Discovery of a Highly Potent and Selective ENL Degrader

Abstract

The eleven-nineteen leukemia protein (ENL), a YEATS domain-containing acyl-lysine reader, represents a critical dependency in acute myeloid leukemia (AML). We previously reported our first-generation ENL proteolysis targeting chimera (PROTAC) degrader MS41. Here, via a comprehensive structure-activity relationship (SAR) study, we discovered MS108 (compound 124), the most potent ENL degrader to date, which recruits the von Hippel-Lindau (VHL) E3 ligase and achieved 5.8-fold higher ENL degradation potency (DC₅₀ = 0.6 ± 0.05 nM) and 18-fold stronger antiproliferation potency (GI₅₀ = 1.19 ± 0.03 nM) over MS41 in MV4;11 cells. Compound 124 induced robust and highly selective degradation of ENL in a concentration-, time-, VHL- and ubiquitin-proteasome system (UPS)-dependent manner while displaying improved pharmacokinetic properties. Collectively, we discovered a highly potent and selective ENL degrader, providing a useful chemical tool for the research community and a compelling lead for further development of ENL degraders into therapeutics to treat AML.

Graphical Abstract

INTRODUCTION

Post-translational modifications (PTMs) of histone proteins play a fundamental role in the regulation of gene expression.^1–2 These site-specific covalent modifications are installed by epigenetic writer enzymes and removed by epigenetic erasers, contributing to the dynamic regulation of transcriptional programs.³ In addition, these histone markers are recognized by epigenetic reader proteins, which interpret these marks to modulate chromatin structure and gene expression.⁴ Among the key histone PTMs, lysine acetylation (Kac) is one of the most investigated and well-characterized processes.^5–8 Targeting epigenetic reader proteins of acylated histones via occupying their acyl-lysine recognition pockets has emerged as a promising therapeutic strategy in epigenetic drug discovery for cancer.^7,9 For example, bromodomains, the most extensively studied acetyl-lysine readers, are frequently dysregulated in cancer, and several bromodomain inhibitors have advanced to clinical evaluation.^10,11

More recently, the YEATS domain, named after its five founding members (Yaf9, ENL, AF9, Taf14, and Sas5), has been recognized as a novel acetyl/acyl-lysine reader^12,13, capable of recognizing not only histone acetylation but also acylation such as crotonylation, propionylation, butyrylation, and succinylation.^14–16 The human genome encodes four YEATS domain-containing proteins: ENL, AF9, YEATS2, and GAS41.^12,13,16 Eleven-nineteen leukemia protein (ENL), also known as MLLT1, is a chromatin-associated epigenetic reader that recognizes acetyl- and crotonyl-lysine modifications on histone H3 tail through its conserved YEATS domain and shares a high sequence homology with its human paralog AF9.¹⁷ Upon binding to these acylation marks, ENL recruits transcriptional machinery to chromatin, thereby promoting the activation of target gene expression. Specifically, the C-terminal ANC-1 homology domain (AHD) of ENL has been shown to directly interact with histone methyltransferase DOT1L and the components of the Super Elongation Complex (SEC).^18–20 At the ENL N-terminal, YEATS domain can interact with the polymerase-associated factor 1 complex (PAF1c) and the histone acetyltransferase MOZ complex, which plays a key role in coordinating epigenetic deposition and transcriptional activation.^21,22

Genome-wide CRISPR-Cas9 screening has revealed ENL as a critical co-transcriptional factor that regulates oncogenic genes expression and supports the proliferation of acute myeloid leukemia (AML) cells.^23,24 Genetic depletion of ENL or disruption of its YEATS domain interaction with acetylated histones suppress leukemic cell proliferation.^23,24 Recurrent hotspot mutations within the YEATS domain of ENL have been identified in patients with Wilms’ tumor, further underscoring its cancer relevance.^25–27 Moreover, in leukemia, ENL is frequently fused to the mixed-lineage leukemia protein (MLL1) via chromosomal translocations. Importantly, the YEATS domain of ENL is retained in approximately 84% of MLL-ENL relevant patients and is critical for leukemogenesis.^28,29 MLL-ENL positive AML represents a highly aggressive leukemia subtype associated with poor clinical prognosis.^17,28

Given the roles of ENL YEATS domain in human AML, identifying its small-molecule inhibitors has attracted significant interest in epigenetic drug discovery.^16,30–31 However, compared with bromodomain inhibitors, the development of highly selective and potent small-molecule inhibitors of ENL YEATS reader domain has progressed slowly and remained challenging mainly due to its long, narrow tunnel-like binding site and the high sequence homology of YEATS family members (ENL and AF9 share 88% sequence identity).³² In 2018, Moustakim et al. reported the first dual small-molecule chemical probe, SGC-iMLLT, targeting the YEATS domains of ENL and AF9 (Figure 1).³³ Subsequently, several additional small-molecule inhibitors of the ENL/AF9 YEATS domains have been developed (Figure 1, left).^34–41 However, the efficacy of these inhibitors is limited by their reliance on stochiometric target occupancy, incomplete inhibition of ENL functions outside the YEATS domain, and the potential emergence of drug resistance.^33–41 In contrast, proteolysis-targeting chimera (PROTAC) degraders act catalytically by forming a transient ternary complex between the target protein, the PROTAC, and an E3 ligase, resulting in complete elimination of the target protein and its associated functions. Thus, the development of ENL-targeting PROTACs represents a potential strategy to achieve more sustained and potent ENL suppression, broader cellular activity and reduced likelihood of resistance.⁴² To date, four ENL degraders have been independently developed by the Erb group,^35,40 the Song group,⁴³ and our group.⁴⁴ These heterobifunctional molecules induces ENL degradation by recruiting an E3 ubiquitin ligase to ENL, leading to proximity-induced polyubiquitination of ENL and its subsequent proteasomal degradation (Figure 1, right). Our previous work has successfully demonstrated MS41, a PROTAC that recruits the von Hippel–Lindau (VHL) E3 ubiquitin ligase, as an effective ENL degrader while avoiding potential off-target degradation of cereblon (CRBN) neo-substrates such as IKZF1/3 and GSPT1 that has been associated with CRBN-recruiting PROTACs.⁴⁴

Figure 1.

Chemical structures of previously reported representative ENL inhibitors (left) and four reported degraders (right).

Here, we report our comprehensive structure-activity relationship (SAR) study to develop potent and selective ENL degraders. We designed, synthesized and evaluated 126 putative PROTACs to systematically explore various linker attachment points based on the crystal structure of PFI-6 in complex with AF9 YEATS domain, as well as various CRBN and VHL E3 ligase ligands. From this SAR study, we discovered MS108 (compound 124) as the most potent VHL-recruiting ENL degrader to date with DC₅₀ of 0.6 ± 0.05 nM and D_max > 97% in MV4;11 cells with excellent selectivity demonstrated by global proteomics study. Compound 124 induced rapid and robust degradation of ENL in a concentration-, time-, VHL- and ubiquitin-proteasome system (UPS)-dependent manner, achieving a 5.8-fold improvement in ENL degradation compared to MS41. Furthermore, compound 124 also induced more potent ENL degradation compared to previously reported CRBN-recruiting PROTACs^35,43 in MV4;11 cells. Importantly, compound 124 potently suppressed proliferation and clonogenic growth across multiple cancer cell lines, with 6- to 20-fold lower GI₅₀ values compared to MS41, thereby exhibiting substantially enhanced potency. In addition, compound 124 displayed sufficient plasma exposure in a mouse pharmacokinetic (PK) study via intraperitoneal (IP) administration.

RESULTS AND DISCUSSION

Design of Putative ENL PROTAC Degraders.

To develop potent and selective ENL degraders, we employed PFI-6³⁷ as the ENL binder and conducted a comprehensive SAR study. Our SAR study included systematic evaluation of different linker attachment points of PFI-6, variations in linker length and composition, and screening of various CRBN and VHL recruiting E3 ligase ligands. Unlike the acetyl-lysine (Kac) binding pocket of bromodomains, which typically features a deep, enclosed binding site, the acyl-lysine recognition site of the ENL YEATS domain forms a narrow, channel-like groove with two solvent-exposed termini. As shown in the co-crystal structure of PFI-6 in complex with the YEATS domain of AF9, the ENL paralog, both ends of PFI-6 are solvent-exposed, enabling linker attachment and derivatization for the design of PROTAC degrader (PDB: 8PJ7, Figure 2A). Based on this structural insight, we explored two principal strategies for linker attachment on PFI-6: (1) via the left-hand side (LHS) N,N-dimethylbenzamide moiety, and (2) via the right-hand side (RHS) 2,3-dihydro-1H-indene, using either an ether or amide linkage as the functional group (Figure 2B). In parallel, we examined both CRBN- and VHL-recruiting E3 ligase ligands using carbon- and polyethylene glycol (PEG)-based linkers (Figure 2B). For CRBN recruitment, 4-OH-thalidomide was selected as the ligand (Figure 2B). For VHL-based PROTACs, we evaluated four ligands: VHL1 (also known as (a.k.a.) VH032, amine), VHL7 (a.k.a. VH101, acid), VHL4 (a.k.a. VH101, phenol),⁴² and the recently reported VHL-1 ligand 10 with potent binding to VHL (Figure 2B).^45,46 We designed, synthesized and evaluated 126 putative ENL PROTACs, categorized into three classes based on their linker attachment site and linkage: (1) PROTACs with linker attached via the LHS N,N-dimethylbenzamide of PFI-6 (compounds 1–43); (2) PROTACs with linker attached via the RHS 2,3-dihydro-1H-indene, using an ether linkage (compounds 44–87); and (3) PROTACs with linker attached via the RHS 2,3-dihydro-1H-indene, using an amide linkage (compounds 88–126).

Figure 2. Design of putative ENL degraders.

(A) Cocrystal structure of AF9 YEATS domain in complex with PFI-6 (PDB: 8PJ7). (B) Chemical structures of PFI-6 derivatives, CRBN ligand (4-OH-Thalidomide) and VHL ligands (VHL1; VHL7; VHL4 and VHL-1 ligand 10) utilized in the design and synthesis of putative ENL PROTAC degraders.

Structure-Activity Relationship Study of ENL PROTAC Degraders.

To assess the degradation potency of newly designed ENL degraders, we monitored ENL protein levels via Western blot (WB) analysis in the human MLL-r leukemia cell line MV4;11 stably expressing 3xFlag–hemagglutinin (HA)–tagged ENL (3Flag-HA-ENL). ENL degradation was quantified as the percentage of ENL degraded (%D), calculated from WB band intensities following 24-hour treatment with degraders at 1 μM and 10 μM. First, we explored the LHS N,N-dimethylbenzamide of PFI-6 as a linker attachment point. This design was intended to investigate whether the “left face” of ENL is more favorable for ternary complex formation, leading to ENL degradation. PFI-6 was conjugated to the CRBN ligand 4-OH-thalidomide through this linking strategy, yielding compounds 1–11. Furthermore, the oxyacetamide functional group, which is known to abrogate CRBN neosubstrate degradation,⁴⁷ was used as a connecting handle to the CRBN ligand (Table 1). As shown in Table 1 and Figure S1, CRBN-recruiting PROTACs (compounds 1–11) bearing PEG linkers showed low levels of ENL degradation even at the high concentration of 10 μM. Although compound 1 with a short alkyl linker showed higher ENL degradation with 85% at 10 μM, its overall ENL degradation activity is far from satisfying. Similarly, VHL-recruiting PROTACs (compounds 12–43), which utilize the same linker attachment strategy, failed to induce appreciable ENL degradation (Tables 2, 3 & 4; Figures S2, S3 & S4). This was consistent across PROTACs incorporating multiple VHL ligands, including VHL1, VHL7 and VHL4. These results suggest that linker installation at the LHS of PFI-6 does not promote a favorable ternary complex formation and effective ENL polyubiquitination and degradation in general.

Table 1.

ENL Protein Degradation Activity of Compounds 1–11^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 1–11 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. For compounds exhibiting > 50% degradation, results are reported as the mean ± SD of two independent experiments. ND = no degradation. Corresponding representative WBs are shown in Figure S1.

Table 2.

ENL Protein Degradation Activity of Compounds 12–24^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 12–24 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. ND = no degradation. Corresponding WBs are shown in Figure S2.

Table 3.

ENL Protein Degradation Activity of Compounds 25–34^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 25–34 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. ND = no degradation. Corresponding WBs are shown in Figure S3.

Table 4.

ENL Protein Degradation Activity of Compounds 35–43^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 35–43 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. ND = no degradation. Corresponding WBs are shown in Figure S4.

We next investigated compounds 44–87, in which the linker was installed on the RHS 2,3-dihydro-1H-indene moiety of PFI-6 via an ether linkage (Tables 5, S1, S2, and S3). As shown in Table S1 and Figure S5, CRBN-recruiting degraders 49, 52, and 53 displayed moderate ENL degradation, with degradation levels ranging from 56% to 84% at 10 μM. We also evaluated VHL-recruiting PROTACs 55–87 (Tables 5, S2, and S3). Using VHL1 as the E3 ligase ligand, there is no obvious improvement in ENL degradation, as the best compounds from this group, compounds 63 and 64, featuring a long alkyl linker (9-carbon or 10-carbon), achieved about 67% ENL degradation at 10 μM (Table 5, Figure S6). The use of VHL7 and VHL4 as VHL ligands led to some improvement. Compounds 75 (Table S2) and 85 (Table S3), both featuring a 1-PEG linker, reduced the ENL protein levels by 96% at 10 μM. Even at 1 μM, they retained moderate degradation levels of 73% and 62%, respectively (Tables S2 & S3; Figures S7 & S8). Nevertheless, the ENL degradation induced by these compounds is less profound than our previously reported ENL degrader MS41. These results suggest that beyond the linker attachment position, the functional group near the solvent-exposed region, used as a handle for linker attachment, may also play a pivotal role in leading to effective ENL degradation. In addition, E3 ligase ligands with higher binding affinity (such as VHL7 and VHL4)⁴⁵ appeared to enhance ENL degradation.

Table 5.

ENL Protein Degradation Activity of Compounds 55–69^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 55–69 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. For compounds exhibiting > 50% degradation, results are reported as the mean ± SD of two independent experiments. ND = no degradation. Corresponding representative WBs are shown in Figure S6.

Based on the above results, we turned our attention to compounds 88–126, which also use the RHS 2,3-dihydro-1H-indene as the attachment point, but feature an amide group instead of an ether for the linkage. Twelve CRBN-recruiting putative ENL degraders incorporating carbon- and PEG-based linkers of various lengths were evaluated for ENL degradation (Table 6, Figure S9). To our delight, compounds 92, 93, and 94, bearing moderately long alkyl linkers (6, 7, and 8-carbons respectively), markedly improved ENL degradation activity, achieving about 95% ENL degradation at 1 μM. These results underscore the critical role of the amide linkage on the 2,3-dihydro-1H-indene in promoting ENL degradation. In contrast, compounds 95–99 incorporating PEG linkers displayed less profound ENL degradation activity. Overall, CRBN-recruiting ENL degraders containing moderately long carbon chain linkers with an amide linkage are more favorable for promoting ENL degradation.

Table 6.

ENL Protein Degradation Activity of Compounds 88–99^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 88–99 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. For compounds exhibiting > 50% degradation, results are reported as the mean ± SD of two independent experiments. ND = no degradation. Corresponding representative WBs are shown in Figure S9.

We next evaluated VHL-recruiting compounds 100–116, which use VHL7 and VHL4 as VHL ligands and feature carbon linkers (Tables 7 & 8, Figures S9 & S10). While compounds 104–107 with 6-9 carbon linkers showed considerable ENL degradation (> 90% at 1 μM), either shortening (compounds 100–103) or lengthening (compounds 108–110) the linker greatly attenuated ENL degradation (Table 7, Figure S9). A similar trend was observed for compounds 111–116, which displayed substantially weaker ENL degradation (Table 8, Figure S10).

Table 7.

ENL Protein Degradation Activity of Compounds 100–110^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 100–110 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. For compounds exhibiting > 50% degradation, results are reported as the mean ± SD of two independent experiments. ND = no degradation. Corresponding representative WBs are shown in Figure S10.

Table 8.

ENL Protein Degradation Activity of Compounds 111–116^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 111–116 at 1 and 10 μM in MV4;11 cells for 24 h. For compounds exhibiting < 50% degradation, data represent a single determination. For compounds exhibiting > 50% degradation, results are reported as the mean ± SD of two independent experiments. ND = no degradation. Corresponding representative WBs are shown in Figure S11.

Additionally, we applied the recently reported high-affinity VHL-1 ligand 10^45,46 to determine whether this modification would further boost ENL degradation. Remarkably, the use of VHL-1 ligand 10 resulted in highly effective ENL degraders. As shown in Table 9 and Figure S12, nearly all PROTACs incorporating VHL-1 ligand 10 achieved high levels of ENL degradation (> 95%) at 1 μM. Surprisingly, compounds bearing either shorter (117) or longer (123–126) carbon linkers demonstrated no hook effect at concentrations up to 10 μM, making them promising hits for subsequent characterization.

Table 9.

ENL Protein Degradation Activity of Compounds 117–126^a

^aThe percentage of ENL degradation was determined by immunoblotting and normalized to DMSO controls after treatment with compounds 117–126 at 1 and 10 μM in MV4;11 cells for 24 h. The results are reported as the mean ± SD of two independent experiments. Corresponding representative WBs are shown in Figure S12.

Compound 124 is the Most Potent ENL PROTAC Degrader.

With these promising results in hand, we next sought to identify the most potent ENL degrader from these 126 compounds. To determine the best ENL degradation induced by promising candidates (compounds 94, 104, 105, 106, 107, 117, 118, 123, 124, 125, 126), we conducted WB analyses following 24-hour treatment of MV4;11 cells with multiple concentrations (1 nM, 10 nM, 100 nM and 1 μM) (Figure 3). For comparison, the three previously reported ENL degraders were synthesized and evaluated as controls. As shown in Figure 3, compound 124 emerged as the most potent ENL degrader, achieving near-complete degradation at concentrations as low as 1 nM, outperforming MS41, the most effective degrader reported previously. Overall, our design with the RHS 2,3-dihydro-1H-indene as the linker attachment site, amide as the linkage functional group, and VHL-1 ligand 10 as the high-affinity VHL ligand yielded compound 124, the most potent ENL degrader to date.

Figure 3. ENL degradation induced by selected candidates and reported ENL degraders.

MV4;11 cells were treated with DMSO or compounds at indicated concentrations for 24 h. ENL protein levels in cell lysates were determined by Western blotting. β-Actin was used as the loading control. All immunoblots are representative of three biological repeats.

Compound 124 Effectively and Rapidly Induces ENL Degradation in a Concentration- and Time-dependent Manner.

We first conducted detailed characterization of ENL degradation induced by compound 124. MV4;11 and Jurkat cells were treated with various concentrations of compound 124 for 24 hours. Compound 124 robustly induced ENL degradation in a concentrationdependent manner and achieved a DC₅₀ of 0.6 ± 0.05 nM and a D_max of 97.2 ± 2.8% in MV4;11 cells, representing a 5.8-fold higher potency (lower DC₅₀) compared with MS41 (Figures 4A & B). In addition, ENL degradation was also observed in Jurkat cells, with a slightly higher DC₅₀ of 1.13 ± 0.19 nM and a D_max of 98.4 ± 3.3% (Figures 4A & B). In subsequent time-course experiments, treatment with 30 nM compound 124 induced ENL degradation in a time-dependent manner, with detectable degradation within 10 minutes in MV4;11 cells and within 20 minutes in Jurkat cells, highlighting its rapid degradation kinetics (Figure 4C). Near-complete ENL degradation was achieved within 30 minutes in MV4;11 cells and substantial degradation was maintained for up to 72 hours in both cell lines. Collectively, these results demonstrate that compound 124 effectively and rapidly degrades ENL in concentration- and time-dependent manner.

Figure 4. Compound 124 degrades ENL in a concentration- and time-dependent manner.

(A) Concentration-dependent degradation of ENL mediated by compound 124. Immunoblots for ENL and β-Actin in MV4;11 and Jurkat cells treated with DMSO, the indicated concentrations of compound 124 for 24 hours. (B) Measurement of DC₅₀ and D_max values of compound 124 in MV4;11 and Jurkat cells. The band intensity in (A) was determined by image J software. Values and error bars are presented as means ± SEM from three independent experiments. (C) Time-dependent ENL degradation mediated by compound 124. Immunoblots for ENL in MV4;11 and Jurkat cells treated with DMSO or compound 124 (30 nM) for the indicated times. β-Actin was used as a loading control. All immunoblots are representative of three biological repeats.

Compound 124-mediated ENL Degradation Requires Binding to VHL and Occurs through the UPS.

To evaluate the mechanism-of-action (MOA) of compound 124, we designed a negative control compound 152 (MS108N). Compound 152, a close analog of compound 124, retains the same ENL-binding moiety and linker as compound 124 but incorporates a diastereomer of VHL-1 ligand 10,⁴⁵ with two stereocenters of hydroxy-proline reversed, thereby disrupting its ability to recruit VHL (Figure 5A). We next compared the effects of compound 124, its negative control compound 152, and the parental ENL inhibitor PFI-6 on ENL protein levels (Figure 5B). As expected, compound 152 failed to induce ENL degradation, indicating that the degradation of ENL by compound 124 requires binding to VHL (Figure 5B). The parental ENL inhibitor, PFI-6, showed no degradation effect, underscoring the requirement for a bifunctional degrader to achieve target degradation (Figure 5B). To further validate the MOA of compound 124, we performed competition and rescue experiments in MV4;11 cells. First, we pre-treated cells with the ENL parental inhibitor PFI-6 or VHL ligand VHL-298 and evaluated their effect on compound 124-mediated ENL degradation. Pretreatment with PFI-6 or VHL-298, which competitively binds ENL or VHL, respectively, abolished compound 124-induced ENL degradation (Figures 5C & D). Moreover, to determine whether compound 124 acts through the UPS, we pre-treated MV4;11 cells with the neddylation inhibitor MLN4924⁴⁸ or the proteasome inhibitor MG132.⁴⁹ Both treatments effectively blocked compound 124-induced ENL degradation, highlighting the necessity of an active cullin-RING ubiquitin ligase complex and proteasome for ENL degradation (Figures 5E & F). In addition, CRISPR-Cas9-mediated genetic ablation of VHL in MV4;11 cells completely abolished compound 124-induced ENL degradation (Figure 5G), further confirming the requirement for VHL-mediated ubiquitination. A washout experiment demonstrated that ENL protein levels gradually recovered within ~12 h and were nearly fully restored within 24 h following compound 124 removal (Figure 5H), suggesting that compound 124-induced ENL degradation is a reversible process. Together with RT-qPCR analysis showing unchanged ENL mRNA levels (Figure 5I), these results clearly demonstrated that compound 124 degraded ENL in a VHL- and UPS-dependent manner, rather than by transcriptional regulation.

Figure 5. Compound 124-mediated ENL degradation requires binding to both ENL and VHL and occurs through the UPS in human leukemia cells.

(A) Chemical structures of compound 124 and its negative control compound 152, which cannot bind VHL. (B) Immunoblots for ENL in MV4;11 cells treated with the indicated concentrations (1 nM, 10 nM, 100 nM, 1 μM and 10 μM) of compound 124, compound 152 or PFI-6 for 24 h. (C to F) Immunoblots for ENL in MV4;11 cells pretreated with PFI6 (C), VHL ligand VHL-298 (D), neddylation inhibitor MLN4924 (E) or proteasome inhibitor MG132 (F) at indicate concentrations for 1 h, followed by 30 nM of compound 124 treatment for 1 h. D is DMSO in panels C to H. (G) Immunoblots for VHL and ENL in MV4;11 cells expressing a control sgRNA (sgGFP) or VHL sgRNAs treated with compound 124 at concentrations 10 nM, 30 nM, or 100 nM for 24 h. (H) Immunoblots for ENL in MV4;11 cells pretreated with 30 nM of compound 124 for 24 h, followed by washout for the indicated times. (I) Analysis of relative ENL mRNA levels in MV4;11 cells treated with DMSO, compound 124 (30 nM) or compound 152 (30 nM) for the indicated times. Values were normalized against RPS15. P-values were determined using two-way ANOVA, ns: no significant difference. Error bars represent means ± SEM from three independent experiments. Immunoblots in panels B to H are representative of three independent biological repeats. β-Actin was used as a loading control.

Compound 124 Is a Highly Selective ENL/AF9 Degrader.

Furthermore, compound 124 efficiently degraded both ENL and its paralog AF9, which shares a highly conserved YEATS domain with ENL, in a concentration-dependent manner in MV4;11 cells (Figure 6A). This result is expected since it has already been shown that PFI-6 binds both ENL and AF9 indiscriminately and our previous ENL degrader MS41 also featuring PFI-6 as the parental inhibitor degraded both ENL and AF9.^37,44 Again, not surprisingly, no apparent degradation of other YEATS domaincontaining proteins, including GAS41 and YEATS2, was observed even at concentrations as high as 1 μM (Figure 6A). It is worth mentioning that the degradation of AF9 was assessed using MV4;11 cells stably expressing 3×Flag-HA-tagged AF9, as endogenous AF9 levels in these cells are too low to be detected by immunoblotting. To access the selectivity of compound 124 on a proteome-wide scale, we performed an unbiased mass spectrometry (MS)-based global proteomic study. The samples for MS analysis were prepared by treating MV4;11 cells with DMSO, 30 nM of compound 124 or negative control 152 for 2 hours. WB analysis confirmed that ENL protein levels were significantly depleted by compound 124, but not by the negative control compound 152 (Figure 6B). Among more than 5,837 proteins quantified, ENL was the only protein exhibited profound reduction in abundance following treatment with compound 124 relative to the negative control compound 152 (Figure 6C). These findings provide compelling evidence that compound 124 functions as a highly selective degrader of ENL/AF9.

Figure 6. Compound 124 is a highly selective ENL degrader in a global proteomic study.

(A) Immunoblots for endogenous ENL, GAS41, and YEATS2 and ectopically expressed HA-AF9 in MV4;11 cells treated with the indicated concentrations of compound 124, compound 152 (1000 nM), or PFI-6 (1000 nM). (B) WB analysis of ENL protein levels in samples processed for proteomic study. MV4;11 cells were treated with DMSO or 30 nM of compound 124 or 152 (negative control) for 2 h. Immunoblots results shown are representative of three independent repeats. (C) Volcano plot of the −Log10 (p-value) versus the Log2 fold change for compound 124 versus 152 in MV4;11 samples shown in panel C. P values were calculated from the data of 2 biological replicates. Dotted lines indicate Log2 fold change > 1 or < −1 and −Log10 p-value > 1.3.

Compound 124 Exhibits Strong Antiproliferative Activity in Leukemia Cells.

ENL is well-established therapeutic target in cancer.^17,24,50 Previous studies have demonstrated that the proliferation of human acute leukemia cells, particularly MLL-r leukemia cells, is highly sensitive to ENL depletion via CRISPR/Cas9.^23–24,36 To access the antiproliferative effects of the most potent ENL degrader, compound 124, we evaluated its ability to inhibit cell growth in a panel of human leukemia cell lines, using compound 152 as the control. As expected, compound 124 showed strong proliferation inhibition against three MLL-r leukemia cell lines containing MLL-AFF1 fusions, including MV4;11, RS4;11, and SEMK2, whereas the negative control compound 152 did not display any antiproliferative effect on these cell lines and cytotoxicity was observed only at high concentrations (Figures 7A & B). These findings underscore that ENL degradation is a major driver of the growth inhibition observed with compound 124. The half-maximal growth inhibition concentration (GI₅₀) values of compound 124 in these cell lines are summarized in Figure 7C. Remarkably, compound 124 exhibited substantially enhanced growth-inhibitory activity in MV4;11 cells (GI₅₀ = 1.19 ± 0.03 nM), representing an approximately 18-fold improvement compared with our previously reported MS41 (GI₅₀ = 21.28 ± 1.36 nM) (Figures 7A & C). Similarly, compound 124 exhibited potent antiproliferative activity in RS4;11 and SEMK2 cells, with GI50 values of 4.68 ± 0.64 nM and 43.16 ± 4.78 nM, corresponding to 5.3- and 9.4-fold enhancements in potency, respectively, relative to MS41 (Figures 7A & C). Meanwhile, compound 124 also potently inhibited the proliferation of KASUMI1 cells harboring the AML1-ETO translocation, achieving GI₅₀ value of 5.89 ± 0.41 nM and a 12-fold improvement compared to MS41 (GI₅₀ = 69.05 ± 11.45 nM) (Figures 7A & C). In contrast, compound 124 displayed negligible antiproliferative activity in Jurkat and K-562 cells (GI50 > 50 μM), which are non-MLL-r leukemia cell lines and insensitive to ENL knockout (Figures 7A & C).

Figure 7. Compound 124 exhibits strong antiproliferative activity in leukemia cells.

(A & B) Growth inhibition curves of compounds 124 (A) and 152 (B) in human leukemia cells: MV4;11, RS4;11, SEMK2, KASUMI1, Jurkat and K-562. Y axis shows the relative live cell ratio upon treatment with the indicated concentrations (X axis) of compound 124 or 152 for 12 days, normalized to DMSO-treated cells. Error bars represent means ± SEM from three independent biological experiments. (C) GI₅₀ of compound 124 in human leukemia cells. (D) Representative image of colony-forming cell (CFC) assay in MV4;11 cells treated with DMSO, compound 124 (30 nM) or 152 (30 nM) for 14 days. Scale bars, 2 mm (top) and 500 μm (bottom). (E) Quantification of colony numbers in CFC assay in MV4;11 cells. Error bars represent means ± SEM from three independent experiments. Student’s t test, compared with DMSO treatment, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and ns: no significant difference. (F) RT-qPCR analysis showing mRNA expression levels of selected ENL target genes in MV4;11 cells treated with DMSO, compound 124 (30 nM) or 152 (30 nM) for 12 hours. Values were normalized to RPS15. P-values were determined using two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and ns: no significant difference. Error bars represent means ± SEM from three independent biological experiments.

Colony formation assays in MV4;11 cells further confirmed the on-target activity of compound 124, with both colony number and size significantly reduced upon treatment, but not with the negative control compound 152, suggesting that the growth-inhibitory effect of compound 124 is dependent on ENL degradation (Figures 7D & E). To investigate the mechanism of compound 124-mediated anti-leukemia activity, RT-qPCR was performed in MV4;11 cells treated with 124 or its inactive analog compound 152 for 12 hours. Compound 124 significantly downregulated the expression of ENL-dependent oncogenic targets, including HOXA9, HOXA10, MEIS1, MYB, and MYC, whereas compound 152 had no significant effect (Figure 7F). Collectively, these results demonstrate that compound 124 induces potent, selective growth inhibition in multiple leukemia cell lines through targeted ENL degradation and suppression of downstream oncogenic transcriptional programs.

Compound 124 Induces Apoptosis and Cell Cycle Arrest in Leukemia Cells.

To elucidate the antiproliferative mechanism of the ENL degrader compound 124, flow cytometry analyses were performed to assess its effects on apoptosis and cell cycle in MV4;11 cells. Compound 124 significantly induced a time-dependent increase in both early and late apoptotic populations, with the total proportion reaching approximately 33% after 6 days of treatment at 30 nM, whereas the inactive analog compound 152 showed no such effects (Figures 8A & B). Given that leukemia cell proliferation may be suppressed through cell cycle arrest, we next examined the impact of compound 124 on cell cycle progression using flow cytometry analysis. As shown in Figures 8C & D, compound 124 effectively induced G1-phase arrest at the indicated time points. Collectively, these findings indicate that compound 124 exhibits antiproliferative activity by simultaneously promoting apoptosis and blocking cell cycle progression.

Figure 8. Compound 124 induces apoptosis and cell cycle G1 arrest.

(A to B) Apoptosis analysis of MV4;11 cells treated with DMSO, 30 nM of compound 124 or 152 for 3 or 6 days. Collected cells were analyzed using flow cytometry with annexin V-FITC/PI. (C to D) Cell cycle analysis of MV4;11 cells treated with DMSO, 30 nM of compound 124 or 152 for 3 or 6 days. Error bars represent means ± SEM. Cell apoptosis and cell cycle results shown are representative of three independent biological experiments. P-values were determined using two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and ns: no significant difference.

Compound 124 Exhibits Favorable Pharmacokinetic Properties in Vivo.

Finally, we assessed in vivo mouse PK properties of compound 124. Following a single intraperitoneal (IP) injection of compound 124 at 50 mg/kg dose in male Swiss albino mice, the plasma concentrations of compound 124 were monitored up to 24 hours (Figure 9). No adverse clinical signs were observed in the tested mice during the PK study. The peak plasma concentration (C_max = 387.7 nM) was achieved at 4 h post-treatment and the exposure of compound 124 maintained above 230 nM after 12 h. Even after 24 h, the concentration of compound 124 was still above 58 nM. Notably, compound 124 showed a superior systemic exposure than MS41.⁴⁴ Given its excellent DC₅₀ (0.6 ± 0.05 nM in MV4;11) and GI₅₀ (1.19 ± 0.03 nM in MV4;11) values in cellular assays, the measured plasma concentrations and exposure suggest that compound 124 can be readily used for in vivo animal studies.

Figure 9. Pharmacokinetic profile of compound 124 in mice.

Plasma concentrations of compound 124 over a 24-hour period in mice following a single intraperitoneal (IP) injection at 50 mg/kg. Data represent mean ± SEM from three mice per time point.

Chemical synthesis

The key precursors 127, 128 and 129 that are used for the syntheses of compounds 1–126 were prepared following our previously reported procedures.^44,51 CRBN-based (4-OH-thalidomide) or VHL-based (VHL1, VHL7 and VHL4) linkers were synthesized following our previously reported method.^52–54

Under the classic amide coupling condition, precursor 127 was coupled with carbon-based or PEG-based linkers that are attached to the CRBN or VHL ligands (compounds 153–195) to give putative ENL PROTACs (compounds 1–43, Scheme 1). Putative ENL PROTACs (compounds 44–87, Scheme 2) were synthesized by coupling precursor 128 with compounds 196–239. Following the same route, putative ENL PROTACs (compounds 88–99, Scheme 3A) was obtained from amide coupling of precursor 129 and compounds 196–201, 240, and 202–206. Similarly, amide coupling between precursor 129 and compounds 241–242, 222, 243, 223–225, 244, 226, 245–246, 232, 234, 247, 235, 248 and 236 yielded putative ENL PROTACs (compounds 100–116, Scheme 3A). The final putative ENL PROTACs (compounds 117–126, Scheme 3A) were prepared through amide coupling between precursor 129 and triazole-based VHL Linkers 130– 140 (Scheme 3A). The synthetic route for the preparation of Linkers 130–140 is outlined in Scheme 3B. Intermediate 142 is prepared according to published procedures (Scheme 3B).⁵⁵ Click reactions between intermediate 142 and commercially available linkers, followed by Boc group deprotection, afforded triazole-based VHL Linkers 130–140 (Scheme 3B).

Scheme 1.

Synthesis of Compounds 1–43^a

^aReagents and conditions: (a) EDCI, HOAt, NMM, DMSO, rt.

Scheme 2.

Synthesis of Compounds 44–87^a

^aReagents and conditions: (a) EDCI, HOAt, NMM, DMSO, rt.

Scheme 3.

Synthesis of Compounds 88–126^a

^aReagents and conditions: (a) EDCI, HOAt, NMM, DMSO, rt; (b) DMAP, DCM, 0 °C −rt, 30 min; (c) CuSO₄, (+)−Sodium L-ascorbate, tBuOH/H₂O (v/v: 1:1), overnight; (d) TFA, DCM, rt, 1 h.

The synthetic route for the negative control compound 152 is outlined in Scheme 4. Intermediate 145 was synthesized using the Suzuki–Miyaura coupling reaction between commercially available compound 143 and 144, followed by Boc-deprotection (Scheme 4A). Amide coupling between commercially available compound 146 and 147 and subsequent hydrolysis led to intermediate 148, which was then coupled with intermediate 145 and followed Boc-deprotection to yield intermediate 149. Intermediate 150 was prepared from 149 by Azide transfer reaction. Intermediate 151 was obtained via a click reaction and a final Boc-deprotection. Subsequent amide coupling between intermediate 151 and precursor 129 finished the negative control compound 152 (Scheme 4B).

Scheme 4.

Synthesis of Negative Control Compound 152^a

^a Reagents and conditions: (a) Pd(OAc)₂, K₂CO₃, N₂, DMF, 90 °C, 18 h; (b) TFA/DCM, rt; (c) EDCI, HOAt, NMM, DMSO, rt; (d) LiOH, MeOH/H₂O, rt; (e) DMAP, DCM, 0 °C −rt, 30 min; (f) CuSO₄, (+)−Sodium L-ascorbate, tBuOH/H₂O.

CONCLUSIONS

In summary, through an extensive SAR study via design, synthesis, and evaluation of 126 PROTAC degraders, we discovered compound 124, the most potent ENL degrader to date, which has sub-nM potency in degrading ENL (DC₅₀ = 0.6 ± 0.05 nM). This comprehensive optimization campaign also revealed valuable SAR insights such as the optimal linker attachment site, the key linkage functional group, and optimal E3 ligase ligand selection, which could provide general guidelines for further optimization and development of highly potent and selective ENL PROTAC degraders. Notably, compared with our previous ENL PROTAC degrader MS41, compound 124 displayed 5.8-fold improvement in ENL degradation potency with low DC₅₀ value of 0.6 ± 0.05 nM in MV4;11 cells and 5- to 18-fold stronger growth-inhibitory activity in multiple leukemia cell lines through ENL degradation-induced cell apoptosis and cell cycle arrest. The investigation of MOA indicated that compound 124 effectively and rapidly degrades ENL in a concentration-, time-, VHL- and UPS-dependent manner. In addition, WB analyses and MS-based global proteomic study indicated that compound 124 is a highly selective ENL degrader. Through induced ENL degradation, compound 124 downregulated the expression of ENL-dependent oncogenic transcriptional programs. Furthermore, compound 124 displayed higher plasma exposure than MS41 in a mouse PK study. Collectively, considering the exceptionally low DC₅₀ and GI₅₀ values, excellent selectivity together with improved plasma exposure in vivo, compound 124 is a valuable chemical biology tool for the scientific community to further investigate the role of ENL in health and disease as well as a potential therapeutic to treat ENL-dependent AML.

Experimental Section

General chemistry methods.

All chemical reagents were purchased from commercial vendors and used without further purification. A Teledyne ISCO CombiFlash Rf⁺ instrument equipped with a variable wavelength UV detector and a fraction collector was used for flash column chromatography. RediSepRf HP C18 and flash silica columns were used for purification of compounds. High-performance liquid chromatography (HPLC) spectra for all compounds were acquired using an Agilent 1200 series system with a diode array detector. Chromatography was performed on a 2.1 × 150 mm Zorbax 300SB-C18 5 μm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0–1 min), 1–99% B (1–4 min), and 99% B (4–8 min). High-resolution mass spectrometry (HRMS) data were acquired in positive ion mode using an Agilent G1969A API-TOF system with an electrospray ionization (ESI) source. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DRX-400 spectrometer with 400 MHz for proton (¹H NMR) or 101 MHz for carbon (¹³C NMR) or a Bruker DRX-600 spectrometer with 600 MHz for proton (¹H NMR). Chemical shifts are reported in ppm (δ). Preparative HPLC was performed on Agilent Prep 1200 series with a UV detector set to 254 nm or 220 nM. Samples were injected into a Phenomenex Luna 5 μM C18 column, with dimensions of 250 × 30 mm, at room temperature. The flow rate was 40 mL/min. A linear gradient was used with 10% of MeOH (A) in H2O (with 0.1% trifluoroacetic acid (TFA)) (B) to 100% of MeOH (A). All final compounds had > 95% purity using the HPLC methods described above.

Method A: Preparation of ENL SAR Putative degraders 1–43.

To a solution of precursor 127 (Scheme 1) (4.2 mg, 0.01 mmol) in DMSO (1 mL) were added each type of linker-attached CRBN or VHL ligands (0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixtures were purified by preparative HPLC (5%-60% acetonitrile / 0.1% TFA in H2O) to afford the final compounds 1–43 as white solids.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)propanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (1).

White solid (5.1 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.83 – 7.74 (m, 1H), 7.56 – 7.46 (m, 1H), 7.44 – 7.21 (m, 8H), 7.12 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 5.14 (ddd, J = 12.5, 5.6, 3.7 Hz, 1H), 4.74 (s, 2H), 3.76 – 3.36 (m, 7H), 3.16 – 3.06 (m, 2H), 3.03 – 2.83 (m, 3H), 2.80 – 2.71 (m, 2H), 2.66 – 2.58 (m, 1H), 2.55 – 2.39 (m, 2H), – 2.13 (m, 1H), 2.13 – 2.04 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₁H₄₀N₇O₁₁⁺ 806.2780, found 806.2776.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (2).

white solid (5.1 mg, 62%).¹H NMR (600 MHz, Methanol-d₄) δ 7.80 (t, J = 7.9 Hz, 1H), 7.55 – 7.51 (m, 1H), 7.47 – 7.39 (m, 2H), 7.36 – 7.20 (m, 6H), 7.13 (s, 1H), 5.68 – 5.62 (m, 1H), 5.17 – 5.10 (m, 1H), 4.78 (s, 2H), 3.76 – 3.63 (m, 1H), 3.57 – 3.50 (m, 1H), 3.45 – 3.36 (m, 5H), 3.16 – 3.07 (m, 2H), 3.05 – 2.83 (m, 3H), 2.80 – 2.69 (m, 2H), 2.64 – 2.56 (m, 1H), 2.35 – 2.03 (m, 4H), 1.94 – 1.75 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₂H₄₂N₇O₁₁⁺ 820.2937, found 820.2938.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(5-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pentanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (3).

White solid (5 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.79 (t, J = 7.9 Hz, 1H), 7.55 – 7.49 (m, 1H), 7.44 – 7.37 (m, 2H), 7.36 – 7.21 (m, 6H), 7.16 – 7.10 (m, 1H), 5.66 (t, J = 7.3 Hz, 1H), 5.14 (ddd, J = 12.8, 5.5, 3.0 Hz, 1H), 4.75 (s, 2H), 3.74 – 3.65 (m, 1H), 3.56 – 3.48 (m, 1H), 3.45 – 3.38 (m, 2H), 3.19 – 3.07 (m, 3H), 3.03 – 2.83 (m, 5H), 2.81 – 2.69 (m, 2H), 2.66 – 2.58 (m, 1H), 2.30 – 2.23 (m, 1H), 2.20 – 2.04 (m, 3H), 1.76 – 1.53 (m, 4H). HRMS m/z [M + H]⁺ calcd for C₄₃H₄₄N₇O₁₁⁺ 834.3093, found 834.3088.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (4).

White solid (5.6 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.79 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 7.43 – 7.39 (m, 2H), 7.35 – 7.18 (m, 6H), 7.12 (s, 1H), 5.66 – 5.60 (m, 1H), 5.15 – 5.11 (m, 1H), 4.74 (s, 2H), 3.73 – 3.65 (m, 1H), – 3.46 (m, 1H), 3.44 – 3.36 (m, 2H), 3.15 – 3.06 (m, 3H), 3.02 – 2.82 (m, 5H), 2.80 – 2.68 (m, 2H), 2.63 – 2.55 (m, 1H), 2.26 – 2.02 (m, 4H), 1.71 – 1.49 (m, 4H), 1.43 – 1.27 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₄H₄₆N₇O₁₁⁺ 848.3250, found 848.3245.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(7-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)heptanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (5).

White solid (6 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.79 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 7.43 – 7.38 (m, 2H), 7.36 – 7.18 (m, 6H), 7.12 (s, 1H), 5.63 (t, J = 7.6 Hz, 1H), 5.17 – 5.08 (m, 1H), 4.73 (s, 2H), 3.73 – 3.65 (m, 1H), 3.57 – 3.47 (m, 1H), 3.44 – 3.36 (m, 2H), 3.15 – 3.05 (m, 3H), 3.03 – 2.82 (m, 4H), 2.79 – 2.67 (m, 2H), 2.63 – 2.55 (m, 1H), 2.24 – 2.01 (m, 5H), 1.71 – 1.49 (m, 4H), 1.44 – 1.25 (m, 4H). HRMS m/z [M + H]⁺ calcd for C₄₅H₄₈N₇O₁₁⁺ 862.3406, found 862.3403.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(9-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)nonanamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (6).

White solid (6.5 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.39 – 7.31 (m, 3H), 7.30 – 7.19 (m, 3H), 7.15 (s, 1H), 5.65 (t, J = 7.7 Hz, 1H), 5.14 (dd, J = 12.6, 5.5 Hz, 1H), 4.75 (s, 2H), 3.75 – 3.66 (m, 1H), 3.59 – 3.50 (m, 1H), 3.47 – 3.38 (m, 2H), 3.17 – 3.06 (m, 3H), 3.04 – 2.84 (m, 4H), 2.81 – 2.70 (m, 2H), 2.64 – 2.57 (m, 1H), 2.25 – 2.03 (m, 5H), 1.69 – 1.48 (m, 4H), 1.41 – 1.24 (m, 8H). HRMS m/z [M + H]⁺ calcd for C₄₇H₅₂N₇O₁₁⁺ 890.3719, found 890.3722.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)propanamido)ethyl)(methyl)-carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (7).

White solid (5.2 mg, 62%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.77 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.35 – 7.19 (m, 6H), 7.10 (s, 1H), 5.67 – 5.61 (m, 1H), 5.17 – 5.08 (m, 1H), 4.73 (s, 2H), 3.80 – 3.71 (m, 1H), 3.70 – 3.63 (m, 2H), 3.62 – 3.45 (m, 5H), 3.40 – 3.33 (m, 2H), 3.13 – 3.05 (m, 3H), 3.00 – 2.82 (m, 4H), 2.78 – 2.66 (m, 2H), 2.64 – 2.56 (m, 1H), 2.52 – 2.35 (m, 2H), 2.18 – 2.10 (m, 1H), 2.10 – 2.01 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₃H₄₄N₇O₁₂⁺ 850.3042, found 850.3037.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,12-dioxo-6,9-dioxa-3,13-diazapentadecan-15-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (8).

White solid (5.1 mg, 57%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.80 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 7.3 Hz, 1H), 7.44 – 7.40 (m, 2H), 7.38 – 7.31 (m, 3H), 7.31 – 7.20 (m, 3H), 7.13 (s, 1H), 5.65 (t, J = 7.6 Hz, 1H), 5.18 – 5.10 (m, 1H), 4.77 (s, 2H), 3.79 – 3.57 (m, 8H), 3.55 – 3.45 (m, 3H), 3.44 – 3.38 (m, 2H), 3.15 – 3.07 (m, 3H), 3.04 – 2.85 (m, 4H), 2.81 – 2.69 (m, 2H), 2.65 – 2.57 (m, 1H), 2.50 – 2.35 (m, 2H), 2.20 – 2.12 (m, 1H), 2.12 – 2.04 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₅H₄₈N₇O₁₃⁺ 894.3305, found 894.3301.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,15-dioxo-6,9,12-trioxa-3,16-diazaoctadecan-18-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (9).

White solid (5.6 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.45 – 7.41 (m, 2H), 7.39 – 7.32 (m, 3H), 7.31 – 7.20 (m, 3H), 7.14 (s, 1H), 5.65 (t, J = 7.6 Hz, 1H), 5.16 – 5.11 (m, 1H), 4.77 (s, 2H), 3.78 – 3.48 (m, 14H), 3.44 – 3.36 (m, 3H), 3.17 – 3.07 (m, 3H), 3.04 – 2.85 (m, 4H), 2.80 – 2.67 (m, 2H), 2.65 – 2.57 (m, 1H), 2.51 – 2.36 (m, 2H), 2.19 – 2.13 (m, 1H), 2.12 – 2.03 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₇H₅₂N₇O₁₄⁺ 938.3567, found 938.3570.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosan-21-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (10).

White solid (6.5 mg, 66%). ¹H NMR (600 MHz, Methanol-d4) δ 7.79 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 7.43 – 7.39 (m, 2H), 7.36 – 7.29 (m, 3H), 7.28 – 7.18 (m, 3H), 7.13 (s, 1H), 5.63 (t, J = 7.7 Hz, 1H), 5.15 – 5.09 (m, 1H), 4.75 (s, 2H), 3.75 – 3.45 (m, 18H), 3.44 – 3.34 (m, 3H), 3.16 – 3.05 (m, 3H), 3.02 – 2.82 (m, 4H), 2.78 – 2.68 (m, 2H), 2.62 – 2.56 (m, 1H), 2.49 – 2.34 (m, 2H), 2.17 – 2.12 (m, 1H), 2.09 – 2.02 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₉H₅₆N₇O₁₅⁺ 982.3829, found 982.3830.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,21-dioxo-6,9,12,15,18-pentaoxa-3,22-diazatetracosan-24-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (11).

White solid (6.4 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.39 – 7.32 (m, 3H), 7.30 – 7.20 (m, 3H), 7.15 (s, 1H), 5.65 (t, J = 7.8 Hz, 1H), 5.14 (ddd, J = 12.9, 5.6, 2.1 Hz, 1H), 4.78 (s, 2H), 3.79 – 3.48 (m, 22H), 3.44 – 3.36 (m, 3H), 3.17 – 3.07 (m, 3H), 3.04 – 2.85 (m, 4H), 2.81 – 2.70 (m, 2H), 2.64 – 2.57 (m, 1H), 2.52 – 2.36 (m, 2H), 2.19 – 2.13 (m, 1H), 2.12 – 2.03 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₀N₇O₁₆⁺ 1026.4091, found 1026.4095.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁴-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)succinimide (12).

White solid (5.8 mg, 62%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.99 (s, 1H), 7.49 – 7.20 (m, 11H), 7.18 – 7.10 (m, 1H), 5.68 – 5.61 (m, 1H), 4.64 – 4.49 (m, 4H), 4.37 (d, J = 15.5 Hz, 1H), 3.93 (d, J = 11.0 Hz, 1H), 3.84 – 3.79 (m, 1H), 3.77 – 3.54 (m, 1H), 3.51 – 3.37 (m, 1H), 3.18 – 3.06 (m, 3H), 3.03 – 2.90 (m, 4H), 2.65 – 2.41 (m, 8H), 2.28 – 2.21 (m, 1H), 2.12 – 2.03 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₄₉H₅₇N₈O₉S⁺ 933.3964, found 933.3966.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁵-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)glutaramide (13).

White solid (6.4 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.05 (s, 1H), 7.49 – 7.40 (m, 6H), 7.38 – 7.31 (m, 2H), 7.31 – 7.20 (m, 3H), 7.15 (s, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.66 – 4.60 (m, 2H), 4.56 – 4.51 (m, 2H), 4.36 (d, J = 15.5 Hz, 1H), 3.97 (d, J = 11.0 Hz, 1H), 3.83 (dd, J = 10.9, 3.9 Hz, 1H), 3.79 – 3.39 (m, 2H), 3.18 – 3.06 (m, 3H), 3.05 – 2.90 (m, 4H), 2.65 – 2.58 (m, 1H), 2.49 (s, 3H), 2.39 – 2.06 (m, 7H), 2.00 – 1.79 (m, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₀H₅₉N₈O₉S⁺ 947.4120, found 947.4123.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁶-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)adipamide (14).

White solid (6.7 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.50 – 7.41 (m, 5H), 7.39 – 7.20 (m, 6H), 7.15 (s, 1H), 5.65 (t, J = 7.8 Hz, 1H), 4.67 – 4.49 (m, 4H), 4.37 (d, J = 15.5 Hz, 1H), 3.93 (d, J = 11.1 Hz, 1H), 3.82 (dd, J = 11.0, 3.9 Hz, 1H), 3.56 – 3.50 (m, 1H), 3.45 – 3.37 (m, 1H), 3.18 – 3.05 (m, 3H), 3.03 – 2.91 (m, 4H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.37 – 2.03 (m, 8H), 1.72 – 1.52 (m, 3H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₁N₈O₉S⁺ 961.4277, found 961.4277.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁷-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)heptanediamide (15).

White solid (6.3 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.92 (s, 1H), 7.49 – 7.39 (m, 6H), 7.40 – 7.31 (m, 2H), 7.30 – 7.20 (m, 3H), 7.16 (s, 1H), 5.65 (t, J = 7.6 Hz, 1H), 4.65 (s, 1H), 4.60 (t, J = 8.6 Hz, 1H), 4.57 – 4.49 (m, 2H), 4.36 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.82 (dd, J = 10.9, 3.9 Hz, 1H), 3.74 – 3.68 (m, 1H), 3.56 – 3.49 (m, 1H), 3.18 – 3.06 (m, 3H), 3.03 – 2.91 (m, 4H), 2.65 – 2.57 (m, 1H), 2.48 (s, 3H), 2.32 – 2.19 (m, 4H), 2.17 – 2.04 (m, 2H), 1.71 – 1.50 (m, 4H), 1.43 – 1.28 (m, 3H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₂H₆₃N₈O₉S⁺ 975.4433, found 975.4437.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁸-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)octanediamide (16).

White solid (7.2 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.93 (s, 1H), 7.48 – 7.39 (m, 5H), 7.37 – 7.18 (m, 6H), 7.14 (s, 1H), 5.64 (t, J = 7.8 Hz, 1H), 4.63 (s, 1H), 4.60 – 4.47 (m, 3H), 4.34 (d, J = 15.4 Hz, 1H), 3.90 (d, J = 11.1 Hz, 1H), 3.80 (dd, J = 11.0, 3.9 Hz, 1H), 3.71 – 3.59 (m, 1H), 3.52 – 3.35 (m, 1H), 3.17 – 3.04 (m, 3H), 3.02 – 2.88 (m, 4H), 2.62 – 2.56 (m, 1H), 2.47 (s, 3H), 2.32 – 2.02 (m, 4H), 1.66 – 1.46 (m, 5H), 1.43 – 1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₅N₈O₉S⁺ 989.4590, found 989.4594.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N⁹-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nonanediamide (17).

White solid (7.1 mg, 71%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.98 (s, 1H), 7.50 – 7.46 (m, 2H), 7.46 – 7.41 (m, 3H), 7.39 – 7.31 (m, 3H), 7.30 – 7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 4.65 (s, 1H), 4.62 – 4.48 (m, 3H), 4.37 (d, J = 15.4 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.74 – 3.67 (m, 1H), 3.54 – 3.48 (m, 1H), 3.16 – 3.06 (m, 3H), 3.03 – 2.90 (m, 4H), 2.64 – 2.57 (m, 1H), 2.49 (s, 3H), 2.34 – 2.20 (m, 3H), 2.16 – 2.03 (m, 2H), 1.68 – 1.50 (m, 4H), 1.40 – 1.24 (m, 8H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₇N₈O₉S⁺ 1003.4746, found 1003.4741.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N¹⁰-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)decanediamide (18).

White solid (6.5 mg, 64%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.51 – 7.42 (m, 5H), 7.39 – 7.31 (m, 3H), 7.30 – 7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J = 7.8 Hz, 1H), 4.65 (s, 1H), 4.61 – 4.48 (m, 3H), 4.37 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.74 – 3.64 (m, 1H), 3.56 – 3.49 (m, 1H), 3.17 – 3.07 (m, 3H), 3.04 – 2.90 (m, 4H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.33 – 2.20 (m, 4H), 2.16 – 2.05 (m, 2H), 1.68 – 1.48 (m, 4H), 1.40 – 1.26 (m, 9H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₉N₈O₉S⁺ 1017.4903, found 1017.4902.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N¹¹-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)undecanediamide (19).

White solid (7.0 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.97 (s, 1H), 7.50 – 7.42 (m, 5H), 7.40 – 7.31 (m, 3H), 7.30 – 7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 4.65 (s, 1H), 4.62 – 4.48 (m, 3H), 4.37 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.82 (dd, J = 11.0, 4.0 Hz, 1H), 3.73 – 3.68 (m, 1H), 3.56 – 3.50 (m, 1H), 3.17 – 3.06 (m, 3H), 3.04 – 2.90 (m, 4H), 2.65 – 2.57 (m, 1H), 2.49 (s, 3H), 2.33 – 2.19 (m, 4H), 2.14 – 2.03 (m, 2H), 1.68 – 1.50 (m, 5H), 1.39 – 1.25 (m, 10H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₇₁N₈O₉S⁺ 1031.5059, found 1031.5063.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(3-hydroxy-4-((2-(3-(3-(((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)-3-oxopropoxy)propanamido)ethyl)(methyl)carbamoyl)-phenyl)isoxazole-3-carboxamide (20).

White solid (8.7 mg, 78%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.48 – 7.39 (m, 5H), 7.38 – 7.31 (m, 3H), 7.29 (d, J = 7.4 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.15 (s, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.70 – 4.58 (m, 2H), 4.57 – 4.48 (m, 2H), 4.36 (t, J = 18.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 10.9, 3.8 Hz, 1H), 3.79 – 3.59 (m, 8H), 3.56 – 3.41 (m, 2H), 3.16 – 3.07 (m, 3H), 3.03 – 2.91 (m, 4H), 2.65 – 2.57 (m, 1H), 2.49 (s, 3H), 2.44 – 2.37 (m, 1H), 2.28 – 2.22 (m, 1H), 2.13 – 2.03 (m, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₁N₈O₁₀S⁺ 977.4226, found 977.4230.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(3-hydroxy-4-(((S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-4,13-dioxo-7,10-dioxa-3,14-diazaheptadecyl)(methyl)carbamoyl)phenyl)isoxazole-3-carboxamide (21).

White solid (7.8 mg, 77%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.00 (s, 1H), 7.49 – 7.40 (m, 5H), 7.39 – 7.35 (m, 2H), 7.34 – 7.20 (m, 4H), 7.15 (s, 1H), 5.65 (t, J = 7.8 Hz, 1H), 4.67 (s, 1H), 4.61 (t, J = 8.3 Hz, 1H), 4.54 (d, J = 15.4 Hz, 1H), 4.52 – 4.49 (m, 1H), 4.36 (d, J = 15.5 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.78 – 3.48 (m, 10H), 3.44 – 3.36 (m, 2H), 3.17 – 3.07 (m, 3H), 3.03 – 2.91 (m, 4H), 2.64 – 2.52 (m, 2H), 2.49 (s, 3H), 2.42 – 2.33 (m, 1H), 2.27 – 2.21 (m, 1H), 2.13 – 2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₅N₈O₁₁S⁺ 1021.4488, found 1021.4485.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(3-hydroxy-4-(((S)-18-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-19,19-dimethyl-4,16-dioxo-7,10,13-trioxa-3,17-diazaicosyl)(methyl)carbamoyl)phenyl)isoxazole-3-carboxamide (22).

White solid (6.7 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.50 – 7.47 (m, 2H), 7.45 – 7.41 (m, 3H), 7.39 – 7.35 (m, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J = 7.8 Hz, 1H), 4.66 (s, 1H), 4.60 (t, J = 8.3 Hz, 1H), 4.55 (d, J = 15.5 Hz, 1H), 4.52 – 4.49 (m, 1H), 4.37 (d, J = 15.5 Hz, 1H), 3.91 (d, J = 10.9 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.78 – 3.66 (m, 6H), 3.64 – 3.50 (m, 10H), 3.18 – 3.06 (m, 3H), 3.04 – 2.91 (m, 4H), 2.64 – 2.54 (m, 2H), 2.50 (s, 3H), 2.49 – 2.45 (m, 2H), 2.44 – 2.37 (m, 1H), 2.26 – 2.21 (m, 1H), 2.12 – 2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₉N₈O₁₂S⁺ 1065.4750, found 1065.4745.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N¹⁶-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-4,7,10,13-tetraoxahexadecanediamide (23).

White solid (7.3 mg, 66%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.05 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.46 – 7.41 (m, 3H), 7.39 – 7.36 (m, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J = 7.8 Hz, 1H), 4.66 (s, 1H), 4.62 – 4.57 (m, 1H), 4.55 (d, J = 15.5 Hz, 1H), 4.52 – 4.49 (m, 1H), 4.37 (d, J = 15.5 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.78 – 3.50 (m, 18H), 3.18 – 3.07 (m, 3H), 3.04 – 2.91 (m, 4H), 2.65 – 2.54 (m, 2H), 2.50 (s, 3H), 2.49 – 2.45 (m, 2H), 2.43 – 2.34 (m, 1H), 2.27 – 2.20 (m, 1H), 2.13 – 2.03 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₇₃N₈O₁₃S⁺ 1109.5012, found 1109.5016.

N¹-(2-(4-(3-(((R)-2,3-dihydro-1H-inden-1-yl)carbamoyl)isoxazol-5-yl)-2-hydroxy-N-methylbenzamido)ethyl)-N¹⁹-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-4,7,10,13,16-pentaoxanonadecanediamide (24).

White solid (6.5 mg, 56%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.07 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.46 – 7.42 (m, 3H), 7.39 – 7.36 (m, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 7.4 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 4.69 – 4.65 (m, 1H), 4.61 – 4.58 (m, 1H), 4.56 (d, J = 15.5 Hz, 1H), 4.52 – 4.49 (m, 1H), 4.37 (d, J = 15.5 Hz, 1H), 3.90 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.78 – 3.51 (m, 22H), 3.18 – 3.07 (m, 3H), 3.03 – 2.91 (m, 4H), 2.64 – 2.55 (m, 2H), 2.51 (s, 3H), 2.50 – 2.46 (m, 2H), 2.43 – 2.37 (m, 1H), 2.26 – 2.21 (m, 1H), 2.12 – 2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₇N₈O₁₄S⁺ 1153.5274, found 1153.5270.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(2-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)acetamido)-ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (25).

White solid (6.3 mg, 61%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.51 – 7.20 (m, 11H), 7.13 (s, 1H), 5.66 (t, J = 7.9 Hz, 1H), 5.45 – 5.37 (m, 1H), 4.76 (d, J = 9.1 Hz, 1H), 4.68 – 4.56 (m, 1H), 4.47 (s, 1H), 3.89 – 3.73 (m, 2H), 3.60 – 3.51 (m, 1H), 3.44 – 3.37 (m, 1H), 3.18 – 3.06 (m, 2H), 3.03 – 2.83 (m, 7H), 2.65 – 2.57 (m, 1H), 2.48 (s, 3H), 2.23 (dd, J = 13.2, 7.6 Hz, 1H), 2.11 – 2.04 (m, 2H), 2.02 – 1.97 (m, 2H), 1.41 – 1.24 (m, 4H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₁FN₉O₁₀S⁺ 1034.4241, found 1034.4245.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(4-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)butanamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (26).

White solid (5.8 mg, 55%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.07 (s, 1H), 7.52 – 7.39 (m, 5H), 7.36 – 7.18 (m, 6H), 7.13 (s, 1H), 5.64 (t, J = 7.8 Hz, 1H), 5.35 – 5.29 (m, 1H), 4.73 (d, J = 9.2 Hz, 1H), 4.62 – 4.55 (m, 1H), 4.44 (s, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.70 – 3.62 (m, 1H), 3.50 – 3.43 (m, 1H), 3.18 – 3.04 (m, 6H), 3.01 – 2.90 (m, 4H), 2.84 (dd, J = 14.2, 6.4 Hz, 1H), 2.74 (dd, J = 14.2, 8.1 Hz, 1H), 2.62 – 2.56 (m, 1H), 2.48 (s, 3H), 2.19 (dd, J = 13.4, 7.7 Hz, 1H), 2.12 – 1.91 (m, 3H), 1.76 – 1.53 (m, 2H), 1.39 – 1.24 (m, 4H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₅FN₉O₁₀S⁺ 1062.4554, found 1062.4557.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(5-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)pentanamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (27).

White solid (6.2 mg, 58%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.51 – 7.41 (m, 5H), 7.40 – 7.28 (m, 4H), 7.28 – 7.20 (m, 2H), 7.15 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 5.32 (dd, J = 8.1, 6.2 Hz, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.61 (t, J = 8.8 Hz, 1H), 4.46 (s, 1H), 3.84 (d, J = 11.1 Hz, 1H), 3.78 (dd, J = 11.1, 3.8 Hz, 1H), 3.72 – 3.63 (m, 1H), 3.55 – 3.45 (m, 1H), 3.18 – 3.05 (m, 6H), 3.03 – 2.91 (m, 4H), 2.87 – 2.81 (m, 1H), 2.75 (dd, J = 14.2, 8.2 Hz, 1H), 2.66 – 2.57 (m, 1H), 2.50 (s, 3H), 2.23 – 2.14 (m, 1H), 2.11 – 2.03 (m, 2H), 2.00 – 1.93 (m, 1H), 1.60 – 1.50 (m, 2H), 1.49 – 1.28 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₀S⁺ 1076.4710, found 1076.4713.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(6-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)hexanamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (28).

White solid (6.6 mg, 61%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.03 (s, 1H), 7.51 – 7.39 (m, 5H), 7.38 – 7.26 (m, 4H), 7.25 – 7.18 (m, 2H), 7.13 (s, 1H), 5.64 (t, J = 7.7 Hz, 1H), 5.30 (dd, J = 8.1, 6.2 Hz, 1H), 4.73 (d, J = 9.2 Hz, 1H), 4.58 (t, J = 8.5 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.73 – 3.65 (m, 1H), 3.50 – 3.47 (m, 1H), 3.15 – 3.03 (m, 6H), 3.00 – 2.89 (m, 4H), 2.83 (dd, J = 14.1, 6.2 Hz, 1H), 2.73 (dd, J = 14.3, 8.2 Hz, 1H), 2.64 – 2.55 (m, 1H), 2.48 (s, 3H), 2.22 – 2.14 (m, 1H), 2.10 – 2.01 (m, 2H), 1.98 – 1.91 (m, 1H), 1.62 – 1.43 (m, 2H), 1.42 – 1.14 (m, 8H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₆₉FN₉O₁₀S⁺ 1090.4867, found 1090.4872.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(7-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)heptanamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (29).

White solid (7.2 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.50 – 7.38 (m, 4H), 7.37 – 7.18 (m, 7H), 7.13 (s, 1H), 5.64 (t, J = 7.8 Hz, 1H), 5.30 (dd, J = 8.3, 6.0 Hz, 1H), 4.73 (d, J = 9.2 Hz, 1H), 4.58 (t, J = 8.5 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.72 – 3.65 (m, 1H), 3.52 – 3.46 (m, 1H), 3.15 – 2.90 (m, 10H), 2.83 (dd, J = 14.1, 6.0 Hz, 1H), 2.73 (dd, J = 14.0, 8.4 Hz, 1H), 2.64 – 2.55 (m, 1H), 2.49 (s, 3H), 2.21 – 2.12 (m, 1H), 2.09 – 2.02 (m, 2H), 1.98 – 1.91 (m, 1H), 1.60 – 1.51 (m, 1H), 1.49 – 1.40 (m, 1H), 1.38 – 1.13 (m, 10H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₀S⁺ 1104.5023, found 1104.5024.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(8-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)octanamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (30).

White solid (5.9 mg, 53%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.00 (s, 1H), 7.49 – 7.38 (m, 4H), 7.38 – 7.17 (m, 6H), 7.13 (s, 1H), 5.64 (t, J = 7.7 Hz, 1H), 5.30 (dd, J = 8.3, 6.0 Hz, 1H), 4.73 (d, J = 9.3 Hz, 1H), 4.58 (dd, J = 9.2, 7.7 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.71 – 3.56 (m, 4H), 3.50 (d, J = 5.8 Hz, 2H), 3.23 – 3.18 (m, 1H), 3.17 – 2.88 (m, 7H), 2.83 (dd, J = 14.1, 5.9 Hz, 1H), 2.73 (dd, J = 14.1, 8.4 Hz, 1H), 2.62 – 2.56 (m, 1H), 2.48 (s, 3H), 2.21 – 2.12 (m, 2H), 2.09 – 2.02 (m, 1H), 1.98 – 1.92 (m, 1H), 1.63 – 1.50 (m, 1H), 1.48 – 1.42 (m, 3H), 1.38 – 1.11 (m, 8H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₃FN₉O₁₀S⁺ 1118.5180, found 1118.5178.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-(((S)-1-((2S,4R)-1-((S)-2-(1-fluoro-cyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,12-trioxo-9-oxa-2,6,13-triazapentadecan-15-yl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (31).

White solid (6.2 mg, 57%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.96 (s, 1H), 7.51 (dd, J = 9.3, 3.4 Hz, 1H), 7.47 – 7.35 (m, 6H), 7.34 – 7.20 (m, 4H), 7.14 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 5.33 (t, J = 7.1 Hz, 1H), 4.75 (d, J = 9.3 Hz, 1H), 4.63 – 4.57 (m, 1H), 4.45 (s, 1H), 3.83 (d, J = 11.1 Hz, 1H), 3.77 (dd, J = 11.1, 3.8 Hz, 1H), 3.72 – 3.57 (m, 3H), 3.56 – 3.51 (m, 1H), 3.49 – 3.36 (m, 3H), 3.18 – 3.07 (m, 3H), 3.03 – 2.91 (m, 4H), 2.85 (dd, J = 14.2, 6.8 Hz, 1H), 2.76 (dd, J = 14.2, 7.7 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.48 (s, 3H), 2.47 – 2.33 (m, 2H), 2.24 – 2.16 (m, 1H), 2.12 – 2.04 (m, 1H), 2.00 – 1.93 (m, 1H), 1.42 – 1.26 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₁S⁺ 1092.4659, found 1092.4662.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,15-trioxo-9,12-dioxa-2,6,16-triazaoctadecan-18-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (32).

White solid (7 mg, 62%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.99 (s, 1H), 7.50 (dd, J = 9.3, 3.4 Hz, 1H), 7.48 – 7.41 (m, 4H), 7.39 – 7.20 (m, 6H), 7.15 (s, 1H), 5.66 (t, J = 7.8 Hz, 1H), 5.33 (t, J = 7.2 Hz, 1H), 4.75 (d, J = 9.0 Hz, 1H), 4.63 – 4.57 (m, 1H), 4.47 – 4.44 (m, 1H), 3.84 (d, J = 11.1 Hz, 1H), 3.80 – 3.38 (m, 12H), 3.18 – 3.07 (m, 3H), 3.02 – 2.91 (m, 4H), 2.90 – 2.83 (m, 1H), 2.81 – 2.72 (m, 1H), 2.66 – 2.57 (m, 1H), 2.49 (s, 3H), 2.41 – 2.38 (m, 1H), 2.21 (dd, J = 13.3, 7.8 Hz, 1H), 2.12 – 2.03 (m, 1H), 2.00 – 1.92 (m, 1H), 1.42 – 1.27 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₂S⁺ 1136.4921, found 1136.4917.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,18-trioxo-9,12,15-trioxa-2,6,19-triazahenicosan-21-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (33).

White solid (8 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.53 – 7.42 (m, 5H), 7.39 – 7.35 (m, 2H), 7.34 – 7.20 (m, 4H), 7.15 (s, 1H), 5.66 (t, J = 7.8 Hz, 1H), 5.34 (t, J = 7.1 Hz, 1H), 4.75 (d, J = 9.1 Hz, 1H), 4.63 – 4.58 (m, 1H), 4.46 (s, 1H), 3.84 (d, J = 11.1 Hz, 1H), 3.80 – 3.65 (m, 4H), 3.62 – 3.38 (m, 12H), 3.18 – 3.07 (m, 3H), 3.04 – 2.91 (m, 4H), 2.86 (dd, J = 14.2, 6.2 Hz, 1H), 2.77 (dd, J = 14.1, 8.1 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.50 (s, 3H), 2.43 – 2.38 (m, 1H), 2.21 (dd, J = 13.0, 7.7 Hz, 1H), 2.11 – 2.04 (m, 1H), 2.00 – 1.93 (m, 1H), 1.42 – 1.25 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₃S⁺ 1180.5184, found 1180.5181.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,21-trioxo-9,12,15,18-tetraoxa-2,6,22-triazatetracosan-24-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (34).

White solid (7.5 mg, 61%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.53 – 7.41 (m, 5H), 7.39 – 7.36 (m, 2H), 7.34 – 7.20 (m, 4H), 7.16 (s, 1H), 5.66 (t, J = 7.7 Hz, 1H), 5.34 (t, J = 7.1 Hz, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.60 (t, J = 8.5 Hz, 1H), 4.46 (s, 1H), 3.84 (d, J = 11.1 Hz, 1H), 3.80 – 3.40 (m, 19H), 3.19 – 3.07 (m, 3H), 3.02 – 2.90 (m, 4H), 2.86 (dd, J = 14.2, 6.1 Hz, 1H), 2.78 (dd, J = 14.1, 8.1 Hz, 1H), 2.66 – 2.57 (m, 1H), 2.51 (s, 3H), 2.49 – 2.45 (m, 1H), 2.41 – 2.38 (m, 1H), 2.22 (dd, J = 13.0, 7.8 Hz, 1H), 2.13 – 2.03 (m, 1H), 2.01 – 1.93 (m, 1H), 1.42 – 1.27 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₂H₇₉FN₉O₁₄S⁺ 1224.5446, found 1224.5443.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)acetamido)ethyl)-(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (35).

White solid (6 mg, 57%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.54 – 7.43 (m, 2H), 7.42 – 7.20 (m, 5H), 7.14 – 7.00 (m, 4H), 5.70 – 5.61 (m, 1H), 4.78 – 4.55 (m, 5H), 4.50 – 4.42 (m, 2H), 4.06 – 3.89 (m, 2H), 3.87 – 3.67 (m, 3H), 3.61 – 3.39 (m, 1H), 3.17 – 3.07 (m, 3H), 3.02 – 2.90 (m, 4H), 2.65 – 2.57 (m, 1H), 2.49 (s, 3H), 2.21 (dd, J = 13.3, 7.6 Hz, 1H), 2.14 – 2.03 (m, 2H), 1.41 – 1.23 (m, 4H), 1.00 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₁FN₉O₁₁S⁺ 1050.4190, found 1050.4188.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(3-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)propanamido)-ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (36).

White solid (6.9 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.89 (s, 1H), 7.42 – 7.36 (m, 2H), 7.32 – 7.07 (m, 5H), 7.03 – 6.93 (m, 3H), 6.84 (s, 1H), 5.56 – 5.50 (m, 1H), 4.62 (d, J = 9.2 Hz, 1H), 4.53 – 4.32 (m, 4H), 3.76 – 3.66 (m, 2H), 3.63 – 3.56 (m, 2H), 3.53 – 3.35 (m, 5H), 3.04 – 2.94 (m, 3H), 2.89 – 2.79 (m, 4H), 2.53 – 2.46 (m, 2H), 2.38 (s, 3H), 2.15 – 2.06 (m, 1H), 2.00 – 1.91 (m, 2H), 1.29 – 1.11 (m, 4H), 0.90 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₃FN₉O₁₁S⁺ 1064.4346, found 1064.4349.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(5-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)pentanamido)-ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (37).

White solid (7.6 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.99 (s, 1H), 7.49 (d, J = 7.7 Hz, 2H), 7.42 (s, 1H), 7.37 – 7.20 (m, 5H), 7.17 – 7.06 (m, 2H), 6.96 (s, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.74 (d, J = 9.2 Hz, 1H), 4.65 – 4.55 (m, 4H), 4.53 – 4.45 (m, 2H), 3.85 (d, J = 11.1 Hz, 1H), 3.80 (dd, J = 11.1, 3.8 Hz, 1H), 3.74 – 3.60 (m, 2H), 3.56 – 3.38 (m, 2H), 3.19 – 3.06 (m, 3H), 3.03 – 2.89 (m, 4H), 2.65 – 2.58 (m, 1H), 2.50 (s, 3H), 2.29 – 2.02 (m, 4H), 1.72 – 1.50 (m, 5H), 1.43 – 1.25 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₁S⁺ 1092.4659, found 1092.4657.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(6-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)hexanamido)-ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (38).

White solid (6.9 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.97 (s, 1H), 7.52 – 7.46 (m, 2H), 7.43 (dd, J = 7.9, 1.6 Hz, 1H), 7.40 – 7.20 (m, 5H), 7.15 (s, 1H), 7.10 (dd, J = 7.7, 1.6 Hz, 1H), 6.99 – 6.96 (m, 1H), 5.67 – 5.62 (m, 1H), 4.74 (d, J = 9.3 Hz, 1H), 4.66 – 4.57 (m, 4H), 4.52 – 4.45 (m, 2H), 3.85 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 11.0, 3.9 Hz, 1H), 3.74 – 3.67 (m, 1H), 3.55 – 3.38 (m, 3H), 3.18 – 3.06 (m, 3H), 3.04 – 2.91 (m, 4H), 2.69 – 2.56 (m, 1H), 2.50 (s, 3H), 2.26 – 2.17 (m, 2H), 2.14 – 2.03 (m, 2H), 1.69 – 1.51 (m, 5H), 1.42 – 1.24 (m, 6H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₆₉FN₉O₁₁S⁺ 1106.4816, found 1106.4817.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((2-(3-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethoxy)pro-panamido)ethyl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (39).

White solid (7.8 mg, 71%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.94 (s, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.42 – 7.20 (m, 6H), 7.12 (s, 1H), 7.09 (dd, J = 7.7, 1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.66 – 4.59 (m, 2H), 4.56 (d, J = 15.2 Hz, 1H), 4.52 – 4.46 (m, 2H), 3.86 (d, J = 11.1 Hz, 1H), 3.80 (dd, J = 11.2, 3.7 Hz, 1H), 3.76 – 3.63 (m, 2H), 3.61 – 3.43 (m, 6H), 3.16 – 3.07 (m, 3H), 3.00 – 2.91 (m, 4H), 2.64 – 2.58 (m, 1H), 2.49 (s, 3H), 2.47 – 2.33 (m, 2H), 2.26 – 2.19 (m, 1H), 2.13 – 2.03 (m, 1H), 1.41 – 1.25 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₂S⁺ 1108.4608, found 1108.4611.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,12-dioxo-6,9-dioxa-3,13-diazapentadecan-15-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (40).

White solid (7.8 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.00 (s, 1H), 7.53 – 7.49 (m, 1H), 7.42 (dd, J = 7.9, 1.5 Hz, 1H), 7.37 – 7.34 (m, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.14 (s, 1H), 7.09 (dd, J = 7.7, 1.6 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.75 (d, J = 9.1 Hz, 1H), 4.67 – 4.58 (m, 3H), 4.56 – 4.47 (m, 2H), 3.85 (d, J = 11.1 Hz, 1H), 3.81 (dd, J = 11.1, 3.7 Hz, 1H), 3.75 – 3.63 (m, 2H), 3.62 – 3.38 (m, 9H), 3.15 – 3.06 (m, 3H), 3.02 – 2.90 (m, 4H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.49 – 2.42 (m, 1H), 2.41 – 2.34 (m, 1H), 2.26 – 2.20 (m, 1H), 2.13 – 2.03 (m, 2H), 1.40 – 1.24 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₃S⁺ 1152.4871, found 1152.4870.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,15-dioxo-6,9,12-trioxa-3,16-diazaoctadecan-18-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (41).

White solid (8.2 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.43 (dd, J = 7.8, 1.5 Hz, 1H), 7.38 – 7.34 (m, 2H), 7.32 (d, J = 7.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.15 (s, 1H), 7.10 (dd, J = 7.7, 1.6 Hz, 1H), 6.99 (d, J = 1.6 Hz, 1H), 5.65 (t, J = 7.8 Hz, 1H), 4.75 (d, J = 9.1 Hz, 1H), 4.67 – 4.59 (m, 3H), 4.56 (d, J = 15.2 Hz, 1H), 4.53 – 4.48 (m, 1H), 3.85 (d, J = 11.1 Hz, 1H), 3.81 (dd, J = 11.0, 3.8 Hz, 1H), 3.75 – 3.64 (m, 2H), 3.62 – 3.52 (m, 11H), 3.49 (t, J = 5.6 Hz, 2H), 3.17 – 3.06 (m, 3H), 3.03 – 2.91 (m, 4H), 2.64 – 2.57 (m, 1H), 2.51 (s, 3H), 2.49 – 2.37 (m, 2H), 2.23 (dd, J = 13.1, 7.8 Hz, 1H), 2.13 – 2.03 (m, 2H), 1.41 – 1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₄S⁺ 1196.5133, found 1196.5130.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosan-21-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (42).

White solid (7.9 mg, 64%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.97 (s, 1H), 7.53 – 7.49 (m, 2H), 7.45 – 7.42 (m, 1H), 7.39 – 7.34 (m, 1H), 7.32 (d, J = 7.3 Hz, 1H), 7.30 – 7.20 (m, 3H), 7.15 (s, 1H), 7.10 (d, J = 7.7 Hz, 2H), 6.99 (d, J = 1.5 Hz, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.67 – 4.54 (m, 4H), 4.50 (d, J = 15.0 Hz, 1H), 3.85 (d, J = 11.1 Hz, 1H), 3.81 (dd, J = 11.0, 3.8 Hz, 1H), 3.75 – 3.64 (m, 2H), 3.63 – 3.52 (m, 15H), 3.51 – 3.46 (m, 2H), 3.22 – 3.20 (m, 1H), 3.17 – 3.06 (m, 3H), 3.03 – 2.91 (m, 4H), 2.64 – 2.57 (m, 1H), 2.50 (s, 3H), 2.49 – 2.36 (m, 2H), 2.25 – 2.20 (m, 1H), 2.13 – 2.03 (m, 2H), 1.41 – 1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₂H₇₉FN₉O₁₅S⁺ 1240.5395, found 1240.5398.

N-((R)-2,3-dihydro-1H-inden-1-yl)-5-(4-((1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,21-dioxo-6,9,12,15,18-pentaoxa-3,22-diazatetracosan-24-yl)(methyl)carbamoyl)-3-hydroxyphenyl)isoxazole-3-carboxamide (43).

White solid (7.7 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.94 (s, 1H), 7.52 – 7.49 (m, 2H), 7.45 – 7.42 (m, 1H), 7.38 – 7.35 (m, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.29 (d, J = 7.4 Hz, 1H), 7.27 – 7.20 (m, 2H), 7.16 (s, 1H), 7.11 – 7.08 (m, 1H), 6.99 (d, J = 1.6 Hz, 1H), 5.65 (t, J = 7.7 Hz, 1H), 4.75 (d, J = 9.4 Hz, 1H), 4.66 – 4.64 (m, 2H), 4.63 – 4.55 (m, 2H), 4.53 – 4.48 (m, 1H), 3.85 (d, J = 11.2 Hz, 1H), 3.81 (dd, J = 11.0, 3.8 Hz, 1H), 3.63 – 3.55 (m, 20H), 3.52 – 3.48 (m, 2H), 3.22 – 3.20 (m, 1H), 3.17 – 3.07 (m, 3H), 3.03 – 2.91 (m, 4H), 2.64 – 2.58 (m, 1H), 2.50 (s, 3H), 2.49 – 2.37 (m, 2H), 2.26 – 2.19 (m, 1H), 2.12 – 2.04 (m, 2H), 1.42 – 1.25 (m, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₄H₈₃FN₉O₁₆S⁺ 1284.5657, found 1284.5653.

Method B: Preparation of ENL SAR Putative degraders 44–87.

To a solution of precursor 128 (Scheme 2) (4.65 mg, 0.01 mmol) in DMSO (1 mL) were added each type of linker attached CRBN or VHL ligands (0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixtures were purified by preparative HPLC (5%-60% acetonitrile / 0.1% TFA in H₂O) to afford the final compounds 44–87 as white solids with >95% purity.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (44).

White solid (6.5 mg, 79%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.76 (dd, J = 8.4, 7.3 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.42 – 7.36 (m, 2H), 7.35 – 7.32 (m, 2H), 7.16 (d, J = 8.3 Hz, 1H), 7.12 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 8.3, 2.5 Hz, 1H), 5.59 (t, J = 7.9 Hz, 1H), 5.01 (dd, J = 12.2, 5.3 Hz, 1H), 4.69 (d, J = 6.2 Hz, 2H), 4.47 (d, J = 4.1 Hz, 2H), 3.52 – 3.43 (m, 4H), 3.17 – 2.93 (m, 7H), 2.89 – 2.81 (m, 1H), 2.72 – 2.57 (m, 4H), 2.13 – 2.04 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₁H₄₀N₇O₁₂⁺ 822.2729, found 822.2726.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)propyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (45).

White solid (6.2 mg, 74%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.78 – 7.73 (m, 1H), 7.47 (dd, J = 7.3, 5.9 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.35 – 7.30 (m, 2H), 7.16 (dd, J = 8.4, 3.9 Hz, 1H), 7.12 (d, J = 3.4 Hz, 1H), 6.95 (t, J = 2.7 Hz, 1H), 6.89 – 6.85 (m, 1H), 5.59 (t, J = 7.9 Hz, 1H), 5.12 (ddd, J = 12.6, 7.6, 5.5 Hz, 1H), 4.77 – 4.69 (m, 2H), 4.47 (s, 2H), 3.38 – 3.33 (m, 1H), 3.32 – 3.25 (m, 2H), 3.18 – 2.95 (m, 8H), 2.89 – 2.79 (m, 2H), 2.77 – 2.68 (m, 2H), 2.63 – 2.56 (m, 1H), 2.18 – 2.05 (m, 2H), 1.80 – 1.73 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₂H₄₂N₇O₁₂⁺ 836.2886, found 836.2886.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (46).

White solid (5.9 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.74 (m, 1H), 7.55 – 7.29 (m, 5H), 7.24 – 7.06 (m, 2H), 7.02 – 6.83 (m, 2H), 5.64 – 5.54 (m, 1H), 5.14 – 5.07 (m, 1H), 4.75 – 4.70 (m, 2H), 4.51 – 4.45 (m, 2H), 3.33 – 3.23 (m, 4H), 3.18 – 2.93 (m, 8H), 2.89 – 2.65 (m, 3H), 2.62 – 2.56 (m, 1H), 2.17 – 2.02 (m, 2H), 1.64 – 1.49 (m, 4H). HRMS m/z [M + H]⁺ calcd for C₄₃H₄₄N₇O₁₂⁺ 850.3042, found 850.3041.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((5-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pentyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (47).

White solid (6.4 mg, 74%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.83 – 7.70 (m, 1H), 7.55 – 7.23 (m, 5H), 7.22 – 7.08 (m, 2H), 7.02 – 6.75 (m, 2H), 5.71 – 5.50 (m, 1H), 5.28 – 5.10 (m, 1H), 4.72 (s, 2H), 4.50 – 4.40 (m, 2H), 3.31 – 2.54 (m, 16H), 2.23 – 2.01 (m, 2H), 1.69 – 1.26 (m, 6H). HRMS m/z [M + H]⁺ calcd for C₄₄H₄₆N₇O₁₂⁺ 864.3199, found 864.3194.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (48).

White solid (5.9 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.84 – 7.74 (m, 1H), 7.55 – 7.29 (m, 5H), 7.23 – 7.09 (m, 2H), 7.00 – 6.84 (m, 2H), 5.60 (t, J = 7.9 Hz, 1H), 5.17 – 5.05 (m, 1H), 4.73 (s, 2H), 4.50 – 4.42 (m, 2H), 3.30 – 3.19 (m, 3H), 3.18 – 2.53 (m, 13H), 2.23 – 2.00 (m, 2H), 1.61 – 1.20 (m, 8H). HRMS m/z [M + H]⁺ calcd for C₄₅H₄₈N₇O₁₂⁺ 878.3355, found 878.3357.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((7-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)heptyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (49).

White solid (5.6 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.77 (m, 1H), 7.51 (d, J = 7.3 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.37 – 7.32 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.3, 2.5 Hz, 1H), 5.64 – 5.58 (m, 1H), 5.14 (dd, J = 12.5, 5.5 Hz, 1H), 4.75 (s, 2H), 4.47 (d, J = 2.4 Hz, 2H), 3.30 (t, J = 6.7 Hz, 2H), 3.23 (t, J = 7.1 Hz, 2H), 3.16 – 2.94 (m, 8H), 2.91 – 2.83 (m, 2H), 2.78 – 2.70 (m, 2H), 2.64 – 2.58 (m, 1H), 2.18 – 2.11 (m, 1H), 2.11 – 2.04 (m, 1H), 1.58 – 1.52 (m, 2H), 1.51 – 1.45 (m, 2H), 1.37 – 1.23 (m, 5H). HRMS m/z [M + H]⁺ calcd for C₄₆H₅₀N₇O₁₂⁺ 892.3512, found 892.3510.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-(2-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (50).

White solid (5.9 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.79 – 7.68 (m, 1H), 7.52 – 7.26 (m, 4H), 7.19 – 7.03 (m, 2H), 6.98 – 6.86 (m, 2H), 6.85 – 6.70 (m, 1H), 5.60 – 5.48 (m, 1H), 5.15 – 5.08 (m, 1H), 4.74 – 4.63 (m, 2H), 4.43 (s, 2H), 3.70 – 3.38 (m, 7H), 3.23 – 2.91 (m, 8H), 2.88 – 2.53 (m, 5H), 2.22 – 2.01 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₃H₄₄N₇O₁₃⁺ 866.2992, found 866.2989.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (51).

White solid (6.5 mg, 72%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.70 (m, 1H), 7.51 – 7.28 (m, 4H), 7.24 – 7.10 (m, 3H), 6.99 – 6.80 (m, 2H), 5.64 – 5.55 (m, 1H), 5.17 – 5.08 (m, 1H), 4.70 (s, 2H), 4.45 (s, 2H), 3.71 – 3.38 (m, 11H), 3.20 – 2.94 (m, 8H), 2.91 – 2.80 (m, 2H), 2.73 (t, J = 15.2 Hz, 2H), 2.64 – 2.54 (m, 1H), 2.21 – 2.02 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₅H₄₈N₇O₁₄⁺ 910.3254, found 910.3257.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecyl)-oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (52).

White solid (6.3 mg, 66%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 – 7.75 (m, 1H), 7.49 (dd, J = 7.3, 1.7 Hz, 1H), 7.42 – 7.37 (m, 2H), 7.36 – 7.31 (m, 2H), 7.20 – 7.16 (m, 1H), 7.14 (d, J = 2.6 Hz, 1H), 6.96 (t, J = 3.1 Hz, 1H), 6.90 – 6.85 (m, 1H), 5.60 (t, J = 7.9 Hz, 1H), 5.11 (ddd, J = 12.7, 5.5, 2.1 Hz, 1H), 4.73 (s, 2H), 4.48 (s, 2H), 3.63 – 3.52 (m, 11H), 3.49 – 3.45 (m, 2H), 3.43 (t, J = 5.4 Hz, 2H), 3.16 – 2.95 (m, 8H), 2.90 – 2.82 (m, 2H), 2.79 – 2.68 (m, 2H), 2.63 – 2.57 (m, 1H), 2.18 – 2.12 (m, 1H), 2.12 – 2.05 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₇H₅₂N₇O₁₅⁺ 954.3516, found 954.3513.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((20-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)-oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (53).

White solid (6.9 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 – 7.75 (m, 1H), 7.50 (dd, J = 7.3, 2.8 Hz, 1H), 7.44 – 7.30 (m, 4H), 7.25 – 7.10 (m, 2H), 6.99 – 6.84 (m, 2H), 5.60 (t, J = 7.8 Hz, 1H), 5.11 (dd, J = 13.1, 5.4 Hz, 1H), 4.74 (s, 2H), 4.48 (s, 2H), 3.73 – 3.39 (m, 21H), 3.20 – 2.95 (m, 8H), 2.91 – 2.83 (m, 2H), 2.79 – 2.67 (m, 2H), 2.65 – 2.54 (m, 1H), 2.22 – 2.04 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₉H₅₆N₇O₁₆⁺ 998.3778, found 998.3781.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((23-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,22-dioxo-6,9,12,15,18-pentaoxa-3,21-diazatricosyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (54).

White solid (7.6 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.76 (m, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.37 – 7.32 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.18 – 7.14 (m, 1H), 6.97 – 6.94 (m, 1H), 6.91 – 6.85 (m, 1H), 5.61 (t, J = 7.9 Hz, 1H), 5.12 (dd, J = 12.9, 5.5 Hz, 1H), 4.75 (s, 2H), 4.55 – 4.44 (m, 2H), 3.72 – 3.41 (m, 23H), 3.20 – 2.95 (m, 8H), 2.92 – 2.83 (m, 2H), 2.81 – 2.67 (m, 2H), 2.65 – 2.57 (m, 1H), 2.20 – 2.13 (m, 1H), 2.12 – 2.05 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₀N₇O₁₇⁺ 1042.4040, found 1042.4043.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(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-oxoethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (55).

White solid (5.9 mg, 63%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.16 (s, 1H), 7.52 – 7.33 (m, 7H), 7.25 – 7.12 (m, 2H), 7.03 – 6.91 (m, 2H), 5.68 – 5.58 (m, 1H), 4.65 (d, J = 5.7 Hz, 1H), 4.62 – 4.48 (m, 5H), 4.45 – 4.33 (m, 1H), 4.01 (s, 2H), 3.94 – 3.76 (m, 2H), 3.22 – 2.95 (m, 7H), 2.94 – 2.82 (m, 1H), 2.67 – 2.55 (m, 1H), 2.51 (s, 3H), 2.28 – 2.18 (m, 1H), 2.16 – 2.03 (m, 2H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₄₈H₅₅N₈O₁₀S⁺ 935.3756, found 935.3755.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((3-(((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)-3-oxopropyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (56).

White solid (6.7 mg, 71%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.54 – 7.29 (m, 7H), 7.25 – 7.12 (m, 2H), 7.06 – 6.87 (m, 2H), 5.68 – 5.55 (m, 1H), 4.64 – 4.45 (m, 6H), 4.42 – 4.32 (m, 1H), 3.93 (d, J = 10.7 Hz, 1H), 3.79 (d, J = 11.5 Hz, 1H), 3.60 – 3.50 (m, 2H), 3.49 – 3.37 (m, 1H), 3.24 – 2.92 (m, 7H), 2.91 – 2.81 (m, 1H), 2.64 – 2.55 (m, 1H), 2.51 (s, 3H), 2.29 – 2.20 (m, 1H), 2.15 – 2.03 (m, 3H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₄₉H₅₇N₈O₁₀S⁺ 949.3913, found 949.3915.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((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-oxobutyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (57).

White solid (7 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.16 (s, 1H), 7.51 – 7.47 (m, 2H), 7.45 – 7.40 (m, 3H), 7.37 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.18 (s, 1H), 6.97 – 6.95 (m, 1H), 6.93 – 6.90 (m, 1H), 5.63 (t, J = 7.9 Hz, 1H), 4.64 – 4.53 (m, 3H), 4.52 – 4.46 (m, 3H), 4.37 (d, J = 15.6 Hz, 1H), 3.94 – 3.89 (m, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.31 – 3.26 (m, 2H), 3.19 – 2.93 (m, 7H), 2.91 – 2.83 (m, 1H), 2.65 – 2.56 (m, 1H), 2.50 (s, 3H), 2.31 – 2.19 (m, 3H), 2.13 – 2.06 (m, 2H), 1.84 – 1.75 (m, 2H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₀H₅₉N₈O₁₀S⁺ 963.4069, found 963.4071.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((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)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (58).

White solid (6.8 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.21 – 9.10 (m, 1H), 7.60 – 7.29 (m, 7H), 7.26 – 7.11 (m, 2H), 7.01 – 6.82 (m, 2H), 5.66 – 5.56 (m, 1H), 4.65 – 4.32 (m, 7H), 3.91 (d, J = 11.0 Hz, 1H), 3.83 – 3.74 (m, 1H), 3.30 – 3.21 (m, 2H), 3.18 – 2.80 (m, 8H), 2.67 – 2.55 (m, 1H), 2.51 (s, 3H), 2.37 – 2.17 (m, 3H), 2.16 – 2.02 (m, 2H), 1.71 – 1.43 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₁N₈O₁₀S⁺ 977.4226, found 977.4229.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((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)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (59).

White solid (6.6 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.58 – 7.33 (m, 7H), 7.23 – 7.18 (m, 2H), 6.95 – 6.89 (m, 2H), 5.66 – 5.59 (m, 1H), 4.68 – 4.33 (m, 7H), 3.94 – 3.75 (m, 2H), 3.29 – 3.21 (m, 2H), 3.17 – 2.79 (m, 7H), 2.49 (s, 3H), 2.33 – 2.20 (m, 3H), 2.14 – 2.05 (m, 2H), 1.75 – 1.44 (m, 4H), 1.40 – 1.27 (m, 2H), 1.17 – 1.11 (m, 2H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₂H₆₃N₈O₁₀S⁺ 991.4382, found 991.4383.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((7-(((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)-7-oxoheptyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (60.

White solid (7.6 mg, 76%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.16 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.46 – 7.42 (m, 3H), 7.37 (d, J = 1.5 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 7.17 (s, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 8.2, 2.5 Hz, 1H), 5.61 (t, J = 7.8 Hz, 1H), 4.64 (s, 1H), 4.62 – 4.53 (m, 2H), 4.52 – 4.49 (m, 1H), 4.47 (s, 2H), 4.37 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.24 (t, J = 7.1 Hz, 2H), 3.17 – 2.92 (m, 7H), 2.91 – 2.83 (m, 1H), 2.65 – 2.56 (m, 1H), 2.51 (s, 3H), 2.32 – 2.20 (m, 3H), 2.14 – 2.04 (m, 2H), 1.62 – 1.54 (m, 2H), 1.54 – 1.46 (m, 2H), 1.34 – 1.25 (m, 4H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₅N₈O₁₀S⁺ 1005.4539, found 1005.4535.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((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)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (61).

White solid (7.1 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.51 – 7.46 (m, 2H), 7.46 – 7.42 (m, 3H), 7.39 – 7.34 (m, 2H), 7.22 – 7.15 (m, 2H), 6.94 (d, J = 2.5 Hz, 1H), 6.91 – 6.88 (m, 1H), 5.62 (t, J = 7.8 Hz, 1H), 4.65 (s, 1H), 4.62 – 4.44 (m, 5H), 4.37 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 1H), 3.28 – 3.21 (m, 2H), 3.16 – 2.95 (m, 7H), 2.91 – 2.83 (m, 1H), 2.64 – 2.58 (m, 1H), 2.49 (s, 3H), 2.31 – 2.19 (m, 3H), 2.14 – 2.04 (m, 2H), 1.62 – 1.55 (m, 2H), 1.52 – 1.46 (m, 2H), 1.34 – 1.23 (m, 6H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₇N₈O₁₀S⁺ 1019.4695, found 1019.4700.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((9-(((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)-9-oxononyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (62).

White solid (7.2 mg, 77%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.21 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.47 – 7.41 (m, 3H), 7.39 – 7.33 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.90 (dd, J = 8.3, 2.5 Hz, 1H), 5.61 (t, J = 7.8 Hz, 1H), 4.65 (s, 1H), 4.62 – 4.45 (m, 5H), 4.38 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.81 (dd, J = 11.0, 3.9 Hz, 1H), 3.24 (t, J = 7.1 Hz, 2H), 3.17 – 2.94 (m, 7H), 2.91 – 2.82 (m, 1H), 2.65 – 2.57 (m, 1H), 2.51 (s, 3H), 2.34 – 2.20 (m, 3H), 2.13 – 2.06 (m, 2H), 1.64 – 1.56 (m, 2H), 1.53 – 1.44 (m, 2H), 1.38 – 1.22 (m, 8H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₉N₈O₁₀S⁺ 1033.4852, found 1033.4849.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((10-(((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)-10-oxodecyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (63).

White solid (7.6 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.05 (s, 1H), 7.56 – 7.30 (m, 7H), 7.25 – 7.12 (m, 2H), 6.99 – 6.85 (m, 2H), 5.61 (t, J = 7.8 Hz, 1H), 4.70 – 4.43 (m, 6H), 4.37 (d, J = 15.7 Hz, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.81 (d, J = 10.5 Hz, 1H), 3.28 – 2.94 (m, 9H), 2.91 – 2.81 (m, 1H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.36 – 2.20 (m, 3H), 2.09 (t, J = 10.7 Hz, 2H), 1.71 – 1.46 (m, 5H), 1.40 – 1.20 (m, 9H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₇₁N₈O₁₀S⁺ 1047.5008, found 1047.5004.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((11-(((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)-11-oxoundecyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (64).

White solid (7 mg, 66%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.15 (s, 1H), 7.62 – 7.30 (m, 7H), 7.26 – 7.11 (m, 2H), 7.02 – 6.84 (m, 2H), 5.67 – 5.56 (m, 1H), 4.72 – 4.31 (m, 7H), 4.02 – 3.76 (m, 2H), 3.28 – 3.19 (m, 2H), 3.18 – 2.80 (m, 9H), 2.68 – 2.56 (m, 2H), 2.51 (s, 3H), 2.40 – 2.01 (m, 5H), 1.70 – 1.42 (m, 5H), 1.40 – 1.20 (m, 11H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₇₃N₈O₁₀S⁺ 1061.5165, found 1061.5167.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((2-(3-(((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)-3-oxopropoxy)ethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (65).

White solid (6.1 mg, 62%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.19 (s, 1H), 7.50 – 7.29 (m, 7H), 7.23 – 7.13 (m, 2H), 6.98 – 6.84 (m, 2H), 5.56 (t, J = 8.0 Hz, 1H), 4.68 – 4.60 (m, 2H), 4.54 – 4.41 (m, 4H), 4.33 (d, J = 15.5 Hz, 1H), 3.86 (d, J = 11.1 Hz, 1H), 3.73 (dd, J = 11.0, 3.9 Hz, 1H), 3.69 – 3.62 (m, 1H), 3.60 – 3.51 (m, 2H), 3.50 – 3.39 (m, 3H), 3.21 – 2.89 (m, 7H), 2.87 – 2.79 (m, 1H), 2.61 – 2.53 (m, 1H), 2.49 (s, 3H), 2.44 – 2.33 (m, 1H), 2.27 – 2.20 (m, 1H), 2.11 – 2.01 (m, 3H), 1.01 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₁H₆₁N₈O₁₁S⁺ 993.4175, found 993.4179.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-14-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-2,12-dioxo-6,9-dioxa-3,13-diazahexadecyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (66).

White solid (7.1 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.19 (s, 1H), 7.50 – 7.45 (m, 2H), 7.43 – 7.40 (m, 3H), 7.37 – 7.32 (m, 2H), 7.20 – 7.15 (m, 2H), 6.93 (d, J = 2.5 Hz, 1H), 6.87 (dd, J = 8.2, 2.5 Hz, 1H), 5.59 (t, J = 7.8 Hz, 1H), 4.65 (s, 1H), 4.61 – 4.51 (m, 2H), 4.51 – 4.42 (m, 3H), 4.34 (d, J = 15.5 Hz, 1H), 3.86 (d, J = 11.0 Hz, 1H), 3.76 (dd, J = 10.9, 3.9 Hz, 1H), 3.74 – 3.62 (m, 2H), 3.60 – 3.50 (m, 6H), 3.42 (t, J = 5.5 Hz, 2H), 3.18 – 2.92 (m, 7H), 2.89 – 2.80 (m, 1H), 2.62 – 2.55 (m, 1H), 2.52 – 2.39 (m, 5H), 2.24 – 2.17 (m, 1H), 2.12 – 2.02 (m, 2H), 1.01 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₅N₈O₁₂S⁺ 1037.4437, found 1037.4442.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-17-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-18,18-dimethyl-2,15-dioxo-6,9,12-trioxa-3,16-diazanonadecyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (67).

White solid (6.5 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.14 (s, 1H), 7.57 – 7.29 (m, 7H), 7.25 – 7.13 (m, 2H), 7.03 – 6.84 (m, 2H), 5.69 – 5.56 (m, 1H), 4.68 – 4.45 (m, 6H), 4.36 (d, J = 15.0 Hz, 1H), 3.89 (d, J = 10.9 Hz, 1H), 3.83 – 3.77 (m, 1H), 3.75 – 3.51 (m, 12H), 3.47 – 3.42 (m, 2H), 3.19 – 2.83 (m, 8H), 2.64 – 2.41 (m, 6H), 2.26 – 2.19 (m, 1H), 2.12 – 2.03 (m, 2H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₉N₈O₁₃S⁺ 1081.4699, found 1081.4696.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-20-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-21,21-dimethyl-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (68).

White solid (6.4 mg, 57%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.27 (s, 1H), 7.59 – 7.32 (m, 7H), 7.24 – 7.13 (m, 2H), 7.05 – 6.88 (m, 2H), 5.75 – 5.54 (m, 1H), 4.72 – 4.45 (m, 6H), 4.37 (d, J = 15.3 Hz, 1H), 3.90 (d, J = 11.1 Hz, 1H), 3.83 – 3.78 (m, 1H), 3.73 – 3.49 (m, 16H), 3.49 – 3.41 (m, 2H), 3.20 – 2.81 (m, 8H), 2.67 – 2.42 (m, 6H), 2.29 – 2.20 (m, 1H), 2.14 – 2.04 (m, 2H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₇₃N₈O₁₄S⁺ 1125.4961, found 1125.4964.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-23-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-24,24-dimethyl-2,21-dioxo-6,9,12,15,18-pentaoxa-3,22-diazapentacosyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (69).

White solid (7.6 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.30 (s, 1H), 7.57 – 7.41 (m, 5H), 7.39 – 7.33 (m, 2H), 7.24 – 7.15 (m, 2H), 6.99 – 6.87 (m, 2H), 5.62 (t, J = 7.8 Hz, 1H), 4.66 (s, 1H), 4.61 – 4.47 (m, 5H), 4.37 (d, J = 15.6 Hz, 1H), 3.90 (d, J = 11.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.8 Hz, 1H), 3.75 – 3.68 (m, 2H), 3.67 – 3.52 (m, 20H), 3.49 – 3.42 (m, 2H), 3.19 – 2.92 (m, 7H), 2.92 – 2.83 (m, 1H), 2.66 – 2.44 (m, 4H), 2.27 – 2.20 (m, 1H), 2.14 – 2.06 (m, 2H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₇N₈O₁₅S⁺ 1169.5224, found 1169.5227.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((3-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)propyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (70).

White solid (7.2 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.14 (s, 1H), 7.51 – 7.41 (m, 5H), 7.38 – 7.32 (m, 2H), 7.21 (d, J = 8.3 Hz, 1H), 7.16 (s, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.91 (dd, J = 8.3, 2.5 Hz, 1H), 5.62 (t, J = 7.9 Hz, 1H), 5.33 (dd, J = 8.1, 6.2 Hz, 1H), 4.75 (d, J = 8.6 Hz, 1H), 4.64 – 4.58 (m, 1H), 4.47 – 4.43 (m, 3H), 3.84 (d, J = 11.1 Hz, 1H), 3.77 (dd, J = 11.1, 3.8 Hz, 1H), 3.19 – 2.93 (m, 12H), 2.91 – 2.82 (m, 2H), 2.75 (dd, J = 14.2, 8.2 Hz, 1H), 2.65 – 2.57 (m, 1H), 2.49 (s, 3H), 2.24 – 2.17 (m, 1H), 2.15 – 2.06 (m, 1H), 1.99 – 1.92 (m, 1H), 1.57 – 1.51 (m, 2H), 1.41 – 1.24 (m, 3H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₅FN₉O₁₁S⁺ 1078.4503, found 1078.4501.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((6-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)hexyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (71).

White solid (6.7 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.51 – 7.42 (m, 6H), 7.38 – 7.34 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90 (dd, J = 8.3, 2.5 Hz, 1H), 5.62 (t, J = 7.9 Hz, 1H), 5.32 (dd, J = 8.3, 6.0 Hz, 1H), 4.77 – 4.73 (m, 1H), 4.60 (dd, J = 9.3, 7.6 Hz, 1H), 4.48 – 4.43 (m, 3H), 3.84 (d, J = 10.9 Hz, 1H), 3.78 (dd, J = 11.1, 3.8 Hz, 1H), 3.21 – 2.95 (m, 12H), 2.91 – 2.82 (m, 2H), 2.76 (dd, J = 14.1, 8.3 Hz, 1H), 2.64 – 2.57 (m, 1H), 2.49 (s, 3H), 2.23 – 2.18 (m, 1H), 2.13 – 2.06 (m, 1H), 2.01 – 1.93 (m, 1H), 1.45 – 1.26 (m, 7H), 1.23 – 1.11 (m, 4H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₁S⁺ 1120.4972, found 1120.4973.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((7-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)heptyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (72).

White solid (7.9 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.03 (s, 1H), 7.52 – 7.41 (m, 5H), 7.39 – 7.33 (m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.15 (s, 1H), 6.96 – 6.94 (m, 1H), 6.92 – 6.88 (m, 1H), 5.62 (t, J = 7.8 Hz, 1H), 5.32 (dd, J = 8.4, 5.9 Hz, 1H), 4.75 (d, J = 9.1 Hz, 1H), 4.62 – 4.58 (m, 1H), 4.49 – 4.43 (m, 3H), 3.84 (d, J = 11.1 Hz, 1H), 3.80 – 3.75 (m, 1H), 3.21 – 2.94 (m, 12H), 2.92 – 2.82 (m, 2H), 2.75 (dd, J = 14.0, 8.4 Hz, 1H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.24 – 2.17 (m, 1H), 2.14 – 2.05 (m, 1H), 2.00 – 1.94 (m, 1H), 1.46 – 1.26 (m, 7H), 1.23 – 1.15 (m, 4H), 1.14 – 1.09 (m, 2H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₃FN₉O₁₁S⁺ 1134.5129, found 1134.5125.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((8-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)octyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (73).

White solid (8.6 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.59 – 7.31 (m, 7H), 7.27 – 7.13 (m, 2H), 7.02 – 6.88 (m, 2H), 5.62 (t, J = 7.9 Hz, 1H), 5.35 – 5.30 (m, 1H), 4.75 (d, J = 8.9 Hz, 1H), 4.63 – 4.57 (m, 1H), 4.54 – 4.43 (m, 3H), 3.84 (d, J = 11.1 Hz, 1H), 3.78 (dd, J = 11.1, 3.7 Hz, 1H), 3.26 – 2.94 (m, 12H), 2.93 – 2.84 (m, 2H), 2.75 (dd, J = 13.9, 8.6 Hz, 1H), 2.65 – 2.58 (m, 1H), 2.50 (s, 3H), 2.24 – 2.18 (m, 1H), 2.14 – 2.05 (m, 1H), 2.01 – 1.94 (m, 1H), 1.47 – 1.26 (m, 9H), 1.23 – 1.14 (m, 6H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₁S⁺ 1148.5285, found 1148.5283.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((9-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)nonyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (74).

¹H NMR (400 MHz, Methanol-d₄) δ 8.88 (s, 1H), 7.51 – 7.45 (m, 1H), 7.43 (d, J = 2.4 Hz, 3H), 7.40 (d, J = 1.8 Hz, 1H), 7.36 – 7.31 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.13 (s, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.3, 2.5 Hz, 1H), 5.61 (q, J = 7.8 Hz, 1H), 5.33 – 5.28 (m, 1H), 4.85 – 4.79 (m, 2H), 4.74 (d, J = 9.2 Hz, 1H), 4.62 – 4.54 (m, 1H), 4.46 (d, J = 11.5 Hz, 3H), 3.83 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.7 Hz, 1H), 3.24 – 3.16 (m, 2H), 3.14 – 2.94 (m, 8H), 2.91 – 2.80 (m, 2H), 2.73 (dd, J = 14.0, 8.5 Hz, 1H), 2.66 – 2.56 (m, 1H), 2.47 (s, 3H), 2.19 (dd, J = 13.2, 7.7 Hz, 1H), 2.13 – 2.03 (m, 1H), 1.99 – 1.92 (m, 1H), 1.48 – 1.35 (m, 3H), 1.35 – 1.25 (m, 5H), 1.22 – 1.07 (m, 9H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₁H₇₇FN₉O₁₁S⁺ 1162.5442, found 1162.5440.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,13-trioxo-9-oxa-2,6,12-triazatetradecan-14-yl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (75).

White solid (7.6 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.07 (s, 1H), 7.57 – 7.30 (m, 7H), 7.22 – 7.14 (m, 2H), 7.00 – 6.83 (m, 2H), 5.61 (t, J = 7.9 Hz, 1H), 5.36 – 5.30 (m, 1H), 4.75 (d, J = 8.9 Hz, 1H), 4.64 – 4.57 (m, 1H), 4.53 – 4.41 (m, 3H), 3.84 (d, J = 11.1 Hz, 1H), 3.77 (dd, J = 11.1, 3.9 Hz, 1H), 3.52 – 3.35 (m, 5H), 3.29 – 3.22 (m, 2H), 3.19 – 2.92 (m, 8H), 2.90 – 2.82 (m, 2H), 2.75 (dd, J = 14.2, 8.0 Hz, 1H), 2.64 – 2.56 (m, 1H), 2.49 (s, 3H), 2.24 – 2.18 (m, 1H), 2.14 – 2.04 (m, 1H), 2.02 – 1.90 (m, 1H), 1.45 – 1.22 (m, 4H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₂S⁺ 1108.4608, found 1108.4606.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,16-trioxo-9,12-dioxa-2,6,15-triazaheptadecan-17-yl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (76).

White solid (7.5 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.14 (s, 1H), 7.52 – 7.41 (m, 5H), 7.40 – 7.33 (m, 2H), 7.25 – 7.15 (m, 2H), 7.01 – 6.85 (m, 2H), 5.67 – 5.59 (m, 1H), 5.35 – 5.30 (m, 1H), 4.75 (d, J = 8.5 Hz, 1H), 4.63 – 4.58 (m, 1H), 4.53 – 4.42 (m, 3H), 3.84 (d, J = 10.7 Hz, 1H), 3.76 (dd, J = 11.2, 3.9 Hz, 1H), 3.63 – 3.36 (m, 9H), 3.31 – 3.26 (m, 2H), 3.21 – 2.96 (m, 8H), 2.92 – 2.83 (m, 2H), 2.79 – 2.71 (m, 1H), 2.66 – 2.58 (m, 1H), 2.50 (s, 3H), 2.27 – 2.19 (m, 1H), 2.16 – 2.05 (m, 1H), 2.03 – 1.93 (m, 1H), 1.44 – 1.26 (m, 4H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₃S⁺ 1152.4871, found 1152.4874.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,19-trioxo-9,12,15-trioxa-2,6,18-triazaicosan-20-yl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (77).

White solid (7.2 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.95 (s, 1H), 7.57 – 7.31 (m, 7H), 7.25 – 7.14 (m, 2H), 6.99 – 6.86 (m, 2H), 5.62 (t, J = 7.8 Hz, 1H), 5.36 – 5.30 (m, 1H), 4.78 – 4.72 (m, 1H), 4.60 (t, J = 8.6 Hz, 1H), 4.53 – 4.43 (m, 3H), 3.84 (d, J = 11.1 Hz, 1H), 3.77 (dd, J = 11.2, 3.7 Hz, 1H), 3.68 – 3.39 (m, 13H), 3.31 – 3.23 (m, 2H), 3.19 – 2.96 (m, 8H), 2.91 – 2.82 (m, 2H), 2.80 – 2.73 (m, 1H), 2.65 – 2.57 (m, 1H), 2.48 (s, 3H), 2.25 – 2.18 (m, 1H), 2.13 – 2.06 (m, 1H), 2.01 – 1.93 (m, 1H), 1.45 – 1.23 (m, 4H), 1.07 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₄S⁺ 1196.5133, found 1196.5135.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,22-trioxo-9,12,15,18-tetraoxa-2,6,21-triazatricosan-23-yl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (78).

White solid (9 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.60 – 7.30 (m, 7H), 7.25 – 7.13 (m, 2H), 6.99 – 6.86 (m, 2H), 5.62 (t, J = 7.8 Hz, 1H), 5.37 – 5.31 (m, 1H), 4.75 (d, J = 9.0 Hz, 1H), 4.60 (t, J = 8.6 Hz, 1H), 4.54 – 4.42 (m, 3H), 3.84 (d, J = 11.2 Hz, 1H), 3.77 (dd, J = 11.1, 3.8 Hz, 1H), 3.71 – 3.34 (m, 17H), 3.31 – 3.22 (m, 2H), 3.18 – 2.93 (m, 8H), 2.91 – 2.82 (m, 2H), 2.81 – 2.74 (m, 1H), 2.64 – 2.57 (m, 1H), 2.49 (s, 3H), 2.25 – 2.18 (m, 1H), 2.14 – 2.05 (m, 1H), 2.01 – 1.93 (m, 1H), 1.43 – 1.24 (m, 4H), 1.08 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₂H₇₉FN₉O₁₅S⁺ 1240.5395, found 1240.5393.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5,25-trioxo-9,12,15,18,21-pentaoxa-2,6,24-triazahexacosan-26-yl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (79).

White solid (8.5 mg, 66%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.63 – 7.30 (m, 7H), 7.26 – 7.12 (m, 2H), 6.98 – 6.85 (m, 2H), 5.63 (t, J = 8.5 Hz, 1H), 5.44 – 5.31 (m, 1H), 4.75 (d, J = 9.0 Hz, 1H), 4.63 – 4.57 (m, 1H), 4.56 – 4.42 (m, 3H), 3.84 (d, J = 11.3 Hz, 1H), 3.80 – 3.75 (m, 1H), 3.70 – 3.41 (m, 21H), 3.34 – 3.22 (m, 2H), 3.20 – 2.94 (m, 8H), 2.90 – 2.83 (m, 2H), 2.81 – 2.74 (m, 1H), 2.65 – 2.56 (m, 1H), 2.50 (s, 3H), 2.25 – 2.20 (m, 1H), 2.13 – 2.04 (m, 1H), 2.03 – 1.92 (m, 1H), 1.49 – 1.23 (m, 4H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₄H₈₃FN₉O₁₆S⁺ 1284.5657, found 1284.5654.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (80).

White solid (7 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.14 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.45 (dd, J = 9.4, 3.3 Hz, 1H), 7.42 – 7.39 (m, 1H), 7.37 – 7.31 (m, 2H), 7.18 – 7.15 (m, 2H), 7.08 (dd, J = 7.7, 1.6 Hz, 1H), 6.97 (d, J = 1.7 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.86 (dd, J = 8.2, 2.5 Hz, 1H), 5.59 (t, J = 8.0 Hz, 1H), 4.72 (d, J = 8.6 Hz, 1H), 4.62 – 4.56 (m, 2H), 4.54 – 4.44 (m, 3H), 4.35 (d, J = 1.9 Hz, 2H), 3.84 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 11.1, 3.8 Hz, 1H), 3.52 – 3.41 (m, 4H), 3.17 – 2.94 (m, 8H), 2.87 – 2.79 (m, 1H), 2.63 – 2.55 (m, 1H), 2.49 (s, 3H), 2.21 – 2.14 (m, 1H), 2.11 – 2.02 (m, 2H), 1.40 – 1.22 (m, 4H), 1.00 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₃FN₉O₁₂S⁺ 1080.4295, found 1080.4297.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((3-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)propyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (81).

White solid (7.5 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.09 (s, 1H), 7.58 – 7.27 (m, 5H), 7.25 – 7.04 (m, 3H), 7.01 – 6.85 (m, 3H), 5.59 (t, J = 8.1 Hz, 1H), 4.77 – 4.39 (m, 8H), 3.87 – 3.71 (m, 2H), 3.40 – 3.22 (m, 4H), 3.20 – 2.81 (m, 8H), 2.63 – 2.54 (m, 1H), 2.48 (s, 3H), 2.24 – 2.00 (m, 3H), 1.80 – 1.67 (m, 2H), 1.41 – 1.16 (m, 6H), 0.99 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₅FN₉O₁₂S⁺ 1094.4452, found 1094.4456.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((4-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)butyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (82).

White solid (7.1 mg, 64%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.11 (s, 1H), 7.53 – 7.45 (m, 2H), 7.44 – 7.40 (m, 1H), 7.38 – 7.32 (m, 2H), 7.21 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 1.5 Hz, 1H), 7.10 (dd, J = 10.0, 3.8 Hz, 1H), 7.02 – 6.86 (m, 3H), 5.62 (t, J = 7.9 Hz, 1H), 4.73 (d, J = 9.3 Hz, 1H), 4.66 – 4.44 (m, 7H), 3.84 (d, J = 11.0 Hz, 1H), 3.78 (dd, J = 11.4, 4.0 Hz, 1H), 3.31 – 3.21 (m, 4H), 3.18 – 2.93 (m, 8H), 2.91 – 2.83 (m, 1H), 2.64 – 2.57 (m, 1H), 2.50 (s, 3H), 2.23 – 2.17 (m, 1H), 2.13 – 2.04 (m, 2H), 1.64 – 1.46 (m, 4H), 1.42 – 1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₂S⁺ 1108.4608, found 1108.4606.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((6-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)hexyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (83).

White solid (6.8 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.05 (s, 1H), 7.54 – 7.31 (m, 5H), 7.21 (d, J = 8.3 Hz, 1H), 7.17 – 7.13 (m, 1H), 7.12 – 7.08 (m, 1H), 7.01 – 6.96 (m, 2H), 6.90 (dd, J = 8.3, 2.6 Hz, 1H), 5.62 (t, J = 7.8 Hz, 1H), 4.74 (d, J = 9.2 Hz, 1H), 4.67 – 4.55 (m, 3H), 4.51 – 4.45 (m, 4H), 3.85 (d, J = 10.9 Hz, 1H), 3.78 (dd, J = 11.1, 3.8 Hz, 1H), 3.29 – 3.25 (m, 2H), 3.21 (t, J = 7.1 Hz, 2H), 3.17 – 2.95 (m, 8H), 2.91 – 2.83 (m, 1H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.25 – 2.18 (m, 1H), 2.14 – 2.04 (m, 2H), 1.60 – 1.42 (m, 4H), 1.40 – 1.21 (m, 8H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₂S⁺ 1136.4921, found 1136.4924.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((8-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)octyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (84).

White solid (8.1 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.00 (s, 1H), 7.55 – 7.31 (m, 5H), 7.21 (d, J = 8.3 Hz, 1H), 7.17 – 7.08 (m, 2H), 7.01 – 6.94 (m, 1H), 6.90 (dd, J = 8.4, 2.4 Hz, 1H), 5.62 (t, J = 7.8 Hz, 1H), 4.75 (d, J = 9.3 Hz, 1H), 4.66 – 4.56 (m, 3H), 4.51 – 4.45 (m, 4H), 3.86 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 11.1, 3.8 Hz, 1H), 3.28 (t, J = 7.0 Hz, 2H), 3.23 (t, J = 7.2 Hz, 2H), 3.18 – 2.94 (m, 8H), 2.91 – 2.83 (m, 1H), 2.65 – 2.57 (m, 1H), 2.50 (s, 3H), 2.25 – 2.19 (m, 1H), 2.14 – 2.04 (m, 2H), 1.58 – 1.44 (m, 6H), 1.42 – 1.19 (m, 10H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₂S⁺ 1164.5234, found 1164.5232.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(2-((2-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethoxy)ethyl)amino)-2-oxoethoxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (85).

White solid (8.1 mg, 72%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.55 – 7.29 (m, 5H), 7.22 – 7.05 (m, 3H), 7.00 – 6.82 (m, 3H), 5.63 – 5.54 (m, 1H), 4.78 – 4.42 (m, 8H), 3.90 – 3.73 (m, 2H), 3.69 – 3.36 (m, 8H), 3.20 – 2.94 (m, 8H), 2.89 – 2.80 (m, 1H), 2.66 – 2.56 (m, 1H), 2.48 (s, 3H), 2.25 – 2.16 (m, 1H), 2.15 – 2.04 (m, 2H), 1.42 – 1.19 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₃S⁺ 1124.4558, found 1124.4562.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((14-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,13-dioxo-6,9-dioxa-3,12-diazatetradecyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (86).

White solid (7.9 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.05 (s, 1H), 7.52 – 7.47 (m, 2H), 7.42 (dd, J = 7.9, 1.6 Hz, 1H), 7.37 – 7.32 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 7.09 (dd, J = 7.7, 1.6 Hz, 1H), 6.96 (dd, J = 14.8, 2.0 Hz, 2H), 6.89 (dd, J = 8.3, 2.5 Hz, 1H), 5.60 (t, J = 7.8 Hz, 1H), 4.74 (d, J = 9.3 Hz, 1H), 4.63 – 4.46 (m, 7H), 3.85 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 11.1, 3.8 Hz, 1H), 3.61 – 3.52 (m, 8H), 3.49 – 3.41 (m, 4H), 3.18 – 2.95 (m, 8H), 2.90 – 2.81 (m, 1H), 2.64 – 2.56 (m, 1H), 2.50 (s, 3H), 2.22 (dd, J = 13.3, 7.7 Hz, 1H), 2.13 – 2.03 (m, 2H), 1.41 – 1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₄S⁺ 1168.4820, found 1168.4816.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((17-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecyl)oxy)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (87).

White solid (7.4 mg, 61%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.10 (s, 1H), 7.53 – 7.48 (m, 2H), 7.42 (dd, J = 7.9, 1.6 Hz, 1H), 7.38 – 7.32 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 7.10 (dd, J = 7.8, 1.6 Hz, 1H), 6.99 (d, J = 1.6 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.2, 2.5 Hz, 1H), 5.61 (t, J = 7.8 Hz, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.64 – 4.58 (m, 3H), 4.54 – 4.46 (m, 4H), 3.85 (d, J = 11.0 Hz, 1H), 3.80 (dd, J = 11.0, 3.8 Hz, 1H), 3.60 – 3.51 (m, 12H), 3.49 – 3.41 (m, 4H), 3.17 – 2.94 (m, 8H), 2.90 – 2.82 (m, 1H), 2.64 – 2.56 (m, 1H), 2.51 (s, 3H), 2.22 (dd, J = 13.3, 7.7 Hz, 1H), 2.13 – 2.04 (m, 2H), 1.41 – 1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₅S⁺ 1212.5082, found 1212.5087.

Method C: Preparation of ENL SAR Putative degraders 88–116.

To a solution of precursor 129 (Scheme 3A) (4.35 mg, 0.01 mmol) in DMSO (1 mL) were added each type of linker-attached CRBN or VHL ligands (0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixtures were purified by preparative HPLC (5%-60% acetonitrile / 0.1% TFA in H₂O) to afford the final compounds 88–116 as white solids with >95% purity.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (88).

White solid (4.6 mg, 58%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.73 (d, J = 8.0 Hz, 1H), 7.71 – 7.64 (m, 2H), 7.44 – 7.29 (m, 6H), 7.10 (d, J = 9.6 Hz, 1H), 5.71 – 5.64 (m, 1H), 5.02 (dd, J = 12.9, 5.4 Hz, 1H), 4.79 – 4.72 (m, 2H), 3.63 – 3.50 (m, 4H), 3.20 – 2.93 (m, 8H), 2.86 – 2.76 (m, 1H), 2.71 – 2.54 (m, 3H), 2.18 – 2.03 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₀H₃₈N₇O₁₁⁺ 792.2624, found 792.2619.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)propyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (89).

White solid (5.1 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.75 (m, 2H), 7.74 – 7.68 (m, 1H), 7.54 – 7.49 (m, 1H), 7.45 – 7.39 (m, 2H), 7.37 – 7.29 (m, 3H), 7.17 (s, 1H), 5.68 – 5.62 (m, 1H), 5.14 – 5.07 (m, 1H), 4.77 – 4.74 (m, 2H), 3.51 – 3.39 (m, 4H), 3.19 – 2.92 (m, 8H), 2.89 – 2.80 (m, 1H), 2.78 – 2.60 (m, 3H), 2.17 – 2.08 (m, 2H), 1.92 – 1.84 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₁H₄₀N₇O₁₁⁺ 806.2780, found 806.2782.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (90).

White solid (5.8 mg, 71%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.80 – 7.69 (m, 3H), 7.49 (t, J = 7.2 Hz, 1H), 7.44 – 7.30 (m, 5H), 7.15 (d, J = 8.5 Hz, 1H), 5.70 (t, J = 8.0 Hz, 1H), 5.09 (dd, J = 12.7, 5.4 Hz, 1H), 4.76 – 4.68 (m, 2H), 3.50 – 3.35 (m, 4H), 3.18 – 2.94 (m, 8H), 2.85 – 2.62 (m, 4H), 2.18 – 2.04 (m, 2H), 1.76 – 1.59 (m, 4H). HRMS m/z [M + H]⁺ calcd for C₄₂H₄₂N₇O₁₁⁺ 820.2937, found 820.2933.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((5-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pentyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (91).

White solid (5 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 – 7.73 (m, 2H), 7.68 (ddd, J = 17.9, 7.9, 1.7 Hz, 1H), 7.49 (dd, J = 11.0, 7.3 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.38 – 7.33 (m, 2H), 7.29 (dd, J = 14.8, 7.9 Hz, 1H), 7.16 (s, 1H), 5.69 – 5.63 (m, 1H), 5.16 – 5.07 (m, 1H), 4.75 – 4.70 (m, 2H), 3.43 – 3.34 (m, 3H), 3.19 – 2.93 (m, 8H), 2.91 – 2.82 (m, 1H), 2.78 – 2.60 (m, 3H), 2.18 – 2.09 (m, 2H), 1.69 – 1.61 (m, 4H), 1.51 – 1.44 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₃H₄₄N₇O₁₁⁺ 834.3093, found 834.3091.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (92).

White solid (5.5 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 – 7.75 (m, 2H), 7.75 – 7.69 (m, 1H), 7.50 (dd, J = 7.3, 4.4 Hz, 1H), 7.44 – 7.32 (m, 5H), 7.16 (s, 1H), 5.70 (t, J = 7.9 Hz, 1H), 5.15 – 5.06 (m, 1H), 4.76 – 4.71 (m, 2H), 3.38 – 3.34 (m, 3H), 3.17 – 2.94 (m, 8H), 2.90 – 2.81 (m, 1H), 2.76 – 2.62 (m, 3H), 2.16 – 2.10 (m, 2H), 1.65 – 1.57 (m, 4H), 1.47 – 1.39 (m, 4H). HRMS m/z [M + H]⁺ calcd for C₄₄H₄₆N₇O₁₁⁺ 848.3250, found 848.3251.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((7-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)heptyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (93).

white solid (6.2 mg, 72%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.81 – 7.76 (m, 2H), 7.73 – 7.68 (m, 1H), 7.48 (dd, J = 8.9, 7.3 Hz, 1H), 7.44 – 7.38 (m, 2H), 7.37 – 7.32 (m, 3H), 7.16 (d, J = 4.3 Hz, 1H), 5.69 (t, J = 8.0 Hz, 1H), 5.16 – 5.11 (m, 1H), 4.76 – 4.71 (m, 2H), 3.32 – 3.28 (m, 3H), 3.18 – 2.94 (m, 8H), 2.91 – 2.82 (m, 1H), 2.79 – 2.63 (m, 3H), 2.19 – 2.10 (m, 2H), 1.63 – 1.54 (m, 4H), 1.43 – 1.34 (m, 6H). HRMS m/z [M + H]⁺ calcd for C₄₅H₄₈N₇O₁₁⁺ 862.3406, found 862.3405.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((8-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)octyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (94).

White solid (5.3 mg, 61%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.01 (t, J = 7.9 Hz, 1H), δ 7.81 – 7.74 (m, 2H), 7.73 – 7.69 (m, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.42 – 7.37 (m, 2H), 7.37 – 7.30 (m, 3H), 7.14 (d, J = 2.8 Hz, 1H), 5.69 (q, J = 8.1 Hz, 1H), 5.12 (dd, J = 12.6, 5.5 Hz, 1H), 4.72 (s, 2H), 3.33 (d, J = 6.7 Hz, 2H), 3.27 (t, J = 6.4 Hz, 2H), 3.18 – 2.90 (m, 8H), 2.89 – 2.81 (m, 1H), 2.77 – 2.60 (m, 3H), 2.18 – 2.02 (m, 2H), 1.59 – 1.50 (m, 4H), 1.33 (s, 8H). HRMS m/z [M + H]⁺ calcd for C₄₆H₅₀N₇O₁₁⁺ 876.3563, found 876.3562.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)-ethyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (95).

White solid (5 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.80 – 7.75 (m, 2H), 7.70 – 7.67 (m, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.40 (dd, J = 7.9, 1.6 Hz, 1H), 7.37 – 7.31 (m, 3H), 7.21 (d, J = 7.9 Hz, 1H), 7.13 (s, 1H), 5.58 (t, J = 7.9 Hz, 1H), 5.04 (dd, J = 5.5 Hz, 1H), 4.73 – 4.61 (m, 2H), 3.73 – 3.59 (m, 5H), 3.57 – 3.49 (m, 3H), 3.20 – 2.96 (m, 8H), 2.92 – 2.85 (m, 1H), 2.80 – 2.56 (m, 4H), 2.16 – 2.04 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₂H₄₂N₇O₁₂⁺ 836.2886, found 836.2889.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)-ethoxy)ethyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (96).

White solid (5.6 mg, 64%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.80 (d, J = 7.3 Hz, 1H), 7.78 – 7.74 (m, 1H), 7.74 – 7.69 (m, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.42 – 7.39 (m, 1H), 7.38 – 7.31 (m, 4H), 7.14 (d, J = 5.1 Hz, 1H), 5.68 (t, J = 8.0 Hz, 1H), 5.11 (dd, J = 12.8, 5.5 Hz, 1H), 4.70 – 4.67 (m, 2H), 3.66 – 3.57 (m, 8H), 3.55 – 3.51 (m, 2H), 3.49 – 3.39 (m, 2H), 3.18 – 2.93 (m, 8H), 2.90 – 2.82 (m, 1H), 2.79 – 2.70 (m, 3H), 2.69 – 2.62 (m, 1H), 2.19 – 2.09 (m, 1H). HRMS m/z [M + H]⁺ calcd for C₄₄H₄₆N₇O₁₃⁺ 880.3148, found 880.3152.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (97).

White solid (6.2 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.79 (d, J = 3.9 Hz, 1H), 7.77 – 7.74 (m, 1H), 7.74 – 7.70 (m, 1H), 7.46 (dd, J = 7.3, 3.3 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.36 – 7.30 (m, 3H), 7.13 (d, J = 4.6 Hz, 1H), 5.67 (t, J = 8.0 Hz, 1H), 5.13 – 5.07 (m, 1H), 4.71 (s, 2H), 3.63 – 3.50 (m, 14H), 3.44 – 3.39 (m, 2H), 3.15 – 2.93 (m, 8H), 2.89 – 2.81 (m, 1H), 2.77 – 2.60 (m, 3H), 2.16 – 2.07 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₆H₅₀N₇O₁₄⁺ 924.3410, found 924.3414.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (98).

White solid (6.7 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 (d, J = 4.6 Hz, 1H), 7.78 (dd, J = 8.4, 7.3 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.50 (dd, J = 7.3, 1.9 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.38 – 7.33 (m, 3H), 7.16 (d, J = 2.3 Hz, 1H), 5.70 (t, J = 7.9 Hz, 1H), 5.13 (dd, J = 12.8, 5.5 Hz, 1H), 4.76 – 4.73 (m, 2H), 3.66 – 3.53 (m, 18H), 3.47 (t, J = 5.3 Hz, 2H), 3.17 – 2.95 (m, 8H), 2.92 – 2.84 (m, 1H), 2.79 – 2.62 (m, 3H), 2.18 – 2.09 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₄₈H₅₄N₇O₁₅⁺ 968.3672, found 968.3671.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((1R)-6-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12,15,18-pentaoxa-3-azaicosan-20-yl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (99).

White solid (6.1 mg, 61%). ¹H NMR (600 MHz, Methanol-d₄) δ 7.82 (d, J = 3.8 Hz, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.77 – 7.74 (m, 1H), 7.50 (dd, J = 7.3, 2.3 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.38 – 7.33 (m, 3H), 7.16 (d, J = 1.8 Hz, 1H), 5.70 (t, J = 8.0 Hz, 1H), 5.16 – 5.11 (m, 1H), 4.76 – 4.73 (m, 2H), 3.69 – 3.51 (m, 22H), 3.49 (t, J = 5.3 Hz, 2H), 3.18 – 2.95 (m, 8H), 2.92 – 2.84 (m, 1H), 2.80 – 2.71 (m, 2H), 2.70 – 2.62 (m, 1H), 2.19 – 2.09 (m, 2H). HRMS m/z [M + H]⁺ calcd for C₅₀H₅₈N₇O₁₆⁺ 1012.3935, found 1012.3938.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((2-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)ethyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (100).

White solid (6.2 mg, 60%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.00 (s, 1H), 7.66 (s, 1H), 7.56 (dd, J = 7.9, 1.7 Hz, 1H), 7.38 – 7.29 (m, 3H), 7.27 – 7.19 (m, 5H), 7.05 (s, 1H), 5.56 (t, J = 7.9 Hz, 1H), 5.23 (t, J = 7.0 Hz, 1H), 4.64 – 4.59 (m, 1H), 4.49 (dd, J = 9.3, 7.6 Hz, 1H), 4.35 – 4.31 (m, 1H), 3.72 (d, J = 11.2 Hz, 1H), 3.65 (dd, J = 11.1, 3.8 Hz, 1H), 3.32 – 3.26 (m, 4H), 3.06 – 2.81 (m, 8H), 2.71 (dd, J = 14.2, 6.8 Hz, 1H), 2.64 (dd, J = 14.2, 7.3 Hz, 1H), 2.56 – 2.49 (m, 1H), 2.35 (s, 3H), 2.12 – 2.07 (m, 1H), 2.04 – 1.97 (m, 1H), 1.87 – 1.81 (m, 1H), 1.30 – 1.10 (m, 4H), 0.93 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₁FN₉O₁₀S⁺ 1034.4241, found 1034.4243.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((3-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)propyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (101).

White solid (6.9 mg, 66%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.06 (s, 1H), 7.77 (s, 1H), 7.70 (dd, J = 8.0, 1.7 Hz, 1H), 7.48 – 7.45 (m, 2H), 7.44 – 7.39 (m, 3H), 7.37 – 7.31 (m, 2H), 7.18 (s, 1H), 5.70 (t, J = 8.0 Hz, 1H), 5.33 (dd, J = 8.1, 6.0 Hz, 1H), 4.75 – 4.70 (m, 1H), 4.63 (dd, J = 9.2, 7.6 Hz, 1H), 4.45 – 4.41 (m, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.25 – 3.18 (m, 1H), 3.17 – 2.91 (m, 11H), 2.85 (dd, J = 14.1, 6.0 Hz, 1H), 2.75 (dd, J = 14.1, 8.2 Hz, 1H), 2.69 – 2.62 (m, 1H), 2.44 (s, 3H), 2.25 – 2.09 (m, 2H), 1.98 – 1.92 (m, 1H), 1.64 – 1.56 (m, 2H), 1.40 – 1.23 (m, 4H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₃FN₉O₁₀S⁺ 1048.4397, found 1048.4401.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((4-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)butyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (102).

White solid (7.3 mg, 69%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.03 (s, 1H), 7.75 (s, 1H), 7.69 (dd, J = 7.9, 1.7 Hz, 1H), 7.50 – 7.38 (m, 5H), 7.37 – 7.32 (m, 3H), 7.15 (s, 1H), 5.68 (t, J = 7.9 Hz, 1H), 5.30 (dd, J = 8.3, 6.1 Hz, 1H), 4.74 – 4.71 (m, 1H), 4.60 – 4.55 (m, 1H), 4.45 – 4.41 (m, 1H), 3.82 (d, J = 11.1 Hz, 1H), 3.75 (dd, J = 11.1, 3.8 Hz, 1H), 3.27 (t, J = 6.7 Hz, 2H), 3.19 – 2.92 (m, 11H), 2.82 (dd, J = 14.1, 6.1 Hz, 1H), 2.73 (dd, J = 14.1, 8.4 Hz, 1H), 2.68 – 2.61 (m, 1H), 2.44 (s, 3H), 2.21 – 2.09 (m, 2H), 1.98 – 1.92 (m, 1H), 1.47 – 1.23 (m, 7H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₅FN₉O₁₀S⁺ 1062.4554, found 1062.4550.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((5-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)pentyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (103).

White solid (8.1 mg, 75%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.88 (s, 1H), 7.67 (s, 1H), 7.62 (dd, J = 7.9, 1.7 Hz, 1H), 7.37 (dd, J = 9.4, 3.4 Hz, 1H), 7.34 – 7.21 (m, 7H), 7.06 (s, 1H), 5.59 (t, J = 8.0 Hz, 1H), 5.17 (dd, J = 8.5, 6.0 Hz, 1H), 4.65 – 4.60 (m, 1H), 4.48 (dd, J = 9.3, 7.7 Hz, 1H), 4.36 – 4.31 (m, 1H), 3.72 (d, J = 10.9 Hz, 1H), 3.66 (dd, J = 11.1, 3.8 Hz, 1H), 3.16 (t, J = 7.1 Hz, 2H), 3.07 – 2.82 (m, 11H), 2.72 (dd, J = 14.1, 6.0 Hz, 1H), 2.61 (dd, J = 14.1, 8.5 Hz, 1H), 2.58 – 2.50 (m, 1H), 2.36 (s, 3H), 2.11 – 2.00 (m, 2H), 1.88 – 1.82 (m, 1H), 1.45 – 1.38 (m, 2H), 1.33 – 1.05 (m, 7H), 0.95 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₀S⁺ 1076.4710, found 1076.4715.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((6-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)hexyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (104).

White solid (7.8 mg, 72%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.72 (dd, J = 8.1, 1.7 Hz, 1H), 7.47 (dd, J = 9.4, 3.4 Hz, 1H), 7.44 – 7.41 (m, 4H), 7.37 – 7.32 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 8.0 Hz, 1H), 5.29 (dd, J = 8.4, 5.8 Hz, 1H), 4.73 (dd, J = 9.3, 1.2 Hz, 1H), 4.62 – 4.56 (m, 1H), 4.45 – 4.43 (m, 1H), 3.86 – 3.79 (m, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.26 (t, J = 7.1 Hz, 2H), 3.15 – 3.08 (m, 4H), 3.04 – 2.95 (m, 5H), 2.83 (dd, J = 14.1, 5.9 Hz, 1H), 2.73 (dd, J = 14.1, 8.4 Hz, 1H), 2.67 – 2.62 (m, 1H), 2.47 (s, 3H), 2.20 – 2.16 (m, 1H), 2.14 – 2.09 (m, 1H), 1.98 – 1.92 (m, 1H), 1.51 (q, J = 7.3 Hz, 2H), 1.41 – 1.24 (m, 9H), 1.20 – 1.15 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₆₉FN₉O₁₀S⁺ 1090.4867, found 1090.4870.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((7-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)heptyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (105).

White solid (7.4 mg, 67%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.16 (s, 1H), 7.77 (s, 1H), 7.72 (dd, J = 7.9, 1.7 Hz, 1H), 7.50 – 7.45 (m, 1H), 7.45 – 7.41 (m, 4H), 7.37 – 7.32 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.30 (dd, J = 8.4, 5.9 Hz, 1H), 4.75 – 4.72 (m, 1H), 4.58 (dd, J = 9.3, 7.7 Hz, 1H), 4.45 – 4.43 (m, 1H), 3.82 (d, J = 11.2 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.35 (s, 1H), 3.29 – 3.26 (m, 2H), 3.15 – 3.08 (m, 4H), 3.04 – 2.95 (m, 4H), 2.83 (dd, J = 14.1, 5.9 Hz, 1H), 2.73 (dd, J = 14.0, 8.5 Hz, 1H), 2.67 – 2.62 (m, 1H), 2.49 (s, 3H), 2.21 – 2.15 (m, 1H), 2.15 – 2.10 (m, 1H), 1.98 – 1.93 (m, 1H), 1.54 – 1.49 (m, 2H), 1.39 – 1.22 (m, 11H), 1.14 – 1.12 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₀S⁺ 1104.5023, found 1104.5023.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((8-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)octyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (106).

White solid (7 mg, 63%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.08 (s, 1H), 7.78 (s, 1H), 7.75 – 7.70 (m, 1H), 7.50 – 7.45 (m, 1H), 7.45 – 7.40 (m, 4H), 7.37 – 7.32 (m, 3H), 7.14 (s, 1H), 5.69 (t, J = 8.0 Hz, 1H), 5.30 (dd, J = 8.5, 5.8 Hz, 1H), 4.76 – 4.72 (m, 1H), 4.58 (dd, J = 9.3, 7.7 Hz, 1H), 4.45 – 4.43 (m, 1H), 3.82 (d, J = 11.0 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.35 (s, 1H), 3.30 – 3.25 (m, 2H), 3.15 – 3.06 (m, 4H), 3.02 – 2.96 (m, 4H), 2.83 (dd, J = 14.0, 5.8 Hz, 1H), 2.72 (dd, J = 14.0, 8.6 Hz, 1H), 2.68 – 2.62 (m, 1H), 2.48 (s, 3H), 2.21 – 2.17 (m, 1H), 2.15 – 2.09 (m, 1H), 1.99 – 1.93 (m, 1H), 1.53 – 1.48 (m, 2H), 1.39 – 1.16 (m, 13H), 1.13 – 1.09 (m, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₃FN₉O₁₀S⁺ 1118.5180, found 1118.5182.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((9-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)nonyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (107).

White solid (7.7 mg, 68%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.10 (s, 1H), 7.79 (s, 1H), 7.73 (dd, J = 7.8, 1.7 Hz, 1H), 7.49 – 7.46 (m, 1H), 7.45 – 7.40 (m, 4H), 7.37 – 7.32 (m, 3H), 7.13 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.30 (dd, J = 8.5, 5.8 Hz, 1H), 4.76 – 4.71 (m, 1H), 4.58 (dd, J = 9.3, 7.7 Hz, 1H), 4.45 – 4.43 (m, 1H), 3.83 (d, J = 11.0 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.34 (d, J = 15.8 Hz, 3H), 3.15 – 3.07 (m, 4H), 3.02 – 2.96 (m, 4H), 2.83 (dd, J = 14.0, 5.8 Hz, 1H), 2.73 (dd, J = 14.0, 8.5 Hz, 1H), 2.67 – 2.62 (m, 1H), 2.48 (s, 3H), 2.21 – 2.17 (m, 1H), 2.16 – 2.09 (m, 1H), 1.99 – 1.93 (m, 1H), 1.56 – 1.51 (m, 2H), 1.40 – 1.22 (m, 11H), 1.18 – 1.16 (m, 4H), 1.08 (d, J = 8.0 Hz, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₀H₇₅FN₉O₁₀S⁺ 1132.5336, found 1132.5336.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((10-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)decyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (108).

White solid (8.1 mg, 71%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.03 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 8.0, 1.7 Hz, 1H), 7.51 – 7.39 (m, 5H), 7.38 – 7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.30 (dd, J = 8.5, 5.8 Hz, 1H), 4.76 – 4.72 (m, 1H), 4.58 (dd, J = 9.2, 7.5 Hz, 1H), 4.46 – 4.42 (m, 1H), 3.83 (d, J = 11.0 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.36 – 3.32 (m, 2H), 3.16 – 2.92 (m, 11H), 2.83 (dd, J = 14.0, 5.8 Hz, 1H), 2.77 – 2.70 (m, 1H), 2.69 – 2.61 (m, 1H), 2.48 (s, 3H), 2.23 – 2.16 (m, 1H), 2.16 – 2.07 (m, 1H), 1.98 – 1.92 (m, 1H), 1.56 (p, J = 7.2 Hz, 2H), 1.38 – 1.13 (m, 15H), 1.10 – 1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₁H₇₇FN₉O₁₀S⁺ 1146.5493, found 1146.5490.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((11-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)undecyl)-carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (109).

White solid (8.9 mg, 77%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.11 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 8.0, 1.7 Hz, 1H), 7.51 – 7.40 (m, 5H), 7.38 – 7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.31 (dd, J = 8.5, 5.8 Hz, 1H), 4.76 – 4.71 (m, 1H), 4.58 (dd, J = 9.2, 7.6 Hz, 1H), 4.46 – 4.42 (m, 1H), 3.83 (d, J = 11.1 Hz, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.36 – 3.32 (m, 2H), 3.16 – 2.94 (m, 11H), 2.84 (dd, J = 14.0, 5.8 Hz, 1H), 2.73 (dd, J = 14.0, 8.6 Hz, 1H), 2.68 – 2.61 (m, 1H), 2.49 (s, 3H), 2.23 – 2.16 (m, 1H), 2.15 – 2.07 (m, 1H), 1.99 – 1.92 (m, 1H), 1.58 (p, J = 7.2 Hz, 2H), 1.41 – 1.25 (m, 9H), 1.23 – 1.12 (m, 8H), 1.11 – 1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₂H₇₉FN₉O₁₀S⁺ 1160.5649, found 1160.5652.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((12-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)dodecyl)-carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (110).

White solid (8.6 mg, 73%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 7.9, 1.7 Hz, 1H), 7.52 – 7.40 (m, 5H), 7.38 – 7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.31 (dd, J = 8.5, 5.8 Hz, 1H), 4.74 (dd, J = 9.3, 1.3 Hz, 1H), 4.58 (dd, J = 9.2, 7.5 Hz, 1H), 4.47 – 4.42 (m, 1H), 3.85 – 3.80 (m, 1H), 3.76 (dd, J = 11.1, 3.8 Hz, 1H), 3.37 – 3.32 (m, 2H), 3.16 – 2.94 (m, 11H), 2.84 (dd, J = 14.0, 5.8 Hz, 1H), 2.74 (dd, J = 14.0, 8.5 Hz, 1H), 2.68 – 2.60 (m, 1H), 2.48 (s, 3H), 2.23 – 2.16 (m, 1H), 2.15 – 2.07 (m, 1H), 1.99 – 1.92 (m, 1H), 1.59 (p, J = 7.2 Hz, 2H), 1.42 – 1.13 (m, 19H), 1.12 – 1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₆₃H₈₁FN₉O₁₀S⁺ 1174.5806, found 1174.5809.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (111).

White solid (8 mg, 76%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.12 (s, 1H), 7.75 (s, 1H), 7.70 (dd, J = 8.0, 1.7 Hz, 1H), 7.54 – 7.44 (m, 3H), 7.37 – 7.32 (m, 3H), 7.18 (s, 1H), 6.96 (dd, J = 5.4, 1.6 Hz, 2H), 5.69 (t, J = 7.9 Hz, 1H), 4.75 – 4.71 (m, 1H), 4.66 – 4.43 (m, 6H), 3.87 – 3.75 (m, 2H), 3.60 – 3.48 (m, 4H), 3.18 – 2.93 (m, 8H), 2.70 – 2.63 (m, 1H), 2.46 (s, 3H), 2.21 – 2.10 (m, 2H), 2.09 – 2.00 (m, 2H), 1.41 – 1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₁FN₉O₁₁S⁺ 1050.4190, found 1050.4188.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((4-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)butyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (112).

White solid (8.4 mg, 78%). ¹H NMR (600 MHz, Methanol-d₄) δ 8.98 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 7.9, 1.7 Hz, 1H), 7.47 – 7.39 (m, 3H), 7.38 – 7.30 (m, 3H), 7.15 (s, 1H), 7.06 (dd, J = 7.7, 1.6 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 5.69 (t, J = 7.9 Hz, 1H), 4.71 – 4.67 (m, 1H), 4.63 – 4.53 (m, 4H), 4.47 – 4.40 (m, 2H), 3.78 (d, J = 11.1 Hz, 1H), 3.73 (dd, J = 11.1, 3.8 Hz, 1H), 3.37 – 3.32 (m, 2H), 3.17 – 2.93 (m, 8H), 2.69 – 2.61 (m, 1H), 2.47 (s, 3H), 2.19 – 2.09 (m, 2H), 2.05 – 1.99 (m, 1H), 1.65 – 1.57 (m, 4H), 1.38 – 1.22 (m, 6H), 0.98 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₅FN₉O₁₁S⁺ 1078.4503, found 1078.4501.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((5-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)pentyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (113).

White solid (7.6 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.02 (s, 1H), 7.80 (s, 1H), 7.74 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 – 7.41 (m, 3H), 7.38 – 7.33 (m, 3H), 7.16 (s, 1H), 7.08 (dd, J = 7.7, 1.6 Hz, 1H), 6.96 (d, J = 1.6 Hz, 1H), 5.69 (t, J = 7.9 Hz, 1H), 4.74 – 4.71 (m, 1H), 4.64 – 4.52 (m, 4H), 4.48 (dd, J = 4.8, 2.5 Hz, 1H), 4.43 (d, J = 15.0 Hz, 1H), 3.84 (d, J = 10.9 Hz, 1H), 3.77 (dd, J = 11.0, 3.8 Hz, 1H), 3.40 – 3.34 (m, 2H), 3.18 – 2.94 (m, 8H), 2.68 – 2.62 (m, 1H), 2.50 (s, 3H), 2.23 – 2.03 (m, 3H), 1.65 – 1.57 (m, 4H), 1.42 – 1.24 (m, 8H), 1.00 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₆H₆₇FN₉O₁₁S⁺ 1092.4659, found 1092.4658.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((6-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)hexyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (114).

White solid (7.9 mg, 71%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.02 (s, 1H), 7.79 (s, 1H), 7.73 (dd, J = 7.9, 1.7 Hz, 1H), 7.49 – 7.43 (m, 2H), 7.40 (dd, J = 7.9, 1.6 Hz, 1H), 7.38 – 7.31 (m, 3H), 7.14 (s, 1H), 7.07 (dd, J = 7.7, 1.6 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 5.69 (t, J = 7.9 Hz, 1H), 4.72 – 4.69 (m, 1H), 4.62 – 4.53 (m, 4H), 4.48 – 4.42 (m, 2H), 3.82 (d, J = 11.1 Hz, 1H), 3.75 (dd, J = 11.1, 3.8 Hz, 1H), 3.36 – 3.32 (m, 2H), 3.27 (t, J = 7.0 Hz, 2H), 3.16 – 2.91 (m, 8H), 2.68 – 2.61 (m, 1H), 2.48 (s, 3H), 2.21 – 2.16 (m, 1H), 2.14 – 2.09 (m, 1H), 2.07 – 2.01 (m, 1H), 1.57 – 1.50 (m, 4H), 1.40 – 1.23 (m, 8H), 0.99 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₆₉FN₉O₁₁S⁺ 1106.4816, found 1106.4813.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((7-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)heptyl)carbamoyl)-yl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (115).

White solid (7.3 mg, 65%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.07 (s, 1H), 7.80 (s, 1H), 7.74 (dd, J = 8.0, 1.7 Hz, 1H), 7.50 – 7.46 (m, 2H), 7.43 (dd, J = 7.9, 1.6 Hz, 1H), 7.40 – 7.33 (m, 3H), 7.16 (s, 1H), 7.09 (dd, J = 7.8, 1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 5.70 (t, J = 7.9 Hz, 1H), 4.76 – 4.71 (m, 1H), 4.64 – 4.54 (m, 4H), 4.52 – 4.45 (m, 2H), 3.85 (d, J = 11.1 Hz, 1H), 3.79 (dd, J = 11.0, 3.8 Hz, 1H), 3.37 – 3.34 (m, 2H), 3.30 – 3.26 (m, 2H), 3.18 – 2.94 (m, 8H), 2.70 – 2.63 (m, 1H), 2.50 (s, 3H), 2.24 – 2.19 (m, 1H), 2.17 – 2.04 (m, 2H), 1.62 – 1.51 (m, 4H), 1.41 – 1.24 (m, 10H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₁FN₉O₁₁S⁺ 1120.4972, found 1120.4970.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((8-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)octyl)carbamoyl)-2,3-hydro-1H-inden-1-yl)isoxazole-3-carboxamide (116).

White solid (7.9 mg, 70%). ¹H NMR (600 MHz, Methanol-d₄) δ 9.01 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 8.0, 1.7 Hz, 1H), 7.50 – 7.45 (m, 2H), 7.41 (dd, J = 7.9, 1.6 Hz, 1H), 7.38 – 7.31 (m, 3H), 7.14 (s, 1H), 7.08 (dd, J = 7.8, 1.6 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 5.69 (t, J = 7.9 Hz, 1H), 4.74 – 4.70 (m, 1H), 4.62 – 4.53 (m, 4H), 4.50 – 4.43 (m, 2H), 3.83 (d, J = 10.8 Hz, 1H), 3.77 (dd, J = 11.1, 3.8 Hz, 1H), 3.36 – 3.32 (m, 2H), 3.25 (t, J = 7.0 Hz, 2H), 3.16 – 2.92 (m, 8H), 2.68 – 2.61 (m, 1H), 2.48 (s, 3H), 2.23 – 2.17 (m, 1H), 2.15 – 2.02 (m, 2H), 1.60 – 1.47 (m, 4H), 1.39 – 1.22 (m, 12H), 1.00 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₉H₇₃FN₉O₁₁S⁺ 1134.5129, found 1134.5129.

(2S,4R)-1-((S)-2-azido-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methyl-thiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (142).

To a solution of the (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrolidine-2-carboxamide (444.59 mg, 1.0 mmol, 1.0 eq) and N,N-dimethyl-4-aminopyridine (DMAP) (366.51 mg, 3.0 mmol, 3.0 eq) in DCM (2.0 mL) at 0 °C, a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP) (327.90 mg, 1.15 mmol, 1.15 eq) in DCM (2.0 mL) was added at 0 °C. The reaction mixture was stirred for 30 min at room temperature. Upon completion, the reaction was quenched with saturated aqueous sodium bicarbonate (20.0 mL) and extracted with dichloromethane (3 × 30.0 mL). The combined organic layers were washed successively with water (30.0 mL) and brine (30.0 mL), then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to afford the crude product and purified by using reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to yield the intermediate 142 as a white solid (329.62 mg, 70% yield). ¹H NMR (400 MHz, Methanol-d₄) δ 8.65 (s, 1H), 7.34 (t, J = 6.3 Hz, 4H), 4.98 (q, J = 6.9 Hz, 1H), 4.60 (t, J = 7.8 Hz, 1H), 4.44 (q, J = 3.9 Hz, 1H), 3.68 (s, 1H), 3.63 – 3.56 (m, 2H), 2.45 (s, 3H), 2.21 – 2.13 (m, 1H), 2.09 – 2.03 (m, 1H), 1.45 (d, J = 7.0 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₃H₃₁N₆O₃S⁺ 471.2173, found 471.2175.

(2S,4R)-1-((S)-2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (130).

To a solution of the intermediate 142 (23.5 mg, 0.05 mmol, 1.0 eq), commercially available N-Boc-propargylamine (9.3 mg, 0.06 mg, 1.2 eq) in tBuOH (1.0 mL) and water (1.0 mL) was added CuSO₄ (12.0 mg, 0.075 mmol, 1.5 eq) and (+)-Sodium L-ascorbate (29.7 mg, 0.15 mmol). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-70% acetonitrile / 0.1% TFA in H₂O) to afford intermediate as white solid in TFA salt form. The obtained intermediate was dissolved in DCM (0.5 mL) followed by TFA (0.5 mL). After being stirred for 1 h at room temperature, the reaction mixture was concentrated and purified by preparative HPLC (5%-70% acetonitrile / 0.1% TFA in H₂O) to afford intermediate 130 as white solid in TFA salt form. (13.92 mg, 53% yield for two steps). ¹H NMR (400 MHz, Methanol-d₄) δ 8.85 (s, 1H), 8.27 (s, 1H), 7.35 (s, 4H), 5.38 (s, 1H), 5.03 (q, J = 6.9 Hz, 1H), 4.51 (t, J = 8.4 Hz, 1H), 4.42 – 4.38 (m, 1H), 4.19 (s, 2H), 3.75 – 3.70 (m, 2H), 2.48 (s, 3H), 2.16 (dd, J = 13.4, 7.8 Hz, 1H), 2.05 – 1.98 (m, 1H), 1.48 (d, J = 7.0 Hz, 3H), 1.01 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₆H₃₆N₇O₃S⁺ 526.2595, found 526.2592.

Linkers 131 to 139 were synthesized following the same procedure for preparing Linker 130 (Schem 3B).

(2S,4R)-1-((S)-2-(4-(2-aminoethyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (131).

White solid, 13.48 mg, 50% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.81 (s, 1H), 8.04 (s, 1H), 7.35 (s, 4H), 5.35 (s, 1H), 5.02 (q, J = 6.9 Hz, 1H), 4.49 (t, J = 8.4 Hz, 1H), 4.40 (d, J = 4.3 Hz, 1H), 3.74 – 3.66 (m, 2H), 3.29 – 3.18 (m, 2H), 3.07 – 2.99 (m, 2H), 2.47 (s, 3H), 2.15 (dd, J = 13.5, 7.8 Hz, 1H), 2.05 – 1.98 (m, 1H), 1.48 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₇H₃₈N₇O₃S⁺ 540.2751, found 540.2749.

(2S,4R)-1-((S)-2-(4-(3-aminopropyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutano-yl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (132).

White solid, 14.67 mg, 53% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.80 (s, 1H), 7.95 (s, 1H), 7.34 (s, 4H), 5.33 (s, 1H), 5.02 (q, J = 6.9 Hz, 1H), 4.47 (t, J = 8.3 Hz, 1H), 4.40 (d, J = 4.7 Hz, 1H), 3.74 – 3.63 (m, 2H), 2.88 (t, J = 7.6 Hz, 2H), 2.75 (d, J = 7.4 Hz, 2H), 2.47 (s, 3H), 2.12 (dd, J = 13.5, 7.8 Hz, 1H), 2.06 – 2.01 (m, 1H), 2.00 – 1.94 (m, 2H), 1.47 (d, J = 7.0 Hz, 3H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₈H₄₀N₇O₃S⁺ 554.2908, found 554.2911.

(2S,4R)-1-((S)-2-(4-(4-aminobutyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrolidine-2-carboxamide (133).

White solid, 14.49 mg, 51% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.78 (s, 1H), 7.93 (s, 1H), 7.34 (s, 4H), 5.34 (s, 1H), 5.03 (t, J = 6.9 Hz, 1H), 4.48 (t, J = 8.3 Hz, 1H), 4.44 – 4.39 (m, 1H), 3.75 – 3.67 (m, 2H), 2.84 (t, J = 7.3 Hz, 2H), 2.74 – 2.64 (m, 2H), 2.47 (s, 3H), 2.14 (dd, J = 13.4, 7.8 Hz, 1H), 2.05 – 1.98 (m, 1H), 1.73 – 1.66 (m, 2H), 1.62 – 1.56 (m, 2H), 1.48 (d, J = 7.0 Hz, 3H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₉H₄₂N₇O₃S⁺ 568.3064, found 568.3067.

(2S,4R)-1-((S)-2-(4-(5-aminopentyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (134).

White solid, 14.85 mg, 51% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.70 (s, 1H), 7.93 (s, 1H), 7.36 – 7.30 (m, 4H), 5.36 (s, 1H), 5.02 (q, J = 6.9 Hz, 1H), 4.48 (t, J = 8.4 Hz, 1H), 4.42 (s, 1H), 3.73 (d, J = 2.8 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H), 2.68 (q, J = 7.8 Hz, 2H), 2.46 (s, 3H), 2.15 (dd, J = 13.6, 7.8 Hz, 1H), 2.04 – 1.97 (m, 1H), 1.70 – 1.62 (m, 2H), 1.61 – 1.55 (m, 2H), 1.48 (d, J = 7.0 Hz, 3H), 1.31 (q, J = 7.6 Hz, 2H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₀H₄₄N₇O₃S⁺ 582.3221, found 582.3225.

(2S,4R)-1-((S)-2-(4-(6-aminohexyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (135).

White solid, 16.10 mg, 54% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.78 (s, 1H), 7.92 (s, 1H), 7.34 (s, 4H), 5.36 (s, 1H), 5.02 (q, J = 6.9 Hz, 1H), 4.48 (t, J = 8.3 Hz, 1H), 4.44 – 4.40 (m, 1H), 3.79 – 3.70 (m, 2H), 2.79 (t, J = 7.6 Hz, 2H), 2.68 – 2.62 (m, 2H), 2.47 (s, 3H), 2.14 (dd, J = 13.5, 7.8 Hz, 1H), 2.05 – 1.98 (m, 1H), 1.66 – 1.60 (m, 2H), 1.57 (dd, J = 12.4, 5.4 Hz, 2H), 1.48 (d, J = 7.0 Hz, 3H), 1.34 – 1.27 (m, 4H), 1.02 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₁H₄₆N₇O₃S⁺ 596.3377, found 596.3372.

(2S,4R)-1-((S)-2-(4-(7-aminoheptyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (136).

White solid, 15.87 mg, 52% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.86 (s, 1H), 7.99 (s, 1H), 7.34 (d, J = 2.2 Hz, 4H), 5.38 (s, 1H), 5.03 (q, J = 6.9 Hz, 1H), 4.50 (t, J = 8.3 Hz, 1H), 4.43 (s, 1H), 3.80 (d, J = 10.9 Hz, 1H), 3.73 (dd, J = 10.9, 3.4 Hz, 1H), 2.80 (t, J = 7.6 Hz, 2H), 2.73 – 2.60 (m, 2H), 2.47 (s, 3H), 2.16 (dd, J = 13.5, 7.7 Hz, 1H), 2.07 – 2.00 (m, 1H), 1.69 – 1.61 (m, 2H), 1.54 (d, J = 7.7 Hz, 2H), 1.48 (d, J = 7.0 Hz, 3H), 1.28 (s, 6H), 1.03 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₂H₄₈N₇O₃S⁺ 610.3534, found 610.3529.

(2S,4R)-1-((S)-2-(4-(8-aminooctyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (137).

White solid, 15.61 mg, 50% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.70 (s, 1H), 7.90 (s, 1H), 7.32 (d, J = 1.8 Hz, 4H), 5.35 (s, 1H), 5.01 (q, J = 6.9 Hz, 1H), 4.47 (t, J = 8.3 Hz, 1H), 4.41 (d, J = 4.8 Hz, 1H), 3.78 – 3.70 (m, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.65 – 2.61 (m, 2H), 2.45 (s, 3H), 2.13 (dd, J = 13.5, 7.8 Hz, 1H), 2.06 – 1.99 (m, 1H), 1.62 – 1.53 (m, 4H), 1.47 (d, J = 7.0 Hz, 3H), 1.25 (s, 8H), 1.01 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₃H₅₀N₇O₃S⁺ 624.3690, found 624.3691.

(2S,4R)-1-((S)-2-(4-(9-aminononyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (138).

White solid, 17.24 mg, 54% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.64 (s, 1H), 7.87 (s, 1H), 7.34 – 7.29 (m, 4H), 5.01 (t, J = 6.9 Hz, 1H), 4.44 (dd, J = 19.3, 11.0 Hz, 2H), 3.77 – 3.68 (m, 2H), 2.79 (t, J = 7.7 Hz, 2H), 2.62 (t, J = 7.4 Hz, 2H), 2.44 (s, 3H), 2.13 – 2.02 (m, 2H), 1.62 – 1.50 (m, 5H), 1.46 (d, J = 7.0 Hz, 3H), 1.28 – 1.21 (m, 10H), 0.99 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₄H₅₂N₇O₃S⁺ 638.3847, found 638.3850.

(2S,4R)-1-((S)-2-(4-(10-aminodecyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (139).

White solid, 15.98 mg, 49% yield for two steps. ¹H NMR (400 MHz, Methanol-d₄) δ 8.74 (s, 1H), 7.89 (s, 1H), 7.32 (s, 4H), 5.01 (q, J = 6.9 Hz, 1H), 4.47 (t, J = 8.2 Hz, 1H), 4.43 – 4.38 (m, 1H), 3.79 (d, J = 11.1 Hz, 1H), 3.69 (dd, J = 11.0, 3.6 Hz, 1H), 2.80 (t, J = 7.7 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.45 (s, 3H), 2.13 – 2.01 (m, 2H), 1.56 (q, J = 7.9 Hz, 5H), 1.46 (d, J = 7.0 Hz, 3H), 1.28 – 1.19 (m, 12H), 0.99 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₅H₅₄N₇O₃S⁺ 652.4003, found 652.4007.

Method D: Preparation of ENL SAR Putative degraders 117–126.

To a solution of precursor 129 (Scheme 3A) (4.35 mg, 0.01 mmol) in DMSO (1 mL) were added triazole-based VHL ligands (130-139) (0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixtures were purified by preparative HPLC (5%-60% acetonitrile / 0.1% TFA in H₂O) to afford the final compounds 117-126 as white solids.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-(((1-((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)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (117).

White solid (5.47 mg, yield 58%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.00 (s, 1H), 8.22 (s, 1H), 7.83 – 7.76 (m, 2H), 7.46 – 7.41 (m, 4H), 7.40 – 7.31 (m, 4H), 7.15 (s, 1H), 5.70 (t, J = .0 Hz, 1H), 5.50 (s, 1H), 5.01 (d, J = 7.0 Hz, 1H), 4.64 (d, J = 6.1 Hz, 2H), 4.54 – 4.49 (m, 1H), 4.42 (s, 1H), 3.83 (dd, J = 11.0, 3.8 Hz, 1H), 3.73 (d, J = 11.1 Hz, 1H), 3.17 – 3.06 (m, 4H), 2.98 (t, J = 8.4 Hz, 4H), 2.69 – 2.61 (m, 1H), 2.49 (s, 3H), 2.22 – 2.08 (m, 2H), 1.99 – 1.90 (m, 1H), 1.66 – 1.60 (m, 1H), 1.50 (d, J = 7.0 Hz, 2H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₄₉H₅₅N₁₀O₈S⁺ 943.3920, found 943.3921.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((2-(1-((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)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (118).

White solid (5.16 mg, yield 54%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.04 (s, 1H), 8.11 (s, 1H), 7.75 – 7.69 (m, 2H), 7.44 (dd, J = 7.5, 5.0 Hz, 4H), 7.38 – 7.31 (m, 4H), 7.18 (s, 1H), 5.69 (t, J = 7.8 Hz, 1H), 5.45 (s, 1H), 5.03 (d, J = 7.0 Hz, 1H), 4.53 (t, J = 8.4 Hz, 1H), 4.42 (s, 1H), 3.81 (dd, J = 11.1, 3.8 Hz, 1H), 3.72 – 3.63 (m, 3H), 3.16 – 3.08 (m, 4H), 3.00 (dd, J = 15.8, 8.9 Hz, 6H), 2.68 – 2.60 (m, 1H), 2.50 (s, 3H), 2.20 (dd, J = 13.2, 7.8 Hz, 1H), 2.15 – 2.08 (m, 1H), 1.97 – 1.90 (m, 1H), 1.66 – 1.60 (m, 1H), 1.50 (d, J = 7.0 Hz, 2H), 1.00 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₀H₅₇N₁₀O₈S⁺ 957.4076, found 957.4074.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((3-(1-((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)-1H-1,2,3-triazol-4-yl)propyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (119).

White solid (5.44 mg, yield 56%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.94 (s, 1H), 8.10 (s, 1H), 7.80 – 7.74 (m, 2H), 7.47 – 7.42 (m, 4H), 7.38 – 7.31 (m, 4H), 7.16 (s, 1H), 5.70 (t, J = 7.9 Hz, 1H), 5.48 (s, 1H), 5.02 (d, J = 7.0 Hz, 1H), 4.54 (dd, J = 9.9, 7.0 Hz, 1H), 4.43 (s, 1H), 3.84 (dd, J = 11.1, 3.8 Hz, 1H), 3.72 (d, J = 11.1 Hz, 1H), 3.42 – 3.37 (m, 2H), 3.10 (s, 4H), 2.97 (q, J = 8.7 Hz, 4H), 2.78 (t, J = 7.6 Hz, 2H), 2.69 – 2.61 (m, 1H), 2.49 (s, 3H), 2.22 – .09 (m, 2H), 1.99 – 1.93 (m, 3H), 1.51 (d, J = 7.0 Hz, 3H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₁H₅₉N₁₀O₈S⁺ 971.4233, found 971.4236.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((4-(1-((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)-1H-1,2,3-triazol-4-yl)butyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (120).

White solid (5.91 mg, yield 60%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.98 (s, 1H), 8.06 (s, 1H), 7.79 – 7.70 (m, 2H), 7.43 (dd, J = 9.8, 3.2 Hz, 5H), 7.37 – 7.31 (m, 3H), 7.16 (s, 1H), 5.68 (q, J = 7.7 Hz, 1H), 5.47 (s, 1H), 5.03 (q, J = 7.0 Hz, 1H), 4.55 (dd, J = 9.0, 7.4 Hz, 1H), 4.44 (s, 1H), 3.83 (dd, J = 11.1, 3.8 Hz, 1H), 3.76 – 3.65 (m, 1H), 3.39 (t, J = 6.8 Hz, 2H), 3.17 – 3.05 (m, 4H), 2.98 (q, J = 8.2 Hz, 4H), 2.75 (t, J = 7.3 Hz, 2H), 2.68 – 2.60 (m, 1H), 2.49 (s, 3H), 2.21 (dd, J = 13.2, 7.8 Hz, 1H), 2.14 – 2.06 (m, 1H), 1.98 – 1.92 (m, 1H), 1.79 – 1.69 (m, 2H), 1.68 – 1.60 (m, 3H), 1.52 (d, J = 7.0 Hz, 2H), 1.04 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₂H₆₁N₁₀O₈S⁺ 985.4389, found 985.4387.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((5-(1-((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)-1H-1,2,3-triazol-4-yl)pentyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (121).

White solid (5.30 mg, yield 53%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.97 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.76 – 7.72 (m, 1H), 7.43 (dd, J = 8.4, 5.4 Hz, 5H), 7.37 – 7.32 (m, 3H), 7.16 (s, 1H), 5.69 (t, J = 7.7 Hz, 1H), 5.47 (s, 1H), 5.04 (t, J = 7.0 Hz, 1H), 4.54 (t, J = 8.4 Hz, 1H), 4.43 (s, 1H), 3.83 (dd, J = 11.0, 3.8 Hz, 1H), 3.73 (d, J = 11.1 Hz, 1H), 3.36 (d, J = 6.9 Hz, 1H), 3.15 – 3.06 (m, 4H), 2.98 (t, J = 7.8 Hz, 4H), 2.73 (d, J = 7.5 Hz, 2H), 2.68 – 2.62 (m, 1H), 2.49 (s, 3H), 2.20 (dd, J = 13.2, 7.7 Hz, 1H), 2.14 – 2.07 (m, 1H), 1.98 – 1.91 (m, 1H), 1.71 (t, J = 7.5 Hz, 2H), 1.67 – 1.60 (m, 3H), 1.52 (d, J = 7.0 Hz, 3H), 1.42 (q, J = 8.1, 7.7 Hz, 2H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₃H₆₃N₁₀O₈S⁺ 999.4546, found 999.4544.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((6-(1-((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)-1H-1,2,3-triazol-4-yl)hexyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (122).

White solid (5.78 mg, yield 57%). ¹H NMR (400 MHz, Methanol-d₄) δ 8.94 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.73 (dd, J = 7.9, 1.7 Hz, 1H), 7.46 – 7.40 (m, 5H), 7.37 – 7.34 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 8.0 Hz, 1H), 5.48 (s, 1H), 5.03 (t, J = 6.9 Hz, 1H), 4.58 – 4.52 (m, 1H), 4.44 (s, 1H), 3.88 – 3.81 (m, 1H), 3.73 (d, J = 11.2 Hz, 1H), 3.35 – 3.34 (m, 1H), 3.15 – 3.07 (m, 4H), 3.02 – 2.95 (m, 4H), 2.70 (t, J = 7.5 Hz, 2H), 2.66 – 2.62 (m, 1H), 2.48 (s, 3H), 2.20 (dd, J = 13.2, 7.7 Hz, 1H), 2.14 – 2.08 (m, 1H), 1.99 – 1.92 (m, 1H), 1.70 – 1.65 (m, 2H), 1.64 – 1.56 (m, 3H), 1.52 (d, J = 7.0 Hz, 3H), 1.42 – 1.37 (m, 4H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₄H₆₅N₁₀O₈S⁺ 1013.4702, found 1013.4700.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((7-(1-((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)-1H-1,2,3-triazol-4-yl)heptyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (123).

White solid (5.65 mg, yield 55%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.16 (s, 1H), 8.06 (s, 1H), 7.77 (s, 1H), 7.73 (dd, J = 7.9, 1.7 Hz, 1H), 7.49 – 7.38 (m, 5H), 7.34 (q, J = 7.9, 7.3 Hz, 3H), 7.15 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.49 (s, 1H), 5.03 (d, J = 7.0 Hz, 1H), 4.58 – 4.53 (m, 1H), 4.44 (s, 1H), 3.83 (dd, J = 11.1, 3.7 Hz, 1H), 3.77 – 3.67 (m, 1H), 3.35 (d, J = 7.1 Hz, 1H), 3.18 – 3.05 (m, 4H), 2.98 (q, J = 8.2 Hz, 4H), 2.70 (t, J = 7.5 Hz, 2H), 2.67 – 2.61 (m, 1H), 2.51 (s, 3H), 2.23 – 2.16 (m, 1H), 2.14 – 2.08 (m, 1H), 2.03 – 1.93 (m, 1H), 1.70 – 1.63 (m, 3H), 1.62 – 1.56 (m, 2H), 1.52 (d, J = 7.0 Hz, 3H), 1.36 (d, J = 5.4 Hz, 6H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₅H₆₇N₁₀O₈S⁺ 1027.4859, found 1027.4861.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((8-(1-((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)-1H-1,2,3-triazol-4-yl)octyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (124, MS108).

White solid (6.04 mg, yield 58%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.17 (s, 1H), 8.07 (s, 1H), 7.77 (s, 1H), 7.73 (dd, J = 8.0, 1.7 Hz, 1H), 7.50 – 7.39 (m, 5H), 7.37 – 7.31 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.50 (s, 1H), 5.04 (t, J = 7.0 Hz, 1H), 4.60 – 4.51 (m, 1H), 4.44 (s, 1H), 3.84 (dd, J = 11.0, 3.7 Hz, 1H), 3.75 (d, J = 11.1 Hz, 1H), 3.35 (d, J = 7.1 Hz, 1H), 3.18 – 3.06 (m, 4H), 2.98 (q, J = 8.4 Hz, 4H), 2.72 – 2.60 (m, 3H), 2.51 (s, 3H), 2.21 (dd, J = 13.2, 7.7 Hz, 1H), 2.17 – 2.07 (m, 1H), 1.98 – 1.91 (m, 1H), 1.70 – 1.62 (m, 3H), 1.59 (t, J = 8.2 Hz, 2H), 1.52 (d, J = 7.0 Hz, 3H), 1.38 – 1.31 (m, 8H), 1.06 (s, 9H). ¹³C NMR (101 MHz, Methanol-d₄) δ 171.54, 170.50, 169.82, 168.66, 166.60, 159.72, 159.29, 154.13, 151.82, 147.24, 144.41, 143.19, 133.25, 129.83, 129.11, 128.79, 128.76, 127.00, 126.27, 126.17, 126.02, 124.49, 122.54, 116.93, 112.25, 99.38, 69.52, 68.43, 59.42, 56.89, 54.35, 53.43, 48.80, 39.65, 37.51, 36.37, 32.65, 29.77, 29.04, 29.01, 28.88, 28.85, 28.52, 26.59, 25.76, 25.71, 24.79, 21.03, 16.14, 14.21. HRMS m/z [M + H]⁺ calcd for C₅₆H₆₉N₁₀O₈S⁺ 1041.5015, found 1041.5014.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((9-(1-((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)-1H-1,2,3-triazol-4-yl)nonyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (125).

White solid (5.70 mg, yield 54%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.22 (s, 1H), 8.08 (s, 1H), 7.77 (s, 1H), 7.74 – 7.70 (m, 1H), 7.50 – 7.38 (m, 5H), 7.38 – 7.31 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.50 (s, 1H), 5.04 (t, J = 7.0 Hz, 1H), 4.60 – 4.49 (m, 1H), 4.49 – 4.41 (m, 1H), 3.84 (dd, J = 11.0, 3.7 Hz, 1H), 3.78 – 3.63 (m, 1H), 3.36 – 3.34 (m, 1H), 3.19 – 3.05 (m, 4H), 2.97 (q, J = 8.6 Hz, 4H), 2.73 – 2.60 (m, 3H), 2.51 (s, 3H), 2.21 (dd, J = 13.3, 7.7 Hz, 1H), 2.15 – 2.08 (m, 1H), 2.00 – 1.92 (m, 1H), 1.69 – 1.57 (m, 5H), 1.53 (d, J = 7.0 Hz, 3H), 1.32 (d, J = 5.9 Hz, 10H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₇H₇₁N₁₀O₈S⁺ 1055.5172, found 1055.5169.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((10-(1-((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-obutan-2-yl)-1H-1,2,3-triazol-4-yl)decyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (126).

White solid (5.99 mg, yield 56%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.16 (s, 1H), 8.07 (s, 1H), 7.77 (s, 1H), 7.72 (dd, J = 7.9, 1.7 Hz, 1H), 7.49 – 7.37 (m, 5H), 7.37 – 7.31 (m, 3H), 7.15 (s, 1H), 5.69 (t, J = 7.9 Hz, 1H), 5.50 (s, 1H), 5.04 (d, J = 7.0 Hz, 1H), 4.58 – 4.52 (m, 1H), 4.44 (s, 1H), 3.84 (dd, J = 11.0, 3.7 Hz, 1H), 3.79 – 3.67 (m, 1H), 3.35 (d, J = 7.2 Hz, 1H), 3.15 – 3.08 (m, 4H), 2.97 (q, J = 8.5 Hz, 4H), 2.71 – 2.60 (m, 3H), 2.51 (s, 3H), 2.21 (dd, J = 13.2, 7.8 Hz, 1H), 2.16 – 2.06 (m, 1H), 2.00 – 1.92 (m, 1H), 1.70 – 1.57 (m, 5H), 1.53 (d, J = 7.0 Hz, 3H), 1.32 (dd, J = 16.8, 6.4 Hz, 12H), 1.06 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₅₈H₇₃N₁₀O₈S⁺ 1069.5328, found 1069.5328.

(S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethan-1-amine (145).

To a stirred solution of tert-butyl (S)-(1-(4-bromophenyl)ethyl)carbamate 143 (600.4 mg, 2.0 mmol), 4-methylthiazole 144 (396.6 mg, 4.0 mmol, 2.0 equiv), Pd(OAc)₂ (4.49 mg, 0.02 mmol, 0.01 equiv), and K₂CO₃ (276.42 mg, 2.0 mmol, 1.0 equiv) in DMF (60 mL) was added under a nitrogen atmosphere. The reaction mixture was heated at 90 °C for 18 h. After cooling to rt, the mixture was diluted with EA (100ml) and wash with water (3 x 300 ml) and saturated NaCl solution (50 ml). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified with by silica gel column chromatography (Hexane/EA = 4:1) to afford the intermediate as brown oil. This obtained intermediate was dissolved in DCM (2 mL) and TFA (1 mL) was added. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford intermediate 145 as brown solid in TFA salt form (255.42 mg, 58.5% yield for two steps). ¹H NMR (400 MHz, Methanol-d₄) δ 8.64 (s, 1H), 7.43 – 7.37 (m, 4H), 4.32 (q, J = 6.9 Hz, 1H), 2.42 (s, 3H), 1.58 (d, J = 6.9 Hz, 3H). HRMS m/z [M + H]⁺ calcd for C₁₂H₁₅N₂S⁺ 219.0950, found 219.0955.

(2R,4S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxy-pyrrolidine-2-carboxylic acid (148).

To a solution of commercially available (S)-2-((tert-butoxycarbonyl)-amino)-3,3-dimethylbutanoic acid 146 (693.87 mg, 3.0 mmol) in DMSO (10 mL) were added commercially available methyl (2R,4S)-4-hydroxypyrrolidine-2-carboxylate 147 (435.48 mg, 3.0 mmol, 1 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide) (862.65 mg, 4.5 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (612.50 mg, 4.5 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (910.35 mg, 9.0 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford the intermediate. To a solution of this intermediate in MeOH (8 mL), was then added the solution of LiOH (118.55 mg, 4.95 mmol, 3.0 equiv) in H₂O (2.5 mL) dropwise. The mixture was stirred at rt overnight. Resulting crude mixture was purified by flash column chromatography by using reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to yield title compound 148 as a white solid in TFA salt form (511.45 mg, two step yield: 49.5%). ¹H NMR (400 MHz, Methanol-d₄) δ 4.48 – 4.43 (m, 1H), 4.26 – 4.20 (m, 2H), 3.84 (dd, J = 10.9, 5.0 Hz, 1H), 3.57 – 3.52 (m, 1H), 2.09 (dd, J = 7.5, 5.0 Hz, 2H), 1.37 (s, 9H), 0.94 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₁₆H₂₉N₂O₆⁺ 345.2020, found 345.2022.

(2R,4S)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methyl-thiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (149).

To a solution of the intermediate 148 (344.40 mg, 1.0 mmol) in DMSO (5 mL) were added the intermediate 145 (218.31 mg, 1.0 mmol, 1 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide) (287.55 mg, 1.5 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (204.17 mg, 1.5 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (303.45 mg, 3.0 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford the intermediate. The obtained intermediate was dissolved in DCM (2.0 mL) followed by TFA (1.0 mL). After being stirred for 1 h at room temperature, the reaction mixture was concentrated and purified by reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to afford the intermediate 149 as a white solid in TFA salt form. (186.10 mg, 41.86% yield for two steps). ¹H NMR (400 MHz, Methanol-d₄) δ 8.67 (s, 1H), 7.39 – 7.32 (m, 4H), 4.99 (q, J = 7.0 Hz, 1H), 4.51 – 4.41 (m, 2H), 3.85 (s, 1H), 3.75 (dd, J = 10.9, 4.1 Hz, 1H), 3.63 (t, J = 13.5 Hz, 1H), 2.45 (s, 3H), 2.31 – 2.22 (m, 1H), 2.05 – 1.99 (m, 1H), 1.44 (d, J = 7.0 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₃H₃₃N₄O₃S⁺ 445.2268, found 445.2265.

(2R,4S)-1-((S)-2-azido-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methyl-thiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (150).

To a solution of the intermediate 149 (88.92 mg, 0.2 mmol) and N,N-dimethyl-4-aminopyridine (DMAP) 141 (73.3 mg, 0.6 mmol, 3.0 eq) in DCM (0.5 mL) at 0 °C, a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADMP) (65.58 mg, 0.23 mmol, 1.15 eq) in DCM (0.5 mL) was added at 0 °C. The reaction mixture was stirred for 30 min at room temperature. Upon completion, the reaction was quenched with saturated aqueous sodium bicarbonate (20.0 mL) and extracted with dichloromethane (3 × 30.0 mL). The combined organic layers were washed successively with water (30.0 mL) and brine (30.0 mL), then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to afford the crude product and purified by using reverse phase C18 column (10% - 100% methanol / 0.1% TFA in water) to yield the intermediate 150 as a white solid (55.53 mg, 57% yield). ¹H NMR (400 MHz, Methanol-d₄) δ 8.82 (s, 1H), 7.42 – 7.37 (m, 4H), 5.02 (q, J = 7.0 Hz, 1H), 4.69 (dd, J = 8.3, 4.9 Hz, 1H), 4.61 – 4.56 (m, 1H), 3.82 – 3.75 (m, 2H), 3.62 (s, 1H), 3.55 (dd, J = 10.5, 4.4 Hz, 1H), 2.51 (s, 3H), 2.44 – 2.38 (m, 1H), 1.51 (d, J = 7.1 Hz, 3H), 1.09 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₂₃H₃₁N₆O₃S⁺ 471.2173, found 471.2178.

(2R,4S)-1-((S)-2-(4-(9-aminononyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethylbutanoyl)-4-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (151).

To a solution of the intermediate 150 (9.41 mg, 0.02 mmol), commercially available tert-butyl dec-9-yn-1-ylcarbamate (6.08 mg, 0.024 mg, 1.2 equiv) in tBuOH (1.0 mL) and water (1.0 mL) was added CuSO₄ (4.8 mg, 0.03 mmol, 1.5 equiv) and (+)-Sodium L-ascorbate (7.92 mg, 0.04 mmol, 2.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-70% acetonitrile / 0.1% TFA in H₂O) to afford intermediate as white solid in TFA salt form. The obtained intermediate was dissolved in DCM (0.5 mL) followed by TFA (0.5 mL). After being stirred for 1 h at room temperature, the reaction mixture was concentrated and purified by preparative HPLC (5%-70% acetonitrile / 0.1% TFA in H₂O) to afford intermediate 151 as white solid in TFA salt form. (5.36 mg, 42% yield for two steps). ¹H NMR (400 MHz, Methanol-d₄) δ 8.72 (s, 1H), 7.51 (s, 1H), 7.18 (dd, J = 7.8, 3.9 Hz, 4H), 4.82 (q, J = 6.9 Hz, 1H), 4.52 (t, J = 7.2 Hz, 1H), 4.35 – 4.32 (m, 1H), 3.50 (d, J = 4.0 Hz, 1H), 3.20 (dd, J = 10.8, 4.8 Hz, 1H), 2.74 (t, J = 7.3 Hz, 2H), 2.45 (t, J = 7.6 Hz, 2H), 2.39 (s, 3H), 2.11 – 2.01 (m, 2H), 1.51 – 1.38 (m, 5H), 1.33 (d, J = 6.9 Hz, 3H), 1.18 – 1.06 (m, 8H), 0.95 (s, 9H). HRMS m/z [M + H]⁺ calcd for C₃₄H₅₂N₇O₃S⁺ 624.3690, found 624.3686.

5-(4-(dimethylcarbamoyl)-3-hydroxyphenyl)-N-((R)-6-((8-(1-((S)-1-((2R,4S)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-obutan-2-yl)-1H-1,2,3-triazol-4-yl)octyl)carbamoyl)-2,3-dihydro-1H-inden-1-yl)isoxazole-3-carboxamide (152, MS108N).

To a solution of the intermediate 129 (3.66 mg, 0.0084 mmol) in DMSO (1mL) were added linker 151 (5.36 mg, 0.0084 mmol, 1.0 equiv), EDCI (2.42 mg, 0.0126 mmol, 1.5 equiv), HOAt (1.71 mg, 0.0126 mmol, 1.5 equiv), and NMM (2.55 mg, 0.0252 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile / 0.1% TFA in H₂O) to afford compound 152 as a white solid (3.94 mg, yield 45%). ¹H NMR (400 MHz, Methanol-d₄) δ 9.04 (s, 1H), 7.78 (d, J = 5.9 Hz, 2H), 7.73 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.0 Hz, 3H), 7.35 (d, J = 9.1 Hz, 3H), 7.13 (d, J = 1.4 Hz, 1H), 5.68 (t, J = 7.9 Hz, 1H), 5.38 (d, J = 2.4 Hz, 1H), 4.64 (t, J = 7.9 Hz, 1H), 4.45 (s, 1H), 3.74 (d, J = 11.0 Hz, 1H), 3.46 (dd, J = 10.9, 4.0 Hz, 1H), 3.16 – 3.07 (m, 4H), 2.97 (q, J = 8.6 Hz, 4H), 2.67 – 2.61 (m, 1H), 2.47 (s, 5H), 2.21 (t, J = 10.7 Hz, 1H), 2.11 (dd, J = 12.6, 8.2 Hz, 1H), 2.06 – 1.98 (m, 1H), 1.53 (q, J = 6.8 Hz, 3H), 1.45 (d, J = 7.1 Hz, 5H), 1.34 – 1.13 (m, 10H), 1.05 (s, 9H). ¹³C NMR (101 MHz, Methanol-d₄) δ 172.23, 170.49, 168.64, 166.19, 159.69, 159.28, 154.12, 147.23, 144.26, 143.20, 133.25, 129.41, 128.78, 128.75, 127.03, 126.55, 126.02, 124.50, 122.53, 116.93, 112.26, 99.38, 69.59, 68.25, 59.34, 55.72, 54.33, 48.78, 39.62, 37.28, 35.63, 32.64, 29.77, 29.04, 28.92, 28.86, 28.61, 26.61, 25.81, 24.83, 21.09, 16.14, 14.36. HRMS m/z [M + H]⁺ calcd for C₅₆H₆₉N₁₀O₈S⁺ 1041.5015, found 1041.5013.

Cell culture and drug treatment

Human leukemia cell lines MV4;11, RS4;11, KASUMI1, SEMK2, Jurkat and K-562 were maintained in RPMI (Corning, Cat. No. 10-040-CV) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 100 U/mL penicillin/streptomycin (Corning, Cat. No. 30-009-CI). All human cell lines were mycoplasma-negative and were tested for authentication by short tandem repeat (STR) profiling performed by American Type Culture Collection (ATCC) or by UT MD Anderson Cancer Center CCSG core.

In the first-round drug screening, MV4;11 cells were treated with compounds 1–126 at concentrations 1 or 10 μM for 24 h. Then cells were collected for Western blot analysis and the band intensity was quantified by image J software. The degradation efficiencies were summarized in Table 1–6 and Table S1–S3.

In the second-round drug screening, MV4;11 cells were treated with selected compounds at indicated concentrations (1 nM, 10 nM, 100 nM and 1 μM) for 24 h. Then cells were collected for Western blot analysis.

For further drug screening, MV4;11 cells were treated with indicated concentrations of compound 124 for 24 h. Then cells were collected for Western blot analysis. For DC₅₀ and D_max determination, leukemia cells were treated with increasing concentrations of compound 124 ranging from 0.049 nM to 50 nM for 24 h. Dose-dependent ENL degradation curves were generated based on immunoblotting of ENL and β-Actin. DC₅₀ values were determined using nonlinear regression of variable slope (four parameters) curve fitting using GraphPad Prism. In washout experiment, MV4;11 cells were first treated with 30 nM compound 124 for 24 h. Remove compound 124-containing medium and wash MV4;11 cells with PBS twice. Then cells were cultured in normal medium and collected for Western blot analysis at the indicated time points.

Immunoblotting

Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH7.4, 250 mM NaCl, 0.5% Triton X-100, 10% glycerol) freshly supplemented with 1 mM DTT and protease inhibitors. Protein concentration was measured by Bradford assay. Equal amounts of protein lysate were used for immunoblotting.

CRISPR-Cas9-Mediated VHL Knockout (KO)

sgRNA sequences targeting the human VHL gene were designed based on a CRISPR sgRNA database (GeneScript) and cloned into lentiCRISPR v2 vector (Addgene #52961).⁵⁶ Lentivirus packaging was performed in HEK293T cells, using X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366546001) in accordance with the manufacturer’s instructions. Medium containing virus was centrifuged and concentrated using Amicon Ultra-15 columns. Spin infection was performed at 1,500 rcf at 25°C for 90 min and transduced cell populations were usually selected or sorted 48 hours after infection.

Proteomics studies

MV4;11 cells were treated with DMSO or 30 nM of compound 124 or 152 for 2 h. Two million cells were collected for each sample and subjected to further sample preparation for liquid chromatography-mass spectrometry (LS-MS) following the previously reported method.⁴⁴

Cell viability assay

A total of 2000 cells were seeded in 96-well plate in 100 μL of medium, treated with DMSO or compounds at the indicated concentrations for 6 days. On day 6, 40 μL of medium containing compounds at 3× the indicated concentrations were added, and the culture was continued for another 6 days. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay kit (Promega, Cat. No. G9241) according to the manufacturer’s instructions. Surviving cells were calculated as percentage relative to the DMSO treated cells. Dose response curves were generated using GraphPad with nonlinear regression of variable slope (four parameters) curve fitting, and GI₅₀ values were determined from the plots.

Clonogenicity assay

Colony formation experiments were performed in Methocult (Stemcell, Cat. No. H4100). 8000 leukemia cells were resuspended in 400 μL RPMI medium and mixed with 4 mL complete Methocult medium. Triplicates of 1.1 mL cell suspension were added to six-well plates. 14 days later, colonies were scanned and counted by Celigo (Nexcelom).

Cell cycle and apoptosis assay

Cells were harvested at the indicated time points. DAPI (4′,6-diamidino-2-phenylindole)-Alcohol fixation method was used for cell cycle analysis. Cells were resuspended in 50 μL of HBSS containing 2% FBS, then 1 mL of ice-cold 70% ethanol was added in a dropwise manner for fixation. Fixed cells were washed twice with HBSS containing 2% FBS and incubated with 1 mL DAPI working solution (10 μg/mL DAPI and 0.1% Triton X-100 in PBS) in dark for 15-30 minutes at RT. The stained cells were then ready for flow cytometry analysis. FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, Cat. No. 556547) was used for cell apoptosis analysis. Cells were collected and washed twice with cold PBS containing 2% FBS and then resuspended in 1x binding buffer at a concentration of 1x10⁶ cells/mL. 100 μL of resuspended cells were stained with 5 μL of FITC-Annexin V and 2 μL of PI at RT for 15 minutes. Following staining, 200 μL of 1x binding buffer was added, and all the samples were analyzed by flow cytometry within 1 hour.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR).

Total RNA was isolated using the RNeasy Mini kit (Qiagen, catalog no. 74134) and reverse transcribed with the iScript cDNA Synthesis Kits (Bio-Rad, catalog no. 1708840) in accordance with the manufacturer’s instructions. Quantitative real-time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, catalog no. A25742) with Bio-Rad CFX96 Real-Time system (C1000Touch Thermal Cycler). Relative gene expression was calculated using the comparative ΔΔCT method, normalizing the target gene’s Ct value to that of the internal control, Ribosomal Protein S15 (RPS15), and expressed as 2^−ΔΔCT. Primers used for RT-qPCR are provided in table S8.

Mouse PK Study

Compound 124 (in its sodium salt form) was dissolved in a solution formulation of 5% NMP, 45% PG, and 50% PEG400. Three male Swiss albino mice were administered intraperitoneally with a solution formulation of compound 124 at 50 mg/kg. Blood samples (approximately 60 μL) were collected under light isoflurane anesthesia from a set of three mice at 0.5, 2, 4, 8, 12 and 24 h in pre-labelled tubes containing 20% v/v K2-EDTA as anticoagulant. Immediately after blood collection, plasma was harvested by centrifugation at 10,000 rpm, 10 min at 4 °C and samples were stored at −70±10 ° C until bioanalysis. The plasma concentration-time of compound 124 was used for the PK analysis. Plasma samples were quantified by the fit-for-purpose LC–MS/MS method (LLOQ: 4.00 ng/mL). PK analysis was performed using GraphPad Prism software in the form of nonlinear regression analysis. The compound concentrations in plasma at each time point are the average values from three test mice. Error bars represent ± SEM. Experiments involving mice were performed according to the Institutional Animal Care and Use Committee (IACUC)-approved protocol (IACUC-2018-0001).

Supplementary Material

The following Supporting Information (SI) is available free of charge on the ACS Publications website at DOI.

WB results of the ENL level in MV4;11 cells treated with compounds 1–11 (Figure S1); WB results of the ENL level in MV4;11 cells treated with compounds 12–24 (Figure S2); WB results of the ENL level in MV4;11 cells treated with compounds 25–34 (Figure S3); WB results of the ENL level in MV4;11 cells treated with compounds 35–43 (Figure S4); ENL Protein Degradation Activity of compounds 44–54 (Table S1); WB results of the ENL level in MV4;11 cells treated with compounds 44–54 (Figure S5); WB results of the ENL level in MV4;11 cells treated with compounds 55–69 (Figure S6); ENL Protein Degradation Activity of compounds 70–79 (Table S2); WB results of the ENL level in MV4;11 cells treated with compounds 70–79 (Figure S7); ENL Protein Degradation Activity of compounds 80–87 (Table S3); WB results of the ENL level in MV4;11 cells treated with compounds 80–87 (Figure S8); WB results of the ENL level in MV4;11 cells treated with compounds 88–99 (Figure S9); WB results of the ENL level in MV4;11 cells treated with compounds 100–110 (Figure S10); WB results of the ENL level in MV4;11 cells treated with compounds 111–116 (Figure S11); WB results of the ENL level in MV4;11 cells treated with compounds 117–126 (Figure S12); LCMS, ¹H NMR spectra for compounds 94, 104, 105, 106, 107, 117, 118, 123, 125, and 126, as well as LC-MS, ¹H NMR and ¹³C NMR spectra of compound 124 and 152 (PDF).

Molecular formula strings for all compounds (CSV).

ABBREVIATIONS USED

AML

acute myeloid leukemia

ADH

ANC-1 homology domain

ADMP

dimethylimidazolinium hexafluorophosphate

CRBN

cereblon

CFC

colony-forming cell

C_max

maximum concentration

DC₅₀

half-maximal degradation concentration

DMSO

dimethyl sulfoxide

DMAP

N,N-dimethyl-4-aminopyridine

DCM

dichloromethane

DMF

N,N-dimethylformamide

ENL

eleven-nineteen leukemia protein

EDCI

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ESI

electrospray ionization

GI₅₀

half-maximal growth inhibition concentration

HA

hemagglutinin

HOAt

1-hydroxy-7-azabenzotriazole

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

IP

intraperitoneal

Kac

lysine acetylation

LHS

left-hand side

MLL1

mixed-lineage leukemia protein

MOA

mechanism-of-action

MS

mass spectrometry

ND

no degradation

NMM

N-methylmorpholine

NMR

nuclear magnetic resonance

PROTAC

proteolysis targeting chimera

PTMs

post-translational modifications

PAF1c

polymerase-associated factor 1 complex

PK

pharmacokinetic

PEG

polyethylene glycol

RHS

right-hand side

RT-qPCR

quantitative reverse transcriptase polymerase chain reaction

rt

room temperature

SAR

structure-activity relationship

SEC

Super Elongation Complex

TFA

trifluoroacetic acid

UPS

ubiquitin-proteasome system

VHL

von Hippel-Lindau

WB

western blotting