3H-1,2-Benzoxaphosphepine 2-oxides as selective inhibitors of carbonic anhydrase IX and XII

Abstract The synthesis of 3H-1,2-benzoxaphosphepine 2-oxides and evaluation of their inhibitory activity against human carbonic anhydrase (hCA) isoforms I, II, IX, and XII are described. The target compounds were obtained via a concise synthesis from commercial salicylaldehydes and displayed low to sub-micromolar inhibition levels against the tumour-associated isoforms hCA IX and XII. All obtained benzoxaphosphepine 2-oxides possess remarkable selectivity for inhibition of hCA IX/XII over the off-target cytosolic hCA isoforms I and II, whose inhibition may lead to side effects.


Introduction
Carbonic anhydrases (CA, EC 4.2.1.1) are a superfamily of metalloenzymes present across all kingdoms of life 1 . These enzymes catalyse a simple yet essential physiological reactionthe reversible hydration of carbon dioxide 2 . To date, 15 different human CA (hCA) isoforms have been identified, out of which hCA IX and XII isoforms are highly overexpressed in different tumour types and may contribute in the survival and progression of tumour cells by regulating intra-and extracellular pH [2][3][4][5][6] . Therefore, the development of selective hCA IX/ XII inhibitors is a potential strategy for designing anti-tumour agents.
Due to the high degree of structural homology and sequence similarities within the active site of the hCA isoforms, the design and development of isoform-selective hCA inhibitors pose a challenge 7 . A variety of compounds have been reported as potent and selective inhibitors of tumour-associated isoforms hCA IX and XII including coumarins [8][9][10][11] , thiocoumarins 8,11 , sulphocoumarins 8,[12][13][14][15] , as well as their congeners, homosulphocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) 16 . In this work, attention was drawn to phosphorus, as phosphorus-containing molecules display a multitude of biological activities relevant in medicinal chemistry 17 . Additionally, several groups have shown the use of organophosphorus compounds as CA inhibitors 18 .
Considering isosteric relationship between sulphonyl derivatives and phosphonates 19 , our research group designed and synthesised a series of benzoxaphosphepine 2-oxides pursuing the development of new classes of selective CA inhibitors. These compounds showed interesting inhibitory activity against hCA IX and XII. Moreover, the results of current study demonstrate the bioisosteric utility of the cyclic phosphonate moiety in the design of novel CA inhibitors.

Chemistry
The air-or moisture-sensitive reactions were performed under argon atmosphere using dry glassware. Toluene was freshly distilled from Na prior to use. DCM and NEt 3 were distilled from CaH 2 . Other reagents, starting materials and solvents were purchased from commercial sources and used as received. TLC was performed on silica gel plates (60 F 254 ) and visualised under UV light (254 and 365 nm). Melting points were determined on an OptiMelt MPA100 apparatus. IR spectra were recorded on a Shimadzu FTIR IR Prestige-21 spectrophotometer. 1 H, 13 C, and 31 P NMR spectra were recorded on a Bruker Avance Neo 400 MHz spectrometer. The chemical shifts (d) were reported in parts per million (ppm) relative to the residual solvent peak as an internal reference (DMSO-d 6 : 1 H 2.50, 13 C 39.52; CDCl 3 : 1 H 7.26, 13 C 77.16; C 6 D 6 : 1 H 7.16, 13 C 128.06). 31 P shifts were referenced externally to H 3 PO 4 . The coupling constants (J) were expressed in Hertz (Hz). HRMS was performed on a Q-TOF Micro mass spectrometer.

Carbonic anhydrase inhibition assay
The CA-catalysed CO 2 hydration activity was assayed by using an applied photophysics stopped-flow apparatus 24 . Phenol red (0.2 mM) was used as indicator following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The indicator worked at the absorbance maximum of 557 nm, with 20 mM HEPES buffer (pH 7.4) and 20 mM NaClO 4 for maintaining constant ionic strength. For the determination of the kinetic parameters and inhibition constants, the CO 2 concentrations were varied from 1.7 to 17 mM. For each inhibitor, at least six traces of the initial 5-10% of the reaction were used for determining the initial velocity. The uncatalysed rates were determined in the same fashion and subtracted from the total observed rates. The stock solutions of inhibitor were prepared as 1 mM solutions in distilled, deionised water. Afterwards, dilutions down to 0.01 nM were prepared in distilled and deionised water. Inhibitor and enzyme were preincubated together for 6 h at room temperature in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were acquired by the non-linear least squares method using PRISM 3 and the Cheng-Prusoff equation, whereas the kinetic parameters of uninhibited enzymes were obtained from Lineweaver-Burk plots and represent the mean from at least three different determinations. All CA isoforms were recombinant, obtained in-house as reported earlier 25-27 .

Chemistry
The synthetic strategy for the synthesis of 3H-1,2-benzoxaphosphepine 2-oxides is outlined in Scheme 1. The synthesis commenced with the Wittig reaction of commercially available 2hydroxybenzaldehydes 1, which provided olefins 2 in high yields. In the following step, compounds 2 were treated with ethyl allylphosphonochloridate (3, the reagent was prepared according to the literature procedure 23 ) to give diolefins 4 in good to excellent yields. These key intermediates 4 were successfully cyclised by ring-closing metathesis, utilising commercially accessible Ru-based catalyst 5. The reaction furnished corresponding cyclic ethyl phosphonates 6 in good yields. Finally, compounds 6 were treated with TMSBr to afford hydroxy derivatives 7 in very good yields.

Carbonic anhydrase inhibition
The newly synthesised compounds 6 and 7 were evaluated for their CA inhibition activity by using the stopped-flow CO 2 hydrase assay 24 . The study was carried out against four human CA isoformsthe ubiquitous cytosolic CA I and II as well as trans-membrane tumour-associated CA IX and XII 1-7 . The clinically used acetazolamide (AAZ) was used as the reference drug. The results of this study are shown in Table 1, and the following inferences could be drawn: i. All synthesised benzoxaphosphepine2-oxide derivatives 6-7 have no inhibitory activity towards cytosolic isoforms hCA I and hCA II (K I > 100 mM), whose inhibition in most cases is undesirable, as hCA I and II isoforms are found in many tissues of the organism 1,2,7 . It should be mentioned that AAZ is a highly effective inhibitor of all the four hCA isoforms considered here, which explains the many side effects of that drug 28,29 . ii. The tumour-associated hCA IX isoform was inhibited by all synthesised compounds 6-7 with inhibition constants in the sub-micromolar to low micromolar range (K I : 0.67-11.3 mM).
The compound 7g was found to be the most potent hCA IX inhibitor among tested compounds with K I ¼ 0.67 mM. iii. The other tumour-associated isoform hCA XII was also notably inhibited by all the synthesised derivatives 6-7 with K I values in the low micromolar and sub-micromolar range (K I : 0.51-7.4 mM). Among all tested compounds, compound 7a was the most effective inhibitor against hCA XII with K I ¼ 0.51 mM. iv. Overall, hydroxy derivatives 7 showed slightly higher inhibition potency against tumour-associated hCA IX and XII than the corresponding ethoxy derivatives 6. In the case of hydroxy derivatives 7, the range of K I values was found to be from 0.67 to 2.5 mM for hCA IX and from 0.51 to 1.8 mM for hCA XII. Regarding ethoxy derivatives 6, the range of K I values was from 0.76 to 11.3 mM for hCA IX and from 0.95 to 7.4 mM for hCA XII.  Albeit the efficacy of the synthesised compounds 6-7 was lower in comparison to the reference drug AAZ, these compounds displayed desirable isoform-selective inhibition activity for tumourassociated isoforms hCA IX and hCA XII. The establishing of the selectivity is necessary to prevent possible side effects from inhibition of cytosolic hCA I and II isoforms 7,28,29 .

Conclusions
Herein we report the synthesis of novel benzoxaphosphepine 2oxide derivatives as a new class of tumour-associated CA IX and XII inhibitors. These compounds were investigated against four human CA isoforms with pharmacological applications (hCA I, hCA II, hCA IX, and hCA XII). All tested compounds exhibited selective inhibition of the tumour-associated hCA isoforms IX and XII with activities in the sub-micromolar or low micromolar range, whereas the off-target cytosolic isoforms hCA I and II were not significantly inhibited by these compounds. Considering that hCA IX and XII are implicated in processes connected to tumourigenesis 2-6 , present findings give an insight towards development of new selective anti-tumour agents.

Disclosure statement
No potential competing interest was reported by all authors 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.