Synthesis, spectroscopic identification and molecular docking of certain N-(2-[2-(1H-Indol-2-ylcarbonyl) hydrazinyl](oxo)acetyl phenyl)acetamides and N-[2-(2-[2-(Acetylamino)phenyl](oxo)acetyl hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides: New antimicrobial agents

Article abstract of DOI:10.3390/molecules23051043

N-(2-[2-(1H-Indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl phenyl)acetamides (5a–h) and N-[2-(2-[2-(acetylamino)phenyl](oxo)acetyl hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides (5i–l) were synthesized and characterized with different analytical tools. N-Acetylisatines 4a–d were subjected to ring opening at their C2 carbons with the aid of different indole-bearing hydrazides 3a,b and 7 to afford the respective glyoxylamides 5a–l. The antimicrobial activity of the target compounds 5a–l was assessed with the aid of Diameter of the Inhibition Zone (DIZ) and Minimum Inhibitory Concentration (MIC) assays against a panel of Gram-positive and Gram-negative bacteria and certain fungal strains. The antimicrobial screening revealed that Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans are the most sensitive microorganisms towards the synthesized compounds 5a–l. In addition, compounds 5c and 5h emerged as the most active congeners towards Staphylococcus aureus and Candida albicans, respectively. Molecular docking studies revealed the possible binding mode of compounds 5c and 5h to their target proteins.

Full text of DOI:10.3390/molecules23051043

molecules
Article
Synthesis, Spectroscopic Identification and Molecular
Docking of Certain N-(2-{[2-(1H-Indol-2-ylcarbonyl)
hydrazinyl](oxo)acetyl}phenyl)acetamides and
N-[2-(2-{[2-(Acetylamino)phenyl](oxo)acetyl}
hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides:
New Antimicrobial Agents
Maha S. Almutairi 1 ID , Azza S. Zakaria 2, Reem I. Al-Wabli 1, I. Hubert Joe 3,
Ali S. Abdelhameed 1 and Mohamed I. Attia 1,4,
*
ID
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; malmutbiri@ksu.edu.sa (M.S.A.); ralwabli@ksu.edu.sa (R.I.A.-W.);
asaber@ksu.edu.sa (A.S.A.)
2
3
4
Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University,
Alexandria 21500, Egypt; azzazakaria@yahoo.com
Centre for Molecular and Biophysics Research, Department of Physics, Mar Ivanios College,
Thiruvananthapuram 695 015, Kerala, India; hubertjoe@gmail.com
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
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 March 2018; Accepted: 27 April 2018; Published: 29 April 2018
Abstract: N-(2-{[2-(1H-Indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl}phenyl)acetamides (5a–h) and
N-[2-(2-{[2-(acetylamino)phenyl](oxo)acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamides (5i–l
were synthesized and characterized with different analytical tools. N-Acetylisatines 4a–d were
subjected to ring opening at their C2 carbons with the aid of different indole-bearing hydrazides 3a,b
)
and
7 to afford the respective glyoxylamides 5a–l. The antimicrobial activity of the target compounds
5a–l was assessed with the aid of Diameter of the Inhibition Zone (DIZ) and Minimum Inhibitory
Concentration (MIC) assays against a panel of Gram-positive and Gram-negative bacteria and
certain fungal strains. The antimicrobial screening revealed that Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, and Candida albicans are the most sensitive microorganisms towards the
synthesized compounds 5a–l. In addition, compounds 5c and 5h emerged as the most active
congeners towards Staphylococcus aureus and Candida albicans, respectively. Molecular docking
studies revealed the possible binding mode of compounds 5c and 5h to their target proteins.
Keywords: indole; N-Acetylisatins; ring opening; antimicrobial; glyoxylamides
1. Introduction
Indole is a hetero-aromatic bicyclic ring system and indoles represent an important class in drug
discovery and development process [
isolated from natural resources and are widely used as precursors in fine organic synthesis to develop
new pharmacological lead pharmaceuticals across a broad range of therapeutic areas [ ]. Biological
], anticancer [ ],
1]. Indole-bearing compounds are commonly identified and
2–4
activities exhibited by different indole derivatives include anti-inflammatory [
antihypertensive [7], antiviral [8], antibacterial [9] and antifungal activities [10].
5
6
Molecules 2018, 23, 1043; doi:10.3390/molecules23051043
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1043
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In the same vein, 5-methoxyindole constitutes the backbone of the natural hormone melatonin,
which helps sleep regulation and wake cycles through manipulation of three different melatoninergic
receptors (M1–M3) [11,12]. Melatonin exhibits various therapeutic applications like anti-inflammatory,
antioxidant [13], and antitumor activities [14]. In addition, 5-methoxyindole fragment was incorporated
in a number of bioactive melatoninergic ligands [15,16].
2,3-Dioxindole (isatin) is another heterocyclic aromatic nucleus that was identified as an
endogenous compound in humans and other mammals [17]. Isatin has a broad synthetic utility owing
to its incorporation of reactive function groups which have been functionalized to prepare diverse
bioactive molecules such as anticancers [18], anticonvulsants [19], and antimicrobials [20]. Moreover,
N-acetylisatins are privileged structures in drug discovery and development processes due to their
facile utility to prepare the corresponding glyoxylamides via attacking their C2-carbonyl functionality
with different nucleophilic amines. The presence of two carbonyl groups with two different spatial
orientations in glyoxylamides significantly enhances their H-bonding with protein targets and hence
improving their biological activities. In addition, glyoxylamide derivatives have broad applications in
organic chemistry and they are incorporated in a vast of bioactive molecules [21–24].
Therefore, the aforementioned premises encouraged us to synthesize the title glyoxylamides
5a–l via opening certain N-acetylisatines 4a–d with different indole-bearing hydrazides 3a,b and 7.
The antimicrobial profile of the title compounds 5a–l was in vitro evaluated against a panel of
microorganisms including Gram-positive and Gram-negative bacteria as well as filamentous and
non-filamentous fungi.
2. Results and Discussion
2.1. Chemistry
The target compounds 5a–l were successfully achieved as portrayed in Schemes 1 and 2. Thus,
N-acetylisatines 4a–d were allowed to react with the appropriate hydrazide 3a,b in acetonitrile, a polar
non-nucleophile solvent, to afford the respective targets 5a–h (Scheme 1). NMR (1H and 13C) as well
as mass spectral data of compounds 5a–h are consistent with their proposed chemical structures.
The single crystal X-ray structure of compound 5c
[25], as a representative example of compounds
5a–h, confirmed doubtlessly the assigned chemical structures of 5a–h.
R
R
R
OCH3
O
NHNH2
O
OH
O
i
ii
N
H
N
H
N
H
1a,b
2a,b
3a,b
O
X
O
iii
N
1-3
a
R
H
OCH3
4
a
b
c
d
X
H3C
O
H
Br
Cl
F
4a-d
b
CH3
HN
O
X
O
R
HN NH
O
N
H
O
5a-h
Scheme 1. Cont.

Molecules 2018, 23, 1043
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Compound No.
R
X
5a
5b
5c
5d
5e
5f
H
H
H
H
Br
Cl
F
H
Br
Cl
F
H
OCH3
OCH3
OCH3
OCH3
5g
5h
Scheme 1. Synthesis of the target compounds 5a–h. Reagents and conditions: (i) Methanol, drops of
H2SO4, reflux, 4 h; (ii) Methanol, H2N-NH2.H2O, reflux, 2 h; and (iii) Acetonitrile, reflux 2 h.
Ethyl glycinate was coupled with the commercially available indole-2-carboxylic acid (1a) in
the presence of carbonyldiimidazole to furnish the coupled product
of the ester functionality of compound with hydrazine hydrate yielded the respective hydrazide
Subsequently, N-acetylisatines 4a–d were allowed to react with hydrazide to give the corresponding
6 (Scheme 2). Hydrazinolysis
6
7.
7
target compounds 5i–l (Scheme 2). The assigned chemical structures of compounds 5i-l were confirmed
via their NMR (1H and 13C) and mass spectral data.
O
O
CH3
NHNH2
O
HN
O
OH
O
HN
O
i
ii
N
H
N
H
N
H
1a
6
7
O
X
4
a
b
c
d
X
O
H
iii
N
Br
Cl
F
H3C
O
H3C
HN
4a-d
O
O
H
N
O
N
H
O
X
HN
O
N
H
5i-l
Compound No.
R
X
5i
5j
5k
5l
H
H
H
H
H
Br
Cl
F
Scheme 2. Synthesis of the target compounds 5i–l. Reagents and conditions: (i) Tetrahydrofuran,
ethyl glycinate hydrochloride, carbonyldiimidazole, rt, 18 h; (ii) Methanol, H2N-NH2.H2O, reflux, 2 h;
and (iii) Acetonitrile, reflux 2 h.
2.2. Antimicrobial Evaluation
The title glyoxylamides 5a–l were divided into two sets, the first set contains compounds 5a–h
in which either indole hydrazide 3a or 5-methoxyindole hydrazide 3b was used for ring opening of
N-acetylisatin derivatives 4a–d. The second set comprises compounds 5i–l in which the hydrazide
was used for ring opening of compounds 4a–d.
7

Molecules 2018, 23, 1043
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Table 1 presented the results of the preliminary antimicrobial activity of the title compounds 5a–l
against certain Gram-positive and Gram-negative bacteria as well as certain fungi using Diameter of
the Inhibition Zone (DIZ) assay. Compounds 5c and 5h manifested the best activity against the tested
Gram-positive bacteria with DIZ values of 21 and 22 mm towards S. aureus and B. subtilis, respectively.
On the other hand, compounds 5b and 5f showed the best activity against the tested Gram-negative
bacteria with DIZ values of 19 and 18 mm against E. coli and Ps. Aeruginosa, respectively. C. albicans
was the most sensitive fungus towards compound 5h with DIZ value of 25 mm.
The results of the Minimum Inhibitory Concentration (MIC) assay for the target compounds 5a–l
are presented in Table 2. S. aureus is the most sensitive Gram-positive bacteria towards the tested
compounds 5a–l with MIC values of 3.9, 31.25, and 62.5
respectively. Also, B. subtilis was sensitive to compound 5h with MIC value of 62.5
the tested strains of Gram-negative bacteria, compounds 5d 5i, and 5k are the best candidates towards
E. coli (compounds 5i and 5k) and compound 5d towards Ps. Aeruginosa being equipotent with
an MIC value of 62.5 g/mL. Compound 5h manifested the best antifungal profile for the whole
µg/mL for compounds 5c
, 5d and 5b or 5i,
µg/mL. Regarding
,
µ
synthesized series 5a–l as it showed MIC values of 7.8, 31.25, and 62.5
A. niger, and P. notatum, respectively.
µg/mL against C. albicans,
In summary, it can be deduced from the above antimicrobial screening tests that S. aureus,
E. coli, Ps. Aeruginosa, and C. albicans are the most sensitive microorganisms towards the synthesized
compounds 5a–l. Compound 5c bearing indole hydrazide 3a fragment and a chloro substituent in
the first set 5a–h, is the best candidate against S. aureus. In the same set, compound 5h bearing
5-methoxyindole hydrazide 3b fragment and a fluoro substituent and is the most active congener
towards both B. subtilis and the tested three fungal strains. On the other hand, compounds 5i and 5k,
bearing indole hydrazide
7 fragment in the second set 5i–l, are the most active compounds against
E. coli being equipotent, while compound 5d is the most active candidate towards Ps. Aeruginosa.
2.3. Molecular Docking
The three-dimensional (3D) structural coordinates of compounds 5c and 5h were sketched by
ChemDraw Ultra 7.0.1 program [26]. The energy of compounds 5c and 5h was minimized using
PRODRG online server on basis of GROMACS force field method [27]. The antibacterial (PDB ID:
4DH6) [28] and antifungal (PDB ID: 1EA1) [29] target proteins have been chosen for the docking study
for compounds 5c and 5h, respectively. The 3D structural coordinates of the target proteins were
downloaded from the RCSB protein data bank [30]. The target proteins manipulation has been carried
out by following steps: (i) all water molecules were removed; (ii) hydrogen atoms were added to the
crystal structure; (iii) Kollaman0s charges were added; and (iv) the docked inhibitors were removed
from the target proteins. The protein (rigid) and ligand (flexible) docking was performed with the aid
of AutoDock 4.2 [31] program interfaced with MGL Tools 1.5.6 rc3 [32] to create affinity grids centered
on the active site with 90
×
90
×
90 grid size. The results of the predicted free binding energy (∆E),
inhibition constant (Ki) as well as the bounded amino acid resides of the complex are given in Table 3.
The docking results were evaluated by sorting the free binding energies predicted by their docking
conformations. The best conformation binding energy is predicted to be –7.88 kcal/mol with Ki value
of 1.67 µM for compound 5c. Its hydrogen bonding interactions were observed with THR232, GLY230,
and PRO70 amino acid residues of 4DH6 target protein (Figure 1).
On the other hand, the best conformation binding energy is predicted to be –7.28 kcal/mol
with Ki value of 4.63 µM for compound 5h. Its hydrogen bonding interactions were noted with
ARG326, HIS392, GLN72, VAL395, and ASN102 amino acid residues of 1EA1 target protein (Figure 1).
The molecular docking investigations manifested the possible binding pose of compounds 5c and 5h
inside their target bacterial and fungal proteins, respectively.

Molecules 2018, 23, 1043
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Table 1. Diameter of the Inhibition Zone (DIZ) of the title compounds 5a–l, AMP, and FLC against Gram-positive bacteria, Gram-negative bacteria, and fungi.
DIZ in mm ± S.D.*
Strain
Compound No.
Gram-Positive Bacteria
Gram-Negative Bacteria
Fungi
B.
Subtilis
E.
S.
Aureus
E.
Coli
K.
P.
Ps.
Aeruginosa
S.
A.
Niger
C.
P.
MRSA
Fecalis
Pneumonia
Vulgaris
Enteridis
Albicans
Notatum
5a
5b
5c
9 ± 0.0
15 ± 1.0
15 ± 0.8
16 ± 0.2
11 ± 0.8
9 ± 0.0
9 ± 0.4
22 ± 1.6
13 ± 0.6
12 ± 0.6
14 ± 0.4
15 ± 0.4
30 ± 0.0
ND
9 ± 1.0
9 ± 0.0
9 ± 0.0
18 ± 1.3
11 ± 0.0
11 ± 0.6
9 ± 0.5
14 ± 0.3
12 ± 0.9
11 ± 0.2
11 ± 0.5
9 ± 0.8
−ve
9 ± 0.0
−ve
11 ± 0.4
11 ± 0.0
−ve
13 ± 1.6
11 ± 0.0
11 ± 0.5
−ve
−ve
−ve
−ve
36 ± 0.7
ND
19 ± 0.0
19 ± 0.0
21 ± 0.7
14 ± 0.3
−ve
16 ± 0.5
14 ± 0.4
14 ± 0.4
19 ± 0.0
9 ± 0.2
9 ± 0.0
9 ± 0.0
−ve
13 ± 0.9
19 ± 1.1
13 ± 0.3
14 ± 0.8
13 ± 0.44
14 ± 0.0
15 ± 1.0
14 ± 0.3
17 ± 0.6
13 ± 0.2
14 ± 0.0
14 ± 0.0
45 ± 1.0
ND
−ve
11 ± 0.6
−ve
11 ± 0.3
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
−ve
11 ± 0.7
−ve
11 ± 0.2
−ve
13 ± 0.4
14 ± 0.3
9 ± 0.0
11 ± 0.3
9 ± 0.2
18 ± 0.5
9 ± 0.0
11 ± 0.6
14 ± 0.5
9 ± 0.7
9 ± 0.6
12 ± 0.8
35 ± 1.0
ND
9 ± 0.0
9 ± 0.0
9 ± 0.0
9 ± 0.0
9 ± 0.0
15 ± 0.7
9 ± 0.0
9 ± 0.0
16 ± 0.2
9 ± 0.0
9 ± 0.1
9 ± 0.0
30 ± 0.5
ND
8 ± 0.0
8 ± 0.0
17 ± 0.4
8 ± 0.0
15 ± 0.7
8 ± 0.0
17 ± 0.1
11 ± 0.1
8 ± 0.0
16 ± 0.5
8 ± 0.0
8 ± 0.0
ND
11 ± 0.4
10 ± 0.3
8 ± 0.0
15 ± 0.1
15 ± 0.7
18 ± 0.3
8 ± 0.0
9 ± 0.5
15 ± 1.6
14 ± 1.0
16 ± 0.5
18 ± 1.2
14 ± 0.5
16 ± 0.4
14 ± 0.5
ND
5d
5e
14 ± 0.2
14 ± 1.1
18 ± 0.0
18 ± 0.0
25 ± 1.6
11 ± 0.2
13 ± 0.3
13 ± 1.0
15 ± 0.7
ND
5f
5g
5h
5i
5j
5k
5l
−ve
32 ± 0.4
ND
−ve
18 ± 0.4
ND
AMP
FLC
ND
ND
21 ± 0.5
16 ± 0.8
15 ± 0.0

Molecules 2018, 23, 1043
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Table 2. Minimum Inhibitory Concentrations (MICs) of the title compounds 5a–l, AMP, and FLC against Gram-positive bacteria, Gram-negative bacteria, and fungi.
MIC Values (µg/mL)
Strain Name
Gram-Negative Bacteria
K. P.
Pneumonia Vulgaris
Gram-Positive Bacteria
Fungi
Compound No.
B.
E.
Fecalis
S.
Aureus
E.
Coli
Ps.
S.
A.
Niger
C.
P.
MRSA
Subtilis
Aeruginosa Enteridis
Albicans
Notatum
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
AMP
FLC
500
125
250
250
250
250
250
62.5
500
500
500
250
15.6
ND
500
500
500
250
500
500
500
125
1000
500
500
500
500
>1000
500
500
500
500
500
500
125
62.5
3.9
31.25
500
250
250
250
62.5
500
125
125
125
125
125
125
125
250
62.5
125
62.5
125
<7.8
ND
500
500
500
500
500
500
500
500
500
500
500
250
250
ND
250
250
250
250
250
250
250
250
250
250
1000
125
500
ND
125
250
125
62.5
250
250
250
250
125
125
125
125
3.9
500
250
125
250
250
500
250
500
125
500
250
250
1000
ND
250
62.5
250
15.6
250
250
125
31.25
125
125
250
250
ND
15.6
250
250
250
62.5
125
125
31.25
7.8
250
250
250
250
ND
31.25
125
250
250
62.5
125
250
125
62.5
250
250
250
250
ND
250
500
500
500
<7.8
ND
500
500
>1000
ND
1000
>1000
ND
ND

Molecules 2018, 23, 1043
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Table 3. Binding energies and inhibition constants results of compounds 5c and 5h with their
target proteins.
Binding Energy (∆E)
Estimated Inhibition Constant (Ki)
[µM]
Protein ID
Bounded Residues
[kcal/mol]
4DH6
1EA1
–7.88
1.67
4.63
THR232, GLY230 and PRO70
–7.28
ARG326, HIS392, GLN72, VAL395, and ASN102
Figure 1. Hydrogen bonding projection of protein-ligand interactions and binding pose for compound 5c
(left) and compound 5h (right).
3. Experimental
3.1. General
A Gallenkamp device was used to measure melting points and they are uncorrected. Bruker NMR
spectrometer (Bruker, Reinstetten, Germany) was used to record the NMR spectra of the synthesized
1
compounds 5a–l in DMSO-d6 at 500 MHz for H and 125.76 MHz for 13C 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. Agilent Quadrupole 6120 LC/MS with ESI
(Electrospray ionization) source (Agilent Technologies, Palo Alto, CA, USA) was used to record mass
spectra of the synthesized compounds. High-resolution mass spectrometry (HR-MS) measurements
were performed on an LTQ-Orbitrap XL coupled to matrix-assisted laser desorption ionization
(MALDI). Compounds 2a,b [33], 3a,b [34], 4a–d [21], and 6 [35] were prepared according to literature
procedures. Ampicillin (AMP) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) and
fluconazole (FLC) was purchased from Shouguang-Fukang Pharmaceutical Ltd. (Shandong, China).
3.2. Chemistry
Methyl 1H-indole-2-carboxylate (2a): White powder; melting point (m.p.) 150–151 ◦C [33].
Methyl 5-methoxy-1H-indole-2-carboxylate (2b): Yellow powder; m.p. 176–177 ◦C [36].
1H-Indole-2-carbohydrazide (3a): Off-White powder; m.p. 251–253 ◦C [37].
5-Methoxy-1H-indole-2-carbohydrazide (3b): Off-White powder; m.p. 266–268 ◦C [38].
1-Acetyl-1H-indole-2,3-dione (4a): Yellow crystals; m.p. 141–143 ◦C [39].
1-Acetyl-5-bromo-1H-indole-2,3-dione (4b): Brown powder; m.p. 167–169 ◦C [21].
1-Acetyl-5-chloro-1H-indole-2,3-dione (4c): Light brown powder; m.p. 240–242 ◦C [40].
1-Acetyl-5-fluoro-1H-indole-2,3-dione (4d): Yellow powder; m.p. 147-149 ◦C [21].

Molecules 2018, 23, 1043
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3.2.1. General Procedure for the Synthesis of the Target Compounds 5a–h
The appropriate N-acetylisatin 4a–d (1 mmol) was added to a suspension containing the proper
acid hydrazide 3a,b (1 mmol) in acetonitrile (15 mL). The reaction mixture was heated to reflux for
two hours, cooled to room temperature, and filtered. The collected solid was dried and re-crystallized
from ethanol to give the title compounds 5a–h.
N-(2-{[2-(1H-Indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl}phenyl)acetamide (5a): Yellow powder;
m.p. 249–250 ◦C (yield 58%); 1H-NMR (DMSO-d6):
δ (ppm) 2.20 (s, 3H, CH3), 7.09 (t, J = 7.5 Hz, 1H,
Ar-H), 7.24 (t, J = 7.5 Hz, 1H, Ar-H), 7.27 (d, J = 1.5 Hz, 1H, CH-3-indole), 7.31 (t, J = 7.5 Hz, 1H, Ar-H),
7.47 (d, J = 8.5 Hz, 1H, Ar-H), 7.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.73 (dd, J = 1.5, 8.5 Hz, 1H, Ar-H), 8.11
(dd, J = 1.0, 8.0 Hz, 1H, Ar-H), 8.21 (d, J = 8.0 Hz, 1H, Ar-H), 10.71 (s, 1H, NH), 10.75 (s, 1H, NH), 10.90
(s, 1H, NH), 11.85 (s, 1H, NH-indole); 13C-NMR (DMSO-d6):
δ (ppm) 24.9 (CH3), 104.2, 112.9, 120.5,
121.5, 121.9, 122.3, 123.6, 124.4, 127.4, 129.5, 133.5, 135.9, 137.3, 140.3 (Ar-CH and Ar-C), 158.3, 164.5,
−
169.6, 192.7 (4
found: 363.1028 (M − H).
N-(4-Bromo-2-{[2-(1H-indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl}phenyl)acetamide (5b): Yellow
×
C=O); MS m/z (ESI): 363 [M
−
H] ; HR-MS (MALDI) calcd for C19H16N4O4: 363.1093,
◦
powder; m.p. 261–263 C (yield 56%); 1H-NMR (DMSO-d6):
δ (ppm) 2.19 (s, 3H, CH3), 7.08 (t, J = 7.5 Hz,
1H, Ar-H), 7.24 (t, J = 7.5 Hz, 1H, Ar-H), 7.27 (d, J = 1.5 Hz, 1H, CH-3-indole), 7.49 (d, J = 8.5 Hz, 1H,
Ar-H), 7.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.89 (dd, J = 2.0, 8.5 Hz, 1H, Ar-H), 7.96 (d, J = 8.5 Hz, 1H, Ar-H),
8.08 (d, J = 2.5 Hz, 1H, Ar-H), 10.65 (s, 1H, NH), 10.68 (s, 1H, NH), 10.91 (s, 1H, NH), 11.83 (s, 1H,
NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.7 (CH3), 104.3, 112.9, 114.0, 115.4, 120.2, 122.3, 123.7,
124.4, 127.3, 129.5, 133.4, 134.6, 137.5, 138.6 (Ar-CH and Ar-C), 158.3, 160.6, 169.4, 192.6 (4× C=O);
MS m/z (ESI): 441 [M
H]−, 442 [(M + 1) H]−, 443[(M + 2) H]−; HR-MS (MALDI) calcd for
−
−
−
C19H15BrN4O4: 441.0198, found: 441.0206 (M − H).
N-(4-Chloro-2-{[2◦-(1H-indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl}phenyl)acetamide (5c): Yellow
δ (ppm) 2.19 (s, 3H, CH3), 7.08 (t, J = 7.5 Hz,
powder; m.p. 259–260 C (yield 77%); 1H-NMR (DMSO-d6):
1H, Ar-H), 7.24 (t, J = 7.5 Hz, 1H, Ar-H), 7.27 (d, J = 1.5 Hz, 1H, CH-3-indole), 7.48 (d, J = 8.0 Hz, 1H,
Ar-H), 7.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.77 (dd, J = 2.5, 8.5 Hz, 1H, Ar-H), 7.99 (d, J = 2.5 Hz, 1H, Ar-H),
7.02 (d, J = 8.5 Hz, 1H, Ar-H), 10.66 (s, 1H, NH), 10.69 (s, 1H, NH), 10.91 (s, 1H, NH), 11.83 (s, 1H,
NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.7 (CH3), 104.3, 112.9, 120.5, 122.3, 123.8, 124.4, 124.8,
127.4, 127.7, 129.5, 131.7, 134.9, 137.2, 138.2 (Ar-CH and Ar-C), 160.9, 163.4, 169.7, 190.2 (4 C=O);
MS m/z (ESI): 397 [M
H]−, 398 [(M + 1) H]−, 399 [(M + 2) H]−; HR-MS (MALDI) calcd for
C19H15ClN4O4: 397.0704, found: 397.0738 (M − H).
N-(4-Fluoro-2-{[2-(1H-indol-2-ylcarbonyl)hydrazinyl](oxo)acetyl}phenyl)acetamide (5d): Yellow
×
−
−
−
◦
powder; m.p. 268–270 C(yield 73%); 1H-NMR (DMSO-d6):
δ (ppm) 2.17 (s, 3H, CH3), 7.08 (t, J = 7.5 Hz,
1H, Ar-H), 7.24 (t, J = 8.0 Hz, 1H, Ar-H), 7.27 (d, J = 1.5 Hz, 1H, CH-3-indole), 7.47 (d, J = 8.5 Hz, 1H,
Ar-H), 7.60 (ddd, J = 2.5, 3.0, 8.5 Hz, 1H, Ar-H), 7.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.84 (dd, J = 3.0, 9.5 Hz,
1H, Ar-H), 7.97–7.99 (m, 1H, Ar-H), 10.55 (s, 1H, NH), 10.69 (s, 1H, NH), 10.89 (s, 1H, NH), 11.85 (s, 1H,
NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.5 (CH3), 104.3, 112.9, 118.4, 118.5, 120.5, 122.2, 122.3,
124.4, 127.4, 129.5, 130.4, 133.2, 135.6, 137.7 (Ar-CH and Ar-C), 161.1, 163.6, 169.6, 190.1 (4 C=O);
H]−; HR-MS (MALDI) calcd for C19H15FN4O4: 381.0999, found: 381.0951
×
MS m/z (ESI): 381 [M
(M − H).
−
N-{2-[{2-[(5-Methoxy-1H-indol-2yl)carbonyl]hydrazinyl}(oxo)acetyl]phenyl}acetamide (5e): Pale
yellow powder; m.p. 228-230 ◦C (yield 52%); 1H-NMR (DMSO-d6):
δ (ppm) 2.19 (s, 3H, CH3), 3.78 (s,
3H, OCH3), 6.90 (dd, J = 2.5, 8.5 Hz, 1H, Ar-H), 7.15 (d, J = 1.0 Hz, 1H, Ar-H), 7.19 (s, 1H, CH-3-indole),
7.31(t, J = 7.5 Hz, 1H, Ar-H), 7.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.72 (t, J = 8.0 Hz, 1H, Ar-H), 8.11 (d,
J = 8.0 Hz, 1H, Ar-H), 8.20 (d, J = 8.5 Hz, 1H, Ar-H), 10.66 (s, 1H, NH), 10.75 (s, 1H, NH), 10.89 (s, 1H,
NH), 11.71 (s, 1H, NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.9 (CH3), 55.7 (OCH3), 102.6, 103.9,
113.7, 115.8, 121.5, 121.9, 123.6, 127.8, 129.8, 132.5, 133.5, 135.9, 140.3, 154.4 (Ar-CH and Ar-C), 160.9,
164.5, 169.6, 192.7 (4 C=O); MS m/z (ESI): 393 [M
H]−; HR-MS (MALDI) calcd for C20H18N4O5:
393.1199, found: 393.1139 (M − H).
×
−

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N-{4-Bromo-2-[{2-[(5-methoxy-1H-indol-2-yl)carbonyl]hydrazinyl}(oxo)acetyl]phenyl}acetamide
5f): Light brown powder; 248–250 ◦C (yield 65%); 1H-NMR (DMSO-d6):
(ppm) 2.18 (s, 3H, CH3),
(
δ
3.78 (s, 3H, OCH3), 6.89 (dd, J = 2.5, 9.0 Hz, 1H, Ar-H), 7.14 (d, J = 2.0 Hz, 1H, Ar-H), 7.19 (d, J = 1.5 Hz,
1H, CH-3-indole), 7.36 (d, J = 8.5 Hz, 1H, Ar-H), 7.89 (dd, J = 2.0, 8.5 Hz, 1H, Ar-H), 7.97 (d, J = 9.0 Hz,
1H, Ar-H), 8.08 (d, J = 2.5 Hz, 1H, Ar-H), 10.62 (s, 1H, NH), 10.65 (s, 1H, NH), 10.88 (s, 1H, NH),
11.68 (s, 1H, NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.7 (CH3), 55.7 (OCH3), 102.5, 103.9, 113.7,
115.4, 115.7, 123.9, 125.0, 127.7, 129.8, 132.5, 134.5, 137.8, 138.6, 154.4 (Ar-CH and Ar-C), 160.9, 163.4,
169.7, 190.1 (4 C=O); MS m/z (ESI): 471 [M
H]−, 472 [(M + 1) H]−, 473 [(M + 2) H]−; HR-MS
(MALDI) calcd for C20H17BrN4O5: 471.0304, found: 471.0365 (M − H).
N-{4-Chloro-2-[{2-[(5-methoxy-1H-indol-2-yl)carbonyl]hydrazinyl}(oxo)acetyl]phenyl}acetamide
5g): Yellow powder; 255–257 ◦C (yield 77%); 1H-NMR (DMSO-d6):
(ppm) 2.18 (s, 3H, CH3), 3.78 (s,
×
−
−
−
(
δ
3H, OCH3), 6.89 (dd, J = 2.0, 9.0 Hz, 1H, Ar-H), 7.14 (d, J = 2.5 Hz, 1H, Ar-H), 7.19 (d, J = 2.0 Hz, 1H,
CH-3-indole), 7.36 (d, J = 8.5 Hz, 1H, Ar-H), 7.78 (dd, J = 2.5, 9.0 Hz, 1H, Ar-H), 7.99 (d, J = 2.5 Hz,
1H, Ar-H), 8.03 (d, J = 9.0 Hz, 1H, Ar-H), 10.63 (s, 1H, NH), 10.65 (s, 1H, NH), 10.89 (s, 1H, NH),
11.68 (s, 1H, NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.7 (CH3), 55.7 (OCH3), 102.5, 103.9, 113.7,
115.7, 123.8, 124.7, 127.6, 127.7, 129.7, 131.7, 132.6, 134.9, 138.3, 154.4 (Ar-CH and Ar-C), 160.9, 163.5,
169.7, 190.2 (4 C=O); MS m/z (ESI): 427 [M
H]−, 428 [(M + 1) H]−, 429 [(M + 2) H]−; HR-MS
(MALDI) calcd for C20H17ClN4O5: 427.0809, found: 427.0845 (M − H).
N-{4-Fluoro-2-[{2-[(5-methoxy-1H-indol-2-yl)carbonyl]hydrazinyl}(oxo)acetyl]phenyl}acetamide
5h): Yellow powder; 238–240 ◦C (yield 60%); 1H-NMR (DMSO-d6):
(ppm) 2.17 (s, 3H, CH3), 3.78 (s,
×
−
−
−
(
δ
3H, OCH3), 6.89 (dd, J = 2.5, 9.0 Hz, 1H, Ar-H), 7.14 (d, J = 2.0 Hz, 1H, Ar-H), 7.18 (d, J = 1.5 Hz, 1H,
CH-3-indole), 7.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.60 (ddd, J = 2.5, 3.0, 9.0 Hz, 1H, Ar-H), 7.85 (dd, J = 3.0,
9.0 Hz, 1H, Ar-H), 7.99 (dd, J = 1.0, 9.0 Hz, 1H, Ar-H), 10.55 (s, 1H, NH), 10.64 (s, 1H, NH), 10.87 (s, 1H,
NH), 11.70 (s, 1H, NH-indole); 13C-NMR (DMSO-d6):
δ
(ppm) 24.5 (CH3), 55.8 (OCH3), 102.6, 103.9,
113.7, 115.8, 122.2, 122.4, 124.4, 124.5, 127.8, 129.7, 132.6, 135.9, 142.4, 154.4 (Ar-CH and Ar-C), 160.9,
163.7, 169.6, 190.3 (4 C=O); MS m/z (ESI): 411 [M
H]−; HR-MS (MALDI) calcd for C20H17FN4O5:
×
−
411.1105, found: 411.1148 (M − H).
Ethyl [(1H-indol-2-ylcarbonyl)amino]acetate (6): White powder; m.p. 222-224 ◦C [35].
3.2.2. Synthesis of N-(2-Hydrazinyl-2-oxoethyl)-1H-indole-2-carboxamide (7)
Hydrazine hydrate (50 mmol) was added to a suspension containing compound
6 (5 mmol) in
methanol (15 mL). The reaction mixture was heated to reflux for three hours under stirring. The cooled
reaction mixture was filtered off and dried to furnish compound
7
in 79% yield as a white powder, m.p.
1
227–229 ◦C, which was pure enough to be used for further reactions. H-NMR (DMSO-d6)
δ
(ppm):
3.89 (d, J = 6.0 Hz, 2H, CH2), 4.52 (s, 2H, NH2), 7.02–7.06 (m, 1H, Ar-H), 7.10 (s, 1H, CH-3-indole),
7.16–7.30 (m, 1H, Ar-H), 7.43–7.46 (m, 1H, Ar-H), 7.60 (d, J = 7.7 Hz, 1H, Ar-H), 9.76 (t, J = 6.0 Hz, 1H,
-CH2-NH), 9.80 (s, 1H, NH), 11.63 (s, 1H, NH); 13C-NMR (DMSO-d6)
120.2, 121.9, 123.6, 127.5, 130.9, 136.8 (Ar-CH and Ar-C), 161.7, 168.8 (2
HR-MS (MALDI) calcd for C11H12N4O2: 231.0882, found: 231.0808 (M − H).
δ
(ppm): 41.3 (CH2), 102.3, 112.−8,
×
C=O); MS m/z: 231 [M
−
H] ;
3.2.3. General Procedure for the Synthesis of the Target Compounds 5i–l
Compounds 5i–l were prepared by adopting the aforementioned procedure for the synthesis for
compounds 5a–h.
N-[2-(2-{[2-(Acetylamino)phenyl](ox◦o)acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-carboxamide
1
(
5i): Pale yellow powder; m.p. 228–230 C (yield 61%); H-NMR (DMSO-d6):
δ
(ppm) 2.19 (s, 3H,
CH3), 4.08 (d, J = 6.0 Hz, 2H, CH2), 7.07 (t, J = 7.0 Hz, 1H, Ar-H), 7.16–7.24 (m, 1H, Ar-H), 7.27 (s,
1H, CH-3-indole), 7.29–7.32 (m, 1H, Ar-H), 7.46 (t, J = 7.5 Hz, 1H, Ar-H), 7.62–7.68 (m, 1H, Ar-H),
7.69–7.74 (m, 1H, Ar-H), 8.11 (d, J = 8.0 Hz, 1H, Ar-H), 8.20 (d, J = 8.5 Hz, 1H, Ar-H), 8.88 (t, J = 6.0 Hz,
1H, -CH2-NH), 10.70 (s, 1H, NH), 10.74 (s, 1H, NH), 11.63 (s, 1H, NH), 11.85 (s, 1H, NH); 13C-NMR
(DMSO-d6):
δ (ppm) 24.9 (CH3), 63.4 (CH2), 103.6, 104.2, 112.9, 120.5, 122.3, 123.6, 124.4, 127.4, 127.6,

Molecules 2018, 23, 1043
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129.5, 131.8, 135.9, 136.9, 140.2 (Ar-CH and Ar-C), 161.0, 161.9, 168.7, 169.6, 192.7 (5
×
C=O); MS m/z
(ESI): 420 [M − H]−; HR-MS (MALDI) calcd for C21H19N5O5: 420.1307, found: 420.1353 (M − H).
N-[2-(2-{[2-(Acetylamino)-5-bromophenyl](ox◦o)acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-car
boxamide (5j): Green-yellow powder; m.p. 238-240 C (yield 47%); 1H-NMR (DMSO-d6):
δ (ppm) 2.13
(s, 3H, CH3), 4.07 (d, J = 6.0 Hz, 2H, CH2), 7.04–7.09 (m, 1H, Ar-H), 7.19–7.24 (m, 1H, Ar-H), 7.27 (s, 1H,
CH-3-indole), 7.45–7.49 (m, 1H, Ar-H), 7.64–7.69 (m, 1H, Ar-H), 7.84–7.86 (m, 1H, Ar-H), 7.89–7.95 (m,
1H, Ar-H),7.98 (d, J = 2.0 Hz, 1H, Ar-H), 8.89 (t, J = 6.0 Hz, 1H, -CH2-NH), 10.27 (s, 1H, NH), 10.59 (s,
1H, NH), 11.63 (s, 1H, NH), 11.82 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ
(ppm) 24.7 (CH3), 63.4 (CH2),
103.6, 112.8, 115.4, 122.1, 122.3, 123.9, 124.0, 125.2, 127.5, 129.8, 131.8, 134.5, 137.7, 138.5 (Ar-CH and
−
−
H] , 500
Ar-C), 161.9, 162.9, 168.7, 169.7, 189.9 (5
×
C=O); MS m/z (ESI): 498 [M
−
H] , 499 [(M + 1)
−
[(M + 2) − H]–; HR-MS (MALDI) calcd for C21H18BrN5O5: 498.0413, found: 498.0467 (M − H).
N-[2-(2-{[2-(Acetylamino)-5-chlorophenyl](oxo)acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-car
◦
1
boxamide (5k): Yellow powder; m.p. 237–239 C (yield 72%); H-NMR (DMSO-d6):
δ (ppm) 2.14
(s, 3H, CH3), 4.07 (d, J = 6.0 Hz, 2H, CH2), 7.04–7.09 (m, 1H, Ar-H), 7.19–7.24 (m, 1H, Ar-H), 7.27
(s, CH-3-indole), 7.45–7.49 (m, 1H, Ar-H), 7.64–7.69 (m, 1H, Ar-H), 7.73–7.78 (m, 1H, Ar-H), 7.88 (d,
J = 2.0 Hz, 1H, Ar-H),7.97–8.03 (m, 1H, Ar-H), 8.89(t, J = 6.0 Hz, 1H, -CH2-NH),10.59 (s, 1H, NH), 10.66
(s, 1H, NH), 11.63 (s, 1H, NH), 11.83 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ
(ppm) 24.6 (CH3), 63.4 (CH2),
103.6, 104.3, 112.9, 122.1, 122.3, 123.8, 124.4, 127.4, 127.7, 129.5, 131.8, 134.8, 137.3, 138.2 (Ar-CH and
Ar-C), 160.9, 161.9, 168.7, 169.7, 190.0 (5
×
C=O); MS m/z (ESI): 454 [M
−
H]–, 455 [(M + 1)
−
H]–, 456
[(M + 2) – H]–; HR-MS (MALDI) calcd for C21H18ClN5O5: 454.0918, found: 454.0973 (M − H).
N-[2-(2-{[2-(Acetylamino)-5-fluorophenyl](oxo)acetyl}hydrazinyl)-2-oxoethyl]-1H-indole-2-car
boxamide (5l): Pale yellow powder m.p. 239–241 C (yield 58%); 1H-NMR (DMSO-d6):
δ (ppm) 2.12 (s,
◦
3H, CH3), 4.07 (d, J = 6.0 Hz, 2H, CH2), 7.04–7.09 (m, 1H, Ar-H), 7.19–7.24 (m, 1H, Ar-H), 7.27 (s, 1H,
CH-3-indole), 7.45–7.48 (m, 1H, Ar-H), 7.54–7.60 (m, 1H, Ar-H), 7.64–7.69 (m, 1H, Ar-H), 7.84 (dd,
J = 9.0, 3.0 Hz, 1H, Ar-H), 7.97–7.99 (m, 1H, Ar-H), 8.88 (t, J = 6.0 Hz, 1H, -CH2-NH), 10.48 (s, 1H, NH),
10.55 (s, 1H, NH), 11.62 (s, 1H, NH), 11.85 (s, 1H, NH); 13C-NMR (DMSO-d6):
δ
(ppm) 24.4 (CH3), 63.1
(CH2), 100.0, 103.6, 112.9, 120.5, 122.3, 123.9, 124.4, 127.4, 127.5, 129.5, 131.8, 135.6, 136.9, 140.9 (Ar-CH
and Ar-C), 161.0, 161.9, 168.7, 169.5, 192.8 (5
C=O); MS m/z (ESI): 438 [M – H]–; HR-MS (MALDI)
×
calcd for C21H18FN5O5: 438.1213, found: 438.1261 (M − H).
3.3. Antimicrobial Activity
3.3.1. Isolates
The common pathogenic microorganisms were selected: four Gram-positive isolates, namely
Bacillus subtilis (B. subtilis), Enterococcus fecalis (E. fecalis), Methicillin resistant Staphylococcus aureus
(MRSA), and Staphylococcus aureus (S. aureus); five Gram-negative organisms, namely Escherichia
coli (E. coli), Klebsiella pneumonia (K. pneumonia), Proteus vulgaris (P. vulgaris), Pseudomonas aeruginosa
(Ps. Aeruginosa), and Salmonella enteridis (S. enteridis); and three fungal isolates, namely Asperagillus
niger (A. niger), Candida albicans (C. albicans), and Penicillum notatum (P. notatum). All isolates were
obtained from King Khaled Hospital, Riyadh, Saudi Arabia.
3.3.2. Disk Diffusion Assay
Disk diffusion assay for the title compounds 5a–l was carried out at 1000 µg/mL concentration as
previously reported [34] (Supplementary Materials: Antimicrobial Activity).
3.3.3. Determination of Minimum Inhibitory Concentrations (MICs)
The MIC values for the title compounds 5a–l and the reference compounds were determined by
adopting the previously reported method [34].

Molecules 2018, 23, 1043
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4. Conclusions
Opening N-acetylisatins 4a–d with the aid of different hydrazides 3a,b and/or
7
has been
successfully achieved to furnish the corresponding glyoxylamides 5a–l. The new acetamides 5a–h and
carboxamides 5i–l were characterized with various spectroscopic techniques. In vitro antimicrobial
potential of the title glyoxylamides 5a–l was examined using DIZ and MIC assays towards a panel
of Gram-positive and Gram-negative bacteria as well as filamentous and non filamentous fungi.
S. aureus, E. coli, Ps. Aeruginosa, and C. albicans are the most sensitive microorganisms towards the
synthesized compounds 5a–l. Compounds 5b–d bearing indole hydrazide 3a fragment are the most
active candidates towards S. aureus. Compound 5h bearing 5-methoxyindole hydrazide 3b moiety
manifested the best antifungal profile against the tested three fungal strains being about three-fold
more potent than fluconazole. Compounds 5i and 5k bearing indole hydrazide
most active congeners against E. coli being equipotent with MIC value of 62.5
7
fragment are the
µg/mL. Molecular
docking investigations predicted the possible binding pose of compounds 5c and 5h to their target
proteins. It is believed that the results of the current investigation could support the development of
new indole-based bioactive glyoxylamides.
Supplementary Materials: Supplementary materials are available online.
Author Contributions: Maha S. Almutairi, Reem I. Al-Wabli, and Mohamed I. Attia designed, prepared,
and interpreted the spectra of the title compounds. Azza S. Zakaria conducted the microbiological assays
and analyzed their results. I. Hubert Joe performed molecular modeling study. Ali S. Abdelhameed carried out
high resolution mass analysis. All authors discussed the contents of the manuscript.
Acknowledgments: 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. RG-1438-083.
Conflicts of Interest: The authors have declared 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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