Bioorganic & Medicinal Chemistry Letters 24 (2014) 275–279

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Sulfonamide inhibition studies of the d-carbonic anhydrase from the diatom Thalassiosira weissflogii Daniela Vullo a, Sonia Del Prete b, Sameh M. Osman c, Viviana De Luca b, Andrea Scozzafava a, Zeid AlOthman c, Claudiu T. Supuran a,c,d,⇑, Clemente Capasso b,⇑ a

Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italy Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia d Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEIROFABA, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy b c

a r t i c l e

i n f o

Article history: Received 29 October 2013 Accepted 10 November 2013 Available online 20 November 2013 Keywords: Carbonic anhydrase d-Class enzyme Sulfonamide Enzyme inhibitor Thalassiosira weissflogii

a b s t r a c t The d-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a d-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with KIs of 49.6–118 nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (KIs of 375–897 nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the d-class enzyme, with KIs of 4.27–9.20 lM. The inhibition profile of the d-CA is very different from that of a-, b- and c-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the d-CA active site. Ó 2013 Elsevier Ltd. All rights reserved.

There are five distinct classes of carbonic anhydrases (CAs, EC 4.2.1.1) in organisms all over the phylogenetic tree, denominated a-, b-, c-, d- and f-CA.1–3 The d-CA class, although reported in 1997 by Morel’s group4 in the diatom Thalassiosira weissflogii, was the least investigated one5 due to difficulties connected with the production of pure recombinant protein (in Escherichia coli).4,5 Only recently we have reported6 the preparation of this enzyme by using the artificial gene encoding the d-CA from Thalassiosira weissflogii (denominated TweCA), the kinetic properties of this enzyme as well as an inhibition study with inorganic anions and small molecules such as sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid. We observed that TweCA is an efficient catalyst for the CO2 hydration to bicarbonate and protons (the physiological reaction in which CAs are involved), possessing kinetic parameters in the same range as enzymes belonging to the other CA classes, with a kcat of 1.3  105 s 1 and a kcat/KM of 3.3  107 M 1 s 1.6 Furthermore, the best TweCA inhibitors detected in the earlier study were anions such as hydrogensulfide, or the small molecules sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which ⇑ Correspondence authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.); tel.: +39 081 6132559; fax: +39 081 6132712 (C.C.). E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), [email protected] (C. Capasso). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.021

showed KIs in the range of 9–90 lM, whereas acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide) inhibited the enzyme with a KI of 83 nM.6 As acetazolamide was the best TweCA inhibitor detected so far, it appeared of interest to investigate in more detail sulfonamides and their isosteres (e.g., sulfamates) as d-CA inhibitors. Indeed, the primary sulfonamides constitute the classical, best investigated type of inhibitors of all classes of CAs, and their mechanism of inhibition is also well understood: they bind in deprotonated state to the metal ion from the enzyme active site, interfering thus with its catalytic activity.2,3,7,8 Sulfonamides (and sulfamates, sulfamides, which are bioisosteres of the sulfonamides) were investigated in fact as inhibitors of all other classes of CAs, except the d-CAs.7–11 Indeed, compounds such as acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA and dichlorophenamide DCP, are clinically used, systemically acting antiglaucoma CA inhibitors (CAIs).2,11 Dorzolamide DZA and brinzolamide BRZ are topically-acting antiglaucoma agents, benzolamide BZA is an orphan drug belonging to this class of pharmacological agents, whereas topiramate TPM, zonisamide ZNS and sulthiame SLT are widely used antiepileptic drugs.2,11 Sulpiride SLP and indisulam IND were also shown by our group to belong to this class of pharmacological agents,2 together with the COX2 ‘selective’ inhibitors celecoxib CLX and valdecoxib VLX.2,12 Saccharin and the diuretic hydrochlorothiazide HCT are also known to act as CAIs.2,13

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SO2NH2

SO2NH2

SO2NH2

SO2NH2

SO2NH2

NH2

NH2

1

2

4

3

SO2NH2

SO2NH2

SO2NH2

CH2NH2

CH2CH2NH2

NH2

SO2NH2

F 6

5

7

SO2NH2

SO2NH2

Cl

Cl

Cl

SO2NH2 NH2

10

9

H2N

SO2NH2

S

N HN

SO2NH2

(CH2)nOH

COOH

15: n = 0 16: n = 1 17: n = 2

18

N S

SO2NH2

14

H N

SO2NH2

O H2N

N NH2

S N H O HO

N

S N H

O

19 O

O2N

12

SO2NH2

H3C

N

SO2NH2 NH2

11

13

N

SO2NH2

CF3

NH2

N

8

SO2NH2

OH Br

Cl NH2

N S

SO2NH2

20 O

SO2NH2 H2N

21

Thus, a library of 40 compounds, comprising 39 sulfonamides and one sulfamate were included in our study of TweCA inhibition. Derivatives 1–24 (in addition to AAZ–HCT) were included in the study, as they are simple aromatic/heterocyclic sulfonamides widely used as building blocks for obtaining new families of such pharmacological agents.14 Data of Table 1 show the inhibition data of TweCA with the 40 derivatives mentioned above, as obtained by a stopped-flow CO2 hydrase assay monitoring the physiologic reaction catalyzed by CAs.15 Inhibition data of the human (h), possibly off target a-CA isoform hCA II,2 of a b-class CA, LdcCA (from the protozoan pathogen Leishmania donovani chagasii)11b and of PgiCA, a c-CA from the oral pathogen Porphyromonas gingivalis,16 are also presented in Table 1, for comparison reasons of sulfonamide inhibition of a-, b-, c- and d-CAs (such a comparison has never been done until now). The following structure-activity relationship (SAR) data can be observed for the inhibition of TweCA with this panel of sulfonamides/sulfamates: (i) A first group of sulfonamides, including compounds 13–17, 20–22 and SAC, HCT, showed very ineffective TweCA inhibitory

( )n S N H O 22: n = 0 23: n = 1 24: n = 1

SO2NH2

power, with inhibition constants in the range of 3.74–15.2 lM. They include the heterocyclic amines/imines 13 and 14, the 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides 15–17, as well as aminobenzolamide 20/sulfonylated–sulfanilamide type of derivatives (21, 22). Considering also the saccharine/hydrochlorothiazide scaffolds here, it is obvious that the inefficient TweCA inhibitors belong to quite heterogeneous classes of compounds, for which a real SAR is difficult if not impossible to define. (ii) Medium potency TweCA inhibition was observed for a large number of the investigated sulfonamides, such as 1–8, 10–12, 18, 19, 23, 24, MZA, DCP, BZA–SLT (Table 1). These derivatives showed inhibition constants in the range of 167–897 nM against TweCA. Although they are again rather heterogeneous, a more clear-cut SAR was possible to delineate for these medium potency TweCA inhibitors. Thus, it may be observed the 3- or 4-substituted and 3,4-disubstituted benzenesulfonamides incorporating amino, methyl, as well as chloro and amino groups, are rather effective TweCA inhibitors, with KIs of 167–295 nM. However bulkier such substituents (as in 3, with 1,3-disulfamoyl moieties) or 5 and 6 (incorporating aminomethyl and aminoethyl moieties instead of

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N CH3CONH

H3C

N SO2NH2

S

N

CH3CON

N SO2NH2

S

AAZ

EtO

EZA NHEt

NHEt

SO2NH2

Me

Cl

O

DCP

S

SO2NH2

S

N

MeO(CH2)3

O

O DZA

N

O S N H O

S

S

SO2NH2

O

BRZ

N

O NH2 O S O O

O SO2NH2

S

SO2NH2

S

MZA

SO2NH2

Cl

N

O

O O

BZA

SO2NH2 N O

TPM

ZNS

OMe O H N

N H

N

O O S N H

Cl

SO2NH2

SO2NH2 SLP

IND SO2NH2

SO2NH2

H3C

N O N

CH3

N

S

N

SO2NH2

O O SLT

F F F

VLX

CLX

O NH O

S

O

SAC

the NH2 group present in 2) lead to a decrease of the TweCA inhibitory power, to KIs of 424–896 nM (Table 1). The same is true for the fluoro-substituted sulfanilamide 7, the 4-substituted sulfanilamides incorporating a COOH moiety (18) or the aminopyrimidine scaffold (19), as well as the trisubstituted benzenesulfonamides/ disulfonamides 10–12 (KIs in the range of 534–871 nM). Thus, very small changes in the substitution pattern or in the nature of the other group(s) present in the sulfanilamide scaffold (e.g., compare 7 and 8 in which a F was substituted by a Cl atom), lead to drastic changes in the inhibitory power of these compounds against TweCA. The same is true for the compoounds incorporating the elongated, sufanilated sulfanilamide scaffolds (23 and 24) which were anyhow more effective as TweCA inhibitors compared to the structurally related compounds with shorter spacers (20–22)

H N HN O

Cl

S O

SO2NH2

HCT

discussed above. Among the clinically used sulfonamides/sulfamates, the most ineffective TweCA inhibitors were the 1,3-benzenedisulfonamide derivative dichlorophenamide, benzolamide (a long molecule compound similar to 20–22), the sulfamate topiramate, the aliphatic sulfonamide zonisamide, indisulam and valdecoxib (KIs in the range of 375–458 nM) whereas the remaining derivatives in this category showed a rather compact behavior (KIs varying between 169 and 265 nM)—see Table 1. (iii) The most effective TweCA inhibitors detected here were 3-bromosulfanilamide 9, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide, with KIs in the range of 49.6–118 nM. From the SAR viewpoint, it is interesting to note that for the halogenosulfanilamides 7–9, the inhibition of TweCA steeply increased with the tomic weight of the halogen atom. Indeed, the

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Table 1 Inhibition of human isoforms hCA I and hCA II, of the protozoan one from L. donovani chagasi (LdcCA) as well as the bacterial enzyme from Porphyromonas gingivalis (PgiCA) and the d-CA from T. weissflogii (TweCA) with sulfonamides 1–24 and the clinically used drugs AAZ–HCT, by a CO2 hydrase, stopped-flow assay15 KI* (nM)

Inhibitor/enzyme class LdcCA b

PgiCAc

a

c

TweCAd d

300 240 8 320 170 160 60 110 40 54 63 75 60 19 80 94 125 46 33 2 11 46 33 30 12 14 8 38 9 3 9 10 35 40 15 43 21 9 5959 290

5960 9251 8910 >100,000 >100,000 >100,000 15,600 9058 8420 9135 9083 4819 584 433 927 389 227 59.6 >100,000 95.1 50.2 136 87.1 73.4 91.7 87.1 51.5 189 806 764 236 >100,000 >100,000 >100,000 316 338 705 834 >100,000 50.2

4220 893 >100,000 945 945 945 680 662 201 218 711 1040 510 595 326 223 178 560 685 1450 3540 4100 4650 3400 324 343 613 1035 685 722 741 >100,000 157 418 131 755 169 424 273 380

295 181 424 424 424 424 534 167 94.6 762 725 662 10,600 5525 4410 15,200 4700 871 615 12,350 7880 3740 897 862 83 170 85.1 458 49.6 118 394 392 404 259 375 378 265 169 9200 4270

hCA II 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 AAZ MZA EZA DCP DZA BRZ BZA TPM ZNS SLP IND VLX CLX SLT SAC HCT

a

b

*

Errors in the range of 5–10% of the shown data, from three different assays. Human recombinant isozymes, stopped flow CO2 hydrase assay method, from Ref. 2. b Recombinant protozoan enzyme, stopped flow CO2 hydrase assay method, from Ref. 11b. c Recombinant bacterial enzyme, from Ref. 16. d Recombinant diatom enzyme, this work. a

fluoroderivative 7 was a weak inhibitor, the chloro-derivative 8 was almost 4-times more effective than 7, whereas the bromoderivative 9 was the only aromatic sulfonamide investigated here with a KI