Synthesis and biological evaluation of dihydroindeno and indeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazines as antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2011.08.019

Two series of compounds namely, dihydroindeno and indeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadizines (9a-l & 11a-l) were synthesized by cyclocondensation between α-bromoindanones (7a-b) or/and α,α-dibromoindanones (8a-b) and various 3-alkyl/aryl-4-

Full text of DOI:10.1016/j.ejmech.2011.08.019

European Journal of Medicinal Chemistry 46 (2011) 5065e5073
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of dihydroindeno and indeno [1,2-e]
[1,2,4]triazolo [3,4-b] [1,3,4]thiadiazines as antimicrobial agents
,
b
b
b
b
b
Om Prakash a , Deepak K. Aneja , Khalid Hussain , Poonam Lohan , Pooja Ranjan , Sanjiv Arora ,
*
Chetan Sharma c, Kamal R. Aneja c
a Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, Haryana, India
b Department of Chemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India
c Department of Microbiology, Kurukshetra University, Kurukshetra 136119, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of compounds namely, dihydroindeno and indeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thia-
Received 6 July 2011
Received in revised form
10 August 2011
Accepted 15 August 2011
Available online 22 August 2011
dizines (9ael & 11a-l) were synthesized by cyclocondensation between
a-bromoindanones (7aeb) or/
and -dibromoindanones (8aeb) and various 3-alkyl/aryl-4-amino-5-mercapto-1,2,4-s-triazoles (3aef)
a,a
in methanol with an aim to explore their effect on in vitro growth of microorganism causing microbial
infection. In vitro antibacterial activity was performed against four strains namely, Staphylococcus aureus,
Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and antifungal activity against three fungal
strains namely, Aspergillus niger, Aspergillus flavus, Penicillium species. Of all the compounds screened for
activity some of the compounds were associated with considerably higher antibacterial and antifungal
activity than commercial antibiotics.
Keywords:
Thiadiazine
Triazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
a
a
-Bromoindanone
-Dibromoindanone
,
a
Antibacterial activity
Antifungal activity
1. Introduction
These results inspired us to synthesize compounds containing
a system which involves combination of these pharmacophores in
Synthesis of nitrogen containing heterocyclic systems has been
attracting increasing interest over the past decade because of their
utility in various applications. In recent years, a special attention
has been given to nitrogen bridged heterocyclic systems derived
from 1,2,4-triazoles owing to their promising biological activities
as antihypertensive, antimicrobial, anticonvulsant, antidepressant,
analgesic and antitumour [1e13] etc. The fused ring of triazoles and
thiadiazines called triazolothiadiazines represent an important
class of nitrogen bridgehead heterocyclic compounds with wide
range of activities such as antifungal, antibacterial, antiviral, anti-
helmintic, antitumour, antitubercular, diuretics, analgesic, anti-
inflammatory, antiviral, CNS-stimulant, PDE4 inhibitors and hypo-
glycemic agents [14e29], etc.
one molecular framework to give the title structure for screening
their antimicrobial activities. Synthesis of 1,3,4-thiadiazines is
mainly based on cyclocondensation of heterocyclic amino thiols
with bifunctional reagents such as
a
-halo/tosyloxy carbonyl
-Halo ketones are
compounds, dihalides, -halo nitriles [30e38].
a
a
among the most versatile intermediates in organic synthesis
[39e44] and their high reactivity makes them prone to react with
large number of nucleophiles to provide a variety of biologically
active heterocyclic compounds. In an important development, it
has been observed that
a,
a-dihalo ketones behave as synthetic
equivalents of their corresponding
a
-halo ketones [45e51]. Being
non-lachrymatory, generally solid at room temperature and soluble
in commonly used reaction solvents, these compounds are easy to
work with and can be handled easily.
In view of the above facts, it was considered worthwhile to
Abbreviations: MIC, Minimum inhibitory concentration; B. subtilis, Bacillus
subtilis; E. coli, Escherichia coli; P. aeruginosa, Pseudomonas aeruginosa; S. aureus,
Staphylococcus aureus; A. niger, Aspergillus niger; A. flavus, Aspergillus flavus;
Penicillium sp., Penicillium species; s, singlet; d, doublet; t, triplet; dd, doublet of
doublet; m, multiplet; dec, decomposition.
* Corresponding author. Tel.: þ91 1744 239617; fax: þ91 1744 238277.
E-mail addresses: dromprakash50@rediffmail.com (O. Prakash), dk_aneja@
rediffmail.com (D.K. Aneja).
extend the application of
a-halo and a,a-dihalo ketones based
methodology to the synthesis of two series of polycyclic fused
thiadiazines containing 1,2,4-triazoles namely, dihydroindeno and
indeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazines of the type
9 and 11 to evaluate their antimicrobial activity. The structure of
the compounds 9 and 11 were modified from 6-phenyl-7H-[1,2,4]
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.019

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O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
triazolo [3,4-b] [1,3,4]thiadiazines (A), which were known to
the reaction of a-bromoindanones (7aeb) with 3-alkyl/aryl-
possess good antimicrobial activity (Scheme 1).
4-amino-5-mercapto-1,2,4-s-triazoles (3aef) in methanol. IR
spectra of the products showed disappearance of bands corre-
sponding to CO and NH2 stretch and characterization by the spec-
tral (1H NMR, 13C NMR, IR) and elemental analytical data of the
products confirmed the formation of 3-alkyl/aryl-9-methyl-
10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
zines (9ael) (Scheme 2) [59]. For example, 1H NMR of the
R'
N
N
N
R'
N
N
N
N
S
N
compound 9h showed dd of three protons at
d value ranges
Close ring having
single bond
between 2.850 and 4.236 ppm apart from other aliphatic and
aromatic signals (see Supplementary materials).
S
9
R'
Then, we carried out reaction of a,a-dibromoindanones (8aeb)
N
with 3-alkyl/aryl-4-amino-5-mercapto-1,2,4-s-triazoles (3aef).
During the progress of the reaction, it was observed that colour of
the reaction mixture changed from colourless to red. The reactions
were monitored by tlc and the formation of two products were
observed in each case. Column chromatography using silica gel
(100e200 mesh) as stationary phase and pet ether (boiling range
60e80 ꢀC)/ethyl acetate as mobile phase was run for separation of
mixture of products. From tlc comparison, data analysis and melting
points determination it was found that the one of the product 9 in all
cases was identical to that obtained from cyclocondensation of
N
Open ring
N
A
N
S
Close ring having
double bond
11
Scheme 1. Modification in the series.
2. Results and discussion
a
-bromoindanones (7aeb) and substituted 3-alkyl/aryl-4-amino-
5-mercapto-1,2,4-s-triazoles (3aef). The complete spectral (1H
NMR, 13C NMR, IR) and elemental analytical data of the second
product confirmed the formation of new 3-alkyl/aryl-9-
methylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazines
(11ael) rather than expected alkoxy derivatives (10) (Scheme 2) as
2.1. Chemistry
First, we synthesized
a
-bromo and
a,
a
-dibromoindanone by
using bromine (1.1 eq) in dichloromethane (DCM) and by refluxing
indanone with bromine (2.2 eq) in acetic acid. Then, we performed
O
O
Br2 (1.1 equiv.)
DCM/ RT/ 12h
Br
R
R
7a: R = H
7b: R = CH3
6a: R = H
6b: R = CH3
N
N
Br2 (2.2 equiv.)
R'
SH
MeOH/reflux
N
NH2
3a-f
Acetic acid/ reflux 1h
O
R'
N
S
Br
N
N
Br
N
R
8a: R = H
8b: R = CH3
R
9a-l
N
N
R'
SH
MeOH/reflux
N
NH2
3a-f
R'
N
S
R'
N
S
R'
N
S
N
N
N
N
N
N
N
N
N
+
O
R
R
R
9a-l
11a-l
10
Scheme 2. Synthesis of the title compounds 9ael and 11ael.

O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
5067
Table 1
reported earlier in the case of
a,
a
-dibromoacetophenones (2) [38]
(Scheme 3). A careful comparison of 1H NMR of 11ael and 9ael
Physical data of the synthesized compounds 9ael and 11ael.
showed that doublet of doublet (dd) of three protons which
R'
4
N
R'
4
N
3
3
appeared at
d value in the range of 3.1e3.3, 3.6e3.8 and 4.1e4.4 of
N 2
N1
N 2
N1
5
N
5N
9ael disappeared in 11ael. Instead, a singlet integrating to one
6
6
proton appeared in the range of 6.9e7.1. For example, 1H NMR of the
7
8
7
8
10aS
S
compound 11h showed a singlet at
d value 7.082 ppm apart from
10a
other characteristic signals (see Supplementary materials).
It is worthwhile here to mention that the reaction of
10
9
10
R9
R
11
9
a
-bromoacetophenone (1) with 4-amino-5-mercapto-s-triazoles
(3) in ethanol under reflux gave triazolothiadiazines (4) which is
reported in the literature [37]. In our previous work dealing with
the reactivity of a,a-dibromoacetophenones (2) with 4-amino-5-
Entry
R
R0
Product
Time
(h)
Yield
(%)
Mp
(ꢀC)
mercapto-s-triazoles (3), we arrived at the result that fate of reac-
tion is decided by the alcohol employed in the reaction (Scheme 3)
[38]. In case of ethanol and i-propanol only alkoxy derivatives 5
were formed. However in case of methanol and propanol, in
addition to alkoxy derivatives, reduced products were also formed
(Scheme 3). In view of this, we continued our studies and per-
1
2
3
4
5
6
7
8
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
Me
Et
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
5.0
5.0
4.5
4.0
4.5
5.0
5.0
5.0
4.5
4.0
4.5
5.0
7.0
6.0
6.5
6.0
6.5
6.5
6.0
6.5
7.0
6.5
6.5
6.5
92a/50b
90a/60b
93a/62b
84a/55b
86a/40b
89a/58b
90a/52b
92a/62b
91a/63b
84a/58b
83a/32b
88a/60b
25b
215e216
154e155
115e116
129e130
114e115
214e215
207e208
219e220
223e225
80e81
187e188
w255 (dec)
154e156
163e165
130e132
133e135
w160 (dec)
169e171
202e204
239e241
167e169
151e152
w165 (dec)
210e212
Pr
i-Pr
Ph
H
Me
Et
formed the reaction of a-bromoindanones and a,a-dibromoinda-
nones (8aeb) with various substituted 3-alkyl/aryl-4-amino-5-
mercapto-1,2,4-s-triazoles (3aef). Based on our earlier observa-
tions, it was anticipated that this reaction might afford
triazolothiadiazines 9 and their alkoxy derivatives 10. However, the
reaction did not follow similar trends and in this case in addition to
expected dihydroindeno triazolothiadiazines 9, we obtained new
indeno triazolothiadiazines 11 (Scheme 2).
Possible mechanistic pathways for the conversions of 8 to 9 and
11 are analogous to the literature report on the reactions of
a,a-dibromoacetophenones (2) (For detailed mechanism see
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Pr
i-Pr
Ph
H
Me
Et
30b
32b
Pr
31b
i-Pr
Ph
H
Me
Et
Pr
i-Pr
Ph
25b
H
30b
Me
Me
Me
Me
Me
Me
27b
28b
30b
Supplementary materials) [45,52,53].
The physical data of all the synthesized compounds are
summarized in Table 1.
32b
22b
28b
a
Isolated yield obtained from the reaction between 3 and 7.
Isolated yield obtained from the reaction between 3 and 8.
b
2.2. Biological results and discussion
2.2.1. In vitro antibacterial activity
Table 2
All the twenty four compounds 9ael and 11ael were screened
for their in vitro antibacterial activity against two Gram-positive
bacteria namely Staphylococcus aureus (MTCC 96), Bacillus subtilis
(MTCC 121) and two Gram-negative bacteria namely Escherichia
coli (MTCC 1652), Pseudomonas aeruginosa (MTCC 741) by agar well
diffusion method (Table 2). Standard antibiotics namely cipro-
floxacin was used for comparison of antibacterial activity shown by
compounds 9ael and 11ael. The results were recorded for each
tested compound as average diameter of zone of inhibition of
bacterial growth adjoining the well in millimetres (Table 2, Fig. 1).
Minimum inhibitory concentration (MIC) measurements were
In vitro antibacterial activity of the compounds 9ael and 11ael.
Entry Compounds
Diameter of growth of inhibition zone (mm)a
Staphylococcus Bacillus Escherichia Pseudomonas
aureus
subtilis
coli
aeruginosa
1
2
3
4
5
6
7
8
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
15.3
14.6
25.3
27.3
28.6
20.6
26.3
27.6
26.6
25.3
28.6
22.3
14.3
14.6
16.3
25.6
15.6
14.6
25.3
20.3
21.6
16.3
17.6
18.6
17.6
16.3
21.6
25.0
24.6
16.3
30.6
31.3
28.0
21.6
27.0
21.6
16.0
15.3
17.6
24.3
17.3
15.0
28.6
18.6
23.0
18.6
19.3
22.6
24.0
e
e
e
e
18.3
19.3
20.6
18.3
22.3
20.6
21.0
19.3
21.6
17.3
e
16.0
18.6
19.3
14.6
20.3
18.6
19.3
17.6
20.3
15.6
e
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
e
e
e
e
18.6
e
16.3
e
e
e
20.6
18.6
19.3
15.6
15.0
15.6
25.0
18.3
15.6
17.3
14.3
14.6
15.3
22.0
Ciprofloxacin 26.6
Scheme 3. Reaction of 3b with
nones 2.
a
-bromoacetophenones 1 and
a
,
a-dibromoacetophe-
e: No activity.
a
Values including diameter of the well (8 mm) are means of three replicates.

5068
O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
S. aureus
B. subtilis
E. coli
P. aeruginosa
35
30
25
20
15
10
5
0
9f
9i
9j
9l
9c
9h
9k
9a
9b
9d
9e
9g
11j
11l
11f
11i
11a
11b
11c
11d
11e
11g
11h
11k
Ciprofloxacin
Compounds/Standard drug
Fig. 1. Comparisons of diameter of growth of inhibition zone (mm) of compounds 9ael and 11ael with standard drug ciprofloxacin.
performed using a modified agar well diffusion method (Table 3,
Fig. 2). MIC of those compounds was determined which were
showing activity in primary screening.
against B. subtilis showing maximum zone of inhibition >20.0 mm
as compared to standard drug ciprofloxacin which showed the zone
of inhibition of 24.0 mm against B. subtilis. In this case also, out of
these thirteen compounds eight compounds 9dee, 9gei, 9k, 11d
and 11g showed greater activity than the standard drug. However,
in case of the Gram-negative bacteria all the compounds showed
moderate to good antibacterial activity against the test bacteria
E. coli and P. aeruginosa as shown in Table 2. In case of E. coli, six
compounds 9e, 9gei, 9k and 11g showed maximum zone of inhi-
bition >20 mm in comparison to ciprofloxacin which showed
25 mm. Furthermore, in case of P. aeruginosa, seven compounds
9dee, 9gei, 9k and 11g sowed the maximum zone of inhibition
>18 mm in comparison to standard drug, ciprofloxacin, which
showed 22 mm (Table 2, Fig. 1).
Results revealed that in general, all the tested compounds
possessed moderate to excellent antibacterial activity against both
Gram-positive (S. aureus, B. subtilis) and Gram-negative bacteria
(E. coli, P. aeruginosa). However, out of the twenty four compounds
tested only seven compounds 9aeb, 11aec and 11eef did not show
activity against Gram-negative bacteria (E. coli, P. aeruginosa). On
the basis of zone of inhibition against test bacterium, eleven
compounds 9cee, 9gel, 11d and 11h showed excellent activity
against S. aureus showing the maximum zone of inhibition
>25.0 mm as compared with standard drug ciprofloxacin which
showed the zone of inhibition of 26.6 mm against S. aureus. It
should be noted that out of these eleven compounds, four
compounds 9dee, 9i and 9k were found to be most potent member
showing zone of inhibition against S. aureus even greater than the
standard drug (Table 2, Fig. 1).
In the whole series, MIC of the synthesized compounds ranged
between 2 and 256
(Table 3, Fig. 2). In case of S. aureus, compounds 9dee, 9gei, 9k are
most potent member having MIC ranged between 4 and 8 g/mL in
comparison to standard drug having MIC of 5 g/mL. Other
compounds showing good activity are 9c, 9j and 11d having MIC of
16 g/mL and the compounds showing moderate activity are 9c, 9j,
9l, 11i and 11l having MIC of 32 g/mL. In case of B. subtilis, most
potent members having MIC ranged between 2 and 8 g/mL are
9gei, 9k and 11g. Other compounds showing good to moderate
activity having MIC ranged between 16 and 32 g/mL are 9cee, 9j,
mg/mL against the Gram-positive bacteria
m
Similarly, on the basis of zone of inhibition thirteen compounds
9cee, 9gel, 11d, 11g, 11i, and 11l were found to be most effective
m
m
m
Table 3
m
MIC of the compounds 9ael and 11ael.
Entry Compounds
Minimum inhibitory concentration (MIC) in
mg/mL
m
9l, 11d, 11i, and 11l. All other compounds showed reasonable
Staphylococcus Bacillus Escherichia Pseudomonas
aureus
subtilis
coli
aeruginosa
activity against Gram-positive bacteria. In case of Gram-negative
bacteria, MIC ranged between 32 and 256
potent members among all the tested compounds are 9g, 9i and 9k
having MIC of 32 g/mL (Table 3, Fig. 2).
A comparison of the activity data of the compounds 9ael and
11ael indicate that the compounds 9ael are more potent than the
compounds 11ael. Further, it is also noteworthy that the
substituted indanones 9gel are more biologically active than
parent indanones (9aef).
mg/mL and the most
1
2
3
4
5
6
7
8
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
128
256
16
8
4
64
8
8
8
16
4
32
256
256
128
16
128
256
16
64
32
128
128
64
5
128
128
32
16
16
128
2
2
4
32
8
32
128
256
128
16
128
256
4
64
32
64
64
32
5
e
e
e
e
64
64
64
64
32
64
32
64
32
128
e
128
64
32
256
32
64
32
64
32
128
e
m
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2.2.2. Antifungal activity
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
Ciprofloxacin
All the twenty four compounds 9a-l and 11a-l were also tested
for their in vitro antifungal activity against three fungal strains,
namely, A. niger, A. flavus and Penicillium species through
poisoned food method. Standard drug fluconazole was used for
comparison with antifungal activity shown by the compounds
9ael and 11ael and results were recorded as percentage (%) of
mycelial growth inhibition. It has been revealed from the Table 4
that all the compounds 9ael and 11ael showed variable anti-
fungal activity against three pathogens. From the careful
comparison of the results, it has been revealed that mainly the
three compounds 9h, 11g and 11j showed excellent antifungal
activity with >65% inhibition of mycelial growth against all the
e
e
e
e
64
e
128
e
e
e
64
64
64
128
128
128
5
128
128
64
256
256
128
5
e: Not tested.

O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
5069
S. aureus
B. subtilis
E. coli
P. aeruginosa
300
250
200
150
100
50
0
9f
9i
9j
9l
9a
9b
9c
9d
9e
9g
9h
9k
11f
11i
11j
11l
11a
11b
11c
11d
11e
11g
11h
11k
Compounds/Standard drug
Ciprofloxacin
Fig. 2. Comparison of MIC of the compounds 9ael and 11ael.
compounds with reference drug in terms of % mycelial growth
inhibition are also shown in Fig. 3.
Table 4
In vitro antifungal activity of the compounds 9ael and 11ael.
Entry Compounds Mycelial growth inhibition (%)
3. Conclusion
Aspergillus niger Aspergillus flavus Penicillium species
1
2
3
4
5
6
7
8
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
51.1
53.3
58.8
55.5
53.3
51.1
63.3
67.7
64.4
58.8
63.3
55.5
53.3
50.0
48.8
51.1
48.8
45.5
71.1
53.3
65.5
68.8
65.5
55.5
52.5
51.1
55.5
52.5
51.1
48.8
65.5
68.8
63.3
55.5
61.1
58.8
50
48.8
45.5
48.8
50
48.8
68.8
50
51.1
55.5
57.7
53.3
51.1
50.0
62.5
67.7
65.5
61.1
65.5
60
51.1
52.5
50.0
47.7
44.4
51.1
66.6
51.1
63.3
65.5
63.3
48.8
83.3
In conclusion, the present study offers an application of
-bromo and a,a-dibromoindanones in an efficient and conve-
a
nient synthesis of dihydroindeno and indeno [1,2-e] [1,2,4]triazolo
[3,4-b] [1,3,4]thiadiazines (9 and 11). Biological evaluation of these
compounds proves them potent antimicrobial agents. Some of the
compounds tested are superior to the reference drug. Thus, the
present work will have a good impact on chemist and bio-chemist
and can be further used in pharmaceutical industry for mankind,
as an antimicrobial agent, after testing its toxicity to human
beings.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4. Experimental
4.1. General
Melting points were taken on slides in an electrical apparatus
Labindia visual melting range apparatus and are uncorrected.
Benzoic acid was taken as reference for calibration of instrument.
The infrared spectra (IR) were recorded on a PerkineElmer 1800
FT-IR spectrophotometer using KBr Pellets technique. The 1H NMR
and 13C NMR spectra were recorded in CDCl3 on Bruker Nuclear
Magnetic Resonance (NMR) spectrometer at 300 and 75 MHz
respectively, using tetramethylsilane (TMS) as an internal standard.
62.5
65.5
61.1
51.1
77.7
Fluconazole 81.1
three fungi in comparison with the standard drug (Aspergillus
niger 81.1%, Aspergillus flavus 77.7% and Penicillium sp. 83.3%). In
addition to these, three more compounds which showed >65%
inhibition are 9g, 9i and 11i against A. flavus, Penicillium sp. and
A. niger respectively. There are many other compounds which
showed good and fair antifungal activities are summarized in
Table 4. Comparisons of antifungal activity of all the synthesized
Chemical shifts are expressed in
d ppm. Purification of the
compounds was done by the column chromatography using silica
gel (100e200 mesh) as stationary phase and petroleum ether/ethyl
acetate mixture as eluent. The purity of the compounds were
checked by elemental analysis and thin layer chromatography (TLC)
using silica gel aluminium plates of Merck using a mixture of
petroleum ether and ethyl acetate as mobile phase. Iodine and
A. niger
A. flavus
Penicillium sp.
90
80
70
60
50
40
30
20
10
0
9f
9i
9j
9l
9c
9k
9a
9b
9d
9e
9g
9h
11i
11j
11l
11f
11c
11k
11a
11b
11d
11e
11g
11h
Fluconazole
Compounds/Standard drug
Fig. 3. Comparisons of antifungal activity of compounds 9ael and 11ael with standard drug fluconazole.

5070
O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
Ultraviolet (UV) lamp was used for visualisation of the compounds.
All other chemicals used as such as procured from supplier. The
physical data of all the synthesized compounds are summarized in
Table 1.
133.90, 132.92, 128.72, 125.85, 123.67, 37.01, 35.06; Anal. Calcd. for
11H8N4S: C 57.88, H 3.53, N 24.54; Found: C 57.80, H 3.50, N 24.44.
C
4.4.1.2. 3-Methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (9b). IR (nmax, cmꢂ1): transparent in the region of
C]O str., 1612, 1535, 1466, 1435, 1373, 1304, 1203, 1149, 1095; 1H
4.2. Synthesis of a-bromoindanones (7aeb)
NMR (d, CDCl3, 300 MHz): 7.981 (bs, 1H, ArH), 7.599e7.489 (m, 3H,
4.2.1. 2-Bromo-2,3-dihydroinden-1-one (7a)
ArH), 4.163 (bs, 1H), 3.759e3.705 (m, 1H), 3.177e3.127 (m, 1H),
2.633 (s, 3H, CH3); 13C NMR: 163.46, 1587.23, 147.42, 133.57, 133.33,
128.53, 125.80, 123.57, 36.28, 34.95, 18.40; Anal. Calcd. for
C12H10N4S: C 59.48, H 4.16, N 23.12; Found: C 57.44, H 4.10, N 23.10.
To a solution of 1-indanone (6a, 5 g, 37.8 mmol) in 50 mL DCM,
bromine (5.5 g, 34.3 mmol) was added and the reaction mixture was
stirred at room temperature for 12 h. Reaction was monitored by
thin layer chromatography. After completion of reaction the reac-
tion mixture was quenched with water and separated the organic
layer. The organic layer was washed with saturated aqueous sodium
metabisulphite solution (30 mL ꢁ 3), brine and finally with water.
After evaporation of solvent a solid was obtained in good yield (7a,
7.29 g, 83%), Mp. 40e42 ꢀC(Lit. Mp.39e40 ꢀC) [39e41].
4.4.1.3. 3-Ethyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (9c). IR (nmax, cmꢂ1): transparent in the region of
C]O str., 1605, 1528, 1458, 1381, 1311, 1273, 1188, 1157, 1095; 1H
NMR (
d
, CDCl3, 300 MHz): 8.005e7.979 (d, 1H, ArH, J ¼ 7.8 Hz),
7.627e7.580 (m, 1H, ArH), 7.496 (m, 2H, ArH), 4.243e4.153 (m, 1H),
3.763e3.677 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 17.4 Hz), 3.193e2.946 (m, 3H),
1.481e1.431 (t, 3H, CH3, J ¼ 7.5 Hz), 13C NMR: 162.36, 158.32, 147.42,
133.57, 133.33, 128.53, 125.80, 123.57, 36.28, 34.95, 18.40, 11.30;
Anal. Calcd. for C13H12N4S: C 60.91, H 4.72, N 21.86; Found: C 60.88,
H 4.67, N 21.78.
The other derivative 7b was prepared in a similar manner.
4.2.2. 2-Bromo-2,3-dihydro-4-methylinden-1-one (7b)
Mp. 69e71 ꢀC; yield 80%; IR (nmax, cmꢂ1): 1720 (C]O); 1H NMR
(
d
, CDCl3, 300 MHz,): 7.707e7.682 (d, 1H, ArH, J ¼ 7.5 Hz),
7.506e7.481 (m, 1H, ArH), 7.393e7.368 (m, 1H, ArH), 4.698e4.663
(dd, 1H, J1 ¼ 3.0 Hz, J2 ¼ 7.5 Hz), 3.797e3.712 (dd, 1H, J2 ¼ 7.5 Hz,
J3 ¼ 18 Hz), 3.349e3.289 (m, 1H), 2.360 (s, 3H, CH3).
4.4.1.4. 3-Propyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (9d). IR (nmax, cmꢂ1): transparent in the region of
C]O str., 1605,1528, 1458, 1389, 1311, 1265, 1196, 1149, 1095, 1057,
4.3. Synthesis of
a,
a-dibromoindanones (8aeb)
1026; 1H NMR (
d, CDCl3, 300 MHz): 7.980 (m, 1H, ArH), 7.578e7.475
(m, 3H, ArH), 4.166 (m, 1H), 3.696 (m, 1H), 3.160e3.127 (m, 1H),
2.975 (m, 2H, CH2),1.886 (m, 2H, CH2), 1.069 (m, 3H, CH3); 13C NMR:
162.61, 154.26, 147.43, 139.32, 133.58, 133.52, 128.52, 125.80, 123.56,
36.29, 34.94, 26.57, 20.48,13.84; Anal. Calcd. for C14H14N4S: C 62.20,
H 5.22, N 20.72; Found: C 62.17, H 5.18, N 20.69.
4.3.1. 2,2-Dibromo-2,3-dihydroinden-1-one (8a)
To a solution of 1-indanone (6a, 5 g, 37.8 mmol) in acetic acid
(50 mL), bromine (13.3 g, 83.3 mmol) was added and the solution
was refluxed for 1 h. After cooling to room temperature, the solu-
tion was poured on the crushed ice (100 g). A solid thus obtained
was filtered and washed with cold water and dried to give the
desired product 8a (9.66 g, 88%). Mp. 133 ꢀC (Lit. Mp. 131e134 ꢀC)
[42e44].
4.4.1.5. 3-Isopropyl-10,10a-dihydroindeno
[1,2-e]
[1,2,4]triazolo
[3,4-b] [1,3,4]thiadiazine (9e). IR (nmax, cmꢂ1): transparent in the
region of C]O str., 1605, 1520, 1458, 1389, 1304, 1196, 1157, 1095; 1H
The other derivative 8b was prepared in a similar manner.
NMR (d, CDCl3, 300 MHz): 7.976e7.951 (d, 1H, ArH, J ¼ 7.5 Hz),
7.585e7.560 (d, 1H, ArH, J ¼ 7.5 Hz), 7.489e7.415 (m, 2H, ArH),
4.195e4.149 (dd, 1H, J1 ¼ 5.4 Hz, J3 ¼ 8.4 Hz), 3.746e3.662 (m, 1H),
3.448e3.378 (m, 1H), 3.161e3.086 (dd, 1H, J1 ¼ 5.4 Hz, J2 ¼ 17.1 Hz),
1.502e1.491 (d, 3H, CH3, J ¼ 7.5 Hz), 1.426e1.402 (d, 3H, CH3,
J ¼ 7.5 Hz); 13C NMR: 162.11, 158.40, 147.47, 139.63, 133.60, 133.31,
128.52, 125.83, 123.52, 36.21, 34.95, 25.50, 20.81, 19.79; Anal. Calcd.
forC14H14N4S:C 62.20, H5.22, N 20.72;Found:C 62.18, H5.18 N20.66.
4.3.2. 2,2-Dibromo-2,3-dihydro-4-methylinden-1-one (8b)
Mp. 66e68 ꢀC; yield 85%; IR (nmax, cmꢂ1): 1728 (C]O str.);1H
NMR (
d
, CDCl3, 300 MHz): 7.807e7.782 (d, 1H, ArH, J ¼ 7.5 Hz),
7.566e7.542 (d, 1H, ArH, J ¼ 7.2 Hz), 7.45e7.4 (m, 1H, ArH), 4.213 (s,
2H, CH2), 2.337 (s, 3H, CH3).
4.4. Synthesis of 3-alkyl/aryl-9-methyl-10,10a-dihydroindeno
[1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazines (9ael)
4.4.1.6. 3-Phenyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (9f). IR (nmax, cmꢂ1): transparent in the region of
C]O str., 1597, 1466, 1366, 1296, 1196, 1165, 1103, 1072, 1026; 1H
4.4.1. Typical procedure
To a solution of 7a (1g, 3.31 mmol) in 30 mL ethanol was added
4-amino-5-mercapto-1,2,4-triazole (3a, 0.384 g, 3.31 mmol) and
the reaction mixture was refluxed for 4e5 h. The reaction mixture
was poured onto crushed ice (100 g), followed by basification with
ammonia solution. The solid product thus obtained was filtered,
washed with water and recrystalized from ethanol to give pure
10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
zine (9a, 0.67 g, 90%).
NMR (d, CDCl3, 300 MHz): 8.209 (m, 2H, ArH), 8.015e7.992 (m, 1H,
ArH), 7.612e7.489 (m, 6H, ArH), 4.232 (bs,1H), 3.815e3.731 (dd,1H,
J1 ¼ 8.1 Hz, J2 ¼ 17.1 Hz), 3.220e3.163 (m, 1H); 13C NMR: 163.05,
152.18, 147.63, 140.97, 133.81, 133.21, 130.23, 128.61, 128.57, 128.25,
126.21, 125.87, 123.78, 35.86, 34.98; Anal. Calcd. for C17H12N4S: C
67.08, H 3.97, N 18.41; Found: C 67.02, H 3.93, N 18.38.
4.4.1.7. 9-Methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (9g). IR (nmax, cmꢂ1): transparent in the region of
C]O str., 1597, 1481, 1443, 1373, 1288, 1149, 1065, 1034; 1H NMR
All other derivatives were synthesized by adopting the similar
procedure.
(d, CDCl3, 300 MHz): 8.637 (s, 1H, triazole H), 7.777e7.620 (bs, 1H,
4.4.1.1. 10,10a-Dihydroindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]
thiadiazine (9a). IR (nmax, cmꢂ1): transparent in the region of C]O
str., 1628, 1605, 1481, 1443, 1366, 1304, 1281, 1287, 1188, 1157, 1095;
ArH), 7.415e7.312 (m, 2H, ArH), 4.250e4.207 (m, 1H), 3.696e3.612
(dd, 1H, J ¼ 8.1 Hz, J ¼ 16.5 Hz), 3.137e3.005 (m, 1H), 2.377 (s, 3H,
CH3); 13C NMR: 163.80, 146.72, 142.25, 139.96, 135.44, 134.48,
132.60, 128.97, 121.01, 36.91, 33.92, 18.35; Anal. Calcd. for
C12H10N4S: C 59.48, H 4.16, N 23.12; Found: C 59.44, H, 4.10, N
23.08.
1H NMR (
d, CDCl3, 300 MHz): 8.758 (s, 1H, triazole H), 7.954 (m, 1H,
ArH), 7.601e7.494 (m, 3H, ArH), 4.414e4.398 (m, 1H), 3.823e3.731
(m, 1H), 3.193e3.026 (m, 1H); 13C NMR: 163.15, 158.45, 147.56,

O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
5071
4.4.1.8. 3-Methyl-9-methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]tri-
azolo [3,4-b] [1,3,4]thiadiazine (9h). IR (nmax, cmꢂ1): transparent in
the region of C]O str., 1597, 1535, 1458, 1373, 1311, 1203, 1057; 1H
ether-ethyl acetate (4:1) as eluent to give 9a (0.413 g, 50%) and 11a
(0.206 g, 25%).
All other derivatives were synthesized by adopting the similar
procedure and purified by column chromatography.
NMR (d, CDCl3, 300 MHz): 7.851e7.802 (m, 1H, ArH); 7.412e7.398
(m, 2H, ArH), 4.184e4.138 (dd, 1H, J1 ¼ 5.4 Hz, J3 ¼ 8.4 Hz),
3.690e3.605 (dd, 1H, J2 ¼ 16.8 Hz, J3 ¼ 8.4 Hz), 3.053e2.979 (dd,1H,
4.5.1.1. Indeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazine
J1 ¼ 5.4 Hz, J2 ¼ 16.8 Hz), 2.636 (s, 3H, CH3), 2.380 (s, 3H, CH3); 13
C
(11a). IR (nmax, cmꢂ1, neat): transparent in the region of C]O str.,
NMR: 162.97, 150.10, 146.56, 138.81, 134.96, 133.68, 132.34, 128.24,
120.25, 35.64, 33.18, 17.85, 9.80; Anal. Calcd. for C13H12N4S: C 60.91,
H 4.72, N 21.86. Found: C 60.85, H 4.67, N 21.77.
1605, 1535, 1450, 1388, 1319; 1H NMR (
d, CDCl3, 300 MHz): 8.791 (s,
1H, triazole H), 7.951e7.432 (m, 4H, ArH), 6.985 (s, 1H,¼CH), 13C
NMR: 151.26, 142.75, 134.91, 132.45, 130.28, 128.05, 126.07, 122.44,
121.13, 114.88; Anal. Calculated for C11H6N4S: C 58.39, H 2.67, N
24.76; Found: C 58.26, H 2.60, N 24.69.
4.4.1.9. 3-Ethyl-9-methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]triazolo
[3,4-b] [1,3,4]thiadiazine (9i). IR (nmax, cmꢂ1): transparent in the
region of C]O str., 1636, 1597, 1528, 1458, 1381, 1350, 1311 1281,
4.5.1.2. 3-Methylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
1203, 1165, 1065, 1034; 1H NMR (
d
, CDCl3, 300 MHz): 7.825e7.799
zine (11b). IR (nmax, cmꢂ1, neat): transparent in the region of C]O
(m, 1H, ArH), 7.403e7.390 (m, 2H, ArH), 4.190e4.145 (m, 1H),
3.683e3.598 (dd, 1H, J1 ¼ 8.4 Hz, J2 ¼ 17.1 Hz), 3.131e2.910 (m, 3H),
2.376 (s, 3H, CH3), 1.470e1.421 (t, 3H, CH3, J ¼ 7.5 Hz), 13C NMR:
162.70, 155.30, 146.51, 139.50, 135.35, 134.13, 133.06, 128.82, 120.92,
36.20, 33.81, 18.41, 18.35, 11.30; Anal. Calcd. for C14H14N4S: C 62.20,
H 5.22, N 20.72; Found: C 62.16, H 5.18, N 20.68.
str., 1605, 1532, 1450, 1389, 1319; 1H NMR (
d, CDCl3, 300 MHz):
7.948e7.923 (d, 1H, ArH, J ¼ 7.5 Hz), 7.502e7.452 (m, 1H, ArH),
7.330e7.306 (m, 2H, ArH), 6.982 (s, 1H, ¼CH), 2.734 (s, 3H, CH3); 13
C
NMR: 152.85, 151.25, 142.75, 139.27, 132.45, 130.28, 128.05, 126.06,
122.43, 121.13, 114.87, 10.13; Anal. Calculated for C12H8N4S: C 59.98,
H 3.36, N 23.32; Found: C 59.92, H 3.28, N 23.29.
4.4.1.10. 3-Propyl-9-methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]tri-
azolo [3,4-b] [1,3,4]thiadiazine (9j). IR (nmax, cmꢂ1): transparent in
the region of C]O str.,1597,1520,1458,1389,1342,1304,1273,1203,
4.5.1.3. 3-Ethylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazine
(11c). IR (nmax, cmꢂ1, neat): transparent in the region of C]O str.,
1605, 1535, 1450, 1388, 1319; 1H NMR (
d, CDCl3, 300 MHz):
1034; 1H NMR (
d, CDCl3, 300 MHz): 7.800e7.293 (m, 3H, ArH),
7.934e7.909 (d, 1H, ArH, J ¼ 7.5 Hz), 7.492e7.442 (m, 1H, ArH),
7.321e7.272 (m, 2H, ArH), 6.971 (s, 1H, ¼CH), 3.166e3.090 (q, 2H,
CH2, J ¼ 7.5 Hz), 1.520e1.470 (t, 3H, CH3, J ¼ 7.5 Hz); 13C NMR:
155.44, 152.67, 142.76, 134.97, 132.42, 130.32, 128.0, 126.03, 122.38,
121.11,114.95,18.19,11.34; Anal. Calculated for C13H10N4S: C 61.40, H
3.96, N 22.03; Found: C 61.35, H 3.89, N 21.95.
4.230e4.201 (m, 1H), 3.720e3.510 (m, 1H), 3.101e2.910 (m, 3H),
2.245 (s, 3H, CH3), 1.928e1.801 (m, 2H, CH2), 1.102e0.912 (m, 3H,
CH3); 13C NMR: 163.42,146.70,139.77,135.42,134.35,132.88,128.85,
121.01, 36.19, 33.85, 26.45, 20.43, 18.36, 13.86; Anal. Calcd. for
C15H16N4S: C 63.35, H 5.67, N 19.70. Found: C 63.30, H 5.64, N 19.65.
4.4.1.11. 3-Isopropyl-9-methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]
triazolo [3,4-b] [1,3,4]thiadiazine (9k). IR (nmax, cmꢂ1): transparent
in the region of C]O str., 1597, 1520, 1458, 1389, 1350, 1311, 1281,
4.5.1.4. 3-Propylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
zine (11d). IR (nmax, cmꢂ1, neat): transparent in the region of C]O
str., 1605, 1535, 1450, 1389, 1319; 1H NMR (
d, CDCl3, 300 MHz):
1203, 1157, 1095, 1065, 1034; 1H NMR (
d
, CDCl3, 300 MHz):
7.800e7.293 (m, 3H, ArH), 4.217 (bs, 1H), 3.682e3.450 (m, 2H),
3.025e2.969 (m, 1H), 1.602e1.501 (m, 6H, (CH3)2), 2.865 (s, 3H); 13
7.946e7.922 (d, 1H, ArH, J ¼ 7.2 Hz), 7.498e7.447 (m, 1H, ArH),
7.328e7.304 (m, 2H, ArH), 6.979 (s, 1H, ¼CH), 3.115e3.064 (t, 2H,
CH2, J ¼ 7.5 Hz), 2.015e1.717 (m, 2H, CH2), 1.129e1.080 (t, 3H, CH3,
J ¼ 7.5 Hz); 13C NMR: 155.41, 152.66, 142.77, 134.85, 132.41, 130.35,
127.97, 126.02, 122.39, 121.11, 114.99, 26.29, 20.53, 13.84; Anal.
Calculated for C14H12N4S: C 62.66, H 4.51, N 20.88; Found: C 62.60,
H 4.44, N 20.83.
C
NMR: 162.76, 158.32, 146.60, 139.76, 135.38, 134.19, 133.01, 128.81,
120.89, 36.13, 33.83, 25.49, 20.80, 19.77, 18.35; Anal. Calcd. for
C15H16N4S: C 63.35, H 6.67, N 19.70; Found: C 63.28, H 6.62, N 19.65.
4.4.1.12. 3-Phenyl-9-methyl-10,10a-dihydroindeno [1,2-e] [1,2,4]tri-
azolo [3,4-b] [1,3,4]thiadiazine (9l). IR (nmax, cmꢂ1): transparent in
the region of C]O str., 1597, 1528, 1458, 1373, 1304, 1203, 1119,
4.5.1.5. 3-Isopropylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thia-
diazine (11e). IR (nmax, cmꢂ1, neat): transparent in the region of
1034; 1H NMR (
d
, CDCl3, 300 MHz): 8.612e7.441 (m, 8H, ArH), 4.274
C]O str., 1605, 1534, 1450, 1389, 1319; 1H NMR (
d, CDCl3,
(bs, 1H), 3.687 (bs, 1H), 3.070e3.019 (m, 1H), 2.384 (s, 3H, CH3); 13
C
300 MHz): 7.939e7.915 (d, 1H, ArH, J ¼ 7.2 Hz), 7.668e7.303 (m,
3H, ArH), 6.978 (s, 1H, ¼CH), 3.582e3.542 (m, 1H, CH),
1.574e1.270 (6H, m, (CH3)2); 13C NMR: 155.87, 154.25, 132.41,
127.97, 126.02, 122.34, 121.12, 114.70, 25.33, 22.68, 20.37; Anal.
Calculated for C14H12N4S: C 62.66, H 4.51, N 20.88; Found: C 62.62,
H 4.45, N 20.81.
NMR: 161.45, 157.22, 146.43, 134.35, 132.96, 130.22, 128.90, 128.57,
128.23, 121.15, 35.79, 33.91, 18.39; Anal. Calcd. for C18H14N4S: C
67.90, H 4.43, N 17.60; Found: C 67.84, H 4.40, N 17.56.
4.5. Synthesis of 3-alkyl/aryl-9-methylindeno [1,2-e] [1,2,4]triazolo
[3,4-b] [1,3,4]thiadiazines (11ael)
4.5.1.6. 3-Phenylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
zine (11f). IR (nmax, cmꢂ1, neat): transparent in the region of C]O
4.5.1. Typical procedure
str., 1605, 1520, 1450, 1373, 1319; 1H NMR (
d, CDCl3, 300 MHz):
To a solution of 8a (1 g, 3.45 mmol) in 50 mL methanol was
added 4-amino-5-mercapto-1,2,4-triazole (3a, 0.79 g, 6.90 mmol)
and the reaction mixture was refluxed for 5 h. The colour of the
reaction mixture changed from colourless to red as the reaction
proceeded. The reaction mixture was poured onto crushed ice
(100 g), followed by basification with ammonia solution to give the
crude product as red solid, filtered, washed with water and dried
(mixture of the two products by tlc). The crude product was
chromatographed over silica gel (100e200 mesh) using petroleum
8.268e8.235 (m, 2H, ArH), 7.930e7.904 (d, 1H, ArH, J ¼ 7.8 Hz),
7.606e7.559 (m, 3H, ArH), 7.501e7.447 (m, 1H, ArH), 7.321e7.271
(m, 2H, ArH), 7.014 (s, 1H, ¼CH); 13C NMR: 153.09, 152.25, 142.78,
135.84, 132.57,130.58, 130.32, 128.79, 128.66,128.29, 126.12, 125.70,
122.58, 121.19, 114.35; Anal. Calculated for C17H10N4S: C 67.53, H
3.33, N 18.53; Found: C 67.47, H 3.29, N 18.51.
4.5.1.7. 9-Methylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thiadia-
zine (11g). IR (nmax, cmꢂ1, neat): transparent in the region of C]O

5072
O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
str., 1620, 1474, 1443, 1381, 1327; 1H NMR (
d
, CDCl3, 300 MHz):
were subcultured on Nutrient agar whereas fungi on Sabouraud
dextrose agar.
8.783 (s, 1H, triazole H), 7.762e7.738 (d, 1H, ArH, J ¼ 7.2 Hz),
7.310e7.194 (m, 2H, ArH), 7.105 (s, 1H, ¼CH), 2.423 (s, 3H, CH3); 13
C
NMR: 154.14, 142.71, 141.28, 135.46, 134.20, 130.72, 129.86, 127.26,
126.37, 120.30, 114.46, 14.11; Anal. Calculated for C12H8N4S: C 59.98,
H 3.36, N 23.32; Found: C 59.95, H 3.32, N 23.28.
4.6.2. Antibacterial activity
The antibacterial activity of twenty four compounds was eval-
uated by the agar well diffusion method. All the microbial cultures
were adjusted to 0.5 McFarland standard, which is visually
4.5.1.8. 3,9-Dimethylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]thia-
comparable to
a
microbial suspension of approximately
diazine (11h). IR (nmax, cmꢂ1, neat): transparent in the region of
1.5 ꢁ 108 cfu/mL 20 mL of Mueller Hinton agar medium was poured
C]O str.,1605,1543,1458, 1381,1327; 1H NMR (
d
, CDCl3, 300 MHz):
into each petri plate and plates were swabbed with 100 mL inocula
7.796e7.772 (d, 1H, ArH, J ¼ 7.2 Hz), 7.3e7.28 (m, 1H, ArH),
7.238e7.188 (m, 1H, ArH), 7.082 (s, 1H, ¼CH), 2.735 (s, 3H, CH3),
2.424 (s, 3H, CH3); 13C NMR: 154.24, 153.16, 142.61, 141.27, 133.91,
130.53, 126.54, 126.12, 120.10, 14.11, 10.15; Anal. Calculated for
C13H10N4S: C 61.40, H 3.96, N 22.03; Found: C 61.37, H 3.91, N 21.98.
of the test microorganisms and kept for 15 min for adsorption.
Using sterile cork borer of 8 mm diameter, wells were bored into
the seeded agar plates and these were loaded with a 100 mL volume
with concentration of 2.0 mg/mL of each compound reconstituted
in the dimethylsulphoxide (DMSO). All the plates were incubated at
37 ꢀC for 24 h. Antibacterial activity of each synthetic compound
was evaluated by measuring the zone of growth inhibition against
the test organisms with zone reader (Hi Antibiotic zone scale).
DMSO was used as a negative control whereas ciprofloxacin was
used as positive control. This procedure was performed in three
replicate plates for each organism [55,56].
4.5.1.9. 3-Ethyl-9-methylindeno [1,2-e] [1,2,4]triazolo [3,4-b] [1,3,4]
thiadiazine (11i). IR (nmax, cmꢂ1, neat): transparent in the region of
C]O str., 1620, 1458, 1389, 1311; 1H NMR (
d, CDCl3, 300 MHz):
7.766e7.742 (d, 1H, ArH, J ¼ 7.2 Hz), 7.255e7.167 (m, 2H, ArH), 7.053
(s, 1H, ¼CH), 3.154e3.04 (q, 2H, CH2, J ¼ 7.5 Hz), 2.406 (s, 3H, CH3),
1.517e1.467 (t, 3H, CH3, J ¼ 7.5 Hz); 13C NMR: 154.22, 153.11142.81,
141.38, 126.23, 120.06, 17.90, 11.34; Anal. Calculated for C14H12N4S:
C 62.66, H 4.51, N 20.88; Found: C 62.62, H 4.46, N 20.84.
4.6.3. Determination of minimum inhibitory concentration (MIC)
MIC is the lowest concentration of an antimicrobial compound
that will inhibit the visible growth of a microorganism after over-
night incubation. MIC of the various compounds against bacterial
strains was tested through a modified agar well diffusion method
[57]. In this method, a two fold serial dilution of each compound was
prepared by first reconstituting the compound in DMSO followed by
dilution in sterile distilled water to achieve a decreasing concen-
4.5.1.10. 9-Methyl-3-propylindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (11j). IR (nmax, cmꢂ1, neat): transparent in the
region of C]O str., 1620, 1458, 1389, 1319; 1H NMR (
d, CDCl3,
300 MHz): 7.788e7.764 (d, 2H, ArH, J ¼ 7.2 Hz), 7.293e7.181 (m, 2H,
ArH), 7.074 (s, 1H, ¼CH), 3.113e3.062 (t, 2H, CH2, J ¼ 7.5 Hz), 2.419
(s, 3H, CH3,), 2.014e1.890 (m, 2H, CH2), 1.129e1.080 (t, 3H, CH3,
J ¼ 7.5 Hz); 13C NMR: 154.38, 153.05, 141.36, 134.96, 133.88, 130.51,
126.50,120.06,114.13, 26.29, 20.52,17.78,13.84; Anal. Calculated for
C15H14N4S: C 63.80, H 5.0, N 19.84; Found: C 63.77, H 4.95, N 19.81.
tration range of 256 to 0.5
was introduced into wells (in triplicate) in the agar plates already
seeded with 100
L of standardized inoculum (106 cfu/mL) of the test
mg/mL. A 100 mL volume of each dilution
m
microbial strain. All test plates were incubated aerobically at 37 ꢀC
for 24 h and observed for the inhibition zones. MIC, taken as the
lowest concentration of the chemical compound that completely
inhibited the growth of the microbe, showed by a clear zone of
inhibition, was recorded for each test organism. Ciprofloxacin was
used as positive control while DMSO as negative control.
4.5.1.11. 3-Isopropyl-9-methylindeno [1,2-e] [1,2,4]triazolo [3,4-b]
[1,3,4]thiadiazine (11k). IR (nmax, cmꢂ1, neat): transparent in the
region of C]O str., 1620, 1458, 1384, 1311; 1H NMR (
d, CDCl3,
300 MHz): 7.923e7.322 (m, 3H, ArH), 6.892 (s, 1H, ¼CH),
3.572e3.531 (m, 1H, CH), 2.5 (s, 3H, CH3), 1.583e1.258 (m, 6H,
(CH3)2); 13C NMR: 155.87, 154.25, 132.41, 127.97, 126.02, 122.34,
121.12, 114.70, 25.33, 22.68, 20.37, 14.24; Anal. Calculated for
C15H14N4S: C 63.80, H 5.0, N 19.84; Found: C 63.76, H 4.95, N 19.80.
4.6.4. Antifungal activity
The antifungal activity of chemical compounds was evaluated by
poisoned food technique. The moulds were grown on Sabouraud
dextrose agar (SDA) at 25 ꢀC for 7 days and used as inocula. The
4.5.1.12. 9-Methyl-3-phenylindeno [1,2-e] [1,2,4]triazolo [3,4-b]
15 mL of molten SDA (45 ꢀC) was poisoned by the addition of 100
mL
[1,3,4]thiadiazine (11l). IR (nmax, cmꢂ1, neat): transparent in the
volume of each compound having concentration of 4.0 mg/mL
reconstituted in the DMSO, poured into a sterile Petri plate and
allowed it to solidify at room temperature. The solidified poisoned
agar plates were inoculated at the centre with fungal plugs (8 mm
diameter) obtained from the colony margins and incubated at
25 ꢀC for 7 days. DMSO was used as the negative control whereas
fluconazole was used as the positive control. The experiments were
performed in triplicates. Diameter of fungal colonies was measured
and expressed as percent mycelial inhibition by applying the
formula [58].
region of C]O str., 1605, 1543, 1458, 1381, 1327; 1H NMR (
d, CDCl3,
300 MHz): 8.081e7.842 (m, 4H, ArH), 7.422e7.396 (m, 4H, ArH),
7.087 (s, 1H, ¼CH) 2.5 (s, 3H, CH3); 13C NMR: 163.54, 152.12, 146.71,
141.03,135.43,134.34,139.95,130.20,128.89,128.53,128.22,126.23,
121.12, 18.35; Anal. Calculated for C18H12N4S: C 68.33, H 3.82, N
17.71; Found: C 68.28, H 3.78, N 17.67.
4.6. Antimicrobial assay
4.6.1. Test microorganisms
Percent inhibition of myelial growth ¼ ðdc ꢂ dtÞ=dc ꢁ 100
Total seven microbial strains were selected on the basis of their
clinical importance in causing diseases in humans. Two Gram-
positive bacteria (S. aureus MTCC 96 and B. subtilis MTCC 121); two
Gram-negative bacteria (E. coli MTCC 1652 and P. aeruginosa MTCC
741) and three fungi, (A. niger, A. flavus and Penicillium sp.) the ear
pathogens isolated from the patients of Kurukshetra [54] were used
in the present study for evaluation of antimicrobial activity of the
compounds. All the bacterial cultures were procured from Microbial
Type Culture Collection (MTCC), IMTECH, Chandigarh. The bacteria
where dc ¼ average diameter of fungal colony in negative control
plates; dt ¼ average diameter fungal colony in experimental plates.
Acknowledgements
We are thankful to Council of Scientific & Industrial Research,
New Delhi for providing financial assistance to carry out this work.

O. Prakash et al. / European Journal of Medicinal Chemistry 46 (2011) 5065e5073
5073
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Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.08.019.
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