Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase isoforms hCA IX and hCA XII

Article abstract of DOI:10.1016/j.bmc.2014.01.055

Three series of novel heterocyclic compounds (3a-3g, 4a-4g and 5a-5g) containing benzenesulfonamide moiety and incorporating a 1,2,4-triazole ring, have been synthesized and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II, compounds of two series (3a-3g and 4a-4g) showed K_i values in the range of 84-868 nM and 5.6-390 nM, respectively whereas compounds of series 5a-5g were found to be poor inhibitors (K_i values exceeding 10,000 nM in some cases). Against hCA IX and XII, all the tested compounds exhibited excellent to moderate inhibitory potential with K_i values in the range of 2.8-431 nM and 1.3-63 nM, respectively. Compounds 3d, 3f and 4f exhibited excellent inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the standard drug acetazolamide (AZA) whereas compound of the series 5a-5g were comparatively less potent but more selective towards hCA IX and XII.

Full text of DOI:10.1016/j.bmc.2014.01.055

Accepted Manuscript
Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tu-
mor associated carbonic anhydrase isoforms hCA IX and hCA XII
Sita Ram, Gulsah Celik, Poonam Khloya, Daniela Vullo, Claudiu T. Supuran,
Pawan K. Sharma
PII:
S0968-0896(14)00092-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.055
BMC 11386
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 December 2013
27 January 2014
27 January 2014
Please cite this article as: Ram, S., Celik, G., Khloya, P., Vullo, D., Supuran, C.T., Sharma, P.K.,
Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase
isoforms hCA IX and hCA XII, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.01.055
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Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of
tumor associated carbonic anhydrase isoforms hCA IX and hCA XII
SitaRama, Gulsah Celikb, Poonam Khloyaa, Daniela Vullob,
Claudiu T. Supuranb,c*, Pawan K. Sharmaa*
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, India
bUniversità degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica,
Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
cUniversità degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and
Nutraceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
*Corresponding authors: PKS – Tel.: +91 9416457355; Fax: +91 1744 238277; e-mail:
pksharma@kuk.ac.in. CTS – Tel.: +39 055 4573005; Fax: +39 055 4573385; e-mail:
claudiu.supuran@unifi.it
Abstract:
Three series of novel heterocyclic compounds (3a-3g, 4a-4g and 5a-5g) containing
benzenesulfonamide moieties and incorporating a 1,2,4-triazole ring, have been synthesized
and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs,
EC 4.2.1.1), comprising hCAs I and II(cytosolic, ubiquitous isozymes) and hCAs IX and XII
(transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II,
compounds of two series (3a-3g and 4a-4g) showed Ki values in the range of 84-868 nM and
5.6-390 nM respectively whereas compounds of series 5a-5g were found to be poor inhibitors
(Ki values exceeding 10000 nM in some cases). Against hCA IX and XII, all the tested
compounds exhibited excellent to moderate inhibitory potential with Ki values in the range of
2.8-431 nM and 1.3-63 nM respectively. Compounds 3d, 3f and 4f exhibited excellent
inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the
standard drug acetazolamide (AZA) whereas compound of the series 5a-5g were
comparatively less potent but more selective towards hCA IX and XII.
Keywords: triazoles, benzenesulfonamide, carbonic anhydrase isoforms 1, II, IX, XII,
acetazolamide
1

1. Introduction:
Carbonic anhydrase is the omnipresent class of metalloenzymes which catalyzes the
reversible hydration and dehydration of carbon dioxide.1 This is a rather simple but essential
reaction as the products of this reaction are involved in crucial physiological processes such
as respiration and transport of CO2/bicarbonate between metabolizing tissues and the lungs,
pH and CO2 homeostasis, electrolyte secretion, gluconeogenesis, lipogenesis and ureagenesis,
bone resorption, calcification, tumorigenicity and many other physiologic or pathologic
processes. 2-7
There are five main classes of these enzymes viz. α, β, γ, δ and ζ-CAs. Out of the 16
different CA isoforms discovered so far in the α-class, human CA isoforms hCA I and II are
cytosolic enzymes that are widespread throughout the human body and are drug targets for
clinically used diuretics, antiglaucoma drugs and anticonvulsants.8-10 Further, dimeric
transmembrane glycoproteins hCA IX and XII are also human associated CA isoforms
having extracellular active site and are marker for a broad spectrum of hypoxic tumor
types.11-13 The overexpression of these isoforms contributes to the increased acidification of
extracellular hypoxic environment (pH = 6.8) in contrast to normal tissues (pH = 7.4) thus
promoting tumor cell survival in an acidic condition by decreasing uptake of weakly basic
anticancer drugs.14 They also help tumors by providing bicarbonate to be used as substrate for
cell growth as bicarbonate is required in the synthesis of pyrimidine nucleotides.15 Thus
specifically targeting the tumor associated isoforms hCA IX and XII over the main off target
isoforms hCA I and II, which have a physiological relevance,16-20using specific inhibitors is
considered to be a promising strategy in the cancer therapy.
Sulfonamides are the most widely investigated class of CA inhibitors possessing
significant inhibitory power against many isoforms. Among the interesting class of such
sulfonamide derivatives, acetazolamide (AZA), methazolamide (MZA), ethoxzolamide
(EZA) etc. are widely used CAIs as clinical drugs (Figure 1). Recently it has also been
reported that many benzenesulfonamide incorporating compounds possessing significant anti-
tumor activity like indisulam (IND) (Figure 1) also exhibited excellent CA inhibitory activity
against a number of isoforms.21 Possessing a wide spectrum of biological activities, indeed
the class of heterocyclic compounds containing 1,2,4-triazole scaffold has been attracting the
attention of researchers for a long time. 1,2,4-Triazole moiety has been found to be present in
the skeleton of various natural products22 and a large number of compounds containing this
2

moiety exhibits antimicrobial, antitubercular, analgesic, anti-inflammatory, anti-convulsant,
antiviral and antidepressant activities.23,24 In addition to the wide range of bioactivities,
compounds possessing such scaffold have also shown potent antitumor effects25-27 and
recently triazolothiadiazoles128 and triazolothiadiazines 229 were reported to possess good
antitumor activity (Figure 1).
Motivated by the above findings coupled with our ongoing interest in the field of
exploring benzenesulfonamide containing heterocycles as potential biologically active
agents,30-33we envisioned to synthesize three series of compounds containing
benzenesulfonamide moiety bearing 1,2,4-triazole scaffold (3a-3g, 4a-4g and 5a-5g) for
evaluating their CA activity profile ( Scheme 1 and 2).
O
O
N
S
N
N
N
N
SO2NH2
N
N
H
SO2NH2
SO2NH2
S
S
O
MZA
AZA
EZA
N
S
N
N
N
Cl
O
O
N
S
N
NH
S
N
HN
N
Cl
SO2NH2
Cl
F
1
IND
S
2
H
N
N
N
N
S
N
S
N
N
N
N
N
N
R
H2NO2S
H2NO2S
H2NO2S
R
R
5
3
4
Figure 1
2. Results and discussion
2.1. Chemistry:
The synthetic route for preparing all the target compounds is outlined in Scheme 1 and 2. The
structures of newly synthesized target compounds were elucidated on the basis of their
spectral (MS, IR and NMR) data. 4-Aminosulfonylphenyl isothiocyanate 8 was the key
precursor for realizing the synthesis of target compounds 3. The only literature procedure
reported till date for the synthesis of 8 requires thiophosgene34 which is a well-known highly
3

toxic chemical. It is pertinent to mention here that we could successfully obtain the target
compound 8 by adapting a recently reported green methodology.35 Refluxing 8 with variously
substituted aromatic hydrazides in anhydrous ethanol resulted in the formation of
thiosemicarbazides (9a-9g) which on further refluxing in aqueous NaOH transformed into the
target compounds (3a-3g)36 (Scheme 1).
S
S- Et3N+H
NH2
NCS
HN
O
H
H
iii
N
N
i
ii
N
H
S
H2NO2S
R
SO2NH2
SO2NH2
SO2NH2
6
9a-9g
7
8
iv
H2NO2S
S
N
NH
N
R
3 a-3g
Compounds
R
3a
3b
3c
OCH3
3d
3e
3f
3g
SO2NH2
H
CH3
F
Cl
Br
Scheme 1. Reagents and conditions: (i) a.CS2, TEA, acetone, (ii) I2, NaHCO3, ethyl acetate:H2O (iii) 4-R-
C6H4CONHNH2, dry ethanol, reflux (iv) Reflux in aq. NaOH solution.
The key intermediate in the preparation of triazolothiadiazines (4a-4g) and
triazolothiadiazoles (5a-5g) was aminotriazole 13, which in turn was prepared from 4-
aminosulfonyl benzoic acid hydrazide11. The later was prepared by refluxing an aqueous
suspension of tolylsulfonamide 10 with KMnO4 to generate the corresponding carboxylic
acid followed by refluxing its methanolic solution firstly in acidic condition and then in
excess of hydrazine hydrate (Scheme 2). Then the required dithiocarbazinate 12 was prepared
by reacting hydrazide 11 with carbondisulfide and potassium hydroxide in anhydrous ethanol.
The salt 12 underwent ring closure on refluxing with an excess of 99% hydrazine hydrate to
give aminotriazole 13. While 4-amino-3-mercapto-1,2,4-triazoles may exist in thione–
thioltautomeric forms, in our study, the proton NMR data showed it to be only thione without
any trace of thiol. The compound 13 thus obtained was treated with variously substituted
4

phenacyl bromides in ethanol under reflux conditions to obtain the final compounds
1
triazolothiadiazines 4a-4g (Scheme 2). The H NMR data of these compounds showed a
characteristic singlet at around δ 4.5 for ring SCH2 protons and SO2NH2 protons appeared at
δ 7.52 as an exchangeable singlet.
We attempted to prepare fused triazolothiadiazoles (5a-5g) from 13 by refluxing it with
variously substituted aromatic carboxylic acids in POCl3, but could not isolate the desired
products. Instead, a mixture of products was obtained and proton NMR did not show any
peak corresponding to free SO2NH2 group probably due to the reaction of this NH2 group
with carboxylic acid in the presence of POCl3. Therefore, we first protected the sulfonamide
group as its formamidine following a green methodology developed recently by our research
group37 which involved stirring of 13 in neat DMFDMA at room temperature followed by a
simple work up in water resulting into the formation of 14 in excellent yield (Scheme 2). A
characteristic singlet of =CH proton of protected sulfonamide group was obtained at around δ
8.28 in proton NMR. Two singlets at δ 2.95 and δ 3.18 for -N(CH3)2 also evidenced the
protection of sulfonamide group. Then the compound 14 was reacted with aromatic acids
under the conditions of reflux in POCl3 which resulted into successful conversion to 15a-15g.
The 1H-NMR data of the compounds 15a-15g showed complete disappearance of singlets for
NH2 as well as SH protons which confirmed the cyclization. The final compounds 5a-5g
were obtained by deprotection of sulphonamide group in 15a-15g under acidic conditions
(Scheme 2). The deprotection was further confirmed by proton NMR which showed a singlet
for SO2NH2 group at δ 7.51.
5

S
H
O
N
CONHNH2
C
N
H
S-K+
CH3
H
N
N
v
i, ii, iii
S
iv
N
NH2
SO2NH2
SO2NH2
H2NO2S
SO2NH2
13
10
11
12
S
N
N
H
N
N
N
N
S
vii
vi
N
13
R
N
NH2
H2NO2S
NO2S
4a-4g
14
x
viii
N
N
N
N
N
N
S
S
ix
N
N
N
NO2S
H2NO2S
R
R
5a-5g
15a-15g
Compounds
R
4a/5a
4b/5b
CH3
4c/5c
4d/5d
4e/5e
4f/5f
Br
4g/5g
H
OCH3
F
Cl
NO2
Scheme 2. Reagents and conditions: (i) a. KMnO4, H2O, reflux (ii)MeOH, H+, reflux (iii) NH2NH2.H2O, MeOH,
reflux (iv) CS2, KOH (v) H2NH2.H2O, reflux (vi) ArCOCH2Br, EtOH, reflux (vii) DMFDMA, stirring, r.t. (viii)
ArCOOH, POCl3, reflux (ix) Conc. HCl, reflux, (x) ArCOOH, POCl3, reflux.
2.2. CA inhibition:
The CA inhibitory potential of all the 21 compounds (3a-3g, 4a-4g, 5a-5g) against
cytosolic isoforms hCA I and II as well as the membrane associated isoforms hCA IX and
XII was assayed by using stopped flow assay method and the results are presented in Table
1. Clinically used acetazolamide (AZA) was the reference drug chosen for the assay.
Inhibition data of the compounds let the following insights regarding CAs inhibitory
properties.
6

(i)
The cytosolic isoform hCA I was moderately inhibited by compounds 3a-3g and
4a-4g, with the inhibition constants in the range of 84-868 nM and mainly the
compounds 3b, 3d, 3e, 3f, 3g, 4c, and 4f were shown to be more efficient
inhibitors than AZA. The compounds of series 3a-3g exhibited almost similar
activities and a remarkable difference was found only in the case of chloro
substituted compound 3e which showed almost three times greater potential than
the reference acetazolamide (AZA) with inhibition constant, KI = 86 nM. Other
two halogensubstituents (F and Br) also show an in increase in potential as
compared to unsubstituted derivative 3a.In case of series of 4a-4g, p-methoxy
and p-bromo substituted compounds (4c and 4f) showed maximum range of
activity i.e. 84 and 89 nM respectively while all other compounds were weakly
efficient against this enzyme. Compounds 5a-5g proved to be much weaker
inhibitors as most of the compounds crossed 10 µM range and none of them was
even near to the AZA range. Thus, it is of great interest to relate this behaviour of
these compounds toward hCA I and it can be concluded that both nuclear
triazoles as well as triazole-fused with six membered thiadiazines are moderate
inhibitors of hCA I while fusing triazoles with five membered thiadiazole ring
leads to a steep decrease in the inhibitory potency of these compounds against
hCA I.
(ii)
The tested compounds in general showed better activity profile against hCA II
than hCA I and compounds 3d, 3f and 4f were better inhibitors of hCA II as
compared to AZA. However being a ubiquitous, housekeeping isoform, this may
not be a valuable property in another context if compounds targeting other
isoforms (hCA IX and hCA XII) should also possess activity against hCA II.
Thus it is interesting to note that some of the novel compounds showed poor
inhibition against hCA II while retaining potent inhibition against hCA IX and
hCA XII. Compounds 5a-5g in which the triazole ring is fused with another 5-
memebered thiadiazole ring showed a highly reduced potential for hCA II.This
again leads to the conclusion that reducing the size of heteroyclic ring fused to
the triazole ring leads to important differences in hCA II inhibitiory potency. The
relative effect of substitution on activity profile was almost similar to that against
hCA I.
(iii)
The tumor associated target isoform hCA IX was highly inhibited by all the
compounds of series 3a-3g and 4a-4g except compounds 4e and 4g which were
7

less efficient than AZA. The compound 3d having fluoro substitution showed
around 10 fold activity as compared to AZA. The activity pattern of compounds
3a-3g shows that both electron donating as well as electron withdrawing groups
enhance the inhibitory potential. The sulfonamide substituted compound 3g was
rather a less efficient inhibitor than halogen substituted compounds, probably due
to large bulky nature of the group. The compounds of 4a-4g series also showed
similar pattern to that of 3a-3g and again compound possessing sulfonamide
group was weaker inhibitor. Compounds 5a-5gwere less effective as hCA IX
inhibitors, with KIs in the range of 41-431 nM.
(iv)
The second tumor-associated isoform, hCA XII, was the most inhibited isoform
by all the compounds investigated here, with many of them being low nanomolar
(3c-3g, 4b, 4c and 4f) CAIs. The least effective hCA XII inhibitors were 4g, 5e
and 5f (KIs of 43-63 nM). Eight compounds 3c, 3d, 3e, 3f,3g, 4b, 4c, and 4f were
more potent inhibitors than the standard AZA. The best results were shown by
compounds 3f with a bromo substitution which was found to be four-fold
efficient than AZA with inhibition constant 1.3 nM.
(v)
Selectivity for inhibiting the tumor-associated isoforms (hCA IX and XII) over
the widespread cytosolic forms (hCA I and II) is a key issue when designing
CAIs. As seen from data in Table 2, where these ratios are provided, none of the
compounds reported here showed uniformity in their inhibitory properties against
all four CAs investigated (hCA I, II, IX and XII, Tables 1 and 2). It is satisfying
to note that all the compounds showed better activity profile against hCA IX and
XII over I and II which is highly desirable when only the tumor-associated
isoforms would be targeted. It was observed that the compounds which showed
excellent inhibition of isoform IX and XII were also shown to be highly selective.
Compound 5g showed maximum selectivity for IX and XII over II with the
selectivity ratio of 164.63 and 758.43 respectively and have potential almost half
to that of AZA. All other compounds of this series i.e. 5a-5f were also shown to
be excellently selective having selectivity ratio close to 10 or more. These
compounds 5a-5g also showed excellent selectivity profile for both tumor
associated isoforms over hCA I. All the compounds of series 3a-3g and 4a-4g
were also more selective in their activity profile as compared with the chosen
standard. Compound 3d being the most efficient inhibitor of hCA IX was about
five-fold more selective over hCAII than AZA. Similarly compound 3f being the
8

most efficient inhibitor of hCA XII, was about four fold more selective over
hCAII. Thus, the new class of sulfonamides reported here may indeed lead to
isoform-selective CAIs targeting the tumor-associated CAs.
Table 1. Inhibitory potency data for compounds 3a-3g, 4a-4g, and 5a-5g against isozymes hCAI, hCAII,
hCAIX, and hCAXII
Ki* (nM)
hCA I
288
Inhibitors
hCA II
31
hCA IX
22
hCA XII
10.4
9.5
4.9
4.9
3.6
1.3
4.5
12.7
4.9
3.8
18
3a
3b
3c
3d
3e
3f
166
20
16
271
27
8.1
2.8
5.7
7.6
9.0
21
170
6.6
86
24
132
6.7
220
37
3g
4a
4b
4c
4d
4e
4f
711
158
73
292
13
84
44
6.5
25
507
93
868
223
5.6
76
45
89
3.7
115
114
356
431
64
3.0
63
463
390
831
3780
6585
8760
885
3615
6750
12.1
4g
5a
5b
5c
5d
5e
5f
4480
>10000
>10000
>10000
6630
8450
>10000
250
18
24
20
21
89
43
104
41
56
8.9
5.7
5g
AZA
25
AZA = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for comparison.
* Mean from 3 diferent assays, errors were in the range of 5-10 % of the reported value.
9

Table 2.Selectivity ratios for the inhibition of the tumor-associated isozymes hCA IX and
hCAXII over the cytosolic isozymes hCA I and hCA II for compounds 3a-3g, 4a-4g, and 5a-5g
Selectivity ratio*
hCA I/hCA IX
13.0
Inhibitors
hCA II/hCA IX hCA I/XII
hCA II/hCAXII
2.9
1.4
27.6
3a
3b
3c
3d
3e
3f
10.3
1.2
17.4
2.1
33.4
3.3
55.3
5.5
60.7
2.3
34.6
1.3
15.0
4.2
23.8
6.6
17.3
0.8
101.5
48.8
5.1
24.4
4.1
8.2
3g
4a
4b
4c
4d
4e
4f
33.8
7.5
55.9
12.4
14.9
11.5
5.1
22.4
5.6
59.5
12.9
6.7
22.1
20.2
3.7
28.1
11.4
3.0
19.2
4.9
24.0
1.5
29.6
1.8
4.0
3.3
7.3
6.1
4g
5a
5b
5c
5d
5e
5f
74.3
7.2
471.1
>416.0
>500.0
>476.0
154.1
150.8
>1123.0
43.8
46.1
157.5
329.2
417.1
20.5
64.5
758.4
2.1
>28.0
>23.0
>156.0
74.4
10.6
15.2
136.8
9.9
81.2
34.7
164.6
0.4
>243.0
10.0
5g
AZA
AZA = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for comparison.
* The Ki ratios are indicative of isozyme selectivity: a weak selective inhibitor is characterized by a low value
ratio..
3. Conclusion:
Three novel series of compounds 3a-3g, 4a-4g and 5a-5g containing 1,2,4-triazole
scaffold have been synthesized and investigated as inhibitors against four of the isomers of α
class of carbonic anhydrases comprising cytosolic, ubiquitous isozymes hCA I and II as well
as the transmembrane, tumor-associated isoforms hCA IX and XII which are validated
10

antitumor targets. In this study, compounds 3c-3g, 4b, 4c and4f showed excellent CAs
inhibitory profile. Compounds 3d and 3f being the most potent inhibitors of hCA IX and XII
were associated with manyfold selectivity over hCA I and II as compared to the standard
drug AZA. The activity profile also revealed that both nuclear triazole scaffold as well as
triazole fused with six membered thiadiazines are moderate inhibitors of hCA I andII and
substitution of thiadiazine ring by a 5-membered thiadiazole leads to a steep decrease in the
inhibitory property of these compounds.Similarly, 5g was the most selective inhibitor of
tumor associated hCA XII over hCA II of all the investigated compound with nearly same
range of activity comparable to the reference compound. As it has been evidenced that
isoforms IX and XII are responsible for tumours and are also potential targets for diagnosis
and treatment of cancers, discovery of potent selective IX and XII inhibitors will be a
promising step in the strategy for an effective cancer therapy.
4. Experimental protocols:
4.1. General
Melting points were determined in open glass capillaries in an electrical melting point
apparatus and are uncorrected. The infrared (IR) spectra were recorded on Shimadzu-21 FT-
IR or Perkine Elmer IR Spectrophotometer using the KBr pellet technique. 1H NMR and 13C
NMR spectra were recorded either in pure DMSO-d6 or in CDCl3/DMSO-d6 mixture on
Bruker NMR spectrometers at 300/400 MHz and 75.5/100 MHz respectively using
tetramethylsilane (TMS) as internal standard. Chemical shifts are expressed in δ ppm. Mass
spectra (DART-MS) were recorded on a JEOL-AccuTOFJMS-T100LC Mass spectrometer
having a DART (Direct Analysis in Real Time) source in ESþ mode. The purity of the
1
compounds was checked by H NMR and thin layer chromatography (TLC) on silica gel
plates using a mixture of petroleum ether and ethyl acetate as eluent. Iodine or UV lamp was
used as a visualizing agent. Abbreviations‘s’ for singlet, ‘m’ for multiplet, ‘ex’ for
exchangeable proton are used for NMR assignments; ‘s’ for strong, ‘m’ for medium for IR
and ‘br’ for broad in NMR as well as IR assignments. ‘d’ stands for decomposition in melting
point data.
4.2. Synthesis of 4-isothiocyanatobenzenesulfonamide (8)
ᵒ
To a well cooled mixture of methanol and triethylamine to 0 C was added carbon
disulphide dropwise and stirred for 10 minutes. Then the amine was added in small lots with
constant stirring and allowed the reaction mixture to stirr for further 15-16 hours when
11

triethyl ammonium dithiocarbamate salt precipitated completely. The solid thus precipitated
was filtered, washed with diethyl ether and air dried. Then to stirred and ice-cooled biphasic
solvent system, water/ethylacetate (1:1, 10 ml) was added the dithiocarbamate salt 7 (1
mmol) followed by sodium bicarbonate (2 mmol). To this stirring solution was then added
iodine (2 mmol) pinch wise over a period of 15-20 minutes. After the completion of reaction,
aqueous solution of sodium thiosulfate was added to neutralize the excess of iodine and then
the ethylacetate layer was separated, filtered to remove precipitated sulphur, washed with
water and dried over anhydrous sodium Sulphate. The ethylacetate was evapourated under
1
reduced pressure yielding pure. Yield 79%, Lit. mp: 212-214 oC, Obs. mp: 209-211oC, H-
NMR (300 MHz, DMSO-d6) δ (ppm): 7.87 (d, J = 7.5 Hz, 2H, Ar), 7.58 (d, J = 7.5 Hz, 2H,
Ar), 7.49 (s, 2H, SO2NH2); 13C NMR (DMSO-d6, 75.5 Hz) δ (ppm): 143.38 (NCS), 133.63,
127.85, 126.91, 122.78.
4.3. Synthesis of hydrazino carbothiamide compounds (9a-9g):
General procedure
A mixture of 8 (1 mmol) and aromatic acid hydrazide was refluxed in absolute
ethanol for 3-4 hours. After the completion of the reaction the solid precipitated was filtered
off, washed with cold ethanol, dried and recrystalized from ethanol which afforded the pure
products 9a-9g.
4.3.1. N-[4-(aminosulfonyl)phenyl]-2-benzoyl-1-hydrazinecarbothioamide (9a)
o
Yield 84%, mp: 198-200 C, IR(KBr) (υ, cm-1), 3333, 3240, 3132 and 3101 (NH), 1643
(CO), 1335, 1157 (SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.38, 9.92, 9.68 (3s, 3H,
3NH, exch. D2O), 7.96 (d, J = 6.9 Hz, 2H, Ar), 7.76 (d, J = 8.4 Hz, 2H, Ar), 7.70 (s, 1H, Ar),
7.58 (d, J = 6.9 Hz, 2H, Ar), 7.52 (d, J = 7.5 Hz, 2H, Ar), 7.31 (s, 2H, SO2NH2); 13C NMR
(75.5 Hz, DMSO-d6) δ (ppm): 166.46, 142.85, 132.87, 132.42, 128.77, 128.32, 126.15.
4.3.2. N-[4-(aminosulfonyl)phenyl]-2-(4-methylbenzoyl)-1-hydrazinecarbothioamide (9b)
o
Yield 87%, mp: 202-204 C, IR(KBr) (υ, cm-1), 3333, 3202 and 3109 (NH), 1659 (CO),
1335, 1157 (SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.42, 9.92, 9.84 (3s, 3H, 3NH,
exch. D2O), 7.88 (d, J = 8.7 Hz, 2H, Ar), 7.47 (m, 4H, Ar), 7.31 (s, 2H, SO2NH2), 7.22 (d, J =
8.7 Hz, 2H, Ar), 2.34 (s, 3H, CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 166.44, 142.89,
142.48, 130.10, 129.30, 128.35, 126.16, 21.51.
12

4.3.3. N-[4-(aminosulfonyl)phenyl]-2-(4-methoxybenzoyl)-1-hydrazinecarbothioamide (9c)
o
Yield 92%, mp: 200-202 C, IR(KBr) (υ, cm-1), 3340, 3202 and 3194 (NH), 1659 (CO),
1335, 1157 (SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.44, 9.96, 9.87 (3s, 3H, 3NH,
exch. D2O), 7.94 (d, J = 8.7 Hz, 2H, Ar), 7.50 (m, 4H, Ar), 7.31 (s, 2H, SO2NH2), 7.04 (d, J =
8.7 Hz, 2H, Ar), 3.82 (s, 3H, OCH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 166.07,
162.65, 142.86, 130.28, 126.11, 125.06, 114.02, 55.91.
4.3.4. N-[4-(aminosulfonyl)phenyl]-2-(4-fluorobenzoyl)-1-hydrazinecarbothioamide (9d)
o
Yield 78%, mp: 172-174 C, IR(KBr) (υ, cm-1), 3325, 3202 (NH), 1666 (CO), 1335, 1157
1
(SO2); H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.62 (1s, 1H, NH, exch. D2O), 9.95 (2s,
2H, 2NH, exch. D2O), 8.02 (m, 2H, Ar), 7.78 (m, 4H, Ar), 7.70 (m, 2H, Ar), 7.30 (s, 2H,
SO2NH2); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 166.41, 142.83, 130.97, 126.89, 126.08,
122.19, 115.90, 115.62.
4.3.5. N-[4-(aminosulfonyl)phenyl]-2-(4-chlorobenzoyl)-1-hydrazinecarbothioamide (9e)
o
Yield 85%, mp: 194-196 C, IR(KBr) (υ, cm-1), 3310, 3202 (NH), 1666 (CO), 1335, 1157
1
(SO2); H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.67 (1s, 1H, NH, exch. D2O), 9.94 (2s,
2H, 2NH, exch. D2O), 7.96 (d, J = 8.4 Hz, 2H, Ar), 7.76 (d, J = 8.4 Hz, 2H, Ar), 7.69 (m, 2H,
Ar), 7.60 (d, J = 8.4 Hz, 2H, Ar), 7.31 (s, 2H, SO2NH2); 13C NMR (75.5 Hz, DMSO-d6) δ
(ppm): 165.58, 142.78, 137.29, 131.70, 130.25, 128.89, 126.20.
4.3.6. N-[4-(aminosulfonyl)phenyl]-2-(4-bromobenzoyl)-1-hydrazinecarbothioamide (9f)
o
Yield 74%, mp: 188-190 C, IR(KBr) (υ, cm-1), 3310, 3202 (NH), 1659 (CO), 1335, 1157
1
(SO2); H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.67 (1s, 1H, NH, exch. D2O), 9.96 (2s,
2H, 2NH, exch. D2O), 7.89 (d, J = 7.8 Hz, 2H, Ar), 7.75 (m, 6H, Ar), 7.30 (s, 2H, SO2NH2);
13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 165.54, 142.98, 137.47, 131.79, 131.36, 128.46,
126.18.
4.3.7 2-[4-(aminosulfonyl)benzoyl]-N-[4-(aminosulfonyl)phenyl]-1-hydrazinecarbothioamide (9g)
o
Yield 78%, mp: 194-196 C, IR(KBr) (υ, cm-1), 3325, 3256 and 3202 (NH), 1666 (CO),
1335, 1157 (SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 10.78 (1s, 1H, NH, exch. D2O),
9.98 (2s, 2H, 2NH, exch. D2O), 7.87 (d, J = 8.1 Hz, 2H, Ar), 7.64 (d, J = 8.4 Hz, 2H, Ar),
7.46 (m, 2H, Ar), 7.35 (d, J = 8.4 Hz, 2H, Ar), 7.31 (s, 2H, SO2NH2), 7.29 (s, 2H, SO2NH2);
13

13C NMR (75.5 Hz, DMSO-d6,) δ (ppm): 165.58, 142.78, 137.29, 131.70, 130.25, 128.89,
126.20.
4.4. Synthesis of thioxotriazolo benzensulfonamide compounds (3a-3g)
General procedure: Compound 9 was dissolved in 2% aq. NaOH solution and refluxed for 4
hrs. Then cooled the solution to room temperature, added ice to it and neutralized with conc.
HCl which resulted into the precipitation of a white solid. The solid was filtered off, washed
with water, dried and recrystallized from appropriate solvent which afforded the pure
products 3a-3g.
4.4.1. 4-(3-phenyl-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)benzenesulfonamide (3a)
o
Yield 78%, mp: 294 C, IR(KBr) (υ, cm-1), 3381, 3278, 3094, 1557, 1512, 1335, 1165 (SO2);
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 14.2 (s, 1H, ring NH, exch. D2O), 7.91 (d, J = 7.8
Hz, 2H, Ar), 7.58 (d, J = 8.1 Hz, 2H, Ar), 7.53 (s, 2H, SO2NH2), 7.43 (m, 2H, Ar), 7.36 (m,
3H, Ar); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 169.02 (C=S), 150.92, 145.15, 137.65,
130.99, 130.00, 129.13, 128.91, 127.13, 126.04; DART MS m/z 333.05 (M+H)+,
C14H12N4O2S2H+, calcd. 332.04.
4.4.2.4-[3-(4-methylphenyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzenesulfonamide (3b)
o
Yield 79%, mp: 276 C, IR(KBr) (υ, cm-1), 3379, 3279, 3094, 1551, 1512, 1335, 1165 (SO2);
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 14.2 (s, 1H, ring NH), 7.91 (d, J = 8.1 Hz, 2H, Ar),
7.56 (d, J = 8.1 Hz, 2H, Ar), 7.53 (s, 2H, SO2NH2), 7.19 (m, 4H, Ar), 2.27 (s, 3H, CH3); 13C
NMR (75.5 Hz, DMSO-d6) δ (ppm): 168.92 (C=S), 151.00, 145.11, 140.92, 137.73, 130.01,
129.69, 128.80, 127.13, 123.18, 21.31 (CH3); DART MS m/z 347.19 (M+H)+,
C15H14N4O2S2H+, calcd. 346.43.
4.4.3.4-[3-(4-methoxyphenyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzenesulfonamide (3c)
o
Yield 82%, mp: 274 C, IR(KBr) (υ, cm-1), 3360, 3287, 3078, 1551, 1512, 1335, 1165 (SO2);
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.91 (d, J = 6.9 Hz, 2H, Ar), 7.57 (d, J = 7.5 Hz,
2H, Ar), 7.53 (s, 2H, SO2NH2), 7.25 (d, J = 7.2 Hz, 2H, Ar), 6.92 (d, J = 7.5 Hz, 2H, Ar),
3.74 (s, 3H, OCH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 168.77 (C=S), 161.21, 150.84,
145.06, 137.82, 130.44, 130.03, 127.15, 118.16, 114.62, 55.78 (OCH3); DART MS m/z
362.87 (M+H)+, C15H14N4O3S2H+, calcd. 362.43.
14

4.4.4. 4-[3-(4-fluorophenyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzenesulfonamide (3d)
o
1
Yield 77%, mp: 282 C, IR(KBr) (υ, cm-1), 3371, 3286, 3094, 1512, 1335, 1165 (SO2); H-
NMR (300 MHz, DMSO-d6) δ (ppm): 14.2 (s, 1H, ring NH), 7.91 (d, J = 7.8 Hz, 2H, Ar),
7.58 (d, J = 8.1 Hz, 2H, Ar), 7.52 (s, 2H, SO2NH2), 7.40 (m, 2H, Ar), 7.25 (m, 2H, Ar); 13C
NMR (75.5 Hz, DMSO-d6) δ (ppm): 168.96 (C=S), 150.20, 145.15, 137.48, 131.63, 130.00,
127.16, 122.62, 116.49, 116.19; DART MS m/z 348.86 (M+H)+, C14H11FN4O2S2H+, calcd.
350.39.
4.4.5. 4-[3-(4-chlorophenyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzenesulfonamide (3e)
o
Yield 76%, mp: 288 C, IR(KBr) (υ, cm-1), 3354, 3264, 3094, 1512, 1335, 1165 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 14.3 (s, 1H, ring NH), 7.90 (d, J = 6.3 Hz, 2H, Ar),
7.58 (d, J = 5.8 Hz, 2H, Ar), 7.52 (s, 2H, SO2NH2), 7.46 (d, 2H, Ar), 7.33 (d, 2H, Ar); 13C
NMR (75.5 Hz, DMSO-d6) δ (ppm): 169.02 (C=S), 150.03, 145.17, 137.40, 135.88, 130.78,
129.97, 129.30, 127.17, 124.94; DART MS m/z 367.13/369.13 (M+H)+/(M+H+2)+,
C14H11ClN4O2S2H+, calcd. 366.85/368.85.
4.4.6. 4-[3-(4-bromophenyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]benzenesulfonamide (3f)
o
1
Yield 71%, mp: 268 C, IR(KBr) (υ, cm-1), 3348, 3225, 1497, 1339, 1149 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 14.2 (s, 1H, ring NH), 7.91 (d, J = 7.8 Hz, 2H, Ar), 7.59 (m,
4H, Ar), 7.52 (s, 2H, SO2NH2), 7.26 (d, J = 8.1 Hz, 2H, Ar); 13C NMR (75.5 Hz, DMSO-d6)
δ (ppm): 169.08 (C=S), 150.14, 145.19, 137.40, 132.22, 130.93, 129.96, 127.19, 125.27,
124.72; DART MS m/z 411.88/413.88 (M+H)+/(M+H+2)+, C14H11BrN4O2S2H+, calcd.
410.55/412.55.
4.4.7.4-{3-[4-(aminosulfonyl)phenyl]-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl}benzenesulfonamide(3g)
o
Yield 74%, mp: 294 C IR(KBr) (υ, cm-1), 3225, 3063, 1535, 1342, 1165 (SO2); 1H-NMR
(300 MHz, DMSO-d6) δ (ppm): 14.45 (s, 1H, ring NH), 7.92 (d, J = 8.4 Hz, 2H, Ar), 7.18 (d,
J = 8.4 Hz, 2H, Ar), 7.62 (d, J = 8.4 Hz, 2H, Ar), 7.53 (s, 2H, SO2NH2), 7.51 (d, J = 8.7Hz,
2H, Ar), 7.45 (s, 2H, SO2NH2); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 169.56 (C=S),
149.88, 146.02, 145.30, 137.37, 130.00, 129.61, 129.06, 127.23, 126.33; DART MS m/z
411.99 (M+H)+, C14H13N5O4S3H+, calcd. 411.48.
4.5 Synthesis of 4-(hydrazinocarbonyl)benzenesulfonamide (11):
15

Tolylsulfonamide 10 (1.00 mM) was first converted to 4-aminosulfonyl benzoic acid by
refluxing its aqueous suspension with KMnO4 (3.00 mM) to generate the corresponding
carboxylic acid followed by refluxing its methanolic solution firstly in acidic condition and
o
then in excess of hydrazine hydrate.38 Yield 74%, Lit. mp: 230-232 C, Obs. mp: 228-230 oC.
4.6 Synthesis of 4-(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-
yl)benzenesulfonamide (13):
To a well cooled and stirring ethanolic solution of KOH (1.00 mM) was added 11 (1.00 mM)
followed by dropwise addition of carbondisulfide (1.20 mM). The reaction mixture was
stirred for overnight and the dithiocarbamate salt precipitated as yellow solid was filtered,
washed with diethyl ether and dried. Then to the aqueous suspension of dithiocarbamate salt
was added excess of hydrazine hydrate and was refluxed for 20 h. To the cooled reaction
mixture was added glacial acetic acid just to neutralize it when aminotriazole 13 was
precipitated as white solid which was filtered, washed with water, dried and recystallized
o
from alcohol.39 Yield 72%, mp: 238-240 C, IR(KBr) (υ, cm-1), 1565, 1342, 1165 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 14.12 (s, 1H, ring NH), 8.22 (d, J = 6.9 Hz, 2H, Ar),
7.95 (d, J = 6.9 Hz, 2H, Ar), 7.51 (s, 2H, SO2NH2), 5.82 (s, 1H, NH2); 13C NMR (75.5 Hz,
DMSO-d6) δ (ppm): 167.78 (C=S), 148.85, 145.89,128.97, 127.13, 126.25; DART MS m/z
272 (M+H)+, C8H9N5O2S2H+, calcd. 271.32.
4.7
Synthesis
of
4-(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-N-[(Z)-
(dimethylamino)methylidene]benzenesulfonamide (14):
A suspension of 13 (1.00 mM) in DMFDMA (1.20 mM) was stirred at 40 ᵒC for 7-8 hours
and then reaction mixture was poured in cold water. The solid separated was filtered, washed
o
with water, dried and recrystallized from alcohol. Yield 83%, mp: 220-222 C IR(KBr) (υ,
cm-1), 1342, 1165 (SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 14.12 (s, 1H, ring NH),
8.24 (s, 1H, =CH), 8.19 (d, J = 8.7 Hz, 2H, Ar), 7.91 (d, J = 8.7 Hz, 2H, Ar), 5.80 (s, 1H,
NH2), 3.16 (s, 3H, N-CH3), 2.92 (s, 3H, N-CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm):
167.78 (C=S), 160.43, 151.38, 150.74, 130.45, 128.82, 127.80, 126.76, 41.50, 35.18.
4.8. Synthesis of triazolothiadiazine compounds (4a-4g)
General procedure: A mixture of 9 (1.00 mM) and variously substituted phenacyl bromides
(1.00 mM) was refluxed in absolute ethanol on water bath for 9-11 hrs and a solid
16

precipitated out while reflux. The reaction mixture was allowed to cool to room temp. and the
solid was filtered off, dried and recrystallized from suitable solvent which afforded the pure
title compounds.
4.8.1. 4-(6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl)benzenesulfonamide (4a)
o
Yield 74%, mp: 264-266 C, IR(KBr) (υ, cm-1), 3294, 2970 and 2839 (CH2), 1697, 1651,
1
1558, 1319, 1157 (SO2); H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (d, J = 8.1 Hz, 2H,
Ar), 8.03 (m, 3H), 7.62 (m, 4H, Ar), 7.49 (s, 2H, SO2NH2), 4.44 (s, 2H, CH2); 13C-NMR
(75.5 MHz, DMSO-d6) δ (ppm): 156.77, 151.21, 145.69, 143.76, 133.78, 132.54, 129.62,
129.34, 128.86, 128.13, 127.51, 127.16, 126.60, 23.31; DART MS m/z 372 (M+H)+,
C16H13N5O2S2H+, calcd. 371.44.
4.8.2.4-[6-(4-methylphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4b)
o
Yield 77%, mp: 264-266 C, IR(KBr) (υ, cm-1), 3317, 3040, 1597, 1528, 1335, 1157 (SO2).
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (d, J = 8.7 Hz, 2H, Ar), 8.02 (d, J = 8.7 Hz,
2H, Ar), 7.93 (d, J = 8.1 Hz, 2H, Ar), 7.50 (s, 2H, SO2NH2), 7.40 (d, J = 8.1 Hz, 2H, Ar),
4.44 (s, 2H, CH2), 2.40 (s, 3H, CH3); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 156.69,
151.09, 145.64, 143.80, 142.88, 130.93, 130.21, 129.37, 128.80, 128.10, 126.58, 23.16,
21.53; DART MS m/z 386 (M+H)+, C17H15N5O2S2H+, calcd. 385.46.
4.8.3.4-[6-(4-methoxyphenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4c)
Yield 71%, mp: 264-266o C, IR(KBr) (υ, cm-1), 3225, 3032, 1705, 1597, 1551, 1327, 1157
(SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (d, J = 8.4 Hz, 2H, Ar), 8.00 (m, 4H,
Ar), 7.52 (s, 2H, SO2NH2), 7.12 (d, J = 9.0 Hz, 2H, Ar), 4.40 (s, 2H, CH2), 3.84 (s, 3H,
OCH3); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 156.73, 151.09, 145.99, 143.86, 142.91,
130.88, 130.22, 129.32, 128.82, 128.10, 126.57, 23.10, 55.67; DART MS m/z 402 (M+H)+,
C17H15N5O3S2H+, calcd. 401.46.
4.8.4.4-[6-(4-fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4d)
o
Yield 73%, mp: 274-276 C, IR(KBr) (υ, cm-1), 3217, 3047, 1690, 1597, 1512, 1335, 1157
(SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (d, J = 8.4 Hz, 2H, Ar), 8.11 (m, 2H,
Ar), 8.02 (d, J = 8.4 Hz, 2H, Ar), 7.51 (s, 2H, SO2NH2), 7.42 (m, 2H, Ar), 4.47 (s, 2H, CH2);
1
13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 164.82 (d, JCF = 251.41 Hz), 155.85, 151.19,
17

145.69, 143.68, 130.83 (d, 3JCF = 9.06 Hz), 130.29, 129.29, 128.86, 126.59, 116.74 (d, 2JCF
=
22.65 Hz), 23.28; DART MS m/z 390 (M+H)+, C16H12FN5O2S2H+, calcd. 389.43.
4.8.5. 4-[6-(4-chlorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4e)
o
Yield 78%, mp: 276-278 C, IR(KBr) (υ, cm-1), 3209, 3047, 1697, 1589, 1335, 1157 (SO2);
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.19 (d, J = 8.1 Hz, 2H, Ar), 8.04 (m, 4H, Ar), 7.67
(d, J = 8.4 Hz, 2H, Ar), 7.52 (s, 2H, SO2NH2), 4.46 (s, 2H, CH2); 13C-NMR (75.5 MHz,
DMSO-d6) δ (ppm): 155.79, 151.21, 145.71, 143.69, 137.45, 132.62, 129.94, 129.71, 129.24,
128.86, 126.60, 23.19; DART MS m/z 406/408 (M+H)+/(M+H+2)+, C16H12ClN5O2S2H+,
calcd. 405.88/407.88.
4.8.6.4-[6-(4-bromophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4f)
o
Yield 74%, mp: 280-282 C, IR(KBr) (υ, cm-1), 3225, 3032, 1705, 1597, 1551, 1327, 1157
(SO2); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.19 (d, J = 8.1 Hz, 2H, Ar), 8.02 (d, J = 8.4
Hz, 2H, Ar), 7.97 (d, J = 8.7 Hz, 2H, Ar), 7.80 (d, J = 8.4 Hz, 2H, Ar), 7.52 (s, 2H, SO2NH2),
4.46 (s, 2H, CH2); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 155.96, 151.20, 145.65,
143.75, 132.94, 132.66, 130.08, 129.18, 128.88, 126.60, 126.42, 23.09; DART MS m/z
451/453 (M+H)+/(M+H+2)+, C16H12BrN5O2S2H+, calcd. 405.88/407.88.
4.8.7.4-[6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl]benzenesulfonamide(4g)
o
Yield 64%, mp: 286-288 C, IR(KBr) (υ, cm-1), 3232, 3063, 1643, 1574, 1512, 1342, 1157
(SO2); .1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.40 (d, J = 8.7 Hz, 2H, Ar), 8.27 (d, J =
8.7 Hz, 2H, Ar), 8.19 (d, J = 8.1 Hz, 2H, Ar), 8.02 (d, J = 8.1 Hz, 2H, Ar), 7.51 (s, 2H,
SO2NH2), 4.53 (s, 2H, CH2); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 155.14, 151.40,
149.72, 145.81, 143.61, 139.71, 129.56, 129.08, 128.94, 126.64, 124.58, 23.42; DART MS
m/z 417 (M+H)+, C16H12N6O4S2H+, calcd. 416.43.
4.9. Synthesis of N-protected triazolothiadiazole compounds (15a-15g)
General procedure: A mixture of 10 (10 mmol) and aromatic acids (10 mmol) in the
presence of POCl3 (15mL) was refluxed for 4–6 h. After the completion of the reaction the
reaction mixture was cooled to room temperature and then slowly added to crushed ice and
neutralized with aq. NaOH solution. A solid was precipitated out which was filtered off,
washed with excess of water, dried and recrystalized from appropriate solvent to afford the
title compounds.
18

4.9.1. N-[(Z)-(dimethylamino)methylidene]-4-(6-phenyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
3-yl)benzenesulfonamide (15a)
o
Yield 88%, mp: 262 C, IR(KBr) (υ, cm-1), 2978, 1705, 1620 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.47 (d, J = 8.1 Hz, 2H, Ar), 8.25 (s, 1H, CH=N),
8.01-8.10 (m, 4H, Ar), 7.68 (m, 3H, Ar), 3.18 (s, 3H, N-CH3), 2.95 (s, 3H, N-CH3); 13C NMR
(75.5 Hz, DMSO-d6) δ (ppm): 167.78, 160.43, 144.73, 133.57, 130.17, 129.35, 128.69,
127.83, 127.28, 126.87, 41.48, 35.64.
4.9.2. N-[(Z)-(dimethylamino)methylidene]-4-[6-(4-methylphenyl)[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-3-yl]benzenesulfonamide (15b)
Yield 84%, mp: 266 oC , IR(KBr) (υ, cm-1), 2978, 1705, 1620 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.51 (d, J = 8.4 Hz, 2H, Ar), 8.25 (s, 1H, CH=N), 8.24
(d, J = 8.4 Hz, 2H, Ar), 7.77 (d, J = 8.1 Hz, 2H, Ar), 7.43 (d, J = 8.1 Hz, 2H, Ar), 3.17 (s, 3H,
N-CH3), 2.95 (s, 3H, N-CH3), 2.40 (s, 3H, CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm):
160.27, 144.45, 143.9, 130.43, 128.68, 127.48, 127.09, 126.48, 41.52, 35.28, 21.71.
4.9.3. N-[(Z)-(dimethylamino)methylidene]-4-[6-(4-methoxyphenyl)[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-3-yl]benzenesulfonamide (15c)
o
Yield 89%, mp: 274 C, IR(KBr) (υ, cm-1), 2978, 1705, 1620 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.46 (d, J = 8.1 Hz, 2H, Ar), 8.27 (s, 1H, CH=N),
8.00-8.04 (m, 4H, Ar), 7.19 (d, J = 8.7 Hz, 2H, Ar), 3.89 (s, 3H, OCH3), 3.17 (s, 3H, N-CH3),
2.95 (s, 3H, N-CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 161.13, 144.47, 142.7, 129.57,
129.18, 127.78, 127.29, 126.32, 55.63, 41.52, 35.28.
4.9.4.N-[(Z)-(dimethylamino)methylidene]-4-[6-(4-fluorophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-
yl]benzenesulfonamide(15d)
o
Yield 83%, mp: 278 C, IR(KBr) (υ, cm-1), 2978, 1705, 1620 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.46 (d, J = 8.4 Hz, 2H, Ar), 8.28 (s, 1H, CH=N), 8.17
(m, 2H, Ar), 8.01 (d, J = 8.4 Hz, 2H, Ar), 7.51 (m, 2H, Ar), 3.18 (s, 3H, N-CH3), 2.95 (s, 3H,
N-CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 160.44, 148.62, 127.24, 126.73, 41.49,
35.26.
4.9.5. 4-[6-(4-chlorophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]-N-[(Z)-
(dimethylamino)methylidene]benzenesulfonamide (15e)
19

o
Yield 81%, mp: 282 C, IR(KBr) (υ, cm-1), 2978, 1705, 1628 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.45 (d, J = 8.1 Hz, 2H, Ar), 8.28 (s, 1H, CH=N), 8.11
(d, J = 8.1 Hz, 2H, Ar), 8.01 (d, J = 8.1 Hz, 2H, Ar), 7.73 (d, J = 8.1 Hz, 2H, Ar), 3.18 (s, 3H,
N-CH3), 2.95 (s, 3H, N-CH3); 13C NMR (75.5 Hz, DMSO-d6) δ (ppm): 159.89, 129.94,
128.89, 127.01, 126.50, 41.54, 35.54.
4.9.6. 4-[6-(4-bromophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]-N-[(Z)-
(dimethylamino)methylidene]benzenesulfonamide (15f)
o
Yield 79%, mp: 286 C, IR(KBr) (υ, cm-1), 2978, 1705, 1628 (C=N), 1342, 1149 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.44 (d, J = 8.4 Hz, 2H, Ar), 8.28 (s, 1H, CH=N), 8.01
(m, 4H, Ar), 7.85 (d, J = 8.4 Hz, 2H, Ar), 3.18 (s, 3H, N-CH3), 2.95 (s, 3H, N-CH3); 13C-
NMR (75.5 MHz, DMSO-d6) δ (ppm): 166.76, 160.46, 145.16, 144.78, 133.18, 129.66,
128.64, 128.59, 127.28, 127.19, 126.86, 41.48, 35.64.
4.9.7. N-[(Z)-(dimethylamino)methylidene]-4-[6-(4-nitrophenyl)[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-3-yl]benzenesulfonamide (15g)
o
Yield 87%, mp: 294 C, IR(KBr) (υ, cm-1), 3078, 1705, 1636 (C=N), 1342, 1142 (SO2); 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.46 (m, 4H, Ar), 8.36 (d, J = 9 Hz, 2H, Ar), 8.30 (s,
1H, CH=N), 8.02 (d, J = 8.4 Hz, 2H, Ar), 3.18 (s, 3H, N-CH3), 2.95 (s, 3H, N-CH3); 13C-
NMR (75.5 MHz, DMSO-d6) δ (ppm): 166.23, 160.49, 145.06, 143.58, 133.18, 128.96,
128.43, 128.47, 127.29, 128.17, 127.76, 41.44, 35.62.
4.10. Synthesis of triazolothiadiazole benzenesulfonamide compounds (5a-5g):
General procedure: The compounds 5a-5g were synthesized by refluxing a mixture of 11a-
11g in conc. hydrochloric acid for 3 hrs. This leads to the deprotection of sulfonamide under
acidic condition. After the completion of reaction the reaction mixture was cooled to room
temperature and a solid was precipitated out which was filtered off, washed with excess of
water, dried and recrystalized from suitable solvent to afford the title compounds.
4.10.1 4-(6-phenyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)benzenesulfonamide(5a)
o
1
Yield 81%, mp: 306 C, IR(KBr) (υ, cm-1), 3217, 3063, 1520, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.51 (d, J = 8.1 Hz, 2H, Ar), 8.08 (m, 4H, Ar), 7.69 (m, 3H,
Ar), 7.54 (s, 2H, SO2NH2); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 167.79, 155.26,
20

145.67, 145.09, 133.58, 130.18, 129.33, 128.76, 127.86, 127.02, 126.84; DART MS m/z 358
(M+H)+, C15H11N5O2S2H+, calcd. 357.41.
4.10.2.4-[6-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide(5b)
o
1
Yield 79%, mp: 312 C, IR(KBr) (υ, cm-1), 3217, 3063, 1520, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.48 (d, J = 8.4 Hz, 2H, Ar), 8.06 (d, J = 8.4 Hz, 2H, Ar),
7.95 (d, J = 8.1 Hz, 2H, Ar), 7.56 (s, 2H, SO2NH2), 7.43 (d, J = 8.1 Hz, 2H, Ar), 2.40 (s, 3H,
CH3); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 167.62, 155.39, 145.56, 145.01, 133.32,
130.45, 129.54, 128.76, 127.86, 127.02, 123.64, 21.48; DART MS m/z 372 (M+H)+,
C16H13N5O2S2H+, calcd. 371.44.
4.10.3.4-[6-(4-methoxyphenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide (5c)
o
1
Yield 83%, mp: 314 C, IR(KBr) (υ, cm-1), 3217, 3063, 1520, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.51 (d, J = 8.4 Hz, 2H, Ar), 8.06 (d, J = 8.4 Hz, 2H, Ar),
8.05 (d, J = 9.0 Hz, 2H, Ar), 7.53 (s, 2H, SO2NH2), 7.19 (d, J = 9.0 Hz, 2H, Ar), 3.89 (s, 3H,
OCH3); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 167.04, 156.34, 145.18, 144.79, 133.32,
130.45, 129.54, 128.76, 127.86, 127.02, 123.74, 21.67; DART MS m/z 388 (M+H)+,
C16H13N5O3S2H+, calcd. 387.44.
4.10.4. 4-[6-(4-fluorophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide (5d)
o
1
Yield 77%, mp: 310 C, IR(KBr) (υ, cm-1), 3225, 3047, 1605, 1558, 1335, 1165 (SO2); H-
NMR (300 MHz, DMSO-d6) δ (ppm): 8.49 (d, J = 8.1 Hz, 2H, Ar), 8.17 (m, 2H, Ar), 8.06 (d,
J = 8.1 Hz, 2H, Ar), 7.55 (s, 2H, SO2NH2), 7.49 (m, 2H, Ar); 13C-NMR (75.5 MHz, DMSO-
d6) δ (ppm): 166.67, 145.73, 130.65, 130.53, 128.74, 127.01, 126.84, 125.95, 117.53, 117.23;
DART MS m/z 376 (M+H)+, C15H10FN5O2S2H+, calcd. 375.40.
4.10.5. 4-[6-(4-chlorophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide(5e)
o
1
Yield 78%, mp: 318 C, IR(KBr) (υ, cm-1), 3240, 3078, 1597, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.50 (d, J = 8.1 Hz, 2H, Ar), 8.13 (d, J = 8.4 Hz, 2H, Ar),
8.06 (d, J = 8.1 Hz, 2H, Ar), 7.73 (d, J = 8.4 Hz, 2H, Ar), 7.54 (s, 2H, SO2NH2); 13C-NMR
(75.5 MHz, DMSO-d6) δ (ppm): 166.66, 145.77, 138.25, 130.53, 129.63, 128.72, 128.24,
127.02, 126.87; DART MS m/z 392/394 (M+H)+/(M+H+2)+, C15H10ClN5O2S2H+, calcd.
391.86/393.86.
21

4.10.6. 4-[6-(4-bromophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide(5f)
o
1
Yield 73%, mp: 304 C, IR(KBr) (υ, cm-1), 3286, 3063, 1589, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.48 (d, J = 8.1 Hz, 2H, Ar), 7.99 (m, 4H, Ar), 7.89 (d, J =
8.1 Hz, 2H, Ar), 7.55 (s, 2H, SO2NH2); 13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 166.80,
145.75, 145.14, 138.25, 129.63, 128.72, 128.24, 127.02, 126.87; DART MS m/z 438/440
(M+H)+/(M+H+2)+, C15H10BrN5O2S2H+, calcd. 436.31/438.31.
4.10.7. 4-[6-(4-nitrophenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]benzenesulfonamide(5g)
o
1
Yield 69%, mp: 302 C, IR(KBr) (υ, cm-1), 3217, 3063, 1520, 1342, 1165 (SO2); H-NMR
(300 MHz, DMSO-d6) δ (ppm): 8.50 (d, J = 8.1 Hz, 2H, Ar), 8.44 (d, J = 8.7 Hz, 2H, Ar),
8.37 (d, J = 8.7 Hz, 2H, Ar), 8.07 (d, J = 8.4 Hz, 2H, Ar), 7.58 (s, 2H, SO2NH2); 13C-NMR
(75.5 MHz, DMSO-d6) δ (ppm): 166.21, 146.24, 145.36, 133.29, 129.09, 128.63, 128.29,
126.90, 126.59, 124.91; DART MS m/z 403 (M+H)+, C15H10N6O4S2H+, calcd. 402.41.
Acknowledgements
One of the authors (SitaRam) is grateful to the Council of Scientific and Industrial
Research, New Delhi, India and the other (Poonam Khloya) to the Haryana State Council for
Science and Technology (HSCST), Panchkula (Haryana), India for the award of Senior
Research Fellowships. The authors are thankful to Sophisticated Analytical Instrument
Facility, Central Drug Research Institute, Lucknow for providing Mass spectra. Work from
CTS lab was financed by an EU project of the FP7 programme (Metoxia).
22

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25

Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of
tumor associated carbonic anhydrase isoforms hCA IX and hCA XII
SitaRam, Gulsah Celik, Poonam Khloya, Daniela Vullo,
Claudiu T. Supuran, Pawan K. Sharma
H2NO2S
H2NO2S
N
N
H2NO2S
N
N
S
S
N
N
N
N
N
S
NH
N
R
R
R
3c, 3d, 3e, 3f, 3g, 4c, 4f KI(nM) < 10 against hCA IX
3c, 3d, 3e, 3f, 3g, 4b, 4c, 4f 4f KI(nM) < 5 against hCA XII
Novel heterocyclic compounds containing benzenesulfonamide moiety bearing 1,2,4-triazole
scaffold showed excellent carbonic anhydrase hCA IX and hCA XII inhibitory efficiency and
also promising selectivity over hCA I and hCA II.

Research Highlights
•
•
•
•
•
Library of novel hCA I, II, IX and XII inhibitors was synthesized.
Benzene sulphonamide incorporated to 1,2,4-triazole scaffold.
Compounds were screened against hCA isoforms I, II, IX and XII.
Tested compounds showed excellent, low nanomolar affinity for CA isozymes.
Some of the tested compounds showed high degree of selectivity for hCA IX and XII.