Synthesis and antibacterial activities of pyrano[3,2-b]pyranones from kojic acid, ethyl cyanoacetate, and benzaldehydes in aqueous K2CO 3

Article abstract of DOI:10.1007/s00706-014-1190-0

Pyrano[3,2-b]pyranones are prepared in good yields from three-component coupling of kojic acid, ethyl cyanoacetate, and benzaldehydes in aqueous K ₂CO₃. The synthetic compounds exert moderate activity against both Gram positive and G

Full text of DOI:10.1007/s00706-014-1190-0

Monatsh Chem
DOI 10.1007/s00706-014-1190-0
ORIGINAL PAPER
Synthesis and antibacterial activities of pyrano[3,2-b]pyranones
from kojic acid, ethyl cyanoacetate, and benzaldehydes in aqueous
K2CO3
•
Sakineh Asghari Robabeh Baharfar Manag Alimi
•
•
•
Mohammad Ahmadipour Mojtaba Mohseni
Received: 1 October 2013 / Accepted: 18 February 2014
Ó Springer-Verlag Wien 2014
Abstract Pyrano[3,2-b]pyranones are prepared in good
yields from three-component coupling of kojic acid, ethyl
cyanoacetate, and benzaldehydes in aqueous K2CO3. The
synthetic compounds exert moderate activity against both
Gram positive and Gram negative bacteria and their
effectiveness is higher against Bacillus subtilis.
simple phase separation techniques [4–7]. Moreover several
reactions undergo a substantial rate acceleration in water
even though a water-insoluble substrate is used in suspension
[4]. In this regard, it is desirable to use water instead of
organic solvents in organic reactions [8].
The synthesis of 4H-pyran derivatives has attracted
great interest, because many of them have useful biological
and pharmological properties, e.g., as anticoagulant, spas-
molytic, anticancer, and antianaphylactic agents [9–11].
Some 2-amino-4H-pyrans can also be used as photoactive
materials [12]. Considering the importance of these com-
pounds, many methods have been reported for their
synthesis [13–30]. The reported methods for the synthesis
of 2-amino-4H-pyran derivatives often involve multistep
reactions, expensive catalysts, organic solvents, and
microwave irradiation. It is therefore important to find
facile, efficient, and environmentally benign synthetic
procedures for the preparation of these compounds.
In this research, we report the three-component reaction
of kojic acid (1), ethyl cyanoacetate (2), and benzaldehydes
3 in the presence of catalytic amounts of K2CO3 in aqueous
media for the preparation of alkyl 2-amino-4-aryl-6-
(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-
carboxylates 4 (Scheme 1). In addition, compounds 4a–4h
were examined for their antibacterial activity against Gram
positive and Gram negative bacteria.
Keywords Green chemistry Á Three-component reaction Á
Kojic acid Á Benzaldehydes Á Michael addition Á
Antibacterial activities
Introduction
Green chemistry relies on the development of environmen-
tally benign chemical processes and technology [1–3]. Water
is an inexpensive, safe, abundant, and environmentally
benign solvent. Organic reactions performed in water have
attracted considerable attention with respect to the unique
properties of water in promoting reactions and enhancing
selectivity as well as allowing easy isolation of products by
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-014-1190-0) contains supplementary
material, which is available to authorized users.
S. Asghari (&)
Nano and Biotechnology Research Group, University of
Mazandaran, Babolsar, Iran
e-mail: s.asghari@umz.ac.ir
Results and discussion
S. Asghari Á R. Baharfar Á M. Alimi Á M. Ahmadipour
Department of Organic Chemistry, Faculty of Chemistry,
University of Mazandaran, 47416-95447 Babolsar, Iran
Chemistry
Initially, we examined the three-component reaction of
kojic acid, ethyl cyanoacetate, and benzaldehyde in the
absence of base at room temperature. In this reaction, the
M. Mohseni
Department of Biology, University of Mazandaran, Babolsar,
Iran
123

S. Asghari et al.
Scheme 1
Table 1 Optimization of the one-pot reaction of kojic acid, ethyl
cyanoacetate, and benzaldehyde in the presence of various bases in
water
Table 3 Reactions of kojic acid, ethyl cyanoacetate, and benzalde-
hydes in the presence of K2CO3 (20 mol%) in water
Entry
Benzaldehyde
Yield/%
Entry
Base
Yield of 4a/%
1
2
3
4
5
6
7
8
3a: X = H
88
95
93
97
94
81
75
94
a
1
2
3
4
a
–
Trace
88
3b: X = 4-CN
3c: X = 4-Br
3d: X = 4-NO2
3e: X = 4-Cl
3f: X = 4-CH3
3g: X = 4-OCH3
3h: X = 2-Cl
K2CO3
NEt3
90
DBU
92
Without catalyst
Table 2 Influence of different catalyst amounts for the reaction of
kojic acid, ethyl cyanoacetate, and benzaldehyde
Entry
Catalyst amount/mol%
Yield of 4a/%
high yields. However, when the reaction was performed in
the presence of 4-methyl- or 4-methoxybenzaldehyde (3f or
3g) which contained electron-donating substituents, the
yields of the corresponding products decreased (Table 3,
entries 6 and 7). Whereas, in the presence of 4-cyano- or
4-nitrobenzaldehyde (3b or 3d), containing electron-with-
drawing substituents, the yields of the reaction products 4b or
4d increased (Table 3, entries 2 and 4).
1
2
3
4
5
6
10
20
30
88
89
90
91
91
40
60
80
100
A proposed mechanism for the reaction is shown in
Scheme 2. Initially, the intermediate 5 forms via initial
Knoevenagel condensation reaction. This intermediate then
reacts with kojic acid enolate (6) via Michael addition to
form intermediate 7. Intermediate 7 is converted to enolate
8 in the presence of K2CO3 and subsequently undergoes
intramolecular cyclization and 1,3-proton transfer to afford
compound 4 exclusively.
starting materials were recovered without the formation of
the desired product 4a. When this reaction was carried out
in the presence of a catalytic amount of K2CO3 (50 mol%),
the product 4a was obtained as colorless crystals in high
yields. Different organic bases (NEt3 and DBU) were also
used in order to improve the yield of the reaction (Table 1).
However, K2CO3 is an inexpensive, safe, and environ-
mentally benign base and it was therefore considered the
most suitable base for this reaction. The effect of the
amount of catalyst on the yield and the rate of the reaction
was also investigated by using different amounts of K2CO3.
The optimal amount of K2CO3 was 20 mol% (Table 2).
On the basis of the above results, the scope and limitation
of these three-component reactions were investigated. A
variety of benzaldehydes with electron-donating or electron-
withdrawing substituents were employed as substrates and
the reactions afforded the corresponding products in good to
The structures of 4a–4h were deduced from IR spectra,
1H, 13C NMR, and mass spectroscopy as well as elemental
analyses. The IR spectrum of 4a showed two broad signals
at 3460 and 3383 cm-1 and a broad signal at 3266 cm-1
corresponding to NH2 and OH functional groups, respec-
1
tively. The H NMR spectrum of 4a exhibited a triplet at
1.00 ppm (3JHH = 7.2 Hz) for the methyl group, a quartet
at 3.93 ppm (3JHH = 7.2 Hz) for the methylene group of
the ethoxy moiety, an AB quartet and doublet at 4.20 ppm
(2JHH = 15.6 and 3JHH = 5.6 Hz) for the methylene group
123

Synthesis and antibacterial activities of pyrano[3,2-b]pyranones
Scheme 2
of CH2OH, a singlet at 4.80 ppm for the CH group, a triplet
at 5.69 ppm (3JHH = 6.0 Hz) for the OH group, a singlet at
6.32 ppm for the CH group of the 4H-pyrone moiety, two
multiplets at 7.21–7.25 and 7.30–7.34 ppm for the aro-
matic protons, and a singlet at 7.84 ppm for the NH2 group.
The 13C NMR spectrum of 4a showed 16 signals in
agreement with the proposed structure. The mass spectrum
of this compound displayed a molecular ion peak at m/
z = 343 (43 %). Fragmentations involved loss of side
chains (–NH2, OH, CH2OH, and CO2Et).
To conclude, K2CO3 is an effective catalyst for the syn-
thesis of 2-amino-substituted pyrano[3,2-b]pyranones by
condensation of benzaldehydes, ethyl cyanoacetate, and
kojic acid in water. The simultaneous formation of several
bonds in only one step, high yields, ease of handling, short
reaction times, environmentally non-harmful media, and the
absence of any hazardous organic solvents are some of the
advantages of this procedure. In addition, compounds 4a and
4c exhibited good antibacterial activity and can be further
developed for application as effective antimicrobial agents.
1
The H and 13C NMR spectra of 4b–4h were similar to
those of 4a except for the substituents in the aromatic
moiety, which displayed characteristic resonances in the
appropriate regions of the spectra.
Experimental
Ethyl cyanoacetate, kojic acid, and various benzaldehydes
were obtained from Fluka (Buchs, Switzerland) and Merck
(Germany) and were used without further purification.
Melting points were measured on an Electrothermal 9100
apparatus. IR spectra were recorded on an FT-IR Bruker
vector 22 spectrometer. Elemental analysis was performed
Antibacterial activity
The antibacterial activity of compounds 4a–4h was eval-
uated against Gram positive (Staphylococcus aureus and
Bacillus subtilis) and Gram negative (Escherichia coli and
Pseudomonas aeruginosa) by the disc diffusion method.
The standard antibiotics gentamicin and chloramphenicol
were used as controls (Table 4). The results revealed that
compounds 4a–4h have moderate to good growth inhibi-
tory effect against some microorganisms; among the
bacteria tested, they exhibited the highest antibacterial
activity against B. subtilis. The activity values obtained for
compounds 4a and 4c represent good activity against all
the tested bacteria, whereas compounds 4f and 4g showed
no activity against P. aeruginosa (Table 4).
using a Heraeus CHN-ORapid analyzer. H and 13C NMR
1
spectra were measured with a Bruker DRX-400 Avance
spectrometer at 400.13 MHz for 1H and 100.6 MHz for 13C.
Mass spectra were measured on a Finnigan-Matt 8430 mass
spectrometer operating at an ionization potential of 30 eV.
General procedure for the preparation
of pyrano[3,2-b]pyranones 4a–4h
To a solution of 0.142 g kojic acid (1 mmol) in 5 cm3 H2O
was added 0.11 cm3 ethyl cyanoacetate (1 mmol),
123

S. Asghari et al.
Table 4 Antibacterial activity of compounds 4a–4h using Kirby–Bauer disc diffusion method
Test strain
Zone of growth inhibition/mm
4a 4b 4c
4d
4e
4f
4g
4h
Gentamicin Chloramphenicol
(10 lg/disc) (30 lg/disc)
E. coli
13.0 1.4 8.5 0.7 12.5 0.7 7.5 0.7 11.5 0.7 11.5 0.7 10.0 1.4 10.5 0.7 19.6 1.1 20.7 1.5
P. aeruginosa 12.5 0.7 9.5 0.7 10.5 0.7 8.5 0.7 11.0 1.4 NE
NE
11.0 1.4 15.6 0.5 NE
S. aureus
B. subtilis
10.0 1.4 9.0 1.4 11.5 0.7 9.5 0.7 12.0 1.4 11.5 0.7 7.5 0.7 12.0 1.4 20.3 1.5 21.7 0.6
12.0 1.4 12.0 1.4 13.5 0.7 8.5 0.7 11.0 1.4 8.5 0.7 NE
9.5 0.7 26.0 1.7 22.3 1.2
The data are expressed as mean SD (n = 3)
NE no effect
150.8, 160.1, 167.8 (5 Cq), 168.7 (C=O, ester), 170.0 (C=O,
pyrone) ppm; IR (KBr): mꢀ = 3355, 3262 (NH2, OH), 2227
(CN), 1671 (C=O), 1631 (C=C), 1210 (Csp2–O), 1033
(Csp3–O) cm-1; MS (70 eV): m/z (%) = 369 (57), 368 (22),
339 (10), 323 (8), 320 (12), 295 (100), 266 (59), 256 (79),
238 (14), 152 (74), 116 (42), 113 (18), 102 (21), 69 (49).
benzaldehydes (1 mmol), and 0.028 g K2CO3 (20 mol%).
The reaction mixture was stirred for 1–2 h at room tem-
perature. The progress of the reaction was monitored by
thin-layer chromatography (TLC). After completion of the
reaction, the solid was collected by filtration and recrys-
tallized from hexane/ethyl acetate (9:1). Pure product was
obtained as colorless crystals.
Ethyl 2-amino-4-(4-bromophenyl)-6-(hydroxymethyl)-8-
oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4c, C18H16BrNO6)
Ethyl 2-amino-6-(hydroxymethyl)-8-oxo-4-phenyl-4,8-
dihydropyrano[3,2-b]pyran-3-carboxylate
(4a, C18H17NO6)
Colorless crystals; 0.39 g (93 %); m.p.: 195–197 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 1.01 (t, 3H, 3JHH = 7.2 Hz,
Colorless crystals; 0.30 g (88 %); m.p.: 194–196 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 1.00 (t, 3H, 3JHH = 7.2 Hz,
CH3), 3.93 (q, 2H, 3JHH = 7.2 Hz, OCH2CH3), 4.20 (2 dd,
2H, 2JHH = 15.6 Hz, 3JHH = 5.6 Hz, CH2OH), 4.80 (s, 1H,
3
CH3), 3.93 (q, 2H, JHH = 7.2 Hz, OCH2CH3), 4.20 (2 d,
2
2H, JHH = 16.0 Hz, CH2OH), 4.81 (s, 1H, CH), 5.78 (s,
3
1H, OH), 6.32 (s, 1H, CH), 7.20 (d, 2H, JHH = 8.4 Hz, 2
3
3
CH), 5.69 (t, 1H, JHH = 6.0 Hz, OH), 6.32 (s, 1H, CH),
Ar–H), 7.52 (d, 2H, JHH = 8.4 Hz, 2 Ar–H), 7.87 (s, 2H,
NH2) ppm; 13C NMR (100.6 MHz, acetone-d6): d = 13.7
(CH3), 39.9 (CH), 59.1, 59.8 (2 OCH2), 75.6 (Cq), 111.4
(CH), 120.5 (Cq), 130.0 (2 CH), 131.4 (2 CH), 136.4, 142.8,
151.0, 160.0, 167.8 (5 Cq), 167.9 (C=O, ester), 169.6 (C=O,
7.21–7.25 (m, 3H, 3 Ar–H), 7.30–7.34 (m, 2H, 2 Ar–H), 7.84
(s, 2H, NH2) ppm; 13C NMR (100 MHz, CDCl3): d = 14.5
(CH3), 40.3 (CH), 59.4, 59.6 (2 OCH2), 75.5 (Cq), 111.8
(CH), 127.6 (CH), 128.0 (2 CH), 129.0 (2 CH), 136.4, 143.5,
152.2, 160.2, 168.0 (5 Cq), 168.6 (C=O, ester), 170.1 (C=O,
ꢀ
pyrone) ppm; IR (KBr): m = 3388, 3272 (NH2, OH), 1663
ꢀ
pyrone) ppm; IR (KBr): m = 3460, 3383 (NH2), 3266 (OH),
(C=O), 1638 (C=C), 1210 (Csp2–O), 1024 (Csp3–O), 549 (C–
Br) cm-1; MS (70 eV): m/z (%) = 423 (15), 421 (15), 394
(12), 392 (12), 375 (15), 373 (15), 350 (65), 348 (65), 311
(100), 309 (100), 266 (98), 238 (33), 220 (30), 157 (16), 155
(16), 76 (62), 69 (98).
1672 (C=O), 1621 (C=C), 1208 (Csp2–O), 1024 (Csp3–O)
cm-1; MS (70 eV): m/z (%) = 343 (43), 314 (13), 298 (7),
270 (75), 266 (89), 231 (100), 129 (2), 113 (7), 91 (36), 77
(33), 67 (24).
Ethyl 2-amino-6-(hydroxymethyl)-4-(4-nitrophenyl)-8-
oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4d, C18H16N2O8)
Ethyl 2-amino-4-(4-cyanophenyl)-6-(hydroxymethyl)-
8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4b, C19H16N2O6)
Colorless crystals; 0.38 g (97 %); m.p.: 203–205 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 0.99 (t, 3H, 3JHH = 7.2 Hz,
Colorless crystals; 0.35 g (95 %); m.p.: 196–198 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 0.97 (t, 3H, 3JHH = 7.2 Hz,
CH3), 3.88–3.96 (m, 2H, OCH2CH3), 4.19 (2 dd, 2H,
3
CH3), 3.92 (q, 2H, JHH = 7.2 Hz, OCH2CH3), 4.19 (2 dd,
2H, 2JHH = 16.0 Hz, 3JHH = 6.0 Hz, CH2OH), 5.00 (s, 1H,
3
2JHH = 16.0 Hz, JHH = 6.0 Hz, CH2OH), 4.93 (s, 1H,
3
CH), 5.69 (t, 1H, JHH = 6.0 Hz, OH), 6.33 (s, 1H, CH),
3
CH), 5.68 (t, 1H, JHH = 6.0 Hz, OH), 6.32 (s, 1H, CH),
3
7.55 (d, 2H, JHH = 8.8 Hz, 2 Ar–H), 7.95 (s, 2H, NH2),
3
7.46 (d, 2H, JHH = 8.4 Hz, 2 Ar–H), 7.80 (d, 2H,
3
8.20 (d, 2H, JHH = 8.8 Hz, 2 Ar–H) ppm; 13C NMR
3JHH = 8.4 Hz, 2 Ar–H), 7.93 (s, 2H, NH2) ppm; 13C
NMR (100.6 MHz, DMSO-d6): d = 14.5 (CH3), 40.5 (CH),
59.5, 59.6 (2 OCH2), 74.6 (Cq), 110.5 (Cq), 111.9 (CH),
119.1 (CN), 129.3 (2 CH), 133.0 (2 CH), 136.5, 149.0,
(100.6 MHz, DMSO-d6): d = 14.5 (CH3), 40.3 (CH), 59.5
(2 OCH2), 74.6 (Cq), 111.9 (CH), 124.3 (2 CH), 129.6 (2
CH), 136.6, 147.1, 150.6, 151.0, 160.1, 167.7 (6 Cq), 168.7
123

Synthesis and antibacterial activities of pyrano[3,2-b]pyranones
(C=O, ester), 170.0 (C=O, pyrone) ppm; IR (KBr):
ꢀ
3H, OCH3), 3.91–3.97 (m, 2H, OCH2CH3), 4.20 (2 dd, 2H,
2JHH = 16.0 Hz, 3JHH = 6.0 Hz, CH2OH), 4.73 (s, 1H, CH),
5.69 (t, 1H, 3JHH = 6.0 Hz, OH), 6.31 (s, 1H, CH), 6.87 (d,
2H, 3JHH = 8.8 Hz, 2 Ar–H), 7.13 (d, 2H, 3JHH = 8.4 Hz, 2
Ar–H), 7.80 (s, 2H, NH2) ppm; 13C NMR (100.6 MHz,
DMSO-d6): d = 14.6 (CH3), 39.4 (CH), 55.5 (OCH3), 59.4,
59.6 (2 OCH2), 75.8 (Cq), 111.8 (2 CH), 114.4(CH), 129.0 (2
CH), 135.5, 136.2, 152.5, 158.8, 160.1, 168.1 (6 Cq), 168.5
m = 3419, 3299 (NH2, OH), 1688, 1672 (C=O), 1619
(C=C), 1513, 1351 (NO2), 1203 (Csp2–O), 1050 (Csp3
–
O) cm-1; MS (70 eV): m/z (%) = 389 (47), 388 (29), 359
(16), 343 (7), 340 (7), 315 (65), 276 (51), 266 (100), 238
(20), 176 (13), 113 (15), 68 (40).
Ethyl 2-amino-4-(4-chlorophenyl)-6-(hydroxymethyl)-
8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4e, C18H16ClNO6)
ꢀ
(C=O, ester), 170.1 (C=O, pyrone) ppm; IR (KBr): m = 3459,
3400 (NH2), 3287 (OH), 1666 (C=O), 1610 (C=C), 1202
(Csp2–O), 1035 (Csp3–O) cm-1; MS (70 eV): m/z (%) = 373
(15), 344 (9), 328 (6), 326 (7), 325 (4), 300 (32), 266 (13),
261 (100), 243 (4), 238 (6), 220 (5), 69 (5).
Colorless crystals; 0.36 g (94 %); m.p.: 195–197 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz,
DMSO-d6):
d = 1.01
(t,
3H,
3JHH = 7.2 Hz, CH3), 3.88–3.97 (m, 2H, OCH2CH3),
4.20 (2 dd, 2H, 2JHH = 15.6 Hz, 3JHH = 5.6 Hz, CH2OH),
4.82 (s, 1H, CH), 5.69 (t, 1H, 3JHH = 6.0 Hz, OH), 6.32 (s,
Ethyl
2-amino-4-(2-chlorophenyl)-6-(hydroxymethyl)-8-
oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4h, C18H16ClNO6)
3
1H, CH), 7.26 (d, 2H, JHH = 8.4 Hz, 2 Ar–H), 7.38 (d,
3
2H, JHH = 8.4 Hz, 2 Ar–H), 7.87 (s, 2H, NH2) ppm; 13C
Colorless crystals; 0.36 g (94 %); m.p.: 197–199 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 0.92 (t, 3H, 3JHH = 7.2 Hz,
NMR (100.6 MHz, acetone-d6): d = 13.6 (CH3), 40.3
(CH), 59.0, 59.8 (2 OCH2), 76.1 (Cq), 111.4 (CH), 127.1
(2 CH), 127.8 (2 CH), 128.4, 136.3, 143.4, 151.8, 160.0,
167.8 (6 Cq), 168.0 (C=O, ester), 170.0 (C=O, pyrone)
CH3), 3.83–3.91 (m, 2H, OCH2CH3), 4.17(2 ddd, 2H, 2JHH
=
3
4
15.9 Hz, JHH = 6.0 Hz, JHH = 0.8 Hz, CH2OH), 5.36
ꢀ
3
(s, 1H, CH), 5.66 (t, 1H, JHH = 6.0 Hz, OH), 6.30 (s, 1H,
ppm; IR (KBr): m = 3387, 3272 (NH2, OH), 1686, 1668
(C=O), 1628 (C=C), 1210 (Csp2–O), 1031 (Csp3–O), 766
(C–Cl) cm-1; MS (70 eV): m/z (%) = 380 (6), 378 (18),
350 (3), 348 (9), 306 (13), 304 (37), 266 (100), 238 (14),
220 (11), 125 (42), 113 (16), 69 (40).
CH), 7.23–7.33 (m, 3H, 3 Ar–H), 7.41–7.43 (m, 2H, 2 Ar–H),
7.91 (s, 2H, NH2) ppm; 13C NMR (100.6 MHz, DMSO-d6):
d = 14.4 (CH3), 39.3 (CH), 59.3, 59.6 (2 OCH2), 74.8 (Cq),
111.8 (CH), 128.2, 129.3, 129.8, 130.6 (4 CH), 133.1, 136.4,
141.1, 151.1, 160.3, 167.9 (6 Cq), 168.7 (C=O, ester), 170.0
Ethyl 2-amino-6-(hydroxymethyl)-4-(4-methylphenyl)-8-
oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4f, C19H19NO6)
ꢀ
(C=O, pyrone) ppm; IR (KBr): m = 3410, 3298 (NH2, OH),
1669 (C=O), 1620 (C=C), 1199 (Csp2–O), 1041 (Csp3–O), 743
(C–Cl) cm-1; MS (70 eV): m/z (%) = 379 (5), 377 (15), 350
(5), 348 (15), 334 (2), 332 (7), 331 (2), 329 (5), 306 (10), 304
(29), 266 (100), 238 (27), 231 (93), 142 (4), 125 (26), 69 (23),
68 (22).
Colorless crystals; 0.29 g (81 %); m.p.: 187–189 °C;
Rf = 0.4 (n-hexane/ethyl acetate 1:9, v/v); 1H NMR
(400.13 MHz, DMSO-d6): d = 1.03 (t, 3H, 3JHH = 7.2 Hz,
CH3), 2.25 (s, 3H, CH3), 3.89–3.97 (m, 2H, OCH2CH3),
2
3
4.20 (2 dd, 2H, JHH = 15.8 Hz, JHH = 6.0 Hz, CH2OH),
4.75 (s, 1H, CH), 5.68 (t, 1H, 3JHH = 6.0 Hz, OH), 6.31 (s,
1H, CH), 7.09–7.13 (m, 4H, 4 Ar–H), 7.80 (s, 2H, NH2)
ppm; 13C NMR (100.6 MHz, DMSO-d6): d = 14.6, 21.1 (2
CH3), 39.8 (CH), 59.4, 59.6 (2 OCH2), 75.6 (Cq), 111.8
(CH), 127.8 (2 CH), 129.6 (2 CH), 136.3, 136.8, 140.6,
152.4, 160.1, 168.0 (6 Cq), 168.5 (C=O, ester), 170.1 (C=O,
General procedure for evaluation of antibacterial
activity
The in vitro antibacterial activities of compounds 4a–4h
were assayed using the Kirby–Bauer disc diffusion method
in which a filter disc is impregnated with the compounds
and placed on the surface of inoculated agar plates [31].
The synthesized compounds were dissolved in DMSO to
achieve 20 mg cm-3 solutions and then filter sterilized
using a 0.22-lm Ministart (Sartorius).
The antibacterial activity of compounds 4a–4h was
investigated against four bacterial species. Test organisms
included E. coli PTCC 1330, P. aeruginosa PTCC 1074, S.
aureus ATCC 35923, and B. subtilis PTCC 1023 [32]. Late
exponential phase growths of the bacteria were prepared by
inoculating 1 % (v/v) of the cultures into fresh Muller–
Hinton broth (Merck) and incubating on an orbital shaker
at 37 °C and 100 rpm overnight. Before using the cultures,
ꢀ
pyrone) ppm; IR (KBr): m = 3404, 3281 (NH2, OH), 1662
(C=O), 1624 (C=C), 1211 (Csp2–O), 1026 (Csp3–O) cm-1
;
MS (70 eV): m/z (%) = 357 (36), 328 (16), 312 (7), 284
(70), 267 (12), 266 (74), 245 (100), 238 (22), 227 (5), 220
(17), 199 (8), 142 (6), 127 (6), 91 (10), 69 (5).
Ethyl 2-amino-6-(hydroxymethyl)-4-(4-methoxyphenyl)-8-
oxo-4,8-dihydropyrano[3,2-b]pyran-3-carboxylate
(4g, C19H19NO7)
Colorless crystals; 0.28 g (75 %); m.p.: 79–81 °C; Rf = 0.4
(n-hexane/ethyl acetate 1:9, v/v); H NMR (400.13 MHz,
1
3
DMSO-d6): d = 1.04 (t, 3H, JHH = 7.2 Hz, CH3), 3.71 (s,
123

S. Asghari et al.
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they were standardized with a final cell density of
approximately 108 cfu cm-3. Muller–Hinton agar (Merck)
was prepared and inoculated from the standardized cultures
of the test organisms and then spread as uniformly as
possible throughout the entire media. Sterile paper discs
(6 mm diameter, Padtan, Iran) were impregnated with
20 mm3 of solution of compounds 4a–4h and then allowed
to dry. The impregnated disc was introduced onto the upper
layer of the seeded agar plate and incubated at 37 °C for
24 h. The antibacterial activities of compounds 4a–4h were
compared with the known antibiotics gentamicin (10 lg/
disc) and chloramphenicol (30 lg/disc) as positive control
and DMSO (20 mm3/disc) as negative control. Antibacte-
rial activity was evaluated by measuring the diameter of
inhibition zone (millimeters) on the surface of plates and
the results were reported as mean SD after three repeats.
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Acknowledgments The authors acknowledge the University of
Mazandaran for financial support of this research
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