Carbonic Anhydrase Inhibitors. the β Carbonic Anhydrases From the Fungal Pathogens Cryptococcus Neoformans and Candida Albicans Are Strongl | Enzyme Inhibitor

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  Carbonic anhydrase inhibitors. The  b -carbonic anhydrases from the fungalpathogens  Cryptococcus neoformans  and  Candida albicans  are strongly inhibited by substituted-phenyl-1  H  -indole-5-sulfonamides Özlen Güzel a,b , Alfonso Maresca b , Rebecca A. Hall c , Andrea Scozzafava b , Antonio Mastrolorenzo d ,Fritz A. Mühlschlegel c,e , Claudiu T. Supuran b, * a Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy c School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK  d Università degli Studi di Firenze, Centro MTS, Dipartimento di Dermatologia, Firenze, Italy e East Kent Hospitals University NHS Foundation Trust, Clinical Microbiology Service, William Harvey Hospital, Ashford, Kent, TN24 0LZ, UK  a r t i c l e i n f o  Article history: Received 26 January 2010Revised 24 February 2010Accepted 26 February 2010Available online 3 March 2010 Keywords: Carbonic anhydrase b -Class enzyme Candida albicansCryptococcus neoformans SulfonamideIndole-5-sulfonamides a b s t r a c t A series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1 H  -indole-5-sulfonamides and 1-({[5-(amino-sulfonyl)-3-phenyl-1 H  -indol-2-yl]carbonyl}amino)-2,4,6 trimethylpyridinium perchlorates possessingvarious 2-, 3- or 4-substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, aswell as the perfluorophenyl moiety, have been evaluated as inhibitors of the  b -carbonic anhydrases(CAs, EC 4.2.1.1) from the pathogenic fungi  Cryptococcus neoformans  (Can2) and  Candida albicans (CaNce103). Both enzymes were potently inhibited by these sulfonamides,  K  I s in the range of 4.4–118nM against Can2, and of 5.1–128 against CaNce103, respectively. Minor structural changes inthe 3-substituted phenyl moiety contribute significantly to the inhibitory activity. Some of the investi-gated sulfonamides showed promising selectivity ratios for inhibiting Can2 over the host, humanenzymes CA I and II.   2010 Elsevier Ltd. All rights reserved. Inpreviouswork 1 fromthislaboratorywehavereportedthat2-(hydrazinocarbonyl)-3-substituted-phenyl-1 H  -indole-5-sulfona-mides  1  and  2  possessing various 2-, 3- or 4-substituted phenylgroups, show interesting inhibitory activity against some isoformsof the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). 2 Indeed,CAs are widespread enzymes in organisms all over the phyloge-netic tree, as they catalyze the interconversion of carbon dioxideand bicarbonate, a simple but physiologically crucial reaction. 2–4 Whereas mammalscontainonly a -class CAs(intheformof 16dif-ferent isoforms), 2 genetically distinct  b -,  c -,  d - and  n -CAs are pres-ent, among others, in bacteria, archaea, fungi, plants, diatoms andother such simpler organisms. 2–4 The five classes of CAs are thus aperfect example of convergent evolution, as their active sites, 3Dfolds of the protein backbone and catalytic mechanisms are verydifferent among the diverse enzyme classes. 2–6 Sulfonamides represent the main class of clinically used CAinhibitors (CAIs). 2 Members of this class including acetazolamide  AZA  or ethoxzolamide  EZA  among others, and are clinically em-ployed for the management of a variety of disorders connected toCA disbalances, such as glaucoma; 2,7 in the treatment of edemadue to congestive heart failure, 8 or for drug-induced edema; asmountainsickness drugs, 8 whereas other agents of this pharmaco- SO 2 NH 2 SNO H 2 NO 2 SNHONHNH 2 SNNCH 3 CONH SO 2 NH 2 H 2 NO 2 SNHONHNH 2 R H 2 NO 2 SNHONH  NR 1AZAEZA2   +ClO 4 - 3 0960-894X/$ - see front matter   2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2010.02.103 *  Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385. E-mail address:  claudiu.supuran@unifi.it (C.T. Supuran).Bioorganic & Medicinal Chemistry Letters 20 (2010) 2508–2511 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl  logical class show applications as anticonvulsants, 9 antiobesity 2 orantitumor drugs/tumor diagnostic agents. 2,10 As there are few iso-form-selective inhibitors to date, new sulfonamides are continu-ously reported to find derivatives with better inhibition profilesas compared to the promiscuous, first generation inhibitors suchas  AZA  or  EZA . 2 Furthermore, enzymes belonging to the  b - and c -CAclasses, some of whichpresent in pathogenic bacteria or fun-gi, 2,5,6,11,12 have also been investigated recently for their interac-tion with this class of derivatives, in the search for antiinfectivespossessingadifferentmechanismofactioncomparedtotheclassi-cal antibiotic/antifungal drugs. 11,12 We have recently shown 11a that the  b -CAs from the fungalpathogens  Cryptococcus neoformans  (Can 2) 5 and  Candida albicans (CaNce103) 6 are inhibited by sulfonamides. Up to now we haveinvestigated only simple sulfonamide scaffolds as well as the 20clinically used sulfonamides, among which acetazolamide  AZA and ethoxzolamide  EZA , for the inhibition of these enzymes, find-ing very feweffective inhibitors against the fungal enzymes. 11a In-deed,  AZA  was a good Can2 inhibitor (with a  K  I  of 10nM) whereasit had a more modest activity against CaNce103 (with a  K  I  of 132nM). 11a EZA  was even less effective, with  K  I s of 87nM againstCan2 and 1070nM against CaNce103, respectively. Thus, in thesearch for more effective sulfonamide compounds targeting thefungal pathogenic enzymes Can2 and CaNce103, we report herean inhibition study with a series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1 H  -indole-5-sulfonamides  1 ,  2  and 1-({[5-(aminosulfonyl)-3-phenyl-1 H  -indol-2-yl]carbonyl}amino)-2,4,6trimethylpyridinium perchlorates  3,  which have been describedearlier for their interaction with the mammalian  a -CAs. 1,2 We have included sulfonamides  1 – 3  in this study as they con-stitute a class of CA inhibitors (CAIs) of potential interest. 1,12e Infact we have observed recently an excellent activity against twoof the three  b -CAs from the bacterial pathogen  Mycobacteriumtuberculosis  for the 2-(pyridiniumaminocarbonyl)-3-substituted-phenyl-1 H  -indole-5-sulfonamides  3 . 12e The presence of varioussubstituents at the 3-phenyl group of the indole rings of deriva-tives  2  may increase lipo- or hydrosolubility of these compounds,and also interact in a positive/negative manner with amino acidresiduespresentintheCAactivesiteallowingthustoobtainstruc-ture activity relationship (SAR) insights for the inhibition of theseenzymes. 12a Thus, incorporation of 2-, 3- or 4-substituted phenylgroupspossessingmethyl-,halogeno-andmethoxy-functionalitiesin the 3-position of the indolesulfonamides  2 , has been contem-plated in order to explore as many as possible scaffolds for detect-ing high affinity and possibly selective inhibitors for the fungal(Can2, CaNce103) over host enzymes (hCA I and hCA II).Inhibition data with sulfonamides  1 – 3  as well as the standarddrugs  AZA  and  EZA  against Can2 and CaNce103 ( b -class enzymes)as well as the ubiquitous, 2 human CA isoforms hCA I and II ( a -CAs) 1a (off-targets) are shown in Table 1. 13,14 ThefollowingSARcanbedrawnby consideringdataof  Table1:(i) Against Can2, the indolesulfonamides  1  and  2  showed goodinhibitoryactivity,with K  I sintherangeof7.2–118nM,mak-ing this entire sulfonamide class among the best sulfon-amide Can2 inhibitors detected so far. Thus, the leadcompound  1 , and derivatives  2i–2l  and  2n  showed med-ium-high potency as Can2 inhibitors, with inhibition con-stants in the range of 62–118nM. These derivativesincorporate the 4-chloro, 2-, 3- and 4-bromo as well aspentafluorophenyl moieties (together with the lead  1 ). Incontrast, the remaining derivatives, incorporating methyl-,fluoro-, 2-/3-chloro- and methoxy-substituted phenyl moie-ties in the position 3 of the indole ring, of types  2a–2h , and 2m , showedamuchstrongerCan2inhibitoryeffect,with K  I sin the range of 7.2–10.5nM. Thus, the nature of the groupsubstituting the 3-phenyl ring present in compounds  1  and 2  strongly influences the Can2 inhibitory activity, with themethyl-, methoxy-, fluoro- and chloro-substituted deriva-tives showing a better activity (around 10-fold) comparedto the lead  1  or the bromosubstituted compounds  2j–2l .The position of the substituent of the phenyl ring is some-how less influential on the inhibitory activity (except forthechloro-derivatives  2g–2i , case inwhichthe4-chlorosub-stituted compound  2i  was around 8.7–9.4 times a weakerinhibitor compared to the 2- or 3-chlorosubstituted isomers 2g   and  2h ). Indeed, the 2-, 3- or 4-methyl-substituted com-pounds  2a–2c , or the 2-, 3- or 4-fluoro-substituted com-pounds  2d–2f  , respectively, showed comparable Can2inhibitory activities (Table 1). The pyridinium-substitutedsulfonamides 3 showedsimilaractivitywiththecorrespond-ing series of sulfonamides  2  (from which they have beenprepared). However they were better Can2 inhibitorscompared to the corresponding carbohydrazides  2 , withinhibition constants in the range of 4.4–60.1nM. Again the  Table 1 Inhibition of human a -CA (hCA) isozymes I and II and fungal  b -CAs from  C. neoformans (Can2) and  C. albicans  (CaNce103) with sulfonamides  1, 2a – n, 3a – p , acetazolamide(  AZA ) and ethoxzolamide ( EZA ) as standards 13 H 2 NO 2 SNHONHNH 2 R H 2 NO 2 SNHONH  NR+ClO 4 - 1, 2 3 Inhibitor R   K  I# (nM)hCA I a hCA II a Can2 b CaNce103 b 1  H 7.5 7.2 72 85 2a  2-Me 107 11.6 9.2 78 2b  3-Me 730 48.4 9.1 43 2c  4-Me 104 60.5 10.5 52 2d  2-F 621 36.0 7.9 47 2e  3-F 116 8.6 7.2 54 2f   4-F 108 15.5 8.6 61 2g   2-Cl 640 38.8 8.0 45 2h  3-Cl 311 9.2 7.4 52 2i  4-Cl 112 11.6 70 55 2j  2-Br 110 48.5 62 81 2k   3-Br 510 54.1 93 87 2l  4-Br 659 40.8 118 94 2m  3-OMe 342 7.4 12.0 128 2n  F 5  110 7.0 103 7.5 3a  H 9.0 71 16.5 42 3b  2-F 8.5 91 16.2 19 3c  3-F 11.3 3380 8.3 24 3d  4-F 7.6 65 8.7 21 3e  2-Cl 25.1 100 15.4 33 3f   3-Cl 113 1800 4.4 31 3g   4-Cl 3.2 77 3.1 29 3h  2-Br 43.4 38 12.1 45 3i  3-Br 30.8 74 14.3 48 3j  4-Br 12.3 85 10.9 53 3k   2-Me 10.5 106 6.5 62 3l  3-Me 110 104 6.1 64 3m  4-Me 5.1 68 5.9 41 3n  3-OMe 8.6 2840 8.3 119 3p  F 5  9.7 0.93 60.1 5.1  AZA  — 250 12 10 132 EZA  — 25 8 87 1070 # Errors in the range of ±5% of the reported data from three different assays by astopped-flow CO 2  hydration method. 13a From Ref. 1a. b This work. Ö. Güzel et al./Bioorg. Med. Chem. Lett. 20 (2010) 2508–2511  2509  bestCan2inhibitorwasachlorine-substitutedderivative, 3f  ,whereastheleasteffectiveonethepentafluorophenyl deriv-ative 3p . Overall, thissubseriesof positively-chargedsulfon-amides showed excellent inhibitory capacity against thefungal enzyme Can2. It should be also mentioned that themost active compounds among the indolesulfonamidesinvestigated here showed a better activity than acetazola-mide  AZA , the most effective Can2 inhibitor detected beforethis study. 11a (ii) CaNce103wasslightlylesssusceptibletobeinhibitedbytheindolesulfonamides  1 – 3 , compared to Can2, a situationalready observed with other classes of sulfonamides. 11a Thus,derivatives 1, 2  showed K  I sof7.5–128nMfortheinhi-bition of CaNce103, being more effective CAIs compared to  AZA  ( K  I  of 132nM, the best sulfonamide inhibitor detectedbefore this study) or  EZA  ( K  I  of 1070nM). The 3-methoxy-substituted derivative  2m  was the least effective CaNce103inhibitorinthisseries,witha K  I of128nM,whereasthepen-tafluorophenyl-substitutedone, 2n ,themosteffectiveinhib-itor ( K  I  of 7.5nM). This is one of the best CaNce103inhibitors detected so far, with efficiency 17.6 times betterthan that of   AZA , and also possessing a quite hydrophobiccharacterduetothepresenceof thepentafluorophenyl moi-ety. This is a positive feature indeed, as sulfonamides arenormally insufficiently lipophilic to penetrate the cell wallsand membranes of some bacteria or fungi, 12f  a fact whichis attributedtothehighlypolar nature of theSO 2 NH 2  group.The remaining derivatives, of types  1  and  2a–2l , showed arather flat SAR, and were only moderately inhibitory againstCaNce103, with inhibition constants of 43–94nM. Both thesubstitution pattern and the nature of the 3-substituted-phenyl moieties influence the CaNce103 inhibitory activityof this series of indolesulfonamides. Thus, the lead  1  wasmoderatelyactive( K  I  of85nM) andall substitutionpatternsat the3-phenylmoiety(exceptthebromophenyl ones, pres-ent in  2j–2l ) lead to an increase of the CaNce103 inhibitorypower. Actually, the bromophenyl derivatives  2j–2l  showedsimilar ( 2j  and  2k  ) or slightly diminished ( 2l ) CaNce103inhibitoryactivitycomparedto 1 .Forthehalogenosubstitut-ed compounds, the 2-halogeno derivative was a betterCaNce103 inhibitor compared to the corresponding 3-halo-geno substituted compound, which in turn was a betterinhibitorcomparedtothe4-halogenosubstitutedderivative.For the methyl substituted compounds, the best CaNce103inhibitor was the 3-substituted compound  2b . Thus, minorstructural changes in the scaffold of compounds  2  stronglyinfluence the CaNce103 inhibitory activity for this series of derivatives. The compounds  3 , bearing the trimethylpyridi-nium moiety instead of the terminal amino one present in 2 , were also effective CaNce103 inhibitors, with inhibitionconstantsintherangeof5.1–119nM(Table1).SARforthesepositively-charged derivatives was rather similar to thecorresponding carbohydrazides from which they were pre-pared, with the pentafluorophenyl derivative  3p  being themost effective CaNce103 inhibitor reported so far ( K  I  of 5.1nM) and the 3-methoxy-substituted one  3n  the leasteffective ( K  I  of 119nM). Generally, all the positively-charged, trimethylpyridinium derivatives  3  were betterCaNce103 inhibitors compared to the corresponding non-charged derivatives  2 .(iii) theinvestigatedsulfonamides  2, 3  were generallyless effec-tive hCA I inhibitors ( K  I s of 110–730nM) except for the lead 1 , which is a very potent hCA I inhibitor ( K  I  of 7.5nM) butmost of them were highly effective hCA II inhibitors ( K  I s of 7.2–60.5nM). However, some interesting selectivity ratiosfor the inhibition of the fungal over the host enzymes havebeen observed for some of the investigated sulfonamides.Thus, compound  2b  had a selectivity ratio of 5.1 for inhibit-ing Can2 over hCA II, and of 80.2 for inhibiting Can2 overhCA I. This is the first example of a fungal pathogenicCA-selective inhibitor (over the off-target hCA II). Similarfeatures were also observed for  2g  , with selectivity ratiosof 4.8(Can2overhCAII) and80(Can2overhCAI). However,no CaNce103 selective inhibitors (over hCA II) have beendetected so far, in this or other studies. 11a Inconclusion,inthisLetterweinvestigatedaseriesof2-(hydraz-inocarbonyl)-3-substituted-phenyl-1 H  -indole-5-sulfonamidespossessing various 2-, 3- or 4-substituted phenyl groups withmethyl-,halogeno-andmethoxy-functionalities,aswellastheper-fluorophenylmoiety,fortheinhibitionoftwo b -CAsfromthefungalpathogens  C. neoformans  (Can 2) and  C. albicans  (CaNce103). Bothenzymes were potently inhibited by these sulfonamides, withinhibition constants in the range of 4.4–118nM against Can2,and of 5.1–128 against CaNce103, respectively. SAR was ratherwell defined, with minor structural changes in the 3-substitutedphenyl moiety being the main contributors to the enzyme inhib-itory activity. Some of the investigated sulfonamides alsoshowed acceptable selectivity ratios for inhibiting Can2 overthe host, human enzymes hCA I and II.  Acknowledgments Ö.G. is grateful to TUBITAK (Ankara, Turkey) for providingfinancing under the Contract No. 2219/2008. This research was fi-nanced in part by a grant of the 6th Framework Programme of theEuropean Union (DeZnIT project, to A.S. and C.T.S). Work in theFAM lab is funded by the bbsrc and MRC. F.A.M. and R.A.H. thankKara Turner for technical support. Thanks are addressed to Profes-sor Dr. Clemens Steegborn (Bochum University, Germany) for thegift of the Can2 plasmid. References and notes 1. (a) Güzel, Ö.; Innocenti, A.; Scozzafava, A.; Salman, A.; Parkkila, S.; Hilvo, M.;Supuran, C. T.  Bioorg. Med. Chem.  2008 ,  16 , 9113; (b) Güzel, Ö.; Temperini, C.;Innocenti, A.; Scozzafava, A.; Salman, A.; Supuran, C. T.  Bioorg. Med. Chem. Lett. 2008 ,  18 , 152.2. (a) Supuran, C. T.  Nat. Rev. Drug Disc.  2008 ,  7  , 168; (b) Supuran, C. T.;Scozzafava, A.  Bioorg. Med. Chem.  2007 ,  15 , 4336.3. (a)Supuran,C.T.;Scozzafava,A.;Casini,A. Med. Res. Rev. 2003 ,  23 ,146–189;(b)Scozzafava, A.;Mastrolorenzo, A.;Supuran, C.T.  Expert Opin. Ther. Patents  2004 , 14 ,667–702;(c)Winum,J.Y.;Montero,J.L.;Scozzafava,A.;Supuran,C.T. Mini-Rev. Med. Chem.  2006 ,  6 , 921–936.4. (a)Tripp,B.C.;Smith,K.S.;Ferry,J.G.  J. Biol. Chem.  2001 ,  276 ,48615;(b)Smith,K. S.;Ferry,J.G.  FEMS Microbiol. Rev.  2000 ,  24 , 335;(c)Zimmerman,S.A.;Ferry, J.G.;Supuran,C.T. Curr. Top. Med. Chem.  2007 , 7  ,901;(d)Rowlett,R.S. Biochim.Biophys. Acta  2010 ,  1804 , 362.5. (a)Mogensen,E.G.;Janbon,G.;Chaloupka,J.;Steegborn,C.;Fu,M.S.;Moyrand,F.; Klengel, T.; Pearson, D. S.; Geeves, M. A.; Buck, J.; Levin, L. R.; Mühlschlegel,F. A.  Eukaryot. Cell  2006 ,  5 , 103; (b) Bahn, Y. S.; Mühlschlegel, F. A.  Curr. Opin.Microbiol.  2006 ,  9 , 572.6. (a) Hall, R. A.; Mühlschlegel, F. A. Fungal and Nematode Carbonic Anhydrases:their Inhibition in Drug Design. In  Drug Design of Zinc-Enzyme Inhibitors:Functional, Structural, and Disease Applications ;Supuran,C.T.,Winum,J.Y.,Eds.; John Wiley & Sons: Hoboken, 2009; pp 301–322; (b) Schlicker, C.; Hall, R. A.;Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.; Muhlschlegel, F. A.;Steegborn, C.  J. Mol. Biol.  2009 ,  385 , 1207.7. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T.  J. Enzyme Inhib. Med.Chem.  2004 ,  19 , 199–229; (b) Supuran, C. T.; Scozzafava, A.; Casini, A.Development of Sulfonamide Carbonic Anhydrase Inhibitors. In  Carbonic  Anhydrase—its Inhibitors and Activators ; Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton, 2004; pp 67–147; (c) Thiry, A.; Dogné, J. M.;Masereel, B.; Supuran, C. T.  Trends Pharmacol. Sci.  2006 ,  27  , 566–573.8. Supuran, C. T.  Curr. Pharm. Des.  2008 ,  14 , 641–648.9. (a)DeSimone,G.;Vitale,R.M.;DiFiore,A.;Pedone,C.;Scozzafava,A.;Montero, J.L.;Winum,J.Y.;Supuran,C.T.  J. Med. Chem.  2006 , 49 ,5544;(b)DeSimone,G.;Di Fiore, A.; Menchise, V.; Pedone, C.; Antel, J.; Casini, A.; Scozzafava, A.; Wurl,M.; Supuran, C. T.  Bioorg. Med. Chem. Lett.  2005 ,  15 , 2315; (c) Winum, J. Y.;2510  Ö. Güzel et al./Bioorg. Med. Chem. Lett. 20 (2010) 2508–2511  Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.; Ciattini, S.; Montero, J. L.;Scozzafava, A.; Supuran, C. T.  J. Med. Chem.  2006 ,  49 , 7024.10. (a) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.;De Simone, G.; Supuran, C. T.  J. Am. Chem. Soc.  2006 ,  128 , 8329; (b) Casini, A.;Antel, J.; Abbate, F.;Scozzafava, A.; David, S.; Waldeck, H.; Schafer, S.; Supuran,C. T.  Bioorg. Med. Chem. Lett.  2003 ,  13 , 841; (c) Weber, A.; Casini, A.; Heine, A.;Kuhn, D.; Supuran, C. T.; Scozzafava, A.; Klebe, G.  J. Med. Chem.  2004 ,  47  , 550;(d)Menchise, V.;DeSimone, G.;Alterio, V.;DiFiore, A.;Pedone, C.;Scozzafava,A.; Supuran, C. T.  J. Med. Chem.  2005 ,  48 , 5721.11. (a) Innocenti, A.; Hall, R. A.; Schlicker, C.; Scozzafava, A.; Steegborn, C.;Mühlschlegel, F. A.; Supuran, C. T.  Bioorg. Med. Chem.  2009 ,  17  , 4503; (b)Innocenti, A.; Mühlschlegel, F. A.; Hall, R. A.; Steegborn, C.; Scozzafava, A.;Supuran, C. T.  Bioorg. Med. Chem. Lett.  2008 ,  18 , 5066; (c) Innocenti, A.; Hall, R.A.;Schlicker, C.; Mühlschlegel, F. A.;Supuran, C. T.  Bioorg. Med. Chem.  2009 ,  17  ,2654; (d) Innocenti, A.; Winum, J.-Y.; Hall, R. A.; Mühlschlegel, F. A.;Scozzafava, A.; Supuran, C. T.  Bioorg. Med. Chem. Lett.  2009 ,  19 , 2642.12. (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)Isik,S.;Kockar, F.; Aydin, M.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Scozzafava,A.; Supuran, C. T.  Bioorg. Med. Chem.  2009 ,  17  , 1158; (c) Minakuchi, T.;Nishimori, I.; Vullo, D.; Scozzafava, A.; Supuran, C. T.  J. Med. Chem.  2009 ,  52 ,2226; (d) Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Innocenti, A.;Supuran, C. T.  J. Med. Chem.  2009 ,  52 , 3116; (e) Güzel, Ö.; Maresca, A.;Scozzafava, A.; Salman, A.; Balaban, A. T.; Supuran, C. T.  J. Med. Chem.  2009 ,  52 ,4063; (f)Carta, F.; Maresca, A.;SuarezCovarrubias, A.;Mowbray, S.L.;Jones, T.A.; Supuran, C. T.  Bioorg. Med. Chem. Lett.  2009 ,  19 , 6649.13. Khalifah, R. G.  J. Biol. Chem.  1971 ,  246 , 2561. AnAppliedPhotophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2  hydrationactivity. Phenol red (at a concentration of 0.2mM) has been used as indicator,working at the absorbance maximum of 557nm, with 10–20mM Hepes (pH7.5for the a -CAs) orTRIS (pH8.3, for the b -CAs)asbuffers, and20mMNa 2 SO 4 or 20mM NaClO 4  (for maintaining constant the ionic strength), following theinitial rates of the CA-catalyzed CO 2  hydration reaction for a period of 10–100s. TheCO 2  concentrationsrangedfrom1.7to17mMforthedeterminationof the kinetic parameters and inhibition constants. For each inhibitor at leastsix traces of the initial 5–10% of the reaction have been used for determiningtheinitialvelocity.Theuncatalyzedratesweredeterminedinthesamemannerand subtracted from the total observed rates. Stock solutions of inhibitor(10mM) were prepared in distilled–deionized water and dilutions up to0.01 l M were done thereafter with distilled–deionized water. Inhibitor andenzymesolutionswerepreincubatedtogether for 15min at roomtemperatureprior to assay, in order to allow for the formation of the E–I complex. Theinhibition constants were obtained by non-linear least-squares methods usingPRISM 3, as reported earlier, 11,12 and represent the mean from at least three different determinations.Human 14 andfungal 5,6 CAisozymeswereprepared inrecombinant form as reported earlier by our groups. Sulfonamides  1, 2  werereported earlier by these groups 1 .14. (a) Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava, A.; Supuran, C. T.  J.Med. Chem.  2004 ,  47  , 1272; (b) Nishimori, I.; Vullo, D.; Innocenti, A.;Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T.  J. Med. Chem.  2005 ,  48 , 7860. Ö. Güzel et al./Bioorg. Med. Chem. Lett. 20 (2010) 2508–2511  2511
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