α,α-Dibromoacetophenones mediated synthesis of some new 7H-7-alkoxy-3-alkyl/phenyl-6-aryl-s-triazolo[3,4-b][1,3,4] thiadiazines and their antimicrobial evaluation

Article abstract of DOI:10.1007/s00044-011-9945-1

Aseriesofnew7H-7-alkoxy-3-alkyl/phenyl-6-aryl-s-triazolo[3,4-b][1,3,4]thiadiazines (3, 4) were synthesized by the reaction of various α,α-dibromoacetophenones 1 with 3-alkyl/phenyl-4-amino-5-mercapto-s-triazoles (2) in different alcoholic solvents in good

Full text of DOI:10.1007/s00044-011-9945-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:4043–4052
DOI 10.1007/s00044-011-9945-1
ORIGINAL RESEARCH
a,a-Dibromoacetophenones mediated synthesis of some new
7H-7-alkoxy-3-alkyl/phenyl-6-aryl-s-triazolo[3,4-b][1,3,4]
thiadiazines and their antimicrobial evaluation
•
Rashmi Pundeer Vijay Kiran Richa Prakash
•
•
•
•
•
Sushma Subhash C. Bhatia Chetan Sharma
Kamal R. Aneja
Received: 5 September 2011 / Accepted: 9 December 2011 / Published online: 25 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of new 7H-7-alkoxy-3-alkyl/phenyl-6-
aryl-s-triazolo[3,4-b][1,3,4]thiadiazines (3, 4) were synthe-
sized by the reaction of various a,a-dibromoacetophenones 1
with 3-alkyl/phenyl-4-amino-5-mercapto-s-triazoles (2) in
different alcoholic solvents in good yields. All the newly
synthesized compounds (3, 4) were screened for their in vitro
antibacterial and antifungal activity. Biological activities of
these compounds were compared with those of the com-
mercially available antibiotic, ciprofloxacin and antifungal
agent, amphotericin-B. The title compounds showed good
activity against the Gram-positive bacteria, Staphylococcus
aureus and Bacillus subtilis and the yeasts, Saccharomyces
cerevisiae and Candida albicans.
2002; Dandia et al., 2006; Jin et al., 2007a, b; Ammar
et al., 2002). Among them, 1,2,4-triazoles fused with thi-
adiazines, incorporating the N–C–S linkage in their skel-
eton, constitute a chemical entity of particular interest. The
broad biological and pharmacological activities of triazolo
thiadiazines have been extensively studied and especially,
s-triazolo[3,4-b][1,3,4]thiadiazines have been shown to
possess a wide spectrum of bioactivities such as antibac-
terial, antifungal (Almajan et al., 2010; Karabasanagouda
et al., 2007; Mohan and Kataria, 1995; Ilhan et al., 1996;
Holla et al., 2006a, b; Chadha et al., 1992; Filmwala et al.,
2002), anti-inflammatory (Zitouni et al., 2007), anthel-
mintic (Nadkarni et al., 2001), antitubercular (Laddi et al.,
2001), plant-growth regulating (Ding et al., 2010; Jin et al.,
2007a, b; Zhou et al., 2007), antiviral (Farghaly et al.,
2006; Holla et al., 2001), and anticancer properties (Holla
et al., 2006a, b). In view of the importance of triazolo[3,4-
b][1,3,4]thiadiazines, our effort in the present study is to
identify some new derivatives of this class that may be
valuable in designing new, potent, selective, and less toxic
antimicrobial agents.
Keywords a,a-Dibromoacetophenones ꢀ
7H-7-alkoxy-3-alkyl/phenyl-6-aryl-s-triazolo[3,4-
b][1,3,4]thiadiazines ꢀ
3-alkyl/phenyl-4-amino-5-mercapto-s-triazoles ꢀ
Antibacterial activity ꢀ Antifungal activity
Introduction
The synthetic utility of a-halocarbonyl compounds in
heterocyclic chemistry is well known for more than a
century and they have been widely used as versatile
intermediates for the synthesis of a variety of heterocyclic
systems (Wilhelm and Schmidt, 1969; Garcia et al., 1973;
Little and Webber, 1994). However, as the a-haloketones
suffer with serious handling problems due to highly lach-
rymatory properties associated with them, there is an
increasing tendency to search a substitute for them.
Recently, a,a-dihaloketones are gaining particular interest
in view of their convenient preparation, high reactivity,
selectivity, and easy handling (Prakash et al., 2007a, b, c;
Boeykens and Kimpe, 1994). In addition, a,a-dihaloke-
tones and a-haloketones show analogous behavior in most
Triazole fused six-membered ring systems have engrossed
substantial interest because of their utility in organic
chemistry, medicine, agriculture, and industry (Holla et al.,
R. Pundeer (&) ꢀ V. Kiran ꢀ R. Prakash ꢀ Sushma ꢀ S. C. Bhatia
Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
e-mail: dr.rashmip@gmail.com
C. Sharma ꢀ K. R. Aneja
Department of Microbiology, Kurukshetra University,
Kurukshetra 136119, India
123

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Med Chem Res (2012) 21:4043–4052
of their reactions and a,a-dihaloketones seem to act as
masked a-haloketones (Ahluwalia et al., 1992, 1987;
Prakash et al., 2007a, b, c; Prakash and Sharma, 2007). In
view of the above facts and in continuation with our
ongoing program on exploring the synthetic utility of a,
a-dihaloketones, it was thought worthwhile to synthesize
fused heterocycles containing 1,2,4-triazole and 1,3,4-thi-
adiazine moieties using a,a-dibromoacetophenones. In this
study, we are extending the application of a,a-dibromo-
acetophenones in the synthesis of some new 7H-7-alkoxy-
3-alkyl/phenyl-6-aryl-s-triazolo [3,4-b][1,3,4]thiadiazines
(3, 4) of potential biological interest.
(Bala et al., 1978). Using MeOH and n-PrOH as a solvent in
this reaction, a mixture of two products, namely 7H-7-
alkoxy-6-phenyl-3-ethyl-s-triazolo[3,4-b][1,3,4]thiadiazine
(4a or 4e) and 7H-3-ethyl-6-phenyl-s-triazolo[3,4-b]
[1,3,4]thiadiazine (5a) (Bala et al., 1978) was obtained.
With i-PrOH, the 7-isopropoxy compound, 7H-6-aryl-3-
ethyl-7-isopropoxy-s-triazolo[3,4-b][1,3,4]thiadiazines (3i)
was obtained as the sole product. With solvents n-BuOH
and i-BuOH, only the 7-unsubstituted product, i.e., the
compound 5a was obtained. Also, by using CH3CN in
place of alcohol, 7-unsubstituted thiadiazine 5a was the
only product.
All these reactions in different solvents were also repeated
with the substrate a,a-dibromo-4-chloroacetophenone (1b).
Similar results were obtained in all the cases as with the
substrate 1a.
Chemistry
The present study was started with a,a-dibromoacetophe-
none 1a (Ar = Ph) as a model substrate. The reaction of 1a
was carried out with 4-amino-3-ethyl-5-mercapto-s-triazole
2a (R = C2H5) in ethanol under reflux conditions. Usual
work-up of the reaction afforded the expected product, 7H-
7-ethoxy-3-ethyl-6-phenyl-s-triazolo [3,4-b][1,3,4] thiadi-
azine (3a) in 78% yield. The product 3a was characterized
by the combined application of IR, NMR (1H, 13C) mea-
surements, and elemental analysis. IR spectrum of the
product showed the absence of any peak in the C=O or
Furthermore, the reaction of a,a-dibromoacetophenone
1a was studied with various 3-substituted triazoles 2 (b;
R = H, c; R = n-Pr and d; R = C6H5). Similar results
were obtained with triazoles 2b and 2c as with the triazole
2a in various alcoholic solvents (MeOH, EtOH, n-PrOH,
and i-PrOH), a single product (7-alkoxy) was obtained in
EtOH and i-PrOH, while a mixture of two products
(7-alkoxy and 7-unsubstituted) was obtained in MeOH and
n-PrOH. However, with the triazole 2d, the reaction pro-
ceeded differently and a mixture of two products was
obtained in all the alcoholic solvents used.
1
–NH2 region. H NMR spectrum of 3a showed a charac-
teristic singlet at d 5.75 for CH of the thiadiazine ring. The
spectrum also produced a triplet at d 1.47 and two multiplets
at d 3.62 and 4.03 corresponding to the –OCH2CH3 group.
The 13C NMR of the product presented all the carbons
corresponding to the triazolothiadiazine nucleus and the
substituents; d 11.09 (–CH2CH3), 14.46 (–OCH2CH3),
18.35 (–CH2CH3), 63.93 (–OCH2–), 73.13 (C7),
127.09–142.46 (C6 and aromatic carbons), 150.25 (C3 of
triazole), 155.05 (C5 of triazole). Structure of the product
was further confirmed by mass spectral data and elemental
analysis. To assess the generality of the reaction, various
a,a-dibromoacetophenones (1b–1f) were treated with the
triazole 2a under similar conditions. The reaction, indeed,
afforded the corresponding 7-ethoxy triazolothiadiazines
3b–3f in all the cases in good yields (65–81%) (Scheme 1).
From the results, it is apparent that the reaction followed
a similar trend by changing the a,a-dibromo substrate 1. In
order to extend scope of the study, it was considered
worthwhile to study the effect of changing the (i) solvent
and (ii) triazole on the reaction.
Mechanistic aspects of this synthetic approach have not
been investigated in the present study. Detailed investiga-
tions regarding the mechanism of the reaction and sepa-
ration of the intermediates are underway.
Biological results and discussion
In vitro antibacterial activity
The newly synthesized 7H-7-alkoxy-3-alkyl/phenyl-6-aryl-
7-s-triazolo[3,4-b][1,3,4]thiadiazines (3a–3n, 4a–4j) were
screened for their antibacterial activity against four strains
of bacteria namely Staphylococcus aureus, Bacillus sub-
tilis, Escherichia coli, and Pseudomonas aeruginosa by
agar well diffusion technique. All the tested chemical
compounds possessed variable antibacterial activity against
both the Gram-positive (S. aureus and B. subtilis) bacteria.
Positive controls produced significantly sized inhibition
zones against the tested bacteria; however, negative control
produced no observable inhibitory effect against any of the
test organism as shown in Tables 1 and 2. The activity of
the title compounds was compared to well-known com-
mercial antibiotic, ciprofloxacin. On the basis of maximum
inhibitory activity shown against Gram-positive bacteria,
compound 4i was found to be most effective against
Accordingly, several experiments were carried out on the
substrate a,a-dibromoacetophenone 1a using the solvents
MeOH, n-PrOH, i-PrOH, n-BuOH, i-BuOH, and CH3CN.
The reaction did not follow a similar trend in all the cases.
However, one of the two products obtained with solvents
MeOH and n-PrOH are similar to those reported in literature
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Med Chem Res (2012) 21:4043–4052
4045
N
N
O
C
Scheme 1 Reaction of a,a-
dibromoacetophenones 1 with
triazoles 2 in different alcohols
+
SH
R
N
CHBr2
Ar
NH2
1
2
Reflux, 6-8 h
R' (for R=Ph) / R"OH
R'OH
N
N
N
N
N
N
S
S
S
R
R
R
N
N
N
N
N
N
+
OR'
OR"
Ar
Ar
Ar
3
4
5
Compound
Ar
R
R’
Compound
Ar
R
R”
3a
3b
3c
3d
3e
3f
C6H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
H
Et
Et
Et
Et
Et
Et
Et
Et
4a
4b
4c
4d
4e
4f
C6H5
C2H5
C2H5
H
Me
Me
Me
Me
4-MeC6H4
4-ClC6H4
4-BrC6H4
4-FC6H4
4-NO2C6H4
C6H5
4-ClC6H4
C6H5
C6H5
n-C3H7
C2H5
C2H5
H
C6H5
n-Pr
n-Pr
n-Pr
n-Pr
Me
4-ClC6H4
C6H5
3g
3h
4g
4h
C6H5
n-C3H7
C2H5
C6H5
n-C3H7
3i
3j
C6H5
i-Pr
i-Pr
4i
4j
C6H5
C6H5
C6H5
4-ClC6H4
C2H5
C6H5
n-Pr
3k
3l
C6H5
C6H5
C6H5
C6H5
H
i-Pr
i-Pr
Et
5a
5b
5c
5d
5e
C6H5
4-ClC6H4
C6H5
C2H5
C2H5
H
Me/n-Pr
Me/n-Pr
Me/n-Pr
Me/n-Pr
Et/Me/ n-
Pr/i-Pr
n-C3H7
C6H5
C6H5
3m
3n
iso-Pr
C6H5
n-C3H7
C6H5
C6H5
S. aureus with zone of inhibition of 25.6 mm, while the
compound 4a was found to be most effective against B.
subtilis showing the maximum zone of inhibition of
21.6 mm, when compared with commercial antibiotic,
ciprofloxacin, which showed maximum zone of inhibition
of 26.6 and 24.0 mm against S. aureus and B. subtilis,
respectively (Table 1). In the whole series, the MIC of
various tested chemical compounds ranged between 16 and
256 lg/ml against Gram-positive bacteria (Table 2).
Compounds 3m and 4i exhibited the lowest MIC of 16 lg/
ml against S. aureus and the compound 4a was found to be
best against B. subtilis with the lowest MIC of 32 lg/ml.
None of the tested compounds possessed any activity
against Gram-negative bacteria.
In vitro antifungal activity
All the title compounds, 3(a–n) and 4(a–j) were screened
for their in vitro antifungal activity against two fungi,
namely Saccharomyces cerevisiae and Candida albicans.
The activity of the compounds were compared to the
standard antifungal agent, amphotericin-B. The compounds
123

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Med Chem Res (2012) 21:4043–4052
Table 1 In vitro antimicrobial activity of tested compounds through
agar well diffusion method
Table 2 MIC (in lg/ml) of tested compounds by using macrodilution
method
Compound nos. Diameter of growth of inhibition zone (mm)a
Compound nos. S. aureus B. subtilis S. cerevisiae C. albicans
S. aureus B. subtilis S. cerevisiae C. albicans
3a
256
[256
–
64
128
–
–
–
3b
[256
32
–
3a
13.6
12.3
–
18.3
17
–
–
3c
64
–
3b
10.6
18.3
–
–
3d
[256
–
256
–
–
3c
–
15.6
–
3e
128
64
128
128
–
3d
12.3
–
13.6
–
3f
–
–
3e
13.6
15.6
13.6
–
13
13.3
13.6
–
3g
128
256
256
[256
256
[256
16
64
128
–
3f
–
–
3h
128
[256
[256
256
256
128
256
32
–
3g
16.3
13.6
13.6
11.3
14.6
12.3
24.3
18.3
22.3
19.3
19.6
16.0
14.0
12.6
15.6
13.6
25.6
20.6
26.6
20.3
17.6
12.3
12.3
13.6
14.6
17.6
15.6
21.6
16.3
18.6
18.3
13.6
16.3
18.3
16.3
18.0
17.3
24.0
Nt
3i
–
–
3h
3j
[256
128
–
–
3i
–
–
3k
–
3j
11.3
13.3
–
–
3l
–
3k
–
3m
32
64
–
3l
–
3n
128
32
128
64
3m
17.6
13.3
15.3
12.6
13.6
12.6
–
15.6
–
4a
64
128
128
–
3n
4b
64
128
64
128
128
256
–
4a
16.3
13.0
13.3
–
4c
64
4b
4d
128
256
[256
128
256
16
64
4c
4e
256
128
64
–
4d
4f
[256
–
–
4e
–
4g
–
4f
10.3
–
–
4h
128
64
64
–
4g
–
4i
32
–
4h
15.3
18.6
17.3
Nt
–
4j
64
128
5
64
–
4i
–
Ciprofloxacin
5
Nt
Nt
100
4j
–
Amphotericin-B Nt
Nt
100
Ciprofloxacin
Nt
14.3
Amphotericin-B Nt
13.6
- no activity, Nt not tested
a
Values, including diameter of the well (8 mm), are means of three
replicates
Conclusions
In this study, application of a,a-dibromoacetophenones in
an efficient and facile synthesis of new 7H-7-alkoxy-3-
alkyl/phenyl-6-aryl-s-triazolo[3,4-b][1,3,4]thiadiazines is
extended. The title compounds showed good activity
against the Gram-positive bacteria, S. aureus and B. subtilis
and the yeasts, S. cerevisiae and C. albicans. Among all the
tested chemical compounds, compounds 3c, 3m, and 4a
showed antifungal activity even better than the commercial
drug amphotericin-B against both the tested fungal patho-
gens and these compounds can further be used as anti-
fungal agents in pharmaceutical industry, after testing their
toxicity to human beings.
3c (18.3 and 15.6 mm), 3m (17.6 and 15.6 mm) and 4a
(15.3 and 16.3 mm), showed antifungal activity even better
than the commercial drug amphotericin-B (13.6 and
14.3 mm) against both the fungal strains, S. cerevisiae and
C. albicans, respectively. Against S. cerevisiae, the activity
of the title compounds 3f (15.6) and 4h–4j (15.3, 18.6, and
17.3, respectively) is better than; while the activity of 3e,
3g, and 4c is comparable with amphotericin-B (Table 1). In
case of yeast, MIC ranged between 32 and 256 lg/ml
(Table 2). The compounds 3c, 3m, and 4a showed lowest
MIC of 64 lg/ml against C. albicans, while compounds 3c,
3m, and 4i showed the lowest MIC of 32 lg/ml against S.
cerevisiae. Thus, these compounds seems to be very
interesting from medicinal point of view as many of them
exhibited MIC values even lower than the standard anti-
fungal drug, amphotericin-B which showed MIC value
100 lg/ml for both fungal strains.
Experimental
Melting points were taken in open capillaries and were
uncorrected. Elemental analyses (C, H, N) and mass
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Med Chem Res (2012) 21:4043–4052
4047
spectra were carried out at University Science Instrumen-
1
tation Centre, University of Delhi, Delhi, India. H NMR
Characterization data of the new compounds
(3a–3n, 4a–4j)
spectra were recorded on a Bruker 300 MHz instrument
using TMS as an internal standard. Infrared (IR) spectra
were recorded on a Perkin-Elmer 1800 FT-IR spectro-
photometer. The purity of the synthesized compounds was
tested by thin-layer chromatography (TLC). The starting
materials, 4-amino-3-alkyl-5-mercapto-s-triazoles (2a–2c)
were prepared (Bala et al., 1978; Dhaka et al., 1974)
starting from carbondisulphide and hydrazine hydrate.
4-amino-3-phenyl-5-mercapto-s-triazole 2d was prepared
according to literature procedure (Chande et al., 1990). The
a,a-dibromoacetophenones needed for the present study
were prepared by stirring different acetophenones with
bromine (2.5 eq.) in CHCl3 (Boeykens and Kimpe, 1994)
and were confirmed by comparison with literature mp. All
chemicals used were taken from the commercial suppliers
and are used as such without further purification.
7H-3-ethyl-7-ethoxy-6-phenyl-s-triazolo[3,4-b]
[1,3,4]thiadiazine (3a)
Light brown; mp 110–112°C; yield 68%; 1H NMR (CDCl3,
300 MHz): d 1.26 (t, 3H, –CH2CH3), 1.47 (t, 3H,
–OCH2CH3), 3.0 (m, 2H, –CH2CH3), 3.62 (m, 1H,
–OCH2CH3), 4.03 (m, 1H, –OCH2CH3), 5.75 (s, 1H,
–CH), 7.59–7.89 (m, 5H, Ar–H); 13C NMR (CDCl3,
300 MHz): d 11.09 (–CH2CH3), 14.46 (–OCH2CH3),
18.35 (–CH2CH3), 63.93 (–OCH2–), 73.13 (C7),
127.09–142.46 (C6 and aromatic carbons), 150.25 (C3 of
triazole), 155.05 (C5 of triazole); Anal. Calculated for
C14H16N4OS: C, 58.31; H, 5.59; N, 19.43. Found: C, 58.27;
H, 5.64; N, 19.32; ESI-MS, m/z: 289.32 [M ? 1]?.
7H-3-ethyl-7-ethoxy-6-(4-methylphenyl)-s-
triazolo[3,4-b][1,3,4]thiadiazine (3b)
Preparation of 7H-7-alkoxy-6-aryl-3-alkyl/phenyl-s-
triazolo[3,4-b][1,3,4]thiadiazines (3a–3n, 4a–4j)
Brown; mp 138–140°C; yield 72%; 1H NMR (CDCl3,
300 MHz): d 1.25 (t, 3H, –CH2CH3), 1.50 (t, 3H,
–OCH2CH3), 2.68 (s, 3H, –CH3), 3.16 (m, 2H, –CH2CH3),
3.67 (m, 1H, –OCH2CH3), 4.05 (m, 1H, –OCH2CH3), 5.94
(s, 1H, –CH), 7.34 (d, 2H, J = 8.4, Ar-H), 7.77 (d, 2H,
J = 7.8, Ar-H); 13C NMR (CDCl3, 300 MHz): d 11.31
(–CH2CH3), 14.46 (–OCH2CH3), 18.40 (–CH2CH3),
21.54 (Ar-CH3), 63.94 (–OCH2–), 73.13 (C7), 127.09-
142.49 (C6 and aromatic carbons), 150.05 (C3 of triazole),
155.18 (C5 of triazole); Anal. Calculated for C15H18N4OS:
C, 59.58; H, 6.00; N, 18.53. Found: C, 59.45; H, 6.07; N,
18.39; ESI-MS, m/z: 303.28 [M ? 1]?.
General procedure
A solution of appropriate a,a-dibromoacetophenone (1a–
1f, 2 mmol) was refluxed with triazole 2 (2 mmol) in
various alcohols (MeOH, EtOH, n-PrOH, iso-PrOH) for
6–8 h. The reaction mixture was cooled and neutralized
with ammonium hydroxide.
(a) With alkyl triazoles 2a–2c (i) in EtOH and iso-PrOH
as solvent, a solid was separated out. The solid was
filtered, washed with water, and recrystallized from
EtOH/H2O to obtain the pure products 3a–3l; (ii) In
MeOH, a gummy mass, containing a mixture of two
products, was obtained which was purified by column
chromatography on silica gel using pet ether-ethyl
acetate (20–25%) as eluent to obtain the pure
products, 4a–4d and 5a–5c; (iii) In n-PrOH, a white
solid, containing a mixture of two products, was
obtained. The mixture was separated by column
chromatography on silica gel using pet ether-ethyl
acetate (20–30%) as eluent to obtain the products, 4e–
4h and 5a–5c
7H-3-ethyl-7-ethoxy-6-(4-chlorophenyl)-s-triazolo
[3,4-b][1,3,4]thiadiazine (3c)
Brown; mp 141–142°C; yield 75%; 1H NMR (CDCl3,
300 MHz): d 1.24 (t, 3H, –CH2CH3), 1.463 (t, 3H,
–OCH2CH3), 3.05 (m, 2H, –CH2CH3), 3.64 (m, 1H,
–OCH2CH3), 4.05 (m, 1H, –OCH2CH3), 5.73 (s, 1H,
–CH), 7.50 (d, 2H, J = 8.4, Ar-H), 7.80 (d, 2H, J = 8.4,
Ar-H); 13C NMR (CDCl3, 300 MHz): d 11.10 (–CH2CH3),
14.45 (–OCH2CH3), 18.38 (–CH2CH3), 64.00 (–OCH2–),
72.94 (C7), 128.50–138.15 (C6 and aromatic carbons),
148.99 (C3 of triazole), 155.23 (C5 of triazole); Anal.
Calculated for C14H15ClN4OS: C, 52.09; H, 4.68; N, 17.36.
Found: C, 51.85; H, 4.76; N, 17.27; ESI-MS, m/z: 323.47
[M ? 1]?, 325. 21 [M ? 3]?.
(b) With 3-phenyl triazole 2d, a mixture of two products
was obtained in all the alcoholic solvents used
(MeOH, EtOH, n-PrOH, iso-PrOH). The mixture
was separated by column chromatography on silica
gel using pet ether-ethyl acetate (30–40%) as eluent
to obtain the pure compounds (3m, 3n, 4i, 4j) and 5d.
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Med Chem Res (2012) 21:4043–4052
–CH), 7.32-7.72 (m, 5H, Ar-H), 8.70 (s, 1H, triazole); 13C
NMR (CDCl3, 300 MHz): d 14.42 (–OCH2CH3), 64.12
(–OCH2–), 74.56 (C7), 126.48–142.76 (C6 and aromatic
carbons), 150.65 (C3 of triazole), 155.35 (C5 of triazole);
Anal. Calculated for C12H12N4OS: C, 55.37; H, 4.65; N,
21.52. Found: C, 55.28; H, 4.68; N, 21.43; ESI-MS, m/z:
261.17 [M ? 1]?.
7H-3-ethyl-7-ethoxy-6-(4-bromophenyl)-s-triazolo
[3,4-b][1,3,4]thiadiazine (3d)
Brown; mp 135–138°C; yield 78%; 1H NMR (CDCl3,
300 MHz): d 1.26 (t, 3H, –CH2CH3), 1.46 (t, 3H,
–OCH2CH3), 3.05 (m, 2H, –CH2CH3), 3.68 (m, 1H,
–OCH2CH3), 4.05 (m, 1H, –OCH2CH3), 5.70 (s, 1H,
–CH), 7.66 (d, 2H, J = 8.7, Ar-H), 7.72 (d, 2H, J = 8.7,
Ar-H); 13C NMR (CDCl3, 300 MHz): d 11.10 (–CH2CH3),
14.45 (–OCH2CH3), 18.38 (–CH2CH3), 64.00 (–OCH2–),
72.88 (C7), 126.59–137.01 (C6 and aromatic carbons),
149.12 (C3 of triazole), 155.23 (C5 of triazole); Anal.
Calculated for C14H15BrN4OS: C, 45.78; H, 4.12; N, 15.26.
Found: C, 45.95; H, 3.98; N, 15.32; ESI-MS, m/z: 368.06
[M ? 1]?, 370.14 [M ? 3]?.
7H-3-propyl-7-ethoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (3h)
Light brown; mp 148–150°C; yield 72%; 1H NMR (CDCl3,
300 MHz): d 1.06 (t, 3H, –CH2CH2CH3), 1.25 (t, 3H,
–OCH2CH3), 1.91 (m, 2H, –CH2CH2CH3), 3.02 (m, 2H,
–CH2CH2CH3), 3.62 (m, 1H, –OCH2CH3), 4.03 (m, 1H,
–OCH2CH3), 5.75 (s, 1H, –CH), 7.52–7.88 (m, 5H, Ar-H);
13C NMR (CDCl3, 300 MHz): d 18.52 (–CH2CH2CH3),
19.34 (–OCH2CH3), 25.06 (–CH2CH2CH3), 31.15
(–CH2CH2CH3), 68.57 (–OCH2–), 77.75 (C7), 132.12–
142.33 (C6 and aromatic carbons), 155.37 (C3 of triazole),
158.54 (C5 of triazole); Anal. Calculated for C15H18N4OS:
C, 59.58; H, 6.0; N, 18.53. Found: C, 59.52; H, 6.04; N,
18.62; ESI-MS, m/z: 303.15 [M ? 1]?.
7H-3-ethyl-7-ethoxy-6-(4-fluorophenyl)-s-triazolo
[3,4-b][1,3,4]thiadiazine (3e)
Light brown; mp 152–154°C; yield 70%; 1H NMR (CDCl3,
300 MHz): d 1.25 (t, 3H, –CH2CH3), 1.46 (t, 3H,
–OCH2CH3), 3.07 (m, 2H, –CH2CH3), 3.62 (m, 1H,
–OCH2CH3), 4.03 (m, 1H, –OCH2CH3), 5.72 (s, 1H,
–CH), 7.20 (d, 2H, J = 8.4, Ar–H), 7.87 (d, 2H, J = 8.4,
Ar–H); 13C NMR (CDCl3, 300 MHz): d 11.11 (–CH2CH3),
14.45 (–OCH2CH3), 18.38 (–CH2CH3), 63.97 (–OCH2–),
73.07 (C7), 116.25–136.96 (C6 and aromatic carbons),
149.04 (C3 of triazole), 155.19 (C5 of triazole) 166.55
(–C5H4C-F); Anal. Calculated for C14H15FN4OS: C, 54.89;
H, 4.94; N, 18.29. Found: C, 55.02; H, 4.83; N, 18.17; ESI-
MS, m/z: 307.18 [M ? 1]?.
7H-3-ethyl-6-phenyl-7-iso-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (3i)
Pale yellow; mp 183–185°C; yield 68%; 1H NMR (CDCl3,
300 MHz): d 1.16 (d, 3H, –OCH(CH3)2), 1.33 (d, 3H,
–OCH(CH3)2), 1.47 (t, 3H, –CH2CH3), 3.03 (m, 2H,
–CH2CH3), 4.28 (m, 1H, –OCH–), 5.85 (s, 1H, –CH),
7.53–7.90 (m, 5H, Ar-H); 13C NMR (CDCl3, 300 MHz): d
11.10 (–CH2CH3), 18.42 (–CH2CH3), 20.60 (–OCH
(CH3)2), 23.04 (–OCH(CH3)2), 69.98 (–OCH–), 70.99
(C7), 127.12-137.35 (C6 and aromatic carbons), 150.47 (C3
of triazole), 155.19 (C5 of triazole); Anal. Calculated for
C15H18N4OS: C, 59.58; H, 6.00; N, 18.53. Found: C, 59.73;
H, 6.07; N, 18.38; ESI-MS, m/z: 303.09 [M ? 1]?.
7H-3-ethyl-7-ethoxy-6-(4-nitrophenyl)-s-triazolo
[3,4-b][1,3,4]thiadiazine (3f)
Yellow–brown; mp 210-212°C; yield 81%; 1H NMR
(CDCl3, 300 MHz): d 1.28 (t, 3H, –CH2CH3), 1.48 (t, 3H,
–OCH2CH3), 3.15 (m, 2H, –CH2CH3), 3.76 (m, 1H,
–OCH2CH3), 4.10 (m, 1H, –OCH2CH3), 6.28 (s, 1H,
–CH), 8.18(d, 2H, J = 8.7, Ar-H), 8.40 (d, 2H, J = 8.7,
Ar-H); 13C NMR (CDCl3, 300 MHz): d 11.07 (–CH2CH3),
14.44 (–OCH2CH3), 18.38 (–CH2CH3), 64.12 (–OCH2–),
72.81 (C7), 124.29–147.92 (C6 and aromatic carbons), 149.53
(C3 of triazole), 155.37 (C5 of triazole); Anal. Calculated for
C14H15N5O3S: C, 50.44; H, 4.54; N, 21.01. Found: C, 50.26;
H, 4.62; N, 21.18; ESI-MS, m/z: 334.24 [M ? 1]?.
7H-3-ethyl-6-(4-chlorophenyl)-7-iso-propoxy-s-
triazolo[3,4-b][1,3,4]thiadiazine (3j)
Brown; mp 146–148°C; yield 74%; 1H NMR (CDCl3,
300 MHz): d 1.15 (d, 3H, –OCH(CH3)2), 1.31 (d, 3H,
–OCH(CH3)2), 1.46 (t, 3H, –CH2CH3), 3.04 (m, 2H,
–CH2CH3), 4.27 (m, 1H, –OCH–), 5.81 (s, 1H, –CH), 7.49
(d, 2H, J = 8.4, Ar–H), 7.81 (d, 2H, J = 8.4, Ar–H); 13C
NMR (CDCl3, 300 MHz): d 11.12 (–CH2CH3), 18.38
(–CH2CH3), 20.63 (–OCH(CH3)2), 23.07 (–OCH(CH3)2),
70.02 (–OCH–), 71.05 (C7), 127.07–137.31 (C6 and aro-
matic carbons), 150.42 (C3 of triazole), 155.18 (C5 of tri-
azole); Anal. Calculated for C15H17ClN4OS: C, 53.49; H,
7H-7-ethoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (3g)
Dark brown; mp 120–122°C; yield 75%; 1H NMR (CDCl3,
300 MHz): d 1.24 (t, 3H, –OCH2CH3), 3.65 (m, 1H,
–OCH2CH3), 4.06 (m, 1H, –OCH2CH3), 5.79 (s, 1H,
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4049
5.09; N, 16.63. Found: C, 53.58; H, 4.99; N, 16.48; ESI-
MS, m/z: 337.52 [M ? 1]?, 339.27 [M ? 3]?.
7.51–8.17 (m, 10H, Ar-H); 13C NMR (CDCl3, 300 MHz): d
21.46 (–OCH(CH3)2), 24.12 (–OCH(CH3)2), 70.18 (–OCH–),
74.39 (C7), 126.12-137.85 (C6 and aromatic carbons),
151.27 (C3 of triazole), 155.79 (C5 of triazole); Anal Cald
for C19H18N4OS: C, 65.12; H, 5.18; N, 15.99. Found: C,
65.21; H, 5.16; N, 15.88; ESI-MS, m/z: 351.35 [M ? 1]?.
7H-6-phenyl-7-iso-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (3k)
Dark brown; mp 153–155°C; yield 76%; 1H NMR (DMSO-
d6, 300 MHz): d 1.18 (d, 3H, –OCH(CH3)2), 1.35 (d, 3H,
–OCH(CH3)2), 4.28 (m, 1H, –OCH–), 6.46 (s, 1H, –CH),
7.66–8.01 (m, 5H, Ar-H), 9.31 (s, 1H, triazole); 13C NMR
(DMSO-d6, 300 MHz): d 20.94 (–OCH(CH3)2), 23.44
(–OCH(CH3)2), 70.24 (–OCH–), 71.86 (C7), 127.34–
137.85 (C6 and aromatic carbons), 150.77 (C3 of triazole),
155.34 (C5 of triazole); Anal. Calculated for C13H14N4OS:
C, 56.91; H, 5.14; N, 20.42. Found: C, 56.83; H, 5.17; N,
20.38; ESI-MS, m/z: 275.17 [M ? 1]?.
7H-3-ethyl-7-methoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (4a)
Dark brown; mp 122–124°C; yield 46%; 1H NMR (CDCl3,
300 MHz): d 1.44 (t, 3H, –CH2CH3), 3.03 (m, 2H,
–CH2CH3), 3.53 (s, 3H, –OCH3), 5.62 (s, 1H, –CH),
7.50–7.87 (m, 5H, Ar-H); 13C NMR (CDCl3, 300 MHz): d
11.10 (–CH2CH3), 18.38 (–CH2CH3), 55.32 (–OCH3),
74.58 (C7), 128.50–138.18 (C6 and aromatic carbons),
149.05 (C3 of triazole), 155.20 (C5 of triazole); Anal. Cal-
culated for C13H14N4OS: C, 56.91; H, 5.14; N, 20.42. Found:
C, 56.78; H, 5.21; N, 20.31; ESI-MS, m/z: 275.14 [M ? 1]?.
7H-3-propyl-6-phenyl-7-iso-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (3l)
Light brown; mp 172–174°C; yield 70%; 1H NMR (CDCl3,
300 MHz): d 1.17 (t, 3H, –CH2CH2CH3), 1.37 (d, 3H,
–OCH(CH3)2), 1.45 (d, 3H, –OCH(CH3)2), 2.10 (m, 2H,
–CH2CH2CH3), 3.07 (m, 2H, –CH2CH2CH3), 4.27 (m, 1H,
7H-3-ethyl-7-methoxy-6-(4-chlorophenyl)-s-
triazolo[3,4-b][1,3,4]thiadiazine (4b)
Reddish brown; mp 130–132°C; yield 54%; 1H NMR
(CDCl3, 300 MHz): d 1.45 (t, 3H, –CH2CH3), 3.04 (m, 2H,
–CH2CH3), 3.53 (s, 3H, –OCH3), 5.61 (s, 1H, –CH), 7.50
(d, 2H, J = 8.4, Ar-H), 7.79 (d, 2H, J = 8.4, Ar-H); 13C
NMR (CDCl3, 300 MHz): d 11.15 (–CH2CH3), 18.36
(–CH2CH3), 55.29 (–OCH3), 74.65 (C7), 128.53-138.23
(C6 and aromatic carbons), 149.07 (C3 of triazole), 155.22
(C5 of triazole); Anal. Calculated for C13H13ClN4OS: C,
50.57; H, 4.24; N, 18.14. Found: C, 50.65; H, 4.35; N,
18.02; ESI-MS, m/z: 309.62 [M ? 1]?, 311.28 [M ? 3]?.
–OCH–), 5.80 (s, 1H, –CH), 7.41–7.83 (m, 5H, Ar-H); 13
C
NMR (CDCl3, 300 MHz): d 18.44 (–CH2CH2CH3),
20.57 (–OCH(CH3)2), 22.98 (–OCH(CH3)2), 24.92
(–CH2CH2CH3), 31.07 (–CH2CH2CH3), 70.14 (–OCH–),
71.35 (C7), 127.26–137.65 (C6 and aromatic carbons),
150.45 (C3 of triazole), 155.24 (C5 of triazole); Anal.
Calculated for C16H20N4OS: C, 60.73; H, 6.37; N, 17.71.
Found: C, 60.78; H, 6.35; N, 17.58; ESI-MS, m/z: 317.12
[M ? 1]?.
7H-3-phenyl-7-ethoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (3m)
7H-7-methoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (4c)
1
Light brown; mp 174–176°C; yield 36%; 1H NMR (CDCl3,
300 MHz): d 1.27 (t, 3H, –OCH2CH3), 3.65 (m, 1H,
–OCH2CH3), 4.07 (m, 1H, –OCH2CH3), 5.76 (s, 1H,
–CH), 7.44–8.17 (m, 10H, Ar-H); 13C NMR (CDCl3,
300 MHz): d 10.45 (–OCH2CH3), 63.76 (–OCH2–), 73.28
(C7), 127.09–142.46 (C6 and aromatic carbons), 150.48 (C3
of triazole), 155.65 (C5 of triazole); Anal. Calculated for
C18H16N4OS: C, 64.26; H, 4.79; N, 16.65. Found: C, 64.18;
H, 4.75; N, 16.77; MS, m/z: 337.22 [M ? 1]?.
Brown; mp 102–104°C; yield 38%; H NMR (DMSO-d6,
300 MHz): d 3.46 (s, 3H, –OCH3), 6.46 (s, 1H, –CH),
7.66–7.98 (m, 5H, Ar-H), 9.31(s, 1H, triazole); 13C NMR
(DMSO-d6, 300 MHz): d 55.32 (–OCH3), 74.58 (C7),
128.50–138.18 (C6 and aromatic carbons), 149.05 (C3 of
triazole), 155.20 (C5 of triazole); Anal. Calculated for
C11H10N4OS: C, 53.64; H, 4.09; N, 22.75. Found: C, 53.76;
H, 4.05; N, 22.62; ESI-MS, m/z: 247.21 [M ? 1]?.
7H-3-propyl-7-methoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (4d)
7H-3-phenyl-6-phenyl-7-iso-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (3n)
Brown; mp 134–136°C; yield 36%; 1H NMR (CDCl3,
300 MHz): d 1.06 (t, 3H, –CH2CH2CH3), 1.92 (m, 2H,
–CH2CH2CH3), 3.02 (m, 2H, –CH2CH2CH3), 3.54 (s, 3H,
Brown; mp 161–164°C; yield 32%; 1H NMR (CDCl3,
300 MHz): d 1.18 (d, 3H, –OCH(CH3)2), 1.35 (d, 3H,
–OCH(CH3)2), 4.28 (m, 1H, –OCH–), 5.76 (s, 1H, –CH),
–OCH3), 5.66 (s, 1H, –CH), 7.52–7.92 (m, 5H, Ar-H); 13
C
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Med Chem Res (2012) 21:4043–4052
7H-3-propyl-6-phenyl-7-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (4h)
NMR (CDCl3, 300 MHz): d 13.80 (–CH2CH2CH3), 20.43
(–CH2CH2CH3), 26.52 (–CH2CH2CH3), 55.30 (–OCH3),
74.91 (C7), 127.12–133.29 (C6 and aromatic carbons),
150.09 (C3 of triazole), 156.28 (C5 of triazole); Anal. Cal-
culated for C14H16N4OS: C, 58.31; H, 5.59; N, 19.43. Found:
C, 58.38; H, 5.51; N, 19.31; ESI-MS, m/z: 289.14 [M ? 1]?.
Light brown; mp 196–198°C; yield 34%; 1H NMR (CDCl3,
300 MHz): d 0.89 (t, 3H, –CH2CH2CH3), 1.05 (t, 3H,
–OCH2CH2CH3), 1.59 (m, 2H, –OCH2CH2CH3), 1.90 (m,
2H, –CH2CH2CH3), 3.03 (m, 2H, –CH2CH2CH3), 3.50 (m,
1H, –OCH2CH2CH3), 3.94 (m, 1H, –OCH2CH2CH3), 5.73
(s, 1H, –CH), 7.52–7.88 (m, 5H, Ar-H); 13C NMR (CDCl3,
300 MHz): d 10.90 (–OCH2CH2CH3), 13.84 (–CH2CH2
CH3), 20.30 (–CH2CH2CH3), 25.06 (–OCH2CH2CH3),
31.15 (–CH2CH2CH3), 69.63 (–OCH2–), 72.90 (C7),
129.15–139.91 (C6 and aromatic carbons), 150.46 (C3 of
triazole), 154.06 (C5 of triazole); Anal. Calculated for
C16H20N4OS: C, 60.73; H, 6.37; N, 17.71. Found: C, 60.62;
H, 6.34; N, 17.82; ESI-MS, m/z: 317.18 [M ? 1]?.
7H-3-ethyl-6-phenyl-7-propoxy-s-triazolo[3,4-
b][1,3,4]thiadiazine (4e)
Light brown; mp 128–130°C; yield 34%; 1H NMR (CDCl3,
300 MHz): d 0.78 (t, 3H, –OCH2CH2CH3), 1.35 (t, 3H,
–CH2CH3), 1.50 (m, 2H, –OCH2CH2–), 2.95 (m, 2H,
–CH2CH3), 3.40 (m, 1H, –OCH2–), 3.83 (m, 1H, –OCH2–
), 5.71 (s, 1H, –CH), 7.46–7.87 (m, 5H, Ar-H); 13C NMR
(CDCl3, 300 MHz): d 10.41 (–OCH2CH2CH3), 11.15
(–CH2CH3), 18.40 (–CH2CH3), 22.16 (–OCH2CH2–),
70.04 (–OCH2–), 73.52 (C7), 127.20–137.10 (C6 and aro-
matic carbons), 150.10 (C3 of triazole), 155.25 (C5 of tri-
azole); Anal. Calculated for C15H18N4OS: C, 59.58; H,
6.00; N, 18.53. Found: C, 59.67; H, 5.89; N, 18.62; ESI-
MS, m/z: 303.27 [M ? 1]?.
7H-3-phenyl-7-methoxy-6-phenyl-s-triazolo
[3,4-b][1,3,4]thiadiazine (4i)
Dark brown; mp 132–135°C; yield 42%; 1H NMR (CDCl3,
300 MHz): d 3.57 (s, 3H, –OCH3), 5.67 (s, 1H, –CH),
7.41–8.20 (m, 10H, Ar-H); 13C NMR (CDCl3, 300 MHz):
d 55.59 (–OCH3), 73.98 (C7), 126.39–137.72 (C6 and
aromatic carbons), 151.34 (C3 of triazole), 155.78 (C5 of
triazole); Anal. Calculated for C17H14N4OS: C, 63.33; H,
4.38; N, 17.38. Found: C, 63.26; H, 4.37; N, 17.49; ESI-
MS, m/z: 323.22 [M ? 1]?.
7H-3-ethyl-6-(4-chlorophenyl)-7-propoxy-s-
triazolo[3,4-b][1,3,4]thiadiazine (4f)
Pale yellow; mp 115–118°C; yield 38%; 1H NMR (CDCl3,
300 MHz): d 0.89 (t, 3H, –OCH2CH2CH3), 1.45 (t, 3H,
–CH2CH3), 1.60 (m, 2H, –OCH2CH2–), 3.05 (m, 2H,
–CH2CH3), 3.46 (m, 1H, –OCH2–), 3.92 (m, 1H, –OCH2–),
5.70 (s, 1H, –CH), 7.50 (d, 2H, J = 8.4, Ar-H), 7.95(d, 2H,
J = 8.4, Ar-H); 13C NMR (CDCl3, 300 MHz): d 10.44
(–OCH2CH2CH3), 11.17 (–CH2CH3), 18.42 (–CH2CH3),
22.19 (–OCH2CH2–), 70.07 (–OCH2–), 73.58 (C7),
127.12–137.16 (C6 and aromatic carbons), 150.07 (C3 of
triazole), 155.19 (C5 of triazole); Anal. Calculated for
C15H17ClN4OS: C, 53.49; H, 5.09; N, 16.63. Found: C,
53.37; H, 5.16; N, 16.41; ESI-MS, m/z: 337.11 [M ? 1]?,
339.26 [M ? 3]?.
H-3-phenyl-6-phenyl-7-propoxy-s-triazolo
[3,4-b][1,3,4]thiadiazine (4j)
Brown; mp 188–190°C; yield 28%; 1H NMR (CDCl3,
300 MHz): d 0.92 (t, 3H, –OCH2CH2CH3), 1.68 (m, 2H,
–OCH2CH2–), 3.53 (m, 1H, –OCH2–), 3.70 (m, 1H,
–OCH2–), 5.75(s, 1H, –CH), 7.52–8.16 (m, 10H, Ar-H);
13C NMR (CDCl3, 300 MHz): d 10.45 (–OCH2CH2CH3),
23.01 (–OCH2CH2–), 70.14 (–OCH2–), 76.81 (C7),
125.91–138.26 (C6 and aromatic carbons), 152.97 (C3 of
triazole), 156.65 (C5 of triazole); Anal. Calculated for
C19H18N4OS: C, 65.12; H, 5.18; N, 15.99. Found: C, 65.05;
H, 5.19; N, 16.09; ESI-MS, m/z: 351.16 [M ? 1]?.
7H-6-phenyl-7-propoxy-s-triazolo[3,4-
b][1,3,4]thiadiazine (4g)
Dark brown; mp 144–146°C; yield 32%; 1H NMR (CDCl3,
300 MHz): d 1.27 (t, 3H, –OCH2CH2CH3), 2.05 (m, 2H,
–OCH2CH2–), 3.68 (m, 1H, –OCH2–), 4.05 (m, 1H,
–OCH2–), 5.84 (s, 1H, –CH), 7.43–7.84 (m, 5H, Ar-H), 8.74
(s, 1H, triazole); 13C NMR (CDCl3, 300 MHz): d 10.52
(–OCH2CH2CH3), 22.92 (–OCH2CH2–), 69.87 (–OCH2–),
74.08 (C7), 127.15–138.62 (C6 and aromatic carbons),
150.32 (C3 of triazole), 153.01 (C5 of triazole); Anal. Cal-
culated for C13H14N4OS: C, 56.91; H, 5.14; N, 20.42. Found:
C, 59.82; H, 5.09; N, 20.54; ESI-MS, m/z: 275.22 [M ? 1]?.
Biological assay
Test microorganisms
Total six 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 two yeast
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Med Chem Res (2012) 21:4043–4052
4051
(C. albicans MTCC 227 and S. cerevisiae MTCC 170)
were screened for evaluation of antibacterial and antifungal
activity of the chemical compounds. All the microbial
cultures were procured from Microbial Type Culture Col-
lection (MTCC), IMTECH, Chandigarh. The bacteria were
sub-cultured on Nutrient agar whereas yeast on Malt yeast
agar.
Ciprofloxacin and amphotericin-B was used as positive
control, while DMSO as negative control.
Acknowledgments The authors are indebted to UGC/CSIR for
providing research fellowship to Ms.Vijay Kiran for carrying out this
study. The authors also thanks USIC, University of Delhi, Delhi, India
for elemental analysis.
Antimicrobial activity (bacteria and yeasts)
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