Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Sulfonamide inhibition studies of the b carbonic anhydrasefrom Drosophila melanogaster

http://dx.doi.org/10.1016/j.bmcl.2014.04.1170960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +39 055 4573729; fax: +39 055 4573385.E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).

Please cite this article in press as: Syrjänen, L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.117

Leo Syrjänen a,b, Seppo Parkkila a,b,c, Andrea Scozzafava d, Claudiu T. Supuran d,e,⇑a School of Medicine, University of Tampere, Tampere, Finlandb BioMediTech, University of Tampere, Tampere, Finlandc Fimlab Laboratories Ltd and Tampere University Hospital, Tampere, Finlandd Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italye Università degli Studi di Firenze, Neurofarba Dipartment, Sezione di Scienza Farmaceutiche e Nutraceutiche, Via Ugo Sciff 6, 50019 Sesto Fiorentino (Firenze), Italy

a r t i c l e i n f o

Article history:Received 31 March 2014Revised 28 April 2014Accepted 29 April 2014Available online xxxx

Keywords:Carbonic anhydraseb-Class enzymeDrosophila melanogasterSulfonamideEnzyme inhibitor

a b s t r a c t

An inibition study of the b-carbonic anhydrase (CA, EC 4.2.1.1) DmBCA from the insect Drosophila mela-nogaster with sulfonamides and sulfamates is reported. Among the panel of 40 investigated compounds,the best DmBCA inhibitors were the sulfonylated benzenesulfonamides and ethoxzolamide, whichshowed inhibition constants in the range of 65.3–138 nM. Methazolamide and sulthiame were also effec-tive inhibitors with KIs ranging between 237 and 249 nM, whereas most of the simple aromatic/hetero-cyclic sulfonamides showed inhibition constants in the range of 0.47–6.40 lM. Topiramate, zonisamideand saccharine did not inhibit DmBCA. As orthologs of this mitochondrial CA are found in many insectspecies involved in the spread of various diseases, inhibitors interfering with their activity may be ofinterest for developing insecticides with an alternative mechanism of action to the presently used agents,for which many insects developed extensive resistance.

� 2014 Elsevier Ltd. All rights reserved.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the reversiblehydration of carbon dioxide: CO2 + H2O M HCO3

� + H+, a reactionof fundamental importance in most organisms.1–4 CAs are metallo-enzymes usually having a zinc(II) ion at the active site, but somecadmium(II)-CAs (which is inter-exchangeable with Zn(II)),3 asan alternative metal cofactor were also reported.3c-CAs may con-tain iron(II) ions within the active site, at least in some anaerobicArchaea.5 The reaction catalyzed by CAs is essential in the regula-tion of acid–base balance in organisms all over the phylogenetictree, which explains why these enzymes are present in a large vari-ety of classes and isoforms in many organisms.1–6 In fact, this reac-tion helps to remove carbon dioxide out of the tissues, being alsoinvolved in biosynthetic reactions such as gluconeogenesis andureagenesis, etc.1

Until now, five classes of CAs have been identified: the a-, b-, c-,d- and f-ones.1–3,7–10 The metal ion is coordinated by three His res-idues in the a-, c- and d-CAs, whereas for the lastly discoveredfamily, the f-CA (isolated from the marine diatom Thalassiosiraweissflogii)3 the coordination of the metal ion within the active siteis very similar to that of the b-CAs, with one His, two Cys and awater molecule acting as ligands of the Cd(II) or Zn(II) ions.3,6–11

b-CAs seem to be the group with the widest distribution, assuch enzymes have been described in various groups of organismsincluding the Archaea and Bacteria domains, as well as in plantsand fungi among Eukaryotes.1,2,12,13 Recently, we reported thewidespread occurrence of at least one single-copy of a b-CA geneamong animal species distinct from chordates.6 Indeed, b-CAs haveso far been reported in various pathogenic organisms including thefungi/yeasts Candida albicans, Candida glabrata, Cryptococcus neo-formans and Saccharomyces cerevisiae7,8 and the bacteria Mycobac-terium tuberculosis, Brucella suis, Salmonella typhimurium,Helicobacter pylori, Streptococcus pneumoniae, Legionella pheumo-phila and Haemophilus influenzae.12–17 The inhibition profiles ofthese enzymes with various agents such as sulfonamides, anions,carboxylates, phenols, dithiocarbamates and boronic acids havealso been explored.1,2,8–17

b-CAs have been reported in two invertebrate species, namelythe insect Drosophila melanogaster and the worm Caenorhabditiselegans.6,16 For the insect, it has been shown that this enzymehas a mitochondrial localization, being probably involved in meta-bolic processes, similar to the mammalian mitochondrial isoforms,which participate in gluconeogenesis, lipogenesis and ureagene-sis.1,2 However, b-CAs are not present in vertebrates, which offersthe opportunity to design specific inhibitors for the b-CAs, thatcould be used against pathogenic, invertebrate organisms or

2 L. Syrjänen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx

microorganisms. For example, a b-CA from protozoa belonging tothe genus Leishmania was recently discovered, cloned and charac-terized, being shown that some thiols, CA inhibitors of this enzyme,kill the pathogen in vivo.14

Sulfonamides are the main class of zinc-binding CA inhibitors(CAIs),1,18–20 but several other classes of such compounds werealso reported recently, such as the thiols, dithiocarbamates, cou-marins, and polyamines among others.21–23 However, b-CAs areeffectively inhibited only by sulfonamides, thiols and dithiocarba-mates, as the coumarins are not acting as inhibitors of this class ofCAs (which do not possess esterase activity).23

Here we report the first inhibition study with sulfonamides ofthe recently discovered b-CA from the model insect Drosophila mel-anogaster, DmBCA.6 As many insects spread various infectiveagents (such as malaria parasites, West Nile virus, yellow fevervirus, etc.)17b,24 these studies may provide lead compounds usefulin the investigation of repellents or insecticides with an alternativemechanism of action compared to the widespread such agents towhich many insects developed resistance.25 It should be men-tioned that only simple inorganic anions as well as sulfamideand sulfamic acid were investigated up until now as inhibitors of

SO2NH2

NH2

SO2NH2

NH2

SO2NH2

CH2NH2

SO2NH2

CH2CH2NH2

SO2NH2

NH2

Br

SO2NH2

OH

ClCl

CF3

S

NN

NH2 SO2NH2S

N

NH SO2NH2

NCH3

12

5 6

9 10

1314

NH2

SO2NH2NH

N

N

NH2

NH

SO

O SO2NH2

OH

O2N

21

19

Please cite this article in press as: Syrjänen, L.; et al. Bioorg. Med. Chem

DmBCA.6 Anions showed millimolar affinity for this enzyme,whereas sulfamide and sulfamate were micromolar inhibitors.6

A series of simple aromatic and heterocyclic sulfonamides (of types1–24) as well as the clinically used agents belonging to the sulfon-amide/sulfamate class, that is, compounds such as acetazolamideAAZ, methazolamide MZA, ethoxzolamide EZA, dichlorophenamideDCP, dorzolamide DZA and brinzolamide BRZ were included in thisstudy.1,2 Topiramate TPM (a sulfamate) and zonisamide ZNS (an ali-phatic sulfonamide), widely used antiepileptic drugs, as well as othercompounds incorporating primary/secondary sulfamoyl moieties suchas sulpiride SLP and indisulam IND were also shown to belong to thisclass of pharmacological agents together with the COX2 selectiveinhibitors celecoxib CLX and valdecoxib VLX, the antiepileptic sulthi-ame SLT, the sweetener saccharin SAC and the diuretic hydrochlorothi-azide HCT, were also included in the present study.1,2 Compounds 1–24used in the assay as well as the clinically used drugs mentioned abovewere commercially available, or prepared as reported earlier by ourgroup.26 In this way it is possible to explore a wider chemical spacefor detecting effective inhibitors of the new enzymes reported here,considering the fact that the 40 compounds tested in our study possessa range of scaffolds and various substitution patterns.

SO2NH2

SO2NH2

SO2NH2

NH2

F

SO2NH2

NH2

Cl

SO2NH2

NH2

SO2NH2

SO2NH2

NH2

SO2NH2

Cl

SO2NH2

SO2NH2

(CH2)nOH

SO2NH2

COOH

3

7 8

11 12

4

15: n = 016: n = 117: n = 2

18

S

NN

NH

SO2NH2SO

O

SO2NH2NH

SO

ONH2

20

22: n = 023: n = 124: n = 2

( )n

. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.117

S SSO2NH2

O OMe

NHEt

S

NN

CH3CONH SO2NH2 S

N

CH3CON SO2NH2

NCH3

NS S

SO2NH2

O OMeO(CH2)3

NHEtSO2NH2

ClSO2NH2Cl

S

N

SO2NH2EtO

S

NN

NH

SO2NH2SO

O O

O

OO

OO

SNH2

O

O

SO2NH2

SOO

NH

NH

ClN

SO2NH2

OMe

NH

O

ON

SO2NH2

AAZ MZA

DZA BRZ

EZA

DCP

BZA TPM

INDSLP

ZNS

NN

SO2NH2

F

FF

CH3

NO

SO2NH2

CH3

SNH

O

O O

SO2NH2N

SO O

NH

NHS

Cl

SO2NH2O O

VLXCLX

SAC

SLT

HCT

L. Syrjänen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 3

Data of Table 1 show the inhibition of the Drosophila enzymeDmBCA with this set of sulfonamides/sulfamates. For comparisonreasons, the inhibition of the two human (h) offtarget isoformshCA I and II (belonging to the a-CA family) as well as the protozoana-/b-CA from the pathogens Trypanosoma cruzi (TcCA) and Leish-mania donovani chagasi (LdcCA) with this set of 40 compounds,are also presented in Table 1. The following structure–activity rela-tionship (SAR) can be observed for the inhibition of DmBCA withthese compounds:

(i) The first group of compounds, including 1, 2, 4–8, 13–15, 18,DZA, BRZ, BZA, SLP, IND, VLX, CLX, and HCT, showed inef-fective inhibitory properties against DmBCA, with inhibition

Please cite this article in press as: Syrjänen, L.; et al. Bioorg. Med. Chem

constants ranging between 1.235 and 6.40 lM. It should bealso noted that three of the investigated compounds, thatis, TPM, ZNS and SAC, did not inhibit DmBCA at all, up until100 lM concentrations of inhibitor in the assay system(Table 1). The SAR for these ineffective inhibitors is not verystraightforward as they belong to many different classes ofsulfonamides, such as the 4-substituted or 3,4-disubstitutedbenzenesulfonamides incorporating amino, aminoalkyl,hydroxymethyl and halogen functionalities, or the heterocy-clic sulfonamides (13, 14, DZA, BRZ, BZA, etc.). More specif-ically, for the 4-amino- and 4-aminoalkyl derivatives 2, 5and 6, the inhibitory power increases with the length ofthe linker (from 0 to 2 carbon atoms) between the amino

. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.117

Table 1Inhibition of human isoforms hCA I and hCA II, the protozoan ones from T. cruzi (TcCA)and L. donovani chagasi (LdcCA) and the insect one DmBCA from D. melanogaster, withsulfonamides 1–24 and the clinically used agents AAZ–HCT18

Inhibitor KI* (nM)

hCA Ia hCA IIa TcCAb LdcCAc DmBCAd

1 28,000 300 25,460 5960 58452 25,000 240 57,300 9251 48103 79c 8 63,800 8910 7854 78,500 320 44,200 >100,000 54905 25,000 170 7231 >100,000 32106 21,000 160 9238 >100,000 16157 8300 60 8130 15,600 28108 9800 110 6925 9058 23459 6500 40 8520 8420 94210 7300 54 9433 9135 31411 5800 63 842 9083 28712 8400 75 820 4819 36013 8600 60 534 584 417514 9300 19 652 433 191015 5500 80 73,880 927 123516 9500 94 71,850 389 87217 21,000 125 66,750 227 61318 164 46 84,000 59.6 640019 109 33 810 >100,000 52520 6 2 88.5 95.1 47121 69c 11 c 134 50.2 93.222 164 46 365 136 13823 109 33 243 87.1 10424 95 30 192 73.4 65.3AAZ 250 12 61.6 91.7 516MZA 50 14 74.9 87.1 237EZA 25 8 88.2 51.5 116DCP 1200 38 128 189 605DZA 50,000 9 92.9 806 3565BRZ 45,000 3 87.3 764 5370BZA 15 9 93.6 236 1565TPM 250 10 85.5 >100,000 >100,000ZNS 56 35 867 >100,000 >100,000SLP 1200 40 87.9 >100,000 6260IND 31 15 84.5 316 5025VLX 54,000 43 82.7 338 4470CLX 50,000 21 91.1 705 5900SLT 374 9 71.9 834 249SAC 18,540 5959 8210 >100,000 >100,000HCT 328 290 134 50.2 5450

* Errors in the range of 5–10% of the shown data, from 3 different assays.a Human recombinant isozymes, stopped flow CO2 hydrase assay method, from

Ref. 26b.b Recombinant bacterial enzyme, stopped flow CO2 hydrase assay method, from

Ref. 24a.c Recombinant enzyme, stopped flow CO2 hydrase assay method, from Ref. 14.d Recombinant insect enzyme, stopped flow CO2 hydrase assay method.

4 L. Syrjänen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx

moiety and the benzenesulfonamide scaffold, with 4-amino-ethylbenzenesulfonamide 6 being 2.97-times a betterDmBCA inhibitor compared to sulfanilamide 1. Comparing4-amino-/4-hydroxybenzenesulfonamides (2 and 15) as wellas the corresponding aminoalkyl/hydroxyalkyl derivatives(5 and 16, or 6 and 17), it may be observed that the hydro-xyl-containing derivatives were more inhibitory comparedto the corresponding amino derivatives, and again, theinhibitory power increased with the length of the spacerbetween the hydroxyl and benzenesulfonamide scaffold,from 0 to 2. It is also obvious that the presence of bulky moi-eties at the benezenesulfonamide scaffold, as in SLP, IND,VLX and CLX, is detrimental to the DmBCA inhibitory activ-ity of these sulfonamides.

(ii) More effective DmBCA inhibitory activity was observed forthe following sulfonamides: 3, 9–12, 16, 17, 19, 20, AAZ,and DCP, which showed inhibition constants ranging

Please cite this article in press as: Syrjänen, L.; et al. Bioorg. Med. Chem

between 287 and 942 nM (Table 1). Some of these deriva-tives incorporated the 1,3-benzene-disulfonamide scaffold(e.g., 3, 11, 12 and DCP). The presence of additional amino,trifluoromethyl or halogeno moieties in the scaffold of 3was beneficial for the inhibitory effects of these compoundsagainst DmBCA. For the halogenosulfanilamides 7–9, theinhibition power against DmBCA increased with the weightof the halogen present in the molecule, the bromoderivative9 being more effective compared to the chloroderivative 8,which in turn was more inhibitory than the fluorine deriva-tive 7. In the case of the 1,3,4-thiadiazole-2-sulfonamides,the aminoderivative 13 showed a weak inhibition, as men-tioned above, but its N-acetylation led to acetazolamidewhich was an order of magnitude more effective as a DmBCAinhibitor. The replacement of the acetyl by the sulfanilylmoiety (as in 20) further increased the inhibitory efficacycompared to AAZ (Table 1).

(iii) The most effective DmBCA inhibitors were compounds 21–24, MZA, EZA and SLT, which had inhibition constants rang-ing between 65.3 and 249 nM (Table 1). It may be observedthat the aromatic derivatives 21–24 and SLT possess anelongated molecule, incorporating either the sulfonylatedaromatic sulfonamide chemotype (21–24) or the 6-mem-bered sultam linked to the benzenesulfonamide scaffold(present in sulthiame). For the homolog series of compounds22–24, SAR was again similar to what observed earlier forsimilar derivatives, that is, the DmBCA inhibition increasedwith the length of the molecule, and more precisely withthe increase of the linker between the two fragments (fromn = 0 to n = 2). In fact, the best DmBCA inhibitor detected sofar was 24, which possessed the longest molecule among thecompounds included in this panel. This may probably becorrelated with the shape of the active site of b-CAs(although DmBCA has not yet been crystallized and analyzedby means of X-ray crystallography). For example for the fun-gal enzyme Can2, for which the X-ray crystal structure isavailable,13d it has been reported that the active site hasthe shape of a rather long channel at the bottom of whichis present the catalytically crucial zinc ion, to which inhibi-tors of the sulfonamide type coordinate.

(iv) From data of Table 1 it may also be observed that the inhibi-tion profile of DmBCA is very different from that of other a-or b-class CAs, such as the human isoforms hCA I and II orthe T. cruzi enzyme TcCA (a-CAs) or the b-CA from Leish-mania, LdcCA. Indeed, these differences of affinity of the sul-fonamides/sulfamates for the various CAs may be of help inthe search of potential selective insect CA inhibitors, usefulas repellents/insecticides.

In conclusion, we investigated the inhibition of the b-CADmBCA from Drosophila melanogaster with sulfonamides and sul-famates. Among the panel of 40 investigated compounds, the bestDmBCA inhibitors were the sulfonylated benzenesulfonamidesand ethoxzolamide, which showed inhibition constants in therange of 65.3–138 nM. Methazolamide and sulthiame were alsoeffective inhibitors with KIs ranging between 237 and 249 nM,whereas most of the simple aromatic/heterocyclic sulfonamidesshowed inhibition constants in the range of 0.47–6.40 lM. Topi-ramate, zonisamide and saccharine did not inhibit DmBCA. Asorthologs of this mitochondrial CA are found in many insect spe-cies involved in the spread of various diseases,27 inhibitors inter-fering with their activity may be of interest for developinginsecticides with an alternative mechanism of action to the pres-ently used agents, for which many insects developed extensiveresistance.

. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.117

L. Syrjänen et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 5

Acknowledgments

The work in our laboratories was supported by the competitiveResearch Funding of Fimlab Ltd and the grants from Academy ofFinland, Sigrid Juselius Foundation, and Jane and Aatos Erkko Foun-dation. CTS was supported by two EU grants (Metoxia andDynano).

References and notes

1. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med.Chem. Lett. 2010, 20, 3467; (c) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran,C. T.; De Simone, G. Chem. Rev. 2012, 112, 4421.

2. (a) Aggarwal, M.; McKenna, R. Expert Opin. Ther. Pat. 2012, 22, 903; (b) Sly, W.S.; Hu, P. Y. Annu. Rev. Biochem. 1995, 64, 375; (c) Supuran, C. T. J. Enzyme Inhib.Med. Chem. 2012, 27, 759.

3. (a) Lane, T. W.; Saito, M. A.; George, G. N.; Pickering, I. J.; Prince, R. C.; Morel, F.M. Nature 2005, 435, 42; (b) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M.Nature 2008, 452, 56.

4. (a) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (b) Aggarwal, M.;Boone, C. D.; Kondeti, B.; McKenna, R. J. Enzyme Inhib. Med. Chem. 2013, 28, 267.

5. (a) Tripp, B. C.; Bell, C. B., 3rd; Cruz, F.; Krebs, C.; Ferry, G. J. J. Biol. Chem. 2004,279, 6683; (b) Macauley, S. R.; Zimmerman, S. A.; Apolinario, E. E.; Evilia, C.;Hou, Y. M.; Ferry, J. G.; Sowers, K. R. Biochemistry 2009, 48, 817; (c)Zimmerman, S. A.; Ferry, J. G. Curr. Pharm. Des. 2008, 14, 716.

6. Syrjanen, L.; Tolvanen, M.; Hilvo, M.; Olatubosun, A.; Innocenti, A.; Scozzafava,A.; Leppiniemi, J.; Niederhauser, B.; Hytonen, V. P.; Gorr, T. A.; Parkkila, S.;Supuran, C. T. BMC Biochem. 2010, 11, 28.

7. (a) Klengel, T.; Liang, W. J.; Chaloupka, J.; Ruoff, C.; Schroppel, K.; Naglik, J. R.;Eckert, S. E.; Mogensen, E. G.; Haynes, K.; Tuite, M. F.; Levin, L. R.; Buck, J.;Muhlschlegel, F. A. Curr. Biol. 2005, 15, 2021; (b) Bahn, Y. S.; Cox, G. M.; Perfect,J. R.; Heitman, J. Curr. Biol. 2013, 2005, 15; (c) Innocenti, A.; Muhlschlegel, F. A.;Hall, R. A.; Steegborn, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.2008, 18, 5066.

8. (a) Isik, S.; Kockar, F.; Arslan, O.; Guler, O. O.; Innocenti, A.; Supuran, C. T. Bioorg.Med. Chem. Lett. 2008, 18, 6327; (b) Zimmerman, S. A.; Ferry, J. G.; Supuran, C. T.Curr. Top. Med. Chem. 2007, 7, 901; (c) Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.;Guler, O. O.; Innocenti, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.2009, 17, 1158.

9. (a) Minakuchi, T.; Nishimori, I.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med.Chem. 2009, 52, 2226; (b) Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.;Innocenti, A.; Supuran, C. T. J. Med. Chem. 2009, 52, 3116.

10. (a) Joseph, P.; Turtaut, F.; Ouahrani-Bettache, S.; Montero, J. L.; Nishimori, I.;Minakuchi, T.; Vullo, D.; Scozzafava, A.; Kohler, S.; Winum, J. Y.; Supuran, C. T. J.Med. Chem. 2010, 53, 2277; (b) Vullo, D.; Nishimori, I.; Minakuchi, T.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 3591.

11. (a) Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.;Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585; (b)Burghout, P.; Vullo, D.; Scozzafava, A.; Hermans, P. W.; Supuran, C. T. Bioorg.Med. Chem. 2011, 19, 243.

12. Cronk, J. D.; Rowlett, R. S.; Zhang, K. Y.; Tu, C.; Endrizzi, J. A.; Lee, J.; Gareiss, P.C.; Preiss, J. R. Biochemistry 2006, 45, 4351.

13. (a) Innocenti, A.; Winum, J. Y.; Hall, R. A.; Muhlschlegel, F. A.; Scozzafava, A.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2009, 19, 2642; (b) Innocenti, A.; Hall, R.A.; Schlicker, C.; Scozzafava, A.; Steegborn, C.; Muhlschlegel, F. A.; Supuran, C.T. Bioorg. Med. Chem. 2009, 17, 4503; (c) Innocenti, A.; Hall, R. A.; Schlicker, C.;Muhlschlegel, F. A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 2654; (d)Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.;Muhlschlegel, F. A.; Steegborn, C. J. Mol. Biol. 2009, 385, 1207.

14. Syrjänen, L.; Vermelho, A. B.; de Almeida Rodrigues, I.; Corte-Real, S.; Salonen, T.;Pan, P.; Vullo, D.; Parkkila, S.; Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56,7372.

Please cite this article in press as: Syrjänen, L.; et al. Bioorg. Med. Chem

15. (a) Innocenti, A.; Leewattanapasuk, W.; Muhlschlegel, F. A.; Mastrolorenzo, A.;Supuran, C. T. Bioorg. Med. Chem. Lett. 2009, 19, 4802; (b) Nishimori, I.;Minakuchi, T.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2011,19, 5023.

16. Fasseas, M. K.; Tsikou, D.; Flemetakis, E.; Katinakis, P. Mol. Biol. Rep. 2010, 37,2941.

17. (a) Krungkrai, J.; Scozzafava, A.; Reungprapavut, S.; Krungkrai, S. R.; Rattanajak,R.; Kamchonwongpaisan, S.; Supuran, C. T. Bioorg. Med. Chem. 2005, 13, 483; (b)Krungkrai, J.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 631; (c) Krungkrai, J.;Krungkrai, S. R.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 5466.

18. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydrationactivity. 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 7.4)or 20 mM Tris (pH 8.4) as buffers, and 20 mM Na2SO4 (for maintaining constantthe ionic strength), following the initial rates of the CA-catalyzed CO2

hydration reaction for a period of 10–100 s. The CO2 concentrations rangedfrom 1.7 to 17 mM for the determination of the kinetic parameters andinhibition constants. For each inhibitor at least six traces of the initial 5–10% ofthe reaction have been used for determining the initial velocity. Theuncatalyzed rates were determined in the same manner and subtracted fromthe total observed rates. Stock solutions of inhibitors (10 mM) were preparedin distilled–deionized water and dilutions up to 0.01 nM were done thereafterwith the assay buffer. Inhibitor and enzyme solutions were preincubatedtogether for 15 min at room temperature prior to assay, in order to allow forthe formation of the E–I complex. The inhibition constants were obtained bynon-linear least-squares methods using PRISM 3, as reported earlier,13,15,17 andrepresent the mean from at least three different determinations. DmBCA was arecombinant protein, obtained in house as reported earlier.6

19. (a) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (b) Supuran, C. T. Future Med.Chem. 2011, 3, 1165; (c) Chohan, Z. H.; Supuran, C. T.; Scozzafava, A. J. EnzymeInhib. Med. Chem. 2005, 20, 303.

20. (a) De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov. 2013, 8,793; (b) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (c)Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; (d) Supuran, C. T.;Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M. A. Eur. J. Med.Chem. 1998, 33, 247.

21. (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran,C. T. Chem. Commun. 2012, 1868; (b) Maresca, A.; Carta, F.; Vullo, D.; Supuran,C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 407; (c) Monti, S. M.; Maresca, A.;Carta, F.; De Simone, G.; Mühlschlegel, F. A.; Scozzafava, A.; Supuran, C. T.Bioorg. Med. Chem. Lett. 2012, 22, 859; (d) Carta, F.; Aggarwal, M.; Maresca, A.;Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. J. Med. Chem. 2012, 55,1721; (e) Supuran, C. T.; Vullo, D.; Manole, G.; Casini, A.; Scozzafava, A. Curr.Med. Chem.-Cardiovasc. Hematol. Agents 2004, 2, 49; (f) Mari, F.; Bertol, E.;Fineschi, V.; Karch, S. B. J. R. Soc. Med. 2004, 97, 397.

22. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.Med. Chem. 2010, 53, 5511.

23. (a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.;Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; (b) Maresca, A.;Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med.Chem. 2010, 53, 335.

24. (a) Pan, P.; Vermelho, A. B.; Capaci Rodrigues, G.; Scozzafava, A.; Tolvanen, M.E.; Parkkila, S.; Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56, 1761.

25. (a) Poupardin, R.; Srisukontarat, W.; Yunta, C.; Ranson, H. PLoS Negl. Trop. Dis.2014, 8, e2743; (b) Zimmer, C. T.; Müller, A.; Heimbach, U.; Nauen, R. Pestic.Biochem. Physiol. 2014, 108, 1.

26. (a) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.; Masini, E.;Impagnatiello, F.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 138; (b)Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.;Supuran, C. T. Bioorg. Med. Chem. 1999, 7, 2397; (c) Bertol, E.; Mari, F.; Milia, M.G.; Politi, L.; Furlanetto, S.; Karch, S. B. J. Pharm. Biomed. Anal. 2011, 55, 1186.

27. Zolfaghari Emameh, R.; Barker, H.; Tolvanen, M. E.; Ortutay, C.; Parkkila, S.Parasites Vectors 2014, 7, 38.

. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.117