Synthesis and antibacterial evaluation of 3,3′-diindolylmethane derivatives

Article abstract of DOI:10.1007/s00044-013-0737-7

Various 3,3′-diindolylmethane (DIM) derivatives were synthesized and the antibacterial activity of these compounds were tested against ten bacterial strains and their minimum inhibitory concentration (MIC) values were determined. The MIC values of derivatives 3a-d and 5a-e were ranging from 125 to 500 μg/mL. Among these derivatives, 2-(di(1H-indol-3-yl)methyl)phenol (5a) and 3-((1H-indol-3-yl)(pyridin-3-yl)methyl)-1H-indole (5d) exhibited potent activity, showing MIC values 6.5-62.5 μg/mL against Gram positive and Gram negative bacteria. Hemolytic assay of these active DIM derivatives did not show considerable toxic effect on the normal human erythrocytes.

Full text of DOI:10.1007/s00044-013-0737-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0737-7
ORIGINAL RESEARCH
0
Synthesis and antibacterial evaluation of 3,3 -diindolylmethane
derivatives
Sutapa Roy
Arumugam Meyyapan
•
Rahul Gajbhiye
•
•
Madhumita Mandal
•
•
Churala Pal
Parasuraman Jaisankar
•
Joydeep Mukherjee
Received: 15 March 2013 / Accepted: 17 August 2013
Ó Springer Science+Business Media New York 2013
0
Abstract Various 3,3 -diindolylmethane (DIM) deriva-
tives were synthesized and the antibacterial activity of these
compounds were tested against ten bacterial strains and
their minimum inhibitory concentration (MIC) values were
determined. The MIC values of derivatives 3a–d and
resistant microorganisms especially bacteria has become a
serious threat to public health (Walsh, 2003). This has not
only made the treatment of infectious disease more prob-
lematic but has also resulted in the reappearance of many
diseases which were thought to be under control. Molecular
manipulations of promising lead compounds provide a
major line of approach to develop new drugs against
multidrug resistance bacteria. For example, some antibi-
otics like Gramicidin S are highly active against both Gram
positive and Gram negative bacteria, but the main side
effect of this drug is its high hemolytic activity which led
the chemists to design and synthesize antibacterial mole-
cules with low-hemolytic activity (Leslie et al., 1996).
Indole and its derivatives possess a wide range of biolog-
ical activities with interesting chemistry. There have been a
continuous efforts by synthetic organic chemists to develop
simple and efficient synthetic methodologies for bis(indo-
lyl)alkanes due to their excellent biological activities
5
a–e were ranging from 125 to 500 lg/mL. Among these
derivatives, 2-(di(1H-indol-3-yl)methyl)phenol (5a) and
-((1H-indol-3-yl)(pyridin-3-yl)methyl)-1H-indole (5d)
3
exhibited potent activity, showing MIC values 6.5–62.5 lg/
mL against Gram positive and Gram negative bacteria.
Hemolytic assay of these active DIM derivatives did not
show considerable toxic effect on the normal human
erythrocytes.
Keywords Diindolylmethane ꢀ Antibacterial activity ꢀ
MIC ꢀ Hemolytic activity ꢀ Cytotoxicity
0
Introduction
(Mandal et al., 2011). 3,3 -Diindolylmethane (DIM) is a
dimeric product of indole-3-carbinol (I3C), formed under
acidic condition and contains bis-indole moiety. DIM has
been the subject of much research due to its potential
ability in promoting healthier estrogen metabolism in
patients with thyroid proliferative disease (Rajoria et al.,
The rates of resistance of pathogenic microorganisms (Ge
et al., 2002) to conventional antibiotics are increasing
alarmingly and the remarkable increase of multidrug-
2
011), prevention of cancer development in transgenic
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0737-7) contains supplementary
material, which is available to authorized users.
mouse prostate adenocarcinoma model (Cho et al., 2011).
0
0
Some of our recent works showed that 2,2 -diphenyl-3,3 -
diindolylmethane induces apoptosis in breast cancer cells
by inhibiting EGFR pathway (Bhowmik et al., 2013). Also
we have reported that DIM and its derivatives are good
plant growth promoters (Pal et al., 2007) and potent
inhibitors of Leishmania donovani topoisomerase I (Roy
et al., 2008, 2009, 2011). Sung and Lee (2007) reported
that I3C, which was found in brassica vegetables, exhibit
antimicrobial activities against human pathogens. Some
S. Roy ꢀ A. Meyyapan ꢀ J. Mukherjee
School of Environmental Studies, Jadavpur University,
Kolkata 700 032, India
R. Gajbhiye ꢀ M. Mandal ꢀ C. Pal ꢀ P. Jaisankar (&)
Department of Chemistry, CSIR-Indian Institute of Chemical
Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032,
India
e-mail: jaisankar@iicb.res.in
123

Med Chem Res
bis-indole derivatives such as pyrazolylbisindoles and bis-
imidazolinylindole) have also been reported their antimi-
crobial properties (Sivaprasad et al., 2006; Panchal et al.,
009). However, to the best of our knowledge, the inhibi-
few minutes. It was then stirred at room temperature for
8 h. Water (10 mL) was added to the reaction mixture
followed by heating at 80 °C for 30 min. It was then
(
2
extracted with CHCl (3 9 25 mL). The organic layer was
3
tory activity of DIM derivatives on pathogenic bacteria has
not been established. We report herein the synthesis and
evaluation of antibacterial activity of some novel bis-
indolylmethane derivatives. Hemolytic activity of the
DIMs is also being reported for the first time to show that
these compounds do not posses considerable toxic effect on
the normal human erythrocytes.
dried over anhydrous Na SO and the solvent removed to
4
2
give a solid mass. Column chromatography of the residue
over silica gel using increasing concentrations of chloro-
form in petroleum ether yielded 3a as white solid (285 mg,
92 %). The same experimental procedure was followed
for the synthesis of the other DIM derivatives
3b–d (Scheme 1; Table 1) (Pradhan et al., 2005).
0
3
,3 -Diindolylmethane (3a) mp 159–161 °C; IR (KBr):
-
1 1
Materials and methods
tmax 3396, 1454, 1343, 741 cm ; H NMR (DMSO-d ,
6
3
00 MHz): d 4.13 (s, 2H, –CH –), 6.92 (t, J = 7.1 Hz, 2H,
2
0
0
Physical measurements
H-6, H-6 ), 7.03 (t, J = 7.1 Hz, 2H, H-5, H-5 ), 7.13 (brs,
0 0
H, H-2, H-2 ), 7.32 (d, J = 8.0 Hz, 2H, H-7, H-7 ), 7.52
2
1
0
13
(d, J = 8.0 Hz, 2H, H-4, H-4 ), 10.73 (s, 2H, 2 9 NH); C
H NMR spectra were recorded on a Bruker DPX
3
00 MHz and Bruker DRX 600 MHz NMR instrument at
NMR (CDCl ? DMSO-d , 75 MHz): d 26.43 (CH ),
116.52 (2 9 1C, C-2, C-2 ), 120.04 (2 9 1C, C-3, C-3 ),
3
6
2
0
0
ambient temperature either in CDCl or CD OD or dime-
3
3
1
3
0
0
thyl sulfoxide (DMSO) DMSO-d6. C NMR spectra were
recorded either at 75 or 150 MHz at ambient temperature
with complete proton decoupling. The chemical shifts were
recorded in parts per million (ppm) with TMS as internal
123.65 (2 9 1C, C-7, C-7 ), 124.07 (2 9 1C, C-6, C-6 ),
0
0
126.36 (2 9 1C, C-5, C-5 ), 127.79 (2 9 1C, C-4, C-4 ),
0
132.67 (2 9 1C, C-3a, C-3a ), 141.84 (2 9 1C, C-7a,
?
C-7a ); ESI–MS m/z: 269 [M?Na] ; Anal. Calcd. for
0
1
reference. H NMR is reported as follows: Chemical shift,
C H N : C, 82.90; H, 5.73; N, 11.37. Found: C, 82.65; H,
17 14 2
multiplicity (s = singlet, d = doublet, dd = doublet of
doublet, brs = broad singlet, t = triplet, m = multiplet),
coupling constant, and integration. Coupling constants
values are given in Hz. EIMS were recorded on a GC–MS
5.80; N, 11.49.
0
0
2,2 -Dimethyl-3,3 -diindolylmethane (3b) mp 127–128 °C;
IR (KBr): t
3396, 3052, 2917, 1681, 1461, 1322, 1230,
max
-1 1
Ò
Shimadzu-QP5050A and ESIMS were done on a waters
TM
744 cm ; H NMR (DMSO-d , 300 MHz): d 2.43 (s, 6H,
6
Ò
Micromass Q-TOF Micro
Mass Spectrometer. Mass
CH ) 3.98 (s, 2H, –CH –), 6.79 (t, J = 7.1 Hz, 2H, H-4,
3
2
0
0
0
spectral data are given in m/z unit. Melting points were
recorded on a SPAC-N SERVICE (India) open capillary
melting point apparatus (Laboratory device) and are
uncorrected. Infrared Spectra were recorded on a JASCO-
FT-IR Model-410. Spectra were calibrated against the
H-4 ), 6.9 (t, J = 7.1 Hz, 2H, H-7, H-7 ), 7.21 (m, 4H, H-5,
13
0
H-5 , H-6, H-6 ), 10.62 (s, 2H, 2 9 NH); C NMR
(CDCl ? DMSO-d , 75 MHz): d 12.44 (2 9 CH ), 19.72
3
6
3
0
0
(CH ), 110.57 (2 9 1C, C-7, C-7 ), 111.06 (2 9 1C, C-3,
2
0
C-3 ), 118.59 (2 9 1C, C-6, C-6 ), 118.69 (2 9 1C, C-4,
-
1
0
0
polystyrene absorption at 1,601 cm
.
C-4 ), 120.51 (2 9 1C, C-5, C-5 ), 129.36 (2 9 1C, C-7a,
Synthesis of compounds
3
Table 1 InCl catalyzed formation of DIM derivatives 3a–d
1
R
2
a
Typical experimental procedure for InCl catalyzed
3
Products
R
Time (h)
Yield (%)
preparation of DIM (3a)
3
3
3
3
a
a
b
c
H
H
8
10
8
72
87
89
90
Me
H
H
Anhydrous InCl3 (67 mg; 0.3 mmol) was added to a
solution of indole (1a; 351 mg; 3 mmol) and hexamethy-
lenetetramine (HMTA) (2; 35 mg; 0.25 mmol) in dry i-
PrOH (5 mL). The reaction mixture turned turbid after a
OMe
H
d
Ph
12
Isolated yield
Scheme 1 Reagent and
conditions (i) InCl (10 mol%),
i-PrOH, 80 °C, 8–12 h
R2
N
R2
R2
3
(i)
+
N
N
N
R1
N
N
N
R1
H
H
R1
H
2
3a-d
1
a-d
1
23

Med Chem Res
0
0
C-7a ), 132.0 (2 9 1C, C-3a, C-3a ), 136.0 (2 9 1C, C-2a,
using catalytic amount of InCl (Scheme 2; Table 2) (Chen
3
0
?
C-2a ); ESI–MS m/z: 297 [M?Na] ; Anal. Calcd. for
et al., 1996; Ezzat et al., 2012; Samad et al., 2012).
C H N : C, 83.16; H, 6.61; N, 10.21. Found: C, 82.54; H,
19 18 2
1
5
.92; N, 11.04. Found: C, 82.37; H, 6.12; N, 10.67.
2-(Di(1H-indol-3-yl)methyl)phenol
(5a)
H
NMR
(
300 MHz, CDCl ): d 5.97 (s, 1H), 6.72 (s, 1H), 6.88 (m,
3
0
0
5
,5 -Dimethoxy-3,3 -diindolylmethane (3c) mp 112–114 °C;
3H), 7.09 (m, 3H), 7.13–7.62 (m, 8H), 7.93 (brs, 2 9 NH);
3
1
0
IR (KBr): tmax 3416, 3387, 2932, 2830, 1620, 1584,1484,
C NMR (CDCl , 75 MHz): d 35.46 (CH), 111.3, (C-3 ,
3
-1 1
294, 1212, 1059, 808 cm ; H NMR (300 MHz, CDCl3):
00 0 00 0 00
1
C-3 ), 116.4 (C-2), 117.1 (C-7 , C-7 ), 119.4 (C-6 , C-6 ),
0
00
0
00
d 3.81 (s, 6H), 4.17 (s, 2H), 6.83–6.91 (m, 4H), 7.06 (s,
119.8 (C-4 , C-4 ), 120.7 (C-4), 122.1 (C-5 , C-5 ), 123.7
1
3
0 00 0 00
2
H), 7.25 (d, J = 8.8 Hz, 2H), 7.81 (s, 2H, 2 9 NH);
C
(C-2 , C-2 ), 126.8 (C-6), 127.9 (C-3 a, C-3 a), 129.3 (C-
0
00
NMR (CDCl , 75 MHz): d 26.53 (CH ), 60.94 (2 9 1C,
2
3), 129.9 (C-5), 136.82 (C-7 a, C-7 a).
3
2
0
9 OCH ), 106.01 (2 9 1C, C-4, C-4 ), 116.40 (2 9 1C,
3
0
0
0
0
C-6, C-6 ), 117.26 (2 9 1C, C-7, C-7 ), 119.58 (2 9 1C,
0
1
-Furyl-3,3 -diindolylmethane (5b) H NMR (300 MHz,
2
C-3, C-3 ), 128.66 (2 9 1C, C-2, C-2 ), 132.97 (2 9 1C,
0
CDCl ): d 5.94 ((s, 1H), 6.05 (d, J = 7.2 Hz, 1H), 6.29 (s,
0
3
C-7a, C-7a ), 137.118 (2 9 1C, C-3a, C-3a ), 158.46
1H), 6.88 (s, 1H), 7.05 (t, J = 7.5 Hz, 2H), 7.16 (t,
0
?
2 9 1C, C-5, C-5 ); ESI–MS m/z: 329 [M?Na] ; Anal.
(
J = 7.5 Hz, 2H), 7.255 (m, 3H), 7.47 (d, J = 7.8 Hz, 3H),
13
Calcd. for C H N O : C, 74.49; H, 5.92; N, 9.14. Found:
2
1
9
18
2
7
.97 (s, 2H, 2 9 NH); C NMR (CDCl , 75 MHz): 35.08
0 00
3
C, 75.09; H, 6.30; N, 9.19.
(
CH), 106.6 (C-3), 110.1 (C-4), 111.13, (C-3 , C-3 ), 117.1
0 00 0 00 0 00
(
C-7 , C-7 ), 119.3 (C-6 , C-6 ), 119.6 (C-4 , C-4 ), 121.9
0 00 0 00 0 00
0
0
2
,2 -Diphenyl-3,3 -diindolylmethane (3d) mp 184–186 °C;
IR (KBr): t
H NMR (300 MHz, CDCl ): d 4.57 (s, 2H), 6.85–6.88 (m,
(
C-5 , C-5 ), 123.07 (C-2 , C-2 ), 126.7 (C-3 a, C-3 a),
0 00
36.50 (C-7 a, C-7 a), 141.2 (C-5), 157.0 (C-2).
-1
3301, 2815, 1640, 1452, 1247, 1050 cm ;
max
1
1
3
2
9 1H), 7.05–7.08 (m, 2 9 1H), 7.19–7.43 (m, 2 9 5H),
1
.56–7.58 (m, 2 9 2H), 8.00 (brs, 2 9 NH); C NMR
3
7
1-[Bis(1H-indol-3-yl)methyl]-4-(dimethylamino)benzene
1
(
(
(
(
(
CDCl , 75 MHz): d 21.70, 110.89 (2 9 1C), 112.47
(5c) H NMR (CDCl 300 MHz): d 5.79 (s, 1H), 6.67 (d,
3
3,
2 9 1C), 119.84 (2 9 1C), 120.42 (2 9 1C), 122.37
2 9 1C), 127.91 (2 9 1C), 128.74 (2 9 2C), 129.09
2 9 2C), 129.78 (2 9 1C), 133.74 (2 9 1C), 134.90
J = 8.7 Hz, 4H), 7.05 (t, J = 7.5 Hz, 2H), 7.13 (m, 4H),
1
7.35 (m, 4H), 7.90 (s, 2H, 2 9 NH); C NMR (CDCl3,
3
75 MHz): 23.36 (2 9 1C, 2 9 CH3), 40.43 (CH), 111.2,
0 00 0 00
(C-3 , C-3 ), 112. 7 (C-3, C-5), 117.7 (C-7 , C-7 ), 118.9
?
2 9 1C), 136.32 (2 9 1C); ESI–MS m/z: 421 [M?Na] .
0 00 0 00 0 00
Anal. Calcd. for C H N : C, 87.41; H, 5.56; N, 7.03.
9 22 2
(C-6 , C-6 ), 119.1 (C-4 , C-4 ), 121.7 (C-5 , C-5 ), 123.41
2
0 00 0 00
(C-2 , C-2 ), 127.0 (C-3 a, C-3 a), 129.18 (C-2, C-6), 136.3
Found: C, 87.32; H, 5.60; N, 6.99.
0 00
Other DIM derivatives were synthesized from their
corresponding benzaldehydes 4a–e and indole (1a) by
(C-7 a, C-7 a), 146.33 (C-4).
1
3
-((1H-Indol-3-yl)(pyridin-3-yl)methyl)-1H-indole (5d)
NMR (300 MHz, CDCl ): d 5.90 (s, 1H), 6.12 (S, 2H), 7.03
H
R
3
(
t, J = 7.8 Hz, 2H), 7.16 (t, J = 7.8 Hz, 2H), 7.35 (m,
(ii)
+
R-CHO
3H), 7.55 (d, J = 7.8 Hz, 1H), 8.20 (s, 2H, 2 9 NH), 8.45
13
(s, 2H), 8.64 (s, 1H); C NMR (CDCl , 75 MHz): 37.34
3
N
N
N
H
H
H
0 00 0 00 0
1
a
4a-e
5a-e
3
(10 mol%), i-PrOH,
(CH), 110, (C-3 , C-3 ), 111.33 (C-7 , C-7 ), 117.7 (C-6 ,
0
0
0
00
0
00
C-6 ), 118.9 (C-4 , C-4 ), 119.15 (C-5), 121.7 (C-5 , C-5 ),
Scheme 2 Reagent and conditions (ii) InCl
0 °C, 9–15 h
0 00 0 00
1
1
23.33 (C-2 , C-2 ), 126.20 (C-3 a, C-3 a), 134.16 (C-6),
0
8
00
36.3 (C-7 a, C-7 a), 146.10 (C-2), 149.10 (C-4).
1
Tris(1H-indol-3-yl)methane (5e) H NMR (300 MHz,
Table 2 InCl
3
catalyzed formation of DIM derivatives 5a–e
a
Aldehydes
Products Time (h) Yield (%)
CDCl ): d 6.16 (s, 1H), 6.76 (s, 3H), 7.69 (t, J = 7.5 Hz,
3
3
H), 7.1 (t, J = 7.5 Hz, 3H), 7.26 (s, 3H), 7.35 (d,
Salicylaldehyde (4a)
5a
5b
10
15
10
12
9
92
87
89
87
90
J = 8.1 Hz, 3H), 7.35 (d, J = 7.8 Hz, 3H), 7.995 (s, 3H,
13
3
Furan-2-carboxaldehyde (4b)
9 NH); C NMR (CDCl , 75 MHz): 40.40 (CH), 110,
3
p-Dimethylaminobenzaldehyde (4c) 5c
0 00 0 00 0
(C-3 , C-3 , C-3), 118.5 (C-6 , C-6 C-6), 119.6 (C-4 ,
Pyridine-3-carboxaldehyde (4d)
Indole-3-carboxaldehyde (4e)
5d
5e
00
0
00
0
00
C-4 , C-4), 121.2 (C-5 , C-5 , C-5), 122.9 (C-2 , C-2 ,
0
00
0
00
C-2), 126.72 (C-3 a, C-3 a, C-3a), 136.3 (C-7 a, C-7 a,
C-7a).
a
Isolated yield
123

Med Chem Res
0
?
The synthesis of bis(3 -indolyl)alditols were achieved by
m/z: 389 [M?Na] Anal. Calcd for C H N O : C,
21 22 2 4
utilizing the direct C-glycosylation method involving a
65.61; H, 6.29; N, 7.29. Found: C, 65.28; H, 5.90; N, 7.12.
catalytic amount of InCl in aqueous media. This method
3
0
permitted the reaction to proceed under mild, simple, and
1-Deoxy-1,1-bis(3 -indolyl)-D-glucitol (7b) mp 108–110 °C;
2
5
environmentally benign conditions. Accordingly, indole 1a
(
[a] ?55 (c1.2, MeOH); IR (KBr): t
1419, 1342, 1089, 747 cm
3406, 2927, 1623,
D
max
-
1
1
233 mg, 1.99 mmol) was added to 2 mL of 1:1 ethanol–
;
H NMR (DMSO-d6,
water followed by D-glucose (6b) (180 mg, 1.0 mmol), and
InCl (44 mg, 0.199 mmol). The solution was then refluxed
300 MHz): d (glucose moiety) 3.38 (br, J = 6.0 and
10.8 Hz, 1H, H-6a), 3.50 (m, 1H, H-5), 3.64 (m, 2H, H-4
and -6b), 3.72 (m, J = 4.2 and 6.8 Hz, 1H, H-3), 4.29 (m,
2H, OH 9 2), 4.46 (dd, J = 4.2 and 7.3 Hz, 1H, H-2),
4.48, 4.49, and 4.50 (m, each 1H, OH 9 3), 4.72 (d,
J = 7.3 Hz, 1H, H-1), (indolyl moiety) 6.80–6.98 (m, 4H,
3
for 6 h and the reaction mixture was evaporated under
reduced pressure. The residue was then purified by column
chromatography over silica gel using increasing concen-
trations of methanol in chloroform to yield 7b as white
solid (285 mg, 72 %). The same procedure was followed
for the synthesis of other DIM derivatives (Scheme 3;
Table 3).
0
00
0
00
0
H-5 , H-5 , H-6 , H-6 ), 7.17 (d, J = 2.1 Hz, 1H, H-2 ),
00
0
7.24 (d, J = 8.0, 2H, H-7 , H-7 ), 7.38 (d, J = 1.7 Hz, 1H,
0
0
H-2 ’), 7.54 (d, J = 8.0 Hz, 1H, H-4 ), 7.63 (d, J = 8.0 Hz,
00
1
H, H-4 ), 10.69 (d, J = 2.1 Hz, 1H, NH), 10.71 (d,
13
0
1
-Deoxy-1,1-bis(3 -indolyl)-D-ribitol (7a) mp 101–103 °C;
D
J = 1.7 Hz, 1H, NH); C NMR (DMSO-d6, 75 MHz): d
(glucose moiety) d 36.2 (C-1), 63.6 (C-6), 69.4, 71.7, 73.8,
2
5
[
a] ?52.4 (c 1.00, MeOH); IR (KBr): tmax 3405,
-
923,1620, 1454, 1422, 1341, 1223, 1084, 748 cm ; H
1
1
0 00
2
75.6, (indolyl moiety) 111.2, 111.3 (C-3 , C-3 ), 115.9,
0 00 0 00
117.4 (C-7 , C-7 ), 117.9, 118.1 (C-6 , C-6 ), 119.3, 119.6
NMR (DMSO-d6, 300 MHz): d (ribose moiety): 3.31 (m,
0 00 0 00 0
1
H, H-3), 3.43 (m, 1H, H-5a), 3.58 (dd, J = 3.6 and
(C-4 , C-4 ), 120.4, 120.7 (C-5 , C-5 ), 123.1, 123.5 (C-2 ,
00 0 00 0
C-2 ), 127.1, 127.9 (C-3 a, C-3 a), 136.3, 136.2 (C-7 a,
1
1.0 Hz, 1H, H-5b), 3.69 (m, 1H, H-4), 4.15 (d, 1H,
0
0
?
C-7 a); ESI–MS m/z: 419 [M?Na] Anal. Calcd for
OH 9 1), 4.44 (s, 1H, OH 9 1), 4.62–4.67 (m, 2H,
OH 9 3); 4.86 (d, J = 8.6 and 2.4 Hz, 1H, H-2), 4.98 (m,
C H N O : C, 66.05; H, 6.15; N, 7.00. Found: C, 66.04;
22 24 2 5
J = 2.4 Hz, 1H, H-1, d (indolyl moiety): 6.85–7.35 (m,
H, 6.18; N, 6.92.
0
8
H, ArH), 7.50 and 7.59 (d, J = 2.0 Hz, each 1H, H-2 and
0
0
H-2 ), 10.69 and 10.77 (d, J = 2.0 Hz, each 1H, NH 9 2);
1
0
1
-Deoxy-1,1-bis(3 -indolyl)-D-mannitol (7c) mp 100–111 °C;
D
3
C NMR (DMSO-d , 75 MHz): d (ribose moiety): 35.4
25
6
[
a] ?59.4 (c1.0, MeOH); IR (KBr): t 3407, 2889, 1624,
max
(
C-1), 63.4 (C-5), 73.13, 74.37, 76.9, (indolyl moiety)
-1
1
1
3
1
341,1216, 1079, 747 cm
;
H NMR (DMSO-d6,
0 00 0 00
1
1
1
1
11.8, 112.06 (C-3 , C-3 ), 114.9, 118.6 (C-7 , C-7 ),
0
00 MHz): d (mannose moiety): 3.51 (m, J = 6.0 and
0.8 Hz, 1H, H-6a), 3.54 (m, 1H, H-5), 3.58 (m, 2H, H-4 and
00
0
00
00
18.7, 118.9 (C-6 , C-6 ), 119.6, 120.3 (C-4 , C-4 ), 121.1,
0
00
0
21.3 (C-5 , C-5 ), 124.09, 125.15 (C-2 , C-2 ), 127.8,
0
-
6b), 3.62 (m, J = 4.2 and 6.8 Hz, 1H, H-3), 4.09–4.30 (m,
H, OH 9 5), 4.42 (d, J = 4.2 and 7.3 Hz, 1H, H-2), 4.99 (s,
J = 7.4 Hz, 1H, H-1), (indolyl moiety): 6.82–6.90 (m, 4H,
00
0
00
29.2 (C-3 a, C-3 a), 136.6, 136.9 (C-7 a, C-7 a);. ESI–MS
5
0
00
0
00
0
H-5 , H-5 , H-6 , H-6 ), 7.14 (d, J = 2.1 Hz, 1H, H-2 ), 7.28
0 00 0
0 00
.54 (t, J = 8.0 Hz, 2H, H-4 , H-4 ), 10.65 (d, J = 2.1 Hz,
13
1H, NH), 10.74 (d, J = 1.7 Hz, 1H, NH); C NMR (DMSO-
(
d, J = 8.0, 2H, H-7 , H-7 ), 7.36 (1, J = 1.7 Hz, 1H, H-2 ’),
Sugar
7
(
iii)
+
Sugar
N
H
N
N
d 75 MHz): d (mannose moiety): d 34.9 (C-1), 64.8 (C-6),
6
,
H
H
7
3
0.64, 70.8, 72.3, 74.16, (indolyl moiety) 111.74, 111.97 (C-
0
1
a
6a-d
7a-d
(10 mol%), EtOH–H
0
00
0
00
, C-3 ), 115.0,118.5 (C-7 , C-7 ), 118.6, 119.4 (C-6 ,
0
0
0
00
0
00
C-6 ), 119.7, 120.3 (C-4 , C-4 ), 121.02,121.3 (C-5 , C-5 ),
Scheme 3 Reagent and conditions (iii) InCl
(
3
2
O
0 00 0 00
1:1), reflux, 6–9 h
124.12, 125.1 (C-2 , C-2 ), 127.9, 129.2 (C-3 a, C-3 a),
?
0
00
1
36.6, 136.9 (C-7 a, C-7 a); ESI–MS m/z: 419 [M?Na]
Anal. Calcd for C H N O : C, 65.67; H, 6.02; N, 7.13.
22 24 2 5
3
Table 3 InCl catalyzed formation of DIM derivatives 7a–d
Found: C, 65.94; H, 6.11; N, 6.97.
a
Sugar
Products
Time (h)
Yield (%)
D-Ribose (6a)
7a
7b
7c
7d
9
6
6
7
72
72
70
70
0
1
-Deoxy-1,1-bis(3 -indolyl)-D-galacitol (7d) mp 128–130 °C;
D-Glucose (6b)
D-Mannose (6c)
D-Galactose (6d)
25
[
a]D -188 (c 1.12, MeOH); IR (KBr): t 3405, 2933,
max
-1 1
622, 1420, 1342, 1220, 1089, 747 cm ; H NMR (DMSO-
1
d6, 300 MHz): d (galactose moiety) 3.30 (d, J = 6.0 and
10.8 Hz, 1H, H-6a), 3.44 (s 1H, H-5), 3.49 (m, J = 4.2 and
a
Isolated yield
1
23

Med Chem Res
6
.8 Hz, 3H, H-3, H-4, 6b), 3.69 (t, 2H, OH 9 2), 4.05 (t, 3H,
Results and discussion
OH 9 3), 4.4 (s, J = 4.2 and 7.3 Hz, 1H, H-2), 4.69 (s,
J = 7.3 Hz, 1H, H-1), (indolyl moiety) 6.83–6.96 (m, 4H,
Chemistry
0
0
00
00
0
00
0
0
H-5 , H-5 , H-6 , H-6 ), 7.27 (t, J = 2.1 Hz, 4H, H-2 , H-2 ’,
0
00
0
H-7 , H-7 ), 7.56 (t, J = 8.0 Hz, 2H, H-4 , H-4 ), 10.67 (d,
1
Various substituted 3,3 -DIM derivatives 3a–d, 5a–e, and
3
J = 2.1 Hz, 1H, NH), 10.72 (d, J = 1.7 Hz, 1H, NH);
C
7a–d were synthesized by using different synthetic meth-
odologies. It was observed that treatment of 12 equivalent
of indoles 1a–d with one equivalent of HMTA (2) in the
presence of catalytic amount of indium trichloride
NMR (DMSO-d 75 MHz): d (galactose moiety): d 37.6 (C-
6
,
1
1
1
), 64.14 (C-6), 70.7,70.75, 70.98, 72.03, (indolyl moiety)
0
00
0
00
11.95, 111.3 (C-3 , C-3 ), 117.5, 118.2 (C-7 , C-7 ), 118.5,
0
0
00
00
0
18.7 (C-6 , C-6 ), 119.3, 120.6 (C-4 , C-4 ), 121.1, 121.3
0
(10 mol%) in i-PrOH at 80 °C for 8–12 h resulted in 3,3 -
00
0
00
0
(
C-5 , C-5 ), 123.4, 123.6 (C-2 , C-2 ), 127.95, 128.5 (C-3 a,
0
DIMs 3a–d with 72–90 % yields (Scheme 1). Other DIM
derivatives (5a–e) were obtained from their corresponding
benzaldehydes 4a–e and indole (1a) by using catalytic
0
0
00
C-3 a), 136.93, 137.07 (C-7 a, C-7 a); ESI–MS m/z: 419
?
[
M?Na] Anal. Calcd for C H N O : C, 66.55; H, 6.19;
22 24 2 5
N, 7.43. Found: C, 66.08; H, 6.26; N, 7.1.
amount of InCl with 87–92 % yields (Scheme 2). When
3
two-equivalent of indole 1a selectively reacted with the
one-equivalent of sugars 6a–d to gave corresponding
0
In vitro bioassays
1-deoxy-1,1-bis(3 -indolyl)alditols 7a–d with 70–72 %
yields (Scheme 3). Detailed mechanism for Schemes 1, 2,
and 3 are discussed in supplemental information. The
structures of the newly synthesized compounds were con-
Antibacterial activity bioassay
1
13
Twenty-four hours cultures of bacterial strains were used
for the antibacterial screening. The organisms were pre-
served at refrigerated condition and revived once in a week
and used for further analysis. Two percent agar was added
to nutrient broth to prepare nutrient agar media. Stock
solutions of DIMs and streptomycin were prepared in
DMSO and stored at -20 °C. Antibacterial activity was
evaluated by measuring minimum inhibitory concentration
firmed by IR, Mass spectroscopy, H NMR, and C NMR
(Supplemental Fig. 2–27).
Antibacterial activity bioassay
0
All the synthesized 3,3 -indolylmethane (DIM) derivatives
3a–d, 5a–e, and 7a–d were evaluated for in vitro antibac-
terial activity against various Gram positive and Gram
negative bacterial strains by agar plate bioassay method.
Bacterial strains were obtained from the Microbial Type
Culture Collection, Chandigarh, India. Ten bacterial strains
were used for antibacterial activity test of which four were
Gram negative bacteria (Pseudomonas aeruginosa, Shigella
flexneri, Proteus mirabilis and Escherichia coli), six were
Gram positive bacteria (Staphylococcus aureus, Bacillus
subtilis, Bacillus subtilis, Rhodococcus sp., Arthrobacter
protophormia, and Mycobacterium smegmatis). Antibacte-
rial activities of the tested compounds were expressed in
terms of MIC as shown in Table 4.
(
MIC) against the test organism in comparison to a nega-
tive and reference standard Streptomycin. Each assay was
repeated twice.
Hemolytic assay
Blood from healthy human volunteers was collected in
1
0 mL vacutainer tubes containing sodium heparin as
anticoagulant. The cells were washed till the supernatant
liquid become clear and colorless. Then 0.5 mL erythro-
cyte suspension diluted with 0.5 mL PBS (erythrocyte
Our focus was to develop DIM derivatives having lower
MIC values against bacterial strains than the parent mole-
cule 3a with least hemolytic activity, because the unsub-
stituted DIM 3a showed MIC in the range of 125–500 lg/
mL (Table 4). We have synthesized DIM derivatives with
substitutions at C-2 and C-5 of indole moiety
3b–d. Unfortunately, none of the DIM derivatives such as
9
concentration 10 cells/mL) was mixed with 0.1 mL test
substance at a series of concentration (12.5–400 lg/mL).
The mixture was incubated at 37 8C for 1 h. After incu-
bation, the tube was centrifuged at 2,0009g for 10 min.
Optical density of the supernatants was then measured at
5
40 nm using a spectrophotometer (Colona Electric,
0
0
0
Japan). The values for 0 and 100 % lysis were determined
2,2 -dimethyl-DIM 3b, 2,2 -diphenyl-DIM 3c, or 5,5 -
dimethoxy-DIM 3d, showed better antibacterial activity
than the parent molecule DIM 3a (Table 4). Whereas
substitution at methylene carbon of DIM 3a had indeed
improved its efficacy to great extent. Thus, (2-hydroxy-
by incubating erythrocytes with PBS and distilled H O,
2
respectively. The percent hemolysis was calculated by the
following formula:
[
(Abs540 for the sample with hemolysin) - (Abs540 for
the control without hemolysin)/(Abs540 for the sample with
00 % lysis)] 9 100.
0
phenyl)-di-3,3 -indolylmethane (5a) and 3-pyridyl-di-3-
indolylmethane (5d) showed good antibacterial activity
1
123

Med Chem Res
Streptomycin
Table 4 Minimum inhibitory concentration (MIC) of DIM derivatives against bacteria
Microorganisms
MIC (lg/mL)
3
a
3b
3c
3d
5a
5b
5c
5d
5e
7a
7b
7c
7d
Gram positive bacteria
S. aureus
250
250
500
125
125
125
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
15.5
15.5
31.2
7.5
125
125
NA
NA
NA
NA
NA
NA
32.2
15.5
15.5
6.5
250
250
500
62.5
125
62.5
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
12.5
1.5
B. subtilis
L. lactis
62.5
1.5
Rhodococcus sp.
A. protophormiae
M. smegmatis
Gram negative bacteria
P. aeroginosa
S. flexneri
31.2
31.2
15.5
31.2
125
31.2
15.5
62.5
31.2
0.75
0.75
1.25
15.5
6.5
15.5
31.2
250
125
250
250
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
62.5
15.5
15.5
15.5
31.2
31.2
31.2
62.5
NA
NA
NA
NA
31.2
15.5
15.5
31.2
125
250
250
125
62.5
31.2
31.2
62.5
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.15
6.25
1.25
6.25
P. mirabilis
E. coli
NA not significant (MIC [500 lg/mL)
with MIC value ranging from 7.5 to 15.5 and 6.5 to 125 lg/
mL, respectively, against both Gram positive and Gram
negative bacterial strains. Our investigation was not
restricted to aromatic methylene-substituted DIMs, other
DIMs with methylene-substituted sugar moieties 7a–
d were also tested. The results confirmed that pentose-
ribose-substituted DIM 7a appeared to be very much safe
as it lysed only 15 % of erythrocytes up to the concentra-
tion of 400 lg/mL, which is almost tenfold greater than its
MIC value. But in case of other substituted DIMs 3a, 5a,
5d, and 5e, we could observe 20–30 % lysis of erythrocytes
up to a concentration of 200 lg/mL. Compound 5b lysed
50 % erythrocytes at a concentration of 12.5 lg/mL.
Whereas ribose-substituted DIM 7a exhibited low-hemo-
lytic property, while pyridine-substituted DIM 5e showed
high hemolytic property up to the concentration as high as
400 lg/mL. However, our template molecule 3a and other
substituted DIMs 5a, 5d exhibited medium hemolytic
properties.
0
substituted DIM, 1-deoxy-1,1-bis(3 -indolyl)-D-ribitol (7a)
has got better MIC in the range of 15.5–125 lg/mL com-
pared to other hexose-substituted DIMs 7b–d. Thus, all
these results suggest that substitution of DIM at the indole
nuclei led to loss of antibacterial activity, whereas substi-
tution at the methylene carbon of the DIM, either with
aromatic groups such as furan, pyridine, etc. or with dif-
ferent sugars resulted in the enhancement of its antibacte-
rial potency against both Gram positive and Gram negative
bacterial strains.
Hemolytic assay
In view of the antibacterial properties of the DIMs, it was
imperative to investigate if the compounds mediate any
toxic effect on the normal human cells. Such type of
cytotoxic studies helps us to develop a highly potent anti-
bacterial drug having less toxic effects. Therefore, a direct
approach was sought by using human red blood cells to
analyze hemolytic activity of the DIMs. Since DIM
derivatives proved to be good antibacterial agents, showing
a strong inhibition of the growth of Gram positive and
Gram negative bacteria, we therefore performed in vitro
hemolytic assay of DIM derivatives at different concen-
tration as a measure of their cell cytotoxicity. Figure 1
shows the percent hemolytic activities of DIM derivatives
at various concentrations. The results showed that only
Fig. 1 Hemolytic activity of DIM derivatives against normal human
erythrocytes
1
23

Med Chem Res
Conclusion
hemolytic activity by ring size in cyclic gramicidin S analogs.
J Biol Chem 41:25261–25268
Mandal M, Kumar D, Roy R, Sen R, Das P, Chatterjee M, Jaisankar P
A novel series of bisindolylmethane derivatives were synthe-
sized and their antibacterial and hemolytic activities were
evaluated. It was found that amongst all the DIM derivatives
tested, 2-(di(1H-indol-3-yl)methyl)phenol (5a), 3-((1H-Indol-
0
(
2011) Regioselective one pot synthesis of 3,3 -diindolylethylene
derivatives and study of their cytotoxic activity. Bioorg Med
Chem Lett 21:3084–3087
Pal C, Dey S, Mahato SK, Vinayagam J, Pradhan PK, Giri VS,
Jaisanka P, Hossain T, Barui S, Ray D, Biswas SM (2007) Eco-
3
-yl)(pyridin-3-yl)methyl)-1H-indole (5d), and 1-deoxy-1,1-
0
friendly synthesis and study of new plant growth promoters: 3,3 -
diindolylmethane and its derivatives. Bioorg Med Chem Lett
17:4924–4928
0
bis(3 -indolyl)-D-ribitol (7a) have stronger antibacterial
activity as compared to other DIM derivatives such as 5b, 5e,
and 3a. Also DIM derivative of D-ribose origin 7a lysed the
normal human erythrocytes the least among the other anti-
bacterial DIM derivatives such as 3a, 5a, and 5d. Compound
Panchal RG, Ulrich RL, Lane D, Butler MM, Houseweart C,
Opperman T, Williams JD, Peet NP, Moir DT, Nguyen T,
Gussio R, Terry B, Bavaril S (2009) Novel broad-spectrum bis-
(imidazolinylindole)derivatives with potent antibacterial activi-
ties against antibiotic-resistant strains. Antimicrob Agents Che-
mother 53:4283–4291
5
b possesses the highest toxic effect on normal human eryth-
rocytes. These results suggest that DIM derivatives may fur-
ther be developed as possible drug candidates for the treatment
of infectious diseases caused by bacteria with low-hemolytic
property.
3
Pradhan PK, Dey S, Giri VS, Jaisankar P (2005) InCl -HMTA as a
methylene donor: one-pot synthesis of diindolylmethane (DIM)
and its derivatives. Synthesis 11:1779–1782
Rajoria S, Suriano S, Parmar PS, Wilson YL, Megwalu U, Moscatello
A, Bradlow HL, Sepkovic DW, Geliebter J, Schantz SP, Tiwari
0
RK (2011) 3,3 -diindolylmethane modulates estrogen metabo-
lism in patients with thyroid proliferative disease: a pilot study.
Thyroid 21:299–304
Acknowledgments Authors Madhumita Mandal and Rahul Ga-
jbhiye thankfully acknowledge CSIR and UGC India for the financial
support in the form of research fellowships. This project was sup-
ported by the Grant from CSIR in the form of Network Projects
(ORIGIN, CSC 0108). Special thanks to Sourav Chatterjee, IICB for
his valuable suggestion.
Roy A, Ganguly A, Dasgupta SB, Das BB, Pal C, Dey S, Jaisankar P,
Majumder HK (2008) Mitochondria dependent ROS-mediated
0
programmed cell death (PCD) induced by 3,3 -diindolylmethane
(
DIM) through inhibition of FoF1-ATP synthase in unicellular
protozoan parasite Leishmania donovani. Mol Pharmacol
4:1292–1307
7
Roy A, BoseDasgupta S, Ganguly A, Jaisankar P, Majumder HK
(2009) Topoisomerase I gene mutations at F270 in the large
subunit and at N184 in the small subunit contribute to the
resistance mechanism of unicellular parasite Leishmania dono-
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