Synthesis and biological evaluation of 3,6-disubstituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as a novel class of potential anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2009.01.003

A new series of 3,6-disubstituted triazolo[3,4-b]thiadiazole derivatives have been synthesized by simple, high yielding routes. The key step in the construction of the triazolo[3,4-d]thiadiazole nucleus involves the reaction of 4-amino-5-substituted [1,2,

Full text of DOI:10.1016/j.ejmech.2009.01.003

European Journal of Medicinal Chemistry 44 (2009) 2776–2781
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 3,6-disubstituted [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole derivatives as a novel class of potential anti-tumor agents
*
D.A. Ibrahim
National Organization for Drug Control and Research, 6 Abou Hazim Street, Pyramid Avenue, P.O. Box 29, Cairo, Gizaa 111321, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 3,6-disubstituted triazolo[3,4-b]thiadiazole derivatives have been synthesized by simple,
high yielding routes. The key step in the construction of the triazolo[3,4-d]thiadiazole nucleus involves
the reaction of 4-amino-5-substituted [1,2,4]triazole-3-thiol with carbon disulphide, 4-amino benzoic
acid, (2-amino[1,3]thiazole-4-one-5-yl) acetic acid, and (1H-pyrazolo[3,4-d]pyrimidine-2,4-dithione-5-
yl) acetonitrile. The newly synthesized compounds were evaluated for their cytotoxic activity against
a panel of 60 human cancer cell lines by the National Cancer Institute (NCI) and some of them
demonstrated inhibitory effects on the growth of a wide range of cancer cell lines generally at 10ꢀ5 M
level and in some cases at 10ꢀ7 M concentrations. In this assay, the anti-tumor activity of the newly
synthesized compounds could not be interpreted in terms of tyrosine kinase inactivation but more likely
as a relatively broad specificity for the ATP-binding domain of other kinases. The pharmacological
mechanism of action for these intriguing compounds has not, as yet, been successful.
Received 1 August 2008
Received in revised form
7 December 2008
Accepted 4 January 2009
Available online 19 January 2009
Keywords:
[1,2,4]Triazolo[3,4-d][1,3,4]thiadiazole
Alkylation
Amination
Cyclization
Anti-tumor
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
and the related acyclic structure 2,4,40-trihydroxyazobenzene
(IV) which have the same overall substitution pattern as
The isoflavone genistein (I) [1] and the flavone quercetin (II) [2]
are competitive inhibitors at the ATP-binding site of kinases, and
synthetic flavones modeled on quercetin have been investigated
recently in an effort to identify compounds with greater inhibitory
selectivity towards tyrosine kinases over serine–threonine kinases
[3,4].
genistein.
During the course of this work, we prepared 4-(3-substituted
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl) aniline derivatives (2),
2-amino-5-substituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl
methyl-1, 3-thiazol-4-ol derivatives (4) and triazolo[3,4-b]thia-
diazole derivatives as analogues to anti-tumor benzothiazoles to
compare between benzothiazole nucleus and triazolothiadiazole
nucleus and optimize the biological response of the new lead
Protein tyrosine kinases occupy a central position in the
control of cellular proliferation. Several transforming oncogenes
(e.g. Src, abl) are known to possess tyrosine kinase activity, and
it is well recognized that the response of many cells to growth
factors is initiated by the activation of receptor tyrosine kinases
(RTKs). Overexpression of certain RTKs shows association with
promotion and maintenance of malignant disease [5]. Thus,
inactivation of the specific tyrosine kinases that are responsible
for the malignant phenotype of certain cancers represents
a potential approach for the design of antiproliferative drugs
[6]. Some benzothiazoles were designed as potential tyrosine
kinase inhibitors modeled on structural comparison with the
natural products which compete at the ATP-binding sites of
tyrosine kinases [7]. The most interesting bioactive compounds
were 4,6-dihydroxy-2-(4-hydroxyphenyl)benzothiazole (III)
triazolothiadiazole
derivatives.
The
newly
synthesized
compounds were evaluated for their cytotoxic activity against
a panel of 60 human cancer cell lines by the National Cancer
Institute.
OH
O
OH
OH
O
OH
OH
OH
HO
O
HO
O
(II)
(I)
OH
OH
OH
N
N
S
N
OH
HO
HO
(IV)
(III)
HO
N
N
S
N
S
N
N
NH2
N
NH2
N
N
S
N
R
R
(2)
(4)
* Tel.: þ202 33365599; fax: þ202 5855582.
E-mail address: dino1421@hotmail.com
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.003

D.A. Ibrahim / European Journal of Medicinal Chemistry 44 (2009) 2776–2781
2777
SH
N
The data in Table 1 show that compounds 2a, 2c, 4a, 4c, 7c, 8c
showed modestly potent (IC50 < 10 M) and the other compounds
did not show any activity against CDKs.
N
S
N
i
N
N
N
m
HOOC
NH2
+
NH2
N
NH2
R
R
3.2. In vitro
1a R = CH3
b R = -CH2Ph
c R = -CH2-O- Ph(p-Cl)
2a R = CH3
b R = -CH2Ph
Evaluation of anticancer activity on [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole derivatives was performed at the National
Cancer Institute (NCI). First the newly synthesized compounds
have been evaluated in primary anticancer assay at 10ꢀ5 M
concentration against three cell lines MCF7 (Breast), NCI-H460
(Lung) and SF-268 (CNS) cell lines Table 2.
c R = -CH2-O- Ph(p-Cl)
Scheme 1. Reagents and conditions (i) PPA, heat at 200 ꢁC.
2. Results and discussion
For NCI criteria compounds, which reduced the growth of any
one of the cell lines to ca. 32% or less, are passed on for evaluation in
the full panel of 60 human tumor cell lines. The anticancer activity
of each compound is deduced from dose–response curves and is
presented in Table 2 according to the data provided by NCI. The
response parameters GI50, TGI refer to the drug concentration that
produced 50% inhibition and total growth inhibition respectively,
A one pot synthesis of 4-(3-substituted [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl) aniline derivatives (2) was achieved when
4-amino-5-substituted [1,2,4]triazole-3-thiol (1) [8,9] and 4-amino
benzoic acid were heated at 180–200 ꢁC in PPA. The reaction
temperature appeared to be crucial for this one-step process. When
the reaction was carried out below 180 ꢁC, no intramolecular
cyclization was observed, and the starting materials were obtained
as the major product (Scheme 1).
and are expressed in micromolar concentrations (mM).
From the analysis of data reported in Table 3, where the activity
of compounds 2a, 2c, 4a, 4c, 7c, and 8c is stated, we can evince that
compounds 2c and 4c maintained the highest growth inhibition
activity at micromolar concentrations in different human tumor
cell lines. Compound 8c maintained the moderate cytotoxic activity
and finally, compounds 2a, 4a, and 7c maintained the lowest
growth inhibition activity against different human tumor cell lines.
Compound 4c exhibited the highest sensitivity against Renal, Colon
and Melanoma Cancer cell lines, the best results being against
Renal Cancer A498 cell line with log GI50 ꢀ7.27. The compound 2c
displayed high activity against NCI-H226 (log GI50 ꢀ5.14) cell line of
Non-small cell lung cancer sub-panel and against CCRF-CEM
(log GI50 ꢀ5.0) cell line of Leukemia sub-panel. Compound 8c
exhibited moderate sensitivity against Leukemia SR cell line with
log GI50 ꢀ4.85. Finally, compounds 2a, 4a and 7c displayed the
lowest sensitivity against CNS, Non-small cell lung and Colon
cancer with log GI50 ꢀ4.67, ꢀ4.11 and ꢀ4.09 respectively.
Compounds 4c, 2c and 8c were selected for in vivo screening test by
the biological committee of NCI.
We also discovered that 2-amino(5-substituted [1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-yl)-1,3-thiazol-4-ol derivatives (4)
could be prepared in one-step synthesis by the reaction of
compound (1) and (2-amino[1,3]thiazol-4-one-5-yl)acetic acid (3)
[10] in PPA at 100 ꢁC. When the reaction temperature was raised
above 100 ꢁC (130–180 ꢁC) the yield was reduced dramatically
(Scheme 2).
To examine further the effect of alteration at the
C-6 position of triazolothiadiazoles, we synthesized 3-(3-
substituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl-1H-
pyrazolo[3,4-d]pyrimidine-4,6-dithione derivatives (6) by the
reaction of 4-amino-5-substituted [1,2,4]triazole-3-thiol (1) with
(1H-pyrazolo[3,4-d]pyrimidine-4,6-dithione-3-yl) acetonitrile (5)
in PPA at 100 ꢁC for 2 h. When the reaction was carried out by fusion
of the starting materials, no product was isolated (Scheme 3).
To complete the synthetic work, we prepared the sulfur and
nitrogen derivatives at C-6 to examine their effects in biological
profile. 3-Substituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol
(7) was prepared by the reaction of 4-amino-5-substituted
[1,2,4]triazole-3-thiol (1) with carbon disulphide in refluxing pyri-
dine. The disulfide derivatives (8) were prepared from the reaction of
compound (7) with iodine in the presence of sodium carbonate. Also,
alkylation of compound (7) with alkyl halides takes place readily in
the presence of alkaline to give S-alkyl derivatives (9), which reacted
with hydrazine hydrate to give 6-hydrazino-3-substituted
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (10) (Scheme 4).
3.3. In vivo
On the basis of the in vitro results three compounds (2c, 4c and
8c) were selected for in vivo anti-tumor activity assays in nude mice
xenografted with HCT-116 tumors. Initially acute toxicity in mice
was performed for each individual compound, despite their relative
high cytotoxic activity in vitro. Compounds (2c and 8c) had no
significant cytotoxic activity in mice, but compound (4c) showed
acute cytotoxicity. Therefore, National Cancer Institute (NCI)
selected it for testing in vivo anti-tumor Hollow Fiber Assays.
3. Pharmacology
3.1. Biochemical assay
The biochemical assay of the newly synthesized compounds
3.3.1. Hollow fiber assay for preliminary in vivo testing
as CDK inhibitors was determined and expressed as IC50
(Table 1).
(
mM)
The three compounds (2c, 4c and 8c) were tested against
a minimum of 12 human cancer cell lines. This represents four
Scheme 2. Reagents and conditions (i) PPA, heat at 100 ꢁC.

2778
D.A. Ibrahim / European Journal of Medicinal Chemistry 44 (2009) 2776–2781
Scheme 3. Reagents and conditions (i) PPA, heat at 100 ꢁC.
Scheme 4. Reagents and conditions (i) CS2, pyridine, reflux; (ii) I2, Na2CO3, stirring; (iii) R0X, alcohol, Na2CO3, stirring; (iv) H2NNH2$H2O, ethyl alcohol, reflux.
experiments since each experiment contains 3-cell lines. The data
are reported as percentage T/C for each of the 2 compound doses
against each of the cell lines with separate values calculated for the
intraperitoneal and subcutaneous samples. Compounds were
found to be active if their combined IP þ SC score > 20, and SC
score > 8 or a net cell kill of one or more cell lines is referred for
xenograft testing. The data of Hollow Fiber Assay for the selected
compound (4c, NSC 723448) are shown in Table 4.
4. Conclusion
Table 1
From the present study, it can be concluded that the newly
synthesized compounds were designed to act as tyrosine kinases
but they were found to be inactive in terms of tyrosine kinase
inactivation. Their mode of action could be interpreted more
likely as a relatively broad specificity for the ATP-binding domain
of other kinases. Also, the anti-tumor activity of [1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazoles appears to be related to some
structural requirements and to the presence of particular
substituents, as matter of fact 4-chlorophenoxymethylene
moiety plays an important role for anti-tumor activity. These
compounds were not active as PTKs so we tested them as CDKs
IC50 Values for the newly synthesized compounds as CDK inhibitors.
Compound
IC50 (mM)
CDK1/B
CDK2/A
CDK4/D
2a
2b
2c
4a
4b
4c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
9d
9e
9f
10a
10b
10c
>120
>120
6.5
0.57
>28
0.33
0.25
14
0.18
24
24
37
6.8
23
0.40
23
15
0.82
11
16
24
>28
24
>28
11
>28
15
>120
14
2.1
12
>48
3
12
>120
0.91
>48
14
29
19
25
0.9
6.8
5.3
10
>48
>48
23
37
0.4
11
25
8.0
14
>120
>48
18
>120
37
>120
>48
>120
Table 2
Results of primary assay of anti-tumor activity of the newly synthesized compounds
on 3-cell lines.a
Compound
Growth percentage at 10ꢀ5 M concentration
Cell line (cancer)
11
>120
>48
>120
25
>48
>120
22
MCF7 (Breast)
NCI-H460 (Lung)
SF-268 (CNS)
2a
2c
4a
4c
7c
8c
119
128
130
null
null
147
null
null
null
null
null
null
null
null
null
null
null
null
>120
28
a
We reported the active compounds only.

D.A. Ibrahim / European Journal of Medicinal Chemistry 44 (2009) 2776–2781
2779
Table 3
based on cell growth characteristics) were added by pipet (100 ml)
into 96-well microtiter plates. Inoculates were allowed a pre-
incubation period of 24 h at 37 ꢁC for stabilization. Dilution at twice
Inhibition of cancer cell lines by compounds 2a, 2c, 4a, 4c, 7c and 8c.
a
Cell line
Cytotoxicity log GI50 (M)
the intended test concentration was added at time zero in 100-ml
2a
2c
4a
4c
7c
8c
aliquots to the microtiter plate wells. Usually, test compounds were
evaluated at five 10-fold dilutions. In a routine testing, the highest
well concentration is 10E-4 M, but for the standard agents the
highest well concentration used depended on the agent. Incubation
lasted for 48 h in 5% CO2 atmosphere and 100% humidity. The cells
were assayed by using the sulforhodamine B assay [11]. A plate
reader was used to read the optical densities [12,13].
Leukemia
CCRF-CEM
RPMI-8226
SR
Nb
N
ꢀ5.0
N
NT
N
NTc
N
ꢀ4.31
ꢀ4.01
N
ꢀ4.02
ꢀ4.12
ꢀ4.35
ꢀ4.85
ꢀ4.11
ꢀ4.04
N
Non-small cell lung
HOP-62
HOP-92
NCI-H226
NCI-H322M
ꢀ4.06
ꢀ4.69
ꢀ4.01
ꢀ5.14
N
N
N
N
N
ꢀ4.18
ꢀ4.74
ꢀ4.06
NT
NT
N
ꢀ4.01
ꢀ4.1
ꢀ4.05
N
ꢀ4.11
NT
N
ꢀ4.06
ꢀ4.95
5.1.2. Hollow fiber assay for preliminary in vivo testing
Colon
In this assay the human tumor cells are cultivated in poly-
vinylidene fluoride (PVDF) hollow fibers, and a sample of each cell
line is implanted into each of two physiologic compartments
(intraperitoneal and subcutaneous) in mice. Each test mouse
receives six fibers (3 intraperitoneally and 3 subcutaneously) rep-
resenting 3 distinct cancer cell lines. Three mice are treated with
potential anti-tumor compounds at each of two test doses by the
intraperitoneal route using a QD ꢂ 4 treatment schedule. Vehicle
controls consist of six mice receiving the compound diluents only.
The fiber cultures are collected on the day following the last day of
treatment. To assess anticancer effects, viable cell mass is deter-
mined for each of the cell lines using a formazan dye (MTT)
conversion assay. From this, the percentage T/C can be calculated
using the average optical density of the compound treated samples
divided by the average optical density of the vehicle controls. In
addition, the net increase in cell mass can be determined for each
sample as a sample of fiber cultures is assessed for viable cell mass
on the day of implantation into mice. Thus, the cytostatic and
cytocidal capacities of the test compound can be assessed [13].
HCC-2998
HCT-116
HCT-15
KM12
N
NT
N
ꢀ4.0
N
N
N
NT
N
ꢀ4.79
N
ꢀ4.46
ꢀ4.08
ꢀ4.0
NT
N
NT
N
ꢀ6.13
ꢀ4.06
ꢀ4.0
ꢀ4.01
ꢀ5.13
N
CNS
SF-268
SF-539
SNB-75
N
N
ꢀ4.67
ꢀ4.33
ꢀ4.46
NT
N
N
N
N
N
N
N
N
N
N
ꢀ4.4
N
Melanoma
LOXIMVI
UACC-62
N
N
N
N
N
N
ꢀ5.44
ꢀ4.3
N
N
NT
ꢀ4.35
Ovarian
OVCAR-3
OVCAR-4
N
N
N
N
N
N
N
NT
ꢀ4.09
ꢀ4.45
ꢀ4.33
N
Renal
A498
ACHN
CAKI-1
TK-10
N
N
N
NT
N
N
N
ꢀ7.27
ꢀ4.45
N
ꢀ4.34
N
N
N
N
ꢀ4.54
N
ꢀ4.27
ꢀ4.31
ꢀ4.07
ꢀ4.2
ꢀ4.11
N
ꢀ4.14
Breast
MCF7
N
ꢀ4.39
ꢀ4.06
NT
ꢀ4.36
N
N
N
ꢀ4.09
ꢀ4.0
ꢀ4.5
N
ꢀ5.75
N
ꢀ4.35
N
5.2. Chemistry
HS578T
BT-549
ꢀ4.05
ꢀ4.0
ꢀ4.16
ꢀ4.06
ꢀ4.1
ꢀ4.05
ꢀ4.32
Mean valued
ꢀ4.36
All melting points are uncorrected and determined by the open
capillary method. IR spectra were recorded using (KBr) disc. 1H
NMR spectra were recorded using DMSO-d6 as a solvent and TMS as
a
Data obtained from NCI’s in vitro disease-oriented tumor cells screen.
In active.
Not tested.
Mean value over all active cell lines tested.
b
c
an internal reference. Chemical shifts are expressed in
d units
d
(ppm). Mass spectra were recorded with a mass spectrometer MS9
(AEI) 70 eV. Elemental combustion analyses were within ꢃ0.4% of
the theoretical values. All the results were in an acceptable range.
(ATP competitive inhibitors). We found that a large hydrophobic
group should be found at position 4 to give a good biological
activity. Compounds with thiazole-ring were found to be active
because we think that the thiazole-ring came at the sugar pocket
of CDKs protein. The pharmacological mechanism of action for
these intriguing compounds is under investigation.
5.2.1. General procedure for the synthesis of 4-(3-substituted
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl) anilines 2
A mixture of 4-amino-5-substituted [1,2,4]triazole-3-thiol
(1) (6.81 mmol) and polyphosphoric acid (20 ml) was heated to
50–60 ꢁC with stirring, then 4-aminobenzoic acid (6.9 mmol)
added portionwise. The mixture was heated at 180–200 ꢁC for
3 h with stirring then poured onto ice. After neutralizing with
concentrated aqueous ammonia solution, the crude product
was collected by filtration, and recrystallized from appropriate
5. Experimental
5.1. Pharmacology
solvent
to
give
4-(3-substituted
[1,2,4]triazolo[3,4-
5.1.1. Anticancer assay for preliminary in vitro testing
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
b][1,3,4]thiadiazol-6-yl) anilines derivatives (2).
5.2.1.1. 4-(3-Methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl) aniline
2a. Compound 2a was obtained as green crystals (80% yield);
recrystallized from ethyl alcohol; M.P. 204–206 ꢁC; IR, 3320–3400,
2 mM
L-glutamine. For a typical screening experiment, cell
suspensions that were diluted according to the particular cell type
and the expected target cell density (5000–40,000 cells per well
2995, 1600 cmꢀ1
;
13C NMR (DMSO-d6)
d
¼ 15, 120, 130, 140, 146,
150, 153, 159; 15NNMR (DMSO-d6)
d
¼ ꢀ60.2, ꢀ68.1, ꢀ80.4, ꢀ120.5,
Table 4
ꢀ311.6; M.S, m/z 232 (Mþ, 10%), 139 (21.9%), 113 (100%), 81 (40%),
and 66 (35.2%). Anal. (C10H9N5S$0.25H2O) C, H, N.
The IP, SC and cell kill data for compound 4c.
Exp id
HF: 876
HF: 877
HF: 878
HF: 879
IP score
11
15
26
N
SC score
Total score
Cell kill
5.2.1.2. 4-(3-Benzyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl) aniline
2b. Compound (2b) was obtained as yellowish white crystals
(85% yield); recrystallized from ethyl alcohol; M.P. 180–183 ꢁC;
IP: intraperitoneal; SC: subcutaneous.

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D.A. Ibrahim / European Journal of Medicinal Chemistry 44 (2009) 2776–2781
1H NMR (DMSO-d6),
d
¼ 7.9–7 (m, 9H), 6.3 (s, 2H), 4 (s, 2H); M.
5.2.3.2. 3-[(3-Benzyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl]
-1H-pyrazolo[3,4-d]-pyramidine-4,6-dithione 6b. Compound 6b was
obtained as yellow crystals (77% yield); recrystallized from DMF; M.P.
230–232 ꢁC;IR, 3400, 3120, 2995,1600,1200 cmꢀ1; M.S, m/z413 (Mþ,
2.77%), 321 (17.7%), 262 (20.05%), 203 (40.61%), 138 (10.74%), 124
(32.0%), 98(100%),and66(5.45%). Anal. (C16H12N8S3$0.25H2O)C,H, N.
S, m/z 307 (Mþ, 100%), 215 (14%), 189 (35%), 157 (20%), and 66
(15.11%). Anal. (C16H13N5S) C, H, N.
5.2.1.3. 4-{3-[(4-Chlorophenoxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]thia-
diazol-6-yl} aniline 2c. Compound (2c) was obtained as buff
crystals (76% yield); recrystallized from ethyl alcohol; M.P.
166–168 ꢁC; 1H NMR (DMSO-d6),
d
¼ 7.5–6.9 (m, 8H), 5.3 (s, 2H),
5.2.3.3. 3-({3-[(4-chlorophenoxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]
thiadiazol-6-yl)methyl]-1H-pyrazolo[3,4-d] pyramidine-4,6-dithione
6c. Compound 6b was obtained as yellow crystals (75% yield);
recrystallized from DMF; M.P. 238–240 ꢁC; 1H NMR (DMSO-d6),
and 4.8 (s, 2H). Anal. (C16H12ClN5OS$0.5H2O) C, H, N.
5.2.2. General procedure for the synthesis of 2-amino-5-[(3-
substituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl]-1,3-
thiazol-4-ol 4
d
¼ 13.4 (s, 1H), 11.9 (s, 1H), 10.5 (s, 1H), 7.5–7.0 (m, 4H), 5.4 (s, 2H),
and 3.8 (s, 2H). Anal. (C16H11ClN8OS3$0.5H2O) C, H, N.
A mixture of (2-amino[1,3]thiazole-4-one-5-yl)acetic acid (3)
(6.81 mmol) and polyphosphoric acid (20 ml) was heated to 50 ꢁC
with stirring, then 4-amino-5-substituted [1,2,4]triazole-3-thiol (1)
(6.81 mmol) was added portionwise. The reaction mixture was
heated at 100 ꢁC for 4 h with stirring, then cooled, and poured into
ice-cold 10% aqueous sodium carbonate. The solid product was
collected, washed with water, and dried to give compound (4) that
was purified by recrystallization from the appropriate solvent.
5.2.4. General procedure for the synthesis of 3-substituted
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thioles 7
A solution of 4-amino-5-substituted [1,2,4]triazole-3-thiol (1)
(0.01 mol) in pyridine (20 ml) was treated with carbon disulfide
(2 ml) and few drops of triethylamine. The reaction mixture was
refluxed at 100 ꢁC with stirring for 6 h, then cooled, poured into ice-
cold water and makes the reaction mixture slightly acidic by adding
HCl. The solid product was collected by vacuum filtration, washed
well with water and recrystallized from the appropriate solvent to
give the desired compound (7).
5.2.2.1. 2-Amino-5-[(3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl)methyl]-1,3-thiazol-4-ol 4a. Compound 4a was obtained as
brown crystals (65% yield); recrystallized from DMF/ethyl alcohol;
M.P. 260–262 ꢁC; IR, 3390–3350, 2980, 1680, 1620 cmꢀ1
,
1H NMR
5.2.4.1. 3-Methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol7a. Compound
7a was obtained as shiny white crystals (82% yield), recrystallized from ethyl
alcohol; M.P. 180–182 ꢁC; M.S, m/z 172 (100%), 139 (20%), 113 (44.55%), 81
(14.82%), and 66 (30.04%). Anal. (C4H4N4S2) C, H, N.
(DMSO-d6),
(C8H8N6OS2$0.25H2O) C, H, N.
d
¼ 6.1 (s, 1H), 4.5 (s, 2H), 3.4 (d, 2H), 1.3 (s, 3H). Anal.
5.2.2.2. 2-Amino-5-[(3-benzyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl)methyl]-1,3-thiazol-4-ol 4b. Compound 4b was obtained as dark
brown crystals (71% yield); recrystallized from ethyl alcohol; M.P.
5.2.4.2. 3-Benzyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol 7b. Compound
7b was obtained as shiny buff (88% yield); recrystallized from ethyl alcohol;
210–212 ꢁC; 13C NMR (DMSO-d6)
d
¼ 30, 33, 100, 126, 128, 130, 134,
M.P.192–194 ꢁC; 1H NMR (DMSO-d6),
d
¼ 7.3–7.1 (m, 5H), 4.9 (s,1H), and 4.3
143, 156, 165, 168, 180; 15N NMR (DMSO-d6)
d
¼ ꢀ55.2, ꢀ60.1, ꢀ78.4,
(s, 2H). Anal. (C10H8N4S2) C, H, N.
ꢀ118.5, ꢀ180.1, ꢀ299.6; M.S, m/z 344 (11%), 253 (41%), 237 (3.15%),
138 (15.5%),124 (20.5%), and 98 (35.71%). Anal. (C14H12N6OS2) C, H, N.
5.2.4.3. 3-[(4-Chlorophenoxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]thia
diazole-6-thiol 7c. Compound 7c was obtained as white crystals
(97% yield); recrystallized from ethyl alcohol; M.P. 166–168 ꢁC; IR,
5.2.2.3. 2-Amino-5-({3-[(4-chlorophenoxy)methyl][1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-6-yl}-methyl)-1,3-thiazol-4-ol 4c. Compound 4c was
obtained as buff powders (59% yield); recrystallized from ethyl
3190, 2990, 1610, 1200 and 1100 cmꢀ1
;
1H NMR (DMSO-d6),
d
¼ 7.4–6.9 (m, 4H), 5.4 (s, 2H), and 3.5 (s, 1H). Anal.
alcohol; M.P. 200–202 ꢁC; 1H NMR (DMSO-d6),
(m, 4H), 5.4 (s, 2H), 4.8 (s, 2H) and 3.4 (s, 2H); M.S, m/z 394.5 (5.3%),
253 (44.5%), 237 (2.5%), 138 (20.1%), and 98 (100%). Anal.
(C14H11ClN6O2S2$0.5H2O) C, H, N.
d
¼ 5.9 (s, 1H), 7.3–6.9
(C10H7ClN4OS2$0.25H2O) C, H, N.
5.2.5. General procedure for the synthesis of 3-substituted-6-
[(3-substituted [1,2,4]triazolo-[3,4-b][1,3,4]thiadiazol-6-yl)
dithio][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives 8
A suspension of compound (7) (0.01 mol) in 10% Na2CO3
solution (20 ml) was treated with iodine (0.01 mol) portionwise.
The reaction mixture was stirred at room temperature for 5 h, and
then the solid so formed was collected by vacuum filtration,
washed well with water and dried at 50 ꢁC for 6 h. The desired
compound (8) was obtained after recrystallization from ethyl
alcohol.
5.2.3. General procedure for the synthesis of 3-[(3-substituted
[1,2,4]triazolo[3,4-b]-[1,3,4]thiadiazol-6-yl)methyl]-1H-
pyrazolo[3,4-d]pyrimidine-4,6-dithione 6
A
mixture of (1H-pyrazolo[3,4-d]pyrimidine-2,4-dithione-5-
yl)acetonitrile (5) (6.81 mmol) and polyphosphoric acid (20 ml) was
heated at 100 ꢁC with stirring, then 4-amino-5-substituted
[1,2,4]triazole-3-thiol (1) (6.81 mmol)was added. The reaction
mixturewasmaintainedatthistemperaturefor2 hwithstirring, then
cooledandpouredintoice-cold10%aqueoussodiumbicarbonate. The
solid product was collected by vacuum filtration, washed with water
and recrystallized from appropriate solvent to give compound (6).
5.2.5.1. 3-Methyl-6-[(3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
6-yl)dithio][1,2,4] triazolo[3,4-b][1,3,4]thiadiazole 8a. Compound 8a
was obtained as shiny white crystals (65% yield); M.P. 160–162 ꢁC;
13C NMR (CDCl3)
d
¼ 15, 143, 146, 153; m/z 342 (2.09%), 171 (100%),
113 (36.16%), 81 (57.15%), and 66 (10.70%); 15N NMR (CDCl3)
¼ ꢀ55.2, ꢀ62.1, ꢀ70.4, ꢀ118.5. Anal. (C8H6N8S4$0.5H2O) C, H, N.
5.2.3.1. 3-[(3-Methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)methyl]
-1H-pyrazolo[3,4-d]-pyramidine-4,6-dithione 6a. Compound 6a was
obtained as yellow crystals (89% yield); recrystallized from DMF; M.P.
d
240–242 ꢁC; 1H NMR (DMSO-d6),
d
¼ 13.4 (s, 1H), 11.4 (s, 1H), 10.9 (s,
1H), 3.5 (s, 2H), and 1.4 (s, 3H); 13C NMR (DMSO-d6)
¼ 15, 25, 110,
143, 148, 152, 155, 158, 163, 182; 15N NMR (DMSO-d6)
¼ ꢀ58.2,
5.2.5.2. 3-Benzyl-6-[(3-benzyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl)dithio][1,2,4] triazolo[3,4-b][1,3,4]thiadiazole 8b. Compound 8b
was obtainedas shiny pale brown crystals (60% yield); M.P.170–172 ꢁC;
M.S, m/z 494 (1.09%), 274 (100%), 215 (10.20%), 189 (33.30%), 98
(15.08%), and 66 (41.15%). Anal. (C20H14N8S4$0.25H2O) C, H, N.
d
d
ꢀ65.1, ꢀ80.4, ꢀ120.5, ꢀ130.2, ꢀ180.1, ꢀ260.6, ꢀ320.6. Anal.
(C10H8N8S3$1H2O) C, H, N.

D.A. Ibrahim / European Journal of Medicinal Chemistry 44 (2009) 2776–2781
2781
5.2.5.3. 3-[(4-Chlorophenoxy)methyl]-6-({3-[(4-chlorophenoxy)methyl]
5.2.7. General procedure for the synthesis of 6-hydrazino-3-
[1,2,4]triazolo-[3,4-b][1,3,4]thiadiazol-6-yl}dithio)[1,2,4]triazolo[3,4-b][1,3,4]
substituted [1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 10
thiadiazole 8c. Compound 8c was obtained as metallic purple crystals
To a solution of 6-alkylthio-3-substituted [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole (9) (0.01 mol) in ethyl alcohol (25 ml) was
added hydrazine hydrate (0.015 mol). The reaction mixture was
refluxed on water-bath for 3 h, and then the excess of alcohol was
removed under reduced pressure. The solid residue was triturated
with water and 2 drops of acetic acid, the solid precipitate was
collected by filtration, washed well with water and recrystallized
from alcohol to give compound (10).
(58% yield); M.P.155–157 ꢁC; 1H NMR (DMSO-d6),
d
¼ 7.7–7.1 (m, 8H),
and 5.2 (s, 4H). Anal. (C20H12Cl2N8O2S4$0.5H2O) C, H, N.
5.2.6. General procedure for the synthesis of 6-alkylthio-3-
substituted [1,2,4]triazolo[3,4-b]-[1,3,4]thiadiazole derivatives 9
To
a stirred solution of 3-substituted [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole-6-thiole (7) (0.01 mol) in ethyl alcohol (10 ml)
at room temperature was added 1 N NaOH (15 ml), then the reac-
tion mixture was treated with appropriate alkyl halide (0.01 mol)
drop-wise with stirring. The reaction mixture was stirred for 4 h,
and then the solid product was collected by filtration, washed with
water and diethyl ether to give the titled compound (9).
5.2.7.1. 6-Hydrazino-3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
10a. Compound 10a was obtained as white crystals (50% yield);
M.P.125–127 ꢁC; IR, 3400, 3300–3340, 2995 cmꢀ1; 1H NMR (DMSO-
d6),
d
¼ 4.7–4.6 (t, 1H), 4.6–4.5 (d, 2H) and 1.7 (s, 3H); M.S, m/z 170
(100%), 139 (30.05%), 113 (20.70%), 81(10.05%), and 66 (14.40%).
5.2.6.1. 6-(Ethylthio)-3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
9a. Compound 9a was obtained as yellow crystals (90% yield);
recrystallized from ethyl alcohol; M.P. 110–112 ꢁC; M.S, m/z 200
(10%), 139 (7.77%), 113 (30.60%), 81 (100%), and 66 (59.78%). Anal.
(C6H8N4S2) C, H, N.
Anal. (C4H6N6S$0.25H2O) C, H, N.
5.2.7.2. 3-Benzyl-6-hydrazino[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
10b. Compound 10b was obtained as white crystals (65% yield);
M.P.130–132 ꢁC; 1H NMR (DMSO-d6),
d
¼ 7.2–6.9 (m, 5H), 4.8–4.7 (t,
1H), 4.5–4.4 (d, 2H), and 4.2 (s, 2H); 13C NMR (DMSO-d6)
d
¼ 30,
5.2.6.2. 6-(Benzylthio)-3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
9b. Compound 9b was obtained as yellow crystals (92% yield);
recrystallized from ethyl alcohol; M.P. 98–100 ꢁC; 13C NMR (DMSO-
126, 128, 130, 136, 146, 151, 158; Anal. (C10H10N6S$1H2O) C, H, N.
5.2.7.3. 3-[(4-Chlorophenoxy)methyl]-6-hydrazino[1,2,4]triazolo[3,4-b]
d6)
d
¼ 14, 35, 125, 127, 129, 135, 145, 149, 156; M.S, m/z 262 (5%),
[1,3,4]thiadiazole 10c. Compound 10c was obtained as white crystals
171(20.1%), 139 (42.2%), 113 (30%), 81 (100%) and 55 (66.1%); 15N
(45% yield); M.P. 118–120 ꢁC; 1H NMR (DMSO-d6),
d
¼ 7.5–7 (m, 4H),
NMR (DMSO-d6)
C, H, N.
d
¼ ꢀ58.2, ꢀ60.1, ꢀ74.4, ꢀ120.5. Anal. (C11H10N4S2)
5.4 (s, 2H), 4.9–4.7 (t, 1H), and 4.7 (d, 2H); 15N NMR (DMSO-d6)
d
¼ ꢀ54.2, ꢀ62.1, ꢀ72.4, ꢀ122.5, ꢀ220.4, ꢀ303.2; M.S, m/z 296.5
(20%), 169 (3.04%), 155 (100%), 124 (19.91%), 98 (40.41), and (22.11%).
5.2.6.3. 3-Benzyl-6-(ethylthio)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
9c. Compound 9c was obtained as yellow crystals (85% yield);
recrystallized from ethyl alcohol; M.P. 115–118 ꢁC; 1H NMR (DMSO-
Anal. (C10H9ClN6OS$0.75H2O) C, H, N.
Acknowledgements
d6),
d
¼ 7.3–6.9 (m, 5H), 4.2 (s, 2H), 2.1–2 (q, 2H) and 1–1.3 (t, 3H);
Anal. (C12H12N4S2) C, H, N.
The author is grateful to Dr. V. L. Narayanan (NCI) NIH, Bethesda,
USA for anticancer screening studies of the newly synthesized
compounds reported herein.
5.2.6.4. 3-Benzyl-6-(benzylthio)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
9d. Compound 9d was obtained as yellow crystals (88% yield);
recrystallized from ethyl alcohol; M.P. 108–110 ꢁC;, 1H NMR (DMSO-
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(C17H14N4S2$0.25H2O) C, H, N.
5.2.6.5. 3-[(4-Chlorophenoxy)methyl]-6-(ethylthio)[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole 9e. Compound 9e was obtained as yellow crystals
(80% yield); recrystallized from ethyl alcohol; M.P. 120–122 ꢁC; 1H
NMR (DMSO-d6),
1.1–0.9 (t, 3H). Anal. (C12H11ClN4OS2$0.75H2O) C, H, N.
d
¼ 7.4–7.0 (m, 4H), 5.3 (s, 2H), 2.1–1.9 (q, 2H), and
5.2.6.6. 6-(Benzylthio)-3-[(4-Chlorophenoxy)methyl][1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole 9f. Compound 9f was obtained as yellow crystals (75%
yield), recrystallized from ethyl alcohol; M.P. 105–107 ꢁC; 1H NMR
(DMSO-d6),
(C17H13ClN4OS2) C, H, N.
d
¼ 7.6–7.2 (m, 9H), 5.1 (s, 2H) and 4.4 (s, 2H); Anal.