Heterobifunctional Ligase Recruiters Enable pan-Degradation of Inhibitor of Apoptosis Proteins

Abstract

Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.

Introduction

In the last decade, significant advancements have been made in the field of proteolysis targeting chimeras (PROTACs). PROTACs are now recognized as one of the most promising modalities with the potential to promote the development of targeted therapy drugs.¹⁻⁶ Classical PROTACs are heterobifunctional compounds comprising a ligand that binds to a target protein of interest, a ligand that binds to and recruits an E3 ubiquitin ligase, and a linker tether. Such molecules can facilitate the formation of a ternary target–PROTAC–E3 ligase complex, followed by ubiquitination of the target protein and its subsequent degradation by the proteasome.⁷ PROTACs possess several advantages over conventional inhibitors as they exert their action catalytically, resulting in a potent intracellular degradation of the target proteins. Moreover, PROTACs can discriminate between similar proteins within the same family or even protein isoforms, thus allowing exclusive target-selective degradation.^8,9

E3 ubiquitin ligases orchestrate protein turnover via facilitating substrate proximity and ubiquitin transfer. They encompass a diverse group of more than 600 enzymes, with most E3 ligases belonging to the really interesting new gene (RING) family. Many have crucial roles in various biological processes¹⁰ but are also implicated in multiple diseases. Therefore, targeting E3 ligases is considered an attractive approach for small-molecule drugs.¹¹⁻¹⁵ Cellular RING E3 ligases are large multi-subunit complexes but usually do not possess a defined ligand-binding site rendering the inhibitor design difficult. Nevertheless, traditional approaches yielded potent compounds targeting murine double minute 2 (MDM2), von Hippel-Lindau (VHL), and inhibitor of apoptosis (IAP) proteins.¹¹ The consequence of binding to these E3 ligases is disrupting protein–protein interactions between ligases and their respective substrates. E3 ligases can also be degraded via proximity-induced ubiquitination. Namely, several homodimeric E3 degraders have been developed by linking two identical E3 ligase ligands.¹⁶⁻¹⁹

Directing different E3 ligases against each other by heterodimeric PROTACs also proved to be a productive strategy for their depletion. We recently reported preferential degradation of cereblon (CRBN) over VHL with molecules assembled from pomalidomide-based CRBN binders and a VHL ligand (CRBN-6-5-5-VHL, Figure 1A).²⁰ The prevalence of VHL over CRBN was also observed in a separate study by Ciulli and colleagues (14a, Figure 1A).²¹ On the contrary, linking MDM2 inhibitors to lenalidomide resulted in MDM2 degradation (MD-224 and PROTAC 32, Figure 1A).^22,23 Of note, CRBN levels were not monitored for the latter two examples, thereby not entirely confirming the unilateral degradation of MDM2. Recently, the compendium of heterobifunctional ligase degraders was extended by KEAP1-CRBN recruiters (PROTAC 14 and NJH-04-087, Figure 1A) that preferentially degrade KEAP1.^24,25

Figure 1.

(A) Examples of heterobifunctional E3 ligase degraders. CRBN-6-5-5-VHL and 14a were shown to be potent CRBN degraders which utilize VHL as the recruited ligase,^20,21 whereas MD-224 and PROTAC 32 cause depletion of MDM2 via CRBN-mediated ubiquitination.^18,23PROTAC 14 and NJH-04-087 exhibited degradation of KEAP1 over CRBN.^24,25 (B) Schematic representation of the designed compounds in this work.

Cellular IAP1 (cIAP1/BIRC2), cellular IAP2 (cIAP2/BIRC3), and X-chromosome-linked IAP (XIAP/BIRC4) have been studied in great detail because of their critical role in controlling the apoptotic machinery.²⁶ Their overexpression has been linked to tumor progression, resistance to anti-cancer therapies, and poor prognosis.²⁷⁻²⁹ This clinical significance has translated to the development of numerous mimetics of the IAP-binding motif (i.e., the N-terminal Ala-Val-Pro-Ile moiety)³⁰⁻³² of the second mitochondria-derived activator of caspases (SMAC), which functions as an endogenous IAP antagonist.^33,34 Several SMAC-mimicking IAP monovalent and bivalent antagonists have entered into clinical trials for the treatment of various cancers (Figure S1).^35,36 However, they demonstrated low efficacy as single agents, and current clinical evaluations are limited to combination studies with other cytotoxic drugs, radiation, and immunotherapy.³⁷

IAP antagonists have profound effects on cIAPs levels. Both monovalent and bivalent SMAC mimetics bind to the baculoviral IAP repeat (BIR) type 3 domain of IAPs. This event promotes a rapid and ubiquitin- and proteasome-dependent loss of cIAP1 and cIAP2.^38,39 It was suggested that BIR3-binding IAP antagonists destabilize a closed/autoinhibited form of cIAP (by blocking the crucial BIR3-RING domain interactions), resulting in dimerization, E3 ubiquitin ligase activation, autoubiquitination, and proteasomal degradation.^40,41 Although most IAP antagonists do also possess an affinity for the BIR3 domain of XIAP, this rarely results in XIAP autodegradation.³⁸ IAP antagonists such as bestatin and more recently the improved LCL161 also represent valuable E3-recruiter moieties for the assembly of heterobifunctional protein degraders.⁴²

Encouraged by our successful outcomes with heterodimerizing PROTACs and the fact that IAPs are validated anti-cancer targets, we decided to include IAPs in the hetero-PROTAC approach for E3 modulation (Figure 1B). We systematically designed three series of bifunctional molecules. Two series were assembled by linking the selected IAP ligand⁴³ to two VHL ligands with differently oriented exit vectors. In the third series, the CRBN ligand pomalidomide was incorporated into hetero-PROTACs (for E3 ligand structures, see Table S1). Our efforts to induce IAP degradation through hijacking another E3 ligase via hetero-PROTACs led to selective depletion of IAPs, including XIAP, and, in one case, also to selective XIAP degradation. Notably, the pan-IAP deficit could translate into very potent inhibitors of cancer cell viability, thus further substantiating the rationale of our strategy.

Results and Discussion

Design and Synthesis of Bifunctional IAP PROTACs

It is widely accepted that linkers play a significant role in PROTAC activity, as even subtle differences in length or composition influence the degradation activity and selectivity.^44,45 Three libraries of heterobifunctional PROTACs were systematically designed with eight linkers of varying length and chemical composition in each library (Tables S2–S6). The synthesis of the first series of IAP-VHL hetero-PROTACs (Series 1) was accomplished by a straightforward approach employing chloro to carboxylic acid (Cl-to-CO₂H) linkers L1a–L8a (Table S2). Most of the linkers were acquired by a BAIB/TEMPO-mediated oxidation of the appropriate primary alcohol precursors. In contrast, L6a, L7a, and L8a were prepared by elongating C6–O–C6 or C6–O–C5 alcohols with tert-butyl bromoacetate, tert-butyl 5-bromopentanoate, or tert-butyl 6-bromohexanoate (Schemes 1 and S1), respectively, followed by deprotection of the tert-butyl ester. Linkers L2a–L8a were first coupled to the VHL ligand VH032 (VHL1 ligand, 68),⁴⁶ and the obtained conjugates 75–81 were applied to alkylate the Boc-protected IAP ligand 65 (Scheme 2A). Because intramolecular cyclization occurred when coupling the C5 linker L1a to the VHL1 ligand, L1a was first attached to the Boc-protected IAP ligand 65 or to the negative control IAP ligand 66. This was followed by deprotection of the terminal carboxylic acid before coupling with either VH032 (68) or a methylated VHL ligand (Me-VHL1, 69) which is known to enhance the VHL-binding affinity.⁴⁷ The desired hetero-PROTACs 1–9 and controls 10a and 11 were obtained following Boc deprotection of the corresponding IAP ligands (Scheme 2A). Additional control compounds were obtained by derivatization of PROTAC 9 into dimethyl- (10b) or acetyl- (10c) bearing analogues.

Scheme 1. Syntheses of Linkers L6a, L8a and L6b, L8b.

Reagents and conditions: (a) 50% NaOH (aq), DMSO, rt, 24 h; (b) tert-butyl bromoacetate, NaH, DMF/THF, 0 °C to rt, 18 h; (c) TFA, CH₂Cl₂, 40 °C, 2 h; (d) tert-butyl 6-bromohexanoate (41), toluene, 50% NaOH (aq), TBAHS, rt, 18 h; (e) MsCl, DIPEA, CH₂Cl₂, rt, 2 h; (f) 3,4-dihydro-2H-pyran, CuSO₄ × 5 H₂O, MeCN, rt, 4 h; (g) 50, TBAHS, 50% NaOH (aq), toluene, rt, 18 h; (h) pTsOH × H₂O, MeOH, rt, 20 h; (i) 56, TBAHS, 50% NaOH (aq), toluene, rt, 18 h.

Scheme 2. (A) Syntheses of the IAP-VHL Series 1 Hetero-PROTACs 2–8, 1, and 9 and Negative Controls 10a–d and 11.

Reagents and conditions: (a) linker L2a–L8a (Table S2), HATU, DIPEA, DMF, rt, 16 h; (b) step 1: NaI, acetone, 60 °C, 48 h; step 2: 65, Cs₂CO₃, DMF, 60 °C, 16 h; (c) 1 M HCl in EtOAc, rt, 4 h; (d) step 1: linker L1a (Table S2), NaI, acetone, 60 °C, 48 h; step 2: Cs₂CO₃, DMF, 60 °C, 16 h; (e) Pd/C, H₂, EtOAc, rt, 18 h; (f) 68 or 69, HATU, DIPEA, DMF, rt, 16 h; (g) 9, formaldehyde, Pd/C, H₂, DMF, rt, 16 h (for compound 10b) or 9, Ac₂O, DIPEA, CH₂Cl₂, 0 °C to rt, 16 h (for compound 10c); (B) synthesis of the IAP-VHL series 2 hetero-PROTACs 12–19. Reagents and conditions: (h) linker L1b–L8b (Table S3), Cs₂CO₃, DMF, rt, 16 h, then 60 °C, 3 h; (i) step 1: NaI, acetone, 60 °C, 48 h and step 2: 65, Cs₂CO₃, DMF, 60 °C, 16 h.

For the second IAP-VHL series (Series 2), a different exit vector at the VHL side was employed, presumably leading to differently oriented ternary complexes.^48,49 For this subseries, methane-sulfonate to chloro (OMs-to-Cl) linkers L1b–L8b (Table S3) were utilized as crucial building blocks. Most of these (L1b–L5b) were prepared by subjecting the selected alcohols to mesylation. At the same time, L6b, L7b, and L8b were obtained through elongation of C6–O–C6 or C6–O–C5 mesylates with the corresponding tetrahydropyranyl (THP)-protected diols. Following cleavage of the THP-protecting group, mesylates were prepared (Schemes 1 and S5–S7). The obtained linkers were attached to the phenolic VHL ligand (VHL2 ligand, 71) through O-alkylation. These ligand–linker conjugates 83–90 were then connected to the IAP ligand 65, followed by removing the Boc-protecting group under acidic conditions to yield hetero-PROTACs 12–19 (Scheme 2B).

In the third series of hetero-PROTACs, OMs-to-Cl linkers were used to alkylate the IAP ligand 65, and the resulting chloro-linker-IAP ligand conjugates 91–98 were converted to amino-functionalized building blocks via azidolysis and hydrogenolysis. Finally, these amine building blocks 99–106 were reacted with 4-fluorothalidomide (72) in a nucleophilic aromatic substitution. Removal of the Boc protecting group yielded the envisioned IAP-CRBN heterobifunctional PROTACs 20–27. For the assembly of negative control compounds 28 and 29, an N-methylated thalidomide derivative 73 was used in place of 4-fluorothalidomide (72) (Scheme 3).

Scheme 3. Synthesis of the IAP-CRBN Series Hetero-PROTACs 20–29.

Reagents and conditions: (a) linkers L1b–L8b (Table S3), K₂CO₃, DMF, 70 °C, 20 h; (b) step 1: NaN₃, DMF, 80 °C, 4 h; step 2: Pd/C, H₂, MeOH, rt, 3 h; (c) 72 or 73, DIPEA, DMSO, 90 °C, 20 h; (d) 1 M HCl in EtOAc, rt, 4 h.

The physicochemical properties of all hetero-PROTACs (Tables S4–S6) and known IAP inhibitors (Table S7) are provided in the Supporting Information. Despite encompassing a wide range in terms of lipophilicity (elog D_7.4 from 3.4 to 5.8), a similar activity window was observed for IAP-VHL Series 1 PROTACs. In Series 2, the most lipophilic hetero-PROTAC 19 showed the lowest IAP degradation levels at 0.1 μM, whereas for IAP-CRBN series, high PROTAC lipophilicity led to XIAP-selective depletion (Figure 2 and Table S6).

Figure 2.

Degradation profiles of Series 1 (left), Series 2 (middle), and Series 3 (right) of hetero-PROTACs on cIAP1, cIAP2, XIAP, VHL30, CRBN, and IKZF3 expression levels. Percentage degradation is indicated as the remaining protein levels after MM.1S cells were subjected to 16 h treatment with each compound at 0.1 μM. Values are normalized to respective loading controls and to DMSO-treated conditions. All data represent an average of at least three independent experiments. CST530: IAP ligand and VH298: VHL ligand.

Hetero-PROTACs Induce Potent pan-IAP Degradation

At the outset of our studies, we were aware of the different outcomes possible, as the E3 ligase crosstalk can result in a favored degradation of one ligase or depletion of both E3s in the case of simultaneous cross- and/or autoubiquitination. In the case of preferred ubiquitination of IAP(s), our compounds might be degraders of either cIAP1, cIAP2, XIAP, or of two or even three IAPs. To evaluate the capability of our panel of hetero-PROTACs for E3 ligase degradation, VHL19, VHL30, CRBN, cIAP1, cIAP2, and XIAP levels were quantified by western blot analyses. For each series, MM.1S cells were treated with 0.1 or 1 μM of each compound for 16 h. Original blots are provided in the Supporting Information (Figures S2–S4), and an overview is given in Figure 2. The IAP antagonists LCL161, AZD5582, birinapant, and BV6 were assessed as comparators (Figure 3). These monovalent and bivalent IAP-targeting compounds led to substantial degradation of cIAP1 and cIAP2 at concentrations as low as 0.1 μM. The bivalent IAP antagonists performed better than the monovalent SMAC mimetics, but all of these compounds failed to degrade XIAP (Figure 3).

Figure 3.

Monovalent (CST530 and LCL161) and bivalent IAP antagonists (AZD5582, birinapant, and BV6) induce potent cIAP autodegradation, whereas the IAP-VHL hetero-PROTAC 1 is a pan-IAP degrader. MM.1S cells were treated at 0.1 or 1 μM for 16 h.

The effect of representatives from Series 1 on IAP depletion was significantly enhanced in comparison to the incorporated IAP ligand CST530 itself (Figures 2, 3, and S2), which caused only potent cIAP1 autodegradation but moderate cIAP2 depletion in MM.1S cells. As expected, treatment with the VHL inhibitor VH298 did not affect the levels of IAPs. Hetero-PROTACs, on the other hand, induced complete cIAP1 and XIAP degradation even at 0.1 μM. In addition, a substantial reduction of cIAP2 levels was observed for these hetero-PROTACs (Figures 2 and S2–S4). We were particularly pleased with the ability of PROTACs to degrade XIAP, which was rarely down-regulated by monovalent or bivalent IAP ligands in all previous studies. Of note, after a 16 h treatment with 0.1 μM concentration, a reduction of VHL30 levels was observed for all Series 1 PROTACs (Figures 2 and S2). However, this effect was less pronounced for compounds containing long and hydrophobic linkers (i.e., PROTACs 6–8). The most profound degradation of VHL30 (71% protein degradation, as quantified by western blot) was observed for hetero-PROTAC 3, meaning that both ligases were degraded simultaneously at 0.1 μM concentration. The effect on VHL30 levels was very similar at 1 μM (Figure S5). Bidirectional degradation was in stark contrast to the properties of our CRBN-VHL hetero-PROTAC CRBN-6-5-5-VHL, which selectively degraded CRBN.^20,50 Similarly, the VHL-targeting homo-PROTAC CM11 only caused a reduction of the long VHL isoform but spared the 19 kDa protein.¹⁷ Comparative analysis between 1, 2, and CM11 confirmed similarities between PROTACs whereby IAP-VHL hetero-PROTAC 1 also notably and dose dependently reduced VHL19 levels (42% remaining VHL19 at 1 μM, Figure S6). We estimate that the effects of IAP-VHL degraders would not be masked by the hypoxia-inducible factor (HIF)-dependent hypoxic response because recent results showed that homo-PROTAC-mediated VHL30 degradation or siRNA-mediated knockdown of VHL leads to almost undetectable stabilization of HIF-1α.^17,51

After the initial PROTAC screening, 1 was selected for further optimization due to its comparatively small size and thus the higher chance to overcome PK/PD penalties.⁵² We modified the compound by incorporating the Me-VHL ligand 69 with improved binding affinity for VHL into the hetero-PROTAC. The resulting compound 9 (Figure 4A) showed enhanced pan-IAP degradation in MM.1S cells at even lower concentrations (Figure 4B). Interestingly, also stronger VHL19 degradation was observed at 1 μM (24% remaining VHL19). Densitometric quantifications of western blotting bands after treatment with hetero-PROTAC 9 in MM.1S cells revealed DC_50,16h values of 2.4 nM (cIAP1), 6.2 nM (cIAP2), and 0.7 nM (XIAP). Maximum cIAP1, cIAP2, and XIAP degradation (Dmax) of 99, 90, and 99%, respectively, at 0.1 μM concentration of 9 was achieved (Figure 4C). A head-to-head comparison of 9 with birinapant demonstrated that the latter caused more pronounced cIAP1 degradation, whereas 9 outperformed birinapant in depleting cIAP2 and XIAP (Figure S7A). On the other hand, AZD5582 showed stronger cIAPs degradation than IAP-VHL hetero-PROTAC 9 but did not influence XIAP levels even at 1 μM (Figure S7B).

Figure 4.

(A) Structure of pan-IAP degrader PROTAC 9. (B) IAP-VHL hetero-PROTAC 9 (CST626) induces cIAP1, cIAP2, XIAP, and VHL30 degradation in a dose-dependent manner. Hetero-PROTAC 11 (impaired VHL binding) and the monovalent IAP inhibitor CST530 induce only cIAP1 degradation. VHL inhibitor VH298 has no effects on the investigated proteins. MM.1S cells were treated with PROTACs or controls at indicated concentrations for 16 h. (C) Quantification of (B) and calculation of the DC₅₀ values from repeats (n = 4).

Profiling the activities of the second series of hetero-PROTACs, where we utilized a different linker exit vector, revealed a degradation profile similar to that of the first series. Namely, hetero-PROTACs 12–14 with C5, C8, and C4–O–C4 linkers, respectively, induced the most potent pan-IAP degradation (Figures 2 and S3). Unidirectional ubiquitination between the two E3 ligases was again observed only for hetero-PROTACs with long linkers (compounds 17–19). In line with this, also no effect on VHL19 levels was seen (Figures 2 and S3). In terms of achieving degradation of a pair of IAPs, 19 seemed interesting as it caused dual cIAP1/XIAP degradation at 0.1 μM concentration in MM.1S. However, profiling of the concentration dependence of 19 showed pan-IAP degradation at 1 μM and no degradation at 10 nM (Figure S8).

To investigate the relative ability of E3 ligases to induce degradation of each other upon treatment with the IAP-CRBN hetero-PROTACs, MM.1S cells were used. For 21–26, consistent, unidirectional, and distinct degradation of all three IAPs was observed already at 0.1 μM concentration (Figure 2). Of these, compounds 22, 25, and 26 showed pronounced pan-IAP depletion, and, concurrently, they induced substantial IKZF3 degradation at 1 μM as an effect of modulating the substrate scope upon pomalidomide binding (Figure S4B). At 0.1 μM, only 22 caused depletion of IKZF3, with approximately 40% of IKZF3 degraded after 16 h treatment. This dual mode could be useful in settings where these secondary effects are desirable. The most intriguing finding within the IAP-CRBN hetero-PROTAC series was observed for compound 27, equipped with the longest linker. An isoform-selective XIAP degradation was indicated after 16 h-treatment at 0.1 μM (Figures 2 and S4A). A significant and selective decrease of XIAP levels compared to cIAP1 and cIAP2 was confirmed on a proteome level, where MM.1S cells were treated with hetero-PROTAC 27 for 3 h (see Figure 6B). This result unveils that IAP selectivity within the IAP-CRBN hetero-PROTAC series can be tuned by linker modifications.

Figure 6.

diaPASEF quantitative proteomics for (A) hetero-PROTAC 9 (CST626), (B) hetero-PROTAC 27 (SAB142), and (C) homobivalent compound AZD5582. MM.1S cells were treated with either DMSO or the mentioned compounds at 0.1 μM for 3 h in four and two biological replicates, respectively. Downstream statistical analysis was performed using Bioconductor’s limma package. The quantified proteins were plotted as log 2-fold change (compound/DMSO) versus −log 10 of p-value using RStudio. Note: dataset for 27 was obtained in an independent/separate proteomics run.

Mechanistic Considerations

To understand the mechanism of E3 recruitment and ubiquitin transfer, we tested a set of control compounds with inactivated IAP- or VHL-binding motifs (Table S4). As little was known about appropriately rendering IAP ligands inoperative, we synthesized a series of putative IAP-non-binding controls 10a–d (Scheme 2A and Figure S9). In 10a, the stereochemistry of the critical N-methyl alanine portion was inverted or substituted with a second methyl group (10b). However, literature data indicated remaining affinity for the XIAP-BIR3 domain.⁵³ Indeed, 10a and 10b were still able to induce pan-IAP or cIAP1 and cIAP2 degradation, respectively (Figure S9). Further increasing the size of the N-terminal substituent and lowering the basicity in 10c (R = acetyl) and 10d (R = Boc) led to inactivated PROTACs. Both methylation and acetylation were performed through a convenient late-stage modification reactions of the final PROTAC 9, highlighting the general utility of these transformations to produce inactivated IAP-recruiting PROTACs. By analogy with series 10, hetero-PROTAC 11 (VHL-ent) possessing a VHL non-binding diastereomer only induced cIAP1 degradation (Figure 4B), which is a common attribute of IAP antagonists.

Next, cellular activities of hetero-PROTACs 2, 4, and 6 were evaluated in chronic lymphocytic leukemia cells (HG3), for which a VHL-knockout cell line was created (Figure 5). In both VHL^+/+ and VHL^–/– cells, cIAP1 autodegradation was observed after treatment with our hetero-PROTACs, demonstrating that ligand binding is the conditio per quam. In contrast, recruitment of a VHL is required for XIAP degradation as this occurred only in HG3 wild-type cells. Thus, VHL knockout confirmed the involvement of E3 ubiquitin ligase CRL2^VHL in the induced degradation of XIAP (and, at least in part, cIAP1) by these IAP-VHL heterobifunctional PROTACs. This provides additional evidence that the degradation of IAPs relies on the formation of a hetero–ternary complex consisting of both ligases and the degraders.

Figure 5.

PROTACs 6, 2, and 4 retain degradation of cIAP1 and XIAP in chronic lymphocytic leukemia wild-type cells (HG3) and HG3 VHL-knockout cells. Cells were treated with compounds at 1 μM for 16 h.

When evaluating the time dependence of hetero-PROTAC 9, we observed complete degradation of cIAP1, cIAP2, and XIAP already after 3 h at 0.1 μM compound concentration. Interestingly, the effect on VHL30 depletion was most pronounced after 6 h of treatment in MM.1S cells (Figure S10A).

Next, we examined the persistence of IAP degradation in MM.1S cells after a single exposure to 1 μM of 9 and subsequent removal of the compound. Results indicated a nearly full and stable pan-degradation of IAPs up to 72 h. VHL19 and VHL30 levels restored more rapidly following drug washout (Figure S11A). Nevertheless, intracellularly cycling quantities of PROTAC that remain inside the cells after washout may be sufficient to generate these characteristics. In contrast, when the system was further challenged with the competing VH298 after the washout, XIAP levels increased more rapidly (Figure S11B), consistent with an IAP antagonist mode and an unleashed resynthesis of XIAP. In a series of experiments where individual IAPs were knocked out, we observed no differences in VHL30 degradation by hetero-PROTAC 9; e.g., in cIAP1 knockout cells, VHL30 degradation could also be mediated by cIAP2 (Figure S12). A set of experiments were performed to demonstrate the involvement of the ubiquitin–proteasome system in degradation. Treatment of cells with a proteasome inhibitor MG132 completely abrogated degradation of IAPs. The reliance on CRL2^VHL was assessed with a neddylation inhibitor MLN4924, which blocks the activity of CRLs (Figure S13A). Similarly, a selective ubiquitin-activating enzyme inhibitor MLN7243 also prevented the PROTAC-induced degradation of IAPs (Figure S13A).

Concentration- and time-dependent degradation of IAPs in MM.1S cells was evaluated for 25 too (Figures S14 and S10B). Complete cIAP1 and XIAP depletion occurred already at 10 nM, whereas 0.1 μM concentration was needed for the complete depletion of all IAPs. The corresponding CRBN-non-binding control 28 failed to degrade XIAP at 1 μM concentration but caused a significant deficit of cIAP2 (32% remaining protein, Figure S14). Pan-depletion of IAPs by 25 was also very rapid as we observed complete degradation already after 3 h at 0.1 μM (Figure S10B). In addition, the proteasome-mediated mechanism of IAP degradation by 25 was confirmed using the same experiments as for hetero-PROTAC 9 (Figure S13B).

To analyze the proteome-wide degradation selectivity of hetero-PROTACs 9, 25, and 27, a diaPASEF-based mass spectrometry approach was employed.⁵⁴ MM.1S cells were treated with 100 nM PROTACs for 3 h. Of the total 7170 unique proteins identified, 9 (Figure 6A) and 25 (Figure S15) degraded cIAP1 and XIAP to levels below the detection level, whereas cIAP2 could not be evaluated in this experiment as it was undetected in DMSO–vehicle treatments. Accordingly, global proteomic plots show the mathematically imputed levels of IAP proteins in treatment groups if the corresponding IAP was detected in the control treatment (see also Experimental Section). AZD5582 also depleted cIAP1 below the detection level but did not cause XIAP degradation (Figure 6C), which is in accordance with the fact that IAP antagonists have no effect on XIAP.

In a separate experiment, global proteome analysis in MM.1S cells after treatment with 27 (Figure 6B) showed selective XIAP degradation with no impact on cIAP1 and cIAP2, rendering this compound significantly more selective for XIAP over cIAP1 and cIAP2 that were only degraded at higher concentrations and after prolonged treatment times. Moreover, we did not observe changes in CRBN, IKZF1, IKZF3, and VHL levels in the proteomic data, thus further substantiating the unilateral effect of our hetero-PROTACs.

pan-IAP Degradation Reduces Cell Viability

To assess the pharmacological consequences of IAP depletion, the pan-IAP degraders 9 and 25, along with appropriate inactivated PROTACs, were evaluated for their cell growth inhibition in nine hematological cell lines (Figures 7 and S17), i.e., three myeloma (RPMI-8226, JJN3, and NCI-H929), three leukemia (HEL, K562, and MOLM13), and three lymphoma cells (SUDHL4, DB, and SUDHL6). We included the monovalent ligase ligands VH298 (VHL), pomalidomide (CRBN), and CST530 (IAP), as well as the homobivalent SMAC mimetic AZD5582 as reference standards. As TNF-α and related signaling cascades represent crucial factors for the single-agent activity of IAP-targeting compounds,^29,38,39,55 the viability inhibition of sensitive cell lines was evaluated in the presence and absence of this inflammatory stimulus. Co-administration of TNF-α potentiated the inhibitory effects of both SMAC mimetics and PROTACs for all cell lines tested, consistent with previous studies.⁵⁶⁻⁵⁹ pan-IAP degraders 9 and 25 were more potent than the positive control IAP monovalent and bivalent antagonists in several cell lines (Figures 7 and S17). PROTACs 9 and 25 demonstrated superior activity over AZD5582 in NCI-H929, reaching IC₅₀ values of 8.5 and 27 nM, respectively (Tables 1 and S8). In addition, PROTAC 9 demonstrated a competitive IC₅₀ profile in MOLM13 cells at 2.1 nM and SUDHL6 cells at 1.6 nM. While the activity of PROTAC 25 in these two cell lines did not supersede PROTAC 9, it was able to induce potent cell viability reduction in other cell lines such as JJN3 and SUDHL4 (Table S8), surpassing that of IAP antagonists. These effects were independent of IAP baseline levels (Figure S16), which is in agreement with the previous studies of IAP antagonists.

Figure 7.

Cell viability screenings in nine different hematological cancer cell lines with pan-IAP degrader 9, its VHL non-binding control 11, as well as the structurally related IAP antagonist CST530 and the VHL inhibitor VH298 as respective controls. In certain cases, viability inhibition was assessed in the presence and absence of TNF-α. Multiple myeloma, acute myeloid leukemia, and lymphoma cell lines were treated with the respective compounds at indicated concentrations for 96 h. Viability is normalized to their respective DMSO controls. Data represent means ± s.d. of at least three independent biological replicates.

Table 1. Cell Viability Profiles (IC₅₀ Values) of the VHL-Recruiting PROTAC 9, the VHL Non-Binding Control 11, as Well as the Monovalent IAP Antagonist CST530 and the VHL Inhibitor VH298 for Comparison^a.

		IC50 (μM)
cell line	diseaseb	9	11	CST530	VH298
RPMI-8826	MM	2.54	>5	2.79	>10
JJN3	MM	1.14	>5	4.73	>10
NCI-H929	MM	0.0085	>5	2.34	>10
HEL	AML	1.17	>10	0.45	>10
K562	AML	0.42	>5	1.71	>10
MOLM13	AML	0.0021	>1	0.42	>10
SUDHL4	DLBCL	1.69	>5	3.44	>10
DB	DLBCL	0.46	>5	2.21	>10
SUDHL6	DLBCL	0.0016	0.17	0.17	>10

^aValues correspond to TNFα-challenged conditions.

^bMM, multiple myeloma; AML, acute myeloid leukemia; DLBCL, diffuse large B-cell lymphoma.

Conclusions

In this study, we designed heterobifunctional compounds assembled from an IAP antagonist linked to either a VHL- or a CRBN-recruiting ligand. The entire set of PROTACs consisted of 32 tailored members, which were subjected to in-depth biological studies. Through appropriate control experiments (chemical controls and impairment of the ubiquitin–proteasome system), we provided significant evidence for the engagement with the proposed E3 ligases and PROTAC-induced ubiquitin transfer. The accompanied heterodimerization approach led to novel E3 modulators with IAP degradation profiles that could not be reached with monomeric or homobivalent SMAC mimetics. Among the set of IAP degraders were compounds that induced depletion of the 19 and 30 kDa VHL isoforms. The described pan-IAP degraders will serve as selective tools to explore the biology of IAPs and thus open up new avenues for apoptosis research in various cellular contexts. In addition, selected compounds from our series warrant further appraisal as anti-cancer agents on account of their ability of depleting validated cancer-related IAPs. Preliminary cell-based evaluations of our lead hetero-PROTAC 9 demonstrated that induced degradation of IAPs supersedes the biological effects of monovalent and bivalent IAP antagonists in certain cases. Therefore, further development of IAP-targeting heterobifunctional compounds may lead to degraders with significant therapeutic benefits in the battle against cancer. Exploiting PROTAC methodology to induce the degradation of therapeutically relevant ligases raises hope to unlock this difficult-to-tackle class of drug targets. We also anticipate that the contest between two different E3s may be extendable to any other ligandable ligase.

Experimental Section

Chemistry General Remarks

Preparative column chromatography was performed using Merck silica gel 60 (0.063–0.200 mm) or using an automated flash chromatography system puriFlash XS 520Plus. Melting points were determined on a Büchi 510 oil bath apparatus or on a Reichelt hot-stage apparatus and were uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer, a Bruker Avance 500 MHz NMR spectrometer, or a Bruker Avance III 600 MHz NMR spectrometer, respectively. NMR spectra were processed and analyzed in MestReNova. Chemical shifts are given in parts per million (ppm) and are referenced to the deuterated solvent used. Coupling constants J are given in Hz, and the splitting patterns are given as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). In the case of overlapping extraneous solvent peaks, multiplet analyses in ¹H NMR spectra were performed using qGSD (quantitative Global Spectral Deconvolution). Resonance assignments were made on the basis of one- and two-dimensional NMR techniques which include ¹H, ¹³C, DEPT, HSQC, and HMBC experiments. Important note: the presence of amide rotamers significantly complicated the appearance and validation of the ¹H and ¹³C NMR spectra associated with synthetic intermediates and final PROTACs. The presence of rotamers was demonstrated by recording a representative ¹H NMR at 80 °C (see the Supporting Information). Thus, reported resonances and integrals may have limited accuracy. High-resolution mass measurements were recorded on a Thermo Scientific Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The purity and identity of compounds were determined on an Infinity Lab LC/MSD system (Agilent) with the ESI source coupled with an HPLC 1260 Infinity II (Agilent) using an EC50/2 Nucleodur C18 Gravity 3 μm column (Macherey-Nagel). The column temperature was 40 °C. HPLC conditions started with 90% H₂O containing 2 mM NH₄Ac. The gradient ramped up to 100% MeCN in 10 min, followed by further flushing with 100% MeCN for 5 min. The flow rate was 0.5 mL/min. The samples were dissolved in H₂O, MeOH, or MeCN (approx. 1 mg/mL), and 2 μL of the sample solution was injected. Positive total ion scans were observed from 100 to 1000 m/z (or more if necessary), and UV absorption was detected from 190 to 600 nm using a diode array detector (DAD). The purity was determined at 220–600 nm. Analytical reversed-phase HPLC for PROTACs 11 and 20–27 was performed on a Thermo Scientific Dionex UltiMate 3000 UHPLC modular system (Thermo Fisher Scientific), equipped with a photodiode array detector set to 254 nm. A Waters Acquity UPLC HSS C18 SB column (1.8 μm, 2.1 mm × 50 mm) was used and thermostated at 40 °C. The mobile phase consisted of 0.1% TFA in H₂O (A) and MeCN (B), employing the following gradient: 95% A to 5% A in 10 min, then 95% B for 4 min, with a flow rate of 0.3 mL/min, and an injection volume of 5 μL. All compounds that were evaluated in biological assays are >95% pure by HPLC analysis.

Note: To provide readers a clearer picture of all synthesized compounds and to enable easier tracking of experimental procedures, a table with structures of all intermediates is given at the end of the Supporting Information.

General Procedures

General Procedure I: Mesylation

To a solution of the corresponding alcohol (7 mmol) in dry CH₂Cl₂ (15 mL), DIPEA (1.36 g, 1.79 mL, 10.5 mmol) was added under an argon atmosphere and the mixture was cooled to 0 °C. Subsequently, methanesulfonyl chloride (1.20 g, 0.81 mL, 10.5 mmol) was added dropwise at 0 °C, followed by stirring of the mixture at rt for 2 h. After the reaction was complete (monitored by TLC), MeOH (20 mL) was added to the mixture carefully. The volatiles were then evaporated, and the crude product was purified by column chromatography.

General Procedure II: Alkylation of the IAP Ligand Using Cl-Bearing Linkers or Conjugates

The corresponding linker or the VHL ligand–linker conjugate (0.30 mmol) was dissolved in dry acetone (15 mL), and NaI (0.45 g, 3.0 mmol) was added. The mixture was stirred at 60 °C for 48 h. After evaporation of the solvent, the residue was suspended in EtOAc (50 mL) and subsequently washed with 10% Na₂SO₃ solution, H₂O, and brine (each 25 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated. This intermediate was dissolved in dry DMF (5 mL), and Cs₂CO₃ and the corresponding IAP ligand 65 or 66 (1.0 equiv based on the yield from the Finkelstein reaction) were added. The mixture was stirred at 60 °C for 18 h. After cooling, it was quenched with half-saturated brine (100 mL) and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were washed with 5% LiCl solution and brine (each 50 mL), dried over Na₂SO₄, filtered, and evaporated. The crude product was purified by column or flash chromatography.

General Procedure III: Coupling of the VHL1 Ligand and Cl-to-COOH Linkers

The corresponding Cl-to-COOH linker L1a–L8a (0.50 mmol) was dissolved in dry DMF (5 mL), and DIPEA (0.35 mL, 2 mmol) was added, followed by the addition of HATU (0.21 g, 0.55 mmol). After stirring for 5 min, the corresponding VHL ligand 68–70 (deprotected amine, 0.62 mmol) dissolved in dry DMF (5 mL) and DIPEA (0.35 mL, 2 mmol) were added to the mixture. The combined mixture was stirred at room temperature for 16 h, after which half-saturated brine (50 mL) was added, and the product was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated NH₄Cl solution, 5% LiCl solution, and brine (each 50 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo.

General Procedure IV: Alkylation of the VHL2 Ligand Using OMs-to-Cl Linkers

The corresponding OMs-to-Cl linker L1b–L8b (1.2 mmol) was dissolved in dry DMF (10 mL), followed by the addition of Cs₂CO₃ (0.49 g, 1.5 mmol). Then, the phenolic VHL ligand 71 (0.55 g, 1.0 mmol) dissolved in dry DMF (5 mL) was added. The combined mixture was stirred at room temperature for 16 h and 3 h at 60 °C. After cooling, half-saturated brine (50 mL) was added, and the product was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with saturated NH₄Cl solution, 5% LiCl solution, and brine (each 50 mL); dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was purified by column or flash chromatography.

General Procedure V: Alkylation of the IAP Ligand Using OMs-to-Cl Linkers

To a solution of IAP ligand 65 (0.17 g, 0.25 mmol) and K₂CO₃ (52 mg, 0.38 mmol) in dry DMF (2 mL), a solution of the corresponding mesylate-bearing linker L1b–L8b (0.30 mmol) in dry DMF (2 mL) was added under an argon atmosphere. The mixture was stirred at 70 °C for 20 h. The volatiles were then evaporated, and the crude product was purified by column chromatography.

General Procedure VI: Synthesis of Alkyl Azides and Subsequent Reduction to Amines

To a solution of the corresponding IAP ligand–linker–chloro conjugate 91–98 (0.19 mmol) in dry DMF (5 mL), NaN₃ (25 mg, 0.38 mmol) was added under an argon atmosphere. After stirring the mixture at 80 °C for 4 h, the volatiles were removed, and H₂O (40 mL) was added. The product was extracted with EtOAc (60 mL). The organic layer was washed with brine (50 mL), dried over Na₂SO₄, filtered, concentrated, and further dried under high vacuum. This azide intermediate was dissolved in dry MeOH (5 mL) and treated with 10% Pd/C (22 mg, 20% w/w). The reaction mixture was stirred under H₂ (1 atm, balloon) for 2 h. The mixture was filtered through Celite and washed with MeOH, and the filtrate was concentrated. The products were used in the next step without further purification.

General Procedure VII: Removal of Boc Protecting Groups

The Boc-protected PROTAC precursor was treated with 1 M HCl in EtOAc (5 mL), and the mixture was stirred at rt for 4 h. After removal of the volatiles, the oily residue was treated with Et₂O (5 mL), and the mixture was stirred at rt for 1 h. If a colorless precipitate appeared, it was collected by suction filtration and washed with Et₂O (2 × 2 mL). Because of sufficient purity, PROTACs 2, 4, 6, 9, 10, and 12–17 were used as hydrochloride salts. For the remaining final PROTACs, additional purification by column chromatography was necessary, and those compounds were transformed into free bases.

General Procedure VIII: Nucleophilic Aromatic Substitution

The corresponding IAP ligand–linker–amine conjugate 99–106 (0.11 mmol) was dissolved in dry DMSO (2 mL), and DIPEA (44 mg, 56 μL, 0.33 mmol) and the corresponding CRBN ligand 72 or 73 (32 mg, 0.11 mmol) were added. The mixture was stirred at 90 °C for 20 h. After cooling, H₂O (30 mL) and saturated NaHCO₃ solution (10 mL) were added, and the mixture was extracted with CH₂Cl₂ (5 × 50 mL). The combined organic layers were washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated. The oily residue was purified by column chromatography.

Syntheses of Linkers

Syntheses of L1a–L8a Linkers (L1a: Cl-to-CO₂Bn and L2a–L8a: Cl-to-CO₂H)

L1a: Benzyl 5-Chloropentanoate (30)

5-Chlorovaleric acid (1.85 g, 13.55 mmol), benzyl bromide (2.32 g, 1.61 mL, 13.55 mmol), and Na₂CO₃ (1.72 g, 16.26 mmol) were mixed in MeCN (20 mL) and heated to 80 °C for 18 h. After cooling, the suspension was filtered and then partitioned between H₂O (100 mL) and EtOAc (2 × 100 mL). The combined organic layers were washed with H₂O, NaHCO₃ solution, and brine (each 50 mL); dried over Na₂SO₄; filtered; and concentrated in vacuo. The crude product was purified by column chromatography (petroleum ether/EtOAc 19:1) to give a colorless oil. Yield (1.87 g, 61%); R_f = 0.43 (petroleum ether/EtOAc 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 1.61–1.76 (m, 4H), 2.39 (t, J = 7.3 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 5.08 (s, 2H), 7.29–7.39 (m, 5H); ¹³C NMR (151 MHz, DMSO-d₆): δ 22.00, 31.46, 32.76, 40.24, 45.08, 65.54, 128.09, 128.14, 128.58, 136.39, 172.67; LC–MS (ESI) m/z: [M + H]⁺ calcd for C₁₂H₁₆ClO₂, 227.08; found, 227.1.

L2a: 8-Chlorooctanoic Acid (31)

This compound was synthesized as we described previously.⁶⁰

4-(4-Chlorobutoxy)butan-1-ol (32)

1,4-Butanediol (33.80 g, 375 mmol) was mixed in DMSO (50 mL) and aqueous NaOH (50%, 19.3 mL, 375 mmol). After stirring for 10 min, 1-bromo-4-chlorobutane (12.86 g, 75 mmol) was added while cooling with a water bath. The resulting suspension was vigorously stirred at rt for 24 h. After the addition of a saturated NH₄Cl solution (150 mL), the mixture was extracted with CH₂Cl₂ (3 × 150 mL). The combined organic layers were washed with H₂O (150 mL) and brine (150 mL), dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (gradient of petroleum ether/EtOAc 2:1 to 3:2) to give a colorless oil. Yield (4.88 g, 36%); R_f = 0.31 (petroleum ether/EtOAc 1:1); ¹H NMR (500 MHz, DMSO-d₆): δ 1.38–1.54 (m, 4H), 1.54–1.63 (m, 2H), 1.70–1.79 (m, 2H), 3.30–3.43 (m, 6H), 3.63 (t, J = 6.7 Hz, 2H), 4.32 (t, J = 5.2 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 26.04, 26.76, 29.34, 29.42, 45.44, 60.71, 69.23, 70.08; LC–MS (ESI) m/z: [M + H]⁺ calcd for C₈H₁₈ClO₂, 181.10; found, 180.9.

L3a: 4-(4-Chlorobutoxy)butanoic Acid (33)

Alcohol 32 (1.08 g, 6 mmol) was dissolved in MeCN (15 mL) and H₂O (15 mL). TEMPO (0.20 g, 1.32 mmol) was then added, followed by the portionwise addition of (diacetoxyiodo)benzene (4.25 g, 13.2 mmol). The orange mixture was stirred at rt for 16 h. It was neutralized by the addition of saturated NaHCO₃ solution (100 mL), and the aqueous layer was washed with EtOAc (2 × 100 mL). The aqueous phase was then acidified by the careful addition of 2 N HCl solution until pH = 1. The mixture was then extracted with EtOAc (2 × 100 mL), and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (gradient of CH₂Cl₂ to CH₂Cl₂/MeOH 9:1) to give a brownish oil. Yield (0.89 g, 76%); R_f = 0.45 (CH₂Cl₂/MeOH 9:1); ¹H NMR (500 MHz, DMSO-d₆): δ 1.54–1.63 (m, 2H), 1.66–1.79 (m, 4H), 2.23 (t, J = 7.3 Hz, 2H), 3.35 (dt, J = 6.3, 8.0 Hz, 4H), 3.63 (t, J = 6.6 Hz, 2H), 11.97 (s, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 24.88, 26.72, 29.28, 30.56, 45.45, 69.17, 69.26, 69.39, 174.38; LC–MS (ESI) m/z: [M + H]⁺ calcd for C₈H₁₆ClO₃, 195.08; found, 195.1.

L4a: 2-(2-(2-Chloroethoxy)ethoxy)acetic Acid (34)

This compound was synthesized as we described previously.⁶⁰

L5a: 2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)acetic Acid (35)

This compound was synthesized as we described previously.⁶⁰

6-((6-Chlorohexyl)oxy)hexan-1-ol (36)

This compound was synthesized as we described previously.²⁰

tert-Butyl 2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)acetate (37)

Linker 36 (1.89 g, 8 mmol), tert-butyl bromoacetate (3.08 g, 3.5 mL, 24 mmol), and TBAHS (2.71 g, 8 mmol) were mixed in toluene (6 mL), followed by the addition of 50% aqueous NaOH solution (4 mL) at 0 °C. The mixture was vigorously stirred at rt for 18 h. The mixture was diluted with H₂O (100 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether/EtOAc 10:1) to give a colorless oil. Yield (1.42 g, 51%); R_f = 0.48 (petroleum ether/EtOAc 10:1); ¹H NMR (600 MHz, DMSO-d₆): δ 1.24–1.39 (m, 8H), 1.41 (s, 9H), 1.44–1.51 (m, 6H), 1.66–1.73 (m, 2H), 3.29–3.34 (m, 4H), 3.40 (t, J = 6.5 Hz, 2H), 3.60 (t, J = 6.6 Hz, 2H), 3.90 (s, 2H); ¹³C NMR (151 MHz, DMSO-d₆): δ 25.15, 25.58, 25.68, 26.24, 27.89, 29.22, 29.27, 29.34, 32.17, 45.47, 68.17, 69.93, 70.03, 70.65, 80.66, 169.63; LC–MS (ESI) m/z: [M + H]⁺ calcd for C₁₈H₃₆ClO₄, 351.23; found, 351.2.

L6a: 2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)acetic Acid (38)

This compound was produced from 37 after acidic cleavage of the tert-butyl ester group in CH₂Cl₂/TFA (1:1) at 40 °C for 2 h and was used in the next step without further purification after evaporation of the volatiles.

tert-Butyl 5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentanoate (39)

This compound was synthesized as we described previously.²⁰

L7a: 5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentanoic Acid (40)

This compound was produced from 39 after acidic cleavage of the tert-butyl ester group in CH₂Cl₂/TFA (1:1) at 40 °C for 2 h and was used in the next step without further purification after evaporation of the volatiles.

tert-Butyl 6-Bromohexanoate (41)

This compound was synthesized as we described previously.²⁰

tert-Butyl 6-((6-((6Chlorohexyl)oxy)hexyl)oxy)hexanoate (42)

This compound was synthesized as we described previously.²⁰

L8a: 6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexanoic Acid (43)

This compound was produced from 42 after acidic cleavage of the tert-butyl ester group in CH₂Cl₂/TFA (1:1) at 40 °C for 2 h and was used in the next step without further purification after evaporation of the volatiles.

Syntheses of L1b–L8b Linkers (OMs-to-Cl)

L1b: 5-Chloropentyl Methanesulfonate (44)

This compound was prepared using general procedure I and 5-chloro-1-pentanol (2.0 g, 16.3 mmol). The crude product was purified by column chromatography (EtOAc) to give a colorless oil. Yield (2.80 g, 85%); R_f = 0.60 (EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 1.52–1.63 (m, 2H), 1.74–1.87 (m, 4H), 3.01 (s, 3H), 3.55 (t, J = 6.5 Hz, 2H), 4.24 (t, J = 6.4 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 22.99, 28.56, 31.97, 37.52, 44.69, 69.72; HRMS (ESI) m/z: [M + Na]⁺ calcd for C₆H₁₃O₃ClSNa, 223.0166; found, 223.0164.

L2b: 8-Chlorooctyl Methanesulfonate (45)

This compound was synthesized as we described previously.⁶⁰

L3b: 4-(4-Chlorobutoxy)butyl Methanesulfonate (46)

This compound was prepared using general procedure I and 32 (0.41 g, 2.29 mmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 1:1) to give a colorless oil. Yield (0.33 g, 56%); R_f = 0.22 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.65–1.74 (m, 4H), 1.79–1.89 (m, 4H), 3.00 (s, 3H), 3.43 (td, J = 6.2, 2.1 Hz, 4H), 3.56 (t, J = 6.6 Hz, 2H), 4.25 (t, J = 6.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.83, 26.37, 27.16, 29.63, 37.50, 45.09, 70.00, 70.07, 70.14; HRMS (ESI) m/z: [M + H]⁺ calcd for C₉H₂₀O₄ClS, 259.0765; found, 259.0764.

L4b: 2-(2-(2-Chloroethoxy)ethoxy)ethyl Methanesulfonate (47)

This compound was synthesized as we described previously.⁶⁰

L5b: 2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)ethyl Methanesulfonate (48)

This compound was synthesized as we described previously.⁶⁰

6-((6-Chlorohexyl)oxy)hexyl Methanesulfonate (49)

This compound was prepared using general procedure I and 36 (1.05 g, 3.38 mmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (0.75 g, 57%); R_f = 0.35 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.32–1.50 (m, 8H), 1.53–1.62 (m, 4H), 1.71–1.82 (m, 4H), 3.00 (s, 3H), 3.39 (t, J = 6.6 Hz, 4H), 3.53 (t, J = 6.7 Hz, 2H), 4.22 (t, J = 6.6 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.44, 25.65, 25.82, 26.84, 29.22, 29.67, 29.70, 32.68, 37.50, 45.21, 70.18, 70.76, 70.86; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₇H₃₆O₅ClS, 387.1967; found, 387.1960.

2-((Tetrahydro-2H-pyran-2-yl)oxy)ethan-1-ol (50)

To a solution of ethylene glycol (3.00 g, 48.34 mmol) in dry MeCN (25 mL), 3,4-dihydro-2H-pyran (4.47 g, 4.82 mL, 53.17 mmol) was added under an argon atmosphere. Subsequently, CuSO₄ × 5 H₂O (2.41 g, 9.67 mmol) was added, followed by stirring of the mixture at rt for 3 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (EtOAc) to give a colorless oil. Yield (1.21 g, 18%); R_f = 0.30 (EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 1.49–1.60 (m, 4H), 1.73–1.89 (m, 2H), 2.80–2.86 (m, 1H), 3.51–3.58 (m, 1H), 3.66–3.81 (m, 4H), 3.88–3.97 (m, 1H), 4.55–4.59 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 20.01, 25.27, 30.81, 62.24, 63.26, 70.72, 100.15; HRMS (ESI) m/z: [M + Na]⁺ calcd for C₇H₁₄O₃Na, 169.0835; found, 169.0837.

2-(2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)ethoxy)tetrahydro-2H-pyran (51)

To a solution of 50 (0.72 g, 4.93 mmol) and 49 (1.55 g, 4.93 mmol) in toluene (20 mL), TBAHS (1.67 g, 4.93 mmol) and 50% NaOH (aq) (2.5 mL) were added. The reaction mixture was stirred at rt for 18 h. H₂O (70 mL) was then added, and the mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were further washed with H₂O and brine (each 200 mL). The organic layers were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (1.08 g, 50%); R_f = 0.45 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.30–1.47 (m, 8H), 1.47–1.65 (m, 10H), 1.67–1.87 (m, 4H), 3.37 (td, J = 6.6, 2.1 Hz, 4H), 3.41–3.55 (m, 5H), 3.55–3.61 (m, 3H), 3.80–3.90 (m, 2H), 4.62 (t, J = 4.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 19.59, 25.55, 25.64, 26.10, 26.18, 26.83, 29.70, 29.73, 29.84, 30.67, 32.68, 45.18, 62.31, 66.73, 70.08, 70.78, 71.00, 71.44, 99.03; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₉H₃₈O₄Cl, 365.2453; found, 365.2445.

2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)ethan-1-ol (52)

To a solution of 51 (1.05 g, 2.88 mmol) in MeOH (10 mL), pTsOH × H₂O (0.27 g, 1.44 mmol) was added. The reaction mixture was stirred at rt for 20 h. Saturated NaHCO₃ solution (50 mL) was added, and it was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic layers were washed with brine (200 mL). The organic layers were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (0.70 g, 87%); R_f = 0.25 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.31–1.41 (m, 6H), 1.40–1.48 (m, 2H), 1.50–1.64 (m, 6H), 1.71–1.81 (m, 2H), 2.22 (t, J = 5.9 Hz, 1H), 3.38 (td, J = 6.6, 1.1 Hz, 4H), 3.42–3.48 (m, 2H), 3.48–3.54 (m, 4H), 3.65–3.74 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.61, 26.09, 26.14, 26.81, 29.67, 29.77, 32.65, 45.17, 61.91, 70.77, 70.93, 71.40, 71.85; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₄H₃₀O₃Cl, 281.1878; found, 281.1873.

L6b: 2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)ethyl Methanesulfonate (53)

This compound was prepared using general procedure I and 52 (0.68 g, 2.41 mmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (0.80 g, 93%); R_f = 0.18 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.29–1.48 (m, 8H), 1.50–1.62 (m, 6H), 1.72–1.81 (m, 2H), 3.04 (s, 3H), 3.38 (td, J = 6.6, 2.2 Hz, 4H), 3.44–3.49 (m, 2H), 3.52 (t, J = 6.7 Hz, 2H), 3.65–3.69 (m, 2H), 4.32–4.37 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.62, 26.02, 26.12, 26.81, 29.60, 29.68, 29.80, 32.66, 37.75, 45.20, 68.54, 69.43, 70.80, 70.91, 71.59; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₅H₃₂O₅ClS, 359.1654; found, 359.1647.

L7b: 5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentyl Methanesulfonate (54)

This compound was synthesized as we described previously.⁶⁰

6-((Tetrahydro-2H-pyran-2-yl)oxy)hexan-1-ol (55)

To a solution of 1,6-hexanediol (6.00 g, 50.77 mmol) in dry MeCN (35 mL), 3,4-dihydro-2H-pyran (4.70 g, 5.08 mL, 55.85 mmol) was added under an argon atmosphere. Subsequently, CuSO₄ × 5 H₂O (2.53 g, 10.15 mmol) was added, followed by stirring of the mixture at rt for 3 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (EtOAc/n-hexanes 1:1) to give a colorless oil. Yield (3.72 g, 36%); R_f = 0.18 (EtOAc/n-hexanes 1:1); ¹H NMR (400 MHz, CDCl₃): δ 1.34–1.42 (m, 5H), 1.47–1.64 (m, 8H), 1.67–1.75 (m, 1H), 1.77–1.88 (m, 1H), 3.38 (dt, J = 9.5, 6.5 Hz, 1H), 3.45–3.52 (m, 1H), 3.63 (t, J = 6.6 Hz, 2H), 3.73 (dt, J = 9.6, 6.8 Hz, 1H), 3.82–3.89 (m, 1H), 4.54–4.58 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 19.86, 25.61, 25.68, 26.16, 29.81, 30.91, 32.83, 62.55, 63.04, 67.65, 99.04; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₁H₂₃O₃, 203.1642; found, 203.1638.

6-((Tetrahydro-2H-pyran-2-yl)oxy)hexyl Methanesulfonate (56)

This compound was prepared using general procedure I and 55 (3.00 g, 14.83 mmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (3.81 g, 92%); R_f = 0.18 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.38–1.47 (m, 4H), 1.51–1.64 (m, 6H), 1.67–1.84 (m, 4H), 3.00 (s, 3H), 3.38 (dt, J = 9.7, 6.4 Hz, 1H), 3.46–3.53 (m, 1H), 3.74 (dt, J = 9.6, 6.7 Hz, 1H), 3.82–3.91 (m, 1H), 4.22 (t, J = 6.6 Hz, 2H), 4.54–4.58 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 19.85, 25.42, 25.59, 25.87, 29.19, 29.65, 30.89, 37.48, 62.58, 67.47, 70.18, 99.08; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₂H₂₅O₅S, 281.1417; found, 281.1413.

2-((6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexyl)oxy)tetrahydro-2H-pyran (57)

To a solution of 36 (2.11 g, 8.92 mmol) and 56 (2.50 g, 8.92 mmol) in toluene (40 mL), TBAHS (3.03 g, 8.92 mmol) and 50% NaOH (aq) (4.5 mL) were added. The reaction mixture was stirred at rt for 18 h. H₂O (70 mL) was then added, and the mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were further washed with H₂O and brine (each 200 mL). The organic layers were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a light yellow oil. Yield (1.80 g, 48%); R_f = 0.50 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.31–1.38 (m, 8H), 1.46–1.64 (m, 16H), 1.66–1.83 (m, 4H), 3.35–3.40 (m, 8H), 3.44–3.55 (m, 4H), 3.72 (dt, J = 9.6, 6.8 Hz, 1H), 3.85 (ddd, J = 11.1, 7.5, 3.2 Hz, 1H), 4.53–4.58 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 19.82, 25.61, 25.66, 26.20, 26.26, 26.85, 29.72, 29.84, 30.89, 32.69, 45.20, 62.48, 67.70, 70.79, 70.98, 71.02, 98.97; MS (ESI) m/z: [M + MeOH + H]⁺ calcd for C₂₄H₅₀O₅Cl, 454.10; found, 454.1.

6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexan-1-ol (58)

To a solution of 57 (1.77 g, 4.20 mmol) in MeOH (20 mL), pTsOH × H₂O (0.40 g, 2.10 mmol) was added. The reaction mixture was stirred at rt for 20 h. Saturated NaHCO₃ solution (50 mL) was added, and it was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic layers were washed with brine (200 mL). The organic layers were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a light yellow oil. Yield (1.00 g, 71%); R_f = 0.20 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.29–1.47 (m, 12H), 1.50–1.62 (m, 10H), 1.72–1.81 (m, 2H), 3.38 (td, J = 6.6, 1.7 Hz, 8H), 3.52 (t, J = 6.7 Hz, 2H), 3.62 (t, J = 6.6 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.64, 25.71, 26.12, 26.18, 26.83, 29.69, 29.81, 32.68, 32.82, 45.20, 63.00, 70.79, 70.90, 70.98, 71.02; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₈H₃₈O₃Cl, 337.2504; found, 337.2497.

L8b: 6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexyl Methanesulfonate (59)

This compound was prepared using general procedure I and 58 (1.00 g, 2.97 mmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 1:2) to give a colorless oil. Yield (0.79 g, 64%); R_f = 0.40 (EtOAc/n-hexanes 1:2); ¹H NMR (400 MHz, CDCl₃): δ 1.30–1.47 (m, 12H), 1.51–1.61 (m, 8H), 1.70–1.82 (m, 4H), 2.99 (s, 3H), 3.34–3.41 (m, 8H), 3.53 (t, J = 6.7 Hz, 2H), 4.22 (t, J = 6.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 25.44, 25.66, 25.82, 26.20, 26.85, 29.21, 29.68, 29.72, 29.84, 32.69, 37.48, 45.22, 70.19, 70.72, 70.80, 71.02, 71.04; HRMS (ESI) m/z: [M + H]⁺ calcd for C₁₉H₄₀O₅ClS, 415.2280; found, 415.2270.

Synthesis of E3 Ligands

tert-Butyl N-[(1S)-2-[[(1S)-1-Cyclohexyl-2-[(2S,4R)-4-hydroxy-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (60)

This compound was synthesized as described previously.⁴³ A colorless solid was obtained. Yield (72%); R_f = 0.18 (petroleum ether/EtOAc 1:2); mp 88–90 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.83–1.29 (m, 8H, CH₂), 1.39 (s, 9H, CH₃), 1.52–1.93 (m, 11H), 1.96–2.04 (m, 1H, CH, CH₂, 2-H, 3-H, 3′-H), 2.65–2.75 (m, 2H, 4-H), 2.73 (s, 3H, CH₃), 3.49–3.64 (m, 1H), 3.64–3.79 (m, 1H, 5′-H), 4.28–4.45 (m, 3H, CH, 2′-H, 4′-H), 4.45–4.69 (m, 1H, CH), 4.88–4.96 (m, 1H, 1-H), 5.07 (d, J = 3.6 Hz, 1H, OH), 7.03–7.20 (m, 3H), 7.29 (d, J = 7.6 Hz, 1H, Ar–H), 7.37–7.83 (m, 1H), 8.21 (d, J = 8.8 Hz, 1H, CONH); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.13 (CH₃), 20.58 (C-3), 25.75, 25.93, 26.04, 27.82 (CH₂), 28.18 (C(CH₃)₃), 28.95 (C-4), 29.14 (CH₂), 30.08 (C-2, CH₃), 38.01 (CH, C-3′), 46.69 (C-1), 53.38, 54.75 (CH), 55.71 (C-5′), 58.97 (C-2′), 68.98 (C-4′), 79.24 (C(CH₃)₃), 125.79, 126.71, 128.41, 128.65 (C-5 to C-8), 137.07, 137.86 (C-4a, C-8a), 155.21, 169.81, 170.51, 171.14 (CO); LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₃₂H₄₉N₄O₆, 585.36; found, 585.2.

tert-Butyl N-[(1R)-2-[[(1S)-1-Cyclohexyl-2-[(2S,4R)-4-hydroxy-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (61)

This compound was synthesized analogously to 60 but using Boc-N-Me-d-Ala-OH. A colorless solid was obtained. Yield (61%); R_f = 0.43 (EtOAc); mp 188–190 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.85–1.21 (m, 5H), 1.24 (d, J = 7.2 Hz, 3H), 1.39 (s, 9H), 1.51–2.04 (m, 12H), 2.65–2.73 (m, 2H), 2.73 (s, 3H), 3.59 (dd, J = 2.7, 10.4 Hz, 1H), 3.62–3.79 (m, 1H), 4.33–4.43 (m, 3H), 4.57–4.83 (m, 1H) 4.88–4.98 (m, 1H), 5.04 (d, J = 3.7 Hz, 1H), 7.03–7.10 (m, 2H), 7.10–7.16 (m, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.64, 20.58, 25.72, 25.90, 26.08, 27.72, 28.19, 28.94, 29.09, 30.05, 30.10, 37.97, 46.67, 53.55, 54.64, 55.54, 58.95, 68.87, 79.13, 125.75, 126.69, 128.43, 128.62, 137.04, 137.89, 155.12, 169.93, 171.11, 171.53; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₃₂H₄₉N₄O₆, 585.36; found, 585.5; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₂H₄₉N₄O₆, 585.3647; found, 585.3626.

3-Benzyloxyphenol (62)

Benzyl bromide (3.42 g, 2.4 mL, 20 mmol), resorcinol (4.40 g, 40 mmol), and K₂CO₃ (2.76 g, 20 mmol) in DMF (20 mL) were stirred at 80 °C for 18 h. The brown oil was filtered, rinsed with EtOAc (100 mL), and washed with half-saturated brine (2 × 100 mL) and 5% LiCl solution (100 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated. The material was purified by column chromatography (petroleum ether/EtOAc 19:1) to give a brown oil. Yield (1.92 g, 48%); R_f = 0.37 (petroleum ether/EtOAc 8:1); ¹H NMR (500 MHz, DMSO-d₆): δ 5.02 (s, 2H), 6.32–6.37 (m, 1H), 6.38 (t, J = 2.3 Hz, 1H), 6.42 (dd, J = 2.4, 7.9 Hz, 1H), 7.04 (t, J = 8.2 Hz, 1H), 7.27–7.34 (m, 1H), 7.34–7.41 (m, 2H), 7.39–7.47 (m, 2H), 9.35 (br s, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 69.16, 102.27, 105.64, 108.19, 127.70, 127.86, 128.53, 129.98, 137.41, 158.70, 159.72; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₁₃H₁₃O₂, 201.09; found, 210.0.

tert-Butyl N-[(1S)-2-[[(1S)-2-[(2S,4S)-4-(3-Benzyloxyphenoxy)-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-1-cyclohexyl-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (63)

Compound 60 (2.92 g, 5.0 mmol) was dissolved in dry toluene (100 mL), and it was sonicated for 10 min to remove any dissolved gases. Subsequently, the solution was transferred into a round-bottom flask equipped with a large stirring bar, and the colorless solution was purged with argon for 5 min. Then, the other substrates were added in the following order: 62 (1.05 g, 5.25 mmol), PS-TPP (1 mmol/g loading, 5.75 g), and finally DEAD (40% in toluene, 2.40 mL, 5.25 mmol). After purging for another 5 min, the vessel was closed, and the yellow suspension was stirred at rt for 18 h. The resin was filtered off, and it was washed with EtOAc (2 × 100 mL). The combined organic layers were washed with H₂O and brine (each 250 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by column chromatography as follows: the column was packed and started with petroleum ether/EtOAc 2:1. Then, the polarity was gradually increased to 1:2, and it was eluted until the complete elimination of the side product. Subsequently, the polarity was further increased to petroleum ether/EtOAc 1:4 and kept at this level. A colorless solid was obtained. Yield (1.76 g, 46%); R_f = 0.50 (petroleum ether/EtOAc 1:4); mp 66–68 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 1.07 (s, 3H), 1.01–1.20 (m, 6H), 1.20–1.31 (m, 1H), 1.31–1.45 (m, 12H), 1.53–1.82 (m, 4H), 2.04–2.11 (m, 1H), 2.44–2.56 (m, 1H), 2.67 (q, J = 7.8 Hz, 2H), 2.70 (s, 3H), 3.59 (dd, J = 4.3, 11.0 Hz, 1H), 4.24 (dd, J = 5.9, 10.8 Hz, 1H), 4.30 (t, J = 7.9 Hz, 1H), 4.44 (dd, J = 5.2, 9.0 Hz, 1H), 4.53 (s, 1H), 4.87–4.94 (m, 1H), 5.02 (q, J = 4.0 Hz, 1H), 5.05 (s, 2H), 6.43–6.51 (m, 1H), 6.54 (t, J = 2.4 Hz, 1H), 6.61 (dd, J = 2.4, 8.2 Hz, 1H), 7.01–7.15 (m, 4H), 7.17 (t, J = 8.2 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 7.28–7.34 (m, 1H), 7.34–7.45 (m, 5H), 7.83 (d, J = 8.6 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 14.82, 16.97, 17.63, 20.14, 21.24, 24.31, 25.66, 25.83, 25.98, 28.22, 28.47, 28.95, 29.13, 29.88, 30.23, 33.72, 34.61, 40.97, 46.85, 52.18, 55.33, 58.77, 69.46, 75.16, 80.73, 102.85, 107.88, 108.30, 125.94, 126.93, 127.86, 128.04, 128.55, 128.64, 128.87, 130.31, 137.22, 137.44, 158.31, 159.78, 169.55, 170.11, 170.51; LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₄₅H₅₈N₄O₇, 767.43; found, 767.4.

tert-Butyl N-[(1R)-2-[[(1S)-2-[(2S,4S)-4-(3-Benzyloxyphenoxy)-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-1-cyclohexyl-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (64)

This compound was synthesized analogously to 63 but using precursor 61 (1.75 g, 3.0 mmol) and on a smaller scale (3.0 mmol). A colorless solid was obtained. Yield (1.06 g, 46%); R_f = 0.50 (petroleum ether/EtOAc 1:1); mp 84–86 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.84–0.95 (m, 2H), 1.01–1.12 (m, 4H), 1.24 (d, J = 7.2 Hz, 3H), 1.31–1.37 (m, 10H), 1.52–1.65 (m, 6H), 1.67–1.81 (m, 5H), 2.10 (br s, 1H), 2.63–2.72 (m, 2H), 2.73 (s, 2H), 3.61 (br s, 1H), 4.24–4.40 (m, 2H), 4.44 (dd, J = 5.0, 9.0 Hz, 1H), 4.88–4.94 (m, 1H), 4.96–5.05 (m, 1H), 5.06 (s, 2H), 6.45–6.51 (m, 1H), 6.52–6.56 (m, 1H), 6.61 (dd, J = 2.4, 8.0 Hz, 1H), 7.01–7.20 (m, 4H), 7.23–7.27 (m, 1H), 7.29–7.34 (m, 1H), 7.33–7.45 (m, 4H), 7.86 (s, 1H), 7.92 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 14.56, 16.06, 20.31, 20.54, 21.22, 25.93, 26.06, 26.31, 28.48, 29.20, 29.36, 30.09, 30.51, 35.00, 47.11, 52.55, 53.22, 54.61, 55.29, 58.93, 60.21, 69.72, 75.44, 79.30, 103.10, 108.14, 108.51, 126.18, 127.18, 128.12, 128.28, 128.88, 129.11, 130.55, 137.49, 137.70, 155.50, 158.56, 160.05, 170.24, 170.90, 172.42; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₄₅H₅₈N₄O₇, 767.43; found, 767.5; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₅H₅₉N₄O₇, 767.4378; found, 767.4350.

tert-Butyl N-[(1S)-2-[[(1S)-1-Cyclohexyl-2-[(2S,4S)-4-(3-hydroxyphenoxy)-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (65)

Compound 63 (1.69 g, 2.2 mmol) was dissolved in dry MeOH (20 mL), and 10% Pd/C (0.17 g, 10% w/w) was added. The vessel was closed, evacuated, and refilled with nitrogen gas (3×), followed by hydrogen gas. The black mixture was stirred for 18 h at rt. The charcoal was removed by filtration, and the title compound was obtained as a colorless solid after evaporation. Yield (1.31 g, 81%); R_f = 0.43 (EtOAc); mp 92–96 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.71–1.24 (m, 9H), 1.38 (s, 9H), 1.52–1.84 (m, 10H), 2.01–2.14 (m, 1H), 2.43–2.53 (m, 1H), 2.64–2.76 (m, 5H), 3.60 (dd, J = 4.4, 10.8 Hz, 1H), 4.18–4.27 (m, 1H), 4.32 (t, J = 7.8 Hz, 1H), 4.44 (dd, J = 5.1, 9.0 Hz, 1H), 4.85–5.00 (m, 2H), 6.23–6.40 (m, 3H), 7.00–7.16 (m, 4H), 7.24 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 9.40 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.11, 20.04, 25.61, 25.78, 25.92, 28.17, 28.90, 29.07, 29.65, 29.81, 30.16, 34.64, 39.52, 46.78, 52.25, 53.27, 55.23, 58.65, 74.96, 79.17, 103.14, 106.14, 108.55, 125.89, 126.86, 128.51, 128.79, 130.06, 137.17, 137.40, 155.27, 158.29, 158.76, 169.40, 169.97, 170.38; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₃₈H₅₃N₄O₇, 677.39; found, 677.6.

tert-Butyl N-[(1R)-2-[[(1S)-1-Cyclohexyl-2-[(2S,4S)-4-(3-hydroxyphenoxy)-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (66)

This compound was synthesized analogously to compound 65 but using precursor 64 (1.02 g, 1.33 mmol) in dry MeOH (13 mL) and 10% Pd/C (0.10 g, 10% w/w). A colorless solid was obtained. Yield (0.87 g, 96%); R_f = 0.57 (EtOAc); mp 92–96 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.82–1.28 (m, 9H), 1.35 (s, 9H), 1.53–1.82 (m, 10H), 2.01–2.21 (m, 1H), 2.27–2.49 (m, 1H), 2.62–2.71 (m, 2H), 2.74 (s, 3H), 3.62 (br s, 1H), 4.31 (p, J = 13.5, 15.6 Hz, 2H), 4.44 (dd, J = 4.8, 9.0 Hz, 1H), 4.88–4.96 (m, 2H), 6.19–6.45 (m, 3H), 6.93–7.19 (m, 4H), 7.25 (d, J = 7.5 Hz, 1H), 7.86 (br s, 1H), 7.91 (br s, 1H), 9.39 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.73, 19.94, 25.59, 25.73, 25.98, 28.15, 28.88, 29.04, 29.65, 29.75, 30.19, 34.77, 46.79, 52.27, 52.84, 54.93, 58.57, 74.96, 79.23, 103.14, 106.09, 108.55, 125.88, 126.87, 128.59, 128.79, 130.06, 137.17, 137.36, 155.19, 158.24, 158.75, 169.41, 170.47, 170.99; LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₃₈H₅₃N₄O₇, 677.39; found, 677.7; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₈H₅₃N₄O₇, 677.3909; found, 677.3896.

tert-Butyl N-[(1S)-2-[[(1S)-1-Cyclohexyl-2-oxo-2-[(2S,4S)-4-phenoxy-2-[[(1R)-tetralin-1-yl]carbamoyl]pyrrolidin-1-yl]ethyl]amino]-1-methyl-2-oxo-ethyl]-N-methyl-carbamate (67)

This compound was synthesized analogously to 63 but using phenol (99 mg, 1.05 mmol) instead of 62 and on a smaller scale (1.0 mmol). A colorless solid was obtained. Yield (185 mg, 28%); R_f = 0.22 (petroleum ether/EtOAc 1:4); mp 70–74 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 0.97–1.26 (m, 8H), 1.38 (s, 9H), 1.50–1.84 (m, 11H), 2.06–2.14 (m, 1H), 2.49–2.58 (m, 1H), 2.64–2.74 (m, 5H), 3.62 (dd, J = 4.4, 10.7 Hz, 1H), 4.26 (dd, J = 6.0, 10.7 Hz, 1H), 4.32 (t, J = 7.8 Hz, 1H), 4.45 (dd, J = 5.1, 9.1 Hz, 1H), 4.87–4.95 (m, 1H), 4.99–5.07 (m, 1H), 6.83–6.98 (m, 3H), 7.01–7.17 (m, 3H), 7.21–7.32 (m, 3H), 7.63 (br s, 1H), 7.82 (d, J = 8.5 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 14.75, 20.01, 25.59, 25.76, 25.91, 28.16, 28.88, 29.09, 29.79, 30.16, 34.54, 36.86, 46.76, 52.18, 53.01, 55.24, 58.71, 75.04, 79.18, 115.70, 121.22, 125.86, 126.86, 128.51, 128.78, 129.70, 137.15, 137.38, 154.94, 157.07, 169.99, 170.43; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₃₈H₅₃N₄O₆, 661.39; found, 661.5; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₈H₅₃N₄O₆, 661.3960; found, 661.3916.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2 (Methylamino)propanamido)acetyl)-4-phenoxy-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (CST530)⁵³

Compound 67 (70 mg, 106 μmol) was stirred with 1 M HCl in EtOAc (3 mL) for 3 h at rt. Subsequently, it was diluted with EtOAc (25 mL) and washed with saturated NaHCO₃ solution and brine (each 10 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The product material was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 29:1) to give the title compound as a colorless solid. Yield (49 mg, 83%); R_f = 0.37 (CH₂Cl₂/MeOH + NH₃ 19:1); mp 80–84 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 0.88–1.18 (m, 9H), 1.52–1.85 (m, 10H), 2.16 (s, 3H), 2.04–2.13 (m, 1H), 2.50–2.59 (m, 1H), 2.62–2.77 (m, 2H), 2.87–3.00 (m, 1H), 3.64 (dd, J = 4.5, 10.8 Hz, 1H), 4.23–4.34 (m, 1H), 4.39 (dd, J = 7.1, 8.6 Hz, 1H), 4.45 (dd, J = 5.2, 9.0 Hz, 1H), 4.87–4.95 (m, 1H), 5.04 (p, J = 5.4 Hz, 1H), 6.88–6.92 (m, 2H), 6.95 (t, J = 7.5 Hz, 1H), 7.02–7.19 (m, 3H), 7.21–7.33 (m, 3H), 7.84 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 19.19, 19.97, 25.55, 25.75, 25.92, 27.91, 28.86, 29.18, 29.77, 34.39, 34.54, 46.73, 52.15, 54.45, 58.63, 59.28, 74.96, 115.69, 121.20, 125.86, 126.84, 128.54, 128.76, 129.69, 137.14, 137.35, 157.06, 169.98, 170.57, 174.56; LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₃₃H₄₅N₄O₄, 561.34; found, 561.3.

(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (68)

This compound was synthesized as we described previously.²⁰

(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (69)

This compound was synthesized as described previously.^61,62

(2S,4S)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (70)

This compound was synthesized as described previously.⁶³

(2S,4R)-4-Hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (71)

This compound was synthesized as we described previously.⁶⁰

2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (72)

This compound was synthesized as we described previously.¹⁶

4-Fluoro-2-(1-methyl-2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (73)

This compound was synthesized as we described previously.²⁰

Synthesis of E3–Linker Conjugates

Benzyl 5-(3-(((3S,5S)-1-((S)-2-((S)-2-((tert-Butoxycarbonyl)(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-3-yl)oxy)phenoxy)pentanoate (74)

This compound was prepared using general procedure II, linker L1a (68 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 39:1) to give a colorless oil. Yield (156 mg, 60%); R_f = 0.40 (CH₂Cl₂/MeOH 39:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.77–1.12 (m, 6H), 1.18 (s, 3H), 1.38 (s, 9H), 1.51–1.83 (m, 14H), 2.05–2.12 (m, 1H), 2.38–2.56 (m, 3H), 2.63–2.75 (m, 5H), 3.55–3.63 (m, 1H), 3.85–3.96 (m, 2H), 4.23 (dd, J = 6.1, 11.0 Hz, 1H), 4.31 (t, J = 7.9 Hz, 1H), 4.44 (dd, J = 5.1, 9.1 Hz, 1H), 4.88–5.05 (m, 2H), 5.07 (d, J = 1.2 Hz, 2H), 6.40–6.54 (m, 3H), 7.00–7.18 (m, 4H), 7.21–7.26 (m, 1H), 7.28–7.39 (m, 5H), 7.81 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.3 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.28, 20.10, 21.38, 25.62, 25.78, 25.94, 28.16, 28.18, 28.90, 29.10, 29.84, 30.18, 33.27, 34.57, 46.80, 52.16, 53.47, 55.26, 58.72, 65.53, 67.20, 75.11, 79.19, 102.50, 107.50, 107.85, 125.88, 126.86, 128.08, 128.14, 128.49, 128.58, 128.81, 130.19, 136.43, 137.16, 137.41, 154.69, 158.26, 160.02, 170.04, 170.46, 172.81; LC–MS (ESI) 96% purity, m/z: [M + H]⁺ calcd for C₅₀H₆₇N₄O₉, 867.49; found, 867.6; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₀H₆₇N₄O₉, 867.4863; found, 867.4861.

(2S,4R)-1-((S)-2-(8-Chlorooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (75)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-1-((S)-2-(4-(4-Chlorobutoxy)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (76)

This compound was prepared using general procedure III, linker L3a (97 mg, 0.50 mmol), and VHL ligand 68 (265 mg, 0.62 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless semi-solid. Yield (191 mg, 63%); R_f = 0.35 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 1.21–1.28 (m, 1H), 1.54–1.62 (m, 2H), 1.61–1.80 (m, 4H), 1.86–1.93 (m, 1H), 1.99–2.06 (m, 1H), 2.13–2.21 (m, 1H), 2.22–2.31 (m, 1H), 2.43 (s, 3H), 3.27–3.40 (m, 3H), 3.56–3.70 (m, 4H), 4.21 (dd, J = 5.5, 15.8 Hz, 1H), 4.31–4.37 (m, 1H), 4.39–4.46 (m, 2H), 4.53 (d, J = 9.3 Hz, 1H), 5.10 (d, J = 3.6 Hz, 1H), 7.35–7.45 (m, 4H), 7.84 (d, J = 9.3 Hz, 1H), 8.53 (t, J = 6.1 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.09, 25.73, 26.53, 26.75, 29.29, 31.78, 35.38, 38.10, 40.24, 41.82, 45.49, 45.51, 56.49, 56.53, 58.86, 69.03, 69.28, 69.61, 127.60, 128.80, 129.81, 131.33, 139.67, 147.89, 151.59, 169.84, 171.95, 172.10; LC–MS (ESI) 95% purity, m/z: [M + H]⁺ calcd for C₃₀H₄₄ClN₄O₅S, 607.27; found, 607.3; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₀H₄₄ClN₄O₅S, 607.2716; found, 607.2707.

(2S,4R)-1-((S)-2-(2-(2-(2-Chloroethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (77)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-1-((S)-2-(tert-Butyl)-14-chloro-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (78)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-1-((S)-2-(2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (79)

This compound was prepared using general procedure III, linker L6a (147 mg, 0.50 mmol), and VHL ligand 68 (265 mg, 0.62 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless oil. Yield (233 mg, 66%); R_f = 0.27 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 1.30–1.62 (m, 14H), 1.70–1.78 (m, 2H), 2.05–2.13 (m, 1H), 2.44–2.52 (m, 1H), 2.49 (s, 3H), 3.33–3.38 (m, 4H), 3.41–3.52 (m, 4H), 3.61 (dd, J = 3.8, 11.2 Hz, 1H), 3.82–3.94 (m, 2H), 4.01–4.06 (m, 1H), 4.32 (dd, J = 5.4, 15.1 Hz, 1H), 4.46 (d, J = 8.7 Hz, 1H), 4.48–4.55 (m, 2H), 4.69 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.33 (s, 4H), 7.40 (t, J = 6.0 Hz, 1H), 8.76 (s, 1H). The signal for OH is missing. ¹³C NMR (151 MHz, DMSO-d₆): δ 15.63, 25.48, 25.90, 25.98, 26.34, 26.67, 29.40, 29.65, 32.51, 34.96, 35.90, 43.17, 45.04, 55.24, 56.62, 56.92, 58.50, 69.81, 70.07, 70.70, 70.77, 71.85, 128.14, 129.45, 130.35, 132.11, 138.40, 147.58, 150.75, 170.51, 170.76, 171.27; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₃₆H₅₆ClN₄O₆S, 707.36; found, 707.6; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₆H₅₆ClN₄O₆S, 707.3604; found, 707.3592.

(2S,4R)-1-((S)-2-(5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (80)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-1-((S)-2-(6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (81)

This compound was prepared using general procedure III, linker L8a (175 mg, 0.50 mmol), and VHL ligand 68 (265 mg, 0.62 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless oil. Yield (225 mg, 59%); R_f = 0.31 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 1.20–1.32 (m, 8H), 1.33–1.40 (m, 2H), 1.40–1.55 (m, 10H), 1.65–1.72 (m, 2H), 1.86–1.93 (m, 1H), 1.98–2.05 (m, 1H), 2.06–2.13 (m, 1H), 2.21–2.29 (m, 1H), 2.43 (s, 3H), 3.24–3.33 (m, 8H), 3.60 (t, J = 6.6 Hz, 2H), 3.63 (d, J = 10.4 Hz, 1H), 3.63–3.69 (m, 1H), 4.20 (dd, J = 5.5, 15.8 Hz, 1H), 4.32–4.36 (m, 1H), 4.39–4.45 (m, 2H), 4.53 (d, J = 9.3 Hz, 1H), 5.10 (d, J = 3.6 Hz, 1H), 7.37 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 9.3 Hz, 1H), 8.52 (t, J = 6.1 Hz, 1H), 8.96 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.10, 25.17, 25.48, 25.58, 25.73, 25.76, 26.26, 26.55, 29.17, 29.25, 29.38, 29.41, 32.19, 35.03, 35.37, 38.11, 41.83, 45.51, 56.47, 56.49, 58.87, 69.04, 69.95, 70.06, 127.60, 128.81, 129.81, 131.34, 139.67, 147.89, 151.59, 169.89, 172.12, 172.23; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₄₀H₆₃ClN₄O₆S, 763.42; found, 763.9; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₀H₆₃ClN₄O₆S, 763.4230; found, 763.4215.

Benzyl 5-(3-(((3S,5S)-1-((S)-2-((R)-2-((tert-Butoxycarbonyl)(methyl)amino)propanamido)-2-cyclohexylacetyl)-5-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-3-yl)oxy)phenoxy)pentanoate (82)

This compound was prepared using general procedure II, linker L1a (68 mg, 0.30 mmol), and IAP ligand 66. The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless oil. Yield (156 mg, 60%); R_f = 0.40 (CH₂Cl₂/MeOH 39:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.82–1.17 (m, 6H), 1.24 (s, 3H), 1.35 (s, 9H), 1.49–1.85 (m, 14H), 2.00–2.19 (m, 1H), 2.33–2.47 (m, 3H), 2.61–2.81 (m, 5H), 3.54–3.69 (m, 1H), 3.85–3.93 (m, 2H), 4.23–4.39 (m, 2H), 4.43 (dd, J = 4.9, 9.0 Hz, 1H), 4.87–5.03 (m, 2H), 5.08 (s, 2H), 6.39–6.58 (m, 3H), 7.00–7.18 (m, 4H), 7.25 (d, J = 7.5 Hz, 1H), 7.29–7.38 (m, 5H), 7.83 (s, 1H), 7.92 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.75, 20.00, 21.34, 25.60, 25.73, 25.98, 28.15, 28.87, 29.02, 29.77, 30.19, 33.26, 34.67, 46.79, 52.41, 53.80, 54.95, 58.63, 65.51, 67.18, 75.13, 78.97, 102.46, 107.52, 107.81, 125.85, 126.86, 128.06, 128.12, 128.57, 128.79, 130.17, 136.42, 137.15, 137.98, 155.18, 158.21, 160.00, 169.88, 170.43, 172.17, 172.78; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₅₀H₆₇N₄O₉, 867.49; found, 867.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₀H₆₇N₄O₉, 867.4863; found, 867.4886.

(2S,4R)-N-(2-((5-Chloropentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (83)

This compound was prepared using general procedure IV and linker L1b (240 mg, 1.20 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (346 mg, 53%); R_f = 0.30 (CH₂Cl₂/MeOH 19:1); mp 84–86 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 1.52–1.62 (m, 2H), 1.71–1.84 (m, 4H), 1.88–1.95 (m, 1H), 2.00–2.09 (m, 1H), 2.27–2.36 (m, 1H), 2.46 (s, 3H), 3.64–3.71 (m, 3H), 3.74–3.81 (m, 1H), 4.05 (t, J = 6.2 Hz, 2H), 4.17–4.36 (m, 3H), 4.37–4.58 (m, 3H), 4.70 (d, J = 10.8 Hz, 1H), 5.08 (d, J = 4.1 Hz, 1H), 6.96–7.03 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.45–7.53 (m, 1H), 7.56–7.64 (m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 8.36 (t, J = 5.9 Hz, 1H), 8.98 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.21, 18.80, 19.06, 23.23, 28.11, 28.57, 31.94, 37.21, 38.28, 45.54, 46.99, 55.59, 57.96, 58.87, 67.71, 68.79, 111.91, 120.98, 123.20, 123.81, 127.15, 127.86, 128.09, 131.17, 131.49, 131.56, 131.77, 142.39, 148.09, 151.65, 156.10, 167.66, 168.28, 171.72; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₃₄H₄₂ClN₄O₅S, 653.26; found, 653.3; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₄H₄₂ClN₄O₅S, 653.2559; found, 653.2548.

(2S,4R)-N-(2-((8-Chlorooctyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (84)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-N-(2-(4-(4-Chlorobutoxy)butoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (85)

This compound was prepared using general procedure IV and linker L3b (310 mg, 1.20 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (302 mg, 60%); R_f = 0.37 (CH₂Cl₂/MeOH 29:1); mp 78–80 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 1.56–1.64 (m, 2H), 1.64–1.83 (m, 6H), 1.88–1.95 (m, 1H), 1.99–2.07 (m, 1H), 2.26–2.37 (m, 1H), 2.46 (s, 3H), 3.34–3.47 (m, 4H), 3.62 (t, J = 6.7 Hz, 2H), 3.65–3.71 (m, 1H), 3.77 (dd, J = 4.4, 10.6 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 4.18–4.35 (m, 3H), 4.37–4.59 (m, 3H), 4.70 (d, J = 10.9 Hz, 1H), 5.08 (d, J = 4.1 Hz, 1H), 6.96–7.01 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.49 (ddd, J = 2.2, 6.2, 8.1 Hz, 1H), 7.56–7.64 (m, 2H), 7.70 (dd, J = 1.0, 7.6 Hz, 1H), 8.36 (t, J = 6.0 Hz, 1H), 8.98 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.20, 18.81, 19.06, 25.86, 26.07, 26.80, 28.57, 29.35, 37.22, 38.27, 45.52, 46.99, 55.60, 57.96, 58.88, 67.74, 68.80, 69.32, 69.85, 111.88, 120.95, 123.20, 123.81, 127.15, 127.88, 128.09, 131.16, 131.49, 131.56, 131.77, 142.39, 148.07, 151.65, 156.09, 167.66, 168.28, 171.71; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₃₇H₄₈ClN₄O₆S, 711.30; found, 711.5; HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₇H₄₈ClN₄O₆S, 711.2978; found, 711.2965.

(2S,4R)-N-(2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (86)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-N-(2-(2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (87)

This compound was synthesized as we described previously.⁶⁰

(2S,4R)-N-(2-(2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)ethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (88)

This compound was prepared using general procedure IV and linker L6b (430 mg, 1.20 mmol). The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂), followed by preparative HPLC (gradient from 70 to 100% MeOH) to separate the inversely attached linker–ligand side product. A colorless resin of the chloroalkane product was obtained. Yield (316 mg, 39%); R_f = 0.29 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 1.24–1.39 (m, 8H), 1.40–1.55 (m, 6H), 1.64–1.72 (m, 2H), 1.88–1.95 (m, 1H), 1.99–2.07 (m, 1H), 2.27–2.37 (m, 1H), 2.46 (s, 3H), 3.26–3.32 (m, 4H), 3.47 (t, J = 6.5 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 3.65–3.80 (m, 4H), 4.13–4.36 (m, 5H), 4.37–4.60 (m, 3H), 4.71 (d, J = 10.8 Hz, 1H), 5.06 (s, 1H), 7.00 (dd, J = 1.6, 7.8 Hz, 1H), 7.05 (d, J = 1.7 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.45–7.53 (m, 1H), 7.57–7.64 (m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 8.32 (t, J = 6.0 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.16, 18.76, 19.03, 25.15, 25.64, 25.72, 26.25, 28.54, 29.23, 29.36, 32.17, 37.21, 38.22, 45.49, 46.96, 55.54, 57.94, 58.86, 68.12, 68.77, 68.82, 69.93, 70.05, 70.61, 112.42, 121.21, 123.16, 123.75, 127.37, 127.85, 128.05, 131.13, 131.41, 131.54, 131.72, 142.34, 148.06, 151.58, 156.08, 167.63, 168.25, 171.68; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₄₃H₆₀ClN₄O₇S, 811.39; found, 811.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₃H₆₀ClN₄O₇S, 811.3866; found, 811.3860.

(2S,4R)-N-(2-((5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (89)

This compound was prepared using general procedure IV and linker L7b (464 mg, 1.20 mmol). The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless resin. Yield (411 mg, 49%); R_f = 0.35 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 1.24–1.40 (m, 6H), 1.41–1.52 (m, 8H), 1.52–1.64 (m, 2H), 1.65–1.72 (m, 2H), 1.73–1.83 (m, 2H), 1.86–1.97 (m, 1H), 1.99–2.07 (m, 1H), 2.27–2.39 (m, 1H), 2.46 (s, 3H), 3.31–3.39 (m, 8H), 3.59 (t, J = 6.6 Hz, 2H), 3.62–3.73 (m, 1H), 3.73–3.82 (m, 1H), 4.04 (t, J = 6.3 Hz, 2H), 4.18–4.59 (m, 6H), 4.71 (d, J = 10.8 Hz, 1H), 5.06 (s, 1H), 6.96–7.02 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.45–7.55 (m, 1H), 7.57–7.64 (m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 8.33 (t, J = 5.9 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.15, 18.76, 19.02, 22.55, 22.68, 25.13, 26.24, 28.53, 28.64, 29.10, 29.19, 29.22, 32.17, 37.17, 38.21, 45.48, 46.96, 55.53, 57.94, 58.84, 67.86, 68.77, 69.92, 70.01, 70.04, 70.08, 111.86, 120.90, 123.16, 123.75, 127.14, 127.84, 128.04, 131.13, 131.46, 131.53, 131.72, 142.35, 148.03, 151.56, 156.10, 167.63, 168.26, 171.66; LC–MS (ESI) 94% purity, m/z: [M + H]⁺ calcd for C₄₅H₆₄ClN₄O₇S, 839.42; found, 839.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₅H₆₄ClN₄O₇S, 839.4179; found, 839.4160.

(2S,4R)-N-(2-((6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (90)

This compound was prepared using general procedure IV and linker L8b (498 mg, 1.20 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless resin. Yield (269 mg, 31%); R_f = 0.27 (CH₂Cl₂/MeOH 19:1); ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 1.24–1.41 (m, 10H), 1.42–1.55 (m, 10H), 1.64–1.71 (m, 2H), 1.71–1.79 (m, 2H), 1.88–1.95 (m, 1H), 1.99–2.06 (m, 1H), 2.27–2.35 (m, 1H), 2.46 (s, 3H), 3.27–3.36 (m, 8H), 3.59 (t, J = 6.6 Hz, 2H), 3.67 (d, J = 10.6 Hz, 1H), 3.77 (dd, J = 4.5, 10.6 Hz, 1H), 4.03 (t, J = 6.3 Hz, 2H), 4.16–4.60 (m, 6H), 4.70 (d, J = 10.8 Hz, 1H), 5.06 (d, J = 4.1 Hz, 1H), 6.96–7.01 (m, 2H), 7.32 (d, J = 7.6 Hz, 1H), 7.46–7.52 (m, 1H), 7.57–7.64 (m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 8.33 (t, J = 6.0 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.17, 18.78, 19.05, 25.17, 25.58, 25.66, 25.74, 26.26, 28.55, 28.83, 29.25, 29.38, 32.20, 37.19, 38.23, 45.51, 46.99, 55.56, 57.97, 58.87, 67.86, 68.79, 69.95, 70.03, 70.06, 111.87, 120.93, 123.19, 123.77, 127.17, 127.88, 128.07, 131.15, 131.49, 131.55, 131.75, 142.37, 148.05, 151.58, 156.13, 167.66, 168.29, 171.68; LC–MS (ESI) 96% purity, m/z: [M + H]⁺ calcd for C₄₇H₆₈ClN₄O₇S, 867.45; found, 867.6; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₇H₆₈ClN₄O₇S, 867.4492; found, 867.4482.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((5-Chloropentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (91)

This compound was prepared using general procedure V and linker L1b (72 mg, 0.36 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a pale yellow oil. Yield (0.18 g, 75%); R_f = 0.20 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.75–1.00 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.37–1.42 (m, 1H), 1.47 (s, 9H), 1.49–1.57 (m, 5H), 1.71–1.88 (m, 8H), 1.98–2.10 (m, 1H), 2.26–2.37 (m, 1H), 2.68–2.81 (m, 5H), 2.92 (s, 1H), 3.52–3.60 (m, 2H), 3.71–3.84 (m, 2H), 3.89 (t, J = 6.3 Hz, 2H), 4.18 (dd, J = 11.3, 4.8 Hz, 1H), 4.42 (t, J = 7.9 Hz, 1H), 4.55–4.70 (m, 1H), 4.76 (d, J = 9.3 Hz, 1H), 4.94 (d, J = 4.8 Hz, 1H), 5.13 (q, J = 5.0 Hz, 1H), 6.33–6.43 (m, 2H), 6.53 (dd, J = 8.2, 2.2 Hz, 1H), 6.59 (d, J = 8.3 Hz, 2H), 7.02–7.19 (m, 4H), 7.27–7.32 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ; 19.89, 23.41, 25.34, 25.45, 25.71, 28.23, 28.42, 28.72, 28.85, 29.10, 29.57, 29.81, 31.27, 33.25, 34.90, 40.31, 42.34, 42.71, 47.42, 48.70, 53.49, 54.65, 59.91, 61.54, 67.42, 72.10, 76.07, 102.59, 107.29, 108.18, 125.99, 126.89, 128.54, 128.89, 129.90, 136.50, 137.25, 157.77, 160.09, 170.94, 172.37; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₃H₆₂O₇N₄Cl, 781.4302; found, 781.4284.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((8-Chlorooctyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (92)

This compound was prepared using general procedure V and linker L2b (87 mg, 0.36 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a colorless oil. Yield (0.20 g, 82%); R_f = 0.22 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.75–0.97 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.32–1.44 (m, 8H), 1.47 (s, 9H), 1.50–1.58 (m, 4H), 1.69–1.86 (m, 8H), 1.98–2.09 (m, 1H), 2.27–2.37 (m, 1H), 2.70–2.80 (m, 5H), 2.90–2.94 (m, 1H), 3.53 (t, J = 6.7 Hz, 2H), 3.75–3.84 (m, 2H), 3.87 (t, J = 6.5 Hz, 2H), 4.17 (dd, J = 11.5, 4.8 Hz, 1H), 4.42 (d, J = 7.9 Hz, 1H), 4.59–4.69 (m, 1H), 4.76 (dd, J = 9.7, 2.1 Hz, 1H), 4.93 (d, J = 5.1 Hz, 1H), 5.13 (q, J = 7.6 Hz, 1H), 6.31–6.41 (m, 2H), 6.51–6.55 (m, 1H), 6.58 (d, J = 8.3 Hz, 1H), 7.01–7.17 (m, 5H), 7.27–7.32 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 22.73, 25.45, 25.57, 25.82, 25.87, 26.77, 28.49, 28.78, 29.14, 29.22, 29.70, 29.93, 31.36, 33.42, 35.07, 40.46, 40.47, 42.56, 47.49, 47.54, 48.80, 53.56, 54.70, 60.04, 60.19, 67.83, 74.19, 102.68, 102.83, 107.73, 107.85, 107.97, 126.14, 127.01, 128.70, 128.99, 129.97, 136.57, 137.34, 160.38, 171.00, 172.54, 175.16; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₆H₆₈O₇N₄Cl, 823.4771; found, 823.4753.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(4-(4-Chlorobutoxy)butoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (93)

This compound was prepared using general procedure V and linker L3b (165 mg, 0.64 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a colorless oil. Yield (0.30 g, 69%); R_f = 0.35 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.75–0.97 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.37–1.44 (m, 1H), 1.47 (s, 9H), 1.49–1.59 (m, 4H), 1.69–1.89 (m, 10H), 1.98–2.08 (m, 1H), 2.28–2.38 (m, 1H), 2.71–2.79 (m, 5H), 2.86–2.93 (m, 1H), 3.42–3.52 (m, 6H), 3.57 (t, J = 6.6 Hz, 2H), 3.73–3.87 (m, 2H), 3.90 (d, J = 6.2 Hz, 2H), 4.18 (dd, J = 11.5, 4.8 Hz, 1H), 4.42 (t, J = 7.9 Hz, 1H), 4.57–4.70 (m, 0H), 4.76 (dd, J = 9.9, 2.2 Hz, 1H), 4.94 (dd, J = 82.8, 5.0 Hz, 1H), 5.09–5.16 (m, 1H), 6.32–6.42 (m, 2H), 6.51–6.55 (m, 1H), 6.58 (d, J = 8.4 Hz, 2H), 7.02–7.18 (m, 4H), 7.27–7.32 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 22.73, 25.46, 25.56, 25.83, 26.08, 26.22, 26.28, 27.01, 28.21, 28.49, 29.22, 29.68, 29.93, 31.37, 33.42, 35.00, 40.48, 42.41, 47.52, 48.81, 53.55, 54.73, 60.01, 67.60, 73.52, 102.74, 102.87, 107.52, 108.15, 126.12, 127.00, 128.73, 128.99, 129.98, 136.60, 137.36, 157.90, 160.29, 171.08, 172.51, 175.11; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₆H₆₈O₈N₄Cl, 839.4720; found, 839.4703.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (94)

This compound was prepared using general procedure V and linker L4b (74 mg, 0.30 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a pale yellow oil. Yield (0.14 g, 66%); R_f = 0.18 (CH₂Cl₂/MeOH 20:1); ¹H NMR (400 MHz, CDCl₃): δ 0.78–0.95 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.37–1.43 (m, 1H), 1.47 (s, 9H), 1.56 (s, 4H), 1.75–1.85 (m, 4H), 1.99–2.08 (m, 1H), 2.27–2.36 (m, 1H), 2.71–2.80 (m, 5H), 2.90 (d, J = 14.1 Hz, 1H), 3.63 (td, J = 5.8, 0.6 Hz, 2H), 3.68–3.82 (m, 7H), 3.82–3.87 (m, 2H), 4.07 (d, J = 4.6 Hz, 2H), 4.19 (dd, J = 11.4, 4.8 Hz, 1H), 4.41 (t, J = 7.9 Hz, 1H), 4.57–4.69 (m, 1H), 4.76 (d, J = 9.6 Hz, 1H), 4.93 (d, J = 4.2 Hz, 1H), 5.12 (q, J = 7.7 Hz, 1H), 6.37–6.42 (m, 2H), 6.56 (d, J = 7.2 Hz, 1H), 6.56–6.62 (m, 2H), 7.02–7.18 (m, 4H), 7.27–7.31 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 20.08, 25.53, 25.64, 25.89, 28.42, 28.52, 29.34, 29.82, 30.01, 33.48, 40.64, 42.84, 47.70, 53.68, 55.33, 60.18, 67.50, 69.83, 70.78, 70.88, 71.47, 76.25, 103.07, 107.72, 108.48, 126.30, 127.22, 128.73, 129.18, 130.18, 136.64, 137.47, 157.89, 160.13, 169.46, 171.68, 172.36; HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₄H₆₄O₉N₄Cl, 827.4356; found, 827.4340.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (95)

This compound was synthesized as we described previously.⁶⁰

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-((6-((6-Chlorohexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (96)

This compound was prepared using general procedure V and linker L6b (118 mg, 0.33 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a colorless oil. Yield (0.15 g, 58%); R_f = 0.30 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.79–0.99 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.33–1.43 (m, 8H), 1.47 (s, 9H), 1.50–1.58 (m, 10H), 1.72–1.86 (m, 6H), 1.99–2.08 (m, 1H), 2.26–2.37 (m, 1H), 2.70–2.81 (m, 5H), 2.90 (d, J = 14.1 Hz, 1H), 3.39 (td, J = 6.6, 1.5 Hz, 4H), 3.46–3.57 (m, 5H), 3.75 (d, J = 3.5 Hz, 2H), 3.80 (d, J = 11.5 Hz, 1H), 4.04 (t, J = 4.6 Hz, 2H), 4.18 (dd, J = 11.4, 4.9 Hz, 1H), 4.42 (t, J = 7.9 Hz, 1H), 4.56–4.69 (m, 1H), 4.76 (dd, J = 9.8, 2.2 Hz, 1H), 4.93 (t, J = 4.8 Hz, 1H), 5.13 (d, J = 6.1 Hz, 1H), 6.36–6.42 (m, 2H), 6.55 (dd, J = 2.3, 0.8 Hz, 1H), 6.56–6.62 (m, 2H), 7.03–7.18 (m, 4H), 7.27–7.31 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 22.74, 25.45, 25.56, 25.81, 25.90, 26.01, 26.73, 28.42, 28.48, 29.16, 29.22, 29.55, 29.58, 29.63, 29.70, 29.93, 31.36, 33.38, 35.08, 40.44, 42.53, 47.54, 48.79, 53.56, 54.72, 60.04, 60.20, 67.38, 69.04, 70.59, 70.80, 74.01, 102.88, 107.64, 108.32, 126.15, 127.04, 128.63, 129.00, 129.99, 136.57, 137.34, 157.81, 160.08, 170.99, 172.54, 175.19; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₂H₈₀O₉N₄Cl, 939.5608; found, 939.5650.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((5-((5-((6-Chlorohexyl)oxy)pentyl)oxy)pentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (97)

This compound was prepared using general procedure V and linker L7b (254 mg, 0.66 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a pale yellow oil. Yield (0.36 g, 67%); R_f = 0.28 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.77–0.98 (m, 5H), 1.29 (d, J = 7.1 Hz, 3H), 1.33–1.44 (m, 5H), 1.46 (s, 9H), 1.50–1.65 (m, 15H), 1.72–1.86 (m, 8H), 1.98–2.10 (m, 1H), 2.27–2.37 (m, 1H), 2.75 (d, J = 4.6 Hz, 5H), 2.89–2.93 (m, 1H), 3.36–3.44 (m, 8H), 3.52 (t, J = 6.7 Hz, 2H), 3.74–3.85 (m, 2H), 3.88 (t, J = 6.5 Hz, 2H), 4.17 (dd, J = 11.4, 4.7 Hz, 1H), 4.41 (t, J = 7.9 Hz, 1H), 4.58–4.69 (m, 1H), 4.75 (dd, J = 9.8, 2.2 Hz, 1H), 4.93 (t, J = 4.8 Hz, 1H), 5.12 (q, J = 6.1 Hz, 1H), 6.34–6.40 (m, 2H), 6.50–6.54 (m, 1H), 6.55–6.71 (m, 2H), 7.02–7.17 (m, 4H), 7.27–7.31 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 22.73, 25.44, 25.55, 25.81, 25.90, 26.74, 28,24, 28.47, 29.04, 29.15, 29.22, 29.45, 29.51, 29.58, 29.70, 29.93, 31.36, 33.42, 35.09, 40.45, 42.53, 47.53, 48.79, 53.56, 54.70, 60.05, 60.20, 67.78, 74.83, 76.17, 102.86, 107.62, 107.82, 107.87, 126.14, 127.02, 128.62, 128.99, 129.96, 136.55, 137.33, 160.35, 167.57, 170.99, 172.53, 175.19; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₄H₈₄O₉N₄Cl, 967.5921; found, 967.5918.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((6-((6-((6-Chlorohexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (98)

This compound was prepared using general procedure V and linker L8b (136 mg, 0.33 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a colorless oil. Yield (0.24 g, 88%); R_f = 0.25 (CH₂Cl₂/MeOH 50:1); ¹H NMR (400 MHz, CDCl₃): δ 0.78–1.01 (m, 5H), 1.30 (d, J = 7.1 Hz, 3H), 1.34–1.44 (m, 12H), 1.47 (s, 9H), 1.51–1.59 (m, 10H), 1.73–1.84 (m, 8H), 1.99–2.09 (m, 1H), 2.28–2.38 (m, 1H), 2.72–2.79 (m, 5H), 2.89 (d, J = 14.1 Hz, 1H), 3.37–3.43 (m, 8H), 3.49 (d, J = 5.2 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H), 3.77–3.85 (m, 2H), 3.87 (t, J = 6.5 Hz, 2H), 4.17 (dd, J = 11.5, 4.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.57–4.69 (m, 1H), 4.76 (d, J = 9.6 Hz, 1H), 4.94 (d, J = 4.3 Hz, 1H), 5.14 (d, J = 4.8 Hz, 1H), 6.31–6.41 (m, 2H), 6.51–6.55 (m, 1H), 6.58 (d, J = 8.4 Hz, 1H), 6.68 (s, 1H), 7.03–7.18 (m, 4H), 7.28–7.31 (m, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 22.74, 25.45, 25.56, 25.81, 25.91, 25.95, 26.02, 26.76, 28.12, 28.48, 29.17, 29.23, 29.60, 29.65, 29.71, 29.93, 31.36, 33.46, 35.04, 40.46, 42.54, 47.54, 48.80, 53.56, 54.73, 60.06, 60.17, 67.85, 70.61, 70.74, 74.62, 102.88, 107.66, 107.81, 126.15, 127.03, 128.63, 129.00, 129.97, 136.56, 137.34, 157.84, 160.39, 170.96, 172.53, 175.12; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₆H₈₈O₉N₄Cl, 995.6234; found, 995.6230.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((5-Aminopentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (99)

This compound was prepared using general procedure VI and 91 (167 mg, 0.21 mmol). The crude product was used in the next step without further purification. R_f = 0.35 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₃H₆₄O₇N₅, 762.4800; found, 762.4781.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((8-Aminooctyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (100)

This compound was prepared using general procedure VI and 92 (200 mg, 0.24 mmol). The crude product was used in the next step without further purification. R_f = 0.12 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₆H₇₀O₇N₅, 804.5270; found, 804.5247.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(4-(4-Aminobutoxy)butoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (101)

This compound was prepared using general procedure VI and 93 (160 mg, 0.19 mmol). The crude product was used in the next step without further purification. R_f = 0.25 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₆H₇₀O₈N₅, 820.5219; found, 820.5204.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (102)

This compound was prepared using general procedure VI and 94 (137 mg, 0.17 mmol). The crude product was used in the next step without further purification. R_f = 0.18 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₄H₆₆O₉N₅, 808.4855; found, 808.4837.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (103)

This compound was prepared using general procedure VI and 95 (180 mg, 0.21 mmol). The crude product was used in the next step without further purification. R_f = 0.35 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₆H₇₀O₁₀N₅, 852.5117; found, 852.5097.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-(2-((6-((6-Aminohexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (104)

This compound was prepared using general procedure VI and 96 (150 mg, 0.16 mmol). The crude product was used in the next step without further purification. R_f = 0.18 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₂H₈₂O₉N₅, 920.6107; found, 920.6074.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((5-((5-((6-Aminohexyl)oxy)pentyl)oxy)pentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (105)

This compound was prepared using general procedure VI and 97 (190 mg, 0.28 mmol). The crude product was used in the next step without further purification. R_f = 0.22 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₄H₈₆O₉N₅, 948.6420; found, 948.6409.

tert-Butyl ((S)-1-(((S)-2-((2S,4S)-4-(3-((6-((6-((6-Aminohexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-1-cyclohexyl-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (106)

This compound was prepared using general procedure VI and 98 (230 mg, 0.23 mmol). The crude product was used in the next step without further purification. R_f = 0.40 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₆H₉₀O₉N₅, 976.6733; found, 976.6696.

Synthesis of Boc-Protected and Final PROTACs

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (107)

The IAP ligand–linker conjugate 74 (147 mg, 0.17 mmol) was dissolved in dry EtOAc (10 mL) and treated with 10% Pd/C (10% w/w). The reaction mixture was stirred under H₂ (1 atm, balloon) for 18 h. The mixture was filtered through Celite, and the filtrate was concentrated. The oily residue was dissolved in dry DMF (5 mL), and DIPEA (59 μL, 0.34 mmol) and HATU (71 mg, 0.187 mmol) were added. After stirring at rt for 5 min, the VHL ligand 68 (90 mg, 0.20 mmol) in dry DMF (2.5 mL) and DIPEA (59 μL, 0.34 mmol) was added. The combined mixture was stirred at rt for 16 h. Subsequently, it was quenched with half-saturated brine (50 mL) and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were washed with saturated NH₄Cl solution, 5% LiCl solution, and brine (each 50 mL); dried over Na₂SO₄; filtered; and evaporated. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless resin. Yield (64 mg, 32%); R_f = 0.22 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₅H₈₉N₈O₁₁S, 1189.6366; found, 1189.6333.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (108)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 75 (177 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (206 mg, 56%); R_f = 0.26 (CH₂Cl₂/MeOH 19:1); mp 112–116 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₈H₉₅N₈O₁₁S, 1231.6836; found, 1231.6679.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutoxy)butoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (109)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 76 (182 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless semi-solid. Yield (202 mg, 54%); R_f = 0.25 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₈H₉₅N₈O₁₂S, 1247.6785; found, 1247.6775.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (110)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 77 (179 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless solid. Yield (263 mg, 71%); R_f = 0.25 (CH₂Cl₂/MeOH 19:1); mp 96–100 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₆H₉₁N₈O₁₃S, 1235.6378; found, 1235.6421.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (111)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 78 (192 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless semi-solid. Yield (134 mg, 35%); R_f = 0.21 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₈H₉₅N₈O₁₄S, 1279.6683; found, 1279.6676.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((6-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (112)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 79 (212 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless solid. Yield (105 mg, 26%); R_f = 0.15 (CH₂Cl₂/MeOH 19:1); mp 70–72 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₄H₁₀₇N₈O₁₃S, 1347.7673; found, 1347.7704.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((5-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)pentyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (113)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 80 (221 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless semi-solid. Yield (235 mg, 57%); R_f = 0.22 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₆H₁₁₁N₈O₁₃S, 1375.7986; found, 1375.7981.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((5-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)pentyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (114)

This compound was prepared using general procedure II, VHL1 ligand–linker conjugate 81 (229 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (CH₂Cl₂/MeOH 19:1) to give a colorless solid. Yield (219 mg, 52%); R_f = 0.23 (CH₂Cl₂/MeOH 19:1); mp 74–76 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₈H₁₁₅N₈O₁₃S, 1403.8299; found, 1403.8300.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (115 = 10d)

This compound was synthesized analogously to 107 but using VHL ligand 69 (92 mg, 0.21 mmol). A colorless solid was obtained after flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂). Yield (143 mg, 70%); R_f = 0.23 (CH₂Cl₂/MeOH 19:1); mp 134–138 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.93 (s, 9H), 1.03–1.10 (m, 1H), 1.18 (s, 4H), 1.24 (s, 1H), 1.34–1.41 (m, 16H), 1.56–1.71 (m, 6H), 1.72–1.83 (m, 2H), 1.96–2.03 (m, 1H), 2.05–2.11 (m, 1H), 2.14–2.22 (m, 1H), 2.28–2.35 (m, 1H), 2.44 (s, 3H), 2.50–2.56 (m, 1H), 2.64–2.75 (m, 7H), 3.56–3.64 (m, 3H), 3.85–3.93 (m, 3H), 4.21–4.30 (m, 3H), 4.31 (t, J = 7.8 Hz, 1H), 4.39–4.47 (m, 2H), 4.52 (d, J = 9.3 Hz, 1H), 4.87–4.95 (m, 3H), 5.00–5.06 (m, 1H), 5.07 (d, J = 3.5 Hz, 1H), 6.44 (t, J = 2.4 Hz, 1H), 6.44–6.55 (m, 2H), 7.00–7.19 (m, 5H), 7.24 (d, J = 7.5 Hz, 1H), 7.35–7.44 (m, 5H), 7.78–7.85 (m, 2H), 8.33 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H); LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₆₆H₉₁N₈O₁₁S, 1203.65; found, 1204.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₆H₉₁N₈O₁₁S, 1203.6523; found, 1203.6508.

tert-Butyl ((R)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (116)

This compound was synthesized analogously to 107 but using IAP ligand–linker conjugate 82 (147 mg, 0.17 mmol). A colorless solid was obtained after flash chromatography (gradient from 0 to 8% MeOH in CH₂Cl₂). Yield (133 mg, 66%); R_f = 0.22 (CH₂Cl₂/MeOH 19:1); mp 132–136 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₅H₈₉N₈O₁₁S, 1189.6366; found, 1189.6315.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-(((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (117)

Compound 74 (225 mg, 0.26 mmol) was dissolved in dry MeOH (8 mL) and treated with 10% Pd/C (45 mg, 20% w/w). The reaction mixture was stirred under H₂ (1 atm, balloon) for 2 h. The mixture was filtered through Celite and washed with MeOH, and the filtrate was concentrated. The product was dissolved in dry DMF (5 mL) along with HATU (109 mg, 0.286 mmol) and DIPEA (101 mg, 133 μL) 0.78 mmol. A solution of 70 (121 mg, 0.28 mmol) in dry DMF (5 mL) was added, followed by stirring of the mixture at rt for 18 h. The volatiles were then evaporated, and the crude product was purified by column chromatography (CH₂Cl₂/MeOH 20:1) to give an off-white resin. Yield (275 mg, 89%); R_f = 0.25 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₅H₈₉O₁₁N₈S, 1189.6366; found, 1189.6350.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 1)

This compound was prepared using general procedure VII and PROTAC precursor 107 (60 mg, 50 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 15:1) to give a colorless solid. Yield (24 mg, 43%); R_f = 0.14 (CH₂Cl₂/MeOH + 7 N NH₃ 19:1); mp 76–80 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 0.94–1.14 (m, 7H), 1.52–1.83 (m, 14H), 1.86–1.93 (m, 1H), 1.96–2.14 (m, 2H), 2.15 (s, 3H), 2.16–2.21 (m, 2H), 2.28–2.40 (m, 1H), 2.42 (s, 3H), 2.50–2.57 (m, 1H), 2.62–2.74 (m, 2H), 2.89–2.98 (m, 1H), 3.46–3.69 (m, 4H), 3.84–3.93 (m, 2H), 4.13–4.60 (m, 8H), 4.87–4.93 (m, 1H), 4.98–5.06 (m, 1H), 5.13 (d, J = 3.6 Hz, 1H), 6.38–6.54 (m, 3H), 7.01–7.18 (m, 4H), 7.23 (d, J = 7.5 Hz, 1H), 7.32–7.44 (m, 4H), 7.80 (d, J = 8.6 Hz, 1H), 7.86 (d, J = 9.4 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 8.53 (t, J = 6.1 Hz, 1H), 8.95 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.15, 19.27, 20.14, 22.35, 25.66, 25.85, 26.02, 26.61, 28.03, 28.50, 28.97, 29.31, 29.91, 34.46, 34.65, 34.77, 35.46, 38.15, 40.23, 41.92, 46.89, 52.26, 54.63, 56.59, 56.63, 58.79, 58.96, 59.31, 67.42, 69.12, 75.17, 102.62, 107.61, 107.87, 125.99, 127.00, 127.68, 128.58, 128.89, 128.92, 129.88, 130.30, 131.43, 137.26, 137.41, 139.72, 147.96, 151.70, 158.32, 160.16, 169.97, 170.17, 170.70, 172.23, 172.26, 174.73; LC–MS (ESI) 95% purity, m/z: [M + H]⁺ calcd for C₆₀H₈₁N₈O₉S, 1089.58; found, 1089.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₀H₈₁N₈O₉S, 1089.5842; found, 1089.5796.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 2)

This compound was prepared using general procedure VII and PROTAC precursor 108 (123 mg, 100 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (93 mg, 80%); mp 162–166 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 1.03–1.19 (m, 3H), 1.21–1.47 (m, 10H), 1.48–1.82 (m, 8H), 1.85–1.91 (m, 1H), 1.99–2.14 (m, 3H), 2.22–2.29 (m, 1H), 2.42–2.47 (m, 7H), 2.63–2.73 (m, 3H), 3.60–3.68 (m, 3H), 3.81–3.93 (m, 4H), 4.17–4.24 (m, 2H), 4.31–4.48 (m, 6H), 4.53 (d, J = 9.4 Hz, 1H), 4.87–4.94 (m, 1H), 5.00–5.09 (m, 1H), 6.39–6.55 (m, 4H), 7.02–7.18 (m, 6H), 7.22 (d, J = 7.5 Hz, 1H), 7.35–7.44 (m, 6H), 7.84 (d, J = 9.4 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 8.57 (t, J = 6.1 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 8.80–8.88 (m, 1H), 9.02 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.00, 16.03, 20.10, 25.58, 25.66, 25.83, 25.91, 26.58, 28.09, 28.71, 28.83, 28.94, 28.98, 29.88, 30.92, 34.68, 35.04, 35.42, 38.16, 40.24, 41.83, 46.81, 52.31, 55.64, 56.03, 56.46, 56.54, 58.76, 58.89, 67.60, 69.05, 75.12, 102.71, 107.52, 107.73, 125.94, 126.94, 127.63, 128.53, 128.84, 128.89, 129.69, 130.26, 131.52, 137.22, 137.40, 139.80, 147.63, 151.85, 158.24, 160.14, 168.72, 169.90, 169.93, 169.96, 172.17, 172.30; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₉S, 1131.63; found, 1131.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₉S, 1131.6311; found, 1131.6272.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutoxy)butoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 3)

This compound was prepared using general procedure VII and PROTAC precursor 109 (80 mg, 64 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 15:1) to give a colorless solid. Yield (43 mg, 58%); R_f = 0.33 (CH₂Cl₂/MeOH + 7 N NH₃ 15:1); mp 84–86 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.91 (s, 9H), 0.95–1.19 (m, 7H), 1.49–1.83 (m, 17H), 1.85–1.93 (m, 1H), 1.96–2.11 (m, 2H), 2.15 (s, 3H), 2.16–2.34 (m, 3H), 2.42 (s, 3H), 2.49–2.56 (m, 1H), 2.62–2.74 (m, 2H), 2.88–2.99 (m, 1H), 3.28–3.35 (m, 2H), 3.44–3.51 (m, 2H), 3.59–3.68 (m, 3H), 3.85–3.94 (m, 2H), 4.17–4.28 (m, 2H), 4.31–4.49 (m, 5H), 4.52 (d, J = 9.4 Hz, 1H), 4.87–5.06 (m, 2H), 5.12 (d, J = 3.6 Hz, 1H), 6.39–6.44 (m, 1H), 6.44–6.54 (m, 2H), 7.04–7.18 (m, 4H), 7.23 (d, J = 7.4 Hz, 1H), 7.34–7.44 (m, 4H), 7.75–7.85 (m, 2H), 7.92 (d, J = 8.6 Hz, 1H), 8.52 (t, J = 6.1 Hz, 1H), 8.95 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.13, 19.25, 20.13, 25.63, 25.80, 25.83, 26.01, 26.09, 26.58, 28.01, 28.95, 29.28, 29.89, 31.89, 34.44, 34.62, 35.44, 38.13, 40.22, 41.89, 46.87, 52.23, 54.60, 56.55, 56.64, 58.75, 58.94, 59.30, 67.52, 69.10, 69.68, 69.80, 75.16, 102.62, 107.57, 107.87, 125.97, 126.97, 127.66, 128.55, 128.87, 128.90, 129.86, 130.28, 131.40, 137.24, 137.39, 139.70, 147.94, 151.67, 158.29, 160.13, 169.92, 170.13, 170.68, 172.13, 172.21, 174.71; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₁₀S, 1147.63; found, 1148.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₁₀S, 1147.6260; found, 1147.6232.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 4)

This compound was prepared using general procedure VII and PROTAC precursor 110 (78 mg, 63 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (71 mg, 47%); mp 164–166 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 0.93–1.17 (m, 7H), 1.33 (d, J = 6.9 Hz, 3H), 1.56–1.82 (m, 6H), 1.84–1.93 (m, 1H), 2.01–2.09 (m, 2H), 2.40–2.48 (m, 7H), 2.49–2.56 (m, 1H), 2.63–2.75 (m, 2H), 3.57–3.68 (m, 7H), 3.69–3.81 (m, 2H), 3.81–3.96 (m, 1H), 3.97 (s, 2H), 4.01–4.09 (m, 2H), 4.19–4.30 (m, 2H), 4.31–4.46 (m, 5H), 4.55 (d, J = 9.6 Hz, 1H), 4.87–4.94 (m, 1H), 5.01 (p, J = 5.5 Hz, 1H), 6.39–6.56 (m, 3H), 7.02–7.17 (m, 5H), 7.22 (d, J = 7.6 Hz, 1H), 7.35–7.45 (m, 6H), 7.90 (d, J = 8.5 Hz, 1H), 8.58 (t, J = 6.0 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.78–8.86 (m, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.92, 16.00, 20.05, 25.62, 25.78, 25.87, 26.34, 26.44, 28.04, 28.89, 28.93, 29.84, 30.88, 34.61, 35.84, 38.06, 40.24, 41.84, 46.77, 52.23, 55.59, 55.88, 56.01, 56.71, 58.67, 58.90, 67.25, 69.01, 69.13, 69.79, 69.89, 70.60, 75.01, 102.57, 107.38, 108.07, 125.88, 126.88, 127.61, 128.47, 128.83, 129.79, 130.22, 131.36, 137.17, 137.37, 139.62, 147.75, 151.65, 158.22, 159.84, 168.66, 168.75, 169.31, 169.84, 169.93, 171.92; LC–MS (ESI) 96% purity, m/z: [M + H]⁺ calcd for C₆₁H₈₂N₈O₁₁S, 1131.59; found, 1135.7; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₁H₈₂N₈O₁₁S, 1135.5897; found, 1135.5877.

(2S,4R)-1-((S)-2-(tert-Butyl)-14-(3-(((3S,5S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-5-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-3-yl)oxy)phenoxy)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (PROTAC 5)

This compound was prepared using general procedure VII and PROTAC precursor 111 (80 mg, 63 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 19:1) to give a colorless solid. Yield (35 mg, 47%); R_f = 0.17 (CH₂Cl₂/MeOH + 7 N NH₃ 19:1); mp 84–86 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 1.03–1.11 (m, 6H), 1.51–1.69 (m, 7H), 1.71–1.81 (m, 3H), 1.85–1.92 (m, 1H), 2.01–2.09 (m, 2H), 2.15 (s, 2H), 2.18 (s, 1H), 2.42 (s, 3H), 2.51–2.54 (m, 1H), 2.63–2.70 (m, 2H), 2.87–3.01 (m, 1H), 3.55–3.71 (m, 12H), 3.93–4.02 (m, 4H), 4.20–4.29 (m, 3H), 4.32–4.46 (m, 6H), 4.54 (d, J = 9.5 Hz, 1H), 4.90 (d, J = 6.5 Hz, 1H), 5.01 (t, J = 5.4 Hz, 1H), 5.13–5.17 (m, 1H), 6.42–6.53 (m, 3H), 7.02–7.18 (m, 5H), 7.22 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 7.6 Hz, 6H), 7.83 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 8.56 (t, J = 6.2 Hz, 1H), 8.94 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.10, 19.27, 20.12, 25.64, 25.83, 26.00, 26.38, 26.48, 28.00, 28.95, 29.26, 29.89, 34.45, 34.61, 35.92, 38.09, 40.22, 41.92, 46.86, 52.22, 54.59, 55.94, 56.77, 58.72, 58.98, 59.31, 67.34, 69.10, 69.12, 69.81, 69.83, 70.09, 70.14, 70.70, 75.11, 102.48, 107.54, 108.13, 125.97, 126.97, 127.69, 128.56, 128.90, 129.91, 130.31, 131.37, 137.24, 137.40, 139.63, 147.96, 151.66, 158.30, 159.93, 168.88, 169.38, 170.13, 170.67, 172.03; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₁₂S, 1179.62; found, 1180.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₃H₈₇N₈O₁₂S, 1179.6159; found, 1179.6122.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 6)

This compound was prepared using general procedure VII and PROTAC precursor 112 (101 mg, 75 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (73 mg, 75%); mp 142–144 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.92 (s, 9H), 0.95–1.15 (m, 5H), 1.26–1.92 (m, 30H), 2.02–2.10 (m, 2H), 2.41–2.47 (m, 6H), 2.50–2.57 (m, 1H), 2.63–2.72 (m, 2H), 3.24–3.34 (m, 4H), 3.42–3.47 (m, 2H), 3.56–3.61 (m, 2H), 3.63–3.67 (m, 2H), 3.81–3.93 (m, 6H), 4.17–4.29 (m, 2H), 4.31–4.46 (m, 5H), 4.54 (d, J = 9.6 Hz, 1H), 4.85–4.94 (m, 1H), 5.01–5.07 (m, 1H), 6.39–6.45 (m, 1H), 6.46–6.54 (m, 2H), 7.02–7.18 (m, 4H), 7.22 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 9.6 Hz, 1H), 7.35–7.46 (m, 4H), 7.89 (d, J = 8.5 Hz, 1H), 8.62 (t, J = 6.1 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 8.80–8.87 (m, 1H), 9.00 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.27, 20.36, 25.85, 25.95, 26.01, 26.10, 26.18, 26.62, 28.37, 29.11, 29.21, 29.25, 29.52, 29.67, 30.14, 31.18, 34.95, 36.33, 38.36, 40.51, 42.13, 47.08, 52.58, 55.90, 56.05, 56.30, 57.07, 59.02, 59.23, 67.82, 69.34, 69.87, 70.32, 71.32, 75.39, 103.00, 107.78, 107.98, 126.21, 127.21, 127.94, 128.80, 129.14, 130.04, 130.52, 131.74, 137.48, 137.67, 139.97, 147.96, 152.06, 158.50, 160.40, 168.99, 169.58, 170.18, 170.22, 172.25; LC–MS (ESI) 95% purity, m/z: [M + H]⁺ calcd for C₆₉H₉₉N₈O₁₁S, 1247.71; found, 1248.1; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₉H₉₉N₈O₁₁S, 1247.7149; found, 1247.7130.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((5-((5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)pentyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 7)

This compound was prepared using general procedure VII and PROTAC precursor 113 (80 mg, 58 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 19:1) to give a colorless solid. Yield (38 mg, 52%); R_f = 0.31 (CH₂Cl₂/MeOH + 7 N NH₃ 19:1); mp 90–92 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.91 (s, 9H), 0.97–1.11 (m, 5H), 1.24–1.83 (m, 22H), 1.85–1.93 (m, 1H), 1.98–2.04 (m, 1H), 2.06–2.13 (m, 2H), 2.15 (s, 3H), 2.17–2.29 (m, 2H), 2.42 (s, 3H), 2.50–2.56 (m, 1H), 2.62–2.74 (m, 2H), 2.94 (q, J = 6.8 Hz, 1H), 3.24–3.33 (m, 10H), 3.40–3.51 (m, 4H), 3.59–3.68 (m, 3H), 3.88 (t, J = 6.4 Hz, 2H), 4.17–4.31 (m, 2H), 4.31–4.46 (m, 6H), 4.52 (d, J = 9.4 Hz, 1H), 4.87–4.93 (m, 1H), 4.98–5.05 (m, 1H), 5.12 (d, J = 3.6 Hz, 1H), 6.40 (t, J = 2.4 Hz, 1H), 6.45–6.53 (m, 2H), 7.01–7.18 (m, 5H), 7.21–7.25 (m, 1H), 7.34–7.44 (m, 5H), 7.79 (d, J = 6.8 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 8.52 (t, J = 6.1 Hz, 1H), 8.95 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.14, 19.28, 20.14, 22.51, 22.74, 25.60, 25.65, 25.71, 25.84, 26.02, 26.60, 28.03, 28.87, 28.97, 29.04, 29.28, 29.42, 29.91, 34.47, 34.63, 34.89, 35.44, 38.14, 40.23, 41.91, 46.89, 52.26, 54.61, 56.57, 58.78, 58.95, 59.33, 67.60, 69.11, 69.88, 70.10, 70.14, 75.20, 102.63, 107.63, 107.82, 125.98, 126.98, 127.67, 128.57, 128.87, 128.91, 129.87, 130.28, 131.42, 137.25, 137.40, 139.71, 147.95, 151.68, 158.29, 160.18, 169.96, 170.14, 170.71, 172.22, 172.34, 174.75; LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₇₁H₁₀₃N₈O₁₁S, 1275.75; found, 1276.1; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₁H₁₀₃N₈O₁₁S, 1275.7462; found, 1275.7422.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-((6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 8)

This compound was prepared using general procedure VII and PROTAC precursor 114 (48 mg, 34 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 19:1) to give a colorless solid. Yield (18 mg, 41%); R_f = 0.20 (CH₂Cl₂/MeOH + 7 N NH₃ 19:1); mp 88–90 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.91 (s, 10H), 0.96–1.14 (m, 7H), 1.14–1.83 (m, 28H), 1.85–1.93 (m, 1H), 1.95–2.05 (m, 1H), 2.05–2.12 (m, 2H), 2.15 (s, 3H), 2.16–2.28 (m, 2H), 2.42 (s, 3H), 2.67 (dd, J = 9.1, 15.4 Hz, 2H), 2.95 (p, J = 7.5 Hz, 1H), 3.22–3.33 (m, 10H), 3.59–3.68 (m, 3H), 3.87 (t, J = 6.4 Hz, 2H), 4.17–4.26 (m, 2H), 4.26–4.46 (m, 6H), 4.52 (d, J = 9.4 Hz, 1H), 4.86–4.93 (m, 1H), 5.02 (h, J = 4.9 Hz, 1H), 5.12 (d, J = 3.5 Hz, 1H), 6.40 (t, J = 2.4 Hz, 1H), 6.49 (ddd, J = 2.3, 8.2, 20.1 Hz, 2H), 7.01–7.19 (m, 5H), 7.20–7.25 (m, 1H), 7.34–7.46 (m, 5H), 7.75–7.82 (m, 2H), 7.92 (d, J = 8.6 Hz, 1H), 8.52 (t, J = 6.1 Hz, 1H), 8.95 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.15, 19.27, 20.15, 25.55, 25.60, 25.64, 25.72, 25.80, 25.82, 25.85, 26.03, 26.62, 28.05, 28.88, 28.98, 29.23, 29.32, 29.42, 29.45, 29.92, 34.46, 34.64, 35.13, 35.44, 38.15, 40.24, 41.92, 46.90, 52.27, 54.63, 56.56, 56.59, 58.80, 58.96, 59.32, 67.61, 69.12, 70.09, 70.14, 75.22, 102.65, 107.65, 107.83, 125.99, 126.99, 127.69, 128.58, 128.89, 128.92, 129.89, 130.30, 131.43, 137.26, 137.40, 139.72, 147.96, 151.70, 158.29, 160.19, 169.98, 170.15, 170.73, 172.24, 172.43, 174.75; LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₇₃H₁₀₇N₈O₁₁S, 1303.77; found, 1304.1; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₃H₁₀₇N₈O₁₁S, 1303.7737; found, 1303.7775.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 9) (CST626)

This compound was prepared using general procedure VII and PROTAC precursor 115 (100 mg, 83 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (90 mg, 95%); mp 64–66 °C (free base, obtained after column chromatography); ¹H NMR (600 MHz, DMSO-d₆): δ 0.93 (s, 9H), 0.96–1.20 (m, 6H), 1.30–1.40 (m, 6H), 1.51–1.83 (m, 14H), 1.96–2.10 (m, 2H), 2.14–2.22 (m, 1H), 2.28–2.39 (m, 1H), 2.42–2.47 (m, 6H), 2.51–2.58 (m, 1H), 2.64–2.74 (m, 2H), 3.58–3.68 (m, 4H), 3.81–3.94 (m, 3H), 4.21 (dd, J = 5.9, 10.9 Hz, 1H), 4.24–4.30 (m, 1H), 4.37–4.47 (m, 3H), 4.51 (d, J = 9.2 Hz, 1H), 4.87–4.95 (m, 2H), 5.04 (h, J = 4.7 Hz, 1H), 6.43 (s, 1H), 6.46–6.56 (m, 2H), 7.03–7.19 (m, 4H), 7.23 (d, J = 7.5 Hz, 1H), 7.35–7.44 (m, 4H), 7.81 (dd, J = 3.7, 9.3 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.80–8.87 (m, 1H), 9.00 (s, 1H), 9.37 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.95, 16.05, 20.08, 22.26, 22.58, 25.63, 25.80, 25.88, 26.62, 28.07, 28.40, 28.44, 28.91, 28.96, 29.85, 30.90, 34.65, 34.69, 35.37, 37.89, 46.80, 47.88, 52.28, 55.63, 56.02, 56.42, 56.58, 58.73, 67.36, 68.91, 75.09, 102.67, 107.50, 107.79, 125.90, 126.57, 126.91, 128.49, 128.85, 128.99, 129.77, 130.23, 131.40, 137.18, 137.38, 144.88, 147.71, 151.77, 158.24, 160.10, 168.69, 169.76, 169.90, 169.96, 170.80, 172.04; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₆₁H₈₃N₈O₉S, 1103.60; found, 1103.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₁H₈₃N₈O₉S, 1103.5998; found, 1103.5980.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((R)-2-(methylamino)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 10a)

This compound was prepared using general procedure VII and PROTAC precursor 116 (100 mg, 84 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (95 mg, 99%); mp 140–142 °C (dec); ¹H NMR (600 MHz, DMSO-d₆): δ 0.93 (s, 9H), 1.00–1.20 (m, 4H), 1.39 (d, J = 6.9 Hz, 3H), 1.51–1.82 (m, 16H), 1.86–1.93 (m, 1H), 1.99–2.06 (m, 1H), 2.06–2.12 (m, 1H), 2.14–2.22 (m, 1H), 2.29–2.37 (m, 1H), 2.44 (d, J = 5.6 Hz, 6H), 2.50–2.58 (m, 1H), 2.64–2.75 (m, 2H), 3.62–3.91 (m, 6H), 4.15–4.31 (m, 2H), 4.31–4.49 (m, 5H), 4.54 (d, J = 9.3 Hz, 1H), 4.85–4.94 (m, 1H), 5.01–5.07 (m, 1H), 6.39–6.45 (m, 1H), 6.45–6.55 (m, 2H), 7.03–7.20 (m, 4H), 7.23 (d, J = 7.5 Hz, 1H), 7.35–7.43 (m, 4H), 7.84–7.89 (m, 2H), 8.55 (t, J = 6.1 Hz, 1H), 8.79–8.90 (m, 2H), 9.00 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 16.02, 16.13, 16.79, 20.09, 22.27, 25.59, 25.73, 25.91, 26.56, 28.04, 28.44, 28.90, 28.94, 29.84, 30.85, 34.68, 35.40, 38.12, 40.24, 41.83, 46.82, 52.35, 55.46, 56.03, 56.51, 58.79, 58.87, 67.36, 69.02, 75.16, 102.68, 107.49, 107.77, 125.90, 126.91, 127.61, 128.48, 128.81, 128.84, 129.72, 130.22, 131.45, 137.18, 137.39, 139.74, 147.69, 151.73, 158.23, 160.09, 168.83, 169.86, 169.90, 169.93, 172.09, 172.12; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₆₀H₈₁N₈O₉S, 1089.58; found, 1089.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₀H₈₁N₈O₉S, 1089.5842; found, 1089.5823.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(dimethylamino)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 10b)

PROTAC 9 (0.11 g, 0.10 mmol) was dissolved in dry DMF (2.5 mL), and 10% Pd/C (11 mg) and formaldehyde 36% in an aqueous solution (25 μL, 0.30 mmol) were added. The mixture was stirred under a hydrogen atmosphere at rt for 16 h. After removal of the catalyst by filtration, it was evaporated, and the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 9:1) to give a colorless solid. Yield (93 mg, 83%); R_f = 0.54 (CH₂Cl₂/MeOH + 7 N NH₃ 9:1); mp 130–132 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.93 (s, 9H), 1.04 (d, J = 6.8 Hz, 3H), 1.36 (d, J = 7.0 Hz, 3H), 1.53–1.83 (m, 17H), 1.96–2.03 (m, 1H), 2.05–2.11 (m, 1H), 2.17 (s, 6H), 2.18 (s, 1H), 2.28–2.35 (m, 1H), 2.44 (s, 3H), 2.50–2.58 (m, 1H), 2.63–2.70 (m, 1H), 2.70 (s, 1H), 2.90–2.99 (m, 1H), 3.56–3.64 (m, 3H), 3.85–3.95 (m, 3H), 4.23–4.36 (m, 2H), 4.33–4.39 (m, 1H), 4.39–4.44 (m, 1H), 4.42–4.50 (m, 1H), 4.52 (d, J = 9.4 Hz, 1H), 4.86–4.95 (m, 3H), 5.03 (h, J = 4.8 Hz, 1H), 5.07 (d, J = 3.6 Hz, 1H), 6.44 (t, J = 2.4 Hz, 1H), 6.46–6.55 (m, 2H), 7.02–7.19 (m, 5H), 7.20–7.25 (m, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.40–7.44 (m, 2H), 7.74–7.84 (m, 3H), 8.33 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 12.79, 16.12, 20.07, 22.26, 22.56, 25.57, 25.77, 25.95, 26.61, 28.06, 28.42, 28.90, 29.25, 29.84, 34.57, 34.69, 35.35, 37.86, 41.89, 41.93, 46.77, 47.85, 52.16, 56.41, 56.55, 58.67, 58.70, 63.37, 67.32, 68.92, 75.06, 102.51, 107.48, 107.81, 125.89, 126.54, 126.87, 128.48, 128.81, 128.97, 129.85, 130.17, 131.26, 137.16, 137.38, 144.79, 147.91, 151.60, 158.26, 160.09, 169.77, 169.99, 170.52, 170.77, 172.00, 172.55; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₂H₈₅N₈O₉S, 1117.62; found, 1117.9; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₂H₈₅N₈O₉S, 1117.6155; found, 1117.6143.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(N-methylacetamido)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 10c)

PROTAC 9 (0.11 g, 0.10 mmol) was dissolved in dry CH₂Cl₂ (2.5 mL) and cooled to 0 °C, and DIPEA (26 μL, 0.15 mmol) and acetic anhydride (15 μL, 0.15 mmol) were added. The mixture was stirred at rt for 16 h. After removal of the volatiles, the crude product was purified by flash chromatography (gradient from 0 to 10% MeOH in CH₂Cl₂) to give a colorless solid. Yield (101 mg, 88%); R_f = 0.44 (CH₂Cl₂/MeOH + 7 N NH₃ 9:1); mp 86–88 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.93 (s, 9H), 1.08 (d, J = 25.9 Hz, 2H), 1.13–1.16 (m, 3H), 1.19–1.26 (m, 1H), 1.36 (d, J = 7.0 Hz, 3H), 1.53–1.74 (m, 14H), 1.77 (s, 3H), 1.95–2.03 (m, 1H), 2.03–2.12 (m, 2H), 2.14–2.22 (m, 1H), 2.28–2.35 (m, 1H), 2.44 (s, 3H), 2.69 (s, 4H), 2.81 (s, 2H), 3.55–3.66 (m, 3H), 3.89–3.93 (m, 2H), 4.21–4.35 (m, 3H), 4.40–4.46 (m, 2H), 4.52 (d, J = 9.4 Hz, 1H), 4.88–4.94 (m, 2H), 4.96–5.05 (m, 2H), 5.08 (d, J = 3.6 Hz, 1H), 6.41–6.57 (m, 3H), 7.04–7.19 (m, 5H), 7.24 (d, J = 7.5 Hz, 1H), 7.33–7.47 (m, 5H), 7.79–7.86 (m, 3H), 8.33 (d, J = 7.8 Hz, 1H), 8.96 (s, 1H); LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₆₃H₈₅N₈O₁₀S, 1145.61; found, 1146.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₃H₈₅N₈O₁₀S, 1145.6104; found, 1145.6100.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-(((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 11)

This compound was prepared using general procedure VII and PROTAC precursor 117 (150 mg, 0.126 mmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1) to give a colorless solid. Yield (64 mg, 47%); R_f = 0.36 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 98–100 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.78–0.98 (m, 6H), 1.06 (s, 9H), 1.27 (d, J = 6.9 Hz, 3H), 1.36–1.70 (m, 10H), 1.73–1.85 (m, 6H), 1.97–2.07 (m, 2H), 2.10–2.20 (m, 1H), 2.35 (s, 3H), 2.42–2.47 (m, 1H), 2.49 (s, 3H), 2.68–2.77 (m, 2H), 2.85 (d, J = 14.0 Hz, 1H), 3.03 (q, J = 7.1 Hz, 1H), 3.59–3.66 (m, 1H), 3.73–3.85 (m, 3H), 4.07–4.13 (m, 1H), 4.21–4.36 (m, 3H), 4.40 (d, J = 7.4 Hz, 1H), 4.49–4.65 (m, 2H), 4.68–4.78 (m, 2H), 4.94–4.98 (m, 1H), 5.11 (d, J = 6.2 Hz, 1H), 6.15 (d, J = 7.1 Hz, 1H), 6.34–6.52 (m, 3H), 6.62 (d, J = 8.4 Hz, 1H), 7.01–7.06 (m, 2H), 7.08–7.15 (m, 2H), 7.27–7.36 (m, 5H), 7.49 (t, J = 6.1 Hz, 1H), 7.64–7.71 (m, 1H), 8.64 (s, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 16.26, 19.69, 20.19, 22.45, 25.62, 25.75, 25.99, 26.34, 26.82, 28.50, 28.68, 29.41, 29.89, 30.12, 33.55, 34.24, 35.30, 35.68, 37.74, 40.63, 42.90, 47.78, 53.79, 54.93, 55.14, 58.71, 59.53, 60.19, 60.36, 67.72, 69.73, 76.33, 102.91, 107.58, 108.61, 126.34, 127.27, 127.68, 128.77, 129.45, 130.25, 130.67, 131.83, 136.71, 137.57, 138.69, 148.49, 150.35, 158.10, 160.28, 169.67, 171.21, 171.31, 172.69, 174.09, 175.37; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 6.50 min, 97% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₀H₈₁O₉N₈S, 1089.5842; found, 1089.5818.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)pentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (118)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 83 (196 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless solid. Yield (70 mg, 18%); R_f = 0.23 (CH₂Cl₂/MeOH 19:1); mp 130–134 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₇₂H₉₃N₈O₁₂S, 1293.66; found, 1294.3; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₂H₉₃N₈O₁₂S, 1293.6628; found, 1293.6611.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((8-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)octyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (119)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 84 (209 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (208 mg, 52%); R_f = 0.24 (CH₂Cl₂/MeOH 19:1); mp 128–130 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₂S, 1335.71; found, 1336.2; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₂S, 1335.7098; found, 1335.7072.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(4-(4-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)butoxy)butoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (120)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 85 (213 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (85 mg, 21%); R_f = 0.19 (CH₂Cl₂/MeOH 19:1); mp 114–118 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 96% purity, m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₃S, 1351.71; found, 1352.2; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₃S, 1351.7047; found, 1351.7051.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-(2-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (121)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 86 (210 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by column chromatography (CH₂Cl₂/MeOH 29:1) to give a colorless solid. Yield (104 mg, 26%); R_f = 0.12 (CH₂Cl₂/MeOH 39:1); mp 96–98 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 97% purity, m/z: [M + H]⁺ calcd for C₇₃H₉₅N₈O₁₄S, 1339.67; found, 1339.7; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₃H₉₅N₈O₁₄S, 1339.6683; found, 1339.6650.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-(2-(2-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (122)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 87 (223 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless solid. Yield (112 mg, 27%); R_f = 0.15 (CH₂Cl₂/MeOH 29:1); mp 96–98 °C. Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 96% purity, m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₅S, 1383.69; found, 1384.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₅H₉₉N₈O₁₅S, 1383.6945; found, 1383.6920.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((6-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (123)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 88 (243 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (gradient from 0 to 6% MeOH in CH₂Cl₂) to give a colorless resin. Yield (100 mg, 23%); R_f = 0.21 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₈₁H₁₁₁N₈O₁₄S, 1451.79; found, 1452.7; HRMS (ESI) m/z: [M + H]⁺ calcd for C₈₁H₁₁₁N₈O₁₄S, 1451.7935; found, 1451.7932.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((5-((5-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)pentyl)oxy)pentyl)oxy)hexyl)oxy)phenoxy)-2-((®-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyrrolidinedin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (124)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 89 (252 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless resin. Yield (120 mg, 27%); R_f = 0.29 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 93% purity, m/z: [M + H]⁺ calcd for C₈₃H₁₁₅N₈O₁₄S, 1479.82; found, 1479.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₈₃H₁₁₅N₈O₁₄S, 1479.8248; found, 1479.8238.

tert-Butyl ((S)-1-(((S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((6-((6-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (125)

This compound was prepared using general procedure II, VHL2 ligand–linker conjugate 90 (260 mg, 0.30 mmol), and IAP ligand 65. The crude product was purified by flash chromatography (gradient from 0 to 5% MeOH in CH₂Cl₂) to give a colorless resin. Yield (109 mg, 24%); R_f = 0.21 (CH₂Cl₂/MeOH 19:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. LC–MS (ESI) 84% purity, m/z: [M + H]⁺ calcd for C₈₅H₁₁₉N₈O₁₄S, 1507.86; found, 1507.5; HRMS (ESI) m/z: [M + H]⁺ calcd for C₈₅H₁₁₉N₈O₁₄S, 1507.8561; found, 1507.8562.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)pentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 12)

This compound was prepared using general procedure VII and PROTAC precursor 118 (35 mg, 27 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (30 mg, 90%); mp 164–168 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.95–1.24 (m, 6H), 1.33 (d, J = 6.9 Hz, 3H), 1.50–1.86 (m, 15H), 1.88–1.95 (m, 1H), 2.00–2.11 (m, 2H), 2.25–2.57 (m, 8H), 2.61–2.74 (m, 2H), 3.66 (dd, J = 5.8, 10.5 Hz, 2H), 3.76 (dd, J = 4.5, 10.6 Hz, 1H), 3.85 (q, J = 6.6 Hz, 1H), 3.94 (t, J = 6.4 Hz, 2H), 4.04–4.09 (m, 2H), 4.15–4.62 (m, 9H), 4.70 (d, J = 10.8 Hz, 1H), 4.86–4.94 (m, 1H), 5.01–5.07 (m, 1H), 6.33–6.58 (m, 3H), 6.92–7.20 (m, 6H), 7.22 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.46–7.52 (m, 1H), 7.60 (d, J = 4.3 Hz, 2H), 7.70 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 8.36 (t, J = 5.9 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.79–8.86 (m, 1H), 8.99 (s, 1H), 9.26–9.33 (m, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.93, 16.12, 18.76, 19.05, 20.07, 22.40, 25.62, 25.79, 25.87, 28.06, 28.54, 28.56, 28.89, 28.96, 29.84, 30.89, 34.63, 37.21, 38.23, 46.79, 46.97, 52.30, 55.54, 55.61, 56.02, 57.96, 58.75, 58.87, 67.57, 67.84, 68.76, 75.14, 102.74, 107.60, 107.73, 111.91, 120.97, 123.18, 123.77, 125.89, 126.89, 127.21, 127.88, 128.07, 128.47, 128.84, 130.22, 131.10, 131.53, 131.75, 137.18, 137.37, 142.35, 147.95, 151.66, 156.11, 158.22, 160.11, 167.66, 168.28, 168.68, 169.89, 169.94, 171.71; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₇H₈₅N₈O₁₀S, 1193.61; found, 1191.9; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₇H₈₅N₈O₁₀S, 1193.6104; found, 1193.6079.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((8-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)octyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 13)

This compound was prepared using general procedure VII and PROTAC precursor 119 (100 mg, 75 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (94 mg, 99%); mp 212–214 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.97–1.18 (m, 6H), 1.31–1.48 (m, 11H), 1.53–1.82 (m, 8H), 1.88–1.95 (m, 1H), 2.00–2.11 (m, 2H), 2.27–2.40 (m, 1H), 2.44–2.47 (m, 6H), 2.63–2.74 (m, 2H), 3.63–3.73 (m, 6H), 3.76 (dd, J = 4.5, 10.6 Hz, 2H), 3.82–3.93 (m, 3H), 4.03 (t, J = 6.4 Hz, 2H), 4.16–4.25 (m, 2H), 4.26–4.34 (m, 2H), 4.32–4.47 (m, 4H), 4.53 (d, J = 18.1 Hz, 1H), 4.70 (d, J = 10.8 Hz, 1H), 4.91 (q, J = 7.1 Hz, 1H), 5.04 (p, J = 5.1 Hz, 1H), 6.40–6.45 (m, 1H), 6.46–6.55 (m, 2H), 6.96–7.01 (m, 2H), 7.01–7.19 (m, 5H), 7.22 (d, J = 7.5 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.45–7.52 (m, 1H), 7.55–7.63 (m, 2H), 7.70 (d, J = 7.5 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H), 8.36 (t, J = 5.9 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.80–8.85 (m, 1H), 9.00 (s, 1H), 9.30–9.35 (m, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.92, 16.08, 18.76, 19.03, 20.05, 25.61, 25.67, 25.71, 25.77, 25.86, 28.05, 28.52, 28.81, 28.88, 28.92, 28.95, 29.83, 30.87, 34.61, 37.16, 38.23, 46.77, 46.96, 52.29, 55.54, 55.59, 56.00, 57.94, 58.74, 58.85, 67.59, 67.90, 68.75, 75.13, 102.74, 107.52, 107.69, 111.87, 120.91, 123.16, 123.75, 125.88, 126.88, 127.20, 127.83, 128.05, 128.47, 128.82, 130.20, 131.03, 131.53, 131.56, 131.72, 137.16, 137.35, 142.33, 147.85, 151.69, 156.11, 158.19, 160.10, 167.63, 168.26, 168.66, 169.90, 171.68; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₀S, 1235.66; found, 1235.7; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₀S, 1235.6573; found, 1235.6530.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(4-(4-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)butoxy)butoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 14)

This compound was prepared using general procedure VII and PROTAC precursor 120 (50 mg, 37 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (42 mg, 88%); mp 170–174 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.96–1.15 (m, 6H), 1.33 (d, J = 6.9 Hz, 3H), 1.50–1.83 (m, 12H), 1.88–1.96 (m, 1H), 2.00–2.11 (m, 2H), 2.27–2.34 (m, 1H), 2.44–2.47 (m, 6H), 2.50–2.59 (m, 1H), 2.61–2.74 (m, 2H), 3.48–3.58 (m, 10H), 3.63–3.69 (m, 2H), 3.73–3.93 (m, 4H), 4.06 (t, J = 6.3 Hz, 2H), 4.17–4.53 (m, 10H), 4.70 (d, J = 10.7 Hz, 1H), 4.87–4.94 (m, 1H), 5.04 (p, J = 5.2 Hz, 1H), 6.42 (q, J = 3.5 Hz, 1H), 6.46–6.54 (m, 2H), 6.99 (s, 1H), 7.04–7.18 (m, 4H), 7.22 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.45–7.53 (m, 1H), 7.60 (d, J = 5.4 Hz, 2H), 7.70 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 8.36 (t, J = 5.9 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.79–8.86 (m, 1H), 8.98 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.93, 16.11, 18.77, 19.04, 20.06, 25.62, 25.79, 25.86, 26.07, 28.06, 28.53, 28.89, 28.96, 29.84, 30.89, 34.63, 37.20, 38.23, 40.23, 46.79, 46.97, 52.30, 55.55, 55.61, 56.02, 57.96, 58.74, 58.87, 67.45, 67.76, 68.77, 69.76, 69.85, 75.13, 102.77, 107.49, 107.74, 111.87, 120.95, 123.18, 123.76, 125.89, 126.89, 127.19, 127.89, 128.07, 128.48, 128.84, 130.21, 131.08, 131.53, 131.74, 137.18, 137.36, 142.35, 147.93, 151.66, 156.08, 158.21, 160.07, 167.65, 168.27, 168.68, 169.90, 169.92, 171.70; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₁S, 1251.65; found, 1252.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₁S, 1251.6523; found, 1251.6488.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-(2-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)ethoxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 15)

This compound was prepared using general procedure VII and PROTAC precursor 121 (58 mg, 43 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (54 mg, 98%); mp 206–210 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.5 Hz, 3H), 0.91–1.13 (m, 7H), 1.31–1.37 (m, 3H), 1.53–1.83 (m, 12H), 1.87–1.94 (m, 1H), 2.00–2.10 (m, 2H), 2.27–2.39 (m, 1H), 2.41–2.47 (m, 6H), 2.51–2.71 (m, 5H), 3.67–3.81 (m, 8H), 3.81–3.94 (m, 2H), 4.02 (t, J = 4.6 Hz, 3H), 4.15–4.27 (m, 5H), 4.31 (dd, J = 5.2, 16.4 Hz, 3H), 4.37–4.46 (m, 4H), 4.70 (d, J = 10.7 Hz, 1H), 4.90 (q, J = 7.4 Hz, 1H), 6.46 (s, 1H), 6.48 (d, J = 8.2 Hz, 1H), 6.53 (d, J = 8.8 Hz, 1H), 6.98–7.18 (m, 7H), 7.22 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.46–7.52 (m, 1H), 7.60 (d, J = 4.4 Hz, 2H), 7.70 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 8.36 (d, J = 6.5 Hz, 1H), 8.73 (d, J = 8.2 Hz, 1H), 8.99 (d, J = 2.7 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.93, 16.10, 18.76, 19.03, 20.05, 25.61, 25.78, 25.86, 28.06, 28.53, 28.88, 28.92, 29.83, 30.87, 34.61, 37.23, 38.23, 40.23, 46.77, 46.96, 52.27, 55.55, 55.61, 56.00, 57.94, 58.70, 58.87, 67.29, 68.09, 68.76, 69.10, 69.19, 70.11, 70.26, 75.06, 102.61, 107.44, 108.01, 112.35, 121.25, 123.16, 123.75, 125.88, 126.88, 127.42, 127.89, 128.05, 128.47, 128.82, 130.23, 131.06, 131.49, 131.53, 131.73, 137.16, 137.37, 142.34, 147.93, 151.70, 156.04, 158.22, 159.88, 167.63, 168.26, 168.66, 169.87, 169.93, 171.71; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₆₈H₈₇N₈O₁₂S, 1239.62; found, 1240.0; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₈H₈₇N₈O₁₂S, 1239.6159; found, 1239.6109.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-(2-(2-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 16)

This compound was prepared using general procedure VII and PROTAC precursor 122 (55 mg, 40 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (51 mg, 99%); mp 144–148 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.6 Hz, 3H), 0.93–1.17 (m, 8H), 1.33 (d, J = 6.9 Hz, 3H), 1.49–1.85 (m, 10H), 1.87–1.94 (m, 1H), 2.00–2.09 (m, 2H), 2.26–2.38 (m, 1H), 2.40–2.48 (m, 6H), 2.50–2.58 (m, 1H), 2.63–2.74 (m, 2H), 3.51–3.72 (m, 13H), 3.73–3.80 (m, 3H), 3.80–3.93 (m, 1H), 3.96–4.05 (m, 2H), 4.14–4.56 (m, 11H), 4.70 (d, J = 10.7 Hz, 1H), 4.85–4.93 (m, 1H), 5.03 (p, J = 5.3 Hz, 1H), 6.36–6.55 (m, 3H), 6.95–7.02 (m, 1H), 7.02–7.24 (m, 6H), 7.33 (d, J = 7.8 Hz, 1H), 7.43–7.52 (m, 1H), 7.54–7.63 (m, 2H), 7.70 (d, J = 7.5 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 8.35 (t, J = 6.0 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.79–8.86 (m, 1H), 8.99 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.93, 16.10, 18.77, 19.04, 20.05, 25.62, 25.79, 25.87, 28.06, 28.54, 28.89, 28.93, 29.83, 30.89, 34.62, 37.22, 38.25, 40.24, 46.78, 46.97, 52.27, 55.56, 55.61, 56.01, 57.95, 58.70, 58.87, 67.31, 68.10, 68.77, 69.06, 69.15, 70.01, 70.07, 70.24, 75.06, 102.60, 107.45, 108.02, 112.35, 121.25, 123.17, 123.76, 125.89, 126.90, 127.42, 127.88, 128.06, 128.49, 128.83, 130.24, 131.07, 131.50, 131.53, 131.74, 137.18, 137.37, 142.35, 147.95, 151.70, 156.05, 158.23, 159.88, 167.65, 168.27, 168.68, 169.88, 169.93, 171.72; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₃S, 1283.64; found, 1283.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₀H₉₁N₈O₁₃S, 1283.6421; found, 1283.6380.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-(2-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)ethoxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 17)

This compound was prepared using general procedure VII and PROTAC precursor 123 (35 mg, 24 μmol). The product possessed sufficient purity after filtration. A colorless solid was obtained. Yield (29 mg, 86%); mp 118–122 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.72 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.96–1.15 (m, 5H), 1.19–1.85 (m, 30H), 1.87–1.95 (m, 1H), 2.00–2.11 (m, 2H), 2.27–2.40 (m, 1H), 2.40–2.48 (m, 6H), 2.50–2.59 (m, 1H), 2.64–2.75 (m, 2H), 3.25–3.33 (m, 4H), 3.46 (t, J = 6.5 Hz, 2H), 3.63–3.93 (m, 8H), 4.14–4.56 (m, 11H), 4.70 (d, J = 10.8 Hz, 1H), 4.87–4.94 (m, 1H), 5.04 (p, J = 5.1 Hz, 1H), 6.39–6.44 (m, 1H), 6.46–6.54 (m, 2H), 7.00 (dd, J = 1.6, 7.8 Hz, 1H), 7.03–7.19 (m, 5H), 7.22 (d, J = 7.4 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.43–7.52 (m, 1H), 7.61 (q, J = 4.1 Hz, 2H), 7.70 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 8.34 (t, J = 6.0 Hz, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.78–8.86 (m, 1H), 8.99 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 15.91, 16.12, 18.76, 19.03, 20.04, 25.53, 25.61, 25.64, 25.72, 25.77, 25.86, 28.05, 28.53, 28.80, 28.89, 28.94, 29.36, 29.83, 30.88, 34.62, 37.20, 38.22, 40.24, 46.77, 46.96, 52.28, 55.53, 55.59, 56.00, 57.94, 58.73, 58.86, 67.53, 68.12, 68.75, 68.81, 70.00, 70.07, 70.61, 75.12, 102.73, 107.50, 107.68, 112.40, 121.22, 123.16, 123.74, 125.87, 126.87, 127.40, 127.85, 128.05, 128.47, 128.82, 130.19, 131.07, 131.47, 131.53, 131.72, 137.16, 137.35, 142.33, 147.95, 151.64, 156.08, 158.19, 160.09, 167.62, 168.24, 168.66, 169.89, 171.69; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₇₆H₁₀₃N₈O₁₂S, 1351.74; found, 1352.1; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₆H₁₀₃N₈O₁₂S, 1351.7411; found, 1351.7349.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((5-((5-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)pentyl)oxy)pentyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 18)

This compound was prepared using general procedure VII and PROTAC precursor 124 (60 mg, 40 μmol). After filtration of the solid material, the crude product was purified by column chromatography (CH₂Cl₂/MeOH + 7 N NH₃ 9:1) followed by preparative HPLC (100% MeOH) to give a colorless solid. Yield (48 mg, 87%); mp 98–102 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.85 (t, J = 6.9 Hz, 2H), 0.95 (d, J = 6.5 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H), 1.19–1.82 (m, 30H), 1.88–1.95 (m, 1H), 1.99–2.11 (m, 2H), 2.16 (s, 3H), 2.17–2.24 (m, 1H), 2.31 (s, 1H), 2.46 (s, 4H), 2.50–2.56 (m, 1H), 2.64–2.75 (m, 2H), 2.87–2.97 (m, 1H), 3.30–3.39 (m, 8H), 3.61 (dd, J = 4.5, 10.7 Hz, 1H), 3.65–3.70 (m, 1H), 3.74–3.79 (m, 1H), 3.82–3.92 (m, 2H), 4.03 (t, J = 6.3 Hz, 2H), 4.19–4.57 (m, 10H), 4.70 (d, J = 10.8 Hz, 1H), 4.88–4.94 (m, 1H), 4.99–5.08 (m, 2H), 6.42 (t, J = 2.4 Hz, 1H), 6.45–6.53 (m, 2H), 6.95–7.01 (m, 2H), 7.01–7.17 (m, 5H), 7.24 (d, J = 7.5 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.46–7.52 (m, 1H), 7.55–7.63 (m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 8.33 (t, J = 6.0 Hz, 1H), 8.97 (s, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 14.10, 16.15, 18.76, 19.03, 19.22, 20.06, 22.21, 22.56, 22.67, 25.52, 25.58, 25.64, 25.76, 25.95, 27.95, 28.54, 28.65, 28.81, 28.90, 29.11, 29.20, 29.35, 29.83, 31.10, 34.41, 34.57, 37.17, 38.21, 40.24, 46.77, 46.96, 52.15, 54.47, 55.54, 57.94, 58.65, 58.85, 59.28, 67.51, 67.85, 68.77, 70.00, 70.05, 70.08, 75.05, 102.51, 107.50, 107.73, 111.86, 120.90, 123.16, 123.74, 125.87, 126.85, 127.15, 127.83, 128.04, 128.51, 128.79, 130.15, 131.12, 131.46, 131.54, 131.71, 137.14, 137.38, 142.34, 148.03, 151.54, 156.10, 158.24, 160.10, 167.62, 168.26, 170.01, 170.60, 171.66, 174.58; LC–MS (ESI) 98% purity, m/z: [M + H]⁺ calcd for C₇₈H₁₀₇N₈O₁₂S, 1379.77; found, 1379.6; HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₈H₁₀₇N₈O₁₂S, 1379.7724; found, 1379.7662.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-((6-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 19)

This compound was prepared using general procedure VII and PROTAC precursor 125 (32 mg, 21 μmol). After filtration of the solid material, the crude product was purified by preparative HPLC (gradient from 60 to 100% ACN +0.05% TFA) to give a colorless solid. Yield (23 mg, 75%); mp 102–104 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 0.73 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.97–1.13 (m, 5H), 1.25–1.29 (m, 5H), 1.31 (d, J = 6.8 Hz, 3H), 1.33–1.82 (m, 25H), 1.87–1.96 (m, 1H), 1.98–2.12 (m, 2H), 2.27–2.43 (m, 2H), 2.45 (s, 3H), 2.50–2.74 (m, 3H), 3.26–3.36 (m, 6H), 3.69 (s, 8H), 3.76 (dd, J = 4.5, 10.6 Hz, 1H), 3.82–3.93 (m, 3H), 4.03 (t, J = 6.3 Hz, 2H), 4.15–4.35 (m, 5H), 4.38–4.57 (m, 5H), 4.70 (d, J = 10.8 Hz, 1H), 4.85–4.94 (m, 1H), 5.00–5.08 (m, 1H), 6.41 (t, J = 2.4 Hz, 1H), 6.45–6.56 (m, 2H), 6.96–7.00 (m, 2H), 7.03–7.20 (m, 6H), 7.22 (d, J = 7.5 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.47–7.52 (m, 1H), 7.57–7.63 (m, 2H), 7.70 (d, J = 7.5 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 8.33 (t, J = 6.0 Hz, 1H), 8.71 (d, J = 8.1 Hz, 1H), 8.74–8.81 (m, 1H), 8.97 (s, 1H).; ¹³C NMR (151 MHz, DMSO-d₆): δ 15.86, 16.14, 18.77, 19.04, 20.04, 25.53, 25.56, 25.61, 25.65, 25.72, 25.78, 25.87, 28.04, 28.54, 28.81, 28.90, 28.97, 29.35, 29.38, 29.84, 30.93, 34.64, 37.18, 38.22, 40.23, 46.79, 46.97, 52.29, 55.54, 55.59, 56.02, 57.96, 58.74, 58.86, 67.54, 67.85, 68.77, 70.01, 70.06, 75.13, 102.78, 107.51, 107.68, 111.86, 120.92, 123.18, 123.76, 125.89, 126.90, 127.17, 127.87, 128.07, 128.48, 128.85, 130.20, 131.13, 131.49, 131.54, 131.74, 137.19, 137.35, 142.35, 148.01, 151.59, 156.12, 158.17, 158.20, 158.41, 160.10, 167.65, 168.28, 168.72, 169.88, 171.68; LC–MS (ESI) 99% purity, m/z: [M + H]⁺ calcd for C₈₀H₁₁₁N₈O₁₂S, 1407.80; found, 1408.8; HRMS (ESI) m/z: [M + H]⁺ calcd for C₈₀H₁₁₁N₈O₁₂S, 1407.8037; found, 1407.8026.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (126)

This compound was prepared using general procedure VIII, 99 (75 mg, 98 μmol), and 72 (27 mg, 98 μmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 4:1) to give a yellow resin. Yield (51 mg, 51%); R_f = 0.30 (EtOAc/n-hexanes 4:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₆H₇₂O₁₁N₇, 1018.5284; found, 1018.5250.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (127)

This compound was prepared using general procedure VIII, 100 (70 mg, 87 μmol) and 72 (24 mg, 87 μmol). The crude product was purified by column chromatography (EtOAc/n-hexanes 4:1) to give a yellow resin. Yield (55 mg, 59%); R_f = 0.32 (EtOAc/n-hexanes 4:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₉H₇₇O₁₁N₇, 1060.5754; found, 1060.5736.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(4-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butoxy)butoxy)phenoxy)-2-((R-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoylpyrrolidin-1-yl)-2-oxoethyl)amino-1-oxopropan-2-yl)-(methyl)carbamate (128)

This compound was prepared using general procedure VIII, 101 (94 mg, 115 μmol) and 72 (32 mg, 115 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 20:1) to give a yellow resin. Yield (72 mg, 58%); R_f = 0.35 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₉H₇₈O₁₂N₇, 1076.5703; found, 1076.5691.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (129)

This compound was prepared using general procedure VIII, 102 (71 mg, 88 μmol), and 72 (24 mg, 88 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 20:1) to give a yellow resin. Yield (72 mg, 58%); R_f = 0.35 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₇H₇₄O₁₃N₇, 1064.5339; found, 1064.5325.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (130)

This compound was prepared using general procedure VIII, 103 (100 mg, 117 μmol), and 72 (32 mg, 117 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 20:1) to give a yellow resin. Yield (30 mg, 23%); R_f = 0.35 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₉H₇₈O₁₄N₇, 1108.5601; found, 1108.5585.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-((6-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-2-((R-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoylpyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (131)

This compound was prepared using general procedure VIII, 104 (95 mg, 103 μmol), and 72 (28 mg 98 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 30:1) to give a yellow resin. Yield (86 mg, 71%); R_f = 0.18 (CH₂Cl₂/MeOH 20:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₅H₉₀O₁₃N₇, 1176.6591; found, 1176.6571.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((5-((5-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)pentyl)oxy)pentyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (132)

This compound was prepared using general procedure VIII, 105 (65 mg, 69 μmol), and 72 (21 mg, 69 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a yellow resin. Yield (47 mg, 57%); R_f = 0.45 (CH₂Cl₂/MeOH 9:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₇H₉₄O₁₃N₇, 1204.6904; found, 1204.6904.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-((6-((6-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (133)

This compound was prepared using general procedure VIII, 106 (142 mg, 145 μmol), and 72 (40 mg, 145 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a yellow resin. Yield (51 mg, 29%); R_f = 0.35 (CH₂Cl₂/MeOH 20:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₉H₉₈O₁₃N₇, 1232.7217; found, 1232.7205.

tert-Butyl ((2S)-1-(((1S)-1-Cyclohexyl-2-((2S,4S)-4-(3-(2-((6-((6-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-2-(((R)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl)pyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (134)

This compound was prepared using general procedure VIII, 104 (92 mg, 100 μmol), and 73 (29 mg, 100 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 20:1) to give a yellow resin. Yield (65 mg, 55%); R_f = 0.23 (CH₂Cl₂/MeOH 20:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₆H₉₂O₁₃N₇, 1190.6748; found, 1190.6743.

tert-Butyl ((2S)-1-(((1S)-1-cyclohexyl-2-((2S,4S)-4-(3-((6-((6-((6-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-2-((R-1,2,3,4-tetrahydronaphthalen-1-yl)carbamoylpyrrolidin-1-yl)-2-oxoethyl)amino)-1-oxopropan-2-yl)(methyl)carbamate (135)

This compound was prepared using general procedure VIII, 106 (125 mg, 128 μmol), and 73 (37 mg, 128 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH 50:1) to give a yellow resin. Yield (83 mg, 52%); R_f = 0.30 (CH₂Cl₂/MeOH 20:1). Due to the presence of the N-Boc protecting group resulting in an additional set of rotamers, NMR data is only provided for the deprotected final PROTAC. HRMS (ESI) m/z: [M + H]⁺ calcd for C₇₀H₁₀₀O₁₃N₇, 1246.7374; found, 1246.7360.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 20)

This compound was prepared using general procedure VII and PROTAC precursor 126 (51 mg, 50 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1) to give a yellow solid. Yield (35 mg, 76%); R_f = 0.45 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 64–69 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.82–1.00 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.43–1.68 (m, 8H), 1.70–1.85 (m, 6H), 1.97–2.14 (m, 2H), 2.35 (s, 3H), 2.69–2.78 (m, 4H), 2.82–2.90 (m, 2H), 3.02–3.10 (m, 1H), 3.30 (q, J = 6.6 Hz, 2H), 3.60–3.67 (m, 2H), 3.74–3.83 (m, 3H), 3.92 (t, J = 6.2 Hz, 1H), 4.22–4.31 (m, 1H), 4.39–4.47 (m, 1H), 4.71–4.78 (m, 1H), 4.84–4.96 (m, 2H), 5.08–5.16 (m, 1H), 6.25 (t, J = 5.7 Hz, 1H), 6.36–6.42 (m, 2H), 6.48–6.55 (m, 1H), 6.61–6.72 (m, 1H), 6.88 (d, J = 8.6 Hz, 1H), 7.02–7.18 (m, 5H), 7.29 (d, J = 7.3 Hz, 2H), 7.45–7.52 (m, 1H), 7.67 (d, J = 8.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.65, 20.20, 22.93, 23.73, 25.66, 25.77, 26.03, 28.73, 29.04, 29.16, 29.42, 29.89, 30.12, 31.59, 33.57, 35.22, 40.63, 42.65, 43.03, 47.74, 49.02, 53.81, 54.96, 60.22, 61.86, 67.73, 71.27, 72.42, 102.90, 107.61, 108.50, 110.06, 111.57, 116.76, 126.31, 127.21, 128.86, 129.21, 130.22, 132.64, 136.27, 136.81, 137.57, 147.08, 158.09, 160.40, 167.77, 168.55, 169.65, 171.25, 172.69, 175.24; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 7.19 min, 95% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₁H₆₄O₉N₇, 918.4760; found, 918.4735.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 21)

This compound was prepared using general procedure VII and PROTAC precursor 127 (54 mg, 51 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (42 mg, 86%); R_f = 0.55 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 83–85 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.83–1.00 (m, 5H), 1.27 (d, J = 6.9 Hz, 3H), 1.34–1.54 (m, 8H), 1.54–1.70 (m, 8H), 1.72–1.84 (m, 6H), 1.98–2.14 (m, 2H), 2.29–2.35 (m, 1H), 2.36 (s, 3H), 2.67–2.81 (m, 4H), 2.82–2.91 (m, 2H), 3.01–3.08 (m, 1H), 3.25 (q, J = 6.6 Hz, 2H), 3.81 (d, J = 11.5 Hz, 1H), 3.85–3.90 (m, 2H), 4.21–4.29 (m, 1H), 4.38–4.46 (m, 1H), 4.71–4.78 (m, 1H), 4.85–4.96 (m, 2H), 5.13 (q, J = 5.9 Hz, 1H), 6.22 (t, J = 5.5 Hz, 1H), 6.36–6.41 (m, 2H), 6.50–6.54 (m, 1H), 6.64 (d, J = 9.2 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 7.02–7.17 (m, 5H), 7.30 (d, J = 7.8 Hz, 1H), 7.45–7.50 (m, 1H), 7.63 (dd, J = 9.0, 3.0 Hz, 1H), 8.38 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.70, 20.21, 22.93, 25.65, 25.77, 26.03, 26.08, 26.98, 28.69, 29.34, 29.43, 29.90, 30.14, 31.56, 33.62, 35.28, 40.67, 42.76, 47.75, 49.00, 53.76, 54.91, 60.24, 60.40, 68.03, 102.88, 103.03, 107.93, 108.17, 109.97, 111.48, 116.77, 126.34, 127.22, 128.90, 129.19, 130.18, 132.63, 136.24, 136.78, 137.54, 147.15, 158.08, 160.58, 167.77, 168.53, 169.66, 171.20, 172.74, 175.37; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 8.04 min, 97% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₄H₇₀O₉N₇, 960.5230; found, 960.5201.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(4-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butoxy)butoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 22)

This compound was prepared using general procedure VII and PROTAC precursor 128 (71 mg, 66 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (59 mg, 92%); R_f = 0.42 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 80–81 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.83–0.99 (m, 5H), 1.28 (d, J = 6.8 Hz, 3H), 1.39–1.65 (m, 7H), 1.65–1.88 (m, 12H), 1.99–2.13 (m, 2H), 2.35 (s, 3H), 2.67–2.89 (m, 6H), 3.02–3.10 (m, 1H), 3.30 (q, J = 6.5 Hz, 2H), 3.44–3.50 (m, 4H), 3.80 (d, J = 11.7 Hz, 1H), 3.91 (d, J = 5.4 Hz, 2H), 4.23–4.29 (m, 1H), 4.39–4.48 (m, 1H), 4.71–4.78 (m, 1H), 4.81–4.96 (m, 2H), 5.13 (q, J = 6.1 Hz, 1H), 6.26 (t, J = 5.7 Hz, 1H), 6.36–6.41 (m, 2H), 6.49–6.54 (m, 1H), 6.59–6.73 (m, 1H), 6.88 (d, J = 8.5 Hz, 1H), 7.02–7.16 (m, 5H), 7.30 (d, J = 7.1 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.61–7.70 (m, 1H), 8.52 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.65, 20.20, 22.93, 25.66, 25.77, 26.04, 26.28, 26.42, 26.48, 27.21, 28.69, 29.42, 29.88, 30.13, 31.58, 33.62, 35.20, 40.68, 42.61, 47.73, 49.01, 53.75, 54.93, 60.22, 67.80, 70.40, 70.67, 76.35, 102.94, 103.07, 107.72, 108.36, 110.01, 111.50, 116.78, 126.32, 127.21, 128.93, 129.19, 130.18, 132.64, 136.25, 136.81, 137.56, 147.08, 158.10, 160.49, 167.78, 168.58, 169.67, 171.28, 172.71, 175.31; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 7.42 min, 97% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₄H₇₀O₁₀N₇, 976.5179; found, 976.5161.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 23)

This compound was prepared using general procedure VII and PROTAC precursor 129 (47 mg, 44 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (40 mg, 94%); R_f = 0.40 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 80–82 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.82–1.01 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.37–1.71 (m, 7H), 1.74–1.84 (m, 4H), 1.99–2.10 (m, 2H), 2.33 (s, 3H), 2.62–2.89 (m, 6H), 3.02–3.13 (m, 1H), 3.40–3.51 (m, 2H), 3.68–3.76 (m, 6H), 3.77–3.82 (m, 1H), 3.82–3.88 (m, 2H), 4.02–4.10 (m, 2H), 4.23–4.32 (m, 1H), 4.40–4.48 (m, 1H), 4.72–4.77 (m, 1H), 4.80–4.93 (m, 2H), 5.09–5.17 (m, 1H), 6.36–6.44 (m, 2H), 6.47–6.56 (m, 2H), 6.70 (s, 1H), 6.90 (dd, J = 8.5, 1.7 Hz, 1H), 7.01–7.17 (m, 5H), 7.27–7.32 (m, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.62–7.75 (m, 1H), 8.69 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 14.26, 19.59, 20.19, 22.86, 25.66, 25.79, 26.03, 28.70, 29.42, 29.84, 30.11, 31.58, 33.57, 35.11, 40.60, 42.53, 47.69, 49.01, 53.76, 54.97, 60.18, 67.61, 69.69, 69.96, 70.97, 102.75, 107.69, 107.88, 110.40, 111.72, 116.94, 126.31, 127.21, 128.86, 129.20, 130.17, 132.61, 136.16, 136.87, 137.58, 146.97, 158.10, 160.22, 167.80, 168.62, 169.41, 169.68, 171.40, 172.67, 175.25; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 6.63 min, 95% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₂H₆₆11N₇, 964.4815; found, 964.4799.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 24)

This compound was prepared using general procedure VII and PROTAC precursor 130 (30 mg, 27 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (22 mg, 81%); R_f = 0.35 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 76–80 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.81–1.02 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.38–1.70 (m, 10H), 1.74–1.84 (m, 4H), 1.99–2.13 (m, 2H), 2.36 (s, 3H), 2.62–2.78 (m, 4H), 2.78–2.90 (m, 2H), 3.05 (q, J = 7.5 Hz, 1H), 3.44 (q, J = 5.5 Hz, 2H), 3.66–3.68 (m, 3H), 3.68–3.74 (m, 5H), 3.83 (t, J = 4.9 Hz, 2H), 4.05 (q, J = 4.7 Hz, 2H), 4.24–4.31 (m, 1H), 4.44 (t, J = 8.0 Hz, 1H), 4.72–4.78 (m, 1H), 4.84–4.94 (m, 2H), 5.09–5.16 (m, 1H), 6.38–6.43 (m, 2H), 6.48 (q, J = 5.4 Hz, 1H), 6.51–6.55 (m, 1H), 6.65 (d, J = 7.8 Hz, 1H), 6.91 (dd, J = 8.5, 2.3 Hz, 1H), 7.02–7.16 (m, 5H), 7.29 (d, J = 7.9 Hz, 1H), 7.44–7.50 (m, 1H), 7.65 (d, J = 8.7 Hz, 1H), 8.57 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.67, 20.20, 22.91, 25.66, 25.78, 26.02, 28.68, 29.42, 29.88, 30.13, 31.57, 33.54, 35.24, 40.64, 42.57, 47.73, 49.02, 53.78, 54.93, 60.20, 60.35, 67.63, 69.69, 69.81, 70.83, 70.88, 70.96, 102.95, 107.73, 108.73, 110.40, 111.77, 116.97, 126.33, 127.23, 128.85, 129.21, 130.21, 132.63, 136.18, 136.81, 137.56, 147.00, 158.08, 160.19, 167.76, 168.59, 169.42, 169.66, 171.29, 172.72, 175.35; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 6.64 min, 98% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₄H₇₀O₁₂N₇, 1008.5077; found, 1008.5049.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-((6-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 25) (SAB 141)

This compound was prepared using general procedure VII and PROTAC precursor 131 (80 mg, 68 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (61 mg, 83%); R_f = 0.42 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 70–73 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.82–1.00 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.34–1.45 (m, 10H), 1.51–1.71 (m, 13H), 1.72–1.84 (m, 4H), 1.98–2.15 (m, 2H), 2.36 (s, 3H), 2.67–2.80 (m, 4H), 2.82–2.90 (m, 2H), 3.01–3.07 (m, 1H), 3.25 (q, J = 5.9 Hz, 2H), 3.38 (dd, J = 6.6, 2.7 Hz, 4H), 3.51 (d, J = 6.7 Hz, 2H), 3.75 (t, J = 4.7 Hz, 2H), 3.81 (d, J = 11.6 Hz, 1H), 4.04 (t, J = 5.1 Hz, 2H), 4.26 (dd, J = 11.2, 4.8 Hz, 1H), 4.42 (t, J = 8.3 Hz, 1H), 4.75 (dt, J = 9.8, 2.5 Hz, 1H), 4.85–4.96 (m, 2H), 5.13 (q, J = 6.1 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 6.37–6.43 (m, 2H), 6.52–6.57 (m, 1H), 6.59–6.64 (m, 1H), 6.84–6.89 (m, 1H), 7.02–7.17 (m, 5H), 7.27–7.31 (m, 1H), 7.45–7.51 (m, 1H), 7.63 (d, J = 8.9 Hz, 1H), 8.35 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.71, 20.21, 22.94, 25.65, 25.77, 26.01, 26.11, 26.22, 26.94, 28.68, 29.36, 29.42, 29.76, 29.78, 29.84, 29.90, 30.13, 31.57, 33.58, 35.29, 40.64, 42.73, 47.74, 49.00, 53.77, 54.92, 60.24, 60.40, 67.58, 69.25, 70.80, 71.00, 71.72, 103.08, 107.84, 108.52, 108.60, 109.97, 111.50, 116.78, 126.36, 127.24, 128.83, 129.21, 130.19, 132.63, 136.26, 136.77, 137.55, 147.14, 158.02, 160.28, 167.79, 168.54, 169.66, 171.19, 172.74, 175.39; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 8.26 min, 96% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₀H₈₂O₁₁N₇, 1076.6067; found, 1076.6042.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((5-((5-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)pentyl)oxy)pentyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 26)

This compound was prepared using general procedure VII and PROTAC precursor 132 (47 mg, 39 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (35 mg, 81%); R_f = 0.20 (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1); mp 63–66 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.81–0.99 (m, 5H), 1.27 (d, J = 6.9 Hz, 3H), 1.34–1.53 (m, 10H), 1.54–1.71 (m, 15H), 1.72–1.84 (m, 6H), 1.99–2.15 (m, 2H), 2.36 (s, 3H), 2.66–2.81 (m, 4H), 2.82–2.91 (m, 2H), 3.00–3.07 (m, 1H), 3.25 (d, J = 5.8 Hz, 2H), 3.36–3.44 (m, 8H), 3.81 (d, J = 11.4 Hz, 1H), 3.87 (d, J = 6.0 Hz, 2H), 4.24 (dd, J = 11.5, 4.9 Hz, 1H), 4.42 (d, J = 8.1 Hz, 1H), 4.75 (dd, J = 9.8, 2.1 Hz, 1H), 4.86–4.96 (m, 2H), 5.13 (q, J = 6.0 Hz, 1H), 6.21 (t, J = 5.6 Hz, 1H), 6.34–6.40 (m, 2H), 6.50–6.53 (m, 1H), 6.61 (dd, J = 8.4, 3.8 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 7.02–7.17 (m, 5H), 7.30 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 7.0 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 8.38 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.71, 20.21, 22.93, 25.64, 25.76, 26.01, 26.11, 26.94, 28.68, 29.24, 29.35, 29.42, 29.66, 29.72, 29.78, 29.91, 30.13, 31.56, 33.63, 35.29, 40.65, 42.73, 47.74, 48.99, 53.76, 54.91, 60.25, 60.40, 67.98, 70.84, 70.97, 76.37, 103.06, 107.83, 108.02, 108.07, 109.96, 111.49, 116.76, 126.34, 127.23, 128.83, 129.20, 130.16, 132.62, 136.24, 136.76, 137.53, 147.13, 158.04, 160.56, 167.77, 168.51, 169.66, 171.19, 172.73, 175.39; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 8.60 min, 99% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₂H₈₆O₁₁N₇, 1104.6380; found, 1104.6396.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 27) (SAB142)

This compound was prepared using general procedure VII and PROTAC precursor 133 (50 mg, 41 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1) to give a yellow solid. Yield (45 mg, 97%); R_f = 0.25 (CH₂Cl₂/MeOH/NH₄OH 15:1:0.1); mp 62–64 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.80–0.99 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.33–1.51 (m, 12H), 1.51–1.70 (m, 17H), 1.72–1.84 (m, 6H), 1.99–2.07 (m, 1H), 2.08–2.15 (m, 1H), 2.36 (s, 3H), 2.69–2.81 (m, 4H), 2.83–2.91 (m, 2H), 3.05 (q, J = 6.8 Hz, 1H), 3.25 (d, J = 6.2 Hz, 2H), 3.38 (dd, J = 6.6, 2.5 Hz, 8H), 3.82 (d, J = 11.0 Hz, 1H), 3.87 (d, J = 6.5 Hz, 2H), 4.24 (dd, J = 11.5, 4.8 Hz, 1H), 4.43 (d, J = 8.2 Hz, 1H), 4.71–4.78 (m, 1H), 4.86–4.97 (m, 2H), 5.13 (d, J = 6.1 Hz, 1H), 6.22 (d, J = 5.6 Hz, 1H), 6.34–6.41 (m, 2H), 6.49–6.54 (m, 1H), 6.59–6.65 (m, 1H), 6.87 (d, J = 8.6 Hz, 1H), 7.02–7.17 (m, 5H), 7.27–7.31 (m, 1H), 7.48 (dd, J = 8.5, 7.1 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 8.31 (br s, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.68, 20.21, 22.94, 25.65, 25.76, 26.02, 26.11, 26.16, 26.23, 26.96, 28.69, 29.37, 29.43, 29.80, 29.85, 29.91, 30.14, 31.57, 33.66, 35.25, 40.66, 42.74, 47.75, 49.00, 53.77, 54.93, 60.27, 60.38, 68.05, 70.82, 70.94, 71.02, 71.05, 76.38, 103.08, 107.86, 108.02, 109.97, 111.50, 116.77, 126.35, 127.24, 128.84, 129.21, 130.17, 132.63, 136.25, 136.76, 137.54, 147.14, 158.04, 160.59, 167.78, 168.51, 169.64, 169.68, 171.17, 172.73, 175.32; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 9.08 min, 99% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₄H₉₀O₁₁N₇, 1132.6693; found, 1132.6669.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-(2-((6-((6-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)ethoxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 28)

This compound was prepared using general procedure VII and PROTAC precursor 134 (65 mg, 55 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1) to give a yellow solid. Yield (41 mg, 69%); R_f = 0.30 (CH₂Cl₂/MeOH/NH₄OH 9:1:0.1); mp 58–61 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.79–0.99 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.33–1.49 (m, 10H), 1.57 (s, 13H), 1.74–1.84 (m, 4H), 2.00–2.12 (m, 2H), 2.36 (s, 3H), 2.69–2.81 (m, 4H), 2.89 (d, J = 14.5 Hz, 1H), 2.94–3.00 (m, 1H), 3.01–3.07 (m, 1H), 3.21 (s, 3H), 3.25 (q, J = 6.7 Hz, 2H), 3.39 (td, J = 6.6, 2.7 Hz, 4H), 3.51 (t, J = 6.7 Hz, 2H), 3.73–3.77 (m, 2H), 3.82 (d, J = 11.4 Hz, 1H), 4.04 (d, J = 4.9 Hz, 2H), 4.25 (dd, J = 11.5, 4.8 Hz, 1H), 4.41 (t, J = 8.2 Hz, 1H), 4.76 (dd, J = 9.8, 2.1 Hz, 1H), 4.87–4.96 (m, 2H), 5.13 (q, J = 7.0 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 6.37–6.44 (m, 2H), 6.53–6.57 (m, 1H), 6.59 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 7.03–7.19 (m, 5H), 7.29 (d, J = 7.7 Hz, 1H), 7.45–7.51 (m, 1H), 7.64 (d, J = 8.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.71, 20.20, 22.29, 25.64, 25.76, 26.01, 26.12, 26.22, 26.98, 27.40, 28.70, 29.39, 29.43, 29.77, 29.81, 29.86, 29.91, 30.14, 32.07, 33.59, 35.30, 40.64, 42.75, 47.74, 49.76, 53.80, 54.92, 60.24, 60.41, 67.57, 69.24, 70.85, 71.04, 71.74, 76.41, 103.15, 107.91, 108.50, 110.07, 111.44, 116.71, 126.37, 127.26, 128.81, 129.22, 130.20, 132.69, 136.19, 136.77, 137.54, 147.11, 157.98, 160.29, 167.94, 169.18, 169.61, 169.83, 171.40, 172.76, 175.27; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 8.53 min, 97% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₁H₈₄O₁₁N₇, 1090.6223; found, 1090.6203.

(2S,4S)-1-((S)-2-Cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-4-(3-((6-((6-((6-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)oxy)hexyl)oxy)hexyl)oxy)phenoxy)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)pyrrolidine-2-carboxamide (PROTAC 29)

This compound was prepared using general procedure VII and PROTAC precursor 135 (82 mg, 66 μmol). The crude product was purified by column chromatography (CH₂Cl₂/MeOH/NH₄OH 20:1:0.1) to give a yellow solid. Yield (44 mg, 58%); R_f = 0.30 (CH₂Cl₂/MeOH/NH₄OH 20:1:0.1); mp 56–60 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.80–0.98 (m, 5H), 1.28 (d, J = 6.9 Hz, 3H), 1.32–1.49 (m, 12H), 1.50–1.71 (m, 18H), 1.72–1.84 (m, 6H), 2.00–2.12 (m, 2H), 2.36 (s, 3H), 2.70–2.81 (m, 4H), 2.85–2.93 (m, 1H), 2.94–2.99 (m, 1H), 3.00–3.05 (m, 1H), 3.20 (s, 3H), 3.22–3.30 (m, 1H), 3.38 (dd, J = 6.6, 2.6 Hz, 8H), 3.78–3.84 (m, 1H), 3.87 (t, J = 6.5 Hz, 2H), 4.23 (dd, J = 11.5, 4.8 Hz, 1H), 4.41 (d, J = 8.4 Hz, 1H), 4.76 (dd, J = 9.8, 2.1 Hz, 1H), 4.87–4.98 (m, 2H), 5.13 (d, J = 6.2 Hz, 1H), 6.22 (d, J = 5.6 Hz, 1H), 6.35–6.40 (m, 2H), 6.51–6.55 (m, 1H), 6.59 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 7.02–7.17 (m, 5H), 7.27–7.33 (m, 1H), 7.45–7.52 (m, 1H), 7.65 (d, J = 8.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃, only the peaks for the major rotamer are given): δ 19.59, 20.07, 22.16, 25.51, 25.62, 25.88, 25.98, 26.03, 26.03, 26.10, 26.85, 27.26, 28.56, 29.25, 29.30, 29.68, 29.74, 29.79, 30.00, 31.94, 33.52, 35.18, 40.53, 42.62, 47.62, 49.63, 53.67, 54.77, 60.14, 60.28, 67.92, 70.71, 70.82, 70.91, 76.29, 102.99, 107.76, 107.86, 111.31, 116.57, 126.23, 127.12, 128.68, 129.08, 130.04, 132.55, 136.06, 136.61, 137.40, 146.98, 157.88, 160.47, 167.81, 169.04, 169.50, 169.69, 171.26, 172.63, 175.17; HPLC (95% H₂O (with 0.1% TFA) to 95% MeCN in 10 min, then 95% MeCN for 4 min), t_R = 8.40 min, 97% purity, detection at 254 nm; HRMS (ESI) m/z: [M + H]⁺ calcd for C₆₅H₉₂O₁₁N₇, 1146.6849; found, 1146.6812.

Cell Lines

All cell lines were obtained from ATCC (Manassas, Virginia, USA) and the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) and maintained in RPMI-1640 medium (Merck KGaA, Darmstadt, Germany) containing 10% fetal bovine serum and supplemented with 1% penicillin/streptomycin and 1% l-glutamine. NCI-H929 cells were cultured in media supplemented with 2-mercaptoethanol and sodium pyruvate. Cells were maintained at 37 °C with 5% CO₂ in a humidified atmosphere.

For generation of lentiviral vectors, HEK293T cells were transfected with constructs along with the packaging and envelope vectors. Viral supernatants were harvested 48 h after transfection and were used to transduce cell lines. MM.1S and HG3 cells were transduced with virus containing pLKO5d.SSF.SpCas9.P2a.BSD, and cells were selected with blasticidin. Selected cells were then transduced with respective sgRNA constructs targeting VHL, cIAP1, cIAP2, XIAP, and negative control luciferase, which were cloned into pLKO5.hU6.sgRNA.dTom. Transduction success was confirmed through FACS analysis 48 h post-transduction with a minimum efficiency of 95% tomato fluorescence.

Immunoblotting

Cells were treated with respective drugs for 16 h, and treated cells were washed and lysed in Pierce IP lysis buffer. SDS-PAGE was performed, and proteins were then transferred onto PVDF membranes. Blotted membranes were blocked with 5% milk in Tris-buffered saline/Tween20 (TBST). Primary antibodies were diluted in 5% BSA in TBST, and incubations were performed overnight at 4 °C. Secondary HRP-conjugated antibodies diluted in 5% milk were incubated for 1 h at room temperature. Detection of proteins on PVDF was carried out using the WesternBright ECL HRP substrate or the WesternBright Sirius HRP substrate (Advansta, San Jose, USA) and imaged with LAS 4000× (Fujifilm). Membranes were subjected to 10 min incubation with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, USA), followed by TBST washes. After brief re-activation with methanol, the membranes were blocked, and further probing of proteins was carried out.

Reagents and Antibodies

LCL-161, AZD5582, birinapant, and BV6 were obtained from MedChemExpress. Human TNF-α was obtained from Miltenyi Biotec. MG132, MLN4924, and MLN7243 were purchased from SelleckChem.

Primary antibodies used for immunoblotting include BIRC2 (BioRad; VMA00532; clone AB01/3B4), cIAP2 (Cell Signaling; 3130S; clone 58C7), XIAP (Cell Signaling; 14334S; clone D2Z8W), VHL (Cell Signaling; 68547S), CRBN (Sigma-Aldrich; SAB2106014), Ikaros (Cell Signaling; 14859S; clone D6N9Y), Aiolos (Cell Signaling; 15103S; clone D1C1E), HIF-1α (BD BioSciences, 610958; clone 54), α-tubulin (Sigma-Aldrich; T5168; clone B512), and beta-actin (Sigma-Aldrich; A1978). Secondary antibodies include anti-rabbit IgG HRP-linked antibody (Cell Signaling; 7074) and anti-mouse IgG HRP-linked antibody (Cell Signaling; 7076).

Cell Viability Assays

Cells were seeded in 384-well plates with respective treatments, and plates were incubated at 37 °C for 96 h. Viability assays were performed with or without the addition of TNF-α at 1 ng/mL. The cell viability readout was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, USA) and measured with a Synergy LX Multi-Mode plate reader (BioTek, Vermont, USA). All conditions were normalized to the DMSO-treated control. Data represents the mean ± SD of biological triplicates.

Statistical Analysis

Statistical and graphical analyses of cell viability experiments were performed with Prism version 9.1.0 (GraphPad Software, San Diego, CA, USA). Quantification of blots was performed using ImageJ software (National Institutes of Health).

diaPASEF-Based Proteomics

Sample Preparation LFQ Quantitative Mass Spectrometry

MM1s cells were treated with DMSO or 0.1 μM of compound for 3 h. The cells were harvested by centrifugation and washed with phosphate-buffered saline before snap-freezing in liquid nitrogen. The cells were lysed by addition of lysis buffer (8 M urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (EPPS) pH 8.5, protease, and phosphatase inhibitors) and homogenization by bead beating (BioSpec) for three repeats of 30 s at 2400. Bradford assay was used to determine the final protein concentration in the clarified cell lysate. 50 μg of protein for each sample was reduced, alkylated, and precipitated using methanol/chloroform as previously described,⁶⁴ and the resulting washed precipitated protein was allowed to air dry. The precipitated protein was resuspended in 4 M urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme/protein) for 12 h at RT. The LysC digestion was diluted to 0.5 M urea with 200 mM EPPS pH 8, followed by digestion with trypsin (1:50; enzyme/protein) for 6 h at 37 °C. Sample digests were acidified with formic acid to a pH of 2–3 prior to desalting using C18 solid-phase extraction plates (SOLA, Thermo Fisher Scientific). Desalted peptides were dried in a vacuum centrifuge and reconstituted in 0.1% formic acid for LC–MS analysis.

Data were collected using a TimsTOF Pro2 (Bruker Daltonics, Bremen, Germany) coupled to a nanoElute LC pump (Bruker Daltonics, Bremen, Germany) via a CaptiveSpray nanoelectrospray source. Peptides were separated on a reversed-phase C₁₈ column (25 cm × 75 μm ID, 1.6 μM, IonOpticks, Australia) containing an integrated captive spray emitter. Peptides were separated using a 50 min gradient of 2–30% buffer B (acetonitrile in 0.1% formic acid) with a flow rate of 250 nL/min and column temperature maintained at 50 °C.

Data-Dependent Acquisition (DDA) was performed in the Parallel Accumulation-Serial Fragmentation (PASEF) mode to determine effective ion mobility windows for downstream diaPASEF data collection.⁵⁴ The diaPASEF parameters included 100% duty cycle using accumulation and ramp times of 50 ms each, 1 TIMS-MS scan, and 10 PASEF ramps per acquisition cycle. The TIMS-MS survey scan was acquired between 100–1700 m/z and 1/K₀ of 0.7–1.3 V × s/cm². Precursors with 1–5 charges were selected, and those that reached an intensity threshold of 20,000 arbitrary units were actively excluded for 0.4 min. The quadrupole isolation width was set to 2 m/z for m/z < 700 and 3 m/z for m/z > 800, with the m/z between 700 and 800 m/z being interpolated linearly. The TIMS elution voltages were calibrated linearly with three points (Agilent ESI-L Tuning Mix Ions; 622, 922, 1222 m/z) to determine the reduced ion mobility coefficients (1/K₀). To perform diaPASEF, the precursor distribution in the DDA m/z-ion mobility plane was used to design an acquisition scheme for DIA data collection, which included two windows in each 50 ms diaPASEF scan. Data was acquired using 16 of these 25 Da precursor double window scans (creating 32 windows), which covered the diagonal scan line for doubly and triply charged precursors, with singly charged precursors able to be excluded by their position in the m/z-ion mobility plane. These precursor isolation windows were defined between 400–1200 m/z and 1/K₀ of 0.7–1.3 V × s/cm².

LC–MS Data Analysis

The diaPASEF raw file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides were performed using library-free analysis in DIA-NN 1.8.⁶⁵ The library-free mode performs an in silico digestion of a given protein sequence database alongside deep learning-based predictions to extract the DIA precursor data into a collection of MS² spectra. The search results are then used to generate a spectral library which is then employed for the targeted analysis of the DIA data searched against the Swiss-Prot human database (January 2021). Database search criteria largely followed the default settings for directDIA including tryptic with two missed cleavages, carbamidomethylation of cysteine, and oxidation of methionine and precursor Q-value (FDR) cut-off of 0.01. The precursor quantification strategy was set to Robust LC (high accuracy) with RT-dependent cross-run normalization. Proteins with poor-quality data were excluded from further analysis (summed abundance across channels of <100 and the mean number of precursors used for quantification <2), and proteins with missing values were imputed by random selection from a Gaussian distribution either with a mean of the non-missing values for that treatment group or with a mean equal to the median of the background (in cases when all values for a treatment group are missing). Protein abundances were scaled using in-house scripts in the R framework (R Development Core Team, 2014), and statistical analysis was carried out using the limma package within the R framework.

Imputation Description

Protein level data output from diaNN was read into R and processed using in-house scripts. Summary statistics were calculated for the replicates of each protein condition group. Missing values for each group were imputed by random selection from a Gaussian distribution with a mean of the non-missing values for that group. For protein condition groups missing all values, the values were imputed by random selection from a Gaussian distribution with a mean equal to the median of the background, defined as the lowest 1% of the dataset. The standard deviation of each distribution was based on the global relative standard deviation of the dataset, and each distribution was truncated to have a minimum value of 100 and a maximum of 1.2 times the maximum value in the entire dataset.

Molecular Descriptor Calculations

Predicted values for the topological polar surface area (TPSA) were calculated using MarvinSketch 17.28.0 (ChemAxon). Predicted values for the number of rotatable bonds were obtained using LigandScout 4.4.3.

Log D Measurements

The determination of the log D_7.4 values was performed by a chromatographic method as described previously.^60,66 The system was calibrated by plotting the retention times of six different drugs (atenolol, metoprolol, labetalol, diltiazem, triphenylene, and permethrin) versus their literature-known log D_7.4 in a calibration line (R² = 0.99). Subsequently, the mean retention times of the analytes were taken to calculate their log D_7.4 values with the aid of the calibration line. At least two independent measurements of each analyte were performed.

Plasma Protein Binding Studies

Plasma protein binding (% PPB) was estimated by correlating the logarithmic retention times of the analytes on a CHIRALPAK HSA 50 × 3 mm, 5 μm column with the literature-known % PPB values (converted into log K values) of the following drugs: warfarin, ketoprofen, budesonide, nizatidine, indomethacin, acetylsalicylic acid, carbamazepine, piroxicam, nicardipine, and cimetidine (for details, see Valko et al.).⁶⁷ The samples were dissolved in MeCN/DMSO 9:1 to achieve a final concentration of 0.5 mg/mL. Mobile phase A was 50 mM ammonium acetate adjusted to pH 7.4 with ammonia solution, while mobile phase B was iPrOH. The flow rate was set to 1.0 mL/min, the UV detector was set to 254 nm, and the column temperature was kept at 30 °C. After injecting 2 μL of the sample, a linear gradient from 100% A to 30% iPrOH in 5.4 min was applied. From 5.4 to 18 min, 30% iPrOH was kept, followed by switching back to 100% A in 1.0 min and a re-equilibration time of 6 min. With the aid of the calibration line (R² = 0.96), the log K values of new substances were calculated and converted to their % PPB values. At least two independent measurements of each analyte were performed.

Glossary

Abbreviations

AML

acute myeloid leukemia

BAIB

(diacetoxyiodo)benzene

BIR

baculoviral IAP repeat

cIAP

cellular IAP

CRBN

cereblon

DCC

N,N′-dicyclohexylcarbodiimide

DEAD

diethyl azodicarboxylate

DIPEA

N,N-diisopropylethylamine

DLBCL

diffuse large B-cell lymphoma

DMAP

4-dimethylaminopyridine

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HIF

hypoxia-inducible factor

HPLC

high-performance liquid chromatography

IAP

inhibitor of apoptosis

IKZF1

zinc finger protein Ikaros

IKZF3

zinc finger protein Aiolos

LC–MS

liquid chromatography–mass spectrometry

MDM2

murine double minute 2

MM

multiple myeloma

NMR

nuclear magnetic resonance

PROTAC

proteolysis targeting chimera

PS-TPP

polymer-bound triphenylphosphine

RING

really interesting new gene

SMAC

second mitochondria-derived activator of caspases

TBAHS

tetrabutylammonium hydrogen sulfate

TEMPO

(2,2,6,6-tetramethylpiperidin-1-yl)oxyl

TFA

trifluoroacetic acid

THP

tetrahydropyranyl

TLC

thin-layer chromatography

TNF-α

tumor necrosis factor alpha

VHL

von Hippel-Lindau

XIAP

X-chromosome-linked IAP

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01817.

• Overview on physicochemical properties of compounds, selected NMR, HPLC, and MS spectra (PDF)

• Recommended compound characterization checklist (XLSX)

• Molecular formula strings (CSV)

• Hitlist AZD5582 vs CTRLs (CSV)

• Hitlist PROTAC 9 vs CTRLs (CSV)

• Hitlist PROTAC 25 vs CTRLs (CSV)

• Hitlist PROTAC 27 vs CTRLs (CSV)

• Normalized raw abundances for 27 and pomalidomide (XLSX)

• Raw precursor abundances for 27 and pomalidomide (XLSX)

• Normalized raw abundances for AZD5582, 9, 25, and pomalidomide (XLSX)

• Raw precursor abundances for AZD5582, 9, 25, and pomalidomide (XLSX)

Author Contributions

^¶ Y.L.D.N. and A.B. contributed equally. M.G., J.K., C.S., and I.S. contributed to conceptualization; Y.L.D.N., A.B., K.A.D., and C.S. contributed to methodology; Y.L.D.N., J.A.J., A.M., K.P., and K.A.D. contributed to validation; Y.L.D.N., J.A.J., A.M., and K.A.D. contributed to formal analysis; Y.L.D.N., J.A.J., A.M., K.P., K.A.D., C.S., and I.S. contributed to investigation; M.G., J.K., C.S., and I.S. contributed to resources; C.S. and I.S. contributed to data curation; C.S. and I.S. contributed to writing—original draft; A.B., C.S., and I.S. contributed to writing—review and editing; Y.L.D.N., A.B., J.A.J., A.M., C.S., and I.S. contributed to visualization; M.G., J.K., C.S., and I.S. contributed to supervision; C.S. and I.S. contributed to project administration; and M.G., J.K., and I.S. contributed to funding acquisition.

We acknowledge the support by the Slovenian Research Agency (ARRS) (Program P1-0208 and grant J1-2485 to I.S.) and the DFG (Emmy-Noether Program Kr-3886/2-1 and SFB-1074 to J.K.).

The authors declare the following competing financial interest(s): K.A.D is a consultant to Kronos Bio and Neomorph Inc. All other authors declare no competing financial interest.

Notes

A preprint version of this study was posted on ChemRxiv preprint server.⁶⁸ While this manuscript was under consideration for publication, related pan-IAP degraders hijacking CRBN were published by S. Park and colleagues.⁶⁹