An effective green and ecofriendly catalyst for synthesis of bis(indolyl)methanes as promising antimicrobial agents

Article abstract of DOI:10.1002/jhet.3729

An effective and suitable meglumine-catalyzed high-yielding process was considered and engaged for the synthesis of new bis(indolyl)methanes at ambient temperature under aqueous conditions. The catalytic reaction proceeds very smoothly. Clean reaction, ea

Full text of DOI:10.1002/jhet.3729

Received: 16 October 2018
DOI: 10.1002/jhet.3729
Revised: 12 April 2019
Accepted: 26 August 2019
A R T I C L E
An effective green and ecofriendly catalyst for synthesis of
bis(indolyl)methanes as promising antimicrobial agents
Bakthavatchala Reddy Nemallapudi1
| Grigory V. Zyryanov1,2 | Balakrishna Avula3 |
Mallikarjuna Reddy Guda1 | Sravya Gundala1
1 Chemical Engineering Institute, Ural
Abstract
Federal University, Yekaterinburg, Russia
An effective and suitable meglumine‐catalyzed high‐yielding process was con-
2 Ural Division of the Russian Academy of
Sciences, I. Ya. Postovskiy Institute of
Organic Synthesis, Yekaterinburg, Russia
3 Rajeev Gandhi Memorial College of
Engineering and Technology
(Autonomous), Nandyal, Andhra Pradsh,
India
sidered and engaged for the synthesis of new bis(indolyl)methanes at ambient
temperature under aqueous conditions. The catalytic reaction proceeds very
smoothly. Clean reaction, ease of product isolation/purification, easily avail-
able reactant and ecofriendly reaction conditions are the notable advantages
of the present methodology. All the title compounds were characterized by
IR, 1H, 13C NMR, and mass spectra. All the synthesized compounds were
tested for antimicrobial activity, and the results indicated that most of the syn-
thesized compounds exhibited excellent activity against the tested microorgan-
isms. In this new series, compound 3l, having nitro substituent on the aromatic
ring, showed exceptional potent inhibitory activity against Pseudomonas
aeruginosa and Penicillium chrysogenum.
Correspondence
Sravya Gundala, Chemical Engineering
Institute, Ural Federal University.
Yekaterinburg 620002, Russia
Email: sravyas72@yahoo.com
1 | INTRODUCTION
developments of BIM's are now driven mainly by the
curiosity in exhibiting broad spectrum bioactivities
Organic N‐heterocycles such as BIM's (bisindole meth-
anes) and their derivatives are advanced molecules and
have become indispensable as potential bioactive mole-
cules in modern medicinal chemistry. In the recent years,
because of their biological versatility, the BIM's have
emerged as potential target of synthesis for medicinal
chemist. Bis(indolyl)methanes (BIMs) and their deriva-
tives are promising nitrogen‐containing compounds that
are present in a variety of natural products and synthetic
compounds that find applications in pharmaceutical drug
research as HT6 receptor antagonist.[1] Derivatives of
indoles find their current applications ranging from design
and development, evaluation of novel drug delivery sys-
tems, catalysis to green chemistry, cosmetics, and agro-
chemicals.[2] Researchers are employing BIM's (bisindole
methanes), Figure 1,[3] to distinguish promising drugs
and avoid failures in potent drugs and have found to pos-
sess wide range of applications in the pharmaceutical
industry.[4] In fact, the continued expansion of
such as antifungal,[5] antiviral,[6] antimicrobial,[7] anti‐
inflammatory[8] and antioxidant.[9] In addition, BIM's are
evidently reported to exhibit anticancer activity, inhibit
the proliferation of cancerous cells, including those of
lung, pancreatic, colon, and cervical prostrate.[10] The
most significant metabolite of indole‐3‐carbinol dimeric
3,3′‐bis(indolyl methane) plays a significant role in
inhibiting breast cancer.[11] Indole carbazole derivatives
are known to function as triplet energy materials, while
oxidized BIM's are used as colorimetric sensors and
dyes.[12] Inferable from their explicit organic and pharma-
cological properties, there is growing interest for the devel-
opment of substantial routes for synthesizing BIM's.[13]
Green chemistry is a transforming technology that pro-
vides access to novel techniques and robust catalysts,
which enables to enhance conventional methodologies.
In past decades, electrophilic substitution of indoles with
carbonyl compounds is usually mediated by Lewis acids
or bronsted acids, hetero polyacids, ionic liquids, use of
J Heterocyclic Chem. 2019;1–9.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

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NEMALLAPUDI ET AL.
FIGURE 1 Bio‐active BIMs derivatives [Color figure can be viewed at wileyonlinelibrary.com]
toxic reagents, volatile organic solvents, excess catalyst
loading, and use of expensive catalysts are drawbacks of
these methods.[14,15] To overcome these problems, in
recent years, researchers are striving for developing
advanced green chemical methodologies to reduce costs,
hazards, waste and energy consumption using ecofriendly
reagents, and economical reaction conditions for achieving
successful synthetic procedures.[16] There are a wide range
of interpretations about the organic practices that are con-
sistent with the objectives of sustainable and green chemis-
try. In this context, the usage of environmentally benign
solvents (ethylene glycol, glycerol, and water), solvent free
conditions, and non‐conventional methods such as micro-
waves, ultrasound and grinding constitute influential and
green chemical protocols for cost effective synthesis.[17]
In this study, we examine meglumine, which is an amino
sugar sequestered from sorbitol‐possessing chemical nota-
tion C7H17NO5, as an efficient and reusable catalyst for
the synthesis of novel BIM's derivatives[18] (Scheme 1).
KHSO4, FeCl3, LiClO4, and sulfamic acid were used.
Finally, the preferred product was obtained in 35% to 67%
yield (Table 1, entries 1‐4). Additional investigation desig-
nated that meglumine was the greatest catalyst for this
conversion and afforded the preferred product in 96% in
0.25 hours (Table 1, entry 6). Some solvents such as H2O,
MeOH, THF, PEG 400, and ethanol‐water mixture were
verified for the model reaction. It was detected that the
mixture of water and ethanol was the most effective sol-
vent, and the present reaction continued strongly, giving
the highest yield. It is notable that when the reaction was
done in solvent‐free conditions, low yield of target product
was acquired (Table 1, entry 7). Then, the influence of cat-
alyst loading was calculated in the model reaction at room
temperature in H2O.
Apart from these, to find out the influence of the cata-
lyst concentration, the reaction of indole (1) and benzal-
dehyde (2) was carried out with different concentrations
of catalyst in H2O at room temperature. The results pre-
sented in Table 2 showed that 10 mol% of meglumine is
best to attain high yield in smaller reaction time. There
is no effect by enhancing the quantity of catalyst on the
product yield. On the other hand, employing a lower frac-
tion of meglumine caused a reduced yield of the preferred
product (Table 2).
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
Primarily, we immersed on improving the reaction condi-
tions for the development of meglumine catalyzed proce-
dure for the synthesis of new bis(indolyl)methanes. We
designated indole 1a (1.0 mmol) benzaldehyde 2a
(0.5 mmol) for the typical reaction (Scheme 1). The above
reaction was carried out at room temperature in water in
presence of KHSO4 catalyst to inaugurate the actual ability
of the catalyst. As presented in Table 1, it was detected that
only a low yield of product was fashioned even after the
reaction time was extended to 13 hours (Table 1, entry 1).
Then, we tried to enhance the reaction conditions with
altered catalysts, which might help to decrease the reaction
time and develop the yield of the objective product. After
observing numerous catalysts, it can be noticed that Cu
(OTf)2 displayed a slight catalytic activity to provide the
product in a low yield (78%, Table 1, entry 5). An improve-
ment was detected when numerous catalysts such as
All compounds were accomplished in open atmo-
sphere and are not sensitive to air and moisture. All the
synthesized compounds have been characterized by ana-
lytical data.
3 | PHARMOCOLOGY
3.1 | Antimicrobial activity
3.1.1 | Antibacterial activity
The compounds 3(a‐m) were evaluated for antibacterial
activity at four different concentrations 12.5, 25, 50, and
100 μg/well in contradiction of Staphylococcus aureus,
Bacillus subtilis (Gram‐positive bacteria), and Pseudomo-
nas aeruginosa, Klebsiella pneumoniae (Gram‐negative

NEMALLAPUDI ET AL.
3
SCHEME 1 Synthesis of bis(indolyl)methane derivatives (3a‐m)
TABLE 1 Optimization of the reaction conditions for the syn-
thesis of 3(a‐m)
TABLE 2 Effect of concentration of meglumine on the reaction
of indole (1) with benzaldehyde (2)
Entry Catalyst
Solvent
Time (hrs) Yield (%)
Entry
Mole % of catalyst
Time (min)
Yield (%)
1
KHSO4
FeCl3
H2O
H2O
H2O
13
12
12
10
6
55
59
35
67
78
96
16
60
65
41
52
1
2
3
4
5
6
7
1
50
45
40
20
15
15
15
50
65
70
75
96
96
96
2
2.5
5
3
LiClO4
4
Sulfamic acid H2O
7.5
10
5
Cu (OTf)2
H2O
6
Meglumine
Meglumine
Meglumine
Meglumine
Meglumine
Meglumine
H2O:EtOH (1:1) 0.25
12.5
15
7
No
5
4
4
4
4
8
MeOH
9
EtOH:H2O (1: 9)
THF
more vulnerable in the direction of the verified com-
pounds than Gram‐positive ones. It was detected that
the compounds (3l and 3m) exhibited slightly higher
activity than the respective. This may be because of the
presence of electron‐withdrawing groups, as well as elec-
tronegative atoms such as NO2, CN, F, Cl, and Br. In fact,
the compound 3l displayed outstanding activity against
10
11
PEG 400
bacteria) by means of chloramphenicol as reference drug.
The results of antibacterial activity revealed in Table 3 &
Figure S1 designated that Gram‐negative bacteria were

4
NEMALLAPUDI ET AL.

NEMALLAPUDI ET AL.
5
P. aeruginosa when associated with the standard drug
chloramphenicol. This may be because of the presence
of more electronegative nitro group on the aromatic ring.
Furthermore, it was perceived that the compounds 3f, 3g,
3h, 3i, and 3k presented considerable activity; 3a, 3b, and
3e demonstrated reasonable activity; and the compounds
3c, 3d, and 3j were inactive.
Aspergillus niger. Among all the compounds, 3l displayed
greater inhibitory activity, particularly against
P. chrysogenum when compared with the standard drug
ketoconazole (Table 4 & Figure S2). Besides, the com-
pounds 3f, 3g, 3h, 3i, and 3 k showed good activity.
3.1.3 | Minimum inhibitory concentra-
tion, minimum bactericidal concentration,
and minimum fungicidal concentration of
the compounds 3f, 3l, and 3m
3.1.2 | Antifungal activity
All the tested compounds reserved the spore germination.
In general, most of the compounds revealed slightly higher
antifungal activity towards Penicillium chrysogenum than
The minimum inhibitory concentration (MIC), minimum
bactericidal concentration (MBC), and minimum fungi-
TABLE 4 The in vitro antifungal activity of compounds 3(a‐m)
Zone of Inhibition (mm)
Aspergillus niger
Penicillium chrysogenum
Compound
12.5 μg/well 25 μg/well 50 μg/well 100 μg/well 12.5 μg/well 25 μg/well 50 μg/well 100 μg/ well
3a
‐
08
09
‐
1
2
10
11
‐
2
1
10
13
‐
3
1
‐
‐
11
14
‐
1
3
14
16
‐
3
2
3b
‐
‐
12
‐
2
3c
‐
‐
3d
‐
‐
‐
‐
‐
‐
‐
‐
3e
‐
09
27
23
24
17
‐
2
1
3
3
3
11
29
25
26
19
‐
2
3
3
2
1
13
31
27
28
21
‐
3
3
3
2
3
12
29
28
25
29
‐
1
3
3
2
2
13
34
30
27
32
‐
3
2
1
3
3
15
36
32
29
34
‐
2
3
2
2
2
17
35
34
31
37
‐
2
2
3
3
3
3f
25
21
22
15
‐
2
2
1
2
3g
3h
3i
3j
3k
23
27
24
29
‐
3
3
1
3
25
29
26
31
‐
2
2
1
2
27
31
28
34
‐
1
1
1
1
29
32
30
37
‐
2
3
2
1
30
34
31
34
‐
2
1
2
1
32
35
33
36
‐
2
2
3
1
34
37
35
37
‐
1
1
3
2
36
41
38
39
‐
1
2
2
2
3l
3m
Ketoconazole
Control (DMSO)
Note. −, no activity; , standard deviation.
TABLE 5 MIC, MBC, and MFC of compounds 3f, 3l, and 3m
Minimum Inhibitory Concentration
MIC (MBC/MFC) μg/mL
Staphylococcus
aureus
Bacillus
subtilis
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
Aspergillus
niger
Penicillium
Chrysogenum
Compound
3f
50(200)
25(>100)
50(>200)
6.25
25(100)
25(100)
50(>200)
6.25
12.5(50)
6.25(12.5)
25(100)
6.25
25(>100)
12.5(50)
25(>100)
12.5
25(100)
12.5(50)
25(100)
‐
12.5(50)
12.5(25)
50(>200)
‐
3l
3m
Chloramphenicol
Ketoconazole
‐
‐
‐
‐
6.25
12.5
Abbreviations: MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration.

6
NEMALLAPUDI ET AL.
cidal concentration (MFC) values of the compounds tested
are listed in Table 5. MIC is the lowest concentration of an
antimicrobial that will inhibit the visible growth of a
microorganism. The MBC/MFC is the lowest concentra-
were recorded in CDCl3/DMSO‐d6 on a Jeol JNM λ‐
400 MHz spectrometer. The 13C NMR spectra were
recorded in CDCl3/DMSO‐d6 on a Jeol JNM spectrometer
operating at λ‐100 MHz. High‐resolution mass spectra
were recorded on Micromass Q‐TOF micro mass spec-
trometer using electro spray ionization. All chemical
shifts were reported in δ (ppm) using TMS as an internal
standard. The microanalyses were performed on a
Perkin‐Elmer 240C elemental analyzer. The temperature
was measured by flexible probe throughout the reaction.
The microanalyses were performed on a Perkin‐Elmer
240C elemental analyzer (Massachusetts). The progress
of the reaction was monitored by TLC using silica gel
plates (silica gel 60 F254 0.25 mm), and components were
visualized by observation under ultraviolet (UV) light
(254 and 365 nm).
tion of antibiotic required to kill
a
particular
bacterium/fungus. The MBC/MFC involves an additional
set of steps performed once the MIC is determined. The
antimicrobials
are
usually
regarded
as
bactericidal/fungicidal if the MBC/MFC is not greater than
four times the MIC.[19] The compound 3l exhibited low
MIC values when compared with 3f and 3m. In addition,
MBC value in 3l is 2 × MIC in case of P. aeruginosa and
MFC value is 2 × MIC in case of P. chrysogenum. However,
the other compounds showed bactericidal and fungicidal
effects greater than 2 × MIC. The nitro substituted
bis(indolyl)methane, 3l exhibited excellent antibacterial
activity against P. aeruginosa with an inhibition zone of
34 mm at 100 μg/well and MIC and MBC of 6.25 and
12.5 μg/mL, respectively. The compound 3l also displayed
strong antifungal activity against P. chrysogenum with an
inhibition zone of 41 mm at 100 μg/well and MIC and
MFC of 12.5 and 25 μg/mL, respectively. Moreover, it was
observed that the compounds having nitro substituent on
aromatic ring enhanced the activity when compared with
all other compounds.
General procedure for the synthesis of bis(indolyl)
methane derivatives (3a‐3m).
To a stimulated solution of substituted indole (1)
(2.0 mmol) and carbonyl compound (2) (1 mmol) in
H2O (10 mL), meglumine (0.10 mmol) was added and
continued the stirring at room temperature for 15 minutes
and monitored by TLC. After completion of the reaction,
precipitate was formed, filtered, and washed with water.
The subsequent product was found to be pure enough
for characterization.
4 | CONCLUSION
5.1 | 3,3′‐(Phenylmethylene)bis(1H‐
In conclusion, we have successfully developed competent
methodology for the synthesis of bis(indolyl)methanes at
room temperature under aqueous conditions in high
yields by reacting aldehydes with indoles in the presence
of meglumine catalyst, which could be beneficial
meriting further investigations. Our findings suggest that
the present methodology will open a new route for the
synthesis of bis(indolyl)methane derivatives. All the enti-
indole) (3a)
Red solid; Yield: 94%; mp: 140°C‐142°C; IR (KBr, cm−1):
3315 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ 7.89 (s,
2H, NH), 7.28‐6.62 (m, 15H, Ar─H), 5.91 (s, 1H, CH)
ppm; 13C NMR (DMSO‐d6, 100 MHz): δ 144.4, 136.9,
129.7, 128.5, 127.4, 126.8, 123.4, 121.7, 120.3, 119.9,
118.6, 111.7 (aromatic carbons), 41.2 (CH) ppm. MS (EI)
m/z: 322.4033 [M+]; Anal. Calcd. for C23H18N2: C, 85.68;
H, 5.63; N, 8.69; Found: C, 85.78; H, 5.61; N, 8.90%.
1
tled compounds were characterized by IR, H, 13C NMR,
mass spectra. All the lead compounds were tested for
antimicrobial activity. The compound 3l having nitro
substituent on the aromatic ring is a potential and prom-
ising bioactive compound against P. aeruginosa and
P. chrysogenum. We anticipate that simple synthetic
accessibility and intriguing biological properties of new
bis(indolyl)methane derivatives could address unmet
challenges in the pharmaceutical industry.
5.2 | 3,3′‐(p‐Tolylmethylene)bis(1H‐
indole) (3b)
White solid; Yield: 96%; mp: 93°C‐95°C; IR (KBr, cm−1):
3362 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ 8.42 (s,
2H, NH), 7.41‐6.97 (m, 14H, Ar─H), 5.91 (s, 1H, CH),
2.73 (s, 3H, CH3) ppm; 13C NMR (DMSO‐d6, 100 MHz):
δ 141.6, 136.6, 135.3, 130.8, 128.1, 127.6, 124.7, 121.3,
119.8, 119.4, 118.1, 111.9 (aromatic carbons), 41.5 (CH),
21.9 (CH3) ppm. MS (EI) m/z: 336.4298 [M+]; Anal.
Calcd. for C24H20N2: C, 85.68; H, 5.99; N, 8.33; Found:
C, 85.81; H, 5.97; N, 8.56%.
5 | EXPERIMENTAL
Melting points were determined in open capillaries on a
Mel‐Temp apparatus and are uncorrected. The homoge-
neity of the compounds was checked by TLC (silica gel
H, BDH, hexane/ethyl acetate, 3:1). The 1H NMR spectra

NEMALLAPUDI ET AL.
7
5.3 | 3,3′‐((4‐Methoxyphenyl)methylene)
bis(1H‐indole) (3c)
5.7 | 3,3′‐((4‐Chlorophenyl)methylene)
bis(1H‐indole) (3g)
1
Brown solid; Yield: 92%; IR (KBr, cm−1): 3359 (NH); H
NMR (DMSO‐d6, 400 MHz): δ 8.64 (s, 2H, NH), 7.73‐
7.14 (m, 14H, Ar─H), 5.71 (s, 1H, CH), 3.05 (s, 3H,
OCH3) ppm; 13C NMR (DMSO‐d6, 100 MHz): δ 158.3,
136.8, 135.4, 130.4, 127.5, 124.1, 122.5, 121.0, 120.6,
119.7, 113.9, 111.6 (aromatic carbons), 55.8 (OCH3), 42.7
(CH) ppm. MS (EI) m/z: 352.1578 [M+]; Anal. Calcd. for
C24H20N2O: C, 81.79; H, 5.72; N, 7.95; Found: C, 81.91;
H, 5.74; N, 8.14%.
Red solid; Yield: mp: 74°C‐76°C, 88%; IR (KBr, cm−1):
3328 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ 7.94 (s,
2H, NH), 7.06‐6.64 (m, 14H, Ar─H), 5.88 (s, 1H, CH)
ppm; 13C NMR (DMSO‐d6, 100 MHz): δ 143.7, 137.4,
132.5, 131.6, 129.1, 128.9, 124.7, 123.3, 119.7, 119.1,
118.6, 111.2 (aromatic carbons), 39.6 (CH) ppm. MS (EI)
m/z: 356.8479 [M+]; Anal. Calcd. for C23H17ClN2: C,
77.41; H, 4.80; N, 7.85; Found: C, 77.55; H, 4.81; N, 8.06%.
5.8 | 3,3′‐((2‐Chlorophenyl)methylene)
bis(1H‐indole) (3h)
5.4 | 3,3′‐((3,5‐dimethoxyphenyl)
methylene)bis(1H‐indole) (3d)
Pink solid; Yield: 91%; mp: 71°C‐73°C, IR (KBr, cm−1):
3332 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ 7.71 (s,
2H, NH), 7.19‐6.54 (m, 14H, Ar─H), 6.52 (s, 1H, CH);
13C NMR (DMSO‐d6, 100 MHz): δ 141.3, 136.2, 134.1,
131.2, 129.5, 126.6, 126.1, 125.7, 124.6, 122.7, 119.7,
119.3, 118.4, 111.3 (aromatic carbons), 36.2 (CH) ppm.
MS (EI) m/z: 356.8479 [M+]; Anal. Calcd. for C23H17ClN2:
C, 77.41; H, 4.80; N, 7.85; Found: C, 77.54; H, 4.82; N,
8.09%.
Red solid; Yield: 89%; mp: 196°C‐198°C; IR (KBr, cm−1):
3323 (NH); H‐NMR (DMSO‐d6, 400 MHz) δ 7.89 (s, 2H,
1
NH), 7.19‐6.68 (s, 16H, Ar─H), 5.85 (s, 1H, CH), 3.76 (s,
6H, 2‐OCH3) ppm; 13C‐NMR (DMSO‐d6, 100 MHz) δ
161.4, 140.8, 136.3, 127.7, 123.9, 121.0, 119.7, 118.9,
116.5, 113.2, 111.8, 105.9, 97.6 (aromatic carbons), 55.8
(OCH3), 42.4 (CH); MS (EI) m/z: 352.1578 [M+]; Anal.
Calcd. for C25H22N2O2: C, 78.51; H, 5.80; N, 7.32; Found:
C, 78.62; H, 5.83; N, 7.57%.
5.9 | 3,3′‐((4‐Bromophenyl)methylene)
bis(1H‐indole) (3i)
5.5 | 3,3′‐((4‐N,N‐dimethylaniline)
methylene)bis(1H‐indole) (3e)
Pink solid; Yield: 87%; mp: 111°C‐113°C, IR (KBr, cm−1):
3341 (NH); H NMR (DMSO‐d6, 400 MHz): δ 7.88 (s, 2H,
Pink solid; Yield: 86%; mp 170°C‐171°C; IR (KBr, cm−1):
3346 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ 8.01 (s,
2H, NH), 7.61‐6.68 (m, 14H, Ar─H), 5.88 (s, 1H, CH),
3.16 (s, 6H, CH3) ppm; 13C NMR (DMSO‐d6, 100 MHz):
δ 151.7, 137.5, 133.0, 130.2, 126.9, 123.7, 121.9, 118.5,
118.1, 117.3, 116.2, 111.1, 108.2 (aromatic carbons), 39.7
(CH), ppm. MS (EI) m/z: 365.4714 [M+]; Anal. Calcd.
for C25H23N3: C, 82.16; H, 6.34 N, 11.50; Found: C,
82.28; H, 6.36; N, 11.75%.
1
NH), 7.07‐6.62 (m, 14H, Ar─H), 5.87 (s, 1H, CH); 13C
NMR (DMSO‐d6, 100 MHz): δ 144.2, 137.8, 132.4, 131.6,
128.4, 124.8, 123.2, 120.7, 119.9, 119.5, 119.1, 111.2 (aro-
matic carbons), 39.9 (CH) ppm. MS (EI) m/z: 401.2979
[M+]; Anal. Calcd. for C23H17BrN2: C, 68.84; H, 4.27; N,
6.98; Found: C, 68.97; H, 4.29; N, 7.19%.
5.10 | 3,3′‐((4‐Hydroxyphenyl)methylene)
bis(1H‐indole) (3j)
5.6 | 3,3′‐((4‐Fluorophenyl)methylene)
bis(1H‐indole) (3f)
Red solid; Yield: 84%; IR (KBr, cm−1): 3318 (NH), 3554
1
1
Red solid; Yield: 95%; IR (KBr, cm−1): 3350 (NH); H
(OH); mp: 121°C‐123°C, H NMR (DMSO‐d6, 400 MHz):
NMR (DMSO‐d6, 400 MHz): δ 7.59 (s, 2H, NH), 7.20‐
6.83 (m, 14H, Ar─H), 5.75 (s, 1H, CH) ppm; 13C NMR
(DMSO‐d6, 100 MHz): δ 162.5, 139.1, 136.2, 131.2, 128.1,
124.3, 123.1, 119.4, 119.1, 118.7, 116.0, 111.9 (aromatic
carbons), 39.6 (CH) ppm. MS (EI) m/z: 340.3932 [M+];
Anal. Calcd. for C23H17FN2: C, 81.16; H, 5.03; N, 8.23;
Found: C, 81.28; H, 5.05; N, 8.46%.
δ 10.8 (s, 2H, NH), 9.04 (s, 1H, OH), 7.06‐6.71 (m, 14H,
Ar─H), 5.92 (s, 1H, CH) ppm; 13C NMR (DMSO‐d6,
100 MHz): δ 156.1, 136.6, 135.4, 129.7, 126.3, 123.9,
120.8, 119.5, 118.2, 117.8, 114.3, 111.2 (aromatic carbons),
38.9 (CH) ppm; MS (EI) m/z: 338.4020 [M+]; Anal. Calcd.
for C23H18N2O: C, 81.63; H, 5.36 N, 8.28; Found: C, 81.76;
H, 5.38; N, 8.50%.

8
NEMALLAPUDI ET AL.
50, 100 μg/well) were added into the wells by using sterile
pipettes. Simultaneously, the standard antibiotics, chlor-
amphenicol for antibacterial activity and ketoconazole
for antifungal activity (as positive control), were tested
against the pathogens. The samples were dissolved in
DMSO, which showed no zone of inhibition acts as nega-
tive control. The plates were incubated at 37°C for
24 hours for bacteria and at 28°C for 48 hours for fungi.
After appropriate incubation, the diameter of the zone
of inhibition of each well was measured. Duplicates were
maintained, and the average values were calculated for
eventual antimicrobial activity.
5.11 | 3,3′‐((4‐Nitrophenyl)methylene)
bis(1H‐indole) (3k)
Yellow solid; Yield: 90%; mp: 218°‐220°C, IR (KBr, cm−1):
1
3366 (NH); H NMR (DMSO‐d6, 400 MHz): δ 7.57 (s, 2H,
NH), 7.09‐6.87 (m, 14H, Ar─H), 5.91 (s, 1H, CH); 13C
NMR (DMSO‐d6, 100 MHz): δ 153.7, 146.7, 135.5, 128.4,
127.2, 122.2, 121.8, 120.9, 118.8, 118.2, 116.3, 114.2 (aro-
matic carbons), 40.1 (CH) ppm. MS (EI) m/z: 367.4006
[M+]; Anal. Calcd. for C23H17N3O2: C, 75.19; H, 4.66 N,
11.44; Found: C, 75.33; H, 4.68; N, 11.65%.
Broth dilution test is used to determine MIC of the pre-
viously mentioned samples.[21,22] Freshly prepared nutri-
ent broth was used as diluents. The 24‐hour‐old culture
of the test bacteria S. aureus, B. subtilis, P. aeruginosa,
and K. pneumoniae and the test fungi A. niger and
P. chrysogenum were diluted 100‐fold in nutrient broth
(100 μL bacterial cultures in 10 mL NB). The stock solu-
tion of the synthesized compounds was prepared in
dimethyl sulfoxide (DMSO) by dissolving 5 mg of the
compound in 1 mL of DMSO. Increasing concentrations
of the test samples (1.25, 2.5, 5, 10, 20, and 40 μL of stock
solution contains 6.25, 12.5, 25, 50, 100, and 200 μg of the
compounds) were added to the test tubes containing the
bacterial and fungal cultures. All the tubes were incu-
bated at 37°C for 24 hours for bacteria and at 28°C for
48 hours for fungi. The tubes were examined for visible
turbidity and using NB as control. Control without test
samples and with solvent was assayed simultaneously.
The lowest concentration that inhibited visible growth
of the tested organisms was recorded as MIC.
5.12 | 3,3′‐((3‐Nitrophenyl)methylene)
bis(1H‐indole) (3l)
Yellow solid; Yield: 93%; mp: 220°C‐222°C, IR (KBr,
cm−1): 3372 (NH); 1H NMR (DMSO‐d6, 400 MHz): δ
7.83 (s, 2H, NH), 7.12‐6.97 (m, 2H, Ar─H), 5.88 (s, 1H,
CH); 13C NMR (DMSO‐d6, 100 MHz): δ 147.5, 145.4,
136.9, 133.9, 129.6, 126.7, 123.9, 124.6, 123.3, 122.5,
119.1, 119.4, 118.3, 111.3 (aromatic carbons), 40.1 (CH).
MS (EI) m/z: 367.4035 [M+]; Anal. Calcd. for
C23H17N3O2: C, 75.19; H, 4.66 N, 11.44; Found: C, 75.29;
H, 4.67; N, 11.64%.
5.13 | 3,3′‐((4‐Phenylnitrile)methylene)
bis(1H‐indole) (3m)
Slight brown solid; Yield: 85%; mp 205°C‐207°C; IR (KBr
cm−1): 3357 (NH), 1592 (C═N); 1H NMR (DMSO‐d6,
400 MHz): δ 7.96 (s, 2H, NH), 7.64‐6.89 (m, 14H, Ar─H),
5.97 (s, 1H, CH) ppm; 13C NMR (DMSO‐d6, 100 MHz): δ
150.9, 136.8, 132.7, 129.5, 126.4, 123.3, 121.5, 118.9,
118.8, 118.3, 116.7, 111.6, 108.9, 38.8 (CH), ppm (aromatic
carbons). MS (EI) m/z: 347.4124 [M+]; Anal. Calcd. for
C24H17N3: C, 82.97; H, 4.93 N, 12.10; Found: C, 83.06;
H, 4.94; N, 12.33%.
To determine the MBC[23] and MFC[24] for each set of
test tubes in the MIC determination, a loopful of broth
was collected from those tubes, which did not show any
growth and inoculated on sterile nutrient broth (for bac-
teria) and PDA (for fungi) by streaking. Plates inoculated
with bacteria and fungi were incubated at 37°C for
24 hours and at 28°C for 48 hours, respectively. After
incubation, the lowest concentration was noted as MBC
(for bacteria) or MFC (for fungi), at which no visible
growth was observed.
6 | ANTIMICROBIAL STUDIES
The compounds 3(a‐m) were dissolved in DMSO at dif-
ferent concentrations of 12.5, 25, 50, and 100 μg/well.
Bacterial strains S. aureus, B. subtilis, P. aeruginosa, and
K. pneumoniae and fungi A. niger and P. chrysogenum.
The in vitro antimicrobial studies were carried out by
agar well diffusion method against test organisms.[20]
Nutrient broth (NB) plates were swabbed with 24‐hour‐
old broth culture (100 μL) of test bacteria. Using the ster-
ile cork borer, wells (6 mm) were made into each
petriplate. The compounds were dissolved in DMSO of
5 mg/mL and from this, 2.5, 5, 10, and 20 μL (12.5, 25,
ACKNOWLEDGMENT
The authors are thankful to Ural Federal University,
Yekaterinburg, Russia for providing Postdoctoral
Fellowship.
ORCID
Bakthavatchala Reddy Nemallapudi
0000-0003-2039-9277
https://orcid.org/

NEMALLAPUDI ET AL.
9
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How to cite this article: Nemallapudi BR,
Zyryanov GV, Avula B, Guda MR, Gundala S. An
effective green and ecofriendly catalyst for synthesis
of bis(indolyl)methanes as promising antimicrobial
agents. J Heterocyclic Chem. 2019;1–9. https://doi.
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