Discovery of IAP-Recruiting BCL-X_L PROTACs as Potent Degraders across Multiple Cancer Cell Lines

Abstract

Targeting BCL-X_L via PROTACs is a promising strategy in reducing BCL-X_L inhibition associated platelet toxicity. Recently, we reported potent BCL-X_L PROTAC degraders that recruit VHL or CRBN E3 ligase. However, low protein expression or mutation of the responsible E3 ligase has been known to result in decreased protein degradation efficiency of the corresponding PROTACs. To overcome these mechanisms of resistance, PROTACs based on recruiting alternative E3 ligases could be generated. Thus, we designed and synthesized a series of PROTACs that recruit IAP E3 ligases for BCL-X_L degradation. Among those PROTACs, compound 8a efficiently degrades BCL-X_L in malignant T-cell lymphoma cell line MyLa 1929 while CRBN-based PROTACs that have high potency in other cancer cell lines show compromised potency, likely due to the low CRBN expression. Moreover, compared with the parent compound ABT-263, PROTAC 8a shows comparable cell killing effects in MyLa 1929 cells whereas the on-target platelet toxicity is significantly reduced. Our findings expand the anti-tumor spectra of BCL-X_L degraders and further highlight the importance of selecting suitable E3 members to achieve effective cellular activity.

Graphical Abstract

1. Introduction

Apoptosis is an essential biological process for the maintenance of tissue homeostasis, normal development, and clearance of abnormal cells [1]. The ability of malignant cells to evade apoptosis is considered a hallmark of cancer and contributes to cancer drug resistance [2]. Thus, targeting apoptotic pathways is a valuable therapeutic strategy for cancer. BCL-2 family proteins play a crucial role in the mitochondria-mediated apoptotic pathway. Several anti-apoptotic members including BCL-2, BCL-X_L, and MCL-1 are well-validated anticancer targets, of which many small-molecule inhibitors have been developed [3]. Among these inhibitors, venetoclax, a selective BCL-2 antagonist, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of a number of hematological malignancies [4]. Compared with BCL-2, BCL-X_L might be a more attractive cancer target with broader potential applications since it is the most common BCL-2 family member overexpressed in solid tumors, as well as in a fraction of leukemia and lymphoma cells [5]. A strong correlation between the levels of BCL-X_L expression and resistance to chemotherapies has been confirmed using bioinformatics analyses [6]. However, BCL-X_L inhibition results in severe thrombocytopenia which limits the clinical applications of BCL-X_L inhibitors [7]. This is because BCL- X_L is required for the viability of platelets [8, 9]. BCL-X_L inhibitors have been converted to phosphate prodrugs, antibody-drug conjugates (ADCs), or novel drug delivery conjugates in order to minimize drug exposure to platelets and thus improve the therapeutic window [10–12].

Recently, we have demonstrated that proteolysis-targeting chimera (PROTAC) technology is a valid strategy to alleviate the on-target toxicity associated with BCL-X_L inhibition because platelets have low levels of E3 ligases [13–16], based on the concept that E3 ligase expression level correlates with PROTAC’s potency in inducing protein degradation. Those PROTAC BCL-X_L degraders were based on von Hippel–Lindau (VHL) cullin-2 or cereblon (CRBN) cullin-4A RING E3 ligase. It is well established that the E3 ubiquitin ligase that induces the most potent degradation is different for each target protein [17–20], highlighting the importance of exploring multiple E3 ligases/ligands for a protein target of interest. In addition, PROTACs may have limited application in tissues with low expression levels of the E3 ligase they recruit. Further, acquired resistance to PROTACs has been observed for both CRBN- and VHL-based PROTACs after chronic treatment [21]. Therefore, to further expand the anti-tumor spectra of BCL-X_L degraders, we investigated the possibility of recruiting inhibitor of apoptosis (IAP) E3 ligases for the degradation of BCL-X_L.

IAP proteins are negative regulators of caspases and cell death characterized by the presence of one to three conserved baculoviral IAP repeat (BIR) domains. Eight IAPs in humans have been identified, including NAIP (BIRC1), cIAP1 (BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), Survivin (BIRC5), BRUCE (BIRC6), ML-IAP (BIRC7), and ILP2 (BIRC8). The second mitochondria-derived activator of caspase (SMAC) is one of the well-characterized endogenous IAP antagonists that interacts with IAPs and prevents their activities. Overexpression of IAPs or loss of endogenous antagonists has been observed in multiple human malignancies and can contribute to tumor progression, drug resistance, and poor prognosis. Therefore, SMAC mimetics have been developed as potent IAP antagonists and some are under clinical evaluation as anticancer drugs (Figure 1) [22–25]. IAP-based PROTACs (also known as SNIPERs---specific and non-genetic IAP-dependent protein erasers) have been developed to target estrogen/androgen receptor, BCR–ABL, CRABP-II, RARα, TACC3, BRD4, PDE4, Notch1, and huntingtin protein for degradation [26–28]. XIAP and cIAP1 are believed to be the major IAPs utilized by IAP-based PROTACs [29]. However, they may not contribute equally in PROTAC-mediated protein degradation. For example, SNIPER(ER)-87, an IAP-based ERα degrader, preferentially recruits XIAP to ERα despite its better binding affinity against cIAP1 [28]. Bioinformatic data from the Human Protein Atlas reveals that XIAP is expressed at different levels in various cell and tissue types [29], which could be utilized to achieve tissue-specific silencing of disease-causing proteins. Importantly, we found that IAP E3 ligases are also low in human platelets. Herein, we report the rational design and synthesis of IAP-based BCL-X_L degraders. The linkerology and the E3 binding moiety were investigated and the biological profile of the designed PROATCs was systematically characterized.

Figure 1.

Chemical structures of representative IAPs antagonists

2. Results and discussion

2.1. Evaluation of antiproliferative activity and BCL-X_L degradation in MyLa 1929 cells

We continued the use of ABT-263 as the BCL-X_L targeting warhead and piperazine as the linker tethering moiety (Table 1). IAP inhibitor LCL161 was used as the E3 ligase binding moiety (Figure 1, Table 1). It has been reported that XIAP has a pronounced effect on SNIPER-induced proteasomal degradation and ligand’s binding affinity to XIAP positively correlates to the degradation efficiency [27]. Thus, we also synthesized BCL-X_L PROTACs based on IAP antagonist 1 (Figure 1, Table 1), which has higher XIAP binding affinity than LCL161 [30]. Compound 1 was converted to a ‘PROTAC-ready’ precursor 2 that contains a phenolic OH for linker attachment (Figure 1). The linker tethering position on 1 was determined based on the proposed binding mode [30].

Table 1.

BCL-X_L degraders with various linkers tethering ABT-263 to IAP ligands

Compd	E3 Ligand	IC50 (nM) MyLa 1929	Compd	E3 Ligand	IC50 (nM) MyLa 1929	Linker
ABT-263	-	50	1	-	>10,000	-
3a	L1	157	6a	L2	153
3b	L1	147	6b	L2	119
4a	L1	143	7a	L2	100
4b	L1	75	7b	L2	70
5a	L1	997	8a	L2	62
5b	L1	1959	8b	L2	76
5c	L1	>10,000	8c	L2	127
5d	L1	>10,000	8d	L2	274

^aIC₅₀ values are the means of at least two independent experiments; reduction of MyLa 1929 cell viability after 72 h treatment.

We first tested the effects of the designed PROTACs on the viability of MyLa 1929, a cutaneous T-cell lymphoma (CTCL) cell line sensitive to BCL-X_L inhibition, with ABT-263 as the positive control (Table 1), followed by evaluating their ability of inducing BCL-X_L degradation in the same cells using Western blotting (Figure 2). Compounds 3a-b, which are based on LCL161 and contain a 1,2,3-triazole group and a polyethylene glycol (PEG) chain, showed equal and robust cell killing effects against MyLa 1929 cells despite the different linker length. Removal of the alkyl triazole portion of the linker in 3a, which results in 4a, had no effect on the cytotoxicity in MyLa 1929 cells. However, extension of the linker in 4a by adding PEG units resulted in a one-fold improvement in cellular activity (4a vs 4b). In the series with linkers containing an alkane chain, we synthesized compounds 5a-d with 3–6 methylene groups in the linker. However, these PROTACs displayed little or no cell killing effects in MyLa 1929 cells (Table 1). The protein degradation data (Figure 2) matched well with the cytotoxicity data. The poor cellular activity of 5c and 5d can be attributed to the lack of BCL-X_L degradation. The most potent PROTAC 4b in this series displayed the highest degree of BCL-X_L degradation.

Figure 2.

Western blot analysis of the effect of IAP-based degraders on BCL-X_L protein levels in MyLa 1929 cells. Cells were treated with 500 nM of the indicated compound for 16 h before harvesting. Degradation activity is reported as % of total protein remaining after compound treatment relative to vehicle treatment after normalization with β-actin by Image J software quantification.

To improve the activity of the PROTACs, we synthesized another series of analogs by replacing IAP ligand LCL161 with compound 1. Compounds 6a, 6b, 7a, and 7b exhibited comparable cytotoxicity to their corresponding LCL161-based analogs 3a, 3b, 4a, and 4b, respectively. However, PROTACs 8a-d with alkane linkers exhibited largely improved cytotoxicity when compared to their corresponding LCL161-based counterparts 5a-d. In addition, ligand 1-based BCL-X_L PROTACs had higher potency in degrading BCL-X_L compared with PROATCs that are based on LCL161. The antiproliferative effect of those PROTACs on MyLa 1929 cell viability was not mediated by the IAP inhibition because IAP ligand 1 did not affect the cell survival. PROTAC 8a with the shortest alkane linker was one of the most potent BCL-X_L degraders and the most potent compound in killing MyLa 1929 cells among all IAP-based BCL-X_L PROATCs. Thus, we selected 8a for further investigation.

2.2. Mechanism of action studies

The catalytic mechanism of PROTACs relies on the cellular availability of the bespoke E3 ligase they recruit. Proteomics data from the Human Protein Atlas revealed that CRBN is widely expressed and present in 76% of the tested cell and tissue types [29]. We previously developed several potent CRBN-based BCL-X_L degraders that can efficiently degrade BCL-X_L and kill various cancer cells [14–16]. PROTAC 8a has similar cell killing effect to XZ424 but much less cytotoxic than XZ739 in MOLT-4 cells (Figure 3A). However, in MyLa 1929 cells, compromised cytotoxicity was observed for the CRBN-based BCL-X_L degraders XZ424 and XZ739. Interestingly, XZ424 exhibited a minimal effect on BCL-X_L degradation while 8a potently and dose-dependently induced BCL-X_L degradation in MyLa 1929 cells (Figure 3B). Western blot analysis revealed that CRBN expression level is relatively low in MyLa 1929 cells than in MOLT-4 T-ALL and RS4;11 B-ALL cells (Figure 3C). In contrast, XIAP is highly expressed across all three cell lines. BCL-X_L degradation induced by 8a in MyLa 1929 was observed after 8 h drug treatment, and most of the protein was degraded with 500 nM of 8a after 16 h drug exposure (Figure 3D). Further, 8a-induced BCL-X_L degradation can be abrogated by proteasome inhibitor MG-132 (Figure 3E), excess competitive IAP ligand 1 (Figure 3F), or BCL-X_L inhibitor ABT-263 (Figure 3G), indicating that the degradation depends on proteasome activity and engaging BCL-X_L and IAP E3 ligase.

Figure 3.

PROTAC-induced BCL-X_L degradation. (A) Antiproliferative activities for 8a and representative CRBN-based PROTACs in MyLa 1929 and MOLT-4 cells. (B) Western blots showing the BCL-X_L protein levels in MyLa 1929 cells treated with the indicated concentrations of 8a or XZ424 for 16 h. (C) CRBN and IAPs expression in MyLa 1929 cells in comparison to other cancer cell lines. (D) BCL-X_L protein levels in MyLa 1929 cells treated with 500 nM of 8a at the indicated time points. (E-G) Pretreatment with MG-132 (10 μM), IAP ligand 1 (20 μM), or ABT-263 (10 μM) for 2 h blocked 8a-induced BCL-X_L degradation.

2.3. PROTAC-induced degradation in various cancer cell lines

BCL-X_L is overexpressed in various human malignancies including breast cancer, colorectal cancer, melanoma, and lung cancer. Bioinformatics analyses also reveal a strong correlation between the levels of BCL-X_L expression and drug resistance to chemotherapies [6]. To characterize the BCL-X_L degradation ability of 8a, we selected six cancer cell lines including A549 (human non-small cell lung cancer), MDA-MB-231 (triple-negative breast cancer), SW620 (colorectal cancer cell ), MeWo (melanoma), SK-MEL-28 (melanoma), and CHL-1 (melanoma) cell lines for further exploration. As shown in Figure 4, 8a was able to induce remarkable BCL-X_L degradation in a dose-dependent manner across all tested cell lines, suggesting the broad application across cancer types. We further examined the correlation between BCL-X_L degradation and XIAP expression (Figure S1). Our preliminary analysis suggested that there is a correlation between the levels of XIAP expression and sensitivity of cells to PROTAC-induced BCL-X_L degradation. For example, cells with lower XIAP expressing, such as MDA-MB-231 cells, were less sensitive to PROTAC-induced BCL-X_L degradation compared to A549 cells that express a higher level of XIAP. However, additional studies will be needed to further investigate the role of different IAP proteins in mediating PROTAC-induced BCL-X_L degradation in various cancer cell lines in our future studies.

Figure 4.

Effects on BCL-X_L degradation in cancer cell lines treated with the indicated concentrations of 8a for 16 h.

2.4. Platelet and normal cell toxicity

Both CRBN- and VHL-based BCL-X_L degraders showed decreased platelet toxicity when compared with ABT-263 because both E3 ligases are poorly expressed in human platelets [13–15]. The toxicity for 8a against human platelets was tested and the IC₅₀ ratio between platelets and MyLa 1929 cells was calculated to evaluate the selectivity. As shown in Figure 5A, ABT-263 has poor selectivity for MyLa 1929 cells over platelets, while 8a achieved high selectivity. Western blot analysis confirmed that BCL-X_L in human platelets was not degraded by PROTAC treatment (Figure 5B), which is likely due to the low expression levels of IAPs in human platelets (Figure 5C). In addition, 8a induced BCL-X_L degradation in WI-38 lung fibroblast cells but had no observed toxicity against these cells (Figure 5A–B), because normal cells do not depend on BCL-X_L for survival [28].

Figure 5.

Cellular toxicity studies. (A) Platelets and normal WI-38 fibroblasts were cultured with the indicated compound for 48 h and 72 h, respectively. IC₅₀ ratio between human platelets and MyLa 1929 cells was calculated. (B) Western blot analysis of BCL-X_L levels after treatment of human platelets or WI-38 cells with the indicated concentration of 8a for 16 h. (C) Differential E3 expressions in MyLa 1929 cells and human platelets.

2.5. ML-IAP E3 ligase knockout experiments

IAP ligand 1 has a strong binding ability against melanoma IAP (ML-IAP, BIRC7) which is enriched in multiple melanoma cell lines [30]. ML-IAP contains one BIR domain and a really interesting new gene (RING) finger domain which can interact with E2 ubiquitin-conjugating enzymes. We hypothesized that if ML-IAP can be hijacked for PROTAC-induced degradation, the efficacy should be much higher in melanoma cells to achieve cell-type selectivity against melanomas. Thus, we compared the protein degradation ability in wild-type and ML-IAP knockout SK-MEL-28 cells. As shown in Figure 6, ML-IAP is highly expressed in wild-type SK-MEL-28. However, BCL-X_L degradation was not affected when ML-IAP was knocked out, indicating ML-IAP was not involved in 8a-mediated BCL-X_L degradation. In addition, both BCL-2 and MCL-1 levels were not affected after the PROTAC treatment, further confirming the specificity of this IAP-based degrader.

Figure 6.

Effects of ML-IAP on BCL-X_L degradation in wild type or ML-IAP knockout (KO) SK-MEL-28 cells treated with the indicated concentrations of 8a for 16 h.

3. Chemistry

The synthesis of precursor 17 is illustrated in Scheme 1. (R)-6-(Benzyloxy)-1,2,3,4-tetrahydronaphthalen-1-amine (9) was treated with ethyl formate at 80 °C to form formamide 10. Dehydration of 10 in the presence of POCl₃ and triethylamine afforded isocyanide 11. Intermediate 14 was formed through an Ugi four-component reaction that involved acid 12, aldehyde 13, isocyanide 11 and ammonia in methanol. The 7,5-heterobicycle 15 was formed as a mixture of diastereomers by treating 14 with TFA. Coupling of 15 with Boc-N-Me-Ala-OH afforded a mixture of diastereomers 16a and 16b, which was separated by flash column chromatography on silica gel [30]. Removal of the benzyl protective group in 16a via hydrogenolysis at 40 °C afforded compound 17.

Scheme 1.

Reagents and conditions: (a) Ethyl formate, 80 °C; (b) POCl₃, Et₃N, DCM, 0 °C then rt; (c) NH₃, MeOH, rt; (d) TFA, DCM, rt; (e) Boc-N-Me-Ala-OH, HATU, DIPEA, DCM, rt; (f) Pd(OH)₂, H₂, MeOH, 40 °C.

The syntheses of PROTACs are depicted in Scheme 2. ABT-263 derivatives 19a and 19b were synthesized following our recent publication [13] whereas LCL161 derivative 18 was prepared according to the procedure reported in the literature [28]. Coupling of 18 with tosylate 20a-b, which gave 21a-b, followed by a click reaction with azide 19a and de-Boc afforded PROATCs 3a-b. PROTACs 6a-b were synthesized using a similar synthetic approach. Acids 24a-b were prepared through nucleophilic substitution between 18 and tosylate 23a-b followed by hydrolysis of ester in the presence of LiOH. PROTACs 4a-b were obtained via amidation between 24a-b and 19b followed by the removal of the Boc protecting group. Similarly, PROTACs 7a-b, 5a-d, and 8a-d were synthesized starting from the corresponding tosylates or bromides.

Scheme 2.

Reagents and conditions: (a) K₂CO₃, NaI, DMF, 70 °C; (b) CuSO₄·5H₂O, sodium L-ascorbate, ^tBuOH, THF, H₂O, 50 °C; (c) TFA, DCM, rt; (d) LiOH, MeOH, H₂O, rt; (e) HATU, Et₃N, DCM, rt.

4. Conclusions

In summary, we developed and evaluated a series of IAP-based BCL-X_L PROTACs. PROTACs that with IAP antagonist 1 as the E3 ligand appeared to be more potent in inducing BCL-X_L degradation than LCL161-based analogs. Compound 1 possesses excellent binding affinity to ML-IAP; however, ML-IAP is unlikely to be involved in 8a-mediated BCL-X_L degradation based on the knockout experiment. While CRBN-based PROTACs showed limited ability of inducing protein degradation due to the low CRBN expression in MyLa 1929 cells, IAP-based PROTAC 8a efficiently degraded BCL-X_L in a proteasome-dependent manner. Compared to ABT-263, 8a is less toxic to human platelets but equally potent against MyLa 1929 cells, confirming that the therapeutic window can be improved by converting a BCL-X_L inhibitor to an IAP-based BCL-X_L degrader. Moreover, 8a was able to induce powerful BCL-X_L degradation in multiple cancer cell lines, indicating that IAP-based BCL-X_L degraders have the potential of broad applications in cancer treatment. Our findings expand the anti-tumor spectra of BCL-X_L degraders and further highlight the importance of selecting suitable E3 ligases to achieve effective cellular activity.

5. Experimental section

5.1. Chemistry

THF and DCM were obtained via a solvent purification system by filtering through two columns packed with activated alumina and 4 Å molecular sieve, respectively. All other chemicals obtained from commercial sources were used without further purification. Flash chromatography was performed using silica gel (230–400 mesh) as the stationary phase. Reaction progress was monitored by thin-layer chromatography (silica-coated glass plates) and visualized by UV light, and/or by LC-MS. NMR spectra were recorded in CDCl_3, CD₃OD, or DMSO-d₆ at 400 or 600 MHz for ¹H NMR. Chemical shifts δ are given in ppm using tetramethylsilane as an internal standard. Multiplicities of NMR signals are designated as singlet (s), broad singlet (br s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), and multiplet (m). All final compounds for biological testing were of ≥95.0% purity as analyzed by LC-MS, performed on an Advion AVANT LC system with the expression CMS using a Thermo Accucore™ Vanquish™ C18+ UHPLC Column (1.5 µm, 50 x 2.1 mm) at 40 °C. Gradient elution was used for UHPLC with a mobile phase of acetonitrile and water containing 0.1% formic acid.

5.1.1. General Methods.

5.1.1.1. General Method A.

A mixture of phenol (1.0 equiv.), an appropriate bromide or tosylate (2.0 equiv.), K₂CO₃ (2.0 equiv.), and NaI (0.2 equiv.) in DMF was stirred at 70 °C overnight. The mixture was poured into water and extracted with ethylacetate. The combined organic layers were washed with NH₄Cl (aq.) × 1, brine × 1, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by column chromatography to afford the corresponding ester intermediate.

5.1.1.2. General Method B.

To a mixture of azide (1.0 equiv.), alkyne (1.2 equiv.) in ^tBuOH-THF (1:1, v/v) under argon was added CuSO₄·5H₂O (0.2 equiv.) and sodium ascorbate (0.2 equiv.) in water. The mixture was stirred at 50 °C for 3 h and cooled to room temperature before poured into water. The resulting mixture was extracted with DCM. The combined organic layers were washed with brine × 1, dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The residue crude product was purified via flash column chromatography using DCM and MeOH as eluents to afford the desired product.

5.1.1.3. General Method C.

To a solution of Boc-protected amine (1.0 equiv.) in DCM was added TFA (20–30 equiv.). The mixture was stirred at room temperature overnight and the solvent was evaporated under reduced pressure. The residue crude product was washed with Et₂O to afford the desired product.

5.1.1.4. General Method D.

To a solution of ester (1.0 equiv.) in MeOH and water was added LiOH monohydrate (5.0 equiv.). The mixture was stirred at room temperature for 2 h and quenched with 1 N HCl (5.0 equiv.). The solvent was removed under reduced pressure and the residue was used directly in the next step.

5.1.1.5. General Method E.

A mixture of acid (1.0 equiv.), amine (1.0 equiv.), HATU (1.05 equiv.) and Et₃N (5.0 equiv.) in DCM was stirred at room temperature for 1 h. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with NH₄Cl (aq.) × 1, brine × 1, dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum. The residue crude product was purified by flash column chromatography to afford the desired compound.

5.1.2.1. (R)-N-(6-(Benzyloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)formamide (10)

A mixture of compound 9 (200 mg, 0.79 mmol) and ethyl formate (75 µL, 0.93 mmol) was heated at 80 °C for 16 h. The reaction mixture was cooled to room temperature and Et₂O was added. The resulting suspension was filtered and the solid was collected to afford the title compound (187 mg, 84% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H), 7.47 – 7.28 (m, 5H), 7.25 – 7.15 (m, 1H), 6.89 – 6.77 (m, 1H), 6.76 – 6.64 (m, 1H), 5.76 – 5.60 (m, 1H), 5.29 – 5.17 (m, 1H), 5.04 (s, 2H), 2.91 – 2.66 (m, 2H), 2.17 – 1.97 (m, 1H), 1.92 – 1.75 (m, 3H). LC-MS (ESI): m/z 282.2 [M+H] ⁺.

5.1.2.2. (R)-6-(Benzyloxy)-1-isocyano-1,2,3,4-tetrahydronaphthalene (11)

To a solution of compound 10 (180 mg, 0.64 mmol) and Et₃N (500 µL, 3.6 mmol) in DCM (2 mL) at 0 °C was added phosphorus oxychloride (100 µL, 1.07 mmol). The mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was poured into a saturated NaHCO₃ aqueous solution and extracted with DCM. The organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel to afford the title compound (150 mg, 89% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46 – 7.29 (m, 6H), 6.86 (dd, J = 8.6, 2.7 Hz, 1H), 6.72 (d, J = 2.7 Hz, 1H), 5.05 (s, 2H), 4.83 – 4.73 (m, 1H), 2.89 – 2.78 (m, 1H), 2.78 – 2.66 (m, 1H), 2.21 – 1.97 (m, 3H), 1.88 – 1.75 (m, 1H). LC-MS (ESI): m/z 237.2 [M+H-HNC] ⁺.

5.1.2.3. (4S,9aS)-4-Amino-N-((R)-6-(benzyloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide trifluoroacetate (15)

A mixture of carboxylic acid 12 (52 mg, 0.24 mmol), aldehyde 13 (38 mg, 0.24 mmol), benzyl isocyanide 11 (62 mg, 0.24 mmol) and 7 M ammonia in MeOH (50 μL, 0.35 mmol) in MeOH (1 mL) was stirred at room temperature for 16 h. The mixture was diluted with water and extracted with DCM. The organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was dissolved in DCM (2 mL) and combined with TFA (180 μL, 2.35 mmol) and stirred at room temperature for 16 h. The mixture was concentrated in vacuo and the resulting crude product was purified by flash chromatography on silica gel to afford the title compound as a diastereomixture (105 mg, 74% yield over 2 steps).

5.1.2.4. tert-Butyl ((S)-1-(((4S,7S,9aS)-7-(((R)-6-(benzyloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)-8,8-dimethyl-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepin-4-yl)amino)-1-oxopropan-2-yl)(methyl)carbamate (16a) and tert-butyl ((S)-1-(((4S,7R,9aS)-7-(((R)-6-(benzyloxy)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)-8,8-dimethyl-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepin-4-yl)amino)-1-oxopropan-2-yl)(methyl)carbamate (16b)

A mixture of Boc-N-Me-Ala-OH (36.5 mg, 0.18 mmol), amine 15 (82 mg, 0.14 mmol), HATU (98 mg, 0.26 mmol) and DIPEA (92 µL, 0.56 mmol) in DCM (5 mL) was stirred at room temperature for 1 h. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum. The crude product was purified by flash column chromatography to afford compounds 16a (45 mg, 38% yield) and 16b (25 mg, 21% yield). ¹H NMR and LC-MSs data for compound 16a. ¹H NMR (600 MHz, DMSO-d₆) δ 7.77 (d, J = 6.7 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.45 – 7.35 (m, 4H), 7.35 – 7.29 (m, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.81 – 6.74 (m, 2H), 5.50 – 5.42 (m, 1H), 5.07 (s, 2H), 4.87 – 4.80 (m, 1H), 4.79 – 4.73 (m, 1H), 4.70 – 4.43 (m, 1H), 3.98 (s, 1H), 3.93 – 3.87 (m, 1H), 3.85 – 3.79 (m, 1H), 2.77 – 2.61 (m, 5H), 2.11 (dd, J = 13.1, 6.5 Hz, 1H), 1.88 – 1.65 (m, 6H), 1.59 – 1.50 (m, 1H), 1.40 (s, 9H), 1.26 (d, J = 7.2 Hz, 3H), 1.02 (s, 3H), 1.01 (s, 3H). LC-MS (ESI): m/z 663.5 [M+H] ⁺. ¹H NMR and LC-MSs data for compound 16b. ¹H NMR (600 MHz, DMSO-d₆) δ 8.32 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 6.4 Hz, 1H), 7.44 – 7.38 (m, 4H), 7.35 – 7.30 (m, 1H), 7.10 (d, J = 9.0 Hz, 1H), 6.82 – 6.71 (m, 2H), 5.44 (d, J = 7.1 Hz, 1H), 5.09 (s, 2H), 4.88 – 4.80 (m, 1H), 4.77 – 4.70 (m, 1H), 4.69 – 4.48 (m, 1H), 4.12 (s, 1H), 4.09 – 4.01 (m, 1H), 3.94 – 3.86 (m, 1H), 2.79 – 2.63 (m, 5H), 2.17 (dd, J = 13.4, 7.0 Hz, 1H), 1.87 – 1.56 (m, 7H), 1.41 (s, 9H), 1.28 (d, J = 7.3 Hz, 3H), 1.21 (s, 3H), 0.99 (s, 3H). LC-MS (ESI): m/z 663.4 [M+H] ⁺.

5.1.2.5. tert-Butyl ((S)-1-(((4S,7S,9aS)-7-(((R)-6-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)-8,8-dimethyl-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepin-4-yl)amino)-1-oxopropan-2-yl)(methyl)carbamate (17)

A mixture of compound 16 (270 mg, 0.41 mmol) and 10% Pd(OH)₂/C (10% w/w, 20 mg) in methanol (10 mL) was stirred at 40 °C for 2 h. After solid was removed by filtration, the filtrate was evaporated to dryness to afford the title compound (233 mg, 100% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 9.21 (s, 1H), 7.78 (d, J = 6.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.57 – 6.51 (m, 1H), 6.48 (d, J = 2.5 Hz, 1H), 5.47 (t, J = 6.4 Hz, 1H), 4.84 – 4.73 (m, 2H), 4.71 – 4.38 (m, 1H), 4.00 – 3.90 (m, 2H), 3.87 – 3.79 (m, 1H), 2.74 (s, 3H), 2.68 – 2.63 (m, 1H), 2.63 – 2.56 (m, 1H), 2.11 (dd, J = 13.0, 6.5 Hz, 1H), 1.88 – 1.64 (m, 6H), 1.62 – 1.51 (m, 1H), 1.41 (s, 9H), 1.26 (d, J = 7.2 Hz, 3H), 1.02 (s, 3H), 1.01 (s, 3H). LC-MS (ESI): m/z 573.4 [M+H] ⁺.

5.1.2.6. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(4-(4-((2-(2-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (3a)

Following general methods A, B and C, compound 3a was obtained from 20a, 18 and 19a (5.7 mg, 37% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.35 (d, J = 2.2 Hz, 1H), 8.10 (s, 1H), 8.06 – 8.00 (m, 1H), 7.87 – 7.81 (m, 2H), 7.80 – 7.63 (m, 3H), 7.59 (s, 1H), 7.40 – 7.34 (m, 3H), 7.33 – 7.27 (m, 3H), 7.26 – 7.22 (m, 2H), 7.15 (dd, J = 8.2, 2.6 Hz, 1H), 7.01 – 6.96 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 9.3 Hz, 1H), 5.55 (dd, J = 8.0, 2.7 Hz, 1H), 4.69 (s, 2H), 4.67 – 4.61 (m, 1H), 4.41 (t, J = 6.5 Hz, 2H), 4.22 – 4.17 (m, 2H), 3.90 – 3.17 (m, 19H), 2.93 (dd, J = 13.7, 7.4 Hz, 1H), 2.79 (s, 2H), 2.48 – 1.98 (m, 26H), 1.76 – 1.58 (m, 7H), 1.46 (t, J = 6.5 Hz, 2H), 1.36 – 1.13 (m, 8H), 0.98 (s, 6H). LC-MS (ESI): m/z 1708.9 [M+H] ⁺.

5.1.2.7. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(4-(4-((2-(2-(2-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)ethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)butanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (3b)

Following general methods A, B and C, compound 3b was obtained from 20b, 18 and 19a (5.4 mg, 42% yield). ¹H NMR (400 MHz, CDCl₃ and CD₃OD) δ 8.33 (d, J = 2.2 Hz, 1H), 8.14 – 8.07 (m, 2H), 7.78 – 7.71 (m, 3H), 7.69 – 7.62 (m, 2H), 7.44 – 7.35 (m, 3H), 7.34 – 7.30 (m, 2H), 7.29 – 7.23 (m, 3H), 7.16 (dd, J = 8.0, 2.8 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.02 – 6.97 (m, 2H), 6.78 (d, J = 8.7 Hz, 2H), 6.65 – 6.53 (m, 1H), 5.53 (dd, J = 7.9, 2.9 Hz, 1H), 4.66 (s, 2H), 4.59 (d, J = 6.4 Hz, 1H), 4.42 (t, J = 6.6 Hz, 2H), 4.24 – 4.16 (m, 2H), 3.92 – 3.33 (m, 22H), 3.15 – 2.99 (m, 4H), 2.66 – 2.07 (m, 26H), 1.81 – 1.59 (m, 7H), 1.50 – 1.03 (m, 10H), 1.00 (s, 6H). LC-MS (ESI): m/z 1752.8 [M+H] ⁺.

5.1.2.8. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(2-(2-(2-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)ethoxy)ethoxy)acetyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (4a)

Following general methods A, D, E and C, compound 4a was obtained from 23a, 18 and 19b (9.0 mg, 33% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.35 (s, 1H), 8.10 – 7.99 (m, 2H), 7.85 – 7.71 (m, 4H), 7.68 (s, 1H), 7.41 – 7.29 (m, 6H), 7.28 – 7.23 (m, 2H), 7.18 – 7.14 (m, 1H), 7.07 – 6.98 (m, 3H), 6.75 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 9.6 Hz, 1H), 5.60 – 5.48 (m, 1H), 4.70 – 4.63 (m, 1H), 4.25 – 4.18 (m, 4H), 3.92 – 3.61 (m, 10H), 3.48 – 3.20 (m, 8H), 3.10 (dd, J = 13.7, 4.8 Hz, 1H), 2.98 (dd, J = 13.6, 7.4 Hz, 1H), 2.82 (s, 2H), 2.50 – 2.00 (m, 22H), 1.75 – 1.61 (m, 7H), 1.48 (t, J = 6.5 Hz, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.26 – 1.09 (m, 5H), 1.00 (s, 6H). LC-MS (ESI): m/z 1600.0 [M+H] ⁺.

5.1.2.9. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(14-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)-3,6,9,12-tetraoxatetradecanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (4b)

Following general methods A, D, E and C, compound 4b was obtained from 23b, 18 and 19b (11.0 mg, 28% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.37 (d, J = 2.3 Hz, 1H), 8.12 – 8.04 (m, 2H), 7.83 – 7.67 (m, 5H), 7.40 – 7.34 (m, 3H), 7.33 – 7.29 (m, 3H), 7.28 – 7.23 (m, 2H), 7.15 (dd, J = 8.3, 2.6 Hz, 1H), 7.03 – 6.98 (m, 3H), 6.78 – 6.73 (m, 2H), 6.55 (d, J = 9.3 Hz, 1H), 5.56 (dd, J = 8.1, 2.8 Hz, 1H), 4.66 (dd, J = 9.2, 6.3 Hz, 1H), 4.22 – 4.15 (m, 4H), 3.91 – 3.61 (m, 18H), 3.47 – 3.23 (m, 8H), 3.11 (dd, J = 13.8, 4.8 Hz, 1H), 2.98 (dd, J = 13.7, 7.6 Hz, 1H), 2.90 – 2.80 (m, 2H), 2.50 – 2.01 (m, 22H), 1.79 – 1.61 (m, 7H), 1.47 (t, J = 6.5 Hz, 2H), 1.36 – 0.98 (m, 14H). LC-MS (ESI): m/z 1688.2 [M+H] ⁺.

5.1.2.10. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(4-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)butanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (5a)

Following general methods A, D, E and C, compound 5a was obtained from 26a, 18 and 19b (17.0 mg, 25% yield). ¹H NMR (600 MHz, CDCl₃ and CD₃OD) δ 8.37 (d, J = 2.4 Hz, 1H), 8.11 (s, 1H), 8.08 – 8.04 (m, 1H), 7.80 – 7.68 (m, 5H), 7.42 – 7.37 (m, 3H), 7.35 – 7.29 (m, 3H), 7.28 – 7.25 (m, 2H), 7.16 – 7.12 (m, 1H), 7.05 (d, J = 8.6 Hz, 1H), 7.02 – 6.98 (m, 2H), 6.80 – 6.74 (m, 2H), 6.59 (d, J = 9.4 Hz, 1H), 5.56 (dd, J = 8.1, 2.8 Hz, 1H), 4.67 (dd, J = 9.1, 6.3 Hz, 1H), 4.10 (t, J = 5.9 Hz, 2H), 3.97 – 3.85 (m, 2H), 3.84 – 3.79 (m, 1H), 3.77 – 3.69 (m, 1H), 3.50 – 3.23 (m, 8H), 3.12 (dd, J = 13.9, 5.0 Hz, 1H), 3.00 (dd, J = 13.9, 7.4 Hz, 1H), 2.83 (s, 2H), 2.57 – 2.02 (m, 26H), 1.81 – 1.60 (m, 7H), 1.48 (t, J = 6.5 Hz, 2H), 1.38 (d, J = 6.9 Hz, 3H), 1.22 – 1.01 (m, 5H), 1.00 (s, 6H). LC-MS (ESI): m/z 1540.1 [M+H] ⁺.

5.1.2.11. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(5-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)pentanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (5b)

Following general methods A, D, E and C, compound 5b was obtained from 26b, 18 and 19b (19.0 mg, 31% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.39 (d, J = 2.3 Hz, 1H), 8.14 – 8.09 (m, 2H), 7.78 – 7.66 (m, 5H), 7.42 – 7.36 (m, 3H), 7.35 – 7.29 (m, 5H), 7.15 – 7.10 (m, 2H), 7.03 – 6.98 (m, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 9.4 Hz, 1H), 5.56 (dd, J = 8.1, 2.6 Hz, 1H), 4.66 (dd, J = 9.2, 6.4 Hz, 1H), 4.10 – 4.04 (m, 2H), 3.99 – 3.90 (m, 2H), 3.87 – 3.79 (m, 1H), 3.75 – 3.66 (m, 1H), 3.50 – 3.16 (m, 8H), 3.13 (dd, J = 13.9, 5.0 Hz, 1H), 3.03 (dd, J = 13.9, 7.4 Hz, 1H), 2.81 (s, 2H), 2.50 – 2.02 (m, 26H), 1.92 – 1.71 (m, 9H), 1.48 (t, J = 6.5 Hz, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.26 – 1.06 (m, 5H), 1.01 (s, 6H). LC-MS (ESI): m/z 1554.1 [M+H] ⁺.

5.1.2.12. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(6-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)hexanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (5c)

Following general methods A, D, E and C, compound 5c was obtained from 26c, 18 and 19b (11.0 mg, 45% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.34 (d, J = 2.3 Hz, 1H), 8.12 – 8.06 (m, 2H), 7.79 – 7.66 (m, 4H), 7.41 – 7.35 (m, 3H), 7.34 – 7.29 (m, 2H), 7.28 – 7.24 (m, 3H), 7.15 – 7.11 (m, 1H), 7.03 (d, J = 8.5 Hz, 1H), 7.01 – 6.97 (m, 2H), 6.80 – 6.74 (m, 2H), 6.59 (d, J = 9.3 Hz, 1H), 5.55 (dd, J = 8.1, 2.8 Hz, 1H), 4.67 – 4.61 (m, 1H), 4.03 (t, J = 6.3 Hz, 2H), 3.96 – 3.60 (m, 5H), 3.47 – 3.22 (m, 8H), 3.12 (dd, J = 13.9, 5.0 Hz, 1H), 3.00 (dd, J = 13.8, 7.4 Hz, 1H), 2.83 (s, 2H), 2.50 – 2.00 (m, 26H), 1.87 – 1.51 (m, 11H), 1.47 (t, J = 6.5 Hz, 2H), 1.39 (d, J = 6.9 Hz, 3H), 1.20 – 1.01 (m, 5H), 0.99 (s, 6H). LC-MS (ESI): m/z 1568.1 [M+H] ⁺.

5.1.2.13. 4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((R)-4-(4-(7-(3-(2-((S)-1-((R)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)pyrrolidin-2-yl)thiazole-4-carbonyl)phenoxy)heptanoyl)piperazin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide (5d)

Following general methods A, D, E and C, compound 5d was obtained from 26d, 18 and 19b (21.0 mg, 37% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.37 (d, J = 2.3 Hz, 1H), 8.13 – 8.08 (m, 2H), 7.78 – 7.68 (m, 5H), 7.42 – 7.36 (m, 3H), 7.35 – 7.29 (m, 3H), 7.28 – 7.26 (m, 2H), 7.16 – 7.12 (m, 1H), 7.07 (d, J = 8.5 Hz, 1H), 7.03 – 6.99 (m, 2H), 6.77 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 9.4 Hz, 1H), 5.63 – 5.52 (m, 1H), 4.67 (dd, J = 9.1, 6.4 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.97 – 3.70 (m, 4H), 3.50 – 3.24 (m, 8H), 3.12 (dd, J = 13.8, 4.9 Hz, 1H), 3.01 (dd, J = 13.8, 7.4 Hz, 1H), 2.83 (s, 2H), 2.48 – 2.03 (m, 26H), 1.86 – 1.40 (m, 15H), 1.37 (d, J = 6.9 Hz, 3H), 1.23 – 1.03 (m, 5H), 1.00 (s, 6H). LC-MS (ESI): m/z 1582.1 [M+H] ⁺.

5.1.2.14. (4S,7S,9aS)-N-((R)-6-(2-(2-((1-(4-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (6a)

Following general methods A, B and C, compound 6a was obtained from 20a, 17 and 19a (7.2 mg, 38% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.34 (d, J = 2.2 Hz, 1H), 8.13 – 8.01 (m, 2H), 7.76 (d, J = 8.6 Hz, 2H), 7.58 (s, 1H), 7.40 – 7.34 (m, 2H), 7.33 – 7.27 (m, 3H), 7.26 – 7.21 (m, 2H), 7.07 – 6.96 (m, 4H), 6.82 (d, J = 8.3 Hz, 1H), 6.74 (d, J = 8.7 Hz, 2H), 6.68 (dd, J = 8.6, 2.6 Hz, 1H), 6.62 – 6.56 (m, 2H), 5.29 – 5.22 (m, 1H), 5.12 – 4.98 (m, 1H), 4.80 – 4.72 (m, 1H), 4.67 (s, 2H), 4.41 (t, J = 6.4 Hz, 2H), 4.20 (s, 1H), 4.10 – 4.00 (m, 2H), 3.95 – 3.77 (m, 5H), 3.75 – 3.60 (m, 5H), 3.42 – 3.20 (m, 8H), 3.10 (dd, J = 13.8, 4.9 Hz, 1H), 2.98 (dd, J = 13.7, 7.2 Hz, 1H), 2.81 (s, 2H), 2.74 – 2.67 (m, 2H), 2.53 – 2.16 (m, 21H), 2.12 – 1.87 (m, 5H), 1.84 – 1.72 (m, 4H), 1.70 – 1.62 (m, 1H), 1.46 (t, J = 6.5 Hz, 2H), 1.35 (d, J = 6.9 Hz, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.98 (s, 6H). LC-MS (ESI): m/z 1683.3 [M+H] ⁺.

5.1.2.15. (4S,7S,9aS)-N-((R)-6-(2-(2-(2-((1-(4-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (6b)

Following general methods A, B and C, compound 6b was obtained from 20b, 17 and 19a (13.8 mg, 45% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.34 (d, J = 2.2 Hz, 1H), 8.14 – 8.03 (m, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.60 (s, 1H), 7.39 – 7.27 (m, 5H), 7.26 – 7.22 (m, 2H), 7.07 – 6.96 (m, 4H), 6.82 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.67 (dd, J = 8.5, 2.7 Hz, 1H), 6.62 – 6.56 (m, 2H), 5.30 – 5.22 (m, 1H), 5.09 – 5.00 (m, 1H), 4.79 – 4.71 (m, 1H), 4.67 (s, 2H), 4.41 (t, J = 6.4 Hz, 2H), 4.20 (s, 1H), 4.08 – 4.01 (m, 2H), 3.92 – 3.77 (m, 5H), 3.73 – 3.62 (m, 9H), 3.42 – 3.18 (m, 8H), 3.11 (dd, J = 13.8, 5.0 Hz, 1H), 2.99 (dd, J = 13.8, 7.3 Hz, 1H), 2.81 (s, 2H), 2.71 – 2.67 (m, 2H), 2.52 – 2.18 (m, 21H), 2.10 – 1.89 (m, 5H), 1.83 – 1.72 (m, 4H), 1.70 – 1.59 (m, 1H), 1.46 (t, J = 6.4 Hz, 2H), 1.35 (d, J = 6.9 Hz, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.98 (s, 6H). LC-MS (ESI): m/z 1727.4 [M+H] ⁺.

5.1.2.16. (4S,7S,9aS)-N-((R)-6-(2-(2-(2-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-2-oxoethoxy)ethoxy)ethoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (7a)

Following general methods A, D, E and C, compound 7a was obtained from 23a, 17 and 19b (7.4 mg, 37% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.35 – 8.28 (m, 1H), 8.06 – 7.97 (m, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.37 – 7.25 (m, 5H), 7.23 – 7.19 (m, 2H), 7.04 – 6.93 (m, 4H), 6.81 – 6.66 (m, 4H), 6.60 – 6.54 (m, 2H), 5.27 – 5.20 (m, 1H), 5.06 – 4.96 (m, 1H), 4.78 – 4.67 (m, 1H), 4.21 – 4.11 (m, 3H), 4.08 – 4.00 (m, 2H), 3.91 – 3.74 (m, 5H), 3.71 – 3.59 (m, 5H), 3.48 – 3.28 (m, 4H), 3.26 – 3.15 (m, 4H), 3.08 (dd, J = 13.8, 5.0 Hz, 1H), 2.96 (dd, J = 13.2, 6.8 Hz, 1H), 2.79 (s, 2H), 2.71 – 2.65 (m, 2H), 2.53 – 2.21 (m, 16H), 2.06 – 1.85 (m, 6H), 1.79 – 1.70 (m, 4H), 1.68 – 1.59 (m, 1H), 1.44 (t, J = 6.5 Hz, 2H), 1.35 (d, J = 6.9 Hz, 3H), 1.13 (s, 3H), 1.07 (s, 3H), 0.96 (s, 6H). LC-MS (ESI): m/z 1574.2 [M+H] ⁺.

5.1.2.17. (4S,7S,9aS)-N-((R)-6-((14-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-14-oxo-3,6,9,12-tetraoxatetradecyl)oxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (7b)

Following general methods A, D, E and C, compound 7b was obtained from 23b, 17 and 19b (13.2 mg, 31% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.33 (d, J = 2.3 Hz, 1H), 8.15 (d, J = 6.7 Hz, 1H), 8.06 – 7.98 (m, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.40 – 7.34 (m, 2H), 7.31 – 7.29 (m, 1H), 7.28 – 7.22 (m, 4H), 7.04 (d, J = 8.5 Hz, 1H), 7.01 – 6.96 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 6.71 (dd, J = 8.6, 2.7 Hz, 1H), 6.59 (d, J = 2.6 Hz, 1H), 6.53 (d, J = 9.3 Hz, 1H), 5.27 (t, J = 6.5 Hz, 1H), 5.08 – 4.99 (m, 1H), 4.79 – 4.68 (m, 1H), 4.22 – 4.14 (m, 3H), 4.08 – 4.04 (m, 2H), 3.90 – 3.80 (m, 5H), 3.72 – 3.57 (m, 13H), 3.45 – 3.31 (m, 4H), 3.25 – 3.18 (m, 4H), 3.13 – 3.07 (m, 1H), 2.96 (dd, J = 13.7, 7.6 Hz, 1H), 2.82 (s, 2H), 2.78 – 2.63 (m, 2H), 2.51 (s, 3H), 2.45 – 2.21 (m, 13H), 2.09 (d, J = 5.1 Hz, 1H), 2.02 – 1.87 (m, 5H), 1.81 – 1.73 (m, 4H), 1.70 – 1.62 (m, 1H), 1.46 (t, J = 6.4 Hz, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.15 (s, 3H), 1.10 (s, 3H), 0.98 (s, 6H). LC-MS (ESI): m/z 1662.3 [M+H] ⁺.

5.1.2.18. (4S,7S,9aS)-N-((R)-6-(4-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-4-oxobutoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (8a)

Following general methods A, D, E and C, compound 8a was obtained from 26a, 17 and 19b (14.6 mg, 67% yield). ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 8.34 (d, J = 2.2 Hz, 1H), 8.06 – 7.99 (m, 1H), 7.87 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.39 – 7.33 (m, 2H), 7.31 – 7.27 (m, 2H), 7.26 – 7.19 (m, 3H), 7.06 – 6.95 (m, 3H), 6.91 (d, J = 8.8 Hz, 1H), 6.80 – 6.66 (m, 4H), 6.62 (d, J = 9.3 Hz, 1H), 6.56 (d, J = 2.6 Hz, 1H), 5.26 (t, J = 6.5 Hz, 1H), 5.10 – 5.00 (m, 1H), 4.80 – 4.71 (m, 1H), 4.18 (s, 1H), 4.01 – 3.74 (m, 6H), 3.47 – 3.38 (m, 2H), 3.29 – 3.16 (m, 6H), 3.10 (dd, J = 13.9, 5.1 Hz, 1H), 2.98 (dd, J = 13.8, 7.1 Hz, 1H), 2.79 (s, 2H), 2.75 – 2.67 (m, 2H), 2.53 (s, 3H), 2.49 – 1.94 (m, 22H), 1.92 – 1.69 (m, 5H), 1.67 – 1.55 (m, 1H), 1.46 (t, J = 6.5 Hz, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.98 (s, 6H). LC-MS (ESI): m/z 1514.1 [M+H] ⁺.

5.1.2.19. (4S,7S,9aS)-N-((R)-6-((5-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-5-oxopentyl)oxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (8b)

Following general methods A, D, E and C, compound 8b was obtained from 26b, 17 and 19b (9.9 mg, 29% yield). ¹H NMR (400 MHz, CDCl₃+ CD₃OD) δ 8.33 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 9.3 Hz, 1H), 8.01 – 7.95 (m, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.40 – 7.33 (m, 2H), 7.31 – 7.27 (m, 3H), 7.26 – 7.20 (m, 2H), 7.04 (d, J = 8.5 Hz, 1H), 7.01 – 6.92 (m, 3H), 6.79 (d, J = 8.1 Hz, 1H), 6.72 (d, J = 8.6 Hz, 2H), 6.69 – 6.65 (m, 1H), 6.61 – 6.53 (m, 2H), 5.26 (t, J = 6.5 Hz, 1H), 5.08 – 5.00 (m, 1H), 4.81 – 4.71 (m, 1H), 4.19 (s, 1H), 3.92 – 3.70 (m, 6H), 3.43 – 3.28 (m, 4H), 3.23 – 3.17 (m, 4H), 3.10 (dd, J = 14.0, 4.9 Hz, 1H), 2.97 (dd, J = 13.8, 7.3 Hz, 1H), 2.80 (s, 2H), 2.75 – 2.66 (m, 2H), 2.49 (s, 3H), 2.44 – 2.10 (m, 17H), 2.01 – 1.62 (m, 13H), 1.46 (t, J = 6.6 Hz, 2H), 1.34 (d, J = 7.0 Hz, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.98 (s, 6H). LC-MS (ESI): m/z 1528.1 [M+H] ⁺.

5.1.2.20. (4S,7S,9aS)-N-((R)-6-((6-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-6-oxohexyl)oxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (8c)

Following general methods A, D, E and C, compound 8c was obtained from 26c, 17 and 19b (13.0 mg, 53% yield). ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 8.35 (d, J = 2.2 Hz, 1H), 8.10 – 8.06 (m, 1H), 8.00 (d, J = 6.4 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.41 – 7.35 (m, 2H), 7.34 – 7.27 (m, 3H), 7.25 – 7.21 (m, 2H), 7.06 – 6.95 (m, 4H), 6.82 – 6.72 (m, 3H), 6.70 – 6.53 (m, 3H), 5.31 – 5.20 (m, 1H), 5.13 – 4.96 (m, 1H), 4.79 – 4.66 (m, 1H), 4.20 (s, 1H), 3.91 – 3.70 (m, 6H), 3.42 – 3.24 (m, 8H), 3.11 (dd, J = 13.8, 5.2 Hz, 1H), 2.99 (dd, J = 13.5, 7.1 Hz, 1H), 2.83 (s, 2H), 2.77 – 2.64 (m, 2H), 2.53 (s, 3H), 2.44 – 2.18 (m, 17H), 2.11 – 1.64 (m, 15H), 1.46 – 1.44 (m, 2H), 1.37 (d, J = 7.0 Hz, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 0.97 (s, 6H). LC-MS (ESI): m/z 1542.2 [M+H] ⁺.

5.1.2.21. (4S,7S,9aS)-N-((R)-6-((7-(4-((R)-3-((4-(N-(4-(4-((4’-Chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1’-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptyl)oxy)-1,2,3,4-tetrahydronaphthalen-1-yl)-8,8-dimethyl-4-((S)-2-(methylamino)propanamido)-5-oxooctahydropyrrolo[2,1-b][1,3]oxazepine-7-carboxamide (8d)

Following general methods A, D, E and C, compound 8d was obtained from 26d, 17 and 19b (3.4 mg, 12% yield). ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 8.34 (d, J = 2.2 Hz, 1H), 8.10 – 8.06 (m, 1H), 8.00 (m, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.41 – 7.35 (m, 2H), 7.34 – 7.27 (m, 3H), 7.25 – 7.21 (m, 2H), 7.06 – 6.96 (m, 4H), 6.82 – 6.72 (m, 3H), 6.70 – 6.53 (m, 3H), 5.31 – 5.20 (m, 1H), 5.13 – 4.96 (m, 1H), 4.79 – 4.64 (m, 1H), 4.20 (s, 1H), 3.91 – 3.70 (m, 6H), 3.42 – 3.24 (m, 8H), 3.10 (dd, J = 13.8, 5.2 Hz, 1H), 2.98 (dd, J = 13.5, 7.1 Hz, 1H), 2.83 (s, 2H), 2.79 – 2.64 (m, 2H), 2.53 (s, 3H), 2.44 – 2.18 (m, 17H), 2.11 – 1.43 (m, 19H), 1.37 (d, J = 6.9 Hz, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 0.97 (s, 6H). LC-MS (ESI): m/z 1556.2 [M+H] ⁺.

5.2. Biology

5.2.1. Cell lines and culture.

A549 (Cat# CCL-185), MDA-MB-231 (Cat# HTB-26), SW620 (Cat# CCL-227), MEWO (Cat# HTB-65), SK-MEL-28 (Cat# HTB-72), CHL-1 (Cat# CRL-9446) and WI-38 (Cat# CCL-75) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in complete Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). MyLa1929 cells were kindly provided by Dr. David Weinstock in the Dana-Farber Cancer Institute and cultured in RPMI 1640 media (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA).

5.2.2. Cell viability assay.

Cell viability was measured by Tetrazolium-based MTS assay (Promega, Madison, WI, USA). 1×10⁵ of MyLa1929 cells were seeded and treated in 96-well plates for 72 h. The EC₅₀ values of individual agents were calculated with GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA).

5.2.3. Immunoblotting.

Cells were collected and lysed in Lysis buffer (Boston Bio Products, Ashland, MA, USA) supplied with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The equal amount of protein lysates was separated on pre-casted 4–20% Tris-glycine gels (Bio-Rad, Hercules, CA, USA). Thereafter, the proteins were transferred to PVDF membranes (MilliporeSigma, Billerica, MA, USA). The membranes were blocked with 5% w/v nonfat dry milk in TBS + Tween-20 (0.1% v/v), and then probed with primary antibodies overnight at 4° C. Next day, the membranes were washed and incubated with appropriate HRP-conjugated secondary antibodies. The signal was detected using ECL substrate (MilliporeSigma) and captured on X-ray films or ChemiDoc MP Imaging System (Bio-Rad).

5.2.4. Human platelet isolation and viability assays.

Platelet-rich plasma (PRP) was purchased from Life South Community Blood Center (Gainesville, FL, USA). Platelets were separated from PRP as previously described [13]. Briefly, PRP was transferred into a 50 mL tube containing 5 mL acid citrate buffer (Cat. No. sc-214744, Santa Cruz Biotechnology). To prevent clotting, prostaglandin E1 (PGE1, Cat. No. sc-201223A, Santa Cruz Biotechnology) and apyrase (Cat. No. A6237, Sigma-Aldrich) were added to final concentrations of 1 µM and 0.2 units/mL, respectively. After centrifugation, platelet number was adjusted to 2 × 10⁸/mL in HEPES Tyrode’s buffer containing 10% FBS, 1 µM PGE1 and 0.2 units/mL apyrase, and treated with various compounds. After 48 h of treatment, platelet viability was measured using the MTS reagent (Cat. No. G1111, Promega, Madison, WI, USA). The data were analyzed by GraphPad Prism 7 software for the calculation of IC₅₀ values.

5.2.5. ML-IAP knockout in SK-MEL-28 cells

To deplete ML-IAP, the sgRNAs targeting human gene Birc7 were designed and cloned into lentiCRISPR v2 vector (a gift from Feng Zhang; Addgene plasmid # 52961). Packaging 293T cells were transfected with Birc7 sgRNA or negative control (non-targeting sgRNA-NC) [31] and helper vectors (pMD2.G and psPAX2; Addgene plasmid #s 12259 and 12260) using Lipofectamine 2000 reagent (Cat# 11668019, Life Technologies). Medium containing lentiviral particles and 8 µg/mL polybrene (Sigma-Aldrich, St. Louis) was used to infect SK-MEL-28 cells. Infected cells were selected in medium containing 2 µg/mL puromycin. The target guide sequences are as follows: sgBirc7: forward (5’-CACCGCCTGGACGGAGCATGCCAAG-3’) and reverse (5’-AAACCTTGGCATGCTCCGTCCAGGC-3’).

• IAP-based PROTAC 8a efficiently degraded BCL-X_L in multiple cancer cell lines

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