Synthesis, spectral characterization and α-chymotrypsin activity of 7-O-substituted derivatives of 7-hydroxy-4-methyl-1-benzopyran-2-one

Article abstract of DOI:10.14233/ajchem.2014.15374

In this work, a series of O-alkyl/arenyl/acyl substituted derivatives of 7-hydorxy-4-methyl-1-benzenpyran-2-one (3a-l) was synthesized. The parent compound 7-hydroxy-4-methyl-1-benzenpyran-2-one (1) was geared up by the coupling of resorcinol (a) with ethylacetoacetate (b) in the presence of conc. sulphuric acid. Further, O-substituted derivatives of parent compound were prepared by treating with different electrophiles (2a-l) using sodium hydride as base and DMF as a solvent. The structure of these synthesized compounds were characterized by IR, EI-MS and ¹H NMR. These derivatives were also screened against α-chymotrypsin enzyme to check their enzyme inhibition activity. All the compounds displayed a-chymotrypsin activity to varying degree.

Full text of DOI:10.14233/ajchem.2014.15374

Asian Journal of Chemistry; Vol. 26, No. 2 (2014), 326-330
http://dx.doi.org/10.14233/ajchem.2014.15374
Synthesis, Spectral Characterization and α-Chymotrypsin Activity of
7-O-Substituted Derivatives of 7-Hydroxy-4-methyl-1-benzopyran-2-one
1,*
1
1
1
AZIZ-UR-REHMAN , TEHSEEN ILYAS , MUHAMMAD ATHAR ABBASI , KHADIJA NAFEESA ,
1
1
1
1
2
SHAISTA KHALIQ , KANIZ RUBAB , GHULAM HUSSAIN , SHAZIA KHURSHID and IRSHAD AHMAD
1Department of Chemistry, Government College University, Lahore-54000, Pakistan
2Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan
*Corresponding author: Tel: +92 42 111000010, Ext. 450; E-mail: azizryk@yahoo.com
Received: 26 February 2013;
Accepted: 8 May 2013;
Published online: 15 January 2014;
AJC-14535
In this work, a series of O-alkyl/arenyl/acyl substituted derivatives of 7-hydorxy-4-methyl-1-benzenpyran-2-one (3a-l) was synthesized.
The parent compound 7-hydroxy-4-methyl-1-benzenpyran-2-one (1) was geared up by the coupling of resorcinol (a) with ethylacetoacetate
(b) in the presence of conc. sulphuric acid. Further, O-substituted derivatives of parent compound were prepared by treating with different
electrophiles (2a-l) using sodium hydride as base and DMF as a solvent. The structure of these synthesized compounds were characterized
1
by IR, EI-MS and H NMR. These derivatives were also screened against α-chymotrypsin enzyme to check their enzyme inhibition
activity. All the compounds displayed α-chymotrypsin activity to varying degree.
Keywords: 7-Hydroxy-4-methyl-1-benzopyran-2-one,
INTRODUCTION
α-Chymotrypsin enzyme, 1H NMR, EI-MS.
pressure regulation. In the last decade, it became obvious that
in invertebrates, serine proteases and their inhibitors are also
involved in parallel physiological processes, for example the
blood clotting cascade in Limulus12 and the innate immune
response13. Therefore, the discovery of potent and safe inhibitors
has been a very important area of pharmaceutical research.
Coumarin and its derivatives have attracted organic chemists
because of its remarkable role in biological activities.
The present work is a successful effort to synthesize such
compounds exhibiting diverse and improved pharmacological
potential. We have synthesized the O-alkyl/arenyl/acyl substi-
tuted derivatives of 7-hydorxy-4-methyl-1-benzenpyran-2-one
with an objective to search new contenders of drug having
significant enhanced activity and could be helpful in controlling
many degenerative diseases.
The coumarin present in nature is a significant member
of benzopyrones1. Coumarin was naturally extracted from
tonka beans2 and it is an important constituent of cinnamon
and dates3-5. Coumarin has the properties of anticoagulant,
insecticide, antibacterial and pharmacological functions6.
Natural and synthesized coumarin is one of the main human
metabolite and in human diet, it acts as dietary antioxidant. It
also reduces the blood glucose level in human body7. Organic
and medicinal chemists have considerable attraction for
coumarin and its derivatives because of its antiinflammatory/
anti-oxidant activities8. In bakery and beverage industry, it is
used as flavoring agents and enhanced the flavors of different
bakery items9. Efforts have been made for the extraction of
naturally occurring coumarin from numerous plants and
artificial synthesis of coumarin compounds, so that it can be
further utilized as the potential drug in medicinal field10.
Among the enzymes involved in extracellular matrix
degradation, certain serine proteases (elastase, collagenase,
cathepsin G and chymotrypsin) are able to solubilize fibrous
proteins such as elastin and collagen11. Potent inhibitors have
the potential to be developed as new therapeutic agents. In
vertebrates, serine protease inhibitors have been studied for
many years and they are known to be involved in phagocytosis,
coagulation, complement activation, fibrinolysis and blood
EXPERIMENTAL
All melting points are in degree centigrade and were
recorded on Griffin and George apparatus by using open
capillary tube. Thin layer chromatography technique was
performed on pre-coated silica gel G-25-UV254 plates using
mixture of n-hexane and ethyl acetate as solvents in different
ratios for each reaction to check the purity of synthesized
compound by giving single spot of product. UV light of 254
nm wavelength and ceric sulphate reagent were used for this
detection. IR technique was performed on instrument Jasco-

Vol. 26, No. 2 (2014)
Synthesis and α-Chymotrypsin Activity of Derivatives of 7-Hydroxy-4-methyl-1-benzopyran-2-one 327
320-A spectrophotometer (wave number in cm-1) using KBr
pellets. Numbers of protons calculated by nuclear magnetic
resonance spectra also helped in identification of structures
and were recorded on a Bruker spectrometer operating at 300
MHz in CD3OD solution. Chemical shifts are quoted in ppm.
Mass spectra (EIMS) were given on JMS-HX-110 spectro-
meter. Resorcinol, ethylacetoacetate and the other electrophiles
(methyl iodide, ethyl iodide, isopropyl iodide, allyl iodide,
2-phenylethyliodide, 2-chlorobenzylchloride, 4-bromobenzyl-
bromide, 4-flurobenzyl chloride and 4-chlorobenzyl chloride)
were purchased from Sigma Aldrich and Alfa Aesar through
local suppliers. All the other employed solvents were of
analytical grade.
m.w. 190; IR (KBr, νmax, cm-1): 3535 (O-H stretching), 3401
(C-H aromatic stretching), 1729 (C=O stretching), 1616 (C=C
stretching of aromatic ring). 1H NMR (300 MHz, CD3OD δ/
ppm): 7.60 (d, J = 8.4 Hz, 1H, H-5), 6.80 (dd, J = 8.7, 2.5 Hz,
1H, H-6), 6.69 (d, J =2.5 Hz, 1H, H-8), 6.09 (s, 1H, H-3),
3.78 (s, 3H, OCH3-1), 2.41 (s, 3H, CH3-4). EI-MS: m/z 190
[M]+, 175 [C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 108
[C6H4O2]+, 51 [C4H3]+.
7-Ethoxy-4-methyl-1-benzopyran-2-one (3b): Buff
amorphous powder; yield 76 %; m.p. 98-100 ºC; m.f.: C12H12O3;
m.w. 204; IR (KBr, νmax, cm-1): 3539 (O-H stretching), 3407
(C-H aromatic stretching), 1727 (C=O stretching), 1614 (C=C
stretching of aromatic ring). 1H NMR (300 MHz, CD3OD δ/
ppm): 7.68 (d, J = 8.7 Hz, 1H, H-5), 6.95 (dd, J = 8.7, 2.4 Hz,
1H, H-6), 6.87 (d, J =2.4 Hz, 1H, H-8), 6.15 (s, 1H, H-3),
4.11 (q, J = 6.9 Hz, 2H, CH2-1), 2.43 (s, 3H, CH3-4), 1.42 (t,
J = 6.9 Hz, 3H, CH3-2). EI-MS: m/z 204 [M]+, 189 [M-CH3]+,
175 [C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 108 [C6H4O2]+,
51 [C4H3]+.
Procedure for the synthesis of 7-hydroxy-4-methyl-1-
benzopyran-2-one (1): Resorcinol (4.4 g; a) and ethylaceto-
acetate (5 mL; b) was taken in iodine flask. Reaction mixture
was shaken well until reaction mass become clear by keeping
the flask on water bath and temperature was maintained at
40 ºC. Then, cooled concentrated sulphuric acid (15 mL) was
added to the iodine flask and the reaction mixture was placed
for overnight. Next day, the chilled water was added in the
solution with continuous stirring. The reaction mixture was
reserved at room temperature for 0.5 h; dark yellow solid was
filtered and washed with distilled water to afford 7-hydroxy-
4-methyl-1-benzopyran-2-one (1) on drying.
7-Isopropoxy-4-methyl-1-benzopyran-2-one (3c): Off-
white amorphous powder; yield 78 %; m.p: 88-90 ºC; m.f.:
C13H14O3; m.w. 218; IR (KBr, νmax, cm-1): 3542 (O-H stretching),
3406 (C-H aromatic stretching), 1728 cm–1 (C=O stretching),
1613 (C=C stretching of aromatic ring). 1H-NMR (300 MHz,
CD3OD δ/ppm): 7.67 (d, J = 8.7 Hz, 1H, H-5), 6.92 (dd, J =
8.7, 2.4 Hz, 1H, H-6), 6.86 (d, J = 2.4 Hz, 1H, H-8), 6.13 (s,
1H , H-3), 4.72 (m, 1H, H-1), 2.43 (s, 3H, CH3-4), 1.34 (d, J =
6.0 Hz, 6H, H-2 & 3). EI-MS: m/z 218[M]+, 175 [C10H7O3]+,
159 [C10H7O2]+, 144 [C9H4O2]+, 108 [C6H4O2]+, 51 [C4H3]+.
7-Allyoxy-4-methyl-1-benzopyran-2-one (3d): Creamy
white amorphous powder; yield 78 %; m.p. 154-157 ºC; m.f.:
C13H12O3; m.w. 216; IR (KBr, νmax, cm-1): 3503 (O-H stretching),
3410 (C-H aromatic stretching), 1729 (C=O stretching), 1617
(C=C stretching of aromatic ring). 1H NMR (300 MHz, CD3OD
δ/ppm): 7.66 (d, J = 9.0 Hz, 1H, H-5), 6.96 (dd, J = 9.0, 2.4,
1H, H-6), 6.90 (d, J =2.4 Hz, 1H, H-8), 6.15 (s, 1H, H-3),
6.11-6.02 (m, 1H, H-2), 5.44 (dd, J =1.5, 17.1 Hz, Hb-3), 5.27
(dd , J = 1.5, 10.5 Hz, Ha-3), 4.66 (d, J = 5.4 Hz, H-1), 2.43 (s,
3H, CH3-4). EI-MS: m/z 216[M]+, 175 [C10H7O3]+, 159
[C10H7O2]+, 144 [C9H4O2]+, 108 [C6H4O2]+, 51 [C4H3]+.
7-(2-Phenylethoxy)-4-methyl-1-benzopyran-2-one (3e):
Buff amorphous powder; yield 64 %; m.p. 96-98 ºC; m.f.:
C18H16O3; m.w. 280; IR (KBr, νmax, cm-1): 3510 (O-H stretching),
3418 (C-H aromatic stretching), 1723 (C=O stretching), 1613
(C=C stretching of aromatic ring). 1H NMR (300 MHz, CD3OD
δ/ppm): 7.64 (d, J = 8.7 Hz, 1H, H-5), 7.32-7.17 (m, 5H, H-2
to 6'), 6.94 (dd, J = 9.0 Hz, 2.4, 1H, H-6), 6.88 (d, J =2.4 Hz,
1H, H-8), 6.14 (s, 1H, H-3), 4.28 (t, J = 6.9 Hz, 2H, H-8'),
3.12 (t, J = 6.6 Hz, 2H, H-7'), 2.42 (s, 3H, CH3-4), EIMS: m/z
280 [M]+, 175 [C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 108
[C6H4O2]+, 91 [C7H7]+, 51 [C4H3]+.
General procedure for the synthesis of O-substituted
derivatives 2a-i: Compound (1) (2.80 mmol, 0.5 g) was taken
in round bottom flask and N,N-dimethyl formamide (around
5 mL) and sodium hydride (2.80 mmol; 0.01g,) were added in
it at room temperature. The reaction mixture was stirred for
15 min and then the electrophiles (2.80 mmol; 2a-i) were added
into the mixture. The reaction mass was further stirred and
monitoring through TLC. After completion of the reaction,
the reaction mixture was quenched with cold water (150 mL).
The received solid was filtered, washed with water and dried to
yield the corresponding O-substituted derivatives of 7-hydroxy-
4-methyl-1-benzopyran-2-one (3a-i).
General procedure for the synthesis of compounds 3j-l:
Compound 1 (2.8 mmol; 0.5 g) and 20 mL of 5 % sodium
hydroxide solution was taken in the round bottom flask. The
mixture was stirred till solution become clear and then the
corresponding electrophiles (2.80 mmol; 2j-l) were added into
the reaction mixture with constant stirring at room temperature
until precipitates were formed. Precipitates were filtered,
washed with distilled water and dried to yield the corresponding
derivatives (3j-l).
Characterization of the synthesized compounds
7-Hydroxy-4-methyl-1-benzopyran-2-one (1): Dark
yellow amorphous powder; yield 92 %; m.p. 188-190 ºC; m.f.:
C10H8O3; m.w. 176; IR (KBr, νmax, cm-1): 3550 (O-H stretching),
3418 (C-H aromatic stretching), 1717 (C=O stretching), 1629
(C=C stretching of aromatic ring); 1H NMR (300 MHz, CD3OD
δ/ppm): 7.58 (d, J = 8.7 Hz, 1H, H-5), 6.82 (dd, J = 8.7, 2.4
Hz, 1H, H-6), 6.70 (d, J =2.4 Hz,1H, H-8), 6.09 (s, 1H, H-3),
2.41 (d, J = 0.9 Hz, 3H, CH3-4) . EI-MS: m/z 176 [M]+, 159
[C10H7O2]+, 144 [C9H4O2]+, 108 [C6H4O2]+, 51 [C4H3]+.
7-(2-Chlorobenzyloxy)-4-methyl-1-benzopyran-2-one
(3f): Creamy white amorphous powder; yield 70 %; m.p. 135-
137 ºC; m.f.: C17H13O3Cl; m.w. 300; IR (KBr, νmax, cm-1): 3515
(O-H stretching), 3416 (C-H aromatic stretching), 1719 (C=O
stretching), 1611 (C=C stretching of aromatic ring). 1H NMR
(300 MHz, CD3OD δ/ppm): 7.72 (d, J = 8.7 Hz, 1H, H-5),
7.58-7.32 (m, 4H, H-3' to 6'), 7.05 (dd, J = 8.7, 2.7 Hz, 1H, H-
6), 6.98 (d, J =2.4 Hz, 1H, H-8), 6.17 (s, 1H, H-3), 5.27 (s,
7-Methoxy-4-methyl-1-benzopyran-2-one (3a): White
amorphous powder; yield 58 %; m.p. 88-90 ºC; m.f.: C11H10O3;

328 Aziz-ur-Rehman et al.
Asian J. Chem.
2H, H-7'), 2.44 (s, 3H, CH3-4), EIMS: m/z 300 [M]+, 175
[C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 125 [C7H6Cl]+, 108
[C6H4O2]+, 51 [C4H3]+.
O-(4-Methyl-2-oxo-1-benzopyran-7-yl)-O-phenylcar-
bonate (3l): Oily liquid; yield 54 %; m.f.: C17H12O5; m.w. 296;
IR (KBr, νmax, cm-1): 3529 (O-H stretching), 3409 cm (C-H
aromatic stretching), 1706, 1727 (C=O stretching), 1621 (C=C
stretching of aromatic ring). 1H NMR (300 MHz, CD3OD δ/
ppm): 7.40 (d, J = 8.7 Hz, 1H, H-5), 7.17 (d, J = 2.1 Hz, 1H,
H-8), 6.70 (dd, J = 8.7, 2.1 Hz, 1H, H-6), 6.75-6.81 (m, 5H,
H-2' to 6'), 6.00 (s, 1H, H-3), 2.30 (s, 3H, CH3-4). EIMS: m/z
296 [M]+, 175 [C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 108
[C6H4O2]+, 93 [C6H5O]+, 51 [C4H3]+.
7-(4-Bromobenzyloxy)-4-methyl-1-benzopyran-2-one
(3g): Light yellow amorphous powder; yield 72 %; m.p. 128-
132 ºC; m.f.: C17H13O3Br; m.w. 345; IR (KBr, νmax, cm-1): 3523
(O-H stretching), 3412 (C-H aromatic stretching), 1725 (C=O
stretching), 1616 (C=C stretching of aromatic ring). 1H NMR
(300 MHz, CD3OD δ/ppm): 7.70 (d, J = 9.0 Hz, 1H, H-5),
7.55 (d, J = 8.4 Hz, 2H, H-3', H-5'), 7.37 (d, J = 8.4 Hz, 2H,
H-2', H-6'), 7.03 (dd, J = 8.7 2.4 Hz, 1H, H-6), 6.97 (d, J =2.4
Hz, 1H, H-8), 6.16 (s, 1H, H-3), 5.15 (s, 2H, H-7'), 2.44 (s,
3H, CH3-4), EI-MS: m/z 345 [M]+, 175 [C10H7O3]+, 170
[C7H6Br]+,159[C10H7O2]+,144[C9H4O2]+,108[C6H4O2]+,51[C4H3]+.
7-(4-Flurobenzyloxy)-4-methyl-1-benzopyran-2-one
(3h): Creamy white amorphous powder; yield 85 %; m.p. 110-
112 ºC; m.f.: C17H13O3F; m.w. 284; IR (KBr, νmax, cm-1): 3511
(O-H stretching), 3417 (C-H aromatic stretching), 1721 (C=O
stretching), 1617 (C=C stretching of aromatic ring). 1H NMR
(300 MHz, CD3OD δ/ppm): 7.67 (d, J = 8.7 Hz, 1H, H-5),
7.65 (d, J = 8.4 Hz, 2H, H-3', H-5'), 7.13 (d, J = 8.4 Hz, 2H, H-
2' and H-6'), 7.01 (dd, J = 8.7, 2.7 Hz, 1H, H-6), 6.95 (d, J =
2.4 Hz, 1H, H-8), 6.13 (d, J = 0.9 Hz, 1H, H-3), 5.17 (s, 2H,
H-7'), 2.42 (s, 3H, CH3-4), EI-MS: m/z 284 [M]+, 175
[C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 109 [C7H6F]+, 108
[C6H4O2]+, 51 [C4H3]+.
in vitro α-Chymotrypsin assay: The chymotrypsin
inhibitory activity of the compounds was performed by the
method of Cannell et al.14. Chymotrypsin (9 units/mL of 50
mM Tris-HCl buffer pH 7.6; Sigma Chemical Co. USA) was
pre-incubated with the compounds for 20 min at 258 ºC. 100
mL of substrate solution (1 mg of N-succinyl-phenylalanine-
p-nitroanilide/mL of 50 mM Tris-HCl buffer pH 7.6) was added
to start the enzyme reaction. The absorbance of released
p-nitroaniline was continuously monitored at 410 nm until a
significant colour change was achieved. The final DMSO
concentration in the reaction mixture was 7 %. The percentage
(%) inhibition was calculated as follows (ES)/E x 100, where
E is the activity of the enzyme without test compound and S is
the activity of enzyme in the presence of the test compound.
The concentrations of test compounds that inhibited the
hydrolysis of substrate upto 50 % (IC50) were determined by
monitoring the effect of various concentrations of these
compounds in the assays on the inhibition values. The IC50
value was then designed using the EZ-Fit Enzyme Kinetics
program (Perrella Scientific Inc., Amherst, USA).
7-(4-Chlorobenzyloxy)-4-methyl-1-benzopyran-2-one
(3i): White amorphous powder; yield 60 %; m.p. 178-180 ºC;
m.f.: C17H13O3Cl; m.w. 300; IR (KBr, νmax, cm-1): 3519 (O-H
stretching), 3423 (C-H aromatic stretching), 1721 (C=O
stretching), 1619 (C=C stretching of aromatic ring). 1H-NMR
(300 MHz, CD3OD δ/ppm): 7.67 (d, J = 9.0 Hz, 1H, H-5),
7.43 (d, J = 8.4 Hz, 2H, H-2' and H-6'), 7.37 (d, J = 8.4 Hz,
2H, H-3' and H-5'), 7.03 (dd, J = 8.7, 2.7 Hz, 2.7, 1H, H-6),
6.97 (d, J = 2.4 Hz, 1H, H-8), 6.15 (s, 1H, H-3), 5.17 (s, 2H,
H-7'), 2.43 (s, 3H, CH3-4), EIMS: m/z 300 [M]+, 175
[C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 125 [C7H6Cl]+, 108
[C6H4O2]+, 51 [C4H3]+.
Statistical analysis: All the measurements were done in
triplicate and statistical analysis was performed by Microsoft
Excel 2003. Results are presented as mean sem.
RESULTS AND DISCUSSION
In the undertaken research, heterocyclic O-substituted
derivatives of coumarin were synthesized. The parent compound
7-hydroxy-4-methyl-1-benzenpyran-2-one (1), was prepared
by the coupling of resorcinol (a) with ethylacetoacetate (b) in
the presence of sulphuric acid. Further, the reaction of 1 with
different electrophiles (2a-l) yielded a series of O-substituted
derivative of 7-hydroxy-4-methyl-1-benzenpyran-2-one (3a-
l) as represented in Scheme-I. Synthesis of all O-substituted
derivatives 3a-i was performed in DMF (N,N-dimethyl-
formamide) and sodium hydride (NaH) which act as a base.
Complete conversion was achieved within 40-120 min by stirring.
The products were isolated by adding cold water in the reaction
mixture and filtering off the precipitated solid. In some cases,
compound was taken out through solvent extraction method
by chloroform/ ethyl acetate. The structure of all the prepared
compounds were characterized by 1H NMR, IR and mass spectral
data as elaborated in experimental section. Parent compound
(1) was synthesized as dark yellow powder. The molecular
formula C10H8O3 was established by molecular ion peak
observed at m/z 296 in EI-MS and by counting the number of
4-Methyl-2-oxo-1-benzopyran-7-yl-acetate (3j): White
amorphous powder; yield 88 %; m.p. 128-130 ºC; m.f.:
C12H10O4; m.w. 218; IR (KBr, νmax, cm-1): 3534 (O-H stretching),
3412 (C-H aromatic stretching), 1715, 1729 (C=O stretching),
1627 (C=C stretching of aromatic ring). 1H NMR (300 MHz,
CD3OD δ/ppm): 7.69 (d, J = 8.4 Hz, 1H, H-5), 7.11 (dd, J =
8.4 Hz, 2.1, 1H, H-6), 6.32 (d, J =1.2 Hz, 1H, H-8), 6.1 (s,
1H, H-3), 2.4 (s, 3H, CH3-4), 2.3 (s, 3H, CH3COO). EIMS: m/z
218 [M]+, 175 [C10H7O3]+, 159 [C10H7O2]+, 144 [C9H4O2]+, 108
[C6H4O2]+, 51 [C4H3]+, 43 [C2H3O]+.
4-Methyl-2-oxo-1-benzopyran-7-yl-benzoate (3k):
Creamy white amorphous powder; yield 78 %; m.p. 136-
138 ºC; m.f.: C17H12O4; m.w. 280; IR (KBr, νmax, cm-1): 3531
(O-H stretching), 3410 (C-H aromatic stretching), 1718, 1734
cm-1 (C=O stretching), 1623 (C=C stretching of aromatic ring).
1H NMR (300 MHz, CD3OD δ/ppm): 8.20-7.84 (m, 5H, H-2'
to H-6'), 7.80 (d, J = 9.0 Hz, 1H, H-5), 7.30 (dd, J = 8.7, 2.1
Hz, 1H, H-6), 6.90 (d, J =2.4 Hz, 1H, H-8), 6.30 (s, 1H, H-3),
2.50 (s, 3H, CH3-4), EIMS: m/z 280 [M]+, 175 [C10H7O3]+,
159 [C10H7O2]+, 144 [C9H4O2]+, 108 [C6H4O2]+, 105 [C7H5O]+,
51 [C4H3]+.
1
protons in its H-NMR spectrum. The IR spectrum showed
absorption peaks at 3550, 3418, 1717 and 1629 cm-1 which
were assigned to O-H (stretching of hydroxyl group), C-H
(aromatic stretching), C=O (stretching of carbonyl bond) and

Vol. 26, No. 2 (2014)
Synthesis and α-Chymotrypsin Activity of Derivatives of 7-Hydroxy-4-methyl-1-benzopyran-2-one 329
O
O
RO
H
, Na
DMF
Stir. at RT
(2a-i
OH
O
O
HO
conc H2SO4
)
R1-X
O
C
O
C
CH3
O
3a-i
+
C2H5O
dist H2O
C
H2
CH3
OH
O
RO
1
CH3
b
a
NaOH Soln.
Stir. at RT
j-l)
R2-X (2
CH3
3j-l
Compound
R
Compound
R
Compound
R
7'
7'
8'
CH2 CH2
1'
CH3
6'
2'
6'
2'
4'
4'
Cl
CH2
3a
3e
3i
7'
6'
H2
C
4'
O
CH2
2'
CH3
2'
3b
3f
3j
H3C
C
1'
Cl
3'
H3C
O
C
7'
6'
2'
6'
2'
4'
Br
F
CH2
CH
1'
H3C
2'
3c
3g
3h
3k
3l
4'
O
C
H
1'
2'
7'
6'
2'
6'
4'
HC
C
3'
CH2
CH2
3d
4'
O
H
2'
Scheme-1: Outline for the synthesis of O-substituted derivatives of 7-hydroxy-4-methyl-1-benzopyarn-2-one
C=C (stretching of aromatic ring), respectively. In the 1H-NMR
Cl
spectrum, the signals appeared at δ 7.58 (d, J = 8.7, 1H, H-5),
6.82 (dd, J = 8.7, 2.4 Hz, 1H, H-6), 6.70 (d, J =2.4 Hz, 1H,
H-8) and 6.09 (d, J = 0.9, 1H, H-3). In the aliphatic region of
the spectrum, signal resonated at δ 2.41 (d, J = 0.9, 3H, CH3-4)
indicated the presence of one methyl group present in the
molecule. On the basis of mentioned cumulative evidences,
the structure of compound 1 was assigned 7-hydroxy-4-methyl-
1-benzenpyran-2-one. Mass fragmentation pattern of one of
the compound, 7-(4-chlorobenzyloxy)-4-methyl-1-benzopyran-
2-one (3i) is clearly elaborated in Fig. 1. Similarly, the structures
of other 7-O-alkyl/arenyl/acyl derivatives were characterized
by IR, 1H NMR and EI-MS data.
m/z = 111
C11H9O3
Cl
O
H2C
O
O
[M+] m/z = 300
CH3
C9H7ClO
C7H6Cl
C10H7O3
O
O
O
O
O
All the compounds were screened against α-chymotrypsin
enzyme and data is given in Table-1. All compounds showed
varying degree of enzyme inhibition. It is clearly evident that
the compounds 4-methyl-2-oxo-1-benzopyran-7-yl benzoate
(3k) and 7-(4-chlorobezyloxy)-4-methyl-1-benzopyran-2-one
(3i) were found to be good inhibitors against a-chymotrypsin
enzyme having IC50 value 58.11 0.22 and 115.11 0.05 µmol/L,
relative to Chymostatin a reference standard with IC50 value of
8.24 0.11 µmoles/L, respectively. This indicates that nature
of the substituent has great effect on inhibitory activity of these
derivatives. These compounds can further be exploited and their
derivatives could be synthesized to get closer to IC50 values of
the standard, chymostatin. In this way, the compounds could be
potential target in the drug discovery and drug development
program. Compounds (3e, 3f) were found to be the least inhibi-
tor having IC50 value > 400. Similarly, the compounds 3a, 3g, 3j
and 3l were remained inactive against α-chymotrpsin enzyme.
Cl
CH3
CH3
m/z = 175
m/z = 134
m/z = 125
CO
O
CH3
C2H2
C2HCl
O
Cl
O
O
CH3
m/z = 147
m/z = 100
m/z = 65
m/z = 119
Fig. 1. Mass fragmentation pattern of 7-(4-chlorobezyloxy)-4-methyl-1-
benzopyran-2-one (3i)
Conclusion
The projected structures of the synthesized compounds
are well supported by spectroscopic data. The synthesized
compounds showed varying degree of chymostatin activity.

330 Aziz-ur-Rehman et al.
Asian J. Chem.
TABLE-1
BIOACTIVITY STUDIES OF O-SUBSTITUTED DERIVATIVES
OF 7-HYDROXY-4-METHYL-1-BENZOPYARN-2-ONE
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65.33 0.11
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52.45 0.15
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