6-Substituted Sulfocoumarins Are Selective Carbonic Anhdydrase IX

Article pubs.acs.org/jmc

6‑Substituted Sulfocoumarins Are Selective Carbonic Anhdydrase IX and XII Inhibitors with Significant Cytotoxicity against Colorectal Cancer Cells Aiga Grandane,† Muhammet Tanc,‡,§ Lorenzo Di Cesare Mannelli,∥ Fabrizio Carta,§ Carla Ghelardini,∥ Raivis Ž alubovskis,*,† and Claudiu T. Supuran*,‡,§ †

Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia NEUROFARBA Department, Section of Pharmaceutical Chemistry, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy § Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy ∥ NEUROFARBA Department, Section of Pharmacology and Toxicology, Università degli Studi di Firenze, Viale Pieraccini 6, 50139 Florence, Italy ‡

ABSTRACT: 6-Substituted sulfocoumarins bearing the carboxamido, trimethylammonium as well as the cyano and methoxy moieties with interesting inhibitory activity/selectivity against the tumor associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and XII are reported. Moieties leading to the best inhibition were tert-butylcarboxamido, phenylcarboxamido, and 4-pyridylcarboxamido, with KI values of 2.1−8.1 nM. No inhibition of the off-target hCA II and I was observed. A number of these compounds were evaluated against HT-29 colon cancer cell lines ex vivo. Compounds 9c and 9e revealed effective cytotoxic effects after 72 h of incubation in both normoxic and hypoxic conditions, unlike sulfonamide CA inhibitors that show such effects only in hypoxia. These results may be of particular importance for the choice of future drug candidates targeting hypoxic tumors and metastases, considering the fact that a sulfonamide CA IX inhibitor (SLC-0111) is presently in phase I clinical trials.

■

INTRODUCTION 1,2-Benzoxathiine 2,2-dioxides, also referred as “sulfocoumarins”, are bioisosters of the coumarins and recently were reported as potent and isoform-selective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs).1 In analogy to the coumarins, their mechanism of action relies on the CA-mediated esterase activity which cleaves the intramolecular sulfonic acid ester to afford, upon geometrical isomerization, a (E)-vinylsulfonic acid species (Figure 1A). Kinetic as well as X-ray crystallographic experiments on CA II adducts revealed the sulfonic acid moiety tightly bound to the zinc-coordinated water molecule within the enzyme active site (Figure 1B).2−4 The sulfonamides and their isosters such as the sulfamates and sulfonates are established CAIs and are in clinical use for almost 70 years for the treatment of glaucoma, obesity, and epilepsy and as diuretics.5 The wide use of CAIs for pharmaceutical applications relies on the wide distribution of the 15 human (h) CA isoforms within different tissues as well as on their implication in many physiological/pathological conditions. Antiglaucoma CAI drugs mainly target CA II, IV, and XII; the diuretics CA II, IV, XII, and XIV; the antiepileptics CA VII and XIV. The selective inhibition of the CA IX and XII isoforms results in antitumor and antimetastatic effects. However, the © XXXX American Chemical Society

main drawback associated with the use of CAIs is represented by the lack of selectivity in inhibiting the various isoforms, thus resulting in a plethora of unfavorable side effects.6,7 In this contest many efforts have been made for the development of specific CAIs, and some remarkable results have been achieved in the past 15 years since the introduction of the tail approach.6−10 Currently a sulfonamide CA IX inhibitor entered phase I clinical studies for the treatment of hypoxic and advanced stage solid tumors.8e Moreover novel CAIs classes such as the polyamines,11 phenols,12 dithiocarbamates,13 xanthates,14 coumarins, thiocoumarins, 2-thioxocoumarins, and coumarine oximes4,15,16,1,17,18 were identified and the inhibition mechanisms of many of these compounds were determined by means of X-ray crystallography CA II adducts.2,6,12,13 The sulfocoumarins represent the latest CAI class introduced,17 and in our previously investigations1,17 we established the CA inhibition profiles of a series of sulfocoumarins bearing 6-tetrazolyl and 6-triazolyl moieties as water-soluble isosters of the carboxylic function.1−4 As extension herein we report the synthesis, characterization, in vitro CA inhibition, and the ex vivo evaluation on HT-29 colon cancer cell Received: February 16, 2015

A

DOI: 10.1021/acs.jmedchem.5b00523 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. CA inhibition mechanism of sulfocoumarins. (A) The sulfocoumarin 1 undergoes an enzyme-mediated hydrolysis with formation of the E-2-hydroxyphenyl-ω-ethenylsulfonic acid 3. (B) The sulfonic acid group binds to the CA II/IX mimic active site by anchoring to the zinc-coordinated water molecule. The Zn(II) ion (central larger sphere), its three His ligands (His94, -96, and -119), water molecule coordinated to the zinc (small sphere), and active site residues Thr200 and Pro201 involved in the binding of the hydrolyzed sulfocoumarin are shown, as determined by X-ray crystallography (PDB entry 4BCW).1

starting materials, respectively. The key intermediate 6iodosulfocoumarin 12a was then subjected to a Sonogashira coupling reaction to afford the desired 6-cyano-substituted sulfocoumarin 13 (Scheme 2). All compounds were extensively characterized by mean of spectroscopic and physicochemical methods which confirmed their structures. Carbonic Anhydrase Inhibition. All compounds reported were investigated as inhibitors against the four human physiologically relevant CA isoforms: the cytosolic hCA I and II and the transmembrane tumor associated hCA IX and XII (Table 1). According to our previous reports,1,17 all new compounds reported here did not inhibit the two cytosolic CAs, which is a desirable feature for compounds designed to target the tumor-associated enzymes. Both hCA IX and XII were potently inhibited by these derivatives, with KI values of 5.6−81.3 nM against hCA IX and 2.1−26.6 nM against hCA XII, respectively. In particular the introduction of a permanent positive charge, as for 8, resulted in a good inhibition profile against the hCA IX and XII (KI values of 9.6 and 8.7 nM, respectively), which also might be due to the membrane-impermeant properties assumed.20 The simple alkylcarboxamido derivatives 9a,b, the phenylcarboxamido 9c−f, and the pyridinecarboxamido derivatives 9j,k showed excellent inhibition data against hCA IX, with KI values far below the clinically used sulfonamide AAZ (Table 1). On the contrary, the introduction of a carbon unit spacer, as for 9g, or a heterocyclic five-membered ring (9h and 9i) spoiled the affinity of such compounds for hCA IX (KI values of 81.3, 29.5, and 33.0 nM, respectively). In analogy the replacement of the 6-carboxamido moiety with a 6-methoxy (compound 12b) and 6-cyano (compound 13) resulted in a slightly weaker inhibitory action (KI values of 33.8−42.1 nM, Table 1). The definition of a proper

lines of another series of 6-substituted sulfocoumarins bearing carboxamido moieties.

■

RESULTS AND DISCUSSION Design and Synthesis of Compounds. The rationale of the work is based on the fact that the nature of moieties present in the 6- or 7-position of the sulfocoumarin ring highly influences the biological activity as CA inhibitors of the obtained derivatives. We showed that the nature and position of such groups lead to a highly different inhibition profile of the obtained sulfocoumarins.1,17 To date, sulfocoumarin-containing compounds incorporating the alkyl-/arylcarboxamido functionalities were not reported; thus, we decided to investigate such substitution patterns in the present work. The synthesis of the new sulfocoumarins was carried out according to Scheme 1 by using Schotten−Baumann coupling reactions of the intermediate 6-amino derivative 717d with the appropriate acyl chlorides to afford amides 9a−k. Alternatively the amino moiety of 7 was quaternarized by reaction with methyl iodide to afford 8 (Scheme 1). The key intermediate 1,2-benzoxathiine 2,2-dioxide 7 was obtained according to the procedure earlier reported by some of us17d and consisted of mesylation of the phenolic hydroxide 4, followed by intramolecular cyclization in the presence of 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) to afford the racemic 4-hydroxy-6-benzyloxy-3,4-dihydrosulfocoumarin (not shown), which in turn was converted to the cyclic scaffold 6 upon treatment with POCl3. Then reduction of the nitro group using iron (Fe0) in acidic conditions afforded the desired compound 7. In addition we introduced the methoxy or cyano groups at the 6-position by means of the same synthetic procedure previously reported for 717d and using the commercially available 2-hydroxy5-methoxyaldehyde 10b and 2-iodo-5-methoxyaldehyde 10a as B



Article

Scheme 1. Synthesis of 6-Substituted Sulfocoumarin Compounds 8 and 9a−k

Scheme 2. Synthesis of 6-Substituted Sulfocoumarin Compounds 12a,b and 13

Table 1. Inhibition Data against Isoforms hCA I, II, IX, and XII with Compounds 8, 9a−k, and 12b, 13 and Acetazolamide (AAZ) as Standard Inhibitor, by a Stopped-Flow CO2 Hydrase Assay19 KI (nM)a

structure−activity-relationship (SAR) for hCA XII is not feasible, as all the compounds showed high affinity for such isoform (Table 1). With the exception of the methoxy 12b and cyano 13 derivatives (KI values of 10.9 and 26.6 nM, respectively), all series reported inhibition values in a very narrow range (2.1−8.7 nM). These data clearly demonstrate that the nature of the substituent in position 6 of the sulfocoumarin ring is the main factor determining the potency (and selectivity) for the inhibition of the transmembrane versus the cytosolic CA isoforms. Cytotoxicity Assay. We focused our attention on the ex vivo activity of compounds 8, 9c, 9e, and 9k for which cell viability over the time in both normoxic and hypoxic conditions were evaluated, working with the human colon cancer HT-29 cells that overexpress high amounts of CA IX. All these compounds are low nanomolar CA IX/XII inhibitors and do not inhibit significantly CA I and II (Table 1). We compared the effects of these sulfocoumarins with those of the standard sulfonamide inhibitor AAZ, working in the same conditions (Figures 2 and 3).

compd

hCA I

hCA II

hCA IX

hCA XII

8 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 12b 13 AAZ

>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 250

>10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 12.0

9.6 17.2 5.6 6.6 9.0 9.7 9.1 81.3 29.5 33.0 10.5 7.7 42.1 33.8 25.0

8.7 3.7 3.2 8.1 2.4 7.0 4.8 4.0 6.1 6.2 6.8 2.1 10.9 26.6 5.7

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

Compounds 8 and 9k did not show any activity in these ex vivo assays (data not reported), whereas in normoxia, a reduced cell viability was observed for compound 9c after a 16 h incubation at 30 μM concentration. (Figure 2) Its efficacy at the same concentration increased over the time reaching a maximum after 72 h of incubation (43% cell viability). The increase of the concentration (100 and 300 μM) and the incubation times (48 and 72h) did not significantly affect the compound toxicity. For instance at 100 μM concentration at 16 h of incubation 9c C



Article

Figure 2. HT-29 cells (1 × 104/well) were treated with increasing concentrations (0, 30, 100, and 300 μM) of compounds 9c, 9e, and AAZ as control. Incubation was allowed for 16, 48, or 72 h in normoxic conditions (20% O2). Cell viability was measured by the reduction of 3-(4,5-dimethylthiozol2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment functionality. Formazan crystals produced by the reaction were dissolved in DMSO, and the absorbance was measured at 550 nm. Control condition was arbitrarily set as 100%, and values are expressed as the mean ± SEM of three experiments: (∗) P < 0.05 and (∗∗) P < 0.01 in comparison to control (0 μM). Errors are in the ±5% (not shown in the pictures).

Figure 3. HT-29 cells (1 × 104/well) were treated with increasing concentrations (0, 30, 100, and 300 μM) of compounds 9c, 9e, and AAZ as control. Incubation was allowed for 16, 48, or 72 h in hypoxic conditions (0.1% O2) . Cell viability was measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment functionality. Formazan crystals produced by the reaction were dissolved in DMSO, and the absorbance was measured at 550 nm. Control condition was arbitrarily set as 100%, and values are expressed as the mean ± SEM of three experiments: (∗) P < 0.05 and (∗∗) P < 0.01 in comparison to control (0 μM). Errors are in the ±5% (not shown in the pictures).

candidates targeting hypoxic tumors and metastases, considering the fact that a sulfonamide CA IX inhibitor is presently in phase I clinical trials.

showed a viability dropping (52%) which did not change over time (60% at 48h and 40% at 72h). Higher concentrations (300 μM) resulted in a significant loss of cytotoxicity. (Figure 2) Interestingly the hypoxic conditions did not significantly affect compound 9c cytotoxicity which reached a maximum of 35% cell viability at 100 μM and 72 h of incubation. Similarly compound 9e showed the maximum efficacy at 72 h with 100 μM in both normoxia and hypoxia (Figures 1 and 2). It should be mentioned that it is difficult to understand why some of these compounds show such significant cytotoxic effects not only in hypoxia (when CA IX is overexpressed) but also in normoxia, when the enzyme is not present in these cells. Thus, it is not improbable that they may exert their cytotoxic effects through multiple mechanisms of action, such as interference with the CA IX/XII activity in hypoxia and other not yet identified pathways in normoxia.

■

EXPERIMENTAL PROTOCOLS

General. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa Aesar, and TCI. Compounds 5, 6, 7,1 11a,3 and 12a2 were sythesized according to the literature procedures. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringe techniques to transfer solutions. Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were recorded using a Varian Mercury 400 MHz spectrometer in DMSO-d6. 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 doublets. 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. Melting points were determined on an OptiMelt automated melting point system in open capillary tubes and are uncorrected. IR spectra were measured on a Shimadzu FTIR IR Prestige-21 spectrometer. HRMS data were obtained with a Q-TOF micro high-resolution mass spectrometer with ESI (ESI+/ESI−). Elemental analyses were performed on Carlo Erba CHNS-O EA-1108 apparatus. All synthesized compounds reported here were >98% pure. HPLC was performed by using a Waters 2690 separation module coupled with a photodiode array dectector (PDA Waters 996) and a Nova-Pak C18 4 μm, 3.9 mm × 150 mm (Waters), silica-based reverse phase column. Sample was dissolved in acetonitrile 10%, and an injection volume of 45 μL was used. The mobile phase, at a flow rate of 1 mL/min, was a gradient of water + trifluoroacetic acid (TFA) 0.1% (A)

■

CONCLUSIONS We report a new series of 6-substituted sulfocoumarins bearing carboxamido, trimethylammonium as well as cyano and methoxy moieties in position 6 of the heterocyclic compounds. The new derivatives were assayed for their inhibitory properties against four physiologically relevant hCA isoforms (cytosolic hCA I and II and the tumor associated hCA IX and XII). All compounds were ineffective in inhibiting the cytosolic enzymes, whereas they showed interesting inhibition profiles against the tumor associated isoforms hCA IX/XII, with KI values in the low nanomolar range. A selection of such compounds was evaluated ex vivo on HT-29 colon cancer cell lines. In particular compounds 9c and 9e revealed a maximum cytotoxic effect after 72 h of incubation, in both normoxia and hypoxia. These results may be of particular importance for the choice of future drug D



Article

8.05 (d, 1H, J = 2.5 Hz), 9.49 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 27.1, 39.2, 118.5, 118.7, 121.0, 122.9, 124.1, 136.7, 137.2, 146.1, 176.7; HRMS (ESI) [M + H]+: m/z calcd for (C13H16NO4S) 282.0800. Found 282.0811. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)benzamide (9c).

and acetonitrile + TFA 0.1% (B), with steps as follows (A %/B %): 0−10 min 90:10, 10−25 min gradient to 60:40, 26−28 min isocratic 20:80, 29−35 min isocratic 90:10. TFA 0.1% in water as well as in acetonitrile was used as counterion. Fractions of 0.5 mL from each tube were collected, with monitoring at 210 nm. (2,2-Dioxo-2H-2λ 6 -benzo[e][1,2]oxathiin-6-yl)trimethylammonium Iodide (8).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), benzoyl chloride (0.13 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9c as a white solid (0.153 g, 50%). Mp 223−224 °C. IR (KBr, cm−1) νmax: 3223 (N−H), 1654 (CO), 1369 (SO), 1345 (SO), 1174 (SO), 1166 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.46 (d, 1H, J = 9.0 Hz), 7.51−7.58 (m, 3H), 7.59−7.64 (m, 1H), 7.76 (d, 1H, J = 10.4 Hz), 7.85 (dd, 1H, J = 9.0, 2.6 Hz), 7.96−8.00 (m, 2H), 8.21 (d, 1H, J = 2.6 Hz), 10.53 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 118.8, 118.9, 121.0, 123.0, 124.2, 127.7, 128.5, 131.9, 134.4, 136.7, 137.1, 146.5, 165.7. HRMS (ESI) [M + H]+: m/z calcd for (C15H11NO4SNa) 324.0306. Found 324.0283. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-4-methylbenzamide (9d).

A solution of 7 (0.200 g, 1.01 mmol) in N-methyl-2-pyrrolidinone (NMP; 15 mL) was treated with iodomethane (0.38 mL, 6.06 mmol), and the mixture was stirred at room temperature for 48 h. Et2O (150 mL) was added and the oily residue formed was washed with dry Et2O (3 × 70 mL) and crystallized from EtOH to afford the title compound 8 as a yellow solid (0.221 g, 60%). Mp 167−169 °C. IR (KBr, cm−1) νmax: 1368 (SO), 1350 (SO), 1156 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 3.65 (s, 9H), 7.75 (app. s, 2H), 7.78 (d, 1H, J = 9.4 Hz), 8.24 (dd, 1H, J = 9.4, 3.2 Hz), 8.43 (d, 1H, J = 3.2 Hz). 13C NMR (100 MHz, DMSO-d6) δ: 56.6, 119.3, 120.1, 122.7, 124.4, 125.1, 135.5, 144.3, 150.7. HRMS (ESI) [M]+: m/z calcd for (C11H14NO3S) 240.0689. Found 240.0703. Anal. Calcd for C11H14IO3S (367.20): C 35.98, H 3.84, N 3.81. Found: C 35.90, H 3.71, N 3.83. General Procedure for the Synthesis of Compounds 9a−k. To a solution of aminoderivative 7 (1.0 equiv) in dry DCM (20 mL per mmol of compound 7) the appropriate acyl chloride (1.1 equiv) and NEt3 (1.0 equiv) were added. The resulting mixture was stirred at room temperature under an argon atmosphere for 2 h. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)acetamide (9a).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), 4-methylenzoyl chloride (0.17 mL, 1.32 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9d as a white solid (0.140 g, 44%). Mp 236−236 °C. IR (KBr, cm−1) νmax: 3240 (N−H), 1646 (CO), 1360 (SO), 1349 (SO), 1167 (SO), 1140 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 3H), 7.33−7.37 (m, 2H), 7.45 (d, 1H, J = 8.9 Hz), 7.52 (d, 1H, J = 10.3 Hz), 7.75 (d, 1H, J = 10.3 Hz), 7.85 (dd, 1H, J = 8.9, 2.6 Hz), 7.87−7.92 (m, 2H), 8.20 (d, 1H, J = 2.6 Hz), 10.43 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 21.0, 118.7, 118.8, 121.0, 123.0, 124.2, 127.7, 129.0, 131.5, 136.8, 137.1, 142.0, 146.4, 165.5. HRMS (ESI) [M + H]+: m/z calcd for (C16H14NO4S) 316.0644. Found 316.0672. 2-Bromo-N-(2,2-dioxido-1,2-benzoxathiin-6-yl)benzamide (9e).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), acetyl chloride (0.08 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The reaction was quenched with water (20 mL), and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na2SO4, and the solvent was evaporated in vacuo to afford 9a as a white solid (0.140 g, 44%). Mp 197−199 °C. IR (KBr, cm−1) νmax: 3303 (N−H), 1668 (CO), 1358 (SO), 1344 (SO), 1181 (SO), 1167 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 2.07 (s, 3H), 7.38 (d, 1H, J = 8.9 Hz), 7.49 (d, 1H, J = 10.3 Hz), 7.62 (dd, 1H, J = 8.9, 2.7 Hz), 7.72 (d, 1H, J = 10.3 Hz), 7.99 (d, 1H, J = 2.7 Hz), 10.22 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 23.9, 118.8, 118.9, 119.5, 122.7, 122.9, 136.7, 137.2, 146.0, 168.6. HRMS (ESI) [M + H]+: m/z calcd for (C10H10NO4S) 240.0331. Found 240.0338. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-2,2-dimethylpropanamide (9b).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), 2-bromobenzoyl chloride (0.15 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9e as a white solid (0.263 g, 68%). Mp 234−235 °C. IR (KBr, cm−1) νmax: 3220 (N−H), 1653 (CO), 1360 (SO), 1350 (SO), 1163 (SO), 1138 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.42−7.48 (m, 2H), 7.49−7.56 (m, 2H), 7.59 (dd, 1H, J = 7.6, 1.7 Hz), 7.71−7.76 (m, 2H), 7.79 (d, 1H, J = 10.3 Hz), 8.19 (d, 1H, J = 2.6 Hz), 10.79 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 118.9, 119.0, 119.1, 120.1, 123.1, 123.4, 127.8, 128.9, 131.4, 132.8, 136.7, 136.8, 138.6, 146.6, 166.0. HRMS (ESI) [M + H]+: m/z calcd for (C15H11NO4SBr) 379.9592. Found 379.9594.

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), pivaloyl chloride (0.14 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The reaction was quenched with water (20 mL), and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na2SO4, and the solvent was evaporated in vacuo to afford 9b as a white solid (0.278 g, 98%). Mp 182−184 °C. IR (KBr, cm−1) νmax: 3313 (N−H), 1664 (CO), 1369 (SO), 1173 (SO), 1153 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 1.23 (s, 9H), 7.38 (d, 1H, J = 9.0 Hz), 7.49 (d, 1H, J = 10.2 Hz), 7.67−7.74 (m, 2H), E



Article

N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)furan-2-carboxamide (9i).

N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-2-iodobenzamide (9f).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), 2-furoyl chloride (0.11 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9i as a light brown solid (0.217 g, 74%). Mp 247−248 °C. IR (KBr, cm−1) νmax: 3229 (N−H), 1654 (CO), 1355 (SO), 1349 (SO), 1164 (SO), 1138 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 6.72 (dd, 1H, J = 3.5, 1.6 Hz), 7.37 (d, 1H, J = 3.5 Hz), 7.44 (d, 1H, J = 9.0 Hz), 7.52 (d, 1H, J = 10.3 Hz), 7.74 (d, 1H, J = 10.3 Hz), 7.84 (dd, 1H, J = 9.0, 2.5 Hz), 7.96−7.98 (m, 1H), 8.15 (d, 1H, J = 2.5 Hz), 10.48 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 112.3, 115.3, 118.8, 118.9, 121.0, 123.0, 124.3, 136.4, 136.7, 146.0, 146.5, 147.1, 156.3. HRMS (ESI) [M + H]+: m/z calcd for (C13H10NO5S) 292.0280. Found 292.0244. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)pyridine-3-carboxamide (9j).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), 2-iodobenzoyl chloride (0.297 g, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9f as a white solid (0.335 g, 77%). Mp 260−261 °C. IR (neat, cm−1) νmax: 3218 (N−H), 1653 (CO), 1357 (SO), 1349 (SO), 1169 (SO), 1163 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.23− 7.28 (m, 1H), 7.46 (d, 1H, J = 8.9 Hz), 7.50−7.56 (m, 3H), 7.74 (dd, 1H, J = 8.9, 2.6 Hz), 7.79 (d, 1H, J = 10.4 Hz), 7.94−7.97 (m, 1H), 8.19 (d, 1H, J = 2.6 Hz), 10.73 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 93.6, 119.0, 119.1, 120.0, 123.1, 123.4, 128.1, 128.2, 131.3, 136.7, 136.9, 139.1, 142.6, 146.5, 167.8. HRMS (ESI) [M + H]+: m/z calcd for (C15H11NO4SI) 427.9454. Found 427.9425. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-2-phenylacetamide (9g).

Pyridine-3-carboxylic acid (0.42 g, 3.38 mmol) was added to thionyl chloride (5 mL). The mixture was stirred at 70 °C for 3 h, then evaporated in vacuo to afford a residue that was treated with 7 (0.20 g, 1.01 mmol) in dry DCM (20 mL) and NEt3 (0.14 mL, 1.01 mmol). The resulting mixture was stirred at room temperature under an argon atmosphere for 2 h. The obtained precipitate was recovered by filtration, dissolved in EtOAc, and washed with a saturated aqueous solution of NaHCO3. The organic layer was dried over Na2SO4 and evaporated in vacuo to afford 9j as a yellow solid (0.168 g, 55%). Mp 232−234 °C. IR (KBr, cm−1) νmax: 3342 (N−H), 1669 (CO), 1363 (SO), 1350 (SO), 1169 (SO), 1144 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.48 (d, 1H, J = 8.8 Hz), 7.54 (d, 1H, J = 10.3 Hz), 7.58−7.63 (m, 1H), 7.77 (d, 1H, J = 10.3 Hz), 7.85 (dd, 1H, J = 9.0, 2.5 Hz), 8.19 (d, 1H, J = 2.5 Hz), 8.30−8.35 (m, 1H), 8.79 (dd, 1H, J = 4.9, 1.6 Hz), 9.12−9.15 (m, 1H), 10.72 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 118.9, 119.0, 121.1, 123.1, 123.6, 124.2, 130.2, 135.7, 136.6, 136.7, 146.7, 148.6, 152.2, 164.2. HRMS (ESI) [M + H]+: m/z calcd for (C14H11N2O4S) 303.0440. Found 303.0452. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)pyridine-4-carboxamide (9k).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), phenylacetyl chloride (0.15 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The reaction was quenched with water (20 mL), and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na2SO4, and the solvent was evaporated in vacuo to afford 9g as a yellow solid (0.280 g, 88%). Mp 184−186 °C. IR (KBr, cm−1) νmax: 3224 (N−H), 1675 (CO), 1363 (SO), 1168 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 3.66 (s, 2H), 7.22−7.27 (m, 1H), 7.31−7.35 (m, 4H), 7.39 (d, 1H, J = 9.0 Hz), 7.49 (d, 1H, J = 10.4 Hz), 7.65 (dd, 1H, J = 9.0, 2.6 Hz), 7.70 (d, 1H, J = 10.4 Hz), 8.01 (d, 1H, J = 2.6 Hz), 10.46 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 43.2, 118.9, 119.0, 119.7, 122.9, 123.0, 126.6, 128.3, 129.1, 135.7, 136.7, 137.0, 146.1, 169.4. HRMS (ESI) [M + H]+: m/z calcd for (C16H14NO4S) 316.0644. Found 316.0599. N-(2,2-Dioxido-1,2-benzoxathiin-6-yl)thiophene-2-carboxamide (9h).

The title compound was obtained according to the general procedure previously described using 7 (0.20 g, 1.01 mmol), 2-thiophenecarbonyl chloride (0.12 mL, 1.12 mmol), and NEt3 (0.14 mL, 1.01 mmol). The precipitate formed was recovered by filtration and dried under vacuum to afford 9h as a white solid (0.155 g, 50%). Mp 224−225 °C. IR (KBr, cm−1) νmax: 3266 (N−H), 1641 (CO), 1358 (SO), 1347 (SO), 1166 (SO), 1137 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.25 (dd, 1H, J = 4.9, 3.9 Hz), 7.46 (d, 1H, J = 8.9 Hz), 7.53 (d, 1H, J = 10.3 Hz), 7.75 (d, 1H, J = 10.3 Hz), 7.82 (dd, 1H, J = 8.9, 2.7 Hz), 7.89 (dd, 1H, J = 4.9, 1.1 Hz), 8.04 (dd, 1H, J = 3.9, 1.1 Hz), 8.12 (d, 1H, J = 2.7 Hz), 10.50 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 118.8, 118.9, 121.1, 123.0, 124.2, 128.2, 129.5, 132.3, 136.6, 136.7, 139.4, 146.5, 160.1. HRMS (ESI) [M + H]+: m/z calcd for (C13H10NO4S2) 308.0051. Found 308.0070.

Pyridine-4-carboxylic acid (0.41 g, 3.31 mmol) was added to thionyl chloride (5 mL). The mixture was stirred at 70 °C for 3 h, then evaporated in vacuo to afford a residue that was treated with 7 (0.20 g, 1.01 mmol) in dry DCM (20 mL) and NEt3 (0.14 mL, 1.01 mmol). The resulting mixture was stirred at room temperature under an argon atmosphere for 2 h. The obtained precipitate was recovered by filtration, dissolved in EtOAc, and washed with a saturated aqueous solution of NaHCO3, dried over Na2SO4 and the solvent was removed in vacuo to give a residue that was recrystallized from EtOH to afford 9k as a light brown crystalline solid (0.179 g, 58%). Mp 200−201 °C. IR (KBr, cm−1) νmax: 3236 (N−H), 1680 (CO), 1363 (SO), 1167 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.46 (d, 1H, J = 9.0 Hz), 7.53 (d, 1H, J = 10.2 Hz), 7.70 (ddd, 1H, J = 7.7, 4.7, 1.3 Hz), 7.74 (d, 1H, J = 10.2 Hz), F



Article

8.00 (dd, 1H, J = 9.0, 2.7 Hz), 8.09 (dt, 1H, J = 7.7, 1.7 Hz), 8.16−8.19 (m, 1H), 8.35 (d, 1H, J = 2.7 Hz), 8.75−8.77 (m, 1H), 10.96 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 118.8, 118.9, 121.1, 122.6, 123.0, 124.5, 127.2, 136.3, 136.7, 138.2, 146.6, 148.5, 149.5, 162.9. HRMS (ESI) [M + H]+: m/z calcd for (C14H11N2O4S) 303.0440. Found 303.0452. 2-Formyl-4-methoxyphenyl Methanesulfonate (11b).

120.0, 124.1, 134.5, 135.3, 136.2, 153.4. Anal. Calcd for C9H5O3S (207.21): C 52.17, H 2.43, N 6.76. Found: C 52.13, H 2.44, N 6.65. 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.19 Inhibitor and enzyme were preincubated for 6 h. IC50 values were obtained from dose−response curves, working at seven different concentrations of test compound (from 10 nM to 10 μM), by fitting the curves using PRISM (www.graphpad.com) and nonlinear least-squares methods, with values representing the mean of at least three different determinations, as described earlier by us.20,21 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.20,21 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. Cell Culture and Treatments. Human colon cancer cell lines HT-29 were obtained from American Type Culture Collection (Rockville, MD). HT-29 were cultured in DMEM high glucose with 20% FBS in 5% CO2 atmosphere at 37 °C. Media contained 2 mM −1 L-glutamine, 1% essential amino acid mix, 100 IU ml penicillin, and 100 μg mL−1 streptomycin (Sigma, Milan, Italy). HT-29 cells were plated in 96-wells cell culture (1 × 104/well) and, 24 h after, treated with the tested compounds (0−300 μM) for 16, 48, and 72 h. Low oxygen conditions were acquired in a hypoxic workstation (Concept 400 anaerobic incubator, Ruskinn Technology Ltd., Bridgend, U.K.). The atmosphere in the chamber consisted of 0.1% O2 (hypoxia), 5% CO2, and residual N2. In parallel, normoxic (20% O2) dishes were incubated in air with 5% CO2. Cell Viability Assay. HT-29 cell viability was evaluated by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment functionality. Cells were plated and treated as described. After treatments, after extensive washing, 1 mg/mL MTT was added into each well and incubated for 30 min at 37 °C. After washing, the formazan crystals were dissolved in 150 μL of DMSO. The absorbance was measured at 550 nm. Experiments were performed in quadruplicate on at least three different cell batches.

A solution of aldehyde 10b (1.50 g, 9.86 mmol) and NEt3 (1.64 mL, 11.8 mmol) in dry DCM (25 mL) at 0 °C was treated with mesyl chloride (1.23 mL, 15.9 mmol). The mixture was stirred at room temperature for 2 h, then quenched with H2O (60 mL) and extracted with EtOAc (3 × 40 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a residue which was purified by silica gel column chromatography, eluting with petroleum ether/EtOAc 1/1 to afford 11b as a yellow oil (2.22 g, 98%). IR (neat, cm−1) νmax: 1695 (CO), 1369 (SO), 1349 (SO), 1155 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 3.54 (s, 3H), 3.85 (s, 3H), 7.33−7.39 (m, 2H), 7.47−7.51 (m, 1H), 10.18 (s, 1H). 13 C NMR (100 MHz, DMSO-d6) δ: 37.5, 55.9, 111.8, 122.0, 125.3, 130.0, 143.3, 158.1, 188.5. HRMS (ESI) [M + Na]+: m/z calcd for (C9H10NaO5S) 253.0147. Found 253.0162. 6-Methoxy-1,2-benzoxathiine 2,2-Dioxide (12b).

A solution of aldehyde 11b (1.50 g, 6.51 mmol) in dry DCM (30 mL) at 0 °C was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.97 mL). The mixture was stirred at 0 °C for 2 h and then quenched with slush containing 10% aqueous HCl, extracted with Et2O (3 × 50 mL). The combined organic layers were dried over Na2SO4 and the solvent was evaporated in vacuo to give an oily residue, which was dissolved in dry pyridine (15 mL) at 0 °C and treated at the same temperature with POCl3 (1.21 mL, 13.02 mmo). The mixture was stirred at room temperature for 7 h, quenched with slush and the precipitate formed was collected by filtration and recrystallyzed from EtOH to afford 12b as a light brown solid (1.05 g, 76%). Mp 115− 116 °C. IR (neat, cm−1) νmax: 1343 (SO), 1165 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 3.80 (s, 3H), 7.14 (dd, 1H, J = 8.9, 2.2 Hz), 7.30 (d, 1H, J = 2.2 Hz), 7.37 (d, 1H, J = 8.9 Hz), 7.49 (d, 1H, J = 10.3 Hz), 7.64 (d, 1H, J = 10.3 Hz). 13C NMR (100 MHz, DMSO-d6) δ: 55.8, 113.7, 118.3, 119.5, 119.6, 123.2, 136.4, 144.5, 156.8. HRMS (ESI) [M]+: m/z calcd for (C9H8NaO4S) 235.0041. Found 235.0070. 1,2-Benzoxathiine-6-carbonitrile 2,2-Dioxide (13).

■

AUTHOR INFORMATION

Corresponding Authors

*R.Z.: phone, +371 67014826; fax, +371 67550338; e-mail, [email protected]. *C.T.S.: phone, +39-055-4573005; fax, +39-055-4573385; e-mail, claudiu.supuran@unifi.it. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the European Regional Development Fund (Project 2014/0019/2DP/2.1.1.1.0/14/APIA/ VIAA/062) for Latvian Institute of Organic Synthesis and the EU FP7 ITN Project Dynano (to M.T. and C.T.S.).

■

A mixture of 12a (0.20 g, 0.649 mmol), CuCN (0.12 g, 1.30 mmol), and Pd(Ph3)4 (0.02 g, 0.016 mmol) in dry DMF (5 mL) was stirred under argon at 145 °C for 20 h. The volatiles were evaporated in vacuo, and the residue was suspended in EtOAc and filtered. The mother liquor was washed with a 10% NH3 aqueous solution, dried over Na2SO4 and the solvent was evaporated in vacuo to give a residue which was purified by silica gel column chromatography, eluting with petroleum ether/EtOAc 2/1 and recrystallyzed from EtOH to afford 13 as a white solid (0.106 g, 79%). Mp 168−170 °C. IR (neat, cm−1) νmax: 2237 (CN), 1368 (SO), 1161 (SO). 1H NMR (400 MHz, DMSO-d6) δ: 7.68 (d, 1H, J = 8.7 Hz), 7.69−7.76 (m, 2H), 8.08 (dd, 1H, J = 8.7, 2.0 Hz), 8.29 (d, 1H, J = 2.0 Hz). 13C NMR (100 MHz, DMSO-d6) δ: 109.2, 117.5, 119.6,

ABBREVIATIONS USED CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor; KI, inhibition constant; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane

■

REFERENCES

(1) Tars, K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis, R.; Scozzafava, A.; Supuran, C. T. Sulfocoumarins (1,2benzoxathiine-2,2-dioxides): a class of potent and isoform-selective inhibitors of tumor-associated carbonic anhydrases. J. Med. Chem. 2013, 56, 293−300.

G



Article

carbonic anhydrases I, II, IV and XII. J. Enzyme Inhib. Med. Chem. 2013, 28, 289−293. (i) Singh, S.; Supuran, C. T. QSARs on human carbonic anhydrase VA and VB inhibitors of some new not yet synthesized, substituted aromatic/heterocyclic sulphonamides as anti-obesity agent. J. Enzyme Inhib. Med. Chem. 2012, 27, 666−672. (j) See more at ClinicalTrials.gov. Safety Study of SLC-0111 in Subjects with Advanced Solid Tumours (Full Text View): clinicaltrials.gov/ct2/show/ NCT02215850. (9) (a) Svastova, E.; Hulíkova, A.; Rafajova, M.; Zatovicova, M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. Hypoxia activates the capacity of tumorassociated carbonic anhydrase IX to acidify extracellular pH. FEBS Lett. 2004, 577, 439−445. (b) Thiry, A.; Dogné, J. M.; Masereel, B.; Supuran, C. T. Targeting tumor-associated carbonic anhydrase IX in cancer therapy. Trends Pharmacol. Sci. 2006, 27, 566−573. (c) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone, G. Crystal structure of the catalytic domain of the tumorassociated human carbonic anhydrase IX. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233−16238. (d) Ahlskog, J. K.; Schliemann, C.; Mårlind, J.; Qureshi, U.; Ammar, A.; Pedleym, R. B.; Neri, D. In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives. Bioorg. Med. Chem. Lett. 2009, 19, 4851−4856. (10) (a) Stander, B. A.; Joubert, F.; Tu, C.; Sippel, K. H.; McKenna, R.; Joubert, A. M. Signaling pathways of ESE-16, an antimitotic and anticarbonic anhydrase estradiol analog, in breast cancer cells. PLoS One 2013, 8, e53853. (b) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.; Dedhar, S.; Supuran, C. T. Ureido-substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis. J. Med. Chem. 2011, 54, 1896−1902. (c) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors. Cancer Res. 2011, 71, 3364−3376. (d) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4substituted-ureido-benzenesulfonamides and correlate to inhibitor potency. Chem. Commun. 2010, 46, 8371−8373. (11) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. Polyamines inhibit carbonic anhydrases by anchoring to the zinc-coordinated water molecule. J. Med. Chem. 2010, 53, 5511− 5522. (12) (a) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors: interactions of phenols with the 12 catalytically active mammalian isoforms (CA I-XIV). Bioorg. Med. Chem. Lett. 2008, 18, 1583−1587. (b) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors: inhibition of mammalian isoforms I-XIV with a series of substituted phenols including paracetamol and salicylic acid. Bioorg. Med. Chem. 2008, 16, 7424− 7428. (c) Davis, R. A.; Innocenti, A.; Poulsen, S. A.; Supuran, C. T. Carbonic anhydrase inhibitors. Identification of selective inhibitors of the human mitochondrial isozymes VA and VB over the cytosolic isozymes I and II from a natural product-based phenolic library. Bioorg. Med. Chem. 2010, 18, 14−18. (d) Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo, D.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. Natural product-based phenols as novel probes for mycobacterial and fungal carbonic anhydrases. J. Med. Chem. 2011, 54, 1682−1692. (13) (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations. Chem. Commun. 2012, 48, 1868−1870. (b) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C. T. Dithiocarbamates strongly inhibit the β-class carbonic anhydrases from Mycobacterium tuberculosis. J. Enzyme Inhib. Med. Chem.

(2) (a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.; Quinn, R. J.; Supuran, C. T. Non-zinc mediated inhibition of carbonic anhydrases: Coumarins are a new class of suicide inhibitors. J. Am. Chem. Soc. 2009, 131, 3057−62. (b) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J. Med. Chem. 2010, 53, 335−344. (3) (a) Maresca, A.; Scozzafava, A.; Supuran, C. T. 7,8-Disubstitutedbut not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II in the low nanomolar/subnanomolar range. Bioorg. Med. Chem. Lett. 2010, 20, 7255−7258. (b) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.; Dedhar, S.; Winum, J. Y.; Supuran, C. T. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors. J. Med. Chem. 2011, 54, 8271−8277. (c) Bonneau, A.; Maresca, A.; Winum, J. Y.; Supuran, C. T. Metronidazole-coumarin conjugates and 3-cyano-7-hydroxy-coumarin act as isoform-selective carbonic anhydrase inhibitors. J. Enzyme Inhib. Med. Chem. 2013, 28, 397−401. (4) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. 5- And 6membered (thio)lactones are prodrug type carbonic anhydrase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 267−270. (5) (a) Carta, F.; Supuran, C. T.; Scozzafava, A. Sulfonamides and their isosters as carbonic anhydrase inhibitors. Future Med. Chem. 2014, 10, 1149−1165. (b) 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. (6) (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem. Rev. 2012, 112, 4421−4468. (b) Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discovery 2008, 7, 168−181. (c) Neri, D.; Supuran, C. T. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10, 767−777. (d) Aggarwal, M.; Kondeti, B.; McKenna, R. Insights towards sulfonamide drug specificity in αcarbonic anhydrases. Bioorg. Med. Chem. 2013, 21, 1526−1533. (7) (a) Aggarwal, M.; McKenna, R. Update on carbonic anhydrase inhibitors: a patent review (2008−2011). Expert. Opin. Ther. Pat. 2012, 22, 903−915. (b) Supuran, C. T. Structure-based drug discovery of carbonic anhydrase inhibitors. J. Enzyme Inhib. Med. Chem. 2012, 27, 759−772. (c) Supuran, C. T.; Scozzafava, A.; Casini, A. Carbonic anhydrase inhibitors. Med. Res. Rev. 2003, 23, 146−189. (d) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J. Enzyme Inhib. Med. Chem. 2004, 19, 199−229. (8) (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Development of sulfonamide carbonic anhydrase inhibitors (CAIs). In Carbonic AnhydraseIts Inhibitors and Activators; Supuran, C.T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton, FL, 2004; pp 67−147. (b) Supuran, C. T. Carbonic anhydrase inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 3467−3474. (c) Supuran, C. T. Carbonic anhydrase inhibitors and activators for novel therapeutic applications. Future Med. Chem. 2011, 3, 1165−1180. (d) Aggarwal, M.; Boone, C. D.; Kondeti, B.; McKenna, R. Structural annotation of human carbonic anhydrases. J. Enzyme Inhib. Med. Chem. 2013, 28, 267−277. (e) Jain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D. W. Identification of two hydrophobic patches in the active-site cavity of human carbonic anhydrase II by solution-phase and solid-state studies and their use in the development of tight-binding inhibitors. J. Med. Chem. 1994, 37, 2100−2105. (f) Allouche, F.; Chabchoub, F.; Carta, F.; Supuran, C. T. Synthesis of aminocyanopyrazoles via a multi-component reaction and anti-carbonic anhydrase inhibitory activity of their sulfamide derivatives against cytosolic and transmembrane isoforms. J. Enzyme Inhib. Med. Chem. 2013, 28, 343−349. (g) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Inhibition of the alpha- and beta-carbonic anhydrases from the gastric pathogen Helycobacter pylori with anions. J. Enzyme Inhib. Med. Chem. 2013, 28, 388−391. (h) Supuran, C. T.; Maresca, A.; Gregáň, F.; Remko, M. Three new aromatic sulfonamide inhibitors of H



Article

2013, 28, 407−411. (c) Monti, S. M.; Maresca, A.; Carta, F.; De Simone, G.; Mühlschlegel, F. A.; Scozzafava, A.; Supuran, C. T. Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases from Cryptococcus neoformans, Candida albicans and Candida glabrata. Bioorg. Med. Chem. Lett. 2012, 22, 859−862. (d) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo. J. Med. Chem. 2012, 55, 1721−1730. (14) Carta, F.; Akdemir, A.; Scozzafava, A.; Masini, E.; Supuran, C. T. Xanthates and trithiocarbonates strongly inhibit carbonic anhydrases and show antiglaucoma effects in vivo. J. Med. Chem. 2013, 56, 4691− 4700. (15) Carta, F.; Vullo, D.; Maresca, A.; Scozzafava, A.; Supuran, C. T. New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors with low affinity for the offtarget cytosolic isoform II. Bioorg. Med. Chem. Lett. 2012, 22, 2182−2185. (16) (a) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Novel coumarins and 2-thioxo-coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII. Bioorg. Med. Chem. 2012, 20, 2266− 2273. (b) Davis, R. A.; Vullo, D.; Maresca, A.; Supuran, C. T.; Poulsen, S. A. Natural product coumarins that inhibit human carbonic anhydrases. Bioorg. Med. Chem. 2013, 21, 1539−1543. (17) (a) Tanc, M.; Carta, F.; Bozdag, M.; Scozzafava, A.; Supuran, C. T. 7-Substituted-sulfocoumarins are isoform-selective, potent carbonic anhydrase II inhibitors. Bioorg. Med. Chem. 2013, 15, 4502−4510. (b) Grandane, A.; Tanc, M.; Zalubovskis, R.; Supuran, C. T. 6-Triazolylsubstituted sulfocoumarins are potent, selective inhibitors of the tumorassociated carbonic anhydrases IX and XII. Bioorg. Med. Chem. Lett. 2014, 5, 1256−1260. (c) Grandane, A.; Tanc, M.; Zalubovskis, R.; Supuran, C. T. Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII. Bioorg. Med. Chem. 2014, 5, 1522−1528. (d) Grandane, A.; Belyakov, S.; Trapencieris, P.; Zalubovskis, R. Facile synthesis of coumarin bioisosteres 1,2benzoxathiine 2,2-dioxides. Tetrahedron 2012, 68, 5541−5546. (18) (a) Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van Zandvoort, M. A. M.; Pastoreková, S.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors. Radiother. Oncol. 2007, 83, 367−373. (b) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Imaging of CA IX with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-oxygenated cells in a xenograft tumour model. Radiother. Oncol. 2009, 92, 423−428. (c) Akurathi, V.; Dubois, L.; Lieuwes, N. G.; Chitneni, S. K.; Cleynhens, B. J.; Vullo, D.; Supuran, C. T.; Verbruggen, A. M.; Lambin, P.; Bormans, G. M. Synthesis and biological evaluation of a 99m Tc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia. Nuclear Med. Biol. 2010, 37, 557−564. (d) Dubois, L.; Peeters, S.; Lieuwes, N. G.; Geusens, N.; Thiry, A.; Carta, F.; Scozzafava, A.; Dogné, J. M.; Supuran, C. T.; Harris, A. L.; Masereel, B.; Lambin, P. Specific inhibition of CA IX activity enhances the therapeutic effect of tumor irradiation. Radiother. Oncol. 2011, 99, 424−431. (e) Groves, K.; Bao, B.; Zhang, J.; Handy, E.; Kennedy, P.; Cuneo, G.; Supuran, C. T.; Yared, W.; Peterson, J. D.; Rajopadhye, M. Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors. Bioorg. Med. Chem. Lett. 2012, 22, 653−657. (19) Khalifah, R. J. The carbon dioxide hydration activity of carbonic anhydrase. J. Biol. Chem. 1971, 246, 2561−2573. (20) (a) Supuran, C. T.; McKenna, R. Carbonic anhydrase inhibitors drug design. In Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications; McKenna, R., Frost, S., Eds.; Springer Verlag: Heidelberg, 2014; pp 291−323. (Subcell. Biochem. 2014, 75, 291.) (b) 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. (c) 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. (21) (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors. Synthesis of sulfonamides incorporating dtpa tails and of their zinc complexes with powerful topical antiglaucoma properties. Bioorg. Med. Chem. Lett. 2001, 11, 575− 582. (b) Vomasta, D.; Innocenti, A.; König, B.; Supuran, C. T. Carbonic anhydrase inhibitors: two-prong versus mono-prong inhibitors of isoforms I, II, IX, and XII exemplified by photochromic cis-1,2-αdithienylethene derivatives. Bioorg. Med. Chem. Lett. 2009, 19, 1283− 1286.

I


6-Substituted Sulfocoumarins Are Selective Carbonic Anhdydrase IX

Recommend Documents