Synthesis and Evaluation of (1,4-Disubstituted)-1,2,3-triazoles as Estrogen Receptor Beta Agonists as Estrogen Receptor Beta Agonists

: Estrogen receptors (ER) are nuclear hormone receptors which are responsible for sex hormone signaling in women. A series of (1,4-disubstituted)-1,2,3-triazoles 5 – 21 were prepared by reaction of azidophenols with terminal alkynes under Fokin reaction conditions. The products were puriﬁed by column chromatography or recrystallization and characterized by NMR and HRMS. The compounds were tested for binding to ER β via a ligand displacement assay, and 1-(4-hydroxyphenyl)-α -phenyl-1,2,3-triazole-4-ethanol ( 21 ) was found to be the most potent analog (EC 50 = 1.59 µ M). Molecular docking of 5 – 21 within the ligand binding pocket of ER β (pdb 2jj3) was performed and the docking scores exhibited a general qualitative trend consistent with the measured EC 50 values. Abstract: Estrogen receptors (ER) are nuclear hormone receptors which are responsible for sex hormone signaling in women. A series of (1,4-disubstituted)-1,2,3-triazoles 5 – 21 were prepared by reaction of azidophenols with terminal alkynes under Fokin reaction conditions. The products were purified by column chromatography or recrystallization and characterized by NMR and HRMS. The compounds were tested for binding to ER β via a ligand displacement assay, and 1-(4-hydroxy-phenyl)- α -phenyl-1,2,3-triazole-4-ethanol ( 21 ) was found to be the most potent analog (EC 50 = 1.59 μ M). Molecular docking of 5 – 21 within the ligand binding pocket of ER β (pdb 2jj3) was performed and the docking scores exhibited a general qualitative trend consistent with the measured EC 50 values.


Introduction
The estrogen receptors α and β (ERα and ERβ) belong to the nuclear hormone family of intracellular receptors for which 17β-estradiol (E2, Figure 1) is the predominant endogenous ligand. The two receptors exhibit overlapping but distinct patterns of tissue distributions as well as different types of transcriptional regulation [1]. ERα is highly expressed in the breast, liver and uterus and contributes to malignant growth in these tissues [2]. ERβ is more highly expressed in the lungs, prostate, colon, brain and gastrointestinal tract, and binding of E2 exerts beneficial effects in these organs/tissues without the risk of breast cancer [3].

Introduction
The estrogen receptors α and β (ERα and ERβ) belong to the nuclear hormone family of intracellular receptors for which 17β-estradiol (E2, Figure 1) is the predominant endogenous ligand. The two receptors exhibit overlapping but distinct patterns of tissue distributions as well as different types of transcriptional regulation [1]. ERα is highly expressed in the breast, liver and uterus and contributes to malignant growth in these tissues [2]. ERβ is more highly expressed in the lungs, prostate, colon, brain and gastrointestinal tract, and binding of E2 exerts beneficial effects in these organs/tissues without the risk of breast cancer [3].
Numerous non-steroidal molecules are known [4,5] to bind to ERα and/or ERβ, including the ERα-selective agonist PPT [6] and the ERβ-selective agonist DPN [7] ( Figure  1). The majority of these molecules contain a phenol moiety which is responsible for hydrogen-bonding interactions between the phenolic hydroxyl and residues Glu353/Arg394 of ERα. Numerous non-steroidal molecules are known [4,5] to bind to ERα and/or ERβ, including the ERα-selective agonist PPT [6] and the ERβ-selective agonist DPN [7] (Figure 1). The majority of these molecules contain a phenol moiety which is responsible for hydrogenbonding interactions between the phenolic hydroxyl and residues Glu353/Arg394 of ERα.
(Glu305/Arg346 of ERβ), and generally between an active site histidine and a second aliphatic or aromatic hydroxyl group located~11Å distant from the phenolic hydroxyl group [8].
The Cu(I)-catalyzed alkyne-azide cycloaddition reaction, pioneered by the groups of Tornoe [9] and Sharpless [10], has revolutionized the preparation of 1,2,3-triazoles and thrust this moiety into prominence as a linkage. A recent search of SciFinder revealed >12,000 journal references containing the topic "azide alkyne cycloaddition". A number of review articles [11][12][13][14][15][16] have covered 1,2,3-triazole as a pharmacological scaffold of importance. This may be attributed to the fact that the triazole is stable to metabolic degradation [17] and imparts improved solubility due to the possibility of hydrogen bonding.
Tron's group synthesized a family of 1,4-di(hydroxyphenyl)-1,2,3-triazoles [18]. All of these were highly toxic to MCF-7 and MDA-MB-231 cells after 5 days of incubation at a concentration of 100 µM; however, only one compound (1, Figure 2) had high affinity (IC 50~4 5 pM) in a radiolabel displacement assay for the estrogen receptor isolated from cytosolic extracts of porcine uterus. Subsequently, the group of de Pascual-Teresa and Ramos reported the synthesis of 1,4-diaryl-1,2,3-triazoles, their affinity for ERα and ERβ, and their effect on MCF-7 proliferation [19]. Their results revealed that compounds 2a and 2b are full agonists of ERβ at 20 µM, though only 2b exhibited no proliferation effect on MCF-7 cells. (Glu305/Arg346 of ERβ), and generally between an active site histidine and a second aliphatic or aromatic hydroxyl group located ~11Å distant from the phenolic hydroxyl group [8].
The Cu(I)-catalyzed alkyne-azide cycloaddition reaction, pioneered by the groups of Tornoe [9] and Sharpless [10], has revolutionized the preparation of 1,2,3-triazoles and thrust this moiety into prominence as a linkage. A recent search of SciFinder revealed >12,000 journal references containing the topic "azide alkyne cycloaddition". A number of review articles [11][12][13][14][15][16] have covered 1,2,3-triazole as a pharmacological scaffold of importance. This may be attributed to the fact that the triazole is stable to metabolic degradation [17] and imparts improved solubility due to the possibility of hydrogen bonding.

General Experimental Outline
Purifications by chromatography were carried out using flash silica gel (32-63 m) Melting points were measured in open capillary tubes on a MelTemp melting point apparatus and are uncorrected. NMR spectra were recorded on either a Varian Mercury+ 300 Hz or a Varian UnityInova 400 MHz instrument. CHCl3, CD3OD and DMSO-d6 were purchased from Cambridge Isotope Laboratories. Hydrogen-1 NMR spectra were calibrated to 7.27 ppm for residual CHCl3, 3.95 ppm for CD2HOD or 2.49 ppm for d5-DMSO. Carbon-13 NMR spectra were calibrated from the central peak at 77.23 ppm for CDCl3, 49.3 ppm for CD3OD or 39.7 ppm for d6-DMSO. Fluorine-19 NMR spectra were calibrated from internal CF3CO2H (−76.55 ppm). Coupling constants are reported in Hz. High-resolution mass spectra were obtained from the COSMIC lab at Old Dominion University.

General Procedure for the Preparation of Azidophenols
To a solution of aminophenol (9.163 mmol) in ethyl acetate (20 mL) and water (2 mL) at 0 °C was added 5 mL of concentrated HCl. The reaction mixture was stirred for 10 min Given our interest in the discovery and application of ERβ-selective agonists [20][21][22][23][24], we undertook to prepare and assess a series of (1,4-disubstituted)-1,2,3-triazoles. The results are reported herein. To a solution of aminophenol (9.163 mmol) in ethyl acetate (20 mL) and water (2 mL) at 0 • C was added 5 mL of concentrated HCl. The reaction mixture was stirred for 10 min and then sodium nitrate (1.309 g, 15.40 mmol) in water (3 mL) was added to the solution over 3 min and stirred for an additional 30 min. A solution of sodium azide (1.000 g, 15.38 mmol) in water (5 mL) was added dropwise and the mixture was stirred for 30 min. The mixture was then diluted with water (20 mL) and extracted several times with ethyl acetate. The combined organic layers were washed with dilute NaOH (20 mL) followed by water (20 mL). The organic layer was dried (MgSO 4 ) and concentrated to afford the desired azide. The crude azidophenols were used without further purification.

General Procedure for Triazole Synthesis
To a solution of alkyne (1.00 mmol), sodium ascorbate (0.40 mmol), and copper (II) sulfate (0.20 mmol) in water/tBuOH (1:1, 10 mL) was added 1.00 mmol of the desired azide. The solution was stirred for 30 h. The reaction was extracted with ethyl acetate (2 × 10 mL). The combined organic layers were washed with brine (15 mL), dried (MgSO 4 ) and concentrated. The resulting triazole compound was purified by column chromatography.

ERβ Assays 2.2.1. TR-FRET Assay
ERβ ligand displacement measurements were performed using the SelectScreen assay service from Thermo Fisher Scientific. The TR-FRET assay involves human ERβ ligandbinding domain (LBD) that is tagged with glutathione-S-transferase (GST), a Tb-anti-GST antibody that binds to the tag, and a fluorescently labeled estrogen bound in the active site pocket. The TR-FRET signal obtained decreases when competitor compounds displace the fluorescently labeled tracer. Individual dose-response curves are found in Figure S1.

Cell-Based Functional Assay
Kits from Indigo Biosciences were used to examine the impact of (±)-21 on agonist and antagonist activity for full-length, native ERβ. In this assay, a luciferase reporter gene was downstream from an ERβ-responsive promoter activated by an agonist. Chemiluminescence resulting from ER-induced luciferase expression was measured in a SpectraMax M5 (Molecular Devices). Vehicle and E2 controls were included. E2 had an agonist IC 50 value of 0.022 ± 0.005 nM. Kit instructions were followed. Data were normalized to controls and EC 50 values were calculated by performing a nonlinear squares fit using Prism 6 (GraphPad).

Computational
Computational docking was performed using the on-line 1-Click Docking tool in Mcule (www.mcule.com, accessed on 3 July 2022) into the ERβ agonist configuration (pdb 2jj3) [28]. The 1-Click Docking tool uses the Vina docking algorithm [29]. As a control experiment, 17β-estradiol was docked into the structure of ERβ (pdb2jj3) and found to adopt a binding mode where the phenolic hydroxyl group was hydrogen bound to Glu305 and Arg 346 and the 17β-hydroxyl group was hydrogen bound to His 475 (binding score = −9.7).

Discussion
The (1,4-disubstituted)-1,2,3-triazoles exhibited a range of potencies in the TR-FRET ligand displacement assay. The 4-phenyl-and 4-benzyl-substituted 1,2,3-triazoles 5-8 were selected to assess whether the presence of two hydroxyl groups at opposite ends of the ligand were crucial for agonist activity. Similarly, the 4-hydroxymethyl-, 4-(1methyl-1-hydroxyethyl)-, 4-(2-hydroxyethyl)-, 4-(2-hydroxypropyl), and 4-(2-hydroxy-2phenylethyl)-substituted 1,2,3-triazoles 9-21 were prepared to assess the effect of a hydroxyalkyl substituent on agonist activity. While 1-(4-hydroxyphenyl)-1,2,3-triazoles 10 and 13 were more potent than their corresponding 1-(3-hydroxyphenyl)-1,2,3-triazole counterparts 9 and 12, respectively, the opposite was found to be the case for 7 and 18 compared to 8 and 19. However a general correlation between the observed EC 50 values and the calculated docking scores was observed; those (1,4-disubstituted)-1,2,3-triazoles with less negative docking scores were found to be less potent. The most potent ERβ agonist was found to be (±)-21 (EC 50 = 1.59 µM). This increased potency might be explained by reference to the computationally generated docking poses (Figure 3). These reveal that the phenolic group is situated in a position similar to the phenolic portion of E2, with hydrogen bonding to both a Glu and Arg residue and a π-π interaction with a Phe residue. Similarly, the aliphatic hydroxyl group of (R)-or (S)-21 is situated in close proximity to the His residue. What perhaps allows the increased binding of (±)-21 is that the aryl group of the α-phenylethanol sidechain is oriented into a cleft between helix 12 and the estradiol binding pocket.

Data Availability Statement:
The data presented in this study are available in Supplementary Materials.

Conflicts of Interest:
Daniel S. Sem and William A. Donaldson are co-founders and shareholders of Estrigenix Therapeutics, Inc., a company which aims to dramatically improve women's health by developing safe, clinically proven treatments for the mental and physical effects of menopause, to enable and empower women to live happier and healthier lives. Since D.S.S. and W.A.D. have disclosed other more potent and highly selective ERβ agonists, they do not anticipate patenting the compounds described in this manuscript.