Synthesis of novel bisindolylmethane Schiff bases and their antibacterial activity

Article abstract of DOI:10.3390/molecules190811722

In an effort to develop new antibacterial drugs, some novel bisindolylmethane derivatives containing Schiff base moieties were prepared and screened for their antibacterial activity. The synthesis of the bisindolylmethane Schiff base derivatives 3-26 was carried out in three steps. First, the nitro group of 3,3′-((4-nitrophenyl)-methylene)bis(1H-indole) (1) was reduced to give the amino substituted bisindolylmethane 2 without affecting the unsaturation of the bisindolylmethane moiety using nickel boride in situ generated. Reduction of compound 1 using various catalysts showed that combination of sodium borohydride and nickel acetate provides the highest yield for compound 2. Bisindolylmethane Schiff base derivatives were synthesized by coupling various benzaldehydes with amino substituted bisindolylmethane 2. All synthesized compounds were characterized by various spectroscopic methods. The bisindolylmethane Schiff base derivatives were evaluated against selected Gram-positive and Gram-negative bacterial strains. Derivatives having halogen and nitro substituent display weak to moderate antibacterial activity against Salmonella typhi, S. paratyphi A and S. paratyphi B.

Full text of DOI:10.3390/molecules190811722

Molecules 2014, 19, 11722-11740; doi:10.3390/molecules190811722
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel Bisindolylmethane Schiff bases and Their
Antibacterial Activity
1
,2
1,2,
1,2,
3
Syahrul Imran , Muhammad Taha *, Nor Hadiani Ismail *, Khalid Mohammed Khan ,
4
5
5
Farzana Naz , Memona Hussain and Saima Tauseef
1
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak
Alam Campus 42300, Malaysia
2
3
Faculty of Applied Science, Universiti Teknologi MARA, Shah Alam 40450, Selangor D.E., Malaysia
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
4
5
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Federal Urdu University of Arts,
Science and Technology, Karachi-75370, Pakistan
Department of Microbiology, Federal Urdu University of Arts, Sciences and Technology,
Gulshan-e-Iqbal Campus, Karachi-75370, Pakistan
*
Authors to whom correspondence should be addressed; E-Mails: taha_hej@yahoo.com (M.T.);
norhadiani@puncakalam.uitm.edu.my (N.H.I.); Tel.: +601-8290-1765 (M.T.);
+
601-3267-5756 (N.H.I.).
Received: 2 July 2014; in revised form: 23 July 2014 / Accepted: 1 August 2014 /
Published: 6 August 2014
Abstract: In an effort to develop new antibacterial drugs, some novel bisindolylmethane
derivatives containing Schiff base moieties were prepared and screened for their
antibacterial activity. The synthesis of the bisindolylmethane Schiff base derivatives 3–26
was carried out in three steps. First, the nitro group of 3,3'-((4-nitrophenyl)-
methylene)bis(1H-indole) (1) was reduced to give the amino substituted bisindolylmethane
2
without affecting the unsaturation of the bisindolylmethane moiety using nickel boride in
situ generated. Reduction of compound 1 using various catalysts showed that combination
of sodium borohydride and nickel acetate provides the highest yield for compound 2.
Bisindolylmethane Schiff base derivatives were synthesized by coupling various
benzaldehydes with amino substituted bisindolylmethane 2. All synthesized compounds
were characterized by various spectroscopic methods. The bisindolylmethane Schiff base
derivatives were evaluated against selected Gram-positive and Gram-negative bacterial

Molecules 2014, 19
11723
strains. Derivatives having halogen and nitro substituent display weak to moderate
antibacterial activity against Salmonella typhi, S. paratyphi A and S. paratyphi B.
Keywords: indole; bisindolylmethane; bisindole Schiff base; antibacterial; sodium
borohydride; nickel acetate
1
. Introduction
As the rate of resistance to current antibacterial therapy has increased alarmingly, the search for
new selective [1] and nontoxic [2] antibacterial agents has become an important area of investigation
in medicinal chemistry. Resistance of pathogenic bacteria to drugs is constantly emerging, even though
various classes of antibacterial agents are available. Chemical diversity and various mechanisms of
action make it difficult to find a definite way of identifying new drugs. This has not only made treating
infectious diseases more problematic, but has also resulted in reappearance of many diseases which
were thought to be under control such as malaria. To overcome this severe medical issue, discovery of
new types of antibacterial agents or improvement for the bioactivity of the existing drugs is an
important task [3]. Hence, an increasing number of current research is oriented towards development
of new antibacterial agents using novel lead compounds to overcome the problem of bacterial
resistance against available drugs.
Synthesis, characterization and structural activity relationship of Schiff bases have been studied
worldwide as it is proven that C=N linkage in Schiff bases is an essential feature for bioactivity [4].
Schiff bases have been reported to possess noteworthy antibacterial [5], antifungal [6], anticancer [7],
urease inhibition [8], antioxidant [9–14] and antiglycation [15–17] activities.
Because of their vast pharmacological activities, indole and their derivatives are an important class
of heterocycles and bioactive intermediates in the pharmaceutical industry and organic synthesis [18].
Chemists are continuously developing simple and efficient synthetic protocols to synthesize indoles
and their derivatives due to their excellent biological activities [19]. The indole ring has become an
important structural requirement in many pharmaceutical drugs because of the structural diversity of
biologically active indoles and their derivatives [20]. Indole is a valuable compound which has become
prominent in medicinal chemistry because of its various biological activities [21] such as
anticonvulsant [22], anti-inflammatory and analgesic activities [23].
The importance of bisindoles as bioactive intermediates in pharmaceutical industry cannot be
underestimated. Bisindoles display a wide range of biological activities such as antimicrobial and
antifungal [24], antibacterial [25], analgesic and anti-inflammatory [26] activities. Naturally occurring
bisindoles such as hamacanthin A isolated from the sponge, Hamacantha sp [27] and Spongosorites sp [28]
have shown potent antibacterial activity against Staphylococcus aureus and Bacillus subtilis [21].
Besides antitumor [29] and anticancer [30] activities, bisindoles have also been reported to be able to
promote beneficial estrogen metabolism, promote useful estrogen, and induce apoptosis in human
cancer cells [31]. Naturally occurring bisindoles such as bisindolylmethane or diindolylmethane are
found mostly in cruciferous plants like broccoli, brussels sprouts, cauliflower, kale, and cabbage [31].
Medical epidemiologists believe that bisindolylmethane plays an important role in lowering the risk of

Molecules 2014, 19
11724
cancer for people consuming plenty of cruciferous vegetables. Bisindolylmethane is also able to
normalize abnormal cell growth associated with cervical dysplasia. Hong [32] and Kedmi [33]
reported on the potential of bisindolylmethanes to proliferate and induce apoptosis in human prostate
and breast cancer cells. Bhowmik [34] claimed that bisindolylmethane induces apoptosis in breast
cancer cells by inhibiting epidermal growth factor receptor pathway. It has been reported that
bisindolylmethane and its derivatives are potent inhibitors for Leishmanial donovani [35].
Schiff bases and bisindolylmethane had been reported to show potent antibacterial activity against
various pathogenic bacteria [13,25]. Therefore, in this study, novel derivatives of bisindolylmethane Schiff
bases have been synthesized and tested against pathogenic bacteria such as Staphylococcus aureus,
Salmonella typhi, S. paratyphi A, S. paratyphi B and Escherichia coli to find out the effect of
combining a Schiff base and bisindolylmethane.
2
. Results and Discussion
2
.1. Chemistry
All compounds in this study were synthesized by the method outlined in Schemes 1 and 2.
Compound 1 was synthesized through acid catalyzed electrophilic substitution reaction of indole and
aldehyde. Compound 1 was reduced to produce 4-(di(1H-indol-3-yl)methyl)aniline (2, Scheme 1).
Acetic acid acts as the activating agent which increases electropositivity of electrophilic center on
p-nitrobenzaldehyde and enables the addition of indole which act as a nucleophile to produce compound 1
which had been reported previously [36].
Scheme 1. Reaction scheme for synthesis of compounds 1 and 2.
4 2 2
Reagents and conditions: (a) Glacial acetic acid, reflux; (b) NaBH , Ni(OAc) .4H O, room temperature.
Scheme 2. Reaction scheme for synthesis of bisindolylmethane Schiff bases (3–26).

Molecules 2014, 19
11725
Reduction of the nitro substituent of compound 1 to an amino moiety was carried out using various
reducing agents, as mentioned in Table 1. Since, there is a tendency for indole to be reduced to
indoline, the reduction of compound 1 was studied using different catalysts to select the best one that
provides the best yield for compound 2 in a reasonably short time. Besides the formation of indoline,
uncontrolled reduction reactions could also result in production of azo compounds [37]. Other
parameters such as the amount of reducing agent used and temperature were kept constant to observe
the effect of different reducing agents on compound 1. It was observed that in situ generation of nickel
boride plays an important role in the reduction process.
Table 1. Synthesis of compound 2 using different reducing agents.
Reducing Agents
Lithium aluminum hydride
Sodium borohydride
Lithium aluminum hydride
Sodium borohydride
Raney nickel
Nickel Acetate
Reaction Time
Yield (%)
+
2 h
3 h
54
95
51
78
70
76
+
−
6 h
−
4 h
−
4 h
Hydrazine hydrate
−
15 h
+
Present; − Not present.
Observation shows that a mixture of sodium borohydride and nickel acetate provides the best yield
in a fairly short reaction time (Table 1). The high yield obtained with sodium borohydride and nickel
acetate evidences the in situ generation of nickel boride by the reaction of these reagents. Longer
reaction times and lower yields were observed for the control experiment which was conducted without
the presence of nickel acetate. This newly formed nickel boride resulting from the combination of
sodium borohydride and nickel acetate is a milder reducing agent and able to selectively reduce the nitro
group to an amino group without affecting the main indole skeleton. It was observed that there is not
much difference in product yield when lithium aluminium hydride was used in the absence of nickel
acetate, which suggest that of lithium aluminium hydride could reduce nitro group independently without
1
the presence of nickel boride. The H-NMR spectrum of compound 2 shows the appearance of a singlet
1
for NH
2
at 4.81 ppm which was not observed in H-NMR spectra of compound 1. Lithium aluminum
hydride gives a shorter reaction time, but at the same time produces a low product yield compared to
other catalysts. The presence of double bonds of bisindolylmethane would be the main reason for the
reduced yield recorded by lithium aluminum hydride. The nonspecific reduction effect of other
catalysts, especially lithium aluminum hydride, on both nitro and double bond of indole might have led
to the formation of bisindoline as the minor product.
Compound 2 was converted to bisindolylmethane Schiff bases 3–26 with various substitution
patterns (Table 2). Bisindolylmethane Schiff bases having imine functionalities were formed through a
dehydration reaction between the aniline of compound 2 with various substituted benzaldehydes
(
Scheme 2).
The first part of the mechanism for Schiff base formation involves reaction of compound 2 with
benzaldehyde which produces an unstable intermediate known as a carbinolamine which then
undergoes dehydration to form an imine or Schiff base. A non-catalyzed reaction was employed in this
study to prevent protonation of the amine. Protonation of the amine will shift the reaction equilibrium

Molecules 2014, 19
11726
to the left, decreasing the formation of carbinolamine. Thus, most of the synthetic methods used for
Schiff base synthesis employ either neutral or mildly acidic media.
Table 2. Structures of bisindolylmethane Schiff base derivatives.
Compound
3
Ar
Compound
15
Ar
4
5
6
7
16
17
18
19
8
9
20
21
22
1
1
0
1
23
1
1
1
2
3
4
24
25
26
All compounds synthesized gave satisfactory spectroscopic information. The results obtained
1
through H-NMR and mass spectrometry showed consistency with the assigned structures and
molecular weight of the synthetic compounds. Functional group transformation was confirmed using
−1
FTIR. Compound 1 showed the presence of two important peaks at 1340 and 1543 cm which
indicates the presence of a NO group. Disappearance of these bands followed by the appearance of a
2
−1
2 2 2
NH absorption band at 3425 cm suggests the successful conversion of NO to NH .

Molecules 2014, 19
11727
1
H-NMR was used to confirm the formation of the final Schiff base derivatives 3–26 by observing
peak at 4.81 ppm followed by the appearance of a singlet due to the C-H
the disappearance of the NH
2
of the imine between 8.28–8.93 ppm. Other characteristic peaks include a singlet of one proton
observed between 5.85–5.89 ppm, which represents the C-H of the bisindolylmethane methine linkage,
and a singlet of two N-H protons on both indole rings, observed between 10.81–10.84 ppm.
2
.2. Antibacterial Activity
As a continuation of our work on bioactive compounds [38–43]. Bisindolylmethane Schiff bases
3
–26 were evaluated for their ability to inhibit both Gram-positive and Gram-negative bacterial strains
using the disc diffusion method [44]. Gram-negative bacterial strains used in this study include
Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, S. parathypi A, S. parathypi B,
Shigella dysenteriae, S. flexneri, Acinobacter baumannii, Vibrio cholerae. Results listed in Table 3
show that these derivatives display weak to moderate inhibitory activity against Gram-negative
bacteria. Decreased or no activity against both Gram-negative and Gram-positive bacterial strains is
generally observed for most compounds having hydroxyl groups substituted in any position, perhaps
due to decrease in lipophilicity [45]. In accordance with Overtone’s concept of cell permeability,
liposolubility plays an important role in manipulating antibacterial activity based on the ability of cell
membranes to allow passage of only lipid-soluble materials [46]. Therefore, compounds having higher
lipophilicity such as compounds with halogen groups have a greater capability of diffusing into
bacteria and result in an increased antibacterial activity of those compounds. Further observation
(
Table 3) shows that derivatives having chloro and fluoro residues possessed the capability of
inhibiting S. typhi, S. parathypi A and S. parathypi B. Compounds having halo, nitro and dimethoxy
substituents displayed significant inhibitory activity. Compound 21 having a chloro group at the ortho
position recorded the highest inhibition for S. typhi and S. para typhi A with inhibition zones of 14 and
1
2 mm, respectively. S. para typhi B inhibition was demonstrated by compound 20 bearing a fluoro
substitution at the para position. Other compounds having chloro, fluoro and nitro substituents (19, 22
and 23) showed comparably moderate activity against S. paratyphi B.
Compound 5, having a hydroxy at the para position inhibited S. dysenteriae and A. baumannii.
Compound 7, 9, and 10 which are dihydroxy-substituted compounds inhibited K. pneumoniae.
Compound 8 which is also a dihydroxy-substituted compound showed inhibitory activity against
S. dysenteriae. Compounds 11 and 12 which are trihydroxy-substituted compounds did not inhibit any
of the tested Gram-negative bacterial strains. Compound 13, 14, 15, and 16 having both hydroxy and
methoxy substituents did not inhibit any Gram-negative bacterial strains. Compound 17 inhibited
S. flexneri, while compound 18 inhibited S. dysenteriae. Compounds 19 and 20 having fluoro at the
meta and para positions were capable of inhibiting three different strains of Salmonella. Compounds
2
1 and 22 having chloro at the ortho and meta position successfully inhibited the three different strains
of Salmonella. Compound 26 which is a dimethoxy-substituted compound inhibited all three different
strains of Salmonella. Compound 23 having a chloro at the para position showed activity against S. typhi
and S. paratyphi B. Compounds 24 and 25 having nitro at ortho and para position displayed inhibition
against S. typhi and S. paratyphi B. Compound 20 inhibited growth of A. baumannii and V. cholerae.

Molecules 2014, 19
11728
Table 3. Antibacterial activity of compounds 3–26 against Gram-negative bacteria
inhibition zones in mm using the disc diffusion method).
(
Inhibition Zone (mm)
Compound
A
-
B
-
C
-
D
-
E
-
F
-
G
-
H
-
I
-
3
4
5
6
7
8
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11
-
-
09
-
-
-
-
-
-
-
-
-
-
07
-
-
-
-
-
-
-
-
-
-
-
-
10
-
-
-
-
-
09
06
-
-
-
-
-
-
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
07
-
-
-
-
-
-
-
-
10
-
-
-
-
-
08
12
14
11
10
09
10
08
25
10
09
12
10
-
15
17
16
15
16
11
09
10
25
-
-
-
10
-
-
-
-
12
-
13
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
-
09
-
-
-
-
-
-
-
-
-
-
-
-
-
-
08
25
-
-
-
-
Gentamicin
29
23
23
28
14
25
A = Escherichia coli, B = Klebsiella pneumoniae, C = Salmonella typhi, D = Salmonella paratyphi A,
E = Salmonella paratyphi B, F = Shigella dysenteriae, G = Shigella flexneri, H = Acinobacter baumannii,
I = Vibrio cholera; - No activity
Result for the inhibition activity of compounds 3–26 against Gram-positive bacterial strains are
presented in Table 4, which shows that most of the derivatives having hydroxy residues were found to
be inactive. Some derivatives having hydroxy substituents such as compounds 5, 6 and 7 demonstrated
weak activity against Staphylococcus aureus. Compound 8, a derivative having a dihydroxy
substituent showed weak inhibition against S. aureus, S. epidermidis, and MRSA. Comparison
between hydroxyl- and non-hydroxy-substituted compounds reveals that compounds with chloro,
fluoro, and nitro substituents exhibited better antibacterial activity in terms of number of bacterial
strains inhibited. Compounds with halogens were found to be active against Bacillus subtilis,
Corynebacterium diphtheriae and S. aureus, while compounds having nitro and dimethoxy
substituents showed potential against S. epidermidis and S. faecalis. Compound 20 with a fluoro
substituted at the para position inhibited all tested Gram-positive bacterial strains. However,
compounds having a chloro substituent, such as 21 and 23 demonstrated inhibitory ability against most

Molecules 2014, 19
11729
of the tested Gram-positive bacterial strains. Compound 21 with a chloro at the ortho position was
found to be inactive against MRSA, while compound 23 with chloro substitution at the para position
was observed to inhibit all the tested Gram-positive bacterial strains, except B. subtilis and MRSA.
Table 4. Antibacterial activity of compounds 3–26 against Gram-positive bacteria
(
inhibition zones in mm using the disk diffusion method).
Inhibition Zone (mm)
Compound
A
-
B
-
C
-
D
-
E
-
F
-
3
4
5
6
7
8
9
-
-
-
-
-
-
-
-
10
11
08
09
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11
-
-
08
-
-
-
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
-
-
-
-
-
-
-
-
-
07
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
08
07
-
08
NA
-
-
-
-
07
-
09
-
08
10
-
-
-
12
-
08
09
10
13
-
09
-
10
09
11
12
-
-
12
11
10
09
12
09
-
09
-
11
09
09
-
09
08
07
08
-
-
-
07
-
08
07
09
08
28
-
-
-
-
-
-
-
-
-
-
08
25
-
Gentamicin
24
25
25
20
A = Bacillus subtilis, B = Corynebacterium diphtheriae, C = Staphylococcus aureus, D = Staphylococcus
epidermidis, E = Staphylococcus faecalis, F = MRSA; - No activity.
3. Experimental Section
3.1. General Information
Melting points were determined on a Stuart Scientific SMP11 Analog melting point apparatus
(
Staffordshire, UK). IR spectra was obtained with a Perkin–Elmer system 2000 FTIR spectrophotometer
Norwalk, CT, USA) using KBr disks. NMR spectroscopy was performed on a Bruker Ultra Shield FT
(
NMR 500 MHz spectrometer (Wissembourg, Switzerland). EI-MS spectroscopic analysis was carried out
using Finnigan-MAT-311-A instrument (Bremen, Germany). Thin layer chromatography (TLC) was
performed using precoated silica gel on aluminum sheets (Kieselgel 60 F-254, 0.20 mm, Merck,

Molecules 2014, 19
11730
Darmstadt, Germany). Chromatograms were visualized using a handheld UV lamp at 254 nm (UVGL-58;
UVP, Upland, CA, USA).
3
.2. Synthesis of 3,3'-((4-Nitrophenyl)methylene)bis(1H-indole) (1)
Acetic acid (40 mL) was added to a mixture of indole (36 mmol) and p-nitrobenzaldehyde
(18 mmol) in a round-bottomed flask and then refluxed. The progress of the reaction was monitored by
TLC and upon completion; water was added to allow precipitate formation. The precipitate was filtered
and dried at room temperature. The precipitate was finally rinsed with diethyl ether to afford pure
−1
product. Light yellow solid; Yield 92%; m.p. 219–221 °C; IR cm : 3420, 1543, 1457, 1342, 1226;
1
6
H-NMR (DMSO-d ): δ 6.03 (s, 1H), 6.87–6.90 (m, 4H), 7.05 (t, J = 7.0 Hz, 2H), 7.29 (d, J = 6.5 Hz,
2
H), 7.37 (d, J = 6.5 Hz, 2H), 7.62 (d, J = 6.5 Hz, 2H), 8.16 (d, J = 7.5 Hz, 2H), 11.05 (s, 2H);
1
3
6
C-NMR (DMSO-d ): δ 40.35, 112.46, 112.46, 117.55, 117.55, 119.29, 119.29, 119.78, 119.78,
1
1
21.97, 121.97, 124.28, 124.28, 124.73, 124.73, 127.24, 130.32, 130.32, 137.47, 137.47, 146.63,
46.63, 154.00. EI MS m/z (% rel. abund.): 367.13 (100.0, M), 368.14 (25.1), 369.14 (3.4), 368.13
(
1.1). Anal. Calcd for C H N O : C, 75.19; H, 4.66; N, 11.44; O, 8.71. Found: C, 75.18; H, 4.69; N,
23 17 3 2
1
1.43; O, 8.74.
3
.3. Synthesis of 4-(di(1H-Indol-3-yl)methyl)aniline (2)
Acetonitrile (50 mL) was added to mixture of compound 1 (16.3 mmol) and nickel acetate
tetrahydrate (3.3 mmol) in a round-bottomed flask and the mixture stirred in an ice bath. While
stirring, sodium borohydride (66 mmol) was added in portions. Progress of the reaction was monitored
using TLC. Upon completion water (90 mL) was added and stirred for another 3 min. The product was
extracted using dichloromethane (100 mL × 3). The dichloromethane layer was collected and washed
2 4
using a small quantity of water, dried over anhydrous Na SO , and evaporated under vacuum to give
the crude product, which was rinsed with diethyl ether to afford pure product. Dark orange solid; Yield
−1
1
9
5%; m.p. 230–232 °C; IR cm : 3425, 3387, 1629, 1276; H-NMR (DMSO-d
6
): δ 4.81 (s, 2H), 5.62
(
(
s, 1H), 6.46 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 2.0 Hz, 2H), 6.84 (t, J = 7.5 Hz, 2H), 6.98–7.03
13
m, 4H), 7.26 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 10.72 (s, 2H); C-NMR (MeOD-d
4
): δ
9.88, 110.72, 110.72, 115.51, 115.51, 118.08, 118.08, 118.48, 118.48, 119.75, 119.75, 120.86,
20.86, 123.40, 123.40, 126.97, 126.97, 129.59, 129.59, 137.10, 137.01, 144.01. EI MS m/z
3
1
(
% rel. abund.): 337.16 (100.0, M), 338.16 (25.1), 339.16 (3.2), 338.15 (1.1). Anal. Calcd for
C H N : C, 81.87; H, 5.68; N, 12.45. Found: C, 81.89; H, 5.70; N, 12.44.
2
3
19
3
General Procedure for Synthesis of Bisindolylmethane Schiff bases 3–26
Methanol (20 mL) was added to a mixture of compound 2 (0.5 mmol) and a substituted
benzaldehyde (0.5 mmol) in round-bottomed flask and the resulting mixture was refluxed until TLC
monitoring of the progress of the reaction indicated its completion. The solvent was then removed
using a rotary evaporator and the crude product was collected and rinsed using hot hexane to afford
pure product.

Molecules 2014, 19
11731
(
E)-2-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)phenol (3). Dark orange solid; Yield 74%;
−1
1
m.p. 209–211 °C; IR cm : 3572, 3389, 1670, 1590, 1361, 1222; H-NMR (DMSO-d ): δ 5.89 (s, 1H),
6
6
2
7
.88 (dd, J = 12.5 Hz, 5.1 Hz, 4H), 6.97 (dd, J = 13.7 Hz, 7.3 Hz, 2H), 7.05 (dd, J = 11.6 Hz, 4.4 Hz,
H), 7.31 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.39–7.42 (m, 1H),
13
.44 (d, J = 8.4 Hz, 2H), 7.62 (dd, J = 7.7 Hz, 1.6 Hz, 1H), 8.96 (s, 1H), 10.84 (s, 2H). C-NMR
): δ 39.84, 111.96, 111.96, 117.02, 118.34, 118.34, 118.71, 118.71 119.57, 119.57, 121.41,
(DMSO-d
6
121.41, 121.55, 124.07, 124.07, 127.06, 127.06, 129.77, 132.95, 132.95, 133.54, 136.89, 137.10, 137.10,
144.53, 146.17, 149.48, 160.77, 160.77, 163.22. EI MS m/z (% rel. abund.): 441.05 (100.0, M), 325.03
(
5.0), 321.01 (4.9), 245.03 (29.0), 196.00 (0.7), 204.01 (6.9). Anal. Calcd for C H N O: C, 81.61; H,
30 23 3
5.25; N, 9.52; O, 3.62. Found: C, 81.63; H, 5.27; N, 9.54; O, 3.59.
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)phenol (4). Light orange solid; Yield 68%; m.p.
−1
1
1
38–140 °C; IR cm : 3565, 3383, 1652, 1602, 1335, 1220; H-NMR (DMSO-d ): δ 5.86 (s, 1H),
6
6
.85–6.90 (m, 4H), 7.05 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.30–7.31 (m, 4H), 7.35–7.40
13
(m, 5H), 8.52 (s, 1H), 10.83 (s, 2H); C-NMR (DMSO-d
6
): δ 39.79, 103.04, 111.92, 111.92, 114.61,
1
1
1
1
5
17.61, 118.51, 118.51, 118.65, 118.65, 119.59, 119.59, 120.63, 121.21, 121.21, 121.34, 124.01,
24.01, 127.09, 127.09, 129.50, 130.26, 130.26, 137.09, 137.09, 158.14, 160.35, 160.35, 162.77,
70.85. EI MS m/z (% rel. abund.): 440.99 (100.0, M), 335.99 (33.8), 323.99 (11.3), 244.99 (45.5),
16.99 (27.8). Anal. Calcd for C H N O: C, 81.61; H, 5.25; N, 9.52; O, 3.62. Found: C, 81.62; H,
3
0
23
3
.24; N, 9.54; O, 3.61.
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)phenol (5). Brownish red solid; Yield 86%;
−1
1
m.p. 118–120 °C; IR cm : 3551, 3386, 1664, 1578, 1365, 1210; H-NMR (DMSO-d
6
): δ 5.84 (s, 1H),
6
.85–6.89 (m, 7H), 7.04 (t, J = 8.0 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.35 (dd,
1
3
J = 2.0 Hz, 8 Hz, 4H), 7.73 (d, J = 8.5 Hz, 2H), 8.45 (s, 1H), 10.82 (s, 2H); C-NMR (MeOD-d
4
): δ
3
1
1
9.84, 110.82, 110.82, 115.37, 118.12, 118.12, 118.67, 118.67, 119.16, 119.16, 120.30, 120.30,
20.90, 120.90, 123.42, 123.42, 127.00, 127.00, 129.31, 130.61, 130.61, 137.09, 137.09 142.82,
49.48, 158.17, 158.17 160.26, 160.26, 162.53. EI MS m/z (% rel. abund.): 441.00 (100.0, M), 337.00
(
23 3
59.7), 244.99 (39.9), 116.99 (9.7). Anal. Calcd for C30H N O: C, 81.61; H, 5.25; N, 9.52; O, 3.62.
Found: C, 81.64; H, 5.23; N, 9.53; O, 3.60.
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-1,2-diol (6). Light orange solid; Yield
−1
1
7
1%; m.p. 157–159 °C; IR cm : 3549, 3378, 1673, 1585, 1359, 1223; H-NMR (DMSO-d
6
): δ 5.89 (s,
1
H), 6.75 (t, J = 8.0 Hz, 1H), 6.87–6.92 (m, 4H), 6.93 (d, J = 1.0 Hz, 1H), 7.03–7.07 (m, 3H), 7.31 (d,
J = 8.0 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 8.90 (s,
1
3
1
1
1
4
H), 10.84 (s, 2H); C-NMR (MeOD-d ): δ 39.89, 110.82, 110.82, 118.02, 118.12, 118.12, 118.43,
18.43, 119.07, 119.28 119.28, 120.39, 120.39, 120.91 120.91, 122.86, 123.42, 123.42, 126.95,
26.95, 129.52, 129.52, 137.11, 137.11, 144.16, 145.37, 145.68, 149.75, 162.13, 171.68. EI MS m/z
(% rel. abund.): 456.98 (100.0, M), 337.00 (49.2), 321.97 (6.7), 244.99 (39.7), 116.97 (2.9). Anal.
Calcd for C H N O : C, 78.75; H, 5.07; N, 9.18; O, 6.99. Found: C, 78.76; H, 5.08; N, 9.16; O, 6.96.
3
0
23
3
2
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-2,4-diol (7). Dark red solid; Yield
−1
1
8
6
8%; m.p. 153–155 °C; IR cm : 3550, 3395, 1667, 1587, 1361, 1220; H-NMR (DMSO-d ): δ 5.86 (s,

Molecules 2014, 19
11732
1
2
8
1
1
H), 6.27 (d, J = 2.5 Hz, 1H), 6.38 (dd, J = 2.5 Hz, 8.5 Hz, 1H), 6.85–6.89 (m, 4H), 7.04 (t, J = 7.0 Hz,
H), 7.26 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.38-7.40 (m, 3H),
1
3
.77 (s, 1H),10.83 (s, 2H); C-NMR (MeOD-d ): δ 39.88, 60.35, 94.16, 107.60, 108.55, 110.79,
4
10.79, 112.21, 118.08, 118.08, 118.46, 118.46, 119.04, 119.04, 119.70, 119.70, 120.88, 120.88,
23.39, 123.39, 126.95, 126.95, 129.55, 129.55, 134.29, 137.11, 137.11, 143.51, 144.31, 159.98. EI
MS m/z (% rel. abund.): 456.98 (28.9), 337.00 (100.0, M), 244.99 (26.0), 116.99 (18.9). Anal. Calcd
for C H N O : C, 78.75; H, 5.07; N, 9.18; O, 6.99. Found: C, 78.73; H, 5.10; N, 9.19; O, 7.01.
3
0
23
3
2
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-2,5-diol (8). Dark red solid; Yield
−1
1
7
6%; m.p. 103–105 °C; IR cm : 3561, 3383, 1664, 1584, 1363, 1223; H-NMR (DMSO-d
6
): δ 5.88 (s,
1H), 6.78 (d, J = 9.0 Hz, 1H), 6.84–6.90 (m, 5H), 7.00 (d, J = 3 Hz, 1H), 7.04 (t, J = 7 Hz, 2H), 7.31
(
(
d, J = 8.5 Hz, 4H), 7.36 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 8.82 (s, 1H), 9.16 (s, 1H), 10.84
1
3
s, 2H), 12.36 (s, 1H); C-NMR (MeOD-d ): δ 39.79, 110.80, 110.80, 116.87, 116.96, 117.60, 118.08,
4
118.08, 118.46, 118.46, 119.04, 119.31, 119.31, 120.41, 120.41 120.47, 120.88, 123.40, 123.40,
1
26.95, 126.95, 129.48, 129.48, 137.11, 137.11, 144.09, 144.09, 146.37, 149.44, 161.91.EI MS m/z (%
rel. abund.): 456.98 (100.0, M),337.00(92.3), 245.03 (53.0), 116.99 (15.8). Anal. Calcd for
C H N O : C, 78.75; H, 5.07; N, 9.18; O, 6.99. Found: C, 78.76; H, 5.06; N, 9.16; O, 6.96.
3
0
23
3
2
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-3,4-diol (9). Dark red solid; Yield
−1
1
8
8%; m.p. 144–145 °C; IR cm : 3554, 3390, 1670, 1585, 1365, 1225; H-NMR (DMSO-d
6
): δ 5.84 (s,
1H), 6.81 (d, J = 8.0 Hz, 1H), 6.85 (s, 2H), 6.87 (t, J = 8.0 Hz, 2H), 7.04 (t, J = 7.5 Hz, 2H), 7.11 (d,
J = 8.5 Hz, 2H), 7.15 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 7.30 (d, J = 7.5 Hz, 2H), 7.34–7.36 (m, 5H), 8.36
1
3
(
s, 1H), 10.81 (s, 2H), 12.01 (s, 1H); C-NMR (MeOD-d
4
): δ 39.89, 56.08, 110.72, 110.81, 110.81,
1
1
1
13.52, 113.62, 114.12, 118.09, 118.09, 118.64, 118.64, 119.11, 120.30, 120.30, 120.88, 120.88,
22.57, 122.57, 122.92, 123.41, 124.71, 126.99, 126.99, 129.28, 129.28, 137.09, 137.09, 153.59,
60.75. EI MS m/z (% rel. abund.): 456.98 (10.2), 336.95 (100.0, M), 244.96 (49.3), 116.96 (94.5).
Anal. Calcd for C30
O, 6.98.
23 3 2
H N O : C, 78.75; H, 5.07; N, 9.18; O, 6.99. Found: C, 78.73; H, 5.09; N, 9.21;
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-3,5-diol (10). Brown solid; Yield 70%;
−1
1
m.p. 222–224 °C; IR cm : 3541, 3376, 1665, 1589, 1357, 1224; H-NMR (DMSO-d
.34 (t, J = 2.0 Hz, 1H), 6.77 (d, J = 2.0 Hz, 2H), 6.85–6.89 (m, 4H), 7.04 (t, J = 7.0 Hz, 2H), 7.15 (d,
J = 8.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.30–7.38 (m, 4H), 8.39 (s, 1H), 10.83 (s, 2H), 12.15 (s,
6
): δ 5.85 (s, 1H),
6
1
3
2
1
1
4
6
H); C-NMR (DMSO-d ): δ 39.96, 107.10, 107.73, 111.80, 111.80, 111.93, 118.44, 118.44, 118.52,
18.52, 118.66, 118.66, 119.47, 119.47, 119.59, 119.59, 119.70, 119.70, 121.20, 121.36, 121.36,
23.76, 124.01, 127.09, 127.09, 129.48, 129.48, 137.08, 137.08, 159.12. EI MS m/z (% rel. abund.):
23 3 2
56.98 (1.1), 336.95 (100.0, M), 244.96 (49.3), 116.96 (94.5). Anal. Calcd for C30H N O : C, 78.75;
H, 5.07; N, 9.18; O, 6.99. Found: C, 78.76; H, 5.05; N, 9.20; O, 7.01.
(
E)-5-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-1,2,3-triol (11). Dark yellow solid;
−1
1
Yield 75%; m.p. 201–202 °C; IR cm : 3564, 3362, 1662, 1603, 1357, 1220; H-NMR (DMSO-d ):
6
δ 5.88 (s, 1H), 6.85–6.89 (m, 6H), 7.05 (t, J = 7.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 7.5
1
3
4
Hz, 2H), 7.35-7.37 (m, 4H), 8.28 (s, 1H), 9.04(s, 3H), 10.82 (s, 2H); C-NMR (MeOD-d ): δ 39.84,

Molecules 2014, 19
11733
1
08.15, 110.78, 110.78, 115.44, 118.05, 118.05, 118.64, 118.64, 119.08, 119.08, 120.11, 120.11,
1
20.85, 120.85, 123.39, 123.39, 126.99, 126.99, 129.31, 129.31, 137.11, 137.11, 142.79, 145.83,
60.88, 160.88, 171.69, 171.69, 191.91. EI MS m/z (% rel. abund.): 472.99 (100.0, M), 336.95 (38.7),
44.99 (40.6), 116.99 (58.8). Anal. Calcd for C H N O : C, 76.09; H, 4.90; N, 8.87; O, 10.14.
1
2
3
0
23
3
3
Found: C, 76.12; H, 4.92; N, 8.85; O, 10.16.
(
E)-5-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)benzene-1,3,5-triol (12). Dark orange solid;
−1
1
Yield 83%; m.p. 215–217 °C; IR cm : 3553, 3364, 1668, 1578, 1360, 1220; H-NMR (DMSO-d ):
6
δ 5.80 (s, 2H), 5.85 (s, 1H), 6.85–6.89 (m, 4H), 7.04 (t, J = 7.0 Hz, 2H), 7.20 (d, J = 10.0 Hz, 2H), 7.30
(
d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 8.90 (s, 1H), 10.82 (s, 2H),
1
3
1
1
1
2.22 (s, 1H); C-NMR (MeOD-d ): δ 39.81, 60.14, 102.65, 110.78, 110.78, 118.09, 118.09, 118.39,
4
18.39, 118.61, 119.01, 119.01, 120.88, 120.88, 123.40, 123.40, 126.52, 126.93, 126.93, 129.76,
29.76, 137.11, 137.11, 143.37, 143.37, 152.85, 162.13, 162.13, 171.68, 171.68. EI MS m/z (% rel.
abund.): 472.96 (100.0, M), 336.95 (32.8), 245.02 (5.8), 116.99 (16.1). Anal. Calcd for C H N O : C,
3
0
23
3
3
7
6.09; H, 4.90; N, 8.87; O, 10.14. Found: C, 76.06; H, 4.88; N, 8.89; O, 10.15.
(
E)-2-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)-5-methoxyphenol (13). Dark orange solid;
-1
1
Yield 74%; m.p. 121–123 °C; IR cm : 3552, 3383, 1665, 1587, 1361, 1225; H-NMR (DMSO-d
6
):
δ 3.85 (s, 3H), 5.88 (s, 1H), 6.48 (d, J = 2.0 Hz, 1H), 6.54 (dd, J = 2.0 Hz, 8.5Hz, 1H), 6.87–6.90 (m,
4
2
1
1
1
3
H), 7.05 (t, J = 7.0 Hz, 2H), 7.31 (t, J = 7.5 Hz, 4H), 7.37 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.5 Hz,
1
3
4
H), 7.50 (d, J = 8.5 Hz, 2H), 8.85 (s, 1H), 10.84 (s, 2H); C-NMR (MeOD-d ): δ 39.87, 54.50,
00.80, 106.66, 110.78, 110.78, 115.46, 115.46, 118.06, 118.06, 118.44, 118.44, 119.02, 119.02,
19.79, 119.79, 120.87, 120.87, 123.38, 123.38, 126.95, 126.95, 129.57, 129.57, 133.81, 137.12,
44.00, 160.06, 165.29, 167.29, 186.82. EI MS m/z (% rel. abund.): 471.00 (100.0, M), 354.05 (21.3),
25 3 2
37.07 (12.4), 245.12 (51.7), 117.08 (10.0). Anal. Calcd for C31H N O : C, 78.96; H, 5.34; N, 8.91;
O, 6.79. Found: C, 78.95; H, 5.32; N, 8.92; O, 6.76.
(
E)-2-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)-4-methoxyphenol (14). Light orange solid;
−1
1
Yield 76%; m.p. 207–209 °C; IR cm : 3548, 3375, 1661, 1571, 1353, 1220; H-NMR (DMSO-d
.74 (s, 3H), 5.89 (s, 1H), 6.86–6.90 (m, 5H), 7.01–7.06 (m, 3H), 7.22 (d, J = 3.0 Hz, 1H), 7.31 (t,
J = 8.0 Hz, 4H), 7.36 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 8.91 (s, 1H), 9.95 (s, 1H), 10.84 (s,
6
): δ
3
13
2
1
1
H); C-NMR (DMSO-d ): δ 39.95, 56.03, 111.96, 111.96, 115.72, 115.72, 117.88, 117.88, 118.35,
6
18.35, 118.70, 119.58, 119.58, 119.68, 119.68, 120.70, 121.40, 121.52, 124.08, 124.08, 127.07,
27.07, 129.77, 129.77, 137.11, 137.11, 144.49, 146.43, 152.33, 154.82, 162.79. EI MS m/z (% rel.
abund.): 471.00 (100.0, M), 354.07 (19.7), 337.13 (48.3), 321.06 (15.7), 117.09 (17.8). Anal. Calcd for
: C, 78.96; H, 5.34; N, 8.91; O, 6.79. Found: C, 78.97; H, 5.36; N, 8.93; O, 6.76.
31 25 3 2
C H N O
(
E)-5-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)-2-methoxyphenol (15). Brown solid; Yield
−1
1
7
3
2
8%; m.p. 132–134 °C; IR cm : 3566, 3382, 1668, 1584, 1362, 1222, H-NMR (DMSO-d
6
): δ 3.83 (s,
H), 5.86 (s,1H), 6.86–6.89 (m, 4H), 7.01–7.06 (m, 4H), 7.14 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz,
13
H), 7.354–7.377 (m, 4H), 7.42 (s, 1H), 8.43 (s, 1H), 9.37 (s, 1H), 10.82 (s, 2H); C-NMR (MeOD-d )
4
δ 39.89, 56.08, 110.72, 110.72, 110.81, 113.52, 113.62, 113.62, 114.12, 118.09, 118.09, 118.64,
18.64, 119.11, 119.11, 120.30, 120.30, 120.88, 122.57, 122.92, 123.41, 123.41, 124.71, 126.99,
1

Molecules 2014, 19
11734
126.99, 129.28, 129.28, 137.09, 137.09, 153.59, 160.75. EI MS m/z (% rel. abund.): 471.00 (100.0,
M), 53.00 (11.3), 337.05 (10.9), 245.09 (27.0), 117.09 (9.3). Anal. Calcd for C H N O : C, 78.96; H,
3
1
25
3
2
5
.34; N, 8.91; O, 6.79. Found: C, 78.98; H, 5.32; N, 8.94; O, 6.76.
(
E)-3-(((4-(di(1H-Indol-3-yl)methyl)phenyl)imino)methyl)-2-iodo-6-methoxyphenol (16). Dark red solid;
−1
1
Yield 86%; m.p. 135–137 °C; IR cm : 3556, 3387, 1673, 1582, 1357, 1222; H-NMR (DMSO-d ):
6
δ 3.89 (s, 1H), 5.86 (s,1H), 6.86–6.89 (m, 4H), 7.05 (t, J = 7.0 Hz, 2H), 7.10 (d, J = 8.5 Hz, 1H), 7.14
(
(
d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.59
1
3
d, J = 8.5 Hz, 2H), 8.62 (d, 2H), 10.83 (s, 2H), 12.13 (s, 1H),; C-NMR (MeOD-d ): δ 38.68,
4
110.11, 110.11, 110.50, 110.81, 113.18, 113.18, 114.21, 118.11, 118.11, 118.58, 118.58, 119.10,
119.10, 120.11, 120.11, 120.47, 120.89, 122.69, 123.05, 123.05, 123.45, 126.98, 126.98, 128.69,
129.39, 129.39, 137.09, 137.09, 152.14, 164.28. EI MS m/z (% rel. abund.): 597.00 (2.7), 471.11
(
80.0), 337.11 (78.4), 245.11 (53.5), 117.08 (100.0, M). Anal. Calcd for C H IN O : C, 62.32; H,
31 24 3 2
4
.05; I, 21.24; N, 7.03; O, 5.36. Found: C, 62.35; H, 4.07; I, 21.25; N, 7.01; O, 5.34.
(
E)-2-Bromo-4-(((4-(di(1H-indol-3-yl)methyl)phenyl)imino)methyl)phenol (17). Light red solid; Yield
−1
1
7
8%; m.p. 139–141 °C; IR cm : 3552, 3384, 1667, 1587, 1350, 1215, 1060; H-NMR (DMSO-d ): δ 5.85
6
(
s,1H), 6.85–6.89 (m, 4H), 7.03–7.06 (m, 3H), 7.15 (d, J = 7.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H),
.35-7.38 (m, 4H), 7.74 (d, J = 8.5 Hz, 2H), 8.03 (s, 1H), 8.47 (s, 1H), 10.82 (s, 2H), 11.87 (s, 1H);
7
1
3
4
C-NMR (MeOD-d ): δ 39.86, 110.43, 110.43, 110.83, 116.11, 118.13, 118.13, 118.60, 118.60,
119.15, 119.15, 120.29, 120.29, 120.91, 123.44, 123.44, 126.98, 126.98, 129.33, 129.33, 129.52,
129.52, 133.49, 137.08, 137.08, 143.16, 148.73, 157.94, 158.91, 190.12. EI MS m/z (% rel. abund.):
520.95 (100.0, M), 441.06 (14.8), 337.05 (81.5), 245.07 (67.3), 117.05 (25.3). Anal. Calcd for
C
30
H22BrN
3
O: C, 69.24; H, 4.26; Br, 15.35; N, 8.07; O, 3.07. Found: C, 69.25; H, 4.25; Br, 15.37; N,
8
.09; O, 3.09.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(2-fluorobenzylidene)aniline (18). Dark orange solid; Yield 64%;
−1
1
m.p. 120–122 °C; IR cm : 3396, 1661, 1591, 1357, 1235 (C–F); H-NMR (DMSO-d
6
): δ 5.88 (s, 1H),
6
.86–6.89 (m, 4H), 7.03 (t, J = 8.0 Hz, 2H), 7.23–7.25 (m, 3H), 7.30–7.36 (m, 7H), 7.40 (d, J = 8.5
13
Hz, 2H), 8.78 (s, 1H), 10.83 (s, 2H); C-HMR (MeOD-d
4
): δ 39.87, 110.68, 110.68, 110.81, 115.52,
17.87, 118.08, 118.08, 118.51, 118.51, 119.05, 119.05, 120.40, 120.40, 120.70, 120.70, 120.88, 123.41,
23.41, 124.37, 126.96, 126.96, 128.29, 129.38, 129.38, 137.10, 137.10, 143.81, 153.30 (d, J = 206.25
1
1
Hz).EI MS m/z (% rel. abund.): 442.95 (100.0, M), 325.98 (34.5), 245.08 (72.5), 117.06 (34.5). Anal.
Calcd for C30 : Elemental Analysis: C, 81.24; H, 5.00; F, 4.28; N, 9.47. Found: Elemental
Analysis: C, 81.25; H, 4.98; F, 4.29; N, 9.49.
H22FN
3
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(3-fluorobenzylidene)aniline (19). Light orange solid; Yield 66%;
−1
1
m.p. 139–141 °C; IR cm : 3392, 1661, 1591, 1364, 1238; H-NMR (DMSO-d
.86–6.89 (m, 4H), 7.00 (t, J = 9.0 Hz, 1H), 7.05 (t, J = 7.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.31 (d,
J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 5.5 Hz, 1H), 7.70 (d,
6
): δ 5.87 (s, 1H),
6
13
J = 9.5 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 8.66 (s, 1H), 10.83 (s, 2H); C-NMR (MeOD-d ): δ 39.88,
4
1
1
10.83, 110.83,113.80, 113.98, 113.98, 118.12, 118.12, 118.54, 118.54, 119.11, 119.11, 120.43,
20.43, 120.91, 120.91, 123.43, 123.43, 124.85, 126.97, 126.97, 129.34, 129.34, 130.24, 130.31,

Molecules 2014, 19
11735
1
37.10, 137.10, 143.71, 148.81, 159.07 (d, J = 203.75 Hz), 174.86. EI MS m/z (% rel. abund.): 442.95
(
100.0, M), 325.98 (22.9), 245.08 (64.4), 117.06 (5.4). Anal. Calcd for C H FN : Elemental
3
0
22
3
Analysis: C, 81.24; H, 5.00; F, 4.28; N, 9.47. Found: Elemental Analysis: C, 81.21; H, 5.01; F, 4.30;
N, 9.50.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(4-fluorobenzylidene)aniline (20). Dark red solid; Yield 78%; m.p.
−1
1
1
1
7
39–141 °C; IR cm : 3397, 1664, 1583, 1359, 1235; H-NMR (DMSO-d ): δ 5.87 (s, 1H), 6.76 (s,
6
H), 6.83–6.89 (m, 4H), 7.00 (t, J = 8.5 Hz ,2H), 7.04 (d, J = 7.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H),
.22 (d, J = 7.5 Hz, 1H), 7.30–7.36 (m, 5H), 7.39 (d, J = 8.0 Hz, 1H), 8.63 (s, 1H), 10.83 (s, 2H);
1
3
4
C-NMR (MeOD-d ): δ 39.86, 110.85, 110.85, 115.30, 115.30, 115.47, 115.47, 118.14, 118.14,
1
1
18.58, 118.58, 119.13, 119.13, 120.37, 120.37, 120.92, 120.92, 123.44, 123.44, 126.97, 126.97,
30.60, 130.67, 130.67, 137.08, 137.08, 143.37, 143.37, 149.07, 159.21 (d, J = 228.75 Hz). EI MS m/z
(
% rel. abund.): 442.95 (100.0, M), 325.92 (14.4), 245.04 (53.4), 117.06 (3.9). Anal. Calcd for
C H FN : Elemental Analysis: C, 81.24; H, 5.00; F, 4.28; N, 9.47. Found: Elemental Analysis: C,
3
0
22
3
81.32; H, 5.02; F, 4.31; N, 9.45.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(2-chlorobenzylidene)aniline (21). Dark red solid; Yield 84%; m.p.
−1
1
1
6
45–147 °C; IR cm : 3386, 1668, 1584, 1363, 1092; H-NMR (DMSO-d ): δ 5.88 (s, 1H), 6.87–6.90
(
m, 4H), 7.05 (t, J = 8.0 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 8.0 Hz, 2H), 7.42(d, J = 8.5 Hz, 2H), 7.48 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.55 (t,
J = 7.5 Hz, 1H), 7.58 (dd, J = 1 Hz, 8.0 Hz, 1H), 8.15 (dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.88 (d, 1H),
1
3
1
1
1
6
0.84 (s, 2H); C-NMR (DMSO-d ): δ 39.79, 103.04, 111.92, 111.92, 114.61, 117.61, 118.51, 118.51,
18.65, 118.65, 119.59, 119.59, 120.63, 120.63, 121.21, 121.34, 124.01, 124.01, 127.09, 127.09,
29.50, 129.50, 130.26, 130.26, 137.09, 137.09, 158.14, 160.35, 162.77, 170.85. EI MS m/z (% rel.
abund.): 458.95 (100.0, M), 337.03 (60.8), 245.05 (85.1), 117.04 (4.9). Anal. Calcd for C30
8.34; H, 4.82; Cl, 7.71; N, 9.14. Found: C, 78.36; H, 4.81; Cl, 7.74; N, 9.16.
3
H22ClN : C,
7
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(3-chlorobenzylidene)aniline (22). Light orange solid; Yield 75%;
−1
1
m.p. 148–150 °C; IR cm : 3390, 1674, 1586, 1365, 1090; H-NMR (DMSO-d
6
): δ 5.87 (s, 1H),
6
.86–6.89 (m, 4H), 7.05 (t, J = 7 Hz, 2H), 7.23 (d, J = 8 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 7.5
13
Hz, 2H), 7.54 (m, 3H), 7.87 (d, J = 7.5 Hz, 2H), 8.65 (s, 1H), 10.83 (s, 2H); C-NMR (MeOD-d
4
):
δ 39.89, 110.80, 110.80, 113.20, 118.07, 118.07, 118.52, 118.52, 119.07, 120.44, 120.44, 120.87,
1
1
20.87, 123.39, 123.39, 126.97, 126.97, 127.70, 129.34, 129.34, 129.98, 129.98, 130.76, 134.55,
37.11, 137.11, 138.16, 143.78, 149.01, 158.82. EI MS m/z (% rel. abund.): 458.95 (100.0, M), 337.10
(
45.3), 245.10 (66.6), 117.06 (3.4). Anal. Calcd for C H ClN : C, 78.34; H, 4.82; Cl, 7.71; N, 9.14.
30 22 3
Found: C, 78.36; H, 4.85; Cl, 7.69; N, 9.13.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(4-chlorobenzylidene)aniline (23). Light orange solid; Yield 89%;
−1
1
m.p. 131–133 °C; IR cm : 3383, 1679, 1584, 1367, 1090; H-NMR (DMSO-d
.84–6.89 (m, 4H), 6.98-7.06 (m, 4H), 7.21 (d, J = 8 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 8.64 (s, 1H), 10.83 (s, 2H);
6
): δ 5.87 (s, 1H),
6
1
3
C-NMR (MeOD-d
4
): δ 39.88, 110.68, 110.68, 110.83, 118.11, 118.11, 118.55, 118.55, 119.11,
1
19.11, 119.22, 119.22, 120.39, 120.39, 120.90, 123.28, 123.28, 123.42, 126.97, 126.97, 128.64,

Molecules 2014, 19
11736
1
29.08, 129.08, 129.33, 129.33, 129.76, 129.76, 137.10, 137.10, 159.08. EI MS m/z (% rel. abund.):
4
59.10 (41.0), 337.10 (100.0, M), 245.09 (52.2), 117.08(3.2). Anal. Calcd for C H ClN : C, 78.34;
3
0
22
3
H, 4.82; Cl, 7.71; N, 9.14. Found: C, 78.36; H, 4.80; Cl, 7.74; N, 9.15.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(2-nitrobenzylidene)aniline (24). Dark yellow solid; Yield 83%;
−1
1
m.p. 124–126 °C; IR cm : 3379, 1664, 1576, 1361, 1340; H-NMR (DMSO-d ): δ 5.88 (s, 1H),
6
6
.87–6.90 (m, 4H), 7.05 (t, J = 7.0 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 8.0 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.76 (t, J = 9.0 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 8.10 (d,
1
3
J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H), 8.88 (s, 1H), 10.84 (s, 2H); C-NMR (MeOD-d ,): δ 39.87,
4
1
10.68, 110.68, 110.81, 115.52, 117.87, 118.08, 118.08, 118.51, 118.51, 119.05, 119.05, 120.40,
20.40, 120.70, 120.70, 120.88, 120.88, 123.41, 123.41, 124.37, 126.96, 126.96, 127.48, 129.10,
29.38, 129.38, 137.10, 143.81, 153.30. EI MS m/z (% rel. abund.): 470.10 (61.1), 337.12 (100.0, M),
45.07 (73.4), 117.09 (3.4). Anal. Calcd for C H N O : C, 76.58; H, 4.71; N, 11.91; O, 6.80. Found:
1
1
2
3
0
22
4
2
C, 76.56; H, 4.73; N, 11.92; O, 6.82.
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(4-nitrobenzylidene)aniline (25). Dark yellow solid; Yield 80%;
−1
1
m.p. 187–189 °C; IR cm : 3383, 1665, 1574, 1362, 1342; H-NMR (DMSO-d ): δ 5.89 (s, 1H),
6
6.87–6.89 (m, 4H), 7.05 (t, J = 7.0 Hz, 2H), 7.30-7.32 (m, 4H), 7.36 (d, J = 8.0 Hz, 2H), 7.43 (d,
J = 8.5 Hz, 2H), 8.17 (d, J = 9.0 Hz, 2H), 8.36 (d, J = 8.5 Hz, 2H), 8.82 (s, 1H), 10.84 (s, 2H);
1
3
6
C-NMR (DMSO-d ): δ 49.07, 111.94, 111.94, 118.36 118.36, 118.67, 118.67, 119.56, 119.56,
1
1
4
4
21.37, 121.37, 121.61, 121.61, 124.04, 124.04, 124.48, 124.48, 127.06, 127.06, 129.63, 129.95,
29.95, 137.09, 137.09, 142.19, 142.19, 144.60, 148.71, 149.21, 158.42. EI MS m/z (% rel. abund.):
69.90 (100.0, M), 336.99 (2.6), 244.99 (44.3), 117.04 (3.2). Anal. Calcd for C30
.71; N, 11.91; O, 6.80. Found: C, 76.60; H, 4.68; N, 11.93; O, 6.81.
22 4 2
H N O : C, 76.58; H,
(
E)-4-(di(1H-Indol-3-yl)methyl)-N-(3,4-dimethoxybenzylidene)aniline (26). Dark red solid; Yield 71%;
−1
1
m.p. 214–216 °C; IR cm : 3395, 1671, 1583, 1358, 1224, 1012; H-NMR (DMSO-d
6
): δ 3.86 (s, 6H),
.85 (s,1H), 6.85–6.89 (m, 4H), 7.04 (t, J = 7.0 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H),
.35–7.38 (m, 4H), 7.42 (dd, J = 1.5 Hz, 8.5 Hz, 1H), 7.52 (d, J = 1.5 Hz, 1H), 8.50 (s, 1H), 10.85 (s,
5
7
2
1
1
1
3
4
H); C-NMR (MeOD-d ): δ 37.95, 56.08, 56.18, 110.81, 110.81, 113.52, 113.62, 114.12, 118.09,
18.09, 118.64, 118.64, 119.11, 119.11, 120.30, 120.30, 120.88, 120.88, 122.57, 122.57, 122.92,
23.41, 124.71, 124.71, 126.99, 126.99, 129.28, 129.28, 137.09, 137.09, 153.59, 160.75. EI MS m/z
(% rel. abund.): 485.00 (100.0, M), 337.03 (3.3), 321.04 (11.0), 245.03 (22.2), 117.09 (1.2). Anal.
Calcd for C32 : C, 79.15; H, 5.60; N, 8.65; O, 6.59. Found: C, 79.16; H, 5.63; N, 8.67; O, 6.57.
27 3 2
H N O
3
.4. Bioassay
The antibacterial activity of compounds 3–26 was evaluated by using the agar diffusion method [44].
Gentamicin was used as positive control. Bacterial cultures which had been subjected through
2
4 h incubation were mixed with sterile physiological saline solution (0.85%). Turbidity of all cultures
6
was adjusted to match standard inoculum of 0.5 Mac-Farland scale [~10 CFU/mL]. Petri dishes
containing 20 mL of Mueller Hinton Agar (MHA) were used for all bacterial strains. Inocula were
swabbed on the surface of solidified media. Whatman No. 1 filter paper discs (6 mm in diameter)

Molecules 2014, 19
11737
impregnated with the test compound (10 µL/disc) were placed on the plates. Plates inoculated with the
bacteria were incubated for 24 h at 37 °C. All tests were replicated thrice and the final readings were
determined by using average diameter recorded.
4
. Conclusions
Nickel boride generated through an in-situ reaction between sodium borohydride and nickel acetate
and found to be a selective as well as mild reducing agent for compound 1. Nickel boride is capable of
converting its nitro group into amino group with very high yield in a shorter reaction time without
affecting the main bisindolylmethane skeleton. The antibacterial activity of bisindolylmethane Schiff
base derivatives 3–26 demonstrated that compounds 19, 20, 21, 22 and 26 specifically inhibit the
Salmonella typhi, S. paratyphi A and Salmonella paratyphi B bacterial strains. Further modification of
the structure or introduction of other substituents could improve the antibacterial potential of these
compounds and help in the discovery of new and antibacterial agents.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/8/11722/s1.
Acknowledgments
The authors would like to acknowledge Universiti Teknologi MARA for the financial support
through research grant, UiTM 600-RMI/5/3 CIFI (142/2013). The first author would like to thank
Ministry of Higher Education Malaysia for the financial funding through MyBrain15 scholarship.
Author Contributions
All authors have contributed equally to this work.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–26 are available from the authors.
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