molecules

Review Phenols and as Carbonic Anhydrase Inhibitors

Anastasia Karioti 1,*, Fabrizio Carta 2 and Claudiu T. Supuran 2,*

1 Laboratory of Pharmacognosy, School of Pharmacy, Aristotle University of Thessaloniki, University Campus, Thessaloniki 54124, Greece 2 Neurofarba Department, Sezione di Chimica Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via U. Schiff 6, I-50019 Sesto Fiorentino (Firenze), Italy; fabrizio.carta@unifi.it * Correspondence: [email protected] (A.K.); claudiu.supuran@unifi.it (C.T.S.); Tel.: +30-231-099-0356 (A.K.); +39-055-457-3729 (C.T.S.); Fax: +39-055-457-3385 (C.T.S.)

Academic Editor: Jean-Yves Winum Received: 6 November 2016; Accepted: 28 November 2016; Published: 2 December 2016

Abstract: Phenols are among the largest and most widely distributed groups of secondary metabolites within the plant kingdom. They are implicated in multiple and essential physiological functions. In humans they play an important role as microconstituents of the daily diet, their consumption being considered healthy. The physical and chemical properties of phenolic compounds make these molecules versatile ligands, capable of interacting with a wide range of targets, such as the Carbonic Anhydrases (CAs, EC 4.2.1.1). CAs reversibly catalyze the fundamental reaction of CO2 hydration to bicarbonate and protons in all living organisms, being actively involved in the regulation of a plethora of patho/physiological processes. This review will discuss the most recent advances in the search of naturally occurring phenols and their synthetic derivatives that inhibit the CAs and their mechanisms of action at molecular level. Plant extracts or mixtures are not considered in the present review.

Keywords: carbonic anhydrase inhibitors (CAIs); metalloenzymes; phenols; polyphenols; flavonoids

1. Introduction

1.1. The Carbonic Anhydrases Family, Occurrence and Role

CO2, bicarbonate and protons are an essential molecule and ions for many important physiologic processes occurring in all living organisms, including Archaea, Bacteria, and Eukarya [1,2]. However the uncatalyzed rate of interconversion of such species is too slow to meet the physiological needs of most biochemical processes [2,3]. This task is efficiently accomplished by the carbonic anhydrases (CAs, EC 4.2.1.1). This superfamily of metalloenzymes possess within their active sites a highly nucleophilic metal hydroxide species, such as zinc(II), cadmium(II) or iron (II) hydroxide, depending on the class [2,3]. To date seven not-genetically related CA families are reported, indicated with the Greek letters α-, β-, γ-, δ-, ζ- η- and θ-[1–9]. All the CA families have been thoroughly characterized by means of kinetic and structural features, with the exception of the δ-, η- and θ-CAs, for which only kinetic data are available so far [8–11]. The α-CAs are mainly distributed among the vertebrates, protozoa, algae, cytoplasm of green plants and in many Bacteria species. Since α-CAs are the only superfamily expressed in humans, they are also the most investigated ones for medicinal chemistry purposes [1–5,8,12–17]. Mammals (except the primates) possess 16 α-CA-isoforms, and among them three are devoid of catalytic activity [1,2]. The distribution of the various isoforms differs within tissues, cellular compartments as well as in modification of physio/pathological conditions [1,2]. The inhibition and activation mechanisms of CAs are well understood processes at a molecular

Molecules 2016, 21, 1649; doi:10.3390/molecules21121649 www.mdpi.com/journal/molecules Molecules 2016, 21, 1649 2 of 27 level. The classical CA inhibitors (CAIs) such as the primary sulfonamides and their bioisosters (the sulfamides and sulfamates) as well as the thiocyanates, tightly bind to the Zn(II) ion within the enzymatic active site, thus interrupting the carbon dioxide hydration cycle [1,2,12]. Although the sulfonamides are in clinical use as CAIs for almost 70 years they usually are associated to various side effects, which among others include hypersensitivity [1,12,13]. In the search for new and more effective drug candidates, natural products are indeed of particular interest. Up to now coumarins [18], polyamines, [19] and phenols [20,21] were revealed as interesting alternatives. The coumarins acts as prodrugs, [18] the simple phenols and polyamines anchor to the Zn(II)-bound water or hydroxide ion [19–21]. These different modes of action remarked many structural differences occurring between the CA isoforms and never explored before, thus paving the way for the design of new CAIs with high isoform selectivity and of particular interest for the future development as drug leads. All the above considerations are of particular interest, as selective CA targeting is the crucial aspect for the successful pharmacological treatment of diseases in which such enzymes are involved. Besides the use of CAI-based drugs for the treatment of glaucoma, oedema and altitude sickness, CAs are established targets for the prognosis and treatment and of hypoxic tumors [22]. To date the hCA IX monoclonal chimeric antibody girentuximab (Rencarex®), and its iodo radiolabeled derivative (Redectane®) are marketed for both the treatment of renal cell carcinoma (RCC) and for imaging purposes respectively. Recent advances account for the introduction in phase I clinical trials of the primary sulfonamide small molecule SLC-0111 for the treatment of metastatic solid tumors in patients showing positive response to hCA IX and XII markers [23]. hCAs are also valid therapeutic targets for the treatment of osteoporosis [24], central-nervous-system (CNS) affecting diseases such as Alzheimer or epilepsy [17,25–27], as well as for the treatment of obesity [28,29]. A research stream of particular interest is also represented by development of new antiinfectives by means of the selective targeting of the CAs expressed in bacteria, fungi and protozoa which represent the etiologic agents of various infectious diseases [30,31]. The development of new and more efficient antiinfectives is a field of prior interest in medicinal chemistry since many resistant bacterial strains are seriously jeopardizing the currently used pharmacological drugs. In this context natural products constitute a remarkable source of highly promising lead compounds for future development as next generation drugs.

1.2. Plant Phenolics Phenols and polyphenols constitute one of the largest and most ubiquitous classes of secondary metabolites in the plant kingdom. They are widely distributed in the Pteridophyta and Spermatophyta taxa (Gymnospermae and Angiospermae), whereas they are less common in bacteria, fungi and algae [32]. Simple phenols comprise structures with one aromatic ring, substituted with one or more hydroxyl moieties (Figure1). Most phenols of natural origin have at least two hydroxyls in their structure and are generated biosynthetically from the shikimate () pathway (C6-C3) through a series of reactions including decarboxylation, dehydration, and oxidative cleavage [33–35]. Polyphenols have at least two phenolic rings in their structure and are characterized by a wide structural diversity (Figures2 and3). They derive from the shikimate (phenylpropanoid) pathway, the polyketide (acetate) pathway or from a combination of both biosynthetic routes. Historically the term referred to tannins (condensed, hydrolysable and elagitannins, Figure2), which are complex polyphenolic compounds of polymeric nature with molecular masses ranging from 500 to 4000 Da, capable of interacting and binding with proteins. This property of tannins to act as protein precipitants has been exploited since antiquity in the processing of animal skin to leather [33,36,37]. Molecules 2016, 21, 1649 3 of 27 Molecules 2016, 21, 1649 3 of 27 Molecules 2016, 21, 1649 3 of 27

Figure 1. Main examples of phenols according to the classification of Harborne [32]. Figure 1. Main examples of phenols according to the classification of Harborne [32]. Figure 1. Main examples of phenols according to the classification of Harborne [32].

Figure 2. Examples of condensed (procyanidins), hydrolysable tannins and elagitannins. Figure 2. Examples of condensed (procyanidins), hydrolysable tannins and elagitannins. Figure 2. Examples of condensed (procyanidins), hydrolysable tannins and elagitannins. With the advent of chromatographic techniques and the development of phytochemistry, the termWith polyphenol the advent was of expanded chromatographic to include techniques a wider range and ofthe molecules development with atof leastphytochemistry, two phenolic the rings termWithin theirpolyphenol the skeleton: advent was xanthones, ofexpanded chromatographic stilb to includeenes and a widerstilbenoids, techniques range depsides, of and molecules the phenylpropanoid development with at least two of derivatives, phytochemistry,phenolic ringslignans the termin andtheir polyphenol lignin skeleton: (Figure wasxanthones, 3) expanded and, stilbmostenes importantly, to includeand stilbenoids, aflavon wider oidsdepsides, range (Figure ofphenylpropanoid 4), molecules which are withby derivatives, far at the least biggest lignans two group phenolic ringsand[32] in lignin their of polyphenolic (Figure skeleton: 3) and, xanthones,compounds. most importantly, stilbenes flavon and stilbenoids,oids (Figure 4), depsides, which are phenylpropanoid by far the biggest group derivatives, lignans[32] of and polyphenolicMore lignin than (Figure 8000 compounds. flavonoids3) and, most and importantly,derivatives thereof flavonoids have been (Figure reported4), which to date are by[38]. far The the term biggest groupflavonoid [More32] of than polyphenolic refers 8000 to yellow compounds. (flavus and =derivatives yellow) pigments thereof have which been in theirreported classical to date form [38]. incorporate The term a refers to yellow (flavus = yellow) pigments which in their classical form incorporate a Morebenzo[ thanγ]pyrone 8000 system. flavonoids However, and under derivatives the same thereof group have several been biogenetically reported torelative date [secondary38]. The term benzo[γ]pyrone system. However, under the same group several biogenetically relative secondary flavonoid refers to yellow (flavus = yellow) pigments which in their classical form incorporate a benzo[γ]pyrone system. However, under the same group several biogenetically relative secondary Molecules 2016, 21, 1649 4 of 27

Molecules 2016, 21, 1649 4 of 27 Molecules 2016, 21, 1649 4 of 27 metabolitesmetabolites are are comprised comprised including including thethe yellowyellow pigments aurones and and chalcones, chalcones, the thecolorless colorless flavanonesflavanonesmetabolites and and theare thered/bluecomprised red/blue anthocyanidinsincluding the yellow (Figure pigments 44).). aurones and chalcones, the colorless and the red/blue anthocyanidins (Figure 4).

FigureFigure 3. 3.Some Some examples examples of polyphenolic polyphenolic classes. classes. Figure 3. Some examples of polyphenolic classes.

Figure 4. Basic flavonoid structures. FigureFigure 4. 4.Basic Basic flavonoidflavonoid structures. structures. In plants phenolics play a key role in many vital physiological processes, such as lignification, pigmentationIn plants ofphenolics flowers andplay fruits, a key pollination, role in many as gr vitaowthl physiological factors, and theyprocesses, also govern such plantas lignification, responses Intowardspigmentation plants environmental phenolics of flowers play andstress, afruits, key such rolepollination, as in protection many as gr vitalowth from physiological factors, excess and UV they radiation, processes, also govern nutrient such plant asdeficiency, responses lignification, pigmentationdroughttowards stress,environmental of flowers and most and stress, fruits,importantly such pollination, as they protection act as as growth chemical from factors,excess defense UV and againstradiation, they also herbivores, nutrient govern plantinsectsdeficiency, responses and towardsmicrobialdrought environmental stress, pathogens and stress, most[36,37]. importantly such For humans, as protection they plant act fromphenolicsas chemical excess are UVdefense important radiation, against constituents nutrient herbivores, deficiency,of many insects edible and drought stress,andmicrobial and medicinal most pathogens importantly plants [36,37]. and they Forwidely humans, act asused chemical plant in the phenolics defensefood industry are against important as herbivores, flavorings, constituents insectsantioxidants of many and edible microbialand pathogensantibacterialand medicinal [36,37 agents]. plants For [39,40]. humans, and Phenolswidely plant andused phenolics polyphenol in the food ares have importantindustry attracted as constituents great flavorings, interest antioxidants ofdue many to their edible wideand and antibacterial agents [39,40]. Phenols and polyphenols have attracted great interest due to their wide medicinalpresence plants in our and daily widely diet and used mo inst the importantly food industry due to as their flavorings, antioxidant antioxidants properties. andMany antibacterial efforts presence in our daily diet and most importantly due to their antioxidant properties. Many efforts agentshave [39 been,40]. done Phenols to estimate and polyphenols the daily intake have in pl attractedant phenolics. great Humans interest seem due to to have their adapted wide their presence enzymatichave been donesystems to estimateand only the a dailysmall intake portion in ofpl antthe phenolics. phenolic contenHumanst in seem foods to reacheshave adapted the tissues their in our daily diet and most importantly due to their antioxidant properties. Many efforts have unalteredenzymatic [41]. systems Although and theonly relationships a small portion between of thefood phenolic phenolics conten and healtht in foods are not reaches fully understood, the tissues been done to estimate the daily intake in plant phenolics. Humans seem to have adapted their epidemiologicalunaltered [41]. Although studies thein relationshipshumans demonstrate between foodthat phenolicsconsumption and healthof food are richnot fullyin polyphenolic understood, enzymaticconstituentsepidemiological systems might andstudies be beneficial only in ahumans small especially portiondemonstrate for some of the thage-relatedat phenolic consumption disorders, content of foodsuch in foods asrich cardiovascular in reaches polyphenolic the and tissues unalteredneurodegenerativeconstituents [41]. Although might bediseases, beneficial the relationships type es IIpecially diabetes between for and some cancers food age-related phenolics [41–43]. disorders, and health such as are cardiovascular not fully understood, and epidemiological neurodegenerative studies diseases, in humans type II diabetes demonstrate and cancers that consumption[41–43]. of food rich in polyphenolic constituents might be beneficial especially for some age-related disorders, such as cardiovascular and neurodegenerative diseases, type II diabetes and cancers [41–43]. The ability of plant phenolics to interact with a plethora of targets and cause such a broad range of activities should be attributed to the physicochemical properties of the main structural unit, the Molecules 2016, 21, 1649 5 of 27

MoleculesThe2016 ability, 21, 1649 of plant phenolics to interact with a plethora of targets and cause such a broad range5 of 27 of activities should be attributed to the physicochemical properties of the main structural unit, the phenolic ring [[41].41]. The phenolic moiety has an amph amphiphiliciphilic character. The presence of the hydrophobic planar aromatic ring is responsibleresponsible for hydrophobic interactions (π-stacking), whereas at the same time the polar hydroxyl groups can participate in hydrogen bonding.bonding. This dual behavior allows these molecules to bind to the amino acid residues of several proteins, enzymes or receptors. Furthermore the occurrence occurrence of of ortho-hydroxyl ortho-hydroxyl groups groups renders renders them them excellent excellent antioxidant antioxidant agents agents and chelating and chelating factors. factors.These properties These properties render renderplant phenolics plant phenolics as excellen as excellentt candidate candidate molecules molecules to study to study the theinhibition inhibition of ofmetalloenzymes. metalloenzymes. One such an enzyme is the family of the carbonic anhydrases (CAs, EC 4.2.1.1) which is the subject of the present present review. review. Simple Simple phenol phenolss and and polyphenols polyphenols of natural of natural origin origin are discussed, are discussed, along along with withtheir theirsynthetic synthetic derivatives. derivatives. The inhibition The inhibition mechanisms mechanisms and possible and possible structure structure activity activityrelationships relationships are also arepresented. also presented. Since coumarins Since coumarins are discussed are discussed in another in article another of articlethis special of this issue special they issue are not they included are not includedin the present in the one. present Only one. reports Only on reports pure compounds on pure compounds were taken were into takenconsideration. into consideration. This is an update This is anof previous update of reviews previous [44,45]. reviews [44,45].

2. Phenols and Polyphenols as Effective Carbonic Anhydrase Inhibitors

2.1. Inhibition Mechanism of Phenol Phenol, the simplest member of this family of compounds was reported to be aa competitivecompetitive inhibitor of human (h) Carbonic AnhydraseAnhydrase II (hCA II)II) [[46,47].46,47]. The X-ray crystal structure of its adduct with hCA II waswas studiedstudied andand revealedrevealed thethe bindingbinding ofof twotwo phenolphenol moleculesmolecules to thethe enzyme.enzyme. The firstfirst one was located in a hydrophobic patch about 15 Å away from the catalytic zinc ion thus it is likely that this binding does not interfere with the enzymatic enzymatic catalytic catalytic activity. activity. However, the second phenol molecule was found within the active site and showedshowed a new binding mode. Indeed, this molecule did not coordinate to the zinc ion, but was anchored to the active site through two hydrogen bonds of the OH moiety with the zinc-bound water/hydroxidewater/hydroxide ion and the NH amide of Thr 199. Furthermore, Furthermore, the aromatic ring was found to lie in a hydrophobic pocket of the active active site, site, delimited delimited by by residues residues Val Val 121, Val 143,143, LeuLeu 198,198, andand 197197 TrpTrp 209,209, thusthus contributingcontributing toto thethe complexcomplex stabilization stabilization [ 48[48]] (Figure (Figure5 ).5).

Figure 5. Schematic representation of the binding mode of phenol within the hCA II active site [[48].48].

2.2. Simple Phenols These findingsfindings opened the way for the explorationexploration of otherother phenolicphenolic andand polyphenolicpolyphenolic natural products. In vitro kinetic studies of a series of phenolsphenols widely available on the market such as salicylicsalicylic acid, resorcinol, resorcinol, p-coumaric,p-coumaric, caffeic, caffeic, ferulic, ferulic, gallic gallic acids, acids, etc., sh etc.,owed showed that these that are these affective are CA affective inhibitors CA inhibitorsin the low inmicromolar the low micromolar range (Figure range 6) (Figure[45,46].6 )[ 45,46]. Molecules 2016, 21, 1649 6 of 27 Molecules 2016, 21, 1649 6 of 27 Molecules 2016, 21, 1649 6 of 27

Figure 6. Some phenolic compounds as CAIs [[45].45]. Figure 6. Some phenolic compounds as CAIs [45]. The presence of free hydroxyl groups at position 4’ in combination with the double bond in the The presence of of free free hydroxyl hydroxyl groups groups at at position position 4’ 4’in incombination combination with with the thedouble double bond bond in the in phenylpropanoid skeleton, as in p-coumaric acid, clearly enhanced the inhibitory activity when thephenylpropanoid phenylpropanoid skeleton, skeleton, as in as inp-coumaricp-coumaric acid, acid, clearly clearly enhanced enhanced the the inhibitory inhibitory activity activity when compared to the simple phenol moiety. Similar results were obtained in a further study with a small compared to the simple phenol mo moiety.iety. Similar results were obtained in a further study with a small group of monophenolic constituents, where trans-cinnamic (14), o-coumaric (15) and chlorogenic (16) group of monophenolic constituents, where trans-cinnamic ( 14), o-coumaric ( (15) and and chlorogenic chlorogenic ( (16)) acids were more effective than simple phenolic constituents such as benzoic acid (9) [45] (Figure 7 acids werewere moremore effectiveeffective than than simple simple phenolic phenolic constituents constituents such such as benzoicas benzoic acid acid (9)[ (459)] [45] (Figure (Figure7 and 7 and Table 1). Tableand Table1). 1).

Figure 7. Structures of tested phenols: benzoic acid (9), m-benzoic acid (10), propylparaben (11), Figure 7. Structures of tested phenols: benzoic acid (9), m-benzoic acid (10), propylparaben (11), Figureprotocatechuic 7. Structures acid (12 of), vanillic tested phenols:acid (13), cinnamic benzoic acidacid ( 149),),m o-coumaric-benzoic acidacid (1015), chlorogenic propylparaben acid ( 1611),), protocatechuic acid (12), vanillic acid (13), cinnamic acid (14), o-coumaric acid (15), chlorogenic acid (16), protocatechuicguaiacol (17), 4-methylguaiacol acid (12), vanillic ( acid18), (4-propylguaiacol13), cinnamic acid (19 (14),), eugenolo-coumaric (20), acid isoeugenol (15), chlorogenic (21), vanillin acid ( (2216),), guaiacol (17), 4-methylguaiacol (18), 4-propylguaiacol (19), eugenol (20), isoeugenol (21), vanillin (22), guaiacolsyringaldehyde (17), 4-methylguaiacol (23), catechol (24 (18),), 3-methylcatechol 4-propylguaiacol ( (2519),), 4-methylcatechol eugenol (20), isoeugenol (26), 3-methoxycatechol (21), vanillin (22), syringaldehyde (23), catechol (24), 3-methylcatechol (25), 4-methylcatechol (26), 3-methoxycatechol syringaldehyde(27) [45]. ( ), catechol ( ), 3-methylcatechol ( ), 4-methylcatechol ( ), 3-methoxycatechol (27)[) [45].45].

Molecules 2016, 21, 1649 7 of 27

Table 1. Inhibition data of compounds 9–16 [45].

a IC50 (µg/mL) Compound hCA I hCA II Benzoic acid (9) 1.31 1.12 m-hydroxybenzoic acid (10) 1.33 1.00 Propylparaben (11) 0.84 0.82 Protocatechuic acid (12) 1.31 1.15 Vanillic acid (13) 0.52 0.36 o-coumaric acid (14) 0.93 0.22 trans-cinnamic (15) 0.11 0.09 Chlorogenic acid (16) 0.98 0.88 a From three different assays, errors ± 5%–10% of the reported value.

In another study several derivatives of guaiacol and catechol (17–27) were considered for their CA inhibitory activity against human CA I, II, IX and XII isoforms [49]. Among the compounds tested in vitro were the volatile constituents eugenol (20), isoeugenol (21) and vanillin (22), which are major components of many aromatic herbs and spices such as clove, basil, anise seed, Vanilla sp., etc. All compounds showed micromolar inhibition potencies spanning between 2.2–10.92 µM with the exception of the catechol (Table2). Among the tested phenolic derivatives, compounds 4-methyl-catechol (26) and 3-methoxycatechol (27) showed potent activity as inhibitors of the tumour-associated transmembrane isoforms hCA IX and XII with KI values in the submicromolar range.

Table 2. Inhibition constants of certain phenolic compounds derived from guaiacol and catechol against four human carbonic anhydrase isoenzymes (hCA I, II, IX and XII) using an esterase bioassay [49].

a KI (µM) Compound hCA I hCA II hCA IX hCA XII Guaiacol (17) 7.50 5.63 9.98 9.83 4-Methylguaiacol (18) 9.15 7.74 9.89 8.55 4-Propylguaiacol (19) 10.34 8.51 9.01 9.12 Eugenol (20) 8.32 7.27 9.62 8.89 Isoeugenol (21) 10.29 6.73 9.32 9.13 Vanillin (22) 11.37 7.15 9.81 8.39 Syringaldehyde (23) 10.92 6.68 9.11 7.70 Catechol (24) 247.50 5.51 373.23 515.98 3-Methylcatechol (25) 5.95 4.69 9.55 7.22 4-Methylcatechol (26) 6.16 2.76 8.11 6.58 3-Methoxycatechol (27) 6.32 2.20 7.83 7.53 a From three different assays, errors ±5%–10% of the reported value.

Combination of the phenolic moiety with other functional groups is a common strategy to obtain products with enhanced activity. For example, in the so-called “sugar approach” the incorporation of a hydrophilic moiety of a sugar permits to the molecule to anchor in a different manner to the enzyme cavity. Furthermore, it increases its selectivity towards the membrane bound CA isoforms over the cytosolic ones [50,51]. This is desirable since selectivity against specific hCA isoforms might reduce side effects. In this concept, a series of synthetic C-cinnamoyl glycosides were synthetized and considered for the inhibition on twelve mammalian isoforms of carbonic anhydrase (Figure8)[ 52]. These compounds combined two functional groups with known activity against the hCAs: the cinnamoyl and the glucose group. In order to avoid undesirable degradation, the O-glycosidic bond was replaced by the more stable C-glycosidic one. The C-cinnamoyl glycosides (28–35) were generally effective CAs, with Molecules 2016, 21, 1649 8 of 27 inhibition constants in the low micromolar range against CA I, II, IV, VA, VB, VI, VII, IX, XII, XIII, XIV Moleculesand ineffective 2016, 21, 1649 inhibitors of CA III (Table3). 8 of 27

Figure 8. StructuresStructures of the cinnamoyl glycosides 28–35 [52].[52].

TableTable 3. Inhibition of mammalian α-CA with the C-cinnamoyl glycosides 28–35 and phenol 1..

I a K (µM)a KI (µM) No. hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA No. hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA I II III IV VA VB VI VII IX XII XIII XIV I II III IV VA VB VI VII IX XII XIII XIV 28 8.5 7.0 >100 5.6 9.8 6.0 8.8 9.5 5.2 6.7 5.1 5.9 28 8.5 7.0 >100 5.6 9.8 6.0 8.8 9.5 5.2 6.7 5.1 5.9 2929 5.75.7 3.93.9 >100>100 4.94.9 8.48.4 4.04.0 6.26.2 6.36.3 5.95.9 6.26.2 4.94.9 5.65.6 3030 5.15.1 7.17.1 >100>100 7.87.8 9.59.5 6.96.9 7.77.7 7.17.1 3.33.3 3.93.9 7.27.2 4.64.6 3131 9.39.3 5.55.5 >100>100 6.76.7 9.39.3 5.25.2 7.97.9 5.85.8 2.92.9 4.24.2 6.76.7 2.32.3 32 6.8 7.8 >100 8.3 7.4 >100 >100 4.9 4.5 7.4 8.8 4.3 3332 3.76.8 8.8 7.8 >100>100 7.18.3 4.4 7.4 >100>100 >100>100 8.54.9 8.2 4.5 8.7 7.4 8.6 8.8 7.1 4.3 3433 3.63.7 3.1 8.8 >100>100 9.27.1 3.4 4.4 >100>100 8.1>100 9.08.5 9.2 8.2 8.4 8.7 8.6 8.6 >100 7.1 3534 5.53.6 6.83.1 8.4>100 8.49.2 8.03.4 >100>100 >1008.1 9.39.0 8.29.2 6.88.4 >1008.6 >100>100 351 10.25.5 5.56.8 2.78.4 9.58.4 2188.0 543>100 208>100 7109.3 8.88.2 9.26.8 >100 697 11.5>100 a 1 From10.2 three different5.5 assays, 2.7 errors 9.5± 5%–10% 218 of the543 reported 208 value, 710 CO2 hydrase, 8.8 stopped-flow 9.2 697 assay. 11.5

a From three different assays, errors ± 5%–10% of the reported value, CO2 hydrase, stopped-flow assay. Another hybrid molecule recently investigated against several hCA isozymes [53] is Dodoneine 36, oneAnother of the hybrid active principlesmolecule recently of the medicinal investigated plant againstAgelanthus several dodoneifolius hCA isozymes(DC) [53] Polhilland is Dodoneine Wiens. 36The, one plant of isthe used active in traditional principlesmedicine of the medicinal for the treatment plant Agelanthus of hypertension. dodoneifolius Structurally (DC) Polhilland36 incorporates Wiens. Theboth plant the phenol is used andin traditional lactone (α medicine-pyrone) fo moieties.r the treatment Dodoneine of hypertension. showed to be Structurally selective in 36 inhibiting incorporates the bothhCA isoformsthe phenol I, III,and IV, lactone XIII and (α-pyrone) XIV with moieties. KIs in the Dodoneine range of 5.5–10.4 showedµM. to CA be I,selective III and XIIIin inhibiting are cytosolic, the hCACA IV isoforms is localized I, III, in IV, plasma XIII and membrane XIV with and KIs hCA in the XIV range is a membraneof 5.5–10.4 μ boundedM. CA I, isozyme.III and XIII Further are cytosolic,in vitro CAand IVin is vivo localizedstudies in in plasma rat aorta membrane and vascular and hCA smooth XIV muscle is a membrane cells showed bounded that hCAisozyme. II, III, Further XIII and in XIVvitro wereand in expressed vivo studies in rat in aortarat aorta while and only vascular the hCA smooth isoforms muscle III, cells XIII showed and XIV that were hCA expressed II, III, XIII in andsmooth XIV musclewere expressed cells. Thus in rat it isaorta reasonable while only to assumethe hCA that isoforms dodoneine III, XIII induced and XIV vasorelaxation were expressed by in a smoothdual mode muscle of action cells. includingThus it is both reasonable block of to the assume L-type that calcium dodoneine channels induced and inhibition vasorelaxation of the hCAby a dualisoforms mode therein of action expressed including [54 both]. A seriesblock of the synthetic L-type analogues calcium channels of dodoneine and inhibition (37–41), reportedof the hCA in isoformsFigure9, weretherein prepared expressed and [54]. tested A for series their of inhibition synthetic properties analogues against of dodoneine the hCAs. (37 Among–41), reported them the in Figurebicyclic 9, compound were prepared40 which and tested is normally for their found inhibition in the properties plant in small against amounts the hCAs. and Among its hydroxylated them the bicyclicderivative compound41 and the 40 fluorinatedwhich is normally analogue found39 (Table in the4 ).plant Compound in small amounts39 inhibited and all its hCA hydroxylated isoforms, whilederivative41 inhibited 41 and the selectively fluorinated hCA analogue III (KI = 5.1 39 nM)(Table and 4). mCA Compound XIII (KI 39= 340 inhibited nM). all hCA isoforms, while 41 inhibited selectively hCA III (KI = 5.1 nM) and mCA XIII (KI = 340 nM). Molecules 2016,, 21, 1649 9 of 27 Molecules 2016, 21, 1649 9 of 27

Figure 9. Structures and hCA inhibition data of dodoneine (36) and its analogues 37–41 by a stopped Figure 9. Structures and hCA inhibition data of dodoneine (36) and its analogues 37–41 by a stopped Figureflow CO 9.2 Structureshydrase assay and [53]. hCA inhibition data of dodoneine (36) and its analogues 37–41 by a stopped flowflow CO 2 hydrasehydrase assay assay [53]. [53]. Table 4. Structures and hCA inhibition data of dodoneine (36) and its analogues 37–41 by a stopped Table 4. Structures and hCA inhibition data of dodoneine (36) and its analogues 37–41 by a stopped Tableflow CO 4. Structures2 hydrase assay and hCA[53]. inhibition data of dodoneine (36) and its analogues 37–41 by a stopped 2 flowflow CO 2 hydrasehydrase assay assay [53]. [53]. KI (µM) a a KI (µM)a No. hCA hCA hCA hCA hCA hCAKI ( µM)hCA hCA hCA hCA hCA hCA No. hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA No. I II III IV VA VB VI VII IX XII XIII XIV b hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCA hCAb 36 5.48II II_II c 10.35IIIIII 9.61IVIV VA _ c VB_ c VI_ c VII_ c IX_ c XII_ c XIII9.27XIII XIV9.34b c c c c c c c 36 5.48 _ c 10.35c 9.61 _ _ _ c _ c _ _ 9.27 9.34 3736 5.480.38 __c 10.35_ 9.614.12 21.6 _ c 13.7_ c __c __ c 32.6_ c 24.5_ c 8.139.27 9.347.24 c c c c 373837 0.380.76 21.8 __c __c 4.124.12 13.7 21.68.55 13.76.32 __ c _ c 32.6 15.732.6 24.510.8 24.5 8.137.89 8.13 7.24 12.5 3838 0.76 21.8 13.7 8.55 6.32 c 15.7 10.8 10.8 7.89 7.89 12.5 39 0.13 36.9 5.36 7.13 1.36 _c 24.9 3.57 1.48 0.96 2.44 39 0.13 36.9 5.36 7.13 1.36 _ c 24.9 3.57 1.48 0.96 2.44 39 0.13c 36.9c 5.36c 7.13c 1.36c _ c 24.9c 3.57c 1.48c 0.96 2.44c 40 __c __c 10.8010.80 __c __c __c __c __ c __ c _ c 0.910.91 _ _ c c c c c c c c c c c 4041 __c c ___c c 10.805.135.13 ___c c ___c c ___ c c ___ c c __ cc __ cc __ cc 0.910.340.34 _ _ cc 41 _ c _ c 5.13 _ c _ c _ c _ c _ c _ c _ c 0.34 _ c a Froma From three three different different assays, assays, errors errors ±± 5%–10%5%–10% ofof thethe reported reported value. value.b mCA; b mCA;c Not c Not active active > 100. > 100. a From three different assays, errors ± 5%–10% of the reported value. b mCA; c Not active > 100. 2.3. Polyphenols 2.3. Polyphenols Recently rosmarinic acid (42) was reported to be a potent inhibitor of the hCA I and II isoenzymes Recently rosmarinic acid (42) was reported to be a potent inhibitor of the hCA I and II isoenzymes at low micromolar range with K II valuesvalues of of 86.0 86.0 μµMM and and 57.0 57.0 μµM, respectively (Figure (Figure 10)10)[ [55].55]. atRosmarinic low micromolar acidacid isis aa powerful rangepowerful with antioxidant antioxidant KI values found foundof 86.0 in in manyμ manyM and aromatic, aromatic, 57.0 μ medicinalM, medicinal respectively and and culinary culinary(Figure herbs 10)herbs of[55]. the of RosmarinicLamiaceaethe Lamiaceae family,acid family, is sucha powerful such as rosemary, as antioxidantrosemary, sage, sage, oreganofound oreg in and anomany thymeand aromatic, thyme [56]. Chemically[56]. medicinal Chemically and it belongs culinary it belongs to theherbs to class the of theofclass depsides, Lamiaceae of depsides, or family, the or so-called the such so-called as labiataetannins rosemary, labiataetannins sage, [41 oreg], which[41],ano which and are obtainedthyme are obta [56].ined by Chemically condensation by condensation it belongs of caffeic of caffeicto acid the classunitsacid unitsof or depsides, by or combination by combination or the so-called of caffeic of caffeiclabiataetannins acid with acid 3,4-dihydroxyphenyl with [41], 3,4-dihydroxyphenyl which are obta lacticined acid, lactic by tocondensation acid, form to dimers, form of dimers,trimers caffeic acidandtrimers tetramers.units and or tetramers. by combination of caffeic acid with 3,4-dihydroxyphenyl lactic acid, to form dimers, trimers and tetramers.

Figure 10. Structure of rosmarinic acid (42). Figure 10. Structure of rosmarinic acid (42).

Based on these results further investigations were carried out with the salvianolic acids 43–45 Based on these results further investigations were carried out with the salvianolic acids 43–45 (Figure 11)11) isolated from from SalviaSalvia miltiorrhiza miltiorrhiza (Lamiaceae)(Lamiaceae) [57,58]. [57,58 ].SalviaSalvia miltiorrhiza miltiorrhiza or orDanshen Danshen is one is one of (Figure 11) isolated from Salvia miltiorrhiza (Lamiaceae) [57,58]. Salvia miltiorrhiza or Danshen is one of ofthe the most most important important herbal herbal medicinal medicinal drugs drugs in in th thee Chinese Chinese Traditional Traditional Medicines Medicines (TCM) (TCM) to treat the most important herbal medicinal drugs in the Chinese Traditional Medicines (TCM) to treat bleeding disorders disorders (e.g., (e.g., menstrual menstrual bleeding) bleeding) and and blood blood stasis stasis [59]. [59 Salvianolic]. Salvianolic acids acids are together are together with bleeding disorders (e.g., menstrual bleeding) and blood stasis [59]. Salvianolic acids are together with withtanshinones tanshinones (lipophilic, (lipophilic, diterpenic diterpenic quinones) quinones) the main the active main activeprinciples principles of the ofherbal the herbaldrug. These drug. tanshinoneshydrophilic derivatives,(lipophilic, abundantditerpenic in quinones) the roots ofthe the main plant, active incorporate principles three of to the four herbal units ofdrug. caffeic/3,4- These hydrophilicdihydroxyphenyl derivatives, lactic acids.abundant They in ha theve rootsattracted of the interest plant, due incorporate to their protective three to four roles units in CNS of caffeic/3,4- neuronal dihydroxyphenyl lactic acids. They have attracted interest due to their protective roles in CNS neuronal Molecules 2016, 21, 1649 10 of 27

These hydrophilic derivatives, abundant in the roots of the plant, incorporate three to four units of caffeic/3,4-dihydroxyphenyl lactic acids. They have attracted interest due to their protective roles in CNSMolecules neuronal 2016, 21 injuries, 1649 and degeneration. The main mechanism involved in such processes10 of 27 is the decrease of reactive oxygen species (ROS) levels. Furthermore, there is evidence of the protective injuries and degeneration. The main mechanism involved in such processes is the decrease of reactive role ofoxygen salvianolic species (ROS) acids levels. in several Furthermore, cardiovascular there is eviden diseases,ce of the osteoporosis, protective role liver of salvianolic fibrosis andacids hepaticin failureseveral [59–61 cardiovascular]. diseases, osteoporosis, liver fibrosis and hepatic failure [59–61].

FigureFigure 11. 11.Structures Structures of of salvianolic salvianolic acids 4343––4545 fromfrom SalviaSalvia miltiorrhiza miltiorrhiza. .

The CAs inhibition studies reported in Table 5 clearly showed that depsides 43–45 were Theineffective CAs inhibition hCA I and studies weak hCA reported II inhibitors. in Table On5 clearlythe other showed hand, they that were depsides inhibitors43–45 of werethe hCA ineffective IV hCAwith I and KI weak values hCA in the II range inhibitors. of 62.2–102.1 On the nM other and hand, comparable theywere to the inhibitors standard drug of the acetazolamide hCA IV with KI valuesAAZ in (74.0 the rangenM). Even of 62.2–102.1 if a proper structure–activity-relationship nM and comparable to the (SAR) standard consideration drug acetazolamide is difficult to be AAZ (74.0drawn nM). Even it is noteworthy if a proper structure–activity-relationshipthat the flexible and more containing (SAR) catechol consideration moieties issalvianolic difficult acids to be A drawn it is noteworthyand B (43 and that 45) thewere flexible better inhibitors and more when containing compared catechol to the lithospermic moieties salvianolic acid 44. Furthermore, acids A and B (43 andsalvianolic45) were acids better A and inhibitors B were whenmedium compared and high to nanomolar the lithospermic inhibitors acid (KI 4439.8. Furthermore, and 453.6 nM salvianolicof the tumor associated CA XII. By contrast, lithospermic acid (44) showed to be a very potent hCA XII acids A and B were medium and high nanomolar inhibitors (KI 39.8 and 453.6 nM of the tumor inhibitor with a KI value of 4.8 nM. The presence of ortho-hydroxyl moieties in these molecules, the associated CA XII. By contrast, lithospermic acid (44) showed to be a very potent hCA XII inhibitor possibility to release in situ caffeic acid or even the presence of free carboxyl groups are some of the with a K value of 4.8 nM. The presence of ortho-hydroxyl moieties in these molecules, the possibility structuralI characteristics that play an important role in the CA inhibitory activity. In any case such to releaseresults in put situ in caffeicevidence acid a new or evenclass of the natural presence products of free which carboxyl deserves groups further are investigation some of the in order structural characteristicsto decipher that a proper play anSAR important correlation. role in the CA inhibitory activity. In any case such results put in evidence a new class of natural products which deserves further investigation in order to decipher a proper SARTable correlation. 5. Inhibition data of human CA isoforms hCA I, II, IV, VII and XII with salvianolic acids 43–45 reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 Tablehydrase 5. Inhibition assay [58]. data of human CA isoforms hCA I, II, IV, VII and XII with salvianolic acids 43–45 AAZ reported here and the standard sulfonamide inhibitor acetazolamideKI (nM) a ( ) by a stopped flow CO2 No. hydrase assay [58]. hCA I hCA II hCA IV hCA VII hCA XII Salvianolic Acid A (43) >10,000 9594.4 66.6 71.4 39.8 a Lithospermic Acid (44) >10,000 >10,000 101.2KI (nM) 268.3 4.8 No. Salvianolic Acid B hCA(45) I>10,000 hCA>10,000 II 65.6 hCA IV35.5 hCA VII453.6 hCA XII AAZ 250 12 74 2.5 5.7 Salvianolic Acid A (43) >10,000 9594.4 66.6 71.4 39.8 a ± Lithospermic AcidFrom (44) three >10,000different assays, >10,000errors 5%–10% of 101.2 the reported value. 268.3 4.8 Salvianolic Acid B (45) >10,000 >10,000 65.6 35.5 453.6 AnotherAAZ recent example is250 constituted 12by a series of 74 2.5 esters (Scheme 5.7 1) incorporating caffeic, ferulic, and p-coumaric acid, and benzyl, m/p-hydroxyphenethyl- as well as a From three different assays, errors ± 5%–10% of the reported value. p-hydroxy-phenethoxy-phenethyl moieties, which were investigated for their inhibitory effects against the mammalian isozymes of human (h) or murine (m) origin, hCA I–hCA XII, mCA XIII and hCA AnotherXIV [62]. These recent enzymes example were inhibited is constituted in the submic byromolar a series range of by phenolic many of these acid derivatives esters (Scheme (with 1) incorporatingKIs of 0.31–1.03 caffeic, μM against ferulic, hCA and VA,p-coumaric VB, VI, VII, acid, IX and and XIV). benzyl, The off-target,m/p-hydroxyphenethyl- highly abundant isoforms as well as p-hydroxy-phenethoxy-phenethylhCA I and II, as well as hCA III, moieties, IV and XII which were po wereorly investigated inhibited by formany their of these inhibitory esters, effectsalthough against Molecules 2016, 21, 1649 11 of 27 the mammalian isozymes of human (h) or murine (m) origin, hCA I–hCA XII, mCA XIII and hCA XIV [62]. These enzymes were inhibited in the submicromolar range by many of these derivatives (with KIs of 0.31–1.03 µM against hCA VA, VB, VI, VII, IX and XIV). The off-target, highly abundant isoforms hCAMolecules I and II, 2016 as, 21 well, 1649 as hCA III, IV and XII were poorly inhibited by many of these esters,11although of 27 the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, possesspossess a CA a inhibitionCA inhibition mechanism mechanism distinct distinct of of thethe sulfonamides/sulfamates, clinically clinically used used drugs drugs for for Molecules 2016, 21, 1649 11 of 27 the treatmentthe treatment of a of multitude a multitude of of pathologies, pathologies, but but withwith severe side side effects effects due due to tohCA hCA I/II I/IIinhibition. inhibition. the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, possess a CA inhibition mechanism distinct of the sulfonamides/sulfamates, clinically used drugs for the treatment of a multitude of pathologies, but with severe side effects due to hCA I/II inhibition.

Scheme 1. Synthesis of phenolicScheme 1. derivativesSynthesis of phenolic46–53 Reagents derivatives and 46 conditions:–53 [62]. (i) THF, Ph3P, DIAD, 0 °C [62]. In an attempt to find new chemotypes, two floroglucinol derivatives 54, 55 were prepared and tested against hCA I and hCA II (Scheme 2). Unfortunately, none of the compounds proved to be active [63]. Scheme 1. Synthesis of phenolic derivatives 46–53 [62]. In an attempt to find new chemotypes, two floroglucinol derivatives 54, 55 were prepared and tested againstIn an attempt hCA I to and find hCA new chemotypes, II (Scheme 2two). Unfortunately,floroglucinol derivatives none of 54 the, 55 were compounds prepared and proved tested to be activeagainst [63]. hCA I and hCA II (Scheme 2). Unfortunately, none of the compounds proved to be active [63].

Scheme 2. Synthesis of floroglucinol derivatives 54 and 55. Reagents and conditions: NaBH4, EtOH,

0 °C, 30 min, 98%; (ii) PBr3, diethylether, 0 °C, 1 h, 85%; (iii) NaH, THF, r.t., 17 h, 26%.

2.4. Flavonoids Scheme 2. Synthesis of floroglucinol derivatives 54 and 55. Reagents and conditions: NaBH4, EtOH, Scheme 2. Synthesis of floroglucinol derivatives 54 and 55. Reagents and conditions: NaBH4, EtOH, ◦ 0Data °C, 30of min,the interaction 98%; (ii) PBr of3, flavonoidsdiethylether, with 0◦ °C, CAs 1 h, indicate85%; (iii) thatNaH, these THF, may r.t., 17be h, another 26%. category of CAIs 0 C, 30 min, 98%; (ii) PBr3, diethylether, 0 C, 1 h, 85%; (iii) NaH, THF, r.t., 17 h, 26%. of interest, which can be used as leads for generating more potent CAIs. Preliminary test of a small 2.4.number Flavonoids of basic flavonoid structures 56–59 (Figure 12) against hCA I, II, IX and XII showed that these 2.4. Flavonoids act asData low ofmicromolar the interaction inhibitors of flavonoids and their with effect CAs is indicate independent that these of the may incubation be another time category [64]. This of CAIs fact Dataofindicates interest, of thethat which interaction flavonoids can be usedinhibit of flavonoids as CAsleads probably for with generati CAsthroughng indicate more a mechanism potent that CAIs. these similar Preliminary may to be the another onetest of a categoryphenol small of CAIsnumberrather of interest, than of basic hydrolysis which flavonoid can of the be structures usedchromenone as 56 leads–59 as (Figure forin the generating 12)case against of coumarins. more hCA I, potent II, IX and CAIs. XII showed Preliminary that these test of a smallact number as low micromolar of basic flavonoid inhibitors structures and their effect56–59 is independent(Figure 12) againstof the incubation hCA I, II,time IX [64]. and This XII fact showed that theseindicates act asthat low flavonoids micromolar inhibit inhibitors CAs probably and their through effect a mechanism is independent similar of to the the incubation one of phenol time [64]. This factrather indicates than hydrolysis that flavonoids of the chromenone inhibit CAs as in probablythe case of throughcoumarins. a mechanism similar to the one of phenol rather than hydrolysis of the chromenone as in the case of coumarins. Molecules 2016, 21, 1649 12 of 27 Molecules 2016, 21, 1649 12 of 27 Molecules 2016, 21, 1649 12 of 27 Molecules 2016, 21, 1649 12 of 27

Figure 12. Structures of compounds 56–59. Figure 12. Structures of compounds 56–59. Figure 12. Structures of compounds 56–59. This hypothesis was further confirmed in several studies including flavonoids lacking the C-4 This hypothesis was further confirmedconfirmed in several studies including flavonoidsflavonoids lacking the C-4 carbonylThis group.hypothesis A series was offurther flavonoids confirmed 60–64 in with several different studies substitution including patternsflavonoids were lacking investigated the C-4 carbonyl group.group. AA seriesseries ofof flavonoids flavonoids60 60–64–64with with different different substitution substitution patterns patterns were were investigated investigated for forcarbonyl their inhibitory group. A effects series againstof flavonoids four α-CA 60– 64isozymes with different from human substitution and bovine patterns (hCA were I, hCA investigated II, bCA III, theirfor their inhibitory inhibitory effects effects against against four fourα-CA α-CA isozymes isozymes from from human human and and bovine bovine (hCA(hCA I,I, hCAhCA II, bCA III, hCAfor their IV) inhibitorytissues: the effects against four α(60-CA) and isozymes luteolin from (61), humanthe and bo quercetinvine (hCA (62 I, )hCA and II,morin bCA ( 63III,) hCA IV) tissues: tissues: the the flavones flavones apigenin apigenin (60 (60) and) and luteolin luteolin (61 (),61 the), the flavonols flavonols quercetin (62) (and62) andmorin morin (63) andhCA the IV) -3-ol, tissues: the catechinflavones (apigenin64) (Figure (60 13)) and [65]. luteolin As it can (61 ),be the observed flavonols from quercetin Table 6,(62 flavonoids) and morin 60 (–6364) (and63) andthe flavan-3-ol, the flavan-3-ol, catechin (64) (Figure (64) (Figure 13) [65]. 13)[ As65 it]. can As itbe can observed be observed from Table from 6, Table flavonoids6, flavonoids 60–64 wereand the quite flavan-3-ol, effective, catechin similar ( 64to ) the(Figure standards 13) [65]. us Ased. it The can fourbe observed hCA isozymes from Table showed 6, flavonoids quite diverse 60–64 60were–64 quitewere quiteeffective, effective, similar similar to the to thestandards standards used. used. The The four four hCA hCA isozymes isozymes showed showed quite quite diverse inhibitionwere quite profiles effective, with similar these tocompounds the standards suggesting used. The that four the hCAtype ofisozymes flavonoid showed and/or quite substitution diverse inhibition profilesprofiles withwith thesethese compoundscompounds suggestingsuggesting thatthat thethe typetype ofof flavonoid flavonoid and/or and/or substitutionsubstitution patterninhibition are profiles important with for these the compoundsactivity. As suggestingbriefly outlined that theabove, type the of C-4flavonoid carbonyl and/or group substitution is not so pattern are important for the activity. As briefly briefly outlined above, the the C-4 carbonyl group is not so importantpattern are for important the inhibition, for the as activity.shown byAs the briefly example outlined of catechin above, ( 64the). ThisC-4 carbonylis another group indication is not that so important for the inhibition, as shown by thethe exampleexample ofof catechincatechin ((64).). This is another indication that theimportant inhibition for mechanismthe inhibition, does as notshown proceed by the via example hydrolysis of catechin(CA esterase (64). activity).This is another Instead, indication the presence that the inhibition mechanism does not proceed via hydrolysis (CA esterase activity). Instead, Instead, the the presence presence ofthe one inhibition or two mechanismneighboring does hydroxyl not proceed groups via is essential,hydrolysis as (CA shown esterase by the activity). example Instead, of morin the 67presence which of one or two neighboring hydroxyl groups is essential, as shown by the example of morin 67 which isof theone less or two potent neighboring compound hydroxyl compared groups to quercetin is essential, 62 or as luteolin shown 61by. theThe example latter, 61 of showed morin 67the which most is the less potent compound compared to quercetin 62 or luteolin 61. The latter, 61 showed the most effectiveis the less inhibitory potent compound activity with compared KIs in the to quercetinlow micromolar 62 or luteolin range. 61 . The latter, 61 showed the most effective inhibitory activity with KIIs in the low micromolar range. effective inhibitory activity with KIs in the low micromolar range.

Figure 13. Structures of compounds 60–64. Figure 13. Structures of compounds 60–64. Figure 13. Structures of compounds 60–64. Comparison with the relative monophenols, catechol, phenol and resorcinol (Figure 14), which Comparison with the relative monophenols, catechol, phenol and resorcinol (Figure 14), which wereComparison considerably with less theactive, relative suggests monophenols, that also catechol,catethe chol,ring Aphenol of flavonoids and resorcinol may be (Figure involved 1414),), whichin the were considerably less active, suggests that also the ring A of flavonoids may be involved in the wereinhibitory considerably mechanism. less Whether active, suggests this is due that to also a di the fferent ring mechanism A of flavonoidsflavonoids or simply may stronger be involved binding in the to inhibitory mechanism. Whether this is due to a different mechanism or simply stronger binding to theinhibitory enzyme mechanism.mechanism. cavity (e.g. Whether Whetherby further this this hydrogen is is due due to tobo a differentands) different is to mechanism be mechanism investigated or or simply insimply the stronger future. stronger binding binding to the to the enzyme cavity (e.g. by further hydrogen bonds) is to be investigated in the future. enzymethe enzyme cavity cavity (e.g., (e.g. by furtherby further hydrogen hydrogen bonds) bonds) is to is be to investigatedbe investigated in the in the future. future.

Figure 14. Structures of basic phenol rings: phenol, catechol and resorcinol. Figure 14. Structures of basic phenol rings: phenol, catechol and resorcinol. Figure 14. Structures of basic phenol rings: phenol, catechol and resorcinol.

Molecules 2016, 21, 1649 13 of 27

Table 6. Inhibition of hCA isozymes I, II and IV and bCA III with compounds 60–64, EZA, ZNA and TableAZA by 6. Inhibitionthe esterase of activity. hCA isozymes I, II and IV and bCA III with compounds 60–64, EZA, ZNA and AZA by the esterase activity. IC50 Value (µM) a No. hCA I hCAIC50 II Value bCA (µ IIM) a hCA IV No. Apigenin (hCA60) I4.1 hCA2.7 II11.6 bCA II 9.1 hCA IV Luteolin (61) 2.2 0.74 5.4 4.4 Apigenin (60) 4.1 2.7 11.6 9.1 Luteolin (61Quercetin) (62 2.2) 3.6 0.742.4 9.1 5.4 13.8 4.4 Quercetin (62)Morin (63) 3.612.8 2.44.4 21.3 9.1 15.7 13.8 Morin (63)Catechin (64 12.8) 6.8 6.2 4.4 b 2.2 21.3 5.6 15.7 b Catechin (64)Phenol 6.8 17.3 6.27.4 4.6 2.214.3 5.6 PhenolCatechol 17.34014 11.4 7.4 6.5 4.6 11.9 14.3 Catechol 4014 11.4 6.5 11.9 ResorcinolResorcinol 828 828 9.4 219 219 634 634 EZA EZA 3.73.7 0.32 9.4 9.4 0.84 0.84 ZNA ZNA 14.814.8 1.1 8.5 8.5 38.4 38.4 AZA AZA 36.236.2 0.37 263 263 0.58 0.58 a b a FromFrom three three different different assays, assays, errors errors ±± 5%–10%5%–10% ofof the the reported reported value; value.Values b Values from from Ref. [Ref.66]. [66].

Two furtherfurther studies studies [46 [46,67],67] with with several several flavonoids flavonoids60, 6260,, 6462–,71 64(Figure–71 (Figure 15, Tables 15, Tables7 and8 )7 against and 8) humanagainst human CA I, II, CA IV, I, VI II, andIV, VI bovine and bovine CA III CA isoforms III isoforms gave gave further further evidence evidence that that the catecholthe catechol moiety moiety on C-2on C-2 enhances enhances the the activity, activity, as as demonstrated demonstrated in in the the case case of of fisetin fisetin ((6666),), rhamnetinrhamnetin (67) and rutin rutin ( (6969).). Although a comparison between resu resultslts of Tables 7 7–9–9 is is notnot feasiblefeasible duedue toto thethe differentdifferent enzymaticenzymatic assays used and to different enzyme purities,purities, it can be observed that catechin (64) andand epicatechinepicatechin (70) have similar activities despite different stereoch stereochemistryemistry on C-3. Similarly, the hydroxyl group of C-3 seems to be of little importance for the activity. This is evident if we compare luteolin (61) with quercetin (62) (Table6 6)) and and apigenin apigenin ( (6060)) withwith kaempferolkaempferol ((6565)) (Table(Table7 ).7). Therefore,Therefore, thethe keto-enolketo-enol system apparently does not take part in thethe inhibitioninhibition mechanism.mechanism. The better inhibitory activity of fisetinfisetin ( 66) and rhamnetin ( 67) should be attributed to to structural structural differences differences in in ring ring A. A. Interestingly, Interestingly, rutin (69) compared to flavonolflavonol aglycones such as quercetin ( 62) or rhamnetin ( 67) is more active suggesting that sugars may play an important role,role, asas discusseddiscussed previously.previously.

Figure 15. Structures of compounds 65–71 [[46,67].46,67]. Molecules 2016, 21, 1649 14 of 27

Table 7. hCA isoforms I and II inhibition with compounds 60, 64–70 by a stopped-flow, CO2 hydration assay [46].

a IC50 Value (µg/mL) No. hCA I hCA II Apigenin (60) 1.22 1.14 Catechin (64) 1.24 0.87 (65) 0.83 0.76 Fisetin (66) 0.72 0.55 Rhamnetin (67) 0.95 0.86 Isorhamnetin (68) 0.52 0.36 Rutin (69) 1.44 0.52 Epicatechin (70) 1.47 1.02 a From three different assays, errors ±5%–10% of the reported value.

Table 8. KI values (µM) for compounds 60, 62, 64, 69–71 and AZA of some human (h) and bovine (b) α-carbonic anhydrase isoforms (by an esterase assay) [67].

a KI (nM) No. hCA I hCA II bCA III hCA IV hCA VI Apigenin (60) 0.97 0.113 2.21 1.12 1.49 Quercetin (62) 2.68 b 2.54 b 3.73 7.89 b 6.17 b Catechin (64) 1.73 0.83 4.77 3.76 7.82 Rutin (69) 2.42 b 1.84 b 9.71 4.09 b 4.91 b Epicatechin (70) 2.32 1.24 8.93 3.98 4.36 Silymarin (71) 1.49 c 2.51 c 5.68 8.96 c 9.70 c AZA 36.2 0.37 263 0.578 0.34 a From three different assays, errors ±5%–10% of the reported value; b Values from Ref. [68]; c Values form Ref. [66].

Table 9. Inhibition constant KI values (µM) of human carbonic anhydrase I, II, VA, IX, and XII isoforms for compounds 60, 62, 65, 68, 72–77 and acetazolamide (AZA).

K (nM) a No. I hCA I hCA II hCA VA hCA IX hCA XII Apigenin (60) 4.10 2.70 b 0.30 0.46 1.00 Quercetin (62) 2.95 0.41 2.82 2.52 0.38 Kaempferol (65) 3.13 0.31 0.15 3.57 2.18 Isorhamnetin (67) >10 0.42 1.92 0.44 0.29 Diosmetin (72) >10 >10 2.10 3.39 0.24 Eriocitrin (73) 3.66 0.34 4.24 0.43 0.19 (74) 3.60 b 2.40 b 2.65 2.22 0.45 Naringin (75) >10 0.41 4.08 3.59 0.17 (76) 2.53 0.26 3.93 0.45 0.11 Quercetin-3-O-glucoside (77) >10 0.44 >10 2.79 0.22 AZA 0.10 0.008 0.38 0.041 0.038 a b From three different assays, errors ±5%–10% of the reported value, CO2 hydrase, stopped-flow assay; Values from Ref. [65].

A possible explanation of the way that flavonoids interact with the CA active site was given in a very interesting docking study by Gidaro et al., [69]. In this work a structure-based virtual screening was performed against five carbonic anhydrase isoforms using, as a ligand library, natural components of Citrus bergamia (Bergamot) and Allium cepa var. tropea (red onion). Both plants are edible and part of the Calabrian (Italy) diet. By use of virtual screening, the flavone apigenin 60 and the flavanone glycoside eriocitrin (eriodictyol-7-O-rutinoside) 73 (Figure 16) were identified together with other eight Molecules 2016, 21, 1649 15 of 27 Molecules 2016, 21, 1649 15 of 27 Molecules 2016, 21, 1649 15 of 27 of the Calabrian (Italy) diet. By use of virtual screening, the flavone apigenin 60 and the of the Calabrian (Italy) diet. By use of virtual screening, the flavone apigenin 60 and the flavanone glycoside eriocitrin (eriodictyol-7-O-rutinoside) 73 (Figure 16) were identified together with other eight glycoside eriocitrin (eriodictyol-7-O-rutinoside) 73 (Figure 16) were identified together with other eight flavonoids.flavonoids. In vitro tests against CA CA I, II, VA, IX,IX, andand XII isoforms showed that eriocitrin was the best flavonoids. In vitro tests against CA I, II, VA, IX, and XII isoforms showed that eriocitrin was the best hCA VA inhibitorinhibitor withwith aa KKII value of 0.15 μµM. On the contrary, the computational prediction failed for hCA VA inhibitor with a KI value of 0.15 μM. On the contrary, the computational prediction failed for apigenin. Docking studies revealed th thatat eriocitrin probably binds by both the catechol and rutinoside apigenin. Docking studies revealed that eriocitrin probably binds by both the catechol and rutinoside portions. The catechol moiety is linked by additionaladditional H-bond interactions with the enzymatic residue portions. The catechol moiety is linked by additional H-bond interactions with the enzymatic residue Thr 199 located in the catalytic site, while the rutino rutinosese group connects by an extended H-bond network Thr 199 located in the catalytic site, while the rutinose group connects by an extended H-bond network with several external mCA VA aminoamino acidacid residuesresidues (Gln(Gln 67,67, GluGlu 69,69, Gln 92, Asp 171) at the entrance of with several external mCA VA amino acid residues (Gln 67, Glu 69, Gln 92, Asp 171) at the entrance of the binding pocket. Apigenin instead, which was not as active as predicted, seems to interact only by the binding pocket. Apigenin instead, which was not as active as predicted, seems to interact only by its ring B which isis involvedinvolved inin thethe zinczinc coordinationcoordination intointo thethe mCAmCA VAVA activeactive site.site. its ring B which is involved in the zinc coordination into the mCA VA active site.

Figure 16. Structures of compounds 72–77 [[69].69]. Figure 16. Structures of compounds 72–77 [69]. The lipophilic furanoflavonoid 78 from the bark of Millettia ovalifolia (Fabaceae) was considered The lipophilic furanoflavonoidfuranoflavonoid 78 from the bark of Millettia ovalifolia (Fabaceae) was considered for inhibition of the cytosolic form of the bovine carbonic anhydrase-II (Figure 17) [70]. Despite the for inhibition of the cytosoliccytosolic form of thethe bovinebovine carboniccarbonic anhydrase-IIanhydrase-II (Figure 1717))[ [70].70]. Despite the lack of free hydroxyl groups in its structure it displayed significant inhibitory activity with IC50 value lack of free hydroxyl groups inin itsits structurestructure itit displayeddisplayed significantsignificant inhibitoryinhibitory activityactivity withwith ICIC50 value of 17.86 ± 0.07 μM (vs. zonisamide, 1.86 ± 0.03). 50 of 17.86 ± 0.070.07 μµMM (vs. (vs. zonisamide, zonisamide, 1.86 1.86 ±± 0.03).0.03).

Figure 17. Structure of furanoflavonoid 78. Figure 17. Structure of furanoflavonoidfuranoflavonoid 78. Molecules 2016, 21, 1649 16 of 27

The above reported results spurred us to investigate a larger number of flavonoids against a line of human CAs. In total, 18 flavonoids (Figure 18) were selected both from commercial sources and Molecules 2016, 21, 1649 16 of 27 lab isolates [71]. The selection of the compounds was done on the basis of the flavonoid subgroup and the substitutionThe above reported pattern. results Flavones, spurred flavonols,us to investigate flavanones, a larger number isoflavones of flavonoids and some against glycosylated a line derivativesof human were CAs. considered In total, 18 for flavonoids assays. Chrysin (Figure (18)79 )were is one selected of the mainboth from flavonoids commercial of Passiflora sources incarnataand , a medicinallab isolates plant [71]. used Thephytotherapy selection of the ascompounds a mild sedative was done and on anxiolyticthe basis of [ 72the]. flavonoid It is a flavone subgroup without and any substitutionthe substitution of ring pattern. B. The Flavones, flavone luteolinflavonols, ( 61flavan), theones, two flavonols and kaempferol some glycosylated (65) and derivatives isorhamnetin (68), aswere well considered as the flavonol for assays. glycosides Chrysin ( quercetin-3-79) is one of theO-glucoside main flavonoids (77), quercetin-3-of Passiflora incarnataO-rhamnoside, a medicinal (87) and kaempferol-3-plant usedO phytotherapy-glucoside (88 as) a are mild ubipresent sedative and flavonoids anxiolytic in [72]. most It is vegetables a flavone without and fruits any dailysubstitution consumed, of ring B. The flavone luteolin (61), the two flavonols kaempferol (65) and isorhamnetin (68), as well as while galanginMolecules (80 2016) is, 21 present, 1649 in honey and propolis [73,74]. Among the flavonoids16 considered of 27 were the flavonol glycosides quercetin-3-O-glucoside (77), quercetin-3-O-rhamnoside (87) and kaempferol- the flavanones (81), eriodictyol (82) and hesperitin (83), which are abundant in edible 3-O-glucosideThe ( 88above) are reported ubipresent results spurredflavonoids us to ininvestigate most vegetables a larger number and of fruits flavonoids daily against consumed, a line while of human CAs. In total, 18 flavonoids (Figure 18) were selected both from commercial sources and Citrusgalanginspp. (e.g., (80) is orange, presentmandarin) in honey and and propolis possess [73,74]. antioxidant, Among the anti-inflammatory flavonoids considered and were antitumoral the properties [75lab]. isolates The [71]. isoflavones The selection84 of– the85 compoundsare abundant was done in on legumes, the basis of the soy flavonoid and show subgroup potent and estrogenic flavanonesthe nsubstitutionaringenin pattern. (81), eriodictyol Flavones, flavonols, (82) and flavan hesperitinones, isoflavones (83), which and some are abundant glycosylated in derivatives edible Citrus spp. activity(e.g. by orange, interactingwere consideredmandarin) with for and assays. the possess estrogenic Chrysin antioxidant, (79) receptorsis one of the an mainti-inflammatory [76, flavonoids77]. Puerarin of Passiflora and (86 antitumoral )incarnata is the, maina medicinal properties active principle[75]. of PuerariaThe isoflavones lobataplant usedroot 84 phytotherapy–85 (Fabaceae) are abundant as a mild and in sedative legumes, it used and insoyanxiolytic clinical and show [72]. practice It po is atent flavone estrogenic in without China anyactivity to substitution treat by cardiovascularinteracting diseases,with cerebrovasculartheof estrogenic ring B. The flavonereceptors disorders, luteolin [76,77]. (61 Parkinson’s), thePuerarin two flavonols (86 disease,) is kaempferol the main Alzheimer’s (65 active) and isorhamnetin principle disease, of (68 Pueraria diabetes), as well aslobata and root diabetic the flavonol glycosides quercetin-3-O-glucoside (77), quercetin-3-O-rhamnoside (87) and kaempferol- (Fabaceae) and it used in clinical practice in China to treat cardiovascular diseases, cerebrovascular complications3-O [78-glucoside]. The flavonols(88) are ubipresent89–91 flavonoidswere isolated in most from vegetables leaves and of fruitsQuercus daily consumed, ilex (Fagaceae) while and belong to thedisorders, groupgalangin of Parkinson’s acylated (80) is present flavonoidsdisease, in honey Alzheimer’s (Figure and propolis 19diseas). [73,74]. Sincee, diabetes Among they the combineand flavonoids diabetic the considered complications flavonol were nucleus the [78]. The and the flavonols 89–91 were isolated from leaves of Quercus ilex (Fagaceae) and belong to the group of acylated coumaric acidflavanones moieties, naringenin they were(81), eriodictyol selected (82 with) and hesperitin the aim (83 to), which see whether are abundant we inwould edible Citrus have spp. an additional flavonoids(e.g. (Figure orange, 19).mandarin) Since andthey possess combine antioxidant, the flavon anti-inflammatoryol nucleus and and the antitumoral coumaric properties acid moieties, [75]. they inhibitory effect on the CAs. For the in vitro inhibitory activities five physiological relevant hCA were selectedThe isoflavones with the 84 aim–85 are to abundantsee whether in legumes, we would soy and have show an po additionaltent estrogenic inhibitory activity by effectinteracting on the CAs. with the estrogenic receptors [76,77]. Puerarin (86) is the main active principle of Pueraria lobata root isoformsFor the were in considered:vitro inhibitory the activities cytosolic five I, II,physiological VII and the relevant membrane hCA boundisoforms CA were IV andconsidered: XII. Results the are displayed in(Fabaceae) Table 10 and. it used in clinical practice in China to treat cardiovascular diseases, cerebrovascular cytosolicdisorders, I, II, VII Parkinson’sand the membrane disease, Alzheimer’s bound CA diseas IV e,and diabetes XII. Results and diabetic are displayedcomplications in Table[78]. The 10. flavonols 89–91 were isolated from leaves of Quercus ilex (Fagaceae) and belong to the group of acylated flavonoids (Figure 19). Since they combine the flavonol nucleus and the coumaric acid moieties, they were selected with the aim to see whether we would have an additional inhibitory effect on the CAs. For the in vitro inhibitory activities five physiological relevant hCA isoforms were considered: the cytosolic I, II, VII and the membrane bound CA IV and XII. Results are displayed in Table 10.

Figure 18. Structures of flavone 79, flavonol 80, flavanones 81–83, isoflavones 84, 85, glucoside Figure 18. Structures of flavone 79, flavonol 80, flavanones 81–83, isoflavones 84, 85, isoflavone 86 and flavonol glycosides 87–88. glucoside 86Figureand flavonol18. Structures glycosides of flavone 7987, flavonol–88. 80, flavanones 81–83, isoflavones 84, 85, isoflavone glucoside 86 and flavonol glycosides 87–88.

Figure 19. Structures of tested acylated flavonol glycosides 89–91 (isolated from Quercus ilex) [71]. FigureFigure 19. Structures 19. Structures of testedof tested acylated acylated flavonol flavonol glycosides 8989–91–91 (isolated(isolated from from QuercusQuercus ilex) ilex[71].)[ 71]. Molecules 2016, 21, 1649 17 of 27

Table 10. Inhibition data of human CA isoforms hCA I, II, IV, VII and XII with flavonoids 59, 63, 66, 74, 75, 76–88 reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped

flow CO2 hydrase assay.

a KI (nM) Entry Name hCA I hCA II hCA IV hCA VII hCA XII 79 Chrysin >10,000 >10,000 537.75 171.41 34.7 61 Luteolin >10,000 3691.6 293.0 b 4.7 60.3 80 Galangin >10,000 >10,000 568.3 24.5 41.8 65 Kaempferol >10,000 9525.7 >10,000 25 145.9 67 Isorhamnetin >10,000 >10,000 212.9 4.4 54.9 81 Naringenin >10,000 >10,000 79.5 4.3 44.3 82 Eriodictyol >10,000 >10,000 72.8 4.3 31.1 83 Hesperitin >10,000 >10,000 102.1 3.3 454.1 76 Taxifolin >10,000 >10,000 9098.2 493.1 32.5 84 >10,000 >10,000 718.7 4.2 56.4 85 Biochanin A >10,000 >10000 7078.5 371.5 52.5 86 Puerarin >10,000 >10,000 >10,000 452.6 515.0 77 Quercetin-3-O-glucoside >10,000 6211.2 75.7 3.8 52.1 87 Quercetin-3-O-rhamnoside >10,000 6367.7 67.3 3.9 43.5 88 Kaempferol-3-O-glucoside >10,000 168.0 5591.2 423.7 4.9 89 Tiliroside >10,000 >10,000 5468.7 4.6 134.3 Kaempferol-3-O-(2”,6”-di-E- 90 >10,000 8790.2 344.2 4.7 408.9 p-coumaroyl)-β-glucopyranoside Kaempferol-3-O-(3”,4”-diacetyl-2”,6”-di-E- 91 >10,000 6863.3 62.2 26.6 399.7 p-coumaroyl)-β-glucopyranoside AAZ 250 12 74 2.5 5.7 a Mean from three different assays, errors were in the range of ±5%–10% of the reported values; b From Ref. [65]. Although a SAR is difficult, some general remarks can be made. As can be observed from Table 10, none of the compounds considered here show a significant inhibitory activity against the cytosolic hCA I, but rather showed selectivity against isoforms hCA IV and hCAV II. Concerning the inhibition of hCA II, the presence of ortho-hydroxyl groups on ring B seems to be important for the activity as long as the double bond in ring C remains intact. This is shown comparing luteolin with eriodictyol and taxifolin. Instead, glycosylation of the C-3 hydroxyl enhances the activity, as demonstrated by Kaempferol-3-O-glucoside which was the most active of all tested compounds with a KI of 168 nM. Results were quite surprising regarding the inhibition hCA IV. The flavanones naringenin, eriodictyol and hesperitin, lacking the C2-3 double bond as well as the glycosides 77, 87, 91 were quite effective against the with KI values close to the standard CAI acetazolamide (74.0 nM). Especially comparing the activity of compounds 65, 89–91 it could be suggested that the optimum activity of compound 91 is not due to the flavonoid scaffold but to the acetyl/coumaric functionalities which may hydrolyze and released in situ. This hypothesis requires further investigation. The kinetic data on the cytosolic hCA VII showed that several of the investigated compounds were low nanomolar inhibitors of the hCA VII with KI very close to the standard acetazolamide. Among the investigated constituents are the flavone luteolin (with ortho-hydroxyl groups) the flavonol isorhamnetin, the flavanones naringenin, eriodictyol and hesperitin, the isoflavone daidzein, the quercetin flavonols 77 and 87 and the acylated glycosides 89 and 90. With the exception of catechin, this is the first time that flavonoids are tested against this particular hCA isoform [68]. The heterogeneity of constituents does not permit any SAR conclusion or it is possible that some other common structural feature may be responsible for the inhibition, such as ring A or combination of rings A and B. Similarly, the inhibition data on the tumor associated hCA XII isoform (Table 10) showed a complex and different SAR. Generally most of the substances were high nanomolar inhibitors with KI values spanning between 515.0 and 134.3 nM. The best inhibitor evidenced was Kaempferol-3-O-glucoside with a KI value of 4.9 nM. The results in both hCAV II and XII show that flavonoids merit further attention and warrant future investigation to comprehend the mechanism of action in order to design new, selective inhibitors. Molecules 2016, 21, 1649 18 of 27

A series of synthetic bischalcones 92–103 bearing a C–N=N–C linkage has been prepared and testedMolecules against 2016 human, 21, 1649hCA I and II (Figure 20)[79]. Most of the compounds had one to three18 methoxyof 27 groups as substituents on their phenolic rings. Compounds 93, 97, 101, and 103 were among the best groups as substituents on their phenolic rings. Compounds 93, 97, 101, and 103 were among the best inhibitors (Table 11). Furthermore, similarly to AZA, some of the investigated bischalcone compounds inhibitors (Table 11). Furthermore, similarly to AZA, some of the investigated bischalcone compounds act as competitive inhibitors with 4-NPA as substrate, that is, they bind to the same regions of the act as competitive inhibitors with 4-NPA as substrate, that is, they bind to the same regions of the active activesite site cavity cavity as the as substrate. the substrate. However, However, the binding the si bindingte of 4-NPA site itself of 4-NPA is unknown, itself isbut unknown, it is presumed but it is presumedto be in to the be same in the region same as region that of as CO that2, the of physiological CO2, the physiological substrate of this substrate enzyme. of this enzyme.

Figure 20. Structures of compounds 92–103. Figure 20. Structures of compounds 92–103.

Table 11. KI values of the compounds 92–103 (CA esterase activity). Table 11. KI values of the compounds 92–103 (CA esterase activity). KI (nM) a Compound hCA I hCA II a KI (nM) Compound 92 274.19 645.39 93 hCA112.34 I 154.45 hCA II 92 94 274.19325.12 418.06 645.39 93 95 112.34165.35 227.56 154.45 94 96 325.12308.88 307.52 418.06 95 97 165.35111.22 193.19 227.56 96 98 308.88177.91 446.62 307.52 97 99 111.22188.56 208.53 193.19 98 100 177.91323.30 182.31 446.62 99 101 188.5672.90 166.54 208.53 100 102 323.30179.11 268.87 182.31 101 103 72.90103.55 319.13 166.54 102 AZA 179.11250.00 12.00 268.87 a Mean from at least103 three determinations.103.55 Errors in the range of ~3% 319.13 of the reported value. AZA 250.00 12.00 2.5. Phenols and Polyphenols as Effective Mycobacterial CA Inhibitors a Mean from at least three determinations. Errors in the range of ~3% of the reported value. The discovery of the α-, β-, and η-families of carbonic anhydrase in parasitic bacteria, fungi 2.5. Phenolsprotozoa and and Polyphenols helminths aswith Effective different Mycobacterial structure than CA human Inhibitors CAs has created new opportunities in the search for new drugs against pathogens [29]. Since many pathogen carbonic anhydrases belong Theto different discovery enzyme of thefamilies,α-, β which-, and areη-families either absent of carbonicin humans anhydrase (like β- and in η-CAs) parasitic or have bacteria, marked fungi protozoastructural and helminthsdifferences with the different human structureisoforms they than consist human attractive CAs has targets created for the new development opportunities of in the searchnew anti-infective for new drugs agents against in terms pathogens of selectivity [29]. and Since efficacy. many Given pathogen the continuous carbonic development anhydrases belongof to differentresistance enzyme of several families, pathogens which to the are existing either absent therapeutic in humans schemes, (like CAβ inhibi- andtionη-CAs) represents or have a new, marked structuralcomplementary differences mechanism with the of human action worth isoforms of investigation. they consist Among attractive the most targets investigated for the CAs, development are the β of new-CAs anti-infective from Helicobacter agents pylori in terms, Brucella of selectivitysuis and Mycobacterium and efficacy. tuberculosis, Given the which continuous have been development cloned β of resistanceand characterized of several [80,81]. pathogens Other to-CAs the which existing have therapeutic been intensely schemes, studied CA are inhibition the CAs from represents the a fungal strains Candida albicans, Candida glabrata and Cryptococcus neoformans: CaNce103 and CgNce103 new, complementary mechanism of action worth of investigation. Among the most investigated CAs, (from C. albida and C. glabrata, respectively) and Can1, Can2 from C. neoformans. In these pathogens CAs are the β-CAs from Helicobacter pylori, Brucella suis and Mycobacterium tuberculosis, which have been regulate the physiological concentrations of CO2/HCO3. Both fungal strains are adapted to extremely β clonedlow and concentrations characterized of [ 80CO,812. ].However, Other -CAsonce whichthe infection have been occurs, intensely elevated studied concentrations are the CAs of CO from2 the fungal strains Candida albicans, Candida glabrata and Cryptococcus neoformans: CaNce103 and CgNce103 (from C. albida and C. glabrata, respectively) and Can1, Can2 from C. neoformans. In these pathogens Molecules 2016, 21, 1649 19 of 27

CAs regulate the physiological concentrations of CO2/HCO3. Both fungal strains are adapted to extremely low concentrations of CO2. However, once the infection occurs, elevated concentrations of CO2 promoteMolecules 2016 distinct, 21, 1649 structural and developmental procedures which influence the survival,19 virulence of 27 and pathogenesis of these fungi [82]. promote distinct structural and developmental procedures which influence the survival, virulence The first evidence that phenols can inhibit pathogenic CAs came from the screening of a natural and pathogenesis of these fungi [82]. product library [83] against β-CAs from M. tuberculosis, C. albicans, and C. neoformans. Screening of The first evidence that phenols can inhibit pathogenic CAs came from the screening of a natural librariesproduct ofMolecules plant library 2016 extracts , [83]21, 1649 against and β isolated-CAs from constituents, M. tuberculosis although, C. albicans time, and consuming, C. neoformans is. Screening a19 valuableof 27 of tool in the exploration of new scaffolds in order to discover new chemotypes to combat diseases. librariespromote of plant distinct extracts structural and andisol ateddevelopmental constituents, procedures although which time influence consuming, the survival, is a valuable virulence tool in The Queenslandthe explorationand pathogenesis Compound of new of these scaffolds Library fungi [82].in reported order to by di Davisscover etnew al., chemotypes [83] comprised to combat a wide diseases. range of The natural and syntheticQueensland productsThe Compound first evidence incorporating Library that phenols reported the can phenol inhibit by Davis pathogenic group. et al., Overall [83] CAs comprised came 21 from constituents thea wide screening range104 of– of 124a naturalnaturalwere and selected and testedsyntheticproduct (Figures products library 21 incorporatingand[83] against 22) including β-CAs the from ph eightenol M. tuberculosisgroup. fungal Overall (compounds, C. albicans 21 constituents, and104 C. neoformans–109a 104, 112–124. Screening, 113 were), two selectedof ascidian (compoundsand testedlibraries107 (Figures ,of108 plant), 21 extracts and and three 22) and in isol plantcludingated (compoundsconstituents, eight fungal although (compounds111–113 time) co secondary 104nsuming,–109a , is112 metabolites.a valuable, 113), two tool ascidianin The tested the exploration of new scaffolds in order to discover new chemotypes to combat diseases. The structures(compounds were 107 diverse, 108), and comprising three plant (compounds a series of 111 simple–113) secondary mono- or metabolites. disubstituted The tested phenols structures104 –108, were Queenslanddiverse comprising Compound a seriesLibrary of reported simple bymono- Davis or et disubstitutedal., [83] comprised phenols a wide 104 range–108 of, natural(−)-xylariamide and (−)-xylariamidesynthetic A products (109a) andincorporating its synthetic the ph enantiomerenol group. Overall (+)-xylariamide 21 constituents A (104109b–124), were polyandrocarpamine selected A (109a) and its synthetic enantiomer (+)-xylariamide A (109b), polyandrocarpamine A (110), A(110), polyandrocarpamineand tested (Figures 21 and B (22)111 in),cluding xanthones eight fungal112 and (compounds113, endiandrin 104–109a, 112 A, (113114), ),two endiandrin ascidian B (115) polyandrocarpamine B (111), xanthones 112 and 113, endiandrin A (114), endiandrin B (115) and and (−)-dihydroguaiaretic(compounds 107, 108), acid and three116. plant The (compounds synthetic phenolic111–113) secondary compounds metabolites.117– The124 tested(Figure structures 22) were eight (−)-dihydroguaiareticwere diverse comprising acid 116 a series. The of synthetic simple mono- phenolic or disubstituted compounds phenols 117– 104124– 108(Figure, (−)-xylariamide 22) were eight secondarysecondaryA amides (109a amides) and inspired inspiredits synthetic by by the theenantiomer fungal fungal metabolite metabolite (+)-xylariamide 108.. A (109b), polyandrocarpamine A (110), polyandrocarpamine B (111), xanthones 112 and 113, endiandrin A (114), endiandrin B (115) and HO (−)-dihydroguaiaretic acid 116HO . The synthetic phenolic compoundsO 117–124 (Figure 22) were eight

secondary amides inspired by the fungalOH metaboliteR 1081 . R2 104 105 R1 =H R2 =OH HO HO 106 R1 =HO R2 =NH2 107 R1 =Cl R2 =OH OH R1 R2 108 R =Cl R =NH HO HO O 104 1 2 2 O 105 R1 =H R2 =OH OCH 106 R1 =H R2 =NH2 Cl 3 107 R1 =ClO R2 =OH O 108 R =Cl R =NH OR1 O OCH3 HO 109aHO O R1O 1 2 2 O NH HO OCH N HO Cl HO O 3 HO O O NH O R1 O 2 OR1 O OCH3 109a OCH R1O 3 HO Cl 110 R1 =CHNH3 112 R =CH N 1 3 HO HO O O HO111 R =H 109b O 1 113 R1 =CH2OH NH2 O R1 OCH Cl 3 110 R =CH 1 3 112 R1 =CH3 O 111 R =H 109b 1 113 R1 =CH2OH

HO OH HO OH HO OH H3CO OCH OCH3 OCH H3CO H3CO 3 3 HO OH HO OH HO OH H3CO OCH OCH3 H3CO H3CO 3 114OCH3 115 116 114 115 116 FigureFigure 21. 21.Structures Structures of compounds 104104–116–116. . Figure 21. Structures of compounds 104–116.

Figure 22. Structures of synthetic derivatives 117–124. Figure 22. Structures of synthetic derivatives 117–124. Figure 22. Structures of synthetic derivatives 117–124. Molecules 2016, 21, 1649 20 of 27

Inhibition studies over human CAs were carried out in parallel in order to assess the selectivity of the tested compounds. As it can be observed from Table 12, several phenolic natural products were selective inhibitors of mycobacterial and fungal β-CAs.

Table 12. Enzyme inhibition of pathogenic M. tuberculosis β-CA isozymes Rv3273 and Rv1284, C. albicans isozyme Nce103, C. neoformans isozyme Can2 and human α-CA isozymes I and II, with the NP-Based Library (104–124), known CA Inhibitors (SA, AZA, ZNS, and TPM) and phenol.

a KI (µM) Compound Rv3273 Rv1284 Nce103 Can2 CA I CA II 104 12.1 0.85 1.10 1.08 430 8.711.4 105 11.4 10.8 1.02 0.90 309 10.3 106 9.12 0.85 0.91 0.84 309 11.2 107 10.8 10.3 1.08 1.12 265 8.6 108 11.2 10.5 1.00 0.85 237 131 109a 11.3 0.84 1.03 1.15 239 8.3 109b 10.9 0.71 1.06 1.11 231 8.0 110 0.91 11.8 0.92 0.89 10.5 9.6 111 0.92 0.91 0.90 0.95 355 13.1 112 11.4 10.5 1.06 1.12 201 8.4 113 10.9 0.99 1.01 1.08 374 9.2 114 8.92 0.82 0.73 0.77 368 11.7 115 0.89 0.80 0.70 0.95 354 12.1 116 9.10 0.85 0.62 0.81 307 230 117 0.98 12.2 0.78 0.72 10.5 11.4 118 0.97 0.80 0.93 0.81 9.6 9.8 119 0.91 1.27 0.72 0.94 11.2 10.8 120 0.90 1.78 0.75 0.86 11.9 11.5 121 0.85 1.16 0.79 0.74 0.70 0.018 122 1.14 11.0 0.99 0.95 158 10.4 123 10.2 12.3 0.96 0.91 11.4 10.8 124 10.4 11.6 0.81 0.73 10.7 9.4 SA 7.11 9.84 7.63 0.77 25.0 0.24 AZA 0.10 0.48 0.13 0.01 0.25 0.012 TPM 3.02 0.61 1.11 0.37 0.25 0.010 ZNS 0.21 286.8 0.94 0.97 0.056 0.035 Phenol 79.0 64.0 17.3 25.9 10.1 5.5 a Errors in the range of ±5% of the reported value, from three determinations.

Among the natural products tested the best inhibitors was (−)-dihydroguaiaretic acid (116) which inhibited β-CA in the submicromolar range with up to 495-fold selectivity over hCA I and 371-fold selectivity over hCA II. From the library of synthetic derivatives, compound 108 was a low micromolar inhibitor of the fungal CAs and displayed 130- to 280-fold selectivity over the two human CAs. These compounds were the first non-sulfonamide inhibitors that displayed β- over α-CA enzyme selectivity. This selectivity could be explained if the different structure of α- and β-isozymes is taken into consideration. Although both enzymes contain a zinc ion in the active site, they have several differences. Unlike α-CAs, the β-class of CAs exist as dimers (Figure 23). Whilst the catalytically important residues in the active site of β-CAs are provided exclusively from one monomer, access to the catalytic center may be constituted by residues from both monomers leaving a cleft of limited space for the inhibitor to enter. Therefore, it seems unlikely that the phenolic hydroxyl groups of the tested constituents directly interact with the active site zinc of the pathogen CAs, especially for the spacious one 116. It was suggested that the compounds might cause either monomerization or considerable conformational changes in the access area to the active site. To prove this concept crystallization of the adduct, and X-ray analysis is necessary. Unfortunately none of the tested compounds gave crystals with Can2, Nce103, Rv3273, and Rv1284 enzymes. Molecules 2016, 21, 1649 21 of 27

Molecules 2016, 21, 1649 21 of 27

Molecules 2016, 21, 1649 21 of 27

Figure 23. Comparison of accessible surface volumes of the active sites of M. tuberculosis Rv1284 (left, Figure 23. Comparison of accessible surface volumes of the active sites of M. tuberculosis Rv1284 PDB accession code 1ylk), C. albicans Nce103 (middle, PDB accession code 3eyx), and human CA II (right, (left, PDB accession code 1ylk), C. albicans Nce103 (middle, PDB accession code 3eyx), and human PDB accession code 3NB5). The individual monomers of the dimeric β-CAs are colored in green and CA II (right,blue, respectively. PDB accession The catalytic code 3NB5). zinc ion Theis shown individual in magenta. monomers The figure of was the prepared dimeric withβ-CAs PyMol are [84]. colored in greenFigure and blue, 23. Comparison respectively. of Theaccessible catalytic surface zinc volumes ion is shown of the active in magenta. sites of M. The tuberculosis figure was Rv1284 prepared (left, with PyMolPDBA [84 seriesaccession]. of phenoliccode 1ylk), acids C. albicans 6–8 and Nce103 some (middle, of their PDB esters accession 125– 132code, derivatives3eyx), and human of p-coumaric CA II (right, (6 ), caffeicPDB ( 7accession) and ferulic code acid 3NB5). (8), wasThe individualinvestigated monomers for the inhibition of the dimeric of three β-CAs β-carbonic are colored anhydrases in green (CAs,and ECblue, 4.2.1.1) respectively. from the The pathogenic catalytic zincbacterium ion is shown Mycobacterium in magenta. tuberculosis The figure was, Rv1248, prepared Rv3588 with PyMoland Rv3273 [84]. Aβ series-CAs (Figure of phenolic 24) [85]. acids Some 6of– 8theseand compounds some of theirwere low esters micromolar125–132 inhibitors, derivatives of the ofpathogenicp-coumaric (6), caffeic (enzymes7)A and series ferulic and of they phenolic acid did (not8), acids show was 6 investigatedinhibitory–8 and some activity forof their theagainst inhibitionesters the human125– of132 threewidesp, derivativesβread-carbonic cytosolic of p-coumaric anhydrases isoforms (6), (CAs, EC 4.2.1.1)caffeicCA I ( fromand7) and II. the ferulicAs pathogenicit can acid be ( observed8), was bacterium investigated from TableMycobacterium for13, thesimple inhibition phenolic tuberculosis of threeacids 6β,–-carbonic Rv1248,8 are active anhydrases Rv3588 against andMtb (CAs, Rv3273 β-CAsECβ (Figure -CAs4.2.1.1) but 24from suffer)[85 the ].of Somelowpathogenic selectivity. of these bacterium The compounds presence Mycobacterium of were additional low tuberculosis micromolarphenolic ,rings Rv1248,inhibitors in the Rv3588 structure of and the makes Rv3273 pathogenic enzymesβ-CAsthe and compounds (Figure they 24) did more [85]. not Someselective show of inhibitory theseas it increases compounds activity the ratiowe againstre oflow β- the micromolarover human α-CA inhibitory widespreadinhibitors activity. of the cytosolic pathogenic This is isoforms enzymespossibly and due they to additional did not show interactions inhibitory with activity the dimeric against structure the human of the widespβ-CA. read cytosolic isoforms CA I and II. As it can be observed from Table 13, simple phenolic acids 6–8 are active against Mtb CA I and II. As it can be observed from Table 13, simple phenolic acids 6–8 are active against Mtb β-CAs but suffer of low selectivity. The presence of additional phenolic rings in the structure makes β-CAs but suffer of low selectivity. The presence of additional phenolic rings in the structure makes the compoundsthe compounds more more selective selective as as it it increases increases thethe ratioratio of of ββ- -over over α-CAα-CA inhibitory inhibitory activity. activity. This Thisis is possiblypossibly due due to additional to additional interactions interactions with with the the dimericdimeric structure of of the the β-CA.β-CA.

Figure 24. Structures of compounds 6–8 and derivatives 125–132.

FigureFigure 24. 24.Structures Structures of of compounds compounds 66––88 andand derivatives derivatives 125125–132–132. . Molecules 2016, 21, 1649 22 of 27

Table 13. Inhibition of human CA isozymes I and II and Mtb CAs Rv1248, Rv3273 and with phenolic

acids 6–8 and esters 122–129 by a stopped flow CO2 hydrase assay.

a,b KI (µM) Compound Molecules 2016, 21, 1649 hCA I hCA II Rv1248 Rv3273 Rv3588 22 of 27

Table 13.6 Inhibition of human1.07 CA isozymes 0.98 I and II and Mtb 6.05 CAs Rv1248, Rv3273 2.69 and with 4.33phenolic Molecules 2016, 21, 1649 22 of 27 acids 6–78 and esters 122–2.38129 by a stopped 1.61 flow CO2 hydrase 5.92 assay. 6.70 5.36 8 2.89 2.40 7.13 2.40 5.64 a,b Table 13.125 Inhibition of human5.23 CA isozymes >50 I and II andKI (µM) Mtb 5.32 CAs Rv1248, Rv3273 5.10 and with phenolic 7.13 Compound acids 6126–8 and esters 122–1299.62 by a stoppedhCA I >50flowhCA CO II 2 hydraseRv1248 3.95 assay. Rv3273 Rv3588 6.83 8.03 127 >506 1.07 >500.98 6.05 4.51 2.69 3.194.33 7.05 KI (µM) a,b 128 Compound>507 2.38 >501.61 5.92 5.67 6.70 3.205.36 7.26 hCA I hCA II Rv1248 Rv3273 Rv3588 129 >508 2.89 >502.40 7.13 4.69 2.40 2.325.64 5.40 130 1256>50 1.075.23 >500.98>50 6.05 5.32 3.78 2.69 5.10 4.33 1.847.13 5.04 131 1263.877 2.389.62 >501.61>50 5.92 3.95 4.67 6.70 6.83 5.36 1.878.03 5.13 132 1273.668 2.89>50 >502.40>50 7.13 4.51 5.74 2.403.19 5.64 2.307.05 6.09 125128 5.23>50 >50>50 5.32 5.67 5.103.20 7.137.26 a Errors in the range of ±5% of the reported data from three different assays; b h = human. 126129 9.62>50 >50>50 3.95 4.69 6.832.32 8.035.40 127130 >50>50 >50>50 4.51 3.78 3.191.84 7.055.04 128 >50 >50 5.67 3.20 7.26 Unfortunately it was not131 possible 3.87 to obtain>50 good 4.67 quality 1.87 crystals 5.13 which would permit the 129132 >503.66 >50>50 4.69 5.74 2.32 2.30 5.40 6.09 130 >50 >50 3.78 1.84 5.04 study of the adducta Errors by in X-ray the range crystallography. of ±5% of the reported data To overcomefrom three different this assays. problem, b h = human. the binding mode of these inhibitors to the bacterial131 enzymes 3.87 Rv3588>50 and 4.67 Rv1248 1.87 was 5.13 investigated by computational 132 3.66 >50 5.74 2.30 6.09 Unfortunately it was not possible to obtain good quality crystals which would permit the study of approaches (Figuresa Errors 25 in and the range 26). of A±5% protocol of the reported consisting data from three of classic different MDassays. and b h = human. SMD simulations with the adduct by X-ray crystallography. To overcome this problem, the binding mode of these inhibitors molecular docking and rescoring was applied. It was proposed that the inhibitors anchor to the to the bacterial enzymes Rv3588 and Rv1248 was investigated by computational approaches (Figures 25 Unfortunately it was not possible to obtain good quality crystals which would permit the study of zinc-coordinatedand 26). A waterprotocol molecule consisting of from classic theMD and CA SMD active simulations site interfering with molecular with docking the and nucleophilic rescoring attack the adduct by X-ray crystallography. To overcome this problem, the binding mode of these inhibitors was applied. It was proposed that the inhibitors anchor to the zinc-coordinated water molecule from of the zincto hydroxidethe bacterial enzymes on the Rv3588 substrate and Rv1248 CO2 was. These investigated compounds by computational may be approaches considered (Figures as 25 interesting the CA active site interfering with the nucleophilic attack of the zinc hydroxide on the substrate CO2. anti-mycobacterialand 26). A protocol lead compounds. consisting of classic MD and SMD simulations with molecular docking and rescoring These compounds may be considered as interesting anti-mycobacterial lead compounds. was applied. It was proposed that the inhibitors anchor to the zinc-coordinated water molecule from the CA active site interfering with the nucleophilic attack of the zinc hydroxide on the substrate CO2. These compounds may be considered as interesting anti-mycobacterial lead compounds.

Figure 25. Predicted binding mode of 2 (panel A), 3 (panel B) and 8 (panel C) towards the active site of Figure 25. Rv3588Predicted β-CAbinding from Mtb. mode Monomer of2 A (panel is coloredA), green, 3 (panel monomerB) and B is 8colored (panel orange.C) towards Small molecules the active site of Rv3588 β-CAare shown from as Mtb. cyan Monomersticks. Residues A within is colored 5 Å from green, the Zn(II) monomer ion and those B is coloredin contact orange.with the inhibitors Small molecules Figure 25. Predicted binding mode of 2 (panel A), 3 (panel B) and 8 (panel C) towards the active site of are shown as lines. H-bond interactions are highlighted by black dashed lines. Residues involved in are shownRv3588 as cyan β-CA sticks. from ResiduesMtb. Monomer within A is5 colored Å from green, the Zn(II)monomer ion B andis colored those orange. in contact Small molecules with the inhibitors H-bonds are labeled. The catalytic Zn(II) ion is shown as a grey sphere [85]. are shownare as shown lines. as H-bond cyan sticks. interactions Residues within are 5 Å highlighted from the Zn(II) by ion black and those dashed in contact lines. with Residues the inhibitors involved in H-bonds areare shown labeled. as lines. The H-bond catalytic interact Zn(II)ions ion are ishighlighted shown as by a black grey dashed sphere lines. [85 Residues]. involved in H-bonds are labeled. The catalytic Zn(II) ion is shown as a grey sphere [85].

Figure 26. Predicted binding mode of 2 (panel A), 4 (panel B) and 8 (panel C) towards the active site of Rv1248 β-CA from Mtb. Monomer A is colored green, monomer B is colored orange. Small molecules are shown as cyan sticks. Residues within 5 Å from the Zn(II) ion and those in contact with the inhibitors Figure 26. Predicted binding mode of 2 (panel A), 4 (panel B) and 8 (panel C) towards the active site of are shown as lines. H-bond interactions are highlighted by black dashed lines. Residues involved in Figure 26.Rv1248Predicted β-CA bindingfrom Mtb.mode Monomer of 2A (panelis coloredA ),green, 4 (panel monomerB) and B is colored 8 (panel orange.C) towards Small molecules the active site of H-bonds are labeled. The catalytic Zn(II) ion is shown as a grey sphere [85]. Rv1248 β-CAare shown from as Mtb. cyan sticks. Monomer Residues A within is colored 5 Å from green, the Zn(II) monomer ion and those B is in colored contact with orange. the inhibitors Small molecules are shownare as shown cyan sticks. as lines. Residues H-bond interact withinions 5 Åare from highlighted the Zn(II) by black ion dashed and those lines.in Residues contact involved with the in inhibitors are shownH-bonds as lines. are H-bondlabeled. The interactions catalytic Zn(II) are ion highlighted is shown as a bygrey black sphere dashed [85]. lines. Residues involved in H-bonds are labeled. The catalytic Zn(II) ion is shown as a grey sphere [85]. Molecules 2016, 21, 1649 23 of 27

3. Conclusions Since the first discovery of activity of simple phenol as a competitive CA inhibitor and the elucidation of its inhibition mechanism, several phenolic derivatives have been investigated in detail for their interactions with the major physiological isoforms of hCAs. Among the natural products screened so far, phenols and polyphenols remain the most investigated ones. Studies including both natural products and synthetic derivatives have allowed the identification of selective inhibitors and demonstrated the enormous potential of this class of compounds. Due to their structural variety, these products are characterized by different selectivity profiles compared to the primary sulfonamides. The latter are the classical CA inhibitors with long use in clinical practice, which nevertheless suffer from pharmacological side effects. In the search for alternative isoform-selective inhibitors phenols and polyphenols are among the most promising constituents. The different mode of mechanism, which in many cases is unknown, creates new opportunities for drug design and development. However, given the immense variety of chemotypes of natural phenolics, there is still much to be explored. Quite recently they emerged as important probes against pathogen CAs with exceptional selectivity, opening a new way for the development of novel anti-infective agents. Further studies including in vitro and in vivo tests and disease models are necessary to confirm these results.

Acknowledgments: This work was financed the 7th framework program (Metoxia, Dynano) to C.T.S. Author Contributions: A.K., F.C. and C.T.S. equally contributed to the work. Conflicts of Interest: The authors declare no conflict of interest.

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