GPER proteolytic targeting chimeras

Abstract

To provide PROteolytic TArgeting Chimeras (PROTACs) capable of inactivating a G-protein coupled estrogen receptor (GPER).SOLUTION: A molecule comprising a G-protein coupled estrogen receptor (GPER) ligand coupled to a linker coupled to an E3 ubiquitin ligase ligand and methods of using the molecule are provided. In one embodiment, the GPER ligand is estradiol and the E3 ubiquitin ligase ligand is (S,R,S)-AHPC.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] Epidemiological, clinical and preclinical evidence suggests that breast cancer is an estrogen-driven malignancy (Germain et al., 2011; Parl et al., 2018; Rothenberger et al., 2018; Rugo et al., 2016; Yue et al., 2016). This explains the widespread success of antiestrogens as effective adjuvant treatment for early-stage ER(+) breast cancer (DePlacido et al., 2018; Tremont et al., 2017). Two classes of drugs are used to antagonize estrogen action: 1) compounds that inhibit aromatase (aromatase inhibitors, AIs), the enzyme that converts androgens to estrogens, and 2) estrogen mimetics (ER antagonists), which competitively block the interaction of estrogen with the ER. To be effective, AIs or ER antagonists must be administered on a prolonged dosing schedule of 3–5 years or more. Finally, the long-term use of anti-estrogen therapy is associated with undesirable and sometimes intolerable side effects, such as menopausal symptoms, osteoporosis, bone pain, and joint pain (Yang et al., 2013). Furthermore, the long-term use of ER antagonists is associated with an increased risk of endometrial cancer (Hu et al., 2015; Jones et al., 2012) and thrombosis (Cosman et al., 2005).

[0002] De novo or acquired drug resistance further limits the use of anti-estrogen therapy, with resistance occurring in more than 20% of treated patients (Augusto et al., 2018; Clarke et al., 2001; Haque et al., 2019; Lei et al., 2019). De novo resistance is due to intratumor heterogeneity in ER expression at diagnosis (Reinhardt et al., 2017), while acquired resistance reflects tumor heterogeneity that evolves due to selective pressure exerted by anti-estrogens during treatment. Examples include: a) selection of mutations that promote the selection of mutations that result in loss of drug-receptor interaction (Fan et al., 2015) or enhanced estrogen-independent ER-mediated gene transactivation (Barone et al., 2010; GRCIA-Becerra et al., 2012), b) epigenetic silencing of the ER promoter (Achinger-Kaecke et al., 2020), and c) transcriptional upregulation of compensatory genes that promote estrogen-independent growth by modulating cell cycle activity (Thngavel et al., 2011) or downstream signaling activity of the epidermal growth factor receptor (EGFR) (Guilano et al., 2011). Yet another mechanism of anti-estrogen resistance is provided by the G protein-coupled estrogen receptor (GPER). This recently evaluated estrogen receptor is clearly important for breast cancer biology and treatment (Rouhimoghadm et al., 2020). Unlike the nuclear estrogen receptor, which is primarily intracellular and functions as a ligand-inducible transcription factor, GPER is a Gs-coupled seven-transmembrane receptor that is located in the plasma membrane and intracellular membranes and promotes rapid pregenomic actions, including activation of adenylate cyclase (Filardo et al., 2002) and transactivation of EGFR (Filardo et al., 2000).Stimulation of GPER contributes to the activation of signaling effectors downstream of EGFR, such as Ras, PI3K, AKT, and Erk1 / 2, which are involved in cell proliferation, survival, invasion, and resistance to endocrine therapy (Peperman et al., 2019). Analysis of GPER expression in primary breast tumors shows that its presence is associated with disease progression (Filardo et al., 2006), breast cancer stem cell survival (Chan et al., 2020), and tamoxifen resistance (Ignatov et al., 2011; Rouhimoghadam et al., 2018; Yin et al., 2017). Moreover, its expression is commonly retained in triple-negative breast cancers that lack ER, PR, and her-2 / neu (Steiman et al., 2013). GPER thus broadens the ER-centric view of estrogen responsiveness ( Filardo et al., 2018 ) and confounds the binary rubrics that guide rational allocation of adjuvant breast cancer treatments. This is particularly important for patients receiving endocrine therapy, as “partial” (tamoxifen) and “pure” (fulvestrant and raloxifene) ER antagonists function as GPER agonists ( Filardo et al., 2000 ; Petrie et al., 2013 ).

[0003] Current clinical guidelines recommend the use of fulvestrant as a second-line treatment for endocrine-resistant breast cancer (Nathan et al., 2017; Sammons et al., 2019). Fulvestrant is an estrogen mimetic drug that functions not only as a competitive inhibitor of the ER but also as a selective ER degrader (SERD) (Pike et al., 2001). Fulvestrant is the only FDA-approved drug in this class, and upon binding to the ER, it destabilizes interactions with associated heat shock chaperone proteins, leading to ER degradation in the 26S proteasome (Callige et al., 2006). Thus, fulvestrant acts to decrease the bioavailability of the drug target, thereby reducing the risk of secondary drug resistance. However, the main limitation of fulvestrant is its poor bioavailability after oral administration, which requires painful monthly intramuscular injections (Robertson et al., 2007).

[0004] A method to selectively degrade the ER has been achieved through the use of Proteolytic Targeting Chimeras (PROTACs) that exploit the ubiquitin-proteasome pathway (Cyrus et al., 2011; Huang et al., 2016). Typically, PROTACs are heterobifunctional compounds consisting of two functional binding domains that act to directly link a protein of interest to the ubiquitination machinery. They consist of a targeting domain linked to an E3 ubiquitin ligase recognition motif via a chemical spacer. Upon binding, PROTACs polyubiquitinate their targets and direct them to the 26S proteasome for degradation. PROTACs offer the advantage that they have been used to target a wide range of cytoplasmic, nuclear, and plasma membrane proteins (Sun et al., 2019). Although efficient PROTAC degradation requires a systematic approach to optimize the spatial arrangement of the targeting and E3 ligase recruitment domains (Bondesonet al., 2018). PROTACs offer the added advantage of providing high specificity and processivity for the target protein of interest; thus, effectively reducing the concentration of the target molecule and lowering the chance of drug-resistant mutants evolving. This strategy has been successfully used to downmodulate the ER, and ER-PROTACs have been developed with targeting domains consisting of various estrogen derivatives, including 17β-estradiol (17b-E2) (Rodriguez-Gonzales et al., 2008), endoxifen (Dragovich et al., 2020), raloxifene (Hu et al., 2019), and tamoxifen (Fan, 2020), each linked with a different chemical spacer to a protein or small molecule E3 ligase recruitment motif. ERα, ERβ, and GPER have different binding affinities for natural and synthetic estrogens ( Prissnitz et al., 2015 ), but each exhibits high binding affinity for 17β-estradiol (E2) as measured by dissociation constants in the low nanomolar range. Summary of the Invention

[0005] The present disclosure provides PROTACs that can inactivate G protein-coupled estrogen receptor (GPER). In one embodiment, the present disclosure provides orally delivered small molecule PROTACs that can inactivate or not activate GPER, which are useful for treating cancers such as, for example, triple-negative breast cancer (TNBC), one of the most difficult to treat breast cancers. TNBC is a type of breast cancer that lacks effective therapeutic targets and commonly expresses GPER (>80% of TNBCs). The GPER-PROTACs disclosed herein delay or inhibit cancer progression, for example by enhancing the proteolysis of GPER in TNBC, thus proving that endocrine therapy can be used for TNBC. GPER-PROTACs may also be useful for other cancers, such as, for example, other types of breast cancer or gynecological cancers in patients currently deemed ineligible for endocrine therapy. GPER-PROTACs can also be used in combination with existing therapies (e.g., aromatase inhibitors). In one embodiment, the GPER-PROTACs are delivered orally. In one embodiment, the GPER-PROTAC is an E2-based PROTAC (e.g., UI-EP001 or UI-EP002) that interacts with and degrades ERα, ERβ, and GPER, e.g., it is a pan-estrogen receptor inhibitor. In one embodiment, the bispecific PROTAC interacts with plasma membrane and intracellular estrogen receptors and promotes ubiquitin-proteasome-dependent degradation.

[0006] PROTACs for inactivating GPER are evaluated, for example, using a competitive radioreceptor binding assay using [3H]-17β-estradiol (17β-E2) as a tracer to determine GPER specific binding in the plasma membrane of target cells, for example, breast cancer cells. PROTACs for inactivating GPER are also evaluated for their ability to promote proteasomal degradation of GPER in cancer cell lines, such as breast cancer cell lines, and for their anti-carcinogenic effects in animal models, such as spontaneous cancer models using small primary TNBC tumors. As disclosed herein, UIEP001, an exemplary PROTAC for inactivating GPER, has been shown to reduce native and recombinant GPER plasma membrane protein and reduce steady-state expression of HA-GPER, but not HA-β1AR.

[0007] In one embodiment, the present disclosure provides a G protein-coupled estrogen receptor (GPER) ligand linked to a ligand of an E3 ligase. In one embodiment, the present disclosure provides a G protein-coupled estrogen receptor (GPER) ligand linked to a linker, e.g., chemically linked via a covalent bond, which is further linked to the ligand of an E3 ligase, e.g., via a covalent bond. In one embodiment, the GPER ligand comprises 17β-estradiol, estrone, phytoestrogen, xenoestrogen, estriol, estriol 3-sulfate, estriol 17-sulfate, G-1, G-15, G-36, genistein, or quercetin. In one embodiment, the phytoestrogen comprises a flavone, an isoflavone, a lignin saponin, a coumestin, or a stilbene. In one embodiment, the GPER ligand comprises a bisphenol, an alkylphenol, a methoxyphenol, a polychlorinated biphenyl, or a dioxin. In one embodiment, the GPER ligand is a GPER antagonist. In one embodiment, the GPER ligand is a GPER agonist. In one embodiment, the GPER ligand is not 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline. In one embodiment, the GPER PROTAC does not have formula (II) or (III). In one embodiment, the GPER ligand comprises 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline. In one embodiment, the GPER ligand does not bind to ER. In one embodiment, the E3 ligase ligand is a von Hippel ligase (VHL) ligand. In one embodiment, the E3 ligase ligand comprises lenalidomide, pomalidomide, iverdimide, (S,R,S)-AHPC, thalidomide, VH-298, CC-885, E3 ligase ligand 8, TD-106, VL285, VH032, VH101, VH298, VHL ligand 4, VHL ligand 7 (see Scheepstra et al., Comout. Struct. Biotech. J., 17:160 (2019), the disclosure of which is incorporated herein by reference), VHL-2 ligand 3, E3 ligase ligand 3, E3 ligase ligand 2, cereblon, CC-122, or BC-1215.In one embodiment, the linker has a chain having 2-200 atoms. In one embodiment, the linker has a chain having 5-25 atoms. In one embodiment, the linker has a chain having 25-50 atoms. In one embodiment, the linker has a chain having 30-50 atoms. In one embodiment, the linker has a chain having 50-100 atoms. In one embodiment, the linker is an alkyl linker. In one embodiment, the linker is a heteroalkyl linker. In one embodiment, the linker comprises a polyethylene glycol (PEG), e.g., 2, 3, 4, or 5 PEG units, and optionally one or more other groups, such as amide groups. In one embodiment, the linker comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG units, and optionally one or more other groups, such as amide groups. In one embodiment, the linker comprises a cleavable PEG linker, e.g., one having a disulfide bond. In one embodiment, the linker is of a length that allows it to span the membrane of a vertebrate cell, e.g., at least 5-10 nm in length. In one embodiment, the linker comprises at least 3 PEG units, e.g., (OCC)3. In one embodiment, the linker comprises at least 4 PEG units, e.g., (OCC)4. In one embodiment, the linker has at least 5 PEG units. In one embodiment, the linker is C1-C. 10 alkyl(PEG)n. Contains at least 4 PEG units, e.g., (OCC)4. In one embodiment, the linker is (PEG) n NH(CO)(PEG) m wherein n and m are independently 1 to 15. In one embodiment, n and m are independently 3 to 10. In one embodiment, n is 5 to 10 and m is 3 to 6.

[0008] In one embodiment, the linker has a backbone having a chain length of 5-200 atoms, counting in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand. In one embodiment, the linker has a backbone having a chain length of 15-50 atoms, counting in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand. In one embodiment, the linker has a backbone having a chain length of 20-50 atoms, counting in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand. In one embodiment, the linker has a backbone having a length of 8-300 angstroms, calculated length determined by summing the bond lengths in the linear path between the GPER ligand and the E3 ubiquitin ligase ligand.

[0009] In one embodiment, the linker has a backbone length of 25-75 angstroms, calculated by summing the bond lengths in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand.

[0010] [ka]

[0011] wherein Q is a divalent group that forms a bond or a covalent bond to a GPER ligand; Z is a linear chain that includes one or more alkyl, aryl, heteroalkyl, heteroaryl, alkyloxy, alkylamino, alkylglycol, carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, amine, oxygen, sulfur, sulfone, or sulfoxide in divalent form; and G is a divalent group that forms a bond or a covalent bond to an E3 ubiquitin ligase ligand. In one embodiment, the linker is

[0012] [ka]

[0013] where Q is a divalent group that forms a bond or a bond to a GPER ligand; R is the divalent form of alkyl, aryl, heteroalkyl, heteroalkyl, heteroaryl, alkyloxy, alkylamino, alkylglycol, carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, amine, oxygen, sulfur, sulfone, or sulfoxide; G is a divalent group that forms a bond or a linkage to an E3 ubiquitin ligase ligand; m, n, p, and q, if present, are each independently integers from 0 to 50, provided that at least one of m, n, p, and q, if present, is an integer greater than 0. In one embodiment, Q and G are independently the divalent form of carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, sulfone, or sulfoxide. In one embodiment, Q and G are independently carbonyl or acyl selected from the group consisting of acetyl, 2-hydroxyacetyl, 2-aminoacetyl, propionyl, 3-hydroxypropanoyl, 3-aminopropanoyl, butanoyl, 4-hydroxybutanoyl, and 4-aminobutanoyl, each of which is in its divalent form.

[0014] In one embodiment, m and n, if present, are each independently an integer from 0 to 2, and Q and G are each independently carbonyl, thiocarbonyl, acetyl, 2-hydroxyacetyl, 2-aminoacetyl, propionyl, 3-hydroxypropanoyl, 3-aminopropanoyl, butanoyl, 4-hydroxybutanoyl, 4-aminobutanoyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, sulfone, or sulfoxide, each of which is in divalent form. In one embodiment, Z is hydrophilic. In one embodiment, the linker is

[0015] [ka]

[0016] which provides a backbone having a length of 13 atoms counted in a linear path. In one embodiment, the linker has the structure

[0017] [ka]

[0018] wherein p and q are each independently an integer between 0 and 50. In one embodiment, the linker comprises one or more of a divalent alkyl group, a divalent heteroalkyl group, or a divalent polyethylene glycol (PEG), or one or more of each. In one embodiment, the linker comprises 5 or more divalent ethoxy (-CH2CHO-) groups. In one embodiment, the linker comprises one or more heteroatoms for every two carbons and does not comprise an alkyl chain longer than butyl. In one embodiment, the linker is attached to the GPER ligand via an oxygen, nitrogen, sulfur, carbonyl, or ethynyl of the GPER ligand, and the linker is attached to the E3 ubiquitin ligase ligand via an oxygen, nitrogen, sulfur, carbonyl, or ethynyl of the E3 ubiquitin ligase ligand. In one embodiment, the GPER ligand is an estrogen steroid and comprises a divalent group selected from oxygen, amine, sulfur, vinyl, ethyne, and carbonyl at C6 or C17 of the estrogen steroid, said divalent group at C6 or C17 being linked to a linker. In one embodiment, the E3 ubiquitin ligase ligand is (S,R,S)-AHPC, which is linked to a linker via the amine of (S,R,S)-AHPC.

[0019] Also provided are methods of using the molecules. In one embodiment, a method is provided for preventing, inhibiting, or treating endocrine-resistant or hormone therapy-resistant cancer in a mammal. The method comprises administering to the mammal a composition having an effective amount of a molecule comprising a G protein-coupled estrogen receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase. In one embodiment, the mammal is a human. In one embodiment, the administration is systemic. In one embodiment, the administration is oral. In one embodiment, the composition is a tablet. In one embodiment, the composition is a liquid. In one embodiment, the composition is injected. In one embodiment, the mammal is a human resistant to aromatase inhibitor therapy.

[0020] Also provided is a method of preventing, inhibiting, or treating triple-negative breast cancer in a mammal, the method comprising administering to the mammal a composition having an effective amount of a molecule comprising a G protein-coupled estrogen receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase. In one embodiment, the mammal is a human. In one embodiment, the administration is systemic. In one embodiment, the administration is oral. In one embodiment, the GPER ligand is a GPER antagonist.

[0021] Further provided is a method of preventing, inhibiting, or treating gynecological cancer, such as cervical cancer, ovarian cancer, or endometrial cancer, in a female mammal, comprising administering to said mammal a composition having an effective amount of a molecule comprising a G protein-coupled estrogen receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase. In one embodiment, the mammal is a human. In one embodiment, the administration is systemic. In one embodiment, the administration is oral.

[0022] Also provided is a method of preventing, inhibiting, or treating cancer, including prostate cancer or colon cancer, or any cancer that is not hormone dependent, in a mammal, comprising administering to the mammal a composition having an effective amount of a molecule comprising a G protein-coupled estrogen receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase. In one embodiment, the mammal is a human. In one embodiment, the administration is systemic. In one embodiment, the administration is oral. In one embodiment, the GPER ligand is a GPER antagonist. [Brief description of the drawings]

[0023] [Figure 1] Proteolytic targeting of GPER by an exemplary PROTAC. [Diagram 2] Structure confirmation of E2-PROTAC, UI-EP001. [Diagram 3] PROTAC prototype UI-EP001 reduces surface GPER. UI-EP001 (100uM, 16h) causes downmodulation of surface recombinant GPER. Right: Results quantified by corrected total red fluorescence measured from three microscopic fields. [Figure 4] The PROTAC prototype UI-EP001 reduces surface GPER. Similar results were obtained for native GPER in her2+ and TNBC breast cancer cells. [Diagram 5] GRER PROTAC for the treatment of triple-negative breast cancer. [Figure 6] E2β-PROTAC (100 uM) can reduce the GPER of the surface. [Figure 7] E2β-PROTAC (100 uM) can reduce the GPER of the surface. [Figure 8] E2β-PROTAC (100 uM) can reduce the GPER of the surface. [Figure 9] E2β-PROTAC (100 uM) can reduce the GPER of the surface. [Figure 10]Assessment of specificity of UI-EP001 downmodulation. [Figure 11] Restoblue assay. [Figure 12] Comparison of viability of HCC1806 and SKBR3 treated with E2-PROTAC and partial PROTAC (12 hours after treatment). [Figure 13] Comparison of viability of HCC1806 and SKBR3 treated with E2-PROTAC and partial PROTAC (24 hours after treatment). [Figure 14] Comparison of viability of HCC1806 cell lines pretreated with estradiol saturation (24 h treatment). [Figure 15] Comparison of viability of SKBR3 cell lines pretreated with estradiol saturation (24 h treatment). [Figure 16] IC50 value. [Figure 17] PROTAC mechanism of action. PROTACs bind target proteins to E3 ligases, leading to their polyubiquitination and proteasomal degradation. [Figure 18]E2-PROTAC (UI-EP001) downmodulates GPER in human breast cancer cells. Structure (A) and NMR spectrum (B) of UI-EP001. Human SKBR3 (Her2+) and HCC-1806 (TNBC) breast cancer cells were exposed to vehicle (untreated) or 100 μM of partial PROTAC or E2β-PROTAC, UI-EP001 for 16 h. Cells were immunostained with GPER antibody for 30 min and surface GPER was detected using goat anti-rabbit Alexa594 IgG (red). Nuclei were counterstained with DAPI (blue). (C) HA-GPER or HA-β1AR cells were treated with UI-EP001 for the indicated times and immunoblotted with HA antibody (D) or immunostained for surface receptors (E). Total corrected red fluorescence (red) (TCRF) of treated and untreated HA-GPER or HA-β1AR cells was measured by subtracting the cellular background. Significant degradation of GPER was observed in the presence of E2-PROTAC, UI-EP001, and HA-GPER, but not in ΗA-β1AR cells (F). No differences in surface GPER expression were measured in cells similarly treated with free 17β-E2, data not shown. [Figure 19] Design of small molecule GPER-PROTACs. 17β-estradiol is conjugated via C6 or C-17 of its estrane ring to chemical linkers of various subunit lengths to the VHL-derived ubiquitin E3 ligase ligand (S,R,S)AHPC. [Figure 20] Synthesis of C-17 linked GPER-PROTACs. Partial PROTACs consisting of (S,R,S)-AHPC-PEG2-COOH; (S,R,S)-AHPCPEG4-COOH or (S,R,S)-AHPC-PEG6-COOH are linked via the 17β-OH group of 17β-estradiol by EDC coupling. [Figure 21] Synthesis of C-6 linked GPER-PROTACs. Partial PROTACs consisting of (S,R,S)-AHPC-PEG2-COOH; (S,R,S)-AHPC-PEG4-COOH or (S,R,S)-AHPC-PEG6-COOH are linked to the C-6 carbon atom in several steps as shown in the diagram above. [Figure 22] Detection of plasma membrane and intracellular GPER by NanoBiT luminescence assay, intact or detergent permeabilized. HEK293 cells or HiBiT-GPER-HEK293 cells were labeled with soluble LgBiT protein and luminescent substrate for 4 min at ambient temperature. Luminescence was measured from quadruplicate samples. Background from empty wells without cells was subtracted and reported as relative luminescence units (RLU). [Figure 23] Modeling of PROTACs on GPER and ERα. A) UI-EP001 (blue) and UI-EP002 (green) are bound within the GPER homology model and designed with linkers exiting the binding pocket at two different lengths. B) GPER binding pocket (grey surface) showing the exit of UI-EP001 (blue) and UI-EP002 (green) from between TM1 and TM7. C) UI-EP001 (blue) and UI-EP002 (green) bound within the ERα ligand domain of the ERα homodimer (PDB:1A52). D) ERα binding pocket (grey surface) showing the exit of UI-EP001 (blue) and UI-EP002 (green). [Figure 24]Synthesis of UI-EP001 and UI-EP002. Scheme 1: Synthesis of partial PROTAC (compound 7), reagents and conditions: (i) NaH, dioxane, rt, overnight; (ii) TFA / DCM, rt, 2 hours; (iii) Pd(OAc)2, KOAc, DMAc, 120°C, 24 hours; (iv) CoCl2, NaBH4, anhydrous methanol, 0°C to rt, 2 hours; (v) HBTU, DIPEA, anhydrous DMF, rt, overnight; (vi) 1. TFA / DCM, rt, 30 minutes; 2. Boc-Tle-OH, HBTU, DIPEA, anhydrous DMF, rt, overnight; (vii) compound 2, HBTU, DIPEA, rt, overnight. Scheme 2: Synthesis of UI-EP001 (compound 8). Reagents and conditions: (viii) DMAP, EDC, anhydrous DMF, rt, 48 hours. Scheme 3: Synthesis of UI-EP002 (compound 10). Reagents and conditions: (ix) Fmoc-NH-PEG-CH2CH2COOH, DMAP, EDC, anhydrous DMF, rt, 72 h; (x) 1. Et3N, anhydrous DMF; 2. compound 7, HATU, DIPEA, anhydrous DMF, rt, 48 h. [Diagram 25] E2-PROTACs act through high affinity binding to GPER and ER. A) Intact HEK-293 (ER-, GPER-), HA-GPER (ER-, GPER+), SKBR3 (ER-, GPER+) and MCF-7 (ER+, GPER+) cells were labeled with rabbit GPER antibody at 4°C. Surface GPER was then visualized with Alexa594-conjugated goat anti-rabbit IgG (red). B) Specific binding in intact SKBR3 cells calculated as a function of E2-FITC concentration. C) Specific binding of 17b-E2, UI-EP001, UI-EP002 and partial PROTACs in intact SKBR3 cells measured by competitive binding assay using E2-FITC as fluorotracer. D) Determination of total E2-FITC binding using 10 nM E2-FITC with increasing concentrations of cytosolic proteins prepared from MCF-7 cells. E) Fluorescence polarization assay using E2-FITC to assess the specific binding of E2, UI-EP001, UI-EP002, and partial PROTACs in cytosolic fractions prepared from MCF-7 cells. Means and SD are obtained from three independent experiments. [Figure 26] E2-PROTACs promote rapid loss of recombinant membrane and intracellular estrogen receptors. HEK-293 cells (ERα, GPER) were transiently transfected with 50 ng of HiBiT-GPER or HiBiT-ER and pcDNA3.1(+) ZeoCarrier plasmid. Concentration response curves of (A) HiBiT-GPER and (B) HiBiT-ERα were measured using a luminescent substrate in extracellular LgBiT and detergent-permeabilized cells after 1 h treatment with either increasing doses of UI-EP001 or UI-EP002. (C) Histograms showing total HiBiT-GPER and HiBiT-ER after 1 h treatment with 100 mM drug or control. Kinetics of (D) HiBiT-GPER and (E) HiBiT-ERα after incubation of cells with 100 μM UI-EP001, UI-EP002, and partial PROTACs for various incubation periods from 1 to 8 h. Results shown represent the mean ± SE of three independent experiments (***, P < 0.0004; ****, P < 0.0001; one-way ANOVA). [Figure 27]E2-PROTACs selectively reduce the expression of native and recombinant GPER. (A) HEK-293 cells stably expressing HA-GPER, HA-β1AR, or HA-CXCR4 were treated with 100 μM UI-EP001, UI-EP002, and partial PROTACs for 1 h. Fixed cells were permeabilized and labeled with rabbit HA antibody, and total receptors were visualized using Alexa Fluor594 anti-rabbit secondary antibody (red). (B) Corrected Total Red Fluorescence (CTRF) from images of HA-GPER, ΗA-β1AR, and HA-CXCR4 cells treated with either vehicle, UI-EP001, UI-EP002, and partial PROTACs was measured using Image J software from three different microscopic fields (***, P<0.0004; ****, P<0.0001; one-way ANOVA). (C) MCF-7 (ERa+, ERb+, PR+, GPER+) cells were incubated with vehicle, UI-EP001, UI-EP002, or partial PROTAC for 1 h at 37 °C, then immunostained with mouse ERα, rabbit ERβ, mouse PR, and rabbit GPER antibodies and detected with Alexa488-conjugated anti-mouse IgG or Alexa488-conjugated anti-rabbit IgG (green). (D) Quantification of the results from the images in (C) measured as CTRF. (E) SKBR3 (ERα+, ERb-, GPER+) cells were incubated with vehicle (1% DMSO), 100 uM UI-EP001, UI-EP002, or partial PROTAC for 1 h. Surface and intracellular GPER were visualized using rabbit GPER antibody and Alexa594-conjugated goat anti-rabbit IgG (red), respectively, in intact or detergent-permeabilized cells. [Figure 28]E2-PROTACs induce proteasome-dependent degradation of GPER and ERα. HEK-293 cells transiently transfected with (A) HiBiT-GPER and (B) HiBiT-ERα were treated with 100 μM UI-EP001 in the presence of increasing concentrations of E2β or aldosterone. Total receptor was then measured by binary luminescence complementation in detergent-permeabilized cells. (C) Effect of 100 μM UI-EP001 alone or in combination with 100 μM E2 or aldosterone in transiently transfected cells. **** indicates statistical significance, P<0.0001 or less. (D) Cells were treated with either (D) 10 μM MG132 or (E) 100 μM chloroquine for 1 h in the presence or absence of 100 μM UI-EP001 or UI-EP002. Total receptors were then quantified by binary luminescence complementation. Representative luminescence data from three independent experiments are shown, along with the mean and SD (***, p<0.0004; **, P<0.001; ns, not significant; one-way ANOVA). [Figure 29] E2-PROTACs induce estrogen receptor-specific cytotoxicity in human breast cancer cells. Cell viability of SKBR3, HCC-1806, MCF-7, and MDA-MB-231 breast cancer cells was measured after 24 h treatment with increasing concentrations of UI-EP001 or UI-EP002 or partial PROTACs. Results shown represent the mean ± SE of three independent experiments. [Diagram 30]E2-PROTACs promote estrogen receptor-dependent G2 / M arrest. E2-PROTACs induced G2 / M arrest in human breast cancer cell lines expressing membrane and / or intracellular estrogen receptors. SKBR3, HCC-1806, MCF-7, and MDA-MB-231 cells growing in 10% FBS were treated with 10 μM UI-EP001 or UI-EP002 or partial PROTACs for 24 h. Fixed cells were permeabilized and stained with propidium iodide (PI). Cell cycle data were acquired on a flow cytometer and analyzed using Flow-Jo software. Data are expressed as mean ± SEM (n=3). The percentage of cells in each phase was plotted using Prism 8.0. [Diagram 31] E2-FITC synthesis scheme. [Diagram 32] Exemplary structures. [Figure 33-1] NMR data. [Figure 33-2] NMR data. [Figure 33-3] NMR data. [Figure 33-4] NMR data. [Figure 33-5] NMR data. [Figure 33-6] NMR data. [Diagram 34] Gating strategy for cell cycle analysis. A first gate was applied to the scatter plot (A) to gate out debris. Once cells were identified, a second gate was applied to the single cell population using pulse treatment (pulse width vs. pulse area) (B) to exclude cell doublets from the analysis. From this cell population, the percentage of each subpopulation in each phase was determined by histogram plot (C). [Diagram 35] Nude mice challenged with MCF7 (100 μl IT treatment every 2 days (7×). [Diagram 36] Nude mice challenged with MCF7 (100 μl IT treatment every 2 days (7×). [Figure 37] Nude mice challenged with SKB3 (100 μl IT treatment every 2 days (7×). [Figure 38]CIMBA-PROTAC degrades recombinant GPER. HEK293 cells stably expressing recombinant HA-tagged GPER were treated with various concentrations of CIMBA-PROTAC-001 or partial PROTAC for 16 hours and then lysed with detergent. Equal amounts of protein lysates were separated on SDS-polyacrylamide gels and then electrotransferred to PVDF nylon membranes. The membranes were probed with rabbit HA antibody. The HA-antibody was stripped from the membrane at low pH, and the membranes were then probed with transferrin receptor (TFNR) antibody as a specificity and loading control and stripped. [Figure 39] Exemplary structures of GPER-PROTACs having 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline as the GPER binder (Formula (II) or (III)). [Figure 40-1] Ligands for exemplary E3 ligases. [Figure 40-2] Same as above. [Figure 40-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description Breast cancer is an estrogen-driven malignancy, and blocking estrogen action is highly effective against breast cancer and is much less toxic than other forms of cancer treatment. However, strategies that inhibit ER function and estrogen biosynthesis are suitable for use only in postmenopausal women with early stage ER-positive (ER+) breast cancer. Furthermore, drug resistance may develop and side effects may be severe (Rugo et al., 2016; Miller et al., 2013). Selective estrogen receptor degraders (SERDs), such as fulvestrant, are effective agents for the treatment of estrogen receptor (ER)-positive breast cancer, especially in patients who develop endocrine-resistant disease. SERDs function by binding to the ER and inducing an allosteric change that leads to its destruction at the 26S-proteasome. Fulvestrant, an ER degrader, is used as a second-line treatment for endocrine-resistant breast cancer, but its poor bioavailability requires painful monthly intramuscular injections (Nathan et al., 2017).

[0025] Estrogen acts through the orphan GPCR homolog GPR30 (also known as GPER) to trigger a Gbg subunit protein-dependent HB-EGF autocrine loop. This discovery provided the first mechanistic explanation for the EGF-like effects of estrogen. GPR30 is a Gs-coupled receptor that has specific estrogen binding and is coupled to adenylyl cyclase. Studies on GPER trafficking have shown that it is ubiquitinated at the plasma membrane and uses clathrin-mediated, b-arrestin-independent retrograde transport to the trans-Golgi network prior to proteasomal degradation.

[0026] Integrin a5b1 mediates transmembrane signaling in GPER-mediated EGFR transactivation. Estrogen action via GPER coordinately promotes fibronectin matrix assembly and EGF release, cellular actions related to cell survival. See, e.g., Filardo et al. (2000); Filardo et al. (2002); Pang et al. (2005).

[0027] GPER is expressed independently of ER in breast tumor specimens. Furthermore, GPER is directly associated with markers of aggressive breast cancer, inversely related to ER with these same prognostic variables. GPER is also associated with poor prognosis in female reproductive cancers. GPER is upregulated in cortical epithelium during estrus, and several groups have collaborated on the role of GPER in the hypothalamus and neural function of luteinizing hormone release. See, for example, Filardo et al. (2006); Noel et al. (2009); Cheng et al. (2014); and Waters et al. (2015).

[0028] Thus, as GPER expression is not restricted to ER+ breast cancers ( Prissnitz et al., 2015 ; Neklesa et al., 2017 ), GPER provides an alternative mechanism by which breast cancer responds to estrogen ( Ignatov et al., 2011 ).

[0029] A recent survey of 121 patients with TNBC suggests that more than 80% of these tumors express this receptor. The expression of GPER in the majority of TNBCs is supported by other smaller studies and contrasts with data from rather limited studies of tumors and cell lines suggesting that GPER is a tumor suppressor in TNBC. Unlike ER expression, which correlates inversely with clinical predictors of aggressive breast cancer, GPER expression is directly associated with these same variables, suggesting that it is involved in metastasis. Thus, the development of this type of therapeutic agent that selectively degrades GPER holds promise for the treatment of TNBC and may also be useful in endocrine-resistant breast cancer. Finally, GPER has been associated with aggressive disease and poor prognosis in gynecological cancers, suggesting that GPER-PROTACs may also benefit these patients.

[0030] Exemplary GPER-PROTACs Treatment algorithms for breast cancer are ER-centric and do not yet consider GPER for allocation of estrogen-targeted therapies, even though GPER is recognized by the International Union of Basic and Clinical Pharmacology (IUPHAR) (Ignatov et al., 2011). PROTACs are a new technology in the development of therapeutics, as they eliminate targets and thereby avoid the selection of drug resistance (Neklesa et al., 2017; Gu et al., 2018). ER-PROTACs are promising for ER-targeted therapy, as they show superior efficacy and improved bioavailability (Flanagan et al., 2018). ER-PROTACs have been developed with the aim of targeting the ER and consist of synthetic ERα-specific ligands (Flanagan et al., 2018; Tria et al., 2018). Therefore, they are not effective for targeting GPER. Although synthetic antagonists of GPER exist (Prissnitz et al., 2015) and show antitumor activity in animal models (Petrie et al., 2013), they are delivered intraperitoneally and chronically and do not overcome the potential development of resistance. PROTAC approaches targeting GPER are particularly attractive because downmodulation of GPER from the plasma membrane occurs via the ubiquitin-proteasome degradation pathway (Cheng et al., 2011).

[0031] ER and GPER bind a diverse group of endogenous, food-derived, and environmental estrogens (Prissnitz et al., 2015). The relative affinities of these receptors for individual estrogens vary, and these affinities cannot be compared because each receptor exists in a different physicochemical environment (Prissnitz et al., 2015; Filardo et al., 2012). Both ER and GPER bind 17α-E2 with high affinity. Therefore, we prepared GPER-PROTACs with 17α-E2 as a GPER targeting ligand. GPER-PROTACs bind the target (GPER ligand) to E3 ligase, which triggers GPER polyubiquitination and proteasomal degradation.

[0032] The G protein-coupled estrogen receptor (GPER) is a member of the GPCR superfamily. Following stimulation, GPER undergoes adaptive changes that lead to its desensitization and endocytosis. The 26S proteasome is the natural destination of endocrine GPER, making it suitable for targeting via PROTACs. GPER is directly associated with aggressive disease and poor prognosis in breast, endometrial, and ovarian cancers. As GPER is expressed in 97 of 121 (>80%) triple-negative breast cancers (TNBC), this breast cancer subtype lacking known therapeutic targets represents an ideal cancer for GPER-PROTAC therapy. Available ER-PROTACs are optimized to bind ERα-specific ligands and therefore do not target GPER. As described herein, GPER-PROTACs UI-EP001 and UI-EP00 promoted down-modulation of GPER in human breast cancer cells. GPER-PROTACs with altered chemical spacer composition and length showed increased potency [ 1 ] for GPER-specific binding. 3 This can be tested using a competitive radioreceptor binding assay with 3H]-17β-estradiol (17-β-E2).

[0033] The PROTACs with the highest relative binding affinity (RBA) for 17β-E2 were assayed for their ability to promote GPER degradation in breast cancer cells using quantitative immunofluorescence and immunochemistry assays. Confirmation of GPER ablation may be achieved using Nanobit™, a highly sensitive binary bioluminescence complementation assay. Polyubiquitination of GPER is confirmed by tandem ubiquitin-binding element (TUBE) assay, and proteasome-specific degradation is determined using the proteasome inhibitor MG132. The ability of GPER to transactivate the erbB1 to erk signaling axis is also assessed.

[0034] The anti-carcinogenic effect, biodistribution, and toxicity of GPER-PROTACs are tested based on their ability to delay TNBC recurrence and metastasis using BRCA-1 mutant mice, which are widely used as a TNBC carcinogenesis model. Orally delivered small molecule PROTACs that can inactivate GPER in breast cancer are identified. These small molecules are useful for the treatment of other cancers where inactivation of GPER is desired, such as ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, or colon cancer.

[0035] Exemplary Formulations The disclosed GPER-PROTACs can be delivered in biodegradable particles that can include or be formed from biodegradable polymer molecules, such as polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (i.e., polylactic-co-glycolic acid (PLGA)), poly-ε-caprolactone (PCL), polyethylene glycol (PEG), poly(3-hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoesters), polyol / diketene acetal addition polymers, poly-alkyl-cyano-acrylates (PAC), poly(sebacic acid free), poly(sodium EDTA ... The polymerizable composition may include, but is not limited to, poly(carboxybiscarboxyphenoxyphenoxyhexone (PCPP) poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of PSA, PCPP and PCPM, poly(amino acids), poly(pseudoamino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] and poly[(organo)phosphazenes], polyhydroxybutyric acid, or S-caproic acid, elastin, or gelatin. (See, e.g., Kumari et al., Colloids and Surfaces B: Biointerfaces 75 (2010) 1-18; and U.S. Patent Nos. 6,913,767; 6,884,435; 6,565,777; 6,534,092; 6,528,087; 6,379,704; 6,309,569; 6,264,987; 6,210,707; 6,090,925; 6,022,564; 5,981,719 See U.S. Patent Application Publication Nos. 5,871,747, 5,723,269, 5,603,960, and 5,578,709; and U.S. Patent Application Publication Nos. 2007 / 0081972; and WO 2012 / 115806; and WO 2012 / 054425, the contents of which are incorporated herein by reference in their entireties.

[0036] The particles can be prepared by methods known in the art. (See, for example, Nagavarma et al., Asian J. of Pharma. And Clin. Res., Vol 5, Suppl 3, 2012, pp. 16-23; Cismaru et al., Rev. Roum. Chim., 2010, 55(8), 433-442; and WO 2012 / 115806; and WO 2012 / 054425, the contents of which are incorporated herein by reference in their entirety). Suitable methods for preparing particles can include methods that utilize preformed polymer dispersions, and can include, but are not limited to, solvent evaporation, nanoprecipitation, emulsification / solvent diffusion, salting out, dialysis, and supercritical fluid techniques. In some embodiments, the particles can be prepared by forming a double emulsion (e.g., water-in-oil-in-water) followed by solvent evaporation. The particles obtained by this method may be optionally subjected to further processing steps such as washing and lyophilization. Optionally, the particles may be combined with a preservative (e.g., trehalose).

[0037] Typically, the particles have an average effective diameter of less than 1 micron, for example, the particles have an average effective diameter of about 25 nm to about 500 nm, e.g., about 50 nm to about 250 nm, about 100 nm to about 150 nm, or about 450 nm to 650 nm, or about 25 μm to about 500 μm, e.g., about 50 μm to about 250 μm, about 100 μm to about 150 μm, or about 450 μm to about 650 μm. The size of the particles (e.g., average effective diameter) can be assessed by methods known in the art, including, but not limited to, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), photon correlation spectroscopy (PCS), nanoparticle surface area monitor (NSAM), condensation particle counter (CPC), differential mobility analyzer (DMA), scanning mobility particle measurer (SMPS), nanoparticle tracking analysis (NTA), X-ray diffraction (XRD), aerosol time-of-flight mass spectrometry (ATFMS), and aerosol particle mass analyzer (APM).

[0038] In one embodiment, the delivery vehicle comprises a polymer, including but not limited to poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), linear and / or branched PEI of different molecular weights (e.g., 2, 22 and 25 kDa), dendrimers such as polyamidoamine (PAMAM) and polymethacrylate; a liposome, an emulsion, a lipid, including but not limited to DOTAP, DOTMA, DMRIE, DOSPA, distearoylphosphatidylcholine (DSPC), DOPE, or DC-cholesterol; a peptide-based vector, including but not limited to poly-L-lysine or protamine; or a poly(β-amino ester), chitosan, PEI-polyethylene glycol, PEI-mannose-dextrose, or DOTAP-cholesterol.

[0039] In one embodiment, the delivery vehicle is a glycopolymer-based delivery vehicle, poly(glycamidoamine) (PGAA). These materials are made by polymerizing methyl ester or lactone derivatives of various carbohydrates (D-glucarate (D), mesogalactarate (G), D-mannarate (M), and L-tartrate (T)) with a series of oligoethyleneamine monomers (containing one to four ethyleneamines (Liu and Reineke, 2006). A subset of these carbohydrates and four ethyleneamines in the polymer repeat unit can be used.

[0040] In one embodiment, the delivery vehicle of GPER-PROTAC comprises polyethyleneimine (PEI), polyamidoamine (PAMAM), PEI-PEG, PEI-PEG-mannose, dextran-PEI, OVA conjugate, PLGA microparticles, or PLGA microparticles coated with PAMAM, or any combination thereof. The polymer may include, but is not limited to, polyamidoamine (PAMAM) dendrimers. Polyamidoamine dendrimers suitable for preparing nanoparticles of the present disclosure may include third, fourth, fifth, or at least sixth generation dendrimers.

[0041] In one embodiment, the delivery vehicle is a lipid, such as N-[1-(2,3-dioleoyloxy)proper]-N,N,N-trimethylammonium (DOTMA), 2,3-dioleyloxy-N-[2-sperminecarboxamido]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA, Lipofectamine); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); N-[1-(2,3-dimyristloxy)propyl]; N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 3-β-[N-(N,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol); dioctadecylamidoglyceryl spermine (DOGS, Transfectam); or imethyldioctadeclyammonium bromide (imethyldioctadecylammonium bromide). Cationic lipids include pyridinium dihydrochloride (DDAB). The positively charged hydrophilic head group of cationic lipids usually consists of monoamines such as tertiary and quaternary amines, polyamines, amidinium, or guanidinium groups. A series of pyridinium lipids have been developed (Zhu et al., 2008; van der Woude et al., 1997; Ilies et al., 2004). In addition to pyridinium cationic lipids, other types of heterocyclic head groups include imidazoles, piperidines, and amino acids. The main function of the cationic head group is to condense negatively charged nucleic acids into slightly positively charged nanoparticles through electrostatic interactions, facilitating cellular uptake and endosomal escape.

[0042] Lipids with two linear fatty acid chains, such as DOTMA, DOTAP, and SAINT-2, or DODAC, can be used as delivery vehicles, as can a tetraalkyl lipid chain surfactant, which is a dimer of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). All trans-oriented lipids are available in a variety of sizes depending on their hydrophobic chain length (C 16:1 , C 18:1 , and C 20:1), appear to enhance transfection efficiency compared to their cis -oriented counterparts.

[0043] Polymeric structures useful as delivery vehicles include, but are not limited to, linear polymers such as chitosan and linear poly(ethyleneimine), branched polymers such as branched poly(ethyleneimine) (PEI), cyclic polymers such as cyclodextrin, network (crosslinked) type polymers such as crosslinked poly(amino acids) (PAA), and dendrimers. Dendrimers are composed of a central core molecule from which several highly branched arms "grow" to form symmetrical or asymmetrical tree-like structures. Examples of dendrimers include polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers.

[0044] DOPE and cholesterol are neutral co-lipids commonly used in the preparation of liposomes. Branched PEI-cholesterol water-soluble lipopolymer complexes self-assemble into cationic micelles. Non-ionic polymers Pluronic® (poloxamers) and SP1017, a combination of Pluronic® L61 and F127, can also be used.

[0045] In one embodiment, PLGA particles are used to increase encapsulation frequency, although complexation with PLL may also improve encapsulation efficiency. Other materials, such as PEI, DOTMA, DC-Chol, or CTAB, can be used.

[0046] In one embodiment, the GPER-PROTAC is embedded in or applied to a material including, but not limited to, a hydrogel of poloxamer, polyacrylamide, poly(2-hydroxyethyl methacrylate), carboxyvinyl polymers (e.g., Carbopol® 934, Goodrich Chemical Co.), cellulose derivatives such as methylcellulose, cellulose acetate and hydroxypropylcellulose, polyvinylpyrrolidone or polyvinyl alcohol, or combinations thereof.

[0047] In some embodiments, the biocompatible polymeric material is derived from a biodegradable polymer such as collagen, e.g., hydroxylated collagen, fibrin, polylactic-polyglycolic acid, or polyanhydrides. Other examples include, but are not limited to, any biocompatible polymer, such as ethylene vinyl acetate copolymer (EVA), polymethyl methacrylate, polyamide, polycarbonate, polyester, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, N-isopropylacrylamide copolymer, poly(ethylene oxide) / poly(propylene oxide) block copolymer, poly(ethylene glycol) / poly(D,L-lactide-co-glycolide) block copolymer, polyglycolide, polylactide (PLLA or PDLA), poly(caprolactone) (PCL), or poly(dioxanone) (PPS), which may be hydrophilic, hydrophobic, or amphiphilic.

[0048] In another embodiment, the biocompatible material includes polyethylene terephthalate, polytetrafluoroethylene, copolymers of polyethylene oxide and polypropylene oxide, combinations of polyglycolic acid and polyhydroxyalkanoates, gelatin, alginate, poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, and polyhydroxyoctanoate, and polyacrylonitrile polyvinyl chloride.

[0049] In one embodiment, the following polymers can be used, such as natural polymers of starch, chitin, glycosaminoglycans, such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate, and microbial polyesters, such as hydroxyalkanoates, such as hydroxyvalerate and hydroxybutyrate copolymers, and synthetic polymers, including poly(orthoesters) and polyanhydrides, and homo- and copolymers of glycolide and lactide (e.g., poly(L-lactide, poly(L-lactide-co-D,L-lactide), poly(L-lactide-co-glycolide, polyglycolide and poly(D,L-lactide), poly(D,L-lactide-coglycolide), poly(collidine lactate), and polycaprolactone).

[0050] In one embodiment, the biocompatible material is derived from isolated extracellular matrix (ECM). ECM can be isolated from various cell populations, tissues and / or endothelial layers of organs, such as any organ or tissue source, including the dermis of the skin, liver, digestive, respiratory, intestinal, urinary or reproductive tract of a warm-blooded vertebrate. The ECM used in the present invention can be derived from a combination of multiple sources. The isolated ECM can be prepared into sheets, particle forms, gel forms, etc.

[0051] The biocompatible polymer may include silk, elastin, chitin, chitosan, poly(d-hydroxy acid), poly(anhydride), or poly(orthoester). More specifically, the biocompatible polymer may be polyethylene glycol, poly(lactic acid), poly(glycolic acid), copolymers of lactic acid and glycolic acid, copolymers of lactic acid, glycolic acid, and polyethylene glycol, poly(E-caprolactone), poly(3-hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoester), polyol / diketene acetal addition polymer, poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxyhexone (PCPP), poly(carboxyphenyl)-1,1-dihydropyridine, poly(hydroxy ... They may be formed from poly[bis(p-carboxyphenoxy)methane] (PCPM), SA, copolymers of CPP and CPM, poly(amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] or poly[(organo)phosphazenes], poly-hydroxybutyric acid, or S-caproic acid, polylactide-co-glycolide, polylactic acid, polyethylene glycol, cellulose, oxidized cellulose, alginate, gelatin or derivatives thereof.

[0052] Thus, the polymer may be formed from any of a wide range of materials, including polymers, including naturally occurring polymers, synthetic polymers, or combinations thereof. In one embodiment, the scaffold comprises a biodegradable polymer. In one embodiment, a naturally occurring biodegradable polymer can be modified to provide a synthetic biodegradable polymer derived from a naturally occurring polymer. In one embodiment, the polymer is poly(lactic acid) ("PLA") or poly(lactic-co-glycolic acid) ("PLGA"). In one embodiment, scaffold polymers include, but are not limited to, alginate, chitosan, poly(2-hydroxyethyl methacrylate), xyloglucan, copolymers of 2-methacryloyloxyethyl phosphorylcholine, poly(vinyl alcohol), silicone, hydrophobic and hydrophilic polyesters, poly(lactide-co-glycolide), N-isopropylacrylamide copolymers, poly(ethylene oxide) / poly(propylene oxide), polylactic acid, poly(orthoesters), polyanhydrides, polyurethanes, copolymers of 2-hydroxyethyl methacrylate and sodium methacrylate, phosphorylcholine, cyclodextrins, polysulfones and polyvinylpyrrolidine, starch, poly-D,L-lactic acid-para-dioxanone-polyethylene glycol block copolymers, polypropylene, poly(ethylene terephthalate), poly(tetrafluoroethylene), poly-ε-caprolactone, or crosslinked chitosan hydrogels.

[0053] A number of liposomal delivery systems can be used in formulations with GPER-PROTACs. Virtually any lipid can be used to form the liposomes. Exemplary lipids for use include, for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-[phosphol-L-serine] (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:1 PEG-2000PE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PEG-2000PE), 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] lauroyl}-sn-glycero-3-phosphocholine (18:1-12:0 NBD PC), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] lauroyl}-sn-glycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol, and mixtures / combinations thereof. Although cholesterol is not technically a lipid, it is presented as a lipid for the purposes of certain embodiments in view of the fact that, according to certain embodiments, cholesterol may be an important component of the lipid bilayer of protocells. In many cases, cholesterol is incorporated to increase the structural integrity of the bilayer. All of these lipids are readily commercially available from Avanti Polar Lipids (Alabaster, Alabama, USA).

[0054] Pharmaceutical Compositions The present disclosure provides compositions comprising, consisting essentially of, or consisting of at least one GPER-PROTAC and a pharma- ceutically acceptable (e.g., physiologically acceptable) carrier. In one embodiment, when the composition consists essentially of at least one GPER-PROTAC and an optional pharma- ceutically acceptable carrier, additional components that do not substantially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.) can be included. In one embodiment, when the composition consists of at least one GPER-PROTAC encapsulated in a particle and a pharma- ceutically acceptable carrier, the composition does not include additional components. Any suitable carrier can be used as part of the present invention, and such carriers are well known in the art. The choice of carrier is determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions may be produced according to conventional techniques, for example as described in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0055] Suitable formulations for the compositions include aqueous and non-aqueous solutions, isotonic sterile solutions, which may contain antioxidants, buffers, and bacteriostatic agents, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be provided in unit-dose or multi-dose sealed containers, such as ampoules or vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, e.g., water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type described above. In one embodiment, the carrier is a buffered saline solution. In one embodiment, the GPER-PROTAC is administered in a composition formulated to protect the GPER-PROTAC from damage prior to administration. For example, the composition may be formulated to reduce loss of GPER-PROTAC in equipment, e.g., glassware, syringes, or needles, used to prepare, store, or administer the GPER-PROTAC. The composition may be formulated to reduce the light sensitivity and / or temperature sensitivity of the GPER-PROTAC. To this end, the compositions may include, for example, a pharma- ceutically acceptable liquid carrier, such as those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.

[0056] The composition may also be formulated to enhance transduction efficiency. Additionally, one skilled in the art will appreciate that at least one GPER-PROTAC may be present in the composition with other therapeutic or bioactive agents. For example, factors that control inflammation, such as ibuprofen or steroids, may be part of the composition to reduce swelling and inflammation. Immune system stimulants or adjuvants, such as interleukins, lipopolysaccharides, and double-stranded RNA, may also be administered. Antibiotics, i.e., bactericides and antifungals, may be present to treat existing infections and / or reduce the risk of future infections.

[0057] Injectable depot preparations are made by forming at least one GPER-PROTAC with a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of GPER-PROTAC to polymer and the nature of the particular polymer used, the release rate of GPER-PROTAC can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injections are also optionally prepared by encapsulating GPER-PROTAC in a complex with a polymer in liposomes or microemulsions that are compatible with body tissues.

[0058] In certain embodiments, the formulation comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic acid esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

[0059] The composition can be administered in or on a device that allows for controlled or sustained release, such as a sponge, a biocompatible mesh, a mechanical reservoir, or a mechanical implant. Implantable devices, such as implants (see, e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No. 4,863,457), for example mechanical reservoirs or implants or devices composed of polymeric compositions, are particularly useful for administering GPER-PROTACs. The composition can also be administered in the form of sustained release formulations (see, e.g., U.S. Pat. No. 5,378,475), including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphate esters, such as bis-2-hydroxyethyl terephthalate (BHET), and / or polylactic-glycolic acid.

[0060] The dose of GPER-PROTAC in the composition administered to the mammal depends on many factors, including the size (mass) of the mammal, the extent of side effects, the particular route of administration, etc. In one embodiment, the method includes administering a "therapeutically effective amount" of a composition comprising a GPER-PROTAC as described herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the extent of the disease or disorder, the age, sex, and weight of the individual, and the ability of the GPER-PROTAC to induce the desired response in the individual.

[0061] In one embodiment, the composition is administered once to the mammal. It is believed that a single administration of the composition can result in sustained expression in the mammal with minimal side effects. However, in some cases, it may be appropriate to administer the composition multiple times during the treatment period to ensure sufficient exposure of cells to the composition. For example, the composition can be administered to the mammal more than once (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 times or more) during the treatment period.

[0062] The present disclosure provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of at least one GPER-PROTAC. Route of administration, dosage and dosage form Administration of the GPER-PROTAC may be continuous or intermittent, depending, for example, on the physiological state of the recipient and other factors known to those skilled in the art. Administration of the GPER-PROTAC may be essentially continuous over a preselected period of time, or may be a series of spaced doses. Both local administration, e.g., intranasal or intrathecal, and systemic administration are contemplated. Any route of administration may be used, e.g., intravenous, intranasal or oral, or topical administration.

[0063] One or more suitable unit dosage forms containing the GPER-PROTAC can be optionally formulated for sustained release and can be administered by a variety of routes, including topically, for example intrathecally, orally, or parenterally, including via rectal, buccal, vaginal and sublingual, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic or pulmonary routes. The formulations may, where appropriate, be conveniently provided in discrete unit dosage forms, which may be prepared by any method well known in the art of pharmacy. Such methods may include the step of combining the vector with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, where appropriate, introducing or shaping the product into the desired delivery system.

[0064] The amount of GPER-PROTAC administered to achieve a particular result will vary depending on a variety of factors, including but not limited to the condition, patient-specific parameters such as height, weight, age, and whether the goal is prevention or treatment.

[0065] The GPER-PROTAC may conveniently be provided in the form of a formulation suitable for administration. The appropriate mode of administration will best be determined by the physician for each individual patient, following standard procedures. Suitable pharmaceutically acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceuticals Sciences. By "pharmaceutically acceptable" it is meant a carrier, diluent, excipient, and / or salt that is compatible with the other components of the formulation and not deleterious to the recipient thereof.

[0066] GPER-PROTAC may be formulated into a solution at neutral pH, e.g., about pH 6.5 to about pH 8.5, or about pH 7 to 8, with excipients to make the solution approximately isotonic, e.g., 4.5% mannitol or 0.9% sodium chloride, pH buffering with buffers known in the art, such as sodium phosphate, generally regarded as safe, and with accepted preservatives, such as 0.1% to 0.75% meta-cresol, or 0.15% to 0.4% meta-cresol. Obtaining the desired isotonicity can be accomplished using sodium chloride or other pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is useful in buffers that contain sodium ions. Solutions of the above compositions may also be prepared to enhance shelf life and stability, if necessary. Therapeutically useful compositions may be prepared by mixing the ingredients according to generally accepted procedures. For example, selected components can be mixed to produce a concentrated mixture and then adjusted to a final concentration and viscosity by adding water and / or a buffer to control pH or additional solutes to control tonicity.

[0067] The GPER-PROTAC may be provided in a dosage form containing an amount effective for single or multiple administrations. The GPER-PROTAC may be administered at a dose of at least about 0.0001 mg / kg to about 1 mg / kg, at least about 0.001 mg / kg to about 0.5 mg / kg, at least about 0.01 mg / kg to about 0.25 mg / kg, or at least about 0.01 mg / kg to about 0.25 mg / kg body weight, although other doses may provide beneficial results. The dosage will vary depending on a variety of factors, including, but not limited to, the disease, weight, condition, health, and / or age of the mammal. Such factors can be readily determined by the clinician using animal models or other test systems available in the art. As stated above, the exact dose to be administered is determined by the attending physician, but may be contained in 1 mL of phosphate buffered saline. In one embodiment, individual or divided doses of 0.0001 to 1 mg or more, for example up to 1 g, for example 0.001 to 0.5 mg, or 0.01 to 0.1 mg of GPER-PROTAC may be administered.

[0068] By way of example, liposomes and other lipid-containing complexes can be used to deliver one or more GPER-PROTACs. Pharmaceutical formulations containing GPER-PROTAC can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the agent can be formulated with common excipients, diluents, or carriers and formed into tablets, capsules, suspensions, powders, etc. GPER-PROTAC can also be formulated as elixirs or solutions suitable for parenteral administration, for example, by intramuscular, subcutaneous, or intravenous routes.

[0069] The pharmaceutical formulations can also be in the form of an aqueous or anhydrous solution, for example a lyophilized formulation or dispersion, or in the form of an emulsion or suspension. In one embodiment, the vector may be formulated for administration, for example, by injection, e.g., bolus injection or continuous infusion via a catheter, and may be provided in unit dosage form in ampoules, prefilled syringes, small volume infusion containers, or multi-dose containers with added preservatives. The active ingredient may take such forms as a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0070] These formulations can contain pharma- ceutically acceptable vehicles and adjuvants well known in the art. For example, it is possible to prepare solutions using one or more organic solvents that are physiologically acceptable.

[0071] For administration to the upper (nasal) or lower respiratory tract by inhalation, the vector is conveniently delivered from an inhaler, nebulizer or pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0072] Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder, for example a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, for example, in capsules or cartridges or, for example, gelatin or blister packs from which the powder may be administered with the aid of an inhaler, insufflator or metered dose inhaler.

[0073] For intranasal administration, the GPER-PROTAC can be administered via nose drops, liquid spray, such as a plastic bottle atomizer, or a metered dose inhaler, representative of which are the Mistometer (Wintrop) and Mezihaler (Riker).

[0074] Localized delivery can also be by using various techniques, such as catheters or needles, to administer the GPER-PROTAC at or near the disease site. Examples of site-specific or targeted localized delivery techniques are not intended to be limiting, but are illustrative of available techniques. Examples include injection or indwelling catheters, localized delivery catheters such as needle injection catheters, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application.

[0075] The formulations and compositions described herein may also include other ingredients, such as antimicrobial agents or preservatives. subject The subject can be any animal, including humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, specifically non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. The subject can also be livestock, such as cows, pigs, sheep, poultry, and horses, or pets, such as dogs and cats.

[0076] The subject includes a human subject suffering from or at risk of suffering from oxidative damage. The subject will generally be diagnosed with a condition of the present invention by a person of ordinary skill in the art, such as a physician. The methods described herein can be used with subjects of any species, sex, age, ethnic group, or genotype.Thus, the term subject includes males and females, and includes elderly people, subjects in the transition age from elderly to adulthood, adults, subjects in the transition age from adulthood to minors, and minors, including adolescents, children, and infants.

[0077] Examples of human ethnic groups include Caucasians, Asians, Hispanics, Africans, African-Americans, Native Americans, Semitic peoples, and Pacific Islanders. The methods of the present invention may be more suitable for some ethnic groups, such as Caucasians, particularly Northern Europeans, and Asians.

[0078] The term subject also includes subjects of any genotype or phenotype, as required by the present invention, as described above. Furthermore, a subject can have any genotype or phenotype of hair color, eye color, skin color, or any combination thereof.

[0079] The term subject includes subjects of any height, weight, or size or shape of any organ or body part. Exemplary Linkers As used herein, the terms "linking group", "linker molecule", "linker" and the like refer to any group of molecules useful for linking at least two separate chemical entities. To effect a bond between chemical entities, it is necessary that each of the reactants contain chemically complementary reactive groups. Examples of complementary reactive groups are amino and carboxyl groups to form amide bonds, carboxy and hydroxy groups to form ester bonds, amino and alkyl halides to form alkylamino bonds, thiols and thiols to form disulfides, or thiols and maleimides or alkyl halides to form thioethers. Hydroxyl, carboxyl, amino, and other functional groups can be introduced by known methods if not already present. If necessary, one or more of the reactive complementary groups can be "protected", in which case the protected reactive groups must be "deprotected" prior to carrying out the chemical reactions necessary to effect the particular conjugation chemistry. Any suitable protection / deprotection scheme can be used in the particular situation. As will be appreciated by those skilled in the art, any suitable molecular group can be used as a linker, and while which molecular group is suitable for a particular situation may vary, it is readily within the skill of the art to select or prepare a suitable molecular group with suitable chemically complementary reactive groups to effect the desired bond. Regardless of the molecular group selected in a particular environment, a stable covalent bond between different chemical entities can be provided to form a conjugate according to the present invention. Specifically, the covalent bond must be stable to the solution conditions to which the linker and linking group are exposed. Generally, linkers of any suitable length or configuration can be used, but linkers containing about 4-80 carbons, preferably about 10-70 carbons, about 10-50 carbons, or about 10-30 carbons, or about 10-20 carbons are contemplated. The linker may also contain one or more heteroatoms (e.g., N, O, S, and P), particularly 0-10 heteroatoms, in the molecular linking group. In various embodiments, the linker can include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 heteroatoms.In yet further embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 divalent oxygen (-O-), divalent nitrogen (-NH-), or both. The molecular linking group can be branched or linear. It will also be understood that in some cases, the conjugate can be formed directly between the purine analog and the targeting moiety or specific binding molecule. In this case, no linker is used. In such cases, the substituents of the purine analog and the specific binding molecule are typically derivatized to provide the complementary reactive groups (one or more of which may be protected, if appropriate) required to carry out the appropriate chemical reaction to link different chemical entities.

[0080] The length of the linker chain in atoms can be determined based on counting the atoms along the linear backbone of the linker from the G protein-coupled estrogen receptor (GPER) ligand to the E3 ubiquitin ligase ligand, but does not include atoms corresponding to atoms of the unlinked GPER ligand or E3 ubiquitin ligase ligand. For example, if β-estradiol is the GPER ligand, then none of the oxygens of β-estradiol are counted when counting the atoms of the linker chain. In yet another embodiment, the linker has a chain length in the backbone that is about, equal to, or greater than the following number of atoms when counted along a linear path between (but not including) the GPER ligand and the E3 ubiquitin ligase ligand: 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, up to 200. In further embodiments, the chain length is no more than 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 atoms. The length of the linker can also be expressed in angstroms based on the calculation of the bond length in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand, including the bonds connecting the linker and each of the ligands. In various further embodiments, the linker can have the above calculated length, when calculated based on the addition of the bond lengths in a linear path between the GPER ligand and the E3 ubiquitin ligase ligand, that is about, equal to, or greater than: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 angstroms, up to 300 angstroms. In further embodiments, the linker has a calculated length of less than or equal to 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 angstroms.

[0081] Non-limiting examples of linkers useful for GPER-PROTACs include oxygen, sulfur, nitrogen and / or carbon atoms (and appropriately added hydrogen atoms if necessary to satisfy valences), as well as linkers with side chains that enhance solubility, such as morpholino, piperidino, pyrrolidino, or piperazino rings; amino acids, polymers of amino acids (proteins or peptides), such as dipeptides or tripeptides; carbohydrates (polysaccharides), nucleotides, such as PNA, RNA, and DNA; polymers of organic materials, such as polyethylene glycol, polylactides, and the like. In one embodiment, the linker can be a divalent aryl or heteroaryl, bis-amidoaryl, bis-amidoheteroaryl, bis-hydrazidearyl, bis-hydrazideheteroaryl, and the like. In one embodiment, the linker has a chain having from about 24 to 50 atoms, where the atoms are selected from the group consisting of carbon, nitrogen, sulfur, non-peroxide oxygen, and phosphorus. In one embodiment, the linker has a chain having from about 4 to about 12 to 20 atoms, or from about 16 to about 48 atoms.

[0082] In one embodiment, the linker is an alkylene linker, a phenylene linker, or an alkyl linker. 48 These linkers may include a carbonyl group. In certain embodiments, the linker may be a -C(Y')(Z')-C(Y"(Z")- linker, where Y', Y", Z' and Z" are each independently hydrogen C1-C 10 Alkyl, substituted C1-C 10 Alkyl, C1-C 10 Alkoxy, substituted C1-C 10 Alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C 10 Aryl, substituted C5-C 10Aryl, C5-C9 heterocycle, substituted C5-C9 heterocycle, C1-C6 alkanoyl, Het, HetC1-C6 alkyl, or C1-C6 alkoxycarbonyl, and the substituents in the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl, or heterocycle are hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocycle, C1-6 alkoxy C 1~6 In one embodiment, the linker is a chain in which the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, and any carbon atom can be substituted, for example, with oxo, and any sulfur atom can be substituted with one or two oxo groups. The chain may be interspersed with one or more cycloalkyl, aryl, heterocyclyl, or heteroaryl rings. In one embodiment, the linker comprises a chain having about 4 to about 50 atoms in the chain, and the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, and any carbon atom can be substituted with oxo, and any sulfur atom can be substituted with one or two oxo groups.

[0083] In one embodiment, the linker is -(Y) y -, -(Y) y -C(O)N-(Z) z -, -(CH2) y -C(O)N-(CH2) z -, -(Y) y -NC(O)-(Z) z -, -(CH2) y -NC(O)-(CH2) z each y (subscript) and z (subscript) is independently 0 to 20; each Y and Z is independently C1 to C 10 Alkyl, substituted C1-C 10 Alkyl, C1-C 10 Alkoxy, substituted C1-C 10 Alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C 10 Aryl, substituted C5-C10 Aryl, C5-C9 heterocycle, substituted C5-C9 heterocycle, C1-C6 alkanoyl, Het, HetC1-C6 alkyl, or C1-C6 alkoxycarbonyl, and the substituents in the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl, or heterocycle are hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocycle, C 1~6 Alkoxy C 1~6 alkenyl, amino, cyano, halogen, or aryl. In certain embodiments, the linker may be a -C(Y')(Z')-C(Y"(Z")- linker, where Y', Y", Z', and Z" are each independently hydrogen C1-C 10 Alkyl, substituted C1-C 10 Alkyl, C1-C 10 Alkoxy, substituted C1-C 10 Alkoxy, C3-C9 cycloalkyl, substituted C3-C9 cycloalkyl, C5-C 10 Aryl, substituted C5-C 10 Aryl, C5-C9 heterocycle, substituted C5-C9 heterocycle, C1-C6 alkanoyl, Het, HetC1-C6 alkyl, or C1-C6 alkoxycarbonyl, and the substituents in the alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, Het, aryl, or heterocycle are hydroxyl, C1-C 10 Alkyl, hydroxyl C1-C 10 Alkylene, C1-C6 alkoxy, C3-C9 cycloalkyl, C5-C9 heterocycle, C1-6 alkoxy C 1~6 It is alkenyl, amino, cyano, halogen or aryl.

[0084] In one embodiment, the linker is an amide linking group (e.g., -C(O)NH- or -NH(O)C-); an alkylamino linking group (e.g., -C1-C6 alkyl-C(O)NH-, -C1-C6 alkyl-NH(O)C-, -C(O)NH-C1-C6 alkyl-, -NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-C(O)NH-C1-C6 alkyl-, or -C(O)NH-(CH2) t -, where t is 1, 2, 3, or 4; a substituted 5- or 6-membered ring (e.g., an aryl ring, a heteroaryl ring (e.g., tetrazole, pyridyl, 2,5-pyrrolidinedione (e.g., 2,5-pyrrolidinedione substituted with a substituted phenyl moiety)), a carbocyclic ring, or a heterocyclic ring) or an oxygen-containing moiety (e.g., -O-, -C1-C6 alkoxy).

[0085] In one embodiment, the linker is a polyethylene glycol (PEG) moiety (R 3 ) r Including R 3 is a PEG unit and r is from about 1 to about 10 (e.g., r is from about 2 to about 6). 3 is -O-CH-CH- or -CH-CH-O-. In some embodiments, R 3 is -O-CH2-CH2- or -CH2-CH2-O-, and r is from about 1 to about 100 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100). In certain related embodiments, r is from about 5 to about 25, from about 10 to about 50, from about 5 to about 15, from about 12 to about 35, from about 25 to about 55, or from about 65 to about 95. In some embodiments, (R 3 ) r The substituents are linear and in certain embodiments, (R 3 ) r The substituents are branched. For linear moieties, s may be smaller than r (e.g., R 3In some embodiments, R 3 is a linear PEG moiety (eg, having from about 1 to about 30 PEG units).

[0086] Other linkers include, but are not limited to, those in WO 19 / 123367 and Sun et al. (Signal Transd. Targeted Ther., 4:64 (2019)), the disclosures of which are incorporated herein by reference.

[0087] As used herein, the terms "alkyl," "alkenyl," and "alkynyl" may include linear, branched, and cyclic monovalent hydrocarbyl radicals, and combinations thereof, which, if unsubstituted, contain only C and H. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The total number of carbon atoms in each such group may be listed herein, for example, 1-10C or C1-C if the group can contain up to 10 carbon atoms. 10 Or C 1~10 When heteroatoms (typically N, O, and S) can replace carbon atoms, such as in a heteroalkyl group, for example, even though the example still states C1-C6, the number describing the group represents the sum of the number of carbon atoms in the group and the number of heteroatoms included as replacements for carbon atoms in the backbone of the described ring or chain.

[0088] Typically, the alkyl, alkenyl and alkynyl substituents of the present invention contain one 10C (alkyl) or two 10C (alkenyl or alkynyl). For example, one 8C (alkyl) or two 8C (alkenyl or alkynyl). In some cases, one 4C (alkyl) or two 4C (alkenyl or alkynyl) are included. A single group can contain more than one type of multiple bond, or more than one multiple bond. Such groups are included within the definition of the term "alkenyl" if they contain at least one carbon-carbon double bond, and are included within the scope of the term "alkynyl" if they contain at least one carbon-carbon triple bond.

[0089] Alkyl, alkenyl, and alkynyl groups are often optionally substituted to the extent that is chemically justifiable. Exemplary substituents include, but are not limited to, halo, =O, =N-CN, =N-OR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRS02R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 heteroaryl ... 10 Aryl or C5-C 10 heteroaryl, each R is optionally substituted with halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'S02R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, and each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C8 heteroaryl ... 10 Aryl or C5-C 10 Heteroaryl. The alkyl group, alkenyl group and alkynyl group are C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10Heteroaryls can also be substituted, each of which can be substituted with substituents suitable for that particular group.

[0090] An "acetylene" substituent can include an optionally substituted 2-10C alkynyl group of the formula -C≡C-Ri, where Ri is H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C8 aryl ... 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Aryl alkyl or C6-C 12 heteroarylalkyl, each R group is optionally substituted with one or more substituents selected from halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'S02R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, where each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C6 heteroaryl ... 10 Aryl, C5-C 10 Heteroaryl, C 7~12 Aryl alkyl or C 6~12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O; two R' can be joined to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O, and S. In some embodiments, Ri in -C≡C-Ri is H or Me.

[0091] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl", etc. are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl, and alkynyl) groups, but the term "hetero" refers to a group containing 1 to 3 O, S, or N heteroatoms or combinations thereof in the backbone residue. Thus, at least one carbon atom of the corresponding alkyl, alkenyl, or alkynyl group is replaced with one of the specific heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group. The typical size of the hetero forms of alkyl, alkenyl, and alkynyl groups is usually the same as the corresponding hydrocarbyl groups, and the substituents that may be present on the hetero forms are the same as those described above for the hydrocarbyl groups. For reasons of chemical stability, unless otherwise specified, it is also understood that such groups do not contain three or more consecutive heteroatoms, except when an oxo group is present on the N or S, such as a nitro or sulfonyl group.

[0092] As used herein, "alkyl" includes cycloalkyl and cycloalkylalkyl groups, and the term "cycloalkyl" can be used herein to represent a carbocyclic non-aromatic group bonded through a ring carbon atom, and "cycloalkylalkyl" can be used to represent a carbocyclic non-aromatic group bonded to a molecule through an alkyl linker. Similarly, "heterocyclyl" can be used to represent a non-aromatic cyclic group that contains at least one heteroatom as a ring member and is bonded to a molecule through a ring atom that may be C or N. "Heterocyclylalkyl" can be used to represent a group bonded to another molecule through a linker. The sizes and substituents suitable for cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. As used herein, these terms also include rings that contain one or two double bonds, as long as the ring is not aromatic.

[0093] As used herein, "acyl" encompasses groups containing an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical attached to one of the two available valence positions of the carbonyl carbon atom, and heteroacyl refers to the corresponding group in which at least one carbon other than the carbonyl carbon is replaced by a heteroatom selected from N, O, and S. Thus, heteroacyl includes, for example, -C(=O)OR and -C(=O)NR2, as well as -C(=O)-heteroaryl.

[0094] Acyl and heteroacyl groups are attached to any group or molecule through the open bond of the carbonyl carbon atom. Typically, they are C1-C8 acyl groups, including formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, including methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms of such groups that comprise the acyl or heteroacyl groups can be substituted with the substituents described herein as generally suitable substituents for each of the corresponding components of the acyl or heteroacyl groups.

[0095] An "aromatic" or "aryl" moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristic of aromaticity, examples of which include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic ring systems containing one or more heteroatoms selected from O, S, and N as ring members. The inclusion of a heteroatom confers aromaticity to both five- and six-membered rings. Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, and C8-C6 aromatic groups such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like, in which one of these monocyclic groups is fused with either a phenyl ring or a heteroaromatic monocyclic group. 10This definition includes fused bicyclic moieties formed by forming a bicyclic group. Any monocyclic or fused ring bicyclic system that has the character of aromaticity in terms of electron distribution throughout the ring system is included in this definition. This also includes bicyclic groups that have the character of aromaticity at least in which the ring is directly attached to the remainder of the molecule. Typically, the ring system contains 5 to 12 ring member atoms. For example, a monocyclic heteroaryl can contain 5 to 6 ring members, and a bicyclic heteroaryl contains 8 to 10 ring members.

[0096] The aryl and heteroaryl moieties are C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C 12 and heteroaryl moieties, each of which may themselves be further substituted. Other substituents for the aryl and heteroaryl moieties include halo, OR, NR2, SR, SO2R, SO2NR2, NRS02R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 heteroaryl, C8-C8 alkynyl, C6-C8 heteroaryl, C8-C8 alkynyl, C8-C8 heteroaryl, C6-C8 heteroaryl, C8 ... 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Aryl alkyl or C6-C 12 Heteroarylalkyl, where each R may be optionally substituted as described above for alkyl groups. Substituents on aryl or heteroaryl groups may, of course, be further substituted with groups described herein as appropriate for each type of such substituent or each component of the substituent. Thus, for example, an arylalkyl substituent may be substituted in the aryl portion with substituents described herein as typical for aryl groups, and may be further substituted in the alkyl portion with substituents described herein as typical or appropriate for alkyl groups.

[0097] Similarly, "arylalkyl" and "heteroarylalkyl" refer to aromatic and heteroaromatic ring systems that are attached to their points of attachment through a linking group, such as alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Typically, the linker is a C1-C8 alkyl or heteroform thereof. These linkers can also include carbonyl groups, thus providing substituents as acyl or heteroacyl moieties. The aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group may be substituted with the same substituents as those described above for aryl groups. For example, arylalkyl groups include a phenyl ring, optionally substituted with groups defined above for aryl groups, and a C1-C4 alkylene that is unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups, the alkyl or heteroalkyl groups optionally being cyclized to form rings such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, heteroarylalkyl groups may include C5-C6 monocyclic heteroaryl groups optionally substituted with groups mentioned above as typical substituents for aryl groups, and C1-C4 alkylene that is unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups, or include optionally substituted phenyl rings or C5-C6 monocyclic heteroaryl and C1-C4 heteroalkylene that is unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups, said alkyl or heteroalkyl groups optionally being cyclized to form rings such as cyclopropane, dioxolane, or oxacyclopentane.

[0098] When an arylalkyl or heteroarylalkyl group is described as being optionally substituted, the substituents may be on either the alkyl or heteroalkyl portion or the aryl or heteroaryl portion of the group. Optional substituents on the alkyl or heteroalkyl portion are generally the same as those described above for alkyl groups. Optional substituents on the aryl or heteroaryl portion are generally the same as those described above for aryl groups.

[0099] As used herein, "arylalkyl" groups, when unsubstituted, are hydrocarbyl groups and are described by the total number of carbon atoms in the ring and alkylene or similar linker. Thus, a benzyl group is a C7-arylalkyl group and a phenylethyl is a C8-arylalkyl group.

[0100] The above "heteroarylalkyl" refers to a moiety containing an aryl group linked via a linking group, and differs from "arylalkyl" in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O, and S. Heteroarylalkyl groups are described herein according to the total number of atoms in the rings and linkers connected, and include aryl groups linked via heteroalkyl linkers; heteroaryl groups linked via hydrocarbyl linkers such as alkylene; and heteroaryl groups linked via heteroalkyl linkers. Thus, for example, C7-heteroarylalkyl includes pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

[0101] As used herein, "alkylene" refers to a divalent hydrocarbyl group. Because it is divalent, it can link two other groups. Typically, it is -(CH2) n -, where n is 1-8, for example, n is 1-4, although where specified, alkylene can be substituted with other groups, can be other lengths, and open bonds that need not be at both ends of the chain. Thus, -CH(Me)- and -C(Me)2- are also referred to as alkylene, as are cyclic groups such as cyclopropane-1,1-diyl. When an alkylene group is substituted, the substituents include those typically found on alkyl groups as described herein.

[0102] In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group contained in a substituent, or any heteroform of one of these groups, may itself be optionally substituted by additional substituents. The nature of these substituents is the same as that described for the primary substituent itself, unless the substituents are otherwise described. Thus, the linker R 2 When an embodiment of R is an alkyl, the alkyl may be selected from R if it is chemically meaningful and does not violate the size restrictions provided for the alkyl itself. 2 It may be optionally substituted by the remaining substituents listed as embodiments of, for example, alkyl substituted with alkyl or alkenyl simply expands the upper carbon atom limit of these embodiments, but is not included. However, alkyl substituted with aryl, amino, alkoxy, =O, etc. are included within the scope of the present invention, and the atoms of these substituents are not counted in the numbers used to describe the alkyl, alkenyl, etc. groups described. When the number of substituents is not specified, each such alkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted with several substituents depending on its available valences. In particular, any of these groups may be substituted, for example, with fluorine atoms at any or all of its available valences.

[0103] In one embodiment, the linker is (R 3 ) r -(R 4 ) or R 3 or ((R 3 ) r -(R 4 )-(R 3 ) t In one embodiment, R 3 is a PEG moiety or a derivative of a PEG moiety. In certain embodiments, R 3is -O-CH2-CH2- or -CH2-CH2-O-. In one embodiment, the PEG moiety can include one or more PEG units. The PEG moiety can include from about 1 to about 1,000 PEG units, and in some embodiments, including but not limited to, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 units. In certain embodiments, the PEG moiety can include about 1 to 5 and up to about 25 PEG units, about 1 to 5 and up to about 10 PEG units, about 10 to up to about 50 PEG units, about 18 to up to about 50 PEG units, about 47 to about 150 PEG units, about 114 to up to about 350 PEG units, about 271 to up to about 550 PEG units, about 472 to up to about 950 PEG units, about 50 to up to about 150 PEG units, about 120 to up to about 350 PEG units, about 250 to up to about 550 PEG units, or about 650 to up to about 950 PEG units. In certain embodiments, the PEG unit is -O-CH2-CH2- or -CH2-CH2-O-. In one embodiment, R 4 is NH(CO), -C1-C6 alkyl, -C1-C6 alkoxy, -NR a R b , -N3, -OH, -CN, -COOH, -COOR 1 , -C1~C6 alkyl-NR a R b , C1~C6 alkyl-OH, C1~C6 alkyl-CN, C1~C6 alkyl-COOH, C1~C6 alkyl-COOR 1 , a 5- or 6-membered ring, a substituted 5- or 6-membered ring, a -C1-C6 alkyl-5- or 6-membered ring, a -C1-C6 alkyl-substituted 5- or 6-membered ring, a C2-C9 heterocycle, or a substituted C2-C9 heterocycle.

[0104] In some embodiments, R 3is -O-CH2-CH2- or -CH2-CH2-O-, and r or t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, r or t is from about 4 to about 15, and sometimes r is about 4 or 11. In some embodiments, R 3 is a PEG unit (PEG) r where r is about 2 to about 4, and t is about 8 to 14.

[0105] In certain embodiments, R4 is an amide linking group (e.g., -C(O)NH- or -NH(O)C-); an alkylamide linking group (e.g., -C1-C6 alkyl-C(O)NH-, -C1-C6 alkyl-NH(O)C-, -C(O)NH-C1-C6 alkyl-, -NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-NH(O)C-C1-C6 alkyl-, -C1-C6 alkyl-C(O)NH-C1-C6 alkyl-, or -C(O)NH-(CH2) t -, t is 1, 2, 3, or 4; a substituted 5- or 6-membered ring (e.g., an aryl ring, a heteroaryl ring (e.g., tetrazole, pyridyl, 2,5-pyrrolidinedione (e.g., 2,5-pyrrolidinedione substituted with a substituted phenyl moiety)), a carbocyclic ring, or a heterocyclic ring) or an oxygen-containing moiety (e.g., -O-, -C1-C6 alkoxy).

[0106] Exemplary embodiments In one embodiment, a molecule is provided that includes a G protein-coupled estrogen receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase. In one embodiment, the GPER ligand includes 17β-estradiol, estrone, phytoestrogens, xenoestrogens, estriol, estriol 3-sulfate, estriol 17-sulfate, G-1, G-15, G-36, genistein, or quercetin. In one embodiment, the GPER ligand includes 17β-estradiol. In one embodiment, the GPER ligand is a GPER antagonist. In one embodiment, the E3 ligase ligand is a von Hippel ligase (VHL) ligand. In one embodiment, the E3 ligase ligand comprises lenalidomide, pomalidomide, iberdomide, (S,R,S)-AHPC, thalidomide, VH-298, CC-885, E3 ligase ligand 8, TD-106, VL285, VH032, VH101, VH298, VHL ligand 4, VHL-2 ligand 3, E3 ligase ligand 3, E3 ligase ligand 2, or BC-1215. In one embodiment, the linker comprises a chain having 5 to 50 atoms, e.g., about 8 to about 35 atoms. In one embodiment, the linker is an alkyl linker. In one embodiment, the linker is a heteroalkyl linker having one or more O, N, or S. In one embodiment, the linker comprises polyethylene glycol (PEG). In one embodiment, the linker comprises 4 to 15 PEG units. In one embodiment, the linker is (PEG) m NH(CO)(PEG) nwherein n and m are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, n is 3, 4, 5, or 6. In one embodiment, m is 7, 8, 9, or 10. In one embodiment, the GPER ligand, linker, or E3 ligand comprises an amine group, a carboxyl group, a carbonyl group, a sulfhydryl group, such as a maleimide, an aldehyde group, such as a hydrazide, or a hydroxyl group useful for forming a covalent bond with another molecule. In one embodiment, the linker comprises a carbodiimide, such as EDC, NHS, ABH, ANB-NOS, APDP, EMCH, EMCS, GMBS, MBS, or SIAB.

[0107] In one embodiment, a method of preventing, inhibiting, or treating endocrine-resistant or hormone therapy-resistant cancer in a human is provided, in one embodiment, the method comprises administering to the human a composition having an effective amount of a molecule comprising a G Protein-Coupled Estrogen Receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase.

[0108] In one embodiment, a method of preventing, inhibiting, or treating triple-negative breast cancer in a human is provided, the method comprising administering to a mammal a composition having an effective amount of a molecule comprising a G Protein-Coupled Estrogen Receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase.

[0109] In one embodiment, a method is provided for preventing, inhibiting, or treating cervical cancer, ovarian cancer, or endometrial cancer in a human, the method comprising administering to a mammal a composition having an effective amount of a molecule comprising a G Protein-Coupled Estrogen Receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase.

[0110] In one embodiment, a method of preventing, inhibiting, or treating prostate cancer or ovarian cancer in a human is provided, the method comprising administering to a mammal a composition having an effective amount of a molecule comprising a G Protein-Coupled Estrogen Receptor (GPER) ligand bound to a linker bound to a ligand of an E3 ligase.

[0111] Exemplary Chimeras A compound having the structure of Formula I, or a salt thereof, is provided: XLY Formula I wherein X is a G protein-coupled estrogen receptor (GPER) ligand; Y is an E3 ubiquitin ligase ligand; L is a linker.

[0112] In one example, X is 17β-estradiol, estrone, phytoestrogens, xenoestrogens, estriol, estriol 3-sulfate, estriol 17-sulfate, G-1, G-15, G-36, genistein, dazin, quercetin, or a derivative thereof. In one example, X is a GPER antagonist. In one example, Y is a von Hippel ligase (VHL) ligand. In one example, Y is cereblon, lenalidomide, pomalidomide, iberdomide, (S,R,S)-AHPC, thalidomide, VH-298, CC-122, CC-885, E3 ligase ligand 8, TD-106, VL285, VH032, VH101, VH298, VHL ligand 4, VHL ligand 7, VHL-2 ligand 3, E3 ligase ligand 3, E3 ligase ligand 2, BC-1215, or a derivative thereof. In one example, L has a backbone having a chain length of 5-200 atoms, counted in a linear path between X and Y. In one example, L has a backbone having a chain length of 15-50 atoms, counted in a linear path between X and Y. In one example, L has a backbone having a chain length of 20-50 atoms, counted in a linear path between X and Y. In one example, L has a backbone length of 8 to 300 angstroms, calculated by summing the bond lengths in a straight line path between X and Y. In one example, L has a backbone length of 25 to 75 angstroms, calculated by summing the bond lengths in a straight line path between X and Y. For example, the structure of L is

[0113] [ka]

[0114] The structure is Q is a bond or a divalent group that forms a covalent bond to X; Z is a linear chain containing one or more alkyl, aryl, heteroalkyl, heteroaryl, alkyloxy, alkylamino, alkylglycol, carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, amine, oxygen, sulfur, sulfone, or sulfoxide in divalent form; G is a bond or a divalent group that forms a covalent bond to Y.

[0115] In one example, L is

[0116] [ka]

[0117] The structure is Q is a bond or a divalent group that forms a bond to X; R is the divalent form of alkyl, aryl, heteroalkyl, heteroalkyl, heteroaryl, alkyloxy, alkylamino, alkylglycol, carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, amine, oxygen, sulfur, sulfone, or sulfoxide; G is a bond or a divalent group that forms a bond to Y; m, n, p, and q are each independently an integer from 0 to 50, provided that at least one of m, n, p, and q, if present, is an integer greater than 0.

[0118] In one example, Q and G are independently the divalent form of carbonyl, thiocarbonyl, acyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, sulfone, or sulfoxide.

[0119] In one example, Q and G are independently carbonyl or acyl selected from the group consisting of acetyl, 2-hydroxyacetyl, 2-aminoacetyl, propionyl, 3-hydroxypropanoyl, 3-aminopropanoyl, butanoyl, 4-hydroxybutanoyl, and 4-aminobutanoyl, each of which is in its divalent form.

[0120] In one example, m and n, if present, are each independently an integer from 0 to 2, and Q and G are each independently carbonyl, thiocarbonyl, acetyl, 2-hydroxyacetyl, 2-aminoacetyl, propionyl, 3-hydroxypropanoyl, 3-aminopropanoyl, butanoyl, 4-hydroxybutanoyl, 4-aminobutanoyl, carbamate, urea, thiocarbamate, thiourea, dithiocarbamate, aminocarbonyl, amide, ester, thioester, thioamide, sulfone, or sulfoxide, each of which is in its divalent form.

[0121] In one example, Z is hydrophilic. In one example, L is

[0122] [ka]

[0123] which provides a main chain having a length of 13 atoms counted in a linear path. In one example, L is

[0124] [ka]

[0125] wherein p and q each independently represent an integer of 0 to 50. In one example, L includes one or more of a divalent alkyl group, a divalent heteroalkyl group, or a divalent polyethylene glycol (PEG), or one or more of each.

[0126] In one example, L contains 5 or more divalent ethoxy (-CH2CH2O-) groups. In one example, L contains 1 or more heteroatoms for every 2 carbons and does not contain any alkyl chains longer than butyl.

[0127] In one example, L is attached to X through an oxygen, nitrogen, sulfur, carbonyl, or ethynyl of X, and L is attached to Y through an oxygen, nitrogen, sulfur, carbonyl, or ethynyl of Y. In one example, X is an estrogen steroid and includes a divalent group selected from oxygen, amine, sulfur, vinyl, ethyne, and carbonyl located at C6 or C17 of the estrogen steroid, and the divalent group at C6 or C17 is bonded to L.

[0128] In one example, Y is (S,R,S)-AHPC, which is attached to L via the amine of (S,R,S)-AHPC. In one example, the compound

[0129] [ka]

[0130] where L is a linker. In one example, the compound

[0131] [ka]

[0132] where L is a linker. In one example, the compound

[0133] [ka]

[0134] It has the structure: In one example, the compound

[0135] [ka]

[0136] It has the structure: In one example, the compound

[0137] [ka]

[0138] It has the structure: In one example, the compound

[0139] [ka]

[0140] It has the structure: The invention is illustrated by the following non-limiting examples. Example 1 GPER has been associated with poor survival and disease progression in breast cancer (Filardo et al., 2000; Filardo et al., 2006; Sjostrom et al., 2014), ovarian cancer (smith et al., 2009) and endometrial cancer (Smith et al., 2007). Importantly, GPER is expressed in over 80% of TNBC, a subtype of breast cancer with poor overall survival due to lack of expression of proteins targeted by available drugs. This estrogen receptor is an independent measure of estrogen action and a druggable target. The goal of this project is to make an important step in the development of PROTACs to target and degrade GPER.

[0141] A study of 121 TNBC patients suggests that this receptor is expressed in more than 80% of these tumors. The expression of GPER in the majority of TNBCs is supported by other smaller studies and contrasts with data from a rather limited study of six tumors and two cell lines suggesting that GPER is a tumor suppressor in TNBC. Unlike ER expression, which correlates inversely with clinical predictors of aggressive breast cancer, GPER expression is directly associated with these same variables (Filardo et al., 2006; Ignatov et al., 2011), suggesting that it is involved in metastasis. This observation, together with the fact that GPCRs are targets of the pharmaceutical industry, makes GPER a promising druggable target in breast cancer. Thus, this type of therapeutic agent that selectively degrades GPER may be useful in the treatment of TNBC and may also be useful in other endocrine-resistant breast cancers. Finally, GPER is associated with aggressive disease and poor prognosis in gynecological cancers, suggesting that GPER-PROTACs may benefit these patients.

[0142] Estrogen-targeted therapy is effective in postmenopausal women. A spontaneous model of TNBC carcinogenesis is used, where tumor formation occurs after menopause. GPER-PROTACs are delivered to mice with early stage TNBC (tumors ≤0.5 cm). GPER-PROTACs may work most effectively in the absence of endogenous estrogen, but are also tested in men, although they represent less than 1% of all breast cancer cases (Anderson et al., 2010).

[0143] The GPER-PROTAC prototype, UI-EP001, selectively degrades recombinant GPER and downmodulates the expression of native GPER in human breast cancer cells (Figures 2 and 3). To screen GPER-PROTACs, a GPER binding assay can be used that measures a high-affinity (Kd=2.7 nm), limited capacity, displaceable single binding site specific for estrogen in the plasma membrane of human SKBR3 breast cancer cells that express GPER but lack nuclear ER (Thomas et al., 2005). GPER undergoes an unusual mechanism of endocytosis that does not require receptor phosphorylation, interaction with β-arrestin, or lysosomal activity. Instead, GPER is polyubiquitinated at the plasma membrane and retrogradely transported to the trans-Golgi network (TGN) via rab11-positive recycling endosomes prior to proteasomal degradation (Cheng et al., 2011). Finally, to quantify the extent of GPER degradation, a highly sensitive Nanobit binary luminescence assay is used to measure the relative efficacy of GPER-PROTACs to proteolyze GPER (Figure 11).

[0144] Identification of GPER-PROTACs with high binding affinity and specificity to GPER principle. Proteolytic targeting of ER is an effective means to treat endocrine-resistant breast cancer. ER-PROTACs promote tumor regression in preclinical models of ER+ breast cancer when administered orally (Flanagan et al., 2019). PROTACs targeting GPER, a distinct estrogen receptor expressed in the majority of ER-negative (Filardo et al., 2006; Ignatov et al., 2011) and triple-negative tumors (97 / 121=80.2%), have been developed (Prissnitz et al., 2015) but are not yet approved for clinical use, and may be more effective.

[0145] Design of GPER-PROTACs.In one embodiment, the PROTAC design is based on the use of 17β-E2 as a targeting ligand and the von Hippel-Lindau (VHL) derived ubiquitin E3 ligase ligand, (2S,4R)-1-((S)-2-amino-3,3-dimethyl-butanoyl)-4 hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, known as (S,R,S)AHPC

[24] . The GPER-PROTAC is assembled using a partial PROTAC consisting of AHPC conjugated to an array of 2, 4, or 6 units of PEGylated chemical linkers (Figure 19). Structure-activity relationship (SAR) data suggests that attachment of the chemical linker to the C6 and C17 atoms of the estrane ring is most likely to maintain the estrogen binding function of GPER. Fulvestrant (ICI182,780), which contains a C6 side chain, has a relative binding activity of 5% and functions as a GPER agonist at 500 nM ( Filardo et al., 2007 ), with a potency of 17β-E2-hemisuccinate-17-BSA similar to that of 17β-E2 in stimulating intracellular cAMP production ( Filardo et al., 2007 ).

[0146] Synthesis, purification, and structural confirmation of GPER-PROTAC.C-17 linked GPER-PROTACs are synthesized by EDC coupling of the 17β-hydroxyl group of 17β-E2 with (S,R,S)-AHPC-PEG2-COOH; (S,R,S)-AHPC-PEG4-COOH or (S,R,S)-AHPC-PEG6-COOH using EDC.HCl and HOBt in the presence of DMF at room temperature. The synthesis of the desired compounds is confirmed by NMR analysis and high-resolution mass spectrometry. C-6 linked GPER-PROTACs can be synthesized in a multi-step reaction. First, reductive amination of the 6 carbonyl group of 13-methyl-6-oxo-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl diacetate (1) using sodium cyanoborohydride in the presence of ammonium acetate in refluxing methanol. Second, coupling of 6-amino-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl diacetate (2) with (S,R,S)-AHPC-PEG2-COOH; (S,R,S)-AHPC-PEG4-COOH or (S,R,S)-AHPC-PEG6-COOH using EDC.HCl and HOBt in the presence of DMF. Purification of the compounds is achieved by C18 reversed phase preparative HPLC column. The synthesis and purity of the desired compounds are confirmed using NMR analysis and LC-MS. All chemicals used in this synthesis are commercially available.

[0147] Assessment of GPER-PROTAC binding. GPER-PROTACs are tested for binding activity using a competitive radioreceptor assay

[22] . Briefly, GPER-PROTACs are dissolved in ethanol and added to a glass reaction tube to a final concentration of 1 nM to 10 uM. The ethanol is evaporated under nitrogen. Membrane protein (100 ug) and 4 nM (89 Ci / mmol) 3 H-17β-estradiol (E2) is added to the tube and incubated for 30 min at 4 °C. Binding is terminated by rapid filtration through presoaked glass fiber filters, allowing the free 3Binding is separated from H-E2. Filters are washed and radioactivity is measured using a liquid scintillation counter. Maximum specific binding is calculated as the difference between total binding (absence of competitor) and non-specific binding in the presence of a 1,000-fold molar excess of 17β-E2 or GPER-PROTAC. The concentration of competitor required to displace 50% of maximum specific binding (EC 50 ) is calculated from the one-site competitive binding curve. The top and bottom of the curve are defined as 100% and 0%, respectively. The relative binding affinity (RBA) is calculated based on the EC 50 EC of 17β-E2 against 50 and expressed as a percentage. In one embodiment, the RBA of one or more GPER-PROTACs relative to 17β-E2 is >10%.

[0148] Testing the ability of GPER-PROTAC candidates to degrade GPER and reduce its signaling activity principle. This is directed at the ability of GPER-PROTACs to recruit E3 ubiquitin ligases, ubiquitinate GPER, and cause destruction by the proteasome. The degradative power of GPER-PROTACs is first assessed by quantitative immunofluorescence and immunochemistry analysis. Drug candidates that show strong degradative ability are then further evaluated using high-throughput and sensitive luminescence assays. Finally, the efficacy of each tested GPER-PROTAC in reducing active GPER is evaluated by measuring GPER-dependent erbB1 tyrosyl phosphorylation and phosphorylation of erk-1 / 2 (Prissnitz et al., 2015). Taken together, these measurements of GPER downmodulation identify GPER-PROTACs that effectively reduce GPER activity.

[0149] In breast cancer cells, the majority of GPER is uncoupled from its heterotrimeric G protein and in a low affinity state, retained in the endoplasmic reticulum. Only a small percentage of the high affinity G protein-bound state is expressed at the plasma membrane (Prissnitz et al., 2015; Smith et al., 2007). When used at high concentrations in intact cells, GPER-PROTACs can degrade both low and high affinity receptors. The effect of GPER-PROTACs on cell surface-associated and total GPER is measured. As described above and detailed below, several quantitative modalities are used to measure GPER degradation and its signaling activity in breast cancer cells treated with GPER-PROTAC candidates, for example, in breast cancer cells with luminal A, luminal B, her2 overexpressing and basal immunophenotype treated with GPER-PROTAC candidates. Finally, the relative efficacy of existing GPER antagonists G15 or G36 in reducing GPER signaling activity is compared.

[0150] Evaluation of the GPER decomposition.The GPER-PROTACs with the highest RBA are tested for their ability to ubiquitinate and degrade GPER. GPER-expressing breast cancer cells are treated with various doses of GPER-PROTACs or vehicle for 5 min to 8 h, and GPER downmodulation is assessed by both immunofluorescence and immunochemistry analysis. The presence of native GPER is detected in fixed intact or detergent-permeabilized breast cancer cells using either N- or C-terminal antibodies. GPER detected by immunofluorescence is quantified by total corrected cell fluorescence (J-Image) and ELISA. Steady-state levels of immature or mature forms of GPER protein are determined by immunoblotting with a GPER-specific antibody. In these experiments, the extent of GPER degradation is determined by measuring band pixel intensity (ImageJ) and normalizing to GAPDH. The specificity of GPER action is confirmed in the presence of excess free 17β-E2 or a control steroid (17β-estradiol, which shows no measurable binding to GPER) (Thomas et al., 2005). Partial PROTACs lacking or bound to 17β-E2 serve as drug controls. Target specificity is assessed in HEK293 cells stably expressing HA-GPER, HA-CXCR4, or HA-β-1AR (Filardo et al., 2018).

[0151] Assessment of GPER polyubiquitination.Ubiquitination status is confirmed by blotting with a GPER-specific antibody in a tandem ubiquitin binding element (TUBE) pull-down assay. Again, the extent of GPER ubiquitination in both immature and surface forms is quantified by measuring pixel band intensity of lysates from untreated, PROTAC-treated, and partial PROTAC-treated cells. Proteasomal degradation is assessed by treating cells with the proteasome inhibitor MG132, while control cells receive chloroquine to inhibit lysosomal hydrolases. Lysless versions of GPER that cannot be ubiquitinated (Lys333Arg, Lys341Arg, Lys351Arg) are also tested for susceptibility to GPER-PROTAC degradation.

[0152] Nanobit complementation assays to assess the degradation of surface and total GPER To quantify the removal of GPER from the cell surface and its overall degradation, we perform a Nanobit™ (Promega) binary luminescence complementation assay. This assay records a luminescence signal with a dynamic range of more than 6 logs. In the presence of luciferin substrate, the luminescence signal is generated by the interaction of the two split components of Ophiopogon luciferase, called HiBit and LgBit. In these experiments, soluble LgBit is delivered together with luciferin into intact or detergent-permeabilized cells expressing N-terminally HiBit-tagged GPER.

[0153] Assessment of GPER signaling. After treatment with GPER-PROTACs for various periods, cells are stimulated with 17β-E2 and GPER activity is measured by monitoring erbB1 tyrosyl phosphorylation or erk-1 / 2 phosphorylation. Measurement of EGF-stimulated erbB1 or erk-1 / 2 serves as a control for the specificity of the GPER-PROTACs. Additional control experiments measuring erbB1 or erk-1 / 2 stimulation are performed using partial PROTACs conjugated to 17β-E2. Data are quantified by pixel band intensity and normalized to untreated cells.

[0154] Determining in vivo efficacy of lead GPER-PROTACs principle. Early studies of efficacy, biodistribution, and toxicity are important in the initial evaluation and subsequent structural modifications of rationally designed pharmaceuticals. These biological responses will be measured following oral administration of GPER-PROTACs.

[0155] GPER acts on cancer cells to promote their survival, as well as affecting cells that determine the tumor microenvironment and promote cancer progression (Filardo et al., 2018). Thus, the anti-carcinogenic or anti-tumor activity of GPER-PROTACs is determined using mouse models simulating human TNBC carcinogenesis, for example by measuring the inhibition of tumor growth or animal survival. Toxicity and biodistribution studies are performed in both wild-type and GPER-null mice. Experiments are performed in ovarian-intact and ovariectomized (OVX) mice.

[0156] Statistical analysis. Twelve mice per group achieves 80% power to detect a mean difference of 1.2 times the group-specific standard deviation. Power is conservatively estimated using a two-tailed two-sample t-test at a significance level of 5% at a given time point. Mixed-effects regression is used to estimate and compare tumor growth rates as a function of time and treatment group. A random-effects model is used to account for longitudinal correlation of repeated tumor measurements. This formal analysis is expected to have higher power because all tumor measurements over time are utilized. For overall survival, curves for each treatment group are constructed using the Kaplan-Meier method and compared using the log-rank test.

[0157] GPER-PROTACs as anticancer drugs. The GPER-PROTACs found to have the highest in vitro efficacy will be investigated for their anti-carcinogenic effects. fl / fl Brca1 fl / fl Mice were crossed with female K14-cre p53 fl / fl Brca1 fl / flMice produce normal litter sizes and transgenic pups born have been shown to be non-embryonic lethal. Approximately 85% of female mice develop mammary tumors with a median survival of 248 days. Tumor formation is closely monitored using digital calipers and, once tumors reach 5 mm in one dimension, mice are randomly divided into control and treatment groups. Initial experiments aim to identify doses and treatment schedules at which effects on tumor growth (regression) are observed, using doses of GPER-PROTAC (100-4,000 mg / kg) based on toxicity studies. Mice receive a single dose of vehicle or GPER-PROTAC by oral gavage. Tumor growth in mice is monitored using calipers. Tumor growth and overall survival (OS) are used as measures of responsiveness to treatment.

[0158] Biodistribution studies. GPER-PROTACs are administered by oral gavage to wild-type and GPER-null mice. Dosing schedules and concentrations can be varied. Tumors and tissues are excised from breast, ileum, duodenum, spleen, heart, lung, and liver, and drug concentrations are measured by LC-MS. Tumors and tissues are harvested at 1 hour, 4 hours, 24 hours, 7 days, 14 days, 21 days, and 28 days after inoculation.

[0159] Toxicology research. In conjunction with the experiments outlined above, to identify toxicity associated with treatment with GPER-PROTAC, liver function (ALT, AST, LDH, bilirubin), kidney function (creatinine, BUN), and inflammatory bowel disease (IBD) (diarrhea, bloody stool) will be monitored weekly in both treated and untreated mice (5 mice / group). Objective criteria indicating toxicity include a 3-fold or greater increase in liver and kidney function values, as well as unabated diarrhea and / or bloody stool.

[0160] As shown in Figure 11, after 24 hours of treatment, HCC1806 and SKBR3 cells showed lower viability when treated with E2-PROTAC compared to partial PROTAC. HCC1806 showed a higher response to E2-PROTAC than SKBR3. IC50 ranged from about 10 to about 50uM. Pretreatment of cells with estradiol significantly reduced cell death in both HCC1806 and SKBR3. Degradation of GPER may result in cell death or reduced cell proliferation. Alternatively, delocalization of GPER from the membrane to the nucleus may recruit ligase to degrade proteins in the cytoplasm / nucleus.

[0161] Example 2 Decisions regarding the allocation of hormonal therapy for breast cancer are based solely on the presence of nuclear estrogen receptor (ER) in biopsied tumor tissue. This is contrary to the fact that G protein-coupled estrogen receptor (GPER) is associated with aggressive breast cancer and is required for the survival of breast cancer stem cells. Observations suggest that effective endocrine therapy should also target this receptor. Here, we describe two ER / GPER-targeting proteolytic chimeras (UI-EP001 and UI-EP002) that effectively degrade ERa, ERb, and GPER. These chimeras form high-affinity interactions with GPER and ER, with binding dissociation constants of approximately 30 nM and 10–20 nM, respectively. Plasma membrane and intracellular GPER and nuclear ER were degraded by UI-EP001 and UI-EP002, but not by partial PROTACs lacking the estrogen targeting domain. Pretreatment of cells with MG132, a proteasome inhibitor, blocked the proteolysis of UI-EP001 and UI-EP002, whereas chloroquine, a lysosomotropic inhibitor, had no effect. No off-target activity was observed against recombinant b1-adrenergic receptor or CXCR4. Target specificity was further demonstrated in human MCF-7 cells, where both drugs effectively degraded ERa, ERβ, and GPER, but not progesterone receptor (PR). UI-EP001 and UI-EP002 induced cytotoxicity and G2 / M cell cycle arrest in MCF-7 breast cancer and human SKBR3 (ERa-ERb-GPER+) breast cancer cells, but not in human MDA-MB-231 breast cancer cells that do not express functional GPER / ER. These results provide a receptor-based strategy for anti-estrogen treatment of breast cancer that targets both plasma membrane and intracellular estrogen receptors.

[0162] Materials and Methods Molecular docking of E2-PROTAC to ERa or GPERThe previously established GPER homology model was used in GLIDE (Schroedinger, Cambridge, MA) docking studies (Amatt et al., 2012; Amatt et al., 2013). In these studies, a grid was first constructed around the previously established binding site of E2 using Receptor Grid Generation within GLIDE. UI-EP001 and UI-EP002 were drawn in Maestro11.3 (Schroedinger, Cambridge, MA) and prepared for docking using the LigPrep function within Schroedinger. Possible ionization states were generated at pH 7 ± 2 using the OPLS3 force field with Epik. All other settings were left at default settings. GLIDE docking of UI-EP001 and UI-EP002 was then performed. The van der Waals radii were set to a scaling factor of 0.8 with a partial charge cutoff of 0.15. XP (extra-precise) docking was performed using flexible ligand sampling with both sample nitrogen inversion and sample ring structure turned on. Biased sampling of amides was set to penalize non-planar conformations. A penalty for epic states was added to the docking score. For docking, 10,000 poses per ligand were kept at the initial stage of docking and the best 1,000 poses per ligand were kept for energy minimization using the OPLS3 force field. Post-docking minimization was performed, keeping 100 poses per ligand. All other settings were left at default settings. Both UI-EP001 and UI-EP002 were also docked into ERα in a similar manner. Using the ERα homodimer crystal structure (PDB: 1A52, Tanenbaum et al., 1998), docking studies were performed with UI-EP001 and UI-EP002 after removing bound E2 molecules and then preparing for docking using Receptor Grid Generation in GLIDE.

[0163] Synthesis of E2-PROTAC and E2-FITC . The chemical synthesis of UI-EP001, UI-EP002 and E2-FITC was carried out according to the schematic diagrams in Figures 24 and 31, respectively. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE AV-300 and a Bruker AVANCE AV-500 instrument at 300 K. 1H NMR spectra are reported in parts per million (ppm) downfield from tetramethylsilane (TMS). All 13 C NMR spectra are reported in ppm and obtained with 1H decoupling. In reported spectral data, the format (δ) chemical shift (multiplicity, J value in Hz, integral) was used with the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. MS analysis was performed using a Waters Q-Tof Premier mass spectrometer. UI-EP001 and UI-EP002 were purified by C18 reversed-phase preparative HPLC column with a Shimadzu Nexera X2 UHPLC system using solvent A (0.1% TFA in HO) and solvent B (0.1% TFA in MeCN) as eluents.

[0164] Cells and culture conditions Human MCF-7, MDA-MB-231, HCC-1806, and SKBR3 breast cancer cells and human embryonic kidney 293 cells (HEK-293) were purchased from the American Tissue Culture Collection (Manassas, VA). HEK-293 cells stably expressing hemagglutinin-tagged GPER (HA-GPER), β1-adrenergic receptor (ΗA-β1AR), and CXCR4 (HA-CXCR4) were previously described (Filardo et al., 2007). All cell lines were cultured in phenol red-free (1:1) DMEM / Ham's F-12 medium (PRF-DF12) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C in a humidified chamber containing 5% CO2.

[0165] Steroids and inhibitors17β-estradiol (17b-E2) and aldosterone were purchased from Sigma-Aldrich (St. Louis, MO). The 26S-proteasome inhibitor, MG132, was purchased from Selleckchem (Pittsburgh, PA), and the lysosomotrophic agent, chloroquine diphosphate, was purchased from Bio-Techne (Minneapolis, MN).

[0166] antibody GPER-specific antibodies were raised in New Zealand White rabbits against a synthetic peptide derived from amino acids 1-62 from the N-terminus of the human GPER polypeptide (Pacific Immunology, Ramona, CA). Commercially available antibodies included: rabbit anti-HA epitope antibody (Cell Signaling Technologies, Beverly, MA), mouse monoclonal ER-α (2Q418), PR (F-4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit monoclonal ER-β antibody, clone 68-4 was purchased from EMD Millipore (Billerica, MA). Goat anti-rabbit Alexa-Fluor 594, goat anti-rabbit Alexa-Fluor 488, and goat anti-mouse Alexa-Fluor 488 secondary antibodies were purchased from Abcam (Cambridge, MA), and goat anti-rabbit IgG and goat anti-mouse horseradish peroxidase (HRP)-conjugated antibodies were purchased from Southern Biotechnology (Birmingham, AL).

[0167] PlasmidsMolecular clones encoding full-length human GPER (Filardo et al., 2007) and Era (deConink et al., 1995) under the transcriptional control of the CMV promoter have been reported. A HiBiT tag (VSGWRLFKKIS) was inserted in frame at the amino terminus of GPER by inverse PCR using the forward 5'-GTTCAAGAAGATTAGCGATGTGACTTCCCAAGCC-3' (SEQ ID NO: 1) and reverse 5'-AGCCGCCAGCCGCTCACCA-TGTCTCTGCACCGTGC-3' (SEQ ID NO: 2) oligonucleotide primers and the Q5 mutagenesis kit (New England Biolabs, Salem, MA). A similar inverse PCR strategy with forward 5'-TGGCGGCTGTTCAAGAAGATTAGCTGAGAGCTCCCTGGCGGA-3' (SEQ ID NO: 3) and reverse 5'-GCCGCTCACAG-AGCCTCCTCCACCGACTGTGGCAGGGAAACCC-3' (SEQ ID NO: 4) oligonucleotide primers was used to insert a HiBiT tag at the carboxyl terminus of ER-α.

[0168] Transient transfection and binary Nano-Bit™ luminescence assay The Nano-Glo HiBiT Extracellular Detection system kit (Promega Corporation, Madison, WI) was used to detect total HiBiT-tagged GPER or ER estrogen receptors in detergent-permeabilized cells by binary emission complementation with recombinant LgBit and substrate. Briefly, HEK293 cells (0.75 × 10 6 ) were seeded into 35 mm tissue culture dishes and 24 hours later, 50 ng of HiBiT-GPER or HiBiT-ERa and 950 ng of pcDNA3.1(+)zeo carrier plasmid were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The next day, transfected cells were harvested by trypsinization and plated at 10 wells in 96-well poly-L-lysine-coated Greiner white-bottom microplates. 4Cells were seeded at a density of 1000 cells / well. The next day, cell culture medium was aspirated and replaced with 100 μL of serum-free PRF-DF12 containing various concentrations of E2-PROTAC, partial PROTAC, or vehicle at 37°C for the indicated time intervals. In some experiments, chloroquine (100 mM) or the proteasome inhibitor MG132 (10 mM) was included. After treatment, total HiBiT-tagged receptors were measured in cells permeabilized with 0.05% Triton® X-100 using HiBiT complementation reagent consisting of LgBiT protein and Nano-Glo® HiBiT substrate. Luminescence was measured using an Infinite 200 PRO multimode microplate reader from Tecan (Raleigh, NC) and reported as relative luminescence units (RLU). All samples were measured in triplicate and expressed as the mean plus or minus the standard deviation.

[0169] Immunocytofluorescence Cells were plated in 12-well cluster plates (CoStar, Corning, NY) on 18 mm glass coverslips coated with poly-L-lysine at 12,500–25,000 cells / cm in PRF-DF12 containing 5% FBS. 2The next day, cells were left untreated or treated with 100 mM UI-EP001, UI-EP002, or partial PROTACs in the presence or absence of chloroquine (100 mM) or the proteasome inhibitor MG132 (10 mM) for 1 h at 37 °C. After treatment, the plates were cooled on ice for 10 min before labeling with GPER N-terminal peptide antibody for 30 min at 4 °C. Excess antibody was removed by washing with cold PBS, and then cells were fixed with 4% paraformaldehyde in PBS for 5 min. Cells were then washed twice with PBS and nonspecific antibody binding sites were blocked for 1 h in PBS containing 5% bovine serum albumin (BSA) and 5% normal goat serum. Total receptors were measured in cells permeabilized with 0.1% Triton® X-100 for 10 min before immunostaining. Fixed and permeabilized cells were washed twice with PBS and incubated with primary antibody for 1 h. Excess primary antibody was removed by washing with PBS before cells were exposed to goat anti-rabbit or anti-mouse secondary antibodies for an additional hour. Following this second incubation, cells were washed once with PBS and once with Tris-buffered saline before mounting coverslips in Vecta-Shield antiquench medium containing DAPI (Vector Laboratories, Burlingame, CA). Immunofluorescence images were visualized on an Eclipse 80i microscope (Nikon, Inc., Melville, NY) equipped with a Nikon Plan Fluor 100× 0.5-1.3 oil iris with differential interference contrast and epifluorescence capabilities using a Qimaging Retiga 2000R digital camera and Nikon imaging software (NIS-Elements-BR3.0). After capture, images were processed using PHOTOSHOP® CS2 (Adobe) with brightness / contrast adjustments.

[0170] Competitive binding assay GPER binding was measured in an intact cell-based competitive binding assay using fluorescein-labeled estradiol (E2-FITC) as a tracer as described (Cao et al., 2017) with minor modifications. SKBR3 cells were incubated at 175 cm2 Cells were grown to near confluence in flasks (Corning, NY, USA) and then placed in serum-free medium overnight. Cells were detached in HBSS containing 5 mM EDTA and harvested by centrifugation at 150 g for 5 min. Cell pellets were washed twice with ice-cold HBSS containing 2 mM CaCl2 and 2 mM MgCl2, and the cells were soaked in the same buffer for 10 min. 6 Cells (100 μL) were mixed with 100 nM E2-FITC and seeded into 96-well conical V-bottom microtiter plates on ice containing an equal volume of HBSS or various concentrations of 17b-E2, partial PROTAC, or E2-PROTAC. Samples were then incubated on ice for 30 min. Cells were pelleted by centrifugation at 4°C, washed once with HBSS containing cations, and analyzed on an Aurora FCM instrument (Cytek Biosciences, USA). At least 10,000 events per sample were analyzed using forward scatter vs. side scatter dot plot gating to resolve primary cell populations. Fluorescence intensity of cells in the fluorescein isothiocyanate (FITC) channel was recorded in log mode for each sample. Each condition was run in triplicate and reported as the median fluorescence intensity plus or minus standard deviation.

[0171] ER binding was measured using cytosolic fractions prepared from MCF-7 cells by fluorescence polarization. 6Cells) were lysed with EDTA, cell homogenates were prepared using a Dounce homogenizer, and subcellular fractions were isolated by differential centrifugation as previously described (Filardo et al, 2002). Protein concentrations of each fraction were determined by bicinchoninic acid (BCA) assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher). For saturation binding assays of the probes, different concentrations of the cytosolic fractions were diluted in assay buffer (10 mM Tris-HCl, pH 7.4; 50 mM KCl, 10% glycerol, 0.1 mM dithiothreitol (DTT), 1 ug / mL BGG, 10 nM protease inhibitor cocktail (Roche), 10 nM E2-FITC in a final volume of 200 uL. The mixture was incubated at ambient temperature for 1 h, and the interaction of the probes with cytosolic proteins was determined by fluorescence polarization (FP). For competitive binding assays, aliquots of the cytosolic fractions were and E2-FITC (final concentration 10 nM) were mixed and incubated for 1 h at 4 °C. Various concentrations of drugs and controls were then added to the mixture. The mixture was incubated for an additional hour and then subjected to FP. All samples were measured in triplicate. Negative controls (cytosolic fraction only or E2-FITC only) and positive controls (100% bound E2-FITC and cytosolic fraction without treatment) were included. Relative binding affinity was expressed as a percentage of FP recorded from treated wells compared to the positive control. FP values ​​in millipolarization units (mP) were measured at ex / em wavelengths of 485 nm / 530 nm in 96-well black flat-bottom microplates (Greiner Bio-One North America, Inc., Monroe, NC) using a Spectra Max plus 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The K of the probes d Values ​​were determined by non-linear regression fitting of the saturation curves and IC50 values ​​of the treatments were determined by non-linear regression fitting of the competition curves.

[0172] Cytotoxicity assayAfter E2-PROTAC treatment, cell viability was measured using the Presto-Blue Viability kit according to the manufacturer's recommended protocol (Thermo Fisher Scientific). Briefly, breast cancer cells were cultured in growth medium for 10 min at 4°C for 1 h. 4 The following day, the contents of the 96-well plates were aspirated and incubated with various concentrations (10 -9 ~10 -3 Treatments were replaced with 90 μL of medium + 10 μL of Prestoblue reagent (range 100 μM) followed by addition of medium to a total volume of 200 μL / well. Untreated controls were incubated with 200 μL / well of fresh medium. After 1 day, medium was aspirated and replaced with 90 μL of medium + 10 μL of Prestoblue reagent followed by incubation at 37°C for 1 h. Fluorescence was excited at 560 nm and emission was recorded at 590 nm using a Spectra Max plus 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Relative cell viability values ​​were expressed as the percentage of fluorescence recorded from wells containing treated cells compared to control wells containing untreated cells.

[0173] Cell cycle analysis 2.5 x 10 cells 5Cells were seeded in 6-well plates at a density of 1000 cells / well and either left untreated or exposed to partial PROTACs, UI-EP001, or UI-EP002 for 24 hours. After treatment, cells were harvested by trypsinization and washed twice with ice-cold PBS. Cells were then fixed in 70% ethanol for 30 minutes at 4°C. Fixed cells were pelleted by centrifugation at 230g for 5 minutes and the cell pellet was incubated in Krishan's solution (3.8 mM sodium citrate (Fisher Scientific), 0.014 mM propidium iodide (AnaSpec, Fermont, Canada), 1% NP-40 (Sigma) and 2.0 mg / mL RNase A (Fisher Scientific)) for 30 minutes at 37°C. Cells were then centrifuged and washed with PBS before analysis using a FacScallibur flow cytometer. Data from flow cytometry was subjected to further analysis by CellQuest software version 3.3. The DNA histograms generated show the percentage of the cell population in sub-G1, G0-G1, S, or G2 / M phases of the cell cycle.

[0174] statistical analysis All data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Normality and homogeneity of variance of all data were analyzed by one-way analysis of variance (ANOVA). Each experiment was performed in triplicate, and differences were considered significant when P < 0.05.

[0175] result Molecular docking analysis of the interaction of ERa or GPER1 with E2-PROTACs Previous studies on the GPER binding pocket and its interaction with E2 have shown that E2 can be modified using PROTAC strategies and still maintain its binding efficacy (Amatt et al., 2012; Amatt et al., 2013). Based on these homology modeling studies, the 17C-hydroxyl group points outside the GPER binding pocket between TM1 and TM7, and N118 2.62 and H3077.37 It is hypothesized that it interacts with the E2 ligand. Previous studies have shown that attachment of a fluorophore at 17C does not adversely affect GPER activity (Filardo et al., 2007). Therefore, two different PROTAC molecules were designed in silico with the von Hippel-Lindau (VHL), E3 ligand (VHL1 / VHL032) attached to the 17-hydroxyl group via a polyethylene glycol (PEG) linker. An ester bond to the 17-hydroxyl group was chosen for ease of synthesis, and two different linker lengths were chosen to explore possible spatial constraints of the GPER-PROTAC. The first molecule, UI-EP001, was designed to have a 14-atom linker between E2 and VHL1, and the second molecule, UI-EP002, was designed to have a 32-atom linker between E2 and VHL1. The linker length of UI-EP001 was based on reported literature, and a linker of 13-14 atoms resulted in high degradation rate and selectivity of the target receptor, whereas the longer linker of UI-EP002 contributed to the improvement of the overall hydrophilicity and flexibility of this compound. Docking studies of UI-EP001 and UI-EP002 with GPER1 show that the original E2 binding pocket is maintained and the linker emerges from the binding pocket between TM1 and TM7 (Figure 23). Based on the crystal structures of known PROTACs and their protein targets, both linker lengths allow UI-EP001 and UI-EP002 to interact with VHL E3 ligase while bound to GPER1 (Famaby et al., 2019; Gadd et al., 2017).

[0176] Although it is known that modification of the 17C position of E2 is tolerant to GPER binding (Thomas et al., 2005), it is unclear whether the designed PROTAC molecules bind well to ERα. Therefore, additional docking studies were performed for UI-EP001 and UI-EP002, along with ERα, using the crystal structure of E2 bound to the ERα ligand-binding domain (PDB:1A52). All known interactions between E2 and ERα are maintained in both UI-EP001 and UI-EP002. Moreover, the linker length of both molecules is long enough for the VHL1 moiety to interact with VHL E3 ligase.

[0177] The synthesis details of partial PROTAC (compound 7) are diagrammed in Figure 24 (Scheme 1). Linker 2 was prepared from triethylene glycol according to the two-step Scheme 1A. VHL E3 ligase ligand (VH032) was synthesized according to the previously published literature (Scheme 1B) (Steinebach et al., 2019; Madak et al., 2017). Palladium-catalyzed cross-coupling between 4-bromobenzonitrile and 4-methylthiazole was performed to give cyanide 3, which was subsequently treated with cobalt(II) chloride and sodium borohydride to give primary amine 4. Compound 7 was subsequently obtained from 4 after amide coupling with Boc-Hyp-OH, Boc-Tle-OH and linker 2. Conjugation between E2 and compound 7 was performed under Steglich conditions to give UI-EP001 or compound 8 (Scheme 2). UI-EP002 (compound 10) was prepared by introducing a PEGS8 linker between E2 and compound 7. First, E2 was esterified with Fmoc-NH-PEG8-CH2CH2COOH, followed by treatment with trimethylamine to remove the Fmoc group to give compound 9. Finally, compound 10 was obtained from the conjugation between compound 9 and compound 7 by careful monitoring by HPLC for 72 h (Scheme 3).

[0178] E2-PROTACs exhibited high-affinity specific binding to plasma membrane and intracellular estrogen receptors GPER and ER each bind E2 with high affinity (Prissnitz et al., 2015). However, each estrogen receptor resides in a different intracellular compartment, which has important implications regarding their relative ability to interact with E2-PROTACs. GPER displays all the characteristics of a plasma membrane receptor, despite the fact that the majority of the receptor is expressed in intracellular membranes (Filardo et al., 2007). In support of its role as a plasma membrane receptor, previous studies have demonstrated that GPER binds to estrogen in sucrose density gradient enriched membrane homogenates suggesting a plasma membrane binding site. 3 Specific binding of H-E2 was measured (Thomas et al., 2005). To directly assess the binding of UI-EP0001 and UI-EP002 to GPER on the plasma membrane, a competitive binding assay was performed using intact SKBR3 cells expressing GPER but not ERa or ERb, using cell-impermeant E2-FITC as a fluorescent tracer (Figure 25). The GPER-specific binding activity of E2-FITC was measured by differential saturation analysis, subtracting the binding activity of cells expressing or not expressing GPER on the cell surface (Figure 25A). Maximum GPER-specific binding reached close to 100 nM E2-FITC (Figure 25B), and this concentration was selected for the competitive binding experiment (Figure 25C). The concentration of E2 required to displace 50% of E2-FITC (IC 50 ) was calculated as the dissociation constant (Kd) of E2 at 100 ± 1.5 nM for 17b-E2, which is consistent with the previously reported IC 50 The IC value of UI-EP001 and UI-EP002 is similar to that of 300 nM (Cao et al., 2017). 50The values ​​were nearly three-fold (RBA=330%) higher than those measured with E2 (30.2±0.9 nM and 30.2±2.3 nM, respectively) (Table 1). This may be explained, in part, by the structural homology shared between E2-FITC and E2-PROTAC, each of which contains a 17C-substituted hydroxyl. In contrast, a partial PROTAC consisting of the chemical spacer of UI-EP001 linked to the VHL E3 ubiquitin ligase recognition motif but lacking the E2 targeting domain was unable to compete for E2-FITC binding even at a concentration as high as 10 mM (Figure 25C).

[0179] [Table 1]

[0180] [Table 2]

[0181] Abbreviations: IC50, concentration for 50% inhibition; all IC50 values ​​were determined from triplicate samples and are reported as the mean ± SD. In contrast to GPER, ERa and ERb are intracellular receptors concentrated in the cytoplasm and nucleus. Therefore, the binding activity of UI-EP001 and UI-EP002ER was assessed using fluorescence polarization (FP) in cytosolic fractions isolated from MCF-7 breast cancer cells. Saturation analysis was performed using 10 nM E2-FITC and increasing concentrations of cytosolic protein (Figure 25D). Based on the near-saturating (80%) FP values, a competitive binding assay was performed using 600 mg / mL of cytosolic protein, where E2 exhibited half-maximal displacement, IC 50 = 5.6 ± 1.4 nM) (Figure 25E). In comparison, the IC 50The calculated values ​​were 17.2±8.2 nM and 11.4±3.3 nM, respectively. These results indicate that UI-EP001 and UI-EP002 exhibit slightly weaker binding affinity compared to E2, with RBAs of 32% and 49%, respectively (Table 1). Taken together, these competitive binding assays, performed on intact cells and cytosolic fractions, show that UI-EP001 and UI-EP002 bind with high affinity to the plasma membrane and intracellular estrogen receptor GPER in the low nanomolar concentration range.

[0182] E2-PROTACs reduce the expression levels of natural and / or recombinant estrogen receptors To assess the efficacy of UI-EP001 and UI-EP002 in degrading plasma membrane and intracellular estrogen receptors, we employed a sensitive Nano-BiT binary luminescence complementation assay that measures the interaction between HiBiT-tagged proteins and soluble Lg-BiT protein. Cells treated with UI-EP001 or UI-EP002 showed a dose-dependent reduction in HiBiT-GPER or HiBiT-ERα (Figures 26A and B), whereas 100 μM of the partial PROTAC showed no significant reduction for either recombinant receptor within the concentrations tested (Figure 26C). These assays revealed DC α-dependent cleavage of GPER (100 μM) and ERα (10-100 μM), respectively. 50 The DC values ​​(concentration required for 50% proteolysis) were calculated. 50 Concentrations were used to measure the degradation rates of HiBiT-GPER and HiBiT-ER by UI-EP001 and UI-EP002. Both UI-EP001 and UI-EP002 induced a time-dependent decrease in the expression levels of either recombinant GPER or ER, with a 50% decrease measured as early as 1 h (Figures 26D and E). In contrast, 83% of the receptors were present after cells were treated with partial PROTACs for 8 h.

[0183] The specificity of UI-EP001 and UI-EP002 for GPER was determined by examining their ability to degrade other GPCRs, including the b1-adrenergic receptor, b1AR, and the chemokine CXC chemokine type 4 receptor, CXCR4 (Figure 27A). To this end, the relative efficacy of UI-EP001 or UI-EP002 or partial PROTACs was evaluated for their efficacy in degrading HA-GPER, -b1AR, or -CXCR4 in stably transfected HEK293 cell lines. Immunofluorescence analysis using HA-specific antibodies measured the specific degradation of HA-GPER without detectable off-target degradation of HA-b1AR and HA-CXCR4 (Figures 27A and B). Next, we evaluated the specificity of UI-EP001 and UI-EP002 to degrade endogenous plasma membrane and intracellular estrogen receptors using human MCF-7 breast cancer cells expressing ERa, ERb, and GPER (Figure 27C and D). MCF-7 cells were exposed to UI-EP001, UI-EP002, partial PROTACs for 1 hour or left untreated, fixed, permeabilized, and immunostained with specific antibodies against GPER, ERa, ERb, or progesterone receptor, PR. A clear site-specific reduction was observed for nuclear ERα and ERβ as well as intracellular GPER (Figure 27C and D). In contrast, treatment of MCF-7 cells with partial PROTACs did not affect the expression of ERa, ERb, and GPER. Neither UI-EP001 nor UI-EP002 showed detectable degradation of PR in MCF-7 cells, further demonstrating the specificity of E2-PROTACs for estrogen receptors (Figure 5D). Moreover, UI-EP001 and UI-EP002, but not partial PROTACs, reduced plasma membrane and total GPER in human SKBR3 breast cancer cells that are negative for ERa and ERb (Figure 27E). Similarly, E2-specific PROTAC-mediated degradation was observed on the surface of MCF-7 cells. The specificity of UI-EP001 and UI-EP002 for their intended targets was further evaluated by examining the ability of exogenous E2 to inhibit E2-PROTAC-mediated ER / GPER degradation (Figures 28A and B).Cells transfected with ER-HiBiT or GPER-HiBiT were treated with 100 μM UI-EP001 and UI-EP002 alone or in combination with increasing doses of E2β or aldosterone (100 nM to 100 μM) for 1 h (Figures 28A and B). E2 effectively inhibited the degradation of UI-EP001 and UI-EP002, whereas no degradation of either estrogen receptor target was measured in the presence of the control steroid, aldosterone (Figure 28C).

[0184] Taken together, these findings suggest that UI-EP001 and UI-EP002 function as estrogen receptor degraders (SERDs) that can selectively degrade plasma membrane and intracellular estrogen receptors.

[0185] Degradation of UI-EP001 and UI-EP002 in the ER and GPER occurs via the 26S-proteasome Finally, we assessed whether UI-EP001 and UI-EP002 could promote degradation of target receptors via the ubiquitin-proteasome pathway. To perform these experiments, HEK293 cells transiently expressing HiBiT-tagged estrogen receptors were pretreated with proteasome inhibitors, MG132, lysosomotropic agents, chloroquine, or vehicle before exposure to UI-EP001 or UI-EP002, after which total receptor expression was assessed as described above. MG132 reversed the degradation of GPER or ERα in UI-EP001 or UI-EP002, whereas chloroquine had no such effect (Figure 28D and E). This result is consistent with the fact that PROTAC-induced protein degradation occurs as a result of receptor polyubiquitination and protein degradation in the 26S-proteasome. Importantly, these results suggest that our first generation E2-PROTACs, UI-EP001 and UI-EP002, interact with estrogen receptors present at the plasma membrane and intracellular compartments and rapidly promote the degradation of membrane (GPER) and nuclear (ER) estrogen receptors in the 26S-proteasome.

[0186] Taken together, these studies suggest that simultaneous engagement of estrogen receptors (ERα and GPER) and VHL E3 ubiquitin ligase by UI-EP001 and UI-EP002 is important for efficient degradation.

[0187] E2-PROTACs inhibit breast cancer cell proliferation by inducing cell cycle arrest To evaluate the cytotoxic potential of E2-PROTACs, we measured the viability of four different human breast cancer cell lines with different estrogen receptor profiles after treatment with UI-EP001, UI-EP002, or partial PROTACs (Figure 29). The study included: i) MCF-7 cells expressing all three estrogen receptors (ERa + , ERb + , GPER + ), ii) SKBR3 cells overexpressing her2 / neu (ERa - , ERb - , GPER + ), iii) triple-negative and GPER-positive HCC1806 cells (ERa - , ERb - , GPER + ) and iv) MDA-MB-231 cells (ERa) that are ERa negative, express low levels of ERb and GPER, and do not induce either ER (Al-Badar et al., 2011) or GPER (Filardo et al., 2000)-dependent signaling activity. - , ERb low , GPER low ). Each breast cancer cell line was treated with various concentrations of E2-PROTAC or partial PROTAC for 24 hours, after which cell viability was assessed using the MTS assay, which measures the mitochondrial oxidoreductase enzyme capable of converting tetrazolium salts into formazan dyes (summarized in Table 2). A dose-dependent decrease in cell viability was measured in all breast cancer cell lines treated with either UI-EP001 or UI-EP002. For UI-EP001, IC values ​​of 9.0, 10.9, and 34.9 mM were observed in MCF-7, SKBR3, and HCC1806 breast cancer cell lines expressing GPER, respectively. 50 Similar IC values ​​were measured.50 IC50 values ​​were measured for UI-EP002 in these three cell lines, respectively (17.0, 11.1, and 7.4 mM). In contrast, IC50 values ​​for UI-EP001 and UI-EP002 were approximately 100-fold higher (>100 mM) in MDA-MB-231 cells expressing low concentrations of functional ERbs or GPER, indicating >80% cell viability. These IC 50 Values ​​were partially similar to those measured in cells treated with PROATAC. These data suggest that ablation of membrane and intracellular estrogen receptors is an important factor determining breast cancer cell viability.

[0188] To investigate biological effects related to reduced cell viability, cell cycle analysis was performed on the same four cell lines using propidium iodide staining and measured by flow cytometry 24 hours after treating cells with 10 μM E2-PROTACs or partial PROTACs (Figure 30). Untreated cells from all four cell lines showed a typical cell cycle distribution pattern with the majority of cells in G1 and a small percentage of cells in S or G2 / M. After treatment with either UI-EP001 or UI-EP002, a dramatic increase in the G2 / M peak was observed in MCF-7, SKBR3, and HCC1806 breast cancer cells, along with a significant decrease in G1. This shift was not observed in MDA-MB-231 cells, which do not express ERa and express only low amounts of functional ERb or GPER, suggesting that E2-PROTACs induce cell cycle arrest at the G2 / M phase by decreasing one or both estrogen receptors. As expected, cells treated with partial PROTACs did not show significant changes in the percentage of the total cell population between G1 and G2 / M. These in vitro results strongly suggest that degradation of ERα and / or GPER may be effective in vivo for the treatment of ER+ as well as ER− breast cancer.

[0189] Consideration The carcinogenic effects of estrogen are manifested through cellular receptors belonging to the nuclear steroid hormone and G protein-coupled receptor superfamily (Filardo et al., 2018). A number of ER-targeting PROTACs have been developed, but their ability to degrade GPER has not yet been tested. Here, proof-of-principle evidence is provided that a single estradiol-based PROTAC can target both receptor classes. Based on in silico modeling and structure-activity relationship data, we designed two first-generation PROTACs (UI-EP001 and UI-EP002) containing estradiol-based targeting domains substituted with 17C-ester-linked 14- or 32-atom-long PEG spacers conjugated to the small molecule VHL E3 ubiquitin ligase recruitment motif (S,R,S)AHPC. Each PROTAC exhibited high binding affinity to nuclear ER and GPER and selective proteasome-dependent degradation activity against GPER and ER without measurable detectable off-target activity. Most importantly, UI-EP001 and UI-EP002 exhibited target-specific cytotoxicity and G2 / M arrest, desirable in vitro properties for a first-generation all-estrogen receptor PROTAC.

[0190] The PROTAC platform has been employed to selectively degrade a wide range of proteins with potential to treat human diseases. These include, but are not limited to, steroid hormone receptors (AR, ERa, ERRa, RAR) (Itoh et al., 2011; Peng et al., 2019; Schneekloth et al., 2008), bromo- and extra-terminal (BET) family proteins (Lu et al., 2015; Winter et al., 2015), kinases (Bondesonet al., 2018; Lai et al., 2016), and microtubule-associated proteins (Tau, TACC3) (Silva et al., 2019). This group has recently been expanded to include GPCRs (Li et al., 2020). The mere fact that PROTACs can efficiently target proteins with unique structural properties that are localized in different subcellular compartments shows their intriguing promise. However, developing a PROTAC that targets a single protein is not a simple task. Ultimately, target optimization is achieved by evaluating various targeting and / or warhead domains and making numerous fine-tuning changes to the length and composition of the chemical spacer, as well as the overall stereochemistry of the entire heterobifunctional molecule. In theory, designing a single PROTAC that can target two structurally and functionally distinct estrogen receptors with unique structures that exist in different physicochemical environments may be a more challenging task, but is necessary for the treatment of ER+ breast cancer, as the majority of these tumors also express GPER. Although various ERa-PROTACs have been developed over the past decade, not a single study has been published that shows whether these compounds can also target the highly related estrogen receptor, ERb, or the more recently described plasma membrane estrogen receptor, GPER.

[0191] The pioneering ER-PROTAC contained a peptide reactive group domain, which resulted in the binding of the E3 ubiquitin ligase SCF b-TCRPThey showed low cell permeability and dependence on cellular enzyme activity to effectively recruit ER (Sakamoto et al., 2003). More recent second generation ER-PROTACs use ER antagonists in their targeting domain and show high cell permeability and degradation capacity at low nanomolar concentrations (Hu et al., 2019: Denizu 2012; Okhira et al., 2013). These latter PROTACs may target GPER as well as ER, but perhaps to a lesser extent due to the expected reduced RBA of E2 compared to ER antagonists of either receptor. Evaluation of drug-target interactions by experimental methodology is a crucial step to evaluate target specificity and selectivity. However, binding properties of ER-PROTACs are not usually measured and in many cases target selectivity is not evaluated. Both UI-EP001 and UI-EP002 have a calculated K d The 17C-substituted E2-FITC showed high affinity for both target estrogen receptors, with K values ​​of 30.2 nM for UI-EP001 and UI-EP002, suggesting that the 17C-substituted E2 targeting domain interacts well with the ligand contact sites of both GPER and ER. Using 17C-substituted E2-FITC and intact SKBR3 breast cancer cells that express GPER and have no detectable ERa or ERb by RT-PCR (Vladusic et al., 1998), the K values ​​for UI-EP001 and UI-EP002 were d values ​​were measured. This is consistent with previous evidence that 1,3,5(10)-estratriene-3,17β-diol 17-hemisuccinate:BSA (E2-BSA) can stimulate GPER-dependent activation of intracellular cAMP (Filardo et al. 2007). However, these findings were not consistent with those of free E2 bound to recombinant ERa or ERb. 125This is in contradiction to a published study showing that I-16a-iodo-E2, but not E2-BSA, was able to displace or induce a gel shift in an electrophoretic mobility assay (Stevis et al., 1999). Free 17b-E2, UI-EP001 or UI-EP002 displaced E2-FITC bound to cytosolic fractions prepared from MCF-7 cell homogenates containing endogenous ERa and ERb, and increased the measured K d The values ​​were 5.6 nM, 17.2 nM, and 11.4 nM, respectively. The reason for the discrepancy between our findings and those of Stevis and colleagues is unclear, but may be related to both the difference in the binding probe and the difference in the multivalency of E2 relative to BSA versus the 1:1 stoichiometry of E2 and FITC. One possibility is that the binding kinetics of E2-FITC to GPER differs from that of 125 Although there are differences compared to I-16a-iodo-E2, which may affect the displacement of the probe, these data suggest that our first generation E2-PROTACs possess the most important characteristic of a new drug: the ability to interact specifically and with high affinity with the intended target.

[0192] By their design, PROTACs are promising cancer therapeutics for selective protein target degradation. The gold standard assay for demonstrating target specificity for a given PROTAC is to compare a global readout of the proteome of treated and untreated cells using mass spectrometry analysis (Beveridge et al., 2020). From a practical perspective, target specificity can best be assessed by evaluating the difference in viability of cancer cells expressing or lacking the cancer therapeutic target. This is particularly important for evaluating bispecific PROTACs designed to selectively degrade ER and GPER. In this study, we evaluated target selectivity using immunofluorescence, biochemical and biological methods. By immunofluorescence analysis, we were able to show selective site-specific reduction of ERa and ERb in the nucleus and for GPER, both at the cell surface and intracellularly. Degradation of intracellular GPER by UI-EP001 or UI-EP002 was observed in the crude membrane fraction and in the plasma membrane fraction enriched by sucrose density centrifugation. 3This is consistent with our previous findings showing GPER-dependent specific displacement of H-17b-E2 (Thomas et al., 2005). Selectivity was demonstrated by the fact that no reduction in PR, CXCR4, and b1AR was measured in E2-PROTAC-treated cells. Similarly, target selectivity was also demonstrated by showing by binary emission complementation that exogenous E2, but not aldosterone, could block E2-PROTAC-mediated degradation of HiBiT-GPER or HiBiT-ER. Finally, the target selectivity of UI-EP001 and UI-EP002 was demonstrated in biological assays comparing their relative effects on viability and cell cycle distribution in various breast cancer cell lines expressing different complements of estrogen receptors. Decreased viability and G2 / M arrest were observed in MCF-7 cells expressing all three estrogen receptors, but not in MDA-MB-231 cells, which lack ERa and express low levels of functional ERb and GPER. Whether this is due to a decrease in ER or GPER, or both, is unclear in these cells. It is noteworthy that others have evaluated the cytotoxicity of ER-PROTACs in ER-positive MCF-7, BT-474, and CAMA-1 breast cancer cell lines, and these cells also express GPER (Kargno et al., 2019; Zhang et al., 2004). Either E2-PROTAC in this study induced cytotoxicity and G2 / M arrest following treatment of ER-negative, GPER-positive SKBR3 and HCC-1806 breast cancer cell lines, which represent her-2 / neu overexpression and TNBC tumor immunophenotypes. While various chemotherapeutic agents exhibit cell cycle arrest at various stages of the cell cycle, the observation that E2-PROTACs induce G2 / M arrest is consistent with previous reports that the androgen receptor (AR)-PROTAC, ARD-61, also induces G2 / M arrest (Zhao et al., 2020). In some respects, the induction of cytotoxic effects and cell cycle arrest by UI-EP001 or UI-EP002 may seem somewhat unexpected given the fact that no complete loss of either ER or GPER was observed.One possible explanation for these results may be related to the fact that GPCRs generally exhibit a hyperbolic relationship between ligand occupancy and receptor response. This relationship is called "fractional occupancy" and suggests why it is not necessary to eliminate the total amount of GPER at the plasma membrane to observe a significant reduction in receptor activity. More importantly, our in vitro findings indicate that E2-PROTACs may have therapeutic potential not only against GPER-expressing breast cancers, but also against some ER-negative breast cancers that are thought to be untreatable by estrogen-targeted therapy.

[0193] Arvinas, LLC has developed PROTACs targeting either the ER (ARV-471) or the AR (ARV-110) with promising success in preclinical mouse models of cancer. Both PROTACs are in Phase I clinical trials (NCT03888612 and NCT04072952 at clinicaltriais.gov) for prostate and breast cancer, respectively (Flanagan et al., 2018; Neklessa et al., 2019). PROTACs have several attractive advantages over current SERDs for the treatment of breast cancer (Neklessa et al., 2019). Because PROTACs are catalytic in nature, they are ideally suited to overcome acquired drug resistance due to overexpression or mutations, one of the main limitations of endocrine therapy (provided the ubiquitination acceptor site is unaltered). Indeed, ARV-471 has been demonstrated to efficiently degrade clinically relevant ERα mutants (Y537S and D538G) in cell lines and PDX models (Flanagan et al., 2018). Because the targeting domain of ARV-471 is private and proprietary, it is unclear whether it can also target GPER. Similarly, many other ER-PROTACs have been developed, and it is unclear whether these PROTACs also target GPER. The development of cancer therapeutics targeting GPER is crucial, since GPER is expressed in 60% of ER-positive breast cancers (Filardo et al., 2008), associated with clinical variables predictive of advanced cancer, commonly expressed in ER-negative breast cancers and TNBC, and associated with resistance to anti-estrogen therapy. Total estrogen receptor PROTACs, such as E2-PROTACS, UI-EP001, and UI-EP002, may be ideal for this purpose and would complement existing anti-estrogen therapies.

[0194] Example 3 Scheme

[0195] [ka]

[0196] Synthesis procedure Synthesis of UI-EP001(8)

[0197] [ka]

[0198] Di-tert-butyl 3,6,9,12-tetraoxatetradecanedioate (1). A mixture of triethylene glycol (1.50 g, 1.36 ml, 10 mmol, 1 eq.), 60% NaH in mineral oil (800 mg, 20 mmol, 2 eq.) and tert-butyl bromoacetate (4.50 g, 3.4 ml, 20 mmol, 2 eq.) was dissolved in 10 mL of dioxane. The reaction mixture was stirred overnight and then quenched with saturated NH4Cl. The mixture was extracted with EtOAc, dried over Na2SO4 and then concentrated in vacuo. The product was subjected to flash chromatography (hexane:EtOAc=8:2) to give compound 1 as a colorless oil. Yield: 3.25 g, 8.60 mmol (92%). 1 H NMR (300MHz, CDCl3): δ3.99 (s, 4H), 3.72-3.66 (m, 16H), 1.46 (s, 9H). C 18 H 34 O8[M+1] + HRMS(ESI+) calculated: 379.23, found: 379.46.

[0199] [ka]

[0200] Di-tert-butyl 3,6,9,12-tetraoxatetradecanedioate (2). A solution of compound 1 (1.74 g, 4.62 mmol) in 50% v / v trifluoroacetic acid (TFA) in DCM (6 mL per mmol) was stirred at room temperature for 2 h. TLC analysis (10% methanol in DCM) showed complete conversion of the starting material. The reaction mixture was then concentrated under vacuum and the crude product was lyophilized to give the desired product 2 (quantitative yield).1 H NMR (300MHz, CDCl3): δ4.03(s,4H),3.85-3.42(m,16H). C 10 H 18 O8[M+1] + HRMS(ESI+) calculated: 267.10, found: 267.56.

[0201] [ka]

[0202] 4-(4-Methylthiazol-5-yl)benzonitrile (3). A mixture of 4-bromobenzonitrile (5.00 g, 27.5 mmol), 4-methylthiazole (4.98 mL, 54.7 mmol), KOAc (5.40 g, 55.0 mmol), and palladium acetate (62.0 mg, 0.27 mmol) was dissolved in 20 mL of DMAc. The mixture was heated to 120° C. overnight, cooled, and diluted with EtOAc. The solution was washed with brine, dried over Na2SO4, and concentrated. The resulting oil was subjected to flash chromatography (hexane:EtOAc=9:1→1:1→1:9). Compound 3 was obtained as a yellow solid (4.49 g, 22.5 mmol, 82%). 1 H NMR (300MHz, CDCl3): δ8.76 (s, 1H), 7.75-7.71 (m, 2H), 7.60-7.55 (m, 2H), 2.58-2.56 (m, 3H). C 11 H8N2S[M+1] + HRMS(ESI+) calculated: 201.04, found: 201.36.

[0203] [ka]

[0204] (4-(4-Methylthiazol-5-yl)phenyl)methanamine (4). Compound 3 (4.49 g, 22.5 mmol) was dissolved in 300 mL of anhydrous MeOH. Cobalt chloride (CoCl2) (4.39 g, 33.75 mmol) was added and the solution was cooled in an ice bath for 30 min. NaBH4 (5.22 g, 138 mmol) was added in portions over 20 min. The mixture was stirred for an additional 90 min and quenched with cold H2O. The mixture was filtered, diluted with H2O, and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, concentrated, and purified by flash chromatography (DCM:MeOH=99:1→90:10, 0.5 M Et3N). Compound 4 was obtained as a white powder (3.44 g, 16.88 mmol, 75%). 1 H NMR (300MHz, DMSO-d6): δ8.94 (s, 1H), 7.42 (m, 4H), 3.77 (s, 2H), 2.45 (s, 3H). 13 C NMR (300MHz, DMSO-d6) δ152.23,147.98,145.01,131.89,129.95,129.01,127.35,45.23,15.98. C 11 H 12 N2S[M+1] + HRMS(ESI+) calculated: 205.07, found: 205.77.

[0205] [ka]

[0206] (4R)-4-Hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-Boc-pyrrolidine-2-carboxamide (5). A mixture of compound 4 (2.04 g, 10.00 mmol), Boo-Hyp-OH (2.31 g, 10.00 mmol), DIPEA (6.95 mL, 40.00 mmol), and HBTU (4.16 g, 11.00 mmol) was dissolved in 50 mL of anhydrous DMF. The mixture was stirred at room temperature overnight, then diluted with H2O and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (DCM:MeOH=99:1→90:10). Compound 5 was obtained as a colorless oil (2.79 g, 6.7 mmol, 67%). 1 H NMR(300MHz,CDCl3)δ8.69(s,1H),8.28-8.04(m,1H),7.45-7.32(m,4H),4.52-4.44(m,3H),4.10(t,1H) ,3.10-3.00(m,1H),2.88-2.77(m,1H),2.54(s,3H),2.41-2.32(m,1H),2.07-1.93(m,1H),1.43(s,9H). 13 C NMR (300MHz, CDCl3) δ172.15,153.88,152.12,148.02,139.75,131.52,130.66,129.05,128.05,77.25,65.48,55.98,53.95,42.16,28.96,14.86. C 21 H 27 N3O4S[M+1] + HRMS(ESI+) calculated: 418.17, found: 418.03.

[0207] [ka]

[0208] (4R)-1-((S)-2-Boc-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6). Compound 5 (2.79, 6.7 mmol) was dissolved in 50% v / v TFA / DCM (6 mL per mmol) and stirred at room temperature for 2 h. The mixture was concentrated and used in the next step without further purification. A mixture of the product of the last step, Boc-L-tert-leucine (1.54 g, 6.7 mmol), HBTU (2.79 g, 7.37 mmol), and DIPEA (4.66 mL, 26.8 mmol), was dissolved in 50 mL of DMF. The mixture was stirred at room temperature overnight, diluted with H2O, and extracted with EtOAc. The organic layer was washed with saturated NaHCO3 solution, brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash chromatography (DCM:MeOH=99:1 to 90:10) to give compound 6 as a white powder (2.09 g, 3.95 mmol, 59% yield after two steps). 1 H NMR(300MHz,DMSO-d6)δ8.98(s,1H),7.56-7.50(m,4H),4.91-4.86(m,4H),4.08(s,1H),3 .36-3.31(m,1H),2.58(s,3H),2.34-2.30(m,1H),2.13-2.07(m,1H),1.20-1.02(m,18H). 13 C NMR(300MHz,DMSO-d6)δ173.12,170.15,157.23,153.48,148.16,139.68,132.36,130.12,12 8.79,125.56,75.69,65.26,58.56,55.23,53.29,42.56,38.26,36.18,27.48,25.42,15.99. C 27 H 38 N4O5S[M+1] + HRMS(ESI+) calculated: 531.26, found: 531.49.

[0209] [ka]

[0210] Partial PROTAC (7). Compound 5 (2.09, 3.95 mmol) was dissolved in 50% v / v TFA / DCM (6 mL per mmol) and stirred at room temperature for 2 h. The mixture was concentrated and used in the next step without further purification. A mixture of the product of the last step (1 eq), compound 2 (2.1 g, 7.9 mmol, 2 eq), HBTU (1.65 g, 4.30 mmol), and DIPEA (2.75 mL, 15.8 mmol) was dissolved in 50 mL of DMF. The mixture was stirred at room temperature overnight. TLC indicated complete reaction of the starting material, so the mixture was diluted with DCM, washed with brine, dried, and concentrated. The product was purified by HPLC reverse phase column (Zorbax300SB-C18, 21.2 x 150 mm, 5 uCrt) to give the desired product 7. Yield: (1.21 g, 46% after two steps). 1 H NMR(500MHz,MeOD-d4)δ9.68(s,1H),7.56-7.50(m,4H),4.67.-4.69(t,1H),4.58-4.55(m,2H),4.43-4.40(d,1H),4.15-4.11(m,4H),4.08-4 .05(m,17H),3.78-3.80(m,1H),3.85-3.71(m,1H),3.36-3.31(m,1H), 2.58(s,3H),2.34-2.30(m,1H),2.13-2.07(m,1H),1.20-1.02(m,9H). 13 C NMR(500MHz,MeOD-d4)δ172.97,170.73,170.31,151.43,147.69,138.87,132.01,130.15,129.00,127.58,70.93, 70.37,70.24,70.20,69.77,69.73,69.66,59.41,56.75,56.69,50.39,42.33,37.53,37.70,29.26,25.56,14.40. C 31 H 44 N4O 10 S[M+1] + HRMS(ESI+) calculated: 679.29, found: 679.89.

[0211] [ka]

[0212] UI-EP001 (8). Compound 7 (300 mg, 0.46 mmol) was activated by N-hydroxysuccinimide (NHS) (53 mg, 0.46 mmol) in 5 mL of DMF for 3 h, followed by the addition of 17β-estradiol (125 mg, 0.46 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (71.3 mg, 0.46 mmol). The reaction mixture was stirred at room temperature for 48 h and monitored by reverse phase HPLC. The crude product was diluted with DCM:MeOH (90:10), washed with brine, dried and concentrated. The product 8 was obtained by HPLC reverse phase column (Zorbax300SB-C18, 21.2×150 mm, 5 uCrt). Yield (119 mg, 28%). 1 H NMR(500MHz,MeOD-d4)δ9.64(s,1H),7.56-7.49(m,4H),7.09-7.07(d,1H),6.55-6.53(m,1 H),6.48-6.49(d,1H),4.71-4.67(t,1H),4.58-4.55(m,2H),4.12-3.95(m,17H),3.86-3.8 4(d,1H),3.74-3.71(m,1H),3.68-3.65(t,1H),2.78-2.77(m,2H),2.57(s,3H),2.32-2.30 (m,2H),2.14-1.95(m,4H),1.74-1.67(m,1H),1.54-1.18(m,7H),1.15(s,9H),0.78(m,3H). 13 C NMR(500MHz,MeOD-d4)δ173.12,170.15,157.23,153.48,148.16,139.68,132.36,130.12,12 8.79,125.56,75.69,65.26,58.56,55.23,53.29,42.56,38.26,36.18,27.48,25.42,15.99. C 50 H 68 N4O 11 S[M+1] + HRMS(ESI+) calculated: 933.46, found: 933.23.

[0213] Synthesis of UI-EP002(10)

[0214] [ka]

[0215] Compound (9). Fmoc-NH-PEG-CH2CH2COOH (300 mg, 0.3 mmol) was activated with N-hydroxysuccinimide (NHS) (34.5 mg, 0.3 mmol) in 1 mL of DMF for 3 h, followed by the addition of 17β-estradiol (81.6 mg, 0.3 mmol) and EDC (46.6 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 72 h and monitored by reversed-phase HPLC. The crude product was purified by HPLC reversed-phase column (Zorbax300SB-C18, 21.2×150 mm, 5 uCrt) to give compound 9. Yield (41 mg, 16%). 1 H NMR(500MHz,MeOD-d4)δ7.12-7.11(d,1H),6.59-6.57(m,1H),6.52-6.51(d,1H), 4.26-4.24(m,1H),3.77-3.74(t,2H),3.71-3.56(m,31H),3.34-3.32(m,2H),2.8 3-2.80(m,2H),2.59-2.56(t,2H),2.36-2.31(m,1H),2.17-2.14(m,1H),2.09-2. 05(m,1H),2.01-1.97(m,1H),1.92-1.88(m,1H),1.59-1.19(m,8H),0.81(s,3H). 13 C NMR(500MHz,MeOD-d4)δ171.62,152.12,141.58,138.85,135.04,128.85,125.00,123.42,122.38,117.17,112.27,109.95,78.73,67.76,6 7.74,67.67,67.59,67.53,67.14,64.07,63.81,47.54,40.59,38.02 ,36.76,34.26,32.13,26.94,26.93,26.88,24.75,23.83,20.25,7.90 C 51 H 69 NO13 [M+1] + HRMS(ESI+) calculated: 905.11, found: 905.19.

[0216] [ka]

[0217] UI-EP002 (10). The Fmoc group of compound 9 (35.0 mg, 0.39 mmol) was removed by Et3N (1 mL) in DMF (5 mL). Et3N was removed from the reaction mixture by rotary evaporation, followed by addition of partial PROTAC7 (264.4 mg, 0.39 mmol), HATU (178.6 mg, 0.47 mmol) and DIPEA (0.271 mL, 1.56 mmol). The reaction mixture was stirred at room temperature for 48 hours and monitored by reversed-phase HPLC. The product 10 was obtained by HPLC reversed-phase column (Zorbax300SB-C18, 21.2×150 mm, 5 uCrt). Yield (26 mg, 52%). 1 H NMR(500MHz,MeOD-d4)δ9.69(s,1H),7.57-7.51(m,4H),7.12-7.11(d,1H),6.59-6.57(m,1H),6.52-6.51(d,1H),4.71-4.68(m,1H), 4.59-4.56(d,1H),4.44-4.40(d,1H),4.26-4.24(m,1H),4.09(s,1H),3.88-3.86(m,1 H),3.77-3.74(t,2H),3.71-3.56(m,47H),3.36-3.32(m,6H),2.83-2.23(m,2H),2.59- 2.56(m,5H),2.36-2.31(m,1H),2.21-2.14(m,1H),2.10-2.03(m,1H),2.01-1.97(m,1 H),1.92-1.88(m,1H),1.76-1.70(m,1H),1.43-1.30(m,8H),1.15(s,9H),0.81(s,3H). 13C NMR(500MHz,MeOD-d4)δ174.01,167.15,154.52,143.97,141.25,137.43 ,131.25,127.39,126.77,125.81,124.81,124.78,119.56,114.67,112. 34,81.12,70.16,70.13,69.98,69.93,69.54,66.47,66.21,49.94,42.9 8,40.41,39.16,34.52,29.33,29.32,29.17,27.14,26.22,22.64,10.30. C 68 H 103 N5O 20 S[M+1] + HRMS(ESI+) calculated: 1342.69, found: 1342.15.

[0218] E 2 Synthesis of -FITC (12)

[0219] [ka]

[0220] Compound (11). 17α-Ethynylestradiol (592 mg, 2 mmol) was added to a mixture of 4-(t-butyloxycarbonylaminomethyl)iodobenzene (778 mg, 2.33 mmol), tetrakis(triphenylphosphine)palladium Pd(Ph3)4 (5 mol%), and Cul (5 mol%) in Et3N (20 ml) under argon atmosphere. The reaction mixture was stirred at room temperature overnight, and then the solvent was reduced in vacuo. The crude product was purified by flash chromatography (hexane:EtOAc=20:80→DCM:MeOH=90:10) to give a yellow powder product. Yield (756 mg, 78%). 1H NMR(300MHz,MeOD-d4)δ7.43-7.41(m,2H),7.28-7.16(m,2H),6.67-6.64(m,1H),6.59-6.58(d,1H),4 .71-4.67(t,1H),4.33-4.31(d,1H),2.83(m,2H),2.43-1.72(m,15H),1.57-1.38(s,9H),0.94(s,3H). 13 C NMR(500MHz,MeOD-d4)δ155.12,143.25,131.59,130.98,125.16,124.33,113.25,113.21,95.2 3,84.03,77.69,62.22,49.96,45.11,42.68,35.66,33.22,27.22,26.45,25.56,24.21,13.56. C 32 H 39 NO3[M+1] + HRMS(ESI+) calculated: 486.29, found: 486.85.

[0221] [ka]

[0222] E2-FITC (12). A solution of compound 11 (485 mg, 1 mmol) in 50% v / v TFA / DCM was stirred at room temperature for 2 h. After TLC showed complete conversion, the mixture was coevaporated with DCM 5-6 times to remove TFA. The residue was redissolved in DMF (5 mL), followed by addition of FITC (429 mg, 1.1 mmol) and pyridine (0.25 mL). The reaction mixture was stirred overnight at room temperature in the dark. After drying, the crude product was purified by flash chromatography (CHCl3:MeOH=95:5→90:10) to give compound 12 as a yellow oil (355 mg, 45%). 1H NMR(300MHz,MeOD-d4)δ10.23(s,1H),8.05(m,1H),7.83-7.79(m,1H),7.43-7.41(m,2H),7.27(m ,2H),6.68-6.53(m,8H),4.70(t,1H),4.32(m,1H),2.85(m,2H),2.40-1.53(m,15H),0.93(s,3H). C 48 H 42 N2O7S[M+1] + HRMS(ESI+) calculated: 791.27, found: 791.28.

[0223] References

[0224] [Table 3-1]

[0225] [Table 3-2]

[0226] [Table 3-3]

[0227] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with respect to certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that the specific details herein may be varied considerably without departing from the underlying principles of the invention.

Claims

[Claim 1] The invention described in the specification.

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