Synthesis of novel benzenesulfamide derivatives with inhibitory activity against human cytosolic carbonic anhydrase I and II and Vibrio cholerae α- and β-class enzymes

Abstract The synthesis of a new series of sulfamides incorporating ortho-, meta, and para-benzenesulfamide moieties is reported, which were investigated for the inhibition of two human (h) isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I and II, and two Vibrio cholerae enzymes, belonging to the α- and β-CA classes (VchCAα, VchCAβ). The compounds were prepared by using the “tail approach”, aiming to overcome the scarcity of selective inhibition profiles associated to CA inhibitors belonging to the zinc binders. The built structure–activity relationship showed that the incorporation of benzhydryl piperazine tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAα, with several compounds showing KIs < 100 nM. The activity was lower against hCA II and VchCAβ, probably due to the fact that the incorporated tails are quite bulky. The obtained evidences allow us to continue the investigations of different tails/zinc binding groups, with the purpose to increase the effectiveness/selectivity of such inhibitors against bacterial CAs from pathogens, affording thus potential new anti-infectives.


Introduction
Cholera is an infectious human disease of the small intestine and is caused by the gram-negative bacterium Vibrio cholera 1,2 . It is characterised by a massive loss of water and electrolytes which leads to severe dehydration and hypovolemic shock followed by death if the disease is not well treated [2][3][4] . In developing countries, cholera spreads among victims mainly through contaminated water sources, and countries without proper sanitation techniques have greater incidence of this disease 3,5 . In this regard, WHO reported 132,121 cases in 38 countries during 2016, which also included 2420 deaths 6 . It has been demonstrated that a potential inducer of virulence gene expression is sodium bicarbonate, which is present at a high concentration in the upper small intestine 2 . Consequently, bicarbonate is considered the first positive effector for ToxT, the major direct transcription activator of the virulence genes 2 .
Many studies conducted by some of us have shown that specific carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, may control the bicarbonate-mediated virulence induction, suggesting the conversion of CO 2 into bicarbonate by CA plays a crucial role as a virulence factor for Vibrio cholerae 7,8 . Three different CAs have been found in Vibrio cholerae, belonging to the three enzyme classes found in bacteria, VchCAa, b, and c. VchCAs have been suggested as potential targets for anti-infectives development with a novel mechanism of action 9,10 .
We recently reported a set of benzhydrylpiperazine benzenesulfamide showing effective and selective inhibition profiles against the cytosolic isoform hCA I 15 . In the present study, we have oriented our efforts in developing sulfamide CAIs as potential antibacterial agents, continuing the development of the previously reported series of sulfamides 15 , and report the synthesis of new series of such derivatives. In addition to the bacterial enzyme VchCAa and b, these novel compounds were investigated for their property to inhibit the physiologically most important human cytosolic isoforms CA I and II 16,17 .
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 spectra ( 1 H NMR: 400 MHz; 13C NMR: 100 MHz; 19F NMR: 376 MHz) were recorded in DMSO-d 6 using an Avance III 400 MHz spectrometer (Bruker, Milan, Italy). 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; m, multiplet; brs, broad singlet; dd, double of doublets. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D 2 O. Analytical thin-layer chromatography (TLC) was carried out on silica gel F-254 plates (Merck, Milan, Italy). Melting points (m.p.) were carried out in open capillary tubes and are uncorrected. The solvents used in MS measures were acetone, acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich (Milan, Italy) and mQ water 18 MX, obtained from Millipore's Simplicity system (Milan, Italy). The mass spectra were obtained using a 1200 L triple quadrupole system (Varian, Palo Alto, CA) equipped by electrospray source (ESI) operating in both positive and negative ions. Stock solutions of analytes were prepared in acetone at 1.0 mg ml À1 and stored at 4 C. Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of mQ H 2 O/ACN 1:1 (v/v) up to a concentration of 1.0 mg ml À1 . The mass spectra of each analyte were acquired by introducing, via syringe pump at 10 ml min À1 , its working solution. Raw-data were collected and processed by Varian Workstation Vers. 6.8 software (Palo Alto, CA).

2.2.5.
General procedure for the synthesis of compounds 4a-p A stirred solution of compounds 2a-d (1.0 eq) in DCM (10.0 ml) was treated with TFA (3.0 eq) and stirred at r.t. for 2 h. The reaction mixture was concentrated to dry and co-distilled twice with DCM to afford the corresponding amines 3a-d as TFA salts (not isolated), which were readily dissolved in DCM (10.0 ml) and treated with DIPEA (5.0 eq) and the appropriate sulfonyl chloride (1.2 eq). The reaction solutions were stirred at r.t. for 1 h, then concentrated to dry and the residue obtained was purified by silica gel column chromatography using ethyl acetate in n-hexane (20-40% v/v) as eluent to afford the titled compounds 4a-p as off-white solids.

Carbonic anhydrase inhibition assay
An Applied Photophysics (Leatherhead, UK) stopped-flow instrument has been used for assaying the CA-catalysed CO 2 hydration activity 18 . Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, 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 velocity. 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 distilled-deionised 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. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations 19,20 . All CA isoforms were recombinant ones obtained in-house as reported earlier 21 .

Chemistry
The sulfamide moiety is able to establish a more extended network of hydrogen bonds within the enzymatic pocket compared to classical sulfonamides, due to the presence of the additional nitrogen atom, but unfortunately this feature does not confer selectivity against different CA isoforms 16,17 . Therefore, the purpose of this study is not only to explore the activity of the sulfamides against the two CA isoforms of Vibrio cholerae, but also to propose specific structural changes to obviate to the problem of selectivity.
The novel sulfamides reported here feature the sulfamides groups (in ortho-, meta-, or para positions on the benzene ring), connected into a highly flexible alkyl-aryl scaffolds. In particular, we designed two series of compounds which differ by the spacer connecting the 1-benzhydrylpiperazin tail with the benzene-sulfamide zinc binding functionality (Scheme 1) 16 .
The planned synthesis of the compounds 6a-p provides a first step during which the coupling of the benzhydryl piperazines 1a,b with the appropriate acids takes place to give the intermediates 2a-d, followed by treatment with trifluoroacetic acid (TFA) to afford the alkylamines 3a-d. In the second step, the free amines were coupled with commercially available nitrobenzenesulfonyl chlorides and the obtained nitro derivatives 4a-p were reduced with iron, Fe(0), in acidic media, to afford the amines 5a-p. Finally, the desired compounds 6a-p ( Figure 1) were obtained by treatment of amines 5a-p with freshly prepared sulfamoyl chloride 16 . All compounds were purified by trituration from Et 2 O to afford products with high purity in yields between 10 and 80%; the analytical and spectroscopic data of the purified compounds are in agreement with the purposed structures (see section 2 for details).

Carbonic anhydrase inhibition
All synthesised compounds 6a-p, were tested in vitro for their inhibitory activity against the cytosolic isoenzymes hCA I and II and bacterial enzymes VchCAa and VchCAb by means of a stopped-flow carbon dioxide hydration assay 18 , and their activities were compared to the standard CAI acetazolamide (AAZ). The following structure-activity-relationship (SARs) can be drawn from the data of Table 1: i. hCA I was effectively inhibited by most compounds investigated here, with inhibition constants spanning between 43.1 and 839.9 nM. It is interesting to note the case of compounds 6m and 6p that demonstrate the best activity (K I s ¼ 47.8 and 49.1 nM), both having the sulfamide group in para position of the aromatic ring but differing for the glycine and isonipecotic acid spacer, respectively. The removal of the fluorine atoms in the aromatic rings of compound 6g (corresponding to the non-halogenated analogue of 6d), decreased the activity (K I ¼ 601.3 nM), suggesting that substitution with halogens as -F, in this case, is advantageous for the inhibitory activity. The same trend concerned compounds 6j and its corresponding non-halogenated 6b, which showed the following inhibition data K I s of 64.6 and 555.1 nM, respectively. On the other hand, the presence of fluorine atoms in aromatic rings of compound 6i (K I ¼ 75.0 nM), did not have a significant impact on its activity, both compound having a similar activity to that of 6a (K I ¼ 66.2 nM). The inhibition potencies were dependent not only on the presence of halogens but also on the sulfamide regioisomer considered. In fact, the potency ranking for compounds 6j, 6i, and 6h (K I s ¼ 64.6, 75.0, 842.6 nM), 6m, 6l, and 6k (K I s ¼ 47. 8, 71.1, 337.8 nM) and for compounds 6p, 6o, and 6n (K I s ¼ 49.1, 72.0, 682.6 nM) was para>meta>ortho. ii. The ubiquitous hCA II was inhibited by sulfamides 6a-p, with K I s spanning between range 181.7 and 1430.7 nM. Likewise hCA I, the derivatives with sulfamide group in para position of the aromatic ring showed better activity than the corresponding meta and ortho analogues. In fact, compound 6g shows a lower K I ¼ 226.7 nM than its meta-substituted analogue 6f (K I ¼ 576.4 nM) or ortho-substituted derivative 6e (K I ¼ 1360.1 nM). The introduction of fluorine atoms on the phenyl rings of compounds 6g, 6f, and 6e to give derivatives 6o, 6p, and 6n, caused a slight change in the inhibition trend, with a better K I of the ortho-substituted compared to the meta-substituted derivative. It is interesting to underline the case of the compound 6p (K I ¼ 181.7 nM), which is the best inhibitor against hCA II. The substitution of the isonipecotic acid spacer of 6p with the glycine spacer of 6m did not show any impact on the inhibitory activity. The removal of the fluorine atoms in 6g did not substantially altered the activity compared to the perhydro derivative. Equally to hCA I, for hCA II the most considerable differences of inhibitory activity concerned the position of the ZBG in the phenyl ring, as demonstrated for example by compounds 6d (metasubstituted) and 6c (ortho-substituted) with K I s of 396.4 and 850.2 nM, respectively. iii. The general tendencies described above were also applicable for the VchCAa, with this latter being moderately inhibited by benzenesulfamide derivatives prepared in this study (K I s values in the range of 91.4 and 3748.6 nM). As reported for the human isoforms discussed above, also for VchCAa, the worst inhibitors are reconfirmed to be the compounds that Scheme 1. General synthetic scheme of compounds 6a-p.
present the ZBG in the ortho position of the phenyl ring (6c, 6e, 6h, 6k, and 6n with K I s ¼ 930. 8, 879.3, 2797.0, 1747.0, and 3748.6 nM, respectively). The most active compounds were the halogen derivative 6l and the non-halogen analogue 6d, which exhibited K I values of 91.4 and 95.7 nM, respectively. It is interesting to note that the introduction of the rigid linker of isonipecotic acid determined a lower efficacy against this isoform, as shown by the compounds 6f (K I ¼ 654.2 nM) and 6o (K I ¼ 497.2 nM), compared with the above-mentioned derivatives 6d and 6l. This improvement of VchCAa inhibition potency might be due to the flexible glycine spacer, which probably increases the interactions of the inhibitor with active site amino acids residues.
iv. VchCAb was the least inhibited among the enzymes herein considered and showed K I s spanning between 1578.2 and 4676.1 nM. Interestingly, the meta derivatives 6o and 6i (K I s ¼ 1578.2 and 1742.2 nM) were the most potent inhibitors against the VchCAb, compared to the corresponding para analogues 6p and 6j (K I s ¼ 4533.7 and 4349.2 nM). On the contrary, the meta derivatives 6a and 6f (K I s ¼ 4676.1 and 3792.0 nM) were less effective with respect to para derivatives 6b and 6g (K I s ¼ 1880.7 and 2538.1 nM). It is rather difficult to explain this result but we could speculate that the introduction of fluorine atoms leads to a decrease in the inhibitory potency of sulfamides against VchCAb. In fact the fluorination of compounds 6g, 6b, and 6d (K I s ¼ 2538.1, 1880.7, and 3782.8 nM), to afford 6p, 6j, and 6l, respectively, decreased the inhibition efficacy (K I s ¼ 4533.7, 4349.2, and >10,000 nM, respectively), with the potency of compound 6l being very low. Finally, it is important to emphasise the selectivity of some compounds against VchCAa with respect to the b-bacterial isoform. For instance, compounds 6l and 6d showed a nanomolar efficacy against VchCAa (K I s of 91.4 and 95.7 nM) but 6l was totally ineffective against the b-isoform, whereas compound 6d showed a K I of 3782.8 nM against VchCAb.

Conclusions
In this work, we reported the investigation of benzenesulfamide derivatives (-NH-SO 2 NH 2 ) as an alternative zinc binding class of inhibitors to the primary aromatic sulfonamides in the design of new potential bacterial CAIs 17 . The "tail approach", the method that consists in varying the terminal portions of the molecule to selectively enhance the inhibition of the target isoform(s), was applied to the phenyl sulfamide scaffold. All compounds were synthesised by a convenient and efficient method and were screened for in vitro inhibition against cytosolic isoenzymes hCA I, II, VchCAa and VchCAb by using a stopped-flow CO 2 hydrase assay method. The obtained results suggest that the benzhydryl piperazine moiety appended to benzene-sulfamide functionalities are not favourable for the interaction with Vibrio cholerae CA isoforms. Conversely, the same moiety is slightly more favourable for the inhibition of hCA I, as demonstrated by compounds 6f, 6m, 6p, and 6d that showed interesting activities against this isoform, with a nanomolar inhibition constant (K I s ¼ 43.1, 47.8, 49.1, and 58.1 nM, respectively), being thus more potent than AAZ (as standard inhibitor).