Design, Synthesis, and Evaluation of Potent, Selective, and Bioavailable AKT Kinase Degraders

Abstract

The serine/threonine kinase AKT functions as a critical node of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (m-TOR) signaling pathway. Aberrant activation and overexpression of AKT are strongly correlated with numerous human cancers. To date, only two AKT degraders with no structure–activity relationship (SAR) results have been reported. Through extensive SAR studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified two novel and potent AKT proteolysis targeting chimera (PROTAC) degraders: von Hippel–Lindau (VHL)-recruiting degrader 13 (MS98) and cereblon (CRBN)-recruiting degrader 25 (MS170). These two compounds selectively induced robust AKT protein degradation, inhibited downstream signaling, and suppressed cancer cell proliferation. Moreover, these two degraders exhibited good plasma exposure levels in mice through intraperitoneal injection. Overall, our comprehensive SAR studies led to the discovery of degraders 13 and 25, which are potentially useful chemical tools to investigate biological and pathogenic functions of AKT in vitro and in vivo.

INTRODUCTION

The serine/threonine kinase AKT, also known as protein kinase B (PKB), is encoded by three closely related genes in humans: AKT1 (PKB-α), AKT2 (PKB-β), and AKT3 (PKB-γ).^1–3 AKT is an important component of the PI3K/AKT/ mammalian target of rapamycin (m-TOR) signaling pathway and regulates fundamental cellular and physiological processes, such as cell growth and survival, apoptosis, transcription, migration, and protein synthesis.^4–7 PI3K/AKT/m-TOR signaling is one of the most frequently dysregulated pathway in the initiation and propagation of human cancers.^8–10 Hyperactivation or overexpression of AKT is associated with a variety of human malignancies, including breast, prostate, lung, colon, brain, pancreatic, and ovarian cancer; gastric carcinoma; and melanoma.^9,11,12 Consequently, AKT has been recognized as an oncogenic therapeutic target for several decades.^12–14

Several highly potent adenosine triphosphate (ATP)-competitive AKT inhibitors (such as GSK690693,¹⁵ GSK2110183,^16,17 GSK2141795,¹⁶ GDC-0068,¹⁸ and AZD5363¹⁹) and allosteric inhibitors (such as MK-2206^20,21 and ARQ-092²²) are currently being investigated in clinics for oncology indications (Figure 1).^3,14 ATP-competitive AKT inhibitors, such as GDC-0068, paradoxically lead to the elevated hyperphosphorylation of AKT at Thr308 and Ser473 that stabilizes AKT active conformation.^18,23 Moreover, the clinical efficacy of these ATP-competitive inhibitors is compromised by lacking selectivity over closely related AGC kinase family members.²⁴ Allosteric inhibitors, such as MK-2206 and ARQ092, possess a high degree of selectivity for AKT1/2/3 and inhibit the phosphorylation of two regulatory sites: Thr308 and Ser473. However, these allosteric inhibitors did not achieve sufficient antitumor activities in clinical studies.^25,26 Recently, several covalent allosteric AKT inhibitors have been developed by irreversibly targeting AKT at Cys296.^27–29 For example, borussertib exerts potent tumor regression in KRAS-mutant patient-derived xenografts when it is combined with the MEK inhibitor trametinib, but further investigation is needed to explore its therapeutic potential. Moreover, kinase-independent functions of AKT can also promote cancer cell survival.^7,30 Therefore, an alternative therapeutic strategy is highly desirable for the treatment of AKT-associated human cancers.

Figure 1.

Representative ATP-competitive and allosteric inhibitors of AKT.

Targeted protein degradation using the proteolysis targeting chimera (PROTAC) technology has gained substantial attention from both academic institutions and pharmaceutical industry since 2015. This technology has been utilized to degrade a wide range of oncological and clinically relevant targets.^31–35 PROTACs are heterobifunctional small molecules that engage target proteins and E3 ubiquitin ligases, inducing polyubiquitination and subsequent degradation of the target proteins at the proteasome.^36–39 PROTACs are able to temporally eliminate both catalytic and noncatalytic (such as scaffolding) functions of the targeted enzymes. Because functional binders (inhibitors or activators) of the targeted protein are not required for developing effective PROTACs, this technology has the potential to target a broad spectrum of previously inaccessible proteome.^40–42 Moreover, isoform-selective PROTACs can be achieved using the binders that are not isoform-selective.^43–46 Overall, these features highlight a few potential advantages of PROTACs over conventional small-molecule inhibitors.

Two AKT PROTAC degraders have been reported to date.^47,48 You et al. reported a potent and selective GDC-0068-based cereblon (CRBN)-recruiting AKT degrader, INY-03-041, which induced sustained AKT degradation with prolonged inhibition of downstream signaling.⁴⁷ We very recently reported MS21, a potent and selective AZD5363-based von Hippel–Lindau (VHL)-recruiting AKT degrader, which was efficacious in vivo.⁴⁸ However, no structure–activity relationship (SAR) studies have been reported to date. Herein, we report extensive SAR studies for developing AKT PROTACs by exploring various linkers, E3 ligase ligands, and AKT binding moieties. Through these studies, we discovered multiple series of AKT degraders derived from two AKT inhibitors, GDC-0068 and GSK690693, by recruiting either VHL or CRBN E3 ligase. We further characterized two GDC-0068-based degraders: 13 (MS98, which recruits VHL) and 25 (MS170, which recruits CRBN). Both degraders promoted selective AKT protein degradation, reduced downstream signaling, and inhibited cancer cell proliferation in a concentration- and time-dependent manner. In addition, both degraders are bioavailable in mice and could be used for in vivo efficacy studies. Overall, we present multiple promising AKT degraders, which are potentially useful for investigating pathophysiological functions of AKT.

RESULTS AND DISCUSSION

Design, Synthesis, and Evaluation of GDC-0068-Based VHL-Recruiting AKT Degraders.

GDC-0068 is a highly potent and selective ATP-competitive pan-AKT kinase inhibitor.¹⁸ As illustrated in Figure 2A, the cocrystal structure of AKT1 in the complex with GDC-0068 (PDB: 4EKL) revealed that the isopropyl group was solvent-exposed, which offered a suitable exit vector for attaching a linker. In addition, the N–H of the isopropylamino group interacts with Glu 234 and Glu 278 side chains through hydrogen bonds (Figure 2B). On the basis of these observations, we designed two precursors (1 and 2) by replacing the isopropyl moiety with propionic acid and ethylamine groups to serve as bridge groups for linker installation (Figure 2C).

Figure 2.

Schematic design of GDC-0068-based AKT putative degraders. (A) The crystal structure of AKT1 in complex with GDC-0068 (PDB: 4EKL) indicates that the isopropyl group is solvent-exposed. (B) Key hydrogen bonding interactions are indicated by yellow dotted lines. (C) Design of precursors for AKT degraders.

It is known that linker types, physicochemical properties, and lengths play critical roles in the successful development of PROTAC degraders.^49,50 Therefore, we designed and synthesized a series of putative VHL-recruiting AKT degraders (3–11) using either a polyethylene glycol (PEG) or an alkylene linker with different linker lengths (Figure 3A). Their effects on reducing total AKT (T-AKT) and phosphorylated AKT (P-AKT) protein levels were assessed by western blot (WB) analysis in BT474 cells (a PIK3CA^K111N mutant and HER2 positive breast cancer cell line)⁵¹ after 24 h treatment with 1, 5, and 10 μM compound concentrations (Figure 4). We also evaluated their effects on inhibiting the phosphorylation of two downstream targets: PRAS40 (P-PRAS40) and S6 (P-S6). In this assay, DMSO, GDC-0068, VHL ligand (VHL-1), and CRBN ligand pomalidomide (POM) were used as controls. As expected, GDC-0068, VHL-1, and POM had no detectable effect on the T-AKT protein level. Consistent with earlier reports,^18,23 GDC-0068 treatment effectively inhibited downstream signaling but also resulted in AKT hyperphosphorylation. Compound 3, bearing the shortest PEG linker (one PEG unit), induced significant T-AKT and P-AKT protein degradation at concentrations of 5 and 10 μM. However, its inhibition potency against the downstream signaling, P-PRAS40 and P-S6, was less than GDC-0068. Compounds 4 and 5, bearing longer PEG linkers, were less effective in AKT degradation. Compound 6, with a butylene linker, concentration-dependently reduced the protein levels of T-AKT and P-AKT and exhibited a similar inhibitory effect as GDC-0068 against P-S6. Increasing the alkylene linker length (compounds 7–11) resulted in more potent AKT degradation, although an obvious ″hook effect″ was observed for more potent degraders (8–11) at higher concentrations. For example, compound 11, bearing the longest linker (decylene), exhibited near-complete degradation of T-AKT and P-AKT at 1 μM. However, at 5 and 10 μM, a ″hook effect″ was observed. Moreover, compound 11 also inhibited P-PRAS40 and P-S6. However, it was less effective than the parent inhibitor GDC-0068 in inhibiting P-PRAS40 and P-S6 at the same concentration (1 μM).

Figure 3.

Chemical structures of GDC-0068-based VHL-recruiting (A) and CRBN-recruiting (B) AKT putative degraders.

Figure 4.

Effects of GDC-0068-based VHL-recruiting compounds on degrading AKT and inhibiting downstream signaling. BT474 cells were treated with GDC-0068 (1 μM); VHL-1 (1 μM); pomalidomide (POM, 1 μM); compounds 3–11 at 1, 5, and 10 μM; or compounds 12–13 at 1, 3, and 10 μM for 24 h. The cell lysates were analyzed by western blotting to examine the protein levels of total AKT (T-AKT) and phosphorylated AKT at serine 473 (P-AKT (S473)), and downstream signaling inhibition of P-PRAS40 (T246) and P-S6 (S240/244). β-Actin was used as the loading control.

It has been reported that the VHL ligand (S,R,S)-AHPC-Me (VHL-2), which adds a benzylic methyl group on VHL-1, could increase the binding affinity to the VHL E3 ligase and lead to more effective PROTACs.^52–54 Therefore, we replaced VHL-1 in compound 11 with VHL-2^55,56 and obtained compound 12. In addition, we designed an analog of 12, compound 13 with a reversed amide group (Figure 3A). Both 12 and 13 displayed remarkable effects on degrading T-AKT and P-AKT, and more profound inhibition of P-PRAS40 and P-S6 than 11 (Figure 4). Because 13 exhibited slightly better T-AKT and P-AKT degradation effects and less ″hook effect″ than 12, we selected compound 13 for further biological characterization.

Design, Synthesis, and Evaluation of GDC-0068-Based CRBN-Recruiting AKT Degraders.

In addition to exploring VHL-recruiting degraders, we also designed CRBN-recruiting degraders 14–25 by conjugating GDC-0068 to pomalidomide through various PEG and alkylene linkers (Figure 3B). Similar to the VHL-recruiting degraders, the effects of these putative CRBN-recruiting degraders on degrading T-AKT and P-AKT and inhibiting downstream signaling were evaluated in BT474 cells (Figure 5). Among the five compounds with PEG linkers, compounds 15–18 (with two to four PEG unit linkers) were effective in reducing T-AKT and P-AKT protein levels at 1 μM, while compound 14 (with one PEG unit linker) was not very effective at all concentrations tested. However, compounds 15 to 18 were less effective than GDC-0068 in inhibiting P-PRAS40 and P-S6 (Figure 5). Among the compounds with alkylene linkers, compounds with ethylene (19), propylene (20), butylene (21), and pentylene (22) linkers were marginally effective in inducing AKT degradation at 1–10 μM. On the other hand, compounds with relatively longer linkers, such as 23 (hexylene), 24, (heptylene) and 25 (octylene), were highly effective in reducing T-AKT and P-AKT protein levels. Among these three compounds, 25 was the most effective AKT degrader without a ″hook effect″ and was also the most effective in inhibiting the downstream signaling, P-PRAS40 and P-S6.

Figure 5.

Effects of GDC-0068-based CRBN-recruiting compounds on degrading AKT and inhibiting downstream signaling. BT474 cells were treated with GDC-0068 (1 μM), VHL-1 (1 μM), POM (1 μM), or indicated compounds at 1, 5, and 10 μM for 24 h. The cell lysates were analyzed by western blotting to examine the protein levels of T-AKT, P-AKT (S473), P-PRAS40, (T246) and P-S6 (S240/244). β-Actin was used as the loading control.

We further designed four degraders, 26–29, which have the same linker length as 25 but different linker types or CRBN binders (Figure 3B). While compound 26, which has a reverse amide linker, did not reduce the P-AKT level, it partially degraded T-AKT (Figure 5). Surprisingly, compound 26 inhibited P-PRAS40 at 1 μM as effectively as GDC-0068. It is unclear what the major contributor to this strong downstream signaling inhibition is. One possibility is that the partial T-AKT degradation may contribute to the observed inhibition of P-PRAS40. This warrants further investigation. In addition, removing one of the two carbonyl oxygen atoms in the phthalimide moiety (28) completely abolished AKT degradation activity. On the contrary, changing the NH-group at the linker attachment site of POM to a methylene group (27) or oxygen atom (29) maintained the excellent AKT degradation and downstream signaling inhibition effects. Overall, compounds 25, 27, and 29 were very effective in inducing T-AKT and P-AKT degradation (>95% degradation at 1 μM) and inhibiting P-PRAS40 and P-S6. To rank these three compounds, we compared their cell growth inhibition effect using a colony formation assay with BT474 cells. From this study, compound 25 exhibited slightly better potency than 27 and much better potency than 29 in inhibiting the colony formation of these cells (Figure S1). We therefore selected compound 25 for further characterization.

Design, Synthesis, and Evaluation of GSK690693-Based AKT Degraders.

To explore other AKT binding moieties, we next designed and synthesized a set of putative AKT degraders (31–60) with a modified AKT binding moiety based on GSK690693¹⁵ (Figure 6). Analysis of the cocrystal structure of GSK690693-AKT2 (PDB: 3D0E, Figure S2) and the reported SAR results of the amine-containing side chain moiety revealed that the piperidinyl ring is solvent-exposed.¹⁵ Based on synthesis considerations, intermediate 30 was designed as a degrader precursor by replacing the piperidinyl group with a propylamino group, which was coupled with a butyric acid bridge group (Figure S2). This modified AKT binding moiety was then linked to VHL-1 or POM via various linkers (Figure 6).

Figure 6.

Chemical structures of GSK690693-based AKT degraders.

The effects of putative degraders 31–60 on degrading T-AKT and P-AKT and inhibiting P-PRAS40 and P-S6 were assessed in PC3 cells (a PETN loss prostate cancer cell line)⁵⁷ treated with 1 μM of the test compound for 24 h (Figure 7). Western blotting analysis showed that compound 48 from the VHL series and compound 60 from the CRBN series were the most effective in degrading T-AKT and P-AKT. Compared to GDC-0068-based degraders with alkylene linkers, a similar SAR trend on the linker length was observed for GSK690693-based degraders with alkylene linkers. That is, compounds 48 and 60 have the longest alkylene linker in VHL- and CRBN-recruiting series, respectively. On the other hand, GSK690693-based VHL-recruiting degraders with a PEG linker, 31–38, were completely ineffective in degrading T-AKT and P-AKT, while CRBN-recruiting degraders with a PEG linker, 49–53, were marginally effective or ineffective. Interestingly, degraders 48 and 60 displayed a similar effect as GSK690693 on suppressing P-S6 but were less effective than GSK690693 in inhibiting P-PRAS40. Mainly due to this, we did not select degraders 48 and 60 for further characterization in this study. Nevertheless, compounds 48 and 60 are interesting AKT degraders, which could be useful for the research community to study AKT functions.

Figure 7.

Effects of GSK690693-based AKT degraders on degrading AKT and inhibiting downstream signaling. PC3 cells were treated with GSK690693 or indicated compounds at 1 μM for 24 h. The cell lysates were analyzed by western blotting to examine the protein levels of T-AKT, P-AKT (S473), P-PRAS40 (T246), and P-S6 (S240/244). β-Actin was used as the loading control.

After extensively exploring a variety of linkers, E3 ligase ligands, and AKT binding moieties and identifying multiple highly effective AKT degraders, we next sought to further characterize AKT degraders 13 and 25 in a battery of biochemical and cellular assays. To help elucidate the mechanism of action of AKT degradation and characterize cellular effects, we developed two negative control compounds, 61 (MS98N) and 62 (MS170N), for degraders 13 and 25, respectively (Figure 8). Compound 61 was designed to abolish VHL engagement by incorporating a diastereoisomer of VHL-2.⁵² Similarly, compound 62 was designed to abolish CRBN engagement by incorporating a methyl group at the glutarimide moiety.⁵⁸ In addition, we also used the previously reported AKT degrader INY-03–041 as a positive control (Figure 8).

Figure 8.

Chemical structures of negative control compounds 61 and 62 and the previously reported AKT degrader INY-03–041.

Binding Affinities of Compounds 13, 25, 61, and 62 to Three AKT Isoforms.

Using a competitive binding assay, we assessed binding affinities of the lead degraders 13 and 25 and their negative controls 61 and 62 to AKT1/2/3 in comparison to the parental inhibitor GDC-0068 (Figure 9). In this assay, inhibitor GDC-0068 displayed very high binding affinities to AKT1 (K_d = 0.64 ± 2.6 nM) and AKT3 (K_d = 2.5 ± 1.4 nM) and lower binding affinity to AKT2 (K_d = 35 ± 2.4 nM). Compared to GDC-0068, degraders 13 and 25 and their corresponding negative control compounds 61 and 62 exhibited 2- to 6-fold lower binding affinities to AKT1 (13: K_d = 4.0 ± 4.6 nM, 25: K_d = 1.3 ± 1.6 nM, 61: K_d = 1.9 ± 2.7 nM, and 62: K_d = 1.8 ± 2.6 nM). As for AKT2, all tested compounds (13: K_d = 140 ± 4.5 nM, 25: K_d = 77 ± 6.9 nM, 61: K_d = 84 ± 3.1 nM, and 62: K_d = 74 ± 4.2 nM) showed 2- to 4-fold decreased binding affinities compared to GDC-0068. Similarly, binding affinities of degraders 13 (K_d = 8.1 ± 2.7 nM), 25 (K_d = 6.5 ± 3.1 nM), and negative controls 61 (K_d = 4.2 ± 2.6 nM) and 62 (K_d = 12 ± 2.4 nM) to AKT3 decreased by 2- to 5-fold compared with GDC-0068. Overall, while these degraders and negative controls displayed 2- to 6-fold lower binding affinities to AKT isomers compared with the parent inhibitor, all of these compounds still retained high binding affinities for all three AKT isoforms, indicating that the attachment of linkers and E3 ligase ligands at the solvent-exposed moiety of GDC-0068 is tolerated.

Figure 9.

GDC-0068, 13, 61, 25, and 62 bind AKT1 (A), AKT2 (B), and AKT3 (C) with high affinities. The K_d determinations were performed in a competitive binding assay in duplicate. Error bars represent ± SEM from duplicated experiments.

Compounds 13 and 25 Concentration- and Time-Dependently Induced AKT Degradation through the Ubiquitin-Proteasome System (UPS).

We next determined the potency of degraders 13 and 25 in reducing AKT protein levels in BT474 cells (Figure 10A–D). We found that compounds 13 and 25 concentration-dependently depleted cellular T-AKT with DC₅₀ values of 78 ± 64 and 32 ± 18 nM, respectively. Compound 13 induced T-AKT degradation substantially at 100 nM and achieved near-complete depletion of T-AKT at 1 μM. Compound 25 exhibited a slightly higher potency than compound 13 with a significant decrease of the AKT protein level at 30 nM and complete degradation of T-AKT at 300 nM. No ″hook effect″ was observed for both compounds at concentrations up to 10 μM. As anticipated, negative control compounds 61 and 62 did not degrade T-AKT (Figure 10,FE). In addition, we found that the previously reported CRBN-recruiting AKT degrader INY-03–041 was slightly more potent than degrader 25 (Figure 10G). However, a very significant ″hook effect″ was observed for INY-03–041 at high concentrations. In addition, we assessed the effect of 13, 25, and INY-03–041 on degrading AKT in PC3 and MDA-MB-468 cells and found that both 13 and 25 effectively induced AKT degradation in a concentration-dependent manner (Figure S3). Interestingly, 13 exhibited similar potency as INY-03–041, while 25 was less potent than 13 and INY-03–041 in PC3 cells (Figure S3A–C). In addition, while both 13 and 25 were less potent than INY-03–041 in MDA-MB-468 cells (Figure S3D–F), INY-03–041 displayed an obvious ″hook effect″ at higher concentrations in MDA-MB-468 cells (Figure S3F), similar to what was observed in BT474 cells (Figure 10G). We also confirmed that the negative controls 61 and 62 had no effect on the AKT protein levels in MDA-MB-468 cells (Figure S3G–H).

Figure 10.

Compounds 13 and 25, but not 61 and 62, potently induced AKT degradation in BT474 cells. BT474 cells were treated with 13 (A, C), 25 (B, D), 61 (E), or 62 (F) at indicated concentrations for 24 h. (G) Effects of CRBN-recruiting AKT degraders 25 and INY-03–041 on AKT degradation and downstream signaling inhibition in BT474 cells treated with the test compound at indicated concentrations.

We also investigated the kinetics of AKT degradation and downstream signaling inhibition induced by compounds 13 and 25 in BT474 cells, with GDC-0068 as a control (Figure 11). As expected, GDC-0068 had no effect on the T-AKT protein level and increased the P-AKT protein level. On the other hand, compound 13 (at 1 μM) induced rapid T-AKT and P-AKT degradation (Figure 11A). Obvious degradation occurred at 4 h, and near-complete degradation was achieved at 8 h. Similarly, compound 25 degraded T-AKT and P-AKT in a time-dependent manner (Figure 11B). Significant P-AKT and T-AKT degradation occurred as early as 4 and 8 h, respectively. Both compounds maintained T-AKT and P-AKT degradation for at least 24 h. In addition, compounds 13 and 25 inhibited the downstream signaling, albeit the inhibition induced by 13 and 25 was not as rapid as that induced by GDC-0068. Taken together, these data indicate that 13 and 25 can induce rapid and sustained AKT degradation.

Figure 11.

Compounds 13 and 25 induced AKT degradation and inhibited downstream signaling in a time-, E3 ligase- and proteasome-dependent manner in BT474 cells. BT474 cells were treated with 1 μM of compound 13 (A) or compound 25 (B) for the indicated time, with GDC-0068 as a control. Cell lysates were collected at the indicated time point for western blotting. MOA studies of 13 (C) or 25 (D) induced AKT degradation and downstream signaling inhibition. BT474 cells were pretreated with DMSO or the indicated compound (Ac-VHL-2 (10 μM), POM (10 μM), MLN4924 (1 μM), MG132 (10 μM), or GDC-0068 (1 μM)) for 2 h and subsequently treated with 13 or 25 for an additional 22 h. Levels of the indicated proteins were accessed by western blotting. β-Actin was used as the loading control.

To demonstrate that the AKT downregulation induced by 13 and 25 was through the UPS system, we next performed a set of competition experiments (Figure 11C,D). In case of the VHL-recruiting degrader 13 (Figure 11C), pretreatment with an excess of acetyl-capped VHL-2 (Ac-VHL-2) partially rescued AKT degradation and P-PRAS40 inhibition, suggesting that VHL binding is required for 13-induced AKT degradation and downstream signaling inhibition. Pretreatment with the NEDD8-activating enzyme (NAE) inhibitor MLN4924⁵⁹ or proteasome inhibitor MG132 completely prevented 13-mediated degradation of T-AKT and P-AKT and inhibition of P-PRAS40, indicating that the cullin-ring ubiquitin ligase complexes and proteasome are necessary for the observed AKT degradation and downstream signaling inhibition. These results also suggest that compound 13’s AKT degradation activity, instead of its AKT inhibition activity, is the primary contributor to compound 13’s downstream signaling inhibition effect. Additionally, pretreatment with GDC-0068 also blocked the AKT degradation induced by 13. Similarly, pretreatment with the CRBN ligand POM, MLN4924, MG132, or GDC-0068 rescued 25-induced AKT degradation (Figure 11D). Pretreatment with POM, MLN4924, or MG132 also blocked 25-mediated downstream signaling inhibition. Collectively, these results, together with that negative controls 61 and 62 were unable to degrade AKT, support that the AKT degradation induced by 13 and 25 is mediated by the UPS and requires the engagement of AKT and the corresponding E3 ligase (VHL or CRBN).

Compounds 13 and 25 Exhibited Remarkable Selectivity for AKT in Unbiased Global Proteomic Studies.

To assess whether the expression levels of other proteins are affected by AKT degraders 13 and 25, we conducted unbiased quantitative tandem mass tag (TMT) labeling mass spectrometry (MS)-based proteomic studies. The samples for the proteomic studies were prepared by the treatment of BT474 cells with 13, 25, 61, or 62 at 1 μM for 24 h. Immunoblot analysis confirmed that AKT1, AKT2, and T-AKT protein levels were greatly depleted by degraders 13 and 25, but not the negative controls 61 and 62, in these samples (Figure S4). We did not detect AKT3 in these samples, which is consistent with the previous report that the expression of AKT3 in BT474 cells is too low to be detected.⁶⁰ This TMT-labeled proteomic approach enabled quantification of over 8000 proteins (Figure 12). Compared to the negative control 61, compound 13 significantly and selectively downregulated the abundance of AKT1 and AKT2, out of over 8000 quantified proteins, demonstrating that degrader 13 is highly selective for AKT1 and AKT2 (Figure 12A). Similarly, compared to the negative control 62, CRBN-recruiting compound 25 significantly reduced the protein levels of AKT1 and AKT2, as well as 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and the CRBN neo-substrate ZFP91, again suggesting that 25 is a selective degrader of AKT1 and AKT2 (Figure 12B). We did not detect AKT3 in these proteomic studies, in agreement with our WB analysis results (Figure S4). Overall, the results from our MS-based proteomic studies suggest that 13 and 25 are highly selective AKT degraders.

Figure 12.

Compounds 13 (A) and 25 (B) selectively degraded AKT1 and AKT2 in unbiased global MS-based proteomic studies. BT474 cells were treated with 13, 61, 25, or 62 at 1 μM for 24 h before they were collected for mass spectrometry analysis. The log₂ fold change is shown on the x axis and the negative log₁₀ p value is shown on the y axis for two independent biological replicates of each treatment.

Compounds 13 and 25 Effectively Inhibited the Proliferation in Multiple Human Cancer Cell Lines.

We first evaluated the antiproliferative activities of 13 and 25 in BT474 cells, with GDC-0068, 61, 62, and INY-03–041 as controls (Figure 13A). Both VHL-recruiting degrader 13 (GI₅₀ = 1.3 ± 0.3 μM) and CRBN-recruiting degrader 25 (GI₅₀ = 0.7 ± 0.2 μM) effectively inhibited the proliferation in BT474 cells. Their potencies were slightly weaker than those of GDC-0068 (GI₅₀ = 0.3 ± 0.03 μM) and INY-03–041 (GI₅₀ = 0.4 ± 0.2 μM). Of note, 13 was 2- to 3-fold more potent than its control compound, 61 (GI₅₀ = 3.1 ± 0.4 μM), and 25 showed 5-fold improved potency over its control compound, 62 (GI₅₀ = 3.3 ± 0.8 μM), suggesting that the antiproliferation activities of 13 and 25 are partially due to their AKT degradation effect. We next examined their antiproliferation activities in two additional cancer cell lines: PC3 and MDA-MB-468 (a triple-negative breast cell line). Both 13 and 25 effectively inhibited the growth in these two cancer cell lines, with comparable potencies to GDC-0068 and INY-03–041 (Figure 13B,C). Next, we evaluated the effect of these compounds on cell apoptosis in MDA-MB-468 cells and found that neither the AKT inhibitor GDC-0068 nor degraders 13 and 25 induced more than 3% apoptosis (Figure S5). Therefore, these AKT degraders inhibited the growth of MDA-MB-468 cells mainly through an antiproliferative effect. Collectively, these results indicate that AKT degraders 13 and 25 can suppress the growth in multiple human cancer cell lines.

Figure 13.

AKT degraders 13 and 25 effectively inhibited the proliferation in multiple cancer cell lines. BT474 (A), PC3 (B), and MDA-MB-468 (C) cells were treated with the indicated compound at indicated concentrations for 5 days. Cell growth was determined using calculations derived from phase-contrast images in IncuCyte, and error bars indicate the standard errors from three independent experiments.

Compounds 13 and 25 Were Bioavailable in Mice.

We next examined in vivo mouse pharmacokinetic (PK) properties of compounds 13 and 25 following a single intraperitoneal (IP) injection of each compound at a dose of 50 mg/kg. The maximum plasma concentration (C_max) of 13 reached approximately 3.5 μM at 2 h, and the plasma concentrations remained above 3 μM over 8 h (Figure 14A). Similarly, compound 25 was also bioavailable in mice via IP injection (Figure 14B). Although the C_max (1.4 μM at 2 h) of 25 was about 2.5-fold less than that of 13, its exposure levels in plasma were still good. The sufficient in vivo mouse PK properties of 13 and 25 make them suitable for in vivo efficacy studies. The in vivo PK properties of INY-03–041 have not been reported.⁴⁷ It is also worth noting that both 13 and 25 were well tolerated in the mouse PK studies. No clinical signs were observed in the test mice.

Figure 14.

AKT degraders 13 and 25 were bioavailable in mouse PK studies. Plasma concentrations of 13 (A) and 25 (B) following a single 50 mg/kg IP injection over 8 h in male Swiss albino mice. Experiments were carried out in biological triplicates, with points representing mean concentrations ± SEM.

Synthesis.

Synthetic routes for intermediates 1 and 2 are outlined in Scheme 1. The amide coupling reaction between known compounds 63 and 64 and subsequent deprotection of the Boc group under acidic conditions yielded intermediate 65.¹⁸ The nucleophilic substitution of commercially available ethyl 3-bromopropanoate (66) with 65 followed by the hydrolysis of the ethyl ester afforded the carboxylic acid precursor 1. The amine intermediate 2 was prepared in a similar manner from benzyl (2-iodoethyl)carbamate (67) and 65 through nucleophilic substitution, Boc protection, and Cbz deprotection.

Scheme 1. Synthesis of Intermediates 1 and 2^a.

^aReaction conditions: (a) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxy-7-azabenzo-triazole (HOAt), N-methylmorpholine (NMM), DMSO, rt, 12 h; (b) TFA/DCM, rt, 30 min; (c) ethyl 3-bromopropanoate (66), K₂CO₃, DMF, 80 °C, 12 h; (d) LiOH, THF, H₂O, rt, 12 h; (e) benzyl (2-iodoethyl)carbamate (67), K₂CO₃, CH₃CN, 80 °C, 8 h; (f) Boc₂O, Et₃N, DCM, rt, 2 h; and (g) H₂, Pd/C, MeOH, rt, 1 h.

Compounds 3–11 were prepared by amide coupling reactions between linker-attached VHL ligands 68–76⁶¹ and intermediate 1 (Scheme 2A). The synthesis of 12 and 13 is outlined in Scheme 2B. Briefly, the amide coupling reaction between the VHL-2 ligand (77) and Boc protected amino acid 78 followed by Boc deprotection afforded amine intermediate 79, which was coupled with intermediate 1 to provide 12. Similarly, the amide coupling between 77 and diacid 80 provided acid intermediate 81, which was converted to 13 by an amide coupling reaction followed by Boc deprotection.

Scheme 2. Synthesis of GDC-0068-Based VHL-Recruiting Compounds 3–11 (A) and 12 and 13 (B)^a.

^aReaction conditions: (a) EDCI, HOAt, NMM, DMSO, rt, 12 h and (b) TFA/DCM, rt, 30 min.

Synthetic routes for compounds 14–29 are included in Scheme 3. Compounds 14–25 were synthesized using the amide coupling reactions between intermediate 1 and different linker-attached pomalidomide analogs 82–93⁶¹ (Scheme 3A). Similarly, compound 26 was synthesized through the amide coupling reaction between intermediate 2 and 94 (Scheme 3B). The synthesis of compound 27 commenced with the conversion of oct-7-yn-1-ol (95) to its corresponding oct-7-yn-1-yl 4-methylbenzenesulfonate (96), which was subjected to the substitution with sodium cyanide to generate non-8-ynenitrile (97). The Sonogashira coupling reaction between 97 and commercially available 4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione followed by the hydrogenation reaction to reduce alkynyl and cyanide groups furnished intermediate 99. Amide coupling between 99 and intermediate 1 yielded compound 27 (Scheme 3C). Compounds 28 and 29 were prepared from tert-butyl (8-iodoctyl)carbamate (100) and 101 or 102 through a sequence of nucleophilic substitution, Boc-deprotection, and amide coupling reaction with intermediate 1 (Scheme 3D).⁶¹

Scheme 3. Synthesis of GDC-0068-Based CRBN-Recruiting Compounds 14–25 (A), 26 (B), 27 (C), and 28 and 29 (D)^a.

^aReaction conditions: (a) EDCI, HOAt, NMM, DMSO, rt, 12 h; (b) TFA/DCM, rt, 30 min; (c) 4-toluenesulfonyl chloride, NEt₃, DCM, 0 °C to rt, 4 h; (d) NaCN, NaI, DMF, 50 °C, 8 h; (e) 4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, Pd(PPh₃)₂Cl₂, CuI, NEt₃, DMF, 70 °C, 2 h; (f) Raney Ni, H₂, MeOH/NH₃.H₂O (3/1), rt, 1 h; and (g) NaHCO₃, KI, DMF, 60 °C, overnight.

GSK690693-based degraders 31–60 were prepared following the synthetic route outlined in Scheme 4. Reductive amination between 105¹⁵ and methyl 4-oxobutanoate followed by Boc-protection afforded intermediate 106. The subsequent Sonogashira coupling of 106 and 2-methylbut-3-yn-2-ol yielded 107, which was converted to compound 30 through an ester hydrolysis reaction. Amide coupling reactions between intermediate 30 and linker-attached E3 ligase ligands 68–76, 82–93, and 108–116 followed by Boc deprotection afforded the designed bifunctional compounds 31–60.

Scheme 4. Synthesis of Compounds 31–60^a.

^aReaction conditions: (a) methyl 4-oxobutanoate, NaBH(OAc)₃, DCM, rt; (b) Boc₂O, Et₃N, DCM, rt; (c) 2-methylbut-3-yn-2-ol, Pd(PPh₃)₄, Zn powder, DBU, NaI, DMSO, 80 °C; (d) NaOH (3 N), MeOH, 60 °C; (e) 68–76, 108–116, and 82–93, EDCI, HOAt, NMM, DMSO, rt, 12 h; and (f) TAF/DCM, rt, 30 min.

Synthetic routes for compounds 61 and 62 are outlined in Scheme 5. Intermediate 118 was prepared by amide coupling between intermediate 117 and commercially available dodecanedioic acid (80). Amide coupling between intermediate 2 and 118 followed by Boc removal provided 61. The nucleophilic aromatic substitution (S_NAr) of intermediate 119 with tert-butyl(8-aminoctyl)carbamate (120) and the subsequent Boc-deprotecting reaction afforded intermediate 121.⁶¹ Amide coupling of 121 and intermediate 1 provided compound 62.

Scheme 5. Synthesis of Negative Control Compounds 61 and 62^a.

^aReaction conditions: (a) EDCI, HOAt, NMM, DMSO, rt, 12 h; (b) TFA/DCM, rt, 30 min; and (c) NMP, DIPEA, MW, 100 °C, 1 h.

CONCLUSIONS

To date, only two AKT PROTAC degraders and no SAR studies have been reported. We conducted comprehensive SAR studies by the design, synthesis, and evaluation of a large set of novel compounds to explore various AKT binders, linkers, and E3 ligase ligands. Through these extensive SAR studies, we discovered multiple promising AKT PROTAC degraders derived from AKT inhibitors GDC-0068 and GSK690693, including compounds 13, 25, 48, and 60. Among them, the GDC-0068-based VHL-recruiting compound 13 and CRBN-recruiting compound 25 were further characterized in biochemical, cellular, proteomic, and PK studies. We also developed the corresponding negative controls of 13 and 25, compounds 61 and 62, which maintained good binding affinity to AKT but were unable to bind VHL and CRBN, respectively. We show that 13 and 25, but not 61 and 62, concentration- and time-dependently reduced the protein levels of T-AKT and P-AKT and inhibited the downstream signaling such as P-PRAS40 and P-S6 in BT474 cells. Through a series of rescue experiments, we demonstrate that the AKT degradation and downstream signaling inhibition induced by 13 and 25 were mediated by the UPS and required the engagement with AKT and the corresponding E3 ligase (VHL or CRBN). Using unbiased quantitative MS-based global proteomic studies, we show that both 13 and 25 are highly selective AKT degraders. Moreover, compounds 13 and 25 effectively suppressed the proliferation in multiple human cancer cell lines. Lastly, both 13 and 25 were bioavailable in mouse PK studies via IP injection and could be suitable for in vivo efficacy studies. Overall, our comprehensive SAR studies resulted in the discovery of multiple novel, potent ,and selective AKT PROTAC degraders.

EXPERIMENTAL SECTION

Chemistry General Procedures.

All commercially available chemical reagents were used directly in syntheses without further purification. Microwave-heated reactions were performed with a Discover SP microwave system with an Explorer 12 Hybrid Autosampler by CEM (Buckingham, UK). A Teledyne ISCO CombiFlash Rf⁺ instrument and HP C18 RediSep Rf reverse phase columns equipped with UV detector were used to conduct flash chromatography. All final compounds for biological evaluation were purified with preparative high-performance liquid chromatography (HPLC) on an Agilent Prep 1200 series with the UV detector set to 254 or 220 nm with a flow rate of 40 mL/min at room temperature. Crude samples were injected into a Phenomenex Luna 750 × 30 mm, 5 μm C18 column, with the gradient program set to 10% of methanol or acetonitrile (B) in H₂O containing 0.1% TFA (A) progressing from 10 to 100% of methanol or acetonitrile (B). The purity of all compounds for biological activity was >95% as assessed by HPLC-HRMS. All HPLC spectra was obtained by using an Agilent 1200 series system with a DAD detector and a 2.1 × 150 mm Zorbax 300SB-C18 5 μm column for chromatography. Samples (0.5 μL) were injected onto a C18 column at room temperature with the flow rate of 0.4 mL/min. Chromatography was performed with the solvent as follows: water containing 0.1% formic acid was designated as solvent A, while acetonitrile containing 0.1% formic acid was designated as solvent B. The linear gradient was set such that 1% B was used from 0 to 1 min, 1–99% B from 1 to 4 min, and 99% B from 4 to 8 min. High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. All compounds were also characterized using either a Bruker (Billerica, MA) DRX Nuclear Magnetic Resonance (NMR) spectrometer (400, 500, and 600 MHz, ¹H NMR) or a Bruker DXI 800 MHz spectrometer (800 MHz ¹H NMR, 201 MHz ¹³C NMR). Chemical shifts for all compounds are reported in units of parts per million (ppm, δ) relative to residual solvent peaks. ¹H NMR data are reported in the following format: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), coupling constant, and integration.

Synthetic route, procedures, and characterization of compounds 68–76, 82–93, and 108–116 are depicted in the Supporting Information.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)propanoic Acid (1).

To a solution of intermediate 65¹⁸ (360 mg, 0.86 mmol) in DMF (10 mL) was added potassium carbonate (358 mg, 2.6 mmol, 3 equiv). The resulting suspension was stirred at 80 °C for 15 min before ethyl 3-bromopropanoate (66, 310 mg, 1.72 mmol, 2 equiv) was added. After the reaction was stirred overnight, water was added and the mixture was extracted with ethyl acetate (3 × 10 mL), dried over Na₂SO₄, filtered, and evaporated. The resulting mixture was purified by flash chromatography (DCM/ MeOH = 10:1) to afford the product ethyl 3-(((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-propanoate (257 mg, yield 58%). ESI [M + H]⁺ (m/z): 416.2. The obtained intermediate was dissolved in THF/H₂O (1:1). To the solution was added lithium hydroxide (24 mg, 1 mmol). After stirring overnight at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase column (10–100% methanol/0.1% TFA in H₂O) to afford intermediate 1 as a white solid in TFA salt form (238 mg, yield 97%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (d, J = 4.5 Hz, 1H), 7.47 (dt, J = 8.7, 2.3 Hz, 2H), 7.36 (dd, J = 8.4, 6.0 Hz, 2H), 5.28 (t, J = 7.9 Hz, 1H), 4.50 (ddd, J = 9.7, 6.6, 4.1 Hz, 1H), 4.37 (td, J = 7.9, 4.6 Hz, 1H), 4.24–4.10 (m, 1H), 4.09–4.01 (m, 1H), 3.95–3.81 (m, 4H), 3.65 (dd, J = 12.9, 8.9 Hz, 3H), 3.52–3.38 (m, 2H), 3.21 (dd, J = 12.8, 4.6 Hz, 1H), 2.78 (t, J = 6.4 Hz, 2H), 2.28 (dd, J = 12.9, 7.4 Hz, 1H), 2.17 (ddt, J = 12.6, 8.3, 4.1 Hz, 1H), 1.19 (dd, J = 21.1, 7.0 Hz, 3H). HRMS (m/z) for C₂₄H₃₁ClN₅O ⁺ [M + H]⁺: calculated 488.2059, found 488.2057.

tert-Butyl (2-aminoethyl)((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)carbamate (2).

To a solution of intermediate 65 (467 mg, 1.12 mmol, 1.1 equiv) in CH₃CN was added potassium carbonate (706 mg, 5 mmol, 5 equiv). After the resulting suspension was stirred at 80 °C for 15 min, benzyl (2-iodoethyl)carbamate (67, 305 mg, 1.0 mmol) was added. After the reaction was stirred at 80 °C for 8 h, the reaction mixture was filtered, and the filtrate was concentrated. The resulting residue was purified by preparative HPLC to afford the desired intermediate as a white solid (201 mg, yield 30%). The white solid (201 mg, 0.34 mmol) was dissolved in DCM (5 mL). To the resulting solution were added triethylamine (92 μL, 0.68 mmol, 2 equiv) and di-tert-butyl dicarbonate (89 mg, 0.4 mmol, 1.2 equiv). After the reaction was stirred at room temperature for 2 h, the solvent was removed under reduced pressure. The resulting residue was purified by flash chromatography (MeOH/DCM = 1:9). The product was obtained as a white solid (160 mg, yield 68%). This product was dissolved in methanol (6 mL) followed by the addition of 10% palladium on carbon (16 mg, 10% of weight). After stirring under H₂ for 4 h, the mixture was filtered, and the filtrate was concentrated. The resulting mixture was purified by preparative HPLC to afford the title compound 2 as a white solid in TFA salt form (91 mg, yield 71%). 1H NMR (600 MHz, CD₃OD) δ 8.58 (d, J = 3.7 Hz, 1H), 7.44–7.35 (m, 2H), 7.32 (s, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.32 (s, 1H), 4.21 (s, 1H), 3.98 (d, J = 8.2 Hz, 1H), 3.85 (s, 2H), 3.79–3.65 (m, 6H), 3.57 (d, J = 13.4 Hz, 2H), 3.51–3.41 (m, 2H), 3.03 (s, 1H), 2.32–2.25 (m, 1H), 2.21–2.16 (m, 1H), 1.41 (s, 9H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₂₈H₄₀ClN₆O₄⁺ [M + H]⁺: calculated 559.2794, found 559.2782.

(2S,4R)-1-((2S,15S)-2-(tert-Butyl)-15-(4-chlorophenyl)-16-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-pyrimidin-4-yl)piperazin-1-yl)-4,10,16-trioxo-6-oxa-3,9,13-triaza-hexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (3).

To a solution of intermediate 1 (12 mg, 0.02 mmol) in DMSO (1 mL) were added linker 68 (11.3 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole, 4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-methylmorpholine, 6.1 mg, 0.06 mmol, 3.0 equiv). After stirring overnight at room temperature, the reaction mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H O) to afford title compound 3 as a white solid in TFA salt form (18.2 mg, yield 91%). ¹H NMR (600 MHz, CD₃OD) δ 8.97 (s, 1H), 8.58 (s, 1H), 7.54–7.39 (m, 6H), 7.39–7.30 (m, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.71 (s, 1H), 4.61–4.45 (m, 4H), 4.42–4.36 (m, 1H), 4.17 (s, 1H), 4.08–4.03 (m, 1H), 3.99–3.76 (m, 7H), 3.71–3.54 (m, 7H), 3.47–3.36 (m, 3H), 3.28–3.23 (m, 1H), 2.80–2.64 (m, 3H), 2.48 (s, 3H), 2.33–2.22 (m, 2H), 2.17 (dt, J = 12.7, 8.2 Hz, 1H), 2.09 (ddd, J = 13.4, 9.4, 4.4 Hz, 1H), 1.17 (d, J = 6.9 Hz, 3H), 1.04 (s, 9H). HRMS (m/z) for C₅₀H₆₆ClN₁₀O₈S⁺ [M + H]⁺: calculated 1001.4469, found 1001.4472.

(2S,4R)-1-((2S,18S)-2-(tert-Butyl)-18-(4-chlorophenyl)-19-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-pyrimidin-4-yl)piperazin-1-yl)-4,13,19-trioxo-6,9-dioxa-3,12,16-tri-azanonadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (4).

Compound 4 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 69 (12.3 mg, 0.02 mmol, 1.0 equiv). Compound 4 was obtained as a white solid in TFA salt form (9.4 mg, yield 45%). ¹H NMR (600 MHz, CD₃OD) δ 8.95 (s, 1H), 8.57 (s, 1H), 7.53–7.37 (m, 6H), 7.34 (dd, J = 8.5, 2.1 Hz, 2H), 5.31 (t, J = 7.9 Hz, 1H), 4.75 (s, 1H), 4.63–4.45 (m, 4H), 4.40 (d, J = 15.5 Hz, 1H), 4.17 (s, 1H), 4.03 (d, J = 2.0 Hz, 2H), 3.96–3.79 (m, 6H), 3.74–3.47 (m, 13H), 3.39 (t, J = 9.5 Hz, 1H), 3.28–3.22 (m, 3H), 2.68 (t, J = 6.3 Hz, 1H), 2.47 (s, 3H), 2.32–2.24 (m, 2H), 2.17 (dt, J = 12.7, 8.1 Hz, 1H), 2.08 (ddd, J = 13.5, 9.6, 4.3 Hz, 1H), 1.17 (dd, J = 7.0, 2.0 Hz, 3H), 1.04 (s, 9H). HRMS (m/z) for C₅₂H₇₀ClN₁₀O₉S⁺ [M + H]⁺: calculated 1045.4731, found 1045.4738.

(2S,4R)-1-((2S,25S)-2-(tert-Butyl)-25-(4-chlorophenyl)-26-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-pyrimidin-4-yl)piperazin-1-yl)-4,20,26-trioxo-7,10,13,16-tetraoxa-3,19,23-triazahexacosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5).

Compound 5 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 70 (14.3 mg, 0.02 mmol, 1.0 equiv). Compound 5 was obtained as a white solid in TFA salt form (11.2 mg, yield 48%). ¹H NMR (600 MHz, CD₃OD) δ 8.95 (s, 1H), 8.58 (s, 1H), 7.54–7.39 (m, 6H), 7.39–7.28 (m, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.65 (s, 1H), 4.60–4.47 (m, 4H), 4.36 (d, J = 15.5 Hz, 1H), 4.18 (s, 1H), 3.98–3.77 (m, 6H), 3.76–3.57 (m, 19H), 3.53 (t, J = 5.4 Hz, 2H), 3.44–3.33 (m, 4H), 3.29–3.23 (m, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.58 (ddd, J = 15.0, 7.5, 5.2 Hz, 1H), 2.48 (s, 3H), 2.33–2.26 (m, 1H), 2.25–2.15 (m, 2H), 2.12–2.04 (m, 1H), 1.17 (d, J = 6.9 Hz, 3H), 1.04 (s, 9H). HRMS (m/z) for C₅₇H₈₀ClN₁₀O₁₁S⁺ [M + H]⁺: calculated 1147.5412, found 1147.5434.

(2S,4R)-1-((S)-2-(5-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino)propanamido)pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-benzyl)pyrrolidine-2-carboxamide (6).

Compound 6 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 71 (11.3 mg, 0.02 mmol, 1.0 equiv). Compound 6 was obtained as a white solid in TFA salt form (7.6 mg, yield 38%). ¹H NMR (600 MHz, CD₃OD) δ 8.93 (s, 1H), 8.57 (s, 1H), 7.51–7.39 (m, 6H), 7.39–7.32 (m, 2H), 5.31 (t, J = 7.9 Hz, 1H), 4.62 (s, 1H), 4.59–4.48 (m, 4H), 4.36 (d, J = 15.4 Hz, 1H), 4.18 (s, 1H), 3.96–3.86 (m, 4H), 3.81 (dt, J = 10.9, 6.1 Hz, 2H), 3.70–3.60 (m, 5H), 3.40 (t, J = 8.9 Hz, 1H), 3.27 (dd, J = 12.6, 3.8 Hz, 2H), 3.22–3.16 (m, 2H), 2.67–2.63 (m, 2H), 2.47 (s, 3H), 2.33–2.26 (m, 3H), 2.23–2.16 (m, 2H), 2.08 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 1.65–1.57 (m, 2H), 1.51 (t, J = 7.4 Hz, 2H), 1.17 (d, J = 7.3 Hz, 3H), 1.03 (s, 9H). HRMS (m/z) for C₅₁H₆₈ClN₁₀O₇S⁺ [M +H]⁺: calculated 999.4676, found 999.4678.

(2S,4R)-1-((S)-2-(6-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino)propanamido)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-benzyl)pyrrolidine-2-carboxamide (7).

Compound 7 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 72 (11.6 mg, 0.02 mmol, 1.0 equiv). Compound 7 was obtained as a white solid in TFA salt form (12.7 mg, yield 63%). ¹H NMR (600 MHz, CD₃OD) δ 8.97 (s, 1H), 8.58 (d, J = 4.4 Hz, 1H), 7.54–7.38 (m, 6H), 7.38–7.32 (m, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.63 (s, 1H), 4.60–4.47 (m, 4H), 4.37 (d, J = 15.4 Hz, 1H), 4.18 (s, 1H), 3.97–3.78 (m, 6H), 3.72–3.54 (m, 5H), 3.40 (dd, J = 10.8, 7.1 Hz, 1H), 3.29–3.24 (m, 2H), 3.18 (t, J = 7.1 Hz, 2H), 2.67–2.61 (m, 2H), 2.48 (s, 3H), 2.34–2.14 (m, 5H), 2.08 (ddd, J = 13.3, 9.2, 4.5 Hz, 1H), 1.66–1.57 (m, 2H), 1.50 (q, J = 7.3 Hz, 2H), 1.38–1.30 (m, 2H), 1.17 (d, J = 7.1 Hz, 3H), 1.03 (s, 9H). HRMS (m/z) for C₅₂H₇₀ClN₁₀O₇S⁺ [M + H]⁺: calculated 1013.4833, found 1013.4847.

(2S,4R)-1-((S)-2-(7-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino)propanamido)heptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-benzyl)pyrrolidine-2-carboxamide (8).

Compound 8 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 73 (11.9 mg, 0.02 mmol, 1.0 equiv). Compound 8 was obtained as a white solid in TFA salt form (8.6 mg, yield 42%). ¹H NMR (600 MHz, CD₃OD) δ 8.95 (s, 1H), 8.58 (s, 1H), 7.54–7.38 (m, 6H), 7.36 (t, J = 6.6 Hz, 2H), 5.38–5.26 (m, 1H), 4.64 (s, 1H), 4.61–4.48 (m, 4H), 4.37 (d, J = 15.6 Hz, 1H), 4.18 (s, 1H), 4.02–3.81 (m, 6H), 3.73–3.53 (m, 5H), 3.41 (s, 1H), 3.27 (d, J = 6.0 Hz, 2H), 3.23–3.10 (m, 2H), 2.72–2.57 (m, 2H), 2.48 (s, 3H), 2.33–2.17 (m, 5H), 2.10 (s, 1H), 1.66–1.58 (m, 2H), 1.53–1.46 (m, 2H), 1.39–1.31 (m, 4H), 1.17 (d, J = 7.5 Hz, 3H), 1.03 (s, 9H). HRMS (m/z) for C₅₃H₇₂ClN₁₀O₇S⁺ [M + H]⁺: calculated 1027.4989, found 1027.4983.

(2S,4R)-1-((S)-2-(9-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino)propanamido)nonanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-benzyl)pyrrolidine-2-carboxamide (9).

Compound 9 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 74 (12.4 mg, 0.02 mmol, 1.0 equiv). Compound 9 was obtained as a white solid in TFA salt form (9.9 mg, yield 47%). ¹H NMR (600 MHz, CD₃OD) δ 8.96 (s, 1H), 8.58 (d, J = 3.7 Hz, 1H), 7.55–7.39 (m, 6H), 7.39–7.32 (m, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.63 (s, 1H), 4.60–4.48 (m, 4H), 4.36 (d, J = 15.5 Hz, 1H), 4.18 (s, 1H), 3.97–3.78 (m, 6H), 3.73–3.60 (m, 5H), 3.40 (t, J = 8.9 Hz, 1H), 3.29–3.23 (m, 2H), 3.17 (dd, J = 7.8, 6.3 Hz, 2H), 2.67–2.61 (m, 2H), 2.48 (s, 3H), 2.34–2.14 (m, 5H), 2.08 (s, 1H), 1.59 (d, J = 7.0 Hz, 2H), 1.48 (d, J = 7.1 Hz, 2H), 1.35–1.29 (m, 8H), 1.18 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H). HRMS (m/z) for C₅₅H₇₆ClN₁₀O₇S⁺ [M + H]⁺: calculated 1055.5302, found 1055.5303.

(2S,4R)-1-((S)-2-(10-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino)propanamido)decanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)-benzyl)pyrrolidine-2-carboxamide (10).

Compound 10 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 75 (12.7 mg, 0.02 mmol, 1.0 equiv). Compound 10 was obtained as a white solid in TFA salt form (2.8 mg, yield 13%). ¹H NMR (600 MHz, CD₃OD) δ 8.93 (s, 1H), 8.58 (s, 1H), 7.54–7.40 (m, 6H), 7.36 (d, J = 8.5 Hz, 2H), 5.31 (d, J = 8.2 Hz, 1H), 4.63 (s, 1H), 4.61–4.43 (m, 4H), 4.36 (d, J = 15.3 Hz, 1H), 4.19 (s, 1H), 4.03–3.78 (m, 6H), 3.76–3.56 (m, 5H), 3.49–3.39 (m, 1H), 3.17 (d, J = 7.7 Hz, 2H), 3.03–2.95 (m, 1H), 2.91–2.83 (m, 1H), 2.65 (d, J = 8.9 Hz, 2H), 2.47 (s, 3H), 2.36–2.14 (m, 5H), 2.13–2.06 (m, 1H), 1.69–1.56 (m, 2H), 1.56–1.46 (m, 2H), 1.41–1.25 (m, 10H), 1.18 (d, J = 7.0 Hz, 3H), 1.04 (s, 9H). HRMS (m/z) for C₅₆H₇₈ClN₁₀O₇S⁺ [M + H]⁺: calculated 1069.5459, found 1069.5464.

(2S,4R)-1-((S)-2-(11-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino) propanam ido)-undecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11).

Compound 11 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12 mg, 0.02 mmol) and 76 (13.0 mg, 0.02 mmol, 1.0 equiv). Compound 11 was obtained as a white solid in TFA salt form (11.3 mg, yield 52%). ¹H NMR (600 MHz, CD₃OD) δ 8.96 (s, 1H), 8.58 (d, J = 3.9 Hz, 1H), 7.53–7.38 (m, 6H), 7.38–7.28 (m, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.63 (s, 1H), 4.60–4.44 (m, 4H), 4.36 (d, J = 15.5 Hz, 1H), 4.18 (s, 1H), 3.97–3.78 (m, 6H), 3.72–3.59 (m, 5H), 3.45–3.36 (m, 1H), 3.29–3.24 (m, 2H), 3.17 (t, J = 7.1 Hz, 2H), 2.67–2.62 (m, 2H), 2.48 (s, 3H), 2.34–2.16 (m, 5H), 2.12–2.04 (m, 1H), 1.60 (dt, J = 15.2, 7.4 Hz, 2H), 1.49 (t, J = 7.0 Hz, 2H), 1.36–1.26 (m, 12H), 1.18 (d, J = 6.9 Hz, 3H), 1.03 (s, 9H). HRMS (m/z) for C₅₇H₈₀ClN₁₀O₇S⁺ [M + H]⁺: calculated 1083.5615, found 1083.5637.

(2S,4R)-1-((S)-2-(11-(3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-piperazin-1-yl)-3-oxopropyl)amino) propanam ido)-undecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (12).

Compound 12 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.01 mg, 0.01 mmol) and 79 (7.4 mg, 0.01 mmol, 1.0 equiv). Compound 12 was obtained as a white solid in TFA salt form (6.5 mg, yield 59%). ¹H NMR (600 MHz, CD₃OD) δ 8.87 (s, 1H), 8.52 (s, 1H), 7.43 (dt, J = 15.8, 8.5 Hz, 6H), 7.37 (dd, J = 8.6, 6.8 Hz, 2H), 5.19 (t, J = 7.5 Hz, 1H), 5.03–4.98 (m, 1H), 4.62 (s, 1H), 4.59–4.50 (m, 2H), 4.43 (s, 1H), 4.13–4.03 (m, 1H), 4.02–3.90 (m, 1H), 3.91–3.70 (m, 7H), 3.62 (t, J = 11.0 Hz, 4H), 3.59–3.51 (m, 1H), 3.43–3.37 (m, 1H), 3.26 (dd, 1H), 3.17 (t, J = 7.2 Hz, 2H), 2.65 (t, J = 6.0 Hz, 2H), 2.47 (s, 3H), 2.32–2.27 (m, 1H), 2.26–2.14 (m, 3H), 1.98–1.90 (m, 1H), 1.50 (d, J = 7.0 Hz, 3H), 1.31 (s, 16H), 1.15 (d, J = 7.0 Hz, 3H), 1.04 (s, 9H). HRMS (m/z) for C₅₈H₈₂ClN₁₀O₇S⁺ [M + H]⁺: calculated 1097.5772, found 1097.57567.

N¹-(2-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)ethyl)-N¹²-((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)dodecanediamide (13).

Compound 13 was synthesized following the standard amide coupling procedure for preparing compound 3 from intermediate 2 (11.2 mg, 0.01 mmol) and 81 (13.1 mg, 0.01 mmol, 1.0 equiv). The resulting product was dissolved in TFA (1 mL) and DCM (1 mL). The mixture was stirred at room temperature for 30 min. The solvent was removed, and the mixture was purified by preparative HPLC. Compound 13 was obtained as a white solid in TFA salt form (17.4 mg, yield 79%). ¹H NMR (600 MHz, CD₃OD) δ 8.98 (s, 1H), 8.58 (s, 1H), 7.49–7.41 (m, 6H), 7.36 (dd, J = 8.5, 1.8 Hz, 2H), 5.31 (t, J = 8.0 Hz, 1H), 5.00 (q, J = 7.0 Hz, 1H), 4.62 (s, 1H), 4.59–4.50 (m, 2H), 4.46–4.42 (m, 1H), 4.24–4.14 (m, 1H), 4.13–3.99 (m, 1H), 3.99–3.90 (m, 2H), 3.90–3.78 (m, 3H), 3.75 (dd, J = 11.0, 4.0 Hz, 1H), 3.73–3.59 (m, 4H), 3.51–3.46 (m, 2H), 3.46–3.36 (m, 1H), 3.35–3.27 (m, 2H), 3.19 (t, J = 5.7 Hz, 2H), 2.48 (s, 3H), 2.36–2.14 (m, 5H), 2.00–1.91 (m, 1H), 1.67–1.53 (m, 4H), 1.50 (d, J = 7.0 Hz, 3H), 1.39–1.24 (m, 12H), 1.24–1.12 (m, 3H), 1.04 (s, 9H). ¹³C NMR (151 MHz, CD₃OD) δ 176.70, 174.64, 171.83, 170.92, 169.36, 159.67, 151.49, 149.25, 147.64, 144.28, 134.38, 133.49, 131.98, 130.11, 129.62 (2C), 129.50 (2C), 129.10 (2C), 126.22 (2C), 126.06, 121.02, 70.78, 69.56, 67.64, 59.19, 57.60, 56.57, 50.23, 48.74, 48.64, 48.17, 45.49, 45.42, 44.99, 44.39, 41.40, 37.39, 36.43, 36.19, 35.74, 35.44, 35.27, 35.11, 29.16, 29.14, 29.04, 28.94, 28.93, 25.66 (3C), 25.30, 20.98, 19.10, 14.36. t_R = 4.07 min; HRMS (m/z) for C₅₈H₈₂ClN₁₀O₇S⁺ [M + H]⁺: calculated 1097.5772, found 1097.5756.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)propenamide (14).

Compound 14 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.0 mg, 0.01 mmol) and 82 (4.8 mg, 0.01 mmol, 1.0 equiv). Compound 14 was obtained as a yellow solid in TFA salt form (2.1 mg, yield 25%). ¹H NMR (600 MHz, CD₃OD) δ 8.56 (s, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.43 (d, J = 6.5 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.16–6.96 (m, 2H), 5.29 (t, J = 7.8 Hz, 1H), 5.07 (d, J = 12.1 Hz, 1H), 4.49 (s, 1H), 4.22–3.93 (m, 3H), 3.92–3.75 (m, 4H), 3.71 (s, 2H), 3.67–3.53 (m, 5H), 3.50 (s, 2H), 3.46–3.33 (m, 3H), 3.27–3.14 (m, 2H), 2.87 (t, J = 14.8 Hz, 1H), 2.79–2.62 (m, 4H), 2.31–2.22 (m, 1H), 2.22–2.05 (m, 2H), 1.16 (d, J = 6.9 Hz, 3H). HRMS (m/z) for C₄₁H₄₉ClN₉O₈⁺ [M + H]⁺: calculated 830.3387, found 830.3385.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)propenamide (15).

Compound 15 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.0 mg, 0.01 mmol) and 83 (5.0 mg, 0.01 mmol, 1.0 equiv). Compound 15 was obtained as a yellow solid in TFA salt form (5.8 mg, yield 64%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 7.59–7.53 (m, 1H), 7.45 (dd, J = 8.5, 1.8 Hz, 2H), 7.39–7.29 (m, 2H), 7.13–7.03 (m, 2H), 5.30 (t, J = 7.9 Hz, 1H), 5.07 (dd, J = 12.7, 5.5 Hz, 1H), 4.58–4.45 (m, 1H), 4.20–3.97 (m, 2H), 3.96–3.77 (m, 4H), 3.74–3.70 (m, 2H), 3.71–3.58 (m, 8H), 3.56 (t, J = 5.5 Hz, 2H), 3.51 (t, J = 5.2 Hz, 2H), 3.43–3.33 (m, 3H), 3.29–3.22 (m, 2H), 2.88 (ddd, J = 17.7, 13.8, 5.3 Hz, 1H), 2.80–2.67 (m, 2H), 2.67–2.60 (m, 2H), 2.28 (dd, J = 12.8, 7.5 Hz, 1H), 2.22–2.08 (m, 2H), 1.23–1.12 (m, 3H). HRMS (m/z) for C₄₃H₅₃ClN₉O₉⁺ [M + H]⁺: calculated 874.3649, found 874.3650.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy) ethoxy) ethoxy)ethyl)-propenamide (16).

Compound 16 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 84 (5.4 mg, 0.01 mmol, 1.0 equiv). Compound 16 was obtained as a yellow solid in TFA salt form (4.7 mg, yield 50%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 7.56 (ddd, J = 8.5, 7.0, 1.3 Hz, 1H), 7.49–7.41 (m, 2H), 7.39–7.29 (m, 2H), 7.15–6.99 (m, 2H), 5.30 (t, J = 7.9 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.56–4.48 (m, 1H), 4.16 (s, 1H), 4.05 (s, 1H), 3.95–3.78 (m, 4H), 3.72 (t, J = 5.1 Hz, 2H), 3.70–3.55 (m, 12H), 3.56–3.48 (m, 4H), 3.43–3.37 (m, 1H), 3.35 (q, J = 3.9, 2.6 Hz, 2H), 3.29–3.20 (m, 2H), 2.92–2.83 (m, 1H), 2.80–2.68 (m, 2H), 2.68–2.61 (m, 2H), 2.29 (dd, J = 12.8, 7.5 Hz, 1H), 2.21–2.09 (m, 2H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₅H₅₇ClN₉O₁₀⁺ [M + H]⁺: calculated 918.3911, found 918.3916.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)propenamide (17).

Compound 17 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (9.9 mg, 0.0165 mmol) and 85 (9.3 mg, 0.0165 mmol, 1.0 equiv). Compound 17 was obtained as a yellow solid in TFA salt form (3.5 mg, yield 22%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 7.60–7.52 (m, 1H), 7.49–7.41 (m, 1H), 7.37–7.24 (m, 3H), 7.10 (d, J = 8.6 Hz, 1H), 7.06 (d, J = 7.1 Hz, 1H), 5.29 (s, 1H), 5.13–5.00 (m, 1H), 4.58–4.42 (m, 1H), 4.16 (s, 1H), 4.03 (s, 1H), 3.95–3.74 (m, 4H), 3.72 (t, J = 5.2 Hz, 2H), 3.70–3.54 (m, 14H), 3.56–3.50 (m, 4H), 3.44–3.32 (m, 4H), 3.30–3.22 (m, 3H), 2.93–2.80 (m, 1H), 2.79–2.68 (m, 2H), 2.68–2.59 (m, 2H), 2.34–2.25 (m, 1H), 2.22–2.08 (m, 2H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₇H₆₁ClN₉O₁₁⁺ [M + H]⁺: calculated 962.4174, found 962.4167.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-propenamide (18).

Compound 18 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (9.9 mg, 0.0165 mmol) and 86 (10.0 mg, 0.0165 mmol, 1.0 equiv). Compound 18 was obtained as a yellow solid in TFA salt form (5.0 mg, yield 30%). ¹H NMR (600 MHz, CD₃OD) δ 8.58 (d, J = 3.6 Hz, 1H), 7.56 (dd, J = 8.6, 7.0 Hz, 1H), 7.49–7.42 (m, 1H), 7.39–7.22 (m, 3H), 7.10 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 7.1 Hz, 1H), 5.30 (t, J = 8.0 Hz, 1H), 5.10–5.05 (m, 1H), 4.56–4.44 (m, 1H), 4.16 (s, 1H), 4.11–3.97 (m, 1H), 3.96–3.77 (m, 4H), 3.72 (t, J = 5.2 Hz, 2H), 3.70–3.55 (m, 18H), 3.56–3.49 (m, 4H), 3.43–3.32 (m, 4H), 3.30–3.22 (m, 3H), 2.94–2.82 (m, 1H), 2.79–2.69 (m, 2H), 2.67 (dd, J = 10.2, 4.0 Hz, 2H), 2.34–2.23 (m, 1H), 2.22–2.06 (m, 2H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₉H₆₅ClN₉O₁₂⁺ [M + H]⁺: calculated 1006.4436, found 1006.4449.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)propenamide (19).

Compound 19 was synthesized following the standard procedure for preparing compound 3 from intermediate 2 (6.1 mg, 0.01 mmol) and 87 (4.3 mg, 0.01 mmol, 1.0 equiv). Compound 19 was obtained as a yellow solid in TFA salt form (5.8 mg, yield 74%). ¹H NMR (600 MHz, CD₃OD) δ 8.55 (s, 1H), 7.62–7.54 (m, 1H), 7.42–7.37 (m, 4H), 7.13 (d, J = 8.6 Hz, 1H), 7.05 (dd, J = 7.0, 2.3 Hz, 1H), 5.26 (t, J = 7.7 Hz, 1H), 5.10–5.06 (m, 1H), 4.57 (d, J = 9.1 Hz, 1H), 4.06 (s, 2H), 3.85 (t, J = 14.7 Hz, 3H), 3.76–3.56 (m, 4H), 3.56–3.40 (m, 4H), 3.39–3.32 (m, 2H), 3.29–3.24 (m, 2H), 2.92–2.79 (m, 1H), 2.73 (t, J = 18.1 Hz, 2H), 2.66 (t, J = 5.6 Hz, 2H), 2.25 (t, J = 10.2 Hz, 1H), 2.20–2.08 (m, 2H), 1.14 (dd, J = 7.0, 1.7 Hz, 3H). HRMS (m/z) for C₃₉H₄₅ClN₉O ⁺ [M + H]⁺: calculated 786.3125, found 786.3130.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)propenamide (20).

Compound 20 was synthesized following the standard procedure for preparing compound 14 from intermediate 1 (6.1 mg, 0.01 mmol) and 88 (4.4 mg, 0.01 mmol, 1.0 equiv). Compound 20 was obtained as a yellow solid in TFA salt form (7.8 mg, yield 97%). ¹H NMR (600 MHz, CD₃OD) δ 8.51 (s, 1H), 7.57–7.53 (m, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 7.08–7.02 (m, 2H), 5.20 (t, J = 7.6 Hz, 1H), 5.13–5.02 (m, 1H), 4.61–4.48 (m, 1H), 4.11–3.89 (m, 2H), 3.90–3.67 (m, 4H), 3.68–3.46 (m, 4H), 3.41 (t, J = 6.5 Hz, 2H), 3.39–3.31 (m, 3H), 3.28–3.21 (m, 2H), 2.91–2.81 (m, 1H), 2.81–2.55 (m, 4H), 2.28–2.02 (m, 3H), 1.85 (t, J = 6.6 Hz, 2H), 1.11 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₀H₄₇ClN₉O₇⁺ [M + H]⁺: calculated 800.3281, found 800.3284.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)propenamide (21).

Compound 21 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 89 (4.6 mg, 0.01 mmol, 1.0 equiv). Compound 21 was obtained as a yellow solid in TFA salt form (5.7 mg, yield 70%). ¹H NMR (600 MHz, CD₃OD) δ 8.53 (s, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.35 (dd, J = 8.4, 4.1 Hz, 2H), 7.05 (t, J = 9.5 Hz, 2H), 5.23 (s, 1H), 5.10–5.02 (m, 1H), 4.52 (s, 1H), 4.07 (d, J = 11.6 Hz, 1H), 4.03–3.89 (m, 1H), 3.89–3.71 (m, 4H), 3.71–3.50 (m, 4H), 3.42–3.33 (m, 3H), 3.26 (d, J = 15.5 Hz, 4H), 2.91–2.80 (m, 1H), 2.80–2.56 (m, 4H), 2.30–2.02 (m, 3H), 1.79–1.55 (m, 4H), 1.14 (d, J = 6.8 Hz, 3H). HRMS (m/z) for C₄₁H₄₉ClN₉O₇⁺ [M + H]⁺: calculated 814.3438, found 814.3443.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)propenamide (22).

Compound 22 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 90 (4.7 mg, 0.01 mmol, 1.0 equiv). Compound 22 was obtained as a yellow solid in TFA salt form (4.7 mg, yield 57%). ¹H NMR (600 MHz, CD₃OD) δ 8.54 (s, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.35 (dd, J = 8.4, 2.8 Hz, 2H), 7.04 (t, J = 7.8 Hz, 2H), 5.25 (s, 1H), 5.13–5.00 (m, 1H), 4.58–4.45 (m, 1H), 4.16–3.91 (m, 2H), 3.91–3.72 (m, 4H), 3.70–3.54 (m, 4H), 3.38–3.31 (m, 4H), 3.28–3.17 (m, 3H), 2.91–2.82 (m, 1H), 2.79–2.68 (m, 2H), 2.65 (d, J = 6.2 Hz, 2H), 2.28–2.21 (m, 1H), 2.21–2.14 (m, 1H), 2.13–2.07 (m, 1H), 1.73–1.65 (m, 2H), 1.57 (t, J = 7.5 Hz, 2H), 1.46 (d, J = 7.7 Hz, 2H), 1.22–1.07 (m, 3H). HRMS (m/z) for C₄₂H₅₁ClN₉O₇ ⁺ [M + H]⁺: calculated 828.3594, found 828.3597.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)propenamide (23).

Compound 23 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 91 (4.9 mg, 0.01 mmol, 1.0 equiv). Compound 23 was obtained as a yellow solid in TFA salt form (4.7 mg, yield 57%). ¹H NMR (600 MHz, CD₃OD) δ 8.55 (s, 1H), 7.55 (dd, J = 8.6, 7.1 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.39–7.27 (m, 2H), 7.04 (t, J = 7.2 Hz, 2H), 5.28 (t, J = 7.8 Hz, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.56–4.48 (m, 1H), 4.14 (s, 1H), 4.09–3.97 (m, 1H), 3.92–3.73 (m, 4H), 3.70–3.56 (m, 4H), 3.42–3.32 (m, 4H), 3.28–3.23 (m, 1H), 3.19 (t, J = 7.0 Hz, 2H), 2.90–2.81 (m, 1H), 2.77–2.68 (m, 2H), 2.68–2.60 (m, 2H), 2.27 (dd, J = 12.9, 7.4 Hz, 1H), 2.21–2.13 (m, 1H), 2.13–2.06 (m, 1H), 1.71–1.65 (m, 2H), 1.58–1.51 (m, J = 7.2 Hz, 2H), 1.49–1.43 (m, 2H), 1.40 (d, J = 7.2 Hz, 2H), 1.16 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₃H₅₃ClN₉O₇⁺ [M + H]⁺: calculated 842.3751, found 842.3758.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptyl)propenamide (24).

Compound 24 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 92 (5.2 mg, 0.01 mmol, 1.0 equiv). Compound 24 was obtained as a yellow solid in TFA salt form (8.8 mg, yield 98%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (d, J = 4.4 Hz, 1H), 7.55 (dd, J = 8.6, 7.1 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.39–7.29 (m, 2H), 7.04 (dd, J = 7.8, 6.1 Hz, 2H), 5.30 (t, J = 7.9 Hz, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.54–4.50 (m, 1H), 4.16 (s, 1H), 4.04 (d, J = 22.0 Hz, 1H), 3.97–3.76 (m, 4H), 3.71–3.58 (m, 4H), 3.40 (t, J = 8.9 Hz, 1H), 3.34 (d, J = 6.9 Hz, 2H), 3.29–3.25 (m, 2H), 3.18 (t, J = 6.8 Hz, 2H), 2.86 (ddd, J = 17.6, 14.0, 5.4 Hz, 1H), 2.79–2.67 (m, 2H), 2.63 (t, J = 6.3 Hz, 2H), 2.28 (dd, J = 12.7, 7.5 Hz, 1H), 2.21–2.15 (m, 1H), 2.14–2.08 (m, 1H), 1.69–1.63 (m, 2H), 1.54–1.50 (m, 2H), 1.47–1.30 (m, 6H), 1.17 (d, J = 6.9 Hz, 3H). HRMS (m/z) for C₄₄H₅₅ClN₉O₇⁺ [M + H]⁺: calculated 856.3907, found 856.3912.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)propenamide (25).

Compound 25 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (6.1 mg, 0.01 mmol) and 93 (5.3 mg, 0.01 mmol, 1.0 equiv). Compound 25 was obtained as a yellow solid in TFA salt form (2.8 mg, yield 32%). ¹H NMR (600 MHz, CD₃OD) δ 8.56 (s, 1H), 7.55 (dd, J = 8.6, 7.0 Hz, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.41–7.29 (m, 2H), 7.04 (dd, J = 7.8, 3.6 Hz, 2H), 5.28 (t, J = 7.8 Hz, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 1H), 4.52 (dd, J = 9.3, 4.1 Hz, 1H), 4.15 (s, 1H), 4.10–3.97 (m, 1H), 3.94–3.74 (m, 4H), 3.74–3.50 (m, 4H), 3.44–3.32 (m, 4H), 3.28–3.24 (m, 1H), 3.17 (t, J = 7.2 Hz, 2H), 2.90–2.81 (m, 1H), 2.78–2.67 (m, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.27 (dd, J = 12.9, 7.4 Hz, 1H), 2.21–2.14 (m, 1H), 2.14–2.06 (m, 1H), 1.71–1.61 (m, 2H), 1.53–1.25 (m, 10H), 1.16 (d, J = 7.0 Hz, 3H). ¹³C NMR (201 MHz, CD₃OD) δ 173.30, 171.00, 170.35, 169.44, 169.35, 167.92, 161.30, 159.61, 148.93, 146.92, 135.90, 134.30, 133.61, 132.49, 129.58 (2C), 129.47 (2C), 120.97, 116.66, 110.38, 109.54, 70.68, 50.20, 48.80, 45.53, 45.42, 44.96, 44.44, 44.36, 41.98, 41.45, 41.33, 39.06, 36.48, 30.84, 29.62, 28.82 (2C), 28.77 (2C), 26.40 (2C), 22.41, 19.15. t_R = 4.03 min; HRMS (m/z) for C₄₅H₅₇ClN₉O₇⁺ [M + H]⁺: calculated 870.4064, found 870.4078.

N-(2-(((S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)ethyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)nonanamide (26).

Compound 26 was synthesized following the standard procedure for preparing compound 13 from intermediate 2 (19.2 mg, 0.034 mmol) and 9-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)nonanoic acid (94, 14.7 mg, 0.034 mmol, 1.0 equiv). Compound 26 was obtained as a yellow solid in TFA salt form (22.1 mg, yield 74%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (d, J = 3.9 Hz, 1H), 7.58–7.32 (m, 5H), 7.12–6.90 (m, 2H), 5.30 (t, J = 7.9 Hz, 1H), 5.06 (ddd, J = 12.9, 5.6, 3.4 Hz, 1H), 4.64–4.52 (m, 1H), 4.24–3.31 (m, 15H), 3.24–3.15 (m, 2H), 2.94–2.79 (m, 2H), 2.76–2.57 (m, 1H), 2.34–2.02 (m, 5H), 1.71–1.49 (m, 4H), 1.41–1.27 (m, 8H), 1.16 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₅H₅₇ClN₉O₇⁺ [M + H]⁺: calculated 870.4064, found 870.4056.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(9-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)nonyl)propenamide (27).

Compound 27 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (9.7 mg, 0.02 mmol) and 4-(9-aminononyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (99, 8 mg, 0.025 mmol, 1.0 equiv). Compound 27 was obtained as a white solid in TFA salt form (11.2 mg, yield 64%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 7.74–7.68 (m, 2H), 7.65–7.59 (m, 1H), 7.47–7.42 (m, 2H), 7.36 (dd, J = 8.4, 1.2 Hz, 2H), 5.31 (t, J = 8.0 Hz, 1H), 5.17–5.10 (m, 1H), 4.54 (dd, J = 9.4, 4.1 Hz, 1H), 3.97–3.75 (m, 3H), 3.71–3.59 (m, 3H), 3.47–3.23 (m, 6H), 3.16 (t, J = 7.2 Hz, 2H), 3.12–3.06 (m, 2H), 2.94–2.84 (m, 1H), 2.80–2.69 (m, 2H), 2.67–2.62 (m, 2H), 2.32–2.25 (m, 1H), 2.21–2.11 (m, 2H), 1.69–1.62 (m, 2H), 1.48 (t, J = 7.1 Hz, 2H), 1.36 (s, 5H), 1.30 (d, J = 3.4 Hz, 5H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₆H₅₇ClN₉O₇⁺ [M + H]⁺: calculated 869.4112, found 869.4123.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)octyl)propenamide (28).

Compound 28 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12.2 mg, 0.025 mmol) and 3-(4-((8-aminooctyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (103, 9.7 mg, 0.025 mmol, 1.0 equiv). Compound 28 was obtained as a yellow solid in TFA salt form (8.8 mg, yield 41%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (s, 1H), 7.51–7.42 (m, 2H), 7.38–7.33 (m, 2H), 7.27 (ddd, J = 8.9, 6.9, 1.3 Hz, 1H), 7.21–7.12 (m, 1H), 7.00–6.90 (m, 1H), 5.30 (t, J = 7.9 Hz, 1H), 5.13 (dd, J = 13.4, 5.1 Hz, 1H), 4.57–4.48 (m, 1H), 4.36 (dd, J = 16.7, 1.1 Hz, 1H), 4.27 (d, J = 16.7 Hz, 1H), 4.19–4.12 (m, 1H), 3.97–3.46 (m, 8H), 3.44–3.23 (m, 6H), 3.21–3.10 (m, 2H), 3.02–2.81 (m, 2H), 2.68–2.59 (m, 2H), 2.51–2.43 (m, 1H), 2.32–2.24 (m, 1H), 2.21–2.10 (m, 2H), 1.56–1.43 (m, 4H), 1.39–1.24 (m, 8H), 1.24–1.08 (m, 3H). HRMS (m/z) for C₄₅H₅₉ClN₉O₆⁺ [M + H]⁺: calculated 856.4271, found 856.4264.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)octyl)propenamide (29).

Compound 29 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (12.2 mg, 0.025 mmol) and 4-((8-aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (104, 10 mg, 0.025 mmol, 1.0 equiv). Compound 29 was obtained as a white solid in TFA salt form (10.2 mg, yield 47%). ¹H NMR (600 MHz, CD₃OD) δ 8.57 (d, J = 2.2 Hz, 1H), 7.81–7.74 (m, 1H), 7.49–7.38 (m, 4H), 7.38–7.33 (m, 2H), 5.30 (t, J = 8.0 Hz, 1H), 5.10 (dd, J = 12.8, 5.5 Hz, 1H), 4.61–4.49 (m, 1H), 4.25–4.13 (m, 3H), 3.98–3.76 (m, 3H), 3.73–3.58 (m, 4H), 3.43–3.36 (m, 1H), 3.35–3.28 (m, 2H), 3.21–3.16 (m, 2H), 2.96–2.82 (m, 1H), 2.79–2.67 (m, 2H), 2.64 (t, J = 6.2 Hz, 2H), 2.31–2.24 (m, 1H), 2.23–2.03 (m, 2H), 1.86–1.81 (m, 2H), 1.59–1.45 (m, 4H), 1.46–1.33 (m, 8H), 1.17 (d, J = 7.0 Hz, 3H). HRMS (m/z) for C₄₅H₅₆ClN₈O₈⁺ [M + H]⁺: calculated 871.3904, found 871.3938.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-(tert-butoxycarbonyl)amino)butanoic acid (30).

The intermediate 105 was synthesized following a known procedure.¹⁵ To the suspension of intermediate 105 (119 mg × 3, 0.5 mmol) and methyl-4-oxobutanonate (58 mg × 3, 0.5 mmol) in 5 mL of DCM was added sodium triacetoxyborohydride (211 mg × 3, 1.0 mmol) in three portions. Once the reaction mixture became a clear solution, a saturated NaHCO₃ solution was added. The aqueous phase was extracted with DCM (10 mL × 3), dried over Na₂SO₄, filtered, and concentrated. The resulting residue was dissolved in DCM (15 mL). To this solution were added di-tert-butyl decarbonate (272 mg, 1.25 mmol) and triethylamine (188 mg, 1.86 mmol). The reaction was stirred for 30 min before the reaction mixture was concentrated. The residue was purified by silica gel column (hexane/EA = 1:3) to afford intermediate 106 as a yellow solid (178 mg, yield 53%). ESI m/z 538.8 [M + H]⁺. To the solution of intermediate 106 (178 mg, 0.34 mmol) in DMSO (5 mL) were added 2-methylbut-3-yn-2-ol (371 μL, 4.1 mmol), zinc powder (68 mg, 1.02 mmol), NaI (16 mg, 0.11 mmol), DBU (153 μL,1.02 mmol), and triethylamine (207 μL, 1.02 mmol). The reaction was degassed for 5 min, before the catalyst Pd(PPh₃)₄ (40 mg, 10 mol %) was added. The reaction was purged with nitrogen, sealed, and heated to 80 °C for 1 h. Saturated NH₄Cl solution was added, and the aqueous layer was extracted with ethyl acetate, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford the intermediate 107, which was used in the next step without further purification. The intermediate 107 (146 mg, 0.25 mmol) was dissolved in methanol (5 mL). To the resulting solution was added NaOH (0.5 mL, 3 N). The reaction was heated at 60 °C for 1 h before the reaction mixture was concentrated. The residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford title compound 30 as a white solid in TFA salt form (128.3 mg, yield 90%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.15 (s, 1H), 7.03 (s, 2H), 4.93–4.74 (m, 2H), 4.40–4.26 (m, 2H), 3.39 (t, J = 7.1 Hz, 2H), 3.22 (t, J = 7.2 Hz, 2H), 2.21 (t, J = 7.2 Hz, 2H), 2.13–2.02 (m, 2H), 1.82–1.69 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H), 1.41–1.30 (m, 15H). HRMS (m/z) for C₂₇H₃₈N₇O₇⁺ [M + H]⁺: calculated 572.2827, found 572.2838.

(2S,4R)-1-((S)-17-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,10-dioxo-6-oxa-3,9,14-triazaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (31).

To a solution of intermediate 30 (9.1 mg, 0.016 mmol) in DMSO (1 mL) were added 68 (9.1 mg, 0.016 mmol, 1.0 equiv), EDCI (4.6 mg, 0.024 mmol, 1.5 equiv), HOAt (3.3 mg, 0.024 mmol, 1.5 equiv), and NMM (4.8 mg, 0.048 mmol, 3.0 equiv). After stirring overnight at room temperature, the resulting mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the desired product. After this product was dissolved in DCM (1 mL), the reaction mixture was treated with TFA (1 mL) for 30 min. After the solvent was evaporated, the residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford compound 31 as a white solid in TFA salt form (6.1 mg, yield 39%). ¹H NMR (500 MHz, CD₃OD) δ 8.97 (d, J = 5.7 Hz, 1H), 8.23 (s, 1H), 7.51–7.34 (m, 4H), 5.05 (q, J = 7.0 Hz, 2H), 4.74–4.65 (m, 1H), 4.64–4.28 (m, 6H), 4.13–3.93 (m, 2H), 3.93–3.77 (m, 2H), 3.68–3.49 (m, 2H), 3.43–3.35 (m, 4H), 3.24–3.07 (m, 2H), 2.58–2.33 (m, 7H), 2.27 (dd, J = 13.2, 7.6 Hz, 1H), 2.10 (ddd, J = 13.5, 9.4, 4.4 Hz, 1H), 2.00 (h, J = 7.2 Hz, 2H), 1.69 (s, 6H), 1.58 (t, J = 7.0 Hz, 3H), 1.05 (s, 9H). HRMS (m/z) for C₄₈H₆₅N₁₂O₉S⁺ [M + H]⁺: calculated 985.4713, found 985.4728.

(2S,4R)-1-((S)-18-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,11-dioxo-7-oxa-3,10,15-triazaoctadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (32).

Compound 32 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 108 (12.4 mg, 0.016 mmol, 1.0 equiv). Compound 32 was obtained as a white solid in TFA salt form (11.8 mg, yield 74%). ¹H NMR (500 MHz, CD₃OD) δ 8.97 (s, 1H), 8.24 (s, 1H), 7.55–7.32 (m, 4H), 5.05 (q, J = 7.1 Hz, 2H), 4.71–4.63 (m, 1H), 4.64–4.48 (m, 5H), 4.41 (d, J = 15.4 Hz, 1H), 3.96–3.80 (m, 2H), 3.74–3.68 (m, 2H), 3.50 (t, J = 5.4 Hz, 2H), 3.38–3.33 (m, 4H), 3.16 (t, J = 7.0 Hz, 2H), 2.56–2.36 (m, 9H), 2.29–2.24 (m, 1H), 2.13–2.08 (m, 1H), 2.03–1.95 (m, 2H), 1.69 (s, 6H), 1.58 (t, J = 7.1 Hz, 3H), 1.05 (s, 9H). HRMS (m/z) for C₄₉H₆₇N₁₂O₉S⁺ [M + H]⁺: calculated 999.4869, found 999.4875.

(2S,4R)-1-((S)-20-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,13-dioxo-6,9-dioxa-3,12,17-triazaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (33).

Compound 33 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 69 (9.8 mg, 0.016 mmol, 1.0 equiv). Compound 33 was obtained as a white solid in TFA salt form (10.6 mg, yield 64%). 1H NMR (500 MHz, CD₃OD) δ 9.01 (d, J = 2.9 Hz, 1H), 8.26 (d, J = 3.0 Hz, 1H), 7.53–7.44 (m, 4H), 5.06 (q, J = 7.0 Hz, 2H), 4.95–4.71 (m, 1H), 4.64–4.31 (m, 6H), 4.03–4.01 (m, 2H), 3.94–3.78 (m, 2H), 3.80–3.58 (m, 4H), 3.56–3.48 (m, 2H), 3.46–3.26 (m, 4H), 3.22–3.08 (m, 2H), 2.59–2.35 (m, 7H), 2.36–2.23 (m, 1H), 2.19–2.06 (m, 1H), 2.03–1.93 (m, 2H), 1.70 (s, 6H), 1.59 (t, J = 7.0 Hz, 3H), 1.06 (s, 9H). HRMS (m/z) for C₅₀H₆₉N₁₂O₁₀S⁺ [M + H]⁺: calculated 1029.4975, found 1029.4983.

(2S,4R)-1-((S)-21-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,14-dioxo-7,10-dioxa-3,13,18-triazahenicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (34).

Compound 34 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 109 (13.1 mg, 0.016 mmol, 1.0 equiv). Compound 34 was obtained as a white solid in TFA salt form (6.4 mg, yield 38%). ¹H NMR (500 MHz, CD₃OD) δ 9.01 (s, 1H), 8.26 (s, 1H), 7.54–7.36 (m, 4H), 5.07 (q, J = 7.1 Hz, 2H), 4.67 (d, J = 6.2 Hz, 1H), 4.64–4.47 (m, 5H), 4.39 (d, J = 15.4 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 3.9 Hz, 1H), 3.80–3.69 (m, 2H), 3.66–3.56 (m, 4H), 3.50 (t, J = 5.4 Hz, 2H), 3.36–3.31 (m, 4H), 3.17 (t, J = 7.0 Hz, 2H), 2.59 (ddd, J = 15.0, 7.3, 5.2 Hz, 1H), 2.55–2.34 (m, 8H), 2.28–2.20 (m, 1H), 2.10 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.03–1.96 (m, 2H), 1.70 (s, 6H), 1.59 (t, J = 7.1 Hz, 3H), 1.05 (s, 9H). HRMS (m/z) for C₅₁H₇₁N₁₂O₁₀S⁺ [M + H]⁺: calculated 1043.5131, found 1043.5145.

(2S,4R)-1-((S)-23-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,16-dioxo-6,9,12-trioxa-3,15,20-triazatricosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (35).

Compound 35 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 110 (13.5 mg, 0.016 mmol, 1.0 equiv). Compound 35 was obtained as a white solid in TFA salt form (8.1 mg, yield 47%). ¹H NMR (500 MHz, CD₃OD) δ 9.00 (s, 1H), 8.25 (s, 1H), 7.49–7.41 (m, 4H), 5.06 (q, J = 6.9 Hz, 2H), 4.75–4.64 (m, 1H), 4.64–4.45 (m, 5H), 4.48–4.30 (m, 1H), 4.15–3.97 (m, 2H), 3.93–3.78 (m, 2H), 3.78–3.57 (m, 8H), 3.49 (t, J = 5.4 Hz, 2H), 3.34–3.24 (m, 4H), 3.17 (d, J = 7.1, 2.4 Hz, 2H), 2.54–2.33 (m, 7H), 2.33–2.21 (m, 1H), 2.17–2.05 (m, 1H), 2.04–1.92 (m, 2H), 1.75–1.63 (m, 6H), 1.59 (t, J = 7.1 Hz, 3H), 1.06 (s, 9H). HRMS (m/z) for C₅₂H₇₃N₁₂O₁₁S⁺ [M + H]⁺: calculated 1073.5237, found 1073.5231.

(2S,4R)-1-((S)-24-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,17-dioxo-7,10,13-trioxa-3,16,21-triazatetracosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (36).

Compound 36 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 111 (13.8 mg, 0.016 mmol, 1.0 equiv). Compound 36 was obtained as a white solid in TFA salt form (10.7 mg, yield 61%). ¹H NMR (500 MHz, CD₃OD) δ 9.02 (s, 1H), 8.27 (s, 1H), 7.59–7.33 (m, 4H), 5.07 (q, J = 7.2 Hz, 2H), 4.73–4.62 (m, 1H), 4.62–4.47 (m, 5H), 4.39 (dd, J = 15.6, 2.6 Hz, 1H), 3.94–3.87 (m, 1H), 3.87–3.66 (m, 3H), 3.70–3.54 (m, 8H), 3.50 (t, J = 5.4 Hz, 2H), 3.37–3.30 (m, 4H), 3.17 (t, J = 7.0 Hz, 2H), 2.60 (ddd, J = 15.0, 7.5, 5.2 Hz, 1H), 2.52–2.30 (m, 8H), 2.27–2.21 (m, 1H), 2.10 (ddd, J = 13.3, 9.2, 4.4 Hz, 1H), 2.04–1.97 (m, 2H), 1.70 (s, 6H), 1.60 (t, J = 7.1 Hz, 3H), 1.06 (s, 9H). HRMS (m/z) for C₅₃H₇₅N₁₂O₁₁S⁺ [M + H]⁺: calculated 1087.5393, found 1087.5383.

(2S,4R)-1-((S)-27-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,20-dioxo-7,10,13,16-tetraoxa-3,19,24-triazaheptacosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (37).

Compound 37 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 70 (11.4 mg, 0.016 mmol, 1.0 equiv). Compound 37 was obtained as a white solid in TFA salt form (12.6 mg, yield 70%). ¹H NMR (500 MHz, CD₃OD) δ 9.02 (s, 1H), 8.27 (s, 1H), 7.56–7.33 (m, 4H), 5.07 (q, J = 7.1 Hz, 2H), 4.65 (s, 1H), 4.62–4.46 (m, 5H), 4.38 (d, J = 15.5 Hz, 1H), 3.90 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 3.9 Hz, 1H), 3.77–3.71 (m, 2H), 3.67–3.55 (m, 12H), 3.51 (t, J = 5.4 Hz, 2H), 3.40–3.32 (m, 4H), 3.17 (t, J = 7.1 Hz, 2H), 2.60 (ddd, J = 15.0, 7.3, 5.3 Hz, 1H), 2.55–2.29 (m, 8H), 2.26–2.22 (m, 1H), 2.10 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.03–1.98 (m, 2H), 1.70 (s, 6H), 1.60 (t, J = 7.1 Hz, 3H), 1.05 (s, 9H). HRMS (m/z) for C₅₅H₇₉N₁₂O₁₂S⁺ [M + H]⁺: calculated 1131.5656, found 1131.5653.

(2S,4R)-1-((S)-30-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)-oxy)-2-(tert-butyl)-4,23-dioxo-7,10,13,16,19-pentaoxa-3,22,27-triazatriacontanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (38).

Compound 38 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 112 (15.2 mg, 0.016 mmol, 1.0 equiv). Compound 38 was obtained as a white solid in TFA salt form (9.4 mg, yield 50%). ¹H NMR (500 MHz, CD₃OD) δ 8.99 (s, 1H), 8.25 (s, 1H), 7.53–7.36 (m, 4H), 5.07 (q, J = 7.1 Hz, 2H), 4.66 (s, 1H), 4.61–4.47 (m, 5H), 4.38 (d, J = 15.5 Hz, 1H), 3.90 (d, J = 11.1 Hz, 1H), 3.86–3.70 (m, 3H), 3.66–3.56 (m, 16H), 3.50 (t, J = 5.4 Hz, 2H), 3.36–3.28 (m, 4H), 3.18 (t, J = 7.0 Hz, 2H), 2.60 (ddd, J = 14.8, 7.4, 5.1 Hz, 1H), 2.52–2.32 (m, 8H), 2.27–2.21 (m, 1H), 2.15–2.02 (m, 1H), 1.99 (q, J = 6.8 Hz, 2H), 1.69 (s, 6H), 1.59 (t, J = 7.1 Hz, 3H), 1.06 (s, 9H). HRMS (m/z) for C₅₇H₈₃N₁₂O₁₃S⁺ [M + H]⁺: calculated 1175.5918, found 1175.5911.

(2S,4R)-1-((S)-2-(2-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (39).

Compound 39 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 113 (11.4 mg, 0.016 mmol, 1.0 equiv). Compound 39 was obtained as a white solid in TFA salt form (9.3 mg, yield 62%). ¹H NMR (500 MHz, CD₃OD) δ 9.01 (d, J = 6.5 Hz, 1H), 8.25 (d, J = 6.2 Hz, 1H), 7.56–7.39 (m, 4H), 5.06 (dd, J = 12.7, 5.7 Hz, 2H), 4.83–4.31 (m, 7H), 4.08–3.78 (m, 4H), 3.46–3.35 (m, 2H), 3.21 (q, J = 7.2, 5.9 Hz, 2H), 2.80–2.29 (m, 7H), 2.33–2.18 (m, 1H), 2.18–1.90 (m, 3H), 1.70 (s, 6H), 1.64–1.51 (m, 3H), 1.06 (d, J = 10.6 Hz, 9H). HRMS (m/z) for C₄₆H₆₁N₁₂O₈S⁺ [M + H]⁺: calculated 941.4451, found 941.4458.

(2S,4R)-1-((S)-2-(3-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (40).

Compound 40 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 114 (11.7 mg, 0.016 mmol, 1.0 equiv). Compound 40 was obtained as a white solid in TFA salt form (11.3 mg, yield 72%).¹H NMR (500 MHz, CD₃OD) δ 9.02 (s, 1H), 8.26 (s, 1H), 7.51–7.28 (m, 4H), 5.07 (q, J = 7.0 Hz, 2H), 4.62 (s, 1H), 4.59–4.47 (m, 5H), 4.40 (d, J = 15.5 Hz, 1H), 3.95 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 3.9 Hz, 1H), 3.47–3.33 (m, 4H), 3.17 (t, J = 7.2 Hz, 2H), 2.57–2.33 (m, 9H), 2.26 (dd, J = 13.3, 7.6 Hz, 1H), 2.11 (ddd, J = 13.4, 9.3, 4.3 Hz, 1H), 2.02–1.96 (p, J = 6.8 Hz, 2H), 1.70 (s, 6H), 1.60 (t, J = 7.1 Hz, 3H), 1.05 (s, 9H). HRMS (m/z) for C₄₇H₆₃N₁₂O₈S⁺ [M + H]⁺: calculated 955.4607, found 955.4613.

(2S,4R)-1-((S)-2-(4-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (41).

Compound 41 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 115 (11.9 mg, 0.016 mmol, 1.0 equiv). Compound 41 was obtained as a white solid in TFA salt form (9.3 mg, yield 60%). ¹H NMR (500 MHz, CD₃OD) δ 9.02 (s, 1H), 8.27 (s, 1H), 7.58–7.34 (m, 4H), 5.07 (q, J = 7.3, 6.3 Hz, 2H), 4.75–4.48 (m, 6H), 4.40 (dd, J = 15.5, 4.9 Hz, 1H), 3.96–3.77 (m, 2H), 3.40–3.32 (m, 4H), 3.18 (q, J = 7.2, 6.4 Hz, 2H), 2.63–2.19 (m, 10H), 2.11 (ddd, J = 13.3, 9.2, 4.6 Hz, 1H), 2.00 (p, J = 6.6 Hz, 2H), 1.85–1.64 (m, 8H), 1.60 (t, J = 7.0 Hz, 3H), 1.06 (d, J = 4.6 Hz, 9H). HRMS (m/z) for C₄₈H₆₅N₁₂O₈S⁺ [M + H]⁺: calculated 969.4764, found 969.4754.

(2S,4R)-1-((S)-2-(5-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (42).

Compound 42 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 71 (9.1 mg, 0.016 mmol, 1.0 equiv). Compound 42 was obtained as a white solid in TFA salt form (6.7 mg, yield 43%). ¹H NMR (500 MHz, CD₃OD) δ 8.98 (s, 1H), 8.24 (s, 1H), 7.52–7.37 (m, 4H), 5.06 (q, J = 7.1 Hz, 2H), 4.63 (s, 1H), 4.58–4.51 (m, 5H), 4.39 (d, J = 15.4 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 4.0 Hz, 1H), 3.41–3.26 (m, 2H), 3.22–3.04 (m, 4H), 2.51–2.35 (m, 7H), 2.35–2.18 (m, 3H), 2.17–2.05 (m, 1H), 2.03–1.96 (m, J = 6.9 Hz, 2H), 1.69 (s, 6H), 1.65–1.40 (m, 7H), 1.04 (s, 9H). HRMS (m/z) for C₄₉H₆₇N₁₂O₈S⁺ [M + H]⁺: calculated 983.4920, found 983.4927.

(2S,4R)-1-((S)-2-(6-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)hexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (43).

Compound 43 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 72 (9.3 mg, 0.016 mmol, 1.0 equiv). Compound 43 was obtained as a white solid in TFA salt form (6.7 mg, yield 42%). ¹H NMR (500 MHz, CD₃OD) δ 8.97 (s, 1H), 8.23 (s, 1H), 7.54–7.35 (m, 4H), 5.06 (q, J = 7.0 Hz, 2H), 4.64 (s, 1H), 4.61–4.47 (m, 5H), 4.38 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.83 (dd, J = 10.9, 3.9 Hz, 1H), 3.39–3.32 (m, 2H), 3.17 (t, J = 7.0 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.50 (s, 3H), 2.48–2.35 (m, 4H), 2.34–2.20 (m, 3H), 2.11 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.03–1.96 (m, 2H), 1.69 (s, 6H), 1.65–1.54 (m, 5H), 1.49–1.45 (m, J = 7.3 Hz, 2H), 1.39–1.25 (m, 2H), 1.05 (s, 9H). HRMS (m/z) for C₅₀H₆₉N₁₂O₈S⁺ [M + H]⁺: calculated 997.5077, found 997.5079.

(2S,4R)-1-((S)-2-(7-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)heptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (44).

Compound 44 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 73 (9.5 mg, 0.016 mmol, 1.0 equiv). Compound 44 was obtained as a white solid in TFA salt form (13.9 mg, yield 86%). ¹H NMR (500 MHz, CD₃OD) δ 9.11–8.85 (m, 1H), 8.25 (s, 1H), 7.58–7.32 (m, 4H), 5.09–5.06 (m, J = 9.3, 4.8 Hz, 2H), 4.72–4.48 (m, 6H), 4.39 (dd, J = 15.4, 3.3 Hz, 1H), 3.92 (d, J = 10.7 Hz, 1H), 3.83 (d, J = 10.9, 3.6 Hz, 1H), 3.33–3.27 (m, 2H), 3.22–3.14 (m, 2H), 3.11–2.95 (m, 2H), 2.57–2.17 (m, 10H), 2.15–2.09 (m, 1H), 2.04–1.97 (m, 2H), 1.73–1.51 (m, 9H), 1.51–1.41 (m, 2H), 1.41–1.23 (m, 6H), 1.05 (s, 9H). HRMS (m/z) for C₅₁H₇₁N₁₂O₈S⁺ [M + H]⁺: calculated 1011.5233, found 1011.5239.

(2S,4R)-1-((S)-2-(8-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)octanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (45).

Compound 45 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 116 (12.8 mg, 0.016 mmol, 1.0 equiv). Compound 45 was obtained as a white solid in TFA salt form (8.6 mg, yield 52%). ¹H NMR (500 MHz, CD₃OD) δ 8.97 (s, 1H), 8.22 (s, 1H), 7.57–7.19 (m, 4H), 5.14–4.97 (m, 2H), 4.67–4.43 (m, 6H), 4.38 (dd, J = 15.5, 2.6 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 3.9 Hz, 1H), 3.32 (d, J = 2.2 Hz, 2H), 3.19–3.15 (m, 2H), 3.10–3.06 (m, 2H), 2.54–2.35 (m, 8H), 2.34–2.18 (m, 2H), 2.13–2.06 (m, 1H), 2.04–1.93 (m, 2H), 1.77–1.52 (m, 9H), 1.48–1.40 (m, J = 7.2 Hz, 2H), 1.39–1.12 (m, 8H), 1.05 (s, 9H). HRMS (m/z) for C₅₂H₇₃N₁₂O₈S⁺ [M + H]⁺: calculated 1025.5390, found 1025.5387.

(2S,4R)-1-((S)-2-(9-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)nonanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (46).

Compound 46 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 74 (10 mg, 0.016 mmol, 1.0 equiv). Compound 46 was obtained as a white solid in TFA salt form (7 mg, yield 42%). ¹H NMR (500 MHz, CD₃OD) δ 8.95 (s, 1H), 8.21 (s, 1H), 7.54–7.31 (m, 4H), 5.06 (q, J = 7.1 Hz, 2H), 4.65 (s, 1H), 4.62–4.48 (m, 5H), 4.38 (d, J = 15.5 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.83 (dd, J = 11.0, 3.9 Hz, 1H), 3.37–3.29 (m, J = 2.8 Hz, 2H), 3.17 (t, J = 6.9 Hz, 2H), 3.05 (t, J = 7.1 Hz, 2H), 2.52–2.37 (m, 7H), 2.34–2.19 (m, 3H), 2.15–2.04 (m, 1H), 2.03–1.95 (m, 2H), 1.69 (s, 6H), 1.65–1.54 (m, 3H), 1.48–1.42 (m, 2H), 1.30 (d, J = 13.7 Hz, 10H), 1.05 (s, 9H). HRMS (m/z) for C₅₃H₇₅N₁₂O₈S⁺ [M + H]⁺: calculated 1039.5546, found 1039.5534.

(2S,4R)-1-((S)-2-(10-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)decanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (47).

Compound 47 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 75 (13.2 mg, 0.016 mmol, 1.0 equiv). Compound 47 was obtained as a white solid in TFA salt form (3.9 mg, yield 23%). ¹H NMR (500 MHz, CD₃OD) δ 8.92 (d, J = 4.5 Hz, 1H), 8.17 (d, J = 5.8 Hz, 1H), 7.62–7.29 (m, 4H), 5.15–5.00 (m, 2H), 4.65 (d, J = 2.5 Hz, 1H), 4.63–4.46 (m, 5H), 4.42–4.29 (m, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.83 (dd, J = 11.0, 3.9 Hz, 1H), 3.35–3.29 (m, 2H), 3.17 (dd, J = 7.8, 5.9 Hz, 2H), 3.08–3.03 (m, J = 7.3, 3.5 Hz, 2H), 2.58–2.18 (m, 10H), 2.11 (ddd, J = 13.2, 9.0, 4.5 Hz, 1H), 2.04–1.89 (m, 2H), 1.76–1.50 (m, 9H), 1.42 (t, J = 7.0 Hz, 2H), 1.38–1.12 (m, 12H), 1.05 (s, 9H). HRMS (m/z) for C₅₄H₇₇N₁₂O₈S⁺ [M + H]⁺: calculated 1053.5703, found 1053.5711.

(2S,4R)-1-((S)-2-(11-(4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]-pyridin-7-yl)oxy)propyl)amino)butanamido)undecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-pyrrolidine-2-carboxamide (48).

Compound 48 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 76 (10.4 mg, 0.016 mmol, 1.0 equiv). Compound 48 was obtained as a white solid in TFA salt form (4.8 mg, yield 26%). ¹H NMR (500 MHz, CD₃OD) δ 8.96 (d, J = 2.6 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.58–7.32 (m, 4H), 5.13–5.00 (m, 2H), 4.65 (s, 1H), 4.63–4.50 (m, 5H), 4.37 (dd, J = 15.4, 2.8 Hz, 1H), 3.92 (d, J = 11.0 Hz, 1H), 3.83 (dd, J = 11.0, 3.9 Hz, 1H), 3.33–3.30 (m, 2H), 3.21–3.14 (m, 2H), 3.06 (dd, J = 9.4, 4.5 Hz, 2H), 2.54–2.38 (m, 7H), 2.36–2.20 (m, 3H), 2.11 (ddd, J = 13.3, 9.1, 4.5 Hz, 1H), 2.06–1.91 (m, 2H), 1.75–1.57 (m, 9H), 1.43 (t, J = 7.1 Hz, 2H), 1.39–1.22 (m, 14H), 1.05 (s, 9H). HRMS (m/z) for C₅₅H₇₉N₁₂O₈S⁺ [M + H]⁺: calculated 1067.5859, found 1067.5853.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)butanamide (49).

Compound 49 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 82 (7.6 mg, 0.016 mmol, 1.0 equiv). Compound 49 was obtained as a yellow solid in TFA salt form (9.6 mg, yield 26%). ¹H NMR (500 MHz, CD₃OD) δ 8.17 (s, 1H), 7.50 (dd, J = 8.6, 7.1 Hz, 1H), 7.01 (t, J = 8.2 Hz, 2H), 5.06 (dd, J = 12.7, 5.5 Hz, 1H), 4.99 (q, J = 7.1 Hz, 2H), 4.50 (t, J = 5.8 Hz, 2H), 3.66 (t, J = 5.1 Hz, 2H), 3.52 (t, J = 5.3 Hz, 2H), 3.44 (t, J = 5.1 Hz, 2H), 3.40–3.26 (m, 4H), 3.14 (t, J = 6.8 Hz, 2H), 2.88 (ddd, J = 17.3, 13.8, 5.3 Hz, 1H), 2.81–2.63 (m, 2H), 2.48 (t, J = 6.4 Hz, 2H), 2.39–2.35 (m, 2H), 2.17–2.10 (m, 1H), 2.01–1.94 (m, 2H), 1.69 (s, 6H), 1.53 (t, J = 7.1 Hz, 3H). HRMS (m/z) for C₃₉H₄₈N₁₁O₉⁺ [M + H]⁺: calculated 814.3631, found 814.3611.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)butanamide (50).

Compound 50 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 83 (8.3 mg, 0.016 mmol, 1.0 equiv). Compound 50 was obtained as a yellow solid in TFA salt form (7.8 mg, yield 57%). ¹H NMR (500 MHz, CD₃OD) δ 8.16 (s, 1H), 7.53 (dd, J = 8.5, 7.1 Hz, 1H), 7.05 (dd, J = 15.6, 7.8 Hz, 2H), 5.09–4.97 (m, 3H), 4.51 (t, J = 5.9 Hz, 2H), 3.73 (t, J = 5.2 Hz, 2H), 3.70–3.60 (m, 4H), 3.55–3.46 (m, 4H), 3.34–3.24 (m, 4H), 3.14 (t, J = 7.0 Hz, 2H), 2.87 (ddd, J = 17.6, 14.0, 5.3 Hz, 1H), 2.80–2.64 (m, 2H), 2.46–2.34 (m, 4H), 2.17–2.11 (m, 1H), 1.99–1.95 (m, 2H), 1.69 (s, 6H), 1.56 (t, J = 7.1 Hz, 3H). HRMS (m/z) for C₄₁H₅₂N₁₁O₁₀⁺ [M + H]⁺: calculated 858.3893, found 858.3879.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)butanamide (51).

Compound 51 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 84 (9 mg, 0.016 mmol, 1.0 equiv). Compound 51 was obtained as a yellow solid in TFA salt form (11.3 mg, yield 78%). ¹H NMR (500 MHz, CD₃OD) δ 8.23 (s, 1H), 7.51 (dd, J = 8.6, 7.1 Hz, 1H), 7.02 (dd, J = 19.2, 7.8 Hz, 2H), 5.09–4.99 (m, 3H), 4.52 (t, J = 5.9 Hz, 2H), 3.73 (t, J = 5.2 Hz, 2H), 3.68 (s, 4H), 3.69–3.63 (m, 2H), 3.66–3.55 (m, 2H), 3.49 (m, 4H), 3.37–3.25 (m, 4H), 3.15 (t, J = 7.0 Hz, 2H), 2.88 (ddd, J = 17.6, 14.0, 5.3 Hz, 1H), 2.82–2.65 (m, 2H), 2.47–2.38 (m, 4H), 2.16–2.10 (m, 1H), 2.01–1.96 (m, 2H), 1.70 (s, 6H), 1.57 (t, J = 7.1 Hz, 3H). HRMS (m/z) for C₄₃H₅₆N₁₁O₁₁⁺ [M + H]⁺: calculated 902.4155, found 902.4134.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)butanamide (52).

Compound 52 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 85 (9 mg, 0.016 mmol, 1.0 equiv). Compound 52 was obtained as a yellow solid in TFA salt form (5.2 mg, yield 34%). ¹H NMR (500 MHz, CD₃OD) δ 8.19 (s, 1H), 7.52 (dd, J = 8.6, 7.1 Hz, 1H), 7.04 (dd, J = 19.6, 7.8 Hz, 2H), 5.09–4.98 (m, 3H), 4.51 (t, J = 5.8 Hz, 2H), 3.73 (t, J = 5.2 Hz, 2H), 3.70–3.60 (m, 10H), 3.58 (dd, J = 6.1, 3.4 Hz, 2H), 3.49 (t, J = 5.3 Hz, 4H), 3.34–3.25 (m, 4H), 3.16 (t, J = 7.0 Hz, 2H), 2.88 (ddd, J = 17.5, 14.0, 5.3 Hz, 1H), 2.81–2.65 (m, 2H), 2.46 (t, J = 6.5 Hz, 2H), 2.41–2.37 (m, 2H), 2.17–2.10 (m, 1H), 2.01–1.96 (m, 2H), 1.69 (s, 6H), 1.57 (t, J = 7.1 Hz, 3H). HRMS (m/z) for C₄₅H₆₀N₁₁O₁₂⁺ [M + H]⁺: calculated 946.4417, found 946.4412.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)butanamide (53).

Compound 53 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 86 (9.6 mg, 0.016 mmol, 1.0 equiv). Compound 53 was obtained as a yellow solid in TFA salt form (12.8 mg, yield 81%). ¹H NMR (500 MHz, CD₃OD) δ 8.24 (d, J = 3.6 Hz, 1H), 7.60–7.45 (m, 1H), 7.07–7.01 (m, 2H), 5.07–5.03 (m, 3H), 4.54 (q, J = 5.8, 5.0 Hz, 2H), 3.82–3.56 (m, 18H), 3.49 (q, J = 5.3 Hz, 4H), 3.37–3.27 (m, 4H), 3.23–3.15 (m, 2H), 2.93–2.82 (m, 1H), 2.83–2.72 (m, 2H), 2.47–2.37 (m, 4H), 2.15–2.10 (m, 1H), 2.01–1.97 (m, 2H), 1.70 (d, J = 3.7 Hz, 6H), 1.62–1.53 (m, 3H). HRMS (m/z) for C₄₇H₆₄N₁₁O₁₃⁺ [M + H]⁺: calculated 990.4680, found 990.4668.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)ethyl)butanamide (54).

Compound 54 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 87 (7 mg, 0.016 mmol, 1.0 equiv). Compound 54 was obtained as a yellow solid in TFA salt form (12.1 mg, yield 98%). ¹H NMR (500 MHz, CD₃OD) δ 8.26 (d, J = 13.2 Hz, 1H), 7.55–7.44 (m, 1H), 7.02 (dd, J = 14.2, 7.8 Hz, 2H), 5.09–4.98 (m, 3H), 4.62–4.51 (m, 2H), 3.36–3.32 (m, 6H), 3.15 (t, J = 6.6 Hz, 2H), 2.87 (ddd, J = 17.2, 13.8, 5.3 Hz, 1H), 2.81–2.62 (m, 2H), 2.51–2.43 (m, 2H), 2.43–2.37 (m, 2H), 2.18–2.12 (m, 1H), 2.01–1.94 (m, 2H), 1.69 (s, 6H), 1.64–1.52 (m, 3H). HRMS (m/z) for C₃₇H₄₄N₁₁O₈⁺ [M + H]⁺: calculated 770.3369, found 770.3356.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)propyl)butanamide (55).

Compound 55 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 88 (7.1 mg, 0.016 mmol, 1.0 equiv). Compound 55 was obtained as a yellow solid in TFA salt form (11.7 mg, yield 93%). ¹H NMR (500 MHz, CD₃OD) δ 8.25 (s, 1H), 7.51 (dd, J = 8.6, 7.1 Hz, 1H), 6.99 (dd, J = 7.8, 4.6 Hz, 2H), 5.08–4.98 (m, 3H), 4.54 (t, J = 5.8 Hz, 2H), 3.39–3.29 (m, 4H), 3.31–3.22 (m, 2H), 3.18 (t, J = 6.6 Hz, 2H), 2.87 (ddd, J = 17.5, 14.0, 5.4 Hz, 1H), 2.80–2.64 (m, 2H), 2.51 (t, J = 6.4 Hz, 2H), 2.45–2.38 (m, 2H), 2.16–2.08 (m, 1H), 2.00 (q, J = 6.5 Hz, 2H), 1.77 (q, J = 6.4 Hz, 2H), 1.70 (s, 6H), 1.56 (t, J = 7.1 Hz, 3H). HRMS (m/z) for C₃₈H₄₆N₁₁O₈⁺ [M + H]⁺: calculated 784.3525, found 784.3534.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)butyl)butanamide (56).

Compound 56 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 89 (7.3 mg, 0.016 mmol, 1.0 equiv). Compound 56 was obtained as a yellow solid in TFA salt form (9.6 mg, yield 75%). ¹H NMR (500 MHz, CD₃OD) δ 8.25 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.03–6.96 (m, 2H), 5.04 (q, J = 7.1, 6.3 Hz, 3H), 4.54 (t, J = 5.8 Hz, 2H), 3.47–3.01 (m, 8H), 2.93–2.62 (m, 3H), 2.59–2.31 (m, 4H), 2.18–2.07 (m, 1H), 2.01–1.95 (m, J = 6.4 Hz, 2H), 1.82–1.45 (m, 13H). HRMS (m/z) for C₃₉H₄₈N₁₁O₈⁺ [M + H]⁺: calculated 798.3682, found 798.3667.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)pentyl)butanamide (57).

Compound 57 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 90 (7.6 mg, 0.016 mmol, 1.0 equiv). Compound 57 was obtained as a yellow solid in TFA salt form (10.6 mg, yield 82%). ¹H NMR (500 MHz, CD₃OD) δ 8.18 (s, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 6.98 (dd, J = 7.8, 6.4 Hz, 2H), 5.08–4.96 (m, 3H), 4.51 (t, J = 5.8 Hz, 2H), 3.34–3.23 (m, 4H), 3.20–3.07 (m, 4H), 2.88 (ddd, J = 17.0, 13.7, 5.2 Hz, 1H), 2.81–2.64 (m, 2H), 2.47 (t, J = 6.5 Hz, 2H), 2.41–2.36 (m, 2H), 2.18–2.08 (m, 1H), 2.01–1.95 (m, J = 6.7 Hz, 2H), 1.69 (s, 6H), 1.69–1.60 (m, 2H), 1.58–1.47 (m, 5H), 1.44–1.38 (m, 2H). HRMS (m/z) for C₄₀H₅₀N₁₁O₈⁺ [M + H]⁺: calculated 812.3838, found 812.3854.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)hexyl)butanamide (58).

Compound 58 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 91 (6.5 mg, 0.016 mmol, 1.0 equiv). Compound 58 was obtained as a yellow solid in TFA salt form (11.9 mg, yield 90%). ¹H NMR (500 MHz, CD₃OD) δ 8.24 (s, 1H), 7.60–7.48 (m, 1H), 7.15–6.86 (m, 2H), 5.07–5.01 (m, 3H), 4.54 (q, J = 5.8, 4.9 Hz, 2H), 3.34–3.16 (m, 8H), 2.98–2.67 (m, 3H), 2.43–2.35 (m, 4H), 2.19–2.10 (m, 1H), 2.01–1.97 (m, 2H), 1.83–1.15 (m, 17H). HRMS (m/z) for C₄₁H₅₂N₁₁O₈⁺ [M + H]⁺: calculated 826.3995, found 826.3987.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)heptyl)butanamide (59).

Compound 59 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 92 (8 mg, 0.016 mmol, 1.0 equiv). Compound 59 was obtained as a yellow solid in TFA salt form (12.5 mg, yield 93%). ¹H NMR (500 MHz, CD₃OD) δ 8.22 (s, 1H), 7.53 (ddd, J = 8.5, 7.2, 2.0 Hz, 1H), 7.05–7.02 (m, 2H), 5.09–5.00 (m, 3H), 4.53 (t, J = 5.9 Hz, 2H), 3.39–3.27 (m, 4H), 3.16 (t, J = 6.9 Hz, 2H), 3.09 (t, J = 7.1 Hz, 2H), 2.93–2.82 (m, 1H), 2.81–2.66 (m, 2H), 2.46–2.39 (m, 4H), 2.18–2.11 (m, 1H), 2.01–1.96 (m, 2H), 1.72–1.62 (m, 8H), 1.58 (t, J = 7.1 Hz, 3H), 1.49–1.29 (m, 8H). HRMS (m/z) for C₄₂H₅₄N₁₁O₈⁺ [M + H]⁺: calculated 840.4151, found 840.4157.

4-((3-((2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methylbut-1-yn-1-yl)-1H-imidazo[4,5-c]pyridin-7-yl)oxy)propyl)-amino)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-amino)octyl)butanamide (60).

Compound 60 was synthesized following the standard procedure for preparing compound 31 from intermediate 30 (9.1 mg, 0.016 mmol) and 93 (8.2 mg, 0.016 mmol, 1.0 equiv). Compound 60 was obtained as a yellow solid in TFA salt form (12.6 mg, yield 98%). ¹H NMR (500 MHz, CD₃OD) δ 8.25 (d, J = 2.8 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 2.3 Hz, 1H), 7.02 (dd, J = 7.8, 3.0 Hz, 2H), 5.10–5.01 (m, 3H), 4.54 (t, J = 5.8 Hz, 2H), 3.34–3.27 (m, 4H), 3.17 (td, J = 7.3, 2.6 Hz, 2H), 3.09–3.07 (m, 2H), 2.92–2.82 (m, 1H), 2.81–2.66 (m, 2H), 2.48–2.36 (m, 4H), 2.18–2.10 (m, 1H), 2.03–1.92 (m, 2H), 1.75–1.62 (m, 8H), 1.65–1.55 (m, 3H), 1.49–1.25 (m, 10H). HRMS (m/z) for C₄₃H₅₆N₁₁O₈⁺ [M + H]⁺: calculated 854.4308, found 854.4313.

N¹-(2-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)ethyl)-N¹²-((S)-1-((2R,4S)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)dodecanediamide (61).

Compound 61 was synthesized following the standard procedure for preparing compound 13 from intermediate 2 (48.1 mg, 0.073 mmol) and 12-(((S)-1-((2R,4S)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)-phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecanoic acid (118, 40.9 mg, 0.073 mmol, 1.0 equiv). Compound 61 was obtained as a white solid in TFA salt form (32.2 mg, yield 40%). ¹H NMR (800 MHz, CD₃OD) δ 8.98 (s, 1H), 8.59 (s, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.1 Hz, 4H), 7.39 (dd, J = 8.8, 3.1 Hz, 2H), 5.34 (t, J = 7.9 Hz, 1H), 5.05 (q, J = 6.9 Hz, 1H), 4.61–4.53 (m, 2H), 4.51 (s, 1H), 4.49–4.44 (m, 1H), 4.24–4.15 (m, 1H), 4.14–4.02 (m, 1H), 4.00–3.91 (m, 3H), 3.91–3.80 (m, 1H), 3.77–3.63 (m, 5H), 3.55–3.48 (m, 2H), 3.48–3.40 (m, 1H), 3.22 (t, J = 5.9 Hz, 2H), 2.52 (s, 3H), 2.35–2.27 (m, 2H), 2.27–2.16 (m, 4H), 2.16–2.06 (m, 1H), 1.65–1.57 (m, 4H), 1.48 (d, J = 7.1 Hz, 3H), 1.37–1.26 (m, 14H), 1.20 (d, J = 7.0 Hz, 3H), 1.09 (s, 9H). ¹³C NMR (201 MHz, CD₃OD) δ 176.58, 175.02, 172.10, 170.87, 169.37, 160.62, 159.54, 151.66, 148.41, 147.26, 144.19, 134.36, 133.53, 132.35, 129.78, 129.60 (2C), 129.50 (2C), 129.02 (2C), 126.45 (2C), 120.93, 70.55, 69.12, 59.36, 58.17, 55.49, 50.23, 48.54 (2C), 45.55, 45.49, 45.00, 44.44, 44.33, 41.40 (2C), 37.58, 36.58, 35.70, 35.46, 35.18, 34.29, 29.14, 29.03, 29.00, 28.94 (2C), 25.66 (3C), 25.51, 25.29, 21.20, 19.17, 14.35. t_R = 3.90 min; HRMS (m/z) for C₅₈H₈₂ClN₁₀O₇S⁺ [M + H]⁺: calculated 1097.5772, found 1097.5772.

3-(((S)-2-(4-Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)amino)-N-(8-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)propenamide (62).

Compound 113 was synthesized following the standard procedure for preparing compound 3 from intermediate 1 (19.8 mg, 0.04 mmol) and 4-((8-aminooctyl)amino)-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (121, 16.8 mg, 0.04 mmol, 1.0 equiv). Compound 62 was obtained as a yellow solid in TFA salt form (15.5 mg, yield 44%). ¹H NMR (800 MHz, CD₃OD) δ 8.60 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.07 (dd, J = 7.9, 5.1 Hz, 2H), 5.34 (t, J = 8.0 Hz, 1H), 5.11 (dd, J = 13.0, 5.6 Hz, 1H), 4.56 (dd, J = 9.6, 4.1 Hz, 1H), 4.19 (d, J = 10.8 Hz, 1H), 3.99–3.80 (m, 4H), 3.75–3.61 (m, 4H), 3.48–3.40 (m, 1H), 3.38–3.26 (m, 8H), 3.17 (s, 3H), 2.95–2.87 (m, 2H), 2.77–2.61 (m, 3H), 2.32 (dd, J = 12.6, 7.4 Hz, 1H), 2.25–2.16 (m, 1H), 2.15–2.08 (m, 1H), 1.74–1.63 (m, 2H), 1.56–1.50 (m, 2H), 1.50–1.44 (m, 2H), 1.44–1.35 (m, 5H), 1.20 (d, J = 6.9 Hz, 3H). ¹³C NMR (201 MHz, CD₃OD) δ 172.28, 171.00, 170.11, 169.48, 169.36, 167.93, 160.71, 159.59, 148.46, 146.96, 135.89, 134.38, 133.53, 132.53, 129.62 (2C), 129.46 (2C), 120.94, 116.63, 110.38, 109.58, 70.56, 50.18, 49.46, 45.58, 45.46, 44.96 (2C), 44.45, 44.32, 41.99, 41.43 (2C), 39.04, 36.58, 31.10, 29.54, 28.85, 28.82 (2C), 26.42 (2C), 25.95, 21.66, 19.15. t_R = 4.08 min; HRMS (m/z) for C₄₆H₅₉ClN₉O₇⁺ [M + H]⁺: calculated 884.4220, found 884.4215.

(2S,4R)-1-((S)-2-(11-Aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-pyrrolidine-2-carboxamide (79).

To a solution of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide⁵³ (77, 22.2 mg, 0.05 mmol) in DMSO (1 mL) were added commercially available 11-((tert-butoxycarbonyl)amino)undecanoic acid (78, 15.1 mg, 0.05 mmol, 1.5 equiv), EDCI (14.4 mg, 0.075 mmol, 1.5 equiv), HOAt (10.2 mg, 0.075 mmol, 1.5 equiv), and NMM (15.2 mg, 0.15 mmol, 3.0 equiv). After stirring overnight at room temperature, the reaction mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the desired intermediate, which was dissolved in TFA/DCM (1 mL/1 mL). The mixture was stirring for 30 min and concentrated. The residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the title compound 79 as a white solid (21.8 mg, yield 53%). ¹H NMR (400 MHz, CD₃OD) δ 8.97–8.80 (m, 1H), 7.48–7.42 (m, 4H), 5.02 (d, J = 6.9 Hz, 1H), 4.64 (s, 1H), 4.58 (t, J = 8.3 Hz, 1H), 4.45 (s, 1H), 3.92–3.71 (m, 2H), 2.96–2.83 (m, 2H), 2.49 (d, J = 7.6 Hz, 3H), 2.34–2.13 (m, 2H), 1.62 (d, J = 16.1 Hz, 4H), 1.52 (d, J = 7.8, 2.9 Hz, 3H), 1.35 (s, 14H), 1.05 (s, 9H). HRMS (m/z) for C₃₄H₅₄N₅O₄S⁺ [M + H]⁺: calculated 628.3891, found 628.3896.

12-(((S)-1-((2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)-phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecanoic acid (81).

Compound 81 was synthesized following the standard procedure for preparing compound 79 from intermediate 77 (58.4 mg, 0.13 mmol) and dodecanedioic acid (80, 36 mg, 0.16 mmol, 1.2 equiv). Compound 81 was obtained as a white solid (39.4 mg, yield 46%). ¹H NMR (600 MHz, CD₃OD) δ 9.09 (s, 1H), 7.44 (q, J = 8.0 Hz, 4H), 5.00 (q, J = 7.0 Hz, 1H), 4.62 (s, 1H), 4.57 (t, J = 8.5 Hz, 1H), 4.43 (s, 1H), 3.88 (d, J = 11.0 Hz, 1H), 3.74 (dd, J = 11.0, 3.9 Hz, 1H), 2.50 (s, 3H), 2.35–2.22 (m, J = 6.9 Hz, 4H), 2.19 (dd, J = 13.4, 8.4 Hz, 1H), 1.94 (ddd, J = 13.5, 9.0, 4.5 Hz, 1H), 1.62–1.55 (m, 4H), 1.50 (d, J = 7.0 Hz, 3H), 1.34–1.29 (m, 14H), 1.04 (s, 9H). HRMS (m/z) for C₃₅H₅₃N₄O₆S⁺ [M + H]⁺: calculated 657.3680, found 657.3667.

4-(9-Aminononyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (99).

To a solution of oct-7-yn-1-ol (95, 1.0 g, 7.94 mmol) in DCM (20 mL) were added NEt₃ (3.2 mL, 23.8 mmol, 3 equiv) and 4-toluenesulfonyl chloride (2.3 g, 11.9 mmol, 1.5 equiv). The reaction was stirring at room temperature for 2 h, and solvent was removed. The residue was purified by flash chromatography (hexane/EA = 8:1), and oct-7-yn-1-yl 4-methylbenzenesulfonate (96, 1.86 g, yield 84%) was obtained. To a solution of 4-methylbenzenesulfonate (96, 280 mg, 1 mmol) in DMF (5 mL) were added sodium cyanide (250 mg, 5 mmol, 5.0 equiv) and sodium iodide (15 mg, 0.1 mmol, 0.1 equiv). The reaction was heated at 60 °C for 3 h, and water was added, extracted with EtOAc (15 mL × 3), washed with brine, and dried over Na₂SO₄. The solvent was removed, and the residue was purified by flash chromatography (hexane/EA = 9:1). The non-8-ynenitrile (97, 126 mg) was obtained in 93% yield. To a solution of commercially available 4-bromo-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (33.7 mg, 0.1 mmol) and non-8-ynenitrile (97, 27 mg, 0.2 mmol, 2 equiv) in DMA (1.5 mL) were added tetrakis(triphenylphosphine)-palladium (0) (2.3 mg, 2 mol %), copper iodide (1 mg, 4 mol %), and triethylamine (22 mg, 0.3 mmol, 3.0 equiv). The reaction was heated at 100 °C for 12 h, cooled to rt, and filtered through Celite. The filtrate was evaporated, and the mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the corresponding product (31.4 mg, yield 80%). This intermediate was dissolved in MeOH, and Raney nickel (5 mg) was added. After the reaction was stirred at room temperature under hydrogen for 1 h, it was filtered and concentrated. The resulting residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the title product 99 as a white solid (29.8 mg, yield 94%). ¹H NMR (600 MHz, CD₃OD) δ 7.72–7.70 (m, 2H), 7.62 (dd, J = 5.8, 2.9 Hz, 1H), 5.12 (dd, J = 12.6, 5.5 Hz, 1H), 3.09 (dd, J = 8.8, 6.8 Hz, 2H), 2.92–2.87 (m, 2H), 2.79–2.69 (m, 2H), 2.18–2.05 (m, 1H), 1.69–1.59 (m, 5H), 1.41–1.32 (m, 10H). HRMS (m/z) for C₂₂H₃₀N₃O₄⁺ [M + H]⁺: calculated 400.2231, found 400.2257.

3-(4-((8-Aminooctyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (103).

To a solution of 3-(4-amino-1-oxoisoindolin-2-yl)-piperidine-2,6-dione (101, 52 mg, 0.2 mmol) in DMF (2 mL) were added tert-butyl (8-iodooctyl)carbamate (100, 85.2 mg, 0.24 mmol, 1.2 equiv) and NaHCO₃ (40 mg, 0.46 mmol, 2.3 equiv). After the reaction was stirred at 60 °C overnight, water was added to quench the reaction. The reaction mixture was extracted with EtOAc (5 mL × 3), dried over Na₂SO₄, filtered, and concentrated. The residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the desired intermediate as a yellow solid (68.8 mg, yield 71%). This intermediate (68.8 mg, 0.14 mmol) was dissolved in TFA/DCM (1/1 mL). After the reaction solution was stirred at room temperature for 30 min, the solvent was evaporated and the resulting residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the title compound 103 as a yellow solid (56.4 mg, yield 99%). ¹H NMR (600 MHz, CD₃OD) δ 7.42 (d, J = 3.7 Hz, 2H), 7.21 (dd, J = 5.9, 3.0 Hz, 1H), 5.14 (dd, J = 13.5, 5.0 Hz, 1H), 4.52–4.29 (m, 2H), 3.83–3.65 (m, 2H), 2.95–2.90 (m, 2H), 2.56–2.35 (m, 1H), 2.24–2.09 (m, 1H), 1.73–1.58 (m, 2H), 1.58–1.46 (m, 2H), 1.43–1.26 (m, 10H). HRMS (m/z) for C H N O ⁺ [M + H]⁺: calculated 387.2391, found 387.2376.

4-((8-Aminooctyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (104).

Compound 104 was synthesized following the standard procedure for preparing compound 103 from 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (102, 55 mg, 0.2 mmol) and tert-butyl (8-iodooctyl)carbamate (100, 85.2 mg, 0.24 mmol, 1.2 equiv). Compound 104 was obtained as a white solid (28.2 mg, 70% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 7.75 (t, J = 7.9 Hz, 1H), 7.41 (dd, J = 8.4, 3.9 Hz, 2H), 5.09 (dd, J = 12.9, 5.4 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 2.98–2.81 (m, 3H), 2.81–2.62 (m, 2H), 2.12 (dd, J = 12.8, 6.1 Hz, 1H), 1.89–1.82 (m, 2H), 1.64 (q, J = 7.5 Hz, 2H), 1.54 (q, J = 7.4 Hz, 2H), 1.48–1.31 (m, 6H). HRMS (m/z) for C₂₁H₂₈N₃O₅⁺ [M + H]⁺: calculated 402.2023, found 402.2034.

12-(((S)-1-((2R,4S)-4-Hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)-phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecanoic acid (118).

To the solution of (2R,4S)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide⁵⁵ (117, 22.0 mg, 0.05 mmol) in DMSO (1 mL) were added dodecanedioic acid (80, 17.3 mg, 0.075 mmol, 1.5 equiv), EDCI (14.4 mg, 0.075 mmol, 1.5 equiv), HOAt (10.2 mg, 0.075 mmol, 1.5 equiv), and NMM (15.2 mg, 0.15 mmol, 3.0 equiv). After stirring overnight at room temperature, the reaction mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the title compound 118 as a white solid (12.4 mg, yield 38%). ¹H NMR (600 MHz, CD₃OD) δ 9.07 (s, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.50–7.38 (m, 2H), 5.02 (q, J = 7.0 Hz, 1H), 4.55 (dd, J = 8.3, 6.4 Hz, 1H), 4.48 (s, 1H), 4.46–4.41 (m, 1H), 3.94 (dd, J = 10.8, 4.9 Hz, 1H), 3.68 (dd, J = 10.8, 3.5 Hz, 1H), 2.51 (s, 3H), 2.33–2.22 (m, 3H), 2.22–2.15 (m, 2H), 2.13–2.05 (m, 1H), 1.61–1.53 (m, 4H), 1.45 (d, J = 7.1 Hz, 3H), 1.33–1.19 (m, 12H), 1.06 (s, 9H). HRMS (m/z) for C₃₅H₅₃N₄O₆S⁺ [M + H]⁺: calculated 657.3680, found 657.3693.

4-((8-Aminooctyl)amino)-2-(1-methyl-2,6-dioxopiperidin-3-yl)-isoindoline-1,3-dione (121).

To a solution of 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione⁶¹ (119, 58 mg, 0.2 mmol) in 1-methyl-2-pyrrolidinone (1 mL) were added commercially available tert-butyl (8-aminooctyl)carbamate (120, 49 mg, 0.2 mmol) and N,N-diisopropylethylamine (77 mg, 0.6 mmol, 3 equiv). After stirring at 100 °C under microwave for 1 h, the reaction mixture was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford the desired intermediate, which was dissolved in TFA/DCM (1/1 mL). The mixture was stirred for 30 min at room temperature before it was concentrated. The resulting residue was purified by preparative HPLC (10–100% methanol/0.1% TFA in H₂O) to afford title compound 121 as a yellow solid (66.8 mg, yield 81%). ¹H NMR (600 MHz, CD₃OD) δ 7.52 (dd, J = 8.5, 7.1 Hz, 1H), 7.06–6.91 (m, 2H), 5.06 (dd, J = 13.0, 5.4 Hz, 1H), 3.12 (s, 3H), 2.94–2.81 (m, 4H), 2.74–2.56 (m, 1H), 2.14–1.99 (m, 1H), 1.64 (q, J = 7.3 Hz, 4H), 1.50–1.28 (m, 10H). HRMS (m/z) for C₂₂H₃₁N₄O₄⁺ [M + H]⁺: calculated 415.2340, found 415.2345.

Cell Culture.

All cell lines were purchased from ATCC and authenticated. Cell lines were regularly tested in the lab for mycoplasma. All cells were cultured at 37 °C and 5% CO₂. Cell lines were cultured in 1× DMEM (Corning, 10–013-CV) with 10% fetal bovine serum (Atlanta Biologicals S11150) and 1× penicillin/ streptomycin. Cells were split using 0.05 or 0.25% trypsin (Corning 25–051-Cl or 25–053-Cl, respectively) before they reached full confluence, and media were changed every 3–4 days.

Western Blotting Analysis.

Total cell lysates were collected with an immunoprecipitation (IP) lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail). Whole cell lysates were obtained by sonication followed by centrifugation, and protein concentration was measured by a BCA Protein Assay Kit (Pierce). Cell lysates were boiled with 2× sample buffer (125 mM Tris–HCl at pH 6.8, 10% β-ME, 2% SDS, 20% glycerol, 0.05% bromophenol blue, and 8 M urea). Protein lysates were loaded into 4–12% Bis–Tris gels and resolved by electrophoresis. Samples were then blotted on a PVDF membrane (Millipore IPVH00010) using the wet transfer technique (Invitrogen). Membranes were blocked in 5% milk–TBST for 1 h, washed in TBST for 10 min, and incubated in primary antibody in 5% milk–TBST or 5% BSA–TBST at 4 °C for 16 h. Membranes were rinsed (3 × 6 min) in TBST, incubated in horseradish peroxidase-conjugated secondary antibodies in 5% milk–TBST for 1 h, and rinsed again in TBST (3 × 6 min). Membranes were visualized using the chemiluminescence system (Thermo 34080, 37075) on an autoradiography film (Denville E3018). Primary antibodies were as follows: β-actin (Sigma A5316), p-AKT (Ser473 CST-9271), total AKT (CST-9272), p-S6 (Ser240/244, CST-5364), and p-PRAS40 (Thr246, CST-2997). Secondary antibodies were as follows: mouse (Thermo 31432) and rabbit (Thermo 31460).

Cell Colony Formation Assay.

Cells were cultured for 14 days in the presence of different compounds at indicated concentrations. Media with compound were replenished every 2 days. At the end of the experiment, media were aspirated and viable cells were stained with 0.5% crystal violet dye. Quantification analysis was performed as previously described.⁶²

AKT Binding Assay.

Binding affinities of GDC0068, 13, 25, 61, and 62 to three AKT isoforms (AKT1, AKT2 and AKT3) were determined by DiscoverX using the KINOMEscan assay, a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay was performed by combining three components: DNA-tagged AKT, immobilized ligand, and a test compound. The ability of the test compound to compete with the immobilized ligand was measured by quantitative PCR of the DNA tag. The K_d values were determined by using an 11-point 3-fold compound dilution (the top concentration of 10 μM for GDC-0068 and 30 μM for the other four compounds) with three DMSO control points in duplicates.

Cell Proliferation and Apoptosis Assays.

Experiments were carried out in 96-well plates in triplicates (Corning 720089). A total of 1–3 × 10³ cells per well were grown in the presence of indicated compounds, and cells were then monitored for 5 days using the IncuCyte live cell imaging system (Essen BioScience, Ann Arbor, MI, USA), which was placed in a cell culture incubator operated at 37 °C and 5% CO₂. Cell confluence was determined using calculations derived from phase-contrast images readings on an IncuCyte ZOOM (Essen Biosciences) on live cells over time. For the measurement of cell apoptosis, DRAQ7 (Cell Signaling # 7406) at 1.5 μM was included in the medium and apoptotic red counts were measured in an IncuCyte FLR automated incubator microscope.

TMT-Based Global Proteomic Profiling Sample Preparation.

Trypsin was purchased from Promega. All chemicals were HPLC-grade unless specifically indicated. The TMT11plex Isobaric Label Reagent was purchased from Thermofisher (cat. # A34808). The cell pellets were resuspended in 8 M urea and 50 mM Tris–HCl (pH 8.0), reduced with dithiothreitol (5 mM final) for 30 min at room temperature, and alkylated with iodoacetamide (15 mM final) for 45 min in the dark at room temperature. Samples were diluted fourfold with 25 mM Tris–HCl (pH 8.0) and 1 mM CaCl₂ and digested with trypsin at a 1:100 (w/w, trypsin/protein) ratio overnight at room temperature. Peptides were desalted on homemade C18 stage tips, and 100 μg of each peptide sample was labeled with the TMT reagent following the manufacturer’s instruction. The mixture of labeled peptides was desalted and fractionated into 24 fractions in 10 mM trimethylammonium bicardonate (TMAB) buffer containing 5–40% acetonitrile.

Mass Spectrometry Analysis.

Dried peptides were dissolved in 0.1% formic acid and 2% acetonitrile. For global profiling samples, peptide concentration was measured with a Pierce Quantitative Colorimetric Peptide Assay (Thermofisher). A total of 0.5 μg of each fraction were analyzed on a Q-Exactive HF-X coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded onto a nanoEase MZ HSS T3 Column (100 Å, 1.8 μm, 75 μm × 150 mm, Waters). Analytical separation of all peptides was achieved with a 100 min gradient. A linear gradient of 5 to 10% buffer B over 5 min, 10 to 31% buffer B over 70 min, and 31 to 75% buffer B over 15 min was executed at a 300 nL/min flow rate followed by a ramp to 100% B in 1 min and 9 min wash with 100% B, where buffer A was aqueous 0.1% formic acid and buffer B was 80% acetonitrile and 0.1% formic acid. LC–MS experiments were also carried out in a data-dependent mode with full MS (externally calibrated to a mass accuracy of <5 ppm and a resolution of 120,000 for TMT-labeled samples at m/z 200) followed by high energy collision-activated dissociation-MS/MS with a resolution of 30,000 for TMT-labeled global samples at m/z 200. High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 32 eV (for TMT-labeled sample) in the presence of nitrogen bath gas atoms. Dynamic exclusion was 45 or 20 s.

Raw Proteomics Data Processing and Analysis.

Mass spectra were processed, and peptide identification was performed using the MaxQuant software version 1.6.10.43 (Max Planck Institute, Germany). All protein database searches were performed against the UniProt human protein sequence database (UP000005640). A false discovery rate (FDR) for both peptide-spectrum match (PSM) and protein assignment was set at 1%. Search parameters included up to two missed cleavages at Lys/Arg on the sequence, oxidation of methionine, and protein N-terminal acetylation as a dynamic modification. Carbamidomethylation of cysteine residues was considered as a static modification. Peptide identifications are reported by filtering of reverse and contaminant entries and assigning to their leading razor protein. The TMT reporter intensity found in MaxQuant was for quantitation. Data processing and statistical analysis were performed on Perseus (Version 1.6.10.50). Protein quantitation was performed using the TMT reporter intensity found in MaxQuant, and a two-sample t test on two biological replicates was used with a p value of 1% to report statistically significant protein abundance fold-changes.

Mouse Pharmacokinetic Study.

Compounds 13 and 25 (in their HCl salt form) were dissolved in 5% DMA, 25% Solutol HS-15, and 70% normal saline as formulation. Three male Swiss albino mice were administered intraperitoneally with a solution formulation of each compound at a 50 mg/kg dose. The formulation vehicle was 5% v/v NMP, 5% v/v Solutol HS-15, and 90% v/v normal saline. Blood samples (approximately 60 μL) were collected from the three test mice at 0.5, 2, and 8 h. Plasma was harvested by centrifugation of the blood and stored at −70 ± 10 °C until analysis. Plasma samples were quantified by the fit-for-purpose LC–MS/MS method. 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.

ABBREVIATIONS USED

ATP

adenosine triphosphate

Boc

tert-butyloxycarbonyl

Cbz

carboxybenzyl

CRBN

cereblon

CRLs

cullin-ring ubiquitin ligases

EDCI

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

HOAt

1-hydroxy-7-azabenzo-triazole

IMiDs

immunomodulatory drugs

MOA

mechanism of actions

MS

mass spectrometry

m-TOR

mammalian target of rapamycin

NAE

NEDD8-activating enzyme

NMM

N-methylmorpholine

P-AKT

phosphorylated AKT

PEG

polyethylene glycol

PI3K

phosphatidylinositol 3-kinase

PK

pharmacokinetic

POM

pomalidomide

PROTACs

proteolysis targeting chimeras

SAR

structure–activity relationships

VHL

von Hippel–Lindau

T-AKT

total AKT

TMT

tandem mass tag