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

analogues are, probably because of steric impairment of activator access into the enzymatic site. On the other hand, 2,6-dialkyl-4-phenyl derivatives were highly active, supporting the previous QSAR finding that the molecular elongation along the axis passing through the enzyme Zn(II) ion, the CAA molecule itself and the active-site exit is the most beneficial substitution pattern for activating properties.

(3) The complete derivatization of the ω-amino alkyl group eliminates the activating effect. Furthermore, these studies (Supuran et al. 1993b, 1996a) first provided the suggestion that selective CAAs could be obtained. Because of their cationic nature, pyridinium derivatives are membrane impermeant, so that theoretically they should discriminate between the membrane-bound CA IV and the very similar cytosolic CA II (Ilies et al. 2002).

12.3.3 CA ACTIVATION WITH AZOLE DERIVATIVES

Imidazole is widely used as a buffer, and the imidazole moiety is present in both histamine (the prototypical CAA) and histidine (an amino acid of crucial importance for the CA catalytic cycle, and also a CAA). Consequently, research in the field began by investigating CA activation by imidazoles. A major difference between isozymes I and II was thus unveiled: imidazole is a unique competitive inhibitor with CO2 as substrate for hCA I (Khalifah 1971), whereas it behaves as a very efficient activator for isozyme II (Parkes and Coleman 1989; Supuran 1992). Subsequent studies (Supuran et al. 1993a, 1996b) afforded CA II activation data for a large series of azole derivatives (12.7 to 12.11) as well as some interesting SARs.

A strong relationship between activatory efficiency of these compounds and the pKa value of their proton shuttle moiety was observed (Table 12.2). pKa values of 5.5 to 8.0 were optimal for the activator potency. The same empirical conclusion was found for compounds of types 12.1 and 12.4 (Supuran and Balaban 1994). The graphical representation of pKa of the activator vs. % CA activation showed a Gaussian-type variation, with a maximum in the previously mentioned pKa range (Figure 12.6). These facts can be correlated either with a pKa value of 7.1, which characterizes His 64 — the native CA proton shuttle, or with a pKa value of 6.8, which is that of the zinc-bound water molecule in the CA catalytically inactive form.

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N

R′′

N

R

(12.11a,b)

Taking into account that (1) amino-pyridinium derivatives (Supuran et al. 1993b, 1996a) as well as azole compounds (Supuran et al. 1993a, 1996b) are efficient activators and (2) the salt-like character induced by the pyridinium group has been exploited successfully in generating CA IV-specific inhibitors (Supuran et al. 1998; Scozzafava et al. 2000a), Ilies and coworkers (1997, 2002) extended the investigated CAAs series to pyridinium azoles of types 12.12 to 12.15. This research has led to

(1) the first investigation of the activation of the bovine isozyme IV (bCA IV; Ilies et al. 1997); (2) a study of comparative activation for several CA isozymes (hCA I, hCA II, and bCA IV; Ilies et al. 1997); and (3) the first membrane-impermeant CAAs (Ilies et al. 2002; Ilies 2002).

Two structural variations in the choice of the reactants were followed to obtain insights into the SARs for this class of activators: (1) the number of azole nitrogen atoms (two, three or four) and their position (pyrazole/imidazole), and (2) the

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TABLE 12.2

CA II Activation Data with Compounds 12.7 to 12.11 at 10–5 M for the Hydrolysis of 4-Nitrophenyl Acetate and the Corresponding pKa Values of the Activator

aBis-azoles pKa values are for the second deprotonation step. (From Catalan et al. (1987) Advances in Heterocyclic Chemistry 41, 187.)

bCA II activity in the absence of activator is taken as 100%.

pyridinium ring substitution pattern (alkyl/aryl moieties as well as different combinations of them), because this was one of the main parameters that strongly influenced the efficacy of some previously reported pyridinium derivatives, both as CAAs (Supuran et al. 1993a, 1996b) and CAIs (Supuran et al. 1998; Scozzafava et al. 2000a). The following findings emerged (Ilies et al. 1997, 2002; Ilies 2002):

1.Presuming an identical pyridinium ring substitution pattern and the same investigated isozyme, the nature of the azole ring determined the following order of the CA activation effect: imidazole > triazole > pyrazole » tetrazole. In keeping with earlier assumptions (Supuran et al. 1996b;

Supuran and Balaban 1994), the pKa values of the azolyl moieties in aqueous system might provide an explanation. Thus, it seems that Mother Nature always makes the best choice by giving to the His 64 imidazolyl residue the proton shuttle role in the native CA, and the imidazole ring system has been proved to be the best option for designing CAAs of the

azole type. Proceeding to triazole and pyrazole, the pKa becomes lower, reaching a quite acidic range for tetrazole, which has an inappropriate

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FIGURE 12.6 Percent CA activation vs. pKa value for amino acid and amine derivatives of type 12.1 and 12.4 (Curve 1) and for azole derivatives of type 12.7 to 12.11 (Curve 2; enzyme concentration of 10 µM). [Adapted from Supuran, C.T., and Balaban, A.T. (1994) Revue Roumaine de Chimie 39, 107–113. With permission.]

value for proton acceptance. Triazole and tetrazole were found to behave as CAIs (Alberti et al. 1981). It can be concluded that polarization of the molecule induced by the electron-attracting pyridinium group is practically responsible for the activating properties in triand tetrazole derivatives. A strong argument for this claim is the zwitterionic form in which pyridinium tetrazoles of type 12.15 have been isolated. By acquiring for the NH proton a pKa value similar to that for compounds 12.13 (pKa 6 to 8), most of the triazole derivatives (12.14) were efficient CAAs, although they were only micromolar activators, whereas the major part of their analogues (12.13) showed activatory effects in the nanomolar range.

2.The influence of the pyridinium moiety substituents on the biological activity has confirmed the corresponding SAR established previously for amino-pyridinium CAAs of types 12.4 and 12.6 (Supuran et al. 1993b, 1996a). Interesting extensions of such correlations have been made:

a.Among the most active 2,6-dialkyl-4-phenyl-pyridinium-azoles, those substituted with the isopropyl radical have arguably been the best in

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TABLE 12.3

In Vitro Activation Pattern of hCA I, hCA II and bCA IV with Histamine and Compounds 12.12 to 12.15

vitro activators in all the four new series (12.12 to 12.15) and against all three assayed isozymes.

b.Newly introduced in this study (Ilies et al. 2002), the substitution of the 2,6-dialkyl-pyridinium ring with a high lipophilic and flexible looped side chain the of 3,5-nonamethylen type has led to a remarkable enhancement in CA activity, such compounds being only slightly less effective than the 2,6-dialkyl-4-phenyl substituted azoles. These results show that the activator efficacy can be successfully fine-tuned by modulating its structural hydrophilic–hydrophobic balance through an appropriate substitution pattern.

3.Significant differences in isozyme specificity with both classical CAAs, such as histamine, and the newly synthesized compounds have been revealed, the in vitro activation pattern Shown in Table 12.3. The particular histidine cluster in the hCA II active site (Briganti et al. 1997b) explains well its constant lowest sensitivity to activation. The triazoles derivatives 12.14 maintain the general histamine activation pattern (probably a con-

sequence of their similar pKas for the NH proton), except for the mem- brane-bound bCA IV, which is much more susceptible to the new type of activators (presumably because of their cationic nature).

The best in vitro activators (i.e., 2,6-di-i-propyl-4-phenyl-pyridinium azoles) strongly enhance ex vivo red cell lysate CA activity, being about twice more efficient than the standard drug histamine. The same type of experiments performed with unlyzed erythrocytes clearly demonstrate that pyridinium pyrazole/imidazole derivatives have no influence on the cytosolic hCA I and hCA II, whereas the zwitterionic nature of the corresponding pyridinium tetrazoles permits them to cross the plasma membrane and thus activate the CA isozymes with an internal cellular localization, similar to that by histamine.

12.3.4 CA ACTIVATION WITH HISTAMINE DERIVATIVES

Histamine belongs to the amino-azole class of CAAs. Therefore, the CAAs derived from this lead molecule can be considered an independent and particular category of such compounds.

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recent structural aspects of the hCA II–histamine interaction revealed by x-ray crystallography: (1) the unique histidine cluster of the hCA II active site surrounds its opening and consists of six residues, some of them possessing flexible conformations, which can easily participate in hydrogen bonds with activators or inhibitors bound within the active site (Briganti et al. 1997b, 1998); (2) the histamine molecule binds at the hCA II active-site entrance, being anchored to amino acid side chains and to water molecules through hydrogen bonds involving only the nitrogen atoms of the imidazole moiety, whereas its aliphatic amino group extends into the solvent, making no contact with the enzyme (Briganti et al. 1997b). Thus, it was of interest to derivatize the amino aliphatic end of the histamine molecule with highly polarized moieties. Such groups might interfere in an energetically favorable way with the polar amino acid residues at the rim of the active site. By decreasing the activators’ whole energy of binding, these additional contacts will lead to more stable enzyme–activator adducts and hence to a more efficient activation process. This approach, based on improvement in the energy of binding of certain compounds to the enzyme through chemically modified units of a given parent structure, was also reported both by the groups of Whitesides (Gao et al. 1995) and Supuran (Scozzafava et al. 1999) for the design of tight-binding sulfonamide CAIs.

To obtain a much better affinity than histamine for the protein, the following concepts have been considered while choosing the polar derivatizing groups: (1) as shown by earlier OSAR calculations (Clare and Supuran 1994), the oxygen and nitrogen atoms have a stimulating effect on the activating properties; (2) a peptidetype side chain, fulfilling the previous condition, will additionally confer to the compound a specific embedded structure through which it can better interact with amino acid residues at the active site (Supuran and Scozzafava 1999); (3) according to the activation mechanism described previously, a free amino or imidazole moiety acting as a second proton transferring group (besides the imidazole ring from the lead histamine) might positively influence efficacy of the CAAs (Supuran and Scozzafava 1999, 2000c).

The synthetic strategy involved treating histamine with tetrabromophthalic anhydride and protecting its imidazole residue with tritylsulfenyl chloride, followed by hydrazinolysis of the phthalimido moiety under mild conditions. The obtained N-1- tritylsulfenyl histamine, the key intermediate, was further derivatized at its terminal amino ethyl function by a variety of procedures to introduce in the molecule the desired polar group, as summarized in Table 12.4. The final removal of the tritylsulfenyl moiety in an acidic medium afforded the target compounds 12.16 to 12.22 (Supuran and Scozzafava 1999, 2000c; Briganti et al. 1999; Scozzafava and Supuran 2000a, 2000b; Scozzafava et al. 2000b).

Thus, a large series of alkyl/arylsulfonamido derivatives of type 12.16 was obtained by reacting the key intermediate with the corresponding sulfonyl halides, whereas an alternative route involving its treatment with arylsulfonylisocyanates gave some arylsulfonylureido compounds with the general formula 12.17 (Briganti et al. 1999). Alkyl/arylcarboxamido structures of type 12.18 were generated by reacting the same key intermediate with acyl chlorides or carboxylic acid anhydrides

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TABLE 12.4

General Derivatizing Procedures for the Key Intermediate N-1-Tritylsulfenyl Histamine

Note: R = alkyl; substituted-alkyl; aryl; substituted-aryl; hetaryl; Ar = substitutedphenyl; Boc = t-butoxy-carbonyl; X = 2-Me, 4-Me, 4-F, 4-Cl; aa, aa1, aa2 = amino acid/dipeptide residues.

R = alkyl; substituted-alkyl; aryl; substituted-aryl; hetaryl;

Ar = substituted-phenyl;

X = O, NH, S.

in the presence of triethylamine, or carboxylic acids in the presence of diisopropylcarbodiimide (DIPC; Scozzafava and Supuran 2000a). The use of aryl/alkyl isocyanates/isothiocyanates, cyanamide or dicyandiamide as derivatizing reagents led to

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R, R1, R2 = amino acid/dipeptide residues;

X = 2-Me; 4-Me; 4-F; 4-Cl.

aryl/alkylureido/thioureido or guanidino compounds of type 12.19 (Scozzafava and Supuran 2000a). Novel CAAs with a 12.20 scaffold were prepared by treating the previously mentioned N-1-tritylsulfenyl histamine with Boc-protected amino acids/dipeptides in the presence of DIPC (Supuran and Scozzafava 1999). The previous strategies were combined and extended by coupling the key intermediate with arylsulfonylureido amino acids/dipeptides resulting from the reaction of the corresponding amino acids/dipeptides with arylsulfonyl isocyanates. A large series of arylsulfonylureido amino acyl/dipeptidyl histamine derivatives of types 12.21 and 12.22 were thereby obtained (Scozzafava and Supuran 2000b; Supuran and Scozzafava 2000c; Scozzafava et al. 2000b). This last synthetic approach yielded some very efficient activators, such as compounds 12.23, 12.25 (Supuran and Scozzafava 1999) or 12.24 and 12.26 (Scozzafava and Supuran 2000b; Supuran and Scozzafava 2000c; Scozzafava et al. 2000b). This was explained by the authors as being because such molecules, in addition to the histamine imidazole, possess multiple moieties able to shuttle protons (i.e., NH from imidazole ring or free amino groups).

Different histamine-derived CAAs, i.e., compounds 12.27 and 12.28, were obtained through another synthetic method involving direct acylation of the lead molecule with thylenediaminotetraacetic and diethylenetriaminopentaacetic acid dianhydride, respectively, in a molar ratio of 2:1 (Scozzafava and Supuran 2000a).

Enzymatic assays of the novel activators 12.21 to 12.28 against hCA I, hCA II and bCA IV led to some new SARs (Supuran and Scozzafava 1999, 2000c; Briganti et al. 1999; Scozzafava and Supuran 2000a, 2000b; Scozzafava et al. 2000b):

1.The most interesting finding in these series was the constant high affinity of hCA II (in the low nanomolar range) for many of the new derivatives, i.e., ca. 1000 times more affinity than the lead histamine. This might be explained by the stronger interactions with the hCA II histidine cluster assured by the specific structure of these compounds. Isozymes I and IV were also very susceptible to the new CAAs, in the nanomolar range for

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