Developing Orally Active Estrogen Receptor Degraders by Conjugation of Boronic Tamoxifen and Cereblon Ligands

Abstract

Degradation of estrogen receptor has been clinically proven effective in treating ER+ breast cancer. Proteolysis-Targeting Chimeras (PROTACs) can degrade a target protein by engaging both the target protein and the E3 ligase, bringing them into close physical proximity to effect ubiquitination and proteasomal degradation. We report the design and construction of ER PROTACs that link tamoxifen derivatives to a CRBN E3 ligand to achieve ER protein degradation. The ER degrading efficacy of the synthesized PROTACs was found to depend on the ER binding affinity, the linker structure, and the E3 ligand. Tamoxifen-4-boronic acid conjugated to lenalidomide through a rigid piperidine-methylene-piperazine linker was found to yield degraders that show nanomolar antiestrogenic potency and excellent oral bioavailability. The most active ER degrader with good pharmacokinetic profile, 4r, showed remarkable in vivo efficacy in blocking ER+ (both non-mutant and Y537S mutant) breast tumor growth as a monotherapy or in combination with CDK4/6 inhibitors.

Graphic Abstract

1. Introduction

Degradation of a disease-driving protein as a therapeutic modality has been actively pursued in the clinic for a variety of oncology and immune disorders [1–5]. Unlike an inhibitor which blocks the function of the target protein by occupying its active site, a degrader works by removing the target protein altogether to achieve a more durable blockade of its function. The only clinically validated degrader is a small molecule antagonist of the estrogen receptor which upon binding causes destabilization and subsequent degradation of the ER [6–7]. This compound, fulvestrant, known as a selective estrogen receptor degrader (SERD), has been successfully used for over 20 years to treat ER dependent breast cancer at various stages [8–9].

Clinical effectiveness of ER degradation motivated the development of orally bioavailable SERD molecules in the hope that oral SERDs can improve and extend the limited clinical benefits currently achieved by the injection-only fulvestrant. A well-known fulvestrant limitation lies in the sub optimal level of drug exposure, due in large part to its poor bioavailability, and the month-long time to reach steady-state plasma concentration, leading to incomplete receptor degradation and possibly reduced clinical response [10–11]. To address the challenges facing fulvestrant, another novel approach has recently gained momentum where a proteolysis targeting chimera (PROTAC) molecule is used to degrade a target protein by linking a ligand that binds the target protein with an E3 ligase to achieve ubiquitin proteasome system (UPS) mediated degradation. PROTAC degraders are heterobifunctional small molecules that bind to a target protein on one end of the molecule and an E3-ubiquitin ligase on the other end, thus bringing the two to close proximity and enabling ubiquitination and degradation of the target protein by the proteasome [12–13]. The bifunctional PROTAC molecules consist of three moieties with each empirically optimized to achieve degradation of the target protein with high selectivity and potency. Such PROTAC molecules have been reported to effectively degrade the estrogen receptor and demonstrate therapeutic efficacy in breast cancer models [14–15].

We report the rational design and synthesis of a series of PROTAC degraders of the estrogen receptor by conjugating tamoxifen-4-boronic acid binding motif and a CRBN E3 ligand. We explored the effect of boronic acid modification in the ER binding core structure on the ER degrading and antagonizing activities. We also investigated the effect of linker structure and length on the overall PROTAC properties. Our work identified highly potent ER degrading PROTAC molecules that are effective in blocking ER transcriptional activities, inhibiting ER+ breast cancer cell growth, and degrading ER protein levels. Moreover, in vivo studies of a lead ER degrader showed potent efficacy in inhibiting tumor growth in multiple xenograft breast cancer models.

2. Results and discussion

2.1. Structural Analysis of the ER Targeting Warhead and E3-Recruting Ligand.

As our target ER PROTACs are based on the ER-targeting warhead tamoxifen/triphenylethylene and the CRBN E3 ligase ligand thalidomide/lenalidomide, we analyzed the crystal structures of ER in complex with 4-hydroxytamoxifen (PDB ID: 3ERT) and E3-ligase CRBN in complex with lenalidomide (PDB ID: 4CI2) to find the solvent exposed positions on both ligands for linker attachment. Figure 1 illustrates the crystal structures of these complexes within their respective binding sites. In the case of lenalidomide, the solvent exposed benzene ring on the isoindoline group of the molecule, especially the carbon atoms at positions 5 and 6, marked in red circle, are found to be ideal positions for linker attachment to the CRBN ligand. Similarly, in the case of 4-hydroxytamoxifen, the solvent exposed dimethylamine group of the molecule was found to be a good position for linker attachment to the ER warhead. In this work, we used the proposed linker attachment sites on ER warhead and CRBN ligand to connect linkers of varying length, composition, and functionality for developing a series of PROTAC molecules.

Figure 1.

Binding of E3 recruiting ligand and ER targeting ligand to CRBN and ER, respectively. (A) Crystal structure of CRBN E3 ligase in complex with lenalidomide (PDB: 4CI2), and (B) crystal structure of ER in complex with 4-hydroxytamoxifen (PDB: 3ERT). Parts of the ligand molecules exposed to the solvent and marked in red circles are the sites for linker attachment. Important amino acids in the binding pockets are displayed in stick models and labeled.

2.2. Construction of ER degraders by conjugating tamoxifen derivatives and CRBN ligands

We selected triphenylethylene (tamoxifen) as the warhead for the targeted protein, estrogen receptor. Tamoxifen is a well-established ER-binding ligand that acts as a selective estrogen receptor modulator (SERM) in the treatment of ER+ breast cancer. The 4-hydroxylated metabolites, 4-hydroxytamoxifen (4-OHT) and endoxifen bind the ER with much higher affinity (up to 100-fold), hence are mainly responsible for tamoxifen’s therapeutic effect in ER-positive breast cancer. Thus using 4-hydroxylated tamoxifen structure in the warhead would conceivably increase the potency of the resulting degrader. However, the phenolic (hydroxyl) group make the compounds more susceptible to rapid conjugation (glucuronidation/sulfation) in the liver, resulting in significant first-pass metabolism and poor oral bioavailability. With this consideration, we also seek to modify the 4-hydroxy group to a boronic acid so as to maintain the overall binding affinity to the LBD of ER while improving the metabolic stability and bioavailability of the warhead moiety [16–18].

To evaluate ER-targeting activities we used three essential screening assays. First, we employed the estrogen receptor response element (ERE) luciferase assay to measure the transcriptional activity of estrogen receptor (ERα) in response to antiestrogens or ER degraders that modulate estrogen receptor signaling. By placing a luciferase reporter gene under the control of a promoter/enhancer region containing an ERE, receptor inhibition can be quantified based on the luminescent signal produced. Second, we measured degradation activity of the ER degraders in ER+ breast cancer cells (e.g., MCF-7) by Western blot analysis. Cells were grown to 70% confluence before being treated with the ER degrader compounds at various concentrations for 4–24 hours. Extracted proteins were analyzed by Western blotting for ER levels in response to treatment. Third, cell growth inhibition assays were conducted on select ER degraders in MCF-7 breast cancer cells.

Starting with the triphenylethylene core, we built a series of chimeric molecules with rigid linkers and thalidomide or lenalidomide as CRBN ligands (Table 1). In this series (1a – 1e), the degrader with tamoxifen warhead (R = H, 1b) exhibited the weakest antiestrogen activity (IC₅₀ = 364 nM), consistent with its ER degradation potency (DC₅₀ =104 nM). 4-Hydroxy substitution increased the antiestrogen potency and ER degradation activity by 10 fold (1c compared to 1b). Similarly, boronic acid substituted 1d (Z isomer) and 1e (E isomer) showed enhanced inhibition of ER activity and degradation of the receptor. For comparison, the antagonist activities of the warhead molecules (tamoxifen, 4-OHT, and tamoxifen-4-boronic acid) were also measured in the ERE luciferase assay. As reported, both 4-OHT and tamoxifen-4-boronic acid showed 50–60-fold higher in vitro potency than tamoxifen [18–20].

Table 1.

Anti-estrogen and ER degradation activities of ER PROTACs using a triphenylethylene warhead

Compound	R	X	Linker	ERE Luciferase IC50 (nM)	ER DC50 (nM)	ER Dmax %
1a	OH	C=O		44.2±6.5	12	90
1b	H	CH2		364±102	104	86
1c	OH	CH2		59.8±35.1	9.5	89
1d	B(OH)2	CH2		33.1±3.5	15	92
1e	B(OH)2	CH2		12.1±3.4	19	90
2a	OH	C=O		82.6±20.5	9.3	94
2b	OH	CH2		9.2±0.2	12	96
2c	B(OH)2	CH2		13.7±3.4	10.9	92
3a	OH	C=O		28.7±2.8	1.5	95
3b	B(OH)2	C=O		37.9±4.9	1.3	96
3c	H	CH2		24.4±3.4	10.8	93
3d	OH	CH2		4.0±0.7	1.6	97
3e	B(OH)2	CH2		3.7±0.8	1.5	95
Tamoxifen				262
4-Hydroxytamoxifen				4.3
Tamoxifen-4-boronic acid				5.7

Increasing the linker length to an ethyl group appears to have little effect on the antiestrogenic activity as seen in 2a - 2c. However, when the ethyl linker becomes methyl amine, significant improvements in ER degradation activities were achieved. For example, compared to 2a, the methyl amino linked 3a showed over 5-fold greater degradation potency (DC₅₀=1.5 nM). When E3 ligase binder is lenalidomide (X = CH₂), both antiestrogen activity and ER degradation were further enhanced to reach low nanomolar potency. As in series 1 and 2, despite varying potency as antiestrogens and ER degraders, 3 compounds all showed similar maximum degradation efficiency (D_max = 86–95%).

To determine how ER degradation affect ER-dependent breast cancer cell growth, we conducted cell growth inhibition assays on select ER degraders in MCF-7 breast cancer cells. As shown in Figure 2, compound 1b, 1c, and 1d, representing H, -OH, and -B(OH)₂ substituted warhead, inhibited MCF-7 cell growth in a dose dependent manner that is well in line with their respective degradation activities. Similarly, more potent degrader compound 3c, 3d, and 3e, showed proportionally stronger activities against ER-dependent cell growth.

Figure 2.

Anti-proliferative and ER degradation activity profiles of select ER degraders 1b, 1c, 1d, 3c, 3d, and 3e.

We next used a tamoxifen motif with the ethoxy side chain retained to preserve maximal structural fidelity in the warhead. Linkers were varied to include a greater scope of length, rigidity and polarity while the E3 ligand remains thalidomide or lenalidomide. Compared to 1a – 3e, series 4 compounds have generally greater antiestrogenic and ER degradation activities, suggesting a significant contribution of the ethoxy group to effective ER engagement. The ER-targeting activities and degrader structures are summarized in Table 2.

Table 2.

Antagonist and ER degradation activities of tamoxifen-based ER PROTACs

Compound	R	X	Linker	ERE Luciferase IC50 (nM)	ER DC50 (nM)	ER Dmax %
4a	OH	C=O		68.8±19.9	23	71
4b	B(OH)2	CH2		51.4±6.4	12	92
4c	H	C=O		148.9±27.8	66	78
4d	OH	C=O		14.2±0.8	53	84
4e	B(OH)2	C=O		29.9±3.1	51	83
4f	OH	C=O		36.1±6.3	>1000	<10
4g	B(OH)2	C=O		72.3±7.7	>1000	<10
4h	OH	C=O		19.2±3.0	14	91
4i	B(OH)2	C=O		16.5±1.8	19	73
4j	OH	C=O		20.8±2.9	5.3	90
4k	B(OH)2	C=O		23.0±3.1	8.2	88
4l	OH	C=O		45.0±5.1	17	79
4m	B(OH)2	C=O		81.1±16.2	24	89
4n	OH	C=O		7.1±0.6	13	76
4o	B(OH)2	C=O		15.1±1.5	15	80
4p	H	CH2		29.5±5.7	50	85
4q	OH	CH2		1.8±0.2	1.3	98
4r	B(OH)2	CH2		1.9±0.3	1.4	98
4s	OH	C=O		14.6±2.4	4.8	94
4t	B(OH)2	C=O		13.8±5.9	5.2	91
4u	OH	CH2		35.5±6.2	4.4	97
4v	B(OH)2	CH2		44.1±3.8	10	96
4w	OH	C=O		10.2±2.4	1.8	93
4x	B(OH)2	C=O		63.3±8.8	5.3	95
4y	OH	C=O		78.8±8.7	1.6	98
4z	B(OH)2	C=O		79.8±22.3	1.5	98
4aa	OH	C=O		26.4±1.9	1.4	94
4bb	B(OH)2	C=O		71.4±18.5	1.6	95

Flexible alkyl linker with ample length range afforded moderate anti-estrogenic activities, in line with their ER-degrading potencies as measured by DC₅₀ and D_max values (4a – 4e). Minimal linker length requirement for degradation activity was apparent in compounds 4f and 4g where a single piperazine linker was insufficient to accommodate the formation of a ternary complex, leading to no degradation activity. Optimal linker length appeared to be reached in 4l – 4r where methylene linked piperidine and piperazine afforded some of the most active compounds. However, this optimal linker length would lose significant potency when the two heterocyclic rings switch position, as in 4l and 4m. This small but meaningful SAR suggests an important feature unique to PROTAC mode of action where all three components of the degrader molecule coordinate to achieve optimal activity. Further increase in linker length and flexibility, as in compounds 4s – 4bb, did not yield greater antiestrogen activities, instead, approximately 5 to 10-fold decline in ERE activity and ER degrading potency was observed. Here, the most potent degraders 4q and 4r, comprise a lenalidomide CRBN ligand and a 4-hydroxy or 4-boronic acid derivative. They are about 5-fold more potent than thalidomide ligand (4n, 4o), and about 10-fold more potent than tamoxifen ligand (R = H, 4p).

2.3. Comparison of the binding of ER warheads in 4p, 4q and 4r to the LBD of Erα.

Figure 3 shows the ER ligand part of 4p, 4q and 4r with no functional group, a hydroxy group, and a boronic acid group, respectively, on the phenyl ring at position 4 (C-4) and their binding to the ligand binding domain of ER. When the substitution was an -OH group (4q), positioning of the triphenylethylene scaffold in the binding site was found to be identical to that of 4p with no substitution at C-4, but slightly shifted when the substitution was a -B(OH)₂ group (4r). However, both substitutions -OH in 4q and -B(OH)₂ in 4r were found to make the conserved hydrogen bond interactions with Glu353 and Arg394, as commonly observed in many ER antagonists and agonists, including 4-hydroxytamoxifen [21–25]. The observed hydrogen bonding in 4r was made possible because of the smaller size of boron, requiring a slight shifting of the whole molecule by about 1 Å along the binding pocket. In our previous study also, we observed the boronic acid groups in ZB716 (an analog of ER ligand fulvestrant in which the 3-OH group was replaced by a -B(OH)₂) and in GLL398 (an analog of ER ligand GW-7604 where the 4-OH was replaced by a -B(OH)₂) retained the conserved hydrogen bonding with Glu353 and Arg394 in the ER ligand binding pocket [26–27]. The docking scores indicate that the boronic acid derivative of tamoxifen (4r) has a better binding affinity (−10.87 kcal/mol) than tamoxifen (4p) without any substitution (−9.38 kcal/mol), as reflected in the observed IC₅₀ in ERE luciferase assays.

Figure 3:

Binding of ER ligands to the ligand binding domain of Erα. ER ligand part of PROTACs 4p, 4q and 4r; blue – ER ligand in 4p with R = H, green – ER ligand in 4q with R = OH, and purple – ER ligand in 4r with R = B(OH)₂. Important amino acids in the binding pockets are shown in stick models.

2.4. Antiproliferative activity of ER degraders

The antiestrogen and ER degradation activities are ultimately translated to cellular efficacy of inhibiting ER driven cell growth. We next determined the IC₅₀ values of select series 4 degraders in inhibiting MCF-7 cell proliferation. Figure 4 shows the effect of linker length on degrader’s cellular potency where linker varied from a single piperazine (4g), piperidine and piperazine (4i), methylene-linked piperidine and piperazine (4m), two ethyl-linked piperazines (4t), to two propyl-linked piperazines (4bb). As the linker progressively increased in length and flexibility, the inhibition of cell growth and degradation of the estrogen receptor increased proportionally. Importantly, 4g with the shortest linker and no ER degradation activity, retained moderate antiproliferative potency (IC₅₀ = 250 nM), indicating that the antiestrogenic activity stems entirely from ER binding, but not from ER degradation, consistent with its activity observed earlier in ERE luciferase assays (IC₅₀ = 72.3 nM). Replacing thalidomide with lenalidomide as CRBN ligand resulted in more potent degrader compounds 4p (R=H), 4q (R=OH), and 4r (R=B(OH)₂), showed proportionally stronger activities against ER-dependent cell growth (Figure 4).

Figure 4.

Anti-proliferative and ER degradation activity profiles of select ER degraders 4g, 4i 4m, 4t, 4bb, 4p, 4q, and 4r.

We next tested the most active compound 4r for its antiproliferative and ER degrading activity in CAMA-1 breast cancer cells (Figure 5). Compared to fulvestrant and ARV-471, a clinical stage ER PROTAC, 4r exhibits similar potency in inhibiting ER-dependent cell growth (Figure 5A) and degrading the ER protein (Figure 5C). In contrast, the ER degrader, like ARV-471 and fulvestrant, shows low cytotoxicity in MCF-10A, a non-tumorigenic cell line (Figure 5B).

Figure 5.

A. Dose dependent effect of ER degraders on the cell viability of CAMA-1 breast cancer cells, B. Effect of ER degraders on the viability of non-tumorigenic, MCF-10A cells, C. ER degradation by compound 4r in comparison with clinical ER PROTAC ARV-471 in CAMA-1 breast cancer cells.

2.5. Pharmacokinetics and oral bioavailability

Adequate bioavailability, especially oral bioavailability has been a challenging aspect of protein degraders, due to three different moieties that constitute a relatively large degrader molecule that often has poor cell permeation and poor oral bioavailability. We conducted pharmacokinetic profiling studies of the three degraders that showed high potency (3e, 4q, 4r). The three compounds were administered to SD rats in a single oral dose to track plasma drug levels over the course of 24 hours. An intravenous administration group was also included to measure the absolute oral bioavailability (F%) of each compound. As summarized in Table 3, at an oral dose of 10 mg/kg, 3e afforded 113 ng/mL C_max and AUC of 1360 h*ng/mL, with a modest oral availability of 9.8%, demonstrating meaningful oral drug exposure in rats. Compound 4q which has 4-hydroxytamoxifen as warhead where the phenol group is susceptible to first-pass metabolism, showed significantly lower oral bioavailability with 1.5 % F. 4r, which differs from 4q by a substitution of -OH with -B(OH)₂, showed dramatically improved oral bioavailability: at 10 mg/kg dose, C_max reached 523 ng/mL, AUC at 6102 h*ng/mL, and an F value at 31.4%.

Table 3.

Pharmacokinetic profiles of select ER degraders

Compound	Dose (mg/kg, iv)	AUC (0-t) (h*ng/mL)	Dose (mg/kg, po)	Cmax (ng/mL)	Tmax (hr)	T1/2 (hr)	AUC (0-t) (h*ng/mL)	%F
3e	1.0	1419	10	113	2.0	4.6	1390	9.8
4q	2.0	3152	20	39	1.0	3.8	468	1.5
4r	1.0	1943	10	523	4.0	6.5	6102	31.4

2.6. In vivo efficacy

To test the in vivo efficacy of the most potent ER degrader with high oral bioavailability, we utilized three xenograft models of hormone receptor positive breast cancer in mice. In the first in vivo experiment, mice bearing MCF-7 cell derived xenograft tumors were treated with oral dose of 4r or subcutaneous fulvestrant, the FDA approved SERD. Figure 6A demonstrates the superior efficacy of 4r in blocking tumor growth compared to fulvestrant. Body weight change over the course of treatment indicates that the ER degrader, like fulvestrant, was well tolerated (Figure 6B). In the second breast cancer model based on patient derived xenograft (PDX), ST1799, mice were treated with a daily oral dose of 20 mg/kg 4r. Also included was a treatment group of combination therapy with a CDK4/6 inhibitor, abemaciclib. As shown in Figure 7, treatment with oral 4r inhibited the PDX tumor growth. Combination of 4r with a CDK4/6 inhibitor, abemaciclib led to complete tumor regression.

Figure 6.

Nude mice bearing MCF-7 breast cancer xenograft were treated with either fulvestrant by s.c. injection or 4r by oral gavage at 20 mg/kg. (A) tumor volumes were plotted vs. days of drug treatment; (B) body weight changes during treatment period.

Figure 7.

Nude mice bearing MCF-7 breast cancer xenograft were treated with 4r by oral gavage at 20 mg/kg or 4r in combination with a CDK4/6 inhibitor, abemaciclib. (A) tumor volumes were plotted vs. days of drug treatment; (B) body weight changes during treatment period.

In another PDX breast tumor model (ST941) harboring the clinically relevant ESR1 mutant Y537S and is resistant to tamoxifen treatment, the ER degrader 4r inhibited tumor growth more effectively than the standard of care SERD, fulvestrant (Figure 8). Combination treatment with the CDK4/6 inhibitor palbociclib further enhanced its efficacy in blocking tumor growth in mice.

Figure 8.

Nude mice were transplanted with ST941 PDX breast tumor harboring ESR1 (Y537S) mutation and resistant to hormone treatment. Animals were treated with either fulvestrant by s.c. injection, 4r by oral gavage at 20 mg/kg, or 4r (20 mg/kg PO) and palbociclib (50 mg/kg PO). (A) tumor volumes were plotted vs. days of drug treatment; (B) body weight changes during treatment period.

2.7. A putative in silico model of the degrader 4r

A representative in silico model of the PROTAC mediated ternary complex consisting of ERα, CRBN E3 ligase and the most active degrader 4r, is depicted in Figure 9. Binding modes of the ER ligand and the CRBN ligand parts in PROTAC 4r are similar to the ones observed when they are individually bound to their respective target proteins. The boronic acid group on the ER ligand maintained the conserved hydrogen bonding interactions with R394 and E353. Similarly, the triphenylethylene structure has retained its position in the hydrophobic packet within the ligand binding domain. The CRBN ligand part of 4r also retained hydrogen bonding interactions with H380, N353 and W382 as seen in CRBN in complex with free CRBN ligand (lenalidomide). The linker part of 4r bridges the CRBN and ER, making it a closely interacting PROTAC mediated ternary complex. The ternary structure provided a detailed molecular interaction between the degrader 4r and its two binding partners. Although we report no mutagenesis or X-ray crystallographic studies, binding of the degrader is more likely based on the ternary model obtained by the in silico studies, which can further be used to optimize and design ER degraders with high potency and efficacy.

Figure 9.

A putative model of the most potent degrader 4r based ternary complex involving ERα and CRBN E3 ligase: (A) full view of the complex and (B) a close-up view of the binding: green – 4r, purple ribbon – ERα, teal ribbon – CRBN E3 ligase. Important amino acids interacting with the proteins are shown in orange stick models.

3. Conclusion

By conjugating a clinically approved SERM molecule to a CRBN binding ligand, we have constructed a series of ER targeting PROTACs that exhibited a wide range of degradation activity against the estrogen receptor protein. As all degrader molecules contain an ER binding moiety, there is a baseline anti-estrogen activity in the form of antiestrogen transcriptional and anti-proliferative activities that are present in our ER PROTACs that show little to no degradation activity. However, PROTAC molecules that are effective ER degraders showed greater overall anti-estrogen activities, confirming that inhibition and degradation both contribute to blocking ER-driven activities in breast cancer cells.

The ER degrading efficacy of synthesized PROTACs was impacted by three major structural features that constitute the degrader molecules: the ER binding moiety, the linker composition, and the CRBN E3 ligand. The effect of ER ligand binding affinity on degrader efficacy was clear in all series (1 – 4) where the 4-hydroxy and 4-boronic acid derivatives of tamoxifen consistently showed 5–10-fold greater ER degradation potency. When all other parameters are the same, the well-established binding affinity difference between tamoxifen and its 4-OH derivative becomes the sole contributing factor to enhanced ER degrading activity of the PROTAC. The linker structure also showed an important role in the degrader activity. In the 4 series where the ER-binding moiety retained the ethoxy side chain, the optimal linker length was found to be the methylene-linked piperidine/piperazine variants. In 1-3 series where the ER-binding moiety is a triphenylethylene without the ethoxy side chain, the optimal linker-length becomes the longer, piperidine-ethylene-piperazine structure, highlighting the effect of physical distance between the ER and E3 binding ligand that is required for effective ternary complex formation. In addition, flexible alkyl linker structure, while providing sufficient length necessary for ternary complex, was inferior to rigid, heterocyclic ring structures. Finally, the effect of E3 binding affinity on degrader activity was simply manifested in the thalidomide vs lenalidomide variation.

The metabolic susceptibility of the phenolic functional group in the 4-hydroxy tamoxifen warhead was carried through to their corresponding degrader molecules as seen in paired pharmacokinetic profile comparison of 4-hydroxy to 4-boronic acid substituted ER degraders. In this regard, the boronic acid functional group in the warhead enabled significant gain in oral bioavailability for the degrader molecule (4r) by overcoming first-pass metabolism of the 4-OH containing degrader molecule (4q).

4. Experimental

4.1. Chemistry

General method A: SNAr addition. A mixture of R-X, secondary amine and DIEA in DMSO was stirred at 100 °C for 16 hours. After completion, the mixture was poured into ice-water and extracted with EA 3x. Combined organic layers were washed with Brine, dried over Na₂SO₄, filtered, evaporated in vacuo. The residue was subjected onto silica gel column chromatography.

General method B: McMurry reaction. To a suspension of Zn powder in dry THF was added dropwise TiCl₄ under N₂ at 0 °C. When the addition was completed, the mixture was warmed to room temperature and brought to reflux for 2 hours. To the cooled suspension of the Ti-reagent was added a solution of propiophenone and substituted benzophenone from general method A in dry THF at 0 °C. The mixture was refluxed in the dark for another 2 hours. After being cooled to room temperature, the reaction mixture was quenched with brine and extracted with EA 3x. Combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was subjected onto silica gel column chromatography.

General method C: Oxidation reaction by using SO₃∙Py condition. A solution of obtained primary alcohol in DMSO was added triethylamine, followed by SO₃∙Py complex at room temperature. After completion, DCM 2x and aq. NaHCO₃ solution was added for extracting. Combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was subjected onto silica gel column chromatography.

General method D: Reductive amination. To a mixture of aldehyde and amine in DCM was added a drop of HOAc, followed by STAB portion-wise at room temperature. After completion, the solvent was removed in vacuo and the residue was subjected onto silica gel column chromatography.

General method E: Miyaura Borylation Reaction. A mixture of bromo or chloro substrate, B₂Pin₂, Pd(dppf)Cl₂ or Pd₂(dba)₃-PCy₃ and KOAc in 1,4-dioxane was heated to 90–100 °C under nitrogen for 2 hours. After completion, the mixture was filtered through celite. The filtrate was evaporated to dryness. The residue was subjected onto silica gel column chromatography. The product was further purified by Prep-HPLC (column: Waters xbridge 150*25mm 10um; mobile phase: [water (0.3%FA)-ACN]) and freeze-drying.

General method F: Oxidating boronic acid to be phenol. To a solution of boronic acid/ester in THF-H₂O was added NaBO₃∙4H₂O at 0 °C. The mixture was stirred at room temperature for 5 hours. EA 3x were added to extract the product. Combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was subjected onto silica gel column chromatography. The product was further purified by Prep-HPLC (column: Waters xbridge 150*25mm 10um; mobile phase: [water (0.3%FA)-ACN]) and freeze-drying.

Preparation of 1b and 1e

To a stirred mixture of 4-bromobenzoyl chloride (25 g, 114 mmol) and fluorobenzene (54.7 g, 570 mmol) was added AlCl₃ (17.6 g, 130 mmol) portion-wise at 0 °C. The resulting mixture was stirred at 85 °C overnight. The mixture was cooled to room temperature and poured into 400 mL of 5% aqueous HCl solution. The product was extracted by 300 mL of DCMx2. The organic layers were combined and concentrated to yield (4-bromophenyl)(4-fluorophenyl)methanone (27.3 g, 85% yield) as light yellow solid. ¹H NMR: (300 MHz, CDCl₃) δ 7.83 (d, J = 7.2 Hz, 2H), 7.62–7.70 (m, 4H), 7.16 (d, J = 12 Hz, 2H).

By using General method A: A stirred solution of (4-bromophenyl)(4-fluorophenyl)methanone (8.3 g, 30 mmol), piperidin-4-ylmethanol (5.2 g, 45 mmol) and DIEA (5.2 g, 60 mmol) in 30 mL of DMSO was heated in an oil bath at 100 °C overnight. After cooling to room temperature, the mixture was poured into ice-water and extracted with 100 mL of EAx3. Combined organic layers were dried over MgSO₄, filtered, evaporated to dryness. The residue was subjected onto silica gel column chromatography to provide (4-bromophenyl)(4-(4-(hydroxymethyl)piperidin-1-yl)phenyl)methanone (7.5 g, 67%) as white solid. LC-MS: 374; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 7.74 (d, J = 12 Hz, 2H), 7.57–7.63 (m, 4H), 6.87 (d, J = 12 Hz, 2H), 3.96 (d, J = 16 Hz, 2H), 3.54 (d, J = 8 Hz, 2H), 2.92 (t, J = 16 Hz, 2H), 2.62 (s, 2H), 1.76–1.90 (m, 1H). 1.36 (t, J = 10 Hz, 2H).

By using General method B: To a suspension of Zn powder (17.3 g, 266 mmol) in 130 mL of dry THF was added dropwise TiCl₄ (22.0 g, 116 mmol) under N₂ at 0 °C. When the addition was completed, the mixture was warned to room temperature and brought to reflux for 2 hours. To the cooled suspension of the Ti-reagent was added a solution of propiophenone (8.0 g, 60 mmol) and (4-bromophenyl)(4-(4-(hydroxymethyl)piperidin-1-yl)phenyl)methanone (7.5 g, 20 mmol) in 240 mL of dry THF at 0 °C. The mixture was refluxed in the dark for another 2 hours. After being cooled to room temperature, the reaction mixture was quenched with brine and extracted with 100 mL of EA 3x. Combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was subjected onto silica gel column chromatography to provide (1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methanol (6.1 g, 64%) as light yellow oil. LC-MS: 476/478; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 7.47 (d, J = 8.6 Hz, 2H), 7.09–7.21 (m, 7 H), 6.69 (d, J = 9.2 Hz, 2H), 6.56 (d, J = 12 Hz, 2H), 3.62–3.80 (m, 1H), 3.47–3.69 (m, 6H), 2.58 (t, J = 14Hz, 2H), 2.43 (q, J = 6.7 Hz, 1H), 1.70–1.93 (m, 3H), 0.92 (t, J = 6.7 Hz, 3H).

By using General method C: A solution of provide (1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methanol (1.4, 3 mmol) in 5 mL of DMSO was added triethylamine (0.9 g, 9 mmol), followed by SO₃∙Py (0.9 g, 6 mmol) at room temperature. TLC was used to determine the end of the reaction. After completion, 50 mL of DCM and 50 mL of aqueous NaHCO₃ solution were added. The organic layer was dried over MgSO₄, filtered and evaporated to dryness. The residue was subjected onto silica gel column chromatography to provide 1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidine-4-carbaldehyde (1.1 g, 78%) as colorless oil. LC-MS: 474/476; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 9.66 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.02–7.24 (m, 7H), 6.71 (d, J = 12 Hz, 2H), 6.56 (d, J = 12 Hz, 2H), 3.42–3.70 (m, 4H), 2.69–2.80 (m, 2H), 2.43 (q, J = 6.7 Hz, 2H), 1.62–1.93 (m, 3H), 0.92 (t, J = 6.7 Hz, 3H).

By using General method D: A solution of 1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidine-4-carbaldehyde (1.1 g, 2.3 mmol), 3-(1-oxo-5-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (0.5 g, 1.6 mmol) and a drop of HOAc in 10 mL of DCM was added STAB (0.7 g, 3.1 mmol) portion-wise. After stirring for 2 hours, the solvent was removed in vacuo and the residue was subjected onto silica gel column chromatography to provide 3-(5-(4-((1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (570 mg, 47%) as white foam. LC-MS: 786/788; [M+H]⁺, ESI⁺.

By using General method E: A mixture of 3-(5-(4-((1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.2 g, 0.25 mmol), B₂Pin₂ (0.13 g, 0.5 mmol), Pd(dppf)Cl₂ (9 mg, 0.0125 mmol) and KOAc (74 mg, 0.75 mmol) in 5 mL of 1,4-dioxane was stirred and heated in an oil bath under nitrogen for 16 hours. After cooling to room temperature, the solvent was evaporated in vacuo. The residue was subjected onto silica gel column chromatography. The crude product was purified by reversed-phase HPLC (Column : Phenomenex Synergi C18 150*25mm* 10um ; Condition: water(0.1%FA)-CAN), the eluent was lyophilized to provide 3-(5-(4-((1-(4-(1,2-diphenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1b) (12 mg) as white solid. LC-MS: 708; [M+H]⁺, ESI⁺, HRMS (ESI) m/z: calcd, 707.3835 for C₄₅H₄₉N₅O₃ [M + H]⁺; found, 708.3920; ¹H NMR: (300 MHz, DMSO-d₆) δ 10.95 (s, 1H), 7.04 (s, 1H), 7.70 (s, 1H), 7.40–7.70 (m, 4H), 6.93–7.44 (m, 8H), 6.46–6.76 (m, 2H), 5.05 (d, J = 15 Hz, 1H), 4.24–4.70 (m, 3H), 3.11–3.78 (m, 8H), 2.43–2.54 (m, 4H), 2.15–2.25 (m, 8H), 1.64–1.99 (m, 4H), 0.84(t, J = 6.7 Hz, 3H) and (E)-(4-(1-(4-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (1e) (85 mg) as white solid. LC-MS: 752; [M+H]⁺, ESI⁺, HRMS (ESI) m/z: calcd, 751.3905 for C₄₅H₅₀BN₅O₅ [M + H]⁺; found, 752.3998; ¹H NMR: (300 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.15 (s, 1H), 8.05 (s, 1H), 7.70 (s, 1H), 7.44–7.72 (m, 4H), 6.92–7.35 (m, 8H), 6.53–6.69 (m, 2H), 5.05 (d, J = 15 Hz, 1H), 4.21–4.78 (m, 3H), 3.05–3.62 (m, 8H), 2.29–2.49 (m, 4H), 2.13–2.20 (m, 8H), 1.62–1.90 (m, 4H), 0.83(t, J = 6.7 Hz, 3H).

Preparation of 1a, 1c, 1d

By using General method F: To a solution of (4-(1-(4-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (50 mg, 0.06 mmol) in THF-H₂O (1 mL/1mL) was added excess of NaBO₃∙4H₂O at 0°C. The mixture was stirred at room temperature for 5 hours. EA 3x were added to extract the product. Combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was subjected onto silica gel column chromatography. The product was further purified by Prep-HPLC (column: Waters xbridge 150*25mm 10um; mobile phase: [water (0.3%FA)-ACN]) and freeze-drying to provide 3-(5-(4-((1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1c) (22 mg) as white solid. LC-MS: 724; [M+H]⁺, ESI⁺, HRMS (ESI) m/z: calcd, 723.3785 for C₄₅H₄₉N₅O₄ [M + H]⁺; found, 724.3869; ¹H NMR: (300 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.05 (s, 1H), 7.55 (s, 1H), 7.42–7.69 (m, 4H), 6.92–7.28 (m, 8H), 6.53–6.69 (m, 2H), 5.05 (d, J = 15 Hz, 1H), 4.20–4.57 (m, 3H), 3.05–3.62 (m, 8H), 2.29–2.49 (m, 4H), 2.13–2.20 (m, 8H), 1.62–1.90 (m, 4H), 0.84(t, J = 6.7 Hz, 3H).

2-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (1a) 32.1 mg, white solid. LC-MS: 738, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 737.3577 for C₄₅H₄₇N₅O₅ [M + H]⁺; found, 738.3655; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 7.66 (d, J = 8 Hz, 1H), 6.89–7.43 (m, 10 H), 6.73 (d, J = 8 Hz, 1H), 6.52–6.73 (m, 4H), 6.38 (d, J = 8.4 Hz, 1H), 5.06 (d, J = 15 Hz, 1H), 3.46–3.73 (m, 5H), 3.56–3.01 (m, 6H), 2.04–2.51 (m, 4H), 1.77–1.90 (m, 4H), 1.22–1.54 (m, 4H), 0.72–0.91 (m, 3H).

(Z)-(4-(1-(4-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (1d) 21 mg, white solid. LC-MS: 752, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 751.3905 for C₄₅H₅₀BN₅O₅ [M + H]⁺; found, 752.4001; ¹H NMR (300 MHz, DMSO-d₆) δ 7.65–7.79 (m, 1H), 7.40–7.62 (m, 1H), 7.01–7.43 (m, 8H), 6.72–6.95 (m, 2H), 6.51–6.71 (m, 2H), 4.61–4.83 (m, 1H), 4.35–4.48 (m, 2H), 3.11–3.78 (m, 8H), 2.43–2.54 (m, 4H), 2.15–2.25 (m, 8H), 1.64–1.99 (m, 4H), 0.78–0.94 (m, 3H).

Preparation of 2a~2c

By using General method A, (4-bromophenyl)(4-(4-(2-hydroxyethyl)piperidin-1-yl)phenyl)methanone (3.7 g, 95%) was obtained as light yellow solid. LC-MS: 388/390; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 7.74 (d, J = 6 Hz, 2H), 7.60 (s, 4H), 6.88 (d, J = 6Hz, 2H), 3.83–3.96 (m, 2H), 3.70–3.77 (m, 2H), 2.91 (t, 12 Hz, 2H), 1.83 (d, J = 12 Hz, 2H), 1.64–1.79 (m, 1H), 1.50–1.62 (m, 2H), 1.22–1.45 (m, 3H).

By using General method B, (Z/E)-2-(1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)ethan-1-ol (3.6 g, 73%) was obtained as light yellow oil. LC-MS: 490/492; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 7.47 (d, J = 8.6 Hz, 2H), 7.09–7.21 (m, 10H), 6.69 (d, J = 9.2 Hz, 2H), 6.56 (d, J = 12 Hz, 2H), 3.62–3.79 (m, 2H), 3.51–3.59 (m, 2H), 2.38–2.80 (m, 5H), 1.83 (d, J = 12 Hz, 2H), 1.64–1.79 (m, 1H), 1.50–1.62 (m, 2H), 1.22–1.38 (m, 3H), 0.90 (t, 7.5 Hz, 3H).

By using General method C, (Z/E)-2-(1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)acetaldehyde (2.6 g, 72%) was obtained as yellow oil. LC-MS: 488/490; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 9.78 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.14–7.28 (m, 10 H), 6.81–6.92 (m, 1H), 6.69 (d, J = 9.2 Hz, 2H), 6.66 (d, J = 12 Hz, 2H), 3.62–3.79 (m, 2H), 3.51–3.59 (m, 2H), 2.38–2.80 (m, 5H), 1.83 (d, J = 12 Hz, 2H), 1.64–1.79 (m, 1H), 1.50–1.62 (m, 2H), 1.22–1.38 (m, 3H), 0.90 (t, 7.5 Hz, 3H).

By using General method D, (Z/E)-3-(5-(4-(2-(1-(4-(1-(4-bromophenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)ethyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.74 g, 93%) was obtained as red solid. LC-MS: 800/802; [M+H]⁺, ESI⁺, ¹H NMR: (300 MHz, CDCl₃) δ 10.96 (s, 1H), 7.42–7.61 (m, 2H), 6.98–7.25 (m, 12 H), 6.70–6.92 (m, 2H), 6.53–6.61 (m, 4H), 5.05 (d, 12 Hz, 1H), 4.25 (dd, J = 21 Hz, 15 Hz, 2H), 3.15–3.52 (m, 8H), 2.83–2.97 (m, 2H), 2.15–2.51 (m, 6H), 1.64–1.84 (m, 2H), 1.33–1.51 (m, 4H), 1.12–1.28 (m, 3H), 0.79–0.91 (m, 3H).

By using General method E and F, (Z/E)-3-(5-(4-(2-(1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)ethyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2b) was obtained as white solid. 25 mg, LC-MS: 738, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 737.3941 for C₄₆H₅₁N₅O₄ [M + H]⁺; found, 738.4020; ¹H NMR (300 MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.18 (s, 1H), 7.30–7.45 (d, 6.8 Hz, 1H), 6.84–7.29 (m, 10 H), 6.75 (d, J = 8.4 Hz, 1H), 6.50–6.65 (m, 3H), 6.40 (d, J = 8.4Hz, 1H), 5.06 (d, J = 15 Hz, 1H), 4.22 (dd, 2H), 3.15–3.78 (m, 9H), 2.72–2.90 (m, 3H), 2.32–2.68 (m, 5H), 1.89–2.03 (m, 2H), 1.65–1.89 (m, 2H), 1.09–1.58 (m, 5H), 0.78–0.92 (m, 3H).

(Z/E)-(4-(1-(4-(4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (2c) was obtained as white solid, 15 mg, LC-MS: 766, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 765.4062 for C₄₆H₅₂BN₅O₅ [M + H]⁺; found, 766.4147; ¹H NMR (300 MHz, DMSO-d₆) δ 10.96 (s, 1H), 8.16 (s, 1H), 7.69–7.82 (m, 2H), 7.41–7.55 (m, 2H), 6.99–7.32 (m, 9H), 6.72–6.98 (m, 1H), 6.51–6.63 (m, 3H), 5.05 (d, 1H), 4.22 (dd, 2H), 3.15–3.78 (m, 9H), 2.81–2.93 (m, 2H), 2.32–2.68 (m, 5H), 1.89–2.03 (m, 2H), 1.65–1.89 (m, 2H), 1.09–1.58 (m, 5H), 0.82–0.92 (m, 3H).

By using the above process, (Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)ethyl)piperazin-1-yl)isoindoline-1,3-dione (2a) was obtained as white solid, 22 mg, LC-MS: 752, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 751.3734 for C₄₆H₄₉N₅O₅ [M + H]⁺; found, 752.3812; ¹H NMR (300 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.14 (s, 2H), 7.67 (s, 1H), 7.02–7.41 (m, 7H), 6.77–7.01 (m, 4H), 6.75 (d, 1H), 6.53–6.66 (m, 3H), 6.39 (d, 1H), 5.05 (d, 1H), 3.55–3.76 (m, 4H), 2.79–3.03 (m, 4H), 2.55–2.89 (m, 6H), 1.91–2.10 (m, 3H), 1.60–1.87 (m, 5H), 1.13–1.57 (m, 8H), 0.83 (t, 3H).

Preparation of 3a~3e

By using General method D and E, (Z/E)-(4-(1-(4-(4-(((1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperidin-4-yl)amino)methyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (3e) was obtained as tan solid. 35 mg, LC-MS: 766, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 765.4062 for C₄₆H₅₂BN₅O₅ [M + H]⁺; found, 766.4149; ¹H NMR (300 MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.32 (s, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.38–7.56 (m, 2H), 6.81–7.30 (m, 14H), 6.77 (d, J = 8.1 Hz, 2H), 6.52–6.68 (m, 4H), 5.01–5.13 (m, 1H), 4.26 (d, J = 14.7Hz, 2H), 3.28–3.53 (m, 8H), 2.23–2.98 (m, 7H), 1.54–2.01 (m, 11H), 0.86 (t, 3H).

(Z/E)-3-(5-(4-(((1-(4-(1,2-diphenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)amino)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (3c) was obtained as tan solid. 12 mg, LC-MS: 722, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 721.3992 for C₄₆H₅₁N₅O₃ [M + H]⁺; found, 722.4075; ¹H NMR (300 MHz, DMSO-d₆) δ 10.96 (s, 1H), 8.24 (s, 1H), 7.25–7.51 (m, 2H), 6.88–7.21 (m, 10H), 6.54–6.68 (m, 4H), 5.02–5.14 (m, 1H), 4.26 (d, J = 14.7Hz, 2H), 3.28–3.53 (m, 8H), 2.23–2.98 (m, 7H), 1.54–2.01 (m, 11H), 0.79–0.90 (m, 3H).

By using General method F, (Z/E)-3-(5-(4-(((1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)amino)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (3d) was obtained as brown solid. 42 mg, LC-MS: 738, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 737.3941 for C₄₆H₅₁N₅O₄ [M + H]⁺; found, 738.4013; ¹H NMR (300 MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.33 (s, 1H), 7.35–7.62 (m, 2H), 6.89–7.37 (m, 10H), 6.47–6.81 (m, 4H), 5.02–5.12 (m, 1H), 4.26 (d, J = 14.7Hz, 2H), 3.28–3.53 (m, 8H), 2.23–2.98 (m, 7H), 1.54–2.01 (m, 11H), 0.76–0.80 (m, 3H).

By using the above process, (Z/E) -(4-(1-(4-(4-(((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)amino)methyl)piperidin-1-yl)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (3b) was obtained as yellow solid, 16 mg, LC-MS: 780, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 779.3854 for C₄₆H₅₀BN₅O₆ [M + H]⁺; found, 780.3943; ¹H NMR (300 MHz, DMSO-d₆) δ 11.11 (s, 1H), 8.21 (s, 2H), 7.61–7.82 (m, 2H), 7.10–7.45 (m, 10H), 6.91–7.08 (m, 2H), 6.55–6.63 (m, 2H), 5.05 (dd, J = 3 Hz,, 2.4 Hz, 1H), 4.02–4.18 (m, 4H), 3.44–3.76 (m, 4H), 3.12–3.40 (m, 2H), 2.72–3.11 (m, 4H), 2.24–2.67 (m, 4H), 1.92–2.11 (m, 4H), 1.38–1.59 (m, 4H), 0.85–0.92 (m, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(((1-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)piperidin-4-yl)methyl)amino)piperidin-1-yl)isoindoline-1,3-dione (3a) was obtained as yellow solid, 25 mg, LC-MS: 752, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 751.3734 for C₄₆H₄₉N₅O₅ [M + H]⁺; found, 752.3812; ¹H NMR (300 MHz, DMSO-d₆) δ 11.13 (s, 1H), 8.22 (s, 2H), 7.52–7.70 (m, 2H), 7.05–7.39 (m, 10H), 6.91–7.03 (m, 2H), 6.72 (d, 1H), 6.55–6.62 (m, 4H), 6.37 (d, 1H), 5.05 (d, 1H), 4.02–4.18 (m, 4H), 3.44–3.76 (m, 4H), 3.12–3.40 (m, 2H), 2.72–3.11 (m, 4H), 2.24–2.67 (m, 4H), 1.92–2.11 (m, 4H), 1.38–1.59 (m, 4H), 0.85–0.92 (m, 3H).

Preparation of 4a and 4b

By using General method B, starting from (4-chlorophenyl)(4-hydroxyphenyl)methanone (9.3 g, 40 mmol), 4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenol (9.1 g, 68%) was obtained as yellow oil.

A mixture of 4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenol (1.0 g, 3 mmol), 5-hydroxypentyl 4-methylbenzenesulfonate (0.93 g, 3.6 mmol) and K₂CO₃ (0.62 g, 4.5 mmol) in 10 mL of MeCN was stirred at reflux for 24 hours. After cooling to room temperature, the solids were filtered out. The filtrate was evaporated in vacuo. The residue was subjected onto silica gel column chromatography by using n-hexane-EA as eluent. (Z/E)-5-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)pentan-1-ol (0.46 g, 37% yield) was obtained as yellow oil. LC-MS: 421, [M+H]⁺, ESI⁺.

By using General method C, D and E, (Z/E)-(4-(1-(4-((5-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)pentyl)oxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4b) was obtained as brown solid. LC-MS: 741, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 740.3745for C₄₄H₄₉BN₄O₆ [M + H]⁺; found, 741.3840; ¹H NMR (300 MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.14–8.42 (m, 1H), 7.92–8.14 (m, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.23–7.47 (m, 2H), 7.07–7.30 (m, 14H), 6.79 (d, J = 8.1 Hz, 2H), 6.52–6.68 (m, 4H), 5.01–5.13 (m, 1H), 4.26 (d, 2H), 3.28–3.53 (m, 8H), 2.23–2.98 (m, 7H), 1.54–2.01 (m, 11H), 0.83–0.92 (m, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(5-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)pentyl)piperazin-1-yl)isoindoline-1,3-dione (4a), yellow solid, 33 mg, LC-MS: 727, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 726.3417 for C₄₄H₄₆N₄O₆ [M + H]⁺; found, 727.3506; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.17 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.02–7.28 (m, 6H), 6.89–7.02 (m, 2H), 6.63–6.79 (m, 2H), 6.58 (t, J = 8.4 Hz, 2H), 6.40 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 3.83–3.99 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.33–1.79 (m, 7H), 0.84 (t, J = 7.2 Hz, 3H).

Preparation of 4c~4g, 4n, 4o, 4s, 4t, 4w, 4x, 4aa and 4bb

A mixture of (Z/E)-4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenol (8.0 g, 24 mmol), 1,2-dibromoethane (45 g, 239 mmol) and Cs₂CO₃ (31 g, 95.5 mmol) in 300 mL of MeCN was stirred at reflux overnight. After cooling to room temperature, the mixture was filtered through celite.

The filtrate was evaporated to dryness. The residue was subjected onto silica gel column chromatography by using n-hexane-EA as eluent to provide (Z/E)-1-(2-bromoethoxy)-4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)benzene (5.7 g, 54%) as light yellow oil. LC-MS: 441/443, [M+H]⁺, ESI⁺; ¹H NMR (300 MHz, CDCl₃) δ 7.32 (d, J = 8.4 Hz, 1H), 7.05–7.19 (m, 6H), 6.96 (d, J = 8.8 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.70–6.81 (m, 2H), 6.54 (d, J = 8.8 Hz, 1H), 4.31 (t, J = 6.4 Hz, 1H), 4.18 (t, J = 6.4 Hz, 1H), 3.66 (t, J = 6.4 Hz, 1H), 3.55 (t, J = 6.4 Hz, 1H), 2.40–2.52 (m, 2H), 0.85–0.97 (m, 3H). A mixture of (Z/E)-1-(2-bromoethoxy)-4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)benzene (5.7 g, 12.8 mmol), tert-butyl piperazine-1-carboxylate (5.7 g, 25.6 mmol) and K₂CO₃ (5.3 g, 38.4 mmol) in 200 mL of MeCN was stirred at reflux for 16 hours. After cooling to room temperature, the mixture was filtered through celite. The filtrate was evaporated to dryness. The residue was subjected onto silica gel column chromatography by using n-hexane-EA as eluent. (Z/E)-tert-butyl 4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine-1-carboxylate (5.9 g, 84%) was obtained as light yellow oil. LC-MS: 547, [M+H]⁺, ESI⁺; ¹H NMR (300 MHz, CDCl₃) δ 7.32 (d, J = 8.4 Hz, 1H), 7.05–7.19 (m, 6H), 6.96 (d, J = 8.8 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.70–6.81 (m, 2H), 6.54 (d, J = 8.8 Hz, 1H), 3.92–4.30 (m, 3H), 3.39–3.62 (m, 4H), 2.68–2.91 (m, 2H), 2.40–2.62 (m, 4H), 2.05–2.23 (m, 2H), 1.42 (s, 9H), 0.88–1.03 (m, 3H).

It can be prepared by one step process: A mixture of (Z/E)-4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenol (6.7 g, 20 mmol), tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (6.0 g, 24 mmol), KI (6.6 g, 40 mmol) and K₂CO₃ (0.28 g, 2 mmol) in 100 mL of EtOH was stirred at reflux overnight. After cooling to room temperature, the mixture was filtered through celite. The filtrate was evaporated to dryness. The residue was subjected onto silica gel column chromatography by using n-hexane-EA as eluent. (Z/E)-tert-butyl 4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine-1-carboxylate (7.0 g, 71 %) was obtained as light yellow oil. LC-MS: 547, [M+H]⁺, ESI⁺.

A solution of (Z/E)-tert-butyl 4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine-1-carboxylate (7.0 g, 12.8 mmol) in TFA/DCM (10 mL/10 mL) was stirred at room temperature for 2 hours. After evaporated to dryness, (Z/E)-1-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine was obtained as quantitative yield and used in the next step without further purification. LC-MS: 447, [M+H]⁺, ESI⁺.

Starting from (Z/E)-1-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine (1.3 g, 30 mmol), and following general method D, (Z/E)-tert-butyl 4-((4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidine-1-carboxylate (1.7 g, 88% yield) was obtained as yellow oil. LC-MS: 644, [M+H]⁺, ESI⁺. A solution of (Z/E)-tert-butyl 4-((4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidine-1-carboxylate (1.7g, 26.4 mmol) in 10 mL of 1,4-dioxane was added 5 mL of 4M HCl in 1,4-dioxane at room temperature. After stirring for 5 hours, the volatiles were removed in vacuo. (Z/E)-1-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)-4-(piperidin-4-ylmethyl)piperazine (1.9 g, quant. HCl salt) was obtained as white solid. LC-MS: 544, [M+H]⁺, ESI⁺. ¹H NMR (300 MHz, CDCl₃) δ 8.98 (br, 2H), 7.46 (d, 1H), 7.01–7.25 (m, 9H), 6.83 (d, 1H), 6.76 (d, 1H), 6.68 (d, J = 8.8 Hz, 1H), 4.11–4.52 (m, 3H), 3.23–3.70 (m, 11H), 2.91–3.20 (m, 2H), 2.71–2.90 (m, 2H), 2.33–2.49 (m, 2H), 1.95–2.15 (m, 2H), 1.15–1.50 (m, 4H), 0.84–0.91 (m, 3H).

By using General method A, (Z/E)-5-(4-((4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione was obtained as tan solid. LC-MS: 800, [M+H]⁺, ESI⁺.

By using general method E and F, (Z/E)- (4-(1-(4-(2-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4o) was obtained as yellow solid. LC-MS: 810, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 809.3960 for C₄₇H₅₂BN₅O₇ [M + H]⁺; found, 810.4042; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.18 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.05–7.32 (m, 6H), 6.85–7.02 (m, 1H), 6.67–6.83 (m, 2H), 6.56–6.64 (m, 1H), 5.05 (d, 1H), 3.82–4.12 (m, 4H), 3.77–3.83 (m, 2H), 3.50–3.83 (m, 4H), 2.81–2.96 (m, 2H), 2.01–2.25 (m, 3H), 1.67–1.87 (m, 2H), 1.33–1.79 (m, 3H), 0.85 (t, J = 7.2 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-((4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidin-1-yl)isoindoline-1,3-dione (4n) was obtained as yellow solid. LC-MS: 782, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 781.3839 for C₄₇H₅₁N₅O₆ [M + H]⁺; found, 782.3936; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.20 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.01–7.25 (m, 6H), 6.87–7.02 (m, 2H), 6.62–6.73 (m, 2H), 6.56–6.64 (m, 1H), 5.05 (d, 1H), 3.92–4.09 (m, 4H), 3.77–3.91 (m, 2H), 3.50–3.83 (m, 4H), 2.81–2.96 (m, 2H), 2.01–2.25 (m, 3H), 1.67–1.87 (m, 2H), 1.33–1.79 (m, 3H), 0.85 (t, J = 7.2 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-((3-(4-(2-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)propyl)amino)isoindoline-1,3-dione (4c), yellow solid, LC-MS: 726, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 725.3577 for C₄₄H₄₇N₅O₅ [M + H]⁺; found, 726.3673; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.18 (s, 1H), 7.03–7.59 (m, 11H), 6.60–7.02 (m, 5H), 5.05 (d, 1H), 4.27–4.40 (m, 2H), 3.86–4.12 (m, 4H), 2.50–3.99 (m, 14H), 2.23–2.48 (m, 4H), 1.90–2.15 (m, 2H), 1.67–1.82 (m, 2H), 0.85 (t, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-((3-(4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)propyl)amino)isoindoline-1,3-dione (4d), yellow solid, LC-MS: 742, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 741.3526 for C₄₄H₄₇N₅O₆ [M + H]⁺; found, 742.3613; ¹H NMR (300 MHz, DMSO-d₆) δ 11.07 (s, 1H), 8.20 (s, 1H), 7.54–7.80 (m, 2H), 7.01–7.32 (m, 8H), 6.80–6.97 (m, 2H), 6.52–6.79 (m, 4H), 6.38 (d, 1H), 5.05 (d, 1H), 4.27–4.40 (m, 2H), 3.86–4.12 (m, 4H), 2.50–3.99 (m, 14H), 2.23–2.48 (m, 4H), 1.90–2.15 (m, 2H), 1.67–1.82 (m, 2H), 0.85 (t, 3H).

(Z/E)-(4-(1-(4-(2-(4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)propyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4e), yellow solid, LC-MS: 770, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 769.3647 for C₄₄H₄₈BN₅O₇ [M + H]⁺; found, 770.3729; ¹H NMR (300 MHz, DMSO-d₆) δ 11.11 (s, 1H), 8.21 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.02–7.25 (m, 5H), 6.51–7.01 (m, 9H), 5.05 (d, 1H), 4.31–4.62 (m, 2H), 3.92–3.98 (m, 2H), 3.77–3.83 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.33–1.79 (m, 7H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)isoindoline-1,3-dione (4f), yellow solid, LC-MS: 685, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 684.2948 for C₄₁H₄₀N₄O₆ [M + H]⁺; found, 685.3031; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.17 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 6.89–7.37 (m, 10 H), 6.53–6.79 (m, 2H), 6.40 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 4.10–4.21 (m, 1H), 3.88–4.01 (m, 1H), 3.50–3.83 (m, 11H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.33–1.79 (m, 7H), 0.85 (t, J = 7.2 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4g), yellow solid, LC-MS: 713, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 712.3068 for C₄₁H₄₁BN₄O₇ [M + H]⁺; found, 713.3147; ¹H NMR (300 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.05–7.36 (m, 10 H), 6.92–6.98 (m, 1H), 6.65–6.80 (m, 2H), 6.62 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 4.10–4.21 (m, 1H), 3.88–4.01 (m, 1H), 3.50–3.83 (m, 11H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.15–1.30 (m, 7H), 0.74–0.92 (m, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)ethyl)piperazin-1-yl)isoindoline-1,3-dione (4s), yellow solid, LC-MS: 797, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 796.3948 for C₄₇H₅₂N₆O₆ [M + H]⁺; found, 797.4026; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.20 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.333 (s, 1H), 7.02–7.30 (m, 6H), 6.89–7.01 (m, 2H), 6.63–6.79 (m, 2H), 6.58 (t, J = 8.4 Hz, 2H), 6.40 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 3.92–3.98 (m, 2H), 3.77–3.83 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.93–2.04 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4t), yellow solid, LC-MS: 825, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 824.4069 for C₄₇H₅₃BN₆O₇ [M + H]⁺; found, 825.4167; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.16 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.65 (s, 1H), 7.67 (s, 1H), 7.34 (s, 1H), 7.02–7.28 (m, 6H), 6.89–7.01 (m, 2H), 6.63–6.79 (m, 2H), 6.58 (t, J = 8.4 Hz, 2H), 6.39 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 4.01–4.11 (m, 2H), 3.87–3.95 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.94–2.10 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)ethyl)piperidin-1-yl)isoindoline-1,3-dione (4w), yellow solid, LC-MS: 796, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 795.3996 for C₄₈H₅₃N₅O₆ [M + H]⁺; found, 796.4084; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.23 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.02–7.28 (m, 6H), 6.89–7.01 (m, 2H), 6.63–6.79 (m, 2H), 6.58 (t, J = 8.4 Hz, 2H), 6.41 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 3.87–4.13 (m, 2H), 3.77–3.83 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.10–1.45 (m, 7H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)ethyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4x), yellow solid, LC-MS: 824, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 823.4116 for C₄₈H₅₄BN₅O₇ [M + H]⁺; found, 824.4200; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.79 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.42 (s, 1H), 7.02–7.37 (m, 6H), 6.89–7.01 (m, 2H), 6.77 (m, 2H), 6.73 (t, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 3.92–4.13 (m, 2H), 3.77–3.83 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.89–2.13 (m, 2H), 1.33–1.79 (m, 7H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(3-(4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)propyl)piperazin-1-yl)isoindoline-1,3-dione (4aa), yellow solid, LC-MS: 811, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 810.4105 for C₄₈H₅₄N₆O₆ [M + H]⁺; found, 811.4189; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.22 (s, 1H), 7.05–7.35 (m, 9 H), 6.89–7.03 (m, 2H), 6.63–6.79 (m, 2H), 6.58 (t, J = 8.4 Hz, 2H), 6.39 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 4.02–4.12 (m, 2H), 3.87–3.92 (m, 2H), 3.50–3.83 (m, 11 H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 2.01–2.13 (m, 2H), 1.51–1.90 (m, 7H), 0.84 (t, J = 7.2 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)propyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4bb), yellow solid, LC-MS: 839, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 838.4225 for C₄₈H₅₅BN₆O₇ [M + H]⁺; found, 839.4326; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.15 (s, 1H), 8.05 (s, 1H), 7.80 (s, 1H), 7.78 (d, 1H), 7.67 (d, 1H), 7.04–7.53 (m, 9H), 6.91–6.96 (m, 2H), 6.65–6.79 (m, 4H), 6.59 (d, J = 8.4 Hz, 1H), 5.05 (d, 1H), 4.02–4.12 (m, 2H), 3.87–3.92 (m, 2H), 3.50–3.83 (m, 11H), 2.81–2.96 (m, 2H), 2.25–2.68 (m, 5H), 1.92–2.05 (m, 1H), 1.67–1.80 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H).

Preparation of 4h-4m, 4u, 4v, 4y and 4z

Synthesis of 4j, 4k for representative example: By using General method A, 1-(2-bromoethoxy)-4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)benzene (440 mg, 1 mmol), 5-([4,4'-bipiperidin]-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (440 mg, 1 mmol) and DIEA (0.39 g, 3 mmol) in 2 mL of NMP was stirred at 100 °C for 2 hours. After cooling to room temperature, the mixture was subjected onto silica gel column chromatography by-EA-MeOH as eluent. (Z/E)-5-(1'-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)-[4,4'-bipiperidin]-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (0.1 g) was obtained as yellow solid. LC-MS: 786, [M+H]⁺, ESI⁺. By using General method E, (Z/E)-(4-(1-(4-(2-(1'-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-[4,4'-bipiperidin]-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4k) was obtained as yellow solid. LC-MS: 795, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 794.3851 for C₄₇H₅₁BN₄O₇ [M + H]⁺; found, 795.3937; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.25 (s, 2H), 7.90–8.10 (s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.02–7.40 (m, 7H), 6.56–7.00 (m, 4H), 5.05 (dd, 1H), 3.85–4.21 (m, 4H), 3.50–3.83 (m, 3H), 2.33–2.50 (m, 2H), 2.52–3.12 (m, 8H), 1.90–2.12 (m, 3H), 1.60–1.89 (m, 4H), 1.01–1.50 (m, 6H), 0.86 (t, 3H).

By using General method F, (Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(1'-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)-[4,4'-bipiperidin]-1-yl)isoindoline-1,3-dione (4j) was obtained as yellow solid. LC-MS: 767, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 766.3731 for C₄₇H₅₀N₄O₆ [M + H]⁺; found, 767.3819; ¹H NMR (300 MHz, DMSO-d₆) δ 11.11 (s, 1H), 8.30 (s, 2H), 7.60–7.90 (m, 2H), 7.10–7.55 (m, 9H), 6.35–7.01 (m, 4H), 5.05 (dd, 1H), 3.85–4.21 (m, 2H), 3.50–3.83 (m, 5H), 2.33–2.50 (m, 2H), 2.52–3.12 (m, 8H), 1.90–2.12 (m, 3H), 1.65–1.84 (m, 4H), 1.11–1.56 (m, 6H), 0.85 (t, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(1-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperidin-4-yl)piperazin-1-yl)isoindoline-1,3-dione (4h), yellow solid, LC-MS: 768, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 767.3683 for C₄₆H₄₉N₅O₆ [M + H]⁺; found, 768.3773; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.22 (s, 1H), 7.50–7.72 (m, 2H), 7.03–7.35 (m, 9H), 6.90–7.02 (m, 1H), 6.69–6.71 (m, 1H), 6.56–6.64 (m, 1H), 6.39 (d, J = 8.4 Hz, 1H), 5.05 (dd, 1H), 4.52 (s, 1H), 2.89–3.15 (m, 4H), 2.68–2.73 (m, 2H), 2.35–2.53 (m, 6H), 2.12–2.33 (m, 2H), 1.91–2.11 (m, 4H), 1.67–1.83 (m, 2H), 1.35–1.51 (m, 3H), 0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)piperidin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4i), yellow solid, LC-MS: 796, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 795.3803 for C₄₆H₅₀BN₅O₇ [M + H]⁺; found, 796.3887.

(Z/E)-(4-(1-(4-(2-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4l), yellow solid, LC-MS: 810, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 809.3960 for C₄₇H₅₂BN₅O₇ [M + H]⁺; found, 810.4046; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.21 (s, 1H), 8.05 (s, 1H), 7.40–7.89 (m, 3H), 7.06–7.37 (m, 8H), 6.55–7.02 (m, 4H), 5.05 (dd, 1H), 4.02–4.12 (m, 1H), 3.87–3.92 (m, 1H), 3.13–3.35 (m, 4H), 2.79–2.98 (m, 2H), 2.12–2.79 (m, 14H), 1.89–2.08 (m, 2H), 1.23–1.75 (m, 5H),0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (4m), yellow solid, LC-MS: 782, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 781.3839 for C₄₇H₅₁N₅O₆ [M + H]⁺; found, 782.3920; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.44 (s, 1H), 7.50–7.81 (m, 2H), 7.05–7.41 (m, 8H), 6.87–7.01 (m, 1H), 6.39–6.85 (m, 4H), 5.05 (dd, 1H), 4.02–4.12 (m, 1H), 3.87–3.92 (m, 1H), 3.13–3.35 (m, 4H), 2.79–2.98 (m, 2H), 2.12–2.79 (m, 14H), 1.89–2.08 (m, 2H), 1.23–1.75 (m, 5H),0.84 (t, J = 6.8 Hz, 1H).

(Z/E)-3-(5-(4-(2-(1-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperidin-4-yl)ethyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (4u), yellow solid, LC-MS: 782, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 781.4203 for C₄₈H₅₅N₅O₅ [M + H]⁺; found, 782.4290; ¹H NMR (300 MHz, DMSO-d₆) δ 8.16 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.12 (dd, J = 25.0, 8.2 Hz, 5H), 6.95 (dd, J = 14.8, 8.6 Hz, 2H), 6.83 – 6.65 (m, 1H), 6.65 – 6.50 (m, 1H), 6.39 (d, J = 8.5 Hz, 1H), 5.16 – 4.91 (m, 1H), 4.49 – 4.14 (m, 2H), 3.99 (d, J = 49.5 Hz, 3H), 3.23 – 2.82 (m, 12H), 2.82 – 2.58 (m, 3H), 2.48 – 2.18 (m, 6H), 2.02 (d, J = 28.9 Hz, 4H), 1.64 (s, 2H), 1.38 (s, 2H), 1.29 – 0.98 (m, 3H), 0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4v), white solid, LC-MS: 810, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 809.4324 for C₄₈H₅₆BN₅O₆ [M + H]⁺; found, 810.4408; ¹H NMR (300 MHz, DMSO-d₆) δ 8.15 (s, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.66 – 7.36 (m, 4H), 7.34 – 7.05 (m, 4H), 7.05 – 6.93 (m, 2H), 6.93 – 6.68 (m, 2H), 6.60 (d, J = 8.6 Hz, 1H), 6.40 (d, J = 8.4 Hz, 1H), 5.07 (d, J = 5.4 Hz, 1H), 4.73 (s, 2H), 4.48 – 4.27 (m, 4H), 4.27 – 4.09 (m, 5H), 3.01–3.29 (m, 6H), 2.01–2.87 (m, 12H), 1.65–1.86 (m, 6H), 1.29 – 0.98 (m, 3H), 0.86 (q, J = 6.9 Hz, 3H).

(Z/E)-2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-(1-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperidin-4-yl)ethyl)piperazin-1-yl)isoindoline-1,3-dione (4y), yellow solid, LC-MS: 796, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 795.3996 for C₄₈H₅₃N₅O₆ [M + H]⁺; found, 796.4082; ¹H NMR (300 MHz, DMSO-d₆) δ 11.10 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.05–7.37 (m, 8H), 6.89–7.01 (m, 2H), 6.65–6.78 (m, 2H), 6.53–6.61 (m, 2H), 6.39 (d, J = 8.8 Hz, 1H), 5.05 (dd, 1H), 4.08 (t, 1H), 3.89 (t, 1H), 2.78–2.96 (m, 4H), 2.30–2.71 (m, 12H), 1.87–2.08 (m, 4H), 1.53–1.70 (m, 2H), 1.11–1.42 (m, 7H), 0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4z), white solid, LC-MS: 824, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 823.4116 for C₄₈H₅₄BN₅O₇ [M + H]⁺; found, 824.4190; ¹H NMR (300 MHz, DMSO-d₆) δ 11.09 (s, 2H), 8.04 (s, 1H), 7.88 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.35 (s, 2H), 7.30 – 7.08 (m, 15H), 6.97 (d, J = 7.2 Hz, 3H), 6.77 (dd, J = 14.8, 8.4 Hz, 4H), 6.67 – 6.55 (m, 3H), 5.07 (dd, J = 12.5, 5.3 Hz, 2H), 2.54 (s, 11H), 2.38 (s, 6H), 1.33 – 1.20 (m, 8H), 0.85 (t, J = 6.7 Hz, 7H).

Preparation of 4p, 4q, 4r

By using General method D, a solution of (Z/E)-tert-butyl 4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazine-1-carboxylate (0.55 g, 1 mmol) and 3-(5-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.51 g, 1.25 mmol) in TFA/DCM (5 mL/5 mL) was stirred at room temperature for 5 hours. After completion, the solvents were removed in vacuo. The residue was added 20 mL of DCM, followed by excess of NEt₃. Then, STAB (0.42 g, 2 mmol) was added portionwise. After stirring for additional 2 hours, 100 mL of aqueous NaHCO₃ solution was added to quench the reaction. DCM x3 was used to extract the intermediate. Combined organic layers were dried over Na₂SO₄, filtered, evaporated. The residue was subjected onto silica gel column chromatography by-EA-MeOH as eluent to provide (Z/E)-3-(5-(4-((4-(2-(4-(1-(4-chlorophenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione as yellow solid. LC-MS: 786, [M+H]⁺, ESI⁺. By using General method E and F, 3-(5-(4-((4-(2-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (4p) was obtained as white solid. LC-MS: 752, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 751.4098 for C₄₇H₅₃N₅O₄ [M + H]⁺; found, 752.4177; ¹H NMR (300 MHz, DMSO-d₆) δ 10.90 (s, 1H), 7.48 (d, 1H), 7.35 (t, 1H), 7.02–7.33 (m, 10H), 6.93–7.01 (m, 2H), 6.70–6.89 (m, 4H), 6.59 (d, J = 8.8 Hz, 1H), 5.05 (d, 1H), 4.25 (dd, 2H), 4.07 (t, 1H), 3.91 (t, 1H),2.73–3.03 (m, 6H), 2.30–2.69 (m, 6H), 2.11–2.25 (m, 2H), 1.91–2.01 (m, 2H), 1.62–1.89 (m, 5H), 1.01–1.32 (m, 6H), 0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-3-(5-(4-((4-(2-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (4q), white solid, LC-MS: 768, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 767.4047 for C₄₇H₅₃N₅O₅ [M + H]⁺; found, 768.4138; ¹H NMR (300 MHz, DMSO-d₆) δ 10.97 (s, 1H), 7.32–7.55 (m, 2H), 6.89–7.30 (m, 10H), 6.52–6.81 (m, 4H), 6.43 (d, J = 8.8 Hz, 1H), 5.05 (d, 1H), 4.25 (dd, 2H), 4.07 (t, 1H), 3.91 (t, 1H),2.73–3.03 (m, 6H), 2.30–2.69 (m, 6H), 2.11–2.25 (m, 2H), 1.91–2.01 (m, 2H), 1.62–1.89 (m, 5H), 1.01–1.32 (m, 6H), 0.84 (t, J = 6.8 Hz, 3H).

(Z/E)-(4-(1-(4-(2-(4-((1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenyl)boronic acid (4r), white solid, LC-MS: 796, [M+H]⁺, ESI⁺; HRMS (ESI) m/z: calcd, 795.4167 for C₄₇H₅₄BN₅O₆ [M + H]⁺; found, 796.4246; ¹H NMR (300 MHz, DMSO-d₆) δ 11.92 (s, 1H), 7.99 (s, 2H), 7.78 (d, 8.0 Hz, 2H), 7.49 (d, 9.2 Hz, 1H), 7.10–7.26 (m, 7H), 7.04 (d, 1H), 6.72 (d, J = 8.4 Hz, 2H), 6.59 (d, J = 8.8Hz, 2H), 5.05 (dd, J = 18, 6.8Hz, 1H), 4.25 (dd, 2H), 3.80–3.96 (m, 4H), 4.57–4.63 (m, 4H), 3.52–3.61 (m, 4H), 2.71–2.96 (m, 4H), 2.52–2.63 (m, 4H), 2.31–2.50 (m, 11 H), 1.91–2.16 (m, 4H), 1.70–1.82 (m, 3H), 1.11–1,22 (m, 2H), 0.85 (t, J = 6.8 Hz, 3H).

Cell Culture.

The MCF-7 and CAMA-1 cell lines were originally purchased from ATCC (Manassas, VA) and were routinely maintained in phenol red-free DMEM medium supplemented with 5% FBS, 4 mM glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin. Cultures were maintained in 5% carbon dioxide at a temperature of 37°C.

For growth assays in the presence of E2 and ER degraders, MCF-7 cells were plated in six-well plates at a density of 50,000 each well in 5% FBS DMEM medium. The cells were treated with ER degrader compounds at 5 different doses ranging from 10⁻¹⁰ M to 10⁻⁶ M for 3 days, while equal volumes of DMSO were used as vehicle controls. Viable cell numbers were counted with a Z Series Coulter Counter instrument (Beckman-Coulter) following manufacturer’s instructions. The ratio of drug treated viable cell numbers to vehicle treated viable cell numbers was defined as survival ratio where the control has the survival ratio of 100%. IC₅₀ values were obtained from dose-response curves for all treatments.

Luciferase Assay.

T47d-kb-Luc stably transfected human breast cancer cell reporter gene assay was used to determine the anti-estrogenic activities of all synthesized ER degrader molecules. The T47d-kb-Luc cells are stably transfected with an artificial gene from the firefly that is only induced in the cells if estrogens bind and activate the ER to induce the gene product (Luciferase) that is then measured with a quantitative enzyme assay that produces light. Antagonist activities were measured by the samples ability to inhibit the activity of estradiol, the natural estrogen. Data were then normalized relative to the activity of the estradiol control and determinations were performed for three concentrations of the samples in quadruplicate in at least three separate experiments.

Western Blot of ER Downregulation Assay.

MCF-7 cells were plated at a density of 200,000 cells/60mm dish. Media containing the same drug concentrations as the growth curve assay were added on the day following plating (day 0) and allowed to incubate for 5 days for western blot. Media with the tested compound was changed every other day. Cells were lysed, snapped frozen in liquid nitrogen, and stored at −80 °C until assay for ERα. All procedures for lysates were performed on ice.

Media were removed and dishes were washed with 1xDPBS. Lysates were made by adding 150μL complete lysis solution and scraping cells into a 1.5 mL microcentrifuge tube. Lysates were placed on a rotisserie at 4 °C for 30 min and then spun at 4 °C at 12,000 rcf for 10 min. Supernatants were assayed for protein content, snap-frozen, and stored at −80 °C if not run immediately. 50 μg of protein was subjected to western blot protocol. Membranes were blocked and then incubated with 1:200 dilution of ERα antibody at 4 °C overnight followed by 1:10,000 dilution of secondary antibody for 1 hr at room temperature. They were then imaged on a LICOR infrared scanner.

Mouse efficacy study

All the procedures related to animal handling, care, and treatment in this study were performed according to guidelines approved by the IACUC of Xavier University of Louisiana following the guidance of the AAALAC. For the cell-derived xenograft (CDX) model, MCF-7 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Female NOD/SCID mice, 6–8 weeks of age were allowed to adapt to laboratory conditions for 10 days prior to transplant and were allowed food and water ad libitum. Each mouse was inoculated subcutaneously in the mammary fat pad with MCF-7 cells (1 × 10⁶) in 0.2 ml of PBS mixed with Matrigel (50:50) for tumor development. For patient derived xenograft models, tumor fragments were harvested from host animals and implanted into immune-deficient mice. ST1799 and ST941 studies were initiated at a mean tumor volume of approximately 175–300 mm³. Tumor bearing mice were dosed with compound 4r at 20 mg/kg QD po, alone or in combination with palbociclib or abemaciclib, fulvestrant at 200 mg/kg once a week by s.c injection, or vehicle (Propylene glycol/Solutol/20% HP-β-CD in water (20:5:75) for three to four weeks. Body weight and tumor volume were measured every 2–3 days using a caliper and other clinical observations for treatment and tumor burden effects were made daily. At the endpoint of the efficacy study, the mice were euthanized according to the Institutional Animal Care and Use Committee (IACUC) guidelines. Tumor volume collected over the course of the study were used to calculate tumor growth inhibition (TGI) using the formula: TGI% = [1- ΔT/ΔC]*100, where ΔT is the volume change for treated tumors, and ΔC is the volume change for control tumors.

Pharmacokinetic Study (Sampling and Analysis).

Four to six weeks old Sprague Dawley rats (Crl: CD (SD)) (SPF/VAF) were purchased from Charles River (Wilmington MA). Animals were quarantined/acclimated for 2–6 weeks prior to dose initiation. Sprague Dawley rats were given oral administration of test compounds (20% PG, 5% Solutol, and 75% of 20% HP-β-CD in water) at different dose levels. After treatment with the compounds, whole blood was collected from each study animal by puncture of the tail vein (0.4 mL of whole blood from rats) into tubes containing K2EDTA at each timepoint. The plasma samples were divided into 2 aliquots (approximately equal in volume) and stored frozen at −75 ± 15 °C within 2 h of the collection until analysis.

The stock solution of ER degrader compounds (0.01 M) in DMSO was prepared and stored at −20 ± 5 °C. The desired serial concentrations of working reference analyte solutions were achieved by diluting a stock analyte solution with 50% acetonitrile (0.1% formic acid) in water solution. The bioanalytical LC–MS/MS method was developed for quantitative determination of each test compound in Sprague Dawley rat plasma with acceptable selectivity, sensitivity, calibration curve, precision, and accuracy. The analytical method is applicable for the determination of test compounds in Sprague Dawley rat plasma with a lower limit of quantification of 10.0 ng/mL, and an upper limit of quantification of 5,000 ng/mL. Five microliters of diluted supernatant was injected into the LC–MS/MS system (TSQ Quantis) and a SynergiTM 4 μm Fusion-RP 80 Å (50 × 2 mm) column using gradient elution and a Triple Quad mass spectrometer) for quantitative analysis. The mobile phases used were 0.1% formic acid in deionized water and 0.1% formic acid in acetonitrile. The pharmacokinetic parameters T1/2 (the biological half-life), Cmax (maximal concentration), Tmax (time at which Cmax is observed), and AUC (area under the plasma concentration–time curve) were calculated from the plasma concentration versus time data using WinNonlin (PhoenixTM, version 8.1, Mountain View, CA, USA).

Molecular Modeling.

The crystal structures of 4-hydroxytamoxifen in complex with ER (PDB ID: 3ERT) and lenalidomide in complex with cereblon E3 ligase (PDB ID: 4CI2) were utilized for the structural analyses of ER targeting warhead and E3-recruiting ligand and their binding to ER and cereblon, respectively. To compare the effect of -H, -OH and -B(OH)₂ functional groups at position C-4 of 4-hydroxytamoxifen, as in the ER warheads of degraders 4p, 4q, and 4r, respectively, molecular docking studies were conducted. The protein structure was prepared by removing the crystallographic water molecules and adding hydrogen atoms consistent with physiological pH followed by energy minimization with an RMSD cutoff value of 0.3 Å for all heavy atoms using the Maestro (13.3) interface on Schrodinger Suite 2022–3. The ER ligands were built using the builder module of Schrodinger suite followed by energy minimization. All the docking studies were performed using the Glide program of Schrodinger Suite 2022–3 [28] with the default parameters under the standard precision (SP) mode. Drummond et al [29, 30] have developed an in silico tool with multiple protocols and methods for modeling PROTAC-mediated ternary complexes. We used Method 4B which yielded superior prediction in their case studies [29,30] to model the ternary structure using MOE 2022–2. In this method, two protein-ligand structures are initially required, and they are ERα-ligand and Cereblon-ligand complexes, where the ligands are the corresponding PROTAC binder moieties. We chose the degrader 4r and crystal structures for ER (PD code: 3ERT) and CRBN E3 ligase (PDB code: 4CI2) to build the ternary complex. Initial structures of the protein-ligand complexes were prepared using the QuickPrep module of MOE software. Method 4B generated a large number of ternary complexes. Finally, a ternary structure belonging to the largest double cluster population was chosen as a representative model of the PROTAC mediated ternary complex.

Highlights.

• Tamoxifen and CRBN based ER degraders synthesized and optimized

• Boronic substitution in the tamoxifen warhead enhanced oral bioavailability of the overall PROTAC molecule

• High in vitro and in vivo potency achieved