An efficient one-pot synthesis of some new 2,4-diaryl pyrido[3,2-c] coumarins as potent antimicrobial agents

Article abstract of DOI:10.1002/jhet.234

(Chemical Equation Presented) Poly(ethylene glycol) (PEG-400) has been used as sustainable, nonvolatile, and environmental friendly reaction solvent for the synthesis of title compounds. Various diaryl pyrido[3,2-c]coumarins have been synthesized in one s

Full text of DOI:10.1002/jhet.234

January 2010
An Efficient One-Pot Synthesis of Some New 2,4-Diaryl
Pyrido[3,2-c]coumarins as Potent Antimicrobial Agents
237
Bhaskar S. Dawane,a* Shankaraiah G. Konda,a* Ragini G. Bodade,b
and Raghunath B. Bhosalec
aOrganic Research Laboratory, Department of Chemistry, Yeshwant Mahavidyalaya,
Nanded 431602, Maharashtra, India
bSchool of Life sciences, Swami Ramanand Teerth Marathwada University,
Nanded 431606, Maharashtra, India
cSchool of Chemical Sciences, Solapur University, Solapur 413255, Maharashtra, India
*E-mail: bhaskardawane@rediffmail.com or kondasg@rediffmail.com
Received May 2, 2009
DOI 10.1002/jhet.234
Published online 21 December 2009 in Wiley InterScience (www.interscience.wiley.com).
Poly(ethylene glycol) (PEG-400) has been used as sustainable, nonvolatile, and environmental
friendly reaction solvent for the synthesis of title compounds. Various diaryl pyrido[3,2-c]coumarins
have been synthesized in one step by reacting 4-hydroxy-7-methyl-coumarin with a,b-unsaturated
ketones in the presence of ammonium acetate. Further, in vitro antimicrobial (antibacterial and antifun-
gal) activities of the compounds were screened against different pathogens. The results revealed that
most of them showed potent activity.
J. Heterocyclic Chem., 47, 237 (2010).
INTRODUCTION
anticoagulant [9], antidiabetic [10], and analgesic [11]
properties, being characterized by a phenanthrene like
structure as found in tetrahydrocannabinol. Pyrido[3,2-
c]coumarin, the back bone of naturally occurring alka-
loid, santiagonamine [12] isolated from Berberis Dawi-
nii (Barberidaca). This alkaloid has interesting wound
healing properties [13]. Owing to such interesting prop-
erties, synthesis of pyrido-coumarins has remained an
active subject of interest. However, a survey of these
literatures quotes reveals that the most of the methods
are multistep or difficult starting materials. Hence, it
Coumarins are well-known natural products and may
exhibit high level of biological activity [1]. Coumarins
are also used as food additives, in cosmetics [2], as op-
tical brightening agents [3], dispersed fluorescent and
laser dyes [4]. For instance, coumarin nucleus is present
in promising drug candidates as nonpeptidic HIV
protease inhibitors [5], topoisomerase II [6], and tyro-
sine kinase [7] inhibitors. Coumarins fused with pyri-
dines have also been reported to posses antiallergic [8],
C
V
2009 HeteroCorporation

238
B. S. Dawane, S. G. Konda, R. G. Bodade, and R. B. Bhosale
Vol 47
Scheme 1
and of low toxicity [16]. Many organic reactions have
been carried out using PEGs as solvent or cosolvent
such as Heck reaction [17], asymmetric dihydroxylation
[18], Suzuki crosscoupling reaction [19], oxydehydroge-
nation of alcohols and cyclic dienes, oxidation of sul-
fides and the Wacker reaction [20], and partial reduction
reaction of alkynes [21]. The use of PEG as a recyclable
solvent system for the metal mediated radical polymer-
ization of methyl methacrylate and styrene has also
been reported [22].
As quoted by Kroehnke [23], reaction of a,b-unsatu-
rated ketones with the active methylene function of
phenacyl bromide pyridinium salt of ammonium acetate
and acetic acid yields pyridine. This methodology has
been successfully utilized by us for the synthesis of vari-
ety of pyridyl substituted coumarins. In this article,
here, we wish to report the expeditious synthesis of
novel pyrido-fused coumarin derivatives (3a–l) under
benign reaction solvent.
The starting 4-hydroxy-7-methyl-coumarin (1) was
prepared by the literature procedure using an appropri-
ately m-cresol and malonic acid, a Lewis acid (ZnCl2)
and as condensing agent phosphorous oxychloride
(POCl3), whereas the a,b-unsaturated carbonyl com-
pounds (2a–l) were already reported [24]. Thus, various
2,4-diaryl pyrido[3,2-c]coumarins 3(a–l) have been syn-
thesized by reacting 4-hydroxy-7-methyl-coumarin 1
with a,b-unsaturated ketones 2(a–l) in the presence of
ammonium acetate in PEG-400 (Scheme 1).
was thought worthwhile to envisage a synthesis of
pyrido-fused coumarins.
RESULTS AND DISCUSSION
In view of the current emphasis on green chemistry
[14], our approach is to reduce the use of solvents that
are volatile organic compounds (VOCs), which are
potentially toxic and hazardous [15]. Recently, liquid
polymers or low-melting polymers have emerged as al-
ternative green reaction media with unique properties
such as thermal stability, commercial availability, non-
volatility, immiscibility with a number of organic sol-
vents, and recyclability. Poly(ethylene glycols) (PEGs)
are preferred over other polymers because they are inex-
pensive, completely nonhalogenated, easily degradable,
Mechanism of pyrido-coumarin formation. The
formation of products 3(a–l) involves the Kroehnke’s
mechanism. Mannich bases provide the required a,b-un-
saturated ketones in situ during the course of reaction,
to which the Micheal addition of coumarioyl methyl
pyridinium salts takes place resulting in a 1,5-dionyl
pyridinium (not isolated) derivative, which subsequently
cyclization in the presence of ammonium acetate afford
the 2,4-diaryl pyrido[3,2-c]coumarins (Scheme 2). The
Scheme 2
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

January 2010
An Efficient One-Pot Synthesis of Some New 2,4-Diaryl
Pyrido[3,2-c]coumarins as Potent Antimicrobial Agents
239
Table 1
Physical and analytical data of 2,4-diaryl-pyrido[3,2-c]coumarins.
Product
R1
R2
R3
R4
R5
Timea
Yield (%)b
MP (ꢁC)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
Br
I
Cl
H
H
Cl
Cl
Cl
H
Cl
Cl
2
3
2
2
3
3
2
3
2
2
3
3
85
89
83
85
82
86
81
85
86
85
81
85
240
266
258
230
172
260
243
251
265
170
255
225
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
Cl
Cl
H
H
Cl
I
H
Br
I
OH
Me
OH
Me
H
Cl
Cl
Cl
Cl
OH
OH
OH
OH
Cl
Cl
Cl
Cl
Cl
Cl
Me
3j
3k
3l
H
H
I
H
a Time in hours.
b Pure isolated yields of products.
an internal standard. The mass were recorded on EI-Shimazdu-
GC-MS spectrometer. Elemental analyses were performed on a
Carlo Erba 106 Perkin-Elmer model 240 analyzer.
presence of IR absorption bands in the region 1660–
1690 cmꢀ1 clearly indicates that >C¼¼O group of chal-
1
cone has been transformed into coumarin (lactone). H
Chemistry
NMR revealed that singlet of methyl proton at d 2.30–
2.50 ppm. The multiplet at d 7.00–8.20 is due to aro-
matic protons. A singlet of phenolic proton appeared at
d 11.00–12.50 ppm (D2O exchangeable).
General procedure for the synthesis of a,b-unsaturated
carbonyl compounds (2a–l)24. A mixture of substituted 2-
hydroxy acetophenone (5 mmol), 1-phenyl-3-(4-sustituted phe-
nyl) pyrazol-4-carboxaldehyde (5 mmol), KOH (1 mmol), and
PEG-400 (20 mL) was stirred at 40ꢁC for 1 h. After comple-
tion of the reaction (monitored by TLC), the crude mixture
was worked up in ice cold water (100 mL). Separated product
was filtered out. The PEG-400 was recovered from water and
utilized to synthesize further chalcones.
Typical procedure for the synthesis of 2,4-diaryl pyr-
ido[3,2-c]coumarins (3a–l). To a well-stirred solution of 4-
hydroxy-7-methyl-coumarin 1 (0.528 g; 6 mmol) in PEG-400
(20 mL) was added ammonium acetate (6.0 g) followed by chal-
cone 2a (2.514 g; 6 mmol) in PEG-400 (10 mL). The reaction
mixture was stirred at room temperature for 30 min and then
refluxed gently on oil-bath at 130ꢁC for the period as shown in
Table 1. After completion of the reaction (checked by TLC), the
reaction mixture was allowed to reach room temperature and
was poured into ice cold water (100 mL). The resultant solid
was filtered, washed with 2 ꢂ 5 mL ice cold water and recrys-
tallized by chloroform to give pure product 3a.
In summary, we have demonstrated an efficient,
one-pot method toward the expeditious synthesis of 2,4-
diaryl pyrido[3,2-c]coumarins using PEG-400 as an
alternative reaction solvent. The advantages of the present
protocol are the simplicity of operation, the high yields of
products, the recyclability of PEG-400 and preclusion of
the usage of volatile organic solvents. Antimicrobial and
antifungal activities of the all synthesized compounds
were summarized in Table 2. The compounds 3b, 3d, 3e,
3f, 3h, 3j, and 3l showed good antibacterial activity
against one or more bacteria. Compounds 3a, 3c, 3d, 3i,
and 3k were found to be active against Escherichia coli.
On the other hand, the compounds 3i, 3k was found to be
active against Salmonella typhi. The compounds 3i, 3j,
and 3k were also found to be active against Bacillus subti-
lis. Most of the compounds showed inhibitory effect
against fungi. The compounds 3a, 3c, 3d, 3g, 3j, and 3l
showed most potent activity against all pathogens than
other tested compounds. The substitution of hydroxyl
group in position 2 and presence of halo groups in 3 and 5
positions of aryl nucleus, which may enhance the antimi-
crobial activity of the products against various pathogens.
To check the reusability of the solvent, the filtrate was
evaporated to remove water leaving PEG behind in the con-
tainer and subjected for second run by charging the same sub-
strates. The results of the first and second experiments were
almost consistent in yield.
3-(4-Chloro-phenyl)-1-[3-(4-chloro-phenyl)-1-phenyl-1H-pyr-
azol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-10-one (3a). IR
(KBr cmꢀ1/tmax): 1668, 1598 cmꢀ1 1H NMR (DMSO-d6): d
;
2.35 (s, 3H, CH3), 7.10–8.15 (m, 17H, ArAH), 8.90 (s, 1H,
-5H of pyrazole) ppm; MS (m/z): 574 [Mþ], 576 [M þ 2],
578 [M þ 4]; Anal. Found for C34H21O2N3Cl2: C, 71.02; H,
3.61; N, 7.34 requires: C, 71.19; H, 3.78; N, 7.21%.
EXPERIMENTAL
Melting points were determined by open capillary method and
were uncorrected. IR spectra were recorded (in KBr pallets) on
Shimadzu spectrophotometer. 1H NMR spectra were recorded
(in DMSO-d6) on Avance-300 MHz spectrometer using TMS as
3-(5-Chloro-2-hydroxy-phenyl)-1-[3-(4-chloro-phenyl)-1-phe-
nyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-10-one
(3b). IR (KBr cmꢀ1/tmax): 3413, 1671, 1605 cmꢀ1 1H NMR
;
(DMSO-d6): d 2.33 (s, 3H, CH3), 7.16–8.24 (m, 16H, ArAH),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

240
B. S. Dawane, S. G. Konda, R. G. Bodade, and R. B. Bhosale
Vol 47
Table 2
Antimicrobial activities of synthesized products (3a–l).
Bacteriaa
Fungi germination
Product
Ec
St
Sa
Bs
An
Af
Pc
Fm
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
21
12
19
18
19
08
22
09
21
19
20
18
–
19
10
22
14
12
10
20
12
20
12
16
12
–
16
08
21
16
10
07
18
15
19
18
19
15
–
19
10
20
08
14
14
22
12
18
16
22
18
–
RD
ꢀve
ꢀve
RD
RD
ꢀve
ꢀve
RD
ꢀve
RD
ꢀve
RD
ꢀve
þve
RD
ꢀve
þve
RD
RD
ꢀve
þve
ꢀve
þve
ꢀve
RD
ꢀve
ꢀve
þve
NA
þve
ꢀve
þve
ꢀve
RD
þve
þve
ꢀve
þve
ꢀve
RD
RD
þve
ꢀve
ꢀve
ꢀve
ꢀve
þve
NA
ꢀve
ꢀve
3l
RD
RD
Control
Penicillin
Nystatin
þve
NA
þve
NA
22
NA
22
NA
24
NA
24
NA
ꢀve
ꢀve
ꢀve
ꢀve
Ec, Escherichia coli; St, Salminella typhi; Sa, Staphylococcus aureus; Bs, Bacillus subtilis; An, Aspergillus niger; Af, Aspergillus flavus; Fm, Fusar-
ium moniliforme, Pc, Penicillium chrysogenum. þve, Growth; ꢀve, No growth; RD, reduced growth, NA, not applicable.
a Zone of inhibition in millimeters.
8.89 (s, 1H, -5H of pyrazole), 11.26 (s, 1H, AOH, D2O
exchangeable) ppm; MS (m/z): 590 [Mþ], 592 [M þ 2], 594
[M þ 4]; Anal. Found for C34H21O3N3Cl2: C, 69.16; H, 3.51;
N, 7.08 requires: C, 69.21; H, 3.68; N, 7.22%.
3-(3-Bromo-5-chloro-2-hydroxy-phenyl)-1-[3-(4-chloro-phe-
nyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanth-
1H NMR (DMSO-d6): d 2.24 (s, 3H, CH3), 2.48 (s, 3H, CH3),
7.14–8.21 (m, 15H, ArAH), 8.91 (s, 1H, -5H of pyrazole),
11.31 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z): 604
[Mþ], 606 [M þ 2], 608 [M þ 4]; Anal. Found for
C35H23O3N3Cl2: C, 69.51; H, 3.86; N, 6.91 requires: C, 69.64;
H, 3.97; N, 6.85%.
ren-10-one (3c). IR (KBr cmꢀ1/tmax): 3433, 1679, 1606 cmꢀ1
;
3-(3,5-Dichloro-2,4-dihydroxy-phenyl)-1-[3-(4-chloro-phenyl)-
1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-
10-one (3g). IR (KBr cmꢀ1/tmax): 3412, 3168, 1681, 1606
1H NMR (DMSO-d6): d 2.38 (s, 3H, CH3), 7.18–8.25 (m,
15H, ArAH), 8.90 (s, 1H, -5H of pyrazole), 11.64 (s, 1H,
AOH, D2O exchangeable) ppm; MS (m/z): 669 [Mþ], 671
[M þ 2], 673 [M þ 4], 675 [M þ 6]; Anal. Found for
C34H20O3N3Cl2Br: C, 61.06; H, 3.05; N, 6.21 requires: C,
60.88; H, 3.19; N, 6.34%.
1
cmꢀ1; H NMR (DMSO-d6): d 2.38 (s, 3H, CH3), 6.26 (s, 1H,
OH), 7.15–8.25 (m, 14H, ArAH), 8.90 (s, 1H, -5H of pyraz-
ole), 12.28 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z):
641 [Mþ], 643 [M þ 2], 645 [M þ 4], 647 [M þ 6]; Anal.
Found for C34H20O4N3Cl3: C, 63.76; H, 3.11; N, 6.58 requires:
C, 63.62; H, 3.25; N, 6.65%.
3-(5-Chloro-2-hydroxy-3-iodo-phenyl)-1-[3-(4-chloro-phenyl)-
1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-
1
10-one (3d) IR (KBr cmꢀ1/tmax): 3421, 1681, 1604 cmꢀ1; H
3-(5-Chloro-2-hydroxy-3-iodo-4-methyl-phenyl)-1-[3-(4-chloro-
phenyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenan-
thren-10-one (3h). IR (KBr cmꢀ1/tmax): 3433, 1682, 1605
NMR (DMSO-d6): d 2.43 (s, 3H, CH3), 7.15–8.25 (m, 15H,
ArAH), 8.91 (s, 1H, -5H of pyrazole), 11.68 (s, 1H, AOH,
D2O exchangeable) ppm; MS (m/z): 716 [Mþ], 718 [M þ 2],
720 [M þ 4]; Anal. Found for C34H20O3N3Cl2I: C, 57.04; H,
2.81; N, 5.85 requires: C, 57.18; H, 2.72; N, 5.97%.
1
cmꢀ1; H NMR (DMSO-d6): d 2.26 (s, 3H, CH3), 2.52 (s, 3H,
CH3), 7.18–8.26 (m, 14H, ArAH), 8.90 (s, 1H, -5H of pyraz-
ole), 12.06 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z):
730 [Mþ], 732 [M þ 2], 734 [M þ 4]; Anal. Found for
C35H22O3N3Cl2I: C, 57.61; H, 3.01; N, 5.78 requires: C,
57.56; H, 3.04; N, 5.75%.
3-(2,4-Dihydroxy-phenyl)-1-[3-(4-chloro-phenyl)-1-phenyl-
1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-10-one
(3e). IR (KBr cmꢀ1/tmax): 3398, 3152, 1676, 1602 cmꢀ1 1H
;
NMR (DMSO-d6): d 2.38 (s, 3H, CH3), 5.68 (s, 1H, OH),
7.16–8.22 (m, 16H, ArAH), 8.90 (s, 1H, -5H of pyrazole),
12.22 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z): 572
[Mþ], 574 [M þ 2]; Anal. Found for C34H22O4N3Cl: C,
71.36; H, 3.81; N, 7.31 requires: C, 71.49; H, 3.95; N,
7.38%.
3-(5-Chloro-2-hydroxy-phenyl)-1-[3-(4-hydroxy-phenyl)-1-
phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanthren-
10-one (3i). IR (KBr cmꢀ1/tmax): 3398, 3169, 1666, 1599
cmꢀ1 1H NMR (DMSO-d6): d 2.38 (s, 3H, CH3), 7.14–8.18
;
(m, 16H, ArAH), 8.86 (s, 1H, -5H of pyrazole), 10.48 (s,
1H, OH), 11.78 (s, 1H, AOH, D2O exchangeable) ppm; MS
3-(5-Chloro-2-hydroxy-4-methyl-phenyl)-1-[3-(4-chloro-phe-
nyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanth-
(m/z): 572 [Mþ], 574 [M
C34H22O4N3Cl: C, 71.36; H, 3.91; N, 7.31 requires: C,
71.51; H, 3.78; N, 7.39%.
þ
2]; Anal. Found for
ren-10-one (3f). IR (KBr cmꢀ1/tmax): 3410, 1670, 1602 cmꢀ1
;
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

January 2010
An Efficient One-Pot Synthesis of Some New 2,4-Diaryl
Pyrido[3,2-c]coumarins as Potent Antimicrobial Agents
241
Acknowledgments. The authors gratefully thank the Principal,
Yeshwant Mahavidyalaya, Nanded, for providing laboratory
facilities. We also thank the Director, IICT, Hyderabad, for pro-
viding necessary instrumental facilities.
3-(3-Bromo-5-chloro-2-hydroxy-phenyl)-1-[3-(4-hydroxy-phe-
nyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanth-
ren-10-one (3j). IR (KBr cmꢀ1/tmax): 3405, 3188, 1672, 1602
cmꢀ1 1H NMR (DMSO-d6): d 2.36 (s, 3H, CH3), 7.16–8.22
;
(m, 15H, ArAH), 8.71 (s, 1H, -5H of pyrazole), 10.61 (s, 1H,
OH), 12.23 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z):
650 [Mþ], 652 [M þ 2], 654 [M þ 4]; Anal. Found for
C34H21O4N3ClBr: C, 62.72; H, 3.21; N, 6.51 requires: C,
62.84; H, 3.35; N, 6.46%.
REFERENCES AND NOTES
3-(5-Chloro-2-hydroxy-3-iodo-phenyl)-1-[3-(4-hydroxy-phe-
nyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanth-
ren-10-one (3k). IR (KBr cmꢀ1/tmax): 3416, 3196, 1675, 1608
[1] Murray, R. D. H.; Mendey, J.; Brown, S. A. The Natural
Coumarins; Wiley: New York, 1982, p 147.
[2] Kennedy, R. O.; Tharnes, R. D. Coumarins: Biology, Appli-
cation and Mode of Action; Wiley: Chichester, 1997, p 1.
[3] Zahradink, M. The Production and Application of Fluores-
cent Brightening Agents; Wiley: New York, 1992, p 284.
[4] Maeda, M. Laser Dyes; Academic Press: New York, 1984,
p 19.
cmꢀ1 1H NMR (DMSO-d6): d 2.38 (s, 3H, CH3), 7.15–8.26
;
(m, 15H, ArAH), 8.84 (s, 1H, -5H of pyrazole), 10.68 (s, 1H,
OH), 12.32 (s, 1H, AOH, D2O exchangeable) ppm; MS (m/z):
698 [Mþ], 700 [M þ 2]; Anal. Found for C34H21O4N3ClI: C,
58.54; H, 3.05; N, 6.01 requires: C, 58.62; H, 3.23; N, 6.14%.
3-(2-Hydroxy-3-iodo-4-methyl-phenyl)-1-[3-(4-hydroxy-phe-
nyl)-1-phenyl-1H-pyrazol-4-yl]-7-methyl-9-oxa-4-aza-phenanth-
ren-10-one (3l). IR (KBr cmꢀ1/tmax): 3396, 3159, 1678, 1604
[5] Thaisrivongs, S.; Janakiraman, M. N.; Chong, K. T.;
Tomich, P. K.; Dolack, L. A.; Turner, S. R.; Strohbach, J. W.; Lynn,
J. C.; Horng, M. M.; Hinshaw, R. R.; Watenpugh, K. D. J Med Chem
1996, 39, 2400.
1
cmꢀ1; H NMR (DMSO-d6): d 2.24 (s, 3H, CH3), 2.42 (s, 3H,
[6] Rappa, G.; Shyam, K.; Lorico, A.; Fodstad, O.; Sartorelli,
A. C. Oncol Res 2000, 12, 113.
CH3), 7.16–8.25 (m, 15H, ArAH), 8.88 (s, 1H, -5H of pyraz-
ole), 10.51 (s, 1H, OH), 12.36 (s, 1H, AOH, D2O exchange-
able) ppm; MS (m/z): 677 [Mþ]; Anal. Found for
C35H24O4N3I: C, 62.01; H, 3.59; N, 6.18 requires: C, 62.18;
H, 3.68; N, 6.11%.
[7] Yang, E. D.; Zhao, Y. N.; Zhang, K.; Mack, P. Biochem
Biophys Res Commun 1999, 260, 682.
[8] Ukawa, K.; Ishiguro, T.; Wada, Y.; Nohara, A. Hetero-
cycles 1986, 24, 1931.
Biology. All the synthesized compounds 3(a-l) were
screened for their in vitro antimicrobial activity by agar well
diffusion method [25]. Antibacterial activity was checked
against bacteria Escherichia coli, Salmonella typhi, Staphylo-
coccus aureus, and Bacillus subtilis. The culture strains of bac-
teria maintained on nutrient agar slant at 37 6 2ꢁC tempera-
ture for 24–48 h. Antifungal activity was studied against As-
pergillus niger, Aspergillus flavus, Penicillium chrysogenum,
and Fusarium moniliforme. The results were compared with
Penicillin and Nystatin, respectively. All the culture strains of
fungi maintained on potato dextrose agar (PDA) slant at 27 6
2ꢁC temperature for 24–28 h, till sporulation. Spore of strains
were transferred in 5 mL of sterile water containing 1%
Tween-80 (to suspend the spore properly). The spores were
counted by Hemocytometer (106 CFU/mL). Sterile plate PDA
was prepared containing 2% agar 0.1 mL of each fungal spore
suspension was spread on each plate and incubated at 27 6
2ꢁC temperature for 12 h. After incubation well prepared using
sterile cork borer and each agar well was filled with 0.1 mL
pyrido coumarin solution of concentration 50, 100, and 250
lg/mL for screening minimum inhibitory concentration (MIC).
Dimethyl sulfoxide (DMSO) was used as control without
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet