Identification of Potent and Selective Human Carbonic Anhydrase VII (hCA VII) Inhibitors

Carbonic anhydrases (CA, EC 4.2.1.1) are a family of metalloenzymes comprising different isoforms with different tissue and cellular localization. They play an important role in many physiological processes linked to the catalytic hydration of carbon dioxide to bicarbonate, regulating respiration, pH, electrolyte secretion, biosynthetic reactions, bone resorption, calcification, etc. Furthermore, these enzymes are involved in several pathological pathways, suggesting the development of carbonic-anhydrase inhibitors (CAIs) as new tools, such as diuretics, anticonvulsants, antiobesity and antitumor drugs. 2] The most active CAIs are characterized by the presence of a sulfonamide/sulfamate functionality coordinating the zinc ion and interacting with specific amino-acid residues in the catalytic site. Although these chemical features are important requirements for potent CA inhibition, the lack of specific isoform selectivity for therapeutically relevant over ubiquitous CA isoforms (such as CA I and CA II) is often considered responsible for some undesirable side effects that limit the development of CAIs. Moreover, the inhibition of specific isoforms that play a role in physiological/pathological processes could drive pharmacological activities and avoiding off-target effects. In fact, it is well known that the antiglaucoma agents target CA II, CA IV and CA XII, the antiobesity agents inhibit CA VA and CA VB, 8] the anticonvulsant agents act against CA II, CA VII and CA XIV, and the antitumor drugs target CA IX and CA XII. 10–13] In previous works, we rationally designed, synthesized and evaluated isoquinoline-sulfonamides as CAIs, testing their affinity towards different human CA isoforms (hCA I, hCA II, hCA IX, and hCA XIV). 15a–b] As a result of these studies, we identified some isoquinolines (1–10 ; Tables 1 and 2) that showed nanomolar binding affinities (Ki values) ; these compounds were also characterized by significant selectivity for hCA IX and hCA XIV over ubiquitous cytosolic hCA II of up to approximately three orders of magnitude (i.e. , compound 8). Since the hCA IX and hCA XIV isoforms are emerging targets for antitumor and neuroprotective agents, we considered the Ki values and selectivity profiles of these isoquinolines particularly useful for the design of new CAIs with minor side effects. Moreover, a crystal structure of one of the most active inhibitors bound to the human dominant CA II was prepared, thus confirming that the sulfonamide moiety of compound 2 coordinates to the zinc ion and Thr 199 located in the catalytic binding pocket. In addition, using preliminary docking experiments, we proposed the main protein–inhibitor interactions within the catalytic site, thereby rationalizing the inhibitory properties. The main structure–affinity relationship identified was that the nature of the C-1 substituent on the isoquinoline scaffold controlled the affinity, as well as the CA isoform selectivity. In order to evaluate the influence of stereochemistry at the C-1 position, the enantiomeric resolution for the most active molecules is in progress. Continuing our efforts to identify potent and selective CAIs, herein we report the inhibitory effects of 3,4-dihydroisoquinoline-2(1H)-sulfonamides 1–10 against these interesting and druggable CA isoforms, including the neuronal hCA VII (Table 2) as an important target for the development of anticonvulsant agents and neuropathic pain killers. Moreover, we prepared and tested a new class of sulfonamides 11–14, which we designed on the basis of previously published findings that the ArSO2NH2 fragment is a crucial structural requirement of CAIs. 18] Scheme 1 shows the synthetic pathway to obtain the benzenesulfonamides 11–14. Initially, 2-(3’,4’-dimethoxyphenyl)ethylamine (15) was converted into intermediate 16 by reaction with 4-(aminosulfonyl)benzoic acid (11) and using HBTU as the coupling reagent. The obtained 4-(aminosulfonyl)-N-[2-(3’,4’-dimethoxyphenyl)ethyl]benzamide (11) was cyclized by treatment with phosphorus chloride oxide to give the 3,4-dihydroisoquinoline derivative 12 ; reduction of this intermediate with sodium borohydride gave the 1,2,3,4-tetrahydroisoquinoline 13. Finally, treatment with sulfamide yielded 1-[4-(aminosulfonyl)phenyl]-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfoTable 1. Isoquinoline sulfonamides 1–10.