Isatin based Schiff bases as inhibitors of α-glucosidase: Synthesis, characterization, in vitro evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2015.03.005

Isatin base Schiff bases (1-20) were synthesized, characterized by ¹H NMR and EI/MS and evaluated for α-glucosidase inhibitory potential. Out of these twenty (20) compounds only six analogs showed potent α-glucosidase inhibitory potential with IC₅₀ value ranging in between 2.2 ± 0.25 and 83.5 ± 1.0 μM when compared with the standard acarbose (IC₅₀ = 840 ± 1.73 μM). Among the series compound 2 having IC₅₀ value (18.3 ± 0.56 μM), 9 (83.5 ± 1.0 μM), 11 (3.3 ± 0.25 μM), 12 (2.2 ± 0.25 μM), 14 (11.8 ± 0.15 μM), and 20 (3.0 ± 0.15 μM) showed excellent inhibitory potential many fold better than the standard acarbose. The binding interactions of these active analogs were confirmed through molecular docking.

Full text of DOI:10.1016/j.bioorg.2015.03.005

Accepted Manuscript
Isatin based Schiff bases as Inhibitors of α-glucosidase: Synthesis, Characteri-
zation, In Vitro Evaluation and Molecular Docking Studies
Fazal Rahim, Fazal Malik, Hayat Ullah, Abdul Wadood, Fahad Khan,
Muhammad Tariq Javid, Muhammad Taha, Wajid Rehman, Ashfaq Ur Rehman,
Khalid Mohammed Khan
PII:
S0045-2068(15)00031-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.03.005
YBIOO 1803
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
5 January 2015
Please cite this article as: F. Rahim, F. Malik, H. Ullah, A. Wadood, F. Khan, M.T. Javid, M. Taha, W. Rehman,
A.U. Rehman, K.M. Khan, Isatin based Schiff bases as Inhibitors of α-glucosidase: Synthesis, Characterization, In
Vitro Evaluation and Molecular Docking Studies, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bioorg.2015.03.005
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Isatin based Schiff bases as Inhibitors of α-glucosidase: Synthesis, Characterization, In
Vitro Evaluation and Molecular Docking Studies
a
a
a
b
a
Fazal Rahim, Fazal Malik, Hayat Ullah, Abdul Wadood, Fahad Khan, Muhammad Tariq
a
c
a
b
d
Javid, Muhammad Taha, Wajid Rehman, Ashfaq Ur Rehman, and Khalid Mohammed Khan
a
Depatment of Chemistry, Hazara University, Mansehra-21120, Pakistan.
b
Department of Biohemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
c
Atta-ur-Rahman Institute for Natural Product Discovery, UniversitiTeknologi MARA (UiTM),
PuncakAlam Campus, 42300 Bandar PuncakAlam, Selangor, Malaysia,
d
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan.
Abstract:
1
Isatin base Schiff bases (1-20) were synthesized, characterized by HNMR and EI/MS and
evaluated for α-glucosidase inhibitory potential. Out of these twenty (20) compounds only six
analogs showed potent α-glucosidase inhibitory potential with IC50 value ranging in between 2.2
±
0.25 to 83.5 ± 1.0µM when compared with the standard acarbose (IC50 = 840±1.73 µM).
Among the series compound 2 having IC50 value (18.3 ± 0.56 µM), 9 (83.5 ± 1.0 µM), 11 (3.3 ±
.25 µM), 12 (2.2 ± 0.25 µM), 14 (11.8 ± 0.15 µM), and 20 (3.0 ± 0.15µM) showed excellent
0
inhibitory potential many fold better than the standard acarbose. The binding interactions of
these active analogs were confirmed through molecular docking.
Keywords: Isatin, Schiff bases, Synthesis, α-Glucosidase Inhibition, Molecular Docking.
*Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343;
1

1
. Introduction
α-Glucosidase enzyme is located in the brush-border surface membrane of intestinal cells,
responsible for catalyzing the final step in the digestive process of carbohydrates [1]. It
specifically hydrolyzed the α-glucopyranoside bond, thereby releasing α-D-glucose from the
non-reducing end of the sugar [2].The inhibitors of this enzyme have applications in modern
medicinal chemistry. Its might be used to treat diabetics, HIV and cancers [3-5].The biological
importance of α-glucosidase inhibitors ensures its uses as a vital tool to understand the
mechanisms of action of α-glucosidase, and as therapeutic agents for some degenerative diseases
[
6-8]. Its inhibitors like acarbose, voglibose, and miglitol are clinically used in the effective
treatment of type-2 diabetes mellitus [9].
Isatin heterocycles acts as a potent endogenous neurochemical regulator in mammalian brain [10,
1
1]. It’s concentration in urine become a diagnostic marker for the clinical severity of
Parkinson’s disease in humans [12, 13]. It is a component of tribulin, a selective inhibitor of
monoamine oxidase-B enzyme [14]. Isatin is well protective against certain types of infections
[
[
15]. Its derivatives exhibited activities like antibacterial [16], anti-inflammatory [17], analgesic
18], anti-viral [19], antifungal [20], anti-tubercular [21] and anti-depressant [22].Schiff bases of
isatin derivatives have been reported as antiprotozoal, antibacterial, anti-HIV, anticonvulsant,
antitumor, and anthelminthic activities, influence neurodegenerative diseases, participate in
metabolism, acetyl cholinesterase inhibitors, and stimulate plants growth [23,24].
Herein we are going to report synthesis of isatin based Schiff bases, their α-glucosidase
inhibitory potentials, SAR and molecular docking studies.
2
2
. Result and Discussion
.1. Chemistry
Isatin based Schiff bases (1-20) were synthesized in three steps, first an esterification carried out
by reacting different carboxylic acid with methanol in sulphuric acid (2-3ml) under reflux
condition for 12-16 hrs. The completion of reaction was monitored by TLC. After completion of
reaction, reaction mixture was extracted with hexane to obtained pure esters. Then esters were
refluxed with hydrazine hydrate in methanol with few drops of glacial acetic acid for 3 h. After
completion of reaction, reaction mixture was washed with chloroform to obtained different
hydrazides. These hydrazides (1mmole) each were than treated with different isatin (1mmole) in
methanol having catalytic amount of glacial acetic acid for 2-4h. Reaction completion was
2

monitored through periodic TLC. After completion of reaction, reaction mixture were washed
with n-hexane to obtain our desired products (1-20).
Insert Scheme-1 Here
Insert Table-1 Here
2
.2. Biological activity
Compounds 1-20 showed variable degree of α-glucosidase inhibitory potential when compared
with standard acarbose (IC50 value 840±1.73µM). Among the series only compounds 2, 9, 11,
1
2, 14 and 20 showed potent inhibitory potential with IC50 values of 18.3 ± 0.56, 83.5 ± 1.0, 3.3
0.25, 2.2 ± 0.25, 11.8 ± 0.15 and 3.0 ± 0.15 µM respectively. The most active compounds are
±
1
1 (IC50 3.3 ± 0.25) with 3, 4-dichloro substitution at phenyl part of hydrazide and 12 (IC50 2.2 ±
0
.25) with 2,4-dichloro substitution at phenyl part of hydrazide and 6-bromo on phenyl part of
isatin. The compound 2 (IC50 18.3 ± 0.56) with di-chloro substitution at phenyl part of hydrazide
but have no bromo group at isatin and have extra alkyl group at nitrogen of isatin. The slight
activity difference among the two compounds 11 and 12 is may be due to difference in position
of chloro groups, while compound 2 have some decline in activity which might be due to extra
alkyl group at isatin nitrogen. Compound 9 (IC50 83.5 ± 1.0) with 5-chloro substitution and 14
(
IC50 11.8 ± 0.15) with 6-bromo substitution on the phenyl part of isatin also showed potent
inhibitory activity as compared to standard acarbose. Compounds 20 was unique having no
substitution at phenyl group but also showed potent inhibitory activity.
The results showed that the compounds having more electron withdrawing groups i.e. chloro or
bromo such as 11 and 12 are more potent inhibitor, while those with less number of electron
withdrawing groups 2, 9 and 14 show comparatively less potent inhibitory activity. The binding
interaction of these compounds was confirmed through molecular docking.
2
.3. Homology modeling and docking simulation
The crystallographic structure for α-glucosidase enzyme has not been solved up-to yet. However,
only few homology models have been reported [25-28] so, we developed the 3D model for α-
glucosidase by comparative homology modeling technique using the same etiquette as described
by Burke et al [29]. The primary sequence of α-glucosidase was retrieved from UniProt protein
3

resource data bank (http://www.uniprot.org/) under the access code P53341. Template search
was performed by means of MOE-Search tools against the PDB data bank implemented in MOE
2
010.11. The 1.30 Å resolving crystallographic structure of Saccharomyces cerevisiae
isomaltase (PDB code 3AJ7,) [30] with 72.4% of sequence identity with the target were selected
as the template for modeling. The 3D structure of α-glucosidase for Saccharomyces cerevisiae
was built by means of MOE homology modeling tools. The developed 3D model was subjected
to energy minimization up to 0.05 gradients. Before docking, ligands and protein were prepared
using MOE 2010.11. 3D structure of all synthesized compounds was built using the Molecular
Builder program implemented in MOE. Finally, a database was created in which all the ligands
were converted into their respective 3D structures and this database was used as input file MOE-
docking. Subsequently, the energy of compounds present in the database was minimized up to
0
.05 Gradient using MMFF94x force field. Energy minimization of the database was followed by
the preparation of protein for docking purposes. Most macromolecular crystal structures contain
little or no hydrogen coordinate data due to limited resolution and thus protonation was
accomplish prior to docking using Protonate 3D tools implemented in MOE. Protonation was
followed by energy minimization up to 0.05 Gradient using Amber99 force field. The database
was docked into the active site of protein using the Triangular Matching docking method and 30
conformations of each Ligand protein complex were generated with docking score (S). Each
complex was analyzed for interactions and their 3D images were taken.
2
.4. Molecular docking
From the docking simulation and in vitro validation study, it was observed that compound 12
IC50 = 2.2±0.25 µM) are the most active analog among the series which are mention in this
(
study. Compound 20 (IC50 3.0±0.15 µM) also the second ranked most active compound against
α-glucosidase inhibitory assay. These compounds were bound extremely into the binding cavity
of α-glucosidase making interactions with the active site residues Glu276, His239 and Asp349
as described by Khan, M et al [31].
The most active compound 12 is fit intensely into the catalytically active residues of the enzyme
making interactions with the most important active site residues Arg212, His239, Glu276 and
Thr307 (Figure-01). Arg212 was found in hydrogen interaction with the oxygen of indolenone
group. His239 make π-interaction with the Di-chloro substituted phenyl ring. Glu276 establish
4

interaction with the hydrogen of the amino group of the tested compound 12. Thr307 was found
in interaction with the oxygen of carbonyl group of the compound [32].
Insert Figure-1 Here
Similarly compound 20 also show best inhibitory activity against α-glucosidase enzyme and
establish interaction with the active site residue which is readily involved in inhibition Phe157
and Arg212 (Figure-02). Phe157 was found in making interaction with the hydrogen next attach
to the nitrogen group and Arg212 establish two interactions with the compound, one with the
oxygen of the indolenone group and other with the nitrogen of the hydrazide group.
Insert Figure-2 Here
Compound 11 is ranked third most active compound among the series, which have IC50 value 3.3
±
1.0. This compound established two hydrogen interactions with the active site residues of the
α-glucosidase enzyme Asn241 and His239. Asn241 is most important residue and involved in
inhibition, here found in making interaction with the nitrogen (Figure-03). His239 make
hydrogen interaction with the hydrogen next attach to nitrogen of the hydrazide moiety.
Insert Figure-3 Here
Similarly compound 14 and 02 are involved in making interaction with the active site residue
Arg212 and His245 (Figure-04), and all the remaining profile of the compounds reflects no
interaction (e.g. compound 01, as shown in Figure-04), therefore these compounds were not
further evaluated for further study.
Insert Figure-4 Here
2
.5. α-Glucosidase assay
α-Glucosidase inhibitory activities was determined as per reported methods [33].10 µL of test
samples (5 mg/mL DMSO solution)were reconstituted in 100 µL of 100 mM-phosphate buffer
(
pH6.8) in 96-well microplate and incubated with 50 µL of crude intestinal α-glucosidase for 5
min before 50 µL substrate (5 mM, p-nitrophenyl-α-D-glucopyranoside prepared in same buffer)
was added. Release of p-nitrophenol was measured at 405 nm spectrophotometrically
®
(
SpectraMax plus384) for5 min after incubation with substrate. Individual blanks for test
5

samples were prepared to correct background absorbance where substrate was replaced with 50
µL of buffer. Control sample contained 10 µL DMSO in place of test samples. Percentage of
ꢀ
enzyme inhibition was calculated as (1- ) x 100 whereA represents absorbance of control
ꢁ
without test samples, and B represents absorbance in presence of test samples.
3
. Conclusion
Twenty compounds were synthesized and evaluated for α-glucosidase inhibition. Six analogs
showed varied α-glucosidase inhibitory potential with IC50 value ranging between 2.2 ± 0.25 to
8
3.5 ± 1.0 µM when compared with standard acarbose (IC50 = 840 ± 1.73 µM). Compound 2 IC50
value (18.3 ± 0.56 µM), 9 (83.5 ± 1.0 µM), 11 (3.3 ± 0.25 µM), 12 (2.2 ± 0.25 µM), 14 (11.8 ±
.15 µM), and 20 (3.0 ± 0.15 µM) showed excellent inhibitory potential much better than the
0
standard acarbose. The binding interaction of these active compounds was confirmed through
molecular docking. All other analogs were found inactive.
4
4
. Experimental Section
.1. General methods
Finnegan MAT-311A, Germany, spectrometer was used for electron impact mass spectra
EI/MS) analysis. An internal standard cesium iodide (CsI) was used for mass measurement.
(
Advance Bruker AM 300 MHZ and AMX-400 MHZ spectrometers have used for NMR analysis.
DMSO and acetone were used for accurate NMR analysis. All the above characterizations were
performing at H.E.J Research Institute of Chemistry, Karachi University Pakistan. Column
chromatography was performed on silica gel (E. Merck, type 60, 70-230 mesh). Pre-coated silica
gel aluminum plates (Kieselgel 60, 2020 and 0.5 mm thick, E. Merck, Germany) were used for
TLC analysis. Light of wavelength 254 and 265 nm were used to visualize the chromatogram.
4
.2. General Procedure for the synthesis of isatin based Schiff bases derivatives (1-20)
Isatin based Schiff bases (1-20) were synthesized in three steps, first an esterification carried out
by reacting different carboxylic acid with methanol in sulphuric acid (2-3ml) under reflux
6

condition for 12-16 hrs. The completion of reaction was monitored by TLC. After completion of
reaction, reaction mixture was extracted with hexane to obtained pure esters. Then esters were
refluxed with hydrazine hydrate in methanol with few drops of glacial acetic acid for 3 h. After
completion of reaction, reaction mixture was washed with chloroform to obtained different
hydrazides. These hydrazides (1mmole) each were than treated with different isatin (1mmole) in
methanol having catalytic amount of glacial acetic acid for 2-4h. Reaction completion was
monitored through periodic TLC. After completion of reaction, reaction mixture was washed
with n-hexane to obtain our desired products (1-20). The structure of all compounds was
1
established through EI-MS and HNMR.
4
.2.1. (E)-2-hydroxy-N'-(2-Oxo-1-propylindolin-3-ylidene) benzohydrazide (1)
1
Yield:79%; H-NMR: (DMSO-d
6
, 300 MHz): δ 14.3 (s, 1H, NH), 12.2 (s, 1H, OH), 8.0 (dd, J7,5
=
),
1
.5 Hz, J7,6 = 8.1 Hz,1H, H-7), 7.99 (d, J6’,5’ = 7.5 Hz,1H,H-6’), 7.51 (m, 2H, H-4/6), 7.2(d, J3’,4’
=
7.8 Hz,1H, H-3’), 7.15 (m, 1H, H-5’), 7.0 (m, 1H, H-5), 6.9 (m, 1H, H-4’), 3.71 (t, 2H, NCH
2
+
1
.64 (m, 2H, NCH
2
CH
2
), 0.91 (t, 3H, CH
2
CH
3
).; EI-MS: m/z (rel. int. %): 323 (M , 80), 308
(
69), 230 (100), 94 (35);
4
.2.2. (E)-2-(2,4-Dichlorophenyl)-N'-(2-oxo-1-propylindolin-3-ylidene)acetohydrazide (2)
1
Yield:82%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 8.0 (d, J3’,5’ = 1.2 Hz,1H, H-3’),
7
.7(d, J4,5 = 7.5 Hz,1H, H-4), 7.6 (m, 3H, H-6/4’/5), 7.2 (d, J6’,5’ = 8.1 Hz, 1H, H-6’), 7.1 (m, 1H,
H-3), 3.7 (t, 2H, NCH
2
), 1.6 (m, 2H, NCH
2
CH
2
), 0.9 (t, 3H, CH
2
CH ),; EI-MS: m/z (rel. int. %):
3
+
3
89 (M , 60), 374 (100), 250 (78), 93 (44);
4
.2.3. (E)-2-chloro-N'-(2-Oxo-1-propylindolin-3-ylidene) benzohydrazide (3)
1
Yield:82%; H-NMR: (DMSO-d
6
, 300 MHz):δ13.3 (s, 1H, NH), 7.6 (m, 4H, H-4/7/3’/6), 7.5 (m,
), 1.6 (m, 2H, NCH CH ),
),; EI-MS: m/z (rel. int. %): 341 (M , 35), 202 (100), 174 (55), 139 (58);
2
0
H, H-5/5’), 7.2 (d, J6’,5’/4’,5’= 7.8 Hz, 2H, H-6’/4’), 3.6 (t, 2H, NCH
2
2
2
+
.9 (t, 3H, CH
2
CH
3
4
.2.4. (E)-3-methyl-N'-(2-Oxo-1-propylindolin-3-ylidene) benzohydrazide (4)
1
Yield:90%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.3 (s, 1H, NH), 8.0 (d, J4,5 = 7.2 Hz,1H, H-4),
7
.7(d, J7,6 = 7.5 Hz,1H, H-7), 7.6 (m, 3H, H-6/6’/2’), 7.2 (d, J4’,5’ = 8.1 Hz, 1H, H-4’), 7.1 (m,
7

2
H, H-5/5’), 3.7 (t, 2H, NCH
2
), 1.6 (m, 2H, NCH
2
CH
2
), 0.8 (t, 3H, CH
2
CH ),; EI-MS: m/z (rel.
3
+
int. %): 321(M , 45), 290 (100), 187 (60), 138 (45);
4
.2.5. (E)-4-methyl-N'-(2-Oxo-1-propylindolin-3-ylidene) benzohydrazide (5)
1
Yield:87.5%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 8.0 (d, J4,5 = 7.2 Hz,1H, H-4),
7
.7(d, J7,6 = 7.5 Hz,1H, H-7), 7.6 (m, 3H, H-6/6’/2’), 7.2(d, J3’,2’/5’,6’= 8.0 Hz, 2H, H-3’/5’), 7.1
m, 1H, H-5), 3.7 (t, 2H, NCH ), 1.6 (m, 2H, NCH CH ), 0.9 (t, 3H, CH CH ),; EI-MS: m/z (rel.
int. %): 321(M , 45), 290 (100), 187 (60), 138 (45);
(
2
2
2
2
3
+
4
.2.6. (E)-2,5-dimethoxy-N'-(2-Oxo-1-propylindolin-3-ylidene)benzohydrazide (6)
1
Yield:76%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.2 (s, 1H, NH), 7.6 (m, 2H, H-4/7), 7.4 (t, J6/5,7
), 1.6 (m, 2H, NCH CH ), 0.9 (t,
),; EI-MS: m/z (rel. int. %): 367 (M , 67), 220 (100), 167 (58), 139 (45);
=
7.5 Hz,1H,H-6), 7.2(m, 4H, H-5/3’/4’/6’), 3.7 (m, 2H, NCH
2
2
2
+
3
H, CH
2
CH
3
4
.2.7.(E)-N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(3,4-dichlorophenyl)acetohydrazide(7)
1
Yield:74.5%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.1 (s, 1H, NH), 7.6 (s, 1H,
H-4), 7.4 (s, 1H, H-2’), 7.23 (t, J7,6= 8.1 Hz,1H, H-7), 7.2(m, 3H, H-5’/6/6’), 1.6 (s, 2H,
+
CH
2
),;EI-MS: m/z (rel. int. %): 381 (M , 60), 242 (100), 93 (60).
4
.2.8. (E)-N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenyl)acetohydrazide (8)
1
Yield:78%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.1 (s, 1H, NH), 7.6 (s, 1H, H-
);EI-
4
), 7.4 (s, 1H, H-3’), 7.2 (d, J7,6 = 8.0 Hz,1H, H-7), 7.2(m, 3H, H-5’/6/6’), 1.6 (s, 2H, CH
2
+
MS: m/z (rel. int. %): 381 (M , 60), 242 (100), 198 (76), 93 (60);
4
.2.9. (E)-N'-(5-Chloro-2-oxoindolin-3-ylidene)-4-methylbenzohydrazide (9)
1
Yield:90%; H-NMR: (DMSO-d
6
, 300 MHz): δ 14.3 (s, 1H, NH), 13.2 (s, 1H, NH), 7.5 (s, 1H,
H-4), 7.4 (d, J7,6 = 8.0 Hz,1H, H-7), 7.2 (d, J6,7 = 8.0 Hz,1H,H-6), 7.1 (d, J6’,5’/2’,3’ = 8.0 Hz,2H,
+
H-2’/6’), 7.1(m, 2H, H-3’/5’), 1.6 (s, 3H, CH
06 (100), 162 (45);
3
),;EI-MS: m/z (rel. int. %): 313 (M , 60), 298 (56),
2
8

4
.2.10. (E)-N'-(5-Bromo-2-oxoindolin-3-ylidene) dodecanehydrazide (10)
1
Yield:82%; H-NMR: (DMSO-d
6
, 300 MHz):δ14.2 (s, 1H, NH), 13.3 (s, 1H, NH), 7.4 (s, 1H, H-
); EI-
4
), 7.3 (d, J7,6 = 7.5 Hz,1H, H-7), 7.2 (d, J6,7 = 7.5 Hz,1H, H-6), 1.2 (m, 23H, (CH
2
)
10CH
3
+
MS:m/z (rel. int. %): 377 (M , 67), 342 (85), 232 (100), 145 (77);
4
.2.11. (E)-N'-(6-Bromo-2-oxoindolin-3-ylidene)-2-(3,4-dichlorophenyl)acetohydrazide (11)
1
Yield:80%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.1 (s, 1H, NH), 7.8 (s, 1H, H-
2
’), 7.4 (s, 1H, H-7), 7.3 (d, J4,5 = 8.1 Hz,1H, H-4), 7.2 (d, J5’,6’ = 7.5 Hz,1H, H-5’), 7.1(m, 2H,
+
H-4/6’), 1.6 (s, 2H, CH ),;EI-MS: m/z (rel. int. %): 425 (M , 43), 336 (78), 277 (100), 148 (89);
2
4
.2.12. (E)-N'-(6-Bromo-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenyl)acetohydrazide (12)
1
Yield: 82%; H-NMR: (DMSO-d , 300 MHz):δ 14.2 (s, 1H, NH), 12.9 (s, 1H, NH), 7.8 (s, 1H,
6
H-3’), 7.6 (s, 1H, H-7), 7.4 (d, J4,5 = 7.1 Hz,1H, H-4), 7.3 (d, J5’,6’ = 7.5 Hz,1H, H-5’), 7.1(d,
+
J5,4/6’,5’ = 7.5 Hz, 2H, H-5/6’), 1.6 (s, 2H, CH
2
); EI-MS: m/z (rel. int. %): 425 (M , 49), 336
(
100), 277 (88), 148 (75);
4
.2.13. (E)-N'-(6-Bromo-2-oxoindolin-3-ylidene)-4-methylbenzohydrazide (13)
1
Yield:87%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.2 (s, 1H, NH), 7.5 (s, 1H, H-
4
2
2
), 7.4 (d, J7,6 = 8.1 Hz,1H, H-7), 7.3 (d, J5,4 = 8.1 Hz,1H, H-5), 7.2 (d, J6’,5’/2’,3’ = 8.0 Hz, 2H, H-
+
’/6’), 7.1(m, 2H, H-3’/5’), 1.6 (s, 3H, CH ); EI-MS: m/z (rel. int. %): 357 (M , 56), 342 (75),
3
68 (100), 89 (66);
4
.2.14. (E)-N'-(6-Bromo-2-oxoindolin-3-ylidene)-2-hydroxybenzohydrazide (14)
1
Yield:83%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.2 (s, 1H, NH), 10.2 (s, 1H,
OH), 7.5 (s, 1H, H-7), 7.4 (d, J4,5 = 8.0 Hz,1H, H-4), 7.3 (d, J5,4 = 8.0 Hz,1H, H-5), 7.2 (d,
+
J
3’,4’/6’,5’ = 8.0 Hz, 2H, H-3’/6’), 7.1(m, 2H, H-4’/5’); EI-MS: m/z (rel. int. %): 359 (M , 100),
3
42 (65), 270 (78), 89 (34);
4
.2.15. (E)-N'-(6-Bromo-2-oxoindolin-3-ylidene) dodecanehydrazide (15)
9

1
Yield:76%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.2 (s, 1H, NH), 7.5 (s, 1H, H-
), 1.2 (m, 23H,
7
), 7.3 (d, J4,5 = 7.1 Hz,1H, H-7), 7.2 (d, J5,6 = 7.1 Hz,1H, H-5), 1.8 (m, 2H, CH
2
+
(
CH
2
)
10CH ),;EI-MS: m/z (rel. int. %): 421 (M , 69), 332 (100), 276 (85), 93 (70);
3
4
.2.16. (E)-2-(3,4-Dichlorophenyl)-N'-(2-oxoindolin-3-ylidene)acetohydrazide (16)
1
Yield: 85%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.2 (s, 1H, NH), 13.2 (s, 1H, NH), 7.7 (s, 1H,
H-2’), 7.5 (d, J5’,6’ = 8.0 Hz,1H, H-5’), 7.4 (d, J6’,5’ = 8.0 Hz,1H, H-6’), 7.3 (d, J4,5 = 7.5 Hz,1H,
+
H-4), 7.2(m, 3H, H-5/6/7), 1.6 (s, 2H, CH
2
),;EI-MS: m/z (rel. int. %): 347 (M , 67), 312 (100),
2
02 (80), 145 (70);
4
.2.17. (E)-2-(2,4-Dichlorophenyl)-N'-(2-oxoindolin-3-ylidene)acetohydrazide (17)
1
Yield: 83%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 12.7 (s, 1H, NH), 7.7 (s, 1H,
H-3’), 7.6 (d, J5’,6’ = 7.5 Hz,1H, H-5’), 7.5 (d, J6’,5’ = 7.5 Hz,1H, H-6’), 7.4 (d, J4,5 = 7.1 Hz,1H,
H-4), 7.3 (d, J7.6= 7.1 Hz,1H, H-7), 7.1(m, 2H,H-5/6), 1.6 (s, 2H, CH ),;EI-MS: m/z (rel. int. %):
2
+
3
47 (M , 65), 312 (87), 202 (100), 145 (71);
4
.2.18. (E)-4-methyl-N'-(2-Oxoindolin-3-ylidene) benzohydrazide (18)
1
Yield: 84%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.1 (s, 1H, NH), 13.2 (s, 1H, NH), 7.5 (d, J4,5
=
8
.0 Hz,1H, H-4), 7.4 (d, J7,6 = 7.5 Hz,1H, H-7), 7.3 (d, J2’,3’/6’,5’= 8.1 Hz,2H, H-2’/6’), 7.2 (d,
J
3’,2’/5’,6’ = 8.0 Hz,2H, H-3’/5’), 7.1(m, 2H, H-5/6), 1.6 (s, 3H, CH ),;EI-MS: m/z (rel. int. %): 279
3
+
(
M , 88), 264 (100), 188 (50), 91(78);
4
.2.19. (E)-2-hydroxy-N'-(2-Oxoindolin-3-ylidene) benzohydrazide (19)
1
Yield: 80%; H-NMR: (DMSO-d
6
, 300 MHz):δ 14.3 (s, 1H, NH), 13.1 (s, 1H, NH), 10.4 (s, 1H,
OH), 7.5 (s, J4,5 = 8.0 Hz,1H, H-4), 7.4 (d, J7,6 = 8.1 Hz,1H, H-7), 7.3 (d, J6’,5’ = 7.5 Hz,1H, H-
+
6
’), 7.2 (m, 3H, H-3’/5/6), 7.1(m, 2H, H-4’/5’); EI-MS: m/z (rel. int. %): 281 (M , 80), 264
(
100), 198 (60), 83 (79);
4
.2.20. (E)-N'-(2-Oxoindolin-3-ylidene) dodecanehydrazide (20)
10

1
Yield: 88%; H-NMR: (DMSO-d
6
, 300 MHz): δ 14.3 (s, 1H, NH), 13.1 (s, 1H, NH), 7.5 (d, J4,5
=
7
2
.1 Hz,1H, H-4), 7.3 (d, J7,6 = 7.1 Hz,1H, H-7), 7.2 (m,1H,H-5/6), 1.8 (m, 2H, CH ), 1.2 (m,
2
+
3H, (CH
2
)
10CH ); EI-MS: m/z (rel. int. %): 343 (M , 60), 228 (76), 198 (100), 93 (70);
3
Acknowledgments
The authors are thankful to Higher Education Commission of Pakistan for financial support.
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12

Caption
Scheme-1: synthesis of Schiff based isatin derivatives (1-20)
Table-1: α-glucosidase inhibitor activity of isatin Schiff bases (1-20)
Figure-1: The binding modes and molecular interactions of Isatin Schiff basescompound12 in
the active sites of α-glucosidase.
Figure-2: The binding modes and molecular interactions of isatin Schiff basescompound20 in
the active sites of α-glucosidase.
Figure-3: The binding modes and molecular interactions of Isatin Schiff basescompound11 in
the active sites of α-glucosidase.
Figure-4: 2-Dimensional pose of the compound 01, 02 and 14 in the active site of α-glucosidase
enzyme
13

Scheme-1:
14

Figure-1:
15

Figure-2:
16

Figure-3:
17

Figure-4:
18

Table-1
IC50 ± SEM
Compounds
R1
R2
R3
R4
[
µM]
H2
C
C
CH3
1
H
H
H
NA
2
1
8.3 ± 0.56
2
3
H
H
H
H
NA
4
5
6
H
H
H
H
NA
NA
NA
H
H
19

7
Cl
Cl
H
H
H
H
NA
NA
8
9
Cl
Cl
H
H
H
H
83.5 ± 1.0
NA
1
0
1
1
H
Br
H
3
.3 ± 0.25
1
1
2
3
H
H
Br
Br
H
H
2.2 ± 0.25
NA
20

1
1
4
5
H
H
Br
Br
H
H
11.8 ± 0.15
NA
H
1
1
6
7
NA
NA
H
H
H
H
H
H
1
8
H
H
NA
1
2
9
0
H
H
H
H
H
H
NA
3.0 ± 0.15
Acarbose
840±1.73
SEM is stand error of the mean, NA not active; acarbose is standard for α-glucosidase inhibition activity
21

Graphical abstract
Cl
H
N
HN
Cl
N
O
O
Compound 12
IC50, 2.2 ± 0.25 μM)
Potent Inhibitors of α-glucosidase
(
22

Highlights:





Synthesis of Isatin based Schiff bases
In vitro α-glucosidase inhibitory activity
Identification of a novel class of α-glucosidase inhibitors
Structure-activity Relationship established
Molecular docking
23