Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I, II, IX and XII

Article abstract of DOI:10.1016/j.bmc.2016.01.018

Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmem

Full text of DOI:10.1016/j.bmc.2016.01.018

Accepted Manuscript
Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory
action against carbonic anhydrase isoforms I, II, IX and XII
Elisabetta Barresi, Silvia Salerno, Anna Maria Marini, Sabrina Taliani,
Concettina La Motta, Francesca Simorini, Federico Da Settimo, Daniela Vullo,
Claudiu T. Supuran
PII:
S0968-0896(16)30018-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.018
BMC 12761
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 November 2015
4 January 2016
8 January 2016
Please cite this article as: Barresi, E., Salerno, S., Marini, A.M., Taliani, S., Motta, C.L., Simorini, F., Settimo, F.D.,
Vullo, D., Supuran, C.T., Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action
against carbonic anhydrase isoforms I, II, IX and XII, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.01.018
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Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against
carbonic anhydrase isoforms I, II, IX and XII
Elisabetta Barresi,1 Silvia Salerno,1 Anna Maria Marini,*1 Sabrina Taliani,1 Concettina La
Motta,1 Francesca Simorini,1 Federico Da Settimo,1 Daniela Vullo,2 and Claudiu T. Supuran*3
1Università di Pisa, Dipartimento di Farmacia, Via Bonanno 6, 56126 Pisa, Italy
2Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
3Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche
e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. Three series of polycyclic compounds possessing either primary sulfonamide or
carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four
physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane
hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects
against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the
other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7 nM)
without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating
polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or
dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring
the design of isoform-selective CAIs with various pharmacologic applications.
Keywords: carbonic anhydrase; sulfonamide; carboxylic acid; benzothiopyranopyrimidine;
pyridothiopyranopyrimidine; dihydrbenzothiopyrano[4,3-c]pyrazole.
___
* Corresponding authors. Phone: +39-050-2219555; e-mail: marini@unipi.it; Phone: +39-055-
4573005; Fax: +39-055-4573385; e-mail: claudiu.supuran@unifi.it (CTS).

1. Introduction
Sulfonamides possessing the general formula RSO2NH2 constitute the historically most
important class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs).1-8 They are in clinical use
for more than 70 years as diuretics, antiglaucoma, antiepileptic, and antiobesity agents,1-7 and, more
recently, they have shown applications as antitumor/antimetastatic agents or diagnostic tools, with
at least one sulfonamide in Phase I clinical trials for the treatment of advanced, metastatic solid
tumors.8
Recently,1 we have disclosed a new class of sulfonamides 1a-e featuring the classical
benzenesulfonamide moiety for binding to the catalytically crucial zinc ion forming the enzyme
active site. The head part of these molecules is connected to a pyrazole moiety, further annealed
with a bulky heterocyclic ring, benzothiopyrano[4,3-c]pyrazole (1a-c) and pyridothiopyrano[4,3-
c]pyrazole (compounds 1d-e), (Chart 1) in order to explore both alternative chemotypes and the
possibility to further enhance the isoform selectivity observed with celecoxib (CLX) and valdecoxib
(VLX) as CAIs.2-8 Actually, compounds 1a-e may be regarded as geometrically constrained
analogues of the two reference leads CLX and VLX.
The most interesting feature of this new class of sulfonamides was their capability to
predominantly exert strong inhibition of only human (h) isoforms hCA I and II, as well as of the
mycobacterial β-class enzymes (Rv1284, Rv3273, and Rv3588c), whereas their inhibitory activity
against hCA III, IV, VA, VB, VI, VII, IX, XII, XIII, and XIV was at least 2 orders of magnitude
lower. The combination of X-ray crystal structure of the hCA II- 1e adduct, and homology
modeling allowed us to explain this peculiar inhibition profile, which was also quite different from
those of the reference coxibs, CLX and VLX. Thus, the benzenesulfonamides 1a-e constitute a
highly interesting class of compounds which, inhibiting only a restricted number of physiologically
relevant CA isoforms among the 12 catalytically active human enzymes, should lead to fewer side
effects. In addition, the good inhibition profile of some of the new derivatives (1d-e) against
mycobacterial CAs is also to be considered as relevant because these enzymes are less inhibited by
other classes of sulfonamides.
As a further development of this project, in the present work we aim to study three novel
series of compounds in search for selective CAIs. In the first class (compounds 2a-d, Chart 1), we
decided to move the benzenesulfonamide function from position 1 to position 2 of the pyrazole
system of benzothiopyrano[4,3-c]pyrazole (2a,b) and pyridothiopyrano[4,3-c]pyrazole (2c,d), to
assess whether this change of position could lead to selective compounds for some CA isoforms.

Substituents at 7-position (R) were chosen by considering the groups that most contributed to the
activity of the above described compounds 1a-e.1
Chart 1: Sulfonamide CAIs of types 1-4 discussed in the paper.
Then, with the aim to identify novel chemotypes to further expand the chemical diversity of
CAIs, we searched among our in-house database of heteropolycyclic scaffold-based derivatives.9-13
Benzothiopyranopyrimidines (3a-c) and pyridothio-pyranopyrimidines (3d-e), reported by us
earlier,11,13attracted our attention due to their structural similarity to the pyrazoles of types 1 and 2.
Thus, in this work, we considered compounds 3a-e, featuring a carboxylic group. Actually, it was
reported that some carboxylates14-16 share with dithiocarbamates17-19 the CA inhibition mechanism
of sulfonamides and their bioisosteres (sulfamates, sulfamides),20,21 representing one of the main
class of pharmacologically relevant CAIs.20-23
Finally, the benzothiopyranopyrimido and the pyridothiopyrano-pyrimido scaffolds were
further investigated, by the design of compounds 4a-e (Chart1), that bear a benzenesulfonamide
moiety, typical of the classical CAIs, at the 2-position of the tricyclic system and spaced by an NH
linker.

2. Results and Discussion
2.1. Chemistry.
The preparation of the 2-(p-sulfonamidophenyl)substituted-pyrazole derivatives 2a-d was
performed following the synthetic route described in Scheme 1. The starting key intermediates 7-
substituted-3-hydroxymethylenebenzothiopyranones
5a-b9
or
3-hydroxymethylene-
pyridothiopyranones 5c-d10,11 were converted into the corresponding p-toluensulfonates 6a-d. In
these intermediates the reactive methine functionality was protected as previously reported by us.12
The subsequent condensation of 6a-d with 4-sulfonamidophenylhydrazine hydrochloride, in
anhydrous dimethylformamide at room temperature, involved, as a first step, only the carbonyl
moiety functionalization at the 4-position, affording the intermediate arylhydrazones.12These latter
compounds easily cyclized in situ by heating the reaction mixture at 100 °C, to afford the desired 2-
substituted derivatives 2a-d. All compounds were purified by flash chromatography and
characterized by analytical and spectral data (see Experimental protocols for details) which were in
accordance with analogue compounds reported earlier.1, 9, 12
Scheme 1. Preparation of sulfonamides 2a-d.
The synthetic pathway leading to the target 2-(p-benzensulfonamido)-benzothiopyranopyrimidines
4a-c and 2-(p-benzensulfonamido)pyridothiopyranopyrimidines 4d-e took advantage of the use of
the 7-substituted benzothiopyrano or pyridothiopyrano moiety as “synthetic tool”, which
demonstrated to be versatile intermediates in many synthetic procedures.13 The 1,3-bielectrophile

reactivity of the 7-substituted-3-dimethylaminomethylene derivatives 7a-e13,24-26 in the reaction
with the 4-guanidinobenzenesulfonamide (as bicarbonate salt) 827 in butanol at reflux, in the
presence of sodium hydroxide,28 allowed to easily obtain the desired 2-benzenesulfonamidophenyl
substituted pyrimidines 4a-e, as shown in Scheme 2. The N-(4-aminosulfonylphenyl) guanidine
carbonate 8 was obtained in good yield by reaction of a 50% cyanamide water solution and a
solution of sulfanilamide in the presence of concentrated HCl. The mixture was heated at 100 °C for
20 minutes, as described in the literature.27
1
The purity of the target compounds was assessed by physicochemical methods, analytical and H
13
NMR and C NMR spectral data, which were in agreement with the proposed structures and with
other previously reported results. (see Experimental protocols for details).
Scheme 2. Synthesis of sulfonamides 4a-e.
2.2. CA inhibition
The compounds 2a-d, 3a-e and 4a-e reported here were investigated for their enzyme inhibitory
capacity against four physiologically relevant CA isoforms, the human (h) hCA I, II, IX and XII

(Table 1). Acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide) was used as
standard drug in the assay.28
Table 1. Inhibition of isoforms hCA I, II, IX and XII with sulfonamides 2a-d, by a stopped-flow
CO2 hydrase assay.28 Acetazolamide (AAZ) used as standard inhibitor.
2a-d
N
X
R
KI (nM)*
hCA IX
hCA I
41.5
68.4
22.5
37.1
250
hCA II
8.5
hCA XII
68.6
2a
CH
CH
N
Cl
6.1
7.4
6.3
7.7
25
2b
OCH3
H
7.4
38.1
2c
7.1
66.7
2d
N
CH3
-
7.6
62.5
AAZ
-
12
5.6
* Mean from 3 different assays. Errors in the range of 10 of the reported values.
The data listed in Table 1 show that benzenesulfonamides 2a-d, bearing the bulky annealed tricyclic
tails, significantly inhibited all four investigated CA isoforms, with inhibition constants in the range
of 22.5 - 68.4 nM against hCA I, 7.1 - 8.5 nM against hCA II, 6.1 - 7.7 nM against hCA IX and
38.1 - 68.6 nM against hCA XII, respectively. The structure-activity relationship (SAR) against all
four isoforms was rather flat, since the substituent R and the X moiety showed a small influence on
the inhibitory activity. The other salient feature was that the isoform-selectivity, observed with
compounds 1 reported earlier,1 is lost for the sulfonamides 2. This may presumably be attributed to
the capacity of the quite bulky scaffold present in the derivatives investigated here, to interact
favorably with the active sites of all four CA isoforms.

Thus, as the shift of the benzenesulfonamide moiety from position 1 to position 2 of the pyrazole
system (compounds 2a-d) led to less isoform selective compounds, we decided to continue the
study in this area by changing the starting pyrazole with a pyrimidine ring fused on the
benzothiopyrano or pyridothiopyrano moieties, thus obtaining compounds 3 and 4, whose inhibition
data are reported in Tables 2 and 3.
Table 2. Inhibition of isoforms hCA I, II, IX and XII with carboxylic acids 3a-e, by a stopped-flow
CO2 hydrase assay.28 Acetazolamide (AAZ) used as standard inhibitor.
3a-e
N
X
R
R1
R2
KI (nM)*
hCA I
510
hCA II hCA IX hCA XII
3a
CH
CH
CH
N
OCH3 COOH
OCH3 COOH
CH3
H
5390
4220
40.8
54.1
8390
5975
9030
7170
6185
5.6
3b
871
3c
OCH3 (CH2)2CH(NH2)COOH H
>50000
440
>50000 335
3d
H
COOH
COOH
-
CH3
CH3
638
7450
12
67.8
92.7
25
3e
N
CH3
-
370
AAZ
-
250
* Mean from 3 different assays. Errors in the range of 10 of the reported values.
Data of Table 2 show that the carboxylic acids 3 (except the one with the longer aliphatic chain, 3c)
possess a more interesting inhibition profile against the four CA isoforms compared to
sulfonamides 2. Indeed, compounds 3a,b,d and e behaved as hCA IX-selective inhibitors (KIs in
that range of 40.8 - 92.7 nM) with much lower affinity for hCA I, II and XII, for which KIs in the
range of 370 - 9030 nM were measured (Table 2). The lysine derivative 3c did not significantly
inhibit hCA I, II and XII, being a weak hCA IX inhibitor too (KI of 335 nM).
Table 3. Inhibition of isoforms hCA I, II, IX and XII with sulfonamides 4a-e, by a stopped-flow
CO2 hydrase assay.28 Acetazolamide (AAZ) used as standard inhibitor.

4a-e
N
X
R
KI (nM)*
hCA IX
hCA I
80.5
66.9
84.0
68.3
68.9
250
hCA II
96.0
63.2
218
9.4
hCA XII
75.1
4a
CH
CH
CH
N
H
7.0
4b
OCH3
Cl
22.5
8.7
48.9
4c
15.5
4d
H
27.6
8.1
17.8
4e
N
CH3
-
8.0
34.4
AAZ
-
12
25
5.6
* Mean from 3 different assays. Errors in the range of 10 of the reported values.
Data of Table 3 show that all CA isoforms investigated here were inhibited by sulfonamides 4a-e.
The slow cytosolic isoform hCA I was inhibited moderately, with KIs ranging between 66.9 and
84.0 nM. Again the SAR was rather flat for this isoform, proving that the nature of the X and R
fragments of the molecule do not significantly influence the biological activity. This is however not
the case for the inhibition of the physiologically dominant isoform hCA II, for which a more
diversified inhibitory behavior has been evidenced for these sulfonamides. Indeed, 4d and 4e were
low nanomolar, highly effective hCA II inhibitors (KIs ranging between 8.0 and 9.4 nM), being thus
more effective than the standard drug AAZ, whereas 4a-c showed a reduced efficacy, with KIs
ranging between 63.2 and 218 nM. Thus, the best substitution pattern was that incorporating
pyridine and not benzene in the terminal ring of the polycyclic system. The presence of H or Me as
R moieties was not highly influential on the hCA II inhibition profile of these compounds.
The transmebrane isoform hCA IX, a validated antitumor target,8 was also effectively inhibited by
sulfonamides 4, which showed KIs ranging between 7.0 and 27.6 nM (Table 3). In this case both
benzene- and pyridine in the third ring of the polycyclic system led to effective inhibitors (e.g.,
compare 4a and 4e which show very similar KI values). The methoxy moiety (in 4b) led to an
almost three-fold loss of inhibitory efficacy (compared to 4a) whereas chlorine (in 4c) was better

tolerated, with the last compound having a similar activity as 4a. The presence of the methyl moiety
in 4e was this time beneficial for the hCA IX inhibitory effects, with this compound being 3-times
more potent compared to4d, which has a hydrogen in place of the methyl group (Table 3).
The second transmembrane isoform investigated here, hCA XII, was also effectively inhibited by
these sulfonamides, which had KIs ranging between 15.5 and 75.1 nM. The best inhibitors were the
chlorine derivative in the benzene series (4c) and the unsubstituted derivative (R = H) in the
pyridine series (4d) with KIs of 15.5 nM and 17.8 nM, respectively (Table 3).
3. Conclusions
Primary sulfonamides 2 and 4 or carboxylic acids 3 incorporating polycyclic ring systems such as
benzothiopyranopyrimidine,
pyridothiopyranopyrimidine
or
dihydrobenzothiopyrano[4,3-
c]pyrazole were investigated as inhibitors of four physiologically relevant CA isoforms, the
cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. An excellent inhibitory
activity against isoforms hCA II, IX and XII was shown by most of the new sulfonamides 2 and 4,
although with a low selectivity profile among the various isoforms. The carboxylates 3 on the other
hand, acted as hCA IX-selective inhibitors (KIs in that range of 40.8 - 92.7 nM), with much lower
affinity for hCA I, II and XII. Thus, sulfonamide or carboxylic acid moieties as zinc-binding groups
connected to a pyrazole or a pyrimidine moiety annealed with a bulky heterocyclic ring may be
considered as interesting candidates for exploring the design of novel isoform-selective CAIs with
various pharmacologic applications. Such a “compromise” between a lower affinity but higher
selectivity of enzyme inhibitors might be acceptable, as reported for example in the field of MMP
inhibitors,29a or for some carboxylate-based CAIs.29b,c As a consequence, exploration of diverse
scaffolds and zinc-binding groups may lead to inhibitors with the desired properties for the various
pharmacologic applications of this class of derivatives.
4. Experimental Section
4.1. Chemistry
General. The uncorrected melting points were determined using a Reichert Köfler hot-stage
apparatus. NMR spectra were obtained on a Bruker AVANCE 400 (1H, 400 MHz, 13C, 100 MHz) in
DMSO-d6 or CDCl3(internal standard tetramethylsilane). The coupling constants are given in Hertz.
Magnesium sulphate was used as the drying agent. Evaporations were made in vacuo (rotating

evaporator). Analytical TLC have been carried out on Merck 0.2 mm precoated silica gel
aluminium sheets (60 F-254). Silica gel 60 (230-400 mesh) was used for column chromatography.
Purity of the target inhibitors 2a-d, 3a-e, and 4a-e was determined, using a Shimadzu LC- 20AD SP
liquid chromatograph equipped with a DDA Detector at 196 nm(column C18 (250 mm × 4.6 mm, 5
μm, Shim-pack)). The mobile phase, delivered at isocratic flow, consisted of acetonitrile (40-60%)
and water (40-60%) and a flow rate of 1.0 mL/ min.
All the compounds showed percent purity values of ≥ 95%.
Reagents, starting materials, and solvents were purchased from commercial suppliers and used as
received. The following intermediates were obtained according to methods previously described: 7-
Methoxy-2,3-dihydro-3-hydroxymethylene-1-benzothiopyran-4(4H)-one 5a9 and 7-chloro-2,3-
dihydro-3-hydroxymethylene-1-benzothiopyran-4(4H)-one 5b,9 2,3-dihydro-3-hydroxymethylene-
thiopyrano[2,3-b]-pyridin-4(4H)-one
pyrano[2,3-b]-pyridin-4(4H)-one 5d,11 3-[(p-Methylphenyl)sulfonyl]oxymethylen-2,3-dihydro-
benzothiopyran-4(4H)-one 3-[(p-Methylphenyl)sulhonyl]oxymethylen-2,3-dihydrothio-
6a,9
pyrano[2,3-b]pyridin-4(4H)-one 6c12 and 7-Methyl-3-[(p-Methylphenyl)sulhonyl]oxymethylen-2,3-
dihydrothiopyrano[2,3-b]pyridin-4(4H)-one 3-Dimethylaminomethylen-2,3-dihydro-
6d,12
benzo[3',2':5,6]thiopyran-4(4H)-one 7a,26 7-Methoxy-3-dimethylaminomethylen-2,3-dihydro-
benzo[3',2':5,6]thiopyran-4(4H)-one 7-Chloro-3-dimethylaminomethylen-2,3-dihydro-
7b,13
benzo[3',2':5,6]thiopyran-4(4H)-one 7c,26 2,3-Dihydro-3-dimethylaminomethylenethiopyrano[2,3-
b]pyridin-4(4H)-one 7d,
117-Methyl-2,3-Dihydro-3-dimethylaminomethylenethiopyrano[2,3-
5c,10
7-Methyl-2,3-dihydro-3-hydroxymethylenethio-
b]pyridin-4(4H)-one 7e,11 and N-(4-Aminosulfonylphenyl)guanidine carbonate 8.27 2-Glycinyl-
3a,13 2-(N-methyl)glycinyl- 3b,13 2-(5N-ornithinyl)-8-methoxy- 5H-benzo[3',2':5,6]thiopyrano[4,3-
d]pyrimidine 3c,13 N-Methyl-N-(5H-pyrido[3',2';5,6]thiopyrano[4,3-d]pyrimidin-2-yl)glycine 3d,11
andN-Methyl-N-(8-Methyl-5H-pyrido[3',2';5,6]thiopyrano[4,3-d]pyrimidin-2-yl)glycine 3e11 were
prepared in accordance with reported procedures.
3-[(p-Methylphenyl)sulfonyl]chloromethylen-2,3-dihydrobenzothiopyran-4(4H)-one 6b.
p-Toluensulphonylchloride (0.801 g, 4.8 mmol) was added to a solution of 5b (0.903 g, 2.4mmol)
in 8 mL of anhydrous tetrahydrofurane in the presence of potassium carbonate (1.161 g, 9.6 mmol).
The reaction mixturewas stirred at room temperature, under nitrogen atmosphere, for 15 hours and
then refluxed for 3 hours. After cooling, the suspension was concentrated under reduced pressure,
and the residue obtained was treated with water and then extracted with dichloromethane. The
organic layers were dried and evaporated to give the crude product 6b which was purified by flash
chromatography on a silica gel column (60/0.040-0.063 mm) using petroleum ether 40-60°C/ethyl

1
acetate 8/2 as the eluting system. Yield 22%, mp 80-82 °C. H NMR (400 MHz, CDCl3): δ (ppm)
2.50 (s, 3H, CH3),3.83 (s, 2H, CH2-S), 7.20 (dd, 1H, Ar-H, Jmax = 8.4 Hz, Jmin = 2.0 Hz), 7.29 (d,
1H, Ar-H, J = 2.0 Hz),7.43 (m, 2H, Ar-H), 7.67-7.68 (m, 1H, CH), 7.87-7.89 (m, 2H, Ar-H), 8.04
(d, 1H, Ar-H, J = 8.4 Hz).
General Procedure for the synthesis of 7-Substituted-2-(p-sulfonamidophenyl)-1,4-
dihydrobenzothiopyrano[4,3-c]pyrazoles 2a-b and 7-Substituted-2-(p-sulfonamidophenyl)-1,4-
dihydropyrido[3′,2′:5,6]thiopyrano[4,3-c]pyrazoles 2c-d.
4-Hydrazinobenzenesulfonamide hydrochloride (0.300g, 1.3 mmol) in
9 ml of N,N-
dimethylformamide was added dropwise to a solution of the opportune derivative 6a-d (2.0 mmol)
in 6 mL of the same solvent. The reaction mixture was stirred at room temperature for 15 hours,
then refluxed for 2 hours. After cooling, the suspension was poured into a saturated aqueous
potassium carbonate solution and extracted with dichloromethane. The organic layers were dried
and evaporated under reduced pressure to give desired crude pyrazoles 2a-d which were purified by
flash chromatography on a silica gel column (60/0.040-0.063 mm) using petroleum ether 40-60
°C/ethyl acetate in various ratio as the eluting system.
7-Methoxy-2-(p-sulfamidophenyl)-1,4-dihydrobenzothiopyranopyrazole 2a.
Yield 35%, mp 230-232 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 3.74 (s, 3H, OCH3), 3.99 (s,
2H, CH2-S), 6.67 (dd, 1H, Ar-H, Jmax = 8.8 Hz; Jmin = 2.4 Hz), 6.76-6.74 (m, 1H,Ar-H), 7.09 (d,
1H, Ar-H,J=2.4Hz.), 7.53(s, 2H, NH2 exch.), 77.61 (d, 2H, Ar-H J=8.8 Hz), 7.71 (s, 1H, Ar-H),
7.95 (d, 2H, Ar-H, J= 8.8 Hz). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 23.24, 55.87, 112.53,
114.30, 117.06, 118.54, 126.23, 126.30, 127.52, 135.63, 137.87, 137.90, 143.16, 143.98, 159.33.
7-Cloro-2-(p-sulfamidophenyl)-1,4-dihydrobenzothiopyranopyrazole 2b.
Yield 51%, mp: 210-212 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.04 (s, 2H, CH2-S), 6.81 (d,
1H, Ar-H, J = 8.8 Hz), 7.16 (dd, 1H, Ar-H, Jmax = 8.4, Jmin = 2.0 Hz), 7.53 (bs, 2H, NH2exch.),
7.60-7.64 (m, 3H, Ar-H), 7.78 (s, 1H, Ar-H), 7.94-7.96 (m, 2H, Ar-H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 23.12, 118.81, 124.48, 126.21, 126.34, 126.40, 127.63, 128.61, 133.12,
136.29, 137.06, 138.04, 142.81, 144.20.
2-(p-Sulfamidophenyl)-1,4-dihydropyridothiopyranopyrazole 2c.
1
Yield30%, mp 213-215 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) 4.19 (s, 2H, CH2-S), 7.05-
7.07 (m, 2H, Ar-H), 7.54 (bs, 2H, NH2exch.), 7.61-7.63 (m, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 7.94-
7.96 (m, 2H, Ar-H), 8.29-8.30 (m, 1H, Ar-H).13C NMR (100 MHz, DMSO-d6): δ (ppm) 22.90,
118.13, 120.77, 121.69, 126.35, 127.64, 131.52, 136.75, 138.20, 142.64, 144.35, 148.76, 157.44.
7-Methyl-2-(p-sulfamidophenyl)-1,4-dihydropyridothiopyranopyrazole 2d.

1
Yield 25%, mp235-238 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) 2.38 (s, 3H, CH3), 4.15 (s,
2H, CH2-S), 6.90 (d, 1H, Ar-H, J = 8.0 Hz), 6.95 (d, 1H, Ar-H, J = 8.8 Hz), 7.52 (s, 2H, NH2exch.),
7.59-7.61 (m, 2H, Ar-H), 7.75 (s, 1H, Ar-H), 7.93-7.95 (m, 2H, Ar-H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 22.99, 24.03, 117.41, 118.97, 120.15, 126.28, 127.61, 131.80, 137.00, 138.13,
142.70, 144.29, 156.73, 157.74.
General procedure for the synthesis of 4-((8-substituted-5H-thiochroman[4,3-d]pyrimidin-2-
yl)amino)benzenesulfonamides 4a-c and 4-((8-substituted-5H-pyrido[3',2':5,6]thiopyrano[4,3-
d]pyrimidin-2-yl)amino)benzenesulfonamides 4d-e.
N-(4-Aminosulfonylphenyl)guanidine carbonate 8 (0.332 g, 1.2 mmol) and NaOH (0.096 g, 2.4
mmol) were added to
a
solution of the appropriate 3-dimethylaminomethylen-2,3-
7a-c or2,3-Dihydro-3-dimethylamino-
dihydrobenzo[3',2':5,6]thiopyran-4(4H)-one
methylenethiopyrano[2,3-b]pyridin-4(4H)-one 7d-e (1.2 mmol) in 10 mL of n-BuOH. The solution
was heated at 120 °C for 4 hours.After cooling, a precipitate was obtained in the case of compounds
4a-c, which was collected by vacuum filtration. Instead, starting from compounds 7d-e, it was not
observed any formation of precipitate, thus, the solvent of reaction was evaporated under reduced
pressure.All compounds were purified by flash chromatography on a silica gel column (60/0.040-
0.063 mm) using ethyl acetate/ petroleum ether 40-60 °C in various ratio as the eluting system.
4-((5H-thiochroman[4,3-d]pyrimidin-2-yl)amino)benzenesulfonamide 4a.
Yield 44%, mp 275-278 °C.1H-NMR (400 MHz, DMSO-d6): δ (ppm) 4.07 (s, 2H, CH2-S), 7.20 (s,
2H, NH2exch.), 7.41-7.47 (m, 3H, Ar-H), 7.76-7.78 (m, 2H, Ar-H), 7.97-8.00 (m, 2H, Ar-H), 8.32-
8.34 (m, 1H, Ar-H), 8.56 (s, 1H, Ar-H), 10.15 (bs, 1H, NHexch). 13C NMR (100 MHz, DMSO-d6):
δ (ppm) 26.42, 116.66, 118.21, 126.83, 127.10, 127.67, 128.43, 131.83, 132.40, 136.58, 137.12,
144.16, 157.00, 158.41, 159.43.
4-((8-Methoxy-5H-thiochroman[4,3-d]pyrimidin-2-yl)amino)benzenesulfonamide 4b.
Yield 31%, mp 228-230 °C.1H-NMR (400 MHz, DMSO-d6): δ (ppm) 3.85 (s, 3H, -OCH3), 4.05 (s,
2H, CH2-S), 6.98-7.01 (m, 2H, Ar-H), 7.19 (bs, 2H, NH2exch.), 7.76 (d, 2H, Ar-H, J=8.8 Hz), 7.98
(d, 2H, Ar-H,J = 9.2 Hz), 8.27 (d, 1H, Ar-H, J= 8.8 Hz), 8.48 (s, 1H, Ar-H), 10.07 (bs, 1H,
NHexch.).13C NMR (100 MHz, DMSO-d6): δ (ppm) 26.64 56.09, 112.68, 113.63, 115.47, 118.10,
125.11, 127.08, 129.36, 136.40, 139.0, 144.26, 156.50, 158.42, 159.33, 161.93.
4-((8-Chloro-5H-thiochroman[4,3-d]pyrimidin-2-yl)amino)benzenesulfonamide 4c.
Yield 45%, mp 268- 270 °C.1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.10 (s, 2H, CH2-S), 7.21 (s,
2H, NH2exch.), 7.47-7.50 (dd, 1H, Ar-H, Jmax = 8.6, Jmin = 2.4Hz), 7.60 (d, 1H, Ar-H, J = 2.4Hz),
7.77 (d, 2H, Ar-H, J = 8.8 Hz), 7.97 (d, 2H, Ar-H, J = 8.8 Hz), 8.30 (d, 1H, Ar-H, J = 8.8 Hz),

8.57(s, 1H, Ar-H), 10.19 (s, 1H, NHexch.). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 26.40,
116.27, 118.25, 126.94, 127.12, 127.68, 129.20, 131.22, 136.41, 136.67, 139.46, 144.05, 157.24,
157.59, 159.43.
4-((5H-Pyrido[3',2':5,6]thiopyrano[4,3-d]pyrimidin-2-yl)amino)benzenesulfonamide 4d.
Yield 45%, mp 283-285 °C.1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.23 (s, 2H, CH2-S), 7.19 (s,
2H, NH2exch.), 7.41-7.44 (m, 1H, Ar H), 7.78 (d, 2H, Ar-H, J = 8.8 Hz), 7.97 (d, 2H, Ar-H, J = 8.8
Hz), 8.52-8.54 (m, 1H, Ar-H), 8.57-8.59 (m, 1H, Ar-H), 8.60 (s, 1H, Ar-H), 10.18 (bs, 1H,
NHexch.). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 26.31, 115.62, 118.36, 121.84, 127.14,
128.66, 134.75, 136.76, 143.95, 151.90, 157.57, 157.75, 159.41, 160.13.
4-((8-Methyl-5H-pyrido[3',2':5,6]thiopyrano[4,3-d]pyrimidin-2-yl)amino)benzenesulfonamide
4e.
Yield 45%, mp 283-285 °C.1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.20 (s, 2H, CH2-S), 7.21 (s,
2H, NH2exch.), 7.27 (d, 2H, Ar-H, J = 8.8 Hz), 7.96 (d, 2H, Ar-H, J = 8.8 Hz), 8.46 (d, 2H, Ar-H, J
= 8.0 Hz), 8.56 (s, 1H, Ar-H), 10.17 (s, 1H, NHexch.).13C NMR (400 MHz, DMSO-d6): δ (ppm)
24.42, 26.36, 115.20, 118.27, 121.38, 125.97, 127.13, 135.01, 136.66, 144.01, 157.31, 157.98,
159.36, 159.46, 161.28.
4.2. CA inhibition. An Applied Photophysics stopped-flow instrument has been used for assaying
the CA catalysed CO2 hydration activity.28 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,29-31and
represent the mean from at least three different determinations. All CA isofoms were recombinant
enzymes obtained in-house as reported earlier.30-32

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Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against
carbonic anhydrase isoforms I, II, IX and XII
Elisabetta Barresi,1 Silvia Salerno,1 Anna Maria Marini,*1 Sabrina Taliani,1 Concettina La
Motta,1 Francesca Simorini,1 Federico Da Settimo,1 Daniela Vullo,2 and Claudiu T. Supuran*3
2
2
KIs (hCA IX) = 6.1 - 92.7 nM