Carbonic anhydrase I, II, IV and IX inhibition with a series of 7-amino-3,4-dihydroquinolin-2(1H)-one derivatives

Abstract A series of new derivatives was prepared by derivatisation of the 7-amino moiety present in 7-amino-3,4-dihydroquinolin-2(1H)-one, a compound investigated earlier as CAI. The derivatisation was achieved by: i) reaction with arylsulfonyl isocyanates/aryl isocyanates; (ii) reaction with fluorescein isothiocyanate; (iii) condensation with substituted benzoic acids in the presence of carbodiimides; (iv) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate; (v) reaction with methylsulfonyl chloride and (vi) reaction with maleic anhydride. The new compounds were assayed as inhibitors of four carbonic anhydrases (CA, EC 4.2.1.1) human (h) isoforms of pharmacologic relevance, the cytosolic hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX. hCA IX was the most inhibited isoform (KIs ranging between 243.6 and 2785.6 nm) whereas hCA IV was not inhibited by these compounds. Most derivatives were weak hCA I and II inhibitors, with few of them showing KIs < 10 µm. Considering that the inhibition mechanism with these lactams is not yet elucidated, exploring a range of such derivatives with various substitution patterns may be useful to identify leads showing isoform selectivity or the desired pharmacologic action.


Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich (Milan, Italy). All reactions involving air-or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d 6 . 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; q, quadruplet; dd, double of doublet. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D 2 O.

CA assay
A stopped-flow method 44 has been used for assaying the CA catalysed CO 2 hydration activity with Phenol red as an indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalysed CO 2 hydration reaction for 10-100 s. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.01 mm) were prepared in distilled-deionised water with 5% DMSO and dilutions up to 0.1 nm were done thereafter with the assay buffer. Enzyme and inhibitor were incubated for 6 h 45-48 . The inhibition constant (K I ) was obtained by considering the classical Michaelis-Menten equation which has been fitted by using non-linear least squares with PRISM 3 (La Jolla, CA). All CA isozymes used in the experiments were purified, recombinant proteins obtained as reported earlier by our group [49][50][51][52][53][54][55][56][57][58][59] .

Chemistry
In a previous report from this group 43 , we showed that 7-amino-3,4-dihydroquinolin-2(1H)-one (1) (Scheme 1) possesses interesting CA inhibitory properties against many human isoforms such as hCA VII, IX, XII and XIV, some of which are important drug targets for various applications of the CAIs. The lactam 1 was investigated as a CAI due to its structural similarity with the coumarins, a class of CAIs reported by this group [45][46][47][48] . Indeed, unlike other classes of such pharmacological agents, the coumarins act as prodrug inhibitors, being hydrolysed by the CA esterase activity to substituted 2hydroxy-cinnamic acids, which thereafter bind at the entrance of the active site cavity, far away from the catalytic Zn(II) ion with which most CAIs interact 13,45 . That region is the most variable among the 15 human CAs, and this explains why coumarins and their derivatives are among the most isoform-selective CAIs reported so far 1,13,[45][46][47][48] . In fact, a large number of substitution patterns at the coumarin ring, isosteric replacements or various other modifications were done on this chemotype, leading to a large number of CAIs possessing interesting properties 13,[45][46][47][48] . Thus, the rationale of this work was to derivatise the 7-amino moiety of the lead 1, by reacting it with a variety of agents used earlier for the design of sulfonamide or dithiocarbamate CAIs (Scheme 1) [13][14][15][16][22][23][24][25][35][36][37]60,61 .
As shown in Scheme 1, a multitude of derivatisations of the amino moiety of compound 1 were achieved, such as: (i) reaction with arylsulfonyl isocyanates (leading to arylsulfonylureido derivatives 2-6); (ii) reaction with isocyanates, leading to ureas 7-19; (iii) reaction with fluoresceine isothiocyanate, leading to the fluorescent thiourea 20; (iv) condensation with substituted benzoic acids in the presence of carbodiimides, leading to the amides 21 and 22; (v) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate, leading to the pyridinium salt 26; (vi) reaction with methylsulfonyl chloride leading to the secondary sulfonamide 24, which was subsequently methylated with methyl iodide, leading to the 1-Nmethyl derivative 25, and (vii) reaction with maleic anhydride leading to the monoamide 23 (Scheme 1). All these compounds were thoroughly characterised by physicochemical procedures which confirmed their structures (see "Materials and methods" for details).

CA inhibition
Compounds 2-26 were assayed for their CA inhibitory activity by a stopped-flow, CO 2 hydrase method 44 against four isoforms of  pharmacologic relevance, the cytosolic human (h) hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumourassociated hCA IX (Table 1). The following structure-activity relationship can be observed from the inhibition data of Table 1: i. hCA I was poorly inhibited by most derivatives 2-26, with only seven of them showing K I s in the micromolar range (i.e. 3.20-8.75 mm), the remaining ones having K I s > 10 mm ( Table 1). The more effective inhibitors were 2-4, 6, 17 and 18, which incorporate arylsulfonylureido and ureido moieties. The other substitution patterns led to compounds with much weaker hCA I inhibitory activity. ii. hCA II, the dominant cytosolic isoform was generally also poorly inhibited by these derivatives (K I s > 10 mm) except the arysulfonylureido ones 2-6 (K I s of 4.43-7.46 mm) the ureas 15-18 (K I s of 4.97-7.88 mm) and the thiourea 20 (K I of 3.37 mm), which was the best hCA II inhibitor in the series. iii. hCA IV was the least sensitive isoform to these compounds with only one of them (17, K I of 3.80 mm) having an activity <10 mm (Table 1). It is rather difficult to explain this result considering that the inhibition mechanism with these lactams is not yet elucidated. iv. The tumour-associated hCA IX was the most inhibited isoform among the four investigated ones, with K I s ranging between 243.6 and 2758.6 nm (Table 1). Only four derivatives (8, 10, 16 and 21) had K I s > 10 mm, whereas the best hCA IX inhibitors were 15 and 24 with K I s of 243.6-292.8 nm. These compounds rather different as the first one is a urea incorporating a pentafluorophenyl moiety, whereas the second one has the secondary sulfonamide functionality. It should be noted that small variations in the structures of such derivatives (as the N1-methylation of 24 leading to 25) or the reduction of the number of fluorine atoms on the phenyl ring, as in 14, led to a rather important reduction of the hCA IX inhibitory power compared to 24 and 15, respectively. Generally, all other arylsulfonylureas/ureas 2-19 (except the two compounds mentioned above as weak inhibitors and 15 which is one of the best) showed a similar behaviour of medium potency hCA IX inhibitors with K I s of 1.05-2.48 mm. v. All the derivatives reported here showed much weaker CA inhibitory activity compared to the clinically used sulfonamide acetazolamide AAZ (Table 1).

Conclusions
A series of derivatives was prepared by derivatisation of the 7amino moiety of 7-amino-3,4-dihydroquinolin-2(1H)-one, a compound investigated earlier as CAI. The derivatisation was achieved by: (i) reaction with arylsulfonyl isocyanates (ii) reaction with aryl isocyanates; (iii) reaction with fluoresceine isothiocyanate; (iv) condensation with substituted benzoic acids in the presence of carbodiimides; (v) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate; (vi) reaction with methylsulfonyl chloride and (vii) reaction with maleic anhydride. The new compounds were assayed as inhibitors of four CA human isoforms of pharmacologic relevance, the cytosolic hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX. hCA IX was the most inhibited isoform (K I s ranging between 243.6 and 2658.3 nm) whereas hCA IV was not inhibited by these compounds. Most derivatives were weak hCA I and II inhibitors, with few of them showing K I s < 10 mm. Considering that the inhibition mechanism with these lactams is not yet elucidated, exploring a large range of derivatives with various substitution patterns may be useful to identify leads showing isoform selectivity. Errors were in the range of ±5-10% of the reported data, from three different assays.