Carbonic Anhydrase Inhibitors with Dual-Tail Moieties To Match the

Jan 12, 2015 - Carbonic Anhydrase Inhibitors with Dual-Tail Moieties To Match the. Hydrophobic and Hydrophilic Halves of the Carbonic Anhydrase...
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Carbonic Anhydrase Inhibitors with Dual-Tail Moieties To Match the Hydrophobic and Hydrophilic Halves of the Carbonic Anhydrase Active Site Rajendra P. Tanpure,† Bin Ren,‡ Thomas S. Peat,‡ Laurent F. Bornaghi,† Daniela Vullo,§ Claudiu T. Supuran,*,§ and Sally-Ann Poulsen*,† †

Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia § Polo Scientifico, Neurofarba Department and Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy ‡

S Supporting Information *

ABSTRACT: We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

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(PDB).6 Although the inhibition of CAs has been of therapeutic interest for several decades, the development of isozyme-selective inhibitors has been addressed only much more recently, facilitated in part by improving knowledge of the CA active site topography. Amino acid residues in and around the upper rim of the active site of CAs have been shown to form a number of selective binding pockets, whereas, overall, the CA active site cavity is partitioned into two conserved environments comprising a lining of hydrophobic amino acids (I91, V121, F131, V135, L141, V143, L198, P202, L204, V207, and W209) on one side and hydrophilic amino acids (Y7, N62, H64, N67, Q92, T199, and T200) lining the other side (residue numbering based on hCA II).6,9 It is noteworthy that all of the clinically used CA inhibitors exhibit broad CA isozyme inhibition; thus, their use is associated with side effects. These compounds are compact and do not extend to form contacts with the identified selective CA binding regions.10 Different hCA isozymes exhibit variable tissue distribution, subcellular location, and expression profiles in healthy versus diseased cells.5,11 Together, these differences enable opportunities for medicinal chemistry programs to inhibit specific CA

INTRODUCTION Carbonic anhydrases (CA, EC 4.2.1.1) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton: CO2 + H2O ⇆ HCO3− + H+. An understanding of the impact of this critical equilibrium to human health continues to develop; for example, the CAmediated regulation of pH in the hypoxic tumor microenvironment is now proposed to be used as a therapeutic target.1−4 Humans encode 12 catalytically active α-CA isozymes, many of which have been studied both functionally and structurally. These CAs comprise CA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, and XIV, all of which contain a Zn2+ cation located at the base of a 15 Å deep funnel-shaped active site cavity that is coordinated to the imidazole groups of three histidine residues and to the substrate H2O/hydroxide that reacts with CO2.5 Most reported small molecule CA inhibitors comprise a primary sulfonamide moiety as a zinc binding group (ZBG). The sulfonamide anion (−SO2NH−) is known to coordinate to the active site Zn2+ and to block catalysis. Protein X-ray crystal structures are available for the majority of human CA isozymes, and they show a highly conserved secondary structure despite amino acid differences.6−8 hCA II (h = human) is the most well-studied, with >100 unique sulfonamide ligand (R− SO2NH2) complexes reported in the Protein Data Bank © XXXX American Chemical Society

Received: November 20, 2014

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Journal of Medicinal Chemistry isozymes by designing compounds with a combination of structure−activity relationships (SAR) and structure−property relationships (SPR); this approach is now at the forefront of most promising medicinal chemistry campaigns.12,13 There have been two widely employed approaches toward the development of carbonic anhydrase inhibitors: (i) the ring approach and (ii) the tail approach.14,15 In the ring approach, an aromatic ring is attached directly to a primary sulfonamide group: most clinically used CA inhibitors are examples of this approach. In the tail approach, the aromatic sulfonamide ring incorporates further substitution with tail moieties that are selected to modulate interactions with the enzyme active site (SAR), the physicochemical properties of the molecule (SPR), or, ideally, both SAR and SPR. A flexible hydrophobic tail group will generally adopt a conformation to interact with the hydrophobic half of the CA active site; similarly, a flexible hydrophilic tail substituent will generally interact with the hydrophilic half of the CA active site.10 Amino acid F131 has been shown to play a key role in orienting the inhibitor molecules within the CA II active site,6,9,10 and the position of residue 131 is variable among CA isozymes. A specific example of the tail approach, known as click tailing, combines the tail approach with click chemistry (copper-catalyzed azide−alkyne cycloaddition, CuAAC) for the straightforward covalent linking of diverse tail fragments onto a known CA pharmacophore scaffold via formation of a linking 1,2,3-triazole moiety.12,16−18 The 1,2,3-triazole is a nonclassical peptide bond bioisostere and may participate either passively (linker only) or actively (linker plus interactions with biological target) in the resulting hybrid compound.19,20 The 1,2,3-triazole-bridged tail substitution of these inhibitors is positioned to interrogate the outer rim of the CA active site cavity, a region where there is the greatest difference in amino acid residues among the different CA isozymes.6,9 We have designed and synthesized dual-tail CA inhibitors using the click tail approach, i.e., inhibitors with two distinct tail moieties that are oriented in opposing directions.

Figure 1. (A) Target CA inhibitors with dual-tail moieties derived from acetazolamide (AZA). The tail combinations include (i) two hydrophobic moieties (blue) 1, (ii) two hydrophilic moieties (red) 2, and (iii) one hydrophobic (blue) and one hydrophilic (red) moiety 3. (B) Corresponding single-tail CA inhibitors 8 and 9.

enable a comparative analysis of the single-tail versus dual-tail enzyme inhibition profile (Figure 1). The target dual-tail compounds 1−3 were synthesized starting with commercially available AZA. Upon treatment of AZA with HCl in EtOH, the acetamide moiety is removed to form 5-amino-1,3,4-thiadiazole-2-sulfonamide 4. Next, the primary amine is bis-alkylated with propargyl bromide to install two terminal alkyne groups and thus two synthetic handles for click tailing (compound 5). Reaction with the selected hydrophobic (phenyl azide) and hydrophilic (glucose azide) complementary azide building blocks was implemented using CuAAC to synthesize 1 and the per-O-acetylated precursors for 2 and 3, compounds 6 and 7, respectively (Scheme 1). Standard conditions (methanolic NaOMe) to remove acetate groups caused ring opening of the 1,3,4-thiadiazole of 6 and 7 and so could not be employed. Instead, the acetates of compounds 6 and 7 were removed under acidic conditions to give the final compounds, glycoconjugates 2 and 3, respectively. The homogeneous dual-tail compounds 1 and 2 proved to be straightforward to synthesize; however, removal of copper residues during purification was difficult owing to the likely coordination of copper to the triazole−triazole−thiadiazole system.22 The heterogeneous dual-tailed compound 3 was obtained as a mixture of the three possible expected products, and, as was the case for compounds 1 and 2, removal of copper was difficult; however, the mixture of products presented an added layer of purification difficulty for compound 3, lowering the recovered yield of this compound. For the single-tail compounds, AZA was alkylated directly with propargyl bromide to prepare the alkyne 10; this compound was then reacted with either phenyl azide or glucosyl azide to form 11 and 12, respectively, by CuAAC. Compounds 11 and 12 were then deacetylated using basic (for 11) and acidic (for 12) conditions to form 8 and 9, respectively (Scheme 2).

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RESULTS AND DISCUSSION 5-Acetamido-1,3,4-thiadiazole-2-sulfonamide, or acetazolamide (AZA), is considered the par excellence CA inhibitor and is approved for the treatment of a range of conditions including glaucoma, epilepsy, and altitude sickness.21 The core structure of AZA comprises the 1,3,4-thiadiazole heterocycle, and to this core heterocycle is attached a primary sulfonamide substituent at the 2 position (the ZBG) and an acetamide substituent at the 5 position. AZA has a compact 3D structure and interacts similarly with different CA isozymes; hence, although it is a good CA inhibitor, AZA is also considered to be a broadspecificity CA inhibitor owing to its widespread inhibition of CAs. By adding dual tail groups to the AZA primary sulfonamide warhead, compounds 1−3 can be made to interrogate the outer region of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. The tail combinations selected include (i) two hydrophobic moieties, compound 1; (ii) two hydrophilic moieties, compound 2; and (iii) one hydrophobic and one hydrophilic moiety, compound 3, Figure 1. We expected that this dual-tail approach could provide an opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the CA active site. In addition to the dual-tail compounds, a corresponding set of single-tail analogues, compounds 8 and 9, was also designed and synthesized to B

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Journal of Medicinal Chemistry Scheme 1. Synthetic Route for the Synthesis of Target CA Inhibitors 1−3 with Dual-Tail Moietiesa

Reagents and conditions: (i) 6.25 equiv HCl, EtOH, reflux, 16 h; (ii) 2.2 equiv propargyl bromide, 2.0 equiv Cs2CO3, DMF, 0 °C → rt, 16 h; (iii) 2.0 equiv azide, 0.4 equiv CuSO4·5H2O, 0.8 equiv sodium ascorbate, tBuOH/water 1:1, 40 °C, 8 h; (iv) 0.9 equiv of each azide, 0.4 equiv CuSO4· 5H2O, 0.8 equiv sodium ascorbate, tBuOH/water 1:1, 40 °C, 8 h; (v) 8% HCl in MeOH, rt, 3 days.

a

Scheme 2. Synthetic Route for the Synthesis of CA Inhibitors 8 and 9 with Single-Tail Moietiesa

Reagents and conditions: (i) 1.4 equiv propargyl bromide, 1.0 equiv Cs2CO3, DMF, rt, 16 h; (ii) 1.0 equiv azide, 0.4 equiv CuSO4·5H2O, 0.8 equiv sodium ascorbate, tBuOH/water 1:1, 40 °C, 8 h; (iii) (a) NaOMe (25% w/v in MeOH), rt, 4 h; (b) 1.0 M HCl; (iv) 8% HCl in MeOH, rt, 5 days. a

inhibitors, possibly attributed to the loss of the AZA NH proton. Compensation by the additional interactions of the two hydrophobic tail moieties of 1 (Ki of 83 nM) partly offset the loss of activity of 5, indicating that the hydrophobic pocket of CA II is being accessed by this compound. Similarly, compound 3 with one hydrophobic tail moiety has less, but also improved, activity (Ki of 114 nM) compared to that of 5. The two hydrophilic tail moieties of 2 (Ki of 201 nM) lead to inhibition that is equipotent with that of 5, demonstrating that the interactions with the hydrophilic half of the CA active site are not optimal; however, they also do not hamper compound

The CA inhibition data (to block the interconversion of CO2 with HCO3− and a H+) for compounds 1−3, the corresponding single-tail derivatives 8 and 9, synthetic intermediate 5, and AZA as a reference compound was measured for CA I and II, and the results are presented in Table 1. At hCA II, the Ki values for compounds 1, 2, and 3 were 83, 201, and 114 nM, respectively. This inhibition is weaker than that for the parent compound AZA (Ki of 12 nM); however, it is similar to that of the bis-alkyne synthetic intermediate 5 (Ki of 201 nM), suggesting that the bis-alkylation of AZA impairs binding interactions with CA II and lowers the Ki’s of dual-tail C

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Journal of Medicinal Chemistry Table 1. Inhibition of Human CA Isozymes I and II by DualTail Compounds 1−3, Corresponding Single-Tail Derivatives 8 and 9, Synthetic Intermediate 5, and Parent Compound AZAa compd

CA I Ki (nM)

CA II Ki (nM)

1 2 3 5 8 9 AZA

7445 >20 000 3190 5665 141 87.0 250

83 201 114 201 9.1 2.7 12

Errors are in the range of ±5% of the reported value, from three determinations.

a

binding further, so it should be possible to improve the structure−activity relationship through the design of secondgeneration dual-tail inhibitors, for example, with the incorporation of hydrophobic substituents that are larger than the phenyl moiety. The Ki values for single-tail compounds 8 and 9 are 9.1 and 2.7 nM, respectively. This is substantially improved inhibition compared to that of compounds 1−3 and adds further support to the idea that retention of the NH proton of AZA in second-generation compounds is sensible; however, importantly, it also demonstrates that both tail groups (hydrophilic glucosyl moiety and hydrophobic phenyl moiety) are independently well-tolerated by the CA II active site. The mode of binding of the heterogeneous dual-tailed compound 3 to CA II was of substantial interest and was studied using X-ray crystallography. The data extend to 1.53 Å resolution, and most protein residues and most of compound 3 are seen in good density. The electron density for compound 3 becomes weaker with increasing distance from the sulfonamide group, with the hydrophobic phenyl and hydrophilic glucosyl tail substituents beyond the triazole linkers having broken density. Nonetheless, on one side, there is planar density, and this is where the phenyl moiety has been placed; reversing the orientation results in higher R factors after refinement. The compound makes multiple interactions with the protein as it extends out of the catalytic pocket (Figure 2). The sulfonamide functional group interacts with the protein similar to that for >100 sulfonamide/CA II complexes reported in the PDB, with the canonical sulfonamide zinc interactions including the NH− moiety in the sulfonamide group participating in the coordination interactions with the zinc, which is also within hydrogen-bonding distance with the OG1 atom of Thr198 (2.69 Å), whereas one oxygen atom of the sulfonamide group forms another hydrogen bond with the main chain nitrogen of Thr198 (3.00 Å). The two nitrogens in the 1,3,4-thiadiazole are within hydrogen-bonding distance of the OG1 atom of Thr199 (2.89 and 3.08 Å). In the tail region of 3, the N3 nitrogen of the 1,2,3-triazole of the hydrophilic tail forms a hydrogen bond with the NE2 atom of Gln92 (2.73 Å). A glycerol molecule is located on top of the 1,3,4-thiadiazole heterocyclic ring system. Although not having direct binding interactions in the structure, the hydrophilic glucosyl tail group of 3 is in close proximity to a number of polar or charged residues, including Gln92, Asn67, Glu69, Asp72, and Arg58. In contrast, the phenyl group of the hydrophobic tail is neighbored by a number of hydrophobic residues, including Phe130, Val134, Leu203, Pro201, and Leu197. A surface representation of hCA II with the binding of the heterogeneous

Figure 2. Interactions and environment at the binding site in the structure of hCA II/3 complex. Hydrogen bonds are shown as broken lines, and the Zinc ion, as a sphere. The picture was produced with PyMOL.23

dual-tailed compound 3 is shown in Figure 3. This representation shows that the interactions with the hydro-

Figure 3. Surface representation of hCA II with the binding of the heterogeneous dual-tailed compound 3. Hydrophobic half, red; hydrophilic half, blue. The picture was prepared with PyMOL.23

phobic half of the CA II active site dominate the binding orientation of 3, whereas the hydrophilic glucosyl moiety is not well aligned to form close interactions with the hydrophilic half of the CA active site. This initial study has successfully provided a novel dual-tail approach to CA inhibitor design, wherein compounds exploit a combination of hydrophobic and hydrophilic substituents to complement the distinct active site architecture of CAs. The design of next-generation dual-tail inhibitors will build on the valuable SAR obtained in this study. Notably, the enzyme inhibition profile and hCA II/3 complex structure affirm the proof-of-concept of this dual-tail approach, and future efforts from our groups will aim to optimize the interactions with both active site halves through the design of new inhibitor structures.

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EXPERIMENTAL SECTION

Compound Synthesis. All reagents were purchased from commercial suppliers. The synthesis of the β-D-glucopyranosyl azide has been reported by us previously.24 All reactions were monitored using TLC: TLC plates were visualized with UV fluorescence (λ = 254 D

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Journal of Medicinal Chemistry nm). Silica gel flash chromatography was performed using silica gel 60 Å (230−400 mesh). 1H NMR were acquired at 500 MHz, and 13C NMR, at 125 MHz at 30 °C. For 1H and 13C NMR acquired in DMSO-d6, chemical shifts (δ) are reported in ppm relative to the solvent residual peak: proton (δ 2.50 ppm) and carbon (δ 39.5 ppm) signals, respectively. Assignments for 1H NMR were confirmed by 1 H−1H gCOSY, and assignments for 13C NMR were confirmed by 1 H−13C HSQC. Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublet), and b (broad). Coupling constants are reported in hertz (Hz). Melting points are uncorrected. High- and low-resolution electrospray ionization mass spectra were acquired using electrospray as the ionization technique in positive ion and/or negative ion modes as stated. All MS analysis samples were prepared as solutions in MeOH. Purity of all compounds was ≥95%, as determined by HPLC with UV detection. 5-Amino-1,3,4-thiadiazole-2-sulfonamide (4). A suspension of acetazolamide (22.23 g, 100 mmol, 1 equiv) in ethanol (200 mL) and HCl (32%, 60 mL, 625 mmol, 6.25 equiv) was prepared in a 500 mL round-bottomed flask. The reaction was refluxed at 95 °C; upon reflux, the suspension became a colorless homogeneous solution. After 16 h, the reaction was cooled to room temperature, and the solvent was removed under reduced pressure. The concentrate was diluted with water (100 mL) and neutralized with NaOH (5.0 M, ∼20 mL). The precipitate that formed was filtered and dried in vacuo to obtain 1 (12.4 g, 69%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 8.04 (s, 2H, NH2), 7.79 (s, 2H, SO2NH2). 13C NMR (125 MHz, DMSOd6): δ 171.6 (C5), 157.9 (C2). Consistent with literature.25 5-N,N-Bis(prop-2-yn-1-yl)amino-1,3,4-thiadiazole-2-sulfonamide (5). 5-Amino-l,3,4-thiadiazolo-2-sulfonamide 4 (1.84 g, 10.2 mmol, 1.0 equiv), Cs2CO3 (6.56 g, 20.4 mmol, 2.0 equiv), and DMF (20 mL) were combined in a 100 mL flask at 0 °C to form a suspension. Propargyl bromide (2.5 mL, 22.4 mmol, 2.2 equiv) was added, and the reaction mixture was stirred overnight until the reaction was complete, as evidenced by TLC using 50% EtOAc/ hexane. The reaction was quenched with water (50 mL) and extracted with EtOAc (3 × 50 mL). The organic fractions were combined and dried over MgSO4, and the solvent was evaporated in vacuo to obtain a pale brown liquid. Column chromatography (EtOAc/hexane 1:1) of this crude product gave title compound 2 (1.00 g, 38%) as a pale yellow solid. Rf = 0.32 (EtOAc/hexane 1:1). mp 183−185 °C (dec.). 1 H NMR (500 MHz, DMSO-d6): δ 8.06 (s, 2H, SO2NH2), 4.21 (d, J = 2.5 Hz, 4H, CH2), 3.38 (t, J = 2.5 Hz, 2H, ≡CH). 13C NMR (125 MHz, DMSO-d6): δ 172.9 (C5), 152.6 (C2), 77.5 (≡CH), 76.7 (C≡), 37.4 (CH2). LRMS (ESI+): m/z = 257.5 [M + H]+. HRMS (ESI+): m/ z calcd for C8H8N4O2S2Na1 [M + Na+], 278.9981; found, 278.9983. 5-N-(Prop-2-yn-1-yl)acetamido-1,3,4-thiadiazole-2-sulfonamide (10). Acetazolamide (11.26 g, 50.7 mmol, 1.0 equiv) and Cs2CO3 (16.51 g, 50.7 mmol, 1.0 equiv) were combined in DMF (30 mL) in a 250 mL flask to form a suspension. Propargyl bromide (8 mL, 71.8 mmol, 1.4 equiv) was added, and the reaction mixture was stirred at room temperature overnight. The reaction was monitored using TLC (EtOAc/hexane 1:1). On completion, the reaction was quenched with water (100 mL) and extracted with EtOAc (2 × 100 mL). The organic fractions were combined and dried over MgSO4, and the solvent was evaporated under reduced pressure to obtain title compound 10 (5.98 g, 45.3%) as a white solid. Rf = 0.29 (EtOAc/ hexane 1:1). mp 178−180 °C (dec.). 1H NMR (500 MHz, DMSOd6): δ 8.37 (s, 2H, SO2NH2), 5.13, (d, J = 2.5 Hz, 2H, CH2), 3.45 (t, J = 2.5 Hz, 1H, ≡CH), 2.56 (s, 3H, COCH3). 13C NMR (125 MHz, DMSO-d6): δ 171.1 (CO), 167.0 (C2), 161.1 (C5), 78.4 (C≡), 76.2 (≡CH), 38.3 (CH2), 22.2 (COCH3). LRMS (ESI+): m/z = 261.5 [M + H]+. HRMS (ESI+): m/z calcd for C7H8N4O3S2Na1 [M + Na+], 282.9930; found, 282.9932. General Procedure 1: CuAAC Reaction Conditions. A mixture of azide (1.0 or 2.0 equiv) and alkyne (1.0 equiv) was suspended in a H2O/tBuOH mixture (1:1, 0.2−0.5 M final concentration). A solution of sodium ascorbate (0.8 equiv) and CuSO4·5H2O (0.4 equiv) in water (1 mL) was added to the reaction mixture. The bright yellow suspension was stirred vigorously at 40 °C until either full

consumption of the azide or no further change was observed, as evidenced by TLC. The reaction mixture was cooled, diluted with water (50 mL), and extracted with EtOAc (3 × 50 mL). The combined organic fractions were dried over MgSO4, and the solvent was evaporated under reduced pressure. Purification of the product triazoles were carried out as described below. 5-N,N-Bis[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]amino1,3,4-thiadiazole-2-sulfonamide (1). Title compound 1 was synthesized from alkyne 5 (0.260 g, 1.01 mmol, 1 equiv) and phenylazide (0.602 g, 5.05 mmol, 5 equiv) according to general procedure 1 in 8 h. Purification of the crude product by flash chromatography (65% EtOAc/hexane) gave the product, bistriazole 1 (0.276 g, 55%), as a white solid. Rf = 0.11 (acetone/hexane 2:3). mp 202−203 °C (dec.). 1H NMR (500 MHz, DMSO-d6): δ 8.70 (s, 2H, CHtriazole), 7.95 (s, 2H, SO2NH2), 7.84 (d, J = 7.8 Hz, 4H, H2′,6′), 7.60 (t, J = 7.8 Hz, 4H, H3′,5′), 7.51 (t, 7.8 Hz, 2H, H4′), 4.77 (s, 4H, CH2). 13 C NMR (125 MHz, DMSO-d6): δ 172.5 (C5), 154.4 (C2), 143.1 (Ctriazole), 137.0 (C1′), 130.3 (C3′,5′), 129.2 (C4′), 123.1 (CHtriazole), 120.7 (C2′,6′), 43.2 (CH2). LRMS (ESI+): m/z = 495.3 [M + H]+. HRMS (ESI+): m/z calcd for C20H18N10O2S2Na1 [M + Na+], 517.0948; found, 517.0947. 5-N,N-Bis([1-{2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosyl}1H-1,2,3-triazol-4-yl]methyl)amino-1,3,4-thiadiazole-2-sulfonamide (6). Title compound 6 was synthesized from alkyne 5 (0.513 g, 2.002 mmol, 1 equiv) and β-D-glycopyranosyl azide (1.512 g, 4.05 mmol, 2 equiv) according to general procedure 1 in 8 h. Purification of the crude product by flash chromatography (EtOAc/hexane 4:1) gave product bis-triazole 6 (1.4 g, 70%) as an off white solid. Rf = 0.18 (EtOAc/hexane 4:1). mp 155−157 °C (dec.). [α]32 D −1.62 (c = 0.115, CH3OH). 1H NMR (500 MHz, DMSO-d6): δ 8.38 (s, 2H, CHtriazole), 7.90 (s, 2H, SO2NH2), 6.34 (d, J = 9.1 Hz, 2H, H1′), 5.65 (t, J = 9.4 Hz, 2H, H2′), 5.57 (t, J = 9.4 Hz, 2H, H3′), 5.19 (t, J = 9.7 Hz, 2H, H4′), 4.60 (d, J = 16.0 Hz, 2H, H6′), 4.40 (d, J = 16.0 Hz, 4H, H5′, H6′), 4.18 (dd, J = 5.5, 12.6 Hz, 2H, NCH2), 4.10 (dd, J = 2.4, 12.6 Hz, 2H, NCH2), 2.04 (s, 6H, OCOCH3), 2.02 (s, 6H, OCOCH3), 1.98 (s, 6H, OCOCH3), 1.82 (s, 6H, OCOCH3). 13C NMR (125 MHz, DMSOd6): δ 172.4 (C5), 170.5 (OCOCH3), 170.0 (OCOCH3), 169.8 (OCOCH3), 169.1 (OCOCH3), 154.6 (C2), 142.2 (Ctriazole), 123.9 (CHtriazole), 84.4 (C1′), 73.8 (C5′), 72.5 (C3′), 70.6 (C2′), 68.1 (C4′), 62.2 (C6′), 41.9 (CH2), 21.0 (OCOCH3), 20.8 (OCOCH3), 20.7 (OCOCH3), 20.4 (OCOCH3). HRMS (ESI+): m/z calcd for C36H46N10O20S2Na1 [M + Na+], 1025.2223; found, 1025.2241. 5-N,N-Bis([1-{β- D -glucopyranosyl}-1H-1,2,3-triazol-4-yl]methyl)amino-1,3,4-thiadiazole-2-sulfonamide (2). Title compound 2 was synthesized from per-O-acetylated precursor 6 (0.400 g, 0.399 mmol, 1 equiv) upon treatment with 8% HCl in MeOH (4 mL). The reaction mixture was sonicated for 1 min, the colorless solution turned cloudy, and a suspension was formed. The reaction was allowed to stir for 3 days and was monitored by normal-phase silica gel TLC using EtOAc as eluent and RP-18 silica gel using 10% ACN/H2O as eluent. The solvent was evaporated under reduced pressure. Column chromatography using RP-18 silica gel with 10% ACN/H2O gave title compound 2 (0.162 g, 61%) as a hygroscopic off-white solid. Rf = 0.92 1 (ACN/H2O 9:1). [α]25 D −7.21 (c = 0.125, CH3OH). H NMR (500 MHz, DMSO-d6): δ 8.24 (s, 2H, CHtriazole), 7.89 (s, 2H, SO2NH2), 5.54 (d, J = 9.2 Hz, 2H, H1′), 4.60 (s, 4H, CH2), 3.79−3.72 (m, 4H, H2′, H6′), 3.50−3.45 (m, 4H, H5′, H6′), 3.40 (t, J = 9.2 Hz, 2H, H3′), 3.25 (t, J = 9.2 Hz, 2H, H4′). 13C NMR (125 MHz, DMSO-d6): δ 172.7 (C5), 154.5 (C2), 141.9 (Ctriazole), 124.0 (CHtriazole), 88.0 (C1′), 80.4 (C5′), 77.3 (C3′), 72.6 (C2′), 70.1 (C4′), 61.3 (C6′), 42.5 (NCH2). LRMS (ESI+): m/z = 667.33. [M + H]+. HRMS (ESI+): m/z calcd for C20H30N10O12S2Na1 [M + Na+], 689.1378; found, 689.1388. 5-(N-[1-Phenyl-1H-1,2,3-triazol-4-yl]methyl-N-[1-{2′,3′,4′,6′tetra-O-acetyl-β- D -glucopyranosyl}-1H-1,2,3-triazol-4-yl]methyl)amino-1,3,4-thiadiazole-2-sulfonamide (7). Title compound 7 was synthesized from alkyne 5 (1.410 g, 5.50 mmol, 1.0 equiv), phenylazide (0.600 g, 5.04 mmol, 0.9 equiv), and β-Dglycopyranosyl azide (1.860 g, 4.98 mmol, 0.9 equiv) according to general procedure 1 in 8 h. The reaction mixture contained the desired mixed triazole 7 as well as the other two possible bis triazoles. E

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Journal of Medicinal Chemistry

−3.3 (c = 0.12, CH3OH). 1H NMR (500 MHz, DMSO-d6): δ 8.49 (s, 1H, CHtriazole), 8.33 (s, 2H, SO2NH2), 6.32 (d, J = 9.1 Hz, 1H, H1′), 5.61 (t, J = 9.4 Hz, 1H, H2′), 5.57 (s, 2H, CH2), 5.52 (t, J = 9.4 Hz, 1H, H3′), 5.16 (t, J = 9.7 Hz, 1H, H4′), 4.35 (m, 1H, H5′), 4.10 (m, 2H, H6′), 2.57 (s, 3H, NCOCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.73 (s, 3H, COCH3). 13C NMR (125 MHz, DMSO-d6): δ 171.5 (NCOCH3), 170.5 (OCOCH3), 170.0 (OCOCH3), 169.8 (OCOCH3), 168.8 (OCOCH3), 166.7 (C5), 161.5 (C2), 143.1 (Ctriazole), 123.4 (CHtriazole), 84.4 (C1′), 73.8 (C5′), 72.5 (C3′), 70.7 (C2′), 68.0 (C4′), 62.2 (C6′), 43.9 (NCH2), 22.4 (NCOCH3), 21.0 (OCOCH3), 20.8 (OCOCH3), 20.7 (OCOCH3), 20.2 (OCOCH3). LRMS (ESI+): m/z = 634.6 [M + H]+. HRMS (ESI+): m/z calcd for C21H27N7O12S2Na1 [M + Na+], 656.1051; found, 656.1052. 5-(1-[β-D-Glucopyranosyl]-1H-1,2,3-triazol-4-yl)methylamino-1,3,4-thiadiazole-2-sulfonamide (9). Title compound 9 was synthesized from per-O-acetylated precursor 12 (0.205 g, 0.324 mmol, 1 equiv) upon treatment with 8% HCl in MeOH (8 mL). The reaction mixture was sonicated for 1 min, the colorless solution turned cloudy, and a suspension was formed. The reaction was allowed to stir for 5 days and was monitored by normal-phase silica gel TLC using EtOAc as eluent and RP-18 silica gel using 10% ACN/H2O as eluent. The solvent was evaporated under reduced pressure. Column chromatography using RP-18 silica gel with 10% ACN/H2O gave title compound 9 (0.100 g, 73%) as a hygroscopic off-white solid. Rf = 0.92 1 (ACN/H2O 9:1). [α]32 D −0.14 (c = 0.12, CH3OH). H NMR (500 MHz, D2O): δ 8.94 (s, 1H, NH), 8.25 (s, 1H, CHtriazole), 8.10 (s, 2H, SO2NH2), 5.54 (d, J = 9.3 Hz, 1H, H1′), 4.62 (s, 2H, NCH2), 3.76 (t, J = 9.1 Hz, 1H, H2′), 3.70 (d, J = 10.2 Hz, 1H, H6′), 3.45 (m, 2H, H5′, H6′), 3.38 (t, J = 9.1 Hz, 1H, H3′), 3.23 (t, J = 9.1 Hz, 1H, H4′). 13C NMR (125 MHz, DMSO-d6): δ 171.1 (C5), 158.7 (C2), 143.8 (Ctriazole), 122.9 (CHtriazole), 87.9 (C1′), 80.4 (C5′), 77.5 (C3′), 72.6 (C2′), 70.0 (C4′), 61.2 (C6′), 40.1 (NCH2). LRMS (ESI+): m/z = 424.29 [M + H]+. HRMS (ESI+): m/z calcd for C11H17N7O7S2Na1 [M + Na+], 446.0523; found, 446.0523. Protein X-ray Crystallography. The hCA II protein was expressed, purified, and crystallized as reported previously.26 Briefly, the protein was expressed in Escherichia coli and purified using ion exchange and gel filtration chromatography; it was then concentrated to 14 mg/mL and crystallized in 2.9 M ammonium sulfate with 0.1 M Tris buffer, pH 8.5, at 20 °C. Crystal soaking with compound 3 lasted for 8 days and was done by sprinkling the compound directly into the crystallization drop. The crystals were subsequently cryo-cooled in liquid nitrogen before obtaining data from the Australian Synchrotron MX-2 beamline. The data were indexed using XDS27 and scaled using SCALA.28 Molecular replacement was done using Phaser29 using the structure of hCA II in complex with the saccharin ligand (PDB code 4cq026) as the initial starting model. The model was manually rebuilt using Coot30 and refined using Phenix.31 Density was found for compound 3, and the initial structure and cif dictionary were generated using the eLBOW program within Phenix.32 CA Inhibition Assay. An Applied Photophysics stopped-flow instrument was used for assaying the CA-catalyzed CO2 hydration activity.33 IC50 values were obtained from dose−response curves working at seven different concentrations of test compound by fitting the curves using PRISM (www.graphpad.com) and nonlinear leastsquares methods; values represent the mean of at least three different determinations, as described by us previously.34 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 maximum. All enzymes used were recombinant, produced in E. coli as reported earlier.35,36 The concentration of enzymes used in the assay were as follows: hCA I, 10.4 nM; hCA II, 8.3 nM. Coordinates. The atomic coordinates and structure factors of hCA II/3 complex have been deposited in the RCSB Protein Data Bank with the access number 4RN4.

Compound 7 could not be readily separated, and the reaction mixture was used as-is for the next reaction. LRMS (ESI+): m/z = 749.3 [M + H]+. 5-(N-[1-{β-D-Glucopyranosyl}-1H-1,2,3-triazol-4-yl]methyl-N[1-phenyl-1H-1,2,3-triazol-4-yl]methyl)amino-1,3,4-thiadiazole-2-sulfonamide (3). Title compound 3 was synthesized from the reaction mixture comprising the per-O-acetylated precursor 7 (0.063 g, 0.084 mmol, 1 equiv) upon treatment with 8% HCl in MeOH (5 mL). The reaction mixture was sonicated for 1 min, the colorless solution turned cloudy, and a suspension was formed. The reaction was allowed to stir for 3 days and was monitored by normal-phase silica gel TLC using EtOAc as eluent and RP-18 silica gel using 10% ACN/H2O as eluent. The solvent was evaporated under reduced pressure. Column chromatography using RP-18 silica gel with 10% ACN/H2O (25 mL) followed by 40% ACN/H2O (100 mL) gave title compound 3 (0.015 g, 31%) as a hygroscopic off-white solid. Rf = 0.43 (ACN/H2O 4:6). 1 [α]25 D +1.77 (c = 0.1, CH3OH). H NMR (500 MHz, DMSO-d6): δ 8.62 (s, 1H, CHtriazole‑Ar), 8.33 (s, 1H, CHtriazole‑Glc), 7.92 (s, 2H, SO2NH2), 7.84 (d, J = 7.5 Hz, 2H, H2′′,6′′), 7.61 (t, J = 7.5 Hz, 2H, H3′′,5′′), 7.52 (t, J = 7.5 Hz, 1H, H4′′), 5.56 (d, J = 9.3 Hz, 1H, H1′), 4.70 (s, 2H, NCH2‑triazole‑Ar), 4.68 (s, 2H, NCH2‑triazole‑Glc), 3.78 (t, J = 9.1 Hz, 1H, H2′), 3.72 (m, 1H, H6′), 3.47 (m, 2H, H5′, H6′), 3.40 (t, J = 8.9 Hz, 1H, H3′), 3.26 (t, J = 8.9 Hz, 1H, H4′). 13C NMR (125 MHz, DMSO-d6): δ 172.6 (C5), 154.5 (C2), 142.6 (Ctriazole‑Ar), 142.2 (Ctriazole‑Glc), 137.0 (C1′′), 130.3 (C3′′,5′′), 129.2 (C4′′), 123.9 (CHtriazole‑Glc), 123.1 (CHtriazole‑Ar), 120.8 (C2′′,6′′), 88.0 (C1′), 80.4 (C5′ ), 77.3 (C 3′ ), 72.6 (C 2′ ), 70.0 (C 4′ ), 61.2 (C 6′ ), 43.0 (NCH2triazole‑Ar), 42.6 (NCH2triazole‑Glc). LRMS (ESI+): m/z = 581.18 [M+1]+. HRMS (ESI): m/z calcd for C20H24N10O7S2Na1 [M + Na+], 603.1163; found, 603.1178. 5-N-[(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl]acetamido1,3,4-thiadiazole-2-sulfonamide (11). Title compound 11 was synthesized from alkyne 10 (2.67 g, 10.26 mmol, 1 equiv) and phenylazide (1.27 g, 10.66 mmol, 1 equiv) according to general procedure 1 in 8 h. Purification of crude product by flash chromatography (15% MeOH/CH2Cl2) gave the product, triazole 11 (2.98 g, 77%), as a pale yellow solid. Rf = 0.62 (EtOAc/hexane 4:1). mp 233−235 °C (dec.). 1H NMR (500 MHz, DMSO-d6): δ 8.88 (s, 1H, Htriazole), 8.32 (s, 2H, SO2NH2), 7.89 (d, J = 7.8 Hz, 2H, H2′,6′), 7.59 (t, J = 7.8 Hz, 2H, H3′,5′), 7.49 (t, J = 7.8 Hz, 1H, H4′), 5.65 (s, 2H, NCH2), 2.67 (s, 3H, COCH3). 13C NMR (125 MHz, DMSO-d6): δ 171.7 (C5), 166.7 (CO), 161.5 (C2), 143.5 (Ctriazole), 136.9 (C1′), 130.3 (C3′,5′), 129.2 (C4′), 122.4 (CHtriazole), 120.6 (C2′,6′), 44.0 (NCH2), 22.7 (COCH3). LRMS (ESI+): m/z = 380.6 [M + H]+. HRMS (ESI+): m/z calcd for C13H13N7O3S2Na1 [M + Na+], 402.0413; found, 402.0413. 5-(1-Phenyl-1H-1,2,3-triazol-4-yl)methylamino-1,3,4-thiadiazole-2-sulfonamide (8). To a suspension of compound 11 (0.770 g, 2.029 mmol, 1 equiv) in MeOH (20 mL) was added methanolic sodium methoxide (25% w/v, 0.700 mL, 3.06 mmol, 1.5 equiv), and the reaction was stirred at room temperature for 4 h. On completion, the reaction was neutralized with HCl (1.0 M), and the resulting suspension was filtered to obtain title compound 8 (0.605 g, 88%) as a solid. Rf = 0.74 (EtOAc). mp 265−267 °C (dec.). 1H NMR (500 MHz, DMSO-d6): δ 8.84 (br s, 1H, NH), 8.80 (s, 1H, CHtriazole), 8.10 (br s, 2H, SO2NH2), 7.90 (d, J = 7.5 Hz, 2H, H2′,6′), 7.61 (t, J = 7.5 Hz, 2H, H3′,5′), 7.50 (t, J = 7.5 Hz, 1H, H4′), 4.70 (s, 2H, CH2). 13C NMR (125 MHz, DMSO-d6): δ 171.2 (C5), 158.8 (C2), 145.0 (Ctriazole), 137.1 (C1′), 130.4 (C3′,5′), 129.2 (C4′), 122.1 (CHtriazole), 120.5 (C2′,6′), 40.2 (CH2). LRMS (ESI+): m/z = 338.8 [M + H]+. HRMS (ESI+): m/z calcd for C11H11N7O2S2Na1 [M + Na+], 360.0308; found, 360.0313. 5-N-([1-{2′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyranosyl}-1H1,2,3-triazol-4-yl]methyl)acetamido-1,3,4-thiadiazole-2-sulfonamide (12). Title compound 12 was synthesized from alkyne 10 (0.520 g, 1.998 mmol, 1 equiv) and β-D-glycopyranosyl azide (0.756 g, 2.025 mmol, 1 equiv) according to general procedure 1 in 8 h. Purification of the crude product by flash chromatography (EtOAc/ hexane 1:1) gave the product, triazole 12 (0.90 g, 71%), as a white solid. Rf = 0.45 (EtOAc/hexane 4:1). mp 129−131 °C (dec.). [α]32 D F

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(12) Lopez, M.; Salmon, A.; Supuran, C.; Poulsen, S. Carbonic anhydrase inhibitors developed through ‘click tailing’. Curr. Pharm. Des. 2010, 16, 3277−3287. (13) Winum, J.-Y.; Poulsen, S.-A.; Supuran, C. T. Therapeutic applications of glycosidic carbonic anhydrase inhibitors. Med. Res. Rev. 2009, 29, 419−435. (14) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors. Synthesis of water-soluble, topically effective, intraocular pressure-lowering aromatic/heterocyclic sulfonamides containing cationic or anionic moieties: is the tail more important than the ring? J. Med. Chem. 1999, 42, 2641−2650. (15) Supuran, C. T. Carbonic anhydrases: from biomedical applications of the inhibitors and activators to biotechnologic use for CO2 capture. J. Enzyme Inhib. Med. Chem. 2013, 28, 229−230. (16) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Supuran, C. T.; Poulsen, S.-A. A novel class of carbonic anhydrase inhibitors: glycoconjugate benzene sulfonamides prepared by “clicktailing”. J. Med. Chem. 2006, 49, 6539−6548. (17) Salmon, A. J.; Williams, M. L.; Hofmann, A.; Poulsen, S.-A. Protein crystal structures with ferrocene and ruthenocene-based enzyme inhibitors. Chem. Commun. 2012, 48, 2328−2330. (18) Wilkinson, B. L.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S.-A. Inhibition of carbonic anhydrases with glycosyltriazole benzene sulfonamides. J. Med. Chem. 2008, 51, 1945−1953. (19) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genazzani, A. A. Click chemistry reactions in medicinal chemistry: applications of the 1,3-dipolar cycloaddition between azides and alkynes. Med. Res. Rev. 2008, 28, 278−308. (20) Massarotti, A.; Aprile, S.; Mercalli, V.; Del Grosso, E.; Grosa, G.; Sorba, G.; Tron, G. C. Are 1,4- and 1,5-disubstituted 1,2,3-triazoles good pharmacophoric groups? ChemMedChem 2014, 9, 2497−2508. (21) Kayser, B.; Dumont, L.; Lysakowski, C.; Combescure, C.; Haller, G.; Tramèr, M. R. Reappraisal of acetazolamide for the prevention of acute mountain sickness: a systematic review and metaanalysis. High Alt. Med. Biol. 2012, 13, 82−92. (22) Wu, H.; Li, H.; Kwok, R. T. K.; Zhao, E.; Sun, J. Z.; Qin, A.; Tang, B. Z. A recyclable and reusable supported Cu(I) catalyzed azide−alkyne click polymerization. Sci. Rep. 2014, 4, 5107. (23) The PyMOL Molecular Graphics System; Schrödinger, LLC: New York. (24) Carroux, C. J.; Moeker, J.; Motte, J.; Lopez, M.; Bornaghi, L. F.; Katneni, K.; Ryan, E.; Morizzi, J.; Shackleford, D. M.; Charman, S. A.; Poulsen, S.-A. Synthesis of acylated glycoconjugates as templates to investigate in vitro biopharmaceutical properties. Bioorg. Med. Chem. Lett. 2013, 23, 455−459. (25) Waghorn, P. A.; Jones, M. W.; McIntyre, A.; Innocenti, A.; Vullo, D.; Harris, A. L.; Supuran, C. T.; Dilworth, J. R. Targeting carbonic anhydrases with fluorescent BODIPY-labelled sulfonamides. Eur. J. Inorg. Chem. 2012, 17, 2898−2907. (26) Moeker, J.; Peat, T. S.; Bornaghi, L. F.; Vullo, D.; Supuran, C. T.; Poulsen, S.-A. Cyclic secondary sulfonamides: unusually good inhibitors of cancer-related carbonic anhydrase enzymes. J. Med. Chem. 2014, 57, 3522−3531. (27) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125−132. (28) Evans, P. Scaling and assessment of data quality. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72−82. (29) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658−674. (30) Emsley, P.; Cowton, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (31) Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 352−367.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra for compounds 1−3, 6, and 8−12. Data collection and structure refinement statistics for the hCA II/3 crystal structure. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(C.T.S.) Telephone: +39 055 457 3729; E-mail: claudiu. supuran@unifi.it. *(S.-A.P.) Telephone: +61 7 3735 7825; E-mail: s.poulsen@ griffith.edu.au. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Australian Research Council (grant nos. DP110100071 and FT10100185 to .-A.P.) and two EU grants of the Seventh Framework Program (Metoxia and Dynano projects to C.T.S.) for support. We thank OpenEye Scientific Software for a license to use Afitt, the CSIRO Collaborative Crystallization Centre (www.csiro.au/c3) for crystallization, and the Australian Synchrotron and the beamline scientists at the MX-2 beamline for their support in data collection.

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ABBREVIATIONS USED CA, carbonic anhydrase; CuAAC, copper-catalyzed azide− alkyne cycloaddition; SAR, structure−activity relationship; Ki, inhibition constant; ACN, acetonitrile

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REFERENCES

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DOI: 10.1021/jm501798g J. Med. Chem. XXXX, XXX, XXX−XXX