New regioselective synthesis and biological activity of substituted 1H-[1,2,4]triazolo[3,4-c][1,2,4]triazoles

Article abstract of DOI:10.1002/jccs.200400054

Two regioselective synthetic approaches for the title compounds 7 via reaction of hydrazonoyl halides 1 with 3-methylthio-5-phenyl-1,2,4-triazole 3 and base-catalyzed cyclization of N-phenyl-N-(5-phenyl-s-triazol-3-yl) thiohydrazides 6 are described. The mechanisms of the reactions studied and the biological activity of the isolated products 6 and 7 are pointed out.

Full text of DOI:10.1002/jccs.200400054

Journal of the Chinese Chemical Society, 2004, 51, 351-357
351
New Regioselective Synthesis and Biological Activity of Substituted
1H-[1,2,4]Triazolo[3,4-c][1,2,4]triazoles
A. S. Shawali*, M. A. Abdallah, I. M. Abbas and G. M. Eid
Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
Two regioselective synthetic approaches for the title compounds 7 via reaction of hydrazonoyl halides 1
with 3-methylthio-5-phenyl-1,2,4-triazole 3 and base-catalyzed cyclization of N-phenyl-N-(5-phenyl-s-
triazol-3-yl)thiohydrazides 6 are described. The mechanisms of the reactions studied and the biological activ-
ity of the isolated products 6 and 7 are pointed out.
Keywords: Hydrazonoyl halides; Nitrilimines; s-Triazolo[3,4-c]-s-triazoles; Antimicrobial activity.
INTRODUCTION
A literature search revealed that many derivatives of
1,2,4-triazole-3(2H)-thione 2 in ethanol in the presence of so-
dium ethoxide at room temperature afforded, in each case,
only one isolable product as evidenced by tlc analysis of the
crude products. Both microanalysis and mass spectral data
were consistent with either the thiohydrazonate structure 4 or
the thiohydrazide structure 6 (Scheme I). A comparison of
the mass spectra of the isolated products with those of typical
thiohydrazonate esters immediately ruled out the thiohydra-
zonate type structure 4. This is because the mass spectra of
both aryl and heteroaryl thiohydrazonates were reported to be
characterized by elimination of the elements of the corre-
sponding arenethiol and heteroaryl thiol from their molecular
ions, respectively.7 Such peaks were absent in the mass spec-
tra of the isolated products. Instead, the mass spectra of 6 ex-
hibited a characteristic peak at m/z corresponding to (M+ -
RCSN) species. This finding indicates that the isolated prod-
ucts have the thiohydrazide structure 6. Further evidence in
support of the assigned structure 6 for the isolated products
was derived from their 13C NMR spectra which revealed the
1,2,4-triazoles and annelated 1,2,4-triazoles have received
considerable attention among medicinal chemists because
such compounds have been found to display a wide range of
antihypertensive, antifungal, and antibacterial activities as
well as being potential agents in agricultural chemistry.1,2 In
the light of these considerations and in continuation of our in-
terest in developing new syntheses of functionally substi-
tuted heterocycles utilizing hydrazonoyl halides 1,3-6 we in-
vestigated the reactivity of the latter halides with 5-phenyl-
1,2,4-triazole-3(2H)-thione 2 and its 3-methylthio derivative
3 with the following objectives in mind. Firstly, we thought it
interesting to explore the utility of the halides 1 as synthons
for new derivatives of 1,2,4-triazole and fused 1,2,4-tria-
zolo-1,2,4-triazole ring systems. The second objective of the
present study was to shed some light on the regiochemistry of
the target reactions. This is because both of 2 and 3 have more
than one reactive site so that their reactions with 1 can lead to
isomeric products depending on which nitrogen (N2 or N4)
of the 1,2,4-triazole ring will participate. For example, con-
densation of 1 with 3 can lead to the formation of [1,2,4]tri-
azolo[3,4-c][1,2,4]triazole derivatives 7 and/or [1,2,4]tri-
azolo[4,3-b][1,2,4]triazole 8 (Scheme IV). Furthermore, it
was thought worthwhile to explore the biological activity of
the products that will be isolated from the reactions to be
studied.
Scheme I
RC(Cl):NNHAr
1
EtONa / EtOH
H
N
2
N
N
N
N
N
N
R
4
+
-
H
N
R-C = N-N-Ar
+
Ph
N
H
S
Ph
Ph
N
H
S
N
S
Ar
H
N
S
H
N
R
R
N
H
N
Ph
N
H
N
N
Ar
Ar
6
5
RESULTS AND DISCUSSION
R : A, Ph; B, PhNHCO; C, C2H5OCO; D, PhCH=CH
Ar = XC6H4 : X : a, H; b, 4-Me; c, 4-Cl; d, 4-NO2
Treatment of the hydrazonoyl halides 1 with 5-phenyl-

352 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Shawali et al.
-
presence of a thiohydrazide carbon signal near d 155-156 and
the absence of the signal near d 141-142 due to the carbon
atom of the -S-C=N-NH- group.8
® 6] and [11 + EtO ® 6] involve the formation of the
thiohydrazonates 4 as intermediates which undergo in situ
Smiles rearrangement7,10 as soon as they are formed to yield
the thiohydrazides 6 as end products (Schemes I and II).
Next, cyclization of the thiohydrazides 6 was exam-
ined. Refluxing each of the thiohydrazides 6 in pyridine for
24 h resulted in elimination of hydrogen sulfide and the for-
mation of a single product, in each case as evidenced by tlc
analysis of the crude product. Elemental analysis and the
mass spectral data were compatible with each of the two iso-
meric structures namely 1,3,5-trisubstituted 1H-[1,2,4]tri-
azolo[3,4-c][1,2,4]triazoles 7 and 1,3,6-trisubstituted 1H-
[1,2,4]triazolo[4,3-b][1,2,4]triazoles 8 (Scheme III). For ex-
ample, the mass spectra of the latter products were character-
ized by molecular ion, base peaks of the spectra, abundant
[RCN], [RCNNPh] and [PhN2] ions (Chart I). The [RCNNPh]
ions confirm the integrity of the original 1,2,4-triazole ring.
Thus, on the basis of these data together with the IR data, it
was not possible to distinguish between these two regioiso-
meric structures 7 or 8. Also, the 1H NMR spectra of the iso-
lated products were not of much help to distinguish between
the two structures 7 and 8. The structural assignment of the
isolated products was, however, secured as 7 on the basis of
the identity (mp, m.mp, IR and 1H NMR spectra) of the prod-
uct, isolated from cyclization of 6Aa, with an authentic sam-
ple of 1,3,5-triphenyl[1,2,4]triazolo[3,4-c][1,2,4]triazole
7Aa prepared by an unequivocal method via cyclization of
3-benzoylhydrazino-2,5-diphenyl[1,2,4]triazole 12Aa with
POCl3 as previously described11 (Scheme III).
The structure of the thiohydrazides 6 was further sup-
ported by their alternate synthesis. Thus, treatment of 5-
phenyl-1,2,4-triazole-3(4H)-thione 2 with the appropriate
active chloromethylene compounds 9B,C in ethanol in the
presence of sodium ethoxide at room temperature afforded
the respective (1,2,4-triazol-3-yl)thiomethylene compounds
10B,C, respectively (Scheme II). The formation of the latter
products 10 from 2 and 9 is analogous to the S-alkylation ex-
hibited by 1,2,4-triazole-3(2H)-thione derivatives when treated
with a-halo derivatives of ketones, acids or esters.9 The
structures of the products 10 isolated were established by
their microanalyses and their mass, 1H NMR and IR spectra
which showed all the expected signals (see Experimental).
Scheme II
N
N
N
N
R
H
R-CH(Cl)-COCH3
9
EtONa
EtOH
+
Ph
N
SH
Ph
Ph
N
S
COCH3
H
H
2
10
+
ArN2
N
N
R
N
N
R
H
N
Ph
N
S
COCH3
N=N-Ar
N
S
4
N
Ar
R
H
H
11
H
N
N
6
H
EtONa / EtOH
Ph
N
N
S
N
R
Ph
N
N
N
H
N
5
N
Ar
S
H
Ar
Scheme III
R : B, PhNHCO; C, C2H5OCO
Ar = C6H5
N
N H
N
N
NHCSR
NHCSR
Ph
Ph
N
N
Ph
N
H
N
Ar
6
Ar
When 10B,C were treated with benzenediazonium
chloride in aqueous ethanol containing sodium acetate, they
afforded the azo products 11B,C, respectively (Scheme II). In
no case the expected Japp-Klingemann product, namely the
thiohydrazonate ester 4, was produced. Structural assign-
ments for the isolated products 11 were made on the basis of
their mass, 1H NMR and IR data (see Experimental).
pyridine
R
N
N
N
N
8
N
Ar
Ph
N
N
N
N
N
Ar
H
R
N
7
NH
RCO
Ar
N
N
N
POCl3
or CCl4 / Ph3P
Treatment of the azo products 11 with sodium ethoxide
in ethanol at room temperature, to effect the Japp-Klingemann
cleavage of the acetyl group, was found to afford products
that proved identical in all respects with the products 6 ob-
tained above from reaction of 1 with the thione 2. This find-
ing indicates that both reactions namely [1 + thione 2 + EtO-
R
12
In an attempt to develop a new one-step strategy for
synthesis of 7, reactions of 3-methylthio-5-phenyl-1,2,4-
triazole 3 with each of N-aryl hydrazonoyl halides 1A-D

New Regio-Synthesis and Bio-Activity of Triazoles
J. Chin. Chem. Soc., Vol. 51, No. 2, 2004 353
Scheme IV
Chart I
.
H
+
N
N
a
a
b
b
RCNNPh
c
R-C(X):NNHAr
1
c
+
R
Ph
SMe
N
a
N
N
+
b
N
N
N
N
b
Ph
3
- HX
PhN2
Ph
N
N
Ph
N
Ph
N
.
R
+
c
a
c
R
PhCN
8
7
N
N
C=NNHAr
N
N
Ph
SMe
N
were examined in ethanol in the presence of sodium ethoxide
at reflux (Scheme IV). In our hands, each of such reactions
was found to give a single product as evidenced by tlc analy-
sis of the crude product. The isolated products were assigned
structure 7 as they proved to be identical in all respects with
those obtained above from cyclization of the respective
thiohydrazides 6 (Scheme III). This finding indicates that re-
action of 1 with 3 is also regioselective and it proceeds via the
formation of the amidrazones 13 as intermediates rather than
14 which cyclize in situ through elimination of methanethiol
as soon as they are formed to give 7 as end products (Scheme
IV). The regioselectivity of the reaction of 1 with 3 seems
consistent with literature reports which indicate that N-4 is
the site of preference for cyclization of N-(5-phenyl-1,2,4-
triazol-3-yl) arenecarbohydrazonoyl bromides leading to 3-
aryl-5-phenyltriazolo[3,4-c][1,2,4]triazoles.12
Ph
SMe
N
N
R
C=NNHAr
14
13
-MeSH
R
- MeSH
N
N
N
Ar
Ph
N
N
N
N
N
Ph
N
8
R
Ar
7
R : A, Ph; B, PhNHCO; C, EtOCO; D, PhCH=CH
Ar = XC6H4; X : a, H; b, 4-Me; c, 4-Cl; d, 4-NO2
aeruginosa Pa and Staphylococcus aureus Sa. Also, five
fungi species namely Candida spp. Cs, Syncephalastrum
racemosum Sr, Penicillium marneffei Pm, Aspergillus
fumigatus Af and Alternaria alternata Aa were tested. The or-
ganisms were tested against the activity of solutions of con-
centration of 1.0 mg/mL of each compound and using inhibi-
tion zone diameter in cm (IZD) as criterion for the anti-
microbial activity. The fungicide Terbinafine and the bacte-
ricide Chloramphenicol were used as references to evaluate
the potency of the tested compounds under the same condi-
tions. The results are depicted in Table 1. They revealed that
compound 6Aa exhibited the highest degree of inhibition
against all tested organisms. Furthermore, while the data
In conclusion, the foregoing results indicate that reac-
tions of hydrazonoyl halides 1 with either thione 2 or its
methylthio derivative 3 provide facile and novel regioselec-
tive routes for synthesis of 1H-[1,2,4]triazolo[3,4-c][1,2,4]-
triazoles.
The compounds 6A-C and 7A-C were tested for their
antimicrobial activities using four bacteria species namely
Escherichia coli Ec, Bacillus subtilis Bs, Pseudomonas
Table 1. Antimicrobial Activity of the Products 6 and 7
Micro-organism / IZD (cm)*
Compd. no.
Ec
Bs
Pa
Sa
Cs
Sr
Pm
Af
Aa
6Aa
6Ba
6Ca
7Aa
7Ba
7Ca
CAa
TEb
+
+
-
++
+
-
++
+
-
+
+
++
+
+
+
+
++
+
-
+
+
++
++
+
-
+
++
+
+
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
2.6
+
2.8
-
2.4
+
2.6
4.7
5.0
3.6
3.0
3.6
¦ 50 mL of solution whose concentration 1.0 mg/mL was tested.
a Chloramphenicol; b Terbinafine
* IZD beyond control / (sign): 1.1-1.5 cm / (+++); 0.6-1.0 cm / (++); 0.1-0.5 cm / (+); 0 cm / (-).

354 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Shawali et al.
showed that all other compounds exhibited lower activity
against the tested microorganisms, they revealed that all
compounds exhibited no inhibition of the three fungi species
Pm, Af and Aa.
identical in all respects with those obtained by method A
above. The physical constants of the products 6A-D are given
below.
Thiobenzoic acid N¢-phenyl-N¢-[5-phenyl-4H-1,2,4]-
triazol-3-yl])hydrazide (6Aa)
1
EXPERIMENTAL SECTION
M.p. 139 °C (MeOH), Yield 60%; n (cm-1) 3126; H
NMR d 6.86-7.99 (m, 16H), 9.25 (s, 1H). Anal. calcd. for
C21H17N5S (371): C, 67.90; H, 4.61; N, 18.85. Found: C,
67.50; H, 4.50; N, 18.9%.
Melting points were determined on a Gallenkamp appa-
ratus and are uncorrected. IR spectra were recorded in potas-
sium bromide using Fourier Transform Infrared and Pye
1
Unicam SP300 Infrared spectrophotometers. H- and 13C-
Thiobenzoic acid N¢-(4-nitrophenyl)-N¢-(5-phenyl-4H-
1,2,4-triazol-3-yl)hydrazide (6Ad)
NMR spectra were recorded using a Varian Mercury VXR-
300 NMR spectrometer (300 MHz) in CDCl3 or DMSO-d6.
Chemical shifts were related to that of the solvent used. Mass
spectra were recorded on a GCMS-QP 1000 EX Varian MAT
711 and SSQ 7000 spectrometers. Elemental analyses were
carried out at the Microanalytical Laboratory of Cairo Uni-
versity, Giza, Egypt.
M.p. 182 °C (EtOH-dioxane), Yield 75%; n (cm-1)
1
3421; H NMR d 7.26-8.25 (m, 15H), 9.77 (s, 1H). Anal.
calcd. for C21H16N6O2S (416): C, 60.57; H, 3.84; N, 20.19.
Found: C, 60.48; H, 3.81; N, 20.14%.
N-Phenyl-2-[N-phenyl-N¢-(5-phenyl-4H-1,2,4-triazol-3-yl)-
hydrazino]-2-thioxoacetamide (6Ba)
The biological evaluation of the studied compounds 6
and 7 was carried out at the Regional Center for Mycology
and Biotechnology at Alazhar University, Cairo, Egypt.
The hydrazonoyl chlorides 1A-D,13-17 5-phenyl-1,2,4-
triazole-3(2H)thione 2,18 3-methylthio-5-phenyl-1,2,4-tri-
azole 318 and the active chloromethylene compounds 9A-
C19,20 were prepared as previously described.
M.p. 208-9 °C (MeOH), Yield 70%; n (cm-1) 3155,
3215, 3386, 1654, 1640; 1H NMR d 6.86-7.86 (m, 15H), 8.89
(s, 1H), 9.80 (s, 1H). MS m/z (%) 415 (M++1, 5.4), 414 (5.8),
236 (100), 119 (25), 91 (46), 77 (98). Anal. calcd. for
C22H18N6OS (414): C, 63.78; H, 4.38; N, 20.28. Found: C,
63.69; H, 4.30; N, 20.25%.
Synthesis of the thiohydrazides (6)
2-[N¢-(4-methylphenyl)-N¢-(5-phenyl)-4H-1,2,4-triazol-3-
yl)-hydrazino]-N-phenyl-2-thioxoacetamide (6Bb)
Method A
To a stirred ethanolic sodium ethoxide solution, pre-
pared by dissolving sodium metal (0.23 g, 10 mg atom) in ab-
solute ethanol (20 mL), was added 5-phenyl-1,2,4-triazole-
3(4H)-thione 2 (1.77 g, 10 mmoles). After 15 min, the appro-
priate hydrazonoyl chloride 1 (10 mmoles) was added, and
the resulting mixture was stirred at room temperature for 12
h. The crude product that precipitated was collected by filtra-
tion, washed with water, dried and finally crystallized from
the appropriate solvent to give the respective 6.
M.p. 174 °C (AcOH-water), Yield 70%; n (cm-1) 3352,
1674; 1H NMR d 2.32 (s, 3H), 7.25 - 8.01 (m, 14H), 10.06 (s,
1H), 10.51 (s, 1H), 13.26 (s, 1H). MS m/z (%) 429 (M++1, 2),
428 (3), 336 (13), 250 (97), 146 (31), 118 (29), 91 (100), 77
(59). Anal. calcd. for C23H20N6OS (428): C, 64.48; H, 4.67;
N, 19.62. Found: C, 64.39; H, 4.55; N, 19.54%.
Ethyl 2-[N¢-phenyl-N¢-(5-phenyl-4H-1,2,4-triazol-3-yl)-
hydrazino](thioxo)acetate (6Ca)
Method B
M.p. 172 °C (EtOH), Yield 60%; n(cm-1) 3122, 1703,
1667; 1H NMR d 1.35 (t, J = 7 Hz, 3H), 4.45 (q, J = 7 Hz, 2H),
7.0-7.45 (m, 10H), 8.3 (s, 1H), 9.9 (s, 1H). Anal. calcd. for
C18H17N5O2S (367): C, 58.84; H, 4.66; N, 19.06. Found: C,
58.70; H, 4.70; N, 19.0%.
To a stirred ethanolic sodium ethoxide solution, pre-
pared by dissolving sodium metal (0.23 g, 10 mg atom) in ab-
solute ethanol (20 mL) was added the appropriate azo deriva-
tive 11, and the mixture was stirred at room temperature for
12 h. During this period the reactant 11 dissolved and the
crude product 6 precipitated. The latter was filtered, washed
with water, dried and finally crystallized from the appropriate
solvent. The products 6, prepared by this method proved
3-Phenylthioacrylic acid N¢-phenyl-N¢-(5-phenyl-4H-1,2,4-
triazol-3-yl)hydrazide (6Da)
M.p. 186 °C (aq. dioxane), Yield 70%; n (cm-1) 3275;

New Regio-Synthesis and Bio-Activity of Triazoles
J. Chin. Chem. Soc., Vol. 51, No. 2, 2004 355
1H NMR d 6.88 (d, J = 17 Hz, 1H), 7.15 (d, J = 17 Hz, 1H),
7.24-7.93 (m, 16H), 10.09 (s, 1H). Anal. calcd. for C23H19N5S
(397): C, 69.52; H, 4.78; N, 17.63. Found: C, 69.35; H, 4.66;
N, 17.53%.
21.93%.
1-(4-Methylphenyl)-5-phenyl-3-phenylaminocarbonyl-
[1,2,4]triazolo[3,4-c][1,2,4]triazole (7Bb)
M.p. 220 °C (AcOH), Yield 65%; (cm-1) 3348, 1697; 1H
NMR d 2.42 (s, 3H), 7.16 - 8.33 (m, 14H), 9.22 (s, 1H). MS
m/z (%) 395 (M++1, 27.6), 394 (100), 274 (58), 144 (18), 117
(31), 91 (33), 77 (32). Anal. calcd. for C23H18N6O (394): C,
70.00; H, 4.56; N, 21.31. Found: C, 69.80; H, 4.4o; N,
21.18%.
Synthesis of 1,2,4-Triazolo[3,4-c][1,2,4]triazoles (7)
Method A
To a stirred ethanolic sodium ethoxide solution, pre-
pared from sodium metal (0.23 g, 10 mg atom) and absolute
ethanol (20 mL), was added 5-phenyl-3-methylthio-1,2,4-
triazole 3 (1.91 g, 10 mmoles). To the resulting solution was
added the appropriate hydrazonoyl chloride 1 (10 mmoles)
portionwise while stirring the mixture. After the addition was
complete, the reaction mixture was refluxed till methanethiol
ceased to evolve (18 h), then cooled. The solid that precipi-
tated was filtered off, washed with water, air dried and finally
crystallized from the appropriate solvent to give the respec-
tive triazolotriazole 7 in 62% yield. The latter products
proved identical in all respects with those obtained above.
Method B
Ethyl 1,5-diphenyl-[1,2,4]triazolo[3,4-c][1,2,4]triazole-3-
carboxylate (7Ca)
M.p. 200 °C (EtOH/DMF), Yield, 55%, n (cm-1) 3070,
1720; 1H NMR d 1.53 (t, J = 7 Hz, 3H), 4.63 (q, J = 7 Hz, 2H),
7.26-8.34 (m, 10H). MS m/z (%) 334 (M++1, 16), 333 (30),
260 (19), 103 (100), 91 (7), 77 (29). Anal. calcd. for
C18H15N5O2 (333): C, 64.86; H, 4.54; N, 21.01. Found: C,
65.00; H, 4.60; N, 21.2%.
A solution of the appropriate thiohydrazide 6 (1 g) in
pyridine (5 mL) was refluxed till all hydrogen sulfide ceased
to evolve (30 h) while being stirred. The mixture was then
cooled and acidified with hydrochloric acid. The solid that
precipitated was filtered off, washed with water and air dried.
Crystallization from the appropriate solvent gave the respec-
tive product 7 in 55-65% yield.
Ethyl 1-(4-chlorophenyl)-5-phenyl-[1,2,4]triazolo[3,4-c]-
[1,2,4]triazole-3-carboxylate (7Cc)
M.p. 166 °C (DMF-EtOH), Yield 60%; n (cm-1) 1740,
1597; 1H NMR d 1.53 (t, J = 7 Hz, 3H), 4.65 (q, J = 7 Hz, 2H),
7.26-8.31 (m, 9H). MS m/z (%) 368 (M++1, 14), 367 (72),
294 (18), 137 (100), 102 (24), 77 (14). Anal. calcd. for
C18H14ClN5O2 (367): C, 58.77; H, 3.80; N, 19.04. Found: C,
58.70; H, 3.67; N, 19.12%.
1,3,5-Triphenyl[1,2,4]triazolo[3,4-c][1,2,4]triazole (7Aa)
M.p. 246 °C (EtOH/DMF) (Lit.11 m.p. 240-245 °C);
Yield 62%.
1,5-Diphenyl-3-(2-phenylethenyl)-[1,2,4]triazolo[3,4-c]-
[1,2,4]triazole (7Da)
M.p. 165 °C (DMF-EtOH), Yield 70%; n (cm-1) 1600,
1597; 1H NMR d 7.16-8.34 (m). MS m/z (%) 364 (M++1, 2),
363 (6), 286 (2), 260 (1), 235 (9), 155 (7), 103 (100), 77 (13).
Anal. calcd. for C23H17N5 (363): C, 76.03; H, 4.68; N, 19.28.
Found: C, 75.90; H, 4.48; N, 19.15%.
3,5-Diphenyl-1-(4-nitrophenyl)-[1,2,4]triazolo[3,4-c][1,2,4]-
triazole (7Ad)
M.p. 260 °C (Dioxane), Yield 60%; n (cm-1) 1593; 1H
NMR d 7.25 (d, J = 11 Hz, 2H), 7.90 (d, J = 11 Hz, 2H),
8.31-8.54 (m, 10H). Anal. calcd. for C21H14N6O2 (382): C,
65.96; H, 3.66; N, 21.98. Found: C, 65.77; H, 3.54; N,
21.80%.
Synthesis of 3-Oxo-2-[(5-phenyl-1,2,4-triazol-3-yl)thio]-
butanoic acid derivatives (10B,C)
General Procedure
1,5-Diphenyl-3-phenylaminocarbonyl[1,2,4]triazolo[3,4-c]-
[1,2,4]triazole (7Ba)
To a stirred ethanolic sodium ethoxide solution, pre-
pared from sodium metal (0.23 g sodium metal, 10 mg atom)
and absolute ethanol (20 mL), was added equimolar quanti-
ties of 5-phenyl-1,2,4-triazole-3(4H)thione 2 and the appro-
priate chloromethylene compound 9 (10 mmoles, each). The
resulting mixture was stirred for 12 h at room temperature.
During this period the reactants dissolved and a new solid
M.p. 238 °C (aq. AcOH), Yield, 60%, n (cm-1) 3323,
1
1676; H NMR d 7.22-8.97 (m, 15H), 9.32 (s, 1H). MS m/z
(%): 381 (M++1, 67.8), 380 (74.1), 260 (74.3), 144 (30.6),
103 (100), 77 (82.9). Anal. calcd. for C22H16N6O (380): C,
69.46; H, 4.24; N, 22.09. Found: C, 69.31; H, 4.23; N,

356 J. Chin. Chem. Soc., Vol. 51, No. 2, 2004
Shawali et al.
precipitated. The latter was filtered, washed with water, dried
and finally crystallized from the appropriate solvent to give
the respective product 10. The physical constants of the prod-
ucts 10Ba and 10Ca are given below.
Ethyl 2-phenylazo-2-[(5-phenyl-1,2,4-triazol-3-yl)thio]-3-
oxobutanoate (11Ca)
M.p. 180-2 °C (EtOH), Yield 69%; n (cm-1) 3201, 1745,
1693; 1H NMR d 1.34 (t, J = 7 Hz, 3H), 2.78 (s, 3H), 4.35 (q, J
= 7 Hz, 2H), 7.05-7.50 (m, 10H), 9.12 (s, 1H). Anal. calcd. for
C20H19N5O3S (409): C, 58.67; H, 4.68; N, 17.11. Found: C,
58.55; H, 4.55; N, 16.92%.
N-Phenyl-2-[(5-phenyl-1,2,4-triazol-3-yl)thio]-3-oxobutan-
amide (10Ba)
M.p. 134 °C (MeOH), yield 65%, n (cm-1) 3356, 3371,
1687, 1658; 1H NMR d 2.43 (s, 3H), 6.05 (s, 1H), 7.04-7.91
(m, 10H), 8.9 (s, 1H), 9.8 (s, 1H). Anal. calcd. for
C18H16N4O2S (352): C, 61.35; H, 4.58; N, 15.90. Found: C,
61.30; H, 4.20; N, 15.80%.
Antimicrobial assay
Cultures of five fungi species namely Candida spp. Cs,
Syncephalastrum racemosum Sr, Penicillium marneffei Pm,
Aspergillus fumigatus Af and Alternaria alternata Aa were
tested as well as four bacteria species namely Escherichia
coli Ec, Bacillus subtilis Bs, Pseudomonas aeruginosa Pa and
Staphylococcus aureus Sa. were used to investigate the anti-
microbial activity of the compounds 6 and 7. The antimicro-
bial activity was assayed biologically using diffusion plate
technique. The latter technique was carried out by pouring a
spore suspension of the fungal species (one ml of sterile wa-
ter contains approximately 108 conidia) or spreading bacte-
rial suspension over a solidified malt agar medium. The layer
is allowed to set for 30 min. A solution of the test compound 6
or 7 (1.0 mg/mL) was placed onto sterile 5 mm filter paper
discs and allowed to dry, then the discs were placed on the
centre of the malt agar plate and incubated at optimum incu-
bation temperature 28 ± 2 °C. Test organism growth may be
affected by the inhibitory action of the test compound, so a
clear zone around the disc appears as an indication of the in-
hibition of test organism growth. The size of the clearing
zone is proportional to the inhibitory action of the compound.
The fungicide Terbinafine and the bactericide Chloram-
phenicol were used as standards under the same conditions.
Measurements were considered after 72 h for fungi and 24 h
for bacteria. The results are summarized in Table 1.
Ethyl 2-[(5-phenyl-1,2,4-triazol-3-yl)thio]-3-oxobutanoate
(10Ca)
M.p. 108 °C (Acetone/H2O); Yield 60%; n (cm-1) 3130,
1741, 1666; 1H NMR d 1.24 (t, J = 7 Hz, 3H), 2.42 (s, 3H),
4.27 (q, J = 7 Hz, 2H), 7.26 (s, 1H), 7.42-8.0 (m, 5H), 13.83
(s, 1H). Anal. calcd. for C14H15N3O3S (305): C, 55.07; H,
4.95; N, 13.76. Found: C, 55.03; H, 4.90; N, 13.66%.
Synthesis of 2-Arylazo-3-Oxo-2-[(5-phenyl-s-triazol-3-
yl)thio]-butanoic acid derivatives (11Ba and 11Ca)
General Procedure
To a solution of the appropriate 10 (10 mmoles) in etha-
nol (40 mL) was added sodium acetate trihydrate (3 g), and
the mixture was cooled in an ice bath to 0-5 °C while being
stirred. To the resulting cold solution was added portionwise
a cold solution of benzenediazonium chloride, prepared as
usual by diazotizing aniline (10 mmoles) in hydrochloric acid
(6 mL, 6 M) with sodium nitrite (0.7 g, 10 mmoles) in water
(10 mL). After all diazonium salt solution was added, the re-
action mixture was stirred for a further 30 min while cooling
in an ice bath and left overnight in a refrigerator. The solid
that precipitated was filtered, washed with water, air dried
and finally crystallized from the appropriate solvent to give
the respective arylazo derivative 11. The products 11Ba and
11Ca prepared together with their physical constants are
listed below.
Received May 23, 2003.
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N-Phenyl-2-phenylazo-2-[(5-phenyl-1,2,4-triazol-3-yl)thio]-
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