Mechanism of unusual formation of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones and 4-oxo-4H-chromene-3-carbothioic acid N-phenylamides and their antimicrobial evaluation

Article abstract of DOI:10.1016/j.ejmech.2009.03.030

6/6,7-Substituted 3-formylchromones (9a-e) react with 2 equivalents of 2-phenyl-4-dimethylamino-1-thia-3-azabuta-1,3-diene (10) or thiobenzamide (11) in refluxing toluene to furnish novel substituted 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (12

Full text of DOI:10.1016/j.ejmech.2009.03.030

European Journal of Medicinal Chemistry 44 (2009) 3209–3216
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Mechanism of unusual formation of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-
yl)chromen-4-ones and 4-oxo-4H-chromene-3-carbothioic acid
N-phenylamides and their antimicrobial evaluation
Tilak Raj a, Richa Kaur Bhatia a, Rakesh Kumar Sharma b, Vivek Gupta c,
,
Deepak Sharma c, Mohan Paul Singh Ishar a
*
a Bio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143 005, Punjab, India
b Department of Microbiology, Guru Nanak Dev University, Amritsar 143 005, Punjab, India
c Department of Physics, University of Jammu, Jammu Tawi 180 006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
6/6,7-Substituted 3-formylchromones (9a–e) react with 2 equivalents of 2-phenyl-4-dimethylamino-1-
thia-3-azabuta-1,3-diene (10) or thiobenzamide (11) in refluxing toluene to furnish novel substituted
3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (12a–e). However, reactions of substituted 2-
anilino-3-formylchromones (15a–d) with thiobenzamide (11, 2 equivalents) in refluxing xylene furnish
4-oxo-4H-chromene-3-carbothioic acid N-phenylamide (17a–d) in high yields. A mechanistic ration-
alization of the conversion of 2-anilino-3-formylchromones (15a–d) to N-phenylamides (17a–d), and
3-formylchromones (9a–e) to the corresponding thioaldehydes, is proffered. All the compounds (12a–e,
17a–d) display very high antifungal and antibacterial activities against a number of strains. Dithiazole
Received 2 January 2009
Received in revised form
14 February 2009
Accepted 19 March 2009
Available online 28 March 2009
Keywords:
Dithiazoles
N-Phenylthioamide
Antibacterial activity
Antifungal activity
12d exhibits a very high antifungal activity (MIC 5
m
g/ml) against Geotrichum candidum, better than
fluconazole (MIC 09
m
g/ml) and also possesses good antibacterial activity (MIC 52 g/ml) against Shigella
m
flexneri.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
methylene-bisdithiocarbamate (Nabam) [12]. Compounds such
as N-3-(1,2,4-dithiazole-5-thione)-b-resorcylcarbothioamide (5)
Heterocycles, both natural and synthetic, exhibit a wide range of
interesting biological activities [1–6]. Among five-membered
heterocycles dithiazoles, both 1,2,3- and 1,2,4-, are endowed with
interesting biological activities, in particular, antimicrobial activity
[7,8]. For instance 4-chloro-5-heteroimmino-1,2,3-dithiazoles (1a–
e) have been evaluated against fungal species and a number of
cancer cell lines; among these compounds (1b–d) exhibit the
highest antifungal activity [9]. Similarly, N-arylimino-l,2,3-dithia-
zoles (2) possess antifungal activity against yeasts [10], whereas,
5-(4-chloro-[1,2,3]dithiazol-5-ylideneamino)-naphthalen-1-ol (3)
possesses significant bactericidal as well as fungicidal activities;
these are postulated to be potent inhibitors of enzymes such as
serine proteases [11]. In fact, 1,2,4-dithiazole is the active moiety of
the antifungal compound 5,6-dihydroimidazo[2,l-c]-l,2,4-dithia-
zole-3-thione (ethylenethiuram monosulfide 4), which is obtained
by auto-oxidation of the well-known fungicide disodium
display significant antifungal activity against various fungal strains
(Fig. 1). These molecules are reported to possess diverse modes of
action and their targets include enzymes such as leucine arylami-
dase, esterases,
a-glucosidase, N-acetyl-b-glucosaminidase, lipases,
and alkaline phosphatase [13].
At the same time, chromones of both synthetic and natural origin
are also known to display valuable antifungal activity. Some recently
discovered antifungal chromones includes 8-hydroxy-6-methyl-2,3-
dihydro-cyclopenta[b]chromen-1,9-dione (6, Fig. 2), isolated from
a carpophilus fungus [14] and synthetic 2-phenylchromone deriva-
tive 2-benzo[1,3]dioxol-5-yl-7-benzyloxy-chromen-4-one (7) [15].
Another important synthetic chromone based antifungal agent is N-
isobutyl-2-(4-oxo-2-phenyl-4H-chromen-6-yl)-1H-benoimidazole-
5-carboxamidine (8), which displays valuable antimycotic proper-
ties against Candida albicans and Candida krusei.
2. Chemistry
Recently, we had reported [16] a serendipitously discovered route
to 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (12a–e).
* Corresponding author. Tel.: þ91 183 2258802x3321; fax: þ91 183 2258820.
E-mail address: mpsishar@yahoo.com (M.P.S. Ishar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.030

3210
T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
Cl
S
NC
N
N
x3
x2
a) R=Me,
X1=N, X2= N, X3= CH
X1=N, X2= N, X3= CH
X1=N, X2= N, X3= CH
X1=N, X2= N, X3= CH
N
b) R=Ph(CH2)3
c) R=C(CH2)3
d) R=Bn
S
Cl
x1
R
R
S
N
S
e) R=Ph(CH2)2, X1=N, X2= H, X3= N
R=H, 2-OMe
2
1
S
S
N
N
S
S
H
Cl
N
S
S
N
OH
S
N
N
S
R
R=H, OH
S
OH
5
3
4
Fig. 1. Some antifungal and antibacterial dithiazole derivatives.
thinking that possibly the reaction may be starting with an attack
on C2, the 2-N-methylanilino-3-formylchromone (14) [21] was
reacted with two equivalents of thiobenzamide (11)/thioazadiene
(10) under similar conditions; no reaction occurred as indicated by
TLC and NMR analysis.
However, when the substituted 2-anilino-3-formylchromones
(15a–d) were reacted with 2 equivalents of thiobenzamide (11),
4-oxo-4H-chromene-3-carbothioic-N-phenylamides (17a–d) were
obtained in high yields (Scheme 3, Table 1), but no formation of
(2-phenylamino-3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)-chromen-4-
one (16) was observed.
O
O
OH O
O
i.but
NH
O
O
Ph
Ph
N
N
O
O
HN
O
7
O
6
8
Fig. 2. Some chromone based antifungal and antibacterial agents
Thus, attempted cycloaddition of 2-phenyl-4-dimethylamino-1-thia-
3-azabuta-1,3-diene (10) with substituted 3-formylchromones 9, in
sealed tube, led to the isolation of (12a–e) in good yields and the
yields improved considerably using two equivalents of 10. Subse-
quently, it was discovered that same dithiazoles (12a–e) were also
obtained on reacting 9 with two equivalents of thiobenzamide (11) in
refluxing toluene (Scheme 1).
Based on the above observations and detection of benzonitrile
and DMF (the latter in the case of reaction with thioazadiene 10), by
HPLC, a plausible mechanism was postulated (Scheme 2); conver-
sion of thioaldehydes to dithiazoles has precedent in literature
[17–19].
Keeping in view the anticipated antifungal and antibacterial
activities of these molecules, which encompass chromone and
dithiazole moieties, it was decided to synthesize these molecules
and evaluate their antifungal and antibacterial activity. It has been
postulated that 3-formylchromone react with thiobenzamide/
thiazadienebyinitialattackonthealdehydefunction[16](Scheme2),
although more electron deficient C2 position is also present [20]
therefore, it was also of interest to probe the mechanism of the
formation of dithiazoles.
All the products (17a–d) were characterized by detailed spec-
troscopic analysis (1H and 13C NMR, IR and mass) and microana-
lytical data. The structure of 17c was further confirmed X-ray
crystallographically (Fig. 3) [22].
4. Discussion
Formation of 17a–d clearly indicates that the reaction indeed
starts with an initial attack on C2, instead of earlier proposed
involvement of aldehyde function [16]. Mechanistically, the
formation of 17 can be rationalized as outlined in Scheme 4; such
opening and recyclization of chromone ring is precedented [21].
Apparently, reactions of 2-N-methylanilino-3-formylchromones
Me
Me
N
N
S
Ph
O
O
S
S
O
O
10
X1
X2
X1
X2
N
H
Toluene
Sealed tube
3. Results
S
O
9
a X1 = H,
X2 = H
12
H2N
Because in the proposed mechanism (Scheme 2) the first step is
thionation of the aldehyde moiety, reactions of benzaldehyde (13)
with one and two equivalents of thioazadine (10)/thiobenzamide
(11) in refluxing xylene were carried out. However, these reactions
failed and no thionation was observed, indicating that the reaction
doesn’t start with aldehyde function. As the C2 of the chromone
moiety has been reported to be highly electrophilic [20] and
Ph
b X1 = Cl, X2 = H
c X1 = F, X2 = H
d X1 = F, X2 = Cl
e X1= CH3, X2 = H
11
Scheme 1. Synthesis of 1,2,4-dithiazole derivatives.

T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
3211
CH3
H
N
H3C
Step-I, Thionation
N
S
H3C CH3
N
O
O
H
O
O
O
N
Ph
+
H
S
Ph
O
A
- PhCN - DMF
H
O
O
O
O
S
N
O
OH
Ph
- H2O
H
+
S
Ph
H
..
NH2
S
- PhCN
O
O
B
Thioaldehyde
Step-II, Formation of Dithiazoles
O
S
O
SH
S
S
O
O
S
S
N
H
Ph
Ph
H2N
H
Ph
N
oxidative cyclization
O
C
O
Thioaldehyde
Scheme 2. Mechanism of the 1,2,4-dithiazole formation.
O
In view of the known antimicrobial properties of chromone
derivatives and heterocyclic thioamides [23,24], it was decided to
synthesize the N-phenylthioamides (17a–d) in addition to chro-
manyl-1,2,4-dithiazole (12a–e) and evaluate their antimicrobial
activity.
S
S
x
Ph
N
H
O
O
N
O
16
N
Ph
H
S
H
H
i
+
H2N
Ph
O
O
N
Ph
5. Pharmacolology
S
15
11
O
17
All the synthesized compounds (12a–e) and (17a–d) were
evaluated for both antifungal and antibacterial activities.
a X1=H, X2=H
b X1=F, X2=H
c X1=H, X2=Cl
d X1=Cl, X2=Cl
5.1. Antifungal activity
Scheme 3. Reaction conditions: (i) xylene reflux 4 h.
In vitro antifungal investigations on 3-(5-phenyl-3H-[1,2,4]di-
thiazol-3-yl)chromen-4-ones (12a–e) and 4-oxo-4H-chromene-3-
with thiobenzamide (10)/thioazadinene (11) did not occur due to
steric reasons.
Therefore, the thionation of substituted 3-formylchromones (9)
on reaction with thiobenzamide (11) or thioazadiene (10) may be
similarly involving the attack on C2 (Scheme 5).
After thionation, the thioaldehyde further reacts with another
molecule of the thiobenzamide (11) to yield 3-(5-phenyl-3H-
[1,2,4]dithiazol-3-yl)chromen-4-ones (12) whereas 4-oxo-4H-
chromene-3-carbothioic acid phenylamides (17a–d) fail to react
with thiobenzamide.
Table 1
Reaction yield (%) of products 17.
Chromone
Product
% Yield
15a
15b
15c
15d
17a
17b
17c
17d
71
76
78
72
Fig. 3. ORTEP view of 17c.

3212
T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
Route-I
O
O
O
N
O
O
O
Ph
H
O
HN
O
N
C
H
H
H
H
S
Ph
2 N
Ph
Ph
O
H
S
H
N
S
H
H
Ph
N
+
Ph
Ph
H
O
O
HN
N
H
C
O
S
N
Ph
OH
-PhCN
-H2O
S
O
17
Scheme 4. Mechanism of formation of N-phenylthioamides.
carbothioic acid N-phenylamides (17a–d) were carried out on
fungal strains Aspergillus niger (MTCC-281), Geotrichum candidum
(MTCC-3993), C. albicans (MTCC-227) and Candida tropicalis
(MTCC-230). Fluconazole was used as positive control. Minimum
inhibitory concentration (MIC) is the concentration mg/ml required
to inhibit bacterial cell proliferation by 50% after exposure of cells
to test compounds. Inhibitory potential of the compounds was
determined in terms of MIC (mg/ml) using turbidimetry method
Route -I Thionation with thioazadiene
CH3
N
O
O
O
O
O
O
N
O
N
CH3
H
H
O
S
H H
Ph
O
O
H
S
Ph
Ph
S
N
Moisture
N
H3C
CH3
:
N
H3C CH3
H
CH3
N
O
N
O
CH3
S
O
H
H
C N
S
Ph
CH3
H
H2O
O
Ph C N
OH
CH3
O
O
Thioaldehyde
Route-II Thionation with thiobenzamide
O
O
O
O
O
O
H
O
H
N
C
H
H
S
Ph
O
S
O
H
H
H
N
+
-
S
Ph
H
H
N
+
Ph
H
O
O
H
N
O
O
C
S
H
S
OH
Ph
-PhCN
-H2O
Thioaldehyde
Scheme 5. Mechanism of thionation with both thioazadiene and thiobenzamide.

T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
3213
Table 2
In vitro antifungal activities of compounds 12a–e and 17a–d.
Table 3
In vitro antibacterial % growth inhibition by compounds 12a–e and 17a–d.
Compound Conc. ( g/ml) % Growth inhibition
E. coli S. flexneri
(MTCC-119) (MTCC-1457) (MTCC-741) (MTCC-740)
Compound
MIC (
m
g/ml)
m
Asperagillus
niger
Geotrichum
candidum
Candida
albicans
Candida
tropicalis
P. aeruginosa S. aureus
(MTCC-281)
(MTCC-3993)
(MTCC-227)
(MTCC-230)
12a
12b
12c
12d
12e
17a
17b
17c
17d
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
2.5 ꢀ 10ꢁ5
5 ꢀ 10ꢁ5
1 ꢀ 10ꢁ4
24.83
56.68
100.00
21.96
49.60
100.00
–
12.28
80.90
–
14.00
30.50
–
30.00
72.50
38.46
85.50
100.00
–
73.75
100.00
–
42.60
87.60
20.36
78.28
100.00
44.26
85.50
100.00
27.34
55.80
100.00
28.36
62.32
100.00
48.30
98.55
100.00
21.01
60.86
100.00
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
30.20
73.88
100.00
26.42
51.50
100.00
–
23.88
48.80
32.86
68.65
100.00
35.04
92.72
100.00
35.60
76.25
100.00
–
12a
12b
12c
12d
12e
17a
17b
17c
90
125
50
90
150
90
120
80
–
32
30
32
05
95
–
–
–
–
09
30
28
30
40
60
–
40
28
42
28
52
–
–
60
–
–
60
75
24
17d
Fluconazole
49
25
(Table 2). For 12c maximum inhibition (MIC 50) was observed
against A. niger followed by 17c (MIC 80). Compound 12d with MIC
5 mg/ml was found to be the most active compound against
G. candidum (Fig. 4). Against C. albicans compound 12b was most
active (MIC 28) followed by 12a,c (MIC 30). Compounds 12b,d
showed maximum activity (MIC 28) against C. tropicalis. It may be
mentioned here that compound 12d showed very high inhibition
55.34
100.00
–
–
–
–
–
–
–
52.18
100.00
8.82
60.86
100.00
43.62
81.00
3.78
90.90
100.00
(MIC 5 mg/ml), which is more than that of fluconazole (MIC 9) used
as positive control.
5.2. Antibacterial activity
In vitro antibacterial studies of various compounds (12a–e) and
(17a–d) were carried out on Gram negative and Gram positive
bacterial strains by disk-diffusion assay. The Gram negative strains
used were Escherichia coli (MTCC-119, facultative anaerobic),
Pseudomonas aeruginosa (MTCC-741, aerobic) and Shigella flexneri
(MTCC-1457, facultatively anaerobic). Gram positive strain used is
Staphylococcus aureus (MTCC-740, facultatively anaerobic). The
antibacterial activity was determined at concentrations of
2.5 ꢀ10ꢁ5, 5 ꢀ10ꢁ5 and 1 ꢀ10ꢁ4 mg/ml (Table 3). Ciprofloxacin and
chloramphenicol were used as positive controls. MIC values were
determined using turbidimetry method (Table 4). In the case of
anaerobic Gram negative bacteria E. coli the maximum inhibition of
85.5% (5 ꢀ10ꢁ5, MIC 62) was observed for 17a followed by 78.28%
(5 ꢀ10ꢁ5, MIC 64) for 17d and 73.7% (5 ꢀ10ꢁ5, MIC 68) for 17b.
(5 ꢀ10ꢁ5 mg/ml, MIC 54) was observed for 12e followed by 90.90%
(MIC 55) for 17d.
5.3. Structure activity relationships
In vitro antifungal testing revealed compounds bearing electron
withdrawing groups at C6 i.e., 12c and 12b show maximum activity
against A. niger and C. albicans, respectively, while maximum
inhibition was observed in the case of Candidum geotrichum (MIC
05) for compound 12d, which bears electron withdrawing (F, Cl)
substituents at C6 and C7. The 4-oxo-4H-chromene-3-carbothioic
acid N-phenylamide derivatives (17a,b,d) are found to be more
active against E. coli. On the other hand, 1,2,4-dithiazole-chromen-
4-one derivative 12d showed maximum inhibition against S. flex-
neri, when compounds possess electron withdrawing group (Cl) at
C6 or C7 positions. Compound 12e bearing electron releasing group
Compound 12a also showed significant activity 56.68% (5 ꢀ10ꢁ5
,
MIC 88). In the case of S. flexneri, maximum inhibition observed was
98.55% (MIC 52) followed by 85.50% (MIC 58) for 12d and 12a
respectively, at same concentration. It is pertinent to mention here
that none of the compounds 12a–e and 17a–d were found active
against aerobic Gram negative P. aeruginosa bacteria. For Gram
positive bacteria S. aureus, maximum growth inhibition of 92.72%
Table 4
In vitro antibacterial activity MIC (mg/ml) of compounds 12a–e and 17a–d.
Compound code
MIC (
mg/ml)
E. coli
S. flexneri
P. aeruginosa
S. aureus
(MTCC-119)
(MTCC-119)
(MTCC-119)
(MTCC-119)
12a
12b
12c
12d
12e
17a
17b
88
100
120
350
132
62
58
90
80
52
82
92
–
–
–
–
–
–
–
–
68
98
205
72
54
70
68
–
17c
17d
115
64
2.5
1.2
96
62
5.6
1.4
–
–
2.7
1.8
125
55
13.6
0.9
Chloramphenicol
Ciprofloxacin
Fig. 4. Zone of inhibition of 12d against fluconazole on Geotrichum candidum.

3214
T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
Antifungal activity
H
Electron donating
O
O
S
S
O
O
Electron withdrawing group
at C6 position
N
group at C6 position
decrease activity
enhance activity
N
S
Electron withdrawing group
at C7 inhances the activity
aginst Candida albicans and
C. tropicalis
Compounds with
electron withdrawing group
at C7 show high activity
Antibacterial activity
Electron withdrawing
group at C6 position
enhance activity against
gram +ve and gram -ve
strains
H
O
Electron releasing
group at C6 position
enhance activity against
gram +ve strains
N
S
S
O
O
N
S
O
Electron withdrawing group
at C7 enhance active against
gram -ve as well as gram +ve
strains
Electron withdrawing group
at C7 ehances activity
against gram -ve as well as
gram +ve strains
(–CH3) at C-6 position possesses significant activity against
S. aureus but lesser than compounds bearing electron withdrawing
groups at C7.
recorded with Shimadzu FT-IR-8400S spectrophotometer on KBr
pellets. Mass spectra, ESI-method, were recorded on Bruker Dal-
tonics Esquire 300 mass spectrometer. Elemental Analyses were
carried out on a Thermoelectron EA-112 elemental analyzer and are
reported in percent atomic abundance. All melting points are
uncorrected and measured in open glass-capillaries on a Veego
(make) MP-D digital melting point apparatus. X-ray analysis was
recorded at Bruker SMART APEX diffractometer equipped with low-
temperature device and the structure was solved by direct methods
using SHELXS 97 software (Sheldrick, 1997).
6. Conclusion
Conversion of 2-anilino-3-formylchromones 15 to correspond-
ing thioamides (17) establishes that the reaction of the former with
thiobenzamide 11 proceeds through attack on more electrophilic
C2. Therefore, in the light of failure of benzaldehyde 13 and 2-N-
methylanilino-3-formylchromone 14 to react with thiobenzamide
11 or thiazadiene 12, it is concluded that thionation of 3-for-
mylchromones also proceeds through initial attack on more elec-
trophilic C2 rather than aldehydic carbon, thereby establishing the
mechanism of thionation and hence formation of 3-(5-phenyl-3H-
[1,2,4]dithiazol-3-yl)chromen-4-ones (12a–e). All the synthesized
compounds (12a–e) and (17a–d) were evaluated for antifungal and
antibacterial activities. All the compounds (12a–e) and (17a–d)
display good antifungal activity. However, the dithiazole derivative
(12d) bearing electron withdrawing (F, Cl) groups at C6 and C7
positions shows high antifungal activity (MIC 05) in comparison to
fluconazole (MIC 09) against G. candidum. All the compounds (12a–
e and 17a–d) display useful antibacterial activity, but the thioamide
derivatives (17a–d) are relatively more active. Chromone moiety
has been shown to be a good replacement for quinolone moiety
present in fluroquinolone antibacterials [23] i.e., tertiary nitrogen
of quinolones is replaced by isosteric oxygen, therefore, thioamides
(17a–d) may be having the same mode of action as that of fluo-
roquinolones; the latter are known bacterial DNA gyrase inhibitors
[25–31]. These molecules are being developed further.
7.2. General procedure – synthesis of 1,2,4-dithiazoles (12a–d)
The reactions were carried out by reacting substituted 3-for-
mylchromone (500 mg, 2.8 mmol) with thiobenzamide (767 mg,
5.6 mmol) in RBF using toluene (20 mL) as the solvent at an oil bath
temperature of 120 ꢂC. The completion of the reactions was
monitored by TLC. After completion of the reaction, the solvent was
evaporated under reduced pressure. The crude mixture was puri-
fied by column chromatography over silica 60–120 mesh (Loba
Cheme 20 g packed in hexane) and eluted with 1% EtOAc in hexane.
The purified products (12a–e) were characterized [16] by various
spectroscopic techniques (UV, IR, 1H and 13C NMR, mass).
7.3. Synthesis of 4-oxo-4H-chromene-3-carbothioic
acid N-phenylamides (17a–d)
The reactions were carried out by refluxing substituted 2-
anilino-3-formylchromones (500 mg, 1.8 mmol) with thio-
benzamide (656 mg, 3.7 mmol) in a 100 ml round bottom flask
using xylene (20 mL) at the solvent at an oil bath temperature of
145 ꢂC. Progress of reactions was monitored by TLC. After comple-
tion of the reaction the solvent was evaporated under reduced
pressure. The crude mixture was purified by column chromatog-
raphy over silica 60–120 mesh (Loba Cheme 20 g packed in hexane)
and eluted with 5% EtOAc in hexane. The purified products (17a–d)
were characterized by various spectroscopic techniques (IR, 1H and
13C NMR, mass) and by elemental analysis.
7. Experimental
7.1. Chemistry
Starting materials, reagents and solvents were purchased from
commercial suppliers and purified/distilled/crystallized before use.
JEOL AL-300FT (300 MHz) NMR spectrometers were used to record
1H and 13C NMR (75 MHz) spectra. Chemical shifts (
d) are reported
as downfield displacements from TMS used as internal standard
and coupling constants (J) are reported in Hz. IR spectra were

T. Raj et al. / European Journal of Medicinal Chemistry 44 (2009) 3209–3216
3215
7.3.1. 4-Oxo-4H-chromene-3-carbothioic acid
N-phenylamide (17a)
different concentrations. Dimethylsulfoxide was used as a negative
control and antifungal (Fluconazole) as positive controls.
Yield: 71%; orange crystalline solid, mp 121–127 ꢂC (chlor-
oform:hexane, 1:1); IR (KBr) nmax (cmꢁ1): 3360 (NH), 1650(C]O);
7.4.2. Antifungal activity
The disk-diffusion assay was applied to determine the growth
inhibition of fungi by compounds to be tested. Overnight fungal
1H NMR (300 MHz CDCl3):
d
¼ 13.65 (s, 1H, NH), 9.71 (s, 1H, C2H),
8.35 (dd, 1H, J ¼ 7.8 and 1.8 Hz, ArH), 8.33–7.30 (m, 8H, ArH); 13C
NMR (75 MHz, CDCl3):
d
¼ 187.5 (C]S), 177.7 (C4), 166.1 (q), 155.5
cultures (100 mL) were spread onto SDA. The compounds were
(C2), 139.0 (q), 134.9 (CH), 129.3 (CH), 128.8 (CH), 126.8 (CH), 126.6
(CH), 124.2 (CH), 123.3 (q), 120.2 (CH), 118.5 (CH); MS (ESI): m/z 282
(M þ H)þ, 281 (Mþ); Anal. calcd. for C16H11NO2S %: C, 68.31; H, 3.94;
N, 4.98. Found %: C, 67.99; H, 3.75; N, 4.31.
applied to 8 mm disks (Whatman paper No. 1). After 48 h of incu-
bation at 25 ꢂC, the diameter of growth inhibition zones was
measured.
7.4.3. MIC determination
7.3.2. 6-Fluoro-4-oxo-4H-chromene-3-carbothioic acid
N-phenylamide (17b)
The broth dilution test was performed in test tubes. The conidial
suspension, which gave the final concentration of 1 ꢀ105 CFU/ml,
was prepared. A growth control tube and sterility control tube were
used in each test. After 24–72 h incubation at 25 ꢂC, the MIC was
determined visually as the lowest concentration that inhibits
growth, evidenced by the absence of turbidity.
Yield: 76%; light yellow crystalline solid, mp 128–131 ꢂC
(chloroform:hexane, 1:1); IR (KBr): nmax 3370(NH), 1645(C]O)
cmꢁ1; 1H NMR (300 MHz, CDCl3):
d
¼ 13.55 (s, 1H, NH), 9.74 (s, 1H,
ArH), 8.00–7.30 (m, 8H, ArH); 13C NMR (75 MHz, CDCl3):
d
¼ 187.7
(C]S),176.9 (C4), 166.1 (q), 151.3 (C2), 139.3 (q), 135.3 (CH), 129.4
(CH), 128.8 (CH), 126.9 (CH), 126.3 (CH), 124.2 (CH), 123.0 (q), 120.7
(CH),118.4 (CH); MS (ESI): Fragment m/z 338 (M þ Caþ); Anal. calcd.
for C16H10FNO2S %: C, 64.20; H, 3.37; N, 4.68. Found %: C, 64.01; H,
3.06; N, 4.21.
7.5. Antibacterial activity – materials and methods
The following bacterial strains were used: E. coli MTCC-119, S.
aureus MTCC-740, P. aeruginosa MTCC-741, S. flexneri MTCC-1457
from Microbial Type Culture Collection, IMTECH, Chandigarh, India.
Bacteria were cultivated at 37 ꢂC in Nutrient Agar. For MIC
determination Mueller Hinton Broth (MHB) and Mueller Hinton
agar (MHA) was used. The compounds (12a–e) and (17a–d) and
standards were dissolved in dimethylsulfoxide (1/10) and applied
in different concentrations. Dimethylsulfoxide was used as a nega-
tive control and antibiotics (chloramphenicol and ciprofloxacin) as
positive controls.
7.3.3. 7-Chloro-4-oxo-4H-chromene-3-carbothioic acid
N-phenylamide (17c)
Yield: 78%; light orange crystalline solid, mp 165–168 ꢂC
(chloroform:hexane, 1:1); IR (KBr): nmax 3350(NH), 1655 (C]O),
1517, 1220 (S–S) cmꢁ1; 1H NMR (200 MHz, CDCl3):
d
¼ 13.53 (s, 1H,
NH), 9.68 (s, 1H, C2H), 8.64 (dd, 1H, J ¼ 8.4 and 1.8 Hz, ArH), 8.26–
7.32 (m, 7H, ArH); 13C NMR (75 MHz, CDCl3):
d
¼ 187.7 (C]S), 176.0
(C4), 166.1 (q), 151.3 (C2), 139.8 (q), 131.9 (CH), 129.4 (CH), 128.8
(CH), 126.9 (CH), 126.3 (CH), 124.2 (CH), 123.0 (q), 120.7 (CH), 118.4
(CH); Fragment m/z 338 (M þ Naþ); Anal. calcd. for C16H10ClNO2S %:
C, 61.86; H, 3.19; N, 4.44. Found %: C, 61.10; H, 2.96; N, 4.09.
7.5.1. Antibacterial activity
The disk-diffusion assay was applied to determine the growth
inhibition of bacteria by compounds to be tested. 4-h grown
bacterial cultures in Nutrient Broth (100 ml) were spread onto MHA
7.3.4. 6,7-Dichloro-4-oxo-4H-chromene-3-carbothioic acid
N-phenylamide (17d)
and the compounds were applied to 8 mm disks (Whatman paper
No.1). After 24 h of incubation at 37 ꢂC, the diameter of growth
inhibition zones was measured.
Yield: 72%; light orange crystalline solid, mp 131–135 ꢂC
(chloroform:hexane, 1:1); UV (MeOH): 307, 247 nm; IR (KBr): nmax
3372(NH), 1653(C]O) cmꢁ1; 1H NMR (200 MHz, CDCl3):
d
¼ 13.1 (s,
1H, NH), 9.17 (s, 1H, C2), 9.16 (dd, 1H, J ¼ 8.4 and 0.9 Hz, ArH), 8.53–
7.63 (m, 6H, ArH); 13C NMR (75 MHz, CDCl3):
7.5.2. MIC determination
The broth dilution test was performed in test tubes. In two-
fold serial dilutions of the compounds, a standardized suspen-
d
¼ 187.2 (C]S), 176.0
(C4), 166.4 (q), 153.4 (C2), 139.4 (q), 131.9 (CH), 128.8 (CH), 127.7
(CH), 127.6 (CH), 127.1 (CH), 124.1 (CH), 123.4 (q), 120.4 (CH), 120.2
(CH); MS (ESI): Fragment m/z 348 (Mþ2); Anal. calcd. For
C16H9Cl2NO2S %: C, 54.87; H, 2.59; N, 4.00 Found %: C, 54.61; H,
3.27; N, 3.76.
sion (McFarland turbidity standard) of test bacteria (100 ml) was
added to obtain
a
final concentration of 5 ꢀ105 CFU/ml.
A growth control tube and sterility control tube were used in
each test. After overnight incubation at 37 ꢂC, the MIC was
determined by measuring optical density at 600 nm as the
lowest concentration that inhibits growth, evidenced by the
absence of turbidity [34].
7.4. Pharmacology
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