5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigations

Accepted Manuscript 5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigations Jekaterīna Ivanova, Agnese Balode, Raivis Žalubovskis, Janis Leitans, Andris Kazaks, Daniela Vullo, Kaspars Tars, Claudiu T. Supuran PII: DOI: Reference:

S0968-0896(16)31116-6 http://dx.doi.org/10.1016/j.bmc.2016.11.045 BMC 13412

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Accepted Date:

2 November 2016 23 November 2016

Please cite this article as: Ivanova, J., Balode, A., Žalubovskis, R., Leitans, J., Kazaks, A., Vullo, D., Tars, K., Supuran, C.T., 5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigations, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.045

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5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: solution and crystallographic investigations

Jekaterīna Ivanova,1,2 Agnese Balode,1,2 Raivis Žalubovskis,*1 Janis Leitans,3 Andris Kazaks,3 Daniela Vullo,4 Kaspars Tars,3,5 Claudiu T. Supuran*6 1

Latvian Institute of Organic Synthesis, 21 Aizkraukles Str., Riga LV-1006, Latvia.

2

Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied

Chemistry, Riga Technical University, 3/7 Paula Valdena Str., Riga LV-1048, Latvia. Latvian Biomedical Research and Study Center, Rātsupītes 1, LV-1067, Riga, Latvia

3 4

Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica

Bioinorganica, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. 5

Faculty of Biology, Department of Molecular Biology, University of Latvia, Jelgavas 1,

LV1004, Riga, Latvia. 6

Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di

ScienzeFarmaceutiche e Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.

Abstract. A series of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans. The new compounds were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The cytosolic human (h) isoforms hCA I was poorly inhibited by the new sulfonamides (KIs in the range of 683-4250 nM), whereas hCA II, and the transmembrane, tumor associated isoforms hCA IX and XII were effectively inhibited in the subnanomolar – nanomolar range. A high resolution X-ray crystal structure of the adduct of hCA II with one of the new sulfonamides allowed us to rationalize the excellent inhibitory activity of these heterocyclic sulfonamides.

Keywords. Carbonic anhydrase; thiophene-2-sulfonamide; mercaptan; inhibitor. ____ *Corresponding authors: Tel: +371 67014826, Fax: +371 67550338, E-mail: [email protected] (Raivis Žalubovskis); Tel/Fax: +39-055-4573729, E-mail: [email protected] (Claudiu T. Supuran).

1. Introduction

Primary sulfonamides are a well-known class of potent inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), 1-3 some of which are used clinically for decades.4,5 The CA inhibitors (CAIs) have applications as diuretics, antiglaucoma, antiobesity, antiepileptic and antitumor agents, 6,7 and they target diverse CA isoforms of the 15 such proteins described so far in humans. 8 Despite the fact that at least 25 such agents are present in pharmacopeias,1a their use is limited due to side effects connected with the inhibition of isoforms not involved in the pharmacological responses of interest.1,2 Thus, a lot of research in this field was dedicated in the last years to the development of isoform-selective CAIs, belonging both to the sulfonamide5-7 as well as to the non-sulfonamide chemotypes.9,10 Although the most isoform-selective CAIs known to date are non-sulfonamide inhibitors (e.g., coumarins, sulfocoumarins, polyamines, etc.),9,10 several classes of sulfonamides were also discovered to show higher affinity for some isoforms (e.g, the transmembranetumor associated CA IX and XII) compared to the widespread, cytosolic ones (such as CA I and II).5 Thiophene-2-sulfonamides were already investigated in the early 90s by Shepard et al.11 in the search of topically-acting antiglaucoma CAIs. More recently, our groups12 reported a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, which showed very effective inhibitory properties against cytosolic and transmembrane CA isoforms with pharmacological applications. Furthermore, and X-ray crystallographic study of several of these sulfonamides bound to human (h) isoform hCA II revealed a very particular binding mode, as predominantly the hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the tight binding of these compounds. Considering that this scaffold (thiophene-2-sulfonamide) was not thoroughly investigated up until now for designing CAIs (except the two studies mentioned above11,12), we report here the synthesis and CA inhibitory data for a series of such derivatives incorporating 5-substituted-benzylsulfanyl (ArCH2S) moieties.

2. Results and Discussion

2.1. Chemistry

The rationale for introducing substituted-benzylsulfanyl moieties in position 5 of the thiophene-2-sulfonamide scaffold was that the SCH2 linker between the two aromatic fragments may lead to a good flexibility of the inhibitor tail, which has been demonstrated earlier to lead to isoform-selective sulfonamide CAIs.13 Thus, we have elaborated several strategies for introducing these functionalities in the position 5 of the thiophene-2-sulfonamide scaffold.14 NH HBr Method B

Method A Ar

Br

thiourea

Ar

1

S

NH2

thiourea

3

Ar

OH 2

HBr K2CO3

Ar SK 4 Br

S

Ar

SO2NH2

S

5

S

SO2NH2

6-17

Comp ound

Ar

Method

Yield (%)

6

Ph

-

20

7

(3,5-OMe)C6H3

-

18

8

(2-Ph)C6H4

A

12

9

(3-Ph)C6H4

A

8

10

2-Naphthyl

A

18

11

(4-CF3)C6H4

A

20

12

(4- CF3O)C6H4

A

17

13

(4-CN)C6H4

A

14

14

(4-Me)C6H4

A

25

15

(4-MeO)C6H4

B

29

16

(4-F)C6H4

B

29

17

(4-Cl)C6H4

B

35

Scheme 1. Synthesis of sulfonamides 6-17 reported in this paper.

Aryl bromides 1 or alcohols 2 were first converted to thiouronium salts 3 as shown in Scheme 1. Thiolates 4 were generated by treatment 3 with potassium carbonate and further in situ reacted with 5-bromothiophene-2-sulfonamide 5.15 Inhibitors 6 and 7 were isoleated in 20% and 18% respectively. For the synthesis of sulfonamides 6-17 reported here, the relevant step was a nucleophilic substitution reaction of bromide by thiolate in the key intermediate 5-bromothiophene-2-sulfonamide 5 (Scheme 1). It is worth noting that in these approaches the use of volatile and toxic thiols was avoided

by in situ generation of thiolates 4 from corresponding thiouronium salts 3. For the synthesis of compounds 6 and 7 the corresponding benzyl and dimethoxybenzyl thiouronium salts 3 were separately synthesized according literature procedure.14 To avoid time consuming synthesis of thiouronium salts 3, the literature reported but rarely applied one-pot procedure was used for the synthesis of inhibitors 8-17. In the Method A,16 the thiouronium salts 3 were prepared from corresponding substituted benzyl bromides 1 and after solvent replacement and treatment with potassium carbonate thiolates 4 were generated, which thereafter were reacted with 5bromothiophene-2-sulfonamide 5. This method afforded compounds 8-17 in 8% to 25% yields. By using method B,17 the thiouronium salts 3 were generated from substituted benzyl alcohols by treatment with hydrobromic acid in the presence of thiourea. The thiolates 4 were thereafter reacted whith 5-bromothiophene-2sulfonamide 5, leading to compounds 15-17 in 29% - 35% yields. All compounds were extensively characterized by physico-chemical methods which confirmed their structures (see Experimental part for details)

2.2. Carbonic anhydrase inhibition

As shown in Table 1, the new sulfonamides 6-17 were tested in vitro for their inhibition profiles against four physiologically relevant hCA enzymes, the cytosolic isoforms I and II and the trans-membrane, tumor associated IX and XII, by using a stopped-flow CO2 hydrase assay.18

Table 1. CA inhibition data against isoforms hCA I, II, IX and XII with sulfonamides 6-17 and acetazolamide (AAZ) as standard, by a stopped-flow CO2 hydrase assay.18 _____________________________________________________________________ KI (nM)*

Compound hCA I

hCA II

hCA IX

hCA XII

_____________________________________________________________________ 6

683

100.9

5.4

7.8

7

1280

31.6

7.5

12.9

8

3600

280

10.5

8.2

9

1870

316

25.4

80.1

10

965

24.1

8.3

5.9

11

4250

3.7

0.94

1.5

12

983

5.1

1.7

0.82

13

2470

20.1

39.8

12.9

14

716

9.3

10.5

8.4

15

982

5.5

6.1

12.7

16

737

4.4

10.5

17.1

17

826

2.9

7.0

5.2

AAZ

250

12

25

5.6

_____________________________________________________________________ *

Errors in the range of ±5 % of the reported values, from three different assays.

The following structure-activity relationship (SAR) may be observed by considering data of Table 1: (i) Medium - low inhibitory potency was observed for sulfonamides 6-17 against the slow cytosolic isoform hCA I, with KIs ranging between 683 and 4250 nM. Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ), a clinically used compound was a much more effective inhibitor of this isoform, with a KI of 250 nM. Thus, both compact arylsulfanyl groups such as benzyl (5) or substituted benzyls (13-17), or bulkier ones such as naphthylmethyl (10) or biphenylmethyl (8 and 9) lead to ineffective hCA I inhibitors in the series of investigated sulfonamides. (ii) In contrast to hCA I, the other investigated cytosolic isoform, the physiologically dominant one hCA II, was generally highly inhibited by sulfonamides 6-17 reported here. Apart the biphenylmethyl-substituted derivatives 8 and 9, which were poor hCA II inhibitors (KIs in the range of 280 - 316 nM, Table 1), all the other derivatives showed excellent inhibitory activity, with inhibition constants ranging between 2.9 and 31.6 nM. The 3,5-dimethoxy-phenyl (7), 2-naphthyl (10) and 4-cyanophenyl (13) derivatives showed the “worst” activity (KIs of 20.1-31.6 nM) whereas the remaining ones had inhibition constants < 10 nM, being more effective than the standard drug AAZ (Table 1). Thus, a range of substitution patterns lead to highly effective hCA II inhibitors, the main factor influencing activity being the nature of the Ar substituent present in the sulfanyl tail. Considering the phenyl derivative 6 as the parent compound of the whole series, which is already a very good hCA II inhibitor, introduction of substituents in the para position of the phenyl (e.g., halogens, methyl,

methoxy, trifluoromethyl, trifluoromethoxy) led to an enhanced inhibitory activity, the best compound being the 4-chlorophenyl derivative 17 (KI of 2.9 nM). (iii) The transmembrane, tumor-associated hCA IX was highly inhibited by all sulfonamides 6-17 investigated here, with KIs in the range of 0.94 – 39.8 nM (Table 1). The least effective inhibitors were 9 (biphenyl derivative) and 13 (4-cyano-phenyl derivative) with KIs of 25.4 – 39.8 nM (inhibition profile comparable to that of AAZ, KI of 25 nM), whereas the remaining derivatives showed a very high affinity for this isoform, with KIs in the range of 0.94 – 10.5 nM. SAR is thus very flat, since all the substitution patterns present in the tail led to highly potent hCA IX inhibitors (Table 1). (iv) Only compound 9 showed medium hCA XII inhibitory activity (KI of 80.1 nM) whereas all the other sulfonamides in the series were highly effective inhibitors of this transmembrane isoform involved in tumorigenesis and glaucoma-genesis. Indeed, inhibition constants ranging between 0.82 and 17.1 nM were measured for the investigated sulfonamides, as shown in Table 1. Again, as for hCA IX, the SAR is flat, with almost all investigated substitution patterns in position 5 of the thiophene ring leading to very effective CAIs. (v) With few exceptions, there was not isoform selectivity observed in this series of compounds. The biphenyl-substituted derivatives 8 and 9 were however better inhibitors of the transmembrane, tumor-asssociated isoform hCA IX compared to the cytosolic hCA I and II, and 8 was also effective in inhibiting hCA XII over the cytosolic enzymes. The remaining compounds effectively inhibited hCA II, IX and XII; which makes them of interest as candidates for antiglaucoma and antitumor applications of the CAIs.

2.3. X-Ray Crystallography

In order to rationalize the above results and to explore the binding mode of these sulfonamides within the CA active site, we resolved the crystal structure of the hCA II – 17 complex at 1.17 Å resolution (Table 2). As expected for a sulfonamide derivative, 17 was bound to enzyme active site with its sulfonamide group coordinated to the zinc ion (Fig. 1). The deprotonated sulfonamide amino group was bound to the Zn2+ ion, also forming a hydrogen bond with Thr199. Both these interactions are typical for all sulfonamide-CA complexes investigated so far.12,13,19

Compound 17 was not involved in other polar interactions with the enzyme active site, and its orientation was largely dominated by the hydrophobic and van der Waals contacts with residues Val121, Phe131, Val135, Leu198, Thr200 and Pro202 (Fig. 1). Interestingly, 17 was oriented towards the hydrophobic part of the active site, towards a region which is the most diverse one among all CA isoforms.20 Electron density of the ligand was good for all its moieties, as seen from Fig. 1.

Figure 1. Binding mode of compound 17 within the active site of hCA II. The Fo-Fc OMIT electron density map was calculated in the absence of the ligand and contoured at 3σ. The zinc ion is shown as a gray sphere and its coordinating resides (His94, 96, 119) are also shown. Oxygen atoms are indicated in red, carbon in green, nitrogen in blue, sulfur in yellow and chlorine in purple. Sulfonamide 17 carbon atoms are shown in black. Residues participating in hydrogen bonding, hydrophobic and van der Waals interactions with the ligand are also shown. The figure was generated using Pymol.21

Figure 2. Superimposition of the sulfonamide 17 and acetazolamide AAZ in complex with hCA II. Compound 17 is shown in thin lines (carbon atoms in black, heteroatoms colored), whereas acetazolamide (PDB code 3HS4) is shown with thick lines and light blue carbons. Amino acid residues involved in the binding of the inhibitors, the Zn(II) ion (gray sphere) and its three His ligands (His 94, 96 and 119) are also shown.

The binding mode of the thiophene/sulfonamide moieties from 17 is fairly similar to the binding mode of the thiadiazole/sulfonamide moieties from acetazolamide AAZ, as shown from the superposition of Fig. 2. The main difference is that the thiophene ring of 17 cannot participate to the hydrogen bond with Thr200, as there is no nitrogen in the thiophene ring (Fig. 2). It may be observed that the sulfamoyl and 5membered heterocyclic rings of the two inhibitors were completely superimposable, whereas the sulfanyl ring extended towards the hydrophobic part of the active site, as in the cotrresponding thiophene-2-sulfonamides incorporating triazole moieties reported earlier.12

Table 2. Data scaling, refinement and validation statistics for the hCA II-17 complex. ____________________________________________________________________ Structure hCA II-17 Space group P21 Cell dimensions

a (Å) b (Å) c (Å) b (o) Resolution (Å) Highest resolution shell (Å) Number of reflections Number of reflections in test set Completeness (%) Rmerge Average multiplicity R-factor Rfree Average B factor (Å2) Average B factor for inhibitor (Å2) from Wilson plot (Å2) Number of protein atoms Number of inhibitor atoms

42.4 41.3 72.3 104.5 18-1.17 1.17-1.23 77608 3989 99.6 (98.3*) 0.10 (0.45*) 6.3 (2.3*) 3.3 (3.0*) 0.13 (0.13*) 0.13 (0.14*) 15.7 31.6 6.4 2093 18

_____________________________________________________________________

3. Conclusions

A small library of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans (as potassium salts) or their equivalents, generated in situ. The new compounds were investigated as CAIs, against several pharmacologically relevant isoforms. The cytosolic hCA I isoform was poorly inhibited by the new sulfonamides (KIs in the range of 683-4250 nM), whereas another cytosolic enzyme, hCA II, and the transmembrane, tumor associated ones hCA IX and XII, were effectively inhibited in the subnanomolar – nanomolar range. A high resolution X-ray crystal structure of the adduct of hCA II with one of the new sulfonamides allowed us to rationalize the excellent inhibitory activity of these heterocyclic derivatives, which belong to a poorly investigated class of CAIs. The interesting inhibition profiles of these derivatives make them good candidates for antiglaucoma or antitumor studies.

4. Experimental part

4.1. Chemistry. Reagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatography was performed on silica gel, spots were visualized with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were measured on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Varian Mercury (400 MHz) spectrometer with chemical shifts values (δ) in ppm relative to TMS using the residual DMSO-d6 signal (1H 2.50; 13C 39.52) as an internal standard. HRMS data were obtained with a Q-TOF micro high resolution mass spectrometer with ESI (ESI+/ESI-).

General procedure from thiouronium salt To a solution of corresponding thiouronium salt 314 (1 equiv) in dry DMF (4.5 mL) was added potassium carbonate (2.2 equiv) dissolved in 0.5 mL water. The reaction mixture was stirred at room temperature for 45 minutes, then 5bromothiophene-2-sulfonamide (5) (1 equiv) was added and the mixture was stirred at 150 °C for 3.5 h, cooled to room temperature, diluted with water (25 mL), extracted with diethyl ether (3 × 30 mL), washed again with water (2 × 25 mL), dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by reversed phase chromatography (C-18, H2O-MeCN gradient MeCN 10-90 %), followed by recrystallization from MeOH/H2O.

O S

S

S

O NH2

5-(Benzylsulfanyl)thiophene-2-sulfonamide (6) Compound 6 was prepared according to the general procedure from amino(benzylthio)methaniminium bromide14 (0.50 g, 2.02 mmol), potassium carbonate (0.62 g, 4.49 mmol), 5-bromothiophene-2-sulfonamide (5) (0.49 g, 2.02 mmol) as a white solid (0.12 g, 20%). Mp 131-132 °C.

IR (KBr, cm-1) νmax= 3338 (NH), 3239 (NH), 1334 (S=O), 1150 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.20 (s, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.23-7.34 (m, 5H), 7.38 (d, 1H, J = 3.9 Hz), 7.68 (br s, 2H);

13

C NMR (100 MHz, DMSO-d6) δ=

41.5, 127.5, 128.5, 128.9, 130.1, 132.0, 137.0, 139.6, 146.8; HRMS (ESI) m/z [M+Na]+ calcd for C11H11NO2S3Na: 307.9850, found 307.9830. O O

S

S

S

O NH2

O

5-[(3,5-Dimethoxybenzyl)sulfanyl]thiophene-2-sulfonamide (7) Compound 7 was prepared according to the general procedure from amino[(3,5dimethoxybenzyl)thio]methaniminium bromide14 (0.50 g, 1.63 mmol), potassium carbonate (0.50 g, 3.62 mmol), 5-bromothiophene-2-sulfonamide (5) (0.39 g, 1.63 mmol) as a white solid (0.10 g, 18%). Mp 79-80 °C. IR (KBr, cm-1) νmax= 3360 (NH), 3258 (NH), 1316 (S=O), 1301 (S=O), 1164 (S=O), 1153 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 3.69 (s, 6H), 4.12 (s, 2H), 6.39 (t, 1H, J = 2.3 Hz), 6.44 (d, 2H, J = 2.3 Hz), 7.08 (d, 1H, J = 3.9 Hz), 7.40 (d, 1H, J = 3.9 Hz), 7.70 (br s, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 41.7, 55.2, 99.4, 106.8, 130.1, 132.1, 139.3, 139.7, 146.9, 160.4; HRMS (ESI) m/z [M+1]+ calcd for C13H16NO4S3: 346.0241, found 346.0283. General procedure for the synthesis of 5-(arylthio)thiophene-2-sulfonamides from arylmethylbromides (Method A) To a solution of thiourea (1 equiv) in acetonitrile (4 mL), the corresponding bromide (1 equiv) was added. The mixture was stirred at 40 °C overnight (16 h). Solvent was evaporated under reduced pressure and the resulting thiouronium salt 3 was dissolved in dry DMF (4.5 mL), a solution of potassium carbonate (2.2 equiv) in water (0.5 mL) was added and reaction mixture was stirred at room temperature for 45 minutes. Then 5-bromothiophene-2-sulfonamide (5) (0.8 equiv) was added and the reaction mixture was stirred at 150 °C for 3.5 h, cooled to room temperature, diluted with water (25 mL), extracted with diethylether (3 × 30 mL), washed with water (2 × 25 mL), dried over Na2SO4, evaporated under reduced pressure. The crude product

was purified by reversed phase chromatography (C-18, H2O-MeCN gradient MeCN 10-90 %), followed by crystallization from MeOH/H2O. O S

S

S

O NH2

5-[(Biphenyl-2-ylmethyl)sulfanyl]thiophene-2-sulfonamide (8)

Compound 8 was prepared according to the general procedure from thiourea (0.15 g, 2.02 mmol), 2-(bromomethyl)-1,1'-biphenyl (0.50 g, 2.02 mmol), potassium carbonate (0.62 g, 4.45 mmol), 5-bromothiophene-2-sulfonamide (5) (0.39 g, 1.62 mmol) as a white solid (0.07 g, 12%). Mp 123-124 °C. IR (KBr, cm-1) νmax= 3349 (NH), 3269 (NH), 1343 (S=O), 1165 (S=O), 1157 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.13 (s, 2H), 6.91 (d, 1H, J = 3.9 Hz), 7.21-7.24 (m, 1H), 7.27-7.31 (m, 2H), 7.34-7.47 (m, 7H), 7.68 (br s, 2H);

13

C NMR

(100 MHz, DMSO-d6) δ= 40.1, 127.3, 127.7, 127.8, 128.3, 128.9, 130.0, 130.1, 130.2, 132.5, 133.7, 139.2, 140.1, 141.9, 147.3; HRMS (ESI) m/z [M+1]+ calcd for C17H16NO2S3: 362.0343, found 362.0354. O S

S

S

O NH2

5-[(Biphenyl-3-ylmethyl)sulfanyl]thiophene-2-sulfonamide (9)

Compound (9) was prepared according to the general procedure from thiourea (0.15 g, 2.02 mmol), 3-(bromomethyl)-1,1'-biphenyl (0.50 g, 2.02 mmol), potassium carbonate (0.62 g, 4.45 mmol), 5-bromothiophene-2-sulfonamide (5) (0.39 g, 1.62 mmol) as a white solid (0.05 g, 8%). Mp 74-75 °C. IR (KBr, cm-1) νmax= 3342 (NH), 3252 (NH), 1345 (S=O), 1152 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.28 (s, 2H), 7.08 (d, 1H, J = 3.9 Hz), 7.27-7.31 (m, 1H), 7.34-7.49 (m, 5H), 7.53-7.62 (m, 4H), 7.67 (br s, 2H);

13

C NMR (100 MHz,

DMSO-d6) δ= 41.5, 125.8, 126.7, 127.3, 127.6, 127.9, 129.0, 129.1, 130.2, 132.3, 137.8, 139.6, 139.8, 140.3, 146.9; HRMS (ESI) m/z [M+Na]+ calcd for C17H15NNaO2S3: 384.0163, found 384.0145. O S

S

S

O NH2

5-[(Naphthalen-2-ylmethyl)sulfanyl]thiophene-2-sulfonamide (10) Compound 10was prepared according to the general procedure from thiourea (0.17 g, 2.26 mmol), 2-(bromomethyl)naphthalene (0.50 g, 2.26 mmol), potassium carbonate (0.69 g, 4.98 mmol), 5-bromothiophene-2-sulfonamide (5) (0.44 g, 1.81 mmol) as a white solid (0.11 g, 18%). Mp 173-174 °C. IR (KBr, cm-1) νmax= 3338 (NH), 3243 (NH), 1335 (S=O), 1150 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.38 (s, 2H), 7.04 (d, 1H, J = 3.8 Hz), 7.36 (d, 1H, J = 3.8 Hz), 7.47-7.53 (m, 3H), 7.66 (br s, 2H), 7.74-7.76 (m, 1H), 7.82-7.86 (m, 1H), 7.87-7.92 (m, 2H);

13

C NMR (100 MHz, DMSO-d6) δ= 41.8, 126.1, 126.4, 127.0,

127.5, 127.6, 127.7, 128.2, 130.2, 132.1, 132.2, 132.7, 134.6, 139.5, 146.8; HRMS (ESI) m/z [M+Na]+ calcd for C15H13NNaO2S3: 358.0006, found 357.9988. O S

S

S

O NH2

F F

F

5-{[4-(Trifluoromethyl)benzyl]sulfanyl}thiophene-2-sulfonamide (11) Compound 11 was prepared according to the general procedure from thiourea (0.16 g, 2.09 mmol), 1-(bromomethyl)-4-(trifluoromethyl) (0.50 g, 2.09 mmol), potassium carbonate (0.64 g, 4.60 mmol), 5-bromothiophene-2-sulfonamide (5) (0.41 g, 1.67 mmol) as a white solid (0.12 g, 20%). Mp 124-125 °C. IR (KBr, cm-1) νmax= 3369 (NH), 3263 (NH), 1330 (S=O), 1320 (S=O), 1170 (S=O), 1145 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.30 (s, 2H), 7.05 (d, 1H, J = 3.9 Hz), 7.39 (d, 1H, J = 3.9 Hz), 7.47-7.52 (m, 2H), 7.66-7.71 (m, 4H);

13

C NMR

(100 MHz, DMSO-d6) δ= 40.7, 124.3 (q, J = 272.0 Hz), 125.3 (q, J = 3.9 Hz), 127.9

(q, J = 31.8 Hz), 129.7, 130.2, 132.6, 138.8, 142.2 (q, J = 1.6 Hz), 147.2; HRMS (ESI) m/z [M+Na]+ calcd for C12H10F3NNaO2S3: 375.9723, found 375. 9704. O

F F

S

S

S

F

O NH2

O

5-{[4-(Trifluoromethoxy)benzyl]sulfanyl}thiophene-2-sulfonamide (12) Compound 12 was prepared according to the general procedure from thiourea (0.15 g, 1.96 mmol), 1-(bromomethyl)-4-(trifluoromethoxy)benzene (0.50 g, 1.96 mmol), potassium carbonate (0.60 g, 4.31 mmol), 5-bromothiophene-2-sulfonamide (5) (0.38 g, 1.57 mmol) as a white solid (0.10 g, 17%). Mp 108-109 °C. IR (KBr, cm-1) νmax= 3369 (NH), 3262 (NH), 1302 (S=O), 1169 (S=O), 1145 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.24 (s, 2H), 7.05 (d, 1H, J = 3.9 Hz), 7.28-7.33 (m, 2H), 7.37-7.42 (m, 3H), 7.67 (br s, 2H); 13C NMR (100 MHz, DMSOd6) δ= 40.5, 120.1 (q, J = 256.6 Hz), 121.0, 130.2, 130.8, 132.4, 136.8, 139.1, 147.1, 147.5; HRMS (ESI) m/z [M+Na]+ calcd for C12H10F3NNaO3S3: 391.9673, found 391.9690. O S

S

S

O NH2

N

5-[(4-Cyanobenzyl)sulfanyl]thiophene-2-sulfonamide (13) Compound 13 was prepared according to the general procedure from thiourea (0.19 g, 2.55 mmol), 4-(bromomethyl)benzonitrile (0.50 g, 2.55 mmol), potassium carbonate (0.78 g, 5.61 mmol), 5-bromothiophene-2-sulfonamide (5) (0.49 g, 2.04 mmol) as a yellow solid (0.11 g, 14 %). Mp 80-81 °C. IR (KBr, cm-1) νmax= 3329 (NH), 3237 (NH), 2234 (CN), 1347 (S=O), 1164 (S=O), 1153 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.28 (s, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.38 (d, 1H, J = 3.9 Hz), 7.43-7.47 (m, 2H), 7.70 (br s, 2H), 7.76-7.80 (m, 2H);

13

C NMR (100 MHz, DMSO-d6) δ= 40.9, 110.1, 118.7, 129.9, 130.2, 132.4,

132.9, 138.5, 143.3, 147.4; HRMS (ESI) m/z [M+Na]+ calcd for C12H10N2NaO2S3: 332.9802, found 332.9819.

O S

S

S

O NH2

5-[(4-Methylbenzyl)sulfanyl]thiophene-2-sulfonamide (14) Compound 14 was prepared according to the general procedure from thiourea (0.21 g, 2.70 mmol), 1-(bromomethyl)-4-methylbenzene (0.50 g, 2.70 mmol), potassium carbonate (0.82 g, 5.94 mmol), 5-bromothiophene-2-sulfonamide (5) (0.52 g, 2.16 mmol) as a white solid (0.16 g, 25 %). Mp 143-144 °C. IR (KBr, cm-1) νmax= 3344 (NH), 3255 (NH), 1322 (S=O), 1155 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 2.27 (s, 3H), 4.16 (s, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.10-7.13 (m, 2H), 7.15-7.18 (m, 2H), 7.39 (d, 1H, J = 3.9 Hz), 7.69 (br s, 2H);

13

C

NMR (100 MHz, DMSO-d6) δ= 20.7, 41.3, 128.8, 129.0, 130.2, 131.8, 133.9, 136.6, 139.9, 146.6; HRMS (ESI) m/z [M+Na]+ calcd for C12H13NNaO2S3: 322.0006, found 322.0015.

General procedure for the synthesis of 5-(arylthio)thiophene-2-sulfonamides from aryl methanol (Method B) To a solution of thiourea (1 equiv) in H2O (0.75 mL/mmol phenylmethanol) and 48% HBr (0.70 mL/mmol arylmethanol) corresponding arylmethanol (1 equiv) was added. The mixture was stirred at 60 °C overnight (16 h). Solvent was evaporated under reduced pressure and the resulting thiouronium salt was dissolved in dry DMF (4.5 mL), a solution of potassium carbonate (2.2 equiv) in water (2.0 mL) was added and reaction mixture was stirred at room temperature for 1 hour. Then 5bromothiophene-2-sulfonamide (5) (0.5 equiv) was added and reaction mixture was stirred at 150 °C for 4 h, cooled to room temperature, diluted with saturated NH 4Cl (40 mL), extracted with EtOAc (3 × 30 mL), washed with water (2 × 25 mL), dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by reversed phase chromatography (C-18, H2O-MeCN gradient MeCN 10-90 %). O S O

S

S

O NH2

5-[(4-Methoxybenzyl)sulfanyl]thiophene-2-sulfonamide (15) 11a Compound 15 was prepared according to the general procedure from thiourea (0.27 g, 3.62 mmol), (4-methoxyphenyl)methanol (0.50 g, 3.62 mmol), potassium carbonate (1.09 g, 7.96 mmol), 5-bromothiophene-2-sulfonamide (5) (0.44 g, 1.81 mmol) as a white solid (0.16 g, 29%). Mp 137-138 °C. IR (KBr, cm-1) νmax= 3325 (NH), 3246 (NH), 1318 (S=O), 1155 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 3.73 (s, 3H), 4.15 (s, 2H), 6.84-6.89 (m, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.17-7.22 (m, 2H), 7.39 (d, 1H, J = 3.9 Hz), 7.68 (br s, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 41.1, 55.1, 113.9, 128.7, 130.1, 130.2, 131.8, 139.9, 146.6, 158.6; HRMS (ESI) m/z [M+Na]+ calcd for C12H13NNaO3S3: 337.9955, found 337.9941. O S

S

S

O NH2

F

5-[(4-Fluorobenzyl)sulfanyl]thiophene-2-sulfonamide (16) Compound 16 was prepared according to the general procedure from thiourea (0.30 g, 3.96 mmol), (4-fluorophenyl)methanol (0.50 g, 3.96 mmol), potassium carbonate (1.20 g, 8.71 mmol), 5-bromothiophene-2-sulfonamide (5) (0.48 g, 1.98 mmol) as a white solid (0.17 g, 29%). Mp 111-112 °C. IR (KBr, cm-1) νmax= 3339 (NH), 3245 (NH), 1345 (S=O), 1151 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.19 (s, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.10-7.17 (m, 2H), 7.28-7.33 (m 2H) 7.39 (d, 1H, J = 3.9 Hz), 7.69 (br s, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 40.6, 115.3 (d, J = 21.0 Hz), 130.2, 130.9 (d, J = 8.6 Hz), 132.3, 133.4, (d, J = 3.1 Hz), 139.2, 147.0, 161.4 (d, J = 244.0 Hz); HRMS (ESI) m/z [M+Na]+ calcd for C11H10FNNaO2S3: 325.9755, found 325.9745. O S

S

S

O NH2

Cl

5-[(4-Chlorobenzyl)sulfanyl]thiophene-2-sulfonamide (17)

Compound 17 was prepared according to the general procedure from thiourea (0.27 g, 3.51 mmol), (4-chlorophenyl)methanol (0.50 g, 3.51 mmol), potassium carbonate (1.07 g, 7.72 mmol), 5-bromothiophene-2-sulfonamide (5) (0.42 g, 1.75 mmol) as a white solid (0.20 g, 35%). Mp 109-110 °C. IR (KBr, cm-1) νmax= 3338 (NH), 3254 (NH), 1327 (S=O), 1157 (S=O); 1H NMR (400 MHz, DMSO-d6) δ= 4.19 (s, 2H), 7.04 (d, 1H, J = 3.9 Hz), 7.27-7.31 (m, 2H), 7.35-7.40 (m, 3H), 7.69 (br s, 2H);

13

C NMR (100 MHz, DMSO-d6) δ= 40.6,

128.4, 130.2, 130.7, 132.0, 132.4, 136.3, 139.1, 147.1; HRMS (ESI) m/z [M+Na]+ calcd for C11H10ClNNaO2S3: 341.9460, found 341.9453.

4.2. CA inhibition assay An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes. 18 Phenol Red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na 2SO4 or NaClO4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration),12 following the CA-catalyzed CO2 hydration reaction for a period of 5-10 s. Saturated CO2 solutions in water at 25 °C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSOwater 1:1, v/v) and dilutions up to 1 nM done with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using the Cheng-Prusoff equation, as reported earlier,12 and represent the mean from at least three different determinations. All CA isozymes used here were recombinant proteins obtained as reported earlier by our group.22-25

4.3. X-ray crystallography

4.3.1. Crystallization and data collection

hCA II was concentrated to 10 mg/ml in 20 mM tris-HCl pH 8.0 using a 10 kDa cutoff Amicon concentrator. Crystallization was performed by the hanging drop technique in EasyXtal 15 well plates (QIAGEN). 5 µl of protein was mixed with 5 µL of bottom solution (1.4 M Na citrate, 100 mM tris–HCl pH 9.0) and 0.1 µL of 100 mM compound 17 in 100% DMSO. Data were collected on beamline I911−3 at the MAX-Lab Synchrotron, Lund, Sweden.

Structure Determination Data were processed with MOSFLM

26

and scaled with SCALA.27 Structures were

solved by molecular replacement method using 4BF112 as initial model in program REFMAC.28 The parameter files of the compound was generated by LIBCHECK.29 Ligand modeling in electron density and structure validation was done using COOT,30 followed by further REFMAC runs. Data scaling, refinement and validation statistics are listed in Table 2. Coordinates and experimental structure factors were deposited in Protein Data Bank with accession code XXXX.

Acknowledgements. The study was supported by grant 7869 from Biostruct-X. We are thankful to the MAX-lab staff (Lund, Sweden) for their support at the synchrotron.

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