The clinical benzothiophene SERM (BT-SERM), raloxifene, was compared with estrogens in protection of primary rat neurons against oxygen-glucose deprivation (OGD). Structure-activity relationships for neuroprotection were determined for a family of BT-SERMs displaying a spectrum of ERα and ERβ binding affinity and agonist/antagonist activity, leading to discovery of a neuroprotective pharmacophore, present in the clinically relevant SERMS, raloxifene and desmethylarzoxifene (DMA), for which submicromolar potency was observed for neuroprotection. BT-SERM neuroprotection did not correlate with binding to ER nor classical ER activity, however, both the neuroprotective SERMs and estrogens were shown, using pharmacological probes, to activate the same kinase signaling cascades. The antiestrogen ICI 182,780 inhibited the actions of estrogens, but not those of BT-SERMs, whereas antagonism of the G-protein coupled receptor, GPR30, was effective for both SERMs and estrogens. Since SERMs have antioxidant activity, ER-independent mechanisms were studied using the classical phenolic antioxidants, BHT and Trolox, and the Nrf2-dependent cytoprotective electrophile, sulforaphane. However, neuroprotection by these agents was not sensitive to GPR30 antagonism. Collectively, these data indicate that the activity of neuroprotective BT-SERMs is GPR30-dependent and ER-independent and not mediated by antioxidant effects. Comparison of novel BT-SERM derivatives and analogs identified a neuroprotective pharmacophore of potential use in design of novel neuroprotective agents with a spectrum of ER activity.

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has re-emerged as a potential treatment option following exhaustive use of tamoxifen (TAM) or aromatase inhibitors although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEMs) as an alternative to E2 for the treatment of TAM-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, TAM-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of TAM-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of TAM-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of ERα to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. Additionally, unlike E2 or TAM, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.

o-Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o-quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o-quinone (t1/2 = 3.9 ± 0.1 h) which like 4-hydroxyequilenin-o-quinone (t1/2 = 2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o-quinones, which were characterized by LC-MS/MS. Incubations of LY2066948 or equilenin with rat liver microsomes also gave detectable o-quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N-dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o-quinone GSH conjugates were detected in equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, noo-quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o-quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer’s disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.

The bioactivation of both endogenous and equine estrogens to electrophilic quinoid metabolites has been postulated as a contributing factor in carcinogenic initiation and/or promotion in hormone sensitive tissues. Bearing structural resemblance to estrogens, extensive studies have shown that many selective estrogen receptor modulators (SERMs) are subject to similar bioactivation pathways. Lasofoxifene (LAS), a third generation SERM which has completed Phase III clinical trials for the prevention and treatment of osteoporosis, is currently approved in the European Union for this indication. Previously, Prakash et al. (Drug Metab. Dispos. 2008, 36, 1218-26) reported that similar to estradiol, two catechol regioisomers of LAS are formed as primary oxidative metabolites, accounting for roughly half of total LAS metabolism. However, the potential for further oxidation of these catechols to electrophilic o-quinones has not been reported. In the present study, LAS was synthesized and its oxidative metabolism investigated in vitro under various conditions. Incubation of LAS with tyrosinase, human liver microsomes, or rat liver microsomes in the presence of GSH as a trapping reagent resulted in formation of two mono-GSH and two di-GSH catechol conjugates which were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar conjugates were also detected in incubations with P450 3A4, P450 2D6, and P450 1B1 supersomes. Interestingly, these conjugates were also detected as major metabolites when compared to competing detoxification pathways such as glucuronidation and methylation. The 7-hydroxylasofoxifene (7-OHLAS) catechol regioisomer was also synthesized and oxidized either chemically or enzymatically to an o-quinone that was shown to form depurinating adducts with DNA. Collectively, these data show that analogous to estrogens, LAS is oxidized to catechols and o-quinones which could potentially contribute to in vivo toxicity for this SERM.