7-Hydroxy-2-substituted-4-H-1-benzopyran-4-one derivatives as aldose reductase inhibitors: A SAR study

Article abstract of DOI:10.1016/S0223-5234(01)01272-7

On the basis of the results of molecular modelling studies performed on the aldose reductase (ALR2) inhibitor 7-hydroxy-2-(4′-hydroxybenzyl)-4H-1-benzopyran-4-one (compound A) bound at the active site of the enzyme, we synthesised and tested on bovine and human ALR2 several derivatives modified at position 2 of the benzopyran moiety, in order to confirm the hypothesised binding mode of this compound. The substitution of the methylene bridge with the isosteric sulphur substituent gives an active derivative, while substitution with a polar NH causes a decrease in inhibitory activity; this is in accordance to the previously reported structure in which the methylene linker was found to be adjacent to a hydrophobic aminoacid (Leu300). Among the substituents at 4′ position examined, the most favourable for inhibitory activity are those able to act as hydrogen bond donors, supporting the hypothesis of the importance of the interaction with Thr113 for the inhibition of the enzyme.

Full text of DOI:10.1016/S0223-5234(01)01272-7

Eur. J. Med. Chem. 36 (2001) 697–703
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01272-7/FLA
Original article
7-Hydroxy-2-substituted-4-H-1-benzopyran-4-one derivatives as aldose reductase
inhibitors: a SAR study
Luca Costantinoa,*, Antonella Del Corsob, Giulio Rastellia, J. Mark Petrashc, Umberto Murab
aDipartimento di Scienze Farmaceutiche, Modena e Reggio Emilia University, Via Campi 183, I-41100 Modena, Italy
bDipartimento di Fisiologia e Biochimica, Pisa University, Via S. Maria 55, I-56100 Pisa, Italy
cDepartment of Ophthalmology and Visual Sciences, Washington University, School of Medicine, St. Louis, MO 63110, USA
Received 16 May 2001; accepted 23 July 2001
Abstract – On the basis of the results of molecular modelling studies performed on the aldose reductase (ALR2) inhibitor
7-hydroxy-2-(4%-hydroxybenzyl)-4H-1-benzopyran-4-one (compound A) bound at the active site of the enzyme, we synthesised and
tested on bovine and human ALR2 several derivatives modified at position 2 of the benzopyran moiety, in order to confirm the
hypothesised binding mode of this compound. The substitution of the methylene bridge with the isosteric sulphur substituent gives
an active derivative, while substitution with a polar NH causes a decrease in inhibitory activity; this is in accordance to the
previously reported structure in which the methylene linker was found to be adjacent to a hydrophobic aminoacid (Leu300). Among
the substituents at 4% position examined, the most favourable for inhibitory activity are those able to act as hydrogen bond donors,
´
supporting the hypothesis of the importance of the interaction with Thr113 for the inhibition of the enzyme. © 2001 Editions
scientifiques et me´dicales Elsevier SAS
aldose reductase inhibitors / benzopyran-4-one derivatives / diabetic complications
1. Introduction
formula A (IC50: 2.50 mM) [2], starting from the
activity of the flavonoid Quercetin.
Aldose reductase (ALR2) is the first enzyme of the
The 7-hydroxyl substituent was identified as the
polyol pathway; it converts glucose to sorbitol. Sor-
most important structural requirement for ALR2 in-
bitol is then converted to fructose by the second
hibition, because in its dissociated form it hydrogen
enzyme of this pathway, sorbitol dehydrogenase. Glu-
bonds to Tyr48 and His110. The 2-benzyl substituent
cose, metabolised through this pathway, has been
was found, from molecular docking experiments, to
linked to long-term diabetic complications such as
fit optimally an additional hydrophobic pocket of the
cataract, retinopathy, nephropathy and neuropathy.
enzyme, lined by Trp111 and Leu300, with the 4%-hy-
droxy group hydrogen bonding to Thr113 [2, 3].
Thus aldose reductase inhibitors (ARIs) have been
proposed as therapeutic agents able to prevent the
Given the interest in this class of compounds, ow-
development of diabetic complications [1].
ing to their lower acidity with respect to carboxylic
acids (a well known class of ARIs), which potentially
In a recent publication, we reported the discovery
of a new and more potent ARI, a compound of
make for better pharmacokinetic properties, we de-
cided to expand structure–activity relationships by
modification of compound A in two different ways:
first, in order to investigate the importance of the
hydrogen bond between the 4%-hydroxyl and Thr113,
we introduced different functional groups at 4% posi-
tion; then the methylene connecting the phenyl to the
benzopyran-4-one nucleus was changed to sulphur
and amine analogues.
* Correspondence and reprints
E-mail address: costantino.luca@unimo.it (L. Costantino).

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L. Costantino et al. / European Journal of Medicinal Chemistry 36 (2001) 697–703
Moreover, while IC50s were determined only on
160 °C in the presence of a catalytic amount of
p-toluenesulfonic acid yielded 1, while compound 2
was obtained by treatment of the corresponding sulf-
oxide 2a with 4-hydroxythiophenol, followed by de-
protection of the 7-hydroxyl.
7-Hydroxy-2-(4%-chlorobenzyl)-4H-1-benzopyran-4-
one (3) was synthesised by the reaction between 2-hy-
droxy-4-[2-(tetrahydropyranyl)oxy]acetophenone (1a)
and methyl 4-chlorophenylacetate in the presence of
NaH–pyridine (figure 2). Treatment of the intermedi-
ate 1,3-diketone with hydrochloric acid–acetic acid
enabled the cyclisation and removal of the protecting
group simultaneously, to yield 3.
partially purified bovine ALR2 in our previous study
and the modelling experiments were performed on
human ALR2 [2], here inhibition constants (Ki) are
determined also for both highly-purified bovine and
human enzyme, so as to investigate possible differ-
ences between the inhibition of the two enzymes.
2. Chemistry
7-Hydroxy-2-(4%-hydroxyphenylamino)-4H-1-
benzopyran-4-one (1) and 7-hydroxy-2-(4%-hydroxy-
phenylthio)-4H-1-benzopyran-4-one (2) were ob-
tained following general methods [4, 5] starting from
the intermediate 2-methylthio-7-[2-(tetrahydropy-
ranyl)oxy]-4H-1-benzopyran-4-one (1c) (figure 1).
Treatment of this intermediate with 4-aminophenol at
7-Hydroxy-2-(4%-nitrobenzyl)-4H-1-benzopyran-4-
one (4) was synthesised starting from 2-hydroxy-4-
methoxy(methoxycarbonyl)acetophenone (4a) by re-
action with 4-nitrophenyl acetylchloride in the
presence of Mg(OEt)2 (figure 3) following a general
t
Figure 1. (a): CS2, BuOK; (b): CH3I; (c): mCIPBA; (d): 4-aminophenol, D; (e): 4-hydroxythiophenol, K2CO3; (f): p-toluenesulfonic
acid.
Figure 2. (a): ClC6H4CH2COOCH3, NaH; (b): CH3COOH/HCl.

L. Costantino et al. / European Journal of Medicinal Chemistry 36 (2001) 697–703
699
Figure 3. (a): NO2C6H4CH2COCl; Mg(OEt)2; (b): HBr 48%; (c): D; (d): HBr 48%; (e) SnCl2.
Table I. Inhibitory activity toward ALR2.
Compound
X
R4%
IC50 (bovine enzyme) a
Ki (bovine enzyme) b
Ki (human enzyme) b
1
2
3
4
5
NH
S
CH2
CH2
CH2
OH
OH
Cl
NO2
NH2
9.08 (7.21–11.43)
1.17 (0.98–1.39)
12.89 (11.23–14.80)
18.70 (15.92–21.97)
2.74 (2.23–3.37)
2.5290.17
0.2990.01
N.T. c
0.7690.17
0.3090.02
N.T. c
N.T. c
N.T. c
0.5390.05
0.4390.03
a Partially purified enzyme; IC50 values (mM) (95% C.L.).
b Highly purified enzyme; Ki values (mM) (9S.D.).
c N.T.: not tested.
purified bovine lens ALR2 [2]) with the isosteric sul-
phur substituent (compound 2) gives a compound
with similar activity (IC50: 1.17 mM, table I) while
substitution with a polar NH (compound 1) causes a
decrease in the inhibitory activity of about one order
of magnitude (IC50: 9.08 mM, table I). The same
trend is obtained from the Ki values (uncompetitive
inhibition) determined on highly purified bovine and
human ALR2 (table I). These conclusions are consis-
tent with the previously reported structure of the
complex between compound A and ALR2 [2], in
which the methylene linker was found to be adjacent
to a hydrophobic aminoacid (Leu300).
procedure [6]. The methyl 7-methoxy-2-(4%-nitroben-
zyl)-4H-1-benzopyran-4-one-3-carboxylate (4b) thus
obtained was saponified using HBr 48%, then decar-
boxylated by heating in quinoline. Subsequent
treatment with HBr 48% yielded the desired com-
pound 4. Reduction of 4 with SnCl2 afforded com-
pound 5.
3. Results
The substitution of the methylene bridge in the lead
compound A (IC50: 2.50 mM towards partially

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L. Costantino et al. / European Journal of Medicinal Chemistry 36 (2001) 697–703
The effect of the substituents at 4% position able to
DMSO-d6 solution. J values are given in Hz. Micro-
analyses were carried out in the Microanalysis Labora-
tory of the Dipartimento di Scienze Farmaceutiche,
Modena University. Analyses indicated by the symbol
of the elements were within 0.4% of the theoretical
values. TLCs were performed on precoated silica gel
F254 plates (Merck). Silica gel (Merck, 70-230 Mesh)
was used for column chromatography.
act as hydrogen bond donors to Thr113 (OH, com-
pound A, NH2, compound 5) was compared with that
of substituents which cannot act as donors like Cl and
NO2 (compounds 3 and 4). While the derivative with
the NH2 moiety at 4% (compound 5) retains activity
toward ALR2 in all inhibitory assays here performed
(table I), the derivatives with Cl and NO2 at 4% (com-
pounds 3 and 4) are considerably less active (table I).
Similar conclusions were obtained for 4%-OCH3 (IC50:
7.01 mM, bovine enzyme [2]), and for 4%-CH3 or 4%-
CF3, which are one order of magnitude less active
than A on both bovine and human ALR2 [unpub-
lished results]. Taken together, these results support
the hypothesis that a hydrogen bond donor sub-
stituent interacting with Thr113 is important for
activity.
In conclusion, the results obtained from this limited
number of derivatives seem to confirm the hypothe-
sised binding mode of this class of compounds, given
the effects of substituents on the hydrophobic linker
region and on the hydrogen bond donor region of
Thr113. These conclusions hold for both bovine (par-
tially or highly purified) enzyme and for human en-
zyme (table I). This last finding is consistent with the
high sequence identity between the bovine and human
enzymes [7, 8] (86% identity), with particular empha-
sis on the binding site, where the aminoacidic residues
in contact with the inhibitors are completely con-
served between the two related enzymes [2]. However,
it should be noted that compound 1 is more active in
the presence of human than bovine ALR2 (table I),
and other studies are needed in order to explain this
difference.
4.1.1. 4-Hydroxy-7-[2-tetrahydropyranyl)oxy]-
2-thio-2H-1-benzopyran (1b)
A
solution of 2-hydroxy-4-[2-(tetrahydropyranyl)-
oxy]acetophenone (1a) [9] (6.62 g, 28.1 mmol) and CS2
(1.69 mL, 28.1 mmol) in anh. THF (20 mL) was slowly
added to a stirred suspension of potassium t-butoxide
(9.40 g, 83.8 mmol) in anh. THF (20 mL) at 15 °C. The
red mixture was stirred overnight at r.t. under N2 atmo-
sphere and then poured into water, extracted with
CH2Cl2 (2×20 mL), acidified to pH 5 and extracted with
EtOAc (2×25 mL). The solvent was dried (Na2SO4) and
removed under reduced pressure. Yield 4.00 g (51%),
1
m.p. 121–123 °C (EtOAc), H-NMR: 7.82 (1H, d, J=
8.78), 7.19 (1H, d, J=2.81), 7.10 (1H, dd, J=8.78,
J=2.81), 6.59 (1H, s), 5.70 (1H, broad s), 3.70 (2H, m),
1.75 (6H, m).
4.1.2. 2-Methylthio-7-[2(tetrahydropyranyl)oxy]-
4H-1-benzopyran-4-one (1c)
A stirred mixture of 1b (4.00 g, 14.4 mmol), K2CO3
(2.24 g, 16.2 mmol) in anh. acetone (50 mL) and
iodomethane (4.00 mL, 64.3 mmol) was refluxed for 30
min. The mixture was then filtered, water was added to
the filtrate and the mixture was extracted with CHCl3
(3×25 mL); the solvent was dried (Na2SO4) and re-
moved under reduced pressure and the residue purified
by column chromatography (CH2Cl2:CH3OH 97.5:2.5).
These data will be useful in the future development
of new inhibitors based on the benzopyran-4-one
scaffold.
1
Yield 2.20 g (52%) (viscous oil); H-NMR (CDCl3): 8.07
(1H, m), 7.05 (2H, m), 6.16 (1H, s), 5.55 (1H, m), 3.80
(2H, m), 2.54 (3H, s), 1.80 (6H, m).
4. Experimental protocols
4.1.3. 7-Hydroxy-2-(4%hydroxyphenylamino)-
4H-1-benzopyran-4-one (1)
4.1. Chemistry
A mixture of 1c (0.070 g, 0.24 mmol), 4-aminophenol
(0.50 g, 4.58 mmol), p-toluenesulfonic acid monohy-
drate (0.010 g, 0.05 mmol) and ethylene glycol (1 mL)
was heated at 160 °C for 20 min. The mixture was then
cooled, water was added and the precipitate was filtered,
washed with water and purified by means of column
chromatography (cyclohexane:EtOAc 87.5:12.5, then
acetone 100%). Yield 0.010 g (16%), m.p. 254 °C (dec.);
Melting points were determined on a Buchi 510 melt-
ing point apparatus and are uncorrected. 1H-NMR
spectra were recorded on a Bruker AC200 spectrometer
(Centro Interdipartimentale Grandi Strumenti, Modena
University) in DMSO-d6 solution. Chemical shifts are
reported in ppm from tetramethylsilane as internal stan-
dard. Unless otherwise stated, spectra were recorded in

L. Costantino et al. / European Journal of Medicinal Chemistry 36 (2001) 697–703
701
1H-NMR: 10.46 (1H, s), 9.51 (2H, s), 7.75 (1H, d,
J=8.54), 7.10 (2H, m), 6.75 (4H, m), 5.14 (1H, s).
Analysis: C15H11NO4: C, H, N.
12.6 mmol) in anh. pyridine (8 mL). When the reaction
subsided, the mixture was heated at 90 °C for 15 min.
After cooling, the mixture was decomposed in 2N HCl
and extracted with CH2Cl2 (3×25 mL). The combined
organic layers were washed with 1N HCl (2×30 mL)
and water (30 mL) and dried (Na2SO4); then the solvent
was removed under reduced pressure. The residue was
then purified by means of column chromatography (cy-
clohexane:EtOAc 75:25). Yield 0.76 g (63%), m.p. 216–
4.1.4. 2-Methylsulphinyl-7-[2-(tetrahydropyranyl)oxy]-
4H-1-benzopyran-4-one (2a)
A solution of m-chloroperbenzoic acid (1.11 g, 85%
pure, 5.47 mmol) in CHCl3 (10 mL) was slowly added to
a solution of 1c (1.60 g, 5.5 mmol) in CHCl3 (10 mL)
maintained at −10 °C. After 2 h, the suspension was
filtered and the filtrate was washed with 5% NaHCO3
solution, dried (Na2SO4) and the solvent evaporated
under reduced pressure. The residue was then purified
by means of column chromatography (CH2Cl2:CH3OH
1
217 °C, H-NMR: 10.60 (1H, s), 7.83 (1H, d, J=8.21),
7.40 (4H, m), 6.90 (1H, dd, J=8.21, J=2.30), 6.80 (1H,
d, J=2.30), 6.08 (1H, s), 4.00 (2H, s). Analysis:
C16H11ClO3: C, H.
1
97.5:2.5). Yield 0.90 g (53%) (oil), H-NMR (CDCl3):
4.1.8. Methyl 7-methoxy-2-(4%-nitrobenzyl)-4H-1-
benzopyran-4-one 3-carboxylate (4b)
8.05 (1H, m), 7.00 (2H, m), 6.78 (1H, s), 5.50 (1H, broad
s), 3.70 (2H, m), 2.92 (3H, s), 1.70 (6H, m).
A
solution of 2-hydroxy-4-methoxy-(methoxycar-
bonyl)acetophenone (4a) [6] (6.00 g, 26.8 mmol) in anh.
EtOH (30 mL) was added to a suspension of Mg(OEt)2
(3.07 g, 26.8 mmol) in anh. EtOH (100 mL). A white
precipitate formed. EtOH was then removed under re-
duced pressure, and anh. benzene was added (70 mL),
followed by 4-nitrophenylacetyl chloride (5.35 g, 26.9
mmol) dissolved in benzene (30 mL). The mixture was
refluxed for 3 h, then it was cooled and poured in 10%
acetic acid in water (300 mL) at 0 °C. The suspension
was extracted with EtOAc (3×100 mL), the organic
layer was dried (Na2SO4) and the solvent removed under
reduced pressure. Finally, the resultant residue was
purified by column chromatography (cyclohex-
ane:EtOAc 50:50). Yield 6.00 g, (61%), m.p. 146–147
°C, 1H-NMR (CDCl3): 8.26 (2H, m), 8.12 (1H, d,
J=8.92), 7.60 (2H, m), 7.00 (1H, dd, J=8.92, J=
2.38), 6.77 (1H, d, J=2.38), 4.20 (2H, s), 3.99 (3H, s),
3.92 (3H, s).
4.1.5. 2-(4-Hydroxyphenylthio)-7-[2-tetrahydropyranyl)-
oxy]-4H-1-benzopyran-4-one (2b)
4-Hydroxythiophenol (0.13 g, 1.03 mmol) in anh.
acetone (2 mL) and K2CO3 (0.14 g, 1.02 mmol) were
added at r.t. to a stirred solution of 2a (0.20 g, 0.65
mmol) in anh. acetone (10 mL). After 8 h, HCl 1N was
added until pH 5. The precipitate was filtered and
washed with water. Yield 0.15 g (63%), m.p. 182–183
1
°C; H-NMR: 10.31 (1H, s), 7.86 (1H, m), 7.55 (2H, m),
7.15 (2H, m), 6.95 (2H, m), 5.70 (1H, broad s), 5.56 (1H,
s), 3.77 (2H, m), 1.80 (6H, m).
4.1.6. 7-Hydroxy-2-(4-hydroxyphenylthio)-4H-
1-benzopyran-4-one (2)
p-Toluenesulfonic acid monohydrate (0.010 g, 0.053
mmol) was added to a suspension of 2b (0.15 g, 0.41
mmol) in CH3OH (20 mL), and refluxed for 15 min.
Water was then added to the solution thus obtained and
the precipitate was filtered and washed with water. Yield
0.10 g (86%), m.p. 278–280 °C (acetone), 1H-NMR:
10.80 (1H, s), 10.26 (1H, s), 7.80 (1H, d, J=8.69), 7.52
(2H, m), 6.97 (2H, m), 6.88 (1H, dd, J=8.69, J=2.43),
6.75 (1H, d, J=2.43), 5.51 (1H, s). Analysis C15H10O4S:
C, H.
4.1.9. 7-Methoxy-2-(4%-nitrobenzyl)-4H-
1-benzopyran-4-one-3-carboxylic acid (4c)
A suspension of 4b (0.50 g, 1.4 mmol) in HBr 48% (15
mL) was heated at 110–120 °C for 30 min. After
cooling, the precipitate was filtered and washed with
1
water. Yield 0.40 g (83%), m.p. 185–186 °C, H-NMR:
8.22 (2H, m), 8.05 (1H, m), 7.69 (2H, m), 7.11 (2H, m),
4.51 (2H, s), 3.90 (3H, s).
4.1.7. 7-Hydroxy-2-(4%-chlorobenzyl)-4H-1-benzopyran-
4-one (3)
A
solution of 2-hydroxy-4-[2-(tetrahydropyranyl)-
4.1.10. 7-Methoxy-2-(4%-nitrobenzyl)-4H-1-benzopyran-
4-one (4d)
A solution of 4c (0.40 g, 1.12 mmol) in quinoline (65
mL) was heated at 180 °C for 10 min, then cooled and
poured in water (100 mL) containing conc. HCl (12
oxy]acetophenone (1a) [9] (1.0 g, 4.2 mmol) and methyl
4-chlorophenyl acetate (0.86 g, 4.6 mmol) in anh. pyri-
dine (8 mL) was added dropwise to a well-stirred sus-
pension of NaH (60% dispersion in mineral oil) (0.50 g,

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L. Costantino et al. / European Journal of Medicinal Chemistry 36 (2001) 697–703
mL). The suspension was extracted with EtOAc (3×40
mL), the organic phase was dried (Na2SO4) and the
solvent removed under reduced pressure. Yield 0.25 g
(71%), m.p. 153–155 °C (EtoAc), 1H-NMR (CDCl3):
8.25 (2H, m), 8.10 (1H, d, J=8.90), 7.51 (2H, m), 6.95
(1H, dd, J=2.91, J=8.90), 6.80 (1H, d, J=2.91), 6.16
(1H, s), 4.04 (2H, s), 3.91 (3H, s).
Protein concentration was evaluated by the method of
Bradford [12]. The electrophoretic homogeneity of en-
zyme preparations was assessed by SDS-PAGE accord-
ing to the method of Laemmli [13], and gels were stained
according to the method of Wray et al. [14].
The assay for aldose reductase activity was performed
at 37 °C using 4.7 mM
D,L-glyceraldehyde as substrate,
in 0.25 M sodium phosphate buffer pH 6.8 containing
0.11 mM NADPH, 0.38 M ammonium sulphate and 0.5
mM EDTA. One unit of enzyme activity is the amount
of enzyme which catalyses the oxidation of 1 mmol of
NADPH/min.
4.1.11. 7-Hydroxy-2-(4%-nitrobenzyl)-4H-1-benzo-
pyran-4-one (4)
A solution of 4d (0.5 g, 1.6 mmol) in HBr 48% (15
mL) was heated at 140 °C for 5 h. After cooling, the
precipitate was collected and washed with water. Yield
0.22 g (46%), m.p. 210 °C (dec.) (acetone–petroleum
The sensitivity of aldose reductase to different com-
pounds was tested in the above assay conditions in the
presence of the inhibitor dissolved at appropriate con-
centration in DMSO. The concentration of DMSO in
the assay mixture was kept constant at 1%. IC50 values
on partially purified bovine lens aldose reductase were
obtained using crude enzyme after the ion exchange
chromatography on DEAE-DE52 purification step [2].
Each log dose–inhibition curve was generated using at
least four concentrations of inhibitor causing an inhibi-
tion between 20 and 80%, with two replicates at each
concentration; the 95% confidence limits (95% CL) were
calculated from T values for n=2, where n is the total
number of determinations [2]. For the determination of
1
ether); IR (cm−1) (Nujol): 1639, 1555, 1299; H-NMR:
10.76 (1H, s), 8.25 (2H, m), 7.85 (1H, d, J=8.90), 7.66
(2H, m), 6.90 (1H, dd, J=2.33, J=8.90), 6.75 (1H, d,
J=2.33), 6.18 (1H, s), 4.18 (2H, s). Analysis:
C16H11NO5: C, H, N.
4.1.12. 7-Hydroxy-2-(4%-aminobenzyl)-4H-1-benzo-
pyran-4-one (5)
SnCl2·2H2O (0.76 g, 3.4 mmol) was added to a solu-
tion of 4 (0.20 g, 0.7 mmol) in EtOH. The resulting
suspension was stirred and refluxed for 20 min, then the
solvent was removed under reduced pressure. The
residue was diluted with water (7 mL), filtrated and the
filtrate was brought to pH 6 with NaOH 0.1N and
extracted with EtOAc (3×30 mL); the organic phase
was dried (Na2SO4) and the solvent removed under
reduced pressure. Yield 0.1 g (56%), m.p. 215–218 °C;
Ki, double reciprocal plots with
D,L-glyceraldehyde as
variable substrate at fixed concentrations of NADPH
were obtained. For each inhibitor at least three different
concentrations were used.
1
IR (cm−1) (Nujol): 3465, 3358, 1643; H-NMR: 10.70
(1H, s), 7.86 (1H, d, J=8.60), 7.04 (2H, m), 6.88 (1H,
dd, J=8.60, J=2.20), 6.80 (1H, d, J=2.20), 6.55 (2H,
m), 6.00 (1H, s), 5.00 (2H, broad s), 3.75 (2H, s).
Analysis: C16H13NO3: C, H, N.
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