Ординатура / Офтальмология / Английские материалы / Carbonic Anhydrase Its Inhibitors and Activators_Supuran, Scozzafava, Conway_2004

4.196: Mr = 3.5 kDa

OH

N N

OCH2CH2NHCOCH2CH2CONH S SO2NH2

OCH2CH2NH2

OH

Aminoethyldextran

4.197: Mr = 6.7- 99 kDa

4.199: QAS

for both CA II and CA IV, and, more importantly, were unable to cross the plasma membranes in vivo (Supuran et al. 2000b; Scozzafava et al. 2000c). In two model systems (human red cells and perfusion experiments in rats), this new class of potent, positively charged CAIs was able to discriminate the membrane-bound from the cytosolic isozymes, selectively inhibiting CA IV only (Supuran et al. 2000b; Scozzafava et al. 2000c). Such data are important both for specific in vivo inhibition of membrane-associated isozymes and for the eventual development of some novel anticancer therapies, because it has been shown that some tumor cells predominantly express only some membrane-associated CA isozymes, such as CA IX and CA XII.

This type of selective CAI might be of great relevance to different physiological studies. For example, Sterling et al. (2002) investigated the functional and physical

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relationship between the downregulated, in adenoma, bicarbonate transporter and CA II, by using membrane-impermeant inhibitors of type 4.210 (in addition to classical inhibitors such as acetazolamide), which could clearly discriminate between the contribution of the cytosolic and membrane-associated isozymes in these physiological processes.

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4.6 ANTITUMOR SULFONAMIDES

There are many connections between CA and cancer; for example, some CA isozymes (CA IX and XII) are predominantly found in cancer cells and not in the normal counterparts (Pastorek et al. 1994; Pastorekova et al. 1997; Chegwidden et al. 2001). Teicher et al. (1993) reported that acetazolamide (4.44) functions as a modulator in anticancer therapies in combination with different cytotoxic agents, such as alkylating agents, nucleoside analogs or platinum derivatives. It was hypothesized that the anticancer effects of acetazolamide (alone or in combination with such drugs) might be due to acidification of the intratumoral environment occurring after CA inhibition, although other mechanisms of action of this drug were not excluded (Treicher et al. 1993). Chegwidden et al. (2001) hypothesized that in vitro inhibition of growth in cell cultures of human lymphoma cells with two other potent, clinically used sulfonamide CAIs, methazolamide (4.48) and ethoxzolamide (4.81), is probably due to the reduced provision of bicarbonate for nucleotide synthesis (HCO3– is the substrate of carbamoyl phosphate synthetase II) as a consequence of CA inhibition.

The development of CAIs possessing potent tumor cell growth inhibitory properties was reported by this group (Supuran and Scozzafava 2000b, 2000c; Scozzafava and Supuran 2000a; Supuran et al. 2001). Such compounds were discovered in a large screening program [in collaboration with the National Institutes of Health (NIH)] of sulfonamide CAIs. Several hundred aromatic/heterocyclic sulfonamides were assessed in vitro as potential inhibitors of growth of a multitude of tumor cell lines, such as leukemia, nonsmall cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancers. The active compounds (most of them nanomolar inhibitors of CA II and CA IV), of types 4.212 to 4.223, belong to both the aromatic and the heterocyclic sulfonamide classes and showed GI50 values (molarity of inhibitor producing a 50% inhibition of tumor cell growth after a 48-h exposure to the drug) in the micromolar range (Supuran and Scozzafava 2000b, 2000c). Better antitumor compounds were then developed by an original strategy, and they incorporated in their molecules N,N-dialkylthiocarbonylsulfenylamino moieties (Scozzafava and Supuran 2000a; Supuran et al. 2001). Thus, aromatic/heterocyclic sulfonamides possessing free amino, imino or hydrazino groups of types A–Y (see the tail approach) were transformed to the corresponding N-morpholyl-thiocarbonyl- sulfenyl or N,N-dimethyl/diethyl-thiocarbonylsulfenylamino derivatives 4.224 to 4.226 by reaction with dithiocarbamates in the presence of oxidizing agents (NaClO or iodine).

Sulfonamides of the types 4.224 to 4.226 showed nanomolar affinity for CA II and CA IV, but, more importantly, some of them inhibited the growth of several tumor cell lines at concentrations as low as 10 nM (Scozzafava and Supuran 2000a; Supuran et al. 2001), thus showing a highly increased antitumor efficacy as compared with classical CAIs (acetazolamide, methazolamide) or compounds 4.212 to 4.218.

The antitumor sulfonamide indisulam, E7070 (4.227), is in Phase II clinical trials in Europe and the U.S. as a novel anticancer agent to treat solid tumors (Supuran 2003). This compound is also a very potent inhibitor of many CA isozymes, including CA I, II and IX (unpublished results from our laboratory).

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4.7SULFONAMIDES WITH MODIFIED MOIETIES AND OTHER ZINC-BINDING GROUPS

Krebs (1948) reported that substitution of the sulfonamido moiety to give compounds of type ArSO2NHR drastically reduced the CA inhibitory properties compared with those of the corresponding derivatives possessing primary sulfonamido groups, ArSO2NH2. As a consequence, other zinc-binding functions except for the SO2NH2 group have rarely been considered in the design of CAIs, although many other zinc enzymes are inhibited by a multitude of derivatives possessing an entire range of zinc-binding functions, such as thiols, phosphonates, carboxylates and hydroxamates (Supuran and Scozzafava 2002a, 2002b). Only recently, several detailed studies on the possible modifications of the sulfonamido moiety, compatible with the retention of strong binding to the enzyme, have been reported (Briganti et al. 1996; Mincione

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4.227: E7070 (indisulam)

et al. 1998; Supuran et al. 1999a; Scozzafava and Supuran 2000b; Scozzafava et al. 2000b; Fenesan et al. 2000). Compounds of types 4.228 to 4.230 were studied kinetically for inhibition of reactions catalyzed by CA I and II (CO2 hydration and ester hydrolysis), but their binding to the enzyme has also been monitored spectroscopically by studying the electronic and 1H-NMR spectra of adducts of such inhibitors with Co(II)–CA II (Briganti et al. 1996).

Thus, for the series of derivatives with modified sulfonamido moieties 4.228 to 4.230 (Table 4.21), it has been observed that the presence of bulky substituents at the sulfonamido moiety (such as phenylhydrazino, ureido, thioureido or guanidino) led to compounds with weak inhibitory properties, whereas moieties present in inorganic anion CAIs (such as NO, NCS or N3) or compact moieties substituting the sulfonamide nitrogen (such as OH, NH2, CN or halogeno) led to compounds with appreciable inhibitory properties. Thus, the N-hydroxy sulfonamide 4.228b, the N-chloro-substituted derivatives 4.228j, k and the nitrosoand thiocyanato derivatives 4.228d, e possessed the same affinity for the two investigated isozymes as did the unsubstituted sulfonamide 4.228a. Interestingly, the thiosulfonic acid (as sodium salt) 4.228q was one of the best inhibitors in this series, in contrast to the sulfonate (as sodium salt) 4.228p, which behaved as a very weak inhibitor. Sulfamide 4.229 (the most simple compound containing a sulfonamido moiety) behaves as a weak inhibitor, but it binds to the Zn(II) ion, as shown by electronic spectroscopic studies on the Co(II)-substituted enzyme, whereas sulfamic acid 4.230 is a much stronger inhibitor. Furthermore, this compound has a much higher affinity for CA I than for CA II, and this might be exploited for designing CA I-specific inhibitors based on this type of zinc-binding function. Recently, the binding of sulfamide and sulfamic acid to hCA II has been investigated in detail by x-ray crystallography (Abbate et al. 2002). The binegatively charged (NH)SO32– sulfamate ion and the monoanion of sulfamide NHSO2NH2– bound to the Zn(II) ion within the enzyme

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TABLE 4.21

Inhibition of hCA I and hCA II by Compounds Incorporating Modified Sulfonamide Moieties 4.228a–t, Sulfamide 4.229 and Sulfamic Acid 4.230

Source: From Briganti, F. et al. (1996) European Journal of Medicinal

Chemistry 31, 1001–1010. With permission.

active site (Abbate et al. 2002). These two structures provided some close insights into why the sulfonamide functional group appears to have unique properties for CA inhibition: (1) it exhibits a negatively charged, most likely monoprotonated nitrogen coordinated to the Zn(II) ion; (2) simultaneously this group forms a hydrogen bond as donor to the oxygen Oγ of the adjacent Thr 199; and (3) a hydrogen bond is formed between one of the SO2 oxygens to the backbone NH of Thr 199. Thus, the basic structural elements explaining the strong affinity of the sulfonamide moiety for the Zn(II) ion of CAs were demonstrated in detail by using these simple compounds as prototypical CAIs, without the need to analyze the interactions of the organic scaffold usually present in other inhibitors (generally belonging to the aromatic/heterocyclic sulfonamide class; Abbate et al. 2002). Despite important similarities for the binding of these two inhibitors to the enzyme with that of

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aromatic/heterocyclic sulfonamides of the type RSO2NH2 previously investigated, the absence of a C–SO2NH2 bond in sulfamide/sulfamic acid leads to a different hydrogen bond network in the neighborhood of the catalytical Zn(II) ion, which was shown to be useful for the drug design of more potent CA inhibitors possessing zinc-binding functions different from those of the classical sulfonamide group (Abbate et al. 2002). By using such compounds as leads, several series of much stronger inhibitors were subsequently reported, possessing modified sulfonamido moieties as zinc-binding functions of the type SO2NHOH, SO2NHCN, SO2NHPO3H2, SO2NHSO2NH2, SO2NHSO3H or SO2NHCH2CONHOH, among others (Mincione et al. 1998; Supuran et al. 1999a; Scozzafava et al. 2000b; Fenesan et al. 2000).

Thus, compounds such as 4.231 to 4.240 possessing N-cyano, N-hydroxy or N-phosphoryl-sulfonamido moieties, or the related modified sulfamide/sulfamic acid zinc-binding functions, and diverse alkyl, aryl or heterocyclic moieties in their molecules showed affinities in the low nanomolar range for hCA II (except 4.237), being equipotent or better inhibitors than the corresponding unsubstituted sulfonamides (Mincione et al. 1998; Supuran et al. 1999a; Scozzafava et al. 2000b; Fenesan

et al. 2000). Compound 4.237 is a weak inhibitor of hCA II (affinity constant of 1.2 μM), but has a much higher affinity (50 nM) for hCA I, thus being one of the

most selective hCA I inhibitors reported to date (Scozzafava et al. 2000b).

Ki = 50 nM (hCA I)

4.238: Ki = 20 nM (hCA II)

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F

4.239

(Ki hCA I= 8 nM; hCA II = 11 nM; bCA IV = 13 nM)

HO

NH

H O

O N S

O

OMe

4.240

(Ki hCA I= 50 nM; hCA II = 5 nM; bCA IV = 39 nM)

Sulfonylated amino acid hydroxamates were also shown to possess strong CA inhibitory properties (Scozzafava and Supuran 2000b). Such hydroxamates generally act as potent inhibitors of metalloproteases containing catalytic zinc ions, such as matrix metalloproteinases (MMPs) or bacterial collagenases (Scozzafava and Supuran 2002a). They bind to the Zn(II) ions present in these enzymes bidentately, coordinating through the hydroxamate (ionized) moiety (Supuran and Scozzafava 2002b). Scolnick et al. (1997) showed that two simple hydroxamates of the type RCONHOH (R = Me, CF3) act as micromolar inhibitors of hCA II and bind to the Zn(II) ion of this enzyme, as demonstrated by x-ray crystallography. By using these two derivatives as lead molecules, Scozzafava and Supuran (2000b) designed a series

of sulfonylated amino acid hydroxamate derivatives possessing the general formula RSO2NHCH(R′)CONHOH and showed by electron spectroscopic studies on the

Co(II)-substituted CA that they bind to the Zn(II) ion of CA. Some of these compounds, such as 4.239 and 4.240, showed affinity in the low nanomolar range for the major CA isozymes (CA I, II and IV), but substitution of the sulfonamide nitrogen by a benzyl or a substituted-benzyl moiety led to a drastic reduction of the CA inhibitory properties and to an enhancement of the MMP inhibitory properties. Thus, between the two types of zinc enzymes, the zinc proteases and the CAs, there exist some cross-reactivity as regards hydroxamate inhibitors, but generally strong MMP inhibitors are weak CAIs and vice versa.

All these data demonstrate that in addition to the classical CAIs of the aromatic/heterocyclic sulfonamide type, other compounds can be designed with very strong affinity for the active site of different isozymes, a fact that might be relevant for obtaining diverse pharmacological agents that modulate the activity of these widespread enzymes.

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4.8ANTIEPILEPTIC SULFONAMIDES AND OTHER MISCELLANEOUS INHIBITORS

Several sulfonamide CA inhibitors such as acetazolamide 4.44, methazolamide 4.48, topiramate 4.241 or zonisamide 4.242 were and are still used as antiepileptic drugs (Masereel et al. 2002). The anticonvulsant effects of these or related sulfonamides are probably due to CO2 retention following inhibition of the red cell and brain enzymes, but other mechanisms of action such as blockade of sodium channels and kainate/AMPA receptors as well as enhancement of GABA-ergic transmission were also hypothesized or proved for some of these drugs (Masereel et al. 2002). Acetazolamide and methazolamide are still clinically used at present in some forms of epilepsy, but are considered to belong to a minor class of antiepileptic agents, whereas the more recently developed drug topiramate 4.241, a very effective antiepileptic, was also shown to act as a strong CA inhibitor, with a potency similar to that of acetazolamide against the physiologically important isozyme CA II (Masereel et al. 2002). Furthermore, its x-ray crystal structure in complex with hCA II has recently been reported by Casini et al. (2003) and reveals a very tight association of the inhibitor with a network of seven strong hydrogen bonds fixing topiramate within the active site, in addition to Zn(II) coordination through the ionized sulfamate moiety. (See also Chapter 3 of this book.)

Recently, a series of aromatic/heterocyclic sulfonamides incorporating valproyl moieties was prepared to design antiepileptic compounds possessing in their structure two moieties known to induce such a pharmacological activity: valproic acid (4.243), one of the most widely used antiepileptic drugs, and the sulfonamide residue included in acetazolamide and topiramate, two CAIs with antiepileptic properties (Masereel et al. 2002). The valproyl derivative of acetazolamide (5-valproylamido- 1,3,4-thiadiazole-2-sulfonamide, 4.244) was one of the best hCA I and hCA II inhibitors in the series and exhibited very strong anticonvulsant properties in the MES test in mice (Masereel et al. 2002). Consequently, other 1,3,4-thiadiazole- sulfonamide derivatives possessing potent CA inhibitory properties (of types 4.245

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O

to 4.253) and substituted with different alkyl/arylcarboxamido/sulfonamido/ureido moieties in position 5 have been investigated for their anticonvulsant effects in the same animal model. Some structurally related derivatives such as 5-benzoylamido-, 5-toluenesulfonylamido-, 5-adamantylcarboxamido- and 5-pivaloylamido-1,3,4-thi- adiazole-2-sulfonamide also showed promising in vivo anticonvulsant properties, and these compounds can be considered interesting leads for developing anticonvulsant or selective cerebrovasodilator drugs (Masereel et al. 2002).

Other miscellaneous CAIs possessing interesting properties have also been reported. Thus, in an attempt to obtain gastric mucosa CA-specific CAIs, a group of sulfenamido sulfonamides of type 4.254 – 4.257 have been reported that possess powerful CA II and CA IV inhibitory properties (Supuran et al. 1997a; Scozzafava and Supuran 1998b). These compounds were designed in such a way as to liberate aromatic/heterocyclic sulfonamides and sulfenyl chlorides in the presence of gastric

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