5.6 ENZYME INHIBITOR EXAMPLES FOR THE TREATMENT OF BREAST CANCER
5.6.5.1 The Enzyme and Breast Cancer
The enzyme steroid sulfatase (E.C. 3.1.6.2, STS) catalyzes the hydrolysis of sulfate esters of 3-hydroxy steroids, which are the biologically inactive forms, to their active 3-hydroxy steroids (Figure 5.8). Major substrates for STS include estrone sulfate (E1S), dehydroepiandrosterone (DHEA) sulfate, pregnenolone sulfate, and choles- terol sulfate, indicating that the A-ring of the steroid can be aromatic or alicyclic.
STS is a 65-kDa membrane-bound protein predominantly associated with the endo-
plasmatic reticulum and present in almost all mammalian tissues. It is one of the eleven mammalian sulfatases that have been identified, and its amino acid sequence has been resolved. In the past decade, STS has been the subject of intensive research due to its potential involvement in the pathogenesis of a number of diseases such as breast cancer, androgen-dependent skin disorders, cognitive dysfunction, and immune function. Among these potential indications, the inhibition of STS as endo- crine therapy for HDBC has been the most investigated.
Much of the estrone (E1) synthesized from androstenedione via the aromatase pathway can be converted to estrone sulfate (E1S) by estrone sulfotransferase.
Plasma and tissue concentrations of E1S are considerably higher than those of unconjugated estrogens. The half-life of E1S (10 to 12 h) is significantly longer than that (20 to 30 min) of unconjugated E1 and estradiol (E2). Hence it has been postulated that the high tissue and circulating levels of E1S could act as a reservoir for the formation of biologically active estrogens via the action of STS. Furthermore, the STS activity in breast tumors has been shown to be much higher than that of the aromatase enzyme. There is evidence to show that as much as a 10-fold greater amount of E1 may originate from the sulfatase pathway than via the aromatase route.
More recently, studies at the molecular level have revealed that in 87% of breast cancer patients investigated, STS mRNA levels were higher in malignant than in nonmalignant tissues.
In addition, the production of androstenediol (Adiol), and to a lesser extent E2, via DHEA (Figure 5.8) could also significantly contribute to the estrogenic stimu- lation of hormone-dependent breast tumors. Adiol, an androgen that binds to estrogen receptors and has estrogenic properties, originates from DHEA sulfate once it has been hydrolyzed to DHEA (Figure 5.8) by DHEA-sulfatase. Therefore, STS inhib- itors, when used alone or in conjuction with an aromatase inhibitor, may enhance the response of hormone-dependent breast tumors to this type of endocrine therapy by reducing not only the formation of E1 from E1S but also the synthesis of other estrogenic steroids such as Adiol from DHEA-S.
5.6.6 INHIBITION OF STEROID SULFATASE AS ENDOCRINE THERAPY 5.6.6.1 Steroidal STS Inhibitors
An initial attempt to design STS inhibitors had resulted in the synthesis of several steroidal 3-O-sulfates, of which Adiol 3-sulfate (5.155,Ki= 2 mM) was shown to be the most potent, although this inhibitor would be of little value clinically because it was a substrate for, and hence hydrolyzed by, STS with the release of the estrogenic Adiol. Continual search for STS inhibitors led to the discovery of danazol (5.156), a drug used for treating endometriosis, as a weak STS inhibitor. However, the main strategy employed by various groups for generating a reasonable lead STS inhibitor was to replace the sulfate group (OSO3-) of E1S with surrogates or mimics such as methylthiophosphonates [–OP(=S)(OH)Me], phosphonates [–OP(=O)(OH)R], sul- fonates (–OSO2R), sulfonyl halides (–SO2Cl and –SO2F), sulfonamide (–SO2NH2), and methylsulfonyl (–SO2CH3). These E1 derivatives were designed to compete with E1S for the enzyme active site without acting as substrates for the enzyme. Despite
the efforts involved, these agents were found to be only weak inhibitors of STS.
However, the breakthrough came when the sulfate group of E1S was replaced by a sulfamate moiety (OSO2NH2). The resulting analog, estrone 3-O-sulfamate (EMATE, IC50 = 18 nM, human placental microsomes), was found to inhibit STS not only potently but also in a time- and concentration-dependent manner, indicating that EMATE is an irreversible active site-directed inhibitor. Unexpectedly, despite its high potency, EMATE is not suitable for use in the treatment of HDBC because it was shown subsequently to be more estrogenic than ethinylestradiol when admin- istered orally in rats. The reason for the estrogenicity of EMATE is most likely due to release of E1 as a consequence of its inactivation of STS. In order to overcome this undesirable property of EMATE, nearly all steroidal inhibitors that followed were designed to be less estrogenic than EMATE but with similar or superior inhibitory activities to that of EMATE.
The initial strategy was to introduce substituents such as allyl, n-propyl, nitro, cyano, and halogens (F, Cl, Br, I) to the A-ring of EMATE at the 2- and/or 4- positions. Analogs of EMATE with electron-withdrawing substituents on the A-ring showed higher potency than EMATE in vitro. In comparison, those analogs with bulkier aliphatic substituents were found to be weaker STS inhibitors. The most successful A-ring modified analog of EMATE was 2-methoxyestrone 3-O-sulfamate (2-MeOEMATE, 5.157). This inhibitor was as potent as EMATE in inhibiting the activity of STS in vitro but, in contrast, was completely devoid of estrogenicity.
More significantly, 2-methoxyEMATE was found to induce apoptosis (programmed cell death) in a few cancer cell lines and inhibit tumor angiogenesis. 2-MethoxyE- MATE is therefore potentially useful for treating both HDBC and some hormone- independent cancers.
The D-ring of EMATE has been targeted for reducing the estrogenicity of the inhibitor. Early work had seen the reduction of the 17-carbonyl of EMATE to the methylene derivative NOMATE (5.158), which was as potent as EMATE but less
estrogenic. Enlargement of the D-ring from a cyclopentanone to a lactone gave estralactone 3-O-sulfamate (5.159), which almost completely inhibited STS in MCF- 7 (human breast cancer) cells at 10 mM.
Introduction of hydrophobic substituents into the D-ring of EMATE increased its potency and significantly reduced its estrogenicity. 17b-(N-Alkylcarbamoyl) estradiol-3-O-sulfamates and 17b-(N-alkanoyl) estradiol-3-O-sulfamates are highly potent STS inhibitors with optimal inhibitory activities shown by their n-heptyl derivatives 5.160 and 5.161, respectively (both IC50 values = 0.4 nM in MDA-MB- 231 cells). Using the estrogen-sensitive MCF-7 cell line that proliferates upon stimulation by estrogens, no significant estrogenic potential was observed in either inhibitor at a concentration of 1 mM, a dose that is about 2000 times higher than their IC50 values against STS. It is evident that the hydrophobic side-chains of these inhibitors have the dual effect of increasing binding to the enzyme active site around the D-ring of EMATE and abolishing estrogenicity of the parent phenolic steroid.
A stereoselective addition of Grignard reagents to the C17-carbonyl group of E1 gave a series of 17a-derivatives of E2 of which the 4¢-t-butylbenzyl 5.162and the 3¢-bromobenzyl5.163derivatives were among the most potent inhibitors of this series, showing IC50values (JEG-3 cells) of 28 nMand 24 nM, respectively. Because of the absence of a sulfamate group, both 5.162and5.163are reversible inhibitors, but they are only seven times less potent than EMATE. In common with 5.160and 5.161, the reversible inhibitors 5.162and 5.163 exploit the hydrophobic binding areas in the vicinity of the D-ring of EMATE. Sulfamoylation of 5.162gave the corresponding sulfamate 5.164, which was found to be even more potent as an STS inhibitor than EMATE (IC50 = 0.15 nMvs. 2.1 nMfor EMATE, recombinant STS in cell extract with E1S as substrate). Because of the potency of 5.162, it was reasoned that this phenolic steroid, released after the inactivation of enzyme by the irreversible STS inhibitor 5.164, would still be exhibiting reversible inhibition against any unreacted STS.
The most recent D-ring modification of EMATE was a series of N-substituted piperidinedione derivatives. This ring structure was designed to serve as a versatile anchor for introducing a variety of side-chains that can exploit the hydrophobic pockets and regions known to be present in the vicinity of the D-ring of EMATE.
Two compounds, the N-(propyl)- (5.166) and N-(1-pyridin-3-ylmethyl)- (5.167) derivatives, showed exceptionally high potency, with both sharing the same IC50 value of 1 nMin a human placental microsomes preparation. EMATE in the same assay gave an IC50value of 18 nM. The N-unsubstituted derivative 5.165showed potency similar (IC50 = 20 nM) to EMATE, indicating that the six-membered piperidinedione ring is a good mimic of the D-ring of EMATE. After an oral dose of 10 mg/kg per day for 5 d, 5.166and5.167were found to inhibit rat liver STS by 99%. Both compounds were devoid of estrogenic activity in the rat uterine weight-gain assay.