Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene

Abstract

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.

Figure 1

Chemical structures of various ER modulators.

Figure 2

Chemical structures of guaiazulene, azulene, and novel ER modulator Az-01.

Scheme 1

Synthesis of azulene-containing ER modulator candidate Az-01. Reagents and conditions: (a) NaBH₄, THF, MeOH, rt, quantitative; (b,c). HCl, MeOH, 60 ^oC, 99%; (c) BBr₃, CH₂Cl₂, room temperature, quantitative.

Figure 3

Assay for competitive binding of Az-01, tam, and 4-Hotam with [³H]estradiol at Erα LBD. Binding assays were performed using the test compounds and unlabeled E2 (0.4–4 μM) in the presence of [2,4,6,7-³H]17β-estradiol (4 nM) in duplicate.

Figure 4

Estrogenic and anti-estrogenic activities of Az-01, 4-hydroxytamoxifen, and tamoxifen in MCF-7 cell proliferation assay. (A) Estrogenic activity of the test compounds. (B) Anti-estrogenic activity of test compounds on E2-induced MCF-7 cell proliferation. The test compounds were prepared in DMSO solution by making a series dilution so that the final concentration of DMSO was 0.5%. E2 was used at the lowest concentration (0.1 nM) that showed the highest estrogenic activity, as determined from the concentration-dependent curve. All assays were performed using sFBS from which various growth factors had been removed. The vertical axis shows the ratio of the number of proliferating cells in the presence of each test compound to the number of cells in the presence of 0.1 nM E2 taken as 1. All assays were performed in triplicate (n = 3). The two-tailed Student’s t-test was used for statistical analyses (* p < 0.001).

Figure 5

Effect of Az-01 on the growth of MCF-7 cells (ER-positive) and MDA-MB-453 cells (ER-negative). Az-01 was prepared in DMSO solution by making a series dilution so that the final concentration of DMSO was 0.5%. All assays were performed using activated-charcoal-untreated FBS. All assays were performed in triplicate (n = 3). The two-tailed Student’s t-test was used for statistical analyses (* p < 0.001).

Figure 6

Docking modes of Az-01 and 4-HOtam in ERα LBD. (A,B) Binding modes of Az-01. (C) Binding mode of 4-HOtam.

Figure 6

Docking modes of Az-01 and 4-HOtam in ERα LBD. (A,B) Binding modes of Az-01. (C) Binding mode of 4-HOtam.