Derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide as selective inhibitors of human carbonic anhydrases IX and XII over the cytosolic isoforms I and II

Abstract A series of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides with various substituents in 5, 6 or 7 positions was obtained from corresponding 2’-hydroxyacetophenones in their reaction with sulphamoyl chloride. 6- and 7-aryl substituted 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides were obtained from aryl substituted 2’-hydroxyacetophenonesprepared from 4- or 5-bromo-2’-hydroxyacetophenones via two-step protocol. 4-Methyl-1,2,3-benzoxathiazine-2,2-dioxides were investigated as inhibitors of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, off-target cytosolic hCA I and II, and target transmembrane, tumour-associated hCA IX and XII. Twenty derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide were obtained. With one exception (compound2a), they mostly act as nanomolar inhibitors of target hCA IX and XII. Basically, all screened compounds express none or low inhibitory properties towards off-target hCA I. hCA II is inhibited in micromolar range. Overwhelming majority of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides express excellent selectivity towards CA IX/XII over hCA I as well as very good selectivity towards CA IX/XII over hCA II.

Sixteen different a-CA isoforms have been identified in mammals so far 1,3 . hCA I and II are widely distributed isoforms which may serve as targets for some diseases and off-targets for others 7 . hCA IX and XII are two transmembrane, multi-domain, hypoxiainduced tumour-associated proteins discovered by Pastorek et al. 8 in 1994 and T€ ureci et al. 9 in 1998 that have received attention as diagnostic markers and potential drug targets for cancer 10,11 .
Tumour growth, angiogenesis, proliferation and metastasis are ascribed to the overexpressed levels of hCA IX and XII. This can be used as a strategy for targeting of these enzymes as a new approach in cancer treatment. Selective inhibition of the tumourassociated hCA IX and XII over the other isoforms, especially the most prevalent cytosolic hCA I and II is important and will result in cancer treatment with fewer side effects 7 .
hCA IX is not significantly expressed in the majority of normal tissues and is present only in the stomach and gallbladder epithelia, but is overexpressed in variety of solid tumors 10,11 . On the other hand, hCA XII is abundant in many healthy tissues, like kidney, prostate, pancreas, intestine and lymphocytes, but overexpressed in a certain number of malignant tumours, and associated with less-aggressive, well-differentiated tumour phenotypes as compared to the hCA IX expressing tumours thus CA IX is the preferred isoform for pharmacological intervention 7,10-12 . hCA IX has been considered as a valuable marker for cancer, and the development of hCA IX inhibitors with selectivity over ubiquitous isoforms hCA I/II is a potential strategy for designing anticancer agents 3,12 .

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 visualised with UV light (254 and 365 nm). Melting points were determined on an OptiMelt automated melting point system. IR spectra were recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR spectra were recorded on Bruker Avance Neo (400 MHz) spectrometer with chemical shifts values (d) in ppm relative to TMS using the residual CDCl 3 signal ( 1 H 7.26; 13 C 77.16) as an internal standard. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyser using the ESI technique. Elemental analyses were performed on Carlo Erba CHNS-O EA-1108 apparatus. GC-MS analyses were performed on Agilent Technologies 7890 A gas chromatograph, column -HP-5HS (df ¼ 0,25 lm, ID ¼ 0,25 mm, length À 30 m) with Agilent Technologies 5975 C masselective detector.

Sulphamoyl chloride 30
Chlorosulphonylisocyanate (3.80 ml, 43.3 mmol) was cooled to þ5 C. Formic acid (1.67 ml, 43.3 mmol) was added dropwise in temperature range between þ5 and þ13 C. Reaction mixture was stirred at 0 C for additional 45 min, then allowed to warm to room temperature. Toluene (30 ml) was added to the reaction mixture, precipitate was filtered, and filtrate was evaporated. Product was obtained as yellow oily solid (6.58 g, 98%). Mp 36-37 C.

Ca inhibitory assay
An applied photophysics stopped-flow instrument has been used for assaying the CA catalysed CO 2 hydration activity 40 .
Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5), and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10 -100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5 -10% of the reaction have been used for determining the initial rate. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilleddeionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay in order to allow for the formation of the E -I complex. Data from Table 1 were obtained after 6 h incubation of enzyme and inhibitor. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng -Prusoff equation, as reported earlier [41][42][43][44][45][46] and represent the mean Table 1. Inhibition data of human CA isoforms hCA I, II, IX, XII with compounds 2a-j and 5a-j reported here and the standard inhibitor acetazolamide (AAZ) by a stopped flow CO 2 hydrase assay. from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier 15,[47][48][49][50][51][52][53][54][55] .
5-, 7-and 8-aryl substituted 4-methylbenzo[1,2,3]oxathiazine-2,2-dioxides 5a-j were obtained from aldehydes 4a-j (Scheme 2). The first step was Suzuki-Miyaura coupling of compounds 3 with various boronic acids followed by cyclisation using sulfamoylphchloride. Yields of the intermediates were from moderate to high but yields of the products are quite low due to the loss during purification process.
None of screened derivatives showed significant inhibitory activity towards off-target hCA I. 7-Aryl substituted compounds 5f-j as well as some others (2a, 2e, 2j, 5d) do not inhibit hCA I at all, whereas other ones showed micromolar inhibitory activity (K I ¼ 10 -82 mM).
Compound 2a was the only one which did not inhibit cytosolic off-target hCA II. Other ones showed slight inhibitory properties. In general, aryl substituted derivatives inhibited hCA II less effectively than compounds 2a-j with small substituents in positions 5, 6 or 7.
Target transmembrane isoenzyme hCA IX was significantly inhibited by most derivatives in nanomolar range (K I ¼ 7.0 À 99.8 nM), except compound 2a which K I ¼ 75 mM.
Another target tumour-associated isoenzyme hCA XII was effectively inhibited by most derivatives (K I ¼ 10.5 -390 nM) as well, although compound 2a did not express hCA XII inhibitory properties also in this case.
Compound 2a was the only one which showed very low or did not show any inhibitory activity against target as well as non-target hCAs. This is probably due to the sterical hindrance provided by the substituent in position 5. Perhaps 5-methoxy group limits binding of the compound 2a to the enzyme.
The derivatives were assayed as inhibitors of four hCA isoforms, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX and XII.
Only compound 2a did not inhibit any of investigated hCA isoforms.
Transmembrane target isoform hCA IX was significantly inhibited by most derivatives in nanomolar range.
Another tumour-associated target isoform hCA XII was effectively inhibited by almost all derivatives as well.

Disclosure statement
No potential conflict of interest was reported by all author(s) except CTS. CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.