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

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Sulfonamide inhibition studies of the b carbonic anhydrase from Drosophila melanogaster 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, Finland BioMediTech, University of Tampere, Tampere, Finland c Fimlab Laboratories Ltd and Tampere University Hospital, Tampere, Finland d Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy e Università degli Studi di Firenze, Neurofarba Dipartment, Sezione di Scienza Farmaceutiche e Nutraceutiche, Via Ugo Sciff 6, 50019 Sesto Fiorentino (Firenze), Italy b

a r t i c l e

i n f o

Article history: Received 31 March 2014 Revised 28 April 2014 Accepted 29 April 2014 Available online xxxx Keywords: Carbonic anhydrase b-Class enzyme Drosophila melanogaster Sulfonamide Enzyme 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 melanogaster with sulfonamides and sulfamates is reported. Among the panel of 40 investigated compounds, the best DmBCA inhibitors were the sulfonylated benzenesulfonamides and ethoxzolamide, which showed inhibition constants in the range of 65.3–138 nM. Methazolamide and sulthiame were also effective inhibitors with KIs ranging between 237 and 249 nM, whereas most of the simple aromatic/heterocyclic sulfonamides showed inhibition constants in the range of 0.47–6.40 lM. Topiramate, zonisamide and saccharine did not inhibit DmBCA. As orthologs of this mitochondrial CA are found in many insect species involved in the spread of various diseases, inhibitors interfering with their activity may be of interest for developing insecticides with an alternative mechanism of action to the presently used agents, for which many insects developed extensive resistance. Ó 2014 Elsevier Ltd. All rights reserved.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the reversible hydration of carbon dioxide: CO2 + H2O M HCO3 + H+, a reaction of fundamental importance in most organisms.1–4 CAs are metalloenzymes usually having a zinc(II) ion at the active site, but some cadmium(II)-CAs (which is inter-exchangeable with Zn(II)),3 as an alternative metal cofactor were also reported.3c-CAs may contain iron(II) ions within the active site, at least in some anaerobic Archaea.5 The reaction catalyzed by CAs is essential in the regulation of acid–base balance in organisms all over the phylogenetic tree, which explains why these enzymes are present in a large variety of classes and isoforms in many organisms.1–6 In fact, this reaction helps to remove carbon dioxide out of the tissues, being also involved in biosynthetic reactions such as gluconeogenesis and ureagenesis, 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 residues in the a-, c- and d-CAs, whereas for the lastly discovered family, the f-CA (isolated from the marine diatom Thalassiosira weissflogii)3 the coordination of the metal ion within the active site is very similar to that of the b-CAs, with one His, two Cys and a water molecule acting as ligands of the Cd(II) or Zn(II) ions.3,6–11 ⇑ Corresponding author. Tel.: +39 055 4573729; fax: +39 055 4573385.

b-CAs seem to be the group with the widest distribution, as such enzymes have been described in various groups of organisms including the Archaea and Bacteria domains, as well as in plants and fungi among Eukaryotes.1,2,12,13 Recently, we reported the widespread occurrence of at least one single-copy of a b-CA gene among animal species distinct from chordates.6 Indeed, b-CAs have so far been reported in various pathogenic organisms including the fungi/yeasts Candida albicans, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae7,8 and the bacteria Mycobacterium tuberculosis, Brucella suis, Salmonella typhimurium, Helicobacter pylori, Streptococcus pneumoniae, Legionella pheumophila and Haemophilus influenzae.12–17 The inhibition profiles of these enzymes with various agents such as sulfonamides, anions, carboxylates, phenols, dithiocarbamates and boronic acids have also been explored.1,2,8–17 b-CAs have been reported in two invertebrate species, namely the insect Drosophila melanogaster and the worm Caenorhabditis elegans.6,16 For the insect, it has been shown that this enzyme has a mitochondrial localization, being probably involved in metabolic processes, similar to the mammalian mitochondrial isoforms, which participate in gluconeogenesis, lipogenesis and ureagenesis.1,2 However, b-CAs are not present in vertebrates, which offers the opportunity to design specific inhibitors for the b-CAs, that could be used against pathogenic, invertebrate organisms or

E-mail address: claudiu.supuran@unifi.it (C.T. Supuran). http://dx.doi.org/10.1016/j.bmcl.2014.04.117 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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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 types 1–24) as well as the clinically used agents belonging to the sulfonamide/sulfamate class, that is, compounds such as acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA, dichlorophenamide DCP, dorzolamide DZA and brinzolamide BRZ were included in this study.1,2 Topiramate TPM (a sulfamate) and zonisamide ZNS (an aliphatic sulfonamide), widely used antiepileptic drugs, as well as other compounds incorporating primary/secondary sulfamoyl moieties such as sulpiride SLP and indisulam IND were also shown to belong to this class of pharmacological agents together with the COX2 selective inhibitors celecoxib CLX and valdecoxib VLX, the antiepileptic sulthiame SLT, the sweetener saccharin SAC and the diuretic hydrochlorothiazide HCT, were also included in the present study.1,2 Compounds 1–24 used in the assay as well as the clinically used drugs mentioned above were commercially available, or prepared as reported earlier by our group.26 In this way it is possible to explore a wider chemical space for detecting effective inhibitors of the new enzymes reported here, considering the fact that the 40 compounds tested in our study possess a range of scaffolds and various substitution patterns.

microorganisms. For example, a b-CA from protozoa belonging to the genus Leishmania was recently discovered, cloned and characterized, 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 were also reported recently, such as the thiols, dithiocarbamates, coumarins, and polyamines among others.21–23 However, b-CAs are effectively inhibited only by sulfonamides, thiols and dithiocarbamates, as the coumarins are not acting as inhibitors of this class of CAs (which do not possess esterase activity).23 Here we report the first inhibition study with sulfonamides of the recently discovered b-CA from the model insect Drosophila melanogaster, DmBCA.6 As many insects spread various infective agents (such as malaria parasites, West Nile virus, yellow fever virus, etc.)17b,24 these studies may provide lead compounds useful in the investigation of repellents or insecticides with an alternative mechanism of action compared to the widespread such agents to which many insects developed resistance.25 It should be mentioned that only simple inorganic anions as well as sulfamide and sulfamic acid were investigated up until now as inhibitors of

SO2NH2

SO2NH2

SO2NH2

SO2NH2

SO2NH2

NH2

NH2

1

2

4

3

SO2NH2

SO2NH2

SO2NH2

CH2NH2

CH2CH2NH2

NH2

SO2NH2

F 6

5

7

SO2NH2

SO2NH2

OH Cl

Br

Cl SO2NH2 NH2

10 H3C

N N SO2NH2

HN

N S

SO2NH2

SO2NH2

(CH2)nOH

COOH

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

18

SO2NH2

O H2N

N NH2

O O2N

S N H O HO

21

N N

S N H

O

19

12

SO2NH2 N

14

H N

SO2NH2 NH2

11

13

N

SO2NH2

CF3

Cl

9

S

8

SO2NH2

NH2

H2N

Cl NH2

S

SO2NH2

20 O

SO2NH2 H2N

( )n S N H O 22: n = 0 23: n = 1 24: n = 2

SO2NH2

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

H3C

N N CH3CONH

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

S N H O

S

SO2NH2

O

O NH2 O S O O

O SO2NH2

S

S

BRZ

N N

O

SO2NH2

S

MZA

SO2NH2

Cl

N

SO2NH2

O

O O

BZA

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

Data of Table 1 show the inhibition of the Drosophila enzyme DmBCA with this set of sulfonamides/sulfamates. For comparison reasons, the inhibition of the two human (h) offtarget isoforms hCA I and II (belonging to the a-CA family) as well as the protozoan a-/b-CA from the pathogens Trypanosoma cruzi (TcCA) and Leishmania donovani chagasi (LdcCA) with this set of 40 compounds, are also presented in Table 1. The following structure–activity relationship (SAR) can be observed for the inhibition of DmBCA with these 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 ineffective inhibitory properties against DmBCA, with inhibition

H N HN O

Cl

S O

SO2NH2

HCT

constants ranging between 1.235 and 6.40 lM. It should be also noted that three of the investigated compounds, that is, TPM, ZNS and SAC, did not inhibit DmBCA at all, up until 100 lM concentrations of inhibitor in the assay system (Table 1). The SAR for these ineffective inhibitors is not very straightforward as they belong to many different classes of sulfonamides, such as the 4-substituted or 3,4-disubstituted benzenesulfonamides incorporating amino, aminoalkyl, hydroxymethyl and halogen functionalities, or the heterocyclic sulfonamides (13, 14, DZA, BRZ, BZA, etc.). More specifically, for the 4-amino- and 4-aminoalkyl derivatives 2, 5 and 6, the inhibitory power increases with the length of the linker (from 0 to 2 carbon atoms) between the amino

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Table 1 Inhibition 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, with sulfonamides 1–24 and the clinically used agents AAZ–HCT18 KI* (nM)

Inhibitor a

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

hCA I

hCA II

TcCAb

LdcCAc

DmBCAd

28,000 25,000 79c 78,500 25,000 21,000 8300 9800 6500 7300 5800 8400 8600 9300 5500 9500 21,000 164 109 6 69c 164 109 95 250 50 25 1200 50,000 45,000 15 250 56 1200 31 54,000 50,000 374 18,540 328

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

25,460 57,300 63,800 44,200 7231 9238 8130 6925 8520 9433 842 820 534 652 73,880 71,850 66,750 84,000 810 88.5 134 365 243 192 61.6 74.9 88.2 128 92.9 87.3 93.6 85.5 867 87.9 84.5 82.7 91.1 71.9 8210 134

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

5845 4810 785 5490 3210 1615 2810 2345 942 314 287 360 4175 1910 1235 872 613 6400 525 471 93.2 138 104 65.3 516 237 116 605 3565 5370 1565 >100,000 >100,000 6260 5025 4470 5900 249 >100,000 5450

*

Errors in the range of 5–10% of the shown data, from 3 different assays. 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. a

moiety and the benzenesulfonamide scaffold, with 4-aminoethylbenzenesulfonamide 6 being 2.97-times a better DmBCA inhibitor compared to sulfanilamide 1. Comparing 4-amino-/4-hydroxybenzenesulfonamides (2 and 15) as well as the corresponding aminoalkyl/hydroxyalkyl derivatives (5 and 16, or 6 and 17), it may be observed that the hydroxyl-containing derivatives were more inhibitory compared to the corresponding amino derivatives, and again, the inhibitory power increased with the length of the spacer between the hydroxyl and benzenesulfonamide scaffold, from 0 to 2. It is also obvious that the presence of bulky moieties at the benezenesulfonamide scaffold, as in SLP, IND, VLX and CLX, is detrimental to the DmBCA inhibitory activity of these sulfonamides. (ii) More effective DmBCA inhibitory activity was observed for the following sulfonamides: 3, 9–12, 16, 17, 19, 20, AAZ, and DCP, which showed inhibition constants ranging

between 287 and 942 nM (Table 1). Some of these derivatives 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 3 was beneficial for the inhibitory effects of these compounds against DmBCA. For the halogenosulfanilamides 7–9, the inhibition power against DmBCA increased with the weight of the halogen present in the molecule, the bromoderivative 9 being more effective compared to the chloroderivative 8, which in turn was more inhibitory than the fluorine derivative 7. In the case of the 1,3,4-thiadiazole-2-sulfonamides, the aminoderivative 13 showed a weak inhibition, as mentioned above, but its N-acetylation led to acetazolamide which was an order of magnitude more effective as a DmBCA inhibitor. The replacement of the acetyl by the sulfanilyl moiety (as in 20) further increased the inhibitory efficacy compared to AAZ (Table 1). (iii) The most effective DmBCA inhibitors were compounds 21– 24, MZA, EZA and SLT, which had inhibition constants ranging between 65.3 and 249 nM (Table 1). It may be observed that the aromatic derivatives 21–24 and SLT possess an elongated molecule, incorporating either the sulfonylated aromatic sulfonamide chemotype (21–24) or the 6-membered sultam linked to the benzenesulfonamide scaffold (present in sulthiame). For the homolog series of compounds 22–24, SAR was again similar to what observed earlier for similar derivatives, that is, the DmBCA inhibition increased with the length of the molecule, and more precisely with the increase of the linker between the two fragments (from n = 0 to n = 2). In fact, the best DmBCA inhibitor detected so far was 24, which possessed the longest molecule among the compounds included in this panel. This may probably be correlated with the shape of the active site of b-CAs (although DmBCA has not yet been crystallized and analyzed by means of X-ray crystallography). For example for the fungal enzyme Can2, for which the X-ray crystal structure is available,13d it has been reported that the active site has the shape of a rather long channel at the bottom of which is present the catalytically crucial zinc ion, to which inhibitors of the sulfonamide type coordinate. (iv) From data of Table 1 it may also be observed that the inhibition profile of DmBCA is very different from that of other aor b-class CAs, such as the human isoforms hCA I and II or the T. cruzi enzyme TcCA (a-CAs) or the b-CA from Leishmania, LdcCA. Indeed, these differences of affinity of the sulfonamides/sulfamates for the various CAs may be of help in the search of potential selective insect CA inhibitors, useful as repellents/insecticides. In conclusion, we investigated the inhibition of the b-CA DmBCA from Drosophila melanogaster with sulfonamides and sulfamates. Among the panel of 40 investigated compounds, the best DmBCA inhibitors were the sulfonylated benzenesulfonamides and ethoxzolamide, which showed inhibition constants in the range of 65.3–138 nM. Methazolamide and sulthiame were also effective inhibitors with KIs ranging between 237 and 249 nM, whereas most of the simple aromatic/heterocyclic sulfonamides showed inhibition constants in the range of 0.47–6.40 lM. Topiramate, zonisamide and saccharine did not inhibit DmBCA. As orthologs of this mitochondrial CA are found in many insect species involved in the spread of various diseases,27 inhibitors interfering with their activity may be of interest for developing insecticides with an alternative mechanism of action to the presently used agents, for which many insects developed extensive resistance.

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Acknowledgments The work in our laboratories was supported by the competitive Research Funding of Fimlab Ltd and the grants from Academy of Finland, Sigrid Juselius Foundation, and Jane and Aatos Erkko Foundation. CTS was supported by two EU grants (Metoxia and Dynano). 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.

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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 stoppedflow instrument has been used for assaying the CA catalysed CO2 hydration activity. 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 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 inhibitors (10 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, as reported earlier,13,15,17 and represent the mean from at least three different determinations. DmBCA was a recombinant 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. Enzyme Inhib. 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.

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