Bioorganic & Medicinal Chemistry 23 (2015) 4181–4187

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Inhibition studies of bacterial, fungal and protozoan b-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties Mariangela Ceruso a,⇑, Fabrizio Carta a, Sameh M. Osman b, Zeid Alothman b, Simona Maria Monti c, Claudiu T. Supuran a,d,⇑ a

Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 16, 80134 Napoli, Italy d Università degli Studi di Firenze, Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy b c

a r t i c l e

i n f o

Article history: Received 29 April 2015 Revised 15 June 2015 Accepted 18 June 2015 Available online 26 June 2015 Keywords: Pathogenic microorganism Anti-infective Bacteria Beta-carbonic anhydrase Selective inhibitor Fungi Protozoa Schiff base Sulfonamide

a b s t r a c t A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three b-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high b-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Prokaryotic (i.e., bacteria and Cyanobacteria) and eukaryotic (i.e., fungi, protozoa and simple algae) microorganisms are defined as pathogens if they represent the cause of diseases or illnesses to their host.1 Although most of such microorganisms are harmless in healthy normal people, they can cause infections and/or diseases in patients with a compromised immune system such as individuals affected by acquired immunodeficiency syndrome (AIDS), cancer etc. Microbes reveal their pathogenicity through their ability to attack and enter a host, grow in the host environments by assimilating nutrients from the host, elude host defences or neutralize immune responses.2,3 A wide numbers of enzymes are involved in all the microorganism abilities above listed which are also considered as virulence factors. Therefore, cloning of genomes of many pathogenic microorganisms gave us the important tool to explore different pathways for inhibiting proteins crucial for their life cycle ⇑ Corresponding authors. Tel.: +39 055 4573143 (M.C.), +39 055 4573005 (C.T.S.). E-mail addresses: mariangela.ceruso@unifi.it (M. Ceruso), claudiu.supuran@ unifi.it (C.T. Supuran). http://dx.doi.org/10.1016/j.bmc.2015.06.050 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

or virulence factors. Numerous Carbonic Anhydrases (CAs, EC 4.2.1.1) were thus isolated recently from prokaryotic and eukaryotic microorganism and used for this purpose. Further studies of CA from bacteria, fungi and protozoa may definitely explain important and novel aspects of microbial virulence. The large family of carbonic anhydrases is involved in a variety of physiological and pathological processes of remarkable interest both in human and microorganism, They are considered ubiquitous metallo-enzymes which mainly catalyse the reversible hydration of CO2 to HCO3 and H+.4 This simple but physiologically relevant reaction is essential for the microorganism life due to its role in many vital processes, such as photosynthesis, CO2 transport as well as respiration.5–7 Six distinct evolutionarily non-related gene families have been discovered to date: alfa, beta, gamma, delta, zeta and eta.8 Although all 16 human CAs (hCAs) belong to the alfa-class, bacteria encode for enzymes belongings to the alfa, beta and gamma classes, whereas in protozoa and fungi only alfa and beta CAs have been reported so far.6,9 The most studied and widely used as diuretic, antiglaucoma, antiepileptic or anticancer drugs,4,10–16 are the classical carbonic anhydrase inhibitors (CAIs) containing a primary sulfonamide

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RSO2NH2 group in their structure.4,11,12 The presence of this effective zinc binding group makes these drugs ideal inhibitors of all human mammalian a-CAs. Only recently, a large range of such derivatives started to be investigated as anti-infectives, which target fungal/bacterial or protozoan CAs (generally belonging to b-CA family). Since the CAs belonging to the b-class are only present in pathogenic microorganism (such as fungi, bacterial and protozoa), and they lack from mammals, in which a-CAs class are physiologically relevant,4 these enzymes started to be widely accepted as possible drug targets for developing new antibacterial agents avoiding thus the important resistance problems, which are affecting most classes of antibiotics in clinical use. An exhaustive study on the antibacterial, antifungal and cytotoxic properties of Schiff base sulfonamide derivatives and their metal complexes has been recently carried out by Cohan et al.17 These assays led to the identification of the sulphonamide/metal complexes as an effective antimicrobial agent with low cytotoxicity. Indeed, it has been suggested that some functional groups such as azomethine (AC@NA) or heteroatoms present in these compounds play an important role in antibacterial and antifungal activity, most probably due to the enhancement of liposolubility of the molecules.17 Therefore, it appeared of great interest to further explore this type of scaffold for obtaining new class of b-CAIs. Here we extend our earlier investigations on Schiff base sulfonamides and report the inhibition studies against three b-CAs (Can 2, bsCA I and II, Ldc CA) over the off-target human (h) isoforms hCA I and II, with the series of a such sulfonamides already tested against the human CAs I, II, IX and XII. In order to obtain a broad and wide screening of the biological activities, the three b-carbonic anhydrases used in current studies for the inhibition assays were obtained from various microorganism sources. In particular, Can 2 was isolated from the pathogenic fungus Cryptococcus neoformans,18 bsCA I and bsCA II from the bacterial pathogen Brucella suis5,6 and LdcCA from Leishmania donovani chagasi, the protozoan parasite responsible for Leishmaniasis.19 Cryptococcus neoformans is a ubiquitous human pathogen fungus and the most commonly isolated agent of fungal meningitis in humans.18 It is presumed that human infection starts with the inhalation of desiccated yeasts or basidiospores with consequently formation of a local pulmonary infection. The organism can then spread to extra-pulmonary tissues, most likely using the blood stream. CO2 plays many different essential roles in growth, differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Indeed, the fungus can live both in a natural environment with 0.03% CO2 and in presence of high CO2 concentration such as during transitions to its mammalian host (5% CO2). The increase in CO2 promotes biosynthesis of a polysaccharide capsule, which represent an important virulence factor of Cryptococcus neoformans.20 The regulation of the Cryptococcus neoformans CO2-sensing system involved two important enzymes, the carbonic anhydrase (belonging to the b-class18) Can 2 and the fungal adenylyl cyclise Cac1. Can 2 catalyzes the formation of bicarbonate and a proton from CO2 and water. The bicarbonate activates the adenylyl cyclase Cac1, which promotes capsule biosynthesis.20 Therefore, fungal b-CAs such as Can 2 was found to be essential for survival of C. neoformans serotype A in its natural environment. Thus, they may represent attractive targets for development of antifungals that target the transmission of the borne disease. The b-carbonic anhydrases have recently emerged also as promising antibacterial targets.4,11 Indeed, several bacterial b-CA class have been cloned and characterized in some pathogens such as, Helicobacter pylori and Mycobacterium tuberculosis.21 Inhibition or genetic silencing studies demonstrated that the b-CA from

Helicobacter pylori is essential for the growth and the virulence of this pathogen.21 Therefore, inhibition of b-CA may be considered as a new possible approach for designing antibacterial agents considering the widespread and alarming emergence of multiresistance to antibiotics among bacterial pathogens. Therefore, two b-carbonic anhydrases from the bacterial pathogen Brucella suis, as denominated bsCA 1 and bsCA II (which are encoded by the gene BRA0788 and BR1829 of B. suis, respectively) has been recently cloned, purified, and characterized kinetically from our group.5,6 Brucella spp. are intracellular pathogens responsible of the most widespread animal infection disease, known as brucellosis or Malta fever. Brucellae are small Gram-negative R-proteobacteria, with several species infecting various vertebrates, from fish to primates, among which Brucella suis (the primary host being the pig) is one of the most infectious for humans. The incidence of human brucellosis may be as high as 200 per 100,000 inhabitants. Human brucellosis is a highly disabling disease which can last for weeks, resulting in chronic forms especially when not treated.21 Brucella is also extremely infectious by aerosol (only 10 bacteria are sufficient to contract disease) and is considered as a potential bioterrorism agent, especially because no human vaccine is available up to now. Therefore, bsCA inhibitors may have relevant applications in the fight against brucellosis, an endemic disease producing invalidating infection in humans and animals. In this study we use as well the protozoan b-CA from L. donovani (subspecies chagasi), denominated here LdcCA, as a new promising diagnostic and therapeutic antileishmanial drug target. Leishmaniasis is a tropical disease caused by unicellular protozoan organisms belonging to the genus Leishmania. The parasites are transmitted to the mammalian host organism through a contaminated sand fly. Among the many species and subspecies of such protozoa, Leishmania donovani chagasi causes visceral leishmaniasis associated to fatal visceral infections. This type of leishmaniasis produces between 20,000 and 40,000 deaths every year.22 It has been reported that Leishmania encodes for one a- and one b-CA. They are probably present in mitochondria where they might contribute to cell metabolism by providing bicarbonate for biosynthetic reactions and regulating intra-mitochondrial pH.19b Taking the above into account, in the current studies we intent to investigate the inhibition profile of a new series of Schiff base sulfonamides against three b-CAs (Can 2, bsCA I and II, Ldc CA) from different pathogenic microorganisms. We report hereby the inhibition studies against three parasite carbonic anhydrases with the main propose to develop antibacterial, antifungal or antiprotozoan agents possessing a different mechanism of action compared with the clinically used drugs to which a considerable level of drug resistance has been reported.4,6,7

2. Results and discussion 2.1. Chemistry Novel Schiff bases incorporating a sulfanilamide/3-fluorosulfonamide or 4-(2-aminoethyl)-benzenesulfonamide moiety in their molecules have been recently synthesized and investigated as inhibitors of human carbonic anhydrase (hCA I, II, IX and XII) from our group.25 A large range of these new derivatives 1–17 was thus obtained by a straightforward and high yield reaction between aromatic sulfonamides with aromatic/heterocyclic aldehydes in methanol (Scheme 1) using the routine procedure of obtaining imines.14–16,23 We previously reported23 that the inhibition profiles of sulphonamide-based CAIs against the mammalian isoforms are mainly influenced by two factors. The first is the nature of the

M. Ceruso et al. / Bioorg. Med. Chem. 23 (2015) 4181–4187

Ar-CHO X

dry MeOH n

NH2

human a-CA isoforms as potential antifungal, antibacterial or antiprotozoan drugs.

SO2NH2

SO2NH2

n =0,2 X= H, F

X n

4183

N Ar 1-17

Scheme 1. Preparation of Schiff’s bases 1–17.

arylsulfonamide moiety which directly binds to the zinc ion from the enzyme active site and, the second is the chemical nature of the tail attached to the inhibitor molecule, which usually binds the middle part or exit of the cleft that constitutes the active site. Indeed, X-ray crystallography studies have demonstrated that the presence of a substituent in the phenylsulfomoyl ring (such as a fluoride atom in compounds 4–6) leads to conformation changes of the arylsulfomoyl moiety when the inhibitor was bound within the enzyme active site.24 Since the most variable regions of the 16 mammalian carbonic anhydrase isoforms are represented by the pockets of the active site,1–3 changes of the arylsulfomoyl/tail moieties of the derivatives may lead effectively to the selectivity of the inhibitors towards various isoforms. Therefore, three different arylsulfonamides (sulphanilamide, 3-fluorosulfanilamide and 4-aminoethylbenzenesulfonamide) were used to obtain the new Schiff’s base sulfonamides here investigated (Scheme 1). On the other hand, the nature of the tail can be changed by using a variety of aldehydes as starting material. Therefore, in order to investigate whether the presence of different tails would lead to isoform-selective CAIs, both aromatic and heterocyclic aldehydes incorporating a wide range of substituents in their molecules were employed for synthesizing the new compounds reported here (Table 1). Indeed, benzaldehyde, fluoro and bromosubstituted benzaldehydes, as well as aldehydes including hydroxyl/methoxy/pyridyl/nitro/methylthio or cyano moieties in their structures were used in the study. Among the heterocyclic derivatives, benzothiophen-3-aldehyde, 2-furylaldehyde, furyl-2-aldehyde-4-sulfonic acid, and 5-bromo-pyrrole-3-aldehyde represent same examples. The chemical structures of the novel Schiff base sulfonamides 1–17 were confirmed by physico-chemical and spectral procedures and fully described elsewhere.25 In the earlier work,25 the Schiff base sulfonamides above mentioned were extensively investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, the cytosolic hCA I and II, as well as the two transmembrane tumor-associated ones, hCA IX and XII. The inhibition studies showed that most of these derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. Considering that a large number of Schiff’s bases incorporating aromatic/heterocyclic aldehydes and aromatic sulfonamides were already known as effective antibacterial for their ability to bind tightly the metal present in enzymes such as those of the pathogen microorganism,17 together with the fact that the earlier compounds showed lower affinity for the widespread, off-target cytosolic isoforms hCA I and II, we thought interesting to further extend the previous inhibition studies and investigate them as well as CA inhibitors against three different parasite b-CAs. Our intent is thus to discover a new lead compounds which will show a selectivity against the b-CAs present only in the pathogenic organisms such bacteria, fungi and protozoa over the off-target

2.2. CA inhibition The inhibition profile of three b-CAs from the pathogenic fungus Cryptococcus neoformans, from the bacterial pathogen Brucella suis and from the protozoan parasite Leishmania donovani chagasi, that is, Can 2, bsCA I/bsCA II and LdcCA has been investigated with derivatives 1–17 reported here. After determination of their inhibition potency by a stopped-flow CO2 hydrase assay method (Table 1), the KI values were compared to those of some standard sulfonamide CA inhibitors such as acetazolamide (AAZ), benzolamide (BZA) (an orphan drug4) and dichlorophenamide (DCP) (Fig. 1), reported earlier by our group.5,6,18,19,25 For comparison reasons, data for the inhibition of two of the dominant human off-target CA isoforms (hCA I and II) with these compounds are also included in Table 1. The structure–activity relationships (SARs) can be observed in this studies (Tables 1 and 2) and summarized as follows. 2.2.1. Human a-CA Inhibition The slow cytosolic isoform hCA I was weakly inhibited by sulfonamides 1–17 reported here, with inhibition constants in the range of 215–7005 nM. The best hCA I inhibitors were the 2-furyl substituted fluorosulfanilamide derivative (5) and the 2-methoxy-4-nitrophenylidene 4-aminoethylbenzensulfonamide (12), with equal KIs values of the clinically used agent acetazolamide AAZ, showing inhibition constants of 215 and 248 nM, respectively. Also in the case of the second cytosolic isoform hCA II, all the sulfonamides investigated here behaved as low potency inhibitors (KIs in the range of 168–713 nM) against this isoform. Only one derivative, 5, showed a rather effective inhibition of hCA II (KI of 63.5 nM, anyhow about five times higher compared to AAZ), on the other hand compound 9 was not inhibitory up to concentrations of 10 mM (Table 1). The structure–activity relationship (SAR) is rather flat as most of them have the inhibition constants in a similar micromolar range (Table 1). 2.2.2. Cryptococcus neoformans b-CA inhibition The first b-CA investigated here with the new series of Schiff base sulfonamides was the isoform Can 2 from the pathogenic fungus Cryptococcus neoformans. Against this beta isoform, Table 1 shows that most of the derivatives behaved as very strong and effective Can 2 inhibitors with KI in sub-nanomolar/nanomolar range between 0.56 and 22.31 nM. They also revealed to be similar or slightly better Can 2 inhibitors compared to AAZ and BZA possessing KIs only in nanomolar range (10.5 and 23 nM, respectively), whereas they showed almost two order of magnitude higher inhibition constant compared to that of DCP, whose KI is in micromolar range (1203 nM). However, only derivatives 4, 7, 16 and 17 were the best Can 2 inhibitors showing KIs in a narrow low range between 0.56 and 2.26 nM against this beta isoform. Indeed, this fungal b-CA isoform was strongly inhibited by the 3-fluorosulfanilamide phenyl substituted derivative 4 with KI of 0.56 nM. Although the active compounds inhibited efficiently Can 2, no significant change of KI values has been observed. Therefore, a very flat SAR was noticed in this case. 2.2.3. Brucella Suis b-CA inhibition Between the two bacterial b-CAs obtained from the pathogen Brucella suis, the bsCA II isoform was the most inhibited by the Schiff bases derivatives 1–17 here reported.

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Table 1 Inhibition data of b-CA isoforms from the pathogenic fungus Cryptococcus neoformans (Can 2), from the bacterial pathogen Brucella suis (bsCA I, bsCA II) and from the protozoan parasite Leishmania donovani chagasi (LdcCA), with sulfonamides 1–17 reported here and the standard sulfonamide inhibitors acetazolamide (AAZ), benzolamide (BZA) and dichlorphenamide (DCP) by a stopped flow CO2 hydrase assay (27)

SO2NH2

X n

N Ar

1-17 No

X

n

KI (nM)*

Ar a

1

H

0

2

H

0

3

H

0

4-Br-2-OH-C6H3

a

hCA I

hCA II

Can 2

bsCA I

bsCA II

Ldc CA

4320

707

16.89

625.36

23.66

>2500

4372

682

21.69

940.25

254.96

1252.7

3055

664

16.68

1032.7

206.69

1421.6

499 248 1511 7005

710 63.5 498 701

0.56 11.63 9.5 1.44

11.5 307.85 522.97 59.74

2.31 161.91 160.8 15.07

>2500 168.02 155.28 541.9

2850

465

12.07

682.49

25.7

1429.6

5335 4400 4235 215

>10000 697 666 168

15.69 17.03 22.31 21.19

45.15 109.58 760.71 329.94

24.3 21.91 31.96 162.52

1269.2 189.78 1984.8 1942.7

1345

403

19.24

528.6

24.8

1236.3

2940 2190

709 713

21.79 20.62

79.37 85.25

28.71 3.31

216.39 1397.7

1750

596

0.95

24.93

2.45

2033.8

2630 250 15b 1200b

652 12 9b 38b

2.26 10.5b 23b 1203b

29.18 63c 75c 58c

2.56 303d 117d 112d

107.38 91.7e 236e 189e

N

S 4 5 6 7

F F F H

0 0 0 2

8

H

2

9 10 11 12

H H H H

2 2 2 2

13

H

2

14 15

H H

2 2

16

H

2

Ph 2-Furyl Biphen-4-yl Biphen-4-yl

NaO3S

O

3-Br-2-OH-C6H3 2-OH-4-MeO-C6H3 4-Br-2-OH-C6H3 2-MeO-4-O2N-C6H3

N 4-MeS-C6H4 4-NC-C6H4

Br N H 17 AAZ BZA DCP * a b c d e

H –

2 – – –

2,3,5,6-C6HF4 – – –

Errors within the range of ±5–10% of the reported values, from 3 different assays (data not shown). From Ref. 25. From Ref. 18. From Ref. 5. From Ref. 6. From Ref. 19.

SO2NH2 O

O

N N N H

S

SO2NH2

AAZ (Acetazolamide)

S N O H

N N S

BZA (Benzolamide)

SO2NH2

Cl

SO2NH2 Cl

DCP (Dichlorphenamide)

Figure 1. Clinically used CA inhibitors with sulfonamide or sulfamate zinc binding group.

(i) The first bacterial isoform, bsCA I, was in fact, poorly inhibited by the majority of the compounds reported in current study, with KIs in the range of 522.97–1032.7 nM. The sulfonamides which behaved as moderate bsCA I inhibitors were the 2-furyl-fluorosulfanilamide (5) and the 2-methoxy-4-nitrophenylidene (12) derivatives containing the

longer 4-aminoethyl-benzenesulfonamide scaffold with KIs of 307.85 and 329.94 nM, respectively. Derivatives 7, 9, 10, 14 and 15 showed inhibitory properties in the same range as the standard and clinically used agents AAZ, BZA and DCP, with inhibition constants between 45.15 and 109.58 nM.

M. Ceruso et al. / Bioorg. Med. Chem. 23 (2015) 4181–4187 Table 2 Selectivity Ratios between KIs of b-CAs and Human a-CA I isoforms obtained with sulfonamides 1–17 reported here and the standard sulfonamide inhibitors acetazolamide (AAZ), benzolamide (BZA) and dichlorphenamide (DCP) Selectivity ratio of KIs*

No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 AAZ BZA DCP

hCA I/Can 2

hCA I/bsCA I

hCA I/bsCA II

hCA I/Ldc CA

255.8 201.6 183.2 891.1 21.3 159.1 4864.6 236.1 340 258.4 189.8 10.1 69.9 134.9 106.2 1842.1 1163.7 23.8 0.7 1

6.9 4.6 3 43.2 0.8 2.9 117.3 4.2 118.2 40.2 5.6 0.7 2.5 37 25.7 70.2 90.1 4 0.2 20.7

182.6 17.1 14.8 216 1.5 9.4 464.8 110.9 219.5 200.8 132.5 1.3 54.2 102.4 661.6 714.3 1027.3 0.8 0.1 10.7

221.5 6.4 0.9 0.5 0.8 8.9 8.4 23.9 22.3 0.2 0.1 0.7

29.9 2.7 3.2 307.4 0.4 3.1 46.5 18.1 >411.5 31.8 20.8 1 16.3 24.7 215.4 243.3 254.7 0.04 0.08 0.3

7.9 3.7 0.3 0.09 0.3 3.3 0.5 0.3 6.1 0.1 0.04 0.2

* The KI ratios are indicative of isoenzyme selectivity for pathogen versus human a CA II CAs and are calculated as: KK II human . pathogen bCA

the hCA I isoform, since they showed a maximum of 25-fold selectivity ratio or no selectivity for the pathogen CAs (Table 2). On the other hand, as reported in Table 3 many effective b-CA inhibitors also possessed excellent selectivity ratios for inhibiting the pathogenic isoforms over the human CA II. Almost all compounds 1–17 discriminated between the fungal b-CA (Can 2) and the human CA II isoform up to 4 orders of magnitude better than between the protozoan CA (Ldc CA) and hCA II isoforms. Although, they were not very selective Ldc CA inhibitors over the human isoform CA II showing low selectivity ratios in the range of 0.3–6.1 against this b-CA isoform, they still represent a slightly better solution compared to the standard the clinically used sulfonamides AAZ, BZA and DCP which revealed low selectivity against Ldc CA over hCA II in the range between 0.04 and 0.2. The sulfonamides reported here, were about 1 or 2 orders of magnitude more selective for the bacterial CA isoform (bsCA II) over the human CA II one (except for derivatives 2, 3, 5, 6 and 12 which showed selectivity ratios in the range of 0.4–3.2). The best performing compound in this analysis was 9 which showed to be a low potency human CA II inhibitors (KI > 10 lM) and an efficient fungal and bacterial b-CAs inhibitors with consequently high selectivity ratios of about 2 orders of magnitude for the inhibition of all b-CAs over hCA II. Furthermore, the phenyl derivative incorporating 3-fluorosulfanilamide4 was a sub-nanomolar Can 2 inhibitor with up to 891.1-fold selectivity over human CA I isoform and 1267.9-fold selectivity over the human CA II one. The consistency of the inhibition results obtained here against three different pathogenic b-CAs, provides a convincing opportunity to explore the Schiff base sulfonamide as scaffold in the development of potent and selective inhibitors for the b-family of CAs. 3. Conclusions A series of Schiff base sulfonamides has been investigated for the inhibition of three b-CAs earlier isolated and cloned isoforms from different pathogenic organisms, in particular from fungus Cryptococcus neoformans (Can 2), from bacterium Brucella suis (bsCA I and bsCA II) and from protozoon Leishmania donovani

chagasi (LdcCA). The inhibition constants so determined were then compared to those obtained against the off-target and physiologically relevant human isoforms CA I and II. The selectivity ratios between the b-class CAs over the human a-CAs were used as tool to understand if the new derivatives can prevalently discriminate the microorganisms (pathogens) which cause diseases to their host. Therefore, the inhibition studies of three b-CAs form different pathogenic microorganism with these new compounds revealed that they possess moderate-weak inhibition potency against the human ubiquitous and physiologically relevant a-CAs I and II, rather efficient inhibitory power against the protozoan Ldc CA, and excellent inhibition of the fungal b-CA isoform, as well as of the two bacterial isoforms bsCA I and II. In particular, few of them significantly inhibited the fungal enzyme Can 2, with efficacy in the sub-nanomolar range, and shown high selectivity for inhibiting the fungal over the human enzymes. Therefore, we can conclude that among the novel Schiff base sulfonamides here studied, some of them may constitute interesting tools for better understanding the physiologic/pathologic roles of b-CAs in the life cycle of bacteria. Furthermore, compared to the classical CA inhibitors, the remarkable b/a selectivity profile shown by these compounds, demonstrates that these new derivatives may have different mechanism of action compared to the standard and clinically used sulfonamides.

4. Experimental protocols 4.1. Chemistry 4.1.1. General procedure for preparation of compounds 1–17 (14b, f) The preparation of compounds 1–17 investigated in this study was carried out according to the procedure described by Supuran et al. (1996).26 Sulfonilamide/3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide (1.0 equiv) was dissolved in dry MeOH and stoichiometrical amount of the appropriate aromatic/herocyclic aldehydes containing either hydrophobic or hydrophilic moieties was added to the reaction. The solution was stirred until the formation of a precipitate was completed which was collected by filtration, washed with ice cold MeOH and dried under vacuo to afford the desired compounds 1–17. The synthesis and the full characterization of all compounds 1–17 investigated here as CA inhibitors are reported elsewhere.25 4.2. CA inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity.27 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4), 10 mM Tris. HCl and 0.1 M NaSO4 (for maintaining constant the ionic strength), following the initial rates of the CAcatalyzed 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 inhibitor (10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the

M. Ceruso et al. / Bioorg. Med. Chem. 23 (2015) 4181–4187

formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver–Burk plots, as reported earlier4,11 and represent the mean from at least three different determinations. All CAs were recombinant proteins obtained as reported earlier by these groups.28–30

16.

17.

Acknowledgments 18.

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Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties.

A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic ...
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