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Inhibition of tumor-associated human carbonic anhydrase isozymes IX and XII by a new class of substituted-phenylacetamido aromatic sulfonamides
Bioorganic & Medicinal Chemistry 21 (2013) 5228–5232
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Inhibition of tumor-associated human carbonic anhydrase isozymes IX and XII by a new class of substituted-phenylacetamido aromatic sulfonamides Atilla Akdemir a, Özlen Güzel-Akdemir b,⇑, Andrea Scozzafava c, Clemente Capasso d, Claudiu T. Supuran c,e,⇑ a
Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazit, Istanbul, Turkey c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy d Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italy e Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, 50019 Sesto Fiorentino, Florence, Italy b
a r t i c l e
i n f o
Article history: Received 3 February 2013 Revised 14 May 2013 Accepted 12 June 2013 Available online 20 June 2013 Keywords: Carbonic anhydrases Cytosolic isoforms I and II Tumor-associated isoforms IX and XII Sulfonamides Docking
a b s t r a c t Here, we investigate 28 structurally new sulfonamides and their subsequent testing for enzyme inhibition of cytosolic and tumor-associated carbonic anhydrases (CAs, EC 4.2.1.1). The compounds showed very potent inhibition of four physiologically relevant human (h) CA isoforms, namely hCA I, II, IX and XII. Interestingly, the KI values were in the nanomolar range for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the investigated CA isoforms. As there are reported cases of tumors overexpressing both CA II and IX, such potent inhibitors for the two isoforms as those detected in this work, may have applications for targeting more than one CA present in tumors. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that mainly catalyze a simple but physiologically important reaction, i.e., the interconversion between carbon diox1–4 ide (CO2) and bicarbonate ðHCO This reaction is so important 3 Þ. that five evolutionarily unrelated subclasses of this superfamily exist (a-, b-, c-, d-, and f-CAs).1–5 The human CA enzymes (hCA) as well as the mammalian enzymes all belong to the a-class CA family that shares similarity in the overall structure and active site composition. Several hCA isoforms have been found to be important in various (patho)physiological processes and are therefore considered important drug targets. The human CA isoforms I and II (hCA I and hCA II, respectively) are cytosolic proteins. These enzymes, especially hCA II, are widespread in the body. Nevertheless, clinically used drugs, such as some diuretics, antiglaucoma and anticonvulsant agents, show their effect by blocking these enzymes. ⇑ Corresponding authors. Tel.: +39 055 457 3005; fax: +39 055 457 3385. E-mail addresses: [email protected] (Ö. Güzel-Akdemir), claudiu.supuran@ unifi.it (C.T. Supuran). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.06.029
In contrast, the tumor-associated hCA IX and hCA XII are mainly, but not exclusively, located on hypoxic tumor cells. These isoforms contain an extracellular catalytic domain (CA domain) and a transmembrane domain that anchors the enzymes to the cell membrane. Both hCA IX and hCA XII seem to be necessary for proliferation and survival of tumor cells. Inhibition of these enzymes by well-known CA inhibitors (CAIs) of the sulfonamide or coumarin type has been shown to stop primary tumor and metastases growth.4 A large number of such agents, mostly benzenesulfonamide derivatives, have been reported in the last years, as some of them possess not only strong antitumor activity in vivo, in animal models of the diseases, but also antiepileptic, antiobesity or antiglaucoma activities and are clinically used.5 These pharmacological actions are correlated with the inhibition of precise CA isoforms of the 16 presently known in mammals. In this study, we report the synthesis and the subsequent testing against several hCA isoforms of 28 new sulfonamides incorporating substituted phenylacetamido tails and aromatic (benzenesulfonamide) zinc anchoring moieties. These compounds were potent inhibitors of the cytosolic hCA II and the tumor-associated hCA IX and hCA XII.
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2. Results and discussion 2.1. Chemistry The drug design hypothesis of the compounds reported here is based on the observation we made earlier,6 that benzenesulfonamides incorporating thienylacetamido, phenacetamido and pyridinylacetamido tails in their molecules, of the type Ar-CH2CONH(CH2)n-C6H4-SO2NH2 show potent and isoform-selective inhibition profiles against some mammalian CA isoforms, such as CA VA and VB among others (n = 0–2, Ar = phenyl, pyridyl, thienyl).6 It appeared thus of interest to investigate in more detail compounds incorporating such tails, of the substituted phenylacetamido type, which were prepared as outlined in Scheme 1. We have attached halogen-/methoxy-phenacetamido tails to the molecules of aromatic or heterocyclic sulfonamides such as sulfanilamide, 3-halogenosulfanilamides, 4-aminomethyl/ethyl-benzenesulfonamide or 5-amino-1,3,4-thia-diazole-2-sulfonamide, as illustrated in Scheme 1. Reaction of the substituted-phenacetyl chlorides 1a–1d with the amino-containing aromatic sulfonamides 2a–2f afforded the aromatic derivatives 3a–3x, whereas the same reaction involving the heterocyclic derivative 4 afforded the substituted 1,3,4-thiadiazole-2-sulfonamides 5a–5d. We have chosen various acyl chlorides 1a–1d in order to generate chemical diversity in this new class of sulfonamides (Scheme 1). The new compounds were characterized by physicochemical procedures which confirmed their structure Materials and Methods.
determined for the tumor-associated hCA IX and hCA XII (Table 1). The KI values for the cytosolic hCA I and hCA II enzymes are also shown, although they constitute offtargets when antitumor CAIs are searched for. It may be observed that compounds 3 and 5 show excellent CA inhibitory properties (Table 1). All obtained KI values for hCA II, hCA IX and hCA XII were similar to or lower than 10 nM, with the exception of compound 3b. In addition, the values are similar to or lower than the KI value obtained for the reference compound AZ. Interestingly, compound 3b showed the highest KI value for all four isoforms. A fluorine atom (compound 3a) or a methoxy group (compound 3d) instead of the para-chlorine atom seems to be preferred at this position as the KI values for all four enzymes decreased significantly. The KI values for hCA I were generally in the lower nanomolar range (KI range: 2.9–346 nM). In all cases, a bromine atom at the 2-position of the phenyl moiety was well-tolerated. In general, a chlorine atom at the p-position of the benzenesulfonamide moiety was well-tolerated (compounds 3j–3l). By increasing the spacer length (n; Table 1) from 0 to 2 was beneficial for the enzyme inhibitory action of these compounds. Finally, replacing the benzenesulfonamide moiety by a thiadiazole-2-sulfonamide moiety also led to good inhibitors. The KI values for the cytosolic hCA II and the tumor-associated hCA IX and hCA XII were close to each other (with the exception of
Table 1 Inhibition of human CA isoforms hCA I, hCA II, hCA IX and hCA XII with compounds 3 and 5
2.2. Carbonic anhydrase inhibition
O
The inhibition values (KIs) of the reference compound acetazolamide (AZ) and the compounds 3 and 5 reported here have been
O N H
X
n
Y
O
+ Cl
X
Compd
2a-d: n=0, Y=H, F, Cl, Br 2e: n=1, Y=H 2f: n=2, Y=H N H2N
N
SO 2 NH 2
S
Cl–
4
O N H
X NEt3/MeCN
N S
SO2NH2
SO2NH2
5a-d
n
H2 N
X, Y, n
Y
1a: X = 4-F 1b: X = 4-Cl 1c: X = 2-Br 1d: X = 4-OCH3
N H
X
3a-x
SO2NH2
N
3a-x
n
SO2NH2
Y
O
N
N
SO2NH2 N H
X
S
5a-d Scheme 1. Preparation of sulfonamides 3a–x and 5a–d.
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3s 3t 3u 3v 3w 3x 5a 5b 5c 5d AZ
4-F, H, 0 4-Cl, H, 0 2-Br, H, 0 4-MeO, H, 0 4-F, F, 0 4-Cl, F, 0 2-Br, F, 0 4-MeO, F, 0 4-F, Cl, 0 4-Cl, Cl, 0 2-Br, Cl, 0 4-MeO, Cl, 0 4-F, Br, 0 4-Cl, Br, 0 2-Br, Br, 0 4-MeO, Br, 0 4-F, H, 1 4-Cl, H, 1 2-Br, H, 1 4-MeO, H, 1 4-F, H, 2 4-Cl, H, 2 2-Br, H, 2 4-MeO, H, 2 4-F, —, — 4-Cl, —, — 2-Br, —, — 4-MeO, —, — —
KIa (nM) hCA I
hCA II
hCA IXb
hCA XIIb
102 346 4.8 116 104 236 8.6 106 254 9.8 4.1 8.5 246 166 6.5 8.7 109 109 4.3 4.9 101 9.7 8.2 3.7 223 4.9 7.6 2.9 250.0
7.9 104 0.96 0.79 8.7 6.9 1.9 0.78 9.5 7.3 0.91 0.74 8.9 0.94 0.80 0.69 9.3 0.81 0.77 0.88 3.8 6.4 0.72 0.81 3.2 0.70 0.70 0.76 12.1
4.7 72.5 0.44 0.32 7.2 7.8 1.6 1.8 10.1 6.3 0.74 0.41 5.0 1.2 0.52 0.62 3.1 0.53 1.0 0.45 6.5 1.2 0.57 0.25 2.1 1.3 0.61 0.31 25.4
8.0 72.4 2.1 0.64 6.3 4.2 0.62 1.8 6.6 3.1 1.3 0.24 4.8 2.3 0.91 0.54 4.5 0.57 1.5 0.94 3.9 3.4 0.83 0.43 3.4 2.5 0.74 0.63 5.6
a Mean ± standard error, from three different assays, by a CO2 hydration stoppedflow assay. b Human, recombinant isozymes, pH 7.5, 20 mM TRIS–HCl buffer.
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compound 3b, the KI value range for hCA II: 0.69–9.5 nM; for hCA IX: 0.31–10.1 nM; for hCA XII: 0.24–6.6 nM) and in all cases lower than the respective KI values for hCA I (Table 1). The compounds investigated here have selectivity ratios for inhibiting CA IX and XII over CA I and II better or comparable to compounds in preclinical evaluation as anticancer/antimetastatic CAIs.2,3
His64
Gln67
Zn2+
Gln92
Trp5
2.3. Structural comparisons of hCA I, hCA II, hCA IX and hCA XII isozymes The crystal structures of hCA I (PDB: 3LXE), hCA II (PDB: 3B4F), hCA IX (PDB: 3IAI) and hCA XII (PDB: 1JD0) superpose well on their Ca-atoms (RMSD: 1.561 Å). In general, the backbone fold of the four enzymes is very similar, especially in the active site, and the Zn2+-binding residues (His94, His96 and His119), Thr199 and Zn2+ superpose well. Nevertheless, minor differences in the backbone fold are present near the active site near the conserved His64 (Val62-Ser65 of hCA I) and near Leu131 of hCA I (Trp123Leu141). The binding pocket residues of the four enzymes, which are defined as all residues within 4.5 Å of topiramate (hCA I), have been compared (Table 2). His64 (proton shuttle) and Gln92 (hydrogen bond formation to ligands) are conserved amongst the four enzymes. Other binding pocket residues that are at contact distance to bound ligands are not conserved (Table 2). At position 67 a positively charged lysine is present in hCA XII, while hydrogen bond forming Asn67 and Gln67 are present in hCA II and hCA IX, respectively. At position 131, a phenylalanine is present in hCA II, which can form hydrophobic stackings with the ligand. This interaction is not thought to introduce extra stabilizing ligand-protein interactions and it is absent in the other three enzymes.
Figure 1. Binding interactions of compounds 3q (brown), 3x (purple) and 5d (turquoise) with hCA IX. For clarity, only Trp5, His64, Gln67, Gln92 and Zn2+ are shown. Hydrogen bonds are depicted in red dotted lines.
Lys67
Zn2+
Asn62 His64
Gln92
Trp5 Thr91
2.4. Docking studies Compounds 3 and 5 and the reference compound AZ have lower KI values for hCA II, hCA IX and hCA XII compared to hCA I (Table 1). In addition, the KI values of the compounds for hCA II, hCA IX and hCA XII were generally below 10 nM. To understand the difference in KI values between these isoforms, we docked compounds 3 and 5 into the crystal structures of hCA I, hCA II, hCA IX and hCA XII using the GOLD Suite docking program and the ChemScore scoring function (25 dockings per ligand). The sulfonamide moiety of the ligands was assigned a negative charge (–SO2NH) and was forced to form hydrogen bonding interactions with Thr199 (which is observed in all X-ray crystal structures of CA sulfonamide adducts).1–4 The docking results for hCA IX and hCA XII indicate the importance of the conserved Gln92 in both enzymes. Many ligands formed hydrogen bonding interactions with the side chain of Gln92 through their carbonyl group (Figs 1 and 2). In addition, the ligands interacted with the side chains of Trp5, His64 and Gln67 of hCA IX and Trp5, Asn62, Lys67 and Thr91 of hCA XII. Figures 1 and 2 depict representative ligands that adopt the most
Figure 2. Binding interactions of compounds 3m (turquoise), 3v (green) and 3h (light brown) with hCA XII. The ligands mainly form hydrogen bonding (red dotted line) with Gln92, but to a lesser extend also with Trp5, Asn62, His64, Lys67 and Thr91. For clarity, only the hydrogen bonding interactions with Lys67 and Trp5 are shown. All three compounds form hydrogen bonds with Gln92, but these are not shown for clarity.
Asn67 Gln92 His64 Zn2+ Phe91 Ile91
Table 2 Binding pocket residues of hCA I, hCA II, hCA IX and hCA XII hCA I
hCA II
hCA IX
hCA XII
Trp5 Val62 His64 His67 Phe91 Gln92 Leu131 Ala135 His200
Trp5 Asn62 His64 Asn67 Ile91 Gln92 Phe131 Val135 Thr200
Trp5 Asn62 His64 Gln67 Leu91 Gln92 Val131 Leu135 Thr200
Trp5 Asn62 His64 Lys67 Thr91 Gln92 Ala131 Ser135 Thr200
Leu131 Phe131
Trp5
Figure 3. Binding interactions of compounds 5a (purple) with hCA II. It forms hydrogen bonds with Gln92 (red dotted lines) and hydrophobic stackings with Phe131. In hCA I, Leu131 (brown) is present instead of Phe131 and the stackings cannot be formed. In addition, Phe91 (brown) is present instead of Ile91.
A. Akdemir et al. / Bioorg. Med. Chem. 21 (2013) 5228–5232
commonly observed binding poses within the hCA IX and hCA XII active sites, respectively. Trp5 and His64 are conserved in all four isoforms investigated here, whereas Asn62 is conserved in hCA II, hCA IX and hCA XII (Table 2). However, our docking studies suggest that only Trp5 and Gln92 are involved in binding interactions in both enzymes (hCA IX aand XII). In general, the binding interactions with His64 and Gln67 of hCA IX and Asn62, Lys67 and Thr91 of hCA XII were less frequently observed. The docking studies also indicated that Phe131 in hCA II forms hydrophobic stackings with the halophenyl or methoxyphenyl tails of compounds 3 and 5 (Fig. 3). This residue is not conserved amongst the other three enzymes (Table 2). In addition, the ligands can form hydrogen bonding interactions with Gln92. In hCA I, a bulky Leu131 is positioned at the Phe131 (hCA II) position. In addition, Phe91 is present close to Leu131 in hCA I. Phe91 is not correctly positioned with respect to the ligands to form hydrophobic stackings with the ligands and it is expected to sterically hinder the ligands to adopt similar poses as observed for hCA II, hCA IX and hCA XII. In summary, the docking studies indicate that various hydrogen bonds with the active site residues of hCA IX and hCA XII and hydrophobic stacking with Phe131 of hCA II allow favorable binding interactions with these isoforms. Probably, the absence of Phe131 and the presence of Phe91 and Leu131 residues in hCA I (see Table 2) restrict the ligands to form similar binding interactions with hCA I. 3. Conclusions The tested sulfonamides show very potent inhibition of four physiologically relevant CA isoforms, hCA I, II, IX and XII, with KI values in the nanomolar range, for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the CA isoforms investigated here. As there are reported cases of tumors overexpressing both CA II and IX,7 such potent inhibitors for the two isoforms may have clinical importance.
4. Materials and methods 4.1. Chemistry Compounds 3–5 investigated here as CAIs were recently described by our group as Trypanosoma cruzi CA inhibitors.18 4.2. CA inhibition assay An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes as reported by Khalifah.8 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nM, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration),9 following the CA-catalyzed CO2 hydration reaction for a period of 5–10 s. Saturated CO2 solutions in water at 25 °C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO–water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. Trip-
5231
licate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier,10,11 and represent the mean from at least three different determinations. Human CA isozymes were prepared in recombinant form as reported earlier by our groups.12,13 4.3. Ligand preparation Compounds 3, 5 and the reference ligand AZ were built and converted to three-dimensional structures using the MOE software package (version 2011.10, Chemical Computing Group, Montreal, Canada). The sulfonamide substituent of the phenyl group was assigned a negative charge (–SO2NH) because it was expected to interact with the Zn2+ ion of the CA active sites. Subsequently, partial atomic charges were calculated and the molecules were energy-minimized according to a steepest-descent protocol using the MMFF94x force field in MOE. The ligands were saved as a multi-mol2 file. 4.4. Template preparation Crystal structures of hCA I (PDB: 3LXE; 1.90 Å),14 hCA II (PDB: 3B4F; 1.89 Å),13 hCA IX (PDB: 3IAI; 2.20 Å)15 and hCA XII (PDB: 1JD0; 1.50 Å)16 were obtained from the protein databank. Only 1 protein chain (chain A) and its corresponding Zn2+ ion and ligand (CA inhibitor) was retained per PDB file and all other protein chains, ions, buffer molecules and water molecules were deleted. Hydrogen atoms and charges were added to the protein using the protonate 3D tool of the MOE software package (version 2011.10, CCG, Montreal, Canada) and a steepest descent energy minimization was applied (MMFF94x forcefield). All proteins were superposed on the hCA I structure using their Ca-atoms (RMSD: 1.561 Å) and the proteins were saved as mol2-files. 4.5. Docking studies Compounds 3, 5 and the reference ligand AZ were docked into the protein models of hCA I, hCA II, hCA IX and hCA XII using the GOLD Suite docking package (version 5.1, CCDC, Cambridge, UK)17 and the ChemScore scoring function (25 docking per ligand). The binding pocket was defined as all residues within 12 Å of the central carbon atom of the CA inhibitor topiramate (atom CAL of topiramate in complex with hCA I; PDB: 3LXE). No restrictions were applied for the ligand conformations and binding poses during the docking procedure except for the requirement to form hydrogen bonding with Thr199 (or its counterparts in other CA isozymes). Acknowledgments This project was in part financed by the Istanbul University Scientific Research Projects Department under project number UDP22409 and by an FP7 EU project (Metoxia). References and notes 1. (a) Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. R.; Gitlin, I.; Gudiksen, K. L.; Weibel, D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946; (b) Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336; (c) Domsic, J. F.; Avvaru, B. S.; Kim, C. U.; Gruner, S. M.; Agbandje-McKenna, M.; Silverman, D. N.; McKenna, R. J. Biol. Chem. 2008, 283, 30766. 2. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767. 3. (a) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 8371; (b) Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna,
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Inhibition of tumor-associated human carbonic anhydrase isozymes IX and XII by a new class of substituted-phenylacetamido aromatic sulfonamides Atilla Akdemir a, Özlen Güzel-Akdemir b,⇑, Andrea Scozzafava c, Clemente Capasso d, Claudiu T. Supuran c,e,⇑ a
Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazit, Istanbul, Turkey c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy d Istituto di Biochimica delle Proteine – CNR, Via P. Castellino 111, 80131 Napoli, Italy e Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, 50019 Sesto Fiorentino, Florence, Italy b
a r t i c l e
i n f o
Article history: Received 3 February 2013 Revised 14 May 2013 Accepted 12 June 2013 Available online 20 June 2013 Keywords: Carbonic anhydrases Cytosolic isoforms I and II Tumor-associated isoforms IX and XII Sulfonamides Docking
a b s t r a c t Here, we investigate 28 structurally new sulfonamides and their subsequent testing for enzyme inhibition of cytosolic and tumor-associated carbonic anhydrases (CAs, EC 4.2.1.1). The compounds showed very potent inhibition of four physiologically relevant human (h) CA isoforms, namely hCA I, II, IX and XII. Interestingly, the KI values were in the nanomolar range for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the investigated CA isoforms. As there are reported cases of tumors overexpressing both CA II and IX, such potent inhibitors for the two isoforms as those detected in this work, may have applications for targeting more than one CA present in tumors. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes that mainly catalyze a simple but physiologically important reaction, i.e., the interconversion between carbon diox1–4 ide (CO2) and bicarbonate ðHCO This reaction is so important 3 Þ. that five evolutionarily unrelated subclasses of this superfamily exist (a-, b-, c-, d-, and f-CAs).1–5 The human CA enzymes (hCA) as well as the mammalian enzymes all belong to the a-class CA family that shares similarity in the overall structure and active site composition. Several hCA isoforms have been found to be important in various (patho)physiological processes and are therefore considered important drug targets. The human CA isoforms I and II (hCA I and hCA II, respectively) are cytosolic proteins. These enzymes, especially hCA II, are widespread in the body. Nevertheless, clinically used drugs, such as some diuretics, antiglaucoma and anticonvulsant agents, show their effect by blocking these enzymes. ⇑ Corresponding authors. Tel.: +39 055 457 3005; fax: +39 055 457 3385. E-mail addresses: [email protected] (Ö. Güzel-Akdemir), claudiu.supuran@ unifi.it (C.T. Supuran). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.06.029
In contrast, the tumor-associated hCA IX and hCA XII are mainly, but not exclusively, located on hypoxic tumor cells. These isoforms contain an extracellular catalytic domain (CA domain) and a transmembrane domain that anchors the enzymes to the cell membrane. Both hCA IX and hCA XII seem to be necessary for proliferation and survival of tumor cells. Inhibition of these enzymes by well-known CA inhibitors (CAIs) of the sulfonamide or coumarin type has been shown to stop primary tumor and metastases growth.4 A large number of such agents, mostly benzenesulfonamide derivatives, have been reported in the last years, as some of them possess not only strong antitumor activity in vivo, in animal models of the diseases, but also antiepileptic, antiobesity or antiglaucoma activities and are clinically used.5 These pharmacological actions are correlated with the inhibition of precise CA isoforms of the 16 presently known in mammals. In this study, we report the synthesis and the subsequent testing against several hCA isoforms of 28 new sulfonamides incorporating substituted phenylacetamido tails and aromatic (benzenesulfonamide) zinc anchoring moieties. These compounds were potent inhibitors of the cytosolic hCA II and the tumor-associated hCA IX and hCA XII.
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2. Results and discussion 2.1. Chemistry The drug design hypothesis of the compounds reported here is based on the observation we made earlier,6 that benzenesulfonamides incorporating thienylacetamido, phenacetamido and pyridinylacetamido tails in their molecules, of the type Ar-CH2CONH(CH2)n-C6H4-SO2NH2 show potent and isoform-selective inhibition profiles against some mammalian CA isoforms, such as CA VA and VB among others (n = 0–2, Ar = phenyl, pyridyl, thienyl).6 It appeared thus of interest to investigate in more detail compounds incorporating such tails, of the substituted phenylacetamido type, which were prepared as outlined in Scheme 1. We have attached halogen-/methoxy-phenacetamido tails to the molecules of aromatic or heterocyclic sulfonamides such as sulfanilamide, 3-halogenosulfanilamides, 4-aminomethyl/ethyl-benzenesulfonamide or 5-amino-1,3,4-thia-diazole-2-sulfonamide, as illustrated in Scheme 1. Reaction of the substituted-phenacetyl chlorides 1a–1d with the amino-containing aromatic sulfonamides 2a–2f afforded the aromatic derivatives 3a–3x, whereas the same reaction involving the heterocyclic derivative 4 afforded the substituted 1,3,4-thiadiazole-2-sulfonamides 5a–5d. We have chosen various acyl chlorides 1a–1d in order to generate chemical diversity in this new class of sulfonamides (Scheme 1). The new compounds were characterized by physicochemical procedures which confirmed their structure Materials and Methods.
determined for the tumor-associated hCA IX and hCA XII (Table 1). The KI values for the cytosolic hCA I and hCA II enzymes are also shown, although they constitute offtargets when antitumor CAIs are searched for. It may be observed that compounds 3 and 5 show excellent CA inhibitory properties (Table 1). All obtained KI values for hCA II, hCA IX and hCA XII were similar to or lower than 10 nM, with the exception of compound 3b. In addition, the values are similar to or lower than the KI value obtained for the reference compound AZ. Interestingly, compound 3b showed the highest KI value for all four isoforms. A fluorine atom (compound 3a) or a methoxy group (compound 3d) instead of the para-chlorine atom seems to be preferred at this position as the KI values for all four enzymes decreased significantly. The KI values for hCA I were generally in the lower nanomolar range (KI range: 2.9–346 nM). In all cases, a bromine atom at the 2-position of the phenyl moiety was well-tolerated. In general, a chlorine atom at the p-position of the benzenesulfonamide moiety was well-tolerated (compounds 3j–3l). By increasing the spacer length (n; Table 1) from 0 to 2 was beneficial for the enzyme inhibitory action of these compounds. Finally, replacing the benzenesulfonamide moiety by a thiadiazole-2-sulfonamide moiety also led to good inhibitors. The KI values for the cytosolic hCA II and the tumor-associated hCA IX and hCA XII were close to each other (with the exception of
Table 1 Inhibition of human CA isoforms hCA I, hCA II, hCA IX and hCA XII with compounds 3 and 5
2.2. Carbonic anhydrase inhibition
O
The inhibition values (KIs) of the reference compound acetazolamide (AZ) and the compounds 3 and 5 reported here have been
O N H
X
n
Y
O
+ Cl
X
Compd
2a-d: n=0, Y=H, F, Cl, Br 2e: n=1, Y=H 2f: n=2, Y=H N H2N
N
SO 2 NH 2
S
Cl–
4
O N H
X NEt3/MeCN
N S
SO2NH2
SO2NH2
5a-d
n
H2 N
X, Y, n
Y
1a: X = 4-F 1b: X = 4-Cl 1c: X = 2-Br 1d: X = 4-OCH3
N H
X
3a-x
SO2NH2
N
3a-x
n
SO2NH2
Y
O
N
N
SO2NH2 N H
X
S
5a-d Scheme 1. Preparation of sulfonamides 3a–x and 5a–d.
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3s 3t 3u 3v 3w 3x 5a 5b 5c 5d AZ
4-F, H, 0 4-Cl, H, 0 2-Br, H, 0 4-MeO, H, 0 4-F, F, 0 4-Cl, F, 0 2-Br, F, 0 4-MeO, F, 0 4-F, Cl, 0 4-Cl, Cl, 0 2-Br, Cl, 0 4-MeO, Cl, 0 4-F, Br, 0 4-Cl, Br, 0 2-Br, Br, 0 4-MeO, Br, 0 4-F, H, 1 4-Cl, H, 1 2-Br, H, 1 4-MeO, H, 1 4-F, H, 2 4-Cl, H, 2 2-Br, H, 2 4-MeO, H, 2 4-F, —, — 4-Cl, —, — 2-Br, —, — 4-MeO, —, — —
KIa (nM) hCA I
hCA II
hCA IXb
hCA XIIb
102 346 4.8 116 104 236 8.6 106 254 9.8 4.1 8.5 246 166 6.5 8.7 109 109 4.3 4.9 101 9.7 8.2 3.7 223 4.9 7.6 2.9 250.0
7.9 104 0.96 0.79 8.7 6.9 1.9 0.78 9.5 7.3 0.91 0.74 8.9 0.94 0.80 0.69 9.3 0.81 0.77 0.88 3.8 6.4 0.72 0.81 3.2 0.70 0.70 0.76 12.1
4.7 72.5 0.44 0.32 7.2 7.8 1.6 1.8 10.1 6.3 0.74 0.41 5.0 1.2 0.52 0.62 3.1 0.53 1.0 0.45 6.5 1.2 0.57 0.25 2.1 1.3 0.61 0.31 25.4
8.0 72.4 2.1 0.64 6.3 4.2 0.62 1.8 6.6 3.1 1.3 0.24 4.8 2.3 0.91 0.54 4.5 0.57 1.5 0.94 3.9 3.4 0.83 0.43 3.4 2.5 0.74 0.63 5.6
a Mean ± standard error, from three different assays, by a CO2 hydration stoppedflow assay. b Human, recombinant isozymes, pH 7.5, 20 mM TRIS–HCl buffer.
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compound 3b, the KI value range for hCA II: 0.69–9.5 nM; for hCA IX: 0.31–10.1 nM; for hCA XII: 0.24–6.6 nM) and in all cases lower than the respective KI values for hCA I (Table 1). The compounds investigated here have selectivity ratios for inhibiting CA IX and XII over CA I and II better or comparable to compounds in preclinical evaluation as anticancer/antimetastatic CAIs.2,3
His64
Gln67
Zn2+
Gln92
Trp5
2.3. Structural comparisons of hCA I, hCA II, hCA IX and hCA XII isozymes The crystal structures of hCA I (PDB: 3LXE), hCA II (PDB: 3B4F), hCA IX (PDB: 3IAI) and hCA XII (PDB: 1JD0) superpose well on their Ca-atoms (RMSD: 1.561 Å). In general, the backbone fold of the four enzymes is very similar, especially in the active site, and the Zn2+-binding residues (His94, His96 and His119), Thr199 and Zn2+ superpose well. Nevertheless, minor differences in the backbone fold are present near the active site near the conserved His64 (Val62-Ser65 of hCA I) and near Leu131 of hCA I (Trp123Leu141). The binding pocket residues of the four enzymes, which are defined as all residues within 4.5 Å of topiramate (hCA I), have been compared (Table 2). His64 (proton shuttle) and Gln92 (hydrogen bond formation to ligands) are conserved amongst the four enzymes. Other binding pocket residues that are at contact distance to bound ligands are not conserved (Table 2). At position 67 a positively charged lysine is present in hCA XII, while hydrogen bond forming Asn67 and Gln67 are present in hCA II and hCA IX, respectively. At position 131, a phenylalanine is present in hCA II, which can form hydrophobic stackings with the ligand. This interaction is not thought to introduce extra stabilizing ligand-protein interactions and it is absent in the other three enzymes.
Figure 1. Binding interactions of compounds 3q (brown), 3x (purple) and 5d (turquoise) with hCA IX. For clarity, only Trp5, His64, Gln67, Gln92 and Zn2+ are shown. Hydrogen bonds are depicted in red dotted lines.
Lys67
Zn2+
Asn62 His64
Gln92
Trp5 Thr91
2.4. Docking studies Compounds 3 and 5 and the reference compound AZ have lower KI values for hCA II, hCA IX and hCA XII compared to hCA I (Table 1). In addition, the KI values of the compounds for hCA II, hCA IX and hCA XII were generally below 10 nM. To understand the difference in KI values between these isoforms, we docked compounds 3 and 5 into the crystal structures of hCA I, hCA II, hCA IX and hCA XII using the GOLD Suite docking program and the ChemScore scoring function (25 dockings per ligand). The sulfonamide moiety of the ligands was assigned a negative charge (–SO2NH) and was forced to form hydrogen bonding interactions with Thr199 (which is observed in all X-ray crystal structures of CA sulfonamide adducts).1–4 The docking results for hCA IX and hCA XII indicate the importance of the conserved Gln92 in both enzymes. Many ligands formed hydrogen bonding interactions with the side chain of Gln92 through their carbonyl group (Figs 1 and 2). In addition, the ligands interacted with the side chains of Trp5, His64 and Gln67 of hCA IX and Trp5, Asn62, Lys67 and Thr91 of hCA XII. Figures 1 and 2 depict representative ligands that adopt the most
Figure 2. Binding interactions of compounds 3m (turquoise), 3v (green) and 3h (light brown) with hCA XII. The ligands mainly form hydrogen bonding (red dotted line) with Gln92, but to a lesser extend also with Trp5, Asn62, His64, Lys67 and Thr91. For clarity, only the hydrogen bonding interactions with Lys67 and Trp5 are shown. All three compounds form hydrogen bonds with Gln92, but these are not shown for clarity.
Asn67 Gln92 His64 Zn2+ Phe91 Ile91
Table 2 Binding pocket residues of hCA I, hCA II, hCA IX and hCA XII hCA I
hCA II
hCA IX
hCA XII
Trp5 Val62 His64 His67 Phe91 Gln92 Leu131 Ala135 His200
Trp5 Asn62 His64 Asn67 Ile91 Gln92 Phe131 Val135 Thr200
Trp5 Asn62 His64 Gln67 Leu91 Gln92 Val131 Leu135 Thr200
Trp5 Asn62 His64 Lys67 Thr91 Gln92 Ala131 Ser135 Thr200
Leu131 Phe131
Trp5
Figure 3. Binding interactions of compounds 5a (purple) with hCA II. It forms hydrogen bonds with Gln92 (red dotted lines) and hydrophobic stackings with Phe131. In hCA I, Leu131 (brown) is present instead of Phe131 and the stackings cannot be formed. In addition, Phe91 (brown) is present instead of Ile91.
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commonly observed binding poses within the hCA IX and hCA XII active sites, respectively. Trp5 and His64 are conserved in all four isoforms investigated here, whereas Asn62 is conserved in hCA II, hCA IX and hCA XII (Table 2). However, our docking studies suggest that only Trp5 and Gln92 are involved in binding interactions in both enzymes (hCA IX aand XII). In general, the binding interactions with His64 and Gln67 of hCA IX and Asn62, Lys67 and Thr91 of hCA XII were less frequently observed. The docking studies also indicated that Phe131 in hCA II forms hydrophobic stackings with the halophenyl or methoxyphenyl tails of compounds 3 and 5 (Fig. 3). This residue is not conserved amongst the other three enzymes (Table 2). In addition, the ligands can form hydrogen bonding interactions with Gln92. In hCA I, a bulky Leu131 is positioned at the Phe131 (hCA II) position. In addition, Phe91 is present close to Leu131 in hCA I. Phe91 is not correctly positioned with respect to the ligands to form hydrophobic stackings with the ligands and it is expected to sterically hinder the ligands to adopt similar poses as observed for hCA II, hCA IX and hCA XII. In summary, the docking studies indicate that various hydrogen bonds with the active site residues of hCA IX and hCA XII and hydrophobic stacking with Phe131 of hCA II allow favorable binding interactions with these isoforms. Probably, the absence of Phe131 and the presence of Phe91 and Leu131 residues in hCA I (see Table 2) restrict the ligands to form similar binding interactions with hCA I. 3. Conclusions The tested sulfonamides show very potent inhibition of four physiologically relevant CA isoforms, hCA I, II, IX and XII, with KI values in the nanomolar range, for the tumor-associated hCA IX and hCA XII. Docking studies have revealed details regarding the very favorable interactions between the scaffolds of this new class of inhibitors and the active sites of the CA isoforms investigated here. As there are reported cases of tumors overexpressing both CA II and IX,7 such potent inhibitors for the two isoforms may have clinical importance.
4. Materials and methods 4.1. Chemistry Compounds 3–5 investigated here as CAIs were recently described by our group as Trypanosoma cruzi CA inhibitors.18 4.2. CA inhibition assay An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes as reported by Khalifah.8 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nM, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration),9 following the CA-catalyzed CO2 hydration reaction for a period of 5–10 s. Saturated CO2 solutions in water at 25 °C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO–water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. Trip-
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licate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier,10,11 and represent the mean from at least three different determinations. Human CA isozymes were prepared in recombinant form as reported earlier by our groups.12,13 4.3. Ligand preparation Compounds 3, 5 and the reference ligand AZ were built and converted to three-dimensional structures using the MOE software package (version 2011.10, Chemical Computing Group, Montreal, Canada). The sulfonamide substituent of the phenyl group was assigned a negative charge (–SO2NH) because it was expected to interact with the Zn2+ ion of the CA active sites. Subsequently, partial atomic charges were calculated and the molecules were energy-minimized according to a steepest-descent protocol using the MMFF94x force field in MOE. The ligands were saved as a multi-mol2 file. 4.4. Template preparation Crystal structures of hCA I (PDB: 3LXE; 1.90 Å),14 hCA II (PDB: 3B4F; 1.89 Å),13 hCA IX (PDB: 3IAI; 2.20 Å)15 and hCA XII (PDB: 1JD0; 1.50 Å)16 were obtained from the protein databank. Only 1 protein chain (chain A) and its corresponding Zn2+ ion and ligand (CA inhibitor) was retained per PDB file and all other protein chains, ions, buffer molecules and water molecules were deleted. Hydrogen atoms and charges were added to the protein using the protonate 3D tool of the MOE software package (version 2011.10, CCG, Montreal, Canada) and a steepest descent energy minimization was applied (MMFF94x forcefield). All proteins were superposed on the hCA I structure using their Ca-atoms (RMSD: 1.561 Å) and the proteins were saved as mol2-files. 4.5. Docking studies Compounds 3, 5 and the reference ligand AZ were docked into the protein models of hCA I, hCA II, hCA IX and hCA XII using the GOLD Suite docking package (version 5.1, CCDC, Cambridge, UK)17 and the ChemScore scoring function (25 docking per ligand). The binding pocket was defined as all residues within 12 Å of the central carbon atom of the CA inhibitor topiramate (atom CAL of topiramate in complex with hCA I; PDB: 3LXE). No restrictions were applied for the ligand conformations and binding poses during the docking procedure except for the requirement to form hydrogen bonding with Thr199 (or its counterparts in other CA isozymes). Acknowledgments This project was in part financed by the Istanbul University Scientific Research Projects Department under project number UDP22409 and by an FP7 EU project (Metoxia). References and notes 1. (a) Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. R.; Gitlin, I.; Gudiksen, K. L.; Weibel, D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946; (b) Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336; (c) Domsic, J. F.; Avvaru, B. S.; Kim, C. U.; Gruner, S. M.; Agbandje-McKenna, M.; Silverman, D. N.; McKenna, R. J. Biol. Chem. 2008, 283, 30766. 2. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767. 3. (a) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 8371; (b) Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna,
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