Bicyclic peptide compounds as il-23 receptor inhibitors

Abstract

The present invention relates to bicyclic peptide compounds, pharmaceutical compositions containing the same and their use as inhibitors of interleukin-23 receptor (IL-23R). More specifically, the present invention provides novel bicyclic peptide compounds as inhibitors of IL-23R, pharmaceutical compositions containing such compounds and methods of using the compounds to treat or prevent relevant diseases and disorders mediated by IL-23R. The present invention also relates to methods of preparing the compounds.

Description

Technical Field

[0001] This invention relates to bicyclic peptide compounds, pharmaceutical compositions containing the same, and their use as inhibitors of interleukin-23 receptor (IL-23R). More specifically, the invention provides novel bicyclic peptide compounds as IL-23R inhibitors, pharmaceutical compositions containing such compounds, and methods of using said compounds to treat or prevent IL-23R-mediated diseases and functional disorders. The invention also relates to methods for preparing said compounds. Background Technology

[0002] Interleukin IL-23 is a member of the interleukin IL-12 cytokine family. It is a heterodimer composed of the IL-23p19 subunit and the IL-12p40 subunit, which is shared with IL-12. IL-12Rβ1 is the receptor for the IL-12 subunit, while IL-23R primarily binds to the p19 subunit. IL-12Rβ1 is mainly expressed on T cells, NK cells, and dendritic cells, while IL-23R is expressed only on T cells, NK cells, monocytes, and dendritic cells. IL-23 binds to IL-23R and IL-12Rβ1 to form a cytokine-receptor ternary complex, including IL-23, IL-23R and IL-12Rβ1, which activates a series of Janus kinases (JAKs; e.g. JAK2, TYK2) and signal transduction and transcription activators (STATs; e.g. STAT3, STAT4) (Floss, DMe et al., Mol. Biol. Cell. 2016, 27, 2301–2316).

[0003] IL-23 is associated with a range of immune-mediated inflammatory diseases (IMIDs), including psoriasis (PsO), psoriatic arthritis (PsA), and inflammatory bowel disease (IBD). Early data suggested that IL-23 enhances IFN-γ production by memory CD4+ T cells; later, it was found that IL-23 can also induce these cells to produce IL-17 (Aggarwal, S. et al., J. Biol. Chem. 2003, 278, 1910–1914.). In the IL-23p19 knockout mouse EAE model, mice were protected from autoimmune inflammation, confirming the crucial role of IL-23 in inflammatory diseases (Cua, DJ et al., Nature, 2003, 421, 744–748.). Subsequent genome-wide association studies have shown that the IL-23 signaling pathway plays an important role in the pathogenesis of several chronic inflammatory diseases, including Crohn's disease (Duerr, RH, et al., Science, 2006, 314, 1461–1463.), spondylitis (Burton, PR, et al., Nat. Genet. 2007, 39, 1329–1337.), and psoriasis (Cargill, M. et al., Am. J. Hum. Genet. 2007, 80, 273–290.).

[0004] IL-23R is expressed on various adaptive and innate immune cells: Th17 cells, γδT cells, natural killer (NK) cells, dendritic cells, macrophages, and innate lymphoid cells. These cells are abundant in the gut, and elevated gene expression and protein levels of IL-23R have been found on the intestinal mucosal surface of IBD patients. It is believed that IL-23R mediates this effect by promoting the development of pathogenic CD4+ T cell populations that produce IL-6, IL-17, and tumor necrosis factor (TNF).

[0005] Psoriasis, a chronic skin disease, has been shown to be mediated by the body's helper T-cell inflammatory response. Approximately 40 million people worldwide suffer from psoriasis, with a prevalence ranging from 0.09% to 3.34% in different countries. Extensive basic and clinical research has demonstrated that the TNF-α / IL-23 / Th17 cytokine transmission axis explains the pathological mechanisms of psoriasis inflammation. The presence of pro-inflammatory cytokines TGF-β, IL-6, and IL-1β induces the initial differentiation of CD4+ T cells into Th17 cells and leads to the upregulation of IL-23R expression on the surface of Th17 cells. IL-23 released by dendritic cells and antigen-presenting cells connects to receptors on the surface of Th17 cells, activating and stimulating the expansion of Th17 subsets. Activated Th17 cells induce the production of cytokines IL-17A, IL-17F, IL-22, IL-21, and TNF-α. IL-17A stimulates neutrophil recruitment and activation, directly activates keratinocytes, and synergistically enhances inflammation with TNF-α; IL-22 induces excessive proliferation of keratinocytes and is associated with the severity of psoriasis.

[0006] Therapeutic drugs that inhibit the IL-23 pathway have been developed for the treatment of IL-23-related diseases and dysregulations. For example, ustekinumab, which targets IL-12 / 23 (IL-12p40 subunit), is currently approved for the treatment of moderate to severe plaque psoriasis (PsO), active psoriatic arthritis (PsA), moderate to severe active Crohn's disease (CD), and moderate to severe active ulcerative colitis (UC). Guselkumab and Risankizumab, which target the IL-23p19 subunit, are approved for the treatment of plaque psoriasis and psoriatic arthritis, pustular psoriasis, erythrodermic psoriasis and psoriatic arthritis, as well as inflammatory bowel disease. Despite the significant breakthroughs achieved in the clinical treatment of psoriasis and inflammatory bowel disease by IL-23-targeting antibody therapies, such as ustekinumab and ranitazine, antibody therapy still faces many challenges, including poor patient compliance and high cost.

[0007] Currently, there are no marketed small molecule or peptide oral inhibitors that selectively inhibit IL-23 signaling. Only the IL-23 receptor (IL-23R) inhibitor peptide JNJ-2113, co-developed by Johnson & Johnson and Protagonist Therapeutics, is in Phase III clinical trials. Therefore, there remains a significant need in the field for more effective small molecule, peptide oral medications to treat and / or prevent IL-23-related diseases and functional impairments, providing treatment options that do not require infusion delivery. Summary of the Invention

[0008] Generally speaking, the present invention relates to novel bicyclic peptide inhibitors of interleukin-23 receptor (IL-23R) or their pharmaceutically acceptable salts, solvates and / or other forms; pharmaceutical compositions containing the thereof; methods and / or uses of IL-23R inhibitors for treating autoimmune inflammatory diseases and / or related disorders.

[0009] Specifically, the present invention relates to compounds of formula (I) or pharmaceutically acceptable salts, solvates and / or other forms thereof; pharmaceutical compositions containing the same; methods and / or uses for treating autoimmune inflammatory diseases and related disorders.

[0010] The bicyclic peptide inhibitor described in this invention comprises the amino acid sequence of formula (I):

[0011] R1-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15

[0012] Formula (I)

[0013] Wherein: the linear form of the structure of formula (I) is intended for illustrative and non-limiting purposes, as will be apparent from the examples set forth and illustrated throughout this specification, i.e., for example, where each such structure may be longer or shorter than the length of the fourteen amino acids and / or other corresponding chemical moieties or functional group substituents as defined herein.

[0014] Specifically, in equation (I)

[0015] R 1 Selected from Wherein ring A is selected from C6-C10 aryl, C5-C10 heteroaryl, C3-C10 cycloalkyl and 3- to 8-membered heterocyclic groups, wherein the C6-C10 aryl, C5-C10 heteroaryl, C3-C10 cycloalkyl and 3- to 8-membered heterocyclic groups are optionally unsubstituted or substituted by one or more substituents B, wherein substituent B is selected from halogens, C1-C10 alkoxy, C3-C10 cycloalkyl, N,N-dimethylamino-C1-C6 alkoxy and N,N,N-trimethylammonium-C1-C6 alkoxy, wherein n is an integer from 0 to 4;

[0016] R a Selected from hydrogen or C1-C4 alkyl groups;

[0017] Preferably, wherein the R 1 Selected from the following groups, either unsubstituted or optionally substituted by one or more substituents C: The substituent C is selected from halogens, Where m = 1 to 4, n = 0 to 4, and r = 1 to 4 are integers;

[0018] R a Selected from hydrogen or methyl;

[0019] More preferably, wherein the R 1 Selected from

[0020] X3 is selected from Cys, Pen, or Abu;

[0021] X4 is selected from Asp or

[0022] X5 is selected from Thr, Ser, or does not exist; it is preferred to be Thr or does not exist.

[0023] X6 is selected from unsubstituted Trp or Trp substituted with C1-C4 alkyl groups; preferably W(7-Me);

[0024] X7 is selected from Lys(Ac), Lys(NMeAc), Pro,

[0025] X8 is selected from Cys or Pen;

[0026] X9 is selected from unsubstituted Tyr or Phe or substituted with one or more substituents D, wherein the substituents D are selected from halogens, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, carboxyl, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy, and N,N,N-trimethyl-3-ammoniumpropoxy;

[0027] Preferably, X9 is selected from Phe or Where R 2 Selected from halogen, ethynyl, C1-C4 alkoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy or N,N,N-trimethyl-3-ammoniumpropoxy;

[0028] More preferably, X9 is selected from Phe or Where R 2 Selected from halogens, methoxy groups, 2-aminoethoxy groups, N,N-dimethyl-2-aminoethoxy groups, N,N,N-trimethyl-5-ammonium-pentoxyamine groups, N,N-dimethyl-5-aminopentoxyamine groups, and N,N,N-trimethyl-3-ammonium-propoxyamine groups.

[0029] X10 is 2Nal;

[0030] X11 is selected from the following α-disubstituted amino acids: 4-amino-4-carboxy-tetrahydropyran (THP), α-MeLys,

[0031] X12 can be any amino acid; preferably, X12 is selected from Glu, Asp, Lys, D-Glu, or D-Asp.

[0032] X13 is any amino acid; preferably, X13 is selected from Glu, Asp, Asn, Ala, Val, Ser, Thr, Arg, D-Ala, D-Asn, D-Asp, D-Glu, D-Ser, D-Thr, D-Arg.

[0033] More preferably, X13 is selected from Glu, Asp, Asn, Ala, Val, Thr,

[0034] X14 is 3Pal;

[0035] X15 is unsubstituted or optionally substituted with one or more substituents of Gly; preferably, X15 is selected from...

[0036]

[0037] in:

[0038] The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond:

[0039] • X3 and X8 are connected by disulfide bonds between Cys and Cys, Pen and Pen, or Cys and Pen; or by thioether bonds between Abu and Cys or Pen, and

[0040] • An amide bond formed between R1 and the carboxylic acid on the side chain of X12, or an amide bond formed between R1 and the amino group on the side chain of X12;

[0041] The cyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

[0042] The present invention also relates to a bicyclic peptide inhibitor of the interleukin-23 receptor, a pharmaceutically acceptable salt or solvate thereof, wherein the bicyclic peptide inhibitor comprises the amino acid sequence of formula (I).

[0043] in:

[0044] R 1 Selected from unsubstituted or substituted by one or more C substituents.

[0045] The substituent C is selected from halogens, Where m = 1 to 4, n = 0 to 4, and r = 1 to 4 are integers;

[0046] R a Selected from hydrogen or methyl;

[0047] X3 is selected from Cys, Pen, or Abu;

[0048] X4 is selected from Asp or

[0049]

[0050] X5 is selected from Thr, Ser, or does not exist;

[0051] X6 is W(7-Me);

[0052] X7 is selected from Lys(Ac), Lys(NMeAc), Pro,

[0053] X8 is selected from Pen or Cys;

[0054] X9 is selected from Phe or Where R 2 Selected from halogen, ethynyl, C1-C4 alkoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy or N,N,N-trimethyl-3-ammoniumpropoxy;

[0055] X10 is 2Nal;

[0056] X11 is selected from 4-amino-4-carboxy-tetrahydropyran (THP), α-MeLys,

[0057] X12 is selected from Glu, Asp, Lys, D-Glu, or D-Asp;

[0058] X13 is selected from Glu, Asp, Asn, Ala, Val, Ser, Thr, Arg, D-Ala, D-Asn, D-Asp, D-Glu, D-Ser, D-Thr, D-Arg,

[0059]

[0060] X14 is 3Pal;

[0061] X15 is selected from

[0062] in:

[0063] The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond:

[0064] • X3 and X8 are connected by disulfide bonds between Cys and Cys, or Pen and Pen; or by thioether bonds between Abu and Cys or Pen, and

[0065] The amide bond formed between R1 and the carboxylic acid on the side chain of X12 or the amide bond formed between R1 and the amino group on the side chain of X12;

[0066] The cyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

[0067] The present invention also relates to a bicyclic peptide inhibitor of the interleukin-23 receptor, a pharmaceutically acceptable salt or solvate thereof, wherein the bicyclic peptide inhibitor comprises the amino acid sequence of formula (I).

[0068] in:

[0069] R 1 Selected from:

[0070]

[0071] X3 is selected from Cys, Pen, or Abu;

[0072] X4 is selected from Asn,

[0073] X5 is selected from Thr or does not exist;

[0074] Where X4 is selected from Asn, At that time, X5 is Thr;

[0075] When X4 is At that time, X5 did not exist;

[0076] X6 is W(7-Me);

[0077] X7 is selected from Lys(Ac), Lys(NMeAc), Pro,

[0078] X8 is selected from Pen or Cys;

[0079] X9 is Where R 2 Selected from methoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy or N,N,N-trimethyl-5-ammoniumpentoxyamino;

[0080] X10 is 2Nal;

[0081] X11 is selected from 4-amino-4-carboxy-tetrahydropyran (THP).

[0082]

[0083] X12 is either Glu or Lys;

[0084] X13 is selected from Glu, Asn, Ala, Val, Thr.

[0085] X14 is 3Pal;

[0086] X15 is selected from

[0087] in:

[0088] The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond:

[0089] • X3 and X8 are connected by disulfide bonds between Cys and Cys, or Pen and Pen; or by thioether bonds between Abu and Cys or Pen, and

[0090] • An amide bond formed between R1 and the carboxylic acid on the side chain of X12, or an amide bond formed between R1 and the amino group on the side chain of X12;

[0091] The cyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

[0092] This invention also relates to a bicyclic peptide inhibitor of the interleukin-23 receptor, and a pharmaceutically acceptable salt or solvate thereof, comprising the following bicyclic polypeptide:

[0093] Table 1:

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] This invention further relates to a bicyclic peptide inhibitor of the interleukin-23 receptor, and a pharmaceutically acceptable salt or solvate thereof, comprising the following bicyclic polypeptide:

[0101] Table 2:

[0102]

[0103] The cyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to the IL-23 receptor (IL-23R).

[0104] This invention also relates to pharmaceutical compositions comprising a bicyclic peptide inhibitor conforming to the amino acid sequence of formula (I) as described herein; isomers, prodrugs, solvates, stable isotopic derivatives or pharmaceutically acceptable salts thereof; and pharmaceutically acceptable carriers, diluents or excipients.

[0105] This invention also relates to pharmaceutical compositions comprising a bicyclic peptide inhibitor conforming to formula (I) as described herein; isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts thereof; or the use of the pharmaceutical composition in the preparation of a medicament for the treatment or prevention of IL23 / IL23R-mediated inflammatory, autoimmune inflammatory diseases and / or related disorders, such as psoriasis, psoriatic arthritis, ulcerative colitis, Crohn's disease, etc.

[0106] This invention also relates to the use of a medicament for treating or preventing IL23 / IL23R-mediated inflammatory, autoimmune inflammatory diseases and / or related disorders selected from: multiple sclerosis, asthma, rheumatoid arthritis, intestinal inflammation, inflammatory bowel disease (IBD), juvenile IBD, young adult IBD, Crohn's disease, ulcerative colitis, celiac disease (non-tropical stomatitis), microscopic colitis, collagenous colitis, eosinophilic gastroenteritis / esophagitis, colitis associated with radiotherapy or chemotherapy, colitis associated with congenital immune disorders such as leukocyte adhesion defect-1, sarcoidosis, systemic lupus erythematosus, ankylosing spondylitis (axial spondyloarthritis), psoriatic arthritis, psoriasis (e.g., plaques). This includes treatment for plaque psoriasis, guttate psoriasis, reversal psoriasis, pustular psoriasis, palmoplantar pustulosis, psoriasis vulgaris, or erythrodermic psoriasis; atopic dermatitis; acne atopic; enteropathy associated with seronegative arthropathy; chronic granulomatous disease; glycogen storage disease type 1b; Hermansky-Pudlak syndrome; Chediak-Higashi syndrome; Wiskott-Aldrich syndrome; pouchitis; pouchitis following rectocele and ileoanal anastomosis; gastrointestinal cancer; pancreatitis; insulin-dependent diabetes mellitus; mastitis; cholecystitis; cholangitis; primary biliary cirrhosis; virus-associated enteropathy; pericholangitis; chronic bronchitis; chronic sinusitis; asthma; uveitis; or graft-versus-host disease. It includes administering a therapeutically effective amount of the compound or its isomers, prodrugs, stable isotopic derivatives, or pharmaceutically acceptable salts, or the pharmaceutical composition thereof, to patients in need.

[0107] This invention also relates to pharmaceutical compositions comprising a bicyclic peptide inhibitor of the amino acid sequence conforming to formula (I) as described herein; isomers, prodrugs, solvates, stable isotope derivatives or pharmaceutically acceptable salts thereof; or the use of the pharmaceutical composition in the preparation of a medicament for the treatment or prevention of IL23 / IL23R-mediated inflammatory, autoimmune inflammatory diseases and / or related disorders selected from: multiple sclerosis, asthma, rheumatoid arthritis, intestinal inflammation, inflammatory bowel disease (IBD), juvenile IBD, young adult IBD, Crohn's disease, ulcerative colitis, celiac disease (non-tropical stomatitis), microscopic colitis, collagenous colitis, eosinophilic gastroenteritis / esophagitis, colitis associated with radiotherapy or chemotherapy, colitis associated with congenital immune disorders such as leukocyte adhesion defect-1, sarcoidosis, etc. Systemic lupus erythematosus, ankylosing spondylitis (axial spondyloarthritis), psoriatic arthritis, psoriasis (e.g., plaque psoriasis, guttate psoriasis, reversal psoriasis, pustular psoriasis, palmoplantar pustulosis, psoriasis vulgaris, or erythrodermic psoriasis), atopic dermatitis, acne atopic, enteropathy associated with seronegative arthropathy, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Wiskott-Aldrich syndrome, pouchitis, pouchitis following rectocele and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, primary biliary cirrhosis, virus-associated enteropathy, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, uveitis, or graft-versus-host disease, etc.

[0108] This invention also relates to pharmaceutical compositions comprising a bicyclic peptide inhibitor conforming to formula (I) as described herein; isomers, prodrugs, solvates, stable isotopic derivatives or pharmaceutically acceptable salts thereof; or the use of the pharmaceutical composition in the preparation of a medicament for the treatment or prevention of IL23 / IL23R-mediated inflammatory, autoimmune inflammatory diseases and / or related disorders, wherein said disease or disorder is associated with an autoimmune inflammatory disease selected from ulcerative colitis (UC), Crohn's disease (CD), psoriasis (PsO), or psoriatic arthritis (PsA). Attached Figure Description

[0109] Figure 1 The inhibitory effect of compound 1 on an IL-23-induced rat skin inflammation model (intravenous administration) was demonstrated.

[0110] Figure 2The inhibitory effects of compounds 1 and 50 on an IL-23-induced rat skin inflammation model (oral administration) were demonstrated. Detailed Implementation

[0111] 1. Overview

[0112] This invention relates to novel bicyclic peptide inhibitors of interleukin-23 receptor (IL-23R) or their pharmaceutically acceptable salts, solvates and / or other forms; pharmaceutical compositions containing the same; methods and / or uses of IL-23R inhibitors for treating autoimmune inflammatory diseases and / or related disorders.

[0113] This invention relates to bicyclic peptide inhibitors of IL-23R. Compared to the corresponding monocyclic peptide inhibitors of IL-23R, the bicyclic peptide inhibitors of this invention can exhibit enhanced properties, such as lower in vivo clearance and longer in vivo half-life.

[0114] 2. Definition

[0115] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art.

[0116] When referring to values, "about" includes the value plus or minus 10%. For example, about 50% includes a range of 45% to 55%, while about 20 molar equivalents includes a range of 18 molar equivalents to 22 molar equivalents. Therefore, when referring to a range, "about" means the value at each end of the range plus or minus 10%. For example, a ratio of about 1 to about 3 (weight / weight) includes a range of 0.9 to 3.3.

[0117] The terms "patient" and "subject" are used interchangeably and refer to a living organism, including but not limited to human subjects who have or are susceptible to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Further non-limiting examples may include, but are not limited to, humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, horses, and other mammals. In some respects, a patient is a human being.

[0118] Unless otherwise stated, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions recommended by the IUPAC Committee on Organic Chemistry Nomenclature and the IUPAC-IUB Committee on Biochemistry Nomenclature, as shown in "Nomenclature of α-Amino Acids (Recommendations, 1974)," Biochemistry, Vol. 14, No. 2 (1975). Any differences in the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims from those recommendations will be made clear to the reader. In the amino acid sequences representing IL-23 inhibitors, individual amino acids are separated by hyphens "-".

[0119] Throughout this specification, unless naturally occurring amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). Unless otherwise stated, the three-letter and one-letter abbreviations for amino acids refer to the L-isomer of the amino acid in question. As used herein, the term "L-amino acid" refers to the "L" isomer of the peptide, and conversely, the term "D-amino acid" refers to the "D" isomer of the peptide (e.g., (D)Asp or D-Asp; (D)Phe or D-Phe). Any L-amino acid residue in the D-isomer can substitute for any L-amino acid residue, provided the peptide retains the desired function. When referred to using one-letter abbreviations, D-amino acids may be indicated by lowercase by convention. For example, L-arginine may be represented as "Arg" or "R," while D-arginine may be represented as "arg" or "r." Similarly, L-lysine can be represented as "Lys" or "K", while D-lysine can be represented as "lys" or "k". Alternatively, a lowercase "d" preceding the amino acid can be used to indicate that it is a D isomer; for example, D-lysine can be represented by dK. When "gE" appears in a modified aa residue, specifically a modified lysine residue (e.g., KPEG2PEG2gEC20OH or KPEG6PEG6gEC18OH), it indicates isoglutamic acid.

[0120] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g., sarcosine, ornithine, etc.), their residues are usually represented by a commonly used three- or four-character code, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutyric acid), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutyric acid), etc.

[0121] The D-isomer of an amino acid can be located at any of the positions described herein in the IL-23R inhibitors (any of X3-X15 appearing in the molecule). In one aspect, the D-isomer can be located at only one or more of X4, X5, X12, and X13, and optionally at an additional position. In other aspects, the D-isomer can be located at only one or more of X13, and optionally at an additional position.

[0122] As is conventionally understood by those skilled in the art, the peptide sequences disclosed herein are shown from left to right, with the left end of the sequence being the N-terminus of the peptide and the right end of the sequence being the C-terminus of the peptide.

[0123] The sequences disclosed herein are sequences incorporating a "-OH" or "-NH2" portion at the carboxyl terminus (C-terminus). In such cases, and unless otherwise stated, the "-OH" or "-NH2" portion at the C-terminus of the sequence indicates a hydroxyl or amino group corresponding to a carboxylic acid (COOH) or amide (CONH2) group present at the C-terminus, respectively. In each sequence of the invention, the C-terminal "-OH" portion may replace the C-terminal "-NH2" portion, and vice versa.

[0124] Those skilled in the art will understand that certain amino acids and other chemical moieties are modified when bound to another molecule. For example, an amino acid side chain can be modified when it forms an intramolecular bridge with another amino acid side chain; for instance, one or more hydrogen atoms may be removed or replaced by bonds.

[0125] "Compounds of the present invention", "inhibitors of the present invention", "IL-23R inhibitors of the present invention", "compounds described herein" and "compounds described herein" include novel compounds disclosed herein, such as compounds of any of the embodiments in the examples, including compounds of formula (I), such as those seen in Table 1.

[0126] "Pharmaceutical effective amount" refers to the amount of the compound of the invention in a composition or combination thereof that provides the desired therapeutic or pharmaceutical outcome.

[0127] The term "pharmaceuticalally acceptable" means that the carrier, diluent, salt, or excipient must be compatible with the other components or ingredients of the composition of the present invention, i.e., useful, safe, and non-toxic for pharmaceutical use. According to the present invention, pharmaceutically acceptable means approved or permitted for use in animals, and more specifically in humans, as listed in the United States Pharmacopeia or other recognized pharmacopoeias.

[0128] "Pharmaceutical acceptable excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for acceptable use in humans or livestock.

[0129] "Absorption enhancer" refers to a component that improves or promotes the mucosal absorption of a drug in the gastrointestinal tract, such as a permeation enhancer or intestinal permeation enhancer. As conventionally understood in the art, a permeation enhancer (PE) is an agent designed to improve the oral delivery of a therapeutic drug with poor bioavailability. PE can increase the intercellular and / or transcellular channels of the drug.

[0130] Drug excipients that can increase permeability have been referred to as “absorption-modifying excipients” (AMEs). AMEs can be used in oral compositions, for example as wetting agents (sodium dodecyl sulfate), antioxidants (e.g., EDTA), and emulsifiers (e.g., polyethylene glycol glyceride), and can be included in compositions, particularly as PEs, to improve bioavailability. PEs can be classified according to how they alter barrier integrity via paracellular or transcellular pathways. “Intestinal permeability enhancers (IPEs)” refer to components that improve the bioavailability of a component. Suitable representative IPEs for use in this invention include, but are not limited to, various surfactants, fatty acids, medium-chain glycerides, steroidal detergents, acylcarnitines and alkylcholines, N-acetylated α-amino acids and N-acetylated non-α-amino acids, as well as deacetylated chitosans, other mucosal adhesion polymers, etc. For example, suitable IPEs for use in this invention may be sodium decanoate (NaC10), sodium caprylate (NaC8), or polyethylene glycol glyceride caprylate (Labrasol).

[0131] As used herein, the terms "composition" or "pharmaceutical composition" are intended to cover inventions or products containing a specific active product ingredient (API) that may contain specific amounts of pharmaceutically acceptable excipients, carriers, or diluents as described herein, such as those defined throughout the invention. A composition or pharmaceutical composition is produced by a combination of specific components, such as specific amounts of specific ingredients as described herein.

[0132] The compositions or pharmaceutical compositions of the present invention can be in various pharmaceutically acceptable forms, including but not limited to liquid compositions, tablet or matrix compositions, capsule compositions, etc. When the composition is a tablet composition, the tablet may include, but is not limited to, different layers, two or more different phases, including an inner phase and an outer phase that may contain a core. The tablet composition may also include, but is not limited to, one or more coatings.

[0133] As used herein, "solvent" means the physical association of the compound of the present invention with one or more solvent molecules. This physical association involves varying degrees of bonding, including hydrogen bonding. In some cases, the solvate will be separable. The term "solvent" is intended to cover both solution-phase solvates and separable solvates. Non-limiting examples of suitable solvates include hydrates.

[0134] Pharmaceutically acceptable salts and tautomer forms of the compounds described herein are also provided. “Pharmaceutically acceptable” or “physiologically acceptable” means compounds, salts, compositions, dosage forms and other materials that can be used to prepare pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0135] The IL-23R inhibitors of the present invention, or their pharmaceutically acceptable salts or solvates, may contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomers, which, in absolute stereochemistry, may be defined as (R)- or (S)-, or for amino acids, as (D)- or (L)-. The present invention is intended to include all such possible isomers of the IL-23R inhibitors of the present invention, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Likewise, all tautomeric forms are also intended to be included. When a compound is expressed in its chiral form, it should be understood that this aspect covers, but is not limited to, a specific diastereomer or enantiomer-enriched form. When chirality is not specified but present, it should be understood that this aspect relates to a specific diastereomer or enantiomer-enriched form; or a racemic or isotropic mixture of such compounds.

[0136] A "racemate" is a mixture of enantiomers. The mixture may contain equal or unequal amounts of each enantiomer.

[0137] One or more “stereoisomers” refer to compounds with one or more stereocenters that are chiral. Stereoisomers include enantiomers and diastereomers. If a compound has one or more asymmetric centers or has asymmetrically substituted double bonds, it can exist in stereoisomeric form and thus can be produced as individual stereoisomers or as mixtures. Unless otherwise stated, this description is intended to include individual stereoisomers as well as mixtures. Methods for determining stereochemistry and separating stereoisomers are well known in the art (see, for example, Chapter 4 of Advanced Organic Chemistry, 4th Edition, J. March, John Wiley and Sons, New York, 1992).

[0138] "Tautomers" refer to alternating forms of compounds with different proton positions, such as enol-ketone and imine-enamine tautomers, or tautomers of heteroaryl groups containing ring atoms connected to both ring-NH- and ring=N-, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetraazoles.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly or conventionally understood by one of ordinary skill in the art. In the field of chemistry, dashes at the beginning or end of chemical groups are for convenience; chemical groups may or may not be depicted with one or more dashes without losing their ordinary meaning. Wavy lines drawn through the structure indicate the junctions of groups. Dashed lines indicate optional bonds. Unless chemically or structurally required, the order in which chemical groups are written or the points where they connect to the rest of the molecule do not indicate or imply directionality. For example, the group “-SO2CH2-” is equivalent to “-CH2SO2-” and both can be connected in either direction. Similarly, an “arylalkyl” group may, for example, be connected to the rest of the molecule at the aryl or alkyl portion of the group. Prefixes such as “Cu-v” or (Cu-Cv) indicate that the following group has u to v carbon atoms. For example, both “C1-6 alkyl” and “C1-C6 alkyl” indicate that the alkyl group has 1 to 6 carbon atoms.

[0140] As used herein, "fatty acid" is a non-branched alkyl acid with a length of at least six carbons, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more carbons. Fatty acids may contain 1, 2, 3 or more carboxylic acid groups. Fatty acids may contain other functional groups, such as, but not limited to, amides and phenyl rings. Exemplary fatty acids include hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 1,6-adipic acid, 1,8-octanoic acid, 1,10-sebacic acid, 1,12-dodecanoic acid, 1,14-tetradecanoic acid, 1,16-hexadecanoic acid, and 1,18-octadecanoic acid.

[0141] In this invention, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 20 carbon atoms, such as straight-chain and branched groups with 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, and various branched isomers thereof. The alkyl group may be optionally substituted or unsubstituted.

[0142] In this invention, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic hydrocarbon group comprising 3 to 12 ring atoms, for example, 3 to 12, 5 to 10, 3 to 10, 3 to 8, or 3 to 6 ring atoms. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2,2,1]heptane, etc. Cycloalkyl can be optionally substituted or unsubstituted.

[0143] In this invention, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group comprising 3 to 20 ring atoms, for example, 3 to 16, 3 to 12, 3 to 10, 3 to 8, or 4 to 6 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), but excluding ring portions of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 10 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, the heterocyclic group comprises 3 to 8 ring atoms, more preferably 4 to 6 ring atoms, and most preferably a 4-membered, 5-membered, or 6-membered ring; wherein 1 to 4 are heteroatoms, more preferably 1 to 3 are heteroatoms, and most preferably 1 to 2 are heteroatoms. Non-limiting examples of heterocyclic groups include oxetane, oxetanehexane, azirone, morpholino, 2-morpholino, dihydropyrazolyl, etc. The heterocyclic group can be optionally substituted or unsubstituted.

[0144] In this invention, the term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group, a polycyclic (i.e., a ring with adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being the most preferred. The aryl group can be substituted or unsubstituted.

[0145] In this invention, the term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms include oxygen, sulfur, and nitrogen. Preferably, the heteroaryl comprises 1 to 3 heteroatoms, and the contained heteroatoms include at least one nitrogen atom. Preferably, the heteroaryl is 5 to 10-membered. More preferably, the heteroaryl is 5- or 6-membered. Preferred heteroaryl groups include, for example, pyrazolyl, imidazolyl, triazolyl (including 1,2,3-triazolyl, 1,2,4-triazolyl, etc.), thiazolyl, pyrazinyl, oxazolyl, isoxazolyl, pyridinyl, etc. The heteroaryl can be optionally substituted or unsubstituted.

[0146] In this invention, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0147] "Polyethylene glycol" or "PEG" is a monovalent polyether group of the general formula -(O-CH2-CH2)n-OH, or a divalent group of the formula -(O-CH2-CH2)nO-, where n is an integer greater than 1. When followed by a number, PEG indicates the number of repeating units in the linking radical portion. For example, PEG3 may correspond to the divalent group of the formula -(O-CH2-CH2)3-O-, while PEG8 may correspond to the monovalent group of the formula -(O-CH2-CH2)8-OH.

[0148] PEG is prepared by the polymerization of ethylene oxide and is commercially available in the molecular weight range of 300 Da to 10,000,000 Da. Low molecular weight PEG is typically obtained as pure oligomers, referred to as monodisperse, homogeneous, or discrete. These are used in some aspects of the present invention. In some aspects, PEG is PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG18, or PEG24. In some aspects, PEG is PEG2, PEG6, or PEG24.

[0149] As used herein, “treatment / treat / treating” refers to a method for achieving a beneficial or desired outcome. For the purposes of this invention, beneficial or desired outcomes include, but are not limited to, relieving symptoms and / or reducing the severity of symptoms and / or preventing the worsening of symptoms associated with a disease or condition. In one aspect, “treatment / treating” includes one or more of the following: (a) suppressing a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition, and / or reducing the severity of a disease or condition); (b) slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, delaying the worsening or progression of a disease or condition); and (c) alleviating a disease or condition, such as causing the disappearance of clinical symptoms, improving the disease state, delaying the progression of the disease, improving quality of life, and / or prolonging survival.

[0150] As used herein, “therapeutic effective amount” or “effective amount” means an amount that effectively elicits the desired biological or medical response, including amounts of compounds sufficient to affect such treatment of a disease when administered to a subject. Effective amounts will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. Effective amounts may include a range of amounts. As understood in the art, an effective amount may be one or more doses; that is, a single or multiple doses may be required to achieve the desired therapeutic endpoint. Effective amounts may be considered in the case of administration of one or more therapeutic agents, and may be considered in combination with one or more other agents if the desired or beneficial outcome can be achieved or would be achieved. Due to the combined effects of compounds (e.g., additive or synergistic effects), the appropriate dose of any co-administered compounds may be optionally reduced.

[0151] As used herein, “co-administration” means administering a unit dose of the compound disclosed herein before or after administering a unit dose of one or more adjunctive therapeutic agents, for example, within seconds or minutes of administering one or more adjunctive therapeutic agents. For example, in some aspects, a unit dose of the compound of the invention is administered first, followed by a unit dose of one or more adjunctive therapeutic agents within seconds or minutes. Alternatively, in other aspects, a unit dose of one or more adjunctive therapeutic agents is administered first, followed by a unit dose of the compound of the invention (occurring in the enteric phase) or parenteral administration (usually by injection, infusion, or implantation, etc.) within seconds or minutes.

[0152] When referring to this invention, “systemically active” peptide pharmacology generally refers to treatment performed by means of a pharmaceutical composition comprising a peptide active ingredient, wherein the peptide resists immediate metabolism and / or excretion, resulting in its exposure to various body tissues and organs, such as the cardiovascular, respiratory, gastrointestinal, nervous, or immune systems.

[0153] The systemic pharmacological activity in this invention also refers to the use of substances that travel through the bloodstream, reach and affect cells in various body tissues and organs for treatment. Systemic active drugs are transported to their sites of action and exert their effects throughout the body to attack the physiological processes that cause inflammatory diseases.

[0154] Bioavailability refers to the extent and rate at which the active portion (drug or metabolite) enters the systemic circulation and reaches its site of action. The bioavailability of a drug is influenced by the properties of the dosage form, which in part depends on its design and manufacturing.

[0155] As used in this article, “digestive tract tissues” refers to all tissues that make up the digestive tract organs. For example, but not limited to, “digestive tract tissues” includes the tissues of the mouth, esophagus, stomach, small intestine, large intestine, duodenum, and anus.

[0156] 3. Compounds

[0157] This invention relates to novel bicyclic peptide inhibitors of interleukin-23 receptor (IL-23R) or pharmaceutically acceptable salts thereof.

[0158] Specifically, the present invention relates to bicyclic peptide inhibitor compounds of interleukin-23 receptor (IL-23R) or pharmaceutically acceptable salts thereof, wherein the structure of each compound is as identified in Table 1 of this specification.

[0159] On the one hand, bicyclic peptide inhibitors of interleukin-23 receptor (IL-23R) compounds or pharmaceutically acceptable salts thereof have the structures of the compounds in Table 1.

[0160] 4. Synthesis

[0161] The compounds described herein can be synthesized using many techniques known to those skilled in the art. In some respects, the monomeric subunits are synthesized and purified using the techniques described in the appended examples.

[0162] In some aspects, the present invention provides a method for generating the compounds of the invention (or their monomeric subunits), the method comprising chemically synthesizing a peptide having the amino acid sequences described herein, including but not limited to any amino acid sequences of formula (I) herein, or those set forth in the compounds of Table 1. In some aspects, the method for generating the compound further comprises cyclizing the compound precursor after the component subunits have been linked. In certain aspects, cyclization is performed via any of the various methods described herein.

[0163] The present invention may include, but is not limited to, polynucleotides and vectors (e.g., expression vectors) encoding a portion of the amino acid sequence of compounds described herein, such as those in the appended examples and Table 1.

[0164] This invention also describes the synthesis of bicyclic compounds such as those of formula (I) and the compounds in Table 1. In some aspects, one or more amino acid residues or amino acid monomers are esterified and then covalently linked to each other to form the compounds of this invention.

[0165] In some respects, one or more of the amino acid residues or amino acid monomers are covalently linked to each other and esterified in an intermediate oligomer stage prior to the attachment of additional amino acids and cyclization to form the compounds of the present invention.

[0166] In some aspects, cyclic peptides are synthesized and then bicyclized to form the compounds of the present invention. Exemplary synthetic methods are described in the following examples.

[0167] The present invention also describes the synthesis of compounds such as those of formula (I) described herein. Exemplary synthetic methods are described in the following examples.

[0168] 5. Pharmaceutical Composition

[0169] This invention relates to pharmaceutical compositions comprising the IL-23R inhibitor of the present invention.

[0170] The present invention includes pharmaceutical compositions comprising one or more inhibitors of the present invention, and pharmaceutically acceptable carriers, diluents or excipients.

[0171] Pharmaceutically acceptable carriers, diluents, or excipients can be solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation adjuvant. Antimicrobial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolic sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride.

[0172] The pharmaceutical composition can be administered orally, parenterally, intracisionally, intravaginally, intraperitoneally, rectally, topically (e.g., via powder, ointment, drops, suppositories, or transdermal patches), by inhalation (e.g., nasal spray), by ocular application (e.g., intraocular), or buccally. As used herein, the term "parenterical" refers to a mode of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injection and infusion. Therefore, in some embodiments, the composition is formulated for delivery via any of these routes of administration. The pharmaceutical composition can be formulated for oral and oral administration. The pharmaceutical composition can be formulated for parenterical and parenteral administration.

[0173] In a particular aspect, the IL-23R inhibitor of the present invention is suspended in a sustained-release matrix. As used herein, the sustained-release matrix is ​​a matrix made by enzymatic or acid-base hydrolysis or by dissolving a biodegradable material (typically a polymer). Once inserted into the body, the matrix is ​​acted upon by enzymes and body fluids. The sustained-release matrix is ​​preferably selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolic acid (polymer of glycolic acid), polylactide-co-glycolic acid (copolymer of lactic acid and glycolic acid), polyanhydride, poly(orthocyanin), polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids (such as phenylalanine, tyrosine, isoleucine), polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and silicone. One embodiment of the biodegradable matrix is ​​a matrix of any one of polylactide, polyglycolic acid, or polylactide-co-glycolic acid (copolymer of lactic acid and glycolic acid).

[0174] As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterionic form of a compound or peptide of the present invention that is water-soluble, oil-soluble, or dispersible, suitable for treating diseases without adverse toxicity, irritation, or allergic reactions; that satisfies a reasonable benefit / risk ratio, and that is effective for its intended use. The salt may be prepared during the final isolation and purification of the compound or by reacting the amino group separately with a suitable acid. "Pharmaceutically acceptable salt" includes salts derived from inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, phosphoric acid, formic acid, acetic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glycolic acid, salicylic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, malonic acid, trifluoroacetic acid, trichloroacetic acid, naphthalene-2-sulfonic acid, etc. Other examples of pharmaceutically acceptable salts are described in Berge et al., “Pharmaceutically acceptable salts”, J. Pharm. Sci., 1977, 66, 1-19, and are obvious to medicinal chemists that the salts are substantially nontoxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion.

[0175] The pharmaceutically acceptable salts of this invention can be synthesized by conventional chemical methods.

[0176] Generally, salts can be prepared by reacting a free base or acid with an equistoichiometric or excess amount of an acid (inorganic or organic) or base in a suitable solvent or solvent combination.

[0177] This invention relates to pharmaceutical compositions comprising the IL-23R inhibitor of the present invention or a pharmaceutically acceptable salt, isomer, or mixture thereof, wherein one to n hydrogen atoms bonded to a carbon atom may be replaced by a deuterium atom or D, where n is the number of hydrogen atoms in the molecule. As is known in the art, a deuterium atom is a non-radioactive isotope of hydrogen. Such compounds can increase resistance to metabolism and are therefore used to increase the half-life of the compounds described herein or their pharmaceutically acceptable salts, isomers, or mixtures when administered to mammals. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized by methods known in the art, for example by using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0178] Examples of isotopes that can be incorporated into the compounds disclosed in this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as... 2H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、 123 I and 125 I. Positron-emitting isotopes (such as...) 11 C 18 F, 15 O and 13 N) substitution can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of formula (I) can generally be prepared using conventional techniques known to those skilled in the art or by methods similar to those described in the examples set forth below, using a suitable isotopically labeled reagent instead of the previously employed unlabeled reagent.

[0179] In some respects, pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders intended for reconstitution into sterile injectable solutions or dispersions prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, β-cyclodextrin, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Suitable flowability can be maintained, for example, by utilizing coating materials such as lecithin in the case of dispersions, and by utilizing surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Extended absorption in injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0180] Injectable reservoir forms include those made by forming a microencapsulated matrix of peptide inhibitors within one or more biodegradable polymers, such as polylactide-polyglycol, poly(orthoester), poly(anhydride), and (poly)diols, such as PEG. The release rate of the peptide inhibitor can be controlled depending on the peptide-to-polymer ratio and the properties of the specific polymer used. Long-acting injectable formulations can also be prepared by encapsulating the peptide inhibitor in tissue-compatible liposomes or microemulsions.

[0181] Injectable formulations may be sterilized, for example, by filtration through a bacterial retention filter or by incorporation of a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media just before use.

[0182] Topical application includes application to the skin or mucous membranes (including the surfaces of the lungs and eyes). For topical pulmonary application, physically acceptable and metabolizable lipids are used. In addition to the peptide inhibitors of this invention, the compositions of this invention in liposome form may also contain stabilizers, preservatives, excipients, etc. In some embodiments, the lipids comprise phospholipids, including natural and synthetic phosphatidylcholine (lecithin) and serine. Methods for forming liposomes are known in the art.

[0183] The pharmaceutical compositions suitable for parenteral administration in the methods or uses described herein may include sterile aqueous solutions and / or suspensions of IL-23R inhibitors that are isotonic with the recipient’s blood, typically prepared using sodium chloride, glycerol, glucose, mannitol, sorbitol, etc.

[0184] This invention provides pharmaceutical compositions for oral delivery. The compositions and peptide inhibitors of this invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery media described herein. Furthermore, those skilled in the art will understand that the peptide inhibitors of this invention can be modified or integrated into systems or delivery media not disclosed herein, but are well known in the art and compatible for oral delivery of peptides.

[0185] Formulations intended for oral administration may contain adjuvants (e.g., resorcinol and / or nonionic surfactants such as polyoxyethylene oleyl ether and hexadecyl polyethylene ether) to artificially increase intestinal permeability, and / or enzyme inhibitors (e.g., trypsin inhibitors, diisopropyl fluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. In some embodiments, the peptide inhibitor in a solid dosage form for oral administration may be mixed with at least one additive, such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. These formulations intended for oral administration may also contain other types of additives, such as inactive diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, α-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, thickeners, buffers, pH adjusters, sweeteners, flavorings, or aromas.

[0186] In certain aspects, oral dosage forms or unit doses compatible with the peptide inhibitors of the present invention may include mixtures of peptide inhibitors and non-pharmaceutical components or excipients, as well as other non-reusable materials that can be considered ingredients or packaging. Oral compositions may include at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, an oral dosage form is provided comprising an effective amount of the peptide inhibitor, wherein the dosage form includes at least one of pills, tablets, capsules, gels, pastes, beverages, syrups, ointments, and suppositories. In some cases, an oral dosage form is provided that is designed and configured to achieve delayed release of the peptide inhibitor in the small intestine and / or colon of a subject.

[0187] Tablets may contain excipients, flow aids, fillers, binders, etc. Aqueous compositions are prepared aseptically and are typically isotonic when intended for delivery via non-oral administration. The composition may optionally contain excipients, such as those described in the "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, etc. The pH of the composition is, for example, in the range of about 3 to about 11. The pH of the composition may be, for example, in the range of about 5 to 7 or about 7 to 10.

[0188] The oral pharmaceutical compositions of the present invention may comprise the IL-23R inhibitor of the present invention and may comprise an enteric coating designed to delay the release of the IL-23R inhibitor in the small intestine. The present invention relates to a pharmaceutical composition comprising the IL-23R inhibitor of the present invention and a protease inhibitor, such as aprotinin, in a delayed-release pharmaceutical formulation. The pharmaceutical composition (e.g., an oral pharmaceutical composition) may comprise an enteric coating soluble in gastric juice at a pH of about 5.0 or higher. Such enteric coatings may comprise polymers having dissociable carboxyl groups, such as cellulose derivatives including hydroxypropyl methyl phthalate, cellulose acetate phthalate, and cellulose trimellitate, as well as similar derivatives of cellulose and other carbohydrate polymers.

[0189] Oral pharmaceutical compositions comprising the IL-23R inhibitor of the present invention may include an enteric coating designed to protect and release the pharmaceutical composition in a controlled manner in the lower gastrointestinal system of a subject, avoiding systemic side effects. In addition to enteric coatings, the peptide inhibitors of the present invention may be encapsulated, coated, conjugated, or otherwise associated with any compatible oral drug delivery system or component. For example, in some embodiments, the IL-23R inhibitor of the present invention is provided in a lipid carrier system comprising at least one of the following: polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0190] To overcome peptide degradation of the IL-23R inhibitor of the present invention in the small intestine, pharmaceutical compositions may comprise a hydrogel polymer carrier system containing the peptide inhibitor of the present invention, whereby the hydrogel polymer protects the IL-23R inhibitor from the effects of proteolytic activity in the small intestine and / or colon. The IL-23R inhibitor may also be formulated for use in harmony with the carrier system, which is designed to increase dissolution kinetics and enhance intestinal absorption of the peptide. These methods include using liposomes, micelles, and nanoparticles to increase the GI channel permeability of the peptide.

[0191] Various bioreactive systems can also be combined with one or more IL-23R inhibitors of the present invention to provide agents for oral delivery. For example, the IL-23R inhibitors of the present invention can be used in combination with bioreactive systems, such as hydrogels and mucosal adhesion polymers having hydrogen-bonded groups (e.g., PEG, poly(methacrylic acid) [PMAA], cellulose, chitosan, and alginate) to provide therapeutic agents for oral administration.

[0192] In some aspects, pharmaceutical compositions and formulations may comprise the IL-23R inhibitor of the present invention and one or more absorption enhancers, enzyme inhibitors, or mucosal adhesion polymers. In one embodiment, the absorption enhancer may be an intestinal permeability enhancer.

[0193] The IL-23R inhibitor of the present invention can be formulated in a formulation medium (such as, for example, an emulsion, liposome, microsphere or nanoparticle).

[0194] This invention provides a method of treating a subject with an IL-23R inhibitor of the invention having an increased half-life. In one aspect, the invention provides a peptide inhibitor having a half-life of at least several hours to several days in vitro or in vivo (e.g., when administered to a human subject), sufficient for a therapeutically effective dose to be administered once daily (qd) or twice daily (bid).

[0195] When used in at least one of the treatment or delivery systems described herein, the peptide inhibitors of the present invention may be employed in pure form or, in the presence of such forms, in a pharmaceutically acceptable salt form.

[0196] The total daily dosage of the IL-23R inhibitor and composition of the present invention can be determined by the attending physician within a reasonable medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a number of factors, including: a) the disorder being treated and its severity; b) the activity of the specific compound used; c) the specific composition used, the patient's age, weight, general health condition, sex, and diet; d) the timing, route of administration, and excretion rate of the specific peptide inhibitor used; e) the duration of treatment; f) the drugs used in combination with or in combination with the specific peptide inhibitor used, and similar factors well known in the medical field.

[0197] In a particular embodiment, the total daily dose of the IL-23R inhibitor of the present invention, administered in single or multiple doses to a human or other mammalian host, may be, for example, an amount from 0.0001 mg / kg body weight to 300 mg / kg body weight per day or from 1 mg / kg body weight to 300 mg / kg body weight per day.

[0198] The composition can be readily available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. Techniques and compositions are commonly found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods involve the step of associating the active ingredient with a carrier constituting one or more auxiliary components. Typically, the composition is prepared by homogeneously and tightly associating the active ingredient with a liquid carrier or a finely chopped solid carrier, or both, and then shaping the product if necessary.

[0199] Compositions suitable for oral administration may exist as discrete units, each containing a predetermined amount of the active ingredient, such as as capsules, pouches, or tablets; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions. The active ingredient may also be administered as a pellet, syrup, or paste. The active ingredient may also be administered as an oral or sublingual formulation. Oral or sublingual formulations may contain the active ingredient in a matrix that releases the active ingredient for transport across the oral cavity and / or sublingual membrane. Oral or sublingual formulations may also contain a rate-controlled matrix that releases the active compound at a predetermined rate for transport across the oral cavity and / or sublingual membrane. Oral or sublingual formulations may also contain one or more compounds selected from: (i) taste maskers, (ii) enhancers, (iii) complexing agents, and mixtures thereof; and (iv) other pharmaceutically acceptable carriers and / or excipients. Enhancers may be penetration enhancers.

[0200] Tablets are prepared by compression or molding, optionally together with one or more excipients. Compressed tablets are prepared by compressing the active ingredient in a free-flowing form (such as powder or granules) optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant in a suitable machine. Molded tablets are prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and optionally formulated to provide a slow or controlled release of the active ingredient therefrom.

[0201] 6. Non-invasive detection of intestinal inflammation

[0202] The IL-23R inhibitor of the present invention can be used for the detection, assessment, and diagnosis of intestinal inflammation via microPET imaging, wherein the peptide inhibitor is labeled with a chelating group or a detectable marker as part of a non-invasive diagnostic procedure. In some embodiments, the IL-23R inhibitor of the present invention is conjugated with a bifunctional chelating agent. In some embodiments, the IL-23R inhibitor of the present invention is radiolabeled. The labeled IL-23R inhibitor is then administered to the subject orally or rectally. In some embodiments, the IL-23R inhibitor is contained in drinking water. Following ingestion of the IL-23R inhibitor, inflammation in the entire intestinal tract and digestive tract of the subject can be visualized using microPET imaging.

[0203] 7. Treatment methods and / or uses

[0204] This invention relates to methods for treating subjects with conditions or indications associated with IL-23 or IL-23R (e.g., activation of the IL-23 / IL-23R signaling pathway), wherein these methods include administering to the subject an IL-23R inhibitor disclosed herein. In one aspect, this invention relates to a method for treating subjects with conditions or indications characterized by inappropriate, deregulated, or increased IL-23 or IL-23R activity or signaling, the method comprising administering to the individual an amount of the peptide inhibitor of the invention sufficient to inhibit (partially or completely) the binding of IL-23 to IL-23R in the subject. Inhibition of IL-23 to IL-23R binding may specifically occur in a particular organ or tissue of the subject, such as the stomach, small intestine, large intestine / colon, intestinal mucosa, lamina propria, Peyer's patches, mesenteric lymph nodes, or lymphatic vessels.

[0205] This invention relates to a method of providing the peptide inhibitor described herein to a subject in need of it. The subject in need of it may be a subject who has been diagnosed with a disease or disorder related to IL-23 / IL-23R or who has been identified as being at risk of developing a disease or disorder related to IL-23 / IL-23R. The subject may be a mammal. Specifically, the subject may be a human.

[0206] Diseases or disorders that can be treated with the IL-23R inhibitor of the present invention can be autoimmune inflammatory diseases and related disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, intestinal inflammation, inflammatory bowel disease (IBD), juvenile IBD, young adult IBD, Crohn's disease, ulcerative colitis, sarcoidosis, systemic lupus erythematosus, ankylosing spondylitis (axial spondyloarthritis), psoriatic arthritis, or psoriasis. Specifically, the disease or disorder can be psoriasis (e.g., plaque psoriasis, guttate psoriasis, reversal psoriasis, pustular psoriasis, palmoplantar pustulosis, psoriasis vulgaris, or erythrodermic psoriasis), atopic dermatitis, acne atopic, ulcerative colitis, Crohn's disease, celiac disease (non-tropical stomatitis), enteropathy associated with seronegative arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis / esophagitis, colitis associated with radiation or chemotherapy, and congenital immune disorders such as leukocyte adhesion defect-1. Colitis, chronic granulomatous disease, glycogen storage disease type 1b, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Wiskott-Aldrich syndrome, pouchitis, pouchitis following rectocolic resection and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, primary biliary cirrhosis, virus-associated enteropathy, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, uveitis, or graft-versus-host disease.

[0207] This invention relates to a method or use of an IL-23R inhibitor for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of the IL-23R inhibitor of the present invention or a pharmaceutically acceptable solvate or salt thereof, or a composition thereof containing the IL-23 inhibitor of the present invention disclosed herein. In some aspects, the present invention provides a method for treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of the IL-23R inhibitor of the present invention or a pharmaceutically acceptable solvate or salt thereof, or a composition thereof. Suitable inflammatory diseases to be treated with the compounds of the present invention or pharmaceutically acceptable salts thereof, or compositions thereof, may include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), psoriasis (PsO), or psoriatic arthritis (PsA). The inflammatory disease to be treated may be inflammatory bowel disease (IBD), Crohn's disease, or ulcerative colitis. The inflammatory disease to be treated may be selected from psoriasis or psoriatic arthritis. The inflammatory disease to be treated may be psoriasis. The inflammatory disease to be treated may be psoriatic arthritis. The inflammatory disease to be treated may be IBD.

[0208] This invention relates to methods for treating inflammatory diseases in subjects in need, methods comprising administering to the subject an IL-23R inhibitor disclosed herein (e.g., a peptide inhibitor or an IL-23R of formula (I) or any of those in Table 1). The inflammatory disease may be IBD, Crohn's disease, or ulcerative colitis. In one aspect, IBD may be ulcerative colitis. In another aspect, IBD may be Crohn's disease. In another aspect, the inflammatory disease may be psoriasis (PsO) or psoriatic arthritis (PsA).

[0209] This invention relates to methods for treating inflammatory diseases in subjects in need of treatment, the methods comprising administering an IL-23R inhibitor of formula (I) to the subject. The inflammatory disease may be IBD, Crohn's disease, or ulcerative colitis. In one aspect, IBD may be ulcerative colitis. In another aspect, IBD may be Crohn's disease. In another aspect, the inflammatory disease may be psoriasis (PsO) or psoriatic arthritis (PsA).

[0210] This invention relates to methods for treating inflammatory diseases in subjects in need of treatment, the methods comprising administering an IL-23R inhibitor of formula (I) to the subject. The inflammatory disease may be IBD, Crohn's disease, or ulcerative colitis. In one aspect, IBD may be ulcerative colitis. In another aspect, IBD may be Crohn's disease. In another aspect, the inflammatory disease may be psoriasis (PsO) or psoriatic arthritis (PsA).

[0211] This invention relates to methods for treating inflammatory bowel disease (IBD) in subjects in need of treatment, comprising administering the bicyclic peptide IL-23R inhibitor of the present invention to the subject. The inflammatory disease may be IBD, Crohn's disease, or ulcerative colitis. IBD may be ulcerative colitis. IBD may be Crohn's disease. The inflammatory disease may be psoriasis (PsO) or psoriatic arthritis (PsA).

[0212] This invention relates to methods for inhibiting the binding of IL-23 to IL-23R on cells, methods comprising contacting IL-23R with a peptide inhibitor of the receptor disclosed herein. The cells may be mammalian cells. The method may be performed in vitro or in vivo. Inhibition of binding can be determined by a variety of conventional experimental methods and assays known in the art.

[0213] This invention relates to a method for selectively inhibiting IL-23 or IL-23R signaling (or the binding of IL-23 to IL-23R) in a subject (e.g., a subject in need of such inhibition), the method comprising administering to the subject a peptide inhibitor of IL-23R as described herein. The invention includes and provides a method for selectively inhibiting IL-23 or IL-23R signaling (or the binding of IL-23 to IL-23R) in the GI tract of a subject (e.g., a subject in need of such inhibition), the method comprising administering to the subject the peptide inhibitor of IL-23R of the invention by oral administration. The exposure of the GI tissue (e.g., the small intestine or colon) to the administered peptide inhibitor may be at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than the exposure level in the blood. In certain embodiments, the present invention includes a method for selectively inhibiting IL23 or IL23R signaling (or the binding of IL23 to IL23R) in the GI pathway of a subject (e.g., a subject in need), the method comprising providing the subject with a peptide inhibitor that does not block the interaction between IL-6 and IL-6R or antagonize the IL-12 signaling pathway. In another related embodiment, the present invention includes a method for inhibiting GI inflammation and / or neutrophil infiltration into the GI pathway, the method comprising providing the subject with the peptide inhibitor of the present invention. In some embodiments, the method of the present invention comprises providing the subject (e.g., a subject in need) with a combination of the peptide inhibitor of the present invention (i.e., a first therapeutic agent) and a second therapeutic agent. In some embodiments, the second therapeutic agent is provided to the subject before and / or simultaneously and / or after administration of the peptide inhibitor. In certain embodiments, the second therapeutic agent is an anti-inflammatory agent. In some embodiments, the second therapeutic agent is a nonsteroidal anti-inflammatory drug, a steroid, or an immunomodulator. In some embodiments, the method comprises administering a third therapeutic agent to the subject. In some implementations, the second therapeutic agent is an antibody that binds to IL-23 or IL-23R.

[0214] This invention relates to methods for inhibiting IL-23 signaling in cells, methods comprising contacting IL-23R with a peptide inhibitor described herein. In some embodiments, the cell is a mammalian cell. In particular embodiments, the method is performed in vitro or in vivo. In particular embodiments, inhibition of IL-23 signaling can be determined by measuring changes in phosphate-STAT3 levels in the cell.

[0215] In any of the foregoing methods, administration of an IL-23R inhibitor to a subject may be oral, but other routes of administration are not excluded. Other routes of administration include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, local, buccal, or ocular routes. The dose of the peptide inhibitor or IL-23R (e.g., a compound of formula (I) or any of those in Table 1) or its salts or solvates to be administered to the subject may be determined by a person skilled in the art considering the disease or condition being treated (including its severity) and factors (including age, weight, sex, etc.). Exemplary dose ranges include, but are not limited to, about 1 mg to about 1000 mg, or about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 10 mg to about 50 mg, about 20 mg to about 40 mg, or about 20 mg to about 30 mg. The dose range of the peptide inhibitor or IL-23R described herein may be about 600 mg to about 1000 mg. The dose range of the peptide inhibitor or IL-23R described herein may be about 300 mg to about 600 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 5 mg to about 300 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 25 mg to about 150 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 25 mg to about 100 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 1 mg to about 100 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 20 mg to about 40 mg. The dosage range of the peptide inhibitors or IL-23R described herein can be from about 20 mg to about 30 mg.

[0216] 8. Examples

[0217] The following examples illustrate the present invention. These examples are not intended to limit the scope of the invention, but rather to provide guidance to those skilled in the art for preparing and using the compounds, compositions, and methods of the invention. While specific aspects of the invention have been described, those skilled in the art will understand that various changes and modifications can be made without departing from the spirit and scope of the invention.

[0218] The following Table 3 defines some abbreviations that can be used to describe the present invention.

[0219] Table 3. Abbreviation Structure Comparison Table for Substituents

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] General peptide synthesis procedure 1

[0227] Peptides were assembled using ethyl 2-oxime cyanoacetate (Oxym) and N,N'-diisopropylcarbodiimide (DIC) coupling conditions. For some amino acid couplings, PyBOP (benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate) and DIEA (N,N-diisopropylethylamine) conditions were used. For peptides with C-terminal amides, Rink amide MBHA resin (100-200 mesh, 0.5–0.7 mmol / g) was used; for peptides with C-terminal acids, pre-packed CTC resin with N-α-Fmoc-protected amino acids was used. A 100 mmol concentration of coupling reagent (Oxym and DIC premixed) was prepared. The bicyclic peptide inhibitors of this invention were optimized based on solid-phase synthesis and medicinal chemistry, and those with superior binding and / or inhibitory properties were screened for identification.

[0228] Preparation of certain modified amino acids

[0229] Certain modified amino acids appear in the sequence of the IL-23R inhibitors described herein. Those modified amino acids suitable for synthesizing the inhibitors described herein, and their precursors, are available from commercial sources, as described in the art, or synthesized by any suitable route. The synthesis of certain additional modified amino acids is described below.

[0230] Example 1: Synthesis of the compound

[0231] Intermediate 1

[0232] 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)butyric acid

[0233]

[0234] first step

[0235] 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)butyrate methyl ester

[0236] A mixture of compound 4-((tert-butoxycarbonyl)aminomethyl)piperidine 1-1 (0.86 g, 4.00 mmol), methyl 4-bromobutyrate (1.09 g, 6.00 mmol), cesium carbonate (1.95 g, 6.00 mmol), and N,N-dimethylformamide (15 mL) was heated to 50 °C and stirred for 2 hours. After cooling to room temperature, the reaction solution was diluted with ethyl acetate (50 mL), washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was dissolved under reduced pressure. The residue was purified by silica gel column chromatography (30-70% petroleum ether / ethyl acetate) to give the target product methyl 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidine-1-yl)butyrate 1-2 (0.71 g), yield: 57%.

[0237] MS m / z(ESI): 315[M+1];

[0238] 1 H NMR(400MHz, CDCl3)δ4.60(brs,1H),3.67(s,3H),3.02-2.88(m,4H),2.35-2.3 1(m,4H),1.93-1.77(m,4H),1.71-1.65(m,2H),1.44(s,9H),1.28-1.19(m,3H).

[0239] Step 2

[0240] 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)butyric acid

[0241] 1-2 (0.71 g, 2.30 mmol) of methyl 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)butyrate were dissolved in 2 M lithium hydroxide methanol / tetrahydrofuran / water (v / v 1 / 1 / 1) solution (9 mL) and reacted at room temperature for 2 hours. The mixture was concentrated under reduced pressure, and the residue was dissolved in water (10 mL). The pH was adjusted to approximately 5 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (20 mL × 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was desolvated under reduced pressure to obtain the target product, intermediate 1 (0.48 g) of 4-(4-(((tert-butoxycarbonyl)amino)methyl)piperidin-1-yl)butyrate. Yield: 71%. MS m / z (ESI): 301 [M+1];

[0242] Intermediate 2

[0243] (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyric acid

[0244]

[0245] first step

[0246] Methyl (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyrate

[0247] A mixture of compound (S)-2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine 2-1 (0.20 g, 1.00 mmol), methyl 4-bromobutyrate (0.36 g, 2.00 mmol), cesium carbonate (0.65 g, 2.00 mmol), and tetrahydrofuran (5 mL) was heated to 40 °C and stirred for 6 hours. After cooling to room temperature, the reaction solution was diluted with ethyl acetate (15 mL), washed with saturated brine (15 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was dissolved under reduced pressure. The residue was purified by silica gel column chromatography (30-70% petroleum ether / ethyl acetate) to give the target product (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyrate 2-2 (0.18 g), yield: 60%. MS m / z (ESI): 301 [M+1];

[0248] Step 2

[0249] (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyric acid

[0250] Methyl (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyrate 2-2 (0.18 g, 0.60 mmol) was dissolved in 3 mL of 2 M lithium hydroxide methanol / tetrahydrofuran / water (1 / 1 / 1 v / v) solution and reacted at room temperature for 2 hours. The solution was concentrated under reduced pressure, and the residue was dissolved in water (5 mL). The pH was adjusted to approximately 5 with dilute hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (10 mL × 2). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was dissolved under reduced pressure to obtain the target product (S)-4-(2-(((tert-butoxycarbonyl)amino)methyl)pyrrolidine-1-yl)butyrate intermediate 2 (0.15 g). Yield: 85%.

[0251] MS m / z(ESI): 287[M+1];

[0252] 1 H NMR(400MHz, CDCl3)δ5.68(s,1H),3.49-3.27(m,3H),3.18-3.01(m,2H),2.85-2. 80(m,1H),2.63-2.42(m,3H),2.09-1.99(m,1H),1.92-1.69(m,5H),1.40(s,9H).

[0253] Intermediate 3

[0254] 5-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-(2-carbonylethyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium

[0255]

[0256] first step:

[0257] 3-Bromo-5-((methylamino)methyl)phenol

[0258] 3-Bromo-5-hydroxybenzaldehyde 3-1 (10.00 g, 0.05 mol), methylamine hydrochloride (6.70 g, 0.10 mol), and triethylamine (12.60 g, 0.12 mol) were dissolved in methanol (200 mL). Sodium borohydride (3.00 g, 0.08 mol) was added at 0 °C, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water (200 mL), and most of the methanol was removed by rotary evaporation under reduced pressure. The aqueous phase was extracted with ethyl acetate (50 mL x 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and dissolved under reduced pressure to obtain the target product, crude 3-bromo-5-((methylamino)methyl)phenol 3-2 (10.00 g). MS m / z (ESI): 216 [M+1];

[0259] Step Two:

[0260] (3-Bromo-5-hydroxybenzyl)(methyl)carbamate tert-butyl ester

[0261] 3-Bromo-5-((methylamino)methyl)phenol 3-2 (10.00 g, 50 mmol) and di-tert-butyl dicarbonate (10.80 g, 50.00 mmol) were dissolved in tetrahydrofuran (150 mL), and the reaction mixture was stirred at room temperature for 16 hours. The solvent was removed by rotary evaporation under reduced pressure, and the residue was dissolved in water (200 mL). The aqueous phase was extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated brine (50 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and desolventized under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1 to 5:1) to give the target product (3-bromo-5-hydroxybenzyl)(methyl)carbamate tert-butyl 3-3 (15.00 g). Yield: 95%.

[0262] MS m / z (ESI): 260 [M-55];

[0263] 1H NMR (400MHz, CDCl3) δ6.94(s,1H),6.85(s,1H),6.66(s,1H),4.31(s,2H),2.81(s,3H),1.48(s,9H).

[0264] Step 3:

[0265] 3-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)methyl acrylate

[0266] (3-bromo-5-hydroxybenzyl)(methyl)carbamate tert-butyl 3-3 (12.50 g, 39.70 mmol), methyl acrylate (17.10 g, 198.80 mmol), palladium acetate (1.78 g, 7.95 mmol), sodium acetate (3.60 g, 43.90 mmol), and triphenylphosphine (11.40 g, 43.50 mmol) were dissolved in N,N-dimethylformamide (150 mL). The reaction mixture was stirred at 90 °C for 16 hours. The residue was dissolved in water (600 mL), and the aqueous phase was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (200 mL × 2). The organic phase was dried with anhydrous sodium sulfate, the desiccant was removed by filtration, and the crude product was obtained by desolvation under reduced pressure. The crude product was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1–5:1) to give the target product, methyl 3-(3-(((tert-butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)acrylate 3-4 (9 g). Yield: 70%.

[0267] MS m / z (ESI): 266 [M-55];

[0268] 1 H NMR (400MHz, CDCl3) δ7.59 (d, J = 15.8Hz, 1H), 6.91 (s, 1H), 6.87 (s, 1H), 6.78 (s, 1H), 6 .37(d,J=16.0Hz,1H),4.37(s,2H),3.80(s,3H),2.81(s,3H),1.48(d,J=12.0Hz,9H).

[0269] Step 4:

[0270] Methyl 3-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)propionate

[0271] Methyl 3-(3-((((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)acrylate 3-4 (9.00 g, 28.00 mmol) was dissolved in methanol (150 mL), and palladium on carbon (10% supported on activated carbon) was added. The reaction mixture was stirred at room temperature under hydrogen (15 psi) for 16 hours. The palladium on carbon was removed by filtration, and the solution was removed under reduced pressure to obtain crude methyl 3-(3-((((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)propionate 3-5 (9.00 g).

[0272] MS m / z (ESI): 224 [M-99];

[0273] 1 H NMR (400MHz, CDCl3) δ6.62-6.60(m,2H),6.55(s,1H),4.32(s,2H),3.67(s,3H),2.95-2.70(m,5H),2.59(m,2H),1.48(s,9H).

[0274] Step 5:

[0275] Methyl 3-(3-((5-bromopentyl)oxy)-5-(((tert-butyloxycarbonyl)(methyl)amino)methyl)phenyl)propionate

[0276] Methyl 3-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-hydroxyphenyl)propionate 3-5 (9.00 g, 28.00 mmol), 1,5-dibromopentane (25.50 g, 0.11 mol), and potassium carbonate (7.73 g, 56.00 mmol) were dissolved in acetonitrile (100 mL). The reaction mixture was stirred at 65 °C for 16 hours. The organic solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in water (200 mL). The aqueous phase was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 2). The organic phase was dried with anhydrous sodium sulfate, the desiccant was removed by filtration, and the crude product was dissolved under reduced pressure. The crude product was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1–10:1) to obtain the target product, methyl 3-(3-(((5-bromopentyl)oxy)-5-(((tert-butyloxycarbonyl)(methyl)amino)methyl)phenyl)propionate 3-6 (9.60 g). Yield: 73%.

[0277] MS m / z (ESI): 372 [M-99];

[0278] 1H NMR (400MHz, CDCl3) δ6.64-6.62(m,3H),4.35(s,2H),3.94(t,J=6.4Hz,2H),3.67(s,3H),3.44(t,J=6.8Hz,2H),2.90(t, J=7.8Hz,2H),2.82(m,3H),2.61(t,J=7.8Hz,2H),1.97-1.90(m,2H),1.87-1.73(m,2H),1.71-1.56(m,2H),1.48(s,9H).

[0279] Step 6:

[0280] 5-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-(3-methoxy-3-propionyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium

[0281] Methyl 3-(3-(((5-bromopentyl)oxy)-5-(((tert-butyloxycarbonyl)(methyl)amino)methyl)phenyl)propionate 3-6 (9.60 g, 20.40 mmol) and trimethylamine (30.6 mL, 61.2 mmol) were dissolved in acetonitrile (100 mL), and the reaction mixture was stirred at 50 °C for 16 hours. The organic solvent was removed by rotary evaporation under reduced pressure, and the residue was dissolved in water (200 mL). The aqueous phase was extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated brine (50 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and dissolved under reduced pressure to obtain the target product 5-(3-(((tert-butyloxycarbonyl)(methyl)amino)methyl)-5-(3-methoxy-3-propionyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium 3-7 crude product (9.6 g). MS m / z (ESI): 451 [M];

[0282] Step 7:

[0283] 5-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-(2-carbonylethyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium

[0284] 9.60 g (20.40 mmol) of 5-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-(3-methoxy-3-propionyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium 3-7 was dissolved in tetrahydrofuran (20 mL), and 4 M lithium hydroxide aqueous solution (20 mL) was added. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was concentrated under reduced pressure, and the residue was adjusted to pH approximately 5 with 1 M hydrochloric acid. The aqueous phase was extracted with ethyl acetate (50 mL × 3), and the combined organic phases were washed with saturated brine (50 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and dissolved under reduced pressure to obtain the crude product. The target product, 5-(3-(((tert-Butoxycarbonyl)(methyl)amino)methyl)-5-(2-carbonylethyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium intermediate 3 (10.00 g), was prepared by liquid chromatography.

[0285] MS m / z (ESI): 437 [M];

[0286] 1 H NMR(400MHz,CD3OD)δ6.71(s,1H),6.68(s,1H),6.61(s,1H),4.36(s,2H),4.00(t,J=6.4Hz,2H),3.40-3.32(m,2H),3 .13(s,9H),2.89-2.85(m,2H),2.81(s,3H),2.58(t,J=7.6Hz,2H),1.90-1.83(m,4H),1.61-1.53(m,2H),1.47(s,9H).

[0287] The synthesis of intermediates 4-5 follows the same process as intermediate 3.

[0288] Intermediate 4

[0289] 3-(3-(((tert-Butoxycarbonyl)amino)methyl)-5-(2-carbonylethyl)phenoxy)-N,N,N-trimethylpentyl-1-ammonium

[0290]

[0291] MS m / z (ESI): 423 [M];

[0292] 1H NMR (400MHz, DMSO) δ6.66-6.63(m,3H),4.06(d,J=6.0Hz,2H),3.95(t,J=6.2Hz,2H),3.35-3.31(m,2 H),3.07(s,9H),2.76(t,J=7.6Hz,2H),2.51(t,J=7.6Hz,2H),1.78-1.71(m,4H),1.47-1.33(m,11H).

[0293] Intermediate 5

[0294] 3-(3-((tert-Butoxycarbonyl)amino)methyl)-5-((5-(dimethylamino)pentyl)oxy)phenyl)propionic acid

[0295]

[0296] MS m / z (ESI): 409 [M+1];

[0297] 1 H NMR (400MHz, DMSO) δ6.65-6.62(m,3H),4.05(d,J=6.0Hz,2H),3.96-3.91(m,2H),3.10-3 .01(m,2H),2.83-2.67(m,8H),2.55-2.44(m,2H),1.79-1.61(m,4H),1.50-1.33(m,11H).

[0298] Intermediate 6

[0299] (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-carboxy-N,N,N-trimethylhexane-1-ammonium

[0300]

[0301] Compound (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(dimethylamino)-2-methylhexanoic acid (synthetic reference WO2024003313 A1) (0.50 g, 1.22 mmol), N,N-diisopropylethylamine (0.39 g, 3.66 mmol), acetonitrile (15 mL), and methanol (15 mL) were mixed and stirred at 0 °C for 10 min. Then, iodomethane (1.73 g, 12.20 mmol) was added, and the mixture was heated to room temperature and stirred for 15 min. The solution was removed under reduced pressure to obtain the crude product, which could be used directly in the next step without purification. MS m / z (ESI): 425 [M];

[0302] Intermediate 7

[0303] (S)-N,N,N,2,2,8-Hexamethyl-4,7,10-trioxo-6-(pyridin-3-ylmethyl)-3,14,17-trioxa-5,8,11-triazanonane-19-ammonium chloride

[0304]

[0305] first step

[0306] (2-(2-(2-dimethylaminoethoxy)ethoxy)ethyl)tert-butyl carbamate

[0307] (2.48 g, 10.00 mmol) of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate 7-1 was dissolved in methanol (30 mL), and 10.00 g of 36% formaldehyde aqueous solution was added. The mixture was stirred at room temperature for 5 minutes, cooled to 0°C in an ice bath, and 6.30 g, 100 mmol of sodium cyanoborohydride was added. The mixture was then slowly heated to room temperature and reacted for 3 hours. The reaction was quenched with 20 mL of saturated ammonium chloride aqueous solution, and methanol was removed by concentration under reduced pressure. The mixture was extracted with dichloromethane (30 mL × 4), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (dichloromethane:methanol = 30:1) to give the target compound (2.20 g) of tert-butyl (2-(2-(2-dimethylaminoethoxy)ethoxy)ethyl)carbamate 7-2, with a yield of 80%.

[0308] MS m / z(ESI): 277[M+1];

[0309] 1 H NMR (400MHz, CDCl3) δ5.10-5.08(m,1H),3.83-3.63(m,6H),3.57-3.54(m,2H),3.32-3.30(m,2H),3.00-2.95(m,2H),2.66(s,6H),1.45(s,9H).

[0310] Step 2

[0311] N,N,N,2,2-Pentamethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-ammonium iodide

[0312] (2.20 g, 7.90 mmol) tert-butyl (2-(2-(2-dimethylaminoethoxy)ethoxy)ethyl)carbamate 7-2 was dissolved in acetonitrile (10 mL) and tetrahydrofuran (20 mL). Iodomethane (3.36 g, 23.80 mmol) was added, and the mixture was heated in an oil bath to 70 °C for 18 hours. Iodomethane (3.36 g, 23.80 mmol) was added again, and the reaction was continued at 70 °C for another 18 hours. The mixture was then cooled to room temperature, and N,N-diisopropylethylamine (300 mg) was added and stirred for 2 hours. The reaction solution was directly concentrated under reduced pressure to obtain the target compound N,N,N,2,2-pentamethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-ammonium iodide 7-3 (3.6 g, crude product).

[0313] MS m / z(ESI):291[M];

[0314] 1 H NMR (400MHz, DMSO-d6) δ6.84-6.81(m,1H),3.86-3.83(m,2H),3.62-3.52(m,8H),3.40-3.36(m,2H),3.12-3.11(m,9H),1.37(s,9H).

[0315] Step 3

[0316] 2-(2-Aminoethoxy)ethoxy-N,N,N-trimethylethyl-1-ammonium chloride hydrochloride

[0317] N,N,N,2,2-pentamethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-iodammonium iodide 7-3 (3.50 g crude, 7.90 mmol) was dissolved in dioxane (10 mL), and dioxane hydrochloride solution (4 M, 20 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and lyophilized to give the target compound 2-(2-aminoethoxy)ethoxy-N,N,N-trimethylethyl-1-ammonium chloride hydrochloride 7-4 (2.80 g crude). MS m / z (ESI): 191 [M];

[0318] Step 4

[0319] N,N,N,2,2,5-Hexamethyl-4,7-dioxo-3,11,14-trioxa-5,8-diazahexadecane-16-ammonium chloride

[0320] 2-(2-aminoethoxy)ethoxy-N,N,N-trimethylethyl-1-iodine hydrochloride 7-4 (2.5 g, 7.2 mmol) and 2-((tert-butoxycarbonyl)(methyl)amino)acetic acid (1.36 g, 7.2 mmol) were dissolved in N,N-dimethylformamide (25 mL), and triethylamine (1.45 g, 14.4 mmol) was added. After stirring at room temperature for 10 minutes, O-(7-azabenzene) was added. (3.28 g, 8.6 mmol)-N,N,N',N'-tetramethylurea hexafluorophosphate was reacted at room temperature for 30 min. The reaction solution was then directly purified by high performance liquid chromatography to obtain 1.85 g of the target compound N,N,N,2,2,5-hexamethyl-4,7-dioxo-3,11,14-trioxa-5,8-diazahexadecane-16-ammonium chloride 7-5 (1.85 g), yield 64%. MS m / z (ESI): 362 [M];

[0321] Step 5

[0322] N,N,N-Trimethyl-4-oxo-8,11-dioxa-2,5-diazatetane-13-ammonium chloride hydrochloride

[0323] N,N,N,2,2,5-hexamethyl-4,7-dioxo-3,11,14-trioxa-5,8-diazatetane-16-ammonium chloride 7-5 (1.85 g, 4.60 mmol) was dissolved in dioxane (10 mL), and dioxane hydrochloride solution (4 M, 10 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and lyophilized to obtain 1.7 g of the target compound N,N,N-trimethyl-4-oxo-8,11-dioxa-2,5-diazatetane-13-ammonium chloride hydrochloride 7-6 (crude product). MS m / z (ESI): 262 [M];

[0324] Step 6

[0325] (S)-N,N,N,2,2,8-Hexamethyl-4,7,10-trioxo-6-(pyridin-3-ylmethyl)-3,14,17-trioxa-5,8,11-triazanonane-19-ammonium chloride

[0326] N,N,N-trimethyl-4-oxo-8,11-dioxa-2,5-diazatridecane-13-ammonium chloride hydrochloride 7-6 (1.70 g, 4.60 mmol) and tert-butoxycarbonyl-3-(3-pyridyl)-L-alanine (1.22 g, 4.60 mmol) were dissolved in N,N-dimethylformamide (15 mL), and triethylamine (1.40 g, 14.00 mmol) was added. After stirring at room temperature for 5 minutes, O-(7-azabenzene) was added. (S)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.19 g, 5.75 mmol) was reacted at room temperature for 1 hour. The reaction solution was directly purified by reversed-phase chromatography (formic acid) to give 1.2 g of the target (S)-N,N,N,2,2,8-hexamethyl-4,7,10-trioxo-6-(pyridin-3-ylmethyl)-3,14,17-trioxa-5,8,11-triazanonane-19-ammonium chloride intermediate 7, yield 48%. MS m / z (ESI): 510 [M];

[0327] Compound 1

[0328] 3AMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E(1)-Ala-3Pal-Sarc-NH2 (hydrochloride)

[0329]

[0330] Expanding the resin: 3 g of Rink amide MBHA solid-phase resin (0.615 mmol / g loading) was transferred to a 150 mL graduated gravity column and the resin was washed three times with N,N-dimethylformamide.

[0331] first step

[0332] Coupling of Fmoc-Sarc-OH

[0333] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-Sarc-OH (1.72 g, 5.53 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) together in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 311 [M+1].

[0334] Step 2

[0335] Coupling of Fmoc-3Pal-OH

[0336] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-3Pal-OH (2.15 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.60 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 459 [M+1].

[0337] Step 3

[0338] Coupling of Fmoc-Ala-OH

[0339] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-Ala-OH (1.73 g, 5.56 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.60 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 530 [M+1].

[0340] Step 4

[0341] Coupling of Fmoc-E(tBu)-OH

[0342] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-E(tBu)-OH (2.35 g, 5.53 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.60 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 659 [M+1].

[0343] Step 5

[0344] Coupling of Fmoc-THP-OH

[0345] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-THP-OH (2.03 g, 5.53 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.60 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 786 [M+1].

[0346] Step 6

[0347] Coupling of Fmoc-2Nal-OH

[0348] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-2Nal-OH (2.42 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) together in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was sampled for pyrolysis to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 983 [M+1].

[0349] Step 7

[0350] Coupling of Fmoc-F(4-AEN(Me)2)-OH

[0351] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-F(4-AEN(Me)2)-OH (2.62 g, 5.53 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.60 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 609 [M / 2+1].

[0352] Step 8

[0353] Coupling of Fmoc-C(Trt)-OH

[0354] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-C(Trt)-OH (3.24 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 660.5 [M / 2+1].

[0355] Step 9

[0356] Coupling of Fmoc-K(Ac)-OH

[0357] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-K(Ac)-OH (2.27 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 745.5 [M / 2+1].

[0358] Step 10

[0359] Coupling of Fmoc-W(7-Me)-OH

[0360] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-W(7-Me)-OH (2.43 g, 5.52 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 845.5 [M / 2+1].

[0361] Step 11

[0362] Coupling of Fmoc-T(t-Bu)-OH

[0363] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-T(t-Bu)-OH (2.2 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 896 [M / 2+1].

[0364] Step Twelve

[0365] Coupling of Fmoc-Ala(3-cyclopropane)-OH

[0366] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-Ala(3-cyclopropane)-OH (1.94 g, 5.53 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 951.5 [M / 2+1].

[0367] Step Thirteen

[0368] Coupling of Fmoc-C(Trt)-OH

[0369] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve Fmoc-C(Trt)-OH (3.24 g, 5.54 mmol) and ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol) in 50 mL of N,N-dimethylformamide. Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was taken for pyrolysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next step of deprotection / coupling cycle was started. MS m / z (ESI): 1003 [M / 2+1].

[0370] Step Fourteen

[0371] Coupling of 3-(3-((tert-Butoxycarbonylamino)methyl)phenyl)propionic acid

[0372] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve 3-(3-((tert-butoxycarbonylamino)methyl)phenyl)propionic acid (1.55 g, 5.54 mmol) in 50 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (1.18 g, 8.31 mmol). Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.93 g, 7.38 mmol) under shaking for 15 minutes, then add it to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.58 g, 4.6 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was pyrolyzed to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, followed by five washes with dichloromethane. The resin was placed under vacuum for 1.5 hours, and then pyrolyzed with trifluoroacetic acid. MS m / z (ESI): 972.5 [M / 2+1].

[0373] Step 15

[0374] 3AMPh2EtCO-C-Ala(3-cyclopropyl)-TW(7-Me)-K(Ac)-CF(4-AEN(Me)2)-2Na l-THP-E-Ala-3Pal-Sarc-NH2

[0375] 100 mL of trifluoroacetic acid lysis mixture (90 / 5 / 2.5 / 2.5, trifluoroacetic acid / water / triisopropylsilane TIS / 2,2′-(1,2-ethylenedioxy)diethylthiol DODT) was added to a tube containing the protected resin-bound peptide and shaken for two hours. The used resin was filtered off, and the filtrate was evaporated to dryness and transferred to a centrifuge tube for precipitation. Isopropyl ether (50 mL) was added to the centrifuge tube, forming a white precipitate, which was then centrifuged. The isopropyl ether was decanted into waste, and the precipitate was washed with isopropyl ether (50 mL x 2). The resulting white precipitate filter cake was evaporated to dryness to obtain the crude reduced peptide. MS m / z (ESI): 972.4 [M / 2+1].

[0376] Step Sixteen

[0377] 3AMPh2EtCO-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E-Ala-3Pal-Sarc-NH2

[0378] Dissolve approximately 3g of crude peptide in 1000mL of 20% acetonitrile / water. While stirring, add dropwise a saturated acetic acid / methanol solution of iodine to the 1000mL peptide solution until the yellow / brown color of elemental iodine is maintained and does not gradually disappear. Allow the pale yellow solution to stand for 5 minutes, then quench any excess iodine with a small amount of ascorbic acid.

[0379] Reverse chromatography purification of monocyclic peptides (disulfide bonds)

[0380] 1 L of quenched oxidized peptide was directly loaded onto the equilibrated reverse-phase column at a flow rate of 50 mL / min using 100% mobile phase A (0.1% aqueous trifluoroacetic acid). The column was washed for 5 min with 5% mobile phase B (100% acetonitrile) / A (0.1% aqueous trifluoroacetic acid). Separation was achieved over 60 min using a linear gradient of 5%–50% at 50 mL / min. The desired oxidized peptide eluted at a gradient of approximately 35%. The products were combined and lyophilized to give 1.6 g of purified oxidized peptide in the form of trifluoroacetate, with a yield of 44.7%.

[0381] Characterization

[0382] After lyophilization, the purity was >78% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a tricharged ion [M / 3+1] of 648.+ And the double-charged ion [M / 2+1] of 971.3 + .

[0383] Step Seventeen

[0384] 3AMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E(1)-Ala-3Pal-Sarc-NH2

[0385] The purified oxidized intermediate peptide (1.6 g, 0.82 mmol) was dissolved in 500 mL of N,N-dimethylformamide. 1H-benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (853 mg, 1.64 mmol) was added, followed by N,N-diisopropylethylamine (530 mg, 4.10 mmol). The mixture was stirred at room temperature, and the reaction was monitored by low-resolution liquid chromatography-mass spectrometry (LC-MS). The reaction was completed within 10 minutes, and the sample was then loaded onto a reverse-phase column for purification.

[0386] Reverse-phase column purification of dicyclic peptides (disulfide and lactam bonds)

[0387] Purification was performed using the same procedure as described previously in step 17. The products were combined and lyophilized to give 320 mg of purified bicyclic peptide in the form of trifluoroacetate, with a lactam bond formation step yield of 20.2% and an overall yield of 9%.

[0388] Trifluoroacetate is converted into hydrochloride.

[0389] The reverse-phase column was equilibrated with 5% acetonitrile / water at 50 mL / min (A = 0.01 M HCl aqueous solution, B = 100% acetonitrile, C = 0.04 M ammonium bicarbonate aqueous solution). The purified peptide TFA salt was dissolved in 20% acetonitrile / water. The solution was loaded onto the equilibrated column at 50 mL / min, and the solvent front was eluted. The column was washed with 5% B in solution A for 5 min. Then, it was washed with 5% B in solution C for 40 min at 50 mL / min to remove all trifluoroacetate ions from the system. It was then washed with 5% B in solution A for 10 min at 50 mL / min to remove all ammonium bicarbonate ions from the system. Finally, the peptide was eluted with a gradient of 5%–70% B in solution A over 60 min and collected in fractions. The products were combined and lyophilized to give 310 mg of the product in hydrochloride form.

[0390] Characterization

[0391] After lyophilization, the purity was >95% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a [M / 3+1] of 642.2. + And 962.5 [M / 2+1] + .

[0392] Table 4A: Synthesis of compounds 2-59 refers to the synthesis process of compound 1 (compounds 2-11 are trifluoroacetates, and compounds 12-59 are hydrochlorides).

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421] Compound 60

[0422] 3AMPh2EtCO(1)-Abu(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E(1)-Ala-3Pal-Sarc-NH2 (hydrochloride)

[0423]

[0424] first step

[0425] Coupling of Fmoc-Abu(Cl)-OH

[0426] A 20% N,N-dimethylformamide solution of 4-methylpiperidine (2 times the resin bed volume) was added to the expanded resin Fmoc-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E(1)-Ala-3Pal-Sarc-Rink (synthesized according to the experimental procedure of compound 1) and shaken for 3 to 5 minutes. The solution was then drained, and a second 2-times resin bed volume of 4-methylpiperidine solution was added and shaken again for 20 to 30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide under shaking. Fmoc-Abu(Cl)-OH (0.19 g, 0.55 mmol) and ethyl 2-oxime cyanoacetate (0.11 g, 0.83 mmol) were dissolved together in 5 mL of N,N-dimethylformamide. The acid was pre-activated by adding N,N'-diisopropylcarbodiimide (0.093 g, 0.73 mmol) under shaking for 15 minutes, and then added to the deprotected resin. After coupling for approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.05 g, 0.46 mmol) was added. Shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, and then the next deprotection / coupling cycle was started. MS m / z (ESI): 1010 [M / 2+1].

[0427] Step 2

[0428] Coupling of 3-(3-((tert-Butoxycarbonylamino)methyl)phenyl)propionic acid

[0429] Add two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the expanded resin and shake for 3 to 5 minutes. Then drain and add a second two resin bed volumes of 4-methylpiperidine solution, shaking for another 20-30 minutes. After deprotection, wash the resin three times with N,N-dimethylformamide under shaking. Dissolve 3-(3-((tert-butoxycarbonylamino)methyl)phenyl)propionic acid (0.15 g, 0.55 mmol) in 5 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.11 g, 0.83 mmol). Pre-activate the acid by adding N,N'-diisopropylcarbodiimide (0.09 g, 0.73 mmol) under shaking for 15 minutes, then add it to the deprotected resin. After coupling for approximately 15 minutes, add N,N'-diisopropylcarbodiimide (0.05 g, 0.46 mmol). Continue shaking for 8 hours. Finally, a small amount of resin was pyrolyzed to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide under shaking, followed by five washes with dichloromethane. The resin was placed under vacuum for 1.5 hours, followed by pyrolysis with trifluoroacetic acid. MS m / z (ESI): 980 [M / 2+1].

[0430] Step 3

[0431] 3AMPh2EtCO-Abu-Ala(3-cyclopropyl)-TW(7-Me)-K(Ac)-CF(4-AEN(Me)2)-2Nal-THP-E-Ala-3Pal-Sarc-NH2

[0432] 10 mL of trifluoroacetic acid lysis buffer (90 / 5 / 2.5 / 2.5, trifluoroacetic acid / water / TIS / DOTS) was added to a tube containing the protected resin-bound peptide and shaken for two hours. The used resin was filtered off, and the filtrate was evaporated to dryness and transferred to a centrifuge tube for precipitation. Isopropyl ether (20 mL) was added to the centrifuge tube, forming a white precipitate, which was then centrifuged. The isopropyl ether was decanted into waste, and the precipitate was washed with isopropyl ether (20 mL x 2). The resulting white precipitate filter cake was evaporated to dryness to obtain the crude reduced peptide. MS m / z (ESI): 980 [M / 2+1].

[0433] Step 4

[0434] 3AMPh2EtCO-Abu(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E-Ala-3Pal-Sarc-NH2

[0435] Approximately 100 mg of crude peptide was dissolved in 5 mL of 10% water / DMF. NaI (0.02 g, 0.07 mmol), Na₂CO₃ (0.50 g, 0.5 mmol), and EDTA (0.02 g, 0.07 mmol) were added at room temperature. The mixture was stirred at room temperature, and the reaction was monitored by low-resolution liquid chromatography-mass spectrometry (LC-MS). The reaction was completed within 4 hours, and the sample was then loaded onto a reverse-phase column for purification.

[0436] 1 L of quenched oxidized peptide was directly loaded onto the equilibrated reverse-phase column at a flow rate of 50 mL / min using 100% mobile phase A (0.1% aqueous trifluoroacetic acid). The column was washed for 5 min with 5% mobile phase B (100% acetonitrile) / A (0.1% aqueous trifluoroacetic acid). Separation was achieved over 60 min using a linear gradient of 5%–50% at 50 mL / min. The desired oxidized peptide was eluted at approximately 35% gradient. The products were combined and lyophilized to yield 50 mg of purified oxidized peptide in the form of trifluoroacetate.

[0437] Characterization

[0438] After lyophilization, the purity was >78% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a tricharged ion [M / 3+1] of 648. + And the double-charged ion [M / 2+1] of 971.3 + .

[0439] Step 5

[0440] 3AMPh2EtCO(1)-Abu(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-C(2)-F(4-AEN(Me)2)-2Nal-THP-E(1)-Ala-3Pal-Sarc-NH2

[0441] The purified oxidized intermediate peptide (50 mg, 0.025 mmol) was dissolved in 500 mL of N,N-dimethylformamide. 1H-benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (20 mg, 0.037 mmol) was added, followed by N,N-diisopropylethylamine (5 mg, 0.037 mmol). The mixture was stirred at room temperature, and the reaction was monitored by low-resolution liquid chromatography-mass spectrometry (LC-MS). The reaction was completed within 10 minutes, and the sample was then loaded onto a reverse-phase column for purification.

[0442] Reverse-phase column purification of dicyclic peptides (disulfide and lactam bonds)

[0443] Purification was performed using the same procedure as described previously in step 17. The products were combined and lyophilized to obtain 10 mg of purified bicyclic peptide in the form of trifluoroacetate.

[0444] Trifluoroacetate is converted into hydrochloride.

[0445] The reverse-phase column was equilibrated with 5% acetonitrile / water at 50 mL / min (A = 0.01 M HCl aqueous solution, B = 100% acetonitrile, C = 0.04 M ammonium bicarbonate aqueous solution). The purified peptide TFA salt was dissolved in 20% acetonitrile / water. The solution was loaded onto the equilibrated column at 50 mL / min, and the solvent front was eluted. The column was washed with 5% B in solution A for 5 min. Then, it was washed with 5% B in solution C at 50 mL / min for 40 min to remove all trifluoroacetate ions from the system. It was then washed with 5% B in solution A at 50 mL / min for 10 min to remove all ammonium bicarbonate ions from the system. Finally, the peptide was eluted with a gradient of 5%–70% B in solution A over 60 min and collected in fractions. The products were combined and lyophilized to give 6 mg of the product in hydrochloride form.

[0446] Characterization

[0447] After lyophilization, the purity was >95% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a [M / 3+H] concentration of 642.2. + And 962.5 [M / 2+H] + .

[0448] Compound 61

[0449] 3MAMPh2EtCO(1)-Pen(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-Pen(2)-F(4-AEN(Me)2)-2Nal-K(αMeNMe2)-E(1)-N-3Pal-Sar(CONMe2) (hydrochloride)

[0450]

[0451] first step

[0452] Coupling of Fmoc-Asp-OAll

[0453] The Rink resin (0.25 g, 0.14 mmol) in the reaction flask was washed twice with 4 mL of N,N-dimethylformamide, followed by treatment with 2.5 mL of 20% 4-methylpiperidine in N,N-dimethylformamide (Fmoc deprotected) for 10 min. The resin was then filtered and washed twice with N,N-dimethylformamide (4 mL), followed by treatment with N-methylpiperidine for 30 min. The resin was washed three more times with N,N-dimethylformamide (4 mL). Fmoc-Asp-OAll (0.17 g, 0.42 mmol) and 1-hydroxyphenyl-4,5-diphenylimidazole (90 mg, 0.62 mmol) were dissolved in N,N-dimethylformamide, and then N,N'-diisopropylcarbodiimide (71 mg, 0.56 mmol) was added. The reaction was stirred at room temperature for 15 min. The mixture was then added to the resin and shaken for 15 min. Add another batch of N,N'-diisopropylcarbodiimide (45 mg, 0.35 mmol). Shake the mixture for 5 hours. Finally, take a small amount of resin for lysis to confirm the completion of the reaction. Filter and wash three times with N,N-dimethylformamide, shaking for 5 minutes each time to obtain the resin, which can be used without further purification.

[0454] Step 2

[0455] Deprotection of allyl groups

[0456] Under nitrogen protection, 5 mL of N,N-dimethylformamide, phenylsilane (0.23 g, 2.10 mmol), and tetrakis(triphenylphosphine)palladium (17 mg, 0.02 mmol) were added to the obtained resin, and the reaction was carried out at room temperature for 12 hours. Finally, a small amount of resin was taken for lysis to confirm the completion of the reaction. The resin was filtered and washed three times with N,N-dimethylformamide, and shaken for 5 minutes to obtain the resin, which could be used directly without further purification. MS m / z (ESI): 354.1 [M+1].

[0457] Step 3

[0458] Coupling of (S)-2-amino-N-(2-(dimethylamino)-2-oxoethyl)-N-methyl-3-(pyridin-3-yl)propionamide

[0459] The resin obtained in the previous step (0.14 mmol) was washed twice with 4 mL of N,N-dimethylformamide, followed by treatment with 2.5 mL of 20% 4-methylpiperidine (Fmoc deprotection) for 10 minutes. The resin was then filtered and washed twice with 4 mL of N,N-dimethylformamide, and then treated with N-methylpiperidine for 30 minutes. The resin was then washed three more times with 4 mL of N,N-dimethylformamide. (S)-2-amino-N-(2-(dimethylamino)-2-carbonylethyl)-N-methyl-3-(pyridin-3-yl)propionamide (0.11 g, 0.42 mmol, synthetic reference WO2023288017) and 1-hydroxyphenyl-4,5-diphenylimidazole (90 mg, 0.62 mmol) were dissolved in N,N-dimethylformamide, and then N,N'-diisopropylcarbodiimide (71 mg, 0.56 mmol) was added. The reaction was stirred at room temperature for 15 minutes. The mixture was added to the resin and shaken for 15 minutes. Then, another batch of N,N'-diisopropylcarbodiimide (45 mg, 0.35 mmol) was added. The reaction was shaken for 5 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and shaken for 5 minutes to obtain the resin, which could be used without further purification. MS m / z (ESI): 600.2 [M+1].

[0460] Step 4

[0461] Coupling of Fmoc-E(tBu)-OH

[0462] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-E(tBu)-OH (0.77 g, 1.80 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.38 g, 2.70 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.30 g, 2.40 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.60 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.19 g, 1.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly in the next step. MS m / z (ESI): 730.3 [M+1].

[0463] Step 5

[0464] Coupling of Fmoc-K(αMeNMe2)-OH

[0465] Deprotection of the FMOC groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-K(αMeNMe2)-OH (0.74 g, 1.80 mmol) and ethyl 2-oxime cyanoacetate (0.38 g, 2.70 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Acid pre-activation with N,N'-diisopropylcarbodiimide (0.30 g, 2.40 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.6 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.19 g, 1.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly in the next step. MS m / z (ESI): 899.8 [M+1].

[0466] Step 6

[0467] Coupling of Fmoc-2Nal-OH

[0468] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-2Nal-OH (0.79 g, 1.80 mmol) and ethyl 2-oxime cyanoacetate (0.38 g, 2.70 mmol) were dissolved together in 10 mL of N,N-dimethylformamide. Acid pre-activation with N,N'-diisopropylcarbodiimide (0.30 g, 2.40 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.6 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.19 g, 1.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 549.3 [M / 2+1].

[0469] Step 7

[0470] Coupling of Fmoc-F(4-AEN(Me)2)-OH

[0471] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-F(4-AEN(Me)2)-OH (0.85 g, 1.80 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.38 g, 2.70 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.30 g, 2.40 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.6 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.19 g, 1.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 666.3 [M / 2+1].

[0472] Step 8

[0473] Coupling of Fmoc-Pen(Trt)-OH

[0474] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-Pen(Trt)-OH (0.37 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol, 4.5 equivalence) were dissolved in 10 ml of N,N-dimethylformamide. Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 731.9 [M / 2+1].

[0475] Step 9

[0476] Coupling of Fmoc-K(Ac)-OH

[0477] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-K(Ac)-OH (0.25 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 816.9 [M / 2+1].

[0478] Step 10

[0479] Coupling of Fmoc-W(7-Me)-OH

[0480] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-W(7-Me)-OH (0.26 g, 0.60 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 917.0 [M / 2+1].

[0481] Step 11

[0482] Coupling of Fmoc-T(tBu)-OH

[0483] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-T(tBu)-OH (0.24 g, 0.60 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 967.5 [M / 2+1].

[0484] Step Twelve

[0485] Coupling of Fmoc-Ala(3-cyclopropane)-OH

[0486] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-Ala(3-cyclopropane)-OH (0.21 g, 0.60 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 1023.0 [M / 2+1].

[0487] Step Thirteen

[0488] Coupling of Fmoc-Pen(Trt)-OH

[0489] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-Pen(Trt)-OH (0.37 g, 0.60 mmol) was dissolved in 10 mL of N,N-dimethylformamide along with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.2 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 1088.5 [M / 2+1].

[0490] Step Fourteen

[0491] Coupling of Boc-MAMPh2EtCO-OH

[0492] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution to the swollen resin and shaking for 3–5 minutes, then draining. This process was repeated by adding a second two resin bed volumes of 20% 4-methylpiperidine N,N-dimethylformamide solution and shaking for another 20–30 minutes, followed by draining. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Boc-MAMPh2EtCO-OH (0.09 g, 0.30 mmol) and ethyl 2-oxime cyanoacetate (0.07 g, 0.45 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Acid pre-activation with N,N'-diisopropylcarbodiimide (0.05 g, 0.40 mmol) was added for approximately 15 minutes, followed by addition to the resin (0.1 mmol). After shaking for 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed three times with N,N-dimethylformamide and used directly for the next step. MS m / z (ESI): 1064.8 [M / 2+1].

[0493] Step 15

[0494] TFA cleavage and ether precipitation

[0495] Prepare 5 mL of a TFA lysis mixture (90 / 5 / 2.5 / 2.5, TFA / water / TIPS / DODT). Add 5 mL of the mixture to a test tube containing the peptide bound to the protective resin and shake for two hours. Filter off the waste resin, add isopropyl ether to the filtrate to form a white precipitate, and then centrifuge the white precipitate. Discard the isopropyl ether into the waste and wash the precipitate twice more with isopropyl ether. Desolvate under reduced pressure to obtain the crude reduced peptide, which is used directly in the next reaction without purification. MS m / z (ESI): 1064.8 [M / 2+1].

[0496] Step Sixteen

[0497] Oxidation of disulfides

[0498] Dissolve 0.20 g of crude reduced peptide in acetonitrile (10 mL) and water (30 mL). While stirring, add a saturated solution of iodine in acetic acid / methanol dropwise to 40 mL of peptide solution until the yellow / brown color of elemental iodine is maintained and does not fade. Then quench the excess elemental iodine with a small amount of ascorbic acid.

[0499] Step Seventeen

[0500] Reverse chromatography purification of monocyclic peptides (disulfide bonds)

[0501] 40 mL of quenched oxidized peptide was directly loaded onto the equilibrated reverse-phase column at a flow rate of 50 mL / min using 100% mobile phase A (0.1% aqueous trifluoroacetic acid). The column was washed for 5 min with 5% mobile phase B (100% acetonitrile) / A (0.1% aqueous trifluoroacetic acid). Separation was achieved over 60 min using a linear gradient of 5%–50% at 50 mL / min. The desired oxidized peptide eluted at a gradient of approximately 35%. The products were combined and lyophilized to give 110.0 mg of purified oxidized peptide in the form of trifluoroacetate, with a yield of 55.0%.

[0502] Step 18

[0503] Characterization

[0504] After lyophilization, the purity was >90% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a tricharged ion [M / 3+1] at 709.5. + And the double-charged ion [M / 2+1] of 1063.7 + .

[0505] Step 19

[0506] Synthesis of 3MAMPh2EtCO(1)-Pen(2)-Ala(3-cyclopropane)-TW(7-Me)-K(Ac)-Pen(2)-F(4-AEN(Me)2)-2Nal-K(αMeNMe2)-E(1)-N-3Pal-Sar(CONMe2)

[0507] The purified oxidized intermediate peptide (110.0 mg, 0.05 mmol) was dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (10 mg, 0.08 mmol) and benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (32 mg, 0.06 mmol) were added. The mixture was stirred at room temperature, and the reaction was monitored by low-resolution liquid chromatography-mass spectrometry (LC-MS). The reaction was completed within 10 minutes, and then the sample was loaded onto a reverse-phase column for purification.

[0508] Step 20

[0509] Reverse-phase column purification of dicyclic peptides (disulfide and lactam bonds)

[0510] Purification was performed using the same procedure as described previously in step 17. The products were combined and lyophilized to give 18 mg of purified bicyclic peptide in the form of trifluoroacetate, with a lactam bond formation step yield of 16.4% and an overall yield of 9%.

[0511] Step Twenty-One

[0512] Trifluoroacetate is converted into hydrochloride.

[0513] The reverse-phase column was equilibrated with 5% acetonitrile / water at 50 mL / min (A = 0.01 M HCl aqueous solution, B = 100% acetonitrile, C = 0.04 M ammonium bicarbonate aqueous solution). The purified peptide TFA salt was dissolved in 20% acetonitrile / water. The solution was loaded onto the equilibrated column at 50 mL / min, and the solvent front was eluted. The column was washed with 5% B in solution A for 5 min. Then, it was washed with 5% B in solution C for 40 min at 50 mL / min to remove all trifluoroacetate ions from the system. It was then washed with 5% B in solution A for 10 min at 50 mL / min to remove all ammonium bicarbonate ions from the system. Finally, the peptide was eluted with a gradient of 5%–70% B in solution A over 60 min and collected in fractions. The products were combined and lyophilized to give 15.0 mg of the product in hydrochloride form.

[0514] Step Twenty-Two

[0515] Characterization

[0516] After lyophilization, the purity was >95% as determined by analytical HPLC. Low-resolution liquid chromatography-mass spectrometry (LC-MS) yielded a tricharged ion [M / 3+1] at 703.4. + And the double-charged ion [M / 2+1] at 1054.4. + .

[0517] Table 4B: Synthesis of compounds 62-81 (Refer to the synthesis process of compound 61; compounds 62-81 are hydrochloride salts).

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529] Compound 82

[0530] 3MAMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E(1)-Ala-3Pal-Sar(CONMe2) (hydrochloride)

[0531]

[0532] first step

[0533] Fmoc-Ala-3Pal-Sar(CONMe2)

[0534] Compound 3Pal-Sar(CONMe2)1 (1.06 g, 4.00 mmol), Fmoc-alanine (1.86 g, 6.00 mmol), N,N-diisopropylethylamine (1.04 g, 8.00 mmol), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (2.60 g, 5.00 mmol), and N,N-dimethylformamide (20 mL) were mixed. The mixture was stirred at room temperature for 30 minutes. This mixture was purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution = 5:95–55:45) to give the target product Fmoc-Ala-3Pal-Sar(CONMe2)2 (1.12 g). Yield: 50%.

[0535] MS m / z(ESI): 558[M+1];

[0536] 1 H NMR(400MHz,DMSO-d6)δ8.90(s,1H),8.60-8.56(m,1H),8.40-8.28(m,1H),7.78-7.72(m,2H),7.70- 7.68(m,1H),7.60-8.56(m,2H),7.42-7.38(m,2H),7.34-7.30(m,2H),5.86-5.82(m,1H),5.34-5.31( m,1H),4.54-4.50(m,1H),4.43-4.40(m,1H),4.33-4.30(m,1H),4.22-4.20(m,2H),3.88-3.84(m,1H) ,3.50-3.45(m,1H),3.35-3.31(m,1H),3.19(s,3H),3.02(s,3H),2.98(s,3H),1.38(d,J=8.0Hz,3H).

[0537] Step 2

[0538] Ala-3Pal-Sar(CONMe2)

[0539] The compound Fmoc-Ala-3Pal-Sar(CONMe2)2 (1.12 g, 2.00 mmol), lithium hydroxide (0.24 g, 10.00 mmol), and methanol (20 mL) were mixed. The mixture was stirred at room temperature for 30 minutes. This mixture was purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution = 5:95–55:45) to give the target product Ala-3Pal-Sar(CONMe2)3 (0.67 g). Yield: 99%. MS m / z (ESI): 336 [M+1].

[0540] Step 3: Fmoc-E(t-Bu)-Ala-3Pal-Sar(CONMe2)

[0541] Compound Ala-3Pal-Sar(CONMe2)3 (0.67 g, 2.00 mmol), Fmoc-O-tert-butyl-L-glutamic acid (0.85 g, 2.00 mmol), N,N-diisopropylethylamine (0.65 g, 5.00 mmol), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (1.04 g, 2.00 mmol), and N,N-dimethylformamide (15 mL) were mixed. The mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated, and the residue was purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution = 5:95–55:45) to give the target product Fmoc-E(t-Bu)-Ala-3Pal-Sar(CONMe2)4 (0.80 g). Yield: 54%. MS m / z (ESI): 743 [M+1].

[0542] Step 4

[0543] Fmoc-E-Ala-3Pal-Sar(CONMe2)

[0544] Compound Fmoc-E(t-Bu)-Ala-3Pal-Sar(CONMe2)4 (0.80 g, 1.08 mmol) was mixed with trifluoroacetic acid (10 mL). The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the residue was purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution = 5:95–55:45) to obtain the target product Fmoc-E-Ala-3Pal-Sar(CONMe2)5 (0.60 g). Yield: 81%. MS m / z (ESI): 687 [M+1].

[0545] Step 5

[0546] Fmoc-E (CTC-resin)-Ala-3Pal-Sar (CONMe2)

[0547] Solid-phase CTC-resin (0.6 g, 0.60 mmol) was transferred to a 50 mL peptide synthesis tube. The resin was washed with anhydrous dichloromethane and shaken for 15 minutes. The resin was then washed four times with anhydrous N,N-dimethylformamide. Fmoc-E-Ala-3Pal-Sar(CONMe2)5 (0.60 g, 0.9 mmol) and N,N-diisopropylethylamine (0.26 g, 2.00 mmol) were dissolved in 10 mL of anhydrous N,N-dimethylformamide and added to the resin. After shaking for 15 hours, the resin was washed four times with a dichloromethane / methanol / N,N-diisopropylethylamine (6:3:1) solution. Finally, a small amount of resin was taken for lysis to confirm the completion of the reaction. The resin was washed four times with anhydrous N,N-dimethylformamide and used directly in the next step. MS m / z (ESI): 687 [M+1].

[0548] Step 6

[0549] Fmoc-THP-E (CTC-resin)-Ala-3Pal-Sar (CONMe2)

[0550] Deprotection of the FMOC groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. 4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)tetrahydro-2H-pyran-4-carboxylic acid (0.22 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL). Acid pre-activation with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol) was added for approximately 15 minutes, followed by addition to the resin. After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 814 [M+1].

[0551] Step 7

[0552] Fmoc-2Nal-THP-E (CTC-resin)-Ala-3Pal-Sar (CONMe2)

[0553] Deprotection of the FMOC groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-3-(2-naphthyl)-L-alanine (0.26 g, 0.60 mmol) was dissolved together with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 1011 [M+1].

[0554] Step 8

[0555] Fmoc-TMA5F-2Nal-THP-E (CTC-resin)-Ala-3Pal-Sar (CONMe2)

[0556] Deprotection of the FMOC groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-4-(N,N,N-trimethyl-5-ammoniumpentoxy)-L-phenylalanine (0.32 g, 0.60 mmol) was dissolved together with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 1301 [M].

[0557] Step 9

[0558] Fmoc-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0559] Deprotection of the FMOC groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-S-triphenylmethyl-L-cysteine ​​(0.35 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 824 [M / 2].

[0560] Step 10

[0561] Fmoc-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0562] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-(S)-6-acetyl-6-methylamino-2-aminohexanoic acid (0.25 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction mixture, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and proceeded directly to the next step. MS m / z (ESI): 916 [M / 2]. (Trt)

[0563] Step 11

[0564] Fmoc-W(7-Me)-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0565] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, then draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(7-methyl-1H-indol-3-yl)propionic acid (0.26 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved together in N,N-dimethylformamide (5 mL). Acid pre-activation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), followed by addition to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction mixture, and shaking was continued for 8 hours. The reaction was confirmed to be complete by pyrolysis of a small amount of resin. The resin was washed four times with N,N-dimethylformamide, and proceeded directly to the next step. MS m / z (ESI): 895 [M / 2].

[0566] Step Twelve

[0567] Fmoc-T(tBu)-W(7-Me)-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0568] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-O-tert-butyl-L-threonine (0.24 g, 0.60 mmol) was dissolved together with ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 945 [M / 2].

[0569] Step Thirteen

[0570] Fmoc-Ala(3-cyclopropane)-T(tBu)-W(7-Me)-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0571] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. (R)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-cyclopropylpropionic acid (0.21 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 1001 [M / 2].

[0572] Step Fourteen

[0573] Fmoc-C(Trt)-Ala(3-cyclopropane)-T(tBu)-W(7-Me)-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0574] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% 4-methylpiperidine solution in N,N-dimethylformamide to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of 4-methylpiperidine solution and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. Fmoc-S-triphenylmethyl-L-cysteine ​​(0.35 g, 0.60 mmol) and ethyl 2-oxime cyanoacetate (0.13 g, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.10 g, 0.80 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.06 g, 0.50 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 1108 [M / 2].

[0575] Step 15

[0576] Boc-3MAMPh2EtCO-C(Trt)-Ala(3-cyclopropane)-T(tBu)-W(7-Me)-K(NMeAc)-C(Trt)-TMA5F-2Nal-THP-E(CTC-resin)-Ala-3Pal-Sar(CONMe2)

[0577] Deprotection of the Fmoc groups bound to the resin was achieved by adding two resin bed volumes of a 20% N,N-dimethylformamide solution of 4-methylpiperidine to the swollen resin and shaking for 3–5 minutes, followed by draining and adding a second two resin bed volumes of the N,N-dimethylformamide solution of 4-methylpiperidine and shaking for another 20–30 minutes. After deprotection, the resin was washed three times with N,N-dimethylformamide by shaking. 3-(3-(((tert-butoxycarbonyl)(methyl)amino)methyl)phenyl)propionic acid (0.09 g, 0.30 mmol) and ethyl 2-oxime cyanoacetate (0.06 g, 0.45 mmol) were dissolved in N,N-dimethylformamide (3 mL). Acid preactivation was performed for approximately 15 minutes with N,N'-diisopropylcarbodiimide (0.05 g, 0.40 mmol), and then added to the resin (0.20 mmol). After approximately 15 minutes, N,N'-diisopropylcarbodiimide (0.03 g, 0.25 mmol) was added to the reaction, and shaking was continued for 8 hours. Finally, a small amount of resin was taken for lysis detection to confirm the completion of the reaction. The resin was washed four times with N,N-dimethylformamide, and the reaction proceeded directly to the next step. MS m / z (ESI): 1029 [M / 2].

[0578] Step Sixteen

[0579] 3MAMPh2EtCO-C-Ala(3-cyclopropane)-TW(7-Me-K(NMeAc)-C-TMA5F-2Nal-THP-E-Ala-3Pal-Sar(CONMe2)

[0580] Prepare 5 mL of TFA lysis mixture (90 / 5 / 2.5 / 2.5, TFA / water / TIPS / DODT). Add 5 mL of the lysis mixture to a test tube containing the peptide bound to the protective resin and shake for two hours. Filter off the waste resin, add isopropyl ether to the filtrate to form a white precipitate, and then centrifuge the white precipitate. Discard the isopropyl ether into the waste and wash the precipitate twice more with isopropyl ether. Dry the resulting white precipitate filter cake under reduced pressure to give 3MAMPh2EtCO-C-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C-TMA5F-2Nal-THP-E-Ala-3Pal-Sar(CONMe2) (0.20 g). MS m / z (ESI): 1029 [M / 2].

[0581] Step Seventeen

[0582] 3MAMPh2EtCO-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E-Ala-3Pal-Sar(CONMe2)

[0583] 3MAMPh2EtCO-C-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C-TMA5F-2Nal-THP-E-Ala-3Pal-Sar(CONMe2) (0.20 g, 0.1 mmol) was dissolved in 20% MeCN / water (80 mL). While stirring, a saturated solution of iodine in acetic acid / methanol was added dropwise to the peptide solution until the yellow / brown color was maintained and did not fade. The pale yellow solution was allowed to stand for 5 minutes, and then excess iodine was quenched with a small amount of ascorbic acid. The mixture was directly purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution = 5:95–55:45). Lyophilization gave the TFA salt product. MS m / z (ESI): 1028 [M / 2].

[0584] Step 18

[0585] 3MAMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E(1)-Ala-3Pal-Sar(CONMe2)

[0586] The purified oxidized intermediate peptide 3MAMPh2EtCO-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E-Ala-3Pal-Sar(CONMe2) (0.035 g, 0.015 mmol) was dissolved in 3 mL of N,N-dimethylformamide. 1H-benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (0.015 g, 0.03 mmol) was added, followed by N,N-diisopropylethylamine (0.013 g, 0.10 mmol). The mixture was stirred at room temperature for 30 minutes and then purified by pre-HPLC to obtain the target product 3MAMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E(1)-Ala-3Pal-Sar(CONMe2) trifluoroacetate (0.015 g).

[0587] Step 19

[0588] Trifluoroacetate is converted into hydrochloride.

[0589] The reverse-phase column was equilibrated with 5% acetonitrile / water at 50 mL / min (A = 0.01 M HCl aqueous solution, B = 100% acetonitrile, C = 0.04 M ammonium bicarbonate aqueous solution). The purified peptide TFA salt was dissolved in 20% acetonitrile / water. The solution was loaded onto the equilibrated column at 50 mL / min, and the solvent front was eluted. The column was washed with 5% B in solution A for 5 min. Then, it was washed with 5% B in solution C for 40 min at 50 mL / min to remove all trifluoroacetate ions from the system. It was then washed with 5% B in solution A for 10 min at 50 mL / min to remove all ammonium bicarbonate ions from the system. Finally, the peptide was eluted with a gradient of 5%–70% B in solution A over 60 min and collected in fractions. The products were combined and lyophilized to obtain 0.012 g of 3MAMPh2EtCO(1)-C(2)-Ala(3-cyclopropane)-TW(7-Me)-K(NMeAc)-C(2)-TMA5F-2Nal-THP-E(1)-Ala-3Pal-Sar(CONMe2) hydrochloride. MS m / z (ESI): 1019 [M / 2].

[0590] Table 4C: Synthesis of compounds 83-87 (Refer to the synthesis process of compound 1; compounds 83-87 are hydrochloride salts).

[0591]

[0592]

[0593]

[0594] Example 2: Inhibition of interleukin-23 binding to its receptor by the bicyclic peptide

[0595] Example 2A: IL23-IL23R Binding Experiment

[0596] Human IL23 and Human IL23R proteins were purchased from Sinocare. The reaction buffer consisted of 25 mM HEPES (pH 7.2), 0.1% BSA, and 0.01% Tween-20. The test compound was dissolved in DMSO to a concentration 200-fold higher than the maximum test concentration, then serially diluted 4-fold with DMSO to eight concentration points. Each concentration point was then diluted 50-fold with the reaction buffer. Human IL23R protein was diluted to 2 nM with the reaction buffer. 5 μL of diluted Human IL23R protein was added to each well of a 384-well plate (Corning, catalog number 3572), followed by 5 μL of the diluted compound or DMSO. The plate was centrifuged at 2000 rpm for 1 min at room temperature and incubated at 25°C for 30 min. Then, 5 μL of 8 nM Human IL23 was added, centrifuged at 2000 rpm for 1 min at room temperature, and incubated at 25°C for 60 min. After incubation, add 5 μL of a mixture of HTRF Streptavidin-Eu cryptate (Cisbio, catalog number 610SAKLA) and HTRF Human PAb Anti IgG-d2 (Cisbio, catalog number 61HFCDAA) to each well. Centrifuge at 2000 rpm for 1 min at room temperature, and incubate the plate at 25°C for 60 min. After incubation, use a microplate reader (EnSpire, Perkin Elmer) to detect the Em665 / Em615 ratio according to the HTRF principle, and calculate the inhibition rate (%).

[0597] In this experiment, the group without IL23 protein was designated as the 100% inhibition group, and the group with IL23, IL23R protein, and DMSO was designated as the 0% inhibition group. The percentage of inhibition of protein binding by the compounds was calculated using the following formula:

[0598] Inhibition percentage = 100 - 100 * (Signal) 化合物 -Signal 100％抑制 ) / (Signal 0％抑制 –Signal 100％抑制 )

[0599] Compound IC 50The values ​​are derived from eight concentration points and calculated using XLfit (ID Business Solutions Ltd., UK) software using the following formula:

[0600] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)×Slope factor))

[0601] Where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-curve, Top is the top plateau value of the S-curve, X is the logarithm of the concentration of the analyte, and Slope factor is the curve slope coefficient.

[0602] Example 2B: HEK blue experiment

[0603] HEK-Blue IL-23 cells (InvivoGen, hkb-il23) were cultured in DMED medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were sputtered at a rate of 1×10⁻⁶ cells / mL. 4 Cells were seeded per well in a 384-well plate. The compound was serially diluted 5-fold at 8 wells, with a final starting concentration of 10 μM. Cells were incubated with the specified concentration of the compound or DMSO for 1 h, followed by the addition of IL23 (ACRO Biosystem, ILB-H5219) to a final concentration of 2 ng / ml. DMSO was used for control wells. The plates were then incubated in a cell culture incubator for 24 h. 5 μL of cell culture supernatant from each test well was transferred to a clear-bottomed 384-well plate, and 45 μL of QUANTI-Blue was added to each well. TM The solution (InvivoGen, rep-qbs) was incubated at 37°C for 10 min. The absorbance was measured at 630 nm using a microplate reader, and the readings were analyzed using analytical software.

[0604] In this experiment, the group without IL23 was designated as the 100% inhibition group, and the group with IL23 and DMSO was designated as the 0% inhibition group. The percentage of inhibition of the IL23 pathway by the compounds was calculated using the following formula:

[0605] Inhibition percentage = 100 - 100 * (Signal) 化合物 -Signal 100％抑制 ) / (Signal 0％抑制 –Signal 100％抑制 )

[0606] Compound IC 50The values ​​are derived from eight concentration points and calculated using XLfit (ID Business Solutions Ltd., UK) software using the following formula:

[0607] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)×Slope factor))

[0608] Where Y is the inhibition percentage, Bottom is the bottom plateau value of the S-curve, Top is the top plateau value of the S-curve, X is the logarithm of the concentration of the analyte, and Slope factor is the curve slope coefficient. JNJ-2113 (Reference: Scientific Reports (2024), 14(1), 17515).

[0609] Table 5: Inhibition of interleukin-23 binding to its receptor by bicyclic peptides

[0610]

[0611]

[0612]

[0613]

[0614] The compounds of the present invention listed in Table 5 have in vitro activities (IL23-IL23R binding and IL-23-induced bioactivity in HEK-Blue IL-23 cells) that are substantially equivalent to the control compound JNJ-2113.

[0615] Example 3: Stability study of the compound in simulated gastric and intestinal fluids

[0616] The residual amount of the compound in artificial gastric and intestinal fluids at different time points was determined by LC-MS / MS, the residual percentage was calculated, and the half-life t was finally obtained. 1 / 2 .

[0617] Experimental conditions and procedures:

[0618] 1. Preparation of stock solution

[0619] Accurately weigh an appropriate amount of the test sample, add an appropriate amount of DMSO to dissolve it, and prepare a stock solution with a concentration of 5 mM.

[0620] 2. Preparation of working solution

[0621] Use 5mM stock solution and add DMSO to prepare 2mM working solution.

[0622] 3. Preparation of artificial gastric juice and artificial intestinal juice

[0623] Artificial gastric juice: Accurately weigh 200mg sodium chloride and dissolve it in 50mL of water. Add an appropriate amount of hydrochloric acid to adjust the pH to 1.2, and make up to 100mL. The final pH is 1.2±0.1. Accurately weigh 64mg pepsin and add it to 20mL of the above solution. This is the artificial gastric juice.

[0624] Artificial intestinal fluid: Accurately weigh 680 mg of potassium dihydrogen phosphate and dissolve it in 50 mL of water. Add an appropriate amount of sodium hydroxide to adjust the pH to 6.8, and bring the volume to 100 mL. The final pH is 6.8 ± 0.1. Accurately weigh 200 mg of trypsin and add it to 20 mL of the above solution. This is the artificial intestinal fluid.

[0625] 4. Stability tests in artificial gastric and intestinal fluids

[0626] Artificial gastric and intestinal fluids were preheated to 37°C. An appropriate amount of the compound working solution was accurately pipetted into a 1.1 mL tube, and blank artificial gastric and intestinal fluids were added. The mixture was vortexed to mix. Stability was investigated at 37°C by accurately sampling at 0 h, 1 h, 6 h, and 24 h. The reaction was terminated by adding a quantitative internal standard solution (5 ng / mL terfenadine containing 0.1% formic acid). The mixture was vortexed for 5 min, centrifuged at 3200 rpm for 15 min at 4°C, and the supernatant was diluted with water, vortexed, and then injected for analysis.

[0627] 5. LC-MS / MS method

[0628] Based on the precise molecular weight of the compound, the corresponding parent ion and fragment ions are identified, and suitable mass spectrometry conditions and liquid chromatography methods are optimized.

[0629] 6. Sample Testing

[0630] Analyze the sample using a suitable LC-MS / MS method.

[0631] 7. Data Processing

[0632] The main computerized system used in this experiment:

[0633] Microsoft Office Excel 2010: Input, calculation, and statistical data;

[0634] Analysis 1.6.3, Data Acquisition and Processing;

[0635] GraphPad Prism 6.0, Drug Residue Rate Time Curve

[0636] Table 6: Stability results of the compounds of the present invention in artificial gastric fluid (SGF) and artificial intestinal fluid (SIF)

[0637] Compound numbering <![CDATA[SIF t 1 / 2 (hr)]]> <![CDATA[SGF t 1 / 2 (hr)]]> 1 91.8 166 16 234 203 17 72.5 183 18 61.2 336 26 187 141 27 223 221 29 66 278 33 110 139 34 69.5 455 38 73.9 253 39 86.4 187 40 55.9 197 JNJ-2113 hydrochloride 102 115

[0638] The compounds of the present invention listed in Table 6 exhibit stability in artificial gastric and intestinal fluids that is substantially equivalent to that of the control compound JNJ-2113.

[0639] Example 4: In vivo pharmacokinetic study in SD rats

[0640] The concentration of compounds in the plasma of SD (Sprague-Dawley) rats was determined by LC-MS / MS, and the pharmacokinetic parameters of the plasma were calculated using Phoenix WinNolin to screen for compounds with good pharmacokinetic properties.

[0641] Experimental conditions and procedures:

[0642] 1. Dispensing medicine

[0643] Formula 1: Weigh an appropriate amount of the test sample into a weighing bottle, and add 10% DMA / 10% Solutol / 80% 50mM PBS pH 7.4 (v / v / v) in sequence. Sonicate and stir at room temperature until a homogeneous solution is obtained, and administer intravenously.

[0644] Formula 2: Weigh an appropriate amount of the test sample into a weighing bottle, add 30% Labrasol / 70% 50mM PBS (v / v) in sequence, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage and colon.

[0645] Formula 3: Weigh an appropriate amount of the test sample into a weighing bottle, add 40 mg / mL sodium decanoate dissolved in 50 mM PBS solution, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage and colon.

[0646] 2. Administration

[0647] Three male SD rats were prepared for each group and weighed. The drug dosage was 1 mg / kg, and the administration volume was 2 mL / kg, administered intravenously to the SD rats. Plasma was collected at 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration.

[0648] Three male SD rats were prepared for each group and weighed. The drug dosage was 10 mg / kg, and the administration volume was 10 mL / kg, administered to the SD rats by gavage. Plasma was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.

[0649] Three male SD rats were prepared for each group and weighed. The drug dosage was 50 mg / kg, and the administration volume was 10 mL / kg, administered to the SD rats by gavage. Plasma was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.

[0650] Five male SD rats were prepared for each group and weighed. The drug dosage was 5 mg / kg, and the administration volume was 2.5 mL / kg, administered to the SD rats via the colon. Plasma was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.

[0651] 3. LC-MS / MS method

[0652] Based on the precise molecular weight of the compound, the corresponding parent ion and fragment ions are identified, and suitable mass spectrometry conditions and liquid chromatography methods are optimized.

[0653] 4. Preparation of standard curve and quality control samples

[0654] Preparation of working solutions: Accurately weigh an appropriate amount of the test sample, add an appropriate amount of DMSO to dissolve it, and prepare a standard stock solution with a concentration of 2 mg / mL. Using the standard stock solution, add acetonitrile-water (50:50, v / v) to prepare a series of standard curves for the test sample and quality control working solutions.

[0655] Preparation of standard curve and quality control plasma samples: Take 20 μL of blank plasma from SD rats, add 2 μL of standard curve of test sample of series concentration and quality control working solution to prepare standard curve plasma sample and quality control plasma sample.

[0656] 5. Sample Analysis Process

[0657] Take 20 μL of plasma sample, add 2 μL of acetonitrile-water (50:50, v / v), place in a 1.1 mL high-capacity container, add 200 μL of internal standard solution (a mixed solution of 5 ng / mL terfenadine and propranolol containing 0.1% formic acid), vortex for 1 minute, centrifuge at 3500 rpm for 10 minutes at 4℃, take the supernatant, dilute with 0.1% formic acid aqueous solution, vortex to mix, and then inject for analysis. Standard curve samples and quality control samples treated in the same way were used concurrently when determining sample concentration.

[0658] 6. Sample Testing

[0659] Analyze the sample using a suitable LC-MS / MS method.

[0660] 7. Data Processing

[0661] The main computerized system used in this experiment:

[0662] Microsoft Office Excel 2010: Input, calculation, and statistical data;

[0663] Analysis 1.6.3, Data Acquisition and Processing;

[0664] Phoenix WinNolin 8.3.1 for calculating pharmacokinetic parameters

[0665] GraphPad Prism 6.0, drug concentration-time curve

[0666]

[0667] Table 7B: Pharmacokinetic results (IDPK) of the IL-23 receptor inhibitor of the present invention in SD rats.

[0668]

[0669] a Formula 1; b Formula 2; c Formula 3

[0670] The compounds of the present invention listed in Tables 7A and 7B generally showed better rat IDPK (colonic administration) performance than the reference compound JNJ-2113 hydrochloride. Among them, compound 1 showed that its rat IDPK, IVPK, and PO exposure (AUC) was 3 to 4 times that of JNJ-2113, demonstrating its excellent pharmacokinetic properties.

[0671] Example 5: In vivo pharmacokinetic study in ICR mice

[0672] The concentration of compounds in the plasma of ICR mice was determined by LC-MS / MS, and the pharmacokinetic parameters of the plasma were calculated using Phoenix WinNolin to screen for compounds with good pharmacokinetic properties.

[0673] Experimental conditions and procedures:

[0674] 1. Dispensing medicine

[0675] Formula 2: Weigh an appropriate amount of the test sample into a weighing bottle, add 30% Labrasol / 70% 50mM PBS (v / v) in sequence, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage and colon.

[0676] Formula 3: Weigh an appropriate amount of the test sample into a weighing bottle, add 40 mg / mL sodium decanoate dissolved in 50 mM PBS solution, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage and colon.

[0677] 2. Administration

[0678] Three male ICR mice were prepared for each group and weighed. The dosage was 50 mg / kg, and the administration volume was 10 mL / kg, administered to the ICR mice by gavage. Plasma was collected at 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration.

[0679] 3. LC-MS / MS method

[0680] Based on the precise molecular weight of the compound, the corresponding parent ion and fragment ions are identified, and suitable mass spectrometry conditions and liquid chromatography methods are optimized.

[0681] 4. Preparation of standard curve and quality control samples

[0682] Preparation of working solutions: Accurately weigh an appropriate amount of the test sample, add an appropriate amount of DMSO to dissolve it, and prepare a standard stock solution with a concentration of 2 mg / mL. Using the standard stock solution, add acetonitrile-water (50:50, v / v) to prepare a series of standard curves for the test sample and quality control working solutions.

[0683] Preparation of plasma standard curve and quality control samples: Take 20 μL of blank plasma from ICR mice, add 2 μL of test sample standard curve and quality control working solution of various concentrations to prepare standard curve plasma sample and quality control plasma sample.

[0684] 5. Sample Analysis Process

[0685] Take 20 μL of plasma sample, add 2 μL of acetonitrile-water (50:50, v / v), place in a 1.1 mL high-capacity container, add 200 μL of internal standard solution (5 ng / mL terfenadine and propranolol solution containing 0.1% formic acid), vortex for 1 minute, centrifuge at 3500 rpm for 10 minutes at 4℃, collect the supernatant, dilute with 0.1% formic acid aqueous solution, vortex to mix, and then inject for analysis. The same method is used to process standard curve samples and quality control samples when determining sample concentrations.

[0686] 6. Sample Testing

[0687] Analyze the sample using a suitable LC-MS / MS method.

[0688] 7. Data Processing

[0689] The main computerized system used in this experiment:

[0690] Microsoft Office Excel 2010: Input, calculation, and statistical data;

[0691] Analysis 1.6.3, Data Acquisition and Processing;

[0692] Phoenix WinNolin 8.3.1 for calculating pharmacokinetic parameters

[0693] GraphPad Prism 6.0, drug concentration-time curve

[0694] Table 8: Pharmacokinetic results of the IL-23 receptor inhibitor of the present invention in ICR mice

[0695]

[0696]

[0697] b Formula 2; c Formula 3;

[0698] The compound 1 of the present invention listed in Table 8 has an AUC (Active Cell Activated Urine) in mice that is ~2.2 times that of JNJ-2113.

[0699] Example 6: Pharmacokinetic Study in Beagle Dogs

[0700] The concentration of compounds in beagle plasma was determined by LC-MS / MS, and the pharmacokinetic parameters of plasma were calculated using Phoenix WinNolin to screen for compounds with good pharmacokinetic properties.

[0701] Experimental conditions and procedures:

[0702] 1. Dispensing medicine

[0703] Formula 1: Weigh an appropriate amount of the test sample into a weighing bottle, add 30% Labrasol / 70% 50mM PBS (v / v) in sequence, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage.

[0704] Formula 2: Weigh an appropriate amount of the test sample into a weighing bottle, add 40 mg / mL sodium decanoate dissolved in 50 mM PBS solution, sonicate and stir at room temperature until a homogeneous solution is obtained, and administer by gavage.

[0705] 2. Administration

[0706] Three male beagle dogs were prepared for each group and weighed. The dosage was 3 mg / kg, and the administration volume was 5 mL / kg, administered to the beagle dogs by gavage. Timing was started after administration, and plasma was collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.

[0707] 3. LC-MS / MS method

[0708] Based on the precise molecular weight of the compound, the corresponding parent ion and fragment ions are identified, and suitable mass spectrometry conditions and liquid chromatography methods are optimized.

[0709] 4. Preparation of standard curve and quality control samples

[0710] Preparation of working solutions: Accurately weigh an appropriate amount of the test sample, add an appropriate amount of DMSO to dissolve it, and prepare a standard stock solution with a concentration of 2 mg / mL. Using the standard stock solution, add acetonitrile-water (50:50, v / v) to prepare a series of standard curves for the test sample and quality control working solutions.

[0711] Preparation of standard curve and quality control plasma samples: Take 20 μL of blank beagle plasma, add 2 μL of the standard curve of test sample of various concentrations and the quality control working solution to prepare the standard curve plasma sample and the quality control plasma sample.

[0712] 5. Sample Analysis Process

[0713] Take 20 μL of plasma sample, add 2 μL of acetonitrile-water (50:50, v / v), place in a 1.1 mL high-capacity container, add 200 μL of internal standard solution (a mixed solution of 5 ng / mL terfenadine and propranolol containing 0.1% formic acid), vortex for 1 minute, centrifuge at 3500 rpm for 10 minutes at 4℃, take the supernatant, dilute with 0.1% formic acid aqueous solution, vortex to mix, and then inject for analysis. The same method is used to process standard curve samples and quality control samples when determining sample concentrations.

[0714] 6. Sample Testing

[0715] Analyze the sample using a suitable LC-MS / MS method.

[0716] 7. Data Processing

[0717] The main computerized system used in this experiment:

[0718] Microsoft Office Excel 2010: Input, calculation, and statistical data;

[0719] Analysis 1.6.3, Data Acquisition and Processing;

[0720] Phoenix WinNolin 8.3.1 for calculating pharmacokinetic parameters

[0721] GraphPad Prism 6.0, drug concentration-time curve

[0722] Table 9: Pharmacokinetic results of the IL-23 receptor inhibitor of the present invention in beagle dogs

[0723]

[0724] a Formula 1; b Formula 2;

[0725] The compound 1 of the present invention listed in Table 9 has an AUC (Active Cell Activated Urine) in beagle dogs that is ~3.6 times higher than that of JNJ-2113. The exposure levels of compound 1 in various genera (rats, mice, beagle dogs, etc.) are significantly higher than those of JNJ-2113.

[0726] Example 7: An IL-23-induced rat skin inflammation model was constructed, and the in vivo efficacy of compound 1 was evaluated by measuring the thickness of rat ears (administered via tail vein injection).

[0727] Female SD rats aged 6-8 weeks were acclimatized for one week and then randomly divided into four groups based on their body weight and ear thickness: a normal control group, a model control group, a JNJ-2113 administration group, and a compound 1 administration group. To establish an IL-23-induced skin inflammation model, recombinant IL-23 protein (Yiqiao Shenzhou, catalog number: CT045-R08H, batch number: MB18JA0307) was diluted to 75 μg / mL with sterile PBS, and 20 μL (1.5 μg) was injected intradermally into the right ear of each rat once daily. Drug administration began on the day of the first IL-23 injection, defined as day 0 after administration. Compound 1 was dissolved in 10% PEG400 + 90% 50mM PBS and administered via tail vein injection at a dose of 1 mg / kg once daily for 5 consecutive days. During the experiment, the ear thickness of the rat's right ear was measured daily, and the ear thickness increase relative to day 0 and the ear thickness inhibition rate (ETI) were calculated for each group. ETI (%) = [1-(T i -T0) / (V i -V0)]×100%, T i V is the average ear thickness measured at each step in the treatment group, T0 is the average ear thickness measured at the start of drug administration in the treatment group, and V is the average ear thickness measured at each step. i The model control group and T i The average ear thickness measured on the same day, V0 is the average ear thickness measured in the model control group at the start of drug administration.

[0728] Results of rat ear thickness analysis showed that the ears of rats in the model group began to thicken 2 days after IL-23 injection, reaching their maximum thickness on day 5, an increase of 0.216 mm relative to day 0. In contrast, the ear thickness of the normal control group remained essentially unchanged, indicating successful model establishment. On day 5 post-administration, the ears of rats in the JNJ-2113 and compound 1 treatment groups thickened by 0.137 mm and 0.070 mm, respectively, with inhibition rates of 36.90% and 64.72%, respectively. These results indicate that, compared to the model group, compound 1 significantly inhibited IL-23-induced ear thickening in rats, and its inhibitory effect was stronger than that of the same dose of the positive control JNJ-2113 (e.g., ...). Figure 1 (As shown).

[0729] In summary, since compound 1 exhibits similar in vitro activity to JNJ-2113, while its pharmacokinetic properties are significantly superior to those of JNJ-2113, compound 1 is far more effective than JNJ-2113 at the same dose in the IL-23-induced rat skin inflammation pharmacodynamic model.

[0730] Example 8: An IL-23-induced rat skin inflammation model was constructed, and the in vivo efficacy of compounds 1 and 50 (oral administration) was evaluated by measuring rat ear thickness.

[0731] Female SD rats aged 6-8 weeks were acclimatized for 3 days and then randomly divided into 5 groups based on their body weight and ear thickness: normal control group, model control group, JNJ-2113 administration group, compound 1 administration group, and compound 50 administration group. The day of grouping was defined as day 0. JNJ-2113, compound 1, and compound 50 were dissolved in Labrasol (Tianrun Pharmaceutical, batch number: 190913) / 50mM PBS, 30 / 70, v / v, and administered orally at a dose of 15 mg / kg twice daily for 5 consecutive days. To establish an IL-23-induced rat skin inflammation model, recombinant rat IL-23 protein (Yiqiao Shenzhou, catalog number: CT045-R08H, batch number: MB18JA0307) was diluted to 75 μg / mL with sterile PBS, and 20 μL (1.5 μg) was injected intradermally into the right ear of each rat, starting from day 1, for a total of 4 injections. During the experiment, the ear thickness of the rat's right ear was measured daily, and the ear thickness increase relative to day 0 and the ear thickness inhibition rate (ETI) were calculated for each group. ETI (%) = [1-(T i -T0) / (V i -V0)]×100%, T i V is the average ear thickness measured at each step in the treatment group, T0 is the average ear thickness measured at the start of drug administration in the treatment group, and V is the average ear thickness measured at each step. i The model control group and Ti The average ear thickness measured on the same day, V0 is the average ear thickness measured in the model control group at the start of drug administration.

[0732] Results of rat ear thickness analysis showed that the ear thickness of rats in the model group increased continuously after IL-23 injection, reaching its maximum on day 5, with an increase of 0.219 mm relative to day 0. The ear thickness of the normal control group remained essentially unchanged, indicating successful model establishment. On day 5 after drug administration, the ears of rats in the JNJ-2113, compound 1, and compound 50 administration groups increased by 0.136 mm, 0.144 mm, and 0.106 mm, respectively, with inhibition rates of 37.61%, 34.17%, and 51.41%. These results indicate that, compared with the model group, oral administration of compounds 1 and 50 significantly inhibited IL-23-induced ear thickening in rats (P<0.01, P<0.0001), and the inhibitory efficacy was comparable to or stronger than that of the positive control JNJ-2113 at the same dose (e.g., ...). Figure 2 (As shown).

[0733] In summary, since compounds 1 and 50 exhibit similar in vitro activity to JNJ-2113, while having significantly superior oral pharmacokinetic properties, compounds 1 and 50 are as effective as or stronger than JNJ-2113 in an IL-23-induced rat skin inflammation model when administered orally.

Claims

1. A bicyclic peptide inhibitor of the interleukin-23 receptor, and a pharmaceutically acceptable salt or solvate thereof, wherein the bicyclic peptide inhibitor comprises the amino acid sequence of formula (I): R1-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 Formula (I), Where R 1 for Wherein ring A is selected from C6-C10 aryl, C5-C10 heteroaryl, C3-C10 cycloalkyl and 3- to 8-membered heterocyclic groups, wherein the C6-C10 aryl, C5-C10 heteroaryl, C3-C10 cycloalkyl and 3- to 8-membered heterocyclic groups are unsubstituted or optionally substituted by one or more substituents B, wherein substituent B is selected from halogens, C1-C10 alkoxy, C3-C10 cycloalkyl, N,N-dimethylamino-C1-C6 alkoxy and N,N,N-trimethylammonium-C1-C6 alkoxy, wherein n is an integer from 0 to 4; R a Selected from hydrogen or C1-C4 alkyl groups; Preferably, wherein the R 1 Selected from the following groups, either unsubstituted or optionally substituted by one or more substituent C groups: The substituent C is selected from halogens, Where m = 1 to 4, n = 0 to 4, and r = 1 to 4 are integers; R a Selected from hydrogen or methyl; More preferably, wherein the R 1 Selected from X3 is selected from Cys, Pen, or Abu; X4 is selected from X5 is selected from Thr, Ser, or does not exist; it is preferred to be Thr or does not exist. X6 is selected from unsubstituted Trp or Trp substituted with C1-C4 alkyl groups; preferably W(7-Me); X7 is selected from Lys(Ac), Lys(NMeAc), Pro, X8 is selected from Cys or Pen; X9 is selected from unsubstituted Tyr or Phe or substituted with one or more substituents D, wherein the substituents D are selected from halogens, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, carboxyl, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy, and N,N,N-trimethyl-3-ammoniumpropoxy; Preferably, X9 is selected from Phe or Where R 2 Selected from halogen, ethynyl, C1-C4 alkoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy or N,N,N-trimethyl-3-ammoniumpropoxy; More preferably, X9 is selected from Phe or Where R 2 Selected from halogens, methoxy groups, 2-aminoethoxy groups, N,N-dimethyl-2-aminoethoxy groups, N,N,N-trimethyl-5-ammonium-pentoxyamine groups, N,N-dimethyl-5-aminopentoxyamine groups, and N,N,N-trimethyl-3-ammonium-propoxyamine groups. X10 is 2Nal; X11 is selected from the following α-disubstituted amino acids: 4-amino-4-carboxy-tetrahydropyran (THP), α-MeLys, X12 can be any amino acid; preferably, X12 is selected from Glu, Asp, Lys, D-Glu, or D-Asp. X13 is any amino acid; preferably, X13 is selected from Glu, Asp, Asn, Ala, Val, Ser, Thr, Arg, D-Ala, D-Asn, D-Asp, D-Glu, D-Ser, D-Thr, D-Arg. More preferably, X13 is selected from Glu, Asp, Asn, Ala, Val, Thr, X14 is 3Pal; X15 is unsubstituted or optionally substituted with one or more substituents of Gly; preferably, X15 is selected from... in: The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond: • X3 and X8 are connected by disulfide bonds between Cys and Cys, Pen and Pen, or Cys and Pen; or by thioether bonds between Abu and Cys or Pen, and • An amide bond formed between R1 and the carboxylic acid on the side chain of X12, or an amide bond formed between R1 and the amino group on the side chain of X12; The bicyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

2. The bicyclic peptide inhibitor of interleukin-23 receptor according to claim 1, and its pharmaceutically acceptable salt or solvate, wherein: R 1 Selected from unsubstituted or substituted by one or more C substituents. The substituent C is selected from halogens, Where m = 1 to 4, n = 0 to 4, and r = 1 to 4 are integers; R a Selected from hydrogen or methyl; X3 is selected from Cys, Pen, or Abu; X4 is selected from Asn, Asp or X5 is selected from Thr, Ser, or does not exist; X6 is W(7-Me); X7 is selected from Lys(Ac), Lys(NMeAc), Pro, X8 is selected from Pen or Cys; X9 is selected from Phe or Where R 2 Selected from halogen, ethynyl, C1-C4 alkoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy, N,N,N-trimethyl-5-ammoniumpentoxy, N,N-dimethyl-5-aminopentoxy or N,N,N-trimethyl-3-ammoniumpropoxy; X10 is 2Nal; X11 is selected from 4-amino-4-carboxy-tetrahydropyran (THP), α-MeLys, X12 is selected from Glu, Asp, Lys, D-Glu, or D-Asp; X13 is selected from Glu, Asp, Asn, Ala, Val, Ser, Thr, Arg, D-Ala, D-Asn, D-Asp, D-Glu, D-Ser, D-Thr, D-Arg, X14 is 3Pal; X15 is selected from in: The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond: • X3 and X8 are connected by disulfide bonds between Cys and Cys, or Pen and Pen; or by thioether bonds between Abu and Cys or Pen, and • An amide bond formed between R1 and the carboxylic acid on the side chain of X12, or an amide bond formed between R1 and the amino group on the side chain of X12; The bicyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

3. The bicyclic peptide inhibitor of interleukin-23 receptor according to claim 1, and its pharmaceutically acceptable salt or solvate, wherein: R 1 Selected from: X3 is selected from Cys, Pen, or Abu; X4 is selected from X5 is selected from Thr or does not exist; Where X4 is selected from Asn, When X5 is Thr; when X4 is At that time, X5 did not exist; X6 is W(7-Me); X7 is selected from Lys(Ac), Lys(NMeAc), Pro, X8 is selected from Pen or Cys; X9 is Where R 2 Selected from methoxy, 2-aminoethoxy, N,N-dimethyl-2-aminoethoxy or N,N,N-trimethyl-5-ammoniumpentoxyamino; X10 is 2Nal; X11 is selected from 4-amino-4-carboxy-tetrahydropyran (THP). X12 is either Glu or Lys; X13 is selected from Glu, Asn, Ala, Val, Thr. X14 is 3Pal; X15 is selected from in: The bicyclic peptide inhibitor of the interleukin-23 receptor is cyclized by forming the following bond: • X3 and X8 are connected by disulfide bonds between Cys and Cys, or Pen and Pen; or by thioether bonds between Abu and Cys or Pen, and • An amide bond formed between R1 and the carboxylic acid on the side chain of X12, or an amide bond formed between R1 and the amino group on the side chain of X12; The bicyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

4. A bicyclic peptide inhibitor of the interleukin-23 receptor, and a pharmaceutically acceptable salt or solvate thereof, selected from the following bicyclic peptides: The cyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

5. A bicyclic peptide inhibitor of the interleukin-23 receptor, a pharmaceutically acceptable salt or solvate thereof, selected from the following bicyclic peptides: The bicyclic peptide inhibitor inhibits the binding of interleukin-23 (IL-23) to its receptor (IL-23R).

6. A pharmaceutical composition comprising a bicyclic peptide inhibitor of the interleukin-23 receptor according to any one of claims 1-5, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

7. Use of a bicyclic peptide inhibitor of the interleukin-23 receptor, a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament, wherein the medicament is used to treat or prevent IL-23 / IL23-R mediated inflammatory diseases, autoimmune inflammatory diseases, and / or related disorders.

8. The use according to claim 7, wherein the inflammatory disease, autoimmune inflammatory disease and / or related disorder is selected from: multiple sclerosis, asthma, rheumatoid arthritis, intestinal inflammation, inflammatory bowel disease (IBD), juvenile IBD, young adult IBD, Crohn's disease, ulcerative colitis, celiac disease (non-tropical stomatitis), microscopic colitis, collagenous colitis, eosinophilic gastroenteritis / esophagitis, colitis associated with radiotherapy or chemotherapy, such as Colitis associated with congenital immune disorders, sarcoidosis, systemic lupus erythematosus, ankylosing spondylitis (axial spondyloarthritis), psoriatic arthritis, psoriasis (e.g., plaque psoriasis, guttate psoriasis, reversal psoriasis, pustular psoriasis, palmoplantar pustulosis, psoriasis vulgaris, or erythrodermic psoriasis) associated with leukocyte adhesion defect-1, atopic dermatitis, acne atopic, enteropathy associated with seronegative arthropathy, chronic granulomatous disease, and glycogen storage disease type 1b. Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Wiskott-Aldrich syndrome, pouchitis, pouchitis following rectocele and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, primary biliary cirrhosis, virus-associated enteropathy, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, uveitis, or graft-versus-host disease, comprising administering to patients in need a therapeutically effective amount of a bicyclic peptide inhibitor of the interleukin-23 receptor according to any one of claims 1-5, a pharmaceutically acceptable salt or solvation thereof, or a pharmaceutical composition according to claim 6.

9. The use according to claim 7 or 8, wherein the autoimmune inflammatory disease is selected from ulcerative colitis, Crohn's disease, psoriasis, or psoriatic arthritis.

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