Design,Synthesis,and Structure-activity Relationship Studies Of Novel Selective Estrogen Receptor Degraders

Estrogen receptor positive（ER+）/human epidermal growth factor receptor 2negative（HER2-）breast cancer（BC）is the main type of female BC,and the treatment has intensively relied on selective estrogen receptor modulators（SERMs）and aromatase inhibitors（AIs）.However,most ER+/HER2-BC would eventually develop to an endocrine-resistant disease,leading to progression,recurrence and metastasis.Even though,estrogen signaling pathway still plays an important role in the setting of endocrine resistant.Fulvestrant is a selective estrogen receptor degrader（SERD）which exhibits superior anti-tumor activities to existing endocrine therapy drugs in ER+BC,due to its unique mechanisms.Unfortunately,resulting from the poor pharmacokinetic properties,fulvestrant has to be injected intramuscularly,which leading to poor patient compliance,and exhibits low drug exposure,suboptimal in vivo efficacy.Therefore,the development of novel and effective oral SERDs is an urgent unmet clinical need.Nevertheless,there is still no orally effective SERD approved for marketing.LSZ102 was a promising oral SERD under clinical investigation.Considering its insufficient in vivo ERαdegradation,moderate anti-tumor effect and unsatisfied pharmacokinetic properties,we carried out systematic studies on structural optimization,structure-activity relationship,structure-pharmacokinetics relationship and structure-toxicity relationship,based on the prevalent benzothiophene skeleton.Consequently,eighty-seven novel SERDs were designed and synthesized for discovering more active compounds with favorable druggability.To address the metabolic liabilities of LSZ102,the phenolic hydroxyl group was replaced by some routine bioisosteres to maintain the hydrogen bonds,among which the 6H-thieno[2,3-e]indazole was the preferred structure.As for the important acrylic acid degron optimization,various degradation motifs were introduced to achieve the balance of in vitro activity,metabolic stability and safety.It was found that 3-fluoromethylazetidine rather than acrylic acid,resulted in II-11 a nearly 4-fold increase in anti-MCF-7 cell proliferation activity(IC₅₀=1.1 n M vs 4.2 n M)while maintaining comparable ERαdegradation to LSZ102.Reversing the azetidine ring allowed us to obtain a series of 1-（3-fluoropropyl）azetidine substituted derivatives,of which V-2 and V-10 performed superior properties to LSZ102 in ERαantagonism(IC₅₀=0.9-1.1 n M),ERαdegradation(EC₅₀=1.1-1.2 n M/92.2%-93.5%)and anti-MCF-7 cell proliferation(IC₅₀=1-1.1 n M).Even though great efforts were made,the investigations around the linkage types and substitution patterns on 2-/3-position aryl groups were unsuccessful to produce a better alternative than V-2 and V-10.In addition,all N-linker compounds at the 3-aryl group displayed comparable in vitro activities to its O-linker analogs,but had moderate h ERG inhibition.On the contrast,O-linker compound V-2 stood out not only for its heart safety profile(h ERG IC₅₀>40μM),but also acting as a full antagonist and inhibiting proliferation of MCF-7 Tam1 and multiple ER+breast cancer cells.What’s more,V-2 strongly inhibited the transcriptional activity of Y537S/D538G ERαmutants,as well as favorable oral bioavailability in mice（F%=67%）.MCF-7 cell line derived xenograft model further demonstrated 5 mg/kg V-2 was as potent as 15 mg/kg LSZ102 both in anti-tumor activity and proteasome-mediated ERαlevel reduction.To improve the water solubility of V-2,we set a panel of acids for screening,and finally V-2 hydrochloride was selected（>50 mg/m L）.Pharmacokinetic study results showed that V-2 hydrochloride had a dose-proportional oral exposure,moderate clearance（CL=28.1 m L/min/kg）and half-life(T_1/2=3.4 h,i.v.),and an oral bioavailability of 27%-37%in rats.Moreover,the concentration of V-2 in tissues expressing ER（breast,uterus,ovary）was 3-8 times higher than that in plasma and was able to penetrate the blood-brain barrier（B/P=0.7）,indicating the potential for ERα+brain metastatic BC treatment.Unfortunately,CYP enzyme inhibition assay presented V-2 to be a strong inhibitor of CYP3A4 enzyme(IC₅₀<1μM),with a potential risk of drug-drug interactions.Although the subsequently evaluated V-10 was proved a 10-fold reduced inhibitory activity(IC₅₀=1-10μM),which was comparable to the phase III clinical drug AZD9833,more comprehensive in vitro and in vivo evaluations are still needed.In summary,our structure optimization effort of LSZ102 generates oral SERDs with potent anti-tumor activities,which are expected to overcome endocrine resistance.Though the development of V-2 is halted due to the strong CYP3A4 inhibition,comprehensive druggability evaluations of V-10,whose overall properties are almost equivalent to V-2,is in progress.Our research is supposed to provide guidance for discovering novel oral SERDs with full antagonistic activity,subsequent structural optimization and biological evaluation will create high-quality drug candidates.