Synthesis and molecular docking studies of potent α-glucosidase inhibitors based on biscoumarin skeleton

Article abstract of DOI:10.1016/j.ejmech.2014.05.010

In our effort directed toward the discovery of new anti-diabetic agent for the treatment of diabetes, a library of biscoumarin derivative 1-18 was synthesized and evaluated for α-glucosidase inhibitory potential. All eighteen (18) compounds displayed asso

Full text of DOI:10.1016/j.ejmech.2014.05.010

Accepted Manuscript
Synthesis and Molecular Docking Studies of Potent #-Glucosidase Inhibitors Based
on Biscoumarin Skeleton
Khalid Mohammed Khan, Fazal Rahim, Abdul Wadood, Naveen Kosar, Muhammad
Taha, Salima Lalani, Aisha Khan, Muhammad Imran Fakhri, Muhammad Junaid,
Wajid Rehman, Momin Khan, Shahnaz Perveen, Muhammad Sajid, M. Iqbal
Choudhary
PII:
S0223-5234(14)00424-3
10.1016/j.ejmech.2014.05.010
EJMECH 6966
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 January 2014
Revised Date: 4 March 2014
Accepted Date: 2 May 2014
Please cite this article as: K.M. Khan, F. Rahim, A. Wadood, N. Kosar, M. Taha, S. Lalani, A. Khan,
M.I. Fakhri, M. Junaid, W. Rehman, M. Khan, S. Perveen, M. Sajid, M.I. Choudhary, Synthesis and
Molecular Docking Studies of Potent #-Glucosidase Inhibitors Based on Biscoumarin Skeleton,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.010.
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ACCEPTED MANUSCRIPT
Synthesis and Molecular Docking studies of Potent α-Glucosidase Inhibitors Based on Biscoumarin Skeleton
d,h
Khalid Mohammed Khan,a Fazal Rahim,bAbdul Wadood,c Naveen Kosar,b Muhammad Taha,
Salima Lalani,a
Aisha Khan,b Muhammad Imran Fakhri,a Muhammad Junaid,c Wajid Rehman,c Momin Khan,e Shahnaz Perveen,f
Muhmmad Sajid,g and M. Iqbal Choudharya
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan
bDepartment of Chemistry, Hazara University, Mansehra, Pakistan
cComputational Medicinal Chemistry Laboratory, Department of Biochemistry, Abdul Wali Khan University,
Mardan, Mardan-23200, Pakistan
dAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam
Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
eDepartment of Chemistry, Abdul Wali Khan University, Mardan, Mardan-23200, Pakistan
fPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
gDepatment of Biochemistry, Hazara University, Mansehra, Pakistan
hFaculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
*Corresponding author : khalid.khan@iccs.edu
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Synthesis and Molecular Docking Studies of Potent α-Glucosidase Inhibitors Based on
Biscoumarin Skeleton
Khalid Mohammed Khan,a Fazal Rahim,b Abdul Wadood,c Naveen Kosar,b Muhammad Taha,d,h
Salima Lalani,a Aisha Khan,b Muhammad Imran Fakhri,a Muhammad Junaid ,c Wajid Rehman,b
Momin Khan,e Shahnaz Perveen,f Muhammad Sajidg and M. Iqbal Choudharya
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepatment of Chemistry, Hazara University, Mansehra, Pakistan
cComputational Medicinal Chemistry Laboratory, Department of Biochemistry, Abdul Wali
Khan University, Mardan, Mardan-23200, Pakistan
dAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
eDepartment of Chemistry, Abdul Wali Khan University, Mardan, Mardan-23200, Pakistan
fPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280,
Pakistan
gDepatment of Biochemistry, Hazara University, Mansehra, Pakistan
hFaculty of Applied Science Universiti Tecknologi MARA, 40450 Shah Alam,Selangor D. E.
Malaysia
*Corresponding author: khalid.khan@iccs.edu
Abstract:
In our effort directed toward the discovery of new anti-diabetic agent for the treatment of
diabetes, a library of biscoumarin derivative 1-18 was synthesized and evaluated for α-
glucosidase inhibitory potential. All eighteen (18) compounds displayed assorted α-glucosidase
activity with IC50 values 16.5-385.9 µM, if compared with the standard acarbose (IC50 = 906 ±
6.387 µM). In addition, molecular docking studies were carried out to explore the binding
interactions of biscoumarin derivatives with the enzyme. This study has identified a new class of
potent α-glucosidase inhibitors.
Keywords: Biscoumarin, α-glucosidase inhibition, molecular docking
Coressponding authors: Tel.: (+92-21) 34824910, E-mail: khalid.khan@iccs.edu and hasssaan2@super.net.pk
(Khalid Mohammed Khan)
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1. Introduction
Mammalian α-glucosidases inhibitors, which impede with enzymatic action in small intestine,
might slow the release of D-glucose from oligosaccharides and disaccharides, causing in
delaying glucose absorption and decreasing postprandial blood glucose levels [1]. The acarbose
[2] and voglibose [3] from microorganisms, and nojirimycin [4] and 1-deoxynojirimycin [5]
from plants have been reported as potent α-glucosidase inhibitors. The effects of all these
compounds on blood glucose levels after food uptake have been reported [6-9].
Glucosidases are liable for the catalytic cleavage of α-glycosidic bond with specificity depending
on the position of cleavage site, the number of monosaccharides, and the configuration of the
hydroxyl groups on the substrate [10]. The pharmaceutical research community has a special
interest in α-glucosidases (EC 3.2.1.20) because the inhibition of its catalytic activity caused in
the impedance of glucose absorption and decreases the postprandial blood glucose level. Thus,
effective α-glucosidases inhibitors may help as chemotherapeutic agents for clinical use in the
treatment of obesity and diabetes [11-13]. The α-glucosidase inhibitors has also been well
appreciated as a therapeutic target for the other carbohydrate mediated diseases including viral
infections [14,15], cancer [16] and hepatitis [17].
In the course of our efforts in the development of biologically important synthetic compounds,
we observed α-glucosidase inhibitory potential in substituted new biscoumarin derivatives. It is
obvious that polyphenols well intermingle with proteins and lead to inhibit enzyme activities
[18]. Natural as well as synthetic biscoumarin have become an important class of oxygenated
heterocycle mainly due to their wide variety of biological activities such as urease inhibition,
anti-inflammatory, antioxidant, CYP3A and antifungal activities [19-21].
There is few reported synthetic and natural coumarin derivatives showed potent α-glucosidase
inhibition [22-25]. Surprisingly no biscoumarin was tested for α-glucosidase inhibition.
Therefore, we designed our project to synthesize biscoumarin and test them for α-glucosidase
inhibitory properties.
In the continuation of our work, on biological potent small molecules [26] and their evaluation
for enzyme inhibition [27], we herein report synthesis of substituted biscoumarin analogs, their
α-glucosidase inhibitory potentials. The molecular docking was also performed in order to study
their binding affinity.
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2. Results and Discussion
2.1 Chemistry
In synthesis of biscoumarin analogs 1-18 (Scheme-1), to a stirred mixture of coumarin
derivatives (4.0 mmol) and substituted aromatic aldehydes (2.0 mmol) in water and a catalytic
amount of tetraethylammonium bromide (TEAB) was added. The reaction mixture was stirred at
60 °C for 1-2 h. Completion of the reaction was monitored by periodic TLC. After completion of
reaction, it was filtered and washes with distilled water affording a pure product in high yields.
In some cases the compounds purified through column chromatography using 3:7 acetone and n-
hexane as eluent afforded pure products in good yield. The structures of compounds 1-18 were
deduced by using different spectroscopic techniques such as 1H NMR and EI mass spectroscopy.
All compounds gave a satisfactory elemental analysis.
Insert Scheme 1 here
Insert table 1 here
2.2 Pharmacology
Glucosidase are responsible for the catalytic cleavage of a glycosidic bond of complex molecules
of carbohydrates. Enzyme inhibition is one of the most significant tools in pharmaceutical
research as well as in the field of drug discovery. During this study, we have synthesized
eighteen (18) derivatives and evaluated for α-glucosidase enzyme inhibitory activity. All
compounds showed a potent inhibition superior to the standard inhibitor of α-glucosidase.
Compounds 1-18 exhibited a varying degree of α-glucosidase inhibitory activity with IC50 values
between 16.54 ± 0.36-385.99 ± 0.65 µM when compared with standard acarbose (IC50 = 906 ±
6.387 µM). Although the main skeleton for all compounds are same, the slight difference in their
inhibitory potential might be due to the different substitution pattern on benzaldehyde.
Compound
3,3′-((2-4-Dichlorophenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(12) showed an excellent activity (IC50 = 16.54 ± 0.36 µM) 54-fold more active than the standard
acarbose (IC50 = 906 ± 6.387 µM). Similarly, compound 3,3′-((3-nitrophenyl) methylene)bis(6-
chloro-4-hydroxy-2H-chromen-2-one) (13) showed an excellent activity with an IC50 value of
27.07 ± 0.13 µM, 33-fold better than the standard. The remaining all compounds also exhibited
potent inhibitory activities.
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To know the mechanism of α-glucosidase inhibition and binding mode of biscoumarin analogs
inside the binding pocket of α-glucosidases, molecular docking studies were performed.
2.2 Molecular Docking studies
The molecular docking study was carried out to explore the binding mode of biscoumarin
derivatives within the binding pocket of α-glucosidase and to understand their structure activity
relationship using MOE-Dock as docking software (www.chemcomp.com). As a means of
testing the adopted protocols, the known inhibitor acarbose (the first α-glucosidase inhibitor
approved for type 2 diabetes treatments) was docked into the binding pocket of a developed
homology model, the acarbose fit well in the binding pocket and showed interaction to the
important active site residues (Figure-1).
Although the x-ray crystallographic structures of α-glucosidase have been reported from some
bacteria, the three-dimensional structural information is still not available for the eukaryotic α-
glucosidase enzyme from Sacchromyces species (the enzyme use in our biological assay).
However, only a few homology models have been reported for this enzyme previously [28-30].
The three dimensional coordinates of none of these model are publically available. Therefore, we
construct the 3D structure of α-glucosidase by homology modeling using the same protocol as
described by Burke et al [31], based on the crystal structure of Saccrhromyces cerevisiae
(3AJ7.pdb) [26].
All the compounds were docked into the binding pocket of a developed homology model of
α-glucosidase enzyme. From the molecular docking it was observed that the top ranked
conformation of the most active compound 12 (IC50 =16.54 ± 0.36 µM) (Figure-2) established
six hydrogen bonds between the hydroxyl group on coumarin ring of the compound and the
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active site residues (Asp 214, Glu 276, Arg 312, Asp 408 and Arg 439). Additionally the aryl
group of the compound formed an arene-cation interaction with the Arg 439. Furthermore,
several hydrophobic interactions were observed between the compound and the active site
residues, e.g., Val 108, Phe 157, Phe 177 and Phe 300 are the other residues that stabilized the
binding of the compound 12 in the active site of α-glucosidase. The strong hydrogen bonding
network observed for compound 12 by the hydroxyl groups attached to the coumarin ring might
be due to chloro groups, particularly dichlorobenzene in which the chloride moiety has strong
electron withdrawing inductive effect. This effect of chloride moieties increases the ionizing
ability of hydroxyl group that might be one of the reasons for its highest activity showed in the
series (Table-1). These observations can be verified in case of compounds 2 (Figure-3), 15
(Figure-4) and 17 (Figure-5) as they have low biological activities as well as less interaction
with active site residue as compared to compound 12. As these compounds have groups with
electron donating inductive effect instead of chloride present as in compound 12. Similarly
compounds 6 to 11 have two side chain chloride groups, but lacking the dichlorobenzene group
that makes them slightly less active as compare to compound 12. From the docking conformation
of compounds 13 it was observed that its m-nitrobenzene showed almost similar behavior as
dichlorobenzene in compound 12 because the nitro group has also electron withdrawing
inductive effect.
Insert Figure 1 here
Insert Figure 2 here
Insert Figure 3 here
Insert Figure 4 here
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Insert Figure 5 here
2.3 Molecular docking
Homology modeling of α-glucosidase for Saccharomyces cerevisiae was performed to predict its
three dimensional (3D) structure. The amino acid sequence of α-glucosidase from
Saccharomyces cerevisiae was retrieved from UniProt protein resource data bank
(http://www.uniprot.org/) under the access code P53341. Similarity search was carried out in
MOE-2010.11 using default parameter against PDB Databank implemented in MOE2010.11.
The crystallographic structure of Saccharomyces cerevisiae isomaltase (PDB code 3AJ7, 1.30 Å
resolution) with 72.4% of sequence identity with the target was selected as the template. 3D
structure was built using homology modeling tool implemented in MOE. The developed
structure was subjected to energy minimization up to 0.05 RMS gradients and the minimized
structure was then refined by MD simulation up to 500 picoseconds. The final refine structure
was then used for the molecular docking purpose.
Prior to docking ligand and protein were prepared using MOE2010.11. All synthetic compounds
were modeled using Builder program implemented in MOE, finally a database was created in
which all the compound structures were present in 3D format. Subsequently, their energies were
minimized up to 0.05 Gradient, using the MMFF94x force field. Energy minimization of
compound database was followed by the preparation of protein for docking purposes. Most of
macromolecular crystal structures contain little or no hydrogen coordinate data due to limited
resolution, thus protonation should be done prior to docking using the Protonate 3D Option. The
protonation was followed by energy minimization up to 0.05 gradient, using Amber99 force
field. The database was docked into the binding pocket of a protein using the Triangular
Matching docking method and 30 conformations for each ligand protein complex were generated
with docking score. Each complex was analyzed for interactions and the 3D image was taken.
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2.4 α-Glucosidase Inhibitory Assay
Rat intestinal acetone powder in typical saline (100:1; w/v) was sonicated appropriately and the
supernatant was used as a source of basic intestinal α-glucosidase after centrifugation. In short,
10 mL of test samples of 5 mg/mL in DMSO solution were reconstituted in 100 mL of 100 mM-
phosphate buffer at pH 6.8 in 96-well micro-plate and incubated with 50 mL of basic intestinal
α-glucosidase for 5 min before 50 mL substrate (5 mM, p-nitrophenyl-α-D-glucopyranoside
prepared in same buffer) was added. p-Nitrophenol released was measured at 405 nm
spectrophotometrically (SpectraMax plus384), Molecular Devices Corporation, Sunnyvale, CA,
USA) 5 min after incubation with the substrate. Individual blanks for test samples were prepared
to accurate background absorbance where the substrate was changed with 50 mL of buffer.
Control sample contained 10 mL DMSO beside test samples. Percentage of enzyme inhibition
was measured as (1 - B/A) x100 where [A] represents absorbance of control exclusive of test
samples, and [B] corresponding to absorbance in presence of test samples [33].
3. Conclusions
Synthesis of biscoumain analogs and their α-glucosidase inhibitory potential was evaluated. All
these eighteen (18) derivatives showed potent α-glucosidase inhibitory potential. Consequently,
in silico studies were performed to recognize the binding mode of these compounds. The planned
scaffold of α-glucosidase inhibitors offers the possibility of expedient additional modifications
that could give rise to lead structures with enhanced inhibitory activity and selectivity towards
the enzyme.
4. Material and Methods
NMR experiments were performed on an Avance Bruker AM 300 MHz machine. CHN Analyses
were carried out a Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI
MS) were recorded on a Finnigan MAT-311A (Germany) mass spectrometer. Thin layer
chromatography (TLC) was carried out on pre-coated silica gel aluminum plates (Kieselgel 60,
254, E. Merck, Germany). Chromatograms were visualized by iodine vapors or UV at 254 and
365 nm.
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4.1 General procedure for the synthesis of compounds 1-18
In synthesis of biscoumarin analogs 1-18 (Scheme-1), to a stirred mixture of coumarin
derivatives (4.0 mmol) and substituted aromatic aldehyde (2.0 mmol) in water and 1o mol%
triethylammonium bromide (TEAB) was added. The reaction mixture was stirred at 60 °C for 1-2
h. Completion of reaction was monitored by periodic TLC. After completion of reaction, it was
filtered, and then washed with distilled water affording a pure product in high yields. In some
cases pure products were obtained through column chromatography (silica gel) using 3:7 acetone
and n-hexane as eluent. The structures of compounds 1-18 were deduced by using different
spectroscopic techniques, including 1H NMR and EI mass spectroscopy. All synthetic
compounds 1-18 gave satisfactory elemental analyses.
4.1.1 3,3'-((4-Nitrophenyl)methylene)bis(4-hydroxy-6-methyl-2H-chromen-2-one) (1)
Yield: 0.23 g (84%); 1H-NMR: (DMSO-d6, 300 MHz): δ 8.05 (d, 2H, J3’’,2’’/6’’,5’’ = 8.7 Hz, H-
3’’/5’’), 7.59 (br s, 2H, H-5/5’), 7.47 (d, 2H, J2’’,3’’/6’’,5’’ = 8.6 Hz, H-2’’/6’’), 7.34 ((dd, 1H,
,
,
, ,
J7,8/7 ,8 = 8.1 , J7,5/7 ,5 = 2.6 Hz, H-7/7’), 7.17 (d, 1H, J8,7/8’,7’ = 8.2 Hz, H-8/8’), 6.31 (s, 1H,
Ar3CH), 2.32 (s, 6H, 2 x CH3); Anal. Calcd for C27H19NO8, C = 66.80, H = 3.95, N = 2.89,
Found C = 66.78, H = 3.97, N = 2.91; EI-MS: m/z (rel. int. %): 485 (M+, 42), 308 (15), 292(45),
134 (100), 106 (20).
4.1.2 3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(6-methyl-4-hydroxy-2H-chromen-2-one
(2)
Yield: 0.22 g (82%); 1H-NMR: (DMSO-d6, 300 MHz, ): δ 7.64 (br, s, 2H, H-5/5’), 7.35 (dd, 2H,
,
,
J7,8/7 ,8, = 8.4 , J7,5/7 ,5, = 1.8 Hz, H-7/7’), 7.19 (d, 2H, J8,7/8’,7’ = 8.1 Hz, H-8/8’), 6.39 (s, 2H, H-
2’’/6’’), 6.21 (s, 1H, Ar3CH), 3.61 (s, 3H, OCH3), 3.54 (s, 6H, OCH3), 2.33 (s, 6H, 2 x CH3);
Anal. Calcd for C30H26O9, C = 67.92, H = 4.94, Found C = 67.94, H = 4.96; EI-MS: m/z (rel. int.
%): 530 (M+, 65), 353 (63), 322 (100), 175 (28), 134 (13).
4.1.3 3,3′-((4-(Methylthio)phenyl)methyl)6-methyl-2H-chromen-2-one) (3)
Yield: 0.23 g (84%); 1H-NMR: (DMSO-d6, 300 MHz): δ 7.61 (br. s, 2H, H-5/5’), 7.35 (dd, 2H,
,
,
J7,8/7 ,8, = 8.4 Hz, J7,5/7 ,5, = 1.8 Hz, H-7/7’), 7.19 (d, 2H, J8,7/8’,7’ = 8.4 Hz, H-8/8’), 7.09 (m, 4H,
H-2’’/3’’/5’’/6’’), 6.21 (s, 1H, Ar3CH), 2.39 (s, 3H, -SCH3), 2.33 (s, 6H, CH3); Anal. Calcd for
8

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C28H22O6S, C = 69.12, H = 4.56, S = 6.59, Found C = 69.14, H = 4.55, S = 6.57; EI-MS: m/z (rel.
int. %): 486 (M+, 42), 308 (59), 263 (100), 176 (42), 134 (61).
4.1.4 3,3′-((3-Hydroxyphenyl)methylene)bis(6-methyl-4-hydroxy-2H-chromen-2-one) (4)
1
Yield: 0.24 g (86%); H-NMR: (DMSO-d6 300 MHz): δ 7.58 (br.s, 2H, H-5/5’), 7.30 (dd, 2H,
,
,
J7,8/7 ,8, = 8.7, J7,5/7 ,5, = 2.1 Hz, H-7/7’), 7.13 (d, 2H, J8,7 = 8.1 Hz, H-8/8’), 6.92 (t, 1H, J5’’(4’’,6’’)
= 7.8 Hz, H-5’’), 6.52 (m, 3H, H-2’’/4’’/6’’), 6.14 (s, 1H, Ar3CH), 2.32 (s, 6H, 2 x CH3); Anal.
Calcd for C27H20O7, C = 71.05, H = 4.42, Found C = 71.06, H = 4.40; EI-MS: m/z (rel. int. %):
456 (M+, 42), 345 (18), 279 (37), 176 (52), 134 (100);
4.1.5 3,3′-((2-Nitrophenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one) (5)
’
Yield: 0.21 g (80%); 1H-NMR (DMSO-d6, 300 MHz); δ 7.69 (d, 2H, J5,7/5 ,7’= 2.7 Hz, H-5/5’),
7.52 (m, 4H, H-7/7’/3’’/6’’), 7.33 (m, 4H, H-8/8’/4’’/5’’), 6.42 (s, 1H, Ar3CH); Anal. Calcd for
C25H13Cl2NO8, C = 57.05, H = 2.49, Found C = 57.07, H = 2.51; EI-MS m/z (rel. int. %): 526
(M+, 42), 298 (67), 283 (58), 196 (27), 154 (100);
4.1.6 3,3′-((3-Methoxy,4-hydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-
one) (6)
,
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz); δ 7.73 (d, 2H, J5,7/5’,7 = 2.7Hz, H-5/5’),
7.54 (dd, 2H, J7,8/7 ,8, = 8.7, J7,5/7 ,5, = 2.4 Hz, H-7/7’),7.31 (d, 2H, J8,7/8 ,7 = 8.7 Hz, H-8/8’), 6.59
,
,
’ ’
(d, 1H, J6 ,5’’ = 6.0 Hz, H-6’’), 6.54 (s, 1H, H-2’’),6.49 (d, 1H, J5 ,6’’= 8.1Hz, H-5’’), 6.12(s, 1H,
Ar3CH), 5.61(s, 1H, OH), 3.53 (s, 3H, OCH3); Anal. Calcd for C26H16Cl2O8, C = 59.22, H =
3.06, Cl = 13.45, Found C = 59.23, H = 3.04; EI-MS m/z (rel. int. %): 527 (M+, 42), 329 (100),
313 (56), 299(73), 196 (51).
’'
’’
4.1.7 3,3′-((3-Hydroxyphenyl) methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one) (7)
’
’
Yield: 0.22 g (82%); 1H-NMR (DMSO-d6, 300 MHz); δ 7.74 (d, 2H, J5,7/5 ,7 = 2.7 Hz, H-5/5’),
’
’
’
7.55 (dd, 2H, J7,8/7 ,8’ = 8.7, J7,5/7 ,5’ = 2.7 Hz, H-7/7’), 7.32 (d, 2H, J8,7/8 ,7’= 8.7 Hz, H-8/8’), 6.92
’’ ’’ ’’
(t, 1H, J5 (4 ,6 ) = 7.8 Hz, H-5’’), 6.51 (m, 3H, H-2’’/4’’/6’’), 6.14 (s, 1H, Ar3CH); Anal. Calcd for
C25H14Cl2O7, C = 60.38, H = 2.84, Found C = 60.36, H = 2.82; EI-MS m/z (rel. int. %): 497
(M+, 42), 299 (95), 283 (74), 196 (41), 154 (100).
4.1.8 3,3′-((3-Hydroxy-4-methoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-
one) (8)
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’
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.73 (d, 2H, J5,7/5 ,7’= 2.7 Hz, H-5/5’),
’
’
’
7.54 (dd, 2H, J7,8/7 ,8’ = 8.7, J7,5/7 ,5’ = 2.7 Hz, H-7/7’),7.32 (d, 2H, J8,7/8 ,7’ = 9.0 Hz, H-8/8’), 6.69
’’
’’
(d, 1H, J5 ,6’’= 8.4 Hz, H-5’’),6.52 (s, 1H, H-2’’), 6.43 (d, 1H, J6 ,5’’ = 8.4 Hz H-6’’), 6.09 (d, 1H,
Ar3CH); Anal. Calcd for C26H16Cl2O8, C = 59.22, H = 3.06, Found C = 59.24, H = 3.04; EI-MS
m/z (rel. int. %): 527 (M+, 42), 329 (89), 313 (100), 299 (53), 196 (48).
4.1.9 3,3’-((3-Ethoxy-4-hydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-
one) (9)
Yield: 0.22 g (82%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.73 (d, 2H, J5,7/5’7’ = 2.4 Hz, 2 x H-5),
’
’
’
7.54 (dd, 2H, J7,8/7 ,8’ = 8.7, J7,5/7 ,5’ = 2.7 Hz, H-7/7’),7.31 (d, 2H, J8,7/8 ,7’ = 8.7 Hz, H-8/8’), 6.58
’’ ’’ ’’ ’’
’’
(d, 2H, J5 ,6 /2 ,6 = 8.1 Hz, H-5’’/2’’),6.48 (d, 1H, J6 ’’= 8.1 Hz, H-6’’), 6.10 (s, 1H, Ar3CH),
,5
3.80 (q, 2H, OCH2CH3), 1.17 (t, 3H, OCH2CH3); Anal. Calcd for C27H18Cl2O8, C = 59.91, H =
3.35, Found C = 59.92, H = 3.34; EI-MS m/z (rel. int. %):541 (M+, 42), 299 (100), 287 (25), 154
(74), 126 (38).
4.1.10
3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-
one) (10)
’
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.75 (d, 2H, J5,7/5 ,7’ = 2.7 Hz, 2 x H-5),
’
’
’
7.54 (dd, 2H, J7,8/7 ,8’ = 8.7, J7,5/7 ,5’ = 2.4 Hz, H-7/7’),7.32 (d, 2H, J8,7/8 ,7’ = 8.7 Hz, H-8/8’), 6.37
(s, 2H, H-2’’/6’’), 6.16 (s, 1H, Ar3CH), 3.60 (s, 3H, OCH3), 3.54 (s, 6H, 2 x OCH3); Anal. Calcd
for C28H20Cl2O9, C = 58.86, H = 3.53; Found C = 58.84, H = 3.54; EI-MS m/z (rel. int. %): 571
(M+, 42), 374 (89), 343 (100), 196 (37), 154 (61).
4.1.11 3,3′-((3,4-Dimethoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(11)
’
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.74 (d, 2H, J5,7/5 ,7’= 1.5 Hz, H-5/5’),
’
’
’
’
’ ’
7.54 (dd, 2H, J7,8/7 ,8 = 5.1, J7,5/7 ,5 = 1.5 Hz, H-7/7’), 7.32 (d, 2H, J8,7/8 ,7 = 5.4 Hz, 2 x H-8),
’’ ’’
’’ ’’
6.75 (d, 1H, J5 ,6 = 5.1Hz, H-5’’), 6.63 (s, 1H, H-2’’), 6.61 (d, 1H, J6 ,5 = 5.1 H-6’’), 6.16 (s,
1H, Ar3CH), 3.67 (s, 3H, 3-OCH3 ), 3.52 (s, 3H, 4-OCH3); Anal. Calcd for C27H18Cl2O8, C =
59.91, H = 3.35; Found C = 59.93, H = 3.33; EI-MS m/z (rel. int. %): 541 (M+, 42), 343 (34),
313 (100), 196 (36), 154 (65).
10

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4.1.12 3,3’-((2-4-Dichlorophenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(12)
’
’
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.76 (s, 1H, H-3’’), 7.73 (d, 2H, J5,7/5 ,7
’
’
’
’
’ ’
= 2.4 Hz, H-5/5’), 7.54 (dd, 2H, J7,8/7 ,8 = 8.7, J7,5/7 ,5 = 2.4 Hz, H-7/7’), 7.36 (d, 2H, J8,7/8 ,7
=
8.7Hz, 2 x H-8/8’), 7.40 (m, 2H, H-5’’/6’’), 6.07 (s, 1H, Ar3CH); Anal. Calcd for C25H12Cl4O6, C
= 54.58, H = 2.20, Found C = 54.56, H = 2.19; EI-MS m/z (rel. int. %):550 (M+, 42), 317 (100),
196 (37), 154 (72), 126 (37).
4.1.13 3,3′-((3-Nitrophenyl) methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one) (13)
’
’
Yield: 0.22 g (82%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.73 (d, 2H, J5,7/5 ,7 = 2.4 Hz, H-5/5’),
’
’
7.57 (dd, 2H, J7,8/7 ,8’ = 8.7, J7,5/7 ,5’ = 2.7 Hz, H-7/7’), 7.48 (m, 4H, H-2’’/4’’/5’’/6’’), 7.32 (d, 2H,
’
’
J8,7/8 ,7 = 8.7Hz, H-8/8’), 6.31 (s, 1H, Ar3CH); Anal. Calcd for C25H13Cl2NO8, C = 57.05, H =
2.49, N = 2.66, Found C = 57.07, H = 2.48, N = 2.67; EI-MS m/z (rel. int. %): 511 (M+, 42), 313
(100), 283 (69), 196 (23), 154 (44);
4.1.14 3,3′-((3-Nitrophenyl)methylene)bis(4-hydroxy-6-methyl-2H-chromen-2-one) (14)
1
Yield: 0.24 g (86%); H-NMR: (DMSO-d6, 300 MHz): δ 8.00 (d, 1H, J4’’,5’’ = 8.1 Hz, H-4’’),
7.86 (br s, 1H, H-2’’), 7.61 (s, 2H, H-5/5’), 7.56 (d, 1H, J6’’,5’’ = 7.2 Hz, H-6’’), 7.48 (t, 1H, J5’’
,
,
, ,
(4’’,6’’) = 7.2 Hz, H-5’’), 7.36 (dd, 2H, J7,8/7 ,8, = 8.7, J7,5/7 ,5, = 2.4 Hz, H-7/7’), 7.19 (d, 2H, J8,7/8 ,7
= 8.4 Hz, 2H-8/8’), 6.33 (s, 1H, Ar3CH), 2.33 (s, 6H, CH3); Anal. Calcd for C27H19NO8, C =
66.80, H = 3.95, N = 2.89, Found C = 66.82, H = 3.97, N = 2.87; EI-MS m/z (rel. int. %): 485
(M+, 42), 309 (24), 176 (31), 134 (100), 106 (24);
4.1.15 3,3′-((3-Ethoxy-4-hydroxyphenyl)methylene)bis(6-methyl-4-hydroxy-2H-chromen-2-
one) (15)
Yield: 0.22 g (82%); 1H-NMR: (DMSO-d6, 300 MHz): δ 7.63 (br s, 2H, H-5/5’), 7.36 (dd, 2H,
,
,
, ,
J7,8/7 ,8 = 8.4, J7,5/7 ,5 = 1.5 Hz, H-7/7’), 7.20 (d, 2H, J8,7/8’,7’ = 8.4 Hz, H-8/8’), 6.61 (d, 2H,
J5’’,6’’/6’’,5’’ = 8.4 Hz, H-5’’/6’’), 6.50 (d, 1H, J2’’,6’’ = 7.8 Hz, H-3’’/5’’), 6.16 (s, 1H, Ar3CH3),
2.35 (s, 6H, CH3), 2.31 (q,2H,OCH2CH3), 1.18 (t,3H,OCH2CH3); Anal. Calcd for C29H24O8, C =
69.59, H = 4.83, Found C = 69.61, H = 4.84; EI-MS: m/z (rel. int. %):500 (M+, 42), 324 (95),
295 (25), 279(60), 267(100).
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4.1.16 3,3′-((2-Nitrophenyl)methylene)bis(6-methyl-4-hydroxy-2H-chromen-2-one) (16)
1
Yield: 0.22 g (82%); H-NMR: (DMSO-d6, 300 MHz ): δ 7.56 (m, 4H,H-5/5’/3’’/6’’), 7.37 (m,
,
,
4H, H-7/7’/3’’/6’’), 7.15(d, 2H, J8,7/8 ,7 = 8.4 Hz, H-8/8’), 6.46 (s, 1H, Ar3CH), 2.31 (s, 6H,
CH3); Anal. Calcd for C27H19NO8, C = 66.80, H = 3.95, N = 2.89, Found C = 66.82, H = 4.83, N
= 2.91; EI-MS: m/z (rel. int. %): 485 (M+, 42), 263 (100), 176 (29), 134 (81), 106 (17).
4.1.17 3,3′-((4-Methoxyphenyl)methylene)bis(4-hydroxy-6-methyl-2H-chromen-2-one)
(17)
1
Yield: 0.23 g (84%); H-NMR: (DMSO-d6, 300 MHz): 7.65 (br s, 2H, H-5/5’), 7.38 (dd, 2H,
,
,
, ,
J7,8/7 ,8 = 8.4 , J7,5/7 ,5 = 2.4 Hz, H-7/7’), 7.22 (d, 2H, J8,7/8’,7’ = 8.4 Hz, H-8/8’), 7.01 (d, 2H,
J2’’,3’’/6’’,5’’ = 8.4 Hz, H-2/6), 6.75 (d, 2H, J3’’,2’’/5’’,6’’ = 8.7 Hz, H-3’’/5’’), 6.22 (s, 1H, Ar3CH),
3.68 (s, 3H, OCH3) 2.34 (s, 6H, 2CH3); Anal. Calcd for C28H22O7, C = 71.48, H = 4.71, Found C
= 71.46, H = 4.73; EI-MS m/z (rel. int. %): 470 (M+, 42), 293 (100), 263 (52), 176(16), 134 (47).
4.1.18 3,3′-((4-Methoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(18)
’
’
Yield: 0.23 g (84%); 1H-NMR (DMSO-d6, 300 MHz): δ 7.73 (d, 2H, J5,7/5 ,7 = 2.4 Hz, H-5/5’),
7.54 (dd, 2H, J7,8/7 ,8 = 8.7, J7,5/7 ,5 = 2.4 Hz, H-7/7’),7.32 (d, 2H, J8,7/8 ,7 = 8.7Hz, H-8/8’), 6.98
’
’
’
’
’ ’
= 8.1Hz, H-2’’/6’’),6.73 (d, 2H, J3 ,2 /5 ,6 = 8.7Hz, H-3’’/5’’), 6.15 (s, 1H,
’’ ’’ ’’ ’’
,3 /6 ,5
’’ ’’ ’’ ’’
(d, 2H, J2
Ar3CH), 3.66 (s, 3H,OCH3); Anal. Calcd for C26H16Cl2O7, C = 61.07, H = 3.15, Found C =
61.06, H = 3.16; EI-MS m/z (rel. int. %): 511 (M+, 42), 313 (100), 283 (69), 196(23), 154 (44);
Acknowledgement: The authors are thankful to Higher Education Commission (HEC) Pakistan
for providing financial support under “National Research Program for Universities” to Project
No. 20-1394.
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Scheme Caption
Synthesis of biscoumarin derivatives 1-18
Table Caption
α-Glucosidase inhibitory potential of biscoumarin analogs 1-18
Figure Captions
(Figure-1) Binding mode of acarbose (known inhibitor) in the binding pocket of a developed
homology model of α-glucosidase.
(Figure-2) Binding mode of compound 12 in the binding pocket of developed homology model
of α-glucosidase.
(Figure-3) Binding mode of compound 2 in the binding pocket of developed homology model of
α-glucosidase.
(Figure-4) Binding mode of compound 15 in the binding pocket of developed homology model
of α-glucosidase
(Figure-5) Binding mode of compound 17 in the binding pocket of developed homology model
of α-glucosidase.
16

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Scheme-1
17

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Table-1:
Compound No.
R1
R2
IC50 ± SEMa (µM)
79.18 ± 2.7
6-Me
1
2
NO2
OMe
6-Me
6-Me
385.99 ± 0.65
37.38 ± 0.69
MeO
OMe
3
S
Me
6-Me
6-Cl
52.6 ± 0.21
4
5
OH
NO2
80.94 ± 0.62
6-Cl
6-Cl
6-Cl
113.05 ± 3.43
84.06 ± 5.7
6
7
8
OMe
OH
OH
83.64 ± 3.39
OH
OMe
OH
6-Cl
57.14 ± 0.35
9
OEt
18

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6-Cl
128.14 ± 2.04
91.36 ± 1.16
10
11
MeO
OMe
OMe
6-Cl
6-Cl
OMe
OMe
Cl
16.54 ± 0.36
12
Cl
6-Cl
27.07 ± 0.13
67.96 ± 2.44
13
14
NO2
6-Me
NO2
6-Me
6-Me
6-Me
221.6 ± 2.47
128.6 ± 1.16
106.63 ± 1.61
15
16
17
OEt
OH
NO2
OMe
6-Cl
75.74 ± 1.11
18
OMe
Acarbose
-
906 ± 6.387
-
SEMa is the standard error of the mean, Acarbose is standard inhibitor for α-glucosidase
19

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Figure-1:
20

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Figure-2:
21

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Figure-3:
22

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Figure-4:
23

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Figure-5:
24

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Highlights:
ꢀ Synthesis of biscoumarion derivatives
ꢀ In vitro -glucosidase inhibitory activity
α
ꢀ Identification of a novel class of
ꢀ Molecular Docking studies
α-glucosidase inhibitors
ꢀ All compounds found to be potent

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Representative 1HNMR, 13CNMR and EI MS spectrum
1HNMR of Compound 3

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13 CNMR of Compound 3

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13 CNMR (90) of Compound 3