Catalytic Selectivity of CYP2C9

Substrates for CYP2C9 include many non-steroidal anti-inflammatory drugs plus a reasonably diverse set of compounds including phenytoin, (S)-warfarin and tolbutamide. All the substrates with routes of metabolism attributable to CYP2C9 have hydrogen bond donating groups a discrete distance from a lipophilic region which is the site of hydroxylation. The hydrogen bond donating groups and sites of metabolism on each of the substrates have been overlaid with those of phenytoin to produce a putative template of the active site of CYP2C9 (Figure 7.7).

Fig. 7.7 Template model of CYP2C9; Y is the site of oxidation, a is the distance from Y to a heteroatom which can act as a H-bond donor and c defines the angle of the H-bond.

The mean dimensions (± SD) for the eight compounds (a = 6.7 ± 0.8 A, C = 133 ± 20°) illustrates the degree of overlap achieved. Like CYP2D6 the catalytic selectivity of CYP2C9 is dominated by substrate-protein interactions.

Tolbutamide (Figure 7.8) is metabolized via the benzylic methyl group by CYP2C9 as the major clearance mechanism. Chlorpropamide is a related compound incorporating a chlorine function in this position. The resultant metabolic stability gives chlorpropamide a lower clearance and a longer half-life (approximately 35 h compared to 5 h) than tolbutamide, resulting in a substantial increase in duration of action [5].

Fig. 7.8 Structures of tolbutamide and the metabolically more stable analogue chlorpropamide.

The mechanism of action of CYPs is radical rather than electrophilic and the actual substitution pattern is important: the role of chlorine is one of blocking rather than deactivation. Many non-steroidal anti-inflammatory drugs are substrates for the

Fig. 7.9 Structures of diclofenac and fenclofenac. Fenclofenac is much more resistant to aromatic hydroxyla-tion.

Fig. 7.9 Structures of diclofenac and fenclofenac. Fenclofenac is much more resistant to aromatic hydroxyla-tion.

fenclofenac

diclofenac fenclofenac

CYP2C9 enzyme and analogous structures show how metabolic stability to p-hydrox-ylation is achieved with only small changes in substitution.

Diclofenac, with ortho substitution in the aromatic ring (Figure 7.9) is metabolized principally to 4-hydroxydiclofenac by CYP2C9. In man, the drug has a short half-life of approximately 1 h due to the relatively high metabolic (oxidative) clearance. In contrast, the analogous compound, fenclofenac, is considerably more meta-bolically stable, due to the p-halogen substitution pattern, and exhibits a half-life of over 20 h [6].

Catalytic Selectivity of CYP3A4

CYP3A4 attacks lipophilic drugs in positions largely determined by their chemical lability: that is, the ease of hydrogen or electron abstraction. CYP3A4 SAR is dominated therefore by substrate-reactant interaction. Binding of substrates seems to be essentially due to lipophilic forces and results in the expulsion of water from the active site. Such an expulsion of water provides the driving force for the spin state change and hence the formation of the (FeO)3+ unit. However, the lipophilic forces holding the substrate in the active site are relatively weak (~ 1 kcal mole-1) and would allow motion of the substrate in the active site. Hence, since the substrate is able to adopt more than one orientation in the active site, the eventual product of the reaction is a product of the interaction between one of these orientations and the (FeO)3+ unit - a substrate-reactant interaction. This lack of apparent substrate structure similarity (apart from chemical reactivity) indicates a large active site that allows substrate molecules considerable mobility. The selectivity of CYP3A4 to its substrates may also be directed by the conformation they adopt within a lipophilic environment such as we are suggesting for the access channel and active site of CYP3A4. We have previously illustrated this point with cyclosporin A. In an aprotic (lipophilic) solvent cyclosporin A adopts a conformation which allows the major allylic site of CYP3A4 metabolism to extend out away from the bulk of the molecule. This is a different conformation from the one adopted in aqueous solution where the lipophilic sites are internalized and thus shielded from the solvent. As a general rule this "spreading out" of apparently sterically hindered molecules as judged by X-ray or aqueous solution structure, may help to further understand the selectivity of CYP3A4. The principle of extension of lipophilic functions normally hidden from solvent is further supported by the

Thc Cyp3a4

Fig. 7.10 Substrates for CYP3A4 illustrating quinidine; C, L-696229; D, indinavir; E, the diversity of structure and the "selectivity" lovastatin; F, D-THC; G, zatosetron H, for attack at allylic and benzylic positions pioglitazone; I, progesterone; J, testosterone;

(major sites of metabolism indicated by K, budesonide and L, salmeterol. asterisks). Substrates are A, cyclosporin A; B,

Fig. 7.10 Substrates for CYP3A4 illustrating quinidine; C, L-696229; D, indinavir; E, the diversity of structure and the "selectivity" lovastatin; F, D-THC; G, zatosetron H, for attack at allylic and benzylic positions pioglitazone; I, progesterone; J, testosterone;

(major sites of metabolism indicated by K, budesonide and L, salmeterol. asterisks). Substrates are A, cyclosporin A; B, study of the soluble bacterial P450BM-3. In this case the substrates are fatty acids, which in aqueous solution adopt a "globular" conformation. However, upon entering the lipophilic access channel, the fatty acid opens out in an extended conformation with the lipophilic head group directed at the haem and the polar acid function directed at the solvent.

The enzyme is the principal participant in N-demethylation reactions where the substrate is a tertiary amine. The list of substrates includes erythromycin, ethylmor-phine, lidocaine, diltiazem, tamoxifen, toremifene, verapamil, cocaine, amiodarone, alfentanil and terfenadine. Carbon atoms in the allylic and benzylic positions, such as those present in quinidine, steroids and cyclosporin A, are also particularly prone to oxidation by CYP3A4, a range of substrates is illustrated in Figure 7.10.

Both these routes of metabolism reflect the ease of hydrogen or electron abstraction from these functions. As with conventional radical chemistry, reactivity needs to be combined with probability. Thus, in molecules such as terfenadine the tertiary butyl group will be liable to oxidation due to its "maximum number" of equivalent primary carbons. Thus, although not a specially labile function, the site of metabolism becomes dominated by statistical probability. Terfenadine, as expected, also undergoes N-dealkylation by CYP3A4, illustrating the ability of the enzyme to produce multiple products (as for cyclosporin A, midazolam, etc.) and underlining the "flexibility" of CYP3A4 substrate binding.

Overcoming metabolism by CYP3A4 is difficult due to the extreme range of substrates and the tolerance of the enzyme to variations in structure. Two strategies are available: removal of functionality and reduction of lipophilicity.

Allylic and benzylic positions are points of metabolic vulnerability. SCH48461 is a potent cholesterol absorption inhibitor [7]. Metabolic attack occurred at a number of positions including benzylic hydroxylation. Dugar and co-workers substituted oxygen for the C-3' carbon to remove this site of metabolism. This step however, produces an electron-rich phenoxy moiety in comparison to the original phenyl group and possibly makes this function more amenable to aromatic hydroxylation. Blocking of the aromatic oxidation with fluorine introduced in the para-position was required to produce the eventual more stable substitution. These steps are shown in Figure 7.11.

Cholesterol Position
Fig. 7.11 Synthetic strategies to overcome benzylic hydroxylation in a series of cholesterol absorption inhibitors. Positions of metabolism are marked with an asterisk.

The lability of benzylic positions to cytochrome P450 metabolism has been exploited to decrease the unacceptably low clearance and resultant long half-life of various compounds. For example celecoxib, a selective cyclooxygenase inhibitor, has a half-life of 3.5 h in the rat. Early structural leads, represented by compounds in

Fig. 7.12 Structures of early long half-life COX2 inhibitor (A) and the candidate compound celecoxib (B) with a moderate half-life.

which the benzylic methyl in celecoxib was substituted with a halogen (Figure 7.12), resulted in compounds with half-life values (in the male rat) of up to 220 h [8].

Diltiazem (Figure 7.13), a calcium channel blocker, is a drug that is extensively metabolized by at least five distinct pathways including N-demethylation, deacetyla-tion, O-demethylation, ring hydroxylation and acid formation. The enzyme responsible for at least the major route (N-demethylation), has been shown to be CYP3A4 [8]. Although widely used in therapy, the compound has a relatively short duration of action. In the search for superior compounds, Floyd et al. [9] substituted the ben-zazepinone ring structure for the benzothiazepinone of diltiazem. Metabolic studies on this class of compound showed that the principal routes of metabolism were similar to that for diltiazem with N-demethylation, conversion to an aldehyde (precursor of an acid), deacetylation and O-demethylation all occurring. It was also noted that the N-desmethyl derivative was equipotent to the parent but much more stable meta-bolically. This can be rationalized as the decreased substitution on the nitrogen (secondary versus tertiary) stabilizing the nitrogen to electron abstraction (decreased radical stability). This stabilization is particularly important, since electron abstraction is the first step to both the N-desmethyl and aldehyde products (a total of 84 % of the total metabolism). The evidence of stability of secondary amines was capitalized on by synthesis of N-1 pyrrolidinyl derivatives, which were designed to achieve metabolic stability both by the decreased radical stability of secondary compounds to tertiary amines and steric hindrance afforded by p-substitution (Figure 7.11). The success of this strategy indicates how even the vulnerable alkyl substituted nitrogen grouping can be stabilized against attack.

Fig. 7.13 Structures of diltiazem and a benzozapepinone analogue resistant to metabolism.

The predominant interaction of CYP3A4 is via hydrophobic forces and the overall lowering of lipophilicity can reduce metabolic lability to the enzyme. Figure 7.14 shows the relationship between unbound intrinsic clearance in man and lipophilici-ty for a variety of CYP3A4 substrates. The substrates are cleared by a variety of metabolic routes including N-dealkylation, aromatization and aromatic and aliphatic hy-droxylation. The trend for lower metabolic lability with lower lipophilicity is maintained regardless of structure or metabolic route.

7.3 Oxidative Metabolism and Drug Design 85

Fig. 7.14 Unbound intrinsic clearance of CYP3A4 substrates and relationship with lipophilic-ity. The data has been calculated from various clinical studies with the drugs listed in order of decreasing lipophilicity.

7.3 Oxidative Metabolism and Drug Design 85

Fig. 7.14 Unbound intrinsic clearance of CYP3A4 substrates and relationship with lipophilic-ity. The data has been calculated from various clinical studies with the drugs listed in order of decreasing lipophilicity.

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