Synthesis of new urease enzyme inhibitors as antiulcer drug and computational study

Article abstract of DOI:10.1080/07391102.2021.1910072

In search of potent urease inhibitor indole analogues (1–22) were synthesized and evaluated for their urease inhibitory potential. All analogues (1–22) showed a variable degree of inhibitory interaction potential having IC₅₀ value ranging between 0.60 ± 0.05 to 30.90 ± 0.90 μM when compared with standard thiourea having IC₅₀ value 21.86 ± 0.90 μM. Among the synthesized analogues, the compounds 1, 2, 3, 5, 6, 8, 12, 14, 18, 20 and 22 having IC₅₀ value 3.10 ± 0.10, 1.20 ± 0.10, 4.60 ± 0.10, 0.60 ± 0.05, 5.30 ± 0.20, 2.50 ± 0.10, 7.50 ± 0.20, 3.90 ± 0.10, 3.90 ± 0.10, 2.30 ± 0.05 and 0.90 ± 0.05 μM respectively were found many fold better than the standard thiourea. All other analogues showed better urease interaction inhibition. Structure activity relationship (SAR) has been established for all analogues containing different substituents on the phenyl ring. To understand the binding interaction of most active analogues with enzyme active site docking study were performed. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2021.1910072

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Synthesis of new urease enzyme inhibitors as
antiulcer drug and computational study
Muhammad Taha, Sukinah Ismail, Syahrul Imran, Noor Barak Almandil,
Munther Alomari, Fazal Rahim, Nizam Uddin, Shawkat Hayat, Khalid Zaman,
Mohamad Ibrahim, Bandar Alghanem, Imadul Islam, Rai Khalid Farooq,
Mohamed Boudjelal & Khalid Mohammed Khan
To cite this article: Muhammad Taha, Sukinah Ismail, Syahrul Imran, Noor Barak Almandil,
Munther Alomari, Fazal Rahim, Nizam Uddin, Shawkat Hayat, Khalid Zaman, Mohamad Ibrahim,
Bandar Alghanem, Imadul Islam, Rai Khalid Farooq, Mohamed Boudjelal & Khalid Mohammed
Khan (2021): Synthesis of new urease enzyme inhibitors as antiulcer drug and computational study,
Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2021.1910072
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1910072
Synthesis of new urease enzyme inhibitors as antiulcer drug and
computational study
Muhammad Tahaa , Sukinah Ismaila,b, Syahrul Imranc,d , Noor Barak Almandila , Munther Alomarie
,
,
g
f
f
a
h
Fazal Rahimf , Nizam Uddin , Shawkat Hayat , Khalid Zaman , Mohamad Ibrahim , Bandar Alghanem
ꢀ
Imadul Islamh, Rai Khalid Farooqi, Mohamed Boudjelalh and Khalid Mohammed Khanj
aDepartment of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
b
c
Dammam, Saudi Arabia; College of clinical pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia; Atta-ur-Rahman
d
Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Bandar Puncak Alam, Selangor, Malaysia; Faculty of Applied
Science, UiTM Shah Alam, Shah Alam, Selangor, Malaysia; Department of Stem Cell Biology, Institute for Research and Medical
Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia; Department of Chemistry, Hazara University,
Mansehra, Khyber Pakhtunkhwa, Pakistan; Department of Chemistry, University of Karachi, Karachi, Pakistan; Medical Research Core
Facility and Platforms (MRCFP, King Abdullah International Medical Research Center/King Saud bin Abdulaziz University for Health Sciences
(KSAU-HS), King Abdulaziz Medical City (KAMC), NGHA, Riyadh, Saudi Arabia; Department of Neuroscience Research, Institute for Research
and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia; H. E. J. Research Institute of
e
f
g
h
i
j
Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 14 December 2020
Accepted 23 March 2021
In search of potent urease inhibitor indole analogues (1–22) were synthesized and evaluated for their
urease inhibitory potential. All analogues (1–22) showed a variable degree of inhibitory interaction
potential having IC50 value ranging between 0.60 0.05 to 30.90 0.90 mM when compared with stand-
ard thiourea having IC50 value 21.86 0.90 mM. Among the synthesized analogues, the compounds 1,
2, 3, 5, 6, 8, 12, 14, 18, 20 and 22 having IC50 value 3.10 0.10, 1.20 0.10, 4.60 0.10, 0.60 0.05,
5.30 0.20, 2.50 0.10, 7.50 0.20, 3.90 0.10, 3.90 0.10, 2.30 0.05 and 0.90 0.05 mM respectively
were found many fold better than the standard thiourea. All other analogues showed better urease
interaction inhibition. Structure activity relationship (SAR) has been established for all analogues con-
taining different substituents on the phenyl ring. To understand the binding interaction of most active
analogues with enzyme active site docking study were performed.
KEYWORDS
Urease inhibition; urease
interaction with inhibitors;
molecular docking
1. Introduction
been reported includes disulphide derivatives (Khan et al.,
2014), hydrazones (Taha et al., 2019), hydroxamic acids
(Odake et al., 1994), and imidazoles like rabeprazole etc
(ParkImamura & Kobashi, 1996).
In recent past, many papers had reported on urease inhib-
ition by indole-based inhibitors (Naureen et al., 2015). A
study conducted by Isaac et. al (Aslam et al., 2011). suggests
that hydrazone derivatives were among the most active
inhibitors of Jack bean urease that has been discovered.
Some metal complexes of Sn (IV), Cu, Ni, Zn and Co with
hydrazone have been reported as urease inhibitors (de
Urease is an important nickel containing metalloenzyme that
catalyse the hydrolysis of urea into ammonium and carbon
dioxide (Krajewska, 2009). Often found in plants, fungi and
bacteria play a pivotal role in nitrogen metabolism of plants
during the germination process (Mobley et al., 1995).
However, ammonia produced by urease can cause hepatic
coma urolithiasis, hepatic encephalopathy, gastric and peptic
ulcers in human (Li & Mobley, 2002; Mobley et al., 1995).
Urease is known to cause virulence as well as determinant in
pathogenicity of many human related diseases (Kwon-Chung
et al., 2014). The urease activity of Helicobacter pylori at low
pH in stomach plays a vital role in the pathogenesis of the
gastric ulcers (Mobley, 1996). Therefore, urease inhibitors
have been identified as first line to treatureolytic bacteria
(Olivera-Severo et al., 2006). Due to this reason, urease inhibi-
tors have special attention over the past decade and many
ꢀ
Fatima et al., 2018).
Indole is nitrogen containing aromatic bicyclic planar mol-
ecule in which pyrrole ring is fused with benzene ring at 2
and 3 position. Indole ring are present in several natural
products like fungal metabolites alkaloids and marine natural
products. Numerous plants yielded indole like Jasmine
compounds have been reported. Among those that had (Bouchikhi et al., 2008; Douglass et al., 2011; Gribble, 2000)
CONTACT Muhammad Taha
taha_hej@yahoo.com; mtaha@iau.edu.sa
Department of clinical pharmacy, Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
ꢀ
Present address: Chemistry Department, Faculty of science, Assiut University, Assiut, Egypt
Supplemental data for this article can be accessed online at http://dx.doi.org/10.1080/07391102.2021.1910072
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
M. TAHA ET AL.
orange blossoms, citrus plants (AmirJaved & Kumar, 2008) evaluated. All synthesized compounds exhibited a variable
and Robiniapseudacacia (Van Order & Lindwall, 1942).
degree of inhibitory potential having IC50 value ranging
In animal body it also presents in brain (Dharmendra between 0.60 0.05 to 30.90 0.90 mM when compared with
et al., 2010), bile (Lucas et al., 1985), in pancreas, liver standard thiourea having IC50 value 21.86 0.90 mM. Among
(Pandeya et al., 1998), and where pus formation occurs the synthesized analogues, compounds 1, 2, 3, 5, 6, 8, 12,
(Siddiqui et al., 2008) and found in coal tar (Omar et al., 14, 18, 20 and 22 having IC50 value 3.10 0.10, 1.20 0.10,
1996). Indole ring system is a valuable structural moiety with 4.60 0.10, 0.60 0.05, 5.30 0.20, 2.50 0.10, 7.50 0.20,
a broad spectrum biological activities including antifungal 3.90 0.10, 3.90 0.10, 2.30 0.05 and 0.90 0.05 mM were
found many folds better than the standard thiourea. All
other analogues showed better urease inhibition. Structure
activity relationships are primarily based on substituted phe-
nyl ring.
The most potent analogue among the synthesized ana-
logues is compound 5 (IC50 ¼ 0.60 0.05 mM) having 3, 4-
dihydroxy substitution on one of the phenyl rings. The
greater inhibition shown by this scaffold may be due to the
presence of OH moiety on one of the phenyl rings which is
possibly involved in hydrogen bonding.
If we compare compound 5 with other dihydroxy substi-
tuted analogues like compound 2 (IC50 ¼ 1.20 0.10 mM)
having hydroxyl moiety present at position2and 4 on the
phenyl ring, compound 3 (IC50 ¼ 4.60 0.10 mM)having
hydroxyl moiety present at position3 and 5 on the phenyl
ring, compound 6 (IC50 ¼ 5.30 0.20 mM) having hydroxyl
moiety present at position2 and 5 on the phenyl ring and
compound 22 (IC50 ¼ 0.90 0.05 mM) having hydroxyl moiety
present at position 2and 3 on the phenyl ring. The difference
in the potential of these analogues seems due to varied pos-
ition of substituent on phenyl ring (Figure 2).
Comparison of compound 5 (IC50 ¼ 0.60 0.05 mM) with
compound 20 (IC50 ¼ 5.30 0.20 mM) showed that replacing
a hydroxyl group with a methoxy group decrease the activity
to 10 folds which is an indication that intermolecular hydro-
gen bonding is a significant factor for enzyme inhibition.
Furthermore, when compound 2 (IC50 ¼ 1.20 0.10 mM) was
compared with compound 21 (IC50 ¼ 30.90 0.90 mM) having
methoxy group at position 2 and 4 instead of hydroxyl
group decreases its activity to 26 folds which suggests us
(Przheval’skii et al., 1997), antioxidant (Poeggeler et al., 1993),
antimicrobial (Hiari et al., 2006), anticonvulsant (Ali et al.,
2013), anti-HIV (SuzenBuyukbingol, 1998), plant growth regu-
lator (Karadeniz et al., 2011), antihistaminic (Battaglia et al.,
1999), anticancer (Chen et al., 1996), analgesic and anti-
inflammatory (Guerra et al., 2011). The N’-benzylidene-2-(1H-
indol-3-yl)acetohydrazide showed cytotoxic, antimicrobial
activities (Choppara et al., 2019), thymidine phosphorylase
(Taha et al., 2020), FAK inhibition (Kassab & Hassan, 2018),
COX-2 inhibitors (de Oliveira Moraes et al., 2018; Sharma
et al., 2019), Dual IGF-1R/SRC inhibitors (Schmidt et al., 2011),
selective novel tubulin inhibitors (Tantak et al., 2017), and
cyclooxygenase inhibitor (Wani et al., 2019).
We are working on new inhibitors for urease and in
recent past we have reported different classes of compound
against urease inhibition (Alomari et al., 2019; Seraj et al.,
2021; Wahid et al., 2020; Zaman et al., 2019) (Figure 1). We
synthesized indole derivatives which are not directly
attached with the ring. The most of inhibitors reported hav-
ing aromatic ring directly attached with other functional. The
acetyl chain which will give the free movement to the other
part of the molecules and will give better results.
2. Result and discussion
2.1. Chemistry
Indole derivatives were synthesized by refluxing methyl 2-
(1H-indol-3-yl)acetate with hydrazine hydrate in methanol for
6 hrs yielded 2-(1H-indol-3-yl)acetohydrazide as intermediate
product (I). The intermediate product (I) was recrystallized
using methanol. 2-(1H-indol-3-yl)acetohydrazide was then
treated with varied aldehyde (1–22) (Table 1) in methanol
for 3–4 hrs yielded indole derivatives (1–22) (Scheme 1). The
reaction completion was monitored using thing layer chro-
matography (TLC). The crude product was recrystallized in
methanol to yield pure compounds (1–22). The compounds
1, 2, 4, 6, 7, 10, 11–13, 17, 18, 20 and 22 are reported ear-
lier (Hanna et al., 2007; Ju et al., 2019; Liu et al., 2019;
Mirfazli et al., 2016). The compounds 3, 5, 8, 9, 14, 15, 16,
19 and 21 are new and all scaffold are reported for urease
activity first time.
that the hydroxyl group play
a key role in activity.
Comparison of compound 2 with compound 8, showed a
very interesting result (IC50 ¼ 2.50 0.10 mM) that adding
more hydroxyl group to compound 2 also decreases its activ-
ity hence position and numbers of hydroxyl play the key role
in activity (Figure 2).
Similarly by comparing scaffold 1(IC50 ¼ 3.10 0.10 mM)
possess nitro group at position 4 of the phenyl ring with
scaffold 10 (IC50 ¼ 12.30 0.30 mM) having nitro group at
position
2
of phenyl ring and scaffold 11(IC50
¼
12.20 0.30 mM) having nitro group at position 3 of phenyl
ring. The slight difference was observed in the potential of
these three-scaffolds, which may be due to the interaction
with enzyme (Figure 3).
2.2. In vitro urease interaction study with inhibitors
Similarly, by comparing analogue 13 (IC50
¼
Our research group is continuously working to explore
potent bioactive compounds (Ali et al., 2018; Saify et al.,
17.40 0.40 mM) having 4-chloro substitution on phenyl ring
with analogue 17 (IC50 ¼ 12.20 0.30 mM) having 3-chloro
2014; Taha et al., 2015; 2015; Taha & Wadood, 2018). In cur- substitution on phenyl ring. The little bit difference was
rent study we have synthesized a new class of indole ana- observed in the potential of these two analogues may be
logues (1–22) and their urease inhibitory potential were due to way of interaction with enzyme (Figure 4).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Table 1. Continued.
Table 1. In vitro urease inhibitory activity of compounds (1–22).
S. NO
R
IC50 SEMa S#
R
IC50 SEMa
30.90 0.90
S. NO
R
IC50 SEMa S#
R
IC50 SEMa
7.50 0.20
10
12.30 0.30 21
1
3.10 0.10 12
11
12.20 0.30 22
0.90 0.05
2
3
1.20 0.10 13
17.40 0.40
Standard Thiourea
21.86 0.40
4.60 0.10 14
3.90 0.10
By comparing compound 15 (IC50 ¼ 26.20 0.60 mM) hav-
ing 2-methyl substitution on phenyl ring with compound 16
(IC50 ¼ 18.20 0.50 mM) having 4-methyl substitution on phe-
nyl ring and compound 19 (IC50 ¼ 20.90 0.50 mM) having 3-
methyl substitution on phenyl ring. The observed difference
in potential among these analogues may be due to way of
interaction with active side of enzyme in a different way
(Figure 5).
4
5
16.60 0.20 15
26.20 0.60
18.20 0.50
The analogues 7 (IC50 ¼ 13.70 0.40 mM), 12 (IC50
¼
0.60 0.05 16
7.50 0.20 mM) and 18 (IC50 ¼ 5.90 0.10 mM) having mono-
hydroxy at position 3, 4, 2 respectively. The most active
among them is 18 which may be due to better fit with the
enzyme active as compared to other analogues 7 and 8 but
still the compound 12 also showed a better activity then
compound 7 may be due to same reason. If we compare
compound 18 with the compound 4 (IC50 ¼ 16.60 0.20 mM)
which is 2-methoxy instead of 2-hydroxy its activity dropped
two-folds which may be due to the possible hydrogen bond-
ing ability of compound 18 with enzyme (Figure 6).
The compound 9 (IC50 ¼ 11.10 0.30 mM) having dime-
thylamine showed good activity this may be due to good
interaction with enzyme. On the other hand, compound 14
(IC50 ¼ 3.90 0.10 mM) showed potent activity having ester
moiety at position 4 this may be enzyme active side inter-
action with ester (Figure 7).
6
7
8
5.30 0.20 17
13.70 0.40 18
2.50 0.10 19
12.20 0.30
5.90 0.10
20.90 0.50
In the above study it was observed that position, nature
and number of substituents on phenyl ring greatly affected
the inhibition of the synthesized analogues. To further
explore the binding interaction of most active analogues
with active site of enzyme, molecular docking study
was performed.
9
11.10 0.30 20
2.30 0.05
2.3. Computational study of interaction of urease
enzyme with most active inhibitors
All compounds were docked into the binding cavity of ure-
ase enzyme (PDB ID: 4ubp). The results showed that active
compounds 2, 5, and 22 can fit well in the urease binding
(continued)

4
M. TAHA ET AL.
Scheme 1. Synthesis of indole derivatives (1–22)
Figure 1. In recent past we published few urease inhibitors (a–e).
cavity. Binding affinity, residues involved, interactions, and interact with the nickel ion (NI798) with the cavity of urease
distance of ligand-residue interactions obtained from the enzyme. The hydroxyl group at meta position also forms a
docking results for selected compounds are summarized in further interaction with the oxygen on the side chain of
Table 2.
The most potent compound
nickel-binding residue Asp363.Hydroxyl group at the ortho
0.60 0.05) position of benzylidene ring forms a hydrogen bonding with
5
(IC50
¼
showed good interactions pattern as shown in (Figure 8). nitrogen on the imidazole ring of His222 at 1.8 Å. The NH of
The hydroxyl groups in compound 5 which are located at hydrazone linkage forms a hydrogen bonding with the side
meta and para positions over the aromatic ring of the ben- chain (OD2) of Asp224 at 1.7 Å. The benzylidene ring for
zylidene moiety. The docking results revealed that hydroxyl compound 22 is sandwiched between two hydrophobic
group at para position of the benzylidene moiety was p-alkyl interactions involving the backbone (C) of both
observed to act as an acceptor and interact with nickel ion Ala366 and guanidine moiety Arg339 while indole is stabi-
(NI798) at 3.2 Å thus retarding the catalytic activity of ure- lized by two hydrophobic p-alkyl interactions with the back-
ase. The same hydroxyl group also acts a hydrogen bond bone of Ala170 (Figure 9).
donor that forms interaction with oxygen on the side chain
Compound 2 which is the third most active displayed
(OD2) of nickel-binding residue Asp363 at 1.8 Å. The benzyli- form a metal interaction through hydroxyl at para position
dene ring interactions were stabilized by an electrostatic of the benzylidene moiety with nickel ion(NI798) at 2.1 Å. It
p-cation interaction with NH2 on the guanidine moiety of was also found to form a hydrogen bonding with oxygen
Arg339. Another interaction of hydrogen bonding was (OD2) on the backbone of nickel-binding residue Asp363 at
observed between the NH of the hydrazine linkage and a distance of 2.9 Å. Furthermore, the other hydroxyl group at
imidazole on the side chain of catalytic residue His323 ortho position forms a hydrogen bonding with the nitrogen
which takes place through a p-donor hydrogen bond at a (Ne2) on the imidazole moiety of catalytic residue His323 at
distance of 2.7 Å. As a result, the interaction of compound 5 a distance of 3.0 Å. Another hydrogen bonding was observed
with Arg339 and catalytic residue His323 had also influ- between NH of the hydrazone linkage and oxygen on the
enced the decrease in catalytic activity of urease enzyme. backbone of residue Cys322 present in the catalytic core of
Other interaction includes indole ring being stabilized by the enzyme at a distance of 2.8 Å. The results also showed
two hydrophobic p-alkyl interactions with the side chain (C) that Cys322 stabilizes the aromatic ring on indole through
of Lys169 (Figure 8).
hydrophobic p-alkyl interaction. Another hydrophobic p-alkyl
Docking results for compound 22, the hydroxyl at meta interaction was observed between Lys169 and aromatic ring
position of the benzylidene moiety acts as an acceptor to of indole moiety (Figure 10).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Figure 2. Dihydroxy, trihydroxy and dimethoxy substituted analogues.
Figure 3. Nitro substituted analogues1, 10 and 11.
Figure 4. Chloro substituted analogues 13 and 17.
Figure 5. Methyl substituted analogues 15, 16 and 19.
Docking studies had been used to further visualize the on the docking results in Figure 11, compound 12 that is the
importance of substituents (R) of each compound and how least active among these 3 compounds was observed to form
these substituents affect the activity. Comparison between minimum interaction in the active site as compare to the other
compounds 12, 2, and 5 showed the significance of hydroxyl 2 compounds. Compound 12 displayed a single interaction
substituents with reference to their position on the ring. Based with nickel ion, while compounds 2 and 5 demonstrated the

6
M. TAHA ET AL.
Figure 6. Monohydroxy substituted analogues 15, 16 and 19and methoxy analogue 4.
Figure 7. Ester and amino substituted analogues 9 and 14.
Table 2. Binding affinity, residues involved, interactions, and distance of ligand-residue interactions.
Comp. No.
IC50 (mM)
Binding affinity (kcal/mol)
Residues
Interaction
Distance (Å)
5
0.6
ꢁ11.8
NI798
Metal acceptor
Hydrogen bond
Hydrogen bond
Metal acceptor
Hydrogen bond
Hydrogen bond
Hydrogen bond
Metal acceptor
Hydrogen bond
Hydrogen bond
Hydrogen bond
Metal acceptor
Hydrogen bond
Metal acceptor
Electrostatic
Hydrogen bond
Hydrogen bond
Metal acceptor
Hydrogen bond
Hydrogen bond
Metal acceptor
Hydrogen bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
Metal acceptor
Metal acceptor
Metal acceptor
Hydrogen bond
3.2
1.8
2.7
3.5
1.9
1.8
1.7
2.1
2.9
3.0
2.8
4.3
2.3
2.7
2.3
2.8
3.1
3.5
2.3
2.5
3.5
2.5
2.6
2.9
2.4
3.4
3.7
5.6
2.8
Asp363
His323
NI798
Asp363
His222
Asp224
NI798
Asp363
His323
Cys322
Ni798
Asp224
Ni798
His249
Cys322
Glu166
Ni798
His222
Asp224
Ni798
Asp363
His249
Cys322
Cys322
Ni798
22
2
0.9
1.2
ꢁ10.7
ꢁ10.5
20
1
2.3
3.1
ꢁ10.2
ꢁ10.3
18
12
5.9
7.5
ꢁ9.8
ꢁ9.6
9
11.1
12.2
12.3
16.6
30.9
ꢁ9.3
ꢁ9.4
ꢁ9.4
ꢁ9.4
ꢁ8.0
11
10
4
Ni798
Ni798
Cys322
21
ability to form multiple metal-acceptor interactions with nickel compared. The results showed in Figure 12 explain how pro-
ion as well as hydrogen bonding with the enzyme active resi- tecting hydroxyl group affect the activity. Compound 20,
dues like Asp363 and His323. It is very important for the which is a derivative of compound 5, displayed significant
hydroxyl to be able to interact well with nickel ion as well as reduction in activity, signifying effect of converting hydroxyl
group at para position to methoxy. Comparison between
residues within the active site.
Moving on further with the activity relationship of docking results of compound 5 and 20 (Figure 12) showed
hydroxyl substituents towards the inhibition activity, docking that methoxy at para position is not able to interact well
results of compounds 2, 4, 5, 12, 18, 20, and 21 are being with nickel ion over a distance apart due to steric factor.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 8. Molecular docking and 2D interaction diagram for compound 5 with urease enzyme (4UBP).
be positioned properly within the active site, thus allowing
nitro substituent to interaction with nickel ion, as can be
seen for compound 1. The importance of oxygen on nitro
substituent as metal-acceptor can also be seen through inter-
action of compound 9, in which the oxygen atoms are now
replaced with two methyl groups and this has resulted in
decrease of inhibition activity.
2.4. In silico ADMET analysis
Most of drug failures at early and late pipeline occur due to
undesired pharmacokinetics and toxicity problems. If these
issues could be addressed early, it would be extremely
advantageous for the drug discovery process.
Figure 9. Molecular docking and 2D interaction diagram for compound 22
In silico ADMET analysis was conducted to predict the
pharmacokinetic properties of the compounds and address
undesired pharmacokinetics and toxicity problems. The com-
pounds were evaluated for various parameters like aqueous
solubility, blood brain barrier (BBB) penetration, CYP2D6
binding, hepatotoxicity, human intestinal absorption (HIA),
and plasma protein binding. Overall observation on the
ADMET biplot showed that all compounds are predicted to
be within the ellipses of HIA and BBB (Figure 14). It is
expected that more than 90% of all compounds will be
absorbed into the bloodstream. This is based on the results
that showed all compounds are well within the absorption
level for both 95 and 99% confidence ellipses. The results in
Table 3 indicates that all compounds have very good HIA
level 0. For blood brain barrier (BBB), all compounds are
with urease enzyme (4UBP).
Similar observation for compound 2 and 21, in which both
hydroxyl groups of compounds 2 were being converted into
methoxy group, causing the great activity loss. Docking
results displayed that presence of two methoxy groups
causes much steric hindrance thus preventing the methoxy
substituents from interacting with nickel ion. Another similar
observation was compounds 4 and 18. Compound 4 which
possess a methoxy substituent at ortho position displayed
less inhibition compared to compound 18 with hydroxyl
group at the same position. Investigating these further using
docking results showed that compound 4 is not able to fit in
well in the cavity and has formed significantly less inter-
action with active site.
As for compounds with nitro substituents, compounds 1, within the 99% confidence ellipse except for compound 8.
10, and 11, docking results suggest that the activity is On the other hand, only 13 compounds are within the BBB
mainly due to the ability of nitro substituents to form inter- 95% confidence ellipse.
Table 3 shows a detailed prediction that majority of the
compounds have high to low BBB penetration levels (levels
1–3) except for compounds 8 which could not penetrate the
action with nickel ion, besides the other residue in the cavity
(Figure 13). Compound 1 that has nitro substituent at pos-
ition is able to interact well within the binding site due to its
linear shape, which allows the nitro substituent to be posi- blood brain barrier (Level 4). Compounds 13, 15, 16, 17, and
tioned deep in the cavity and close to the nickel ion. It was 19 are high BBB penetrants (level 1) while compounds 4, 9,
also well observed that compounds 10 and 11, having nitro and 21 displayed medium BBB penetration (level 2). The rest
substituent at ortho and meta position losses their ability to of the compounds displayed low BBB penetration (level 3). As
inhibit urease activity, as nitro substituent at ortho and meta for the aqueous solubility, all compounds were predicted to
positions tend to be too bulky to allow the aromatic ring to have moderate and good aqueous solubility level (2 and 3).

8
M. TAHA ET AL.
Figure 10. Molecular docking and 2D interaction diagram for compound 2 with urease enzyme (4UBP).
Figure 11. The 2D interaction diagram of compounds 2, 5 and 12 displaying binding position and interaction with nickel ion and residues within the active site of
urease enzyme.
Prediction indicate that all compounds are hepatotoxic protein binding property prediction showed that 7 com-
(level 1, Table 3) and therefore requires extra precaution. On pounds displayed the ability to bind to protein plasma (9,
the other hand, all compounds are noninhibitors of CYP2D6 10, 13, 15, 16, 17). On contrary, prediction for other com-
and this suggests that these compounds are well metabo- pounds suggests that they will not bind to protein plasma.
lized in Phase-I metabolism. Finally, the ADMET plasma Observations suggest that active compounds 2, 5, 22 have

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 12. The 2D interaction diagram of compounds 2, 4, 5, 18, 20 and 21 displaying effect of protecting hydroxyl group on interaction with nickel ion and
important residues within the active site of urease enzyme.

10
M. TAHA ET AL.
Figure 13. Interaction of compounds 1, 9, 10 and 11 that are bearing nitro and amino substituents within the active site of urease enzyme.
high potential. Through the in silico ADMET analysis com-
pounds 2, 5 and 22 was found have good solubility in aque-
ous solution and high intestinal absorptivity. These
compounds are also noninhibitors of CYP2D6 and they do
not interfere with drug metabolism process taking place in
the liver. Their ability not to bind to protein plasma indicates
good bioavailability and are not likely to be highly bound to
carrier proteins in the blood. However, these compounds
need to be further evaluated for their low BBB penetration
and hepatoxicity.
3. Assay protocol
The reaction mixtures containing solution of 25 mL of enzyme
(jack bean urease) and 55 mL of buffer at pH 6.8, having
100 mM urea, were incubated with 5 mL of test compounds
concentration (from 0.5 to 0.00625 mM) for 15 min at 30 ꢂC
in 96-well plates. Urea concentration were changed from 2
to 25 mM for assessment. Urease activity was determined by
Figure 14. ADMET plot for all compounds against Absorption 95% (Red),
Absorption 98% (Yellow), Blood Brain Barrier 95% (Blue), and Blood Brain
Barrier 98% (Black).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Table 3. ADMET prediction of 22 synthesized indole derivatives.
Hepatotoxic
prediction
PPB
Comp. No.
Solubility levela
BBB levelb
Absorption levelc
CYP2D6 predictiond
predictione
1
2
3
4
5
6
7
8
2
3
3
2
3
3
3
3
2
2
2
3
2
2
2
2
2
3
2
3
2
3
3
3
3
2
3
3
3
4
2
3
3
3
1
3
1
1
1
3
1
3
2
3
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
Toxic
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
9
10
11
12
13
14
15
16
17
18
19
20
21
22
TRUE
FALSE
FALSE
TRUE
FALSE
TRUE
TRUE
TRUE
FALSE
TRUE
FALSE
FALSE
FALSE
aSolubility of compounds in aqueous solution (2¼ Moderate, 3 ¼ High).
bBlood brain barrier penetration level (1¼ Good, 2 ¼ Moderate, 3 ¼ Low, 4¼ No penetration).
cHuman intestinal absorptivity level.
dAbility of compounds to inhibit CYP2D6.
eAbility of compounds to bind to protein plasma.
measuring ammonia production using the indophenol (5 mL) in methanol (15 mL) for 6 hrs yielded 2-(1H-indol-3-
yl)acetohydrazide as intermediate product (I). The completion
of reaction was monitored using TLC. The excess solvent
were evaporated under vacuum to obtain the crude product
and recrystallized in methanol to obtained pure product.
Equimolar intermediate product (I) then further treated with
different aldehydes (1–22) in methanol (15 mL) for 3–4 hrs in
the presence of catalytic amount of acetic acid yielded
indole derivatives (1–22). The completion of reaction was
monitored with the help of TLC. The crude was then recrys-
tallized in methanol to obtained pure product (Hanna et al.,
2007; Ju et al., 2019; Liu et al., 2019; Mirfazli et al., 2016).
method as described by Weatherburn. Briefly 70 mL of alkali
reagent (0.5%w/v NaOH and 0.1% active chloride NaOCl) and
45 mL of phenol reagent (1% w/v phenol and 0.005% w/v
sodium nitroprusside) were added to each other well. After
50 min increasing absorbance at 630 nm was measured,
using microplate reader (Molecular device, USA). All reactions
were performed in triplicate in a final volume of 200 mL. The
results (change in absorbance per min) were processed by
using software Soft Max Pro (molecular device, USA). Entire
assay was performed at PH 6.8. percent inhibition was calcu-
lated from the formula below.
% inhibition ¼ 100 -(ODtest/ODcontrol) ꢃ 100.
Thiourea was used as standard inhibitor for urease (Wani
et al., 2019).
4.3. 2-(1h-indol-3-yl)-N’-(4-
nitrobenzylidene)acetohydrazide (1)
Yield: (88%); mp 289–291 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR;
(400 MHz, DMSO-d6): d 11.70 (s, 1H, NH), 11.52 (s, 1H, NH),
8.70 (s, 1H), 8.36 (d, 2H, J ¼ 8.0 Hz), 7.89 (d, 2H, J ¼ 8.0 Hz),
7.60 (t, 1H, J ¼ 7.0 Hz), 7.22 (d, 1H, J ¼ 7.5 Hz), 6.92–6.86 (m,
4. Material and method
4.1. General experiment
Nuclear Magnetic Resonance (NMR) analysis was performed
on JNM ꢁ ECS 400 MHz spectrometer. High Resolution
Electron Impact Mass Spectra (HREI-MS) were carried on
Finnigan MAT-311A (Germany) mass spectrometer. Pre-
coated silica gel aluminium plates (Kieselgel 60, 254, Merck,
Germany) were used for Thin Layer Chromatography (TLC).
Ultraviolet Visible (UV) spectroscopy was conceded to visual-
ize the chromatogram at 254 and 365 nm.
3H), 3.69 (s, 2H, CH2);13CNMR (100, MHz, DMSO)
d
171.0,150.5, 144.3, 139.5, 136.3, 127.2, 125.0, 125.0, 124.2,
124.2, 123.5, 121.4, 119.5,118.6, 111.2, 109.5, 36.0, HR-EIMS
m/z, calcd for C17H14N4O3 [M]þ 322.1066 found 322.1053.
4.4. N’-(2,4-dihydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide (2)
Yield: (89%); mp 279–281 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.62 (s, 1H, NH), 11.28 (s, 1H, NH),
11.28 (s, 2H, OH), 7.75 (m, 1H), 7.34 (t, 2H, J ¼ 7.0 Hz), 7.22 (d,
4.2. General procedure for the synthesis of indole
derivatives (1–22)
Indole derivatives were synthesized by refluxing methyl 2-
(1H-indol-3-yl) acetate (5 mmol) with hydrazine hydrate 2H, J ¼ 7.5 Hz), 6.92–6.86 (m, 2H), 3.39 (s, 2H, CH2);13CNMR

12
M. TAHA ET AL.
(100, MHz, DMSO) d 171.0, 162.1, 162.1, 146.2, 136.3, 133.5,
127.2, 123.3, 122.0, 120.3, 120.0, 119.0,111.5, 111.0, 109.0,
103.5, 36.0, HR-EIMS m/z, calcd for C17H15N3O3 [M]þ 309.1113
found 309.1102.
4.9. N’-(3-hydroxybenzylidene)-2-(1H -indol-3-
yl)acetohydrazide (7)
Yield: (82%); mp 261–263 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.46 (s, 1H, NH), 11.21 (s, 1H, NH),
9.42 (s, 1H, OH), 8.08 (s, 1H), 7.88 (s, 1H), 7.79 (d, 2H,
J ¼ 8.0 Hz), 7.32 (t, 1H, J ¼ 7.0 Hz), 7.24–7.15 (m, 3H),
6.80–6.76 (m, 1H), 3.48 (s, 2H, CH2);13CNMR (100, MHz,
DMSO) d 171.0, 158.5, 146.2, 138.5,136.1, 130.3, 127.2, 123.3,
121.5, 121.3, 119.8, 118.5, 118.0, 114.8, 111.0, 109.0, 36.0, HR-
EIMS m/z, calcd for C17H14N3O2 [M]þ 293.1164
found 293.1152.
4.5. N’-(3,5-dihydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide (3)
Yield: (84%); mp 284–286 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.50 (s, 1H, NH), 11.12 (s, 1H, NH), 10.90 (s, 2H, OH), 7.92 (s,
1H), 7.72 (s, 1H), 7.35 (d, 2H, J ¼ 8.0 Hz), 7.27 (t, 2H,
J ¼ 7.0 Hz), 6.92–6.86 (m, 3H), 3.69 (s, 2H, CH2);13CNMR (100,
MHz, DMSO) d171.0, 160.5, 160.5, 146.2, 136.3, 136.3, 127.2,
123.3, 122.0, 120.3, 119.0, 111.0, 109.0, 107.2, 107.2, 105.6,
36.0, HR-EIMS m/z, calcd for C17H15N3O3 [M]þ 309.1113 found
309.1101.
4.10. 2-(1 h-indol-3-yl)-N’-(2,4,6-
trihydroxybenzylidene)acetohydrazide (8)
Yield: (80%); mp 294–296 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.62 (s, 1H, NH), 10.92 (s, 2H, NH, OH), 10.26 (s, 2H, 2xOH),
8.32 (s, 1H), 7.64 (m, 2H), 7.42 (t, 2H, J ¼ 7.0 Hz), 7.13 (d, 1H,
J ¼ 7.5 Hz), 6.62–6.86 (m, 2H), 3.69 (s, 2H, CH2);13CNMR (100,
MHz, DMSO) d 171.0, 164.0, 164.0, 163.7, 143.5, 136.1, 127.2,
123.3, 121.5, 119.5, 118.6, 111.0, 109.0, 106.4, 96.5, 96.5, 36.0,
HR-EIMS m/z, calcd for C17H15N3O4 [M]þ 325.1063
found 325.1051.
4.6. 2-(1h-indol-3-yl)-N’-(2-
methoxybenzylidene)acetohydrazide (4)
Yield: (87%); mp 229–231 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.64 (s, 1H, NH), 11.32 (s, 1H, NH),
8.35 (s, 1H), 7.94(s, 1H), 7.89 (d, 1H, J ¼ 8.0 Hz), 7.71 (t, 1H,
J ¼ 7.0 Hz), 7.52 (t, 2H, J ¼ 7.5 Hz), 6.85–6.80 (m, 4H), 3.92 (s,
3H, CH3), 3.69 (s, 2H, CH2);13CNMR (100, MHz, DMSO) d 171.0,
157.5, 146.2, 136.1, 132.1, 131.5, 127.2, 123.3, 122.0, 121.5,
120.3,119.0, 116.8, 111.2, 111.0, 109.0, 55.6, 36.0, HR-EIMS m/
z, calcd for C18H17N3O2 [M]þ 307.1321 found 307.1313.
4.11. N’-(4-(dimethylamino)benzylidene)-2-(1H-indol-3-
yl)acetohydrazide (9)
Yield: (80%); mp 295–296 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.18 (s, 1H, NH), 10.93 (s, 1H, NH), 8.09 (s, 1H), 7.84(s, 1H),
7.54–7.47 (m, 2H), 7.41–7.32 (m, 2H), 7.29 (t, 2H, J ¼ 7.0 Hz),
7.16 (d, 1H, J ¼ 7.0 Hz), 3.54 (s, 2H, CH2), 2.96 (s, 6H,
2XCH3),13CNMR (100, MHz, DMSO) d 171.0, 153.5, 144.3,
136.1, 128.5, 128.5, 127.2, 123.6, 123.3, 121.5, 120.0, 119.5,
118.6, 112.0, 111.0, 109.0, 41.5, 41.5, 36.0, HR-EIMS m/z, calcd
for C19H20N4O [M]þ320.1637 found 320.1624.
4.7. N’-(3,4-dihydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(5)
Yield: (80%); mp 251–253 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.29 (s, 1H, NH), 11.05 (s, 1H, NH), 10.92 (s, 1H, OH), 10.87
(s, 1H, OH), 7.95 (s, 1H), 7.74 (s, 1H), 7.53(d, 2H, J ¼ 8.0 Hz),
7.36 (d, 2H, J ¼ 8.0 Hz), 6.87–6.83 (m, 3H), 3.49 (s, 2H,
CH2);13CNMR (100, MHz, DMSO) d 171.0, 149.3, 146.8, 146.2,
136.1, 131.5, 127.2, 123.3, 123.0, 121.5, 120.3, 119.0, 117.5,
116.8, 111.0, 109.0, 36.0, HR-EIMS m/z, calcd for C17H15N3O3
[M]þ 309.1113 found 309.1104.
4.12. 2-(1 h-indol-3-yl)-N’-(2-
nitrobenzylidene)acetohydrazide(10)
Yield: (86%); mp 272–274 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.84 (s, 1H, NH), 11.54 (s, 1H, NH),
8.60 (s, 1H), 8.07–7.94 (m, 3H), 7.75–7.71(m, 2H), 7.54–7.50
(m, 2H), 7.02–6.96 (m, 2H), 3.56 (s, 2H, CH2);13CNMR (100,
MHz, DMSO) d 171.0, 148.0,143.5, 136.1, 134.7, 131.6, 130.4.
128.5, 127.2, 124.2,123.3, 121.5, 119.5, 118.6,111.0, 109.0,
36.0, HR-EIMS m/z, calcd for C17H14N4O3 [M]þ 322.1066 found
322.1055.
4.8. N’-(2,5-dihydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide (6)
Yield: (88%); mp 265–266 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.71 (s, 1H, NH), 11.18 (s, 1H, NH),
10.27 (s, 1H, OH), 9.92 (s, 1H, OH), 7.95 (s, 1H), 7.60 (t, 1H,
J ¼ 7.0 Hz), 7.34–30 (m, 2H), 7.12 (t, 2H, J ¼ 8.0 Hz), 6.99–6.92
(m, 3H), 3.69 (s, 2H, CH2);13CNMR (100, MHz, DMSO) d 171.0,
153.5, 151.3, 146.2, 136.1, 127.2, 123.3, 121.5, 120.3, 120.0,
119.8, 119.7, 119.0, 116.5, 111.0, 109.0, 36.0, HR-EIMS m/z,
calcd for C17H15N3O3 [M]þ 309.1113 found 309.1124.
4.13. 2-(1 h-indol-3-yl)-N’-(3-
nitrobenzylidene)acetohydrazide(11)
Yield: (85%); mp 278–281 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.89 (s, 1H, NH), 11.64 (s, 1H, NH),
8.54 (s, 1H), 8.34 (s, 1H), 8.07–7.94 (m, 3H), 7.79 (t, 2H,

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
J ¼ 8.0 Hz), 7.60 (t, 1H, J ¼ 7.0 Hz), 6.92–6.86 (m, 2H), 3.58 (s,
2H, CH2);13CNMR (100, MHz, DMSO) d 171.0, 148.2, 143.0,
136.1, 134.5, 132.8, 129.5, 127.2, 126.4, 123.3, 122.0, 121.6,
120.0, 119.5, 111.0, 109.0, 36.0, HR-EIMS m/z, calcd for
C17H14N4O3 [M]þ 322.1066 found 322.1053.
4.18. 2-(1 h-indol-3-yl)-N’-(4-
methylbenzylidene)acetohydrazide(16)
Yield: (84%); mp 232–233 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.52 (s, 1H, NH), 11.18 (s, 1H, NH), 8.16 (s, 1H), 7.84 (d, 2H,
J ¼ 8.0 Hz), 7.69–7.62 (m, 4H), 7.06 (t, 1H, J ¼ 7.0 Hz), 6.94 (d,
2H, J ¼ 8.0 Hz), 3.68 (s, 2H, CH2), 2.26 (m, 3H);13CNMR (100,
MHz, DMSO) d 171.0, 144.3, 140.3, 136.8, 130.5, 129.4, 129.4,
127.2, 126.5, 126.5, 123.3, 121.6, 119.5, 118.6, 111.0, 109.0,
36.0. 21.5, HR-EIMS m/z, calcd for C18H17 N3O [M]þ 291.1372
found 291.1356.
4.14. N’-(4-hydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(12)
Yield: (82%); mp 296–298 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.29 (s, 1H, NH), 11.05 (s, 1H, NH),
9.89 (s, 1H, OH), 8.08 (s, 1H),. 7.57–7.44 (m, 4H), 7.42 (t, 2H,
J ¼ 7.0 Hz), 7.26 (d, 2H, J ¼ 7.5 Hz), 6.97–6.94 (m, 1H), 3.64 (s,
2H, CH2);13CNMR (100, MHz, DMSO) d 171.0, 160.5, 144.3,
136.1, 130.4, 130.4, 127.2,126.5, 123.3, 121.6, 119.5, 118.6,
116.1, 116.1, 111.0, 109.0, 36.0, HR-EIMS m/z, calcd for
C17H14N3O2 [M]þ 293.1164 293.1164 found 293.1151.
4.19. N’-(3-chlorobenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(17)
Yield: (85%); mp 288–291 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.65 (s, 1H, NH), 11.45 (s, 1H, NH),
8.16 (s, 1H), 7.74 (d, 2H, J ¼ 8.0 Hz),7.50–7.42 (m, 4H),
7.24–7.18 (m, 2H), 3.62 (s, 2H, CH2);13CNMR (100, MHz,
DMSO) d 171.0, 146.5, 136.8, 135.4, 134.6, 131.0, 130.5, 127.2,
127.2, 127.0, 123.3, 121.6, 119.5, 118.6, 111.0, 109.0, 36.0, HR-
EIMS m/z, calcd for C17H14 Cl N3O [M]þ 311.0825
found 311.0814.
4.15. N’-(4-chlorobenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(13)
Yield: (82%); mp 265–267 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.64 (s, 1H, NH), 11.44 (s, 1H, NH),
8.14 (s, 1H), 7.96 (s, 1H), 7.76–7.68 (m, 3H), 7.76–7.68 (m, 3H),
7.53–7.45 (m, 4H), 6.88 (t, 1H, J ¼ 7.0 Hz), 3.67 (s, 2H,
CH2);13CNMR (100, MHz, DMSO) d 171.0, 144.3, 136.8, 136.4,
131.5, 130.5, 130.5, 128.4, 128.4, 127.2, 123.3, 121.6, 119.5,
118.6, 111.0, 109.0, 36.0, HR-EIMS m/z, calcdC17H14ClN3O for
[M]þ 311.0825 found 311.0811.
4.20. N’-(2-hydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(18)
Yield: (82%); mp 256–259 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.82 (s, 1H, NH), 11.23 (s, 1H, NH),
10.91 (s, 1H, OH), 8.14 (s, 1H), 7.57–7.49 (m, 2H), 7.46 (d, 1H,
J ¼ 7.0 Hz), 7.38–7.35 (m, 2H), 7.4–6.97 (m, 3H), 3.67 (s, 2H,
CH2);13CNMR (100, MHz, DMSO) d 171.0, 157.5, 146.3, 136.8,
132.0, 127.5, 127.2, 123.3, 121.6, 121.4, 119.5, 118.6, 118.4,
117.6, 111.0, 109.0, 36.0, HR-EIMS m/z, calcd for C17H14N3O2
[M]þ 293.1164 found 293.1153.
4.16. Methyl 4-((2-(2-(1H-indol-3-
yl)acetyl)hydrazono)methyl)benzoate(14)
Yield: (87%); mp 302–304 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.72 (s, 1H, NH), 11.43 (s, 1H, NH), 8.33 (s, 1H), 7.98–7.92 (m,
2H), 7.63 (d, 1H, J ¼ 7.0 Hz), 7.22 (d, 2H, J ¼ 8.0 Hz), 7.02 (t,
2H, J ¼ 7.0 Hz), 3.82 (s, 3H, CH3), 3.62 (s, 2H, CH2);13CNMR
(100, MHz, DMSO) d 171.0, 165.6, 144.3, 138.2, 136.8, 132.8,
130.3, 130.3, 129.4, 129.4, 127.2, 123.3, 121.6, 119.5, 118.6,
111.0, 109.0, 51.2, 36.0, HR-EIMS m/z, calcd for C19H17 N3O3
[M]þ 335.1270 found 335.1257.
4.21. 2-(1 h-indol-3-yl)-N’-(3-
methylbenzylidene)acetohydrazide(19)
Yield: (83%); mp 226–228 ꢂC; 1HNMR (400 MHz, DMSO-d6):
d11.51 (s, 1H, NH), 11.32 (s, 1H, NH), 8.14 (s, 1H), 7.53–7.49
(m, 3H), 7.38–7.35 (m, 4H), 6.84–6.86 (m, 2H), 3.57 (s, 2H,
CH2), 2.25 (s, 3H, CH3); 13CNMR (100, MHz, DMSO) d 171.0,
146.5, 138.2, 136.8, 133.5, 131.8, 129.6, 128.4, 127.2, 126.6,
123.3, 121.6, 119.5, 118.6,111.0, 109.0, 36.0, 21.5, HR-EIMS m/
z, calcd for C18H17 N3O [M]þ 291.1372 found 291.1359.
4.17. 2-(1 h-indol-3-yl)-N’-(2-
methylbenzylidene)acetohydrazide(15)
4.22. N’-(3-hydroxy-4-methoxybenzylidene)-2-(1H-indol-
3-yl)acetohydrazide (20)
Yield: (83%); mp 224–226 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.46 (s, 1H, NH), 11.16 (s, 1H, NH), 8.54 (s, 1H), 8.35 (s, 2H),
7.83–7.780 (m, 2H), 7.32–7.25 (m, 5H), 6.92–6.88 (m, 1H), 3.52
(s, 2H, CH2), (s, 3H, CH3); 13CNMR (100, MHz, DMSO) d 171.0,
143.0, 136.8, 135.5, 131.3, 130.5, 129.3, 127.2, 126.7, 125.9,
123.3, 121.6, 119.5, 118.6, 111.0, 109.0, 36.0, 19.0, HR-EIMS m/
z, calcd for C18H17 N3O [M]þ 291.1372 found 291.1359.
Yield: (81%); mp 248–250 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.06 (s, 1H, NH), 11.32 (s, 1H, NH),
9.48 (s, 1H, OH), 8.05 (s, 1H), 7.53 (d, 1H, J ¼ 7.0 Hz), 7.32–7.28
(m, 4H), 7.03–6.92 (m, 3H), 3.82 (s, 3H, CH3), 3.54 (s, 2H,
CH2);13CNMR (100, MHz, DMSO) d 171.0, 152.6, 147.6, 146.5,

14
M. TAHA ET AL.
136.8, 131.2, 127.2, 123.3, 122.0, 121.6, 119.5, 118.6, 115.7, based on their number, position and nature on phenyl ring
112.5, 111.0, 109.0, 56.3, 36.0. HR-EIMS m/z, calcd for has been elaborated through structure activity relationship
C18H17N3O3 [M]þ 323.1270 found 323.1257.
(SAR) study.
4.23. N’-(2,4-dimethoxybenzylidene)-2-(1H-indol-3-
Acknowledgements
yl)acetohydrazide(21)
The authors acknowledged the financial supports By Deanship of scien-
tific research through Project no. 2019-211-IRMC, Imam Abdulrahman
Bin Faisal University (IAU), Saudi Arabia.
Yield: (83%); mp 304–306 ꢂC; 1HNMR (400 MHz, DMSO-d6): d
11.43 (s, 1H, NH), 11.12 (s, 1H, NH), 8.43 (s, 1H), 7.74 (d, 1H,
J ¼ 7.5 Hz), 7.65 (d, 1H, J ¼ 7.5 Hz), 7.56–7.51 (m, 2H), 7.10 (d,
2H, J ¼ 7.5 Hz), 6.59–6.53 (m, 2H), 3.81 (s, 6H, 2xCH3), 3.56 (s,
2H, CH2);13CNMR (100, MHz, DMSO) d 171.0, 163.4, 159.1,
146.5, 136.8, 133.2, 127.2, 123.3, 121.6, 119.5, 118.6, 111.0,
109.3, 109.0, 106.5, 101.8, 56.3, 56.3, 36.0, HR-EIMS m/z, calcd
for C19H19N3O3 [M]þ 337.1426 found 337.1413.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Muhammad Taha
Syahrul Imran
Noor Barak Almandil
Munther Alomari
http://orcid.org/0000-0003-3047-9289
http://orcid.org/0000-0001-7802-7359
http://orcid.org/0000-0002-7049-3563
http://orcid.org/0000-0001-5705-2926
4.24. N’-(2,3-dihydroxybenzylidene)-2-(1H-indol-3-
yl)acetohydrazide(22)
Yield: (85%); mp 229–231 ꢂC match (Hanna et al., 2007; Ju
et al., 2019; Liu et al., 2019; Mirfazli et al., 2016); 1HNMR
(400 MHz, DMSO-d6): d 11.74 (s, 2H, 2xNH), 11.26 (s, 1H, OH),
9.42 (s, 1H, OH) 8.24 (s, 1H), 7.62–7.58 (m, 2H), 7.52–7.47 (m,
4H), 7.13–7.08 (m, 2H), 3.55 (s, 2H, CH2);13CNMR (100, MHz,
DMSO) d 171.0, 151.5, 147.0, 146.5, 136.8, 127.2, 124.6, 123.3,
123.0, 121.6, 120.0, 120.0, 119.5, 118.6, 111.0, 109.0, 36.0, HR-
EIMS m/z, calcd for C17H15N3O3 [M]þ 309.1113
found 309.1103.
Fazal Rahim
Bandar Alghanem
http://orcid.org/0000-0001-5856-4886
http://orcid.org/0000-0002-3414-6580
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In conclusion indole analogues (1–22) have been synthesized
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