Sorting protein binding agents and bifunctional compounds thereof

Abstract

The present disclosure provides compounds capable of binding Sortilin, compositions comprising the compounds, and uses thereof. Also disclosed herein are bifunctional compounds having a moiety that binds Sortilin linked to a binding that binds a target protein of interest, compositions comprising the bifunctional compounds, and uses thereof. The bifunctional compounds described herein can be used to treat a disease or condition by removing the target protein of interest from the plasma or extracellular space of a patient in need thereof.

Description

Technical Field

[0001] This disclosure relates to compounds capable of binding sortilin, compositions comprising said compounds, and uses thereof. This document also discloses bifunctional compounds, compositions comprising said bifunctional compounds, and uses thereof, said bifunctional compounds having a sortilin-binding moiety, the group being linked to the moiety binding a target protein. The bifunctional compounds described herein can be used to treat diseases or conditions by removing the target protein from the plasma or extracellular space of a patient in need. Background of the Invention

[0003] Protein degradation is a crucial component of biomolecular turnover and renewal, a process that occurs naturally in all cells. Cytoplasmic proteins are typically degraded in the proteasome after ubiquitination, while extracellular biomolecules are degraded in the lysosomal compartment. Specific ubiquitination of cytoplasmic disease-associated proteins (DAPs) is achieved through bifunctional molecules called protein degradation-targeting chimeras (PROTACs) (Sun et al.). PROTACs are bifunctional molecules with a DAP-binding warhead at one end, which is linked to an E3 ubiquitin ligase-binding molecule at the other end. PROTACs thus link the E3 ligase to DAP, leading to ubiquitination and subsequent degradation in the proteasome, effectively performing chemical DAP knockdown. However, because PROTACs rely on the proteasome for function, they only work with intracellular proteins. Data suggest that by using bifunctional molecules to bind to the protein sorting mannose-6-phosphate receptor (M6P-R), non-cytoplasmic proteins can be guided to degrade in the lysosomal compartment (Banik et al.). Banik et al. demonstrated that antibodies labeled with the sugar motif of M6P-R promote lysosomal degradation of extracellular and membrane-bound targets; these types of molecules are termed lysosomal-targeting chimeras (LYTACs). This provides the first preclinical proof-of-concept (PoC) demonstrating the therapeutic potential of enhancing lysosomal delivery and degradation of DAP. The M6P-R binding motif is a phosphorylated sugar polymer, thus developing orally available modalities will require significant effort. Both PROTACs and LYTACs are designed to utilize natural cellular mechanisms, thus eliminating the need for the warhead itself to provide functionality. This is particularly advantageous compared to traditional drug development, where warhead development only requires optimizing binding affinity and linker conjugation strategies. Furthermore, these requirements are no longer necessary, further enabling binding to targets currently considered "undruggable" due to limitations.

[0004] LYTAC function depends on the successful recruitment of lysosomal transport receptors, such as M6P-R (Banik et al.). Another lysosomal receptor protein is the sorting protein. Sorting proteins share many characteristics with M6P-R, including rapid internalization from the cell surface and transport of cargo to lysosomes (Braulke et al.). Sorting proteins are expressed in most tissues and promote lysosomal degradation of several known ligands (Lefrancois et al., Ni et al., Hu et al.). The internalization capacity of sorting proteins is demonstrated by the plasma accumulation of the native ligand and frontotemporal lobar dementia (FTD)-associated protein, progranulin; in the plasma of mice lacking sorting proteins, the protein level is increased 3.5-fold (Hu et al., Lee et al.). Increasing extracellular progranulin levels by inhibiting sorting protein-mediated progranulin degradation is considered a therapeutic / preventive approach to FTD, and several independent studies have explored inhibiting this interaction. As a result, a number of high-affinity small-molecule sorting protein binders with different pharmacological characteristics have emerged (Schroder et al., Andersen et al., Stachel et al.), including orally bioavailable compounds and compounds with CNS exposure (Schroder et al.).

[0005] Interleukins (ILs)-17 are a family of cytokines, including IL-17A through IL-17F, which play important roles in regulating inflammatory and immune responses. These cytokines function by binding to the IL-17 receptor (IL-17R) family, which includes multiple members. A functional IL-17R is a transmembrane receptor complex, typically composed of one IL-17RA subunit and a second, other subunit, forming a hybrid receptor that binds to different ligands. IL-17A binds to the hybrid IL-17RA / RC receptor complex (Toy et al.).

[0006] IL-17A recruits and activates immune cells, promotes the production of other cytokines and chemokines, and facilitates the recruitment of neutrophils to sites of infection or inflammation. While IL-17A is crucial for immune defense, dysregulation of its production or activity can lead to the development of various autoimmune and inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, spondyloarthritis, and multiple sclerosis.

[0007] Therefore, IL-17A has become a target for therapeutic interventions in autoimmune and inflammatory diseases, and drugs that block IL-17A or its receptor have been developed and approved for clinical use. Strategies targeting IL-17A focus on inhibiting protein-protein interactions that mediate IL-17A signaling. For example, binding to IL-17A to block downstream effects, targeting IL-17RA, or indirectly targeting via the IL-17 pathway (Beringer et al.). These strategies are primarily represented by anti-IL-17A antibodies, such as secukinumab, which is approved for the treatment of psoriasis and psoriatic arthritis.

[0008] Antibody-mediated therapies suffer from poor oral availability and, in some cases, carry the risk of inducing an immune response to the therapy. To date, small molecule inhibitors of IL-17A have not been successful in treating IL-17A-mediated conditions. Currently, no alternative strategies have been proposed to address IL-17A-mediated conditions.

[0009] Developing compounds capable of reliably inducing protein degradation targeting multiple target molecules mediated by the lysosomal pathway remains a challenge. For example, there is a strong unmet need in the art for alternative therapeutic strategies to overcome these shortcomings and expand the scope of IL-17A-targeted therapies for IL-17A-mediated conditions. Summary of the Invention

[0010] This invention provides a solution to the aforementioned problems by offering compounds capable of binding to sorting proteins and inducing the degradation of extracellular target proteins. Therefore, this invention, as an alternative therapeutic strategy, transcends the inhibition of protein-protein interactions. The inventors have developed small molecule bifunctional compounds capable of inducing sorting protein-mediated target protein degradation. In one main aspect, this disclosure relates to a bifunctional compound of formula (I) or a pharmaceutically acceptable salt thereof: T L –L I – S L (I) in: S L This refers to the protein sorting part based on the combination of AI formulas: Formula (AI), Where R L Indicates with L I The connection; L I It is a connector or key; and T L It is the part that binds to extracellular target molecules.

[0011] Sorting proteins are lysosomal receptor proteins that induce rapid internalization from the cell surface and transport cargo to lysosomes (Barulke et al.). Sorting proteins are expressed in most tissues and promote lysosomal degradation of several known ligands (Lefrancois et al., Ni X. et al., Hu F. et al.). Human sorting proteins are encoded by the SORT1 gene.

[0012] The inventors have demonstrated that the bifunctional compound according to formula (I) can bind to the extracellular target moiety and induce sorting protein-mediated internalization in cells expressing sorting proteins via the lysosomal pathway. Its advantage lies in utilizing a novel mechanism focused on protein degradation rather than directly inhibiting the target protein to address target protein-mediated conditions. The bifunctional compound of this disclosure can simultaneously bind to the target molecule and the sorting protein to form ternary complexes, thereby mediating the internalization and degradation of the target molecule within intracellular compartments.

[0013] Sorting proteins are high-affinity receptors with low-abundance ligands, which have an advantage over other receptors used for lysosomal targeted degradation, such as low-affinity lysosomal sorting receptors with high-abundance ligands, like the low-density lipoprotein receptor (LDLR), receptor LRP2 / megalin, and mannose-6-phosphate receptor (M6PR).

[0014] The inventors have discovered that bifunctional compounds derived from the sorting protein binder of formula AI provide high affinity for binding to sorting proteins and mediate targeted protein degradation, while exhibiting improved drug-like properties, such as reduced lipophilicity and decreased hepatic microsomal clearance, compared to analogs of formula AI (where tert-butyl ether is replaced by a neopentyl group ((CH3)3-CO- is replaced by (CH3)3-C-CH2-)). These properties indicate a surprisingly reduced first-pass metabolism, which is beneficial for oral administration.

[0015] In another aspect, this disclosure provides a pharmaceutical composition comprising the bifunctional compound described herein.

[0016] On the other hand, this disclosure provides the bifunctional compounds described herein, which are used as pharmaceuticals.

[0017] On the other hand, this disclosure provides a compound according to formula (A-II) or a pharmaceutically acceptable salt thereof, or an enantiomer thereof, or a mixture thereof: Equation (A-II), where: R 1 C is H, halogen, alkoxy, -CF3, or optionally substituted.1-5 Hydrocarbon chains, wherein one or more carbon groups of the C1-C5 hydrocarbon chain are optionally and independently replaced by one or more groups selected from the group consisting of: –O-, -NH-, -C(O)-, esters, amides, carbamates, thioureas, sulfonamides, ureas, , , Optionally substituted carbocyclic rings, Optionally substituted heterocyclic rings, or Where X is NH or O.

[0018] Compounds of formula (A-II) bind to sorting proteins with high affinity and possess favorable biophysical properties, thus showing potential in the treatment of sorting protein-mediated conditions. Examples demonstrate that compounds of formula (A-II) bind to sorting proteins with higher affinity than analogs of formula A-II (where the tert-butyl ether is substituted with a neopentyl group ((CH3)3-CO- is replaced with (CH3)3-C-CH2-)).

[0019] In one aspect, this disclosure provides compounds of formula (A-II) which are used as pharmaceuticals.

[0020] The inventors have discovered small molecule bifunctional compounds capable of inducing sorting protein-mediated degradation of the IL-17A protein. Therefore, in another respect, this disclosure relates to a bifunctional compound according to formula (X) or a pharmaceutically acceptable salt thereof: T A-L –L I –S A-L (X) in: S A-L It is a group that binds to sorting proteins; L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

[0021] The inventors have unexpectedly demonstrated that compounds according to formula (X) can generate IL-17A degradation by forming a ternary complex by simultaneously binding IL-17A and the receptor sorting protein. This complex induces IL-17A to be internalized into the lysosomal space and degraded.

[0022] The examples described herein demonstrate that the bifunctional compound according to this disclosure simultaneously binds to IL-17A and the sorting protein, and forms a ternary complex with IL-17A and the sorting protein. Furthermore, the examples show that the bifunctional compound of formula (X) described herein induces the uptake of IL-17A from the extracellular space into cells expressing the sorting protein receptor, and confirm that internalized IL-17A is degraded in lysosomes.

[0023] Therefore, this invention discloses a unique approach to addressing protein-mediated conditions (e.g., IL-17A-mediated conditions) by employing a novel mechanism that focuses on protein degradation rather than directly or indirectly inhibiting protein mediators. The advantages of this invention include one or more of the following: It can induce the targeted degradation of protein mediators (such as IL-17A); Targeting disease protein mediators (e.g., IL-17A) with strategies different from direct inhibition; It can target disease-mediated proteins (such as IL-17A) in a wide range of body compartments that express sorting proteins: blood flow, CNS, PNS, CSF, immune system cells, and tumor subtypes; The compounds disclosed herein are small molecules, thus providing a novel class of compounds that differ from antibody- or fragment-based therapeutics (e.g., anti-IL-17A antibodies or fragments thereof) for addressing protein-mediated conditions. In treatment, T is bound L or T A-L It has enhanced treatment outcomes in aspects of disease mediated by extracellular target molecules; It has improved biophysical properties and improved administration, distribution, metabolism, and excretion (ADME) properties; Compared to other known therapies for conditions involving selected extracellular target molecules, it has improved pharmacokinetics and reduced toxicity or side effects.

[0024] In one aspect, this disclosure provides a composition comprising a compound of formula (VI) as described herein. Attached Figure Description

[0025] Figure 1 The formation of a ternary complex between a bifunctional compound, a sorting protein, and IL-17A was measured by HTRF FRET.

[0026] Figure 2 Cellular uptake curves of IL-17A entering cells expressing sorting proteins mediated by the studied bifunctional compound in the concentration range of 2 nM to 2 µM.

[0027] Figure 3The luminescent signal of cell lysates from cells expressing sorting proteins after incubation with nanoluc-IL-17A and 100 nM bifunctional compound X-065 for 24 hours in the presence of three monofunctional IL-17A binders that act as competitive inhibitors.

[0028] Figure 4 The case of removing IL-17A from extracellular culture medium after incubating cells expressing sorting proteins with a series of diluted concentrations of the bifunctional compound X-017 and different concentrations of IL-17A for 48 hours.

[0029] Figure 5 : In the presence of the lysosomal protease inhibitor leupeptin and a control without leupeptin, the luminescent signal in cell lysates of cells expressing sorting proteins after incubation with nanoluc-IL-17A and the bifunctional compound X-062 for 24 hours was observed.

[0030] definition

[0031] As used in this article, the term "alkyl" refers to the straight-chain or branched hydrocarbon moiety.

[0032] As used herein, the term "alkoxy" refers to a group of the formula -O-alkyl, where the alkyl group is as defined above. In particular, C1-C3-alkoxy is intended to represent such hydrocarbons having 1, 2, or 3 carbon atoms. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, and isopropoxy.

[0033] As used herein, the term "halogenated alkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by halogen atoms, for example, one or more hydrogen atoms being replaced by any of F, Cl, Br or I.

[0034] As used herein, the terms “cycloalkyl” or “carbocyclic” refer to monocyclic or polycyclic systems. The term “cycloalkyl” as used herein may also optionally include one or more unsaturated groups or substituents.

[0035] The terms “heterocyclic” or “heterocyclic” as used alone or in combination herein refer to a saturated or unsaturated aromatic or non-aromatic ring containing 3 to 7 ring atoms, wherein at least one ring atom is a heteroatom. The terms “heteroaromatic” or “heteroaryl” as used alone or in combination herein refer to an aromatic ring containing 5 to 6 ring atoms, wherein at least one ring atom is a heteroatom. “Heteroatom” is intended to refer to sulfur, oxygen, or nitrogen.

[0036] The term "aromatic" or "aryl" refers to a cyclic or polycyclic part of a conjugated unsaturated (4η+2)π-electron system (where n is a positive integer), sometimes referred to as a delocalized π-electron system.

[0037] The term "alkenyl" includes groups having at least one carbon-carbon double bond.

[0038] The terms “substituent” or “substituted” as used alone or in combination herein refer to a group that can be used to replace hydrogen. In some embodiments of the invention, the substituted molecule itself may be further substituted. The term “substituent derived from” as used herein refers to a group of atoms derived from a particular molecule or formula at any position therein. In some embodiments, a substituent derived from a molecule is a corresponding molecule in which one hydrogen atom has been removed. For example, –CH3 is an example of a substituent derived from CH4.

[0039] dissociation constant (K) D) Binding affinity is an indicator of the degree of reversible association between two molecular species. The smaller the dissociation constant, the stronger the binding affinity.

[0040] As described herein, a ternary complex is a complex comprising three distinct molecules bonded together. As described herein, bifunctional compounds can form ternary complexes between a sorting protein and a target molecule. This implies a three-membered complex in which the sorting protein is bonded to both the target protein and the bifunctional compound simultaneously.

[0041] The TNF-alpha used in this article may be referred to as TNFa, TNF-a, TNF-α, TNFα, or TNFalpha. Invention Details

[0043] Bifunctional compound

[0044] In one key aspect, this disclosure relates to a bifunctional compound of formula (I), or a pharmaceutically acceptable salt thereof: T L –L I – S L (I) in: S L This refers to the protein sorting part based on the combination of AI formulas: Formula (AI), Where R L Indicates with L I The connection; L I It is a connector or key; and T L It is the part that binds to extracellular target molecules.

[0045] In one implementation, S L According to equation (A-Ia): Formula (A-Ia), Where R L Indicates with L I The connection.

[0046] In one implementation, S L According to equation (A-Ib): Equation (A-Ib), Where R L Indicates with L I The connection.

[0047] connector

[0048] The adapter connects the sorting protein-binding portion to the targeting portion. In one embodiment, the adapter is as shown in formula (II):

[0049] Equation (II)

[0050] in: Indicates with T L or S L The connection; L 1 and L 2 Each group is independently selected from: bond, -C(H2)-, -O-, -N(H)-; functional groups selected from carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; and C1-C3 hydrocarbon chain, wherein one or more methylene groups are independently and optionally replaced by carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; Z is selected from: divalent, saturated or unsaturated, straight or branched C1-C 30 A hydrocarbon chain in which one or more methylene groups are independently and optionally replaced by one or more groups selected from: -O-, –N(H)-, -N(R)-. L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, –N(H)C(=O)-, -N(R L1 )C(=O)-, –C(=O)N(H)-, -NHC(O)NH-, -NHC(O)O-, -C(=O)N(R L1 )-, -S-, -S(=O)-, –S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 )-; optionally substituted aromatic groups; optionally substituted carbocyclic rings; optionally substituted heterocyclic rings; optionally substituted aromatic heterocyclic rings; ; , , , ;–C(R L2 H- and -N(R) L2 )-; R L1 Selected from C 1-5 Alkyl; R L2 For –(CH2) L -R X ;R x It is -OH or –C(=O)NH2; L is an integer from 0 to 3; n and w are each an independent integer from 1 to 9.

[0051] In one implementation, C1-C 30 The hydrocarbon chain is C5-C. 30 Hydrocarbon chains, such as C8-C 30 Hydrocarbon chains, such as C 10 -C 30 Hydrocarbon chains, such as C 12 -C 30 Hydrocarbon chain. In one embodiment, C1-C 30 The hydrocarbon chain is C 10 -C 25 Hydrocarbon chains, such as C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 Or C 25 Hydrocarbon chain. In one embodiment, C1-C 30 The hydrocarbon chain is C 14 -C 20 Hydrocarbon chain. In one embodiment, C1-C 30 The hydrocarbon chain is C7-C. 13 Hydrocarbon chain.

[0052] In one embodiment, one or more methylene groups of Z (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 methylene groups of the hydrocarbon chain in Z) are independently and optionally replaced by one or more groups selected from: -O-, –N(H)-, -N(R-). L1)-, -OC(=O)-, –C(=O)O-, -C(=O)-, –N(H)C(=O)-, -N(R L1 )C(=O)-, –C(=O)N(H)-, –C(=O)N(R L1 )-, -S-, –S(=O)-, –S(=O)2-, -N(R L1 )S(=O)2-、–S(=O)2N(R L1 )-, –CH2-CH2-O-, optionally substituted carbocyclic rings, optionally substituted heterocyclic rings, and triazoles; wherein R L1 C 1-5 alkyl.

[0053] In one implementation, Z comprises one or more –NH-SO 2 - Group. In one embodiment, Z comprises one or more triazole groups. In one embodiment, Z comprises one or more groups selected from the group consisting of optionally substituted carbocyclic groups and optionally substituted heterocyclic groups.

[0054] In one embodiment, Z comprises two groups, each independently selected from: a triazole, an optionally substituted carbocyclic group, and an optionally substituted heterocyclic group. In another embodiment, Z comprises three groups, each independently selected from: a triazole, an optionally substituted carbocyclic group, and an optionally substituted heterocyclic group.

[0055] In one embodiment, the carbon ring is based on As shown, n is an integer selected from 0, 1, 2 or 3.

[0056] In one embodiment, Z comprises one or more heterocyclic groups.

[0057] In one embodiment, the heterocyclic group may be an optionally substituted 3- to 6-membered ring, wherein one or two carbon atoms of the ring have been replaced by N.

[0058] In one embodiment, the heterocyclic group is based on As shown, n is an integer selected from 0, 1, 2 or 3.

[0059] In one embodiment, Z comprises three groups, each independently selected from the groups shown in Table Z: Table Z

[0060] In one embodiment, Z comprises 1, 2, or 3 groups selected from any group in Table Z. In one embodiment, Z comprises 1 group selected from any group in Table Z. In one embodiment, Z comprises 2 groups selected from any group in Table Z. In one embodiment, Z comprises 3 groups selected from any group in Table Z.

[0061] In one embodiment, Z comprises one or more groups selected from the groups shown in Table ZI: Table ZI

[0062] Where n and / or n' are each independent integers from 1 to 10, and t, t' and / or w are each independent integers from 1 to 20.

[0063] In one embodiment, Z comprises one group selected from any group in Table ZI.

[0064] In one implementation, Z includes or , where n is an integer from 1 to 10.

[0065] In one implementation, Z includes or , where n is an integer from 1 to 10, and t or w is an integer from 1 to 20.

[0066] In one implementation, Z includes ], where n is an integer from 1 to 10, and t and t' are each an integer from 1 to 20 independently.

[0067] In one implementation, Z includes , where n and n' are each an integer from 1 to 10, and t is an integer from 1 to 20.

[0068] In one embodiment, Z comprises one or more (e.g., one, two, or three) branches with polar groups, each branch being independently -C(R) L2 )H- or -N(R L2 )-, where R L2 For –(CH2) L -R X ;R x Z is -OH or -C(=O)NH2; and L is an integer from 0 to 3. In one embodiment, Z comprises a branch with a polar group, said branch being -C(R L2 )H- or -N(R L2) -; where R L2 For –(CH2) L -R X;R x Z is -OH or -C(=O)NH2; and L is an integer from 0 to 3. The inventors have shown that when Z contains a branched chain with a polar group, nonspecific protein binding in plasma is reduced.

[0069] In one implementation, Z includes -C(R) L2 H-, where R L2 For –(CH2) L -R X ;R x Z is -OH, -C(=O)NH2; and L is an integer from 0 to 3. In one embodiment, Z includes -N(R L2 )-, where R L2 For –(CH2) L -R X ;R x It is -OH or -C(=O)NH2; and L is an integer from 0 to 3.

[0070] In one implementation, L is 0, 1, 2, or 3. In one implementation, L is 0, 1, or 2. In one implementation, L is 0 or 1. In one implementation, L is 0. In one implementation, L is 1. In one implementation, L is 2.

[0071] In one implementation, R x For -OH. In one embodiment, R x It is –C(=O)NH2.

[0072] In one embodiment, Z comprises one or more groups selected from the following:

[0073] In one embodiment, Z comprises one and only one group selected from the following:

[0074] In one implementation, Z includes , , , , , or .

[0075] In one implementation, Z includes or .

[0076] In one implementation, L 1 or L 2 It is a triazole group. In one embodiment, L1 and L 2 It has a triazole group.

[0077] In one implementation, L 1 or L 2 For –O-. In one implementation, L 1 and L 2 It is –O-.

[0078] In one implementation, L 1 or L 2 For –NH-. In one implementation, L 1 and L 2 It is –NH-.

[0079] In one implementation, L 1 or L 2 It is –S(=O)2-.

[0080] In one implementation, L 1 and L 2 It is –S(=O)2-.

[0081] In one implementation, L 1 or L 2 for .

[0082] In one implementation, L 1 and L 2 for .

[0083] In one implementation, L 1 or L 2 for .

[0084] In one implementation, L 1 and L 2 for .

[0085] In one implementation, L 1 and / or L 2 for .

[0086] In one implementation, L 1 and / or L 2 for .

[0087] In one implementation, L 1 and / or L 2 for , where X is an atom selected from N or O.

[0088] In one implementation, L 1 and / or L 2 for , where X is an atom selected from N or O.

[0089] In one implementation, L 1 and L 2 These are different groups.

[0090] In one implementation, L 1 and L 2 same.

[0091] In one implementation, the connector (L I According to any one of the structures II-1 to II-98 shown in Table Z-II: Table Z-II:

[0092] in Indicates with T L or S L The connection.

[0093] As illustrated herein, when any structure in Table Z-II contains a chiral center, the center may have any configuration, either R or S, according to the Cahn-Ingold-Prelog rule. For example, in one embodiment, the chiral center has an R configuration. In another embodiment, the chiral center has an S configuration.

[0094] In one implementation, connector L I According to any of formulas II-1 to II-98 in Table Z-II. The connectors shown in Table Z-II (e.g., formulas II-1 to II-98) can be marked with... Any connection point with S L and T LConnection. For example, in one embodiment, the connector shown in Table Z-II appears on the left side of the formula shown in Table Z-II with... The connection of the marker and T L Connect, and appear on the right side of the formula shown in Table Z-II with The connection of the marker and S L connect.

[0095] In one embodiment, the bifunctional compound according to this disclosure is capable of operating at less than 50 The dissociation constant of M (K) D) Combined with sorting proteins, such as those less than 40 M, for example, less than 30 M, for example, less than 20 M, for example, less than 10 M, for example, less than 5 M, for example, less than 4 M, for example, less than 3 M, for example, less than 2 M, for example, less than 1 M, for example, less than 0.8 M, for example, less than 0.6 M, for example, less than 0.5 M, for example, less than 0.4 M, for example, less than 0.3 M, for example, less than 0.2 M, for example, less than 0.1 M, for example, less than 0.05 M, for example, less than 0.04 M, for example, less than 0.03 M, for example, less than 0.02 M, for example, less than 0.01 M.

[0096] In one embodiment, the bifunctional compound according to this disclosure is capable of

[50] M to 0.001 The dissociation constant (K) between M and D) Combined with sorting proteins, such as 50 M to 40 Between M, for example, 40 M to 30 Between M, for example, 30 M to 20 Between M, for example, 20 M to 10 Between M, for example, 10 M to 5 Between M, for example, 5 M to 4 Between M, for example, 4 M to 3 Between M, for example, 3 M to 2 Between M, for example 2 M to 1 Between M, for example, 1 M to 0.9 Between M, for example, 0.9 M to 0.8 Between M, for example 0.8 M to 0.7 Between M, for example, 0.7 M to 0.6 Between M, for example 0.5 M to 0.4 Between M, for example, 0.4 M to 0.3 Between M, for example, 0.3 M to 0.2 Between M, for example, 0.2 M to 0.1 Between M, for example, 0.1 M to 0.05 Between M, for example, 0.05 M to 0.01 Between M, for example, 0.01 M to 0.001 Between M.

[0097] Binding to target or sorting proteins can be measured using various methods known to those skilled in the art. For example, microscale thermophoresis (MST).

[0098] Targeted components and target extracellular molecules

[0099] The bifunctional compounds disclosed herein comprise portions capable of binding extracellular target molecules or proteins such as growth factors, cytokines, hormones, lipoproteins, neurotransmitters, capsids, extracellular secretory proteins, and antibodies.

[0100] In one implementation, the extracellular target molecule is a protein. Extracellular proteins, as described herein, refer to proteins that are not completely enclosed within the cell. This means, for example, proteins located entirely outside the cell, as well as membrane-bound or membrane-associated proteins having extracellular domains.

[0101] In one implementation, T LIt is a substituent of a small organic molecule (i.e., a non-biological agent) that fully binds to the target molecule, or a substituent of a drug-active compound that binds to the target extracellular protein, or a peptide, protein, or biological agent or its binding fragment that fully binds to the extracellular target molecule.

[0102] T L Some may be derived, for example but not limited to, from substituents of approved or clinically-stage drugs, or from substituents of compounds that will be reviewed as drugs by regulatory agencies (such as the FDA or EMA).

[0103] Extracellular target proteins can be any amino acid sequence, including T. L The bifunctional compound can bind to this sequence and produce beneficial therapeutic effects through its degradation. In one embodiment, the target protein is a non-endogenous peptide, such as a peptide derived from a pathogen or toxin. In another embodiment, the target protein can be an endogenous protein mediating the disease. The endogenous protein can be either the normal or abnormal form of the protein. For example, the target protein can be an extracellular mutant protein, or a protein, for example, encoded by a nucleotide polymorphism as partially or completely gain-of-function or loss-of-function. In some embodiments, the bifunctional compound targets the abnormal form of the protein instead of the normal form.

[0104] According to the bifunctional compound of this disclosure, the targeting ligand (T) L The first part is a ligand that binds covalently or non-covalently to a target protein selected for lysosomal degradation.

[0105] Many of the exemplary extracellular proteins targeted for medical therapies described below have characteristic structural information in the well-known Protein Database (“PDB”), a database of three-dimensional structural information of biological macromolecules such as proteins and nucleic acids. The PDB contains X-ray crystallography and other information submitted by scientists worldwide and is freely accessible. For the codes provided below, see, for example, www.rcsb.org, wwwrwwpdb.org, and www.uniprot.org.

[0106] For example, those skilled in the art can use existing visualization tools (including those available on the PDB website) to determine T. L The site of docking into the extracellular protein. Those skilled in the art can also interpret the crystal structure and the selected target T... LImport modeling software (including, for example, PyMOL, Glide, Maestro, RasMol, Visual Molecular Dynamics, Jrnol, and AutoDock) to determine which part of the extracellular protein targeting ligand binds to the extracellular protein. Then, the bifunctional compound T... L At points where binding to extracellular proteins does not have an excessively adverse effect on the junction with the linker (L I ) or sorting protein-binding groups (S L (Combined)

[0107] Non-limiting examples of extracellular proteins: In one embodiment, the extracellular target protein is selected from one of the following: PCSK9, TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgA, IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-β (TGF-β), and platelet-reactive protein-1. (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

[0108] Immunoglobulin G (IgG)

[0109] In some implementations, the target protein is human immunoglobulin G (IgG). IgG accounts for approximately 75% of human serum antibodies. IgG is the most common type of antibody circulating in the blood. IgG antibodies are large globular proteins with a molecular weight of approximately 150 kDa, composed of four peptide chains. It contains two identical gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus exhibiting a tetrameric quaternary structure. The two heavy chains are linked to each other and to each of the light chains by disulfide bonds. The resulting tetramer has two identical halves that together form a Y-shaped structure. Each end of the bifurcation contains an identical antigen-binding site. The regions and domains of a typical IgG are shown in the left figure. The Fc region of IgG has a highly conserved N-glycosylation site at asparagine 297 in the heavy chain constant region. The N-glycan attached to this site is primarily a complex type of core fucosylated dual-antenna structure. In addition, a small amount of such N-glycan also carries a dichotomous GlcNAc and α-2,6-linked sialic acid residues. The N-glycan composition of IgG is associated with a variety of autoimmune, infectious, and metabolic diseases. Furthermore, IgG4 overexpression is associated with IgG4-related diseases, which typically involve multiple organs and include type 1 autoimmune pancreatitis, interstitial nephritis, Riedel's thyroiditis, Mikulicz's disease, Kuttner's tumor, inflammatory pseudotumors (located in various parts of the body), mediastinal fibrosis and retroperitoneal fibrosis in some cases, aortitis, retroperitoneal fibrosis, proximal biliary strictures, tubulointerstitial nephritis, pachymeningitis, pancreatic enlargement, and pericarditis.

[0110] The Protein Data Bank website provides IgG crystal structures searchable via 1H3X (Krapp, S., et al., J. Mol. Biol., 2003, 325: 979) and 5V43 (Lee, CH, et al., Nat. Immunol., 2017, 18: 889-898); and via 5YC5 (Kiyoshi M., et al., Sci.Rep., 2018, 8: 3955-3955), 5XJE (Sakae Y., et al., Sci. Rep., 2017, 7:13780-13780), 5GSQ (Chen, CL, et al., ACS Chem Biol, 2017, 12:1335-1345) and 1HZH (Saphire EO, et al., Science, IgG crystal structures bound to various compounds were retrieved from the database (2001, 293:1155-1159). Furthermore, Kiyoshi, M. et al. provided insights into the structural basis of human IgG1 binding to its high-affinity human receptor FcyRI (Kiyoshi M., et al., Nat Commun., 2015, 6, 6866).

[0111] The bifunctional compound according to the present invention can sort protein-binding moieties (S) L ) binds to sorting proteins and targets the protein-targeting region (T L () binds to target proteins.

[0112] Therefore, in one embodiment, the bifunctional compound of the present invention is capable of simultaneously binding to both the sorting protein and the target protein. This means that the bifunctional compound according to the present disclosure forms a ternary complex with both the sorting protein and the target protein. In the ternary complex, both the sorting protein and the target protein bind to the bifunctional compound simultaneously.

[0113] The formation of ternary complexes can be measured in various ways known in the art. For example, the formation of ternary complexes can be measured by Förster resonance energy transfer (FRET) or by time-resolved Förster resonance energy transfer (TR-FRET). As measured by these methods, an increase in the homogeneous time-resolved fluorescence (HTRF) ratio indicates the formation of ternary complexes.

[0114] As shown in the embodiments, the bifunctional compound according to this disclosure can bind to sorting proteins and target proteins to form a ternary complex.

[0115] In one embodiment, the bifunctional compound according to this disclosure is capable of binding to sorting proteins on the cell surface. In another embodiment, the bifunctional compound is capable of forming a ternary complex with the sorting protein and the target molecule on the cell surface.

[0116] In one implementation scheme, in S L Binding to sorting proteins located on the cell surface and T L Upon binding to the target protein, the target protein is internalized into the cell. In one embodiment, the target protein is degraded after internalization into the cell. In another embodiment, the degradation of the target protein occurs in the lysosomal compartment.

[0117] In one embodiment, the bifunctional compound according to this disclosure has a dissociation constant of less than 50 when it binds to the sorting protein. M (e.g., less than 40) M, for example, less than 30 M, for example, less than 20 M, for example, less than 10 M, for example, less than 5 M, for example, less than 4 M, for example, less than 3 M, for example, less than 2 M, for example, less than 1 M, for example, less than 0.8 M, for example, less than 0.6 M, for example, less than 0.5 M, for example, less than 0.4 M, for example, less than 0.3 M, for example, less than 0.2 M, for example, less than 0.1 M, for example, less than 0.05 M, for example, less than 0.01 M), and the dissociation constant of binding to the target protein is less than 50. M (e.g., less than 40) M, for example, less than 30 M, for example, less than 20 M, for example, less than 10 M, for example, less than 5 M, for example, less than 4 M, for example, less than 3 M, for example, less than 2 M, for example, less than 1 M, for example, less than 0.8 M, for example, less than 0.6 M, for example, less than 0.5 M, for example, less than 0.4 M, for example, less than 0.3 M, for example, less than 0.2 M, for example, less than 0.1 M).

[0118] TNF-alpha

[0119] In some implementations, the target protein is human TNF-α (UniProtKB-PC) 1375 (TNFA_HUMAN)). TNF-α is a pro-inflammatory cytokine active in the body's immune response and in severe inflammatory diseases. TNF-α is associated with a variety of conditions, including but not limited to rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, hidradenitis suppurativa, refractory asthma, systemic lupus erythematosus, diabetes, and induction of cachexia. As used herein, TNF-alpha may be referred to as TNFa, TNF-a, TNF-α, TNFα, or TNFalpha.

[0120] The Protein Data Bank website provides TNF-α crystal structures searchable via 6RMJ (Valentinis, B., et al., Int. J. Mol. Sci., 2019, 20), 5UUI (Carrington et al., Biophys J., 2017, 113 371-380), 600Y, 600Z, and 60PO (O'Connell, J., et al., Nat. Commun., 2019, 10 5795-5795), and 5TSVV (Cha, SS, J Biol Che ., 1998, 273 2153-2160); and 2AZ5 (He., M. VI. et af, Science, TNF-α crystal structures bound to various compounds were retrieved from 2005, 310: 1022-1025; 5WUX (Lee, JU, Int J Mol Sci., 2017, 18); 5MU8 (Blevitt et al., J Med Chem., 2017, 603511-3517); 4Y60 (Feldman J. I,., et al., Biochemistry, 2015, 543037-3050); 3WD5 (Hu, S., et al., J Biol Chem., 2013, 28827059-27067); and 4G3Y (Liang, SY, J Biol Chem., 2013, 288 13799-13807).

[0121] Preprotein convertase subtilisin / kexin 9 (PCSK-9)

[0122] In some implementations, the target protein is the pre-human protein convertase subtilisin / kexin 9 (PCSK-9) (UniProtKB - Q8NBP7 (PCSK9_HUMAN)). PCSK-9 is a key player in the regulation of plasma cholesterol homeostasis. PCSK-9 binds to members of the low-density lipid receptor family: low-density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1 / APOER), and apolipoprotein receptor 2 (LRP8 / APOER2), and promotes their degradation in the acidic compartment of the cell. It functions through a non-proteolytic mechanism, enhancing the degradation of hepatic LDLR via the clathrin-LDLRAP1 / ARH-mediated pathway, and may prevent LDLR from recirculating from endosomes to the cell surface or guide it to lysosomes for degradation. PCSK-9 has been identified as being associated with the development of hypercholesterolemia and cardiovascular disease.

[0123] The Protein Data Bank website provides the crystal structure of PCSK-9 searchable via 2P4E (Cunningham, D., et al., Nat Struct Mol Biol., 2007, 14 413-419); and via 3BPS (Kwon, H. J, et al., Proc Natl Acad Sei USA, 2008, 105 1820-1825); 6U26, 6U2N, 6U2P, 6U36, 6U38 and 6U3X (Petrilli, WL, et al., Ceil Chem Biol., 2019, 27 32-40. e3); 50CA (Gustafsen, C., et al., Nat Commun., 2017, 8 503-503); 4NE9 (Schroeder, C. L, et al.). The crystal structures of PCSK-9 bound to various compounds were retrieved from Chem Biol., 2014, 21 284-294; 40V6 (Mitchell, T., et. al., J Pharmacol Exp Ther., 2014, 350412-424); and 4NMX (Zhang, Y., et. al., J Biol Chem., 2014, 289942-955). Furthermore, Piper et al. provided insights into the crystal structure of PCSK-9 (Piper, DE, etah, Structure, 2007, 15(5), 545-52).

[0124] In one implementation, T L According to either formula BI or B-II:

[0125] Where R L Indicates with L I The connection.

[0126] TNFalpha

[0127] In one embodiment, the target protein is TNFα. Therefore, in one embodiment, the bifunctional compound according to this disclosure is capable of forming a ternary complex with the sorting protein and TNFα. In one embodiment, the bifunctional compound is capable of binding both the sorting protein and TNFα simultaneously.

[0128] In one embodiment, the bifunctional compound according to this disclosure has: S L The dissociation constant of the protein binding to sorting proteins is less than 50. M (e.g., less than 2) M, for example, less than 0.5 M, preferably less than 0.1 M), and T L The dissociation constant of TNFα binding is less than 100. M (e.g., less than 0.5) M, less than 0.1 M).

[0129] In one embodiment, the bifunctional compound according to this disclosure has a dissociation constant of less than 50 when it binds to the sorting protein. M (e.g., less than 2) M, for example, less than 0.5 M, preferably less than 0.1 M), and the dissociation constant of TNFα binding is less than 100. M (e.g., less than 0.5) M, for example, less than 0.1 M).

[0130] In one embodiment, the bifunctional compound makes: in S L Binding to sorting proteins located on the cell surface and T LUpon binding with TNFα, TNFα is internalized into the cells. To assess whether the target protein has been internalized, methods such as detecting the target protein in the cell culture supernatant can be used. Any suitable method can be used, such as ELISA, conjugate fluorescence detection, HPLC, gel electrophoresis with conjugation staining (e.g., SDS-PAGE or Western blotting), or other techniques well-known in the art. The presence of the target protein or fragments thereof within the cells can also be assessed using similar methods after cell lysis.

[0131] Examples demonstrate that the bifunctional compounds according to this disclosure can promote the internalization of target proteins into cells through sorting protein-mediated binding. Examples also demonstrate that internalization leads to the degradation of target proteins.

[0132] In one implementation, TNFα is degraded after being internalized into the cells.

[0133] The bifunctional compound according to any one of the preceding claims, wherein T L According to any one of formulas (B-III-1) to (B-III-8):

[0134] Where R L Indicates with L I The connection.

[0135] In one implementation, T L According to formula (B-III-1). In one implementation, T L According to formula (B-III-2). In one implementation, T L According to formula (B-III-3). In one implementation, T L According to equation (B-III-4). In one implementation, T L According to formula (B-III-5). In one implementation, T L According to formula (B-III-6). In one embodiment, TL is according to formula (B-III-7). In one embodiment, T L According to formula (B-III-8).

[0136] In one embodiment, the bifunctional compound is according to any one of formulas IV-B1 to IV-B8:

[0137] In one embodiment, the bifunctional compound is according to formula IV-B1. In one embodiment, the bifunctional compound is according to formula IV-B2. In one embodiment, the bifunctional compound is according to formula IV-B3. In one embodiment, the bifunctional compound is according to formula IV-B4. In one embodiment, the bifunctional compound is according to formula IV-B5. In one embodiment, the bifunctional compound is according to formula IV-B6. In one embodiment, the bifunctional compound is according to formula IV-B7. In one embodiment, the bifunctional compound is according to formula IV-B8. In one embodiment, this disclosure provides a bifunctional compound according to any one of formulas IV-B1, IV-B2, IV-B3, IV-B4, IV-B5, IV-B6, IV-B7, and IV-B8, wherein the connector (L I ) is selected from any of the formulas II-1 to II-98 shown in Table Z-II.

[0138] In one embodiment, the compound is according to any one of formulas V-B1 to V-B8:

[0139] In one embodiment, the bifunctional compound is according to formula V-B1. In one embodiment, the bifunctional compound is according to formula V-B2. In one embodiment, the bifunctional compound is according to formula V-B3. In one embodiment, the bifunctional compound is according to formula V-B4. In one embodiment, the bifunctional compound is according to formula V-B5. In one embodiment, the bifunctional compound is according to formula V-B6. In one embodiment, the bifunctional compound is according to formula V-B7. In one embodiment, the bifunctional compound is according to formula V-B8. In one embodiment, this disclosure relates to a bifunctional compound according to any one of formulas V-B1, V-B2, V-B3, V-B4, V-B5, V-B6, V-B7, and V-B8, wherein the connector (L I ) is selected from any of the formulas II-1 to II-98 shown in Table Z-II.

[0140] In one embodiment, the bifunctional compound is according to formula IV-B7.

[0141] In one embodiment, the bifunctional compound is according to formula V-B7.

[0142] In one implementation, the compound is able to bind both sorting proteins and TNFα simultaneously.

[0143] The bifunctional compound according to any one of the preceding claims, wherein S L Binding to sorting proteins located on the cell surface and TL Upon binding with TNFα, TNFα is internalized into the cells.

[0144] The bifunctional compound according to any one of the preceding claims, wherein TNFα is degraded after being internalized into the cell.

[0145] In one embodiment, the bifunctional compound is any one of compounds A-001 to A-003 described in Table A of the "Detailed Description of the Invention", or a pharmaceutically acceptable salt thereof.

[0146] Table A

[0147] In one embodiment, the bifunctional compound is A-003, or a pharmaceutically acceptable salt thereof.

[0148] IL-17A

[0149] In one embodiment, the target protein is interleukin-17A (IL-17A). In one embodiment, T L It is the part that combines IL-17A.

[0150] The inventors have produced small-molecule bifunctional compounds capable of inducing sorting protein-mediated degradation of the IL-17A protein. Therefore, in another respect, this disclosure relates to a bifunctional compound according to formula (X) or a pharmaceutically acceptable salt thereof: T A-L – L I –S A-L (X) in: S A-L It is the part that binds and sorts proteins; L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

[0151] In one embodiment, the bifunctional compound of formula X described herein has an S according to formula XI or X-II. A-L : Formula (XI) Formula (X-II) Where R L Indicates with L I The connection.

[0152] In one embodiment, the bifunctional compound of formula X described herein has S according to formula XI.A-L : Formula (XI) Where R L Indicates with L I The connection.

[0153] In one embodiment, the bifunctional compound of formula X described herein has an S according to formula X-II. A-L : Formula (X-II) Where R L Indicates with L I The connection.

[0154] In one embodiment, the bifunctional compound described herein has T A-L It is a substituent derived from any of the following:

[0155] Where R L Indicates with L I The connection.

[0156] In one implementation, T A-L It has a structure according to formula XBI. In one implementation, T A-L It has a structure according to formula XB-II. In one embodiment, T A-L It has a structure according to formula XB-III. In one embodiment, T A-L It has a structure according to formula XCI. In one embodiment, T A-L It has the structure according to formula XDI. In one embodiment, T A-L It has a structure according to formula XD-II. In one embodiment, T A-L It has a structure according to formula XD-III. In one embodiment, T A-L It has a structure according to formula XD-IV.

[0157] In one implementation, the L of formula X I According to any embodiment of the connector as defined in the "Connectors" section. For example, in some embodiments, L of formula X I According to equation (II), where L 1 L 2 Z and X are each defined as described in any of the embodiments in the “Connectors” section of this document. In one embodiment, L of formula X IAccording to any of the structures II-1 to II-98 shown in Table Z-II in the "Connectors" section. Connectors as shown in Table Z-II (e.g., structures II-1 to II-98) can be marked with... Any connection point with S A-L and T A-L Connection. For example, in one embodiment, the connector shown in Table Z-II is indicated by the label appearing on the left side of the formula shown in Table Z-II. The connection point with T A-L Connect, and through the formula shown on the right side of Table Z-II marked with The connection point with S A-L connect.

[0158] In one embodiment, the bifunctional compound is according to any one of formula X-III: Formula X-III, Or its pharmaceutically acceptable salt; wherein: L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

[0159] In one embodiment, the bifunctional compound is according to any one of the following formulas: X-III-B1 to X-III-D4.

[0160] In one embodiment, the bifunctional compound is according to formula X-III-B1. In one embodiment, the bifunctional compound is according to formula X-III-B2. In one embodiment, the bifunctional compound is according to formula X-III-B3. In one embodiment, the bifunctional compound is according to formula X-III-C1. In one embodiment, the bifunctional compound is according to formula X-III-D1. In one embodiment, the bifunctional compound is according to formula X-III-D2. In one embodiment, the bifunctional compound is according to formula X-III-D3. In one embodiment, the bifunctional compound is according to formula X-III-D4.

[0161] In one embodiment, this disclosure provides a bifunctional compound according to any one of formulas X-III-B1, X-III-B2, X-III-B3, X-III-C1, X-III-D1, X-III-D2, X-III-D3, and X-III-D4, wherein the connector (L I ) is selected from any of the formulas II-1 to II-98 shown in Table Z-II.

[0162] In one embodiment, the bifunctional compound is according to any one of formula X-IV: Formula (X-IV) Or its pharmaceutically acceptable salt; wherein: L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

[0163] In one embodiment, the bifunctional compound is according to any one of the following formulas: X-IV-B1 to X-IV-D4.

[0164] In one embodiment, the bifunctional compound is according to formula X-IV-B1. In one embodiment, the bifunctional compound is according to formula X-IV-B2. In one embodiment, the bifunctional compound is according to formula X-IV-B3. In one embodiment, the bifunctional compound is according to formula X-IV-C1. In one embodiment, the bifunctional compound is according to formula X-IV-D1. In one embodiment, the bifunctional compound is according to formula X-IV-D2. In one embodiment, the bifunctional compound is according to formula X-IV-D3. In one embodiment, the bifunctional compound is according to formula X-IV-D4.

[0165] In one embodiment, this disclosure provides a bifunctional compound according to any one of formulas X-IV-B1, X-IV-B2, X-IV-B3, X-IV-C1, X-IV-D1, X-IV-D2, X-IV-D3, and X-IV-D4, wherein the connector (L I ) is selected from any of the formulas II-1 to II-98 shown in Table Z-II.

[0166] In one embodiment, the bifunctional compound is according to any one of formula XV: Formula (XV) Or its pharmaceutically acceptable salt; wherein: L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

[0167] In one embodiment, the bifunctional compound is according to any one of the following formulas: XV-B1 to XV-D4:

[0168] In one embodiment, the bifunctional compound is according to formula XV-B1. In one embodiment, the bifunctional compound is according to formula XV-B2. In one embodiment, the bifunctional compound is according to formula XV-B3. In one embodiment, the bifunctional compound is according to formula XV-C1. In one embodiment, the bifunctional compound is according to formula XV-D1. In one embodiment, the bifunctional compound is according to formula XV-D2. In one embodiment, the bifunctional compound is according to formula XV-D3. In one embodiment, the bifunctional compound is according to formula XV-D4.

[0169] In one embodiment, this disclosure provides a bifunctional compound according to any one of formulas XV-B1, XV-B2, XV-B3, XV-C1, XV-D1, XV-D2, XV-D3, and XV-D4, wherein the connector (L I ) is selected from any of the formulas II-1 to II-98 shown in Table Z-II.

[0170] In one embodiment, the bifunctional compound is any one of compounds X-001 to X-098 as described in Table X, or a pharmaceutically acceptable salt thereof.

[0171] Table X

[0172] As illustrated herein, when any structure in Table X contains a stereocenter with an unspecified stereochemistry, the center may have any R or S configuration according to the Cahn-Ingold-Prelog rule. For example, in one embodiment, the stereocenter has an R configuration. In another embodiment, the stereocenter has an S configuration.

[0173] In one embodiment, the bifunctional compound is capable of forming a ternary complex with the sorting protein and IL-17A. The formation of the ternary complex can be measured using various methods known in the art. For example, the formation of the ternary complex can be measured by Förster resonance energy transfer (FRET) assay or by time-resolved Förster resonance energy transfer (TR-FRET) assay. As measured by these assays, an increase in the homogeneous time-resolved fluorescence (HTRF) ratio indicates the formation of the ternary complex.

[0174] In one implementation, the bifunctional compound is capable of binding both sorting proteins and IL-17A simultaneously.

[0175] In one implementation, the bifunctional compound is able to bind to sorting proteins on the cell surface.

[0176] In one embodiment, the bifunctional compound is able to induce IL-17A internalization into cells expressing sorting proteins.

[0177] In one implementation scheme, in S A-L Binding to sorting proteins located on the cell surface and T A-L Upon binding to IL-17A, the target protein is internalized into the cells. Cellular uptake can be determined by a cellular uptake assay, in which IL-17A can be detected using a tag (e.g., a fluorescent tag or a tag capable of producing or reacting to emit light). The tagged IL-17A can then be detected using methods known to those skilled in the art (e.g., measurement in cell lysates, flow cytometry, imaging techniques, etc.). Removal of IL-17A from the extracellular medium can also be used as a method for cellular uptake assays.

[0178] In one implementation, IL-17A is degraded after being internalized into the cell.

[0179] In one embodiment, IL-17A is degraded in lysosomes. Examples demonstrate that the bifunctional compound induces the degradation of IL-17A in lysosomes by using a control employing inhibition of lysosomal proteases.

[0180] Binding to the target protein or sorting protein can be measured using various methods known to those skilled in the art. For example, micro-thermophoresis (MST).

[0181] In one implementation, S A-L Dissociation constant (K) of the sorting protein D The T value is less than 50 µM (e.g., less than 2 µM, e.g., less than 0.5 µM, preferably less than 0.1 µM), and T A-L The dissociation constant of its binding to its target is less than 100 µM (e.g., less than 0.5 µM, e.g., less than 0.1 µM).

[0182] In one implementation, T A-L The dissociation constant (K) of the IL 17-A binding D Below 500 nM (e.g., below 250 nM, below 100 nM, below 50 nM).

[0183] This invention demonstrates several bifunctional compounds capable of binding IL-17A. As shown in the examples, these compounds exhibit an affinity for IL-17A. Those skilled in the art will understand that compounds containing T... A-L and some or all of L I The bifunctional compound fragment will also act as an IL-17A binder. Therefore, in one embodiment, this disclosure provides a T-17A-based compound as described above. A-L -L I Any combination or fragment thereof of the IL-17A binder described.

[0184] In one embodiment, this disclosure provides a compound selected from any of the compounds shown in Table B, or a pharmaceutically acceptable salt thereof: Table B

[0185] Medical Use

[0186] This invention focuses on mediating the degradation of circulating extracellular proteins in diseases such as those involving immunity, inflammation, hematopoietic / blood disorders (including those caused or exacerbated by angiogenesis), and abnormal cell proliferation (such as tumors and cancer).

[0187] The bifunctional compounds of the present invention can be administered in any manner that allows the bifunctional compounds to bind to extracellular proteins (typically in the bloodstream) and be delivered to cells with sorting proteins for internalization and degradation. Therefore, examples of methods for delivering the degrading agents of the present invention include, but are not limited to, oral, intravenous, buccal, sublingual, subcutaneous, and nasal administration.

[0188] In one aspect, the present invention provides bifunctional compounds for targeting the lysosomal degradation of extracellular target molecules mediated by sorting proteins. In one embodiment, the target molecule is a disease or symptom-related protein.

[0189] On the other hand, this disclosure provides the use of the bifunctional compounds described herein as pharmaceuticals. This disclosure is intended to provide pharmaceutical compositions comprising the bifunctional compounds described herein.

[0190] In another aspect, this disclosure relates to a method for targeting the lysosomal degradation of extracellular target proteins, comprising administering the bifunctional compound described herein to a subject in need.

[0191] In another aspect, this disclosure relates to a method for reducing plasma levels of a target molecule, comprising administering the bifunctional compound described herein to a subject in need.

[0192] In one aspect, this disclosure provides the bifunctional compounds described herein for the treatment of conditions or illnesses mediated by extracellular proteins in subjects of need.

[0193] In one aspect, this disclosure relates to the bifunctional compounds described herein for the removal of extracellular target molecules from the plasma of a subject.

[0194] In one embodiment, the condition or disease is associated with abnormal levels of extracellular proteins. In another embodiment, the condition or disease is associated with abnormally high levels of extracellular target proteins.

[0195] In one implementation, the condition or disease is associated with a mutated or misfolded extracellular protein.

[0196] In one embodiment, the extracellular target protein is selected from: TNF-α, PCSK9, ANGPTL-3, antibody light chain, IgG, IgE, IgA, IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-β (TGF-β), and platelet-reactive protein-1. (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

[0197] In one embodiment, the extracellular protein is TNF-α. In a further embodiment, the extracellular target protein is TNF-α, and the condition or illness is selected from: rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, hidradenitis suppurativa, refractory asthma, systemic lupus erythematosus, diabetes, and induced cachexia. In a further embodiment, the condition is associated with abnormal TNF-α levels. In one embodiment, the condition or illness is an inflammatory disease. In one embodiment, the condition or illness is an autoimmune disease. In one embodiment, the condition or illness is cancer.

[0198] Therefore, in one aspect, this disclosure provides a method for targeted lysosomal degradation of TNFα, comprising administering an effective amount of the bifunctional compound described herein.

[0199] In another aspect, this disclosure provides a method for removing TNFα from the plasma of a patient or subject in need, comprising administering the bifunctional compound described herein.

[0200] On the other hand, this disclosure provides the use of the bifunctional compounds described herein in the preparation of medicaments for treating diseases or conditions.

[0201] On the other hand, this disclosure provides the use of the bifunctional compounds described herein in the preparation of medicaments for treating TNFα-mediated conditions or illnesses.

[0202] In one aspect, this disclosure provides a method for treating a disease or condition, comprising administering the bifunctional compound described herein to a subject in need.

[0203] In one aspect, this disclosure provides a method for treating a TNFα-mediated condition or disease, comprising administering a bifunctional compound according to this disclosure to a subject in need.

[0204] The embodiments show that, in the T described herein A-L Bifunctional compounds that bind to IL-17-A can form a ternary complex with IL-17-A and sorting proteins, inducing cellular uptake of IL-17A and its degradation in lysosomes.

[0205] Therefore, in one aspect, this disclosure provides a method for removing IL-17A from the plasma of a subject in need using a bifunctional compound of formula (X) described herein, the method comprising administering an effective amount of the bifunctional compound to the subject.

[0206] In one embodiment, the method for removing IL-17A includes forming a ternary complex with a bifunctional compound, a sorting protein, and IL-17A. In another embodiment, the method for removing IL-17A includes simultaneously binding the sorting protein and IL-17A to the bifunctional compound.

[0207] In one implementation, the method for removing IL-17A includes binding a bifunctional compound to the sorting protein on the cell surface of cells expressing the sorting protein.

[0208] In one implementation scheme, in S A-L Binding to sorting proteins located on the cell surface and T A-L Upon binding to IL-17A, the target protein is internalized into the cell. In one embodiment, IL-17A is degraded after internalization into the cell. In another embodiment, IL-17A is degraded in lysosomes.

[0209] High levels of IL-17A are known to be associated with a variety of chronic inflammatory diseases or conditions.

[0210] Therefore, in one aspect, this disclosure provides a bifunctional compound of formula (X) described herein or a pharmaceutically acceptable salt thereof for the treatment or prevention of inflammation or inflammatory conditions.

[0211] In one aspect, this disclosure provides a method for treating or preventing inflammation or inflammatory conditions using a bifunctional compound of formula (X) described herein or a pharmaceutically acceptable salt thereof, said method comprising administering an effective amount of the bifunctional compound to a subject.

[0212] This disclosure relates to a method for treating or preventing inflammation or inflammatory conditions, the method comprising administering to a subject an effective amount of a bifunctional compound of formula (X) or a pharmaceutically acceptable salt thereof.

[0213] In one aspect, the present invention relates to the use of the bifunctional compound described herein in the preparation of a medicament for treating or preventing inflammation or inflammatory conditions, the method comprising administering to a subject an effective amount of the bifunctional compound of formula (X) or a pharmaceutically acceptable salt thereof.

[0214] In one embodiment, the inflammation or inflammatory condition is characterized by high levels of IL-17A.

[0215] Therefore, in one aspect, this disclosure provides a method for treating or preventing a condition characterized by IL-17A overexpression using a bifunctional compound of formula (X) described herein or a pharmaceutically acceptable salt thereof, said method comprising administering an effective amount of the bifunctional compound to a subject.

[0216] Therefore, in one aspect, this disclosure provides a bifunctional compound of formula (X) described herein or a pharmaceutically acceptable salt thereof for the treatment or prevention of diseases selected from: psoriasis, rheumatoid arthritis, spondyloarthritis, multiple sclerosis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, hidradenitis suppurativa, systemic lupus erythematosus, palmoplantar pustulosis (PPP), and atopic dermatitis.

[0217] In one aspect, this disclosure provides a method for treating or preventing psoriasis, rheumatoid arthritis, spondyloarthritis, multiple sclerosis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, hidradenitis suppurativa, systemic lupus erythematosus, palmoplantar pustulosis (PPP), and atopic dermatitis; said method comprising administering an effective amount of the bifunctional compound to a subject.

[0218] A method for treating or preventing psoriasis, rheumatoid arthritis, spondyloarthritis, multiple sclerosis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, hidradenitis suppurativa, systemic lupus erythematosus, palmoplantar pustulosis (PPP), and atopic dermatitis; said method comprising administering to a subject an effective amount of the bifunctional compound of formula (X) described herein.

[0219] The use of the bifunctional compound of formula (X) described herein in the preparation of medicaments for the treatment or prevention of psoriasis, rheumatoid arthritis, spondyloarthritis, multiple sclerosis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, hidradenitis suppurativa, systemic lupus erythematosus, palmoplantar pustulosis (PPP), and atopic dermatitis.

[0220] In one embodiment, the subject is a mammal. In another embodiment, the mammal is a human.

[0221] Preparation of bifunctional compounds

[0222] Generally, the compounds of formula (I) or formula (X) and related formulas of this invention can be prepared from readily available starting materials. If such starting materials are not commercially available, preparation can be carried out using standard synthetic techniques. Typically, the synthetic route for any individual compound of formula (I) and related formulas will depend on the specific substituents of each molecule, factors that should be understood by those skilled in the art. The following general methods and procedures described in the examples below can be used to prepare compounds of formula (I) and related formulas. The reaction conditions (e.g., temperature, solvent, or co-reagent) depicted in the following schemes are given by way of example only and are not limiting. It should be understood that, given typical or preferred experimental conditions (i.e., reaction temperature, time, molar amounts of reagents, solvent, etc.), other experimental conditions may be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using conventional optimization procedures. For all methods of protection and deprotection, please refer to Philip J. Kocienski, “Protecting Groups”, Georg Thieme Verlag Stuttgart, New York, 1994, and Theodora W. Greene and Peter GM Wuts, “Protective Groups in Organic Synthesis”, Wiley Interscience, 3rd edition, 1999.

[0223] According to T L (or T) A-L L I and S L (or S) A-L Depending on the properties of (I), different synthetic strategies can be chosen to synthesize compounds of formula (I). Unless otherwise stated, in the methods shown in the following schemes, T L L I and S L As defined above in the instruction manual.

[0224] Compound of formula (I) (where T) L L I and S L (As defined above) methods and reactions known to those skilled in the art can be employed, by means comprising T L – L I Some compounds and S L Prepared by reaction, or from a substance containing L I – S L Some compounds and T L The reaction is used to prepare the product. Such reactions may include, but are not limited to: amide bond formation, ether bond formation, aromatic substitution, alkylation, metal-catalyzed cross-coupling reactions, and click chemistry, using conditions known to those skilled in the art and described in the following example (Scheme 1).

[0225] Option 1

[0226] As those skilled in the art will understand, T L precursor (pT) L ) or S L precursor (pS) L ) can first be with L I or L I precursor (pL) I Coupling. Subsequently, different reaction steps can be used to complete T. L S L or L I Partial. Such reactions may include, but are not limited to: amide bond formation, ether bond formation, aromatic substitution, alkylation, metal-catalyzed cross-coupling reactions, and click chemistry, using conditions known to those skilled in the art and described in the following example (Scheme 1). Suitable protecting groups may be used to avoid functional group incompatibility. For example, preferred protecting groups are: benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ) group, tert-butoxycarbonyl (BOC) group, 9-fluorenylmethoxycarbonyl (FMOC) group, alkyl acyl group (e.g., acetyl (Ac) group), benzoyl (Bz) group, benzyl (Bn) group, carbamate group, p-methoxybenzyl (PMB), 4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP) group, arylsulfonyl (e.g., toluenesulfonyl (Ts) or benzenesulfonyl group).

[0227] For example, illustrative methods for preparing bifunctional compounds include the following: Option 2:

[0228] Option 3:

[0229] As used in Schemes 2 and 3, “→…→…→” indicates a series of synthetic steps that transform the precursor through any suitable chemical reaction or combination of reactions. For example, any number of the above reactions or combinations thereof can be used.

[0230] In schemes 2 and 3, pT L pS L and pL I They refer to T respectively L S L or L I The precursor. T L S L or L I The precursor refers to the substance that can be converted into T using the common reactions described in this article. L S L or L I . group.

[0231] Compound (X) can be prepared in a similar manner as described above and as shown in schemes 1, 2 and 3, using appropriate Tx. A-L and S A-L (or pT) A-L and pS A-L ) group replaces T L and S L (or pT) L and pS L ) group. For example, when T A-L When applying formula CI, those skilled in the art will find information about T in WO 2020 / 146194. A-L This provides guidance on the appropriate synthesis of intermediates and precursors, as well as suitable reaction conditions. Modifications are made using the commonly used reagents and reactions described above to align with L... I ,pL I S L -L I pS L -pL I pS L -L I or S L -pL I Connections are known to those skilled in the art.

[0232] When T A-L When applying formulas BI, B-II, B-III, DI, D-II, D-III, or D-IV, those skilled in the art will find information regarding T in WO2020 / 127685 and WO 2021 / 250194. LGuided by the appropriate synthesis of intermediates and precursors. Modifications are made using the commonly used reagents and reactions described above to align with L. I ,pL I S L -L I pS L -pL I pS L -L I or S L -pL I Connections are known to those skilled in the art.

[0233] compound

[0234] In another respect, this disclosure provides a compound of formula (A-II) or a pharmaceutically acceptable salt thereof, or an enantiomer thereof, or a mixture thereof: Formula (A-II) in: R 1 It is H, halogen, alkoxy, -CF3, or optionally substituted C. 1-5 A hydrocarbon chain, wherein one or more carbon groups of the C1-C5 hydrocarbon chain are optionally and independently replaced by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea, , , Optionally substituted carbocyclic rings; Optionally substituted heterocyclic rings, or ; where X is NH or O.

[0235] In one embodiment, the compound is according to formula (A-IIa): Or, or a pharmaceutically acceptable salt thereof.

[0236] In one embodiment, the compound is according to formula (A-IIb): Or, or a pharmaceutically acceptable salt thereof.

[0237] In one embodiment, the compound is according to formula (A-II-1): Or, or a pharmaceutically acceptable salt thereof.

[0238] In one implementation, R 1 It is H. In one implementation, R 1 Selected from halogens or -CF3.

[0239] In one implementation, R 1 It is an optionally substituted C1-C5 alkyl group, wherein C 1-5One or more methylene groups of the alkyl group may optionally be independently replaced by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, and .

[0240] In one embodiment, the compound is any of the compounds in Table C, or a pharmaceutically acceptable salt thereof.

[0241] Table C

[0242] In one embodiment, the compound is O-(tert-butyl)-N-(6-phenoxynicotinyl)serine (B-001). An alternative name for the compound is O-(tert-butyl)-N-(6-phenoxynicotinyl)-L-serine.

[0243] In one embodiment, the compound is (2S)-3-tert-butoxy-2-[[6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl]amino]propionic acid (B-002). An alternative name for the compound is O-(tert-butyl)-N-(6-(3-(2-methoxyethoxy)phenoxy)nicotinyl)-L-serine.

[0244] The compounds of formula (A-II) or (VI-a) described herein can be prepared from commercially available starting materials by a reaction according to the following scheme (using compound C-001 as an example):

[0245] First, in step 1, an alkyl ester of 2-amino-3-tert-butoxy-propionic acid (e.g., compound 2, O-(tert-butyl)serine methyl ester) is reacted with 6-(3-tert-butoxycarbonylphenoxy)pyridine-3-carboxylic acid (compound 1) to form an amide (compound 3). Then, in step 2, the ester is deprotected to obtain a sorting protein binding agent compound 4.

[0246] As those skilled in the art will know, different reaction conditions can be used to form the amide in compound 3. For example, in an organic solvent (i.e., DMF, DMSO), in the presence of a tertiary amine (such as triethylamine, N,N-diisopropylethylamine (DIPEA or DIEA)), using a coupling agent (such as... N,N' Common conditions for peptide bond formation using dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), or aziridinetriazole tetramethylurea hexafluorophosphate (HATU) are used.

[0247] In step 1, other suitable esters of 2-amino-3-tert-butoxy-propionic acid may be used, such as any C1-C of 2-amino-3-tert-butoxy-propionic acid.10 Alkyl esters.

[0248] In step 2, deprotection of the compound can be carried out by hydrolysis of the ester under acidic or basic conditions. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using conventional optimization procedures. For all protection and deprotection methods, see Philip J. Kocienski, “Protecting Groups,” Georg Thieme Verlag Stuttgart, New York, 1994, and Theodora W. Greene and Peter GM Wuts, “Protective Groups in Organic Synthesis,” Wiley Interscience, 3rd edition, 1999. In one embodiment, deprotection is carried out under aqueous conditions or in a mixture of an organic solvent and an aqueous medium using an alkali metal hydroxide (such as LiOH).

[0249] Compound 4 contains a stereocenter. Compound 4 can be produced as an enantiomer with a high enantiomeric excess by using optically pure or optically enriched starting materials (e.g., an enantiomeric pure alkyl ester of 2-amino-3-tert-butoxy-propionic acid).

[0250] For example, in one embodiment, the compound is O-(tert-butyl)-N-(6-phenoxynicotinyl)-L-serine, and the starting material used is O-(tert-butyl)-L-serine.

[0251] In one aspect, this disclosure provides a compound of formula (A-II) or a pharmaceutically acceptable salt thereof, or an enantiomer thereof or a mixture thereof, for use as a medicament.

[0252] Compounds of formula (A-II) bind to sorting proteins with high affinity, thus showing potential in the treatment of sorting protein-mediated conditions. Furthermore, formula (A-II) has been shown to possess improved drug-like properties, such as reduced lipophilicity and liver microsome clearance. These properties indicate a surprisingly low first-pass metabolism, which is beneficial for oral administration.

[0253] In one aspect, this disclosure provides a composition comprising a compound of formula (A-II) described herein, a pharmaceutically acceptable salt thereof, an enantiomer thereof, or a mixture thereof.

[0254] This invention demonstrates several bifunctional compounds capable of binding to sorting proteins. As shown in the examples, these compounds exhibit affinity for sorting proteins. As those skilled in the art will understand, compounds containing S... L and some or all of L I The bifunctional compound fragment will also act as a sorting agent for protein binders. Therefore, in one embodiment, this disclosure provides a fragment of S as described herein. L -L I Any combination or fragment thereof described as a sorting protein binder.

[0255] Furthermore, this disclosure provides a compound capable of binding to sorting proteins, wherein the compound is an intermediate for preparing the bifunctional compound described in the “Synthetic Scheme” section of this document.

[0256] Example

[0257] Example 1: Binding of small molecule compounds to sorting proteins

[0258] Purpose

[0259] In this embodiment, the equilibrium dissociation constant (Ki) describing the binary binding between the small molecule compound and the soluble extracellular domain of sorting protein-6his was measured using microthermophoresis (MST). D ).

[0260] Materials and Methods

[0261] Using the LV Mosquito pipetting robot (SPT Labtech), 12-point titration series of compound solutions were prepared in a total volume of 1.2 µL in a TTP LVDS 384-well plate (SPT Labtech) in a buffer containing 16.7% DMSO (v / v) and 0.05% Tween 20 (v / v).

[0262] Before adding 8.8 µL of 57 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris Propane) pH 9, 57 mM NaCl, and 0.05% Tween 20 (v / v) containing 114 nM sorting protein-6his and 28.4 nM RED-tris-NTA (Nanotemper) to each well, the plate was centrifuged. The plate was sealed with an adhesive strip and incubated at 600 rpm for 3 min on an Eppendorf thermostatic mixer, followed by centrifugation for 15 sec on a Fisherbrand plate centrifuge and further incubation at room temperature (RT) for 1 h. MST was measured using a Monolith NT. Automated (Nanotemper) in standard-treated capillaries at 5% LED power and high MST power. Dose-response curves were extracted at 20-second hot-time. All experiments were performed with duplicate replication.

[0263] result

[0264] The results are shown in Table 1.

[0265] Table 1: Kd values ​​(nM) of compounds binding to sorting proteins

[0266] Table 1a: Kd values ​​of compounds binding to sorting proteins. D <0.2 nM: K D >0.2 nM:

[0267] The sorting protein binder C-002 was studied and compared with its analogue C-002carb, as shown below. The two differ only in a single atom: (C-002) (C-002carb) Table 1b: KB of the binding of sorting proteins to compound C-002 and one of its atomic substitution analogues D

[0268] in conclusion

[0269] The compounds of this invention bind to sorting proteins.

[0270] Example 2: Bifunctional compounds mediate the internalization of extracellular target proteins into cells

[0271] Purpose

[0272] This embodiment explores the sorting of proteins through S... L Bifunctional compounds derived from small molecules have the ability to promote the internalization of extracellular targets.

[0273] Materials and Methods

[0274] Cellular uptake of neutral avidin-650 (NA650) was investigated in HEK293 cells expressing the full-length sorting protein receptor (Petersen et al.). Cells were seeded in poly-L-lysine-coated 96-well plates (Perkin Elmer) (40K / well) containing 50 μL of medium per well (DMEM (Lonza) + 10% FBS (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco) + 100 μg / mL Zeocin (Invitrogen)) and incubated overnight in a cell culture incubator (37 °C, humidified, 5% CO2).

[0275] The culture medium was replaced with assay medium containing NA650 (Invitrogen) (100 nM) (Thermo Fischer) and the bifunctional compound of this invention (9.8 nM to 10 μM) (DMEM (Lonza) + 10% fetal bovine serum (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco)). Cells were incubated in a cell culture incubator for 3 hours, and then washed in dPBS (Bionordika). The DyLight650 signal in the cell layer was detected using a fluorescence microplate reader (BMG Labtech Clariostar).

[0276] The internalization of fluorescently labeled anti-AlexaFluor488-anti-DNP antibody (ThermoFisher) was evaluated using a similar experimental protocol.

[0277] For the assessment of TNF-α cellular uptake, a similar approach was used in HEK293 cells, detecting mouse anti-TNFα (Invitrogen, MA5-23720) by Western blot. Control cells were incubated either without the compound and with TNFα or with only TNFα.

[0278] The efficacy of internalization of bifunctional compounds is evaluated by the area under the curve of the signal response.

[0279] result

[0280] The results are shown in Table 2.

[0281] Table 2: AUC of target protein internalization. Target internalization AUC score: Neutravidin (AUC score - AUC < 100: +; AUC > 100: ++). Anti-DNP (AUC score - AUC < 10000: +; AUC > 10000: ++). TNFα (AUC > 25000: ++++; AUC 10000-25000: +++; AUC 1000-10000: ++; AUC < 1000: +)

[0282] in conclusion

[0283] The bifunctional compound of this invention mediates the cellular uptake of target proteins.

[0284] Example 3: Binding of small molecule compounds with IL-17A

[0285] Purpose

[0286] In this embodiment, the equilibrium dissociation constant (Ki) describing the binary binding between the small molecule compound and IL-17A was measured using microthermophoresis (MST). D ).

[0287] Materials and Methods

[0288] Using a 16-channel multichannel pipette, a 12-point titration series of compound solutions was prepared in 5 µL total volumes in a TTP LVDS 384-well plate (SPT Labtech) in a buffer consisting of 20 mM Bis-Tris Propane (1,3-bis(tris(hydroxymethyl)methylamino)propane) pH 9.5, 300 mM NaCl, 0.1% Tween 20 (v / v), and 0.1% protease-free BSA. The plate was spun down, and then 5 μL of 40 nM IL17A-NHS dissolved in 20 mM Bis-Tris Propane pH 9.5, 300 mM NaCl, 0.1% T20, and 0.1% protease-free BSA was added. The plates were sealed with adhesive strips and vortexed at 600 rpm for 3 minutes on an Eppendorf thermostat, followed by centrifugation for 15 seconds on a Fisherbrand plate centrifuge and further incubation at RT for 1 hour. MST was measured using a Monolith NT. Automated (Nanotemper) in standard-treated capillaries at 20% LED power and medium MST power. Dose-response curves were extracted at 20-second on-time. All experiments were repeated twice.

[0289] Table 3: K+ of compounds binding to IL-17A D Value. K D < 50 nM: K D 50-250 nM: K D > 250 nM:

[0290] in conclusion

[0291] The compound of this invention binds to IL-17A.

[0292] Example 4: S L Bifunctional compounds derived from small molecules promote the formation of ternary complexes.

[0293] Purpose

[0294] The aim is to demonstrate that bifunctional compounds can form ternary complexes with sorting proteins and IL-17A.

[0295] Materials and Methods

[0296] In these experiments, bifunctional compounds were titrated 12 times in 384-well TTP LVDS plates (SPT Labtech) with a total volume of 5 µL using a 16-channel multichannel pipette. The final buffer composition was 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, 0.1% protease-free BSA (w / v), and 10% DMSO.

[0297] Add the following substances to a black 384-well plate (Corning): 4 µL of 10 nM anti-6HIS Tb monoclonal antibody (mAb Anti-6HIS Tb) (Cisbio) dissolved in 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1% proteinase-free BSA (w / v); 4 µL of 250 µL of streptavidin-d2 (Cisbio) dissolved in 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1% proteinase-free BSA (w / v); 4 µL of 750 nM IL17A-biotin dissolved in 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1% proteinase-free BSA (w / v); 4 µL of 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1% proteinase-free BSA (w / v); 250 nM sorting protein-6his (Sortilin-6his) was prepared in HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, and 0.1% proteinase-free BSA (w / v); and 4 µL of a bifunctional compound was prepared in 50 mM HEPES pH 7.5, 1 mM EDTA, 300 mM NaCl, 0.1% proteinase-free BSA (w / v), and 10% DMSO. After incubation at room temperature for 16 hours, the HTRF signal ratio at 665 / 620 nm was measured on a ClarioStar microplate reader (BMG Labtech). All reactions were run twice.

[0298] Figure 1 The HTRF (665 / 620 nm) signals (Gaussian fit curves) are shown, plotted relative to the lowest local signal after baseline correction for compound concentrations (40 µM to 9.5 pM). Bell-shaped response curves were observed for the formation of ternary complexes between the bifunctional compound, the sorting protein, and IL-17A, as seen with X-017 (…). Figure 1 A) and X-018 ( Figure 1 As shown in B).

[0299] Table 4: Formation of ternary complexes calculated using AUC of HTRF

[0300] in conclusion

[0301] The bifunctional compound of the present invention mediates the formation of a ternary complex between the sorting protein and IL-17A.

[0302] Example 5: Bifunctional compound mediates the internalization of extracellular IL-17A in cells

[0303] Purpose

[0304] This embodiment aims to explore the ability of sorting proteins to promote the internalization of extracellular IL-17A through bifunctional compounds.

[0305] Materials and Methods

[0306] Cellular uptake of IL-17A was investigated in HEK293 cells expressing the sorting protein receptor (Petersen et al.). Cells were seeded in poly-L-lysine-coated 96-well plates (Perkin Elmer) (40K / well) containing 50 µL of medium per well (DMEM (Lonza) + 10% FBS (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco) + 100 µg / mL Zeocin (Invitrogen)) and incubated overnight in a cell culture incubator (37 °C, humidified, 5% CO2).

[0307] The culture medium was replaced with assay medium (DMEM (Lonza) + 10% fetal bovine serum (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco)), which contained a bifunctional compound (2 nM to 2 μM) and leupeptin (80 μM) (Sigma), as well as conditioned medium from cells transiently expressing IL-17A (Q16552.1) with a nanoluc tag at the N-terminus (nanoluc-IL-17A), produced using the ExpiCHO™ expression system (ThermoFisher). Cells were incubated in a cell culture incubator for 24 hours, then washed with dPBS (Bionordika) and lysed in Passive Lysis Buffer (Promega) Nanoluc substrate from a substrate buffer (Nano-Glo luciferase assay system (Promega)) was added to cell lysates, and the luminescence signal was evaluated (BMG Labtech Clariostar).

[0308] Figure 2 The luminescence signal of cell lysates (expressed as %RLU, with Gaussian fitted curves) is shown after HEK293 / sorting protein cells were co-incubated with a series of concentrations (2 nM to 2 µM) of compounds X-062, X-064 and X-065 and nanoluc-IL-17A for 24 hours.

[0309] Table 5: Target internalization calculated as the area under the peak-2-peak (P2P) curve (AUC) of % RLU. AUC > 500: AUC 100-500: AUC < 100:

[0310] in conclusion

[0311] The compounds of this invention mediate the internalization of IL-17A in cells.

[0312] Example 6: Cellular uptake of extracellular targets was inhibited by competitive binding.

[0313] In another experiment, cells were co-incubated for 24 hours with nanoluc-IL-17A and a bifunctional compound (100 nM) (as described in Example 4) and a series of dilutions (2 nM to 2 µM) of a monofunctional IL-17A binder as a IL-17A binding competitor.

[0314] Figure 3 The luminescent signals of cell lysates after 24 hours of co-incubation of HEK293 / sorting protein with nanoluc-IL-17A, 100 nM X-065, and a series of concentrations (2 nM to 2 µM) of the monofunctional IL-17A binders M005, M008, and M009 are shown.

[0315] in conclusion

[0316] The IL-17A internalization mediated by the bifunctional compound of this invention can be inhibited by the addition of a monofunctional target binder.

[0317] Example 7: Bifunctional compound-mediated depletion of target from extracellular cell culture medium

[0318] Purpose

[0319] This embodiment aims to investigate the ability of bifunctional compounds to deplete IL-17A from the extracellular space. The study was conducted by evaluating the presence of IL-17A in the supernatant of cell cultures after co-incubation of cells with the bifunctional molecule and IL-17A.

[0320] Materials and Methods

[0321] HEK293 / sorting protein cells (40,000 cells / 96-well plate) were seeded in culture medium and incubated for 24 hours (as described in Example 4). The culture medium was then replaced with assay medium supplemented with purified recombinant IL-17A and a series of dilutions (0.3 nM to 2 µM) of the bifunctional compound (as described in Example 4). After incubation for 48 hours, residual IL-17A in the cell culture supernatant was assessed using the hIL-17A ELISA (BioLegend) according to the manufacturer's protocol.

[0322] Figure 4 Gaussian curves showing the IL-17A clearance rate (%) of HEK293 / sorting protein cell culture supernatant after co-incubation with IL-17A (0.1, 1.0, 10 or 100 nM) and X-017 dilution series for 48 hours are displayed.

[0323] in conclusion

[0324] The data provided in this embodiment demonstrate that bifunctional compounds can mediate the depletion of targets from cell culture supernatants.

[0325] Example 8: Degradation of internalized IL-17A in lysosomes

[0326] Purpose

[0327] This embodiment aims to investigate the ability of bifunctional molecules to induce the degradation of IL-17A in lysosomes after internalization, and to conduct the study by evaluating IL-17A in cell lysates after inhibiting lysosomal function.

[0328] Materials and Methods

[0329] Intracellular IL-17A levels after lysosomal inhibition were investigated in HEK293 / sorting protein cells. 40,000 (40K) cells were seeded in 96-well plates and incubated overnight as described in Example 4. The medium was then replaced with assay medium containing nanoluc-IL-17A (as described in Example 5), serial dilutions of the bifunctional compound (1.5 nM to 10 µM), and leucopeptide (80 μM) (Sigma Aldrich). Control cells were incubated for 24 hours without leucopeptide, and intracellular IL-17A was then assessed as described in Example 5.

[0330] Figure 5 Gaussian fitted curves of the luminescence signal in the harvested HEK293 / sorted protein lysate after 24 hours of co-incubation with nanoluc-IL-17A and X-062 with and without the leucopeptide peptide are shown. Cells incubated with the leucopeptide peptide showed a significantly increased luminescence signal compared to control cells.

[0331] in conclusion

[0332] The data provided in this example demonstrate that, compared to control cells, IL-17A accumulates intracellularly in cells co-incubated with the lysosomal cysteine ​​protease inhibitor leucopeptide. This indicates that internalization mediated by the bifunctional compound leads to lysosomal degradation of IL-17A.

[0333] Example 9: Protein binding

[0334] Purpose

[0335] This study investigates the protein binding of different bifunctional compounds.

[0336] Materials and Methods

[0337] The test compound or positive control warfarin was spiked into 10% plasma of CD1 mice. The test concentrations of the test compounds and warfarin were 2 µM. Spiked plasma samples were pre-incubated at 37°C and then centrifuged to precipitate plasma proteins, thereby separating them from the free compounds in the supernatant. The concentrations of the test compounds and warfarin in the spiked plasma and supernatant samples were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0338] 10% Plasma: On the day of the experiment, thaw the plasma under running cold tap water and centrifuge at 3220 ×g for 5 minutes to remove any clots. Check and record the pH value. Measure the pH value of the resulting plasma and adjust it to 7.4 ± 0.1 as needed using 1% phosphate or 1 M sodium hydroxide. Then add 1 mL of plasma to 9 mL of PBS as the test matrix.

[0339] result

[0340] The results are shown in Table 6.

[0341] Table 6. Protein binding fraction of different bifunctional binders

[0342] in conclusion

[0343] Adding side chains with polar groups to the linker can reduce protein binding.

[0344] Example 10: Study on lipophilicity and hepatic clearance

[0345] Purpose

[0346] This study investigates the effects of different sorting protein binders on the bifunctional compounds of this invention.

[0347] Materials and Methods

[0348] Microsome clearance: 1.0 μM of the test compound was incubated with liver microsomes (collected from multiple donors) at a concentration of 0.5 mg / mL microsomal protein in the presence of an NADPH regeneration system (approximately 1.0 mM) at 37°C. Reference controls included testosterone (substrate 3A4), propafenone (2D6), and diclofenac (2C9). These were co-incubated with microsomes in the presence of the NADPH regeneration system.

[0349] Time samples (0, 5, 15, 30, 45, and 60 minutes) were removed and immediately mixed with cold acetonitrile containing an internal standard (IS). This also included test compounds incubated with microparticles for 60 minutes without a NADPH regeneration system. Single-point experiments (n=1) were performed for each test condition. Samples were analyzed by LC-MS / MS; the disappearance of test compounds was assessed based on the analyte / IS peak area ratio.

[0350] The microsomal clearance rate was calculated using the following equation:

[0351] To calculate liver clearance, CL was used in the following equation. int(mic) :

[0352] The protein content of microsomal protein per g of liver weight is 45 mg / g.

[0353] Liver weight / body weight: 20 g / kg for humans and 88 g / kg for mice.

[0354] LogD: Log D was determined using a micro-1-octanol / buffered flask method, followed by LC / MS / MS analysis. Since logD is pH-dependent, the pH of the aqueous phase must be specified, typically measured at pH 7.4 (the physiological pH of body fluids).

[0355] A suitable test compound was dissolved in 100% DMSO to prepare a 10 mM solution. The test compound (10 mM DMSO solution; 2 µL / well) and the control sample (10 mM DMSO solution; 2 µL / well) were transferred from the storage tube to 96-well polypropylene cluster tubes. Buffer-saturated 1-octanol (149 µL / well) and 1-octanol-saturated buffer (149 µL / well) were then added to the wells. Each tube was placed sideways and vigorously mixed for 3 minutes, followed by shaking at 880 rpm for 1 hour at room temperature. The tubes were then centrifuged at 4000 rpm for 5 minutes. The buffer layer sample was diluted 20-fold using an internal standard (IS) solution, and the 1-octanol layer sample was diluted 200-fold. The samples were analyzed using a triple quadrupole mass spectrometer. Peak areas were corrected for by dilution factor and internal standard, and the result (Log D value) was calculated using the ratio of the corrected peak areas. The control sample was used to monitor the Log D determination process.

[0356] The Log D value for each compound was calculated using the following formula:

[0357] The following compounds were studied:

[0358] These compounds differ by only one atom in the sorting protein binders (O vs. C).

[0359] result

[0360] Table 7 shows the results for Log D, human and mouse liver clearance. It can be seen that the lipophilicity of X-098 is reduced by more than one log unit, and it exhibits lower clearance in liver microsomes. These properties suggest an unexpectedly reduced first-pass metabolism, which is beneficial for oral administration.

[0361] Table 7. Comparison of studies on lipophilicity and hepatic clearance

[0362] in conclusion

[0363] Modification of a single atom in sorting protein binders significantly improves the drug-like properties of bifunctional compounds.

[0364] Example S1: Synthesis Scheme

[0365] As used herein, the term "rac" in a chemical name immediately preceding the stereocenter designation indicates that the stereocenter can have any configuration (R or S) according to the Cahn-Ingold-Prelog rule. For example, "rac-(2S)-2-amino-3-tert-butoxypropionate" indicates that the reagent used contains both (2S)-2-amino-3-tert-butoxypropionate and (2R)-2-amino-3-tert-butoxypropionate.

[0366] A-001: (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy ...

[0367] 1. A general procedure for preparing tert-butyl benzoate of 3-[[5-[[rac-(1S)-1-(tert-butoxymethyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-2-pyridyl]oxy]benzoate:

[0368] At 0°C, HATU (2.41 g, 6.34 mmol, 1 eq) and DIEA (1.23 g, 9.51 mmol, 1.66 mL, 3 eq) were added to a DMF (15 mL) solution of 6-(3-tert-butoxycarbonylphenoxy)pyridine-3-carboxylic acid (1 g, 3.17 mmol, 1 eq) and methyl rac-(2S)-2-amino-3-tert-butoxypropionate (555.70 mg, 3.17 mmol, 1 eq). The resulting mixture was stirred at 15°C for 2 hours. The reaction mixture was filtered, and the resulting filtrate was purified by preparative HPLC (column: C18 20-40um 100 A 330 g; mobile phase: [water-ACN]; B%: 0%-82% @ 100 mL / min) to obtain tert-butyl benzoate (700 mg, 1.48 mmol, yield 46.71%), as a yellow oil.

[0369] data: LCMS (ESI + ): m / z 473.1 (M+H)+

[0370] 2. A general procedure for preparing 3-[[5-[[rac-(1S)-1-(tert-butoxymethyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-2-pyridyl]oxy]benzoic acid:

[0371] TFA (3.07 g, 26.92 mmol, 2 mL, 18.18 eq) was added to a solution of tert-butyl benzoate (700 mg, 1.48 mmol, 1 eq) in DCM (8 mL). The resulting mixture was stirred at 0 °C for 6 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent and purified by preparative HPLC (column: C18 20-40 μm 100 A 120 g; mobile phase: [water-ACN]; B%: 0%-45% @ 80 mL / min) to give 3-[[5-[[rac-(1S)-1-(tert-butoxymethyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-2-pyridyl]oxy]benzoic acid (197 mg, 473.08 μmol, yield 31.93%) as a white solid.

[0372] data: LCMS (ESI + ): m / z 417.3 (M+H) +

[0373] 3. General procedure for the preparation of methyl rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy ... At 0°C, DIEA (74.49 mg, 576.34 mg) was added to a DMF (5 mL) solution of 3-[[5-[[rac-(1S)-1-(tert-butoxymethyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-2-pyridyl]oxy]benzoic acid (80 mg, 192.11 μmol, 1 eq) and N-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]pentanamide (144.63 mg, 192.11 μmol, 1 eq, TFA). The mixture was prepared by stirring at 15°C for 1 hour. The initial concentrations were 100.39 μL (146.09 mg, 100.39 μL, 3 eq) and HATU (146.09 mg, 384.22 μmol, 2 eq). The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: C18 20-40 μm 100 A 80 g; mobile phase: [water-ACN]; B%: 0%-48% @ 50 mL / min) to obtain methyl rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy ... It is a yellow oily substance (μmol, yield 69.76%).

[0374] data: LCMS (ESI + ): m / z 1037.7 (M+H) +

[0375] 4. General procedure for preparing rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy ... LiOH·H2O (11.25 mg, 268.02 μmol, 2 eq) was added to a solution of methyl rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carbonyl]amino]propionate (139 mg, 134.01 μmol, 1 eq) in THF (1 mL) and H2O (1 mL). The resulting mixture was stirred at 0 °C for 0.5 h. THF was removed under reduced pressure, and the residue was acidified with FA to pH=2. The resulting mixture was purified by preparative HPLC (column: C18 20-40um 100 A 40 g; mobile phase: [water-ACN]; B%: 0%-34% @ 80 mL / min) to obtain rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[rac-(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy ... μmol (yield 43.64%, purity 97.940%), is a yellow solid.

[0376] The product was then purified using supercritical fluid chromatography (SFC) under conditions that allowed for the separation of chiral compounds, yielding the final pure product A-001.

[0377] data: LCMS (ESI) + ): m / z 1023.5(M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.63 (d, J = 2.4 Hz, 1H), 8.54 (br t, J= 5.1 Hz, 1H), 8.28 (dd, J = 2.5, 8.6 Hz, 1H), 8.02 - 7.96 (m, 1H), 7.75 (d,J = 7.9 Hz, 1H), 7.66 (t, J = 1.9 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.39 -7.31 (m, 1H), 7.10 (d, J = 8.6 Hz, 1H), 4.76 (t, J = 4.4 Hz, 1H), 4.48 (dd, J= 4.8, 7.8 Hz, 1H), 4.30 (dd, J = 4.5, 7.9 Hz, 1H), 3.90 (dd, J = 5.1, 9.4Hz, 1H), 3.79 (dd, J = 3.8, 9.4 Hz, 1H), 3.69 - 3.56 (m, 32H), 3.55 - 3.51(m, 2H), 3.38 - 3.33 (m, 2H), 3.24 - 3.15 (m, 1H), 2.92 (dd, J = 4.9, 12.7Hz, 1H), 2.70 (d, J = 12.6 Hz, 1H), 2.21 (t, J = 7.4 Hz, 2H), 1.79 - 1.53 (m,4H), 1.44 (q, J = 7.4 Hz, 2H), 1.20 (s, 9H) A-002: (2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carbonyl]amino]propionic acid

[0378] 1. A general procedure for preparing 6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid ester:

[0379] To a solution of N-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate tert-butyl ester (500 mg, 1.07 mmol, 1 eq) in DCM (10 mL), 3-[(5-methoxycarbonyl-2-pyridyl)oxy]benzoic acid (320.72 mg, 1.17 mmol, 1.1 eq), HATU (608.59 mg, 1.60 mmol, 1.5 eq), and DIEA (206.86 mg, 1.60 mmol, 278.79 μL, 1.5 eq) were added. The mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with saturated H2O (15 mL) and precipitated with ethyl acetate (50 mL). 3) Extraction. The combined organic phases were rinsed with brine (50 mL) 1) Wash, dry with anhydrous Na2SO4, filter and concentrate to obtain methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (600 mg, crude product), which is a yellow oily substance and is used directly in the next step.

[0380] data: LCMS (ESI + ): m / z 724.4(M+H)

[0381] 2. A general procedure for preparing methyl 6-[3-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid: Add TFA (5 mL) to a DCM (10 mL) solution of methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (600 mg, 828.95 μmol, 1 eq). Stir the mixture at 25 °C for 1 hour. Adjust the pH of the reaction mixture to 8-9 with NaHCO3 (5 mL). Separately precipitate the aqueous phase with ethyl acetate (30 mL). 3) Extraction. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated to obtain methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (680 mg, crude product), a yellow oil, which was used directly in the next step.

[0382] data: LCMS (ESI+): m / z 624.4 (M+H)

[0383] 3. A general procedure for preparing methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid: At 0 °C and N2, K2CO3 (177.27 mg, 1.28 mmol, 2 eq) and 1-fluoro-2,4-dinitrobenzene (119.35 mg, 641.34 μmol, 80.53 μL, 1 eq) were added to a DMF (5 mL) solution of methyl 6-[3-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid ester (400 mg, 641.34 μmol, 1 eq) and 1-fluoro-2,4-dinitrobenzene (119.35 mg, 641.34 μmol, 80.53 μL, 1 eq) at 20 °C under N2 atmosphere. The mixture was stirred for 12 hours at N2 atmosphere and 20 °C. The reaction mixture was added to H2O (50 mL) and stirred with EA (30 mL) 3) Extraction. The combined organic layers were rinsed with brine (30 mL). 2) The product was washed, dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (290 mg, 367.19 μmol, yield 57.25%), which was a yellow oil.

[0384] Data: LCMS (ESI) + ): m / z 790.5 (M+H) +

[0385] 4. General procedure for the preparation of 6-[3-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid: LiOH·H2O (69.07 mg, 1.65 mmol, 5 eq) was added to a solution of methyl pyridine-3-carboxylic acid ester (260 mg, 329.21 μmol, 1 eq) in THF (3 mL) and H2O (0.5 mL). The mixture was stirred at 25°C for 4 hours. The pH of the reaction mixture was adjusted to 5-6 using 1 M HCl (5 mL). The aqueous phase was separated by ethyl acetate (15 mL). 3) Extraction. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by preparative HPLC (FA conditions) to give 6-[3-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (180 mg, 232.03 μmol, yield 70.48%), as a yellow oil.

[0386] data: LCMS (ESI + ): m / z 776.4 (M+H)

[0387] 5. General procedure for preparing rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carbonyl]amino]propionic acid: To a DMF (3 mL) solution of 6-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carboxylic acid (90 mg, 116.02 μmol, 1 eq), HATU (66.17 mg, 174.02 μmol, 1.5 eq) and DIEA (22.49 mg, 174.02 μmol, 30.31 μL, 1.5 eq) were added. The mixture was stirred for 30 min, and then rac-(2S)-2-amino-3-tert-butoxypropionic acid (28.05 mg, 174.02 μmol, 1.5 eq) was added. The mixture was stirred at 25 °C for 18 h. The reaction mixture was filtered and the filtrate was concentrated to obtain the residue. The residue was purified by preparative HPLC (FA conditions) to obtain rac-(2S)-3-tert-butoxy-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,4-dinitroaniline)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoyl]phenoxy]pyridine-3-carbonyl]amino]propionic acid (13.9 mg, 15.08 μmol, yield 13.00%, purity 99.7%), as a yellow oil.

[0388] The product was then purified using supercritical fluid chromatography (SFC) under conditions that allowed for the separation of chiral compounds, yielding the final pure product A-002.

[0389] data: LCMS (ESI) + ): m / z 919.4(M+H) 1 H NMR (400 MHz, methanol-) d 4) δ ppm 9.01 (d, J =2.75 Hz, 1 H) 8.61 (d, J =2.50 Hz, 1 H) 8.29 - 8.24 (m, 2 H) 7.76 - 7.70 (m, 1 H) 7.65 - 7.62 (m, 1 H)7.53 (t, J =7.94 Hz, 1 H) 7.36 - 7.30 (m, 1 H) 7.20 (d, J =9.51 Hz, 1 H) 7.10 -7.07 (m, 1 H) 4.75 (t, J=4.32 Hz, 1 H) 3.92 - 3.87 (m, 1 H) 3.82 - 3.76 (m, 3H) 3.68 - 3.55 (m, 30 H) 1.20 (s, 9 H) A-003: (2S)-3-tert-butoxy-2-[[6-[3-[[1-[2-[2-[2-[[1-[2-[(2R)-4-[5-[7-chloro-8-[[(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-3-fluoro-6-methyl-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carbonyl]amino]propionic acid

[0390] 1. A general procedure for preparing methyl 6-[3-[(1-tert-butoxycarbonyl-4-piperidinyl)oxy]phenoxy]pyridine-3-carboxylic acid: Cs₂CO₃ (2.66 g, 8.16 mmol, 2 eq) was added to a DMF (25 mL) solution of methyl 6-(3-hydroxyphenoxy)pyridine-3-carboxylate (1 g, 4.08 mmol, 1 eq) and tert-butyl 4-(p-toluenesulfonyloxy)piperidine-1-carboxylate (1.45 g, 4.08 mmol, 1 eq). The resulting mixture was stirred at 80 °C for 13 hours. The mixture was quenched with H₂O (20 mL) and extracted with EA (90 mL). The combined organic layers were washed with brine (40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to give methyl 6-[3-[(1-tert-butoxycarbonyl-4-piperidinyl)oxy]phenoxy]pyridine-3-carboxylic acid (1.1 g, 2.57 mmol, yield 62.96%) as a colorless oil.

[0391] data: LCMS (ESI) + ): m / z 429.2(M+H) +

[0392] 2. General procedure for preparing methyl 6-[3-(4-piperidinyloxy)phenoxy]pyridine-3-carboxylic acid:

[0393] TFA (1.54 g, 13.46 mmol, 1 mL, 1 eq) was added to a DCM (5 mL) solution of methyl 6-[3-[(1-tert-butoxycarbonyl-4-piperidinyl)oxy]phenoxy]pyridine-3-carboxylic acid (500 mg, 1.17 mmol, 1 eq). The resulting mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated to give methyl 6-[3-(4-piperidinyloxy)phenoxy]pyridine-3-carboxylic acid (500 mg, crude, TFA), as a brown oil.

[0394] data: LCMS (ESI + ): m / z 329.1 (M+H) +

[0395] 3. A general procedure for preparing methyl 6-[3-[[1-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid: At 0 °C, HATU (859.50 mg, 2.26 mmol, 2 eq) and DIEA (584.29 mg, 4.52 mmol, 787.45 μL, 4 eq) were added to a DMF (8 mL) solution of methyl 6-[3-(4-piperidinyloxy)phenoxy]pyridine-3-carboxylate (500 mg, 1.13 mmol, 1 eq) and 2-[2-(2-prop-2-alkynyloxyethoxy)ethoxy]acetic acid (228.54 mg, 1.13 mmol, 1 eq). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35um 100A 40g; mobile phase: [water-ACN]; B%: 0%-75% @ 60mL / min) to obtain methyl 6-[3-[[1-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (263.8 mg, 514.68 μmol, yield 45.54%), as a brown oil.

[0396] data: LCMS (ESI + ): m / z 513.4 (M+H) +

[0397] 4. A general procedure for preparing methyl 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid: Methyl pyridine-3-carboxylate (140 mg, 273.14 μmol, 1 eq) and 4-fluoro-3-[rac-(1R)-1-[[3-chloro-7-fluoro-2-methyl-6-[2-[rac-(3R)-4-(2-azidoacetyl)-3-methyl-piperazin-1-yl]pyrimidin-5-yl]-1,5-naphthidin-4-yl]amino]ethyl]benzonitrile (168.81 mg, 273.14 μmol, 1 eq) in t-BuOH (1.5 mL) and H2O (1.5 mL) were added to a solution of 6-[3-[[1-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylate (140 mg, 273.14 μmol, 1 eq) and 4-fluoro-3-[rac-(1R)-1-[[3-chloro-7-fluoro-2-methyl-6-[2-[rac-(3R)-4-(2-azidoacetyl)-3-methyl-piperazin-1-yl]pyrimidin-5-yl]-1,5-naphthidin-4-yl]amino]ethyl]benzonitrile (168.81 mg, 273.14 μmol, 1 eq) in t-BuOH (1.5 mL) and H2O (1.5 mL). (eq) and (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (54.11 mg, 273.14 μmol, 1.00 eq). The mixture was stirred at 50 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35um 100A 40g; mobile phase: [water-ACN]; B%: 0%-75% @ 60mL / min) to obtain methyl 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (103.2 mg, 91.28 mg). It is a yellow solid (μmol, yield 33.42%).

[0398] data: LCMS (ESI + ): m / z 1130.4 (M+H) +

[0399] 5. General procedure for the preparation of 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid: LiOH·H2O (13.84 mg, 329.74 μmol, 4 eq) was added to a solution of methyl 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-1,5-naphthid-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid ester (93.2 mg, 82.43 μmol, 1 eq) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 0 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35um 100A 40g; mobile phase: [water-ACN]; B%: 0%-75% @ 60mL / min) to obtain 6-[3-[[1-[2-[2-[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluoro-phenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (55 mg, 49.26 μmol, yield 59.75%), as a green solid.

[0400] data: LCMS (ESI + ): m / z 1118.3 (M+H) +

[0401] 6. A general procedure for the preparation of 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (2,5-dioxopyrrolidine-1-yl) ester: At 0 °C, EDCI (15.45 mg, 80.60 μmol, 2 eq) was added to a DMF (1 mL) solution of 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (45 mg, 40.30 μmol, 1 eq) and HOSu (4.64 mg, 40.30 μmol, 1 eq). The mixture was stirred at 25 °C for 1 hour. A DMF (1 mL) solution (50 mg, crude product) of 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthid-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (2,5-dioxopyrrolidine-1-yl) ester was obtained as a brown liquid.

[0402] 7. General procedure for the preparation of rac-(2S)-3-tert-butoxy-2-[[6-[3-[[1-[2-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carbonyl]amino]propionic acid: At 0 °C, DIEA was added to a DMF (1 mL) solution of 6-[3-[[1-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carboxylic acid (2,5-dioxopyrrolidine-1-yl) ester (50 mg, 41.20 μmol, 1 eq) and rac-(2S)-2-amino-3-tert-butoxypropionic acid (6.64 mg, 41.20 μmol, 1 eq). (10.65 mg, 82.40 μmol, 14.35 μL, 2 eq). The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was filtered, and the filtrate was passed through a preparative HPLC system (column: Waters Xbridge BEH C18 100). 30mm 10 μm; Mobile phase: [H2O (10 mMNH4HCO3)-ACN]; Gradient: 35%-65% B) Purification yielded rac-(2S)-3-tert-butoxy-2-[[6-[3-[[1-[2-[2-[2-[[1-[2-oxo-2-[rac-(2R)-4-[5-[7-chloro-3-fluoro-6-methyl-8-[[rac-(1R)-1-(5-cyano-2-fluorophenyl)ethyl]amino]-1,5-naphthidin-2-yl]pyrimidin-2-yl]-2-methyl-piperazin-1-yl]ethyl]triazol-4-yl]methoxy]ethoxy]ethoxy]acetyl]-4-piperidinyl]oxy]phenoxy]pyridine-3-carbonyl]amino]propionic acid (16 mg, 11.76) It is a pale yellow solid (μmol, yield 29.63%, purity 92.595%).

[0403] The product was then purified using supercritical fluid chromatography (SFC) under conditions that allowed for the separation of chiral compounds, yielding the final pure product A-003.

[0404] data: LCMS (ESI + ): m / z 1259.5 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.85 (d, J = 1.1 Hz, 2H), 8.61 (d, J =2.4 Hz, 1H), 8.22 (dd, J = 2.4, 8.6 Hz, 1H), 8.03 - 7.94 (m, 1H), 7.84 - 7.80(m, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.62 (m, J = 2.1, 4.7, 8.4 Hz, 1H), 7.32(t, J = 8.2 Hz, 1H), 7.20 (dd, J = 8.7, 10.1 Hz, 1H), 6.98 (d, J = 8.6 Hz,1H), 6.87 (dd, J = 2.1, 8.3 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.72 (dd, J =1.6, 8.0 Hz, 1H), 6.42 (q, J = 6.7 Hz, 1H), 5.71 - 5.52 (m, 1H), 5.49 - 5.37(m, 1H), 4.85 - 4.67 (m, 4H), 4.66 (s, 2H), 4.61 (m, J = 3.8, 7.0 Hz, 1H), 4.38 (d, J = 8.2 Hz, 1H), 4.26 (d, J = 2.0 Hz, 2H), 3.91 - 3.63 (m, 14H),3.54 - 3.46 (m, 1H), 3.45 - 3.37 (m, 2H), 3.23 - 3.03 (m, 1H), 2.67 (s, 3H),2.03 - 1.91 (m, 2H), 1.72 (d, J = 6.8 Hz, 5H), 1.40 - 1.27 (m, 2H), 1.21 (s,1H), 1.18 (s, 9H) X-001: (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid.

[0405] 5. General procedure for the preparation of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[2-[2-[2-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]methyl hexanoate: At 0 °C, HATU (272.32 mg, 716.20 μmol, 2 eq) was added to a DMF (2 mL) solution of 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (200 mg, 358.10 μmol, 1 eq, TFA) and 2-[2-[2-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamine (125.81 mg, 393.91 μmol, 1.1 eq) (1.1 eq). Then, DIEA (138.85 mg, 1.07 mmol, 187.12 μL, 3 eq) was added to the reaction mixture at 0 °C. The resulting mixture was stirred at 15 °C for 1 hour. The reaction was quenched by adding 2 mL of H2O and then quenched with 9 mL of EtOAc (3 mL). 3) Extraction. The combined organic layers were rinsed with 20 mL of brine (10 mL of...). 2) The product was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (SiO2, ethyl acetate:methanol = 10:1) to give methyl (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[2-[2-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate (250 mg, 335.19 μmol, yield 93.60%), as a yellow oil.

[0406] Data: LCMS (ESI) + ): m / z 746.2 (M+H) +

[0407] 6. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: t-BuOH (1 mL) and H2O were added to N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]-2-methyl-pyrazol-3-carboxamide (83.25 mg, 120.67 μmol, 0.9 eq) and (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[2-[2-[2-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]methyl hexanoate (100 mg, 134.07 μmol, 1 eq). Copper sulfate (10.70 mg, 67.04 μmol, 10.29 μL, 0.5 eq) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (26.56 mg, 134.07 μmol, 1 eq) were added to a 1 mL solution. The resulting mixture was stirred at 50 °C for 0.5 h. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The crude product was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-77% @ 80). Purification was performed at a flow rate of mL / min to obtain compound (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester (96 mg, 66.86 μmol, yield 49.87%), which was a pale yellow oil.

[0408] Data: LCMS (ESI) + ): m / z 1435.4(M+H) +

[0409] 7. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: The mixture of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (96 mg, 66.86 μmol, 1 eq) in DCM (2 mL) and TFA (1 mL) was stirred at 15 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give compound (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (100 mg, crude, TFA), as a yellow oil.

[0410] Data: LCMS (ESI) + ): m / z 1305.2 (M+H) +

[0411] 8. A general procedure for preparing (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O (5.91 mg, 140.89 μmol, 2 eq) to a solution of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (100 mg, 70.45 μmol, 1 eq, TFA) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 15 °C for 0.5 hours to remove THF. The pH of the aqueous layer was adjusted to 5 using FA. The residue was then analyzed by preparative HPLC (neutral conditions; column: Waters Xbridge Prep OBD C18 150). 40mm 10 μm; mobile phase: [H2O(10 mMNH4HCO3)-ACN]; gradient: 25%-65% B) purification to obtain compound X-001, namely (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[4-[4-[4-[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (46.6 mg, 36.08 mg). It is a white solid (μmol, yield 51.22%, purity 100%).

[0412] data: LCMS (ESI + ): m / z1291.4 (M+H) + 1 H NMR (400 MHz, methanol-d4) δ = 8.62 (d, J = 2.0 Hz, 1H), 8.23 ​​(dd, J =2.5, 8.6 Hz, 1H), 7.88 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 2.1Hz, 1H), 7.40 - 7.30 (m, 3H), 6.98 (d, J = 8.8 Hz, 1H), 6.91 - 6.86 (m, 2H), 6.82 (t, J = 2.1 Hz, 1H), 6.78 (br d, J = 8.1 Hz, 1H), 4.91 (d, J = 7.3 Hz,1H), 4.59 (s, 2H), 4.53 (s, 2H), 4.52 - 4.48 (m, 1H), 4.42 (s, 2H), 4.33 (t,J = 7.0 Hz, 2H), 4.10 (s, 3H), 3.63 - 3.53 (m, 22H), 3.49 - 3.42 (m, 4H),2.30 (s, 3H), 2.03 - 1.73 (m, 4H), 1.51 (quin, J = 6.9 Hz, 2H), 1.39 - 1.29(m, 2H), 0.91 (s, 9H), 0.89 - 0.74 (m, 3H), 0.58 - 0.51 (m, 1H), 0.51 - 0.33(m, 4H), 0.32 - 0.21 (m, 3H).

[0413] X-017: (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3, [5-Dimethyl-1H-pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazole-4- [By]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino 5,5-Dimethylhexanoic acid

[0414] 1. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: At 20 °C, CuSO4·5H2O (9.36 g / mL) was added to a solution of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate (50 mg, 74.99 μmol, 1 eq) and 2-(6-azidohexyl)-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazole-3-carboxamide (51.59 mg, 74.99 μmol, 1 eq) in t-BuOH (1 mL) and H2O (1 mL). (mg, 37.49 μmol, 0.5 eq) and (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (14.86 mg, 74.99 μmol, 1 eq). The mixture was stirred at 50 °C for 1 hour. The residue was purified by reversed-phase HPLC with a C18 column (ISCO; 40 g SepaFlash C18 column, eluent 0-100% MeCN / H2O, flow rate 60 mL / min) to obtain compound (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (50 mg, 36.91 mg). It is a white solid (μmol, yield 49.22%).

[0415] Data: LCMS (ESI) + ): m / z 1354.4 (M+H) +

[0416] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Methyl (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (50 mg, 36.91 μmol, 1 eq) was stirred at 20 °C for 3.5 h in a mixture of DCM (1.5 mL) and TFA (0.7 mL). The reaction mixture was concentrated under reduced pressure to give compound (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (49 mg, crude, TFA), as a yellow oil.

[0417] Data: LCMS (ESI) + ): m / z 1224.3 (M+H) +

[0418] 3. General procedure for preparing (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: At 0 °C, LiOH·H2O (38.41 mg, 915.22 μmol, 25 mL) was added to a solution of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (49 mg, 36.61 μmol, 1 eq, TFA) in THF (1.2 mL) and H2O (0.6 mL). (eq) until the pH is approximately 8. The mixture is stirred at 20 °C for 0.5 hours. The residue is concentrated under reduced pressure to remove THF, and then the pH of the residue is adjusted to approximately 4 using formic acid to obtain a solution. The residue was purified by reversed-phase HPLC using a rapid C18 column (ISCO; 40g SepaFlash C18 column, eluent 0-100% MeCN / H2O, flow rate 60 mL / min) to obtain compound X-017, namely (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (34.7 mg, 27.48 mg). It is a white solid (μmol, yield 75.08%, purity 95.872%).

[0419] data: LCMS (ESI + ): m / z 1210.5 (M+H) + .

[0420] 1 H NMR (400 MHz, methanol-d4) δ = 8.61 (d, J = 2.2 Hz, 1H), 8.25 - 8.21(m, 1H), 7.93 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 2.1 Hz, 1H), 7.33 (t, J= 8.2 Hz, 1H), 7.23 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.89 - 6.85 (m, 2H), 6.81 - 6.75 (m, 2H), 4.63 - 4.41 (m, 8H), 4.29 (t, J =7.0 Hz, 2H), 4.21 (s, 2H), 3.74 - 3.66 (m, 1H), 3.55 - 3.49 (m, 4H), 3.45 (brs, 2H), 3.23 - 3.15 (m, 1H), 2.22 (s, 6H), 2.01 - 1.91 (m, 1H), 1.88 - 1.69 (m, 8H), 1.52 - 1.43 (m, 2H), 1.35 (dt, J = 4.9, 11.9 Hz, 2H), 1.29 - 1.22(m, 4H), 0.91 (s, 9H), 0.88 - 0.75 (m, 3H), 0.58 - 0.52 (m, 1H), 0.50 - 0.43(m, 2H), 0.42 - 0.35 (m, 2H), 0.32 - 0.23 (m, 3H).

[0421] X-024:

[0422] (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0423] 1. A general procedure for preparing (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Copper sulfate (7.49 g / mL) was added to a solution of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[2-[2-[2-[2-(2-prop-2-alkynoxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate (70 mg, 93.85 μmol, 1 eq) and 2-(6-azidohexyl)-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (51.65 mg, 75.08 μmol, 0.8 eq) in t-BuOH (0.5 mL) and H2O (0.5 mL). (mg, 46.93 μmol, 7.20 μL, 0.5 eq) and (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (18.59 mg, 93.85 μmol, 1 eq). The mixture was stirred at 50 °C for 0.5 h. The reaction mixture was filtered and concentrated under reduced pressure to give the residue. The crude product was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-80% @ 80). Purification was performed at a flow rate of mL / min to obtain compound (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester (80 mg, 55.80 μmol, yield 59.45%), as a colorless oil.

[0424] data: LCMS (ESI + ): m / z 1433.4 (M+H) +

[0425] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: The mixture of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (160 mg, 111.59 μmol, 1 eq) in DCM (2 mL) and TFA (1 mL) was stirred at 15 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give compound (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (290 mg, crude, TFA), as a colorless oil.

[0426] data: LCMS (ESI + ): m / z 1303.3 (M+H) +

[0427] 3. A general procedure for preparing (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: At 15 °C, LiOH·H2O (17.17 mg, 409.15 mL) was added to a solution of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (290 mg, 204.58 μmol, 1 eq, TFA) in THF (2 mL) and H2O (1 mL). μmol, 2 eq). The mixture was stirred at 15 °C for 1 hour. THF was removed. The pH of the aqueous layer was adjusted to 5 using FA. The residue was subjected to preparative HPLC (neutral conditions; column: Waters Xbridge Prep OBD C18 150). 40mm 10 μm; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 40%-70% B) purification over 8.0 min yielded compound X-024, namely (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (61.6 mg, 47.77 μmol, It is a white solid (yield 23.35%, purity 100%).

[0428] data: LCMS (ESI + ): m / z 1289.4 (M+H) + 1 H NMR (400 MHz, methanol-d4) δ = 8.63 (d, J = 2.2 Hz, 1H), 8.24 (dd, J =2.4, 8.7 Hz, 1H), 7.89 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 2.1Hz, 1H), 7.35 (t, J = 8.3 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.7Hz, 1H), 6.89 (dd, J = 2.1, 8.3 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.83 (t, J= 2.2 Hz, 1H), 6.78 (dd, J = 1.7, 8.1 Hz, 1H), 4.89 (d, J = 7.5 Hz, 1H), 4.58(s, 2H), 4.56 - 4.42 (m, 5H), 4.29 (t, J = 7.0 Hz, 2H), 3.61 (s, 4H), 3.59 -3.53 (m, 18H), 3.49 - 3.41 (m, 2H), 2.22 (s, 6H), 2.06 - 1.91 (m, 1H), 1.88 -1.73 (m, 5H), 1.35 (ddd, J = 5.1, 8.8, 11.8 Hz, 2H), 1.30 - 1.24 (m, 4H),0.91 (s, 9H), 0.89 - 0.74 (m, 3H), 0.59 - 0.51 (m, 1H), 0.51 - 0.43 (m, 2H), 0.43 - 0.34 (m, 2H), 0.33 - 0.21 (m, 3H).

[0429] X-008: (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-3-methyl-1H-pyrazole-5-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0430] 1. General procedure for preparing 4-azidobut-1-ol:

[0431] At 20°C, NaN3 (4.44 g, 68.29 mmol, 1.05 eq) was slowly added to a DMF (150 mL) solution of 4-bromobut-1-ol (10 g, 65.35 mmol, 1 eq), followed by stirring at 20°C for 12 hours. The reaction mixture was diluted with H2O (100 mL) and treated with EtOAc 300 mL (100 mL) 3) Extraction. The combined organic layers were rinsed with 400 mL (200 mL) of brine. 2) Wash, dry with Na2SO4, filter and concentrate under reduced pressure to obtain 4-azidobut-1-ol (9 g, crude product), which is a yellow oily substance and is used directly in the next step without purification.

[0432] data: 1 ¹H NMR (400 MHz, chloroform-d) δ = 3.68 (t, J = 6.1 Hz, 1H), 3.33 (t, J = 6.5 Hz, 2H), 1.76 - 1.60 (m, 4H).

[0433] 2. General procedure for preparing 4-methylbenzenesulfonic acid 4-azidobutyl ester:

[0434] At 0 °C, TEA (7.03 g, 69.48 mmol, 9.67 mL, 2 eq) and TosCl (9.94 g, 52.11 mmol, 1.5 eq) were added to a DCM (100 mL) solution of 4-azidobut-1-ol (4 g, 34.74 mmol, 1 eq), and the reaction was stirred at 15 °C for 2 hours. The reaction mixture was diluted with H2O (30 mL) and EA (90 mL, 30 mL) 3) Extraction. The combined organic layers were rinsed with brine (80 mL). 2) The residue was washed, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to give 4-methylbenzenesulfonic acid 4-azidobutyl ester (2.5 g, 9.28 mmol, yield 26.72%) as a yellow oil.

[0435] data: 1H NMR (400 MHz, chloroform-d) δ = 7.80 (d, J = 8.3 Hz, 2H), 7.36 (d, J =8.0 Hz, 2H), 4.07 (t, J = 6.1 Hz, 2H), 3.27 (t, J = 6.6 Hz, 2H), 2.46 (s,3H), 1.81 - 1.71 (m, 2H), 1.70 - 1.59 (m, 2H).

[0436] 3. A general procedure for preparing 2-[[3-(4-azidobutoxymethyl)-5-methyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyl-trimethyl-silane: At 0°C, NaH (660.22 mg, 16.51 mmol, 60% purity, 1.5 eq) was added to a DMF (70 mL) solution of [5-methyl-4-(4-nitrophenyl)-1-(2-trimethylsilylethoxymethyl)pyrazol-3-yl]methanol (4 g, 11.00 mmol, 1 eq), and the reaction was stirred at 0°C for 0.5 h. A DMF (5 mL) solution of 4-methylbenzenesulfonic acid-4-azidobutyl ester (2.96 g, 11.00 mmol, 1 eq) was then added, and the reaction was stirred at 15°C for 3 h. The reaction mixture was then slowly added to a saturated NH4Cl solution (100 mL) at 0°C, followed by EtOAc (3... Extracted by 100 mL. The organic layer was dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give 2-[[3-(4-azidobutoxymethyl)-5-methyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyl-trimethyl-silane (2 g, 4.34 mmol, yield 39.46%) as a yellow oil.

[0437] data: LCMS (ESI + ): m / z 461.3 (M+H) + 1H NMR (400 MHz, chloroform-d) δ = 8.36 - 8.22 (m, 2H), 7.66 - 7.52 (m,2H), 5.47 (s, 2H), 4.44 (s, 2H), 3.68 - 3.61 (m, 2H), 3.52 (br t, J = 5.8 Hz,2H), 3.26 (t, J = 6.4 Hz, 2H), 2.42 (s, 3H), 1.70 - 1.62 (m, 4H), 0.97 - 0.90(m, 2H), 0.04 - 0.02 (m, 9H)

[0438] 4. A general procedure for preparing 4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline:

[0439] Fe (1.15 g, 20.63 mmol, 5 eq) and NH4Cl (1.10 g, 20.63 mmol, 5 eq) were added to a solution of 2-[[3-(4-azidobutoxymethyl)-5-methyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyl-trimethyl-silane (1.9 g, 4.13 mmol, 1 eq) in EtOH (20 mL) and H2O (2 mL). The reaction was stirred at 50 °C for 72 hours. The mixture was filtered, and the filtrate was distilled using EA (100 mL, 50 mL) 2) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to give 4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline (1.5 g, 3.48 mmol, yield 84.44%) as a yellow oil.

[0440] data: 1H NMR (400 MHz, chloroform-d) δ = 7.17 (d, J = 8.3 Hz, 2H), 6.76 (d, J =8.3 Hz, 2H), 5.43 (s, 2H), 4.41 (s, 2H), 3.66 - 3.58 (m, 2H), 3.49 (br t, J =5.3 Hz, 2H), 3.28 - 3.19 (m, 2H), 2.34 (s, 3H), 1.69 - 1.60 (m, 4H), 0.96 -0.86 (m, 2H), 0.02 - 0.03 (m, 9H).

[0441] 5. General procedure for preparing N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]tert-butyl carbamate:

[0442] To a DMF (30 mL) solution of (2S)-2-(tert-butoxycarbonylamino)-3,3-dicyclopropyl-propionic acid (965.40 mg, 3.58 mmol, 1.05 eq) and 4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline (1.47 g, 3.41 mmol, 1 eq), DIEA (1.32 g, 10.24 mmol, 1.78 mL, 3 eq) and HATU (1.95 g, 5.12 mmol, 1.5 eq) was added, and the reaction was stirred at 15°C for 12 hours. The reaction mixture was diluted with H2O (30 mL) and EA (90 mL, 30 mL) 3) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to give N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]tert-butyl carbamate (1.75 g, 2.57 mmol, yield 75.17%) as a yellow oil.

[0443] data: 1H NMR (400 MHz, chloroform-d) δ = 7.58 (d, J = 8.5 Hz, 2H), 7.36 (d, J =8.5 Hz, 2H), 5.44 (s, 3H), 4.45 - 4.35 (m, 3H), 3.66 - 3.59 (m, 2H), 3.53 -3.46 (m, 2H), 3.24 (br t, J = 6.4 Hz, 2H), 2.36 (s, 3H), 1.67 - 1.60 (m, 4H), 1.49 (s, 9H), 1.00 - 0.86 (m, 4H), 0.83 - 0.73 (m, 2H), 0.60 - 0.43 (m, 4H), 0.38 - 0.24 (m, 4H), -0.01 (s, 9H).

[0444] 6. A general procedure for preparing (2S)-2-amino-N-[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]-3,3-dicyclopropyl-propionamide:

[0445] A mixture of N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]tert-butyl carbamate (1.75 g, 2.57 mmol, 1 eq) in 4M HCl / MeOH (20 mL) was stirred at 15 °C for 1 hour. The reaction solution was concentrated to give (2S)-2-amino-N-[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]-3,3-dicyclopropyl-propionamide (1.59 g, crude product, HCl salt), a yellow solid. The product was used directly in the next step without purification.

[0446] data: LCMS (ESI + ): m / z 582.4 (M+H) +

[0447] 7. A general procedure for preparing N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]-2-methyl-pyrazol-3-carboxamide:

[0448] To a solution of (2S)-2-amino-N-[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]-3,3-dicyclopropylpropionamide (1.49 g, 2.41 mmol, 1 eq, HCl salt) and 2-methylpyrazol-3-carboxylic acid (364.70 mg, 2.89 mmol, 1.2 eq) in DMF (20 mL), DIEA (934.37 mg, 7.23 mmol, 1.26 mL, 3 eq) and HATU (1.37 g, 3.61 mmol, 1.5 eq) were added, and the reaction was stirred at 15°C for 0.5 h. The reaction mixture was diluted with H2O (30 mL) and EA (90 mL, 30 mL) 3) Extraction. The combined organic layers were rinsed with brine (100 mL). 3) The residue was washed, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1) to give N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]-2-methyl-pyrazol-3-carboxamide (1.6 g, 2.32 mmol, yield 96.23%), as a yellow solid.

[0449] data: LCMS (ESI + ): m / z 690.4 (M+H) + 1H NMR (400 MHz, chloroform-d) δ = 7.94 (s, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 2.1 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.11 (br d, J = 8.0 Hz,1H), 6.62 (d, J = 2.0 Hz, 1H), 5.45 (s, 2H), 4.83 (dd, J = 4.8, 8.0 Hz, 1H),4.42 (s, 2H), 4.19 (s, 3H), 3.66 - 3.59 (m, 2H), 3.49 (br t, J = 5.5 Hz, 2H),3.23 (br t, J = 6.3 Hz, 2H), 2.36 (s, 3H), 1.66 - 1.58 (m, 4H), 0.96 - 0.85 (m, 5H), 0.71 - 0.63 (m, 1H), 0.62 - 0.53 (m, 3H), 0.47 - 0.35 (m, 2H), 0.27(q, J = 4.8 Hz, 2H), -0.01 (s, 9H).

[0450] 8. General procedure for preparing 4-[2-(tert-butoxycarbonylamino)ethoxy]piperidine-1-carboxylic acid benzyl ester:

[0451] At 0°C, NaH (1.02 g, 25.50 mmol, 60% purity, 1.2 eq) was added to a DMF (100 mL) solution of 4-hydroxypiperidine-1-carboxylic acid benzyl ester (5 g, 21.25 mmol, 1 eq) and the reaction was stirred at 0°C for 1 hour. Then, 2,2-dioxoxazolidin-3-carboxylic acid tert-butyl ester (5.69 g, 25.50 mmol, 1.2 eq) was added to the mixture, and the reaction was stirred at 25°C for 11 hours. The reaction mixture was quenched with saturated NH4Cl (100 mL) and quenched with ethyl acetate (500 mL, 100 mL). 5) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 0 / 1 to 8 / 1) to give 4-[2-(tert-butoxycarbonylamino)ethoxy]piperidine-1-carboxylic acid benzyl ester (8.22 g, 21.72 mmol, yield 82.20%) as a colorless oil.

[0452] data: LCMS (ESI + ): m / z 279.2 (M-100+H) +

[0453] 9. A general procedure for preparing N-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethyl]carbamate tert-butyl ester:

[0454] Under N2 protection, Pd / C (4.5 g, 10% purity) was added to a THF (100 mL) solution of 4-[2-(tert-butoxycarbonylamino)ethoxy]piperidine-1-carboxylic acid benzyl ester (8 g, 21.14 mmol, 1 eq). The reaction was degassed and purged three times with H2, then stirred at 15°C for 1 hour under H2 atmosphere (15 psi). The catalyst was removed by filtration through diatomaceous earth, which was then washed with MeOH (300 mL). The filtrate was concentrated to give N-[2-(4-piperidinyloxy)ethyl]carbamate tert-butyl ester (4.5 g, crude), as a colorless oil.

[0455] 10. General procedure for preparing N-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethyl]carbamate tert-butyl ester: At 0 °C, DIEA (3.17 g, 24.56 mmol, 4.28 mL, 3 eq) and HATU (4.67 g, 12.28 mmol, 1.5 eq) were added to a DMF (35 mL) solution of N-[2-(4-piperidinyloxy)ethyl]carbamate (2 g, 8.19 mmol, 1 eq), 2-prop-2-alkynyloxyacetic acid (933.98 mg, 8.19 mmol, 1 eq), and stirred at 15 °C for 2 hours. The mixture was diluted with water (100 mL) and then with EtOAc (600 mL, 200 mL). 3) Extraction. The combined organic layers were washed with saturated NaCl (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO; 120 g SepaFlash silica gel column, eluent gradient of 0–93% ethyl acetate / petroleum ether, flow rate 100 mL / min) to give N-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethyl]carbamate tert-butyl ester (1.8 g, 5.29 mmol, yield 90.00%) as a brown oil.

[0456] data: 1¹H NMR (400 MHz, chloroform-d) δ = 4.89 - 4.79 (m, 1H), 4.29 (d, J = 2.4Hz, 2H), 4.26 (s, 2H), 3.93 - 3.81 (m, 1H), 3.73 - 3.61 (m, 1H), 3.59 - 3.46 (m, 3H), 3.43 - 3.21 (m, 4H), 2.52 - 2.44 (m, 1H), 1.91 - 1.79 (m, 2H), 1.65 - 1.52 (m, 2H), 1.45 (s, 9H).

[0457] 11. A general procedure for preparing 1-[4-(2-aminoethoxy)-1-piperidinyl]-2-prop-2-alkynoxy-acetone:

[0458] A solution of N-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethyl]carbamate tert-butyl ester (1.7 g, 4.99 mmol, 1 eq) in DCM (10 mL) and TFA (5 mL) was stirred at 15 °C for 3 hours. DCM and TFA were removed under reduced pressure to give compound 1-[4-(2-aminoethoxy)-1-piperidinyl]-2-prop-2-alkynoxy-acetone (1.2 g, crude, TFA), as a colorless oil.

[0459] 12. A general procedure for the preparation of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate:

[0460] At 0 °C, HATU (510.60 mg, 1.34 mmol, 1.5 eq) and DIEA (347.12 mg, 2.69 mmol, 467.81 μL, 3 eq) were added to a DMF (20 mL) solution of 1-[4-(2-aminoethoxy)-1-piperidinyl]-2-prop-2-alkynyloxy-acetone (258.15 mg, 728.59 μmol, TFA) and 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridinyl]oxy]phenoxy]acetic acid (500 mg, 895.26 μmol, 1 eq, TFA). The mixture was stirred at 15 °C for 1.5 hours. The mixture was diluted with water (10 mL) and then with EtOAc (60 mL, 20 mL). 3) Extraction. The combined organic layers were washed with saturated NaCl (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-54% @ 65 mL / min) to give methyl (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[[1-(2-prop-2-alkynoxyacetyl)-4-piperidinyl]oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate (143 mg, 214.47 μmol, yield 23.96%), as a colorless oil.

[0461] data: LCMS (ESI + ): m / z 667.5 (M+H) +

[0462] 13. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0463] Methyl hexanoate (65 mg, 97.49 μmol, 1 eq) and N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]-2-methyl-pyrazol-3-carboxamide (67.26 mg, 97.49 μmol, 1 eq) were reacted with t-BuOH (1 mL) and H2O (1 mL) to form t-BuOH (1 mL) and H2O (1 mL) of N-[(1S)-1-[[4-[3-(4-azidobutoxymethyl)-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]carbamoyl]-2,2-dicyclopropyl-ethyl]-2-methyl-pyrazol-3-carboxamide (67.26 mg, 97.49 μmol, 1 eq). Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (19.31 mg, 97.49 μmol, 1 eq) and copper sulfate pentahydrate (12.17 mg, 48.74 μmol, 11.70 μL, 0.5 eq) were added to a solution, and the reaction was stirred at 50 °C for 1 hour. The reaction mixture was filtered, and the filtrate was subjected to reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-75% @ 50). Purification at a rate of mL / min yielded (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (100 mg, 73.71 μmol, yield 75.61%), a white solid.

[0464] data: LCMS (ESI + ): m / z 1356.8 (M+H) +

[0465] 14. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0466] A solution of (2S)-2-[[6-[3-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1-(2-trimethylsilylethoxymethyl)pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (100 mg, 73.71 μmol, 1 eq) in DCM (0.9 mL) and TFA (460.50 mg, 4.04 mmol, 0.3 mL, 54.79 eq) was stirred at 15 °C for 3 hours. The reaction solution was concentrated with N2 to obtain (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazol-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (90 mg, crude product), which is a yellow oil and is used directly in the next step without purification.

[0467] data: LCMS (ESI + ): m / z 1226.7 (M+H) +

[0468] 15. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-3-methyl-1H-pyrazol-5-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0469] A solution of (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazole-3-carbonyl)amino]propionyl]amino]phenyl]-5-methyl-1H-pyrazole-3-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (90 mg, 73.38 μmol, 1 eq) and LiOH·H2O (6.16 mg, 146.77 μmol, 2 eq) in THF (0.5 mL) and H2O (0.5 mL) was stirred at 0°C for 0.5 h. Add 1M HCl to the reaction mixture until the pH reaches approximately 6. Filter the mixture and pass the filtrate through a preparative HPLC system (neutral conditions; column: Waters XbridgePrep OBD C18 150). 40mm 10 μm; Mobile phase: [H2O (10 mM NH4HCO3)-ACN]; Gradient: 25%-55% over 8.0 min B) Purification yielded compound X-008 (2S)-2-[[6-[3-[2-[2-[[1-[2-[[1-[4-[[4-[4-[[(2S)-3,3-dicyclopropyl-2-[(2-methylpyrazol-3-carbonyl)amino]propionyl]amino]phenyl]-3-methyl-1H-pyrazol-5-yl]methoxy]butyl]triazol-4-yl]methoxy]acetyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (41.4 mg, 34.10 mg). It is a white solid (μmol, yield 46.46%, purity 99.85%).

[0470] data: LCMS (ESI + ): m / z 1212.4 (M+H) + 1 H NMR (400 MHz, methanol-d4) δ = 8.61 (d, J = 2.2 Hz, 1H), 8.23 ​​(dd, J =2.5, 8.6 Hz, 1H), 8.13 - 8.07 (m, 0.5H), 7.89 (s, 1H), 7.63 (d, J = 8.6 Hz,2H), 7.48 (d, J = 2.1 Hz, 1H), 7.38 - 7.30 (m, 3H), 6.98 (d, J = 8.7 Hz, 1H), 6.91 - 6.84 (m, 2H), 6.83 - 6.75 (m, 2H), 4.91 (d, J = 7.2 Hz, 1H), 4.63 (s, 2H), 4.54 (s, 2H), 4.51 (dd, J = 5.1, 8.5 Hz, 1H), 4.43 (s, 2H), 4.32 (t, J =7.1 Hz, 2H), 4.22 (s, 2H), 4.09 (s, 3H), 3.75 - 3.67 (m, 1H), 3.56 - 3.40 (m,9H), 3.24 - 3.15 (m, 1H), 2.29 (s, 3H), 2.02 - 1.91 (m, 1H), 1.90 - 1.80 (m,3H), 1.79 - 1.71 (m, 2H), 1.55 - 1.44 (m, 4H), 1.39 - 1.29 (m, 2H), 0.91 (s,9H), 0.88 - 0.74 (m, 3H), 0.57 - 0.51 (m, 1H), 0.51 - 0.42 (m, 2H), 0.41 -0.32 (m, 2H), 0.32 - 0.19 (m, 3H).

[0471] X-023: (2S)-2-[[6-[3-[2-[2-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0472] 1. General procedure for the preparation of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-(2-prop-2-alkynoxyethoxy)ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]hexanoate: At 0°C, HATU (816.97 mg, 2.15 mmol, 2 eq) and DIEA (694.23 mg, 5.37 mmol, 935.62 μL, 5 eq) were added to a DMF (6 mL) solution of 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (600 mg, 1.07 mmol, 1 eq, TFA) and 2-(2-prop-2-alkynyloxyethoxy)ethylamine (123.06 mg, 859.45 μmol, 0.8 eq) and added to a mixture of acetic acid (600 mg, 1.07 mmol, 1 eq, TFA) and 2-(2-prop-2-alkynyloxyethoxy)ethylamine (123.06 mg, 859.45 μmol, 0.8 eq ... 3) Extraction. The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (ISCO®; 40 g SepaFlash® silica gel column, eluent 0–87% ethyl acetate / petroleum ether gradient, flow rate 100 mL / min) to give methyl (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-(2-prop-2-alkynoxyethoxy)ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]hexanoate (529 mg, 928.65 μmol, yield 86.44%) as a light brown oil.

[0473] data: LCMS (ESI + ): m / z 570.3 (M+H) +

[0474] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0475] Methyl hexanoate (99.36 mg, 174.43 μmol, 1.2 eq) and 2-(6-azidohexyl)-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (100 mg, 145.36 μmol, 1 eq) were reacted with t-BuOH (1 mL) and H2O (1 mL) to form (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-(2-prop-2-alkynoxyethoxy)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (100 mg, 145.36 μmol, 1 eq). Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (28.80 mg, 145.36 μmol, 1 eq) and copper sulfate (11.60 mg, 72.68 μmol, 11.15 μL, 0.5 eq) were added to the solution, and the reaction was then stirred at 50°C for 3 hours. The product was purified by reversed-phase HPLC (column: C18 20-35um 100A 80g; mobile phase: [water-ACN]; B%: 0%-89% @ 80mL / min) to obtain (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (150mg, 119.28g). It is a yellow oily substance (μmol, yield 82.06%).

[0476] data: LCMS (ESI + ): m / z 688.5 (M+H)

[0477] 3. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0478] A solution of (2S)-2-[[6-[3-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (150 mg, 119.28 μmol, 1 eq) in DCM (1 mL) and TFA (0.5 mL) was stirred at 25°C for 1 hour. The mixture was concentrated and the pH was adjusted to 7 with NH3·H2O. The mixture was diluted with water (10 mL) and then with dichloromethane (5 mL). 3) Extraction. The combined organic layers were washed with saturated NaCl (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give methyl (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (134 mg, 118.86 μmol, yield 99.66%), as a yellow solid.

[0479] data: LCMS (ESI + ): m / z 1128.5 (M+H)

[0480] 4. General procedure for preparing (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0481] To a solution of (2S)-2-[[6-[3-[2-[2-[2-[[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (134 mg, 118.86 μmol, 1 eq) in H2O (1 mL) and THF (1 mL), LiOH·H2O (9.98 mg, 237.73 μmol, 2 eq) was added, and the reaction was stirred at 25°C for 1 h. The residue was adjusted to pH 5 using FA. The mixture was filtered, and the filtrate was passed through a preparative HPLC system (neutral conditions; column: Waters Xbridge Prep OBDC18150). 40mm 10 μm; Mobile phase: [H2O (10 mM NH4HCO3)-ACN]; Gradient: 30%-60% over 8.0 min. B) Purification yielded (2S)-2-[[6-[3-[2-[2-[2-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethylamino]-2-oxoethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (X-023) (30.7 mg, 27.58 μmol, yield 23.20%, purity 100%), a white solid.

[0482] data: LCMS (ESI + ): m / z 1113.4 (M+H) 1 H NMR (400 MHz, DMSO-d6) δ = 10.43 - 10.24 (s, 1H), 8.66 - 8.58 (m,2H), 8.52 (br d, J = 6.1 Hz, 1H), 8.27 (dd, J = 2.4, 8.6 Hz, 1H), 8.11 (br t, J = 5.6 Hz, 1H), 8.01 (s, 1H), 7.66 (d, J= 8.6 Hz, 2H), 7.46 (d, J = 2.0 Hz, 1H), 7.33(t, J = 8.1 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 1.8, 8.1Hz, 1H), 6.79 - 6.74 (m,2H), 4.87 - 4.77 (m, 1H), 4.54 - 4.38 (m, 6H), 4.29 - 4.19 (m, 3H), 3.50 (brd, J = 3.5 Hz,4H), 3.43 - 3.41 (m, 2H), 3.27 (br d, J = 5.7 Hz, 2H), 2.17 (s,6H), 1.79 (br dd, J = 5.9, 11.7 Hz, 1H), 1.75 - 1.62 (m,5H), 1.31 - 1.15 (m,6H), 0.85 (s, 9H), 0.80 (br d, J = 5.7 Hz, 3H), 0.51 - 0.41 (m, 1H), 0.40 -0.32 (m, 2H), 0.30 -0.10 (m, 5H) X-038: (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0483] 1. A general procedure for preparing (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxoethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0484] Methyl (2S)-5,5-dimethyl-2-[[6-[3-[1-[3-(2-prop-2-alkynoxyethoxy)propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]hexanoate (65 mg, 104.05 μmol, 1 eq), 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (73.14 mg, 104.05 μmol, 1 eq), and copper sulfate (8.30 mg, 52.02 μmol, 7.98 μL, 0.5 μL) were administered. A mixture of sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol) (20.61 mg, 104.05 μmol, 1 eq) in t-BuOH (2 mL) and H2O (0.5 mL) was degassed and purged three times with N2, followed by stirring at 50°C for 0.5 h under N2 atmosphere. LC-MS showed several new peaks and detection of 61% of the target compound. The reaction mixture was filtered and concentrated under reduced pressure to give the residue. The crude product was purified by reversed-phase HPLC (column: C18 20-35um 100A 40g; mobile phase: [water-ACN]; B%: 0%-60% @ 60mL / min) to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxoethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (80 It is a white solid (mg, 60.26 μmol, yield 57.91%).

[0485] data: LCMS (ESI + ): m / z 1328.7 (M+H)

[0486] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0487] Add TFA (1.54 g, 13.46 mmol, 1 mL, 223.41 eq) to a DCM (3 mL) solution of (2S)-2-[[6-[3-[1-[3-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxoethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (80 mg, 60.26 μmol, 1 eq). The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to give (2S)-2-[[6-[3-[1-[3-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (140 mg, crude) as a yellow oil.

[0488] data: LCMS (ESI + ): m / z 1197.7 (M+H)

[0489] 3. General procedure for preparing (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0490] At 0°C, LiOH·H2O (9.11 mg, 217.14 μmol, 2 eq) was added to a solution of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (130 mg, 108.57 μmol, 1 eq) in THF (1 mL) and H2O (1 mL). The mixture was stirred at 20°C for 1.5 hours. THF was removed under reduced pressure, and the residue was acidified with FA to pH 2. The mixture was filtered, and the filtrate was passed through a preparative HPLC system (FA conditions: HPLC: column: 3-Phenomenex Luna C18 75). 30mm 3µm; Mobile phase: [H2O(0.2%FA)-ACN]; Gradient: 35%-65% over 8.0 min. B) Purification yielded (2S)-2-[[6-[3-[1-[3-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (20.5 mg, 16.91 μmol, yield 15.57%, purity 97.604%), a white solid.

[0491] data: LCMS (ESI + ): m / z 1184.6 (M+H).

[0492] 1 1H NMR (400 MHz, methanol-d4) δ ppm 8.61 (d, J = 2.32 Hz, 1 H) 8.20 - 8.28 (m, 2 H) 7.97 (s, 1 H) 7.66 (d, J = 8.68 Hz, 2 H) 7.53 (d, J = 2.08 Hz, 1 H) 7.33 (t, J = 8.25 Hz, 1 H) 7.25 (d, J = 8.56 Hz, 2 H) 6.97 (d, J = 8.68 Hz, 1 H) 6.88 (d, J = 2.08 Hz, 1 H) 6.73 (ddd, J = 15.04, 8.31, 1.71 Hz, 2 H) 6.65 (t, J = 2.26 Hz, 1 H) 5.09 (s, 2 H) 4.94 - 5.01 (m, 1 H) 4.89 (br s, 1 H) 4.56 - 4.64 (m, 3 H) 4.47 - 4.56 (m, 2 H) 4.24 - 4.32 (m, 2 H) 3.88 (dd, J = 9.23, 3.85 Hz, 2 H) 3.61 - 3.66 (m, 2 H) 3.54 - 3.59 (m, 2 H) 3.44 - 3.53 (m, 2 H) 3.11 - 3.23 (m, 4 H) 2.27 (s, 6 H) 1.99 - 2.08 (m, 2 H) 1.91 - 1.99 (m, 1 H) 1.76 - 1.88 (m, 1 H) 1.71 (quin, J = 6.39 Hz, 2 H) 1.27 - 1.43 (m, 2 H) 0.91 (s, 9 H) 0.75 - 0.90 (m, 3 H) 0.56 (dt, J = 8.16, 4.29 Hz, 1 H) 0.43 - 0.52 (m, 2 H) 0.34 - 0.43 (m, 2 H) 0.28 (ddt, J = 13.89, 9.46, 4.95, 4.95 Hz, 3 H).

[0493] X-043: (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0494] 1. A general procedure for preparing (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[(1-pent-4-ynylsulfonyl-4-piperidinyl)oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate:

[0495] At 0°C, DIEA (499.13 mg, 3.86 mmol, 672.69 μL, 3 eq) and HATU (1.47 g, 3.86 mmol, 3 eq) were added to a DMF (5 mL) solution of 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (718.97 mg, 1.29 mmol, 1 eq, TFA) and 2-[(1-pent-4-ynylsulfonyl-4-piperidinyl)oxy]ethylamine (0.5 g, 1.29 mmol, 1 eq, TFA). The mixture was stirred at 25°C for 0.5 h. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35μm 100A 120 g; mobile phase: [water-ACN]; B%: 0%-70% @ 100 mL / min) to obtain (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[(1-pent-4-alkynylsulfonyl-4-piperidinyl)oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]methyl hexanoate (370 mg, 527.94 μmol, yield 41.01%), as a colorless oil.

[0496] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Copper sulfate (36.44 mg, 228.30 g) was added to a solution of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[(1-pent-4-ynylsulfonyl-4-piperidinyl)oxy]ethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]methyl hexanoate (0.32 g, 456.59 μmol, 1 eq) and 2-(6-azidohexyl)-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (314.11 mg, 456.59 μmol, 1 eq) in t-BuOH (1 mL) and H2O (1 mL). (35.04 μL, 0.5 eq) and (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (90.45 mg, 456.59 μmol, 1 eq). The mixture was stirred at 50 °C for 1 hour. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 120 g; mobile phase: [water-ACN]; B%: 0%-70% @ 100 mL / min) to obtain (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (0.5 It is a white solid (360.03 μmol, yield 78.85%).

[0497] data: LCMS (ESI + ): m / z 1389.9 (M+H)+

[0498] 3. General procedure for preparing (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: TFA (1.54 g, 13.46 mmol, 1 mL, 38.16 eq) was added to a DCM (3 mL) solution of (2S)-2-[[6-[3-[2-[2-[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (0.49 g, 352.83 μmol, 1 eq). The mixture was stirred at 25 °C for 12 hours. DCM and TFA were removed under reduced pressure to obtain (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (1 g, crude product, TFA), which is a colorless oil.

[0499] 4. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O (82.55 mg, 1.97 mmol, 3 eq) to a solution of (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (0.9 g, 655.71 μmol, 1 eq, TFA) in THF (0.5 mL) and H2O (0.5 mL). Stir the mixture at 0 °C for 1 hour. The aqueous layer was acidified to pH 4 using FA (1 M). The mixture was filtered, and the filtrate was passed through a preparative HPLC system (neutral conditions: column: Waters Xbridge Prep OBD C18 150). 40mm Purification was performed using a mobile phase of [H2O(10mM NH4HCO3)-ACN] and a gradient of 25%-55% B over 8.0 min to obtain (2S)-2-[[6-[3-[2-[2-[[1-[3-[1-[6-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]propylsulfonyl]-4-piperidinyl]oxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (193.4 mg, 155.40 μmol, yield 23.70%, purity 100%), a white solid.

[0500] data: LCMS (ESI + ): m / z 1244.6 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.62 (d, J = 2.4 Hz, 1H), 8.23 ​​(dd, J =2.5, 8.6 Hz, 1H), 8.15 - 8.10 (m, 1H), 7.70 (s, 1H), 7.64 (d, J = 8.6 Hz,2H), 7.50 (d, J = 2.1 Hz, 1H), 7.34 (t, J = 8.2 Hz, 1H), 7.24 (d, J = 8.6 Hz,2H), 6.98 (d, J = 8.6 Hz, 1H), 6.89 - 6.85 (m, 2H), 6.82 - 6.74 (m, 2H), 4.89(br s, 1H), 4.59 - 4.42 (m, 5H), 4.26 (t, J = 7.0 Hz, 2H), 3.57 - 3.50 (m,2H), 3.46 - 3.40 (m, 3H), 3.39 - 3.32 (m, 2H), 3.06 (ddd, J = 3.6, 7.8, 11.9Hz, 2H), 3.00 - 2.95 (m, 2H), 2.78 (t, J = 7.4 Hz, 2H), 2.22 (s, 6H), 2.07(quin, J = 7.5 Hz, 2H), 2.01 - 1.91 (m, 1H), 1.88 - 1.73 (m, 7H), 1.63 - 1.52(m, 2H), 1.42 - 1.21 (m, 6H), 0.90 (s, 9H), 0.86 (br d, J = 5.4 Hz, 3H), 0.59- 0.51 (m, 1H), 0.50 - 0.44 (m, 2H), 0.43 - 0.35 (m, 2H), 0.32 - 0.22 (m, 3H).

[0501] X-049: (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0502] 1. A general procedure for preparing methyl 2-[3-(tert-butoxycarbonylamino)propyl]pyrazole-3-carboxylic acid: A mixture of N-(3-hydroxypropyl)carbamate tert-butyl ester (10 g, 57.07 mmol, 9.76 mL, 1 eq), methyl 1H-pyrazole-5-carboxylate (7.20 g, 57.07 mmol, 1 eq), and PPh3 (19.46 g, 74.19 mmol, 1.3 eq) in THF (300 mL) was degassed and purged three times with N2. DEAD (12.92 g, 74.19 mmol, 13.49 mL, 1.3 eq) was then added dropwise at 0°C, followed by stirring at 20°C for 12 hours under N2 atmosphere. The reaction mixture was quenched by adding H2O (200 mL) and precipitated with EA (600 mL, 200 mL). 3) Extraction. The combined organic layers were rinsed with 300 mL of brine (100 mL). 3) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 80g SepaFlash® silica gel column, eluent of 0-31% ethyl acetate / petroleum ether gradient, flow rate 100mL / min) to obtain methyl 2-[3-(tert-butoxycarbonylamino)propyl]pyrazole-3-carboxylic acid (15.2 g, 53.65 mmol, yield 94.01%), as a colorless oil.

[0503] data: LCMS (ESI + ): m / z 284.1 (M+H) + 1 HNMR: 1H NMR (400 MHz, chloroform-d) δ = 7.50 (d, J = 2.0 Hz, 1H), 6.88 -6.80 (m, 1H), 4.98 (br s, 1H), 4.64 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 3.07 (q, J = 6.1 Hz, 2H), 2.03 (quin, J = 6.5 Hz, 2H), 1.45 (s, 9H).

[0504] 2. General procedure for preparing methyl 2-(3-aminopropyl)pyrazole-3-carboxylic acid: TFA (50 mL) was added to a DCM (150 mL) solution of methyl 2-[3-(tert-butoxycarbonylamino)propyl]pyrazole-3-carboxylate (15 g, 52.94 mmol, 1 eq). The mixture was stirred at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give methyl 2-(3-aminopropyl)pyrazole-3-carboxylate (15 g, crude, TFA) as a yellow oil.

[0505] data: LCMS (ESI + ): m / z 184.1 (M+H) +

[0506] 3. A general procedure for preparing methyl 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid: At 0 °C, HATU (5.12 g, 13.46 mmol, 2 eq) and DIEA (1.74 g, 13.46 mmol, 2.34 mL, 2 eq) were added to a DMF (40 mL) solution of methyl 2-(3-aminopropyl)pyrazole-3-carboxylate (2 g, 6.73 mmol, 1 eq) and 2-azidoacetic acid (680.04 mg, 6.73 mmol, 1 eq). The mixture was stirred at 20 °C for 1 hour. The reaction mixture was quenched by adding H2O (50 mL) and DIEA (150 mL, 50 mL) to the solution. 3) Extraction. Combine the organic layers and extract with brine (150 mL, 50 mL). 3) Wash, dry with Na2SO4, filter, and concentrate under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 40 g SepaFlash® silica gel column, eluent 0-69% ethyl acetate / petroleum ether gradient, flow rate 100 mL / min) to obtain compound 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid methyl ester (2 g, crude product), which is a yellow oil.

[0507] data: LCMS (ESI + ): m / z 267.1 (M+H) + 1 HNMR: 1 ¹H NMR (400 MHz, chloroform-d) δ = 7.52 (d, J = 1.9 Hz, 1H), 6.85 (d,J = 1.9 Hz, 1H), 4.65 (t, J = 6.4 Hz, 2H), 3.96 (s, 2H), 3.89 (s, 3H), 3.25(q, J = 6.2 Hz, 2H), 2.13 - 2.04 (m, 2H).

[0508] 4. A general procedure for preparing 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid:

[0509] LiOH·H2O (472.82 mg, 11.27 mmol, 2 eq) was added to a solution of methyl 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (1.5 g, 5.63 mmol, 1 eq) in THF (10 mL) and H2O (10 mL). The mixture was stirred at 15 °C for 1 h. THF was removed under reduced pressure, and the residue was acidified to pH 4 with 2 M HCl. The mixture was purified by preparative HPLC (column: C18 20-40 μm 100 A 330 g; mobile phase: [water-ACN]; B%: 0%-5% @ 100 mL / min) to give 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (570 mg, 2.26 mmol, yield 40.11%) as a colorless oil.

[0510] data: LCMS (ESI + ): m / z 253.1 (M+H) +

[0511] 5. General procedure for the preparation of 3,5-dimethyl-4-(4-nitrophenyl)-1H-pyrazole:

[0512] A mixture of 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxapentoborane-2-yl)-1H-pyrazole (7.43 g, 33.47 mmol, 1 eq), 1-iodo-4-nitrobenzene (10 g, 40.16 mmol, 1.2 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane complex (2.73 g, 3.35 mmol, 0.1 eq), and K2CO3 (11.56 g, 83.67 mmol, 2.5 eq) in dioxane (200 mL) and H2O (20 mL) was degassed and purged three times with N2. The mixture was then stirred at 100 °C for 36 hours under N2 atmosphere. The reaction mixture was cooled to 20 °C, diluted with water (70 mL), and then precipitated with EtOAc (300 mL, 100 mL). 3) Extraction. Combine the organic layers and extract with saturated NaCl (2... The sample was washed with 100 mL of water, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to give compound 3,5-dimethyl-4-(4-nitrophenyl)-1H-pyrazole (5 g, 23.02 mmol, yield 68.78%) as a pale yellow solid.

[0513] data: 1 H NMR (400 MHz, DMSO-d6) δ = 12.54 (br s, 1H), 8.41 - 8.10 (m, 2H),7.64 - 7.47 (m, 2H), 2.26 (br s, 6H)

[0514] 6. A general procedure for preparing 2-[[3,5-dimethyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyltrimethylsilane:

[0515] At 0 °C, NaH (1.84 g, 46.04 mmol, 60% purity, 2 eq) was added to a stirred solution of 3,5-dimethyl-4-(4-nitrophenyl)-1H-pyrazole (5 g, 23.02 mmol, 1 eq) in DMF (60 mL), and the reaction was stirred at 0 °C for 0.5 h. SEMCl (5.76 g, 34.53 mmol, 6.11 mL, 1.5 eq) was added at 0 °C, and the reaction was stirred at 20 °C for 1 h. The mixture was quenched at 0 °C with saturated NH4Cl solution (100 mL) and treated with EtOAc (450 mL, 150 mL). 3) Extraction. Combine the organic layers and extract with saturated NaCl (100 mL). 2) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 7 / 1) to give compound 2-[[3,5-dimethyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyltrimethylsilane (7.5 g, 21.58 mmol, yield 93.77%), a pale yellow oil.

[0516] data: 1 H NMR (400 MHz, DMSO-d6) δ = 8.27 (d, J = 8.8 Hz, 2H), 7.58 (d, J =8.8 Hz, 2H), 5.39 (s, 2H), 3.57 (t, J = 7.9Hz, 2H), 2.33 (s, 3H), 2.21 (s,3H), 0.88 - 0.83 (m, 2H), -0.04 (s, 9H)

[0517] 7. A general procedure for preparing 4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline:

[0518] Fe (6.03 g, 107.92 mmol, 5 eq) and NH4Cl (5.77 g, 107.92 mmol, 5 eq) were added to a solution of 2-[[3,5-dimethyl-4-(4-nitrophenyl)pyrazol-1-yl]methoxy]ethyltrimethylsilane (7.5 g, 21.58 mmol, 1 eq) in EtOH (80 mL) and H2O (16 mL). The mixture was stirred at 50 °C for 2 hours. The reaction mixture was cooled to 20 °C, filtered, and the filtrate was concentrated under reduced pressure; the mixture was diluted with water (70 mL) and then with EtOAc (300 mL, 100 mL). 3) Extraction. Combine the organic layers and extract with saturated NaCl (2... The sample was washed with 100 mL of the solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to give compound 4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline (5.5 g, 17.32 mmol, yield 80.26%) as a yellow solid.

[0519] data: 1 H NMR (400 MHz, DMSO-d6) δ = 6.90 (d, J = 8.3 Hz, 2H), 6.61 (d, J =8.4 Hz, 2H), 5.30 (s, 2H), 5.05 (s, 2H), 3.54(t, J = 7.9 Hz, 2H), 2.20 (s,3H), 2.09 (s, 3H), 0.83 (t, J = 7.9 Hz, 2H), -0.04 (s, 9H)

[0520] 8. A general procedure for the preparation of N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]tert-butyl carbamate:

[0521] At 0 °C, HATU (4.52 g, 11.88 mmol, 1 eq) and DIEA (1.54 g, 11.88 mmol, 2.07 mL, 2 eq) were added to a DMF (40 mL) solution of 4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]aniline (1.89 g, 5.94 mmol, 1 eq) and (2S)-2-(tert-butoxycarbonylamino)-3,3-dicyclopropyl-propionic acid (1.6 g, 5.94 mmol, 1 eq). The mixture was stirred at 15 °C for 1 hour. The mixture was quenched with saturated H2O solution (50 mL) and DIEA (150 mL, 50 mL) was added. 3) Extraction, using saline (150 mL, 50 mL) 3) Washing. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]tert-butyl carbamate (2.8 g, 4.92 mmol, yield 82.86%), as a colorless oil.

[0522] data: 1H NMR (400 MHz, DMSO-d6) δ = 9.93 (s, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 6.82 (br d, J = 9.1 Hz, 1H), 5.34 (s, 2H), 4.30 (brs, 1H), 3.55 (t, J = 7.9 Hz, 2H), 2.25 (s, 3H), 2.13 (s, 3H), 1.37 (br s,9H), 0.88 - 0.81 (m, 3H), 0.79 - 0.72 (m, 1H), 0.56 (br d, J = 7.0 Hz, 1H),0.48 - 0.42 (m, 1H), 0.37 - 0.23 (m, 3H), 0.18 (td, J = 4.7, 8.9 Hz, 4H), -0.02 - -0.06 (m, 9H)

[0523] 9. A general procedure for the preparation of (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]propionamide:

[0524] A solution of N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]tert-butyl carbamate (2.7 g, 4.75 mmol, 1 eq) in 4M HCl / MeOH (30 mL) was stirred at 15 °C for 2 hours. The solution was concentrated under reduced pressure to give (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]phenyl]propionamide (2.1 g, crude product, HCl salt), as a pale yellow solid.

[0525] data: LCMS (ESI + ): m / z 469.5 (M+H) +

[0526] 10. A general procedure for the preparation of 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide:

[0527] At 0 °C, HATU (1.31 g, 3.44 mmol, 2 eq) and DIEA (667.75 mg, 5.17 mmol, 899.93 μL, 3 eq) were added to a DMF (15 mL) solution of 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (434.39 mg, 1.72 mmol, 1 eq) and (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazole-4-yl]phenyl]propionamide (870 mg, 1.72 mmol, 1 eq, HCl salt) and added to a solution of HCl salt of 2-[3-[(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (434.39 mg, 1.72 mmol, 1 eq) and (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazole-4-yl]phenyl]propionamide (870 mg, 1.72 mmol, 1 eq, HCl salt). The mixture was stirred at 20 °C for 1 hour. The reaction mixture was quenched by adding H2O (20 mL) and EDTA (45 mL, 15 mL) and added to a solution of EDTA (45 mL, 15 mL). 3) Extraction. Combine the organic layers and extract with brine (45 mL, 15 mL). 3) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (ISCO®; 12 g SepaFlash® silica gel column, eluent 0–100% ethyl acetate / petroleum ether gradient, flow rate 100 mL / min) to give compound 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (930 mg, 1.32 mmol, yield 76.82%), a white solid.

[0528] data: LCMS (ESI + ): m / z 703.5 (M+H) + 1H NMR (400 MHz, chloroform-d) δ = 8.07 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 2.0 Hz, 1H), 7.40 - 7.32 (m, 1H), 7.24 (d, J = 8.6 Hz, 2H), 7.14(d, J = 8.2 Hz, 1H), 6.62 (d, J = 2.0 Hz, 1H), 5.40 (s, 2H), 4.85 (dd, J =5.0, 8.2 Hz, 1H), 4.75 - 4.66 (m, 1H), 4.61 - 4.50 (m, 1H), 3.91 (s, 2H),3.67 - 3.59 (m, 2H), 3.23 (br d, J = 6.1 Hz, 2H), 2.31 (s, 3H), 2.26 - 2.21(m, 3H), 2.16 - 2.08 (m, 2H), 1.02 - 0.90 (m, 3H), 0.89 - 0.78 (m, 2H), 0.67(dt, J = 4.0, 8.4 Hz, 1H), 0.62 - 0.52 (m, 3H), 0.46 - 0.34 (m, 2H), 0.28 (brd, J = 3.7 Hz, 2H), -0.01 (s, 9H)

[0529] 11. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0530] Add copper hexafluorophosphate acetonitrile (I) (194.32 mg, 521.37 μmol) to a DMSO (1 mL) solution of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]hexanoate (90 mg, 140.91 μmol) and 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (79.24 mg, 112.73 μmol). The mixture was stirred at 20 °C for 1 hour. The mixture was filtered, and the filtrate was subjected to reversed-phase HPLC (column: C1820-35 μm 100A 330 g; mobile phase: [water-ACN]; B%: 0%-65% @ 100). Purification at a rate of mL / min yielded compound (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (22 mg, 16.40 μmol, yield 11.64%), a white solid.

[0531] data: LCMS (ESI + ): m / z 1341.6 (M+H) +

[0532] 12. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0533] Add TFA (30.70 mg, 269.24 μmol, 0.02 mL) to a DCM (0.1 mL) solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (22 mg, 16.40 μmol). The mixture was stirred at 0 °C for 0.5 hours. The mixture was concentrated under reduced pressure to give compound (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (20 mg, 15.09 μmol, yield 92.02%, TFA), as a yellow oil.

[0534] data: LCMS (ESI + ): m / z 606.6 (M / 2+H) +

[0535] 13. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0536] Add LiOH·H2O (2.22 mg, 52.81 μmol) to a solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (35 mg, 26.41 μmol, TFA) in THF (0.1 mL) and H2O (0.1 mL). The mixture was stirred at 0°C for 0.5 hours. The mixture was then filtered, and the filtrate was passed through a preparative HPLC system (column: Waters Xbridge BEH C18100). 30mm 10 μm; Mobile phase: [H2O(10mM NH4HCO3)-ACN]; Gradient: 19%-49% over 8.0 min B) Purification yielded compound X-049 (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (5.1 mg, 4.19 mg). It is a white solid (μmol, yield 15.87%, purity 98.38%).

[0537] data: LCMS (ESI + ): m / z 1197.5 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.61 (d, J = 2.4 Hz, 1H), 8.23 ​​(dd, J =2.5, 8.7 Hz, 1H), 8.03 (s, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 2.0Hz, 1H), 7.37 - 7.30 (m, 1H), 7.22 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.7 Hz,1H), 6.91 - 6.85 (m, 2H), 6.81 (t, J = 2.1 Hz, 1H), 6.79 - 6.74 (m, 1H), 5.11(s, 2H), 4.87 (br d, J = 7.4 Hz, 1H), 4.64 - 4.57 (m, 3H), 4.56 - 4.48 (m,4H), 4.37 - 4.31 (m, 1H), 4.29 - 4.23 (m, 1H), 4.13 (br d, J = 10.0 Hz, 1H),4.05 - 3.98 (m, 3H), 3.79 - 3.72 (m, 1H), 3.49 - 3.43 (m, 4H), 3.23 - 3.11(m, 2H), 2.22 (s, 6H), 2.08 - 1.92 (m, 3H), 1.87 - 1.75 (m, 1H), 1.41 - 1.28(m, 2H), 0.90 (s, 9H), 0.90 - 0.71 (m, 3H), 0.60 - 0.53 (m, 1H), 0.52 (br s,2H), 0.43 - 0.35 (m, 2H), 0.33 - 0.22 (m, 3H).

[0538] X-059: (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0539] 1. A general procedure for preparing methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate:

[0540] The reaction was carried out in five parallel batches.

[0541] At 0 °C, DBU (3.68 g, 24.15 mmol, 3.64 mL, 1 eq) was added to a solution of methyl 2-(benzyloxycarbonylamino)-2-dimethoxyphosphoryl-acetate (8 g, 24.15 mmol, 1 eq) in NMP (200 mL), and the reaction was stirred for 0.5 h. Then, a solution of 4,4-difluorocyclohexanone (4.05 g, 30.19 mmol, 1.25 eq) in NMP (50 mL) was added to the mixture. The mixture was stirred at 15 °C for 3 h. The mixture was quenched at 0 °C with a saturated H2O solution (300 mL) and treated with EtOAc (900 mL, 300 mL). 3) Extraction, using saline solution (900 mL, 300 mL) 3) Washing. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate (40 g, 117.88 mmol, yield 97.62%) as a white solid.

[0542] data: 1 H NMR (400 MHz, DMSO-d6) δ = 7.42 - 7.29 (m, 5H), 5.11 - 5.03 (m,2H), 3.63 (s, 3H), 2.61 (br t, J = 6.3 Hz, 2H), 2.37 (br t, J = 6.3 Hz, 2H),2.00 - 1.93 (m, 4H)

[0543] 2. General procedure for preparing methyl 2-amino-2-(4,4-difluorocyclohexyl)acetate:

[0544] The reaction was carried out in two parallel batches.

[0545] Pd / C (3.76 g, 3.54 mmol, 10% purity, 0.15 eq) was added to a solution of methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate (8 g, 23.58 mmol, 1 eq) in EtOAc (50 mL) under a nitrogen atmosphere. The suspension was degassed and purged three times with H2. The mixture was stirred at 20 °C for 6 hours under a H2 (15 Psi) atmosphere. The reaction mixtures were combined, filtered, and the filtrate was concentrated under reduced pressure to give methyl 2-amino-2-(4,4-difluorocyclohexyl)acetate (12 g, crude product) as a colorless oil.

[0546] 3. General procedure for preparing methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate:

[0547] The reaction was carried out in two parallel batches.

[0548] At 0 °C, TEA (5.86 g, 57.91 mmol, 8.06 mL, 2 eq) and Cb2Cl (7.41 g, 43.43 mmol, 6.20 mL, 1.5 eq) were added to a DCM (90 mL) solution of methyl 2-amino-2-(4,4-difluorocyclohexyl)acetate (6 g, 28.96 mmol, 1 eq). The mixture was stirred at 20 °C for 4 hours. At 0 °C, the mixture was acidified to pH 5–6 using HCl (2 M). The mixture was then rinsed with 450 mL of DCM (150 mL of TEA). 3) Extraction. The combined organic layers were treated with 300 mL of saturated NaCl (150 mL of saturated NaCl). 2) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 4 / 1; TLC showing the target product as the spot with Rf = 0.5 at PE / EA = 3:1) to obtain methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate (10 g, crude), a colorless oil. The crude product was purified by reversed-phase HPLC (column: C18 20-40 μm 100 A 330 g; mobile phase: [water-ACN]; B%: 0%-85%; flow rate: 100 mL / min) to obtain methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate (5.5 g, 16.11 mmol, yield 55.00%), a colorless oil.

[0549] data: 1H NMR (400 MHz, DMSO-d6) δ = 7.82 (br d, J = 8.2 Hz, 1H), 7.40 - 7.30(m, 5H), 5.04 (s, 2H), 4.09 - 4.05 (m, 1H), 3.64 (s, 3H), 2.04 - 2.00 (m,1H), 1.93 - 1.82 (m, 2H), 1.81 - 1.69 (m, 2H), 1.66 (br d, J = 8.9 Hz, 2H), 1.45 - 1.33 (m, 1H), 1.32 - 1.23 (m, 1H)

[0550] 4. General procedure for preparing 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetic acid:

[0551] At 0 °C, LiOH·H₂O (1.35 g, 32.23 mmol, 2 eq) was added to a solution of methyl 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetate (5.5 g, 16.11 mmol, 1 eq) in THF (55 mL) and H₂O (55 mL). The mixture was stirred at 20 °C for 1 hour. The mixture was acidified with HCl (1 M) to pH 5–6. Subsequently, the mixture was treated with EtOAc 300 mL (100 mL) 3) Extraction. The combined organic layers were washed with 100 mL of saturated NaCl, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 2 / 1; TLC showing the spot with Rf = 0.2 at PE / EA = 3:1 as the target product) to give compound 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetic acid (3.3 g, 10.08 mmol, yield 62.57%) as a white solid.

[0552] 5. General procedure for preparing tert-butyl 4-(benzyloxycarbonylamino)-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate: At 20 °C, CDI (1.53 g, 9.41 mmol, 1.1 eq) was added to a 45 mL THF solution of 2-(benzyloxycarbonylamino)-2-(4,4-difluorocyclohexyl)acetic acid (2.8 g, 8.55 mmol, 1 eq), and the mixture was stirred at 20 °C for 1.5 h (to form solution A). Under a N2 atmosphere and at -70 °C, LiHMDS (1 M, 27.37 mL, 3.2 eq) was added to a 45 mL THF solution of tert-butyl acetate (3.18 g, 27.37 mmol, 3.67 mL, 3.2 eq), and the mixture was stirred for 1 h. Solution A was then added to the mixture at -70 °C. The resulting mixture was stirred at -70 °C for 1 h. The reaction mixture was added to 200 mL of ice-saturated NH4Cl solution, and then 300 mL of EtOAc (100 mL) was added. 3) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 120 g SepaFlash® silica gel column, eluent 0-30% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 3:1) to obtain compound 4-(benzyloxycarbonylamino)-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate tert-butyl ester (2.75 g, crude product), as a yellow oil.

[0553] data: LCMS (ESI-): m / z 424.2 (MH) - 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (s, 9 H) 1.52 (br d, J=15.65 Hz,1 H) 1.62 - 1.87 (m, 4 H) 1.99 (s, 4 H) 3.46 - 3.59 (m, 2 H) 4.16 (dd, J=8.62, 6.17 Hz, 1 H) 5.03 - 5.06 (m, 2 H) 7.31 - 7.39 (m, 5 H) 7.81 (d, J=8.68Hz, 1 H)

[0554] 6. General procedure for preparing tert-butyl 4-(benzyloxycarbonylamino)-2-bromo-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate:

[0555] At 20 °C, 2,6-dimethylpyridine (55.41 mg, 517.08 μmol, 60.22 μL, 0.08 eq) and NBS (1.06 g, 5.95 mmol, 0.92 eq) were added to a MeOH (65 mL) solution of 4-(benzyloxycarbonylamino)-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate tert-butyl ester (2.75 g, 6.46 mmol, 1 eq). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was added to 200 mL of ice water, and then 400 mL of EtOAc (200 mL) was added. 2) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 120 g SepaFlash® silica gel column, eluent 0-30% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 3:1) to obtain compound 4-(benzyloxycarbonylamino)-2-bromo-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate tert-butyl ester (2.55 g, crude product), as a pale yellow oil.

[0556] data: LCMS (ESI-): m / z 502.2 (MH) - 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (d, J=3.42 Hz, 9 H) 1.41 - 1.91 (m, 5 H) 1.95 - 2.17 (m, 4 H) 4.44 - 4.54 (m, 1 H) 5.00 - 5.12 (m, 2 H) 5.59- 5.73 (m, 1 H) 7.30 - 7.40 (m, 5 H) 7.92 (dd, J=9.41, 3.30 Hz, 1 H)

[0557] 7. General procedure for the preparation of N-[3-bromo-1-(4,4-difluorocyclohexyl)-2-oxo-propyl]carbamate:

[0558] At 20 °C, TFA (3.25 mL) was added to a solution of tert-butyl 4-(benzyloxycarbonylamino)-2-bromo-4-(4,4-difluorocyclohexyl)-3-oxo-butyrate (2.55 g, 5.06 mmol, 1 eq) in toluene (30 mL). The mixture was stirred at 80 °C for 2 hours. The reaction mixture was added to 50 mL of water, and then 80 mL of EtOAc (40 mL) was added. 2) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 80 g SepaFlash® silica gel column, eluent 0–30% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 3:1) to give compound N-[3-bromo-1-(4,4-difluorocyclohexyl)-2-oxo-propyl]carbamate (1.6 g, 3.96 mmol, yield 78.28%) as a white solid.

[0559] 8. A general procedure for the preparation of N-[(1S)-3-bromo-1-(4,4-difluorocyclohexyl)-2-oxo-propyl]carbamate: Residue was passed through SFC (column: ChiralPak IH, 250). Separation was performed using a mobile phase of [CO2-IPA] (30 mm, 10 μm; B%: 12%, isocratic elution mode) to obtain compound N-[(1S)-3-bromo-1-(4,4-difluorocyclohexyl)-2-oxo-propyl]carbamate (820 mg, crude product), which was a white solid.

[0560] data: 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.31 (q, J=12.51 Hz, 2 H) 1.48 - 1.65 (m, 2 H) 1.67 - 1.88 (m, 2 H) 2.00 (br s, 3 H) 4.34 (dd, J=8.31, 6.36 Hz, 1H) 4.41 - 4.54 (m, 2 H) 5.05 (s, 2 H) 7.28 - 7.41 (m, 5 H) 7.86 (d, J=8.56Hz, 1 H)

[0561] 9. A general procedure for preparing N-(6-chloro-5-methylpyridazin-3-yl)-2,2-dimethylpropionamide:

[0562] The reaction was carried out in three parallel batches.

[0563] Under a nitrogen atmosphere and at 15 °C, Py (27.41 g, 346.51 mmol, 27.97 mL, 1.99 eq) and 2,2-dimethylpropionyl chloride (27.50 g, 228.11 mmol, 28.07 mL, 1.31 eq) were added to a solution of 6-chloro-5-methylpyridazin-3-amine (25 g, 174.13 mmol, 1 eq) in NMP (100 mL). The mixture was stirred for 1 hour under a nitrogen atmosphere and at 15 °C. The mixture was then heated to 35 °C and stirred for another 0.5 hours until the reaction mixture became clear. The three batches of reaction were cooled to 15 °C and combined for post-processing. 405 mL of H2O was added to the combined reaction mixture, followed by stirring at 5 °C for 1 hour. The resulting thick slurry was filtered to collect the solid, and washed with cold water (315 mL) to give compound N-(6-chloro-5-methyl-pyridazin-3-yl)-2,2-dimethyl-propionamide (121 g, crude product), which is a white solid.

[0564] data: LCMS (ESI + ): m / z 228.2 (M+H) + 1 ¹H NMR (400 MHz, chloroform-d) δ = 8.73 (br s, 1H), 8.42 (s, 1H), 2.39 (s, 3H), 1.32 (s, 9H)

[0565] 10. A general procedure for the preparation of N-[6-chloro-5-[(E)-2-(dimethylamino)vinyl]pyridazin-3-yl]-2,2-dimethylpropionamide:

[0566] The reaction was carried out in three parallel batches.

[0567] A mixture of N-(6-chloro-5-methylpyridazin-3-yl)-2,2-dimethylpropionamide (40 g, 175.68 mmol, 1 eq) and 1,1-diethoxy-N,N-dimethyl-methylamine (103.45 g, 702.71 mmol, 120.43 mL, 4 eq) in DMF (16 mL) was degassed and purged three times with N2, followed by stirring at 120 °C for 12 h under N2 atmosphere. The three batches of reaction were cooled to 20 °C and combined for post-treatment. The combined reaction mixture was allowed to stand at 20 °C for 2 h, during which time a large amount of yellow crystals precipitated at the bottom of the flask. The obtained solid was filtered and washed with TBME (500 mL) to give compound N-[6-chloro-5-[(E)-2-(dimethylamino)vinyl]pyridazin-3-yl]-2,2-dimethyl-propionamide (35 g, crude product), which was a yellow solid.

[0568] data: LCMS (ESI + ): m / z 283.3 (M+H) + ¹H NMR (400 MHz, chloroform-d) δ = 8.31 - 8.24 (m, 2H), 7.24 (d, J = 3.5 Hz, 1H), 5.13 (d, J = 13.2 Hz, 1H), 2.97 (s, 6H), 1.31 (s, 9H)

[0569] 11. A general procedure for the preparation of N-(6-chloro-5-formyl-pyridazin-3-yl)-2,2-dimethyl-propionamide:

[0570] To a solution of N-[6-chloro-5-[(E)-2-(dimethylamino)vinyl]pyridazin-3-yl]-2,2-dimethylpropionamide (17.5 g, 61.89 mmol, 1 eq) in THF (110 mL) and H₂O (110 mL), NaIO₄ (46.33 g, 216.61 mmol, 12.00 mL, 3.5 eq) was added. The mixture was stirred at 15 °C for 45 minutes. The reaction solution was filtered and washed with EtOAc (300 mL). The resulting filtrate was rinsed with EtOAc 400 mL (200 mL) 2) Extraction. The combined organic layers were rinsed with 800 mL (400 mL) of brine. 2) The product was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (ISCO®; 330 g SepaFlash® silica gel column, eluent 0–40% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 1:1) to give N-(6-chloro-5-formyl-pyridazin-3-yl)-2,2-dimethyl-propionamide (14.5 g, crude), a yellow solid. The crude product was ground at 20 °C with PE:EA = 10:1 (150 mL) for 1 hour to give N-(6-chloro-5-formyl-pyridazin-3-yl)-2,2-dimethyl-propionamide (25.5 g, crude), a yellow solid.

[0571] data: 1 H NMR (400 MHz, chloroform-d) δ ppm 1.37 (s, 9 H) 8.71 (br s, 1 H) 8.88 (s, 1 H) 10.39 (s, 1 H)

[0572] 12. A general procedure for preparing N-[6-chloro-5-(epoxyethylene-2-yl)pyridazin-3-yl]-2,2-dimethylpropionamide:

[0573] The reaction was carried out in two parallel batches.

[0574] At 0 °C, NaH (1.89 g, 47.30 mmol, 788.25 μL, 60% purity, 1.8 eq) was added to a solution of trimethyl sulfoxide (11.58 g, 56.75 mmol, 2.16 eq) in THF (70 mL) and DMSO (70 mL). After the addition was complete, the mixture was stirred at 0 °C for 0.5 h, followed by the addition of N-(6-chloro-5-formyl-pyridazin-3-yl)-2,2-dimethyl-propionamide (6.35 g, 26.28 mmol, 1 eq) at 0 °C. The resulting mixture was stirred at 20 °C for 1.5 h. The reaction mixture was quenched at 0 °C by the addition of 150 mL of NH4Cl, followed by dilution with 50 mL of H2O and 600 mL of EtOAc (200 mL of NH4Cl). 3) Extraction. The combined organic layers were rinsed with 600 mL (200 mL) of brine. 3) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (ISCO®; 120 g SepaFlash® silica gel column, eluent 0–25% ethyl acetate / petroleum ether gradient; flow rate: 120 mL / min; PE:EA = 2:1) to give N-[6-chloro-5-(epoxyethylene-2-yl)pyridazin-3-yl]-2,2-dimethylpropionamide (6.5 g, 25.42 mmol, yield 48.37%), a yellow oil.

[0575] data: LCMS (ESI + ): m / z 256.2 (M+H) + 1 H NMR (400 MHz, chloroform-d) δ ppm 1.35 (s, 9 H) 2.75 (dd, J=5.75, 2.38Hz, 1 H) 3.29 (dd, J=5.63, 4.25 Hz, 1 H) 4.10 (br d, J=2.25 Hz, 1 H) 8.40 (s,1 H) 8.50 (br s, 1 H)

[0576] 13. A general procedure for preparing N-[6-chloro-5-(1-hydroxy-2-methoxy-ethyl)pyridazin-3-yl]-2,2-dimethyl-propionamide: The reaction was carried out in two parallel batches.

[0577] At 20 °C, NaOMe (9.51 g, 52.80 mmol, 2 eq) was added to a solution of N-[6-chloro-5-(epoxyethylene-2-yl)pyridazin-3-yl]-2,2-dimethylpropionamide (6.75 g, 26.40 mmol, 1 eq) in DMA (60 mL). The mixture was stirred at 50 °C for 2 hours. The reaction mixture was quenched by adding 100 mL of H2O and 100 mL of EtOAc (300 mL of DMA) was added. 3) Extraction. The combined organic layers were rinsed with 600 mL (300 mL) of brine. 2) The residue was washed, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by rapid silica gel column chromatography (ISCO®; 120 g SepaFlash® silica gel column, eluent gradient of 0–36% ethyl acetate / petroleum ether; flow rate: 120 mL / min, PE:EA = 1:1) to give N-[6-chloro-5-(1-hydroxy-2-methoxy-ethyl)pyridazin-3-yl]-2,2-dimethyl-propionamide (5.01 g, 17.41 mmol, yield 33.40%) as a white solid.

[0578] data: LCMS (ESI + ): m / z 288.3 (M+H) + 1 H NMR (400 MHz, chloroform-d) δ ppm 1.34 - 1.36 (m, 9 H) 3.39 - 3.48 (m, 4H) 3.76 (dd, J=9.83, 3.04 Hz, 1 H) 5.13 (dd, J=7.09, 3.04 Hz, 1 H) 8.58 (brd, J=9.42 Hz, 1 H) 8.77 (s, 1 H)

[0579] 14. A general procedure for preparing N-[6-chloro-5-(2-methoxyacetyl)pyridazin-3-yl]-2,2-dimethylpropionamide:

[0580] At 20 °C, DMP (9.39 g, 22.14 mmol, 6.86 mL, 1.3 eq) was added to a solution of N-[6-chloro-5-(1-hydroxy-2-methoxy-ethyl)pyridazin-3-yl]-2,2-dimethyl-propionamide (4.9 g, 17.03 mmol, 1 eq) in DCM (150 mL). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was then added to 150 mL of water and 400 mL of DCM (200 mL) was added. 2) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 120 g SepaFlash® silica gel column, eluent 0-35% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 2:1) to obtain compound N-[6-chloro-5-(2-methoxyacetyl)pyridazin-3-yl]-2,2-dimethylpropionamide (3.5 g, crude product), as a yellow solid.

[0581] data: LCMS (ESI + ): m / z 286.2 (M+H) + 1 H NMR (400 MHz, chloroform-d) δ ppm 1.37 (s, 9 H) 3.47 (s, 3 H) 4.49 (s, 2H) 8.62 (s, 1 H) 8.68 (br s, 1 H)

[0582] 15. A general procedure for the preparation of N-[5-[1-[[(2S)-2-amino-3,3,3-trifluoro-propyl]amino]-2-methoxy-ethyl]-6-chloro-pyridazin-3-yl]-2,2-dimethyl-propionamide:

[0583] The reaction was carried out in 10 parallel batches.

[0584] Ambersep 900 (OH) anion exchange resin was added to a MeOH solution of (2S)-3,3,3-trifluoropropane-1,2-diamine (77.39 mg, 384.98 μmol, 1.1 eq, 2HCl salt) until the pH was approximately 8. The mixture was then stirred at 20 °C for 0.5 h and filtered to obtain the filtrate. N-[6-chloro-5-(2-methoxyacetyl)pyridazin-3-yl]-2,2-dimethylpropionamide (100 mg, 349.99 μmol, 1 eq) and 4A MS (300 mg, 349.99 μmol) were added to the filtrate, and the mixture was stirred at 20 °C for 3 min. AcOH (31.53 mg, 524.98 μmol, 30.05 μL, 1.5 eq) was then added until the pH was approximately 5. The reaction mixture was stirred at 65 °C for 5 h. NaBH3CN (109.97 mg, 1.75 mmol, 5 eq) was then added to the reaction mixture at 20 °C, and the mixture was stirred at 65 °C for 12 hours. The combined reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the residue. 50 mL of saturated NaHCO3 solution was added to the residue, and 150 mL of DCM (50 mL...) was used. 3) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1; petroleum ether: ethyl acetate = 0 : 1) to give compound N-[5-[1-[[(2S)-2-amino-3,3,3-trifluoro-propyl]amino]-2-methoxy-ethyl]-6-chloro-pyridazin-3-yl]-2,2-dimethyl-propionamide (680 mg, crude), as a brown oil.

[0585] data: LCMS (ESI + ): m / z 398.3 (M+H) +

[0586] 16. A general procedure for the preparation of N-[6-chloro-5-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]pyridazin-3-yl]-2,2-dimethylpropionamide:

[0587] The reaction was carried out in nine parallel batches.

[0588] A mixture of N-[5-[1-[[(2S)-2-amino-3,3,3-trifluoropropyl]amino]-2-methoxy-ethyl]-6-chloro-pyridazin-3-yl]-2,2-dimethylpropionamide (0.1 g, 251.37 μmol, 1 eq) and CDI (81.52 mg, 502.74 μmol, 2 eq) in THF (2.5 mL) was stirred at 60 °C for 12 hours. The combined reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was analyzed by preparative HPLC (FA conditions; column: Phenomenex luna C18 100). 40mm 3 μm; mobile phase: [H2O(0.2%FA)-ACN]; gradient: 20%-50% over 8.0 min. B) Purification yielded compound N-[6-chloro-5-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]pyridazin-3-yl]-2,2-dimethyl-propionamide (235 mg, crude), a pale yellow solid, and compound N-[6-chloro-5-[(1R)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]pyridazin-3-yl]-2,2-dimethyl-propionamide (220 mg, crude), a pale yellow solid.

[0589] data: LCMS (ESI + ): m / z 424.1 (M+H) +

[0590] 17. A general procedure for the preparation of (4S)-1-[(1S)-1-(6-amino-3-chloro-pyridazin-4-yl)-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidine-2-one:

[0591] To a solution of N-[6-chloro-5-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]pyridazin-3-yl]-2,2-dimethylpropionamide (200 mg, 471.90 μmol, 1 eq) in MeOH (0.84 mL), HCl (3 mL) (5 M) was added. The mixture was stirred at 110 °C for 0.5 h. At 0 °C, 15% NaOH solution was added to the reaction mixture until the pH was approximately 9, followed by EtOAc 20 mL (10 mL) 2) Extraction. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound (4S)-1-[(1S)-1-(6-amino-3-chloro-pyridazin-4-yl)-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidine-2-one (180 mg, crude product), which was a yellow solid.

[0592] data: LCMS (ESI + ): m / z 340.2 (M+H) +

[0593] 18. A general procedure for the preparation of N-[(S)-[6-chloro-7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]-(4,4-difluorocyclohexyl)methyl]benzyl carbamate: To a THF (5 mL) solution of (4S)-1-[(1S)-1-(6-amino-3-chloro-pyridazin-4-yl)-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidine-2-one (180 mg, 529.88 μmol, 1 eq) and [(1S)-3-bromo-1-(4,4-difluorocyclohexyl)-2-oxo-propyl]carbamate (214.20 mg, 529.88 μmol, 1 eq), NaHCO3 (111.28 mg, 1.32 mmol, 51.54 μL, 2.5 eq) was added. The mixture was stirred at 70 °C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, ethyl acetate: methanol = 10:1) to give compound [(S)-[6-chloro-7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]-(4,4-difluorocyclohexyl)methyl]carbamate (245 mg, crude product), as a yellow solid.

[0594] data: LCMS (ESI + ): m / z 645.3 (M+H) +

[0595] 19. A general procedure for the preparation of (4S)-1-[(1S)-1-[2-[(S)-amino-(4,4-difluorocyclohexyl)methyl]imidazo[1,2-b]pyridazin-7-yl]-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidin-2-one:

[0596] At 20 °C, Pd / C (50 mg, 10% purity) was added to a MeOH (13 mL) solution of [(S)-[6-chloro-7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]-(4,4-difluorocyclohexyl)methyl]carbamate (245 mg, 379.83 μmol, 1 eq). The reaction was then degassed and purged three times with H2. The resulting mixture was stirred at 20 °C for 12 hours under an H2 atmosphere (15 PSI). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound (4S)-1-[(1S)-1-[2-[(S)-amino-(4,4-difluorocyclohexyl)methyl]imidazo[1,2-b]pyridazin-7-yl]-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidin-2-one (195 mg, crude, HCl salt), as a yellow solid.

[0597] data: LCMS (ESI + ): m / z 477.3 (M+H) +

[0598] 20. A general procedure for the preparation of 2-(6-azidohexyl)-N-[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]pyrazol-3-carboxamide:

[0599] At 20 °C, HATU (50 mg, 97.48 μmol, 1 eq, HCl salt) and DIEA (37.80 mg, 292.45 μmol, 50.94 μL, 3 eq) were added to a DMF (2 mL) solution of (4S)-1-[(1S)-1-[2-[(S)-amino-(4,4-difluorocyclohexyl)methyl]imidazo[1,2-b]pyridazin-7-yl]-2-methoxy-ethyl]-4-(trifluoromethyl)imidazolidin-2-one (50 mg, 97.48 μmol, 1 eq, HCl salt) and 2-(6-azidohexyl)pyrazole-3-carboxylic acid (27.75 mg, 116.98 μmol, 1.2 eq). The mixture was stirred at 20 °C for 1 hour. The reaction mixture was filtered to obtain a filtrate. The filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 60 mL / min) to obtain compound 2-(6-azidohexyl)-N-[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]pyrazol-3-carboxamide (56 mg, 80.50 μmol, yield 82.57%), as a white solid.

[0600] data: LCMS (ESI + ): m / z 696.5 (M+H) +

[0601] 21. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0602] At 20 °C, methyl hexanoate (53 mg, 71.06 μmol, 1 eq) and 2-(6-azidohexyl)-N-[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]pyrazol-3-carboxamide (49.44 mg, 71.06 μmol, 1 eq) were reacted with t-BuOH (1 CuSO4·5H2O (8.87 mg, 35.53 μmol, 0.5 eq) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (14.08 mg, 71.06 μmol, 1 eq) were added to a solution of CuSO4·5H2O (1 mL) and H2O (1 mL). The mixture was stirred at 50 °C for 1 hour. The reaction mixture was filtered to obtain a filtrate. The filtrate was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 55 rpm). Purification was performed at a flow rate of mL / min to obtain compound (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester (96 mg, crude product), which is a pale yellow solid.

[0603] data: LCMS (ESI + ): m / z 1441.9 (M+H) +

[0604] 22. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0605] At 0 °C, methyl 5,5-dimethylhexanoate (96 mg, 66.60 μmol, 1 eq) was subjected to THF (1 mL) and H2O (0.5 mL). LiOH·H₂O (5.59 mg, 133.19 μmol, 2 eq) was added to a 1 mL solution. The mixture was stirred at 0 °C for 0.5 h. The residue was concentrated under reduced pressure to remove THF, and then the pH of the residue was adjusted to approximately 5 using formic acid to obtain a solution. The residue was analyzed by preparative HPLC (neutral conditions; column: Waters Xbridge BEH C18 100). 30mm 10 μm; Mobile phase: [H2O(10mMNH4HCO3)-ACN]; Gradient: 40%-70% within 8.0 min B) Purification yielded compound X-059, namely (2S)-2-[[6-[3-[2-[2-[2-[2-[2-[2-[2-[1-[6-[5-[[(S)-(4,4-difluorocyclohexyl)-[7-[(1S)-2-methoxy-1-[(4S)-2-oxo-4-(trifluoromethyl)imidazolidine-1-yl]ethyl]imidazo[1,2-b]pyridazin-2-yl]methyl]carbamoyl]pyrazol-1-yl]hexyl]triazol-4-yl]methoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (45.5 mg, 31.87 mg). It is a white solid (μmol, yield 47.86%, purity 100.000%).

[0606] data: LCMS (ESI + ): m / z 1427.7 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.63 (d, J = 2.1 Hz, 1H), 8.38 (d, J =2.1 Hz, 1H), 8.25 (dd, J = 2.4, 8.7 Hz, 1H), 8.11 (s, 1H), 7.92 (s, 1H), 7.85(s, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.35 (t, J = 8.2 Hz, 1H), 7.00 (d, J = 8.7Hz, 1H), 6.89 (dd, J = 2.1, 8.1 Hz, 1H), 6.85 - 6.81 (m, 2H), 6.78 (dd, J =1.5, 8.1 Hz, 1H), 5.24 - 5.17 (m, 2H), 4.61 (s, 2H), 4.54 (s, 2H), 4.52 -4.44 (m, 3H), 4.41 - 4.33 (m, 1H), 4.30 (t, J = 7.1 Hz, 2H), 4.02 - 3.90 (m,3H), 3.65 - 3.60 (m, 4H), 3.60 - 3.50 (m, 19H), 3.48 - 3.42 (m, 5H), 2.26 -2.15 (m, 1H), 2.12 - 1.91 (m, 4H), 1.87 - 1.68 (m, 7H), 1.68 - 1.59 (m, 1H),1.53 - 1.30 (m, 4H), 1.28 - 1.17 (m, 4H), 0.90 (s, 9H) X-082: (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0607] 1. A general procedure for preparing 1-[3-[2-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]ethoxy]azacyclobutane-1-yl]-2-prop-2-alkynoxy-acetone: At 50°C, DIEA (237.67 mg, 1.84 mmol, 320.32 μL) was added to a DMF (2 mL) solution of 1-[3-(2-aminoethoxy)azacyclobutan-1-yl]-2-prop-2-alkynoxy-acetone (TFA) (200 mg, 612.99 μmol) and 2-bromoethoxy-tert-butyl-dimethyl-silane (146.64 mg, 612.99 μmol). The mixture was stirred for 12 hours. This mixture was used directly in the next step without purification to give 1-[3-[2-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]ethoxy]azacyclobutan-1-yl]-2-prop-2-alkynoxy-acetone (200 mg, crude), a brown liquid in DMF (2 mL).

[0608] data: LCMS (ESI) + ): m / z 371.3(M+H) +

[0609] 2. General procedure for the preparation of (2S)-2-[[6-[3-[2-[[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethyl]-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester:

[0610] At 0°C, DIEA (209.27 mg, 1.62 mmol, 282.03 μL) and HATU (307.83 mg, 809.59 μmol) were added to a DMF (2 mL) solution of 1-[3-[2-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]ethoxy]azacyclobutane-1-yl]-2-prop-2-alkynyloxy-acetone (200 mg, 539.73 μmol) and 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (301.44 mg, 539.73 μmol, TFA). The mixture was stirred at 20°C for 1 hour. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-80%; flow rate: 80 mL / min) to obtain (2S)-2-[[6-[3-[2-[[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethyl]-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (75 mg, 94.10 μmol, yield 17.43%), as a white solid.

[0611] data: LCMS (ESI + ): m / z 797.6 (M+H) +

[0612] 3. General procedure for the preparation of (2S)-2-[[6-[3-[2-[[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0613] Methyl 5,5-dimethyl hexanoate (70 mg, 87.83 μmol) and 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (92.60 mg, 131.74 μmol) were reacted with t-BuOH (0.2 mL) and H2O (0.2 mL). CuSO4·5H2O (10.96 mg, 43.91 μmol) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (17.40 mg, 87.83 μmol) were added to a solution of (mL). The mixture was stirred at 50°C for 1 hour. The mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-85%; flow rate: 80). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 20.00 μmol, yield 22.77%), as a white solid.

[0614] data: LCMS (ESI + ): m / z 1499.9 (M+H) +

[0615] 4. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0616] Add TFA (153.50 μmol) to a DCM (0.5 mL) solution of (2S)-2-[[6-[3-[2-[[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (27 mg, 18.00 μmol). mg, 1.35 mmol, 0.1 mL). The mixture was stirred for 3 hours. DCM and TFA were removed under reduced pressure to give (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (24 mg, crude, TFA), as a yellow oil.

[0617] data: LCMS (ESI + ): m / z 1255.8 (M+H) +

[0618] 5. A general procedure for preparing (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0619] At 0°C, LiOH·H2O (1.47 ppm) was added to a solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (24 mg, 17.53 μmol, TFA) in THF (0.2 mL) and H2O (0.2 mL). mg, 35.05 μmol). The mixture was stirred at 20°C for 1 hour. THF was removed under reduced pressure, and the residue was acidified to pH 2 with FA. The mixture was filtered, and the filtrate was passed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-54%; flow rate: 65 mg / L). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl-(2-hydroxyethyl)amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (11.7 mg, 9.34 μmol, yield 53.30%, purity 99.116%), as a pale yellow solid.

[0620] data: LCMS (ESI + ): m / z 1241.6 (M+H) + 1 1H NMR (400 MHz, methanol-d4) δ = 8.62 (d, J J = 2.1 Hz, 1H), 8.21 (td, J J = 3.1, 8.6 Hz, 1H), 8.00 (d, J J = 14.1 Hz, 1H), 7.67 - 7.58 (m, 2H), 7.53 (d, J J = 1.9 Hz, 1H), 7.36 - 7.26 (m, 1H), 7.22 (dd, J J = 2.2, 8.6 Hz, 2H), 6.94 (dd, J J = 2.7, 8.7 Hz, 1H), 6.89 - 6.81 (m, 2H), 6.80 - 6.75 (m, 1H), 6.72 (dd, J J = 2.6, 7.9 Hz, 1H), 5.10 (d, J J = 4.8 Hz, 2H), 4.96 (d, J J = 3.3 Hz, 2H), 4.65 - 4.56 (m, 3H), 4.56 - 4.46 (m, 2H), 4.40 - 4.21 (m, 2H), 4.18 - 4.09 (m, 1H), 4.08 - 3.97 (m, 3H), 3.84 - 3.47 (m, 10H), 3.25 - 3.09 (m, 2H), 2.22 (s, 6H), 2.04 (quin, J J = 6.5 Hz, 2H), 2.00 - 1.90 (m, 1H), 1.88 - 1.74 (m, 1H), 1.40 - 1.28 (m, 2H), 0.91 (s, 9H), 0.90 - 0.76 (m, 3H), 0.61 - 0.52 (m, 1H), 0.47 (ddd, J J = 4.8, 8.6, 13.3 Hz, 2H), 0.43 - 0.34 (m, 2H), 0.33 - 0.20 (m, 3H) X-083: (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0621] 1. A general procedure for preparing 2-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethylamino]acetamide:

[0622] DIEA (222.60 mg, 1.72 mmol, 0.3 mL) was added to a solution of 1-[3-(2-aminoethoxy)azacyclobutan-1-yl]-2-prop-2-alkynoxy-acetone (200 mg, 612.99 μmol, TFA) and 2-bromoacetamide (126.86 mg, 919.49 μmol) in EtOH (3 mL). The mixture was stirred for 12 hours. EtOH was removed under reduced pressure to give 2-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutan-3-yl]oxyethylamino]acetamide (140 mg, 519.87 μmol, yield 84.81%) as a yellow oil.

[0623] data: LCMS (ESI + ): m / z 270.3 (M+H) +

[0624] 2. General procedure for the preparation of (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0625] At 0°C, DIEA (92.56 mg, 716.20 μmol, 124.75 μL) and HATU (204.24 mg, 537.15 μmol) were added to a DMF (3 mL) solution of 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (200 mg, 358.10 μmol, TFA) and 2-[2-[1-(2-prop-2-alkynyloxyacetyl)azacyclobutane-3-yl]oxyethylamino]acetamide (96.44 mg, 358.10 μmol) (96.44 mg, 358.10 μmol). The mixture was stirred at 20°C for 1 hour. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 120g; mobile phase: [water-ACN]; B%: 0%-68%; flow rate: 80 mL / min) to obtain (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (60 mg, 86.24 μmol, yield 24.08%), as a white solid.

[0626] data: LCMS (ESI + ): m / z 696.5 (M+H) +

[0627] 3. A general procedure for preparing (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0628] Methyl 5,5-dimethyl hexanoate (55 mg, 79.05 μmol) and 2-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (55 mg, 79.05 μmol) were added to the H2O (0.5 mL) and t-BuOH. CuSO4·5H2O (9.87 mg, 39.53 μmol) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (15.66 mg, 79.05 μmol) were added to a 0.5 mL solution. The mixture was stirred at 50°C for 1 hour. The mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-75%, flow rate: 80). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (80 mg, 57.20 μmol, yield 72.35%), as a white solid.

[0629] data: LCMS (ESI + ): m / z 1398.9 (M+H) +

[0630] 4. General procedure for the preparation of (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0631] Add TFA (767.50 mg, 6.73 μmol) to a DCM (1 mL) solution of (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (80 mg, 57.20 μmol) (mmol, 0.5 mL). The mixture was stirred for 3 hours. DCM and TFA were removed under reduced pressure to give (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (78 mg, crude, TFA), as a yellow oil.

[0632] data: LCMS (ESI + ): m / z 1268.8 (M+H) +

[0633] 5. A general procedure for preparing (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0634] Add LiOH·H2O to a solution of (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (75 mg, 54.25 μmol, TFA) in THF (0.5 mL) and H2O (0.5 mL). (4.55 mg, 108.50 μmol). The mixture was stirred at 0°C for 1 hour. The mixture was filtered, and the filtrate was passed through a preparative HPLC system (FA conditions; column: Phenomenex luna C18 100). 40mm 3 μm; mobile phase: [H2O(0.2%FA)-ACN]; gradient: 30%-60% B) purification over 8.0 min yielded (2S)-2-[[6-[3-[2-[(2-amino-2-oxo-ethyl)-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (5.4 mg, 4.23 mg). It is a white solid (μmol, yield 7.79%, purity 98.188%).

[0635] data: LCMS (ESI + ): m / z 1276.5 (M+Na) + 1 H NMR (400 MHz, methanol-d4) δ = 8.61 (d, J J = 1.6 Hz, 1H), 8.25 - 8.17(m, 1H), 8.02 (d, J J = 15.4 Hz, 1H), 7.69 - 7.58 (m, 2H), 7.53 (d, J J = 2.1 Hz,1H), 7.30 (q, J J = 8.1 Hz, 1H), 7.22 (dd, J J = 1.9, 8.6 Hz, 2H), 6.94 (d, J J =8.7 Hz, 1H), 6.89 (d, J J = 2.2 Hz, 1H), 6.87 - 6.81 (m, 1H), 6.77 (s, 1H),6.75 - 6.69 (m, 1H), 5.10 (d, J J = 5.5 Hz, 2H), 5.00 (s, 2H), 4.69 - 4.47 (m,6H), 4.43 - 4.24 (m, 2H), 4.23 - 3.98 (m, 6H), 3.87 - 3.75 (m, 1H), 3.70 -3.42 (m, 4H), 3.25 - 3.06 (m, 2H), 2.22 (s, 6H), 2.10 - 1.99 (m, 2H), 1.99 -1.90 (m, 1H), 1.88 - 1.74 (m, 1H), 1.43 - 1.28 (m, 2H), 0.93 - 0.90 (m, 9H),0.89 - 0.74 (m, 3H), 0.61 - 0.53 (m, 1H), 0.48 (dt, J J = 4.2, 8.7 Hz, 2H),0.44 - 0.35 (m, 2H), 0.28 (tdd, J J = 4.7, 9.0, 13.1 Hz, 3H) X-084: (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0636] 1. A general procedure for preparing methyl 2-[3-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]propyl]pyrazole-3-carboxylic acid:

[0637] TEA (102.13 mg, 1.01 mmol, 1 eq, TFA) was added to a MeOH (3 mL) solution of methyl 2-(3-aminopropyl)pyrazole-3-carboxylate (300 mg, 1.01 mmol, 1 eq, TFA) until pH = 8–9, and the reaction was stirred at 20°C for 5 minutes. Then, 2-[tert-butyl(dimethyl)silyl]oxyacetaldehyde (123.16 mg, 706.52 μmol, 134.60 μL, 0.7 eq) was added, and AcOH (60.61 mg, 1.01 mmol, 57.78 μL, 1 eq) was added to adjust the pH < 5, and the reaction was stirred at 20°C for 5 minutes. Finally, NaBH3CN (126.86 mg, 2.02 mmol, 2 eq, TFA) was added to the reaction mixture. The reaction was carried out by stirring at 20°C for 20 minutes. The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-42%; flow rate: 80 mL / min) to give methyl 2-[3-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]propyl]pyrazole-3-carboxylic acid (0.2 g, 585.62 μmol, yield 58.02%) as a yellow oil.

[0638] data: LCMS (ESI + ): m / z 342.3 (M+H) +

[0639] 2. A general procedure for preparing methyl 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid:

[0640] At 0°C, HATU (317.30 mg, 834.50 μmol, 1 eq) and DIEA (215.71 mg, 1.67 mmol, 290.71 μL, 3 eq) were added to a DMF (2 mL) solution of methyl 2-[3-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]propyl]pyrazole-3-carboxylic acid (0.19 g, 556.34 μmol, 1 eq) and 2-azidoacetic acid (112.45 mg, 1.11 mmol, 2 eq), and the reaction was then stirred at 20°C for 0.5 h. The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 80 mL / min) to give methyl 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid (130 mg, 306.19 μmol, yield 55.04%), as a yellow oil.

[0641] data: LCMS (ESI + ): m / z 425.3 (M+H) +

[0642] 3. A general procedure for the preparation of 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid:

[0643] LiOH·H₂O (19.27 mg, 459.29 μmol, 1.5 eq) was added to a solution of methyl 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid ester (0.13 g, 306.19 μmol, 1 eq) in H₂O (0.5 mL) and THF (0.5 mL). The mixture was stirred at 0°C for 0.5 h. The aqueous layer was acidified to pH 3 using HCl (1 M). The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 80 mL / min) to obtain 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid (0.15 g, crude product), which is a yellow oil.

[0644] data: LCMS (ESI + ): m / z 411.4 (M+H) +

[0645] 4. A general procedure for preparing 2-[3-[(2-azidoacetyl)-(2-hydroxyethyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide:

[0646] At 0°C, HATU (166.71 mg, 438.44 μmol, 1.5 eq) and DIEA (113.33 mg, 876.89 μmol, 152.74 μL, 3 eq) were added to a DMF (2 mL) solution of 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]pyrazole-3-carboxylic acid (0.12 g, 292.30 μmol, 1 eq) and (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazole-4-yl]phenyl]propionamide (118.13 mg, 233.84 μmol, 0.8 eq, HCl salt) and HCl salt respectively. The reaction was then stirred at 20°C for 1.5 hours. The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-62%; flow rate: 80 mL / min) to give 2-[3-[(2-azidoacetyl)-[2-[tert-butyl(dimethyl)silyl]oxyethyl]amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (40 mg, 46.45 μmol). Both 2-[3-[(2-azidoacetyl)-(2-hydroxyethyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (28 mg, 37.48 μmol, yield 12.82%) are yellow oils.

[0647] data: LCMS (ESI + ): m / z 747.6 (M+H) +

[0648] 5. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: t-BuOH (0.5 mL) and H2O were added to methyl 2-[3-[(2-azidoacetyl)-(2-hydroxyethyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (28 mg, 37.48 μmol, 1 eq) and (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]hexanoate (23.94 mg, 37.48 μmol, 1 eq). Copper sulfate (5.98 mg, 37.48 μmol, 5.75 μL, 1 eq) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (7.43 mg, 37.48 μmol, 1 eq) were added to a 0.5 mL solution, and the reaction was stirred at 50°C for 0.5 h. The residue was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 80). Purification at a rate of mL / min yielded (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 21.65 μmol, yield 57.76%), a yellow oil.

[0649] data: LCMS (ESI + ): m / z 1385.9 (M+H) +

[0650] 6. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Add 0.2 mL of TFA to a solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 21.65 μmol, 1 eq) in DCM (0.8 mL). Stir the mixture at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, crude, TFA), as a yellow oil.

[0651] data: LCMS (ESI + ): m / z 1255.6 (M+H) +

[0652] 7. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O (1.15 mg, 27.38 mL) to a solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (25 mg, 18.26 μmol, 1 eq, TFA) in H2O (0.5 mL) and THF (0.5 mL). μmol, 1.5 eq). The mixture was stirred at 0 °C for 0.5 h. The aqueous layer was acidified to pH 3 using HCl (1 M). The residue was analyzed by preparative HPLC (neutral conditions; column: Phenomenex luna C18 100). 40mm 3 μm; mobile phase: [H2O(0.2%FA)-ACN]; gradient: 30%-60% B) purification over 8.0 min yielded (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (4.6 mg, 3.67 mg). μmol (yield 20.08%, purity 98.930%), is a yellow resinous substance.

[0653] data: LCMS (ESI + ): m / z 1241.6 (M+H) + 1 ¹H NMR (400 MHz, methanol-d⁴) δ = 8.63 - 8.58 (m, ¹H), 8.22 (dd, J = 2.5,8.6 Hz, 1H), 7.95 - 7.89 (m, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.58 - 7.49 (m,1H), 7.34 (t, J = 8.2 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.7 Hz,1H), 6.92 - 6.84 (m, 2H), 6.84 - 6.74 (m, 2H), 5.52 - 5.35 (m, 2H), 4.75 -4.41 (m, 8H), 4.39 - 4.23 (m, 2H), 4.18 - 4.09 (m, 1H), 4.07 - 3.97 (m, 3H), 3.82 - 3.73 (m, 1H), 3.71 - 3.59 (m, 2H), 3.56 - 3.38 (m, 8H), 2.23 (s, 7H), 2.14 - 2.04 (m, 1H), 2.02 - 1.90 (m, 1H), 1.89 - 1.74 (m, 1H), 1.41 - 1.27 (m, 2H), 0.91 (s, 10H), 0.86 - 0.75 (m, 2H), 0.60 - 0.44 (m, 3H), 0.39 (dt, J = 5.0, 8.6 Hz, 2H), 0.34 - 0.22 (m, 3H) X-085: (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0654] 1. A general procedure for preparing (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[2-[tert-butyl(dimethyl)silyl]oxyethyl-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Methyl hexanoate (30 mg, 48.02 μmol, 1.03 eq) and 2-[3-[(2-prop-2-alkynoxyethoxy)propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]hexanoate (30 mg, 48.02 μmol, 1.03 eq) were reacted with t-BuOH (0.5 mL) and H2O (0.5 mL) to form (2S)-5,5-dimethyl-2-[[6-[3-[3-[1-[3-(2-prop-2-alkynoxyethoxy)propyl]oxyethyl]amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (40 mg, 46.45 μmol, 1 eq). Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (9.20 mg, 46.45 μmol, 1 eq) and copper sulfate pentahydrate (5.80 mg, 23.22 μmol, 0.5 eq) were added to a solution. The mixture was stirred at 50 °C for 0.5 h. The reaction mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-78%; flow rate: 100 mL). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[2-[tert-butyl(dimethyl)silyl]oxyethyl-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (40 mg, 26.92 μmol, yield 57.96%), as a colorless oil.

[0655] data: LCMS (ESI + ): m / z 1486.0 (M+H) +

[0656] 2. A general procedure for the preparation of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: A solution of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[2-[tert-butyl(dimethyl)silyl]oxyethyl-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (35 mg, 23.55 μmol, 1 eq) in DCM (0.5 mL) and TFA (0.1 mL) was stirred at 25 °C for 0.5 h. DCM and TFA were removed under reduced pressure to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, crude, TFA), as a yellow oil.

[0657] data: LCMS (ESI + ): m / z 1241.4 (M+H) +

[0658] 3. General procedure for the preparation of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O (1.86 mg, 44.27 μmol, 2 eq) to a solution of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 22.13 μmol, 1 eq, TFA) in THF (0.5 mL) and H2O (0.5 mL). Stir the mixture for 20 minutes. Remove THF under reduced pressure and acidify the residue with FA to pH 2. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-53%; flow rate: 80 mL / min) to obtain (2S)-2-[[6-[3-[1-[3-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl-(2-hydroxyethyl)amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (8.7) It is a yellow oily substance (mg, 6.75 μmol, yield 30.48%, purity 95.185%).

[0659] data: LCMS (ESI + ): m / z 1227.8 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.64 - 8.58 (m, 1H), 8.22 (dd, J = 2.4,8.7 Hz, 1H), 7.91 - 7.86 (m, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.57 - 7.49 (m,1H), 7.33 (t, J = 8.2 Hz, 1H), 7.27 - 7.19 (m, 2H), 6.96 (d, J = 8.7 Hz, 1H), 6.92 - 6.83 (m, 1H), 6.79 - 6.69 (m, 2H), 6.67 - 6.61 (m, 1H), 5.49 - 5.37(m, 2H), 5.01 - 4.93 (m, 1H), 4.66 - 4.41 (m, 6H), 4.28 (td, J = 6.6, 9.1 Hz,2H), 3.92 - 3.83 (m, 2H), 3.69 - 3.54 (m, 6H), 3.53 - 3.45 (m, 4H), 3.45 -3.34 (m, 2H), 3.24 - 3.16 (m, 2H), 2.23 (s, 6H), 2.14 - 2.05 (m, 1H), 2.02 -1.90 (m, 1H), 1.88 - 1.76 (m, 1H), 1.76 - 1.67 (m, 2H), 1.44 - 1.24 (m, 3H),0.91 (s, 9H), 0.90 - 0.75 (m, 3H), 0.60 - 0.45 (m, 3H), 0.44 - 0.35 (m, 2H),0.34 - 0.21 (m, 3H) X-086: (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0660] 1. A general procedure for preparing methyl 2-[3-[(2-amino-2-oxo-ethyl)amino]propyl]pyrazole-3-carboxylic acid:

[0661] DIEA (652.24 mg, 5.05 mmol, 879.02 μL, 3 eq) was added to a solution of methyl 2-(3-aminopropyl)pyrazole-3-carboxylate (0.5 g, 1.68 mmol, 1 eq, TFA) and 2-bromoacetamide (46.42 mg, 336.44 μmol, 0.2 eq) in EtOH (5 mL). The mixture was stirred at 25 °C for 12 h. The residue was purified by reversed-phase HPLC (column: C1820-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-0%; flow rate: 80 mL / min) to give methyl 2-[3-[(2-amino-2-oxo-ethyl)amino]propyl]pyrazole-3-carboxylate (1 g, crude), as a yellow oil.

[0662] data: LCMS (ESI + ): m / z 241.1 (M+H) +

[0663] 2. A general procedure for preparing methyl 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid:

[0664] At 0°C, HATU (2.14 g, 5.62 mmol, 1.5 eq) and DIEA (1.45 g, 11.24 mmol, 1.96 mL, 3 eq) were added to a DMF (10 mL) solution of methyl 2-[3-[(2-amino-2-oxo-ethyl)amino]propyl]pyrazole-3-carboxylic acid ester (0.9 g, 3.75 mmol, 1 eq) and 2-azidoacetic acid (757.16 mg, 7.49 mmol, 2 eq), and the reaction was stirred at 20°C for 0.5 h. The residue was analyzed by preparative HPLC (neutral conditions; column: Waters XbridgePrep OBD C18 150). 40mm 10 μm; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 10%-45% over 8.0 min. B) Purification yielded methyl 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (70 mg, 216.51 μmol, yield 5.78%), a yellow oil.

[0665] data: LCMS (ESI + ): m / z 324.1 (M+H) + 1 ¹H NMR (400 MHz, methanol-d⁴) δ = 7.53 (dd, J = 2.0, 11.7 Hz, 1H), 6.89(dd, J = 2.0, 10.7 Hz, 1H), 4.59 (q, J = 7.1 Hz, 2H), 4.09 - 4.00 (m, 4H), 3.89 (d, J = 1.5 Hz, 3H), 3.44 - 3.32 (m, 2H), 2.19 - 2.05 (m, 2H)

[0666] 3. A general procedure for preparing 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid:

[0667] LiOH·H₂O (9.73 mg, 231.98 μmol, 1.5 eq) was added to a solution of methyl 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (50 mg, 154.65 μmol, 1 eq) in H₂O (0.5 mL) and THF (0.5 mL). The mixture was stirred at 0 °C for 0.5 h. The aqueous layer was acidified to pH 3 using HCl (1 M). The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-0%; flow rate: 60 mL / min) to give 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (45 mg, 145.50 μmol, yield 94.08%), as a yellow oil.

[0668] data: LCMS (ESI + ): m / z 310.2 (M+H) +

[0669] 4. A general procedure for preparing 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide:

[0670] At 0°C, HATU (64.54 mg, 169.75 μmol, 1.5 eq) and DIEA (43.88 mg, 339.50 μmol, 59.13 μL, 3 eq) were added to a DMF (2 mL) solution of 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (35 mg, 113.17 μmol, 1 eq, HCl salt) and DIEA (43.88 mg, 339.50 μmol, 59.13 μL, 3 eq) and DIEA were added to a DMF (2 mL) solution of 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]pyrazole-3-carboxylic acid (35 mg, 113.17 μmol, 1 eq) and (2S)-2-amino-3,3-dicyclopropyl-N-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazole-4-yl]phenyl]propionamide (57.17 mg, 113.17 μmol, 1 eq, HCl salt). The reaction was then stirred at 20°C for 0.5 h. The residue was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 70 mL / min) to obtain 2-[3-[(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (80 mg, 105.27 μmol, yield 93.02%), as a yellow oil.

[0671] data: LCMS (ESI + ): m / z 760.4 (M+H) +

[0672] 5. A general procedure for preparing (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0673] Methyl hexanoate (25.21 mg, 39.48 μmol, 1 eq) and 2-[3-[(2-amino-2-oxo-2-[2-[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxyethylamino]ethoxy]phenoxy]pyridine-3-carbonyl]amino]amino]hexanoate (25.21 mg, 39.48 μmol, 1 eq) were reacted with t-BuOH (0.5 mL) and H2O (0.5 mL) of (2S)-5,5-dimethyl-2-[[6-[3-[2-oxo-2-[2-[1-(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (30 mg, 39.48 μmol, 1 eq). Copper sulfate (6.30 mg, 39.48 μmol, 6.06 μL, 1 eq) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (7.82 mg, 39.48 μmol, 1 eq) were added to a solution of 1000 mL. The reaction was stirred at 50°C for 0.5 h. The mixture was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-68%; flow rate: 80 rpm). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 21.45 μmol, yield 54.33%), as a yellow oil.

[0674] data: LCMS (ESI + ): m / z 1398.5 (M+H) +

[0675] 6. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0676] Add 0.5 mL of TFA to a DCM (1 mL) solution of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (25 mg, 17.87 μmol, 1 eq). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (25 mg, crude, TFA), as a yellow oil.

[0677] data: LCMS (ESI + ): m / z 1268.7 (M+H) +

[0678] 7. General procedure for the preparation of (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0679] Methyl 5,5-dimethyl hexanoate (20 mg, 14.47 μmol, 1 eq, TFA) was reacted with H₂O (0.5 mL) and THF (0.5 mL) to form (2S)-2-[[6-[3-[2-[2-[1-[2-[1-[2-[2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (20 mg, 14.47 μmol, 1 eq, TFA). LiOH·H₂O (910.64 μg, 21.70 μmol, 1.5 eq) was added to a 1 mL solution. The mixture was stirred at 0 °C for 0.5 h. The aqueous layer was acidified to pH 3 using FA (1 M). The residue was analyzed by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-68%; flow rate: 70 rpm). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[2-[1-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxyethylamino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (6.9 mg, 5.47 μmol, yield 37.82%, purity 99.477%), as a yellow resinous substance.

[0680] data: LCMS (ESI + ): m / z1254.5 (M+H) + 1 H NMR (400 MHz, methanol-d4) δ = 8.61 (t, J J = 2.7 Hz, 1H), 8.22 (dd, J J =2.5, 8.6 Hz, 1H), 7.95 (d, J J = 4.3 Hz, 1H), 7.64 - 7.58 (m, 2H), 7.54 (dd, J J = 2.0, 15.9 Hz, 1H), 7.34 (t, J J = 8.2 Hz, 1H), 7.22 (dd, J J = 2.0, 8.6 Hz,2H), 6.97 (d, J J = 8.7 Hz, 1H), 6.92 - 6.85 (m, 2H), 6.83 - 6.79 (m, 1H), 6.77(dd, J J = 1.8, 8.0 Hz, 1H), 5.54 - 5.21 (m, 2H), 4.70 - 4.42 (m, 8H), 4.38 -4.23 (m, 2H), 4.19 - 4.07 (m, 2H), 4.06 - 3.97 (m, 4H), 3.82 - 3.73 (m, 1H),3.55 - 3.34 (m, 6H), 2.23 (s, 7H), 2.14 - 2.04 (m, 1H), 2.01 - 1.91 (m, 1H),1.88 - 1.77 (m, 1H), 1.40 - 1.28 (m, 2H), 0.91 (s, 10H), 0.87 - 0.74 (m, 2H),0.61 - 0.43 (m, 3H), 0.43 - 0.34 (m, 2H), 0.33 - 0.21 (m, 3H) X-087: (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0681] 1. A general procedure for preparing (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Methyl hexanoate (24.66 mg, 39.48 μmol, 1 eq) and 2-[3-[(2-prop-2-alkynoxyethoxy)propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]hexanoate (24.66 mg, 39.48 μmol, 1 eq) were reacted with t-BuOH (0.5 mL) and H2O (0.5 mL) to form (2S)-5,5-dimethyl-2-[[6-[3-[3-[1-[3-(2-amino-2-oxo-ethyl)-(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (30 mg, 39.48 μmol, 1 eq). Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (7.82 mg, 39.48 μmol, 1 eq) and copper sulfate pentahydrate (4.93 mg, 19.74 μmol, 0.5 eq) were added to a solution. The mixture was stirred at 50 °C for 0.5 h. The mixture was filtered, and the filtrate was passed through reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-70%; flow rate: 70). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester (24 mg, 17.33 μmol, yield 43.91%), as a yellow oil.

[0682] data: LCMS (ESI + ): m / z 1384.8 (M+H) +

[0683] 2. A general procedure for preparing (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: A solution of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (24 mg, 17.33 μmol, 1 eq) in DCM (1 mL) and TFA (0.5 mL) was stirred at 25 °C for 2 hours. DCM and TFA were removed under reduced pressure to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (24 mg, crude, TFA), as a yellow oil.

[0684] data: LCMS (ESI + ): m / z 628.2 (M / 2+H) +

[0685] 3. General procedure for the preparation of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O to a solution of (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (24 mg, 17.54 μmol, 1 eq, TFA) in THF (0.5 mL) and H2O (0.5 mL); (1.47 mg, 35.08 μmol, 2 eq). The mixture was stirred at 0 °C for 20 minutes. The mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C1820-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-45%; flow rate: 70). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[1-[3-[2-[[1-[2-[(2-amino-2-oxo-ethyl)-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propyl]amino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]propylcarbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (4.2 mg, 3.39 μmol, yield 19.31%, purity 100%), as a pale yellow solid.

[0686] data: LCMS (ESI + ): m / z 1240.6 (M+H) + 11H NMR (400 MHz, methanol-d4) δ = 8.61 (t, J = 2.8 Hz, 1H), 8.21 (dd, J = 2.3, 8.7 Hz, 1H), 7.91 (d, J = 5.6 Hz, 1H), 7.61 (dd, J = 1.7, 8.5 Hz, 2H), 7.53 (dd, J = 2.0, 16.0 Hz, 1H), 7.33 (t, J = 8.2 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H), 6.88 (dd, J = 2.1, 15.5 Hz, 1H), 6.73 (ddd, J = 2.0, 8.2, 12.9 Hz, 2H), 6.67 - 6.60 (m, 1H), 5.50 - 5.24 (m, 2H), 4.96 (br d, J = 4.0 Hz, 1H), 4.69 - 4.40 (m, 6H), 4.33 - 4.24 (m, 2H), 4.14 (d, J = 3.5 Hz, 1H), 4.01 (s, 1H), 3.87 (br d, J = 8.7 Hz, 2H), 3.66 - 3.54 (m, 4H), 3.54 - 3.41 (m, 4H), 3.20 (t, J = 6.6 Hz, 2H), 2.23 (s, 7H), 2.13 - 2.04 (m, 1H), 2.02 - 1.91 (m, 1H), 1.88 - 1.76 (m, 1H), 1.72 (quin, J = 6.2 Hz, 2H), 1.42 - 1.27 (m, 2H), 0.91 (s, 9H), 0.88 - 0.74 (m, 3H), 0.54 (br dd, J = 4.5, 7.5 Hz, 1H), 0.52 - 0.43 (m, 2H), 0.38 (br dd, J = 4.3, 9.0 Hz, 2H), 0.33 - 0.20 (m, 3H) X-089: (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0687] 1. A general procedure for preparing 3-[2-(tert-butoxycarbonylamino)-3-methoxy-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester: BF3·Et2O (705.34 mg, 4.97 mmol, 611.21 μL, 0.2 eq) was added to a CHCl3 (50 mL) solution of 3-hydroxyazacyclobutane-1-carboxylic acid benzyl ester (10.30 g, 49.70 mmol, 2 eq) and 1-tert-butyl-2-methyl-aziridinium-1,2-dicarboxylic acid ester (5 g, 24.85 mmol, 1 eq). The mixture was stirred at 25 °C for 20 hours. The mixture was filtered, and the filtrate was purified by preparative HPLC. The residue was purified by preparative HPLC (neutral conditions, column: Waters Xbridge Prep OBD C18 150). 40 mm 10 μm; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 45%-75% over 8.0 min. B) Purification yielded 3-[2-(tert-butoxycarbonylamino)-3-methoxy-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (630 mg, 1.54 mmol, yield 6.21%), as a colorless oil.

[0688] data: 1 ¹H NMR (400 MHz, chloroform-d) δ = 7.39 - 7.31 (m, 5H), 5.39 - 5.32 (m, 1H), 5.10 (s, 2H), 4.46 (s, 1H), 4.26 - 4.22 (m, 1H), 4.13 - 4.09 (m, 1H), 3.92 - 3.81 (m, 2H), 3.79 - 3.74 (m, 3H), 3.61 (dd, J = 3.2, 9.4 Hz, 1H), 1.46 (s, 9H)

[0689] 2. A general procedure for preparing 3-[3-amino-2-(tert-butoxycarbonylamino)-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester: A solution of 3-[2-(tert-butoxycarbonylamino)-3-methoxy-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (0.5 g, 1.22 mmol, 1 eq) in NH3 / MeOH (3 mL) was stirred at 25 °C for 12 hours. The mixture was filtered, and the filtrate was purified by preparative HPLC. The crude product was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 120 g; mobile phase: [water-ACN]; B%: 0%-40%; flow rate: 100 mL / min) to give 3-[3-amino-2-(tert-butoxycarbonylamino)-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (330 mg, 838.77 μmol, yield 68.52%) as a white solid.

[0690] 3. General procedure for the preparation of 3-(2,3-diamino-3-oxo-propoxy)azacyclobutane-1-carboxylic acid benzyl ester:

[0691] TFA (1.54 g, 13.46 mmol, 1 mL, 1 eq) was added to a solution of 3-[3-amino-2-(tert-butoxycarbonylamino)-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (0.3 g, 762.52 μmol, 1 eq) in DCM (3 mL). The mixture was stirred at 25 °C for 3 hours. DCM and TFA were removed under reduced pressure to give 3-(2,3-diamino-3-oxo-propoxy)azacyclobutane-1-carboxylic acid benzyl ester (300 mg, crude product, TFA) as a colorless oil.

[0692] 4. A general procedure for the preparation of 3-[3-amino-2-[[2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetyl]amino]-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester:

[0693] HATU (541.40 mg, 1.42 mmol, 2 eq) and DIEA (276.04 mg, 2.14 mmol, 372.02 μL, 3 eq) were added to a DMF (1 mL) solution of 3-(2,3-diamino-3-oxo-propoxy)azacyclobutane-1-carboxylic acid benzyl ester (0.29 g, 711.93 μmol, 1 eq, TFA) and 2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetic acid (397.62 mg, 711.93 μmol, 1 eq, TFA) at 0°C. The mixture was stirred at 25°C for 0.5 h. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC. The crude product was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 100 mL / min) to obtain 3-[3-amino-2-[[2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetyl]amino]-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (0.26 g, 361.22 μmol, yield 50.74%), as a white solid.

[0694] data: LCMS (ESI + ): m / z 720.4 (M+H) +

[0695] 5. A general procedure for preparing (2S)-2-[[6-[3-[2-[[2-amino-1-(azacyclobutane-3-yloxymethyl)-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0696] PdCl2 (49.27 mg, 277.86 μmol, 2 eq) and TEA (72.70 mg, 718.46 μmol, 0.1 mL, 5.17 eq) were added to a DCM (2 mL) solution of 3-[3-amino-2-[[2-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]acetyl]amino]-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (100 mg, 138.93 μmol, 1 eq) and then ...acetyl]amino]-3-oxo-propoxy]azacyclobutane-1-carboxylic acid benzyl ester (2 mL), followed by the addition of triethylsilane (48.46 mg, 416.79 μmol, 66.57 μL, 3 eq). After 0.5 hours, TFA (47.52 mg, 416.79 μmol, 30.96 μL, 3 eq) was added to the reaction mixture, and the mixture was stirred at 25 °C for 0.5 hours. The mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 80 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 100 mL / min) to obtain (2S)-2-[[6-[3-[2-[[2-amino-1-(azacyclobutane-3-yloxymethyl)-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (45 mg, 76.84 μmol, yield 55.31%) as a white solid.

[0697] 6. General procedure for the preparation of (2S)-2-[[6-[3-[2-[[2-amino-2-oxo-1-[[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxymethyl]ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0698] HATU (51.94 mg, 136.60 μmol, 2 eq) and DIEA (17.65 mg, 136.60 μmol, 23.79 μL, 2 eq) were added to a DMF (2 mL) solution of (2S)-2-[[6-[3-[2-[[2-amino-1-(azacyclobutan-3-yloxymethyl)-2-oxo-ethyl]amino]-2-[-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (40 mg, 68.30 μmol, 1 eq) and 2-prop-2-alkynyloxyacetic acid (7.79 mg, 68.30 μmol, 1 eq). The mixture was stirred at 25°C for 0.5 h. The mixture was filtered, and the filtrate was purified by reversed-phase high-performance liquid chromatography. The crude product was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-60%; flow rate: 100 mL / min) to obtain (2S)-2-[[6-[3-[2-[[2-amino-2-oxo-1-[[1-(2-prop-2-alkynoxyacetyl)azacyclobutane-3-yl]oxymethyl]ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 44.01 μmol, yield 64.43%), as a colorless oil.

[0699] data: 1H NMR (400 MHz, chloroform-d) δ = 8.66 - 8.60 (m, 1H), 8.19 (br d, J = 8.3Hz, 1H), 7.42 - 7.34 (m, 2H), 7.05 - 6.75 (m, 5H), 6.25 (br d, J = 1.1 Hz,1H), 5.60 - 5.47 (m, 1H), 4.78 (q, J = 6.6 Hz, 1H), 4.68 - 4.63 (m, 1H), 4.58(s, 2H), 4.48 - 4.28 (m, 2H), 4.26 - 4.19 (m, 3H), 4.17 - 4.12 (m, 2H), 3.94- 3.84 (m, 2H), 3.79 (s, 3H), 3.60 - 3.48 (m, 1H), 2.49 (br d, J = 2.0 Hz,1H), 2.51 - 2.44 (m, 1H), 1.99 - 1.87 (m, 1H), 1.82 - 1.70 (m, 1H), 1.32 (brd, J = 5.1 Hz, 1H), 1.18 (br s, 1H), 1.25 - 1.17 (m, 1H), 0.88 (s, 9H)

[0700] 7. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0701] Methyl 5,5-dimethylhexanoate (25 mg, 36.67 μmol, 1 eq) and 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (27.38 mg, 36.67 μmol, 1 eq) were reacted with t-BuOH (1 mL) and H2O (1 mL) to form (2S)-2-[[6-[3-[2-amino-2-oxo-1-[[1-(2-prop-2-alkynoxyacetyl)azonyl-3-yl]amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (27.38 mg, 36.67 μmol, 1 eq). Sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (7.26 mg, 36.67 μmol, 1 eq) and copper sulfate pentahydrate (4.58 mg, 18.34 μmol, 0.5 eq) were added to a solution. The mixture was stirred at 50°C for 0.5 hours. The mixture was filtered, and the filtrate was passed through reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-70%; flow rate: 100 mL). Purification at a rate of mL / min yielded (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 21.67 μmol, yield 59.08%), a colorless oil.

[0702] 8. A general procedure for preparing (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate:

[0703] Add TFA (767.50 mg) to a DCM (1.5 mL) solution of (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 21.67 μmol, 1 eq). mg, 6.73 mmol, 0.5 mL, 310.67 eq). The mixture was stirred at 25 °C for 2 hours. DCM and TFA were removed under reduced pressure to obtain (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, crude, TFA), as a colorless oil.

[0704] 9. A general procedure for the preparation of (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid:

[0705] Add LiOH·H2O to a solution of (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (25 mg, 18.27 μmol, 1 eq, TFA) in THF (0.5 mL) and H2O (0.5 mL). (1.53 mg, 36.54 μmol, 2 eq). The mixture was stirred at 0 °C for 20 minutes. The mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-50%; flow rate: 60 rpm). Purification was performed at a flow rate of mL / min to obtain (2S)-2-[[6-[3-[2-[[2-amino-1-[[1-[2-[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-oxo-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (3.4 mg, 2.57 μmol, yield 14.05%, purity 93.672%), as a pale yellow solid.

[0706] data: LCMS (ESI + ): m / z 1240.6 (M+H) + 1H NMR (400 MHz, methanol-d4) δ = 8.61 (s, 1H), 8.24 - 8.19 (m, 1H), 8.01(d, J = 4.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 2.0 Hz, 1H), 7.34(t, J = 8.2 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 6.97 (dd, J = 2.1, 8.7 Hz,1H), 6.91 - 6.86 (m, 2H), 6.83 - 6.80 (m, 1H), 6.77 (br d, J = 8.2 Hz, 1H),5.11 (d, J = 2.1 Hz, 2H), 4.66 (br s, 3H), 4.60 - 4.57 (m, 3H), 4.55 (br s,3H), 4.36 - 4.24 (m, 2H), 4.15 - 4.07 (m, 1H), 4.04 - 3.97 (m, 3H), 3.80 -3.70 (m, 2H), 3.68 - 3.61 (m, 1H), 3.23 - 3.11 (m, 2H), 2.22 (s, 6H), 2.03(br t, J = 6.7 Hz, 2H), 1.98 - 1.90 (m, 1H), 1.86 - 1.76 (m, 1H), 1.40 - 1.27(m, 2H), 0.91 (s, 9H), 0.89 - 0.77 (m, 3H), 0.59 - 0.52 (m, 1H), 0.47 (ddd, J= 4.8, 8.4, 13.1 Hz, 2H), 0.43 - 0.35 (m, 2H), 0.33 - 0.21 (m, 3H) X-090: (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0707] 1. A general procedure for preparing 3-(2-prop-2-acetylacetoxy)prop-1-ene:

[0708] At 0°C, NaH (1.20 g, 29.97 mmol, 60% purity, 1.5 eq) was added to a DMF (30 mL) solution of 2-prop-2-alkynoxyethanol (2 g, 19.98 mmol, 1 eq), and the reaction was stirred at 0°C for 0.5 h. Subsequently, 3-bromoprop-1-ene (2.42 g, 19.98 mmol, 1 eq) was added to the mixture. The mixture was stirred at 25°C for 0.5 h. The reaction mixture was then added to saturated NH4Cl (30 mL) and diluted with ethyl acetate (30 mL). 3) Extract and wash with brine (50 mL). The organic layer was dried over Na2SO4 and concentrated to obtain the crude product. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to give 3-(2-prop-2-alkynoxyethoxy)prop-1-ene (1.62 g, 11.56 mmol, yield 57.85%) as a colorless oil.

[0709] data: 1 ¹H NMR (400 MHz, chloroform-d) δ = 5.92 (tdd, J = 5.6, 11.0, 16.9 Hz, 1H), 5.37–5.12 (m, 2H), 4.22 (d, J = 2.1 Hz, 2H), 4.03 (d, J = 5.5 Hz, 2H), 3.76–3.68 (m, 2H), 3.66–3.58 (m, 2H), 2.43 (t, J = 2.1 Hz, 1H)

[0710] 2. A general procedure for preparing 2-(2-prop-2-alkynoxyethoxymethyl)ethylene oxide:

[0711] To a solution of 3-(2-prop-2-alkynoxyethoxy)prop-1-ene (1.52 g, 10.84 mmol, 1 eq) in DCM (20 mL), m-CPBA (3.30 g, 16.26 mmol, 85% purity, 1.5 eq) was added. The mixture was stirred at 25 °C for 12 hours. The reaction mixture was quenched with saturated Na₂SO₃ (20 mL) and treated with EtOAc (30 mL). 3) Extraction. The organic layer was dried over Na2SO4 and concentrated to obtain the crude product. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to give 2-(2-prop-2-alkynoxyethoxymethyl)ethylene oxide (1.7 g, crude product), which was a colorless oil.

[0712] data: 1 H NMR (400 MHz, chloroform-d) δ = 4.15 (d, J = 2.3 Hz, 2H), 3.74 (dd, J =3.1, 11.6 Hz, 1H), 3.70 - 3.57 (m, 4H), 3.37 (dd, J = 5.9, 11.7 Hz, 1H), 3.11(tdd, J = 2.9, 4.1, 5.8 Hz, 1H), 2.74 (t, J = 4.6 Hz, 1H), 2.55 (dd, J = 2.8,5.0 Hz, 1H), 2.37 (t, J = 2.3 Hz, 1H)

[0713] 3. General procedure for preparing 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol:

[0714] A mixture of 2-(2-prop-2-alkynoxyethoxymethyl)ethylene oxide (400 mg, 2.56 mmol, 1 eq) in NH3 / MeOH (4 mL, 7 M) was stirred at 50 °C for 5 hours. The reaction mixture was concentrated to give 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol (440 mg, crude product) as a colorless oil.

[0715] 4. General procedure for preparing N-[2-hydroxy-3-(2-prop-2-alkynoxyethoxy)propyl]carbamate tert-butyl ester:

[0716] TEA (514.10 mg, 5.08 mmol, 707.15 μL, 2 eq) and Boc2O (831.61 mg, 3.81 mmol, 875.38 μL, 1.5 eq) were added to a DCM (10 mL) solution of 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol (440 mg, 2.54 mmol, 1 eq). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was then added to water (2 mL) and mixed with DCM (3 mL). 3) Extraction. The combined organic layers were washed with brine (8 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, petroleum ether: ethyl acetate = 1:1) to give N-[2-hydroxy-3-(2-prop-2-alkynoxyethoxy)propyl]carbamate tert-butyl ester (312 mg, 1.14 mmol, yield 44.94%), as a colorless oil.

[0717] data: 1 H NMR (400 MHz, chloroform-d) δ = 5.12 - 5.01 (m, 1H), 4.22 (d, J = 2.3Hz, 2H), 3.94 - 3.85 (m, 1H), 3.77 - 3.65 (m, 4H), 3.60 - 3.53 (m, 1H), 3.51- 3.43 (m, 1H), 3.37 (br d, J = 13.6 Hz, 1H), 3.20 - 3.11 (m, 1H), 2.47 (t, J= 2.3 Hz, 1H), 1.46 (s, 9H)

[0718] 5. General procedure for preparing 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol:

[0719] TFA (0.3 mL) was added to a DCM (1 mL) solution of N-[2-hydroxy-3-(2-prop-2-alkynoxyethoxy)propyl]carbamate tert-butyl ester (150 mg, 548.80 μmol, 1 eq). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated to give 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol (150 mg, crude, TFA) as a colorless oil.

[0720] 6. A general procedure for preparing (2S)-2-[[6-[3-(azacyclobutane-3-yloxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester: TFA (0.2 mL) was added to a DCM (1 mL) solution of 3-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]azacyclobutane-1-carboxylic acid tert-butyl ester (200 mg, 369.25 μmol, 1 eq). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated to give (2S)-2-[[6-[3-(azacyclobutane-3-yloxy)phenoxy]pyridin-3-carbonyl]amino]-5,5-dimethyl-hexanoate methyl ester (400 mg, crude, TFA), as a brown oil.

[0721] data: LCMS (ESI + ): m / z 442.1 (M+H) +

[0722] 7. A general procedure for preparing 3-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]azacyclobutane-1-carboxylic acid (4-nitrophenyl) ester: Add DIEA (122.14 mg, 945.02 μmol, 164.61 μL, 3 eq) and bis(4-nitrophenyl) carbonate (95.83 mg, 315.01 μmol, 1 eq) to a DMF (4 mL) solution of (2S)-2-[[6-[3-(azacyclobutan-3-yloxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (350 mg, 630.02 μmol, 2 eq, TFA) and bis(4-nitrophenyl) carbonate (95.83 mg, 315.01 μmol, 1 eq). Stir the mixture at 25 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-75%; flow rate: 80 mL / min) to give 3-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]azacyclobutane-1-carboxylic acid (4-nitrophenyl) ester (105 mg, 173.09 μmol, yield 54.95%), as a white solid.

[0723] data: LCMS (ESI + ): m / z 607.2 (M+H) +

[0724] 8. A general procedure for preparing (2S)-2-[[6-[3-[1-[[2-hydroxy-3-(2-prop-2-alkynoxyethoxy)propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: To a DMF (1 mL) solution of 1-amino-3-(2-prop-2-alkynoxyethoxy)prop-2-ol (40.25 mg, 140.12 μmol, 1 eq, TFA) and 3-[3-[[5-[[(1S)-1-methoxycarbonyl-4,4-dimethyl-pentyl]carbamoyl]-2-pyridyl]oxy]phenoxy]azacyclobutane-1-carboxylic acid (4-nitrophenyl) ester (85 mg, 140.12 μmol, 1.00 eq), DIEA (54.33 mg, 420.36 μmol, 73.22 μL, 3 eq) and HOBt (28.40 mg, 210.18 μmol, 1.5 eq) were added. The mixture was stirred at 50 °C for 4 hours. The mixture was then stirred at 50 °C for 12 hours. The reaction mixture was filtered, and the filtrate was purified by reversed-phase HPLC (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-75%; flow rate: 60 mL / min) to give (2S)-2-[[6-[3-[1-[[2-hydroxy-3-(2-prop-2-alkynoxyethoxy)propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (40 mg, 62.43 μmol, yield 44.55%) as a white solid.

[0725] data: LCMS (ESI + ): m / z 641.2 (M+H) +

[0726] 9. A general procedure for preparing (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Methyl 5,5-dimethyl hexanoate (40.00 mg, 62.43 μmol, 1 eq) and 2-[3-[(2-azidoacetyl)amino]propyl]-N-[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]pyrazol-3-carboxamide (43.88 mg, 62.43 μmol, 1 eq) were reacted with t-BuOH (0.5 mL) and H2O (0.5 mL). CuSO4·5H2O (15.59 mg, 62.43 μmol, 1 eq) and sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol (12.37 mg, 62.43 μmol, 1 eq) were added to a solution of 1 mL. The mixture was stirred at 50 °C for 1 hour. The reaction mixture was filtered, and the filtrate was passed through a reversed-phase HPLC system (column: C18 20-35 μm 100A 40 g; mobile phase: [water-ACN]; B%: 0%-75%; flow rate: 60 rpm). Purification at a rate of mL / min yielded (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (56 mg, 41.68 μmol, yield 66.76%), as a yellow solid.

[0727] data: LCMS (ESI + ): m / z 1343.5 (M+H) +

[0728] 10. A general procedure for preparing (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate: Add TFA (307.00 mg, 2.69 mmol, 0.2 mL, 120.59 mg) to a DCM (0.6 mL) solution of (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-[3,5-dimethyl-1-(2-trimethylsilylethoxymethyl)pyrazol-4-yl]anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 22.33 μmol, 1 eq) to a solution of methyl hexanoate (30 mg, 22.33 μmol, 1 eq). The mixture was stirred at 0 °C for 1 hour. Then the mixture was stirred at 25 °C for 11 hours. The reaction mixture was concentrated to give (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, crude, TFA), as a colorless oil.

[0729] data: LCMS (ESI + ): m / z 1213.5 (M+H) +

[0730] 11. General procedure for the preparation of (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid: Add LiOH·H2O (4.15 mg, 98.90 μmol, 4 eq) to a solution of (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoate (30 mg, 24.72 μmol, 1 eq) in THF (0.5 mL) and H2O (0.5 mL). The mixture was stirred at 0 °C for 1 hour. The reaction mixture was filtered, and the filtrate was passed through a preparative HPLC system (FA conditions; column: Phenomenexluna C18 100). 40mm 3 μm; mobile phase: [H2O(0.2%FA)-ACN]; gradient: 35%-65% over 8.0 min. B) Purification yielded (2S)-2-[[6-[3-[1-[[3-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]ethoxy]-2-hydroxy-propyl]carbamoyl]azacyclobutane-3-yl]oxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (10 mg, 8.10). It is a white solid (μmol, yield 32.78%, purity 97.202%).

[0731] data: LCMS (ESI + ): m / z 1221.5 (M+Na) + 1H NMR (400 MHz, methanol-d4) δ = 8.62 (d, J = 2.2 Hz, 1H), 8.29 - 8.18(m, 1H), 7.99 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 1.7 Hz, 1H),7.33 (t, J = 8.3 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.7 Hz, 1H),6.88 (d, J = 1.7 Hz, 1H), 6.80 - 6.67 (m, 2H), 6.64 (s, 1H), 5.10 (s, 2H), 4.98 (br d, J = 3.4 Hz, 1H), 4.66 - 4.45 (m, 5H), 4.31 (br t, J = 7.6 Hz,2H), 3.91 (br d, J = 8.8 Hz, 2H), 3.76 (quin, J = 5.4 Hz, 1H), 3.63 (br dd, J= 5.2, 14.2 Hz, 4H), 3.49 - 3.39 (m, 2H), 3.29 - 3.09 (m, 4H), 2.22 (s, 6H), 2.09 - 2.00 (m, 2H), 1.99 - 1.90 (m, 1H), 1.87 - 1.73 (m, 1H), 1.40 - 1.27(m, 2H), 0.90 (s, 9H), 0.89 - 0.73 (m, 3H), 0.62 - 0.52 (m, 1H), 0.48 (ddd, J= 4.6, 8.5, 13.1 Hz, 2H), 0.43 - 0.34 (m, 2H), 0.33 - 0.20 (m, 3H) X-092: (2S)-2-[[6-[3-[2-[[1-[[1-[2-[[1-[2-[3-[5-[[(1S)-1-(dicyclopropylmethyl)-2-[4-(3,5-dimethyl-1H-pyrazol-4-yl)anilino]-2-oxo-ethyl]carbamoyl]pyrazol-1-yl]propylamino]-2-oxo-ethyl]triazol-4-yl]methoxy]acetyl]azacyclobutane-3-yl]oxymethyl]-2-hydroxy-ethyl]amino]-2-oxo-ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[0732] 1. A general procedure for preparing 3-[tert-butyl(dimethyl)silyl]oxy-2-(dibenzylamino)prop-1-ol:

[0733] Imidazole (551.94 mg, 8.11 mmol, 1.1 eq) and TBSCl (999.80 mg, 6.63 mmol, 816.16 μL, 0.9 eq) were added to a DMF (35 mL) solution of 2-(dibenzylamino)propane-1,3-diol (2 g, 7.37 mmol, 1 eq) at 25 °C. The mixture was stirred at 25 °C for 48 hours. The reaction mixture was added to ice water (100 mL) and then diluted with EtOAc (40 mL). 2) Extraction. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel column chromatography (ISCO®; 80 g SepaFlash® silica gel column, eluent 0%-5% ethyl acetate / petroleum ether gradient; flow rate: 100 mL / min; petroleum ether: ethyl acetate = 5:1) to give 3-[tert-butyl(dimethyl)silyl]oxy-2-(dibenzylamino)prop-1-ol (1.28 g, crude product), as a colorless oil.

[0734] data: LCMS (ESI + ): m / z 386.4 (M+H) +

[0735] 2. A general procedure for preparing N,N-dibenzyl-1-[tert-butyl(dimethyl)silyl]oxy-3-iodo-prop-2-amine:

[0736] Imidazole (264.81 mg, 3.89 mmol, 1.5 eq), PPh3 (816.21 mg, 3.11 mmol, 1.2 eq), and I2 (756.92 mg, 2.98 mmol, 600.73 μL, 1.15 eq) were added to a DCM (35 mL) solution of 3-[tert-butyl(dimethyl)silyl]oxy-2-(dibenzylamino)prop-1-ol (1 g, 2.59 mmol, 1 eq) at 25 °C. The mixture was stirred at 25 °C for 12 hours. LCMS showed that the target mass was detected. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica column chromatography (ISCO®; 40 g SepaFlash® silica column, eluent: 0%-5% ethyl acetate / petroleum ether gradient; flow rate: 80 mL / min; petroleum ether: ethyl acetate = 20:1) to give N,N-dibenzyl-1-[tert-butyl(dimethyl)silyl]oxy-3-iodo-prop-2-amine (1 g, crude product), as a colorless oil.

[0737] data: 1 H NMR (400 MHz, chloroform-d) δ = 7.40 - 7.35 (m, 4H), 7.29 (t, J = 7.5Hz, 4H), 7.24 - 7.18 (m, 2H), 4.18 - 4.11 (m, 1H), 4.07 - 4.01 (m, 2H), 3.83- 3.74 (m, 8H), 3.54 (d, J = 5.9 Hz, 2H), 2.95 (quin, J = 5.7 Hz, 1H), 1.45(s, 9H), 0.89 (s, 9H), 0.03 (d, J = 6.5 Hz, 6H)

[0738] 3. A general procedure for preparing tert-butyl 3-[3-[tert-butyl(dimethyl)silyl]oxy-2-(dibenzylamino)propoxy]azacyclobutane-1-carboxylic acid:

[0739] Under a nitrogen atmosphere and at 0 °C, NaH (117.08 mg, 2.93 mmol, 60% ...

Claims

1. A bifunctional compound according to formula (I) or a pharmaceutically acceptable salt thereof: T L –L I – S L (I) in S L This refers to the protein sorting part based on the combination of AI formulas: Formula (AI), Where R L Indicates with L I The connection; L I It is a connector or key; and T L It is the part that binds to extracellular target molecules.

2. The bifunctional compound according to claim 1, wherein S L According to equation (A-Ia): Equation (A-Ia), Where R L Indicates with L I The connection.

3. The bifunctional compound according to any of the preceding claims, wherein S L According to equation (A-Ib): Formula (A-Ib), Where R L Indicates with L I The connection.

4. The bifunctional compound according to any of the preceding claims, wherein the connector is according to formula (II): Equation (II) in: Indicates with T L or S L The connection; L 1 and L 2 Each group is independently selected from: bond, -C(H2)-, -O-, -N(H)-; functional groups selected from carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; and C1-C3 hydrocarbon chain, wherein one or more methylene groups are independently and optionally replaced by carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; Z is selected from: divalent, saturated or unsaturated, straight or branched C1-C 30 A hydrocarbon chain in which one or more methylene groups are independently and optionally replaced by one or more groups selected from: -O-, –N(H)-, -N(R)-. L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -NHC(O)NH-, -NHC(O)O-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 )-; optionally substituted aromatic groups; optionally substituted carbocyclic rings; optionally substituted heterocyclic rings; optionally substituted aromatic heterocyclic rings; ; , , , -C(R) L2 H- and -N(R) L2 )-; R L1 Selected from C 1-5 Alkyl; R L2 For –(CH2) L -R X ;R X It is -OH or -C(=O)NH2; L is an integer from 0 to 3; n and w are each an independent integer from 1 to 9.

5. The bifunctional compound according to any of the preceding claims, wherein the C1-C 30 The hydrocarbon chain is C5-C. 30 Hydrocarbon chains, such as C8-C 30 Hydrocarbon chains, such as C 10 -C 30 Hydrocarbon chains, such as C 12 -C 30 Hydrocarbon chain.

6. The bifunctional compound according to any of the preceding claims, wherein the C1-C 30 The hydrocarbon chain is C 10 -C 25 Hydrocarbon chains, such as C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 Or C 25 Hydrocarbon chain.

7. The bifunctional compound according to any of the preceding claims, wherein the C1-C 30 The hydrocarbon chain is C 14 -C 20 Hydrocarbon chain.

8. The bifunctional compound according to any of the preceding claims, wherein the C1-C 30 The hydrocarbon chain is C7-C. 13 Hydrocarbon chain.

9. The bifunctional compound according to any of the preceding claims, wherein one or more methylene groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 methylene groups) of the hydrocarbon chain Z are independently and optionally replaced by one or more groups selected from: -O-, –N(H)-, –N(R-). L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 -, -CH2-CH2-O-, optionally substituted carbocyclic rings, optionally substituted heterocyclic rings and triazoles; and R L1 C 1-5 alkyl.

10. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or more –NH-SO42- groups. 2 - group.

11. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or more triazole groups.

12. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or more groups selected from the group consisting of optionally substituted carbocyclic groups and optionally substituted heterocyclic groups.

13. The bifunctional compound according to any of the preceding claims, wherein Z comprises two groups each independently selected from: triazole, optionally substituted carbocyclic group and optionally substituted heterocyclic group.

14. The bifunctional compound according to any of the preceding claims, wherein Z comprises three groups each independently selected from: triazole, optionally substituted carbocyclic group and optionally substituted heterocyclic group.

15. The bifunctional compound according to any of the preceding claims, wherein the carbocyclic ring is based on... As shown, n is an integer selected from 0, 1, 2 or 3.

16. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or more heterocyclic groups.

17. The bifunctional compound according to any of the preceding claims, wherein the heterocyclic group may be an optionally substituted 3- to 6-membered ring, wherein one or two carbon atoms of the ring have been replaced by N.

18. The bifunctional compound according to any of the preceding claims, wherein the heterocyclic group is based on As shown, n is an integer selected from 0, 1, 2 or 3.

19. The bifunctional compound according to any of the preceding claims, wherein Z comprises one, two, or three groups each independently selected from: 、 、 、 、 、 、 、 、 、 、 、 、 or .

20. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or two groups, each independently selected from: Where n and / or n' are each independent integers from 1 to 10, and t, t' and / or w are each independent integers from 1 to 20.

21. The bifunctional compound according to any of the preceding claims, wherein Z comprises or , where n is an integer from 1 to 10.

22. The bifunctional compound according to any of the preceding claims, wherein Z comprises or , where n is an integer from 1 to 10, and t or w is an integer from 1 to 20.

23. The bifunctional compound according to any of the preceding claims, wherein Z comprises , where n is an integer from 1 to 10, and t and t' are each an integer from 1 to 20 independently.

24. The bifunctional compound according to any of the preceding claims, wherein Z comprises , where n and n' are each an integer from 1 to 10, and t is an integer from 1 to 20.

25. The bifunctional compound according to any of the preceding claims, wherein Z comprises one or more branches, such as one, two, or three, bearing polar groups, each branch being independently -C(R) L2 )H- or -N(R L2 )-; where R L2 For –(CH2) L -R X ;R X It is -OH or -C(=O)NH2; and L is an integer from 0 to 3.

26. The bifunctional compound according to any of the preceding claims, wherein Z comprises a branch with a polar group, said branch being –C(R L2 )H- or -N(R L2 )-; where R L2 For –(CH2) L -R X ;R X It is -OH or -C(=O)NH2; and L is an integer from 0 to 3.

27. The bifunctional compound according to any of the preceding claims, wherein Z comprises: 。 28. The bifunctional compound according to any of the preceding claims, wherein Z comprises , , , , , or .

29. The bifunctional compound according to any of the preceding claims, wherein Z comprises or .

30. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 It is a triazole group.

31. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All of them are triazole groups.

32. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 It is –O-.

33. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All are –O-.

34. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 It is –NH-.

35. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All are –NH-.

36. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 It is –S(=O)2-.

37. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All are –S(=O)2-.

38. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 yes .

39. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All are .

40. The bifunctional compound according to any of the preceding claims, wherein L 1 or L 2 yes .

41. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 All are .

42. The bifunctional compound according to any of the preceding claims, wherein L 1 and / or L 2 yes .

43. The bifunctional compound according to any of the preceding claims, wherein L 1 and / or L 2 yes .

44. The bifunctional compound according to any of the preceding claims, wherein L 1 and / or L 2 yes , where X is an atom selected from N or O.

45. The bifunctional compound according to any of the preceding claims, wherein L 1 and / or L 2 yes , where X is an atom selected from N or O.

46. ​​The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 They are different groups.

47. The bifunctional compound according to any of the preceding claims, wherein L 1 and L 2 They are the same.

48. The bifunctional compound according to any of the preceding claims, wherein the connector L 1 It is based on any of the structures in equations II-1 to II-98: 。 49. The bifunctional compound according to any of the preceding claims, wherein the target molecule is a protein.

50. The bifunctional compound according to any of the preceding claims, wherein the compound is capable of forming a ternary complex between the sorting protein and the target protein.

51. The bifunctional compound according to any of the preceding claims, wherein the compound is capable of simultaneously binding sorting proteins and target proteins.

52. The bifunctional compound according to any of the preceding claims, wherein S L The dissociation constant of the protein binding to the sorting protein is less than 50 µM, for example less than 2 µM, for example less than 0.5 µM, preferably less than 0.1 µM, and T L The dissociation constant of its binding to its target is less than 100 µM, for example less than 0.5 µM, for example less than 0.1 µM.

53. The bifunctional compound according to any of the preceding claims, wherein the compound is capable of binding to sorting proteins on the cell surface.

54. The bifunctional compound according to any of the preceding claims, wherein in S L Binding to sorting proteins located on the cell surface and T L After binding to the target protein, the target protein is internalized into the cell.

55. The bifunctional compound according to any of the preceding claims, wherein the target protein is degraded after internalization into the cell.

56. The bifunctional compound according to any of the preceding claims, wherein T L According to either formula BI or B-II: Where R L Indicates with L I The connection.

57. The bifunctional compound according to any of the preceding claims, wherein the target protein is selected from: PCSK9, TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgA, IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-β (TGF-β), platelet-reactive protein-1. (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

58. The bifunctional compound according to any of the preceding claims, wherein the target protein is TNF-α.

59. The bifunctional compound according to any of the preceding claims, wherein T L According to any one of formulas (B-III-1) to (B-III-8): Where R L Indicates with L I The connection.

60. The bifunctional compound according to any of the preceding claims, wherein the compound is according to any one of formulas IV-B1 to IV-B8: 。 61. The bifunctional compound according to any of the preceding claims, wherein the compound is according to any one of formulas V-B1 to V-B8: 。 62. The bifunctional compound according to any of the preceding claims, wherein the compound is according to formula IV-B7.

63. The bifunctional compound according to any of the preceding claims, wherein the compound is according to formula V-B7.

64. The compound according to any one of claims 60 to 63, wherein L I As defined in any one of claims 4 to 48.

65. The bifunctional compound according to any one of claims 60 to 63, wherein L I As defined in claim 48.

66. The bifunctional compound according to any of the preceding claims, wherein the compound is capable of forming a ternary complex between the sorting protein and TNF-α.

67. The bifunctional compound according to any of the preceding claims, wherein the compound is capable of simultaneously binding sorting proteins and TNF-α.

68. The bifunctional compound according to any of the preceding claims, wherein in S L Binding to sorting proteins located on the cell surface and T L Upon binding with TNF-α, TNF-α is internalized into the cells.

69. The bifunctional compound according to any of the preceding claims, wherein TNF-α is degraded after being internalized into cells.

70. The bifunctional compound according to any of the preceding claims, wherein the bifunctional compound is: Or its pharmaceutically acceptable salt.

71. A pharmaceutical composition comprising a bifunctional compound according to any of the preceding claims.

72. The bifunctional compound according to any of the preceding claims, used as a medicament.

73. The bifunctional compound according to any of the preceding claims, used to treat a symptom or condition in a subject in need.

74. The bifunctional compound used according to claim 73, wherein the symptom or condition is mediated by an extracellular protein.

75. The bifunctional compound used according to any of the preceding claims, wherein the extracellular protein is selected from: PCSK9, TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgA, IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-β (TGF-β), and platelet-reactive protein-1. (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

76. The bifunctional compound used according to any of the preceding claims, wherein the extracellular protein is TNF-α.

77. The bifunctional compound used according to any of the preceding claims, wherein the condition or disease is an inflammatory disease.

78. The bifunctional compound used according to any of the preceding claims, wherein the condition or disease is an autoimmune disease.

79. The bifunctional compound used according to any of the preceding claims, wherein the condition or disease is cancer.

80. The bifunctional compound used according to any of the preceding claims, wherein the subject is a mammal.

81. The bifunctional compound used according to any of the preceding claims, wherein the mammal is a human.

82. A method for targeting lysosomes to degrade extracellular proteins, comprising administering an effective amount of the bifunctional compound according to any one of claims 1 to 70.

83. A method for removing an extracellular target protein from the plasma of a patient or subject in need, comprising administering a bifunctional compound according to any one of claims 1 to 70.

84. Use of the bifunctional compound according to any of the preceding claims in the manufacture of a medicament for treating a disease or condition.

85. A method of treating a disease or condition, comprising administering to a subject in need a bifunctional compound according to any one of claims 1 to 70.

86. Use of the bifunctional compound according to any one of claims 1 to 70 in the manufacture of a medicament for treating a disease or condition mediated by extracellular proteins.

87. A method of treating a disease or condition mediated by extracellular proteins, comprising administering to a subject in need a bifunctional compound according to any one of claims 1 to 70.

88. A compound of formula (A-II) or a pharmaceutically acceptable salt thereof, or an enantiomer thereof, or a mixture thereof: Equation (A-II), Where R 1 C is H, halogen, alkoxy, -CF3, or optionally substituted. 1-5 Hydrocarbon chains, wherein one or more carbon groups of the C1-C5 hydrocarbon chain are optionally and independently replaced by one or more groups selected from: –O-, -NH-, -C(O)-, esters, amides, carbamates, thioureas, sulfonamides, ureas, , , Optionally substituted carbocyclic rings, Optionally substituted heterocyclic rings and Where X is NH or O.

89. The compound according to claim 88, wherein the compound is according to formula (A-IIa): Formula (A-IIa), or a pharmaceutically acceptable salt thereof.

90. The compound according to any one of claims 88 to 89, wherein the compound is according to formula (A-II-1): Or, or a pharmaceutically acceptable salt thereof.

91. The compound according to any one of claims 88 to 90, R 1 For H.

92. The compound according to any one of claims 88 to 90, R 1 Selected from halogens or –CF3.

93. The compound according to any one of claims 88 to 90, R 1 The C1-C5 alkyl group is optionally substituted, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally and independently replaced by one or more groups selected from the following: –O-, -NH-, -C(O)-, esters, amides, carbamates, thioureas, and .

94. The compound according to any one of claims 88 to 90, wherein the compound is (C-001) (C-002), or a pharmaceutically acceptable salt thereof.

95. A composition comprising the compound according to any one of claims 88 to 94.

96. The compound according to any one of claims 88 to 94, or the composition according to claim 95, is used as a pharmaceutical.

97. A bifunctional compound of formula (X), or a pharmaceutically acceptable salt thereof: T A-L –L I –S A-L (X) in: S A-L For binding and sorting protein portions; L I For connectors or keys; and T A-L This is the portion that binds to interleukin-17-A (IL-17A).

98. The bifunctional compound according to claim 97, wherein S A-L According to equation X-III: Formula X-III, or a pharmaceutically acceptable salt thereof; wherein L I For connectors or keys; and T A-L It is a group that binds to interleukin-17-A (IL-17A).

99. The bifunctional compound according to claim 98, wherein the bifunctional compound is according to any one of formulas X-III-B1 to X-III-D4: 。 100. The bifunctional compound according to claim 97, wherein S A-L According to equation X-IV: or its pharmaceutically acceptable salt; wherein L I It is a connector or key; and T A-L It is the part that binds to interleukin-17-A (IL-17A).

101. The bifunctional compound according to claim 100, wherein the bifunctional compound is according to any one of formulas X-IV-B1 to X-IV-D4: 。 102. The bifunctional compound according to claim 97, wherein S A-L According to equation XV: Formula (XV), or a pharmaceutically acceptable salt thereof; wherein: L I It is a connector or key; and T A-L It is a group that binds to interleukin-17-A (IL-17A).

103. The bifunctional compound according to claim 102, wherein the bifunctional compound is according to any one of formulas XV-B1 to XV-D4: 。 104. The bifunctional compound according to any one of claims 97 to 103, wherein the connector (L I As defined in any one of claims 4 to 48.

105. The bifunctional compound according to any one of claims 97 to 104, wherein the connector (L I As defined in claim 48.

106. The bifunctional compound according to any one of claims 97 to 105, wherein the bifunctional compound is any one of compounds X-001 to X-098, or a pharmaceutically acceptable salt thereof: 。 107. The bifunctional compound according to any one of claims 97 to 106, wherein the compound is capable of forming a ternary complex with sorting protein and IL-17A.

108. The bifunctional compound according to any one of claims 97 to 106, wherein the compound is capable of simultaneously binding sorting proteins and IL 17-A.

109. The bifunctional compound according to any one of claims 97 to 108, wherein the compound is capable of inducing IL-17A internalization into cells expressing sorting proteins.

110. The bifunctional compound according to any one of claims 97 to 109, wherein in S A-L Binding to sorting proteins located on the cell surface and T A-L Upon binding with IL-17A, IL-17A is internalized into the cells.

111. The bifunctional compound according to any one of claims 97 to 110, wherein IL 17-A is degraded after being internalized into the cell.

112. The bifunctional compound according to any one of claims 97 to 111, wherein T A-L The dissociation constant (K) of the IL 17-A binding D Below 500 nM, for example, below 250 nM, for example, below 100 nM, for example, below 50 nM.

113. The bifunctional compound according to any one of claims 97 to 112, wherein S A-L Dissociation constant (K) of the sorting protein D Less than 50 µM, for example less than 2 µM, for example less than 0.5 µM, preferably less than 0.1 µM, and T A-L The dissociation constant of its binding to its target is less than 100 µM, for example less than 0.5 µM, for example less than 0.1 µM.

114. A composition comprising a bifunctional compound according to any one of claims 97 to 113 and a pharmaceutically acceptable excipient.

115. A method for removing IL-17A from the plasma of a subject in need, using the bifunctional compound according to any one of claims 97 to 113, or the composition according to claim 114, the method comprising administering an effective amount of the bifunctional compound to the subject.

116. A method for treating or preventing inflammation or inflammatory conditions using a bifunctional compound according to any one of claims 97 to 113, or a composition according to claim 114, the method comprising administering an effective amount of the bifunctional compound to a subject.

117. The bifunctional compound or composition according to claim 116, wherein the inflammation or inflammatory condition is characterized by a high level of IL-17A or overexpression of IL-17A.

118. The bifunctional compound or composition used according to claim 117, wherein the condition is psoriasis, rheumatoid arthritis, spondyloarthritis, multiple sclerosis, psoriatic arthritis, axial spondyloarthritis, ankylosing spondylitis, hidradenitis suppurativa, systemic lupus erythematosus, palmoplantar pustulosis (PPP), or atopic dermatitis.

119. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from: 。

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