Synthesis, spectroscopic investigations, DFT studies, molecular docking and antimicrobial potential of certain new indole-isatin molecular hybrids: Experimental and theoretical approaches

Article abstract of DOI:10.1016/j.molstruc.2017.10.025

Indole-isatin molecular hybrids 5a-i have been synthesized and characterized by different spectroscopic methods to be evaluated as new antimicrobial agents against a panel of Gram positive bacteria, Gram negative bacteria, and moulds. Compound 5h was selected as a representative example of the prepared compounds 5a-i to perform computational investigations. Its vibrational properties have been studied using FT-IR and FT-Raman with the aid of density functional theory approach. The natural bond orbital analysis as well as HOMO and LUMO molecular orbitals investigations of compound 5h were carried out to explore its possible intermolecular delocalization or hyperconjugation and its possible interactions with the target protein. Molecular docking of compound 5h predicted its binding mode with the fungal target protein.

Full text of DOI:10.1016/j.molstruc.2017.10.025

Accepted Manuscript
Synthesis, spectroscopic investigations, DFT studies, molecular docking and
antimicrobial potential of certain new indole-isatin molecular hybrids: Experimental
and theoretical approaches
Maha S. Almutairi, Azza S. Zakaria, P. Primsa Ignasius, Reem I. Al-Wabli, Isaac
Hubert Joe, Mohamed I. Attia
PII:
S0022-2860(17)31356-X
10.1016/j.molstruc.2017.10.025
MOLSTR 24396
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 July 2017
Revised Date: 4 October 2017
Accepted Date: 6 October 2017
Please cite this article as: M.S. Almutairi, A.S. Zakaria, P.P. Ignasius, R.I. Al-Wabli, I.H. Joe, M.I. Attia,
Synthesis, spectroscopic investigations, DFT studies, molecular docking and antimicrobial potential
of certain new indole-isatin molecular hybrids: Experimental and theoretical approaches, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.10.025.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, spectroscopic investigations, DFT studies, molecular docking
and antimicrobial potential of certain new indole-isatin molecular hybrids:
Experimental and theoretical approaches
a
b
c,d
a
Maha S. Almutairi , Azza S. Zakaria , P. Primsa Ignasius , Reem I. Al-Wabli , Isaac Hubert
c
a,e*
Joe and Mohamed I. Attia
a
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
b
Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University,
2
1521 Alexandria, Egypt
c
Centre for Molecular and Biophysics Research, Mar Ivanios College, Thiruvananthapuram
6
95015, Kerala, India.
d
Department of Physics, Government College, Nedumangad, Thiruvananthapuram 695541,
Kerala, India
e
Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries
Research Division, National Research Centre (ID: 60014618), El Bohooth Street, Dokki,
Giza 12622, Egypt
Keywords: 5-Methoxyindole; Isatin, FT-IR; FT-Raman; DFT; Antimicrobial
1

ACCEPTED MANUSCRIPT
Abstract
Indole-isatin molecular hybrids 5a-i have been synthesized and characterized by different
spectroscopic methods to be evaluated as new antimicrobial agents against a panel of Gram
positive bacteria, Gram negative bacteria, and moulds. Compound 5h was selected as a
representative example of the prepared compounds 5a-i to perform computational
investigations. Its vibrational properties have been studied using FT-IR and FT-Raman with
the aid of density functional theory (DFT) approach. The natural bond orbital (NBO) analysis
as well as HOMO and LUMO molecular orbitals investigations of compound 5h were carried
out to explore its possible intermolecular delocalization or hyper-conjugation and its possible
interactions with the target protein. Molecular docking of compound 5h predicted its binding
mode with the fungal target protein.
1
. Introduction
Indole (benzopyrrole) is an aromatic bicyclic structure consisting of fused benzene and
pyrrole rings through the 2- and 3-positions of the pyrrole fragment. Since its first synthesis
in 1866, indole was found in many natural products like fungal metabolities, vinca alkaloids,
and marine natural products in addition to its incorporation in various bioactive
agrochemicals and pharmaceuticals [1, 2]. In the last few years it has been documented that
indole myriad derivatives and its bioisosters have a wide spectrum of bioactivities ranging
from antimicrobial to anticancer activities [2-5]. Moreover, 5-methoxyindole constitutes the
scaffold of the natural hormone melatonin (MLT). MLT and its derivatives have diverse
pharmaceutical applications such as treatment of depression, headache, sleep disorders, and
as anticancer agents [6-8].
On the other hand, 2,3-dioxindole (isatin) is an oxidised form of indole and it has been
identified as an endogenous molecule in humans and other mammals [9]. It contains different
functionalizable groups and it possesses privileged electronic characteristics with unique
molecular size giving it the ability to exhibit diverse beneficial biological properties. Isatin
constitutes the backbone of a relatively large number of bioactive compounds endowed with
various useful biological activities such as antibacterial [10, 11], antifungal [12],
anticonvulsant [13], and anticancer activities [14-16]. Consequently, isatin-bearing
compounds have captured the attention in different research fields particularly in the medical
research area.
Molecular hybridization is a well known strategy in medicinal chemistry to get new potent
bioactive molecules [17, 18]. Therefore, the molecular hybrids 5a-i incorporating both isatin
and 5-methoxyindole moieties were designed and synthesized to be evaluated as new
2

ACCEPTED MANUSCRIPT
antimicrobial agents. Single crystal X-ray structure of compound 5h, as a representative
example of the synthesized compounds 5a-i, confirmed their imine double bond
configuration. In addition, the vibrational properties of compound 5h have been investigated
in the current study using FT-IR and FT-Raman characterizations aided by density functional
theory (DFT) computations in order to gain insight into its inter- and intra-molecular
interactions as well as the equilibrium structural geometry of this type of compounds. Whilst,
the natural bond orbital (NBO) analysis of compound 5h has been performed to determine its
possible intermolecular delocalization or hyper-conjugation. Also, compound 5h was
subjected to HOMO and LUMO molecular orbitals investigations to explore its possible
interactions with the target protein. Molecular docking studies predicted the binding mode of
compound 5h with its target protein.
2
. Experimental details
2
.1. General
The melting points were measured using a Gallenkamp melting point device and are
uncorrected. The FT-IR spectrum of compound 5h was recorded using Perkin-Elmer RXL
-
1
spectrometer (Waltham, Massachusetts, USA) in the region 4000-500 cm , with samples in
-1
the KBr pellet method with resolution of 2 cm . The Raman spectrum of compound 5h was
-1
recorded in the region 3500-50 cm using Bruker RFS-27 spectrophotometer (Ettlingen,
-1
Germany) having resolution of 2 cm . The NMR samples of the synthesized compounds 5a-i
were dissolved in DMSO-d and the NMR spectra were recorded using Bruker NMR
6
1
13
spectrometer (Bruker, Reinstetten, Germany) at 500 MHz for H and 125.76 MHz for C at
the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. Chemical
shifts are expressed in δ-values (ppm) relative to TMS as an internal standard. Elemental
analyses were carried out at Microanalysis Laboratory, Cairo University, Cairo, Egypt and
the results agreed favorably with the proposed structures within ± 0.4 % of the theoretical
values. Mass spectra were recorded using Agilent Quadrupole 6120 LC/MS with ESI
(Electrospray ionization) source (Agilent Technologies, Palo Alto, CA, USA). Synthesis of
compounds 4a-i has been previously reported [19-21].
2
2
.2. Synthesis
.2.1. Synthesis of methyl 5-methoxy-1H-indole-2-carboxylate (2)
Compound 2 was prepared according to the literature procedure and its spectral data were
consistent with the published ones [22].
3

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2
.2.2. Synthesis of 5-methoxy-1H-indole-2-carbohydrazide (3)
Hydrazine hydrate (0.32 g, 10 mmol) was added to a suspension of compound 2 (0.21 g, 1
mmol) in methanol (15 mL). The reaction mixture was heated under reflux for three hours,
cooled to room temperature and filtered. The collected solid was dried to give 0.19 g (90%)
of the carbohydrazide derivative 3 [23] as a white powder m.p. 266-268 °C which was pure
enough to be used in the subsequent reactions.
2
.2.3. General procedure for the synthesis of target compounds 5a-i
A mixture of compound 3 (1 mmol) and the appropriate isatin derivative 4a-i (1 mmol) in
absolute ethyl alcohol and catalytic amount of glacial acetic acid was heated under reflux for
4
5
5
2
h, filtered while hot and the collected solid was washed with ethanol. The crude products
a-i were re-crystallized from ethanol/DMF mixture (3:1) to furnish the pure title compounds
a-i in 43-94% yields.
.2.3.1. 5-Methoxy-N'-[(3Z)-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-
1
carbohydrazide (5a): Yellow powder m.p. > 300 °C (yield 55%); H NMR (DMSO-d ) ppm:
6
3
1
.23 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 6.93 (s, 1H, H ), 7.13-7.18 (m, 3H, H ), 7.22 (s,
3 3 ar. ar.
1
3
H, H ), 7.39-7.49 (m, 2H, H ), 8.09 (s, 1H, H ), 11.71 (s, 1H, NH), 11.85 (s, 1H, NH); C
ar.
ar.
ar.
NMR (DMSO-d ) ppm: 26.5 (NCH ), 55.7 (OCH ), 102.6, 109.8, 112.4, 113.9, 115.4, 116.7,
6
3
3
1
22.8, 126.8, 127.8, 129.2, 129.5, 133.1, 139.8, 145.4, 154.5 (C , CH , C=N), 161.5, 163.9
ar. ar.
+
(
2 x C=O); MS m/z: 349 [M+1] .
2
.2.3.2. N'-[(3Z)-5-Bromo-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-
1
indole-2-carbohydrazide (5b): Orange powder m.p. > 300 °C (yield 71%); H NMR (DMSO-
d ) ppm: 3.21 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 6.94 (dd, J = 8.5, 2.0 Hz, 1H, H ), 7.09
6
3
3
ar.
(
d, J = 8.5 Hz, 1H, H ), 7.18 (d, J = 1.5 Hz, 1H, H ), 7.39 (d, J = 8.5 Hz, 1H, H ), 7.57 (s,
ar. ar. ar.
1
1
1
1
2
H, H ), 7.67 (dd, J = 8.0, 0.5 Hz, 1H, H ), 8.36 (s, 1H, H ), 11.83 (s, 1H, NH), 11.89 (s,
ar. ar. ar.
1
3
H, NH); C NMR (DMSO-d ) ppm: 26.6 (NCH ), 55.8 (OCH ), 102.6, 111.6, 113.9, 114.5,
6
3
3
15.5, 116.9, 117.0, 121.7, 123.2, 127.8, 128.9, 132.5, 134.9, 144.5, 154.5 (C , CH C=N),
ar.
ar.,
+
60.4, 163.9 (2 x C=O); MS m/z: 428 [M+1] .
.2.3.3. N'-[(3Z)-5-Chloro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-
1
indole-2-carbohydrazide (5c): Yellow powder m.p. > 300 °C (yield 43%); H NMR (DMSO-
d ) ppm: 3.22 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 6.94 (dd, J = 8.5, 2.0 Hz, 1H, H ), 7.14
6
3
3
ar.
(
d, J = 8.5 Hz, 1H, H ), 7.18 (d, J = 2.0 Hz, 1H, H ), 7.39 (d, J = 8.5 Hz, 1H, H ), 7.55 (dd,
ar. ar. ar.
J = 8.5, 1.5 Hz, 1H, H ), 7.56 (s, 1H, H ), 8.24 (s, 1H, H ), 11.83 (s, 1H, NH), 11.88 (s,
ar.
ar.
ar.
1
3
1
H, NH); C NMR (DMSO-d ) ppm: 26.7 (NCH ), 55.8 (OCH ), 102.6, 111.1, 113.9, 116.4,
6 3 3
4

ACCEPTED MANUSCRIPT
1
1
2
16.9, 120.5, 126.3, 126.8, 127.8, 129.2, 131.2, 132.2, 133.0, 144.1, 154.5 (C , CH C=N),
ar.
ar.,
+
59.9, 163.8 (2 x C=O); MS m/z: 384 [M+1] .
.2.3.4. N'-[(3Z)-5-Fluoro-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-
1
indole-2-carbohydrazide (5d): Yellow powder m.p. > 300 °C (yield 71%); H NMR (DMSO-
d ) ppm: 3.22 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 6.94-6.96 (m, 1H, H ), 7.12-7.15 (m, 1H,
6
3
3
ar.
H ), 7.19 (d, J = 2.0 Hz, 1H, H ), 7.36-7.40 (m, 2H, H ), 7.57 (s, 1H, H ), 8.05 (d, J = 8.0
ar.
ar.
ar.
ar.
1
3
Hz, 1H, H ), 11.82 (s, 1H, NH), 11.84 (s, 1H, NH); C NMR (DMSO-d ) ppm: 26.7
ar.
6
(
NCH ), 55.8 (OCH ), 102.6, 113.9, 116.9, 127.8, 129.2, 133.1, 138.0, 141.7, 154.5 (C ,
3
3
a
r
.
CH C=N), 110.6 (C , J = 8.0 Hz, C ), 114.1 (C , J = 26.4 Hz, C ), 115.8 (C , J = 9.0
ar.,
3'-F
ar.
2'-F
ar.
3'-F
Hz, C ), 118.9 (C , J = 23.1 Hz, C ), 129.2 (C , J = 3.0 Hz, C ), 158.3 (C , J = 235.4
ar.
2'-F
ar.
4'-F
ar.
1'-F
+
Hz, C ) 161.7, 163.9 (2 x C=O); MS m/z: 367 [M+1] .
ar.
2
.2.3.5. 5-Methoxy-N'-[(3Z)-5-methoxy-1-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-
1
indole-2-carbohydrazide (5e): Orange powder m.p. > 300 °C (yield 94%); H NMR (DMSO-
d ) ppm: 3.20 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 6.92-6.95 (m, 1H,
6
3
3
3
H ), 7.09-7.12 (m, 2H, H ), 7.23 (d, J = 1.5 Hz, 1H, H ), 7.38 (d, J = 9.0 Hz, 1H, H ), 7.53
ar.
ar.
ar.
ar.
1
3
(
s, 1H, H ), 7.74 (s, 1H, H ), 11.82 (s, 1H, NH), 12.02 (s, 1H, NH); C NMR (DMSO-d )
ar. ar. 6
ppm: 26.6 (NCH ), 55.7 (OCH ), 56.4 (OCH ), 102.6, 110.2, 111.4, 113.3, 115.9, 116.9,
3
3
3
1
20.4, 127.8, 128.6, 129.5, 132.9, 137.8, 140.8, 154.5, 154.7 (C , CH C=N), 160.8, 162.5
ar. ar.,
+
(
2 x C=O); MS m/z: 379 [M+1] .
2
.2.3.6. N'-[(3Z)-1-(4-Fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-
1
indole-2-carbohydrazide (5f): Yellow powder m.p. 258-260 °C (yield 84%); H NMR
(
DMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.01 (s, 2H, CH ), 6.94-6.95 (m, 1H, H ), 7.09 (d, J =
6 3 2 ar.
8
.0 Hz, 1H, H ), 7.18-7.21 (m, 4H, H ), 7.39-7.46 (m, 4H, H ), 7.56 (s, 1H, H ), 8.12 (d, J
ar. ar. ar. ar.
1
3
=
7.5 Hz, 1H, H ), 11.74 (s, 1H, NH), 11.86 (s, 1H, NH); C NMR (DMSO-d ) ppm: 42.5
ar.
6
(
CH ), 55.7 (OCH ), 102.6, 110.3, 113.9, 116.8, 121.2, 123.1, 127.0, 127.8, 129.8, 129.9,
2 3
1
1
1
2
32.9, 133.1, 142,9, 144.1, 154.5 (Car. and CH , C=N), 115.9 (C2',6'-F, J = 21.4 Hz, Car.),
ar.
30.1 (C3',5'-F, J = 8.1 Hz, C ), 132.4 (C , J = 2.0 Hz, C ), 161.9 (C , J = 241.9 Hz, C ),
ar.
4'-F
ar.
1'-F
ar.
+
63.1, 164.1 (2 x C=O); MS m/z: 443 [M+1] .
.2.3.7.
N'-[(3Z)-5-Bromo-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-
1
methoxy-1H-indole-2-carbohydrazide (5g): Yellow powder m.p. 238-240 °C (yield 60%); H
NMR (DMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.04 (s, 2H, CH ), 6.97-7.88 (m, 11H, H ),
6
3
2
ar.
13
1
2.07 (s, 1H, NH), 13.82 (s, 1H, NH); C NMR (DMSO-d ) ppm: 42.5 (CH ), 55.8 (OCH ),
6 2 3
1
02.7, 110.9, 112.9, 113.9, 115.8, 115.9, 116.1, 117.2, 122.1, 123.5, 127.8, 130.1, 130.2,
5

ACCEPTED MANUSCRIPT
1
32.1, 133.5, 134,1, 141.9, 154.7, 161.4 (C and CH , C=N), 161.8, 162.8 (2 x C=O), MS
ar.
ar.
+
m/z: 522 [M+1] .
.2.3.8.
2
N'-[(3Z)-5-Chloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-
1
methoxy-1H-indole-2-carbohydrazide (5h): Orange powder m.p. 250-252 °C (yield 59%); H
NMR (DMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.00 (s, 2H, CH ), 6.83 (d, J = 8.5 Hz, 1H, H ),
6
3
2
ar.
6
8
.95-6.98 (m, 1H, H ), 7.12-7.25 (m, 4H, H ), 7.35-7.51 (m, 3H, H ), 7.73 (s, 1H, H ),
ar. ar. ar. ar.
1
3
.52 (s, 1H, H ), 11.37 (s, 1H, NH), 12.04 (s, 1H, NH); C NMR (DMSO-d ) ppm: 42.2
ar.
6
(
CH ), 55.8 (OCH ), 102.5, 111.5, 112.5, 113.5, 115.8, 115.9, 116.1, 117.5, 121.7, 121.9,
2 3
1
1
2
28.2, 129.7, 129.8, 130.1, 130.2, 132,1, 140.5, 154.9, 162.5 (Car. and CH , C=N), 162.6,
ar.
+
63.1 (2 x C=O); MS m/z: 478 [M+1] .
.2.3.9.
N'-[(3Z)-5-Fluoro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-
1
methoxy-1H-indole-2-carbohydrazide (5i): Yellow powder m.p. 258-260 °C (yield 68%); H
NMR (DMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.04 (s, 2H, CH ), 6.95 (dd, J = 2.0, 9.0 Hz, 1H,
6
3
2
H ), 7.07-7.09 (m, 2H, H ), 7.17-7.21 (m, 4H, H ), 7.41 (d, J = 9.0 Hz, 1H, H ), 7.42-7.45
ar
ar.
ar.
ar.
(
m, 2H, H ), 7.49-7.51 (m, 1H, H ), 8.11 (d, J = 8.0 Hz, 1H, H ), 11.85 (s, 2H, 2 x NH);
ar. ar. ar.
1
3
C NMR (DMSO-d ) ppm: 43.1 (CH ), 55.7 (OCH ), 102.7, 111.0, 111.1, 113.9, 116.5,
6
2
3
1
1
1
2
2
16.9, 117.2, 120.8, 121.3, 127.9, 129.8, 129.9, 133,1, 140.4, 154.5 (Car. and CH , C=N),
ar.
16.0 (C2',6'-F, J = 21.3 Hz, C ), 130.2 (C
, J = 8.1 Hz, C ), 132.8 (C , J = 2.6 Hz, C ),
3',5'-F ar. 4'-F ar.
ar.
+
58.4 (C , J = 236.0 Hz, C ), 161.1, 162.9 (2 x C=O); MS m/z: 461 [M+1] .
1
'-F
ar.
.3. Antimicrobial evaluation
.3.1. Antimicrobial agents
Ampicillin (AMP) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and
fluconazole (FLC) from Shouguang-Fukang Pharmaceutical Ltd. (Shandong, China). AMP
was used as positive control for bacteria while FLC was used as positive control for fungi.
The antimicrobial discs (containing 10 ꢀg AMP or 25 ꢀg FLC) were purchased from ROSCO
(
Neo-Sensitabs, Taastrup, Denmark). 100% Dimethyl sulfoxide (DMSO) was used to
dissolve FLC and/or the tested compounds 5a-i to obtain an initial concentration of 1000
g/mL. These stock solutions were then diluted to the desired concentrations with sterile
ꢀ
distilled water. AMP was dissolved in water to obtain an initial concentration of 1000 ꢀg/mL.
AMP and FLC antifungal discs were stored at -80°C until used.
2
.3.2. Media
Muller Hinton broth (MHB) and Muller Hinton agar (MHA) were purchased from Sigma-
Aldrich Co. (St. Louis, MO, USA) and were used for the antimicrobial assay of the bacterial
isolates. Liquid RPMI 1640 medi um supplemented with L-glutamine was purchased from
6

ACCEPTED MANUSCRIPT
Sigma-Aldrich Co. (St. Louis, MO, USA) and was added to 2% sodium bicarbonate and
0
.165 M 3-morpholinepropanesulfonic acid (MOPS) from Dojindo Laboratories (Kumamoto,
Japan) then adjusted to pH 7.0 and was used for the assay of the yeast and moulds. Nutrient
agar, MacConkey’s agar, mannitol salt agar, cetrimide agar, sabouraud dextrose agar (SDA)
and Brain heart infusion broth (BHI) from Difco Laboratories (Detroit, MI, USA). Potato
dextrose agar (PDA) was purchased from Eiken Chemical Co. Ltd. (Tokyo, Japan).
2
.3.3. Organisms
Five Gram negative organisms, namely Escherichia coli (E. coli), Pseudomonas aeruginosa
Ps. aeruginosa), Proteus vulgaris (p. vulgaris), Klebsiella pneumonia (K. pneumonia) and
(
Salmonella enteridis (S. enteridis), four Gram positive isolates, namely Staphylococcus
aureus (S. aureus), Methicillin resistant Staphylococcus aureus (MRSA), Enterococcus
fecalis (E. fecalis) and Bacillus subtilis (B. subtilis) and three fungal isolates; one Candida
albicans (C. albicans) and two mould isolates, Asperagillus niger (A. niger) and Penicillum
notatum (P. notatum). All isolates were obtained from King Khaled Hospital, Riyadh, Saudi
Arabia.
2
.3.4. Culture conditions
All clinical samples were first inoculated onto Sheep blood agar (SPML Co. Ltd, Riyadh,
Saudi Arabia), The plates were incubated at 37 °C for 24-48 h. Identification of isolates was
done according to the standard methods described elsewhere [24, 25] and Clinical Laboratory
Standards Institute (CLSI) [26]. Isolates were stored in BHI broth containing 16% (w/v)
glycerol at −80 °C until further use.
2
.3.5. Growth of the tested microorganisms
Staphylococcal isolates were re-inoculated onto mannitol salt agar and then the plates were
incubated at 37 °C for 24-48 h. Mannitol fermentation was observed and recorded. Gram
negative isolates were re-inoculated onto MacConkey’s agar and then the plates were
incubated at 37 °C for 24-48 h. Lactose fermentation was observed and recorded. Ps.
aeruginosa strains were further re-inoculated on cetrimide agar at 37 °C for 24 h.
2
2
.3.6. Determination of Minimum Inhibitory Concentrations (MICs)
.3.6.1. Antibacterial susceptibility studies
Concerning the bacterial isolates (either Gram positive or Gram negative), AMP was used as
a reference control. MICs of AMP and the test compounds were carried out in cation-
adjusted MHB by means of microdilution broth method in accordance to National Committee
for Clinical Laboratory Standards [27] and CLSI documents [28, 29] Stock solutions of AMP
pure drug was prepared in sterile distilled water while stock solution of each of the test
7

ACCEPTED MANUSCRIPT
compounds was prepared in DMSO to reach an initial concentration of 1000 mg/mL.
Preparation of inocula for broth microdilution testing was carried out in accordance with
CLSI standard procedures. Briefly, 0.5 McFarland equivalent inoculum of each strain was
prepared in normal saline from 18-24 h agar plate culture. The suspension was further diluted
5
to achieve desired inoculums concentration of 10 CFU/mL. 100 ꢀL of aliquot of each strain
were then added to a 96-well microtiter plate containing gradient concentrations of either
AMP pure drug or any of the test compounds diluted in double strength MHB. The plates
were then incubated at 37 °C for 24 h. The turbidity of each well was measured at 490 nm
with a microplate ELISA reader. The MIC was defined as the lowest concentration of the
antibiotic or the test compound that prevented bacterial growth.
2
.3.6.2. Antifungal susceptibility studies
Broth microdilution testing was carried out in accordance with CLSI documents M27-A3
30] and M38-A2 [31] with RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS for
[
all organisms. Stock inoculum suspensions of all yeasts were obtained from 24-h-old cultures
grown on PDA at 35 °C. Stock inoculum suspensions of the filamentous fungi were prepared
from cultures grown on PDA at 30 °C. The final inoculum concentrations of the yeasts and
3
3
4
the filamentous fungi ranged from 0.65 x 10 to 2.5 x 10 CFU/mL and 0.87 x 10 to 3.8 x
4
1
0 CFU/mL, respectively. The microplates were incubated at 35 °C for 48 h. In case of
yeasts, the MICs of the examined compounds and the reference control, FLC, were recorded
as the lowest concentration at which 50% decrease in turbidity relative to the turbidity of the
growth control was observed. Whereas, in case of the filamentous fungi, the MICs of the test
compounds and FLC were recorded as the lowest concentrations at which a prominent
decrease in turbidity was observed.
2
.3.7. Disk diffusion assay
The disk diffusion assay was performed as described previously [32]. Colonies obtained from
the bacterial or the fungal isolates under test were suspended in sterile saline and adjusted to a
6
0
.5 McFarland standard corresponding to 5 × 10 CFU/mL. An aliquot of 100 ꢀL of each
isolate suspension was spread uniformly onto MHA and SDA plates for bacteria and fungi,
respectively. Six mm Whatmann filter paper disks were impregnated with 1000 ꢀg of the test
compounds and were allowed to dry. Then they were placed onto the surface of the
inoculated agar plates together with the standard reference discs which were then incubated at
3
5 °C. All tests were performed for three replicates. Plates were examined for evidence of
antimicrobial activities, represented by a zone of inhibition of microorganism’s growth
around the holes at 24 h., and diameters of clear zones were expressed in millimetres [33].
8

ACCEPTED MANUSCRIPT
2
.3.8. Scanning electron microscopy
7
Overnight diluted (10 CFU/mL) S. aureus was cultured for 24 h in MHB containing 5b
sample (2X MIC). Control bacterial isolate was simultaneously done for the sake of better
comparison. Primary fixation of samples was done by buffered Glutaraldehyde 2.5 % over
night in refrigerator, washed by phosphate buffer (pH = 7.2) and centrifuged (3000 g, 15 min,
4
°C). Secondary fixation was done by buffered Osmium Tetroxide 1 % for one hour, then
dehydration by series concentration of ethanol, embedding by resin mixture from SPI (SPI-
Pon™ - Araldite® Epoxy Embedding Kit). The bacterial pellets were mounted on membrane
filters (Anodisc; Whatman International Ltd, Maidstone, UK). Before examination under a
scanning electron microscope (SEM, JEOL, JSM-6060 LV), specimens were coated with 100
Å of a gold-palladium mix in an ion sputter (JEOL JFC 1100) [34].
2
.4. Computational methods
Geometrical parameters, atomic charges and vibrational wavenumbers have been carried out
in both gas and solvent phases using Gaussian’09 program package [35] with B3LYP/6-
3
11++G(d,p) level of basis set [36-38]. The vibrational wavenumbers were then scaled by a
factor of 0.9673 to compensate the errors due to vibrational anharmonicities and the
incompleteness of basis set [39]. Potential energy distribution (PED) has been conducted for
the computed wavenumbers of compound 5h using VEDA4 program [40]. Natural bond
orbital analysis was performed using NBO 3.1 program [41]. The molecular docking analysis
for compound 5h was carried out using AutoDock 4.2 program [42] to find out the binding
mode of compound 5h in its target protein.
3
. Results and discussion
3
.1. Chemistry
Scheme 1 illustrates the synthetic pathway which was adopted to prepare the target
compounds 5a-i. The synthesis was started by esterification of the commercially available 5-
methoxyindole-2-carboxylic acid (1) according to the literature procedure [43]. The obtained
methyl ester 2 was allowed to react with hydrazine hydrate to afford the corresponding acid
hydrazide 3. Subsequently, the appropriate isatin derivative 4a-i was reacted with compound
3
to give the respective title compounds 5a-i.
9

ACCEPTED MANUSCRIPT
H CO
H CO
3
OCH3
O
H CO
3
NHNH2
O
3
OH
O
i
ii
N
H
N
H
N
H
1
2
3
O
X
O
iii
N
R
H CO
4
a-i
3
NH
H
N
N
O
X
O
N
R
5a-i
Compound No.
a, 5a
b, 5b
c, 5c
d, 5d
e, 5e
X
H
Br
Cl
F
R Compound No.
X
H
R
4-F-C
4
4
4
4
H
H
H
H
H
4f, 5f
4g, 5g
4h, 5h
4i, 5i
6
6
6
6
H
4
H
4
H
4
H
4
Br 4-F-C
Cl 4-F-C
F
4-F-C
4
OCH
3
Scheme 1. Synthesis of the target compounds 5a-i. Reagents and conditions: (i) Methanol,
drops of H SO , reflux, 4 h; (ii) Methanol, H N-NH .H O, reflux, 2 h; (iii) Absolute ethanol,
2
4
2
2
2
drops of acetic acid, reflux 4 h.
3
3
.2. Antimicrobial evaluation
.2.1. Antimicrobial spectrum
Tables 1 and 2 showed the MICs and DIZs of the test samples 5a-i, respectively, and the
reference agents against different Gram positive, Gram negative, yeasts, and filamentous
fungi. Table 1 illustrates that all of the tested samples showed almost no activity against the
tested Gram negative isolates as compared to the standard antimicrobial agent except toward
the Ps. aeruginosa isolate. Most of the tested samples exhibited more Gram positive activity
than Gram negative spectrum and even potent activity against a variety of fungal species. All
the tested compounds showed a clear pseudomonal activity with variable MIC values of
which compounds 5c and 5d being eight and six fold more potent than AMP, respectively.
1
0

ACCEPTED MANUSCRIPT
On the other hand, none of the tested compounds showed antibacterial activity against E.
fecalis.
All the tested compounds were highly active against the mould P. notatum. Although A. niger
isolate was resistant to compounds 5c and 5d, it showed comparable MIC activity to that of
FLC to the rest of the tested compounds and even a threefold higher activity than FLC by
applying compound 5i.
The DIZs of the tested compounds 5a-i against the Gram-negative organisms confirmed the
lack of activity of the samples against the tested isolates but for the Ps. aeruginosa isolate
where some of the samples gave clear zones ranged from 10 to 14 mm in diameter compared
to absolutely no zones with AMP. Against Gram positive organisms however, the obtained
results were different, Compounds 5a, 5b, and 5g showed some antibacterial activity giving a
zone diameter of 22, 24, and 22 mm, respectively, though much less than that of AMP (32
mm) MD 16, 19 and 21showed some antibacterial activity giving a zone diameter of 22, 24
and 22 mm, respectively though much less than that of AMP (32 mm). Against MRSA
isolate, however, many of the DIZs obtained on application of different samples produced
comparable results with that of the standard antibiotic. The diameter of the inhibition zones
of some of the tested compounds against either the yeast or the filamentous fungi was almost
equivalent to that of FLC.
3
.2.2. Scanning electron microscope
S. aureus cells were photographed by electron microscopy to compare morphological
alterations after addition of compound 5b sample to the cells with subsequent incubation for
2
4 h. (Figure 1). The most representative photograph was chosen even if morphologically
normal organisms were also observed. Normal mounting of S. aureus cells under scanning
electron microscope showed a clear spherical cell with grape like arrangement (Figure 1).
Upon application of sample 5b on the Gram positive cells, the obtained results showed total
destruction and clumping of the bacterial cells. Abnormal forms were visible with clear
change in the coccoid morphology of the bacterial cells. Some cells were shrunk while others
fused together losing their normal morphology. In the mount, very few bacterial cells were
found retaining their normal shape (Figure 1).
1
1

ACCEPTED MANUSCRIPT
Table 1. Minimum Inhibitory Concentrations (MICs) of the title compounds 5a-i, AMP and FLC towards Gram negative bacteria, Gram positive
bacteria and fungi.
MIC values (µg/mL)
Strain name
Comp.
No.
Gram negative organisms
Ps. p. K.
aeruginosa vulgaris pneumonia enteridis aureus
Gram positive organisms
Fungi
S.
S.
E.
B.
C.
A.
P.
notatum
15.6
7.8
15.6
31.3
250
15.6
15.6
7.8
31.25
ND
E. coli
MRSA
fecalis subtilis albicans niger
5a
>1000
500
500
250
250
250
500
500
500
15.6
ND
250
125
500
500
500
500
250
500
500
500
500
< 7.8
ND
>1000
500
500
500
500
500
500
1000
500
500
500
< 7.8
ND
15.6
15.6
15.6
15.6
500
500
500
500
250
250
ND
15.6
15.6
31.3
62.5
500
500
500
500
500
500
ND
500
500
500
250
500
>1000
500
500
500
3.9
31.3
31.3
31.3
250
500
62.5
500
250
125
1000
ND
62.5
250
62.5
62.5
250
15.6
31.25
500
62.5
15.6
31.3
15.6
15.6
7.8
5b
5c
3.9
500
5d
15.6
500
500
500
5e
5f
62.5
250
500
500
250
5g
62.5
62.5
15.6
ND
5h
500
500
5i
250
500
AMP
FLC
>1000
ND
>1000
ND
ND
31.3
ND
15.6
250
ND: not determined.
12

ACCEPTED MANUSCRIPT
Table 2. Diameter of the Inhibition Zone (DIZ) of the title compounds 5a-i, AMP and FLC towards Gram negative bacteria, Gram positive bacteria
and fungi.
DIZ in mm ± S.D.*
Strain name
Comp.
No.
Gram negative organisms
Ps. p. K.
aeruginosa vulgaris pneumonia enteridis
Gram positive organisms
Fungi
S.
-ve
E.
MRSA
B.
C.
P.
notatum
E. coli
S. aureus
A. niger
fecalis
subtilis albicans
5a
12 ± 0.8
12 ± 0.43
10 ± 0.4
16 ± 1.0
-ve
14 ± 0.7
18 ± 0.0
16 ± 0.0
14 ± 0.4
30 ± 0.0
ND
10 ± 0.8
14 ± 2.0
10 ± 0.9
14 ± 2.4
14 ± 0.1
12 ± 0.0
14 ± 1.6
10 ± 0.4
10 ± 0.0
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
22 ± 0.8
20 ± 0.9
-ve
-ve
-ve
-ve
14 ± 0.8
14 ± 0.0
18 ± 0.4
12 ± 0.5
-ve
-ve
-ve
-ve
13 ± 1.0 10 ± 0.0
-ve 14 ± 0.0
11 ± 0.0 11 ± 0.4
5b
10 ± 0.0 24 ± 0.43 18 ± 0.6
5c
-ve
-ve
11 ± 0.5
-ve
10 ± 0.0
-ve
-ve
45 ± 1.0
ND
16 ± 0.8
18 ± 0.0
11 ± 0.7
18 ± 0.0
22 ± 1.0
14 ± 0.4
12 ± 0.0
32 ± 0.4
ND
18 ± 0.0
14 ± 0.5
9 ± 0.0
-ve
5d
-ve
-ve
-ve
-ve
-ve
5e
11 ± 0.3
12 ± 1.7
-ve
9 ± 0.0 11 ± 0.6 12 ± 0.8 18 ± 0.0
-ve 12 ± 0.0 10±0.0 -ve
12 ± 0.4 12 ± 0.7
5f
5g
-ve
-ve
-ve
11 ± 0.8 11 ± 1.8
14 ± 1.0 10 ± 0.4
14 ± 0.3 14 ± 0.8
ND ND
5h
-ve
-ve
-ve
-ve
-ve
12 ± 0.0
12 ± 0.0
-ve
-ve
5i
10 ± 0.7
36 ± 0.7
ND
-ve
ND
AMP
FLC
18 ± 0.4 35 ± 1.0 30 ± 0.5
ND ND ND
ND
ND
21 ± 0.5 16 ± 0.8 15 ± 0.0
*
Arithmetic mean ± standard deviation; ND: not determined.
13

ACCEPTED MANUSCRIPT
Figure 1. Scanning electron microscopy of compound 5b after 24 h incubation with S.
aureus.
3
3
.3. Computational studies
.3.1. Molecular geometry
Figure 2 illustrates the optimized structure of compound 5h. Optimized parameters, bond
lengths, bond angles and torsional angles calculated by B3LYP/6-311++G(d,p) are listed in
Table 3. The global minimum energy for optimization of compound 5h was found to be -
1
968.65 Hartree. The bond length C -C in the indole ring manifested the highest value of
1 2
1
.4194 and 1.4213 Å in the gas and solution phases respectively, due to its attachment to the
methoxy group. The calculated bond length C -C in the indole ring showed good agreement
8
10
with the experimental X-ray results (1.467/1.467 Å). Whereas, the bond lengths of C -N ,
1
7
16
C -N , C -N , C -N , C -N , and C -N are 1.4123, 1.3791, 1.4568, 1.3775, 1.3726, and
15
16
20
16
8
7
4
7
10
12
1
.3992 Å respectively. Of all the C-N bonds C -N is comparatively higher due to the
15 16
attachment of oxygen atom to the carbon atom. The bond lengths N12-H (1.0214/0.790 Å)
4
0
and N -H (1.0092/0.847 Å) exhibited considerable deviations from the experimental X-ray
7
38
data, due to the intermolecular interactions in the crystalline state. The bond angles C -C -N
3
4
7
(130.78°/130.36°), C -C -C (121.48°/121.81°), C -C -C (120.72°/120.61°), N -C -C
3 4 5 17 18 24 16 17
18
(
109.74°/109.96°), and N -C -H
41
(108.71°/109.19°) showed agreement with the
1
6
20
experimental X-ray results, respectively. The calculated dihedral angles in solution phase of
N -C -C -N and O -C -C -C are 179.61 and 180.00, respectively and they are in a
1
3
14 15
16
33
1
2
3
satisfactory agreement with the experimental XRD results. The deviations in the calculated
dihedral angle values from the experimental XRD results reveal the occurrence of steric
effect in the crystalline state. In general, the statistical linear regression plots between the
experimental XRD values [44] and theoretical geometrical parameters showed good
2
agreement with R values in the range of 0.972-998 in the gas and solution phases (Figure
1
4

ACCEPTED MANUSCRIPT
S1). The root-mean square deviation (RMSD) values were calculated for the predicted bond
lengths to be 0.0300 Å (solution phase) and 0.0304 Å (gas phase) with their experimental
XRD values. Similarly, the RMSD values for the predicted bond angles are 4.7308° and
7
.0373° in solution and gas phases, respectively, with their XRD results. This indicates that
the calculated values in solution phase are in a better agreement with the experimental values.
Figure 2. Optimized molecular structure of compound 5h.
Table 3. Structural geometry parameters of compound 5h along with the recorded XRD results.
Bond lengths (Å)
Bond Angles (°)
Dihedral angles (°)
Gas
Phase
Solution
Phase
Gas
Solution
Phase
Gas
Phase
0.003
-180
-180
-0.003
-0.01
-179.99
180
Solution
Phase
-0.02
179.98
-180
-0.004
0.01
-179.97
179.99
0.01
Parameters
XRD
Parameters
XRD
121.46 C -C -C -C
3
Parameters
XRD
1.54
-178.48
-179.63
0.34
Phase
121.07
114.22
124.71
C -C2
1.4194
1.3843
1.3686
1.3790
1.0831
1.4016
1.0838
1.4202
1.3726
1.4127
1.4254
1.0815
1.3775
1.0089
1.3852
1.4682
1.0796
1.2156
1.3992
1.3319
1.0214
1.2932
1.4980
1.4562
1.3791
1.4213
1.3844
1.3696
1.3797
1.0835
1.4026
1.0834
1.4211
1.3714
1.4139
1.4243
1.0813
1.3787
1.0092
1.3869
1.4651
1.0792
1.2239
1.3906
1.3372
1.0212
1.2916
1.5028
1.4561
1.3736
1.414 C -C -C
1.378 C -C -O
1.379 C -C -O
1.385 C -C -C
0.950 C -C -H
1.392 C -C -H
0.950 C -C -C
1.415 C -C -H
1.379 C -C -H
1.409 C -C -C
1.420 C -C -N
0.950 C -C -N
1.367 C -C -C
0.847 C -C -C
1.382 C -C -C
1.467 C -C -C
0.951 C -C -H
1.222 C -C -H
1.379 C -N -C
1.348 C -N -H
0.790 C -N -H
1.295 N -C -C
1.505 N -C -C
1.455 C -C -C
121.10
114.32
124.58
121.68
117.88
120.44
117.63
120.90
121.47
121.48
130.78
107.74
120.02
106.84
133.14
118.09
121.98
119.93
109.30
127.13
123.57
109.06
118.08
132.86
107.06
1
2
1
6
6
1
2
C -C6
115.23 C -C -C -H
35
1
2
1
33
33
3
6
1
2
C -O
123.30 O -C -C -C
33 1 2 3
1
33
6
1
C -C3
121.57
117.62
120.81
117.77
120.70
121.53
121.46
130.91
107.62
119.88
106.86
133.26
118.25
121.87
119.88
109.39
128.13
122.48
109.05
117.36
133.59
107.07
120.60 O -C -C -H
2
1
2
33
1
2
35
C -H
119.63 C -C -C -C
-2.13
2
35
1
2
35
35
4
2
1
6
5
C -C4
119.68 C -C -C -H
177.85
178.95
-1.07
3
3
2
2
1
6
37
C -H
118.05 O -C -C -C
3
36
2
3
33 1 6
5
C -C5
120.96 O -C -C -H
0.01
4
2
3
36
36
5
33
1
6
37
C -N7
120.99 C -C -C -C
0.002
-179.99
-180.00
0.01
-0.002
179.99
179.99
-0.02
-0.003
180.00
-179.99
0.01
-179.99
0.002
0.003
179.99
0.01
0.002
179.97
-179.99
-0.03
0.73
4
4
3
1
2
3
4
C -C6
121.81 C -C -C -H
-179.25
-179.24
0.78
5
3
4
1
2
3
36
C -C9
130.36 H -C -C -C
35 2 3
107.82 H -C -C -H
35 2 3
5
3
4
7
7
6
9
9
5
4
C -H
6
37
5
4
36
N -C8
119.38 C -C -C -C
5
0.02
-2.36
7
4
5
2
3
4
N -H
106.85 C -C -C -N
7
179.97
-179.94
0.01
177.60
177.62
-2.42
7
38
4
5
2
3
4
C -C9
133.76 H -C -C -C
36 3 4
118.54 H -C -C -N
36 3 4
8
6
5
5
C -C
8
10
1
6
7
C -H
120.74 C -C -C -C
-0.03
1.76
9
39
1
6
37
37
8
3
4
5
6
9
C =O
120.72 C -C -C -C
179.95
-179.99
-0.01
-179.95
0.16
0.01
-179.88
0.02
-179.05
-178.20
0.99
179.79
-4.01
-0.25
175.95
0.52
1
0
11
5
6
3
4
5
C -N
108.69 N -C -C -C
7 4 5
1
0
12
4
7
6
9
8
N -N
124.88 N -C -C -C
1
2
13
4
7
38
38
9
7 4 5
N -H
126.32 C -C -N -C
1
2
40
8
7
3
4
7
N =C
109.80 C -C -N -H
3 4 7
1
3
14
7
8
38
8
C -C
119.68 C -C -N -C
1
4
15
7
8
10
10
8
5
4
7
C -C
130.51 C -C -N -H
38
1
4
18
9
8
5
4
7
C -N
1.366 C -C -C
106.82 C -C -C -C
4 5 6 1
1
5
16
5
9
1
5

ACCEPTED MANUSCRIPT
C =O
1.2287
1.4123
1.4568
1.4084
1.3856
1.3888
1.5184
1.0943
1.0930
1.3993
1.0821
1.3936
1.0822
1.3941
1.7595
1.0821
1.3965
1.3984
1.3943
1.0853
1.3847
1.0828
1.3870
1.3544
1.3916
1.0828
1.0841
1.4193
1.0888
1.0959
1.0959
1.9742
1.2297
1.4117
1.4590
1.4080
1.3858
1.3892
1.5193
1.0927
1.0921
1.3994
1.0818
1.3933
1.0821
1.3936
1.7651
1.0820
1.3976
1.3984
1.3945
1.0846
1.3843
1.0828
1.3857
1.3627
1.3932
1.0828
1.0838
1.4271
1.0883
1.0944
1.0944
1.9798
1.233 C -C -H
126.31
126.62
122.52
114.02
123.46
120.21
121.23
118.56
118.11
127.37
126.07
106.56
106.45
127.29
126.26
110.50
123.34
126.06
109.61
129.02
121.37
106.87
132.42
120.71
114.39
108.87
105.50
109.94
110.34
107.51
117.95
126.15
126.79
122.42
114.59
123.00
119.96
121.51
118.53
118.28
127.54
126.05
106.42
106.51
126.81
126.68
110.54
123.82
125.56
109.74
128.81
121.45
106.78
132.50
120.72
114.23
108.71
105.85
110.14
110.30
107.32
117.92
126.58 C -C -C -H
179.99
-179.99
-0.01
179.99
-179.96
0.02
-179.47
-178.41
1.60
15
19
5
9
39
4
5
6
37
N -C
1.412 C -C -H
126.60 C -C -C -C
9 5 6
1
6
17
8
9
39
1
N -C
1.470 C -C -O
123.93 C -C -C -H
1
6
20
8
10
11
9 5 6
37
8
C -C
1.404 C -C -N
111.91 C -C -C -C
-0.01
0.01
-1.34
1
7
18
8
10
12
4
5
9
C =C
1.379 O -C -N
124.16 C -C -C -H
-179.99
179.99
0.01
-0.01
179.91
-180
-179.96
179.99
0.02
-0.003
179.85
179.89
-0.26
-0.003
179.97
-179.82
0.15
-179.92
-0.01
179.61
-0.46
-0.44
179.48
-179.92
0.46
0.13
-179.48
0.59
177.55
-179.33 -178.35
-2.38
-0.53
179.86
178.61
177.60
-2.36
1
7
21
11
10
12
13
40
4
5
9
39
C -C
1.387 C -N -N
122.16 C -C -C -C
1
8
24
10
12
6 5 9
8
C -C
1.511 C -N -H
120.26 C -C -C -H
6 5 9
2
0
25
10
12
39
C -H
0.990 N -N -H
117.41 C -N -C -C
-0.62
2
0
41
42
22
13
12
40
14
15
18
18
4
7
8
9
C -H
0.990 N -N -C
114.89 C -N -C -C
178.51
-176.75
2.38
20
12
13
4
7
8
10
C -C
1.403 N -C -C
126.95 H -N -C -C
21
13
14
38 7 8
9
C -H
0.950 N -C -C
127.18 H -N -C -C
-0.08
0.02
2
1
43
23
13
14
38
7
8
10
C -C
1.380 C -C -C
105.87 N -C -C -C
5
1.23
22
15
14
7
8
9
C -H
0.950 C -C -N
107.09 N -C -C -H
39
179.99
-179.89
0.09
179.92
0.24
179.74
-0.3
-0.35
179.62
-179.91
0.38
0.17
-179.54
0.39
-178.73
-177.78
2.26
-178.61
-3.36
179.03
-1.96
-1.47
177.55
-178.82
0.45
1.68
-179.06
0.69
2
2
44
24
14
15
16
19
7
8
9
C -C
1.395 C -C -O
127.44 C -C -C -C
10 8 9 5
125.46 C -C -C -H
10 8 9
23
14
15
C -Cl
1.741 N -C -O
2
3
32
16
15
19
17
20
20
18
21
21
17
24
24
39
C -H
0.951 C -N -C
110.16 C -N -N -C
14
2
4
45
15
16
10
12
13
C -C
1.391 C -N -C
122.46 H -N -N -C
2
5
26
30
27
15
16
40 12 13
14
16
19
16
19
17
24
17
24
C -C
1.389 C -N -C
127.28 N -C -C -N
25
17
16
13
14
15
C -C
1.390 N -C -C
109.96 N -C -C -O
26
16
17
13 14 15
C -H
0.950 N -C -C
127.95 C -C -C -N
2
6
46
28
16
17
18
14
15
C -C
1.371 C -C -C
122.09 C -C -C -O
27
18
17
18
14
15
C -H
0.950 C -C -C
106.90 N -C -C -C
2
7
47
29
31
14
18
13 14 18
C -C
1.369 C -C -C
132.49 N -C -C -C
13 14 18
28
14
18
C -F
1.364 C -C -C
120.61 C -C -C -C
28
17
18
15
14
18
C -C
1.390 N -C -C
111.94 C -C -C -C
2
9
30
16
20
25
15
14
18
C -H
0.950 N -C -H
109.19 C -C -N -C
29
48
49
34
50
51
52
16
20
41
42
41
42
14 15 16
17
20
C -H
0.950 N -C -H
109.24 C -C -N -C
176.90
-179.57
-3.06
-0.29
179.63
177.22
30
16
20
14
15
16
O -C
1.431 C -C -H
109.23 O -C -N -C
3
3
25
20
19
15
16
17
20
18
C -H
0.980 C -C -H
109.25 O -C -N -C
-1.82
0.39
-179.98
34
25
20
19
15
16
C -H
0.980 H -C -H
107.91 C -N -C -C
34
41
20
42
15
16
17
C -H
0.980 C -C -C
117.24 O -C -C -C
3
4
17
21
22
33
1
2
3
.
. .
O
19
H
40
3
.3.2 Natural bond orbital analysis
Natural bond orbital (NBO) analysis produces a chemical picture of (hyper)conjugative
interactions from the bonding to anti-bonding orbitals of a compound to understand its
intramolecular charge-transfer (ICT) interactions. The results of NBO analysis of compound
5
h are presented in Table 4. The larger E(2) value indicates more intensive interaction
between electron donors and acceptors. The interaction of the lone pair of the nitrogen
-1
LP(1)N with π*(C -O ) yielded an energy value of 52.87 kcal mol which is higher than
1
6
15
19
-1
the energy value (39.08 kcal mol ) of the interaction with the adjacent pair π*(C -C ) due
1
7
21
to the presence of electro negative oxygen in the former pair. Also, the charge transfer
interaction between LP(1) N12→π*(C -O ) exhibited high energy than the interaction
1
0
11
LP(1) N12→π*(N -C ) due to the presence of oxygen atom connected to C . The lone pair
1
3
14
10
of oxygen LP(2)O interacts with σ*(C -C ) and σ*(C -N ) with energy values of 16.85
1
1
8
10
10
12
-1
and 27.28 kcal mol respectively. While, LP(3)Cl interacts with σ*(C -H ) with an energy
3
2
24
45
-1
value 63.25 kcal mol . The low electron density (0.0389 e) value of the antibonding orbital
N -H reveals the more ability to make interactions with the other acceptor motifs. These
1
2
40
1
6

ACCEPTED MANUSCRIPT
results can identify the efficiency of donor and acceptor groups within the compound, which
helps the interaction with its target protein.
Table 4. Selective donor acceptor interactions results of compound 5h based on second-order
perturbation theory in Fock matrix.
b
c
d
a
E(2)
E(j)-E(i)
F(i,j)
Donor (i)
ED (i)e
Acceptor (i)
ED (j) e
(
kcal/mol)
19.64
15.70
21.10
37.92
39.95
18.87
38.79
36.92
16.85
27.28
42.23
42.19
10.73
12.10
52.87
39.08
74.40
27.47
116.92
32.77
51.50
18.83
15.07
55.37
14.03
19.60
45.64
63.25
51.75
65.56
22.34
22.38
11.16
27.20
34.67
(arb.units) (arb.units)
π (C -C )
1.57391 π*(C -C )
0.33686
0.28170
0.37198
0.02664
1.65607
0.28572
0.49729
0.37198
0.05788
0.08610
0.30522
0.28572
0.03893
0.04195
0.30742
0.39926
0.36353
0.08946
0.02212
0.01369
0.02664
0.01369
0.01334
0.02664
0.01369
0.02664
0.36353
0.01369
0.02664
0.36353
0.01309
0.36353
0.01309
0.33686
0.36353
c
0.28
0.29
0.28
1.31
0.03
0.27
0.29
0.28
0.72
0.63
0.30
0.28
0.76
0.86
0.29
0.29
0.07
0.68
0.18
0.72
0.77
0.70
0.95
0.74
0.58
0.63
0.17
0.77
0.81
0.35
4.59
0.34
4.58
0.34
0.09
0.068
0.062
0.069
0.200
0.056
0.064
0.097
0.092
0.100
0.119
0.102
0.099
0.082
0.092
0.112
0.096
0.065
0.124
0.132
0.138
0.177
0.104
0.109
0.183
0.081
0.099
0.086
0.200
0.185
0.147
0.290
0.085
0.203
0.091
0.051
4
5
1
6
π (C -C )
1.57391 π*(C -C )
2 3
4
5
π (C -C )
1.57391 π*(C -C )
8 9
4
5
σ (N -N ) 1.98814 σ*(C -C )
1
2
13
27
28
π (C -C ) 1.67568 π*(C -C )
1
7
21
22
23
π (C -C ) 1.67839 π*(N -C )
1
8
24
13
14
LP(1)N7
LP(1)N7
1.59925 π*(C -C )
4 5
1.59925 π*(C -C )
8
9
LP(2)O11
LP(2)O11
LP(1) N12
LP(1) N12
LP(1) N13
LP(1) N13
LP(1) N16
LP(1) N16
LP(1) N16
LP(2) O19
LP(1) F31
LP(2) F31
LP(2) F31
LP(3) F31
LP(3) F31
LP(3) F31
LP(2)Cl32
LP(2)Cl32
LP(2) Cl32
LP(3) Cl32
LP(3) Cl32
LP(3) Cl32
LP(3) Cl32
LP(1) O33
LP(1) O33
LP(2) O33
LP(2) O33
1.85846 σ*(C -C )
8 10
1.85846 σ*(C -N )
10
12
1.62155 π*(C -O )
1.62155 π*(N -C )
10
11
13
14
1.90641 σ*(N -H )
1.90641 σ*(C -C )
12
40
14
18
1.64010 π*(C -O )
1.64010 π*(C -C )
15
19
17
21
1.64010 π*(C -C )
1.84605 σ*(C -N )
27
28
15
16
1.98960 σ*(C -C )
1.97242 σ*(C -H )
25
26
24
45
1.97242 σ*(C -C )
1.92663 σ*(C -H )
27
28
24
45
1.92663 σ*(C -C )
1.92663 σ*(C27-C28)
1.96603 σ*(C -H )
26
27
24
45
1.96603 σ*(C -C )
27
28
1.96603 π*(C -C )
1.93242 σ*(C -H )
27
28
24
45
1.93242 σ*(C -C )
1.93242 π*(C -C )
27
28
27
28
1.93242 σ*(C -C )
1.96348 π*(C -C )
29
30
27
28
1.96348 σ*(C -C )
1.84916 π*(C -C )
29
30
1
6
1.84916 π*(C -C )
27
28
a
b
Electron density; Energy of stabilization interactions; Energy difference between donor and acceptor i and j
d
NBO orbital; Fock matrix element between i and j NBO orbital.
3
.3.3 Natural population analysis (NPA)
The atomic charge of compound 5h was calculated by natural population analysis (NPA). It
is an effective method to calculate the atomic charges within the molecule. The plot of charge
1
7

ACCEPTED MANUSCRIPT
distribution is shown in Figure 3. The C (0.669 e) and C (0.646 e) atoms showed more
1
5
10
positive charge than the other carbon atoms due to the presence of electronegative oxygen
atom directly attached to them. Whereas, The C (0.032 e), C (0.100 e), and C (0.188 e)
8
14
17
atoms showed positive charge because of their bonding with nitrogen atoms. On the other
hand, the other carbon atoms manifested negative charges. The N (-0.532 e) is more negative
7
than the other nitrogen atoms N (-0.482 e), N (-0.389 e) and N (-0.204 e). All hydrogen
1
6
12
13
atoms displayed positive charges in which H38 (0.446 e) and H40 (0.378 e) are the most
positive owing to their direct attachment to nitrogen atoms.
Figure 3. Charge distribution chart for compound 5h according to the natural population
analysis.
3
.3.4 Frontier molecular orbital analysis
The chemical reactivity and kinetic stability of the molecules are determined by the highest
occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO).The
former represents the ability to donate an electron while the latter represents the ability to
accept an electron [45]. A molecule is more reactive for a small HOMO-LUMO energy gap
[
46]. The energies have been calculated by B3LYP/6-311++G(d,p) methods and are depicted
in the Figure 4. The energy gap between HOMO and LUMO of the test compound 5h is 2.79
1
8

ACCEPTED MANUSCRIPT
eV. The lowering of HOMO-LUMO energy gap reveals the possibility of charge transfer
interaction within the molecule.
Figure 4. HOMO (upper) and LUMO (lower) energy plots of compound 5h.
3
.3.5. Vibrational analysis
The vibrational spectral analysis of compound 5h was carried out on the basis of the
characteristic vibrations of indole ring, phenyl ring, methoxy group, as well as methylene and
carbonyl groups. The observed and simulated FT-IR and FT-Raman spectra are shown in
Figures 5 and 6, respectively. The computed and experimental wavenumbers as well as their
assignments with PED were presented in Table 5.
1
9

ACCEPTED MANUSCRIPT
Figure 5. (a) Experimental (b) Simulated infrared spectra of compound 5h in the region
-1
4
000-500 cm .
Figure 6. (a) Experimental (b) Simulated Raman spectra of compound 5h in the region 3500-
-
1
5
0 cm .
2
0

ACCEPTED MANUSCRIPT
Table 5. Tentative vibrational assignments of compound 5h predicted based on PED
analysis.
Calculated
Experimental
Wavenumber
Wavenumber
Assignment with PED (%)
-
1
-1
(
cm )
(cm )
Unscaled Scaled
IR
3293 vw
Raman
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
593
206
181
162
131
040
031
729
662
636
612
553
541
518
503
490
484
394
331
273
251
244
218
195
143
124
3476
3101
3077
3058
3029
2940
2933
1672
1607
1583
1559
1502
1490
1469
1454
1441
1436
1348
1287
1231
1210
1203
1178
1156
1106
1087
ν N -H (99)
7
38
3101 w ν C -H (98)
2
4
45
3076 w ν C -H (25), ν C -H (75)
2
35
3
36
3057 w
3043 w ν C -H (96)
26 46
3008 w ν C -H (92)
3
4
50
2941w ν C -H (76)
3
4
51
ν C -H (45)
as 20
41
1665 m
1608 w
1579 vw
1560 vw
1509 m
1666 s ν O =C (77)
11 10
νC =C (16), ν C =C (17),ν C -C (13)
2
3
1
6
3
4
1581vs ν C =C (32), ν C -C (20)
2
7
28
25
26
ν C -C (16), ν C -C (19)
2
2
23
17
18
1525 m ν C -C (20),ν C -C (10)
8
9
3
4
1484 m ν C-H ph
sci H -C -H (12)
1479 vw
1445 w
1349 m
4
1
20
42
1449 m ν C -H (63)
as 34
52
ν C -H (76)
as 34
51
1436 m d C -H -H -H (31)
3
4
51
52
50
1344 w ν C =C (13), ν C =C (17), ν C -C (11)
2
3
1
6
5
6
1297 w β H -C -C (23),H -C -C (22)
4
6
26
27
49
30
29
1229 w
1210 m
1204 s
1181 m
1159 s
1228 m ν N -C (12)
7 4
ρ CH (38) methoxy
3
ν F -C (45)Ph
3
1
28
1182 m βC-H, H -C -C (11)
46 26 27
1158 m ν O33-C34 (59)
1112 m
1088 w
1110 m ρ –O-CH (42)
3
1089 m β H -C -C (18),H -C -C (15),H -C -C (11),H -
4
7
27
26
48
29
30
46
26
27
49
C -C (10)
3
0
29
1
1
1
9
8
8
8
8
7
7
5
5
4
4
4
2
101
055
031
1065
1020
998
945
832
819
815
803
759
730
562
508
478
466
424
210
1050 vw
1024 w
985 w
948 vw
839 vw
820 m
1052 w βC-H,H -C -C (27)Ph
48 29 30
ρ –O-CH (15)
3
987 w γ C -C -C -H (43),C -C -C -H (16)Ph
2
8
29
30
49
27
28
29
48
77
60
47
43
30
84
55
81
26
94
82
39
18
942 w γ C -C -C -H (19), H -C -C -H (59)Ph
26 25 27 46 48 29 30 49
b Ph
b Ph
b Ph
b Ph
811w
808 m
759 vw
729 w
563 vw
512 w
477 vw
466 vw
422w
754 w ν C23-Cl32(41)
731w ρ –CH (23)
2
562 w d -OCH (18)
3
d -OCH (22)
3
d -OCH (28)
3
d -OCH (19)
3
d -OCH (20)
3
203 w τ -OCH (26)
3
ν-stretching; ν -asymmetric stretching; δ-bending; τ-torsion; β-in-plane bending; γ-out-of-plane bending; sci-
as
scissoring; ⍴-rocking; d-deformation; b-ring breathing; vs-very strong; s-strong; m-medium; vw-very weak; w-
weak; ph: phenyl ring.
2
1

ACCEPTED MANUSCRIPT
3
.3.5.1. Indole ring vibrations
-1
In solution, the N-H stretching vibration occurs near 3400 cm while in solid state, the
frequency shifts towards lower values [47]. A band was observed due to the N-H stretching
-1
vibration in the IR spectrum of compound 5h at 3293 cm . A band occurred in the Raman
-1
spectrum of compound 5h at 3076 cm which was assigned to C-H stretching of indole. The
-1
-1
indole ring C-C stretching vibrations are expected to be in the region 1625 cm to 1300 cm
-1
[
47] and it was observed in the IR spectrum of compound 5h at 1608 cm . The C-N
-1
stretching mode is reported at 1244 and 1227cm for indole [48] which occurred at 1229 and
-1
1
228 cm in the IR and Raman spectra of compound 5h, respectively.
3
.3.5.2. Phenyl ring
-1
The C-H stretching bands occur in the region of 3080-3010 cm . The weak bands observed at
-1
3
057 and 3043 cm in the IR and Raman spectra, respectively, of compound 5h correspond
to the C-H stretching mode. The ring C-C stretching vibrations occur in the region of 1625-
-1
-1
1
430 cm [47]. A weak band was observed at 1579 cm in the IR spectrum of compound 5h
-1
and a very strong band at 1581 cm in its Raman spectrum which were assigned for the C-C
stretching mode. The fluorine atom attached to an aromatic ring gives absorption band in the
-1
-1
region of 1270-1100 cm [49] which was noted as a strong band at 1204 cm in the IR
spectrum of compound 5h. The stretching modes for para substituted fluoro phenyl ring are
-1
expected to be in the range 1620-1280 cm [50]. A medium band was observed in the Raman
-1
spectrum of compound 5h at 1484 cm which was assigned to the para substituted fluoro
phenyl ring.
3
.3.5.3. Methoxy group vibrations
-1
The methoxy asymmetric and symmetric stretching bands occur around 2960 and 2846 cm ,
-1
respectively. A weak band was observed at 2941 cm in the Raman spectrum of compound
h which was assigned to methoxy group symmetric stretching vibration. The asymmetric C-
5
-1
H bending vibrations appear in the region of 1470-1440 cm [51]. These vibrations were
-1
noted as a medium intense band at 1449 cm in the Raman spectrum of compound 5h and at
-1
-1
1
445 cm in its IR spectrum. The strong band at 1159 cm in the IR spectrum of compound
-1
5
h and a medium band at 1158 cm in its Raman spectrum were assigned to the C-O
stretching [52].
.3.5.4. Methylene vibrations
Asymmetric stretching vibration for methylene group appears about 2930 cm [49]. A weak
3
-1
-1
band was observed at 2941 cm in the IR spectrum of compound 5h which was assigned for
this mode. Scissoring and rocking vibrations of methylene occur in the region of 1480-1440
2
2

ACCEPTED MANUSCRIPT
-1
-1
-1
cm and 730-710 cm , respectively [53]. The observed weak intensity band at 1479 cm in
the IR spectrum of compound 5h corresponds to its scissoring vibration. While, the noticed
-1
bands of weak intensity at 729 and 731 cm in the IR and Raman spectra of compound 5h,
respectively, were assigned for its rocking vibrations.
3
.2.5.5. Carbonyl vibration
-1
The C=O stretching vibrations appear as intense bands between 1870 and 1540 cm . The
position of these bands is determined by the neighbouring substituents, conjugations and
-1
hydrogen bonding [54]. Strong and medium bands were observed at 1666 and 1665 cm in
the Raman and IR spectra of compound 5h which were assigned to its C=O stretching mode.
3
.3.5.6. Chloro-Indole vibrations
-1
The chloro-indole ring shows strong absorption bands in the range of 760-505 cm for C-Cl
-1
stretching vibration. A shift might occur in the upper limit to 840 cm , if there is a
vibrational coupling with other vibrations [47]. Compound 5h manifested a stretching
-1
absorption band in its IR and Raman spectra at 759 and 754 cm , respectively.
.3.6. Molecular docking studies
3
Molecular docking was performed using AutoDock 4.2 software interfaced with AutoDock
Tools v1.5.6rc3 based on Lamarckian Genetic algorithm [42]. Compound 5h was energy
minimized using Gaussian′09 program package at DFT level. The C. albicans target protein
(PDB code: 1IYL) was selected for the present investigation. Its XRD structural coordinates
were downloaded from research collaboratory for structural bioinformatics (RCSB) protein
data bank, with a resolution of 3.2 Å. The rigid docking of flexible ligand was carried out into
the target protein by AutoDockTools program. Partial atomic charges were added using
Kollman’s method. The grid box was set as 90 x 90 x 90 points with spacing 0.913 Å over
the target protein binding pocket. Visualization (Figure 7) was performed with PyMOL
v1.2r1 program [55] and it manifested that the amidic NH of compound 5h interacts with the
amino acid residue Tyr354 of the target protein. The protein-ligand complex has been
.
..
stabilized by the formation of this N-H O hydrogen bond interaction. This hydrogen bond
interaction (ligand (N-H) : protein (O)) has been predicted by NBO analysis based on the low
electron occupancy on the N-H orbital. The binding energy of the best docked conformation
-1
of compound 5h is -7.79 kcal.mol with estimated inhibition constant of 1.44 µM and it has
been selected from 100 conformations (100 GA run). Moreover, docking of the antifungal
drug, fluconazole, into the target protein (1IYL) in a similar manner like 5h, gave protein-
-1
ligand binding energy of -4.99 kcal.mol indicating the possible anti-Candida activity of
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compound 5h. The docking results showed the possible binding mode of compound 5h with
its target protein (1IYL) and predicted its anti-C. albicans activity.
Figure 7. Binding pose of compound 5h with its target protein.
4
. Conclusion
Nine indole-isatin molecular hybrids 5a-i have been successfully prepared and characterized
using various spectroscopic techniques. The title compounds 5a-i were estimated for their
antimicrobial activity using DIZ and MIC assays. Compound 5c emerged as the most active
-1
congener towards Ps. aeruginosa with MIC value of 3.9 µg mL . Whereas, compound 5i is
the most active congener against A. niger and compounds 5b and 5h are the most active and
-1
equipotent towards P. notatum with MIC value of 7.8 µg mL . The vibrational spectroscopic
analysis of N'-[(3Z)-5-chloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-
methoxy-1H-indole-2-carbohydrazide (5h) was performed experimentally using FT-IR and
FT-Raman and theoretically by DFT/B3LYP method. The optimized geometric parameters,
vibrational harmonic frequencies, PED assignments and molecular orbital energies of the
compound 5h have been calculated by DFT/B3LYP method with 6-311++G(d,p) basis set.
The theoretical optimized geometric parameters and vibrational frequencies are compared
with the experimental data and their level of correlation is fairly good. The detailed PED
analysis of compound 5h showed a good agreement with the experimental data. The charge-
transfer occurring within the molecule can be understood by its HOMO and LUMO energy
values. Molecular docking study manifested the possible binding mode of compound 5h with
its fungal target protein. The results of the current exploration could be harnessed to develop
new bioactive indole-based antimicrobial molecules.
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Supplementary material
Figure S1 and the NMR spectra of the synthesized compounds 5a-i are provided as
supplementary materials.
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through the Research Group
Project no. RGP-196.
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