Discovery of ERD-1233 as a Potent and Orally Efficacious Estrogen Receptor PROTAC Degrader for the Treatment of ER+ Human Breast Cancer

Abstract

Despite the development of highly effective therapies for the treatment of estrogen receptor α (ERα)-positive human breast cancer, clinical resistance to current therapies requires the development of novel therapeutic strategies. Herein, we report the discovery of ERD-1233 as a potent and orally efficacious ERα degrader designed using the PROTAC technology. ERD-1233 was developed based on Lasofoxifene as the ER binding moiety and a novel cereblon ligand through extensive optimization of the linker. ERD-1233 potently and effectively reduces the ERα protein in vitro and achieves excellent oral bioavailability in mice and rats. Oral administration of ERD-1233 effectively reduces ER protein in ER+ tumors and achieves tumor regression in the ER wild-type MCF-7 xenograft tumor model and strong tumor growth inhibition in the ESR1^Y537S mutated model in mice. Our data demonstrate that ERD-1233 is a promising ER PROTAC degrader for extensive evaluation as a new therapy for the treatment of ER+ human breast cancer.

Graphical Abstract

INTRODUCTION

Breast cancer affects about 2 million people each year worldwide and accounts for 15% of all cancer-related deaths in women.¹⁻³ There are three main subtypes of breast cancer: estrogen receptor-positive (ER+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative (breast cancer lacking the expression of ER, progesterone receptor (PR) and HER2).⁴ Approximately 70% of newly diagnosed breast cancer cases are ER+ breast cancer.⁵

Estrogen receptors are steroid hormone receptors, which belong to a large nuclear receptor family and are transcriptional factors regulating gene expression by binding to estrogens.⁶ The estrogen receptors consist of two isoforms: ERα and ERβ, which have different tissue distributions. ERα is overexpressed in the majority of human breast cancers and has been pursued as one of the primary therapeutic targets of breast cancer.⁷ Currently, three types of endocrine therapies have been developed for the treatment of ER+ breast cancer,⁸⁻¹¹ namely, aromatase inhibitors (AIs) that prevent the production of estrogens, selective ER modulators (SERMs) that antagonize ERα activity, and selective ER degraders (SERDs) that inhibit and degrade ERα. In addition, Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, in combination with AIs and SERDs, have been developed for the treatment of ER+ breast cancers.¹²

The inhibition of estrogen synthesis by AIs such as letrozole, anastrozole, and exemestane, and the inhibition of ER pathway signaling by SERMs have shown remarkable clinical benefits in the treatment of ER+ breast cancer.¹³ As a representative SERM, tamoxifen (1, Figure 1) was developed in 1966 and was¹⁴ approved for metastatic breast cancer treatment in 1973. Although tamoxifen, which is a partial ER agonist, is very effective for the treatment of ER+ breast cancer, clinical resistance develops in 40% of cases after prolonged treatment.

Figure 1.

Representative SERMs, SERDs, and ER PROTACs.

SERD molecules were developed to address the resistance issue encountered by SERMs. SERDs not only fully antagonize ER but also promote¹⁵ proteosome-dependent degradation of the ERα protein. Fulvestrant (2, Figure 1) was the first approved SERD molecule for the treatment of postmenopausal women with advanced ER+ breast cancer.¹⁶ One major limitation of fulvestrant is that it is administered via intramuscular injection due to its poor oral bioavailability.¹⁷ The clinical success of fulvestrant nevertheless demonstrates that degradation of ER is beneficial to patients with ER+ breast cancer, even after patients develop resistance to SERMs. In addition, the major limitation of fulvestrant has motivated researchers to develop orally bioavailable SERD molecules.^9,18−43 Indeed, there have been major efforts in the development of orally bioavailable SERD molecules for the treatment of ER+ BC.²⁰⁻⁴³ To date, a large number of orally active SERDs have been identified and a number of them are being evaluated in different stages of clinical trials.⁴⁴⁻⁴⁷ In 2023, elacestrant (3) was approved by the FDA as the first oral SERD for the treatment of advanced or metastatic ER+ breast cancer.⁴⁸ However, there are still limitations associated with oral SERD molecules, including intrinsic and acquired resistance, and considerable side effects experienced by patients in the clinic. Therefore, new therapeutic strategies are clearly needed for ER+ breast cancer.

In recent years, induced targeted protein degradation using the proteolysis targeting chimera (PROTAC) technology has gained major momentum for its promise to develop a completely new type of therapy for the treatment of human cancers and other human diseases. The concept of PROTAC technology was first introduced in 2001 with the objective to induce targeted protein degradation (TPD).⁴⁹ A PROTAC molecule is a heterobifunctional compound that binds to a protein of interest (POI) and an E3 ligase or an E3 ligase complex to promote ubiquitination of the POI, followed by proteasome-dependent degradation of the POI.^50,51 To date, PROTAC molecules have been reported for more than 130 proteins.⁵²⁻⁵⁹ Importantly, more than 20 PROTAC degraders have entered human clinical trials.⁶⁰

The first PROTAC molecules against ER were designed by using estradiol as the ER ligand and an IκB-α phosphopeptide as the E3 ligase ligand in 2003.⁶¹ Since then, several classes of ER PROTAC molecules have been reported using various ER ligands and ligands for different E3 ligases with representative compounds shown in Figure 1. For example, in 2019 our group reported a highly potent VHL-based PROTAC ER degrader (ERD-308, 5, Figure 1) with a half maximal degradation potency (DC₅₀) = 0.17 nM and >99% maximal degradation efficiencies (D_max) in the MCF-7 cell line.⁶²⁻⁶⁶ In 2021, Arvinas disclosed the chemical structure of an orally bioavailable ER PROTAC molecule named ARV-471 (Vepdegestrant, 4, Figure 1), which is currently in Phase 3 clinical development for locally advanced or metastatic ER+/HER2−breast cancer.⁶⁷ Our group has recently reported ERD-3111 (6, Figure 1) as a highly potent and orally efficacious ER PROTAC.⁶⁸

Although ARV-471 has been advanced into Phase 3 clinical development and has demonstrated initial promising efficacy in the clinic, there are considerable side effects, including fatigue, nausea, arthralgia, hot flushes, and increased aspartate aminotransferase.^69,70 In addition, ARV-471 was found to have drug−drug interaction with Palbociclib, which may limit its clinical efficacy in combination with Palbociclib.^71,72 Importantly, similar to the clinical experiences with other ER-targeted therapies, it is fully expected that clinical resistance will develop for ARV-471. Accordingly, there is a clear need for the discovery and development of new, highly potent, orally efficacious, and safer PROTAC ER degraders to achieve the full potential of PROTAC ER degraders for the treatment of ER+ breast cancer. In the present study, we report our design, synthesis, and extensive evaluation of highly potent and orally efficacious ER PROTAC degraders using a potent and novel cereblon ligand RR-11055 (8).

RESULT AND DISCUSSION

Design of High-Affinity Cereblon Ligands.

To date, all orally bioavailable PROTAC degraders advanced into clinical development are developed using cereblon ligands.⁷³ For the purpose of developing new, orally efficacious ER PROTAC degraders, we decided to first focus on the design of novel cereblon ligands with high binding affinities to cereblon and excellent drug-like properties.

Thalidomide and its derivatives, including lenalidomide and pomalidomide are cereblon ligands (Figure 2 and Table 1) and these ligands have been extensively used for the design of PROTAC degraders. We decided to design novel cereblon ligands starting from these known cereblon ligands.

Figure 2.

Design and modeling of new cereblon ligands 8 and 9. (A) Predicted binding models for cereblon ligands 8 displayed in yellow, (B) Comparison of cocrystal structure for lenalidomide in complex with cereblon (PDB ID: 4CI2) with predicted binding models for cereblon ligands 8 and 9. (B1). Co-crystal structure for lenalidomide in complex with cereblon (PDB ID: 4CI2); (B2). Predicted binding model for ligand 8; (B3). Predicted binding model for ligand 9; (B4−B6). Key interactions of cereblon with lenadomide, ligand 8 and 9.

Table 1.

Binding Affinities of Different Ligands to Cereblon

ligands	IC50 (μM)a	ligands	IC50 (μM)a
thalidomide	3.26 ± 0.9	4-OH-Lenalidomide (7)	0.69 ± 0.19
pomalidomide	2.06 ± 0.68	RR-11055 (8)	0.38 ± 0.09
4-hydroxy thalidomide	0.95 ± 0.17	RR-11163 (9)	1.42 ± 0.67
lenalidomide	1.71 ± 0.6
microsome stability T1/2 (min) of compound 8 in mice/rat/human		plasma stability T1/2 (min) of compound 8 in mice/rat/human
>60/ >60/ >60		>120/ >120/ >120

^aIC₅₀ values were determined by a HTRF cereblon binding assay; values reported are the mean ± SD of three experiments.

In our binding assay, thalidomide binds to cereblon with a half-maximal inhibitory activity (IC₅₀) value of 3.2 μM. Pomalidomide and 4-hydroxy thalidomide showed a 1.5-fold and 3.4-fold improvement in their cereblon binding affinities as compared to thalidomide. Removal of one carbonyl group from pomalidomide and 4-hydroxy thalidomide generated lenalidomide, and 4-hydroxy-lenalidomide (7), which have cereblon binding IC₅₀ = 1.7 and 0.7 μM respectively. Based upon 4-hydroxy-lenalidomide, we designed ligands 8 and 9, by introducing piperazine into the C-5 position onto the phenyl ring in 4-hydroxy-lenalidomide and cyclization of the hydroxyl group to the piperazine ring. Our modeling showed that these two new ligands adopt similar binding models as compared to lenalidomide (Figure 2B1−B3). In addition to those key interactions observed for lenalidomide with cereblon (Figure 2B4), ligands 8 and 9 capture additional interactions with His355 in cereblon (Figure 2B5,B6).

We developed a synthetic procedure for chiral ligands 8 and 9. Our binding data showed that ligand 8 has an IC₅₀ value of 0.38 μM, whereas ligand 9 has an IC₅₀ value of 1.42 μM. Hence, ligand 8 (RR-11055) is 3.7 times more potent than ligand 9, 4.5 times more potent than lenalidomide, and 8.6 times more potent than thalidomide. Based upon its improved binding affinities over other thalidomide and lenalidomide, we evaluated RR11055 (8) for its microsomal and plasma stability. Our data showed that RR-11055 (8) has excellent plasma and microsomal stability in human, mouse, and rat species (Table 1 and SI). We decided to employ RR-11055 (8) for the design of new ER PROTAC degraders.

Design of ER PROTAC Degraders Using Lasofoxifene and RR-11055.

In our previous study, we employed three different ER ligands for our design of orally bioavailable ER PROTAC degraders.⁶⁸ Using lasofoxifene as the ER ligand and TX-16 as the cereblon ligand, we have identified ERD-2217.⁶⁸ Although ERD-2217 is less potent than ARV-471 in inducing ER degradation, it has an excellent pharmacokinetic (PK) profile, including good oral bioavailability in mice and rats.⁶⁸ In our cereblon binding assay, TX-16 has an IC₅₀ = 2.6 μM. Hence, RR-11055 (8) is 6.8-times more potent than TX-16. We posited that using our new cereblon RR-11055, which has an improved cereblon binding affinity over TX-16 would provide us with an opportunity to discover highly potent and orally bioavailable ER degraders.

In our design of ERD-3111, we have shown that an amide group in the linker improved the ER degradation potency.⁶⁸ Therefore, we have synthesized a series of degraders with a flexible linker containing an amide group to determine the optimal linker length. Compounds 10, 11, 12, and 13 containing linkers with one, three, four, and five methylene units achieved similar DC₅₀ values of 0.1−0.3 nM. Interestingly, with increased linker lengths, the degradation efficiencies (D_max values) were improved from 56% to 89% in compounds 11−13.

We replaced the connecting oxygen atom with a N-Me in compounds 11 and 12, which yielded compounds 14 and 15, respectively. Compound 14 displayed DC₅₀ = 1.6 nM and D_max = 55%, which is 10 times less potent than compound 11. Compound 15 displayed DC₅₀ = 0.46 nM, and D_max = 63%. Hence, both compounds 14 and 15 have moderate D_max values.

In our previous studies, we have demonstrated that the employment of conformationally constrained, rigid linkers in PROTAC degraders can significantly improve their DC₅₀ and D_max values.⁷⁴⁻⁷⁶ Accordingly, we next designed and synthesized a series of degraders using rigid linkers with the data summarized in Table 3.

Table 3.

Degraders with Rigid Linkers^a

CPD No	Rigid linker	ERα degradationa
		DC50 (nM)b	Dmax (%)c
16 (ERD-3121)		0.01 ± 0.008	67 ± 2
17 (ERD-3118)		<0.01	80 ± 8
18 (ERD-3128)		0.01 ± 0.003	102 ± 1
19 (ERD-3119)		0.02 ± 0.005	100 ± 3
20 (ERD-3120)		0.19 ± 0.03	99 ± 2
21 (ERD-3129)		0.07 ± 0.01	108 ± 1
22 (ERD-3130)		0.24 ± 0.04	107 ± 0.3
23 (ERD-3117)		0.1 ± 0.03	86 ± 8
24 (ERD-3132)		0.06 ± 0.008	104 ± 2
25 (ERD-3116)		0.1 ± 0.01	106 ± 2
26 (ERD-3115)		0.05 ± 0.009	89 ± 2
27 (ERD-3123)		0.4 ± 0.09	101 ± 3

^aa,b,c Same legend as in Table 2.

We synthesized compound 16 containing a piperidine ring connecting to the ER ligand portion and an amide group connecting to the cereblon ligand. While compound 16 is a potent ER degrader with DC₅₀ = 0.01 nM, it only has D_max = 67%, indicating a moderate degradation efficiency. Converting the piperidine ring in compound 16 to the piperazine ring resulted in compound 17 (Table 3). Compound 17 achieves DC₅₀ = < 0.01 nM and D_max = 80% and is thus more potent and effective than compound 16. Based on compound 17, we have designed and synthesized a series of degraders using spiro rings as the linker and maintaining a positively charged nitrogen in the linker, which resulted in compounds 18−22. Gratifyingly, each of these compounds is highly potent and effective in inducing ER degradation, achieving DC₅₀ values of 0.01– 0.2 nM and D_max values of 99−108%.

Encouraged by the potent and effective degradation activities achieved by compounds 18−22, we synthesized compounds 23−27 by changing the amide bond in the linker from the cereblon ligand side to the ER ligand side (Table 3). In general, these compounds are also highly potent and effective ER degraders, achieving DC₅₀ values of 0.01−0.4 nM and D_max values of 86−106%.

Since our objective was to discover potent and orally bioavailable ER degraders for clinical development, we selected several highly potent and effective ER degraders for evaluation of their oral bioavailability. Our previous studies have shown that for PROTAC molecules, it was more challenging to achieve good oral bioavailability in rats than in mice.^75,77 Accordingly, we evaluated their oral exposures for three representative ER degraders from Table 3 in rats, with data summarized in Table 4.

Table 4.

Oral Exposures of Three Representative ER Degraders in Rats

		concentration in rat plasma (ng/mL)a
CPD no.	PO dose (mg/kg)	1 h	3 h	6 h
18 (ERD-3128)	3	<5	<5	<5
20 (ERD-3120)	3	<5	<5	<5
26 (ERD-3115)	3	6.0 ± 3.2	6.0 ± 4.2	<5

^aThe plasma drug concentration data for each compound was independently collected from 3 rats and provided as the mean ± SD.

Unfortunately, all these three compounds displayed very low oral bioavailability in rats (Table 4), highlighting the challenge in designing orally bioavailable ER PROTAC degraders.

In our design of orally bioavailable androgen receptor (AR) degraders, we have shown that the removal of an amide bond in the linker can significantly improve the oral bioavailability of the resulting PROTAC degraders in mice and rats.^77,78 Accordingly, we have synthesized and evaluated a series of ER PROTAC degraders using conformationally constrained linkers without an amide bond in the linker but containing a positively charged amine group. The data are summarized in Table 5.

Table 5.

ER PROTAC Degraders Using a Spiro Rigid Linker without an Amide Bond^a

CPD No	linker	ERα degradationa
		DC50 (nM)b	Dmax (%)C
28 (ERD-3137)		1.1 ± 0.07	92 ± 3
29 (ERD-12019)		1.2 ± 0.18	97 ± 3
30 (ERD-12020)		2.2 ± 0.5	82 ± 3
31 (ERD-2037)		4.9 ± 0.85	86 ± 2
32 (ERD-520)		4.7 ± 0.72	99 ± 0.2
2 (Fulvestrant)	NA	0.29 ± 0.1	100 ± 1
4 (ARV-471)	NA	0.82 ± 0.2	98 ± 1

^aa,b,c Same legend as in Table 2.

While these compounds (28−32) are less potent than those ER PROTAC degraders containing an amide bond in the linker, they are still quite potent and effective in inducing ER degradation with DC₅₀ = 1.1−4.9 nM and D_max = 82−99%. In particular, compounds 28 and 29 are similarly potent and effective in inducing ER degradation as compared to ARV-471.

We evaluated the oral exposure of representative compounds 29 and 31 in rats with limited time points (Table 6). Our data showed that both compounds 29 and 31 have excellent oral exposure in rats, much improved over those three degraders evaluated in Table 4.

Table 6.

Assessment of Oral Exposures of ER Degraders in Rats

		concentration in rat plasma (ng/mL)a
CPD no.	PO dose (mg/kg)	1 h	3 h	6 h	24 h
29 (ERD-12019)	3	211.2 ± 96.9	309.0 ± 93.0	244.6 ± 131.1	ND
31 (ERD-2037)	3	152.3 ± 33.9	237 ± 97.7	182.7 ± 79.8	36.4 ± 22
33 (ERD-1233)	3	820.5 ± 51.6	1145 ± 176.7	977.5 ± 88.3	274 ± 10
34 (ERD-5284)	3	145.4 ± 111.1	105.8 ± 37	66.4 ± 7.4	4.3 ± 0.12

^aThe plasma drug concentration data for each compound was independently collected from 3 rats and provided as the mean ± SD; ND = not determined.

Next, we synthesized a series of ER PROTAC degraders by inserting one or two oxygen atoms into the 6,5-spiro and 6,6-spiro linker rings in compound 31 and compound 32, with their degradation potency data summarized in Table 7. Insertion of one oxygen atom in the 5-membered ring of the 6,5-spiro linker at different positions in compound 31 resulted in compounds 33, 34, and 35, respectively. Compounds 33 and 34 showed 5-times improvement in degradation potency compared to compound 31 with DC₅₀ = 0.9 nM and achieved D_max values of 100%. Compound 35 displays an improved DC₅₀ value by 2.5 times (DC₅₀ = 2 nM) but has no improvement on D_max (86%). Inserting two oxygen atoms in the 5-membered ring in compound 31, which generated compound 36, enhances the degradation potency by 9 times (DC₅₀ = 0.5 nM) but has no beneficial effect on D_max (84%). Insertion of one oxygen atom in the 6-membered ring of the 6,6-spiro linker in compound 32 yielded compound 37, which shows no improvement in degradation potency and D_max values as compared to compound 32. Inserting two oxygen atoms in the 6,6-spiro linker in compound 32 produced compound 38, which is similarly potent and effective as compared to compound 32.

Table 7.

ER PROTAC Degraders Containing One or Two Oxygen Atoms in the Spiro Linker

CPD No	Rigid linker	ERα degradationa
		DC50 (nM)b	Dmax (%)c
31 (ERD-2037)		4.9 ± 0.8	86 ± 2
33 (ERD-1233)		0.9 ± 0.1	100 ± 2
34 (ERD-5284)		0.9 ± 0.1	100 ± 2
35 (ERD-5311)		2.0 ± 0.4	86 ± 4
36 (ERD-3131)		0.5 ± 0.01	84 ± 1
37 (ERD-12184)		5.9 ± 0.02	98 ± 6
38 (ERD-1277)		3.8 ± 0.03	95 ± 5
33a (ERD-1233A)d		0.4 ± 0.04	99 ± 1
33b (ERD-1233B)d		0.7 ± 0.1	100 ± 2

^aa,b,c Same legend as in Table 2.

^bd The absolute stereochemistry is not determined and is arbitrarily assigned. 33a was made using the faster-moving intermediate and 33b was made the slower-moving intermediate.

Based upon their excellent degradation potency and efficiency data for compounds 33 and 34, we evaluated their oral exposures in rats with the data summarized in Table 6. Interestingly, while compound 33 (ERD-1233) displays an excellent oral exposure in rats, which is more than 5 times higher than compound 31 in each of the four time points evaluated, compound 34 has an inferior oral exposure to compound 31. Based on the excellent oral exposure data for compound 33, its two isomers (33a and 33b) were synthesized and evaluated for their ER degradation potency, although the absolute stereochemistry for the linker in these two compounds was not determined. Compound 33a was made using the faster-moving aldehyde intermediate (Scheme 5) and 33b was made using the slower-moving aldehyde. Compound 33a is 2 times more potent than compound 33, whereas compound 33b shows a similar degradation potency as compared to compound 33. Both 33a and 33b showed similar D_max values as compound 33. The degradation potency data for compounds 33, 33a, and 33b showed that the chiral and racemic compounds demonstrate similar degradation potencies. Accordingly, compound 33 was selected for further extensive evaluation.

Scheme 5. Synthesis of Compounds 33−38 in Table 7^a.

^aReagent and conditions: (a) conc. HCl, EtOAc, rt, 2 h then CbzOSu, Na₂CO₃, EtOAc, rt, 5 h; (b) Ph₃P⁺CH₂OCH₃Cl⁻, NaHMDS, THF, 0-rt, 2 h then formic acid, rt, 2 h; (c) HC(OMe)₃, TsOH, MeOH, 60 °C, 4 h; (d) Pd/C, H₂ balloon, rt, 2 h; (e) TFA, DCM, rt, 1 h; (f) 2-(Hydroxymethyl)propane-1,3-diol, pTSA, Toluene, 80 °C, 8 h; (g) Pd(OAc)₂, XPhos, NaOt-Bu, toluene, 100 °C, 60−70%. (h) 4 (N) H₂SO₄, THF, 60 °C, 4 h; (i) 8, NaOAc, NaBH₃CN, DCM:MeOH, rt, 1 h; (j) t-BuXphosPdG3, NaOt-Bu, amyl alcohol, 100 °C, 2 h, 50%; (k) DMP, NaHCO₃, DCM, 0 °C, 1 h; (l) TsCl, Et₃N, DCM, rt, 2 h; (m) 8, DIPEA, DMF, 60 °C, 4 h.

Pharmacokinetics of ERD-1233 in Mice and Rats.

Based upon the initial promising oral exposure data for ERD-1233 in rats, we evaluated its full PK properties in mice and rats with the data summarized in Table 8.

Table 8.

Summary of PK Profiles in Mice and Rats for ERD-1233 and ARV-471^a

ID	species	IV/PO (mg/kg)	Clc (mL/min/kg)	Vssc (L/kg)	T1/2b (h)	Cmaxb (ng/mL)	AUC(0−24h)b (h·ng/mL)	Fb (%)
ERD-1233	ICR mice	1/3	3.2	0.6	2.8	926	7901	50
	SD rats	1/3	1.4	0.5	4.5	1283	12,747	37
ARV-471	ICR mice	1/3	21.9	1.8	2.5	156.3	684	31
	SD rats	1/3	18.6	2.4	4.0	46.5	244	10

^aThe definitions are as follows: IV, intravenous administration; T_1/2, elimination half-life; AUC, area-under-the-curve; V_ss, volume of distribution at steady state; Cl, clearance; PO, oral administration; C_max, maximum drug concentration; F, oral bioavailability.

^bPO.

^cIV.

In mice, ERD-1233 displays a slow clearance (Cl = 3.2 mL/min/kg) and a moderate volume of distribution (V_ss = 0.6 L/kg) with intravenous administration. ERD-1233 achieves excellent oral exposure with C_max = 926 ng/mL and AUC_0−24h = 7901 h· ng/mL with 3 mg/kg PO dose and an overall oral bioavailability of 50%. In rats, ERD-1233 has a slow clearance (Cl = 1.4 mL/min/kg) and a moderate volume of distribution (V_ss = 0.5 L/kg) with intravenous administration. ERD-1233 achieves excellent oral exposure with C_max = 1283 ng/mL and AUC_0−24h = 12,747 h·ng/mL and an overall oral bioavailability of 37% in rats.

We also evaluated ARV-471 for its PK profiles in mice and rats with the data summarized in Table 8. In comparison, ERD-1233 achieves 11.5 and 52 times higher oral plasma exposures than ARV-471 in mice and rats, respectively.

Profiling of ERD-1233 for Its Plasma and Metabolic Stability, Inhibition of hERG and Cytochrome P450 (CYP).

We evaluated ERD-1233 for its plasma and microsomal stability, and inhibition of hERG and CYP, with the data summarized in Table 9. ERD-1233 exhibits excellent plasma and microsomal stability. ERD-1233 has no significant hERG inhibition with an IC₅₀ value >30 μM. ERD-1233 has no significant CYP inhibition against all the CYP isoforms up to 10 μM, the highest concentration tested.

Table 9.

Plasma and Metabolic Stability, CYP and hERG Inhibition of ERD-1233

	plasma and microsomal stability T1/2 (min)					CYP inhibition IC50 (μM)
	human	dog	monkey	rat	mouse	1A2/2C8/2C9/2C19/2D6	3A4Midazolam	3A4 Testosterone	hERG inhibition IC50 (μM)
plasma	>240	>240	>240	>240	>240	>10	>10	>10	>30
microsome	>60	>60	>60	>60	>60

Evaluation of ERD-1233 in MCF-7 and T47D Cell Lines Using Traditional Western Blot.

We evaluated ER degradation of ERD-1233 in ER+ MCF-7 and T47D cell lines using traditional Western blotting analysis, with ARV-471 included as the control.

Our data (Figure 3) showed that both compounds effectively reduce the levels of ER protein in a dose-dependent manner in both cell lines. ERD-1233 achieves DC₅₀ values of ~1 nM with a D_max of ~75% in both cell lines at the concentration of 3−10 nM. ARV-471 also achieves DC₅₀ values of ~1 nM in both cell lines but has a lower D_max in the T47D cell line as compared to ERD-1233.

Figure 3.

Western blot analysis of the concentration-dependent ERα degradation by ERD-1233 and ARV-471 in MCF-7 cell line (A) and T47D cell line (B).

Pharmacodynamic Studies of ERD-1233 in ER Wild-Type and ESR1^Y537S MCF-7 Xenograft Mouse Models.

Based upon its excellent oral bioavailability and in vitro ER degradation activity, we evaluated ERD-1233 for its ability to reduce the levels of ER protein in both ER wild-type MCF-7 and ESR1^Y537S mutant MCF-7 tumors in mice in pharmacodynamic (PD) experiments, with the data summarized in Figure 4.

Figure 4.

PK/PD studies of ERD-1233 in ER wild-type MCF-7 and ESR1^Y537S MCF-7 xenograft tumor mouse models. (A) Western blotting analysis for wild-type MCF-7 protein level; (B) Drug concentration of ERD-1233 in tumor and plasma for wild-type MCF-7 PD experiment; (C) Western blotting analysis for ESR1^Y537S mutant protein level; (B) Drug concentration of ERD-1233 and ARV-471 in tumor and plasma for ESR1^Y537S mutant PD experiment; Animals were dosed once daily for 3 days in the wild-type MCF-7 model and a single oral dose in the ESR1^Y537S MCF-7 model. Data were provided as mean ± SD.

ERD-1233 was administered to mice bearing MCF-7 xenograft tumors via oral gavage at 3 or 10 mg/kg, once daily for 3 days, and plasma and tumor tissue were collected at 6 and 24 h after the last dose for analysis. Western blotting analysis of the MCF-7 tumor tissue showed that ERD-1233 effectively reduced the ER protein levels by 42 and 43%, respectively, at 6 and 24 h time-points at the 3 mg/kg dose level, and by 62 and 50%, respectively, at 6 and 24 h time-points at the 10 mg/kg dose level (Figure 4A).

We analyzed the drug concentrations for ERD-1233 in both plasma and tumor tissue with the data shown in Figure 4B. Our data showed that ERD-1233 has reasonable drug exposures in both plasma and tumor at the 6 h time-point but low drug levels at 24 h time-point, indicating a lack of drug accumulation. There was a dose-proportional increase of drug concentrations in both plasma and tumor tissue when the dose was increased from 3 to 10 mg/kg.

We next performed a PK/PD study of ERD-1233 in the ESR1^Y537S mutant MCF-7 model. We dosed mice bearing the ESR1^Y537S MCF7 tumors with ERD-1233 at 10 mg/kg, a single oral dose, and tumor and plasma samples were collected for analysis. Western blotting analysis of the tumor tissues (Figure 4C) showed that ERD-1233 at 10 mg/kg effectively reduced the levels of the mutant ERα protein at 3 and 24 h time-points by 43 and 78%, respectively. In comparison, a single oral dose of ARV-471 at 10 mg/kg reduced the levels of the mutant ERα protein by 77% at 24 h time-point.

Analysis of drug concentrations in tumor and plasma tissues in the ESR1^Y537S mutant MCF-7 model showed that ERD-1233 dosed at 10 mg/kg achieved a high plasma concentration of 5365 ng/mL and a reasonable drug concentration of 312 ng/mL in the tumor tissue at 3 h time point. At 24 h time-point, ERD-1233 has 16 ng/mL in plasma and 157 ng/mL in tumor tissues. In comparison, ARV-471 has a drug concentration of 9 ng/mL in plasma and 286 ng/mL in the tumor at 24 h time point. Hence, ERD-1233 achieved a sufficiently high drug level in the tumor tissue at both 3 and 24 h time points, consistent with its good PD effect in the tumor tissue.

Antitumor Efficacy of ERD-1233 in the Wild-Type MCF-7 and the ESR1^Y537S Mutant Xenograft Mouse Models.

We evaluated the antitumor activity of ERD-1233 in the MCF-7 xenograft tumor model, with ARV-471 included as the control. The data are summarized in Figure 5A,B.

Figure 5.

Antitumor efficacy of ERD-1233 in ER wild-type and ESR1^Y537S MCF-7 xenograft mouse models. ARV-471 was included as the control. (A) Wild-type MCF-7 tumor growth; (B) Animal body weight changes in the ER wild-type MCF-7 model; (C) ESR1^Y537S mutant MCF-7 tumor growth; (D) ESR1^Y537S MCF-7 body weight change. For the wild-type MCF-7 model, each group of animals was dosed once-daily (PO) starting from day 70 post-tumor implantation up to day 98. For the ESR1^Y537S mutant MCF-7 model, each group of animals was orally dosed 1−5 days a week starting from day 34 post-tumor implantation up to day 63. The 5 mg/kg dose of ERD-1233 was changed to 30 mg/kg after 2nd week. A method of one-tailed unpaired t test with Welch’s correction was used for statistical analysis of the tumor volumes between groups. “*”, P < 0.05; “***”, P < 0.001; “****”, P < 0.0001.

When the MCF-7 tumors grew to 275 mm³ on average, mice were randomized and dosed with ERD-1233, once daily at 10 and 20 mg/kg, and with ARV-471 at 30 mg/kg via oral gavage, and the vehicle control for 4 weeks. ARV-471 at 30 mg/kg was very effective in inhibition of tumor growth and in fact, induced 32% of tumor regression at the end of the treatment. In comparison, ERD-1233 achieved tumor regression of 34% at 10 mg/kg and 68% at 20 mg/kg, respectively. Hence, ERD-1233 at 20 mg/kg achieved greater tumor regression than ARV-471 at 30 mg/kg (p = 0.01) in the MCF-7 tumor xenograft model. Of significance, both compounds induced minimal animal weight losses or other signs of toxicity during the entire experiment.

Based on the excellent efficacy of ERD-1233 in the ER wild-type MCF-7 mouse model, we next performed an efficacy experiment in the ESR1^Y537S mutant MCF-7 model with the data shown in Figure 5C,D. In this experiment, animals were dosed with ERD-1233 at 5 and 10 mg/kg via oral gavage daily, 5 days a week. While ERD-1233 at 10 mg/kg effectively inhibited tumor growth in the entire experiment, ERD-1233 at 5 mg/kg displayed minimal antitumor activity after 2 weeks of drug administration (Figure 5C). To evaluate if ERD-1233 at a higher dose can achieve a stronger antitumor activity, we decided to change the 5 mg/kg dose to 30 mg/kg and dosed the animals for another 2 weeks. Our data showed that ERD-1233 at 30 mg/kg was capable of achieving tumor regression during the treatment period (Figure 5C).

Chemistry.

The synthesis of new cereblon ligands 8 and 9 was summarized in Scheme 1. The synthesis started with the commercially available compound 5-bromoisobenzofuran-1(3H)-one (39). Triflic acid-mediated iodination of compound 39 in the presence of NIS yielded two isomeric iodo intermediates. The isomeric iodo compound mixture was then converted to corresponding hydroxy compounds, and the desired compound 40 was isolated in a 42% yield as a major product after two steps. A Mitsunobu reaction between compound 40 and chiral piperazine alcohol in the presence of DIAD and triphenylphosphine produced an ether intermediate. Subsequent Fmoc deprotection and intramolecular Buchwald amination reaction resulted in intermediate lactone compounds 41a or 41b with a combined yield of 30% over three steps. Saponification of lactone 41a or 41b with NaOH furnished the corresponding acid alcohol compound which was oxidized to the corresponding acid aldehyde intermediate 42a or 42b with manganese(IV) oxide in DCM in 30% yield. The acid aldehyde compounds 42a or 42b were then subjected to reductive amination with (S)-3-aminopiperidine-2,6-dione, using sodium acetate and sodium cyanoborohydride. The resulting amine acid intermediate was immediately cyclized into the corresponding lactam through a HATU-mediated acid-amine coupling reaction. Subsequent removal of the Boc protecting group from the lactam intermediates, using TFA in DCM, yielded chiral ligands 8 and 9 (as TFA salts) with an overall yield of 30−45% over three steps. Compound 8 was converted to compound 43 following a two-step protocol; substitution reaction with tert-butyl 2-bromoacetate in the presence of DIPEA as a base, followed by removal of tert-butyl group using TFA in DCM at room temperature.

Scheme 1. Synthesis of Compounds 8, 9, and 43^a.

^aReagents and conditions: (a) NIS, Triflic acid, rt; (b) Cu₂O, NaOH, DMA, 70 °C, 42% (two steps); (c) 1-((9H-fluoren-9-yl)methyl) 4-(tert-butyl) (S) or (R)-2-(hydroxymethyl)piperazine-1,4-dicarboxylate, TPP, DIAD, THF, 0 °C, 2 h; (d) 20% Piperidine in DMF, DMF, rt, 1 h; (e) Pd₂(dba)₃, XantPhos, Cs₂CO₃, 1,4-dioxane, 100 °C, 6 h; (f) NaOH, MeOH: H₂O, rt, 2 h; (g) MnO₂, DCM, rt, 2 h, 30%; (h) (S)-3-aminopiperidine-2,6-dione, NaOAc, NaBH₃(CN), 3 h; (i) HATU, DIPEA, DMF, 0 °C, 2 h; (j) TFA, DCM, rt, 2 h, 45% (three steps); (k) tert-butyl 2-bromoacetate, DIPEA, DMF, rt, 2 h.

The synthesis of compounds 10−15 is outlined in Scheme 2. Compound 44 was prepared using a reported synthetic procedure.⁷⁹ Compound 44 was converted into ester intermediates 46a-46d involving a substitution reaction with various bromo esters in the presence of potassium carbonate in DMF. These ester intermediates were subsequently hydrolyzed to yield the corresponding acid groups, followed by deprotection of the tert-butyl protecting group using TFA in DCM. The resulting acids were then coupled with the cereblon ligand 8, using HATU and DIPEA, to synthesize the final compounds 10−13.

Scheme 2. Synthesis of Compounds 10−15, in Table 2^a.

^aReagents and conditions: (a) K₂CO₃, DMF, rt, 8 h, 60−80% (b) LiOH then TFA, DCM or TFA, DCM (c) HATU, DIPEA, DMF, 1 h (d) 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonyl fluoride, K₂CO₃, THF/MeCN, rt, 95%; (e) Pd(OAc)₂, XPhos, NaOt-Bu, toluene, 100 °C, 60−70%.

Furthermore, compound 44 was converted into the corresponding perfluoro butanesulfonate 45 using a reported protocol.⁷⁹ Compound 45 was used as Buchwald amination precursor in different amination reactions. Compounds 46e and 46f were synthesized from intermediate 45 by Buchwald amination reactions using corresponding substituted amines. These intermediates were then converted to compounds 14 and 15 through tert-butyl deprotection using TFA, followed by an acid-amine coupling reaction with compound 8 using HATU and DIPEA at room temperature.

Synthesis of compounds 16−27 was outlined in Scheme 3. Starting with compound 45, a Buchwald amination reaction was carried out with tert-butyl 2-(piperidin-4-yl)acetate using Pd(OAc)₂ as a catalyst in toluene to yield compound 47 with a 60% yield. Removing the tert-butyl protecting groups from compound 47, followed by an acid-amine coupling with intermediate 8, led to the successful synthesis of compound 16 with an 80% yield. Similarly, the Buchwald amination reaction between compound 45 with tert-butyl piperazine-1-carboxylate yielded the desired intermediate, which, upon deprotection of both tert-butyl and Boc groups, resulted in intermediate 48 with an 85% yield. The amine, 48, was then coupled with acid 43 to give compound 23 in 80% yield. Intermediate 49 was synthesized using a two-step protocol, first substitution reaction between compound 48 with tert-butyl 2-bromoacetate followed by deprotection of tert-butyl groups, resulting in intermediate 49 in 80% yield. An acid-amine coupling reaction of acid 49 with amine 8 yielded compound 17 in 75% yield.

Scheme 3. Synthesis of Compounds 16−27, in Table 3^a.

^aReagents and conditions: (a) Pd(OAc)₂, XPhos, NaOt-Bu 100 °C; (b) TFA, DCM, rt, 6 h; (c) HATU, DIPEA, DMF; (d) tert-butyl piperazine-1-carboxylate, Pd(OAc)₂, XPhos, NaOt-Bu, 100 °C; (e) tert-butyl 2-bromoacetate, DIPEA, DMF, rt, 1 h.

Additionally, Buchwald amination of 45 with Boc-protected spiro amines followed by deprotection of Boc and tert-butyl groups produced intermediates 50a−50e in 60−80% yields over two steps.⁶⁸ The subsequent acid-amine coupling reaction between intermediates 50a−50d with acid 43 yielded compounds 24−27 in 70−80% yields. Intermediate 50a−50e were also converted into intermediate 51a−51e by substitution reaction with tert-butyl 2-bromoacetate followed by tert-butyl deprotection with TFA in 60−70% yields over two steps. Finally, intermediates, 51a−51e, were coupled with compound 8 through an acid-amine coupling reaction to produce compounds 18−22 in 70−80% yields.

Synthesis of compounds 28−32 was summarized in Scheme 4. Buchwald amination of compound 45 with ethyl 2-(7-azaspiro[3.5]nonan-2-yl)acetate produced compound 52 in 55% yield. Then, compound 52 underwent a selective reduction of its ester group to an aldehyde using DIBAL-H at a low temperature. The subsequent deprotection of the tert-butyl group using 4 (N) H₂SO₄, followed by reductive amination with the cereblon ligand 8, led to the synthesis of compound 28 with an overall yield of 45% after completing the three-step process. Acetal-protected spiro amines 54a−54d were synthesized from commercially available starting keto compounds 53a−53d by following the reported protocol.⁸⁰ Buchwald amination of 45 with spiro amines 54a−54d followed by subsequent removal of the acetal and tert-butyl protecting groups using 4 (N) H₂SO₄ produced aldehydes 55a−55d in 50−60% yields in two steps. Finally, reductive amination of compounds 55a−55d with amine 8 in the presence of sodium acetate and sodium cyanoborohydride at room temperature furnished compounds 29–32 with yields ranging from 55 to 80%.

Scheme 4. Synthesis of Compounds 28−32, in Table 5^a.

^aReagent and conditions: (a) Ethyl 2-(7-azaspiro[3.5]nonan-2-yl)acetate, Pd(OAc)₂, XPhos, NaOt-Bu, toluene, 100 °C, 70%; (b) DIBAL-H (25% in toluene), DCM, −78 °C, 72%; (c) 4 (N) H₂SO₄, THF, 60 °C, 4 h; (d) 8, NaOAc, NaBH₃CN, DCM: MeOH, rt, 1 h; (e) conc. HCl, EtOAc, rt, 2 h then CbzOSu, Na₂CO₃, EtOAc, rt, 5 h; (f) Ph₃P⁺CH₂OCH₃Cl⁻, NaHMDS, THF, 0-rt, 2 h then formic acid, rt, 2 h; (g) HC(OMe)₃, TsOH, MeOH, 60 °C, 4 h; (h) Pd/C, H₂ balloon, rt, 2 h; (i) Pd(OAc)₂, XPhos, NaOt-Bu, toluene, 100 °C, 60−70%.

Synthesis of compounds 33−38 is summarized in Scheme 5. Intermediates 56a and 56b were synthesized using a reported protocol starting from corresponding keto compounds.⁸⁰ Buchwald amination of 56a and 56b with intermediate 45 and subsequent removal of acetal and tert-butyl groups in the presence of 4 (N) H₂SO₄ furnished aldehydes 58a and 58b. Aldehydes 58a and 58b were next subjected to reductive amination with cereblon ligand 8 to provide compounds 33 and 37 in 80 and 68% respectively. Buchwald amination of compound 45 with amines 57a−57d in the presence of t-BuXphosPdG3 and sodium tert-butoxide in amyl alcohol furnished intermediates 59a−59d in 40−50% yields. Intermediates 59a−59d were subjected to Dess-Martin oxidation and tert-butyl deprotection to furnish intermediate aldehydes. Aldehydes were then converted to corresponding final compounds 34, 35, 36, and 38 using a reductive amination protocol with cereblon ligand 8 in 30−40 overall yields in three steps. Compound 45 on Buchwald amination reaction with amine 57e furnished compound 60 in 50% yields. Intermediate 60 was separated into two isomers 60a (faster-moving fraction) and 60b (slower-moving fraction) using the SFC separation protocol. Both isomers were converted into corresponding tosylates to provide 61a and 61b respectively. Substitution reaction of intermediates 61a and 61b with cereblon ligand 8 in the presence of DIPEA followed by tert-butyl deprotection with TFA furnished chiral isomers 33a (ERD-1233A) and 33b (ERD-1233B) respectively.

Summary.

In the present study, we described the design, synthesis, and biological evaluations of novel, potent and orally efficacious ERα PROTAC degraders.

We first designed and evaluated RR-11055 (8) as a new cereblon ligand, which binds to cereblon with an affinity 4.5 and 8.6 times better than lenalidomide and thalidomide. In addition, RR-11055 has excellent microsomal and plasma stability. Employing our new CRBN ligand RR-11055 (8) and lasofoxifene as the ER ligand and through extensive optimization of the linker, we have successfully discovered several highly potent and orally bioavailable ER degraders, with ERD-1233 being the best compound. ERD-1233 showed potent and efficient degradation activity (DC₅₀ = ~ 1 nM) in both MCF-7 and T47D ER+ human breast cancer cell lines with D_max values of 100%, using fulvestrant as the control. ERD-1233 exhibits excellent microsomal and plasma stability and no significant hERG or CYP inhibition. PK/PD experiments showed that the oral administration of ERD-1233 effectively reduces the levels of wild-type ERα protein, as well as the ESR1^Y537S mutated protein in xenograft tumors in mice. Oral administration of ERD-1233 at 10 and 20 mg/kg was capable of achieving tumor regression in the ER wild-type MCF-7 xenograft tumor model. In direct comparison, ERD-1233 at 20 mg/kg was more efficacious than ARV-471 at 30 mg/kg in the MCF-7 xenograft tumor model. In addition, oral administration of ERD-1233 at 10 and 30 mg/kg also achieved strong antitumor activity in the ESR1^Y537S mutant MCF-7 model in mice with tumor regression observed at 30 mg/kg.

Taken together, our data showed that ERD-1233 has a number of advantages over ARV-471. First, ERD-1233 has a much better oral bioavailability than ARV-471 in both mice and rats. Second, ERD-1233 achieves stronger antitumor efficacy and greater tumor regression than ARV-471 in the MCF-7 xenograft tumor model. Third, ERD-1233 does not display any liability in CYP inhibition, hERG inhibition, and microsomal and plasma stability. Because ARV-471 showed a drug−drug interaction with Palbociclib,^71,72 the lack of CYP inhibition by ERD-1233 may represent an advantage in combination with Palbociclib or other CDK4/6 inhibitors. Furthermore, ERD-1233 is also capable of achieving tumor regression in the MCF-7 ESR1^Y537S mutated xenograft tumor model, suggesting its therapeutic potential for the treatment of human breast cancer with mutated ER in the clinic. ERD-1233 was found to be well tolerated in all of our efficacy experiments. Hence, ERD-1233 is a highly promising PROTAC ERα degrader for extensive evaluations for the treatment of ER+ human breast cancer.

EXPERIMENTAL SECTION

General Information for Chemistry.

Unless otherwise noted, all commercial materials were used as received. NMR spectra were recorded on a Bruker Ascend 400 MHz spectrometer and calibrated using residual solvent peaks as an internal reference. In reported spectral data, the format (δ) chemical shift (multiplicity, J values in Hz, integration) was used with the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, hept = heptet, dd = doublet of doublets, and m = multiplet. Low-resolution mass spectrometric (MS) analysis was carried out with a Waters UPLC ACQUITY QDa mass spectrometer. High-resolution mass experiments were operated on an Agilent Technologies 6230 TOF LC/MS instrument with APCI ionization. Flash column chromatography was performed by Teledyne CombiFlash RF+ using RediSep Rf silica gel flash column. The final compounds were all purified by a C18 reverse phase preparative HPLC column (SunFire Prep C18 OBD 5 μm, 50 × 100 mm) with solvent A (0.1% TFA or formic acid in H₂O) and solvent B (0.1% TFA or formic acid in MeCN) as eluents at 60 mL/min flow rate. The purity of all the final compounds was measured and confirmed to be >95% by UPLC-MS analysis (10−100% MeCN in H₂O containing 0.1% formic acid in 5 min, 1.0 mL/min flow rate) with a C18 column (ACQUITY UPLC BEH C18 1.7 μm, 2.1 × 50 mm).

Synthetic Procedure for Compounds 8 and 9.

To a solution of 5-Bromo-3H-isobenzofuran-1-one (39, 5 g, 23.4 mmol, 1 equiv) in trifluoromethanesulfonic acid (68 g, 40 mL, 19.30 equiv) was added NIS (5.5 g, 24.6 mmol, 1.05 equiv) at 0 °C in portions. The mixture was allowed to warm to room temperature and stirred overnight. TLC (hexane: ethyl acetate = 5:1) showed no starting material remained and two new spots (R_f = 0.4, 0.5) formed. The reaction mixture was poured into ice water (100 mL) and yellow solid precipitated. The mixture was filtered, and the filter cake was washed with ice-cold water. The filter cake was dissolved in DCM (500 mL) and washed with 1 (M) Na₂S₂O₃ followed by dried over sodium sulfate. The mixture was filtered, and the filtrate was concentrated to afford a yellow solid. The crude product was purified on a 120 g silica column running a 0−10% EtOAc/hexane gradient over 70 min.

To a mixture of 5-bromo-4-iodo-3H-isobenzofuran-1-one (4 g, 1 equiv), sodium hydroxide (2.3 g, 5 equiv) in water (40 mL, 1.5 M) and N,N-dimethylacetamide (20 mL) was added cuprous oxide (0.338 g, 0.2 equiv). The reaction mixture was heated to 80 °C and held for 12 h. TLC (hexane: ethyl acetate = 1:1, R_f = 0.3) showed that the reaction was completed. The reaction mixture was neutralized using 1 (N) hydrochloride solution and extracted with ethyl acetate (40 mL × 2), washed with brine (150 mL), and then dried over sodium sulfate. The crude product was purified by silica gel column chromatography using 0−100% EtOAc/hexane. 5-Bromo-4-hydroxy-3H-isobenzofuran-1-one (40, 42% yield in two steps) was obtained as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 5.35 (s, 2H).

To a solution of 5-bromo-4-hydroxyisobenzofuran-1(3H)-one (40, 700 mg, 3 mmol, 1 equiv) in 12 mL of THF/DCM, 1-((9H-fluoren-9-yl)methyl) 4-(tert-butyl) (R)-2-(hydroxymethyl)piperazine-1,4-dicarboxylate (2 g, 4.5 mmol, 1.5 equiv) and PPh₃ (1.17 g, 4.5 mmol, 1.5 equiv) was added. The reaction mixture was cooled to 0 °C and DIAD (0.9 mL, 4.5 mmol, 1.5 equiv) was added dropwise. The resultant mixture was then stirred for 2 h at room temperature. The solvent was evaporated at reduced pressure; the crude product was purified by silica gel column chromatography using 0−100% EtOAc/hexane. UPLC−MS (ESI) m/z: calcd, 649.15 for C₃₃H₃₃BrN₂O₇ [M + H]⁺; found, 649.18.

To a solution of 1-((9H-fluoren-9-yl)methyl) 4-(tert-butyl) (R)-2-(((5-Bromo-1-oxo-1,3-dihydroisobenzofuran-4-yl)oxy)methyl)-piperazine-1,4-dicarboxylate (1 g) was added 20% (v/v) piperidine in DMF (5 mL/g of SM). The resulting mixture was stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate and washed with water. The combined organic phases were washed with brine, dried over Na₂SO_4, and the solvent was removed in vacuo to give an oil. The crude product was purified by silica gel column chromatography using 0−5% DCM in methanol. Yield 70%. UPLC−MS (ESI) m/z: calcd, 427.08 for C₁₈H₂₃BrN₂O₅ [M + H]⁺; found, 427.12.

A vial was charged with tert-butyl (R)-3-(((5-bromo-1-oxo-1,3-dihydroisobenzofuran-4-yl)oxy)methyl)piperazine-1-carboxylate (170 mg, 0.38 mmol, 1 equiv), Pd₂(dba)₃ (0.1 equiv), XantPhos (0.2 equiv), Cs₂CO₃ (3 equiv) and dioxane (5 mL). The mixture was purged with nitrogen and heated to 100 °C for 6 h. TLC (ethyl acetate: petroleum ether = 1:2) showed the reaction was complete. The mixture was diluted with ethyl acetate and washed with water. The organic layer was washed with brine and dried over sodium sulfate. The crude product was purified by silica gel column chromatography using 0−50% EtOAc/hexane to furnish compound 41a in 60% yields. UPLC−MS (ESI) m/z: calcd, 347.15 for C₁₈H₂₂N₂O₅ [M + H]⁺; found, 347.18. ¹H NMR (400 MHz, Chloroform-d) δ 7.43 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 5.19 (s, 2H), 4.33 (dd, J = 10.9, 2.9 Hz, 1H), 4.27−4.09 (m, 2H), 4.05 (dd, J = 10.9, 8.2 Hz, 1H), 3.80 (t, J = 6.0 Hz, 1H), 3.29 (ddt, J = 11.3, 8.2, 3.1 Hz, 1H), 3.05 (s, 1H), 2.91 (td, J = 12.1, 3.4 Hz, 1H), 2.68 (s, 1H), 1.51 (s, 9H).

To a solution of tert-butyl (R)-1-oxo-1,3,5a,6,8,9-hexahydroisobenzofuro[4,5-b]pyrazino[1,2-d][1,4]oxazine-7(5H)-carboxylate (41a, 346 mg, 1 mmol, 1 equiv) in tetrahydrofuran (4 mL) and water (4 mL) was added sodium hydroxide (200 mg, 5 equiv). The mixture was stirred at 20 °C for 2 h. TLC (ethyl acetate: hexane = 1:1) showed the reaction was complete. The mixture was adjusted to pH = 5 with aq. hydrochloric acid (1 M) and extracted with ethyl acetate (10 mL × 3). The organic layer was washed with brine (10 × 2 mL) and dried over sodium sulfate. The crude material was not further purified and used as crude for the next steps. UPLC−MS (ESI) m/z: calcd, 365.16 for C₁₈H₂₄N₂O₆ [M + H]⁺; found, 365.16.

To a solution of (R)-3-(tert-butoxycarbonyl)-7-(hydroxymethyl)-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino[1,2-d][1,4]oxazine-8-carboxylic acid (1 equiv) in dichloromethane (10 mL) was added manganese dioxide (15 equiv). The mixture was stirred at 20 °C for 2 h. TLC (ethyl acetate: hexane = 1:1) showed the reaction was complete. The mixture was diluted with dichloromethane (10 mL) and filtered through a pad of Celite. The filtrate was concentrated in a vacuum. The crude product was purified by silica gel column chromatography to get compound 42a in 30% yields. UPLC−MS (ESI) m/z: calcd, 363.15 for C₁₈H₂₂N₂O₆ [M + H]⁺; found, 363.16. ¹H NMR (400 MHz, Methanol-d₄) δ 7.32 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.64−6.40 (m, 1H), 4.42 (dd, J = 11.0, 3.0 Hz, 1H), 4.23−4.01 (m, 3H), 3.95 (d, J = 12.4 Hz, 1H), 3.34−3.23 (m, 1H), 3.08 (brs, 1H), 2.87 (td, J = 12.2, 3.5 Hz, 1H), 2.74 (s, 1H) 1.50 (S, 9H).

To a mixture of (S) 3-aminopiperidine-2,6-dione (HCl salt, 246 mg, 1.5 mmol, 1.5 equiv) in methanol (2 mL) and dichloromethane (4 mL) was added sodium acetate (332 mg, 4 mmol, 4 equiv). The mixture was stirred at 20 °C for 15 min, then tert-butyl (5aR)-3-hydroxy-1-oxo−-1,3,5a,6,8,9-hexahydroisobenzofuro[4,5-b]pyrazino[1,2-d][1,4]-oxazine-7(5H)-carboxylate (42a, 362 mg, 1 mmol, 1 equiv) was added and the mixture was stirred for 30 min. Sodium cyanoborohydride (126 mg, 2 mmol, 2 equiv) was added and the mixture was further stirred for 3 h. LCMS showed the reaction was complete. The mixture was adjusted to pH = 4−5 with an aqueous hydrochloric acid solution (1 M) and extracted with ethyl acetate (10 mL × 3). The crude material was not further purified and used as crude for the next steps. UPLC−MS (ESI) m/z: calcd, 475.21 for C₂₃H₃₀N₄O₇ [M + H]⁺; found, 475.25.

To a solution of (R)-3-(tert-butoxycarbonyl)-7-((((S)-2,6-dioxopiperidin-3-yl)amino)methyl)-1,2,3,4,4a,5-hexahydrobenzo[b]pyrazino-[1,2-d][1,4]oxazine-8-carboxylic acid (426 mg, 0.9 mmol, 1 equiv) in dimethylformamide (5 mL) was added HATU (342 mg, 0.9 mmol, 1.0 equiv) followed by addition of DIPEA (0.47 mL, 2.7 mmol, 3 equiv). The solution was stirred for 2 h at 0 °C. The residue was purified by reverse-phase HPLC to get a Boc-protected intermediate tert-butyl (R)-2-((S)-2,6-dioxopiperidin-3-yl)-1-oxo-2,3,5a,6,8,9-hexahydro-1H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindole-7(5H)-carboxylate. UPLC−MS (ESI) m/z: calcd, 457.20 for C₂₃H₂₈N₄O₆ [M + H]⁺; found, 457.27. ¹H NMR (400 MHz, Methanol-d₄) δ 7.32 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.10 (dd, J = 13.3, 5.2 Hz, 1H), 4.46−4.30 (m, 3H), 4.23−3.98 (m, 3H), 3.93 (d, J = 12.4 Hz, 1H), 3.22 (ddd, J = 11.2, 8.2, 3.0 Hz, 1H), 3.07 (s, 1H), 2.99−2.61 (m, 4H), 2.59−2.42 (m, 1H), 2.21−2.07 (m, 1H), 1.51 (s, 9H).

(S)-3-((R)-1-Oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (8).

To a stirred solution of tert-butyl (R)-2-((S)-2,6-dioxopiperidin-3-yl)-1-oxo-2,3,5a,6,8,9-hexahydro-1H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindole-7(5H)-carboxylate (456 mg, 1.0 mmol, 1 equiv) in DCM (10 mL), TFA (1 mL) was added at room temperature and the reaction was stirred for 2 h. The reaction mixture was concentrated to afford compound 8 TFA salt (400 mg, quantitative) as a white solid. UPLC−MS (ESI) m/z: calcd, 357.15 for C₁₈H₂₀N₄O₄ [M + H]⁺; found, 357.20. ¹H NMR (400 MHz, Methanol-d₄) δ 7.36 (dt, J = 9.1, 2.5 Hz, 1H), 7.14 (dd, J = 8.6, 3.4 Hz, 1H), 5.10 (dd, J = 13.4, 5.0 Hz, 1H), 4.94 (dd, J = 4.5, 2.5 Hz, 1H), 4.50−4.28 (m, 3H), 4.28−4.09 (m, 2H), 3.66 (d, J = 14.5 Hz, 1H), 3.59−3.43 (m, 2H), 3.33−3.11 (m, 3H), 3.02 (t, J = 12.1 Hz, 1H), 2.91−2.72 (m, 1H), 2.50 (qd, J = 13.2, 4.8 Hz, 1H), 2.16 (dtd, J = 12.9, 5.4, 2.4 Hz, 1H). ¹³C NMR (101 MHz, Methanol-d₄) δ 173.3, 170.9, 170.1, 139.3, 136.7, 129.6, 116.8, 114.0, 65.9, 52.3, 49.6, 48.0, 47.8, 47.6, 47.4, 47.2, 45.1, 43.0, 42.9, 42.3, 31.0, 22.7.

(S)-3-((S)-1-Oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino-[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (9).

A similar synthetic procedure for compound 8 was followed to make compound 9 starting from 5-bromo-3H-isobenzofuran-1-one and using 1-((9H-fluoren-9-yl)methyl) 4-(tert-butyl) (S)-2-(hydroxymethyl)-piperazine-1,4-dicarboxylate as chiral precursor. Compound 9 was obtained as a white solid with a 30% overall yield. UPLC−MS (ESI) m/z: calcd, 357.15 for C₁₈H₂₀N₄O₄ [M + H]⁺; found, 357.30. ¹H NMR (400 MHz, Methanol-d₄) δ 7.36 (dt, J = 9.1, 2.5 Hz, 1H), 7.14 (dd, J = 8.6, 3.4 Hz, 1H), 5.10 (dd, J = 13.4, 5.0 Hz, 1H), 4.94 (dd, J = 4.5, 2.5 Hz, 1H), 4.50−4.28 (m, 3H), 4.28−4.09 (m, 2H), 3.66 (d, J = 14.5 Hz, 1H), 3.59−3.43 (m, 2H), 3.33−3.11 (m, 3H), 3.02 (t, J = 12.1 Hz, 1H), 2.91−2.72 (m, 1H), 2.50 (qd, J = 13.2, 4.8 Hz, 1H), 2.16 (dtd, J = 12.9, 5.4, 2.4 Hz, 1H). ¹³C NMR (101 MHz, Methanol-d₄) δ 173.29, 170.98, 170.10, 139.31, 136.71, 129.60, 116.79, 114.02, 65.98, 52.33, 49.64, 48.04, 47.82, 47.61, 47.40, 47.18, 45.10, 43.01, 42.89, 42.35, 31.00, 22.69.

2-((R)-2-((S)-2,6-Dioxopiperidin-3-yl)-1-oxo-2,3,5a,6,8,9-hexahydro-1H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-7(5H)-yl)acetic Acid (43).

A stirred solution of compound 8 (350 mg, 1 mmol, 1 equiv) and tert-butyl 2-bromoacetate (230 mg, 1.2 mmol, 1.2 equiv) in dry DMF, DIPEA (0.26 mL, 1.5 mmol, 1.5 equiv) was added and the mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by UPLC chromatography), the product was purified with reverse phase column chromatography. The solvent was evaporated to dryness and the product dissolved in DCM (3 mL). TFA (1 mL) was added to the mixture and the reaction was stirred at room temperature for another 4 h. After completion of the reaction, the solvent was evaporated to dryness and the compound was dried by lyophilization to get compound 43 (298 mg, 72%). UPLC−MS (ESI) m/z: calcd, 415.15 for C₂₀H₂₂N₄O₆ [M + H]⁺; found, 415.27. ¹H NMR (400 MHz, Methanol-d₄) δ 7.37 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 5.11 (dd, J = 13.3, 5.2 Hz, 1H), 4.49−4.42 (m, 1H), 4.38 (d, J = 8.7 Hz, 1H), 4.33−4.23 (m, 1H), 4.24−4.11 (m, 3H), 3.76 (tdd, J = 15.7, 10.7, 6.1 Hz, 3H), 3.40−3.33 (m, 2H), 3.28 (dd, J = 12.8, 2.1 Hz, 1H), 3.18−3.05 (m, 1H), 2.97−2.84 (m, 1H), 2.79 (ddd, J = 17.6, 4.7, 2.4 Hz, 1H), 2.50 (qd, J = 13.2, 4.7 Hz, 1H), 2.16 (dtd, J = 12.8, 5.3, 2.5 Hz, 1H). ¹³C NMR (101 MHz, Methanol-d₄) δ 173.2, 170.9, 170.0, 139.3, 136.3, 129.6, 123.9, 116.8, 114.1, 65.8, 55.7, 52.3, 51.6, 51.2, 49.6, 45.0, 43.1, 30.9, 26.8, 22.7.

Synthetic Procedure for Compounds 10−38.

(S)-3-((R)-7-(2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino-[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (10).

To a stirred solution of compound 44 (100 mg, 0.27 mmol, 1 equiv), in DMF (3 mL), K₂CO₃ (75 mg, 0.54 mmol, 2.0 equiv) and methyl 2-bromoacetate (60 mg, 0.4 mmol, 1.5 equiv) were added, and the mixture was stirred for 8 h at rt. After completion of the reaction, it was diluted with water and extracted with diethyl ether. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The product was purified using flash column chromatography to get methyl 2-(4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)acetate (46a, 84 mg, 70%) as a colorless liquid. UPLC−MS (ESI) m/z: calcd, 445.23 for C₂₉H₃₂O₄ [M + H]⁺; found, 445.23; ¹H NMR (400 MHz, Chloroform-d) δ 7.23−7.12 (m, 3H), 6.92−6.72 (m, 5H), 6.60−6.52 (m, 2H), 6.34 (dt, J = 8.8, 2.2 Hz, 2H), 4.54 (d, J = 1.2 Hz, 2H), 4.28 (d, J = 5.1 Hz, 1H), 3.88−3.78 (m, 3H), 3.46−3.36 (m, 1H), 3.22−2.95 (m, 2H), 2.18 (tdd, J = 13.5, 11.2, 6.9 Hz, 1H), 1.99−1.75 (m, 1H), 1.48−1.35 (m, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.49, 155.92, 153.57, 144.22, 137.15, 135.82, 134.55, 131.46, 130.83, 128.17, 127.77, 126.03, 123.83, 122.06, 113.06, 78.09, 65.39, 52.17, 50.39, 45.28, 29.99, 28.96, 21.88.

To a stirred solution of compound 46a (80 mg, 0.18 mmol, 1.0 equiv) in MeOH (2 mL) and H₂O (2 mL) at rt, LiOH (8 mg, 0.36 mmol, 2.0 equiv) was added, and the reaction was stirred for 2 h. After completion of the reaction (monitored by TLC analysis), it was acidified with 2 (N) HCl up to pH = 5, and the mixture was extracted with Ethyl acetate. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was then dissolved in DCM (3 mL) and TFA (20.0 equiv) was added to the mixture at rt. After the starting crude material disappeared, organic solvents were evaporated under reduced pressure, and the crude material was used for the next step.

General procedure for acid-amine coupling (General procedure A): To a stirred solution of crude acid (37 mg, 0.1 mmol, 1.0 equiv), compound 8 (35 mg, 0.1 mmol, 1.0 equiv), and HATU (38 mg, 0.1 mmol, 1.0 equiv) in DMF (2 mL) at 0 °C, DIPEA (26 μL, 0.15 mmol, 1.5 equiv) was added dropwise. After 1 h, the reaction was diluted with water and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The product was purified using prep. HPLC chromatography to get compound 10 (53 mg, 75%) as a white solid. UPLC−MS: 1.88 min, purity >95%; UPLC−MS (ESI) m/z: calcd, 713.29 for C₄₂H₄₀N₄O₇ [M + H]⁺; found, 713.30; ¹H NMR (400 MHz, Acetone-d₆) δ 9.72 (s, 0H), 7.26 (d, J = 8.2 Hz, 1H), 7.20−7.04 (m, 4H), 6.90−6.81 (m, 2H), 6.74−6.69 (m, 2H), 6.65−6.56 (m, 3H), 6.40−6.33 (m, 2H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.74 (t, J = 14.5 Hz, 2H), 4.62−4.40 (m, 2H), 4.32−4.19 (m, 3H), 4.11 (t, J = 9.5 Hz, 1H), 4.00 (d, J = 12.2 Hz, 1H), 3.43−3.28 (m, 4H), 3.11−2.92 (m, 5H), 2.77 (ddd, J = 17.3, 4.5, 2.4 Hz, 2H), 2.56 (qd, J = 13.3, 4.5 Hz, 2H), 2.25−2.13 (m, 2H), 1.77 (s, 1H).

(S)-3-((R)-7-(4-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)butanoyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)-piperidine-2,6-dione (11).

Compound 11 was made from 46b using a similar synthetic procedure for compound 10 as white solid (51 mg, 70%). UPLC−MS: 2.0 min, purity >95%; MS (ESI) m/z: calcd, 741.32 for C₄₄H₄₄N₄O₇ [M + H]⁺; found, 741.33; ¹H NMR (400 MHz, Methanol-d₄) δ 7.32 (d, J = 8.4 Hz, 1H), 7.21−7.00 (m, 4H), 6.79 (d, J = 6.0 Hz, 2H), 6.73−6.61 (m, 2H), 6.61−6.44 (m, 3H), 6.31 (d, J = 8.6 Hz, 2H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.61 (dd, J = 25.2, 13.0 Hz, 1H), 4.47−4.26 (m, 3H), 4.19 (d, J = 5.1 Hz, 1H), 4.16−4.02 (m, 2H), 4.02−3.82 (m, 3H), 3.16 (d, J = 10.5 Hz, 1H), 3.08−2.82 (m, 4H), 2.82−2.39 (m, 5H), 2.26−1.97 (m, 4H), 1.73 (d, J = 12.6 Hz, 1H).

(S)-3-((R)-7-(5-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)pentanoyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)-piperidine-2,6-dione (12).

A similar synthetic procedure for compound 10 was followed to get compound 12 (60 mg, 80%) as white solid. UPLC−MS: 2.05 min, purity >95%; MS (ESI) m/z: calcd, 755.34 for C₄₅H₄₆N₄O₇ [M + H]⁺; found, 755.31; ¹H NMR (400 MHz, Acetone-d₆) δ 7.29−7.21 (m, 1H), 7.20−7.10 (m, 3H), 7.06 (t, J = 10.1 Hz, 1H), 6.90−6.83 (m, 2H), 6.75−6.69 (m, 2H), 6.60 (dd, J = 8.3, 2.6 Hz, 1H), 6.57−6.51 (m, 2H), 6.37−6.31 (m, 2H), 5.15 (dd, J = 13.4, 5.1 Hz, 1H), 4.63 (dd, J = 23.9, 12.7 Hz, 1H), 4.47 (d, J = 10.3 Hz, 1H), 4.37−4.20 (m, 3H), 4.20−4.03 (m, 2H), 4.01−3.93 (m, 1H), 3.89 (d, J = 5.6 Hz, 2H), 3.35 (ddd, J = 12.8, 5.0, 2.2 Hz, 3H), 3.16 (s, 1H), 3.10−2.89 (m, 5H), 2.77 (ddd, J = 17.5, 4.4, 2.4 Hz, 1H), 2.55 (dtd, J = 23.1, 13.9, 7.2 Hz, 4H), 2.29−2.13 (m, 2H), 1.78 (d, J = 5.7 Hz, 5H).

(S)-3-((R)-7-(6-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)hexanoyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)-piperidine-2,6-dione (13).

A similar synthetic procedure for compound 10 was followed to get compound 13 (46 mg, 60%) as white solid. UPLC−MS: 2.05 min, purity >95%; MS (ESI) m/z: calcd, 769.35 for C₄₆H₄₈N₄O₇ [M + H]⁺; found, 769.32; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (d, J = 8.3 Hz, 1H), 7.18−7.02 (m, 4H), 6.87−6.75 (m, 2H), 6.74−6.62 (m, 2H), 6.51 (td, J = 9.3, 2.6 Hz, 3H), 6.35−6.26 (m, 2H), 5.10 (dd, J = 13.3, 5.2 Hz, 1H), 4.61 (dd, J = 26.9, 13.0 Hz, 1H), 4.46−4.38 (m, 1H), 4.38−4.27 (m, 2H), 4.20 (d, J = 5.1 Hz, 1H), 4.15−4.00 (m, 2H), 3.94 (d, J = 10.6 Hz, 1H), 3.90−3.80 (m, 2H), 3.66−3.59 (m, 1H), 3.29−3.21 (m, 1H), 3.21−3.13 (m, 1H), 3.10−2.96 (m, 3H), 2.96−2.84 (m, 2H), 2.78 (ddd, J = 17.6, 4.8, 2.5 Hz, 2H), 2.61−2.41 (m, 4H), 2.28−2.08 (m, 2H), 1.72 (dp, J = 27.8, 6.8 Hz, 5H), 1.60−1.46 (m, 2H).

(S)-3-((R)-7-(4-((4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)(methyl)amino)butanoyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (14).

To a stirred solution of compound 42 in tetrahydrofuran (15 mL) and MeCN (15 mL), potassium carbonate (3.7 g, 2.0 equiv) and 1,1,2,2,3,3,4,4,4-non-afluorobutane-l-sulfonyl fluoride (6.0 g, 1.5 equiv) was added. The reaction mixture was stirred at rt for 16 h and monitored by TLC analysis. After the complete conversion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography. The desired compound 4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl 4,4,4,4,4,4,4,4,4-nonafluoro-4l12-buta-1,3-diyne-1-sulfonate (45)⁷⁹ (8.3 g, 95% yield) was obtained as a white solid. The spectroscopic data were matched with the literature.

General Procedure for Pd-Catalyzed Coupling of 45 with Amines (General Procedure B).

A solution of compound 45 (131 mg, 0.2 mmol, 1 equiv), tert-butyl 4-(methylamino)butanoate (41 mg, 0.24 mmol, 1.2 equiv), Xphos (19 mg, 0.04 mmol, 0.2 equiv), and NaOt-Bu (58 mg, 0.6 mmol, 3 equiv) in toluene (4 mL) was degassed with nitrogen gas for 10 min. Pd(OAc)₂ (5 mg, 0.02 mmol, 0.1 equiv) was then added to the reaction mixture, and the mixture was stirred at 100 °C for 2 h. After complete conversion of compound 45 (monitored by TLC analysis), the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography to get tert-butyl 4-((4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)(methyl)amino)butanoate (46e, 74 mg, 70%) as colorless oil in 70% yields. UPLC−MS (ESI) m/z: calcd, 528.34 for C₃₅H₄₅NO₃ [M + H]⁺; found, 528.30; ¹H NMR (400 MHz, Chloroform-d) δ 7.25−7.12 (m, 3H), 6.93−6.82 (m, 4H), 6.75 (dd, J = 8.3, 2.5 Hz, 1H), 6.41−6.33 (m, 2H), 6.29−6.21 (m, 2H), 4.23 (d, J = 4.9 Hz, 1H), 3.38 (ddd, J = 13.1, 5.0, 2.3 Hz, 1H), 3.24 (td, J = 6.9, 1.7 Hz, 2H), 3.14−2.97 (m, 2H), 2.83 (s, 3H), 2.31−2.14 (m, 3H), 1.81 (p, J = 7.2 Hz, 3H), 1.45 (s, 9H), 1.39 (s, 9H).

General Procedure for t-Butyl Deprotection (General Procedure C).

Compound 46e (70 mg, 0.13 mmol, 1 equiv) was dissolved in DCM (1.5 mL), TFA (0.2 mL, 0.26 mmol, 20.0 equiv) was added to the mixture at rt and stirred for 4 h. After complete conversion of the starting crude material, organic solvents were evaporated under reduced pressure, and the crude acid was used for the next step.

Using crude acid (60 mg, 0.14 mmol, 1 equiv) and compound 8 (51 mg, 0.14 mmol, 1 equiv) by following general acid-amine coupling protocol, compound 14 (54 mg, 72%) was made as a white solid; UPLC−MS (ESI) m/z: calcd, 754.35 for C₄₅H₄₇N₅O₆ [M + H]⁺; found, 754.32; ¹H NMR (400 MHz, Methanol-d₄) δ 7.32 (dd, J = 8.3, 4.7 Hz, 1H), 7.24−7.02 (m, 6H), 6.88−6.79 (m, 2H), 6.68 (dt, J = 14.2, 5.3 Hz, 4H), 6.54 (dt, J = 8.4, 2.4 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.69−4.54 (m, 1H), 4.45−4.26 (m, 4H), 4.15−3.90 (m, 3H), 3.63−3.52 (m, 2H), 3.49−3.40 (m, 1H), 3.37 (s, 1H), 3.28−3.14 (m, 4H), 3.13−2.99 (m, 2H), 2.99−2.84 (m, 2H), 2.84−2.74 (m, 2H), 2.74−2.37 (m, 4H), 2.24−2.08 (m, 2H), 1.86−1.70 (m, 3H).

(S)-3-((R)-7-(5-((4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)(methyl)amino)pentanoyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (15).

A similar synthetic procedure for compound 14 was followed to get compound 15 (52 mg, 68%) as a white solid. UPLC−MS (ESI) m/z: calcd, 768.35 for C₄₆H₄₉N₅O₆ [M + H]⁺; found, 768.33; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (d, J = 8.3 Hz, 1H), 7.23−7.10 (m, 5H), 7.06 (dd, J = 8.5, 3.9 Hz, 1H), 6.89−6.79 (m, 2H), 6.73−6.62 (m, 4H), 6.55 (dd, J = 8.3, 2.5 Hz, 1H), 5.10 (dt, J = 13.3, 5.2 Hz, 1H), 4.69−4.50 (m, 1H), 4.46−4.25 (m, 4H), 4.15−3.90 (m, 3H), 3.59−3.49 (m, 2H), 3.45 (ddd, J = 13.2, 5.8, 2.3 Hz, 1H), 3.40−3.34 (m, 1H), 3.19 (d, J = 1.4 Hz, 4H), 3.02 (ddd, J = 24.9, 10.3, 3.7 Hz, 3H), 2.96−2.67 (m, 3H), 2.66−2.35 (m, 4H), 2.16 (ddt, J = 13.3, 10.3, 5.1 Hz, 2H), 1.90−1.76 (m, 1H), 1.64 (dt, J = 18.6, 9.4 Hz, 2H), 1.49 (ddt, J = 15.4, 9.9, 6.0 Hz, 2H).

(S)-3-((R)-7-(2-(1-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperidin-4-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (16).

Using compound 45 (131 mg, 0.2 mmol, 1 equiv) and tert-butyl 2-(piperidin-4-yl)acetate (48 mg, 0.24 mmol, 1.2 equiv) by following the general Buchwald coupling protocol, tert-butyl 2-(1-(4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperidin-4-yl)acetate (47, 69 mg, 60%) was made as a white solid. UPLC−MS (ESI) m/z: calcd, 554.36 for C₃₇H₄₇NO₃ [M + H]⁺; found, 554.32. ¹H NMR (400 MHz, Chloroform-d) δ 7.23−7.13 (m, 3H), 6.93−6.80 (m, 4H), 6.76 (dd, J = 8.3, 2.5 Hz, 1H), 6.62−6.55 (m, 2H), 6.33−6.26 (m, 2H), 4.26 (d, J = 4.9 Hz, 1H), 3.61−3.50 (m, 2H), 3.39 (ddd, J = 13.1, 5.0, 2.2 Hz, 1H), 3.20−2.96 (m, 2H), 2.63 (td, J = 12.1, 2.4 Hz, 2H), 2.28−2.13 (m, 3H), 1.94−1.74 (m, 4H), 1.48 (s, 9H), 1.39 (s, 11H).

Compound 47 (69 mg, 0.12 mmol, 1 equiv) was next subjected to tert-butyl deprotection using TFA (0.1 mL), followed by general acid-amine coupling protocol with compound 8 (42 mg, 0.12 mmol, 1 equiv) to provide compound 16 (74 mg, 80%) as a white solid. UPLC−MS (ESI) m/z: calcd, 780.37 for C₄₇H₄₉N₅O₆ [M + H]⁺; found, 780.38; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (d, J = 8.3 Hz, 1H), 7.26−7.19 (m, 2H), 7.19−7.11 (m, 3H), 7.08 (dd, J = 8.5, 3.0 Hz, 1H), 6.85 (dd, J = 7.6, 1.9 Hz, 2H), 6.74−6.60 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.10 (ddd, J = 13.3, 5.2, 2.1 Hz, 1H), 4.63 (dd, J = 28.1, 13.2 Hz, 1H), 4.48−4.26 (m, 4H), 4.18−4.02 (m, 2H), 4.02−3.93 (m, 1H), 3.65−3.42 (m, 5H), 3.29−3.16 (m, 1H), 3.16−2.98 (m, 3H), 2.98−2.84 (m, 2H), 2.79 (ddd, J = 15.3, 4.7, 2.5 Hz, 1H), 2.64−2.40 (m, 4H), 2.29−2.07 (m, 5H), 1.92−1.80 (m, 1H), 1.72 (d, J = 13.5 Hz, 2H).

(S)-3-((R)-7-(2-(4-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperazin-1-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (17).

Using compound 45 (200 mg, 0.3 mmol, 1 equiv) and tert-butyl piperazine-1-carboxylate (74 mg, 0.36 mmol, 1.2 equiv) by using general Pd-catalyzed Buchwald coupling protocol followed by the tert-butyl deprotection protocol, compound 48 (141 mg, 0.255 mmol, 85%) was made as white solid. UPLC−MS (ESI) m/z: calcd, 385.22 for C₂₆H₂₈N₂O [M + H]⁺; found, 385.27.

To a stirred solution of 48 (80 mg, 0.14 mmol, 1 equiv) in DMF (3 mL) at 0 °C, DIPEA (38 μL, 0.22 mmol, 1.5 equiv) and tert-butyl 2-bromoacetate (30 mg, 0.15 mmol, 1.1 equiv) were added and the reaction was stirred for 1 h. After the complete disappearance of 48, the mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was dissolved in DCM (3 mL) and TFA (1 mL) was added and stirred for another 4 h. After the complete disappearance of the starting material organic solvent was evaporated under reduced pressure to get compound 49 as a white solid (50 mg, 0.11 mmol, 80%). UPLC−MS (ESI) m/z: calcd, 443.23 for C₂₈H₃₀N₂O₃ [M + H]+; found, 443.26.

Using compound 49 (50 mg, 0.11 mmol, 1 equiv) and compound 8 (39 mg, 0.11 mmol, 1 equiv), by following an acid-amine coupling protocol, compound 17 (64 mg, 75%) was synthesized as a white solid. UPLC−MS (ESI) m/z: calcd, 781.36 for C₄₆H₄₈N₆O₆ [M + H]⁺; found, 781.39; ¹H NMR (400 MHz, Acetonitrile-d₃) δ 7.29 (d, J = 8.3 Hz, 1H), 7.25−7.11 (m, 3H), 7.04 (d, J = 8.4 Hz, 1H), 6.93−6.84 (m, 2H), 6.77−6.67 (m, 2H), 6.67−6.59 (m, 2H), 6.57 (dd, J = 8.3, 2.7 Hz, 1H), 6.45−6.35 (m, 2H), 5.03 (ddd, J = 13.4, 5.1, 3.4 Hz, 1H), 4.58−4.45 (m, 1H), 4.40 (ddd, J = 18.3, 11.0, 2.9 Hz, 1H), 4.32−4.24 (m, 2H), 4.24−4.13 (m, 2H), 4.13−4.02 (m, 2H), 4.01−3.88 (m, 1H), 3.67 (dd, J = 25.4, 13.4 Hz, 1H), 3.57−3.21 (m, 8H), 3.09−2.90 (m, 3H), 2.90−2.60 (m, 4H), 2.44 (qd, J = 13.2, 4.9 Hz, 2H), 2.13 (dtd, J = 7.5, 5.0, 2.5 Hz, 3H), 2.07−2.00 (m, 1H), 1.87−1.77 (m, 2H).

(S)-3-((R)-7-(2-(6-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,6-diazaspiro[3.3]heptan-2-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (18).

Using compound 45 (200 mg, 0.3 mmol, 1 equiv) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (72 mg, 2.36 mmol, 1.2 equiv) by following general Buchwald coupling protocol followed by general tert-butyl deprotection procedure, (5R,6S)-5-(4-(2,6-diazaspiro[3.3]-heptan-2-yl)phenyl)-6-phenyl-5,6,7,8-tetrahydronaphthalen-2-ol (50a, 83 mg, 70%) was made as white solid. UPLC−MS (ESI) m/z: calcd, 397.22 for C₂₇H₂₈N₂O [M + H]⁺; found, 397.29.

To a stirred solution of 50a (70 mg, 0.17 mmol, 1 equiv) in DMF (3 mL) at 0 °C, DIPEA (46 μL, 0.26 mmol, 1.5 equiv) and tert-butyl 2-bromoacetate (36 mg, 0.19 mmol, 1.1 equiv) were added and the reaction was stirred for 1 h at room temperature. After the complete disappearance of 50a, the mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was then treated with TFA (0.1 mL) in DCM (2 mL) to get compound 51a (50 mg, 65%) as a white solid. UPLC−MS (ESI) m/z: calcd, 455.23 for C₂₉H₃₀N₂O₃ [M + H]⁺; found, 455.35. ¹H NMR (400 MHz, Methanol-d₄) δ 7.11 (tdd, J = 4.6, 3.4, 1.5 Hz, 3H), 6.80 (dt, J = 6.9, 2.2 Hz, 2H), 6.74−6.62 (m, 2H), 6.52 (dd, J = 8.3, 2.6 Hz, 1H), 6.34−6.25 (m, 2H), 6.21−6.10 (m, 2H), 4.42 (s, 4H), 4.17 (d, J = 3.3 Hz, 3H), 3.94 (s, 4H), 3.32−3.25 (m, 1H), 3.10−2.92 (m, 2H), 2.19 (tdd, J = 13.0, 11.3, 6.7 Hz, 1H), 1.82−1.67 (m, 1H).

Using 51a (45 mg, 0.1 mmol, 1 equiv) and 8 (36 mg, 0.1 mmol, 1 equiv), by following the general acid amine coupling protocol (General procedure A), compound 18 (63 mg, 80%) was synthesized as a white solid. UPLC−MS (ESI) m/z: calcd, 793.36 for C₄₇H₄₈N₆O₆ [M + H]⁺; found, 793.6; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (dd, J = 8.4, 5.0 Hz, 1H), 7.20−7.03 (m, 4H), 6.87−6.74 (m, 2H), 6.68 (d, J = 8.6 Hz, 2H), 6.52 (dd, J = 8.3, 2.7 Hz, 1H), 6.30 (d, J = 8.5 Hz, 2H), 6.19 (d, J = 8.1 Hz, 2H), 5.11 (ddd, J = 13.2, 8.0, 5.1 Hz, 1H), 4.52 (t, J = 17.0 Hz, 4H), 4.45−4.25 (m, 6H), 4.18 (d, J = 5.0 Hz, 1H), 4.13−3.86 (m, 6H), 3.74 (dd, J = 27.2, 12.9 Hz, 1H), 3.42 (d, J = 12.9 Hz, 1H), 3.24 (d, J = 8.1 Hz, 1H), 3.13−2.85 (m, 5H), 2.84−2.62 (m, 2H), 2.55−2.39 (m, 1H), 2.20 (dt, J = 19.4, 12.5 Hz, 2H), 1.77 (d, J = 12.8 Hz, 1H).

(S)-3-((R)-7-(2-(2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-7-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]-oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (19).

Compound 19 was made from 50b using a similar synthetic procedure for compound 18 as a white solid (45 mg, 74%). UPLC−MS (ESI) m/z: calcd, 821.39 for C₄₉H₅₂N₆O₆ [M + H]⁺; found, 821.42; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (dd, J = 8.0, 4.0 Hz, 1H), 7.11 (q, J = 7.5 Hz, 4H), 6.90−6.75 (m, 2H), 6.74−6.61 (m, 2H), 6.53 (dd, J = 8.6, 2.6 Hz, 1H), 6.31 (d, J = 8.2 Hz, 2H), 6.23 (d, J = 7.9 Hz, 2H), 5.11 (dt, J = 11.1, 5.1 Hz, 1H), 4.69−4.50 (m, 1H), 4.48−4.16 (m, 6H), 4.15−3.92 (m, 2H), 3.88−3.52 (m, 7H), 3.43 (d, J = 12.0 Hz, 1H), 3.01 (tt, J = 50.4, 13.8 Hz, 8H), 2.74 (dd, J = 42.8, 13.9 Hz, 2H), 2.57−2.38 (m, 1H), 2.19 (t, J = 20.2 Hz, 6H), 1.77 (d, J = 12.7 Hz, 1H).

(S)-3-((R)-7-(2-(7-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-2-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]-oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (20).

Compound 20 was made from 50c using a similar synthetic procedure for compound 18 as a white solid (52 mg, 71%). UPLC−MS (ESI) m/z: calcd, 821.39 for C₄₉H₅₂N₆O₆ [M + H]⁺; found, 821.42; ¹H NMR (400 MHz, Methanol-d₄) δ 7.32 (t, J = 7.8 Hz, 1H), 7.21−7.01 (m, 4H), 6.88−6.75 (m, 2H), 6.74−6.62 (m, 2H), 6.53 (dd, J = 8.5, 2.5 Hz, 1H), 6.37−6.17 (m, 4H), 5.12 (td, J = 12.5, 5.0 Hz, 1H), 4.58 (t, J = 15.8 Hz, 1H), 4.49−4.15 (m, 6H), 4.15−3.91 (m, 2H), 3.88−3.48 (m, 7H), 3.48−3.37 (m, 1H), 3.27−2.84 (m, 8H), 2.84−2.60 (m, 2H), 2.60−2.37 (m, 1H), 2.36−1.97 (m, 6H), 1.85−1.69 (m, 1H).

(S)-3-((R)-7-(2-(8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,8-diazaspiro[4.5]decan-2-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]-oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (21).

Compound 21 was made from 50d using a similar synthetic procedure for compound 18 as a white solid (55 mg, 78%). UPLC−MS (ESI) m/z: calcd, 835.41 for C₅₀H₅₄N₆O₆ [M + H]⁺; found, 835.10; ¹H NMR (400 MHz, Methanol-d₄) δ 7.33 (dd, J = 8.4, 2.4 Hz, 1H), 7.19−7.11 (m, 5H), 7.09 (dd, J = 8.6, 2.3 Hz, 1H), 6.85 (dd, J = 7.6, 1.8 Hz, 2H), 6.73−6.65 (m, 2H), 6.63−6.57 (m, 2H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.11 (dt, J = 13.3, 5.0 Hz, 1H), 4.65−4.51 (m, 2H), 4.51−4.22 (m, 6H), 4.14−3.90 (m, 3H), 3.76 (dt, J = 27.4, 15.5 Hz, 3H), 3.57−3.40 (m, 6H), 3.29−3.18 (m, 1H), 3.17−3.01 (m, 3H), 3.01−2.85 (m, 2H), 2.84−2.58 (m, 2H), 2.49 (qd, J = 13.1, 4.7 Hz, 2H), 2.24−2.06 (m, 7H), 1.84 (d, J = 12.2 Hz, 1H).

(S)-3-((R)-7-(2-(9-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)acetyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (22).

Compound 22 was made from 50e using a similar synthetic procedure for compound 18 as a white solid (60 mg, 74%). UPLC−MS (ESI) m/z: calcd, 849.43 for C₅₁H₅₆N₆O₆ [M + H]⁺; found, 848.85; ¹H NMR (400 MHz, Methanol-d₄) δ 7.34 (dd, J = 8.4, 3.0 Hz, 1H), 7.23−7.18 (m, 2H), 7.18−7.07 (m, 4H), 6.88−6.83 (m, 2H), 6.72−6.66 (m, 2H), 6.66−6.60 (m, 2H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.11 (dt, J = 13.3, 5.1 Hz, 1H), 4.60 (dd, J = 20.0, 12.9 Hz, 1H), 4.47−4.35 (m, 4H), 4.35−4.23 (m, 2H), 4.16−3.98 (m, 2H), 3.80 (dd, J = 27.2, 13.1 Hz, 1H), 3.66−3.40 (m, 8H), 3.30−3.16 (m, 2H), 3.10−3.03 (m, 2H), 2.99−2.84 (m, 2H), 2.84−2.65 (m, 2H), 2.49 (qd, J = 13.2, 4.9 Hz, 2H), 2.23−2.04 (m, 6H), 1.98−1.81 (m, 5H).

(S)-3-((R)-7-(2-(4-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperazin-1-yl)-2-oxoethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (23).

To a stirred solution of compound 48 (380 mg, 0.1 mmol, 1 equiv), compound 43 (41 mg, 0.1 mmol, 1 equiv), and HATU (38 mg, 0.1 mmol, 1 equiv) in DMF at °C, DIPEA (26 μL, 0.15 mmol, 1.5 equiv) was added dropwise and stirred for 1 h. The product was then purified using preparative HPLC chromatography to get compound 23 (62 mg, 80%) as a white solid. UPLC−MS (ESI) m/z: calcd, 781.36 for C₄₆H₄₈N₆O₆ [M + H]⁺; found, 781.35; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.3 Hz, 1H), 7.22−7.05 (m, 4H), 6.90−6.79 (m, 2H), 6.75−6.63 (m, 4H), 6.53 (dd, J = 8.3, 2.6 Hz, 1H), 6.42−6.32 (m, 2H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.50−4.31 (m, 5H), 4.25 (dd, J = 14.4, 7.6 Hz, 2H), 4.16 (dd, J = 11.2, 7.1 Hz, 1H), 3.85−3.65 (m, 5H), 3.56 (t, J = 5.1 Hz, 2H), 3.34 (s, 3H), 3.23−2.98 (m, 7H), 2.98−2.69 (m, 2H), 2.49 (qd, J = 13.2, 4.7 Hz, 1H), 2.33−2.09 (m, 2H), 1.79 (dd, J = 12.3, 5.2 Hz, 1H).

(S)-3-((R)-7-(2-(6-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,6-diazaspiro[3.3]heptan-2-yl)-2-oxoethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (24).

Using compound 50a (40 mg, 0.1 mmol) and compound 8 (35 mg, 0.1 mmol) by following acid amine coupling procedure, compound 24 (47 mg, 60%) was made as a white solid. UPLC−MS (ESI) m/z: calcd, 793.36 for C₄₇H₄₈N₆O₆ [M + H]⁺; found, 793.6; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.4 Hz, 1H), 7.20−7.04 (m, 4H), 6.82 (dd, J = 7.4, 2.1 Hz, 2H), 6.74−6.64 (m, 2H), 6.52 (dd, J = 8.4, 2.6 Hz, 1H), 6.30 (d, J = 8.5 Hz, 2H), 6.22 (d, J = 8.7 Hz, 2H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.48−4.41 (m, 1H), 4.38 (d, J = 7.9 Hz, 4H), 4.24 (s, 3H), 4.22−4.12 (m, 2H), 4.06 (s, 2H), 3.98−3.85 (m, 4H), 3.71 (q, J = 12.5 Hz, 3H), 3.40−3.34 (m, 1H), 3.29 (s, 2H), 3.14−2.97 (m, 3H), 2.91 (ddd, J = 18.3, 13.5, 5.4 Hz, 1H), 2.84−2.73 (m, 1H), 2.50 (qd, J = 13.2, 4.8 Hz, 1H), 2.28−2.09 (m, 2H), 1.77 (d, J = 12.7 Hz, 1H).

(S)-3-((R)-7-(2-(2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-oxoethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (25).

Using compound 50b (42 mg, 0.1 mmol) and compound 43 (35 mg, 0.1 mmol) by following acid amine coupling procedure, compound 25 (64 mg, 78%) was made as a white solid. UPLC−MS (ESI) m/z: calcd, 821.39 for C₄₉H₅₂N₆O₆ [M + H]⁺; found, 821.49; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.4 Hz, 1H), 7.14 (td, J = 7.4, 2.0 Hz, 6H), 6.89−6.81 (m, 2H), 6.73−6.66 (m, 2H), 6.64−6.57 (m, 2H), 6.54 (dd, J = 8.3, 2.6 Hz, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.45 (dd, J = 11.2, 2.8 Hz, 1H), 4.41−4.31 (m, 3H), 4.31−4.21 (m, 1H), 4.16 (dd, J = 11.1, 7.1 Hz, 1H), 4.07 (d, J = 4.6 Hz, 4H), 3.94 (s, 2H), 3.77−3.63 (m, 3H), 3.51−3.39 (m, 5H), 3.31−3.21 (m, 2H), 3.13−2.97 (m, 3H), 2.91 (ddd, J = 17.6, 13.4, 5.4 Hz, 1H), 2.79 (ddd, J = 17.6, 4.7, 2.5 Hz, 1H), 2.50 (qd, J = 13.2, 4.7 Hz, 1H), 2.26−2.10 (m, 6H), 1.90−1.78 (m, 1H).

(S)-3-((R)-7-(2-(7-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-2-yl)-2-oxoethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (26).

Using compound 50c (42 mg, 0.1 mmol) and compound 43 (35 mg, 0.1 mmol) by following acid amine coupling procedure, compound 26 (65 mg, 80%) was made as a white solid. UPLC−MS (ESI) m/z: calcd, 821.39 for C₄₉H₅₂N₆O₆ [M + H]⁺; found, 821.42; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.3 Hz, 1H), 7.14 (dt, J = 8.2, 4.9 Hz, 4H), 6.91−6.81 (m, 2H), 6.74−6.64 (m, 4H), 6.59−6.45 (m, 3H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.51−4.21 (m, 7H), 4.15 (dd, J = 11.1, 7.1 Hz, 1H), 4.00 (s, 4H), 3.87−3.58 (m, 5H), 3.41 (s, 4H), 3.22−2.71 (m, 6H), 2.50 (qd, J = 13.2, 4.7 Hz, 1H), 2.32−2.10 (m, 2H), 2.08−1.73 (m, 5H).

(S)-3-((R)-7-(2-(8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,8-diazaspiro[4.5]decan-2-yl)-2-oxoethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (27).

Using compound 50d (44 mg, 0.1 mmol) and compound 43 (35 mg, 0.1 mmol) by following acid amine coupling procedure, compound 27 (58 mg, 70%) was made as a white solid. UPLC−MS (ESI) m/z: calcd, 418.21 for C₅₀H₅₄N₆O₆ [M + 2H]²⁺/2; found, 418.51; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (dd, J = 8.3, 1.6 Hz, 1H), 7.14 (dddd, J = 8.8, 7.1, 5.2, 2.7 Hz, 6H), 6.85 (ddd, J = 7.4, 3.7, 1.9 Hz, 2H), 6.69 (dd, J = 8.6, 2.3 Hz, 2H), 6.61 (dd, J = 8.4, 4.8 Hz, 2H), 6.55 (ddd, J = 8.4, 2.7, 1.1 Hz, 1H), 5.11 (ddd, J = 13.3, 5.2, 2.3 Hz, 1H), 4.48−4.42 (m, 1H), 4.40−4.34 (m, 2H), 4.20 (td, J = 19.6, 9.7 Hz, 4H), 3.79−3.59 (m, 5H), 3.58−3.39 (m, 7H), 3.20−3.14 (m, 1H), 3.13−3.00 (m, 3H), 2.98−2.85 (m, 2H), 2.84−2.70 (m, 2H), 2.50 (qd, J = 13.1, 4.7 Hz, 2H), 2.23−2.12 (m, 2H), 2.08 (dd, J = 13.7, 6.6 Hz, 1H), 1.97 (d, J = 6.9 Hz, 5H), 1.85 (d, J = 12.7 Hz, 2H).

(S)-3-((R)-7-(2-(7-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-7-azaspiro[3.5]nonan-2-yl)ethyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (28).

Using compound 45 (200 mg, 0.3 mmol, 1 equiv) and ethyl 2-(7-azaspiro[3.5]nonan-2-yl)acetate (96 mg, 0.46 mmol, 1.5 mmol) by following a Buchwald coupling protocol (General procedure B), compound 52 (96 mg, 55%) was made as colorless oil. UPLC−MS (ESI) m/z: calcd, 566.36 for C₃₈H₄₇NO₃ [M + H]⁺; found, 566.32; ¹H NMR (400 MHz, Chloroform-d) δ 7.22−7.14 (m, 3H), 6.89−6.80 (m, 4H), 6.75 (dd, J = 8.3, 2.5 Hz, 1H), 6.61−6.55 (m, 2H), 6.31−6.25 (m, 2H), 4.24 (d, J = 4.9 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.39 (ddd, J = 13.2, 5.0, 2.2 Hz, 1H), 3.15−2.97 (m, 4H), 2.94 (dd, J = 7.0, 4.5 Hz, 2H), 2.72−2.58 (m, 1H), 2.43 (d, J = 7.6 Hz, 2H), 2.29−2.12 (m, 1H), 2.11−2.00 (m, 2H), 1.89−1.77 (m, 1H), 1.77−1.69 (m, 2H), 1.63 (dd, J = 4.9, 2.7 Hz, 2H), 1.56−1.45 (m, 2H), 1.39 (s, 9H), 1.27 (t, J = 7.2 Hz, 4H).

To a stirred solution of compound 52 (95 mg, 0.168 mmol, 1.0 equiv) in DCM (4 mL) at −78 °C, DIBAL-H (0.2 mL, 0.2 mmol, 1 M in DCM, 1.2 equiv) was added dropwise. After the reaction was stirred for 2 h at the same temperature, the reaction was quenched with 1 (N) HCl, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na₂SO_4, and concentrated under reduced pressure to get crude aldehyde. The crude aldehyde was dissolved in THF and 4 (N) H₂SO₄ was added at 60 °C. The reaction was stirred for 4 h at the same temperature. After complete deprotection of tert-butyl (monitored by UPLC chromatography), the mixture was quenched with sodium bicarbonate and extracted with ethyl acetate. The organic phase was separated, and the solvent was evaporated under reduced pressure to get the crude aldehyde. The crude aldehyde was used for the next step.

General Procedure for Reductive Amination (General Procedure D).

A solution of crude aldehyde (46 mg, 0.1 mmol, 1 equiv), compound 8 (40 mg, 0.11 mmol, 1.1 equiv), and sodium acetate (33 mg, 0.4 mmol, 4 equiv) in DCM (2 mL) and methanol (0.5 mL) was stirred for 10 min at room temperature. Sodium cyanoborohydride (13 mg, 0.2 mmol, 2 equiv) was added to the reaction in portions and stirred for 1 h. After complete consumption of aldehyde, the reaction mixture was diluted with 1 (N) HCL (0.5 mL). The organic solvent was evaporated under reduced pressure and the crude was purified with preparative HPLC chromatography to get compound 28 (68 mg, 85%) as a white solid. UPLC−MS (ESI) m/z: calcd, 806.42 for C₅₀H₅₅N₅O₅ [M + H]⁺; found, 806.16; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.3 Hz, 1H), 7.27−7.09 (m, 6H), 6.85 (dd, J = 7.6, 1.9 Hz, 2H), 6.75−6.61 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.50−4.34 (m, 4H), 4.34−4.23 (m, 1H), 4.23−4.09 (m, 1H), 3.78−3.61 (m, 3H), 3.55−3.37 (m, 5H), 3.23 (d, J = 9.4 Hz, 2H), 3.14 (d, J = 9.7 Hz, 2H), 3.10−2.93 (m, 3H), 2.93−2.84 (m, 1H), 2.79 (ddd, J = 17.5, 4.7, 2.4 Hz, 1H), 2.50 (qd, J = 13.1, 4.7 Hz, 1H), 2.38 (p, J = 7.9 Hz, 1H), 2.27−2.11 (m, 4H), 2.06 (t, J = 5.6 Hz, 2H), 2.02−1.90 (m, 4H), 1.90−1.80 (m, 1H), 1.67 (dd, J = 11.8, 8.3 Hz, 2H).

(S)-3-((R)-7-((2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2-azaspiro[3.3]heptan-6-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (29).

To a stirred degassed solution of compound 45 (200 mg, 0.3 mmol, 1.0 equiv), 2-(dimethoxymethyl)-7-azaspiro[3.5]nonane (54a, 72 mg, 0.36 mmol, 1.2 equiv), NaOt-Bu (85 mg, 0.9 mmol, 3 equiv), and Xphos (28 mg, 0.06 mmol, 0.2 equiv) in toluene (3 mL) at rt, Pd(OAc)₂ (7 mg, 0.03 mmol, 0.1 equiv) was added and the mixture was heated at 100 °C using an oil bath for 2 h. After the complete disappearance of compound 45 (monitored by UPLC chromatography analysis), the mixture was filtered through a pad of Celite and washed with ethyl acetate. The filtrate was washed with brine and the organic layer was dried over Na₂SO₄ and concentrated under reduced pressure.

The crude product was next dissolved in THF (2 mL) and charged with 2 (N) H₂SO₄ (2 mL). The mixture was heated at 60 °C using an oil bath for 4 h. After complete conversion of the crude starting material (monitored by UPLC chromatography) into aldehyde, the mixture was diluted with water (2 mL) and the organic solvent was evaporated under reduced pressure. The mixture was extracted with ethyl acetate. The organic phase was washed with sodium bicarbonate, and brine and dried over Na₂SO₄. The solvent was evaporated under reduced pressure. The product was purified using flash column chromatography to get compound 7-(4-((1R,2S)-6-hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-7-azaspiro[3.5]nonane-2-carbaldehyde (55a, 81 mg, 60%) as a white solid. UPLC−MS (ESI) m/z: calcd, 452.25 for C₃₁H₃₃NO₂ [M + H]⁺; found, 452.25. ¹H NMR (400 MHz, Chloroform-d) δ 9.81 (d, J = 1.3 Hz, 1H), 7.21−7.11 (m, 5H), 6.79 (dd, J = 6.6, 2.9 Hz, 2H), 6.76−6.70 (m, 2H), 6.61 (dd, J = 8.3, 2.7 Hz, 1H), 6.57−6.51 (m, 2H), 4.32 (d, J = 5.4 Hz, 1H), 3.50−3.18 (m, 7H), 3.06 (dd, J = 11.8, 6.0 Hz, 2H), 2.25−1.98 (m, 7H), 1.87 (d, J = 12.5 Hz, 2H).

A solution of aldehyde (55a, 45 mg, 0.1 mmol, 1 equiv), compound 8 (40 mg, 0.11 mmol, 1.1 equiv), and sodium acetate (33 mg, 0.4 mmol, 4 equiv) in DCM (2 mL) and methanol (0.5 mL) was stirred for 10 min at room temperature. Sodium cyanoborohydride (13 mg, 0.2 mmol, 2 equiv) was added to the reaction in portions and stirred for 1 h. After complete consumption of aldehyde, the reaction mixture was diluted with 1 (N) HCL (0.5 mL). The organic solvent was evaporated under reduced pressure and the crude was purified with preparative HPLC chromatography to get compound 29 (63 mg, 80%) as a white solid. UPLC−MS (ESI) m/z: calcd, 792.40 for C₄₉H₅₃N₅O₅ [M + H]⁺; found, 792.02; ¹H NMR (400 MHz, Methanol-d₄) δ 7.37 (d, J = 8.4 Hz, 1H), 7.30−7.20 (m, 2H), 7.20−7.08 (m, 4H), 6.90−6.81 (m, 2H), 6.75−6.62 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.09 (dd, J = 13.3, 5.2 Hz, 1H), 4.50−4.31 (m, 4H), 4.26 (d, J = 10.9 Hz, 1H), 4.16 (dd, J = 11.2, 7.0 Hz, 1H), 3.73−3.58 (m, 3H), 3.58−3.40 (m, 5H), 3.40−3.34 (m, 2H), 3.29−3.16 (m, 3H), 3.16−2.95 (m, 3H), 2.95−2.73 (m, 3H), 2.50 (qd, J = 13.1, 4.8 Hz, 1H), 2.30 (dd, J = 11.9, 8.3 Hz, 2H), 2.23−2.08 (m, 4H), 2.04−1.92 (m, 2H), 1.90−1.76 (m, 3H).

(S)-3-((R)-7-((2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2-azaspiro[3.5]nonan-7-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (30).

Compound 30 was made from 55b using a similar synthetic procedure for compound 29 as white solid (60 mg, 78%). UPLC−MS (ESI) m/z: calcd, 792.40 for C₄₉H₅₃N₅O₅ [M + H]⁺; found, 792.12; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (dd, J = 8.3, 1.2 Hz, 1H), 7.14 (dd, J = 9.6, 5.5 Hz, 4H), 6.92−6.77 (m, 2H), 6.77−6.63 (m, 2H), 6.54 (td, J = 9.5, 4.8 Hz, 2H), 6.42 (d, J = 24.0 Hz, 3H), 5.11 (dt, J = 13.3, 5.8 Hz, 1H), 4.51−4.41 (m, 1H), 4.41−4.33 (m, 2H), 4.33−4.10 (m, 3H), 3.84−3.61 (m, 6H), 3.39 (d, J = 8.4 Hz, 1H), 3.27 (d, J = 10.1 Hz, 2H), 3.17−2.84 (m, 6H), 2.79 (ddd, J = 17.6, 4.7, 2.4 Hz, 1H), 2.50 (ddt, J = 18.4, 13.3, 6.6 Hz, 1H), 2.35−2.00 (m, 4H), 2.00−1.71 (m, 5H), 1.71−1.53 (m, 2H), 1.32 (d, J = 11.1 Hz, 1H), 1.18 (q, J = 12.1 Hz, 2H).

(3S)-3-((5aR)-7-((8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-8-azaspiro[4.5]decan-2-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]-oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (31).

Compound 31 was made from 55c using a similar synthetic procedure for compound 29 as white solid (52 mg, 65%). UPLC−MS (ESI) m/z: calcd, 806.42 for C₅₀H₅₅N₅O₅ [M + H]⁺; found, 806.46; ¹H NMR (400 MHz, Methanol-d₄) δ 7.25−7.11 (m, 7H), 6.90−6.81 (m, 2H), 6.75−6.67 (m, 2H), 6.64 (d, J = 8.4 Hz, 2H), 6.55 (dd, J = 8.4, 2.6 Hz, 1H), 5.14−5.07 (m, 1H), 4.38 (q, J = 4.0 Hz, 3H), 4.27 (d, J = 13.6 Hz, 1H), 4.12 (dt, J = 11.1, 7.2 Hz, 1H), 3.72 (d, J = 11.2 Hz, 3H), 3.56−3.40 (m, 6H), 3.28 (dt, J = 3.3, 1.7 Hz, 3H), 3.06 (dd, J = 11.8, 5.9 Hz, 2H), 3.03−2.94 (m, 1H), 2.94−2.84 (m, 1H), 2.83−2.75 (m, 1H), 2.65−2.53 (m, 1H), 2.53−2.40 (m, 1H), 2.26−2.02 (m, 5H), 1.92 (t, J = 5.8 Hz, 3H), 1.88−1.73 (m, 3H), 1.59−1.48 (m, 1H), 1.39−1.29 (m, 2H).

(S)-3-((R)-7-((3-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-3-azaspiro[5.5]undecan-9-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]-oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (32).

A similar synthetic procedure for compound 29 was followed to make compound 32 (45 mg, 55%) as white solid. UPLC−MS (ESI) m/z: calcd, 410.72 for C₅₁H₅₇N₅O₅ [M + 2H]⁺/2; found, 411.01; ¹H NMR (400 MHz, Methanol-d₄) δ 7.37 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.15 (tdd, J = 6.4, 3.8, 2.3 Hz, 4H), 6.89−6.82 (m, 2H), 6.74−6.63 (m, 4H), 6.55 (dd, J = 8.4, 2.6 Hz, 1H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.42−4.30 (m, 3H), 4.30−4.10 (m, 2H), 3.73 (s, 3H), 3.59−3.41 (m, 5H), 3.21 (d, J = 46.8 Hz, 4H), 3.10−2.95 (m, 3H), 2.88 (dt, J = 13.4, 5.0 Hz, 1H), 2.78 (ddd, J = 17.6, 4.8, 2.4 Hz, 1H), 2.50 (qd, J = 13.1, 4.7 Hz, 1H), 2.26−2.09 (m, 2H), 2.09−1.89 (m, 5H), 1.82 (t, J = 8.7 Hz, 6H), 1.50−1.20 (m, 5H).

(3S)-3-((5aR)-7-((8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-8-azaspiro[4.5]decan-3-yl)-methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (33).

Using compound 45 (200 mg, 0.3 mmol, 1 equiv) and 3-(dimethoxymethyl)-1-oxa-8-azaspiro[4.5]decane (56a, 77 mg, 0.36 mmol, 1.2 mmol) following similar synthetic procedure for compound 29, compound 33 (64 mg, 80%) was made as white solid. UPLC−MS (ESI) m/z: calcd, 808.40 for C₄₉H₅₃N₅O₆ [M + H]⁺; found, 808.41; ¹H NMR (400 MHz, Methanol-d₄) δ 7.37 (d, J = 8.4 Hz, 1H), 7.26−7.21 (m, 2H), 7.18−7.11 (m, 4H), 6.87−6.82 (m, 2H), 6.72−6.63 (m, 4H), 6.55 (dd, J = 8.4, 2.6 Hz, 1H), 5.08 (dd, J = 13.3, 5.1 Hz, 1H), 4.49−4.42 (m, 1H), 4.42−4.30 (m, 3H), 4.30−4.12 (m, 3H), 3.80−3.61 (m, 7H), 3.47 (ddt, J = 13.4, 7.8, 3.0 Hz, 3H), 3.25 (dd, J = 15.8, 12.5 Hz, 2H), 3.14−2.92 (m, 5H), 2.92−2.84 (m, 1H), 2.78 (ddd, J = 17.6, 4.7, 2.5 Hz, 1H), 2.49 (qd, J = 13.1, 4.8 Hz, 1H), 2.30 (dd, J = 12.8, 8.0 Hz, 1H), 2.17 (ddtd, J = 17.9, 10.8, 5.6, 2.1 Hz, 3H), 2.11−1.99 (m, 3H), 1.89−1.80 (m, 1H), 1.71 (dd, J = 12.9, 8.9 Hz, 1H). ¹³C NMR (101 MHz, Methanol-d₄) δ 173.2, 170.9, 170.1, 155.6, 145.7, 143.7, 139.6, 139.3, 137.6, 136.3, 132.0, 131.0, 129.6, 129.4, 127.6, 127.5, 125.9, 123.8, 118.9, 116.9, 114.3, 114.2, 113.5, 77.11, 70.0, 65.8, 53.4, 53.1, 52.4, 52.2, 50.4, 49.7, 45.0, 44.9, 43.4, 41.1, 34.3, 33.9, 33.4, 31.0, 29.4, 22.7, 21.7.

(3S)-3-((5aR)-7-((8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (34).

To a stirred degassed solution of compound 45 (200 mg, 0.3 mmol, 1.0 equiv), (2-oxa-8-azaspiro[4.5]decan-3-yl)methanol (57a, 61 mg, 0.36 mmol, 1.2 equiv), NaOt-Bu (85 mg, 0.0.9, 3 equiv) in amyl alcohol (3 mL) at rt, t-BuXphosPd G3 (24 mg, 0.03 mmol, 0.1 equiv) was added, and the mixture was heated at 100 °C using an oil bath for 2 h. After the complete disappearance of compound 45 (monitored by UPLC chromatography analysis), the mixture was concentrated and filtered through a pad of Celite and washed with ethyl acetate. The filtrate was washed with brine and the organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified with flash column chromatography to get compound 59a (79 mg, 50%) as a white solid. UPLC−MS (ESI) m/z: calcd, 526.32 for C₃₅H₄₃NO₃ [M + H]⁺; found, 526.23; ¹H NMR (400 MHz, Chloroform-d) δ 7.24−7.12 (m, 3H), 6.94−6.80 (m, 4H), 6.75 (dd, J = 8.4, 2.4 Hz, 1H), 6.60 (d, J = 9.1 Hz, 2H), 6.36−6.23 (m, 2H), 4.26 (d, J = 4.9 Hz, 1H), 4.21−4.06 (m, 1H), 3.79−3.61 (m, 3H), 3.60−3.46 (m, 1H), 3.40 (ddd, J = 12.9, 5.1, 2.0 Hz, 1H), 3.26−2.93 (m, 6H), 2.28−2.13 (m, 1H), 1.96−1.65 (m, 6H), 1.62−1.48 (m, 1H), 1.39 (s, 9H).

To a stirred solution of compound 59a (87 mg, 0.165 mmol, 1.0 equiv) in DCM (5 mL) at 0 °C, NaHCO₃ (35 mg, 0.41 mmol, 2,5 equiv) and Dess Martin’s reagent (105 mg, 0.25 mmol, 1.5 equiv) were added, and the reaction was stirred at the same temperature for 2 h. After the complete disappearance of compound 59a, the mixture was diluted with water (3 mL) and extracted with DCM (3 mL). The organic layer was washed with sodium thiosulfate and brine dried over Na₂SO₄ and concentrated under reduced pressure. The crude aldehyde was dissolved in THF (3 mL) and 4 (N) H₂SO₄ (3 mL) was added, and the mixture was stirred for 4 h at 60 °C. After complete deprotection of the tert-butyl group (monitored by UPLC), the solvent was evaporated under reduced pressure, and the crude aldehyde was extracted with ethyl acetate. The organic layer was washed with brine and dried over Na₂SO₄. The solvent was concentrated under reduced pressure, and the crude aldehyde was used for reductive amination reaction with compound 8.

A general procedure of reductive amination was followed using crude aldehyde (48 mg, 0.1 mmol, 1 equiv) and compound 8 (40 mg, 0.12 mmol, 1.2 equiv) to get compound 34 (52 mg, 65%) as a white solid. UPLC−MS (ESI) m/z: calcd, 808.40 for C₄₉H₅₃N₅O₆ [M + H]⁺; found, 808.5; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.4 Hz, 1H), 7.28−7.20 (m, 2H), 7.15 (dt, J = 7.6, 4.5 Hz, 4H), 6.91−6.80 (m, 2H), 6.75−6.59 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.51 (s, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.56 (q, J = 10.1 Hz, 1H), 4.50−4.41 (m, 1H), 4.38 (dd, J = 6.7, 3.4 Hz, 2H), 4.28 (dd, J = 23.5, 12.2 Hz, 1H), 4.17 (ddd, J = 11.1, 7.1, 2.1 Hz, 1H), 3.92−3.63 (m, 6H), 3.62−3.37 (m, 7H), 3.31−3.27 (m, 1H), 3.23 (q, J = 7.3 Hz, 2H), 3.17−3.02 (m, 3H), 2.99−2.84 (m, 2H), 2.79 (ddd, J = 17.5, 4.7, 2.4 Hz, 1H), 2.50 (qd, J = 13.2, 4.7 Hz, 1H), 2.44−2.10 (m, 4H), 1.90−1.79 (m, 1H), 1.63 (dd, J = 13.0, 8.5 Hz, 1H).

(3S)-3-((5aR)-7-((8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-8-azaspiro[4.5]decan-2-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (35).

A similar synthetic procedure for 34 was carried out using compound 45 and (1-oxa-8-azaspiro[4.5]decan-2-yl)methanol to get compound 35 (44 mg, 55%) as a white solid. UPLC−MS (ESI) m/z: calcd, 808.40 for C₄₉H₅₃N₅O₆ [M + H]⁺; found, 808.2; ¹H NMR (400 MHz, Methanold₄) δ 7.39 (dd, J = 8.3, 1.7 Hz, 1H), 7.25 (dd, J = 8.6, 1.6 Hz, 2H), 7.14 (td, J = 8.2, 5.5 Hz, 4H), 6.93−6.81 (m, 2H), 6.78−6.61 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.10 (dd, J = 13.3, 5.2 Hz, 1H), 4.65−4.31 (m, 6H), 4.31−4.04 (m, 3H), 3.77 (td, J = 24.6, 8.9 Hz, 5H), 3.63−3.40 (m, 6H), 3.19−2.99 (m, 3H), 2.97−2.73 (m, 2H), 2.58−2.42 (m, 1H), 2.42−2.23 (m, 2H), 2.24−2.06 (m, 6H), 1.93−1.73 (m, 2H).

(3S)-3-((5aR)-7-((8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetra-hydronaphthalen-1-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decan-2-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (36).

A similar synthetic procedure for 34 was carried out using (1,3-dioxa-8-azaspiro[4.5]decan-2-yl)methanol to get compound 36 (44 mg, 55%) as a white solid. UPLC−MS (ESI) m/z: calcd, 810.38 for C₄₈H₅₁N₅O₇ [M + H]⁺; found, 810.42; ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (d, J = 8.3 Hz, 1H), 7.14 (dt, J = 7.3, 5.8 Hz, 4H), 7.05 (d, J = 8.4 Hz, 2H), 6.85 (dd, J = 7.5, 2.1 Hz, 2H), 6.70 (dd, J = 5.5, 2.9 Hz, 2H), 6.55 (dq, J = 8.1, 2.7 Hz, 3H), 5.10 (dd, J = 13.3, 5.2 Hz, 1H), 4.73 (dd, J = 6.7, 2.9 Hz, 1H), 4.47 (d, J = 11.0 Hz, 1H), 4.41−4.30 (m, 4H), 4.30−4.23 (m, 1H), 4.18 (ddd, J = 10.7, 7.0, 3.2 Hz, 1H), 3.82 (ddd, J = 38.1, 26.4, 11.9 Hz, 4H), 3.59−3.38 (m, 7H), 3.29 (s, 2H), 3.06 (q, J = 10.0 Hz, 3H), 2.97−2.84 (m, 1H), 2.84−2.74 (m, 1H), 2.57−2.42 (m, 1H), 2.27−2.03 (m, 6H), 1.83 (d, J = 12.8 Hz, 1H).

(3S)-3-((5aR)-7-((9-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-9-azaspiro[5.5]undecan-3-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (37).

Title compound 37 was prepared from compound 45 using 3-(dimethoxymethyl)-1-oxa-9-azaspiro[5.5]undecane (56b) by following similar synthetic procedure for compound 29 as a white solid (56 mg, 0.068 mmol, 68%). UPLC−MS (ESI) m/z: calcd, 411.71 for C₅₀H₅₅N₅O₆ [M + 2H]⁺/2; found, 411.7. ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (dd, J = 8.3, 2.6 Hz, 1H), 7.25 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 6.5 Hz, 4H), 6.85 (d, J = 6.9 Hz, 2H), 6.74−6.62 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.10 (dd, J = 13.3, 4.9 Hz, 1H), 4.52−4.33 (m, 4H), 4.26 (d, J = 11.3 Hz, 1H), 4.17 (s, 1H), 3.91 (s, 1H), 3.67 (d, J = 49.1 Hz, 5H), 3.47 (d, J = 17.2 Hz, 4H), 3.23 (d, J = 24.7 Hz, 4H), 3.11−2.93 (m, 3H), 2.93−2.73 (m, 2H), 2.50 (dd, J = 13.1, 5.0 Hz, 2H), 2.28−2.07 (m, 4H), 2.07−1.91 (m, 2H), 1.90−1.78 (m, 2H), 1.78−1.51 (m, 3H).

(S)-3-((R)-7-((9-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1,5-dioxa-9-azaspiro[5.5]undecan-3-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (38).

Title compound 38 was prepared from compound 59d (108 mg, 0.2 mmol) by following a similar synthetic procedure as compound 34 as a white solid (53 mg, 32%). UPLC−MS (ESI) m/z: calcd, 824.39 for C₄₉H₅₃N₅O₇ [M + H]⁺; found, 824.20. ¹H NMR (400 MHz, Methanold₄) δ 7.37 (dd, J = 8.4, 2.4 Hz, 1H), 7.27−7.08 (m, 6H), 6.90−6.79 (m, 2H), 6.73−6.60 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.09 (dd, J = 13.3, 5.0 Hz, 1H), 4.50−4.34 (m, 4H), 4.32−4.11 (m, 4H), 3.88 (dt, J = 11.4, 5.2 Hz, 2H), 3.75 (q, J = 9.5 Hz, 3H), 3.57 (q, J = 5.0 Hz, 4H), 3.51−3.37 (m, 3H), 3.30−3.19 (m, 2H), 3.04 (dt, J = 22.5, 9.2 Hz, 3H), 2.96−2.71 (m, 2H), 2.49 (tt, J = 13.0, 6.2 Hz, 1H), 2.36 (d, J = 27.2 Hz, 3H), 2.28−2.08 (m, 4H), 1.84 (d, J = 12.3 Hz, 1H).

(S)-3-((R)-7-(((S)-8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-8-azaspiro[4.5]decan-3-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (33a).

Compound 45 (1.2 g, 1.9 mmol, 1 equiv) was subjected to the general Buchwald amination reaction with amine 57e (490 mg, 2.8 mmol, 1.5 equiv) to provide 60 (500 mg, 50% yields) as a white solid. UPLC−MS (ESI) m/z: calcd, 526.32 for C₃₅H₄₃NO₃ [M + H]⁺; found, 526.20.

The compound 60 (1.2 g) was further separated by SFC (Instrument: WATERS 150 preparative SFC(SFC-26); column: ChiralPak AD, 250 × 30 mm I.D., 10m; mobile phase: A for CO₂ and B for Methanol; Gradient: B 20%; Flow rate: 150 mL/min; Back pressure: 100 bar) to afford a pure product 60a (faster-moving fraction, 500 mg, 87%) and a pure product 60b (slower-moving fraction, 500 mg, 87%).

To a solution of compound 60a (500 mg, 0.95 mmol, 1 equiv) in DCM (5 mL) was added TEA (0.2 mL, 1.42 mmol, 2 equiv) and DMAP (12 mg, 0.1 mmol, 0.11 equiv). TsCl (216 mg, 1.14 mmol, 1.2 equiv) was added, and the mixture was stirred at room temperature overnight. TLC indicated the starting material was consumed completely. The reaction mixture was quenched by the addition of saturated NH₄Cl solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by flash silica chromatography to give compound 61a (516 mg, 80%). UPLC−MS (ESI) m/z: calcd, 680.33 for C₄₂H₄₉NO₅S [M + H]⁺; found, 680.32. ¹H NMR (400 MHz, CDCl₃) δ 7.86−7.75 (m, 2H), 7.43−7.34 (m, 2H), 7.17 (dd, J = 5.2, 1.9 Hz, 3H), 6.93−6.78 (m, 4H), 6.75 (dd, J = 8.3, 2.5 Hz, 1H), 4.26 (d, J = 4.8 Hz, 1H), 4.08−3.96 (m, 2H), 3.96−3.88 (m, 1H), 3.58 (t, J = 7.8 Hz, 1H), 3.40 (d, J = 12.6 Hz, 1H), 3.20−2.95 (m, 5H), 2.69 (hept, J = 7.4 Hz, 1H), 2.48 (s, 3H), 2.26−2.09 (m, 1H), 1.93 (dd, J = 12.9, 8.6 Hz, 1H), 1.82 (d, J = 12.2 Hz, 1H), 1.57 (d, J = 27.0 Hz, 6H), 1.38 (s, 9H).

Following the same procedure, the title compound 61b was yielded as a white solid from 60b. UPLC−MS (ESI) m/z: calcd, 680.33 for C₄₂H₄₉NO₅S [M + H]⁺; found, 680.39. ¹H NMR (400 MHz, CDCl₃) δ 7.86−7.75 (m, 2H), 7.43−7.34 (m, 2H), 7.17 (dd, J = 5.2, 1.9 Hz, 3H), 6.93−6.78 (m, 4H), 6.75 (dd, J = 8.3, 2.5 Hz, 1H), 4.26 (d, J = 4.8 Hz, 1H), 4.08−3.96 (m, 2H), 3.96−3.88 (m, 1H), 3.58 (t, J = 7.8 Hz, 1H), 3.40 (d, J = 12.6 Hz, 1H), 3.20−2.95 (m, 5H), 2.69 (hept, J = 7.4 Hz, 1H), 2.48 (s, 3H), 2.26−2.09 (m, 1H), 1.93 (dd, J = 12.9, 8.6 Hz, 1H), 1.82 (d, J = 12.2 Hz, 1H), 1.57 (d, J = 27.0 Hz, 6H), 1.38 (s, 9H).

To a solution of compound 61a (340 mg, 0.5 mmol, 1 equiv) in DMF (2 mL) was added compound 8 (196 mg, 0.55 mmol, 1.1 equiv), KI (83 mg, 0.5 mmol, 1.0 equiv) and diisopropylethylamine (0.13 mL, 0.75 mmol, 1.5 equiv). The mixture was stirred at 60 °C for 4 h. LC-MS showed the reaction was completed. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried with anhydrous sodium sulfate, filtered and the filtrate was concentrated in a vacuum. The product was separated using reverse-phase column chromatography. The product was concentrated under reduced pressure. The crude product was next dissolved in DCM (2 mL) treated with TFA (0.2 mL) and stirred for another 2 h. UPLC analysis showed complete removal of the tert-butyl group. The final compound was concentrated and dried under lyophilizer to obtain compound 33a with 64% yields as a white solid (258 mg). UPLC−MS (ESI) m/z: calcd, 808.40 for C₄₉H₅₃N₅O₆ [M + H]⁺; found, 808.41; ¹H NMR (400 MHz, Methanol-d₄) δ 7.37 (d, J = 8.3 Hz, 1H), 7.28−7.21 (m, 2H), 7.17−7.10 (m, 4H), 6.88−6.81 (m, 2H), 6.73−6.63 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.52−4.42 (m, 2H), 4.38 (t, J = 5.9 Hz, 3H), 4.31− 4.11 (m, 3H), 3.82−3.63 (m, 6H), 3.55−3.41 (m, 3H), 3.41−3.34 (m, 2H), 3.25 (q, J = 12.5 Hz, 2H), 3.11−2.93 (m, 4H), 2.92−2.73 (m, 2H), 2.50 (qd, J = 13.1, 4.8 Hz, 1H), 2.33 (ddd, J = 11.5, 7.9, 3.2 Hz, 1H), 2.27−1.97 (m, 6H), 1.91−1.79 (m, 1H), 1.73 (ddd, J = 12.6, 8.9, 3.5 Hz, 1H).

(S)-3-((R)-7-(((R)-8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-8-azaspiro[4.5]decan-3-yl)methyl)-1-oxo-1,3,5,5a,6,7,8,9-octahydro-2H-pyrazino[1′,2′:4,5][1,4]oxazino[2,3-e]isoindol-2-yl)piperidine-2,6-dione (33b).

Compound 33b was made using similar synthetic route as 33a using alcohol 60b (slow moving isomer) as a white solid (250 mg, 62%). UPLC−MS (ESI) m/z: calcd, 808.40 for C₄₉H₅₃N₅O₆ [M + H]⁺; found, 808.42; ¹H NMR (400 MHz, MeOD) δ 7.37 (d, J = 8.4 Hz, 1H), 7.29−7.20 (m, 2H), 7.20−7.12 (m, 4H), 6.90−6.81 (m, 2H), 6.74−6.63 (m, 4H), 6.55 (dd, J = 8.3, 2.6 Hz, 1H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.50−4.30 (m, 4H), 4.30−4.10 (m, 3H), 3.82−3.61 (m, 6H), 3.54−3.41 (m, 3H), 3.35 (d, J = 6.6 Hz, 2H), 3.26 (t, J = 13.6 Hz, 2H), 3.14−2.73 (m, 6H), 2.50 (qd, J = 13.1, 4.8 Hz, 1H), 2.30 (dd, J = 12.8, 8.0 Hz, 1H), 2.26−1.97 (m, 6H), 1.91−1.78 (m, 1H), 1.72 (dd, J = 12.9, 8.9 Hz, 1H).

Characterization of Intermediates.

Methyl 4-(4-((1R,2S)-6(tert-Butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)butanoate (46b).

Compound 46b was prepared from compound 44 following similar synthetic route as 46a using methyl 4-bromobutanoate. UPLC−MS (ESI) m/z: calcd, 473.26 for C₃₁H₃₆O₄ [M + H]⁺; found, 473.26. ¹H NMR (400 MHz, Chloroform-d) δ 7.23−7.14 (m, 3H), 6.88−6.79 (m, 4H), 6.75 (dd, J = 8.3, 2.5 Hz, 1H), 6.59−6.49 (m, 2H), 6.38−6.28 (m, 2H), 4.28 (d, J = 5.0 Hz, 1H), 3.90 (t, J = 6.1 Hz, 2H), 3.69 (s, 3H), 3.40 (ddd, J = 13.2, 5.1, 2.3 Hz, 1H), 3.15−2.98 (m, 2H), 2.50 (t, J = 7.3 Hz, 2H), 2.28−2.12 (m, 1H), 2.07 (tt, J = 7.3, 6.0 Hz, 2H), 1.90−1.79 (m, 1H), 1.39 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 173.70, 156.92, 153.53, 144.36, 137.13, 134.84, 134.72, 131.41, 130.84, 128.21, 127.78, 126.01, 123.84, 122.09, 112.88, 78.06, 66.48, 51.60, 50.42, 45.34, 30.60, 30.04, 28.99, 24.70, 21.93.

Methyl 5-(4-((1R,2S)-6-(tert-Butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)pentanoate (46c).

Compound 46c was prepared from compound 44 following similar synthetic route as 46a using methyl 5-bromopentanoate. UPLC−MS (ESI) m/z: calcd, 487.28 for C₃₂H₃₈O₄ [M + H]⁺; found, 487.20. ¹H NMR (400 MHz, Chloroform-d) δ 7.24−7.12 (m, 3H), 6.90−6.81 (m, 4H), 6.75 (dd, J = 8.3, 2.4 Hz, 1H), 6.56−6.49 (m, 2H), 6.31 (d, J = 8.5 Hz, 2H), 4.27 (d, J = 5.0 Hz, 1H), 3.90−3.83 (m, 2H), 3.75−3.66 (m, 5H), 3.48−3.36 (m, 2H), 3.06 (dd, J = 12.0, 6.0 Hz, 2H), 2.38 (td, J = 7.1, 2.9 Hz, 3H), 2.20 (ddt, J = 19.8, 12.8, 5.9 Hz, 1H), 1.99−1.87 (m, 1H), 1.87−1.75 (m, 6H), 1.39 (d, J = 0.9 Hz, 8H). ¹³C NMR (101 MHz, CDCl₃) δ 173.87, 157.05, 153.51, 144.37, 137.12, 134.86, 134.57, 131.38, 130.84, 128.21, 127.76, 125.99, 123.82, 122.07, 112.86, 78.03, 67.13, 51.61, 51.52, 50.41, 45.35, 33.71, 33.07, 33.00, 32.02, 30.04, 28.99, 28.73, 23.52, 21.94, 21.68.

tert-Butyl 6-(4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenoxy)hexanoate (46d).

Compound 46d was prepared from compound 44 following similar synthetic route as 46a using tert-butyl 6-bromohexanoate. UPLC−MS (ESI) m/z: calcd, 543.34 for C₃₆H₄₆O₄ [M + H]⁺; found, 543.37. ¹H NMR (400 MHz, Chloroform-d) δ 7.24−7.14 (m, 3H), 6.92 (d, J = 2.4 Hz, 1H), 6.90−6.83 (m, 3H), 6.78 (dd, J = 8.3, 2.5 Hz, 1H), 6.60−6.53 (m, 2H), 6.38−6.31 (m, 2H), 4.30 (d, J = 5.0 Hz, 1H), 3.87 (t, J = 6.4 Hz, 2H), 3.43 (ddt, J = 13.1, 5.2, 2.6 Hz, 1H), 3.19−3.00 (m, 2H), 2.28 (td, J = 7.4, 2.9 Hz, 3H), 1.96−1.82 (m, 1H), 1.82−1.75 (m, 2H), 1.71−1.65 (m, 2H), 1.51 (d, J = 0.9 Hz, 2H), 1.50 (s, 9H), 1.42 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 173.00, 157.17, 153.54, 144.38, 137.11, 134.89, 134.45, 131.38, 130.88, 128.23, 127.77, 126.00, 123.82, 122.08, 112.89, 79.98, 77.99, 67.44, 63.76, 50.44, 45.39, 35.49, 30.07, 29.02, 28.18, 25.63, 24.90, 21.97.

tert-Butyl 5-((4-((1R,2S)-6-(tert-butoxy)-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)(methyl)amino)pentanoate (46f).

Similar synthetic route for compound 46e was followed to make 46f (81 mg, 75%) as white solid; UPLC−MS (ESI) m/z: calcd, 542.36 for C₃₆H₄₇NO₃ [M + H]⁺; found, 542.32; ¹H NMR (400 MHz, Chloroform-d) δ 7.26−7.13 (m, 3H), 6.92−6.82 (m, 4H), 6.76 (dd, J = 8.4, 2.4 Hz, 1H), 6.40 (d, J = 8.6 Hz, 2H), 6.32−6.24 (m, 2H), 4.25 (d, J = 4.9 Hz, 1H), 3.39 (ddd, J = 13.1, 5.0, 2.2 Hz, 1H), 3.23 (t, J = 7.0 Hz, 2H), 3.15−2.98 (m, 2H), 2.85 (s, 3H), 2.30−2.14 (m, 3H), 1.88−1.79 (m, 1H), 1.57 (dddt, J = 19.7, 13.9, 8.3, 2.7 Hz, 4H), 1.46 (s, 9H), 1.40 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 172.87, 153.34, 146.98, 144.67, 137.10, 135.24, 131.24, 130.86, 128.33, 127.66, 125.87, 123.78, 122.00, 111.32, 80.16, 78.01, 77.38, 77.06, 76.75, 52.93, 50.33, 45.50, 38.60, 35.37, 30.05, 28.97, 28.13, 26.05, 22.70, 22.03.

2-(7-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-2-yl)acetic Acid (51b).

Compound 51b was prepared from compound 45 following a similar synthetic route as 51a using tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate as a white solid (60 mg, 66%). UPLC−MS (ESI) m/z: calcd, 483.26 for C₃₁H₃₄N₂O₃ [M + H]⁺; found, 483.32; ¹H NMR (400 MHz, Methanol-d₄) δ 7.12 (ddd, J = 7.5, 5.9, 4.5 Hz, 3H), 6.84 (ddd, J = 9.7, 6.7, 1.9 Hz, 2H), 6.68 (td, J = 7.4, 3.1 Hz, 3H), 6.53 (dt, J = 8.3, 3.0 Hz, 1H), 6.43−6.27 (m, 2H), 6.27−6.18 (m, 1H), 4.25 (s, 1H), 4.08 (d, J = 4.4 Hz, 2H), 3.64−3.35 (m, 7H), 3.02 (dt, J = 11.3, 7.1 Hz, 4H), 2.34−1.68 (m, 6H).

2-(2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,7-diazaspiro[3.5]nonan-7-yl)acetic Acid (51c).

A similar synthetic route for compound 51a was followed to make compound 51c as a white solid (50 mg, 65%). UPLC−MS (ESI) m/z: calcd, 483.26 for C₃₁H₃₄N₂O₃ [M + H]⁺; found, 483.30; ¹H NMR (400 MHz, Methanol-d₄) δ 7.20−7.05 (m, 3H), 6.86−6.77 (m, 2H), 6.73−6.61 (m, 2H), 6.52 (dd, J = 8.3, 2.6 Hz, 1H), 6.31 (q, J = 8.7 Hz, 4H), 4.20 (d, J = 5.1 Hz, 1H), 4.08 (s, 2H), 3.69−3.60 (m, 6H), 3.34 (s, 1H), 3.32 (d, J = 4.2 Hz, 1H), 3.02 (ddd, J = 11.8, 7.2, 4.2 Hz, 3H), 2.34−2.02 (m, 5H), 1.84−1.70 (m, 1H).

2-(8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-2,8-diazaspiro[4.5]decan-2-yl)acetic Acid (51d).

A similar synthetic route for compound 51a was followed to make compound 51d as a white solid (50 mg, 65%). UPLC−MS (ESI) m/z: calcd, 497.27 for C₃₂H₃₆N₂O₃ [M + H]⁺; found, 497.32; ¹H NMR (400 MHz, Methanol-d₄) δ 7.12 (dq, J = 4.7, 3.1, 2.2 Hz, 3H), 6.88−6.78 (m, 2H), 6.78−6.62 (m, 4H), 6.53 (dd, J = 8.3, 2.6 Hz, 1H), 6.36 (dd, J = 8.6, 2.5 Hz, 2H), 4.22 (d, J = 5.1 Hz, 1H), 3.97 (s, 2H), 3.57 (d, J = 8.7 Hz, 2H), 3.43−3.34 (m, 3H), 3.12 (t, J = 5.0 Hz, 4H), 3.02 (dt, J = 11.9, 4.4 Hz, 2H), 2.21 (ddd, J = 13.0, 7.0, 2.9 Hz, 1H), 2.05 (td, J = 7.3, 3.8 Hz, 2H), 1.94−1.69 (m, 5H).

2-(9-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-3,9-diazaspiro[5.5]undecan-3-yl)acetic Acid (51e).

A similar synthetic route for compound 51a was followed to make compound 51e as a white solid (50 mg, 60%). UPLC−MS (ESI) m/z: calcd, 511.29 for C₃₃H₃₈N₂O₃ [M + H]⁺; found, 511.35; ¹H NMR (400 MHz, Methanol-d₄) δ 7.26−7.03 (m, 5H), 6.90−6.78 (m, 2H), 6.74−6.65 (m, 2H), 6.65−6.58 (m, 2H), 6.54 (dd, J = 8.3, 2.6 Hz, 1H), 4.37 (d, J = 5.3 Hz, 1H), 4.05 (s, 2H), 3.71−3.33 (m, 9H), 3.17−2.95 (m, 2H), 2.27−1.76 (m, 10H).

2-(Dimethoxymethyl)-7-azaspiro[3.5]nonane (54a).

A reported synthetic procedure was followed to make compound 54a as a white solid (200 mg, 24%) in four steps.⁸⁰ UPLC−MS (ESI) m/z: calcd, 200.16 for C₁₁H₂₁NO₂ [M + H]⁺; found, 200.12; ¹H NMR (400 MHz, Methanol-d4) δ 4.32 (d, J = 6.7 Hz, 1H), 3.33 (s, 6H), 2.95−2.83 (m, 2H), 2.83−2.74 (m, 2H), 2.58 (pd, J = 8.7, 6.8 Hz, 1H), 1.97−1.81 (m, 2H), 1.78−1.64 (m, 4H), 1.64−1.49 (m, 2H).

7-(Dimethoxymethyl)-2-azaspiro[3.5]nonane (54b).

A reported synthetic procedure was followed to make compound 54a as a white solid (200 mg, 20%) in four steps.⁸⁰ UPLC−MS (ESI) m/z: calcd, 200.16 for C₁₁H₂₁NO₂ [M + H]⁺; found, 200.10; ¹H NMR (400 MHz, Methanol-d₄) δ 4.03 (d, J = 6.8 Hz, 1H), 3.36 (s, 6H), 2.42−2.35 (m, 2H), 2.17−2.00 (m, 3H), 1.97−1.81 (m, 4H), 1.74 (d, J = 13.5 Hz, 2H), 1.16−0.97 (m, 2H).

2-(Dimethoxymethyl)-8-azaspiro[4.5]decane (54c).

A reported synthetic procedure was followed to make compound 54a as a white solid (200 mg, 25%) in four steps.⁸⁰ UPLC−MS (ESI) m/z: calcd, 214.17 for C₁₂H₂₃NO₂ [M + H]⁺; found, 214.25; ¹H NMR (400 MHz, Methanol-d₄) δ 4.16 (d, J = 7.6 Hz, 1H), 3.35 (d, J = 0.9 Hz, 6H), 2.81 (q, J = 7.2, 6.5 Hz, 4H), 2.35 (dt, J = 9.4, 7.9 Hz, 1H), 1.83−1.66 (m, 2H), 1.58−1.44 (m, 8H).

9-(Dimethoxymethyl)-3-azaspiro[5.5]undecane (54d).

A reported synthetic procedure was followed to make compound 54a as a white solid (200 mg, 32%) in four steps.⁸⁰ UPLC−MS (ESI) m/z: calcd, 228.19 for C₁₃H₂₅NO₂ [M + H]⁺; found, 228.15; ¹H NMR (400 MHz, Chloroform-d) δ 4.10−3.97 (m, 1H), 3.48 (d, J = 4.3 Hz, 4H), 3.35 (s, 6H), 1.60 (dddd, J = 39.4, 35.6, 13.4, 8.8 Hz, 7H), 1.40−1.00 (m, 6H).

2-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen1-yl)phenyl)-2-azaspiro[3.5]nonane-7-carbaldehyde (55b).

Intermediate 55b was prepared from 45 by following a similar synthetic protocol as compound 55a described earlier. UPLC−MS (ESI) m/z: calcd, 452.25 for C₃₁H₃₃NO₂ [M + H]⁺; found, 452.19; ¹H NMR (400 MHz, Chloroform-d) δ 9.64 (d, J = 0.8 Hz, 1H), 7.25−7.09 (m, 3H), 7.04−6.87 (m, 2H), 6.86−6.67 (m, 4H), 6.61 (dd, J = 8.3, 2.7 Hz, 1H), 6.57−6.45 (m, 2H), 4.32 (d, J = 5.3 Hz, 1H), 4.16 (d, J = 12.7 Hz, 4H), 3.45 (ddd, J = 13.2, 5.4, 2.4 Hz, 1H), 3.15−2.93 (m, 2H), 2.29 (dq, J = 9.8, 4.9, 4.1 Hz, 1H), 2.20−2.00 (m, 2H), 2.00−1.81 (m, 3H), 1.71 (ddd, J = 13.9, 11.0, 3.7 Hz, 2H), 1.51 (dq, J = 22.4, 10.3 Hz, 3H).

8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-8-azaspiro[4.5]decane-2-carbaldehyde (55c).

Intermediate 55c was prepared from 45 by following a similar synthetic protocol as compound 55a described earlier. UPLC−MS (ESI) m/z: calcd, 466.27 for C₃₂H₃₅NO₂ [M + H]⁺; found, 466.32; ¹H NMR (400 MHz, Chloroform-d) δ 9.70 (d, J = 1.4 Hz, 1H), 7.17 (ddd, J = 7.4, 3.5, 2.2 Hz, 5H), 6.85−6.64 (m, 4H), 6.64−6.45 (m, 3H), 4.32 (d, J = 5.4 Hz, 1H), 3.45 (ddd, J = 13.2, 5.4, 2.4 Hz, 5H), 3.16−2.84 (m, 3H), 2.19−1.51 (m, 12H).

3-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-3-azaspiro[5.5]undecane-9-carbaldehyde (55d).

Intermediate 55d was prepared from 45 by following a similar synthetic protocol as compound 55a described earlier. UPLC−MS (ESI) m/z: calcd, 480.28 for C₃₃H₃₇NO₂ [M + H]⁺; found, 480.20; ¹H NMR (400 MHz, Chloroform-d) δ 9.68 (s, 1H), 7.29−7.09 (m, 5H), 6.89−6.65 (m, 4H), 6.56 (dd, J = 17.5, 7.9 Hz, 3H), 4.31 (d, J = 5.2 Hz, 1H), 3.41 (td, J = 17.6, 17.0, 8.6 Hz, 5H), 3.04 (dd, J = 10.6, 6.3 Hz, 2H), 2.32 (s, 1H), 2.15−1.00 (m, 15H).

3-(Dimethoxymethyl)-1-oxa-8-azaspiro[4.5]decane (56a).

A reported synthetic procedure was followed to make compound 56a as a white solid (200 mg, 30%) in four steps.⁸⁰ UPLC−MS (ESI) m/z: calcd, 216.15 for C₁₁H₂₁NO₃ [M + H]⁺; found, 216.26; ¹H NMR (400 MHz, Methanol-d₄) δ 4.10−3.96 (m, 2H), 3.71 (s, 6H), 3.40−3.22 (m, 2H), 3.00 (td, J = 10.5, 9.8, 5.3 Hz, 2H), 2.90 (dd, J = 11.5, 6.4 Hz, 2H), 2.16−1.99 (m, 2H), 1.80−1.56 (m, 4H).

8-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)-1-oxa-8-azaspiro[4.5]decane-3-carbaldehyde (58a).

Intermediate 58a was prepared from compound 45 using 56a by following a similar synthetic procedure for 55a. UPLC−MS (ESI) m/z: calcd, 468.25 for C₃₁H₃₃NO₃ [M + H]⁺; found, 468.30; ¹H NMR (400 MHz, Chloroform-d) δ 9.71 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 5.2, 2.0 Hz, 3H), 6.90−6.76 (m, 3H), 6.72 (d, J = 2.7 Hz, 1H), 6.65−6.50 (m, 3H), 6.36−6.23 (m, 2H), 4.22 (d, J = 5.0 Hz, 1H), 4.17 (dd, J = 9.5, 5.1 Hz, 1H), 4.05 (dd, J = 9.5, 7.7 Hz, 1H), 3.36 (ddd, J = 13.1, 5.1, 2.2 Hz, 1H), 3.22−2.94 (m, 6H), 2.20 (dd, J = 11.8, 6.9 Hz, 1H), 2.13−1.93 (m, 2H), 1.76 (qq, J = 13.5, 6.4, 5.9 Hz, 6H).

Cereblon Binding Assay.

The binding to cereblon was determined using the Cereblon Binding Kit (Cisbio, #64BDCRBNPEG) following the manufacturer’s instructions. Briefly, serially diluted compounds were incubated with GST-tagged wild-type human cereblon protein, XL665-labeled Thalidomide, and Europium Cryptate labeled GST antibody at room temperature for ~3 h. Time-resolved fluorescence resonance energy transfer (TR-FRET) measurements were acquired on a CALRIOstar plate reader with MARS data analysis software (BMG Labtech), with the following settings: 665/10 and 620/10 nm emission, 60 μs delay, and 400 μs integration. The TR-FRET ratio was taken as the 665/620 nm intensity ratio. The readings were normalized to the control (0.5%) and the IC₅₀ was calculated by nonlinear regression (four parameters sigmoid fitted with variable slope) analysis using the GraphPad Prism 8 software.

Computational Modeling.

The DDB1:cereblon complex with lenalidomide (PDB ID: 4CI2) was utilized for conducting docking experiments on cereblon.⁸¹ The protein was prepared using Schrödinger’s Protein Preparation Wizard in Schrödinger 2022−4,⁸² which included steps such as assigning bond orders, adding hydrogens, refining the loop region, optimizing hydrogen bonds, removing water molecules, and performing restrained energy minimization with the OPLS4 force field.⁸³ Following preparation, the DDB1 protein component was removed. Docking was conducted using the extra precision (XP) Glide docking procedure,⁸⁴ which involved setting up a receptor grid centered on lenalidomide and defining a docking box to accommodate ligands of similar size. Default settings in XP Glide docking were applied, allowing for flexible ligand sampling and retaining up to 10 poses per ligand, without setting any constraints, rotatable groups, or excluded volumes.⁸⁴ First, lenalidomide was redocked into the cereblon binding site. This step was undertaken to assess the accuracy of the procedure in reproducing the crystallographically determined pose of lenalidomide. The results indicate that the best-docked pose closely aligns with the crystal structure pose, with the RMSD of 0.4 Å. Subsequently, the ligands ligand 8 (RR-11055) and ligand 9 (RR-11163) were prepared using LigPrep, predicting ionization states at pH 7.0 ± 2.0 with Epik⁸⁵ and generating their 3D structures optimized by the OPLS4 force field.⁸³ Ligands RR-11055 and RR-11163 were docked using the same receptor grid established for lenalidomide, following the identical XP Glide protocol, ensuring consistency.

In-Cell Western Blot Analysis.

The protocol is as follows: (a) seeded cells in black-sided/clear bottom 384-well plates at 40,000 or 10,000 cells/well, overnight; b. added diluted compounds (final 0.5% DMSO), incubated for 12 h, removed medium, added 50 μL of 3.7% formaldehyde (PBS:FA = 9:1) and kept at room temp (RT) for 20 min without shaking; (c) washed with PBS, and permeabilized with 70 μL/well of 1× PBS + 0.1% Triton X-10010 min for 5 times; d. blocked with 50 μL Intercept blocking buffer (LI-COR), kept at RT for 1h with moderate shaking; (d) added 50 μL of anti-ER (cs-8644, 1:1−000) + GAPDH (Millipore MAB374, 1:1000) in Intercept block buffer, and kept at RT for 2 h with gentle shaking. Negative control: cells plus secondary antibodies (no primary antibodies); (e) washed with PBS containing 0.1% Tween 20 with gentle shaking 10 min for 4 times; (f) Blot with goat antirabbit-680 and goat antimouse-800 (1:1000 dilution with 0.2% Tween 20 containing Intercept blocking buffer) (g) washed 5 min with PBS + 0.1% Tween 20 for four times; (h) added 70 μL of PBS to each well and scan with ODYSSEY CLX machine from LI-COR. The data were collected based on triplicate experiments unless otherwise stated. The maximum level of ERα degradation achieved by ARV-471 was set as 100% to minimize the effect of nonspecific signal. The definitions for the degradation profiling are as follows: DC₅₀ = the concentration needed to reduce ERα protein by 50%; D_max = maximal ERα degradation. The data were provided as mean ± SE.

Western Blot Analysis.

Western blot analysis was performed as described previously.⁸⁶ The cells treated with indicated compounds were lysed in Radioimmunoprecipitation Assay Protein Lysis and Extraction Buffer (25 mmol/L Tris. HCl, pH 7.6, 150 mmol/L NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Twenty ug amounts of total protein were separated in 10% SDS-polyacrylamide gels after being quantified by BCA assay (Fisher Scientific, Pittsburgh, PA). The separated protein bands were transferred onto PVDF membranes (GE Healthcare Life Sciences, Marlborough, MA) and blotted against different antibodies, as indicated. The blots were scanned, and the band intensities were quantified with the Image Studio software (Version 5.2). The relative mean intensity of target proteins was expressed after normalization to the intensity of glyceraldehyde-3-phosphate dehydrogenase or beta-actin bands.

Pharmacokinetic (PK) Studies in Rats and Mice.

PK studies were performed in Shanghai Medicilon Inc. Shanghai, China. Mice and rats were purchased from Sino-British SIPPR/BK Lab Animal Ltd., Shanghai, China. The animals were divided into two groups, with one group receiving an intravenous (IV) dose of 1 mg/kg and the other group receiving an oral dose of 3 mg/kg after fasting. The drug solution was freshly prepared before administration, and each compound was formulated in 5% DMSO + 10% solutol +85% saline as a clear solution. Blood samples were collected at the following time points: 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 and 24 h post-dose administration. 200 μL (for rat) or 30 μL (for mouse) of blood was collected and the samples were placed in tubes containing heparin sodium and stored on ice. The samples were centrifuged at ~6800 G for 6 min at 2−8 °C and the resulting plasma was transferred to appropriately labeled tubes within 1 h of blood collection/centrifugation and then stored frozen at −80 °C.

Method development and biological sample analysis for the test articles (Sodium heparin anticoagulant) were performed by the testing facility by means of LC-MS/MS. The analytical results were confirmed using quality control samples for intra-assay variation. The accuracy of >66.7% of the quality control samples was between 80 and 120% of the known value(s). Standard set of parameters including T_1/2 (elimination half-life), AUC(0−t) (area-under-the-curve), V_ss (volume of distribution at steady state), Cl (clearance), C_max (maximum drug concentration), F (oral bioavailability) were calculated using Phoenix WinNonlin 7.0 (Pharsight, USA) by the Study Director.

Determination of Oral Plasma Exposures in Rats.

Oral plasma exposures of selected compounds were evaluated in female SD rats. A group of three rats was dosed orally with each compound at a dose level of 3 mg/kg. The drug solution was freshly prepared using 5% DMSO + 10% solutol + 85% saline as the dosing vehicle before administration. Blood samples were collected at the time points of 1, 3, 6, and 24 h post-dose, and 250−300 μL of blood was collected at each time point from the saphenous vein. The blood was collected into 1.5 mL microfuge tubes pretreated with Heparin and put on ice immediately. Samples were centrifuged at 15,000 rpm for 10 min. A minimum of 100 μL of blood plasma was collected from the upper layer, leaving the blood cells behind in the microfuge tube. The plasma was transferred into a fresh 1.5 mL microfuge tube and frozen at −80 °C for future LC-MS analysis. For the LC-MS experiments, the chromatographic conditions are as follows: column, 50 × 2.1 mm I.D., packed with 3.5 μm C18 (Waters XBridge); mobile phase A, 0.1% formic acid in purified deionized water; mobile phase B, 0.1% formic acid in MeCN; flow rate: 0.5 mL/min; Injection Volume: 15 μL; run time: 5.7 min. MS/MS Conditions in 4500Q: electrospray, Turbo-Ionspray Interface used in the positive ion-mode.

Microsomal Metabolic Stability Studies.

Pooled human, mouse, and rat microsomes (10 μL aliquot) were prepared and stored at −80 °C prior to use. Master-mix containing microsome, phosphate buffer and test compound solution was made as follows: (1) 10 μL of microsome (20 mg/mL) was diluted with 330 μL 0.1 M Phosphate buffer (3.3 mM MgCl₂); (2) about 3.3 mg of NADPH was dissolved in 200 μL of 0.1 M phosphate buffer (3.3 mM MgCl₂); (3) 40 μL of 10 μM ERD-1233 PBS solution, was added to microsome; (4) the master solution was prewarmed at 37 °C for 5 min. NADPH (20 μL) was added to the above master solution to initiate the reaction. The final concentration of ERD-1233 in the reaction system was 1 μM. Aliquot of 40 μL was pipetted from the reaction solution and stopped by the addition of 160 μL cold MeCN containing 25 nM of CE302 as an internal standard at the designated time points (0, 5, 10, 15, 30, 45, and 60 min). The incubation solution was vortexed-mixed (800 rpm/10 min) and centrifuged at 3500 rpm for 10 min to precipitate proteins. The supernatant was collected and used for the LC/MS/MS analysis. The natural log peak area ratio (compound peak area/internal standard peak area) was plotted against time and the gradient of the line was determined.

hERG Channel Inhibition Assay.

The hERG channel inhibition of ERD-1233 was evaluated in HEK293 cells that expressed hERG channel using IonWorks Barracuda system (Molecular Devices Corporation, Union City, CA) at Charles River, 14656 Neo Parkway, Cleveland, OH 44128, United States. HEPES-buffered intracellular solution (Charles River proprietary) for whole-cell recordings was loaded into the intracellular compartment of the Population Patch Clamp planar electrode. Extracellular buffer (HB-PS) was loaded into PPC planar electrode plate wells (11 μL per well). The cell suspension was pipetted into the wells of the PPC planar electrode (9 μL per well). After the establishment of a whole-cell configuration (the perforated patch), membrane currents were recorded using a patch clamp amplifier in the IonWorks Barracuda system. The current recordings were performed one time before the test article application to the cells (baseline) and one time after the application of the test article. Tested compound concentrations were applied to naïve cells (n = 4, where n = replicates/concentration). Each application consisted of the addition of 20 mL of 2× concentrated test article solution to the total 40 mL of the final volume of the extracellular well of the PPC plate. Duration of exposure to each compound concentration was 5 min hERG test voltage protocol: hERG current was measured using a pulse pattern with fixed amplitudes (the first conditioning prepulse to 10 mV for 60 s, the second conditioning prepulse: −90 mV for 20 ms; test pulse: +40 mV for 100 ms) from a holding potential of 0 mV (“zero holding” procedure). hERG current was measured as a difference between the peak current at 1 ms and at the end of the step to +40 mV. Data analysis: data acquisition and analyses were performed using the IonWorks Barracuda system operation software (version 2.0.2). The decrease in current amplitude after the test article application was used to calculate the percent block relative to the control. Results for each test article concentration (n ≥ 2) were averaged; the mean and standard deviation values were calculated and used to generate dose−response curves. Block effect was calculated as % Block = (1 − I_TA/I_Baseline) × 100%, where I_Baseline and I_TA were the currents measured before and after the addition of a test article, respectively. The data were corrected for rundown: % Block’ = 100% − ((% Block − % PC) × (100%/(% VC − % PC)), where % VC and % PC were the mean values of the current block with the vehicle and positive controls, respectively. Concentration−response data were fitted to an equation of the following form: % Block = % VC + (% PC − % VC)/[1 + ([Test]/IC₅₀)^N], where [Test] was the concentration of test article, IC₅₀ was the concentration of the test article producing half-maximal block, N was the Hill coefficient, % VC was the mean current block at the vehicle control and % Block was the percentage of ion channel current inhibited at each concentration of a test article. Nonlinear least-squares fits were solved with the XLfit addin for Excel (Microsoft, Redmond, WA).

Cytochrome P450 (CYP) Enzyme Inhibition Assay.

The CYP inhibition of ERD-1233 was evaluated in Shanghai Medicilon Inc. Shanghai, 201200, China. The protocol for the experiment is as follows. (1) Preheat 0.1 M K-Buffer with 5 mM MgCl₂ (K/Mg-buffer), pH 7.4; (2) Prepare serial dilution for test compound and reference inhibitors in a 96-well plate: (a) Transfer 8 μL of 10 mM test compounds to 12 μL of MeCN; (b) Prepare individual inhibitor spiking solution for CYPs 1A2, 2C8, 2C19, 2C9, 2D6 and 3A4 from 8 μL of DMSO stock to 12 μL of MeCN; (c) Perform 1:2 serial dilutions in DMSO:MeCN mixture (v/v: 40:60). (3) Prepare NADPH cofactor (66.7 mg NADPH in 10 mL 0.1 M K/Mg-buffer, pH7.4). (4) Prepare substrate (2 mL for each enzyme isoform) as indicated (add HLM where required on ice). (5) Prepare 0.2 mg/mL HLM solution (10 μL of 20 mg/mL to 990 μL of 0.1 M K/Mg-buffer). (6) Add 400 μL of 0.2 mg/mL HLM to the assay wells and then add 2 μL of the test compound set (serially diluted, see step 2.1) into the designated wells. (7) Add 200 μL of 0.2 mg/mL HLM to the assay wells and then add 1 μL of serially diluted reference inhibitor solution (see steps 2a and 2b) into the designated wells. (8) Add the following solutions (in duplicate) in a 96-well assay plate on ice: (a) Add 30 μL of test compound and reference compound in 0.2 mg/mL HLM solution (see steps 6 and 7); (b) Add 15 μL of substrate solution (see step 4). (9) Preincubate the 96-well assay plate and NADPH solution at 37 °C for 5 min. (10) Add 15 μL of prewarmed 8 mM NADPH solution into the assay plates to initiate the reaction (See step 3). (11) Incubate the assay plate at 37 °C. Five min for 3A4, 10 min for 2B6. (12) Stop the reaction by adding 180 μL of acetonitrile containing IS. (13) After quenching, shake the plates for 10 min (600 rpm/min) and then centrifuge at 6000 rpm for 15 min. (14) Transfer 80 μL of the supernatant from each well into a 96-well sample plate containing 120 μL of ultrapure water for LC/MS analysis. The LC-MS analysis was conducted using LC-MS/MS-20(TQ-6500+) ACQUITY UPLC HSS T3 1.8 μm(50 mm × 2.10 mm) or LC-MS/MS-11(8050) ACQUITY UPLC BEH C18 1.7 μm(50 mm × 2.10 mm). Perform curve-fitting to calculate IC50 using a Sigmoidal (nonlinear) dose−response model (GraphPad Prism 5.0 or Xlfit model 205) based on data calculation using the formula below: Y = Bottom + (Top−Bottom)/(1 + 10^{((LogEC50‑X)} × HillSlope)), where X is the logarithm of concentration, Y is the response starting from Bottom to Top in a sigmoid shape in response to inhibitor concentration from high to low. The results generated for the reference compounds were consistent with the historic values.

PK/PD and Efficacy Studies in Mice.

All in vivo studies were performed under an animal protocol (PRO00009463 and PRO00011174) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan, in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

To develop ER wild-type MCF-7 xenograft tumors, mice were given 4 μg/mL 17β-Estradiol in 0.05% ETOH drinking water for 1 week, followed by 8 μg/mL 17β-Estradiol in 0.1% EtOH drinking water thereafter. For the development of ESR1^Y537S MCF-7 breast cancer xenografts, mice were not treated with 17β-Estradiol. Ten million cells suspended in 50% Matrigel were injected subcutaneously into female SCID mice (Charles River Laboratories).

For PK/PD studies, tumor-bearing female SCID mice were once-daily administered with vehicle control, ARV-471, or ERD-1233 via oral gavage using 5% DMSO + 10% solutol +85% Saline as the dosing vehicle (dosing volume/mouse weight = 10 μL/g) when tumors reached 100−400 mm³. After continuous dosing for 3 days, mice were sacrificed at indicated time points, and blood samples and tumor tissues were harvested for analysis. At each time point, mice were euthanized with CO_2, and 250−300 μL of blood was collected by cardiac puncture. The blood samples were put into 1.5 mL microfuge tubes containing Heparin sodium stored on ice and then centrifuged at 15,000 rpm for 10 min. A minimum of 100 μL of blood plasma was collected from the upper layer, leaving the blood cells behind in the microfuge tube. The plasma was transferred into a fresh 1.5 mL microfuge tube and placed on wet ice at −80 °C. The tumor samples from each mouse were divided into two parts. One part was immediately frozen in liquid nitrogen (LN2), ground into fine powder, placed on dry ice, and stored at −80 °C for Western blot analysis. Western blots were performed as detailed in the previous section. The other part of the tumor was placed in tared Precellys 2 mL Hard Tissue tubes with Homogenizing Ceramic Beads 16859 (Cayman Chem), weighed, snap-frozen in LN2, and stored at −80 °C for drug concentration analysis.

To prepare tumor samples for LC-MS analysis, mixed ultrapure water, and MeCN solution (4:1) were added to the defrosted tumor tissue samples 5:1, v/w, in order to facilitate homogenization with a Precellys evolution homogenizer at 4 °C. The homogenized tissue solution was denatured using cold MeCN (1:3, v/v) with vortex and centrifuged at 13000 rpm 4 °C for 10 min. Following protein precipitation, the final supernatants were collected for LC−MS analysis.

To determine drug concentrations in plasma and tumor samples, an LC−MS/MS method was developed and validated. The LC−MS/MS method consisted of a Shimadzu HPLC system, and chromatographic separation of a test compound was achieved using a Waters XBridge C18 column (5 cm × 2.1 mm, 3.5 μm). An AB Sciex QTrap 5500 mass spectrometer equipped with an electrospray ionization source (Applied Biosystems, Toronto, Canada) in the positive-ion multiple reaction monitoring mode was used for detection. For example, the precursor/product ion transitions were monitored at m/z 855.3 for ERD-1233 and internal standard, respectively, in the positive electrospray ionization mode. The mobile phases used on HPLC were 0.1% formic acid in purified water (A) and 0.1% formic acid in MeCN (B). The gradient (B) was held at 10% (0−0.3 min), increased to 95% at 0.7 min, then kept at isocratic 95% B for 2.3 min, and then immediately stepped back down to 10% for 2 min re-equilibration. The flow rate was set at 0.4−0.5 mL/min and the injection volume was 5−10 μL.

For the in vivo efficacy experiments, when tumors reached an average volume of 80−200 mm³, mice were tumor-size-matched and randomly assigned to different experimental groups with 7−8 mice for each group. Drugs or vehicle control were given at the dosing schedule as indicated using 5% DMSO + 10% solutol + 85% saline as the dosing vehicle. Tumor sizes and animal weights were measured 2−3 times per week. Tumor volume (mm³) = (length × width²)/2. Tumor growth inhibition was calculated as TGI (%) = [1 − (V_t − V₀)/(V_c − V₀)] × 100, where V_c and V_t are the mean tumor volume of the vehicle control and treated group at the end of treatment (or the last monitored time point), respectively, and V₀ and V₀′ are the mean tumor volume of the vehicle control and treated group at the start, respectively. Tumor regression was calculated as regression (%) = (V₀′ − V_t)/(V₀′) × 100, where V_t is the mean of treated groups at the end of treatment (or the last monitored time point) and V₀′ is that at the start. The tumor volumes at the end of treatment (or the last monitored time point) were statistically analyzed using a one-tailed, unpaired t-test with Welch’s correction (GraphPad Prism 8.0).

Supplementary Material

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01521.

Metabolic stability data of RR-11055, ERD-1233, detailed PK profile in mice and rats for ERD-1233, hERG inhibition data, detailed tissue distribution data for ERD-1233; in-cell Western data for all final compounds; UPLC spectra for all final compounds; NMR spectra of intermediates and all final compounds; and predicted binding models for cereblon ligands 8 and 9 (PDB files) (PDF)

RR-11055 complex with cereblon (PDB)

RR-11163 complex cereblon (PDB)

Molecular string file for all the final compounds (CSV)

Table 2.

Degraders with Flexible, Linear Linkers

	linker		ERα degradationa
CPD no.	n	X	DC50 (nM)b	Dmax (%)c
10 (ERD-3109)	0	O	0.3 ± 0.38	25 ± 11
11 (ERD-3108)	2	O	0.1 ± 0.03	56 ± 4.6
12 (ERD-4111)	3	O	0.12 ± 0.02	79 ± 2
13 (ERD-3124)	4	O	0.16 ± 0.04	89 ± 3.6
14 (ERD-3126)	2	NMe	1.6 ± 0.05	55 ± 4
15 (ERD-3127)	3	NMe	0.46 ± 0.17	63 ± 2
2 (Fulvestrant)	NA		0.29 ± 0.1	100 ± 1
4 (ARV-471)	NA		0.82 ± 0.2	98 ± 1

^aERα degradation potency was tested in the MCF-7 cell line using an in-cell western (ICW) assay. We included fulvestrant and ARV-471 as control compounds in our in vitro experiments. In our summarized data table, we considered the maximum % degradation (D_max) of fulvestrant as 100%. Values reported are the mean ± SE of three experiments.

^bThe concentration needed to reduce ERα protein by 50%.

^cMaximal ERα degradation relative to that (100%) achieved by fulvestrant; Std. Error = SE, NA = Not applicable.

ABBREVIATIONS

ERα

estrogen receptor alpha

ET

endocrine therapy

AI

aromatase inhibitor

SERM

selective estrogen receptor modulator

SERD

selective estrogen receptor degrader

UPS

ubiquitin-proteasome system

CDK4/6

cyclin-dependent kinase 4 and 6

ESR1

the gene encoding ERα

LBD

ligand binding domain

PFS

progression-free survival

PROTAC

proteolysistargeting chimera

CRBN

cereblon

P _app

apparent permeability coefficient

IV

intravenous administration

T _1/2

elimination half-life

AUC

area-under-the-curve

V _ss

volume of distribution at steady state

Cl

clearance

PO

oral administration

C _max

maximum drug concentration

F

oral bioavailability

ADME

absorption, distribution, metabolism, and excretion

ICW

incell western

DC₅₀

the concentration needed to reduce protein by 50%

D _max

maximal ERα degradation

PK

pharmacokinetic

DMPK

drug metabolism and pharmacokinetics

hERG

human Ether-à-go-go-Related Gene

CYP

Cytochromes P450

PD

pharmacodynamic