Synthesis and evaluation of α-glucosidase and pancreatic lipase inhibition by quinazolinone-coumarin hybrids

Article abstract of DOI:10.1007/s10593-017-2002-3

A new series of 2-substituted quinazolin-4(3H)-one derivatives including coumarin nucleus has been synthesized and screened for their lipase and α-glucosidase inhibition properties. Among the synthesized compounds, N'-{2-[2-(3,4-dichlorobenzyl)-4-oxoquinazolin-3(4H)-yl]acetyl}-2-oxo-2H-chromene-3-carbohydrazide and N'-{2-[2-(4-bromobenzyl)-4-oxoquinazolin-3(4H)-yl]acetyl}-2-oxo-2H-chromene-3-carbohydrazide showed the best inhibitory effect against α-glucosidase with IC₅₀ values of 6.11 ± 0.40 and 7.34 ± 0.37 μM, respectively. These compounds also showed strong anti-lipase activity (IC₅₀ 3.52 ± 0.49 and 2.85 ± 0.27 μM, respectively).

Full text of DOI:10.1007/s10593-017-2002-3

DOI 10.1007/s10593-017-2002-3
Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
Synthesis and evaluation of α-glucosidase and pancreatic
lipase inhibition by quinazolinone-coumarin hybrids
Emre Menteşe1*, Nesrin Karaali1, Gülay Akyüz1,
Fatih Yılmaz1, Serdar Ülker2, Bahittin Kahveci3
1 Department of Chemistry, Faculty of Art and Sciences, Recep Tayyip Erdogan University,
Fener St., 53100 Rize, Turkey; e-mail: emre.mentese@erdogan.edu.tr
2 Department of Biology, Faculty of Art and Sciences, Recep Tayyip Erdogan University,
Fener St., 53100 Rize, Turkey; e-mail: serdar.ulker@erdogan.edu.tr
3 Department of Nutrition and Dietetics, Faculty of Health Sciences,
Karadeniz Technical University,
Farabi St., 61080 Trabzon, Turkey; e-mail: bahittin@ktu.edu.tr
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2016, 52(12), 1017–1024
Submitted July 21, 2016
Accepted after revision September 8, 2016
O
N
O
O
O
N
O
H
N
H
N
N
N
H
N
N
H
O
O
O
O
O
Cl
Br
IC50 7.34 0.37 µM
-glucosidase inhibitory activity
Cl
IC50 3.52 0.49 µM
lipase inhibitory activity
A new series of 2-substituted quinazolin-4(3H)-one derivatives including coumarin nucleus has been synthesized and screened for their
lipase and α-glucosidase inhibition properties. Among the synthesized compounds, N'-{2-[2-(3,4-dichlorobenzyl)-4-oxoquinazolin-3(4H)-
yl]acetyl}-2-oxo-2H-chromene-3-carbohydrazide and N'-{2-[2-(4-bromobenzyl)-4-oxoquinazolin-3(4H)-yl]acetyl}-2-oxo-2H-chromene-
3-carbohydrazide showed the best inhibitory effect against α-glucosidase with IC50 values of 6.11
0.40 and 7.34
0.37 µM,
respectively. These compounds also showed strong anti-lipase activity (IC50 3.52 0.49 and 2.85 0.27 µM, respectively).
Keywords: coumarins, quinazolin-4(3H)-one, anti-lipase activity, α-glucosidase inhibition, microwave, ultrasonication.
Obesity is a significant health problem in today's world.1
It causes clinical disorders such as hypertension, diabetes
mellitus,2 and cardiovascular disease.3 Some approaches
for the prevention and treatment of obesity have been
described in the literature. Among these, both natural and
synthetic pancreatic lipase inhibitors, such as orlistat,4,5
have been effective in obesity prevention. However, the use
of orlistat is frequently associated with adverse gastro-
intestinal effects, such as diarrhea and cholestatic
hepatitis.6 Thus, the synthesis of new anti-lipase com-
pounds remains a remarkable area of medicinal chemistry
in the fight against obesity.
Another important area with potential for drug discovery
is the synthesis of new α-glucosidase inhibitors. Today,
there are 366 million people affected by diabetes mellitus
all around the world.7 Inhibitors of α-glucosidase can be of
key importance for the treatment of various diseases, such
as type 2 diabetes and obesity. Inhibition of this enzyme
reduces the postprandial blood glucose level by delaying
the digestion.8 Besides, α-glucosidase inhibitors have
shown promise as potential antiviral (including antiHIV)
agents, because glycosylation of viral envelope proteins is
necessary for the infectivity.9,10
Heterocycles and their derivatives play a major role in
the field of medicinal chemistry. Quinazolinone is a fused
heterocyclic system that exhibits a wide range of biological
activity.10–14 Recently, substituted 2-arylquinazolines have
been synthesized and evaluated for their cytotoxicity and
inhibition of topoisomerases.15 In addition, very recently a
series of quinazoline derivatives has been synthesized and
characterized as novel antioxidants and anti-inflammatory
agents.16
Furthermore, a wide range of heterocyclic compounds
containing the coumarin moiety has also been found to
0009-3122/16/52(12)-1017©2016 Springer Science+Business Media New York
1017

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
Scheme 1
exhibit many useful properties, such as antibacterial,17
antifungal,18 antimicrobial,19 antioxidant,20 analgesic,21
antimutagenic,22 anticancer,23 antiHIV,24 and antitumor
activity.25,26 Literature survey showed that there is limited
evidence about coumarin derivatives as α-glucosidase and
lipase inhibitors.27-31
synthetic applications.31,35 In this context, the reaction of
3-(1H-benzotriazol-1-ylcarbonyl)-2H-chromon-2-one (6)
and compounds 4a–g afforded the target quinazolinone
derivatives containing coumarin nucleus 7a–g.
Quinazolinone derivatives have important applications
in medicinal chemistry. Because of their wide range of
biological activitiy, the preparation of quinazolinone
derivatives has attracted considerable attention.36–40 For
example, compounds 2a–g have been synthesized by using
microwave irradiation, ultrasonication, and conventional
heating procedures, with the results compared in Table 1.
This method represents the first application of ultra-
Molecular hybrid-based approach is one of the fastest
growing areas in medicinal chemistry. In our work, we report
the synthesis and biological evaluation of α-glucosidase
and lipase inhibition properties of some new 2-substituted
quinazolin-4(3H)-one derivatives that contain a coumarin
nucleus. The synthetic strategy for the intermediates and
target compounds consisted of the reactions outlined in
Scheme 1. Compounds 1a–g were prepared according to
the literature procedures.32,33 Quinazolinones 2a–g were
obtained by the reaction of 2-aminobenzamide and
compounds 1a–g in methanol. Esters 3a–g were prepared
by simple alkylation of compounds 2a–g with ethyl
bromoacetate and K2CO3 in anhydrous acetone and were
transformed to acetohydrazides 4a–g by treatment with
hydrazine monohydrate in ethanol. These compounds
served as the first intermediates on the route to the target
carbohydrazides. Coumarin-3-carboxylic acid (5) and
3-(1H-benzotriazol-1-ylcarbonyl)-2H-chromon-2-one (6),
which also served as key intermediates for the synthesis of
the target compounds, were prepared according to the
literature.31,34,35 Literature sources have shown that benzo-
triazole group is a good leaving group, enabling many
Table 1. Comparison of the yields and reaction times
for the preparation of compounds 2a–g
Microwave method Ultrasonic method Conventional method
Com-
pound
Yield,
%
Time,
min
Yield,
%
Time,
min
Yield,
%
Time,
h
92
91
90
93
88
89
94
7
7
7
7
7
7
7
75
76
75
77
71
74
76
15
15
15
15
15
15
15
97
96
99
85
85
88
98
12
12
12
12
13
13
12
2a
2b
2c
2d
2e
2f
2g
1018

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
Table 2. Inhibition of α-glucosidase and pancreatic lipase
sonication technique for the synthesis of such compounds,
by the newly synthesized compounds*
and showed the shortest reaction time compared to
microwave and classical heating procedures, while micro-
wave heating gave the best yield.
α-Glucosidase
Pancreatic lipase
Com-
pound
Inhibition, %
IC50, µM
Inhibition, % IC50, µM
Spectral investigation of the target compounds was used
1
–
–
–
24
12
9
6
2a
2b
2c
2d
2g
3a
3d
3f
for proving the proposed structures. H NMR spectra of
these compounds showed two NH signals (exchangeable
with D2O) at about 11.00 and 10.50 ppm, respectively, and
a singlet characteristic for coumarin C-4 proton was
observed at about 8.90 ppm. The NCH2 signals were
observed at about 4.80 ppm and benzylic CH2 signals were
observed at about 4.15 ppm. The other signals of aromatic
and aliphatic protons matched the proposed structures.
13C NMR spectra of these compounds featured four C=O
signals at about 165, 162 (hydrazide), 161 (C-4 quinazo-
line), and 159 (C-2 coumarin) ppm. The characteristic
quinazoline C=N, coumarin C-3, and coumarin C-4 carbon
atoms resonated at about 155, 154, and 149 ppm, respec-
tively. The other aromatic and aliphatic carbon signals
were in agreement with the proposed structures. Also, these
compounds gave suitable results of elemental analysis.
The presence of coumarin ring is known to enhance the
biological activity of many compounds.31 In particular, the
addition of coumarin to one heterocycle positively affected
the biological activity. In this study, it was obvious that the
addition of coumarin ring to quinazolin-4(3H)-ones
increased the lipase and α-glucosidase inhibition properties.
α-Glucosidase inhibitory activity. All compounds were
evaluated with regard to inhibitory activity against
α-glucosidase, and compounds 7a,b,d,f,g showed anti-
α-glucosidase activity at various concentrations (Table 2).
Compounds 7f,g exhibited stronger inhibitory effect than
acarbose, a known α-glucosidase inhibitor that is used as
antidiabetic drug. No significant inhibitory effect was
detected for other compounds. Compounds 7f,g showed the
best α-glucosidase inhibition among all tested compounds.
At the concentration of 300 µM, these compounds inhibited
α-glucosidase by 100 10 and 100 1%, respectively.
Acarbose showed a 100 1% inhibitory effect at the same
concentration (IC50 26.12 2.38 µM). The IC50 values of
–
100
100
5
6
225.33 58.16
235.71 54.66
14 10
18
50
42
80
29
24
5
1
1
2
7
9
–
–
44
27
4
3.85 0.06
–
–
3g
4e
4f
9
–
100
87
9
4
99.81 17.98
56.28 6.65
39
70
42
81
45
95
90
1
3
4
2
3
1
3
7a
7b
7c
7d
7e
7f
–
100 10
51 16
100 10
38.94 10.77
6.49 1.45
6.11 0.40
7.34 0.37
26.12 2.38
3.52 0.49
2.85 0.27
100
1
1
7g
Acarbose 100
Orlistat
99.2 0.4 0.85 0.042 nM
* All compounds were assayed at 300 µM concentration for α-glucosidase
and 10 µM concentration for pancreatic lipase.
the synthesized quinazolinone-coumarin hybrids showed
significant inhibitory activities. It is clear that the addition
of a coumarin ring to quinazolin-4(3H)-ones increased the
inhibitory properties. The best inhibitory effects against
α-glucosidase was observed in the case of N'-{[2-(3,4-di-
chlorobenzyl)-4-oxoquinazolin-3(4H)-yl]acetyl}-2-oxo-2H-
chromene-3-carbohydrazide and N'-{[2-(4-bromobenzyl)-
4-oxoquinazolin-3(4H)-yl]acetyl}-2-oxo-2H-chromene-
3-carbohydrazide. These compounds also showed the best
anti-lipase activity. These results contribute to the develop-
ment of new enzyme inhibitors containing coumarin
nucleus.
Experimental
compounds 7f,g were calculated as 6.11
7.34 0.37 µM, respectively.
0.40 and
1H and 13C NMR spectra were acquired on a Varian
Mercury 400 spectrometer (400 and 100 MHz,
respectively) in DMSO-d6, with TMS as internal standard.
The elemental compositions were determined on a Carlo
Erba 1106 CHN analyzer; the experimental values were in
agreement ( 0.4%) with the calculated ones. Melting
points were taken in capillary tubes on a Büchi melting
point apparatus and are uncorrected. A monomode CEM
Discover Microwave system was used in the standard
configuration as delivered, including proprietary software.
All experiments were carried out in microwave process
vials (30 ml) with control of the temperature by infrared
temperature sensor. It was monitored by a computer and
maintained at a constant value by a discrete modulation of
delivered microwave power. After completion of the
reaction, the vial was cooled to 60°C via air jet cooling.
All the chemicals were supplied from Merck, Sigma-
Aldrich, and Fluka.
Pancreatic lipase inhibition. All the compounds were
evaluated with regard to the inhibition of pancreatic lipase
activity and some compounds showed anti-lipase activity at
various concentrations (Table 2). Compounds 7f,g showed
the best anti-lipase activity among all tested compounds.
These compounds at 10 µM concentration inhibited
pancreatic lipase by 95 1 and 90
3%, respectively.
Orlistat, a known pancreatic lipase inhibitor used as anti-
obesity drug, showed inhibitory effect by 99% at 300 nM
concentration (IC50 0.85 nM). The IC50 values for
compounds 7f,g were calculated as 3.52
2.85 0.27 µM, respectively.
0.49 and
In conclusion, a new series of quinazolinone-coumarin
hybrid compounds have been synthesized and screened for
inhibitory activity against lipase and a-glucosidase.
Quinazolin-4(3H)-one derivatives lacking a coumarin ring
system displayed very low inhibitory activity. However,
1019

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
Synthesis of compounds 2a–g. Conventional method.
vessel and anhydrous K2CO3 (3.45 g, 0.025 mol) was
added. The mixture was stirred for 15 min at room
temperature. Ethyl bromoacetate (1.67 g, 0.010 mol) was
then added and the mixture was irradiated in the micro-
wave system for 7 min at 90°C, using 300 W maximum
power. After the reaction was complete (monitoring by
TLC, ethyl acetate–hexane, 3:1), the mixture was cooled to
room temperature. The product was precipitated by the
addition of water, filtered off, washed with water, and
recrystallized from ethanol–water, 3:2.
Ethyl [2-(4-methylbenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetate (3a). Yield 75% (conventional method), 81%
(microwave method). Mp 102–103°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.27 (1H, dd, J = 8.0, J = 1.2, H Ar); 8.01
(1H, t, J = 8.0, H Ar); 7.84 (1H, d, J = 8.0, H Ar); 7.70
(1H, t, J = 8.0, H Ar); 7.34 (2H, d, J = 8.0, H Ar); 7.29
(2H, d, J = 8.0, H Ar); 4.97 (2H, s, NCH2); 4.35 (2H, s,
OCH2); 4.13 (2H, q, J = 7.8, OCH2); 2.41 (3H, s, CH3);
1.26 (3H, t, J = 7.8, CH3). 13C NMR spectrum, δ, ppm:
167.9; 162.0 (C=O); 156.3 (C=N); 147.5; 136.8; 135.5;
132.7; 129.9 (2C); 129.3 (2C); 127.7; 127.6; 126.9; 120.3
(C Ar); 61.9 (OCH2); 46.1 (CH2); 21.3 (CH3); 14.5 (CH3).
Found, %: C 71.34; H 5.96; N 8.26. C20H20N2O3.
Calculated, %: C 71.41; H 5.99; N 8.33.
The appropriate compound 1 (0.011 mol) was added to a
solution of 2-aminobenzamide (1.36 g, 0.010 mol) in
methanol (40 ml). The mixture was stirred for 12 h at room
temperature. The end of the reaction was monitored by
TLC (ethyl acetate–hexane, 3:1). The mixture was poured
into water and the precipitated product was filtered off,
washed with water, and recrystallized from ethanol–water,
3:1.
Microwave method. A mixture of the appropriate
compound 1 (0.01 mol) and 2-aminobenzamide (1.36 g,
0.010 mol) in methanol (10 ml) was irradiated in a
microwave process vial at 65°C for 7 min at 300 W
maximum power. After the reaction was completed
(monitoring by TLC, ethyl acetate–hexane, 3:1), the
mixture was cooled to room temperature and poured into
water. The same purification methods as described above
were applied to obtain the pure product.
Ultrasonication method. A mixture of the appropriate
compound 1 (0.013 mol) and 2-aminobenzamide (1.36 g,
0.010 mol) in methanol (30 ml) was ultrasonicated for
15 min at 45°C. After the reaction was complete
(monitoring by TLC, ethyl acetate–hexane, 3:1), the same
purification methods as described above were applied to
obtain the pure product.
Ethyl [2-(4-fluorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetate (3b). Yield 86% (conventional method), 88%
(microwave method). Mp 164–165°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.11 (1H, d, J = 8.0, H Ar); 7.87 (1H, t, J = 8.0,
H Ar); 7.68 (1H, d, J = 8.0, H Ar); 7.54 (1H, t, J = 8.0,
H Ar); 7.35 (2H, d, J = 8.0, H Ar); 7.17 (2H, d, J = 8.0,
H Ar); 4.86 (2H, s, NCH2); 4.25 (2H, s, CH2); 4.01 (2H, q,
J = 7.2, OCH2); 1.14 (3H, t, J = 7.2, CH3). 13C NMR
spectrum, δ, ppm: 168.3; 162.3 (C=O); 162.2 (d, J = 241.4,
C–F); 156.4 (C=N); 147.8; 135.8; 132.3; 131.8; 128.1;
127.2; 120.6; 116.4; 116.2 (C Ar); 62.2 (OCH2); 46.5
(CH2); 14.9 (CH3). Found, %: C 67.00; H 4.96; N 8.17.
C19H17FN2O3. Calculated, %: C 67.05; H 5.03; N 8.23.
Ethyl [2-(4-methoxybenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetate (3c). Yield 95% (conventional method), 94% (micro-
wave method). Mp 88–89°C. 1H NMR spectrum, δ, ppm
(J, Hz): 8.36 (1H, d, J = 7.6, H Ar); 8.11 (1H, t, J = 7.6,
H Ar); 7.95 (1H, d, J = 7.6, H Ar); 7.80 (1H, t, J = 7.6,
H Ar); 7.48 (2H, d, J = 8.0, H Ar); 7.14 (2H, d, J = 8.0,
H Ar); 5.07 (2H, s, NCH2); 4.42 (2H, s, CH2); 4.28 (2H, q,
J = 6.8, OCH2); 3.96 (3H, s, OCH3); 1.38 (3H, t, J = 6.8,
CH3). 13C NMR spectrum, δ, ppm: 168.8; 162.9 (C=O);
159.8 (C=N); 157.3; 148.4; 136.3; 131.3 (2C); 128.6;
128.4; 127.7; 121.4; 115.9 (2C); 115.5 (C Ar); 62.7
(OCH2); 56.6 (OCH3); 46.9 (CH2); 15.4 (CH3). Found, %:
C 68.08; H 5.64; N 7.85. C20H20N2O4. Calculated, %: C 68.17;
H 5.72; N 7.95.
2-(4-Methylbenzyl)quinazolin-4(3H)-one (2a). Mp 232–
233°C (mp 231–233°C (EtOAc–petroleum ether, 3:1)41).
2-(4-Fluorobenzyl)quinazolin-4(3H)-one (2b). Mp 265–
266°C (mp 227–229°C (EtOAc–petroleum ether, 3:1)41).
2-(4-Methoxybenzyl)quinazolin-4(3H)-one (2c). Mp 228–
229°C (mp 219–220°C (EtOAc–petroleum ether, 3:1)41).
2-(4-Chlorobenzyl)quinazolin-4(3H)-one (2d). Mp 263–
264°C (mp 242–244°C (EtOAc–petroleum ether, 3:1)41).
2-(2,4-Dichlorobenzyl)quinazolin-4(3H)-one (2e). Mp
204–206°C (mp 203°C (EtOAc–petroleum ether, 3:1)41).
2-(3,4-Dichlorobenzyl)quinazolin-4(3H)-one (2f). Mp 262–
263°C (mp 263–265°C (EtOAc–petroleum ether, 3:1)41 ).
2-(4-Bromobenzyl)quinazolin-4(3H)-one (2g). Mp 276–
1
277°C. H NMR spectrum, δ, ppm (J, Hz): 12.40 (1H, s,
NH); 8.05 (1H, d, J = 7.6, H Ar); 7.70 (1H, t, J = 7.6,
H Ar); 7.56 (1H, d, J = 7.6, H Ar); 7.47 (2H, d, J = 8.4,
H Ar); 7.40 (1H, t, J = 7.6, H Ar); 7.31 (2H, d, J = 8.4,
H Ar); 3.90 (2H, s, CH2). 13C NMR spectrum, δ, ppm:
162.3 (C=O); 156.0 (C=N); 149.3; 136.3; 134.8 (2C);
132.1; 131.8; 131.6; 127.4; 126.7; 126.2; 121.2; 120.5
(C Ar); 52.2 (CH2). Found, %: C 57.10; H 3.46; N 8.81.
C15H11BrN2O. Calculated, %: C 57.16; H 3.52; N 8.89.
Synthesis of compounds 3a–g. Conventional method.
Anhydrous K2CO3 (3.45 g, 0.025 mol) was added to a
solution of the appropriate compound 2 (0.010 mol) in
acetone and the mixture was stirred for 15 min at room
temperature. Ethyl bromoacetate (1.67 g, 0.010 mol) was
then added and the mixture was stirred for 12 h. The end of
the reaction was monitored by TLC (ethyl acetate–hexane,
3:1). The product was precipitated by the addition of water,
filtered off, washed with water, and recrystallized from
ethanol–water, 3:2.
Ethyl [2-(4-chlorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetate (3d). Yield 90% (conventional method), 94%
(microwave method). Mp 155–156°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.23 (1H, d, J = 8.0, H Ar); 7.95 (1H, t,
J = 8.0, H Ar); 7.78 (1H, d, J = 8.4, H Ar); 7.65 (1H, t,
J = 7.6, H Ar); 7.50–7.44 (4H, m, H Ar); 4.98 (2H, s,
NCH2); 4.37 (2H, s, CH2); 4.15 (2H, q, J = 6.4, OCH2);
1.25 (3H, t, J = 6.4, CH3). 13C NMR spectrum, δ, ppm:
Microwave method. A solution of the appropriate com-
pound 2 (0.010 mol) in acetone (15 ml) was charged into a
1020

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
168.3; 162.3 (C=O); 156.2 (C=N); 147.7; 135.8; 135.2;
2-[2-(4-Methylbenzyl)-4-oxoquinazolin-3(4H)-yl]aceto-
hydrazide (4a). Yield 73% (conventional method), 74%
(microwave method). Mp 252–253°C. 1H NMR spectrum,
δ, ppm (J, Hz): 9.56 (1H, s, NH); 8.28 (1H, d, J = 8.0,
H Ar); 8.00 (1H, t, J = 8.0, H Ar); 7.80 (1H, d, J = 8.4,
H Ar); 7.70 (1H, d, J = 8.4, H Ar); 7.36–7.29 (4H, m,
H Ar); 4.78 (2H, s, NCH2); 4.46 (2H, s, NH2); 4.27 (2H, s,
CH2); 2.44 (3H, s, CH3). 13C NMR spectrum, δ, ppm:
168.7; 162.1 (C=O); 156.7 (C=N); 147.6; 136.8; 135.2;
132.1; 130.0 (2C); 129.1 (2C); 127.6; 127.4; 126.9; 120.5
(C Ar); 45.0 (NCH2); 41.4 (CH2); 21.3 (CH3). Found, %:
C 66.98; H 5.54; N 17.30. C18H18N4O2. Calculated, %:
C 67.07; H 5.63; N 17.38.
132.6; 131.9; 129.5; 129.1; 128.1; 127.2; 120.6 (C Ar);
62.2 (OCH2); 46.5 (CH2); 14.8 (CH3). Found, %: C 63.90;
H 4.71; N 7.77. C19H17ClN2O3. Calculated, %: C 63.96;
H 4.80; N 7.85.
Ethyl [2-(2,4-dichlorobenzyl)-4-oxoquinazolin-3(4H)-
yl]acetate (3e). Yield 78% (conventional method), 83%
(microwave method). Mp 135–136°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.35 (1H, d, J = 7.6, H Ar); 8.03 (1H, t,
J = 7.6, H Ar); 7.82–7.40 (7H, m, H Ar); 5.13 (2H, s,
NCH2); 4.53 (2H, s, CH2); 4.19 (2H, q, J = 7.8, OCH2);
1.32 (3H, t, J = 7.8, CH3). 13C NMR spectrum, δ, ppm:
169.0; 163.4 (C=O); 157.1 (C=N); 148.6; 139.2; 136.3;
135.8; 132.7; 131.0; 130.0; 128.3; 128.0; 126.7; 125.7;
123.6 (C Ar); 65.1 (OCH2); 47.4 (CH2); 14.0 (CH3). Found, %:
C 58.25; H 4.03; N 7.06. C19H16Cl2N2O3. Calculated, %:
C 58.33; H 4.12; N 7.16.
Ethyl [2-(3,4-dichlorobenzyl)-4-oxoquinazolin-3(4H)-
yl]acetate (3f). Yield 79% (conventional method), 82%
(microwave method). Mp 151–152°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.27 (1H, d, J = 7.6, H Ar); 7.98 (1H, t,
J = 7.6, H Ar); 7.82–7.44 (7H, m, H Ar); 5.07 (2H, s,
NCH2); 4.45 (2H, s, CH2); 4.16 (2H, q, J = 7.6, OCH2);
1.29 (3H, t, J = 7.6, CH3). 13C NMR spectrum, δ, ppm:
169.3; 163.3 (C=O); 157.3 (C=N); 148.6; 138.7; 136.5;
134.8; 132.6; 131.6; 130.0; 129.0; 128.2; 126.8; 123.5
(C Ar); 65.1 (OCH2); 47.5 (CH2); 14.8 (CH3). Found, %:
C 58.26; H 4.06; N 7.11. C19H16Cl2N2O3. Calculated, %:
C 58.33; H 4.12; N 7.16.
2-[2-(4-Fluorobenzyl)-4-oxoquinazolin-3(4H)-yl]aceto-
hydrazide (4b). Yield 77% (conventional method), 80%
(microwave method). Mp 264–265°C. 1H NMR spectrum,
δ, ppm (J, Hz): 9.39 (1H, s, NH); 8.09 (1H, d, J = 7.6,
H Ar); 7.80 (1H, t, J = 7.6, H Ar); 7.60 (1H, d, J = 8.4,
H Ar); 7.50 (1H, d, J = 7.6, H Ar); 7.36–7.29 (2H, m,
H Ar); 7.16–7.11 (2H, m, H Ar); 4.66 (2H, s, NCH2); 4.27
(2H, s, NH2); 4.14 (2H, s, CH2). 13C NMR spectrum,
δ, ppm: 166.6; 161.8 (C=O); 161.6 (d, J = 120.7, C-F);
156.4 (C=N); 147.3; 135.0; 132.2; 131.3 (2C); 131.2 (2C);
127.4; 127.2; 127.1; 120.3; 115.9; 115.7 (C Ar); 45.9
(NCH2); 40.4 (CH2). Found, %: C 62.50; H 4.56; N 17.10.
C17H15FN4O2. Calculated, %: C 62.57; H 4.63; N 17.17.
2-[2-(4-Methoxybenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetohydrazide (4c). Yield 77% (conventional method),
1
82% (microwave method). Mp 226–227°C. H NMR
Ethyl [2-(4-bromobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetate (3g). Yield 93% (conventional method), 95%
(microwave method). Mp 152–153°C. 1H NMR spectrum,
δ, ppm (J, Hz): 8.37 (1H, d, J = 8.0, H Ar); 8.10 (1H, t, J = 8.0,
H Ar); 7.93 (1H, d, J = 7.6, H Ar); 7.82–7.72 (3H, m,
H Ar); 7.55 (1H, d, J = 8.0, H Ar); 5.11 (2H, s, NCH2);
4.45 (2H, s, CH2); 4.25 (2H, q, J = 7.2, OCH2); 1.40 (3H, t,
J = 7.2, CH3). 13C NMR spectrum, δ, ppm: 168.1; 162.1
(C=O); 156.0 (C=N); 147.5; 135.6; 135.4; 132.2 (2C);
132.1; (2C); 132.0; 127.9; 127.0; 121.0; 120.4 (C Ar); 62.0
(OCH2); 46.3 (CH2); 14.7 (CH3). Found, %: C 56.80;
H 4.22; N 6.91. C19H17BrN2O3. Calculated, %: C 56.87;
H 4.27; N 6.98.
Synthesis of compounds 4a–g. Conventional method.
Hydrazine monohydrate (2.50 g, 0.050 mol) was added to a
solution of the appropriate compound 3 (0.01 mol) in
ethanol (10 ml). The mixture was stirred for 12 h. The end
of the reaction was monitored by TLC (ethyl acetate–hexane,
3:1). The product was precipitated by the addition of water,
filtered off, dried, and recrystallized from ethanol.
Microwave method. A solution of the appropriate
compound 3 (0.01 mol) in anhydrous ethanol (10 ml) and
hydrazine monohydrate (2.50 g, 0.050 mol) was transferred
to a microwave process vial and the mixture was irradiated
in the microwave system at 120°C for 8 min, using 200 W
microwave power. After the reaction was complete (moni-
toring by TLC, ethyl acetate–hexane, 3:1), the mixture was
cooled to room temperature. The product was then
precipitated by the addition of water, filtered off, dried, and
recrystallized from ethanol.
spectrum, δ, ppm (J, Hz): 9.75 (1H, s, NH); 8.46 (1H, d,
J = 7.6, H Ar); 8.19 (1H, t, J = 8.0, H Ar); 8.01 (1H, d,
J = 8.0, H Ar); 7.98 (1H, t, J = 7.6, H Ar); 7.58–7.54 (2H,
m, H Ar); 7.26–7.24 (2H, m, H Ar); 4.98 (2H, s, NCH2);
4.65 (2H, s, NH2); 4.44 (2H, s, CH2); 4.08 (3H, s, OCH3).
13C NMR spectrum, δ, ppm: 167.5; 162.7 (C=O); 159.6;
157.6 (C=N); 148.3; 135.9; 131.1; 128.6; 128.3; 128.0;
127.6; 121.2; 115.4 (C Ar); 56.4 (OCH3); 45.6 (NCH2);
41.6 (CH2). Found, %: C 63.80; H 5.31; N 16.50.
C18H18N4O3. Calculated, %: C 63.89; H 5.36; N 16.56.
2-[2-(4-Chlorobenzyl)-4-oxoquinazolin-3(4H)-yl]aceto-
hydrazide (4d). Yield 77% (conventional method), 83%
(microwave method). Mp 260–261°C. 1H NMR spectrum,
δ, ppm (J, Hz): 9.42 (1H, s, NH); 8.12 (1H, d, J = 8.0,
H Ar); 7.83 (1H, t, J = 8.0, H Ar); 7.62 (1H, d, J = 8.0,
H Ar); 7.53 (1H, t, J = 7.6, H Ar); 7.40 (2H, d, J = 7.6,
H Ar); 7.39–7.31 (2H, m, H Ar); 4.67 (2H, s, NCH2); 4.30
(2H, s, NH2); 4.17 (2H, s, CH2). 13C NMR spectrum,
δ, ppm: 166.7; 161.9 (C=O); 156.9 (C=N); 147.4; 135.3;
135.2; 132.2; 131.5; 131.4; 129.2; 127.6; 127.4; 126.8;
120.5 (C Ar); 45.1 (NCH2); 40.7 (CH2). Found, %:
C 59.51; H 4.34; N 16.25. C17H15ClN4O2. Calculated, %:
C 59.57; H 4.41; N 16.34.
2-[2-(2,4-Dichlorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetohydrazide (4e). Yield 74% (conventional method),
1
81% (microwave method). Mp 270–271°C. H NMR
spectrum, δ, ppm (J, Hz): 9.53 (1H, s, NH); 8.83 (1H, d,
J = 8.2, H Ar); 7.98 (1H, t, J = 8.2, H Ar); 7.76–7.53 (4H,
m, H Ar); 7.36 (1H, d, J = 7.6, H Ar); 4.82 (2H, s, NCH2);
4.37 (2H, s, NH2); 4.11 (2H, s, CH2). 13C NMR spectrum,
1021

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
δ, ppm: 167.4; 162.0 (C=O); 157.0 (C=N); 148.2; 138.3;
(1H, d, J = 8.0, H Ar); 8.00 (1H, d, J = 8.0, H Ar); 7.80
(1H, t, J = 7.2, H Ar); 7.75 (1H, t, J = 7.2, H Ar); 7.65 (1H,
d, J = 8.0, H Ar); 7.52 (2H, q, J = 8.0, H Ar); 7.44 (1H, t,
J = 8.0, H Ar); 7.20 (2H, d, J = 8.0, H Ar); 7.15 (2H, d,
J = 8.0, H Ar); 4.80 (2H, s, NCH2); 4.14 (2H, s, CH2); 3.11
(3H, s, CH3). 13C NMR spectrum, δ, ppm: 165.0; 161.8;
160.2; 159.5 (C=O); 156.4 (C=N); 154.4 (C-3 coumarin);
148.7 (C-4 coumarin); 136.6; 135.2; 134.9; 132.7; 130.9;
129.8 (2C); 129.0 (2C); 127.5; 127.3; 126.8; 125.7; 120.2;
118.8; 118.4; 116.7 (C Ar); 44.4 (NCH2); 41.1 (CH2); 21.1
(CH3). Found, %: C 67.93; H 4.42; N 11.27. C28H22N4O5.
Calculated, %: C 68.01; H 4.48; N 11.33.
N'-{[2-(4-Fluorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7b). Yield
52% (conventional method), 57% (microwave method).
Mp >300°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.13 (1H,
s, NH); 10.59 (1H, s, NH); 8.87 (1H, s, CH); 8.08 (1H, d,
J = 8.0, H Ar); 7.99 (1H, d, J = 8.0, H Ar); 7.81–7.72 (3H,
m, H Ar); 7.60 (1H, d, J = 8.0, H Ar); 7.50–7.40 (6H, m,
H Ar); 4.80 (2H, s, NCH2); 4.14 (2H, s, CH2). 13C NMR
spectrum, δ, ppm: 165.3; 162.1 (d, J = 240.4, C-F); 161.6;
160.2; 159.6 (C=O); 155.8 (C=N); 154.4 (C-3 coumarin);
148.5 (C-4 coumarin); 136.5; 135.1; 134.5; 132.1; 130.1;
129.7 (2C); 127.9 (2C); 127.5; 127.3; 126.8; 125.7; 120.2;
118.7; 118.3 (C Ar); 44.4 (NCH2); 41.1 (CH2); 21.1 (CH3).
Found, %: C 64.98; H 3.76; N 11.16. C27H19FN4O5.
Calculated, %: C 65.06; H 3.84; N 11.24.
N'-{[2-(4-Methoxybenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7c). Yield
50% (conventional method), 56% (microwave method).
Mp > 300°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.10
(1H, s, NH); 10.61 (1H, s, NH); 8.91 (1H, s, CH); 8.11
(1H, d, J = 7.2, H Ar); 8.00 (1H, d, J = 7.2, H Ar); 7.83
(1H, t, J = 7.2, H Ar); 7.76 (1H, t, J = 8.4, H Ar); 7.65 (1H,
d, J = 8.4, H Ar); 7.53–7.48 (2H, m, H Ar); 7.43 (1H, t,
J = 8.0, H Ar); 7.25 (1H, d, J = 8.4, H Ar); 6.90 (1H, d,
J = 8.4, H Ar); 4.83 (2H, s, NCH2); 4.13 (2H, s, CH2); 3.71
(3H, s, OCH3). 13C NMR spectrum, δ, ppm: 165.1; 161.8;
160.2; 159.5 (C=O); 158.7 (C Ar); 156.6 (C=N); 154.4
(C-3 coumarin); 148.7 (C-4 coumarin); 147.4; 135.2; 134.9;
130.8; 130.2; 127.6; 127.5; 127.3; 126.7; 125.7; 120.2;
118.8; 118.4; 116.7; 114.6 (C Ar); 55.5 (OCH3); 44.4
(NCH2); 40.6 (CH2). Found, %: C 65.81; H 4.28; N 10.93.
C28H22N4O6. Calculated, %: C 65.88; H 4.34; N 10.97.
N'-{[2-(4-Chlorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7d). Yield
54% (conventional method), 60% (microwave method).
Mp 291–292°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.13
(1H, s, NH); 10.62 (1H, s, NH); 8.91 (1H, s, CH); 8.11
(1H, d, J = 8.0, H Ar); 8.01 (1H, d, J = 7.2, H Ar); 7.83–
7.74 (2H, m, H Ar); 7.61 (1H, d, J = 8.0, H Ar); 7.56–7.28
(7H, m, H Ar); 4.86 (2H, s, NCH2); 4.21 (2H, s, CH2).
13C NMR spectrum, δ, ppm: 165.0; 161.7; 160.2; 159.4
(C=O); 156.1 (C Ar); 154.4 (C=N); 148.7 (C-3 coumarin);
147.2 (C-4 coumarin); 146.2; 135.2; 135.0; 132.0; 131.3;
130.8; 129.0; 127.4; 127.3; 126.7; 125.7; 120.2; 118.7;
118.4; 116.7 (C Ar); 44.5 (NCH2); 40.6 (CH2). Found, %:
C 62.90; H 3.65; N 10.81. C27H19ClN4O5. Calculated, %:
C 62.98; H 3.72; N 10.88.
135.8; 132.3; 131.4; 131.0; 130.2; 129.9; 127.3; 126.6;
123.1; 120.2 (C Ar); 44.9 (NCH2); 40.0 (CH2). Found, %:
C 54.05; H 3.68; N 14.76. C17H14Cl2N4O2. Calculated, %:
C 54.13; H 3.74; N 14.85.
2-[2-(3,4-Dichlorobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetohydrazide (4f). Yield 75% (conventional method),
1
81% (microwave method). Mp 245–246°C. H NMR
spectrum, δ, ppm (J, Hz): 9.42 (1H, s, NH); 8.08 (1H, d,
J = 8.0, H Ar); 7.81 (1H, t, J = 8.0, H Ar); 7.64 (1H, s,
H Ar); 7.56–7.43 (3H, m, H Ar); 7.26 (1H, d, J = 8.0,
H Ar); 4.70 (2H, s, NCH2); 4.27 (2H, s, NH2); 4.17 (2H, s,
CH2). 13C NMR spectrum, δ, ppm: 166.6; 161.7 (C=O);
156.9 (C=N); 147.1; 137.3; 134.9; 131.7; 131.4; 131.0;
130.9; 129.9; 127.4; 127.2; 126.6; 120.3 (C Ar); 45.0
(NCH2); 40.0 (CH2). Found, %: C 54.07; H 3.65; N 14.79.
C17H14Cl2N4O2. Calculated, %: C 54.13; H 3.74; N 14.85.
2-[2-(4-Bromobenzyl)-4-oxoquinazolin-3(4H)-yl]aceto-
hydrazide (4g). Yield 78% (conventional method), 85%
(microwave method). Mp 246–247°C. 1H NMR spectrum,
δ, ppm (J, Hz): 9.46 (1H, s, NH); 8.15 (1H, t, J = 8.0,
H Ar); 7.86–7.78 (2H, m, H Ar); 7.76–7.64 (3H, m, H Ar);
7.39–7.28 (2H, m, H Ar); 4.71 (2H, s, NCH2); 4.35 (2H, s,
NH2); 4.19 (2H, s, CH2). 13C NMR spectrum, δ, ppm:
166.5; 161.7 (C=O); 156.2 (C=N); 147.2; 135.5; 134.9;
131.9 (2C); 131.6 (2C); 127.4; 127.1; 126.7; 121.2; 120.5
(C Ar); 44.9 (NCH2); 40.5 (CH2). Found, %: C 52.68;
H 3.85; N 14.42. C17H15BrN4O2. Calculated, %: C 52.73;
H 3.90; N 14.47.
2-Oxo-2H-chromene-3-carboxylic acid (5) was synthe-
sized according to a literature procedure.34 Yield 1.63 g (86%).
Mp 190–191°C (EtOH) (mp 190°C (EtOH)34).
3-(1H-Benzotriazol-1-ylcarbonyl)-2H-chromen-2-one
(6) was synthesized according to a literature procedure.35
Yield 2.21 g (76%). Mp 185–186°C (mp 186–187°C
(CH2Cl2–hexane)35).
Synthesis of compounds 7a–g. Conventional method.
A solution of the appropriate compound 4 (0.010 mol) in
ethanol (15 ml) and compound 6 (0.012 mol) were placed
in a round-bottom flask. The mixture was refluxed for 6 h.
After the completion of the reaction (monitored by TLC,
ethyl acetate–hexane, 3:1), the mixture was cooled to the
room temperature and a solid appeared. This crude product
was filtered off and washed with ethanol to obtain the pure
product.
Microwave method. A mixture of the appropriate
compound 4 (0.010 mol) and compound 6 (0.012 mol) in
ethanol was transferred to a microwave process vial and
irradiated in microwave system for 30 min at 135°C. After the
completion of the reaction (monitored by TLC, ethyl
acetate–hexane, 3:1) the mixture was cooled to the room
temperature and a solid appeared. This crude product was
filtered off and washed with ethanol to obtain the pure
product.
N'-{[2-(4-Methylbenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7a). Yield
50% (conventional method), 54% (microwave method).
Mp 285–286°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.06
(1H, s, NH); 10.57 (1H, s, NH); 8.91 (1H, s, CH); 8.11
1022

Chemistry of Heterocyclic Compounds 2016, 52(12), 1017–1024
N'-{[2-(2,4-Dichlorobenzyl)-4-oxoquinazolin-3(4H)-
pancreatic lipase (Applichem, Germany) (15 ng/ml). Lipase
activity assay was performed according to Kurihara et al.43
The lipase activity was measured using 4-methylumbel-
liferyl oleate (4-MU oleate) as a substrate. The solutions of
compounds were mixed with porcine pancreatic lipase
(PPL) solution in 1:3 (v/v) ratio and incubated for 30 min.
The microtiter plates containing 50 µl of 0.1 mM 4-MU
oleate solution, 25 µl of diluted compound–lipase solution,
25 µl of deionized H2O and buffer (13 mM Tris–HCl,
150 mM NaCl, and 1.3 mM CaCl2, pH 8.0) were incubated
at 37°C for 20 min. After incubation, in order to stop the
reaction, 0.1 ml of 0.1 M pH 4.2 citrate buffer was added to
the reaction mixture. The amount of 4-methylumbelliferone
released by the lipase was measured by using a Molecular
Devices SpectraMax M5 spectrofluorometer at the exci-
tation wavelength of 355 nm and the emission wavelength
of 460 nm. The inhibitory activity of the study compounds
and orlistat (Xenical, Hoffmann-La Roche) against
pancreatic lipase were measured at various concentrations.
The residual activities were calculated by comparing to the
control without inhibitor. The assays were done in
triplicate. The IC50 value was determined as the
concentration of compound that gave 50% inhibition of the
maximal activity.
yl]acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7e). Yield
49% (conventional method), 53% (microwave method).
Mp > 300°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.13 (1H, s,
NH); 10.59 (1H, s, NH); 8.86 (1H, s, CH); 8.10 (1H, d,
J = 8.0, H Ar); 8.05 (1H, d, J = 7.4, H Ar); 7.85–7.70 (2H,
m, H Ar); 7.58 (1H, d, J = 8.0, H Ar); 7.59–7.21 (6H, m,
H Ar); 4.85 (2H, s, NCH2); 4.39 (2H, s, CH2). 13C NMR
spectrum, δ, ppm: 165.3; 162.0; 160.9; 159.9 (C=O); 156.0
(C Ar); 155.6 (C=N); 149.6 (C-3 coumarin); 146.8 (C-4
coumarin); 145.9; 136.3; 135.0; 132.4; 131.7; 130.8; 128.0;
127.4; 126.8; 125.9; 124.4; 121.7; 118.9; 118.2; 115.7 (C Ar);
44.6 (NCH2); 40.1 (CH2). Found, %: C 58.95; H 3.22; N 10.14.
C27H18Cl2N4O5. Calculated, %: C 59.03; H 3.30; N 10.20.
N'-{[2-(3,4-Dichlorobenzyl)-4-oxoquinazolin-3(4H)-
yl]acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7f). Yield
49% (conventional method), 52% (microwave method).
Mp > 300°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.17
(1H, s, NH); 10.63 (1H, s, NH); 8.90 (1H, s, CH); 8.12
(1H, d, J = 7.6, H Ar); 8.02 (1H, d, J = 7.2, H Ar); 7.85–
7.65 (3H, m, H Ar); 7.53 (1H, d, J = 7.6, H Ar); 7.40–7.17
(5H, m, H Ar); 4.92 (2H, s, NCH2); 4.41 (2H, s, CH2).
13C NMR spectrum, δ, ppm: 165.1; 162.1; 160.9; 159.8
(C=O); 155.9 (C Ar); 155.5 (C=N); 149.3 (C-3 coumarin);
146.5 (C-4 coumarin); 145.3; 136.2; 134.1; 132.5; 131.0;
130.2; 128.4; 127.6; 126.3; 125.2; 124.3; 120.5; 118.5;
117.1; 113.4 (C Ar); 44.4 (NCH2); 40.1 (CH2). Found, %:
C 58.95; H 3.22; N 10.14. C27H18Cl2N4O5. Calculated, %:
C 59.03; H 3.30; N 10.20.
This work was supported by the Scientific and
Technological Research Council of Turkey (TUBITAK)
under the project number of 115Z885. The authors
gratefully thank TUBITAK for financial support.
N'-{[2-(4-Bromobenzyl)-4-oxoquinazolin-3(4H)-yl]-
acetyl}-2-oxo-2H-chromene-3-carbohydrazide (7g). Yield
55% (conventional method), 62% (microwave method).
Mp >300°C. 1H NMR spectrum, δ, ppm (J, Hz): 11.13 (1H,
s, NH); 10.60 (1H, s, NH); 8.90 (1H, s); 8.11 (1H, d,
J = 7.2, H Ar); 8.00 (1H, d, J = 8.0, H Ar); 7.83–7.73 (2H,
m, H Ar); 7.60 (1H, d, J = 8.0, H Ar); 7.53–7.42 (5H, m,
H Ar); 7.31–7.24 (2H, m, H Ar); 4.86 (2H, s, NCH2); 4.20
(2H, s, CH2). 13C NMR spectrum, δ, ppm: 165.0; 161.8;
160.3; 159.4 (C=O); 156.0 (C Ar); 154.4 (C=N); 148.7
(C-3 coumarin); 147.2 (C-4 coumarin); 135.4; 135.1; 134.9;
131.9; 131.7; 130.9; 127.4; 127.3; 126.8; 125.7; 120.6; 120.2;
118.7; 118.4; 116.7 (C Ar); 44.5 (NCH2); 40.5 (CH2).
Found, %: C 57.91; H 3.34; N 9.93. C27H19BrN4O5. Calcu-
lated, %: C 57.97; H 3.42; N 10.02.
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α-Glucosidase inhibition assay. α-Glucosidase inhibi-
tion assay was performed spectrophotometrically. α-Gluco-
sidase from Saccharomyces cerevisiae (Sigma-Aldrich)
was dissolved in phosphate buffer (pH 6.8, 50 mM). Test
compounds were dissolved in 80% methanol. In 96-well
microtiter plates, 20 μl of test sample, 20 μl of enzyme
(200 mU/ml), and 135 μl of buffer were added and
incubated for 15 min at 37°C. After incubation, 25 μl of
p-nitrophenyl-α-D-glucopyranoside (2 mM, Sigma-Aldrich)
was added and change in absorbance at 400 nm was
monitored for 30 min. The test compound was replaced by
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