Synthesis and in-vitro antimicrobial activity of some heterocyclic compounds via 7H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl acetic acid hydrazide

Article abstract of DOI:10.1080/10426500601090768

{7 H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl} acetic acid hydrazide was utilized by different reagent, namely isothiocyanates, formic acid, triethyl orthoformate, and carbon disulfide, to yield the corresponding compounds, which were cyclized to constr

Full text of DOI:10.1080/10426500601090768

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Phosphorus, Sulfur, and Silicon
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Synthesis and In-Vitro
Antimicrobial Activity of Some
Heterocyclic Compounds
via 7H-1,2,4-triazolo[1,5-
d]tetrazol-6-ylsulfanyl Acetic
Acid Hydrazide
M. A. M. Taha a & S. M. El-Badry b
a Chemistry Department , Faiyoum University ,
Faiyoum, Egypt
b Physics and Chemistry Department , Alexandria
University , Alexandria, Egypt
Published online: 20 Apr 2007.
To cite this article: M. A. M. Taha & S. M. El-Badry (2007) Synthesis and In-Vitro
Antimicrobial Activity of Some Heterocyclic Compounds via 7H-1,2,4-triazolo[1,5-
d]tetrazol-6-ylsulfanyl Acetic Acid Hydrazide, Phosphorus, Sulfur, and Silicon and the
Related Elements, 182:5, 1011-1021, DOI: 10.1080/10426500601090768
To link to this article: http://dx.doi.org/10.1080/10426500601090768
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Phosphorus, Sulfur, and Silicon, 182:1011–1021, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500601090768
Synthesis and In-Vitro Antimicrobial Activity of Some
Heterocyclic Compounds via
7H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl Acetic
Acid Hydrazide
M. A. M. Taha
Chemistry Department, Faiyoum University, Faiyoum, Egypt
S. M. El-Badry
Physics and Chemistry Department, Alexandria University,
Alexandria, Egypt
{7 H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl} acetic acid hydrazide was utilized
by different reagent, namely isothiocyanates, formic acid, triethyl orthoformate, and
carbon disulfide, to yield the corresponding compounds, which were cyclized to con-
struct 1,2,4-triazoles, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, and 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazoles. The structure of the products was deduced through physico-
1
chemical as well as spectral data (IR, H NMR, and MS). Representative members
of the prepared compounds were tested for antimicrobial activity.
Keywords {7H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl} acetic acid hydrazide; antimi-
crobial activity; structure elucidation; synthesis; various heterocyclic systems
In pursuance of our work on the synthesis1−3 of some heterocyclic
systems with isolated 1,2,4-triazolo[1,5-d ]tetrazole nucleus, we pro-
posed the synthesis of some 1,2,4-triazoles, 1,3,4-oxadiazoles, 1,3,4-
thiadiazoles, and 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles starting from
readily available {7H-1,2,4-triazolo[1,5-d ]tetrazol-6-ylsulfanyl} acetic
acid hydrazide (1) through different reagents. In view of the potential
biological activity of members of previous heterocyclic moieties,4−8 it
was of interest to us to prepare new compounds to test their antimicro-
bial activity.
The synthetic routes followed for preparation of the designed
compounds are depicted in Scheme 1. Reaction of {7H-1,2,4-
triazolo[1,5-d]tetrazol-6-ylsulfanyl} acetic acid hydrazide3 (1) with
Received March 14, 2006; accepted October 6, 2006.
This article is Part III of a series. For Part II, see ref. 1.
Address correspondence to M. A. M. Taha, Chemistry Department, Faculty of Science,
Faiyoum University, Faiyoum, Egypt. E-mail: mamdouhamtaha@yahoo.com
1011

1012

Activity of Heterocyclic Compounds
1013
methyl or phenyl isothiocyanate in ethanol at ambient temperature9
afforded 4-methyl(phenyl)-1-{7H-1,2,4-triazolo[1,5-d]-tetrazol-6-ylsul-
fanylacetyl}thiosemicarbazide (2a,b). 1-acyl-4-arylthiosemicarbazides
are known to undergo dehydrocyclization in acidic medium to yield
2-alkyl-5-arylamino-1,3,4-thiadiazoles.10,11 Applying such dehydrocy-
clization to 2a,b with phosphoryl chloride gave the corresponding 5-
methyl(phenyl)amino-2-methylthio {7H-1,2,4-triazolo[1,5-d]tetrazol-
6-yl}-1,3,4-thiadiazole (3a,b). The IR spectra of (3a,b) showed the
disappearance of the amide absorption bands present in the spectra
of the parent thiosemicarbazides (2a,b).
1-acyl-4-arylthiosemicarbazides were reported to undergo dehydro-
cyclization in basic medium to obtain 3-alkyl-4-aryl-5-thioxo-1,2,4-
triazoloes.11,12 Cyclization of thiosemicarbazides (2a,b) by treatment
with aqueous sodium carbonate solution afforded 3-methylthio
{7H-1,2,4-triazolo[1,5-d ]tetrazol-6-yl}-4-methyl(phenyl)-1,4-dihydro-
5H-1,-2,4-triazole-5-thione (4a,b). 1H NMR spectra of compounds (4a,b
)
revealed two exchangeable iminoproton signals of triazolo tetrazole
and triazole rings. Further support of the structure of 1,2,4-triazoles
(4a,b) was obtained from their mass spectra (cf. Experimental section).
Furthermore, the alkylation of structures (4a,b) with methyl (ethyl)
iodide led to the direct formation of S-alkylated derivatives (5a−d). The
1H NMR spectra of those products revealed signals characteristic of
the 5-methyl (ethyl) thio at 2.65 (4.29, 1.92) ppm. This assignment is
in harmony with the reported results.13
On the other hand, the reaction of hydrazide (1) with formic acid
resulted
1-formyl-2-{7H-1,2,4-triazolo[1,5-d]tetrazol-6-ylsulfanyl}
acetylhydrazine (6). Ring closure of the latter compound by refluxing
with phosphorus pentoxide in toluene yielded the 1,3,4-oxadiazole
structure (8a). In an alternative route compound (8a) was obtained
by reaction of (1) with triethyl orthoformate, which afforded ethoxy-
formaldehyde hydrazone structure (7) followed by thermal cyclization.
Similarly, 1,3,4-thiadiazole 8b was also directly obtained by refluxing
compound 6 with phosphorus pentasulfide in toluene.
In addition, the 1,3,4-oxadiazole moiety was also synthesized14 by the
requisite starting hydrazide (1) and carbon disulfide in a base catalyzed
to produce 1,3,4-oxadiazole ring system (9). Hydrazinolysis15 of the lat-
ter compound yielded 4-amino-4H-1,2,4-triazole-3-thiol structure (10).
The 1H NMR spectrum of the latter compound revealed the existance of
SH and NH2 proton signals. Moreover, we found that 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazole derivatives (11a,b) can easily be obtained directly
from aminothiol (10) by adding carboxylic acid derivatives. These struc-
1
tures were inferred on the basis of H NMR spectra of (11a,b), which

1014
M. A. M. Taha and S. M. El-Badry
TABLE I Antimicrobial Activity of the Prepared
Compounds
S. aureus
E. coli
MIC
C. albicans
Compound No.
I.Z.
MIC
I.Z.
I.Z.
MIC
3b
4b
5a
5d
8a
10
14
18
14
17
15
19
14
40
—
>200
50
100
>200
50
100
>200
12.5
—
20
19
23
14
21
17
15
36
—
>200
50
>200
>200
50
100
100
25
19
13
22
12
22
20
14
—
38
50
25
100
>200
50
100
100
—
11b
Ampicillin
Clotrimazole
—
12.5
IZ = inhibition zone.
revealed the disappearance of the SH and NH2 signals present in the
spectrum of parent compound (10).
The antimicrobial activity (determined in Extension Laboratory, Fac-
ulty of Agriculture, Alexandria University, Alexandra, Egypt) of pre-
pared compounds 3b, 4b, 5a,d, 8a, 10, and 11b (Table I) was evaluated
against Staphylococcus aureus, Escherichia coli, and Candida albicans
using the cup diffusion technique.16,17 The results showed that com-
pounds 4b and 8a were 25% comparable to the activity of ampicillin
against S. aureus. The activity of 4b and 8a was 50% of the ampicillin
against E. coli. Moreover, compounds 4b and (3b, 8a) were 50% and 25%
against C. albicans comparable to clotrimazole, respectively. The rest
of the compounds showed lower activity than the reference standards
(ampicillin and clotrimazole) against the test organisms.
In conclusion, this investigation demonstrated the utility of {7H-
1,2,4-triazolo[1,5-a]tetrazol-6-ylsulfanyl}acetic acid hydrazide (1) as
a synthon for the construction of 1,2,4-triazoles, 1,3,4-oxadiazoles,
1,3,4-thiadiazoles, and 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles by choos-
ing the proper cyclizing reagents. The antibacterial and antifungal ac-
tivities of the prepared compounds were comparable to ampicillin and
clotrimazole.
EXPERIMENTAL
Melting points were determined in capillary tubes in a MEL-TEMP II
melting apparatus and are uncorrected. The infrared spectra (IR) were
recorded on a Perkin-Elmer FT Paragon 1000 and Pye-Unicam SP-300
1
spectrometers. H NMR spectra were scanned on a Varian Mercury

Activity of Heterocyclic Compounds
1015
VXR-3000 spectrometer using tetramethyl silane (TMS) as an internal
standard. MS were recorded on a Shimadzu GCMS-Q 1000 EX mass
spectrometer at 70ev. Microanalyses were performed by the Microana-
lytical Unit, Cairo University, Giza, Egypt.
4-Methyl-1-{7H-1,2,4-triazolo[1,5-d]tetrazol-6-
ylsulfanylacetyl}thiosemicarbazide (2a)
To a solution of compound 1 (1 g, 4.67 mmoles) in ethanol (10 mL),
methyl isothiocyanate (0.34 g, 4.67 mmoles) was added and the mixture
was stirred for 6 h at ambient temperature. The precipitate was filtered
and crystallized from ethanol-water to give (60%) of 2a, m.p. 170◦C; IR
(KBr, ν cm−1): 3340, 3250, 3055 (NH), 1670 (CON), 1630 (C N); 1H NMR
(DMSO-d6, δ ppm): 12. 41, 11.52, 11.04, 11.00 (s, 1 H each, exchangeable,
4 NH), 4.15 (s, 2 H, CH2), 2.91 (s, 3 H, NCH3). Found: C, 25.5; H, 3.5;
N, 44.3%. C6H9N9OS2 (287) required: C, 25.1; H, 3.1; N, 43.9%.
4-Phenyl-1-{7H-1,2,4-triazolo[1,5-d]tetrazol-6-
ylsulfanylacetyl}thiosemicarbazide (2b)
To a solution of compound 1 (1 g, 4.67 mmoles) and phenyl isothio-
cyanate (0.63 g, 4.67 mmoles) as previously described for the procedure
1
of (2a) in 55% yield, m.p. 190◦C (ethanol-water); H NMR (DMSO-d6,
δ ppm): 13.01, 12.42, 11.41, 11.22 (s, 1 H each, exchangeable, 4 NH),
8.24–7.52 (m, 5 H, aromatic H), 4.25 (s, 2 H, CH2). Found: C, 38.2; H,
3.1; N, 35.9%. C11H11N9OS2 (349) required: C, 37.8; H, 3.2; N, 36.1%.
5-Methylamino-2-methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-
6-yl}-1,3,4-thiadiazole (3a)
A mixture of 2a (0.6 g, 2.09 mmoles) and phosphoryl chloride (15 mL)
was heated under reflux for 1 h. The excess of phosphoryl chloride was
removed under reduced pressure, the residue added crushed ice, and
the mixture was stirred at r.t. for 1 h. During this time the solution
was gradually neutralized with a cold saturated solution of sodium bi-
carbonate, and the product that separated was filtered, washed with
water, dried, and crystallized from ethanol to give (54%) of 3a, m.p.
200◦C; IR (KBr, ν cm−1): 3300, 3040 (NH), 1600 (C N); MS: m/z (%) 270
(M++1,10). Found: C, 27.1; H, 2.3; N, 46.5%. C6H7N9S2 (269) required:
C, 26.8; H, 2.6; N, 46.8%

1016
M. A. M. Taha and S. M. El-Badry
2-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-5-
phenylamino-1,3,4-thiadiazole (3b)
A mixture of 2b (0.6 g, 1.72 mmoles) and phosphoryl chloride (15 mL)
as previously described for the preparation of (3a) in 53% yield, m.p.
215◦C (ethanol-water), 1H NMR (DMSO-d6 δ ppm): 13.10, 12.01 (s, 1 H
each, exchangeable, 2 NH), 8.24–7.50 (m, 5H, aromatic H), 4.15 (s, 2H,
CH2). Found: C, 39.6; H, 3.1; N, 37.9%. C11H9N9S2 (331) required: C,
39.9; H, 2.7; N, 38.1%.
4-Methyl-3-methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-
1,4-dihydro-5H-1,2,4-triazole-5-thione (4a)
A stirring mixture of compound 2a (0.6 g, 2.09 mmoles) and 5% aqueous
sodium carbonate solution (15 mL) was refluxed for 5 h. After cooling,
the resulting solution was acidified with hydrochloric acid, and the pre-
cipitate was filtered and crystallized from ethanol to give (71%) of 4a,
1
m.p. 190◦C; IR (KBr, ν cm−1): 3250, 3045 (NH), 1600 (C N); H NMR
(DMSO-d6 δ ppm): 12.15, 11.56 (s, 1H each exchangeable, 2NH), 4.20
(s, 2 H, CH2), 3.09 (s, 3 H, NCH3); MS: m/z (%) 269 (M+, 25), 255 (15),
227 (23), 213 (100). Found: C, 26.5; H, 3.0; N, 46.4%. C6H7N9S2 (269)
required: C, 26.8; H, 2.6; N, 46.8%.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-4-phenyl-
1,4-dihydro-5H-1,2,4-triazole-5-thione (4b)
A mixture of 2b (0.6 g, 1.72 mmoles) and 5% aqueous sodium carbon-
ate (15 mL) as previously deacribed for method of 4a in 61% yield,
1
m.p. 230◦C (ethanol); H NMR (DMSO-d6 δ ppm): 12.20, 11.70 (s, 1
H each, exchangeable, 2NH), 8.50–7.60 (m, 5 H, aromatic H), 4.20 (s,
2 H, CH2); MS: m/z(%) 331 (M+, 56), 317 (25), 289 (100). Found: C,
40.2; H, 2.5; N, 38.4%. C11H9N9S2 (331) required: C, 39.9; H, 2.7; N,
38.1%.
General procedure for the preparation of 3-methylthio{7H-
1,2,4-triazolo [1,5-d]tetrazol-6-yl}-4-substituted-5-alkylthio-
4H-1,2,4-triazoles (5a-d
)
To a solution of compound 4a,b (0.6 g, 2.21 mmoles) in sodium exthox-
ide (10 mL) was added, and the solution was refluxed for 20 min. The
appropriate alkyl iodide (2.21 mmoles) was then added, and refluxing
was continued for an additional 1 hour. The reaction mixture was then
cooled and poured onto cold water, whereby the solid that formed was
filtered off, dried, and crystallized from ethanol.

Activity of Heterocyclic Compounds
1017
The following compounds were prepared.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-4-methyl-
5-methylthio-4H-1,2,4-triazole (5a)
1
Yield: 56%, m.p. 145◦C; IR (KBr, ν cm−1): 3040 (NH), 1600 (C N); H
NMR (DMSO-d6 δ ppm): 12.07 (s, 1 H, NH), 4.25 (s, 2 H, CH2), 3.27
(s, 3 H, NCH3), 2.65 (s, 3 H, SCH3). Found: C, 30.0; H, 2.9; N, 44.9%.
C7H9N9S2 (283) required: C, 29.7; H, 3.2; N, 44.5%.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-4-phenyl-
5-methylthio-4H-1,2,4-triazole (5b)
Yield: 48%, m.p. 170◦C; MS: m/z (%) 346 (M++ 1, 20). Found: C, 41.3;
H, 3.6; N, 36.9%. C12H11N9S2 (345) required: C, 41.7; H, 3.2; N, 36.5%.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-4-methyl-
5-ethylthio-4H-1,2,4-triazole (5c)
Yield: 61%, m.p. 160◦C; 1H NMR (DMSO-d6 δ ppm): 12.91 (s, 1 H, NH),
4.29 (q, 2 H, SCH2CH3), 4.15 (s, 2 H, CH2), 2.88 (s, 3 H, NCH3), 1.92
(t, 3 H, SCH2CH3). Found: C, 31.9; H, 3.8; N, 42.5%. C8H11N9S2 (297)
required: C, 32.3; H, 3.7; N, 42.4%.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-4-phenyl-
5-ethylthio-4H-1,2,4-triazole (5d)
Yield: 46%, m.p. 190◦C; MS: m/z (%) 361 (M++ 2, 40). Found: C,
43.0; H, 3.2; N, 35.6%. C13H13N9S2 (359) required: C, 43.5; H, 3.6; N,
35.1%.
1-Formyl-2-{7H-1,2,4-triazolo[1,5-d]tetrazol-6-
ylsulfanylacetyl}hydrazine (6)
A solution of compound 1 (1 g, 4.67 mmoles) in formic acid (15 mL)
was refluxed for 1 h. The solvent was evaporated and the residue was
crystallized from ethanol to afford (47%) of 6, m.p. 180◦C; IR (KBr, ν
cm−1): 3320, 3240, 3020 (NH), 1690, 1660 (CON), 1H NMR (DMSO-d6,
δ ppm): 12.42, 11.50, 10.99 (s, 1 H each, exchangeable, 3 NH), 8.40 (s,
1 H, formyl H), 4.30 (s, 2 H, CH2). Found: C, 25.2; H, 2.8; N, 45.9%.
C5H6N8O2S (242) required: C, 24.8; H, 2.5; N, 46.3%.

1018
M. A. M. Taha and S. M. El-Badry
Ethoxyformaldehyde{7H-1,2,4-triazolo[1,5-d]tetrazol-6-
ylsulfanylacetyl} hydrazone (7)
The title compound was prepared from 1 (1 g, 4.67 mmoles), and tri-
ethyl orthoformate (10 mL) was heated at reflux for 5 h and then
evaporated under reduced pressure. The obtained residue was crys-
tallized from ethanol to the result (56%) of 7, m.p. 175◦c; IR (KBr, ν
cm−1): 3340, 3120 (NH), 1670 (CON), 1630 (C N); 1H NMR (DMSO-d6,
δ ppm): 13.01, 12.12 (s, 1 H each, 2 NH), 7.01 (s, 1 H, HC N), 4.31 (q,
2 H, CH2CH3), 4.15 (s, 2 H, CH2), 1.85 (t, 3 H, CH2CH3). Found: C,
31.5; H, 3.5; N, 41.2%. C7H10N8O2S (270) required: C, 31.1; H, 3.7; N,
41.5%.
2-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-1,3,4-
oxadiazole (8a)
Method A
To a solution of compound 6 (1 g, 4.13 mmoles) in toluene (20 mL),
phosphorus pentoxide (4.13 mmoles) was added. The mixture was re-
fluxed for 2 h. The solvent was evaporated, and water (5 mL) was added
and extracted with chloroform. The solvent was evaporated and the
residue was crystallized from ethanol to yield (44%) of 8a, m.p. 130◦C;
1
IR (KBr, ν cm−1): 3160 (NH); H NMR (DMSO-d6, δ ppm): 12.31 (s, 1
H, NH), 8.35 (s, 1 H, oxadiazole H), 4.25 (s, 2 H, CH2); MS: m/z (%) 224
(M+, 28). Found: C, 27.1; H, 2.3; N, 50.3%. C5H4N8OS (224) required:
C, 26.8; H, 1.8; N, 50.0%.
Method B
Compound 7 (1 g, 3.70 mmoles) was heated at 10◦C above its melting
point for 20 min in an oil bath. The mass obtained after cooling was
crystallized from ethanol to give (56%) of 8a.
2-Methylthio{7H-1,2,4-triazolo[1,5-d ]tetrazol-6-yl}-1,3,4-
thiadiazole (8b)
A solution of compound 6 (1 g, 4.13 mmoles) in toluene (20 mL) was
treated with phosphorus pentasulfide (0.004 mole) and heated under
reflux for 1 h. The solvent was evaporated, water (5 mL) was added and
the obtained product was crystallized from ethanol to give (61%) of 8b,
m.p. 150◦C; MS: m/z (%) 242 (M++ 2, 43). Found: C, 24.6; H, 2.1; N,
47.1%. C5H4N8S2 (240) required: C, 25.0; H, 1.7; N, 46.7%.

Activity of Heterocyclic Compounds
1019
2-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-1,3,4-
oxadiazole-5-thiol (9)
Carbon disulfide (4.67 mmoles) was added to a mixture of 1 (1 g, 4.67
mmoles) and potassium hydroxide (4.67 mmoles) in ethanol (30 mL).
The reaction mixture was refluxed for 5 h and then, poured onto water
followed by the addition of hydrochloric acid untill the solution became
slightly acidic. The formed solid was filtered, dried, and crystallized
1
from ethanol to yield (59%) of 9, m.p. 180◦C: H NMR (DMSO-d6, δ
ppm): 12.01 (s, 1 H, NH), 4.30 (s, 2 H, CH2), 3.82 (s, 1 H, SH). Found:
C, 23.7; H, 2.1; N, 43.3%. C5H4N8OS2 (256) required: C, 23.4; H, 1.6; N,
43.8%.
4-Amino-4H-5-methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-
6-yl}-1,2,4-triazole-3-thiol (10)
A solution of compound 9 (0.5 g, 1.95 mmoles), in ethanol (10 mL),
was treated with 95% hydrazine (5 mL) was refluxed for 3 h, diluted
with cold water, and acidified by hydrochloric acid. The solid mass was
filtered, washed with water, and crystallized from ethanol to give (48%)
1
of 10, m.p. 160◦C; IR (KBr, ν cm−1): 3230 (NH2), 3130 (NH); H NMR
(DMSO-d6, δ ppm): 12.70 (s, 1 H, NH), 5.72 (s, 2 H, NH2), 4.25 (s, 2 H,
CH2), 3.80 (s, 1 H, SH). Found: C, 22.5; H, 2.6; N, 52.3%. C5H6N10S2
(270) required: C, 22.2; H, 2.2; N, 51.9%.
3-Methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazole (11a)
A mixture of aminothiol 10 (0.5 g, 1.85 mmoles) and formic acid (10 mL)
was refluxed for 2 h. The mixture was evaporated under reduced pres-
sure, and the obtained residue was crystallized from ethanol to give
(65%) of 11a, m.p. 125◦C; IR (KBr, ν cm−1): 3090 (NH); 1H NMR (DMSO-
d6, δ ppm): 12.33 (s, 1 H, NH), 8.23 (s, 1 H, CH), 4.15 (s, 2 H, CH2), MS:
m/z (%) 281 (M++ 1, 56). Found: C, 26.1; H, 1.9; N, 50.4%. C6H4N10S2
(280) required: C, 25.7; H, 1.4; N, 50.0%.
6-Methyl-3-methylthio{7H-1,2,4-triazolo[1,5-d]tetrazol-6-yl}-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole (11b)
Compound 11b was prepared from 10 (0.5 g, 1.85 mmoles) and acetic
acid (10 mL) as previously described for the preparation of 11a. It was
crystallized from ethanol, yield (62%), m.p. 140◦C, 1H NMR (DMSO-d6,
δ ppm): 12.01 (s, 1 H, NH), 4.20 (s, 2 H, CH2), 1.94 (s, 3 H, CH3). Found:

1020
M. A. M. Taha and S. M. El-Badry
C, 29.1; H, 2.4; N, 48.1%. C7H6N10S2 (294) required: C, 28.6; H, 2.0; N,
47.6%.
Antimicrobial Screening
The products were in vitro screened for activity against a variety of
Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacte-
ria (Escherichia coli) and yeast-like fungi (Candida albicans), using
the cup diffusion technique16 to determine the inhibition zones (IZ,
in mm). Furthermore, the microdilution suceptibility test in Mu¨ller-
Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) were used
for the determination of antibacterial and antifungal activities in the
form of the MIC (in µg).17 Ampicillin trihydrate and clotrimazole were
used as standard antibacterial and antifungal agents, respectively.
Inhibition Zone Measurement
The compounds were dissolved in propylene glycol at a concentration of
1 mg/mL. Sterile nutrient agar (Oxoid) was incubated with the tested
organism, so as each 100 mL of the medium received 1 mL of a 24-h
boroth culture, 3 drops of the tested compounds were placed separately
in cups (8 mm diameter), cut in the agar. The plates were incubated at
37◦C for 24 h, and the resultant IZs were measured in mm. Propylene
glycol alone showed no inhibition to any of the tested organisms. Ampi-
cillin trihydrate and clotrimazole at a concentration of 0.1% solution in
propylene glycol were used as standards.
Minimal Inhibitory Concentration Measurement
Solutions of the test compounds, ampicillin trihydrate and clotrimazole,
were prepared in DMSO at a concentration of 1600 µg/mL. The twofold
dilution of the compounds were prepared (800, 400, . . . 6.25 µg/mL). The
microorganism suspensions at 106 CFU/mL (Colony Forming Unit/mL)
concentration were inoculated to the corresponding wells. Plates were
incubated at 36◦C for 24 h to 48 h. The incubation chamber was kept
sufficiently humid. At the end of the incubation period, the MICs were
determined. Controls for the DMSO microorganisms and media mi-
croorganisms were also done.
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Activity of Heterocyclic Compounds
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