Synthesis and antibacterial evaluation of new heterocyclic system: [1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-e][1,3,4]thiadiazine

Article abstract of DOI:10.1515/hc-2011-0051

A series of 1,2,4-triazolo[3′,4′:6,1]pyridazino[4,3-e][1,3,4] thiadiazines have been synthesized by intermolecular cyclization of a hydrazide derivative with various triethyl orthoesters. The antibacterial activity of various derivatives of this new heterocyclic system was evaluated. Copyright by Walter de Gruyter.

Full text of DOI:10.1515/hc-2011-0051

Heterocycl. Commun., Vol. 18(1), pp. 39–42, 2012 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/hc-2011-0051
Synthesis and antibacterial evaluation of new heterocyclic
system: [1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-e]
[1,3,4]thiadiazine
Results and discussion
Chemistry
Mohammad Rahimizadeh*, Ali Shiri, Mahsa Ranaei
and Mehdi Bakavoli
Department of Chemistry, Faculty of Science, Ferdowsi
University of Mashhad, 91775-1436 Mashhad, Iran
4-Bromo-3,6-dichloropyridazine, which was quantitatively
prepared as previously reported (Alazawe and Elvidge, 1974),
was treated with methylhydrazine in dry chloroform at room
temperature to yield 3,6-dichloro-4-(1-methylhydrazino)
pyridazine (Bakavoli et al., 2007). Treatment of the result-
ing compound with phenylisothiocyanate in the presence of
triethylamine in boiling acetonitrile afforded the 3-anilino-
7-chloro-1-methyl-1H-pyridazino[4,3-e][1,3,4]thiadiazine
(1). The reaction of compound 1 with hydrazine hydrate
in refluxing ethanol gave 3-anilino-7-hydrazino-1-methyl-
1H-pyridazino[4,3-e][1,3,4]thiadiazine (2). This product
subsequently underwent cyclocondensation with triethyl
orthoesters in acetic acid under reflux conditions to give the
substituted [1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-e][1,3,4]
thiadiazine 3a–d (Scheme 1).
*Corresponding author
e-mail: rahimizh@yahoo.com
Abstract
A series of 1,2,4-triazolo[3′,4′:6,1]pyridazino[4,3-e][1,3,4]
thiadiazines have been synthesized by intermolecular cycli-
zation of a hydrazide derivative with various triethyl ortho-
esters. The antibacterial activity of various derivatives of this
new heterocyclic system was evaluated.
Keywords: heterocyclization; phenylisothiocyanate; tria-
zolopyridazinothiadiazine; triethylorthoester.
All compounds 3a–d were characterized by physical, spec-
tral, and analytical data. For example, the 1H NMR spectrum
of 3b does not show the NH2 and NH signals of hydrazine
moiety of compound 2 at δ 4.31 and 7.83 ppm, but instead
exhibits a sharp signal at δ 2.54 belonging to the methyl group
of the triazole ring indicating the ring closure. The IR spec-
trum also does not show the NH2 and NH vibration bands
of hydrazine substituent at ν 3310, 3300, and 3290 cm-1 of
the hydrazino derivative 2. The presence of the molecular
ion peak of compound 3b in its mass spectrum at m/z 311
and satisfactory microanalytical data fully support the given
structure.
Introduction
Fused 1,2,4-triazoles express antifungal (El-Hawash et al.,
2006), bactericidal (Brown and Iwai, 1979), anxiolytic (Tarzia
et al., 1988), and herbicidal (Peignier et al., 1991) activities,
and can be applied as antidepressants (Sarges et al., 1990).
By contrast, pyridazinothiadiazines are of interest as potential
inhibitors of cyclic nucleotide phosphodiesterase (Naka and
Furukawa, 1979), dyestuff (Elliot, 1977), and precursors to
herbicides (Nissan Chemical Industries, 1981). Therefore, the
synthesis of triazolopyridazinothiadiazines could be interest-
ingfrompharmacologicalandsyntheticpointofviews.Several
methods for the preparation of triazoles have been reported
in the literature, and most of them are based on heterocyclic
hydrazones and hydrazides as precursors (Bower and Doyle,
1957; Gibson, 1963; Pollak and Tisler, 1966; Crljenak et al.,
1983; Bourgeois et al., 1993; Moreau et al., 1994; Sadana
et al., 2003; Kumar et al., 2004). A review of the literature
also showed that there is no report for the synthesis of fused
triazolopyridazinothiadiazine systems.
As part of our ongoing studies dealing with the synthe-
sis of new biologically active heterocyclic compounds
(Bakavoli et al., 2007, 2008, 2009, 2010a,b, 2011a,b,c), we
now describe the synthesis of the novel heterocyclic system
[1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-e][1,3,4] thiadiazine.
The antibacterial activities of the synthesized compounds
against Gram-positive and Gram-negative bacteria are also
reported.
Antibacterial evaluations
The tested microorganisms were Gram-positive and Gram-
negative bacteria. The sensitivity of the selected micro-
organisms, Staphylococcus aureus PTCC 1074, Bacillus
subtilis PTCC 1365, Escherichia coli HB101 BA 7601C and
Pseudomonas aeruginosa PTCC 1431, to compounds 3a–d
was determined using in vitro cultures that were dissolved in
DMSO, and the tests were carried out using the disk diffusion
method (Reeves and White, 1983).
Mueller-Hinton agar media were sterilized (15 min at
121°C) and poured into the plates to a uniform depth of 5 mm
to solidify. The microbial suspension (1.2×108 CFU/mL)
(0.5 McFarland Nephelometery Standards) was streaked over
the surface of media using a sterile cotton swab (15 min at
180°C) to ensure confluent growth of the organisms. The
tested compounds were dissolved in DMSO and the solution
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40 M. Rahimizadeh et al.
CH3
CH3
Elemental analysis was performed on a Thermo Finnigan Flash
EA1112 microanalyzer.
H
N
Cl
N
N
N2H4
EtOH
N
H2N
N
N
N
N
S
N
H
N
S
N
H
N-(7-Chloro-1-methyl-1H-pyridazino[4,3-e][1,3,4]
thiadiazin-3-yl)-N-phenylamine (1)
(1)
(2)
RC(OEt)3
HOAc
To a magnetically stirred solution of 3,6-dichloro-4-(1-methyl-
hydrazino)pyridazine(3mmol, 0.58g)andtriethylamine(3.1mmol,
0.4 mL) in acetonitrile (25 mL), phenylisothiocyanate (3 mmol,
0.87 g) was added under an atmosphere of nitrogen. The solu-
tion was then heated under reflux for 4 h and the progress of the
reaction was monitored by thin layer chromatography (TLC) using
CHCl3/MeOH (9:1). After cooling the mixture to room tempera-
ture, the resulting solid was filtered off and crystallized from etha-
CH3
N
N
3a: R=H
N
N
3b: R=CH3
3c: R=C2H5
3d: R=Ph
N
N
S
N
H
R
(3a-d)
1
nol to give 1 as yellow needles: yield 70%; mp 235°C; H NMR:
δ 3.22 (s, 3H), 6.91 (s, 1H), 7.02–7.53 (m, 5H), 9.42 (br s, 1H, D2O
exchangeable); IR: ν 3340, 3245, 1636, 1596, 746 cm-1; MS: m/z
291 (M+), 293 (M++2). Anal. Calcd. for C12H10ClN5S: C, 49.40;
H, 3.45; N, 24.00; S, 10.99. Found, C, 49.24; H, 3.41; N, 24.03;
S, 11.01.
Scheme 1 Synthesis of compounds 3a–d.
was diluted with ethanol to a concentration of 100–400
µg/mL. The discs measuring 6.25 mm in diameter (Whatman
no. 1 filter paper) were impregnated with the prepared solu-
tion of compounds 3a–d. Specifically, 1 mL of the solution
was added to each bottle containing 12 discs and the discs
were placed on Muller-Hinton agar media previously inoc-
ulated with bacterial suspension. The inhibition zones as a
criterion for antimicrobial activity were measured in millime-
ters at the end of an incubation period of 24 h at 37°C. The
biological activity results of compounds and streptomycin as
a reference bactericidal antibiotic are shown in Table 1. These
results were obtained in triplicate and the values with differ-
ences >5% were discarded and the measurement repeated.
It can be concluded that compounds 3a–d are highly active
against Bacillus subtilis and Staphylococcus aureus and less
active against Pseudomonas aeruginosa and Escherichia coli
Gram-negative bacteria.
N-(7-Hydrazino-1-methyl-1H-pyridazino[4,3-e][1,3,4]
thiadiazin-3-yl)-N-phenylamine (2)
A solution of compound 1 (1 mmol, 0.29 g) in ethanol (15 mL) was
treated with hydrazine hydrate (3 mmol) and the mixture was heated
under reflux for 5–6 h. After completion of the reaction, as monitored
by silica gel TLC, the solvent was removed under reduced pressure
and the resulting solid was washed with water and crystallized from
1
ethanol: yield 60%; mp 210–211°C; H NMR: δ 3.33 (s, 3H), 4.31
(s, 2H, D2O exchangeable), 6.24 (s, 1H), 6.81–7.62 (m, 5H), 7.83
(s, 1H, D2O exchangeable), 9.22 (s, 1H, D2O exchangeable); IR: ν
3310, 3300, 3290, 1600, 1550 cm-1; MS: m/z 287 (M+). Anal. Calcd.
for C12H13N7S: C, 50.16; H, 4.56; N, 34.12; S, 11.16. Found, C,
50.04; H, 4.41; N, 34.03; S, 11.01.
General procedure for the preparation of [1,2,4]
triazolo[3′,4′:6,1]pyridazino[4,3-e] [1,3,4]thiadiazines
3a–d
Experimental
The 1H NMR (100 MHz, DMSO-d6) spectra were recorded on
a Bruker AC 100 spectrometer. The IR spectra (KBr discs) were
obtained on a 4300 Shimadzu spectrometer. The electron impact
mass spectra were recorded on a Varian Mat CH-7 at 70 eV.
A solution of compound 2 (1 mmol, 0.28 g) in acetic acid (2 mL)
was treated with a triethyl orthoester (1.1 mmol) and the mixture
was heated under reflux for 5 h. After completion of the reaction, as
monitored by silica gel TLC using CHCl3/MeOH (9:1) as an eluent,
Table 1 Antibacterial data of the synthesized compounds 3a–d.
Compound
Gram-negative bacteria
Gram-positive bacteria
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
Bacillus subtilis
HB101 BA 7601C
PTCC 1431
PTCC 1074
PTCC 1365
3a
3b
3c
3d
15a (-)b (400)c
17 (+) (400)
15 (-) (300)
14 (-) (400)
17
13 (+) (500)
12 (+) (300)
12 (+) (300)
13 (+) (400)
13
18 (++) (100)
18 (++) (100)
17 (++) (200)
17 (++) (100)
15
15 (++) (200)
15 (++) (300)
14 (++) (200)
14 (++) (100)
10
Streptomycin
(standard)
aZones of inhibition in millimeters.
b(++) Highly sensitive; (+) moderately sensitive; (-) slightly sensitive.
cConcentration in µg/mL and the maximum inhibition zone for each compound has been shown. Discs of each concentration were placed in
triplicate in Muller-Hinton agar medium seeded with fresh bacteria separately and the average was reported.
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Synthesis and antibacterial evaluation of new heterocyclic system 41
the solvent was removed under reduced pressure. Water (5 mL) was
as potential antibacterial agents. Eur. J. Med. Chem. 2010a, 45,
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added to the residue and the mixture was neutralized using 10%
NaHCO3 solution. The resulting solid was filtered off, washed with
water, and crystallized from ethanol.
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thiadiazin-3-yl)-N-phenylamine(3a) Yield60%;mp281–282°C;
1H NMR: δ 3.25 (s, 3H), 6.91–7.62 (m, 5H), 9.21 (s, 1H), 9.52 (s, 1H,
D2O exchangeable), IR: ν 3290, 1605, 1550 cm-1; MS: m/z 297 (M+).
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Found, C, 52.84; H, 3.81; N, 32.59; S, 10.53.
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Synthesis of new derivatives of 3-aryl-1,5-dimethyl-1H[1,2,4]
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183–187.
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S.; Pordeli, P.; Nikpour, M. Synthesis and evaluation of antibac-
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N-(1,7-Dimethyl-1H-[1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-e]
[1,3,4]thiadiazin-3-yl)-N-phenylamine (3b) Yield 55%; mp
270–271°C; 1H NMR: δ 2.54 (s, 3H), 3.27 (s, 3H), 6.91–7.63
(m, 5H), 9.41 (s, 1H, D2O exchangeable); IR: ν 3310, 1605, 1553
cm-1; MS: m/z 311 (M+). Anal. Calcd. for C14H13N7S: C, 54.00; H,
4.21; N, 31.49; S, 10.30. Found, C, 54.30; H, 4.28; N, 31.53; S,
10.25.
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N-(7-Ethyl-1-methyl-1H-[1,2,4]triazolo[3′,4′:6,1]pyridazino[4,3-
e][1,3,4]thiadiazin-3-yl)-N-phenylamine (3c) Yield 70%; mp
250–251°C; H NMR: δ 1.31 (t, J=7.4 Hz, 3H), 2.96 (q, J=7.4 Hz,
2H), 3.30 (s, 3H), 6.92–7.62 (m, 5H), 9.36 (s, 1H, D2O exchange-
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for C15H15N7S: C, 55.37; H, 4.65; N, 30.13; S, 9.85. Found, C, 55.11;
H, 4.48; N, 29.93; S, 9.65.
1
N-(1-Methyl-7-phenyl-1H-[1,2,4]triazolo[3′,4′:6,1]pyridazino
[4,3-e][1,3,4]thiadiazin-3-yl)-N-phenylamine (3d) Yield 50%;
1
mp 245–246°C; H NMR: δ 3.26 (s, 3H), 6.89–8.11 (m, 10H), 9.43
(s, 1H, D2O exchangeable); IR: ν 3310, 1605, 1550 cm-1; MS: m/z 373
(M+). Anal. Calcd. for C19H15N7S: C, 61.11; H, 4.05; N, 26.26; S, 8.59.
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Received September 25, 2011; accepted November 24, 2011;
previously published online January 13, 2012
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