Synthesis, spectroscopic characterization and antimicrobial potential of certain new isatin-indole molecular hybrids

Article abstract of DOI:10.3390/molecules22111958

Molecular hybridization has a wide application in medicinal chemistry to obtain new biologically active compounds. New isatin-indole molecular hybrids 5a-n have been synthesized and characterized by various spectroscopic tools. The in vitro antimicrobial potential of the prepared compounds 5a-n was assessed using diameter of the inhibition zone (DIZ) and minimum inhibitory concentration (MIC) assays against a panel of Gram-negative bacteria, Gram-positive bacteria and fungi. Most of the synthesized compounds 5a-n showed weak activities against Gram-negative bacteria while compounds 5b and 5c exhibited good activities against Gram-positive bacteria. On the other hand, compound 5j emerged as the most active compound towards Candida albicans (C. albicans), with an MIC value of 3.9 μg/mL, and compound 5g as the most active congener towards Asperagillus Niger (A. Niger), with an MIC value of 15.6 μg/mL. Moreover, compound 5h manifested the best anti-P. notatum effect, with an MIC value of 7.8 μg/mL, making it equipotent with compound 5g.

Full text of DOI:10.3390/molecules22111958

molecules
Article
Synthesis, Spectroscopic Characterization and
Antimicrobial Potential of Certain New Isatin-Indole
Molecular Hybrids
Reem I. Al-Wabli , Azza S. Zakaria and Mohamed I. Attia 1,3,*
1
2
ID
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; ralwabli@KSU.EDU.SA
Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University,
Alexandria 21500, Egypt; azzazakaria@yahoo.com
2
3
Medicinal and Pharmaceutical Chemistry Department,
Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618),
El Bohooth Street, Dokki, Giza 12622, Egypt
*
Correspondence: mattia@ksu.edu.sa; Tel.: +966-146-77337
Received: 25 September 2017; Accepted: 8 November 2017; Published: 15 November 2017
Abstract: Molecular hybridization has a wide application in medicinal chemistry to obtain new
biologically active compounds. New isatin-indole molecular hybrids 5a
and characterized by various spectroscopic tools. The in vitro antimicrobial potential of the prepared
compounds 5a was assessed using diameter of the inhibition zone (DIZ) and minimum inhibitory
concentration (MIC) assays against a panel of Gram-negative bacteria, Gram-positive bacteria and
fungi. Most of the synthesized compounds 5a showed weak activities against Gram-negative
–n have been synthesized
–
n
–
n
bacteria while compounds 5b and 5c exhibited good activities against Gram-positive bacteria. On the
other hand, compound 5j emerged as the most active compound towards Candida albicans (C. albicans),
with an MIC value of 3.9
µg/mL, and compound 5g as the most active congener towards Asperagillus
niger (A. niger), with an MIC value of 15.6
µg/mL. Moreover, compound 5h manifested the best
anti-P. notatum effect, with an MIC value of 7.8 µg/mL, making it equipotent with compound 5g.
Keywords: 5-methoxyindole; isatin; antibacterial; antifungal
1
. Introduction
Resistance to the currently available chemotherapeutic antimicrobial agents has become an
attractive subject of interest over the past 10 years. Understanding antimicrobial drug resistance
mechanisms plays a crucial role in developing effective therapeutic and prophylactic strategies
to preclude the problems that are encountered with resistant pathogens. The development of
antimicrobial resistance microbes might occur via upregulation of genes managing drug efflux,
alteration of the structure or levels of the molecular targets, or decreased affinity of the antimicrobial
agents for their targets. Potential strategies to overcome antimicrobial resistance include the
development of new antimicrobial candidates with better activity, combination therapy and adjunctive
immune therapy with cytokines [1–3].
Isatin (
nearly 170 years ago by Erdmann and Laurent [
endogenous multifunctional compound in plants [ ], fungi [
I
, 1H-indole-2,3-dione; Figure 1) is a heterocyclic compound that was first synthesized
]. Thereafter, it has been identified as an
], marine organisms [ ], and mammals [ ].
4,5
6
7
8
9
Isatin contains a number of chemically reactive function groups that broaden its synthetic utility.
Therefore, it is considered as a privileged substrate in both organic and medicinal chemistry, and it has
been extensively used for the construction of diverse bioactive compounds endowed with a broad
Molecules 2017, 22, 1958; doi:10.3390/molecules22111958
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1958
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spectrum of biological activities [10–17]. Among these activities, the antiprotozoal activity exhibited
certain isatin-
β lactame conjugates [18]. Recently, the potent anti-Trichomonas vaginalis activity of an
N-propyl tethered 5-bromo bis-isatin derivative has been reported [19].
(
I)
(II)
(III)
(5a–n)
Figure 1. Chemical structures of isatin (I), indole (II), melatonin (III), and the target compounds 5a–n.
On the other hand, indole (II; Figure 1) is another privileged bicyclic structure that is found in
diverse molecules with broad range of pharmacological activities representing various important
classes in drug discovery [20,21]. During the past decade, hundreds of published articles have
reported on the isolation or synthesis of indole-bearing natural products, including complex indole
alkaloids, particularly those with novel skeletons or potent bioactivities or those possessing synthetic
value [22]. Among these alkaloids, indole alkaloids with the 3,3-dimethylallyl (prenyl) substituent
on their scaffold constitute outstanding secondary metabolites in cyanobacteri, fungi and certain
marines [23]. Indole-bearing bioactive compounds can modulate multiple receptors with a high
affinity, and hence their application is to a wide range of therapeutic areas, such as analgesics [24],
anti-inflammatories [25], antitumors [26], antimicrobials [27], cyclooxygenase (COX)-2 inhibitors [28],
efflux pump inhibitors [29] and GABA agonists [30].
A
Melatonin (MLT, III; Figure 1) is a naturally occurring hormone incorporating 5-methoxyindole
moiety, and it binds to three types of melatonin receptors to exert its diverse biological activities [31–33].
Moreover, examination of the literature reveals that a number of bioactive indole-bearing compounds
have the 5-methoxyindole fragment in their structure [34–37].
Pharmacophore hybridization is a well-known beneficial approach in medicinal chemistry to
obtain new bioactive compounds [38,39]. The bioactive molecular hybrids usually result from the
hybridization of either two complementary pharmacophores or from pharmacophores with different
modes of action. The combined pharmacophores may address the active site of different targets with a
possible overcoming of drug resistance and exertion of synergetic effects [40]. In the present report,
certain isatin-indole molecular hybrids, compounds 5a–n, have been successfully synthesized to be
evaluated as new antimicrobial agents.
2
. Results and Discussion
2
.1. Chemistry
The title compounds 5a
–
n
have been successfully achieved as portrayed in
Scheme 1. The synthesis was commenced by esterification of the commercially available
-methoxyindole-2-carboxylic acid ( ) in methanol in the presence of a catalytic amount of
sulfuric acid according to the previously reported method [34]. Subsequently, the methyl
-methoxyindole-2-carboxylate ( ) was allowed to react with hydrazine hydrate in methanol to afford
the acid hydrazide derivative . The isatins 4a were reacted with compound in the presence of a
catalytic amount of glacial acetic acid to yield the title compounds 5a–n. The (Z)-configuration of the
5
1
5
2
3
–n
3

Molecules 2017, 22, 1958
3 of 16
imine double bond of the title compounds 5a
–
n
was previously confirmed via single crystal X-ray
analysis of an analogous compound [41].
4
4
4
4
4
4
4
4
,5
a, 5a
X
H
R
H
H
H
H
H
4,5
4h, 5h
4i, 5i
4j, 5j
4k, 5k
4l, 5l
4m, 5m
4n, 5n
X
Cl
F
OCH3
H
H
H
H
R
C H -CH
6
5
2
b, 5b Br
c, 5c Cl
d, 5d
e, 5e OCH3
f, 5f
g, 5g Br
C H -CH
C H -CH
6 5 2
6 5
2
F
4-Cl-C H -CH
6 4 2
4-CN-C H -CH
6 4 2
H
C H -CH
4-CH -C H -CH
6
5
2
3 6 4 2
C H -CH
6
C H
6 5
5
2
Scheme 1. Synthetic pathway to achieve the target compounds 5a–n. Reagents and conditions:
(
i
) Methanol and drops of H SO under reflux for 4 h; (ii) methanol and H N–NH2·H O under reflux
2
4
2
2
for 2 h; (iii) absolute ethanol and 10 drops of acetic acid under reflux for 4 h.
2
.2. Antimicrobial Evaluation
2
.2.1. Antimicrobial Susceptibility Testing
Table 1 shows the diameter of the inhibition zone (DIZ) values after the application of the test
samples 5a and the challenging of these with various bacterial and fungal pathogens. The results
–n
indicated a significant increase in the DIZs on the application of samples 5h and 5i against a
Pseudomonas aeruginosa (Ps. aeruginosa) isolate, as the DIZ values were 18 and 16 mm, respectively,
as compared to 0 mm for the reference standard, ampicillin (AMP). Additionally, compounds 5f, 5h
and 5i displayed a DIZ value of 12 mm toward Klebsiella pneumoniae (K. pneumoniae) isolate, while the
reference standard AMP was inactive.
Against Gram-positive isolates however, clear inhibition zones were observed upon the
application of most test samples, but these were weaker than those of the reference standard, AMP.
On the other hand, various test samples showed a DIZ value against different fungal isolates that
was comparable to that of the reference standard, antifungal fluconazole (FLC). Compound 5j was
the most active congener toward Candida albicans (C. albicans), with a DIZ value of 29 mm, while this
was 21 mm for FLC. Compounds 5e, 5k and 5n, however, exhibited a DIZ value of 18 mm against
Asperagillus niger (A. niger), as compared with 16 mm for FLC. Moreover, compounds 5e and 5j gave
DIZ values comparable to that of FLC against Penicillum notatum (P. notatum).
Table 2 illustrates the MIC values of the test samples 5a
AMP for bacterial isolates and FLC for fungal isolates, against the tested pathogens. Generally, all of the
tested compounds 5a had a clear inhibitory effect on the Bacillus subtilis (B. subtilis) isolate, ranging
–n along with the reference standards,
–n
from 2- to 6-fold less than AMP. Compounds 5b and 5c manifested a clear anti-Gram-positive
activity that was about 4–5-fold more potent than AMP, except against Enterococcus fecalis (E. fecalis).
Although compound 5i showed almost no activity against the tested Gram-positive isolates, except for

Molecules 2017, 22, 1958
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the B. subtilis isolate, it showed better activity than AMP against Ps. aeruginosa with an MIC value of
6
2.5 µg/mL as compared with AMP (MIC value of 1000 µg/mL).
On the other hand, compound 5g displayed a potent antifungal activity against the tested yeast
and the filamentous fungi, as its MIC value was half that of FLC against C. albicans and A. niger.
Moreover, compounds 5g and 5h were equipotent toward P. notatum with an MIC value of 7.8 g/mL,
making them approximately 32 times more potent than FLC (MIC value of 250 g/mL). Compound
g/mL, making it about
µ
µ
5
j was the most active anti-C. albicans candidate with an MIC value of 3.9 µ
4
times more potent than FLC (MIC value of 15.6 µg/mL).
2
.2.2. Scanning Electron Microscopy
Bacterial and fungal cells were photographed using electron microscopy to compare
morphological alterations after the addition of compounds 5b and/or 5j to the pathogens with
subsequent incubation for 24 h (Figure 2). For each isolate, the most illustrative photograph was
chosen even if morphologically normal organisms were still observed in the mount.
The scanning electron microscopy (SEM) appearance of untreated B. subtilis cells is shown
in Figure 2A.
The bacteria appeared as a long, thick bacilli chain-like structure, and even some oval spores were
clear in the mount. However, after the application of compound 5b, the cells were totally deformed,
having a spindle distorted shape with aggregation and exhibiting fusion of the whole cells together
(
Figure 2B). Figure 2C,D show the Staphylococcus aureus (S. aureus) isolate before and after treatment,
respectively. The bacteria were roughly spherical and smooth with a clear grape-like arrangement.
Compound 5b exhibited a profound alteration of the morphological structure of the cells and clumping
of the cocci cells. Moreover, bacterial lysis was observed for most of the cells (Figure 2D).
On the other hand, upon the application of compound 5j on C. albicans cells, the obtained results
were a little different. The normal mount of C. albicans cells under SEM showed clear oval cells
having a scattered arrangement, in addition to few budding cells (Figure 2E). However, after adding
compound 5j, no clumping of the fungal cells was shown, but abnormal forms were visible with a
clear change in their oval morphology (Figure 2F). Some cells were destructed and fused, while others
shrunk with clear pores on their outer surface, which resulted in the loss of their normal oval
morphology (Figure 2F).

Molecules 2017, 22, 1958
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Table 1. Diameter of the inhibition zone (DIZ) of the title compounds 5a–n, ampicillin (AMP) and fluconazole (FLC) toward Gram-negative and -positive bacteria
and fungi.
DIZ in mm ± S.D. *
Strain
Comp. No.
Gram-Negative Bacteria
Gram-Positive Bacteria
Fungi
E. coli
Ps.aeruginosa
P. vulgaris
K. pneumonia
S. enteridis
S. aureus
MRSA
E. fecalis
B. subtilis
C. albicans
A. niger
P. notatum
5
a
14 ± 0.6
10 ± 1.0
12 ± 2.0
10 ± 0.5
−ve
8 ± 0.8
8 ± 0.4
8 ± 0.4
10 ± 0.0
9 ± 0.6
10 ± 1.0
14 ± 1.8
18 ± 0.4
16 ± 0.9
11 ± 0.4
9 ± 0.0
9 ± 0.0
9 ± 0.0
12 ± 0.4
−ve
−ve
−ve
−ve
−ve
−ve
−ve
14 ± 0.8
18 ± 0.4
18 ± 0.4
12 ± 0.0
9 ± 0.0
−ve
−ve
−ve
−ve
−ve
−ve
−ve
11 ± 0.3
13 ± 0.0
−ve
5
b
18 ± 0.5
18 ± 0.0
−ve
18 ± 1.2
12 ± 1.6
11 ± 0.9
13 ± 0.4
14 ± 0.6
12 ± 0.0
12 ± 0.0
12 ± 1.0
18 ± 0.8
16 ± 0.12
11 ± 0.8
20 ± 0.6
16 ± 1.0
30 ± 0.5
ND
12 ± 0.7
10 ± 0.0
12 ± 0.0
20 ± 0.2
10 ± 0.0
18 ± 0.0
18 ± 0.5
14 ± 0.7
29 ± 0.0
18 ± 1.0
16 ± 0.0
18 ± 0.0
18 ± 0.0
ND
5
c
−ve
−ve
−ve
−ve
−ve
5
d
−ve
−ve
−ve
−ve
−ve
−ve
5
5
e
f
−ve
−ve
9 ± 0.3
−ve
9 ± 0.0
14 ± 0.7
−ve
9 ± 0.2
−ve
18 ± 0.4
11 ± 0.0
16 ± 0.5
11 ± 0.0
−ve
15 ± 0.9
9 ± 0.6
13 ± 0.0
11 ± 0.12
−ve
10 ± 0.8
20 ± 0.9
12 ± 1.2
10 ± 1.4
−ve
−ve
12 ± 1.9
−ve
20 ± 1.0
22 ± 0.43
18 ± 0.4
18 ± 0.9
15 ± 0.6
15 ± 0.0
21 ± 0.5
18 ± 1.1
18 ± 0.4
32 ± 0.4
ND
5
g
12 ± 0.7
−ve
10 ± 0.4
−ve
12 ± 0.0
−ve
5
h
12 ± 1.76
12 ± 1.6
−ve
12 ± 0.3
14 ± 0.6
9 ± 0.0
9 ± 0.6
9 ± 0.2
9 ± 0.0
16 ± 0.6
18 ± 0.4
ND
5
5
i
j
−ve
−ve
−ve
−ve
9 ± 0.1
11 ± 0.0
11 ± 0.1
11 ± 0.0
11 ± 0.3
45 ± 1.0
ND
9 ± 0.5
9 ± 0.0
14 ± 0.5
9 ± 0.0
9 ± 0.6
35 ± 1.0
ND
15 ± 1.1
18 ± 0.0
8 ± 0.0
14 ± 0.0
18 ± 0.0
ND
14 ± 0.2
−ve
5
k
−ve
−ve
−ve
5
l
−ve
−ve
−ve
−ve
5
5
m
n
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
AMP
FLC
30 ± 0.0
ND
36 ± 0.7
ND
−ve
ND
ND
ND
21 ± 0.5
16 ± 0.8
15 ± 0.0
*
Arithmetic mean ± standard deviation; −ve: 0.0 mm; ND: not determined.

Molecules 2017, 22, 1958
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Table 2. Minimum inhibitory concentrations (MICs) of the title compounds 5a–n, ampicillin (AMP) and fluconazole (FLC) toward Gram-negative and -positive
bacteria and fungi.
MIC Values (µg/mL)
Strain
Comp. No.
Gram-Negative Bacteria
Gram-Positive Bacteria
Fungi
E. coli
Ps. aeruginosa
P. vulgaris
K. pneumonia
S. enteridis
S. aureus
MRSA
E. fecalis
B. subtilis
C. albicans
A. niger
P. notatum
5
a
250
250
500
250
250
250
500
250
250
250
250
250
250
250
15.6
ND
500
500
500
500
125
500
125
500
62.5
250
250
250
500
250
>1000
ND
500
500
500
500
250
500
125
250
125
250
1000
1000
1000
250
<7.8
ND
500
500
500
500
500
500
500
500
1000
500
500
500
1000
500
>1000
ND
500
500
500
500
500
500
1000
500
500
1000
500
>1000
500
500
<7.8
ND
1000
15.6
15.6
1000
1000
>1000
500
500
500
500
250
62.5
125
250
250
ND
1000
31.25
15.6
500
1000
>1000
500
500
500
>1000
500
500
1000
125
62.5
500
500
500
250
15.6
15.6
500
500
125
250
250
250
250
250
250
500
250
1000
ND
125
7.8
31.25
7.8
15.6
31.25
7.8
31.25
62.5
3.9
62.5
125
62.5
62.5
ND
15.6
500
>1000
>1000
500
15.6
31.25
15.6
31.25
62.5
31.25
62.5
250
125
125
31.25
31.25
62.5
62.5
15.6
7.8
5b
5
c
5
5
d
e
5
f
5
g
500
5h
>1000
>1000
1000
250
125
250
250
3.9
ND
7.8
5
5
i
j
31.25
62.5
125
250
250
250
ND
250
5k
5
l
5
5
m
n
500
1000
500
62.5
ND
31.25
AMP
FLC
ND
ND: not determined.

Molecules 2017, 22, 1958
7 of 16
(
A)
(B)
(D)
(F)
(
C)
(
E)
Figure 2.
) SEM of compound 5b after 24 h incubation with B. subtilis; (
S. aureus) cells; ( ) SEM of compound 5b after 24 h incubation with S. aureus; (E
(
A
) Scanning electron microscopy (SEM) image of normal Bacillus subtilis (B. subtilis) cells;
) SEM of normal Staphylococcus aureus
) normal mount of
(B
C
(
D
Candida albicans (C. albicans) cells; (F) SEM of compound 5j after 24 h incubation with C. albicans.
3
. Experimental
3
.1. General
The melting points were measured using a Gallenkamp melting point device and are
uncorrected. The NMR samples of the synthesized compounds 5a–n were dissolved in dimethyl
sulfoxide (dMSO)-d , and the NMR spectra were recorded using a Bruker NMR spectrometer
6
1
13
(
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 -values
ppm) relative to TMS as an internal standard. Elemental analyses were carried out at the Microanalysis
δ
(

Molecules 2017, 22, 1958
8 of 16
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 an electrospray ionization (ESI) source (Agilent Technologies, Palo Alto,
CA, USA). Compounds 4a–e and 4n are commercially available.
3
.2. Chemistry
3
.2.1. Synthesis of Methyl 5-Methoxy-1H-Indole-2-Carboxylate (2)
5-Methoxy-1H-indole-2-carboxylic acid (2) was subjected to esterification in methanol and a
catalytic amount of sulfuric acid according to the reported method [42]. Its spectral data were in
accordance with those in the literature [43].
3
.2.2. Synthesis of 5-Methoxy-1H-Indole-2-Carbohydrazide (3)
Compound (5 mmol) was suspended in methanol, and hydrazine hydrate (50 mmol) was added.
The reaction mixture was refluxed for 3 h under stirring and was then cooled to ambient temperature.
2
The precipitated solid was filtered and dried to afford compound
3
[
44] in 90% yield as a white solid
◦
1
(m.p. 266–268 C), which was pure enough to be used for further reactions. H-NMR (dMSO-d )
6
ppm: 3.76 (s, 3H, OCH ), 4.52 (s, 2H, NH ), 6.84 (dd, J = 2.5, 8.5 Hz, 1H, Har.), 7.04 (d, J = 1.0 Hz,
3
2
1
H, Har.), 7.07 (d, J = 2.5 Hz, 1H, Har.), 7.36 (d, J = 8.5 Hz, 1H, Har.), 9.75 (s, 1H, NH), 11.46 (s, 1H, NH);
C-NMR (dMSO-d ) ppm: 55.2 (OCH ), 101.7, 101.9, 113.0, 114.2, 127.2, 130.8, 131.6, 153.7 (Car., CHar.),
61.2 (C=O); MS m/z: 206 [M + 1] , 228 [M + 23] .
1
3
6
3
+
+
1
3
.2.3. General Procedure of the Synthesis of the N-Benzylated isatins 4f–m
Benzyl bromide/chloride (10 mmol) was added to a stirred suspension containing the appropriate
isatin (10 mmol) and potassium carbonate (2.76 g, 20 mmol) in dimethylformamide (10 mL).
The reaction mixture was stirred at room temperature for 18 h, poured onto ice-cold water, filtered and
dried to give the respective N-benzylated isatins 4f–m.
◦
1
1
1
1
1
1
4
1
-Benzyl-1H-indole-2,3-dione (4f): Orange powder; m.p. 138–140 C [45].
◦
◦
-Benzyl-5-bromo-1H-indole-2,3-dione (4g): Orange powder; m.p. 148–150 C [46].
-Benzyl-5-chloro-1H-indole-2,3-dione (4h): Orange powder; m.p. 138–140 C [47].
-Benzyl-5-fluoro-1H-indole-2,3-dione (4i): Light-red powder; m.p. 135–137 C [48].
-Benzyl-5-methoxy-1H-indole-2,3-dione (4j): Light brown powder; m.p. 123–125 C [49].
-(4-Chlorobenzyl)-1H-indole-2,3-dione (4k): Orange powder; m.p. 168–170 C [50].
-[(2,3-Dioxo-2,3-dihydro-1H-indol-1-yl)methyl]benzonitrile (4l): Orange powder; m.p. 217–219 C [50].
-(4-Methylbenzyl)-1H-indole-2,3-dione (4m): Orange powder; m.p. 143–145 C [51].
◦
◦
◦
◦
◦
3
.2.4. General Procedure for the Synthesis of the Target Compounds 5a–n
The appropriate isatin derivative (4a–n, 5 mmol) was added to a stirred suspension containing
the carbohydrazide derivative (5 mmol) and a catalytic amount of glacial acetic acid in absolute
3
ethanol (10 mL). The reaction mixture was stirred under reflux for 4 h and the produced solid was
filtered while hot. The collected solid was washed with warm ethanol and dried to afford the title
compounds 5a–n in moderate to good yields. Analytical samples of compounds 5a–n were obtained
via their re-crystallization from a dimthylformamide/ethanol (1:3) mixture.
5
-Methoxy-N’-[(3Z)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide (5a): Yellow powder;
◦
−1
1
m.p. > 300 C (yield 80%); IR (KBr,
ppm: 3.79 (s, 3H, OCH ), 7.12–7.14 (m, 2H, Har.), 7.18 (d, J = 2.0 Hz, 1H, Har.), 7.39 (s, 1H, Har.),
ν
cm ): 3361 (NH) and 1691, 1656 (2
×
C=O); H-NMR (dMSO-d )
6
3
7
.41 (s, 1H, Har.), 7.42–7.44 (m, 1H, Har.), 7.52 (s, 1H, Har.), 8.04 (d, J = 8.0 Hz, 1H, Har.), 10.89 (s, 1H, NH),
13
11.64 (s, 1H, NH), 11.85 (s, 2H, NH); C-NMR (dMSO-d ) ppm: 55.2 (OCH ), 102.1, 110.7, 113.4, 113.5,
6 3

Molecules 2017, 22, 1958
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1
15.6, 116.2, 120.9, 121.8, 122.7, 126.6, 127.3, 132.5, 132.7, 143.9, 154.1 (Car., CHar., C=N), 161.8, 164.8
+
+
(2 × C=O); MS m/z: 335 [M + H] , 357 [M + Na] .
N’-[(3Z)-5-Bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (5b): Yellow
◦
−1
powder; m.p. > 300 C (yield 47%); IR (KBr,
ν
cm ): 3356 (NH) and 1695, 1654 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 7.14 (d, J = 8.5 Hz, 2H, Har.), 7.19 (s, 1H, Har.),
6
3
7
.44 (d, J = 8.5 Hz, 1H, Har.), 7.45 (d, J = 8.0 Hz, 1H, Har.), 7.57 (s, 1H, Har.), 8.20 (s, 1H, Har.),
1
1
1.00 (s, 1H, NH), 11.49 (s, 1H, NH), 11.82 (s, 2H, NH); 13C-NMR (dMSO-d ) ppm: 57.7 (OCH ), 102.6,
6
3
08.2, 112.4, 113.9, 116.9, 117.1, 122.1, 126.2, 126.6, 127.8, 129.2, 132.4, 132.9, 143.1, 154.5 (Car. and CHar.
,
+
C=N), 162.1, 165.1 (2 × C=O); MS m/z: 413 [M] .
N’-[(3Z)-5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (5c): Yellow
◦
−1
powder; m.p. > 300 C (yield 65%); IR (KBr,
ν
cm ): 3360 (NH) and 1691, 1654 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 6.94–6.99 (m, 2H, Har.), 7.23 (s, 1H, Har.), 7.34–7.47 (m, 2H,
ar.), 7.57 (s, 1H, Har.), 8.20 (s, 1H, Har.), 11.00 (s, 1H, NH), 11.83 (br. s, 2H, NH); C-NMR (dMSO-d6)
6
3
13
H
ppm: 57.7 (OCH ), 102.6, 108.3, 112.4, 113.3, 113.4, 116.5, 117.1, 126.3, 127.3, 128.7, 132.5, 133.7, 134.7,
3
+
1
42.9, 153.9 (Car., CHar., C=N), 161.4, 164.5 (2 × C=O); MS m/z: 369 [M] .
N’-[(3Z)-5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (5d): Yellow
◦
−1
powder; m.p. > 300 C (yield 61%); IR (KBr,
ν
cm ): 3371 (NH) and 1695, 1653 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 6.92–6.96 (m, 2H, Har.), 7.19 (d, J = 2.5 Hz, 1H,
6
3
H
ar.), 7.28 (ddd, J = 2.5, 9.0, 9.0 Hz, 1H, Har.), 7.40 (dd, J = 2.5, 9.0 Hz, 1H, Har.), 7.58 (s, 1H, Har.),
.01 (d, J = 8.5 Hz, 2H, Har.), 10.89 (s, 1H, NH), 11.75 (s, 1H, NH), 11.83 (s, 1H, NH); 13C-NMR (dMSO-d6)
ppm: 55.2 (OCH ), 102.1, 113.4, 113.5, 116.4, 127.3, 128.7, 132.5, 138.5, 140.2, 153.9 (Car., CHar., C=N),
8
3
1
63.2, 164.9 (2
×
C=O), 111.3 (C3’-F, J = 7.6 Hz, Car.), 113.7 (C2’-F, J = 26.3 Hz, Car.), 115.8 (C3’-F
,
+
J = 9.0 Hz, Car.), 118.8 (C2’-F, J = 23.8 Hz, Car.), 157.4 (C1’-F, J = 234.0 Hz, Car.); MS m/z: 353 [M + H] ,
3
+
75 [M + Na] .
5
-Methoxy-N’-[(3Z)-5-methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide
(
5e):
◦
−1
Orange powder; m.p. > 300 C (yield 94%); IR (KBr,
ν
cm ): 3348 (NH) and 1718, 1654 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 6.86 (d, J = 8.5 Hz, 1H, Har.),
6
3
3
6
7
.94 (dd, J = 2.5, 9.0 Hz, 1H, Har.), 7.01 (dd, J = 2.5, 8.5 Hz, 1H, Har.), 7.18 (d, J = 2.0 Hz, 1H, Har.),
.39 (d, J = 9.0 Hz, 1H, Har.), 7.54 (s, 1H, Har.), 7.69 (s, 1H, Har.), 10.68 (s, 1H, NH), 11.76 (s, 1H, NH),
1
1
1.82 (s, 2H, NH); 13C-NMR (dMSO-d ) ppm: 55.7 (OCH ), 56.3 (OCH ), 102.6, 111.6, 112.5, 113.2,
6 3 3
13.8, 113.9, 116.4, 116.8, 118.6, 127.7, 127.8, 132.9, 138.0, 154.5, 154.9 (Car., CHar., C=N), 163.8, 165.7
−
(2 × C=O); MS m/z: 363 [M − H] .
N’-[(3Z)-1-Benzyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (5f): Yellow
◦
−1
powder; m.p. 266–268 C (yield 69%); IR (KBr,
ν
cm ): 3338 (NH) and 1718, 1654 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.02 (s, 2H, CH ), 6.95 (d, J = 7.5 Hz, 1H, Har.),
6
3
2
7
.05 (d, J = 7.5 Hz, 1H, Har.), 7.16–7.19 (m, 2H, Har.), 6.28–7.30 (m, 1H, Har.), 7.36–7.37 (m, 2H, Har.),
.39–7.45 (m, 4H, Har.), 7.56 (s, 1H, Har.), 8.11 (d, J = 7.5 Hz, 1H, Har.), 11.75 (s, 1H, NH), 11.87 (s, 1H, NH);
C-NMR (dMSO-d ) ppm: 43.2 (CH ), 57.7 (OCH ), 102.6, 110.4, 113.9, 115.7, 116.8, 122.9, 126.9, 127.6,
7
13
6
2
3
1
27.8, 127.9, 129.1, 129.2, 129.4, 132.9, 133.1, 133.5, 136.7, 144.3, 154.5 (Car., CHar., C=N), 163.4, 164.1
+
+
(2 × C=O); MS m/z: 425. [M + H] , 447 [M + Na] .
N’-[(3Z)-1-Benzyl-5-bromo-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide
◦
−1
(
5g): Yellow powder; m.p. 262–264 C (yield 63%); IR (KBr,
ν
cm ): 3313 (NH) and 1722, 1662
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.02 (s, 2H, CH ), 6.94–7.05 (m, 3H, Har.),
6
3
2
7
8
5
.19–7.32 (m, 2H, Har.), 7.36–7.43 (m, 4H, Har.), 7.60 (d, J = 9.0 Hz, 1H, Har.), 7.82 (s, 1H, Har.),
.42 (s 1H, Har.), 11.84 (s, 1H, NH), 11.94 (s, 1H, NH); 13C-NMR (dMSO-d ) ppm: 43.3 (CH ),
6
2
5.7 (OCH ), 102.6, 112.2, 113.9, 114.7, 115.8, 117.2, 127.7, 127.8, 127.9, 128.0, 129.1, 129.2, 133.0, 134.0,
35.3, 135.8, 136.4, 142.0, 154.5 (Car., CHar., C=N), 164.4, 164.9 (2 × C=O); MS m/z: 503 [M] .
3
+
1

Molecules 2017, 22, 1958
10 of 16
N’-[(3Z)-1-Benzyl-5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide
◦
−1
(
5h): Yellow powder; m.p. 256–258 C (yield 79%); IR (KBr,
ν
cm ): 3315 (NH) and 1726, 1649
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.02 (s, 2H, CH ), 6.94–7.12 (m, 3H, Har.),
6
3
2
7
2
1
1
.19–7.31 (m, 2H, Har.), 7.32–7.48 (m, 4H, Har.), 7.61–7.71 (m, 2H, Har.), 8.31 (s, 1H, Har.), 11.83 (br. s, 2H,
×
NH); 13C-NMR (dMSO-d ) ppm: 43.4 (CH ), 55.7 (OCH ), 102.8, 112.5, 113.1, 113.9, 114.7, 116.9,
6
2
3
27.7, 127.8, 127.9, 128.0, 129.1, 129.2, 133.0, 135.7, 135.9, 136.2, 136.5, 141.7, 154.3 (Car., CHar., C=N),
58.3, 161.7 (2 × C=O); MS m/z: 459 [M + H] , 481 [M + Na] .
+
+
N’-[(3Z)-1-Benzyl-5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide (5i):
◦
−1
Yellow powder; m.p. 236–238 C (yield 93%); IR (KBr,
ν
cm ): 3329 (NH) and 1728, 1653 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.02 (s, 2H, CH ), 6.95 (dd, J = 2.5, 9.0 Hz, 1H, Har.),
6
3
2
7
7
.03–7.06 (m, 2H, Har.), 7.19 (d, J = 1.5 Hz, 1H, Har.), 7.24–7.35 (m, 2H, Har.), 7.36–7.44 (m, 4H, Har.),
.62 (s, 1H, Har.), 8.12 (d, J = 8.5 Hz, 1H, Har.), 11.85 (br. s, 2H, 2
×
NH); 13C-NMR (dMSO-d6)
ppm: 43.4 (CH ), 55.7 (OCH ), 102.8, 111.2, 112.1, 113.8, 113.9, 116.1, 116.9, 117.2, 127.7, 127.8, 127.9,
2
3
1
4
28.0, 129.1, 129.2, 133.1, 135.9, 136.5, 140.6, 154.3 (Car., CHar., C=N), 157.3, 164.0 (2
43 [M + H] , 465 [M + Na] .
×
C=O); MS m/z:
+
+
N’-[(3Z)-1-Benzyl-5-methoxy-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide
◦
−1
(
5j): Orange powder; m.p. 248–250 C (yield 84%); IR (KBr,
ν
cm ): 3346 (NH) and 1705, 1668
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 4.99 (s, 2H, CH ),
6
3
3
2
6
.94–6.97 (m, 2H, Har.), 7.19 (d, J = 2.5 Hz, 1H, Har.), 7.26–7.31 (m, 2H, Har.), 7.36–7.37 (m, 4H,
ar.), 7.40 (d, J = 9.0 Hz, 1H, Har.), 7.58 (s, 1H, Har.), 7.77 (s, 1H, Har.), 11.84 (br. s, 2H, 2 NH);
C-NMR (dMSO-d ) ppm: 43.1 (CH ), 55.7 (OCH ), 56.4 (OCH ), 102.8, 110.9, 113.4, 113.9, 114.9, 116.9,
H
×
1
3
6
2
3
3
1
1
17.9, 127.7, 127.8, 127.9, 129.2, 132.9, 134.1, 135.5, 136.8, 138.6, 142.6, 154.5, 155.5 (Car., CHar., C=N),
58.3, 161.7 (2 × C=O); MS m/z: 453 [M − H] .
−
N’-[(3Z)-1-(4-Chlorobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide
◦
−1
(
5k): Yellow powder; m.p. 273–275 C (yield 72%); IR (KBr,
ν
cm ): 3327 (NH) and 1724, 1654
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.02 (s, 2H, CH ), 6.94–6.97 (m, 1H, Har.),
6
3
2
7
7
1
1
1
.06–7.09 (m, 1H, Har.), 7.16–7.21 (m, 2H, Har.), 7.23 (d, J = 2.5 Hz, 1H, Har.), 7.39–7.42 (m, 4H, Har.),
.45–7.49 (m, 2H, Har.), 7.54 (s, 1H, Har.), 7.72 (d, J = 7.5 Hz, 1H, Har.), 8.13 (d, J = 7.5 Hz, 1H, Har.),
1
3
1.76 (s, 1H, NH), 11.87 (s, 1H, NH); C-NMR (dMSO-d ) ppm: 42.7 (CH ), 55.7 (OCH ), 102.6, 110.4,
6 2 3
13.8, 113.9, 115.7, 116.8, 119.9, 123.1, 127.8,129.2, 129.7, 129.9, 132.6, 132.8, 133.1, 135.2, 135.7, 142.9,
54.5 (Car., CHar., C=N), 161.8, 164.1 (2 × C=O); MS m/z: 457 [M − H] .
−
N’-[(3Z)-1-(4-Cyanobenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-5-methoxy-1H-indole-2-carbohydrazide
◦
−1
(
5l): Yellow powder; m.p. 297–299 C (yield 92%); IR (KBr,
ν
cm ): 3344 (NH) and 1720, 1660
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 3.79 (s, 3H, OCH ), 5.13 (s, 2H, CH ), 6.94–6.97 (m, 1H, Har.),
6
3
2
7
7
1
.04–7.07 (m, 1H, Har.), 7.18–7.22 (m, 1H, Har.), 7.23 (d, J = 2.5 Hz, 1H, Har.), 7.39–7.45 (m, 1H, Har.),
.56 (d, J = 8.5 Hz, 2H, Har.), 7.64 (d, J = 8.5 Hz, 2H, Har.), 7.84 (d, J = 2.5 Hz, 1H, Har.), 7.85 (d, J = 2.5 Hz,
H, Har.), 8.16 (d, J = 8.0 Hz, 1H, Har.), 11.77 (s, 1H, NH), 11.86 (s, 1H, NH); 13C-NMR (dMSO-d6)
ppm: 42.9 (CH ), 55.7 (OCH ), 102.7, 110.8, 113.8, 113.9, 115.8, 117.0, 119.1, 120.0, 121.3, 123.2, 127.8,
2
3
1
28.6, 128.8, 131.9, 133.1, 133.2, 141.9, 142.5, 142.8, 154.5 (Car., CHar., C=N, CN), 157.9, 161.9 (2
×
C=O);
−
MS m/z: 448 [M − H] .
5
-Methoxy-N’-[(3Z)-1-(4-methylbenzyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide
◦
−1
(5m): Yellow powder; m.p. 218–220 C (yield 80%); IR (KBr,
ν
cm ): 3348 (NH) and 1718, 1662
1
(2
×
C=O); H-NMR (dMSO-d ) ppm: 2.27 (s, 3H, CH ), 3.79 (s, 3H, OCH ), 4.97 (s, 2H, CH ),
6
3
3
2
6
H
.94–6.96 (m, 1H, Har.), 7.04 (d, J = 7.5 Hz, 1H, Har.), 7.15–7.19 (m, 4H, Har.), 7.26 (d, J = 8.0 Hz, 2H,
ar.), 7.40 (d, J = 8.0 Hz, 2H, Har.), 7.56 (s, 1H, Har.), 8.11 (d, J = 7.5 Hz, 1H, Har.), 11.75 (s, 1H, NH),
1.87 (s, 1H, NH); 13C-NMR (dMSO-d ) ppm: 21.3 (CH ), 42.9 (CH ), 55.7 (OCH ), 102.6, 110.4, 111.0,
1
1
6
3
2
3
13.4, 113.9, 115.4, 115.7, 121.2, 122.9, 126.9, 127.7, 127.8, 127.9, 129.8, 133.1, 133.6, 137.2, 144.3, 154.5
−
(Car., CHar., C=N), 163.8, 164.1 (2 × C=O); MS m/z: 437 [M − H] .

Molecules 2017, 22, 1958
11 of 16
5
-Methoxy-N’-[(3Z)-2-oxo-1-phenyl-1,2-dihydro-3H-indol-3-ylidene]-1H-indole-2-carbohydrazide
(
5n):
◦
−1
Yellow powder; m.p. > 300 C (yield 85%); IR (KBr,
ν
cm ): 3327 (NH) and 1728, 1660 (2
×
C=O);
1
H-NMR (dMSO-d ) ppm: 3.77 (s, 3H, OCH ), 6.88 (d, J = 8.0 Hz, 1H, Har.), 6.96 (d, J = 2.0 Hz, 1H, Har.),
6
3
7
.19 (d, J = 2.0 Hz, 1H, Har.), 7.27 (d, J = 7.5 Hz, 1H, Har.), 7.40–7.43 (m, 2H, Har.), 7.52–7.55 (m, 2H, Har.),
.58–7.65 (m, 3H, Har.), 7.80 (d, J = 7.5 Hz, 1H, Har.), 8.21 (d, J = 7.5 Hz, 1H, Har.), 11.84 (s, 1H, NH),
7
1
1
1
1.87 (s, 1H, NH); 13C-NMR (dMSO-d ) ppm: 57.7 (OCH ), 102.8, 110.7, 113.9, 115.7, 116.8, 119.9, 121.4,
6 3
23.4, 124.2, 127.2, 127.4, 127.9, 129.2, 130.1, 131.9, 133.3, 134.3, 143.9, 154.6 (Car., CHar., C=N), 161.2,
−
63.4 (2 × C=O); MS m/z: 409 [M − H] .
3
.3. Antimicrobial Activity
3
.3.1. Antimicrobial Agents
Stock solutions (1000
µg/mL) of AMP (Sigma-Aldrich Co., St. Louis, MO, USA) and FLC
(Shouguang-Fukang Pharmaceutical Ltd., Shouguang, Shandong, China) were used as a positive
control for bacteria and fungi, respectively. AMP was dissolved in water, while the test compounds as
well as FLC were prepared in 100% dimethyl sulfoxide (dMSO) and were diluted with sterile distilled
water. The antimicrobial discs (containing 25
µg of FLC or 10 µg of AMP) were purchased from ROSCO
◦
(Neo-Sensitabs, Taastrup, Denmark) and were stored at −80 C until use.
3
.3.2. Media
The bacteria were slanted on Nutrient agar (difco Laboratories, Detroit, MI, USA), yeast was
slanted on Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI, USA), and the fungi were
slanted on potato dextrose agar (PDA; Eiken Chemical Co. Ltd., Tokyo, Japan). Muller Hinton broth
(
MHB) and Muller Hinton agar (MHA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA)
and were used following the manufacturer’s instructions for the antimicrobial assay. Liquid RPMI
640 medium supplemented with L-glutamine was purchased from Sigma-Aldrich Co. (St. Louis,
MO, USA), added to 2% sodium bicarbonate and 0.165 M 3-(N-morpholino)propanesulfonic acid
MOPS) from Dojindo Laboratories (Kumamoto, Japan), adjusted to pH 7.0, and used for the assay of
1
(
the yeast and moulds. MacConkey’s agar, mannitol salt agar, cetrimide agar, and brain heart infusion
broth (BHI) were obtained from Difco Laboratories (detroit, MI, USA).
3
.3.3. Isolates
The following common pathogenic microorganisms were selected: Five Gram-negative organisms,
namely, Ps. aeruginosa, Escherichia coli (E. coli), K. pneumoniae, Proteus vulgaris (P. vulgaris),
and Salmonella enteridis (S. enteridis); four Gram-positive isolates, namely, E. fecalis, S. aureus, B. subtilis,
and methicillin-resistant Staphylococcus aureus (MRSA); three fungal isolates; two mould isolates,
P. notatum and A. niger; and C. albicans. All the isolates were obtained from King Khaled Hospital,
Riyadh, Saudi Arabia.
3
.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. The identification of isolates was performed
according to the standard methods described elsewhere [52] and by the Clinical Laboratory Standards
◦
Institute [53]. Isolates were stored in BHI containing 16% (w/v) glycerol at −80 C until further use.
3
.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

Molecules 2017, 22, 1958
12 of 16
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.
3
.3.6. Determination of Minimum Inhibitory Concentrations
The MICs of AMP and/or the synthesized compounds 5a–n against the bacterial isolates were
determined with a microdilution test, according to the reference method of the CLSI [54]. A stock
solution of pure AMP drug was prepared in sterile distilled water, while a stock solution of each of
the samples under testing was prepared in DMSO to reach an initial concentration of 1000 µg/mL.
The preparation of inocula for broth microdilution testing was performed in accordance with CLSI
standard procedures [55], and the MIC was defined as the lowest concentration of the antibiotic
or the test sample that prevented bacterial growth. The preparation of fungal inocula for the broth
microdilution testing was performed in accordance with CLSI documents M27-A3 [56] and M38-A2 [57
]
with RPMI 1640 medium buffered to pH 7.0 and a 0.165 M MOPS buffer for all organisms. In the case
of yeasts, the MICs were recorded as the lowest concentration at which a 50% decrease in turbidity
relative to the turbidity of the growth control was observed. In the case of the filamentous fungi,
the MICs of the test samples and FLC were recorded as the lowest concentrations at which a prominent
decrease in turbidity was observed.
3
.3.7. Disk Diffusion Assay
The antibacterial and antifungal screenings were conducted by the disk diffusion agar methods as
described previously [58]. Bacterial and fungal suspensions were adjusted to a 0.5 McFarland standard
6
corresponding to 5
×
10 CFU/mL. A 100
µ
L aliquot of each isolate suspension was uniformly spread
onto MHA and SDA plates for the bacteria and fungi, respectively. All the test samples were dissolved
in DMSO at a 1000 g/mL concentration; AMP (10 g) was used as a positive control for bacteria and
FLC (25 g) was used as a positive control for fungi. Sterilized paper discs with only DMSO were
µ
µ
µ
used as negative controls for both the bacteria and fungi. The plates were incubated under aerobic
◦
conditions at 35 C for 24 and 48 h for the bacteria and fungi, respectively. To obtain comparable
results, all prepared solutions were treated under the same conditions. The experiments were carried
out in triplicate. The plates were examined for evidence of antimicrobial activities, represented by a
zone of inhibition of the microorganism’s growth around the discs, and diameters of clear zones were
expressed in millimeters (mm) [59].
3
.3.8. Scanning Electron Microscopy
Overnight S. aureus, B. subtilis and C. albicans cultures were diluted to obtain 10 CFU/mL.
The prepared suspensions were then cultured for 24 h in MHB containing 32 g/mL of compound
for the bacterial isolates and 7.8 g/mL of compound 5j for the Candida isolate (2 times their
7
µ
5
b
µ
MIC values). Then these cells were prepared to be photographed before and after treatment by SEM
following the procedure described elsewhere [60].
4
. Conclusions
A new series of isatin-indole molecular hybrids 5a
chemical structures of compounds 5a have been confirmed by different spectroscopic tools.
The antimicrobial assessment of the target compounds 5a was performed against a panel of
–n has been successfully achieved. The assigned
–
n
–
n
Gram-positive and -negative bacteria as well as filamentous and non-filamentous fungi using both
DIZ and MIC assays. Compound 5c was the most active congener toward most of the tested
Gram-positive isolates, while compound 5i was the most active candidate against the Gram-negative
isolate, Ps. aeruginosa. Compounds 5g and 5h were equipotent toward P. notatum, with an MIC value
of 7.8
Compound 5j showed the best anti-C. albicans activity, with an MIC value of 3.9
about 4 times more potent than the antifungal reference standard FLC. The antifungal profile of the
µg/mL, making it approximately 32 times more potent than FLC (MIC value of 250
µg/mL).
µg/mL, making it

Molecules 2017, 22, 1958
13 of 16
title compounds 5a
–n
seemed to be better than their antibacterial profile. It is believed that the results
of the current study could aid the development of new bioactive isatin-based antimicrobial molecules
to be harnessed in clinics.
Acknowledgments: The authors would like to extend their 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.
Author Contributions: Reem I. Al-Wabli and Mohamed I. Attia designed, synthesized and characterized the
target compounds. Azza S. Zakaria performed and analyzed the microbiological experiments. All authors
discussed the contents of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the synthesized compounds are available from the corresponding author.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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