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Figure 4 1,2-Diphenylenediamine derivatives.

ligands such as diethylstilbestrol, which consists of an antiperiplanar E-stilbene. In an effort to explore the estrogenic behavior of related structures further, Gust and co-workers prepared a series of conformationally constrained analogs of 4, where the orientation of the aryl rings is defined using a heterocyclic ring.61 Piperazine, imidazoline, and imidazole cores were examined, with the most interesting compounds emanating from the imidazoline series.

The compounds were tested in competition experiments using calf uterine cytosol. All showed very weak binding (RBA<0.1%) in this assay. However, in spite of the weak binding affinity, the compounds were capable of activating transcription in a human breast cancer cell line (MCF-7). For example, imidazoline (5) showed maximal efficacy equivalent to that of E2 at 5 x 10 _ 7 mol L_ 1. The authors postulate that one phenol of 5 forms contacts with the receptor at Glu-353 and Arg-394, while the other phenol interacts with Asp-351. Compounds such as 5 which do not displace E2 from its binding site but do demonstrate gene activation have been classified as type II estrogens, as opposed to type I estrogens, such as diethylstilbestrol, which do bind in a similar mode to E2.62

Gust etal. further optimized the 2,3-diaryl piperazine series by exploring substitution of the aryl rings and the effect of N-alkylation (Figure 5).63 Analogs lacking the N-alkyl substituent, such as 6, were essentially devoid of estrogen receptor-binding activity (RBA<0.02%, calf uterine cytosol, 4°C) while the N-ethyl derivatives displayed marginally higher affinity (e.g., 7, RBA = 0.42%). The compounds were then tested for their ability to activate gene expression in stably transfected MCF-7-2a cells. Both 6 and 7 were shown to activate gene expression at a 1 mmol L _ 1 concentration to the level of 73% and 74%, respectively, relative to E2. Methylation of the phenols significantly abrogated gene activation (<5% relative activation).

The pyrroloindolizine ring structure (Figure 6), has recently been used as a core in the preparation of new estrogen receptor ligands.64 The compounds were assessed using estrogen receptor-rich cytosol from rabbit uterine tissue. Not surprisingly, pra-hydroxylation on the phenyl ring was found to be necessary for potent binding affinity. An additional hydroxy group on the pyrroloindolizine core improved estrogen receptor affinity significantly, as demonstrated by 8 (IC50 = 0.9 nmol L" 1) and 9 (IC50 = 0.9 nmol L" 1). Given the lack of a basic side chain, which is a common feature of estrogen receptor antagonists, these compounds are expected to be agonists in uterine tissue. Indeed, NNC 45-0095 (10, IC50 = 9.5 nmol L"1) was evaluated in an Ishikawa human endometrial adenocarcinoma cell line and was shown to be a full agonist (EC50 = 13 nmol L _ 1) with a maximal efficacy of 105% relative to the agonist moxestrol.65 The in vivo estrogenic nature of 10 was examined in immature mice, where it was shown to be a potent agonist on the uterus (EC50 = 0.96 nmol g_ 1). The uterotrophic nature of pyrroloindolizine (10) was also seen in mature OVX mice at doses of 0.1-10 nmol g_ 1. The ability of 10 to prevent loss of bone mineral density of the distal femur completely was demonstrated at a 5 nmolg_ 1 dose, administered subcutaneously five times per week for 5 weeks.

Using the known estrogen receptor agonist coumestrol (11) as a conceptual lead, Jacquot etal. prepared a series of benzothiazinone-based estrogen receptor ligands (Figure 7).66 The compounds were tested in MCF-7 proliferation and binding assays. Both methyl ether (12) and phenol (13) were shown to be high-affinity estrogen receptor ligands

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