Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl) moieties and evaluation of their carbonic anhydrases I, II, IV and IX inhibitory effects

Article abstract of DOI:10.1016/j.bmc.2017.02.037

Using celecoxib as lead, two novel series of sulfonamides incorporating the pyridotriazolopyrimidine scaffold have been synthesized and evaluated in vitro as inhibitors against four relevant human (h) carbonic anhydrases (CAs, EC 4.2.1.1), the cytosolic and ubiquitous hCA I and II as well as the transmembrane hCA IV and hCA IX. Most of the reported sulfonamides acted as efficient, low micromolar inhibitors of hCAI, II and IV, whereas they displayed higher efficacy in inhibiting the tumor-associated isoform hCA IX. Many derivates herein reported showed better hCA IX versus hCA II selectivity ratios compared to celecoxib or acetazolamide. Considering isoform IX is a validated target for the diagnosis and treatment of hypoxic tumors, discovery of selective CA IX inhibitors represents a promising step to unveil more effective anticancer therapies.

Full text of DOI:10.1016/j.bmc.2017.02.037

Accepted Manuscript
Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]tria-
zolo[4,3-a]pyrimidin-1(5H)-yl) moieties and evaluation of their carbonic anhy-
drases I, II, IV and IX inhibitory effects
Mohamed Fares, Radwa A. Eladwy, Alessio Nocentini, Soha R. Abd El Hadi,
Hazem A. Ghabbour, Ashraf Abdel-Megeed, Wagdy M. Eldehna, Hatem A.
Abdel-Aziz, Claudiu T. Supuran
PII:
S0968-0896(17)30176-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.037
BMC 13567
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 January 2017
15 February 2017
17 February 2017
Please cite this article as: Fares, M., Eladwy, R.A., Nocentini, A., Abd El Hadi, S.R., Ghabbour, H.A., Abdel-
Megeed, A., Eldehna, W.M., Abdel-Aziz, H.A., Supuran, C.T., Synthesis of bulky-tailed sulfonamides incorporating
pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl) moieties and evaluation of their carbonic anhydrases I, II,
IV and IX inhibitory effects, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.02.037
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Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-
1(5H)-yl) moieties andevaluationof their carbonic anhydrases I, II, IV and IX inhibitory effects
c
b
Mohamed Fares a,b,*, Radwa A. Eladwy , Alessio Nocentini d,e, Soha R. Abd El Hadi ,
Hazem A. Ghabbour f,g, Ashraf Abdel-Megeed h, Wagdy M. Eldehna i, Hatem A. Abdel-Aziz
j, Claudiu T. Supuran d*
a School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo, 11829, Egypt
c Department of Pharmacology and Toxicology, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt
d Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence,
Polo Scientifico, Via U. Schiff 6, 50019 Sesto, Fiorentino, Firenze, Italy
eUniversità degli Studi di Firenze, Neurofarba Department., Sezione di Scienze Farmaceutiche, Laboratory of
Molecular Modeling Cheminformatics & QSAR, via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
f Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia
g Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt.
h Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
i
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh
33516, Egypt
j Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622, Egypt.
Abstract. Using celecoxib as lead, two novel series of sulfonamides incorporating the
pyridotriazolopyrimidine scaffold have been synthesized and evaluated in vitro as inhibitors
against four relevant human (h) carbonic anhydrases (CAs, EC 4.2.1.1), the cytosolic and
ubiquitous hCA I and II as well as the transmembrane hCA IV and hCA IX. Most of the
reported sulfonamides acted as efficient, low micromolar inhibitors of hCAI, II and IV,
whereas they displayed higher efficacy in inhibiting the tumor-associated isoform hCA IX.
Many derivates herein reported showed better hCA IX versus hCA II selectivity ratios
compared to celecoxib or acetazolamide. Considering isoform IX is a validated target for the
diagnosis and treatment of hypoxic tumors, discovery of selective CA IX inhibitors
represents a promising step to unveil more effective anticancer therapies.
____________
Keywords: Carbonic anhydrase, Tumour-associated isoforms, Pyridotriazolopyrimidine,
Sulfonamide, Celecoxib.
*Corresponding authors. e-mail: ph.fares@yahoo.com (M. Fares); Tel./fax:+39-055-4573729; e-mail:
claudiu.supuran@unifi.it (CTS)

1. Introduction:
Celecoxib I, a sulfonamide acting as dual cyclooxygenase – carbonic anhydrase (CA,
EC 4.2.1.1) inhibitor,1 was considered to be an attractive target for designing CA inhibitors
(CAIs) which may possess a dual action on these two families of enzymes involved in
various pathologies which cross-freact in some cases (e.g., glaucoma, cancer, arthritis).2-6.
Celecoxib shows a nanomolar affinity for the cytosolic human (h) isoform hCA II (Ki of 21
nM), inhibiting even better the tumour associated enzymes hCA XI (Ki of 16 nM) and hCA
XII (Ki of 18 nM).1 These enzymes were recently validated as antitumor/antimetastatic
targets with one compounds (SLC-0111) in Phase Ib/II clinical trials.2,3 The X-ray crystal
structure of celecoxib bound CA II (PDB code: 6cox) reported by one of our groups,1
afforded the deep understanding of the groups important for interaction with the CA II active
site. The sulfonamide group of I was observed to be coordinated to Zn+2 in a tetrahedral
coordination geometry, whereas the trifluoromethyl group occupied the hydrophobic pocket,
located in one half of the active site.1a Unexpectedly, the p-tolyl moiety of the drug was
observed to point towards the hydrophilic half of the active site, making favourable contacts
with residues Asn67, Glu69 and Gln92.1a
Figure 1. Structure of some reported sulfonamide CAIs of types I-III.
The presence of the 2-phenyl substituent in the triazolobenzenesulfonamide derivative II
(Figure 1) elicited moderate to strong inhibition of four CAs, namely, CA I, CA II, CA IX
and CA XII, with KI ranging between 0.041 to 0.542 µM.7 Modification of the phenyl group
to a ferrocene based moiety, as in compound III, led to a decrease of the activity towards CA
I and CA XII, while retaining a good inhibitory effect against CA II (Ki of 0.036 µM) and
CA IX (Ki of 0.065 µM).7

-
-
-
A
B
C
Figure 2: Schematic representation of possible interactions with CA IX active site (PDB:
4q06)1a of celecoxib I (A); the newly designed scaffold, with suggested interactions with the
enzyme active site (B), and the newly designed PTP derivatives bound to the Zn(II) ion
within the CA active site (C).
An analysis of a modelling study of celecoxib bound to CA IX, showed a similar mode
of interaction between the sulfonamide and the active site, as in the hCA II – celecoxib
complex analyzed by X-ray crystallography (Figure 2A). Our current model of designing
selective CAIs is based on a scaffold that can be substituted with less polar groups (Ar1 and
Ar2 in Figure 2B), in order to fill in the unoccupied hydrophobic pocket (Figure 2B). In view
of the aforementioned facts and in continuation of our earlier work on the design of inhibitors
of these enzymes,1-3 it was envisaged to incorporate the bulky pyridotriazolopyrimidine
(PTP) scaffold as tail to the benzene-sulfonamide motif. We aimed to exploit the unoccupied
hydrophobic region shown in Figure 2A, within the active site of CA IX, hypothesizing that
the aryls at position 6 and 8 of the PTP scaffold may interact in a favorable manner with the
enzyme active site, as shown schematically in Figure 2C. We report herein the synthesis and
the carbonic anhydrases inhibition evaluation of the new such derivatives, incorporating the
bulky PTP scaffold.
2. Results and discussion:
2.1.Chemistry:
Our proposed synthetic methodology to the desired pyridotriazolopyrimidine scaffold
containing sulfonamide moiety is depicted in Scheme 1, Scheme 2 and Scheme 3. The
synthesis of the 6-amino-2-thiouracil (3) was accomplished as reported by refluxing ethyl
cyanoacetate (1) with thiourea (2) in sodium ethoxide.8 The condensation of the 6-amino-2-
thiouracil (3) with different aromatic α-β unsaturated ketones, β-enaminones, diketones and

aldehydes was reported as convenient and efficient strategy to synthesize substituted
pyrido[2,3-d]pyrimidines.9-13 Quiroga et al.12 reported that the reaction of the 6-amino-2-
thiouracil (3) with different chalcones in refluxing DMF resulted in the formation of the
pyrido[2,3-d]pyrimidines 5 and/or 6. When the reaction performed under argon atmosphere
for short period of time, it yielded mainly the 2-thioxo-2,3,5,8-tetrahydropyrido[2,3-
d]pyrimidin-4(1H)-one derivative 6. However, under exposure to air for long time in
refluxing DMF, the oxidized isomer 5 was formed. In addition, the substitutions of phenyl
groups of the chalcone affect the product selectivity where the electron withdrawing group
preferentially favours the formation of the oxidized form 5.12
Scheme 1: Prepatration of compounds 5. Reagents and conditions: (i) NaOEt/Ethanol (ii) dry DMF
/reflux 15h.
The synthesis of the key pyrido[2,3-d]pyrimidines 5a-j was accomplished via reacting
6-amino-2-thiouracil 3 and different α-β unsaturated aromatic ketones 4a-j14 in refluxing dry
DMF under air in 40-65% yields (Scheme 1). The structures assigned to compounds 5a-j
1
1
were confirmed by H and 13C NMR. H NMR spectra of the synthesized pyrido[2,3-
d]pyrimidines showed the presence of a singlet at δ 7.56-8.07 ppm integrated to the pyridine
ring proton and two NH singlet signals at δ 12.23-12.41 and 12.90-13.10 ppm corresponding
to two NHs of the pyrimidine ring. Furthermore, 13C NMR spectra of 5a-c revealed the
signals of the pyridine H at δ 108.20-108.27 ppm and the signal of thioxo carbon at δ 175.41-
175.43 ppm.
The hydrazonoyl chlorides 11a and 11b synthesis was started with the chlorination of
the active methylene group of acetylacetone 7a and ethyl acetoacetate 7b using sulphuryl
chloride to afford the α-chloroacetyl derivatives 8a and 8b, as described by Alihn in 1878.15
Then by applying Japp-Klingemann coupling of the aforementioned α-chloroacetyl

derivatives (8a and 8b) with the diazonium salts of sulfanilamide 10, we obtained the acetyl
derivative 11a and the ester derivative 11b in reasonable yields (65 and 72%, respectively)
(Scheme 2).16
Scheme 2: Synthesis of derivatives 11. Reagents and conditions: (i) SO2Cl2/ 0 °C (ii) NaNO2/HCl (iii)
CH3COONa/ 0 °C
The reaction of the pyrido[2,3-d]pyrimidine thiones and hydrazonoyl halides have been
extensively studied.16-19 This reaction proceeds via 1,3-dipolar cycloaddition of hydrazonoyl
chlorides with dipolarophile thiones.17
A literature review of this reaction almost exclusively prefers isomer 16 over 15
(Scheme 3). This suggest that the reaction proceeds through the cyclo-addition of carbon 2
and nitrogen 3 of the pyrido[2,3-d]pyrimidine thiones to the hydrazonoyl chloride derivative
in the presence of a base (Scheme 3).17 The reaction mechanism was postulated to start with
S-alkylation of the thiouracil derivative to afford the thiohydrazones 12. In the presence of a
base, the nucleophilicity of the terminal hydrazone nitrogen increases and the compound
undergoes a Chapman-like rearrangement to give the corresponding thiohydrazide 13. The
latter compound was cyclized in situ to afford 15 or 16. 1H NMR spectra of compounds 16a-t
showed disappearance of the two NH singlet signals. The acetyl derivatives displayed singlet
1
signals resonating at δ 2.63-2.68 ppm representing the acetyl CH3 protons. Meanwhile, H
NMR of the ester analogs showed a typical triplet-quartet pattern of the ethyl protons at δ
1.24-1.31 and 4.42-4.43 ppm.

Scheme 3: Preparation of compounds 15 and 16. Reagents and conditions: (i) Dioxane, TEA, reflux,
6-14h.
Analysis of the 13C NMR spectra of the newly synthesized derivatives indicated that we
preferentially synthesized isomer 16 rather than isomer 15. It was reported that the chemical
shift of the carbonyl carbon in pyrimidin-4-one derivatives is relying on the adjacent
environment.20 For instance, if the adjacent nitrogen is pyrrole type (sp3-hybridized) (as in
16) the carbonyl carbon signals of the pyrimidinone ranged from 160 to 165 ppm.
Nevertheless, if the neighbouring atom is a pyridine type (as in 15) chemical shift of the
carbonyl carbon usually appears at 170-175 ppm. 13C NMR spectral data of compounds 16a-t
revealed carbonyl carbon signals of the pyrimidinone at 159.90 and 163.21 ppm. This result
is in line with previous reports of the reactions of hydrazonoyl halides with similarly
condensed 2- thioxopyridopyrimidines.16-21
2.2. Carbonic anhydrase inhibition:
All the synthesized derivatives 16a-16t were evaluated for their efficacy in inhibiting four
relevant CA isoforms, i.e., hCA I, II, IV and IX, by using the stopped flow carbon dioxide
hydrase assay22, in comparison to acetazolamide (AAZ) as a standard CAI.

In general, all the assayed compounds displayed good inhibitory action against the reported
hCA isoforms, with KI spanning from the low micromolar to the low-medium nanomolar
−activity
range. In detail, the following structure -relationship (SAR) can be gathered from the
data reported in Table 1, for each tested isoform:
Table 1: Inhibition data of human CA isoforms hCA I, II, IV and IX with sulfonamides 16a-
16t, celecoxib and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped
flow CO2 hydrase assay.22
Compound
Structure
R1
R2
KI (µM)
hCA I
3.79
NA
hCA II hCA IV
hCA IX
0.18
3.26
0.25
3.24
0.35
1.46
0.33
1.45
0.33
2.46
1.12
1.90
0.19
1.62
0.99
I
I
-COCH3
-COOC2H5
-COCH3
-H
-H
0.59
3.54
1.19
4.66
2.03
3.65
0.74
1.58
0.86
3.05
0.73
1.41
0.35
1.48
3.43
3.00
4.62
3.58
4.90
3.56
4.70
3.01
NA
16a
16b
16c
16d
16e
16f
I
-F
6.64
9.35
7.42
9.16
5.50
7.37
5.52
7.29
6.78
6.82
2.65
NA
I
-COOC2H5
-COCH3
-F
I
-Cl
I
-COOC2H5
-COCH3
-Cl
I
-OCH3
-OCH3
-NO2
-NO2
-H
16g
16h
16i
I
-COOC2H5
-COCH3
I
2.92
3.56
4.79
NA
I
-COOC2H5
-COCH3
16j
II
II
II
II
II
16k
16l
-COOC2H5
-COCH3
-H
-F
2.45
3.96
2.52
16m
16n
16o
-COOC2H5
-COCH3
-F
-Cl
NA

II
II
II
II
II
-COOC2H5
-COCH3
-Cl
2.64
9.04
0.85
3.02
8.71
>10
1.71
2.98
0.72
0.50
1.75
0.021
0.01
4.33
2.23
4.25
0.20
2.98
0.29
0.07
0.39
0.42
0.38
0.16
1.10
0.02
0.03
16p
-OCH3
-OCH3
-NO2
16q
-COOC2H5
-COCH3
16r
16s
-COOC2H5
-NO2
16t
Celecoxib
AAZ
0.25
*Mean from 3 different assays, by a stopped flow technique (errors were in the range of
−10% of the reported values).
5
(i) The cytosolic isoform hCA I was inhibited by most sulfonamides herein reported in the
low micromolar range (KIs between 2.64 and 9.35 µM), with the exception of compounds
16b, 16n and 16o which did not inhibit hCA I up to 100 µM. Only derivative 16r showed a
submicromolar hCA I inhibitory efficacy (KIs 0.85 µM). Such results ascribed to the prepared
compounds better inhibitory efficacy than those of celecoxib, although worsened compared to
the clinically used sulfonamide AAZ.
(ii) The physiologically dominant isoform hCA II was efficiently inhibited by all
sulfonamides 16a-16t, with KI values ranging between 0.35 and 4.66 µM (Table 1). Despite
the impossibility to draw a clear SAR, probably elicited by the rather flat inhibitory tendency
revealed against hCA II, it is worth noting the submicromolar inhibitors 16a, 16g, 16i, 16k,
16m, 16r and 16s (KIs between 0.35 and 86 µM). All of them bear the acetyl group in
position R1 (except 16r), thus highlighting the better impact of such a group on the binding
efficacy, compared to the carboxymethyl moiety.
(iii) Likewise hCA I, most of the reported sulfonamides showed comparable effectiveness in
inhibiting the membrane-associated isoform hCA IV, with KI values spanning in the low
micromolar range (2.23- 4.90 µM), except 16h and 16l which did not inhibit hCA I up to 100
µM. Only the nitro derivative 16s, which appended the acetyl and the thienyl groups at the
PTP scaffold, acted as a potent hCA IV inhibitor with a KI of 0.20 µM.
(iv) Finally, the tumor-associated isoform hCA IX was potently inhibited by the most of the
reported sulfonamides with KI reaching the low-medium nanomolar range (0.16 – 3.26 µM).
In particular, the acetyl bearing derivatives 16a, 16c, 16e, 16g and 16i from series I and 16m
and 16s from series II, displayed a nanomolar inhibitory profile (KIs of 0.18, 0.25, 0.35, 0.33,
0.33, 0.19 and 0.16 µM, respectively) likened to the corresponding carboxyethyl analogues

(KIs of 3.26, 3.24, 1.46, 1.45, 2.46, 1.62 and 1.10 µM, respectively). Conversely, only the
carboxyethyl bearing compounds 16p and 16r demonstrated to be better hCA IX inhibitors
(KIs of 0.39 and 0.38 µM) in comparison to the acetyl analogues 16o and 16q (KIs of 0.99
and 0.42 µM). Moreover, the data in Table 1 undeniably highlighted the positive effect on the
inhibitory efficacy against hCA IX obtained by swapping the 4-Cl-phenyl group with the
thienyl one.
(v) Despite the not enviable efficacy that sulfonamides 16a-16t showed against the tumor-
associated isoform (celecoxib and AAZ displayed KIs of 0.02 and 0.03 µM), it is reasonable
to focus the attention on the interesting selective profile they possess against hCA IX versus
hCA II (Table 2). Indeed, it is satisfying to note that most of the reported compounds
displayed a selectivity ratio hCA IX/hCA II from two-fold to fourteen-fold higher than
celecoxib or AAZ. Only 16k, 16l and 16n were non-selective hCA IX over hCA II inhibitors.
On the other hand, all the acetyl PTP sulfonamides from the series I and 16k and 16m from
series II were shown to possess much better selectivity for the tumor-associated isoform hCA
IX over hCA IV, compared to the corresponding carboxyethyl analogues and AAZ, while
exhibited comparable selectivity ratio with celecoxib. A different behaviour may be
highlighted for the remaining derivatives of series II. In fact, the carboxyethyl bearing
sulfonamides 16p, 16r and 16t were more selective inhibitors of hCA IX over hCA IV in
comparison to their acetyl analogues. In addition, 16p and 16r displayed selectivity ratio
hCA IX/IV comparable to celecoxib, being four-fold better than AAZ.
(vi) As more than once highlithed here, the data reported in Table 1 clearly demonstrate that
the replacement of the acetyl group in position R1 by the carboxyethyl moiety generally
worsened the inhibition profiles of derivatives 16a-16t depending on the substituents Ar1, R2
and the considered isoform.It is reasonable to hypothesize that such a diminished inhibitory
efficacy of the carboxyethyl derivatives in comparison to the acetyl ones might derive from
the steric hyndrance aroused by the PTP. Indeed, such a bulky core may lead to a rather rigid
and fixed positioning of the tricyclic scaffold within the active site pocket, obviously
depending on the substituents Ar1 and R2. Hence, in most cases the replacement of the acetyl
group with the bigger carboxyethyl moiety may cause clashes with amino acid residues from
the hydrophobic pocket, worsening thus the inhibitory activity.
Table 2: Selectivity ratios for the inhibition of hCA IX over hCA II and hCA IX over hCA
IV for the compounds 16a-16t reported in the paper:

Selectivity ratio
Compound
16a
hCA IX/hCA II
hCA IX/hCA IV
16.90
1.42
3.35
1.08
4.68
1.44
5.78
2.49
2.23
1.09
2.62
1.24
0.65
0.75
1.84
0.91
3.45
4.37
7.06
1.86
3.13
1.58
1.05
0.48
16b
14.12
1.51
16c
16d
10.14
3.21
16e
16f
9.12
16g
NA
16h
8.86
16i
1.45
16j
4.26
16k
NA
16l
12.83
2.44
16m
16n
2.53
16o
11.08
5.29
16p
16q
11.00
1.23
16r
16s
2.70
16t
14.5
Celecoxib
AAZ
2.96
3. Conclusions
Two novel series of sulfonamides, 16a-16j and 16k-16t, containing the
pyridotriazolopyrimidine scaffold have been synthesized and evaluated in vitro as inhibitors
against four relevant hCAs, comprising the cytosolic and ubiquitous isozymes hCA I and II
as well as the transmembrane hCA IV and hCA IX, the last one a validated antitumor drug
target. Most of the reported sulfonamides acted as good, low micromolar inhibitors of hCA I,
II and IV, whereas they displayed a better efficacy in inhibiting the tumor-associated isoform
hCA IX. Many derivatives herein reported highlighted better selectivity ratios for inhibiting
hCA IX over hCA II than celecoxib and AAZ. Conversely, some sulfonamides among the
two series demostrated a comparable selectivity for hCA IX over hCA IV, similar to
celecoxib, whereas being 3 to 6-fold better than AAZ. Considering that isoform hCA IX is a
validated target for the diagnosis and treatment of cancers, discovery of selective inhibitors
represents a promising step to unveil a more effective cancer therapy, and moreover the
obtained SAR might further help in the design of novel, isoform-selective inhibitors.

4. Experimental
4.1 Chemistry:
4..1.1. General:
Melting points were measured with a Stuart apparatus and were uncorrected. The NMR
spectra were recorded by Varian Gemini-300BB 300, 400 and 500 MHz FT-NMR
spectrometers (Varian Inc., Palo Alto, CA). 1H spectra were run at 300, 400 and 500 MHz
and 13C spectra were run at 75, 100 and 125 MHz, respectively, in deuterated
dimethylsulphoxide (DMSO-d6). Chemical shifts (δH) are reported relative to TMS as internal
standard. All coupling constant (J) values are given in hertz. The abbreviations used are as
follows: s, singlet; d, doublet; t, triplet, m, multiplet. Microanalyses were carried out using
Perkin Elmer PE 2400 CHN Elemental Analyzer and the results were within 0.4%.
Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was
employed routinely to follow the course of reactions and to check the purity of products. All
reagents and solvents were purified and dried by standard techniques. 6-Amino-2-thiouracil
(3),23 hydrazonoyl chlorides 11a, 11b,18 pyrido[2,3-d]pyrimidines 5d, 5e, 5g were prepared
according to the literature procedure.12
4.1.2. General procedure for preparation of 5-aryl-7-(thiophen-2-yl / 4-chlorophenyl)-2-
thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (5a-j) and spectral data are provided in
the supplementary file.
4.1.3. General procedure for preparation of target compounds (16a-t). To a mixture of 5-
aryl-7-(thiophen-2-yl / 4-chlorophenyl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-
one (5a-j) (1 mmol) and the appropriate hydrazonoyl chloride 11a, b (1 mmol) in dioxane (30
mL), triethylamine (0.14 mL, 1 mmol) was added. The reaction mixture was refluxed until
the starting materials were fully consumed or until conversion was observed to stall
(monitored by TLC). The solvent was removed under vacuum and the residue was triturated
with methanol. The formed solid was washed thoroughly with water, filtered then crystallized
from
DMF:EtOH
[v:v,
1:1]
or
subjected
to
flash
chromatography
(methanol/dichloromethane, 1:10) to give 16a-t. Compounds 16k and 16l were previously
synthesized in our laboratory.18
4.1.3.1.
4-(3-Acetyl-8-(4-chlorophenyl)-5-oxo-6-phenylpyrido[2,3-d][1,2,4]triazolo[4,3-
1
a]pyrimidin-1(5H)-yl)benzenesulfonamide (16a): Yield: 46%, m.p. >300 ºC; H NMR (500

MHz, DMSO-d6) δ ppm: 2.66 (s, 3H, -CH3), 7.46-7.48 (m, 5H, Ar-H), 7.54 (br. s, 2H, -
SO2NH2, D2O exchangeable), 7.60 (d, J = 8.5 Hz 2H, Ar-H), 7.78 (s, 1H, pyridine H), 8.10
(d, J = 9.0 Hz 2H, Ar-H), 8.35 (d, J = 8.5 Hz 2H, Ar-H), 8.48 (d, J = 9.0 Hz 2H, Ar-H); 13C
NMR (125 MHz, DMSO-d6) δ ppm: 30.11, 108.04, 119.31, 120.69, 127.71, 128.19, 128.61,
128.93, 129.43, 130.14, 136.20, 136.44, 139.26, 139.58, 142.23, 142.70, 147.67, 155.12,
155.52, 160.27, 160.31, 188.03; Anal. Calcd for C28H19ClN6O4S: C, 58.90; H, 3.35; N, 14.72.
Found: C, 59.20; H, 3.56; N, 14.52.
4.1.3.2.
Ethyl
8-(4-chlorophenyl)-5-oxo-6-phenyl-1-(4-sulfamoylphenyl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16b): Yield: 57%, m.p.
o
1
>300 ºC C; H NMR (500 MHz, DMSO-d6) δ ppm: 1.29 (t, J = 7 Hz, 3H, -COOCH2CH3),
4.43 (q, J = 7 and 7, 2 Hz, -COOCH2CH3), 7.45-7.47 (m, 5H, Ar-H), 7.53 (br. s, 2H, -
SO2NH2, D2O exchangeable), 7.59 (d, J = 8.5 Hz, 2H, Ar-H), 7.77 (s, 1H, pyridine H), 8.09
(d, J = 8.5 Hz, 2H, Ar-H), 8.34 (d, J = 8.5 , 2H, Ar-H), 8.44 (d, J = 8.5 , 2H, Ar-H); 13C NMR
(125 MHz, DMSO-d6) δ ppm: 13.72, 63.63, 107.39, 118.77, 120.26, 127.21, 127.72, 128.18,
128.47, 128.94, 129.65, 135.73, 135.87, 135.98, 138.76, 139.03, 142.24, 146.63, 154.57,
154.76, 156.12, 159.79, 159.90; Anal. Calcd for C29H21ClN6O5S: C, 57.95; H, 3.52; N, 13.98.
Found: C, 57.76; H, 3.87; N, 13.82.
4.1.3.3.
4-(3-Acetyl-8-(4-chlorophenyl)-6-(4-fluorophenyl)-5-oxopyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16c): Yield: 51%, m.p.
>300 ºC; 1H NMR (500 MHz, DMSO-d6) δ ppm: 2.67 (s, 3H, -CH3), 7.28 (t, J = 8.5, 2H, Ar-
H), 7.52-7.55 (m, 4H, 2Ar-H + -SO2NH2, D2O exchangeable), 7.59 (d, J = 8.5 , 2H, Ar-H),
7.79 (s, 1H, pyridine H), 8.09 (d, J = 8.5 , 2H, Ar-H), 8.34 (d, J = 8.5 , 2H, Ar-H), 8.47 (d, J
13
= 8.5 , 2H, Ar-H); C NMR (125 MHz, DMSO-d6) δ ppm: 29.63, 107.56, 114.51 (2JF-C
=
21.38 Hz), 118.88, 120.20, 127.21, 128.93, 129.64, 130.66 (3JF-C = 8.38 Hz), 135.26, 135.29,
135.76, 135.88, 138.74, 141.73, 142.24, 146.81, 153.50 (1JF-C = 198.00 Hz), 159.79, 161.24,
163.19, 187.51; Anal. Calcd for C28H18ClFN6O4S: C, 57.10; H, 3.08; N, 14.27. Found: C,
57.46; H, 3.36; N, 14.56.
4.1.3.4.
Ethyl
8-(4-chlorophenyl)-6-(4-fluorophenyl)-5-oxo-1-(4-sulfamoylphenyl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16d): Yield: 62%, m.p.
>300 ºC; 1H NMR (500 MHz, DMSO-d6) δ ppm: 1.30 (t, J = 7Hz, -COOCH2CH3), 4.43 (q, J
= 7 and 7.5 Hz, 2H, -COOCH2CH3), 7.29 (t, J = 9 Hz, 2H, Ar-H), 7.52-7.55 (m, 4H, 2Ar-H +

-SO2NH2, D2O exchangeable), 7.61 (d, J = 8.5 , 2H, Ar-H), 7.81 (s, 1H, pyridine H), 8.09 (d,
13
J = 8.5 , 2H, Ar-H), 8.35 (d, J = 9 , 2H, Ar-H), 8.44 (d, J = 9 , 2H, Ar-H); C NMR (125
MHz, DMSO-d6) δ ppm: 13.74, 63.65, 107.46, 114.53 (2JF-C = 21.88 Hz), 118.86, 120.28,
127.22, 128.97, 129.68, 130.69, 130.76 (3JF-C = 8.63 Hz), 135.28, 135.77, 135.87, 135.96,
138.74, 142.27, 146.62, 153.46, 154.85 (1JF-C = 159.18 Hz), 159.86, 159.90, 161.27, 163.21;
Anal. Calcd for C29H20ClFN6O5S: C, 56.27; H, 3.26; N, 13.58. Found: C, 56.56; H, 3.59; N,
13.62.
4.4.3.5.
4-(3-Acetyl-6,8-bis(4-chlorophenyl)-5-oxopyrido[2,3-d][1,2,4]triazolo[4,3-
1
a]pyrimidin-1(5H)-yl)benzenesulfonamide (16e): Yield: 40%, m.p. >300 ºC; H NMR (500
MHz, DMSO-d6) δ ppm: 2.66 (s, 3H, -CH3), 7.50-7.55 (m, 6H, 4Ar-H + -SO2NH2, D2O
exchangeable), 7.62 (d, J = 8.5 Hz 2H, Ar-H), 7.82 (s, 1H, pyridine H), 8.10 (d, J = 9.0 Hz
13
2H, Ar-H), 8.36 (d, J = 8.5 Hz 2H, Ar-H), 8.48 (d, J = 9.0 Hz 2H, Ar-H); C NMR (125
MHz, DMSO-d6) δ ppm: 30.36, 108.18, 118.00, 120.96, 127.91, 128.42, 129.67, 130.37,
131.05, 133.76, 136.49, 136.59, 138.57, 139.43, 142.42, 142.95, 147.57, 153.93, 155.80,
160.48, 160.64, 188.26; Anal. Calcd for C28H18Cl2N6O4S: C, 55.55; H, 3.00; N, 13.88.
Found: C, 55.79; H, 3.16; N, 13.59.
4.4.3.6. Ethyl 6,8-bis(4-chlorophenyl)-5-oxo-1-(4-sulfamoylphenyl)-1,5-dihydropyrido[2,3-
1
d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16f): Yield: 52%, m.p. >300 ºC; H NMR
(500 MHz, DMSO-d6) δ ppm: 1.30 (t, J = 7, 3H, -COOCH2CH3), 4.45 (q, J = 7 and 7.5, 2H, -
COOCH2CH3), 7.49-7.54 (m, 5H, Ar-H), 7.61 (d, J = 8 Hz, 2H, Ar-H), 7.80 (s, 1H, pyridine
H), 8.09 (d, J = 8.0 Hz, 2H, Ar-H), 8.35 (d, J = 8.5 Hz, 2H, Ar-H), 8.44 (d, J = 8.5 Hz, 2H,
Ar-H); 13C NMR (125 MHz, DMSO-d6) δ ppm: 14.43, 64.35, 108.00, 119.40, 120.98, 127.91,
128.42, 129.66, 130.36, 131.07, 133.78, 136.51, 136.54, 136.59, 138.53, 139.40, 142.98,
147.32, 153.85, 155.51, 156.78, 160.55, 160.64; Calcd for C29H20Cl2N6O5S: C, 54.81; H,
3.17; N, 13.23. Found: C, 55.09; H, 3.30; N, 13.52.
4.4.3.7.
4-(3-Acetyl-8-(4-chlorophenyl)-6-(4-methoxyphenyl)-5-oxopyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16g): Yield: 35%, m.p.
>300 ºC; 1H NMR (300 MHz, DMSO-d6) δ ppm: 2.68 (s, 3H, -COCH3), 3.85 (s, 3H, -OCH3),
7.02 (d, J = 8.7 Hz, 2H, Ar-H), 7.45-7.48 (m, 4H, 2Ar-H + -SO2NH2, D2O exchangeable),
7.60 (d, J = 8.7 Hz, 2H, Ar-H), 7.76 (s, 1H, pyridine H), 8.09 (d, J = 9.0 Hz, 2H, Ar-H), 8.34

(d, J = 7.8 Hz, 2H, Ar-H), 8.48 (d, J = 8.4 Hz, 2H, Ar-H); Anal. Calcd for C29H21ClN6O5S: C,
57.95; H, 3.52; N, 13.98. Found: C, 58.00; H, 3.76; N, 14.24.
4.4.3.8. Ethyl 8-(4-chlorophenyl)-6-(4-methoxyphenyl)-5-oxo-1-(4-sulfamoylphenyl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16h): Yield: 47%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 1.31 (t, J = 7.2 Hz, 3H, -COOCH2CH3),
3.85 (s, 3H, -OCH3), 4.43 (q, J = 6.9 and 7.2 Hz, 2H, -COOCH2CH3), 7.01 (d, J = 8.7 Hz,
2H, Ar-H), 7.31-7.50 (m, 4H, 2Ar-H + -SO2NH2, D2O exchangeable), 7.60 (d, J = 8.7 Hz,
2H, Ar-H), 7.75 (s, 1H, pyridine H), 8.08 (d, J = 8.4 Hz, 2H, Ar-H), 8.33 (d, J = 8.7 Hz, 2H,
Ar-H), 8.44 (d, J = 8.7 Hz, 2H, Ar-H); Anal. Calcd for C30H23ClN6O6S: C, 57.10; H, 3.67; N,
13.32; Found: C, 57.32; H, 3.86; N, 13.59.
4.4.3.9.
4-(3-Acetyl-8-(4-chlorophenyl)-6-(4-nitrophenyl)-5-oxopyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16i): Yield: 52%, m.p.
1
>300 ºC; H NMR (400 MHz, DMSO-d6) δ ppm: 2.67 (s, 3H, -COCH3), 7.52 (s, 2H, -
SO2NH2, D2O exchangeable), 7.61 (d, J = 8.5 Hz, 2H, Ar-H), 7.72 (d, J = 8.5 Hz, 2H, Ar-H),
7.87 (s, 1H, pyridine H), 8.11 (d, J = 8.5 Hz, 2H, Ar-H), 8.32 (d, J = 8.5 Hz, 2H, Ar-H), 8.36
(d, J = 8.5 Hz, 2H, Ar-H), 8.49 (d, J = 8.5 Hz, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ
ppm: 30.08, 107.70, 118.89, 120.79, 123.29, 127.71, 129.46, 130.15, 130.34, 136.17, 136.44,
139.14, 142.13, 142.84, 146.42, 147.40, 147.68, 152.61, 155.51, 160.08, 160.63, 187.79;
Anal. Calcd for C28H18ClN7O6S: C, 54.60; H, 2.95; N, 15.92; Found: C, 54.86; H, 3.18; N,
15.83.
4.4.3.10.
Ethyl
8-(4-chlorophenyl)-6-(4-nitrophenyl)-5-oxo-1-(4-sulfamoylphenyl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16j): Yield: 63%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 1.24 (t, J = 6.6 Hz, 3H, -COOCH2CH3),
4.35 (q, J = 6.9 and 7.6 Hz, 2H, -COOCH2CH3), 7.54 (s, 2H, -SO2NH2, D2O exchangeable),
7.62 (d, J = 8.1 Hz, 2H, Ar-H), 7.74 (d, J = 7.5 Hz, 2H, Ar-H), 7.89 (s, 1H, pyridine H), 8.09
(d, J = 8.7 Hz, 2H, Ar-H), 8.32 (d, J = 7.5 Hz, 2H, Ar-H), 8.37 (d, J = 8.1 Hz, 2H, Ar-H),
8.44 (d, J = 8.7 Hz, 2H, Ar-H); Anal. Calcd for C29H20ClN7O7S: C, 53.92; H, 3.12; N, 15.18.
Found: C, 54.27; H, 3.36; N, 15.40.
4.4.3.11.
4-(3-Acetyl-6-(4-fluorophenyl)-5-oxo-8-(thiophen-2-yl)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16m): Yield: 53%, m.p.

1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 2.66 (s, 3H, COCH3), 7.23-7.33 (m, 3H,
2Ar-H + H4 thiophene), 7.49 (br. s, 2H, D2O exchangeable SO2NH2), 7.51 (d, J = 8.4 Hz, 2H,
Ar-H), 7.76 (s, 1H, pyridine H), 7.86 (m, 1H, H5 thiophene), 8.11 (d, J = 8.9 Hz, 2H, Ar-H),
8.15-8.2 (m, 1H, H3 thiophene), 8.43 (d, J = 8.9 Hz, 2H, Ar-H); Anal. Calcd for
C26H17FN6O4S2: C, 55.71; H, 3.06; N, 14.99. Found: C, 55.86; H, 3.34; N, 15.28.
4.4.3.12.
Ethyl
6-(4-fluorophenyl)-5-oxo-1-(4-sulfamoylphenyl)-8-(thiophen-2-yl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16n): Yield: 48%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 1.29 (t, J = 7.2 Hz, 3H, -COOCH2CH3),
4.41 (q, J = 7.2 and 7.5 Hz, 2H, -COOCH2CH3), 7.22-7.33 (m, 3H, 2Ar-H + H4 thiophene),
7.45-7.53 (m, 4H, 2Ar-H +2H of SO2NH2, D2O exchangeable), 7.77 (s, 1H, pyridine H),
7.82-7.88 (m, 1H, H5 thiophene), 8.10 (d, J = 8.4 Hz, 2H, Ar-H), 8.15-8.2 (m, 1H, H3
thiophene), 8.40 (d, J = 8.4 Hz, 2H, Ar-H); Anal. Calcd for C27H19FN6O5S2 C, 54.91; H, 3.24;
N, 14.23. Found: C, 55.26; H, 3.53; N, 14.56.
4.4.3.13.
4-(3-Acetyl-6-(4-chlorophenyl)-5-oxo-8-(thiophen-2-yl)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16o): Yield: 73%, m.p.
1
>300 ºC; H NMR (400 MHz, DMSO-d6) δ ppm: 2.67 (s, 3H, COCH3), 7.24 (m, 1H, H4
thiophene), 7.48 (d, J = 8.4 Hz, 2H, Ar-H), 7.54-7.56 (m, 4H, 2Ar-H + 2H of SO2NH2, D2O
exchangeable), 7.79 (s, 1H, pyridine H), 7.88 (d, J = 4.5 Hz, 1H, H5 thiophene), 8.12 (d, J =
8.8 Hz, 2H, Ar-H), 8.17 (d, J = 3.3 Hz, 1H, H3 thiophene), 8.45 (d, J = 8.8 Hz, 2H, Ar-H);
13C NMR (100 MHz, DMSO-d6) δ ppm: 30.18, 107.24, 117.80, 120.95, 127.73, 128.22,
129.47, 129.94, 130.71, 132.49, 133.47, 138.39, 139.16, 142.23, 142.83, 143.63, 147.37,
153.29, 155.32, 157.27, 160.39, 188.08; Anal. Calcd for C26H17ClN6O4S2: C, 54.12; H, 2.97;
N, 14.56. Found: C, 54.36; H, 3.26; N, 14.79.
4.4.3.14.
Ethyl
6-(4-chlorophenyl)-5-oxo-1-(4-sulfamoylphenyl)-8-(thiophen-2-yl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16p): Yield: 63%, m.p.
286-289 oC; 1H NMR (300 MHz, DMSO-d6) δ ppm: 1.29 (t, J = 7.2 Hz, 3H, -COOCH2CH3),
4.42 (q, J = 7.2 and 6.9 Hz, 2H, -COOCH2CH3), 7.24 (m, 1H, H4 thiophene), 7.46-7.51 (m,
4H, 2Ar-H + 2H of SO2NH2, D2O exchangeable), 7.53 (d, J = 8.4 Hz, 2H, Ar-H), 7.77 (s, 1H,
pyridine H), 7.87 (d, J = 4.8 Hz, 1H, H5 thiophene), 8.10 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (m,
1H, H3 thiophene), 8.40 (d, J = 8.7 Hz, 2H, Ar-H); Calcd for C27H19ClN6O5S2: C, 53.42; H,
3.15; N, 13.84. Found: C, 53.66; H, 3.47; N, 14.06.

4.4.3.15.
4-(3-Acetyl-6-(4-methoxyphenyl)-5-oxo-8-(thiophen-2-yl)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16q): Yield: 64%, m.p.
>300 ºC; 1H NMR (300 MHz, DMSO-d6) δ ppm: 2.63 (s, 3H, COCH3), 3.80 (s, 3H, -OCH3),
6.97 (d, J = 9 Hz, 2H, Ar-H), 7.20 (m, 1H, H4 thiophene), 7.37-7.45 (m, 4H, 2Ar-H +2H of
SO2NH2, D2O exchangeable), 7.56 (s, 1H, pyridine H), 7.74-8.06 (m, 3H , 2Ar-H + H5
thiophene), 8.27 (m, 1H, H3 thiophene), 8.39 (d, J = 8.1 Hz, 2H, Ar-H); Calcd for
C27H20N6O5S2: C, 56.63; H, 3.52; N, 14.68. Found: C, 56.93; H, 3.86; N, 14.37.
4.4.3.16. Ethyl 6-(4-methoxyphenyl)-5-oxo-1-(4-sulfamoylphenyl)-8-(thiophen-2-yl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16r): Yield: 60%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 1.31 (t, J = 7.2 Hz, 3H, -COOCH2CH3),
3.85 (s, 3H, -OCH3), 4.42 (q, J = 7.2 and 6.9 Hz, 2H, -COOCH2CH3), 7.01 (d, J = 8.7 Hz,
2H, Ar-H), 7.23 (m, 1H, H4 thiophene), 7.41 (d, J = 8.7 Hz, 2H, Ar-H), 7.48 (br. s, 2H,
SO2NH2, D2O exchangeable), 7.72 (s, 1H, pyridine H), 7.85 (d, J = 4.8 Hz, 1H, H5
thiophene), 8.09 (d, J = 8.7 Hz, 2H, Ar-H), 8.13-8.20 (m, 1H, H3 thiophene), 8.40 (d, J = 8.7
13
Hz, 2H, Ar-H); C NMR (75 MHz, DMSO) δ 14.22, 55.70, 64.08, 107.21, 113.64, 117.84,
118.75, 120.92, 127.50, 129.65, 130.46, 130.77, 131.55, 132.22, 136.37, 139.19, 142.73,
143.80, 147.07, 154.48, 155.09, 156.68, 159.92, 160.64,; Calcd for C28H22N6O6S2: C, 55.81;
H, 3.68; N, 13.95. Found: C, 55.54; H, 3.37; N, 14.09.
4.4.3.17.
4-(3-Acetyl-6-(4-nitrophenyl)-5-oxo-8-(thiophen-2-yl)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide (16s): Yield: 69%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 2.67 (s, 3H, COCH3), 7.25 (m, 1H, H4
thiophene), 7.52-7.70 (m, 4H, 2Ar-H +2H of SO2NH2, D2O exchangeable), 7.86 (s, 1H,
pyridine H), 8.00-8.33 (m, 5H , 4Ar-H + H5 thiophene), 8.45-8.55 (m, 3H , 2Ar-H + H3
thiophene); Calcd for C26H17N7O6S2: C, 53.15; H, 2.92; N, 16.69. Found: C, 53.46; H, 3.24
N, 17.02.
4.4.3.18.
Ethyl
6-(4-nitrophenyl)-5-oxo-1-(4-sulfamoylphenyl)-8-(thiophen-2-yl)-1,5-
dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (16t ): Yield: 72%, m.p.
1
>300 ºC; H NMR (300 MHz, DMSO-d6) δ ppm: 1.28 (t, J = 7.2 Hz, 3H, -COOCH2CH3),
4.40 (q, J = 7.2 and 6.9 Hz, 2H, -COOCH2CH3), 7.24 (t, J = 4.2 Hz, 1H, H4 thiophene), 7.50
(br. s, 2H, SO2NH2, D2O exchangeable), 7.72 (d, J = 8.7 Hz, 2H, Ar-H), 7.83 (s, 1H, pyridine

H), 7.88 (d, J = 4.8 Hz, 1H, H5 thiophene), 8.10 (d, J = 8.7 Hz, 2H, Ar-H), 8.16-8.20 (m, 1H,
H3 thiophene), 8.32 (d, J = 8.4 Hz, 2H, Ar-H), 8.40 (d, J = 8.4 Hz, 2H, Ar-H); C NMR (75
13
MHz, DMSO-d6) δ ppm: 13.74, 63.60, 106.34, 117.00, 120.60, 122.82, 127.20, 128.89,
129.65, 130.00, 132.31, 135.78, 138.54, 142.42, 143.02, 146.00, 146.72, 147.17, 151.64,
154.54, 156.00, 157.00, 159.85; Calcd for C27H19N7O7S2: C, 52.51; H, 3.10; N, 15.88. Found:
C, 52.63; H, 3.31; N, 15.64.
4.1.CA activity and inhibition measurements:
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity.22 Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction
have been used for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1
mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear least-squares methods using
PRISM 3 and the Cheng-Prusoff equation, as reported earlier,24 and represent the mean from
at least three different determinations. All CA isofoms were recombinant ones obtained in-
house as reported earlier. 25
Acknowledgements: The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for its funding of this research
through the Research Group Project no. RGP-1436-038.

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Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-
1(5H)-yl) moieties andevaluationof their carbonic anhydrases I, II, IV and IX inhibitory effects
c
b
Mohamed Fares a,b,*, Radwa A. Eladwy , Alessio Nocentini d,e, Soha R. Abd El Hadi ,
Hazem A. Ghabbour f,g, Ashraf Abdel-Megeed h, Wagdy M. Eldehna i, Hatem A. Abdel-Aziz
j, Claudiu T. Supuran d*
F
F
C
F
O
O
N
N
N
N
N
N
N
Cl
SO2NH2
SO2NH2
hCA IX/hCA II = 3.4
hCA IX/hCA IV = 16.9
hCA IX/hCA II = 1.1
hCA IX/hCA IV = 14.5