Structural Insights on Carbonic Anhydrase Inhibitory Action, Isoform

Article pubs.acs.org/jmc

Structural Insights on Carbonic Anhydrase Inhibitory Action, Isoform Selectivity, and Potency of Sulfonamides and Coumarins Incorporating Arylsulfonylureido Groups Murat Bozdag,†,⊥ Marta Ferraroni,†,⊥ Fabrizio Carta,† Daniela Vullo,† Laura Lucarini,‡ Elisabetta Orlandini,§ Armando Rossello,§ Elisa Nuti,§ Andrea Scozzafava,† Emanuela Masini,‡ and Claudiu T. Supuran*,†,∥ †

Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy ‡ Sezione di Farmacologia, Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale Pieraccini 6, 50139 Florence, Italy § Dipartimento di Farmacia, Università di Pisa, Via Bonanno, 6, 56126 Pisa, Italy ∥ Sezione di Scienze Farmaceutiche, Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy ABSTRACT: Sulfonamides and coumarins incorporating arylsulfonylureido tails were prepared and assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some derivatives incorporating 3-pyridinesulfonamide and arylsulfonylureoido fragments were low nanomolar inhibitors of isoforms CA II and XII (upregulated or overexpressed in glaucoma) and showed effective in vivo intraocular pressure lowering effects in an animal model of the disease, which were several times better compared to those of the antiglaucoma drug dorzolamide. By means of X-ray crystallography of adducts of several sulfonamides with CA II, the effective inhibitory properties were rationalized at the molecular level. The coumarins were ineffective as hCA I and II inhibitors but showed low nanomolar activity for the inhibition of the tumor-associated isoforms hCA IX and XII. The presence of arylsulfonylureido tails in these CA inhibitors possessing quite different mechanisms of action led to highly effective and isoformselective compounds targeting enzymes involved in severe pathologies such as glaucoma or cancer.

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Co(II) (in the δ class), or Fe(II) (for γ-CAs, in anaerobic conditions).11,12 This ping-pong mechanism makes some of the members of the CA superfamily among the most effective enzymes known in nature, with kcat/KM values close to the limit of the diffusion-controlled processes.13 Only α-CAs have been reported in vertebrates, but in most investigated species a large number of different isoforms were described.1−3 For example, in humans, 15 CA isoforms are known, CA I−IV, VA, VB, V− XIV, with 12 of them being catalytically active and three (CA VIII, X, and XI) devoid of activity but still playing significant functions in tumorigenesis and other physiologic as well as pathologic processes.14 Because of the fact that the substrates/reaction products of α-CAs (CO2, bicarbonate, and protons) are simple molecules/ ions involved in a host of physiologic processes, their up- or downregulation is associated with a range of diseases.1−3,15−18 Indeed, CA inhibitors (CAIs) are clinically used for decades as diuretics,15 antiglaucoma agents,1b,d,3d antiepileptics,16 or more

INTRODUCTION Among the metalloenzymes possessing a crucial physiologic function, the carbonic anhydrases (CAs, EC 4.2.1.1) represent an interesting case, as they act on very simple substrates, such as CO2, COS, CS2, and cyanamide,1−3 generating products that are involved in pH regulation (bicarbonate and protons), biosynthetic processes (bicarbonate, urea), or other important phenomena such as chemosensing (in vertebrates and invertebrates),4 sexual development (in pathogenic fungi),5 pH and CO2 sensing, pathogenicity, and survival in ambient air of many bacteria, fungi, and/or protozoa.6−8 There are six genetic families encoding such enzymes in virtually all organisms known to date, the α-, β-, γ-, δ-, ζ-, and η-CAs, with the last class reported very recently.9 All CAs known so far are metal-iondependent enzymes, with a metal hydroxide species within the enzyme cavity acting as a nucleophile in the catalytic cycle and with a second step (usually rate-determining) involving a proton transfer reaction from a water molecule coordinated to the active site metal ion to the environment, for regenerating the nucleophile.10 Metal ions employed at the active site of the different CAs include Zn(II) (in all classes), Cd(II) (in ζ-CAs), © 2014 American Chemical Society

Received: August 28, 2014 Published: October 13, 2014 9152

dx.doi.org/10.1021/jm501314c | J. Med. Chem. 2014, 57, 9152−9167

Journal of Medicinal Chemistry

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recently antiobesity agents,17 whereas compounds targeting the tumor-associated isoforms CA IX and XII are in preclinical development as anticancer agents/diagnostic tools for hypoxic tumors.3,18 CA activators (CAAs) may have potential for developing agents for Alzheimer’s disease or aging, as in these pathologies a diminishing of the activity of some physiologically relevant isoforms (such as CA I and II) has been reported.19 One of the main hurdles connected with the use of CAIs in the treatment of diverse conditions as those mentioned above is related to the off-target inhibition of isoforms other than the desired one.1−3 In fact the various pharmacological applications of the CAIs are due to the high number of isoforms and their involvement in different pathologies.15−18 For example, in glaucoma isoforms CA II and XII are the main drug targets, in obesity the mitochondrial ones CA VA/VB, in epilepsy CA VII and XIV, whereas for antitumor agents/diagnostic tools isoform CA IX and XII are targeted.15−18 As the active site architecture of most α-CAs is rather similar, this leads to promiscuous inhibition profiles for the first generation CAIs such as sulfanilamide (SA), acetazolamide (AAZ), ethoxzolamide (EZA), and dichlorophenamide (DCP).1−3 However, some of these compounds (AAZ, DCP) are still in clinical use as antiglaucoma, systemically acting agents, although they show a range of unwanted side effects due to inhibition of CAs in other tissues than the eye.2 Dorzolamide, DZA, a second generation CAI, was the first topically acting, water-soluble sulfonamide to be used clinically, and it is widely employed nowadays as an antiglaucoma medication.3d In the past period a number of important advances in the field of designing isoform-selective CAIs targeting various isoforms were achieved, mainly by using structure-based drug design approaches.1−3 Among them the so-called tail approach is one of the most employed for drug design purposes.20,21 Initially reported for sulfonamide CAIs20 and consisting of attaching tails (moieties) able to interact with the middle and the rim part of the active site cavity, which is more variable among the 15 isoforms, this approach was then extended to all other classes of inhibitors, such as coumarins,22 sulfocoumarins,23 dithiocarbamates,24 etc. In fact, the first generation sulfonamide inhibitors, such as those mentioned above, are mostly small, compact molecules that bind nearby the metal ion, in an active site region that is highly conserved among the different CA isoforms.1−3

CAIs possessing tosylureido tails, such as the derivative ts-SA, were among the first compounds showing a high affinity and isoform selectivity for inhibiting membrane-bound versus cytosolic CA isoforms.21a More recently, we reported21b a pyridine analog of ts-SA, ts-PySA, in which one CH group from the benzene ring was replaced by a nitrogen atom. The compound showed an even better isoform-selectivity profile compared to the lead compound ts-SA (or classical, clinically used sulfonamides such as AAZ or DCP).21b We explained these features of ts-PySA at the molecular level by reporting the high resolution X-ray crystal structure for its adduct with human (h) hCA II and comparing it with the corresponding adduct of ts-SA.21b Here we continue the exploration of this type of derivative, investigating the structural insights on four CA isoform inhibition with arylsulfonylureido-containing sulfonamides and coumarins (another important class of CAIs)22 and report the synthesis, enzyme inhibitory profile, X-ray crystallography, and antiglaucoma activity (in an animal model of the disease) for a series of such new derivatives.

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RESULTS AND DISCUSSION Compound Design and Synthesis. The drug design strategy for the new CAIs reported here was the following one: both sulfonamides1b,c,2d,3 and coumarins22,23 represent important classes of CAIs, possessing however very different inhibition mechanisms. The sulfonamides bind in a deprotonated state to the zinc ion from the enzyme active site, whereas the organic scaffold participates in multiple interactions with amino acid residues and water molecules, which further stabilize (or destabilize, if clashes occur) the enzyme−inhibitor (E−I) adduct.1b,c,2d,3 In addition, the kinetics of CA inhibition with sulfonamides is quite rapid, and for this reason the enzyme and the inhibitor are preincubated for a period of 15 min1b,c in order to allow the equilibration of the species and the formation of the E−I adduct.1b,c,2d,3,25 In contrast to the sulfonamides, coumarins possess a very different behavior: they act as “prodrug” inhibitors, undergoing a CA-mediated hydrolysis of the lactone ring, with generation of hydroxycinnamic acid derivatives which are the real inhibitors, binding far away from the Zn(II) ion, toward the exit of the enzyme active site, as demonstrated by means of kinetic, mass spectrometric, and X-ray crystallographic techniques.26 Furthemore, the kinetics of the hydrolytic reaction are rather slow, and for this reason the coumarins are preincubated with the enzyme for 6 h when the inhibition constants are being measured.22,26 For both classes of inhibitors (sulfonamides and coumarins), the presence of tails that can interact with amino acid residues from the middle and/or rim parts of the active site was shown to lead to interesting selectivity profiles for the inhibition of all catalytically active mammalian isoforms, CA I−XV.1b,cd,2d,3,26,27 In fact, the most isoform-selective CAIs detected so far belong to such derivatized compounds, in which the tail moieties contribute significantly to these effects.21−24,26,27 As tosylureido tails were shown earlier to lead to potent benzenesulfonamide CAIs (such as ts-SA)21a or the corresponding pyridinesulfonamide derivative ts-PySA,21b we investigated whether other types of arylsulfonylureido tails may lead to similar effects in sulfonamide or coumarin-based compounds. The synthetic strategy for the preparation of the new derivatives reported here is illustrated in Scheme 1. Starting from sulfonamides 1 (reported in ref 21b) and 2 (commercially available) or the coumarin derivative 3, all of them possessing reactive primary amine moieties, and by reaction with arylsulfonylureido isocyanates (ArSO2NCO), a series of new 9153



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Scheme 1. Preparation of Arylsulfonylureidosulfonamides 4−15 (A) and Arylsulfonylureidocoumarins 16−21a

a

ACN: acetonitrile.

sulfonylureas 4−21 were obtained in a one-step procedure. The nature of the Ar moiety in the starting arylsulfonyl isocyanate was chosen in such a way as to explore the structure−activity relationship (SAR) for this series of CAIs, by incorporating phenyl and 2- and 4-substituted phenyl moieties (with R = H, Me, F, and Cl) (Scheme 1). Two aminosulfonamides (1 and 2) and one coumarin (3) were included in the study. The sulfonamides differ by the aromatic ring to which the sulfamoyl moiety is attached (pyridine and benzene, respectively) as well as by the spacer between the amino group and the aromatic ring (no spacer for 1 and an ethylene bridge for 2). These differences may be relevant for exploring SAR and selectivity profiles against diverse CA isoforms. The coumarin derivative 3 was chosen with the derivatizable amino group in position 7, as we showed in earlier works that such derivatives show high activity against the tumor-associated isoforms CA IX and XII, with little or no inhibitory effects against the cytosolic isoforms hCA I and II.28 The new compounds were extensively characterized by spectroscopic and physicochemical methods which confirmed their structures. Carbonic Anhydrase Inhibition. We investigated the CA inhibitory properties of compounds 4−21 reported here, as well as the lead compound ts-SA and acetazolamide (AAZ) as standard inhibitor, against four physiologically significant isoforms, the cytosolic, widespread hCA I and II, as well as the transmembrane, tumor-associated hCA IX and XII (the last isoform is however also present in normal tissues, being upregulated in the eyes of glaucomatous patients).29 The reasons why we decided to investigate these isoforms are rather obvious: hCA II and XII are targets for antiglaucoma drugs, as outlined in the Introduction, whereas hCA IX and XII are targets for obtaining antitumor agents with a novel mechanism of action. hCA I is an abundant protein in the blood and the gastrointestinal tract, being one of the main off-target isoforms when considering both antiglaucoma or anticancer CAI drug design.30 The inhibition data are shown in Table 1. The following SAR can be delineated from the data of Table 1: (i) The sulfonamides 4−15 were medium potency hCA I inhibitors (except one derivative, 10, which like the lead ts-SA was a rather effective inhibitor of this isoform, with a KI of 21.6 nM) possessing inhibition constants ranging between

Table 1. Inhibition Data against Isoforms hCA I, II, IX, and XII with Compounds 4−21, the Lead tsSA, and Acetazolamide (AAZ) as Standard Inhibitor, by a Stopped-Flow, CO2 Hydrase Assay25 KI (nM)a compd

R

n

X

hCA I

hCA II

hCA IX

hCA XII

tsSA 4 5b 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 AAZ

4-Me H 4-Me 2-Me 4-Cl 4-F 2-Cl H 4-Me 2-Me 4-Cl 4-F 2-Cl H 4-Me 2-Me 4-Cl 4-F 2-Cl H

0 0 0 0 0 0 0 2 2 2 2 2 2

CH N N N N N N CH CH CH CH CH CH

7.0 72.8 245 206 86.5 81.3 57.6 21.6 67.1 64.2 57.8 82.6 52.8 >10000 >10000 4970 5735 >10000 2380 250

12.1 33.9 21.0 40.3 35.0 19.1 37.6 6.5 3.8 5.3 4.2 5.8 3.7 >10000 >10000 7380 8160 >10000 >10000 12.0

1.3 74.6 9.8 9.5 9.0 24.3 9.7 9.6 9.2 8.1 9.7 68.4 78.9 90.7 22.8 89.2 20.2 23.1 87.8 25.0

1.5 4.1 6.8 9.6 0.79 0.67 4.6 5.9 6.0 6.4 5.6 6.8 5.1 68.5 9.4 8.9 6.0 7.7 7.6 5.7

a Mean from three different assays (errors were in the range of ±10% of the reported values, data not shown). bTs-PySA.

52.8 and 245 nM. They are generally more inhibitory compared to acetazolamide AAZ (KI of 250 nM). For the sulfonamide series, the 4-aminoethylbenzenesulfonamide derivatives 10−15 were better inhibitors compared to the corresponding pyridinesulfonamides 4−9. The coumarins 16−21 were on the other hand not at all inhibitory (KI > 10 μM for 16, 17, and 20) or were very ineffective hCA I inhibitors (KI values in the range of 2.38−5.73 μM, Table 1). This has been observed earlier for many other 7-substituted coumarin derivatives.26,28 (ii) The physiologically dominant isoform hCA II was generally very well inhibited by the sulfonamides 4−15 reported 9154



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Table 2. Summary of Data Collection and Atomic Model Refinement Statistics for the Three Adductsa hCA II + 6 PDB code wavelength (Å) space group unit cell a (Å) b (Å) c (Å) β (deg) limiting resolution (Å) unique reflections Rsym (%) redundancy completeness overall (%) ⟨I/σ(I)⟩ resolution range (Å) unique reflections, working/free Rfactor (%) Rfree (%) no. of non-hydrogen atoms no. of water molecules no. of compound atoms rmsd bonds (Å) rmsd angles (deg) most favored additionally allowed generously allowed regions all atoms compound solvent a

hCA II + 7

hCA II + 8

4KUY 1.542 P21

4KUV 0.980 P21

4KUW 1.542 P21

42.21 41.19 71.96 104.30 1.65 (1.75−1.65) 28318 (4039) 4.8 (36.2) 3.5 (2.1) 97.5 (87.3) 19.0 (2.2) Refinement Statistics 29.0−1.65 26866/1440 15.74 18.76 2537 361 24 0.006 1.20 Ramachandran Statistics (%) 88.0 11.6 0.0 Average B Factor (Å2) 12.1 12.5 25.2

42.33 41.37 72.21 104.215 1.35 (1.43−1.35) 49343 (5786) 5.7 (29.8) 6.5 (5.2) 91.5 (66.9) 22.3 (7.2)

42.24 41.21 71.98 104.19 1.50 (1.59−1.50) 34424 (4618) 3.7 (32.3) 2.9 (2.4) 89.1 (74.3) 20.9 (3.0)

30.0−1.35 46699/2513 14.94 17.02 2395 261 24 0.009 1.51

29.07−1.55 30277/1631 16.09 18.56 2459 303 24 0.007 1.27

88.8 10.7 0.5

88.9 11.1 0.5

11.9 22.5 23.2

10.8 18.8 21.8

Values in parentheses are for the highest resolution shell.

fact that a more elongated inhibitor molecule (as in 10−15) leads to more effective hCA II inhibitors compared to the shorter compounds ts-SA or the pyridinesulfonamides 4−9. (iii) The SAR for the inhibition of the tumor-associated isoform hCA IX was slightly more complicated compared to what reported above for hCA I and II: all the investigated compounds, sulfonamides, and coumarins showed efficient inhibitory properties, with inhibition constants ranging between 8.1 and 90.7 nM (Table 1). For the pyridinesulfonamide subseries, compound 4 (R = H) was the weakest inhibitor, with a KI of 74.6 nM, whereas the remaining derivatives 5−9 showed an effective inhibition, with KI values ranging between 9.0 and 24.3 nM. The substitution patterns leading to the most effective hCA IX inhibitors in this subseries were 4-Me-, 2-Me-, 4-Cl, and 2-Cl, whereas the fluorine-substituted compound 8 was less inhibitory than 5−7 and 9 (Table 1). For the aminoethylbenzenesulfonamide derivatives 10−15, the inhibition profile was diverse, with the H, 4-Me-, 2-Me-, and 4-Cl derivatives being potent (and very similar inhibitors, KI values of 8.1−9.7 nM) and the last two derivatives of the subseries, 14 (4-F) and 15 (2-Cl), behaving as medium potency inhibitors (KI values of 68.4−78.9 nM). Three of the coumarins reported here (17, 19, and 20), all of them possessing 4-substituents as R moieties (Me, Cl and F), were slightly better hCA IX inhibitors compared to acetazolamide (KI values ranging between 20.2 and 23.1 nM), whereas the unsubstituted (16, R = H) and

here (KI values in the range of 3.7−40.3 nM) and poorly or not at all inhibited by the coumarins 16−21 (compounds 16, 17, 20, and 21 showed a KI > 10 μM, and the remaining derivatives had inhibition constants ranging between 7.38 and 8.16 μM, Table 1). As for hCA I, again the aminoethylbenzenesulfonamides 10−15 were better inhibitors compared to the corresponding pyridinesulfonamides 4−9, the difference of activity being of around 1 order of magnitude, but both chemotypes may be considered as potent hCA II inhibitors. The nature of the R moiety from the original arylsulfonyl isocyanate was an important factor for the inhibitory properties: for example the ortho-substituted methyl derivative 6 was 2 times a less potent hCA II inhibitor compared to the para-substituted regiomer 5. However, the corresponding chlorine isomers 7 and 9 showed a very similar inhibitory behavior. The fluorinesubstituted compound 8 was a better inhibitor compared to the corresponding chlorine derivatives 7 and 9, being almost equipotent to the 4-methyl-substituted compound 5 (derivatives 5 and 8 were the best hCA II inhibitors among the pyridinesulfonamides reported here). The corresponding aminoethylbenzenesulfonamides 10−15 showed a compact behavior of very potent hCA II inhibitors, with little variations of the KI values irrespective of the substitution pattern at the aryl moiety (KI values of 3.7−6.5 nM, Table 1). They were however better inhibitors compared to the lead ts-SA or the clinically used AAZ (KI values of 12.0−12.1 nM), proving the 9155



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Figure 1. Electronic density of sulfonamide 6 bound within the hCA II active site.

(bearing the sulfamoyl group) compared to the benzene analog ts-SA when bound within the active site cavity of the enzyme. This led to a very different orientation of the scaffolds of the two sulfonamides when bound to the enzyme. The tilt also led to a clash between a carbon atom from the pyridine ring of 5 and the OH moiety of Thr200, an amino acid residue crucial for the binding of sulfonamide CAIs,1b27 thus leading to less effective inhibitory properties of 5 compared to the benzenesulfonamide derivative ts-SA (but 5, with a KI of around 20 nM, is still an effective hCA II inhibitor). On the other hand, we showed that ts-SA is a promiscuous, low nanomolar inhibitor of 7 out of 10 hCAs, whereas 5 was a low nanomolar inhibitor only for the tumor-associated hCA IX and XII, effectively inhibiting (KI of 21 nM) hCA II among the other isoforms.21b These differences in the binding of such structurally similar compounds prompted us to perform further X-ray crystallographic work on adducts of some of these sulfonamides bound to hCA II. Here we report the high resolution X-ray crystallographic data for the adducts of hCA II with the following pyridinesulfonamides possessing arylsulfonylureido tails: 6, 7, and 8 (PDB codes 4KUY, 4KUV, and 4KUW, respectively). These derivatives were chosen because of their close structural similarity with 5 on one hand but also because they possess effective hCA II inhibitory properties, which range between 19.1 and 40.3 nM. On the other hand, as it will be shown shortly, two of these compounds (5 and 8) also possess remarkable in vivo intraocular pressure lowering (IOP) antiglaucoma effects in an animal model of the disease.

ortho-substituted compounds (18 and 21, R = Cl and Me, respectively) were weaker hCA IX inhibitors (KI values ranging between 87.8 and 90.7 nM, Table 1). It is thus obvious that small structural differences (length of the linker n, nature, and position of the R group) lead to important variations in the affinity of these compounds, both sulfonamides and coumarins, for their CA targets. (iv) The second transmembrane isoform investigated here, hCA XII, was also potently inhibited by the investigated compounds (KI values ranging between 0.67 and 68.5 nM, Table 1). Except one coumarin (16, R = H) which can be considered a medium potency inhibitor (KI of 68.5 nM), all other compounds reported here showed excellent hCA XII inhibitory activity, with KI values only ranging in a small interval (0.67−9.6 nM). A remark should be made on the two subnanomolar inhibitors, 7 and 8, both belonging to the pyridinesulfonamide subseries and bearing 4-halogeno substituents. Thus, in this case, for this isoform, the shorter molecule inhibitors (7 and 8) were more efficient than the corresponding longer molecule inhibitors 13 and 14 (by almost an order of magnitude). It may be observed that among the four investigated CA isoforms, hCA XII was the one more sensible to inhibition by both sulfonamides and coumarins possessing arylsulfonylureido tails. X-ray Crystallography. In the previous work21b on the synthesis of ts-PySA (compound 5 from this paper) we also reported its high resolution X-ray crystal structure in complex with hCA II (PDB code 4KV0) and compared it to that of the isostructural ts-SA (PDB code 1ZFK). We observed that the pyridine derivative 5 underwent a tilt of its heterocyclic ring 9156



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entrance of the hCA II active site, also participating in T-shaped π-stacking with the aromatic ring of Phe131, as also observed for 521b as well as in many other hCA II−sulfonamide adducts investigated earlier (Figures 1−3).24,27,31 From the superposition of the three inhibitors 6−8 with compound 521b (Figure 4), it may be observed that the 4-aminobenzenesulfonamidate fragment of the four inhibitors is completely superimposable, whereas the arylsulfonylureido tails adopt different orientations. The 4-fluoro and 4-methyl derivatives 8 and 5 showed the most similar orientations of this fragment of the molecule, and in fact they have almost identical KI values of around 20 nM (Table 1). The o-tolyl tail of 6 and 4-chlorophenyl tail of 7 on the other hand were observed tilted as compared to the corresponding moieties of compounds 5 and 8 (Figure 4). This may lead to a less favorable conformation of the two compounds, which is reflected in their slightly higher inhibition constants against hCA II compared to the structurally related derivatives 5 and 8 (in the range of 35−40 nM, Table 1). IOP Lowering Activity. An animal model of glaucoma, i.e., rabbits with high IOP, induced by the injection of 0.1 mL of hypertonic saline solution (5% in distilled water) into the vitreous of both eyes (also denominated hypertensive rabbits)32 has been employed for assessing the potential use of the new compounds reported here for the management of glaucoma. Although systemically acting sulfonamides such as acetazolamide AAZ or dichlorophenamide DCP are effective antiglaucoma agents, leading to a 25−30% drop of the IOP in glaucoma patients,3d their side effects (due to inhibition of CAs present

Data collection and statistics for adducts of compounds 6−8 bound to hCA II are shown in Table 2. The electron density of the three inhibitors in adduct with hCA II was clearly defined for almost the entire molecule (Figures 1−3). In the refined models of these adducts the sulfonamides 6−8 were observed coordinated to the Zn(II) ion from the active site through the sulfamoyl, N1 atom of the deprotonated sulfonamide moiety at a distance of around 2 Å. As in most other hCA II−sulfonamide adducts reported so far,24,27 two additional hydrogen bonds from the conserved Thr199 N and OG atoms to the sulfonamide oxygen atoms were observed in all three adducts (OG1 Thr199−N4 atom of the sulfonamide moiety, of around 2.8 Å, and N Thr199−O3 of the sulfamoyl moiety from the inhibitor, of around 3 Å). These strong hydrogen bonds further stabilized the enzyme−inhibitor adducts. The active site zinc ion was in a slightly distorted tetrahedral geometry also being coordinated by the side chains of His94, His96, and His119, along with the deprotonated sulfonamide ligands 6−8 (Figures 1−3). The scaffolds of the three inhibitors were observed in an extended conformation, lying throughout the active site cavity, with the sulfonylurea functionalities participating in strong hydrogen bond interactions with two water molecules (Wat 214−N12 of the inhibitors molecules of around 2.9 Å, and Wat 214−N9 of the inhibitors, of around 2.7 Å, and Wat 99−O11 of the inhibitors, of 2.8 Å). The terminal substituted-phenyl hydrophobic moieties of the three inhibitors were found in the hydrophobic pocket lined by residues Phe131, Val135, Leu198, and Pro202, toward the 9157



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in other tissues than the eye) leads to a range of side effects. The topically acting drug dorzolamide DZA (administered as 2% solution of the hydrochloride salt) is also effective and does not show systemic side effects but being a rather acidic solution (of pH 5.5) leads to some ocular side effects such as stinging and eye reddening, blurred vision, pruritus, etc.3d In addition, the drug is not long acting (the peak IOP lowering is at 2 h postadministration) and its efficacy is rather limited, with maximal values of 4−6 mmHg IOP drop being the most common effect, in both humans and experimental animals.3d We designed some of the compounds reported here in such a way as to possess enhanced water solubility at more neutral pH values compared to DZA, and this was the reason why the pyridine sulfonamide moiety was incorporated in some of these derivatives. Indeed, the pKa of the pyridine moieties (a tertiary amine) is closer to the physiologic pH compared to that of an aliphatic secondary amine (moiety present in DZA). Some of the best hCA II/XII inhibitors discovered here, such as compounds 5 and 8 (KI of 19−21 nM against hCA II and of 0.67−6.8 nM against hCA XII, Table 1), could easily be formulated as 1% or 2% solutions at pH 7.0 and were administered to hypertensive rabbits (Figure 5). DZA hydrochloride was used as a standard drug, whereas the control experiments were done by using the vehicle (hydroxypropylcellulose at 0.05%). The data of Figure 5 show that the vehicle did not modify IOP in the 4 h time-course (curve 4), whereas DZA hydrochloride at 2% showed the effect already reported in the literature for this drug, with a peak of IOP lowering of 4.5 mmHg, at 2 h postadministration (curve 3 in Figure 5). Compound 5 at 1% (curve 1) and compound 8 at 2% concentration (curve 2) were highly effective IOP lowering agents.

The peak of IOP drop was at 1 h postadministration but was considerably higher compared to that of the standard drug, in the range of 22−27.5 mmHg. The efficacy of the IOP lowering was also considerably longer compared to that of acetazolamide, as at 2 h postadministration the drop of IOP was of 16−16.5 mmHg for both drugs (versus 4.5 mmHg for DZA), and such a drop was maintained at 4 h postadministration (with a value of 14 mmHg for compound 5 and of 16.8 mmHg for 8 (Figure 5).

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CONCLUSIONS We report a series of new sulfonamides and coumarins incorporating arylsulfonylureido moieties, designed in order to obtain CAIs with a better inhibition profile compared to clinically used such drugs and with potential use in the management of glaucoma (targeting CA II and XII) or hypoxic tumors (targeting CA IX and XII). Sulfonamide derivatives incorporating pyridinesulfonamide moieties and arylsulfonyluoreido fragments showed interesting inhibition profiles against the isoforms involved in glaucoma, and the X-ray crystal structures of three of them bound to hCA II help us to understand at the molecular level their inhibitory behavior. Two of these compounds also showed highly effective in vivo antiglaucoma activity in an animal model of the disease being much more effective compared to the clinically used drug dorzolamide. The coumarins possessing the same substitution pattern, i.e., arylsulfonylureido moieties in the 7 position of the heterocyclic ring, were ineffective as CA I and II inhibitors but were highly effective CA IX and XII inhibitors, thus showing a potential use for the management of hypoxic tumors which overexpress these two CA isoforms. 9158



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Figure 4. Superposition of sulfonamides 5−8 when bound to hCA II in the corresponding enzyme−inhibitor adducts.

Figure 5. Drop of intraocular pressure (ΔIOP, mmHg) versus time (h) in hypertensive rabbit eyes treated with sulfonamide 5 (curve 1) at a concentration of 1% and with 8 (curve 2) at a concentration of 2%, in comparison with 2% dorzolamide (DZA, curve 3) as standard drug and with vehicle (curve 4). Standard errors were in the range of 10−15% of the reported IOP values (from three different measurements for each of the four animals in the study group) and were statistically significant (p = 0.045, by Student’s t test).

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moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance (1H NMR, 13C NMR, DEPT-135,

EXPERIMENTAL PROTOCOLS

General. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa Aesar, and TCI. All reactions involving air- or 9159



Article

Synthesis of 5-(3-Tosylureido)pyridine-2-sulfonamide 5.

DEPT-90, HSQC, HMBC) spectra were recorded using a Bruker Avance III 400 MHz spectrometer in DMSO-d6 or MeOD-d4. Chemical shifts are reported in parts per million (ppm), and the coupling constants (J) are expressed in hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet; dd, double of doubles. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck silica gel 60 (230−400 mesh ASTM) as the stationary phase, and MeOH and DCM were used as eluents. Melting points (mp) were carried out in open capillary tubes and are uncorrected. Sulfonamide 1 was prepared as reported earlier.21b ESI spectra were recorded by direct introduction at 5 mL/min flow rate in an LTQ linear ion trap (Thermo, San Jose, CA, USA), equipped with a conventional ESI source. The spectra were acquired in both positive and negative ion mode; the specific conditions used for these experiments were as follows: the spray voltage was 5 kV in both polarity; the capillary voltages were 49 V in positive ion mode and −15 V in negative ion mode; the capillary temperature was kept at 280 °C. The sheath gas was set at 10 (arbitrary units), and the sweep gas and auxiliary gas were kept at 5 (arbitrary units). Scan time was 2 microscans, and the maximun injection time was 50 ms. The ESI spectra were acquired using Xcalibur 2.0 (Thermo), and the spectrum range was 150−500 m/z. A Gallenkamp MPD350.BM3.5 apparatus was used to measure the melting points. Amines 2 and 3 as well as all the arylsulfonyl isocyanates were commercially available (from Sigma-Aldrich, Milan, Italy). All compounds reported here, 4−21, were >98% pure. General Procedure for the Synthesis of Sulfonylureas 4−21.21 A solution of amine or aniline (1.0 equiv) in dry acetone or acetonitrile was treated at room temperature with the corresponding arylsulfonyl isocyanate (1.0 equiv), and the solution was stirred under a nitrogen atmosphere until starting materials were consumed (TLC monitoring). The reaction was quenched with H2O or with a 1.0 M aqueous hydrochloric acid solution and treated accordingly. Synthesis of 5-(3-(Phenylsulfonyl)ureido)pyridine-2-sulfonamide 4.

A suspension of 2-sulfamoyl-5-aminopyridine 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with 4-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue that purified by silica gel column chromatography, eluting with MeOH/DCM (10% v/v) to afford the titled compound 5 as white solid. 5-(3-Tosylureido)pyridine-2-sulfonamide 5. 23% yield; silica gel TLC Rf = 0.10 (MeOH/DCM 10% v/v); mp 220−221 °C (dec); δH (400 MHz, DMSO-d6) 2.36 (3H, s), 7.21 (2H, s, exchange with D2O, SO2NH2), 7.23 (2H, d, J 8.4), 7.71 (3H, dd, J 6.8, 8.4), 8.06 (1H, dd, J 2.4, 9.0), 8.68 (1H, d, J 2.4), 9.09 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 21.8, 121.7, 124.4, 127.6, 129.2, 139.2, 140.6, 142.0, 144.2, 151.4, 158.7. Elemental analysis, calcd: C 42.15, H 3.81, N 15.13, S 17.31. Found: C 42.34, H 3.81, N 14.97, S 17.09. m/z (ESI negative) 369.08 [M − H]−. Synthesis of 5-(3-(2-Tolylsulfonyl)ureido)pyridine-2-sulfonamide 6.

A suspension of 2-sulfamoyl-5-aminopyridine 1 (0.2 g, 1.0 equiv) in dry acetone (3.6 mL) was treated with 2-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue that purified by silica gel column chromatography, eluting with MeOH/DCM (20% v/v) to afford the titled compound 6 as white solid. 5-(3-(2-Tolylsulfonyl)ureido)pyridine-2-sulfonamide 6. 27% yield; silica gel TLC Rf = 0.30 (MeOH/DCM 20% v/v); mp 208−209 °C (dec); δH (400 MHz, MeOD-d4) 2.72 (3H, s), 7.26 (2H, m), 7.38 (1H, dt, J 1.2, 7.4), 7.73 (1H, d, J 8.8), 8.03 (1H, m), 8.17 (1H, dd, J 2.8, 8.8), 8.64 (1H, d, J 2.4); δC (100 MHz, MeOD-d4) 21.6, 123.3, 127.4, 127.6, 129.9, 133.2, 133.8, 139.0, 141.5, 142.5, 144.3, 153.5, 161.8. Elemental analysis, calcd: C 42.15, H 3.81, N 15.13, S 17.31. Found: C 42.22, H 4.20, N 15.07, S 17.47. m/z (ESI negative) 369.08 [M − H]−.

A suspension of 2-sulfamoyl-5-aminopyridine 121b (0.1 g, 1.0 equiv) in dry acetone (2.7 mL) was treated with phenylsulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue that was purified by silica gel column chromatography, eluting with MeOH/DCM (20% v/v) to afford the titled compound 4 as white solid. 5-(3-(Phenylsulfonyl)ureido)pyridine-2-sulfonamide 4. 83% yield; silica gel TLC Rf 0.28 (MeOH/DCM 20% v/v); mp 180−181 °C (dec); δH (400 MHz, DMSO-d6) 7.19 (2H, s, exchange with D2O, SO2NH2), 7.44 (3H, m), 7.70 (1H, d, J 8.8), 7.84 (2H, m), 8.06 (1H, dd, J 2.0, 8.6), 8.69 (1H, d, J 2.0), 9.10 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 121.7, 124.6, 127.5, 128.8, 131.0, 139.4, 142.0, 147.0, 151.5, 159.1. Elemental analysis, calcd: C 40.44, H 3.39, N 15.72, S 17.99. Found: C 40.18, H 3.26, N 15.58, S 17.81. m/z (ESI negative) 355.08 [M − H]−. 9160



Article

Synthesis of 5-(3-(4-Chlorophenylsulfonyl)ureido)pyridine-2sulfonamide 7.

Synthesis of 5-(3-(2-Chlorophenylsulfonyl)ureido)pyridine-2sulfonamide 9.

A suspension of 2-sulfamoyl-5-aminopyridine 1 (0.15 g, 1.0 equiv) in dry acetone (2.7 mL) was treated with 4-chlorophenylsulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue that purified by silica gel column chromatography, eluting with MeOH/DCM (10% v/v) to afford the titled compound 7 as white solid. 5-(3-(4-Chlorophenylsulfonyl)ureido)pyridine-2-sulfonamide 7. 62% yield; silica gel TLC Rf = 0.26 (MeOH/DCM 10% v/v); mp 183−184 °C; δH (400 MHz, DMSO-d6) 7.40 (2H, s, exchange with D2O, SO2NH2), 7.76 (2H, d, J 8.4), 7.87 (1H, d, J 8.8), 8.04 (3H, m), 8.66 (1H, d, J 2.4), 9.60 (1H, exchange with D2O, NH), 11.46 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 122.0, 127.8, 130.3, 130.5, 138.1, 139.4, 139.5, 140.9, 150.7, 154.9. Elemental analysis, calcd: C 36.88, H 2.84, N 14.34, S 16.41. Found: C 36.62, H 2.89, N 14.54, S 16.52. m/z (ESI negative) 389.08 [M − H]−. Synthesis of 5-(3-(4-Fluorophenylsulfonyl)ureido)pyridine-2sulfonamide 8.

A suspension of 2-sulfamoyl-5-aminopyridine 1 (0.1 g, 1.0 equiv) in dry acetone (2.7 mL) was treated with 2-chlorophenylsulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue that was washed with diethyl ether, filtered, and dried under vacuum to afford the titled compound 9 as white solid. 5-(3-(2-Chlorophenylsulfonyl)ureido)pyridine-2-sulfonamide 9. 62% yield; silica gel TLC Rf = 0.25 (MeOH/DCM 10% v/v); mp 186−187 °C; δH (400 MHz, DMSO-d6) 7.39 (2H, s, exchange with D2O, SO2NH2), 7.64 (1H, m), 7.74 (2H, m), 7.85 (1H, d, J 8.4), 8.06 (1H, d, J 8.8), 8.17 (1H, d, J 8.4), 8.66 (1H, s), 9.35 (1H, s, exchange with D2O, NH), 11.53 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 122.0, 127.8, 128.7, 131.5, 132.7, 132.9, 136.0, 137.7, 137.9, 140.9, 150.3, 154.9. Elemental analysis, calcd: C 36.88, H 2.84, N 14.34, S 16.41. Found: C 36.83, H 2.82, N 14.72, S 16.80. m/z (ESI negative) 389.08 [M − H]−. Synthesis of N-(4-Sulfamoylphenethylcarbamoyl)benzenesulfonamide 10.

A suspension of 2-sulfamoyl-5-aminopyridine 1 (0.08 g, 1.0 equiv) in dry acetone (2.7 mL) was treated with 4-fluorophenylsulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with a 1.0 M aqueous hydrochloric acid solution, concentrated under vacuum to give a residue that was washed with diethyl ether, and filtered and dried under vacuum to afford the titled compound 8 as white solid. 5-(3-(4-Fluorophenylsulfonyl)ureido)pyridine-2-sulfonamide 8. 40% yield; silica gel TLC Rf = 1.0 (MeOH/DCM 10% v/v); mp 179−180 °C; δH (400 MHz, DMSO-d6) 7.40 (2H, s, exchange with D2O, SO2NH2), 7.53 (2H, t, J 8.8), 7.87 (1H, d, J 8.4), 8.08 (3H, m), 8.65 (1H, s), 9.58 (1H, s, exchange with D2O, NH), 11.4 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 117.3 (d, J2C−F 23), 122.1, 127.8, 131.7 (d, J3C−F 10), 137.0, 138.1, 140.9, 150.7, 154.8, 165.7 (d, J1C−F 251); δF (376 MHz, DMSO-d6) −105.0 (1F, s). Elemental analysis, calcd: C 38.50, H 2.96, N 14.97, S 17.13. Found: C 38.12, H 3.05, N 14.74, S 17.34. m/z (ESI negative) 373.08 [M − H]−.

4(2-Aminoethyl)benzenesulfonamide 2 (0.20 g, 1.0 equiv) in dry acetone (5 mL) was treated with benzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue which was washed with diethyl ether, filtered, and dried under vacuum to afford the titled compound 10 as a white solid. N-(4-Sulfamoylphenethylcarbamoyl)benzenesulfonamide 10. 68% yield; silica gel TLC Rf = 0.50 (MeOH/DCM 10% v/v); mp 180−181 °C; δH (400 MHz, DMSO-d6) 2.76 (2H, t, J 6.8), 3.27 (2H, q, J 6.8), 6.56 (1H, t, J 6.8, exchange with D2O, NH), 7.32 (2H, s, exchange with D2O, SO2NH2), 7.34 (2H, d, J 8.4), 7.65 (2H, m), 7.74 (3H, m), 7.91 (2H, m), 10.70 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 35.7, 41.2, 126.6, 128.0, 129.9, 130.0, 134.1, 141.1, 143.0, 144.1, 152.2. Elemental analysis, calcd: C 46.99, H 4.47, N 10.96, S 16.72. Found: C 47.20, H 4.43, N 10.93, S 17.06. m/z (ESI negative) 382.17 [M − H]−. 9161



Article

Synthesis of 4-Chloro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 13.

Synthesis of 4-Methyl-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 11.

4(2-Aminoethyl)benzenesulfonamide 2 (0.2 g, 1.0 equiv) in dry acetone (5 mL) was treated with 4-chlorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue which was purified by silica gel column chromatography, eluting with MeOH/DCM (10% v/v) to afford titled compound 13 as a white solid. 4-Chloro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 13. 38% yield; silica gel TLC Rf = 0.35 (MeOH/DCM 10% v/v); mp 171−172 °C; δH (400 MHz, DMSO-d6) 2.77 (2H, t, J 6.8), 3.25 (2H, q, J 6.8), 6.60 (1H, t, J 6.8, exchange with D2O, NH), 7.32 (2H, s, exchange with D2O, SO2NH2), 7.36 (2H, d, J 8.4), 7.75 (4H, m), 7.92 (2H, t, J 8.4), 10.79 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 35.8, 41.3, 126.7, 130.0 (3 × C overlapping), 138.8, 140.4, 143.1, 144.2, 152.8. Elemental analysis, calcd: C 43.11, H 3.86, N 10.06, S 15.35. Found: C 42.96, H 3.80, N 9.86, S 15.11. m/z (ESI negative) 416.08 [M − H]−. Synthesis of 4-Fluoro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 14.

4(2-Aminoethyl)benzenesulfonamide 2 (0.2 g, 1.0 equiv) in dry acetonitrile (5 mL) was treated with 4-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) to give a precipitate which was washed with diethyl ether, filtered, and dried under vacuum to afford the titled compound 11 as white solid. 4-Methyl-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 11. 40% yield; silica gel TLC Rf = 0.40 (MeOH/DCM 10% v/v); mp 201−202 °C; δH (400 MHz, DMSO-d6) 2.43 (3H, s), 2.76 (2H, t, J 6.8), 3.25 (2H, q, J 6.8), 6.52 (1H, t, J 6.8, exchange with D2O, NH), 7.35 (4H, d, J 8.4, 2H Ar−H, 2H exchange with D2O, SO2NH2), 7.44 (2H, d, J 8.4), 7.75 (2H, d, J 8.4), 7.78 (2H, d, J 8.4), 10.60 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 22.0, 35.7, 41.2, 126.6, 128.1, 130.0, 130.4, 138.4, 143.0, 144.2, 144.5, 152.3. Elemental analysis, calcd: C 48.35, H 4.82, N 10.57, S 16.13. Found: C 48.44, H 4.55, N 10.71, S 16.52; m/z (ESI negative) 396.17 [M − H]−. Experimental in agreement with reported data.1 Synthesis of 2-Methyl-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 12.

4(2-Aminoethyl)benzenesulfonamide 2 (0.20 g, 1.0 equiv) in dry acetonitrile (5 mL) was treated with 4- fluorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue which was washed with diethyl ether and then filtered and dried under vacuum to afford the titled compound 14 as a white solid. 4-Fluoro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 14. 93% yield; silica gel TLC Rf = 0.31 (MeOH/DCM 10% v/v); mp 167−168 °C δH (400 MHz, DMSO-d6) 2.76 (2H, t, J 7.2), 3.24 (2H, q, J 7.2), 6.51 (1H, brt, exchange with D2O, NH), 7.32 (2H, s, exchange with D2O, SO2NH2), 7.36 (2H, d, J 8.4), 7.46 (2H, t, J 8.8), 7.76 (2H, d, J 8.4), 7.96 (2H, m), 10.73 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 35.7, 41.3, 117.0 (d, J2C−F 23), 126.6, 130.0, 131.1 (d, J3C−F 10), 137.9, 143.0, 144.2, 152.7, 165.3 (d, J1C−F 250); δF (376 MHz, DMSO-d6) −106.0 (1F, s). Elemental analysis, calcd: C 44.88, H 4.02, N 10.47, S 15.98. Found: C 44.45, H 3.95, N 10.44, S 15.65. m/z (ESI negative) 400.08 [M − H]−.

4(2-Aminoethyl)benzenesulfonamide 2 (0.2 g, 1.0 equiv) in dry acetonitrile (5 mL) was treated with 2-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue which was crystallized with IPA/H2O. 2-Methyl-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 12. 24% yield; silica gel TLC Rf = 0.50 (MeOH/DCM 10% v/v); mp 180−181 °C; δH (400 MHz, DMSO-d6) 2.57 (3H, s), 2.74 (2H, t, J 6.8), 3.22 (2H, q, J 6.8), 6.42 (1H, t, J 6.8, exchange with D2O, NH), 7.33 (4H, m, 2H Ar-H, 2H exchange with D2O, SO2NH2), 7.43 (2H, t, J 7.2), 7.59 (1H, t, J 7.2), 7.75 (2H, d, J 8.0), 7.92 (2H, d, J 8.0), 10.72 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSOd6) 20.5, 35.7, 41.1, 126.6, 127.1, 130.0, 130.6, 133.2, 134.0, 137.4, 139.2, 143.1, 144.1, 152.2. Elemental analysis, calcd: C 48.35, H 4.82, N 10.57, S 16.13. Found: C 48.34, H 4.57, N 10.73, S 16.15. m/z (ESI negative) 396.17 [M − H]−. 9162



Article

Synthesis of 4-Methyl-N-(4-methyl-2-oxo-2H-chromen-7ylcarbamoyl)benzenesulfonamide 17.

Synthesis of 2-Chloro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 15.

A suspension of 7-amino-4-methylcoumarin 3 (0.23 g, 1.0 equiv) in dry acetonitrile (4.3 mL) was treated with 4-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with a 1.0 M aqueous hydrochloric acid solution and the formed precipitate was filtered, washed with diethyl ether, dried under vacuum to obtain the titled compound 17 as a white solid. 4-Methyl-N-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 17. 66% yield; silica gel TLC Rf = 0.36 (MeOH/DCM 10% v/v); mp 260−261 °C (dec); δH (400 MHz, DMSO-d6) 2.41 (3H, s), 2.44 (3H, s), 6.29 (1H, s), 7.34 (1H, d, J 8.6), 7.48 (3H, d, J 8.8), 7.70 (1H, d, J 8.8), 7.90 (2H, d, J 8.4), 9.39 (1H, s, exchange with D2O, NH), 11.06 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 22.0, 106.2, 113.2 (2 × C, overlapping), 115.8, 126.9, 128.5, 130.4, 137.8, 142.5, 144.9, 150.3, 153.9, 154.5, 160.8. Elemental analysis, calcd: C 58.05, H 4.33, N 7.52, S 8.61. Found: C 58.01, H 4.21, N 7.52, S 8.94. m/z (ESI negative) 371.17 [M − H]−. Synthesis of 2-Methyl-N-(4-methyl-2-oxo-2H-chromen-7ylcarbamoyl)benzenesulfonamide 18.

4(2-Aminoethyl)benzenesulfonamide 2 (0.14 g, 1.0 equiv) in dry acetonitrile (5 mL) was treated with 2-chlorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O (1.0 mL) and the solvents were evaporated under vacuum to give a residue which was purified by silica gel column chromatography, eluting with MeOH/DCM (10% v/v) to afford the titled compound 15 as a white solid. 2-Chloro-N-(4-sulfamoylphenethylcarbamoyl)benzenesulfonamide 15. 26% yield; silica gel TLC Rf = 0.26 (MeOH/DCM 10% v/v); mp 168−169 °C (dec); δH (400 MHz, DMSO-d6) 2.76 (2H, t, J 6.8), 3.25 (2H, q, J 6.8), 6.44 (1H, brt, exchange with D2O, NH), 7.33 (2H, s, exchange with D2O, SO2NH2) 7.36 (2H, d, J 8.0), 7.59 (1H, m), 7.70 (2H, m), 7.76 (2H, d, J 8.0), 8.06 (1H, d, J 8.0), 10.91 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 35.8, 41.2, 126.6, 128.4, 130.0, 131.4, 132.5, 132.7, 135.5, 138.4, 143.1, 144.2, 152.3. Elemental analysis, calcd: C 43.11, H 3.86, N 10.06, S 15.35. Found: C 43.49, H 3.81, N 10.16, S 15.68. m/z (ESI negative) 416.08 [M − H]−. Synthesis of N-(4-Methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 16.

A suspension of 7-amino-4-methylcoumarin 3 (0.23 g, 1.0 equiv) in dry acetone (4.0 mL) was treated with 2-methylbenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with a H2O and the formed precipitate was filtered, washed with diethyl ether, dried under vacuum to obtain the titled compound 18 as a white solid. 2-Methyl-N-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 18. 53% yield TLC Rf = 1.00 (ethyl acetate/ n-hexane 60% v/v); mp 257−258 °C (dec); δH (400 MHz, DMSO-d6) 2.40 (3H, s), 2.67 (3H, s), 6.27 (1H, s), 7.34 (1H, m) 7.46 (3H, m), 7.60 (1H, m), 7.69 (1H, d, J 8.0), 8.03 (1H, d, J 8.0), 9.2 (1H, s, exchange with D2O, NH), 11.21 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.9, 20.7, 106.1, 113.1 (2 × C overlapping), 115.8, 126.9, 127.2, 131.0, 133.3, 134.3, 137.7, 138.9, 142.7, 150.5, 154.0, 154.5, 160.8. Elemental analysis, calcd: C 58.05, H 4.33, N 7.52, S 8.61. Found: C 58.31, H 4.41, N 7.34, S 8.36. m/z (ESI negative) 371.17 [M − H]−.

A suspension of 7-amino-4-methylcoumarin 3 (0.1 g, 1.0 equiv) in dry acetonitrile (5.0 mL) was treated with benzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with a H2O and the obtained precipitate was filtered under vacuum, washed with diethyl ether, dried under vacuum to afford the titled compound 16 as a white solid. N-(4-Methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 16. 70% yield; silica gel TLC Rf = 1.00 (ethyl acetate/ n-hexane 50% v/v); mp 248−249 °C (dec); δH (400 MHz, DMSO-d6) 2.41 (3H, s), 6.29 (1H, s), 7.35 (1H, m), 7.47 (1H, m), 7.71 (4H, m), 8.02 (1H, d, J 7.6), 9.42 (1H, s, exchange with D2O, NH), 11.33 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.9, 106.2, 113.2 (2 × C overlapping), 115.8, 126.9, 128.4, 130.0, 134.4, 140.7, 142.5, 150.3, 153.9, 154.5, 160.8. Elemental analysis, calcd: C 56.98, H 3.94, N 7.82, S 8.95. Found: C 56.57, H 3.59, N 7.64, S 8.93. m/z (ESI negative) 357.17 [M − H]−. 9163



Article

Synthesis of 2-Chloro-N-(4-methyl-2-oxo-2H-chromen-7ylcarbamoyl)benzenesulfonamide 21.

Synthesis of 4-Chloro-N-(4-methyl-2-oxo-2H-chromen-7ylcarbamoyl)benzenesulfonamide 19.

A suspension of 7-amino-4-methylcoumarin 3 (0.12 g, 1.0 equiv) in dry acetone (5.0 mL) was treated with 4-chlorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O and the formed precipitate was filtered, washed with diethyl ether, dried under vacuo to obtain the titled compound 19 as a white solid. 4-Chloro-N-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 19. 88% yield; silica gel TLC Rf = 1.0 (ethyl acetate/n-hexane 60% v/v); mp 254−255 °C (dec); δH (400 MHz, DMSO-d6) 2.41 (3H, s), 6.29 (1H, s), 7.35 (1H, d, J 8.4) 7.47 (1H, m), 7.70 (1H, t, J 8.4), 7.77 (1H, d, J 8.4), 8.02 (1H, d, J 8.4), 9.50 (1H, s, exchange with D2O, NH), 11.33 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 106.3, 113.3, 115.8, 115.9, 126.9, 130.2, 130.4, 139.4, 139.5, 142.4, 150.3, 153.9, 154.5, 160.8. Elemental analysis, calcd: C 51.98, H 3.34, N 7.13, S 8.16. Found: C 51.96, H 3.09, N 6.96, S 8.02. m/z (ESI negative) 391.08 [M − H]−. Synthesis of 4-Fluoro-N-(4-methyl-2-oxo-2H-chromen-7ylcarbamoyl)benzenesulfonamide 20.

A suspension of 7-amino-4-methylcoumarin 3 (0.12 g, 1.0 equiv) in dry acetonitrile (5.0 mL) was treated with 2-chlorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with a H2O and the obtained precipitate was filtered under vacuum, washed with diethyl ether, dried under vacuum to afford the titled compound 21 as a pale yellow solid. 2-Chloro-N-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 21. 63% yield; silica gel TLC Rf = 1.0 (ethyl acetate/n-hexane 50% v/v); mp 260−261 °C (dec); δH (400 MHz, DMSO-d6) 2.41 (3H, s), 6.29 (1H, s), 7.34 (1H, d, J 8.4), 7.46 (1H, m), 7.70 (4H, m), 8.17 (1H, d, J 7.6), 9.25 (1H, s, exchange with D2O, NH), 11.38 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 106.3, 113.3, 115.8, 116.0, 126.9, 128.7, 131.5, 132.7, 132.9, 136.0, 137.7, 142.2, 149.9, 153.9, 154.5, 160.8. Elemental analysis, calcd: C 51.98, H 3.34, N 7.13, S 8.16. Found: C 51.62, H 3.03, N 6.82, S 7.93. m/z (ESI negative) 391.08 [M − H]−. CA Inhibition Assay. An SX.18MV-R Applied Photophysics stopped-flow instrument was used for assaying the CA-catalyzed CO2 hydration activity by using the method of Khalifah.25 Inhibitor and enzyme were preincubated for 15 min (for the sulfonamides) and 6 h (for the coumarin derivatives). IC50 values were obtained from dose−response curves working at seven different concentrations of test compound (from 0.1 nM to 50 μM), by fitting the curves using PRISM (www.graphpad.com) and nonlinear least-squares methods, values representing the mean of at least three different determinations, as described earlier by us.24,26 The inhibition constants (KI) were then derived by using the Cheng−Prusoff equation, as follows: Ki = IC50/ (1 + [S]/Km) where [S] represents the CO2 concentration at which the measurement was carried out and Km the concentration of substrate at which the enzyme activity is at half maximal. All enzymes used were recombinant, produced in E. coli as reported earlier.24,26 The concentrations of enzymes used in the assay were the following: hCA I, 12.1 nM; hCA II, 8.0 nM; hCA IX, 8.7 nM; hCA XII, 11.9 nM. Cocrystallization and X-ray Data Collection. Crystals of hCA II complexed with compounds 6−8 were obtained using the sitting drop vapor diffusion method. An equal volume of 0.8 mM solution of hCA II in Tris, pH 8.0, and 0.8 mM of each compounds in 20 mM Hepes, pH 7.4, was mixed and incubated for 15 min. Then 1 μL drops of the complex solution were mixed with a solution 1.5 or 1.6 M sodium citrate, 50 mM Tris, pH 8.0, and were equilibrated against the same solution at 296 K. Crystals of the complexes grew in a few days. The crystals were flash-frozen at 100 K using a solution obtained by adding 25% (v/v) glycerol to the mother liquor solution as cryoprotectant. A data set on a crystal of the complex hCA II−7 was collected using synchrotron radiation at the Xaloc beamline at ALBA (Barcelona, Spain) using a wavelength of 0.980 Å and a DECTRIS Pilatus 6M detector. Data on the other complexes were collected at the Centro di Cristallografia Strutturale (CRIST) in Florence, Italy, using an Oxford Diffraction instrument equipped with a sealed tube Enhance Ultra (Cu) and an Onyx CCD detector. Data were integrated and scaled using the program XDS.33 Data processing statistics are shown in Table 2. Structure Determination. The crystal structure of hCA II (PDB accession code 3P58) without solvent molecules and other heteroatoms was used to obtain initial phases of the structures using Refmac5.34 5% of the unique reflections were selected randomly and excluded from

A suspension of 7-amino-4-methylcoumarin 3 (0.17 g, 1.0 equiv) in dry acetone (5.0 mL) was treated with 4-fluorobenzenesulfonyl isocyanate (1.0 equiv) according to the general procedure previously reported. The reaction was quenched with H2O. The solvents were evaporated under vacuum, and the obtained residue was washed with diethyl ether, dried under vacuum to obtain the titled compound 20 as a white solid. 4-Fluoro-N-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)benzenesulfonamide 20. 80% yield; silica gel TLC Rf = 0.30 (MeOH/DCM 10% v/v); mp 255−256 °C (dec); δH (400 MHz, DMSO-d6) 2.41 (3H, s), 6.29 (1H, s), 7.35 (1h, dd, J 2.0, 8.8), 7.48 (1H, m), 7.52 (2H, t, J 8.8), 7.71 (1H, d, J 8.8), 8.09 (2H, dd, J 5.2, 8.8), 9.46 (1H, s, exchange with D2O, NH), 11.20 (1H, brs, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 106.2, 113.2, 115.8, 115.9, 117.2 (d, J2C−F 23), 126.9, 131.7 (d, J3C−F 9), 137.0, 142.5, 150.4, 153.9, 154.5, 160.8, 165.6 (d, J1C−F 250); δF (376 MHz, DMSO-d6) −105.1 (1F, s). Elemental analysis, calcd: C 54.25, H 3.48, N 7.44, S 8.52. Found: C 54.53, H 3.22, N 7.06, S 8.19. m/z (ESI negative) 375.17 [M − H]−. 9164



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the refinement data set for the purpose of Rfree calculations. The initial |Fo − Fc| difference electron density maps unambiguously showed the inhibitors. Atomic models for inhibitors were calculated and energyminimized using the program JLigand 1.0.39. Refinements proceeded using normal protocols of positional, isotropic atomic displacement parameters alternating with manual building of the models using COOT.35 Solvent molecules were introduced automatically using the program ARP.36 Final rounds of refinement for all the models included hydrogen at calculated positions and refined using a riding model. The quality of the final models were assessed with PROCHECK.37 Crystal and refinement data are summarized in Table 2. Hypertensive Rabbit IOP Lowering Studies. Male New Zealand albino rabbits weighing 1500−2000 g were used in these studies. Animals were anesthetized using zoletil (tiletamine chloride plus zolazepam chloride, 3 mg/kg body weight, im) and injected with 0.1 mL of hypertonic saline solution (5% in distilled water) into the vitreous of both eyes. IOP was determined using a tonometer (Tono-pen Avia tonometer, Reichhert Inc., Depew, NY 14043, USA) prior to hypertonic saline injection (basal) at 1, 2, 3, and 4 h after administration of the drug. Vehicle (phosphate buffer 7.00 plus DMSO 2%) or drugs were instilled immediately after the injection of hypertonic saline. Eyes were randomly assigned to different groups. Vehicle or drug (0.50 mL) was directly instilled into the conjunctive pocket at the desired doses (1−2%).32 The IOP was followed for 4 h after drug administration. Four different animals were used for each tested compound.

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design specific drugs targeting 15 different isoforms? Chem. Rev. 2012, 112, 4421−4468. (c) Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discovery 2008, 7, 168−181. (2) (a) Ogawa, T.; Noguchi, K.; Saito, M.; Nagahata, Y.; Kato, H.; Ohtaki, A.; Nakayama, H.; Dohmae, N.; Matsushita, Y.; Odaka, M.; Yohda, M.; Nyunoya, H.; Katayama, Y. Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the β-carbonic anhydrase family enzymes. J. Am. Chem. Soc. 2013, 135, 3818−3825. (b) Neri, D.; Supuran, C. T. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10, 767−777. (c) Smith, K. S.; Jakubzick, C.; Whittam, T. S.; Ferry, J. G. Carbonic anhydrase is an ancient enzyme widespread in prokaryotes. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 15184−15189. (d) Supuran, C. T. Structure-based drug discovery of carbonic anhydrase inhibitors. J. Enzyme Inhib. Med. Chem. 2012, 27, 759−772. (3) (a) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.; Supuran, C. T.; Neri, D. A Small-Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors. Angew. Chem., Int. Ed. 2014, 53, 4231−4235. (b) Aggarwal, M.; Boone, C. D.; Kondeti, B.; McKenna, R. Structural annotation of human carbonic anhydrases. J. Enzyme Inhib. Med. Chem. 2013, 28, 267−277. (c) De Simone, G.; Alterio, V.; Supuran, C. T. Exploiting the hydrophobic and hydrophilic binding sites for designing carbonic anhydrase inhibitors. Expert Opin. Drug Discovery 2013, 8, 793−810. (d) Masini, E.; Carta, F.; Scozzafava, A.; Supuran, C. T. Antiglaucoma carbonic anhydrase inhibitors: a patent review. Expert Opin. Ther. Pat. 2013, 23, 705−716. (e) Gieling, R. G.; Parker, C. A.; De Costa, L. A.; Robertson, N.; Harris, A. L.; Stratford, I. J.; Williams, K. J. Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro. J. Enzyme Inhib. Med. Chem. 2013, 28, 360− 369. (4) (a) Hirohashi, N.; Alvarez, L.; Shiba, K.; Fujiwara, E.; Iwata, Y.; Mohri, T.; Inaba, K.; Chiba, K.; Ochi, H.; Supuran, C. T.; Kotzur, N.; Kakiuchi, Y.; Kaupp, U. B.; Baba, S. A. Sperm from sneaker male squids exhibit chemotactic swarming to CO2. Curr. Biol. 2013, 23, 775−781. (b) Rummer, J. L.; McKenzie, D. J.; Innocenti, A.; Supuran, C. T.; Brauner, C. J. Root effect hemoglobin may have evolved to enhance general tissue oxygen delivery. Science 2013, 340, 1327−1329. (5) (a) Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.; Supuran, C. T.; Mühlschlegel, F. A.; Steegborn, C. Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans. J. Mol. Biol. 2009, 385, 1207−1220. (b) Lehneck, R.; Pöggeler, S. A matter of structure: structural comparison of fungal carbonic anhydrases. Appl. Microbiol. Biotechnol. 2014, 98, 8433−8441. (6) (a) Cummins, E. P.; Selfridge, A. C.; Sporn, P. H.; Sznajder, J. I.; Taylor, C. T. Carbon dioxide-sensing in organisms and its implications for human disease. Cell. Mol. Life Sci. 2014, 71, 831−845. (b) Cottier, F.; Leewattanapasuk, W.; Kemp, L. R.; Murphy, M.; Supuran, C. T.; Kurzai, O.; Mühlschlegel, F. A. Carbonic anhydrase regulation and CO2 sensing in the fungal pathogen Candida glabrata involves a novel Rca1p ortholog. Bioorg. Med. Chem. 2013, 21, 1549−1554. (c) Capasso, C.; Supuran, C. T. Sulfa and trimethoprim-like drugs antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors. J. Enzyme Inhib. Med. Chem. 2014, 29, 379−387. (7) (a) Supuran, C. T. Bacterial carbonic anhydrases as drug targets: towards novel antibiotics? Front. Pharmacol. 2011, 2, 34. (b) Capasso, C.; Supuran, C. T. Antiinfective carbonic anhydrase inhibitors: a patent and literature review. Expert Opin. Ther. Pat. 2013, 23, 693− 704. (c) Maresca, A.; Vullo, D.; Scozzafava, A.; Manole, G.; Supuran, C. T. Inhibition of the β-class carbonic anhydrases from Mycobacterium tuberculosis with carboxylic acids. J. Enzyme Inhib. Med. Chem. 2013, 28, 392−396. (d) Maresca, A.; Scozzafava, A.; Vullo, D.; Supuran, C. T. Dihalogenated sulfanilamides and benzolamides are effective inhibitors of the three β-class carbonic anhydrases from Mycobacterium tuberculosis. J. Enzyme Inhib. Med. Chem. 2013, 28, 384−387.

ASSOCIATED CONTENT

Accession Codes

Coordinates and structure factors for CA II complexes with 6, 7, and 8 have been deposited with the PDB with accession codes 4KUY, 4KUV, and 4KUW, respectively.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-055-4573005. Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it. Author Contributions ⊥

M.B. and M.F. contributed equally.

Notes

The authors declare the following competing financial interest(s): C.T.S. is coauthor of many patents on carbonic anhydrase inhibitors with use as antiglaucoma/antitumor agents (e.g., U.S. Patent 2013/0274305; U.S. Patent 2013/20,130,231,465; WO 2012/087115; WO 2012/070024; WO 2013/050426; and others).

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ACKNOWLEDGMENTS This research was financed by two EU grants of the Seventh Framework Programme (Metoxia and Dynano projects to A.S. and C.T.S.).

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ABBREVIATIONS USED CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor; IOP, intraocular pressure; KI, inhibition constant; SAR, structure−activity relationship

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REFERENCES

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Structural Insights on Carbonic Anhydrase Inhibitory Action, Isoform

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