Rational design and discovery of potent PROTAC degraders of ASK1: a targeted therapy in MASH

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease marked by hepatic steatosis, inflammation, and fibrosis, with limited therapeutic options. This study introduces a novel PROTAC-based strategy for the selective degradation of apoptosis signal-regulating kinase 1 (ASK1), a key mediator of MASH pathology. We first developed dASK1 (35), a cereblon (CRBN)-based PROTAC, which successfully formed a stable ternary complex with ASK1, facilitating its rapid and sustained degradation via the ubiquitin–proteasome pathway. In vitro evaluations demonstrated potent ASK1 degradation in the 10–100 nM range (70% degradation at 100 nM) in HepG2 and HEK293A cell lines, validating the efficacy of dASK1 (35). To enhance the degradation mechanism and explore broader E3 ligase utility, we designed and synthesized dASK1-VHL (60), leveraging the von Hippel–Lindau (VHL) E3 ligase, known for its regulatory functions in hepatic physiology. We optimized the linker length through molecular docking and MMGBSA calculations, achieving efficient ASK1–VHL engagement and stable ternary complex formation. Detailed ADME and pharmacokinetic studies confirmed that dASK1-VHL (60) exhibited enhanced solubility, moderate clearance, and improved bioavailability, making it suitable for in vivo application. In an MCD diet-induced murine model of MASH, dASK1-VHL (60) effectively reduced ASK1 protein levels, suppressed p38 MAPK activation, and decreased hepatic lipid content, indicating significant therapeutic benefits. This work underscores the importance of rational PROTAC design, precise linker engineering, and innovative E3 ligase selection in optimizing target protein degradation. Our findings pave the way for developing VHL-based PROTACs, offering a novel therapeutic approach for metabolic and inflammatory liver diseases.

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Introduction

Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), is a critical component in cellular stress response pathways and is widely expressed across tissues. It is specifically activated in response to pathophysiological conditions, such as oxidative stress, endoplasmic reticulum (ER) stress, and inflammatory stimuli, including tumor necrosis factor-alpha (TNF-α) and lipopolysaccharides (LPS).^1–3 ASK1 initiates a cascade upon activation by phosphorylating mitogen-activated protein kinase kinases (MAP2Ks), particularly MAP2K-3, -4, -6, and -7. These MAP2Ks then activate downstream effectors, including p38 and c-Jun N-terminal kinase (JNK). These drive key processes like apoptosis, cytokine expression, and the activation of fibrogenic genes critical to liver pathology.^4,5 ASK1 activity is finely regulated by thioredoxin (Trx), a redox-sensitive protein that binds ASK1 and inhibits its activity in its reduced state. During oxidative stress, oxidized Trx detaches from ASK1, facilitating ASK1 autophosphorylation and subsequent activation.⁶ This regulatory mechanism underscores the sensitivity of ASK1 to oxidative changes, a feature central to its involvement in chronic liver diseases such as MASH. Chronic ASK1–p38/JNK activation disrupts hepatic lipid,^5,7–11 and glucose metabolism, mainly via JNK signaling.^12,13 ASK1 activation also requires K63-linked ubiquitination by the E3 ligase FBXW5 promoting MASH severity,¹¹ while deubiquitinases like TNFAIP3 mitigate disease progression by removing ubiquitin chains of ASK1's.¹⁰ These post-translational modifications are essential for regulating ASK1 activity in hepatocytes. Preclinical studies revealed that ASK1 inhibitors effectively reduce hepatic steatosis and inflammation in MASH models of diet-induced obesity and non-human primates.¹⁴ These findings indicate the importance of ASK1 in MASH pathology, positioning it as a promising molecular target for managing metabolic and inflammatory liver diseases.^8,10,15

Selonsertib (GS-4997, developed by Gilead Sciences) is a selective small-molecule inhibitor of ASK1 that competitively binds to its ATP-binding domain, effectively blocking kinase activity.^16,17 In a randomized phase II clinical trial (NCT01672866) involving 72 MASH patients, selonsertib demonstrated efficacy in reducing stage 2–3 liver fibrosis, outperforming the antifibrotic agent simtuzumab.^18,19 However, selonsertib did not meet primary endpoints in phase III trials—STELLAR-3 (focused on bridging fibrosis) and STELLAR-4 (targeting cirrhosis).²⁰ Our recent work demonstrates that targeting the COP1 E3 ubiquitin ligase with quinazolinone and quinazolinedione derivatives effectively reduces the ubiquitination of adipose triglyceride lipase (ATGL), a crucial enzyme in lipid catabolism. Enhanced stabilization of ATGL significantly decreased hepatic triacylglycerol (TAG) accumulation in a murine model of non-alcoholic fatty liver disease (NAFLD).²¹

Modulating the ubiquitin–proteasome system (UPS) to control protein degradation has emerged as a promising strategy in the therapy of liver diseases. A novel approach involves harnessing targeted protein degradation (TPD) through the use of proteolysis targeting chimeras (PROTACs), which have shown great potential in both biomedical research and drug development.^22,23 PROTACs are heterobifunctional molecules comprising a ligand that binds the target protein and another ligand that recruits an E3 ubiquitin ligase, connected by a linker.^24,25 This configuration induces ubiquitination, leading to the proteasomal degradation of the target protein. Unlike traditional small-molecule inhibitors, PROTACs function catalytically and can degrade proteins with structural or scaffolding roles.²⁶ Notably, clinical trials of ARV-110 and ARV-471, targeting the androgen receptor (AR) and estrogen receptor (ER), respectively, have demonstrated the clinical feasibility of this approach.^27,28 While PROTACs have primarily been investigated for cancer, immune disorders, and neurodegenerative diseases, their potential in addressing metabolic disorders, including liver diseases, remains largely unexplored.²⁹ Leveraging PROTAC technology to exploit the UPS, we have successfully designed and optimized a series of PROTACs targeting metabolic liver disorders such as MASH. These molecules integrate a tailored selonsertib-derived moiety as the ASK1-targeting ligand and either a thalidomide or VHL ligand for E3 ubiquitin ligase recruitment, connected via a strategically optimized linker. The engineered PROTACs demonstrated robust, selective degradation of hepatic ASK1 in a time-, dose-, and proteasome-dependent manner and were validated through comprehensive in vitro assays corroborated by efficacy in preclinical murine models.

Results and discussion

Design strategy

For our PROTAC development, we selected a fragment derived from selonsertib (Fig. 1A) as the protein-of-interest (POI)-binding ligand due to its strong affinity for ASK1. Selonsertib is a potent ASK1 inhibitor that binds competitively to the ATP-binding pocket, effectively inhibiting the kinase's catalytic function. Structurally, selonsertib comprises two distinct heterocyclic groups—a triazole and an imidazole—located at its termini, along with hydrophobic substituents such as isopropyl and cyclopropane groups (Fig. 1A). The co-crystal structure of the ASK1–selonsertib complex (Fig. 1B) highlights a key interaction: the central amide of selonsertib forms a hydrogen bond with Val757 in the hinge region of ASK1, anchoring the molecule in place. The planar pyridine ring adjacent to the amide facilitates the alignment of the hinge-binding motif, allowing the isopropyl–triazole moiety to establish a secondary hydrogen bond with the catalytic residue Lys709. This additional interaction, combined with the snug fit of the isopropyl group in a nearby hydrophobic pocket,³⁰ significantly enhances the binding stability of selonsertib. On the opposite end, the cyclopropyl–imidazole moiety extends into the solvent-exposed region (Fig. 1B), providing an ideal site for chemical derivatization without disrupting the core binding interactions. Selonsertib's high inhibitory potency, with an IC₅₀ of 3.0 nM against ASK1, makes it a strong candidate for ligand-based modifications.³¹

Fig. 1 Design of ASK1-recruiting thalidomide-based PROTAC degraders. (A) Chemical structure of ASK1 binding ligand selonsertib. (B) Co-crystal structure (PDB ID: 6OYT) of the ASK1 kinase domain (grey) in complex with selonsertib (blue) with H bonds and and π–sulfur interactions (green and yellow dotted lines, respectively). (C) Structure of the fragment compound 3′, without the cyclopropyl imidazole group of selonserib. (D) Structure of the fragment compound 4′, without the isopropyl–triazole group of selonserib. (E) Design of PROTAC degraders recruiting CRBN to target ASK1.

To further explore the essential structural components for ASK1 inhibition, we evaluated two truncated fragments of selonsertib. Fragment 3′, which lacks the cyclopropyl–imidazole group, retained considerable activity, displaying an IC₅₀ of 55 nM (Fig. 1C). This suggests that the cyclopropyl–imidazole group contributes minimally to ASK1 binding affinity.³¹ In contrast, Fragment 4′, which omits the isopropyl–triazole moiety, exhibited a marked decrease in potency, with an IC₅₀ of 6.0 μM—a 2000-fold reduction relative to the parent compound selonsertib (Fig. 1D).³¹ This substantial loss of activity highlights the critical importance of the isopropyl–triazole group for strong binding interactions with the ASK1 active site. Based on this structure–activity relationship analysis, we selected fragment 3′ as the preferred scaffold for developing our PROTACs. The cyclopropyl–imidazole moiety's orientation toward the solvent-exposed region allows for efficient conjugation of a linker without interfering with key interactions at the ASK1 active site.

This structural feature offers flexibility for further functionalization while maintaining high target affinity. In our PROTAC design, fragment 3′ was extended with a strategically chosen linker and coupled to thalidomide, an E3 ligase-binding ligand that engages CRBN (Fig. 1E). This design was intended to facilitate effective recruitment of the E3 ligase complex, driving the ubiquitination and subsequent proteasomal degradation of ASK1.

In the PROTAC design, the linker's structure and length are critical determinants that govern the spatial orientation, conformational flexibility, and ultimately the formation, stability, and efficiency of the ternary complex essential for target degradation. The linker does more than simply bridge the target protein and the E3 ligase; it actively modulates the PROTAC's pharmacokinetics, selectivity, and overall biological efficacy.³² Polyethylene glycol (PEG) linkers are particularly favorable due to their hydrophilicity, enhanced solubility, low cytotoxicity, and reduced risk of immunogenicity.³³ In this study, we systematically explored PEG linkers of varying chain lengths to refine linker properties, aiming to maximize target engagement, improve ternary complex formation, and optimize the biological activity of the PROTACs.

Design of the PROTAC synthesis platform

We developed two distinct synthetic strategies for constructing PROTAC molecules, as illustrated in Fig. 2. In Strategy 1, we designed an ASK1-targeting ligand based on a selonsertib-derived fragment, incorporating a –Br functional group to facilitate subsequent chemical modifications. One end of the linker was attached to the E3 ligase ligand, thalidomide. The thalidomide moiety, linked via a spacer containing a terminal –NH₂ group, was then conjugated to the –Br group on the ASK1 ligand through nucleophilic substitution, yielding the desired PROTAC construct. In Strategy 2, we employed an alternative approach by synthesizing the ASK1-targeting ligand with a terminal –NH₂ group instead of a –Br handle. This modification enabled amide bond formation with the –COOH terminus of the linker. In this strategy, the linker was first coupled to the selonsertib fragment via amide coupling, followed by the attachment of the thalidomide moiety to the linker through an additional coupling reaction, completing the assembly of the PROTAC molecule. These complementary synthetic routes allowed us to optimize linker flexibility and target engagement, ensuring efficient degradation of ASK1. With the synthetic strategies defined, we initiated the synthesis as detailed in Schemes 1–4, systematically verifying each intermediate step through spectroscopic and chromatographic analyses.

Fig. 2 Strategies for establishing PROTAC synthesis platform.

Strategy 1. Scheme 1 details the synthetic protocols and strategies for the preparation of the ASK1 ligand according to Strategy 1. The synthesis began with commercially available methyl 6-aminopicolinate (1) and 5-bromo-2-fluoro-4-methylbenzoic acid (5). First, compound 1 was dissolved in methanol, forming a light yellow solution. Hydrazine hydrate was then added, and the mixture was refluxed at 100 °C for 12 hours, yielding compound 2. Next, compound 2 was reacted with dimethylformamide dimethyl acetal under reflux for 12 hours, producing compound 3. Compound 3 was dissolved in acetonitrile, followed by the addition of acetic acid and isopropylamine. The mixture was stirred at 100 °C for 12 hours, resulting in compound 4. To convert compound 5 into a suitable intermediate, oxalyl chloride and a catalytic amount of DMF were added to a cold solution of compound 5 (1.1 equiv. relative to compound 4) in dichloromethane (DCM). The reaction mixture was stirred at room temperature for 1 hour, after which the solvent was evaporated under a nitrogen atmosphere. The resulting product, without further purification, was added to a mixture of compound 4 in pyridine and stirred at room temperature for an additional 2 hours, yielding compound 6, the ASK1 ligand.

Scheme 1 Synthesis of ligands for POI ASK1 and E3 ligase CRBN. Reagents and conditions: (a) hydrazine hydrate (3 equiv.), methanol, 100 °C, 12 h (yield: 92%); (b) N,N-dimethylformamide dimethyl acetal, reflux, 12 h (yield: 88%); (c) isopropyl amine (1.2 equiv.), acetic acid, acetonitrile, reflux, 12 h (yield: 76%); (d) oxalyl chloride (2 equiv.), dichloromethane, dimethylformamide (cat.), 0 °C – rt, 1 h, quant.; (e) 4 (1 equiv.), DCM, DIPEA (3 equiv.) rt, 4 h (yield: 42%). (f) Sodium acetate (1.2 equiv.), acetic acid, reflux, 12 h (yield: 74%).

Compound 9, a thalidomide-based warhead that functions as a ligand for the CRBN E3 ligase, was synthesized by refluxing commercially available 3-fluorophthalic anhydride (7) with 3-aminopiperidine-2,6-dione hydrochloride (8) in acetic acid (AcOH) and sodium acetate (NaOAc) for 12 hours (Scheme 1).

A solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (9) and commercially available NH₂-PEG2-t-Bu in N-methylpyrrolidone was treated with N,N-diisopropylethylamine. The mixture was then heated at 90 °C for 10–12 hours, yielding compound 10 (Scheme 2). Subsequently, compound 10 was stirred with trifluoroacetic acid (TFA) in DCM at room temperature for 2 hours, resulting in the formation of compound 11 (Scheme 2). We aimed to synthesize the desired PROTAC compounds by carrying out C–N coupling reactions between compound 11 and the bromo group of the selonsertib derivative (6) (Scheme 2). Various reaction conditions were explored, as detailed in Table S2 of the ESI.†

Scheme 2 Synthesis of the E3 ligase ligand with a PEG linker followed by the synthesis of the target PROTAC. Reagents and conditions: (a) NH₂-PEG₂-t-Bu (1.05 equiv.), N-methylpyrrolidone, N,N-diisopropylethylamine (3 equiv.), 12 h, 90 °C (yield: 45%). (b) Trifluoroacetic acid, DCM, rt, 2 h (quant.). (c) 11, different reagents and reaction conditions undertaken (see Table S2†).

Strategy 2. Scheme 3 outlines the synthetic strategies for preparing PEG linkers of various lengths, POI ligands, and the target PROTACs. The process began with an acid–amine coupling reaction using commercially available Boc-amino-PEG-amine (NH₂-PEG_n-t-Bu) linkers of different lengths (n = 1, 2, 4, 5, 6; compounds 12–16) and succinic anhydride. The reaction was performed in a mixture of DCM, pyridine, and MeCN (1 : 1 : 1) at room temperature for 3.5 hours, yielding compounds 17–21. Next, to synthesize the selonsertib fragment as a ligand for the target protein ASK1, oxalyl chloride and a catalytic amount of DMF were added to a solution of commercially available 2,4-difluoro-5-nitrobenzoic acid (22) in DCM under ice-cold conditions. The reaction mixture was then stirred at room temperature. After 1 hour, the solvent was evaporated under a nitrogen atmosphere, and the resulting crude product was directly added to a solution of compound 4 in pyridine without further purification. This mixture was stirred at room temperature for another 2 hours, yielding compound 23. To synthesize compound 24, compound 23 was dissolved in methanol (MeOH) and treated with SnCl₂ and a catalytic amount of HCl. The mixture was then refluxed at 80 °C for 3 hours, completing the synthesis of the target POI ligand (24).

Scheme 3 Synthesis of PEG linkers, POI ligands, and the target PROTACs. Reagents and conditions: (a) succinic anhydride (1.1 equiv.), DCM, pyridine, MeCN, 3.5 h, rt (yield: 90–95%). (b) Oxalyl chloride (2 equiv.), dichloromethane, dimethylformamide (cat.), 0 °C – rt, 1 h, quant.; (c) 4 (1 equiv.), pyridine, rt, 2 h (yield: 45%); (d) stannous chloride dihydrate (SnCl₂·2H₂O) (4 equiv.), methanol, hydrochloric acid (cat.), reflux, 3 h (yield: 96%). (e) 17–21, HATU (2 equiv.), DIPEA (3 equiv.), DMF, 16 h, rt (yield: 50–55%); (f) 4 M HCl in dioxane, dioxane, rt, 2 h (quant.); (g) 9 (1.1 equiv.), DIPEA (3 equiv.), DMSO, 90 °C, 16 h (40–45%).

To synthesize the target PROTAC molecules with PEG linkers of varying lengths, compounds 17–21 were dissolved in DMF and HATU and stirred at room temperature for 30 minutes. Compound 24 was then added, followed by DIPEA, and the reaction mixture was stirred for 24 hours, yielding compounds 25–29. These intermediates were then dissolved in a 1 : 1 mixture of 4 M HCl in dioxane and dioxane, and the reaction mixtures were stirred at room temperature for 2 hours, producing compounds 30–34. Finally, compound 9 was added to the mixture of compounds 30–34 in DIPEA and DMSO, and the reaction was stirred at 90 °C for 12–16 hours, resulting in the formation of the desired PROTAC molecules, 35–39.

Scheme 4 outlines the synthetic protocols and strategies for synthesizing the VHL-based PROTAC molecules. Commercially available compounds 40–44 (1.1 equiv.) were dissolved in DMF with HATU and stirred at room temperature for 30 minutes. Compound (2S,4R)-1-((R)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (VH032) was then added, followed by DIPEA, and the reaction was stirred for 16 hours, yielding compounds 45–49. Next, compounds 45–49 were dissolved in a 1 : 1 mixture of 4 M HCl in dioxane and pure dioxane, and stirred at room temperature for 2 hours to produce compounds 50–54. Compounds 50–54 were then subjected to an acid–amine coupling reaction with succinic anhydride in a mixture of DCM, pyridine, and MeCN (1 : 1 : 1) at room temperature for 3.5 hours, yielding compounds 55–59. Finally, compounds 55–59 were added to a solution of compound 24 in DIPEA and DMSO and stirred at room temperature for 16 hours, resulting in compounds 60–64, the desired VHL-based PROTAC molecules.

Scheme 4 Synthesis of the VHL-based PROTACs. Reagents and conditions: (a) (2S,4R)-1-((R)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (VH032) (1 equiv.), HATU (2 equiv.), DIPEA (3 equiv.), DMF, 16 h, rt (yield: 70–74%); (b) 4 M HCl in dioxane, dioxane, rt, 2 h (quant.); (c) succinic anhydride (1.1 equiv.), DCM, pyridine, MeCN, 3.5 h, rt (yield: 80–84%); (d) 24 (1.1 equiv.), DIPEA (3 equiv.), DMF, rt, 16 h (yield: 36–42%).

PROTAC-mediated degradation of ASK1 in HepG2 and HEK293A cells

To evaluate the efficiency of our synthesized PROTACs in targeting ASK1, we conducted experiments in HepG2 cells using varying concentrations of thalidomide-based PROTACs 35–39 (Fig. 3) for 8 hours, followed by immunoblot analysis to quantify ASK1 protein levels. The primary objective was to investigate the impact of linker length on ASK1 degradation efficiency. Among the tested compounds, dASK1 (35), featuring the shortest linker, demonstrated the highest degradation efficacy, achieving a maximum reduction of 70% in ASK1 protein levels at 100 nM (Fig. 3A). Beyond this concentration, ASK1 protein levels began to increase, a phenomenon consistent with the Hook effect—a well-documented behaviour in PROTAC systems where excessive concentrations disrupt optimal ternary complex formation.²⁴

Fig. 3 Characterization of PROTAC molecules. (A) HepG2 cells were treated with the indicated dosage of dASK1 (35) for 8 h and cell lysates were analyzed for ASK1 protein expression by western blotting. Right panel: band intensities of ASK1 normalized to actin; n = 3. (B–E) HepG2 cells were treated with the indicated dosage of synthesized PROTAC molecules for 8 h and cell lysates were analyzed for ASK1 protein expression by western blotting. (F) HepG2 cells were treated with indicated doses of dASK1 (35) for 4 h, 8 h and 12 h. The cell lysate was analyzed for ASK1 protein expression by western blotting. (G) Western blot showing dose-dependent effect of dASK1 (35) in HEK293A cells treated for 8 h.

In contrast, PROTACs with longer linkers showed progressively diminished degradation activity, as evident from Fig. 3B–E. This trend highlights the critical role of linker length in modulating the spatial alignment and proximity of the target protein (ASK1) and the E3 ligase. Shorter linkers facilitate tighter and more efficient ternary complex formation, promoting effective ubiquitination and subsequent proteasomal degradation of ASK1.

We further investigated the time-dependency of ASK1 degradation by treating HepG2 cells with dASK1 (35) for 4, 8, and 12 hours. Notably, an 8 hour treatment consistently yielded the most significant reduction in ASK1 protein levels (Fig. 3F). Both shorter (4 hour) and longer (12 hour) incubation times were less effective, underscoring the importance of optimizing treatment duration to balance efficient degradation with minimal off-target effects. These results emphasize the need for precise timing in the experimental and therapeutic applications of PROTACs. To validate these findings, we extended the study to HEK293A cells and observed similar patterns of ASK1 degradation with dASK1 (35) (Fig. 3G). Once again, the hook effect was apparent at higher concentrations, consistent with observations in HepG2 cells. This reiterates that maintaining an optimal dose range is critical to preserving ternary complex stability and maximizing degradation efficiency. These results highlight the consistency of dASK1 (35) towards ASK1 degradation in multiple cells. In addition to examining degradation efficacy, we assessed the cytotoxicity profile of dASK1 (35) across a broad concentration range (1 nM to 10 μM) using standard viability assays. Encouragingly, no detectable toxicity was observed (Fig. S1A†), supporting the compound's safety and therapeutic potential. The lack of cytotoxicity further validates the specificity of dASK1 (35) in targeting ASK1 without significant off-target effects. Proteasome dependence of dASK1 (35)-mediated ASK1 degradation was confirmed by MG132 treatment in HepG2 cells, which blocked degradation and led to ASK1 accumulation (Fig. S2†), indicating involvement of the proteasomal pathway.

Collectively, these data emphasize the central role of linker design in PROTAC development. Shorter linkers, as demonstrated by dASK1 (35), enhance the physical alignment and biochemical interaction between the target protein and the E3 ligase, ensuring efficient ubiquitination and degradation. The inverse relationship between linker length and degradation efficiency highlights the importance of optimizing linker properties during PROTAC design to achieve effective target engagement and maximal therapeutic benefit. Furthermore, the study underscores the critical need for a comprehensive understanding of dose–response behaviour and treatment timing to optimize PROTAC activity. These findings establish dASK1 (35) as a potent and selective PROTAC for ASK1 degradation, paving the way for further development of linker-optimized compounds tailored for specific cellular and disease contexts. This study also provides a framework for refining future PROTAC designs to enhance degradation efficiency, minimize off-target effects, and maintain a favourable safety profile, making this class of molecules a promising therapeutic strategy for targeting ASK1-related diseases.

Computational analysis of ternary complex formation

To elucidate the molecular interactions driving ASK1 degradation by the PROTAC dASK1 (35), we performed an in-depth computational study using induced fit docking within the Schrödinger Maestro Suite.³⁴ The analysis employed structural data from the E3 ligase complex [PDB: 5FQD,³⁵ comprising CRBN, DNA damage binding protein 1 (DDB1), and casein kinase 1 (CK1)] and the ASK1 kinase domain [PDB: 6OYT³⁶]. We initiated the docking by positioning the thalidomide moiety of dASK1 (35) into the CRBN binding site (PDB: 5FQD³⁵). The docked pose closely mirrored the co-crystallized conformation of lenalidomide, forming stabilizing hydrogen bonds with key CRBN residues, including His378, Trp380, Asn351, Ser379, and Trp386. Subsequently, we docked the selonsertib fragment into the ASK1 structure (PDB: 6OYT), where it engaged in critical hydrogen bonding interactions with residues Lys709, Val757, Gln756, and Asp822, integral for ASK1 inhibition. To model the complete ternary complex, we replaced CK1 in the CRBN structure (PDB: 5FQD³⁵) with the ASK1 kinase domain, capitalizing on the structural compatibility of kinase domains.³⁷ This substitution positioned the thalidomide moiety of dASK1 (35) within the CRBN pocket and the selonsertib fragment in the ASK1 active site, allowing for an effective spatial arrangement of the linker. We refined the ternary complex using distance constraints and manually adjusted the linker to ensure optimal alignment with the protein surfaces, preserving the structural integrity of the assembly. The complex underwent energy minimization, and we assessed the binding free energy using the MMGBSA method. Among the conformations analyzed, the structure with an intermediate linker length yielding a separation distance of approximately 12 Å between ASK1 and CRBN showed the most favorable binding free energy (Table S1†), suggesting a stable ternary configuration (Fig. 4). In this conformation, the thalidomide moiety of dASK1 (35) retained strong hydrogen bond interactions with CRBN residues Trp380, His378, Asn351, and Trp386. Concurrently, the selonsertib fragment formed key hydrogen bonds with ASK1 residues Lys709, Gln756, and Asp822 (Table S3†). Additionally, the ethereal oxygen in the linker established a stabilizing interaction with Arg705 of ASK1 (Fig. 4), contributing to the enhanced stability and efficient formation of the ternary complex. The computational analysis correlates with our in vitro assay data, highlighting the pivotal role of linker length in modulating the degradation efficiency of ASK1 by PROTACs. Specifically, dASK1 (35) achieved superior degradation performance, attributed to its finely tuned linker length, which likely optimizes the spatial orientation and stability of the ternary complex. Conversely, extending the linker length resulted in a marked reduction in ASK1 degradation efficiency, suggesting suboptimal engagement between the target and E3 ligase (Fig. 3). These findings emphasize the critical need for precise linker design to enhance PROTAC efficacy, facilitating robust and selective degradation of ASK1.

Fig. 4 The complex of CRBN–ASK1–DDB1 proteins with docked dASK1 (35) and its binding interactions in zoomed view. The DDB1, CRBN and ASK1 are indicated in the blue, grey and dark yellow cartoons, respectively, with corresponding residues in the same colours. The orange, yellow and magenta parts of dASK1 (35) represent the thalidomide, linker and selonsertib fragment, respectively. The hydrogen bonds are shown as green dotted lines and π–sulfur in yellow dotted lines.

Lead optimization guided by in vitro ADME studies

Optimizing PROTACs for therapeutic use presents significant challenges, particularly due to their poor bioavailability and limited cellular uptake, issues primarily stemming from their high molecular weight (600–1400 Da).³⁸ Addressing these limitations is vital for advancing PROTACs into clinical development. A comprehensive evaluation of ADME (absorption, distribution, metabolism, and excretion) properties is crucial for rational design improvements aimed at enhancing their drug-like characteristics. Despite the growing interest in PROTACs, detailed ADME assessments have been relatively scarce, with only a limited number of studies reporting on their physicochemical profiles.³⁹ Our in vitro ADME analysis of the lead compound dASK1 (35) revealed favorable plasma stability but identified significant challenges, including poor aqueous solubility and extensive plasma protein binding (99%) (Table 1). These findings underscore the need for targeted modifications to improve solubility and optimize the pharmacokinetic profile of dASK1 (35), ultimately enhancing its bioavailability and therapeutic potential.

Table 1 In vitro pharmacokinetic (ADME) profile of dASK1 (35) and dASK1-VHL (60)

S. No.	Log D@pH 7.40	CLog P	Aq. solubility pH = 7.4 (μg mL^−1)	Caco-2 permeability			Plasma stability		PPB^c
							Human		Human
				A to B^a (Papp) cm s^−1 (10^−6)	B to A^b (Papp) cm s^−1 (10^−6)	Efflux ratio	% remain after 1 h	% remain after 2 h
a A to B: apical to basal. b B to A: basal to apical. c PPB: plasma protein binding.
dASK1 (35)	1.66	1.00	3.92	0.00	3.46	>3.46	93.58	91.67 (t1/2 > 120 min)	99 (92.43% recovery)
dASK1-VHL (60)	2.94	2.89	133.63	0.33	1.03	3.09	92.47	94.28 (t1/2 > 240 min)	96.98 (87.9% recovery)

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Exploration of alternative PROTAC designs with VHL-based dASK1-VHL (60)

Within the diverse family of nearly 600 E3 ubiquitin ligases, the von Hippel–Lindau (VHL) E3 ligase has gained prominence in PROTAC design, owing to its well-characterized and high-affinity ligand, VH032.^39–42 In hepatic cells, VHL plays a pivotal role in regulating oxygen-sensing transcription factors, HIF-1α and HIF-2α, which are essential for cellular adaptation to hypoxia. Importantly, the downregulation of VHL in the liver has been linked to metabolic dysregulation, including steatosis and fibrosis, highlighting its critical function in maintaining hepatic homeostasis.^43,44

To investigate whether ASK1 degradation is specific to CRBN and to explore alternative E3 ligase engagement strategies, we synthesized a novel VHL-based PROTAC, dASK1-VHL (60), with one PEG linker, while retaining the original linker and ASK1-targeting moiety (Scheme 4). This approach aimed to leverage the regulatory role of the VHL E3 ligase in hepatic physiology and broaden the scope of PROTAC applications by enabling engagement with distinct E3 ligases. By targeting VHL, which plays a pivotal role in oxygen-sensing pathways and metabolic regulation in hepatocytes, dASK1-VHL (60) represents a versatile alternative for achieving efficient ASK1 degradation in a liver-specific context. To further investigate the effect of linker length on degradation efficacy, we synthesized PROTACs (Table 2) with a gradually increasing number of PEG linkers (n = 1–5).

Table 2 List of all synthesized PROTACs along with their efficacy to degrade ASK1

S No.	Compound	R1	R2	Activity
1	35			Active
2	36			Inactive
3	37			Inactive
4	38			Inactive
5	39			Inactive
6	60			Active
7	61			Active
8	62			Inactive
9	63			Inactive
10	64			Inactive

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ADME analysis

Following its synthesis, dASK1-VHL (60) was subjected to an in-depth in vitro ADME analysis to evaluate its pharmacokinetic properties. Compared to its CRBN-based counterpart, dASK1 (35), dASK1-VHL (60) exhibited significant improvements, including enhanced aqueous solubility, ensuring better dissolution and potentially higher systemic exposure (Table 1). The optimized Log D (2.94) and CLog P (2.89) values of dASK1-VHL (60) align well with drug-like properties, suggesting better membrane permeability and distribution compared to dASK1 (35). dASK1-VHL (60) demonstrates moderately reduced PPB compared to dASK1 (35). This translates to a higher free drug concentration for dASK1-VHL (60), likely improving its pharmacological activity and therapeutic potential. These pharmacokinetic enhancements make dASK1-VHL (60) a strong candidate for preclinical development, particularly for therapeutic applications targeting hepatic diseases.

In vitro efficacy of ASK1 degradation

In HepG2 cells, dASK1-VHL (60) demonstrated robust ASK1 degradation within the concentration range of 10–100 nM, achieving a maximum reduction of 60% at 50 nM (Fig. 5A). This degradation efficiency highlights the potential of VHL-based PROTACs in achieving targeted protein degradation. Pre-treatment with the VHL ligand VH032 completely abrogated ASK1 degradation (Fig. 5B), confirming the specificity of dASK1-VHL (60) for VHL-mediated degradation. These results validate the ability of dASK1-VHL (60) to effectively engage VHL and facilitate selective ASK1 degradation. PROTAC 61 featured a slightly longer linker, incorporating two PEG units compared to the single PEG linker in dASK1-VHL (60), retained ASK1 degradation capability but required higher concentrations to achieve effective degradation (Fig. 5C). However, PROTACs with even longer PEG linkers (Fig. 5D–F, Table 2) showed no detectable degradation of ASK1, underscoring the critical role of linker length in maintaining the proximity and alignment of the target protein (ASK1) and the E3 ligase (VHL). These findings emphasize the importance of precise linker optimization in enhancing the degradation efficiency of PROTACs. In addition to its efficacy in ASK1 degradation, dASK1-VHL (60) demonstrated a favourable safety profile, exhibiting no detectable cytotoxicity across a wide concentration range (1 nM to 10 μM) in HepG2 cells (Fig. S1B†).

Fig. 5 Characterization of VHL based PROTAC molecules. (A) HepG2 cells were treated with indicated dosage of dASK1-VHL (60) for 8 h and cell lysates were analyzed for ASK1 protein expression by western blotting. Right panel: densitometric analysis of ASK1 protein levels normalized to actin; n = 3. (B) HepG2 cells were pre-treated for 30 min with 1 μM of VHL ligand VH032 followed by treatment with respective doses of PROTAC dASK1-VHL (60) for 8 h. Cellular ASK1 protein levels were determined by immunoblotting. (C–F) HepG2 cells were treated with indicated dosage of synthesized PROTAC molecules for 8 h and cell lysates were analyzed for ASK1 protein expression by western blotting.

These findings reveal that ASK1 degradation is not confined to CRBN-mediated pathways; the VHL E3 ligase is equally effective in facilitating selective and efficient ASK1 degradation. This expands the utility of PROTAC technology by demonstrating the feasibility of alternative E3 ligases, such as VHL, in achieving targeted protein degradation. The improved pharmacokinetic profile, combined with effective ASK1 degradation and a strong safety profile, positions dASK1-VHL (60) as a promising candidate for metabolic and inflammatory liver diseases, including MASH.

In vivo pharmacokinetic evaluation of dASK1-VHL (60)

To evaluate the in vivo pharmacokinetic properties of dASK1-VHL (60), we conducted a study in male C57BL/6 mice, administering the compound at doses of 20 mg kg⁻¹ orally and 2 mg kg⁻¹ intravenously (Table 3). The compound exhibited rapid systemic absorption, with a T_max of 1 hour following oral administration, indicating efficient uptake into the systemic circulation. For intravenous dosing, the compound displayed a short half-life (t_1/2) of 0.55 hours, reflecting its rapid systemic clearance dynamics. The maximum plasma concentration (C_max) following oral dosing was 9429.30 ng mL⁻¹, demonstrating robust absorption through the gastrointestinal tract. The area under the concentration-time curve (AUC_0–∞) was 12913.87 ng h mL⁻¹ for oral administration and 2719.67 ng h mL⁻¹ for intravenous dosing, signifying substantial systemic exposure relative to the dosing route.

Table 3 In vivo pharmacokinetic parameters of dASK1-VHL (60)

Dose^a (mg kg^−1)	C max (ng mL^−1)	t max (h)	t 1/2 (h)	AUC0–inf (ng h mL^−1)	Plasma CL (mL min^−1 kg^−1)	V ss (L kg^−1)	DNAUC	F (%)
a Compounds were administered 20 mg kg^−1 through the oral route and 2 mg kg^−1 through the intravenous route in mice (n = 3). Here, Cmax is the maximum concentration of the compound in plasma/blood, tmax is the time required to reach Cmax, t1/2 is the half-life, AUC is the area under the curve, CL is the rate of clearance, and Vss is the volume of distribution.
20 (n = 3) po	9429.3	1.00	—	12913.87	—	—	645.69	47.48
2 (n = 3) iv	—	—	0.55	2719.67	11.36	0.40	1359.83	—

---

Crucially, the oral bioavailability (% F) of dASK1-VHL (60) was determined to be 47.48%, reflecting effective gastrointestinal absorption and first-pass metabolism efficiency. Additionally, the dose-normalized area under the curve (DNAUC) values for oral and intravenous administration were 645.69 ng h mL⁻¹ mg⁻¹ and 1359.83 ng h mL⁻¹ mg⁻¹, respectively, highlighting efficient systemic exposure relative to the administered dose. The combination of rapid absorption, moderate clearance, and favourable oral bioavailability underscores the therapeutic potential of dASK1-VHL (60) for preclinical applications. These pharmacokinetic characteristics suggest that the compound provides balanced absorption, distribution, and systemic retention, making it a promising candidate for further investigation in metabolic and inflammatory liver disease models. The results support continued evaluation of dASK1-VHL (60) in in vivo efficacy studies, particularly in disease settings where ASK1 degradation could provide a therapeutic benefit.

Efficacy of PROTAC dASK1-VHL (60) in MCD diet-induced murine model

To evaluate the in vivo efficacy of dASK1-VHL (60), we employed a well-established preclinical model of metabolic dysfunction-associated steatohepatitis (MASH) induced by a methionine choline-deficient (MCD) diet. Male C57BL/6 mice (6–8 weeks old) were fed the MCD diet for 14 days to induce liver injury and steatosis characteristic of MASH. Following which mice were randomized into two groups and one group was treated with vehicle (1% DMSO in PBS) and another with dASK1-VHL (60) at a dose of 2 mg kg⁻¹via intraperitoneal injection for an additional 14 days along with MCD diet (n = 6 per group; Fig. 6A). Western blot analysis of liver tissues collected at the end of the treatment period demonstrated a substantial reduction in ASK1 protein levels in mice treated with dASK1-VHL (60), accompanied by a marked decrease in the phosphorylation of p38 MAPK, a key downstream effector of ASK1 signalling (Fig. 6B). This reduction in p38 phosphorylation indicates effective disruption of the ASK1 signaling cascade, suggesting successful engagement and degradation of the target protein. dASK1-VHL (60) did not affect the protein level of other MAP3K, like TAK1 (Fig. 6B).

Fig. 6 In vivo validation of PROTAC dASK1-VHL (60). (A) Schematic representation of the in vivo experiment. 4–6 week male C57/Bl6 mice were fed with the MCD diet for 14 days, followed by 14 days of IP injection of dASK1-VHL (60) at a dose of 2 mg kg⁻¹ weight along with the MCD diet. (B) Left panels: ASK1, phosphorylated p38, and TAK1 protein levels were determined from the livers of two groups of mice as in; right panels: densitometric analysis of the band intensities of ASK1, p-p38 and TAK1 normalized by actin and total p38, respectively; *p < 0.05. (C) Gene expression of ASK1 in the livers of vehicle-treated and dASK1-VHL (60) treated animals. ns, not significant. (D) Liver homogenates from two groups of animals were immunoprecipitated using ASK1 antibody and immunoblotted with anti-ubiquitin antibody. ASK1 and actin from the homogenates were used as input. IP-immunoprecipitation, IB-immunoblotting. (E) Representative images of hematoxylin and eosin (H&E) staining of liver tissue.

Interestingly, quantitative PCR analysis showed no significant change in ASK1 mRNA expression between the vehicle and treated groups (Fig. 6C), implying that the observed decrease in ASK1 protein levels was due to post-translational degradation rather than transcriptional downregulation. To confirm the mechanism of action, we performed immunoprecipitation of ASK1 from liver lysates, followed by ubiquitination analysis. The results indicated a significant reduction in ASK1 ubiquitination in the dASK1-VHL (60)–treated group compared to vehicle controls (Fig. 6D). This finding supports the hypothesis that dASK1-VHL (60) mediates ASK1 degradation via the ubiquitin–proteasome pathway. In addition to reducing ASK1 protein levels, dASK1-VHL (60) treatment resulted in a modest but notable decrease in hepatic lipid accumulation, as evidenced by histological analysis (Fig. 6E). The reduction in hepatic lipid content is indicative of improved metabolic regulation and suggests that ASK1 degradation by dASK1-VHL (60) may attenuate key pathological features of MASH, including steatosis and inflammation.

Overall, these findings demonstrate that dASK1-VHL (60) effectively degrades ASK1 through VHL E3 ligase-mediated proteasomal pathways, providing a potent mechanism for inhibiting ASK1 signaling. The compound's ability to reduce ASK1 protein levels and ameliorate hepatic lipid accumulation highlights its potential as a novel therapeutic strategy for treating MASH and related metabolic liver disorders. The use of VHL as the E3 ligase in this PROTAC design expands the toolkit of available ligases beyond CRBN, offering a broader approach to target engagement and enhancing the versatility of PROTAC technology in liver disease models.

Conclusion

Despite substantial progress in the development of PROTAC technology, its application in metabolic diseases remains limited, with the majority of research efforts concentrated on oncology, neurodegenerative disorders, and immune regulation. This study addresses a critical gap by introducing a novel PROTAC, dASK1-VHL (60), specifically designed to target metabolic liver disease, focusing on MASH. The design strategy emphasized the rational selection of a high-affinity ASK1-binding ligand, precise linker optimization, and the innovative use of VHL E3 ligase—a deliberate shift from the conventionally employed CRBN ligase. The decision to leverage VHL as the E3 ligase was informed by its pivotal role in hepatic physiology, particularly its involvement in modulating hypoxic responses and maintaining metabolic homeostasis. This shift expands the versatility of PROTAC platforms, demonstrating the feasibility of utilizing alternative E3 ligases beyond CRBN, and highlights the potential of VHL-based PROTACs for targeted liver-specific therapies. Our initial PROTAC, dASK1 (35), successfully formed a stable ternary complex between ASK1, the degrader, and the CRBN E3 ligase, resulting in rapid and sustained ASK1 degradation via the ubiquitin–proteasome pathway. In vitro assays validated the efficacy of CRBN-mediated ASK1 degradation, establishing a strong proof of concept for targeting ASK1 using a PROTAC approach. However, to broaden the therapeutic scope and enhance the specificity of degradation, we developed dASK1-VHL (60), incorporating the VHL E3 ligase for improved target engagement and tissue specificity. Comprehensive ADME and in vivo pharmacokinetic studies revealed that dASK1-VHL (60) exhibits an optimized drug-like profile, characterized by enhanced aqueous solubility, moderate systemic clearance, and favourable bioavailability—parameters critical for achieving sustained therapeutic exposure in chronic metabolic disorders. The improved pharmacokinetic properties of dASK1-VHL (60) support its potential for effective in vivo application. In vivo efficacy was demonstrated using a MCD diet-induced murine model of MASH. Administration of dASK1-VHL (60) resulted in significant degradation of ASK1, as evidenced by reduced ASK1 protein levels and diminished phosphorylation of the downstream effector p38 MAPK, confirming the compound's ability to disrupt ASK1 signaling through a proteasome-dependent mechanism. Importantly, dASK1-VHL (60) treatment led to a notable reduction in hepatic lipid content, suggesting that selective ASK1 degradation can mitigate key pathological hallmarks of MASH, including steatosis and hepatic inflammation.

This study underscores the importance of ‘linkerology’—the length and composition of linkers—which directly influence the physicochemical properties and efficacy of the PROTAC,^32,45 E3 ligase selection and comprehensive pharmacokinetic optimization in the rational development of PROTACs targeting metabolic diseases. By expanding PROTAC applications beyond traditional oncology targets and employing tailored strategies to address the unique challenges of metabolic liver disorders, we establish a promising framework for future therapeutic development. The successful degradation of ASK1 and the observed reduction in liver pathology highlight the therapeutic potential of PROTACs in addressing unmet clinical needs in MASH. Furthermore, this work paves the way for broader exploration of alternative E3 ligases, such as VHL, in the design of next-generation PROTACs for metabolic and inflammatory diseases.

Experimental section

Chemical synthesis and methods

General methods. All starting materials, reagents, and solvents were purchased from commercial suppliers and used without further purification. Dry solvents were either commercially purchased or dried using a standard protocol. Reactions that had sensitivity toward moisture or oxygen were carried out under a dry nitrogen or argon atmosphere. All the TLC experiments were performed on silica gel plates (Merck silica gel 60, F254). The spots were visualized under UV light (λ = 254 and 365 nm) or by using the appropriate stain. Compounds were purified using a Teledyne ISCO CombiFlash R_f system using a 230–400 mesh size silica gel. ¹H NMR was recorded at 300 MHz (Bruker-DPX), 400 MHz (JEOL), and 600 MHz (Bruker Avance) frequencies, and ¹³C NMR spectra were recorded at 75 MHz (Bruker-DPX), 100 MHz (JEOL), and 150 MHz (Bruker Avance) frequencies in CDCl₃ or CD₃OD or d₆-DMSO using tetramethylsilane as the internal standard. The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs. = broad singlet. The coupling constant, J, was reported in the Hertz unit (Hz). High-resolution mass spectroscopy, HRMS (m/z), was carried out using ESI (Q-Tof Micro mass spectrometer), and ESI (LTQ Orbitrap XL mass spectrometer). Reversed phase HPLC analysis was performed for compounds 35, 36, 37, 38 and 39 in Shimadzu LC-20AD BLK instrument (column: Supelco Ascentis C18, 250 × 4.6 cm, 5 μm; wavelength: 254 nm, flow rate: 1.5 mL min⁻¹), using 10 μL of sample volume dissolved in 5% ACN. The gradient mobile phase programme with water and ACN in two channels was as: ACN 25% → 75% (0 to 20 min) → 25% (20 to 21 min) → 25% (up to end): the complementary water channel was adjusted accordingly. For compounds 60, 61, 62, 63, 64 and 65, the same HPLC method was followed but with a flow rate of 0.8 mL min⁻¹ and an isocratic mobile phase of ACN–water (75 : 25 v/v).

General procedure A: linker preparation through amidation reaction (Scheme 3). Commercially available Boc-amino-PEG-amine (1 equiv.) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (1.1 equiv.) was then added to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ and evaporated to dryness multiple times for the complete removal of pyridine to give the product with a yield of 90–95%. The product was carried forward for the next step without further purification.

General procedure B (Scheme 3): adjoining linkers with selonsertib derivative through amidation reaction. PEG linkers with acid functionality at one end (1.5 equiv.) were taken in DMF and HATU (2 equiv.) and allowed to stir at room temperature for 30 min. Compound 24 (1 equiv.) was then added to the reaction mixture, followed by DIPEA (3 equiv.) and the reaction mixture was stirred for 16 h. Reactions were monitored by checking TLC. Upon completion, the reaction mixture was washed thoroughly with ice-cold water to remove excess DMF and extracted with EtOAc. Column chromatography was performed in a Teledyne ISCO Combi flash system using 100–200 mesh size silica gel and 4–10% MeOH in CHCl₃ as eluents to get the pure compounds as a colorless liquid with a yield of 50–55%.

General procedure C (Scheme 3): Boc deprotection. Selonsertib derivatives with Boc-protected linkers were taken in a mixture of 4 M HCl in dioxane and dioxane (1 : 1). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude material (quant.). The crude material was then used for the next step without further purification.

General procedure D (Scheme 3): attachment of selonsertib fragment with Boc deprotected linkers with thalidomide to get the final PROTACs. Compounds 30–34 (1 equiv.) were taken in DMSO under an Ar atmosphere, and then DIPEA (3 equiv.) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (1.1 equiv.) was then added to the reaction mixture and the reaction was stirred for 16 h at room temperature. The reaction was monitored by checking TLC. Upon completion, the reaction mixture was diluted with ice-cold water and extracted with EtOAc. The organic part was then concentrated in a rotary evaporator under reduced pressure to get the crude product. Flash column chromatography was performed in a Teledyne ISCO Combi flash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure PROTAC compounds 35–39 as light yellow solids with a yield of 40–45%.

6-Aminopicolinohydrazide (2). A solution of methyl 6-aminopicolinate (1) (3.0 g, 19.8 mmol), hydrazine hydrate (4.95 g, 98.6 mmol, 4.8 mL) and MeOH (15 mL) was heated at 100 °C for 12 h. After completion of the reaction, confirmed by TLC, the reaction solution was cooled to room temperature and filtered. The filter cake was washed with ethyl acetate (30 mL) and dried on a rotary evaporator under reduced pressure to give the title compound 2 (2.94 g, 98%) and carried forward for the next step of synthesis without further purification. ¹H NMR (600 MHz, DMSO-d₆) δ 9.16 (s, 1H), 7.50 (td, J = 7.7, 2.9 Hz, 1H), 7.10 (dd, J = 7.2, 2.9 Hz, 1H), 6.60 (dd, J = 8.2, 2.9 Hz, 1H), 6.08 (s, 2H), 4.48 (s, 2H). HRMS (ESI) m/z (M + H)⁺ calcd for C₆H₉N₄O, 153.0771; found, 153.0776.

(E)-N′-(6-(2-((E)-(Dimethylamino)methylene)hydrazine-1-carbonyl)pyridin-2-yl)-N,N-dimethylformimidamide (3). Compound 2 (2 g, 6.58 mmol) was added to dimethyl formamide dimethyl acetal (20 mL). The system was refluxed with stirring at 110 °C for 12 h. After the reaction was completed, confirmed by TLC, the reaction solution was dried on a rotary evaporator under reduced pressure to obtain a crude product. To the crude product was added ethyl acetate (500 mL), stirred for 20 min at room temperature and filtered. The filter cake was dried to give the title compound 3 (3.03 g, 88%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.69 (s, 1H), 8.86 (s, 1H), 8.08 (s, 1H), 7.76–7.63 (m, 1H), 7.48 (dd, J = 7.4, 1.0 Hz, 1H), 6.91 (dd, J = 8.0, 1.0 Hz, 1H), 3.14 (s, 3H), 3.00 (s, 3H), 2.86 (s, 6H). HRMS (ESI) m/z (M + H)⁺ calcd for C₁₂H₁₉N₆O, 263.1615; found, 263.1619.

6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (4). To a solution of compound 3 (3 g, 11.44 mmol) in a mixture of CH₃CN/AcOH (30 mL, 5 : 1) was added isopropylamine (3.38 g, 57.2 mmol, 4.8 mL). The resulting mixture was heated at 80 °C for 12 h. Upon completion, confirmed by TLC, the reaction solution was allowed to stand and filtered. The residue was dissolved in water (100 mL) and 2 M NaOH was added to a pH of 8.0. The water part was extracted with EA (100 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting residue was chromatographed over silica gel (3–10% MeOH in DCM) to give the title compound 4 as a light-yellow solid (1.76 g, 76%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (s, 1H), 7.51 (dd, J = 8.3, 7.4 Hz, 1H), 7.16 (dd, J = 7.4, 0.8 Hz, 1H), 6.52 (dd, J = 8.3, 0.8 Hz, 1H), 6.15 (s, 2H), 5.52 (hept., J = 6.7 Hz, 1H), 1.42 (d, J = 6.7 Hz, 6H). HRMS (ESI) m/z (M + H)⁺ calcd for C₁₀H₁₄N₅, 204.1244; found, 204.1252.

5-Bromo-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-methylbenzamide (6). 5-Bromo-2-fluoro-4-methylbenzoic acid (5) (1 g, 11.44 mmol) was taken in DCM. Oxalyl chloride (1089 mg, 8.58 mmol, 736 μL) and a catalytic amount of DMF (31 mg, 0.43 mmol, 33 μL) were added to the reaction mixture in ice-cold conditions, followed by stirring at room temperature for 1 h. The solvent was then evaporated to dryness under an N₂ atmosphere, followed by the addition of the mixture of compound 4 (581 mg, 2.86 mmol) in DCM (2 mL) and DIPEA (1108 mg, 8.58 mmol, 1.49 mL). The final reaction mixture was then stirred at room temperature for another 4 h. Upon completion, confirmed by TLC, volatile materials were removed under vacuum. The residue was dissolved in ice-cold water (50 mL) and extracted with DCM (50 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to get the crude product. Column chromatography was performed using 3–10% MeOH in DCM to get the pure compound 6 as a dark yellow solid (502 mg, 42%). ¹H NMR (600 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.87 (s, 1H), 8.18 (d, J = 8.3 Hz, 1H), 8.03 (t, J = 8.0 Hz, 1H), 7.90 (dd, J = 9.8, 7.1 Hz, 2H), 7.47 (d, J = 10.7 Hz, 1H), 5.64 (p, J = 6.8 Hz, 1H), 2.41 (s, 3H), 1.43 (d, J = 6.6 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.55, 159.90, 157.41, 151.37, 150.25, 146.76, 143.79, 143.59, 143.50, 140.25, 133.42, 133.39, 124.14, 123.99, 119.90, 119.37, 119.12, 115.04, 48.45, 23.73, 23.01. HRMS (ESI) m/z (M + H)⁺ calcd for C₁₈H₁₈BrFN₅O, 418.0673; found, 418.0683.

2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (9). Sodium acetate (1.98 g, 24.08 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (2.38 g, 14.45 mmol) were added to a solution of phthalic anhydride (2 g, 12.04 mmol) in acetic acid (50 mL). The mixture was stirred at 120 °C for 12 h. After cooling it to room temperature, the solution was poured into ice water (200 mL) and stirred for 10 min. The mixture was filtered, and the gray solid was purified by flash column chromatography on silica gel (4–10% MeOH in DCM) to afford compound 9 as an off-white solid (2.46 g, 74%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.14 (s, 1H), 7.95 (ddd, J = 8.4, 7.4, 4.5 Hz, 1H), 7.80–7.70 (m, 2H), 5.16 (dd, J = 12.9, 5.4 Hz, 1H), 2.89 (ddd, J = 17.1, 13.9, 5.5 Hz, 1H), 2.67–2.50 (m, 2H), 2.07 (dtd, J = 13.0, 5.3, 2.2 Hz, 1H). HRMS (ESI) m/z (M + H)⁺ calcd for C₁₃H₁₀FN₂O₄, 277.0619; found, 277.0623.

tert-Butyl(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (10). To a solution of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (750 mg, 2.72 mmol) and tert-butyl(2-(2-(2 aminoethoxy)ethoxy)ethyl)carbamate (707.95 mg, 2.85 mmol, 676 μL) and in NMP (4 mL), was added 265 μL DIPEA (1052.82 mg, 8.15 mmol, 1.42 mL). After heating to 90 °C for 12 h, the reaction mixture was diluted with 60 mL of EtOAc, washed with 10% citric acid (20 mL), saturated NaHCO₃ solution (20 mL) and finally with brine (20 mL) before drying over Na₂SO₄ and concentrating in vacuo. The resulting brown oil was dissolved in DCM and purified by silica chromatography (30–90% EtOAc in pet ether) to give the desired product 10 as a brown oil (616.46 mg, 45%). ¹H NMR (400 MHz, chloroform-d) δ 9.02 (s, 1H), 7.47–7.40 (m, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.46 (s, 1H), 5.00 (d, J = 87.9 Hz, 1H), 3.67 (t, J = 5.4 Hz, 2H), 3.60 (q, J = 4.5 Hz, 4H), 3.51 (t, J = 5.0 Hz, 2H), 3.42 (q, J = 5.4 Hz, 2H), 3.30–3.22 (m, 2H), 2.82–2.67 (m, 3H), 2.40–2.20 (m, 1H), 1.37 (s, 9H). HRMS (ESI) m/z (M + H)⁺ calcd for C₂₄H₃₂N₄O₈Na, 527.2118; found, 527.2120.

4-((2-(2-(2-Aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione TFA salt (11). tert-Butyl(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (330.2 mg 0.203 mmol) was dissolved in TFA (1 mL) in DCM (2 mL) and stirred for 2 h at room temperature. The reaction mixture was concentrated under a stream of nitrogen, followed by a vacuum to give the crude product 11 as a brown oil (264 mg, quant.). This material was used without further purification. HRMS (ESI) m/z (M + H)⁺ calcd for C₂₁H₂₅F₃N₄O₈, 518.1624; found, 518.1632.

2,2-Dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazapentadecan-15-oic acid (17). tert-Butyl(2-(2-aminoethoxy)ethyl)carbamate (12) (1.2 g, 5.87 mmol) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (646.65 mg, 6.46 mmol) was then added to the reaction mixture and the reaction was performed according to the general procedure A. The product 17 appeared as a colorless liquid (1.69 g, 95%) and was used for the next step without purification. ¹H NMR (400 MHz, chloroform-d) δ 7.04 (s, 1H), 6.69 (s, 1H), 3.50–3.40 (m, 6H), 3.33–3.24 (m, 2H), 2.71 (dd, J = 7.0, 5.0 Hz, 2H), 2.59–2.43 (m, 2H), 1.46 (s, 9H). HRMS (ESI) m/z (M + H)⁺ calcd for C₁₃H₂₅N₂O₆, 305.1707; found, 305.1716.

2,2-Dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaoctadecan-18-oic acid (18). tert-Butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (13) (1.1 g, 4.43 mmol) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (487.61 mg, 4.87 mmol) was then added to the reaction mixture and the reaction was performed according to the general procedure A. The product 18 appeared as a colorless liquid (1.54 g, 94%) and was used for the next step without purification. ¹H NMR (600 MHz, chloroform-d) δ 7.59 (s, 1H), 5.60 (s, 1H), 3.57 (s, 4H), 3.49 (t, J = 5.0 Hz, 4H), 3.36–3.31 (m, 2H), 3.25 (s, 2H), 2.63–2.47 (m, 4H), 2.41–2.31 (m, 4H), 1.40 (s, 9H). HRMS (ESI) m/z (M + Na)⁺ calcd for C₁₅H₂₈N₂NaO₇, 371.1789; found, 371.1797.

2,2-Dimethyl-4,21-dioxo-3,8,11,14,17-pentaoxa-5,20-diazatetracosan-24-oic acid (19). tert-Butyl(14-amino-3,6,9,12-tetraoxatetradecyl)carbamate (14) (1.2 g, 3.57 mmol) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (392.63 mg, 3.92 mmol) was then added to the reaction mixture and the reaction was performed according to the general procedure A. The product 19 appeared as a colorless liquid (1.46 g, 94%) and was used for the next step without purification. ¹H NMR (400 MHz, chloroform-d) δ 6.90 (s, 1H), 5.22 (s, 1H), 3.67 (s, 2H), 3.63 (dtd, J = 9.3, 5.2, 4.5, 2.1 Hz, 10H), 3.54 (dt, J = 7.3, 5.1 Hz, 4H), 3.44 (q, J = 5.1 Hz, 2H), 3.34–3.24 (m, 2H), 2.67 (dd, J = 7.2, 5.9 Hz, 2H), 2.52 (dd, J = 7.3, 5.9 Hz, 2H), 1.43 (s, 9H). HRMS (ESI) m/z: (M + H)⁺ calcd for C₁₉H₃₆N₂O₉Na, 459.2326; found, 459.2319.

2,2-Dimethyl-4,24-dioxo-3,8,11,14,17,20-hexaoxa-5,23-diazaheptacosan-27-oic acid (20). tert-Butyl(17-amino-3,6,9,12,15-pentaoxaheptadecyl)carbamate (15) (1.2 g, 3.15 mmol) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (347.17 mg, 3.45 mmol) was then added to the reaction mixture and the reaction was performed according to the general procedure A. Product 20 appeared as a colorless liquid (1.39 g, 92%) and used for the next step without purification. ¹H NMR (400 MHz, chloroform-d) δ 7.24 (s, 1H), 5.27 (s, 1H), 3.69–3.60 (m, 16H), 3.53 (dt, J = 9.0, 4.9 Hz, 4H), 3.44 (q, J = 5.0 Hz, 2H), 3.36–3.21 (m, 2H), 2.67–2.61 (m, 2H), 2.53 (t, J = 6.6 Hz, 2H), 1.43 (s, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₂₁H₄₁N₂O₁₀, 481.2761; found, 481.2752.

2,2-Dimethyl-4,27-dioxo-3,8,11,14,17,20,23-heptaoxa-5,26-diazatriacontan-30-oic acid (21). tert-Butyl(20-amino-3,6,9,12,15,18-hexaoxaicosyl)carbamate (16) (1.25 g, 2.94 mmol) was taken in 3 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (324.11 mg, 3.24 mmol) was then added to the reaction mixture and the reaction was performed according to the general procedure A. Product 21 appeared as a colorless liquid (1.39 g, 90%) and used for the next step without purification. ¹H NMR (400 MHz, chloroform-d) δ 7.04 (s, 1H), 5.16 (s, 1H), 3.64–3.59 (m, 20H), 3.54–3.49 (m, 4H), 3.42 (q, J = 5.1 Hz, 2H), 3.28 (s, 2H), 2.99 (s, 1H), 2.65–2.60 (m, 2H), 2.56–2.48 (m, 2H), 1.42 (s, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₂₁H₄₁N₂O₁₀, 525.3023; found, 525.3021.

2,4-Difluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-nitrobenzamide (23). 2,4-Difluoro-5-nitrobenzoic acid (22) (1.5 g, 7.38 mmol) was taken in DCM. Oxalyl chloride (1.87 g, 14.77 mmol, 1.27 mL) and a catalytic amount of DMF were added to the reaction mixture under ice-cold conditions, followed by stirring at room temperature for 1 h. The solvent was then evaporated to dryness under a N₂ atmosphere, followed by the addition of the mixture of compound 6 (1 g, 4.92 mmol) in pyridine (3 mL). The final reaction mixture was then stirred at room temperature for another 2 h. Upon completion, confirmed by TLC, volatile materials were removed under vacuum. The yellow residue was dissolved in ice-cold water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to get the crude product. Column chromatography was performed using 2–10% MeOH in DCM to get the pure compound 23 as a yellow solid (1.29 g, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 8.84 (s, 1H), 8.56 (t, J = 7.6 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.02 (t, J = 8.0 Hz, 1H), 7.93–7.85 (m, 2H), 5.57 (p, J = 6.6 Hz, 1H), 1.40 (d, J = 6.7 Hz, 6H). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₇H₁₅F₂N₆O₃, 389.1168; found, 389.1162.

5-Amino-2,4-difluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide (24). Compound 23 (1.2 g, 3.09 mmol) was dissolved in MeOH (3 mL). Stannous chloride dehydrate (2.78 g, 12.36 mmol) was added to the reaction mixture with a catalytic amount of HCl. The reaction mixture was refluxed for 3 h. Upon completion, confirmed by TLC, the reaction mixture was neutralized with 4 N NaOH to pH 8 and extracted with EtOAc (20 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to get the crude product. The crude product was then triturated with CHCl₃/hexane to give the pure compound 24 as a yellowish-brown solid (1.06 g, 96%). ¹H NMR (400 MHz, methanol-d₄) δ 8.81 (s, 1H), 8.30 (d, J = 8.1 Hz, 1H), 7.97 (t, J = 7.8 Hz, 1H), 7.87–7.84 (m, 1H), 7.34–7.22 (m, 1H), 6.98 (t, J = 10.7 Hz, 1H), 5.54 (p, J = 6.7 Hz, 1H), 1.52 (d, J = 6.6 Hz, 6H). HRMS (ESI) m/z [M + H]⁺ calcd for C₁₇H₁₇F₂N₆O, 359.1432; found, 359.1426.

tert-Butyl(2-(2-(4-((2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)amino)-4-oxobutanamido)ethoxy)ethyl)carbamate (25). Compound 17 (382 mg, 1.26 mmol) was taken in DMF (2 mL). HATU (948.5 mg, 2.5 mmol) was added and the reaction mixture was allowed to stir at room temperature for 15 min. Compound 24 was added (300 mg, 0.84 mmol) to the reaction mixture, followed by DIPEA (324.7 mg, 2.51 mmol, 437.6 μL) and the reaction was performed according to the general procedure B to get the pure compound 25 (296.84 mg, 55%). ¹H NMR (400 MHz, chloroform-d) δ 8.95 (s, 1H), 8.93–8.88 (m, 1H), 8.41–8.35 (m, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.04–6.94 (m, 1H), 6.56 (s, 1H), 5.49 (p, J = 6.7 Hz, 1H), 4.99 (s, 1H), 3.56–3.44 (m, 6H), 3.30 (q, J = 4.8 Hz, 2H), 2.78 (dd, J = 7.9, 3.8 Hz, 2H), 2.67 (dd, J = 7.7, 4.1 Hz, 2H), 1.58 (s, 3H), 1.57 (s, 3H), 1.43 (s, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₃₀H₃₉F₂N₈O₆, 645.2961; found, 645.2973.

tert-Butyl(2-(2-(2-(4-((2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)amino)-4-oxobutanamido)ethoxy)ethoxy)ethyl)carbamate (26). Compound 18 (437.5 mg, 1.26 mmol) was dissolved in DMF (2 mL). HATU (954.95 mg, 2.51 mmol) was added and the reaction mixture was allowed to stir at room temperature for 15 min. Compound 24 was added (300 mg, 0.84 mmol) to the reaction mixture, followed by DIPEA (324.7 mg, 2.51 mmol, 437.6 μL) and the reaction was performed according to the general procedure B to get the pure compound 26 (311.35 mg, 54%). ¹H NMR (600 MHz, chloroform-d) δ 9.02 (s, 1H), 8.96 (d, J = 13.6 Hz, 1H), 8.88 (t, J = 8.3 Hz, 1H), 8.51 (s, 1H), 8.39 (s, 1H), 8.01–7.98 (m, 1H), 7.90 (t, J = 8.0 Hz, 1H), 7.86 (s, 1H), 6.99 (t, J = 10.4 Hz, 1H), 6.63 (s, 1H), 5.49 (dt, J = 13.2, 6.7 Hz, 1H), 3.72 (td, J = 6.7, 4.2 Hz, 2H), 3.58–3.54 (m, 4H), 3.49–3.47 (m, 2H), 3.34–3.28 (m, 2H), 3.18 (qd, J = 7.4, 4.4 Hz, 2H), 2.95 (s, 1H), 2.87 (s, 1H), 2.76 (d, J = 5.1 Hz, 2H), 2.66 (d, J = 4.9 Hz, 2H), 1.58 (d, J = 6.7 Hz, 6H), 1.46 (d, J = 6.6 Hz, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₃₂H₄₃F₂N₈O₇, 689.3217; found, 689.3224.

tert-Butyl(19-((2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)amino)-16,19-dioxo-3,6,9,12-tetraoxa-15-azanonadecyl)carbamate (27). Compound 19 (548.1 mg, 1.26 mmol) was taken in DMF (2 mL). HATU (954.94 mg, 2.51 mmol) was added and the reaction mixture was allowed to stir at room temperature for 15 min. Compound 24 was added (300 mg, 0.84 mmol) to the reaction mixture, followed by DIPEA (324.7 mg, 2.51 mmol, 437.6 μL) and the reaction was performed according to the general procedure B to get the pure compound 27 (344.68 mg, 53%). ¹H NMR (400 MHz, chloroform-d) δ 9.16 (s, 1H), 8.92 (dd, J = 10.7, 6.8 Hz, 2H), 8.40 (dd, J = 8.3, 0.7 Hz, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.90 (d, J = 14.6 Hz, 1H), 6.99 (dd, J = 11.1, 10.0 Hz, 2H), 5.50 (p, J = 6.8 Hz, 1H), 3.66 (s, 4H), 3.64–3.60 (m, 8H), 3.59–3.55 (m, 2H), 3.53 (t, J = 5.1 Hz, 2H), 3.48 (t, J = 5.1 Hz, 2H), 3.35–3.25 (m, 2H), 2.78–2.71 (m, 2H), 2.70–2.62 (m, 2H), 1.57 (d, J = 6.7 Hz, 6H), 1.42 (s, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₃₆H₅₀N₈O₉F₂Na, 799.3567; found, 799.3586.

tert-Butyl(22-((2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)amino)-19,22-dioxo-3,6,9,12,15-pentaoxa-18-azadocosyl)carbamate (28). Compound 20 (603.45 mg, 1.26 mmol) was taken in DMF (2 mL). HATU (954.94 mg, 2.51 mmol) was added and the reaction mixture was allowed to stir at room temperature for 15 min. Compound 24 was added (300 mg, 0.84 mmol) to the reaction mixture, followed by DIPEA (324.7 mg, 2.51 mmol, 437.6 μL) and the reaction was performed according to the general procedure B to get the pure compound 28 (357.35 mg, 52%). ¹H NMR (400 MHz, chloroform-d) δ 9.41 (s, 1H), 8.97 (d, J = 13.4 Hz, 1H), 8.88 (t, J = 8.5 Hz, 1H), 8.39 (d, J = 9.0 Hz, 1H), 8.36 (s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.04–6.93 (m, 1H), 5.50 (p, J = 6.7 Hz, 1H), 5.34 (t, J = 4.7 Hz, 1H), 3.65 (s, 8H), 3.61 (dt, J = 4.8, 2.2 Hz, 8H), 3.58–3.53 (m, 2H), 3.51 (t, J = 5.1 Hz, 2H), 3.46 (q, J = 5.0 Hz, 2H), 3.34–3.24 (m, 2H), 2.74 (dd, J = 7.8, 3.8 Hz, 2H), 2.67 (dd, J = 7.9, 3.8 Hz, 2H), 1.57 (d, J = 6.7 Hz, 6H), 1.42 (s, 9H). HRMS (ESI) m/z [M + Na]⁺ calcd for C₃₈H₅₄N₈O₁₀F₂Na, 843.3829; found, 843.3795.

tert-Butyl(25-((2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)amino)-22,25-dioxo-3,6,9,12,15,18-hexaoxa-21-azapentacosyl)carbamate (29). Compound 21 (658.77 mg, 1.26 mmol) was taken in DMF (2 mL). HATU (954.94 mg, 2.51 mmol) was added and the reaction mixture was allowed to stir at room temperature for 15 min. Compound 24 was added (300 mg, 0.84 mmol) to the reaction mixture, followed by DIPEA (324.7 mg, 2.51 mmol, 437.6 μL) and the reaction was performed according to the general procedure B to get the pure compound 29 (362 mg, 50%). ¹H NMR (400 MHz, chloroform-d) δ 9.33 (s, 1H), 9.00–8.83 (m, 2H), 8.38 (d, J = 8.3 Hz, 1H), 8.35 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.02–6.93 (m, 1H), 5.49 (p, J = 6.7 Hz, 1H), 5.14 (s, 1H), 3.64 (d, J = 2.9 Hz, 10H), 3.62–3.59 (m, 8H), 3.57–3.54 (m, 2H), 3.51 (t, J = 5.1 Hz, 2H), 3.46 (q, J = 5.0 Hz, 2H), 3.28 (d, J = 5.0 Hz, 2H), 2.75 (dd, J = 8.0, 3.9 Hz, 2H), 2.66 (dd, J = 7.9, 4.0 Hz, 2H), 2.46 (s, 2H), 1.57 (s, 3H), 1.42 (s, 9H). HRMS (ESI) m/z [M + H]⁺ calcd for C₄₀H₅₉F₂N₈O₁₁, 865.4266; found, 865.4202.

N ¹-(2-(2-Aminoethoxy)ethyl)-N⁴-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)succinamide hydrochloride (30). Compound 25 (200 mg, 0.31 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction was performed according to the general procedure D to get the crude product 30 (160 mg, quant.), which was then used for the next step without further purification. HRMS (ESI) m/z [M + H]⁺ calcd for C₂₅H₃₁F₂N₈O₄, 545.2431; found, 545.2436.

N ¹-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-N⁴-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)succinamide hydrochloride (31). Compound 26 (200 mg, 0.29 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction was performed according to the general procedure D to get the crude product 31 (162 mg, quant.), which was then used for the next step without further purification. HRMS (ESI) m/z [M + H]⁺ calcd for C₂₇H₃₅F₂N₈O₅, 589.2693; found, 589.2698.

N ¹-(14-Amino-3,6,9,12-tetraoxatetradecyl)-N⁴-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)succinamide hydrochloride (32). Compound 27 (200 mg, 0.26 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction was performed according to the general procedure D to get the crude product 32 (164 mg, quant.), which was then used for the next step without further purification. HRMS (ESI) m/z [M + H]⁺ calcd for C₃₁H₄₃F₂N₈O₇, 677.3217; found, 677.3215.

N ¹-(17-Amino-3,6,9,12,15-pentaoxaheptadecyl)-N⁴-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)succinamide hydrochloride (33). Compound 28 (200 mg, 0.24 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction was performed according to the general procedure D to get the crude product 33 (163 mg, quant.), which was then used for the next step without further purification. HRMS (ESI) m/z [M + H]⁺ calcd for C₃₃H₄₇F₂N₈O₈, 721.3479; found, 721.3486.

N ¹-(20-Amino-3,6,9,12,15,18-hexaoxaicosyl)-N⁴-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)succinamide hydrochloride (34). Compound 29 (200 mg, 0.23 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction was performed according to the general procedure D to get the crude product 34 (162 mg, quant.), which was then used for the next step without further purification. HRMS (ESI) m/z [M + H]⁺ calcd for C₃₅H₅₁F₂N₈O₉, 765.3742; found, 765.3746.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)succinamide (35). Compound 30 (100 mg, 0.18 mmol) was taken in DMSO under an Ar atmosphere, and then DIPEA (69 mg, 0.55 mmol, 93 μL) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (55.8 mg, 0.20 mmol) was added to the reaction mixture and the reaction was performed according to the general procedure D to get the pure compound 35 as a light-yellow solid (66.17 mg, 45%). ¹H NMR (400 MHz, chloroform-d) δ¹H NMR (400 MHz, CDCl₃) δ 9.35 (s, 1H), 9.04 (d, J = 13.7 Hz, 1H), 8.84 (t, J = 8.5 Hz, 1H), 8.43–8.35 (m, 3H), 8.03 (d, J = 7.6 Hz, 1H), 7.90 (t, J = 8.0 Hz, 1H), 7.51–7.45 (m, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.95 (t, J = 10.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 5.2 Hz, 2H), 5.49 (p, J = 6.7 Hz, 1H), 5.17–5.08 (m, 1H), 3.73 (t, J = 4.6 Hz, 2H), 3.62 (t, J = 4.5 Hz, 2H), 3.57–3.51 (m, 2H), 3.43 (q, J = 5.0 Hz, 2H), 2.84–2.74 (m, 7H), 2.17–2.09 (m, 1H), 1.56 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.4, 172.0, 171.3, 170.2, 169.2, 168.3, 167.6, 150.5, 146.9, 139.8, 136.3, 132.5, 125.6, 120.7, 116.9, 115.2, 112.0, 110.7, 100.0, 83.9, 69.9, 68.6, 50.9, 49.1, 48.8, 41.9, 39.4, 32.5, 31.5, 31.2, 23.7, 23.0. HRMS (ESI) m/z [M + H]⁺ calcd for C₃₈H₃₉F₂N₁₀O₈, 801.2915; found, 801.2911. HPLC purity: 98.9%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)succinamide (36). Compound 31 (100 mg, 0.17 mmol) was taken in DMSO under an Ar atmosphere, and then DIPEA (63.85 mg, 0.51 mmol, 86 μL) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (51.6 mg, 0.19 mmol) was added to the reaction mixture and the reaction was performed according to the general procedure D to get the pure compound 36 as a light-yellow solid (63.15 mg, 44%). ¹H NMR (600 MHz, CDCl₃) δ 9.18 (s, 1H), 9.02 (d, J = 13.2 Hz, 1H), 8.87 (t, J = 8.2 Hz, 1H), 8.67 (s, 1H), 8.45–8.40 (m, 2H), 8.04 (d, J = 7.6 Hz, 1H), 7.92 (t, J = 7.9 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.98 (t, J = 10.4 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.65–6.59 (m, 1H), 6.57 (s, 1H), 5.52 (dt, J = 13.3, 6.7 Hz, 1H), 5.00 (dd, J = 11.6, 5.2 Hz, 1H), 3.76 (t, J = 4.8 Hz, 3H), 3.68 (s, 4H), 3.61 (dt, J = 8.3, 4.0 Hz, 3H), 3.49 (dd, J = 10.1, 4.9 Hz, 4H), 2.81–2.71 (m, 4H), 2.63 (h, J = 9.6 Hz, 2H), 2.13 (dt, J = 11.8, 4.7 Hz, 2H), 1.59 (d, J = 6.7 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 171.9, 171.3, 170.7, 169.1, 168.6, 167.1, 160.2, 150.2, 150.0, 146.2, 145.5, 141.6, 139.3, 135.7, 132.1, 125.1, 120.1, 116.3, 114.6, 111.3, 109.9, 104.0, 71.4, 70.2, 69.7, 69.3, 68.6, 63.7, 48.4, 41.8, 39.2, 32.0, 30.9, 30.7, 23.1, 22.5. HRMS (ESI) m/z [M + H]⁺ calcd for C₄₀H₄₃F₂N₁₀O₉, 845.3183; found, 845.3186. HPLC purity: 97.5%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)succinamide (37). Compound 32 (100 mg, 0.15 mmol) was taken in DMSO under an Ar atmosphere, and then DIPEA (55.54 mg, 0.45 mmol, 75 μL) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (44.9 mg, 0.16 mmol) was added to the reaction mixture and the reaction was performed according to the general procedure D to get the pure compound 37 as a light yellow solid (59.28 mg, 43%). ¹H NMR (600 MHz, CDCl₃) δ 9.11 (s, 1H), 8.99–8.85 (m, 3H), 8.41–8.35 (m, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.90 (t, J = 8.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.21 (t, J = 5.1 Hz, 1H), 7.06 (d, J = 7.1 Hz, 1H), 6.95 (t, J = 10.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.50 (t, J = 5.5 Hz, 1H), 5.50 (p, J = 6.8 Hz, 1H), 4.93 (dd, J = 12.1, 5.5 Hz, 1H), 3.73 (t, J = 5.4 Hz, 2H), 3.68 (s, 6H), 3.66–3.64 (m, 3H), 3.63 (s, 3H), 3.58–3.56 (m, 2H), 3.48–3.44 (m, 4H), 2.88–2.81 (m, 1H), 2.77–2.69 (m, 4H), 2.66–2.61 (m, 2H), 2.16–2.11 (m, 1H), 1.57 (d, J = 6.0 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 174.6, 172.2, 171.1, 170.8, 168.8, 168.5, 167.1, 160.1, 150.3, 150.2, 150.0, 146.3, 145.7, 141.5, 139.3, 135.6, 132.1, 132.0, 120.1, 116.3, 114.5, 111.2, 109.8, 70.3, 70.1, 70.0, 69.9, 69.9, 69.7, 69.2, 68.8, 65.4, 48.4, 48.3, 41.8, 38.9, 32.4, 30.9, 30.7, 23.2, 22.4. HRMS (ESI) m/z [M + H]⁺ calcd for C₄₄H₅₁F₂N₁₀O₁₁, 933.3707; found, 933.3710. HPLC purity: 97.8%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)succinamide (38). Compound 33 (100 mg, 0.14 mmol) was taken in DMSO under an Ar atmosphere, and then DIPEA (52.15 mg, 0.42 mmol, 70 μL) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (42.15 mg, 0.15 mmol) was added to the reaction mixture and the reaction was performed according to the general procedure D to get the pure compound 38 as a light-yellow solid (56.9 mg, 42%). ¹H NMR (400 MHz, CDCl₃) δ 9.19 (s, 1H), 8.95 (s, 1H), 8.91 (s, 1H), 8.87 (t, J = 8.52 Hz, 1H), 8.37 (d, J = 8.32 Hz, 1H), 8.35 (s, 1H), 8.02 (d, J = 8.44 Hz, 1H), 7.87 (t, J = 8.00 Hz, 1H), 7.43 (t, J = 8.40 Hz, 1H), 7.04 (d, J = 7.08 Hz, 1H), 6.93 (t, J = 10.16 Hz, 1H), 6.86 (d, J = 8.56 Hz, 1H), 6.47 (t, J = 5.36 Hz, 1H), 5.44–5.53 (m, 1H), 4.89–4.93 (m, 1H), 3.70 (t, J = 5.28 Hz, 2H), 3.62 (t, J = 11.52 Hz, 18H), 3.54 (t, J = 4.60 Hz, 2H), 3.40–3.46 (m, 4H), 2.69–2.83 (m, 4H), 2.61–2.64 (m, 2H), 1.56 (s, 3H), 1.54 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.8, 171.6, 171.4, 171.4, 169.4, 169.0, 167.7, 150.5, 146.8, 146.8, 146.3, 139.8, 139.8, 136.1, 132.6, 120.6, 118.9, 117.2, 116.8, 116.0, 115.0, 111.7, 110.3, 107.0, 106.8, 100.0, 70.8, 70.7, 70.6, 70.6, 70.5, 70.5, 70.4, 70.1, 69.8, 69.4, 49.0, 48.8, 42.4, 39.5, 31.5, 31.2, 29.8, 29.7, 23.7, 22.9. HRMS (ESI) m/z [M + H]⁺ calcd for C₄₆H₅₅F₂N₁₀O₁₂, 977.3964; found, 977.3950. HPLC purity: 95.9%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(20-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15,18-hexaoxaicosyl)succinamide (39). Compound 34 (100 mg, 0.13 mmol) was taken in DMSO under an Ar atmosphere, and then DIPEA (49.14 mg, 0.39 mmol, 66 μL) was added to the reaction mixture and allowed to stir for 20 minutes at room temperature. Compound 9 (39.72 mg, 0.14 mmol) was added to the reaction mixture and the reaction was performed according to the general procedure D to get the pure compound 39 as a light yellow solid (53.4 mg, 40%). ¹H NMR (400 MHz, chloroform-d) δ 9.29 (s, 1H), 8.95 (d, J = 13.5 Hz, 2H), 8.89 (t, J = 8.2 Hz, 1H), 8.42–8.34 (m, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.52–7.41 (m, 1H), 7.32 (s, 1H), 7.07 (d, J = 7.1 Hz, 1H), 6.96 (t, J = 10.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.49 (t, J = 5.4 Hz, 1H), 5.50 (p, J = 6.7 Hz, 1H), 4.92 (dd, J = 12.1, 5.4 Hz, 1H), 3.71 (t, J = 5.2 Hz, 2H), 3.68–3.60 (m, 20H), 3.57–3.54 (m, 2H), 3.45 (dq, J = 10.5, 5.1 Hz, 4H), 2.80–2.69 (m, 4H), 2.66 (dd, J = 7.8, 3.9 Hz, 2H), 2.20–2.02 (m, 2H), 1.58 (s, 3H), 1.56 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.9, 171.6, 171.6, 171.4, 169.3, 168.9, 167.7, 160.6, 160.6, 160.5, 150.5, 146.8, 146.2, 142.1, 142.1, 139.8, 136.10, 132.6, 120.6, 116.8, 116.0, 115.0, 115.0, 111.7, 110.3, 70.8, 70.7, 70.5, 70.5, 70.5, 70.1, 69.8, 69.4, 49.0, 48.8, 42.4, 39.6, 33.0, 32.9, 31.7, 31.5, 31.2, 31.2, 29.8, 29.7, 23.7, 22.9. HRMS (m/z): [M + H]⁺ calcd for C₄₈H₅₉F₂N₁₀O₁₃, 1021.4231; found, 1021.4250. HPLC purity: 96.9%.

tert-Butyl(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)carbamate (45). Commercially available 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid (40, 330 mg, 1.41 mmol) was taken in DMF and HATU (1.07 g, 2.82 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. VH032 (600 mg, 1.28 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (531 mg, 4.23 mmol, 716 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 100–200 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 45 as a white gummy solid (614 mg, 74%). ¹H NMR (600 MHz, chloroform-d) δ 9.00 (s, 1H), 8.49 (s, 1H), 7.45–7.38 (m, 2H), 7.34 (s, 2H), 7.07 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.67 (s, 1H), 4.76 (s, 1H), 4.62 (d, J = 11.9 Hz, 1H), 4.57–4.51 (m, 2H), 4.31 (s, 1H), 4.05 (dd, J = 54.6, 6.5 Hz, 1H), 3.74–3.66 (m, 2H), 3.55 (ddd, J = 8.5, 5.3, 2.7 Hz, 1H), 3.47–3.33 (m, 2H), 3.28–3.03 (m, 2H), 2.59 (s, 3H), 2.51 (s, 1H), 2.42 (d, J = 11.0 Hz, 2H), 2.17 (s, 1H), 1.37 (s, 9H), 0.96 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₃₂H₄₈N₅O₇S, 646.3269; found, 646.3274.

tert-Butyl(2-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate (46). Commercially available 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (41, 392 mg, 1.41 mmol) was taken in DMF and HATU (1.07 g, 2.82 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. VH032 (600 mg, 1.28 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (531 mg, 4.23 mmol, 716 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 100–200 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 46 as a white gummy solid (638 mg, 72%). ¹H NMR (400 MHz, chloroform-d) δ 8.68 (s, 1H), 7.44–7.40 (m, 1H), 7.34 (s, 4H), 7.09 (d, J = 8.0 Hz, 1H), 5.40 (s, 1H), 4.71 (t, J = 7.6 Hz, 1H), 4.64–4.58 (m, 1H), 4.57–4.59 (m, 2H), 4.32 (dd, J = 14.8 Hz, 4.0 Hz, 1H), 4.10 (d, J = 11.6 Hz, 1H), 3.77–3.66 (m, 3H), 3.63–3.57 (m, 5H), 3.51–3.46 (m, 2H), 3.30–3.18 (m, 2H), 2.50 (s, 3H), 2.49–2.45 (m, 2H), 2.18–2.12 (m, 1H), 1.41 (s, 9H), 0.94 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₃₄H₅₁N₅O₈NaS, 712.3356; found, 712.3359.

tert-Butyl((S)-14-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)carbamate (47). Commercially available 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid (42, 454 mg, 1.41 mmol) was taken in DMF and HATU (1.07 g, 2.82 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. VH032 (600 mg, 1.28 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (531 mg, 4.23 mmol, 716 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 100–200 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 47 as a white gummy solid (678 mg, 72%). ¹H NMR (600 MHz, chloroform-d) δ 8.70 (s, 1H), 7.44–7.40 (m, 1H), 7.37 (dd, J = 10.8 Hz, 2.4 Hz, 1H), 6.99 (d, 7.8 Hz, 1H), 5.24 (s, 1H), 4.73 (t, J = 8.0 Hz, 1H), 4.58 (dd, J = 15.0, 6.6 Hz, 1H), 4.54–4.50 (m, 2H), 4.36 (dd, J = 15.0, 5.4 Hz, 1H), 4.14 (d, J = 11.4 Hz, 1H), 3.80–3.75 (m, 2H), 3.72–3.69 (m, 1H), 3.66–3.63 (m, 4H), 3.62–3.59 (m, 4H), 3.54–3.51 (m, 2H), 3.34–3.28 (m, 2H), 2.53 (s, 3H), 2.51 (t, J = 4.8 Hz, 2H), 2.19–2.15 (m, 1H), 1.45 (s, 10H), 0.96 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₃₆H₅₅N₅O₉NaS, 756.3618; found, 756.3617.

tert-Butyl((S)-17-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-18,18-dimethyl-15-oxo-3,6,9,12-tetraoxa-16-azanonadecyl)carbamate (48). Commercially available 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid (43, 516 mg, 1.41 mmol) was taken in DMF and HATU (1.07 g, 2.82 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. VH032 (600 mg, 1.28 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (531 mg, 4.23 mmol, 716 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL) which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 100–200 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 48 as a white gummy solid (710 mg, 71%). ¹H NMR (400 MHz, chloroform-d) δ 8.66 (s, 1H), 7.40 (s, 1H), 7.34 (dd, J = 10.4 Hz, 1.6 Hz, 4H), 7.01 (d, J = 7.6 Hz, 1H), 5.19 (s, 1H), 4.71 (t, J = 8.2 Hz, 1H), 4.55 (dd, J = 14.8, 6.8 Hz, 1H), 4.51–4.45 (m, 2H), 4.32 (dd, J = 14.8, 5.2 Hz, 1H), 4.11 (d, J = 11.2 Hz, 1H), 3.73–3.70 (m, 2H), 3.62–3.60 (m, 4H), 3.61–3.59 (m, 4H), 3.59–3.57 (s, 4H), 3.50 (t, J = 5.4 Hz, 2H), 3.32–3.25 (m, 2H), 2.50 (s, 3H), 2.49–2.46 (m, 2H), 2.13 (dd, J = 14.2, 8.4 Hz, 1H), 1.41 (s, 9H), 0.93 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₃₈H₅₉N₅O₁₀NaS, 800.3880; found, 800.3865.

tert-Butyl((S)-20-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-21,21-dimethyl-18-oxo-3,6,9,12,15-pentaoxa-19-azadocosyl)carbamate (49). Commercially available 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatricosan-23-oic acid (44, 578 mg, 1.41 mmol) was taken in DMF and HATU (1.07 g, 2.82 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. VH032 (600 mg, 1.28 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (531 mg, 4.23 mmol, 716 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 100–200 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 49 as a white gummy solid (739 mg, 70%). ¹H NMR (400 MHz, chloroform-d) δ 8.66 (s, 1H), 7.41 (s, 1H), 7.33 (d, J = 1.2 Hz, 4H), 7.01 (d, J = 8.3 Hz, 1H), 5.17 (s, 1H), 4.70 (t, J = 8.0 Hz, 1H), 4.54 (dd, J = 15.1, 6.5 Hz, 1H), 4.47 (d, J = 8.5 Hz, 2H), 4.32 (dd, J = 15.0, 5.3 Hz, 1H), 4.09 (d, J = 11.5 Hz, 1H), 3.69 (dq, J = 6.8, 3.6 Hz, 2H), 3.61–3.61 (m, 6H), 3.60 (s, 4H), 3.58 (s, 4H), 3.50 (t, J = 4.9 Hz, 2H), 3.32–3.25 (m, 2H), 2.49 (s, 3H), 2.46 (dt, J = 5.1, 2.6 Hz, 2H), 2.12 (dd, J = 13.2, 8.1 Hz, 3H), 1.41 (s, 9H), 0.92 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₄₀H₆₄N₅O₁₁S, 822.4323; found, 822.4305.

(2S,4R)-1-((S)-2-(3-(2-Aminoethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (50). Compound 45 (300 mg, 0.46 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude product 50 (228 mg, quant.), which was then used for the next step without further purification. ¹H NMR (400 MHz, chloroform-d) δ 8.66 (s, 1H), 8.03 (t, J = 6.0 Hz, 1H), 7.36–7.33 (m, 4H), 6.85 (d, J = 9.0 Hz, 1H), 4.78–4.61 (m, 1H), 4.57–4.47 (m, 3H), 4.37 (ddd, J = 14.5, 8.6, 5.7 Hz, 1H), 4.03 (t, J = 10.5 Hz, 1H), 3.79–3.67 (m, 2H), 3.67–3.58 (m, 3H), 3.56–3.50 (m, 1H), 2.96 (s, 2H), 2.51 (s, 1H), 2.50 (s, 3H), 2.43 (ddd, J = 15.1, 5.9, 3.9 Hz, 2H), 2.28–2.24 (m, 2H), 0.99 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₂₇H₄₀N₅O₅S, 546.2745; found, 546.2747.

(2S,4R)-1-((S)-2-(3-(2-(2-Aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (51). Compound 46 (300 mg, 0.43 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude product 51 (231 mg, quant.), which was then used for the next step without further purification. ¹H NMR (400 MHz, chloroform-d) δ 8.65 (s, 1H), 7.76–7.69 (m, 1H), 7.34 (s, 4H), 6.96 (d, J = 8.8 Hz, 1H), 4.67 (t, J = 8.2 Hz, 1H), 4.59–4.53 (m, 2H), 4.48–4.45 (m, 1H), 4.31 (dd, J = 15.2, 5.2 Hz, 1H), 4.04 (d, J = 11.2 Hz, 1H), 3.80–3.73 (m, 2H), 3.68–3.65 (m, 1H), 3.63–3.61 (m, 1H), 3.60–3.57 (m, 4H), 3.47–3.44 (m, 2H), 2.79–2.75 (m, 2H), 2.49 (s, 3H), 2.48–2.45 (m, 2H), 2.19–2.12 (m, 1H), 0.94 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₂₉H₄₄N₅O₆S, 590.3012; found, 590.3014.

(2S,4R)-1-((S)-1-Amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (52). Compound 47 (300 mg, 0.41 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude product 52 (234 mg, quant.), which was then used for the next step without further purification. ¹H NMR (400 MHz, chloroform-d) δ 8.66 (s, 1H), 7.66 (t, J = 5.4 Hz, 1H), 7.34 (s, 4H), 7.19 (d, J = 8.4 Hz, 1H), 4.70 (t, J = 8.2 Hz, 1H), 4.55 (dd, J = 15.0, 6.6 Hz, 1H), 4.51–4.46 (m, 2H), 4.33 (dd, 15.0, 5.4 Hz, 1H), 4.09 (d, J = 12.4 Hz, 1H), 3.79–3.73 (m, 2H), 3.68–3.64 (m, 2H), 3.62–3.60 (m, 4H), 3.59–3.57 (m, 4H), 3.53 (t, 5.2 Hz, 2H), 2.87 (t, 5.0 Hz, 2H), 2.49 (s, 3H), 2.18 (m, 1H), 0.96 (s, 9H). HRMS (m/z): [M + H]⁺ calcd for C₃₁H₄₈N₅O₇S, 634.3274; found, 634.3281.

(2S,4R)-1-((S)-1-Amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (53). Compound 48 (300 mg, 0.39 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude product 53 (236 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₃H₅₂N₅O₈S, 678.3537; found, 678.3518.

(2S,4R)-1-((S)-1-Amino-20-(tert-butyl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (54). Compound 49 (300 mg, 0.37 mmol) was taken in a mixture of 4 M HCl in dioxane (1 mL) and dioxane (1 mL). The reaction mixture was then stirred at room temperature for 2 h. After checking the completion of the reaction by TLC, the volatile materials were removed under reduced pressure to give the crude product 54 (240 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₅H₅₆N₅O₉S, 722.3799; found, 722.3785.

4-((2-(3-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)amino)-4-oxobutanoic acid (55). Compound 50 (200 mg, 0.36 mmol) was taken in 1 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (40.34 mg, 0.40 mmol) was then added to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ (5 mL × 3) and evaporated to dryness to give the crude product 55 (208 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₁H₄₄N₅O₈S, 646.2905; found, 646.2902.

(S)-3-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-2,2-dimethyl-5,15-dioxo-8,11-dioxa-4,14-diazaoctadecan-18-oic acid (56). Compound 51 (200 mg, 0.34 mmol) was taken in 1 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (37.33 mg, 0.37 mmol) was then added to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ (5 mL × 3) and evaporated to dryness to give the crude product 56 (198 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₃H₄₈N₅O₉S, 690.3173; found, 690.3160.

(S)-3-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-2,2-dimethyl-5,18-dioxo-8,11,14-trioxa-4,17-diazahenicosan-21-oic acid (57). Compound 52 (200 mg, 0.32 mmol) was taken in 1 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (34.73 mg, 0.35 mmol) was then taken to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ (5 mL × 3) and evaporated to dryness to give the crude product 57 (192 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₅H₅₂N₅O₁₀S, 734.3435; found, 734.3422.

(S)-3-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-2,2-dimethyl-5,21-dioxo-8,11,14,17-tetraoxa-4,20-diazatetracosan-24-oic acid (58). Compound 53 (200 mg, 0.30 mmol) was taken in 1 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (32.48 mg, 0.32 mmol) was then added to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ (5 mL × 3) and evaporated to dryness to give the crude product 58 (188 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₇H₅₆N₅O₁₁S, 778.3692; found, 778.3690.

(S)-3-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-2,2-dimethyl-5,24-dioxo-8,11,14,17,20-pentaoxa-4,23-diazaheptacosan-27-oic acid (59). Compound 54 (200 mg, 0.28 mmol) was taken in 1 mL of pyridine–DCM–ACN mixture (1 : 1 : 1). Succinic anhydride (30.50 mg, 0.30 mmol) was then added to the reaction mixture, followed by stirring at room temperature for 3.5 h. The reaction was monitored by checking TLC and upon completion, the reaction mixture was diluted with CCl₄ (5 mL × 3) and evaporated to dryness to give the crude product 59 (182 mg, quant.), which was then used for the next step without further purification. HRMS (m/z): [M + H]⁺ calcd for C₃₉H₆₀N₅O₁₂S, 822.3954; found, 822.3952.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethyl)succinamide (60). Compound 55 (200 mg, 0.31 mmol) was taken in DMF (2 mL) and HATU (235.52 mg, 0.62 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. Compound 24 (122 mg, 0.34 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (116 mg, 0.93 mmol, 157 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 60 as a white solid (128 mg, 42%). ¹H NMR (400 MHz, methanol-d₄) δ 8.80 (d, J = 12.7 Hz, 2H), 8.39 (t, J = 8.1 Hz, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.01–7.95 (m, 1H), 7.86 (dt, J = 7.7, 0.8 Hz, 1H), 7.41–7.39 (m, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.20 (t, J = 10.3 Hz, 1H), 5.68 (p, J = 6.9 Hz, 1H), 4.86 (d, J = 0.9 Hz, 2H), 4.79 (d, J = 0.9 Hz, 1H), 4.59 (d, J = 8.6 Hz, 1H), 4.57–4.54 (m, 1H), 4.51 (d, J = 15.5 Hz, 1H), 4.46 (s, 1H), 4.30 (d, J = 15.4 Hz, 1H), 3.87 (d, J = 10.9 Hz, 1H), 3.77 (dd, J = 11.0, 3.8 Hz, 1H), 3.68 (t, J = 5.7 Hz, 2H), 3.49 (dp, J = 15.4, 5.1 Hz, 2H), 3.36 (d, J = 5.1 Hz, 1H), 3.32 (d, J = 0.9 Hz, 1H), 2.69 (dt, J = 14.2, 7.7 Hz, 2H), 2.56 (dd, J = 7.6, 4.8 Hz, 2H), 2.54–2.45 (m, 2H), 2.42 (d, J = 0.7 Hz, 3H), 2.21 (dd, J = 13.2, 7.6 Hz, 1H), 2.04 (ddd, J = 13.3, 9.2, 4.4 Hz, 1H), 1.51 (d, J = 6.7 Hz, 6H), 1.01 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 172.4, 172.1, 171.8, 171.6, 171.4, 171.3, 150.3, 148.4, 146.3, 146.3, 146.2, 142.1, 139.9, 139.8, 138.8, 138.6, 131.7, 130.8, 129.3, 128.0, 127.9, 120.7, 115.0, 100.0, 70.4, 66.9, 48.8, 39.8, 36.5, 36.3, 34.7, 31.7, 31.0, 30.9, 29.1, 26.5, 25.3, 23.7, 22.7, 16.1, 14.2, 11.5. HRMS (m/z): [M + H]⁺ calcd for C₄₈H₅₈F₂N₁₁O₈S, 986.4153; found, 986.4146. HPLC purity: 99.9%.

N ¹-(2,4-difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-(2-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)ethyl)succinamide (61). Compound 56 (424 mg, 0.61 mmol) was taken in DMF (2 mL) and HATU (467 mg, 1.23 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. Compound 24 (200 mg, 0.56 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (230 mg, 1.84 mmol, 311 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 61 as a white solid (119 mg, 40%). ¹H NMR (400 MHz, chloroform-d) δ 9.02 (d, J = 14.0 Hz, 1H), 8.97 (s, 1H), 8.79 (t, J = 8.6 Hz, 1H), 8.64 (s, 1H), 8.37 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.69 (t, J = 5.8 Hz, 1H), 7.56 (t, J = 5.2 Hz, 1H), 7.30 (dd, 14.4 Hz, 8.8 Hz, 4H), 7.19 (d, J = 8.8 Hz, 1H), 6.96–6.91 (m, 1H), 5.45 (quin, J = 6.8 Hz, 1H), 4.71 (t, J = 8.4 Hz, 1H), 4.61 (dd, J = 12.4, 7.9 Hz, 1H), 4.56 (m, 1H), 4.33 (dd, J = 15.2, 5.2 Hz, 1H), 4.09 (d, J = 11.2 Hz, 1H), 3.75–3.67 (m, 3H), 3.65–3.55 (m, 4H), 3.53–3.49 (m, 2H), 3.31–3.25 (m, 1H), 2.71–2.66 (m, 2H), 2.63–2.59 (m, 1H), 2.47 (s, 3H), 2.35–2.28 (m, 2H), 2.26–2.15 (m, 4H), 1.55 (d, 6.8 Hz, 3H), 0.99 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 188.8, 188.1, 185.6, 171.6, 150.4, 150.32, 148.4, 148.0, 146.3, 139.8, 138.6, 135.2, 130.7, 129.4, 127.9, 120.7, 116.2, 114.9, 111.1, 110.1, 100.0, 80.8, 70.3, 70.1, 69.8, 67.0, 59.1, 57.6, 48.9, 43.1, 41.3, 39.5, 38.6, 36.8, 35.6, 34.2, 31.2, 26.5, 25.5, 23.7, 22.4, 16.1, 14.1. HRMS (m/z): [M + H]⁺ calcd for C₅₀H₆₂F₂N₁₁O₉S, 1030.4421; found, 1030.4427. HPLC purity: 99.6%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-((S)-14-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)succinamide (62). Compound 57 (451 mg, 0.61 mmol) was taken in DMF (2 mL) and HATU (467 mg, 1.23 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. Compound 24 (200 mg, 0.56 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (231 mg, 1.84 mmol, 311 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 62 as a white solid (111 mg, 38%). ¹H NMR (400 MHz, chloroform-d) δ 9.04 (s, 1H), 8.97 (d, J = 13.6 Hz, 1H), 8.83 (t, J = 8.6 Hz, 1H), 8.67 (s, 1H), 8.44 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.53 (t, J = 5.6 Hz, 1H), 7.32 (s, 4H), 7.27 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 10.4 Hz, 1H), 5.47 (quin, J = 6.8 Hz, 1H), 4.71 (t, J = 8.2 Hz, 1H), 4.59–4.50 (m, 3H), 4.33 (dd, J = 15.2, 5.4 Hz, 1H), 4.08 (d, J = 11.6 Hz, 1H), 3.77–3.68 (m, 4H), 3.63–3.60 (m, 4H), 3.59–3.57 (m, 4H), 3.54–3.51 (m, 2H), 3.43–3.38 (m, 2H), 2.74–2.70 (m, 2H), 2.63–2.60 (m, 3H), 2.50–2.49 (m, 1H), 2.48 (s, 3H), 2.47 (s, 1H), 2.21 (dd, J = 13.4, 8.2 Hz, 1H), 1.56 (d, J = 6.8 Hz, 6H), 0.96 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 173.0, 172.4, 171.5, 170.9, 151.5, 151.3, 150.7, 147.7, 145.6, 142.9, 139.6, 138.9, 132.0, 130.2, 129.1, 129.0, 127.7, 127.6, 119.8, 115.2, 100.0, 69.7, 66.8, 66.5, 63.1, 62.2, 59.5, 57.6, 56.7, 42.3, 37.6, 36.6, 35.9, 35.4, 25.7, 22.2, 14.5. HRMS (m/z): [M + Na]⁺ calcd for C₅₂H₆₅F₂N₁₁O₁₀NaS, 1096.4502; found, 1096.4479. HPLC purity: 99.8%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-((S)-17-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-18,18-dimethyl-15-oxo-3,6,9,12-tetraoxa-16-azanonadecyl)succinamide (63). Compound 58 (478 mg, 0.61 mmol) was taken in DMF (2 mL) and HATU (467 mg, 1.23 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. Compound 24 (200 mg, 0.56 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (230 mg, 1.84 mmol, 311 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 63 as a white solid (109 mg, 38%). ¹H NMR (500 MHz, chloroform-d) δ 9.20 (s, 1H), 8.97 (d, J = 13.6 Hz, 1H), 8.84 (t, J = 8.0 Hz, 1H), 8.67 (d, J = 2.8 Hz, 2H), 8.41 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.54–7.48 (m, 1H), 7.34–7.32 (m, 4H), 7.09–7.03 (m, 1H), 7.00–6.93 (m, 1H), 5.51–5.43 (m, 1H), 5.40–5.31 (m, 1H), 4.72–4.62 (m, 2H), 4.55–4.46 (m, 3H), 4.38–4.29 (m, 2H), 3.64–3.61 (s, 8H), 3.60–3.56 (m, 3H), 3.44–3.40 (m, 1H), 3.11–3.05 (m, 2H), 2.73–2.70 (m, 1H), 2.65–2.60 (m, 2H), 2.49 (d, J = 2.4 Hz, 4H), 1.57 (s, 2H), 1.55 (s, 2H), 1.51 (d, J = 6.8 Hz, 6H), 1.42 (d, J = 6.8 Hz, 4H), 0.94 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 171.8, 171.5, 171.3, 150.4, 148.3, 148.3, 148.2, 146.1, 146.1, 142.1, 139.8, 131.8, 130.7, 129.5, 128.1, 120.7, 115.0, 77.3, 70.5, 70.2, 70.2, 70.1, 70.1, 69.7, 67.3, 58.8, 57.7, 57.1, 49.0, 43.1, 43.1, 39.6, 36.8, 36.7, 36.7, 36.7, 35.4, 32.7, 32.7, 31.2, 26.5, 23.7, 22.7, 16.1, 14.2. HRMS (m/z): [M + H]⁺ calcd for C₅₄H₇₀F₂N₁₁O₁₁S, 1118.4945; found, 1118.4934. HPLC purity: 99.9%.

N ¹-(2,4-Difluoro-5-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N⁴-((S)-20-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-21,21-dimethyl-18-oxo-3,6,9,12,15-pentaoxa-19-azadocosyl)succinamide (64). Compound 59 (505 mg, 0.61 mmol) was taken in DMF (2 mL) and HATU (467 mg, 1.23 mmol) was added and stirred for 0.5 h at room temperature to obtain a reaction mixture. Compound 24 (200 mg, 0.56 mmol) was then added to the reaction mixture, followed by the addition of DIPEA (230 mg, 1.84 mmol, 311 μL), and the reaction mixture was stirred for another 16 h at room temperature. The progress of the reaction was thoroughly monitored through TLC. After completion of the reaction, the reaction mixture was poured into ice-cold water (10 mL), extracted with CHCl₃ (10 mL × 3) and washed with brine (15 mL), which was then dried over Na₂SO₄. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain the crude, which was purified in a Teledyne ISCO CombiFlash system using 230–400 mesh size silica gel and 2–10% MeOH in CHCl₃ as eluents to get the pure compound 64 as a white solid (102 mg, 36%). ¹H NMR (400 MHz, chloroform-d) δ 8.95 (d, J = 13.6 Hz, 1H), 8.88–8.83 (m, 2H), 8.68 (s, 1H), 8.41 (d, J = 6.0 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.03–8.00 (m, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.33 (dd, J = 7.4, 2.2 Hz, 2H), 7.29–7.27 (m, 2H), 7.10–7.05 (m, 1H), 6.95 (dd, J = 10.8, 4.6 Hz, 1H), 5.57–5.41 (m, 2H), 5.42–5.36 (m, 1H), 4.64–4.54 (m, 2H), 4.52–4.43 (m, 1H), 4.35–4.26 (m, 2H), 4.18 (d, J = 11.2 Hz, 1H), 3.74–3.68 (m, 4H), 3.63–3.61 (m, 8H), 3.59–3.57 (s, 4H), 3.56–3.52 (t, J = 4.8 Hz, 2H), 3.46–3.43 (m, 2H), 2.83–2.77 (m, 2H), 2.74–2.71 (m, 2H), 2.66–2.61 (m, 2H), 2.50 (dd, J = 5.6, 2.0 Hz, 2H), 2.48 (s, 3H), 1.56 (d, J = 6.4 Hz, 6H), 1.54 (dd, J = 6.6, 2.2 Hz, 4H), 0.95 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 172.2, 171.7, 171.4, 171.3, 171.1, 171.1, 170.9, 150.4, 148.4, 138.4, 130.8, 129.5, 129.5, 128.2, 128.1, 120.9, 115.0, 101.1, 70.6, 70.5, 70.3, 70.3, 70.2, 67.5, 67.2, 67.0, 58.9, 58.6, 57.7, 57.0, 56.8, 56.5, 49.0, 43.2, 43.2, 37.4, 37.3, 36.9, 36.5, 36.2, 35.5, 35.4, 35.1, 33.6, 26.5, 23.7, 23.7, 16.1. HRMS (m/z): [M + H]⁺ calcd for C₅₆H₇₄F₂N₁₁O₁₂S, 1162.5207; found, 1162.5208. HPLC purity: 99.8%.

Computational analysis

The computational study was performed in Schrödinger Maestro v13.1.141, (Release 2022-1, Platform Windows-×64), operated in a Windows 10, 64-bit workstation with Intel(R) Xeon(R) Silver 4214R CPU 2 × 2.40 GHz processors, 128 GB RAM. The protein structures of 5FQD and 6OYT were downloaded from the Protein Data Bank (https://www.rcsb.org) in .pdb format.

Protein preparation. The downloaded protein structure files were prepared through protein preparation⁴⁶ workflow of Schrödinger Maestro at a pH of 7.4, where the bond orders were assigned, disulfide bonds were generated, hydrogen atoms were added, missing loops were filled using PRIME⁴⁷ and protonation states of the residues were generated with Epik,⁴⁸ at the same pH. Heavy atoms were minimized abiding force-field OPLS2005^49,50 up to a RMSD of 0.30 Å.

Ligand preparation. Two-dimensional structures of the thalidomide and selonsertib fragments were imported into the Schrödinger Maestro interface. A built-in protocol of LigPrep⁵¹ in Schrödinger Maestro was employed to prepare the ligands and generate tautomers at a target pH of 7.4 using Ionizer.

Molecular docking with induced-fit. All water molecules were removed from the prepared protein structures. In the standard protocol of induced fit docking^52–54 in Schrödinger Maestro, the prepared ligands (thalidomide and selonsertib fragments) were selected to dock onto the prepared protein structure of 5FQD and 6OYT, respectively. To indicate the docking region box, the centroid of the workspace ligand (co-crystallized lenalidomide and selonsertib in the case of 5FQD and 6OYT, respectively) was picked. Ring conformations of the ligands were sampled within the energy window of 2.5 Kcal mol⁻¹. For Primary Glide docking, the side chains were trimmed based on B-factor, and the van der Waals scaling of relevant receptor and ligand atoms was kept at 0.70 and 0.50, respectively. Through Prime, the residues within 5.0 Å of the ligand poses were refined, with side chain optimization. Glide SP^55,56 settings were applied to re-dock the ligands into 20 least-energy induced fit receptor structures, having the lowest Prime energy or within a range of lowest energy plus 30 Kcal mol⁻¹. A maximum of 20 structures were generated, which were ranked according to their IFD scores. In the case of the selonsertib fragment docked to 6OYT, the structure with an IFD score of −2252.73 and docking score of −9.296 was selected as it ranked among the top 5 models and also showed pose-similarity with the co-crystallized ligand. Similarly, in the case of the thalidomide fragment, docked to CRBN of 5FQD, the selected structure had IFD and docking scores of −3372.55 and −12.981, respectively and had a pose identical to that of the co-crystallized ligand.

Preparation of the complex (DDB1–CRBN–ASK1). From the selected structure of the IFD of selonsertib moiety–6OYT, the ASK1 domain was structurally aligned to the CK1 domain of the selected IFD structure of thalidomide–5FQD. All other protein chains were deleted except the CRBN and the ASK1, with their docked corresponding ligands and the adjacent DDB1. The two protein structures were merged into one file.

Optimization of the complex. The merged consolidated complex was minimized using the Prime Minimize^57,58 application in Schrödinger Maestro, with a VSGB solvation model and an additional distance constraint of 8 Å between the nitrogen atoms of the terminal primary amines of both docked ligands at solvent-exposed interphases of CRBN and ASK1. This step was repeated five times with different distance constraints of 10, 12, 13, 15 and 17 Å, leading to the generation of six different protein complexes.

Manual modelling of the linker and further energy minimization. In each of the six protein complexes generated, a PEG linker was manually modelled joining the nitrogen atoms of the terminal amines of two docked ligands, in such a way that it follows the surface topology of the protein. These complexes with the modelled linker were further energy minimized using the Prime Minimize application, without any constraints.

Binding free energy estimation. To understand which one of these six protein complexes is most stable and best represents the biological binding pattern, free energy for binding (ΔG_Binding) was estimated by Prime MMGBSA⁵⁹ in Schrödinger Maestro with VSGB solvation model.

Log D determination assay

1.56 g of NaH₂PO₄·2H₂O was dissolved in 0.5 liters of water in a 1 L beaker. The pH was adjusted to 7.4 using NaOH solution, and then the volume was adjusted to 1 L. Equal volumes of sodium phosphate buffer (10 mM, pH 7.4) and n-octanol were mixed thoroughly in a separation funnel by shaking and inverting it several times. The two layers were allowed to separate overnight and then transferred into two separate glass bottles. A 10 mM stock solution was prepared in 100% DMSO and stored at 4 °C. For the experiment, 495 μL of organic phase (1-octanol) was added to each well of a 2 mL deep well plate, followed by 495 μL of buffer. Then, 10 μL of test compounds were added to each well. The plate was incubated for 3 h at room temperature on a plate shaker set at 500 rpm. After incubation, the samples were allowed to equilibrate for 20 minutes and then centrifuged at 4000 rpm for 30 minutes to ensure complete phase separation. Finally, the samples were analyzed by HPLC-UV.^21,60

Log D = Log(area of octanol/area of buffer)

Aqueous solubility assay

The PBS sachet was dissolved in 0.9 L of Milli-Q water, and the pH was adjusted to 7.4. The volume was then adjusted to 1 L with water and stored at an ambient temperature of 21–25 °C. A stock solution (50 mM) of respective test compounds was prepared in DMSO and stored at 4–8 °C. For the experiment, 4 μL from the 50 mM stock solution was added to a deep well plate containing 396 μL of pH 7.4 phosphate buffer. The mixture was thoroughly mixed and then incubated for 24 h at room temperature with constant mixing at 300 rpm. The plate was sealed tightly during the incubation process. After incubation, the samples were centrifuged for 20 minutes at 4000 rpm. The supernatant was then analysed by HPLC-UV. The DMSO content in the sample was 1.0%. The final concentration of the compound in the deep well plate was 500 μM. The spectrum was read using a Shimadzu UV spectrophotometer at 254 nm.^61,62

Plasma stability assay

1 mM stock of test compound was prepared in acetonitrile : water by diluting from 10 mM stock (i.e. 10 μL of 10 mM stock solution was added to 90 μL of acetonitrile : water (50 : 50)). 50 μM stock of test compound was prepared in acetonitrile : water by diluting from 1 mM stock (i.e. 5 μL of 1 mM stock solution was added to 95 μL of acetonitrile : water (50 : 50)). The frozen plasma was thawed at room temperature and centrifuged at 1400 rpm at 4 °C, for 15 minutes. Approximately 90% of the clear supernatant fraction was transferred to a separate tube and was used for the assay. The final working stock of 2 μM was prepared by diluting in plasma (i.e., 16 μL of 50 μM Acetonitrile: water stock was added to 384 μL of plasma). 300 μL of plasma containing the test compound was incubated for 120 minutes at 37 °C in a shaker water bath with gentle shaking. 25 μL aliquot of the sample at 0, 10, 30, 60 and 120 min was precipitated immediately with 200 μL of acetonitrile containing internal standard and centrifuged at 4000 × RCF, 4 °C for 20 minutes. 150 μL of supernatant was diluted with 150 μL of water and analyzed by LC–MS/MS.^63,64

In vivo pharmacokinetic experiment

Six 8–10 week-old male C57BL/6 mice were taken for the in vivo pharmacokinetic experiment. They were divided into two groups, each having nine mice. dASK1-VHL (60) was reconstituted in 1% DMSO in PBS (0.01 M, pH = 7.4). The mice of the first group were orally administered dASK1-VHL (60) at 20 mg kg⁻¹ dose, and the mice belonging to the second group were given intravenous injections of 2 mg kg⁻¹ compound. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after oral administration of the compound, and serum was isolated with the compound. On the other hand, blood samples were drawn at 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous administration of the compound and serum was isolated. For the pharmacokinetic study, blank plasma and subject samples (Plasma) were retrieved from the deep freezer and were allowed to thaw. The calibration curve with standard samples was prepared using blank plasma. The thawed samples were then vortexed to ensure complete mixing of the contents. A 100 μL of the corresponding samples was then transferred to the vials and 200 μL of acetonitrile containing internal standard was added to all the samples and vortexed. Samples were then kept on the shaker for 5 min to ensure complete mixing of contents. The samples were then centrifuged at 10 000 rpm at 20 °C for 10 min. The supernatant was transferred into auto injector vials and loaded into auto sampler vials and injected 10 μL onto the analytical system for analysis. The calibration curve range was 31.25 to 5000 ng mL⁻¹. Calibration curve standards samples, which were run along with samples, met the acceptance criteria, demonstrating satisfactory performance of the method during the analysis of samples. Mass spectrometry was performed in LC–MS/MS ESI-QTOF Mass Spectrometry (Waters Corporation) using the XBridge C18 column having a diameter of 50 × 4.6 mm with a particle size of 5 μm. The column formed the stationary phase, and a mixture of solution A (10 mM ammonium acetate) and solution B (HPLC-grade acetonitrile) formed the mobile phase. Liquid chromatography (injection volume 10 μL, flow rate 1 mL min⁻¹) was performed using acetonitrile containing the internal standard Telmisartan. The intact compound had a retention time of 4.21 min and (m/z) of 986.37, while the (m/z) of the fragmented compound after MS/MS was 669.29. All data were analyzed using Thermo Xcalibur software.

Culture of HepG2 and HEK 293A cells

HepG2 and HEK293A cells were cultured in minimal Eagle medium (MEM) and Dulbecco's modified Eagle medium (DMEM) [HiMedia and Gibco], respectively. The media is supplemented with 10% fetal bovine serum and 1% antibiotic solution containing penicillin and streptomycin. 1× trypsin–EDTA solution [Gibco] was used for trypsinization. 4.5–5 × 10⁵ cells were plated in 12-well plates for the experiment. The cells were maintained in an incubator at 37 °C, humidified with 5% CO₂. All PROTAC treatment was given at a cell no. of 4–5 × 10⁵ in a 12-well plate.

Treatment of HepG2 cells with small molecules

The PROTAC molecules were dissolved in the required volume of DMSO to prepare a 10 mM solution. The PROTAC molecules were further diluted in MEM or DMEM in the required concentration to prepare different doses for treatment. In order to validate the proteasomal degradation of the PROTAC dASK1 (35), 5 × 10⁵ HepG2 cells were pretreated for 30 min with 5 μM of proteasomal inhibitor MG132 (Sigma-Aldrich, cat no M7449). This was followed by treatment with dASK1 (35) as per the indicated dose for 8 h, followed by western blot analysis for ASK1.

Cell harvesting, protein estimation and preparation

After PROTAC treatment, HepG2 cells were harvested in cell lysis buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and 0.5% protease inhibitor cocktail (Millipore, Billerica, MA, USA). The cell lysate was extracted after centrifugation at 20 000 g for 20 min at 4 °C. BCA reagent was used to estimate the protein concentration. A total of 45 μg of protein sample was prepared per well of SDS-PAGE.

Western blot

Proteins were resolved in 8% SDS PAGE in 1× running buffer. The resolution of the proteins was achieved at 90 V. Transfer was performed using a PVDF membrane (Millipore) having a pore size of 0.45 μm in 1× transfer buffer. Transfer was achieved at 90 V for 4 h. Following the transfer, the PVDF membrane was blocked for 1 h at room temperature in 5% skim milk powder diluted in 1× TBST. Following this, the required primary antibody was prepared in 1× TBST containing 5% bovine serum albumin and 0.04% sodium azide. The membrane was incubated overnight at 4 °C with primary antibody (ASK1) [Cell Signaling Technology, cat no. 8662], TAK1 [Abclonal, cat no. A19077], actin [Sigma-Aldrich, cat no. A5316], p-p38 [Cell Signaling Technology, cat no. 4511], p38 [Cell Signaling Technology, cat no. 8690], ubiquitin [Cell Signaling Technology, cat no. 58395]. Following that, the membrane was again washed thoroughly with 1× TBST. The membrane was further incubated with goat anti-rabbit (1 : 1000–2500) and anti-mouse (1 : 10000) secondary antibodies for 1 h at room temperature, followed by washing multiple times with 1× TBST. The blot was developed using Clarity™ ECL western blotting substrate (BioRad) and viewed in ChemiDoc (Invitrogen).

MTT assay

1 × 10⁴ HepG2 cells were plated in 96-well plates. The cells were maintained in an incubator at 37 °C, humidified with 5% CO₂ for 24 h. Cells were treated with different concentrations of lead compounds for 24 h. 50 μL of 5 mg mL⁻¹ MTT (Sigma Aldrich) was added to each well and incubated for 4 h. The resultant product of the reaction, formazan crystal, was dissolved in 200 μL of DMSO, followed by absorbance measurement at 550 nm.

Animal study

Protocols for animal experiments were approved by the Institutional Animal Ethics Committee at CSIR-IICB under the aegis of the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. 6–8 weeks old C57BL/6 male mice (Mus musculus) were divided into two groups (n = 7 per group). Both groups received an MCD diet (MP Biomedicals, cat no. 960439) for 14 days. Following this, one group received an intraperitoneal injection (IP) of 2 mg Kg⁻¹ body weight of dASK1-VHL (60) in 1% DMSO for another 14 days with simultaneous feeding on MCD diet. On the other hand, the other group only received IP injection of vehicle (1% DMSO) along with MCD diet for another 14 days.

Immunoprecipitation assay

To check the ubiquitination status of ASK1 in the liver lysate of dASK1-VHL (60) treated mice, 1000 μg of liver lysate protein was used for immunoprecipitation using PureProteome Protein A/G Mix Magnetic Beads (Merck Millipore cat no. LSKMAGA02) and anti-ASK1 antibody at dilution of 1 : 100. This mixture was incubated overnight at 4 °C in a rotospin shaker. The next day, the unbound lysate was discarded and the beads were washed thrice with PBS containing 0.1% TritonX-100. The immunoprecipitated proteins were finally eluted using 2× loading dye and lysis buffer, followed by heating at 95 °C. Western blot analysis was performed with the eluted sample, followed by immunoblotting with total ubiquitin antibody.

Histological study

Liver specimens collected from MCD-fed and dASK1-VHL (60) treated mice were formalin-fixed and embedded in paraffin. The tissue section thus obtained (5 μm) was deparaffinized in xylene, followed by rehydration in a series of ethanol concentrations. The deparaffined section was further processed for hematoxylin and eosin staining. The image was taken in Evos XL Core (Invitrogen by Thermo Fisher Scientific).

Abbreviations

ASK1	Apoptosis signal-regulating kinase 1
ADME	Absorption, distribution, metabolism, and excretion
BSA	Bovine serum albumin
COP1	Constitutive photomorphogenic 1
CRBN	Cereblon
DIPEA	N,N-Diisopropylethylamine
DMEM	Dulbecco's modified Eagle medium
DMF	Dimethylformamide
ER	Endoplasmic reticulum
FDA	Food and drug administration
HepG2	Human hepatocellular carcinoma cell line
H&E	Hematoxylin and eosin stain
HRMS	High-resolution mass spectrometry
HATU	Hexafluorophosphate azabenzotriazoletetramethyl uronium
LC–MS	Liquid chromatography–mass spectrometry
MAPK	Mitogen-activated protein kinase
MASH	Metabolic dysfunction-associated steatohepatitis
MEM	Minimum essential medium
MMGBSA	Molecular mechanics with generalised born and surface area solvation
PBS	Phosphate-buffered saline
PCR	Polymerase chain reaction
PROTAC	Proteolysis targeting chimera
TAG	Triacylglycerol
TEA	Triethylamine
THF	Tetrahydrofuran
TLC	Thin-layer chromatography
UPS	Ubiquitin–proteasome system
VHL	von Hippel–Lindau

Ethical statement

Protocols for animal experiments were approved by the Institutional Animal Ethics Committee at CSIR-IICB under the aegis of the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India.

Data availability

ESI† figures and tables, further details of the experimental procedures, NMR spectroscopic data (¹H NMR and ¹³C NMR spectra of compounds 2–64), and HR-MS are available in the ESI.†

Author contributions

H. S. S. and A. S. contributed equally. A. T. and H. S. S. have conceptualized the PROTAC design, synthesis strategy and the protocol for evaluation. H. S. S. and I. H. have synthesized and characterized all the compounds. H. S. S., A. S., S. P. and D. S. carried out all the in vitro biology experiments. U. G. D. performed molecular docking analysis and free energy estimation using the MMGBSA method. P. C. designed the in vivo experiment and in vitro validation. A. S. carried out the in vivo experiment. H. S. S. and A. T. wrote the manuscript, and P. C. joined in finalizing it. A. T. and P. C. supervised the project. A. T. oversaw the progress of each component of the project as PI. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

The authors have no conflicts to declare.

Acknowledgements

The project was supported by the Council of Scientific and Industrial Research (CSIR) Fast Track Translational (FTT) project FTT070504 to A. T. and P. C. The author H. S. S. would like to thank the DST-SERB (NPDF/2020/001487) and CSIR for the postdoctoral fellowships. A. S. is thankful to ICMR for the research fellowship. Authors I. H. and D. S. want to thank the University Grant Commission (UGC), Govt. of India, for the fellowship. U. G. D. and S. P. would like to acknowledge the CSIR for the fellowship. The authors would like to acknowledge Mr. Soumik Laha, S. Khan for helping with LC–MS, Acuabiosys Private Limited, Hydrabad, India for ADME, in vivo pharmacokinetics in mice, Gautom Karmakar and Mr. Sandip Kundu for helping with NMR and the Central Instrument Facility of CSIR-IICB. Indian/World patent application for the design and synthetic strategy of our PROTACs with the patent application number: 202311034982, PCT Int. Appl.: WO2024236595A1 has been filed.⁶⁵

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5md00252d

‡ Authors contributed equally.

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