Pancreatic α-Amylase inhibition and free radical scavenging activity of substituted pyranochromenone derivatives

Article abstract of DOI:10.1007/s00044-013-0867-y

Pyranochromenone derivatives 3a-d, 6a-j and 2H-chromenones 8a-b were synthesized and screened for their in vitro α-Amylase inhibitory and ABTS^?+ [2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] free radical scavenging activities. Compounds 3a, 3c, and 6d displayed dual function of ABTS^?+ radical scavenging as well as α-Amylase inhibition. Compound 6h was found to be most potent α-Amylase inhibitor in present series of compounds. Docking studies suggest that these compounds occupy active site of the human pancreatic α-Amylase similar to that of acarbose which inhibits enzyme by hydrophobic interactions. These compounds have potential to be developed as therapeutics targeted against diet-induced hyperglycemia in diabetes. Graphical abstract: Series of pyranochromenone derivatives 3a-d, 6a-j, and 8a-b were synthesized, among these compound 6h shown potent intestinal α-Amylase inhibitory activity. Compounds 3a, 3c, and 6d were shown dual properties such as α-Amylase inhibitory and antioxidant activities. These derivatives may serve as a model compounds for design and development of therapeutics based agents.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0867-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2821–2833
DOI 10.1007/s00044-013-0867-y
ORIGINAL RESEARCH
Pancreatic a-amylase inhibition and free radical scavenging
activity of substituted pyranochromenone derivatives
•
J. Ashok Kumar Ashok K. Tiwari G. Saidachary Chandan Kishor
•
•
•
•
•
•
•
D. Anand Kumar Zehra Ali B. Sridhar Anthony Addlagatta
B. China Raju
Received: 1 April 2013 / Accepted: 22 October 2013 / Published online: 13 November 2013
ꢀ Springer Science+Business Media New York 2013
Abstract Pyranochromenone derivatives 3a–d, 6a–j and
2H-chromenones 8a–b were synthesized and screened for
their in vitro a-amylase inhibitory and ABTS•? [2,20-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] free
radical scavenging activities. Compounds 3a, 3c, and 6d
displayed dual function of ABTS•? radical scavenging as
well as a-amylase inhibition. Compound 6h was found to
be most potent a-amylase inhibitor in present series of
compounds. Docking studies suggest that these compounds
occupy active site of the human pancreatic a-amylase
similar to that of acarbose which inhibits enzyme by
hydrophobic interactions. These compounds have potential
to be developed as therapeutics targeted against diet-
induced hyperglycemia in diabetes.
Introduction
Incidence of diabetes is continuously increasing world-
wide. Postprandial hyperglycemia (PPHG) has emerged as
a prominent and early defect in ensuing type 2 diabetes
mellitus (Carrol et al., 2003) and identified as an inde-
pendent predictor for the development of future cardio-
vascular diseases (O’Keefe et al., 2008). PPHG has also
been reported as an important and independent risk factor
contributing to the development of atherosclerosis and
amplifying generation of oxidative stress (Ceriello, 2000).
Therefore, development of therapeutics that mitigate
postprandial hyperglycemia and scavenge free radicals
may become mode of treatment in the future. Indian eth-
nicity has been observed to posses increased prevalence of
PPHG than other ethnic groups around the world (Milic-
evic et al., 2008). Proportion of high carbohydrate in the
diet of Asians and South Asian in particular, has been
identified as major contributory factor for this increase
(Misra and Khurana, 2011; Tiwari et al., 2012). Inhibitors
of carbohydrate hydrolyzing enzymes like pancreatic a-
amylase and intestinal a-glucosidase, recently have been
advocated suitable therapeutic targets to control the
increase in postprandial hyperglycemic excursion in such
populations (Scheen, 2009).
Keywords Pyranochromenone derivatives ꢀ
Chroman-6-carbaldehyde ꢀ Chroman-7-ol ꢀ
a-Amylase inhibition ꢀ ABTS•? scavenging ꢀ
Molecular modeling
J. Ashok Kumar ꢀ G. Saidachary ꢀ B. C. Raju
Natural Products Chemistry Division, CSIR-Indian Institute of
Chemical Technology, Hyderabad 500 007, India
e-mail: chinaraju@iict.res.in
A. K. Tiwari (&) ꢀ D. A. Kumar ꢀ Z. Ali
Recently, we have identified a-glucosidase inhibitors
and established their structure activity relationship (SAR)
(Rao et al., 2012). In another study, three different activi-
ties such as a-glucosidase inhibition, free radical scav-
enging, and antihyperglycemic potentials were explored for
the chromenone derivatives (Raju et al., 2010; Ashok
Kumar et al., 2011). In the present study we report syn-
thesis of pyranochromenone derivatives, their a-amylase
inhibition and ABTS•? radical scavenging activities. In
addition, we employed docking studies to understand the
Medicinal Chemistry and Pharmacology Division, CSIR-Indian
Institute of Chemical Technology, Hyderabad 500 007, India
e-mail: tiwari@iict.res.in
C. Kishor ꢀ A. Addlagatta (&)
Centre for Chemical Biology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India
e-mail: anthony@iict.res.in
B. Sridhar
Center for X-ray Crystallography, CSIR-Indian Institute of
Chemical Technology, Hyderabad 500 007, India
123

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Med Chem Res (2014) 23:2821–2833
SAR in identifying the groups responsible for biological
activities.
ethyl 4-chloro-3-oxobutanoate, and ethyl 4,4,4-trifluoro-3-
oxobutanoate in H2SO4 afforded corresponding pyrano-
chromenone derivatives 6a–j (Scheme 2).
Compounds 8a–b were prepared by Pechmann con-
densation of 7a–b with ethyl 4,4,4-trifluoro-3-oxobutano-
ate in presence of H2SO4 (Scheme 3).
Chemistry
Results and discussion
Thus synthesized compounds 3a–d, 6a–j, and 8a–
b were characterized by NMR and IR spectral methods and
the data presented in Table 1. Further, 19F NMR was
recorded for compounds 6d and 6h (Figs. 1, 2). Two
compounds 3c and 6h were crystallized in CHCl3 and their
structures were determined by X-ray crystallography
(Figs. 3, 4; Table 2) (Smart and Saint, 2001; Sheldrick,
2008).
Scheme 1 illustrates the synthetic pathway to obtain
3-substituted pyranochromenone derivatives 3a–d. Con-
densation of 2,4-dihydroxybenzaldehyde 1 and 2-methyl-
but-3-ene-2-ol in ascorbic acid with 5 % citric acid
(Molyneux and Jurd, 1970) afforded 7-hydroxy-2,2-dime-
thylchromancarbaldehyde 2 (Method-A) in low yield.
However, condensation (Sharma et al., 1980) of 2,4-dihy-
droxybenzaldehyde 1 and 2-methylbut-3-ene-2-ol with
BF3.O(C2H5)2 (Method-B) in dry 1,4-dioxane afforded 2 in
moderate yield. Knoevenagel condensation of compound 2
with various b-ketoesters such as ethyl 3-oxobutanoate,
ethyl 3-oxo-3-phenylpropanoate and diethyl malonate,
ethyl 2-cyanoacetate in piperidine (20 mol%) (Raju et al.,
2010) at room temperature (RT) afforded target com-
pounds 3a–d.
Biological activity
ABTS•? Scavenging activity
Synthesized compounds 3a–d, 6a–j, and 8a–b were
screened for their 2,20-azino-bis(3-ethylbenzothiazoline-6-
CF3
Substituted 7,8-dihydro-2H,6H-pyrano[3,2-g]chromene-
2-ones 6a–j were prepared by the condensation (Molyneux
and Jurd, 1970) of resorcinols 4a–b with 2-methylbut-3-
ene-2-ol in ascorbic acid with 5 % citric acid (Method-A)
provided 2,2-dimethylchromans 5a–b. Pechmann conden-
sation (Raju et al., 2010; Ahluwalia and Jolly, 1982; Pathak
et al., 1982) of 5a–b with various b-ketoesters such as
ethyl 3-oxobutanoate, ethyl 3-oxo-3-phenylpropanoate,
O
O
F3C
OC2H5
H2SO4
HO
OH
7a-b
HO
O
O
R
R
8a-b
8a R = H,
8b R = CH3
Scheme 3 Synthesis of compounds 8a–b
Scheme 1 Synthesis of
pyranochromenones 3a–d
O
O
5% Citric acid
Ascorbic acid
or
BF3.O(C2H5)2
1,4-Dioxane
R
O
H
H
C
O
H
H
C
O
H
O
R
R1
+
Piperidine
O
O
O
O
H
O
O
2
3a-d
1
=
a
b
c
3
3
3
R
R
R
H
COC ,
3
CO2C2
=
=
=
H5
,
,
H5
COC6
3d R
N
C
R1
Scheme 2 Synthesis of
pyranochromenones 6a–j
O
O
R2
O
5% Citric acid
Ascorbic acid
OH
R1
OC2H5
+
R2
H2SO4
O
O
O
OH
HO
OH
4a-b
R
R
R
6a-j
5a-b
4a R = H
4b R = CH3
5a R = H
5b R = CH3
6a R = R2 = H, R1 = CH3,
6b R = R2 = H, R1 = C6H5,
6c R = R2 = H, R1 = CH2Cl,
6d R = R2 = H, R1 = CF3,
6e R = R1 = CH3, R2 = H,
6f R = CH3, R1 = C6H5, R2 = H,
6g R = CH3, R1 = CH2Cl, R2 = H,
6h R = CH3, R1 = CF3, R2 = H,
6i R = H, R1 = CH3, R2 = Cl,
6j R = CH3, R1 = CH3, R2 = Cl,
123

Med Chem Res (2014) 23:2821–2833
2823
Table 1 Synthesis of 7,8-dihydro-2H,6H-pyrano[3,2-g]chromenone derivatives and their activity profiles
Entry
1
Compound
ABTS scavenging (%) (IC50, lM)
a-Amylase inhibition (%) (IC50, lM)
90.8 (8.3)
42.29 (11.8)
O
O
O
O
O
O
3a
3b
2
3
4
5
6
7
NS
39.30 (NF)
42.30 (9.9)
9.43 (NF)
OC2H5
O
O
O
89.94 (8.1)
Ph
O
O
O
O
O
3c
3d
NS
CN
O
10.93
12.05
NS
35.70 (15.5)
36.56 (19.7)
31.43 (NF)
O
O
O
O
O
6a
6b
Ph
O
CH2Cl
O
O
O
6c
123

2824
Med Chem Res (2014) 23:2821–2833
Table 1 continued
Entry
8
Compound
ABTS scavenging (%) (IC50, lM)
a-Amylase inhibition (%) (IC50, lM)
90.62 (5.6)
49.3 (7.8)
CF3
O
O
O
O
O
6d
9
NS
31.54 (NF)
51.34 (NF)
30.59 (11.8)
44.92 (4.9)
46.40 (45.0)
40.88 (NF)
O
6e
10
11
12
13
14
NS
Ph
O
O
O
O
6f
34.79 (29.7)
CH2Cl
O
O
6g
20.30
CF3
O
O
O
O
6h
6i
58.41 (14.9)
Cl
O
O
NS
Cl
O
O
O
6j
123

Med Chem Res (2014) 23:2821–2833
2825
Table 1 continued
Entry
15
Compound
ABTS scavenging (%) (IC50, lM)
a-Amylase inhibition (%) (IC50, lM)
98.26 (3.6)
9.13 (NF)
CF3
HO
O
O
O
8a
8b
16
97.57 (4.1)
3.18 (NF)
CF3
HO
O
Compounds displaying both the activities more than 10 % at primary screening concentration were followed for determination of radical
scavenging concentration 50 % (SC50) and enzyme inhibitory concentration 50 % (IC50
)
NS no scavenging activity, NF not followed, % scavenging or inhibition at 5 mg/ml concentration of each compound
sulfonic acid) (ABTS•?) radical scavenging activity
a-Amylase inhibitory activity
described by Walker and Everette method (2009) with
The ABTS•? activity encouraged us to evaluate a-amylase
suitable modifications. Percentage ABTS•? scavenging
activity of compounds and their IC50 values are presented
in Table 1. The IC50 values ranged from 3.6 to 29.7 lM
and the order of compounds potency in decreasing order
was 8a (3.6 lM), 8b (4.1 lM), 6d (5.6 lM), 3c (8.1 lM),
3a (8.3 lM), and 6i (14.9 lM). Compound 8a displayed
potent ABTS•? scavenging activity, whereas 3b, 3d, 6a–
c, 6e–f, and 6j did not. The presence of acetyl and ben-
zoyl substitution on pyranochromenone at 3rd position
substantially increased the ABTS•? scavenging activity;
however, carbethoxy and nitrile substitution could not
display the activity. Trifluoromethyl substitution at 4th
position on pyranochromenone 6d displayed potent
activity when compared to methyl 6a, phenyl 6b, and
chloromethyl 6c. However, the disubstituted pyrano-
chromenones 6e–6h, chloromethyl substitution 6g dis-
played better ABTS•? scavenging activity when com-
pared to trifluoromethyl 6h. This fact is further supported
by the observation that the absence of methyl substitution
at 10th position on pyranochromenone 6i enhanced the
activity along with 3-chloro-4-methyl substitution when
compared to 6j. Compounds without pyran ring on
chromenone 8a–b having trifluoromethyl at 4th position,
hydroxyl at 7th position enhanced the ABTS•? activity.
We observed that these compounds were identified as
potent ABTS•? scavengers in present series. It is inter-
esting to note that in general in present series of com-
pounds, trifluoromethyl containing 6d, 8a, and 8b have
better ABTS•? scavenging activity compared to chloro-
methyl substitution 6c and 6g.
inhibitory activity (Ali et al., 2006) for chromenones 3a–d,
6a–j, and 8a–b. Based on IC50 values (Table 1) acetyl 3a
(11.8 lM) and benzoyl 3c (9.9 lM) substitution at 3rd
position on pyranochromenone displayed better a-amylase
inhibitory activity when compared to carbethoxy 3b and
nitrile 3d. Further, trifluoromethyl substitution at 4th
position on pyranochromenone 6d (7.8 lM) improved a-
amylase inhibitory activity compared to chloromethyl
substitution 6c. However, methyl 6a (15.5 lM) and phenyl
6b (19.7 lM) substitutions displayed moderate activity.
Presence of methyl substitution on pyranochromene 6h
(4.9 lM) having trifluoromethyl substitution at 4th position
increased a-amylase inhibitory activity and was observed
to be the most potent a-amylase inhibitor in present series
of compounds. Whereas chloromethyl substitution 6g dis-
played moderate a-amylase inhibitory activity; however,
methyl 6e and phenyl 6f could not display. Compound 6i
(45 lM) displayed mild a-amylase inhibitory activity with
3-chloro and 4-methyl substitution on pyranochromene;
however, the presence of methyl at 10th position 6j did not
show inhibition.
Inthe a-amylaseinhibitory activity, the presenceof methyl
substitution at 10th position on 6g and 6h enhanced the
activity when compared to 6c and 6d without methyl substi-
tution, however, methyl (6e) and phenyl (6f) could not inhibit
enzyme activity when compared to 6a and 6b without methyl
substitution. Explanations may be that the two different
aspects are ruling the binding of these eight molecules in
displaying a-amylase activity. Regarding compounds 6a and
123

2826
Med Chem Res (2014) 23:2821–2833
Fig. 1 19F NMR of compound
6d
Fig. 2 19F NMR of compound
6h
6b versus 6e and 6f, substitutions at 4th position are hydro-
phobic (methyl and phenyl) and would prefer to be buried in
the enzyme’s hydrophobic environment. The methyl substi-
tution at 10th position in compounds 6e and 6f prevents the
molecules to be buried in the pocket (based on the modeling)
hence they displayed low activity. However, in the case of
compounds 6a and 6b, the absence of methyl at 10th position
may present better opportunity to be buried in the enzyme
pocket for better inhibition of the a-amylase. In the case of
compoundshavingchloromethylortrifluoromethylgroupson
123

Med Chem Res (2014) 23:2821–2833
2827
Table 2 Crystal data and structure refinement
Compound reference
3c
6h
Chemical formula
Formula mass
C21H18O4
334.35
C16H15F3O3
312.28
Crystal system
Monoclinic
30.791(3)
8.1137(6)
13.6677(10)
90.00
Triclinic
˚
a/A
8.0741(14)
8.8963(16)
11.1140(19)
75.157(3)
82.725(3)
87.948(3)
765.5(2)
˚
b/A
˚
c/A
a/ꢁ
b/ꢁ
c/ꢁ
101.786(2)
90.00
3
˚
Unit cell volume/A
3,342.6(4)
294(2)
Fig. 3 The molecular structure of 3c with atom-numbering. Dis-
placement ellipsoids are drawn at the 30 % probability level
Temperature/K
294(2)
ꢀ
P 1
Space group
C2/c
No. of formula units per unit cell, Z
No. of reflections measured
No. of independent reflections
Rint
8
2
15,568
7,106
2,676
0.0180
0.0434
0.1164
0.0465
0.1206
874167
2,947
0.0218
Final R1 values (I [ 2r(I))
Final wR(F2) values (I [ 2r(I))
Final R1 values (all data)
Final wR(F2) values (all data)
CCDC number
0.0658
0.2046
0.0727
0.2146
874166
Molecular modeling results
Although compounds 3c and 6d displayed both a-amy-
lase inhibition and antioxidant activity, 6h was identified
to have potent a-amylase inhibition in present series of
compounds. Therefore, we selected compounds 6d and
6h to study the mechanism of their binding. Molecular
docking of 6d and 6h was carried out against the human
pancreatic a-amylase (HPA) based on the crystal struc-
ture (PDB code: 1CPU; Fig. 5) (Brayer et al., 2000).
AutoDock results suggest that both compounds occupy
active sites of HPA domain similar to known a-amylase
inhibitor Acarbose (Brayer et al., 2000). In the active
site of HPA, 6d and 6h were surrounded by Trp58,
Trp59, Tyr62, Gln63, His101, Thr163, Leu165, Asp197,
His299, His305, and Asp300. The dihydropyran moiety
Fig. 4 The molecular structure of 6h with atom-numbering. Dis-
placement ellipsoids are drawn at the 30 % probability level
chromenone (6c, 6d vs. 6g, 6h) at 4th position, the presence of
electron withdrawing groups may have dominating role
masking the methyl substitution at 10th position.
In contrast to 2H-chromenones 8a–b, pyranochrome-
nones displayed significant a-amylase inhibition. In the
order of potency, 6h was found to be most potent a-amylase
inhibitor followed by 6d and 3c in present series of com-
pounds. Interestingly in contrast to the structural require-
ment for better ABTS•? scavenging activity, presence of
methyl substitution on pyranochromene 6h appears advan-
tageous in improving the a-amylase inhibitory activity.
Presence of a-amylase inhibition as well as antioxidant
(ABTS•? free radical scavenging activity) in one molecule
may hold better therapeutic strategy in mitigating develop-
ment of PPHG and oxidative stress in diabetics. Compounds
3c, 6d, and 6h may hold promise for development of better
therapeutics with these biological activities.
´
˚
of both compounds form hydrogen bonds (3.0 A) with
Gln63 and also are involved in p–p interaction with
indole ring of Trp59. The chromenone moieties of these
compounds were found sandwiched between Leu165 and
Tyr62. From the packing of these molecules it is clear
that there is more space in the enzyme pocket near the
chromenone binding region.
123

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Med Chem Res (2014) 23:2821–2833
Fig. 5 Molecular modeling of
pancreatic alpha-amylase and
interaction with compounds
Conclusion
instrument using CDCl3 as solvent (unless otherwise indi-
cated) and TMS as internal standard. The chemical shift (d)
are given in units of parts per million (ppm). Coupling
constants (J) are quoted in Hz. 13C NMR spectra were
recorded on Avance 300 MHz instrument and the chemical
shift data for each signal are given d in units of ppm. 19F
NMR spectra were recorded with a Varian Innova
300 MHz instrument using internal standard. The chemical
shift data for each signal are given as d in units of ppm
relative to an external standard CF3COOH. IR spectra were
recorded with a Thermo Nicollet 740 FTIR spectrometer as
thin films are reported in wavenumbers (cm-1). Mass
spectra were obtained on Agilent LCMS instrument.
Melting points were determined in open glass capillary
tubes on a Metler FP 51 melting point apparatus and are
uncorrected. Flash column chromatography was carried out
using on silica gel (Merck 60–120 and 100–200 mesh).
Compounds 3a–d, 6a–j, and 8a–b were synthesized
applying Knoevenagel and Pechmann condensation
approaches. ABTS•? [2,20-azino-bis(3-ethylbenzothiazo-
line-6-sulfonic acid)] free radical scavenging and in vitro
a-amylase inhibitory activities to all these chromenone
derivatives is new in the present study. Compound 6d
reported in this study possess intestinal a-amylase inhibi-
tion as well as ABTS•? free radical scavenging activities.
Therefore, it may serve as a model compound for design
and development of therapeutic applications both for free
radical scavenging and a-amylase inhibition. This study
further presents pyranochromenone derivatives as new
class of a-amylase inhibitors and ABTS•? free radical
scavengers.
Experimental section
Typical procedure for the preparation of 7-hydroxy-2,
2-dimethyl-6-chromanecarbaldehyde (2)
General
Method-A
Chemicals were purchased from Sigma-Aldrich and the
reactions were carried out using reagent-grade solvents
used without purification. Thin layer chromatography
(TLC) was carried out on pre-coated Merck silica gel 60
F254 plates and visualized under a UV lamp. 1H NMR
spectra were recorded with a Varian Innova 300 MHz
Resorcinaldehyde (1, 0.5 g, 3.6 mmol) and 2-methyl-3-
buten-2-ol (0.22 g, 2.5 mmol) were refluxed in ascorbic
acid (50 mg) with 5 % citric acid (10 ml) for 18 h. The
contents were diluted with ethyl acetate (3 9 30 ml) and
extracted, the organic layer dried over anhydrous Na2SO4,
123

Med Chem Res (2014) 23:2821–2833
2829
solvent was removed under reduced pressure, and the crude
product was purified by column chromatography afforded
7-hydroxy-2,2-dimethyl-6-chromanecarbaldehyde 2 as color-
less solid in 32 % yield.
14), 26.93 (acetyl CH3), 21.77 (C-6); Mass (ESI): m/z 273
(M ? H)?, 295 (M ? Na)?.
Ethyl-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]
chromene-3-carboxylate (3b)
Method-B
Pale yellow solid; 82 % yield; m.p, 154–156 ꢁC; IR (KBr)
m (cm-1): 3056, 2970, 2919, 1732, 1697, 1622, 1561, 1229,
BF3.O(C2H5)2 (1.2 eq) was added to a stirred solution of
resorcinaldehyde (0.5 g, 3.6 mmol), 2-methylbut-3-ene-2-
ol (0.2 g, 2.5 mmol) in dry 1,4-dioxane at 0 ꢁC. The con-
tents were stirred for another 30 min at same temperature;
slowly the reaction mixture was brought to RT and stirred
for another 4 h. After completion of reaction (TLC), the
reaction mixture was cooled to 0 ꢁC, quenched with water
(10 ml), the contents were extracted with chloroform
(3 9 30 ml). The layers were separated, the organic layer
was dried over anhydrous Na2SO4, solvent was removed
under reduced pressure and the crude product was puri-
fied by column chromatography afforded 7-hydroxy-2,
2-dimethyl-6-chromanecarbaldehyde 2 as colorless solid in
38 % yield. IR (KBr) m (cm-1): 3400, 2976, 1638, 1585,
1
1120, 1026 cm-1; H NMR (CDCl3, 300 MHz): d 8.40 (s,
1H, aromatic), 7.28 (s, 1H, aromatic), 6.70 (s, 1H, aro-
matic), 4.38 (q, 2H, OCH2), 2.85 (t, 2H, J = 6.79 Hz,
CH2), 1.88 (t, 2H, J = 6.79 Hz, CH2), 1.38 (s, 6H, 2CH3),
1.40 (t, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d 163.59
(C=O, ester), 160.43 (C=O, lactone), 157.45 (C-9a), 155.49
(C-10a), 148.96 (C-4), 130.21 (C-5), 119.29 (C-5a), 104.23
(C-10), 76.58 (C-8), 61.51 (C-12, OCH2), 32.15 (C-7),
26.90 (C-13, 14), 21.75 (C-6), 14.23 (C-13, OCH2CH3);
Mass (ESI): m/z 303 (M ? H)?. 325 (M ? Na)?.
3-Benzoyl-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]
chromen-2-one (3c)
1
1490, 1252, 1114 cm-1; H NMR (CDCl3, 300 MHz): d
11.00 (s, 1H, OH), 9.62 (s, 1H, CHO), 7.08 (s, 1H, aro-
matic), 6.22 (s, 1H, aromatic), 2.78 (t, 2H, J = 6.79 Hz,
CH2), 1.82 (t, 2H, J = 6.79 Hz, CH2), 1.40 (s, 3H, CH3);
13C NMR (CDCl3, 75 MHz): d 194.19 (CHO), 162.14 (C-
9), 162.04 (C-7), 135.30 (C-5), 115.23 (C-6), 113.78 (C-
10), 104.38 (C-8), 76.27 (C-2), 32.57 (C-3), 26.98 (C-2,
2CH3), 21.45 (C-4); Mass (ESI): m/z 207 (M ? H)?.
Pale yellow solid; 86 % yield; IR (KBr) m (cm-1): 2968,
2923, 1729, 1653, 1623, 1563, 1371, 1227, 1115 cm-1; 1H
NMR (CDCl3, 300 MHz): d 8.06 (s, 1H, aromatic), 7.85 (d,
2H J = 8.20 Hz, aromatic), 7.55–7.62 (m, 1H, aromatic),
7.48–7.50 (m, 2H, aromatic), 7.38 (s, 1H, aromatic), 6.78
(s, 1H, aromatic), 2.88 (t, 2H, J = 6.79 Hz, CH2), 1.88 (t,
2H, J = 6.79 Hz, CH2), 1.42 (s, 6H, 2CH3); 13C NMR
(CDCl3, 75 MHz): d 192.17 (C=O, benzyloxy), 159.90
(C=O, lactone), 158.99 (C-9a), 155.17 (C-10a), 146.46 (C-
4), 136.94 (C-12), 133.22 (C-15), 129.96 (C-13,17), 129.44
(C-14,16), 128.36 (C-3), 122.46 (C-5), 119.35 (C-5a),
111.49 (C-4a), 104.51 (C-10), 76.50 (C-8), 32.26 (C-7),
26.92 (C-18,19), 21.83 (C-6); Mass (ESI): m/z 335
(M ? H)?.
General procedure for the preparation of 7,8-dihydro-
2H,6H-pyrano[3,2-g]chromene-3-carboxylate (3a–d)
Piperidine (20 mol%) was added to a stirred solution of
7-hydroxy-2,2-dimethyl-6-chromanecarbaldehyde (2, 1 mmol),
and ethyl 3-oxobutanoate (0.143 g, 1.1 mmol) in CH3CN
(4 ml) at RT. The contents were stirred for 4–6 h at the
same temperature. After completion of the reaction (TLC),
the solvent was removed under reduced pressure and the
crude product was purified by column chromatogra-
phy afforded 3-acetyl-8,8-dimethyl-7,8-dihydro-2H,6H-
pyrano[3,2-g]chromen-2-one 3a as pale yellow solid in
90 % yield; m.p, 158–160 ꢁC; IR (KBr) m (cm-1): 2924,
2853, 1720, 1681, 1621, 1552, 1430, 1225, 1024 cm-1. 1H
NMR (CDCl3, 300 MHz): d 8.40 (s, 1H, aromatic), 7.35 (s,
1H, aromatic), 6.70 (s, 1H, aromatic), 2.88 (t, 2H,
J = 6.79 Hz, CH2), 2.70 (s, 3H, COCH3), 1.88 (t, 2H,
J = 6.79 Hz, CH2), 1.45 (s, 3H, CH3), 1.40 (s, 3H, CH3);
13C NMR (CDCl3, 75 MHz): d 195.72 (C=O, keto), 160.65
(C=O, lactone), 157.32 (C-9a), 155.76 (C-10a), 147.76 (C-
4), 131.02 (C-3), 120.23 (C-5), 119.55 (C-5a), 111.65 (C-
4a), 104.18 (C-10), 76.71 (C-8), 32.16 (C-7), 30.61 (C-13,
8,8-Dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]
chromene-3-carbonitrile (3d)
Pale yellow solid; 74 % yield; m.p, 203–204 ꢁC; IR (KBr)
m (cm-1): 2975, 2934, 2231, 1723, 1713, 1617, 1556, 1371,
1
1287, 1117 cm-1; H NMR (CDCl3, 300 MHz): d 8.02 (s,
1H, aromatic), 7.20 (s, 1H, aromatic), 6.72 (s, 1H, aro-
matic), 2.85 (t, 2H, J = 6.79 Hz, CH2), 1.88 (t, 2H,
J = 6.79 Hz, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 161.45 (C=O, lactone), 154.94 (C-9a), 151.34
(C-10a), 148.37 (C-5), 129.99 (C-5a), 124.00 (C-4), 120.23
(CN), 114.45 (C-4a), 110.58 (C-3), 105.01 (C-10), 70.00
(C-8), 31.99 (C-7), 26.92 (C-12,13), 21.73 (C-6); Mass
(ESI): m/z 278 (M ? Na)?.
123

2830
Med Chem Res (2014) 23:2821–2833
Typical procedure for the preparation of 2,2-dimethyl-
125.05 (C-5), 117.98 (C-5a), 113.10 (C-3), 111.66 (C-4a),
104.51 (C-10), 75.61 (C-8), 32.44 (C-7), 26.81 (C-12,13),
22.05 (C-6), 18.59 (C-11, CH3); Mass (ESI): m/z 255
(M ? H)?.
7-chromanol (5a)
Resorcinol (4a, 0.5 g, 4.5 mmol) and 2-methyl-3-buten-2-ol
(0.27 g, 3.1 mmol) were refluxed in ascorbic acid (50 mg)
with 5 % citric acid (10 ml) for 12 h. The contents were
diluted with ethyl acetate (3 9 30 ml) and extracted, the
organic layer dried over anhydrous Na2SO4, solvent was
removed under reduced pressure, and the crude product
was purified by column chromatography afforded 2,2-
dimethyl-7-chromanol 5a as colorless solid in 78 % yield.
1H NMR (CDCl3, 300 MHz): d 6.82 (d, 1H, J = 8.30 Hz,
aromatic), 6.26 (dd, 1H, J = 8.03, 2.26 Hz, aromatic), 6.18
(d, 1H, J = 3.02 Hz, aromatic), 4.98 (bs, 1H, OH), 2.68 (t,
2H, J = 6.79 Hz, CH2), 1.76 (t, 2H, J = 6.79 Hz, CH2),
1.32 (s, 6H, 2CH3); 13C NMR (CDCl3, 75 MHz): d 158.80
(C-7), 157.20 (C-9), 131.00 (C-5), 120.60 (C-10), 110.10
8,8-Dimethyl-4-phenyl-7,8-dihydro-2H,6H-pyrano[3,2-g]
chromen-2-one (6b)
Colorless solid; 76 % yield; IR (KBr) m (cm-1): 2972,
2928, 1723, 1621, 1555, 1443, 1369, 1286, 1145, 1113,
1
952 cm-1; H NMR (CDCl3, 300 MHz): d 7.40–7.52 (m,
5H, aromatic), 7.05 (s, 1H, aromatic), 6.78 (s, 1H, aro-
matic), 6.12 (s, 1H, aromatic), 2.74 (t, 2H, J = 6.79 Hz,
CH2), 1.80 (t, 2H, J = 6.79 Hz, CH2), 1.38 (s, 6H, 2CH3);
13C NMR (CDCl3, 75 MHz): d 192.17 (C=O, lactone),
159.90 (C-4), 155.17 (C-9a), 146.46 (C-10a), 136.94 (C-
11), 133.22 (C-13,15), 129.96 (C-14), 129.44 (C-5), 128.36
(C-12,16), 122.46 (C-5a), 119.35 (C-3), 111.49 (C-4a),
104.51 (C-10), 76.50 (C-8), 32.26 (C-7), 26.92 (C-17,18),
21.83 (C-6); Mass (ESI): m/z 307 (M ? H)?.
(C-6), 98.40 (C-8), 73.9 (C-2), 32.7 (C-3), 26.5 (C-2,
?
methyls), 22.3 (C-4); Mass (ESI): m/z 179 (M ? H)
.
2,2,8-Trimethyl-7-chromanol (5b)
1
Pale yellow solid; 82 % yield; m.p, 168–170 ꢁC; H NMR
(CDCl3, 300 MHz): d 6.70 (d, 1H, J = 8.24 Hz, aromatic),
6.24 (d, 1H, J = 8.24 Hz, aromatic), 4.45 (s, 1H, OH), 2.70
(t, 2H, J = 6.40 Hz, CH2), 2.04 (s, 3H, CH3), 1.75 (t, 2H,
J = 6.40 Hz, CH2), 1.32 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 152.62 (C-9), 152.54 (C-7), 126.38 (C-10),
112.95 (C-5), 114.40 (C-8), 106.53 (C-6), 74.04 (C-2),
32.80 (C-3), 27.02 (C-2, methyls), 22.16 (C-4), 7.94 (C-8,
methyl); Mass (ESI): m/z 193 (M ? H)?.
4-(Chloromethyl)-8,8-dimethyl-7,8-dihydro-2H,
6H-pyrano[3,2-g]chromen-2-one (6c)
Pale yellow solid; 68 % yield; m.p, 179–181 ꢁC; IR (KBr)
m (cm-1): 2972, 2931, 2851, 1713, 1627, 1437, 1404, 1371,
1
1275, 1157, 1080 cm-1; H NMR (CDCl3, 300 MHz): d
7.28 (s, 1H, aromatic), 6.71 (s, 1H, aromatic), 6.29 (s, 1H,
aromatic), 4.56 (s, 2H, CH2Cl), 2.85 (t, 2H, J = 6.79 Hz,
CH2), 1.85 (t, 2H, J = 6.79 Hz, CH2), 1.37 (s, 6H, 2CH3);
13C NMR (CDCl3, 75 MHz): d 161.01 (C=O, lactone),
155.96 (C-4), 151.80 (C-9a), 153.81 (C-10a), 149.38 (C-5),
124.60 (C-5a), 118.50 (C-10), 112.36 (C-3), 110.27 (C-4a),
104.97 (C-9), 75.91 (C-8), 41.37 (C-11, CH2Cl), 32.30 (C-
7), 26.86 (C12,13), 22.11 (C-6). Mass (ESI): m/z 279, 281
(M ? H)?.
General procedure for the preparation of substituted
7,8-dihydro-2H,6H-pyrano[3,2-g]chromene-2-ones
(6a–l)
H2SO4 (10 eq) was added slowly at 0 ꢁC to a stirred
solution of 2,2-dimethyl-7-chromanol (5a, 1.0 mmol) and
ethyl 3-oxobutanoate (1.20 mmol) for 15 min. The con-
tents were slowly brought to RT and stirring continued for
another 10 h. After completion of the reaction (TLC), the
reaction mixture cooled to 0 ꢁC, the ice pieces were added
to the reaction mixture to quench the excess acid. The
compound was fallen out as colorless solid, the solid was
filtered, and washed with cold water, the crude solid was
purified by column chromatography to give 4,8,8-tri-
methyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one
8,8-Dimethyl-4-(trifluoromethyl)-7,8-dihydro-2H,
6H-pyrano[3,2-g]chromen-2-one (6d)
Colorless solid; 74 % yield; m.p, 172–174 ꢁC; IR (KBr) m
(cm-1): 2976, 2934, 1735, 1626, 1563, 1437, 1395, 1287,
1
1169, 1146, 1120 cm-1; H NMR (CDCl3, 300 MHz): d
7.35 (s, 1H, aromatic), 6.78 (s, 1H, aromatic), 6.52 (s, 1H,
aromatic), 2.85 (t, 2H, J = 6.79 Hz, CH2), 1.88 (t, 2H,
J = 6.79 Hz, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 159.67 (C=O, lactone), 158.70 (C-9a), 154.40
(C-4), 125.76 (C-10a), 123.48 (C-5), 119.26 (CF3), 111.92
(C-5a), 111.85 (C-3), 106.50 (C-4a), 105.18 (C-10), 76.32
(C-8), 32.32 (C-7), 26.88 (C-12,13), 22.11 (C-6); 19F NMR
(CDCl3): d -64.89 ppm. Mass (ESI): m/z 299 (M ? H)?.
1
6a as pale yellow solid in 80 % yield; H NMR (CDCl3,
300 MHz): d 7.20 (s, 1H, aromatic), 6.68 (s, 1H, aromatic),
6.02 (s, 1H, aromatic), 2.84 (t, 2H, J = 6.79 Hz, CH2),
2.38 (s, 3H, CH3), 1.85 (t, 2H, J = 6.79 Hz, CH2), 1.38 (s,
6H, 2CH3); 13C NMR (CDCl3, 75 MHz): d 160.54 (C=O,
lactone), 157.45 (C-9a), 153.35 (C-4), 152.33 (C-10a),
123

Med Chem Res (2014) 23:2821–2833
2831
4,8,8,10-Tetramethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]
J = 6.79 Hz, CH2), 2.26 (s, 3H, CH3), 1.88 (t, 2H,
J = 6.79 Hz, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 159.93 (C=O, lactone), 156.50 (C-4), 152.06
(C-9a), 123.59 (C-10a), 122.62 (C-5), 119.94 (C-11, CF3),
118.30 (C-10), 114.09 (C-5a), 111.28 (C-3), 106.03 (C-4a),
76.03 (C-8), 32.20 (C-7), 27.00 (C-12,13), 22.36 (C-6),
8.08 (C-14,CH3); 19F NMR (CDCl3): d -64.59 ppm; Mass
(ESI): m/z 313, 314 (M ? H)?, 335 (M ? Na)?.
chromen-2-one (6e)
1
Colorless solid; 82 % yield; m.p, 147–149 ꢁC; H NMR
(CDCl3, 300 MHz): d 7.08 (s, 1H, aromatic), 6.02 (s, 1H,
aromatic), 2.86 (t, 2H, J = 6.79 Hz, CH2), 2.38 (s, 3H,
CH3), 2.25 (s, 3H, CH3), 1.82 (t, 2H, J = 6.79 Hz, CH2),
1.38 (s, 6H, 2CH3); 13C NMR (CDCl3, 75 MHz): d 161.82
(C=O, lactone), 155.25 (C-9a), 152.71 (C-4), 151.07 (C-
10a), 121.80 (C-5), 121.33 (C-5a), 117.08 (C-3), 111.74
(C-4a), 111.15 (C-9), 75.30 (C-8), 32.30 (C-7), 26.88 (C-
12,13), 22.42 (C-6), 18.70 (C-12, CH3), 8.04 (C-11, CH3).
Mass (ESI): m/z 259 (M ? H)?.
3-Chloro-4,8,8-trimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]
chromen-2-one (6i)
Colorless solid; 66 % yield; m.p, 128–130 ꢁC; IR (KBr) m
(cm-1): 2975, 2930, 1730, 1621, 1553, 1493, 1377, 1350,
1
1158, 1119, 1069 cm-1; H NMR (CDCl3, 300 MHz): d
8,8,10-Trimethyl-4-phenyl-7,8-dihydro-2H,6H-pyrano
[3,2-g]chromen-2-one (6f)
7.22 (s, 1H, aromatic), 6.68 (s, 1H, aromatic), 2.75 (t, 2H,
J = 6.79 Hz, CH2), 2.50 (s, 3H, CH3), 1.88 (t, 2H,
J = 6.79 Hz, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 157.49 (C=O, lactone), 154.32 (C-9a), 151.24
(C-4), 147.82 (C-10a), 125.38 (C-5), 120.64 (C-5a), 118.79
(C-3), 112.75 (C-4a), 104.44 (C-10), 75.86 (C-8), 32.34 (C-
7), 26.83 (C-12,13), 22.11 (C-6), 16.07 (C-11,CH3); Mass
(ESI): m/z 279, 281 (M ? H)?.
Colorless solid; 74 % yield; m.p, 141-143 ꢁC; IR (KBr) m
(cm-1): 2972, 2930, 2851, 1724, 1611, 1564, 1444, 1380,
1
1286, 1214, 1120 cm-1; H NMR (CDCl3, 300 MHz): d
7.45–7.50 (m, 3H, aromatic), 7.38–7.42 (m, 2H, aromatic),
6.92 (s, 1H, aromatic), 6.10 (s, 1H, aromatic), 2.74 (t, 2H,
J = 6.79 Hz, CH2), 2.30 (s, 3H, CH3), 1.80 (t, 2H,
J = 6.79 Hz, CH2), 1.38 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 160.96 (C=O, lactone), 155.64 (C-4), 155.34
(C-9a), 151.92 (C-10a), 136.15 (C-11), 129.14 (C-13,15),
128.57 (C-14), 128.28 (C-12,16), 124.21 (C-5), 116.88 (C-
5a), 113.63 (C-10), 111.59 (C-3), 111.38 (C-4a), 75.25 (C-
8), 32.42 (C-7), 27.08 (C-17,18), 22.31 (C-6), 8.23(C-14,
CH3); Mass (ESI): m/z 321 (M ? H)?.
3-Chloro-4,8,8,10-tetramethyl-7,8-dihydro-2H,
6H-pyrano[3,2-g]chromen-2-one (6j)
Colorless solid; 69 % yield; m.p, 190–192 ꢁC; IR (KBr) m
(cm-1): 2972, 2926, 2862, 1725, 1613, 1563, 1445, 1375,
1
1281, 1152, 1117 cm-1; H NMR (CDCl3, 300 MHz): d
7.10 (s, 1H, aromatic), 2.85 (t, 2H, J = 6.79 Hz, CH2),
2.52 (s, 3H, CH3), 2.25 (s, 3H, CH3), 1.85 (t, 2H,
J = 6.79 Hz, CH2), 1.38 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 157.82 (C=O, lactone), 155.35 (C-9a), 148.39
(C-4), 148.04 (C-10a), 122.21 (C-5), 117.93 (C-5a), 117.67
(C-10), 113.40 (C-3), 112.29 (C-4a), 75.58 (C-8), 32.32 (C-
7), 27.02 (C-12,13), 22.42 (C-6), 16.08 (C-11, CH3), 8.11
(C-14, CH3); Mass (ESI): m/z 293, 295 (M ? H)?.
4-(chloromethyl)-8,8,10-trimethyl-7,8-dihydro-2H,
6H-pyrano[3,2-g]chromen-2-one (6g)
Pale yellow solid; 72 % yield; m.p, 142–144 ꢁC; IR (KBr) m
(cm-1): 2975, 2932, 2851, 1720, 1613, 1573, 1449, 1388,
1286, 1156, 1106 cm-1; 1H NMR (CDCl3, 300 MHz): d 7.14
(s, 1H, aromatic), 6.38 (s, 1H, aromatic), 4.58 (s, 2H, CH2Cl),
2.85 (t, 2H, J = 6.79 Hz, CH2), 2.25 (s, 3H, CH3), 1.86 (t, 2H,
J = 6.79 Hz, CH2), 1.40 (s, 6H, 2CH3); 13C NMR (CDCl3,
75 MHz): d 160.77 (C=O, lactone), 155.73 (C-4), 151.80 (C-
9a), 149.37 (C-10a), 121.39 (C-5), 117.32 (C-5a), 114.08 (C-
10), 112.24 (C-3), 109.92 (C-4a), 75.54 (C-8), 41.47 (C-11,
CH2Cl), 32.51 (C-7), 27.21 (C12,13), 22.55 (C-16), 8.30 (C-
14, CH3); Mass (ESI): m/z 293, 295 (M ? H)?.
7-Hydroxy-4-(trifluoromethyl)-2H-chromen-2-one (6k)
1H NMR (CDCl3, 300 MHz): d 10.41 (s, 1H, Ar-OH), 7.54 (s,
1H, aromatic), 6.93–6.83 (m, 2H, aromatic), 6.53 (s, 1H,
aromatic); 13C NMR (CDCl3, 75 MHz): d 162.30 (C=O,
lactone), 159.72 (C-7), 156.22 (C-4), 142.01 (C-8a), 126.21
(C-9, CF3), 114.59 (C-5), 110.88 (C-8), 106.46 (C-4a), 105.84
(C-3), 103.58 (C-6); Mass (ESI–MS): m/z 229 [M ? H]?.
8,8,10-Trimethyl-4-(trifluoromethyl)-7,8-dihydro-2H,
6H-pyrano[3,2-g]chromen-2-one (6h)
7-Hydroxy-8-methyl-4-(trifluoromethyl)-2H-chromen-2-
Colorless solid; 75 % yield; m.p, 139-141 ꢁC; IR (KBr) m
one (6l)
(cm-1): 2975, 2933, 2857, 1734, 1614, 1573, 1404, 1280,
1
1163, 1145, 1071 cm-1; H NMR (CDCl3, 300 MHz): d
7.22 (s, 1H, aromatic), 6.52 (s, 1H, aromatic), 2.88 (t, 2H,
1H NMR (CDCl3, 300 MHz): d 7.44 (d, 1H, J = 8.30 Hz,
aromatic), 6.88 (d, 1H, J = 8.68 Hz, aromatic), 6.55 (s,
123

2832
Med Chem Res (2014) 23:2821–2833
1H, aromatic), 2.29 (s, 3H, CH3); 0.41 (s, 1H, Ar-OH), 7.54
(s, 1H, aromatic), 6.93–6.83 (m, 2H, aromatic), 6.53 (s, 1H,
aromatic); 13C NMR (CDCl3, 75 MHz): d 160.00 (C=O,
lactone), 154.02 (C-7), 142.21 (q, J = 64.37 Hz, C-4),
123.02 (C-8a), 121.53 (q, J = 275.64 Hz, C-9, CF3),
112.83 (C-5), 112.70 (C-8), 110.24 (C-4a), 110.20 (C-3),
105.85 (C-6); Mass (ESI–MS): m/z 245 [M ? H]?.
spectrophotometrically as above. Individual blanks were
prepared to correct for the background absorbance due to
test samples. In this case DNS solution was added in tubes
before addition of substrate. The percentage inhibition of
enzyme activity was calculated as above. All the tests were
run in triplicate in this assay.
Molecular docking simulation
Bioassay
Porcine pancreatic a-amylase (PPA) is a 496-residue
enzyme sharing more than 83 % similarity with HPA.
Docking simulation was carried out on HPA (PDB code:
1CPU) which is pharmaceutical target against diabetes
mellitus and obesity treatments. After removing the ligand
and solvent molecules, hydrogen atoms and Kollman char-
ges were added to each protein atom. Coordinates of each
compound were generated using Chemdraw11 followed by
MM2 energy minimization. Docking was carried out by
AutoDock4 in active site of HPA (Brayer et al., 2000;
AutoDock, version 4.0, http://www.scripps.edu/mb/olson/
doc/autodock). Grid map in Auto dock that defines the
interaction of protein and ligands in binding pocket was
defined. The grid map was used with 60 points in each x, y,
ABTS•? free radical scavenging assay
Scavenging of the ABTS? [2,20-azino-bis(3-ethylbenzothia-
zoline-6-sulfonic acid)] cation was performed as described by
Walker and Everette (2009) with suitable modifications.
Briefly, 100 mL stock solution of ABTS? (0.5 mM) was
prepared by addition of 1 mL potassium persulfate (6.89 mM
PBS, pH 8.0). The mixture was stored in the dark for 16 h.
Test compounds were dissolved in DMSO (5 mg/ml). Pri-
mary screening was done by mixing 10 lL of test compounds
in 100 lL of methanol followed by 190 lL of ABTS? in a
96-well microplate. Absorbance of decolorized ABTS? was
measured at 734 nm after 15 min incubation in the dark on a
BioTek synergy4 multi-mode microplate reader. For each test
sample a separate blank sample (devoid of ABTS?) was used
for background subtraction. The percentage of ABTS?
scavenging was calculated applying following formula; %
ABTS? scavenging = [(Absorbancecontrol - Absorbancetest)/
Absorbancecontrol 9 100]. Various serial dilutions of active
compounds were prepared and tested for determination of
SC50 values. Suitable regression analysis was applied for
calculation of SC50.
˚
and z direction, equally spaced at 0.375 A. Docking was
performed using the Lamarckian genetic algorithm (Morris
et al., 1998; Hess et al., 2008; Berendsen et al., 1981). Each
docking experiment was performed 100 times, yielding 100
docked conformations. Parameters used for the docking
were as follows: population size of 150; random starting
˚
position and conformation; maximal mutation of 2 A in
translation and 50ꢁ in rotations; elitism of 1; mutation rate of
0.02 and crossover rate of 0.8; and local search rate of 0.06.
Simulations were performed with a maximum of 1.5 million
energy evaluations and a maximum of 50,000 generations.
Final docked conformations were clustered using a tolerance
a-Amylase inhibitory assay
˚
of 1.0 A root mean square deviation. The best model was
The assay method was adopted from (Ali et al., 2006) and
modified accordingly to suite microplate reading. In brief,
40 lL of test samples (5 mg/mL DMSO) were reconstituted
in 160 lL of 20 mM phosphate buffer (pH 6.9) containing
6.7 mM sodium chloride in 2 mL Eppendorf tubes and
incubated with 200 lL of porcine pancreatic a-amylase
(4 U/mL prepared in ice cold distilled water) for 5 min. The
reaction was started by the addition of 400 lL soluble potato
starch solution (0.5 % w/v in 20 mM phosphate buffer, pH
6.9). Exactly 3 min after incubation, 400 lL DNS color
reagent (1.0 g of 3,5-dinitrosalicylic acid, 30 g of sodium
potassium tartrate, and 20 mL of 2 N NaOH to a final vol-
ume of 100 mL in distilled water) was added. Closed tubes
were placed in a water bath (85–90 ꢁC) for 10 min to
develop color and cooled thereafter. 50 lL of reaction
mixtures was diluted with 175 lL of distilled water in a
96-well microplate. a-Amylase activity was determined by
measuring the absorbance of the mixture at 540 nm
picked based on the best stabilization energy.
X-ray data collection and refinement
X-ray data for compounds 3c and 6h was collected at RT
using a Bruker Smart Apex CCD diffractometer with
˚
graphite-monochromated MoKa radiation (k = 0.71073 A)
with x-scan method (Sheldrick, 2008). Preliminary lattice
parameters and orientation matrices were obtained from four
sets of frames. Integration and scaling of intensity data were
accomplished using SAINT program. The structures were
solved by direct methods using SHELXS97 (Sheldrick,
2008) and refinement was carried out by full-matrix least-
squares technique using SHELXL97. Anisotropic displace-
ment parameters were included for all non-hydrogen atoms.
All H atoms were positioned geometrically and treated as
˚
riding on their parent C atoms [C–H = 0.93–0.97 A and
123

Med Chem Res (2014) 23:2821–2833
2833
particular reference to Phyllanthus amarus. J Ethnopharmacol
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Uiso(H) = 1.5Ueq(C) for methyl H or 1.2Ueq(c) for other H
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tip.
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Crystal data for 3c C21H18O4, M = 334.35, colorless
block, 0.21 9 0.17 9 0.08 mm3, monoclinic, space group
C2/c (No. 15), a = 30.791(3), b = 8.1137(6), c = 13.
3
˚
˚
6677(10) A, b = 101.786(2)ꢁ, V = 3342.6(4) A , Z = 8,
Dc = 1.329 g/cm3, F000 = 1408, CCD Area Detector,
˚
MoKa radiation, k = 0.71073 A T = 294(2)K, 15,568
reflections collected, 2,947 unique (Rint = 0.0218). Final
GooF = 1.043, R1 = 0.0658, wR2 = 0.2046, R indices
based on 2,510 reflections with I [ 2r(I) (refinement on
F2), 228 parameters, l = 0.092 mm-1. Intensity data were
measured on Bruker Smart Apex with CCD area detector.
CCDC 874166 contains supplementary Crystallographic
data for the structure. These data can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or
from the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax:
?44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
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diseases: South Asians vreus White Caucasians. Int J Obes
35:167–187
Crystal data for 6h C16H15F3O3, M = 312.28, colorless
needle, 0.12 9 0.10 9 0.06 mm3, triclinic, space group
ꢀ
P 1 (No. 2), a = 8.0741(14), b = 8.8963(16), c = 11.
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Raju BC, Tiwari AK, Ashok Kumar J, Ali AZ, Agawane SB,
Saidachary G, Madhusudana K (2010) a-Glucosidase inhibitory
antihyperglycemic activity of substituted chromenone deriva-
tives. Bioorg Med Chem 18:358–365
Rao RR, Tiwari AK, Reddy PP, Babu KS, Suresh G, Ali AZ,
Madhusudana K, Agawane SB, Badrinarayan P, Sastry GN, Rao
JM (2012) Synthesis of antihyperglycemic, a-glucosidase inhib-
itory and DPPH free-radical scavenging furano chalcones. Med
Chem Res 21:760–774
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mellitus. Lancet 373:1579–1580
Sharma PN, Shoeb A, Kapil RS, Popli SP (1980) Ind J Chem
19B:938–939
˚
1140(19) A, a = 75.157(3), b = 82.725(3), c = 87.948(3)ꢁ,
3
V = 765.5(2) A , Z = 2, Dc = 1.355 g/cm , F000 = 324,
3
˚
˚
CCD Area Detector, MoKa radiation, k = 0.71073 A, T =
294(2)K, 7106 reflections collected, 2676 unique (Rint = 0.
0180). Final GooF = 1.082, R1 = 0.0434, wR2 = 0.1164,
R indices based on 2,403 reflections with I I [ 2(I) (refine-
ment on F2), 203 parameters, l = 0.116 mm-1. CCDC
874166 contains supplementary Crystallographic data for the
structure. These data can be obtained free of charge at www.
ccdc.cam.ac.uk/conts/retrieving.html[orfromthe Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road,
Cambridge CB2 1EZ, UK; fax: ?44(0) 1223 336 033; email:
deposit@ccdc.cam.ac.uk].
Acknowledgments The authors thank Dr. Ahmed Kamal, Director,
and Dr. S. Chandrasekhar, Head, Natural Products Chemistry Division,
CSIR-IICT for their constant encouragement. JAK, CK from UGC and
GS from CSIR, New Delhi are gratefully acknowledged for fellow-
ships. DST and DBT New Delhi is acknowledged for a research grant to
AA. B. China Raju acknowledges CSIR, New Delhi for financial sup-
port through the programme ORIGIN of XII five-year plan (CSC0108).
Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr
A64:112–122
Smart and Saint (2001) Software reference manuals, versions 6.28a
and 5.625. Bruker Analytical X-ray Systems, Madison, WI
Tiwari AK, Kumar MP, Kumar DA, Agawane SB, Madhusudana K,
Ali AZ (2012) Ayurvedic dietary formulations and postprandial
glycemia in rats. Int Food Res J 19:765–773
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antioxidants with ABTS radical cation. J Agric Food Chem
57:1156–1161
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123