3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors

Abstract A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.


Introduction
Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) such as derivatives of type A were discovered by our groups to act as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) 1,2 . A large series of sulfocoumarins derivatives, among which compounds of type B, were thereafter reported, by using click chemistry or other conventional drug design approaches (Figure 1) 3- 6 .
A salient feature of this type of CA inhibitor (CAI) was the fact that they showed a very pronounced isoform selectivity for inhibiting tumour-associated CA isoforms (CA IX and XII) over the widespread, cytosolic ones CA I and II 1-3 . This has been explained when the mechanism of CA inhibition with sulfocoumarins was elucidated, by using kinetic and X-ray crystallographic experiments 1 . Indeed, in the X-ray crystal structure of the adduct of a CA II/IX mimic complexed with the 6-bromosulfocoumarin A2(A, R ¼ Br) (Figure 1), the 2-dihydroxy-5-bromophenyl-vinyl sulfonic acid D was observed within the enzyme active site, probably due to the CA-mediated hydrolysis of A2 to the cis-sulfonic acid C which was thereafter isomerized to the more stable trans-derivative D (Scheme 1) 1 .
This inhibition mechanism is similar to the one observed earlier for coumarins 7,8 the class of CAIs which constituted the lead compounds for the discovery of sulfocoumarins. Finding isoform-selective CAIs for the 15 different human CA isoforms is a challenging task 9,10 , but coumarins and sulfocoumarins (and several families of sulfonamides) do show such properties, which make them of great interest for the design of pharmacological agents useful as diuretics, antiglaucoma, anticonvulsant and/or antitumor drugs 9- 13 .
Here, we report the homo-sulfocoumarins or 3H-1,2-benzoxathiepine 2,2-dioxides, which can be considered as homologs of sulfocoumarins or 1,2-benzoxathiine 2,2-dioxides 1 , where oxathiine ring was expanded by one carbon to form an oxathiepine ring. To the best of our knowledge, there is no reported method for the synthesis of 3H-1,2-benzoxathiepine 2,2-dioxides in the literature. The general strategy for the formation of oxathiepine ring reported in this paper involves a ruthenium-catalysed olefin metathesis as a key step.

Materials and methods
Chemistry Reagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatography was performed on silica gel, spots were visualized with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were measured on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Varian Mercury (400 MHz) spectrometer with chemical shifts values (d) in ppm relative to TMS using the residual DMSOd 6  General procedure for the synthesis of 4-substituted 2-ethenylphenoles (2a-c) 14 To a stirred solution of methyltriphenylphosphonium bromide (2.64 eq.) in dry THF (5 ml/1 mmol of corresponding aldehyde), was added tBuOK (2.86-3.12 eq.) in several portions over 20 min. Reaction mixture was stirred for 1 h at RT. Corresponding 2-hydroxy benzaldehyde (1 eq.) was added and stirring continued at room temperature for 24 h. Reaction mixture was diluted with CH 2 Cl 2 (5 ml/1 mmol aldehyde). Organic layer was collected and washed with water (2 Â 20 ml) and brine (2 Â 20 ml), dried over Na 2 SO 4 , solvent was driven off in vacuum. The crude product was purified by column chromatography (silica gel, EtOAc/PhMe1:5).

CA inhibition assay
An SX.18 MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes 16 . Phenol Red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na 2 SO 4 or NaClO 4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration), 17 following the CA-catalysed CO 2 hydration reaction for a period of 5-10 s. Saturated CO 2 solutions in water at 25 C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to 1 nM done with the assay buffer mentioned above. At least seven different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 6 h at 4 C prior to assay, in order to allow for the formation of the E-I complex.
Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using the Cheng-Prusoff equation, as reported earlier 17 , and represent the mean from at least three different determinations. All CA isozymes used here were recombinant proteins obtained as reported earlier by our group 18 . X-ray structure determination X-Ray diffraction data for compound 6c were collected using a NoniusKappaCCD diffractometre (MoKa radiation, k ¼ 0.71073 Å), equipped with low temperature Oxford CryosystemsCryostream Plus device (Delft, the Netherlands). Data were collected using KappaCCD Server Software, cell refined by SCALEPACK 19 , data reduction performed by DENZO 20 and SCALEPACK 19 , structures solved by direct method using SIR2004 and refined by SHELXL97 21 as implemented in the program package WinGX 22 . Software used to prepare CIF file was SHELXL97 21 and graphics-ORTEP3 22 .

Chemistry
The synthesis of homo-sulfocoumarins began with a Wittig reaction in which salicylic aldehydes 1 were converted to the corresponding mono-olefins 2a-c in good yields (Scheme 2). Treatment of compounds 2a-c with allyl sulfonyl chloride (3) provided bisolefins 4a-c as the key intermediates, again in good yields (see Experimental for details). In the next step, olefin metathesis with the commercially available Ru-catalyst 5 was used, in which bis-olefins 4a-c were converted to 3H-1,2-benzoxathiepine 2,2dioxides 6a-b in 84-96% yields. To obtain a series of 7-substituted homo-sulfocoumarins, the synthesis of 1,4-triazolyl derivatives 9-17 was thereafter performed. For this purpose, 7-nitro derivative 6c was reduced by elemental iron to the corresponding amine 7 in nearly quantitative yield. Further diazotation of amine 7 followed by in situ treatment with sodium azide afforded the azide 8. Treatment of azide 8 with alkynes under click chemistry condition provides a series of 1,4-triazolyl homo-sulfocoumarins 9-17 in good to excellent yields (see Experimental for details).
The structures of all synthesized 3H-1,2-benzoxathiepine 2,2dioxides 6-17 were fully supported by 1 H, 13 C NMR and IR spectroscopy, MS or elemental analysis. Additionally, the final unequivocal identification of the scaffold of 3H-1,2-benzoxathiepine 2,2-dioxide was established by a single-crystal X-ray structure for compound 6c, shown in Figure 2.
Data of Table 1 show that the cytosolic isoforms hCA I and II (widely distributed enzymes, with important physiological roles in many tissues) 9,10 were generally not inhibited by the investigated homo-sulfocoumarins, up to 50 lM concentration of inhibitors in the assay system. Only one derivative, 13, showed a moderate inhibitory profile against hCA II, with an inhibition constant of 5.77 lM.
The tumour associated isoform hCA IX, a validated drug target for antitumor/antimetastatic agents 23,24 , was on the other hand effectively inhibited by the investigated homo-sulfocoumarins, with K I s ranging between 27 nM and 3.59 lM (Table 1). The structure activity relationship (SAR) was very interesting, as the best inhibitor (6c) incorporated a compact, powerful electron attracting moiety (NO 2 ) whereas the remaining derivatives, incorporating substituted 1,2,3-triazole moieties in position 7 of the homo-sulfocoumarin ring were less effective hCA IX inhibitors. Four submicromolar hCA IX inhibitors were however detected apart 6c, derivatives 13, 15, 16 and 17, which incorporate either the compact hydroxymethyl group at the triazole fragment of the molecule, or substituted phenyls with 4-trifluoromethoxy-, 2-amino-, or 4-trifluoromethyl substituents on the aryl fragment. These derivatives showed K I s ranging between 0.34 and 0.87 lM. The remaining homo-sulfocoumarins were low micromolar hCA IX inhibitors.
The SAR for inhibition of the second tumour-associated isoform, hCA XII, was more complex compared to what discussed above for hCA IX (Table 1). Thus, 8 out of 11 derivatives were inactive (K I s > 50 lM) whereas the remaining ones, 6c, 13 and 15, inhibited hCA XII with K I s in the range of 0.64-2.32 lM.
This inhibition profile is rather similar to the one of sulfocoumarins 1-6 and coumarins 7,8 , which are generally selective inhibitors for the tumour-associated over the cytosolic isoforms. However, some homo-sulfocoumarins showed a very specific, and unique up until now inhibition profile among all classes of CAIs known to date 9,10 , as they are highly selective for hCA IX over hCA I, II and XII (e.g. 7-12, 14, 16 and 17).
In conclusion, we report here a new chemotype with effective and isoform-selective CAIs, the homo-sulfocoumarins, which show a unique inhibition profile for the tumour-associated CA isoforms hCA IX (and XII) over the cytosolic ones. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site. Figure 2. Single-crystal X-ray structure of 6c (CCDC deposition number 1526002). Thermal ellipsoids are drawn at the 50% probability level (see Experimental for details).