Antibody-drug conjugates comprising glp1 peptide mimetics and uses thereof

Abstract

Described herein are protein-drug conjugates and compositions thereof that are useful, for example, in targeting the glucagon-like peptide 1 receptor (GLP1R). In certain embodiments, peptide mimetic payloads and linker-payloads and methods of making the same are provided. More specifically, GLP1 peptide mimetics, antibody-drug conjugates, and compositions comprising anti-GLP1R antibodies and GLP1 peptide mimetic payloads and methods of treating GLP1R-related conditions are provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 077,983, filed September 14, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in ASCII format and is hereby incorporated in its entirety by reference. The ASCII copy generated on September 8, 2021, is named 250298_000250_SL.txt and has a size of 6,072 bytes.

[0005] The scope of this disclosure

[0006] This disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods of treating diseases therewith. Peptide mimic payloads and linker-payloads, and methods for their preparation, are also provided. More specifically, this disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates) comprising GLP1 peptide mimics and methods of treating GLP1R-related conditions therewith.

[0007] Background of this public disclosure

[0008] Diabetes mellitus is a chronic disease characterized by abnormal glucose metabolism. An estimated 425 million people worldwide have diabetes. Global diabetes medications include insulin, DPP-4 inhibitors, and glucagon-like peptide-1 receptor (GLP-1R) agonists, but most patients do not achieve the combined treatment goal of controlling hyperglycemia and cardiovascular risk factors.

[0009] Glucagon-like peptide-1 receptor (GLP1R) is the receptor for glucagon-like peptide-1 (GLP1) and is expressed in pancreatic β cells. GLP1R is also expressed in the brain, where it plays a role in controlling appetite, memory, and learning. GLP1R is a member of the G protein-coupled receptor (GPCR) secretin family (class B). Upon binding to its ligand GLP1, GLP1R initiates a downstream signaling cascade via the Gαs G protein (which increases intracellular cyclic AMP (cAMP) levels), leading to transcriptional regulation of genes (Donnelly 2011). Activation of GLP1R results in increased insulin synthesis and release.

[0010] GLP1R and GLP1 are highly effective targets for obesity and type 2 diabetes. Commercially available GLP1R agonists increase insulin secretion, thereby lowering blood sugar levels, but they need to be administered weekly or more frequently.

[0011] Therefore, there is a need in the art for GLP1R agonists with longer duration of action and better safety. In some embodiments, this disclosure meets the need and provides other advantages.

[0012] The foregoing discussion is presented merely to provide a better understanding of the nature of the problems faced in this field and should not be construed in any way as an admission of prior art, nor should any reference cited herein be construed as an admission that such reference constitutes "prior art" of this application.

[0013] Overview of publicly available content

[0014] The following describes various non-limiting aspects and implementations of this disclosure.

[0015] In one respect, compounds having the structure of formula (A):

[0016] BA-(LP) m (A),

[0017] in:

[0018] BA is an antibody or its antigen-binding fragment;

[0019] L is a non-disintegrable connector;

[0020] P is the effective load of a structure that is selected from the following group:

[0021]

[0022]

[0023] in The attachment point where the payload is attached to L;

[0024] X1 is selected from H;

[0025] X2 is selected from

[0026] X3 is selected from the bond, –(CH2). 2-6 –NH–, –(CH2) 2-6 –Tr– and –(CH2) 2-6 –Tr–(CH2) 1-6 –NH, where Tr is the triazole moiety;

[0027] n is 0 or 1;

[0028] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0029] X5 is selected from -OH, -NH2, -NH-OH, and

[0030] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0031] X7 is selected from H,

[0032] X8 is selected from H, -OH, -NH2, and

[0033] Ar selected

[0034] X9 is selected from -NH2, and

[0035] m is an integer from 1 to 4

[0036] Or its pharmaceutically acceptable salt.

[0037] In another aspect, this paper provides compounds having the structure of formula (I):

[0038] BA-LP(I),

[0039] in:

[0040] BA is an antibody or its antigen-binding fragment;

[0041] L is a non-disintegrable connector;

[0042] P is the effective load of a structure that is selected from the following group:

[0043]

[0044] in It is the attachment point where the payload P is attached to L;

[0045] X1 is selected from H;

[0046] X2 is selected from

[0047] X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety;

[0048] n is 0 or 1;

[0049] X4 is selected from H and phenyl;

[0050] X5 is selected from -OH, -NH2, -NH-OH, and

[0051] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0052] X7 is selected from H,

[0053] X8 is selected from H, -OH, -NH2, and

[0054] Or its pharmaceutically acceptable salt.

[0055] In some embodiments of the compounds described herein, BA is an antibody or antigen-binding fragment thereof targeting the glucagon-like peptide-1 receptor (GLP1R). In some embodiments, the antibody targeting GLP1R is a GLP1R agonist antibody. In some embodiments, the antibody targeting GLP1R is 5A10, 9A10, AB9433-I, h38C2, PA5-111834, NLS1205, MAB2814, EPR21819, or glutazumab.

[0056] In some embodiments, connector L is attached to one or both heavy chains of the BA. In some embodiments, connector L is attached to one or both heavy chain variable structural domains of the BA. In some embodiments, connector L is attached to one or both light chains of the BA. In some embodiments, connector L is attached to one or both light chain variable structural domains of the BA.

[0057] In some embodiments, the linker L is attached to the BA via a glutamine residue. In some embodiments, the glutamine residue is introduced into the N-terminus of one or both heavy chains of the BA. In some embodiments, the glutamine residue is introduced into the N-terminus of one or both light chains of the BA. In some embodiments, the glutamine residue is naturally present in the CH2 or CH3 domain of the BA. In some embodiments, the glutamine residue is introduced into the BA by modifying one or more amino acids. In some embodiments, the glutamine residue is Q295 or N297Q.

[0058] In some implementations, the linker L is attached to BA via a lysine residue.

[0059] In some embodiments, the antibody or its antigen-binding fragment is non-glycosylated. In some embodiments, the antibody or its antigen-binding fragment is deglycosylated. In some embodiments, the antigen-binding fragment is a Fab fragment.

[0060] In one implementation, m is 1. In one implementation, m is an integer from 2 to 4. In one implementation, m is 2.

[0061] In some implementations, more than one LP is attached to the BA. In some implementations, two LPs are attached to the BA.

[0062] In some implementations, the connector L has a structure of type (L'):

[0063] -La-Y-Lp-(L'),

[0064] Wherein La is the first connector covalently attached to the BA;

[0065] Y is a group containing triazole, and

[0066] Lp is absent or is a second connector covalently attached to P, wherein Y is also absent when Lp is absent.

[0067] In some implementations, Y has a structure selected from the group consisting of:

[0068] Where Q is C or N.

[0069] In some embodiments, Lp comprises a polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units. In some embodiments, the PEG segment comprises between 2 and 30 EG units. In some embodiments, the PEG segment comprises between 4 and 24 EG units. In some embodiments, the PEG segment comprises 4, 8, 12, or 24 EG units. In some embodiments, the PEG segment comprises 4 EG units. In some embodiments, the PEG segment comprises 8 EG units.

[0070] In some implementations, Y-Lp has a structure selected from the group consisting of:

[0071]

[0072] Or its triazole positional isomer,

[0073] Where p is an integer from 1 to 36.

[0074] In some embodiments, Lp comprises one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof. In some embodiments, Lp comprises 1 to 10 glycines. In some embodiments, Lp comprises 1 to 6 serines. In some embodiments, Lp comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, Lp comprises 4 glycines and 1 serine. In some embodiments, Lp is selected from the group consisting of: Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO:1), Ser-Gly-Gly-Gly-Gly (SG4) (SEQ ID NO:2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (G4S-G4S) (SEQ ID NO:3).

[0075] In some embodiments, Lp comprises a PEG segment having 1 to 36 EG units and a combination of one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof. In some embodiments, the serine residues comprise glycoside groups. In some embodiments, the serine residues comprise glucose groups.

[0076] In some implementations, Lp has a structure selected from the group consisting of:

[0077]

[0078] Where Y is a triazole group, P is the payload, and Rc is selected from H and glucose, g is an integer from 1 to 10, and s is an integer from 0 to 4.

[0079] In some implementations, Y-Lp has a structure selected from the group consisting of:

[0080] as well as Or its triazole positional isomer.

[0081] In some embodiments, La includes a polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units. In some embodiments, the PEG segment includes 4, 8, 12, or 24 EG units. In some embodiments, the PEG segment includes 8 EG units. In some embodiments, La has a structure selected from the group consisting of: and

[0082] In some embodiments, La comprises one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. In some embodiments, La comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, La comprises 4 glycines and 1 serine. In some embodiments, La is selected from the group consisting of: Gly-Gly-Gly-Gly-Ser (G4S), Ser-Gly-Gly-Gly-Gly (SG4), Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (G2S-G2S-G2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (G4S-G4).

[0083] In some embodiments, La comprises a PEG segment having 1 to 36 EG units and a combination of one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. In some embodiments, La is selected from the group consisting of:

[0084]

[0085] In some implementations, La includes –(CH2). 2-24 -Chain. In some implementations, La includes –(CH2). 2-24 - A chain, a PEG segment having 1 to 36 EG units, and a combination of one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. La is selected from the group consisting of:

[0086]

[0087] In various embodiments of the compound described herein, P has the structure

[0088]

[0089] In some implementations, X1 is H; X2 is X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety; n is 1, and X4 is H.

[0090] In some implementations, X1 is X2 is X3 is –(CH2) 2-6–NH–; n is 1; X4 is H, and X5 is selected from -OH, -NH2, -NH-OH, and

[0091] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H.

[0092] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH2OH each time it appears, and X7 is H.

[0093] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –Tr–, where Tr is the triazole moiety; n is 1; X4 is H, and X5 is

[0094] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears; X7 is And X8 is -NH2.

[0095] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H, and X8 is H.

[0096] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is H every time it appears; X7 is And X8 is H.

[0097] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears; X7 is And X8 is

[0098] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H.

[0099] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H.

[0100] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears, and X7 is

[0101] In some implementations, X1 is H; X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H.

[0102] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H, and X5 is

[0103] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 0; X4 is phenyl, and X5 is

[0104] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is phenyl, and X5 is

[0105] In various embodiments of the compound described herein, P has the structure

[0106]

[0107] In some implementations, X1 is X2 is X3 is –(CH2) 2-6 –NH–; X4 is H, and X5 is

[0108] In various embodiments of the compounds described herein, P has a structure selected from the group consisting of:

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] In various embodiments of the compounds described herein, the compounds have a half-life of more than 7 days in plasma.

[0118] In various embodiments of the compounds described herein, the compounds do not bind to G protein-coupled receptors (GPCRs) other than GLP1R.

[0119] In another respect, this document provides pharmaceutical compositions comprising compounds of any of the embodiments described herein.

[0120] In another aspect, this document provides pharmaceutical dosage forms comprising compounds comprising any of the embodiments described herein.

[0121] In another aspect, this document provides a method for selectively targeting GLP1R on the cell surface with a compound according to any of the embodiments described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a pancreatic cell, brain cell, heart cell, vascular tissue cell, kidney cell, adipose tissue cell, hepatocyte, or muscle cell.

[0122] In another aspect, this document provides a method for enhancing the GLP1R activity of individuals with a corresponding need, comprising administering to the individual an effective amount of a compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein.

[0123] In another aspect, this document provides a method for lowering blood glucose levels in individuals with a corresponding need, comprising administering to the individual an effective amount of a compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein.

[0124] In another aspect, this document provides a method for reducing the weight of an individual with a corresponding need, comprising administering to the individual an effective amount of a compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein.

[0125] In another aspect, this document provides a method for treating GLP1R-related conditions in individuals with corresponding needs, comprising administering to the individual an effective amount of a compound of any of the embodiments described herein, a composition described herein, or a dosage form described herein. In some embodiments, the GLP1R-related condition is type II diabetes, obesity, liver disease, coronary artery disease, or kidney disease. In some embodiments, the GLP1R-related condition is type II diabetes and / or obesity.

[0126] In various embodiments of any of the methods described herein, the compounds, compositions, or dosage forms of this disclosure are administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly. In another aspect, this document provides a method for producing compounds having the structure of formula (A) described herein:

[0127] BA-(LP) m (A),

[0128] The method includes the following steps:

[0129] a) In the presence of transglutaminase, BA comprising at least m glutamine residues Gln is contacted with at least m equivalents of compound LP, and

[0130] b) The compound of formula (A) produced by separation.

[0131] In another aspect, this paper provides a method for producing compounds having the structure of formula (I) described herein:

[0132] BA-LP(I),

[0133] The method includes the following steps:

[0134] a) In the presence of transglutaminase, contacting BA, which includes at least one glutamine residue Gln, with compound LP, and

[0135] b) The resulting compound of formula (I) after separation.

[0136] In another aspect, this document provides a method for producing the compound of claim 1 having the structure of formula (A):

[0137] BA-(LP) m (A),

[0138] The connector L has the structure of formula (L'):

[0139] -La-Y-Lp-(L'),

[0140] Wherein La is the first connector covalently attached to the BA;

[0141] Y is a group containing triazole, and

[0142] Lp is a second connector covalently attached to P.

[0143] The method includes the following steps:

[0144] a) In the presence of transglutaminase, BA comprising at least m glutamine residues Gln is contacted with a first linker La comprising an azide or alkyne moiety.

[0145] b) Contact the product of step a) with at least m equivalents of compound Lp-P, wherein the second connector Lp comprises an azide or alkyne moiety, wherein La and Lp are capable of reacting to produce a triazole, and

[0146] c) The resulting compound of formula (A) was isolated.

[0147] In another aspect, this paper provides a method for producing compounds having the structure of formula (I) described herein:

[0148] BA-LP(I),

[0149] The connector L has the structure of formula (L'):

[0150] -La-Y-Lp-(L'),

[0151] Wherein La is the first connector covalently attached to the BA;

[0152] Y is a group containing triazole, and

[0153] Lp is a second connector covalently attached to P.

[0154] The method includes the following steps:

[0155] a) In the presence of transglutaminase, BA comprising at least one glutamine residue Gln is contacted with a first linker La comprising an azide or alkyne moiety;

[0156] b) Contact the product of step a) with compound Lp-P, wherein the second connector Lp comprises an azide or alkyne moiety, wherein La and Lp are capable of reacting to produce a triazole, and

[0157] c) The resulting compound of formula (I) is separated.

[0158] In another aspect, this paper provides compounds having structures selected from the group consisting of formulas (P-IB), (P-IIB), and (P-IIIB):

[0159] (P-IIIB), where:

[0160] X1 is selected from H;

[0161] X2 is selected from

[0162] X3 is selected from -CH3 and –(CH2). 2-6 –NH2、–(CH2) 2-6 –N3 and –(CH2) 2-6 –Tr–(CH2) 1-6 –NH2, where Tr is the triazole moiety;

[0163] n is 0 or 1;

[0164] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0165] X5 is selected from -OH, -NH2, -NH-OH, and

[0166] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0167] X7 is selected from H,

[0168] X8 is selected from H, -OH, -NH2, and

[0169] Ar selected

[0170] X9 is selected from -NH2, and

[0171] m is an integer from 1 to 4

[0172] Or its pharmaceutically acceptable salt.

[0173] In another respect, this paper provides compounds having the structure of formula (II):

[0174]

[0175] in:

[0176] X1 is selected from H;

[0177] X2 is selected from

[0178] X3 is selected from –(CH2) 2-6 –NH2、–(CH2) 2-6 –N3 and -CH3, provided that n is 1 when X3 is -CH3, and Ra is selected from –(CH2) at least once. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0179] n is 0 or 1;

[0180] m is an integer from 0 to 3;

[0181] Ra is independently selected from -CH3 and –(CH2) each time it appears. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0182] X4 is selected from H and phenyl;

[0183] X5 is selected from -OH, -NH2, -NH-OH, and

[0184] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0185] X7 is selected from H,

[0186] X8 is selected from H, -OH, -NH2, and

[0187] And its pharmaceutically acceptable salts.

[0188] In some implementations, P has a structure selected from the group consisting of:

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197] In another aspect, this paper provides compounds having the structure of formula (C):

[0198]

[0199] in:

[0200] L p The following types of connectors are absent or include one or more of the following: Carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units; –(CH2) 2-24 - chain; triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;

[0201] Q is selected from -NH2, -N3. The part where A is C or N;

[0202] X1 is selected from H;

[0203] X2 is selected from

[0204] X3 is selected from -CH3 and –(CH2). 2-6 –NH2、–(CH2) 2-6 –N3 and –(CH2) 2-6 –Tr–(CH2) 1-6 –NH2, where Tr is the triazole moiety;

[0205] n is 0 or 1;

[0206] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0207] X5 is selected from -OH, -NH2, -NH-OH, and

[0208] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0209] X7 is selected from H,

[0210] X8 is selected from H, -OH, -NH2, and

[0211] Ar selected

[0212] X9 is selected from -NH2, and

[0213] m is an integer from 1 to 4

[0214] Or its pharmaceutically acceptable salt.

[0215] In another aspect, this paper provides compounds having the structure of formula (III):

[0216]

[0217] in:

[0218] L p The following types of connectors are absent or include one or more of the following: Carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine and proline and combinations thereof;

[0219] Q is selected from -N3, The part where A is C or N;

[0220] X1 is selected from H;

[0221] X2 is selected from

[0222] X3 is selected from –(CH2) 2-6 –NH2、–(CH2) 2-6 –N3 and -CH3, provided that n is 1 when X3 is -CH3, and Ra is selected from –(CH2) at least once. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0223] n is 0 or 1;

[0224] Ra is independently selected from H, -CH3, and –(CH2) each time it appears. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0225] X4 is selected from H and phenyl;

[0226] X5 is selected from -OH, -NH2, -NH-OH, and

[0227] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0228] X7 is selected from H,

[0229] X8 is selected from H, -OH, -NH2, and

[0230] And its pharmaceutically acceptable salts.

[0231] In some embodiments, the compounds described above have a structure selected from the group consisting of:

[0232] structure

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] Or its pharmaceutically acceptable salt.

[0245] In another aspect, this document provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof targeting the glucagon-like peptide-1 receptor (GLP1R) optionally conjugated to a payload via a linker, said payload having a structure selected from the group consisting of:

[0246] structure

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254] Or its pharmaceutically acceptable salt.

[0255] In another aspect, this article provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment targeting the glucagon-like peptide-1 receptor (GLP1R) and a payload having the following structure:

[0256]

[0257] in The payload is attached to the antibody or its antigen-binding fragment directly or through a connector at the attachment point.

[0258] In one implementation, the payload has the following structure:

[0259]

[0260] In another aspect, this article provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment targeting the glucagon-like peptide-1 receptor (GLP1R) and a linker-payload having the following structure:

[0261]

[0262] in It is the attachment point where the connector-payload is attached to the antibody or its antigen-binding fragment.

[0263] In one embodiment, the joint-payload has the following structure:

[0264]

[0265] These and other aspects of this disclosure will become apparent to those skilled in the art after reading the following detailed description of this disclosure (including the appended claims). Brief description of the attached diagram

[0267] This patent or application document contains at least one color drawing. A published copy of this patent or application with color drawings will be provided by the competent authority upon request and payment of the necessary fees.

[0268] Figure 1A A schematic diagram illustrating the design of an exemplary antibody-tethered drug conjugate (ATDC) and its mechanism of action is shown.

[0269] Figure 1B A schematic diagram of the design and mechanism of action of conventional antibody-drug conjugates (ADCs) is shown.

[0270] Figure 2 A model of a drug conjugate with an antibody tethered to an extracellular domain (ECD) and a payload bound to a transmembrane domain (TMD) is shown.

[0271] Figure 3A A schematic diagram of GLP1(7-36)amide (SEQ ID NO: 4) is shown. The numbers above the sequence correspond to the amino acid positions in the glucagon propeptide. Blue arrows indicate dipeptidyl peptidase-4 (DPP-IV) cleavage sites. Red arrows indicate neutral endopeptidase (NEP) cleavage sites. Dashed arrows indicate cleavage sites caused by one or more unknown endopeptidases. Orange residues are amino acids that, when substituted, reduce GLP1R binding and cAMP production. Green residues are amino acids that, when substituted, reduce GLP1R binding.

[0272] Figure 3B The structure of GLP1R binding to GLP1 (protein database ID: 3IOL) is shown. References to this structure can be found in Zhang et al., Nature 2017, Chepurny et al., JBC 2019, De Graaf et al., Pharmacological reviews 2016, and Manandhar and Ahn, Journal of Medical Chemistry 2014, each of which is incorporated herein by reference in its entirety.

[0273] Figure 4A The sequence and structure of GLP1 peptide mimic peptide 5 (SEQ ID NO:5) are shown. The numbers above the sequence correspond to the amino acid positions in the glucagon propeptide.

[0274] Figure 4BThe superimposed structures of GLP1R binding to peptide 5 (protein database ID: 5NX2) and GLP1R binding to GLP1 (protein database ID: 3IOL) are shown using GLP1R in 5NX2 as a template. A reference to the 5NX2 structure can be found in Jazayeri A, et al., Nature, Vol. 546, pp. 254–258 (2017), which is incorporated herein by reference in its entirety.

[0275] Figure 5 The present disclosure illustrates a synthetic scheme for preparing GLP1 peptide mimic payloads. Solid-phase peptide synthesis on a resin was established, which efficiently produced the payloads of the present disclosure in good yields. Additional GLP1R peptide mimic payloads were generated via systemic modification with R1 / R2 / R3.

[0276] Figures 6A-6D As shown, the GLP1R peptide mimicry payload of this disclosure did not show activation in the relevant GPCR bioassays.

[0277] Figures 7A-7B As shown, the shorter connector GLP1R ATDC exhibits greater efficiency than the control ATDC.

[0278] Figure 8 It was shown that the leader connector-payload exhibited the best in vitro ADME spectrum with no in vitro cardiotoxicity or mutagenic potential, and its ATDC was highly stable in plasma.

[0279] Figure 9A Two methods for conjugating adapter-payload to antibodies of this disclosure are shown.

[0280] Figure 9B Representative hydrophobic interaction chromatograms (HICs) of anti-GLP1R ATDC drug loading profiles are shown. HICs were used for conjugation screening to triage ATDCs with low conjugation yields, low DAR, high aggregates, and poor Biacore binding.

[0281] Figure 10 CRE-dependent luciferase reporter gene activity induced by anti-GLP1R ATDC is shown. Anti-GLP1R ATDC exhibits better in vitro potency than isotype control ATDC. Unconjugated mAbs do not activate hGLP1R cells (not shown). ATDCs do not activate glucagon-like peptide-2 receptor (GLP 2R), glucagon receptor (GCGR), or gastric inhibitory peptide receptor (GIPR) (not shown).

[0282] Figure 11AThis study demonstrates the cyclic AMP response element (CRE)-dependent luciferase reporter gene activity induced by anti-GLP1R ATDC in the presence of unconjugated anti-GLP1R antibody. It shows that concentrations of unconjugated anti-GLP1R mAb <10 nM have no effect on anti-GLP1R ATDC activity. 100 nM of unconjugated anti-GLP1R mAb reduced the potency of anti-GLP1R ATDC by 3.8-fold. Assays were performed by first adding unconjugated anti-GLP1R mAb, then immediately adding anti-GLP1R ATDC, and incubating for 4 hours.

[0283] Figure 11B Showing with Figure 11A The data corresponding to the graph in the image.

[0284] Figure 12 A schematic diagram of an exemplary GLP1R Q-tag mAb-GLP1R agonist conjugate of this disclosure is shown.

[0285] Figure 13 A general synthetic scheme for preparing GLP1 peptide mimics according to this disclosure is shown.

[0286] Figure 14 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P1 and P8 according to this disclosure is shown.

[0287] Figure 15 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P2 and P9 according to this disclosure is shown.

[0288] Figure 16 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P3, P4, P5, P6, P7, P11, P13, P14, P15, P16 and P17 according to this disclosure is shown.

[0289] Figure 17A and Figure 17B The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P10, P12, P18, P19, P25, P26, P27, P28, P29, P30, P31, P36, P37 and P38 according to this disclosure is shown.

[0290] Figure 18 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P20 and P21 according to this disclosure is shown.

[0291] Figure 19 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P22 and P23 according to this disclosure is shown.

[0292] Figure 20 The sequence of solid-supported synthesis of the GLP1 peptide mimic payload P24 according to this disclosure is shown.

[0293] Figure 21 The sequence of solid-supported synthesis of GLP1 peptide mimic payloads P32, P33, P34 and P35 according to this disclosure is shown.

[0294] Figure 22 The sequence of solid-supported synthesis of the GLP1 peptide mimic payload P39 according to this disclosure is shown.

[0295] Figure 23 The sequence of solid-supported synthesis of the GLP1 peptide mimic payload P40 according to this disclosure is shown.

[0296] Figure 24 The sequence of solid-supported synthesis of the GLP1 peptide mimic payload P41 according to this disclosure is shown.

[0297] Figure 25 The sequence of solid-supported synthesis of the GLP1 peptide mimic payload P42 according to this disclosure is shown.

[0298] Figure 26 Synthetic routes for preparing the connector-payloads LP1, LP2, LP3, LP4 and LP5 according to this disclosure are shown.

[0299] Figure 27 The synthetic routes for preparing the connector-payloads LP6 and LP7 according to this disclosure are shown.

[0300] Figure 28 Synthetic routes for preparing connector-payloads LP8, LP9, LP10 and LP11 according to this disclosure are shown.

[0301] Figure 29 A synthetic route for preparing the connector-payload LP12 according to this disclosure is shown.

[0302] Figure 30 The synthetic routes for preparing the connector-payloads LP13 and LP14 according to this disclosure are shown.

[0303] Figure 31 The synthetic routes for preparing the connector-payloads LP15 and LP18 according to this disclosure are shown.

[0304] Figure 32 A synthetic route for preparing the connector-payload LP17 according to this disclosure is shown.

[0305] Figure 33 The synthetic routes for preparing the connector-payloads LP18 and LP20 according to this disclosure are shown.

[0306] Figure 34 A synthetic route for preparing the connector-payload LP19 according to this disclosure is shown.

[0307] Figure 35 A synthetic route for preparing the connector-payload LP21 according to this disclosure is shown.

[0308] Figure 36 A synthetic route for preparing the connector-payload LP22 according to this disclosure is shown.

[0309] Figure 37 A synthetic route for preparing the connector-payload LP23 according to this disclosure is shown.

[0310] Figure 38 A synthetic route for preparing the connector-payload LP24 according to this disclosure is shown.

[0311] Figure 39 A synthetic route for preparing the connector-payload LP25 according to this disclosure is shown.

[0312] Figure 40 A synthetic route for preparing the connector-payload LP26 according to this disclosure is shown.

[0313] Figure 41 The synthetic route for preparing the connector-payloads LP27 and LP28 according to this disclosure is shown.

[0314] Figure 42 A synthetic route for preparing the connector-payload LP29 according to this disclosure is shown.

[0315] Figure 43 A synthetic route for preparing the connector-payload LP30 according to this disclosure is shown.

[0316] Figure 44 A synthetic route for preparing the connector-payload LP31 according to this disclosure is shown.

[0317] Figure 45 A synthetic route for preparing the connector-payload LP32 according to this disclosure is shown.

[0318] Figure 46 A synthetic route for preparing the connector-payload LP33 according to this disclosure is shown.

[0319] Figure 47A synthetic route for preparing the connector-payload LP34 according to this disclosure is shown.

[0320] Figure 48 A synthetic route for preparing the connector-payload LP35 according to this disclosure is shown.

[0321] Figure 49 Synthetic routes for preparing the connector-payloads LP36, LP37, LP38, LP39, LP40 and LP41 according to this disclosure are shown.

[0322] Figure 50 A synthetic route for preparing the connector-payload LP42 according to this disclosure is shown.

[0323] Figure 51 A synthetic route for preparing the connector-payload LP43 according to this disclosure is shown.

[0324] Figure 52 A synthetic route for preparing the connector-payload LP44 according to this disclosure is shown.

[0325] Figure 53 A synthetic route for preparing the connector-payload LP45 according to this disclosure is shown.

[0326] Figure 54 A schematic diagram of a general two-step conjugation procedure for preparing site-specific antibody-drug conjugates is shown.

[0327] Figure 55 A schematic diagram of a general one-step conjugation procedure for preparing site-specific antibody-drug conjugates is shown.

[0328] Figure 56 The voltage command protocol for electrophysiological studies is illustrated. Starting from a holding potential of -80 mV, the voltage is first stepped to -50 mV for 80 ms for leak subtraction, and then stepped to +20 mV for 4800 ms to open the hERG channel. After this, the voltage is stepped back to -50 mV for 5000 ms, inducing a "bounce" or tail current, which is measured and collected for data analysis. Finally, the voltage is stepped back to the holding potential (-80 mV, 1000 ms). The voltage command protocol is repeated every 20 seconds and continuously during the tests (mediate control and test compound).

[0329] Figure 57 The in vitro stability of anti-GLP1R mAB2-LP11 was demonstrated after incubation at 37°C for 7 days in mouse, monkey, and human plasma.

[0330] Figure 58The effect of GLP1R ATDC on the percentage change in body weight in obese GLP1R humanized mice is shown.

[0331] Figure 59 The effect of GLP1R ATDC on blood glucose levels in obese GLP1R humanized mice was demonstrated.

[0332] Figure 60 This is a schematic diagram of an exemplary embodiment of the GLP1R ATDC according to the present disclosure.

[0333] Detailed description

[0334] In some aspects, this disclosure provides antibody-drug conjugates that specifically bind to the glucagon-like peptide-1 receptor (GLP1R) protein. As described in the background section above, GLP1R and its ligand GLP1 are highly effective targets for obesity and type 2 diabetes. However, direct agonist antibodies for the treatment of type 2 diabetes have not been identified. Monopeptides with agonist activity against GLP1R are effective therapeutic agents for glucose control and weight loss, but in-line peptide-antibody fusions are susceptible to proteolysis. In certain embodiments of this disclosure, antibody-drug conjugates are generated by combining an antibody specifically targeting the extracellular domain of GLP1R or an antigen-binding fragment thereof with a GLP1 peptide mimic that functionally activates GLP1R. In some embodiments, the antibody-drug conjugates of this disclosure have a longer duration of action, comparable or better weight and glucose reduction potency, and minimized off-target side effects.

[0335] This document discloses detailed embodiments of the present disclosure; however, it should be understood that the disclosed embodiments are merely illustrative of the present disclosure, which can be manifested in various forms. Furthermore, each example given in connection with the various embodiments of the present disclosure is intended to be illustrative, not restrictive. Therefore, the specific structural and functional details disclosed herein should not be construed as restrictive, but merely as a representative basis for instructing those skilled in the art to employ the present disclosure in different ways.

[0336] definition

[0337] 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 disclosure pertains.

[0338] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, reference to “a method” includes one or more methods and / or types of steps described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0339] The term "treat" or "treatment" for a state, disorder, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or subclinical symptom of a state, disorder, or condition in a subject who may be susceptible to or have a state, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; or (2) suppressing a state, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or the recurrence of at least one clinical or subclinical symptom; or (3) alleviating the disease, i.e., causing the resolution of at least one clinical or subclinical symptom of the state, disorder, or condition. The benefit to the subject treated is statistically significant or at least perceptible to the patient or physician. In some embodiments, treatment includes methods of ablating cells in a manner that indirectly affects the disease. In some embodiments, treatment includes depleting immune cells as a hematopoietic modulatory protocol prior to therapy.

[0340] As used herein, “subject,” “patient,” “individual,” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0341] As used herein, the term "effective" when applied to dosage or amount means an amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject with the appropriate need. Note that when administering a combination of active ingredients, the effective amount of the composition may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject based on species, age and general condition, severity of the condition being treated, one or more specific drugs used, mode of administration, etc.

[0342] The phrase “pharmaceutically acceptable salt” as used in conjunction with the compositions described herein refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci., 1977, 66:1, which is incorporated herein by reference. Examples of salts include, but are not limited to, salts of acid origin, salts of base origin, organic salts, inorganic salts, amine salts, and alkali metal or alkaline earth metal salts, including, but not limited to, salts of calcium, magnesium, potassium, sodium, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. In some instances, the payloads described herein include tertiary amines, wherein the nitrogen atom in the tertiary amine is the atom of the payload bonded to a linker or linker-spacer. In this case, a quaternary amine is formed by bonding with the tertiary amine of the payload in the linker-payload molecule. The positive charge on the quaternary amine can be balanced by an antibalance ion (such as chlorine, bromine, iodine, or any other suitable charged portion, such as those described herein).

[0343] A range may be expressed herein as from “about” or “approximately” a particular value, and / or to “about” or “approximately” another particular value. Another embodiment of expressing such a range includes from one particular value and / or to another particular value.

[0344] "Comprising" or "containing" or "including" means that at least the named compound, element, particle, or method step is present in the composition, article, or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if the other such compounds, materials, particles, or method steps have the same function as the named ones.

[0345] The compounds disclosed herein include those generally described herein, and are further described by the classes, subclasses and species disclosed herein. Unless otherwise indicated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Additionally, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th edition, eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0346] As used herein, the term "alkyl" is given its common meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, the straight-chain or branched alkyl group has about 1-20 carbon atoms (e.g., C1-C1) in its main chain. 20 For straight chains, C2-C 20 For branched chains), and optionally about 1-10 carbon atoms, or about 1-6 carbon atoms. In some embodiments, the cycloalkyl ring has about 3-10 carbon atoms in its ring structure, wherein such a ring is monocyclic or bicyclic, and optionally has about 5, 6, or 7 carbons in the ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight-chain lower alkyl groups).

[0347] As used herein, the term "alkenyl" refers to an alkyl group as defined herein that has one or more double bonds.

[0348] As used herein, the term "alkynyl" refers to an alkyl group as defined herein that has one or more triple bonds.

[0349] The term “heteroatom” refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; any quaternized form of basic nitrogen; or substituted nitrogen of a heterocycle).

[0350] The term "halogen" refers to F, Cl, Br, or I; the term "halogen" refers to a halogen group or substituent, namely -F, -Cl, -Br, or -I.

[0351] The term "adduct" in this disclosure, such as "Diels-Alder adduct," covers any portion of the product of an addition reaction (e.g., a Diels-Alder reaction), regardless of the synthetic steps taken to produce that portion.

[0352] The term "covalent attachment" refers to the formation of a covalent bond, a chemical bond involving two atoms sharing one or more electron pairs. Covalent bonding can include various interactions, including but not limited to σ-bonding, π-bonding, metal-to-metal bonding, agostic interactions, bent bonds, and three-center two-electron bonds. When a first group is described as "capable of covalent attachment" to a second group, it means that the first group can form a covalent bond with the second group directly or indirectly, for example, by using a catalyst or under specific reaction conditions. Non-limiting examples of groups capable of covalent attachment to each other can include, for example, amines and carboxylic acids (forming amide bonds), dienes and dienophiles (via the Diels-Alder reaction), maleimides and thiols (forming thiols-maleimides), and azides and alkynes (forming triazoles via 1,3-cycloaddition reactions).

[0353] As described herein, compounds of this disclosure may include "optionally substituted" portions. Generally, the term "substituted," whether or not preceded by the term "optionally," means that one or more hydrogens of the specified portion are substituted by suitable substituents. Unless otherwise indicated, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a particular group, the substituents may be the same or different at each position. The combinations of substituents contemplated in this disclosure are preferably those that result in the formation of stable or chemically viable compounds.

[0354] As used herein, the term "stable" means a compound that remains substantially unchanged when subjected to conditions that allow for its generation, detection, and, in some embodiments, its recovery, purification, and use for one or more purposes disclosed herein.

[0355] Unless otherwise stated, the structures described herein are also intended to include all isomers of the structure (e.g., enantiomers, diastereomers, and geometric (or conformations)); for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of this disclosure.

[0356] Unless otherwise stated, the cycloadducts described herein, such as products of cycloaddition reactions, including those from azide-acetylene cycloaddition or Diels-Alder reactions, include all positional isomers, i.e., structural isomers that differ only in the position of functional groups or substituents. By way of example, the following structures represent triazole positional isomers that differ only in the position of substituents on the triazole ring:

[0357] Triazole positional isomers can also be represented by the following structures:

[0358] Unless otherwise stated, all tautomer forms of the compounds disclosed herein are within the scope of this disclosure.

[0359] Additionally, unless otherwise stated, the structures described herein also refer to compounds that differ only in the presence of atoms enriched in one or more isotopes. For example, those that, in addition to replacing hydrogen with deuterium or tritium, or those that use... 11 C- or 13 C- or 14 Compounds having the structure of this invention, other than C-enriched carbon substitutes for carbon, are within the scope of this disclosure.

[0360] It should also be understood that referring to one or more method steps does not preclude the existence of other method steps or intermediate method steps between those explicitly identified steps. Similarly, it should be understood that referring to one or more components of an apparatus or system does not preclude the existence of other components or intermediate components between those explicitly identified components.

[0361] Unless otherwise stated, all crystalline forms of the compounds and their salts disclosed herein are also within the scope of this disclosure. The compounds of this disclosure can be isolated in a variety of amorphous and crystalline forms, including but not limited to anhydrous, hydrated, non-solventized, or solvated forms. Exemplary hydrates include hemihydrates, monohydrates, dihydrates, etc. In some embodiments, the compounds of this disclosure are anhydrous and non-solventized. “Anhydrous” means that the crystalline form of the compound contains substantially no bound water in its crystal lattice structure, i.e., the compound does not form crystalline hydrates.

[0362] As used herein, "crystallization form" refers to a specific lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically possess different lattices (e.g., unit cells), resulting in distinct physical properties characteristic of each form. In some cases, different lattice configurations exhibit different water or solvent contents. Different crystalline lattices can be identified using solid-state characterization methods such as X-ray powder diffraction (PXRD). Other characterization methods, such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor adsorption (DVS), and solid-state NMR, further aid in identifying crystalline forms and determining stability and solvent / water content.

[0363] The crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. The hydrated form is a crystalline form that includes water within a crystalline lattice. The hydrated form can be a stoichiometric hydrate, where water exists in the lattice at a given water / molecule ratio, such as a hemihydrate, monohydrate, dihydrate, etc. The hydrated form can also be non-stoichiometric, where the water content is variable and depends on external conditions such as humidity.

[0364] In some embodiments, the compounds of this disclosure are substantially separated. "Substantially separated" means that a particular compound is at least partially separated from impurities. For example, in some embodiments, the compounds of this disclosure include less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not substantially separated from the compound, including, for example, other crystalline forms and other substances.

[0365] Certain groups, parts, substituents, and atoms are depicted with wavy lines. Wavy lines may intersect one or more bonds or cap one or more bonds. Wavy lines indicate the group, part, substituent, or atom through the atoms it is bonded to. For example, a phenyl group substituted with a propyl group as depicted below:

[0366] It has the following structure:

[0367] The term “GLP1R” refers to the glucagon-like peptide-1 receptor and includes recombinant GLP1R protein or fragments thereof. GLP1R has a sequence of 463 residues. Donnelly, Br J Pharmacol, 166(1):27–41 (2011). Glucagon-like peptide-1 (GLP1) is a 31-amino acid peptide hormone released from intestinal L cells after nutrient consumption. The binding of GLP1 to GLP1R enhances glucose-induced insulin secretion from pancreatic β cells, increases insulin expression, inhibits β cell apoptosis, promotes β cell regeneration, reduces glucagon secretion, delays gastric emptying, promotes satiety, and increases peripheral glucose disposal.

[0368] The phrase “antibody that binds to GLP1R” or “anti-GLP1R antibody” includes antibodies that specifically recognize GLP1R and their antigen-binding fragments.

[0369] As used herein, “agonist antibody” (or “antibody that increases or enhances GLP1R activity”) means an antibody that binds to GLP1R, resulting in activation of at least one biological activity of GLP1R. For example, if the cells are mammalian pancreatic β cells, an agonist antibody against GLP1R can stimulate the adenylate cyclase pathway, leading to increased synthesis of cyclic AMP and release of insulin. An agonist antibody against GLP1R can also lower glucose levels after administration to a subject in need.

[0370] All amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as shown in 37C.FR §1.822(B)(J).

[0371] The term "protein" refers to any amino acid polymer having more than 20 amino acids covalently linked via amide bonds. As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, etc. Proteins can be produced using recombinant cell-based production systems, such as bacculovirus systems, yeast systems (e.g., Pichia sp. species), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).

[0372] Unless explicitly specified as originating from a non-human species, all references to proteins, peptides, and protein fragments herein are intended to refer to the human form of the corresponding protein, peptide, or protein fragment. Therefore, unless specified as originating from a non-human species, such as “mouse GLP1R,” “monkey GLP1R,” etc., the expression “GLP1R” refers to human GLP1R.

[0373] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described below: Kabat et al. (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol., 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply the absence of differences in the sequence, and those skilled in the art can readily confirm the sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, when referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported by Kabat et al., ibid.).

[0374] The term "glutamine acyl-modified antibody" refers to an antibody of this disclosure having at least one covalent link from a glutamine side chain to a primary amine compound. In certain embodiments, the primary amine compound is linked via an amide link on the glutamine side chain. In some embodiments, the glutamine is endogenous glutamine. In other embodiments, the glutamine is endogenous glutamine that has been made reactive through peptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the peptide). In yet another embodiment, the glutamine is a peptide engineered with a tag containing an acyl donor glutamine (e.g., a glutamine-containing peptide tag, Q-tag, or TG recognition tag).

[0375] The term "TG recognition tag" refers to an amino acid sequence comprising a recipient glutamine residue, which, upon incorporation (e.g., attachment) into a polypeptide sequence, is recognized by TG under suitable conditions, and cross-linking of TG occurs through a reaction between the amino acid side chain within the amino acid sequence and the reaction partner. The recognition tag can be a peptide sequence not naturally present in the polypeptide comprising the TG recognition tag. In some embodiments, the TG recognition tag comprises at least one Gln. In some embodiments, the TG recognition tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or unconventional amino acids). In some embodiments, the tag containing the acyl donor glutamine includes an amino acid sequence selected from the group consisting of: LLQGG (SEQ ID NO:6), LLQG (SEQ ID NO:7), LSLSQG (SEQ ID NO:8), GGGLLQGG (SEQ ID NO:9), GLLQG (SEQ ID NO:10), GSPLAQSHGG (SEQ ID NO:11), GLLQGGG (SEQ ID NO:12), GLLQGG (SEQ ID NO:13), GLLQ (SEQ ID NO:14), LLQLLQGA (SEQ ID NO:15), LLQGA (SEQ ID NO:16), LLQYQGA (SEQ ID NO:17), LLQGSG (SEQ ID NO:18), LLQYQG (SEQ ID NO:19), LLQLLQG (SEQ ID NO:20), SLLQG (SEQ ID NO:21), LLQLQ (SEQ ID NO:22), LLQLLQ (SEQ ID NO:23), LLQLLQ (SEQ ID NO:24), LLQLQ (SEQ ID NO:25), LLQLLQ (SEQ ID NO:26), LLQLLQ (SEQ ID NO:27), LLQGG (SEQ ID NO:28), LLQLQ (SEQ ID NO:29), LLQLLQ (SEQ ID NO:20), LLQLQ (SEQ ID NO:21), LLQLLQ (SEQ ID NO:22), LLQLLQ (SEQ ID NO:23), LLQLLQ (SEQ ID NO:24), LLQLQ (SEQ ID NO:25), LLQLLQ (SEQ ID NO:26), LLQLQ (SEQ ID NO:27), LLQLQ (SEQ ID NO:28), LLQLQ (SEQ ID NO:29), LLQLLQ (SEQ ID NO:20), LLQLQ (SEQ ID NO:20), LLQLQ (SEQ ID NO:21), LLQL NO:23) and LLQGR (SEQ ID NO:24). See, for example, WO2012059882, the entire contents of which are incorporated herein by reference.

[0376] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM) comprising four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of the antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0377] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. The terms "antigen-binding portion" and "antigen-binding fragment" of an antibody, as used herein, include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from, for example, complete antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulating and expressing DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, arranging one or more variable and / or constant domains into a suitable conformation, or introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0378] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues of a hypervariable region of a mimic antibody (e.g., a separated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed in the term "antigen-binding fragments" as used herein.

[0379] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will typically contain at least one CDR adjacent to or within one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and may contain VH-VH, VH-VL, or VL-VL dimers.

[0380] Optionally, the antigen-binding fragment of the antibody may contain a monomeric VH or VL domain.

[0381] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be present in the antigen-binding fragment of the antibody described herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of the variable and constant domains (including any exemplary configurations listed herein), the variable and constant domains may be directly connected to each other or connected via complete or partial hinge or connector regions. The hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids that result in flexible or semi-flexible connections between adjacent variable and / or constant domains within a single polypeptide molecule.

[0382] In addition, the antigen-binding fragments of the antibodies described herein may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed herein, which are non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via one or more disulfide bonds).

[0383] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically include at least two distinct variable domains, each capable of binding specifically to a single antigen or to different epitopes on the same antigen. Any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed herein, can be adapted for use in the context of the antigen-binding fragments of the antibodies described herein using conventional techniques available in the art.

[0384] In some embodiments, the antibodies of this specification, such as anti-GLP1R antibodies, are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this specification may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutations), said amino acid residues, for example, in the CDR and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human framework sequence.

[0385] In some embodiments, the antibody may be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (described further below), antibodies isolated from recombinant, combined human antibody libraries (described further below), antibodies isolated from animals that are transgenic with respect to human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means including splicing a human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using transgenic animals with human Ig sequences, in vivo somatic cell mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and associated with human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire.

[0386] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, where the dimers are held together by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds and form a molecule of approximately 75-80 kDa (half antibody) comprising covalently coupled light and heavy chains. These forms are extremely difficult to separate even after affinity purification. The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with antibody hinge region isotypes. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. This specification covers antibodies with one or more mutations in the hinge region, CH2 region, or CH3 region, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0387] The antibodies described herein may be isolated or purified antibodies. As used herein, "isolated antibody" or "purified antibody" means an antibody that has been identified and isolated and / or recovered from at least one component of its natural environment. For the purposes of this specification, for example, an antibody that has been isolated or removed from at least one component of an organism or from tissues or cells where the antibody is naturally present or produced is an "isolated antibody." For example, an antibody that has been purified from at least one component of a reaction or reaction sequence is a "purified antibody" or is produced from a purified antibody. Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to some embodiments, isolated or purified antibodies may be substantially free of other cellular material and / or chemicals.

[0388] Compared to the corresponding germline sequence from which the antibody is derived, the antibodies disclosed herein may include one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. This specification includes antibodies and antigen-binding fragments derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework and / or CDR regions are mutated to corresponding residues in one or more of the antibody-derived germline sequence, or one or more of the corresponding residues in another human germline sequence, or conserved amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily generate numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting from the given heavy and light chain variable region sequences. In some embodiments, all framework and / or CDR residues within the VH and / or VL domains are mutated back to residues present in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues present only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues present only in CDR1, CDR2, or CDR3. In other embodiments, one or more frames and / or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated).

[0389] Furthermore, the antibodies described in this specification may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues of a different germline sequence. Once obtained, one or more desired properties of the antibody and antigen-binding fragment containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, improved drug-to-antibody ratio (DAR) of the antibody-drug conjugate, etc. Antibody and antigen-binding fragments obtained in this general manner are covered in this specification.

[0390] The term "non-glycosylated antibody" refers to an antibody that does not contain glycosylated sequences that could interfere with the transglutamine reaction, such as antibodies that do not have a glycosylation group at N297 on one or more heavy chains. In a particular embodiment, the antibody heavy chain has an N297 mutation. In other words, according to the EU numbering system disclosed by Kabat et al., the antibody is mutated to no longer have an asparagine residue at position 297. In a particular embodiment, the antibody heavy chain has an N297Q or N297D mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or disable the glycosylated sequence, or by site-directed mutagenesis to insert a glutamine residue at a site other than any interfering glycosylation site or any other interfering structure. Such antibodies can also be isolated from natural or artificial sources. Non-glycosylated antibodies also include antibodies containing T299 or S298P or other mutations or combinations of mutations that result in the absence of glycosylation.

[0391] The term "deglycosylated antibody" refers to an antibody in which a glycosyl group is removed to facilitate transglutaminase-mediated conjugation. The sugar includes, but is not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation occurs at residue N297. In some embodiments, the removal of the glycosyl group is enzymatic (including, but not limited to, via PNG (Peptide: N-glycosidase, PNGase)).

[0392] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions of the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated from spatially aligned amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated from adjacent amino acid residues in the polypeptide chain. In some cases, an epitope may include portions of a sugar, phosphoryl group, or sulfonyl group on the antigen.

[0393] As used herein, the terms “conjugated protein” or “conjugated antibody” refer to a protein or antibody covalently linked to one or more chemical moieties. The chemical moieties may include amine compounds of the present disclosure. Connectors (L) and payloads (P) suitable for use with the present disclosure are described in detail herein. In a particular embodiment, a conjugated antibody including a therapeutic moieties is an antibody-drug conjugate (ADC), also known as an antibody-payload conjugate or antibody-connector-payload conjugate.

[0394] The term "drug to antibody ratio" or (DAR) is the average of the therapeutic portions (e.g., drugs) conjugated with the binder of this disclosure.

[0395] In some embodiments, the term "connector-antibody ratio" or (LAR), also indicated by lowercase letters, is the average number of reactive primary amine compounds conjugated with the binders of this disclosure. Such binders, such as antibodies, can be conjugated with primary amine compounds comprising, for example, suitable azides or alkynes. The resulting binder, functionalized with an azide or alkyne, can then react with a therapeutic portion comprising the corresponding azide or alkyne via a 1,3-cycloaddition reaction.

[0396] The phrase “pharmaceutically acceptable amount” refers to an amount that is effective or sufficient in treating, reducing, alleviating, or modulating the effects or symptoms of at least one health problem in a subject with an appropriate need. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount that is effective in modulating a biological target using the antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, up to about 0.001% and about 10%, and any amounts in between, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the antibody or antibody-drug-conjugate provided herein.

[0397] The phrase "reaction pH" refers to the pH of the reaction after all reactants or reactants have been added.

[0398] When referring to nucleic acids or fragments thereof, the terms "substantially identical" or "substantially the same" indicate that, upon optimal alignment with appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand), as discussed below, and measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases. In some cases, a nucleic acid molecule substantially identical to a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0399] When applied to peptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when best aligned using procedures such as GAP or BESTFIT with default vacancy weights. Preferably, the dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of amino acid groups with side chains having similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, the conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0400] Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-possibility matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A “moderately conservative” substitution is any change with a non-negative value in the PAM250 log-possibility matrix.

[0401] Sequence similarity of peptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides, such as homologous peptides from different biological species or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (the program in GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions between the query sequence and the search sequence and a percentage of sequence identity (Pearson (2000) ibid.). Another specific algorithm used when comparing sequences in this specification with databases containing a large number of sequences from different organisms is the computer program BLAST, specifically BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402.

[0402] Protein-drug conjugates

[0403] For the foregoing and other purposes, this disclosure provides protein-drug conjugate compounds, such as antibody-drug conjugate compounds, their precursors and intermediates, pharmaceutical compositions, and methods for treating certain diseases in subjects with such therapeutic needs. According to this disclosure, the protein-drug conjugate compounds provided herein comprise a binding agent conjugated to a therapeutic portion, such as a GLP1 peptide mimic as described herein.

[0404] In one aspect, this disclosure provides compounds comprising a binder (e.g., an antibody or a fragment thereof) according to this disclosure, the binder being conjugated to one or more GLP1 peptide mimics via a non-cleavable linker. Illustrative and non-limiting examples include formula (I) described herein. In specific embodiments of protein-drug conjugates according to this disclosure, wherein the binder is an antibody (e.g., a monoclonal antibody), the terms "antibody-drug conjugate" or ADC are optionally used.

[0405] In various embodiments, the antibody-drug conjugates or ADCs of this disclosure are antibody-tethered drug conjugates or ATDCs. ATDCs are antibody-drug conjugates in which the drug is tethered to the antibody via a non-cleavable linker. In some embodiments, the non-cleavable linker in the ATDC of this disclosure is stable after the ATDC is administered in vivo (e.g., to a human body). For example, the non-cleavable linker may be stable in plasma, such as in human plasma, stable when binding to cell surfaces, or stable when the antibody binds to its target antigen and / or a GLP1 peptide mimic binds to GLP1R. In some embodiments, the non-cleavable linker is more stable in vivo than the payload or antibody under the same physiological conditions. In some embodiments, the ATDC of this disclosure may be degraded in lysosomes to release the payload, linker-payload, and / or its ATDC metabolites / catabolites, which in some embodiments are effective for local or systemic GLP1R activation.

[0406] In some embodiments, ATDC is stable in plasma and degrades in lysosomes. In other embodiments, ATDC is stable in plasma and does not degrade in lysosomes.

[0407] In one respect, this paper provides compounds having the structure of formula (A):

[0408] BA-(LP) m (A),

[0409] in:

[0410] BA is an antibody or its antigen-binding fragment;

[0411] L is a non-disintegrable connector;

[0412] P is the effective load of a structure that is selected from the following group:

[0413]

[0414]

[0415] in The attachment point where the payload is attached to L;

[0416] X1 is selected from H;

[0417] X2 is selected from

[0418] X3 is selected from the bond, –(CH2). 2-6 –NH–, –(CH2) 2-6 –Tr– and –(CH2)2-6 –Tr–(CH2) 1-6 –NH, where Tr is the triazole moiety;

[0419] n is 0 or 1;

[0420] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0421] X5 is selected from -OH, -NH2, -NH-OH, and

[0422] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0423] X7 is selected from H,

[0424] X8 is selected from H, -OH, -NH2, and

[0425] Ar selected

[0426] X9 is selected from -NH2, and

[0427] m is an integer from 1 to 4

[0428] Or its pharmaceutically acceptable salt.

[0429] In one implementation, m is 1. In one implementation, m is an integer from 2 to 4. In one implementation, m is 2.

[0430] In one aspect, this disclosure provides compounds having the structure of formula (I):

[0431] BA-LP(I),

[0432] in:

[0433] BA is an antibody or its antigen-binding fragment;

[0434] L is a non-disintegrable connector;

[0435] P is the effective load of a structure that is selected from the following group:

[0436]

[0437] in The attachment point where the payload is attached to L;

[0438] X1 is selected from H;

[0439] X2 is selected from

[0440] X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety;

[0441] n is 0 or 1;

[0442] X4 is selected from H and phenyl;

[0443] X5 is selected from -OH, -NH2, -NH-OH, and

[0444] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0445] X7 is selected from H,

[0446] X8 is selected from H, -OH, -NH2, and

[0447] Or its pharmaceutically acceptable salt.

[0448] Connector L

[0449] In one embodiment, the linker L is a non-cleavable linker, i.e., stable and providing a covalent link between the antibody and the drug, for example, between an antibody targeting GLP1R according to this disclosure and a GLP1 peptide mimic payload P. In some embodiments, the non-cleavable linker L of this disclosure is stable after ATDC is administered in vivo (e.g., to a human body). For example, linker L may be stable in plasma, such as in human plasma, stable when binding to cell surfaces, or stable when the antibody binds to its target antigen and / or the GLP1 peptide mimic binds to GLP1R. In some embodiments, linker L is more stable in vivo than the payload or antibody under the same physiological conditions.

[0450] In one embodiment, the connector L has a structure of formula (L'):

[0451] -La-Y-Lp-(L'),

[0452] La is the first linker that is covalently attached to the antibody or its antigen-binding fragment;

[0453] Y is a group containing triazole, Diels-Alder adduct, or thiol-maleimide adduct, and

[0454] Lp either does not exist or is a second joint covalently attached to the payload P according to this disclosure, wherein Y also does not exist when Lp does not exist.

[0455] In one embodiment, Y is a triazole group.

[0456] In another embodiment, Y is a group containing a Diels-Alder adduct.

[0457] In one embodiment, the connector L has a structure of formula (L'):

[0458] -La-Y-Lp-(L'),

[0459] La is the first linker that is covalently attached to the antibody or its antigen-binding fragment;

[0460] Y is a group containing triazole, and

[0461] Lp is absent or is a second joint covalently attached to the payload P according to this disclosure.

[0462] In one implementation, La includes C 1-6 Alkyl, phenyl, -NH-, -C(O)-, -(CH2) u -NH-C(O)-、-(CH2) u -C(O)-NH-、-(CH2-CH2-O) v -、-(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit comprising 2 to 4 amino acids or a combination thereof, each of which may optionally be substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)- and CO2H, wherein the subscripts u and v are independently integers from 1 to 8.

[0463] In one implementation, La is selected from the group consisting of:

[0464] Where R A It is a group comprising alkynes, azides, tetrazides, trans-cyclooctene, maleimide, amines, ketones, aldehydes, carboxylic acids, esters, thiols, sulfonic acids, toluene sulfonates, halides, silanes, cyano groups, sugar groups, biotin groups, lipid residues, and wherein the subscripts x, n, p, and q are independently integers from 0 to 12, and combinations thereof.

[0465] In one implementation, -La- is selected from the group consisting of:

[0466]

[0467]

[0468] in It is an amino attachment site that attaches to residues (e.g., glutamine residues) of an antibody and / or its antigen-binding fragment.

[0469] In one implementation, -La- is

[0470] In another implementation, -La- is selected from the group consisting of:

[0471]

[0472]

[0473] in It is an amino attachment site that attaches to residues (e.g., glutamine residues) of an antibody and / or its antigen-binding fragment.

[0474] In some embodiments, La includes a polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units. In some embodiments, the PEG segment includes 4, 8, 12, or 24 EG units. In some embodiments, the PEG segment includes 8 EG units. In some embodiments, La has a structure selected from the group consisting of: and

[0475] In some embodiments, La comprises one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. In some embodiments, La comprises 1 to 10 glycines and 1 to 6 serines. In some embodiments, La comprises 4 glycines and 1 serine. In some embodiments, La is selected from the group consisting of: Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO:1), Ser-Gly-Gly-Gly-Gly (SG4) (SEQ ID NO:2), Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (G2S-G2S-G2) and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (G4S-G4) (SEQ ID NO:3).

[0476] In some embodiments, La comprises a PEG segment having 1 to 36 EG units and a combination of one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. In some embodiments, La is selected from the group consisting of:

[0477]

[0478] In some implementations, La includes –(CH2). 2-24 -Chain. In some implementations, La includes –(CH2). 2-24 - A chain, a PEG segment having 1 to 36 EG units, and a combination of one or more amino acids selected from glycine, threonine, serine, glutamine, glutamic acid, alanine, valine, leucine, and proline, and combinations thereof. La is selected from the group consisting of:

[0479]

[0480] In one implementation, Y has a structure selected from the group consisting of:

[0481] Where Q is C or N.

[0482] In one embodiment, the connector L or the first connector La or the second connector Lp includes a polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units.

[0483] In one embodiment, the PEG segment includes between 2 and 30 EG units or between 4 and 24 EG units. In another embodiment, the PEG segment includes 2 EG units, 4 EG units, 6 EG units, 8 EG units, 10 EG units, 12 EG units, 14 EG units, 16 EG units, 18 EG units, 20 EG units, 22 EG units, or 24 EG units.

[0484] In one embodiment, the PEG segment includes 4 EG units. In one embodiment, the PEG segment includes 8 EG units. In one embodiment, the PEG segment includes 12 EG units. In one embodiment, the PEG segment includes 24 EG units.

[0485] In one embodiment, the PEG segment includes 4 to 8 EG units. In another embodiment, the PEG segment includes 4 or 8 EG units.

[0486] In one embodiment, La includes a PEG segment having 3 EG units.

[0487] In one embodiment, Lp includes a PEG segment having 4 EG units. In another embodiment, Lp includes a PEG segment having 8 EG units.

[0488] In one implementation, Y-Lp has a structure selected from the group consisting of:

[0489]

[0490] Where p is an integer from 1 to 36.

[0491] In one implementation, Y-Lp has a structure selected from the group consisting of:

[0492] Or its triazole positional isomer,

[0493] Where p is an integer from 1 to 36.

[0494] In another embodiment, the connector L or the first connector La or the second connector Lp comprises one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof.

[0495] In one embodiment, the connector L or the first connector La or the second connector Lp includes 1 to 10 glycines, or 1 glycine, or 2 glycines, or 3 glycines, or 4 glycines, or 5 glycines, or 6 glycines, or 7 glycines, or 8 glycines, or 9 glycines, or 10 glycines.

[0496] In one embodiment, the connector L or the first connector La or the second connector Lp includes 1 to 6 serines or 1 serine or 2 serines or 3 serines or 4 serines or 5 serines or 6 serines.

[0497] In one embodiment, the connector L or the first connector La or the second connector Lp comprises 1 to 10 glycines and 1 to 6 serines.

[0498] In one embodiment, the connector L or the first connector La or the second connector Lp comprises 4 glycines and 1 serine.

[0499] In one embodiment, the connector L or the first connector La or the second connector Lp is selected from the group consisting of: Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO:1), Ser-Gly-Gly-Gly-Gly (SG4) (SEQ ID NO:2) and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (G4S-G4S) (SEQ ID NO:3).

[0500] In some embodiments, one or more serine residues contain a glycosylation group, such as a glucose group.

[0501] In one embodiment, the connector L or the first connector La or the second connector Lp comprises 1 to 10 glutamic acid and 1 to 10 glycine.

[0502] In some embodiments, the connector L or the first connector La or the second connector Lp comprises a polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units and a combination of one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline and combinations thereof.

[0503] In one embodiment, the connector L, the first connector La, or the second connector Lp comprises a PEG segment having 1 to 36 EG units and a combination of 1 to 10 glycine groups. In another embodiment, the connector L, the first connector La, or the second connector Lp comprises a PEG segment having 1 to 36 EG units and a combination of groups selected from Gly-Gly-Gly-Gly-Ser(G4S) (SEQ ID NO:1), Ser-Gly-Gly-Gly-Gly(SG4) (SEQ ID NO:2), and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(G4S-G4S) (SEQ ID NO:3).

[0504] In one embodiment, the connector L, or the first connector La, or the second connector Lp, has a structure selected from the group consisting of:

[0505]

[0506] Where Y is, for example, a triazole-containing group as shown above, and P is the payload, and Rc is selected from hydrogen (H) and glucose, g is an integer from 1 to 10, and s is an integer from 0 to 4.

[0507] In one implementation, Y-Lp has a structure selected from the group consisting of:

[0508]

[0509] as well as

[0510] In one embodiment, the connector L includes a cyclodextrin portion.

[0511] In some embodiments, the linker L attaches to the antibody or its antigen-binding fragment via a glutamine residue. In some embodiments, the linker L attaches to the antibody or its antigen-binding fragment via a lysine residue. In some embodiments, the linker L attaches to the antibody or its antigen-binding fragment via a cysteine ​​residue.

[0512] Payload P

[0513] In one aspect, the payload P according to this disclosure has a structure selected from the group consisting of formulas (P-IB), (P-IIB), and (P-IIIB):

[0514] (P-IIIB), where:

[0515] X1 is selected from H;

[0516] X2 is selected from

[0517] X3 is selected from -CH3 and –(CH2). 2-6 –NH2、–(CH2) 2-6 –N3 and –(CH2) 2-6 –Tr–(CH2) 1-6 –NH2, where Tr is the triazole moiety;

[0518] n is 0 or 1;

[0519] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0520] X5 is selected from -OH, -NH2, -NH-OH, and

[0521] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0522] X7 is selected from H,

[0523] X8 is selected from H, -OH, -NH2, and

[0524] Ar selected

[0525] X9 is selected from -NH2, and

[0526] m is an integer from 1 to 4.

[0527] Or its pharmaceutically acceptable salt.

[0528] In one embodiment, the payload P according to this disclosure has the structure of formula (II):

[0529]

[0530] in:

[0531] X1 is selected from H;

[0532] X2 is selected from

[0533] X3 is selected from –(CH2) 2-6 –NH2、–(CH2) 2-6 –N3 and -CH3, provided that n is 1 when X3 is -CH3, and Ra is selected from –(CH2) at least once. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0534] n is 0 or 1;

[0535] m is an integer from 0 to 3;

[0536] Ra is independently selected from -CH3 and –(CH2) each time it appears. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0537] X4 is selected from H and phenyl;

[0538] X5 is selected from -OH, -NH2, -NH-OH, and

[0539] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0540] X7 is selected from H,

[0541] X8 is selected from H, -OH, -NH2, and

[0542] And its pharmaceutically acceptable salts.

[0543] In one embodiment, the payload P has a structure selected from the following:

[0544]

[0545] in Indicates the attachment point to the connector.

[0546] In one embodiment, the payload has the structure of formula (PI) shown above, wherein

[0547] X1 is H; X2 is X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety; n is 1, and X4 is H;

[0548] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H, and X5 is selected from -OH, -NH2, -NH-OH, and

[0549] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H;

[0550] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH2OH each time it appears, and X7 is H;

[0551] X1 is X2 is X3 is –(CH2) 2-6 –Tr–, where Tr is the triazole moiety; n is 1; X4 is H, and X5 is

[0552] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears; X7 is And X8 is -NH2;

[0553] X1 is X2 is X3 is –(CH2)2-6 –NH–; n is 1; X4 is H, and X8 is H;

[0554] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is H every time it appears; X7 is And X8 is H;

[0555] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears; X7 is And X8 is

[0556] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H;

[0557] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H;

[0558] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H; X6 is independently selected from H and -CH3 each time it appears, and X7 is

[0559] X1 is H; X2 is X3 is –(CH2) 2-6 –NH–; n is 1, and X4 is H;

[0560] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is H, and X5 is

[0561] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 0; X4 is phenyl, and X5 is and

[0562] X1 is X2 is X3 is –(CH2) 2-6 –NH–; n is 1; X4 is phenyl, and X5 is

[0563] In one embodiment, the payload has a structure as shown above in formula (P-II), where X1 is... X2 is X3 is –(CH2) 2-6 –NH–; X4 is H, and X5 is

[0564] In one embodiment, the payload P according to this disclosure has a structure selected from the following:

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573] In one implementation, the payload as described above has the following characteristics:

[0574]

[0575]

[0576]

[0577] In some implementations, the payload of this disclosure is readily conjugated with a binding agent (e.g., an antibody).

[0578] Connector - Payload (LP)

[0579] In one embodiment, this disclosure provides a reactive adapter-payload, comprising a payload P as described above and an adapter capable of covalently attaching to an antibody or its antigen-binding fragment.

[0580] In one embodiment, the connector-payload according to this disclosure has a structure of formula (C):

[0581]

[0582] in:

[0583] L p The following types of connectors are absent or include one or more of the following: Carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units; –(CH2) 2-24 - chain; triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof;

[0584] Q is selected from -NH2, -N3. The part where A is C or N;

[0585] X1 is selected from H;

[0586] X2 is selected from

[0587] X3 is selected from -CH3 and –(CH2). 2-6 –NH2、–(CH2) 2-6 –N3 and –(CH2) 2-6 –Tr–(CH2) 1-6 –NH2, where Tr is the triazole moiety;

[0588] n is 0 or 1;

[0589] X4 is selected from -NH2, -OH and -N(H)(phenyl);

[0590] X5 is selected from -OH, -NH2, -NH-OH, and

[0591] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0592] X7 is selected from H,

[0593] X8 is selected from H, -OH, -NH2, and

[0594] Ar selected

[0595] X9 is selected from -NH2, and

[0596] m is an integer from 1 to 4

[0597] Or its pharmaceutically acceptable salt.

[0598] In one embodiment, the joint-load according to this disclosure has a structure of formula (III):

[0599]

[0600] in:

[0601] L p The following types of connectors are absent or include one or more of the following: Carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O-(EG) units; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine and proline and combinations thereof;

[0602] Q is selected from -N3, The part where A is C or N;

[0603] X1 is selected from H;

[0604] X2 is selected from

[0605] X3 is selected from –(CH2) 2-6 –NH2、–(CH2) 2-6 –N3 and -CH3, provided that n is 1 when X3 is -CH3, and Ra is selected from –(CH2) at least once. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0606] n is 0 or 1;

[0607] Ra is independently selected from H, -CH3, and –(CH2) each time it appears. 2-6 –NH2 and –(CH2) 2-6 –N3;

[0608] X4 is selected from H and phenyl;

[0609] X5 is selected from -OH, -NH2, -NH-OH, and

[0610] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0611] X7 is selected from H,

[0612] X8 is selected from H, -OH, -NH2, and

[0613] And its pharmaceutically acceptable salts.

[0614] In one embodiment, the linker-payload LP includes a cyclodextrin moiety. In some embodiments, the linker-payload LP including the cyclodextrin moiety exhibits GLP1R agonist activity.

[0615] In one embodiment, the connector-payload LP according to this disclosure has a structure selected from the group consisting of:

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627] In one embodiment, the joint-load capacity as described above has the following characteristics:

[0628]

[0629]

[0630] binder

[0631] In one embodiment, the effectiveness of the protein-drug conjugate embodiments described herein depends on the selectivity with which the binder binds to its binding partner. In one embodiment of this disclosure, the binder is any molecule capable of binding with a given binding partner with some specificity. In one embodiment, the binder is in vivo in a mammal, where the interaction can produce therapeutic use. In an alternative embodiment, the binder is in vitro, where the interaction can produce diagnostic use. In some aspects, the binder is capable of binding to cells or cell populations.

[0632] Suitable binders for this disclosure include proteins that bind to binding partners. Suitable binders include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell-binding or peptide-binding molecules or substances.

[0633] In one embodiment, the binder is an antibody. In some embodiments, the antibody is selected from monoclonal antibodies, polyclonal antibodies, antibody fragments (Fab, Fab', and F(ab)2, microantibodies, biantibodies, triantibodies, etc.). The antibodies described herein can be humanized using the methods described in U.S. Patent No. 6,596,541 and U.S. Publication No. 2012 / 0096572, both of which are incorporated herein by reference in their entirety. In some embodiments of the protein-drug conjugate compounds of this disclosure, BA is a humanized monoclonal antibody. For example, BA may be a monoclonal antibody that binds to GLP1R.

[0634] In this disclosure, the antibody can be any antibody considered suitable for those skilled in the art. In some embodiments, the adapter or adapter-payload is attached to one or both heavy chains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to one or both heavy chain variable domains of the antibody or its antigen-binding fragment.

[0635] In some embodiments, the adapter or adapter-payload is attached to the N-terminus of one or both heavy chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the N-terminus of both heavy chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to one or both light chains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to one or both light chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the N-terminus of one or both light chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the N-terminus of both light chain variable domains of the antibody or its antigen-binding fragment.

[0636] In some embodiments, the adapter or adapter-payload is attached to the C-terminus of one or both heavy chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the C-terminus of both heavy chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to one or both light chains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to one or both light chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the C-terminus of one or both light chain variable domains of the antibody or its antigen-binding fragment. In some embodiments, the adapter or adapter-payload is attached to the C-terminus of both light chain variable domains of the antibody or its antigen-binding fragment.

[0637] In some embodiments, the antibody includes at least one glutamine residue in at least one polypeptide chain sequence. In some embodiments, the antibody includes one or more Gln295 residues. In some embodiments, the antibody includes two heavy chain polypeptides, each having one Gln295 residue. In other embodiments, the antibody includes one or more glutamine residues at sites other than heavy chain 295. Such antibodies can be isolated from natural sources or engineered to include one or more glutamine residues. Techniques for engineering glutamine residues into antibody polypeptide chains are within the skill of those skilled in the art. In some embodiments, glutamine residues are introduced into the N-terminus of the antibody polypeptide chain. In one embodiment, glutamine residues are introduced into the N-terminus of one or both heavy chains of the antibody. In one embodiment, glutamine residues are introduced into the N-terminus of both heavy chains of the antibody. In another embodiment, glutamine residues are introduced into the N-terminus of one or both light chains of the antibody. In one embodiment, glutamine residues are introduced into the N-terminus of both light chains of the antibody. In another embodiment, glutamine residues are introduced into the N-terminus of one or two heavy chains and one or two light chains of the antibody.

[0638] In some embodiments, glutamine residues are introduced into the C-terminus of the antibody polypeptide chain. In one embodiment, glutamine residues are introduced into the C-terminus of one or both heavy chains of the antibody. In one embodiment, glutamine residues are introduced into the C-terminus of both heavy chains of the antibody. In another embodiment, glutamine residues are introduced into the C-terminus of one or both light chains of the antibody. In one embodiment, glutamine residues are introduced into the C-terminus of both light chains of the antibody. In yet another embodiment, glutamine residues are introduced into the C-terminus of one or both heavy chains and one or both light chains of the antibody.

[0639] In some implementations, the antibody or its antigen-binding fragment has been modified to include a TG recognition tag. Suitable TG recognition tags include those described herein.

[0640] In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the N-terminus of one or both antibody light chains. In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the N-terminus of both antibody light chains.

[0641] In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the N-terminus of one or both antibody heavy chains.

[0642] In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the C-terminus of one or both antibody light chains. In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the C-terminus of both antibody light chains.

[0643] In some embodiments, the antibody or its antigen-binding fragment has been modified to include a Q-tag at the C-terminus of one or both antibody heavy chains.

[0644] In some embodiments, the antibody or its antigen-binding fragment is non-glycosylated. In some embodiments, the antibody or its antigen-binding fragment is deglycosylated. In some embodiments, the antigen-binding fragment of the antibody is a Fab fragment.

[0645] Antibodies can be in any form known to those skilled in the art. In some embodiments, antibodies comprise light chains. In some embodiments, the light chain is a κ light chain. In some embodiments, the light chain is a λ light chain.

[0646] In some embodiments, the antibody comprises a heavy chain. In some aspects, the heavy chain is IgA. In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG1. In some aspects, the heavy chain is IgG2. In some aspects, the heavy chain is IgG3. In some aspects, the heavy chain is IgG4. In some aspects, the heavy chain is IgA1. In some aspects, the heavy chain is IgA2.

[0647] In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is an F(ab')2 fragment. In some aspects, the antibody fragment is a Fab' fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

[0648] In one embodiment, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0649] In some implementations, the antibody is a chimeric antibody. In some implementations, the antibody is a humanized antibody. In some implementations, the antibody is a human antibody.

[0650] Antibodies can have binding specificity to any antigen deemed suitable for those skilled in the art. In some embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include, but are not limited to, molecules such as GLP1R.

[0651] Some of the implementation schemes described herein are target-specific for therapeutic or diagnostic purposes.

[0652] In some embodiments, the binder is an anti-glucagon-like peptide-1 receptor (GLP1R), i.e., an anti-GLP1R antibody or its antigen-binding fragment. In some embodiments, the binder is an anti-GLP1R agonist antibody or its antigen-binding fragment.

[0653] In some implementations, suitable anti-GLP1R antibodies are those disclosed in U.S. Publication No. US20060275288A1, which is incorporated herein by reference in its entirety. Exemplary anti-GLP1R antibodies according to this disclosure include 5A10 and 9A10.

[0654] In some implementations, suitable anti-GLP1R antibodies include, but are not limited to, AB9433-I, h38C2, PA5-111834, NLS1205, MAB2814, EPR21819, and glutazumab.

[0655] This disclosure also provides nucleic acid molecules encoding anti-GLP1R antibodies or portions thereof. The scope of this disclosure also includes recombinant expression vectors capable of expressing polypeptides containing heavy or light chain variable regions of anti-GLP1R antibodies. For example, this disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above. The scope of this disclosure also includes host cells into which such vectors have been introduced, and methods for producing antibodies or portions thereof by culturing host cells under conditions allowing for the production of antibodies or antibody fragments and recovering the antibodies and antibody fragments thus produced.

[0656] In some embodiments, this disclosure includes anti-GLP1R antibodies having modified glycosylation patterns. In some embodiments, modifications to remove unwanted glycosylation sites may be useful, or, for example, antibodies lacking the fucose moiety present on the oligosaccharide chain may be used to enhance antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications may be performed to modify complement-dependent cytotoxicity (CDC).

[0657] In another aspect, this disclosure provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically binds to GLP1R and a pharmaceutically acceptable carrier. In a related aspect, this disclosure includes a composition that is a combination of an anti-GLP1R antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent advantageously combined with the anti-GLP1R antibody. Further combination therapies and co-formulations involving the anti-GLP1R antibody of this disclosure are disclosed elsewhere herein.

[0658] In another aspect, this disclosure provides a treatment method for targeting GLP1R-expressing cells using an anti-GLP1R antibody of this disclosure, wherein the treatment method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-GLP1R antibody of this disclosure to a subject in need. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are pancreatic cells, brain cells, heart cells, vascular tissue cells, kidney cells, adipose tissue cells, hepatocytes, or muscle cells. In one embodiment, the cells are pancreatic cells or brain cells.

[0659] In some cases, anti-GLP1R antibodies (or their antigen-binding fragments) can be used to enhance GLP1R activity in cells.

[0660] This disclosure also includes the use of the anti-GLP1R antibody of this disclosure in the preparation of medicaments for treating diseases or disorders associated with GLP1R-expressing cells (e.g., type II diabetes and / or obesity). In one aspect, this disclosure relates to a compound for medical use comprising an anti-GLP1R antibody or antigen-binding fragment as disclosed herein. In another aspect, this disclosure relates to a compound for medical use comprising an antibody-drug conjugate (ADC) as disclosed herein.

[0661] In another aspect, this disclosure provides monospecific anti-GLP1R antibodies for diagnostic applications such as, for example, imaging reagents.

[0662] This disclosure also includes antibody or antigen-binding fragments that compete with the antibodies described herein for binding to human GLP1R.

[0663] This disclosure also includes antibodies or antigen-binding fragments, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human GLP1R as the antibody described herein.

[0664] In one aspect, this disclosure provides antibody-drug conjugates comprising an anti-GLP1R antibody as described above, or an antigen-binding fragment thereof, and a therapeutic agent (e.g., a GLP1 peptide mimic). In some embodiments, the antibody or antigen-binding fragment and the payload are covalently attached via a linker, as discussed above. In many embodiments, the anti-GLP1R antibody or antigen-binding fragment can be any of the anti-GLP1R antibodies or fragments described herein. In some embodiments, the antibody-drug conjugates of this disclosure are stable in plasma. Plasma stability can be determined using in vitro or in vivo plasma stability assays, such as those set forth in Examples 8.2 or 10 below. In some embodiments, the antibody-drug conjugates of this disclosure have the following half-lives in plasma: longer than 4 days, longer than 5 days, longer than 6 days, longer than 7 days, longer than 8 days, longer than 9 days, longer than 10 days, longer than 11 days, longer than 12 days, longer than 13 days, longer than 2 weeks, longer than 3 weeks, longer than 4 weeks, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, and about 6 months. Months, between 5-10 days, between 8-12 days, between 10-15 days, between 12-18 days, between 15-20 days, between 20-30 days, between 1-2 weeks, between 2-3 weeks, between 3-4 weeks, between 4-6 weeks, between 5-8 weeks, between 6-10 weeks, between 1-2 months, between 1.5-3 months, between 2-4 months, between 2.5-5 months, between 3-6 months, or between 4-6 months.

[0665] In some embodiments, the antibody-drug conjugates of this disclosure bind to GLP1R with an affinity at least 10-fold greater than other G protein-coupled receptors (GPCRs). In some embodiments, the antibody-drug conjugates of this disclosure bind to GLP1R with an affinity at least 20, 50, 100, 500, 1000, or 10,000-fold greater than other G protein-coupled receptors (GPCRs). In some embodiments, such antibody-drug conjugates exhibit substantially undetectable binding to GPCRs other than GLP1R. The binding of the antibody-drug conjugate to the target molecule can be measured using standard binding assays available in the relevant art, such as luciferase reporter assays, surface plasmon resonance assays, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), FACS analysis, or Western blot assays. Examples of GPCRs other than GLP1R include, but are not limited to, GIPR, GLP2R, and GCGR.

[0666] In some embodiments, the antibody-drug conjugates of this disclosure comprise an anti-GLP1R antibody or an antigen-binding fragment thereof conjugated to a linker payload, wherein the linker payload and the antibody or antigen-binding fragment thereof are conjugated at the N-terminus of the light chain. In one embodiment, the antibody-drug conjugates of this disclosure comprise an anti-GLP1R antibody or an antigen-binding fragment thereof conjugated to the N-terminus of the light chain, wherein the payload has the following structure:

[0667]

[0668] In some embodiments, the antibody-drug conjugates of this disclosure comprise an anti-GLP1R antibody or an antigen-binding fragment thereof conjugated to two linker payloads, wherein each linker payload is attached to the antibody or antigen-binding fragment thereof at the N-terminus of a light chain. In one embodiment, the antibody-drug conjugates of this disclosure comprise an anti-GLP1R antibody or an antigen-binding fragment thereof conjugated to the N-terminus of each light chain, for a total of two linker payloads for each antibody, wherein the payloads have the following structure:

[0669]

[0670] In another aspect, this article provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment targeting the glucagon-like peptide-1 receptor (GLP1R) and a payload having the following structure:

[0671]

[0672] in The payload is attached to the antibody or its antigen-binding fragment directly or through a connector at the attachment point.

[0673] In one implementation, the payload has the following structure:

[0674]

[0675] In another aspect, this article provides an antibody-drug conjugate comprising an antibody or its antigen-binding fragment targeting the glucagon-like peptide-1 receptor (GLP1R) and a linker-payload having the following structure:

[0676]

[0677] in It is the attachment point where the connector-payload is attached to the antibody or its antigen-binding fragment.

[0678] In one embodiment, the joint-payload has the following structure:

[0679]

[0680] Antibody conjugation

[0681] Techniques and adapters for conjugating residues to antibody or antigen-binding fragments are known in the art. Exemplary amino acid attachments that may be used in the context of this aspect include, for example, lysine (see, for example, US 5,208,020; US2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / 0585592), cysteine ​​(see, for example, US 2007 / 0258987; WO2013 / 055993; WO 2013 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO2011 / 130598 ... 7,750,116), selenocysteine ​​(see, for example, WO 2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (see, for example, Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51; and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-natural amino acids (see, for example, WO 2013 / 068874 and WO 2012 / 166559), and acidic amino acids (see, for example, WO 2012 / 05982). Lysine conjugation can also occur via NHS (N-hydroxysuccinimide). The linker can also conjugate with cysteine ​​residues (including cysteine ​​residues with cleaved interchain disulfide bonds) by forming carbon bridges between thiols (see, for example, US 9,951,141 and US 9,950,076). The linker can also bind to antigen-binding proteins via attachment to sugars (see, for example, US 2008 / 0305497, WO2014 / 065661 and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, for example, WO 2013 / 085925, WO 2010 / 010324, WO 2011 / 018611 and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313).Site-specific conjugation techniques can also be used for direct conjugation to specific residues of antibodies or antigen-binding proteins (see, for example, Schumacher et al., J Clin Immunol (2016) 36(Supplement 1): 100). In specific embodiments discussed in more detail below, site-specific conjugation techniques include glutamine conjugation via transglutaminase (see, for example, Schibli, Angew Chemie Inter, eds. 2010, 49, 9995).

[0682] The payload according to this disclosure is included within the scope of this disclosure by means of lysine and / or cysteine, such as via maleimide or amide conjugation.

[0683] In some embodiments, the protein-drug conjugates of this disclosure are generated according to a two-step method, wherein step 1 is based on the conjugation of a lysine linker, such as conjugation with an NHS-ester linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0684] In some embodiments, the protein-drug conjugates of this disclosure are generated according to a two-step method, wherein step 1 is based on the conjugation of a cysteine ​​linker, such as with a maleimide linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0685] In some embodiments, the protein-drug conjugates of this disclosure are generated according to a two-step method, wherein step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0686] Step 1: Transglutaminase-mediated site-specific conjugation

[0687] In some embodiments, proteins (e.g., antibodies) may be modified according to known methods to provide glutamine acyl-modified proteins. Techniques for conjugating antibodies and primary amine compounds are known in the art. This paper employs a site-specific conjugation technique, using glutamine conjugation via glutamine transaminase to guide conjugation with glutamine (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0688] The primary amine-containing compounds of this disclosure (e.g., linker La) can be conjugated to one or more glutamine residues of a conjugate (e.g., a protein, e.g., an antibody, e.g., an anti-GLP1R antibody) via transglutaminase-based chemical-enzymatic conjugation (see, for example, Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and WO 2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be conjugated to a primary amine linker compound. Briefly, in some embodiments, in the presence of enzymatic transglutaminase, a conjugate having a glutamine residue (e.g., Gln295, i.e., Q295 residue) is treated with a primary amine-containing linker La. In some embodiments, the conjugate is unglycosylated. In some embodiments, the conjugate is deglycosylated.

[0689] In some embodiments, the binder (e.g., a protein, such as an antibody) includes at least one glutamine residue in at least one polypeptide chain sequence. In some embodiments, the binder comprises two heavy chain polypeptides, each having a Gln295 residue. In other embodiments, the binder includes one or more glutamine residues at sites other than heavy chain 295.

[0690] In some embodiments, conjugates such as antibodies can be prepared by site-directed mutagenesis to insert glutamine residues at a specific site without causing antibody function or binding failure. For example, this document includes antibodies carrying one or more Asn297Gln (N297Q) mutations as described herein. In some embodiments, antibodies having Gln295 residues and / or N297Q mutations contain one or more additional naturally occurring glutamine residues in their variable region, which can be accessed by transglutaminase and thus conjugate to a linker or linker payload. Exemplary naturally occurring glutamine residues may be present, for example, at Q55 of the light chain. In such cases, conjugates such as antibodies conjugated via transglutaminase may have a higher than expected LAR value (e.g., a LAR higher than 4). Any such antibody can be isolated from natural or artificial sources.

[0691] Step 2: Payload coupling reaction

[0692] In some embodiments, the linker La according to this disclosure includes at least one first reactive group capable of further reaction after transglutamination. In these embodiments, the glutamine acyl-modified protein (e.g., an antibody) is capable of further reaction with a reactive payload compound P or a reactive linker-payload compound (e.g., Lp-P as disclosed herein) to form a protein-payload conjugate. More specifically, the reactive linker-payload compound Lp-P may include a second reactive group capable of reacting with the first reactive group of the linker La. In some embodiments, the first or second reactive group according to this disclosure includes a moiety capable of undergoing a 1,3-cycloaddition reaction. In some embodiments, the reactive group is an azide. In some embodiments, the reactive group includes an alkyne (e.g., a terminal alkyne or an internally strained alkyne). In some embodiments of this disclosure, the reactive group is compatible with the binding agent and transglutamination reaction conditions.

[0693] In some embodiments of this disclosure, the connector La molecule includes a first reactive group. In some embodiments of this disclosure, the connector La molecule includes more than one reactive group.

[0694] In some embodiments, the reactive connector-payload Lp-P comprises one payload molecule (n = 1). In some other embodiments, the reactive connector-payload Lp-P comprises two or more payload molecules (n ≥ 2).

[0695] In some embodiments of this disclosure, the drug-to-antibody ratio, or DAR, is about 1 to about 30, or about 1 to about 24, or about 1 to about 20, or about 1 to about 16, or about 1 to about 12, or about 1 to about 10, or about 1 to about 8, or about 1, 2, 3, 4, 5, 6, 7, or 8 payload molecules per antibody. In some embodiments, the DAR is 1 to 30. In some embodiments, the DAR is 1 to 16. In some embodiments, the DAR is 1 to 8. In some embodiments, the DAR is 1 to 6. In some embodiments, the DAR is 2 to 4. In some cases, the DAR is 2 to 3. In some cases, the DAR is 0.5 to 3.5. In some embodiments, the DAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the DAR is 2. In some embodiments, the DAR is 4. In some implementations, DAR is 8.

[0696] In one aspect, this disclosure provides a method for producing compounds having the structure of formula (A):

[0697] BA-(LP)m (A),

[0698] The method includes the following steps:

[0699] a) In the presence of transglutaminase, BA comprising at least m glutamine residues Gln is contacted with at least m equivalents of compound LP, and

[0700] b) The compound of formula (A) produced by separation.

[0701] In one aspect, this disclosure provides a method for producing compounds having the structure of formula (A):

[0702] BA-(LP) m (A),

[0703] The connector L has the structure of formula (L'):

[0704] -La-Y-Lp-(L'),

[0705] Wherein La is the first connector covalently attached to the BA;

[0706] Y is a group containing triazole, and

[0707] Lp is a second connector covalently attached to P.

[0708] The method includes the following steps:

[0709] a) In the presence of transglutaminase, BA comprising at least m glutamine residues Gln is contacted with a first linker La comprising an azide or alkyne moiety.

[0710] b) Contact the product of step a) with at least m equivalents of compound Lp-P, wherein the second connector Lp comprises an azide or alkyne moiety, wherein La and Lp are capable of reacting to produce a triazole, and

[0711] c) The resulting compound of formula (A) was isolated.

[0712] In one aspect, this disclosure provides a method for generating compounds having a structure according to formula (I):

[0713] BA-LP(I),

[0714] The method includes the following steps:

[0715] a) In the presence of transglutaminase, contacting BA, which includes at least one glutamine residue Gln, with compound LP, and

[0716] b) The resulting compound of formula (I) after separation,

[0717] BA is an antibody or its antigen-binding fragment;

[0718] L is a non-disintegrable connector;

[0719] P is the effective load of a structure that is selected from the following group:

[0720]

[0721] in The attachment point where the payload is attached to L;

[0722] X1 is selected from H;

[0723] X2 is selected from

[0724] X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety;

[0725] n is 0 or 1;

[0726] X4 is selected from H and phenyl;

[0727] X5 is selected from -OH, -NH2, -NH-OH, and

[0728] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0729] X7 is selected from H,

[0730] X8 is selected from H, -OH, -NH2, and

[0731] Or its pharmaceutically acceptable salt.

[0732] In another aspect, this disclosure provides a method for producing compounds having a structure according to formula (I):

[0733] BA-LP(I),

[0734] The connector L has the structure of formula (L'):

[0735] -La-Y-Lp-(L'),

[0736] Wherein La is the first connector covalently attached to the BA;

[0737] Y is a group containing triazole, Diels-Alder adduct, or thiol-maleimide adduct, and

[0738] Lp is a second connector covalently attached to P.

[0739] The method includes the following steps:

[0740] a) In the presence of transglutaminase, BA comprising at least one glutamine residue Gln is contacted with a first linker La comprising an azide or alkyne moiety;

[0741] b) Contact the product of step a) with compound Lp-P, wherein the second connector Lp comprises an azide or alkyne moiety, wherein La and Lp are capable of reacting to produce a triazole, and

[0742] c) Separate the resulting compound of formula (I),

[0743] BA, L', and P are defined as above.

[0744] In one embodiment, Y is a triazole group.

[0745] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of BA. In another embodiment, the glutamine residue Gln is introduced into BA by modifying one or more amino acids. In one embodiment, Gln is Q295 or N297Q.

[0746] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG). In another embodiment, the transglutaminase is a bacterial transglutaminase (BTG).

[0747] In one embodiment, the compound LP used in any of the above methods for producing the compound of formula (I) has a structure selected from the group consisting of:

[0748] structure

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756] Or its pharmaceutically acceptable salt.

[0757] Therapeutic preparations and administration

[0758] This disclosure provides pharmaceutical compositions comprising protein-drug conjugates.

[0759] In one aspect, this disclosure provides compositions comprising a group of protein-drug conjugates according to this disclosure, said group of protein-drug conjugates having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

[0760] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0761] In one embodiment, the composition has about 2 DAR.

[0762] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0763] In one embodiment, the composition has about 4 DAR.

[0764] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0765] In one embodiment, the composition has about 8 DAR.

[0766] The compositions disclosed herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations are available in formulation collections familiar to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN). TM Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol in various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998), JPharm Sci Technol 52:238-311.

[0767] The dosage of the protein-drug conjugate administered to a patient can vary depending on the patient's age and size, target disease, condition, route of administration, etc. An appropriate dosage is typically calculated based on body weight or body surface area. When the protein-drug conjugate of this disclosure is used for therapeutic purposes in adult patients, it may be advantageous to administer the protein-drug conjugate of this disclosure intravenously in a single dose of about 0.01 mg / kg to about 20 mg / kg body weight, more preferably about 0.02 mg / kg to about 7 mg / kg, about 0.03 mg / kg to about 5 mg / kg, or about 0.05 mg / kg to about 3 mg / kg body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective dosages and regimens for administering the protein-drug conjugate can be determined empirically; for example, by periodically evaluating and monitoring patient progress and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0768] Various delivery systems are known and can be used to administer the pharmaceutical compositions of this disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or rapid concentration, by absorption through mucosal epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0769] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, regarding subcutaneous delivery, pen delivery devices are readily applicable for delivering the pharmaceutical compositions of this disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. After the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0770] Many reusable pens and autoinjector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, a few examples only, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPENJUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices used in the subcutaneous delivery of the pharmaceutical compositions of this disclosure include, but are not limited to, to name just a few, SOLOSTAR. TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL).

[0771] In some cases, the pharmaceutical composition can be delivered using a controlled-release system. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138). Other controlled-release systems are discussed in a review in Langer, 1990, Science 249:1527-1533.

[0772] Injectable products can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable products can be prepared by methods known to the public. For example, injectable products can be prepared by, for instance, dissolving, suspending, or emulsifying the antibodies described above or their salts in a sterile aqueous medium or an oily medium conventionally used for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oily medium, there are, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Injectable preparations thus prepared are preferably filled in suitable ampoules.

[0773] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in dosage forms suitable for the dosage of the active ingredient. Such dosage forms for unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In a unit dose, the amount of the aforementioned antibody contained is typically from about 5 mg to about 500 mg per dosage form; particularly in injectable forms, it is preferred that the aforementioned antibody is contained in about 5 mg to about 100 mg and for other dosage forms in about 10 mg to about 250 mg.

[0774] Protein-drug conjugates, linkers-payloads, and the therapeutic uses of payloads.

[0775] In another respect, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-payloads and / or payloads) are particularly useful for treating, preventing and / or improving diseases, disorders or conditions in need of such treatment.

[0776] In one aspect, this disclosure provides a method of treating a condition in a subject with a corresponding need, comprising administering to the subject a therapeutically effective amount of a compound according to this disclosure (e.g., an antibody-drug conjugate, a linker-loador, and / or a load), or a composition comprising any compound according to this disclosure.

[0777] In some embodiments, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can be used to treat any disease or disorder in which stimulation, activation, and / or targeting of GLP1R would be beneficial. In particular, the anti-GLP1R antibody-drug conjugates of this disclosure can be used to treat, prevent, and / or improve any disease or disorder associated with or mediated by GLP1R expression or activity.

[0778] In some embodiments, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can be used to treat GLP1R-related conditions. In some embodiments, the GLP1R-related condition is type 1 or type 2 diabetes. The applied compounds (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can cause at least one of the following results: induction of insulin secretion, inhibition of glucagon release, reduction of blood glucose, improvement of glycemic control, promotion of islet anogenesis, and delay of gastric emptying or enhancement of glucose-resistant islets.

[0779] In some implementations, GLP1R-related conditions are neurodegenerative disorders, cognitive impairment, memory impairment, or learning disabilities. Neurodegenerative disorders can be, for example, dementia, Alzheimer's disease, mild cognitive impairment, Alzheimer's disease-related dementia, Huntington's chorea, tardive dyskinesia, hyperkinesias, mania, Morbus Parkinson's disease, Steel-Richard syndrome, Down syndrome, myasthenia gravis, neurological trauma, brain trauma, angioamylindness, intracerebral hemorrhage with amyloidosis, encephalitis, Friedrich's ataxia, acute confusional disorder, amyotrophic lateral sclerosis, glaucoma, and Alzheimer's disease.

[0780] In some implementations, the GLP1R-related condition is liver disease. Liver disease can be, for example, non-alcoholic fatty liver disease (NAFLD), fatty liver, non-alcoholic steatohepatitis (NASH), and cirrhosis.

[0781] In some implementations, the GLP1R-related condition is coronary artery disease. Coronary artery disease can be, for example, cardiomyopathy and myocardial infarction.

[0782] In some implementations, the GLP1R-related condition is kidney disease. Kidney disease can be, for example, hypertension or chronic kidney failure.

[0783] In some implementations, the GLP1R-related condition is an eating disorder. An eating disorder can be, for example, binge eating.

[0784] Without being bound by theory, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-loadors, and / or payloads) can be used to attenuate the effects of apoptosis-mediated central nervous system degenerative diseases such as Alzheimer's disease, Creutzfeld-Jakob disease and bovine spongiform encephalopathy, chronic wasting syndrome, and other prion-mediated apoptotic neurological diseases (see, for example, Perry and Grieg (2004) Current Drug Targets 6:565-571). Administration of the compounds disclosed herein (e.g., antibody-drug conjugates, linker-loadors, and / or payloads) can also lead to downregulation of βAPP and thereby improve Aβ monomer or oligomer-mediated pathology associated with Alzheimer's disease (see, for example, Perry et al. (2003) Journal of Neuroscience Research 72:603-612).

[0785] It is also envisioned that the compounds disclosed herein (e.g., antibody-drug conjugates, adapter-payloads, and / or payloads) could be used to improve learning and memory, for example, by enhancing neuronal plasticity and promoting cell differentiation (see During et al. (2003) Nature Medicine 9:1173-1179). Furthermore, the compounds disclosed herein (e.g., antibody-drug conjugates, adapter-payloads, and / or payloads) could also be used to maintain Morbus Parkinson's dopamine neurons and motor function (see, for example, Greig et al. (2005) Abstract 897.6, Society for Neuroscience, Washington, DC).

[0786] In some embodiments, the compounds disclosed herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) may also be used to treat metabolic disorders. Metabolic disorders may include, for example, obesity, dyslipidemia, metabolic syndrome X, and pathologies caused by pancreatic insufficiency.

[0787] Other diseases that can be treated by the compounds of this disclosure (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) include autoimmune diseases, particularly those related to inflammation, including but not limited to autoimmune diabetes, adult-onset diabetes, morbid obesity, metabolic syndrome X, and dyslipidemia. For example, anti-GLP1R antibody-drug conjugates can be used as growth factors to promote pancreatic islet growth in patients with autoimmune diabetes. The compounds described herein (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) can also be used to treat congestive heart failure.

[0788] In one aspect, this disclosure provides a method for selectively targeting an antigen (e.g., GLP1R) on a cell surface with a compound. In one embodiment, the method for selectively targeting an antigen (e.g., GLP1R) on a cell surface with a compound includes linking the compound to a targeting antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a pancreatic cell or a brain cell. In some embodiments, this disclosure provides a method for selectively targeting an antigen on a cell surface with a compound having a structure selected from the group consisting of:

[0789]

[0790]

[0791] in It is the attachment point where the compound attaches to the connector L;

[0792] X1 is selected from H;

[0793] X2 is selected from

[0794] X3 is selected from –(CH2) 2-6 –NH– and –(CH2) 2-6 –Tr–, where Tr is the triazole moiety;

[0795] n is 0 or 1;

[0796] X4 is selected from H and phenyl;

[0797] X5 is selected from -OH, -NH2, -NH-OH, and

[0798] X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears;

[0799] X7 is selected from H,

[0800] X8 is selected from H, -OH, -NH2, and

[0801] Or its pharmaceutically acceptable salt.

[0802] In some embodiments, this disclosure also includes the use of the compounds of this disclosure (e.g., antibody-drug conjugates, linker-payloads, and / or payloads) in the preparation of medicaments for treating diseases or disorders (e.g., cancer) associated with or caused by cells expressing GLP1R. In one aspect, this disclosure relates to protein-drug conjugates comprising anti-GLP1R antibodies or antigen-binding fragments as disclosed herein for use in medicine. In another aspect, this disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein for use in medicine.

[0803] Combination therapies and formulations

[0804] This disclosure provides methods for combining a pharmaceutical composition with one or more additional therapeutic agents, the pharmaceutical composition including any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads described herein.

[0805] Exemplary additional therapeutic agents that can be administered in combination with or in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of this disclosure include other GLP1R agonists (e.g., anti-GLP1R antibodies or small molecule GLP1R agonists or anti-GLP1R antibody-drug conjugates). Non-limiting examples of GLP1R agonists include exenatide (Byetta, Bydureon), liraglutide (Victoza, Saxenda), lixilatide (Lyxumia in Europe, Adlyxin in the United States), abiglutide (Tanzeum), dulaglutide (Trulicity), semaglutide (Ozempic), and tasglutide.

[0806] Exemplary alternative therapeutic agents may include dual or triple agonists, including GLP1R / GIPR dual agonists, such as GLP1R / GCGR dual agonists and GLP1R / GIPR / GCGR triple agonists.

[0807] Other agents that can be beneficially administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of this disclosure include those that can be used to treat diabetes (e.g., type II diabetes), obesity, and / or other related metabolic diseases.

[0808] In some implementations, the additional therapeutic agent is an antidiabetic agent. Any suitable antidiabetic agent may be used. Non-limiting examples of antidiabetic agents include insulin, insulin analogs (including insulin lispro, insulin aspart, insulin glulisine, isophanin insulin, zinc insulin, insulin glargine, and insulin detemir), biguanides (including metformin, phenformin, and buformin), thiazolidinediones or TZDs (including rosiglitazone, pioglitazone, and troglitazone), sulfonylureas (including tolbutamide, acetylhexamide, tolazoline, chlorpromazine, glipizide, glibenclamide, glimepiride, gliclazide, glipizide, and gliquidone), meglitinides (including repaglinide and nateglinide), alpha-glucosidase inhibitors (including miglitol, acarbose, and vogbose), glucagon-like peptide analogs, and agonists. (Including exenatide, liraglutide, semaglutide, tasglutide, lixilatide, abiratelite, and dulaglutide), gastric inhibitory peptide analogs, dipeptidyl peptidase-4 (DPP-4) inhibitors (including vildagliptin, sitagliptin, saxagliptin, linagliptin, alogliptin, septagliptin, teneligliptin, and giglitazone), amylin agonist analogs, sodium / glucose cotransporter 2 (SGLT2) inhibitors, glucokinase activators, squalene synthase inhibitors, other lipid-lowering agents, and aspirin. In some such embodiments, the antidiabetic agent is an oral antidiabetic agent (OAA), such as metformin, acarbose, or TZD. In some such embodiments, the antidiabetic agent is metformin.

[0809] In some implementations, a GLP1R agonist and one or more antidiabetic agents may be formulated into the same dosage form, such as a solution or suspension for parenteral administration.

[0810] One or more additional therapeutically active components may be administered before, during, or shortly after administration of the compound disclosed herein; (for the purposes of this disclosure, such administration regimens are considered as administration of the antigen-binding molecule in combination with the additional therapeutically active component).

[0811] This disclosure includes pharmaceutical compositions in which the protein-drug conjugates (e.g., antibody-drug conjugates), linker-loadors, and / or loads of this disclosure are co-formulated with one or more other additional therapeutically active components described elsewhere herein.

[0812] Application plan

[0813] According to certain embodiments of this disclosure, multiple doses of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-loador, and / or a payload can be administered to a subject over a defined time period. A method according to this aspect of the disclosure includes sequentially administering multiple doses of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-loador, and / or a payload of the disclosure to a subject. As used herein, “sequential administration” means administering each dose of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-loador, and / or a payload to a subject at different time points, such as over different numbers of days separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering to a patient a single initial dose of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-load and / or a load, followed by one or more second doses of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-load and / or a load, and optionally followed by one or more third doses of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-load and / or a load.

[0814] The terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence of administration of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-loador, and / or payload of this disclosure. Thus, the “initial dose” is the dose administered at the start of a treatment regimen (also known as the “baseline dose”); the “second dose” is the dose administered after the initial dose; and the “third dose” is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-loador, and / or payload, but may generally differ from each other in terms of administration frequency. However, in some embodiments, the amounts of the protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), linker-loador, and / or payload contained in the initial, second, and / or third doses differ from each other during the treatment process (e.g., may be increased or decreased as appropriate). In some implementations, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as “loading doses”, followed by subsequent doses based on less frequent administration (e.g., “maintenance doses”).

[0815] In one exemplary embodiment of this disclosure, each second and / or third dose is administered 1 to 26 weeks immediately following the previous dose (e.g., 1, 1...). 1 / 2、2、2 1 / 2, 3, 3 1 / 2、4、4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2、7、7 1 / 2、8、8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2、21、21 1 / 2、22、22 1 / 2、23、23 1 / 2、24、24 1 / 2、25、25 1 / 2, 26, 26 1 (2 or more weeks) administration. As used herein, the phrase “immediately following the previous dose” means, in a sequence of multiple administrations, the dose of the protein-drug conjugate (e.g., anti-GLP1R antibody-drug conjugate), linker-payload, and / or payload administered to the patient before the next dose is administered in the sequence, without intermediate doses.

[0816] Methods according to this aspect of the disclosure may include administering any number of second and / or third doses of a protein-drug conjugate (e.g., an anti-GLP1R antibody-drug conjugate), a linker-payload, and / or a payload to a patient. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0817] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient one to two weeks immediately following the previous dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient two to four weeks immediately following the previous dose. Optionally, the frequency of administration of the second and / or third doses to the patient may be varied during the treatment regimen. Following a clinical examination, the physician may also adjust the administration frequency during the treatment process based on the individual patient's needs. Example

[0818] The following examples illustrate specific aspects of this specification. These examples should not be construed as limiting, as they merely provide a specific understanding and practice of implementation methods and their various aspects.

[0819] The abbreviations used in the embodiments and throughout the specification are as follows:

[0820]

[0821]

[0822]

[0823]

[0824]

[0825] Example 1. Synthesis of small molecule payloads and linker-payload

[0826] 1.1 Solid-phase peptide synthesis with peptide mimicry payload

[0827] General procedure for preparing peptide mimics (payloads) using the SPPS method

[0828] Scheme 1 describes the assembly of peptide mimicry payloads on resin according to this disclosure. Using a polypropylene column equipped with a filter plate, peptides are manually assembled onto Fmoc SPPS (solid-phase peptide synthesis) via a roller mixer. A sufficient amount of Rink amide MBHA resin (loading: 0.5–0.6 mmol / g) is swollen in DMF or CH2Cl2 for 15 min.

[0829] Option 1: General approach for assembling peptide payloads using the SPPS strategy

[0830]

[0831] Step 1: General procedure for removing Fmoc from Fmoc-Rink amide MBHA resin

[0832] The Fmoc groups on the resin were removed by incubating the resin with 20% piperidine in DMF (10-30 ml / 100 mg resin) for 5 to 15 minutes. The deprotected resin was filtered and washed with excess DMF and DCM. After washing three times, the resin was incubated in freshly distilled DMF (1 mL / 100 mg resin) under a nitrogen atmosphere for 5 minutes.

[0833] Step 2: General method for amide coupling on Rink amide MBHA resin

[0834] For amide coupling reactions using SPPS-reactants, a DMF solution containing HATU (1.5–4 equivalents), Fmoc-protected amino acids (1.5–5 equivalents at 0.5 M concentration), and DIPEA (5–10 equivalents) is added to the resin. For amide coupling reactions using natural amino acids as reactants, Fmoc-amino acids (5 equivalents), HATU (4.5 equivalents), and DIPEA (10 equivalents) are mixed with the resin; for amide coupling reactions using non-natural amino acids as reactants, Fmoc-amino acids (1.5–2 equivalents), HATU (1.5 equivalents), and DIPEA (5.0 equivalents) are mixed with the resin. The mixed resin is then shaken under a nitrogen atmosphere for 1–3 hours, and the coupling reaction is qualitatively monitored using a ninhydrin test. After attaching the Fmoc-protected amino acid, the resin is washed with DMF and DCM to produce the corresponding peptide-bound resin.

[0835] Step 3: General procedure for pyrolysis from the resin followed by overall deprotection

[0836] The peptide mimic payload bound to the resin underwent lysis and deprotection with a TFA mixture, as shown below. A solution of TFA / water / triisopropylsilane (95:2.5:2.5) (10 mL per 100 mg peptide-based resin) was added to the peptide-based resin, and the mixture was kept at room temperature. After 2–3 hours, the resin was filtered and washed with the lysis solution. The combined filtrate was treated with cold t-BuOMe to precipitate the peptide. The suspension was centrifuged for 10 min (5000 R). The crude white powder was combined and purified by preparative HPLC.

[0837] As described in Scheme 1, peptide chain elongation is performed through multiple iterations consisting of deprotection, washing, coupling, and washing procedures (i.e., the resin is subjected to reaction conditions for 1 hour each time, and the solution is drained and the resin is subjected to fresh reagents each time). Finally, as described above, the resulting Fmoc-protected peptide resin is deprotected with 20% piperidine and washed four times each with DMF and DCM. The resin-bound peptide is dried under a nitrogen stream for 10–15 minutes and subjected to cleavage / deprotection. Using the above scheme and its suitable variations, the peptide mimics designed in this disclosure are prepared using the Fmoc-SPPS method. Furthermore, the resin-bound peptide mimics are cleaved and deprotected, purified, and characterized using the following scheme.

[0838] 1.2 Preparative HPLC purification of crude peptide mimics:

[0839] Preparative HPLC was performed on a Shimadzu LC-8a liquid chromatograph. A crude peptide solution dissolved in DMF or water was injected into the column and eluted with a linear gradient of ACN in water. Different methods were used (see General Information). The desired eluted product was obtained in fractions, and pure peptide mimics as amorphous powders were obtained by lyophilizing the respective HPLC fractions. Typically, after preparative HPLC purification, overall recoveries were found in the range of 40%–50%.

[0840] Preparative HPLC method A: Using FA conditions (column: Ultimate C 18 150*25mm*5μm; mobile phase: [water (0.225% FA)-ACN]; B%: 40%-70%, 7min) to obtain pure product.

[0841] Preparative HPLC method B: TFA conditions (column: YMC-Exphere C18 10μm 300*50mm 12nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55min) were used to obtain the pure product.

[0842] Preparative HPLC method C: Using neutral conditions (column: Phenomenex Gemini-NX150*30mm*5μm; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 21%-51%, 11min) to obtain pure product.

[0843] Preparative HPLC method D: Using neutral conditions (column: Waters Xbridge 150*255u; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 20%-50%, 7min) to obtain pure product.

[0844] Preparative HPLC method E: Using FA conditions (column: Phenomenex Luna C18 250*50mm*10μm; mobile phase: [water (0.225% FA)-ACN]; B%: 55%-86%, 21 min) to obtain the pure product.

[0845] 1.3 HPLC analysis of purified peptide mimics:

[0846] As described above, after preparative HPLC purification, each peptide is analyzed by analytical HPLC using methods A, B, C, D, E, or F. Typically, chromatographic acquisition is performed at 220 nm using a PDA detector, and the purity of the pure peptide mimics obtained after preparative HPLC purification is found to be >95%.

[0847] HPLC Method A (20 min): Mobile phase: 4.0 mL TFA (solvent A) in 4 L water and 3.2 mL TFA (solvent B) in 4 L acetonitrile, using an elution gradient of 10%-80% (solvent B) over 20 min, and holding at 80% for 3.5 min at a flow rate of 1.0 mL / min; Column: Gemini-NX 5μm 150*4.6 mm, C18, 110A; Wavelength: UV 220 nm, 254 nm; Column temperature: 30℃

[0848] HPLC Method B (15 min): Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: WELCH Ultimate LP-C18 150*4.6 mm 5 μm; Wavelength: UV 220 nm, 215 nm, 254 nm; Column temperature: 40 °C

[0849] HPLC Method C (8 min): Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 7 min, and holding at 80% for 0.48 min at a flow rate of 1.5 mL / min; Column: Ultimate XB-C18, 3 μm, 3.0*50 mm; Wavelength: 220 nm, 215 nm, 254 nm; Column temperature: 40 °C.

[0850] HPLC Method D (15 min): Mobile phase: Water containing 0.04% TFA (solvent A) and acetonitrile containing 0.02% TFA (solvent B), using an elution gradient of 10% to 80% (solvent B) over 15 min, and maintained at 80% for 3.5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm; Wavelength: UV 220 nm, 254 nm; Column temperature: 30 °C.

[0851] HPLC Method E (8 min): Mobile phase: 0.2 mL / 1 L NH3*H2O in water (solvent A) and acetonitrile (solvent B), eluted over 5 minutes using a 0%-60% (solvent B) elution gradient, and held at 60% for 2 minutes at a flow rate of 1.2 mL / min; Column: Xbridge Shield RP-18, 5 μm, 2.1*50 mm; Wavelength: UV 220 nm, 254 nm; Column temperature: 30 °C.

[0852] HPLC Method F (7 min): Mobile phase: 1.5 mL / 4 L TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%–80% (solvent B). Column: Xtimate C18 2.1*30 mm; Wavelength: UV 220 nm, 254 nm; Column temperature: 50 °C.

[0853] 1.4 Characterization by mass spectrometry:

[0854] Each peptide was characterized by electrospray ionization mass spectrometry (ESI-MS) in either flow injection or LC / MS mode. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotope molecular weight. Using the protocol described above, all crude / purified peptide mimics were characterized by mass spectrometry, and generally, the observed peptide mimic mass was consistent with the calculated / theoretical mass, confirming the successful synthesis of the desired peptide mimic.

[0855] LC-MS Method A: MERCK (RP-18e 25-2 mm) column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[0856] LC-MS Method B: Ultimate (C18 2.1*30mm, 3μm) column, flow rate 0.8mL / min, eluted with a gradient of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[0857] LC-MS Method C: Chromolith (Flash RP-18e 25-3 mm) column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.04% TFA and water (solvent A) containing 0.06% TFA.

[0858] LC-MS Method D: Agilent, a Pursuit (5C18 20*2.0mm) column, flow rate 1.5 ml / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[0859] LC-MS Method E: Waters Xbridge C18 30*2.0 mm, 3.5 μm column, flow rate 1.0 mL / min, elution with a gradient from 5% to 95%. Mobile phase: A) 0.05% NH3H2O ​​in water; B) ACN. Gradient: 0% B increased to 95% B over 5.8 min; held at 95% B for 1.1 min; then returned to 0% B at 6.91 min and held for 0.09 min.

[0860] LC-MS method F: XBridge C18 3.5μm 2.1*30mm column, flow rate 1.0mL / min, mobile phase: 0.8mL / 4L NH3·H2O in water (solvent A) and acetonitrile (solvent B), using a gradient of 10%-80% (solvent B) over 2 minutes, and holding at 80% for 0.48 minutes.

[0861] 1.5 HRMS analysis was performed on Agilent 6200 series TOF and 6500 series Q-TOF LC / MS systems.

[0862] Mobile phase: 0.1% FA in water (solvent A) and ACN (solvent B); Elution gradient: 5%-95% (solvent B) over 3 minutes, and maintained at 95% for 1 minute at a flow rate of 1 ml / min; Column: Xbridge Shield RP 18 5μm, 2.1*50mm; Ion source: AJS ESI source; Ion mode: positive; Nebulizing gas: nitrogen; Dry gas (N2) flow rate: 8 L / min; Nebulizer pressure: 35 psig; Gas temperature: 325℃; Sheath gas temperature: 350℃; Sheath gas flow rate: 11 L / min; Capillary voltage: 3.5 kV; Collision voltage: 175 V.

[0863] Example 2. Synthesis of non-natural amino acids

[0864] Table 1. Structure of natural and non-natural amino acids aa1–aa30

[0865]

[0866]

[0867]

[0868]

[0869]

[0870] 2.1 Synthesis of non-natural amino acids (aa1b)

[0871] Scheme 2 outlines the synthesis of non-natural amino acids (aa1b):

[0872] Option 2

[0873]

[0874] Reagents and conditions: a) 1-(benzyloxy)-4-(bromomethyl)benzene (aa1b-4) (1.0 equivalent), t-BuOK (1.0 equivalent), THF, 0℃-10℃, 1h, 67.93%; b) Pd / C (10%), H2 (50Psi), MeOH, 40℃, 44h, 94.24%; c) 1-chloro-4-iodobutane (1.5 equivalent), K2CO3 (2.0 equivalent), DMF, 50℃, 16h, 83.44%; d) NaN3 (2.17 equivalent), K2CO3 (2.0 equivalent) e) DMF, 65℃, 16h, 98.44%; f) LiOH·H2O (2.0 equivalent), THF / H2O, 22℃, 1h, 100%; g) HCl / EtOAc, 22℃, 1h, 85.04%; h) NaHCO3 aqueous solution (0.43M), Fmoc-osu, THF, 0℃-22℃, 16h, 92.11%; h) Boc)2O (3.0 equivalent), DIPEA (3.0 equivalent), Pd / C (10%), H2 (15Psi), 4h, 22.50%.

[0875] aa1b-2 was prepared according to the detailed synthetic procedure described below: Wu, XY; Stockdill, JL; Park, PK; Samuel J. Danishefsky, SJ. Expanding the Limits of Isonitrile-Mediated Amidations: On the Remarkable Stereosubtleties of Macrolactam Formation from Synthetic Seco-Cyclosporins. J. Am. Chem. Soc. 2012, 134, 2378-2384. The preparation of aa1b-1 was referenced to Du, JJ; Gao, XF; Xin, LM; Lei, Z.; Liu, Z.; and Guo, J. Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of ω-Asp p-Nitrophenyl Thioesters in Solution. Org. Lett. 2016, 18, 4828-4831.

[0876] Step 1: Synthesis of methyl (S,Z)-5-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)pent-4-enoate (aa1b-3)

[0877] t-BuOK (3.64 g, 32.43 mmol, 1.0 equivalent) was added to a suspension of aa1b-2 (17.50 g, 32.43 mmol, 1.0 equivalent) in THF (100 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. A solution of aa1b-1 (7.5 g, 32.43 mmol, 1.0 equivalent) in THF (50 mL) was added dropwise to the mixture. The reaction mixture was heated to 10 °C and stirred for 1 h. A pale yellow suspension was observed. The reaction mixture was added to ice water (300 mL) and extracted with EtOAc (200 mL × 2). The organic layers were combined and washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated to give a crude product as a yellow oil. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed for 1.5 h using a silica fast column with an eluent gradient of 0%–18% ethyl acetate / petroleum ether at 50 mL / min, for a total volume of 2.5 L, yielding aa1b-3 as a pale yellow oil (9.7 g, 23.57 mmol, 67.93% yield).

[0878] 1 ¹H NMR (400MHz, chloroform-d) δ=7.45-7.26(m, 6H), 7.18(br d, J=8.6Hz, 1H), 6.92(dd, J=8.7, 11.4Hz, 2H), 6.56-6.34(m, 1H), 5.98-5.83(m, 1H), 5.55-5.42(m, 1H), 5.07(d, J=2.2Hz, 3H), 4.48-4.36(m, 1H), 3.77-3.68(m, 3H), 2.94-2.56(m, 2H), 1.43(s, 9H).

[0879] Step 2: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)valerate (aa1b-4)

[0880] A solution of methyl aa1b-3 (9.7 g, 23.57 mmol, 1.0 equivalent) and Pd / C (10% activated palladium on carbon, 1.0 g, 9.40 mmol) in MeOH (100 mL) was stirred at 40 °C for 44 h under 50 Psi of hydrogen. A black suspension was observed. The reactants were filtered through a diatomaceous earth mat, and the filter cake was washed with MeOH (50 mL × 3). The filtrate was concentrated under vacuum to give aa1b-4 (7.5 g, 22.22 mmol, 94.24% yield, 95.788% purity) as a gray solid.

[0881] 1 ¹H NMR (400MHz, chloroform-d) δ=7.05-6.92(m,J=8.3Hz,2H), 6.79-6.70(m,J=8.3Hz,2H), 5.59(br s,1H), 5.03(br d,J=7.8Hz,1H), 4.37-4.26(m,1H), 3.71(s,3H), 2.61-2.47(m,2H), 1.81(br s,1H), 1.68-1.54(m,3H), 1.44(s,9H). LCMS (ESI): RT=0.927min, for C 17 H 25 The calculated mass of NO5 is 323.17 m / z, while the actual measured mass is 345.9 [M+Na]. + Inverted LC-MS was performed using method C.

[0882] Step 3: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-5-(4-(4-chlorobutoxy)phenyl)valerate (aa1b-5)

[0883] A solution of aa1b-10 (7 g, 21.65 mmol, 1.0 equivalent), 1-chloro-4-iodobutane (7.09 g, 32.47 mmol, 1.5 equivalent), and K2CO3 (5.98 g, 43.29 mmol, 2.0 equivalent) in DMF (70 mL) was stirred at 50 °C for 16 hours. The combined reaction mixture was added to ice water (200 mL) and extracted with EtOAc (150 mL × 3). The organic layers were combined and washed with brine (150 mL × 2), dried over Na2SO4, filtered, and concentrated to give a crude product as a yellow oil. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed for 1.5 h using a silica fast column with an eluent gradient of 0%–20% ethyl acetate / petroleum ether at 50 mL / min, for a total volume of 2.5 L, yielding aa1b-5 as a colorless oil (8 g, 19.33 mmol, 83.44% yield).

[0884] 1 H NMR (400MHz, chloroform-d) δ = 7.04 (d, J = 8.6Hz, 2H), 6.79 (d, J = 8.6Hz, 2H), 4.95 (br d,J=7.3Hz,1H),4.34-4.22(m,1H),3.98-3.92(m,2H),3.70(s,3H),3.60(t,J=6 .2Hz,2H),2.62-2.48(m,2H),2.04-1.84(m,4H),1.83-1.59(m,4H),1.42(s,9H).

[0885] Step 4: Synthesis of methyl (S)-5-(4-(4-azidobutoxy)phenyl)-2-((tert-butoxycarbonyl)amino)valerate (aa1b-6)

[0886] A mixture of aa1b-5 (7.5 g, 18.12 mmol, 1.0 equivalent), NaN3 (2.56 g, 39.32 mmol, 2.17 equivalent), K2CO3 (5.01 g, 36.24 mmol, 2.0 equivalent), and KI (300.78 mg, 1.81 mmol, 0.1 equivalent) in DMF (75 mL) was stirred at 65 °C for 16 hours. The reactants were added to ice water (200 mL) and extracted with EtOAc (100 mL × 3). The organic layers were combined and washed with brine (100 mL × 3), dried over Na2SO4, filtered, and concentrated to give aa1b-6 (7.5 g, 17.84 mmol, 98.44% yield) as a yellow oil.

[0887] Step 5: Synthesis of (S)-5-(4-(4-azidobutoxy)phenyl)-2-((tert-butoxycarbonyl)amino)valeric acid (aa1b-7)

[0888] A solution of aa1b-6 (7.5 g, 17.84 mmol, 1.0 equivalence) and LiOH·H2O (1 M, 35.67 mL, 2.0 equivalence) in THF (70 mL) was stirred at 22 °C for 1 hour. No change was observed. The reactants were concentrated under vacuum to remove THF. The reactants were adjusted to pH 5 with 1 N HCl and extracted with EtOAc (10 mL × 2). The organic layers were combined and washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated to give aa1b-7 (7.25 g, 17.84 mmol, 100.00% yield) as a colorless oil.

[0889] Step 6: Synthesis of (S)-2-amino-5-(4-(4-azidobutoxy)phenyl)valerate (aa1b-8)

[0890] A solution of aa1b-7 (7.25 g, 17.84 mmol, 1.0 equivalent) in 4 M HCl / EtOAc (75 mL) was stirred at 22 °C for 1 hour. The reaction mixture was concentrated under vacuum to give aa1b-8 (5.2 g, 15.17 mmol, 85.04% yield, HCl) as a white solid.

[0891] Step 7: Synthesis of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(4-(4-azidobutoxy)phenyl)valeric acid (aa1b-9)

[0892] NaHCO3 (2.45 g, 29.17 mmol, 2.0 equivalence) in H2O (58 mL) was added to a solution of aa1b-8 (5 g, 14.58 mmol, 1.0 equivalence, HCl) in THF (116 mL) at 0 °C, followed by the addition of Fmoc-OSu (5.41 g, 16.04 mmol, 1.1 equivalence). The reaction mixture was stirred at 22 °C for 16 h. No changes were observed. The reaction mixture was adjusted to pH 6 with 1 N HCl and extracted with EtOAc (50 mL × 3). The organic layers were combined and washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated to give a crude product as a yellow oil. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed for 2.5 h using a silica rapid column with an elution gradient of 0%–5% MeOH / DCM at 50 mL / min, for a total volume of 3 L, yielding aa1b-9 as a yellow oil (7 g, 12.20 mmol, 83.64% yield, 92.113% purity).

[0893] 1 H NMR (400MHz, chloroform-d) δ = 7.75 (br d, J = 7.3Hz, 2H), 7.60-7.49 (m, 2H), 7.41-7.34 (m, 2H), 7.29 (br t, J = 7.5Hz, 2H), 7.05 (br d, J = 8.3Hz, 2H), 6.78 (br d,J=8.3Hz,2H),5.18(br d,J=8.3Hz,1H),4.40(br d,J=6.8Hz,3H),4.21(br t,J=7.0Hz,1H),3.98-3.88(m,2H),3.34(t,J=6.5Hz,2H),2.57(br d,J=6.1Hz,2H),1.98-1.60(m,8H).

[0894] Step 8: Synthesis of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(4-(4-((tert-butoxycarbonyl)amino)butoxy)phenyl)valeric acid (aa1b)

[0895] A mixture of aa1b-15 (7 g, 13.24 mmol, 1.0 equivalent), DIPEA (5.13 g, 39.73 mmol, 6.92 mL, 3.0 equivalent), Boc2O (8.67 g, 39.73 mmol, 9.13 mL, 3.0 equivalent), and Pd / C (10% activated palladium carbide, 3.5 g, 32.9 mmol) in EtOAc (100 mL) was stirred at 25 °C for 4 hours at 15 PSi H2. A black suspension was observed. The reactants were filtered through a diatomaceous earth mat, and the filter cake was washed with EtOAc (50 mL × 3). The filtrate was washed with NH4Cl aqueous solution (100 mL × 3), brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give a crude product as a colorless oil. The crude product was purified by preparative HPLC (column: Phenomenex Luna(2)C18 250*50 10μm; mobile phase: [water (0.225% FA)-ACN]; B%: 45%-78%, 21.5 min) to give aa1b as a yellow oil (1.8 g, 2.99 mmol, 22.50% yield). LCMS (ESI): RT = 0.954 min, for C 35 H42 The calculated mass of N₂O₇Na is 625.29 m / z, while the measured mass is 625.1 [M + Na]. + Inverting LC-MS was performed using method A.

[0896] 1 H NMR (400MHz, chloroform-d) δ = 7.75 (br d, J = 7.6Hz, 2H), 7.60-7.55 (m, 2H), 7.38 (br t, J = 6.7Hz, 2H), 7.29 (t, J = 7.1Hz, 2H), 7.04 (br d,J=8.1Hz,2H),6.78(d,J=7.4Hz,2H),5.24(br s,1H),4.40(br d,J=6.6Hz,2H),4.23-4.18(m,1H),3.92(br s,2H),3.21-3.07(m,2H),2.61-2.51(m,2H),1.97-1.84(m,2H),1.82-1.54(m,8H),1.43(s,9H).

[0897] 2.2 Synthesis of non-natural amino acids (aa2)

[0898] Scheme 3 outlines the synthesis of non-natural amino acids (aa2):

[0899] Option 3

[0900]

[0901] Reagents and conditions: (a) K2CO3, PMB-Cl, DMF, 25℃, 12h; (b) Ph3PCH2Br, t-BuOK, THF, 0℃-25℃, 4h, 71.91%; (c) Pd(dppf)Cl2, KOAc, 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)-1,3,2-dioxacyclopentaborane, 1,4-dioxane, 80℃, 20h, 42.75%; (d) aa 2-6, K2CO3, Pd(PPh3)4, 1,4-dioxane and H2O, 80℃, 2.5h, 76.20%; (e) Pd / C, H2 (15psi), MeOH, 25℃, 48h, 85.01%; (f) K2CO3, 1-chloro-4-iodobutane, DMF, 50℃, 12h; (g) K2CO3, KI, NaN3, DMF, 50℃, 7h;

[0902] Compound aa2-5 was prepared according to the following references: I. Berezowska, N.N. Chung, C. Lemieux, B.C. Wilkes and P.W. Schiller, Agonist vs. Antagonist Behavior of δOpioid Peptides Containing Novel Phenylalanine Analogues in Place of Tyr. J. Med. Chem., Vol. 52, No. 21, 2009, 6941-6945.

[0903] Step 1: Synthesis of 2-bromo-5-((4-methoxybenzyl)oxy)benzaldehyde (aa2-2)

[0904] PMB-Cl (11.69 g, 74.62 mmol, 1.0 equivalent) was added to a solution of aa2-1 (10 g, 49.75 mmol, 1.0 equivalent) and K2CO3 (10.31 g, 74.62 mmol, 1.5 equivalent) in DMF (100 mL). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated under reduced pressure. The residue was diluted with H2O (100 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Compound aa2-2 (21 g, crude) was obtained as a white solid and used directly in the next step without any further purification.

[0905] 1 H NMR (400MHz, DMSO-d6) δ = 10.16 (s, 1H), 7.69 (d, J = 8.8Hz, 1H), 7.43-7.34 (m, 3 H), 7.28 (dd, J=3.3, 8.8Hz, 1H), 6.99-6.90 (m, 2H), 5.10 (s, 2H), 3.75 (s, 3H).

[0906] Step 2: Synthesis of 1-bromo-4-((4-methoxybenzyl)oxy)-2-vinylbenzene (aa2-3)

[0907] t-BuOK (7.16 g, 63.83 mmol, 5.0 equivalent) was added to a solution of Ph3PCH3Br (4.56 g, 12.77 mmol, 1.0 equivalent) in THF (25 mL) under nitrogen atmosphere. The mixture was stirred at 0 °C for 1 hour. Then, aa2-2 (4.1 g, 12.77 mmol, 1.0 equivalent) was added dropwise to THF (25 mL). The mixture was stirred at 25 °C for 3 hours. The reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica rapid column with an eluent gradient of 0%–5% ethyl acetate / petroleum ether at 30 mL / min for 24 min, with a total volume of 0.9 L. Compound aa2-3 was obtained as a white solid (2.93 g, 9.18 mmol, 71.91% yield).

[0908] 1 ¹H NMR (400MHz, chloroform-d) δ=7.46-7.29(m,3H),7.18-7.09(m,1H),7.06-6.96(m,1H),6.95-6.86(m,2H),6.81-6.70(m,1H),5.72-5.59(m,1H),5.40-5.29(m,1H),5.03-4.90(m,2H),3.86-3.74(m,3H).

[0909] Step 3: Synthesis of 2-(4-((4-methoxybenzyl)oxy)-2-vinylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaneborane (aa2-4)

[0910] A solution of aa2-3 (200 mg, 626.58 μmol, 1 equivalent) in 1,4-dioxane (3 mL) was treated with Pd(PPh3)4 (72.41 mg, 62.66 μmol, 0.1 equivalent) and KOAc (122.99 mg, 1.25 mmol, 2 equivalent), and then 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)-1,3,2-dioxacyclopentaborane (477.34 mg, 1.88 mmol, 3 equivalent). The mixture was stirred at 80 °C for 12 hours under nitrogen. The reaction mixture was diluted with brine (30 mL) and extracted with EtOAc (30 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 4g Purification was performed using a silica rapid column with an eluent gradient of 0%–10% ethyl acetate / petroleum ether at 30 mL / min. Compound aa2-4 was obtained as a white solid (190 mg, 518.76 μmol, 82.79% yield).

[0911] 1 H NMR (400MHz, chloroform-d) δ = 7.75 (d, J = 8.4Hz, 1H), 7.55 (dd, J = 10.9, 17.5Hz, 1H), 7.36 (d, J = 8.6Hz, 2H), 7.22 (d, J = 2.4Hz, 1H), 6.97-6 .89(m,2H),6.87(dd,J=2.4,8.4Hz,1H),5.67(d,J=17.4Hz,1H),5.26(d,J=11.0Hz,1H),5.03(s,2H),3.82(s,3H),1.34(s,12H).

[0912] Step 4: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4'-((4-methoxybenzyl)oxy)-2'-vinyl-[1,1'-biphenyl]-4-yl)propionate (aa2-6)

[0913] A solution of aa2-4 (100 mg, 273.03 μmol, 1 equivalent) in 1,4-dioxane (3 mL) and H2O (1 mL) was treated with K2CO3 (56.60 mg, 409.55 μmol, 1.5 equivalent) and Pd(PPh3)4 (31.55 mg, 27.30 μmol, 0.1 equivalent), and then aa2-5 (116.69 mg, 273.03 μmol, 1 equivalent) was added. The mixture was stirred at 80 °C for 2.5 h under nitrogen. The reaction mixture was diluted with brine (30 mL) and extracted with EtOAc (30 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 4g Purification was performed for 14 min using a silica fast column with an eluent gradient of 0%–10% ethyl acetate / petroleum ether at 18 mL / min, for a total volume of 0.3 L. The compound aa2-6 was obtained as a yellow oil (100 mg, 193.20 μmol, 70.76% yield).

[0914] LCMS(ESI): RT = 0.950 min, for C 31 H 35 The calculated mass of NO₆Na is 540.24, [M + Na] + m / z, measured 540.1 [M+Na]+ Inverting LC-MS was performed using method A.

[0915] 1 ¹H NMR (400MHz, chloroform-d) δ=7.39(d,J=8.6Hz,2H),7.27-7.25(m,2H),7.23(s,2H),7.17(dd,J=8.3,16.2Hz,3H),6.97-6.91(m,3H),6.67(dd,J=11.0,17.4Hz,1H),5.67(dd,J=1.1,17.4Hz,1H),5.22-5.15(m,1H),5.05(s,2H),3.83(s,3H),3.74(s,3H),1.47-1.35(m,1H),1.43(s,8H).

[0916] Step 5: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(2'-ethyl-4'-hydroxy-[1,1'-biphenyl]-4-yl)propionate (aa2-7)

[0917] Pd / C (300 mg, 10% activated palladium carbide) was added to a solution of aa2-6 (2.8 g, 5.41 mmol, 1.0 equivalence) in MeOH (25 mL), and the mixture was stirred at 25 °C for 48 hours under hydrogen (15 psi). The reaction mixture was filtered and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica rapid column with an eluent gradient of 0%–30% ethyl acetate / petroleum ether at 35 mL / min for 22 min, with a total volume of 0.9 L. Compound aa2-7 was obtained as a colorless oil (1.85 g, 4.60 mmol, 85.01% yield, 99.3% purity).

[0918] LCMS(ESI): RT = 0.935 min, for C 23 H 29 The calculated mass of NO₅Na is 422.20 [M + Na]. + , m / z, measured 422.1 [M+Na] + Inverting LC-MS was performed using method A.

[0919] 1H NMR (400MHz, chloroform-d) δ = 7.22-7.11 (m, 4H), 7.04 (d, J = 8.2Hz, 1H), 6.78 (d, J = 2.6Hz, 1H), 6.69 (dd, J = 2.6, 8.2Hz, 1H), 5.24 (s, 1H), 5.05 (br d,J=8.4Hz,1H),4.70-4.58(m,1H),3.73(s,3H),3.20-3.11(m,1H),3.11 -3.02(m,1H),2.53(q,J=7.6Hz,2H),1.42(s,9H),1.08(t,J=7.5Hz,3H).

[0920] Step 6: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4'-(4-chlorobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionate (aa2-8)

[0921] 1-Chloro-4-iodobutane (287.11 mg, 1.31 mmol, 1.5 equivalence) was added to a solution of aa2-7 (350 mg, 876.14 μmol, 1 equivalent) and K2CO3 (242.18 mg, 1.75 mmol, 2.0 equivalent) in DMF (5 mL) at 25 °C. The mixture was stirred at 50 °C for 12 hours. The residue was diluted with brine (50 mL) and extracted with EtOAc (50 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica rapid column with an eluent gradient of 0%–30% ethyl acetate / petroleum ether at 35 mL / min for 14 min, with a total volume of 0.4 L. Compound aa2-8 (340 mg, crude) was obtained as a yellow oil.

[0922] LCMS(ESI): RT = 1.145 min, for C 27 H 36 The calculated mass of ClNO₅Na is 512.22 [M + Na]. + m / z, measured 512.2 [M+Na] + Inverting LC-MS was performed using method A.

[0923] Step 7: Synthesis of methyl (S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propionate (aa2-9)

[0924] K₂CO₃ (902.51 mg, 6.53 mmol, 2.0 equivalent), KI (54.20 mg, 326.51 μmol, 0.1 equivalent), and NaN₃ (460 mg, 7.08 mmol, 2.1 equivalent) were added to a solution of aa₂-8 (1.6 g, 3.27 mmol, 1.0 equivalent) in DMF (15 mL). The mixture was stirred at 50 °C for 7 hours. The reaction mixture was diluted with 50 mL of brine and extracted with EtOAc (50 mL × 2). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The aqueous layer was quenched by adding an aqueous solution of NaClO (1.0 M, 100 mL). Compound aa₂-9 (1.7 g, crude) was obtained as a yellow oil.

[0925] LCMS(ESI): RT = 1.140 min, for C 27 H 36 The calculated mass of N₄O₅Na is 519.27 m / z, while the measured mass is 519.3 [M + Na]. + Inverting LC-MS was performed using method A.

[0926] Step 8: Synthesis of (S)-3-(4'-(4-azidobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)-2-((tert-butoxycarbonyl)amino)propionic acid (aa2-10)

[0927] LiOH·H2O (287.31 mg, 6.85 mmol, 2.0 equivalence) in H2O (6 mL) was added to a solution of aa2-9 (1.7 g, 3.42 mmol, 1.0 equivalence) in THF (12 mL) at 0 °C, and the mixture was then allowed to gradually heat to 25 °C and stirred for 2 hours. The mixture was treated with EtOAc (30 mL) and extracted with water (25 mL × 2). The combined aqueous layers were acidified (1 M HCl aqueous solution) and extracted with EtOAc (50 mL × 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Compound aa2-10 (2.01 g, crude) was obtained as a yellow oil, which was used directly in the next step without any further purification.

[0928] Step 9: Synthesis of (S)-2-amino-3-(4'-(4-azidobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionate (aa2-11)

[0929] Compound aa2-10 (2.01 g, 4.17 mmol, 1.0 equivalent) was dissolved in 4.0 M HCl / EtOAc (20 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered. The filter cake was washed with EtOAc (30 mL) and dried under vacuum. Compound aa2-11 (1 g, crude) was obtained as a white solid and used directly in the next step without any further purification.

[0930] LCMS(ESI): RT = 1.121 min, for C 21 H 27 The calculated mass of N4O3 is 383.21 [M+H]. + The measured m / z is 383.2 [M+H]. + Inverting LC-MS was performed using method A.

[0931] Step 10: Synthesis of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-(4-azidobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionic acid (aa2)

[0932] NaHCO3 (401.07 mg, 4.77 mmol, 2.0 equivalence) in H2O (8 mL) was added to a solution of aa2-11 (1 g, 2.39 mmol, 1.0 equivalence) in THF (15 mL) at 0 °C, followed by (2,5-dioxopyrrolidone-1-yl)9H-fluorene-9-ylmethyl carbonate (805.23 mg, 2.39 mmol, 1.0 equivalence). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with brine (100 mL) and acidified (1 M HCl aqueous solution) to pH 2–3. The reaction mixture was extracted with EtOAc (30 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica rapid column with 0%–10% methanol / dichloromethane eluent at 30 mL / min for 16 min, with a total volume of 0.6 L. The product aa2 (1.3 g, 2.14 mmol, 89.52% yield, 99.4% purity) was obtained as a white foam.

[0933] LCMS(ESI): RT = 1.113 min, for C 36 H 36 The calculated mass of N4O5Na is 627.26 m / z, while the measured mass is 627.3 [M+Na]. + Inverting LC-MS was performed using method A.

[0934] 1 H NMR (400MHz, DMSO-d6) δ = 7.88 (d, J = 7.5Hz, 2H), 7.81 (d, J = 8.6Hz, 1H), 7.66 (t, J = 6.9Hz,2H),7.40(dt,J=2.3,7.3Hz,2H),7.34-7.25(m,4H),7.14(d,J=8.2Hz,2H) ,6.97(d,J=8.4Hz,1H),6.83(d,J=2.4Hz,1H),6.76(dd,J=2.5,8.5Hz,1H),4.27- 4.17(m,3H),4.17-4.13(m,1H),4.03-3.98(m,2H),3.42(t,J=6.7Hz,2H),3.13(br dd,J=3.9,13.8Hz,1H),2.96-2.87(m,1H),2.52(d,J=1.8Hz,2H),2.43(q ,J=7.4Hz,2H),1.82-1.74(m,2H),1.73-1.66(m,2H),0.98-0.90(m,3H).

[0935] SFC: ee% = 97.95% - 2.05% = 95.9%; Method notes: Column: Chiralcel OJ-3100 × 4.6 mm I.D., 3 μm; Mobile phase: A: CO2 B: Ethanol (0.05% DEA); Gradient: From 5% B to 40% B over 4 min and hold at 40% for 2.5 min, then hold at 5% B for 1.5 min; Flow rate: 2.8 mL / min; Column temperature: 35 °C; ABPR: 1500 psi.

[0936] 2.3 Synthesis of non-natural amino acids (aa2b)

[0937] Scheme 4 outlines the synthesis of non-natural amino acids (aa1b):

[0938] Option 4

[0939]

[0940] Reagents and conditions: (a) H2, Pd / C, (Boc)2O, DIPEA, MeOH, 20℃, 12h, 47%.

[0941] Step 1: Synthesis of (R)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-(4-((tert-butoxycarbonyl)amino)butoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionic acid (aa2b)

[0942] Pd / C (700 mg, 10% activated palladium carbon), DIPEA (1.37 g, 10.60 mmol, 1.84 mL, 2.0 equivalents), and Boc2O (2.31 g, 10.58 mmol, 2.43 mL, 2.0 equivalents) were added to a solution of aa2 (3.2 g, 5.29 mmol, 1.0 equivalents) in MeOH (30 mL). The mixture was stirred at 20 °C for 12 hours under hydrogen (15 psi). The reaction mixture was filtered through a small diatomaceous earth pad, and the filter cake was washed with 40 x 5 mL EtOAc (40 mL x 5). Then brine (400 mL) was added and extracted with EtOAc (300 mL x 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was passed through a preparative HPLC (column: Phenomenex Luna C18 250*50mm*10μm; mobile phase: [water (0.225% FA)-ACN]; B%: 55%-86%, 21 min). The product was suspended in water (150 mL) and the mixture was frozen in a dry ice / ethanol bath to obtain product aa2b as a white solid (3.4 g, 5.01 mmol, 47.32% yield, 100% purity).

[0943] LCMS(ESI): RT = 0.922 min, for C 41 H 46 The calculated mass of N₂O₇Na is 701.33 [M + Na]. + m / z, measured 701.3 [M+Na] + Inverted LC-MS was performed using method C.

[0944] 1 H NMR (400MHz, DMSO-d6) δ = 7.87 (d, J = 7.6Hz, 2H), 7.69-7.60 (m, 2H), 7.43-7.36 (m, 2H), 7.33-7.20 (m, 4H), 7.15-7.05 (m, 3H), 6.95 (br d,J=8.3Hz,1H),6.89-6.70(m,4H),4.28(br dd,J=5.9,9.0Hz,1H),4.17-4.09(m,2H),4.00-3.91(m,3H),3.19-3.09(m,2H),3.02-2. 88(m,2H),2.46-2.38(m,2H),1.73-1.62(m,2H),1.56-1.48(m,2H),1.37(s,9H),0.93(br t,J=7.6Hz,3H).

[0945] 2.4 Synthesis of non-natural amino acids (aa13)

[0946] Scheme 5 outlines the synthesis of non-natural amino acids (aa13):

[0947] Option 5

[0948]

[0949] Reagents and conditions: (a) HATU (1.5 equivalent), DHP (1.0 equivalent), DIPEA (3.0 equivalent), DCM, 25℃, 2h; (b) 10% Pd / C, MeOH, 25℃, 5h.

[0950] aa13-1 was prepared according to WO 2010 / 052253, the contents of which are incorporated herein by reference in their entirety.

[0951] Step 1: Synthesis of benzyl 2,2-dimethyl-3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxy)amino)propionate (aa13-2)

[0952] A solution of aa13-1 (200 mg, 899.94 μmol, 1 equivalent), HATU (513.28 mg, 1.35 mmol, 1.5 equivalent), and DIPEA (348.93 mg, 2.70 mmol, 470.26 μL, 3 equivalent) in DCM (10 mL) was stirred at 25 °C for 10 min. Tetrahydropyran-2-hydroxylamine (105.42 mg, 899.94 μmol, 1 equivalent) was added to the mixture, and the mixture was stirred at 25 °C for 2 h. DCM (5 mL) was added to the reactants, and the mixture was washed with an aqueous solution of NH4Cl (5 mL). The aqueous layer was separated and extracted with DCM (5 mL). The organic layers were combined and washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give a crude product as a yellow oil, which was obtained by rapid silica gel chromatography (…). 20g Purification was performed using a silica rapid column with 0%–35% ethyl acetate / petroleum ether gradient eluent at 30 mL / min to give aa13-2 as a colorless oil (230 mg, 715.69 μmol, 79.53% yield).

[0953] 1H NMR (400MHz, chloroform-d) δ = 9.28-9.14 (m, 1H), 7.38-7.30 (m, 5H), 5.16 (s, 2H), 4.87 (br s, 1H), 3.93-3.80 (m, 1H), 3.59 (br d, J = 1.5Hz, 1H), 1.76 (br s,3H),1.65-1.58(m,1H),1.56-1.52(m,2H),1.48(s,6H).

[0954] Step 2: Synthesis of 2,2-dimethyl-3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxy)amino)propionic acid (aa13)

[0955] A black suspension of aa13-2 (230 mg, 715.69 μmol, 1 equivalent) and 10% Pd / C (23 mg) in MeOH (5 mL) was stirred at 25 °C for 5 hours under H2 at 15 Psi. The reaction mixture was filtered through a diatomaceous earth mat, and the filter cake was washed with MeOH (5 mL x 3). The filtrate was concentrated under vacuum to give aa13 as a colorless oil (120 mg, 518.93 μmol, 72.51% yield).

[0956] 1 ¹H NMR (400MHz, methanol-d⁴) δ=4.90-4.88(m,1H),4.05(br s,1H),3.59-3.53(m,1H),1.85-1.68(m,3H),1.68-1.51(m,3H),1.40(s,6H).

[0957] 2.5 Synthesis of non-natural amino acids (aa14)

[0958] Scheme 6 outlines the synthesis of non-natural amino acids (aa14):

[0959] Option 6

[0960]

[0961] Reagents and conditions: (a) aa-14-1a (1.0 equivalent), dioxane, 50℃, 15h; (b) LiOH·H2O (2.0 equivalent), THF / H2O = 1 / 1, 50℃, 16h; (c) 10% Pd / C cat.TFA, EtOH

[0962] Compound aa14-1 was prepared according to US2015 / 380666.

[0963] Step 1: Synthesis of methyl 2-(1-benzyl-1H-pyrazole-5-yl)-2-methylpropionate (aa14-3)

[0964] A mixture of AA14-1 (2.76 g, 13.85 mmol, 1 equivalent) and AA14-1A (2.20 g, 13.85 mmol, 1 equivalent) in dioxane (30 mL) was stirred at 50 °C for 15 hours. The reaction mixture was filtered, and the filtrate was concentrated under vacuum to give a crude product as a brown oil. The residue was subjected to rapid silica gel chromatography (…). 20g Purified by silica fast column, eluting with 0%–20% ethyl acetate / petroleum ether gradient @ 25 mL / min, to give aa14-2 as a pale yellow oil (0.9 g, 3.48 mmol, 25.15% yield).

[0965] 1 ¹H NMR (400MHz, chloroform-d) δ=7.53(s,1H), 7.29-7.26(m,1H), 7.23-7.16(m,1H), 6.98(d,J=7.4Hz,2H), 6.22(s,1H), 5.23(s,2H), 3.26(s,3H), 1.56(s,6H).

[0966] Step 2: Synthesis of methyl 2-(1-benzyl-1H-pyrazole-5-yl)-2-methylpropionate (aa14-3)

[0967] A mixture of aa14-2 (700 mg, 2.71 mmol, 1 equivalent) and LiOH·H2O (1 M, 5.42 mL, 2 equivalents) in THF (5.4 mL) was stirred at 50 °C for 16 hours. The reaction mixture was concentrated under vacuum to remove THF. The reaction mixture was diluted with water (5 mL) and MTBE (5 mL). The aqueous layer was separated and adjusted to pH 6 with 1 N HCl. The organic layer was discarded. The aqueous layer was extracted with EtOAc (5 mL x 3). The organic layers were combined and washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to give aa14-3 as a white solid (600 mg, 2.46 mmol, 90.64% yield).

[0968] 1 ¹H NMR (400MHz, chloroform-d) δ=7.53(s,1H),7.26-7.17(m,3H),6.97(d,J=7.4Hz,2H),6.24(s,1H),5.23(s,2H),1.54(s,6H).

[0969] Step 3: Synthesis of 2-methyl-2-(1H-pyrazol-5-yl)propionic acid (aa14)

[0970] A mixture of aa14-3 (500 mg, 2.05 mmol, 1 equivalent), TFA (23.34 mg, 204.68 μmol, 15.15 μL, 0.1 equivalent), and 10% Pd / C (100 mg) in EtOH (5 mL) was stirred at 80 °C for 16 h at 100 Psi H2. The reactants were filtered through a diatomaceous earth mat, and the filter cake was washed with EtOH (5 mL * 3). The filtrate was concentrated under vacuum to give aa14 as a pale yellow oil (300 mg, 1.52 mmol, 74.27% yield, 78.11% purity). LCMS (ESI): RT = 1.036 min, m / z, for C7H 11 N₂O₂, calculated to be 155.07 [M+H] + The measured value was 155.0. Inverting LC-MS was performed using method A.

[0971] 1 ¹H NMR (400MHz, methanol-d⁴) δ=7.57-7.50(m,1H), 6.26(d,J=1.8Hz,1H), 1.56(s,6H).

[0972] 2.6 Synthesis of non-natural amino acids [aa20:(R)-isomer and aa21:(S)-isomer]

[0973] Scheme 7 outlines the synthesis of non-natural amino acids (aa20) and (aa21):

[0974] Option 7

[0975]

[0976] Reagents and conditions: (a) NaH (1.2 equivalents), dibromoethane (2 equivalents), THF (25 ml), 0℃~100℃, 16 h; (b) 2-(1-triphenylmethyl-1H-imidazol-5-yl)ethylamine (0.9 equivalents), DMF (5 ml), 60℃, 16 h; (c) LiOH (4 equivalents), H2O (10 ml), THF (10 ml), 20℃, 16 h; (d) SFC separation.

[0977] Step 1: Synthesis of diethyl 2-(2-bromoethyl)-2-methylmalonic acid (aa20-2)

[0978] A specific volume of THF (10 mL) was added to NaH (252.57 mg, 6.31 mmol, 60% purity, 1.1 equivalents) under a nitrogen atmosphere. The THF solution was cooled to 0 °C. Compound aa20-1 (1 g, 5.74 mmol, 980.39 μL, 1 equivalent) was added to THF (5 mL) over 30 min with stirring. The reaction mixture was allowed to stir at 20 °C for 60 min. The resulting enol ester was added dropwise to 1,2-dibromoethane (2.16 g, 11.48 mmol, 866.23 μL, 2 equivalents) in THF (10 mL) over 60 min with stirring under a nitrogen atmosphere. The reaction mixture was then heated to 100 °C in the solvent for 14 h. TLC indicated that the reactants were consumed and a major new spot with low polarity was detected. The residue was poured into 1N HCl to adjust the pH to 2–4. The aqueous phase was then extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was then subjected to rapid silica gel chromatography (SPCC) with 0.8 L of solvent. 24g Purification was performed using a silica rapid column with an eluent gradient of 0%–30% ethyl acetate / petroleum ether at 20 mL / min for 40 min. The compound aa20-2 (1.2 g, 4.27 mmol, 74.35% yield) was obtained as a pale yellow liquid.

[0979] 1 ¹H NMR (400MHz, chloroform-d) δ 4.18 (q, J = 7.20Hz, 4H), 3.33–3.41 (m, 2H), 2.39–2.47 (m, 2H), 1.43 (s, 3H), 1.22 (s, 6H).

[0980] Step 2: Synthesis of ethyl 3-methyl-2-oxo-1-(2-(1-triphenylmethyl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylate (aa20-3)

[0981] Add aa20-2 (874.95 mg, 3.11 mmol, 1.1 equivalent) to a solution of 2-(1-triphenylmethyl-1H-imidazol-5-yl)ethylamine (1 g, 2.83 mmol, 1 equivalent) in DMF (10 mL). Stir the mixture at 60 °C for 16 h. LCMS showed complete consumption of the material, and the desired product was observed as the major product. Pour the residue into water (100 mL). Extract the aqueous phase with ethyl acetate (50 mL x 3). Wash the combined organic phases with brine (100 mL), dry to anhydrous Na2SO4, filter, and concentrate under vacuum. Perform C-18 reversed-phase chromatography (C-18). The compound was purified to provide a yellow solid using an 80 g C-18 column with an elution gradient of 0%–100% acetonitrile / H₂O at 40 mL / min for 60 min (total volume 2400 mL). The compound aa20-3 was obtained as a yellow solid (650 mg, 903.47 μmol, 31.93% yield, 70.55% purity).

[0982] LCMS(ESI): RT = 0.861 min, m / z, for C 32 H 34 The calculated value of N3O3 is 508.25 [M+H]. + The measured value was 508.3. Inverting LC-MS was performed using method A.

[0983] Step 3: Synthesis of 3-methyl-2-oxo-1-(2-(1-triphenylmethyl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa20-4)

[0984] A mixture of aa20-3 (650 mg, 1.28 mmol, 1 equivalent) was added to LiOH·H2O (268.67 mg, 6.40 mmol, 5 equivalents) in H2O (10 mL) and THF (10 mL). The mixture was stirred at 20 °C for 16 hours. LCMS showed that the material was completely consumed and the desired product was observed as the major product. The residue was poured into H2O (50 mL), and 5% KHSO4 was added to adjust the pH to 2–3. The aqueous phase was extracted with ethyl acetate (50 mL * 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (column: Phenomenex SynergiMax-RP 250*50mm*10μm; mobile phase: water (0.225% FA)-ACN; B%: 30%-60%, 60min) to give aa20-4 as a white solid (180 mg, 375.34 μmol, 29.31% yield).

[0985] LCMS(ESI): RT = 2.391 min, m / z for C 30 H 30 The calculated N3O3 content is 480.6, while the actual measured value is 480.3 [M+H]. + Reversed-phase LC-MS was performed using a Merck RP-18e 25-2 mm column at a flow rate of 1.5 mL / min, with gradient elution using 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[0986] 1H NMR(ES8586-496-P1B1)1H NMR(400MHz, methanol-d4)δ7.92(s,1H),7.35-7.42(m,9H),7.12-7.19(m,6H),6.97(s,1H),3.39-3.64(m,3H),3.32(br d,J=3.75Hz,1H),2.84(br t,J=5.95Hz,2H),2.34-2.42(m,1H),1.86(td,J=7.99,12.90Hz,1H),1.25(s,3H).

[0987] Step 4: Synthesis of (R)-3-methyl-2-oxo-1-(2-(1-triphenylmethyl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa20) and (S)-3-methyl-2-oxo-1-(2-(1-triphenylmethyl-1H-imidazol-5-yl)ethyl)pyrrolidine-3-carboxylic acid (aa21)

[0988] The absolute configurations of aa20(R) and aa21(S) have not yet been confirmed.

[0989] Compound aa20-4 (180 mg, 375.34 μmol) was purified by a preparative SFC (column: DAICL CHIRALPAK IC (250 mm * 30 mm, 10 μm; mobile phase: 0.1% NH3H2O ​​MEOH; B%: 50%-50%, 80 min). The isomer (R)aa20 (86 mg, 177.18 μmol, 94.41% yield, 98.8% purity) was obtained as a white solid, and the isomer (S)aa21 (85 mg, 175.29 μmol, 93.41% yield, 98.9% purity) was also obtained as a white solid.

[0990] Isomer aa20 was run by SFC-HPLC: RT = 2.346 min. HPLC conditions: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm, flow rate 2.8 mL / min. Elution was performed using a gradient of 40% methanol (0.05% DEA) (solvent B) and CO2 (solvent A).

[0991] Isomer aa21 was run by SFC-HPLC: RT = 3.607 min. HPLC conditions: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm, flow rate 2.8 mL / min. Elution was performed using a gradient of 40% methanol (0.05% DEA) (solvent B) and CO2 (solvent A).

[0992] 2.7 Synthesis of non-natural amino acids [aa22(S)-isomer and aa23(R)-isomer]

[0993] Scheme 8 outlines the synthesis of non-natural amino acids (aa22) and (aa23):

[0994] Option 8

[0995]

[0996] Reagents and conditions: (a) TrtCl (1.2 equivalents), Et3N (2 equivalents), DMF, 20℃, 2h, 31%; (b) TsCl (1.5 equivalents), Et3N (3 equivalents), DMAP (0.5 equivalents), DCM, 20℃, 2h, 66%; (c) aa22-3 (1.0 equivalents), aa22-3a (1.2 equivalents), LiI (1.5 equivalents), DIPEA (4 equivalents), 60℃, 2h, 17% yield; (d) aa22-3 (1.0 equivalents), aa22-3b (1.2 equivalents), LiI (1.5 equivalents), K2CO3 (4 equivalents), 20℃, 3h, 58% yield; (e) NaOH (2 equivalents), MeOH / water, 20℃, 2h, 87% yield.

[0997] Step 1: Synthesis of 2-(1-triphenylmethyl-1H-imidazol-4-yl)ethanol (aa22-2)

[0998] TEA (3.61 g, 35.67 mmol, 4.97 mL, 2 equivalents) and [chloro(diphenyl)methyl]benzene (5.97 g, 21.40 mmol, 1.2 equivalents) were added to a solution of aa22-1 (2 g, 17.84 mmol, 1 equivalent) in DMF (40 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 2 hours. The reaction progress was monitored by TLC (DCM:MeOH = 10:1), which indicated that the starting material had been consumed and a new spot was observed. The reaction mixture was quenched by NaHCO3 (saturated aqueous solution, 20 mL) and extracted with EtOAc (50 mL * 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica rapid column with an eluent gradient of 0%–100% ethyl acetate / petroleum ether followed by 0%–10% MeOH (0.5% TEA additive) / DCM at 30 mL / min for 25 min, for a total volume of 1.6 L. Compound aa22-2 was obtained as a white solid (2.2 g, 5.59 mmol, 31.32% yield, 90% purity).

[0999] LCMS (ESI): RT = 1.397 min, m / z, for C24 H 23 The calculated N₂O concentration was 355.2, and the actual measured concentration was 355.2 [M+H]. + Inverted LC-MS was performed using method B.

[1000] 1 ¹H NMR (400MHz, chloroform-d) δ=7.39-7.31(m, 10H), 7.17-7.11(m, 6H), 6.63-6.57(m, 1H), 3.89(t, J=5.6Hz, 2H), 3.64(br s, 1H), 2.76(t, J=5.5Hz, 2H).

[1001] Step 2: Synthesis of 2-(1-triphenylmethyl-1H-imidazol-4-yl)ethyl-4-methylbenzenesulfonate (aa22-3)

[1002] TEA (1.70 g, 16.76 mmol, 2.33 mL, 3 equivalents), 4-methylbenzenesulfonyl chloride (1.60 g, 8.38 mmol, 1.5 equivalents), and DMAP (341.23 mg, 2.79 mmol, 0.5 equivalents) were added to a solution of aa22-2 (2.2 g, 5.59 mmol, 1 equivalent) in DCM (20 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 2 hours. The reaction progress was monitored by LC-MS, which indicated that no starting material remained and the desired product was formed. The reaction mixture was quenched by passing NaHCO3 (50 mL saturated aqueous solution) and extracted with EtOAc (50 mL × 2). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 24g Purification was performed using a silica fast column with an eluent gradient of 0%–5% MeOH (4% TEA additive) / DCM at 20 mL / min for 35 min, for a total volume of 1.2 L. Compound aa22-3 was obtained as a brown solid (2.1 g, 3.72 mmol, 66.52% yield, 90% purity). LCMS (ESI): RT = 0.775 min, m / z, for C 31 H 28 The calculated value of N₂O₃SNa is 531.2 [M+H]. + The measured value was 531.1. Inverting LC-MS was performed using method D.

[1003] 1¹H NMR (400MHz, chloroform-d) δ=7.73(d,J=8.3Hz,2H), 7.38-7.28(m,12H), 7.15-7.08(m,6H), 6.61(s,1H), 4.27(t,J=7.0Hz,2H), 2.91-2.86(m,2H), 2.42(s,3H).

[1004] Step 3: Synthesis of (R)-1-(2-(1-triphenylmethyl-1H-imidazol-4-yl)ethyl)pyrrolidine-3-carboxylic acid (aa22)

[1005] Add aa22-3a (135.81 mg, 1.18 mmol, 1.2 equivalence), LiI (197.36 mg, 1.47 mmol, 56.55 μL, 1.5 equivalence), and DIPEA (508.20 mg, 3.93 mmol, 684.91 μL, 4.0 equivalence) to a solution of aa22-3 (500 mg, 983.03 μmol, 1 equivalent) in DMF (2 mL). The mixture was then heated to 60 °C and stirred at 60 °C for 2 hours. LC-MS showed complete consumption of the reactants and detection of the desired MS. The reaction mixture was quenched by adding water (10 mL), diluted with EtOAc (20 mL), and extracted with EtOAc (20 mL x 2). The aqueous phase was acidified to pH 6 with 2 M HCl. The aqueous phase was then extracted with DCM (20 mL x 5). The combined organic layers were concentrated to give the residue. The crude product was purified by reversed-phase HPLC (neutral conditions). Product aa22 (80 mg, 168.31 μmol, 17.12% yield, 95% purity) was obtained as a grayish-white solid. LCMS (ESI): RT = 1.986 min, for C 29 H 30 The calculated mass of N3O2 is 452.23 m / z, while the actual measured mass is 452.3 [M+H]. + Inverting LC-MS was performed using method A.

[1006] Step 4: Synthesis of (3S)-1-[2-(1-triphenylmethylimidazol-4-yl)ethyl]pyrrolidine-3-carboxylic acid methyl ester (aa23-1)

[1007] K₂CO₃ (815.17 mg, 5.90 mmol, 4 equivalents), aa₂₃b (293.05 mg, 1.77 mmol, 1.2 equivalents, HCl), and LiI (296.04 mg, 2.21 mmol, 84.83 μL, 1.5 equivalents) were added to a solution of aa₂₃ (750 mg, 1.47 mmol, 1 equivalent) in DMF (5 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 3 h. The reaction progress was monitored by LC-MS, indicating that no starting material remained and the desired product was formed. The mixture was cooled to 25 °C and poured into water (30 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (50 mL x 2). The combined organic phases were washed with brine (15 mL x 2), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica rapid column with an eluent gradient of 0%–100% ethyl acetate / petroleum ether followed by 0%–10% MeOH (0.5% TEA additive) / DCM at 30 mL / min for 25 min, for a total volume of 1.6 L. The product aa23-1 (420 mg, 856.99 μmol, 58.12% yield, 95% purity) was obtained as a brown oil.

[1008] LCMS (ESI): RT = 0.717 min, m / z, for C 30 H 31 The calculated value of N3O2 is 466.2 [M+H]. + The measured value was 465.9; LC-MS conditions: reversed-phase LC-MS was performed using method D.

[1009] 1 ¹H NMR (400MHz, chloroform-d) δ=7.40-7.30(m,10H),7.20-7.09(m,6H),6.58(s,1H),3.75-3.64(m,3H),3.10-2.95(m,2H),2.83-2.74(m,5H),2.72-2.64(m,1H),2.55(q,J=8.0Hz,1H),2.14-2.07(m,2H).

[1010] Step 5: Synthesis of (3S)-1-[2-(1-triphenylmethylimidazol-4-yl)ethyl]pyrrolidine-3-carboxylic acid methyl ester (aa23)

[1011] NaOH (67.01 mg, 1.68 mmol, 2 equivalents) was added to a solution of aa23-1 (390.00 mg, 837.66 μmol, 1 equivalent) in MeOH (3 mL) and water (3 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 2 hours. The reaction progress was monitored by LCMS, which indicated that no starting material remained and the desired product was formed. After completion, the reaction mixture was cooled to room temperature. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was adjusted to pH 6 with 1 M HCl and dried by lyophilization. The residue was milled with DCM / MeOH = 10 / 1 (15 mL * 3). The combined organic phases were dried with anhydrous Na2SO4, filtered, and concentrated under vacuum. The product aa23 (350 mg, 736.34 μmol, 87.90% yield, 95% purity) was obtained as a brown solid.

[1012] LCMS (ESI): RT = 0.708 min, m / z, for C 29 H 30 The calculated value of N3O2 is 452.2 [M+H]. + The measured value was 451.9; inverted LC-MS was performed using method D.

[1013] 1 H NMR (400MHz, chloroform-d) δ = 7.38 (s, 1H), 7.36-7.31 (m, 9H), 7.12 (dd, J = 3.3, 6.3Hz, 6H), 6.64 (s, 1H), 3.83 (br s, 1H), 3.56 (br s, 1H), 3.20 (br d,J=18.5Hz,2H),3.05-2.96(m,4H),2.65-2.61(m,1H),2.42-2.30(m,1H),2.18-2.09(m,1H).

[1014] 2.8 Synthesis of non-natural amino acids (aa27)

[1015] Scheme 9 outlines the synthesis of non-natural amino acids (aa27):

[1016] Option 9

[1017]

[1018] Reagents and conditions: (a) HCl / dioxane, 0℃-25℃, 2h, 100%; (b) Cb2Cl (1.2 equivalents), HOSu (1.3 equivalents), DIPEA (2 equivalents), DCM, 0℃-20℃, 12h, 96%; (c) 3a (2 equivalents), K2CO3 (2 equivalents), 60℃, 12h, 56%; (d) LiOH (2.0 equivalents), THF, H2O, 20℃, 12h, 94%; (e) H2, Pd(OH)2, AcOH, MeOH, 20℃, 12h; (f) FmocOSu (1.2 equivalents), NaHCO3 (2.0 equivalents), THF, H2O, 0℃-20℃, 12h, 44%; (g) PhNH2 (1.1 equivalents), DIC (1.0 equivalents), HOBt (1.0 equivalents), DCM, 0℃-20℃, 1h, 76%; (h) Piperidine (5.0 equivalents), DMF, 20℃, 1h, 65%.

[1019] Step 1: Synthesis of methyl (2S)-2-amino-3-[4-(2-ethyl-4-hydroxy-phenyl)phenyl]propionate (aa27-2)

[1020] Compound aa27-1 (3.5 g, 8.76 mmol, 1 equivalent) was dissolved in HCl / dioxane (4 M, 100 mL, 45.65 equivalent) at 0 °C. The mixture was stirred at 25 °C for 2 h. The reaction progress was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to give a crude product. Compound aa27-2 (2.6 g, crude) was given as a colorless oil.

[1021] LCMS(ESI): RT = 0.772 min, for C 18 H 22 The calculated mass of NO3 is 300.16 [M+H]. + Actual m / z measurement: 300.00 [M+H] + Inverting LC-MS was performed using method A.

[1022] Step 2: Synthesis of methyl (2S)-2-(benzyloxycarbonylamino)-3-[4-(2-ethyl-4-hydroxy-phenyl)phenyl]propionate (aa27-3)

[1023] DIPEA (3.24 g, 25.05 mmol, 4.36 mL, 3 equivalents) and CbzCl (1.57 g, 9.19 mmol, 1.31 mL, 1.1 equivalents) were added to a solution of HOSu (1.25 g, 10.86 mmol, 1.3 equivalents) in DCM (50 mL) at 0 °C. After stirring at 25 °C for 2 h, compound aa27-2 (2.5 g, 8.35 mmol, 1 equivalent) was added and the mixture was stirred at 20 °C for 10 h. The reaction progress was monitored by LC-MS and TLC. The mixture was diluted with DCM (30 mL) and washed with H2O (30 mL * 2). The organic layer was dried over Na2SO4, filtered, and concentrated to give the residue, which was then purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 2 / 1). The compound aa27-3 (3.5 g, 8.07 mmol, 96.68% yield) was obtained as a yellow oil.

[1024] LCMS(ESI): RT = 0.992 min, for C26H27NNaO5+, the calculated mass is 456.18 [M+Na]+, m / z, and the measured mass is 456.2 [M+Na]. + Inverting LC-MS was performed using method A.

[1025] 1 H NMR (400MHz, DMSO-d6) δppm 9.35 (s, 1H) 7.88 (br d, J = 8.07Hz, 1H) 7.21-7.38 (m, 7H) 7.15 (br d,J=8.07Hz,2H)6.93(d,J=8.31Hz,1H)6.70(d,J=2.20Hz,1H)6.63(dd,J=8.07,2.45Hz,1H)4.99(br s,2H)4.24-4.36(m,1H)3.60-3.65(m,3H)3.07(br dd,J=13.69,4.89Hz,1H)2.91(br dd,J=13.57,10.39Hz,1H)2.40-2.48(m,2H)0.99(t,J=7.58Hz,3H).

[1026] Step 3: Synthesis of methyl (S)-2-(((benzyloxy)carbonyl)amino)-3-(4'-(4-((tert-butoxycarbonyl)amino)butoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionate (aa27-4)

[1027] K₂CO₃ (2.10 g, 15.23 mmol, 2 equivalents) was added to a solution of aa27-3 (3.3 g, 7.61 mmol, 1 equivalent) and aa27-3a (5.23 g, 15.23 mmol, 2 equivalents) in DMF (30 mL). The mixture was stirred at 60 °C for 12 h. The reaction progress was monitored by LC-MS and TLC. The reaction mixture was diluted with EtOAc (100 mL) and washed with H₂O (80 mL x 3). The organic layer was dried over Na₂SO₄, filtered, and concentrated to give the residue, which was then purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 10 / 1 to 2 / 1). Compound aa27-4 (2.9 g, 4.32 mmol, 56.70% yield, 90% purity) was given as a yellow oil.

[1028] LCMS(ESI): RT = 1.140 min, for C 35 H 44 The calculated mass of N₂NaO₇ is 627.30 [M + Na]. + m / z, measured value 627.2 [M+Na] + Inverting LC-MS was performed using method A.

[1029] Step 4: Synthesis of (S)-2-(((benzyloxy)carbonyl)amino)-3-(4'-(4-((tert-butoxycarbonyl)amino)butoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionic acid (aa27-5)

[1030] A solution of LiOH·H₂O (374.72 mg, 8.93 mmol, 2 equivalents) in H₂O (15 mL) was added to a solution of aa27-4 (2.7 g, 4.46 mmol, 1 equivalent) in THF (30 mL). The mixture was stirred at 20 °C for 2 hours. The reaction progress was monitored by LC-MS. The mixture was adjusted to pH 3–4 with 1 M HCl and extracted with EtOAc (20 mL * 3). The organic layer was dried over Na₂SO₄, filtered, and concentrated to give the product. Compound aa27-5 (2.5 g, 4.23 mmol, 94.79% yield) was given as a yellow oil.

[1031] LCMS(ESI): RT = 1.082 min, for C 34 H 42 The calculated mass of N₂NaO₇ is 613.29 m / z, while the actual measured mass is 613.2 [M + Na]. + Inverting LC-MS was performed using method A.

[1032] 1H NMR (400MHz, chloroform-d) δppm 7.29-7.36 (m, 5H) 7.14-7.21 (m, 4H) 7.07 (br d, J=8.56Hz, 1H) 6.81 (s, 1H) 6.73 (br d, J=7.83Hz, 1H) 5.27 (br d,J=7.58Hz,1H)5.05-5.16(m,2H)4.68-4.75(m,1H)4.50-4.68(m,2H)3.99(br s,2H)3.20-3.28(m,2H)2.53(q,J=7.42Hz,2H)1.62-1.68(m,2H)1.51-1.57(m,2H)1.44(br s,9H)1.07(t,J=7.46Hz,3H).

[1033] Step 5: Synthesis of (2S)-2-amino-3-[4-[4-[4-(tert-butoxycarbonylamino)butoxy]-2-ethyl-phenyl]phenyl]propionic acid (aa27-6)

[1034] Pd(OH)₂ / C (300 mg, 213.62 μmol, 10% purity) and AcOH (105.00 mg, 1.75 mmol, 0.1 mL) were added to a solution of aa27-5 (1.5 g, 2.54 mmol, 1 equivalent) in MeOH (30 mL). The mixture was stirred at 20 °C for 12 hours under H₂. The reaction progress was monitored by LC-MS. The mixture was filtered and concentrated to give a crude product. Compound aa27-6 (1.16 g, crude) was obtained as a yellow oil.

[1035] LCMS(ESI): RT = 0.890 min, for C 26 H 36 The calculated mass of N₂NaO₅ is 479.25 m / z, while the actual measured mass is 479.1 [M + Na]. + Inverting LC-MS was performed using method A.

[1036] Step 6: Synthesis of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4'-(4-((tert-butoxycarbonyl)amino)butoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)propionic acid (aa27-7)

[1037] NaHCO3 (426.64 mg, 5.08 mmol, 197.52 μL, 2 equivalents) and Fmoc-OSu (1.03 g, 3.05 mmol, 1.2 equivalents) were added to a solution of aa27-6 (1.16 g, 2.54 mmol, 1 equivalent) in THF (15 mL) and H2O (8 mL) at 0 °C. The mixture was stirred at 20 °C for 12 h. The reaction progress was monitored by LC-MS. The mixture was adjusted to pH 3–4 and extracted with EtOAc (20 mL x 2). The organic layer was dried over Na2SO4, filtered, and concentrated to give the residue, which was then purified by preparative HPLC (AcOH conditions; MeCN / H2O, 0%–100%). Compound aa27-7 (950 mg, 1.12 mmol, 44.09% yield, 80% purity) was obtained as a colorless oil.

[1038] LCMS(ESI): RT = 1.142 min, for C 41 H 46 The calculated mass of N₂NaO₇ is 701.32 m / z, while the actual measured mass is 701.0 [M + Na]. + Inverting LC-MS was performed using method A.

[1039] HPLC: RT = 4.33 min; mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 6 min, and holding at 80% for 2 min at a flow rate of 1.2 mL / min; column: Ultimate C18 3.0*50 mm, 3 μm.

[1040] SFC: RT = 0.598 min, column: Chiralpak AD-3 50 × 4.6 mm ID, 3 μm; mobile phase: A: CO2 B: ethanol (0.05% DEA) isocratic: 40% B; flow rate: 4 mL / min.

[1041] 1H NMR (400MHz, chloroform-d) δppm 7.29-7.36 (m, 5H) 7.14-7.21 (m, 4H) 7.07 (br d, J=8.56Hz, 1H) 6.81 (s, 1H) 6.73 (br d, J=7.83Hz, 1H) 5.27 (br d,J=7.58Hz,1H)5.05-5.16(m,2H)4.68-4.75(m,1H)4.50-4.68(m,2H)3.99(br s,2H)3.20-3.28(m,2H)2.53(q,J=7.42Hz,2H)1.62-1.68(m,2H)1.51-1.57(m,2H)1.44(br s,9H)1.07(t,J=7.46Hz,3H).

[1042] Step 7: Synthesis of 9H-fluorene-9-ylmethyl N-[(1S)-2-anilino-1-[[4-[4-[4-(tert-butoxycarbonylamino)butoxy]-2-ethyl-phenyl]phenyl]methyl]-2-oxo-ethyl]carbamate (aa27-8)

[1043] A solution of aniline (36.01 mg, 386.71 μmol, 1 equivalent) and DIC (46.48 mg, 368.29 μmol, 57.03 μL, 1 equivalent) in DCM (1.5 mL) was added to a solution of aa27-7 (250 mg, 368.29 μmol, 1 equivalent) and HOBt (49.76 mg, 368.29 μmol, 1 equivalent) in DCM (0.5 mL). The mixture was stirred at 20 °C for 1 hour. The reaction progress was monitored by LC-MS. The mixture was diluted with DCM (20 mL) and washed with H2O (10 mL * 2). The organic layer was dried over Na2SO4, filtered, and concentrated to give the residue. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica rapid column with an eluent gradient of 0%–50% ethyl acetate / petroleum ether at 20 mL / min. Compound aa27-8 was obtained as a white solid (220 mg, 283.05 μmol, 76.86% yield, 97% purity).

[1044] LCMS(ESI): RT = 1.198 min, for C 47 H 51 The calculated mass of N3NaO6 is 776.37 m / z, while the actual measured mass is 777.3 [M+Na]. + Inverting LC-MS was performed using method A.

[1045] HPLC: RT = 8.07 min, reversed-phase HPLC analysis was performed using method D. SFC: RT = 2.186 min; column: Chiralcel OD-3 50×4.6 mm ID, 3 μm; mobile phase: A: CO2, B: ethanol (0.05% DEA); isocratic rate: 40% B; flow rate: 4 mL / min.

[1046] 1 H NMR (400MHz, chloroform-d) δppm 7.74 (br d, J = 7.58Hz, 2H) 7.51-7.58 (m, 3H) 7.30-7.40 (m, 4H) 7.28 (br d,J=7.58Hz,4H)7.17-7.24(m,4H)7.07-7.11(m,1H)7.02(d,J=8.31Hz,1H)6.81(d,J=2.45Hz,1H)6.72(dd,J=8.31,2.45Hz,1H)5.57(br s,1H)4.32-4.69(m,4H)4.20(t,J=6.72Hz,1H)3.99(t,J=6.24Hz,2H)3.06-3.29(m,4H)2.49(q,J =7.42Hz,2H)1.77-1.87(m,2H)1.68(dt,J=14.55,7.15Hz,2H)1.44(s,9H)1.03(t,J=7.58Hz,3H).

[1047] Step 8: Synthesis of N-[4-[4-[4-[(2S)-2-amino-3-anilino-3-oxo-propyl]phenyl]-3-ethyl-phenoxy]butyl] tert-butyl carbamate (aa27)

[1048] A solution of piperidine (124.24 mg, 1.46 mmol, 5 equivalents) was added to a solution of aa27-8 (220 mg, 291.81 μmol, 1 equivalent) in DMF (2 mL). The mixture was stirred at 20 °C for 1 hour. The reaction progress was monitored by LC-MS. The mixture was diluted with EtOAc (30 mL) and washed with H2O (10 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated to give the residue, which was then purified by column chromatography (SiO2, EtOAc:MeOH = 1:0 to 5:1). Compound aa27 (105 mg, 191.56 μmol, 65.65% yield, 97% purity) was obtained as a yellow foam.

[1049] LCMS(ESI): RT = 0.953 min, for C 32 H 41The calculated mass of N3NaO4 is 554.30 m / z, while the actual measured mass is 554.1 [M+Na]. + Reversed-phase LC-MS was performed using method A. HPLC: RT = 9.13 min, followed by reversed-phase HPLC analysis using method C.

[1050] SFC: RT = 3.185 min, column: Chiralpak AS-3 100×4.6 mm ID, 3 μm; mobile phase: A: CO2, B: ethanol (0.1% ethanolamine); gradient: from 5% B to 40% B over 4.5 min, and hold at 40% for 2.5 min, then hold at 5% B for 1 min; flow rate: 2.8 mL / min.

[1051] 1 H NMR (400MHz, chloroform-d) δppm 9.45 (s, 1H) 7.60 (br d,J=7.83Hz,2H)7.33(t,J=7.83Hz,2H)7.20-7.29(m,4H)7.06-7.13(m,2H)6.84(d,J=2.20Hz,1H)6.75(dd,J=8.31,2.45Hz,1H)4.68(br s,1H)4.01(t,J=6.11Hz,2H)3.82(br d,J=5.38Hz,1H)3.41(br dd,J=13.82,3.30Hz,1H)3.13-3.27(m,2H)2.84(br dd,J=13.82,9.90Hz,1H)2.56(q,J=7.50Hz,2H)1.79-1.86(m,2H)1.69(quin,J=7.15Hz,2H)1.45(s,9H)1.09(t,J=7.58Hz,3H).

[1052] 2.9 Synthesis of non-natural amino acids (aa28)

[1053] Scheme 10 outlines the synthesis of non-natural amino acids (aa28):

[1054] Option 10

[1055]

[1056] Reagents and conditions: (a) PhNH2 (1.1 equivalent), DIC (1.0 equivalent), HOBT (1.0 equivalent), DCM, 0℃-20℃, 1h; (b) Piperidine (3.0 equivalent), DMF, 20℃, 12h, 52%; (c) 3a (1 equivalent), DIC (1.0 equivalent), HOBT (1.0 equivalent), DCM, 0℃-20℃, 1h, 61%; (d) Piperidine (15.0 equivalent), DMF, 20℃, 0.5h, 41%.

[1057] Step 1: Synthesis of 9H-fluorene-9-ylmethyl N-[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl)butyl]carbamate (aa28-1)

[1058] Aniline (66.14 mg, 710.21 μmol, 64.84 μL, 1.05 equivalent) was added to a solution of aa1 (300 mg, 676.39 μmol, 1 equivalent) and HOBt (91.40 mg, 676.39 μmol, 1 equivalent) in DCM (1.5 mL) at 0 °C. A solution of DIC (85.36 mg, 676.39 μmol, 1 equivalent) in DCM (0.5 mL) was added to the mixture. The reaction mixture was stirred at 20 °C for 1 hour. The reaction progress was monitored by LC-MS. The mixture was diluted with DCM (20 mL) and washed with H2O (10 mL * 2). The organic layer was dried over Na2SO4, filtered, and concentrated to give a crude product. Compound aa28-1 (400 mg, crude) was given as a yellow solid.

[1059] LCMS(ESI): RT = 1.178 min, for C 34 H 35 N2O3 + The calculated mass is 519.26 [M+H] + The measured m / z is 519.1 [M+H]. + Inverting LC-MS was performed using method A.

[1060] Step 2: Synthesis of (2S)-2-amino-5-(3,5-dimethylphenyl)-N-phenyl-pentanamide (aa28-2)

[1061] Piperidine (197.01 mg, 2.31 mmol, 3 equivalents) was added to a solution of aa28-1 (400 mg, 771.24 μmol, 1 equivalent) in DMF (2 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 12 h. The reaction progress was monitored by LC-MS and TLC. The mixture was diluted with EtOAc (30 mL) and washed with H2O (10 mL * 3). The organic layer was dried over Na2SO4, filtered, and concentrated to give the residue, which was then subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica rapid column with an eluent gradient of 0%–100% ethyl acetate / petroleum ether at 20 mL / min. The compound aa28-2 (150 mg, 399.79 μmol, 51.84% yield, 79% purity) was obtained as a yellow foam. LCMS (ESI): RT = 0.861 min, for C 19 H 25 The calculated mass of N₂O is 297.2 [M+H]. + The measured m / z value is 297.1 [M+H]. + Inverting LC-MS was performed using method A.

[1062] 1 H NMR(400MHz,CD3Cl)δppm 9.46(br s,1H),7.60(d,J=7.83Hz,2H),7.33(t,J=7.95Hz,2H),7.07-7.14(m,1H),6.78-6.86(m ,4H),3.51(dd,J=7.95,4.28Hz,1H),2.57-2.64(m,2H),2.29(s,6H),1.67-1.81(m,4H).

[1063] Step 3: Synthesis of 9H-fluorene-9-ylmethyl N-[(1S)-1-[[4-[4-[4-(tert-butoxycarbonylamino)butoxy]-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl)butyl]amino]-2-oxo-ethyl]carbamate (aa28-3)

[1064] Add aa28-2 (150 mg, 506.06 μmol, 1 equivalent) to a solution of aa2b (343.52 mg, 506.06 μmol, 1 equivalent) and HOBt (68.38 mg, 506.06 μmol, 1 equivalent) in DCM (2.5 mL) at 0 °C. Add a solution of DIC (63.86 mg, 506.06 μmol, 1 equivalent) in DCM (0.5 mL) to the mixture. Stir the reaction mixture at 20 °C for 1 hour. Monitor the reaction progress by TLC. Dilute the mixture with DCM (20 mL) and wash with H2O (10 mL * 2). Dry the organic layer with Na2SO4, filter and concentrate to give the residue. Strain the residue by rapid silica gel chromatography (…). 12g Purification was performed using a silica rapid column with an eluent gradient of 0%–45% ethyl acetate / petroleum ether at 20 mL / min. The compound aa28-3 (400 mg, 309.23 μmol, 61.11% yield, 74% purity) was obtained as a yellow foam. LCMS (ESI): RT = 6.617 min, for C 60 H 68 The calculated mass of N₄NaO₇ is 979.50 m / z, while the actual measured mass is 979.8 [M + Na]. + Inverting LC-MS was performed using method B.

[1065] 1 H NMR(400MHz,CD3Cl)δppm 7.72-7.80(m,2H),7.49-7.57(m,3H),7.35-7.41(m,2H),7.20-7.33(m,5H),7.08-7.20(m ,4H),6.96-7.08(m,2H),6.76-6.84(m,2H),6.68-6.75(m,3H),5.32-5.47(m,1H),4.64(br s,1H),4.52-4.60(m,1H),4.42-4.51(m,2H),4.37(brs,1H),4.13-4.23(m,1H),4.00(t,J=6.24Hz,2H),3.05-3.27( m,4H),2.44-2.61(m,4H),2.20-2.25(m,6H),1.82-1.89(m,2H),1.62-1.75(m,6H),1.46(s,9H),1.01-1.09(m,3H).

[1066] Step 4: Synthesis of N-[4-[4-[4-[(2S)-2-amino-3-[[(1S)-4-(3,5-dimethylphenyl)-1-(phenylcarbamoyl)butyl]amino]-3-oxo-propyl]phenyl]-3-ethyl-phenoxy]butyl] tert-butyl carbamate (aa28)

[1067] Piperidine (507.06 mg, 5.95 mmol, 15 equivalents) was added to a solution of aa28-3 (380 mg, 396.99 μmol, 1 equivalent) in DMF (2.5 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 0.5 h. The reaction progress was monitored by LC-MS. The mixture was diluted with ACN (2 mL) and purified by preparative HPLC (HCl conditions; column: Agela ASB 150*25 mm*5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 55%-85%, 8 min). Compound aa28 (125 mg, 164.97 μmol, 41.56% yield, 97% purity) was obtained as a small amount of yellow foam.

[1068] LCMS(ESI): RT = 1.061 min, for C 45 H 58 The calculated mass of N₄NaO₅ is 757.43 m / z, while the actual measured mass is 757.5 [M + Na]. + Reversed-phase LC-MS was performed using Method A. HPLC: RT = 10.66 min. Reversed-phase HPLC analysis was performed using Method C. SFC: RT = 2.161 min; Column: Chiralpak AD-3 50*4.6mm ID, 3μm; Mobile phase: A: CO2; B: Isopropanol (0.05% DEA); Gradient: from 5% B to 40% B over 2 min and held at 40% for 1.2 min, then 5% B for 0.8 min; Flow rate: 4 mL / min.

[1069] 1H NMR(400MHz,CD3OD)δppm 7.55(br d,J=8.07Hz,2H),7.24-7.31(m,4H),7.14(d,J=8.07Hz,2H),7.05-7.11(m,1H),6.95(d,J=8.31Hz,1H),6.81(d,J=2.45Hz,1H),6 .78(s,3H),6.68-6.72(m,1H),4.54(t,J=6.72Hz,1H),4.20(t,J=6.85Hz,1H),4.00(t,J=6.11Hz,2H),3.25-3.30(m,1H),3.12(br t,J=6.85Hz,3H),2.54-2.63(m,2H),2.51(q,J=7.50Hz,2H),2.22(s,6H),1.62-1.95(m,8H),1.44(s,9H),1.03(t,J=7.58Hz,3H).

[1070] 2.10 Synthesis of non-natural amino acids (aa29)

[1071] Scheme 11 outlines the synthesis of non-natural amino acids (aa29):

[1072] Option 11

[1073]

[1074] Step 1: Synthesis of (E)-3-(1-triphenylmethyl-1H-imidazol-5-yl)acrylic acid (aa29-2)

[1075] Compound aa29-1 (2 g, 14.48 mmol, 1.0 equivalent), TEA (4.40 g, 43.44 mmol, 6.05 mL, 3.0 equivalent), and TrtCl (4.04 g, 14.48 mmol, 1.0 equivalent) were stirred in DMF (34 mL) for 12 hours at 20 °C. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete. The mixture was diluted with CH2Cl2 and washed with H2O (3 x 50 mL) and citric acid solution (3 x 50 mL). The organic phase was evaporated and purified by column chromatography (elution: CH2Cl2 / MeOH, 9:1). Compound aa29–2 (1.6 g, 4.21 mmol, 29.05% yield) was given as a white solid.

[1076] 1H NMR (400MHz, CD3OD) δ7.57 (s, 1H), 7.51 (d, J = 15.77Hz, 1H), 7.38-7.45 (m, 9H), 7.32 (s, 1H), 7.15-7.22 (m, 6H), 6.45 (d, J = 15.65Hz, 1H) ppm.

[1077] Step 2: Synthesis of 3-(1-triphenylmethyl-1H-imidazol-5-yl)propionic acid (aa29)

[1078] Pd / C (1 g, 10% purity) was added to a solution of compound aa29-2 (1.6 g, 4.21 mmol, 1 equivalent) in EtOH (20 mL) under a nitrogen atmosphere. The suspension was degassed and purged three times with H2. The mixture was stirred at 20 °C under H2 (15 Psi.) for 2 hours. After completion, the mixture was filtered and the filtrate was concentrated to give the product. Compound aa29-4 (1.2 g, 3.14 mmol, 74.60% yield) was given as a white solid.

[1079] LCMS(ESI): RT = 0.802 min, for C 25 H 21 The calculated mass of N₂O₂ is 381.17 [MH]. - The measured value is 381.17 [MH]. - The reversed-phase LCMS was performed as follows: using a Waters Xbridge C1830*2.0mm, 3.5μm, flow rate 1.2ml / min, eluted with a gradient of 5% to 95% acetonitrile containing ACN (solvent B) and water containing 0.05% NH3H2O ​​(solvent A).

[1080] 1 H NMR(400MHz,CD3OD)δ7.34-7.39(m,4H),7.22-7.39(m,4H),7.11-7.14(m,3H),7.06-7.20(m,2H ),7.08(d,J=7.25Hz,4H),2.90(t,J=7.32Hz,1H),2.80(t,J=7.38Hz,1H),2.51-2.62(m,2H)ppm.

[1081] 2.11 Synthesis of non-natural amino acids (aa30)

[1082] Scheme 12 outlines the synthesis of non-natural amino acids (aa30):

[1083] Option 12

[1084]

[1085] Step 1: Synthesis of (3-hydroxypropyl)triphenylphosphonium bromide (aa30-2)

[1086] PPh3 (22.65 g, 86.34 mmol, 1.2 equivalent) was added to a solution of 3-bromopropane-1-ol (10 g, 71.95 mmol, 6.49 mL, 1 equivalent) in toluene (100 mL). The mixture was stirred at 100 °C for 12 h under N2. The reaction progress was monitored by LCMS. After completion, the reactants were cooled to 0 °C and filtered, the filter cake was washed with toluene (10 mL x 3), and then dried under reduced pressure. Compound aa3O2 (20.7 g, crude) was given as a white solid.

[1087] LCMS(ESI): RT = 0.758 min, for C 21 H 22 OP + The calculated mass is 321.14 [M]. + The actual measured value is 321.0 [M]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 5%–95% (solvent B) over 0.7 min, and maintained at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursult 5C1820*2.0 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1088] 1 H NMR (400MHz, CD3Cl) δ7.83-7.65(m,15H),4.94(br s,1H),3.87-3.73(m,4H),1.83(m,2H)ppm.

[1089] Step 2: Synthesis of (E)-4-(1-triphenylmethyl-1H-imidazol-4-yl)but-3-en-1-ol (aa30-3)

[1090] LiHMDS (1M, 70.92 mL, 4 equivalents) was added to a solution of aa30-2 (10.67 g, 26.60 mmol, 1.5 equivalents) in THF (50 mL). The mixture was stirred at 0 °C for 0.5 h, and then a solution of aa30-2A (1-trimethylimidazole-4-carboxaldehyde, 6 g, 17.73 mmol, 1.0 equivalents) in THF (60 mL) was added to the mixture, and the resulting mixture was stirred at 25 °C for 12 h. The reaction progress was monitored by LCMS. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue, which was then redissolved in dioxane (50 mL), and the final solution was stirred at 100 °C for 12 h. After this, the solution was concentrated under reduced pressure to give the residue. The residue was analyzed by rapid silica gel chromatography (…). 80g Purification was performed using a silica rapid column with an eluent gradient of 0%–80% ethyl acetate / petroleum ether at 60 mL / min. Compound aa30-3 (650 mg, crude) was obtained as a yellow solid.

[1091] LCMS(ESI): RT = 0.846 min, for C 26 H 24 The calculated mass of N₂ONa is 403.19 [M + Na]. + The measured value was 403.1 [M+Na]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 5%–95% (solvent B) over 0.7 min, and maintained at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursult 5C18 20*2.0 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1092] 1 H NMR (400MHz, CD3OD) δ7.68-7.57(m,1H),7.40-7.37(m,9H),7.18-7.13(m,6H),6.83( d,J=1.0Hz,1H),6.36-6.14(m,2H),3.62(t,J=6.7Hz,2H),2.36(q,J=6.6Hz,2H)ppm.

[1093] Step 3: Synthesis of 4-(1-triphenylmethyl-1H-imidazol-4-yl)but-1-ol (aa30-4)

[1094] Pd / C (50 mg, 489.61 mmol, 10% purity) was added to a solution of aa30-3 (650 mg, 1.71 mmol, 1 equivalent) in MeOH (5 mL). The mixture was stirred at 25 °C for 2 h under H2. The reaction progress was monitored by LCMS. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue. The residue was then subjected to rapid silica gel chromatography (LCMS) in total volume. 25g Purification was performed using a silica rapid column with an eluent gradient of 0%–50% ethyl acetate / petroleum ether at 40 mL / min for 8 min. The compound aa30-4 was obtained as a white solid (450 mg, 1.18 mmol, 68.87% yield, 100% purity).

[1095] LCMS(ESI): RT = 0.850 min, for C 26 H 26 The calculated mass of N₂ONa is 405.20 [M + Na]. + The measured value was 405.2 [M+Na]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 5%–95% (solvent B) over 0.7 min, and maintained at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursult 5C18 20*2.0 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1096] Step 4: Synthesis of 4-(1-triphenylmethyl-1H-imidazol-4-yl)butyric acid (aa30)

[1097] TEMPO (277.51 mg, 1.76 mmol, 1.5 equivalent) and PIDA (iodophenyl diacetate (3240-34-4), 947.35 mg, 2.94 mmol, 2.5 equivalent) were added to a solution of aa30-4 (450 mg, 1.18 mmol, 1 equivalent) in MeCN (5 mL) and H2O (7 mL). The mixture was stirred at 25 °C for 12 h. The reaction progress was monitored by LCMS. After completion, citric acid (15 mL aqueous solution) was added to the mixture to adjust the pH to 4, and then the mixture was extracted with EtOAc (15 mL * 2). The organic layer was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was then ground by MTBE. Compound aa30 (250 mg, crude) was obtained as a white solid.

[1098] LCMS(ESI): RT = 2.484 min, for C 26 H 25 The calculated mass of N₂O₂ is 397.18 [M+H]. + The actual measured value is 397.2 [M+H]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using a gradient of 10%–80% (solvent B) over 6 minutes, and maintaining at 80% for 0.5 minutes at a flow rate of 0.8 mL / min. ESI source, positive ion mode; wavelengths 220 nm & 254 nm; oven temperature 50 °C.

[1099] 1 H NMR (400MHz, CD3OD) δ8.69(d,J=1.6Hz,1H),7.53-7.38(m,9H),7.28-7.14(m,7H),2.75(t,J=7.5Hz,2H),2.34(t,J=7.1Hz,2H),2.00-1.87(m,2H)ppm.

[1100] 2.12 Synthesis of non-natural amino acids (aa31)

[1101] Scheme 13 outlines the synthesis of non-natural amino acids (aa31):

[1102] Option 13

[1103]

[1104] Step 1: Synthesis of (3-carboxypropyl)triphenylphosphonium bromide (aa31-2)

[1105] PPh3 (8.64 g, 32.93 mmol, 1.1 equivalent) was added to a solution of 4-bromobutyric acid (5 g, 29.94 mmol, 6.76 mL, 1 equivalent) in toluene (70 mL). The mixture was stirred at 110 °C for 12 h under N2. The reaction progress was monitored by TLC and LCMS. After completion, the reaction mixture was cooled to 0 °C, filtered, and washed with toluene (10 mL x 3). The filter cake was collected and dried under reduced pressure. Compound aa31-2 (5.6 g, crude) was given as a white solid.

[1106] LCMS(ESI): RT = 1.076 min, for C 22 H 22 O2P + The calculated mass is 349.14 [M]. + The actual measured value is 349.2 [M]. +LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 10%–80% (solvent B) over 1.35 min, and maintained at 80% for 0.9 min at a flow rate of 0.8 mL / min; Column: Ultimate C18 2.1 x 30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1107] 1 H NMR (400MHz, CD3Cl) δ7.91-7.66(m,15H), 3.31-3.22(m,2H), 2.55(t,J=6.3Hz,2H), 1.84(m,2H)ppm.

[1108] Step 2: Synthesis of (E)-5-(1-triphenylmethyl-1H-imidazol-4-yl)pent-4-enoic acid (aa31-3)

[1109] tBuOK (1.99 g, 17.73 mmol, 3 equivalents) was added to a solution of aa31-2 (3.81 g, 8.87 mmol, 1.5 equivalents) in THF (20 mL) at 0 °C under N2. The mixture was stirred at 0 °C for 30 min. Then, a solution of aa31-2A (2 g, 5.91 mmol, 1 equivalent) in THF (20 mL) was added to the mixture at 0 °C, and the final mixture was stirred at 25 °C for 12 h. The reaction progress was monitored by LCMS. After completion, citric acid was added to the mixture to adjust the pH to 4, and then it was extracted with EtOAc (50 mL * 2). The organic layer was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed using a high-speed silica column (elution with 0%–10% MeOH / DCM @ 40 mL / min). Compound aa31-3 (4 g, crude) was obtained as a pale yellow foam.

[1110] LCMS(ESI): RT = 1.487 min, for C 27 H 25 The calculated mass of N₂O₂ is 409.18 [M+H]. + The actual measured value is 409.3 [M+H]. +LCMS conditions: 1.5 mL / 4 L TFA in water (solvent A) and acetonitrile (solvent B), using an elution gradient of 10%–80% (solvent B) over 1.35 min, and holding at 80% for 0.9 min at a flow rate of 0.8 mL / min; Column: Ultimate C18 2.1 x 30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1111] Step 3: Synthesis of 5-(1-triphenylmethyl-1H-imidazol-4-yl)valeric acid (aa31)

[1112] Pd / C (300 mg, 489.61 mmol, 10% purity) was added to a solution of aa31-3 (3 g, 7.34 mmol, crude purity, 1 equivalent) in MeOH (40 mL). The mixture was stirred at 25 °C for 2 h under H2. The reaction progress was monitored by LCMS. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue. The residue was then subjected to rapid silica gel chromatography (LCMS) in total volume. 40g Purification was performed using a silica rapid column (eluting with 0%–10% MeOH / DCM at 40 mL / min) for 7 min. The product was then ground with TBME. Compound aa31 was obtained as a white solid (290 mg, 692.32 mmol, 71.05% yield, 98% purity).

[1113] LCMS(ESI): RT = 1.805 min, for C 27 H 27 The calculated mass of N₂O₂ is 411.20 [M+H]. + The actual measured value is 411.3 [M+H]. + LCMS conditions: 0.8 mL / 4 L NH3·H2O in water (solvent A) and acetonitrile (solvent B), eluted over 6 minutes with a gradient of 10%–80% (solvent B), and maintained at 80% for 0.5 minutes at a flow rate of 0.8 mL / min; Column: XBridge C18 3.5 mm 2.1*30 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1114] 1 H NMR (400MHz, CD3Cl) δ7.65 (s, 1H), 7.44-7.34 (m, 9H), 7.16 (dd, J = 2.9, 6.8Hz, 6H), 6.77 (s, 1H), 2.58 (br t,J=6.8Hz,2H),2.28(t,J=7.1Hz,2H),1.70-1.55(m,4H)ppm.

[1115] 2.13 Synthesis of non-natural amino acids (aa32)

[1116] Scheme 14 outlines the synthesis of non-natural amino acids (aa32):

[1117] Option 14

[1118]

[1119] Step 1: Synthesis of (4-carboxybutyl)triphenylphosphonium bromide (aa32-2)

[1120] PPh3 (15.94 g, 60.76 mmol, 1.1 equivalent) was added to a solution of 5-bromopentanoic acid (10 g, 55.24 mmol, 6.76 mL, 1 equivalent) in toluene (100 mL). The mixture was stirred at 110 °C for 12 h under N2. The reaction progress was monitored by LCMS. After completion, the reaction mixture was cooled to 0 °C, filtered, and washed with toluene (10 mL x 3). The filter cake was collected and dried under reduced pressure. Compound aa32-2 (16.4 g, 36.99 mmol, 66.97% yield) was given as a white solid.

[1121] LCMS(ESI): RT = 1.076 min, for C 22 H 22 O2P + The calculated mass is 349.14 [M+H]. + The actual measured value is 349.2 [M+H]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 10%–80% (solvent B) over 1.35 min, and held at 80% for 0.9 min at a flow rate of 0.8 mL / min; Column: Xtimate C18 2.1 x 30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C;

[1122] 1 H NMR (400MHz, CD3Cl) δ7.91-7.66(m,15H), 3.31-3.22(m,2H), 2.55(t,J=6.3Hz,2H), 1.86-1.82(m,4H)ppm.

[1123] Step 2: Synthesis of (E)-6-(1-triphenylmethyl-1H-imidazol-4-yl)hexa-5-enoic acid (aa32-3)

[1124] t-BuOK (3.98 g, 35.46 mmol, 3 equivalents) was added to a solution of aa32-2 (7.86 g, 17.73 mmol, 1.5 equivalents) in THF (60 mL) at 0 °C under N2. The mixture was stirred at 0 °C for 30 min. A solution of aa32-2A (4 g, 11.82 mmol, 1 equivalent) in THF (40 mL) was added to the above mixture at 0 °C, and the resulting mixture was stirred at 25 °C for 12 h. The reaction progress was monitored by LCMS. After completion, citric acid was added to the mixture to adjust the pH to 4, and then it was extracted with EtOAc (50 mL * 2). The organic layer was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 80g Purification was performed using a high-speed silica column (eluting with 0%–10% MeOH / DCM at 40 mL / min). Compound aa32-3 (4 g, crude) was obtained as a pale yellow foam.

[1125] LCMS(ESI): RT = 2.739 min, for C 28 H 27 The calculated mass of N₂O₂ is 423.20 [M+H]. + The actual measured value is 423.2 [M+H]. + LCMS conditions: 1.5 mL / 4 L TFA in water (solvent A) and acetonitrile (solvent B), using an elution gradient of 10%–80% (solvent B) over 1.35 min, and holding at 80% for 0.9 min at a flow rate of 0.8 mL / min; Column: Ultimate C18 2.1 x 30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1126] Step 3: Synthesis of 6-(1-triphenylmethyl-1H-imidazol-4-yl)hexanoic acid (aa32)

[1127] Pd / C (300 mg, 489.61 mmol, 10% purity) was added to a solution of aa32-3 (4 g, 9.47 mmol, 1 equivalent) in MeOH (40 mL). The mixture was stirred at 25 °C for 2 h under H2. The reaction progress was monitored by LCMS. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 40g Purification was performed using a silica rapid column with 0%–10% MeOH / DCM eluent at 40 mL / min. The product was then ground with TBME. Compound aa32 was obtained as a white solid (660 mg, 1.51 mmol, 15.93% yield, 97% purity).

[1128] LCMS(ESI): RT = 2.807 min, for C 28 H 29 The calculated mass of N₂O₂ is 425.22 [M+H]. + The actual measured value is 425.2 [M+H]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 10%–80% (solvent B) over 6.0 min, and holding at 80% for 0.5 min at a flow rate of 0.8 mL / min; Column: Ultimate C18 2.1 x 30 mm, 3 mm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1129] 1 H NMR(400MHz,CD3OD)δ7.43(d,J=1.3Hz,1H),7.41-7.34(m,9H),7.19-7.11(m,6H),6.65( s,1H),2.52(t,J=7.4Hz,2H),2.24(t,J=7.4Hz,2H),1.60(m,4H),1.38-1.27(m,2H)ppm.

[1130] 2.14 Synthesis of non-natural amino acids (aa33)

[1131] Scheme 15 outlines the synthesis of non-natural amino acids (aa33):

[1132] Option 15

[1133]

[1134] Step 1: Synthesis of (5-carboxypentyl)triphenylphosphonium bromide (aa33-2)

[1135] PPh3 (7.06 g, 26.92 mmol, 1.05 equivalent) was added to a solution of 6-bromohexanoic acid (5 g, 25.63 mmol, 1 equivalent) in toluene (50 mL). The resulting solution was stirred at 120 °C for more than 12 h. After completion, the reaction mixture was cooled to 0 °C, filtered, and washed with toluene (10 mL x 3). The filter cake was collected and dried under reduced pressure. Compound aa33-2 (6.85 g, 14.47 mmol, 56.44% yield, 96.6% purity) was given as a white solid.

[1136] LCMS(ESI): RT = 0.916 min, m / z, for C 24 H 26 PO2 + The calculated value is 377.17, and the actual measured value is 377.1 [M-Br]. + The reversed-phase LCMS was performed as follows: using an Ultimate C18 column, 2.1*30mm 3mm, SN: 3U411301530, at a flow rate of 1.5 mL / min, eluted with a gradient of 10%–80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1137] Step 2: Synthesis of (E)-7-(1-triphenylmethyl-1H-imidazol-4-yl)hept-6-enoic acid (aa33-3)

[1138] tBuOK (1M, 22.16mL, 5 equivalents) was added to a solution of aa33-2 (4.05g, 8.87mmol, 2 equivalents) in THF (20mL) at 0℃ under N2. The mixture was stirred at 0℃ for 30min. Then, a solution of aa33-2A (1.5g, 4.43mmol, 1 equivalent) in THF (20mL) was added to the mixture at 0℃, and the final mixture was stirred at 25℃ for 12h. The reaction progress was monitored by LCMS. After completion, citric acid was added to the mixture to adjust the pH to 4, and then it was extracted with EtOAc (50mL*2). The organic layer was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was used in the next step without any further purification. Compound aa33-3 (3.8g, crude) was obtained as a yellow syrup.

[1139] LCMS(ESI): RT = 2.904 min and 3.068 min, for C 29 H 29 The calculated mass of N₂O₂ is 437.22, and the actual mass is 437.3 [M+H]. +Reversed-phase LCMS was performed as follows: a Chromolith Flash RP-C18 25-3 mm column was used at a flow rate of 1.5 mL / min, with gradient elution of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1140] Step 3: Synthesis of 7-(1-triphenylmethyl-1H-imidazol-4-yl)heptanic acid (aa33)

[1141] Pd / C (300 mg, 489.61 mmol, 10% purity) was added to a solution of aa33-3 (3 g, 6.87 mmol, 1 equivalent) in MeOH (30 mL). The mixture was stirred at 25 °C for 2 h under H2. The reaction progress was monitored by LCMS. After completion, the residue was purified by preparative HPLC (column: Boston Prime C18 150*25 mm*5 mm; mobile phase: [water (0.05% ammonium hydroxide v / v)-ACN]; B%: 22%-45%, 7 min). Compound aa33 (200 mg, 456.04 mmol, 6.64% yield, 100% purity) was obtained as a white solid.

[1142] LCMS(ESI): RT = 2.231 min, for C 29 H 31 The calculated mass of N₂O₂ is 439.23 [M+H]. + The actual measured value was 439.3 [M+H]. + LCMS conditions: Mobile phase: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using a gradient of 10%–80% (solvent B) over 6 minutes, and maintaining at 80% for 0.5 minutes at a flow rate of 0.8 mL / min. ESI source, positive ion mode; wavelengths 220 nm & 254 nm; oven temperature 50 °C.

[1143] 1 H NMR(400MHz,CD3OD)δ7.45-7.31(m,10H),7.19-7.11(m,6H),6.64(s,1H),2.52( t,J=7.3Hz,2H),2.24(t,J=7.4Hz,2H),1.65-1.52(m,4H),1.38-1.26(m,4H)ppm.

[1144] 2.15 Synthesis of non-natural amino acids (aa34)

[1145] Scheme 16 outlines the synthesis of non-natural amino acids (aa34):

[1146] Option 16

[1147]

[1148] Step 1: Synthesis of 7-[BLAH(triphenyl)-λ5-phosphoryl]heptanoic acid (aa34-2)

[1149] PPh3 (13.80 g, 52.61 mmol, 1.1 equivalent) was added to a solution of aa34-1 (10 g, 47.83 mmol, 1 equivalent) in toluene (100 mL). The mixture was stirred at 110 °C for 12 hours. After completion, the reaction mixture was filtered under reduced pressure to give the residue. The crude product was used for the next step without further purification. Compound aa34-2 (22.8 g, 43.73 mmol, 91.43% yield, 90.410% purity) was given as a yellow oil.

[1150] LCMS(ESI): RT = 0.792 min, m / z, for C 25 H 28 O2P + The calculated value is 391.18 [M]. + The measured value is 391.1 [M]. + LC-MS conditions: Mobile phase: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 5%–95% (solvent B) over 0.7 min, and maintained at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursult 5C18 20*2.0 mm.

[1151] Step 2: Synthesis of (E)-8-(1-triphenylmethylimidazol-4-yl)oct-7-enoic acid (aa34-3)

[1152] t-BuOK (4.97 g, 44.33 mmol, 3 equivalents) was added to a solution of aa34-2 (10.45 g, 22.16 mmol, 1.5 equivalents) in THF (150 mL) at 0 °C under N2. The mixture was stirred at 0 °C, and then a solution of aa34-2A (5 g, 14.78 mmol, 1 equivalent) in THF (20 mL) was added to the mixture at 0 °C. The final mixture was stirred at 25 °C for 12 hours. After completion, citric acid was added to the mixture to adjust the pH to 4, and then it was extracted with EtOAc (200 mL * 2). The organic layer was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure to give target aa34-3 (14.5 g, crude) as a white solid.

[1153] LCMS(ESI): RT = 0.870 min, m / z, for C 30 H 31 N₂O₂, calculated to be 451.23 [M+H] + For C 30 H 30 The calculated value of N₂O₂Na is 473.23 [M + Na]. + The measured value was 473.1 [M+Na]. + LC-MS conditions: Mobile phase: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 5%–95% (solvent B) over 0.7 min, and maintained at 95% for 0.4 min at a flow rate of 1.5 mL / min; Column: Agilent Pursult 5C1820*2.0 mm.

[1154] Step 3: Synthesis of 8-(1-triphenylmethylimidazol-4-yl)octanoic acid (aa34)

[1155] Pd / C (4 g, 10% purity) and H2 were added to a solution of aa34-3 (14 g, 15.54 mmol, 50% purity, 1 equivalent) in MeOH (200 mL). The mixture was stirred at 25 °C for 3 hours. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue. The crude product was subjected to C-18 reversed-phase chromatography (C-18). 120g Purification was performed using a C-18 column with an eluent gradient of 60%–70% acetonitrile / H₂O at 80 mL / min for 40 min, total volume 2 mL, to give a residue (neutral). Compound aa34 was given as a white solid (2.1 g, 4.53 mmol, 29.15% yield, 97.6% purity).

[1156] LCMS(ESI): RT = 2.956 min, for C 30 H 33 The calculated mass of N₂O₂ is 453.25 [M+H]. + The actual measured value is 453.3 [M+H]. + LCMS conditions: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 10%–80% (solvent B) over 6.0 min, and holding at 80% for 0.5 min at a flow rate of 0.8 mL / min; Column: Ultimate C18 2.1*30 mm, 3 μm; Wavelength: UV 220 nm & 254 nm; Column temperature: 50 °C; MS ionization: ESI.

[1157] 1 H NMR (400MHz, CD3OD) δ7.47-7.33(m,10H),7.22-7.10(m,6H),6.64(s,1H),2.52( t,J=7.4Hz,2H),2.26(t,J=7.4Hz,2H),1.69-1.48(m,4H),1.37-1.26(m,6H)ppm.

[1158] 2.16 Synthesis of non-natural amino acids (aa35)

[1159] Scheme 17 outlines the synthesis of non-natural amino acids (aa35):

[1160] Option 17

[1161]

[1162] Step 1: Synthesis of (7-carboxyheptyl)triphenylphosphonium bromide (aa35-2)

[1163] PPh3 (20.70 g, 78.92 mmol, 1 equivalent) was added to a solution of aa35-1 (15 g, 71.74 mmol, 1 equivalent) in toluene (150 mL). The mixture was stirred at 110 °C for 20 h under N2. After completion, the reaction mixture was cooled to 0 °C, filtered, and washed with toluene (10 mL x 3). The filter cake was collected and dried under reduced pressure. The residue was ground with THF (200 mL). The mixture was then filtered, and the filter cake was dried to give product aa35-2 (20 g, 40.31 mmol, 56.18% yield, 95% purity) as a white solid.

[1164] LCMS (ESI): Rt = 2.175 min, for C 26 H 30 O2P + [M+H] + The calculated mass was 405.20, and the measured mass was 405.70. The reversed-phase LCMS was performed as follows: a Chromolith Flash RP-C1825-3mm column was used at a flow rate of 0.8 ml / min, and elution was performed using a gradient of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1165] Step 2: Synthesis of (E)-9-(1-triphenylmethyl-1H-imidazol-4-yl)non-8-enoic acid (aa35-3)

[1166] t-BuOK (4.97 g, 44.34 mmol, 3.0 equivalent) was added to a solution of aa35-2 (10.45 g, 22.17 mmol, 1.5 equivalent) in THF (150 mL) at 0 °C. The mixture was then stirred at 0 °C for 30 min. A solution of aa35-2A (5 g, 14.78 mmol, 1 equivalent) in THF (50 mL) was added. The mixture was then stirred at 20 °C for 12 h. After completion, the reaction mixture was quenched at 0 °C by adding H2O (100 mL) and then extracted with EtOAc (150 mL * 2). The combined organic layers were washed with 100 mL (50 mL * 2) of saturated NaCl, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was subjected to rapid silica gel chromatography (…). 80g Special Purification was performed using a silica rapid column with 0%–20% ethyl acetate / petroleum ether gradient eluent at 60 mL / min to give product aa35-3 (1.4 g, crude) as a pale yellow oil.

[1167] LCMS (ESI): Rt = 3.310 min, for C 31 H 33 N₂O₂[M+H] + The calculated mass is 465.25, and the actual measured mass is 465.20 [M+H]. + The reversed-phase LCMS was performed as follows: a Chromolith Flash RP-C18 25-3 mm column was used at a flow rate of 0.8 ml / min, with gradient elution of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1168] Step 3: Synthesis of 9-(1-triphenylmethylimidazol-4-yl)nonanoic acid (aa35)

[1169] Pd / C (0.2 g, 10% purity) was added to a solution of aa35-3 (1.4 g, 3.01 mmol, 1 equivalent) in MeOH (40 mL). The mixture was stirred at 25 °C for 3 hours under H2. After completion, the mixture was filtered and concentrated under reduced pressure to give the residue. The crude product was purified by preparative HPLC (column: C18 spherical 20-35 μm, 100A, 12 g; mobile phase: [water-ACN]; B%: 0%-90%, 15 min) to give aa35 (0.12 g, 244.31 μmol, 8.11% yield, 95% purity) as a white solid.

[1170] LCMS (ESI): Rt = 3.257 min, for C 31 H 35 N₂O₂[M+H] + The calculated mass is 467.26, and the actual measured mass is 467.20 [M+H]. + The reversed-phase LCMS was performed as follows: a Chromolith Flash RP-C18 25-3 mm column was used at a flow rate of 0.8 ml / min, with gradient elution of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1171] 2.17 Synthesis of non-natural amino acids (aa36)

[1172] Scheme 18 outlines the synthesis of non-natural amino acids (aa36):

[1173] Option 18

[1174]

[1175] Step 1: Synthesis of 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-2-one (aa36-2)

[1176] A fractional amount of NaH (2.66 g, 66.58 mmol, 60% purity, 1.1 equivalent) was added to a solution of aa36-1 (6 g, 60.53 mmol, 1 equivalent) in THF (300 mL) at 0 °C. The mixture was stirred at 20 °C for 0.5 h, and aa36-1A (14.48 g, 60.53 mmol, 1 equivalent) was added. The reaction mixture was heated to 70 °C for 12 h. The desired product was observed by LCMS. The mixture was cooled to rt, quenched with water (150 mL), and extracted with EtOAc (150 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1). Compound aa36-2 (2.7 g, 10.49 mmol, 17.33% yield) was given as a colorless oil.

[1177] LCMS(ESI): RT = 0.974 min, for C 13 H 27 The calculated mass of NO2SiH is 258.18, while the actual measured mass is 258.2 [M+H]. +The reversed-phase LCMS was performed as follows: using a Chromolith Flash Agilent Pursult 5C18 20*2.0mm, at a flow rate of 1.5 mL / min, eluted with a gradient of 5%-95% (solvent B) over 0.7 min, and held at 95% for 0.4 min.

[1178] 1 H NMR (400MHz, DMSO-d6)3.64(t,J=6.0Hz,2H),3.32-3.29(m,4H),2.16(t,J=6.0Hz,2H),1.74-1.57(m,4H),0.83(s,9H),0.00(s,6H)ppm.

[1179] Step 2: Synthesis of 1-(2-hydroxyethyl)piperidin-2-one (aa36-3)

[1180] HCl / MeOH (v / v = 30%, 8 mL) was added to a solution of aa36-2 (2.7 g, 10.49 mmol, 1 equivalent) in MeOH (12 mL) at 0 °C. The solution was stirred at 0 °C for 0.5 h. After this, the solution was concentrated under vacuum. The residue was purified by column chromatography (SiO2, DCM / MeOH = 10 / 1). Crude product aa36-3 (1.4 g, 9.73 mmol, 92.76% yield, 99.5% purity) as a yellow solid was obtained and used in the next step without further purification.

[1181] 1 H NMR (400MHz, DMSO-d6) 4.13-4.11 (m, 1H), 3.52-3.44 (m, 2H), 3.34-3.29 (m, 4H), 2.20 (t, J = 6.0Hz, 2H), 1.75-1.61 (m, 4H) ppm.

[1182] LCMS(ESI): RT = 0.448-0.574 min, for C7H 13 The calculated mass of NO2H is 144.09 g / L, while the actual measured mass is 144.2 g / L [M+H]. + The reversed-phase LCMS was performed as follows: using a Chromolith Flash Agilent Pursult 5C18 20*2.0mm, at a flow rate of 1.5 mL / min, eluted with a gradient of 5%-95% (solvent B) over 0.7 min, and held at 95% for 0.4 min.

[1183] Step 3: Synthesis of 2-(2-(2-oxopiperidin-1-yl)ethyl)isoindole-1,3-dione (aa36-4)

[1184] PPh3 (4.12 g, 15.71 mmol, 1 equivalent) and DIAD (3.18 g, 15.71 mmol, 3.06 mL, 1.5 equivalent) were added to a solution of aa36-3 (1.5 g, 10.48 mmol, 1 equivalent) and aa36-3A (1.85 g, 12.57 mmol, 1.2 equivalent) in THF (40 mL) at 0 °C. The resulting mixture was stirred at 20 °C for 2 h. The desired product was observed by LCMS. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1). Compound aa36-4 (0.15 g, 550.87 μmol, 5.26% yield) was given as a white solid.

[1185] LCMS(ESI): RT = 0.764 min, for C 15 H 16 The calculated mass of N₂O₃H is 273.12, while the actual measured mass is 273.1 [M + H]. + The reversed-phase LCMS was performed as follows: using a Chromolith Flash Agilent Pursult 5C18 20*2.0mm, at a flow rate of 1.5 mL / min, eluted with a gradient of 5%-95% (solvent B) over 0.7 min, and held at 95% for 0.4 min.

[1186] 1 H NMR(400MHz,DMSO-d6)7.83-7.65(m,4H),3.68-3.58(m,2H),3.45-3.39(m,2H),3.2 1(t,J=6.0Hz,2H),1.89(t,J=6.4Hz,2H),1.64-1.56(m,2H),1.55-1.47(m,2H)ppm.

[1187] Step 4: Synthesis of 1-(2-aminoethyl)piperidin-2-one (aa36-5)

[1188] Add NH2NH2 to a solution of aa36-4 (0.15 g, 550.87 μmol, 1 equivalent) in MeOH (10 mL). . H₂O (275.76 mg, 5.51 mmol, 267.73 μL, 10 equivalents). The solution was stirred at 45 °C for 2 h. After stirring, the solution was cooled to rt and concentrated under vacuum. Crude product aa36-5 (0.07 g, crude) was obtained as a colorless oil and used in the next step without further purification.

[1189] 1 H NMR (400MHz, DMSO-d6)3.27-3.22(m,6H),2.62(t,J=6.8Hz,2H),2.17(t,J=6.0Hz,2H),1.76-1.58(m,4H)ppm.

[1190] Step 5: Synthesis of 2,2-dimethyl-4-oxo-4-((2-(2-oxopirid-1-yl)ethyl)amino)butyric acid (aa36)

[1191] Add aa36-5A (69.38 mg, 541.50 μmol, 60.86 μL, 1.1 equivalent) to a solution of aa36-5 (0.07 g, 492.27 μmol, 1 equivalent) in DMF (2 mL). Stir the solution at 20 °C for 12 h. LC-MS showed the desired product. Concentrate the solution under vacuum. Purify the residue by preparative HPLC (FA conditions; column: Phenomenex Synergi C18 150*30 mm*4 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 15%-25%, 11 min). Compound aa36 (20 mg, 73.99 μmol, 15.03% yield) was obtained as a white solid.

[1192] LCMS(ESI): RT = 0.19 min, for C 13 H 22 The calculated mass of N2O4H was 271.16, and the actual measured mass was 271.1 [M+H]+. The reversed-phase LCMS was performed as follows: using a Chromolith Flash Agilent Pursult 5C18 20*2.0mm, the flow rate was 1.5mL / min, and the elution was carried out with a gradient of 5%-95% (solvent B) over 0.7 minutes, and then held at 95% for 0.4 minutes.

[1193] 1 ¹H NMR (400MHz, methanol-d⁴) 3.51–3.44 (m, 2H), 3.43–3.35 (m, 4H), 2.47 (s, 2H), 2.35 (t, J = 6.0 Hz, 2H), 1.91–1.76 (m, 4H), 1.25 (s, 6H) ppm.

[1194] 2.18 Synthesis of non-natural amino acids (aa37)

[1195] Scheme 19 outlines the synthesis of non-natural amino acids (aa37):

[1196] Option 19

[1197]

[1198] Step 1: Synthesis of N-[2-(5-methyl-1,3-dioxo-isoindol-2-yl)ethyl]carbamate tert-butyl ester (aa37-2)

[1199] Add aa37-1A (543.46 mg, 3.39 mmol, 532.80 μL, 1.1 equivalent) to a solution of aa37-1 (500 mg, 3.08 mmol, 1 equivalent) in toluene (10 mL). Stir the mixture at 120 °C for 12 h. After this, concentrate the reaction mixture under vacuum to give a crude product, which was milled at 25 °C with MTBE / PE (1:1) for 1 h. Compound aa37-2 (460 mg, 1.44 mmol, 46.56% yield, 95% purity) was obtained as a yellow solid.

[1200] LCMS(ESI): RT = 0.898 min, m / z, for C 16 H 21 N₂O₄, calculated to be 305.14 [M+H] + For C 16 H 21 N₂O₄Na, calculated to be 327.14 [M + Na]. + The measured value was 327.2 [M+Na]. + LC-MS Method A: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1201] Step 2: Synthesis of 2-(2-aminoethyl)-5-methyl-isoindole-1,3-dione (aa37-3)

[1202] HCl / MeOH (4M, 377.87 μL, 1 equivalent) was added to a solution of aa37-2 (460 mg, 1.51 mmol, 1 equivalent). The mixture was stirred at 25 °C for 12 hours. After completion, the reactants were concentrated under vacuum to give crude product aa37-3 (300 mg, crude, HCl salt) as a white solid.

[1203] LCMS(ESI): RT = 0.638 min, m / z, for C 11 H 13 N₂O₂, calculated to be 205.09 [M+H] + Actual measurement: 205.0 [M+H] +LC-MS Method A: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1204] 1 H NMR (400MHz, chloroform-d) δ = 7.99 (br s, 2H), 7.26-7.21 (m, 1H), 3.56 (br s, 2H), 2.77 (br s, 4H), 2.08 (s, 3H) ppm.

[1205] Step 3: Synthesis of 2-[2-[2-(5-methyl-1,3-dioxo-isoindol-2-yl)ethylamino]-2-oxo-ethyl]thioalkylacetic acid (aa37)

[1206] DIPEA (316.42 mg, 2.45 mmol, 426.45 μL, 2 equivalents) was added to a solution of aa37-3 (250 mg, 1.22 mmol, 1 equivalent) and aa37-3A (194.11 mg, 1.47 mmol, 1.2 equivalents) in DMF (4 mL). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was partitioned between EtOAc (50 mL) and water (60 mL). The aqueous layer was acidified to pH 4 with 1 N HCl solution. The organic phase was separated, washed with brine (30 mL × 3), and dried over anhydrous Na2SO4. The resulting solution was concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 12g Purification was performed using a silica rapid column with an eluent gradient of 0%–30% ethyl acetate / petroleum ether at 35 mL / min. Compound aa37 (200 mg, 535.14 μmol, 43.72% yield, 90% purity) was obtained as a white solid.

[1207] LCMS(ESI): RT = 0.757 min, m / z, for C 15 H 16 The calculated value of N₂O₅SNa is 359.08 [M + Na]. + The measured value was 358.9 [M+Na]. + LC-MS Method A: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1208] 1¹H NMR (400MHz, chloroform-d) δ=7.69-7.60(m,1H),7.55(s,1H),7.43(br d,J=7.3Hz,1H),3.82-3.70(m,2H),3.52-3.41(m,2H),2.86(s,2H),2.76(s,2H),2.38(s,3H)ppm.

[1209] Example 3. Synthesis of GLP1 peptide mimics

[1210] A general synthetic scheme for preparing GLP1 peptide mimic payloads according to this disclosure is as follows: Figure 13 As shown in Table 2 below, the structures of Rink amide MBHA resin-bound peptides and intermediates 1-59 are depicted.

[1211] Table 2

[1212]

[1213]

[1214]

[1215]

[1216]

[1217]

[1218]

[1219]

[1220] Rink amide MBHA resin-bound peptides and intermediates (1–59) were analyzed by LC-MS after cleavage from the resin. Table 2B below summarizes these intermediates.

[1221] Table 2B. Intermediates from MBHA resin pyrolysis

[1222]

[1223]

[1224]

[1225]

[1226]

[1227]

[1228]

[1229]

[1230]

[1231]

[1232] Figure 14 The sequence of steps for synthesizing solid supports for GLP1 peptide mimic payloads P1 and P8 according to this disclosure is described.

[1233] 3.1(3S,6S,9R,12S,15S,21S)-21-amino-3-(((S)-1-(((S)-1-amino-5-(3,5-dimethyl) (4'-(4-aminobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)- 1-Oxopropane-2-yl)carbamoyl)-12-(2-fluorobenzyl)-9,15-bis((R)-1-hydroxyethyl)-6-(hydroxymethyl)- 12-Methyl-5,8,11,14,17,20-hexaoxo-22-(2H-tetrazol-5-yl)-4,7,10,13,16,19-hexaazatoctetrazol Preparation of alkyl-1-acids (P1)

[1234] The corresponding Fmoc-protected aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2-aa1 peptide-based Rink amide MBHA resin (9, 16.59 μmol) was prepared as described in the general procedure of SPPS. Resin-bound peptide 9 was cleaved according to the general procedure to give a crude product as a white solid. This crude product was dissolved in DMF (1 mL), and piperidine (14.13 mg, 165.94 μmol, 16.39 μL, 10.0 equivalent) was added in one portion at 20 °C under nitrogen. The mixture was stirred at 20 °C for 2 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by preparative HPLC (column: mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-60%, 60 min) to obtain a pure product. The product was suspended in water (10 mL), the mixture was frozen in a dry ice / ethanol bath, and then lyophilized to dryness to obtain the desired product P1 (1.02 mg, 7.48 e-1 μmol, 4.50% yield, 100% purity) as a white solid.

[1235] HRMS (ESI): For C 65 H 87 FN 17 O 15 The calculated mass is 1364.6552 m / z, and the actual measured mass is 1364.4887 [m+H]. + .

[1236] HPLC: RT = 10.15 min, reversed-phase HPLC was performed using a MERCK RP-18e 25-2 mm column at a flow rate of 1.2 mL / min, with gradient elution of 10% to 80% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1237] 3.2(8S,14S,17S,20S,23S,26S)-8-((2H-tetrazol-5-yl)methyl)-26-(((S)-1-(((S)- 1-Amino-5-(3,5-dimethylphenyl)-1-oxopentane-2-yl)amino)-3-(4'-(4-aminobutoxy)-2'-ethyl- [1,1'-Biphenyl]-4-yl)-1-oxopropane-2-yl)carbamoyl)-17-(2-fluorobenzyl)-14,20-bis((R)-1-hydroxy (Ethyl)-23-(hydroxymethyl)-1-(1H-imidazol-5-yl)-5,5,17-trimethyl-4,6,9,12,15,18,21,24-octaoxo- Preparation of 3,7,10,13,16,19,22,25-octaazaoctacosane-28-acid (P8)

[1238] The corresponding aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2-aa1 peptide-based Rink amide MBHA resin (10, 100.99 μmol) was assembled as described in the general procedure of SPPS. Resin-bound peptide 10 was cleaved following a general procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC using the following: Luna 200*25 mm column, C18, 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%–60%, 60 min, to obtain a pure product. The product was suspended in water (10 mL), the mixture was frozen in a dry ice / acetone bath, and then lyophilized to dryness to obtain the desired product P8 (25.00 mg, 17.49 μmol, 17.32% yield, 100% purity) as a white solid.

[1239] HRMS (ESI): For C 75 H 100 FN 20 O 17 The calculated mass is 1571.7559 m / z, and the actual measured mass is 1571.7589 [m+H]. + .

[1240] HPLC: RT = 10.14 min. Reversed-phase LC-HPLC was performed as follows: a YMC-Pack ODS-A 150*4.6 mm, 5 μm column was used at a flow rate of 1.5 mL / min, with gradient elution of 10% to 80% acetonitrile (solvent B) containing 0.062% TFA and water (solvent A) containing 0.068% TFA.

[1241] Figure 15 The sequence of steps for synthesizing solid supports for GLP1 peptide mimic payloads P2 and P9 according to this disclosure is described.

[1242] 3,3(8S,14S,17S,20R,23S,26S)-8-((2H-tetrazol-5-yl)methyl)-26-(((S)-1-(((S)- 1-Amino-5-(3,5-dimethylphenyl)-1-oxopentane-2-yl)amino)-3-(4'-(4-aminobutoxy)-2'-ethyl- [1,1'-Biphenyl]-4-yl)-1-oxopropane-2-yl)carbamoyl)-17-(2-fluorobenzyl)-14,20-bis((R)-1-hydroxy (Ethyl)-23-(hydroxymethyl)-1-(1H-imidazol-5-yl)-5,5,17-trimethyl-4,6,9,12,15,18,21,24-octaoxo- Preparation of 3,7,10,13,16,19,22,25-octaazaoctacosane-28-acid (P2)

[1243] The corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 74.75 μmol) was prepared as described in the general procedure of SPPS. Resin-bound peptide 18 was cleaved according to the general procedure to give a crude product as a white solid. The crude product was sent to a preparative HPLC (column: mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 60 min) to obtain a pure product. The product was suspended in water (100 mL), the mixture was frozen in a dry ice / acetone bath, and then lyophilized to dryness to provide the desired product P2 (11.56 mg, 8.45 μmol, 11.27% yield, 97.84% purity) as a white solid.

[1244] LC-MS (ESI): RT = 0.830 min. For C65H88FN15O15, the calculated mass was 1337.66[M-4tBu-Boc+6H] + 669.83[M-4tBu-Boc+7H]²⁺, and the measured mass was 670.0[M-4tBu-Boc+7H]²⁺. Reversed-phase LC-MS was performed as follows: a Chromolith Flash RP-18e25-3 mm column was used at a flow rate of 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.04% TFA and water (solvent A) containing 0.06% TFA.

[1245] HPLC: RT = 7.77 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1246] 3,4(8S,14S,17S,20S,23S,26S)-8-((2H-tetrazol-5-yl)methyl)-26-(((S)-1-(((S)- 1-Amino-5-(3,5-dimethylphenyl)-1-oxopentane-2-yl)amino)-3-(4'-(4-aminobutoxy)-2'-ethyl- [1,1'-Biphenyl]-4-yl)-1-oxopropane-2-yl)carbamoyl)-17-(2-fluorobenzyl)-14,20-bis((R)-1-hydroxy (Ethyl)-23-(hydroxymethyl)-1-(1H-imidazol-5-yl)-5,5,17-trimethyl-4,6,9,12,15,18,21,24-octaoxo- Preparation of 3,7,10,13,16,19,22,25-octaazaoctacosane-28-acid (P9)

[1247] The corresponding aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (19) was prepared as described in the general procedure of SPPS. Resin-bound peptide 19 was cleaved according to the general procedure to give a crude product as a white solid. The crude product was sent to preparative HPLC (TFA: mobile phase: [water (0.075% TFA)-ACN]; B%: 15%-45%, 55 min) for purification to obtain a pure product. The product was suspended in water (20 mL), the mixture was frozen in a dry ice / acetone bath, and then lyophilized to dryness to provide the desired product P9 (125 mg, 79.82 μmol, 7.70% yield, 98.7% purity) as a white solid.

[1248] LC-MS (ESI): RT = 0.843 min. For C75H102FN18O17, the calculated mass was 1545.77[M-4tBu-Boc+6H] + 773.385[M-4tBu-Boc+7H]2+, and the measured mass was 773.9[M-4tBu-Boc+7H]2+. Reversed-phase LC-MS was performed as follows: a Chromolith Flash RP-18e25-3 mm column was used at a flow rate of 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.04% TFA and water (solvent A) containing 0.06% TFA.

[1249] Figure 16 The sequence of steps for synthesizing solid supports for GLP1 peptide mimic payloads P3, P4, P5, P6, P7, P11, P13, P14, P15, P16 and P17 according to this disclosure is described.

[1250] 3.5 3-[[(1S)-2-[[2-[[(1S,2R)-1-[[(1S)-2-[[(1S,2R)-1-[[(1S)-2-[[(1S)- 2-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2-[[(1S)-1-carbamoyl] [4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-1-(carboxymethyl)-2-oxo-ethyl]amino [1-(hydroxymethyl)-2-oxo-ethyl]carbamoyl]-2-hydroxy-propyl]amino]-1-[(2-fluorophenyl)methyl]- [1-Methyl-2-oxo-ethyl]carbamoyl]-2-hydroxy-propyl]amino]-2-oxo-ethyl]amino]-2-oxo-1- Preparation of (2H-tetrazol-5-ylmethyl)ethyl]amino]-2,2-dimethyl-3-oxo-propionic acid (P3)

[1251] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 152.96 μmol) and aa11 (57.58 mg, 305.91 μmol, 2.0 equivalents), the corresponding aa11-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (20, 119 μmol) was prepared as described in the general procedure of SPPS.

[1252] The corresponding resin-bound peptide 20 (119 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P3 as a pale yellow solid (8 mg, 5.47 μmol, 3.58% yield, 99.37% purity).

[1253] HPLC: RT = 8.10 min. HPLC conditions: YMC-Pack ODS-A 150*4.6 mm, 5 μm column, flow rate 1.5 mL / min, eluted with a gradient of 10% to 80% acetonitrile (solvent B) containing 0.12% TFA and water (solvent A) containing 0.1% TFA.

[1254] LCMS (ESI): RT = 0.841 min, m / z, calculated as 1452.69 [M+H]+ for C18H21NO2, measured as 727.3 [M+2H]2+; LCMS conditions: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1255] 3,6(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-2-[(3-amino-2,2-dimethyl-3-oxo-propane Acyl)amino]-3-(2H-tetrazole-5-yl)propionyl]amino]acetyl]amino]-3-hydroxy-butyryl]amino]-3-(2- [Fluorophenyl]-2-methyl-propionyl]amino]-3-hydroxy-butyryl]amino]-3-hydroxy-propionyl]amino]-4-oxo-butyryl] Preparation of acid (P4)

[1256] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin compound 18 (192.73 μmol) and aa12 (75.82 mg, 578.18 μmol, 3 equivalents), the corresponding aa12-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (21, 192.73 μmol) was obtained as described in the general procedure of SPPS.

[1257] The corresponding resin-bound peptide 21 (192.73 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P4 as a pale yellow solid (35 mg, 24.03 μmol, 8.73% yield, 99.66% purity).

[1258] LCMS (ESI): RT = 0.861 min, calculated mass of C76H103FN18O17 was 726.36 m / z [M+2H]2+; measured mass was 726.7 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1259] LCMS (ESI): RT = 0.854 min, calculated mass of C76H103FN18O17 was 726.36 m / z [M+2H]2+; measured mass was 726.7 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1260] HPLC: RT = 7.90 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1261] 3,7(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[3-(hydroxy [amino]-2,2-dimethyl-3-oxo-propionyl]amino]-3-(2H-tetrazol-5-yl)propionyl]amino]acetyl]amino] [Butyryl]amino]-2-methyl-propionyl]amino]-3-hydroxy-butyryl]amino]-3-hydroxypropionyl]amino]-4-oxo] Preparation of 1,4-butyric acid (P5)

[1262] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin compound 18 (152.96 μmol) and aa13 (60 mg, 259.47 μmol, 1.7 equivalents), the corresponding aa13-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (22, 152.67 μmol) was obtained as described in the general procedure of SPPS.

[1263] The corresponding resin-bound peptide 22 was cleaved according to a standard procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to give P5 as a pale yellow solid (4 mg, 2.66 μmol, 42.12% yield, 97.7% purity).

[1264] LCMS (ESI): RT = 0.705 min, for C 70 H 96 FN 16 O 18 The calculated mass is 733.85 m / z [M+2H]. 2+ Measured value: 718.2 m / z [M-2OH+4H] 2+ LC-MS: MERCK RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1265] LCMS (ESI): RT = 0.847 min, for C 70 H 96 FN 16 O 18 The calculated mass is 733.85 m / z [M+2H]. 2+ Measured value: 735.2 m / z [M+2H] 2+ LC-MS: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1266] HPLC: RT = 8.00 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1267] 3,8(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[2-methyl- 2-(1H-pyrazol-5-yl)propionyl]amino]-3-(2H-tetrazole-5-yl)propionyl]amino]acetyl]amino]butyryl] [Amino]-2-methyl-propionyl]amino]-3-hydroxy-butyryl]amino]-3-hydroxypropionyl]amino]-4-oxo-butyric acid Preparation of (P6)

[1268] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin compound 18 (82.60 μmol) and aa14 (25.47 mg, 165.19 μmol, 2 equivalents), the corresponding aa14-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (23, 82.60 μmol) was obtained as described in the general procedure of SPPS.

[1269] The corresponding resin-bound peptide 23 (82.60 μmol) was cleaved according to a standard procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to give P6 as a pale yellow solid (9 mg, 6.10 μmol, 7.40% yield, 97.34% purity).

[1270] LC-MS (ESI): RT = 0.856 min, calculated mass for C72H98FN17O16 was 737.87 m / z [M-4tBu-Boc-C17H17NO4+8H]2+, rinkamide (C17H17NO4, exact mass = 299.12); measured mass was 738.2 m / z [M-4tBu-Boc-C17H17NO4+8H]2+, rinkamide (C17H17NO4, exact mass = 299.12); LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1271] LC-MS (ESI): RT = 0.856 min, calculated mass for C72H98FN17O16 was 737.87 m / z [M-4tBu-Boc-C17H17NO4+8H]2+, rinkamide (C17H17NO4, exact mass = 299.12); measured mass was 738.2 m / z [M-4tBu-Boc-C17H17NO4+8H]2+, rinkamide (C17H17NO4, exact mass = 299.12); LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1272] HPLC: RT = 8.16 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1273] 3.9(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-2-[[(2S)-2-amino-3-hydroxy-propionyl] [amino]-3-(2H-tetrazol-5-yl)propionyl]amino]acetyl]amino]-3-hydroxy-butyryl]amino]-3-(2-fluorobenzene [2-methyl-propionyl]amino]-3-hydroxy-butyryl]amino]-3-hydroxy-propionyl]amino]-4-oxo-butyric acid Preparation of (P7)

[1274] The corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 76.48 μmol) and aa4 (87.98 mg, 229.44 μmol, 3 equivalents) were used as the starting point. The corresponding aa4-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (24, 76.48 μmol) was obtained as described in the general procedure of SPPS.

[1275] The corresponding resin-bound peptide 24 (76.48 μmol) was cleaved according to a standard procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 28%-58%, 30 min) to give P7 as a white solid (17 mg, 11.16 μmol, 14.59% yield, 93.57% purity).

[1276] LCMS (ESI): RT = 2.675 min, m / z, calculated as 713.35 [M+2H]2+ for C68H95FN16O17, m / z, measured as 713.8 [M+2H]2+; mobile phase: 1.5 mL / 4 L TFA (solvent A) in water and 0.75 mL / 4 L TFA (solvent B) in acetonitrile, using an elution gradient of 10%-80% (solvent B) over 6 minutes, and maintained at 80% for 0.5 minutes at a flow rate of 0.8 mL / min;

[1277] LCMS (ESI): RT = 0.757 min, m / z. For C68H95FN16O17, the calculated value was 713.35 [M+2H]2+, m / z, and the measured value was 713.8 [M+2H]2+. Reversed-phase LCMS was performed as follows: a MERCK RP-18e 25-2 mm column was used at a flow rate of 1.5 mL / min, with gradient elution using 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1278] HPLC (Rt = 7.58 min, mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min).

[1279] 3.10(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[6-(1H-imidazolium)] [[Azol-5-yl)hexanoylamino]-3-(2H-tetrazol-5-yl)propionyl]amino]acetyl]amino]butyryl]amino]-2-methyl Preparation of [p11]-3-hydroxy-butyryl]amino]-3-hydroxy-propionyl]amino]-4-oxo-butyric acid (P11)

[1280] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 74.75 μmol) and aa17 (70.13 mg, 165.19 μmol, 2 equivalents), the corresponding aa17-aa10-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (27, 74.75 μmol) was obtained as described in the general procedure of SPPS.

[1281] The corresponding resin-bound peptide compound 27 (74.75 μmol) was cleaved according to a standard procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to give P7 as a white solid (9 mg, 5.80 μmol, 7.05% yield, 96.92% purity).

[1282] HPLC: RT = 7.72 min. HPLC conditions: YMC-Pack ODS-A 150*4.6 mm, 5 μm column, flow rate 1.5 mL / min, eluted with a gradient of 10% to 80% acetonitrile (solvent B) containing 0.12% TFA and water (solvent A) containing 0.1% TFA.

[1283] LCMS (ESI): RT = 0.828 min, m / z, calculated as 1502.75 [M+H]+ for C18H21NO2, measured as 752.3 [M+2H]2+; LCMS conditions: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1284] 3.11(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2,5- [diamino-5-oxo-pentanoyl]amino]-3-(1H-imidazol-4-yl)propionyl]amino]propionyl]amino]-3-(2H-tetramethyl] [Azolium-5-yl)propionyl]amino]acetyl]amino]-3-hydroxy-butyryl]amino]-3-(2-fluorophenyl)-2-methylpropionyl] Preparation of [amino]-3-hydroxybutyryl]amino]-3-hydroxypropionyl]amino]-4-oxo-butyric acid (P13)

[1285] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin compounds 18 (82.60 μmol), aa15 (77.14 mg, 247.79 μmol, 3 equivalents), aa16 (93.40 mg, 247.49 μmol, 3 equivalents), and aa19 (40.63 mg, 165.00 μmol, 2 equivalents), the corresponding aa19-aa16-aa15-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (30, 82.50 μmol) was prepared as described in the general procedure of SPPS.

[1286] The corresponding resin-bound peptide 30 (82.50 μmol) was cleaved according to a standard procedure to give a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to give P13 as a white solid (10 mg, 5.69 μmol, 6.90% yield, 95.28% purity).

[1287] LC-MS (ESI): RT = 0.807 min, calculated mass for C79H110FN21O19 was 837.9 m / z [M-4tBu-2Boc-C17H17NO4+9H]2+, rinkamide (C17H17NO4, exact mass = 299.12); measured mass was 838.4 m / z [M-4tBu-Boc-C17H17NO4+9H]2+, rinkamide (C17H17NO4, exact mass = 299.12); LC-MS: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1288] LC-MS (ESI): RT = 0.820 min, calculated mass for C79H110FN21O19 was 837.9 m / z [M-4tBu-2Boc-C17H17NO4+9H]2+, rinkamide (C17H17NO4, exact mass = 299.12); measured mass was 838.3 m / z [M-4tBu-Boc-C17H17NO4+9H]2+, rinkamide (C17H17NO4, exact mass = 299.12); LC-MS: MERCK, RP-18e 25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1289] HPLC: RT = 7.37 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1290] 3.12(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[(3R)-1- [2-(1H-imidazol-5-yl)ethyl]-3-methyl-2-oxo-pyrrolidine-3-carbonyl]amino]-3-(2H-tetrazol-5-yl)propionyl [amino] [acetyl] [amino] [butyryl] [amino] -2-methyl-propionyl] [amino] -3-hydroxy-butyryl] [amino] -3-hydroxy Preparation of [p14]propionyl]amino]-4-oxo-butyric acid

[1291] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 82.60 μmol) and aa20 (79.22 mg, 165.19 μmol, 2 equivalents), the corresponding aa20-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (31, 82.60 μmol) was prepared as described in the general procedure of SPPS.

[1292] The corresponding resin-bound peptide 31 (82.60 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P14 as a white solid (8 mg, 3.85 μmol, 6.2% yield, 93.37% purity).

[1293] LCMS (ESI): RT = 0.840 min, calculated mass of C76H103FN18O17 was 779.39 m / z [M+2H]2+; measured mass was 779.9 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1294] LCMS (ESI): RT = 0.864 min, calculated mass of C76H103FN18O17 was 779.39 m / z [M+2H]2+; measured mass was 779.9 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1295] HPLC: RT = 7.60 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1296] 3.13(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[(3R)-1- [2-(1H-imidazol-5-yl)ethyl]-3-methyl-2-oxo-pyrrolidine-3-carbonyl]amino]-3-(2H-tetrazol-5-yl)propionyl [amino] [acetyl] [amino] [butyryl] [amino] -2-methyl-propionyl] [amino] -3-hydroxy-butyryl] [amino] -3-hydroxy Preparation of [[amino]-4-oxo-butyric acid (P15)]

[1297] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 82.60 μmol) and aa21 (79.22 mg, 165.19 μmol, 2 equivalents), the corresponding aa21-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (32, 82.60 μmol) was prepared as described in the general procedure of SPPS.

[1298] The corresponding resin-bound peptide 32 (82.60 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P15 as a white solid (10.5 mg, 6.54 μmol, 7.91% yield, 96.96% purity).

[1299] LCMS (ESI): RT = 0.828 min, calculated mass of C76H103FN18O17 was 779.39 m / z [M+2H]2+; measured mass was 779.8 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1300] LCMS (ESI): RT = 0.830 min, calculated mass of C76H103FN18O17 was 779.39 m / z [M+2H]2+; measured mass was 779.8 m / z [M+2H]2+; LC-MS: MERCK, RP-18e25-2 mm column, flow rate 1.5 mL / min, eluted with a gradient of 5% to 95% acetonitrile (solvent B) containing 0.02% TFA and water (solvent A) containing 0.04% TFA.

[1301] HPLC: RT = 7.62 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm, 5 μm; Wavelength: UV 220 nm & 215 nm & 254 nm; Column temperature: 40 °C.

[1302] 3.14(3S)-4-[[(1S)-1-[[4-[4-(4-aminobutoxy)-2-ethyl-phenyl]phenyl]methyl]-2- [[(1S)-1-carbamoyl-4-(3,5-dimethylphenyl)butyl]amino]-2-oxo-ethyl]amino]-3-[[(2S)- 2-[[(2S,3R)-2-[[(2S)-3-(2-fluorophenyl)-2-[[(2S,3R)-3-hydroxy-2-[[2-[[(2S)-2-[[(3S)-1- [2-(1H-imidazol-5-yl)ethyl]pyrrolidine-3-carbonyl]amino]-3-(2H-tetrazol-5-yl)propionyl]amino]acetyl] [Amino]butyryl]amino]-2-methyl-propionyl]amino]-3-hydroxy-butyryl]amino]-3-hydroxy-propionyl]amino Preparation of 4-oxo-butyric acid (P16)

[1303] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (18, 141.13 μmol) and aa22 (127.46 mg, 282.27 μmol, 2.0 equivalent), the corresponding aa22-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (33, 141.13 μmol) was prepared as described in the general procedure of SPPS.

[1304] The corresponding resin-bound peptide 33 (141.13 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P16 as a white solid (15 mg, 9.71 μmol, 6.88% yield, and 99% purity).

[1305] LCMS (ESI): RT = 2.361 min, m / z, calculated as 1530.76 M-Boc-4tBu+2H]2+ for C75H103FN18O16, m / z, measured 765.8. Mobile phase: 1.5 mL / 4 L TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using a gradient of 10%-80% (solvent B) over 2.5 min, and held at 80% for 0.5 min at a flow rate of 0.8 mL / min. ESI source, positive ion mode; wavelength 220 nm & 254 nm, oven temperature 50 °C.

[1306] LCMS (ESI): RT = 0.755 min, m / z, calculated as 1530.76 M-Boc-4tBu+2H]2+ for C75H103FN18O16, m / z, measured 765.8; mobile phase: 1.5 mL / 4 L TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 5%-95% (solvent B) over 0.7 min, and held at 95% for 0.4 min at a flow rate of 1.5 mL / min; column: Agilent Pursult 5C18 20*2.0 mm; wavelength: UV 220 nm; column temperature: 50 °C; MS ionization: ESI.

[1307] HPLC: RT = 7.18 min; Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm; Wavelength: UV 220 nm, 215 nm & 254 nm; Column temperature: 40 °C.

[1308] 3.15(3S,9S,12S,15S,18S,21S)-tert-butyl-1-((R)-1-(2-(1H-imidazol-5-yl)ethyl)pyrrole Alkyl-3-yl)-3-((2H-tetrazol-5-yl)methyl)-21-(((S)-1-(((S)-1-amino-5-(3,5-dimethylphenyl)-1- (Oxopentane-2-yl)amino)-3-(4'-(4-aminobutoxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)-1-oxopropyl Alkyl)carbamoyl)-12-(2-fluorobenzyl)-9,15-bis((R)-1-hydroxyethyl)-18-(hydroxymethyl)-12-methyl- Preparation of 1,4,7,10,13,16,19-heptaoxo-2,5,8,11,14,17,20-heptaazatridecane-23-ester (P17)

[1309] Starting with the corresponding aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin compounds (18, 101.97 μmol) and aa23 (80 mg, 177.16 μmol, 1.74 equivalents), the corresponding aa23-aa9-aa8-aa7-aa6-aa5-aa4-aa3-aa2b-aa1 peptide-based Rink amide MBHA resin (34, 101.88 μmol) was prepared as described in the general procedure of SPPS.

[1310] The corresponding resin-bound peptide 34 (101.88 μmol) was further cleaved following a standard procedure to yield a crude product as a white solid. The crude product was purified by preparative HPLC (column: YMC-Exphere C18 10 μm 300*50 mm, 12 nm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 55 min) to provide P17 as a white solid (18 mg, 11.65 μmol, 7.91% yield, 99% purity).

[1311] LCMS (ESI): RT = 0.828 min, m / z, calculated as 1530.76 M-Boc-4tBu+2H]2+ for C75H103FN18O16, m / z, measured 765.8; mobile phase: 1.5 mL / 4 L TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 5%-95% (solvent B) over 0.7 min, and held at 95% for 0.4 min at a flow rate of 1.5 mL / min; column: Agilent Pursult 5C18 20*2.0 mm; wavelength: UV 220 nm; column temperature: 50 °C; MS ionization: ESI.

[1312] HPLC: RT = 7.36 min. Mobile phase: 2.75 mL / 4 L TFA in water (solvent A) and 2.5 mL / 4 L TFA in acetonitrile (solvent B), using an elution gradient of 10%-80% (solvent B) over 10 min, and holding at 80% for 5 min at a flow rate of 1.5 mL / min; Column: YMC-Pack ODS-A 150*4.6 mm; Wavelength: UV 220 nm, 215 nm & 254 nm; Column temperature: 40 °C.

[1313] Figure 17A and Figure 17B The sequence o...

Claims

1. A compound of formula (A): BA-(LP) m (A), in: BA is an antibody or its antigen-binding fragment; L is a connector that includes one or more of the following: ; carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units; -(CH2) 2-24 - chain; triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof; P is the payload selected from the following group: (P-IA) and (P-IIA), in The attachment point where the payload is attached to L; X1 is selected from H. , , , , , , and ; X2 is selected from and ; X3 is selected from the bond, -(CH2). 2-6 -NH-, -(CH2) 2-6 -Tr- and -(CH2) 2-6 -Tr-(CH2) 1-6 -NH, where Tr is the triazole moiety; n is 0 or 1; X4 is selected from -NH2, -OH and -N(H)(phenyl); X5 is selected from -OH, -NH2, -NH-OH, and ; X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears; X7 is selected from H, , , , and ; X8 is selected from H, -OH, -NH2, and ; Ar selected and ; X9 is selected from -NH2, and ;and m is an integer from 1 to 4; Or its pharmaceutically acceptable salt.

2. A compound of formula (I): BA-LP (I), in: BA is an antibody or its antigen-binding fragment; L is a connector that includes one or more of the following: ; carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units; -(CH2) 2-24 - chain; triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof; P is the payload selected from the following group: (PI) and (P-II); in The attachment point where the payload is attached to L; X1 is selected from H. , , , , ,and ; X2 is selected from and ; X3 is selected from -(CH2) 2-6 -NH- and -(CH2) 2-6 -Tr-, where Tr is the triazole moiety; n is 0 or 1; X4 is selected from H and phenyl; X5 is selected from -OH, -NH2, -NH-OH, and ; X6 is independently selected from H, -OH, -CH3, and -CH2OH each time it appears; X7 is selected from H, and ; X8 is selected from H, -OH, -NH2, and ; Or its pharmaceutically acceptable salt.

3. The compound of claim 1, wherein the BA is an antibody or antigen-binding fragment thereof targeting glucagon-like peptide-1 receptor (GLP1R).

4. The compound of claim 3, wherein the antibody targeting GLP1R is a GLP1R agonist antibody.

5. The compound of claim 1, wherein the connector L is attached to one or both heavy chains of the BA or to one or both light chains of the BA.

6. The compound of claim 5, wherein the connector L is attached to one or two heavy chain variable domains of the BA or to one or two light chain variable domains of the BA.

7. The compound of claim 1, wherein the connector L is attached to the BA via a glutamine residue or a lysine residue.

8. The compound according to claim 1, wherein m is 1 or 2.

9. The compound according to claim 1, wherein the connector L comprises a structure of formula (L'): -La-Y-Lp- (L'), Wherein La is the first connector covalently attached to the BA; Y is a group containing a triazole moiety, and Lp is absent or is a second connector covalently attached to P, wherein Y is also absent when Lp is absent.

10. The compound according to claim 9, wherein the Y-Lp is absent.

11. The compound of claim 9, wherein the La comprises a structure selected from the group consisting of: and .

12. The compound according to claim 1, wherein P is (P-IA).

13. A compound of formula (A): BA-(LP) m (A), Or its pharmaceutically acceptable salt, wherein: BA is an antibody or its antigen-binding fragment; L is a connector that includes one or more of the following: ; carbamate group; cyclodextrin; polyethylene glycol (PEG) segment having 1 to 36 -CH2CH2O- (EG) units; -(CH2) 2-24 - chain; triazole; one or more amino acids selected from glycine, serine, glutamic acid, alanine, valine, and proline, and combinations thereof; m is an integer from 1 to 4; and ； ； ； and 。 14. The compound of claim 1, wherein the compound has a plasma half-life of more than 7 days.

15. The compound of claim 1, wherein the compound does not bind to G protein-coupled receptors (GPCRs) other than GLP1R.

16. A pharmaceutical composition comprising the compound according to claim 1.

17. A pharmaceutical dosage form comprising the compound according to claim 1.

18. Use of the compound of claim 1 in the preparation of a medicament for selectively targeting GLP1R on the cell surface.

19. Use of the compound according to claim 1 in the preparation of a medicament for enhancing the GLP1R activity of individuals with the corresponding need.

20. Use of the compound according to claim 1 in the preparation of a medicament for lowering blood glucose levels in individuals with a corresponding need.

21. Use of the compound according to claim 1 in the preparation of a medicament for reducing the weight of an individual with a corresponding need.

22. Use of the compound according to claim 1 in the preparation of a medicament for treating GLP1R-related conditions in individuals with corresponding needs.

23. A method for preparing the compound of formula (A) of claim 1: BA-(LP) m (A), The method includes the following steps: a) In the presence of transglutaminase, BA comprising at least m glutamine residues is contacted with at least an equivalent amount of the compound LP, and b) The resulting compound of formula (A) is isolated; and wherein BA, L, P and m are as defined in claim 1.

24. A compound of formula (P-IB): (P-IB), in: X1 is selected from , , and ; X2 is selected from and ; X3 is selected from -(CH2) 2-6 -NH2、-(CH2) 2-6 -N3 and -(CH2) 2-6 -Tr-(CH2) 1-6 -NH2, where Tr is the triazole moiety; n is 1; X4 is -NH2; X5 is selected from -OH, -NH2, and ; X6 is independently selected from H and -CH3 each time it appears; X7 is selected from or ; X8 is H; Ar selected and ; X9 is selected from -NH2, and ; Or its pharmaceutically acceptable salt.

25. A compound selected from the group consisting of: 。 26. A compound selected from the group consisting of: Or its pharmaceutically acceptable salt.

27. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof targeting a glucagon-like peptide-1 receptor (GLP1R) optionally conjugated to a payload via a linker, said payload being selected from the group consisting of: ； ； ； ； ;and ； Or its pharmaceutically acceptable salt.

28. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof targeting the glucagon-like peptide-1 receptor (GLP1R) and a payload having the following structure: ， in The payload is attached to the antibody or its antigen-binding fragment directly or through a connector at the attachment point.

29. The antibody-drug conjugate according to claim 28, wherein the effective payload is: 。 30. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof targeting the glucagon-like peptide-1 receptor (GLP1R) and a linker-payload having the following structure: ， in It is the attachment point where the connector-payload is attached to the antibody or its antigen-binding fragment.

31. The antibody-drug conjugate according to claim 30, wherein the linker-payload is: 。 32. The compound of claim 3, wherein the antibody targeting GLP1R is glutazumab.

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