New 6-amino-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazin-6-ones: Synthesis, characterization and antibacterial activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2010.02.057

(4-X-Phenylsulfonyl)phenyl] containing 6-amino-[1,2,4]triazolo[3,4-b][1,3, 4]thiadiazines and [1,2,4]triazolo[ 3,4-b][1,3,4]thiadiazin-6-ones were synthesized by intermolecular condensation of 2-chloro-Nphenylacetamide, 2-chloroacetic acid, oxalylchloride

Full text of DOI:10.1016/j.ejmech.2010.02.057

European Journal of Medicinal Chemistry 45 (2010) 3191e3195
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
New 6-amino-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazin-6-ones: Synthesis, characterization and antibacterial
activity evaluation
,
a
a
b
Gabriela Laura Almajan a , Stefania-Felicia Barbuceanu , Ioana Saramet , Constantin Draghici
*
a Organic Chemistry Department, Faculty of Pharmacy, Traian Vuia Street 6, 020956 Bucharest, Romania
b C.D. Nenitescu Institute of Organic Chemistry, Romanian Academy, Splaiul Independentei 202B, 060023, Bucharest, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
(4-X-Phenylsulfonyl)phenyl] containing 6-amino-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazin-6-ones were synthesized by intermolecular condensation of 2-chloro-N-
phenylacetamide, 2-chloroacetic acid, oxalylchloride and bromo-diethylmalonate with 4-amino-5-[4-(4-
X-phenylsulfonyl)phenyl]-4H-1,2,4-triazole-3-thiols (X ¼ H, Cl, Br). The structures of newly synthesized
compounds were confirmed by elemental analysis and IR, NMR spectral data. All the compounds were
screened for their antibacterial activities. Some of them exhibited good activities against Staphylococcus
epidermidis ATCC 14990, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923 and
Escherichia coli ATCC 25922.
Received 16 December 2009
Received in revised form
24 February 2010
Accepted 25 February 2010
Available online 3 March 2010
Keywords:
Amino-triazolothiadiazines
Triazolothiadiazinones
Cyclodehydration
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antibacterial activity
1. Introduction
a single structure, which may lead to compounds with interesting
biological profiles.
In therecentyears, a special attentionwasgiven to 5-substituted-
4-amino-1,2,4-triazole-3-thiols owing to their promising biological
activities such as antimicrobial, anti-inflammatory, analgesic,
hypoglycemic, anticancer [1e8]. In addition to these important
biological applications, 4-amino-1,2,4-triazole-3-thiols are also of
great utility in preparative organic chemistry. The amino and mer-
capto groups are ready-made nucleophilic centers for the synthesis
of condensed heterocyclic rings such as triazolothiadiazoles, tri-
azolothiadiazines, triazolotetrazines and triazolothiadiazepines
[9e11]. 1,2,4-Triazoles fused with six-member ring systems (we
refer here to triazoles fused with pyridine, pyridazine, pyrimidine,
pyrazine and triazine) have diverse applications in the fields of
medicine, agriculture and industry.
Our previous studies on the synthesis of biologically active
compounds have demonstrated that the derivatives with 1,2,4-
triazole nucleus have weaker antimicrobial activity than those
which containing this nucleus condensed with one another
heterocycle (thiadiazine, thiazole) [18,19].
For this reason, we considered interesting to synthesize new
compounds containing [1,2,4]-triazole nucleus fused with
a different substituted [1,3,4]-thiadiazine moiety, in order to study
their antibacterial activity.
The present study describes the synthesis and characterization
of novel amino-triazolothiadiazines and triazolothiadiazinones and
evaluation of their antibacterial activities.
The recent literature survey revealed that a special attention
was given to 1,2,4-triazolo[3,4-b][1,3,4]thiadiazine derivatives
which proved to have promising biological activities: antimicrobial,
antiviral, anti-HIV, CNS-stimulant, antifungal [12e17].
Designing new drugs is based on the development of hybrid
molecules by combining different pharmacophore fragments in
2. Chemistry
Thereactionsequences employedforsynthesisof titlecompounds
are shown in Scheme 1. The key intermediates, 4-amino-5-[4-(4-X-
phenylsulfonyl)phenyl]-4H-1,2,4-triazole-3-thiols1a-c, (X¼ H, Cl, Br)
were prepared from corresponding substituted benzoic acid hydra-
zides according to literature [20].
Triazoles 1b,c (X ¼ Cl, Br) were treated with 2-chloro-N-phenyl-
acetamidein the presence of potassium hydroxide in ethanoland led
to obtaining N-phenyl-2-{4-amino-5-[4-(4-X-phenylsulfonyl)
* Corresponding author. Tel.: þ40 741 095490; fax: þ40 21 3180729.
E-mail address: laura.almajan@gmail.com (G.L. Almajan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.057

3192
G.L. Almajan et al. / European Journal of Medicinal Chemistry 45 (2010) 3191e3195
N
N
N
N
broth dilution method [22] for determination of MIC. Tetracycline
and ampicillin were used as control drugs.
i
H2
C
ii
H
N
Ar
S
C
O
Ar
S
N
N
N
NH2
2b,c
3b,c
4. Results and discussions
X= Cl, Br
X= Cl, Br
HN
N
N
N
N
4.1. Chemistry
N
N
iii
iv
H2
C
Ar
SH
Ar
Ar
N
S
COOH
S
N
N
N
Obtaining of compounds 2b,c; 3b,c; 4a-c; 5a-c; 6c and 7b was
confirmed on the basis of elemental analysis and IR, NMR spectral
data. The elemental analysis data along with some physical prop-
erties of these compounds are reported in Table 1.
NH2
1a-c
HN
4a-c
NH2
v
5a-c
X= H, Cl, Br
N
O
X= H, Cl, Br
X= H, Cl, Br
Ar
S
N
vi
HN
Formation of S-alkyl derivatives 2b,c and 4a-c is supported by
observed changes in IR and NMR spectra. Thus, depending on the
nature of the fragment bound to the thiol sulphur atom, IR spectra
show characteristic bands: an intense band at 1668e1677 cmꢀ1
attributed to the amide carbonyl group for 2b,c and a very strong
band in 1712e1714 cmꢀ1 region, assigned to carboxyl group in 4a-c.
Asymmetric and symmetric stretching frequencies of amino group
appeared at 3370e3374 cmꢀ1 and 3202 cmꢀ1 for 2b,c.
O
6c
O
N
N
X=Br
Ar
S
N
OCH2CH3
HN
O
S
O
O
O
X
Ar =
7b
X=Cl
The 1H NMR spectrum of 2b,c showed
a singlet at
i- PhNHCOCH2Cl/KOH/reflux
ii- POCl3/reflux
4.40e4.25 ppm due to the presence of SCH2 protons, while for 4a-c,
some protons signal appears at 4.20e4.23 ppm. Most important
signals of 13C NMR spectra are those attributed to SCH2 atoms
(w36 ppm for 2b,c and w34 ppm for 4a-c) and C]O atoms
(w164 ppm for 2b,c and w169 ppm for 4a-c, respectively).
The structure of 3b,c derivatives was confirmed by the absence
of the carbonyl bands in its IR spectra, the appearance of the
characteristic singlet signal at 4.66 ppm due to the methylene
protons of thiadiazinic SCH2 group and the appearance of a new
positive signal at 156e158 ppm which corresponding to quaternary
carbon of N]CH group formed by cyclodehydration.
iii- ClCH2COOH/CH3COONa/EtOH/reflux
iv- POCl3/reflux
v- ClCOCOCl/PhH anh./reflux
vi- BrCH(COOEt)2/ CH3COONa/EtOH/reflux
Scheme 1. Synthesis of the title compounds.
phenyl]-4H-1,2,4-triazol-3-yl}thioacetamides 2b,c (X ¼ Cl, Br). If
X ¼ H, S-alkylated derivative could not be properly isolated and
characterized because a complex reaction mixture was obtained
whose components could not be isolated in order to be character-
ized. Therefore the corresponding triazolothiadiazine was not
synthesized. Compounds 2b,c heating with phosphorus oxychloride
underwent intramolecular ring closure with the formation of 6-
phenylamino-3-[4-(4-X-phenylsulfonyl)phenyl]-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazines 3b,c (X ¼ Cl, Br).
Similarly, some changes occur in the spectra of compounds 5a-c
compared with those of corresponding S-alkyl derivatives 4a-c. So,
as a result of overall action of inductive effect and magnetic
anisotropic effect of the aromatic rings, the chemical shift of the
SCH2 group in the thiadiazine moiety appears upfield
(4.05e4.10 ppm in 1H NMR spectra) in comparison with the
obtained values for 4a-c. 13C NMR data of triazolothiadiazinones
5a-c exhibit a new peak at 164e166 ppm which corresponding to
cyclic carbonyl group obtained after intramolecular dehydration.
Transformation of triazoles 1b,c into triazolothiadiazinones 6c
and 7b is supported by the presence of the cyclic amide stretching
frequencies in the IR spectra: 3213 and 1683 cmꢀ1 for 6c and 3116 and
1676 cmꢀ1 for 7b, respectively. In addition, each of these compounds
presents in the IR spectrum, bands characteristic of different func-
tional groups, which are attached on the thiadiazine ring. So, the
intense band at 1750 cmꢀ1 for 6c indicating the presence of other
carbonyl group and the band at 1744 cmꢀ1, together with the bands at
2977, 2941, 2909 cmꢀ1 confirm the presence of ethylcarboxylate
group in 7 position of 7b. The signals recorded in the NMR spectra are
characteristic of these functional groups. The amide NH proton
appears as broadened singlet at 10.87 ppm in 6c and 8.88 ppm in 7b,
respectively.13C NMR spectra exhibit peaks at 172.10 and 160.90 ppm
corresponding to carbonyl groups for 6c and at 168.64, 164.69 and
44.75 ppm, assigned to amide carbonyl, ester carbonyl and SCH
carbons for 7b. In addition, the 1H NMR spectra of 7b contains a two-
proton quadruplet from OCH2CH3 group at 3.32 ppm and a three-
proton triplet from OCH2CH3 group at 1.41 ppm. The signals corre-
sponding to carbon-atoms of this group appear at 62.31 ppm
(OCH2CH3) and 14.20 ppm (OCH2CH3), respectively.
An attempt to transform the aminotriazoles 1a-c into the cor-
responding triazolothiadiazinones 5a-c by refluxing with ethyl-
chloroacetate in basic medium [21] was unsuccessful. Therefore,
this transformation was achieved in two steps. In the first step, S-
alkyl derivatives 4a-c, obtained by reaction of triazoles 1a-c with
chloroacetic acid in presence of sodium acetate, were isolated and
characterized. Finally, we found that the respective tri-
azolothiadiazinone 5a-c was formed by cyclization of these inter-
mediates via elimination of water in presence of phosphorus
oxychloride.
Transformation of aminotriazoles 1a-c in triazolothiadiazinone
was successful only if X ¼ halogen. Thus, treatment of the amino-
triazole 1c with oxalylchloride in dry benzene afforded the corre-
sponding triazolothiadiazin-6,7-dione 6c. Also, the interaction of
1b with bromo-diethylmalonate led to the formation of the 6-oxo-
triazolothiadiazin-7-carboxylate 7b.
3. Antibacterial activity
The synthesized compounds were tested for their in vitro anti-
bacterial activity against the Gram-positive (Enterococcus faecalis
ATCC 29212; Staphylococcus aureus ATCC 25923; Staphylococcus
epidermidis ATCC 14990; Bacillus cereus ATCC 13061) and Gram-
negative (Acinetobacter baumanii ATCC 19606; Citrobacter freundii
ATCC 27853; Enterobacter clocae ATCC 49141; Escherichia coli ATCC
25922; Pseudomonas aeruginosa ATCC 9027) bacteria by using the
4.2. Antibacterial activity
The newly synthesized compounds were tested for in vitro
antibacterial activities compared with “parent” 4-amino-1,2,4-

G.L. Almajan et al. / European Journal of Medicinal Chemistry 45 (2010) 3191e3195
3193
Table 1
Characterization data of the synthesized compounds.
Compd.
X
Molecular formula
Molecular weight (g/mol)
M.p. (ꢁC)
Yield (%)
Elemental analysisa calc. (found)
C
H
N
2b
2c
3b
3c
4a
4b
4c
5a
5b
5c
6c
7b
Cl
Br
Cl
Br
H
Cl
Br
H
Cl
Br
Br
Cl
e
e
e
e
e
e
e
e
e
e
e
e
C22H18ClN5O3S2
C22H18BrN5O3S2
C22H16ClN5O2S2
C22H16BrN5O2S2
C16H14N4O4S2
C16H13ClN4O4S2
C16H13BrN4O4S2
C16H12N4O3S2
499.9
544.4
481.9
526.4
390.4
424.8
469.3
372.4
406.8
451.3
465.3
478.9
217e219
226e228
280e283
248e250
218e220
180e182
231e232
203e205
163e165
197e199
279e282
187e189
68
65
55
57
71
61
64
78
85
72
71
78
52.85 (52.80)
48.53 (48.48)
54.82 (54.78)
59.19 (50.13)
49.22 (49.16)
45.23 (45.17)
40.95 (40.87)
51.60 (51.52)
47.23 (47.17)
42.58 (42.49)
41.30 (41.24)
47.65 (47.60)
3.63 (3.58)
3.33 (3.28)
3.35 (3.30)
3.06 (3.02)
3.61 (3.70)
3.08 (3.14)
2.79 (2.87)
3.25 (3.31)
2.73 (2.80)
2.46 (2.53)
1.95 (1.90)
3.16 (3.12)
14.01 (13.97)
12.86 (12.81)
14.53 (14.47)
13.30 (13.25)
14.35 (14.29)
13.19 (13.11)
11.94 (11.86)
15.04 (14.96)
13.77 (13.69)
12.41 (12.34)
12.04 (11.98)
11.70 (11.65)
C16H11ClN4O3S2
C16H11BrN4O3S2
C16H9BrN4O4S2
C19H15ClN4O5S2
a
Elemental analysis value limit ¼ ꢂ0.4% of the theoretical value.
triazole 1a-c. They were assessed against five Gram-negative and
four Gram-positive bacterial strains and tetracycline and ampicillin
were used as reference drug molecules.
The data generated from this study (Table 2) showed that
compounds displayed low to moderate activity. The obtained
results can be attributed to quite bulky structure of the tested
compounds but they may be associated with the nature of tested
bacterial species.
Thus, we can see that none of the tested compounds has
inhibitory action against A. baumanii, C. freundii, E. cloacae, E. fae-
calis, B. cereus. We can also notice that the exerted action on Gram-
negative bacteria E. coli and P. aeruginosa is better than on Gram-
positive strains S. aureus and S. epidermidis.
4-Amino-5-[4-(4-X-phenylsulfonyl)phenyl]-4H-1,2,4-triazole-
3-thiols 1a-c, (X ¼ H, Cl, Br) displayed low antibacterial activity,
which is in contradiction with the purpose of Ghannoum and co-
workers who believe that the both NH2 and SH groups should be
free for antibacterial activity [23].
sometimes cause an increase of antibacterial activity. Thus, even if
have a linear and bulky structure, 2b showed excellent activity
against P. aeruginosa (MIC ¼ 32
m
g/mL), 2c and 4a-c showed good
activityagainst S. aureus (MIC ¼ 64
m
g/mL) and E. coli (MIC ¼ 64 g/mL),
m
respectively, comparative with triazoles 1a-c. In these cases the
increased activity could be attributed to the presence of CONH
group (for 2b,c) and COOH group (for 4a-c), respectively.
Blocking the NH2 group in S-alkylated derivatives 2b,c and 4a-c,
by thiadiazine ring closure exerts a visible decrease of this
action. If the tested microorganism is A. baumanii there is
a
slight increase of antibacterial activity. The most active
compounds
with
triazolothiadiazine
nucleus
are
5a
g/
(MIC ¼ 32
m
g/mL against S. epidermidis) and 6c ((MIC ¼ 64
m
mL) against P. aeruginosa and S. aureus).
All the tested compounds, which are considered active, are less
effective than drugs taken as a standard.
5. Conclusions
According to our studies, preservation of the free NH2 group and
involvement of the SH group in an alkylation reaction (2b,c and 4a-c)
This study reports the synthesis, characterization and antibac-
terial activity evaluation of new triazolothiadiazines which present
in 6, or 6 and 7 positions different functional groups (carbonyl,
substituted-amino, or ester). The target compounds were obtained
from 4-amino-5-[4-(4-X-phenyl sulfonyl)phenyl]-4H-1,2,4-tri-
azole-3-thiols 1a-c, (X ¼ H, Cl, Br) directly, by intermolecular
condensation with oxalylchloride and bromo-diethylmalonate, or
in two steps, by intramolecular cyclization of intermediaries S-alkyl
derivatives. The antibacterial data given for the compounds pre-
sented in this paper allowed us to state that the variation of anti-
bacterial activity may be associated with the nature of tested
bacterial strains and is due to the chemical structure of the tested
compounds. The most active compound with triazolothiadiazine
nucleus of this series is 3-[4-(phenylsulfonyl)phenyl]-5H-[1,2,4]
Table 2
Antibacterial activities of compounds 1a-c; 2a,b; 3a,b; 4a-c; 5a-c; 6c; 7b as MIC
values (mg/mL).
Compd.
X
Gram-negative bacteriaa
Ab Cf Ebc Ec
512 512 512 512 512 1024
Gram-positive bacteriab
Ef Sa Se Bc
512
Pa
1a
1b
1c
2b
2c
3b
3c
4a
4b
4c
5a
5b
5c
6c
7b
Te
Am
H
Cl
Br
128 1024
512 1024
512 1024
256 1024
128
128
512
512
512
512 1024 128 1024
512 1024 512 1024
512
512
128
64
Cl 1024
Br 1024 1024
Cl
Br
H
Cl 1024
Br 1024
H
Cl
Br
512
256
128
256 128 1024
128 64 1024
256 256 1024 1024 1024
32 1024
256
512
512
512
128 1024
512 512
256 1024 1024
512
triazolo[3,4-b][1,3,4]thiadiazin-6(7H)-one 5a (MIC ¼ 32
mg/mL
1024
512 1024
512 1024
512 1024
64 256
64 256
64 256
512
256
512
256
128
512
512
256
128 1024
256 1024
512 1024
32 1024
256 1024
against S. epidermidis).
256
256
256
512
512
512
512
512
2
512
512
512
512
512
128 128 1024
128 128 512
128 128 1024 1024 1024 1024
512 64 1024 64 512 1024
512 128 1024 1024 1024 1024
6. Experimental protocols
Br 1024
Cl 1024
6.1. Chemistry
2
e
2
2
16
e
32
<2
<2
<2
32
<2
<2
e
2
e
e
Melting points were determined with Boetius apparatus and are
uncorrected. The IR spectra (in KBr pellets) were recorded on the
Vertex 70 Bruker apparatus. The NMR spectra (in DMSO-d6, at room
temperature) were registered on a Varian Gemini 300 BB apparatus
working at 300 MHz for 1H and 75 MHz for 13C, using TMS as internal
standard. The content of C, H, and N were done with ECS-40-10-
Costeh micro-dosimeter, after drying the compounds at 105 ꢁC.
Te ¼ tetracycline; Am ¼ ampicillin.
e ¼ No inhibitory action.
a
Ab (Acinetobacter baumanii ATCC 19606); Cf (Citrobacter freundii ATCC 27853);
Ebc (Enterobacter cloacae ATCC 49141); Ec (Escherichia coli ATCC 25922); Pa (Pseu-
domonas aeruginosa ATCC 9027).
b
Ef (Enterococcus faecalis ATCC 29212); Sa (Staphylococcus aureus ATCC 25923); Se
(Staphylococcus epidermidis ATCC 14990); Bc (Bacillus cereus ATCC 13061).

3194
G.L. Almajan et al. / European Journal of Medicinal Chemistry 45 (2010) 3191e3195
6.1.1. General procedure for synthesis of 2-{4-amino-5-[4-(4-X-
phenylsulfonyl)phenyl}-4H-1,2,4-triazol-3-ylthio}-N-
phenylacetamide 2b,c
ring); 158.63 (C5-triazolic ring); 158.13 (C]N thiadiazine ring);
142.93, 139.31, 138.36, 130.12, 138.06, 129.55, 129.18, 128.28,
1256.72, 121.25, 117.30, 115.94, 115.23 (aromatic ring carbons);
28.25 (SCH2).
2-Chloro-N-phenylacetamide (5 mmol), solution in ethanol
(30 mL) was added to solution of compound 1 (5 mmol) in aqueous
ethanol (10 mL) containing KOH (5 mmol). The reaction mixture
was boiled for 45 min and cooled down and then water (50 mL) was
added. The colorless precipitate was filtered off, washed with water,
dried and purified by recrystallization from ethanol.
6.1.3. General procedure for synthesis of 2-{4-amino-5-[4-(4-X-
phenylsulfonyl)phenyl}-4H-1,2,4-triazol-3-ylthio}acetic acid 4a-c
A mixture of 1 (1 mmol), sodium acetate (6.1 mmol) and
chloroacetic acid (1.1 mmol) in ethanol (10 mL) was heated under
reflux for 12 h. After cooling, the solvent was removed under
reduced pressure, the residue was dissolved in water (50 mL) and
the product was extracted with CH2Cl2 (3 ꢃ 50 mL). The organic
layer was washed with water and dried over Na2SO4. The solvent
was removed under reduced pressure and the solid product was
recrystallized from ethanol.
2b: IR (KBr, cmꢀ1): 3371, 3203 (NH, NH2); 3089 (aromatic CH);
2963, 2923 (SCH2); 1677 (C]O); 1603, 1580 (C]N þ C]Caryl);
1321, 1290, 1156 (SO2); 1011 (NeN); 766 (CeCl); 686 (CeSeC); 1H
NMR (DMSO-d6,
d, ppm): 10.46 (s, 3H, NH, NH2); 8.17 (d, 2H,
J ¼ 7.9 Hz, aromatic protons); 8.14 (d, 2H, J ¼ 8.5 Hz, aromatic
protons); 8.01 (d, 2H, J ¼ 8.5; aromatic protons); 7.72 (dd, 2H,
J ¼ 7.4; 2.7 Hz, aromatic protons); 7.57 (d, 2H, J ¼ 7.9 Hz, aromatic
protons); 7.30 (t, 1H, J ¼ 7.9 Hz, aromatic proton); 7.07 (t, 2H,
J ¼ 7.9 Hz, aromatic protons); 4.40 (s, 2H, SCH2); 13C NMR (DMSO-
4a: IR (KBr, cmꢀ1): 3398 (OH); 3294, 3148 (NH2); 3089 (aromatic
CH); 2956, 2875 (SCH2); 1714 (C]O); 1618, 1583, 1517 (C]N þ C]
Caryl); 1328, 1158 (SO2); 1012 (NeN); 685 (CeSeC); 1H NMR
d6,
d
, ppm): 164.78 (C]O); 164.69 (C3-triazolic ring); 163.96 (C5-
(DMSO-d6,
J ¼ 8.5, 2.1 Hz, aromatic protons); 7.51e7.85 (m, 3H, aromatic
protons); 4.20 (s, 2H, SCH2); 13C NMR (DMSO-d6,
, ppm): 169.30
d, ppm): 8.10 (s, 4H, aromatic protons); 8.05 (dd, 2H,
triazolic ring); 143.12, 139.34, 139.15, 138.62, 130.11, 129.60, 128.87,
128.53, 127.70, 127.60, 123.73, 119.20 (aromatic ring carbons); 36.89
(SCH2).
d
(COOH); 164.58 (C3-triazolic ring); 164.12 (C5-triazolic ring);
141.09, 140.59, 133.61, 131.15, 130.56, 129.59, 128.02, 127.58
(aromatic ring carbons); 34.68 (SCH2).
2c: IR (KBr, cmꢀ1): 3374, 3202 (NH, NH2); 3089 (aromatic CH);
2923, 2860 (SCH2); 1668 (C]O); 1611,1580 (C]N þ C]Caryl); 1323,
1290, 1156 (SO2); 1003 (NeN); 686 (CeSeC); 582 (CeBr); 1H NMR
4b: IR (KBr, cmꢀ1): 3418 (OH); 3301, 3160 (NH2); 3097 (aromatic
CH); 2938, 2840 (SCH2); 1712 (C]O); 1623, 1597, 1529 (C]N þ C]
Caryl); 1318, 1159 (SO2); 1009 (NeN); 765 (CeCl); 685 (CeSeC); 1H
(DMSO-d6,
d
, ppm): 10.41 (s, 3H, NH, NH2); 8.16 (d, 2H, J ¼ 7.2 Hz,
aromatic protons); 8.13 (d, 2H, J ¼ 7.2 Hz, aromatic protons); 8.02
(d, 2H, J ¼ 8.1; aromatic protons); 7.71 (dd, 2H, J ¼ 7.1; 2.7 Hz,
aromatic protons); 7.54 (d, 2H, J ¼ 8.1 Hz, aromatic protons); 7.28 (t,
2H, J ¼ 7.1 Hz, aromatic proton); 7.04 (t, 1H, J ¼ 7.1 Hz, aromatic
NMR (DMSO-d6,
J ¼ 8.5 Hz, aromatic protons); 7.55 (d, 2H, J ¼ 8.5 Hz, aromatic
protons); 4.23 (s, 2H, SCH2); 13C NMR (DMSO-d6,
, ppm): 169.34
d, ppm): 8.03 (s, 4H, aromatic protons); 7.95 (d, 2H,
d
protons); 4.25 (s, 2H, SCH2); 13C NMR (DMSO-d6,
d, ppm): 164.96
(COOH); 163.33 (C3-triazolic ring); 162.91 (C5-triazolic ring);
140.83, 139.60, 139.21, 130.13, 129.59, 129.00, 128.26, 128.21
(aromatic ring carbons); 34.85 (SCH2).
(C]O); 164.62 (C3-triazolic ring); 163.88 (C5-triazolic ring); 143.13,
139.39, 139.15, 138.65, 130.12, 129.73, 128.88, 128.61, 127.72, 127.66,
123.73, 120.17 (aromatic ring carbons); 36.87 (SCH2).
4c: IR (KBr, cmꢀ1): 3427 (OH); 3282, 3176 (NH2); 3092 (aromatic
CH); 2987, 2933 (SCH2); 1717 (C]O); 1632, 1580, 1554 (C]N þ C]
Caryl); 1324, 1158 (SO2); 1011 (NeN); 685 (CeSeC); 579 (CeBr); 1H
6.1.2. General procedure for synthesis of 3-[4-(4-X-phenylsulfonyl)
phenyl]-N-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-
amine 3b,c
NMR (DMSO-d6,
J ¼ 8.7 Hz, aromatic protons); 7.70 (d, 2H, J ¼ 8.7 Hz, aromatic
protons); 4.20 (s, 2H, SCH2); 13C NMR (DMSO-d6,
, ppm): 169.07
d, ppm): 8.15 (s, 4H, aromatic protons); 8.00 (d, 2H,
N-phenylacetamide 2 (2.7 mmol) in phosphorus oxychloride
(8.5 mL) was refluxed for 2e3 h; the excess oxychloride was
evaporated under vacuum to dryness. The oily residue was tritu-
rated with ether and then was neutralized with KOH 10%. The
obtained precipitate was filtered off, washed with water until
pH ¼ 7, dried and recrystallized from ethanol.
d
(COOH); 164.80 (C3-triazolic ring); 164.15 (C5-triazolic ring);
143.30, 139.58, 139.27, 130.31, 129.78, 128.75, 127.93, 127.73
(aromatic ring carbons); 34.48 (SCH2).
6.1.4. General procedure for synthesis of 3-[4-(4-X-phenylsulfonyl)
phenyl]-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6(7H)-one 5a-c
A mixture of 4 (1 mmol) and phosphoryl chloride (10 mL) was
heated under reflux for 3 h. After cooling, the solvent was removed
under reduced pressure and the formed solid residue was poured
into ice-water and neutralized with cold ammonia solution. The
obtained precipitate was filtered off and recrystallized from chlo-
roform:petroleum ether 1:1 (v/v).
3b: IR (KBr, cmꢀ1): 3316, 3142 (NH); 3087 (aromatic CH); 2922,
2852 (SCH2); 1619, 1579, 1502 (C]N þ C]Caryl); 1324, 1157 (SO2);
1010 (NeN); 766 (CeCl); 689 (CeSeC); 1H NMR (DMSO-d6 þ TFA,
d,
ppm): 9.35 (s, 1H, NH); 8.13 (d, 2H, J ¼ 8.8 Hz, aromatic protons);
8.08 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 7.99 (d, 2H, J ¼ 8.5;
aromatic protons); 7.68 (d, 2H, J ¼ 8.8 Hz, aromatic protons); 7.60
(d, 2H, J ¼ 7.7 Hz, aromatic protons); 7.33 (t, 1H, J ¼ 7.7 Hz, aromatic
proton); 7.00 (t, 2H, J ¼ 7.7 Hz, aromatic protons); 4.66 (s, 2H,
5a: IR (KBr, cmꢀ1): 3312 (NH); 3088 (aromatic CH); 2947, 2870
(SCH2); 1768 (C]O); 1621, 1573, 1502 (C]N þ C]Caryl); 1324, 1287,
SCH2); 13C NMR (DMSO-d6 þ TFA,
d, ppm): 159.85 (C3-triazolic
ring); 158.44 (C5-triazolic ring); 156.92 (C]N thiadiazine ring);
142.01139.37, 139.15, 138.35, 138.06, 129.96, 129.04, 128.48, 126.59,
122.14, 120.73, 120.40, 113.09 (aromatic ring carbons); 28.13 (SCH2).
3c: IR (KBr, cmꢀ1): 3326, 3148 (NH); 3087 (aromatic CH); 2918,
2854 (SCH2); 1614, 1579, 1502 (C]N þ C]Caryl); 1326, 1158 (SO2);
1159 (SO2); 1012 (NeN); 685 (CeSeC); 1H NMR (DMSO-d6,
d, ppm):
8.27 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 8.11 (d, 2H, J ¼ 7.8 Hz,
aromatic protons); 8.00 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 7.70
(tt, 1H, J ¼ 7.8, 1.5 Hz, aromatic proton); 7.63 (t, 2H, J ¼ 7.8 Hz,
aromatic protons); 4.07 (s, 2H, SCH2); 13C NMR (DMSO-d6,
d, ppm):
1005 (NeN); 682 (CeSeC); 584 (CeBr); 1H NMR (DMSO-d6,
d
,
166.61 (C]N thiadiazine ring); 162.12 (C3-triazolic ring); 158.70
(C5-triazolic ring); 140.18, 139.60, 133.28, 130.60, 130.24, 129.84,
127.98, 127.54 (aromatic ring carbons); 34.38 (SCH2).
ppm): 9.42 (s, 1H, NH); 8.15 (dd, 2H, J ¼ 8.2, 3.2 Hz, aromatic
protons); 8.08 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 7.99 (d, 2H,
J ¼ 7.9; aromatic protons); 7.33 (d, 2H, J ¼ 8.2 Hz, aromatic protons);
7.63 (d, 2H, J ¼ 7.5 Hz, aromatic protons); 7.40 (d, 2H, J ¼ 7.5 Hz,
aromatic proton); 7.03 (t, 1H, J ¼ 7.5 Hz, aromatic proton); 4.66 (s,
5b: IR (KBr, cmꢀ1): 3288 (NH); 3091 (aromatic CH); 2914, 2868
(SCH2); 1775 (C]O); 1618, 1584, 1498 (C]N þ C]Caryl); 1320,
1283, 1157 (SO2); 1010 (NeN); 763 (CeCl); 688 (CeSeC); 1H NMR
2H, SCH2); 13C NMR (DMSO-d6 þ TFA,
d, ppm): 160.51 (C3-triazolic
(DMSO-d6,
d
, ppm): 8.21 (d, 2H, J ¼ 8.6 Hz, aromatic protons); 8.11

G.L. Almajan et al. / European Journal of Medicinal Chemistry 45 (2010) 3191e3195
3195
(d, 2H, J ¼ 8.6 Hz, aromatic protons); 7.91 (d, 2H, J ¼ 8.5 Hz,
aromatic protons); 7.55 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 4.05 (s,
inhibitory concentrations (MIC) were defined as the lowest
concentration of the compounds that inhibited visible growth of
microorganisms after incubation at 35 ꢁC for 24 h.
2H, SCH2); 13C NMR (DMSO-d6,
d, ppm): 166.18 (C]N thiadiazine
ring); 160.48 (C3-triazolic ring); 158.78 (C5-triazolic ring); 140.75,
139.34, 138.04, 130.20, 129.14, 128.28, 128.18, 127.57 (aromatic ring
carbons); 34.36 (SCH2).
Gram-positive (E. faecalis ATCC 29212, S. aureus ATCC 25923,
S. epidermidis ATCC 14990, B. cereus ATCC 13061) and Gram-
negative (A. baumanii ATCC 19606, C. freundii ATCC 27853, Enter-
obacter cloacae ATCC 49141, E. coli ATCC 25922, P. aeruginosa ATCC
9027) bacteria were used as quality control strains. Bacterial strains
were grown in Mueller-Hinton broth (Merck). The inoculums
densities were 5 ꢃ 105 colony forming units (cfu) in 1 mL. Serial
dilutions of the test compounds, previously dissolved in dime-
thylsulfoxide (DMSO), were prepared in test tubes to final
5c: IR (KBr, cmꢀ1): 3307 (NH); 3087 (aromatic CH); 2939, 2848
(SCH2); 1772 (C]O); 1624,1572,1500 (C]N þ C]Caryl); 1325,1291,
1159 (SO2); 1009 (NeN); 688 (CeSeC); 578 (CeBr); 1H NMR
(DMSO-d6,
d
, ppm): 8.17 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 8.09
(d, 2H, J ¼ 8.5 Hz, aromatic protons); 7.92 (d, 2H, J ¼ 8.5 Hz,
aromatic protons); 7.84 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 4.10 (s,
2H, SCH2); 13C NMR (DMSO-d6,
d, ppm): 164.28 (C]N thiadiazine
concentrations of 1024; 512; 256; 128; 64; 32; 16; 8; 4; 2
mg/mL
ring); 163.52 (C3-triazolic ring); 159.16 (C5-triazolic ring); 144.30,
139.48, 139.05, 133.08, 129.59, 128.68, 128.12, 127.98 (aromatic ring
carbons); 34.42 (SCH2).
100 L of 24 h old inoculums was added to each tube. Tetracycline
m
and ampicillin were used as standard antibiotic. Tests using DMSO
as negative control were carried out in parallel and it was deter-
mined that the solvent had no antimicrobial activity against any of
the test microorganisms. Because the MIC values are not spectac-
ular, no statistical calculations were made.
6.1.5. General procedure for synthesis of 3-[4-(4-X-phenylsulfonyl)
phenyl]-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine-6,7-dione 6c
A mixture of 1c (1 mmol) and oxalylchloride (1 mmol) in dry
benzene (10 mL) was heated under reflux for 8 h. The solvent
was removed under reduced pressure, the formed solid product
was filtered and recrystallized from chloroform:petroleum ether
1:1 (v/v).
Acknowledgements
This work is supported by project ID_226 no. 301/1.10.2007 from
the Exploratory Research Projects of the National University
Research Council (NURC-Romania).
6c: IR (KBr, cmꢀ1): 3213 (NH); 3090 (aromatic CH); 1750, 1683
(C]O); 1631, 1572, 1471 (C]N þ C]Caryl); 1326, 1160 (SO2); 1010
(NeN); 686 (CeSeC); 581 (CeBr); 1H NMR (DMSO-d6,
d, ppm):
References
10.87 (s, 1H, NH); 8.14 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 8.07 (d,
2H, J ¼ 8.5 Hz, aromatic protons); 7.95 (d, 2H, J ¼ 8.5 Hz, aromatic
protons); 7.89 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 13C NMR
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133.08, 129.62, 128.49, 127.44, 127.20 (aromatic ring carbons).
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[1,3,4]thiadiazine-7-carboxylate 7b
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A mixture of 1b (1 mmol), sodium acetate (6.1 mmol) and
bromo-diethylmalonate (1 mmol) in ethanol (10 mL) was heated
under reflux for 8 h. After cooling, the solvent was removed under
reduced pressure and the residue was treated with cold water
(50 mL). The obtained precipitate was filtered off and recrystallized
from chloroform:petroleum ether 1:1 (v/v).
7b: IR (KBr, cmꢀ1): 3116 (NH); 3093 (aromatic CH); 2977, 2941,
2909 (CH, CH2, CH3); 1744 (COO), 1676 (C]O); 1638, 1613, 1582
(C]N þ C] Caryl); 1324, 1157 (SO2); 1012 (NeN); 769 (CeCl); 696
(CeSeC); 1H NMR (DMSO-d6,
d, ppm): 8.88 (s, 1H, NH); 8.04 (d, 2H,
J ¼ 8.5 Hz, aromatic protons); 7.93 (d, 2H, J ¼ 8.8 Hz, aromatic
protons); 7.86 (d, 2H, J ¼ 8.5 Hz, aromatic protons); 7.83 (d, 2H,
J ¼ 8.8 Hz, aromatic protons); 4.89 (s, 1H, SCH); 3.32 (q, 2H,
J ¼ 7.2 Hz, CH2); 1.41 (t, 3H, J ¼ 7.0 Hz, CH3); 13C NMR (DMSO-d6,
d,
ppm): 168.64 (COO), 164.69 (C]O); 163.15 (C3-triazolic ring);
157.10 (C5-triazolic ring); 141.12, 139.62, 139.34, 130.12, 129.13,
128.88, 127.80, 127.62 (aromatic ring carbons); 62.35 (CH2); 44.75
(CH); 14.20 (CH3).
6.2. Antibacterial activity
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Minimal inhibitory concentrations (MICs,
mg/ml) were deter-
mined on different microorganisms using broth micro dilution
procedure according to the recommendations of the National
Committee for Clinical Laboratory Standards [22]. Minimum