In vitro antimicrobial and antimycobacterial activity of some chalcones and their derivatives

Article abstract of DOI:10.1007/s00044-012-0073-3

In this study, novel series of chalcone derivatives, namely, 4-[4-(3-phenyl-acryloyl)-phenylamino]-chromen-2-one (5a-k) have been synthesized from the intermediate 4-(4-acetyl-phenylamino)-chromen-2-one (4). Cyclization reaction of chalcone (5a-k) with hydrazine hydrate, guanidine nitrate, and malononitrile gives the corresponding 4-[4-(1-acetyl-5-phenyl-4,5-dihydro-1H- pyrazol-3-yl)-phenylamino]-chromen-2-one (6a-k), 4-[4-(2-amino-6-phenyl- pyrimidin-4-yl)-phenylamino]-chromen-2-one (7a-k), and 2-amino-6-[4-(2-oxo-2H- chromen-4-ylamino)-phenyl]-4-phenyl-nicotinonitrile (8a-k) derivatives were synthesized. The newly synthesized compounds were evaluated for their antimycobacterial activity and antimicrobial activity against eight bacteria (S. aureus, B. cereus, E. coli, P. aeruginosa, K. pneumoniae, S. typhi, P. vulgaris, and S. flexneri) and four fungi (A. niger, C. albicans, A. fumigatus, and A. clavatus).

Full text of DOI:10.1007/s00044-012-0073-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0073-3
ORIGINAL RESEARCH
In vitro antimicrobial and antimycobacterial activity of some
chalcones and their derivatives
•
Divyesh Patel Premlata Kumari Navin B. Patel
•
Received: 18 January 2012 / Accepted: 19 April 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In this study, novel series of chalcone derivatives,
namely, 4-[4-(3-phenyl-acryloyl)-phenylamino]-chromen-2-
one (5a–k) have been synthesized from the intermediate 4-(4-
acetyl-phenylamino)-chromen-2-one (4). Cyclization reac-
tion of chalcone (5a–k) with hydrazine hydrate, guanidine
nitrate, and malononitrile gives the corresponding 4-[4-(1-
acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-phenylami-
no]-chromen-2-one (6a–k), 4-[4-(2-amino-6-phenyl-pyr-
imidin-4-yl)-phenylamino]-chromen-2-one (7a–k), and
2-amino-6-[4-(2-oxo-2H-chromen-4-ylamino)-phenyl]-4-
phenyl-nicotinonitrile (8a–k) derivatives were synthesized.
The newly synthesized compounds were evaluated for their
antimycobacterial activity and antimicrobial activity against
eight bacteria (S. aureus, B. cereus, E. coli, P. aeruginosa,
K. pneumoniae, S. typhi, P. vulgaris, and S. flexneri) and four
fungi (A. niger, C. albicans, A. fumigatus, and A. clavatus).
wide range of factors leading to the emergence of these
diseases, as well as the increased number of pathogenic
microorganisms with resistance to multiple drugs (Tenover
et al., 2005). To limit the emerging resistance of micro-
organisms, careful use of the existing antimicrobial drugs is
required. However, there is a need for the design of novel
antimicrobial agents, particularly for the treatment of the
infections of the hospitalized patients and the protection of
immunosuppressed or HIV-infected patients. Tuberculosis
(TB) being one of the most common infectious diseases,
mainly caused by Mycobacterium tuberculosis, is a public
health threat in both the developing and the developed
nations. Effective treatment of TB has been hampered by
the emergence of drug-resistant strains of M. tuberculosis,
calling for the search of new drugs (Laughon, 2007).
Chalcones and their derivatives are an attractive
molecular scaffold for the search of new biologically active
molecules. Chalcones or 1,3-diaryl-2-propen-1-ones are
natural or synthetic compounds belonging to the flavonoids
family (Nowakowska, 2007; Zhang et al., 2006). Chalcone
derivatives are very versatile as physiologically active
compounds and substrates for the evaluation of various
organic syntheses. Chalcones have been reported to possess
many useful properties, including anti-inflammatory (Lee
et al., 2006), antimicrobial (Tomar et al., 2007), antifungal
Keywords Coumarin ꢀ Chalcone and their derivatives ꢀ
Antimicrobial activity ꢀ Antimycobacterial activity ꢀ
Structure activity relationship
Introduction
Despite the rapid progress of science, the treatment of
infectious diseases still remains a serious problem of con-
cern to the scientific community, mainly because of the
´
(Lopez et al., 2001), antioxidant (Anto et al., 1995),
antileishmanial, antimalarial (Liu et al., 2003), antitu-
mor (Yi et al., 2000), and anticancer (Hsu et al., 2006)
activities.
D. Patel ꢀ P. Kumari (&)
As part of our continuation study on coumarin-con-
taining derivatives (Divyesh et al., 2011), we have reported
in this article coumarin-based chalcones and their deriva-
tives. The coumarin moiety is distributed widely in nature.
A number of natural products and synthetic analogues
featuring coumarin structural motif display wide-ranging
Department of Applied Chemistry, S.V. National Institute
of Technology, Surat 395007, India
e-mail: premlatakumari1@gmail.com
N. B. Patel
Department of Chemistry, V.N. South Gujarat University,
Surat 395007, India
123

Med Chem Res
biological properties, and they are used as antibacterial
(Khalid et al., 2004), antifungal (Nida et al., 2011), anti-
cancer (Belluti et al., 2010), antiHIV (Bedoya et al., 2005),
antioxidant (Kostova et al., 2011), anti-inflammatory
(Koneni et al., 2011), antibiotics (Hussain et al., 2003),
anticoagulant (Manolov and Danchev, 1995), and antitu-
mor (Al-Soud et al., 2006) agents. Among the wide variety
of heterocycles that have been explored for developing
pharmaceutically important molecules, pyrazoles (Bhat
et al., 2005; Babasaheb et al., 2009), pyrimidines (Kishor
et al., 2008; Amit et al., 2008), and cyanopyridines
(Anjani et al., 2010; Nitin et al., 2011) play an important
role in the medicinal chemistry. Therefore, in light of
above facts, we have reported the reaction of 4-(4-acetyl-
phenylamino)-chromen-2-one with different aldehydes to
form chalcones and their subsequent conversion to pyrazol,
pyrimidine, and cyanopyridine components. All the final
synthesized compounds were tested for their antimicrobial
and antimycobacterial activities.
Furthermore, in the IR spectra, the band at 1,568 cm-1
(C=N) also confirmed the formation of compound 6a. The
presence of singlet signal at 5.31 ppm for two protons of
1
–NH2 in H NMR spectra and the band at 3,385cm-1 for
–NH2 in IR spectrum confirms the formation of pyrimidine
compound 7a. Compound 8a showed singlet peak at
8.05 ppm for one proton of pyridine ring, and the appear-
ance of C:N group at 2,217 cm-1 in IR spectra confirmed
the formation of cyanopyridine ring.
The synthesized compounds 5a–k, 6a–k, 7a–k, and
8a–k were evaluated for in vitro antibacterial and anti-
fungal activities against various Gram-positive, Gram-
negative bacteria, and fungal species. The results are
shown in Tables 1, 2, and 3. Standard antibacterial cipro-
floxacin and antifungal Ketoconazole were also tested
for comparison with synthesized compounds 5a–k, 6a–k,
7a–k, and 8a–k.
A close survey of MIC values indicates that the chalcone
compounds 5a–k exhibited a varied range (25–100 lg/mL)
of antibacterial activity against all the tested bacterial strains
except 5a, which were not having any substituents at ortho,
meta, and para positions of the aryl ring and did not show any
activity against S. aureus, E. coli, P. aeruginosa, P. vulgaris,
andS. flexneri even at a maximum concentrationof100 lg/mL.
Compound 5f with nitro substituent at para position of aryl
ring was also found to be inactive against B. cereus,
P. aeruginosa, and P. vulgaris at 100 lg/mL. Compound 5b
which was having electron-withdrawing chloro substituents
at the ortho-para position of aryl ring shows effective
inhibitory effect in terms of MIC = 25 lg/mL against
S. aureus. Compound 5j which was having electron-donat-
ing methoxy substituent at meta–para position of aryl ring
shows the best activity against S. aureus, E. coli, and
S. flexneri among all the chalcone compounds, 5a–k. Com-
pound 5d with fluoro substituent at the fourth position of aryl
ring showed the highest activity (MIC = 25 lg/mL, 19-mm
zone of inhibition) against B. cereus among compounds,
5a–k. Gram-negative bacteria like P. aeruginosa, K. pneu-
moniae, S. typhi, and P. vulgaris were effectively inhibited
by the compound 5k which contains hydroxy substituent at
ortho–para position of aryl ring.
Results and discussion
Chemistry
The synthetic strategy adopted to obtain the target
compounds is depicted in Scheme 1. 4-Hydroxy-chromen-
2-one (1) on reaction with POCl3 (2) yielded 4-chloro-
chromen-2-one (3) and 4-chloro-3,40,30,400-tercoumarin (3a).
Further on reaction of compound 3 with 1-(4-amino-phenyl)-
ethanone yielded 4-(4-acetyl-phenylamino)-chromen-2-one
(4). Compound 4 when treated with aromatic aldehydes
(a–k) yielded the corresponding chalcone derivatives
5a–k. Cyclization of chalcone derivatives 5a–k with
hydrazine hydrate, guanidine nitrate, and malononitrile led
to the formation of corresponding pyrazol (6a–k), pyrimi-
dine (7a–k), and cyanopyridine (8a–k) derivatives.
The structures of the synthesized compounds were
confirmed by spectral data and elemental analysis, and they
1
were in full agreement with the proposed structures. In H
NMR spectra of compound 4, singlet signal appeared at
8.82 for one proton of –NH and at 2.21 ppm because of
–CH3 of COCH3. Protons corresponding to the coumarin
moiety resonated at 6.11–7.75 ppm. In IR spectra, the
bands at 1,671 cm-1 (C=O of coumarin) and 3,285 cm-1
(–NH) also confirmed the formation of compound 4.
The formation of chalcone 5a was supported by the
appearance of doublet signals at d 8.12 for Ar–CH and d
When chalcones 5a–k were converted to pyrazol (6a–k),
pyrimidine (7a–k), and cyanopyridine (8a–k) derivatives,
then all these derivatives (6a–8k) showed increased anti-
microbial activity.
Among all the synthesized derivatives, cyanopyridine
derivatives (8a–k) showed very good antibacterial activity
compared with chalcones, pyrazol, and pyrimidine deriv-
atives. Some of the pyrazol compounds 6b, 6d, and 6j
displayed the MIC values in the range of 12.5–50 lg/mL
against all the bacterial species. Pyrazol compounds 6b and
6d showed similar MIC = 12.5 lg/mL against S. aureus
and B. cereus, respectively, which is the best MIC value
1
7.05 for –CO–CH in H NMR spectrum, and IR spectrum
for CH=CH–, str. at 1,589 cm-1 also confirmed the for-
mation of compound 5a. The doublet of doublet signals
observed at 3.58 and 3.30 ppm in 1H NMR spectra of
compound 6a was attributed to –CH2 of pyrazol ring.
123

Med Chem Res
Scheme 1 Synthesis of
chalcone and their derivatives
O
Cl
Cl
O
O
O
O
OH
O
Reflux
1h
+
POCl3
2
+
O
O
O
4-Chloro-chromen-2-one
O
4-Hydroxy-chromen-2-one
3
1
4-chloro-3, 4', 3', 4"-tercoumarin
Acetone
Reflux
6-8 h
3a
COCH3
H2N
NH
COCH3
O
O
4-(4-Acetyl-phenylamino)-chromen-2-one
4
DMF/KOH
Room temp.
24 h
Ar-CHO
(a-k)
NH
CO
CH
CH
Ar
O
O
5a-k
Ar
HN
N
N
COCH3
NH2NH2H2O
gl. CH3COOH
O
O
6a-k
NH2
N
N
HN
NH2CNH2 HNO3
Ar
NH
5a-k
Ar
O
O
7a-k
CN
HN
N
NH2
N
N
O
O
8a-k
123

Med Chem Res
Scheme 1 continued
Ar (a-k) – Aromatic aldehydes
OH
CHO
g
a
CHO
Br
b
Cl
CHO
h
CHO
Cl
H3CO
H3CO
CHO
CHO
i
H3C
CHO
c
j
d
CHO
F
H3CO
OH
e
Cl
CHO
HO
CHO
k
f
CHO
O2N
among compounds 6a–k. Only one compound 6k showed
the significant MIC values in the range of 6.25–25 lg/mL
against all Gram-negative bacterial strains. Compound 6k
was also found to be the most active against K. pneumoniae
with excellent MIC = 6.25 lg/mL and maximum inhibi-
tory zone 23 mm among 6a–k.
compound 8j which showed significant MIC values in the
range of 3.12–6.25 lg/mL against all the bacterial species.
Compound 8j showed the highest zone of inhibition of
29 mm with excellent MIC = 3.12 lg/mL against S. aur-
eus along with compound 8b having slightly reduced
inhibition zone (28 mm). Compound 8j was also found to
be the most active against S. flexneri among all the syn-
thesized derivatives. Compound 8d exhibited the greatest
activity (MIC = 3.12 lg/mL, 28-mm inhibitory zone)
against B. cereus. Same compound was found to be the
most potent with similar MIC = 3.12 lg/mL and 29-mm
inhibitory zone against P. aeruginosa and P. vulgaris
among all the synthesized compounds. Compounds 8d and
8i found equipotent in terms of MIC = 3.12 lg/mL against
E. coli, but 8i showed maximum inhibition zone of 28 mm
among all the compounds against E. coli. Strong inhibitory
effect was shown by 8k with MIC = 3.12 lg/mL (30-mm
zone of inhibition) against K. pneumoniae along with
similar efficacy of compounds 8d, 8g, and 8j in terms
of MIC = 3.12 lg/mL against the same bacterial specie.
S. typhi was strongly inhibited by compound 8k with
excellent MIC = 3.12 lg/mL and 28-mm inhibition zone
along with 8j in terms of MIC = 3.12 lg/mL with a quite
reduced inhibition zone of 27 mm.
Pyrimidine derivatives (7a–k) displayed better activity
compared with chalcones and pyrazol derivatives. Pyrimi-
dine compounds 7b, 7d, 7i, and 7k inhibit both the Gram-
positive bacterial strain with similar MIC = 12.5 lg/mL.
Besides compound 7j showed similar activity in terms of
MIC against B. cereus. Compound 7j also found to be the
most potent against S. aureus with the best activity
(MIC = 6.25 lg/mL, 27 mm inhibitory zone) among all the
pyrimidine derivatives (7a–k). In case of activity against
Gram-negative bacterial strains, compounds 7d and 7k
showed excellent MIC = 3.12 lg/mL against K. pneumo-
niae and P. vulgaris, respectively. Compound 7d along with
compound 7j exerted similar activity (MIC = 6.25 lg/mL,
25 mm zone of inhibition) against E. coli.
A close investigation of the MIC values indicates that
cyanopyridine compounds 8b, 8d, 8g, 8i, 8j, and 8k
exhibited a varied range (3.12–12.5 lg/mL) of antibacte-
rial activity against all the tested bacterial species, except
123

Med Chem Res
Table 1 In vitro (Gram-positive) antibacterial activity of newly
synthesized compounds
Table 1 continued
Compound
(100 lg/disk)
Zone of inhibition in mm (MIC in
lg/mL)
Compound
(100 lg/disk)
Zone of inhibition in mm (MIC in
lg/mL)
Gram-positive
Gram-positive
S. aureus
B. cereus
S. aureus
B. cereus
Ciprofloxacin (100 lg/disk)
30 (1.0)
–
31 (1.0)
–
5a
5b
5c
5d
5e
5f
–
\10 (100)
16 (50)
DMSO
20 (25)
Each value is the mean of three independent experiments
14 (100)
18 (50)
15 (100)
19 (25)
– Indicates compound is not active at higher concentration of
100 lg/mL
18 (50)
14 (100)
–
\10 (100)
15 (100)
13 (100)
15(100)
21 (25)
The in vitro antifungal activities of compounds 5a–k,
6a–k, 7a–k, and 8a–k were studied against the fungal
strains viz., A. niger, A. fumigatus, A. clavatus, and
C. albicans. Ketoconazole was used as a standard drug.
The data of antifungal tests are depicted in Table 3. From
the results, we found that chalcone compounds 5a–
k showed less antifungal activity compared with pyrazol
(6a–k), pyrimidine (7a–k), and cyanopyridine (8a–
k) derivatives. Most of the chalcone compounds 5a–k dis-
played MIC in the range of 50–100 lg/mL, except com-
pound 5j which showed MIC at 25 lg/mL against A. niger
and 5k with same MIC against A. fumigatus and A. clav-
atus. In case of pyrazol derivatives, only one compound 6k
displayed good MIC at 12.5 lg/mL against A. fumigatus,
while all other pyrazol compounds showed activity in
terms of MIC in the range of 25–100 lg/mL. Pyrimidine
ring-containing compound 7d inhibits A. clavatus with
MIC = 6.25 lg/mL, 26-mm inhibitory zone along with 7g
and 7k in terms of MIC with quite reduced inhibition zone
(25 mm). Compound 7j found to be the most active against
A. niger and C. albicans among other pyrimidine deriva-
tives, while the best activity was observed against A. fu-
migatus by 7d with MIC = 6.25 lg/mL and 27-mm
inhibitory zone. Cyanopyridine ring-containing compound
8c showed the excellent activity (MIC = 3.12 lg/mL,
28 mm inhibition zone) against C. albicans. Fungal specie
A. fumigatus was strongly inhibited by compounds 8d and
8j with similar activity (MIC = 3.12 lg/mL, 28 mm-
inhibitory zone). Compound 8d was also found to be the
most potent against A. clavatus with MIC = 6.25 lg/mL
and 28-mm inhibitory zone. Besides, the two compounds
8g and 8k showed similar MIC with quite reduced inhib-
itory zone of 27 mm. Among all the cyanopyridine (8a–
k) compounds, only compound 8j inhibited A. niger at
6.25 lg/mL, while others showed MIC in the range of
12.5–25 lg/mL against same fungi.
5g
5h
5i
13 (100)
11 (100)
13 (100)
19 (50)
5j
5k
6a
6b
6c
6d
6e
6f
20 (50)
16 (100)
13 (100)
21 (12.5)
16 (100)
22 (12.5)
17 (100)
11 (100)
16 (100)
14 (100)
15 (100)
18 (50)
\10 (100)
22 (12.5)
16 (100)
23 (12.5)
19 (50)
12 (100)
17 (100)
16 (100)
16 (100)
20 (25)
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
7f
19 (50)
20 (50)
13 (100)
24 (12.5)
18 (50)
15 (100)
22 (12.5)
19 (50)
24 (12.5)
20 (50)
23 (12.5)
19 (50)
15 (100)
18 (50)
16 (100)
19 (50)
7g
7h
7i
17 (100)
22 (12.5)
27 (6.25)
21 (12.5)
17 (50)
16 (100)
21 (12.5)
20 (12.5)
21 (12.5)
19 (50)
7j
7k
8a
8b
8c
8d
8e
8f
28 (3.12)
21 (25)
26 (6.25)
23 (25)
25 (6.25)
24 (12.5)
17 (50)
28 (3.12)
21 (25)
22 (25)
8g
8h
8i
21 (12.5)
23 (25)
22 (12.5)
20 (25)
In vitro antimycobacterial activity of all compounds was
assessed against M. tuberculosis H37 RV. The results
observed from Lowenstein Jensen MIC method along with
the measurement of the potency of the standard drugs are
27 (6.25)
29 (3.12)
22 (12.5)
27 (6.25)
26 (6.25)
26 (6.25)
8j
8k
123

Med Chem Res
Table 2 In vitro (Gram-negative) antibacterial activity of newly synthesized compounds
Compound
(100 lg/disk)
Zone of inhibition in mm (MIC in lg/mL)
Gram-negative
E. coli
P. aeruginosa
K. pneumoniae
S. typhi
P. vulgaris
S. flexneri
5a
5b
5c
5d
5e
5f
–
–
\10 (100)
16 (100)
10 (100)
17 (100)
20 (50)
11 (100)
17 (50)
–
–
14 (100)
\10 (100)
18 (50)
\10 (100)
15 (100)
16 (100)
10 (100)
–
19 (50)
13 (100)
18 (50)
13 (100)
17 (100)
15 (100)
14 (100)
13 (100)
11 (100)
14 (100)
16 (100)
21 (25)
19 (50)
18 (50)
14 (100)
18 (50)
12 (100)
\10 (100)
14 (100)
11 (100)
18 (50)
17 (100)
–
11 (100)
19 (50)
13 (100)
13 (100)
12 (100)
15 (100)
20 (25)
5g
5h
5i
15 (100)
\10 (100)
16 (100)
18 (50)
15 (100)
\10 (100)
18 (50)
14 (100)
16 (100)
17 (100)
21 (25)
5j
22 (25)
19 (50)
5k
6a
6b
6c
6d
6e
6f
19 (50)
19 (25)
20 (25)
17 (100)
\10 (100)
17 (50)
\10 (100)
18 (50)
10 (100)
20 (25)
11 (100)
19 (25)
13 (100)
17 (50)
13 (100)
22 (12.5)
20 (50)
12 (100)
20 (50)
16 (100)
17 (50)
13 (100)
23 (12.5)
21 (50)
14 (100)
18 (50)
20 (50)
21 (25)
20 (25)
15 (100)
14 (100)
16 (100)
14 (100)
19 (50)
14 (100)
11 (100)
16 (100)
12 (100)
18 (50)
18 (50)
19 (50)
20 (50)
13 (100)
20 (50)
17 (100)
16 (100)
15 (100)
17 (100)
21 (25)
\10 (100)
17 (100)
15 (100)
20 (50)
13 (100)
18 (50)
6g
6h
6i
16 (100)
17 (100)
22 (25)
14 (100)
16 (100)
23 (12.5)
20 (25)
6j
21 (25)
20 (50)
20 (50)
6k
7a
7b
7c
7d
7e
7f
20 (25)
22 (25)
23 (6.25)
15 (100)
21 (25)
21 (25)
22 (12.5)
15 (100)
25 (6.25)
21 (25)
14 (100)
19 (50)
12 (100)
22 (25)
17 (50)
14 (100)
21 (25)
20 (25)
15 (100)
25 (6.25)
18 (50)
19 (50)
16 (100)
28 (3.12)
22 (12.5)
18 (50)
18 (50)
22 (25)
24 (12.5)
16 (100)
16 (100)
18 (50)
23 (12.5)
20 (25)
25 (6.25)
23 (12.5)
14 (100)
19 (50)
21 (12.5)
21 (25)
17 (100)
19 (50)
21 (25)
16 (100)
20 (25)
7g
7h
7i
21 (12.5)
18 (50)
17 (100)
20 (50)
16 (100)
22 (25)
15 (100)
21 (12.5)
23 (12.5)
23 (12.5)
18 (50)
17 (100)
24 (12.5)
21 (25)
19 (50)
20 (25)
19 (50)
19 (50)
7j
25 (6.25)
22 (12.5)
19 (50)
23 (12.5)
26 (6.25)
21 (25)
22 (12.5)
24 (12.5)
23 (12.5)
24 (12.5)
21 (25)
24 (12.5)
23 (12.5)
17 (50)
7k
8a
8b
8c
8d
8e
8f
28 (3.12)
19 (25)
20 (12.5)
20 (25)
25 (6.25)
22 (12.5)
29 (3.12)
19 (50)
23 (12.5)
18 (50)
28 (3.12)
24 (12.5)
29 (3.12)
26 (6.25)
19 (50)
23 (12.5)
25 (12.5)
24 (12.5)
23 (12.5)
20 (50)
27 (3.12)
21 (25)
28 (3.12)
25 (6.25)
19 (25)
26 (6.25)
24 (12.5)
23 (12.5)
25 (6.25)
22 (25)
22 (25)
20 (50)
8g
8h
8i
24 (12.5)
18 (50)
24 (12.5)
19 (50)
28 (3.12)
23 (12.5)
22 (12.5)
28 (3.12)
30 (3.12)
22 (12.5)
20 (25)
21 (12.5)
22 (12.5)
24 (12.5)
27 (6.25)
26 (6.25)
28 (3.12)
26 (6.25)
24 (12.5)
25 (6.25)
25 (6.25)
27 (6.25)
22 (12.5)
27 (3.12)
28 (3.12)
26 (6.25)
25 (6.25)
23 (12.5)
8j
8k
123

Med Chem Res
Table 2 continued
Compound
(100 lg/disk)
Zone of inhibition in mm (MIC in lg/mL)
Gram-negative
E. coli
P. aeruginosa
K. pneumoniae
S. typhi
P. vulgaris
S. flexneri
Ciprofloxacin
(100 lg/disk)
32 (1.0)
33 (1.0)
33 (1.0)
30 (1.0)
31 (1.0)
32 (B3)
DMSO
–
–
–
–
–
–
Each value is the mean of three independent experiments
– Indicates compound is not active at higher concentration of 100 lg/mL
1
shown in Table 4. The results revealed that compounds 7d,
7i, 7j, 8b, 8d, 8g, 8i, and 8j showed MIC \100 lg/mL.
Compound 8j which was having methoxy substituents on
meta–para position of aryl ring exerted the best
MIC = 12.5 lg/mL with 99 % inhibition among all the
synthesized compounds. All the remaining derivatives
were found to exert higher MIC ranging from 100 to
[1,000 lg/mL.
and spots were visualized under UV irradiation. H NMR
and 13C NMR spectra were recorded on a Varian 400 MHz
model spectrometer (Varian India Pvt. Ltd., Mumbai,
India) using DMSO as a solvent and TMS as internal
standard with 1H and 13C resonant frequencies of 400 MHz
and 100 MHz, respectively. The H NMR and 13C NMR
1
chemical shifts were reported as parts per million (ppm)
downfield from TMS (Me4Si) and were performed at the
Centre for Excellence, Vapi, India. The splitting patterns
are designated as follows: s, singlet; br s, broad singlet; d,
doublet; t, triplet; q, quartet; and m, multiplet. The mass
spectra were recorded on JEOL SX-102 (EI) model. All
new compounds were subjected to elemental analysis using
a Heraeus Carlo Erba 1180 CHN analyzer (Hanau,
Germany).
Conclusion
In conclusion, the objectives of the present study was to
synthesize and investigate the antimicrobial and antimy-
cobacterial activities of some new chalcones and their
derivatives (pyrazol, pyrimidine, and cyanopyridine) with
the hope of discovering new structure leads serving as
potent antimicrobial and antimycobacterial agents. From
the results of antimicrobial and antimycobacterial activi-
ties, we found that cyanopyridine derivatives (8a–
k) showed better activities compared with chalcone, pyr-
azol, and pyrimidine derivatives. We can also conclude that
biological activities may be associated with the nature of
the tested bacterial, fungal, M. tuberculosis strain, and it
also depends on the substituent on aryl ring at chalcone,
pyrazol, pyrimidine, and cyanopyridine moieties.
Synthesis of 4-chloro-chromen-2-one (3)
4-Hydroxycoumarin 1 (30 g, 0.185 mol) and 60 mL POCl3
were refluxed for 1 h, cooled, and slowly poured into
crushed ice (700 g) with vigorous stirring. The solid was
collected by filtration and washed successively with ice
water. Azeotropic distillation with n-hexane, hot filtration
of the by-product (15 g, 17 %), followed by evaporation of
solvent and crystallization yielded 4-chloro coumarin
´
(21.9 g, 65 %) with m.p. in the range of 87–89 °C (Kovac
et al., 2001).
Experimental section
4-Chloro-3,40,30,400-tercoumarin (by-product) (3a)
Chemicals and solvents were obtained from commercial
sources and used as received throughout the investigation.
Melting points were determined in open capillaries on a
Veego electronic apparatus VMP-D (Veego Instrument
Corporation, Mumbai, India) and are uncorrected. IR
spectra (4,000–400 cm-1) of the synthesized compounds
were recorded on a Shimadzu 8400-S FT-IR spectropho-
tometer (Shimadzu India Pvt. Ltd., Mumbai, India) using
KBr pellets. Thin layer chromatography was performed on
object glass slides (2 9 7.5 cm) coated with silica gel-G,
Crystallization from acetic acid gave yellowish crystals,
´
m.p. in the range of 322–327 °C (Kovac et al., 2001).
Synthesis of 4-(4-acetyl-phenylamino)-chromen-
2-one (4)
4-Chloro-chromen-2-one (10.0 g, 0.055 mol) and 1-(4-
amino-phenyl)-ethanone (7.48 g, 0.055 mol) were dis-
solved in acetone (250 mL). The reaction mixture
was refluxed for 6–8 h. Sodium carbonate was added to
123

Med Chem Res
Table 3 In vitro antifungal
activity of newly synthesized
compounds
Compound
(100 lg/disk)
Zone of inhibition in mm (MIC in lg/mL)
A. niger
A. fumigatus
A. clavatus
C. albicans
5a
5b
5c
5d
5e
5f
\10 (100)
15 (100)
14 (100)
17 (50)
12 (100)
17 (50)
15 (100)
14 (100)
\10 (100)
15 (100)
14 (100)
12 (100)
11 (100)
12 (100)
18 (50)
\10 (100)
12 (100)
11 (100)
13 (100)
15 (100)
10 (100)
13 (100)
18 (50)
13 (100)
19 (50)
12 (100)
11 (100)
16 (100)
12 (100)
20 (50)
14 (100)
\10 (100)
17 (50)
5g
5h
5i
15 (100)
16 (100)
17 (50)
16 (100)
17 (100)
16 (100)
12 (100)
15 (100)
14 (100)
16 (50)
5j
21 (25)
16 (100)
20 (25)
5k
6a
6b
6c
6d
6e
6f
19 (50)
22 (25)
\10 (100)
17 (100)
19 (50)
13 (100)
19 (50)
16 (100)
16 (100)
11 (100)
20 (50)
15 (100)
22 (25)
20 (25)
16 (100)
14 (100)
19 (50)
17 (50)
15 (100)
17 (50)
19 (50)
13 (100)
18 (25)
15 (100)
16 (100)
21 (25)
6g
6h
6i
20 (50)
15 (100)
21 (25)
17 (50)
14 (100)
20 (25)
18 (50)
19 (50)
6j
23 (25)
19 (50)
20 (50)
22 (25)
6k
7a
7b
7c
7d
7e
7f
21 (25)
24 (12.5)
19 (50)
22 (25)
17 (50)
17 (50)
21 (25)
17 (50)
20 (25)
22 (12.5)
18 (50)
19 (50)
21 (25)
21 (25)
16 (100)
26 (6.25)
17 (50)
16 (50)
22 (12.5)
17 (50)
27 (6.25)
19 (25)
20 (25)
21 (12.5)
19 (50)
17 (50)
16 (100)
23 (12.5)
19 (50)
20 (25)
7g
7h
7i
21 (25)
25 (6.25)
17 (50)
18 (50)
20 (50)
23 (25)
23 (12.5)
27 (6.25)
22 (12.5)
21 (25)
20 (25)
23 (12.5)
23 (12.5)
25 (6.25)
23 (12.5)
22 (25)
24 (25)
7j
25 (12.5)
25 (12.5)
21 (25)
26 (6.25)
21 (25)
7k
8a
8b
8c
8d
8e
8f
19 (25)
22 (12.5)
24 (12.5)
23 (12.5)
20 (25)
23 (12.5)
21 (25)
23 (12.5)
28 (3.12)
22 (12.5)
26 (6.25)
23 (12.5)
24 (25)
20 (25)
28 (3.12)
24 (12.5)
20 (25)
28 (6.25)
21 (25)
23 (25)
22 (25)
8g
8h
8i
25 (12.5)
22 (25)
25 (6.25)
22 (12.5)
21 (12.5)
28 (3.12)
25 (6.25)
29 (1.0)
27 (6.25)
19 (25)
24 (25)
25 (12.5)
27 (6.25)
22 (12.5)
30 (B3)
25 (6.25)
25 (6.25)
27 (6.25)
31 (1.0)
25 (12.5)
27 (6.25)
23 (12.5)
33 (1.0)
8j
Each value is the mean of three
independent experiments
8k
Ketoconazole
(100 lg/disk)
– Indicates compound is not
active at higher concentration of
100 lg/mL
DMSO
–
–
–
–
123

Med Chem Res
neutralize HCl evolved during the reaction mixture. After
completion of the reaction, the mixture was cooled and
poured into crushed ice. The solid was separated, filtered,
washed with water, dried, and recrystallized from ethanol
to give compound 4.
Table 4 In vitro antimycobacterial activity of newly synthesized
compounds
Compound
MIC in lg/mL
% Inhibition
5a
1,000
1,000
500
250
1,000
1,000
500
500
250
200
500
1,000
500
200
100
250
500
250
200
100
100
250
500
200
200
62.5
200
500
250
100
62.5
50
95
95
96
97
95
95
97
97
96
97
95
95
96
98
98
97
96
98
98
99
98
97
95
98
98
99
97
96
97
98
98
98
97
96
99
98
97
98
96
97
98
96
99
97
99
99
99
99
Yield: 82%. M.p. [300 °C; IR (KBr, cm-1): 3,285
(–NH), 1,671 (C=O of coumarin), 1,633 (C=O of COCH3);
1H NMR (DMSO-d6, d, ppm): 8.82 (s, 1H, –NH), 7.75 (d,
J = 7.2 Hz, 1H, coumarin), 7.42–7.10 (m, 4H, Ar–H),
6.68–6.49 (m, 3H, coumarin), 6.11 (s, 1H, coumarin), 2.21
(s, 1H, –CH3); 13C NMR (DMSO-d6, d, ppm): 193.21
(C=O of COCH3), 162.37 (C=O of coumarin), 149.57
(C–NH at coumarin), 147.55, 143.27, 133.10, 132.77,
129.67, 129.25, 124.92, 124.89, 119.36, 118.76, 118.70,
117.15, 92.14 (13C, Ar–C), 27.35 (–CH3 of COCH3); EMI-
MS (m/z): 280.19 (M?); Anal. Calcd for C17H13NO3: C,
73.11; H, 4.69; N, 5.02. Found: C, 73.16; H, 4.64; N, 5.08.
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
6a
6b
6c
6d
Synthesis of 4-[4-(3-phenyl-acryloyl)-phenylamino]-
chromen-2-one (5a)
6e
6f
Compound 4 (4.0 g, 0.014 mol) was dissolved in DMF
(50 mL), and benzaldehyde (1.54 g, 0.014 mol) was added
with constant stirring at room temperature. Then KOH
solution (40 wt%) was added to reaction mixture which
was stirred for 24 h at room temperature. After completion
of the reaction, the mixture was poured into crushed ice
and neutralized with HCl. The product was separated out,
filtered, washed with water, dried, and recrystallized from
ethanol to give compound 5a. Similarly, other compounds
5(b–k) were synthesized.
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
Yield: 79 %. M.p. 145–147 °C; IR (KBr, cm-1): 3,281
(–NH), 1,668 (C=O of coumarin), 1,641 (C=O), 1,589
7f
7g
1
(–CH=CH–, str.); H NMR (DMSO-d6, d, ppm): 8.79 (s,
7h
1H, –NH), 8.12 (d, J = 7.5 Hz, 1H, Ar–CH=), 7.78 (d,
J = 7.1 Hz, 1H, coumarin), 7.75–7.15 (m, 9H, Ar–H), 7.05
(d, J = 7.2 Hz, 1H, –CO–CH=), 6.71–6.53 (m, 3H, cou-
marin), 6.15 (s, 1H, coumarin); 13C NMR (DMSO-d6, d,
ppm): 192.87 (C=O), 162.49 (C=O of coumarin), 149.69
(C–NH at coumarin), 141.56 (Ar–CH=), 122.29 (C=O–
CH=), 147.52, 143.36, 133.17, 132.65, 131.25, 130.51,
130.34, 130.13, 129.73, 129.31, 128.29, 128.17, 124.85,
124.77, 119.42, 118.78, 118.63, 117.21, 92.38 (19C, Ar–
C); EMI-MS (m/z): 368.33 (M?); Anal. Calcd for
C24H17NO3: C, 78.46; H, 4.66; N, 3.81. Found: C, 78.38;
H, 4.61; N, 3.88.
7i
7j
7k
200
500
25
8a
8b
8c
100
50
8d
8e
100
250
25
8f
8g
8h
100
62.5
12.5
100
3.12
6.25
0.25
0.20
8i
8j
4-{4-[3-(2,4-Dichloro-phenyl)-acryloyl]-
8k
phenylamino}-chromen-2-one (5b)
Ethambutol
Pyrazinamide
Rifampicin
Isoniazid
Yield: 75 %. M.p. 156–158 °C; IR (KBr, cm-1): 3,283
(–NH), 1,670 (C=O of coumarin), 1,645 (C=O), 1,592
(–CH=CH-, str.), 793 (–Cl); 1H NMR (DMSO-d6, d, ppm):
123

Med Chem Res
4-{4-[3-(4-Chloro-phenyl)-acryloyl]-
8.85 (s, 1H, –NH), 8.15 (d, J = 7.2 Hz, 1H, Ar–CH=), 7.82
(d, J = 7.2 Hz, 1H, coumarin), 7.78–7.12 (m, 7H, Ar–H),
7.08 (d, J = 7.4 Hz, 1H, –CO–CH=), 6.68–6.50 (m, 3H,
coumarin), 6.13 (s, 1H, coumarin); 13C NMR (DMSO-d6,
d, ppm): 192.95 (C=O), 162.57 (C=O of coumarin), 149.55
(C–NH at coumarin), 141.51 (Ar–CH=), 134.15 (C–Cl),
133.19 (C–Cl), 122.43 (C=O–CH=), 147.23, 142.98,
132.94, 132.74, 131.20, 130.43, 130.28, 129.40, 128.15,
128.11, 124.80, 124.69, 119.25, 118.65, 118.55, 117.29,
92.18 (17C, Ar–C); EMI-MS (m/z): 437.16 (M?); Anal.
Calcd for C24H15Cl2NO3: C, 66.07; H, 3.47; N, 3.21.
Found: C, 66.13; H, 3.41; N, 3.29.
phenylamino}-chromen-2-one (5e)
Yield: 76 %. M.p. 163–165 °C; IR (KBr, cm-1): 3,291
(–NH), 1,670 (C=O of coumarin), 1,639 (C=O), 1,590
(–CH=CH–, str.), 810 (–Cl); 1H NMR (DMSO-d6, d, ppm):
8.80 (s, 1H, –NH), 8.11 (d, J = 7.6 Hz, 1H, Ar–CH=), 7.81
(d, J = 7.1 Hz, 1H, coumarin), 7.77–7.12 (m, 8H, Ar–H),
7.10 (d, J = 7.4 Hz, 1H, –CO–CH=), 6.67–6.48 (m, 3H,
coumarin), 6.11 (s, 1H, coumarin); 13C NMR (DMSO-d6,
d, ppm): 192.91 (C=O), 162.70 (C=O of coumarin), 149.61
(C–NH at coumarin), 141.52 (Ar–CH=), 133.45 (C–Cl),
122.58 (C=O–CH=), 147.88, 143.22, 133.24, 132.61,
131.29, 130.69, 130.22, 129.78, 129.25, 128.50, 128.23,
124.80, 124.67, 119.21, 118.86, 118.53, 117.41, 92.45
(18C, Ar–C); EMI-MS (m/z): 402.72 (M?); Anal. Calcd for
C24H16ClNO3: C, 71.73; H, 4.01; N, 3.49. Found: C, 71.66;
H, 4.07; N, 3.44.
4-[4-(3-p-Tolyl-acryloyl)-phenylamino]-chromen-
2-one (5c)
Yield: 68 %. M.p. 183–185 °C; IR (KBr, cm-1): 3,278
(–NH), 1,671 (C=O of coumarin), 1,644 (C=O), 1,587
(–CH=CH–, str.); 1H NMR (DMSO-d6, d, ppm): 8.83 (s, 1H,
–NH), 8.12 (d, J = 7.5 Hz, 1H, Ar–CH=), 7.85 (d,
J = 7.5 Hz, 1H, coumarin), 7.81–7.10 (m, 8H, Ar–H), 7.04
(d, J = 7.2 Hz, 1H, –CO–CH=), 6.65–6.47 (m, 3H, cou-
marin), 6.16 (s, 1H, coumarin), 2.15 (s, 3H, Ph–CH3); 13C
NMR (DMSO-d6, d, ppm): 193.10 (C=O), 162.55 (C=O of
coumarin), 149.43 (C–NH at coumarin), 141.24 (Ar–CH=),
138.54 (C–CH3 of phenyl), 122.47 (C=O–CH=), 147.14,
142.96, 133.29, 132.75, 131.21, 130.59, 129.96, 129.70,
129.26, 128.45, 128.13, 124.81, 124.70, 119.35, 118.63,
118.60, 117.14, 92.58 (18C, Ar–C), 21.29 (C–CH3 of
phenyl); EMI-MS (m/z): 382.57 (M?); Anal. Calcd for
C25H19NO3: C, 78.72; H, 5.02; N, 3.67. Found: C, 78.75;
H, 5.07; N, 3.62.
4-{4-[3-(4-Nitro-phenyl)-acryloyl]-phenylamino}-
chromen-2-one (5f)
Yield: 70 %. M.p. 172–174 °C; IR (KBr, cm-1): 3,287 (–NH),
1,666 (C=O of coumarin), 1,585 (–CH=CH–, str.), 1,637
(C=O), 1,545 (N=O str.); 1H NMR (DMSO-d6, d, ppm): 8.86
(s, 1H, –NH), 8.17 (d, J = 7.2 Hz, 1H, Ar–CH=), 7.79 (d,
J = 7.3 Hz, 1H, coumarin), 7.77–7.16 (m, 8H, Ar–H), 7.09
(d, J = 7.2 Hz, 1H, –CO–CH=), 6.70–6.54 (m, 3H, cou-
marin), 6.13 (s, 1H, coumarin); 13C NMR (DMSO-d6, d,
ppm): 193.17 (C=O), 161.97 (C=O of coumarin), 149.80 (C–
NH at coumarin), 148.19 (C–NO2), 141.15 (Ar–CH=),
122.39 (C=O–CH=), 148.12, 142.96, 132.93, 132.56,
131.20, 130.36, 130.15, 129.62, 129.21, 128.12, 128.10,
124.65, 124.52, 119.18, 118.77, 118.50, 117.10, 91.89 (18C,
Ar–C); EMI-MS (m/z): 412.52 (M?); Anal. Calcd for
C24H16N2O5: C, 69.90; H, 3.91; N, 6.79. Found: C, 69.94; H,
3.87; N, 6.71.
4-{4-[3-(4-Fluoro-phenyl)-acryloyl]-phenylamino}-
chromen-2-one (5d)
Yield: 73 %. M.p. 171–173 °C; IR (KBr, cm-1): 3,287
(–NH), 1,674 (C=O of coumarin), 1,593 (–CH=CH–, str.),
4-{4-[3-(2-Hydroxy-phenyl)-acryloyl]-phenylamino}-
chromen-2-one (5g)
1
1,642 (C=O), 1,155 (C–F); H NMR (DMSO-d6, d, ppm):
Yield: 77 %. M.p. 187–189 °C; IR (KBr, cm-1): 3,284
(–NH), 3,241 (–OH), 1,672 (C=O of coumarin), 1,647
8.77 (s, 1H, –NH), 8.16 (d, J = 7.3 Hz, 1H, Ar–CH=), 7.84
(d, J = 7.2 Hz, 1H, coumarin), 7.82–7.14 (m, 8H, Ar–H),
7.08 (d, J = 7.1 Hz, 1H, –CO–CH=), 6.70–6.52 (m, 3H,
coumarin), 6.13 (s, 1H, coumarin); 13C NMR (DMSO-d6,
d, ppm): 192.95 (C=O), 168.22 (C–F), 162.42 (C=O of
coumarin), 149.66 (C–NH at coumarin), 141.33 (Ar–CH=),
122.25 (C=O–CH=), 147.67, 143.15, 133.56, 132.87,
131.13, 130.63, 129.85, 129.68, 129.20, 128.39, 128.10,
124.77, 124.63, 119.26, 118.73, 118.69, 117.35, 92.16
(18C, Ar–C); EMI-MS (m/z): 386.62 (M?); Anal. Calcd for
C24H16FNO3: C, 74.80; H, 4.18; N, 3.63. Found: C, 74.84;
H, 4.13; N, 3.67.
1
(C=O), 1,593 (–CH=CH–, str.); H NMR (DMSO-d6, d,
ppm): 8.83 (s, 1H, –NH), 8.15 (d, J = 7.3 Hz, 1H,
Ar–CH=), 7.75 (d, J = 7.3 Hz, 1H, coumarin), 7.73–7.10
(m, 8H, Ar–H), 7.03 (d, J = 7.5 Hz, 1H, –CO–CH=),
6.67–6.49 (m, 3H, coumarin), 6.13 (s, 1H, coumarin), 4.51
(s, 1H, –OH); 13C NMR (DMSO-d6, d, ppm): 193.25
(C=O), 162.43 (C=O of coumarin), 158.45 (C–OH), 149.54
(C–NH at coumarin), 141.38 (Ar–CH=), 122.20 (C=O–
CH=), 147.45, 143.49, 133.23, 132.70, 131.45, 130.61,
130.54, 129.86, 129.57, 128.56, 128.13, 124.79, 124.66,
123

Med Chem Res
119.32, 118.80, 118.68, 117.33, 92.24 (18C, Ar–C); EMI-
MS (m/z): 384.39 (M?); Anal. Calcd for C24H17NO4: C,
75.19; H, 4.47; N, 3.65. Found: C, 75.26; H, 4.40; N, 3.69.
(s, 3H, –OCH3), 3.81 (s, 3H, –OCH3); 13C NMR (DMSO-
d6, d, ppm): 193.17 (C=O), 162.29 (C=O of coumarin),
160.67 (C–OCH3), 159.27 (C–OCH3), 149.72 (C–NH at
coumarin), 141.27 (Ar–CH=), 122.68 (C=O–CH=), 147.52,
143.11, 133.39, 132.75, 131.33, 130.87, 129.89, 129.24,
128.56, 128.25, 124.74, 124.62, 119.22, 118.65, 118.54,
117.45, 92.11 (17C, Ar–C), 56.43 (–OCH3); EMI-MS
(m/z): 428.45 (M?); Anal. Calcd for C26H21NO5: C, 73.06;
H, 4.95; N, 3.28. Found: C, 73.13; H, 4.91; N, 3.34.
4-{4-[3-(2-Bromo-phenyl)-acryloyl]-phenylamino}-
chromen-2-one (5h)
Yield: 66 %. M.p. 210–212 °C; IR (KBr, cm-1): 3,279
(–NH), 1,669 (C=O of coumarin), 1,645 (C=O), 1,588
(–CH=CH–, str.), 731 (–Br); 1H NMR (DMSO-d6, d, ppm):
8.81 (s, 1H, –NH), 8.18 (d, J = 7.1 Hz, 1H, Ar–CH=), 7.79
(d, J = 7.5 Hz, 1H, coumarin), 7.76–7.14 (m, 8H, Ar–H),
7.08 (d, J = 7.1 Hz, 1H, –CO–CH=), 6.70–6.51 (m, 3H,
coumarin), 6.15 (s, 1H, coumarin); 13C NMR (DMSO-d6,
d, ppm): 192.95 (C=O), 161.89 (C=O of coumarin), 149.43
(C–NH at coumarin), 141.20 (Ar–CH=), 122.36 (C=O–
CH=), 121.73 (C–Br), 147.57, 143.33, 133.09, 132.85,
131.21, 130.47, 130.32, 129.53, 129.24, 128.21, 127.97,
124.67, 124.35, 119.45, 118.89, 118.61, 117.45, 92.11
(18C, Ar–C); EMI-MS (m/z): 447.23 (M?); Anal. Calcd for
C24H16BrNO3: C, 64.59; H, 3.61; N, 3.14. Found: C, 64.54;
H, 3.67; N, 3.19.
4-{4-[3-(2,4-Dihydroxy-phenyl)-acryloyl]-
phenylamino}-chromen-2-one (5k)
Yield: 80 %. M.p. 213–216 °C; IR (KBr, cm-1): 3,288 (–NH),
3,271 (–OH), 1,674 (C=O of coumarin), 1,648 (C=O), 1,595
(–CH=CH–, str.); 1H NMR (DMSO-d6, d, ppm): 8.77 (s, 1H,
–NH), 8.45 (s, 1H, –OH), 8.15 (d, J = 7.1 Hz, 1H, Ar–CH=),
7.76 (d, J = 7.3 Hz, 1H, coumarin), 7.72–7.13 (m, 7H, Ar–H),
7.10 (d, J = 7.5 Hz, 1H, –CO–CH=), 6.67–6.48 (m, 3H,
coumarin), 6.13 (s, 1H, coumarin), 4.58 (s, 1H, –OH); 13C
NMR (DMSO-d6, d, ppm): 192.97 (C=O), 162.54 (C=O of
coumarin), 157.63 (C–OH), 156.35 (C–OH), 149.77 (C–NH at
coumarin), 141.43 (Ar–CH=), 122.18 (C=O–CH=), 148.13,
143.55, 133.29, 132.39, 131.45, 130.63, 129.70, 129.25,
128.10, 127.87, 124.82, 124.70, 119.31, 118.56, 118.24,
117.56, 92.17 (17C, Ar–C); EMI-MS (m/z): 400.15 (M?);
Anal. Calcd for C24H17NO5: C, 72.17; H, 4.29; N, 3.51. Found:
C, 72.11; H, 4.20; N, 3.54.
4-{4-[3-(4-Methoxy-phenyl)-acryloyl]-phenylamino}-
chromen-2-one (5i)
Yield: 83 %. M.p. 195–197 °C; IR (KBr, cm-1): 3,290
(–NH), 1,666 (C=O of coumarin), 1,647 (C=O), 1,595
1
(–CH=CH–, str.), 1,275 (–OCH3); H NMR (DMSO-d6, d,
ppm): 8.86 (s, 1H, –NH), 8.15 (d, J = 7.5 Hz, 1H,
Ar–CH=), 7.85 (d, J = 7.5 Hz, 1H, coumarin), 7.80–7.16
(m, 8H, Ar–H), 7.11 (d, J = 7.4 Hz, 1H, –CO–CH=),
6.69–6.50 (m, 3H, coumarin), 6.16 (s, 1H, coumarin), 3.81
(s, 3H, –OCH3); 13C NMR (DMSO-d6, d, ppm): 193.33
(C=O), 162.41 (C=O of coumarin), 160.55 (C–OCH3),
149.53 (C–NH at coumarin), 141.79 (Ar–CH=), 122.65
(C=O–CH=), 147.88, 143.93, 132.87, 132.52, 131.46,
130.64, 130.21, 129.79, 129.38, 128.18, 128.11, 124.72,
124.56, 119.15, 118.82, 118.70, 117.54, 91.98 (18C, Ar–
C), 56.13 (–OCH3); EMI-MS (m/z): 398.25 (M?); Anal.
Calcd for C25H19NO4: C, 75.55; H, 4.82; N, 3.52. Found:
C, 75.59; H, 4.80; N, 3.47.
Synthesis of 4-[4-(1-acetyl-5-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)-phenylamino]-chromen-2-one (6a)
A mixture of compound 5a (2.0 g, 0.005 mol) and hydra-
zine hydrate (0.27 g, 0.005 mol) in 1,4-dioxane (30 mL) in
the presence of glacial acetic acid (10 mL) was refluxed
for 6–8 h. After completion of the reaction, mixture was
cooled and poured into crushed ice, and the product
separated out was filtered, washed with water, dried, and
recrystallized from DMF to give compound 6a. Similarly,
other compounds 6(b–k) were synthesized.
Yield: 74 %. M.p. 223–224 °C; IR (KBr, cm-1): 3,284
(–NH), 1,670 (C=O of coumarin), 1,645 (–COCH3), 1,568
1
(C=N); H NMR (DMSO-d6, d, ppm): 8.83 (s, 1H, –NH),
4-{4-[3-(3,4-Dimethoxy-phenyl)-acryloyl]-
7.81 (d, J = 7.2 Hz, 1H, coumarin), 7.77–7.13 (m, 9H,
Ar–H), 6.69–6.51 (m, 3H, coumarin), 6.16 (s, 1H, cou-
marin), 5.48 (dd, J = 7.3, 1.4 Hz, 1H, –CH–CH2), 3.58
(dd, J = 12.2, 6.1 Hz, 1H, –CH2), 3.30 (dd, J = 12.5,
6.3 Hz, 1H, –CH2), 2.33 (s, 3H, –COCH3); 13C NMR
(DMSO-d6, d, ppm): 171.23 (C=O of COCH3), 162.77
(C=O of coumarin), 157.21 (C=N), 149.67 (C–NH at
coumarin), 148.12, 143.32, 133.39, 132.77, 131.34, 131.13,
130.84, 130.45, 129.85, 129.56, 128.38, 128.11, 125.15,
124.89, 119.53, 118.74, 118.61, 117.29, 92.58 (19C,
phenylamino}-chromen-2-one (5j)
Yield: 75 %. M.p. 225–227 °C; IR (KBr, cm-1): 3,285
(–NH), 1,671 (C=O of coumarin), 1,642 (C=O), 1,591
1
(–CH=CH–, str.), 1,278 (–OCH3); H NMR (DMSO-d6, d,
ppm): 8.83 (s, 1H, –NH), 8.11 (d, J = 7.2 Hz, 1H, Ar–
CH=), 7.83 (d, J = 7.3 Hz, 1H, coumarin), 7.79–7.18
(m, 7H, Ar–H), 7.09 (d, J = 7.5 Hz, 1H, –CO–CH=),
6.66–6.45 (m, 3H, coumarin), 6.19 (s, 1H, coumarin), 3.83
123

Med Chem Res
Ar–C), 63.42 (–CH–Ar), 38.25 (–CH2–CH–Ar), 24.45
(–COCH3); EMI-MS (m/z): 424.38 (M?); Anal. Calcd for
C26H21N3O3: C, 73.74; H, 5.00; N, 9.92. Found: C, 73.71;
H, 5.06; N, 9.86.
(C=N), 1,162 (C–F); 1H NMR (DMSO-d6, d, ppm): 8.80 (s,
1H, –NH), 7.79 (d, J = 7.2 Hz, 1H, coumarin), 7.76–7.11
(m, 8H, Ar–H), 6.71–6.48 (m, 3H, coumarin), 6.11 (s, 1H,
coumarin), 5.44 (dd, J = 7.5, 1.1 Hz, 1H, –CH–CH2), 3.52
(dd, J = 12.4, 6.4 Hz, 1H, –CH2), 3.29 (dd, J = 12.6,
6.2 Hz, 1H, –CH2), 2.30 (s, 3H, –COCH3); 13C NMR
(DMSO-d6, d, ppm): 170.83 (C=O of COCH3), 168.76
(C–F), 162.70 (C=O of coumarin), 157.46 (C=N), 149.42
(C–NH at coumarin), 148.15, 143.39, 133.77, 133.07,
132.94, 131.25, 130.24, 129.97, 129.66, 128.68, 128.51,
124.75, 124.69, 119.58, 118.65, 118.42, 117.52, 92.31
(18C, Ar–C), 62.97 (–CH–Ar), 37.75 (–CH2–CH–Ar),
24.52 (–COCH3); EMI-MS (m/z): 442.56 (M?); Anal.
Calcd for C26H20FN3O3: C, 70.74; H, 4.57; N, 9.52. Found:
C, 70.71; H, 4.52; N, 9.59.
4-{4-[1-Acetyl-5-(2,4-dichloro-phenyl)-4,5-dihydro-
1H-pyrazol-3-yl]-phenylamino}-chromen-2-one (6b)
Yield: 71 %. M.p. 211–212 °C; IR (KBr, cm-1): 3,286
(–NH), 1,671 (C=O of coumarin), 1,647 (–COCH3), 1,565
(C=N), 812 (–Cl); 1H NMR (DMSO-d6, d, ppm): 8.88
(s, 1H, –NH), 7.85 (d, J = 7.4 Hz, 1H, coumarin),
7.80–7.17 (m, 7H, Ar–H), 6.70–6.54 (m, 3H, coumarin),
6.15 (s, 1H, coumarin), 5.42 (dd, J = 7.1, 1.5 Hz, 1H,
–CH–CH2), 3.52 (dd, J = 12.4, 6.4 Hz, 1H, –CH2), 3.34
(dd, J = 12.1, 6.2 Hz, 1H, –CH2), 2.37 (s, 3H, –COCH3);
13C NMR (DMSO-d6, d, ppm): 171.45 (C=O of COCH3),
162.68 (C=O of coumarin), 157.34 (C=N), 149.62 (C–NH
at coumarin), 134.37 (C–Cl), 133.42 (C–Cl), 147.92,
143.12, 133.13, 132.85, 131.24, 130.49, 129.55, 129.46,
128.30, 128.16, 124.90, 124.56, 119.47, 118.70, 118.59,
117.33, 92.29 (17C, Ar–C), 63.25 (–CH–Ar), 38.17
(–CH2–CH–Ar), 24.67 (–COCH3); EMI-MS (m/z): 493.19
(M?); Anal. Calcd for C26H19Cl2N3O3: C, 63.43; H, 3.89;
N, 8.53. Found: C, 63.49; H, 3.96; N, 8.45.
4-{4-[1-Acetyl-5-(4-chloro-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6e)
Yield: 73 %. M.p. 262–264 °C; IR (KBr, cm-1): 3,294
(–NH), 1,672 (C=O of coumarin), 1,642 (–COCH3), 1,563
(C=N), 817 (–Cl); 1H NMR (DMSO-d6, d, ppm): 8.87
(s, 1H, –NH), 7.85 (d, J = 7.4 Hz, 1H, coumarin),
7.81–7.16 (m, 8H, Ar–H), 6.70–6.54 (m, 3H, coumarin),
6.16 (s, 1H, coumarin), 5.52 (dd, J = 7.5, 1.2 Hz, 1H,
–CH–CH2), 3.51 (dd, J = 12.1, 6.6 Hz, 1H, –CH2), 3.34
(dd, J = 12.3, 6.5 Hz, 1H, –CH2), 2.39 (s, 3H, –COCH3);
13C NMR (DMSO-d6, d, ppm): 171.67 (C=O of COCH3),
162.55 (C=O of coumarin), 157.36 (C=N), 149.55 (C–NH
at coumarin), 133.80 (C–Cl), 148.22, 143.53, 133.59,
132.75, 131.49, 130.78, 130.15, 129.89, 129.42, 128.48,
128.27, 125.11, 124.84, 119.35, 118.80, 118.49, 117.52,
91.88 (18C, Ar–C), 63.85 (–CH–Ar), 38.41 (–CH2–CH–
Ar), 25.09 (–COCH3); EMI-MS (m/z): 458.11 (M?); Anal.
Calcd for C26H20ClN3O3: C, 68.20; H, 4.40; N, 9.18.
Found: C, 68.26; H, 4.32; N, 9.14.
4-[4-(1-Acetyl-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenylamino]-chromen-2-one (6c)
Yield: 65 %. M.p. 251–253 °C; IR (KBr, cm-1): 3,281
(–NH), 1,673 (C=O of coumarin), 1,645 (–COCH3), 1,572
1
(C=N); H NMR (DMSO-d6, d, ppm): 8.84 (s, 1H, –NH),
7.88 (d, J = 7.1 Hz, 1H, coumarin), 7.83–7.12 (m, 8H, Ar–
H), 6.68–6.49 (m, 3H, coumarin), 6.14 (s, 1H, coumarin),
5.50 (dd, J = 7.6, 1.4 Hz, 1H, –CH–CH2), 3.53 (dd,
J = 12.1, 6.5 Hz, 1H, –CH2), 3.32 (dd, J = 12.4, 6.1 Hz,
1H, –CH2), 2.31 (s, 3H, –COCH3), 2.19 (s, 3H, Ph–CH3);
13C NMR (DMSO-d6, d, ppm): 170.95 (C=O of COCH3),
162.65 (C=O of coumarin), 157.78 (C=N), 149.63 (C–NH
at coumarin), 138.74 (C–CH3 of phenyl), 147.52, 143.11,
133.43, 132.55, 131.39, 130.47, 129.95, 129.89, 129.76,
128.58, 128.41, 124.78, 124.89, 119.40, 118.33, 118.11,
116.90, 92.38 (18C, Ar–C), 62.91 (–CH–Ar), 38.67
(–CH2–CH–Ar), 24.59 (–COCH3), 21.57 (C–CH3 of phe-
nyl); EMI-MS (m/z): 438.62 (M?); Anal. Calcd for
C27H23N3O3: C, 74.12; H, 5.30; N, 9.60. Found: C, 74.18;
H, 5.35; N, 9.53.
4-{4-[1-Acetyl-5-(4-nitro-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6f)
Yield: 68 %. M.p. 243–244 °C; IR (KBr, cm-1): 3,282
(–NH), 1,669 (C=O of coumarin), 1,640 (–COCH3), 1,552
1
(N=O str.), 1,565 (C=N); H NMR (DMSO-d6, d, ppm):
8.83 (s, 1H, –NH), 7.81 (d, J = 7.2 Hz, 1H, coumarin),
7.78–7.14 (m, 8H, Ar–H), 6.72–6.57 (m, 3H, coumarin),
6.19 (s, 1H, coumarin), 5.43 (dd, J = 7.3, 1.1 Hz, 1H,
–CH–CH2), 3.54 (dd, J = 12.3, 6.2 Hz, 1H, –CH2), 3.26
(dd, J = 12.4, 6.1 Hz, 1H, –CH2), 2.31 (s, 3H, –COCH3);
13C NMR (DMSO-d6, d, ppm): 171.65 (C=O of COCH3),
162.19 (C=O of coumarin), 157.78 (C=N), 149.87 (C–NH
at coumarin), 148.85 (C–NO2), 148.25, 143.16, 133.13,
132.78, 131.45, 130.89, 130.55, 129.74, 129.43, 129.02,
128.34, 123.95, 124.88, 119.56, 119.17, 118.48, 116.70,
4-{4-[1-Acetyl-5-(4-fluoro-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6d)
Yield: 76 %. M.p. 239–241 °C; IR (KBr, cm-1): 3,289
(–NH), 1,677 (C=O of coumarin), 1,643 (–COCH3), 1,569
123

Med Chem Res
92.19 (18C, Ar–C), 62.79 (–CH–Ar), 38.13 (–CH2–CH–
Ar), 23.91 (–COCH3); EMI-MS (m/z): 469.25 (M?); Anal.
Calcd for C26H20N4O5: C, 66.66; H, 4.30; N, 11.96. Found:
C, 66.73; H, 4.38; N, 11.91.
(s, 1H, –NH), 7.88 (d, J = 7.4 Hz, 1H, coumarin),
7.83–7.19 (m, 8H, Ar–H), 6.73–6.55 (m, 3H, coumarin),
6.13 (s, 1H, coumarin), 5.46 (dd, J = 7.4, 1.2 Hz, 1H,
–CH–CH2), 3.85 (s, 3H, –OCH3), 3.54 (dd, J = 12.1, 6.3
Hz, 1H, –CH2), 3.33 (dd, J = 12.4, 6.2 Hz, 1H, –CH2),
2.37 (s, 3H, –COCH3); 13C NMR (DMSO-d6, d, ppm):
170.44 (C=O of COCH3), 162.69 (C=O of coumarin),
159.87 (C–OCH3), 157.82 (C=N), 149.51 (C–NH at cou-
marin), 148.18, 144.12, 132.98, 132.66, 132.12, 131.24,
130.46, 129.80, 129.22, 128.37, 128.31, 124.70, 124.42,
119.51, 118.63, 118.60, 117.51, 92.25 (18C, Ar–C), 63.76
(–CH–Ar), 55.80 (–OCH3), 37.79 (–CH2–CH–Ar), 24.13
(–COCH3); EMI-MS (m/z): 454.23 (M?); Anal. Calcd for
C27H23N3O4: C, 71.51; H, 5.11; N, 9.27. Found: C, 71.57;
H, 5.07; N, 9.34.
4-{4-[1-Acetyl-5-(2-hydroxy-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6g)
Yield: 79 %. M.p. 256–258 °C; IR (KBr, cm-1): 3,286
(–NH), 3,252 (–OH), 1,666 (C=O of coumarin), 1,643
1
(–COCH3), 1,574 (C=N); H NMR (DMSO-d6, d, ppm):
8.85 (s, 1H, –NH), 7.78 (d, J = 7.2 Hz, 1H, coumarin),
7.76–7.13 (m, 8H, Ar–H), 6.70–6.54 (m, 3H, coumarin),
6.15 (s, 1H, coumarin), 5.53 (dd, J = 7.3, 1.5 Hz, 1H,
–CH–CH2), 3.57 (dd, J = 12.3, 6.4 Hz, 1H, –CH2), 3.35
(dd, J = 12.2, 6.1 Hz, 1H, –CH2), 4.58 (s, 1H, –OH) 2.40
(s, 3H, –COCH3); 13C NMR (DMSO-d6, d, ppm): 171.75
(C=O of COCH3), 162.71 (C=O of coumarin), 158.64
(C–OH), 157.40 (C=N), 149.66 (C–NH at coumarin),
147.62, 143.53, 133.50, 133.15, 131.40, 130.88, 129.94,
129.82, 129.51, 128.66, 127.83, 125.99, 124.85, 118.92,
118.86, 118.35, 117.55, 92.66 (18C, Ar–C), 63.49 (–CH–
Ar), 38.44 (–CH2–CH–Ar), 24.13 (–COCH3); EMI-MS (m/
z): 440.31 (M?); Anal. Calcd for C26H21N3O4: C, 71.06; H,
4.82; N, 9.56. Found: C, 71.02; H, 4.88; N, 9.50.
4-{4-[1-Acetyl-5-(3,4-dimethoxy-phenyl)-4,5-dihydro-
1H-pyrazol-3-yl]-phenylamino}-chromen-2-one (6j)
Yield: 72 %. M.p. 281–283 °C; IR (KBr, cm-1): 3,280
(–NH), 1,675 (C=O of coumarin), 1,645 (–COCH3), 1,565
(C=N), 1,285 (–OCH3); 1H NMR (DMSO-d6, d, ppm): 8.85
(s, 1H, –NH), 7.87 (d, J = 7.6 Hz, 1H, coumarin),
7.84–7.21 (m, 7H, Ar–H), 6.70–6.49 (m, 3H, coumarin),
6.17 (s, 1H, coumarin), 5.42 (dd, J = 7.4, 1.3 Hz, 1H,
–CH–CH2), 3.88 (s, 3H, –OCH3), 3.85 (s, 3H, –OCH3) 3.51
(dd, J = 12.3, 6.2 Hz, 1H, –CH2), 3.36 (dd, J = 12.5,
6.2 Hz, 1H, –CH2), 2.28 (s, 3H, –COCH3); 13C NMR
(DMSO-d6, d, ppm): 171.54 (C=O of COCH3), 162.45
(C=O of coumarin), 160.75 (C–OCH3), 159.49 (C–OCH3),
156.90 (C=N), 149.35 (C–NH at coumarin), 148.10,
142.85, 133.31, 132.89, 131.20, 131.16, 130.91, 129.84,
128.73, 128.42, 124.77, 124.55, 119.47, 119.15, 118.69,
117.87, 92.06 (17C, Ar–C), 63.11 (–CH–Ar), 55.84
(–OCH3), 38.59 (–CH2–CH–Ar), 24.87 (–COCH3); EMI-
MS (m/z): 484.49 (M?); Anal. Calcd for C28H25N3O5: C,
69.55; H, 5.21; N, 8.69. Found: C, 69.59; H, 5.14; N, 8.73.
4-{4-[1-Acetyl-5-(2-bromo-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6h)
Yield: 63 %. M.p. 220–221 °C; IR (KBr, cm-1): 3,283
(–NH), 1,675 (C=O of coumarin), 1,641 (–COCH3), 1,569
1
(C=N), 745 (–Br); H NMR (DMSO-d6, d, ppm): 8.83 (s,
1H, –NH), 7.82 (d, J = 7.5 Hz, 1H, coumarin), 7.80–7.17
(m, 8H, Ar–H), 6.67–6.50 (m, 3H, coumarin), 6.21 (s, 1H,
coumarin), 5.41 (dd, J = 7.3, 1.3 Hz, 1H, –CH–CH2), 3.55
(dd, J = 12.3, 6.5 Hz, 1H, –CH2), 3.32 (dd, J = 12.1, 6.3
Hz, 1H, –CH2), 2.29 (s, 3H, –COCH3); 13C NMR (DMSO-
d6, d, ppm): 171.86 (C=O of COCH3), 162.15 (C=O of
coumarin), 156.81 (C=N), 149.88 (C–NH at coumarin),
121.35 (C–Br), 148.22, 142.93, 133.27, 133.15, 131.47,
130.68, 130.14, 129.66, 129.45, 128.40, 128.17, 125.10,
124.63, 119.66, 118.75, 118.52, 118.10, 92.54 (18C,
Ar–C), 62.72 (–CH–Ar), 38.79 (–CH2–CH–Ar), 23.80
(–COCH3); EMI-MS (m/z): 503.24 (M?); Anal. Calcd for
C26H20BrN3O3: C, 62.16; H, 4.01; N, 8.36. Found: C,
62.23; H, 4.07; N, 8.31.
4-{4-[1-Acetyl-5-(2,4-dihydroxy-phenyl)-4,5-dihydro-
1H-pyrazol-3-yl]-phenylamino}-chromen-2-one (6k)
Yield: 77 %. M.p. 266–267 °C; IR (KBr, cm-1): 3,291
(–NH), 3,277 (–OH), 1,669 (C=O of coumarin), 1,650
1
(–COCH3), 1,559 (C=N); H NMR (DMSO-d6, d, ppm):
8.82 (s, 1H, –NH), 8.49 (s, 1H, –OH), 7.80 (d, J = 7.2 Hz,
1H, coumarin), 7.75–7.17 (m, 7H, Ar–H), 6.70–6.50 (m,
3H, coumarin), 6.18 (s, 1H, coumarin), 5.41 (dd, J = 7.2,
1.1 Hz, 1H, –CH–CH2), 4.53 (s, 1H, –OH), 3.55 (dd,
J = 12.5, 6.3 Hz, 1H, –CH2), 3.38 (dd, J = 12.3, 6.2 Hz,
1H, –CH2), 2.29 (s, 3H, –COCH3); 13C NMR (DMSO-d6,
d, ppm): 171.60 (C=O of COCH3), 162.88 (C=O of cou-
marin), 157.82 (C–OH), 157.29 (C–OH), 157.11 (C=N),
4-{4-[1-Acetyl-5-(4-methoxy-phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-phenylamino}-chromen-2-one (6i)
Yield: 81 %. M.p. 247–249 °C; IR (KBr, cm-1): 3,294
(–NH), 1,670 (C=O of coumarin), 1,649 (–COCH3), 1,561
(C=N), 1,282 (–OCH3); 1H NMR (DMSO-d6, d, ppm): 8.84
123

Med Chem Res
4-[4-(2-Amino-6-p-tolyl-pyrimidin-4-yl)-
149.33 (C–NH at coumarin), 148.29, 143.67, 133.45,
132.65, 131.49, 131.12, 130.74, 129.56, 128.75, 128.17,
125.12, 124.83, 119.47, 118.61, 118.30, 117.89, 91.87
(17C, Ar–C), 62.82 (–CH–Ar), 37.96 (–CH2–CH–Ar),
24.07 (–COCH3); EMI-MS (m/z): 456.35 (M?); Anal.
Calcd for C26H21N3O5: C, 68.56; H, 4.65; N, 9.23. Found:
C, 68.51; H, 4.60; N, 9.28.
phenylamino]-chromen-2-one (7c)
Yield: 65 %. M.p. 245–246 °C; IR (KBr, cm-1): 3,391
(–NH2), 3,284 (–NH), 1,671 (C=O of coumarin), 1,568
1
(C=N); H NMR (DMSO-d6, d, ppm): 8.81 (s, 1H, –NH),
7.91 (d, J = 7.4 Hz, 1H, coumarin), 7.86–7.16 (m, 9H,
Ar–H), 6.73–6.56 (m, 3H, coumarin), 6.17 (s, 1H, cou-
marin), 5.33 (s, 2H, –NH2), 2.25 (s, 3H, Ph–CH3); 13C
NMR (DMSO-d6, d, ppm): 162.61 (C=O of coumarin),
159.54, 159.32 (2C, pyrimidine), 155.29 (C–NH2), 149.66
(C–NH at coumarin), 138.15 (C–CH3 of phenyl), 113.55
(1C, pyrimidine), 148.43, 142.92, 133.56, 132.41, 131.35,
130.69, 130.05, 129.81, 129.54, 128.44, 128.12, 125.18,
124.70, 119.59, 118.84, 118.62, 117.46, 93.08 (18C,
Ar–C), 22.07 (C–CH3 of phenyl); EMI-MS (m/z): 421.50
(M?); Anal. Calcd for C26H20N4O2: C, 74.27; H, 4.79; N,
13.33. Found: C, 74.22; H, 4.85; N, 13.38.
Synthesis of 4-[4-(2-amino-6-phenyl-pyrimidin-4-yl)-
phenylamino]-chromen-2-one (7a)
A mixture of compound 5a (2.0 g, 0.005 mol), guanidine
nitrate (0.66 g, 0.005 mol), and 1–2 mL of 40 % KOH
solution in ethanol (30 mL) was refluxed for 10–12 h. The
reaction mixture was cooled, then poured into crushed ice,
and the product separated out was filtered, washed
with water, dried, and recrystallized from DMF to give
compound 7a. Similarly, other compounds 7(b–k) were
synthesized.
4-{4-[2-Amino-6-(4-fluoro-phenyl)-pyrimidin-4-yl]-
Yield: 68 %. M.p. 229–231 °C; IR (KBr, cm-1): 3,385
(–NH2), 3,289 (–NH), 1,673 (C=O of coumarin), 1,561
phenylamino}-chromen-2-one (7d)
1
(C=N); H NMR (DMSO-d6, d, ppm): 8.79 (s, 1H, –NH),
Yield: 75 %. M.p. 279–281 °C; IR (KBr, cm-1): 3,388
(–NH2), 3,276 (–NH), 1,672 (C=O of coumarin), 1,564
(C=N), 1,169 (C–F); 1H NMR (DMSO-d6, d, ppm): 8.78 (s,
1H, –NH), 7.82 (d, J = 7.1 Hz, 1H, coumarin), 7.80–7.09
(m, 9H, Ar–H), 6.68–6.52 (m, 3H, coumarin), 6.14 (s, 1H,
coumarin), 5.39 (s, 2H, –NH2); 13C NMR (DMSO-d6, d,
ppm): 168.53 (C–F), 161.88 (C=O of coumarin), 160.12,
159.86 (2C, pyrimidine), 154.92 (C–NH2), 148.54 (C–NH
at coumarin), 113.59 (1C, pyrimidine), 147.70, 143.40,
133.65, 132.17, 132.14, 131.18, 130.57, 129.84, 129.55,
128.86, 128.43, 124.63, 124.54, 119.63, 118.40, 118.32,
117.56, 92.15 (18C, Ar–C); EMI-MS (m/z): 425.34 (M?);
Anal. Calcd for C25H17FN4O2: C, 70.75; H, 4.04; N, 13.20.
Found: C, 70.79; H, 4.10; N, 13.15.
7.86 (d, J = 7.1 Hz, 1H, coumarin), 7.83–7.11 (m, 10H,
Ar–H), 6.65–6.46 (m, 3H, coumarin), 6.13 (s, 1H, cou-
marin), 5.31 (s, 2H, –NH2); 13C NMR (DMSO-d6, d, ppm):
162.55 (C=O of coumarin), 159.88, 159.56 (2C, pyrimi-
dine), 155.13 (C–NH2), 148.86 (C–NH at coumarin),
113.27 (1C, pyrimidine), 146.99, 142.92, 133.77, 133.16,
131.56, 131.33, 130.24, 130.49, 129.65, 129.22, 128.32,
128.20, 124.93, 124.59, 119.44, 118.69, 118.54, 117.13,
93.24 (19C, Ar–C); EMI-MS (m/z): 407.57 (M?); Anal.
Calcd for C25H18N4O2: C, 73.88; H, 4.46; N, 13.78. Found:
C, 73.81; H, 4.52; N, 13.70.
4-{4-[2-Amino-6-(2,4-dichloro-phenyl)-pyrimidin-4-
yl]-phenylamino}-chromen-2-one (7b)
4-{4-[2-Amino-6-(4-chloro-phenyl)-pyrimidin-4-yl]-
Yield: 72 %. M.p. 257–259 °C; IR (KBr, cm-1): 3,381
(–NH2), 3,284 (–NH), 1,669 (C=O of coumarin), 1,568
phenylamino}-chromen-2-one (7e)
1
(C=N), 819 (–Cl); H NMR (DMSO-d6, d, ppm): 8.86 (s,
Yield: 68 %. M.p.[300 °C; IR (KBr, cm-1): 3,380 (–NH2),
3,291 (–NH), 1,675 (C=O of coumarin), 1,562 (C=N), 832
1H, –NH), 7.87 (d, J = 7.2 Hz, 1H, coumarin), 7.79–7.10
(m, 8H, Ar–H), 6.69–6.49 (m, 3H, coumarin), 6.18 (s, 1H,
coumarin), 5.27 (s, 2H, –NH2); 13C NMR (DMSO-d6, d,
ppm): 162.75 (C=O of coumarin), 159.67, 159.43 (2C,
pyrimidine), 154.83 (C–NH2), 149.27 (C–NH at coumarin),
112.96 (1C, pyrimidine), 135.17 (C–Cl), 134.82 (C–Cl),
147.73, 142.92, 133.46, 132.15, 131.39, 130.55, 129.95,
129.76, 128.66, 127.70, 125.10, 124.96, 119.33, 118.68,
118.38, 117.20, 92.75 (17C, Ar–C); EMI-MS (m/z): 476.22
(M?); Anal. Calcd for C25H16Cl2N4O2: C, 63.17; H, 3.39;
N, 11.79. Found: C, 63.21; H, 3.34; N, 11.73.
1
(–Cl); H NMR (DMSO-d6, d, ppm): 8.89 (s, 1H, –NH),
7.83 (d, J = 7.5 Hz, 1H, coumarin), 7.81–7.19 (m, 9H,
Ar–H), 6.67–6.53 (m, 3H, coumarin), 6.15 (s, 1H, couma-
rin), 5.23 (s, 2H, –NH2); 13C NMR (DMSO-d6, d, ppm):
162.70 (C=O of coumarin), 159.51, 159.23 (2C, pyrimi-
dine), 155.39 (C–NH2), 148.35 (C–NH at coumarin),
113.66 (1C, pyrimidine), 134.10 (C–Cl), 147.80, 142.83,
133.66, 132.45, 131.40, 130.56, 130.11, 129.67, 129.32,
128.80, 128.55, 124.16, 123.84, 119.49, 118.87, 118.43,
117.13, 92.19 (18C, Ar–C); EMI-MS (m/z): 441.72 (M?);
123

Med Chem Res
Anal. Calcd for C25H17ClN4O2: C, 68.11; H, 3.89; N, 12.71.
Found: C, 68.16; H, 3.82; N, 12.75.
128.56, 128.11, 124.80, 124.26, 119.69, 118.73, 118.26,
117.90, 93.03 (18C, Ar–C); EMI-MS (m/z): 486.53 (M?);
Anal. Calcd for C25H17BrN4O2: C, 61.87; H, 3.53; N,
11.54. Found: C, 61.93; H, 3.45; N, 11.59.
4-{4-[2-Amino-6-(4-nitro-phenyl)-pyrimidin-4-yl]-
phenylamino}-chromen-2-one (7f)
4-{4-[2-Amino-6-(4-methoxy-phenyl)-pyrimidin-4-yl]-
Yield: 69 %. M.p. 266–267 °C; IR (KBr, cm-1): 3,388
(–NH2), 3,292 (–NH), 1,674 (C=O of coumarin), 1,555
phenylamino}-chromen-2-one (7i)
1
(N=O str.), 1,559 (C=N); H NMR (DMSO-d6, d, ppm):
Yield: 78 %. M.p.[300 °C; IR (KBr, cm-1): 3,387 (–NH2),
3,285 (–NH), 1,674 (C=O of coumarin), 1,566 (C=N), 1,280
(–OCH3); 1H NMR (DMSO-d6, d, ppm): 8.88 (s, 1H, –NH),
7.83 (d, J = 7.4 Hz, 1H, coumarin), 7.81–7.17 (m, 9H, Ar–
H), 6.69–6.51 (m, 3H, coumarin), 6.10 (s, 1H, coumarin),
5.28 (s, 2H, –NH2), 3.79 (s, 3H, –OCH3); 13C NMR
(DMSO-d6, d, ppm): 162.75 (C=O of coumarin), 160.81
(C–OCH3), 159.53, 159.37 (2C, pyrimidine), 155.31 (C–
NH2), 149.17 (C–NH at coumarin), 113.60 (1C, pyrimi-
dine), 147.82, 143.22, 132.91, 132.86, 131.92, 131.04,
130.57, 129.88, 129.15, 128.83, 128.50, 124.66, 124.43,
119.55, 118.21, 118.19, 117.60, 92.64 (18C, Ar–C), 55.93
(–OCH3); EMI-MS (m/z): 437.55 (M?); Anal. Calcd for
C26H20N4O3: C, 71.55; H, 4.62; N, 12.84. Found: C, 71.59;
H, 4.55; N, 12.81.
8.89 (s, 1H, –NH), 7.85 (d, J = 7.3 Hz, 1H, coumarin),
7.80–7.12 (m, 9H, Ar–H), 6.75–6.56 (m, 3H, coumarin),
6.17 (s, 1H, coumarin), 5.36 (s, 2H, –NH2); 13C NMR
(DMSO-d6, d, ppm): 161.10 (C=O of coumarin), 159.47,
159.13 (2C, pyrimidine), 154.44 (C–NH2), 149.15 (C–NH
at coumarin), 148.24 (C–NO2), 113.60 (1C, pyrimidine),
147.36, 143.52, 133.83, 132.19, 131.56, 130.28, 129.85,
129.44, 129.03, 128.55, 128.11, 124.15, 123.88, 119.38,
118.57, 118.15, 116.84, 92.47 (18C, Ar–C); EMI-MS (m/z):
452.31 (M?); Anal. Calcd for C25H17N5O4: C, 66.51; H,
3.80; N, 15.51. Found: C, 66.44; H, 3.71; N, 15.56.
4-{4-[2-Amino-6-(2-hydroxy-phenyl)-pyrimidin-4-yl]-
phenylamino}-chromen-2-one (7g)
Yield: 75 %. M.p. 293–295 °C; IR (KBr, cm-1): 3,383
(–NH2), 3,278 (–NH), 3,248 (–OH), 1,668 (C=O of cou-
4-{4-[2-Amino-6-(3,4-dimethoxy-phenyl)-pyrimidin-
4-yl]-phenylamino}-chromen-2-one (7j)
1
marin), 1,572 (C=N); H NMR (DMSO-d6, d, ppm): 8.83
(s, 1H, –NH), 7.79 (d, J = 7.3 Hz, 1H, coumarin),
7.76–7.10 (m, 9H, Ar–H), 6.72–6.50 (m, 3H, coumarin),
6.13 (s, 1H, coumarin), 5.26 (s, 2H, –NH2), 4.63 (s, 1H,
–OH); 13C NMR (DMSO-d6, d, ppm): 162.23 (C=O of
coumarin), 159.75, 159.21 (2C, pyrimidine), 158.35
(C–OH), 155.48 (C–NH2), 149.77 (C–NH at coumarin),
113.20 (1C, pyrimidine), 147.27, 143.58, 133.67, 132.15,
131.39, 130.65, 129.87, 129.13, 128.81, 128.51, 127.97,
124.82, 124.33, 119.17, 118.13, 118.05, 117.80, 93.16
(18C, Ar–C); EMI-MS (m/z): 423.26 (M?); Anal. Calcd for
C25H18N4O3: C, 71.08; H, 4.29; N, 13.26. Found: C, 71.14;
H, 4.33; N, 13.21.
Yield: 71 %. M.p. 288–289 °C; IR (KBr, cm-1): 3,379
(–NH2), 3,292 (–NH), 1,671 (C=O of coumarin), 1,568
(C=N), 1,280 (–OCH3); 1H NMR (DMSO-d6, d, ppm): 8.87
(s, 1H, –NH), 7.85 (d, J = 7.1 Hz, 1H, coumarin),
7.80–7.18 (m, 8H, Ar–H), 6.72–6.55 (m, 3H, coumarin),
6.19 (s, 1H, coumarin), 3.87 (s, 3H, –OCH3), 3.82 (s, 3H,
–OCH3) 5.30 (s, 2H, –NH2); 13C NMR (DMSO-d6, d,
ppm): 162.89 (C=O of coumarin), 160.75 (C–OCH3),
160.14 (C–OCH3), 159.44, 159.16 (2C, pyrimidine),
155.65 (C–NH2), 148.90 (C–NH at coumarin), 112.79 (1C,
pyrimidine), 147.82, 142.93, 133.20, 132.53, 131.47,
130.16, 129.67, 129.13, 128.70, 128.38, 124.51, 124.36,
119.53, 119.67, 118.54, 117.79, 93.17 (17C, Ar–C), 56.11
(–OCH3); EMI-MS (m/z): 467.49 (M?); Anal. Calcd for
C27H22N4O4: C, 69.52; H, 4.75; N, 12.01. Found: C, 69.56;
H, 4.70; N, 12.09.
4-{4-[2-Amino-6-(2-bromo-phenyl)-pyrimidin-4-yl]-
phenylamino}-chromen-2-one (7h)
Yield: 69 %. M.p. 250–252 °C; IR (KBr, cm-1): 3,395
(–NH2), 3,281 (–NH), 1,670 (C=O of coumarin), 1,562
4-{4-[2-Amino-6-(2,4-dihydroxy-phenyl)-pyrimidin-
1
(C=N), 757 (-Br); H NMR (DMSO-d6, d, ppm): 8.87 (s,
4-yl]-phenylamino}-chromen-2-one (7k)
1H, –NH), 7.81 (d, J = 7.4 Hz, 1H, coumarin), 7.75–7.11
(m, 9H, Ar–H), 6.74–6.58 (m, 3H, coumarin), 6.19 (s, 1H,
coumarin), 5.22 (s, 2H, –NH2); 13C NMR (DMSO-d6, d,
ppm): 161.84 (C=O of coumarin), 160.17, 159.87 (2C,
pyrimidine), 155.49 (C–NH2), 149.33 (C–NH at coumarin),
113.49 (1C, pyrimidine), 121.88 (C–Br), 147.82, 143.18,
133.87, 132.15, 131.56, 130.87, 130.22, 129.43, 129.32,
Yield: 82 %. M.p. [300 °C; IR (KBr, cm-1): 3,390
(–NH2), 3,295 (–NH), 3,271 (–OH), 1,667 (C=O of cou-
1
marin), 1,562 (C=N); H NMR (DMSO-d6, d, ppm): 8.79
(s, 1H, –NH), 8.54 (s, 1H, –OH), 7.83 (d, J = 7.2 Hz, 1H,
coumarin), 7.76–7.10 (m, 8H, Ar–H), 6.68–6.51 (m, 3H,
coumarin), 6.16 (s, 1H, coumarin), 5.26 (s, 2H, –NH2),
123

Med Chem Res
4.49 (s, 1H, –OH); 13C NMR (DMSO-d6, d, ppm): 162.71
(C=O of coumarin), 160.17, 159.88 (2C, pyrimidine),
158.02 (C–OH), 157.85 (C–OH), 154.89 (C–NH2), 149.72
(C–NH at coumarin), 113.40 (1C, pyrimidine), 148.16,
142.87, 133.41, 132.49, 131.53, 130.18, 130.04, 129.86,
128.90, 128.32, 124.56, 124.12, 119.33, 118.51, 118.32,
117.82, 92.18 (17C, Ar–C); EMI-MS (m/z): 439.13 (M?);
Anal. Calcd for C25H18N4O4: C, 68.49; H, 4.14; N, 12.78.
Found: C, 68.54; H, 4.19; N, 12.72.
C, 64.94; H, 3.23; N, 11.22. Found: C, 64.89; H, 3.27; N,
11.17.
2-Amino-6-[4-(2-oxo-2H-chromen-4-ylamino)-
phenyl]-4-p-tolyl-nicotinonitrile (8c)
Yield: 67 %. M.p. 284–285 °C; IR (KBr, cm-1): 3,394
(–NH2), 3,290 (–NH), 2,215 (C:N), 1,668 (C=O of cou-
1
marin); H NMR (DMSO-d6, d, ppm): 8.87 (s, 1H, –NH),
8.11 (s, 1H, pyridine), 7.83 (d, J = 7.2 Hz, 1H, coumarin),
7.78–7.12 (m, 8H, Ar–H), 6.67–6.50 (m, 3H, coumarin),
6.14 (s, 1H, coumarin), 5.37 (s, 2H, –NH2), 2.11 (s, 3H,
Ph–CH3); 13C NMR (DMSO-d6, d, ppm): 162.89 (1C,
pyridine), 162.70 (C=O of coumarin), 156.81 (1C, pyri-
dine), 156.43 (C–NH2), 149.17 (C–NH at coumarin),
138.87 (C–CH3 of phenyl), 116.38 (C:N), 115.41 (1C,
pyridine), 148.12, 143.16, 133.49, 132.40, 131.90, 130.08,
129.85, 129.77, 129.15, 128.67, 128.11, 125.11, 124.79,
119.69, 118.57, 118.44, 117.10, 93.07 (18C, Ar–C), 22.13
(C–CH3 of phenyl), 85.91 (C–C:N); EMI-MS (m/z):
445.20 (M?); Anal. Calcd for C28H20N4O2: C, 75.66; H,
4.54; N, 12.60. Found: C, 75.74; H, 4.61; N, 12.52.
Synthesis of 2-amino-6-[4-(2-oxo-2H-chromen-4-
ylamino)-phenyl]-4-phenyl-nicotinonitrile (8a)
A mixture of 5a (2.0 g, 0.005 mol), malononitrile (0.33 g,
0.005 mol), and ammonium acetate (0.77 g, 0.01) in eth-
anol (30 mL) was refluxed for 8–10 h. After cooling, the
reaction mixture was poured into crushed ice. The product
separated out was filtered, washed with water, dried, and
recrystallized from DMF to give compound 8a. Similarly,
other compounds 8(b–k) were synthesized.
Yield: 73 %. M.p. 280–281 °C; IR (KBr, cm-1): 3,391
(–NH2), 3,287 (–NH), 2,217 (C:N), 1,670 (C=O of cou-
1
marin); H NMR (DMSO-d6, d, ppm): 8.82 (s, 1H, –NH),
8.05 (s, 1H, pyridine), 7.82 (d, J = 7.4 Hz, 1H, coumarin),
7.89–7.17 (m, 9H, Ar–H), 6.74–6.59 (m, 3H, coumarin),
6.20 (s, 1H, coumarin), 5.38 (s, 2H, –NH2); 13C NMR
(DMSO-d6, d, ppm): 163.25 (1C, pyridine), 162.63 (C=O
of coumarin), 157.11 (1C, pyridine), 156.13 (C–NH2),
149.28 (C–NH at coumarin), 116.29 (C:N), 115.69 (1C,
pyridine), 148.56, 143.50, 133.10, 131.95, 131.55, 130.21,
130.17, 130.11, 129.94, 129.53, 128.80, 128.57, 125.15,
124.89, 119.31, 118.70, 118.57, 117.28, 93.15 (19C, Ar–
C), 86.13 (C–C:N); EMI-MS (m/z): 431.52 (M?); Anal.
Calcd for C27H18N4O2: C, 75.34; H, 4.21; N, 13.02. Found:
C, 75.30; H, 4.26; N, 13.08.
2-Amino-4-(4-fluoro-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8d)
Yield: 77 %. M.p. 297–299 °C; IR (KBr, cm-1): 3,383
(–NH2), 3,281 (–NH), 2,206 (C:N), 1,669 (C=O of cou-
marin), 1,167 (C–F); 1H NMR (DMSO-d6, d, ppm): 8.80 (s,
1H, –NH), 8.03 (s, 1H, pyridine), 7.85 (d, J = 7.3 Hz, 1H,
coumarin), 7.83–7.15 (m, 8H, Ar–H), 6.67–6.49 (m, 3H,
coumarin), 6.15 (s, 1H, coumarin), 5.27 (s, 2H, –NH2); 13
C
NMR (DMSO-d6, d, ppm): 168.79 (C–F), 163.10 (1C,
pyridine), 162.50 (C=O of coumarin), 157.23 (1C, pyri-
dine), 155.70 (C–NH2), 149.72 (C–NH at coumarin),
116.47 (C:N), 115.55 (1C, pyridine), 148.15, 142.95,
133.91, 133.06, 131.45, 130.13, 129.48, 129.10, 128.90,
128.56, 127.10, 124.80, 124.51, 119.52, 118.71, 118.66,
117.07, 92.22 (18C, Ar–C), 86.28 (C–C:N); EMI-MS (m/
z): 449.28 (M?); Anal. Calcd for C27H17FN4O2: C, 72.31;
H, 3.82; N, 12.49. Found: C, 72.24; H, 3.73; N, 12.57.
2-Amino-4-(2,4-dichloro-phenyl)-6-[4-(2-oxo-
2H-chromen-4-ylamino)-phenyl]-nicotinonitrile (8b)
Yield: 71 %. M.p. 246–248 °C; IR (KBr, cm-1): 3,386
(–NH2), 3,283 (–NH), 2,211 (C:N), 1,674 (C=O of cou-
marin), 804 (–Cl); 1H NMR (DMSO-d6, d, ppm): 8.89
(s, 1H, –NH), 8.09 (s, 1H, pyridine), 7.84 (d, J = 7.1 Hz,
1H, coumarin), 7.80–7.20 (m, 7H, Ar–H), 6.71–6.53 (m,
3H, coumarin), 6.11 (s, 1H, coumarin), 5.32 (s, 2H, –NH2);
13C NMR (DMSO-d6, d, ppm): 163.57 (1C, pyridine),
162.34 (C=O of coumarin), 157.47 (1C, pyridine), 155.83
(C–NH2), 149.67 (C–NH at coumarin), 134.42 (C–Cl),
133.87 (C–Cl), 116.61 (C:N), 115.52 (1C, pyridine),
147.19, 143.18, 133.27, 132.52, 131.35, 130.88, 130.21,
129.54, 128.86, 128.53, 124.72, 124.66, 119.43, 118.30,
118.15, 117.44, 92.56 (17C, Ar–C), 85.78 (C–C:N); EMI-
MS (m/z): 500.14 (M?); Anal. Calcd for C27H16Cl2N4O2:
2-Amino-4-(4-chloro-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8e)
Yield: 75 %. M.p. [300 °C; IR (KBr, cm-1): 3,393
(–NH2), 3,285 (–NH), 2,215 (C:N), 1,663 (C=O of cou-
1
marin), 819 (–Cl); H NMR (DMSO-d6, d, ppm): 8.82 (s,
1H, –NH), 8.13 (s, 1H, pyridine), 7.84 (d, J = 7.5 Hz, 1H,
coumarin), 7.83–7.16 (m, 8H, Ar–H), 6.69–6.51 (m, 3H,
coumarin), 6.17 (s, 1H, coumarin), 5.40 (s, 2H, –NH2);
13C NMR (DMSO-d6, d, ppm): 163.89 (1C, pyridine),
162.43 (C=O of coumarin), 156.63 (1C, pyridine), 155.78
123

Med Chem Res
1
(C–NH2), 149.44 (C–NH at coumarin), 115.92 (C:N),
115.17 (1C, pyridine), 134.05 (C–Cl), 147.68, 143.56,
133.22, 132.59, 130.97, 130.62, 130.11, 129.90, 129.75,
128.89, 128.05, 123.87, 124.17, 119.86, 118.42, 118.15,
117.66, 93.16 (18C, Ar–C), 86.25 (C–C:N); EMI-MS
(m/z): 465.76 (M?); Anal. Calcd for C27H17ClN4O2: C,
69.75; H, 3.69; N, 12.05. Found: C, 69.83; H, 3.63; N, 12.10.
coumarin), 740 (–Br); H NMR (DMSO-d6, d, ppm): 8.87
(s, 1H, –NH), 8.11 (s, 1H, pyridine), 7.84 (d, J = 7.2 Hz,
1H, coumarin), 7.80–7.15 (m, 8H, Ar–H), 6.71–6.49 (m,
3H, coumarin), 6.17 (s, 1H, coumarin), 5.35 (s, 2H, –NH2);
13C NMR (DMSO-d6, d, ppm): 163.34 (1C, pyridine),
162.15 (C=O of coumarin), 156.91 (1C, pyridine), 156.47
(C–NH2), 149.37 (C–NH at coumarin), 116.88 (C:N),
115.20 (1C, pyridine), 122.16 (C–Br), 147.13, 142.70,
133.18, 133.05, 131.80, 130.56, 129.32, 129.13, 129.04,
128.42, 127.77, 124.62, 124.53, 119.30, 118.87, 118.32,
117.22, 92.67 (18C, Ar–C), 85.63 (C–C:N); EMI-MS (m/z):
510.35 (M?); Anal. Calcd for C27H17BrN4O2: C, 63.67; H,
3.36; N, 11.00. Found: C, 63.74; H, 3.30; N, 11.09.
2-Amino-4-(4-nitro-phenyl)-6-[4-(2-oxo-2H-chromen-
4-ylamino)-phenyl]-nicotinonitrile (8f)
Yield: 70 %. M.p. 236–237 °C; IR (KBr, cm-1): 3,390
(–NH2), 3,288 (–NH), 2,212 (C:N), 1,549 (N=O str.),
1
1,672 (C=O of coumarin); H NMR (DMSO-d6, d, ppm):
8.84 (s, 1H, –NH), 8.10 (s, 1H, pyridine), 7.88 (d,
J = 7.4 Hz, 1H, coumarin), 7.85–7.24 (m, 8H, Ar–H),
6.72–6.51 (m, 3H, coumarin), 6.16 (s, 1H, coumarin), 5.31
(s, 2H, –NH2); 13C NMR (DMSO-d6, d, ppm): 162.85 (1C,
pyridine), 162.60 (C=O of coumarin), 157.09 (1C, pyri-
dine), 155.43 (C–NH2), 149.76 (C–NH at coumarin),
116.55 (C:N), 115.83 (1C, pyridine), 148.87 (C–NO2),
147.16, 143.22, 133.76, 132.87, 131.53, 130.85, 130.11,
129.79, 129.45, 128.23, 128.15, 124.69, 124.44, 119.54,
118.33, 117.83, 117.29, 92.20 (18C, Ar–C), 86.10 (C–
C:N); EMI-MS (m/z): 476.61 (M?); Anal. Calcd for
C27H17N5O4: C, 68.21; H, 3.60; N, 14.73. Found: C, 68.16;
H, 3.69; N, 14.68.
2-Amino-4-(4-methoxy-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8i)
Yield: 84 %. M.p. [300 °C; IR (KBr, cm-1): 3,391
(–NH2), 3,285 (–NH), 2,204 (C:N), 1,676 (C=O of cou-
marin), 1,279 (–OCH3); 1H NMR (DMSO-d6, d, ppm):
8.83 (s, 1H, –NH), 8.07 (s, 1H, pyridine), 7.90 (d, J =
7.5 Hz, 1H, coumarin), 7.86–7.23 (m, 8H, Ar–H),
6.73–6.55 (m, 3H, coumarin), 6.20 (s, 1H, coumarin), 5.25
(s, 2H, –NH2), 3.76 (s, 3H, –OCH3); 13C NMR (DMSO-d6,
d, ppm): 163.11 (1C, pyridine), 162.58 (C=O of coumarin),
160.87 (C–OCH3), 157.18 (1C, pyridine), 156.25 (C–NH2),
149.57 (C–NH at coumarin), 116.33 (C:N), 115.47 (1C,
pyridine), 148.27, 143.83, 132.62, 131.92, 131.13, 130.33,
130.01, 129.80, 129.45, 128.78, 128.27, 125.12, 124.67,
119.53, 118.73, 118.61, 117.22, 92.17 (18C, Ar–C), 55.72
(–OCH3), 86.05 (C–C:N); EMI-MS (m/z): 461.44 (M?);
Anal. Calcd for C28H20N4O3: C, 73.03; H, 4.38; N, 12.17.
Found: C, 73.10; H, 4.28; N, 12.27.
2-Amino-4-(2-hydroxy-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8g)
Yield: 82 %. M.p.[300 °C; IR (KBr, cm-1): 3,395 (–NH2),
3,280 (–NH), 3,252 (–OH), 2,221 (C:N), 1,667 (C=O of
coumarin); 1H NMR (DMSO-d6, d, ppm): 8.81 (s, 1H,
–NH), 8.15 (s, 1H, pyridine), 7.80 (d, J = 7.3 Hz, 1H,
coumarin), 7.77–7.12 (m, 8H, Ar–H), 6.68–6.52 (m, 3H,
coumarin), 6.21 (s, 1H, coumarin), 5.28 (s, 2H, –NH2), 4.61
(s, 1H, –OH); 13C NMR (DMSO-d6, d, ppm): 163.91 (1C,
pyridine), 161.92 (C=O of coumarin), 158.78 (C–OH),
157.18 (1C, pyridine), 156.06 (C–NH2), 149.44 (C–NH at
coumarin), 116.78 (C:N), 115.31 (1C, pyridine), 148.33,
143.51, 133.69, 132.54, 131.16, 130.83, 130.14, 130.06,
129.97, 128.85, 128.67, 125.59, 124.87, 119.13, 118.53,
118.37, 117.46, 93.11 (18C, Ar–C), 85.92 (C–C:N); EMI-
MS (m/z): 447.19 (M?); Anal. Calcd for C27H18N4O3: C,
72.64; H, 4.06; N, 12.55. Found: C, 72.72; H, 4.02; N,
12.47.
2-Amino-4-(3,4-dimethoxy-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8j)
Yield: 78 %. M.p. 285–287 °C; IR (KBr, cm-1): 3,396
(–NH2), 3,290 (–NH), 2,213 (C:N), 1,668 (C=O of cou-
marin), 1,284 (–OCH3); 1H NMR (DMSO-d6, d, ppm):
8.79 (s, 1H, –NH), 8.05 (s, 1H, pyridine), 7.86 (d,
J = 7.4 Hz, 1H, coumarin), 7.77–7.08 (m, 7H, Ar–H),
6.68–6.47 (m, 3H, coumarin), 6.17 (s, 1H, coumarin), 5.33
(s, 2H, –NH2), 3.89 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3);
13C NMR (DMSO-d6, d, ppm): 163.76 (1C, pyridine),
162.55 (C=O of coumarin), 160.43 (C–OCH3), 159.87 (C–
OCH3), 157.28 (1C, pyridine), 156.04 (C–NH2), 149.47
(C–NH at coumarin), 116.13 (C:N), 115.58 (1C, pyri-
dine), 147.64, 143.28, 133.56, 132.30, 131.67, 130.35,
130.19, 129.85, 128.86, 128.39, 124.33, 124.15, 119.49,
118.53, 118.24, 117.22, 92.54 (17C, Ar–C), 85.87 (C–
C:N), 56.15 (–OCH3); EMI-MS (m/z): 491.63 (M?);
2-Amino-4-(2-bromo-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8h)
Yield: 74 %. M.p. 260–270 °C; IR (KBr, cm-1): 3,389
(–NH2), 3,284 (–NH), 2,218 (C:N), 1,662 (C=O of
123

Med Chem Res
Anal. Calcd for C29H22N4O4: C, 71.01; H, 4.52; N, 11.42.
Found: C, 71.05; H, 4.56; N, 11.34.
were incorporated in specified quantity of molten sterile
agar (nutrient agar for antibacterial activity and sabouraud
dextrose agar medium for antifungal activity). A specified
quantity of the medium (40–50 °C) containing the com-
pound was poured into a petri dish to give a depth of
3–4 mm and allowed to solidify. A suspension of the
microorganism was prepared to contain approximately
105 cfu mL-1 and applied to plates with serially diluted
compounds in dimethyl sulfoxide to be tested and incu-
bated at 37 °C for 24 and 48 h for bacteria and fungi,
respectively. The MIC was considered to be the lowest
concentration of the test substance exhibiting no visible
growth of bacteria or fungi on the plate. The observed MIC
is presented in Tables 1, 2, and 3.
2-Amino-4-(2,4-dihydroxy-phenyl)-6-[4-(2-oxo-2H-
chromen-4-ylamino)-phenyl]-nicotinonitrile (8k)
Yield: 73 %. M.p. [300 °C; IR (KBr, cm-1): 3,386
(–NH2), 3,282 (–NH), 3,266 (–OH), 2,216 (C:N), 1,671
(C=O of coumarin); 1H NMR (DMSO-d6, d, ppm): 8.84 (s,
1H, –NH), 8.51 (s, 1H, –OH), 8.12 (s, 1H, pyridine), 7.79
(d, J = 7.6 Hz, 1H, coumarin), 7.76–7.19 (m, 7H, Ar–H),
6.64–6.42 (m, 3H, coumarin), 6.15 (s, 1H, coumarin), 5.36
(s, 2H, –NH2), 4.53 (s, 1H, –OH); 13C NMR (DMSO-d6, d,
ppm): 163.20 (1C, pyridine), 162.17 (C=O of coumarin),
157.83 (C–OH), 157.21 (1C, pyridine), 156.24 (C–OH),
155.85 (C–NH2), 149.69 (C–NH at coumarin), 116.13
(C:N), 115.70 (1C, pyridine), 147.26, 143.32, 133.58,
132.87, 131.40, 130.21, 129.54, 129.05, 128.82, 127.46,
125.02, 124.86, 119.44, 118.32, 118.12, 117.61, 92.44
(17C, Ar–C), 86.08 (C–C:N); EMI-MS (m/z): 463.29
(M?); Anal. Calcd for C27H18N4O4: C, 70.12; H, 3.92; N,
12.12. Found: C, 70.20; H, 3.83; N, 12.05.
Antimycobacterial assay
The antimycobacterial screening for the test compounds
was performed against M. tuberculosis H37Rv using L. J.
(Lowenstein and Jensen) MIC method (Isenberg, 1992) for
the measurement of MIC. Stock solutions of primary: 1000,
500, and 250 lg/mL, and secondary: 200, 100, 62.5, 50,
25, 12.5, 6.25, and 3.25 lg/mL dilutions of each test
compound in dimethylsulfoxide (DMSO) were added in the
liquid L. J. Medium, and then media were sterilized by
inspissation method. A culture of M. tuberculosis H37Rv
growing on L. J. medium was harvested in 0.85 % saline in
bijou bottles. These tubes were then incubated at (37 1)
°C for 24 h followed by streaking of M. tuberculosis
H37Rv (5 9 104 bacilli per tube). These tubes were then
incubated at (37 1) °C. Growth of bacilli was seen after
12 days, 22 days, and finally 28 days of incubation. Tubes
having the compounds were compared with control tubes
where medium alone was incubated with M. tuberculosis
H37Rv. The concentration, at which no development of
colonies occurred or \20 colonies, was taken as MIC
concentration of test compound. The standard strain
M. tuberculosis H37Rv was tested with known drugs rif-
ampicin, isoniazid, ethambutol, and pyrazinamide.
Antimicrobial assay
The synthesized compounds 8a–n, 10a–n, and 12a–n were
examined for antimicrobial activity against several bacteria
(Staphylococcus aureus MTCC 96, Bacillus cereus MTCC
430, Escherichia coli MTCC 739, Pseudomonas aerugin-
osa MTCC 741, Klebsiella pneumoniae MTCC 109, Sal-
monella typhi MTCC 733, Proteus vulgaris MTCC 1771,
and Shigella flexneri MTCC 1457); and fungi (Aspergillus
niger MTCC 282, Aspergillus fumigatus MTCC 343,
Aspergillus clavatus MTCC 1323, and Candida albicans
MTCC 183) species using paper disk diffusion technique
(Gillespie, 1994). The sterilized (autoclaved at 120 °C for
30 min) medium (40–50 °C) was inoculated (1 mL/
100 mL of medium) with the suspension (105 cfu mL-1
)
of the microorganism (matched to McFarland barium sul-
fate standard) and poured into a petri dish to give a depth of
3–4 mm. The paper impregnated with the test compounds
(lg mL-1) in dimethyl sulfoxide) was placed on the
solidified medium. The plates were pre-incubated for 1 h at
room temperature and incubated at 37 °C for 24 and 48 h
for antibacterial and antifungal activities, respectively.
Ciprofloxacin (100 lg/disk) and Ketoconazole (100 lg/
disk) were used as standards for antibacterial and anti-
fungal activities, respectively. The observed zone of inhi-
bition is presented in Tables 1, 2, and 3. MIC of the
compound was determined by agar streak dilution method
(Hawkey and Lewis, 1994). A stock solution of the syn-
thesized compound (100 lg mL-1) in dimethyl sulfoxide
was prepared, and graded quantities of the test compounds
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