Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) β (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERβ)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERβ)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 μM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.


Synthesis of intermediates
Syntheses of GW7604 and methoxy-GW7604 was performed as described previously. 1 1.1.
Synthesis of thioxo-quinazolinones General procedure for synthesis of thioxo-quinazolinone carboxylic acids Modifying the method from Sun et al. 2 , 2-methoxycarbonylphenyl isothiocyanate (1 eq) and the respective amino carboxylic acid (1 eq) were dissolved in EtOH and refluxed for 2-3 days. After cooling the solution to ambient temperature and concentrating to half of the volume, the resulting precipitate was filtered off by suction and washed with ice-cold EtOH.

S 3
General procedure for synthesis of thioxo-quinazolinone carboxamides Similar to the previously mentioned general procedures for amide formation, PyBOP (1.0 eq) was dissolved in dry DCM and DIPEA (2.0 eq) together with the respective acid (1.0 eq) in dry DMF were added at 0 °C.
After 5 min, the respective amine (1.1 eq) in dry DCM or DMF was supplemented dropwise. The mixture was stirred first at 0 °C for 30 min, then at rt for 20 h. The mixture was concentrated to a residue, which was dissolved in EA, washed with water (pH = 3-4; adjusted with 1N HCl) and further extracted twice with EA. The combined organic layers were washed with brine, dried over dry Na2SO4 and evaporated.
Purification was carried out by flash column chromatography.

N-[3-(Boc-amino)propyl]-4-[4-oxo-2-thioxodihydroquinazolin-3-yl]butanamide 3
Synthesis of 3 was carried out according to the general procedure.  (1.25 mmol) in 6 mL of dry DCM were added in that order. The next day, water and 2.5N NaOH were added (to adjust pH = 8-9) and the residue was extracted 3× with EA. Then, the combined organic extracts were washed with H2O/1N HCl (pH = 2-3) whereupon the EA phase turned to cloudy. The aqueous phase was further extracted 2× with EA, then the combined organic extracts were washed with brine, dried over dry Na2SO4 and evaporated. The crude product was washed with water and cold MeOH over a sintered glass funnel to remove salts and organic by-products, giving 6 as a white powder (344 mg, 0.64 mmol, 55%).