Synthesis of some new 1,2,4-triazolo[3,4-b]-thiadiazole derivatives as possible anticancer agents

Article abstract of DOI:10.1080/10426500490464186

3-Substituted-4-amino-5-mercapto-1,2,4-triazoles are versatile synthons for constructing various biologically active heterocycles. Their cyclization with carboxylic acids gives fused five-membered derivatives, whereas with α-bromoketones gives a six-membered heterocycle. In this article we report the synthesis of a series of 1,2,4-triazolo[3,4-b]thiadiazoles 5 starting from 4-amino-3-substituted-5-mercapto-1,2,4-triazoles 3 and fluorobenzoic acids 4 using phosphorous oxychloride as cyclizing agent. Fourteen of the newly synthesized compounds were screened for anticancer properties. Four among them showed in vitro anticancer activity.

Full text of DOI:10.1080/10426500490464186

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SYNTHESIS OF SOME NEW 1,2,4-TRIAZOLO[3,4-b]-
THIADIAZOLE DERIVATIVES AS POSSIBLE ANTICANCER
AGENTS
a
a
a
a
K. Subrahmanya Bhat , D. Jagadeesh Prasad , Boja Poojary & B. Shivarama Holla
a
Department of Chemistry, Mangalore University, Mangalagangotri, India
Version of record first published: 16 Aug 2010.
To cite this article: K. Subrahmanya Bhat, D. Jagadeesh Prasad, Boja Poojary & B. Shivarama Holla (2004): SYNTHESIS OF
SOME NEW 1,2,4-TRIAZOLO[3,4-b]-THIADIAZOLE DERIVATIVES AS POSSIBLE ANTICANCER AGENTS, Phosphorus, Sulfur, and
Silicon and the Related Elements, 179:8, 1595-1603
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Phosphorus, Sulfur, and Silicon, 179:1595–1603, 2004
Copyright ꢀC Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500490464186
SYNTHESIS OF SOME NEW
,2,4-TRIAZOLO[3,4-b]-THIADIAZOLE DERIVATIVES
AS POSSIBLE ANTICANCER AGENTS
1
K. Subrahmanya Bhat, D. Jagadeesh Prasad, Boja Poojary,
and B. Shivarama Holla
Department of Chemistry, Mangalore University,
Mangalagangotri, India
(
Received July 3, 2003; accepted December 5, 2003)
3
-Substituted-4-amino-5-mercapto-1,2,4-triazoles are versatile syn-
thons for constructing various biologically active heterocycles. Their
cyclization with carboxylic acids gives fused five-membered deriva-
tives, whereas with α-bromoketones gives a six-membered heterocycle.
In this article we report the synthesis of a series of 1,2,4-triazolo[3,4-
b]thiadiazoles 5 starting from 4-amino-3-substituted-5-mercapto-1,2,4-
triazoles 3 and fluorobenzoic acids 4 using phosphorous oxychloride
as cyclizing agent. Fourteen of the newly synthesized compounds were
screened for anticancer properties. Four among them showed in vitro
anticancer activity.
Keywords: Anticancer activity; phosphorus oxychloride; triazolothia-
diazoles
INTRODUCTION
Various 1,2,4-triazole derivatives are found to be associated with di-
verse pharmacological activities.1
−4
The 1,2,4-triazole nucleus has
been incorporated into a wide variety of therapeutically interest-
ing drug candidates, including H1/H2 histamine receptor blockers,
cholinesterase-active agents, CNS stimulants, antianxiety agents, and
5
sedatives. It was also found that the thiadiazole nucleus, which in-
corporates a toxophoric N C S linkage, exhibits a large number of
The authors are thankful to RSIC, Punjab University, Chandigarh, and RSIC, IIT,
Chennai for providing elemental analysis/NMR/mass spectra of the compounds reported
herein. KSB is thankful to CSIR, New Delhi for the award of Junior Research Fellowship.
We are also thankful to P. M. Akberali of Strides Laboratories, Mangalore for a gift sample
of 2,4-dichloro-5-fluoroacetophenone.
Address correspondence to B. Shivarama Holla, Department of Chemistry, Mangalore
University, Mangalagangotri 574199, India. E-mail: hollabs@rediffmail.com
1
595

1
596
K. S. Bhat et al.
biological activities.6,7 A number of 1,3,4-thiadiazole derivatives posses
8
antibacterial properties comparable with those of sulfonamide drugs.
Subsequently, thiadiazole derivatives have found applications as anti-
tumor agents and pesticides.9
−12
The cyclocondensation of 4-amino-3-
substituted-5-mercapto-1,2,4-triazole 3 with carboxylic acids 4 using
phosphorous oxychloride gives triazolothiadiazoles. Recently, fluori-
nated heterocycles have attracted attention due to the ability of fluorine
to act as polar hydrogen or hydroxyl mimic. Therefore, substitution of
hydrogen by fluorine has been a strategy in designing molecules for
biological activity studies.1
3,14
Prompted by the varied biological activities of 1,2,4-triazoles and
1,3,4-thiadiazoles, and in continuation of our work on the synthesis
of N-bridged heterocycles derived from 1,2,4-triazoles, a series of 1,2,4-
triazolo[3,4-b]thiadiazoles (Scheme 1) were synthesized. The newly syn-
1
thesized compounds were characterized by analytical, infrared (IR), H
nuclear magnetic resonance (NMR), and mass spectral studies. In ad-
dition, fourteen of them were screened for their anticancer activities.
SCHEME 1 Synthesis of triazolothiadiazoles (5).
RESULTS AND DISCUSSION
The required 4-amino-5-mercapto-1,2,4-triazoles 3 were prepared
by the direct fusion of aliphatic/aromatic carboxylic acids 1, with

Synthesis of Thiadiazole Derivatives
1597
SCHEME 2 Synthesis of 4-amino-3-substituted-5-mercapto-1,2,4-triazoles
3).
(
thiocarbohydrazide 2 (Scheme 2). Then, these triazoles 3 were cyclized
with 4-fluoro/4-fluoro-3-phenoxy/2,4-dichloro-5-fluoro benzoic acids 4,
respectively, using phosphorus oxychloride as the cyclizing agent.
1
5
The new workup procedure reported by Zhang et al. was used.
The cyclization of triazoles with various carboxylic acids 4 affo-
rded triazolothiadiazoles 5. The compounds 5 were confirmed on
the basis of elemental analysis, IR, NMR, and mass spectral stud-
ies. The characterization data of these compounds 5 are given in
Table I.
All of the newly synthesized compounds were analyzed satisfactorily
for their nitrogen content. The IR spectrum of 5c showed an absorp-
−1
tion band at 1580 cm , indicating the presence of C N in the ring. In
the IR spectra of the cyclized products, the absorption bands at 3200–
−1
−1
3
400 cm , and 2300–2400 cm attributed to the NH and SH functional
groups were absent. This confirmed the involvement of the NH2 and SH
groups of the parent triazole unit in the cyclization. A distinct peak at
−1
1
000 cm was assigned to C F stretch. In the IR spectra of the cyclized
products, the absorption band corresponding to carbonyl stretching

1
598

Synthesis of Thiadiazole Derivatives
1599
frequency around 1700–1750 cm 1 was absent, which again gave a con-
−
clusive evidence for the cyclization.
The formation of cyclized products 5 were further supported by
1
recording the H NMR spectra of a few selected compounds. Thus, in
1
the H NMR spectra of triazolothiadiazoles, the characteristic down-
field signal at δ 13.4 attributed to the N C SH moiety was absent.
A sharp signal at δ 5.4 attributable to the N NH2 group in the par-
1
ent triazoles was also absent in the cyclized product. In the H NMR
spectrum of 5b, a singlet that appeared at δ 2.77 was attributed to the
methyl protons. The remaining 8 aromatic protons resonated as mul-
1
tiplets in the range δ 7–7.64. In the H NMR spectrum of 5c, a singlet
that appeared at δ 2.82 was attributed to the methyl protons. The re-
maining aromatic protons appeared as doublet of doublets due to ortho
and meta coupling with fluorine in the range δ 7.6–7.96 integrating for
1
two protons. In the H NMR spectrum of 5k, a singlet was observed
at δ 5.6 due to the presence of OCH2 protons. The remaining protons
resonated as multiplets in the aromatic region δ 6.92–7.61 integrating
for 12 protons.
In the mass spectrum of compound 5b, an intense molecular ion peak
was observed at m/z 326. An intense peak observed at m/z 231 may have
been due to the formation of 4-fluoro-3-(phenoxy)-thiobenzoyl cation.
The base peak observed at m/z 69 may have been due to the formation of
stable fluorocyclobutadienyl cation. In the mass spectrum of compound
5
d, molecular ion peak was observed at m/z 326. The fragmentation
pattern is similar to that of compound 5b, giving m/z values at 231,
39, and 69. The spectral data for some of the compounds are given
1
under Table I.
BIOLOGICAL STUDIES
Anticancer Activity
Fourteen of the newly synthesized compounds were evaluated for their
1
6−18
antitumor activity following the NCI screening program.
They
were first evaluated in a 3-cell line, one-dose preliminary anticancer
assay. The results of this assay are reported in Table II. Compounds
5
k, 5m, 5p, and 5u were considered active.
When these compounds were passed on for evaluation against a full
panel of 60 human cell lines, they showed variable antitumor activi-
ties against most of the tested subpanel tumor cell lines at the GI50
level. The subpanel tumor cell lines’ median growth inhibitory concen-
tration (the average sensitivity of each subpanel towards each of the
test compounds) and the full panel mean graph midpoint (MG-MID;

1
600
K. S. Bhat et al.
TABLE II The 3-Cell Line, One-Dose Primary Anticancer
Assay Results
Growth percentage
(
LUNG)
(Breast)
MCF-7
(CNS)
SF-268
Compd.
NCI-H 460
Activity
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
j
k
m
o
p
q
t
103
97
104
52
46
67
6
3
76
16
86
95
124
37
53
63
24
19
97
45
97
100
98
54
62
60
26
50
106
39
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Active
Inactive
Active
117
107
107
11
100
120
121
17
99
99
84
88
Inactive
Inactive
Inactive
Active
v
u
Samples are tested at 1.00 × 10− Molar concentrations.
04
Growth percentages less than 32 are considered as active.
the average of all cell lines towards each of the test compounds) are
reported in Table III. Compound 5k is active against lung cancer
HOP-92 with a GI50 value of 15.2 µg/ml. Compound 5m is highly active
against leukemia MOLT-4, ovarian cancer OVCAR-3 and prostate can-
cer PC-3 with a GI50 value of 3.51, 8.76, and 0.0735 µg/ml, respectively.
Compound 5p is highly active against leukemia MOLT-4, lung cancer
HOP-92, CNS cancer SF-295, ovarian cancer OVCAR-3 and prostate
cancer PC-3 with a GI50 value of 7.08, 0.25, 6.2, 0.7, and 8.94 µg/ml,
respectively. Compound 5u is active against leukemia RPMT-8226, lung
cancer NCI-H322M, and colon cancer HCC-2998, with a GI50 value of
6.99, 6.22, and 0.58 µg/ml, respectively.
The ratio obtained by dividing the compounds full panel MG-MID
(
µM) by its individual subpanel MG-MID (µM) is considered as a mea-
sure of compound selectivity. Ratios between 3 and 6 refer to moderate
selectivity, ratios >6 indicate high selectivity towards corresponding
cell line, and compounds not meeting either of these criteria are consid-
ered nonselective.19 All the active compounds in the present study prove
to be nonselective against any of the 9 tumor subpanels tested. However,
regarding the selectivity of the tested compounds against the highly
affected individual tumor cell lines, compound 5m exhibits high selec-
tivity towards prostate cancer PC-3 at the GI50 level with a selectivity

Synthesis of Thiadiazole Derivatives
1601
TABLE III Subpanel Tumor Cell Lines Median Growth Inhibitory
Concentration (GI50) and Full-Panel Mean Graph Midpoint (MG-MID)
Subpanel tumor
cell line
5k
5m
5p
5u
Leukamia
>100
15.98
18.77
27.95
27.3
15.85
17.68
26.28
18.73
23.30
18.08
16.86
23.67
23.78
15.85
>100
Lung cancer
Colon cancer
CNS cancer
Melanoma
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
MG-MID
30.29
>100
27.92
>100
41.76
>100
30.8
31.20
25.43
32.35
28.61
43.42
25.5
23.31
15.07
13.82
16.85
17.38
>100
43.8
34.11
30.2
36.19
34.67
MG-MID, average sensitivity of each subpanel and of all cell lines towards each
of the test compounds.
ratio 236.46. On the other hand, compound 5p shows selectivity against
lung cancer HOP-92 subpanel tumor cell line at GI50 level with a se-
lectivity ratio of 63.4. The selectivity ratios for a particular cell line to
other compounds are given in Table IV.
The activity of the tested compounds could be correlated to the struc-
tural variations. The compounds 5k, 5m, and 5p which contained 4-
fluoro-3-phenoxyphenyl moiety exhibited antitumor activities. It is also
interesting to note that compound 5u, with anilinomethyl substituent
on triazole ring and 4-fluorophenyl group on thiadiazole ring, is also
active. These compounds raise the possibility of their further derivati-
zation in order to explore scope and limitations of their activities.
TABLE IV The most Affected Cell Lines and the Corresponding GI50 and
Sensitivity Towards Each of the Active Compounds
Compd.
Most affected cell line
GI50
MG-MID
Sensitivity ratio
5
5
k
m
Lung cancer HOP-92
Leukemia MOLT-4
Ovarian cancer OVCAR-3
Prostate cancer PC-3
Leukemia MOLT-4
15.2 µg/ml
3.51 µg/ml
8.76 µg/ml
0.0735 µg/ml
7.08 µg/ml
0.25 µg/ml
6.2 µg/ml
34.67
17.38
2.28
4.95
1.98
236.46
2.24
63.4
2.56
22.6
1.77
4.32
5
p
u
15.85
30.2
Lung cancer HOP-92
CNS cancer SF-295
Ovarian cancer OVCAR-3
Prostate cancer PC-3
Leukemia RPMT-8226
Lung cancer NCI-H322M
Colon cancer HCC-2998
0.7 µg/ml
8.94 µg/ml
6.99 µg/ml
6.22 µg/ml
0.58 µg/ml
5
4.86
52.07

1
602
K. S. Bhat et al.
EXPERIMENTAL
Melting points were determined in open capillary tubes and were un-
corrected. IR spectra were recorded in KBr pellets on a Perkin-Elemer
1
1
57 IR spectrophotometer. H NMR spectra were recorded in DMSO-d6
on EM-390 (300 MHz) NMR spectrometer. Mass spectra were recorded
on MASPEC low resolution instrument operating at 70 eV. The purity
of compounds was checked by thin layer chromatography (TLC) on sil-
ica gel plates using a hexane: ethylacetate (10:1) solvent system, and
iodine was used as a visualizing agent.
4
-Amino-5-mercapto-1,2,4-triazoles (3)
A mixture of carboxylic acid 1 (0.01 mol) and thiocarbohydrazide 2
0.01 mol) contained in a round-bottomed flask was heated on a mantle
(
until the contents melted. The mixture was maintained at this tem-
perature for 15–20 min (Scheme 2). The product obtained on cooling
was treated with sodium bicarbonate solution to dissolve the unreacted
carboxylic acid if any. It was then washed with water and collected by
filtration. The product was recrystallized from a mixture of dioxane
and ethanol to afford the title compounds 3. Their physical data are in
agreement with the literature values.20
6
-Substituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles
(
5a--w)
To a mixture of suitably substituted triazoles 3 (2 mmol) and fluoro/
-fluoro-3-phenoxy/2,4-dichloro-5-fluoro benzoic acid 4 (2 mmol), phos-
4
phorus oxychloride (10 ml) was added and the contents were heated
under reflux for 6 h in a water bath (Scheme 1). Excess of phospho-
rus oxychloride was then distilled off, and the residue was poured onto
crushed ice and stirred well. It was then washed with sodium bicar-
bonate solution (5%) and the resulting solid product was washed with
water and recrystallized from ethanol:dioxane mixture. The physico-
chemical data for the synthesized compounds are given in Table I. The
prepared compounds were evaluated for their anticancer activities us-
ing the National Cancer Institute (NCI) in vitro anticancer screening
1
7−19
assay.
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