5.6 ENZYME INHIBITOR EXAMPLES FOR THE TREATMENT OF BREAST CANCER
5.6.4.1 Nonsteroidal Aromatase Inhibitors (NSAIs)
The design and development of aromatase inhibitors for the treatment of hormone- dependent cancers began with the discovery of the aromatase inhibitory activity of aminoglutethimide (AG). AG is a simple chemical derivative of the sedative glute- thimide. It was originally introduced as an anticonvulsant, but was found to cause adrenal insufficiency. This unwanted property was due to the ability of AG to block adrenal steroidogenesis by inhibiting the enzymatic conversion of cholesterol to pregnenolone. More interestingly, this agent also blocked the peripheral conversion (aromatization) of androgenic precursors to estrogens, which is the source of estrogen in postmenopausal women. AG binds to aromatase, producing a Type II optical difference spectrum with an absorption peak occurring between 425 and 435 nm and a trough at 390 nm showing competitive inhibition. The commercial product is a mixture of D- and L-isomers, and they have different pharmacological potencies. The D-isomer is 30 times more potent at inhibiting aromatase activity, whereas the L- isomer is more potent at inhibiting cholesterol side-chain cleavage (steroidogenesis).
The discovery of the aromatase inhibitory property of AG in the 1970s led to massive research programs in the following decades that aimed at the design and FIGURE 5.8 The origin of estrogenic steroids in postmenopausal women: CYP 19, aro- matase; ST, sulfotransferase; STS, sulfatase; 17b-HSD, 17b-hydroxysteroid dehydrogenase;
3b-HSD-isomerase, 3b-hydroxysteroid dehydrogenase D5,D4-isomerase; ER, estrogen recep- tor. (Adapted from Woo, L.W.L., Purohit, A., Malini, B. et al. (2000). Potent active site- directed inhibition of steroid sulfatase by tricyclic coumarin-based sulfamates. Chemistry &
Biology, 7, 773–791.)
development of more potent and selective NSAIs. The initial design strategies adopted by several research groups focused on modifying AG structurally, and the modifications included: (1) replacing the p-aminophenyl group with a pyridyl ring, (2) contracting the piperidinedione to a five-membered pyrrolidinedione ring, (3) bridging C3 and C5 of the AG to give a bicyclo derivative, and (4) replacing the C3 ethyl group or substituting at the N-atom with longer alkyl chains or both. Most of these modifications produced inhibitors that were more potent against and selective for aromatase.
With aromatase being a cytochrome P450 enzyme, it had been reasoned that the reversible inhibition observed for AG could involve interaction of its nitrogen atom with the heme iron of the enzyme, in accordance with its Type II spectrum as a competitive inhibitor. This reasoning prompted investigation into compounds that contain aza-heterocycles and led to the discovery that a group of imidazole antifungal agents (miconazole, clotrimazole, and ketoconazole) were better aromatase inhibi- tors than AG. The research efforts that followed produced a series of second- generation NSAIs with S-fadrozole (5.121), liarozole (5.122), and CGS18320B (5.123) showing the most promising biological activities. The replacement of the imidazole ring with a triazole also produced compounds that were highly effective aromatase inhibitors. Substituted triazoles such as vorozole (5.124), letrozole (5.125), and anastrozole (5.126) were prime examples of third-generation NSAIs.
However, not every potent NSAI discovered has successfully reached the clinical development stage. For the second generation of inhibitors, only fadrozole is used clinically, primarily in Japan where a license has been granted for the treatment of postmenopausal women with advanced, hormone-responsive mammary carcinoma.
Fadrozole has been shown to be less effective than the triazole derivatives, and may cause a degree of inhibition of cortisol and aldosterone production at higher doses than normally used clinically.
Being chiral, vorozole (Rivizor®) was developed as its (+)-Risomer. This agent was found to be more potent than anastrozole and letrozole in vitro,but this apparent superiority was not evident in vivo. Vorozole was withdrawn from clinical develop-
ment in the U.S. Phase III trials several years ago, and it is uncertain if this agent will be developed further.
The most successful and highly developed third generation of NSAIs are letrozole and anastrozole, which are highly potent and selective against the aro- matase enzyme. Letrozole (Femara®) and anastrozole (Arimidex®) are currently available in the U.K. for the treatment of advanced breast cancer in postmenopausal women, and both agents are the gold standard NSAIs in the clinics. Recent Phase III trials of letrozole or anastrozole vs. tamoxifen as the first-line therapy for advanced breast cancer in postmenopausal women have shown significant advan- tages of either NSAI over tamoxifen in prolonging the survival of treated patients.
The results of a large head-to-head, multicenter, and randomized trial of letrozole vs. anastrozole as the second-line treatment in postmenopausal women with advanced breast cancer were presented at the 2002 annual meeting of the American Society of Clinical Oncology, which showed an overall response rate of 19.1% for women taking letrozole at a dose of 2.5 mg/d compared with 12.3% for those taking anastrozole at a dose of 1 mg/d. Clinical benefit was seen in 27% of the women treated with letrozole compared with 23% with anastrazole. This prelimi- nary data of a single trial suggests that postmenopausal women with advanced HDBC might respond better to letrozole.
Despite the success in the clinical development of several third-generation NSAIs, new compounds of considerable structural diversity are still being reported in scientific journals as novel inhibitors of aromatase. Like their predecessors, these fourth-generation NSAIs are Type II competitive inhibitors and possess the pharma- cophore for aromatase inhibition, which is a heme-ligating nitrogen heterocycle with substituents that explore favorable interactions with the amino acid residues lining the enzyme active site. It is beyond the scope of this chapter to discuss in detail all the new NSAIs reported to date. Those compounds of particular significance and interest (5.127to5.143) are depicted here.
Phytoestrogens of natural origin such as 7-hydroxyflavone (5.144) and a-naph- thoflavone (5.145) are moderate to good inhibitors of aromatase. Because these agents do not contain a nitrogen heterocycle, their inhibitory activities have been attributed to the heme-ligating potential of their carbonyl moiety.
The most advanced and evaluated fourth-generation NSAIs were compounds 5.142and5.143, developed by Yamanouchi Pharmaceuticals in Japan. Both com- pounds are tertiary amines possessing three different substituents (i.e., a p-cyanophe- nyl group, a halogenated benzyl group, and a heterocyclic group) on a central N- atom, thus avoiding the presence of a tetrahedral center and chirality complications for drug registration purposes. The designing out of chirality is of particular phar- maceutical significance because it has been demonstrated that enantiomers of NSAIs might exhibit different biological activities — both in terms of potency and selec- tivity — against the aromatase enzyme (e.g., AG). Compounds (5.142) and (5.143) were among the most active NSAIs reported to date with respective IC50values of 0.12 nMand 0.04 nM. Both agents were shown to display a high selectivity against the aromatase enzyme and to inhibit insignificantly other enzymes that are respon- sible for steroidogenesis. However, despite their promising biological profiles, Yamanouchi Pharmaceuticals has recently discontinued the clinical development of this class of compounds, probably as a result of competition and the market domi- nance of letrozole and anastrozole.