Synthesis and reactions of new fused heterocycles derived from 5-substituted-4-Amino-3-Mercapto-(4H)-1,2,4-Triazole with biological interest

Article abstract of DOI:10.1080/10426500802470769

The reaction of thiocarbohydrazide with carboxylic acids at the melting temperature allows an improved preparation of 5-substituted-4-amino-3-mercapto 1,2,4-triazoles 1_{a g}. Compound 1_a reacted with 2-bromopropionic acid to give acid

Full text of DOI:10.1080/10426500802470769

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Synthesis and Reactions of New
Fused Heterocycles Derived
from 5-Substituted-4-Amino-3-
Mercapto-(4H)-1,2,4-Triazole
with Biological Interest
A. Y. Hassan a
a Chemistry Department, Faculty of Science (Girls),
Al-Azhar University, Nasr City, Cairo, Egypt
Published online: 03 Nov 2009.
To cite this article: A. Y. Hassan (2009) Synthesis and Reactions of New Fused
Heterocycles Derived from 5-Substituted-4-Amino-3-Mercapto-(4H)-1,2,4-Triazole with
Biological Interest, Phosphorus, Sulfur, and Silicon and the Related Elements, 184:11,
2759-2776, DOI: 10.1080/10426500802470769
To link to this article: http://dx.doi.org/10.1080/10426500802470769
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Phosphorus, Sulfur, and Silicon, 184:2759–2776, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802470769
Synthesis and Reactions of New Fused Heterocycles
Derived from 5-Substituted-4-Amino-3-Mercapto-
(4H)-1,2,4-Triazole with Biological Interest
A. Y. Hassan
Chemistry Department, Faculty of Science (Girls), Al-Azhar University,
Nasr City, Cairo, Egypt
The reaction of thiocarbohydrazide with carboxylic acids at the melting tempera-
ture allows an improved preparation of 5-substituted-4-amino-3-mercapto 1,2,4-
triazoles 1a−g. Compound 1a reacted with 2-bromopropionic acid to give acid
derivative 2. The latter was reacted with a mixture of acetic anhydride and tri-
ethylamine to afford the mesoionic compound 3. Heating of compound 3 in ethanol
gave the ester derivative 4, which on alkaline hydrolysis in methanol gave ke-
tone derivative 5. Substituted 1,2,4-triazolo [3,4-b]-6H-1,3,4-thiadiazine 6h,i and
7 were synthesized by reaction of 1a with acetylacetone, ethyl acetoacetate and
chloroacetamide. Heterocyclic systems 8 and 9 were prepared through the reac-
tion of 1a with 2,3-dichloro-1,4-naphthoquinone and 2,3-dichloroquinoxaline. In
addition, thenoyl isothiocyanate, thenoyl chloride, 2-thiophenecarbaldehyde, and
p-chlorophenyl isocyanate reacted with compound 1a to afford 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazole ring system 10, 11, and urea derivative 12. 1,2,4-Triazolo[3,4-
b]-5H-pyrazole derivatives 14j,k were prepared through the reaction of compound
1a with 3-chloro-2,4-pentandione and ethyl-2-chloroacetoacetate. Compound 14j
was treated with hydrazine to afford products 15, 16, and 17 depending on the
type of hydrazine derivative and reaction conditions. Compound 19 was synthe-
sized by refluxing of compound 14j with hydroxylamine hydrochloride to afford the
corresponding oxime derivative 18 followed by treatment with thenoyl chloride.
Keywords Pyrazoles; 5-substituted-4-amino-3-mercapto-(4H)-1,2,4-triazole; 1,3,4-
thiadiazines; 1,3,4-thiadiazoles
INTRODUCTION
Heterocyclic condensed derivatives posses pharmacological properties
such as analgesic, antibacterial,1 antitumor,2 antineoplastic agent,3
and radioprotective activities.4 1,2,4-Triazolo[3,4-b]-1,3,4-thiadiazole,
1,2,4-triazolo [3,4-b]-6H-1,3,4-thiadiazine, and 1,2,4-triazolo [3,4-b]
Received 4 March 2008; accepted 11 September 2008.
Address correspondence to A. Y. Hassan, Chemistry Department, Faculty of Science
(Girls), Al-Azhar University, Nasr City, Cairo, Egypt. E-mail:helali aisha@yahoo.com
2759

2760
A. Y. Hassan
pyrazole derivatives represent a class of compounds hitherto scarcely
studied. From a biological point of view, it has been reported that 1,2,4-
triazolo [3,4-b]-1,3,4-thiadiazole derivatives possess antibacterial5,7
and antiinflammatory activities8 and interesting CNS-depressant
actions.9 Previously, the preparation and pharmacological evalua-
tion of some 6-substituted-3-(pyridine-4-yl)-1,2,4-triazolo [3,4-b]-1,3,4-
thiadiaz-oles and 1,2,4-triazolo [3,4-b]-1,3,4-thiadiazine have been re-
ported, with some of them exhibiting antitumor activities.10–12
RESULTS AND DISCUSSION
The reaction of thiocarbohydrazide with carboxylic acids at melt-
ing temperature resulted in the formation of 5-substituted-4-amino-3-
mercapto-1,2,4-triazole heterocycles.13 Thus, when equimolar amounts
of carboxylic acids and thiocarbohydrazide were mixed together and
warmed at the melting temperature for 15–60 min, the corresponding 5-
substituted-4-amino-3-mercapto-1,2,4-triazoles 1a−g were obtained in
good to excellent yield (71–96%; Scheme 1, Table I). The Heindel and
Reid synthesis14 was used as a second method for preparation of 1a.
This methodology involved the condensation of a carboxylic acid hy-
drazide with carbon disulfide and potassium hydroxide to yield potas-
sium dithiocarbazates salt, which was converted to compound 1a by
reaction with an excess of hydrazine. Melting point depression was
not observed in a mixed melting point experimental with an authentic
sample from the first method.
4-Amino-3-mercapto-(4H)-1,2,4-triazole derivatives have been
widely utilized as starting materials for the preparation of heterocyclic
compounds15 due to the presence of two adjacent reactive functional
groups as reported for similar compounds,16–20 and this aminothiol
derivative proved to be a versatile intermediate for the preparation of
bridgehead nitrogen heterocycles. The aminothiol derivative 1a reacted
in benzene at room temperature in the presence of triethylamine with
2-bromopropionic acid to give the acid derivative 2. This derivative
SCHEME 1 1a = C5H4N, 1b = C5H4N, 1c = C4H3O, 1d = C6H4-OH-P, 1e =
C6H4-NO2-P, 1f = C6H5CO NH CH2 CH3; 1g = CH2BrCH2.

New Fused Heterocycles
2761
TABLE I Characterization of the Newly Synthesized Compounds
Analysis % Calcd./Found
Comp.
no.
Mp Yield Molecular
(◦C) (%)
formula
R
C
H
N
S
1a
280
96 C7H7N5S
193
43.52
43.10
3.62
3.70
36.26
36.40
16.58
16.30
1b
220
72 C7H7N5S
193
43.52
43.50
3.62
3.13
36.26
36.40
16.58
16.72
1c
1d
>360 70 C6H6N4OS 39.56
182 39.40
3.29
3.11
30.76
30.81
17.58
17.50
220
80 C8H8N4OS 46.15
208 46.00
3.84
4.01
26.92
26.90
15.38
15.11
1e
210
280
85 C8H7N5O2S 40.50
237 40.22
2.95
3.01
29.53
29.20
13.50
13.00
1f
75 C10H11N5OS 48.19
249 48.00
4.41
4.40
28.11
28.00
12.85
12.60
∗1g
CH2BrCH2– >360 75 C4H7BrN4S 21.52
222.98 21.60
3.13
3.00
25.11
25.00
14.35
14.10
∗
1g: Br: Calcd 35.86; Found: 36.01.
2 was stirred at room temperature in benzene with a mixture of
acetic anhydride and triethylamine (V/V-1:1)21 and cyclized to give the
mesoionic compound 3. This mesoionic compound was isolated with a
high degree of purity. Prolonged heating in ethanol of the compound 3
gave the ethyl ester 4. The ketone derivative 5 was only obtained from
the methanolic alkaline hydrolysis of 4. According to their synthetic
route, the reaction of the aminothiol derivative 1a with the appropriate
halo-ester gave a product with a γ -lactam structure.22,23 The 1H-NMR
spectrum of compound 5 showed the presence of an amidic hydrogen
as a signal at δ 12.50 ppm; in the IR spectrum, the presence of an
amidic hydrogen is confirmed by a broad signal at 3210 cm−1
.
Interaction of 1a with acetylacetone or ethyl acetoacetate using
DMSO (dimethyl sulphoxide) as medium caused cyclization to give sub-
stituted 1,2,4-triazole [3,4-b]-6H-1,3-4-thiadiazine 6h,i. The IR spectra

2762
A. Y. Hassan
of compounds 6h,i showed the absence of an absorption band corre-
sponding to the amino group and appearance of a new band correspond-
ing to NH group and 1H-NMR spectra is in agreement with the proposed
structure (Table II). Interaction of 1a with chloroacetamide either by fu-
sion or using dioxan as solvent and triethylamine as catalyst resulted
in the formation of 5-amino-8-(pyridine-4-yl)-1,2,4-triazolo[3,4-b]-4H,
6H-1,3,4-thiadiazine 7 (Scheme 2).
SCHEME 2
On the other hand, treatment of compound 1a with 2,3-
dichloro-1,4-naphthoquinone and 2,3-dichloroquinoxaline afforded
the corresponding naphthoquinon-1,3,4-thiadiazine derivative 8 and
quinoxaline-1,3,4-thiadiazine derivative 9 (Scheme 2). The reaction
pattern is similar to some known reactions of 2,3-dichloro compounds
with binucleophiles such as o-aminothiophenol.24
Thenoyl isothiocyanate25 reacted with 4-amino-5-mercapto-1,2,4-
triazole derivative 1a to give N-[7-(pyridine-4-yl)-1,2,4-triazolo [3,4-b]
1,3,4-thiadiazole-4-yl]-thiopheneamide 10. The IR spectrum of com-
pound 10 showed the absence of absorption band corresponding to the
amino group and appearance of a new band corresponding to NH at
1
3280 cm−1 and H-NMR spectrum is in agreement with the proposed

New Fused Heterocycles
2763
TABLE II Characterization Data of the Newly Synthesized
Compounds
% Analysis: Calcd/Found
Comp.
No.
mp
Yield
%
Mol. formula
M. W
(◦C)
C
H
N
S
2
230
265
82
40
70
50
72
65
55
85
80
73
60
65
72
40
55
80
80
95
75
C10H11N5O2S
265
45.28
45.00
48.58
48.60
49.14
49.00
48.58
48.60
52.74
52.60
51.48
51.60
46.55
46.60
58.78
58.80
56.42
56.30
47.56
47.60
50.52
50.50
48.48
48.50
59.75
59.22
57.56
57.60
52.74
52.70
60.25
60.00
59.09
59.00
56.25
56.20
55.73
55.70
4.15
4.30
3.64
3.30
5.11
5.15
3.64
3.30
4.02
4.00
4.29
4.50
3.44
3.33
2.59
2.72
2.82
2.70
2.43
2.20
2.45
2.11
3.17
3.20
4.56
4.18
4.79
4.88
5.49
5.88
4.60
4.18
4.37
4.30
4.68
4.40
3.82
3.65
26.41
26.60
28.34
28.11
23.89
24.00
28.34
28.00
25.64
25.50
23.10
23.00
36.20
36.00
20.17
20.00
30.72
30.14
25.60
25.61
24.56
24.60
24.24
24.30
29.04
29.18
25.83
25.55
35.89
35.60
35.14
35.00
26.81
26.80
32.81
32.60
22.95
22.80
12.07
12.00
12.95
13.00
10.92
11.00
12.95
13.01
11.72
11.80
10.56
10.80
13.79
14.00
9.22
3
C
C
C
10H9N5OS
247
12H15N5O2S
293
10H9N5OS
247
4
220
5
>360
200
6h
6i
C12H11N5OS
273
C13H13N5O2S
303
220
7
230
C9H8N6S 232
8
210
C
C
C
C
C
17H9N5O2S
347
15H9N7S 319
9.50
9
>360
260
10.03
10.00
19.51
20.00
22.45
22.60
9.23
10
11
12*
14j
14k
15
16
17*
18
19
13H8N6OS2
328
12H7N5S2 285
>360
240
14H11ClN6OS
346.5
9.00
—
160
C12H11N5O 241
130
C13H13N5O2
271
C12H15N7O 273
—
—
—
—
—
180
>360
190
C24H22N12 478
C18H16ClN7
365.5
C12H12N6O 256
>360
110
C17H14N6O2S
366
8.74
8.19
12∗: Cl: Calcd. 10.24; Found: 10.11.
17∗: Cl: Calcd: 9.71; Found: 9.80.

2764
A. Y. Hassan
SCHEME 3
structure (Table II, Scheme 3). In contrast, p−chlorophenyl isocyanate
reacted with 4-amino-5-mercapto-1,2,4-triazole derivative 12a6 to give
5-mercapto 1,2,4-triazole derivative 12. The IR spectrum of compound
12 revealed the appearance of a CO signal at 1708 cm−1. The behav-
ior of 1a toward 2-thiophenecarbaldehyde and thenoyl chloride was
investigated. Thus, interaction of 1a with 2-thiophenecarbaldehyde
using AcOH as medium27 caused cyclization to give 7-(pyridine-4-
yl)-4-thiophene-1,2,4-triazolo[3,4-b] 1,3,4-thiadiazole 11. Compound 11
could be also prepared through ring closure reaction between 1a and
thenoyl chloride.28 The reaction included elimination of one mole of
each HCl and H2O to give 11 (mp, mmp, and TLC; Scheme 3).
In an attempt to synthesize fused 1,3,4-thiadiazine ring with 1,2,4-
triazole, I studied the reaction of 5-substituted-4-amino-3-mercapto-
(4H)-1,2,4-triazole 1a with halogenated 1,3-dicarbonyl active methy-
lene compounds. The products from this reaction were shown to be
1,2,4-triazolo[3,4-b] pyrazole derivatives 14j−k rather than the expected
1,2,4-triazolo[3,4-b] 1,3,4-thiadiazine derivative 13j,k (Scheme 4).
In the IR, 1H-NMR, and 13C-NMR spectra no observable differences
are found capable of differentiating between compounds 14j,k and 13j,k
.
The only differences capable of differentiating between them were ob-
served in their mass spectra, which showed the existence of the molec-
ular ion peaks M+ at m/z 241 and 271 corresponding to 14j and 14k,
respectively, and the elemental microanalysis of these compounds for S
gave 0%. The above results confirm that triazolo[3,4-b]-pyrazole struc-
tures 14j,k were formed and not the expected 1,2,4-triazolo[3,4-b] 1,3,4-
thiadiazine. The mechanism for the formation of compounds 14j,k is

New Fused Heterocycles
2765
SCHEME 4
postulated as shown in Scheme 5. It proceeds by nucleophilic attack
of the amino group of compounds 1a on the carbonyl group of active
methylene compound I to give Ia, followed by elimination of water from
Ia to give Ib, which subsequently undergoes internal nucleophilic at-
tack accompanied by the elimination of hydrogen sulfide to give Ic.
Compound Ic undergoes a redox reaction with eliminated hydrogen
sulfide to afford Id and elemental sulfur. Elimination of hydrogen chlo-
ride from Id afforded the 1,2,4-triazolo [3,4-b] pyrazole derivative Ie.
Compounds 14j,k were prepared directly in good yield by the reaction
of 1a with 3-chloro-2,4-pentandione and/or ethyl-2-chloroaceto-acetate
in refluxing ethanol (Scheme 4).
Treatment of 14j with hydrazine hydrate in 2-propanol at room
temperature afforded the hydrazinium salt 15, which upon reflux-
ing in 2-propanol in the presence of a few drops of acetic acid
furnished N,Nꢀ -bis[4-methyl-7-pyridine-4-yl] 1,2,4-triazolo[3,2-b]-3-yl-
ethylidene) hydrazine-5H-pyrazole 16. The mass spectrum of com-
pound 16 showed a molecular ion peak M+ at 478 m/z and the base
peak (1/2 M+) at m/z 239, which is strong evidence for the structure

2766
A. Y. Hassan
SCHEME 5
of 16. Compound 16 was prepared directly by the reaction of 14j with
hydrazine hydrate in refluxing 2-propanol in the presence of a few
1
drops of acetic acid. The H-NMR spectrum of compound 15 showed
the presence of a broad singlet signal (five protons) at δ 6.90 ppm cor-
responding to the hydrazinium ion (N2H+). The reaction of 14j with
p-chlorophenyl hydrazine in the presence5of a few drops of acetic acid
in refluxing 2-propanol yielded the corresponding hydrazono deriva-
1
tive 17. The H-NMR spectrum of compound 17 showed the presence
of a singlet corresponding to the NH-hydrazono at δ 9.17 ppm and
singlet corresponding to pyrazolo NH at δ 13.00 ppm (Table III). The
mass spectrum of compound 17 showed a molecular ion peak M+ at
364 m/z.
Condensation of 14j with hydroxylamine hydrochloride in refluxing
2-propanol in the presence of triethylamine afforded the correspond-
ing oxime derivative 18, which was reacted with thenoyl chloride in
anhydrous pyridine at 0◦C to give the corresponding 3-(1-thenoylimino

New Fused Heterocycles
2767
TABLE III IR, 1H NMR, 13C NMR, and Mass Spectral Data
Compd.
no.
1H-NMR δ (ppm)/13
C-NMR, δ (ppm)/ms
IR (cm−1
)
1a
3320, 3260 (NH2), 3053 (CHAr),
2562 (SH), 1639 (C N)
5.50 (s, 2H, NH2), 7.25–7.69 (m, 4H,
pyridine-H), 12.80 (br, 1H, SH); m/z
= 193 (10.65%)
1b
1c
3300, 3280 (NH2), 3055 (CHAr),
2500 (SH), 1640 (C N)
3310, 3270 (NH2), 3040 (CHAr),
2530 (SH), 1630 (C N)
5.20 (s, 2H, NH2), 7.25–7.70 (m, 4H,
pyridine-H), 13.01 (br, 1H, SH)
5.00 (s, 2H, NH2), 7.23, 7.40 (2d, 2H,
furan-H, J = 5.80 Hz), 7.66 (s, 1H,
furan-H), 13.00 (br, 1H, SH)
1d
1e
1f
4495 (OH), 3320 (NH2), 3033
(CHAr), 2500 (SH), 1610 (C N)
12.10 (s, 1H, OH), 7.22, 7.56 (2d, 4H,
phenyl-H) AAꢀ XXꢀ system J = 8.44
Hz), 13.04 (br, 1H, SH)
4.80 (s, 2H, NH2) 7.44, 7.68 (2d, 4H,
AAꢀ XXꢀ system, J = 10.22 Hz), 13.37
(s, 1H, SH)
4.11 (s, 2H, CH2), 5.80 (s, 2H, NH2),
7.22–7.80 (m, 4H-phenyl-H), 10.08
(br, s, 1H, NH), 13.55 (s, 1H, SH)
3340 (NH2), 3080 (CHAr), 2560
(SH), 1523 (NO2), 1620 (C N)
3300, 3240 (NH, NH2), 3030
(CHAr), 2900 (CH aliph.), 2560
(SH), 1690 (C O), 1620 (C N)
1g
2
3310 (NH2), 2820–2900 (CH aliph.), 2.94 (m, 4H, CH2–CH2), 5.57 (s, 2H,
2670 (SH), 1620 (C N)
3400 (OH), 3300 (NH2), 1695
(C O), 3063 (CH–pyridine–H),
1635 (C N)
NH2), 13.46 (br.s, 1H, SH)
1.30 (d, 3H, CH3, J = 8.40 Hz), 4.15 (q,
1H, CH, J= 7.00 Hz), 5.70 (s, 2H,
NH2), 12.11 (br.s, 1H, OH)
3
4
3250, 3180 (NH2), 3045 (CHAr),
2912 (CH aliph.), 1695 (C–O−),
1640 (C N)
3330, 3240 (NH2), 3055 (CHAr),
2888–2930 (CH aliph.), 1730
(C O), 1635 (C N)
1.40 (s, 3H, CH3), 6.11 (s, 2H, NH2)
7.20–7.53 (m 4H, pyridine–H); ms:
m/z = 247 (50% M+)
1.41 (t, 3H, CH2CH3, J = 7.80 Hz),
1.62 (d, 3H, CH3, J = 8.91 Hz), 4.48
(q, 1H, C–H, J = 7.20 Hz), 4.75 (q,
2H, CH2CH3, J = 9.80 Hz), 6.22 (s,
2H, NH2), 7.22–7.56 (m, 4H,
pyridine–H)
5
3210 (NH), 3030 (CHAr), 2888 (CH 1.40 (d, 3H, CH3, J = 7.50 Hz), 4.30 (q,
aliph), 1700 (C O), 1630 (C N)
1H, CH, J = 9.00 Hz), 7.30–7.52 (m,
4H, pyridine–H), 12.50 (br s, 1H,
NH); ms: m/z 247 (11.13% M+)
2.30 (s, 3H, CH3), 2.60 (s, 3H, COCH3)
7.22–7.36 (m, 4H, pyridine–H),
12.50 (s, 1H, NH); ms: m/z = 273
(22.40% M+)
6h
6i
3230 (NH), 3060 (CHAr) 2940 (CH
aliph), 1710 (C O), 1640 (C N),
1590 (C C)
3280 (NH), 3080 (CHAr),
2777–2910 (CH-aliph.), 1725
(C O), 1635 (C N), 1590 (C C)
1.33 (t, 3H, CH2CH3, J = 8.11 Hz),
1.82 (s, 3H, CH3), 4.80 (q, 2H,
CH2CH3, J = 10.01 Hz), 7.30–7.52
(m, 4H, pyridine–H), 13.11 (s, 1H,
NH); ms: m/z = 303 (18.07)
(Continued on next page)

2768
A. Y. Hassan
TABLE III (Continued)
Compd.
no.
1H-NMR δ (ppm)/13
C-NMR, δ (ppm)/ms
IR (cm−1
)
7
3380, 3260, 3210 (NH, NH2), 3062
(CHAr), 1630 (C N), 1580 (C C)
5.44 (s, 1H, NH2), 7.22–7.58 (m, 4H,
pyridine–H), 8.24 (s, 1H, CH), 12.08
(s, 1H, NH); ms: m/z = 232 (36.18%
M+)
8
3275 (NH), 3055 (CHAr), 1680
(2C=O), 1640 (C N), 1570
(C C)
7.22–7.86 (m, 8H, Ar–H), 10.01 (s, 1H,
NH); 13C:179.9 (C O),178.6 (C O),
153.50, 153.90, 154.20, 155.00,
158.20, 159.00, 160.70
9
3210 (NH), 3056(CHAr), 1630
(C N), 1600 (C C)
7.28–7.88 (m, 8H, Ar–H), 12.05 (s, 1H,
NH); ms: m/z 305 (15.11M+)
10
3280 (NH), 3055 (CHAr), 1710
(C O), 1640 (C N)
7.22–7.52 (m, 5H, Ar–H), 7.63 (d, 1H,
Ar–H, J = 5.15 Hz), 8.10 (d, 1H,
Ar–H, J = 6.02 Hz), 13.11 (br, s, 1H,
NH); ms: m/z = 328 (28.17% M+)
7.24–7.43 (m, 5H. Ar–H), 7.60 (d, 1H,
J= 8.00Hz), 7.98 (d, 1H, J = 7.99
Hz); ms: m/z=285 (32.00% M+).
7.63, 8.60 (2d, 4H, p-Cl phenyl, AAꢀ
XXꢀ , J = 8.22 Hz), 7.22–7.51 (m, 4H,
pyridine–H), 9.93 (br, s, 1H, NH),
10.04 (br, s, 1H, NH); ms: m/z = 346
(25.00% M+)
11
12
3088 (CHAr), 1640 (C N)
3281(NH), 3045 (CHAr), 1708
(C O), 1635 (C N)
14j
3389 (NH), 3050 (CHAr), 2920 (CH 2.30 (s, 3H, CH3), 2.60 (s, 3H, COCH3),
aliph), 1710 (COCH3), 1644
(C N), 1560 (C C)
7.22–7.54 (m, 4H, pyridine–H),
13.80 (s, 1H, NH); 13C: 15.20 (CH3),
29.50 (COCH3), 139.90, 140.22,
141.00, 146.08, and 168 (C O); ms:
m/z = 241 (11.80 M+)
14k
3400 (NH), 3055 (CHAr),
2887–2900 (CH-aliph.), 1730
(C O) ester, 1640 (C N), 1590
(C C)
2.33 (s, 3H, CH3), 1.30 (t, 3H,
CH3CH2, J = 11.01 Hz), 4.30 (q, 2H,
CH2CH3, J = 9.88 Hz) 12.80 (s, 1H,
NH); 13C: 14.0 (CH3CH2), 16.30
(CH3), 60.60 (OCH2), 162.50 (C O),
139.90, 140.22, 141.00, 146.08. ms:
m/z = 271 (30.08% M+)
15
16
3425, 3247 (NH2), 1711 (C O),
1631 (C N), 1541 (C C)
2.50 (s, 3H, CH3), 2.6 (s, 3H, COCH3),
6.90 (br S, 5H, hydrazinium–Hs),
7.20–7.36 (m, 4H, pyridine–H);13C:
16.40 (CH3), 29.50 (COCH3), 139.50,
149.81, 153.11, 155.70, 191.80
(C O)
3278 (NH), 3055 (CHAr), 2995 (CH 2.1 (s, 3H, CH3), 2.50 (s, 3H,
aliph), 1570 (C C), 1640 (C N) hydrazono-CH3), 7.22–7.77 (m, 8H,
pyridine–H); ms: m/z = 478 (8.12%
M+) and base peak at m/z = 239
(50%)
(Continued on next page)

New Fused Heterocycles
2769
TABLE III (Continued)
Compd.
no.
1H-NMR δ (ppm)/13
C-NMR, δ (ppm)/ms
IR (cm−1
)
17
3437, 3318 (2NH), 2900(CH aliph.)
1635 (C N), 1599 (C C)
2.30 (s, 3H, CH3), 2.50 (s, 3H, CH3),
7.22–7.50 (m, 8H, Ar-H) 7.82, 8.00
(2d, 4H, AAꢀ XXꢀ - system J = 8.10
Hz), 9.17 (s, 1H, hydrazono-NH),
13.0 (s, 1H, pyrazoloe NH), 13C:
14.70 (CH3), 16.10
(hydrazono–CH3), 113.90, 119.90,
130.10, 137.44, 138.80, 139.18,
140.00, 141.33, 141.90 (C=N–NH);
ms: m/z = 364 (17.02% M+)
18
19
3227 (NH), 3042 (CHAr), 2940 (CH 2.00 (s, 3H, CH3), 2.20 (s, 3H,
aliph.), 1636 (C N), 1558 (C C)
CH3–C=NOH), 10.90 (s, 1H, OH);
13C: 14.62 (CH3), 28.11(CH3) 127.70,
129.44, 129.99, 136.50, 142.60
2.3 (s, 3H, CH3), 2.6(s, 3H,
CH3–C=N–OCOC4H3), 7.60–8.13
(m, 7H, Ar–H), 13.00 (s, 1H,
NH-pyrazole). ms: m/z = 366 (18.02
M+)
3461 (NH), 2990 (CH aliph.), 1713
(C O), 1609 (C N), 1543 (C C)
ethyl)-4-methyl-7-(pyridine-4-yl)-1,2,4-triazolo [3,2-b]-5H-pyrazole 19
through protection of the OH group. A singlet corresponding to the
1
OH proton at δ 10.90 ppm was observed in the H-NMR spectrum of
1
compound 18. The H-NMR spectrum of compound 19 shows that the
OH group is no longer present, and the singlet at δ 13.00 ppm shows
that the NH group is present, which confirmed that protection occurred
on the OH group, not the NH group.
Experimental
Melting points were recorded on a Gallen Kamp Melting apparatus and
are uncorrected. Infrared spectra were obtained on a Nexus 470-670,
and 1H-NMR spectra and 13C-NMR were run on JEOL-400 MHz spec-
trometer using DMSO-d6 as a solvent and TMS as an internal standard.
The mass spectra were recorded on Ms-S988 operating at 70eV. Micro-
analysis was performed using a Perkin-Elmer 2400 CHN analyzer. The
newly synthesized compounds were screened for cytotoxic activity (in
vitro study) and antitumor activity (in vitro study) at Cairo University,
National Cancer Institute, Cancer Biology Department, Pharmacology
Unit.

2770
A. Y. Hassan
General Procedure for the Preparation of 5-Substituted-4-
Amino-3-Mercapto-(4H)-1,2,4-Triazoles 1a−h
A mixture of thiocarbohdyrazide (0.1 mmol) and the appropriate car-
boxylic acid (0.1 mmol) was warmed carefully at 160–170◦C until melt-
ing occurred,29 and then it was warmed again after melting occurred
for 15 min. The reaction mixture was cooled, mixed with water (80 mL),
and acidified with concentrated hydrochloric acid (2 mL). The precip-
itate was filtered, washed with water, oven-dried, and recrystallized
from ethanol.
2-[(1-Amino-5-(Pyridine-4-yl)-1,2,4-Triazole-2-yl) Thio]-
Propionic Acid 2
A mixture of aminothiole derivative 1a (1.8 mmol), 2-bromopropionic
acid (1.8 mmol) and triethylamine (0.25 mL) in benzene (30 mL) was
stirred at room temperature for 30 h. After filtration, the resulting solu-
tion was extracted with a sodium bicarbonate 5% solution (30 mL). The
aqueous solution was acidified with drop-wise addition of hydrochloric
acid to pH 5-6; the solid was collected, washed with water, dried, and
recrystallized from ethanol to give 2.
1-Amino-5-Hydroxy-6-Methyl-2-(Pyridine-4-yl)-Thiazolo
[3,2-b]-1,2,4-Triazolium Inner Salt 3
A mixture of acetic anhydride (0.5 mL) and triethylamine (0.5 mL)
was added to a stirred suspension of the acid derivative 2 (0.43 mmol)
in benzene (2 mL); the mixture was stirred at room temperature for 2
h. The yellow resulting mixture was diluted with diethyl ether (10 mL)
and the solid collected, washed with diethyl ether, and dried to give 3
and recrystallized from dioxan/benzene.
Ethyl Ester of 2-[(1-Amino-5-(Pyridine-4-yl) 1,2,4-Triazole-2-yl)
Thio-Propionic Acid 4
The mesoionic compound 3 (35 mg) in ethanol (25 mL) was heated at
reflux for 30 min. The solution was concentrated under vacuum and the
residue was collected, washed with methanol, dried, and recrystallized
from ethanol to give 4.
4-Methyl-4H,6H-8-(Pyridine-4-yl)-1,2,4-Triazolo
[3,4-b]-1,3,4-Thiadiazine-5-one 5
A suspension of ethyl ester 4 (1.0 mmol) in a solution of sodium
hydroxide (1.0 mmol, 10 mL) in methanol (10 mL) and water (2 mL) was
stirred at room temperature for 24 h; the mixture was poured into water
(100 mL); by acidification of the resulting solution with hydrochloric

New Fused Heterocycles
2771
acid until pH 4–5 a white solid separated. This solid was collected,
washed with water, dried, and recrystallized from ethanol to give 5.
4-Acetyl (or Ethoxycarbonyl)-5-Methyl-6H-8(Pyridine-4-yl)
-1,2,4-Triazolo[3,4-b]-1,3,4-Thiadiazine 6h,i
To a stirred suspension of active methylene compounds (0.01 mol,
acetylacetone or ethyl acetoacetate) in DMSO (5 mL) was added of
aminothiol derivative 1a and the solution refluxed for 2 h, concentrated,
and cooled to room temperature. The solid separated out was washed
with a small amount of methanol and recrystallized from methanol to
give pure compounds 6h,i
.
5-Amino-4H,6H-8(Pyridine-4-yl)-1,2,4-Triazolo
[3,4-b]-1,3,4-Thiadiazine 7
r
r
Method A: A mixture of aminothiol derivative 1a (0.01 mmol),
chloro-acetamide (0.01 mol), and triethylamine (0.25 mL) in dioxan
(30 mL) was refluxed for 6 h. After filtration the resulting solid was
collected, dried, and recrystallized from ethanol to give 7.
Method B: The same reactants were fused together at 170◦C for 1
h and then triturated with ethanol and recrystallized to give 7 (mp,
mmp, and TLC).
12-(Pyridine-4-yl)-10-(H)-1,2,4-Triazolo[3,4-b]-1,3,4-Thiadiazino
[2,3-d]-4,9-(Naphthoquinone or Quinoxaline) 8 and 9
Aminothiol derivative 1a (10 mmol) and 2,3-dichloro-1,4-
naphthoquinone or 2,3-dichloroquinoxaline (10 mmol) was refluxed in
ethanol for 6 h. After cooling, the yellow solid was collected and washed
with water and then with ethanol to give 8 and 9.
N-[7-(Pyridine-4-yl)-1,2,4-Triazolo[3,4-b]
1,3,4-Thiadiazol-4-yl]-Thiopheneamide 10
Thenoyl isothiocyanate (0.005 mol) was added to a solution of
aminothiol derivative 1a (0.005 mol) in 20 mL of acetonitrile. The re-
sulting solution was stirred and heated under reflux for 18 h (a solid
formed during the course of the reaction). The reaction mixture was
cooled and the solid was removed by filtration. The crude product was
crystallized from tetrahydrofuran containing a few drops of dimethyl
formamide.
7-(Pyridine-4-yl)-4-Thiophene-1,2,4-Triazolo
[3,4-b]-1,3-4-Thiadiazole 11
r
Method A: Aminothiol derivative 1a (0.01 mol) was refluxed with 2-
thiophene carbaldehyde (0.01 mol), followed by the addition of glacial

2772
A. Y. Hassan
acetic acid (5 mL). The mixture was heated for 3 h and then diluted
with ethanol and cooled. The precipitate was collected and recrys-
tallized from ethanol.
Method B: A solution of 1a (0.01 mol) in thenoyl chloride (10 mL
excess) was refluxed for 1 h. Decomposition over ice-cold water gave
a solid that recrystallized from ethanol to give 11. Melting point
depression was not observed in a mixed melting point experiment
with an authentic sample prepare by method A.
r
N-p-Chlorophenyl-Nꢀ -[3-Mercapto-5-(Pyridine-4-yl)
-1,2,4-Triazole-4-yl] Urea 12
A mixture of aminothiol derivative 1a (0.01 mol) and p-chlorophenyl
isocyanate (0.01 mol) was fused at 140◦C for 2 h. The reaction mixture
was cooled, triturated with light pet. (40–60), washed with ether, and
then recrystallized with ethanol to give 12.
3-Acetyl-4-Methyl-7-(Pyridine-4-yl)-1,2,4-Triazolo-[3,4-b]-5H-
Pyrazole 14j. Ethyl-(4-Methyl-5H-7-(Pyridine-4yl)-1,2,
4-Teriazolo [3,4-b] Pyrazole-3-Carboxylate 14k
To a solution of 1a (0.02 mol) in ethanol (80 mL), 3-chloro-2,4-
pentandione (0.02 mol) was added in one portion and refluxed for 2
h, at which time the starting material was consumed (TLC). On cool-
ing, the solid that separated out was isolated by filtration, dried, and
recrystallized from ethanol.
Hydrazinium-3-Acetyl-4-Methyl-7-(Pyridine-4-yl)
-1,2,4-Teriazolo [3,2-b] Pyrazole-on-5-ide 15
Compound 14j (0.01 mol) and hydrazine hydrate 80% (0.015 mol)
in 2-propanol (50 mL) were stirred at room temperature for 6 h, after
which time the starting material had been consumed (TLC). The solid
product that formed was separated and recrystallized from ethanol.
N,N-bis-(4-Methyl-7-(Pyridine-4-yl)-5H-1,2,4-Triazolo
[3,2-b]-3-yl) Alkylidene-Hydrazine-5H-Pyrazole 16
r
r
Method A: Compound 15 (0.005 mol) and glacial acetic acid (0.5
mol) were refluxed in 2-propanol (50 mL) for 2 h until the starting
material was consumed (TLC). The solid product that formed was
separated and recrystallized from dimethyl formamide.
Method B: Compound 14j (0.01 mol) and hydrazine hydrate (0.0052
mol) in 2-propanol (50 mL) in the presence of acetic acid (1 mL) was
refluxed for 3 h until the starting material was consumed (TLC). The
solid product that formed was separated out and recrystallized from

New Fused Heterocycles
2773
dimethyl formamide. Melting point depression was not observed in
a mixed melting point experimental with authentic sample from
method A.
4-Methyl-3[1-p-Chlorophenylhydrazono]-Ethyl-7-
(Pyridine-4-yl)-1,2,4-Triazolo [2,3-b]-5H-Pyrazole 17
Compound 14j (0.01 mol) and p-chlorophenyl hydrazine (0.01 mol) in
2-propanol (50 mL) in the presence of acetic acid (1 mL) was refluxed for
2 h, until the starting material was consumed (TLC). The solid product
that formed was separated out and recrystallized from ethanol.
3-(1-Hydroxy Iminoethyl)-4-Methyl-7-(Pyridine-4-yl)
-1,2,4-Triazolo [3,2-b]-5H-Pyrazole 18
Compound 14j (0.01 mol) and hydroxylamine hydrochloride (0.01
mol) in 2-propanol (60 mL) in the presence of triethyl amine (0.2 mL)
was heated gradually to 90◦C followed by refluxing for 3 h until the
starting material was consumed (TLC). The solid product that formed
was separated and recrystallized from methanol.
3(1-Thenoyloxyiminoethyl)-4-Methyl-7-(Pyridine-4-yl)
-1,2,4-Triazolo [3,2-b]-5H-Pyrazole 19
A mixture of 18 (0.005 mol) and thenoyl chloride (0.007 mol) was
stirred in anhydrous pyridine (25 mL) at 0◦C for 6 h until the starting
material was consumed (TLC). The reaction mixture was poured on
ice-cold water. The solid product was isolated by filtration and recrys-
tallized from methanol.
Biological Results
Antitumor Activity (In Vitro Study)
EAC were obtained by needle aspiration of ascetic fluid from pre-
inoculated mice under aseptic conditions. The cells were tested for via-
bility and contamination by staining a certain cell volume of this fluid
by an equal volume of the working solution of Trypan blue dye and
examined under a microscope.30 In a set of sterile test tubes, 0.1 mL of
tumor cell suspension, 0.8 mL RPMI 1640 media, and 0.1 mL of each
test compound (corresponding to 100, 50, and 25 µg/mL). The later test
tubes were carried out to calculate the percentage of nonviable cells.
Compounds producing more than 70% nonviable cell are considered
active31 (Table IV).
no of nonviable
Total No. of cell
% of nonviable cells =
× 100

2774
A. Y. Hassan
TABLE IV In Vitro Cytotoxic Activity of Synthesized Compounds
Nonviable cells (%)
concentration (µg/mL)
Compound no.
100
50
25
1a
2
3
4
5
7
8
9
10
11
12
14j
15
16
17
18
90%
70%
85%
60%
40%
−ve
−ve
−ve
80%
50%
80%
60%
60%
70%
90%
50%
−ve
100%
85%
65%
80%
50%
30%
−ve
−ve
−ve
75%
40%
70%
55%
50%
60%
85%
35%
−ve
55%
80%
55%
70%
35%
20%
−ve
−ve
−ve
60%
20%
50%
45%
35%
45%
80%
10%
−ve
20%
19
Doxorubicin32 as reference
Biological Results and Structure Activity Correlation
Antitumor Activity (In Vitro Study)
Potential cytotoxicity of the compounds was tested using the method
of Skehan et al.32 Cells were plated in a 96-well plate (10 cells/well) for
24 h before treatment with the compound to allow attachment of the
cells to the wall of the plate. Different concentrations of the compound
under test (0, 10, 25, 50, and 100 µg/mL) were added to the cells.
Monolayer cells were incubated with the compounds for 48 h at 37◦C
and in an atmosphere of 5% CO2. After 48 h, the cells were fixed,
washed, and stained with sulfo-rhodamine-B stain. Excess stain was
washed with acetic acid and the attached stain was recovered with Tris
EDTA buffer. The color intensity was measured in an ELISA reader.
The relation between surviving fraction and drug concentration was
plotted to obtain this survival curves of tested cell, the response pa-
rameter calculated was IC50 value. The data for the tested compounds
are summarized in Table V.
Compound 1a, which has a thiol group, was more effective than the
positive control (doxorubicin)33 and was able to reduce the magnitude
of activity to 90%, whereas alkylation of compound 1a lowered this

New Fused Heterocycles
2775
TABLE V In Vitro Anti-HEPG2a Testing Results
Comp.
IC50µg/mLb
1a
2
3
10
12
16
17
14.07
50.48
18.55
14.00
25.81
60.27
8.12
Doxorubicinc
43.6
aLiver carcinoma cell line.
bConcentration of compound that causes 50% inhibition of cell growth.
cPositive control.
activity to 70%. Compound 3 showed the highest activity among the
tested compounds and it was able to reduce the activity of EAC to
85%. Compounds 10 and 12 were hypothesized to be active through
intramolecular hydrogen bonding34 and were able to reduce the activity
of cells to 80%. Condensation of compound 14j with hydrazine or p-
chlorophenyl hydrazine afforded compounds 16 and 17, which proved
to be active toward the use tumor cell lines. Compounds 7, 8, 9, and 19
did not show any activity at different concentrations, probably because
of a solubility problem in the culture media used. Compounds 4, 5, 11,
14j, 15, and 1 possess moderate activity against use tumor cell lines
(Table III).
The selectivity of compounds 1a, 2, 3, 10, 12, 16, and 17 were ex-
amined against liver carcinoma cell line (HEPG2) (Table V). On the
basis of the structure of tested compounds, it can be concluded that
structure-activity relationships provided evidence that geometry, size,
and shape of the compound are as important as their substituents.
These heterocycles could be considered as useful templates for future
development and further derivatization or modification to obtain more
potent and selective antitumor agents.
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