Cyclic peptides for capturing interleukin-1 beta

Abstract

Provided are compounds of Formula (I) or pharmaceutically acceptable salts thereof, wherein X 1 , X 2 , X 3 , R 1 , R 9 , R A , R B , R D , A 1 , A 2 and subscripts t and w are as described herein. Formula (I), (I). The compounds and pharmaceutically acceptable salts thereof can trap IL-1β and are expected to have utility as therapeutic agents, for example, for treating cardiovascular diseases and inflammatory disorders. The present disclosure also provides pharmaceutical compositions comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof. The present disclosure also relates to methods of use of the compounds or pharmaceutically acceptable salts thereof in treating or preventing cardiovascular diseases and inflammatory disorders and methods of manufacturing a medicament for this purpose.(I)

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 598,567, filed November 14, 2023, and U.S. Provisional Patent Application No. 63 / 717,339, filed November 7, 2024, the entire contents of which are incorporated herein by reference.

[0002] Reference to the electronically submitted sequence list The contents of the electronic sequence list (25836-WO-PCT_SL.xml; size: 2,104,813 bytes, creation date: October 31, 2024) are incorporated into this document by reference in their entirety.

[0003] Invention Field This disclosure relates to certain cyclic peptides that capture interleukin-1β (IL-1β), pharmaceutical compositions comprising such peptides, and methods of using said compounds to treat, inhibit, or alleviate one or more cardiovascular disease states (including atherosclerosis) that may benefit from capturing IL-1β. Background Technology

[0004] Atherosclerosis is an arterial disease characterized by the buildup of cholesterol plaques on the inner walls of arteries. The progression of atherosclerosis can lead to hardening or narrowing of the arteries and increases the risk of plaque rupture. These ruptures release cholesterol globules and other substances into the bloodstream, which can potentially obstruct blood flow to the brain, heart, or other organs. Medically, these are known as major adverse cardiac events (MACE).

[0005] Risk factors for the development and progression of atherosclerotic cardiovascular disease (ASCVD) include high cholesterol, high blood pressure, a high-saturated-fat diet, smoking, obesity, diabetes, lack of exercise, and elevated levels of C-reactive protein (CRP, an inflammatory marker).

[0006] First-line treatment for preventing the progression of ASCVD is a healthy diet and exercise; however, compliance is often poor. Drug treatment for ASCVD primarily focuses on cholesterol-lowering medications, such as statins, cholesterol absorption inhibitors, and low-density lipoprotein (LDL) receptor inhibitors. These medications are very effective in reducing the accumulation of fatty acid deposits and improving arterial health. Other prescription medications used to treat ASCVD but not to improve the disease state include blood thinners, such as aspirin, to prevent platelet aggregation in narrowed arteries, and blood pressure medications to reduce the risk and severity of heart attacks. Surgical options for more invasive interventions in advanced cases of ASCVD include angioplasty, stent placement, endarterectomy (surgical removal of plaque), and bypass surgery.

[0007] While cholesterol-lowering medications have become an important standard of care for slowing the progression of atherosclerosis, clinical data support an additional crucial role of inflammation in the progression of untreated ASCVD. Inflammatory biomarkers (such as CRP) are associated with an increased risk of cardiovascular events, independent of cholesterol levels. The Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) was the first clinical trial to demonstrate that reducing vascular inflammation without simultaneously lowering lipids reduces the incidence of cardiovascular events. N Engl J Med 2017;377:1119-1131. Cananumab is a human monoclonal antibody against interleukin-1β (IL-1β) approved for clinical use in the treatment of rheumatic diseases. IL-1β is a pro-inflammatory cytokine that induces IL-6, thereby increasing the downstream inflammatory biomarker hypersensitive CRP (hsCRP). Therefore, CANTOS provides a proof-of-concept that IL-1β-targeting therapy may reduce the incidence of MACE in certain patients in a complementary and potentially additive manner to the standard of care for lowering LDL.

[0008] In addition to standard-of-care cholesterol-lowering medications, other non-surgical treatments are needed to slow the progression of atherosclerosis and reduce the risk of MACE. Furthermore, patients with inflammatory conditions may benefit from oral administration of agents that block the same cytokine IL-1β as cannabinoids. Invention Overview This disclosure provides certain cyclic peptides that reduce inflammation and block the involvement of the IL-1 receptor by binding to the IL-1β cytokine, thereby resulting in inhibition of downstream pro-inflammatory signaling. These cyclic peptides could be valuable pharmaceutically active compounds for the treatment of cardiovascular diseases and inflammatory conditions. In one aspect, this disclosure provides compounds of formula (I). (I) And its pharmaceutically acceptable salts.

[0010] The compound can capture IL-1β and thereby affect downstream pro-inflammatory signaling pathways that may be associated with cardiovascular conditions. Therefore, in another aspect, this disclosure provides a method for treating cardiovascular conditions (e.g., atherosclerosis, vascular inflammation) comprising administering a therapeutically effective amount of the disclosed compound to a subject in need of it. In some embodiments, the administration comprises oral administration of the compound.

[0011] Furthermore, this disclosure provides methods for preparing the compounds of this disclosure, as well as pharmaceutical compositions comprising the compounds of this disclosure and pharmaceutically acceptable carriers. Brief description of the attached diagram Figure 1The amino acid sequences and related characteristics of SEQ ID NO: 1-465 are described. Invention Details The compounds disclosed herein In one embodiment, this disclosure provides a compound having the structural formula (I) shown above, wherein: R 1 for (i) R 1a -C(O)N(H)-CH2CH2-O-, where R 1a for (a) C1-C3 alkyl, or (b) (CH3)3N-CH2CH2-M-, where M is -CH2-, -CH2CH2-, -CH2CH2CH2-, -O-CH2CH2-, or -O-CH2CH2-O-CH2CH2-; (ii) (CH3)3N-CH2CH2-O-; (iii) C 1 C 1 for: (a) a phenyl or a 5- to 6-membered monocyclic heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 3 Each heteroatom is independently selected from N, O, and S; (b) A 5- to 6-membered heterocyclic alkyl group, wherein the 5- to 6-membered heterocyclic alkyl group is saturated and contains one or two heteroatoms selected from N, O and S; Where C 1 For unsubstituted or selected independently from the following R C1 Substituent substitutions: halogenated, C1-C3 alkyl, C1-C3 fluoroalkyl, carboxyl, C1-C3 alkoxy, C2-C3 acyl, and C1-C3 alkoxymethyl; (iii) (CH3)3N-CH2CH2-O-; or (iv) group ; R A for: (i) -CO2H; (ii) -CO2NH2; (iii) -S(O)2NH2; or (iv) A five-membered heteroaryl group containing two or three heteroatoms independently selected from N, O and S; The 5-membered heteroaryl group is either unsubstituted or substituted by one or two independent substituents selected from the following: halogenated, C1-C3 alkyl, C1-C3 alkoxy, oxo, and C1-C3 alkoxymethyl; R B It is amino, hydroxyl, or fluorinated; R 2 for (i) naphthyl; or (ii) A 9- or 10-membered bicyclic heteroaryl group, wherein the 9- or 10-membered bicyclic heteroaryl group contains 1 or 2 heteroatoms independently selected from N, O and S; Where R 2 For unsubstituted or selected independently by one or two of the following R 2a Substituents: halogenated, C1-C3 alkyl, and C1-C3 alkoxy; R 3 -(CH2) m CO2H, -(CH2) m CO2NH2、-(CH2) m CO2N(CH3)2、-(CH2) m CH2N(H)C(O)NH2 or -(CH2)mC 3 ; Where C 3 It is a 5-membered heteroaryl group containing 2 to 4 heteroatoms selected from N, O and S; Where C 3 It is either unsubstituted or substituted with one or two substituents selected from halogenated and C1-C3 alkyl groups; R 4 It is a C1-C3 alkyl group; R 5 It is H, C1-C3 alkyl or -CH2CO2H; Alternatively, R 4 and R 5 The atoms bonded to them together form pyrrolidinyl, piperidinyl, or aziridine rings; X 1 X 2 and X 3 It can be C(H) or N independently; R D for: (i) Ring C D Among them, ring C D for: (a) phenyl; or (b) Cyclohexyl; Among them, ring C D By an A 1 Replace; where A 1It can be -CO2H, -CONH2, or -N(H)SO2CH3; (ii) Hydroxyl group; (iii) -CO2NH2; or (iv) -OCH2CH2C(O)N(H)C(O)CH3; A 2 It is H, halogenated, or C1-C3 alkyl; R 6 -H, -NH2, -C(O)N(CH3)2; -(CH2)nNH2; -(CH2)nN(H)CH3; -(CH2)nN(CH3)3; -OH; -(CH2)nOH; -OCH3; -(CH2)nOCH3; -(CH2)nCH3; -(CH2)nN(H)C(O)NH2; -(CH2)nCH2C(O)OH; -(CH2)nN(H)C(O)CH2CH2O(CH2CH2-O)q-CH2CH2N(CH3)3; or -C 6 ; C 6 for (i) 5- to 6-membered monocyclic aryl or heteroaryl, wherein the 5- to 6-membered monocyclic heteroaryl contains 1 to 3 Each heteroatom is independently selected from N, O, and S; (ii) a 9- to 10-membered bicyclic aryl or heteroaryl group, wherein the 9- to 10-membered bicyclic heteroaryl group contains 1 to 4 heteroatoms independently selected from N, O, and S; and (iii) 3 to 8-membered monocyclic or bicyclic cycloalkyl groups; Where C 6 For unsubstituted or selected independently from the following R C6 Substituents: halogenated, amino, hydroxyl, carboxyl, C1-C3 alkyl, C1-C3 fluoroalkyl, C1-C3 alkoxy, and C1-C3 alkoxymethyl; R 7 It can be H, C1-C4 alkyl, -(CH2)uOH, -(CH2)uC(O)N(CH3)2, -(CH2)uN(CH3)3, -(CH2)uOCH3, -(CH2)uC(O)NC(O)NH2, -(CH2)uN(H)C(O)CH2CH2O(CH2CH2-O)v-CH2CH2N(CH3)3 or -CH2-C 7 ; C 7 for: (i) is a 5-membered heteroaryl group containing 2 to 4 heteroatoms selected from N, O and S; or (ii) A 5- or 6-membered heterocyclic alkyl group, wherein the 5- or 6-membered heterocyclic alkyl group is saturated and contains one O atom; Where C 7 It is unsubstituted or substituted with one or two substituents selected from the following: halogenated, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 alkoxymethyl; R 8 The octet is -H, -F, -OH, -NH2, -N(CH3)3, (CH3)3N-(CH2)rC(O)- or (CH3)3N-CH2CH2-O-(CH2CH2-O)s-CH2CH2-C(O)-; R 9 -C 9 or -CH2-C 9 ; C 9 It is a phenyl or a 5- to 6-membered monocyclic heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 heteroatoms independently selected from N, O and S; Where C 9 For unsubstituted or selected independently from the following R C9 Substituents: halogenated, amino, hydroxylated, C1-C3 alkyl, C1-C3 fluoroalkyl, and C1-C3 alkoxy; Where C 9 Optionally bounded by 1 ring R C9C Replacement, ring R C9C for: (a) phenyl; or (b) A 5- to 6-membered monocyclic heteroaryl group containing one or two heteroatoms independently selected from N, O and S; The subscript m is 1 or 2; The subscript n is 1, 2, or 3; and The subscript q is 1, 2, or 3; The subscript r is 3, 4, 5, or 6; The subscript s is 1 or 2; The subscript t is 0 or 1; The subscript u is 0, 1, 2, 3, or 4; The subscript v is 1, 2, or 3; and The subscript w is 0 or 1; or Pharmaceutically acceptable salt.

[0014] In another embodiment, this disclosure provides a compound of formula (I), wherein: C 1 It is phenyl; R 2 It is indole or naphthyl; C 6It is phenyl, indolyl, pyridyl, pyridazinyl, or bicyclic [1.1.1]pentyl; and C 9 It is phenyl or pyrimidinyl.

[0015] In another embodiment, this disclosure provides a compound of formula (I), wherein R 1 It is CH3-C(O)N(H)-CH2CH2-O- or 4-fluorophenyl.

[0016] In another embodiment, this disclosure provides a compound of formula (I), wherein R 1 It is (CH3)3N-CH2CH2-M-, where M is CH2-, -CH2CH2CH2-, -O-CH2CH2-, or -O-CH2CH2-O-CH2CH2-.

[0017] In another embodiment, this disclosure provides a compound of formula (I), wherein R 1 C 1 .

[0018] In another embodiment, this disclosure provides a compound of formula (I), wherein A 2 It can be H, fluorinated, chlorinated, or methyl.

[0019] In another embodiment, this disclosure provides a compound of formula (I), wherein R 2 It can be an unsubstituted or substituted naphthyl or indole group.

[0020] In another embodiment, this disclosure provides a compound of formula (I), wherein R 3 -(CH2) m CO2H.

[0021] In another embodiment, this disclosure provides a compound of formula (I), wherein the subscript m is 1.

[0022] In another embodiment, this disclosure provides a compound of formula (I), wherein R 4 and R 5 It is a methyl group.

[0023] In another embodiment, this disclosure provides a compound of formula (I), wherein: X 1 and X 2 For C(H) and A 1 It is a carboxyl group.

[0024] In another embodiment, this disclosure provides a compound of formula (I), wherein (i) a substituted or unsubstituted or 5- to 6-membered monocyclic aryl or heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 N atoms; or (ii) A substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl group, wherein the bicyclic heteroaryl group contains 1 to 2 N atoms.

[0025] In another embodiment, this disclosure provides a compound of formula (I), wherein R 6 It is an indole group.

[0026] In another embodiment, this disclosure provides a compound of formula (I), wherein R 7 For H.

[0027] In another embodiment, this disclosure provides a compound of formula (I), wherein R 8 It can be -H, -OH, -NH2, (CH3)3N-(CH2)5-C(O)- or (CH3)3N-CH2CH2-O-CH2CH2-O-CH2CH2-C(O)-.

[0028] In another embodiment, this disclosure provides a compound of formula (I), wherein R 8 It can be H or -NH2.

[0029] In another embodiment, this disclosure provides a compound of formula (I), wherein R 9 The pyrrole alkyl ring shown is substituted at position 4.

[0030] In another embodiment, this disclosure provides a compound of formula (I), wherein C 9 It can be a substituted or unsubstituted phenyl or pyrimidinyl group.

[0031] In another embodiment, this disclosure provides a compound of formula (I), wherein R 9 It is -CH2-(4-fluorophenyl) or pyrimidin-5-yl.

[0032] In another embodiment, this disclosure provides a compound of formula (I), wherein: R 1 It is CH3-C(O)N(H)-CH2CH2-O- or 4-fluorophenyl; R 2 It can be an unsubstituted or substituted naphthyl or indole group; R 3 -(CH2) m CO2H; R 4 and R 5 It is methyl; X 1 and X2 For C(H); A 1 It is a carboxyl group; A 2 For H; R 6 for (i) a substituted or unsubstituted or 5- to 6-membered monocyclic aryl or heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 N atoms; or (ii) A substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl group, wherein the 9- or 10-membered bicyclic heteroaryl group contains 1 to 2 N atoms; R 7 For H; R 8 For H or -NH2; and R 9 The pyrrole alkyl ring shown is substituted at position 4.

[0033] In one aspect of this embodiment, the present disclosure provides a compound of formula (I), wherein: R 6 It is an indole group; R 9 It is -CH2-(4-fluorophenyl) or pyrimidin-5-yl, with substitution at the 4-position of the pyrrolidinyl ring shown; and The subscript m is 1.

[0034] In some embodiments, the compound of formula (I) has formula (IA): (IA), in: R 1 for: (i) R 1a -C(O)N(H)-CH2CH2-O-, where R 1a for: (a) Methyl; or (b) (CH3)3N-CH2CH2-O-CH2CH2-; (ii) (CH3)3N-CH2CH2-O-; or (iii) 4-Carboxyphenyl; R 2 as a group or ; R 2a It is either halogenated or methylated; The subscript x is 0 or 1; R 3It is -CH2CO2H or -CH2-tetrazole; R 4 and R 5 It is methyl; Alternatively, R 4 and R 5 Together with the atoms they are attached to, they form a piperidinyl ring; R D It is 4-carboxyphenyl or hydroxy; R 6 The groups are selected from the following: , , , , , or ; R 7 It is H or methyl; R 8 It can be -H, -NH2, or -OH; C 9 The groups are selected from the following: , or .

[0035] In other embodiments, the compound of formula (I) has formula (IB): (IB) in: R 1 for (i) R 1a -C(O)N(H)-CH2CH2-O-, where R 1a for (a) C1-C3 alkyl, or (b) (CH3)3N-CH2CH2-M-, where M is -CH2-, -CH2CH2-, -CH2CH2CH2-, -O-CH2CH2-, or -O-CH2CH2-O-CH2CH2-; or (ii) C 1 C 1 It is a phenyl or a 5- to 6-membered monocyclic aryl or heteroaryl, wherein the 5- to 6-membered monocyclic heteroaryl contains 1 to 2 heteroatoms independently selected from N, O and S; Where C 1 For unsubstituted or selected independently from the following R C1Substituents: halogenated, C1-C3 alkyl, C1-C3 fluoroalkyl, carboxyl, C1-C3 alkoxy, and C2-C3 acyl; R 2 for (i) naphthyl; or (ii) A 9- or 10-membered bicyclic heteroaryl group, wherein the 9- or 10-membered bicyclic heteroaryl group contains 1 or 2 heteroatoms independently selected from N, O and S; Where R 2 For unsubstituted or R-1 independently selected from halogenated compounds 2 α-substituent substitution; R 3 -(CH2) m CO2H, -(CH2) m CO2NH2、-(CH2) m CO2N(CH3)2 or -(CH2)m-tetrazole; R 4 It is a C1-C3 alkyl group; R 5 It is H or C1-C3 alkyl; Alternatively, R 4 and R 5 The atoms bonded to them together form pyrrolidinyl, piperidinyl, or aziridine rings; X 1 and X 2 It can be C(H) or N independently; A 1 It is -CO2H or -N(H)SO2CH3; A 2 It is H, halogenated, or C1-C3 alkyl; R 6 -(CH2)nNH2; -(CH2)nN(H)CH3; -(CH2)nOH; -(CH2)nOCH3; -(CH2)nN(H)CH2CH2O(CH2CH2-O)q-CH2CH2N(CH3)3; or -C 6 ; C 6 for (i) 5- to 6-membered monocyclic aryl or heteroaryl, wherein the 5- to 6-membered monocyclic heteroaryl contains 1 to 2 heteroatoms independently selected from N, O and S; (ii) a 9- to 10-membered bicyclic aryl or heteroaryl group, wherein the 9- to 10-membered bicyclic heteroaryl group contains 1 to 4 heteroatoms independently selected from N, O, and S; and (iii) 3 to 8-membered monocyclic or bicyclic cycloalkyl groups; Where C 6For unsubstituted or selected independently from the following R C6 Substituents: halogenated, amino, hydroxylated, carboxylated, C1-C3 alkyl, C1-C3 fluoroalkyl, and C1-C3 alkoxy; R 7 It is H or C1-C4 alkyl; R 8 For -H, -OH, -NH2, (CH3)3N-(CH2)rC(O)- or (CH3)3N-CH2CH2-O-(CH2CH2-O)s-CH2CH2-C(O)- R 9 -C 9 Or –CH2-C 9 ; C 9 It is a phenyl or a 5- to 6-membered monocyclic heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 heteroatoms independently selected from N, O and S; Where C 9 For unsubstituted or selected independently from the following R C9 Substituents: halogenated, amino, hydroxylated, C1-C3 alkyl, C1-C3 fluoroalkyl, C1-C3 alkoxy, and phenyl; The subscript m is 1 or 2; The subscript n is 1, 2, or 3; and The subscript q is 1 or 2; The subscript r is 3, 4, 5, or 6; The subscript s is 1 or 2; or Its pharmaceutically acceptable salt.

[0036] In some embodiments, this disclosure provides compounds of formula (I), wherein said compounds are selected from... Figure 1 The compound is shown in SEQ ID NO: 1-465. In a particular embodiment, the compound is selected from... Figure 1 The SEQ ID NO: 1-103 shown.

[0037] In a particular embodiment, this disclosure provides compounds of formula (I), wherein the compounds are selected from the following (SEQ ID NO: 1, 2, 78, 80, 82, 92, 94, 95, 98, 99, 100, 101, 102, 215, 216, 217 and 218 respectively): , , , , , , , , , , , , , , , and .

[0038] While not bound by any particular theory, the applicant believes that the compounds disclosed herein capture interleukin-1β, blocking signal transduction via the IL-1 receptor and thereby reducing downstream markers IL-6 and CRP. Therefore, the compounds can be used to treat the inflammatory components of cardiovascular diseases such as ASCVD and heart failure with preserved ejection fraction (HFpEF). The compounds can also be used to treat inflammatory conditions such as hidradenitis suppurativa (acne paradoxically), inflammatory bowel disease, and osteoarthritis.

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] As used throughout this disclosure, the terms “disclosed compound,” “compound of this disclosure,” and “compound disclosed herein” are used interchangeably and should be understood to include the disclosed cyclic peptides and compounds of formula (I). References herein to compounds of formula (I) include compounds of each of formulas (IA) and (IB), and all embodiments and categories thereof. Compounds of formula (I) can form salts, which are also within the scope of this disclosure. References herein to compounds of this disclosure (or compounds of formula (I)) should be understood to include references to their salts unless otherwise stated. As used herein, the term “salt” refers to an acidic salt formed with inorganic and / or organic acids, and a basic salt formed with inorganic and / or organic bases. Furthermore, when a compound of formula (I) contains both a basic moiety (e.g., but not limited to, an amino group, pyrrolidine, or imidazole) and an acidic moiety (e.g., but not limited to, a carboxylic acid), an zwitterion (“internal salt”) can be formed and is included in the term “salt” as used herein. In one embodiment, the salt is a pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salt. In another embodiment, the salt is a salt other than a pharmaceutically acceptable salt. For example, a salt of a compound of formula (I) can be formed, for example, by reacting a compound of formula (I) with a certain amount (e.g., an equivalent) of an acid or base in a medium (e.g., a medium in which the salt is precipitated) or in an aqueous medium, followed by freeze-drying.

[0041] "Acyl" indicates an alkyl-C(O)- group, where the alkyl group is defined as follows. The bond to the parent group is through the carbon atom of the carbonyl group.

[0042] "alkyl" and other groups prefixed with "alk" (e.g., alkoxy groups) indicate a carbon chain containing a specified number of carbon atoms, which can be straight-chain, branched, or a combination thereof. For example, C1-C6 alkyl groups indicate alkyl groups having one (i.e., methyl) to six (i.e., hexyl) carbon atoms. In certain embodiments, straight-chain alkyl groups have 1-6 carbon atoms, and branched alkyl groups have 3-7 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, etc.

[0043] "Alkoxy" and "alkyl-O-" are used interchangeably and refer to alkyl groups bonded to oxygen.

[0044] "Amino" refers to the H2N- group. The bond to the parent group is through a nitrogen atom.

[0045] "Amino acid" refers to naturally occurring α-amino acids and their stereoisomers, as well as non-natural amino acids (e.g., β-amino acids and substituted amino acids) and their stereoisomers. In the sequences given for peptides (compounds) according to this disclosure, amino acid residues have their conventional meanings. Thus, "G" is for glycine, "W" for tryptophan, "A" for alanine, "S" for serine, and so on. It should be understood that "D" isomers are indicated by a "d" preceding a single letter code or amino acid name, such that, for example, dA is the D isomer of L-alanine. Amino acid residues not covered by the foregoing have the definitions provided in the tables in the Examples section below.

[0046] As used in this article, "aryl" represents a monocyclic 6-membered or bicyclic 10-membered ring system in which at least one ring is aromatic and all ring atoms are carbon.

[0047] A “double-ring system” refers to two connected rings. The rings can be fused, meaning they share two adjacent atoms, or “spirocyclic,” meaning they share only a single atom.

[0048] "Carboxyl group" refers to the HO2C- group. The bond to the parent group is through the carbonyl group's carbon atom.

[0049] “Cycloalkyl” indicates a saturated cyclic hydrocarbon group. In certain embodiments, the cycloalkyl group has 3-12 carbon atoms, forming 1-3 carbon rings. The rings can be fused or “spirocyclic,” i.e., sharing only a single atom, or “bridged,” i.e., sharing three or more atoms, wherein two bridgehead atoms are connected by a bridge containing at least one atom. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, bicyclo[1.1.1]pentyl, etc.

[0050] "Fluoroalkyl" includes mono-substituted and poly-fluorinated substituted alkyl groups, up to perfluorinated substituted alkyl groups. Examples include fluoromethyl, 1,1-difluoroethyl, trifluoromethyl, or 1,1,1,2,2-pentafluorobutyl.

[0051] Unless otherwise stated, "halogen" or "halogenated" includes fluorine (fluorinated), chlorine (chlorinated), bromine (brominated), and iodine (iodinated). In one embodiment, halogenation is fluorinated (-F) or chlorinated (-C).

[0052] "Heterocyclic alkyl" or "heterocyclic group" refers to a non-aromatic monocyclic, bicyclic, or tricyclic ring system containing about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, wherein one or more atoms in the ring system are elements other than carbon, such as nitrogen, oxygen, or sulfur alone or in combination. Bicyclic and tricyclic rings can be fused, or "spirocyclic," i.e., sharing only a single atom, or "bridged," i.e., sharing three or more atoms, wherein two bridgehead atoms are connected by a bridge containing at least one atom. Adjacent oxygen and / or sulfur atoms are not present in the ring system. In some embodiments, the heterocyclic alkyl group contains about 5 to about 6 ring atoms. The prefixes nitro, oxo, or thio before the name of the heterocyclic alkyl group indicate at least one nitrogen, oxygen, or sulfur atom present as a ring atom, respectively. In some embodiments, the nitrogen or sulfur atom of the heterocyclic alkyl group may optionally be oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclic groups include piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolyl, 1,4-dioxane, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, etc.

[0053] "Heteroaryl" refers to the monocyclic, bicyclic, and tricyclic ring structures of aromatic compounds, in which one or more atoms, or heteroatoms, within the ring are elements other than carbon. Heteroatoms are typically O, S, or N atoms. Examples of heteroaryl groups include pyridinyl, pyrimidinyl, pyrroloyl, pyridazinyl, isoxazolyl, thiazolyl, oxazolyl, indolyl, benzoxazolyl, benzothiazolyl, and imidazolyl.

[0054] When any variable (e.g., R) C1 When a component appears more than once in any component, in formula (I), or in other general formulas herein, its definition for each occurrence is independent of its definition for each subsequent occurrence. Combinations of substituents and / or variables are only permissible if such combinations produce stable compounds. In selecting the compounds of this disclosure, those skilled in the art will recognize that various substituents (e.g., R...) are... C9 The selection should follow well-known principles of chemical structural connectivity and stability. Unless explicitly stated otherwise, substitution is permitted on any atom of the ring (e.g., aryl, heteroaryl, or saturated heteroaryl rings) by designated substituents, provided that such ring substitution is chemically permissible and yields a stable compound. A “stable” compound is one that can be prepared and isolated, and whose structure and properties remain, or are caused to remain, substantially unchanged for a period of time sufficient to allow the compound to be used for the purposes described herein (e.g., therapeutic or prophylactic administration to a subject).

[0055] The term "substituted" should be considered to include the degree of multiple substitution by the specified substituents. In cases where multiple substituent moieties are disclosed or claimed, the substituted compound may be independently substituted by one or more of the disclosed or claimed substituent moieties in a singular or plural manner. Independent substitution means that (two or more) substituents may be the same or different.

[0056] Unless otherwise explicitly described or depicted, variables described in the structural formula as "floating" bonds are permitted to appear on any available carbon atom in the ring to which the variable is connected. When a portion of Formula (I) or any embodiment thereof is indicated as "optionally substituted," Formula (I) or its embodiments cover compounds containing the indicated substituents (or substituents) in that portion as well as compounds not containing the indicated substituents (or substituents) in that portion.

[0057] As shown in the wavy lines used in this article Indicates the connection point with the rest of the compound.

[0058] Some of the compounds described herein can exist as tautomers, which have different hydrogen connection sites accompanied by one or more double bond transfers. For example, ketones and their enol forms are keto-enol tautomers. The compounds disclosed herein cover individual tautomers and mixtures thereof.

[0059] In the compounds of this disclosure, atoms may exhibit their natural isotopic abundance, or one or more atoms may be artificially enriched with a specific isotope having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. As described and claimed herein, this disclosure means including all suitable isotopic variants of the compounds of this disclosure and embodiments thereof. For example, different isotopic forms of hydrogen (H) include protium (… 1 H) and deuterium ( 2 H (also referred to herein as D). Protium is the dominant hydrogen isotope found in nature. Enrichment of deuterium can provide certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or can provide compounds that can be used as standards for characterizing biological samples. The isotope-enriched compounds of this disclosure can be prepared without extensive experimentation using conventional techniques well known to those skilled in the art, or by methods similar to those described in the schemes and examples herein, using appropriate isotope-enriching reagents and / or intermediates.

[0060] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic base or acid. When the compounds of this disclosure are acidic (or have functional groups that may be anions), their corresponding salts can be conveniently prepared from pharmaceutically acceptable, non-toxic bases (including inorganic and organic bases). Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li. + Na + and K + Alkaline earth metal cations, such as Ca2+ + and Mg2 + and other cations such as Al 3+ and Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+). + The compounds contain substituted ammonium ions and the substituted ammonium ions. Examples of suitable substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, triethylamine, and ethylenediamine. When the compounds of this disclosure are basic, their respective salts can be readily prepared from pharmaceutically acceptable non-toxic acids (including inorganic and organic acids). Examples of such acid addition salts include salts formed from hydrohalic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid), formic acid, acetic acid, decanoic acid, and citric acid. Salts containing acetate, formate, decanoate, chloride, or sodium salts are commonly used with the compounds of this disclosure. In some embodiments, salts of the compounds of this disclosure can be formed by exchanges well known to those skilled in the art, such as by anion exchange, for example, replacing trifluoroacetate ions with chloride ions.

[0061] Furthermore, the compounds of this disclosure may exist in amorphous and / or one or more crystalline forms, and therefore all amorphous and crystalline forms of the compounds of formula (I) and mixtures thereof (including those described in the examples) are intended to be included within the scope of this disclosure. Additionally, some compounds of this disclosure may form solvates with water (i.e., hydrates) or with common organic solvents (e.g., but not limited to acetic acid or acetonitrile). Such solvates and hydrates of the compounds of the present invention, particularly pharmaceutically acceptable solvates and hydrates, together with their non-solventized and anhydrous forms, are also covered within the scope of this disclosure.

[0062] Any pharmaceutically acceptable prodrug modification of a compound of this disclosure that results in its conversion in vivo to a compound within the scope of this disclosure is also within the scope of this disclosure.

[0063] This disclosure also relates to a method for preparing compounds of formula (I), the method described in the following examples, and by which the compounds of this disclosure can be obtained.

[0064] "Treatment" and "performing treatment" refer to all processes that can slow, interrupt, stop, control, or halt the progression of the disease or condition described herein. These terms do not necessarily imply the complete elimination of all symptoms of the disease or condition.

[0065] As used herein, “taking precautions” or “prevention” means reducing the likelihood of exposure to the disease or condition described herein, or reducing the severity of the disease or condition described herein.

[0066] The terms "therapeuticly effective (or effective) amount" and similar descriptions such as "therapeuticly effective amount" or "effective dose" are intended to indicate an amount of the compound of this disclosure that will elicit a biological or medical response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, physician, or other clinician. In a preferred embodiment, the term "therapeuticly effective amount" indicates an amount of the compound of this disclosure that alleviates at least one clinical symptom in a human patient. The terms "preventatively effective (or effective) amount" and similar descriptions such as "preventatively effective amount" are intended to indicate an amount of the compound of this disclosure that will prevent or reduce the risk of biological or medical events that a researcher, veterinarian, physician, or other clinician is seeking to avoid in a tissue, system, animal, or human.

[0067] Dosage of the compounds disclosed herein The dosage regimens using the compounds disclosed herein are selected based on a variety of factors, including patient type, race, age, weight, sex, and medical condition; the severity of the condition to be treated; the potency of the compound chosen for administration; the route of administration; and the patient's renal and hepatic function. These factors are considered within the capabilities of a clinician with ordinary skills in order to determine the therapeutically effective or preventatively effective dosage required to prevent, counteract, or halt disease progression. It should be understood that a specific daily dosage can be both a therapeutically effective dose (e.g., for treating oncological conditions) and a preventatively effective dose (e.g., for preventing oncological conditions).

[0068] Although individual needs vary, the optimal range for determining the effective amount of the compounds of this disclosure is within the scope of the art. For example, for administration to humans in the curative or preventative treatment of conditions and ailments as defined herein, a typical dosage of the compounds of this disclosure may be from about 0.05 mg / kg / day to about 50 mg / kg / day. In some embodiments, the compounds of this disclosure are administered to patients from about 5 mg / day to about 120 mg / day, for example from 10 mg / day, 20 mg / day, 30 mg / day, 40 mg / day, 50 mg / day, 60 mg / day, 70 mg / day, 80 mg / day, 90 mg / day, or 100 mg / day. In some embodiments, the compounds of this disclosure are administered to patients from about 0.2 mg / kg to about 5 mg / kg, for example from 0.5 mg / kg, 0.75 mg / kg, 1.0 mg / kg, 1.25 mg / kg, or 1.5 mg / kg. Such dosages may be administered as a single dose or may be divided into multiple doses.

[0069] Pharmaceutical Composition The compounds disclosed herein and their pharmaceutically acceptable salts may be administered to animals, preferably mammals, and particularly humans, as medicines on their own, in mixtures with each other, or as pharmaceutical compositions. The terms "subject" or "patient" include animals, preferably mammals, and particularly humans, that use the active agents of the invention to prevent or treat a medical condition. Administration of the medicine to a subject includes both self-administration and administration by another person to a patient. A subject may need or expect treatment for an existing disease or medical condition, or may need or expect preventative treatment to prevent or reduce the risk of the disease or medical condition occurring. As used herein, a subject who "needs" treatment for an existing condition or preventative treatment encompasses both a medical professional's determination that such treatment is necessary and a patient's expectation of such treatment.

[0070] Therefore, this disclosure also provides the use of the compounds of this disclosure and their pharmaceutically acceptable salts as medicines, their use in regulating the activity of the cytokine IL-1β, and in particular their use in treating and preventing the following diseases or conditions, and their use in the preparation of medicines for these purposes. In some embodiments, the compounds of this disclosure and their pharmaceutically acceptable salts capture IL-1β.

[0071] Furthermore, this disclosure provides pharmaceutical compositions comprising an effective dose of at least one compound of this disclosure and / or a pharmaceutically acceptable salt thereof as an active component, and conventional pharmaceutically acceptable carriers, i.e., one or more pharmaceutically acceptable carrier substances and / or additives.

[0072] Therefore, this disclosure provides, for example, the use of the compound and its pharmaceutically acceptable salt as a pharmaceutical composition comprising an effective dose of the compound of this disclosure and / or its pharmaceutically acceptable salt as an active ingredient and a conventional pharmaceutically acceptable carrier, and the use of the compound and / or its pharmaceutically acceptable salt in the treatment or prevention of diseases or conditions (e.g., atherosclerosis), and their use in the preparation of medicaments for these purposes.

[0073] The pharmaceutical compositions according to this disclosure can be administered orally, for example, in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, water, alcohol or oil solutions, syrups, emulsions or suspensions, or rectally, for example, in the form of suppositories. They can also be administered parenterally, for example, subcutaneously, intramuscularly or intravenously, in the form of injections or infusions.

[0074] Other suitable forms of application are, for example, transdermal or topical application, such as in the form of ointments, tinctures, sprays, or transdermal treatment systems, or, for example, microcapsules, implants, or rods. Preferred forms of application depend, for example, on the disease to be treated and its severity.

[0075] This disclosure also provides pharmaceutical compositions comprising compounds of formula (I). Compounds of formula (I) can be used in combination with any suitable drug carrier or excipient. Such pharmaceutical compositions comprise a therapeutically effective amount of one or more compounds of formula (I) and a pharmaceutically acceptable excipient and / or carrier. Specific pharmaceutical compositions will be tailored to the administration regimen. In a particular aspect, a pharmaceutically acceptable carrier may be water or a buffer solution.

[0076] Excipients included in pharmaceutical compositions serve various purposes, depending on factors such as the nature of the drug and the mode of administration. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerin, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tension agents, bulking agents, lubricants (e.g., talc or silica, and fats such as vegetable stearin, magnesium stearate, or stearic acid), emulsifiers, suspending agents or viscosity agents, inert diluents, fillers (e.g., cellulose, dicalcium phosphate, vegetable fats and oils, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, and magnesium stearate), disintegrants (e.g., cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethyl cellulose), and binders (e.g., starch, etc.). Gelatin, cellulose, methylcellulose or modified cellulose such as microcrystalline cellulose, hydroxypropyl cellulose, sugars such as sucrose and lactose, or sugar alcohols such as xylitol, sorbitol or maltitol, polyvinylpyrrolidone and polyethylene glycol), wetting agents, antibacterial agents, chelating agents, coating agents (e.g., cellulose membrane coating, synthetic polymers, shellac, corn gluten, corn lysine or other polysaccharides, and gelatin), preservatives (including vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, cysteine, methionine, citric acid and sodium citrate, and synthetic preservatives including methylparaben and propylparaben), sweeteners, flavoring agents, flavoring agents, coloring agents, absorption enhancers, application aids, and combinations thereof.

[0077] A drug delivery vehicle is a compound or substance used in pharmaceutical compositions to improve and / or prolong the delivery of the active ingredient to a subject. Using controlled-release technologies, delivery vehicles can be used to prolong the in vivo activity of a drug or slow drug release in a subject. Delivery vehicles can also reduce drug metabolism and / or decrease drug toxicity in a subject. Delivery vehicles can also be used to target drug delivery to specific cells or tissues in a subject. Common delivery vehicles (both hydrophilic and hydrophobic) include fat emulsions, lipids, PEGylated phospholipids, PEGylated liposomes, PEGylated liposomes coated with cyclic RGD peptides via PEG spacers, liposomes and lipospheres, microspheres (including those made from biodegradable polymers or albumin), polymer matrices, biocompatible polymers, protein-DNA complexes, protein conjugates, erythrocytes, vesicles, nanoparticles, and side chains for hydrocarbon stapling. The aforementioned delivery vehicles can also be used to increase the cell membrane permeability of compounds of formula (I). In addition to its use in the pharmaceutical compositions disclosed herein, the carrier may also be used in compositions for other uses, such as in vitro (e.g., for delivery to cultured cells) and / or in vivo research uses.

[0078] Pharmaceutical compositions suitable for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions, syrups, or suspensions (in aqueous or non-aqueous liquids; or edible foams or whips; or emulsions). Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols. For the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils (e.g., vegetable oils) may be used to provide oil-in-water or water-in-oil suspensions. Excipients that promote absorption from the gastrointestinal tract may be included, such as permeation enhancers, such as sodium decanoate. In some cases, delayed-release articles may be advantageous, and compositions that can deliver the compounds of this disclosure in a delayed or controlled-release manner can also be prepared. Prolonged gastric retention can lead to degradation by enzymes present in the stomach, and therefore, enteric-coated capsules can be prepared using standard techniques in the art, in which the active substance is released at a lower position in the gastrointestinal tract.

[0079] Suitable pharmaceutical compositions for transdermal application can be presented as discrete patches designed to maintain close contact with the recipient's epidermis for extended periods. For example, the active ingredient can be released from the patch via iontophoresis, as is commonly done in… Pharmaceutical Research As described in , 3(6):318 (1986).

[0080] Pharmaceutical compositions suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. When formulated as an ointment, the active ingredient can be used with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical application to the eye include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier (especially an aqueous solvent). Pharmaceutical compositions suitable for topical application in the mouth include lozenges, tablets, and oral washes.

[0081] Pharmaceutical compositions suitable for rectal administration can be presented as suppositories or enemas.

[0082] Suitable pharmaceutical compositions for nasal administration (where the carrier is solid) comprise coarse powder having a particle size, for example, in the range of 20 to 500 micrometers, which are administered by nasal inhalation, i.e., by rapidly inhaling through the nasal passages by bringing the container containing the powder upwards toward the nose. For administration as a nasal spray or as nasal drops, suitable compositions (where the carrier is liquid) comprise aqueous or oily solutions of the active ingredient.

[0083] Suitable pharmaceutical compositions for administration by inhalation include fine particulate dust or mist, which can be generated by various types of metered-dose pressurized nebulizers, sprayers, or blowers.

[0084] Pharmaceutical compositions suitable for vaginal application may be presented as pessaries, suppositories, creams, gels, pastes, foams, or sprays.

[0085] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickeners. Excipients that may be used in injectable solutions include, for example, water for injection, alcohols, polyols, glycerol, and vegetable oils. The compositions may be presented in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water or saline for injection, just before use. Ready-to-use injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets. Pharmaceutical compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts (the substances disclosed herein may be provided themselves in the form of pharmaceutically acceptable salts), buffers, coating agents, or antioxidants. In addition to the compounds disclosed herein, they may also contain therapeutically active agents.

[0086] Methods using the compounds disclosed herein This application provides a method for IL-1-mediated cell signaling, comprising contacting cells with a compound of the present disclosure or a pharmaceutically acceptable salt thereof. Inhibition of IL-1-mediated cell signaling can be assessed by detecting a decrease in the levels of downstream biomarkers IL-6 and CRP (e.g., hsCRP).

[0087] This application also provides methods for treating disease conditions using compounds of this disclosure (or pharmaceutically acceptable salts thereof) or pharmaceutical compositions containing such compounds, including but not limited to conditions related to IL-1β.

[0088] In some embodiments, this disclosure provides a method of treating cardiovascular disease, the method comprising administering to a subject requiring such treatment a therapeutically effective amount of any one of the compounds of this disclosure (or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising such a compound. In some embodiments, the cardiovascular disease is vascular inflammation. In some embodiments, the cardiovascular disease is atherosclerosis. In some embodiments, the cardiovascular disease is heart failure with preserved ejection fraction (HFpEF). In other embodiments, the cardiovascular disease is heart failure with reduced ejection fraction (HFrEF).

[0089] In some embodiments, this disclosure provides a method for treating chronic kidney disease, the method comprising administering to a subject requiring such treatment a therapeutically effective amount of any of the compounds of this disclosure (or pharmaceutically acceptable salts thereof) or any of the aforementioned pharmaceutical compositions comprising such compounds.

[0090] In some embodiments, this disclosure provides a method for treating an inflammatory condition, the method comprising administering to a subject requiring such treatment a therapeutically effective amount of any one of the compounds of this disclosure (or a pharmaceutically acceptable salt thereof) or any of the aforementioned pharmaceutical compositions comprising such compounds. In some embodiments, the inflammatory condition is selected from hidradenitis suppurativa (acne paradoxically), inflammatory bowel disease, arthritis, and nonalcoholic steatohepatitis (NASH).

[0091] In some implementations, the inflammatory condition is hidradenitis suppurativa (acne paradoxica).

[0092] In some implementations, the inflammatory condition is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis.

[0093] In some implementations, the inflammatory condition is arthritis, such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, or gouty arthritis.

[0094] In other implementations, the inflammatory condition is non-alcoholic steatohepatitis (NASH).

[0095] Combination therapy One or more additional pharmacologically active agents may be administered in combination with the compounds of this disclosure. The additional active agents (or agents) are intended to represent pharmaceutically active agents (or agents) that are active in vivo, including prodrugs that are converted to a pharmaceutically active form after administration, which are different from compounds of Formula I, and also include free acids, free bases, and pharmaceutically acceptable salts of said additional active agents. Generally, any suitable additional active agents (or agents) include, but are not limited to, antihypertensive agents, antiatherosclerotic agents (e.g., lipid-modulating compounds), antidiabetic agents and / or antiobesity agents, and anti-inflammatory agents, which may be used in any combination with the compounds of this disclosure in a single dosage form (fixed-dose drug combination), or may be administered to a subject in one or more separate dosage forms that allow simultaneous or sequential administration of the active agents (co-administration of said separate active agents).

[0096] Examples of other active agents that can be used to treat cardiovascular conditions include, but are not limited to, angiotensin-converting enzyme inhibitors (e.g., arapril, benazepril, captopril, siropril, cilazapril, delapril, enalapril, enalapril, fosinopril, imidapril, lisinopril, muvipril, perindopril, quinapril, ramipril, spiropril, temopril, or quindopril), angiotensin II receptor antagonists (e.g., losartan, i.e., COZAAR®, valsartan (including combinations with sacubitril), candesartan, olmesartan, telmesartan, and any of these drugs used in combination with hydrochlorothiazides such as HYZAAR®). One type); sGC activators (e.g., riociguat and vericiguat), PCSK9 inhibitors (e.g., evolocumab, alirocumab, MK-0616, and those disclosed in WO2019 / 246349), neutral endopeptidase inhibitors (e.g., thiorphan and phosphoramidon), aldosterone antagonists, aldosterone synthase inhibitors, renin inhibitors, endothelin receptor antagonists, phosphodiesterase-5 inhibitors (e.g., sildenafil, tadalafil, and vardenafil), vasodilators, calcium channel blockers Depressants (e.g., amlodipine, nifedipine, verapamil, diltiazem, golopamil, niludipine, nimodipine, nicardipine), potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, cloprazolam), diuretics (e.g., hydrochlorothiazide), sympatholytic agents, β-adrenergic blockers (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate), α-adrenergic blockers (e.g., doxazosin, prazosin, or α-methyldopa), central α-adrenergic agonists, external... Peripheral vasodilators (e.g., hydralazine); lipid-lowering agents, such as HMG-CoA reductase inhibitors like simvastatin and lovastatin, which are marketed as lactone prodrugs as ZOCOR® and MEVACOR® and act as inhibitors after administration; and pharmaceutically acceptable salts of dihydroxyocyclocarboxylic acid HMG-CoA reductase inhibitors, such as atorvastatin (especially the calcium salt marketed as LIPITOR®), rosuvastatin (especially the calcium salt marketed as CRESTOR®), pravastatin (especially the sodium salt marketed as PRAVACHOL®), fluvastatin (especially the sodium salt marketed as LESCOL®), crivastatin, and pitavastatin;Cholesterol absorption inhibitors such as ezetimibe (ZETIA®), and their combination with any other lipid-lowering agents (such as the HMG-CoA reductase inhibitors described above) and especially with simvastatin (VYTORIN®) or with atorvastatin calcium; niacin in immediate-release or controlled-release forms and / or with HMG-CoA reductase inhibitors; niacin receptor agonists, such as acilimus and acyclofuran, and partial niacin receptor agonists; metabolic modifiers, including insulin and insulin mimics (e.g., insulin degludec, insulin glargine, insulin lispro), dipeptidyl peptidase-IV (DPP-4) inhibitors (e.g., sitagliptin, alogliptin, omarigliptin, linagliptin). Vildagliptin; insulin sensitizers, including (i) PPARγ agonists, such as glitazones (e.g., pioglitazone, miglitazone, lobeglitazone, rosiglitazone, and baglitazone) and other PPAR ligands, including (1) PPARα / γ dual agonists (e.g., chiglitazar, muraglitazar, alglitazar, sodelglitazar, and naveglitazar); (2) PPARα agonists such as fenofibrate derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate); (3) selective PPARγ modulators (SPPARγM's) (e.g., as in WO (i) those disclosed in WO 02 / 060388, WO 02 / 08188, WO 2004 / 019869, WO 2004 / 020409, WO 2004 / 020408 and WO 2004 / 066963); and (ii) PPARγ partial agonists; (iii) biguanides, such as metformin and its pharmaceutically acceptable salts, especially metformin hydrochloride, and its extended-release formulations, such as Glumetza; TM Fortamet TM and GlucophageXR TM(iii) Protein tyrosine phosphatase-1B (PTP-1B) inhibitors; insulin or insulin analogs (e.g., insulin detemir, insulin glutares, insulin degludec, insulin glargine, insulin lispro and their respective inhaled formulations); leptin and leptin derivatives and agonists; amylin and amylin analogs (e.g., pramlintide); sulfonylureas and non-sulfonylurea insulin secretagogues (e.g., tolbutamide, glibenclamide, glipizide, glimepiride, miglitol, chloropicrin, nateglinide and repaglinide); α-glucosidase inhibitors (e.g., acarbose, voglibose and miglitol); glucagon receptor antagonists; intestinal glucagon mimics, such as GLP-1 GLP-1 analogs, derivatives, and mimics; and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide, including their intranasal, transdermal, and once-weekly formulations); bile acid sequestrants (e.g., colestilan, colestimide, colesevalam hydrochloride). Hydrochloride, cholestyramine, dialkylaminoalkyl derivatives of cross-linked dextran, acyl-CoA: cholesterol acyltransferase inhibitors (e.g., avasimibe); anti-obesity compounds; agents intended for use in inflammatory conditions, such as aspirin, nonsteroidal anti-inflammatory drugs or NSAIDs, glucocorticoids, and selective cyclooxygenase-2 or COX-2 inhibitors; glucosamine activators (GKA); inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (e.g., as disclosed in U.S. Patent No. 6,730,690). Those); inhibitors of fructose-1,6-bisphosphatase (e.g., those disclosed in U.S. Patent Nos. 6,054,587; 6,110,903; 6,284,748; 6,399,782 and 6,489,476); inhibitors of acetyl-CoA carboxylase-1 or 2 (ACC1 or ACC2); AMP-activated protein kinase (AMPK) activators; other agonists of G protein-coupled receptors: (i) GPR-109, (ii) GPR-119, and (iii) GPR-40; SSTR3 antagonists (e.g., those disclosed in WO Those disclosed in WO 2009 / 001836); neurotransmitter U receptor agonists (e.g., those disclosed in WO 2009 / 042053, including but not limited to neurotransmitter S (NMS)); SCD modulators; GPR-105 antagonists (e.g., those disclosed in WO2009 / 000087);SGLT inhibitors (e.g., empagliflozin, dapagliflozin, canagliflozin, ertugliflozin, remogloflozin, tolagliflozin, and etagliflozin); acyl-CoA inhibitors: diacylglycerol acyltransferase 1 and 2 (DGAT-1 and DGAT-2); Inhibitors of fatty acid synthases; inhibitors of acyl-CoA: monoacylglycerol acyltransferases 1 and 2 (MGAT-1 and MGAT-2); agonists of the TGR5 receptor (also known as GPBAR1, BG37, GPCR19, GPR131, and M-BAR); inhibitors of ileal bile acid transporters; PACAP, PACAP mimics, and PACAP receptor 3 agonists; PPAR agonists; protein tyrosine phosphatase-1B (PTP-1B) inhibitors; IL-1β antibodies (e.g., gevokizumab and canakinumab); and bromocriptine mesylate and its rapid-release formulations; or other drugs beneficial for the treatment of the above conditions or symptoms (including chemically feasible free acid, free base, and pharmaceutically acceptable salt forms of the above active agents).

[0097] Examples of other active agents that can be used to treat inflammatory conditions include, but are not limited to, steroidal and nonsteroidal anti-inflammatory agents, glucocorticoids, and therapeutic hormones. In a particular embodiment, in the treatment of hidradenitis suppurativa (acne paradoxically), other active agents may be antibiotics, injectable steroids, therapeutic hormones, TNF inhibitors (e.g., infliximab, adalimumab, etanercept, golimumab, certolizumab), analgesics (e.g., codeine, hydrocodone, morphine, pregabalin, gabapentin, lecithin, corticosteroids, naproxen, ketoprofen, diclofenac, ibuprofen, acetaminophen). In other embodiments, in treating inflammatory bowel disease, additional active agents may be methotrexate, TNF inhibitors, oral sphingosine 1-phosphate receptor modulators (e.g., fingolimod, siponimod, ozanimod, ponesimod) or selective JAK inhibitors (e.g., tofacitinib, baricitinib, upadacitinib). In some embodiments, in treating osteoarthritis, additional active agents may be analgesics (examples listed above). In other embodiments, in treating gouty arthritis, additional active agents may be colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), or glucocorticoids.

[0098] Example Method for preparing the compounds disclosed herein The compounds described herein can be prepared using suitable materials according to the procedures and examples described below, and are further illustrated by the specific examples below. The examples also include methods for testing such compounds in cytoassays. However, the compounds exemplified in the examples should not be construed as forming the only kind considered in this disclosure.

[0099] The examples further illustrate the preparation details of the compounds of this disclosure. Those skilled in the art will readily understand that known modifications to the conditions and processes of the following preparation procedures can be used to prepare these compounds. For example, in some cases, the order of steps in performing the reaction scheme can be altered to promote the reaction or avoid undesirable reaction products. The starting materials and intermediates of the final compounds are purchased, prepared by known procedures, or as otherwise described. The examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0100] On 300MHz or 400MHz instruments, in CDCl3, DMSO- d6 or methanol- d4 NMR data were obtained, and the chemical shifts relative to the tetramethylsilane standard were reported. Resonance signals were reported using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, m = multiplet or overlap of non-equivalent resonances. The coupling constant was reported in Hertz (Hz). ( J).

[0101] Throughout the synthesis scheme and embodiments, unless otherwise indicated, abbreviations and acronyms may be used with the following meanings: abbreviation

[0102] The following examples are illustrative and should not be construed as further limiting. All accompanying drawings, references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0103] Intermediate synthesis: Synthesis Scheme 1 AEFC4acidNMe3 ( S )-4-((2-(4-(2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy) Ethyl)amino)- N , N , N -Trimethyl-4-oxobut-1-ammonium 2,2,2-trifluoroacetate Step 1: At room temperature, NaHCO3 (12.44 g, 148 mmol) was added to a stirred solution of tert-butyl 4-bromobutyrate (3 g, 13.45 mmol) and trimethylamine hydrochloride (10.28 g, 108 mmol) in EtOH (60 mL). The mixture was stirred at 50 °C for 16 hours. The mixture was cooled to room temperature and the solid was filtered off. The filtrate was concentrated under reduced pressure and the residue was suspended in DCM (30 mL). The solid was filtered off and the filtrate was concentrated under reduced pressure to give a colorless oily substance, 4-(tert-butoxy)- N , N , N -Trimethyl-4-oxobut-1-ammonium (2 g, 7.91 mmol, yield 58.8%). MS ESI calculation C 11 H 24 NO2 + [M] + 202.18, actual measurement 202.20.

[0104] Step 2: At 25°C, the solution of 4-(tert-butoxy)-N,N,N-trimethyl-4-oxobut-1-ammonium (2 g, 9.89 mmol) in a dioxane solution of 4 N HCl (50 mL) was stirred for 2 hours. The solvent was concentrated under reduced pressure to obtain a yellowish-white solid of 3-carboxy- N , N , N -Trimethylpropyl-1-ammonium chloride (1.3 g, 8.89 mmol, 90% yield). MSESI calculation of C7H 16 NO2 + [M - Cl] + 146.12, actual measurement 146.15.

[0105] Step 3: At room temperature, add tert-butyl ( S 3-(4-(2-aminoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (5 g, 13.14 mmol) was added to a stirred solution in DMF with a 3-carboxyl- N , N , N-Trimethylpropyl-1-ammonium chloride (5.76 g, 39.4 mmol) and DIEA (8.49 g, 65.7 mmol). The solution was stirred at 0 °C for 10 minutes. HATU (6.00 g, 15.77 mmol) was added to the solution and the solution was stirred at 0 °C for 2 hours. The solution was purified by RP-flash under the following conditions: C 18 Column, 330 g, 5 minutes 1%-1%, 25 minutes 1%-40%, MeCN (0.05% TFA) in water, yielded a yellowish-white solid. S )-4-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)- N , N , N -Trimethyl-4-oxobut-1-ammonium. MS ESI calculation C 27 H 46 N3O6[M-CF3COO] + 508.34, actual measurement 508.25.

[0106] Step 4: At room temperature, towards ( S )-4-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)- N , N , N Trimethyl-4-oxobut-1-ammonium 2,2,2-trifluoroacetate (6.4 g, 10.29 mmol) was added to a stirred solution of TFA (120 mL) in DCM (80 mL). The solution was stirred at room temperature for 3 hours. The solvent was concentrated under reduced pressure to give a yellow oily substance. S )-4-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)- N , N , N -Trimethyl-4-oxobut-1-ammonium 2,2,2-trifluoroacetate (4.7 g, 10.10 mmol, 98% yield). MS ESI calculation C 18 H 30 N3O4[M-CF3COO] + 352.22, actual measurement 352.10.

[0107] Step 5: At 25℃, to ( S )-4-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)- N ,N , N 2,2,2-trifluoroacetate (6.4 g, 13.75 mmol) of trimethyl-4-oxobut-1-ammonium 2,2,2-trifluoroacetate (6.4 g, 13.75 mmol) was added to a mixture of THF (60 mL) and water (60 mL) with NaHCO3 (5.78 g, 68.7 mmol) and Fmoc-OSu (4.64 g, 13.75 mmol). The mixture was stirred at 25 °C for 2 hours. The pH of the reaction mixture was adjusted to 3 with 1 N HCl. The mixture was concentrated under reduced pressure. The residue was purified by RP-flash under the following conditions: C 18 Column, 330 g, 5 minutes 1%-1%, 30 minutes 1%-50%, MeCN (0.05% TFA) in water, yielded a pale yellow semi-solid. S )-4-((2-(4-(2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy)ethyl)amino)- N , N , N -Trimethyl-4-oxobut-1-ammonium 2,2,2-trifluoroacetate (4.7434 g, 6.55 mmol, yield 47.7%). MS ESI calculation C 33 H 40 N3O6[M-CF3COO] + 574.29, actual measurement 574.15. 1 H NMR (300 MHz, CD3OD) δ 7.76 -7.74 (m, 2H), 7.59 -7.56 (m, 2H), 7.40 -7.35 (m, 2H), 7.31 -7.26 (m, 2H), 7.16 -7.14 (m, 2H), 6.84 -6.81 (m,2H), 4.38 -4.12 (m, 4H), 3.98 -3.95 (m, 2H), 3.52 (t, J = 5.4 Hz, 2H), 3.32 -3.24 (m, 2H), 3.13 -3.03 (m, 10H), 2.91 -2.85 (m, 1H), 2.30 (t, J = 6.9 Hz, 2H), 2.05 -1.99 (m, 2H).

[0108] Synthesis Scheme 2 AEFC6acidNMe3 (S)-6-((2-(4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl))phenoxy) Ethyl)amino)-N,N,N-trimethyl-6-oxohex-1-ammonium trifluoroacetate Step 1: At 25°C, add 6-(dimethylamino)hexanoic acid (2 g, 12.56 mmol) to a stirred mixture of DCM (20 mL). N , N 1,256 mmol of dimethylformamide (0.092 g) was added, followed by the addition of a solution of oxaloyl dichloride (4.78 g, 37.7 mmol) in DCM (10 mL) at 0 °C. The resulting mixture was stirred at 30 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give crude 6-(dimethylamino)hexanoyl chloride (2.232 g, 12.56 mmol, 100% yield) as a yellow oil, which was dissolved in DCM (7 mL). At 0 °C, a solution of TEA (5.78 g, 57.1 mmol) and 6-(dimethylamino)hexanoyl chloride (2.232 g, 12.56 mmol) in DCM (7 mL) was added to tert-butyl ( S 3-(4-(2-aminoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (4.34 g, 11.42 mmol) was added to a stirred mixture in DCM (13 mL). After stirring the resulting mixture at 25 °C for 2 hours, it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0–30% MeOH (1% NH4OH) in DCM. The fraction containing the product was collected and evaporated under vacuum to give tert-butyl(...) as a pale white solid. S 2-((tert-butoxycarbonyl)amino)-3-(4-(2-(6-(dimethylamino)hexanoylamino)ethoxy)phenyl)propionate (1.7 g, 3.26 mmol, yield 29%). MS ESI calculation C 28 H 48 N3O6[M+ H] + 522.35, actual measurement 522.30.

[0109] Step 2: At 25°C, tert-butyl ( S 1.7 g (3.26 mmol) of 2-(tert-butoxycarbonyl)amino)-3-(4-(2-(6-(dimethylamino)hexamido)ethoxy)phenyl)propionate (1.7 g, 3.26 mmol) was added to a stirred mixture in MeCN (17 mL) with NaHCO3 (2.74 g, 32.6 mmol) and MeI (1.019 mL, 16.29 mmol). The resulting mixture was stirred at 25 °C for 16 hours. The solid was filtered off and the filtrate was concentrated under reduced pressure to give a crude (yellow oily substance).S )-6-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium iodide (2.163 g, 3.26 mmol, 100% yield). MS ESI calculation C 29 H 50 IN3O6[M - I] + 536.37, actual measurement 536.35.

[0110] Step 3: At 25℃, towards ( S )-6-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)- N , N , N Trimethyl-6-oxohexyl-1-ammonium iodide (2.163 g, 3.26 mmol) was added to a stirred mixture in DCM (10 mL) with TFA (10 mL, 130 mmol). After stirring the resulting mixture at 25 °C for 2 hours, it was concentrated under reduced pressure to give a crude, yellow oily substance. S )-6-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium 2,2,2-trifluoroacetate (1.609 g, 3.26 mmol, 100% yield). MS ESI calculation C 22 H 34 F3N3O6[M-CF3COO] + 380.25, actual measurement 380.15.

[0111] Step 4: At 25℃, to ( S )-6-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)- N , N , N-Trimethyl-6-oxohexyl-1-ammonium 2,2,2-trifluoroacetate (1.609 g, 3.26 mmol) was added to a solution of NaHCO3 (1.369 g, 16.30 mmol) and Fmoc-Osu (1.002 g, 2.93 mmol) in THF (8 mL) and water (8 mL). The resulting mixture was stirred at room temperature for 16 hours, and then the pH was adjusted to 3 with 1 M HCl. The mixture was purified by RP-flash under the following conditions: C 18 Column (120 g); Mobile phase A: water (0.1% TFA), Mobile phase B: ACN; (gradient: 5% B held for 5 min, reaching 38% B within 30 min, 38% B held for 2.6 min; reaching 95% B within 2 min, 95% B held for 5 min); Flow rate: 60 mL / min; Detector: UV 210 nm; RT = 31 min. The fraction containing the product was collected and evaporated under vacuum to obtain a white solid (…). S )-6-((2-(4-(2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy)ethyl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium trifluoroacetate (1.12 g, 1.591 mmol, yield 49%). MS ESI calculation C 37 H 44 F3N3O8[M – CF3COO] + 602.32, actual measurement 602.25. 1 H NMR (400 MHz, CD3OD) δ 7.78 (d, J = 7.6 Hz, 2H), 7.61 -7.57 (m, 2H), 7.41 -7.39 (m, 2H), 7.37-7.28 (m, 2H), 7.17 -7.14 (m, 2H), 6.84 -6.82 (m, 2H), 4.35 -4.14 (m, 4H),3.97 -3.95 (m, 2H), 3.53 -3.50 (m, 2H), 3.31 -3.04 (m, 12H), 2.88 -2.86 (m,1H), 2.24 -2.20 (m, 2H), 1.75 -1.63 (m, 4H), 1.34 -1.32 (m, 2H). 19 F NMR (376MHz, CD3OD) delta -77.401.

[0112] Synthesis Scheme 3 AEFNMe3 ( S )-2-(4-(2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy)- N , N , N -Trimethylethyl-1-ammonium chloride Step 1: At room temperature, add tert-butyl ( S MeI (12.59 g, 89 mmol) and NaHCO3 (7.45 g, 89 mmol) were added to a stirred solution of 3-(4-(2-aminoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (7.5 g, 17.74 mmol) in MeOH (100 mL). The mixture was stirred at 40 °C for 16 hours and then cooled to room temperature. The solid was filtered off and the filtrate was concentrated under reduced pressure to give a pale yellow solid. S )-2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)- N , N , N -Trimethylethyl-1-ammonium iodide (10 g, 16.35 mmol, 92% yield). MS ESI calculation C 23 H 39 N2O5[M - I] + 423.29, actual measurement 423.15.

[0113] Step 2: At room temperature, towards ( S )-2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)- N , N , N Trimethylethyl-1-ammonium iodide (10 g, 18.17 mmol) was added to a stirred solution of TFA (40 mL) in DCM (20 mL). The resulting solution was stirred at 25 °C for 2 hours. The solvent was concentrated under reduced pressure to give a yellow solid. S )-2-(4-(2-amino-2-carboxyethyl))phenoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (7 g, 14.72 mmol, yield 81%). MS ESI calculation C 14 H 23 N2O3[M – CF3COO] + 267.17, actual measurement 267.05.

[0114] Step 3: At room temperature, towards ( S )-2-(4-(2-amino-2-carboxyethyl))phenoxy)- N , N , N 1-Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (8 g, 21.03 mmol) was added to a stirred solution of THF (80 mL) and water (80 mL) with Fmoc-OSu (7.09 g, 21.03 mmol) and NaHCO3 (5.30 g, 63.1 mmol). The resulting solution was stirred at 25 °C for 16 hours. The pH of the solution was adjusted to 3 with 6 N HCl. The resulting solvent was concentrated under reduced pressure, and the residue was purified by RP-flash under the following conditions: 330 g C 18 Column chromatography, 5 minutes 2%-2%, 30 minutes 2%-40%, 5 minutes 98%-98%, MeCN in water (2 mmol HCl), RT = 35 minutes, yielding a yellowish-white solid. S )-2-(4-(2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy)- N , N , N -Trimethylethyl-1-ammonium chloride (5.3489 g, 9.98 mmol, yield 47.5%). MS ESI calculation C 29 H 33 N₂O₅[M - Cl] + 489.24, actual measurement 489.25. 1 H NMR (300 MHz, DMSO- d 6) δ 12.85 (br, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 -7.71 (m, 3H), 7.67 -7.65(m, 2H), 7.44 -7.24 (m, 4H), 6.91 (d, J = 7.5 Hz, 2H), 4.41 -4.40 (m, 2H), 4.24-4.10 (m, 4H), 3.79 -3.76 (m, 2H), 3.17 (s, 9H), 3.08 -3.06 (m, 1H), 2.86 -2.78 (m, 1H).

[0115] Synthesis Scheme 4 AEFPEG1acidNMe3 ( S )-2-(3-((2-(4-(2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)phenoxy (yl)ethyl)amino)-3-oxopropoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate Step 1: At room temperature, NaHCO3 (12.44 g, 148 mmol) was added to a stirred solution of tert-butyl 3-(2-bromoethoxy)propionate (2.5 g, 9.88 mmol) and trimethylamine hydrochloride (9.44 g, 99 mmol) in EtOH (30 mL). The mixture was stirred at 50 °C for 16 hours, then cooled to room temperature, and the solid was filtered off. The filtrate was concentrated under reduced pressure, and the residue was suspended in DCM (30 mL). The solid was filtered off, and the filtrate was concentrated under reduced pressure to give a colorless oily substance of 2-(3-(tert-butoxy)-3-oxopropoxy)- N , N , N -Trimethylethyl-1-ammonium (4.2 g, 9.04 mmol, 92% yield). MS ESI calculation C 12 H 26 NO3[M] + 232.19, actual measurement 232.10.

[0116] Step 2: Stir 2-(3-(tert-butoxy)-3-oxopropoxy)- at 25°C. N , N , N A solution of 1-trimethylethylammonium (4.2 g, 9.04 mmol) in 4 N HCl / dioxane (50 mL) was dissolved for 2 hours. The solvent was concentrated under reduced pressure to give a yellowish-white solid, 2-(2-carboxyethoxy)- N , N , N -Trimethylethyl-1-ammonium chloride (3.8 g, 7.18 mmol, yield 79%). C8H was calculated by MS ESI. 18 NO3[M-Cl] + 176.13, actual measurement 176.15.

[0117] Step 3: At room temperature, react 2-(2-carboxyethoxy)- N , N , N HATU (3.60 g, 9.46 mmol) was added to a stirred solution of trimethylethyl-1-ammonium chloride (5.01 g, 9.46 mmol) and DIEA (6.89 mL, 39.4 mmol) in DMF (40 mL). The solution was stirred at 25 °C for 10 minutes. Tert-butyl ( S3-(4-(2-aminoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (3 g, 7.88 mmol) was obtained, and the resulting solution was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was purified by RP-flash under the following conditions: C 18 Column, 330 g, 5 minutes 1%-1%, 25 minutes 1%-37%, MeCN in water (0.05%), yielded a pale yellow solid. S )-2-(3-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)-3-oxopropoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (5 g, 7.29 mmol, 92% yield). MS ESI calculation C 28 H 48 N3O7[M-CF3COO] + 538.35, actual measurement 538.25.

[0118] Step 4: At room temperature, towards ( S )-2-(3-((2-(4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenoxy)ethyl)amino)-3-oxopropoxy)- N , N , N 40 mL of TFA was added to a stirred solution of 10 mL of trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (5 g, 7.29 mmol) in DCM. The resulting solution was stirred at room temperature for 3 hours. The solvent was concentrated under reduced pressure to give a yellow oily substance. S )-2-(3-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)-3-oxopropoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (3.7 g, 6.72 mmol, 92% yield). MS ESI calculation C 19 H 32 N3O5[M-CF3COO] + 382.23, actual measurement 382.15.

[0119] Step 5: At room temperature, towards ( S )-2-(3-((2-(4-(2-amino-2-carboxyethyl))phenoxy)ethyl)amino)-3-oxopropoxy)-N , N , N Fmoc-OSu (2.267 g, 6.72 mmol) was added to a stirred solution of 1-trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (3.7 g, 6.72 mmol) and Na₂CO₃ (2.137 g, 20.16 mmol) in THF (50 mL) and water (50 mL). The resulting mixture was stirred at 25 °C for 16 h. The pH was adjusted to 3 with 1 M HCl, and the solution was purified by RP-flash under the following conditions: C 18 Column, 330 g, 5 minutes 1%-1%, 30 minutes 1%-37%, MeCN (0.05% TFA) in water, yielded a yellowish-white solid. S )-2-(3-((2-(4-(2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl))phenoxy)ethyl)amino)-3-oxopropoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (4.8452 g, 6.62 mmol, 98% yield). MS ESI calculation C 34 H 42 N3O7[M-CF3COO] - 604.30, actual measurement 604.25. 1 H NMR (400 MHz, DMSO- d 6) δ 8.19 (s, 1H), 7.90 -7.88 (m, 2H), 7.74 -7.72 (m,3H), 7.44 -7.40 (m, 2H), 7.34 -7.32 (m, 2H), 7.30 -7.28 (m, 2H), 7.20 -7.18(m, 2H), 4.23 -4.12 (m, 4H), 3.94 -3.91 (m, 2H), 3.78 -3.77 (m, 2H), 3.68 -3.65 (m, 2H), 3.49 -3.47 (m, 2H), 3.41 -3.39 (m, 2H), 3.04 (s, 9H), 3.00 -2.99 (m, 1H), 2.83 -2.81 (m, 1H), 2.38 (t, J = 6.0 Hz, 2H). 19 F NMR (376 MHz, DMSO- d 6) δ -74.41.

[0120] Synthesis Scheme 5 dProt4NH2C6acidNMe3 6-(((3 S 5 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-5-carboxypyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohex-1-ammonium 2,2,2-trifluoroacetate Step 1: At 0°C, a solution of TEA (9.54 g, 94 mmol) and 6-(dimethylamino)hexanoyl chloride (3.35 g, 18.85 mmol) in DCM (7 mL) was added to 1-(tert-butyl)-2-methyl (2 R 4 S 4-Aminopyrrolidine-1,2-dicarboxylate (4.61 g, 18.85 mmol) was added to a stirred mixture in DCM (13 mL). After stirring at 25 °C for 2 hours, the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0–30% MeOH (1% NH4OH) in DCM. The fraction containing the product was collected and evaporated under vacuum to give 1-(tert-butyl)-2-methyl(2-dicarboxylate) as a white solid. R 4 S 4-(6-(dimethylamino)hexanoylamino)pyrrolidine-1,2-dicarboxylate (2.4 g, 4.98 mmol, yield 26%). MS ESI calculations C 19 H 36 N3O5[M + H] + 386.26, actual measurement 386.35.

[0121] Step 2: At room temperature, apply the mixture to 1-(tert-butyl)-2-methyl(2-methyl)-2-methyl ... R 4 S MeI (8.84 g, 62.3 mmol) was added to a stirred mixture of 4-(6-(dimethylamino)hexanoylamino)pyrrolidine-1,2-dicarboxylate (2.4 g, 6.23 mmol) and NaHCO3 (7.84 g, 93 mmol) in ACN (50 mL). After stirring at 25 °C for 16 hours, the solid was filtered off and the filtrate was concentrated under reduced pressure to give 6-(((3))-4-(6-(dimethylamino)hexanoylamino)pyrrolidine-1,2-dicarboxylate (2.4 g, 6.23 mmol) and NaHCO3 (7.84 g, 93 mmol) in ACN (50 mL). S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium iodide (3.28 g, 5.60 mmol, 90% yield). MSESI calculation of C20 H 38 IN3O5[M - I] + 400.28, actual measurement 400.35.

[0122] Step 3: At room temperature, towards 6-(((3) S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium iodide (3.28 g, 5.60 mmol) was added to a stirred solution of LiOH (16.79 mL, 16.79 mmol, 1 N in water) in 50 mL of THF. After stirring at 25 °C for 1 hour, the pH was adjusted to 6 with 1 N HCl. The solvent was concentrated under reduced pressure to give 6-(((3)) as a yellow solid. S 5 R )-1-(tert-butoxycarbonyl)-5-carboxypyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium chloride (2.36 g, 5.03 mmol, 90% yield). MS ESI calculation C 19 H 36 ClN3O5[M-Cl] + 386.26, actual measurement 386.35.

[0123] Step 4: At room temperature, towards 6-(((3) S 5 R )-1-(tert-butoxycarbonyl)-5-carboxypyrrolidine-3-yl)amino)- N , N , N Trimethyl-6-oxohexyl-1-ammonium chloride (2.36 g, 5.03 mmol) was added to a stirred solution of TFA (50 mL) in DCM (50 mL). The resulting mixture was stirred at 25 °C for 1 hour, and then concentrated under reduced pressure to give 6-(((3))-trimethyl-6-oxohexyl-1-ammonium chloride as a yellow solid. S 5 R )-5-Carboxypyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium 2,2,2-trifluoroacetate (2.01 g, 4.53 mmol, 90% yield). MS ESI calculation C 16 H 28F3N3O5[M - CF3COO] + 286.21, actual measurement 286.15.

[0124] Step 5: At room temperature, towards 6-(((3) S 5 R )-5-Carboxypyrrolidine-3-yl)amino)- N , N , N Fmoc-OSu (1.698 g, 5.03 mmol) was added to a stirred solution of trimethyl-6-oxohexyl-1-ammonium 2,2,2-trifluoroacetate (2.01 g, 5.03 mmol) and NaHCO3 (1.268 g, 15.10 mmol) in THF (50 mL) and water (50 mL). After stirring at 25 °C for 16 h, the pH was adjusted to 3 with 1 N HCl, and the solvent was evaporated under reduced pressure. The residue was dissolved in THF / MeOH (1:1, 50 mL), the solid was filtered off, and the filtrate was purified by RP-flash under the following conditions: C 18 Column, 330 g, 8 minutes 2%-2%, 30 minutes 2%-35%, 10 minutes 35%-95%, 8 minutes 95%-95%, MeCN (0.05% TFA) in water, yielded a pale yellow solid 6-(((3) S 5 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-5-carboxypyrrolidine-3-yl)amino)- N , N , N -Trimethyl-6-oxohexyl-1-ammonium 2,2,2-trifluoroacetate (2.85 g, 4.49 mmol, 89% yield). MS ESI calculation C 31 H 38 F3N3O7[M - CF3COO] + 508.28, actual measurement 508.25. 1 H NMR (300 MHz, DMSO- d 6) δ 8.20 (d, J=6.9 Hz, 1H), 7.93 -7.90 (m, 2H), 7.69 -7.63 (m, 2H), 7.47 -7.41 (m, 2H), 7.36-7.31 (m, 2H), 4.32 -4.15 (m, 5H), 3.68 -3.55 (m, 1H), 3.29 -3.22 (m, 3H), 3.03 (s, 9H), 2.19 -2.08 (m, 4H), 1.69 -1.53 ​​(m, 4H), 1.27 -1.22 (m, 2H). 19 F-NMR (282 MHz, DMSO- d 6) δ 74.261.

[0125] Synthesis Scheme 6 dProt4NHPEG2acidNMe3 2-(2-(3-(((3 S 5 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-5-carboxypyrrolidine-3-yl)amino 3-Oxypropoxyethoxy- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate Step 1: At 0°C under a nitrogen atmosphere, 2-(2-(2-carboxyethoxy)ethoxy)- N , N , N 1-Trimethylethyl-1-ammonium chloride (2.64 g, 10.32 mmol) was added to a stirred solution of DIEA (1.501 mL, 8.60 mmol) and HATU (3.27 g, 8.60 mmol) in DMF (40 mL). The resulting solution was stirred at 0 °C for 10 min, and then 1-(tert-butyl)-2-methyl(2-ethyl)-2-methyl( ... R 4 S 4-Aminopyrrolidine-1,2-dicarboxylate (2.1 g, 8.60 mmol) was added and stirred at 25 °C for 1 hour. The solution was purified by RP-flash under the following conditions: C 18 Column, 330 g, 5 min 2%-2%, 20 min 2%-30%, 5 min 98%-98%, MeCN (0.05% TFA) in water, RT = 35 min, yielding a pale yellow oily substance of 2,2,2-trifluoroacetic acid, 2-(2-(3-(((3) S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N-Trimethylethyl-1-ammonium salt (4.4 g, 7.46 mmol, yield 87%). MS ESI calculation C 21 H 40 N3O7[M-CF3COO] + 446.29, actual measurement 446.35.

[0126] Step 2: At room temperature, add 2,2,2-trifluoroacetic acid, 2-(2-(3-(((3) S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N 4.4 g (7.85 mmol) of trimethylethyl-1-ammonium salt was added to a stirred solution of 2-(2-(3-(((3-))-trimethylethyl-1-ammonium salt) in THF (50 mL) and LiOH (23.55 mL (1 N in water)) was added. The resulting solution was stirred at 25 °C for 1 hour. The pH was adjusted to 3 with 1 N HCl and the solvent was concentrated under reduced pressure to give a yellow oily substance, 2-(2-(3-(((3-)-)-trimethylethyl-1-ammonium salt). S 5 R )-1-(tert-butoxycarbonyl)-5-carboxypyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N -Trimethylethyl-1-ammonium chloride (3.7 g, 7.12 mmol, 91% yield). MS ESI calculation C 20 H 38 N3O7[M-Cl] + 432.27, actual measurement 432.35.

[0127] Step 3: At room temperature, towards 2-(2-(3-(((3) S 5 R )-1-(tert-butoxycarbonyl)-5-carboxypyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N 2-Trimethylethyl-1-ammonium chloride (3.7 g, 7.12 mmol) was added to a stirred solution of TFA (20 mL) in DCM (60 mL). The solution was stirred at 25 °C for 1 hour. The solvent was concentrated under reduced pressure to give a yellow oily substance, 2-(2-(3-(((3) S 5 R )-5-Carboxypyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N, N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (3.2 g, 6.47 mmol, 91% yield). MS ESI calculation C 15 H 30 N3O5[M – CF3COO] + 332.22, actual measurement 332.25.

[0128] Step 4: At room temperature, towards 2-(2-(3-(((3) S 5 R )-5-Carboxypyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (3.8 g, 7.68 mmol) was added to a stirred solution of THF (30 mL) and water (30 mL) with Na2CO3 (2.441 g, 23.03 mmol) and Fmoc-OSu (2.59 g, 7.68 mmol). The resulting mixture was stirred at 25 °C for 16 hours, then the pH was adjusted to 3 with 6 M HCl, and the solution was purified by RP-flash under the following conditions: 330 g C 18 Column, 5 minutes 2%-2%, 20 minutes 2%-30%, 5 minutes 98%-98%, MeCN (0.05% TFA) in water, RT = 25 minutes, yielding a colorless semi-solid 2-(2-(3-((3) S 5 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-5-carboxypyrrolidine-3-yl)amino)-3-oxopropoxy)ethoxy)- N , N , N -Trimethylethyl-1-ammonium 2,2,2-trifluoroacetate (4.1824 g, 6.14 mmol, 80% yield). MS ESI calculation C 30 H 40 N3O7[M – CF3COO] + 554.29, actual measurement 554.20. 1 H NMR (400 MHz, DMSO- d 6) δ8.24 -8.23 (m, 1H), 7.92 -7.89 (m,2H), 7.68 -7.64 (m, 2H), 7.45 -7.32 (m, 4H), 4.48 -4.23 (m, 4H), 4.16 -4.15(m, 1H), 3.83 -3.82 (m, 2H), 3.67 -3.51 (m, 9H), 3.27 -3.25 (m, 1H), 3.09 (s,9H), 2.36 -2.32 (m, 2H), 2.20 -2.11 (m, 2H).

[0129] Synthesis Scheme 7 Prot4CH2Pyrim (2 S 4 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid Step 1: Stir 1-(tert-butyl)-2-methyl( S A solution of 4-methylenepyrrolidine-1,2-dicarboxylate (2.41 g, 9.99 mmol) and 9-BBN (80 mL, 40.0 mmol, 0.5 N THF solution) was prepared for 1 hour. The solution was cooled to room temperature and used directly in the next step. MS ESI calculations were performed for C. 12 H 21 BNO6[M - C8H 14 + H2O2-H] - 286.15, actual measurement 286.25.

[0130] Step 2: At room temperature, apply the mixture to 1-(tert-butyl)-2-methyl(2-methyl)-2-methyl ... S )-4-(((1 R 5 RK₃PO₄ (96 mL, 96 mmol, 1 N in water) was added to a solution of 14.5 g (31.9 mmol) of non-9-yl)methyl)pyrrolidine-1,2-dicarboxylate (320 mL) in THF. The solution was stirred at room temperature for 20 min. 5-Bromopyrimidine (5.08 g, 31.9 mmol) and PdCl₂ (dtbpf) (2.081 g, 3.19 mmol) were added, and the resulting mixture was stirred at 60 °C for 1.5 h. The resulting solution was cooled to room temperature, diluted with brine (50 mL), and extracted with EA (3 × 50 mL). The combined organic layers were washed with brine (2 × 25 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with PE solution of 0–90% EA to give the crude product. The crude product was separated by SFC under the following conditions: column: Chiralpak IG, 3 25 cm, 5 μm; Mobile phase A: CO2, Mobile phase B: IPA:MeCN = 1:1 (0.1% 2 MNH3-MeOH); Flow rate: 100 mL / min; Gradient: 40% B (isoconcentration); Column temperature (°C): 35; Back pressure (bar): 100; Wavelength: 220 nm; RT1 (min): 8.93; RT2 (min): 11.32; Sample solvent: MeOH (0.1% 2 M NH3-MeOH); Injection volume: 3.8 mL; Runs: 5. The fraction at 8.93 min was collected and concentrated under reduced pressure to obtain a yellow oily substance, 1-(tert-butyl)-2-methyl(2-methyl ... S 4 R 4-(pyrimidin-5-ylmethyl)pyrrolidine-1,2-dicarboxylate (4.3 g, 12.71 mmol, yield 40%). MS ESI calculations C 16 H 23 N3O4[M-Boc] + 222.17, actual measurement 222.15. 1 H NMR (400 MHz, CDCl3) δ9.14 (d, J = 4.7 Hz, 1H), 8.60 (s, 2H), 4.39 -4.12 (m, 1H), 3.83 -3.63(m, 4H), 3.17 -3.06 (m, 1H), 2.83 -2.53 (m, 3H), 2.14 -1.88 (m, 2H), 1.44 -1.42 (m, 9H).

[0131] Step 3: At room temperature, apply the mixture to 1-(tert-butyl)-2-methyl(2-methyl)-2-methyl ... S 4 R 4-(pyrimidin-5-ylmethyl)pyrrolidine-1,2-dicarboxylate (4.3 g, 13.38 mmol) was added to a stirred solution of 4-(pyrimidin-5-ylmethyl)pyrrolidine-1,2-dicarboxylate in THF (50 mL) with LiOH (40.1 mL, 40.1 mmol, 1 N in water). The solution was stirred at 25 °C for 2 hours. The pH of the solution was adjusted to 3 with 1 N HCl, and the filtrate was concentrated under reduced pressure to give a yellow solid (2... S 4 R 1-(tert-butoxycarbonyl)-4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid (4.1 g, 12.67 mmol, 95% yield). MS ESI calculation C 15 H 20 N3O4[M - H] - 306.15, actual measurement 306.15.

[0132] Step 4: At room temperature, to (2 S 4 R 4.1 g (12.67 mmol) of 1-(tert-butoxycarbonyl)-4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid was added to a stirred solution in DCM (90 mL) with 30 mL of TFA added. The solution was stirred at 25 °C for 1 hour. The solvent was concentrated under reduced pressure to give a yellow solid (2 S 4 R 4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid (2.6 g, 11.29 mmol, yield 89%). MS ESI calculations for C 10 H 14 N3O2[M + H] + 208.10, actual measurement 208.15.

[0133] Step 5: At room temperature, to (2 S 4 R 2.6 g (12.55 mmol) of 4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid was added to a stirred solution of THF (50 mL) and water (50 mL) with Fmoc-OSu (3.81 g, 11.29 mmol) and NaHCO3 (5.27 g, 62.7 mmol). The resulting mixture was stirred at 25 °C for 16 hours. The pH of the mixture was adjusted to 3 with 1 N HCl, and the solution was purified by RP-flash under the following conditions: 330 g C 18Column, 5 minutes 5%-5%, 25 minutes 5%-40%, 5 minutes 98%-98%, MeCN (0.05% TFA) in water, RT = 30 minutes, yielding a yellowish-white solid (2 S 4 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-4-(pyrimidin-5-ylmethyl)pyrrolidine-2-carboxylic acid (5.0 g, 11.53 mmol, yield 92%). MS ESI calculation C 25 H 24 N3O4[M + H] + 430.17, actual measurement 430.10; 1 H NMR (300 MHz, CD3OD) δ9.18 (d, J = 13.2 Hz, 1H), 8.68 (d, J = 6.9 Hz, 2H), 7.80 -7.75 (m, 2H), 7.61 -7.58 (m, 2H), 7.41 -7.27 (m, 4H), 4.42 -4.31 (m, 3H), 4.23 -4.17 (m, 1H),3.62 -3.50 (m, 1H), 3.18 -3.00 (m, 1H), 2.76 -2.70 (m, 2H), 2.68 -2.66 (m,1H), 2.12 -2.02 (m, 2H).

[0134] Synthesis Scheme 8 Pyrim (2 S 4 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-4-(4-(pyrimidin-5-yl)benzyl)pyrrolidine-2-carboxyl) acid Step 1: At 25°C under argon atmosphere, towards (2 S 4 R )-1-(((9 H 1.2 g (2.370 mmol) of fluorene-9-yl)methoxy)carbonyl)-4-(4-bromobenzyl)pyrrolidine-2-carboxylic acid (1.2 g, 2.370 mmol) was added to a mixture of water (10 mL) and 1,4-dioxane (10 mL) with pyrimidin-5-ylboronic acid (0.294 g, 2.370 mmol), K3PO4 (1.509 g, 7.11 mmol), and Pd(dtbpf)Cl2 (0.232 g, 0.355 mmol). The resulting mixture was stirred at 80 °C for 2 hours, and then the reaction was cooled to room temperature and subjected to... Rp -Flash is purified directly under the following conditions: Column: C 18 Gel column (330 g); Mobile phase A: water (0.01% TFA); Mobile phase B: MeCN; (gradient: 0% B for 5 min, reaching 52% B within 35 min, 52% B for 3 min; reaching 90% B within 2 min, 90% B for 10 min); Flow rate: 60 mL / min; Detector: UV 254 & 210 nm; RT: 35 min. The fraction containing the product was collected and rotary evaporated under vacuum to obtain a yellow solid (2... S 4 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-4-(4-(pyrimidin-5-yl)benzyl)pyrrolidine-2-carboxylic acid (629 mg, 1.244 mmol, yield 52%). MS ESI calculation C 31 H 28 N3O4[M + H] + 506.20, actual measurement 506.15. 1 H NMR (300 MHz, CD3OD) δ 9.11 -9.04 (m,3H), 7.78 -7.76 (m, 2H), 7.69 -7.58 (m, 4H), 7.41 -7.25 (m, 6H), 4.38 -4.19(m, 4H), 3.69 -3.54 (m, 1H), 3.29 -3.08 (m, 1H), 2.79 -2.66 (m, 3H), 2.19 -1.95 (m, 2H).

[0135] Synthesis Scheme 9 PyrimAla4Ph4CO2H ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-(4-(tert-butoxycarbonyl)phenyl)pyrimidine (Pyridine-5-yl)propionic acid Step 1: Under a nitrogen atmosphere at 25°C, 1,10-phenanthroline (0.527 g, 2.432 mmol) was added to a stirred solution of NiBr2-ethylene glycol dimethyl ether (0.951 g, 2.432 mmol) in DMA (100 mL). The resulting solution was stirred at 50°C for 1 hour, then cooled to room temperature. At room temperature, 2-chloro-5-iodopyrimidine (5.85 g, 24.32 mmol) and tert-butyl (...) were added to the solution. R )-2-((((9H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-iodopropionate (6 g, 12.16 mmol), TBAI (4.66 g, 12.16 mmol), and zinc (1.590 g, 24.32 mmol). The resulting mixture was stirred at 25 °C for 2 hours, then quenched with H₂O (200 mL) and extracted with EA (2 × 500 mL). The combined organic layers were washed with brine (3 × 200 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0–20% EA in PE solution to give a yellowish-white solid of tert-butyl( S )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-chloropyrimidin-5-yl)propionate (4 g, 8.35 mmol, 70% yield). MS ESI calculation C 26 H 27 ClN3O4[M + H] + 480.16, actual measurement 480.25.

[0136] Step 2: At room temperature, add tert-butyl ( S )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-chloropyrimidin-5-yl)propionate (6 g, 12.50 mmol) was added to a stirred solution in DCM (15 mL) with TFA (30 mL). The solution was stirred at 25 °C for 3 h and then concentrated under reduced pressure. The residue was purified by RP-flash under the following conditions: column: Flash C 18 (330 g); Mobile phase A: water (0.1% TFA), Mobile phase B: ACN; (Gradient: 5% B for 5 min, reaching 30% B within 15 min, 30% B for 5 min; reaching 95% B within 20 min, 95% B for 10 min); Flow rate: 90 mL / min; Detector: UV 210 nm; RT = 40 min. The fraction containing the product was collected and evaporated under vacuum to obtain a yellow oily substance. S )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-chloropyrimidin-5-yl)propionic acid (2.56 g, 6.04 mmol, yield 51%). MS ESI calculation C 22 H 19 ClN3O4[M + H] + 424.10, actual measurement 424.15.

[0137] Step 3: At room temperature, towards (S )-2-((((9 H Pd(dtbpf)Cl2 (0.590 g, 0.906 mmol) was added to a stirred mixture of fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-chloropyrimidin-5-yl)propionic acid (2.56 g, 6.04 mmol), (4-(tert-butoxycarbonyl)phenyl)boronic acid (1.609 g, 7.25 mmol) and K3PO4 (6.41 g, 30.2 mmol) in water (20 mL) and dioxane (20 mL). The resulting mixture was stirred at 80 °C for 2 hours, then cooled to room temperature and concentrated under reduced pressure. The residue was purified by RP-flash under the following conditions: column: Flash C 18 (330 g); Mobile phase A: water (0.1% TFA), Mobile phase B: ACN; (gradient: 5% B for 5 min, reaching 30% B within 15 min, 30% B for 5 min; reaching 95% B within 20 min, 95% B for 10 min); Flow rate: 90 mL / min; Detector: UV 210 nm; RT = 40 min. The fraction containing the product was collected and evaporated under vacuum to obtain a yellow oily substance. S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(2-(4-(tert-butoxycarbonyl)phenyl)pyrimidin-5-yl)propionic acid (2.5629 g, 4.53 mmol, yield 75%). MS ESI calculation C 33 H 32 N3O6[M + H] + 566.22, actual measurement 566.40. 1 H NMR (300 MHz, CD3OD) δ 8.75 (s, 2H), 8.42 (d, J = 8.3 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 7.75(d, J = 7.5 Hz, 2H), 7.66 -7.45 (m, 2H), 7.42 -7.15 (m, 4H), 4.62 -4.44 (m,1H), 4.38 -4.20 (m, 2H), 4.12 (t, J = 7.0 Hz, 1H), 3.35 -3.33 (m, 1H), 3.11 -2.97 (m, 1H), 1.62 (s, 9H).

[0138] Synthesis Scheme 10 3Pal4Ph4CO2H (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-(4-(tert-butoxycarbonyl)phenyl)pyrene (Pyridine-3-yl)propionic acid Step 1: Under a nitrogen atmosphere at room temperature, add 1,10-phenanthroline (0.905 g, 4.18 mmol) to a stirred solution of NiCl2-ethylene glycol dimethyl ether (0.918 g, 4.18 mmol) in DMA (100 mL). Stir the resulting solution at 50 °C for 1 hour. At room temperature, add 2-chloro-5-iodopyridine (5 g, 20.88 mmol) and tert-butyl... R 9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-iodopropionate (12.36 g, 25.06 mmol), TBAI (8.01 g, 20.88 mmol), and Zn (2.73 g, 41.8 mmol) were added to the above mixture, and the resulting mixture was stirred at 25 °C for 2 hours. The reaction was quenched with H₂O (200 mL) and extracted with EtOAc (2 × 500 mL). The combined organic layers were washed with brine (3 × 200 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with PE solution of 0-30% EtOAc to give tert-butyl( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-chloropyridin-3-yl)propionate. MS ESI calculation C 27 H 28 ClN2O4[M + H] + 479.17, actual measurement 479.20.

[0139] Step 2: At room temperature, add tert-butyl ( S )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-chloropyridin-3-yl)propionate (5 g, 10.48 mmol) was added to a stirred solution of TFA (10 mL) in DCM (5 mL). The solution was stirred at 25 °C for 1 hour. The solvent was concentrated under reduced pressure, and the residue was purified by RP-flash under the following conditions: column: Flash C 18(330 g); Mobile phase A: water (0.1% TFA), mobile phase B: ACN; (gradient: 5% B held for 5 min, reaching 30% B within 15 min, 30% B held for 5 min; reaching 95% B within 20 min, 95% B held for 10 min); flow rate: 90 mL / min; detector: UV 210 nm; RT = 40 min. The fraction containing the product was collected and evaporated under vacuum to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-chloropyridin-3-yl)propionic acid. MS ESI calculation of C 23 H 20 ClN2O4[M + H] + 423.10, actual measurement 423.10.

[0140] Step 3: At 25°C under nitrogen atmosphere, towards ( S )-2-((((9 H (-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-chloropyridin-3-yl)propionic acid (3 g, 7.09 mmol) was added to a stirred solution of (4-(tert-butoxycarbonyl)phenyl)boronic acid (1.890 g, 8.51 mmol) and K3PO4 (7.53 g, 35.5 mmol) in THF (25 mL) and water (5 mL). The resulting solution was stirred at 25 °C for 10 min. Pd(dtbpf)Cl2 (0.694 g, 1.064 mmol) was added to the solution, and the mixture was stirred at 60 °C for 16 h. The reaction was cooled to room temperature, quenched with H2O (200 mL), and extracted with EtOAc (2 × 500 mL). The combined organic layers were washed with brine (3 × 200 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by RP-flash under the following conditions: Column Flash C 18 (330 g); Mobile phase A: water (0.1% TFA), mobile phase B: ACN; (gradient: 15% B held for 15 min, reaching 60% B within 15 min, 60% B held for 15 min; reaching 95% B within 10 min, 95% B held for 10 min); flow rate: 90 mL / min; detector: UV 210 nm; RT = 55 min. The fraction containing the product was collected and evaporated under vacuum to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-(4-(tert-butoxycarbonyl)phenyl)pyridin-3-yl)propionic acid. MS ESI calculation C 34 H 33N₂O₆[M + H] + 565.23, actual measurement 565.15; 1 H NMR (400 MHz, methanol-) d4 ) δ 8.66 (d, J = 1.9 Hz, 1H), 8.12 -8.07 (m,3H), 7.99 -7.92 (m, 3H), 7.77 (d, J = 7.5 Hz, 2H), 7.59 -7.56 (m, 2H), 7.37 -7.33 (m, 2H), 7.30 -7.22 (m, 2H), 4.60 -4.56 (m, 1H), 4.29 -4.27 (m, 2H),4.14 -4.10 (m, 1H), 3.45 -3.41 (m, 1H), 3.14 -3.10 (m, 1H), 1.62 (s, 9H).

[0141] Synthesis Scheme 11 Bip4CO2H (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-(tert-butoxycarbonyl)-[1,1'-bin) [Phenyl]-4-yl)propionic acid Argon gas bubbled through ( S )-2-((((9 HA mixture of (-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-bromophenyl)propionic acid (6 g, 12.87 mmol), (4-(tert-butoxycarbonyl)phenyl)boronic acid (4.29 g, 19.30 mmol), and K3PO4 (8.19 g, 38.6 mmol) in THF (40 mL) was added for 10 minutes, followed by the addition of [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.839 g, 1.287 mmol). After stirring the resulting mixture at 50 °C for 16 hours, it was diluted with EtOAc (300 mL) and washed with saturated NaHCO3 aqueous solution (3 × 80 mL) and brine (2 × 40 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a gradient of 0%–50% EtOAc in PE. The fraction containing the product was collected and rotary evaporated under vacuum. The residue was further purified by combi-Flash under the following conditions: C18 gel column (330 g), 20–35 μm; mobile phase A: 0.5% TFA aqueous solution; mobile phase B: MeCN; (gradient: 0% B for 10 min, reaching 62.3% B within 25 min, holding 62.3% B for 25 min; reaching 90% B within 2 min, holding 90% B for 2 min); flow rate: 90 mL / min; detector: UV 254 & 210 nm; RT = 32.32 min. The fraction containing the product was collected and concentrated under reduced pressure to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-(tert-butoxycarbonyl)-[1,1'-biphenyl]-4-yl)propionic acid. MS ESI calculation C 35 H 34 NO6[M + 1] + 564.23, actual measurement 564.15. 1 H NMR (300 MHz, methanol-) d4 ) δ 7.97 -7.95 (m, 2H), 7.78 -7.76 (m, 2H), 7.61 -7.53 (m, 6H), 7.38 -7.21 (m, 2H), 4.51 -4.11 (m, 4H), 3.32 -3.25 (m,1H), 3.03 -2.95 (m, 1H), 1.61 (s, 9H).

[0142] Synthesis Scheme 12 sbMeW4F (2S,3S)-2-amino-3-(4-fluoro-1H-indol-3-yl)butyric acid To a 1-L, three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 10 g of 4-fluoro-1H-indole (1.00 equivalence), 10.6 g of L-threonine (1.20 equivalence), 100 mL of DMSO, and 300 mL of potassium phosphate buffer (0.2 M, pH 7.4). Heat the reaction mixture to 65 °C, then add 2.5 g of PfTrpB-7E6 (25 wt%) and 0.078 g of 3-hydroxy-2-methyl-5-([phosphonoyloxy]methyl)-4-pyridinecarboxaldehyde (0.004 eq). Stir the resulting solution overnight at 65 °C. Then, cool the mixture to room temperature and use it directly in the next step.

[0143] At 0 °C, THF (100 mL), sodium carbonate (23.56 g, 3.0 equivalents), and 2,5-dioxopyrrolidone-1-yl9H-fluorene-9-ylmethyl carbonate (29.96 g, 1.20 equivalents) were added to the above reaction mixture. The resulting solution was stirred overnight at room temperature. The pH was adjusted to 4 with 3 M HCl, and the solid precipitate was removed by filtration. The resulting solution was extracted with EtOAc (3 × 500 mL). The organic fractions were combined, washed with brine (1 L), dried over anhydrous sodium sulfate, and concentrated under vacuum. The mixture was applied to a silica gel column with MeOH:DCM = 1:5. HPLC-MS: (ES, m / z): [M+1]: 459. 1 H NMR (300 MHz, DMSO- d6 )δ 12.60 (s, 1H), 11.15 (s, 1H), 7.87 (d, J = 7.6 Hz, 2H), 7.76 – 7.49 (m,3H), 7.47 – 7.34 (m, 2H), 7.34 – 7.16 (m, 4H), 7.03 (td, J = 7.9, 5.0 Hz, 1H), 6.73 (dd, J = 11.8, 7.7 Hz, 1H), 4.36 (t, J = 8.5 Hz, 1H), 4.31 – 4.02(m, 3H), 3.51 (q, J = 7.4 Hz, 1H), 1.31 (d, J = 7.0 Hz, 4H), 0.78 (s, 1H).

[0144] Synthesis Scheme 13 sbMeW4Cl (2S,3S)-2-amino-3-(4-chloro-1H-indol-3-yl)butyric acid To a 1-L, three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, add 10 g of 4-chloro-1H-indole (1.00 equivalence), 14.09 g of L-threonine (1.8 equivalence), 100 mL of DMSO, and 300 mL of potassium phosphate buffer (0.2 M, pH 7.4). Heat the reaction mixture to 65 °C, then add 7.5 g of PfTrpB-7E6 (25 wt%) and 174 mg of 3-hydroxy-2-methyl-5-([phosphonoyloxy]methyl)-4-pyridinecarboxaldehyde (174 mg, 0.01 eq). Stir the resulting solution at 65 °C for 36 hours. Then, cool the mixture to room temperature and use it directly for the next step.

[0145] At 0 °C, THF (100 mL), sodium carbonate (20.9 g, 3.0 equivalents), and 2,5-dioxopyrrolidone-1-yl9H-fluorene-9-ylmethyl carbonate (31.0 g, 1.40 equivalents) were added to the above reaction mixture. The resulting solution was stirred overnight at room temperature. The pH was adjusted to 4 with 3 M HCl and the solid precipitate was removed by filtration. The resulting solution was extracted with EtOAc (3 × 500 mL). The organic fractions were combined, washed with brine (1 L), dried over anhydrous sodium sulfate, and concentrated under vacuum. HPLC-MS: (ES, m / z): [M+1]: 475.

[0146] Synthesis Scheme 14 sbMe1Nal (2S,3S)-2-amino-3-(naphth-1-yl)butyric acid Step 1: At 20°C, bromotoluene (250 g, 2.00 eq) was added dropwise to a solution of (2S,3R)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-hydroxybutyric acid (250 g, 1.00 equivalence) in DCM (1.5 L). Then, cesium carbonate (477 g, 2.00 eq) was added, and the solution was stirred at 20°C for 3 hours. The reaction mixture was poured into ice-cold H₂O (3 L) and extracted with EtOAc (500 mL × 3). The organic phase was washed with 3% LiCl solution (500 mL × 2) and brine (500 mL), dried over sodium sulfate, and concentrated under vacuum at 40°C. The crude product was ground with methyl tert-butyl ether:PE = 6:1. 1H NMR (400MHz, CDCl3): δ 7.77 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.31-7.36(m, 10H), 5.65-5.71 m, 1H), 5.13-5.31 (m, 3H), 4.39-4.43 (m, 3H), 4.22-4.25 (m, 1H), 1.25 (d, J = 6.4 Hz, 3H).

[0147] Step 2: Under an inert nitrogen atmosphere, add (2S,3R)-benzyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-hydroxybutyrate (125 g, 1.00 eq) and DCE (750 mL) to a 3-necked round-bottom flask. Cool the reaction to 0°C, then add NIS (195 g, 3.00 eq) and PPh3 (228 g, 3.00 eq). Raise the temperature to 50°C and stir the reaction mixture for 3 hours. Decant the reaction mixture into ice-cold H2O (500 mL) and extract with DCM (500 mL × 2). Dry the organic phase with sodium sulfate and concentrate under vacuum at 40°C. Purify the residue by silica gel column chromatography (PE / EtOAc = 1 / 0 to 0 / 1). 1 HNMR (400 MHz, CDCl3): δ 7.78 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 7.2 Hz, 2H), 7.33-7.43 (m, 9H), 5.27-5.68 (m,1H), 5.21-5.23 (m, 2H), 4.39-4.52 (m, 3H), 4.25-4.38 (m, 1H), 1.91-1.95 (m, 3H).

[0148] Step 3: Add 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-iodobutyrate, 1-iodonaphthalene (42.2 g, 1.20 eq), TBAI (76.7 g, 1.50 eq), Zn (19.0 g, 2.10 eq), and DMA (750 mL) to a three-necked round-bottom flask. At 25°C, add pyridinium imide·2HCl (42.2 g, 1.20 eq), NiCl2·ethylene glycol dimethyl ether (7.61 g, 0.25 eq), and DMA (750 mL) to a second three-necked round-bottom flask. Under argon atmosphere, add the contents of the second flask to the first flask. Then, stir the resulting mixture at 25°C for 12 hours. Pour the reaction mixture into ice-cold H2O (3 L) and extract with EtOAc (1 L × 2). The organic phase was dried over sodium sulfate and concentrated under vacuum at 40 °C. The crude product was purified by reversed-phase HPLC (MeCN:H2O). HPLC-MS: [M+23]: 564. 1 H NMR (400 MHz, CDCl3) δ: 8.17-8.24(m, 1H), 8.15-8.17 (m, 1H), 7.77-7.87 (m, 2H), 7.76-7.77 (m, 4H), 7.30-7.41(m, 10H), 5.30-5.38 (m, 1H), 4.96-5.04 (m, 3H), 4.85-4.87 (m, 1H), 4.30-4.34(m, 1H), 4.18-4.26 (m, 4H), 1.43-1.45 (m, 3H).

[0149] Step 4: 143 g of (2S)-benzyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(naphth-1-yl)butyrate was separated by SFC. The organic phase was concentrated under vacuum at 35 °C.

[0150] Peak 1: (2S,3R)-benzyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(naphth-1-yl)butyrate. 1 H NMR (400 MHz, DMSO- d6): δ 8.11-8.12 (m, 2H), 8.10-8.11 (m, 1H), 7.88-7.90(m, 2H), 7.54-7.88 (m, 1H), 7.44-7.53 (m, 2H), 7.42-7.44 (m, 4H), 7.33-7.42(m, 3H), 7.27-7.33 (m, 6H), 7.08-7.09 (m, 2H), 4.91-4.94 (m, 1H), 4.79-4.82(m, 1H), 4.58 (t, J = 8.0 Hz), 4.17-4.25 (m, 4H), 1.39 (d, J = 6.8 Hz, 3H). Peak 2: (2S,3S)-benzyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(naphth-1-yl)butyrate. 1 H NMR (400MHz, DMSO- d6 ): δ 7.92-8.15 (m, 1H), 7.86-7.92 (m, 1H), 7.84-7.86 (m, 1H), 7.57-7.84 (m, 2H), 7.56-7.57 (m, 1H), 7.41-7.54 (m, 4H), 7.30-7.38 (m, 4H),7.27-7.30 (m, 7H), 5.08-5.14 (m, 2H), 4.65 (t, J = 8.0 Hz), 4.23-4.26 (m, 1H), 4.05-4.18 (m, 3H), 1.30 (d, J = 6.8 Hz, 3H).

[0151] Step 5: Add (2S,3S)-benzyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(naphthyl-1-yl)butyrate (40.0 g, 1.00 eq) and THF (200 mL) to a three-necked round-bottom flask. Add 10% wet Pd / C (7.00 g), purge the reaction three times with H2, and stir at 25 °C under H2 (15 psi) for 12 h. Filter the reaction mixture through a CELITE pad and concentrate it under vacuum at 35 °C. Grind the crude product with PE at 25 °C for 1 h. After filtration, dissolve the filter cake in MeCN (100 mL) and concentrate it under vacuum at 35 °C to remove residual solvent. HPLC-MS: [M+23]: 474. 1 H NMR (400 MHz, DMSO- d6) δ 12.78 (s, 1H), 8.23 ​​(d, J = 7.6 Hz, 1H), 7.86-7.88 (m, 1H), 7.80-7.86 (m, 2H), 7.61-7.80 (m, 2H), 7.55-7.59 (m, 4H), 7.481-7.55 (m, 1H), 7.40-7.48 (m, 3H), 7.27-7.29 (m, 2H), 4.28-4.60 (m, 1H), 4.24-4.28 (m, 1H), 4.17-4.24 (m, 2H), 4.04-4.15 (m, 1H), 1.36 (d, J = 6.8 Hz, 3H).

[0152] Synthesis Scheme 15 TyrEtNAc L-tyrosine O -Ethylacetamide or (S)-3-(4-(2-acetaminoethoxy)phenyl)-2-aminopropionic acid Step 1: At room temperature, potassium carbonate (14.04 g, 102 mmol) was added to a stirred solution of (tert-butoxycarbonyl)-L-tyrosine methyl ester (10.0 g, 33.9 mmol), (2-bromomethyl)carbamate benzyl ester (26.2 g, 102 mmol), and TBAB (5.46 g, 16.93 mmol) in DMF (150 mL). The mixture was then stirred at 50 °C for 24 hours. The mixture was cooled to room temperature, quenched with water (250 mL), and extracted with EtOAc (2 × 500 mL). The combined organic phases were washed with brine (3 × 150 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with PE solution of 0–30% EtOAc. MS ESI calculation C 25 H 32 N₂O₇[M + Na] + 495.22, actual measurement 495.10; 1 HNMR (300 MHz, CDCl3) δ 7.38 -7.32 (m, 5H), 7.04 (d, J = 8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 5.31 (br, 1H), 5.21 (s, 2H), 4.97 (br, 1H), 4.56 -4.53 (m, 1H),4.03 (t, J = 5.0 Hz, 2H), 3.72 (s, 3H), 3.64 -3.58 (m, 2H), 3.06 -3.01 (m, 2H), 1.43 (s, 9H).

[0153] Step 2: At room temperature under a nitrogen atmosphere, towards ( S Methyl 3-(4-(2-(((benzyloxy)carbonyl)amino)ethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (16.0 g, 33.9 mmol) and acetic anhydride (6.39 mL, 67.7 mmol) were added to a stirred solution of methyl 3-(4-(2-(((benzyloxy)carbonyl)amino)ethoxy)phenyl) in 200 mL of THF, followed by the addition of Pd / C (3.60 g, 33.9 mmol, dry, 10% wt). The mixture was degassed three times with hydrogen and stirred at 20 °C for 4 h. DIPEA (17.74 mL, 102 mmol) was added to the mixture and stirred at 20 °C for 1 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0–3% MeOH in DCM. MS ESI calculation of C 19 H 28 N₂O₆[M + Na] + 403.19, actual measurement 403.10; 1 H NMR (300MHz, CDCl3) δ 7.05 (d, J = 8.4 Hz, 2H), 6.85 -6.80 (m, 2H), 5.99 (br, 1H), 5.32(br, 1H), 4.99 -4.97 (m, 1H), 4.02 (t, J = 5.0 Hz, 2H), 3.72 (s, 3H), 3.69 -3.63 (m, 2H), 3.10 -2.89 (m, 2H), 2.02 (s, 3H), 1.42 (s, 9H).

[0154] Step 3: At room temperature, towards ( SMethyl 3-(4-(2-acetamidoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (12.5 g, 32.9 mmol) was added to a stirred solution of THF (100 mL) with lithium hydroxide (65.7 mL, 65.7 mmol, 1 N in water). The solution was stirred at 20 °C for 2 hours. The pH of the solution was adjusted to 3 with 1 N HCl. The aqueous layer was extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. MS ESI [M + H] + : 367.10.

[0155] Step 4: At room temperature, towards ( S 3-(4-(2-acetamidoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionic acid (12.5 g, 30.7 mmol) was added to a stirred solution of 3-(4-(2-acetamidoethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propionic acid in 20 mL of dioxane in 4N HCl. The solution was stirred at 20 °C for 1 hour. The solvent was concentrated under reduced pressure. MS ESI [M + H] + : 267.05.

[0156] Step 5: At room temperature, towards ( S 3-(4-(2-acetaminoethoxy)phenyl)-2-aminopropionate (9.50 g, 25.1 mmol) and NaHCO3 (10.54 g, 126 mmol) were added to a stirred mixture of THF (100 mL) and water (100 mL) with Fmoc-OSu (7.62 g, 22.59 mmol). The mixture was stirred at 20 °C for 1 h. The pH of the solution was adjusted to 3 with 1 N HCl. The aqueous phase was extracted with EtOAc (2 × 500 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium bicarbonate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was recrystallized from EtOAc (200 mL). The solid was collected by filtration and dried under vacuum. MS ESI [M + H] + 489.05; 1 H NMR (300 MHz, methanol-) d4 ) δ7.79 (d, J = 7.6 Hz, 2H), 7.62-7.57 (m, 2H), 7.42-7.26 (m, 4H), 7.17-7.14 (m,2H), 6.83 (d, J= 8.4 Hz, 2H), 4.41-4.31 (m, 2H), 4.29-4.10 (m, 2H), 3.96 (t, J =4.8 Hz, 2H), 3.51 (t, J = 5.4 Hz, 2H), 3.19-3.13 (m, 1H), 2.92-2.84 (m, 1H), 1.94 (s, 3H).

[0157] Prot3Ph3F (2S,3R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-(3-fluorophenyl)pyrrolidine-2-carboxylic acid To a commercially available solution of ((2S,3R)-3-(3-fluorophenyl)pyrrolidine-2-carboxylic acid (495 mg, 2.366 mmol) in acetone (16 mL), water (8 mL) and sodium bicarbonate (2S,3R)-3-(3-fluorophenyl)pyrrolidine-2-carboxylic acid (495 mg, 2.366 mmol) were added. The mixture was stirred for 10 minutes (target pH 9.5), Fmoc-OSu (878 mg, 2.60 mmol) was added, and the resulting mixture was stirred overnight at room temperature. The reaction was acidified with sodium bisulfate (1 M, 75 mL). The aqueous solution was washed with DCM, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The crude material was processed using Redisep Rf silica 80. Column chromatographic purification was performed using a pre-packaged column, eluting with a 0-10% gradient of MeOH in DCM over a 33-minute process. Concentration under reduced pressure yielded the title compound. HPLC-MS: [M + H] + : 432.2; 1 H NMR(500 MHz, CDCl3) δ 7.80 (d, J = 7.5 Hz, 2H), 7.62 (t, J = 7.1 Hz, 2H), 7.44(t, J = 7.4 Hz, 2H), 7.40 -7.25 (m, 3H), 7.05 -6.89 (m, 3H), 4.60 -4.46 (m,2H), 4.35 -4.25 (m, 1H), 3.79 -3.50 (m, 3H), 2.48 -2.30 (m, 1H), 2.10 -1.96(m, 1H).

[0158] Prot4Bn3F4F (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-(3,4-difluorobenzyl)pyrrolidine-2-carboxylic acid To a solution of commercially available (2S,4R)-4-(3,4-difluorobenzyl)pyrrolidine-2-carboxylic acid (1000 mg, 4.15 mmol) in acetone (16 mL), water (8 mL) and sodium bicarbonate (1741 mg, 20.73 mmol) were added. The mixture was stirred for 10 min (target pH 9.5), Fmoc-OSu (1538 mg, 4.56 mmol) was added, and the resulting mixture was stirred overnight at room temperature. The reaction was acidified with sodium bisulfate (1 M, 38 mL). The aqueous solution was washed with DCM, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The crude substance was purified by column chromatography using a RediSep Rf silica 40 g pre-packaged column, eluting with a 0-10% gradient of MeOH in DCM over a 22 min process, and concentrated under reduced pressure to give the title compound. HPLC-MS: [M + H] + : 464.4; 1 H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 7.1 Hz, 2H), 7.56 (d, J = 6.8 Hz, 2H), 7.45 -7.30 (m, 4H), 7.17 -7.05 (m, 1H), 7.00 -6.93(m, 1H), 6.89 -6.82 (m, 1H), 4.57 -4.38 (m, 3H), 4.33 -4.23 (m, 1H), 3.54 -3.47 (m, 1H), 3.03 (t, J = 9.3 Hz, 1H), 2.75 -2.50 (m, 2H), 2.38 -2.32 (m, 1H), 2.0 -1.80 (m, 2H).

[0159] Synthesis Scheme 16 BCP3Ph4CO2H (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(3-(4-(tert-butoxycarbonyl)phenyl)bis Cyclo[1.1.1]pentan-1-yl)propionic acid Step 1: At room temperature, EDCI (6.20 g, 32.3 mmol) was added to a stirred solution of 3-(methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic acid (5 g, 29.4 mmol), 2-hydroxyisoindoline-1,3-dione (5.27 g, 32.3 mmol), and DMAP (0.359 g, 2.94 mmol) in a DCM (65 mL). The resulting mixture was stirred at 25 °C for 16 hours. The solid was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0–30% EA in PE, to give 1-(1,3-dioxoisoindoline-2-yl)3-methylbicyclo[1.1.1]pentane-1,3-dicarboxylic acid ester. 1 H NMR (400MHz, CDCl3) δ 7.96 -7.83 (m, 2H), 7.86 -7.75 (m, 2H), 3.73 (s, 3H), 2.56 (s, 6H).

[0160] Step 2: Under a nitrogen atmosphere at room temperature, add dtbbty (1.906 g, 7.10 mmol) to a stirred solution of NiBr2·3H2O (1.549 g, 5.68 mmol) in DMA (180 mL). Stir the resulting mixture at 50 °C for 30 minutes. Cool the mixture to room temperature. At room temperature, add tert-butyl 4-iodobenzoate (7.2 g, 23.67 mmol), 1-(1,3-dioxoisoindoline-2-yl)3-methylbicyclo[1.1.1]pentane-1,3-dicarboxylate (7.46 g, 23.67 mmol), TMS-Cl (0.303 mL, 2.367 mmol), and zinc (7.74 g, 118 mmol) to the mixture. Stir the resulting mixture at 25 °C for 2 hours. The reaction was quenched with brine (400 mL) and extracted with EA (3 × 800 mL). The combined organic layers were washed with brine (3 × 300 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with 0-40% EA in PE to give methyl 3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentane-1-carboxylic acid ester. 1 H NMR (300 MHz, CDCl3) δ 7.98 -7.88 (m, 2H), 7.29 -7.19 (m, 2H), 3.72 (s, 3H), 2.34 (s, 6H), 1.59 (s, 9H).

[0161] Step 3: At room temperature, LiOH (17.86 mL, 17.86 mmol, 1 N in water) was added to a solution of methyl 3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentane-1-carboxylic acid ester (2.7 g, 8.93 mmol) in THF (17.86 mL). The resulting mixture was stirred at room temperature for 2 hours. The solution was acidified with 1 M HCl (15 mL). The solution was extracted with EA (3 × 150 mL). The combined organic layers were washed with brine (3 × 150 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentane-1-carboxylic acid (2.55 g, crude product). MS ESI calculation C 17 H 19 O4[M - H] - 287.14, actual measurement 287.05.

[0162] Step 4: To ( S Benzyl 2-(tert-butyl)-4-methylene-5-oxooxazolidine-3-carboxylate (602 mg, 2.081 mmol) was added to a stirred solution of 3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentane-1-carboxylic acid (600 mg, 2.081 mmol), 4CzIPN (32.8 mg, 0.042 mmol), and K2HPO4 (906 mg, 5.20 mmol) in DMF (20 mL). The reaction solution was irradiated with a 34 W blue LED and stirred at room temperature under argon for 48 hours. The reaction was quenched with water (100 mL) and extracted with EA (3 × 150 mL). The combined organic layers were washed with brine (3 × 150 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with a 0-25% EA gradient in PE to obtain (2 S 4 S )-4-((3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)methyl)-2-(tert-butyl)-5-oxooxazolidine-3-carboxylic acid benzyl ester. MS ESI calculation C 32 H 40 NO6[M- t Bu + H] - 478.28, actual measurement 478.15. 1 H NMR (400 MHz, CDCl3) δ 7.94 -7.84 (m, 2H), 7.41 -7.35 (m, 5H),7.18 (d, J= 8.0 Hz, 2H), 5.56 (s, 1H), 5.24 -5.12 (m, 2H), 4.34 -4.31 (m, 1H), 2.23 -2.17 (m, 1H), 2.09 -2.01 (m, 1H), 1.99 -1.83 (m, 6H), 1.59 (s, 9H), 0.97 (s, 9H).

[0163] Step 5: At 0℃, towards (2 S 4 S )-4-((3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)methyl)-2-(tert-butyl)-5-oxooxazolidine-3-carboxylic acid benzyl ester (1.6 g, 3.00 mmol) was added to a solution of LiOH (7.50 mL, 7.50 mmol, 1 N in water) in 16 mL of THF. The solution was stirred at room temperature for 4 hours. The solution was cooled to 0 °C and the pH was adjusted to 3–4 with 1 M HCl (7.5 mL). The resulting solution was diluted with water (50 mL) and extracted with EA (3 × 100 mL). The organic layer was washed with brine (3 × 100 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to obtain ( S )-2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)propionic acid. MS ESI calculation C 27 H 31 NO6Na [M + Na] + 488.20, actual measurement 488.10. 1 H NMR (400 MHz, CD3OD) δ 7.88 -7.83 (m, 2H), 7.41 -7.35 (m, 2H), 7.35 -7.25 (m,3H), 7.24 -7.19 (m, 2H), 5.19 -5.04 (m, 2H), 4.24 -4.20 (m, 1H), 2.13 -2.01 (m, 1H), 2.03 -1.87 (m, 7H), 1.58 (s, 9H).

[0164] Step 6: At room temperature under a nitrogen atmosphere, towards ( S1.65 g, 3.54 mmol of 2-((benzyloxy)carbonyl)amino)-3-(3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)propionic acid (40 mL) was added to a solution of Pd-C (0.377 g, 0.354 mmol, dry, 10% wt) in THF. The resulting mixture was degassed three times with hydrogen and stirred at room temperature for 2 hours. The resulting product ((S)-2-amino-3-(3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)propionic acid (1.17 g) was used in the next step without further purification. MS ESI calculations C 19 H 26 NO4[M + H] + 332.17, actual measurement 332.15.

[0165] Step 7: At 0 °C, add NaHCO3 (1.483 g, 17.65 mmol) and Fmoc-OSu (1.191 g, 3.53 mmol) to a reaction mixture of (S)-2-amino-3-(3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)propionic acid (1.17 g, 3.53 mmol) in THF (40 mL) and water (40 mL). Stir the reaction mixture at room temperature for 16 hours. Filter the resulting mixture. Wash the filter cake with THF (2 × 100 mL). Concentrate the solvent under reduced pressure. Adjust the pH of the solution to 4 with 1 N HCl (17.6 mL) and extract with EA (3 × 100 mL). Wash the combined organic layers with brine (3 × 100 mL), dry over anhydrous Na2SO4, and filter. The obtained solution was purified by RP-Flash chromatography, eluting with 2-70% acetonitrile (0.05% TFA) in water to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(3-(4-(tert-butoxycarbonyl)phenyl)bicyclo[1.1.1]pentan-1-yl)propionic acid. MS ESI calculation C 34 H 34 NO6[M - H] – 552.25, actual measurement 552.40. 1H NMR (300 MHz, CD3OD) δ 7.90 -7.76 (m, 2H), 7.73 -7.65 (m, 4H), 7.40-7.24 (m, 6H), 4.52 -4.37 (m, 2H), 4.25 -4.18 (m, 2H), 2.14 -2.03 (m, 1H), 1.99 -1.86 (m, 7H), 1.60 -1.54 (m, 9H).

[0166] Synthesis Scheme 17 Bip4CONH2 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4'-carbamoyl-[1,1'-biphenyl) [4-yl]propionic acid At room temperature, towards ( S )-2-((((9 H 15 g (29.2 mmol) of fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-iodophenyl)propionic acid was added to a stirred solution of fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-iodophenyl)propionic acid in THF (246 mL) with PdCl2 (dtbpf) (2.86 g, 4.38 mmol) and tripotassium phosphate (88 mL, 88 mmol, 1 N in water). The solution was stirred at 50 °C for 2 hours. The resulting solution was cooled to room temperature. The pH was adjusted to 3 with 1 N HCl and extracted with EA (3 × 250 mL). The organic layers were combined, washed with brine (4 × 200 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure and the residue was recrystallized from EtOH (100 mL) to give ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-carbamoyl-[1,1'-biphenyl]-4-yl)propionic acid. MS ESI calculation of C 31 H 27 N₂O₅[M + H] + 507.18, actual measurement 507.45. 1 H NMR (400 MHz, DMSO- d6) δ12.87 (s, 1H), 8.02 (s, 1H), 8.07 -7.96 (m, 2H), 7.94 -7.82 (m, 2H), 7.78 -7.60 (m, 7H), 7.39 -7.29 (m, 5H), 7.29 -7.19 (m, 2H), 4.20 -4.13 (m, 4H), 3.17 -3.13 (m, 1H), 2.93 -2.90 (m, 1H).

[0167] Synthesis Scheme 18 dProt4NMe3 (3S,5R)-1-(((9H-fluorene-9-yl)methoxy)carbonyl)-5-carboxy-N,N,N-trimethylpyrrolidine-3-ammonium chloride compounds Step 1: At ambient temperature, apply 1-(tert-butyl)-2-methyl(2-methyl)-2-methyl ... R 4 S 4-Aminopyrrolidine-1,2-dicarboxylic acid ester (3 g, 12.28 mmol) was added to a mixture of NaHCO3 (8.25 g, 98 mmol) and CH3I (4.61 mL, 73.7 mmol) in MeOH (30 mL). The reaction was stirred at ambient temperature for 16 hours. The resulting solution was filtered. The filtrate was concentrated under vacuum to obtain the crude product. The crude product was dissolved in DCM and filtered. The filtrate was concentrated under vacuum to obtain (3 g, 12.28 mmol) of NaHCO3 (8.25 g, 98 mmol) and CH3I (4.61 mL, 73.7 mmol). S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)- N , N , N -Trimethylpyrrolidine-3-ammonium iodide. MS ESI calculation C 14 H 27 N₂O₄[M - I] + 287.20, actual measurement 287.15.

[0168] Step 2: At ambient temperature, towards (3 S 5 R )-1-(tert-butoxycarbonyl)-5-(methoxycarbonyl)- N , N , N-Trimethylpyrrolidine-3-ammonium iodide (6.5 g, 10.98 mmol) was added to a mixture of LiOH (21.97 mL, 21.97 mmol, 1 M in water) in THF (23 mL). The reaction was stirred at ambient temperature for 4 hours. The resulting solution was acidified to pH 5 with 1 M HCl and concentrated under vacuum to give (3 S 5 R )-1-(tert-butoxycarbonyl)-5-carboxyl- N , N , N -Trimethylpyrrolidine-3-ammonium chloride. MS ESI calculation C 13 H 25 N₂O₄[M - Cl] + 273.18, actual measurement 273.15.

[0169] Step 3: At ambient temperature, towards (3 S 5 R )-1-(tert-butoxycarbonyl)-5-carboxyl- N , N , N 3-Trimethylpyrrolidine-3-ammonium chloride (6.6 g, 10.69 mmol) was added to a mixture in DCM (20 mL) with TFA (20 mL, 260 mmol). The reaction was stirred at ambient temperature for 2 hours. The resulting solution was concentrated under vacuum to give 2,2,2-trifluoroacetic acid, (3 S 5 R )-1-(tert-butoxycarbonyl)-5-carboxyl- N , N , N -Trimethylpyrrolidine-3-ammonium salt. C8H calculated by MS ESI. 17 N2O2[M-CF3COO] + 173.13, actual measurement 173.25.

[0170] Step 4: At room temperature, add 2,2,2-trifluoroacetic acid, (3... S 5 R )-5-carboxyl- N , N , N3-Trimethylpyrrolidine-3-ammonium salt (8.1 g, 14.10 mmol) was added to a mixture of THF (40 mL) and water (40 mL) with NaHCO3 (5.92 g, 70.5 mmol) and Fmoc-OSu (4.28 g, 12.69 mmol). The reaction was stirred at room temperature for 4 hours. The resulting solution was acidified to pH 5 with dilute HCl and concentrated under vacuum. The residue was suspended in DCM (with 10% MeOH) and filtered. The filtrate was concentrated under vacuum. The residue was purified by RP flash under the following conditions: column: Flash C 18 (330 g); Mobile phase A: water (2 mmol HCl), Mobile phase B: ACN; (gradient: 2% B for 5 min, reaching 35% B within 15 min, 35% B for 8 min; reaching 98% B within 5 min, 99% B for 5 min); Flow rate: 80 mL / min; Detector: UV 210 nm; RT = 38 min. The fraction containing the product was collected and concentrated under vacuum to obtain (3... S 5 R )-1-(((9 H -fluorene-9-yl)methoxy)carbonyl)-5-carboxyl- N , N , N -Trimethylpyrrolidine-3-ammonium chloride. MS ESI calculation C 23 H 27 N₂O₄[M - Cl] + 395.20, actual measurement 395.20. 1 H NMR (300 MHz, DMSO- d 6) δ 13.30 (s, 1H), 7.94 -7.89 (m, 2H), 7.72 -7.64 (m, 2H), 7.46 -7.33 (m, 4H), 4.57 -4.14 (m, 5H), 3.86 -3.78 (m, 2H), 3.12 -3.10 (m, 9H), 2.83 -2.73 (m, 1H), 2.50 -2.35 (m, 1H).

[0171] Synthesis Scheme 19 F4SO2 NH2 (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-aminosulfonylphenyl)propionic acid Step 1: At -10℃, acetyl- LA mixture of 1 g (4.83 mmol) of phenylalanine in chlorosulfonic acid (5.34 g, 45.8 mmol) was stirred for 3 hours. The resulting solution was then stirred at 25°C for 2 hours. The reaction mixture was added dropwise to ice while stirring continuously. The resulting mixture was diluted with EA (500 mL), washed with brine (3 × 250 mL), dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure to give the crude product. The crude product was dissolved in NH₄OH (15 mL, 28% wt) and stirred at 100°C for 3 hours. The solvent was concentrated under reduced pressure, and the pH was adjusted to 3 with 1 N HCl. The solid was collected by filtration and lyophilized to give (…). S 2-Acetamido-3-(4-aminosulfonylphenyl)propionic acid. MS ESI calculation of C 11 H 15 N₂O₅S [M + H] + 287.06, actual measurement 287.05. 1 H NMR (300 MHz, DMSO- d 6 ) δ 8.32 -8.30 (m, 1H), 7.75 -7.70 (m,2H), 7.43 -7.41 (m, 2H), 7.28 (s, 2H), 4.49 -4.31 (m, 1H), 3.13 -3.11 (m,1H), 2.92 -2.91 (m, 1H), 1.78 (s, 3H).

[0172] Step 2: Place ( S 2-Acetamido-3-(4-aminosulfonylphenyl)propionic acid (5.1 g, 17.81 mmol) was suspended in water (50 mL), and the pH of the mixture was adjusted to 5 with LiOH (1 M) and then to 7.5 with 0.25 M buffer. Hog renin acylase (510 mg, 17.81 mmol) was added to the mixture at 25 °C. The resulting mixture was then stirred at 25 °C for 20 hours. The reaction was not further purified. S 2-Amino-3-(4-aminosulfonylphenyl)propionic acid solution was used directly in the next reaction. MS ESI calculations of C9H... 13 N₂O₄S [M + H] + 245.05, actual measurement 245.10.

[0173] Step 3: At room temperature, towards ( STHF (50 mL), NaHCO3 (3.95 g, 47.1 mmol), and Fmoc-OSu (3.18 g, 9.42 mmol) were added to a solution of 2-amino-3-(4-aminosulfonylphenyl)propionic acid (2.3 g, 9.42 mmol, dissolved in 50 mL of water). The resulting mixture was stirred at 25 °C for 16 hours. The pH was adjusted to 3 with 1 N HCl. The reaction mixture was extracted with EA (3 × 120 mL). The organic layer was washed with brine (120 mL) and dried over anhydrous Na2SO4. The solvent was concentrated under reduced pressure, and the residue was purified by RP-flash under the following conditions: 330 g C 18 Column, 5 minutes 2%-2%, 30 minutes 2%-30%, ACN (0.05% NH4HCO3) in water, to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-aminosulfonylphenyl)propionic acid. MS ESI calculation C 24 H 26 N3O6S [M + NH4] + 484.12, actual measurement 484.10. 1 H NMR (300 MHz, CD3OD) δ 7.82 -7.73 (m, 4H), 7.68 -7.56 (m, 2H), 7.38 -7.35 (m, 4H), 7.34 -7.23(m, 2H), 4.40 -4.12 (m, 4H), 3.31 -3.30 (m, 1H), 3.01 -2.98 (m, 1H).

[0174] Synthesis Scheme 20 K3CH3NPEG4 (S)-5-carboxy-1-(9H-fluoren-9-yl)-N,N,N-trimethyl-3,11-dioxo-2,14,17,20,23-pentaoxo 4,10-diazapecosano-25-ammonium 2,2,2-trifluoroacetate Step 1: At ambient temperature, NaHCO3 (12.43 g, 148 mmol) and dimethylamine hydrochloride (6.03 g, 74.0 mmol) were added to a stirred solution of tert-butyl 1-bromo-3,6,9,12-tetraoxapentadecan-15-ester (19 g, 49.3 mmol) in MeCN (100 mL). The reaction mixture was then stirred at 80 °C for 3 hours. The reaction progress was monitored by LCMS and TLC. LCMS showed that the product was predominantly the desired product with no residual SM. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 0%–15% MeOH gradient in DCM. Fractions containing the desired product were combined and concentrated under reduced pressure to give tert-butyl 2-methyl-5,8,11,14-tetraoxa-2-azaheptadecane-17-ester. C2 was calculated by MS ESI. 17 H 36 NO6[M + H] + 350.25, actual measurement 350.35. 1 H-NMR (400 MHz, chloroform- d ) δ 3.91 -3.88(m, 2H), 3.73 -3.69 (m, 2H), 3.67 -3.66 (m, 4H), 3.65 -3.64 (m, 4H), 3.63 -3.61 (m, 4H), 3.11 -3.08 (m, 2H), 2.74 (s, 6H), 2.52 -2.49 (m, 2H), 1.45 (s, 9H).

[0175] Step 2: At ambient temperature, 2,2,2-trifluoroacetic acid (30 mL, 47.2 mmol) was added to a stirred solution of tert-butyl 2-methyl-5,8,11,14-tetraoxa-2-azaheptadecane-17-ester (16.5 g, 47.2 mmol) in DCM (100 mL). The reaction solution was then stirred at ambient temperature for 2 hours. The reaction progress was monitored by LCMS and TLC. LCMS showed that the product was predominantly the desired product. The solvent was concentrated under reduced pressure, and the residue was used directly in the next step. CS was calculated by MS ESI. 13 H 28 NO6[M+ H] + 294.19, actual measurement 294.35.

[0176] Step 3: Under a nitrogen atmosphere at -40°C, add N-ethyl-N-isopropylprop-2-amine (12.13 g, 94 mmol) to a stirred solution of 2-methyl-5,8,11,14-tetraoxa-2-azaheptadecane-17-acid (13.77 g, 46.9 mmol) in 80 mL of DMF. Stir the solution at -40°C for 2 minutes. Add HATU (21.42 g, 56.3 mmol) and tert-butyl(((9H-fluorene-9-yl)methoxy)carbonyl)- L -Lysine ester (17.93 g, 42.2 mmol) was added to the solution and stirred at -40 °C for 3 h. The reaction was quenched with saturated NH4Cl (100 mL) and extracted with EtOAc (2 × 300 mL). The combined organic layers were washed with brine (3 × 80 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by RP flash under the following conditions: column, C18 330 g, mobile phase: ACN (0.5% TFA) in water, 5 min 5%-5%, 35 min 5%-95%; detector, UV 254 & 210 nm. RT: 26 min. The collected fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-23-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-2-methyl-17-oxo-5,8,11,14-tetraoxa-2,18-diazatocetane-24-ester. MS ESI calculation C 38 H 59 N3O9[M + H] + 700.42, actual measurement 700.40. 1 H-NMR (300 MHz, methanol-) d 4) δ 7.84 -7.81 (m, 2H), 7.72 -7.68 (m, 2H), 7.44 -7.39 (m, 2H), 7.36 -7.31 (m, 2H), 4.46 -4.32 (m, 2H), 4.27 -4.00 (m, 2H), 3.81 -3.60 (m, 16H), 3.32 -3.28 (m, 2H), 3.22 -3.18 (m, 2H), 2.91 (s,6H), 2.43 (t, J = 6.3 Hz, 2H), 1.83 -1.78 (m, 1H), 1.71 -1.66 (m, 1H), 1.57 -1.52 (m, 2H), 1.47 (s, 9H).

[0177] Step 4: At ambient temperature, apply tert-butyl ( S )-23-((((9 H 13 g (18.57 mmol) of fluorene-9-yl)methoxy)carbonyl)amino)-2-methyl-17-oxo-5,8,11,14-tetraoxa-2,18-diazatetracosane-24-ester (MeCN) was added to a stirred solution in 80 mL of MeCN with NaHCO3 (3.12 g, 37.1 mmol) and methyl iodoform (21.09 g, 149 mmol). The reaction solution was then stirred at 70 °C. The reaction progress was monitored by LCMS and TLC. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was used directly in the next step. C2 was calculated by MS ESI. 39 H 60 N3O9[M] + 714.43, actual measurement 714.41.

[0178] Step 5: At ambient temperature, towards ( S )-5-(tert-butoxycarbonyl)-1-(9 H -fluorene-9-base)- N , N , N -Trimethyl-3,11-dioxo-2,14,17,20,23-pentaoxa-4,10-diazapecopentane-25-ammonium iodide (12 g, 14.25 mmol) was added to a stirred solution in a DCM (50 mL) with 2,2,2-trifluoroacetic acid (30 mL, 14.25 mmol). The reaction solution was then stirred at ambient temperature for 6 hours. The reaction progress was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the residue was purified by RP flash under the following conditions: column, C18 330 g, mobile phase: ACN in water (0.5% TFA), 5 min 5%-5%, 25 min 5%-95%, 21% hold for 8.2 min; detector, UV 254 nm, RT: 35 min. The collected fractions were combined and concentrated under reduced pressure to obtain ( S )-5-carboxy-1-(9 H -fluorene-9-base)- N , N , N -Trimethyl-3,11-dioxo-2,14,17,20,23-pentaoxa-4,10-diazacopeco-25-ammonium 2,2,2-trifluoroacetate. MS ESI calculation C 35 H 52 N3O9[M] + 658.37, actual measurement 658.30. 1¹H NMR (300 MHz, methanol-d⁴) δ 7.82 -7.79 (m, 2H), 7.70 -7.66 (m, 2H), 7.42 -7.29 (m, 4H), 4.39 -4.35 (m, 2H), 4.26 -4.23 (m, 2H), 3.93 -3.88 (m, 2H), 3.72 -3.52 (m, ¹⁶H), 3.20 -3.17 (m, ¹¹H), 2.43 (t, J = 6.3 Hz, 2H), 1.89 -1.87 (m, 1H), 1.79 -1.71 (m, 1H), 1.54 -1.42 (m, 4H).

[0179] Synthesis Scheme 21 OrnMe3 (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-carboxy-N,N,N-trimethylbut-1-ammonium 2,2, 2-Trifluoroacetate Step 1: At ambient temperature, towards ( S MeI (33.7 g, 238 mmol) and KHCO3 (1.983 g, 19.80 mmol) were added to a mixture of 5-amino-2-((tert-butoxycarbonyl)amino)valeric acid (4.6 g, 19.80 mmol) in MeOH (50 mL). The resulting mixture was heated to 50 °C and stirred for 12 hours. The reaction was cooled to room temperature and filtered. The filtrate was concentrated under vacuum, and the residue was dissolved in DCM (80 mL) and then filtered again. The filtrate was concentrated under vacuum to give ( S )-4-((tert-butoxycarbonyl)amino)-5-methoxy- N , N , N -Trimethyl-5-oxopent-1-ammonium iodide. MS ESI calculation of C 14 H 29 N₂O₄[M - I] + 289.21, actual measurement 289.35.

[0180] Step 2: At ambient temperature, towards ( S )-4-((tert-butoxycarbonyl)amino)-5-methoxy- N , N , N-Trimethyl-5-oxopent-1-ammonium iodide (7.4 g, 16.00 mmol) was added to a mixture of MeOH (48 mL) and THF (24 mL) with LiOH (48.0 mL, 48.0 mmol, 1 M in water). The reaction was stirred at ambient temperature for 2 hours, and then concentrated under vacuum. Next, 48 mL of 1N HCl was added. The solvent was concentrated under vacuum to give ( S )-4-((tert-butoxycarbonyl)amino)-4-carboxyl- N , N , N -Trimethylbut-1-ammonium chloride. MS ESI calculation C 13 H 27 N₂O₄[M - Cl] + 275.20, actual measurement 275.20.

[0181] Step 3: At ambient temperature, towards ( S )-4-((tert-butoxycarbonyl)amino)-4-carboxyl- N , N , N Trimethylbut-1-ammonium chloride (7.5 g, 14.48 mmol) was added to a mixture in DCM (50 mL) with TFA (25 mL, 324 mmol). The reaction was stirred at ambient temperature for 1 hour, and then concentrated under vacuum to obtain ( S )-4-amino-4-carboxy- N , N , N -Trimethylbut-1-ammonium 2,2,2-trifluoroacetate. C8H was calculated using MS ESI. 19 N2O2[M-CF3COO] + 175.14, actual measurement 175.20.

[0182] Step 4: At ambient temperature, towards ( S )-4-amino-4-carboxy- N , N , N 10 g (13.88 mmol) of 1-trimethylbut-1-ammonium 2,2,2-trifluoroacetate was added to a mixture of THF (30 mL) and water (30 mL) with NaHCO3 (9.33 g, 111 mmol) and Fmoc-OSu (4.21 g, 12.49 mmol). The reaction was stirred at ambient temperature for 2 hours. The resulting solution was acidified to pH 3–4 with aqueous HCl and then filtered. Rp - The filtrate was purified using the following conditions: Column: Flash C 18(330 g); Mobile phase A: water (0.05% TFA), mobile phase B: ACN; (gradient: 0% B for 7 min, reaching 33% B within 18 min, 33% B for 7 min; reaching 95% B within 5 min, 95% B for 5 min); flow rate: 90 mL / min; detector: UV 210 nm; RT = 40 min. The fraction containing the product was collected and lyophilized to obtain ( S )-4-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-4-carboxyl- N , N , N -Trimethylbut-1-ammonium 2,2,2-trifluoroacetate. MS ESI calculation of C 23 H 29 N₂O₄[M⁻CF₃COO] + 397.21, actual measurement 397.15. 1 H NMR (400 MHz, DMSO- d 6) δ 12.85 (br,1H), 7.92 -7.89 (m, 2H), 7.74 -7.63 (m, 3H), 7.44 -7.40 (m, 2H), 7.33 -7.31(m, 2H), 4.38 -4.22 (m, 3H), 4.02 -3.98 (m, 1H), 3.34 -3.20 (m, 2H), 3.09 (s,9H), 1.79 -1.51 (m, 4H). 19 F-NMR (376 MHz, DMSO- d 6) -73.64.

[0183] Synthesis Scheme 22 Phe4pcCCA (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-((1s,4R)-4-(tert-butoxycarbonyl) Cyclohexylphenylpropionic acid Step 1: At room temperature, tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohexyl-3-en-1-carboxylic acid ester (29.5 g, 96 mmol) was added to a mixture in THF (30 mL). SMethyl 2-((tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate (15.5 g, 38.2 mmol) and Pd(Ph3P)4 (2.210 g, 1.912 mmol). The reaction was heated to 60 °C and held for 4 hours. The resulting solution was quenched with water (100 mL) and extracted with ethyl acetate (3 × 300 mL). The organic layers were combined, washed with brine (2 × 200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum to give the crude product. The residue was purified by silica gel chromatography, eluting with a gradient of ethyl acetate:petroleum ether from 0:1 to 1:4 to give tert-butyl 4'-(( S )-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-carboxylic acid ester. MS ESI calculation of C 26 H 38 NO6Na[M + Na] + 482.26, actual measurement 482.10.

[0184] Step 2: At room temperature, add tert-butyl 4'-(( S )-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-carboxylic acid ester (16 g, 34.8 mmol) was added to a mixture in methanol (160 mL) with Pd-C (10% on carbon, moistened with about 55% water, 5.3 g, 4.98 mmol). The mixture was degassed three times with H2 and stirred for 1 hour at room temperature under an H2 atmosphere (1.5 atm). The resulting mixture was filtered. The filtrate was concentrated under vacuum to give tert-butyl( S )-4-(4-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl)cyclohexane-1-carboxylic acid ester. MS ESI calculation C 26 H 41 NO6[M + H] + 462.28, actual measurement 462.30.

[0185] Step 3: At room temperature, add tert-butyl ( S 15 g (32.5 mmol) of cyclohexane-1-carboxylic acid ester (4-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl)cyclohexane-1-carboxylic acid ester was added to a stirred solution in 300 mL of THF with LiOH (65.0 mL, 65.0 mmol). The solution was stirred at 20 °C for 1 hour. The pH of the solution was adjusted to 3 with 1 N HCl. The reaction was concentrated under reduced pressure to obtain ( S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 25 H 38 NO6Na [M + Na] + 470.26, actual measurement 470.30.

[0186] Step 4: Separate using Prep-SFC under the following conditions ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-(4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (14 g, 31.3 mmol): Column: CHIRAL ART Cellulose-SB, 3 x 25 cm, 5 µm; Mobile phase A: CO2, Mobile phase B: MeOH (0.1% 2M NH3-MEOH); Flow rate: 80 mL / min; Gradient: 10% B; 220 nm; RT1: 7.45; RT2: 8.38; Injection volume: 1.3 ml; Runs: 131; Result: ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-(( 1s , 4R )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (fast elution peak). MSESI calculation C 25 H 38 NO6Na [M + Na] + 470.26, measured: 470.30. ¹H NMR (300 MHz, chloroform-d) δ 7.11 (d, J = 3.0 Hz, 4H), 4.27 (s, 1H), 3.18 -3.12 (m, 1H), 2.91 (s, 1H), 2.60 (s, 1H), 2.49 (s, 1H), 2.20 (d, J = 10.9 Hz, 2H), 1.78 -1.52 (m, 6H), 1.49 (d, J = 0.7 Hz, 9H), 1.36 (s, 9H).

[0187] As a slow elution peak, separation ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-(( 1r , 4S )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 25 H 38 NO6Na [M + Na] + 470.26, actual measurement 470.30. 1¹H NMR (300 MHz, chloroform-d) δ 7.14 -7.06 (m, 4H), 4.25 (s, 1H), 3.14 (d, J = 12.3 Hz, 1H),2.88 (s, 1H), 2.49 -2.41 (m, 1H), 2.29 -2.15 (m, 1H), 2.05 (d, J = 12.2 Hz, 2H), 1.90 (d, J = 12.1 Hz, 2H), 1.69 -1.47 (m, 2H), 1.47 (s, 11H), 1.34 (s, 9H).

[0188] Step 5: At room temperature, towards ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-(( 1s , 4R 4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (8.3 g, 18.54 mmol) was added in portions to a mixture in THF (80 mL) with hydrogen chloride (M / dioxane) (9.27 mL, 18.54 mmol). The reaction was concentrated under reduced pressure to give ( S )-2-amino-3-(4-(( 1s , 4R )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 20 H 30 NO4[M + H] + 348.21, actual measurement 348.25.

[0189] Step 6: At room temperature, towards ( S )-2-amino-3-(4-(( 1s , 4R 4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (6 g, 17.27 mmol) and NaHCO3 (7.25 g, 86 mmol) were added to a stirred solution of THF (60 mL) and water (60.0 mL). n -(9-fluorenylmethoxycarbonyl)succinimide (5.24 g, 15.54 mmol). The mixture was stirred at room temperature for 1 hour. The pH of the solution was adjusted to 3 with 1 N HCl. The aqueous phase was extracted with diethyl ether acetate (2 × 200 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by RP flash to give ( S )-2-((((9H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(( 1s , 4R )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 35 H 41 NO6[M + H] + 570.28, actual measurement 570.15. 1 HNMR (300 MHz, methanol-) d 4) δ 7.81 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.43-7.27 (m, 4H), 7.16 (d, J = 7.8 Hz, 2H), 7.07 (d, J = 7.9 Hz, 2H), 4.45-4.33 (m,2H), 4.22 -4.06 (m, 2H), 3.26 -3.14 (m, 1H), 2.96 -2.88 (m, 1H), 2.59 (s,1H), 2.48 (s, 1H), 2.15 (s, 2H), 1.61 (d, J = 5.5 Hz, 6H), 1.49 (s, 9H).

[0190] Synthesis Scheme 23 Phe4ptCCA (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-((1r,4S)-4-(tert-butoxycarbonyl) Cyclohexylphenylpropionic acid Step 1: Under argon atmosphere, add ( ) to a mixture of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)cyclohexyl-3-en-1-carboxylic acid ester (18.25 g, 59.2 mmol) in THF (200 mL). SMethyl 2-((tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate (20 g, 49.4 mmol), Pd(PPh3)4 (2.85 g, 2.468 mmol), and CsF (30.0 g, 197 mmol) were reacted. The reaction was stirred at 60 °C under argon for 16 h. The reaction was quenched with water (150 mL) and extracted with EA (3 × 500 mL). The combined organic layers were dried over anhydrous MgSO4 and filtered. The residue was purified by silica gel chromatography, eluting with a gradient of ethyl acetate:petroleum ether from 1:1 to 1:5 to give tert-butyl 4'-(( S )-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-carboxylic acid ester. MS ESI calculation of C 26 H 37 NO6Na [M + Na] + 482.26, actual measurement 482.30.

[0191] Step 2: Under argon atmosphere, tert-butyl ( R )-4'-(( S Crabtree's catalyst (14.71 g, 18.28 mmol) was added to a mixture of 2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-2,3,4,5-tetrahydro-[1,1'-biphenyl]-4-carboxylic acid ester (21 g, 45.7 mmol) in dichloromethane (200 mL). The suspension was degassed under vacuum and purged several times with H2. The reaction solution was stirred at room temperature under 2 atm H2 for 24 hours. The reaction was filtered. The filtrate was concentrated under vacuum to give the racemic product. The racemic product was separated using a Prep-SFC column: CHIRALPAK IG, 5 25 cm, 10 μm; Mobile phase A: CO2, Mobile phase B: MeOH (0.1% 2M NH3-MeOH); Flow rate: 200 mL / min; Gradient: isoconcentration 25% B; Column temperature (°C): 35; Back pressure (bar): 100; Wavelength: 220 nm; RT1 (min): 4.15, yielding an orange oily substance of tert-butyl (1 S 4 R )-4-(4-(( S )-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl)cyclohexane-1-carboxylic acid ester. MS ESI calculation of C 26 H 39 NO6Na [M + Na] + 484.28, actual measurement 484.35.

[0192] Step 3: At ambient temperature, apply tert-butyl(1) S 4 R )-4-(4-(( S 10.5 g (22.75 mmol) of cyclohexane-1-carboxylic acid ester (2-(tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl)cyclohexane-1-carboxylic acid ester (THF) in 45.5 mL of water was added to a mixture of 2-(tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl)cyclohexane-1-carboxylic acid ester (45.5 mL). The reaction was stirred at ambient temperature for 2 hours. The resulting solution was acidified with aqueous HCl (1 M) and extracted with EA (4 × 100 mL). The combined organic layers were dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum to give ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-((1 R 4 S )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 25 H 37 NO6Na [M + Na] + 470.26, actual measurement 470.25.

[0193] Step 4: At ambient temperature, towards ( S )-2-((tert-butoxycarbonyl)amino)-3-(4-((1 R 4 S 4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (9.5 g, 21.23 mmol) was reacted with HCl (4 M in 1,4-dioxane) (100 mL, 400 mmol) in a mixture of 4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (9.5 g, 21.23 mmol) and THF (100 mL). The reaction was stirred at ambient temperature for 4 hours. The resulting solution was concentrated under vacuum to give ( S )-2-amino-3-(4-((1 R 4 S )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MSESI calculation of C 20 H 30 NO4[M + H] + 348.21, actual measurement 348.25.

[0194] Step 5: To ( S )-2-amino-3-(4-((1 R 4 S)-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid (7 g, 20.15 mmol) was added to a mixture of THF (30 mL) and water (30 mL) with sodium bicarbonate (8.46 g, 101 mmol) and n -(9-fluorenylmethoxycarbonyloxy)succinimide (6.12 g, 18.13 mmol). The reaction was stirred at ambient temperature for 2 hours. The resulting solution was acidified with aqueous HCl (1 M) and extracted with EA (5 × 100 mL). The combined organic layers were dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under vacuum to give the crude product. The residue was purified by RP flash under the following conditions: column: Flash C 18 (330 g); mobile phase A: water (0.05% TFA), mobile phase B: ACN; (gradient: 2% B for 5 min, reaching 75% B within 15 min, 76.5% B for 6 min; reaching 95% B within 5 min, 95% B for 5 min); flow rate: 90 mL / min; detector: UV 210 nm; RT = 31 min. The fraction containing the product was collected and concentrated under vacuum to give ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-((1 R 4 S )-4-(tert-butoxycarbonyl)cyclohexyl)phenyl)propionic acid. MS ESI calculation C 35 H 40 NO6[M + H] + 570.29, actual measurement 570.25. 1 H NMR (400 MHz, methanol-) d 4)δ 7.80 -7.78 (m, 2H), 7.60 -7.58 (m, 2H), 7.40 -7.38 (m, 2H), 7.37 -7.28 (m,2H), 7.16 -7.14 (m, 2H), 7.06 -7.04 (m, 2H), 4.45 -4.41 (m, 1H), 4.32 -4.28(m, 1H), 4.15 -4.055 (m, 2H), 3.21 -3.17 (m, 1H), 2.91 -2.88 (m, 1H), 2.41 -2.37 (m, 1H), 2.19 -2.17 (m, 1H), 1.96 -1.91 (m, 2H), 1.78 -1.75 (m, 2H), 1.45 -1.42 (m, 10H), 1.40 -1.32 (m, 3H).

[0195] Synthesis Scheme 24 YFN (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)-λ 2 -Azylyl)-3-(4-(5-oxo-4,5-dihydro-1, 2,4-Oxadiazol-3-yl)phenyl)propionic acid Step 1: To ( S 2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionic acid (5 g, 17.22 mmol) was added to the mixture in DCM (10 mL) with tert-butyl N,N' -Diisopropylisourea (carbamimidate) (17.25 g, 86 mmol). The reaction was refluxed at 40 °C for 2 hours. The resulting solution was concentrated under vacuum. The residue was purified by silica gel chromatography, eluting with a gradient of ethyl acetate:petroleum ether from 0:1 to 1:3 to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate. MS ESI calculation of C 19 H 26 N₂O₄Na [M + Na] + 369.19, actual measurement 369.10.

[0196] Step 2: At room temperature, add sodium bicarbonate (10.91 g, 130 mmol) to a solution of hydroxylamine hydrochloride (6.62 g, 95 mmol) in DMSO (120 mL), then raise the temperature to 45-50°C. At this temperature, add tert-butyl ( S2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate (6 g, 17.32 mmol) was added to the resulting solution, and the mixture was heated to 100 °C, followed by stirring at the same temperature for 18 hours. After completion, the reactants were cooled to 15-20 °C and then poured into water. The solution was stirred at 15-20 °C for 15-20 minutes and filtered. The collected solid was washed with water and dried to give tert-butyl( S , E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N '-Hydroxycarbamimidoyl'-phenylpropionate. MS ESI calculation C 19 H 30 N3O5[M + H] + 380.21, actual measurement 380.15.

[0197] Step 3: Add tert-butyl ( S , E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N 5.5 g (14.49 mmol) of '-hydroxymethylammonium)phenyl)propionate was dissolved in tert-butanol (100 mL), and then BOC2O (3.37 mL, 14.49 mmol) was slowly added via a feeding funnel at 25-30 °C. After the addition was complete, the temperature was raised to 50-55 °C, and stirring was continued at the same temperature for 12 hours. After the reaction was complete, the reaction mixture was cooled to 15-20 °C and stirred at 15-20 °C for 60 minutes to obtain a solid. The product was filtered, washed with tert-butanol, and dried under vacuum at 55-60 °C to obtain the crude product. MS ESI calculations C 24 H 38 N3O7[M + H] + 480.26, actual measurement 480.35.

[0198] Step 4: Add tert-butyl ( S , E )-3-(4-( N -(tert-butoxycarbonyl)- N'-Hydroxymethylamidine)phenyl)-2-((tert-butoxycarbonyl)amino)propionate (5.5 g, 11.47 mmol) was dissolved in DMF (60 mL), and the reaction was heated to 110-115 °C. The resulting solution was stirred at the same temperature for 12 hours. The reaction mixture was cooled to room temperature, diluted with 600 mL of EtOAc, washed with saturated NaCl aqueous solution (4 × 100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 0%-70% EtOAc gradient in PE. Fractions containing the desired product were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-(4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)propionate. MS ESI calculation C 20 H 28 N3O6[M + H] + 406.19, actual measurement 406.10. 1 H NMR (400 MHz, DMSO- d 6) δ 12.91 (s, 1H), 7.73 -7.72 (m, 2H), 7.48-7.43 (m, 2H), 7.26 -7.24 (m, 1H), 4.07 -4.05 (m, 1H), 3.03 -3.01 (m, 1H),2.95 -2.89 (m, 1H), 1.33 (s, 9H), 1.32 (s, 9H).

[0199] Step 5: At room temperature under argon atmosphere, tert-butyl ( S 4 g (9.87 mmol) of 2-((tert-butoxycarbonyl)amino)-3-(4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)propionate was added to a mixture in DCM (40 mL) with TFA (40 mL, 519 mmol). The reaction was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give the crude product. Cp was calculated by MS ESI. 11 H 12 N3O4[M + H] + 250.07, actual measurement 249.95.

[0200] Step 6: At room temperature under argon atmosphere, towards ( S2-Amino-3-(4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)propionic acid (2.1 g, 8.43 mmol) was added to a mixture of THF (20 mL) and H2O (20 mL) with sodium bicarbonate (3.54 g, 42.1 mmol) and N -(9-fluorenylmethoxycarbonyloxy)succinimide (2.56 g, 7.58 mmol). The reaction was stirred at room temperature for 1 hour. The pH of the solution was adjusted to 4 with 1 N HCl. The aqueous phase was extracted with ethyl acetate (2 × 200 mL). The combined organic layers were washed with brine (3 × 50 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by RP flash under the following conditions: column: AQ 18 gel column (330 g), 20–35 μm; mobile phase A: 5 mMaq. TFA; mobile phase B: MeCN; (gradient: 0% B for 5 min, reaching 55.3% B within 35 min, 55.3% B for 3.2 min; reaching 95% B within 2 min, 95% B for 10 min); flow rate: 60 mL / min; detector: UV 254 & 210 nm; RT: 35.32 min. The fractions containing the desired product were combined and concentrated under reduced pressure to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)propionic acid. MS ESI calculation C 26 H 20 N3O6[M - H] + 470.14, actual measurement 470.15. 1 H NMR (400 MHz, DMSO- d 6) δ 12.91(s, 1H), 12.82 (s, 1H), 7.93 -7.87 (m, 2H), 7.78 -7.72 (m, 3H), 7.68 -7.62(m, 2H), 7.51 -7.30 (m, 4H), 7.28 -7.26 (m, 2H), 4.27 -4.15 (m, 4H), 3.20 -3.12 (m, 1H), 2.99 -2.92 (m, 1H).

[0201] Synthesis Scheme 25 SbMe1Nal3Cl (2S,3S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(3-chloronaphth-1-yl)butyric acid Step 1: To a mixture of tert-butyl 2-oxobutyrate (80.0 g, 506 mmol, 1.00 eq) and K₂CO₃ (210 g, 1520 mmol, 3.00 eq) in toluene (2.40 L), 1-bromo-3-chloronaphthalene and P(t-Bu)₃.HBF₄ (11.7 g, 40.5 mmol, 0.08 eq) were added, and the mixture was purged three times with N₂. Pd₂(dba)₃ (9.26 g, 10.1 mmol, 0.02 eq) was added, and the reaction mixture was purged three times with N₂ and stirred at 110 °C under N₂ for 3 hours. The reaction mixture was cooled to room temperature, poured into an aqueous solution of NH₄Cl (3.00 L), and extracted with EtOAc (2.00 L × 2). The organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1).

[0202] Step 2: At 0°C, TFA (1.00 L) was added dropwise to a mixture of tert-butyl 3-(3-chloronaphthyl-1-yl)-2-oxobutyrate (100 g, 314 mmol, 1.00 eq) in DCM (1.00 L), and the mixture was stirred at ambient temperature for 2 hours. The reaction mixture was concentrated and analyzed by prep-HPLC (column: Phenomenex luna C18 (250)). 70 mm, 15 μm); Mobile phase: [H2O(0.1% TFA)-ACN]; Gradient: 35%-65% over 20.0 min B) Purify crude product.

[0203] Step 3: At 20°C, add Na₂B₄O₇·10H₂O (28.8 g, 75.4 mmol, 0.30 eq) to a mixture of lysine (114 g, 779 mmol, 3.10 eq) in DMSO (270 mL) and Na₂B₄O₇ (0.1 M, 561 mL), followed by 3-(3-chloronaphthyl-1-yl)-2-oxobutyric acid (66.0 g, 251 mmol, 1.00 eq). At 35°C, add pyridoxal phosphate (2.44 g, 9.85 mmol, 0.05 eq) and transaminase Prozomix TAm-248 (35.0 g) to the reaction mixture. Stir the reaction mixture at 45°C for 12 hours. Cool the reaction mixture to 20°C, adjust the pH to 5.0 with 5 N HCl (60.0 mL), and stir at 20°C for 1 hour. The mixture was filtered, and the filter cake was washed with H2O (500 mL × 3) and dried under reduced pressure to give (2S,3S)-2-amino-3-(3-chloronaphth-1-yl)butyric acid, which was used in the next step without purification.

[0204] Step 4: Add DIPEA (39.7 g, 307 mmol, 1.50 eq) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (82.9 g, 246 mmol, 1.20 eq) to a mixture of (2S,3S)-2-amino-3-(3-chloronaphthyl-1-yl)butyric acid (54.0 g, 205 mmol, 1.00 eq) in EtOAc (540 mL), and stir the mixture for 12 hours. Filter off the precipitate and wash the filtrate with 2.0 L of EtOAc. Concentrate the filtrate to give the crude product, which is then subjected to prep-HPLC (column: Phenomenex luna C18 250). 150mm 15µm; Mobile phase: [H2O (0.04%HCl)-ACN]; Gradient: 50%-95% B) purification over 25 minutes. 1 H NMR: (400 MHz, DMSO- d 6 ) δ 12.8 (s, 1H), 8.22 (d, J = 8.2Hz, 1H), 7.84-8.01 (m, 5H), 7.53-7.72 (m, 4H), 7.31-7.47 (m, 3H), 7.18-7.30(m, 2H), 4.54 (t, J= 8.8 Hz, 1H), 4.21-4.32 (m, 1H), 3.95-4.19 (m, 3H), 3.82(s, 3H), 1.36 (d, J = 6.8 Hz, 3H).

[0205] SbMe1Nal3OMe (2S,3S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(3-methoxynaphth-1-yl)butyric acid The title compound was obtained from a suitable starting material using a method similar to that used in the synthesis of SbMe1Nal3Cl. 1 HNMR: (400 MHz, DMSO- d 6 ) δ 12.77 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.84 (t, J = 8.8Hz, 4H), 7.60 (d, J = 7.4 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.34-7.50 (m, 4H), 7.13-7.29 (m, 3H), 7.05 (s, 1H), 4.53 (t, J = 8.2 Hz, 1H), 4.18-4.32 (m, 1H), 4.07-4.17 (m, 2H), 3.93-4.01 (m, 1H), 3.82 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H).

[0206] SbMe1Nal3Me (2S,3S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(3-methylnaphth-1-yl)butyric acid The title compound was obtained from a suitable starting material using a method similar to that used in the synthesis of SbMe1Nal3Cl. 1 HNMR: (400 MHz, DMSO- d 6 ) δ 8.24 (d, J= 6.8 Hz, 1H),7.83-7.90 (m, 2H), 7.77-7.82(m, 1H), 7.57-7.64 (m, 1H), 7.50-7.56 (m, 2H), 7.34-7.49 (m, 5H), 7.18-7.30(m, 3H), 4.43 (t, J = 7.6 Hz, 1H), 4.22-4.31 (m, 1H), 4.08-4.17 (m, 2H), 3.95-4.04 (m, 1H), 2.40 (s, 3H), 1.32(d, J = 6.8 Hz, 3H).

[0207] SbMe1Nal8F (2S,3S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(8-fluoronaphth-1-yl)butyric acid The title compound was obtained from a suitable starting material using a method similar to that used in the synthesis of SbMe1Nal3Cl. 1 HNMR: (400 MHz, DMSO- d 6 ) δ 7.84 (d, J = 7.5 Hz, 3H), 7.78 (d, J = 8.1 Hz, 1H), 7.71(d, J = 7.0 Hz, 1H), 7.57 (d, J = 7.3 Hz, 1H), 7.43-7.53 (m, 4H), 7.33-7.41 (m,2H), 7.17-7.33 (m, 3H), 4.49 (s, 1H), 4.19-4.37 (m, 2H), 4.04-4.09 (m, 2H),1.32 (d, J = 6.6 Hz, 3H).

[0208] Synthesis Scheme 26 Phe43Oxad1245Me ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,2,4-oxadiazole-3- phenyl propionic acid Step 1: At room temperature, towards ( S2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionic acid (20.0 g, 68.9 mmol) was added to a stirred solution in DCM (200 mL) with tert-butyl ( E )-N,N'-diisopropylisourea (69.0 g, 344 mmol). The reaction mixture was stirred at 40 °C for 5 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera chromatography on a silica column and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate. MS ESI calculation of C 19 H 26 N2O4[MH] + 345.19, actual measurement 345.25.

[0209] Step 2: At room temperature, add sodium bicarbonate (34.6 g, 411 mmol) to a stirred solution of hydroxylamine chloride (21.0 g, 302 mmol) in DMSO (400 mL). Then, at 45-50°C, add tert-butyl ( S 2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate (19.0 g, 54.8 mmol) was added to the reaction mixture. The reaction mixture was stirred at 100 °C for 18 hours. The reaction mixture was cooled to 15-20 °C, quenched with water, and stirred at 15-20 °C for 15-20 minutes. The compound was filtered, washed with water and diethyl ether, and dried under vacuum to give tert-butyl( S , E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N '-Hydroxymethylamidine)phenyl)propionate. MS ESI calculation C 19 H 29 N3O5[M+H] + 380.21, actual measurement 380.29.

[0210] Step 3: Add tert-butyl ( S,E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N' A solution of 19.0 g (50.1 mmol) of hydroxymethylammonium (HMA)phenyl propionate in triethyl orthoacetate (190 mL) was stirred at 150 °C for 5 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera chromatography on a silica column and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl(HMA)phenyl propionate.S )-2-((tert-butoxycarbonyl)amino)-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)propionate. MS ESI calculation C 21 H 29 N3O5[M+H] + 402.21, actual measurement 402.29.

[0211] Step 4: At 0°C, add tert-butyl ( S 2-((tert-butoxycarbonyl)amino)-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)propionate (8.00 g, 19.8 mmol) was added to a stirred solution of TFA (80.0 mL, 1.04 mol) in DCM (80 mL). The reaction mixture was stirred at 24 °C for 8 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was ground with diethyl ether and dried under reduced pressure to give ( S )-2-amino-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)propionic acid. MS ESI calculation C 12 H 13 N3O3[M+H] + 248.10, actual measurement 248.07.

[0212] Step 5: At room temperature, towards ( S Sodium bicarbonate (7.64 g, 91.0 mmol) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidine-1-yl)carbonate (5.53 g, 16.4 mmol) were added to a stirred solution of 2-amino-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)propionic acid (4.50 g, 18.2 mmol) in THF (50 mL) and water (50 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was acidified with 1 N HCl (pH ~4) and extracted with ethyl acetate (2 × 300 mL). The combined organic layers were washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified by reversed-phase rapid chromatography (conditions: column: C18 gel column (100 g), mobile phase A: water; mobile phase B: acetonitrile), and eluted with a gradient of 45% to 100% acetonitrile in water. The pure fractions were combined and concentrated under reduced pressure to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)propionic acid. MS ESI calculation C 27 H 23 N3O5[M+H]+ 470.16, actual measurement 470.25. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm)12.85 (br s, 1H), 7.88 (t, J = 8.0 Hz, 4H), 7.50-7.76 (m, 3H), 7.35-7.47 (m,4H), 7.24-7.33 (m, 2H), 4.10-4.30 (m, 4H), 3.16 (dd, J = 13.8 Hz, 4.2 Hz, 1H), 2.90-3.00 (m, 1H), 2.66 (s, 3H).

[0213] Synthesis Scheme 27 Phe43Oxad1245MeOEt ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,2,4-oxadiazole-3- phenyl propionic acid Step 1: At room temperature, towards ( S 2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionic acid (20.0 g, 68.9 mmol) was added to a stirred solution in DCM (200 mL) with tert-butyl ( E )-N,N'-diisopropylisourea (69.0 g, 344 mmol). The reaction mixture was stirred at 40 °C for 5 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera chromatography on a silica column and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate. MS ESI calculation of C 19 H 26 N2O4[MH] + 345.19, actual measurement 345.25.

[0214] Step 2: At room temperature, add sodium bicarbonate (34.6 g, 411 mmol) to a stirred solution of hydroxylamine chloride (21.0 g, 302 mmol) in DMSO (400 mL). Then, at 45-50°C, add tert-butyl... S19.0 g (54.8 mmol) of 2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propionate was added to the reaction mixture. The reaction mixture was stirred at 100 °C for 18 hours. The reaction mixture was cooled to 15-20 °C, quenched with water, and stirred at 15-20 °C for 15-20 minutes. The compound was filtered, washed with water and diethyl ether, and dried under vacuum to give tert-butyl( S , E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N '-Hydroxymethylamidine)phenyl)propionate. MS ESI calculation C 19 H 29 N3O5[M+H] + 380.21, actual measurement 380.29.

[0215] Step 3: To tert-butyl ( S , E )-2-((tert-butoxycarbonyl)amino)-3-(4-( N '-Hydroxymethylammonium' (HMA)phenyl)propionate (4.0 g, 10.5 mmol) was added to a solution of toluene (100 mL) with triethylamine (3.20 g, 4.41 mL, 31.6 mmol), followed by dropwise addition of 2-ethoxyacetyl chloride (1.55 g, 1.39 mL, 12.6 mmol). The mixture was stirred at 120 °C for 18 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera chromatography on a silica column, eluting with 20% ethyl acetate in hexane. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S 2-((tert-butoxycarbonyl)amino)-3-(4-(5-(ethoxymethyl)-1,2,4-oxadiazol-3-yl)phenyl)propionate. LC-MS measured C2. 23 H 33 N3O6: m / z 448.3 [M + H] + (1.40 min).

[0216] Step 4: At 0°C, add tert-butyl ( S 2-((tert-butoxycarbonyl)amino)-3-(4-(5-(ethoxymethyl)-1,2,4-oxadiazol-3-yl)phenyl)propionate (3.10 g, 6.93 mmol) was added to a stirred solution in DCM (50 mL) with TFA (7.90 g, 5.34 mL, 69.3 mmol). The reaction mixture was stirred at 24 °C for 24 hours. The reaction mixture was concentrated under reduced pressure to give ( S2-Amino-3-(4-(5-(ethoxymethyl)-1,2,4-oxadiazol-3-yl)phenyl)propionic acid. LC-MS measured C1. 14 H 17 N3O4: m / z 292.0 [M + H] + (0.34 min).

[0217] Step 5: At room temperature, towards ( S 2-Amino-3-(4-(5-(ethoxymethyl)-1,2,4-oxadiazol-3-yl)phenyl)propionic acid (2.50 g, 8.58 mmol) was added to a stirred solution of sodium bicarbonate (3.60 g, 42.9 mmol) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidine-1-yl) carbonate (2.89 g, 8.58 mmol) in THF (50 mL) and water (50 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was acidified with 1N HCl (pH ~2) and extracted with ethyl acetate (2 × 200 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by Teledyne Isco (80 g silica gel, eluted with 0 to 100% ethyl acetate in hexane). The purified fractions were combined and concentrated under reduced pressure to give ( S )-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-(ethoxymethyl)-1,2,4-oxadiazol-3-yl)phenyl)propionic acid. LC-MS measured C 29 H 27 N3O6: m / z 514.3 [M + H] + (1.31 min).

[0218] Synthesis Scheme 28 Phe42Oxad1345Me (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,3,4-oxadiazole-2- phenylpropionic acid Step 1: At 80 °C, POCl3 (14.3 mL, 153 mmol) was added dropwise to a stirred solution of 4-bromobenzoylhydrazine (30.0 g, 140 mmol) in DMA (150 mL). The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was diluted with ice water (1000 mL), quenched with Na2CO3 solution, and extracted with DCM (1000 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage column chromatography using silica gel, and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give 2-(4-bromophenyl)-5-methyl-1,3,4-oxadiazole. MS ESI calculation of C9H7BrN2O [M+H] + 238.97, actual measurement 238.95.

[0219] Step 2: At 50°C under argon atmosphere, a solution of nickel chloride, dimethoxyethane adduct (457 mg, 2.08 mmol), and picolinimidamide hydrochloride (656 mg, 4.16 mmol) in DMA (40 mL) was stirred for 40 minutes. Then, at room temperature, TBAI (3.84 g, 10.4 mmol), (… R )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-bromopropionate (5.00 g, 10.4 mmol), 2-(4-bromophenyl)-5-methyl-1,3,4-oxadiazole (2.49 g, 10.4 mmol) and zinc (1.36 g, 20.8 mmol) were added to the reaction mixture. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was quenched with ice-cold water (150 mL) and extracted with EtOAc (3 × 70 mL). The combined organic layers were washed with ice-cold water (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage column chromatography using silica gel, and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give benzyl( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)propionate. MS ESI calculation C 34 H 29 N3O5[M+H] + 560.21, actual measurement 560.33.

[0220] Step 3: At room temperature, apply the compound to the benzyl group ( S )-2-((((9 H 3-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)propionate (5.00 g, 8.93 mmol) was added to a stirred solution of Pd / C (2.35 g, 625 μmol) in THF (75 mL). The reaction mixture was stirred at 25 °C under a hydrogen atmosphere for 16 h. The reaction mixture was filtered through a CELITE bed filter. The filtrate was concentrated under reduced pressure. The crude compound was stirred in DCM (10 mL) and diethyl ether was added. The precipitated solid was filtered and dried under reduced pressure to give ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)propionic acid. MS ESI calculation C 27 H 23 N3O5[M+H] + 470.16, actual measurement 470.22. 1 HNMR (400 MHz, DMSO- d 6 ): δ (ppm) 12.82 (br s, 1H), 7.83-7.94 (m, 4H), 7.77 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 6.4 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.39 (td, J =7.4 Hz, 2.8 Hz, 2H), 7.23-7.35 (m, 2H), 4.07-4.36 (m, 4H), 3.18 (dd, J = 14.0Hz, 4.4Hz, 1H), 2.83-3.02 (m, 1H), 2.57 (s, 3H).

[0221] Synthesis Scheme 29 Ala2Oxad1345Me (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-methyl-1,3,4-oxadiazol-2-yl)propane acid Step 1: At -15℃, towards (S )-4-(benzyloxy)-3-(((benzyloxy)carbonyl)amino)-4-oxobutyric acid (35.0 g, 97.9 mmol) was added to a stirred solution of THF (1 L). N 1-Methylmorpholine (10.8 mL, 97.9 mmol) and ethyl chloroformate (9.41 mL, 97.9 mmol). The reaction mixture was stirred at -15 °C for 15 min. Then, at -15 °C, a solution of acetylhydrazine (7.26 g, 97.9 mmol) in THF (200 mL) was added dropwise to the reaction mixture. The reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was quenched with ice water (500 mL) and extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give ( S , Z )- N -Acetyl-4-(benzyloxy)-3-(((benzyloxy)carbonyl)amino)-4-oxobutanehydrazone. MS ESI calculation C 21 H 23 N3O6[M+H] + 414.16, actual measurement 414.14.

[0222] Step 2: At 0℃, towards ( S , Z )- N Acetyl-4-(benzyloxy)-3-(((benzyloxy)carbonyl)amino)-4-oxobutanehydrazone (10.00 g, 21.77 mmol) was added to a stirred solution in toluene (50 mL) with Burgess reagent (7.781 g, 32.65 mmol). The reaction mixture was stirred at 80 °C for 16 h. The reaction mixture was quenched with cold water (100 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using a 120 g silica cartridge and eluted with 100% EtOAc. The purified fractions were combined and concentrated under reduced pressure to give benzyl( S )-2-(((benzyloxy)carbonyl)amino)-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionate. MS ESI calculation of C 21 H 21 N3O5[M+H] + 396.15, actual measurement 396.26.

[0223] Step 3: At room temperature, apply the compound to the benzyl group ( S10% Pearlman's catalyst (5.00 g, 4.70 mmol) was added to a stirred solution of 11.0 g (25.0 mmol) of 2-((benzyloxy)carbonyl)amino)-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionate in MeOH (250 mL). The mixture was stirred at room temperature for 10 min. The reaction mixture was purged with N2 gas at room temperature for 5 min. The reaction mixture was stirred at 25 °C under an H2 balloon for 16 h. The reaction mixture was filtered through a CELITE filter and washed with MeOH (50 mL) and DCM (100 mL). The organic layer was concentrated under reduced pressure. The crude compound was washed with n-pentane (3 × 50 mL) and dried under reduced pressure to give ( S )-2-amino-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionic acid. 1 H NMR (400 MHz, DMSO-) d 6 ): δ (ppm) 7.50-8.89 (m, 2H), 3.64 (d, J = 6.8 Hz, 1H), 3.56 (dd, J = 9.0 Hz, 4.6 Hz, 1H), 3.37-3.42 (m, 1H), 2.45 (s, 3H).

[0224] Step 4: At 0℃, towards ( S 2-Amino-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionic acid (4.20 g, 22.1 mmol) was added to a stirred solution of 2-amino-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionic acid in THF (35 mL) and H₂O (35 mL) with sodium bicarbonate (2.84 g, 33.1 mmol) and (9... H -fluorene-9-yl)methyl(2,5-dioxopyrrolidone-1-yl)carbonate (11.4 g, 33.1 mmol). The reaction mixture was stirred at 25 °C for 16 h, then diluted with EtOAc (100 mL) and acidified with 1 M HCl (pH ~2-3). The aqueous layer was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage using an 80 g silica cylinder and eluted with 100% EtOAc. The purified fractions were combined, concentrated under reduced pressure, and the separated compound was washed with n-pentane (2 × 100 mL) and then ground with ether and pentane (90:10). A few drops of EtOAc were added to the reaction mixture (three times), and the solid was filtered. The solid was washed with ether and dried under reduced pressure to give ( S)-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(5-methyl-1,3,4-oxadiazol-2-yl)propionic acid. MS ESI calculation C 21 H 19 N3O5[M+H] + 394.13, actual measurement 394.22. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 13.10 (br s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.58-7.82 (m,3H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (td, J = 7.4 Hz, 0.9 Hz, 2H), 4.33-4.45 (m,1H), 4.17-4.32 (m, 3H), 3.27-3.30 (m, 1H), 3.13-3.22 (m, 1H), 2.43 (s, 3H).

[0225] Synthesis Scheme 30 TyrOCOPyrr (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((pyrrolidine-1-carbonyl)oxy)benzene (B)propionic acid Step 1: At room temperature, DMAP (7.241 g, 59.27 mmol), TEA (16.5 mL, 118.5 mmol), and pyrrolidine-1-carbonyl chloride (8.710 g, 65.20 mmol) were added to a stirred solution of tert-butyl(tert-butoxycarbonyl)-L-tyrosine ester (20.00 g, 59.27 mmol) in DCM (100 mL). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated under reduced pressure. The crude compound was diluted with MeOH (100 mL) and stirred at room temperature for 30 minutes. The precipitated solid was filtered, washed with MeOH (2 × 20 mL), and dried under vacuum to give ( S )-4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenylpyrrolidine-1-carboxylic acid ester. MS ESI calculation of C 23 H 34 N₂O₆[M+H] + 435.24, actual measurement 435.31.

[0226] Step 2: At room temperature, towards ( S 12.00 g (27.62 mmol) of 4-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)phenylpyrrolidine-1-carboxylic acid ester was added to a stirred solution in DCM (60 mL) with TFA (63.83 mL, 828.5 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated under reduced pressure, co-distilled with toluene (50 mL), and dried under vacuum to give ( S 2-Amino-3-(4-((pyrrolidine-1-carbonyl)oxy)phenyl)propionic acid. MS ESI calculation of C 14 H 18 N₂O₄[M+H] + 279.13, actual measurement 279.20.

[0227] Step 3: At room temperature, towards ( S 2-Amino-3-(4-((pyrrolidine-1-carbonyl)oxy)phenyl)propionic acid (8.00 g, 28.7 mmol) was added to a stirred solution of acetone (50 mL) and water (50 mL) with sodium bicarbonate (7.24 g, 86.2 mmol) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidine-1-yl) carbonate (10.7 g, 31.6 mmol). The reaction mixture was stirred at 25 °C for 5 hours. The reaction mixture was concentrated under reduced pressure. The aqueous layer was washed with diethyl ether (2 × 100 mL), acidified with 2N HCl (pH ~4), and extracted with 10% MeOH in DCM (2 × 200 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was diluted with 10% diethyl ether in hexane (100 mL) and stirred at 25 °C for 30 minutes. The mixture was filtered and dried under reduced pressure to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-((pyrrolidine-1-carbonyl)oxy)phenyl)propionic acid. MS ESI calculation C 29 H 28 N₂O₆[M+H] + 501.19, actual measurement 501.29. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 7.88 (d, J = 7.6 Hz, 2H), 7.56-7.72(m, 2H), 7.41 (t,J = 7.6 Hz, 2H), 7.27-7.36 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.4 Hz, 3H), 3.71-4.39 (m, 4H), 3.44-3.50 (m, 2H), 3.35-3.37 (m,3H), 3.05 (dd, J = 13.4 Hz, 4.6 Hz, 1H), 2.84-2.97 (m, 1H), 1.79-1.96 (m, 4H).

[0228] Synthesis Scheme 31 Ala3Oxad1245Me (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-methyl-1,2,4-oxadiazol-3-yl)propane acid Step 1: At room temperature, towards ( S 2-((tert-butoxycarbonyl)amino)-3-cyanopropionic acid (24.00 g, 112.0 mmol) was added to a stirred solution in DCM (100 mL) with tert-butyl ( E )- N,N' -Diisopropylisourea (80 mL, 336.1 mmol). A white precipitate formed during the reaction. The reaction mixture was stirred at 45 °C for 5 hours. The reaction mixture was filtered and washed with DCM (2 × 50 mL). The filtrate was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera using a 120 g silica cartridge and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-cyanopropionate. MS ESI calculation of C 13 H 22 N₂O₄[M+H] + 271.16, actual measurement 271.20.

[0229] Step 2: At room temperature, add tert-butyl ( S2-((tert-butoxycarbonyl)amino)-3-cyanopropionate (20.0 g, 74.0 mmol) was added to a stirred solution of sodium bicarbonate (46.6 g, 555 mmol) and hydroxylamine chloride (28.3 g, 407 mmol) in DMSO (150 mL). The reaction mixture was stirred at 100 °C for 16 hours. The reaction mixture was cooled to room temperature, quenched with water (200 mL), and extracted with EtOAc (2 × 300 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-4-(hydroxyamino)-4-iminobutyrate. MS ESI calculation of C 13 H 25 N3O5[M+H] + 304.18, actual measurement 304.22.

[0230] Step 3: At room temperature, add tert-butyl ( S 2-((tert-Butoxycarbonyl)amino)-4-(hydroxyamino)-4-iminobutyrate (9.50 g, 31.3 mmol) was added to a stirred solution of cesium carbonate (20.4 g, 62.6 mmol) and acetyl chloride (2.45 mL, 34.4 mmol) in ACN (100 mL). The reaction mixture was stirred at 25 °C for 3 h. The reaction was monitored by TLC (which showed new spots) and also by LCMS (which showed 93% of the intermediate). The reaction mixture was stirred under reflux for 3 h. The reaction mixture was filtered through a CELITE pad and washed with ACN (2 × 50 mL). The filtrate was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera column chromatography using a silica column and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-(5-methyl-1,2,4-oxadiazol-3-yl)propionate. MS ESI calculation of C 15 H 25 N3O5[M+H] + 328.18, actual measurement 328.25.

[0231] Step 4: At room temperature, add tert-butyl ( S5.80 g (17.7 mmol) of 2-((tert-butoxycarbonyl)amino)-3-(5-methyl-1,2,4-oxadiazol-3-yl)propionate was added to a stirred solution of TFA (20.5 mL, 266 mmol) in DCM (20 mL). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated under reduced pressure, co-distilled with toluene (10 mL), and dried under reduced pressure to give ( S 2-Amino-3-(5-methyl-1,2,4-oxadiazol-3-yl)propionic acid. MS ESI calculation of C6H9N3O3[M+H] + 172.06, actual measurement 171.99.

[0232] Step 5: At room temperature, towards ( S 4.20 g (24.5 mmol) of 2-amino-3-(5-methyl-1,2,4-oxadiazol-3-yl)propionic acid was added to a stirred solution of acetone (20 mL) and H₂O (20 mL) with sodium bicarbonate (4.12 g (49.1 mmol) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (9.11 g (27.0 mmol)). The reaction mixture was stirred at 25 °C for 5 hours. The reaction mixture was concentrated under reduced pressure, subjected to acid-base treatment, and extracted with 10% MeOH (2 × 200 mL) in DCM. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude compound was purified twice using a Biotage-isolera filter in a 120 g silica cylinder, eluting with 10% MeOH in DCM. The purified fractions were combined and concentrated under reduced pressure. The isolated compound was washed with 10% diethyl ether in hexane and dried under reduced pressure to give ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(5-methyl-1,2,4-oxadiazol-3-yl)propionic acid. MS ESI calculation C 21 H 19 N3O5[M+H] + 394.13, actual measurement 394.19. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm)12.95 (br s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.68 (d, J =7.6 Hz, 2H), 7.42 (t,J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 4.33-4.64 (m,1H), 4.10-4.33 (m, 3H), 2.97-3.21 (m, 2H), 2.55 (s, 3H).

[0233] Synthesis Scheme 32 Phe4AcPip (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(4-acetylpiperazin-1-yl)phenyl) propionic acid Step 1: At 25°C under nitrogen atmosphere, towards ( S XPhos Pd G2 (2.057 g, 2.61 mmol) was added to a stirred solution of 3-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)propionic acid (6 g, 17.43 mmol) in toluene (180 mL). The resulting solution was stirred at 100 °C for 10 min. 1-(piperazin-1-yl)ethyl-1-one (2.234 g, 17.43 mmol) and Cs2CO3 (5.04 g, 26.1 mmol) were added, and the resulting solution was stirred at 110 °C for 2 h. The reaction was cooled to room temperature, quenched with H2O (500 mL), and extracted with EtOAc (2 × 500 mL). The combined organic layers were washed with brine (3 × 200 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with 0-60% EtOAc in PE to obtain ( S )-3-(4-(4-acetylpiperazin-1-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionic acid. MS ESI calculation C 20 H 30 N3O5[M + H] + 392.21, actual measurement 392.25. 1 H NMR (400 MHz, methanol-) d4 )δ 7.12 (d, J = 8 Hz, 2H), 6.89 (d, J= 8 Hz, 2H), 4.16 -4.13 (m, 1H), 3.72 -3.65 (m, 4H), 3.14 -3.04 (m, 4H), 2.93 -2.81 (m, 2H), 2.13 (s, 3H), 1.38 -1.29 (m,9H).

[0234] Step 2: At room temperature, towards ( S 10 g (25.5 mmol) of (S)-3-(4-(4-acetylpiperazin-1-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionic acid was added to a stirred solution of (S)-3-(4-(4-acetylpiperazin-1-yl)phenyl)-2-aminopropionic acid in a DCM (30 mL) solution with TFA (30 mL) added. The solution was stirred at 25 °C for 1 hour and then concentrated under reduced pressure. The crude (S)-3-(4-(4-acetylpiperazin-1-yl)phenyl)-2-aminopropionic acid was used directly in the next step without any further purification. MS ESI calculations C 15 H 22 N3O3[M + H] + 292.16, actual measurement 292.20.

[0235] Step 3: At 25°C under nitrogen atmosphere, towards ( S 3-(4-(4-acetylpiperazin-1-yl)phenyl)-2-(carboxyamino)propionic acid (7 g, 20.87 mmol) was added to a stirred solution of THF (25 mL) and water (25 mL) with Fmoc-OSu (6.34 g, 18.79 mmol). Then, NaHCO3 (8.77 g, 104 mmol) was added. The resulting mixture was stirred at 25 °C for 2 hours. The pH was adjusted to 5 with 1N HCl and the mixture was extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography under the following conditions: column: C18 silica gel (330 g); mobile phase A: water (0.1% TFA), mobile phase B: MeCN; (gradient: 5% B for 5 min, reaching 55% B within 15 min, 55% B for 6 min; reaching 95% B within 20 min, 95% B for 5 min); flow rate: 90 mL / min; detector: UV 210 nm; RT = 45 min. The fraction containing the product was collected and concentrated under vacuum to obtain ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(4-acetylpiperazin-1-yl)phenyl)propionic acid. MS ESI calculation C 30 H 32N3O5[M + H] + 514.23, actual measurement 514.30. 1 H NMR (400 MHz, methanol-) d4 ) δ 7.78 -7.76 (m, 2H), 7.61 -7.52 (m, 2H), 7.40 -7.37 (m, 2H), 7.32 -7.22 (m, 4H), 7.08 -6.98 (m, 2H), 4.47 -4.43 (m, 1H), 4.33 -4.31 (m, 1H), 4.14 -4.02 (m,2H), 3.68 -3.63 (m, 4H), 3.23 -3.08 (m, 5H), 2.91 -2.85 (m, 1H), 2.11 (s,3H).

[0236] Synthesis Scheme 33 3AzaPhe4AcPip (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-(4-acetylpiperazin-1-yl)pyridine- 3-yl)propionic acid Step 1: Under argon atmosphere at room temperature, 5-bromo-2-fluoropyridine (15 g, 85 mmol) was added to a mixture of 1-(piperazin-1-yl)ethyl-1-one (21.85 g, 170 mmol) in DMF (150 mL). The reaction was stirred at 100 °C for 2 hours, then diluted with 300 mL of EtOAc, washed with H2O (3 × 80 mL), saturated NaCl aqueous solution (80 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a 0%–100% EtOAc gradient in PE. Fractions containing the desired product were combined and concentrated under reduced pressure to give 1-(4-(5-bromopyridin-2-yl)piperazin-1-yl)ethyl-1-one. MS ESI calculation of C11H15BrN3O [M + H] + 284.03 and 286.03, actual measured values ​​are 283.90 and 285.90.

[0237] Step 2: At 50 °C, a mixture of nickel(II) ethylene glycol dimethyl ether complex (0.696 g, 3.17 mmol) and 1,10-phenanthroline (0.571 g, 3.17 mmol) in DMA (2 mL) was heated for 0.5 h. At 25 °C, a mixture of 1-(4-(5-bromopyridin-2-yl)piperazin-1-yl)ethyl-1-one (4.5 g, 15.84 mmol), tert-butyl(R)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-bromopropionate (7.78 g, 17.42 mmol), and tetrabutylammonium iodide (5.85 g, 15.84 mmol) in DMA (2 mL) was added. Then, zinc powder (2.071 g, 31.7 mmol) was added. The resulting mixture was stirred at 50 °C for 1 h. The resulting mixture was poured into water (300 mL) and extracted with EtOAc (3 × 300 mL). The organic layer was washed with water (100 mL) and brine (2 × 80 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with DCM-MeOH (10:1) to give tert-butyl(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-(4-acetylpiperazin-1-yl)pyridin-3-yl)propionate. MS ESI calculation of C33H39N4O5[M + H] + 571.28, actual measurement 571.40.

[0238] Step 3: Under argon atmosphere at room temperature, TFA (70 mL, 909 mmol) was added to a mixture of tert-butyl(S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-(4-acetylpiperazin-1-yl)pyridin-3-yl)propionate (7.3 g, 12.79 mmol) in DCM (70 mL). The reaction was stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The residue was dissolved in THF (20 mL) and the resulting mixture was purified by reverse-phase Combi-Flash under the following conditions: C18 silica gel column (330 g), 20–35 μm; mobile phase A: 5 mM TFA aqueous solution; mobile phase B: MeCN; (gradient: 0% B for 10 min, reaching 42.3% B within 35 min, 42.3% B for 3.2 min; reaching 95% B within 2 min, 95% B for 10 min); flow rate: 60 mL / min; detector: UV 254 & 210 nm; RT: 35.32 min. The fraction containing the product was collected and concentrated under vacuum to give (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(6-(4-acetylpiperazin-1-yl)pyridin-3-yl)propionic acid. MS ESI calculation of C29H31N4O5 [M + H] + 515.22, actual measurement 515.15. 1 H NMR (300 MHz, methanol-) d4 ) δ 7.87 -7.79 (m, 4H), 7.62 -7.55 (m, 2H), 7.42 -7.27 (m, 4H), 7.11 -7.09 (m, 1H), 4.51 -4.46 (m, 1H), 4.32 -4.09 (m, 3H), 3.65 -3.54 (m, 8H), 3.29 -3.20 (m, 1H), 2.94 -2.89 (m, 1H), 2.12 (s, 3H).

[0239] Synthesis scheme 34 KPEG2acidNMe3 ( S )-5-carboxy-1-(9 H -fluorene-9-base)- N , N , N -trimethyl-3,11-dioxo-2,14,17-trioxa-4, 10-diazanonadecano-19-ammonium 2,2,2-trifluoroacetate Step 1: At room temperature, Me₂NH (5.49 g, 67.3 mmol) was added to a solution of tert-butyl 3-(2-(2-bromoethoxy)ethoxy)propionate (10 g, 33.6 mmol) in ACN (100 mL). The reaction mixture was stirred at 90 °C for 16 hours. The resulting solution was diluted with water (200 mL) and extracted with EA (3 × 300 mL). The combined organic layers were washed with brine (3 × 300 mL), dried over anhydrous Na₂SO₄, and filtered. The residue was purified by silica gel column chromatography, eluting with a 0–10% MeOH gradient in DCM to give tert-butyl 3-(2-(2-(dimethylamino)ethoxy)ethoxy)propionate. C2 was calculated by MS ESI. 13 H 28 NO4[M + H] + 262.20, actual measurement 262.15.

[0240] Step 2: A solution of tert-butyl 3-(2-(2-(dimethylamino)ethoxy)ethoxy)propionate (8 g, 30.6 mmol) in DCM (100 mL) and TFA (100 mL) was prepared at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The resulting solution was concentrated under reduced pressure to give 3-(2-(2-(dimethylamino)ethoxy)ethoxy)propionic acid. C9H was calculated by MS ESI. 20 NO4[M + H] + 206.13, actual measurement 206.10.

[0241] Step 3: At -40°C, add HATU (24.08 g, 63.3 mmol), DIEA (6.08 ml, 34.8 mmol), and tert-butyl (9...) to a stirred solution of 3-(2-(2-(dimethylamino)ethoxy)ethoxy)propionic acid (6.5 g, 31.7 mmol) in DMF (100 mL). H -fluorene-9-yl)methoxy)carbonyl)- L -Lysine ester (13.44 g, 31.7 mmol). After stirring the resulting mixture at -40 °C for 4 hours, it was quenched with water (100 mL) and extracted with EA (3 × 500 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with a gradient of 0%–50% EA in PE to give tert-butyl( S )-17-((((9 H-fluorene-9-yl)methoxy)carbonyl)amino)-2-methyl-11-oxo-5,8-dioxa-2,12-diazaoctadecane-18-ester. MSESI calculation of C 34 H 50 N3O7[M + H] + 612.36, actual measurement 612.45. 1 H NMR (300 MHz, chloroform-) d ) δ 7.79 -7.76(m, 2H), 7.63 -7.61 (m, 2H), 7.44 -7.41 (m, 2H), 7.35 -7.30 (m, 2H), 6.92 (s,1H), 5.56 -5.53 (m, 1H), 4.26 -4.21 (m, 2H), 3.87 -3.85 (m, 2H), 3.73 -3.71(m, 2H), 3.62 -3.53 (m, 5H), 3.30 -3.20 (m, 4H), 2.88 (s, 6H), 2.50 -2.48 (m,2H), 1.84 -1.82 (m, 1H), 1.70 -1.68 (m, 1H), 1.57 -1.40 (m, 13H).

[0242] Step 4: At room temperature, add tert-butyl ( S )-17-((((9 H 1 g (1.635 mmol) of fluorene-9-yl)methoxy)carbonyl)amino)-2-methyl-11-oxo-5,8-dioxa-2,12-diazaoctadecane-18-ester (1 g, 1.635 mmol) was added to a solution in ACN (50 mL) with NaHCO3 (0.137 g, 1.635 mmol) and MeI (0.102 mL, 1.635 mmol). The reaction mixture was stirred at room temperature for 5 hours. The resulting solution was diluted with water (100 mL) and extracted with EtOAc (3 × 300 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhydrous Na2SO4, and filtered. The residue was concentrated under reduced pressure to give ( S )-5-carboxy-1-(9 H -fluorene-9-base)- N , N , N -Trimethyl-3,11-dioxo-2,14,17-trioxa-4,10-diazanonadecan-19-ammonium iodide. MS ESI calculation C 35 H 52 N3O7[M- t Bu]+ 570.32, actual measurement 570.30.

[0243] Step 5: At room temperature, towards ( S )-5-carboxy-1-(9 H -fluorene-9-base)- N , N , N Trimethyl-3,11-dioxo-2,14,17-trioxa-4,10-diazanonadecan-19-ammonium iodide (7 g, 10.03 mmol) was added to a stirred solution of TFA (100 mL) in DCM (100 mL). The reaction mixture was stirred at room temperature for 1 hour. The filtrate was concentrated under reduced pressure, and the residue was purified by RPFlash under the following conditions: (Column: Flash C) 18 330 g; Mobile phase A: water (0.1% TFA); Mobile phase B: ACN; Flow rate: 100 mL / min; 10 min 2% B; 5 min 2% to 20%; 20 min 20% to 50% B; Detector: UV 210 nm. RT: 35 min), to obtain ( S )-5-carboxy-1-(9 H -fluorene-9-base)- N , N , N -Trimethyl-3,11-dioxo-2,14,17-trioxa-4,10-diazanonadecan-19-ammonium 2,2,2-trifluoroacetate. MS ESI calculation C 31 H 44 N3O7 + [M] + 570.32, actual measurement 570.30. 1 H NMR (300 MHz, CD3OD) δ 7.83 -7.80 (m, 2H), 7.71 -7.67 (m, 2H), 7.43-7.30 (m, 4H), 4.44 -4.32 (m, 2H), 4.27 -4.12 (m, 2H), 3.92 -3.89 (m, 2H), 3.74 -3.70 (m, 2H), 3.62 -3.53 (m, 6H), 3.26 -3.17 (m, 11H), 2.44 -2.40 (m,2H), 1.89 -1.70 (m, 2H), 1.59 -1.39 (m, 4H).

[0244] Synthesis Scheme 35 Ala3Oxad1245MeOMe (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(5-(methoxymethyl)-1,2,4-oxadi (Zyr-3-yl)propionic acid Step 1: At room temperature, a solution of DCC (88.84 g, 430.6 mmol) in acetone (500 mL) was added dropwise to a stirred solution of (tert-butoxycarbonyl)-L-asparagine (100.0 g, 430.6 mmol) in pyridine (250 mL). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was filtered and washed with acetone (2 × 100 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and washed with 2N HCl (300 mL) and water (200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by acid-base treatment (using NaHCO3 and 2N HCl) to give ( S )-2-((tert-butoxycarbonyl)amino)-3-cyanopropionic acid. MS ESI calculation of C9H 14 N₂O₄[M+H] + 215.10, actual measurement 215.11.

[0245] Step 2: At room temperature, towards ( S 2-((tert-butoxycarbonyl)amino)-3-cyanopropionic acid (37.00 g, 172.7 mmol) was added to a stirred solution in DCM (200 mL) with tert-butyl ( E )- N , N '-Diisopropylisourea (34.60 g, 172.7 mmol). A white precipitate formed during the reaction. The reaction mixture was stirred at 45 °C for 5 hours. The reaction mixture was filtered and washed with DCM (2 × 100 mL). The filtrate was concentrated under reduced pressure. The crude compound was purified by Biotage-isolera using a 120 g silica cartridge and eluted with 30% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl( S )-2-((tert-butoxycarbonyl)amino)-3-cyanopropionate. MS ESI calculation of C 13 H 22 N₂O₄[M+H] + 271.16, actual measurement 271.16.

[0246] Step 3: At room temperature, add tert-butyl ( S45.0 g (166 mmol) of tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-3-cyanopropionate was added to a stirred solution of sodium bicarbonate (105 g, 1.25 mol) and hydroxylamine chloride (63.6 g, 916 mmol) in DMSO (200 mL). The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was cooled to 25 °C, quenched with water (200 mL), and extracted with EtOAc (2 × 500 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-4-(hydroxyamino)-4-iminobutyrate. MSESI calculation of C 13 H 25 N3O5[M+H] + 304.18, actual measurement 304.30.

[0247] Step 4: At room temperature, cesium carbonate (40.81 g, 125.3 mmol) and 2-methoxyacetyl chloride (8.156 g, 75.16 mmol) were added to a stirred solution of tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-4-(hydroxyamino)-4-iminobutyrate (19.00 g, 62.63 mmol) in ACN (150 mL). The reaction mixture was stirred at 25 °C for 3 hours. Then, the reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 × 150 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by Biotage-isolera using a 120 g silica cartridge and eluted with 40% ethyl acetate in petroleum ether. The purified fractions were combined and concentrated under reduced pressure to give tert-butyl(S)-2-((tert-butoxycarbonyl)amino)-4-(hydroxyamino)-4-iminobutyrate. S )-2-((tert-butoxycarbonyl)amino)-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionate. MS ESI calculation of C 16 H 27 N3O6[M+H] + 358.19, actual measurement 358.41.

[0248] Step 5: At room temperature, add tert-butyl ( S11.0 g (30.8 mmol) of 2-((tert-butoxycarbonyl)amino)-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionate was added to a stirred solution in DCM (60 mL) with TFA (47.4 mL, 616 mmol). The reaction mixture was stirred at 25 °C for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was co-distilled with toluene (20 mL) and dried under reduced pressure to give ( S 2-Amino-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionic acid. C7H was calculated using MS ESI. 11 N3O4[M+H] + 202.07, actual measurement 202.17.

[0249] Step 6: At room temperature, towards ( S 2-Amino-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionic acid (6.20 g, 30.8 mmol) was added to a stirred solution of acetone (30 mL) and H₂O (30 mL) with sodium bicarbonate (5.18 g, 61.6 mmol) and (9H-fluorene-9-yl)methyl(2,5-dioxopyrrolidine-1-yl) carbonate (11.4 g, 33.9 mmol). The reaction mixture was stirred at 25 °C for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was subjected to acid-base treatment and extracted with 10% MeOH (2 × 200 mL) in DCM. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified twice using a Biotage-isolera filter in a 120 g silica cartridge, eluting with 10% MeOH in DCM. The purified fractions were combined and concentrated under reduced pressure. The isolated compound was washed with 10% diethyl ether in hexane and dried under reduced pressure to obtain the compound.

[0250] The compound was further purified by chiral SFC in a Chiralpak-AS-H column at 3°C. 25 cm, 5 μm, with concentrations of 80% CO2 and 20% (0.5% formic acid in IPA), flow rate 90 g / min, temperature 30.0 °C, back pressure 120.0 bar, injection dose 75.0 mg, stacking time 15.0 min.

[0251] The pure fractions were combined, concentrated under reduced pressure, and freeze-dried separately to obtain peak 1 ( R )-2-((((9 H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionic acid (MS ESI calculation C 22 H 21 N3O6[M+H] + 424.14, actual measurement 424.22. 1 H NMR (400 MHz, DMSO- d 6 ) VT, at 90℃: δ (ppm) 12.91 (br s,1H), 7.87 (d, J = 7.6 Hz, 2H), 7.50-7.71 (m, 3H), 7.41 (t, J = 7.4 Hz, 2H), 7.31(td, J = 7.4 Hz, 1.0 Hz, 2H), 4.69 (s, 2H), 4.32-4.50 (m, 1H), 4.15-4.30 (m, 3H), 3.35 (s, 3H), 3.04-3.17 (m, 2H)) and peak 2 ( S )-2-((((9 H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(5-(methoxymethyl)-1,2,4-oxadiazol-3-yl)propionic acid. MS ESI calculation C 22 H 21 N3O6[M+H] + 424.14, actual measurement 424.22. 1 H NMR (400 MHz, DMSO- d 6 ) VT, at 90℃: δ (ppm) 12.80 (br s,1H), 7.87 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.58 (br s, 1H), 7.41(t, J = 7.4 Hz, 2H), 7.25-7.36 (m, 2H), 4.69 (d, J = 2.8 Hz, 2H), 4.38-4.50 (m,1H), 4.14-4.32 (m, 3H), 3.35 (s, 3H), 3.05-3.18 (m, 2H).

[0252] Preparation of the final compound: A. A general procedure for synthesizing linear peptide precursors Standard solid-phase synthesis was used, and Fmoc / tBu chemical substances were used for synthesis. Figure 1 Peptides in , as exemplified by: Chan, WC; White, PD "Fmoc Solid-Phase Synthesis: a Practical Approach", Oxford University Press, Oxford, 2000; Steward, J.; Young, J. "Solid Phase Peptide Synthesis", Pierce Chemical Company, Rockford, 1984.; Benoiton, NL "Chemistry of Peptide Synthesis", CRC Press, New York, 2006; and Lloyd-Williams, P.; Albericio, F.; Giralt, E. “Chemical Approaches to the Synthesis of Peptides and Proteins”, CRC Press, New York, 1997.

[0253] During peptide chain elongation, the α-amino group of each amino acid is protected with a 9H-fluorene-9-ylmethoxycarbonyl (Fmoc). To avoid any side reactions during the chain elongation step, any reactive amino acid side chains also carry acid-labile protecting groups, effectively masking the reactive groups until they are removed by treatment with a strong acid. After completing each coupling step, the Fmoc groups of the N-terminal amino acids are removed with piperidine or 4-methylpiperidine, and the resin is thoroughly washed to prepare for the subsequent coupling of Fmoc-protected amino acid derivatives.

[0254] The side chain protecting groups used are: 3Pal4CO2H, 3Pal4Ph4CO2H, alT, aMeD, aMeS, Bip4CO2H, daMeD, daMeS, hS, Phe4COOH, Phe4Pyrim5CO2H, Proc4OH, PyrimAla4Ph4CO2H, S, and T are tert-butyl ( t Bu); for Ac4cN, AlaPiperaz, aMeDab, aMeK, Dab, daMeK, K, PipH, and Orn, it is tert-butoxy-carbonyl (Boc); for Q, it is triphenylmethyl (Trt); for R, it is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); and for D, it is β-methylpentyl ester (OMpe).

[0255] The amino acids protected by Fmoc are typically obtained from suppliers such as Sigma-Aldrich, Novabiochem, Chem-Impex, and Combi-Block.

[0256] B. Synthetic procedures for preparing cyclic peptides Synthesis scheme 36 Solid-phase synthesis scheme A for peptides As outlined in scheme 36 above, peptides are synthesized using standard solid-phase synthesis with Fmoc / tBu chemicals on a Liberty Blue™ synthesizer from CEMCorporation.

[0257] use N,N' - Diisopropylcarbodiimide (DIC) and ethyl cyano(hydroxyimino)acetate (Oxyma) are used as coupling agents to form an amide bond between the free amino terminus of the resin-bound protected peptide and the carboxylic acid of the Fmoc-protected amino acid.

[0258] The synthesis was performed using 2-chlorotriphenylmethyl resin loaded with H-Gly (200-400 mesh, 0.79 mmol / g loading, 1% cross-linked polystyrene, Novabiochem). All amino acids were dissolved in DCM at a concentration of 0.2 M. N,N The amino acids were activated with an equimolar amount of Oxyma solution (0.5 M in DCM) and a 2-molar excess of DIC solution (1.0 M in DMF). Alternatively, the amino acids were dissolved in DCM at a concentration of 0.125 M. N,N The amino acids were activated with an equimolar amount of Oxyma Pure solution (0.125 M in DMF; with 0.05 M DIEA) and a 2-molar excess of DIC solution (0.25 M in DMF). The reaction was typically carried out on a 25 μmol scale.

[0259] Each synthetic cycle consists of: Fmoc-amino acid deprotection via 20% piperidine in DMF (microwave-assisted heating at 90°C for 2 min) and coupling with Fmoc-protected amino acids / DIC / Oxyma (5, 5, and 10 eq, respectively; microwave-assisted heating at 90°C for 2 or 4 min) (this may be repeated twice for difficult coupling). The cycle of Fmoc deprotection and Fmoc-protected amino acid coupling is repeated with the desired monomer until a complete linear peptide is formed.

[0260] Solid-phase synthesis scheme B for peptides Alternatively, as outlined in Scheme 36 above, peptides are manually synthesized on a Biotage® Syro II peptide synthesizer using standard solid-phase synthesis with Fmoc / tBu chemicals. HATU and DIPEA are used as coupling agents to form an amide bond between the free amino terminus of the resin-bound protected peptide and the carboxylic acid of the Fmoc-protected amino acid. Synthesis is performed using a 2-chlorotriphenylmethyl resin loaded with H-Gly (200-400 mesh, 0.79 mmol / g loading, 1% cross-linked polystyrene, Novabiochem). All amino acids are dissolved in a 1:1 DMF:NMP solution at a concentration of 0.2 M. Reactions are typically carried out at a scale of 12 μmol.

[0261] Each synthetic cycle consisted of: (1) coupling with Fmoc-protected amino acids / HATU / DIPEA (4, 4, and 8 eq, respectively; room temperature; 15 min) (repeated twice). The mixture was filtered and the peptide resin was washed with DMF (2 × 1 mL); (2) Fmoc deprotection (repeated three times): 20% 4-methylpiperidine in DCM (1 mL; room temperature; 3 min). The mixture was filtered and the peptide resin was washed with DMF (4 × 1 mL). The cycles of Fmoc deprotection and Fmoc-protected amino acid coupling were repeated with the desired monomer until a complete linear peptide was formed.

[0262] Selective cleavage and macrolide mating of protected peptides For the cleavage of protected linear peptides from a solid support, peptide resin (~16 mg) was treated with 25% hexafluoroisopropanol (HFIP) in DCM for 20 min at room temperature, filtered, and the solvent was removed under reduced pressure. The resulting residue was dissolved in DMF (5 mL). HATU (0.44 eq) and DIPEA (2.5 eq) were added. The mixture was stirred at room temperature for 5 min. Subsequently, 0.66 eq of HATU was added. After macrocyclic lactamation was complete, the solvent was removed under reduced pressure as monitored by UPLC-MS.

[0263] Final side chain deprotection A solution of TFA / H2O / TIS (90 / 8 / 2, v / v / v, 1 mL) was added to the crude protected cyclic peptide. The mixture was stirred at room temperature for 10 minutes. Cold diethyl ether (15 mL) was added to the solution. The peptide was precipitated by centrifugation (3200 rpm, -10 °C). The precipitate was washed with diethyl ether (2 × 10 mL) and dried under vacuum overnight to obtain a solid crude deprotected cyclic peptide.

[0264] HPLC purification Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used to analyze the results on a Waters X-Bridge Prep C18 OBDPrep column (130 Å, 5 pm, column size 19). Purification was performed on a Waters MS-Directed AutoPurification HPLC / MS system on a 100 mm column. Mobile phase: (A) 0.16% TFA in HPLC water, and (B) 0.16% TFA in HPLC acetonitrile; flow rate: 25 mL / min; UV wavelength λ = 215 nm; gradient: 25% to 50% B over 5 min. Alternatively, purification was performed on a Waters CSH-C18 column (19 × 250 mm, 5 μM) using an Agilent 1290 Infinity II preparative LC system and LC-MSDXT mass spectrometer. Mobile phase: (A) 0.1% formic acid in HPLC water, and (B) 0.1% formic acid in HPLC acetonitrile; flow rate: 25 mL / min; UV wavelength λ = 215 nm; gradient: 20% B over 2.5 min, and 55% B over 2.5 to 20 min. UV absorption fractions containing target m / z ions were collected, and fractions containing products were identified by LC / MS.

[0265] The purity of the fractions was confirmed by UPLC, which was measured using a reverse-phase Waters Acquity UPLC-MS system. Column: Waters XSelect CSH C18 column (130 Å, 2.5 μm, column size 2.1 × 50 mm). Mobile phase: (A) 0.05% TFA in HPLC water, and (B) 0.05% TFA in HPLC acetonitrile; injection volume: 1 μL; flow rate: 1 mL / min; UV wavelength λ = 215 nm; gradient: 5%–100% B over 5 min. The combined fractions containing the purified peptides were lyophilized to obtain the final cyclized product as a powder.

[0266] Biological assay: Procedure for IL-6 assay in MRC5 cells The inhibition of IL-1β-induced IL-6 secretion was evaluated in MRC5 cells. 2X EC was prepared in EMEM (ATCC 30-2003) inoculation medium containing 0.025% BSA (Sigma A9576), 1× penicillin / streptomycin (Gibco 15070-063), 1X NEAA (Gibco 11140-050), 1X GlutaMax (Gibco 35050-061), and 1X sodium pyruvate (Gibco 11360-070). 80Recombinant human IL-1β at a specific concentration (BioLegend 579404). 20 μL of IL-1β was added to 384-well collagen-coated plates (Corning 354664) and pre-incubated at ambient temperature for 1 hour with 200 nL of the compound dispensed using an ECHO 555 liquid processor. Human lung fibroblast MRC5 cells (ATCC CCL-171) were added at a density of 3000 cells / 20 μL per well. Cells were prepared as follows: Collagen-coated T175 flasks (Greiner 661950) were passaged three times in EMEM (ATCC 30-2003) growth medium containing 10% fetal bovine serum (Gibco 16140-071), 1X penicillin / streptomycin (Gibco 15070-063), 1X NEAA (Gibco 11140-050), 1X GlutaMax (Gibco 35050-061), and 1X sodium pyruvate (Gibco 11360-070). Cells were then collected in seeding medium after digestion with 0.25% trypsin EDTA (Gibco 25200-056) for 5 minutes. 384-well collagen-coated plates containing a final volume of 40 μL were incubated overnight at 37°C and 5% CO2. 5 μL of conditioned medium was transferred to a 384-well AlphaLISA plate (PerkinElmer 6005350) for IL-6 detection using the Human AlphaLISA IL-6 Kit (PerkinElmer AL223F) according to the manufacturer's protocol. 20 μL of the receptor bead / biotin-labeled antibody mixture was added to the 384-well AlphaLISA plate and incubated at ambient temperature for 1 hour. The donor bead mixture was protected from light, and 25 μL was added to the plate and incubated at ambient temperature for 30 minutes. The AlphaLISA plate was read using the AlphaScreen setting (laser excitation at 680 nm and emission at 570 nm) on an EnVision multimode plate reader (Perkin Elmer model 2104). The dose-response curve and IC50 were analyzed using a 4-parameter logarithmic equation in Spotfire software (Tibco, Palo Alto, CA). 50 value.

[0267] Amino acid sequence and bioactivity (MRC IC) of example number 1-103 (SEQ ID NO: 1-465) 50 The calculated single isotope mass, molecular formula, calculated molecular weight, and mass spectrometry data ([M+2H] / 2+ or [M+H]+) are provided in [the relevant database]. Figure 1 middle.

[0268] The subject matter disclosed is not limited in scope to the specific embodiments and examples described herein. In fact, various modifications to this disclosure, in addition to those described, will become apparent to those skilled in the art based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0269] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in their entirety, and for all purposes, as if each individual reference (e.g., publications, patents, or patent applications) were specifically and individually indicated to be incorporated herein by reference in their entirety for all purposes. Other embodiments are described in the following claims.

Claims

1. Compounds of formula (I) (I) in: R 1 for (i) R 1a -C(O)N(H)-CH2CH2-O-, where R 1a for (a) C1-C3 alkyl, or (b) (CH3)3N-CH2CH2-M-, where: M is -CH2-, -CH2CH2-, -CH2CH2CH2-, -O-CH2CH2-, or -O-CH2CH2-O-CH2CH2-; (ii) (CH3)3N-CH2CH2-O-; (iii) C 1 C 1 for: (a) a phenyl or a 5- to 6-membered monocyclic heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 3 Each heteroatom is independently selected from N, O, and S; (b) A 5- to 6-membered heterocyclic alkyl group, wherein the 5- to 6-membered heterocyclic alkyl group is saturated and contains one or two heteroatoms selected from N, O and S; Where C 1 For unsubstituted or selected independently from the following R C1 Substituents: Halogenated, C1-C3 alkyl, C1-C3 fluoroalkyl, carboxyl, C1-C3 alkoxy, C2-C 3 Acyl and C1-C3 alkoxymethyl groups; (iii) (CH3)3N-CH2CH2-O-; or (iv) Group ; R A for: (i) -CO2H; (ii) -CO2NH2; (iii) -S(O)2NH2; or (iv) A five-membered heteroaryl group containing two or three heteroatoms independently selected from N, O and S; The 5-membered heteroaryl group is either unsubstituted or substituted by one or two independent substituents selected from the following: halogenated, C1-C3 alkyl, C1-C3 alkoxy, oxo, and C1-C3 alkoxymethyl; R B It is amino, hydroxyl, or fluorinated; R 2 for (i) naphthyl; or (ii) A 9- or 10-membered bicyclic heteroaryl group, wherein the 9- or 10-membered bicyclic heteroaryl group contains 1 or 2 heteroatoms independently selected from N, O and S; Where R 2 For unsubstituted or selected independently by one or two of the following R 2a Substituents: halogenated, C1-C3 alkyl, and C1-C3 alkoxy; R 3 -(CH2) m CO2H, -(CH2) m CO2NH2、-(CH2) m CO2N(CH3)2、-(CH2) m CH2N(H)C(O)NH2 or -(CH2)mC 3 ; Where C 3 It is a 5-membered heteroaryl group containing 2 to 4 heteroatoms selected from N, O and S; Where C 3 It is either unsubstituted or substituted with one or two substituents selected from halogens and C1-C3 alkyl groups; R 4 It is a C1-C3 alkyl group; R 5 It is H, C1-C3 alkyl or -CH2CO2H; Alternatively, R 4 and R 5 The atoms bonded to them together form pyrrolidinyl, piperidinyl, or aziridine rings; X 1 X 2 and X 3 It can be C(H) or N independently; R D for: (i) Ring C D Among them, ring C D for: (a) phenyl; or (b) Cyclohexyl; Among them, ring C D By an A 1 Replace; where A 1 It can be -CO2H, -CONH2, or -N(H)SO2CH3; (ii) Hydroxyl group; (iii) -CO2NH2; or (iv) -OCH2CH2C(O)N(H)C(O)CH3; A 2 It is H, halogen, or C1-C3 alkyl; R 6 -H, -NH2, -C(O)N(CH3)2; -(CH2)nNH2; -(CH2)nN(H)CH3; -(CH2)nN(CH3)3; -OH; -(CH2)nOH; -OCH3; -(CH2)nOCH3; -(CH2)nCH3; -(CH2)nN(H)C(O)NH2; -(CH2)nCH2C(O)OH; -(CH2)nN(H)C(O)CH2CH2O(CH2CH2-O)q-CH2CH2N(CH3)3; or -C 6 ; C 6 for (i) 5- to 6-membered monocyclic aryl or heteroaryl, wherein the 5- to 6-membered monocyclic heteroaryl contains 1 to 3 Each heteroatom is independently selected from N, O, and S; (ii) a 9- to 10-membered bicyclic aryl or heteroaryl group, wherein the 9- to 10-membered bicyclic heteroaryl group contains 1 to 4 heteroatoms independently selected from N, O, and S; and (iii) 3 to 8-membered monocyclic or bicyclic cycloalkyl groups; Where C 6 For unsubstituted or selected independently from the following R C6 Substituents: halogenated, amino, hydroxyl, carboxyl, C1-C3 alkyl, C1-C3 fluoroalkyl, C1-C3 alkoxy, and C1-C3 alkoxymethyl; R 7 It can be H, C1-C4 alkyl, -(CH2)uOH, -(CH2)uC(O)N(CH3)2, -(CH2)uN(CH3)3, -(CH2)uOCH3, -(CH2)uC(O)NC(O)NH2, -(CH2)uN(H)C(O)CH2CH2O(CH2CH2-O)v-CH2CH2N(CH3)3 or -CH2-C 7 ; C 7 for: (i) is a 5-membered heteroaryl group containing 2 to 4 heteroatoms selected from N, O and S; or (ii) A 5- or 6-membered heterocyclic alkyl group, wherein the 5- or 6-membered heterocyclic alkyl group is saturated and contains one O atom; Where C 7 It is unsubstituted or substituted with one or two substituents selected from the following: halogenated, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 alkoxymethyl; R 8 The octet is -H, -F, -OH, -NH2, -N(CH3)3, (CH3)3N-(CH2)rC(O)- or (CH3)3N-CH2CH2-O-(CH2CH2-O)s-CH2CH2-C(O)-; R 9 -C 9 or -CH2-C 9 ; C 9 It is a phenyl or a 5- to 6-membered monocyclic heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 heteroatoms independently selected from N, O and S; Where C 9 For unsubstituted or selected independently from the following R C9 Substituents: halogenated, amino, hydroxylated, C1-C3 alkyl, C1-C3 fluoroalkyl, and C1-C3 alkoxy; Where C 9 Optionally bounded by 1 ring R C9C Replacement, ring R C9C for: (a) phenyl; or (b) A 5- to 6-membered monocyclic heteroaryl group containing one or two heteroatoms independently selected from N, O and S; The subscript m is 1 or 2; The subscript n is 1, 2, or 3; and The subscript q is 1, 2, or 3; The subscript r is 3, 4, 5, or 6; The subscript s is 1 or 2; The subscript t is 0 or 1; The subscript u is 0, 1, 2, 3, or 4; The subscript v is 1, 2, or 3; and The subscript w is 0 or 1; or Its pharmaceutically acceptable salt.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: C 1 It is phenyl; R 2 It is indole or naphthyl; C 6 It is phenyl, indolyl, pyridyl, pyridazinyl, or bicyclic [1.1.1]pentyl; and C 9 It is phenyl or pyrimidinyl.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3-C(O)N(H)-CH2CH2-O- or 4-fluorophenyl.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 2 It can be an unsubstituted or substituted naphthyl or indole group.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 3 -(CH2) m CO2H.

6. The compound of claim 5 or a pharmaceutically acceptable salt thereof, wherein the subscript m is 1.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 4 and R 5 It is a methyl group.

8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: X 1 and X 2 For C(H), and A 1 It is a carboxyl group.

9. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 6 for: (i) a substituted or unsubstituted 5- to 6-membered monocyclic aryl or heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 N atoms; or (ii) A substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl group, wherein the bicyclic heteroaryl group contains 1 to 2 N atoms.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein R 6 It is an indole group.

11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 7 For H.

12. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 8 It can be H or -NH2.

13. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 9 The pyrrole alkyl ring shown is substituted at position 4.

14. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein C 9 It can be a substituted or unsubstituted phenyl or pyrimidinyl group.

15. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 9 It is -CH2-(4-fluorophenyl) or pyrimidin-5-yl.

16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: R 1 It is CH3-C(O)N(H)-CH2CH2-O- or 4-fluorophenyl; R 2 It can be an unsubstituted or substituted naphthyl or indole group; R 3 -(CH2) m CO2H; R 4 and R 5 It is methyl; X 1 and X 2 For C(H); A 1 It is a carboxyl group; A 2 For H; R 6 for: (i) a substituted or unsubstituted 5- to 6-membered monocyclic aryl or heteroaryl group, wherein the 5- to 6-membered monocyclic heteroaryl group contains 1 to 2 N atoms; or (ii) A substituted or unsubstituted 9- or 10-membered bicyclic heteroaryl group, wherein the 9- or 10-membered bicyclic heteroaryl group contains 1 to 2 N atoms; R 7 For H; R 8 For H or -NH2; and R 9 The pyrrole alkyl ring shown is substituted at position 4.

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein: R 6 It is an indole group; R 9 It is -CH2-(4-fluorophenyl) or pyrimidin-5-yl, with substitution at the 4-position of the pyrrolidinyl ring shown; and The subscript m is 1.

18. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has formula (IA). (IA), in: R 1 for: (i) R 1a -C(O)N(H)-CH2CH2-O-, where R 1a for: (a) Methyl; or (b) (CH3)3N-CH2CH2-O-CH2CH2-; (ii) (CH3)3N-CH2CH2-O-; or (iii) 4-Carboxyphenyl; R 2 as a group or ; R 2a It is either halogenated or methylated; The subscript x is 0 or 1; R 3 It is -CH2CO2H or -CH2-tetrazole; R 4 and R 5 It is methyl; Alternatively, R 4 and R 5 Together with the atoms they are attached to, they form a piperidinyl ring; R D It is 4-carboxyphenyl or hydroxy; R 6 The groups are selected from the following: , , , , , or ; R 7 It is H or methyl; R 8 It can be -H, -NH2, or -OH; C 9 The groups are selected from the following: , or .

19. The compound of claim 1, selected from SEQ ID NO: 1-465, or a pharmaceutically acceptable salt thereof.

20. The compound of claim 1, selected from the following (SEQ ID NO: 1, 2, 78, 80, 82, 92, 94, 95, 98, 99, 100, 101, 102, 215, 216, 217 and 218 respectively): 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and ， Or its pharmaceutically acceptable salt.

21. A pharmaceutical composition comprising a compound of any one of claims 1-20 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

22. A method of treating atherosclerosis, comprising administering to a subject in need a therapeutically effective amount of the compound of any one of claims 1-20 or a pharmaceutically acceptable salt thereof.

23. A method of treating vascular inflammation, comprising administering to a subject in need a therapeutically effective amount of the compound of any one of claims 1-20 or a pharmaceutically acceptable salt thereof.

24. A method of treating an inflammatory condition, comprising administering to a subject in need a therapeutically effective amount of the compound of any one of claims 1-20 or a pharmaceutically acceptable salt thereof.

25. The method of any one of claims 22, 23 and 24, wherein an effective amount of the compound or a pharmaceutically acceptable salt thereof is administered orally to the subject.

26. Use of any compound of claims 1-20 or a pharmaceutically acceptable salt thereof in a therapeutic manner.

27. Use of any compound of claims 1-20 or a pharmaceutically acceptable salt thereof for the treatment of atherosclerosis.

28. Use of any compound of claims 1-20 or a pharmaceutically acceptable salt thereof for the treatment of vascular inflammation.

29. Use of any compound of claims 1-20 or a pharmaceutically acceptable salt thereof for the treatment of inflammatory conditions.

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