Synthesis of a new series of 2-(2-oxo-2H-chromen-3-yl)-5H-chromeno[4,3-b] pyridin-5-ones by two facile methods and evaluation of their antimicrobial activity

Article abstract of DOI:10.1007/s00044-013-0489-4

A series of novel 2-(2-oxo-2H-chromen-3-yl)-5H-chromeno[4,3-b]pyridin-5- ones 4a-4l have been synthesized using two methodologies (using Mannich base of 4-hydroxy coumarin and 4-chloro-3-formyl coumarin) (Patel et al., 2012) and assayed for their antibacterial activity against gram-positive bacteria viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96) and gram-negative bacteria viz. Escherichia coli (MTCC 443), Salmonella typhimurium (MTCC 98). The compounds 4a-4l were also assayed for their antifungal activity against Aspergillus niger (MTCC 282) and Candida albicans (MTCC 227). All the compounds 4a-4l exhibited potent inhibitory activity against gram-positive bacteria compared to standard drugs at the tested concentrations. The compounds also showed appreciable activity against gram-negative bacteria as well as fungi. The compounds 4e, 4f, 4i, 4k, and 4l are found to be the most proficient members of the series and emerged as potential molecules for further development. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-013-0489-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4745–4754
DOI 10.1007/s00044-013-0489-4
ORIGINAL RESEARCH
Synthesis of a new series of 2-(2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-ones by two facile methods
and evaluation of their antimicrobial activity
•
Apoorva A. Patel Hemali B. Lad
•
•
•
Kinnar R. Pandya Chirag V. Patel
Dinkar I. Brahmbhatt
Received: 17 August 2012 / Accepted: 11 January 2013 / Published online: 22 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel 2-(2-oxo-2H-chromen-3-yl)-
5H-chromeno[4,3-b]pyridin-5-ones 4a–4l have been syn-
thesized using two methodologies (using Mannich base of
4-hydroxy coumarin and 4-chloro-3-formyl coumarin)
(Patel et al., 2012) and assayed for their antibacterial activity
against gram-positive bacteria viz. Bacillus subtilis (MTCC
441), Staphylococcus aureus (MTCC 96) and gram-negative
bacteria viz. Escherichia coli (MTCC 443), Salmonella
typhimurium (MTCC 98). The compounds 4a–4l were also
assayed for their antifungal activity against Aspergillus niger
(MTCC 282) and Candida albicans (MTCC 227). All the
compounds 4a–4l exhibited potent inhibitory activity
against gram-positive bacteria compared to standard drugs at
the tested concentrations. The compounds also showed
appreciable activity against gram-negative bacteria as well
as fungi. The compounds 4e, 4f, 4i, 4k, and 4l are found to be
the most proficient members of the series and emerged as
potential molecules for further development.
Introduction
The treatment of infectious diseases still remains an impor-
tant and challenging problem because of a combination of
factors including emerging infectious diseases and the
increasing number of multi-drug resistant microbial patho-
gens with particular relevance for gram-positive bacteria
(Tenover and McDonald, 2005; Pfeltz and Wilkinson, 2004;
Roberts, 2004; Kratky et al., 2012; Muroi et al., 2004). On
the other hand, a recent survey of novel small molecule
therapeutics revealed that the majority of them result from an
analog-based approach and that their market value represents
two-thirds of all drug sales (Wermuth, 2006). 2H-Chromen-
2-ones (coumarins) are important oxygen containing het-
erocycles which possess variety of biological activities such
as anti-inflammatory (Timonen et al., 2011), antibacterial
(Song et al., 2012), antioxidant (Xiao et al., 2012), antico-
agulant (Mladenovic et al., 2012), anti-HIV (Olmedo et al.,
2012), antihyperlipidemic (Sashidhara et al., 2010), and as
antitumour (Shen et al., 2012). Besides, 2H-chromen-2-one
nucleus is also present in promising drug candidates as
topoisomerase II (Rappa et al., 2000) and tyrosine kinase
(Yang et al., 1999) inhibitors. 2H-Chromen-2-ones having
pyridyl substitution possess diverse physiological actions
viz. CNS depressant activity (Moffett, 1964), antifungal
(Sardari et al., 1999), and antibacterial (Modranka et al.,
2006) activities. Also, 2H-chromen-2-ones fused with pyri-
dines have been reported to possess antiallergic (Ukawa
et al., 1986), antimicrobial (Patel et al., 2012), analgesic
properties (Heber and Berghaus, 1994) and even antidiabetic
activities (Heber, 1987). Owing to such interesting biologi-
cal properties of both, pyridyl-substituted and pyrido-fused
2H-chromen-2-ones, it was thought worthwhile to synthe-
size molecules which incorporate both of these structural
features. Therefore, the simple and efficient method for
Keywords Krohnke’s reaction ꢀ
3-[(Ethylamino)methyl]-4-hydroxy coumarin ꢀ
4-Chloro-2-oxo-2H-chromene-3-carbaldehyde ꢀ
1-[2-Oxo-2-(2-oxo-2H-chromen-3-yl)ethyl]pyridinium
salt ꢀ Antimicrobial activity
Apoorva A. Patel and Hemali B. Lad contributed equally to this study.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0489-4) contains supplementary
material, which is available to authorized users.
A. A. Patel ꢀ H. B. Lad ꢀ K. R. Pandya ꢀ
C. V. Patel ꢀ D. I. Brahmbhatt (&)
Department of Chemistry, Sardar Patel University,
Vallabh Vidhyanagar 388120, Gujarat, India
e-mail: drdib317@gmail.com
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Med Chem Res (2013) 22:4745–4754
synthesis of such compounds would be a favorable step
towards further investigation in drug discovery.
appropriate salt 3a–3c in the presence of ammonium acetate
in refluxing glacial acetic acid solvent, resulted the new
series of 2-(2-oxo-2H-chromen-3-yl)-5H-chromeno[4,3-b]-
pyridin-5-ones 4a–4l in good yields (Table 1).
Hence, in a view of the biological significance of pyridyl-
substituted 2H-chromen-2-ones, pyrido-fused 2H-chromen-
2-ones, and in continuation of our research work on the
synthesis of such biologically active 2H-chromen-2-one
derivatives (Brahmbhatt et al., 2011), we herein, report the
synthesis of a series of 2-(2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-ones 4a–4l by two facile methods
(A and B), employing different precursors, in one-pot three-
component system (Scheme 1). In method A, 3-[(ethyl-
amino)methyl]-4-hydroxy-2H-chromen-2-ones 1a–1d were
reacted with 1-[2-oxo-2-(2-oxo-2H-chromen-3-yl)ethyl]-
pyridinium salts 3a–3c in the presence of ammonium acetate
and acetic acid, while in method B, 4-chloro-2-oxo-2H-
chromene-3-carbaldehydes 2a–2d were reacted with the
salts 3a–3c in the presence of ammonium acetate for building
up the pyridine ring. Further, all the newly synthesized
compounds were evaluated for their in vitro antimicrobial
activity by broth micro dilution method (NCCLS, 2002)
against different pathogenic strains.
The reaction pathways for methods A and B are shown
in Scheme 2. Here, the starting material Mannich base [1]
undergoes decomposition and results in the formation of
coumarin methide [C]. This coumarin methide [C] then
reacts with salt [3] in the presence of ammonium acetate
and acetic acid, and results in a formation of a 1,5-dicar-
bonyl intermediate [D] which finally gets converted into
expected product [4] by Krohnke’s reaction (Krohnke,
1976). The coumarin methide [C] also undergoes a side
reaction and gives a dicoumarol [E] as a byproduct. The
formation of dicoumarol [E] as a byproduct may be
responsible for the low yield in method A compared to
method B. Similarly in method B, [2] and [3] react to give
the intermediate [F] which finally gets converted into
expected product [4].
Here, we observed that the 4-chloro-3-formyl coumarins
2a–2d reacted much smoothly than Mannich bases of
4-hydroxy coumarins 1a–1d, with salts 3a–3c to afford
target compounds in good yield. In our earlier work (Patel
et al., 2012) also similar observations were made. There
also 4-chloro-3-formyl coumarins reacted smoothly to give
fused chromenone derivatives in appreciable yield. Thus, it
can be said that 4-chloro-3-formyl coumarins are prefera-
ble precursors for the synthesis of pyrido-fused chrome-
none derivatives.
Results and discussion
The synthetic routes adopted to obtain various 2-(2-oxo-2H-
chromen-3-yl)-5H-chromeno[4,3-b]pyridin-5-ones 4a–4l are
shown in Scheme 1. The subsequent one-pot, three-
component reaction of precursors 1a–1d or 2a–2d with
R
R
O
O
O
O
H
CH 3
N
R1
R1
CH O
OH
1a-d
Cl
2a-d
R
7
6
O
O
8
5
2
4
1a : R=R1=H
R1
9
2a : R=R1=H
10
N
1b : R=H; R1=CH3
1c : R=CH3; R1=H
1d : R=H; R1=Cl
Method A
Y
3
2b : R=H; R1=CH3
2c : R=CH3; R1=H
2d : R=H; R1=Cl
Method B
Y
1
3'
2'
O
4'
R4
R3
O
1'
5'
6'
R2
R2
8'
R2
O
O
O
O
O
7'
R3
PyBr
4a-l
R3
PyBr
R4
3a-c
3a : R2=R3=R4=H
O
R4
3a-c
3b : R2=OCH3; R3=R4=H
3c : R2=H; R3+R4=benzo
Scheme 1 Reagent and conditions: (Y) NH4OAc, AcOH, reflux
123

Med Chem Res (2013) 22:4745–4754
4747
Table 1 Isolated yields from
methods A and B of compounds
4a–4l
Compounds
R
R1
R2
R3
R4
Isolated yields (%)
Method A
Method B
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
47
38
43
40
44
46
50
39
54
56
57
52
62
54
64
59
61
58
69
56
73
78
79
76
H
H
OCH3
H
H
H
H
Benzo
H
H
CH3
CH3
CH3
H
H
H
H
H
OCH3
H
H
H
Benzo
H
4g
4h
4i
CH3
CH3
CH3
H
H
H
H
H
OCH3
H
H
H
Benzo
H
4j
Cl
Cl
Cl
H
H
H
4k
4l
H
OCH3
H
H
H
Benzo
O
O
O
O
O
O
Δ
H
N
CH2
H
Coumarin methide
[1]
[C]
O
N
O
O
O
O
O
O
O
O
O
O
NH4OAc/AcOH
[3]
O
O
CH2
O
O
OH
OH
O
[C]
[E]
Side product
O
[4]
[D]
Method A
O
O
O
O
O
O
NH4OAc/AcOH
[3]
N
O
Cl
O
CHO
O
O
O
Cl
[2]
[4]
[F]
Method B
Scheme 2 Reaction pathways for methods A and B
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Med Chem Res (2013) 22:4745–4754
The structures of all the synthesized compounds 1a–1d
and 4a–4l were established on the basis of elemental
activity against gram-positive bacteria S. aureus as com-
pared to ampicillin (MIC = 250 lg/mL). Compounds 4i and
4l (MIC = 62.5 lg/mL) have shown excellent activity against
E. coli as compared to ampicillin (MIC = 100 lg/mL).
As compared to ampicillin (MIC = 250 lg/mL) com-
pounds 4b, 4d, 4f, 4j, and 4l (MIC = 200 lg/mL) showed
better activity against B. subtilis, whereas compounds 4a,
4g, and 4k (MIC = 200 lg/mL) were found to be more
potent against S. aureus as compared to ampicillin (MIC =
250 lg/mL).
Compounds 4a and 4g (MIC = 250 lg/mL) were found
to be equally active against gram-positive bacteria B. subtilis
while compounds 4d and 4h (MIC = 250 lg/mL) showed
equal activity against S. aureus compared to ampicillin
(MIC = 250 lg/mL). Compounds 4i, 4j, and 4l (MIC =
100 lg/mL) have been found to be equipotent as compared
to ampicillin (MIC = 100 lg/mL) against S. typhi.
Compounds 4h (MIC = 200 lg/mL) and 4c (MIC =
250 lg/mL) showed better activity than griseofulvin
(MIC = 500 lg/mL) against fungal pathogen C. albicans.
Compounds 4d, 4g, and 4l have been found to be equi-
potent as compared to griseofulvin (MIC = 500 lg/mL)
against C. albicans. None of the tested compounds showed
better activity against A. niger.
1
analysis, IR, H-NMR, 13C-NMR, APT, and selected mass
spectral data.
In IR spectra, compounds 4a–4l showed a very strong
band between 1,689 and 1,708 cm-1 for the carbonyl
(C=O) stretching of d-lactone ring present in coumarin
nucleus. The strong bands for aromatic C=C and C=N
stretching vibrations, were observed between 1,606–1,632
and 1,479–1,505 cm-1, respectively. The aromatic C–H
stretching vibrations were observed between 3,093 and
3,144 cm-1. The d-lactone carbonyl stretching frequency
was observed somewhat lower than the expected frequency
(*1,720 cm-1). This may be due to the reduction of car-
bonyl character of d-lactone ring by the nitrogen of pyri-
dine ring. Such type of reduction in carbonyl frequency is
observed in coumarin derivatives having nitrogen attached
at 4-position (Stamboliyska et al., 2010).
In the 1H-NMR spectra of all the compounds 4a–4l, the
aromatic proton signals except C10-H were observed in the
region of 6.50d–8.50d. In all the compounds the C10-H and
0
C4 -H signals were observed in the downfield region
between 9.50d and 10.80d. These two proton signals shift
in the downfield region due to the peri effect of the nitrogen
atom of the pyridine ring.
All the compounds showed signals at expected d values
in the 13C-NMR spectra. The aromatic carbon signals were
observed between 95.0d and 176.0d while the two d-lac-
tone carbonyl carbons appeared between 160.0d and
181.0d. The differentiation of quaternary and tertiary car-
bons was done by the help of DEPT-135 spectra. The NMR
spectral data fully supported the structure of the products.
The structure of one representative compound 4a was
further supported by the help of mass spectral data.
Structure–activity relationship
All the compounds 4a–4l possess promising antibacterial
activity against gram-positive bacteria B. subtilis and
S. aureus. Examining the antimicrobial data, it has been
revealed that the derivatization of the parent molecule
increased the antimicrobial potency of the synthesized
analogs.
The observation indicates that the compounds 4b, 4e,
4h, and 4k bearing electron releasing group, i.e., methoxy
(R2 = OCH3) group in the pendent 2-oxo-2H-chromenyl
nucleus showed significant inhibitory activity against
gram-positive bacteria than the parent analogs. Compounds
4d, 4e, and 4f (R1 = CH3) and compounds 4g, 4h, and 4i
(R = CH3) having lipophilic methyl group showed mod-
erate to good antimicrobial activity.
Evaluation of antimicrobial activity
All the compounds 4a–4l were assayed for their in vitro
antimicrobial activity against gram-positive bacteria viz.
Bacillus subtilis (MTCC 441), Staphylococcus aureus
(MTCC 96), gram-negative bacteria viz. Escherichia coli
(MTCC 443), Salmonella typhimurium (MTCC 98), and
antifungal activity against Aspergillus niger (MTCC 282)
and Candida albicans (MTCC 227) by broth dilution
method (NCCLS, 2002).
Among all the compounds 4a–4l, compounds bearing
additional benzene ring, i.e., 4c, 4f, 4i, and 4l in the pen-
dent 2-oxo-2H-chromenyl nucleus have been found to be
more active against gram-positive bacteria than their other
analogs. Compounds 4j, 4k, and 4l (R1 = Cl) having
electron withdrawing group showed good antimicrobial
activity.
It is perceived by examining the antimicrobial activity
data (Table 2) that compounds 4e and 4k (MIC = 62.5 lg/mL)
and compounds 4c, 4h, and 4i (MIC = 125 lg/mL)
showed excellent activity against gram-positive bacteria
B. subtilis as compared to ampicillin (MIC = 250 lg/mL)
where as compounds 4f and 4l (MIC = 62.5 lg/mL),
compounds 4b, 4c, 4e, and 4i (MIC = 100 lg/mL), and
compound 4j (MIC = 125 lg/mL) showed excellent
Compound 4l having both chloro substitution and ben-
zene ring fusion in the pendent 2-oxo-2H-chromenyl
nucleus possesses the highest antibacterial effectiveness
against both gram-positive and gram-negative bacteria.
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Med Chem Res (2013) 22:4745–4754
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Table 2 Antimicrobial activity
of compounds 4a–4l
Compounds
Minimum inhibitory concentration (MIC, lg/mL)
Gram-positive bacteria Gram-negative bacteria
Fungi
B.s.
S.a.
E.c.
S.t.
A.n.
C.a.
4a
250
200
125
200
62.5
200
250
125
125
200
62.5
200
250
100
NT
200
100
100
250
100
62.5
200
250
100
125
200
62.5
250
10
125
200
250
125
250
250
200
250
62.5
125
200
62.5
100
10
200
250
200
125
200
250
250
250
100
100
250
100
100
10
500
1,000
1,000
250
4b
1,000
[1,000
[1,000
500
4c
4d
500
4e
1,000
1,000
500
4f
500
4g
[1,000
[1,000
[1,000
[1,000
500
4h
200
4i
1,000
[1,000
[1,000
500
B.s., Bacillus subtilis; S.a.,
Staphylococcus aureus; E.c.,
Escherichia coli; S.t.,
Salmonella typhi; A.n.,
Aspergillus niger; C.a., Candida
albicans; NT, not tested
4j
4k
4l
1,000
NT
Ampicillin
Norfloxacin
Griseofulvin
Nystatin
NT
NT
NT
Bold values indicate that the
particular compound possesses
better activity than the standard
drug
NT
NT
NT
NT
100
500
NT
NT
NT
100
100
Among all the tested compounds, the compounds 4e, 4f, 4i,
4k, and 4l are found to be the most proficient members of
the series.
(Moorty et al., 1973) 2a–2d were synthesized according to
the literature procedures.
It is interesting to note that the antimicrobial data of
compounds 4a–4l are found to exhibit better activity than
our earlier reported compounds (Patel et al., 2012). The
compounds 4a–4l showed better activity against gram-
positive and gram-negative bacteria than the earlier
reported compounds. No significant change in antifungal
activity was observed. It is noteworthy that here also the
compounds bearing methyl and chloro substitution exhib-
ited better activity than the other analogs.
General procedure for the synthesis of 3-
[(ethylamino)methyl]-4-hydroxy coumarins (1a–1d)
In a round bottom flask formaldehyde (37 % formalin)
(0.025 mol), ethyl amine (0.03 mol), and ethanol (25 mL)
were taken and stirred for 30 min at room temperature.
Then a solution of appropriate 4-hydroxy coumarin
(0.025 mol) in ethanol (15 mL) was added dropwise to the
above solution during 30 min at room temperature with
stirring. The stirring was further continued overnight at
room temperature. The solid product thus obtained was
filtered out, washed with diethyl ether and dried to yield
1a–1d which were pure enough (checked by thin layer
chromatography [TLC]) to use for further reaction. The
purity of compounds was confirmed by TLC.
Experimental section
All the reagents used were of commercial grade. Melting
points were determined through a Thiele apparatus and are
uncorrected. IR spectra were recorded using KBr on Perkin
1
Elmer Spectrum 100 FT-IR spectrometer. The H-NMR
3-((Ethylamino)methyl)-4-hydroxy-2H-chromen-2-one (1a)
(400 MHz) and 13C-NMR (100 MHz) spectra were recor-
ded on Bruker Avance 400 spectrometer. Mass spectrum
was recorded on Shimadzu QP 2010 spectrometer. Ele-
mental analyzes (C, H, N) were carried out by means of
Perkin Elmer 2400 C–H–N–S–O Analyzer Series II, and
the data found were within 0.4 % of theoretical values.
Various 1-[2-oxo-2-(2-oxo-2H-chromen-3-yl)ethyl]py-
ridinium salts (Koelsch, 1950; Rao and Rao, 1986) 3a–3c
and various 4-chloro-2-oxo-2H-chromene-3-carbaldehydes
White solid; Yield: 67 %; m.p. 185 °C (lit. 186 °C)
(Robertson and Link, 1953).
3-((Ethylamino)methyl)-4-hydroxy-6-methyl-2H-
chromen-2-one (1b)
White solid; Yield: 69 %; m.p. 164 °C; IR (KBr, cm-1):
3365 (O–H), 3325 (N–H), 3045 (Ar C–H), 2960 (aliphatic
123

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Med Chem Res (2013) 22:4745–4754
1
C–H), 1720 (C=O), 1605 (Ar C=C); H-NMR (400 MHz,
DMSO-d6, d ppm): 1.19 (3H, t, J = 7.2 Hz, –NHCH2CH3),
2.36 (3H, s, CH3), 2.89 (2H, q, J = 7.2 Hz, –NHCH2CH3),
3.72 (2H, s, –CH2–NH–), 7.17–7.68 (3H, m, Ar–H), 8.33
(2H, br, –NH– and –OH); 13C-NMR (100 MHz, CD3OD-
d4, d ppm): 20.86 (CH3), 30.48 (CH3), 42.69 (CH2), 43.40
(CH2), 113.32 (C), 116.96 (C), 117.37 (CH), 124.18 (CH),
134.90 (CH), 135.36 (C), 153.36 (C), 166.35 (C), 168.44
(C=O); Anal. Calc. for C13H15NO3: C, 66.94; H, 6.48; N,
6.00. Found: C, 67.18; H, 6.56; N, 5.89 %.
mixture was further stirred for 45 min at room temperature,
and then refluxed in an oil bath at 140 °C for 8 h. It was
then allowed to cool to ambient temperature and poured
into ice cold water (75 mL). The gummy mass thus
obtained was extracted with chloroform (3 9 30 mL). The
combined chloroform extract was washed with 10 %
sodium bicarbonate solution (3 9 20 mL) and then with
water (3 9 20 mL). It was dried over anhydrous sodium
sulfate. The removal of chloroform under vacuum gave a
crude solid product. The TLC of the crude product showed
two major spots. The crude product was subjected to col-
umn chromatography using silica gel and ethyl acetate–
petroleum ether as an eluent. By eluting the column using
(1:9) ethyl acetate–petroleum ether as a eluent, the product
obtained, was found to be dicoumarol (E). Then eluting the
column using ethyl acetate–petroleum ether (3:7) as an
eluent, the expected compound 4a was obtained.
3-((Ethylamino)methyl)-4-hydroxy-8-methyl-2H
-chromen-2-one (1c)
Yellow solid; Yield: 72 %; m.p. 157 °C; IR (KBr, cm-1):
3360 (O–H), 3340 (N–H), 3055 (Ar C–H), 2935 (aliphatic
1
C–H), 1710 (C=O), 1600 (Ar C=C); H-NMR (400 MHz,
DMSO-d6, d ppm): 1.19 (3H, t, J = 7.2 Hz, –NHCH2CH3),
2.30 (3H, s, CH3), 2.89 (2H, q, J = 7.2 Hz, –NHCH2CH3),
3.89 (2H, s, –CH2–NH–), 7.01–7.69 (3H, m, Ar–H), 8.16
(2H, br, –NH– and –OH); 13C-NMR (100 MHz, CD3OD-
d4, d ppm): 11.50 (CH3), 15.74 (CH3), 42.80 (CH2), 43.63
(CH2), 123.50 (CH), 123.90 (C), 124.32 (CH), 126.77 (C),
133.86 (CH), 152.49 (C), 153.62 (C), 167.66 (C), 168.38
(C=O); Anal. Calc. for C13H15NO3: C, 66.94; H, 6.48; N,
6.00. Found: C, 66.73; H, 6.41; N, 5.91 %.
Dicoumarol (E): Yield: 20 %; m.p. 289 °C (lit.
288–289 °C Stahmann et al., 1941).
Mixed melting point did not change.
The spectral data of the compound 4a are given below.
2-(2-Oxo-2H-chromen-3-yl)-5H-chromeno[4,3-b]
pyridin-5-one (4a)
Brown solid; Yield 47 %; m.p. 218–220 °C; IR (KBr,
cm-1): 3123 (Ar C–H), 1689 (C=O), 1624 (Ar C=C), 1501
(Ar C=N); 1H-NMR (400 MHz, CDCl3 ? TFA-d1, d
ppm): 7.35–8.12 (9H, m, Ar–H), 8.74 (1H, brs, C10-H),
6-Chloro-3-((ethylamino)methyl)-4-hydroxy-2H
-chromen-2-one (1d)
9.03 (1H, s, C4 -H); 13C-NMR (100 MHz, CDCl3 ? TFA-
0
Yellow solid; Yield: 68 %; m.p. 174 °C; IR (KBr, cm-1):
3370 (O–H), 3315 (N–H), 3040 (Ar C–H), 2950 (aliphatic
d1, d ppm): 110.00 (C), 112.82 (C), 115.63 (C), 117.62
(CH), 117.77 (CH), 118.48 (CH), 119.46 (CH), 125.58
(CH), 125.75 (CH), 126.64 (CH), 131.44 (C), 133.15 (C),
136.24 (CH), 136.51 (CH), 139.18 (C), 142.86 (CH),
147.78 (CH), 154.52 (C), 163.22 (C=O), 163.96 (C=O);
Mass (m/z): 285 (M?-56), 189 (100 %), 172 (17 %), 162
(21 %), 144 (15 %), 133 (36 %), 121 (93 %), 105 (11 %),
92 (68 %), 77 (10 %), 63 (31 %); Anal. Calc. for
C21H11NO4: C, 73.90; H, 3.25; N, 4.10. Found: C, 74.17;
H, 3.19; N, 4.18 %.
1
C–H), 1715 (C=O), 1610 (Ar C=C); H-NMR (400 MHz,
DMSO-d6, d ppm): 1.19 (3H, t, J = 7.2 Hz, –NHCH2CH3),
2.90 (2H, q, J = 7.2 Hz, –NHCH2CH3), 3.87 (2H, s,
–CH2–NH–), 7.18–7.76 (3H, m, Ar–H), 8.14 (2H, br,
–NH– and –OH); 13C-NMR (100 MHz, CD3OD-d4, d
ppm): 11.53 (CH3), 43.00 (CH2), 43.49 (CH2), 119.29
(CH), 122.56 (C), 124.48 (C), 125.21 (CH), 129.77 (C),
132.44 (CH), 152.80 (C), 153.70 (C), 167.91 (C=O); Anal.
Calc. for C12H12ClNO3: C, 56.81; H, 4.77; N, 5.52. Found:
C, 56.53; H, 4.71; N, 5.43 %.
Synthesis of 2-(2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4a)
Synthesis of 2-(2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4a)
Method B
Method A
To a solution of 3a (0.005 mol) in glacial acetic acid
(15 mL), ammonium acetate (0.05 mol) was added with
stirring at room temperature. Then a solution of 2a
(0.005 mol) in glacial acetic acid (15 mL) was added with
stirring at room temperature during 15 min. The reaction
mixture was further stirred for 45 min at room temperature
and then refluxed in an oil bath at 140 °C for 8 h. It was
To a solution of 3a (0.005 mol) in glacial acetic acid
(15 mL), ammonium acetate (0.05 mol) was added with
stirring at room temperature. Then a solution of 1a
(0.005 mol) in glacial acetic acid (15 mL) was added with
stirring at room temperature during 15 min. The reaction
123

Med Chem Res (2013) 22:4745–4754
4751
then allowed to cool to ambient temperature and poured
into ice cold water (75 mL). The gummy mass obtained
was extracted with chloroform (3 9 30 mL). The com-
bined chloroform extract was washed with 10 % sodium
bicarbonate solution (3 9 20 mL) and then with water
(3 9 20 mL). It was dried over anhydrous sodium sulfate.
The removal of chloroform under vacuum gave a crude
solid product. This was further purified by column chro-
matography using silica gel and ethyl acetate–petroleum
ether (3:7) as an eluent, it gave brown solid product 4a in
62 % yield. The product 4a was identical in all aspects
with that obtained by method A.
1H-NMR (400 MHz, DMSO-d6, d ppm): 2.34 (3H, s, CH3),
7.14–8.47 (7H, m, Ar–0H), 9.69–9.87 (2H, m, C4-H and
C10-H), 10.82 (1H, s, C4 -H); 13C-NMR (100 MHz,DMSO-
d6, d ppm): 20.70 (CH3), 96.73 (C), 96.87 (C), 117.17
(CH), 117.31 (CH), 120.49 (CH), 120.61 (CH), 125.40
(CH), 125.83 (CH), 133.58 (C), 133.66 (C), 135.54 (CH),
135.65 (CH), 152.85 (C), 158.60 (C), 160.77 (CH), 162.41
(CH), 162.65 (C), 163.55 (C), 163.65 (C), 178.10 (C=O),
180.39 (C=O); Anal. Calc. for C22H13NO4: C, 74.36; H,
3.69; N, 3.94. Found: C, 74.62; H, 3.65; N, 3.87 %.
2-(8-Methoxy-2-oxo-2H-chromen-3-yl)-9-methyl-5H-
Similarly, the compounds 4b–4l were synthesized using
above two methods.
chromeno[4,3-b]pyridin-5-one (4e)
The spectral data of compounds 4b–4l are given below.
Brown solid; m.p. 219–221 °C; IR (KBr, cm-1): 3144
(Ar C–H), 1703 (C=O), 1620 (Ar C=C), 1490 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 2.35 (3H, s, CH3),
3.92 (3H, s, OCH3), 7.10–8.47 (6H, m, Ar–H), 9.69–9.87
2-(8-Methoxy-2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4b)
(2H, m, C4-H and C10-H), 10.81 (1H, s, C4 -H); 13C-NMR
0
Brown solid; m.p. 211–213 °C; IR (KBr, cm-1): 3137
(Ar C–H), 1696 (C=O), 1631 (Ar C=C), 1502 (Ar C=N);
1H-NMR (400 MHz, CDCl3 ? TFA-d1, d ppm): 3.97 (3H,
s, OCH3), 6.98–8.11 (8H, m, Ar–H), 8.73 (1H, brs, C10-H),
(100 MHz, DMSO-d6, d ppm): 20.71 (CH3), 56.76
(OCH3), 96.68 (C), 96.84 (C), 117.20 (CH), 117.29 (CH),
120.50 (C), 120.58 (C), 125.40 (CH), 125.82 (CH), 133.57
(C), 133.67 (C), 135.56 (CH), 135.65 (CH), 152.86 (C),
160.75 (CH), 162.41 (CH), 163.56 (C), 163.64 (C), 178.08
(C=O), 180.36 (C=O); Anal. Calc. for C23H15NO5: C,
71.68; H, 3.92; N, 3.63. Found: C, 71.45; H, 3.98; N,
3.71 %.
9.01 (1H, s, C4 -H); 13C-NMR (100 MHz, DMSO-d6, d
0
ppm): 56.45 (OCH3), 96.69 (C), 96.81 (C), 115.65 (C),
117.40 (CH), 117.52 (CH), 120.32 (C), 120.88 (C), 120.94
(C), 124.38 (CH), 124.48 (CH), 125.82 (CH), 126.23 (CH),
134.83 (CH), 134.92 (CH), 154.77 (C), 160.80 (CH),
162.50 (CH), 163.37 (C), 163.56 (C), 178.03 (C=O),
180.28 (C=O); Anal. Calc. for C22H13NO5: C, 71.16; H,
3.53; N, 3.77. Found: C, 70.88; H, 3.48; N, 3.70 %.
9-Methyl-2-(3-oxo-3H-benzo[f]chromen-2-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4f)
Brown solid; m.p. 214–216 °C; IR (KBr, cm-1): 3144
(Ar C–H), 1696 (C=O), 1620 (Ar C=C), 1490 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 2.35 (3H, s, CH3),
7.14–8.047 (8H, m, Ar–H), 9.69–9.87 (3H, m, C4-H, C10-H,
2-(3-Oxo-3H-benzo[f]chromen-2-yl)-5H-chromeno
[4,3-b]pyridin-5-one (4c)
Brown solid; m.p. 222–224 °C; IR (KBr, cm-1): 3137
(Ar C–H), 1692 (C=O), 1627 (Ar C=C), 1505 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 7.26–8.48 (9H,
and C5 -H), 10.82 (1H, s, C4 -H); 13C-NMR (100 MHz,
0
DMSO-d6, d ppm): 20.71 (CH3), 96.70 (C), 96.84 (C),
117.20 (CH), 117.29 (CH), 120.50 (C), 120.58 (C), 124.40
(CH), 124.48 (CH), 125.40 (CH), 125.82 (CH), 126.20
(CH), 133.56 (C), 133.66 (C), 135.55 (CH), 135.64 (CH),
152.86 (C), 154.78 (C), 160.74 (CH), 162.41 (CH), 163.57
(C), 163.64 (C), 178.08 (C=O), 180.36 (C=O); Anal. Calc.
for C26H15NO4: C, 77.03; H, 3.73; N, 3.46. Found: C,
77.28; H, 3.67; N, 3.39 %.
0
m, Ar–H), 9.71–9.92 (3H, m, C4-H, C10-H, and C5 -H),
10.83 (1H, s, C4 -H); 13C-NMR (100 MHz, DMSO-d6, d
0
ppm): 96.69 (C), 96.82 (C), 117.39 (CH), 117.49 (CH),
120.88 (C), 120.93 (CH), 124.40 (CH), 124.48 (CH),
125.82 (CH), 126.20 (CH), 134.83 (CH), 134.94 (CH),
152.04 (CH), 154.78 (C), 160.78 (CH), 162.49 (CH),
163.41 (C), 163.53 (C), 163.81 (C), 178.05 (C=O), 180.29
(C=O); Anal. Calc. for C25H13NO4: C, 76.72; H, 3.35; N,
3.58. Found: C, 76.46; H, 3.28; N, 3.51 %.
7-Methyl-2-(2-oxo-2H-chromen-3-yl)-5H-chromeno
[4,3-b]pyridin-5-one (4g)
9-Methyl-2-(2-oxo-2H-chromen-3-yl)-5H-chromeno
Brown solid; m.p. 253–255 °C; IR (KBr, cm-1): 3123
(Ar C–H), 1704 (C=O), 1621 (Ar C=C), 1494 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 2.32 (3H, s, CH3),
7.17–8.48 (7H, m, Ar–H), 90.70–9.92 (2H, m, C4-H, and
C10-H), 10.83 (1H, s, C4 -H); 13C-NMR (100 MHz,
[4,3-b]pyridin-5-one (4d)
Brown solid; m.p. 225–227 °C; IR (KBr, cm-1): 3141
(Ar C–H), 1701 (C=O), 1620 (Ar C=C), 1497 (Ar C=N);
123

4752
Med Chem Res (2013) 22:4745–4754
DMSO-d6, d ppm): 15.77 (CH3), 96.52 (C), 96.71 (C),
117.87 (CH), 120.69 (C), 120.75 (C), 121.05 (C), 123.45
(CH), 123.77 (CH), 123.87 (CH), 126.12 (C), 126.26 (CH),
135.69 (CH), 135.81 (CH), 153.05 (CH), 160.82 (CH),
161.64 (C), 162.46 (CH), 163.29 (C), 163.41 (C), 178.29
(C=O), 180.58 (C=O); Anal. Calc. for C22H13NO4: C,
74.36; H, 3.69; N, 3.94. Found: C, 74.09; H, 3.63; N,
3.84 %.
134.49 (CH), 153.39 (C), 155.41 (C), 161.03 (CH), 161.72
(CH), 162.70 (CH), 162.92 (C), 163.08 (C), 175.62 (C),
176.84 (C=O), 178.82 (C=O), Anal. Calc. for
C21H10ClNO4: C, 67.12; H, 2.68; N, 3.73. Found: C, 67.41;
H, 2.63; N, 3.66 %.
9-Chloro-2-(8-methoxy-2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4k)
2-(8-Methoxy-2-oxo-2H-chromen-3-yl)-7-methyl-5H-
Brown solid; m.p. 261 °C; IR (KBr, cm-1): 3123 (Ar C–
H), 1695 (C=O), 1627 (Ar C=C), 1501 (Ar C=N); 1H-NMR
(400 MHz, DMSO-d6, d ppm): 3.93 (3H, s, OCH3),
7.31–8.48 (6H, m, Ar–H), 9.77–10.06 (2H, m, C4-H and
chromeno[4,3-b]pyridin-5-one (4h)
Brown solid; m.p. 240–242 °C; IR (KBr, cm-1): 3093
(Ar C–H), 1708 (C=O), 1606 (Ar C=C), 1479 (Ar C=N);
1H-NMR (400 MHz, CDCl3, d ppm): 2.50 (3H, s, CH3),
3.99 (3H, s, OCH3), 6.45–7.36 (7H, m, Ar–H), 7.63 (1H,
C10-H), 10.81 (1H, s, C4 -H); 13C-NMR (100 MHz,
0
DMSO-d6, d ppm): 56.15 (OCH3), 96.42 (C), 96.65 (C),
119.73 (CH), 119.84 (CH), 122.28 (C), 124.83 (CH),
125.22 (CH), 128.60 (C), 128.69 (C), 134.37 (CH), 134.48
(CH), 153.38 (C), 156.92 (C), 160.99 (CH), 162.70 (CH),
162.92 (C), 163.09 (C), 176.83 (C=O), 178.84 (C=O);
Anal. Calc. for C22H12ClNO5: C, 65.12; H, 2.98; N, 3.45.
Found: C, 65.38; H, 2.92; N, 3.36 %.
poorly resolved dd, C10-H), 7.94 (1H, s, C4 -H); 13C-NMR
0
(100 MHz, DMSO-d6, d ppm): 16.07 (CH3), 56.33
(OCH3), 105.71 (CH), 111.18 (C), 112.21 (C), 114.31
(CH), 118.55 (CH), 119.28 (C), 119.72 (CH), 124.06 (CH),
124.46 (C), 124.95 (CH), 126.90 (CH), 132.19 (CH),
142.44 (CH), 143.33 (C), 147.26 (C), 151.02 (C), 154.88
(C), 157.80 (C), 157.97 (C), 160.03 (C=O), 169.59 (C=O);
Anal. Calc. for C23H15NO5: C, 71.68; H, 3.92; N, 3.63.
Found: C, 71.44; H, 3.99; N, 3.70 %.
9-Chloro-2-(3-oxo-3H-benzo[f]chromen-2-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4l)
Brown solid; m.p. 258–260 °C; IR (KBr, cm-1): 3123
(Ar C–H), 1695 (C=O), 1631 (Ar C=C), 1501 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 7.31–8.47 (8H,
7-Methyl-2-(3-oxo-3H-benzo[f]chromen-2-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4i)
0
m, Ar–H), 9.80–10.05 (3H, m, C4-H, C10-H, and C5 -H),
Brown solid; m.p. 256–258 °C; IR (KBr, cm-1): 3132
(Ar C–H), 1704 (C=O), 1621 (Ar C=C), 1495 (Ar C=N);
1H-NMR (400 MHz, DMSO-d6, d ppm): 2.32 (3H, s, CH3),
7.17–8.036 (8H, m, Ar–H), 9.70–9.92 (3H, m, C4-H, C10-H,
10.81 (1H, s, C4 -H); 13C-NMR (100 MHz, DMSO-d6, d
0
ppm): 96.45 (C), 96.64 (C), 119.75 (CH), 119.86 (CH),
122.26 (C), 122.53 (C), 124.84 (CH), 125.21 (CH), 128.58
(C), 128.68 (C), 133.77 (CH), 134.37 (CH), 134.46 (CH),
152.45 (C), 153.37 (C), 158.64 (CH), 161.02 (CH), 162.68
(CH), 162.92 (C), 163.07 (C), 176.83 (C=O), 178.80
(C=O); Anal. Calc. for C25H12ClNO5: C, 70.52; H, 2.84; N,
3.29. Found: C, 70.32; H, 2.80; N, 3.22 %.
and C5 -H), 10.83 (1H, s, C4 -H); 13C-NMR (100 MHz,
0
DMSO-d6, d ppm): 15.73 (CH3), 96.50 (C), 96.64 (C),
116.94 (CH), 120.39 (C), 120.68 (C), 120.79 (C), 121.90
(C), 123.48 (CH), 123.80 (CH), 123.86 (CH), 126.15 (C),
126.29 (C), 129.14 (C), 135.73 (CH), 135.84 (CH), 153.04
(C), 160.82 (CH), 161.21 (CH), 161.98 (C), 162.45 (CH),
162.83 (C), 163.30 (C), 163.44 (C), 178.32 (C=O), 180.61
(C=O); Anal. Calc. for C26H15NO4: C, 77.03; H, 3.73; N,
3.46. Found: C, 76.79; H, 3.67; N, 3.38 %.
Minimum inhibitory concentration (MIC) measurement
Antibacterial assay
For the determination of MIC, bacteria were grown over
night in Mueller–Hinton broth as a nutrient medium at
37 °C. DMSO was used as diluents/vehicle to get desired
concentration of synthesized compounds and standard
drugs to test upon standard microbial strains. Serial dilu-
tions were prepared in primary and secondary screening.
Each synthesized compound and standard drugs were
diluted obtaining 2,000 lg/mL concentration, as a stock
solution. In primary screening 1,000, 500, and 250 lg/mL
concentrations of the synthesized drugs were taken. The
active synthesized compounds found in this primary
9-Chloro-2-(2-oxo-2H-chromen-3-yl)-5H-
chromeno[4,3-b]pyridin-5-one (4j)
Brown solid; m.p. 265 °C; IR (KBr, cm-1): 3115 (Ar C–
H), 1696 (C=O), 1632 (Ar C=C), 1502 (Ar C=N); 1H-NMR
(400 MHz, DMSO-d6, d ppm): 7.32–8.48 (7H, m, Ar–H),
0
9.77–10.03 (2H, m, C4-H, and C10-H), 10.81 (1H, s, C4 -
H); 13C-NMR (100 MHz, DMSO-d6, d ppm): 96.43 (C),
96.64 (C), 119.74 (CH), 119.86 (CH), 122.27 (C), 124.84
(CH), 125.23 (CH), 128.57 (C), 128.69 (CH), 134.39 (CH),
123

Med Chem Res (2013) 22:4745–4754
4753
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In conclusion, we have developed a highly efficient and
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3-yl)-5H-chromeno[4,3-b]pyridin-5-ones 4a–4l by two
different methodologies. Our interest in the synthesis of
title compounds was to focus on their study as antimicro-
bial agents as a part of our research work which is aimed at
the development of new heterocyclic compounds as more
potent antimicrobial agents. Compounds 4e, 4f, 4i, 4k, and
4l were found to be the most efficient members of the
series. These compounds were structurally different from
already available inhibitors and we suggest that these
compounds could serve as potential leads for further
development of new antimicrobial agents.
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Acknowledgments The authors are grateful to the Head, Depart-
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research facilities. Financial assistance to AAP, HJP, KRP, and CVP
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