Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis...
Transcript of Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis...
Bioorganic & Medicinal Chemistry Letters 24 (2014) 4006–4010
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier .com/ locate/bmcl
Synthesis of sulfonamides with effective inhibitory action againstPorphyromonas gingivalis c-carbonic anhydrase
http://dx.doi.org/10.1016/j.bmcl.2014.06.0240960-894X/� 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel./fax: +39 055 4573005.E-mail address: [email protected] (C.T. Supuran).
Mariangela Ceruso a, Sonia Del Prete b, Zeid AlOthman c, Sameh M. Osman c, Andrea Scozzafava a,Clemente Capasso b, Claudiu T. Supuran a,c,d,⇑a Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Rm 188, Via della Lastruccia 3, I 50019 Sesto Fiorentino (Firenze), Italyb Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131 Napoli, Italyc Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabiad Università degli Studi di Firenze, Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o a b s t r a c t
Article history:Received 15 May 2014Revised 8 June 2014Accepted 9 June 2014Available online 18 June 2014
Keywords:Carbonic anhydraseEnzyme inhibitorSulfonamideAmino acidPorphyromonas gingivalis
New benzenesulfonamides incorporating GABA or N-a-acetyl-L-lysine scaffolds as well as guanidine func-tionalities as water solubilizing moieties were obtained, using 4-aminoethyl/methyl-benzenesulfonamideand metanilamide/sulfanilamide as zinc-binding motives. The new compounds were medium potencyinhibitors of the widespread cytosolic human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II andmore effective inhibitors (KIs low nanomolar range) of the bacterial c-CA from the oral pathogen Porphyro-monas gingivalis. These sulfonamides may be useful tools for understanding the physiological role ofbacterial CAs in pathogenesis of some infectious disease.
� 2014 Elsevier Ltd. All rights reserved.
Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple butphysiologically crucial reaction in all life kingdoms, the carbondioxide hydration to bicarbonate and protons: CO2 + H2O M HCO3
� +H+.1–3 These enzymes are involved in many physiologic pro-cesses, such as photosynthesis, respiration, CO2 transport,metabolic pathways involving carboxylation reactions, as well asmetabolism of xenobiotics in some bacteria (e.g., cyanate inEscherichia coli).4 Human (h) CAs, of which at least 15 isoformsare presently known belong to the a-CA genetic family, andconstitute drug targets for obtaining diuretics, antiglaucomaagents, antiepileptics, antiobesity and antitumor drugs.2–6 Thismultitude of pharmacologic applications is due to the fact thatthe different isoforms are involved in diverse physiologic orpathologic processes.2,3,6 There are however five distinct geneticfamilies encoding CAs in organisms all over the phylogenetic tree,the a-, b-, c-, d- and f-CAs, and all of them are metalloen-zymes.1,2,6–9 In the last years, the cloning of genomes of manymicroorganisms such as pathogenic bacteria, fungi, protozoa, etc.,progressed immensely. The possibility to explore alternative path-ways for inhibiting virulence factors or proteins essential for thelife cycle of such pathogens emerged, among which the CAs7–15
started to receive considerable attention. Bacteria encode CAsbelonging to three genetic families, the a-, b-, and c-CAs. Someof them were recently shown to be druggable targets.7–15 CAsbelonging to various such classes were investigated in severalpathogenic bacteria such as Helicobacter pylori, Brucella suis,Mycobacterium tuberculosis, Salmonella enterica, Streptococcuspneumoniae, Porphyromonas gingivalis, Clostridium perfringens,Legionella pneumophila, etc., in the search of antibiotics with anovel mechanism of action, since it has been demonstrated thatin many of them, these enzymes are essential for the life cycle ofthe organism.7–15
Among the bacterial species living in the oral cavity, a bacterialcomplex named the ‘red complex’ and composed of Porphyromonasgingivalis, Treponema denticola, and Tannerella forsythia has beenstrongly associated with advanced periodontal lesions.16 Porphyro-monas gingivalis seems to be the major pathogen responsible forthe development of periodontitis, being a highly successful colo-nizer of the oral epithelium. The perturbation of epithelial cellsby bacteria is the first stage in the initiation of inflammatory andimmune processes, eventually causing destruction of the tissuessurrounding teeth, which ultimately leads to tooth loss.16
Recently we have cloned a c-CA from this pathogen, which wasdenominated PgiCA.17a The enzyme was shown to possess asignificant catalytic activity for the reaction that converts CO2 to
M. Ceruso et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4006–4010 4007
bicarbonate and protons, with a kcat of 4.1 � 105 s�1 and a kcat/Km
of 5.4 � 107 M�1 � s�1. Like most enzymes belonging to the CAsuperfamily, PgiCA was also inhibited by acetazolamide (5-acet-amido-1,3,4-thiadiazole-2-sulfonamide, AAZ, a clinically useddrug)2 with an inhibition constant of 324 nM. We have also inves-tigated the inhibition profile of the new enzyme with a range ofinorganic anions such as thiocyanate, cyanide, azide, hydrogen sul-fide, sulfamate, trithiocarbonate, etc. These anions were mediumpotency PgiCA inhibitors with inhibition constants in the rangeof 41–97 lM. More effective inhibitors were diethyldithiocarba-mate, sulfamide, and phenylboronic acid, with KIs of 4.0–9.8 lM,17a whereas most aromatic/heterocyclic sulfonamidesinvestigated up until now also showed moderate inhibitory poweragainst PgiCA.17b Furthermore, some of these active inhibitors arealso selective for the inhibition of the bacterial enzyme over thehuman ones. Indeed, as mentioned earlier, CA inhibitors (CAIs) tar-geting similar bacterial enzymes from Helicobacter pylori,8a Brucellasuis,12 Mycobacterium tuberculosis,14a etc., showed antibacterialactivity in vivo against these species.
In this Letter we continue our interest in sulfonamide CAIs aspotential antibacterial agents, and report the synthesis of benzene-sulfonamides incorporating amino acid scaffolds and guanidinemoieties. These new compounds were investigated for the inhibi-tion of the physiologically dominant, cytosolic isoforms CA I andII, as well as the bacterial enzyme PgiCA.
Although sulfonamides are highly effective CAIs,1–3 one of themain drawbacks of this class of pharmacological agents is the factthat they generally possess a low water solubility.21 This is probablydue to the fact that the highly polar sulfamoyl moiety generally islinked to aromatic/heterocyclic rings, and usually no other watersolubilizing moieties are present in the molecule. This is the reasonwhy we considered the possibility to attach amino acyl moieties astails to the scaffolds of aromatic or heterocyclic sulfonamides, as ameans of increasing their water solubility.21 In this Letter we furtherexplore the tail approach for our drug design purposes, andemployed c-aminobutyric acid (GABA) or N-a-acetyl-L-lysinescaffolds for this purpose. Furthermore, we also explored theconversion of the terminal amino groups of these amino acid deriv-atives to the guanidine moiety, for synthesizing compounds withincreased water solubility which also incorporated 4-aminoethyl/methyl-benzenesulfonamide as well as metanilamide/sulfanil-amide as heads for binding to the zinc ion from the enzyme activesite (Scheme 1–3).
Starting from GABA 1, by protecting the c-amino moiety withthe tert-butyloxycarbonyl (Boc) protecting group, and couplingwith benzenesulfonamides 2 or 3, the c-Boc-GABA-substituted sul-fonamides 4 and 5 were prepared (Scheme 1). After removal of theBoc protecting group in 3 M HCl, the hydrochlorides 6, 7 were trea-ted with N,N0-di-Boc-N00-trifluoromethane-sulfonylguanidine 8 forconverting the terminal amino moiety to the di-Boc-protectedguanidines 9 and 10. Deprotection of the guanidine moiety in thepresence of acid led to the guanidinium-substituted sulfonamides11 and 12 (Scheme 1).22
A quite similar approach has been also adapted for the derivati-zation of N-a-acetyl-L-lysine 13, which has been protected at thee-amino moiety with the Boc group, coupled with sulfonamides2 and 3, and after the deprotection the terminal amino moietywas converted to the guanidine one as shown in Scheme 2. Severalmetanilamide (22, 23), sulfanilamide (24) and 4-carboxy-benzenesulfonamide (25) derivatives incorporating lysyl- or GABAmoieties were also obtained by following the same syntheticprocedure (Scheme 3).
Inhibition data with the set of compounds 4–25 reported here,against the human (h) CA isoforms hCA I, II, and the bacterialenzyme VchCA, are shown in Table 1. The two human isoformswere included in the assay as they may be the main off-targets
in case that bacterial CAs should be inhibited. Indeed, both theseisoforms are abundant, physiologically relevant enzymes.1–3 Thefollowing structure–activity relationship (SAR) can be observedfrom data of Table 1:
(i) Against the cytosolic slow isoform hCA I the new sulfona-mides reported here were generally ineffective or mediumpotency inhibitors, with inhibition constants rangingbetween 88.9–6030 nM. Only two sulfonamides, 4 and 23showed effective inhibitory activity against this isoform,with KIs of 19.8–22.1 nM. Both of them incorporate Boc-GABA moieties. For all other substitution patterns, generallythe 4-aminoethyl-benzenesulfonamides were more effectivehCA I inhibitors compared to the corresponding 4-amino-methylbenzenesulfonamide derivatives (Table 1).
(ii) The physiologically dominant human isoform hCA II waseffectively inhibited by 4, and 23–25, which showed KIs inthe range of 4.4–29.4 nM, of the same order of magnitudeas acetazolamide AAZ (KIs of 12 nM). These compoundsagain incorporate the Boc-GABA moieties, except 25, a deriv-ative of 4-carboxybenzenesulfonamide to which the deriva-tization was achieved at the a-amino moiety, whereas the e-amino one was protected by the benzyloxycarbonyl group.Some of the remaining derivatives, such as 5, 6, 9, 10, 14,15, and 21 were medium potency hCA II inhibitors (KIs inthe range of 44.0–91.9 nM). The other compounds reportedhere showed ineffective hCA II inhibitory activity, with KIsin the range of 197–5100 nM (Table 1).
(iii) Although acetazolamide AAZ, the clinically used sulfon-amide compound is a poor PgiCA inhibitor (KI of 324 nM),in this work we report much more effective PgiCA sulfon-amide inhibitors. Data of Table 1 show that some of thederivatives reported here, such as 6, 12, 22 and 24 showquite effective inhibitory properties against the bacterialenzyme, with KIs in the range of 40.1–56.0 nM. Furthermore,some of these excellent inhibitors are also selective for theinhibition of the bacterial enzyme over the human ones.For example, 12 shows a selectivity ratio for inhibiting PgiCAover hCA I of 52.86, and of PgiCA over hCA II of 21.12 (foracetazolamide the selectivity ratios are of 0.77 and 0.03,respectively, Table 1). An even more PgiCA-selective com-pound was 22, with a selectivity ratio for inhibiting PgiCAover hCA I of 111.25, and of PgiCA over hCA II of 94.09. Beinga quite effective PgiCA inhibitor, this compound may be auseful tool for understanding the physiologic/pathologicrole(s) of this enzyme. It should be noted that both tailsinvestigated here, the derivatized GABA (present in deriva-tives 6, 12 and 24) and the N-a-acetyl-L-lysine one (presentin 22) led to effective PgiCA inhibitors. Also the head groupsfrom these effective inhibitors are variable (sulfanilamide,metanilamide as well as 4-aminomethyl/aminoethyl-benzenesulfonamide).
Other compounds that showed interesting PgiCA inhibitory prop-erties were 7–10, 14, and 16–20, which inhibited the enzyme withKIs ranging between 60.8 and 89.0 nM. Again they incorporate vari-ous structural elements (coming from the amino acid or sulfonamidescaffolds), but minor structural changes also lead to drastic changesin the affinity for the enzyme. For example, 14 and 15 only differ by aCH2 moiety in the linker between the benzenesulfonamide and theamino acyl tail, but their affinity for PgiCA differs by a factor of3.50. Sulfonamides 23 and 24 are regioisomers, only differing bythe position of the sulfamoyl moiety with respect to the amino acyltail, and again there is a huge difference of activity as PgiCA inhibi-tors, with the sulfanilamide derivative 24 being 22-times a betterinhibitor compared to the isomeric metanilamide 23.
NHAcNH2
COOH
SO2NH2
NH2
NHAcBocHN
CONH
SO2NH2
NHAcNH2
CONH
SO2NH2
NHBocBocNH
NS
CF3
O
O
NHAcNH
CONHNBocBocNH
SO2NH2NHAc
NHCONH
NHNH2
SO2NH2
(i) Boc2O
(ii)
( )n
13
2: n =13: n = 2
( )n
14: n =115: n = 2
HCl3M
( )n
HCl
16: n =117: n = 2
8( )n
( )n HCl
6M
18: n =119: n = 2
20: n =121: n = 2
, EDCI
Scheme 2. Synthesis of N-a-acetyl-L-lysine sulfonamides 14–21.
NH2COOH
SO2NH2
NH2
SO2NH2
SO2NH2
NHBocBocNH
NS
CF3
O
O
SO2NH2SO2NH2
BocHNCONH
NH2CONH
NH
NBocBocNH
CONHNH
NHNH2
CONH
(i) Boc2O
(ii)
( )n
1
2: n =13: n = 2
( )n
4: n =15: n = 2
HCl3M
( )n
HCl6: n =17: n = 2
8( )n( )n
HCl
6M9: n =110: n = 2
11: n =112: n = 2
EDCI,
Scheme 1. Synthesis of GABA-containing sulfonamides 1–12.
4008 M. Ceruso et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4006–4010
The least effective PgiCA inhibitors detected here were 4, 5, 11,15, 21 and 23, which inhibited the enzyme with KIs in the range of233–1240 nM (Table 1).
Some physico-chemical parameters of compounds 4–25, suchas CLogP, PSA and Rf (determined in various elution systems) areshown in Table 2 in order to try to rationalize how these
parameters affect the biological activity of the synthesized com-pounds as CAIs (Table 2). However there are no simple, straightfor-ward relationship between these parameters and CA inhibitoryproperties of these sulfonamides.
In conclusion, we report two series of benzenesulfonamidessynthesized by using the tail approach. They incorporate amino
NHAcBocHN
CONH SO2NH2
SO2NH2BocHNCONH
SO2NH2BocHN
CONH
SO2NH2
NH
O
NH
O OCOOMe
Ph
NH2 SO2NH2
NHAcBocHN
COOH
NH2 SO2NH2
BocHNCOOH
SO2NH2
NH2
BocHNCOOH
SO2NH2
COOH
NH2
NH
O OCOOMe
Ph
22
23
24
25
EDCI
EDCI
EDCI
EDCI
Scheme 3. Synthesis of compounds 22–25.
Table 1Inhibition data against human (h) isoforms hCA I, II (cytosolic), and bacterial enzymePgiCA of sulfonamides 4–21 and acetazolamide AAZ (as standard inhibitor) by astopped-flow CO2 hydrase assay18
Compound KI* (nM) Selectivity ratios
hCA I hCA II PgiCA hCA I/PgiCA hCA II/PgiCA
4 19.8 9.8 490 0.04 0.025 253 91.9 233 1.08 0.396 2270 83.5 42.7 53.16 1.967 517 197 65.9 7.85 2.999 193 83.1 74.0 2.60 1.12
10 267 71.3 90.6 2.94 0.7811 972 470 887 1.09 0.5312 2120 847 40.1 52.86 21.1214 484 71.2 89.0 5.43 0.8015 430 75.9 312 1.37 0.2416 2330 563 74.1 31.44 7.6017 626 473 70.5 8.87 6.7018 2310 384 77.6 29.76 4.9419 519 452 60.8 8.53 7.4320 3150 824 80.7 39.03 10.2121 289 44.0 370 0.78 0.1122 6030 5100 54.2 111.25 94.0923 22.1 5.9 1240 0.01 0.00424 151.3 29.4 56.0 2.70 0.5225 88.9 4.4 85.4 1.04 0.05AAZ 250 12 324 0.77 0.03
The selectivity ratios are also reported.* Errors in the range of ±10% of the reported value (from 3 different assays).
Table 2Physical/chemical properties of sulfonamides 4–25 and acetazolamide AAZ
Compound CLogPa PSAb Rfc
4 0.835 135.97 0.88 (n-Butanol/acetic acid/H2O, 3/1/1)5 0.996 135.97 0.69 (n-Butanol/acetic acid/H2O, 3:1:1)6 �0.926 123.66 0.43 (n-Butanol/acetic acid/H2O, 3:1:1)7 �0.386 123.66 0.42 (n-Butanol/acetic acid/H2O, 3:1:1)9 2.689 186.66 0.39 (10% MeOH in DCM)
10 2.850 186.66 0.38 (10% MeOH in DCM)11 �1.63 159.54 0.68 (10% MeOH in DCM)12 �1.475 159.54 0.57 (10% MeOH in DCM)14 0.151 165.07 0.83 (n-Butanol/Acetic acid/H2O, 3:1:1)15 0.367 165.07 0.29 (10% MeOH in DCM)16 �1.552 152.76 0.86 (20% MeOH in DCM)17 �1.336 152.76 0.78 (20% MeOH in DCM)18 1.970 215.76 0.39 (20% MeOH in DCM)19 2.186 215.76 0.37 (20% MeOH in DCM)20 �2.439 188.64 0.5 (n-Butanol/acetic acid/H2O, 3:1:1)21 �2.223 188.64 0.44 (10% MeOH in DCM)22 0.447 165.07 0.7 (10% MeOH in DCM)23 1.22 135.97 0.26 (10% MeOH in DCM)24 1.22 135.97 0.35 (10% MeOH in DCM)25 1.843 162.27 0.57 (10% MeOH in DCM)AAZ 1.131 151.66 nd
a Data determined by ChemDraw Ultra 7.0.b Data determined by ChemSpider (www.chemspider.com).c Ninhydrin was used for staining; DCM = CH2Cl2; nd = not determined.
M. Ceruso et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4006–4010 4009
acid scaffolds (GABA or the N-a-acetyl-L-lysine), which were fur-ther derivatized for achieving increased water solubility, by intro-ducing guanidine groups at the terminal amino moiety from theinitial amino acid. Most of the new compounds were mediumpotency or ineffective inhibitors of two human CAs, hCA I and II,but some of them significantly inhibited the bacterial enzymePgiCA, with efficacy in nanomolar range. Considering also theirhigh selectivity ratios for inhibiting the bacterial over the human
enzymes, some of the compounds may constitute interesting toolsfor better understanding the physiologic/pathologic roles of a-CAsin the life cycle of these pathogens23
Acknowledgments
We thank The Distinguished Scientist Fellowship Program(DSFP) at KSU for funding this project. This work was also fundedin part by an EU FP7 project (Gums and Joints) to A.S.
4010 M. Ceruso et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4006–4010
References and notes
1. Tripp, B. C.; Bell, C. B., 3rd; Cruz, F.; Krebs, C.; Ferry, J. G. J. Biol. Chem. 2004, 279,6683.
2. (a) Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (b) Supuran, C. T.Nat. Rev. Drug Disc. 2008, 7, 168; (c) Alterio, V.; Pan, P.; Parkkila, S.; Buonanno,M.; Supuran, C. T.; Monti, S. M.; De Simone, G. Biopolymers 2014, 101, 769.
3. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem.Rev. 2012, 112, 4421; (b) Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1377; (c)Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467.
4. (a) Aggarwal, M.; Boone, C. D.; Kondeti, B.; McKenna, R. J. Enzyme Inhib. Med.Chem. 2013, 28, 267; (b) Zimmerman, S. A.; Tomb, J. F.; Ferry, J. G. J. Bacteriol.2010, 192, 135; (c) Ferry, J. G. Bioorg. Med. Chem. 2013, 21, 1392.
5. (a) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (b) Krall, N.; Pretto,F.; Decurtins, W.; Bernardes, G. J. L.; Supuran, C. T.; Neri, D. Angew. Chem., Int.Ed. 2014, 53, 4231; (c) Swietach, P.; Patiar, S.; Supuran, C. T.; Harris, A. L.;Vaughan-Jones, R. D. J. Biol. Chem. 2009, 284, 20299.
6. (a) Supuran, C. T. Future Med. Chem. 2011, 3, 1165; (b) Supuran, C. T.; Vullo, D.;Manole, G.; Casini, A.; Scozzafava, A. Curr. Med. Chem. – Cardiovasc. Hematol.Agents 2004, 2, 49.
7. Smith, K. S.; Ferry, J. G. FEMS Microbiol. Rev. 2000, 24, 335.8. (a) Nishimori, I.; Vullo, D.; Minakuchi, T.; Scozzafava, A. M.; Capasso, C.;
Supuran, C. T. Bioorg. Med. Chem. 2014, 22, 2939; (b) Maresca, A.; Vullo, D.;Scozzafava, A.; Manole, G.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28,392; (c) Maresca, A.; Scozzafava, A.; Vullo, D.; Supuran, C. T. J. Enzyme Inhib.Med. Chem. 2013, 28, 384.
9. (a) Supuran, C. T. Front. Pharmacol. 2011, 2, 34; (b) Supuran, C. T. Expert Opin.Ther. Pat. 2013, 23, 677; (c) Capasso, C.; Supuran, C. T. Expert Opin. Ther. Pat.2013, 23, 693.
10. (a) Vullo, D.; Kumar, R. S. S.; Scozzafava, A.; Capasso, C.; Ferry, J. G.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2013, 23, 6706; (b) Syrjänen, L.; Vermelho, A. B.; deAlmeida Rodrigues, I.; Corte-Real, S.; Salonen, T.; Pan, P.; Vullo, D.; Parkkila, S.;Capasso, C.; Supuran, C. T. J. Med. Chem. 2013, 56, 7372; (c) Pan, P.; Vermelho, A.B.; Scozzafava, A.; Parkkila, S.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem.2013, 21, 4472.
11. (a) Supuran, C. T.; Scozzafava, A.; Mastrolorenzo, A. Expert Opin. Ther. Pat. 2001,11, 221; (b) Del Prete, S.; De Luca, V.; Scozzafava, A.; Carginale, V.; Supuran, C.T.; Capasso, C. J. Enzyme Inhib. Med. Chem. 2014, 29, 23.
12. (a) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.; Takeuchi, H.;Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 2117; (b) Joseph,P.; Turtaut, F.; Ouahrani-Bettache, S.; Montero, J. L.; Nishimori, I.; Minakuchi,T.; Vullo, D.; Scozzafava, A.; Köhler, S.; Winum, J. Y.; Supuran, C. T. J. Med. Chem.2010, 53, 2277.
13. (a) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008,14, 622; (b) Nishimori, I.; Minakuchi, T.; Vullo, D.; Scozzafava, A.; Innocenti, A.;Supuran, C. T. J. Med. Chem. 2009, 52, 3116.
14. (a) Buchieri, M. V.; Riafrecha, L. E.; Rodríguez, O. M.; Vullo, D.; Morbidoni, H. R.;Supuran, C. T.; Colinas, P. A. Bioorg. Med. Chem. Lett. 2013, 23, 740; (b) Burghout,P.; Vullo, D.; Scozzafava, A.; Hermans, P. W. M.; Supuran, C. T. Bioorg. Med.Chem. 2011, 19, 243.
15. (a) Del Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg.Med. Chem. 2014, 22, 531; (b) Vullo, D.; Del Prete, S.; Osman, S. M.; De Luca, V.;Scozzafava, A.; AlOthman, Z.; Supuran, C. T.; Capasso, C. Bioorg. Med. Chem.Lett. 2014, 24, 275.
16. (a) Ruggiero, S.; Cosgarea, R.; Potempa, J.; Potempa, B.; Eick, S.; Chiquet, M.Biochim. Biophys. Acta. 2013, 1832, 517; (b) Guo, Y.; Nguyen, K. A.; Potempa,, J.Periodontology 2010, 54, 15; (c) Nguyen, K. A.; Zylicz, J.; Szczesny, P.; Sroka, A.;Hunter, N.; Potempa, J. Microbiology 2009, 155, 328; (d) Veillard, F.;Sztukowska, M.; Mizgalska, D.; Ksiazek, M.; Houston, J.; Potempa, B.; Enghild,J. J.; Thogersen, I. B.; Gomis-Rüth, F. X.; Nguyen, K. A.; Potempa, J. Biochim.Biophys. Acta. 2013, 1830, 4218; (e) de Diego, I.; Veillard, F. T.; Guevara, T.;Potempa, B.; Sztukowska, M.; Potempa, J.; Gomis-Rüth, F. X. J. Biol. Chem. 2013,288, 14287; (f) Bozdag, M.; Dreker, T.; Henry, C.; Tosco, P.; Vallaro, M.; Fruttero,R.; Scozzafava, A.; Carta, F.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 715.
17. (a) Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C.T.; Capasso, C. Bioorg. Med. Chem. Lett. 2013, 23, 4067; (b) Vullo, D.; Del Prete,S.; Osman, S. M.; De Luca, V.; Scozzafava, A.; AlOthman, Z.; Supuran, C. T.;Capasso, C. Bioorg. Med. Chem. Lett. 2014, 24, 240.
18. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed 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)and 20 mM NaBF4 (for maintaining constant the ionic strength), following theinitial 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 determinationof the kinetic parameters and inhibition constants. For each inhibitor, at leastsix traces of the initial 5–10% of the reaction have been used for determiningthe initial velocity. The uncatalyzed rates were determined in the samemanner and subtracted from the total observed rates. Stock solutions ofinhibitor (10 mM) were prepared in distilled–deionized water and dilutions upto 0.01 nM were done thereafter with distilled–deionized water. Inhibitor andenzyme solutions were preincubated together for 15 min at rt prior to assay, inorder to allow for the formation of the E–I complex. The inhibition constantswere obtained by non-linear least-squares methods using PRISM 3, and the
Cheng–Prusoff equation, as reported earlier,19,20 and represent the mean fromat least three different determinations. All CAs were recombinant proteinsobtained as reported earlier by these groups.17,19,20.
19. Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M. A.Eur. J. Med. Chem. 1998, 33, 247.
20. Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D. J. Med.Chem. 2007, 50, 1651.
21. (a) Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2012, 22, 747;(b) Scozzafava, A.; Briganti, F.; Mincione, G.; Menabuoni, L.; Mincione, F.;Supuran, C. T. J. Med. Chem. 1999, 42, 3690; (c) Bozdag, M.; Ferraroni, M.; Nuti,E.; Vullo, D.; Rossello, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.Chem. 2014, 22, 334.
22. Synthesis of tert-butyl-(4-oxo-4((4-sulfamoylbenzyl)amino)butyl)carbamate 4: 4-Tert-Butoxycarbonylamino-butyric acid (1.59 g, 1.0 equiv) was suspended indichloromethane (50 ml) and left stirring at room temperature. 4-Aminomethylbenzenesulfonamide hydrochloride 2 (1.74 g, 1.0 equiv) and 1-hydroxybenzotriazole (1.05 g, 1.0 equiv) were then added to the reactionmixture at the same temperature followed by the additions dropwise ofdiisopropylethylamine (4.00 ml, 3.0 equiv) and dimethylformamide (9 ml). Thereaction mixture was then cooled down to 0 �C in an ice bath and EDCI, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.65 g, 1.1 equiv)was added to the reaction mixture and left stirring for 65 h at roomtemperature. At the end of the reaction time the solution was acidified to pH2 using a 3.0 M HCl solution and extracted with ethyl acetate. The combinedorganic layers were washed with 10% citric acid, NaHCO3 saturated solution,brine and then dried over anhydrous sodium sulfate, filtered, and the solventwas removed under reduced pressure to give 1.83 g of the pure product aswhite crystals.tert-Butyl-(4-oxo-4((4-sulfamoylbenzyl)amino)butyl)carbamate 4: 75% yield; dH
(400 MHz, DMSO-d6): 8.43 (t, 1H, NH, J 6.0, exchange with D2O), 7.80 (d, 2H,CH, J 8.4), 7.45 (d, 2H, CH, J 8.4), 7.33 (br s, 2H, NH2, exchange with D2O), 6.85(br t, 1H, NH, J 5.2, exchange with D2O), 4.35 (d, 2H, CH2, J 6.0), 2.95 (td, 2H,CH2, J 6.5), 2.18 (t, 2H, CH2, J 7.4), 1.66 (tt, 2H, CH2, J 7.3), 1.42 (s, 9H, 3CH3); dC
(100 MHz, DMSO-d6): 172.87 (C@O), 156.51 (C@O), 144.73 (qC aromatic),143.46 (qC aromatic), 128.34 (2 CH aromatic), 126.58 (2 CH aromatic), 78.35(C-Boc), 42.58 (CH2), 33.64 (CH2), 29.19 (CH3-Boc), 26.67 (CH2); m/z (ESInegative), 370.4 [M�H]�.Synthesis of 4-oxo-4-((4-sulfamoylbenzyl)amino)butan-1-aminium chloride 6: Asolution of 4 (0.54 g, 1.0 equiv) and 3.0 M HCl (15 ml) was stirred overnight atroom temperature. The solution was dried under reduced pressure to give ayellowish oil, which was washed with dichloromethane and diethylether. Afterstirring overnight in diethylether the solvent was removed by decantation andthe powder was dried under reduced pressure to give 0.49 g of the pureproduct as a white solid. 4-Oxo-4-((4-sulfamoylbenzyl)amino)butan-1-aminiumchloride 4a: quantitative yield; dH (400 MHz, DMSO-d6): 8.68 (br t, 1H, NH, J5.8, exchange with D2O), 8.06 (br s, 3H, NH3
+, exchange with D2O), 7.80 (d, 2H,CH, J 8.4), 7.46 (d, 2H, CH, J 8.4), 7.37 (br s, 2H, NH2, exchange with D2O), 4.36(d, 2H, CH2, J 6.0), 2.81 (td, 2H, CH2, J 6.8), 2.32 (t, 2H, CH2, J 7.2), 1.85 (tt, 2H,CH2, J 7.4); dC (100 MHz, DMSO-d6): 172.35 (C@O), 144.57 (qC aromatic),143.53 (qC aromatic), 128.40 (2 CH aromatic), 126.58 (2 CH aromatic), 42.62(CH2), 39.27 (CH2), 32.92 (CH2), 24.08 (CH2); m/z (ESI positive), 272.3 [M�Cl]+.Synthesis of N,N0-di-Boc-guanidino-N00-(4-sulfamoyl-benzyl)-butyramide 9: N,N0-Di-Boc-N00-trifluoromethanesulfonylguanidine 8 (0.23 g, 1.0 equiv) was addedto a solution of 6 (0.2 g, 1.1 equiv) and triethylamine (0.07 g, 1.1 equiv) inchloroform, and the mixture was stirred at room temperature until N,N0-di-Boc-N00-trifluoromethanesulfonylguanidine was consumed (TLC control, 10%methanol in DCM). After completion, the mixture was diluted with chloroformand washed with 2.0 M sodium bisulfate, saturated sodium bicarbonate, andbrine. The organic extract was dried over sodium sulfate, the solvent removedunder reduced pressure. Crystallization from petroleum ether and diethylethergave 0.21 g of pure product as a white powder.N,N0-di-Boc-guanidino-N00-(4-sulfamoyl-benzyl)-butyramide 9: 69% yield; dH
(400 MHz, DMSO-d6): 11.55 (s, 1H, NH, exchange with D2O), 8.54 (t, 1H, NH,J 6), 8.38 (br s, H, NH), 7.80 (d, 2H, CH, J 8.4), 7.44 (d, 2H, CH, J 8.4), 7.35 (s, 2H,NH2, exchange with D2O), 4.35 (d, 2H, CH2, J 6.0), 3.32 (m, 2H, CH2), 2.22 (t, 2H,CH2, J 7.2), 1.79 (tt, 2H, CH2, J 7.4), 1.43 (s, 6CH3); dC (100 MHz, DMSO-d6):172.71 (C@O), 156.27 (NAC@N), 153.33 (C@O-boc), 152.93 (C@O-boc), 144.7(qC aromatic), 143.49 (qC aromatic), 128.38 (2 CH aromatic), 126.62 (2 CHaromatic), 83.84 (qC-boc), 42.67 (CH2), 40.45 (CH2), 33.58 (CH2), 28.56 (6CH3),25.82 (CH2); m/z (ESI positive), 514.4 [M�H]+.Synthesis of 4-guanidino-N-(4-sulfamoyl-benzyl)-butyramide hydrochloride 11: Asolution of 9 (0.1 g, 1.0 equiv) and 6.0 M HCl (10 ml) was stirred overnight atroom temperature. The solution was dried under reduced pressure to give ayellowish oil which washed with DCM and diethylether to give 0.07 g of thepure product as a brown gel. 4-Guanidino-N-(4-sulfamoyl-benzyl)-butyramidehydrochloride 8a: 58.9 yield; dH (400 MHz, DMSO-d6): 8.63 (t, 1H, NH, J 6,exchange with D2O), 7.88 (appt, NH), 7.80 (d, 2H, CH, J 8.4), 7.82 (d, 2H, CH, J8.4), 7.37 (s, 5H, NH3
+, exchange with D2O), 4.36 (d, 2H, CH2, J 6.0), 3.14 (m, 2H,CH2), 2.26 (t, 2H, CH2, J 7.2), 1.63 (tt, 2H, CH2, J 7.4); dC (100 MHz, DMSO-d6):172.66 (C@O), 157.92 (NAC@N), 144.57 (qC aromatic), 143.51 (qC aromatic),128.38 (2CH aromatic), 126.59 (2CH aromatic), 42.64 (CH2), 40.51 (CH2), 32.81(CH2), 19.46 (CH2); m/z (ESI positive), 314.4 [M�Cl]+.
23. Capasso, C.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014, in press (doi:10.3109/14756366.2014.910202).