Synthesis and anti-inflammatory activity of N-substituted 2-oxo-2H-1- benzopyran-3-carboxamides and their 2-iminoanalogues

Article abstract of DOI:10.1016/S0223-5234(99)00119-1

A series of N-arylsubstituted 2-imino-2H-1-benzopyran-3-carboxamides 3a and b and 2-oxo-2H-1-benzopyran-3-carboxamides 4a-h were synthesized and evaluated for their anti-inflammatory activity in carrageenan-induced rat paw oedema assays and in acetic acid-induced peritonitis tests in albino rats. The resulting products were found to be active anti-inflammatory agents and their effects were comparable to that of piroxicam as the reference compound. In the consideration of the efficacy of the compounds in these assays, 2- imino/oxo-2H-1-benzopyran-3-carboxamides 3a and b and 4a-h were further studied at graded doses for their acute toxicity (ALD₅₀) in albino mice and were essentially non-toxic at the highest dose tested.

Full text of DOI:10.1016/S0223-5234(99)00119-1

Eur. J. Med. Chem. 34 (1999) 997−1001
1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
997
©
Preliminary communication
Synthesis and anti-inflammatory activity of N-substituted 2-oxo-2H-1-
benzopyran-3-carboxamides and their 2-iminoanalogues
‡
Igor E. Bylov*, Maksym V. Vasylyev, Yaroslav V. Bilokin
Department of Organic Chemistry, Ukrainian Academy of Pharmacy, 310002 Kharkov, Ukraine
(Received 12 November 1999; revised 6 May 1999; accepted 7 May 1999)
Abstract – A series of N-arylsubstituted 2-imino-2H-1-benzopyran-3-carboxamides 3a and b and 2-oxo-2H-1-benzopyran-3-carboxamides
a–h were synthesized and evaluated for their anti-inflammatory activity in carrageenan-induced rat paw oedema assays and in acetic
4
acid-induced peritonitis tests in albino rats. The resulting products were found to be active anti-inflammatory agents and their effects were
comparable to that of piroxicam as the reference compound. In the consideration of the efficacy of the compounds in these assays,
2
-imino/oxo-2H-1-benzopyran-3-carboxamides 3a and b and 4a–h were further studied at graded doses for their acute toxicity (ALD ) in
50
albino mice and were essentially non-toxic at the highest dose tested. © 1999 Éditions scientifiques et médicales Elsevier SAS
coumarins / benzopyrans / amides / Knoevenagel condensation / anti-inflammatory activity
1
. Introduction
ionophore-stimulated mouse peritoneal macrophages. It
was also revealed [7] that compounds containing a ben-
zopyran moiety were potent and selective inhibitors of
cyclooxygenase (COX). Of the 3-substituted coumarin
derivatives, our attention was called to their N-substituted
amide derivatives, since marked anti-inflammatory activ-
ity of structurally related N-substituted amides of
Compounds comprising a coumarin (2-oxo-2H-1-
benzopyran) backbone have a wide range of biological
activities. Thus, among the natural and synthetic cou-
marin derivatives there are compounds possessing anti-
microbial [1], antitumour [2], antiviral [3] and other [4]
activities. Moreover, studies of the hydroalcohol extract
of Justicia pectoralis (Eha) and its main constituents,
coumarin (Cou) and umbelliferone (Umb), showed anal-
gesic and anti-oedema activities on acetic acid-induced
writhing in mice and on the carrageenan end dextran paw
oedema in rats [5]. The Eha, Cou and Umb presented a
significant anti-oedema effect in the carrageenan model
but only Cou decreased the rat paw volume in the dextran
model. Anti-inflammatory activity of coumarins isolated
from Santolina oblongifolia Boiss was also reported [6].
The isolated coumarins identified as 7-methoxycoumarin
4
-hydroxy-2-quinolone-3-carboxylic acids has been re-
ported [8]. Also taking into consideration the fact that
such anti-inflammatory drugs as mefenamic and meclofe-
namic acids [4] constitute derivatives of aromatic amino
acids, N-substituted coumarin-3-carboxamide derivatives
containing aromatic amino acid residues were selected as
targets for our anti-inflammatory studies.
2. Chemistry
The general synthetic strategy employed to prepare
(herniarin),
6,7-dihydroxycoumarin
-methoxy-7-glucosidylcoumarin (scopolin),
-hydroxy-7-methoxycoumarin (scopoletin) showed
(aesculetin),
N-substituted 2-imino/oxo-2H-1-benzopyran-3-carbox-
amide derivatives was based on Knoevenagel condensa-
tion [9, 10] of active methylene compounds. As shown in
figure 1, 2-imino-2H-1-benzopyran-3-N-R-carboxamides
6
6
and
marked activity as inhibitors of eicosanoid-release from
3
a and b were prepared by condensing N-substituted
*
Correspondence and reprints
‡
Present address: Department of Organic Chemistry, The Wei-
zmann Institute of Science, Rehovot 76100, Israel.
cyanoacetamides 1a and b and salicylic aldehyde 2 to
form the expected imino compounds using piperidine as

9
98
condensed with an equivalent amount of salicylic alde-
hydes 2a–f to produce the desired coumarin derivatives
4c–h utilizing piperidine as a catalyst in ethanol at room
temperature. An alternative approach for synthesis of
N-substituted coumarin-3-carboxamides of type 4, based
on rearrangements of 2-imino-2H-1-benzopyran-3-
carboxamides under the action of anthranilic acid as
N-nucleophile, has been recently developed [14] in our
laboratory.
Physicochemical data for the compounds 3a and b and
a–h are given in table I.
4
3
Figure 1. Synthesis of N-arylsubstituted 2-imino/oxo-2H-1-
benzopyran-3-carboxamides 3a and b and 4a and b.
. Pharmacology
The compounds synthesized were evaluated for their
anti-inflammatory activity in carrageenan-induced rat
paw oedema assays [15] and in acetic acid-induced peri-
tonitis tests in albino rats [16]. 2-Imino/oxo-2H-1-
benzopyran-3-carboxamides 3a and b and 4a–h were
further studied for their acute toxicity [17]. Piroxicam
was used as a control compound.
a catalyst in ethanol at room temperature [11, 12]. 2-Oxo-
2
H-1-benzopyran-3-N-R-carboxamides 4a and b were
obtained by acidic hydrolysis of the corresponding imino
analogues 3a and b employing a mixture of ethanol/
water/≈ 32% hydrochloric acid and refluxing for 1 h.
The synthesis of coumarin-3-(N-2-carboxyphenyl)
carboxamides 4c–h was carried out as shown in figure 2.
The precursor 2-carboxymalonanilic acid ester 5 [13] was
4. Results and discussion
Pharmacological results on anti-inflammatory activi-
ties and acute toxicity of the benzopyran derivatives 3a
and b and 4a–h and piroxicam are summarized in
table II.
In carrageenan-induced rat paw oedema assays, the
compounds 4g, 3b, 4b, and 4h were found to be the most
active anti-inflammatory agents at the graded dose
1
0 mg/kg po and exhibited 54 ± 6.50%, 51 ± 5.05%, 48
6.51%, and 47 ± 7.13% inhibition of inflammation,
respectively. Their effects were comparable to that of
±
Figure 2. Synthesis of N-arylsubstituted 2-oxo-2H-1-benzo-
pyran-3-carboxamides 4c–h.
Table I. Physicochemical data of the synthesized N-arylsubstituted 2-imino/oxo-2H-1-benzopyran-3-carboxamides 3a and b and 4a–h.
1
Compound
R
R
Molecular
formula
Yield
(%)
Recryst.
solvent
M.p. (°C)
3
3
4
4
4
4
4
4
4
4
a
b
a
b
c
d
e
f
H
H
H
H
2-CO CH3
C H N O
74
82
87
85
67
59
67
63
71
54
i-PrOH
i-PrOH
i-PrOH
i-PrOH
AcOH
BuOH
BuOH
BuOH
i-PrOH
MeCN
137–138
222–224
205–208
246–247
2
18 14
2
4
4-CO C H
C H N O
2
2
5
19 16 2
4
2-CO CH3
C H NO5
2
18 13
4-CO C H
C H NO5
2
2
5
19 15
a
H
2-CO H
C H NO5
275–276
2
17 11
6-OCH3
8-OCH3
6-NO2
6-Cl
8-allyl
2-CO H
C H NO6
124–125
140–142
147–150
232–235
249–251
2
18 13
2-CO H
C H NO6
2
18 13
2-CO H
C H N O
2
17 10
2
7
g
h
2-CO H
C H ClNO5
2
17 10
2-CO H
C H NO5
20 15
2
a
Lit. [21] m.p. for 4c: 279 °C.

9
99
Table II. Anti-inflammatory activities and acute toxicity of the benzopyran derivatives 3a and b and 4a–h and piroxicam.
Acute toxicity
Anti-inflammatory activity
carrageenan-induced paw oedema acetic acid peritonitis
Compound
Approximate LD50
(mg/kg) (in mice)
(Mean % inhibition ± SE)
po
ip
10 mg/kg po
b
3
3
4
4
4
4
4
4
4
4
a
b
a
b
c
d
e
f
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 1 000
> 700
19 ± 2.11
51 ± 5.05
44 ± 7.21
48 ± 6.51
38 ± 1.01
30 ± 6.96
41 ± 6.42
35 ± 7.11
54 ± 6.50
47 ± 7.13
57 ± 6.61
40 ± 6.43
> 700
> 700
> 700
> 700
> 700
> 700
> 700
> 700
> 700
> 1 000
32 ± 6.89
39 ± 5.09
35 ± 1.02
42 ± 7.20
18 ± 4.63
32 ± 7.01
29 ± 5.58
35 ± 4.88
31 ± 3.31
29 ± 7.24
b
b
b
b
b
g
h
a
a
Piroxicam
360
a
b
Data from ref. [22]; P < 0.05 Student’s t test versus controls.
piroxicam, the reference compound, which showed 57 ±
chromatography (TLC) was performed on aluminium
sheets precoated with silica gel (Merck, Kieselgel 60
F-254). H-NMR spectra were recorded on a Varian
6.61% inhibition at the same dose level.
1
At the same dose level (10 mg/kg po), in acetic acid
peritonitis tests, all compounds exhibited moderate to
good anti-inflammatory activity. The most active com-
pounds 4c, 3a, and 4a showed 42 ± 7.20%, 40 ± 6.43%,
and 39 ± 5.09% inhibition of inflammation, respectively.
In this test, piroxicam revealed a protection of 29 ±
WXR-400 spectrometer in DMSO-d
internal standard (chemical shifts in δ ppm), but a study
on isomerization of benzopyran-2-imines in DMSO-d
6
using TMS as an
6
has to be mentioned [18]. O’Callaghan et al. [18] re-
vealed that when unsubstituted 2-imino-2H-1-benzo-
7
.24%. All tested compounds were essentially non-toxic
pyran-3-carboxamide was dissolved in DMSO-d , NMR
6
at the highest dose graded.
spectra showed that a mixture of 2-imino-2H-1-
benzopyran-3-carboxamide and the isomeric 2-cyano-3-
(
2-hydroxyphenyl)prop-2-ene-1-carboxamide was pre-
sent and other benzopyran-2-imines behaved similarly
and the degree of isomerization varied considerably,
depending on the nature and position of the substituents
presented. In our case, isomerization of N-arylsubstituted
5. Conclusion
The products synthesized were found to be active
anti-inflammatory agents and their effects were compa-
rable to that of piroxicam as the reference compound. The
most active compounds 3b, 4a, 4b and 4g have been
marked for further detailed pharmacological studies to be
evaluated for COX-2 and COX-1 inhibition in micro-
somal and cellular assays.
2
-imino-2H-1-benzopyran-3-carboxamides 3a and b did
not occur in dimethyl sulfoxide-d at room temperature
and only starting materials were present. Mass spectra
6
(MS) were obtained with a Finnigan MAT-4615B spec-
trometer at an ionization potential of 70 eV. Infrared
spectra (IR) were recorded in KBr pellets on an IBM 486
PC computer-controlled Specord M-80 spectrometer. El-
emental analyses were performed at the Microanalysis
Laboratory, Kharkov State University, and the combus-
tion analyses of all compounds synthesized indicated by
the symbols of the elements were within ± 0.4% of
theoretical values. The N-substituted cyanoacetamides 1a
and b, which are key intermediates for synthesis of the
benzopyran-2-imines 3a and b, were prepared according
6. Experimental protocols
6.1. Chemistry
Melting points (°C) were measured with a Büchi
melting point apparatus and were uncorrected. Thin layer

1000
to reported methods [19, 20] from ethyl cyanoacetate and
methyl anthranilate or ethyl 4-aminobenzoate.
6.1.3. N-arylsubstituted 2-oxo-2H-1-benzopyran-3-
carboxamides 4c–h
To a well-stirred solution of 2-carboxymalonanilic acid
ester 5 [13] (4 mmol) in 10 mL of ethanol was added an
equivalent amount of salicylic aldehydes 2a–f and a few
drops of piperidine as a catalyst. The reaction mixture
was stirred at room temperature for ca. 1 day and then
poured into water. The products precipitated were filtered
and recrystallized from the suitable solvent. Yields and
physicochemical data of the synthesized N-substituted
6
.1.1. N-arylsubstituted 2-imino-2H-1-benzopyran-3-
carboxamides 3a and b
To a well-stirred solution of N-substituted cyanoaceta-
mides 1a and b (4 mmol) in 15 mL of ethanol, was added
an equivalent amount of salicylic aldehyde 2 and a few
drops of piperidine as a catalyst. The reaction mixture
was stirred at room temperature for 2 h. The products,
which precipitated in the course of the reactions were
filtered and recrystallized from the proper solvent. Yields
and physicochemical data of the synthesized
2
-imino-2H-1-benzopyran-3-carboxamides 4c–h are
1
listed in table I. 4c: H-NMR: δ 7.15 (dd 1H, J = 8.0, 8.0
Hz, ArH); 7.39 (m, 2H, ArH); 7.56 (dd, 1H, J = 8.0, 8.0
Hz, ArH); 7.73 (dd, 1H, J = 7.9, 7.9 Hz, ArH); 7.94 (d,
N-substituted 2-imino-2H-1-benzopyran-3-carboxamides
1
H, J = 8.2 Hz, ArH); 8.05 (d, 1H, J = 8.2 Hz, ArH); 8.65
1
3
a and b are listed in table I. 3a: H-NMR: δ 3.88 (s, 3H,
(d, 1H, J = 8.3 Hz, ArH); 8.85 (s, 1H, 4-CH); 13.52 (br s,
+⋅
–1
CH ); 7.13–7.25 (m, 3H, ArH); 7.48–7.58 (m, 2H, ArH);
3
1H, NH). MS m/z 309 (M ). IR (KBr), cm : m 3 266
7
.70 (d, 1H, J = 7.9 Hz, ArH); 7.88 (d, 1H, J = 7.9 Hz,
ArH); 8.42 (d, 1H, J = 8.41 Hz, ArH); 8.50 (s, 1H, 4-CH);
.79 (s, 1H, C=NH); 12.94 (s, 1H, NH). MS m/z 322
NH), 3 032 (CH), 1 731 (C=O), 1 696 (C=O), 1 673
17
11
5
8
H-NMR: δ 3.95 (s, 3H, OCH ); 7.14–7.32 (m, 4H, ArH);
3
+⋅
–1
(M ). IR (KBr), cm : m 3 300 (NH), 3 207 (NH), 3 041
7.42 (d, 1H, J = 8.2 Hz, ArH); 8.01 (d, 1H, J = 8.0 Hz,
(
CH), 1 715 (C=O), 1 678 (C=O), 1 641 (C=N), 1 606
ArH); 8.60 (d, 1H, J = 8.0 Hz, ArH); 8.89 (s, 1H, 4-CH);
1
+⋅
–1
(C=C). Anal. C H N O (C, H, N). 3b: H-NMR: δ
18
14
2
4
13.20 (s, 1H, NH). MS m/z 339 (M ). IR (KBr), cm : m
3 287 (NH), 2 952 (CH), 1 726 (C=O), 1 694 (C=O),
1 675 (C=O), 1 614 (C=C). Anal. C H NO (C, H, N).
1
.31 (t, 3H, J = 6.9 Hz, CH ); 4.30 (q, 2H, J = 6.9 Hz,
3
CH ); 7.27–8.04 (m, 8H, ArH); 8.59 (s, 1H, 4-CH); 9.32
2
1
8
13
6
+⋅
1
(s, 1H, C=NH); 13.16 (s, 1H, NH). MS m/z 336 (M ). IR
4
e: H-NMR: δ 3.95 (s, 3H, OCH ); 7.15–7.44 (m, 4H,
3
–1
(
1
KBr), cm : m 3 315 (NH), 2 976 (CH), 1 704 (C=O),
688 (C=O), 1 644 (C=N), 1 593 (C=C). Anal.
C H N O (C, H, N).
ArH); 7.52 (d, 1H, J = 8.3 Hz, ArH); 8.00 (d, 1H, J = 7.9
Hz, ArH); 8.64 (dd, 1H, J = 8.0, 8.0 Hz, ArH); 8.90 (s,
+⋅
1
9
16
2
4
1H, 4-CH); 13.12 (s, 1H, NH). MS m/z 339 (M ). IR
–1
(
KBr), cm : m 3 291 (NH), 2 947 (CH), 1 730 (C=O),
1
689 (C=O), 1 677 (C=O), 1 604 (C=C). Anal.
6
.1.2. N-arylsubstituted 2-oxo-2H-1-benzopyran-3-
1
C H NO (C, H, N). 4f: H-NMR: δ 7.21 (dd, 1H, J =
8
18
13
6
carboxamides 4a and b
.0, 8.0 Hz, ArH); 7.60 (dd, 1H, J = 8.0, 8.0 Hz, ArH);
A solution of the corresponding 2-iminobenzopyran
derivatives 3a and b (4 mmol) in 15–20 mL of a mixture
of ethanol/water/≈ 32% hydrochloric acid (30:1:1, v/v/v)
was refluxed with vigorous stirring for 1 h. After cooling
to room temperature the products precipitated were fil-
tered and recrystallized from the appropriate solvent.
Yields and physicochemical data of the synthesized
7.71 (d, 1H, J = 8.2 Hz, ArH); 8.02 (d, 1H, J = 7.9 Hz,
ArH); 8.46 (d, 1H, J = 8.3 Hz, ArH); 8.72 (d, 1H, J = 7.9
Hz, ArH); 8.95 (s, 1H, 5-CH); 9.10 (s, 1H, 4-CH); 13.21
+⋅
–1
(s, 1H, NH). MS m/z 354 (M ). IR (KBr), cm : m 3 295
(NH), 3 085 (CH), 1 754 (C=O), 1 724 (C=O), 1 683
(C=O), 1 618 (C=C). Anal. C H N O (C, H, N). 4g:
1
7 10 2 7
1
H-NMR: δ 7.20 (t, 1H, J = 7.9 Hz, ArH); 7.60–7.74 (m,
N-substituted 2-oxo-2H-1-benzopyran-3-carboxamides
3H, ArH); 7.98–8.06 (m, 2H, ArH); 8.12 (s, 1H, 5-CH);
9.03 (s, 1H, 4-CH); 12.47 (s, 1H, NH). MS m/z 345, 343
1
4
a and b are listed in table I. 4a: H-NMR: δ 3.92 (s, 3H,
+⋅
–1
CH ); 7.20–8.08 (m, 7H, ArH); 8.61 (d, 1H, J = 8.2 Hz,
(M ). IR (KBr), cm : m 3 389 (OH + NH), 3 047 (CH),
3
ArH); 9.04 (s, 1H, 4-CH); 12.16 (s, 1H, NH). MS m/z 323
1 736 (C=O), 1 689 (C=O), 1 671 (C=O), 1 607 (C=C).
+⋅
–1
1
(M ). IR (KBr), cm : m 3 248 (NH), 3 042 (CH), 1 735
Anal. C H ClNO (C, H, N). 4h: H-NMR: δ 3.63 (m,
17
10
5
(
C=O), 1 713 (C=O), 1 668 (C=O), 1 608 (C=C). Anal.
2H, CH –CH=CH ); 5.14 (m, 2H, CH –CH=CH ); 6.03
(m, 1H, CH –CH=CH ); 7.13 (dd, 1H, J = 8.0, 8.0 Hz,
2 2
2 2 2 2
1
C H NO (C, H, N). 4b: H-NMR: δ 1.38 (t, 3H, J =
1
8
13
5
7
.0 Hz, CH ); 4.34 (q, 2H, J = 7.0 Hz, CH ); 7.42–7.54
ArH); 7.32 (dd, 1H, J = 7.9, 7.9 Hz, ArH); 7.55 (m, 2H,
ArH); 7.78 (d, 1H, J = 8.0 Hz, ArH); 8.03 (d, 1H, J = 7.9
Hz, ArH); 8.76 (dd, 1H, J = 7.9, 7.9 Hz, ArH); 8.98 (s,
3
2
(
4
m, 2H, ArH); 7.72–8.06 (m, 6H, ArH); 9.00 (s, 1H,
-CH); 10.88 (s, 1H, NH). MS m/z 337 (M ). IR (KBr),
+⋅
–
1
+⋅
cm : m 3 250 (NH), 2 984 (CH) 1 704 (C=O), 1 671
C=O), 1 596 (C=C). Anal. C H NO (C, H, N).
1H, 4-CH); 12.53 (s, 1H, NH) MS m/z 349 (M ). IR
–1
(
(KBr), cm : m 3 224 (NH), 2 942 (CH), 1 724 (C=O),
19
15
5

1
001
1
684 (C=O), 1 657 (C=O), 1 600 (C=C). Anal.
difference in results was considered to be significant
when P < 0.05.
C H NO (C, H, N).
2
0
15
5
6
6
6
.2. Pharmacology
Acknowledgements
.2.1. Anti-inflammatory activity
We are grateful to Dr S.N. Kovalenko (Department of
Organic Chemistry) for initial suggestions and helpful
discussions. We thank Dr S.M. Drogovoz (Department of
Pharmacology) for providing the Department’s facilities
for pharmacological tests. We are indebted to Dr I.V.
Ukrainets (Department of Pharmaceutical Chemistry) for
his generous gift of the compound 5.
.2.1.1. Carrageenan-induced rat hind paw oedema test
Experiments were carried out on groups of five
Sprague-Dawley rats (140–160 g). The tested compounds
and reference drug were administered orally (po) in 0.5%
methylcellulose solution in water and 1 h later 0.1 mL of
1
% carrageenan solution was injected under the plantar
aponeurosis of the right hind paw of the rat by the method
of Winter et al. [15]. The volume of the paw was
measured before and 3 h after carrageenan treatment by a
mercury plethysmometer. Anti-inflammatory activity was
given as percentage of inhibition of oedema in treated
groups compared with controls and was calculated ac-
cording to the formula:
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.2.1.2. Acetic acid peritonitis assay
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%
inhibition = 100 × [1 – (V /V )]
t c
[
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[
[
[
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6
.2.1.4. Statistical calculation
Data are expressed as mean ± SE. The Student’s t test
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