.21, 113.34, 83.28,
39.72; IR (CHCl3): nmax 3391, 2923, 1617, 1401, 1022 cmꢀ1; ESI-MS:
m/z 617.9 [M ꢀ H]þ, 641.9 [M þ Na]þ; HRMS: m/z 617.9173 calcd
for C23H15I2N3O2 ꢀ Hþ (617.9170).
d
8.18 (d, J ¼ 8.0 Hz, 2H), 8.10 (s, 2H), 7.66 (d, J ¼ 1.2 Hz,
153.89, 141.47, 133.47, 131.90, 129.09, 127.21, 124.45, 118.47, 116.15,
112.10, 108.81, 101.82, 55.92, 39.36; IR (CHCl3): nmax 3412, 2928,
1584, 1623, 1487, 1210, 1043, 1022 cmꢀ1; ESI-MS: m/z 479 [Mþ1]þ,
501 [M þ Na]þ; HRMS: m/z 477.0590 calcd for C25H20BrFN2O2 ꢀ Hþ
(477.0609).
d
4.2.16. 5,50-Dimethoxy-3,30-(2,6-dichlorophenyl-methanediyl)-
bisindole (9e)
4.2.10. 5,50-Diiodo-3,30-(naphth-1-yl-methanediyl)-bisindole (8h)
Maroon solid; m.p. 224e226 ꢁC; 1H NMR (CDCl3, 400 MHz):
Cream colored solid; m.p. 217e219 ꢁC; 1H NMR (DMSO-d6,
500 MHz):
6.92 (s, 2H), 6.87e6.83 (m, 4H), 6.74 (s, 1H), 3.73 (s, 6H); 13C NMR
(CDCl3, 125 MHz): 153.71, 138.41, 131.45, 130.01, 128.10, 127.51,
d
7.88 (s, 2H), 7.33e7.28 (m, 4H), 7.16 (t, J ¼ 8.0 Hz, 1H),
d
8.05 (d, J ¼ 8.0 Hz, 1H), 7.99 (brs, 2H), 7.91 (d, J ¼ 8.0 Hz, 1H), 7.78
(d, J ¼ 8.0 Hz, 1H), 7.70 (d, J ¼ 1.2 Hz, 2H), 7.50e7.36 (m, 4H), 7.32 (t,
J ¼ 8.0 Hz, 2H), 7.18e7.13 (m, 3H), 6.51 (s, 1H), 6.51 (d, J ¼ 2.4 Hz,
d
125.16, 114.72, 111.82, 111.73, 101.53, 55.75, 37.23; IR (CHCl3): nmax
3412, 2926, 1623, 1582, 1483, 1453, 1210 cmꢀ1; ESI-MS: m/z 451
1H); 13C NMR (CDCl3, 100 MHz):
d 138.60, 135.85, 134.06, 131.65,
130.55, 129.47,128.74,128.36,127.40,126.04,125.92,125.44,125.06,
124.13, 118.46, 113.18, 83.04, 35.54; IR (CHCl3): nmax 3389, 1621,
1384, 1038 cmꢀ1; ESI-MS: m/z 623 [M ꢀ 1]þ; HRMS: m/z 622.9475
calcd for C27H18I2N2 ꢀ Hþ (622.9476).
[Mþ1]þ, 473 [M
þ
Na]þ; HRMS: m/z 473.0800 calcd for
C
25H20Cl2N2O2 þ Naþ (473.0794).
4.2.17. 5,50-Dimethoxy-3,30-(naphth-1-yl-methanediyl)-bisindole
(9h)
4.2.11. 5,50-Diiodo-3,30-(anthracen-10-yl-methanediyl)-bisindole
(8i)
Light brown solid; m.p. 233e235 ꢁC; 1H NMR (CDCl3, 400 MHz):
d
8.16 (d, J ¼ 8.0 Hz, 1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.80 (s, 1H), 7.75 (d,
Brown solid; m.p. 121e123 ꢁC; 1H NMR (CDCl3, 500 MHz):
J ¼ 8.0 Hz, 1H), 7.46e7.40 (m, 3H), 7.30e7.25 (m, 5H), 6.83 (t,
d
8.54e8.48 (m, 2H), 8.05e7.90 (m, 4H), 7.68 (s, 1H), 7.50e7.25 (m,
7H), 7.12 (d, 5.2 Hz, 4H), 6.73 (s, 1H), 6.71 (s, 1H); 13C NMR (DMSO,
100 MHz): 135.10, 134.67, 133.13, 132.75, 130.77, 129.35, 128.89,
J ¼ 6.4 Hz, 4H), 6.58 (d, J ¼ 2.0 Hz, 1H), 6.52 (s, 1H), 3.66 (s, 6H); 13
C
NMR (DMSO-d6, 100 MHz):
d 152.73, 140.24, 133.59, 131.88, 131.38,
d
128.50, 127.01, 126.51, 125.70, 125.47, 125.24, 124.96, 124.14, 117.31,
112.06, 110.50, 101.43, 55.29, 35.49; IR (CHCl3): nmax 3389, 1621,
1384, 1038 cmꢀ1; ESI-MS: m/z 433.2 [Mþ1]þ, 455.2 [M þ Na]þ;
HRMS: m/z 455.1728 calcd for C29H24N2O2þNaþ (455.1730).
128.66, 128.31, 127.72, 126.88, 123.66, 120.13, 117.01, 112.16, 111.57,
82.02, 81.78, 33.38; IR (CHCl3): nmax 3416, 2927, 1622, 1582, 1483,
1453, 1210, 1172, 1028 cmꢀ1; ESI-MS: m/z 673 [M ꢀ 1]þ; HRMS: m/z
672.9621 calcd for C31H20I2N2 ꢀ Hþ (672.9632).
4.2.18. 5,50-Dimethoxy-3,30-(anthracen-10-yl-methanediyl)-
4.2.12. 5,50-Diiodo-3,30-(5-nitrofuran-2-yl-methanediyl)-bisindole
(8j)
bisindole (9i)
Brown solid; m.p. 123e125 ꢁC; 1H NMR (CDCl3, 500 MHz):
d 8.65
Green crystalline solid; m.p. 121e123 ꢁC; 1H NMR (CDCl3,
(d, J ¼ 8.9 Hz, 1H), 8.44 (s, 1H), 8.05 (brs, 2H), 7.81 (brs, 2H), 7.56e
400 MHz):
d
8.15 (s, 2H), 7.75 (s, 2H), 7.52 (m, 2H), 7.21 (t, J ¼ 4 Hz,
7.51 (dd, J ¼ 7.2, 18.3 Hz, 1H), 7.39e7.04 (m, 8H), 6.97e6.67 (m, 4H),
1H), 7.20 (d, J ¼ 8.4 Hz, 2H), 6.91 (d, J ¼ 2.4 Hz, 2H), 6.28 (d,
6.49 (s, 1H), 3.44 (s, 6H); 13C NMR (CDCl3, 125 MHz):
d 153.48,
J ¼ 3.2 Hz, 1H), 5.86 (s, 1H); 13C NMR (CDCl3, 125 MHz):
d
160.19,
135.14, 131.57, 127.75, 127.25, 125.32, 124.68, 118.38, 112.04, 111.61,
101.80, 55.58, 34.71; IR (CHCl3): nmax 3416, 2927, 1622, 1582, 1483,
1453, 1210, 1172, 1028 cmꢀ1; ESI-MS: m/z 483.2 [Mþ1]þ, 505.2
135.63, 130.98, 128.63, 127.90, 124.15, 113.62, 113.46, 112.78, 110.83,
83.45, 34.40; IR (CHCl3): nmax 3418, 2924, 1584, 1455, 1242, 1019,

S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
441
[M þ Na]þ, 521.2 [M þ K]þ; HRMS: m/z 483.2041 calcd for
Recyclability of the Fe-PILC B was checked using a model reac-
tion between 1H-indole (5a) and 4-hydroxy benzaldehyde (6a). To
the solution of indole 5a and aldehyde 6a in water was added
15 mol% Fe-PILC B and reaction mixture was stirred at room tem-
perature for 6 h. After completion of reaction, catalyst was recov-
ered by filtration followed by washing with water. Recovered
catalyst was dried in oven and reused in next cycle. The catalyst was
recycled 3 times and the amount of catalyst recovered and per-
centage yield of the diindolylmethane 7a were determined.
C
33H26N2O2þHþ (483.2067).
4.2.19. 5,50-Dimethoxy-3,30-(thiophen-2-yl-methanediyl)-bisindole
(9j)
Brown solid; m.p. 180e182 ꢁC; 1H NMR (CDCl3, 400 MHz):
d 7.78
(s, 2H), 7.18 (d, J ¼ 8.0 Hz, 2H), 7.08 (t, J ¼ 4.0 Hz, 1H), 6.85 (d,
J ¼ 4.0 Hz, 2H), 6.81e6.75 (m, 6H), 5.98 (s,1H), 3.64 (s, 6H); 13C NMR
(CDCl3, 125 MHz):
d 153.81, 148.22, 131.75, 127.72, 127.20, 125.16,
123.96, 123.63, 119.27, 11.198, 11.70, 56.13, 34.79; IR (CHCl3): nmax
2926, 2515, 1622, 1590, 1445, 1314, 1214, 1110 cmꢀ1; ESI-MS: m/z
387.1 [M ꢀ 1]þ; HRMS: m/z 387.1147 calcd for C23H20N2O2S ꢀ Hþ
(387.1162).
4.4. Assay for in vitro antileishmanial activity [54]
The assays for in vitro antileishmanial activity on cultures of L.
donovani promastigotes and axenic amastigotes were carried out in
96-well plates as reported earlier [55e58]. The promastigotes cul-
ture was maintained at 26 ꢁC in RPMI 1640 pH 7.4 with 10% FBS. The
axenic amastigotes were cultured at 37 ꢁC & 5% CO2 in RPMI-1640
fortified with 4-morpholineethanesulfonic acid (MES) (4.88 g/L),
4.2.20. 5,50-Dimethoxy-3,30-(5-nitrofuran-2-yl-methanediyl)-
bisindole (9k)
Green solid; m.p. 169e171 ꢁC; 1H NMR (CDCl3, 400 MHz):
d 7.91
(s, 2H), 7.23 (m, 2H), 6.88 (s, 2H), 6.83e6.79 (m, 4H), 6.72 (s, 1H),
6.25 (d, J ¼ 4.0 Hz, 1H), 5.81 (s, 1H), 3.69 (s, 6H); 13C NMR (CDCl3,
L-glutamine (298.2 mg/L), adenosine (26.7 mg/L), folic acid
125 MHz):
d
160.19, 135.63, 130.98, 128.63, 127.90, 124.15, 113.62,
(10.1 mg/L), BME vitamin mix, sodium bicarbonate (352.8 mg/L)
and 10% FBS. The pH of the culture medium was 5.5. Compounds at
various concentrations were added to the culture of Leishmania
promastigotes or axenic amastigotes (2 ꢂ 106 cells/mL). Plates were
incubated at 26 ꢁC for promastigotes and 37 ꢁC/5% CO2 for axenic
amastigotes for 72 h and growth of Leishmania promastigotes/
amastigotes was determined by Alamar Blue Assay [54,58]. Pent-
amidine and amphotericin B were used as standard drugs. IC50
values were computed from growth inhibition curves.
113.46, 112.78, 110.83, 83.45, 34.40; IR (CHCl3): nmax 3418, 2924,
1584, 1455, 1305, 1242, 1019 cmꢀ1; ESI-MS: m/z 418 [Mþ1]þ, 440
[M þ Na]þ, 456 [M þ K]þ; HRMS: m/z 418.1395 calcd for
C
23H19N3O5þHþ (418.1398).
4.3. Fe-PILC: preparation, characterization and recyclability
Ferric chloride (FeCl3) was used in Fe-PILC synthesis. A base-
hydrolyzed FeCl3 pillaring solution was prepared using OH/Fe
molar ratio of 2.0. The hydrolysis was carried out at room tem-
perature for 16 h under continuous stirring. Montmorillonite clay
K10 powder was added gradually to Fe pillared solution, providing
a required mmol of Fe per gram of clay. Fe Pillared clay suspension
was stirred for 3 h at room temperature to allow ion exchange
between exchangeable cations of the clay and pillar precursors. The
formed PILCs were centrifuged, washed, dried in air, and calcined in
air at 425 ꢁC for 3 h.
4.5. Assay for in vitro antimalarial activity [36]
The assay is based on the determination of plasmodial LDH ac-
tivity. For the assay, a suspension of red blood cells infected with D6
or W2 strains of P. falciparum (200
hematocrit in RPMI 1640 medium supplemented with 10% human
serum and 60 g/mL Amikacin) was added to the wells of a 96-well
plate containing 10 L of test samples diluted in medium at various
mL, with 2% parasitemia and 2%
m
m
Synthesized Fe-PILC catalyst was characterized using specific
surface area determination, temperature programmed reduction
(TPR), temperature programmed desorption (TPD) and scanning
concentrations. The plate was placed in a modular incubation
chamber (Billups-Rothenberg, CA) flushed with a gas mixture of
90% N2, 5% O2, and 5% CO2 and incubated at 37 ꢁC, for 72 h. Parasitic
LDH activity was determined by using MalstatÔ reagent (Flow Inc.,
Portland, OR) according to the procedure of Makler and Hinrichs
electron microscopy (SEM). The specific surface areas (m2 ꢀ1) of
g
samples were estimated with the N2 adsorption at the liquid ni-
trogen temperature on the Quantachrom Chem-BET 3000. Tem-
perature programmed reduction (TPR) profiles of catalyst
precursors were collected on the Quantachrom Chem-BET 3000
apparatus equipped with a thermal conductivity detector (TCD).
50 mg samples placed in a quartz U-shaped tube were reduced in
5%H2/N2 flow (80 mL minꢀ1) from room temperature to 700 ꢁC at a
temperature rise of 5 ꢁC minꢀ1. Samples were pretreated in N2 flow
(50 mL minꢀ1) at 300 ꢁC for 2 h to remove the adsorbed water and
other contaminants prior to each TPR experiment.
The total acidity and acid distribution of the pillared clays was
determined by ammonia TPD with the help of a CHEM BET-3000
(Quantachrome, USA) instrument in the temperature range of
30e800 ꢁC [53]. About 0.1 g of powdered sample was taken in-
side a quartz U tube and degassed at 350 ꢁC for 1 h with ultra-
pure helium gas. After the sample was cooled to room
temperature, NH3 gas of 1000 ppm with N2 gas was passed
through the sample for 1 h. Then the ammonia adsorbed sample
was purged with helium gas at 40 ꢁC to remove any weakly
adsorbed NH3 on the surface. The temperature-programmed
desorption of ammonia was performed between 80 and 800 ꢁC
at 10 ꢁC/min. The morphology of Fe-PILC B catalyst was studied
using JEOL.JEM100CXII electron microscope with ASID acceler-
ating voltage of 40 kV.
[36]. Briefly, 20 mL of the incubation mixture was mixed with 100 mL
of the MalstatÔ reagent and incubated at room temperature for
30 min. Twenty microliters of a 1:1 mixture of NBT/PES (Sigma, St.
Louis, MO) was then added and the plate is further incubated in the
dark for 1 h. The reaction was then stopped by the addition of
100 mL of a 5% acetic acid solution. The plate was read at 650 nm
using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Ver-
mont). IC50 values were computed from the dose response curves.
Artemisinin and chloroquine were included in each assay as the
drug controls. DMSO (0.25%) was used as vehicle control [55e57].
4.6. Assay for in vitro antimicrobial activity
Microorganisms [fungi, C. albicans (ATCC 90028), Candida krusei
(ATCC 6258), Candida glabrata (ATCC 90030), C. neoformans (ATCC
90113), and A. fumigatus (ATCC 204305) and bacteria, S. aureus
(ATCC 29213), methicillin-resistant S. aureus (MRSA, ATCC 33591),
E. coli (ATCC 35218), P. aeruginosa (ATTCC 27853), and M. intra-
cellulare (ATCC 23068)] were obtained from ATCC. For all organ-
isms, excluding M. intracellulare and A. fumigatus, susceptibility test
was performed using a modified version of the CLSI (formerly
NCCLS) methods [38,39], and optical density was used to monitor
growth. Media supplemented with 5% Alamar Blue (BioSource

442
S.B. Bharate et al. / European Journal of Medicinal Chemistry 63 (2013) 435e443
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[40] and A. fumigatus [37]. Concentrations that afford 50% inhibition
(IC50s) relative to controls were calculated using XLfit 4.2 software
(IDBS) using fit model 201 based on duplicate readings. Minimum
fungicidal or bactericidal concentrations were determined by
removing 5
mL from each clear well, transferring to agar, and
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4.7. Cytotoxicity assay
The in vitro cytotoxicity was determined against mammalian
kidney fibroblasts (VERO) and kidney epithelial (LLC-PK1) cells. The
assay was performed in 96-well tissue culture-treated plates as
described earlier [58]. Briefly, cells were seeded to the wells of the
plate (25,000 cells/well) and incubated for 24 h. Samples were
added and plates were again incubated for 48 h. The number of
viable cells was determined by neutral red assay. IC50 values were
determined from logarithmic graphs of growth inhibition versus
concentration. Doxorubicin was used as a positive control, while
DMSO was used as vehicle control.
Acknowledgments
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RM and RRY thanks CSIR (India) for award of Senior Research
Fellowship. Authors are thankful to analytical department, IIIM for
NMR, MS and IR analysis of our compounds. Surendra Jain, Marsha
Wright and John Trott are acknowledged for technical support in
biological activity testing at NCNPR. United States Department of
Agriculture (USDA), Agricultural Research Service Specific Cooper-
ative Agreement No. 58-6408-2-0009 and US Department of De-
fense CDMRP grant #W81XWH-09 (BLT) are acknowledged for
partial support of this work. Antifungal testing was supported by
NIH, NIAID, Division of AIDS grant # AI27094.
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