Cyclic peptide and covalent warhead-based compounds and uses thereof

Abstract

Provided are cyclic peptides and covalent warhead compounds of Formula (I') that can act as inhibitors of fibroblast activation protein, and tautomers, stereoisomers, solvates, or pharmaceutically acceptable salts thereof, wherein ring W is a cyclic peptide moiety, P is a chelating moiety that can chelate a radionuclide, and Linker is a linker moiety that covalently links ring W to 5P. Also provided are chelates of the compounds with radionuclides and pharmaceutical compositions thereof that can act as inhibitors of fibroblast activation protein for use in diagnosis and treatment of disease.

Description

Cyclic peptide and covalent warhead-based compounds and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN202411170134.8, filed August 23, 2024, entitled “Fibroblast Activation Protein Inhibitors and Uses Thereof,” Chinese Patent Application No. CN202411638778.5, filed November 15, 2024, entitled “Cyclic Peptide and Covalent Warhead-Based Compounds and Uses Thereof,” and Chinese Patent Application No. CN202510424427.2, filed April 7, 2025, entitled “Cyclic Peptide and Covalent Warhead-Based Compounds and Uses Thereof,” the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of medicine, in particular to compounds comprising cyclic peptides and covalent warheads, pharmaceutical compositions comprising the same, and uses thereof in diagnosis or treatment. BACKGROUND

[0004] In recent years, fibroblast activation protein (FAP) has been widely known as a marker of cancer-associated fibroblasts (CAFs). Due to the ubiquitous presence of intratumoral CAFs and stroma, FAP has been found to be a suitable marker for radio pharmaceutical diagnosis and a suitable target for radio pharmaceutical therapy. FAP is a 97 kDa type II transmembrane serine protease containing dipeptidyl peptidase and endopeptidase activities. Under physiological conditions, FAP is not expressed or lowly expressed in normal adult tissues, but is selectively and significantly highly expressed on the surface of CAFs in more than 90% of epithelial malignancies, such as breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, and lung cancer, etc. In addition, FAP is also expressed on some tumor cells, such as sarcoma, mesothelioma, and esophageal cancer, etc. Therefore, FAP has become an important target for tumor clinical diagnosis and treatment, and has attracted more and more attention.

[0005] In recent years, radionuclide conjugated drugs targeting FAP have rapidly developed in tumor diagnosis and treatment, including novel FAPI derivatives with cyclic peptides as binding units. However, there is still a need in the art to provide agents against fibroblast activation protein, which not only need to maintain the excellent tumor tissue targeting of current FAPI, but also need to solve the problem of rapid clearance in tumor tissue, so as to have necessary chemical stability and suitable metabolic kinetics, greatly improve the uptake and retention at the tumor, still keep low uptake at non-target organs, have significantly reduced toxicity compared with traditional strategies for improving tumor retention, and meet the needs of radionuclide therapy and imaging to improve and enhance the effect of tumor treatment.

[0006] Covalent inhibitors are a class of inhibitors that can reversibly or irreversibly bind to target protein residues through covalent bonds to exert their biological functions. Common covalent warheads are electrophiles, which can form reversible or irreversible covalent bonds. Covalent warheads that can be used in vivo and ultimately become drugs are generally "latent electrophiles", that is, they are only activated to accelerate the formation of covalent bonds when combined with specific proteins, have good bio-orthogonality, and thus minimize off-target toxicity related to warhead activity. SUMMARY

[0007] The present application introduces covalent warheads into FAPI derivatives with cyclic peptides as binding units, thereby providing a class of cyclic peptide FAPI derivatives containing covalent warheads. The cyclic peptide FAPI derivatives have beneficial target organ uptake and retention and reduced uptake and retention at non-target organs, optimized sensitivity and / or specificity, improved safety treatment window, and improved bioavailability, thereby improving the therapeutic effect while ensuring safety.

[0008] Therefore, in one aspect of the present disclosure, a compound of Formula (I’), a tautomer, a stereoisomer, a solvate thereof, or a pharmaceutically acceptable salt thereof is provided:

[0009] wherein:

[0010] the ring W is a cyclic peptide moiety comprising 6 to 22 residues of amino acids or amino acid derivatives;

[0011] P is a chelating moiety that can chelate a radionuclide;

[0012] Linker is a linker moiety covalently linking ring W to P;

[0013] at least one of the ring W and Linker comprises a covalent binding warhead selected from -SO2R S , -OSO2R S, -N(R)SO2R S , -S(=N)O(R)(R S ), -OP(O)(R S )N(R)2, -N(R)COCH=CH2, or -N(R)COC≡CCH3;

[0014] R S is halogen; and

[0015] R is H or alkyl.

[0016] In another aspect of the disclosure, there is provided a compound of Formula (I), a tautomer, a stereoisomer, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0017] wherein:

[0018] z is an integer from 6 to 22;

[0019] Xaa1, Xaa2, Xaa3,, Xaaz-2, Xaaz-1, and Xaaz are each independently an amino acid residue selected from Cys, Pro, Thr, Gin, Phe, Tyr, Asn, Gly, Glu, Ala, Val, Leu, lie, Trp, Asp, His, Lys, Met, Arg, Ser, Sec, Pyl, and derivatives thereof;

[0020] Y is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; and

[0021] m and n are each independently an integer from 0 to 6.

[0022] In another aspect of the disclosure, there is provided a compound of Formula (II), a tautomer, a stereoisomer, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0023] In yet another aspect of the disclosure, there is provided a chelate comprising a compound of the disclosure, a tautomer, a stereoisomer, or a solvate thereof, or a pharmaceutically acceptable salt thereof, and a radionuclide.

[0024] In yet another aspect of the disclosure, there is provided a pharmaceutical composition comprising a chelate of the disclosure, and a pharmaceutically acceptable carrier.

[0025] In another aspect of the disclosure, there is provided use of a chelate or a pharmaceutical composition of the disclosure in the manufacture of a medicament for the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in a subject.

[0026] In yet another aspect of the present disclosure, a kit comprising or consisting of a chelate or a pharmaceutical composition of the present disclosure, and instructions for use in the diagnosis or treatment of a disease is provided. DETAILED DESCRIPTION

[0027] Before the application is further described, certain terms used in the specification, examples, and appended claims are described in the following sections. The definitions listed herein are to be read in light of the remainder of the application and are understood to mean what a person of ordinary skill in the art would understand upon reading the remainder of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] DEFINITIONS

[0029] Unless otherwise indicated, when a range of values is disclosed, it is meant to include each and every value and sub-range within the range. For example, a range of 1 to 6 is intended to include each and every integer from 1 to 6, as well as sub-ranges such as 1-6, 2-5, 2-4, etc.

[0030] The specification of this disclosure should be construed to cover all chemically and / or toxicologically possible isomers, as well as their pharmaceutically acceptable salts, where the laws of chemistry and biochemistry permit.

[0031] The use of the terms "include", "includes" or "including" and variants thereof in the specification are meant to encompass the items listed thereafter and equivalents thereof as well as additional items not listed thereafter. The terms "comprise", "comprises" or "comprising" and variants thereof when used in this specification are used to describe one or more features, steps, components, elements, or the like, but do not preclude the presence or addition of one or more other features, steps, components, elements, or the like.

[0032] The term "pharmaceutically acceptable" in the present application means that the compound or composition is chemically and / or toxicologically acceptable, i.e. the compound or composition is compatible chemically and / or toxicologically with the other ingredients of a formulation or with the mammal, human or human tissue to which it is administered.

[0033] The term "subject" or "patient" in the present application includes humans and mammals.

[0034] In the context of the present application, the term "treatment" can also include prevention, unless explicitly indicated to the contrary.

[0035] The terms "alkyl", "alkylene" refer to a saturated straight chain or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably 1 to 6 carbon atoms, most preferably 1 to 3 carbon atoms. Said alkyl is for example methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl. Said alkyl and alkylene groups are optionally substituted.

[0036] The terms "alkenyl", "alkenylene" refer to a straight chain or branched carbon chain containing one or more carbon-carbon double bonds. Suitably, containing 2 to 30 carbon atoms, preferably about 3 to about 25, 5-20 carbon atoms. Said alkenyl and alkenylene groups are optionally substituted.

[0037] The terms "alkynyl", "alkynylene" refer to a straight chain or branched carbon chain containing one or more carbon-carbon triple bonds. Suitably, containing about 3 to about 30 carbon atoms, for example about 3 to about 25, 5-20 carbon atoms. Said alkynyl and alkynylene groups are optionally substituted.

[0038] The term "heteroalkyl" refers to a saturated straight chain or branched carbon chain. Preferably, the chain contains 1 to 9 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms, preferably 1 to 6 carbon atoms, most preferably 1 to 3 carbon atoms, for example methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, which is interrupted one or more than one, for example 1, 2, 3, 4, 5, by the same or different heteroatom, for example -O-CH3, -S-CH3, -CH2-O-CH3, -CH2-O-C2H5, -CH2-S-CH3, -CH2-S-C2H5, -C2H4-O-CH3, -C2H4-O-C2H5, -C2H4-S-CH3, -C2H4-S-C2H5, and the like. Said heteroalkyl groups are optionally substituted.

[0039] The terms "cycloalkyl", "cycloalkylene", "heterocycloalkyl" and "heterocycloalkylene", alone or in combination with other terms, mean, respectively, cyclic versions of "alkyl", "alkylene", "heteroalkyl" and "heteroalkylene", wherein preferably 3 to 10 atoms, i.e. 3, 4, 5, 6, 7, 8, 9 or 10 atoms are in the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, and the like. The terms "cycloalkyl", "cycloalkylene", "heterocycloalkyl" and "heterocycloalkylene" are also meant to include bicyclic, tricyclic and polycyclic versions thereof. The terms "heterocycloalkyl", "heterocycloalkylene" preferably mean a five-membered saturated ring, wherein at least one ring member is an N, O or S atom, and which optionally contains one additional O or one additional N; a six-membered saturated ring, wherein at least one ring member is an N, O or S atom, and which optionally contains one additional O or one additional N or two additional N atoms; or a nine- or ten-membered saturated bicyclic ring, wherein at least one ring member is an N, O or S atom, and which optionally contains one, two or three additional N atoms. "Cycloalkyl", "cycloalkylene", "heterocycloalkyl" and "heterocycloalkylene" are optionally substituted. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like. Examples of heterocycloalkyl groups include 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,4-diazabicyclo[2.2.2]oct-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Examples of heterocycloalkylene groups include piperidinylene, piperazinylene, pyrrolidinylene, 1,4-piperidinylene, 1,4-piperazinylene, 1,2-pyrrolidinylene, and the like.

[0040] The terms "aryl", "arylene" preferably contain an aromatic system of 6 to 14 carbon atoms, including a monocyclic, bicyclic system or tricyclic system, such as an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic system containing 10 carbon atoms or an aromatic tricyclic system containing 14 carbon atoms. Examples are phenyl, naphthyl or anthryl, phenylene, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene. Aryl or arylene are optionally substituted.

[0041] The term "heteroaryl", "heteroarylene" preferably means a 5- to 16-membered aromatic ring system, wherein 1, 2, 3, 4, 5 or 6 carbon atoms are replaced by identical or different heteroatoms, preferably selected from the group consisting of O, N and S; including monocyclic, bicyclic or tricyclic systems; such as a five- or six-membered aromatic monocyclic ring, wherein at least one carbon atom is replaced by 1, 2, 3 or 4 (for a five-membered ring) or 1, 2, 3, 4 or 5 (for a six-membered ring) identical or different heteroatoms, preferably selected from the group consisting of O, N and S; an aromatic bicyclic ring system, wherein 1, 2, 3, 4, 5 or 6 of the 8, 9, 10, 1 1 or 12 carbon atoms are replaced by identical or different heteroatoms, preferably selected from the group consisting of O, N and S; or an aromatic tricyclic ring system, wherein 1, 2, 3, 4, 5 or 6 of the 13, 14, 15, 16 carbon atoms are replaced by identical or different heteroatoms, preferably selected from the group consisting of O, N and S. Heteroaryl or heteroarylene is optionally substituted. Examples are oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1 -benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 2-benzothiophenyl, 1 H-indazolyl, benzimidazolyl, benzoxazolyl, indolizinyl, 2,1 -benzoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1 -benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0042] The term "halogen" means fluorine, chlorine, bromine, iodine and astatine.

[0043] The term "amino acid" as commonly used in the art by those skilled in the art is an organic compound that contains a basic amino group and an acidic carboxyl group, the compound formed by the substitution of the hydrogen atom on the carbon atom of the carboxylic acid with an amino group, including both naturally and non-naturally derived amino acids, having the formula

[0044] Like the hydroxy acids, amino acids can be classified as alpha-, beta-, gamma-, omega-... depending on the position of the amino group on the carbon chain, but the amino acids obtained by hydrolysis of proteins are all alpha-amino acids or imino acids, and there are only twenty-two of them, including glycine, alanine, valine, leucine, isoleucine, methionine (met), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine and pyrrolysine. The amino acids are abbreviated as follows:

[0045] Unless otherwise indicated, all amino acids can be in either the L (laevorotatory) or D (dextrorotatory) configuration.

[0046] As used herein, the term "amino acid residue" refers to the moiety remaining after the dehydration of amino acid molecules to form a peptide bond, typically having the structure -NH-CHR-C(=O)-. In the present application, the term "amino acid residue" includes both the residues of natural amino acids and the residues of modified amino acids, such as dimethylarginine residues and the like.

[0047] As used herein, the term "linker" refers to any chemically suitable linker. Preferably, the linker is not broken or only slowly broken under physiological conditions.

[0048] The expression "optionally substituted" means that one, two, three or more than three hydrogen atoms of a group can be replaced, independently of each other, by a substituent. The substituents can be selected from the group consisting of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halogen, cyano, amino, nitro, -OH, alkoxy, alkylamino or -COOH.

[0049] The compounds of the present application can exist in particular geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis- and trans-forms, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as the racemic mixtures and other mixtures thereof, such as those that are enantiomeric or diastereomeric enriched, all falling within the scope of the application. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the present application.

[0050] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the application is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to yield the pure desired enantiomer. Alternatively, when a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group) is present in the molecule, a diastereomeric salt form of the compound with an appropriate optically active acid or base can be formed, and the desired enantiomer recovered through fractional crystallization or chromatography of the separated diastereomers, followed by recovery of the pure enantiomer. In addition, separation of the enantiomers and diastereomers is often accomplished by the use of chromatography with a chiral stationary phase, optionally in combination with chemical derivatization.

[0051] The term "enantiomer" or "rotamer" means a stereoisomer whose mirror image is not superimposable unless otherwise specified.

[0052] The term "cis-trans isomer" or "geometric isomer" is caused by the inability of a double bond or ring-forming carbon atom single bond to rotate freely unless otherwise specified.

[0053] The term "diastereomer" means a stereoisomer whose molecules have two or more chiral centers and whose molecules are nonmirror images of one another unless otherwise specified.

[0054] "(D)" or "(+)" means dextrorotary, "(L)" or "(-)" means levorotary, and "(DL)" or "(±)" means racemic unless otherwise specified.

[0055] Unless otherwise indicated, a wedged solid line bond and a wedged dashed line bond indicate the absolute configuration of a stereocenter, and a straight solid line bond and a straight dashed line bond indicate the relative configuration of a stereocenter. When the isomeric form of a compound is not explicitly set forth, the chemical formula or structure is intended to encompass all isomeric forms of the compound.

[0056] As used herein, a "radionuclide" is a radioactive isotope of an element that emits alpha particles, beta particles, and / or gamma rays. The radionuclides include, but are not limited to, the following species: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th and 199 Ag.

[0057] The terms "chelator" or "chelate" are used interchangeably in the context of the present application to refer to a molecule, typically an organic molecule, typically a Lewis base, having two or more unshared electron pairs available to a metal ion. The metal ion is typically coordinated to the chelator through two or more electron pairs. The terms "bidentate chelator", "tridentate chelator" and "tetradentate chelator" refer to chelators having two, three and four electron pairs, respectively, which are readily available to a metal ion coordinated through the chelator. Typically, the electron pairs of the chelator form coordinate bonds with a single metal ion. However, in certain instances, the chelator can form coordinate bonds with more than one metal ion, and multiple binding modes are possible. The term "chelating group" refers to the group formed upon removal of one or more hydrogen atoms from a "chelator" or "chelate". Suitable pharmaceutically acceptable salts of the compounds of the present disclosure can be, for example, acid addition salts of the compounds of the present disclosure which carry a sufficiently basic nitrogen atom in a chain or ring, for example, with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxyphenyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentane propionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene disulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid or thiocyanic acid.

[0058] In addition, another suitable pharmaceutically acceptable salt of a compound of the present application having a sufficiently acidic group is a salt with an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium, an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, such as a salt with N-methylglucosamine, dimethylglucosamine, ethylglucosamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxymethylaminomethane, aminopropandiol, 1-amino-2,3,4-butantriol. In addition, a basic nitrogen-containing group can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfate; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides like benzyl and phenethyl bromides and others.

[0059] Those skilled in the art will also recognize that acid addition salts of the claimed compounds can be prepared by reacting a compound of the invention with the appropriate inorganic or organic acid and that alkali and earth alkaline salts of acidic compounds of the disclosure are prepared by reacting an acidic form of the compound with the appropriate base.

[0060] The present application includes all possible salts of the compounds of the present disclosure, which can be either single salts or any mixture of the salts in any ratio.

[0061] The term "solvate" refers to a physical association between a compound of the present application and a solvent molecule, for example a hydrate, a monohydrate, or a hemi- hydrate, where the ratio between the compound of the present application and the solvent molecule is about 2:1, about 1:1, or about 1:2, respectively. This physical association involves varying degrees of ionic and co-ionic bonding (including hydrogen bonding). In some cases (for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), solvates can be isolated. Thus, solvates include solution phases and isolatable solvates. The compounds of the present application can be in solvated forms with pharmaceutically acceptable solvents such as water, methanol, and ethanol, and the present application is intended to cover both solvated and unsolvated forms of the compounds of the present application. One solvate is a hydrate.

[0062] The compounds of the present disclosure can contain one or more asymmetric centers, depending on the positions and nature of the various substituents desired. The asymmetric carbon atoms can exist in the (R)- or (S)-configuration, giving rise to racemic mixtures in the case of one asymmetric center, and diastereomeric mixtures in the case of multiple asymmetric centers. In certain cases, asymmetry can also exist due to hindered rotation about a particular bond, for example, the central bond connecting two substituted aromatic rings of a particular compound.

[0063] Preferred compounds are those that produce more desirable biological activity. Isolated, purified or partially purified isomers and stereoisomers, or racemic or diastereomeric mixtures of the compounds of the present disclosure are included within the scope of the present application. Purification and isolation of such substances can be achieved by standard techniques known in the art.

[0064] The term "pharmaceutical composition" as used herein refers to a substance and / or combination of substances used to identify, prevent, or treat a state of tissue or disease. A pharmaceutical composition is formulated to be suitable for administration to a patient to diagnose, prevent, and / or treat a disease. Additionally, a pharmaceutical composition refers to the combination of an active agent with a non-active or active carrier, which makes the composition suitable for therapeutic use.

[0065] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0066] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such a pharmaceutical carrier can be a sterile liquid, such as a saline solution in water or oil, including those of either vegetable or animal origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0067] The term "optionally" means that the situation can or can not occur.

[0068] The term "not directly linked by a covalent bond" means that there is at least one carbon atom between the two, which carbon atom can be present as C, CH, CH2, or C=0.

[0069] Compound

[0070] In one aspect, the present disclosure provides a compound of Formula (I’), a tautomer, stereoisomer, solvate thereof, or a pharmaceutically acceptable salt thereof:

[0071] wherein:

[0072] Ring W is a cyclic peptide moiety comprising 6 to 22 residues of amino acids or amino acid derivatives;

[0073] P is a chelating moiety that can chelate a radionuclide;

[0074] Linker is a linker moiety covalently linking Ring W to P;

[0075] at least one of Ring W and Linker comprises a covalently bound warhead selected from -SO2R S , -OSO2R S , -N(R)SO2R S , -S(=N)O(R)(RS ), -OP(O)(R S -N(R)2, -N(R)COCH=CH2 or -N(R)COC≡CCH3;

[0076] R S It is halogen; and

[0077] R is H or an alkyl group.

[0078] In some embodiments, the compound represented by formula (I') provided in this disclosure is not

[0079] In some embodiments of the compound of formula (I'), ring W comprises a covalently bonded warhead.

[0080] In some embodiments of the compound of formula (I'), the Linker comprises a covalently bonded warhead.

[0081] In some embodiments, the compounds disclosed herein have formula (I):

[0082] in:

[0083] z is an integer from 6 to 22;

[0084] Xaa1, Xaa2, Xaa3, ..., Xaaz-2, Xaaz-1 and Xaaz are each independently amino acid residues selected from Cys, Pro, Thr, Gln, Phe, Tyr, Asn, Gly, Glu, Ala, Val, Leu, Ile, Trp, Asp, His, Lys, Met, Arg, Ser, Sec, Pyl and their derivatives;

[0085] Y is a cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; and

[0086] m and n are each an independent integer from 0 to 6.

[0087] In some embodiments of the compound of Formula (I), Y is C4-C7cycloalkylene (e.g., C4-C6cycloalkylene or C4-C5cycloalkylene, such as C7cycloalkylene, C6cycloalkylene, C5cycloalkylene, or C4cycloalkylene), 5- to 10-membered heterocycloalkylene (e.g., 5- to 9-membered heterocycloalkylene, 5- to 8-membered heterocycloalkylene, 5- to 7-membered heterocycloalkylene, or 5- to 6-membered heterocycloalkylene, such as 10-membered heterocycloalkylene, 9-membered heterocycloalkylene, 8-membered heterocycloalkylene, 7-membered heterocycloalkylene, 6-membered heterocycloalkylene, or 5-membered heterocycloalkylene), C6-C8arylene (e.g., C6-C7arylene, such as C8arylene, C7arylene, or C6arylene), or 5- to 10-membered heteroarylene (e.g., 5- to 9-membered heteroarylene, 5- to 8-membered heteroarylene, 5- to 7-membered heteroarylene, or 5- to 6-membered heteroarylene, such as 10-membered heteroarylene, 9-membered heteroarylene, 8-membered heteroarylene, 7-membered heteroarylene, 6-membered heteroarylene, or 5-membered heteroarylene).

[0088] In some embodiments of the compound of Formula (I), Y is cyclohexyl, phenyl, thienyl, furanyl, imidazolyl, pyrrolidinyl, pyranyl, pyridyl, piperazinyl, piperidinyl, pyrimidinyl, pyridazinyl, quinolinyl, or naphthyridinyl.

[0089] In some embodiments of the compound of Formula (I), Y is In some embodiments, Y is

[0090] In some embodiments of the compound of Formula (I), m is an integer from 0 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6. In some embodiments, m is 3. In some embodiments, m is 2. In some embodiments, m is 1.

[0091] In some embodiments of the compound of Formula (I), n is an integer from 0 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, 5 to 6. In some embodiments, n is 3. In some embodiments, n is 2. In some embodiments, n is 1.

[0092] In some embodiments of the compound of Formula (I), m is 1 and n is 1.

[0093] In some embodiments of the compound of Formula (I), P is selected from the group consisting of:

[0094] In some embodiments of the compound of Formula (I) and Formula (I’), at least one of the residues of the amino acids or amino acid derivatives of the cyclic peptide moiety comprises a covalently bound warhead. In some embodiments, the covalently bound warhead is -OSO2R S group. In some embodiments, RS halogen. In some embodiments, R S is F.

[0095] In some embodiments of the compound of formula (I), at least one of Xaa2, Xaa3,..., Xaaz-2and Xaaz-1is an amino acid residue having the structure: wherein X1is CH or N. In some embodiments, is selected from

[0096] In some embodiments of the compound of formula (I), Xaaz-1is In some embodiments, R S halogen. In some embodiments, R S is F.

[0097] In some embodiments of the compound of formula (I), at least one of Xaa2, Xaa3,..., Xaaz-2and Xaaz-1is an amino acid residue having the structure:

[0098] In some embodiments of the compound of formula (I), Xaaz-1is

[0099] In some embodiments of the compound of formula (I), Xaa1is a Cys derivative residue having the structure:

[0100] wherein R 1 is alkyl-L 1 -L 2 -; L 1 and L 2 are each independently selected from a direct bond, -CO-, -NH-, -CONH-, -NHCO- or -SO2-; the * end indicates the point of attachment of Xaa1to Xaa2.

[0101] In some embodiments of the compound of formula (I), Xaa1has the structure wherein L 1 is selected from a direct bond, -NH- or -NHCO-, L 2 is -CO-. In some embodiments, R 1 is C1-C6alkyl-CO-, C1-C6alkyl-NHCO- or C1-C6alkyl-NHCOCO-.

[0102] In some embodiments of the compound of formula (I), Xaazis a Cys residue having the structure:

[0103] wherein R2is -OR or -N(R)2, R is H or alkyl (e.g., C1-C6alkyl, C1-C5alkyl, C1-C4alkyl, C1-C3alkyl, or C1-C2alkyl, such as C6alkyl, C5alkyl, C4alkyl, C3alkyl, C2alkyl, or C1alkyl), and the * end indicates the point of attachment of Xaazto Xaaz-i.

[0104] In some embodiments of the compound of formula (I), z is an integer from 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, or 6-7. In some embodiments, z is 8. In some embodiments, z is 7. In some embodiments, z is 6.

[0105] In some embodiments of the compound of formula (I), the compound is of formula (II):

[0106] In some embodiments of the compound of formula (II), R 1 is C3-C5alkyl-CO-, C3-C5alkyl-NHCO-, or C3-C5alkyl-NHCOCO-, and R 2 is -OH or -NH2.

[0107] In some embodiments of the compound of formula (II), Xaa2and Xaa3are Pro, Xaa4is Thr, and Xaa5is Gin.

[0108] In some embodiments of the compound of formula (I) and formula (II), Linker is p -L1-L2-L3-L4-L5-(CH2) e -L6-(CH2) r - wherein the * end is attached to P; L1, L2, L4, and L5are each independently selected from a direct bond, -O-, -CO-, -NH-, -CONH-, or -NHCO-; L3is alkylene (e.g., C1-C6alkylene, C1-C5alkylene, C1-C4alkylene, C1-C3alkylene, or C1-C2alkylene, such as C6alkylene, C5alkylene, C4alkylene, C3alkylene, C2alkylene, or C1alkylene); L6is selected from -S-, -O-, -NH-, or -N(alkyl)-; and p, e, and r are each independently an integer from 0 to 6.

[0109] In some embodiments of the compound of formula (I) and formula (II), Linker is In some embodiments of the compound of formula (I) and formula (II), Linker is

[0110] In some embodiments of the compounds of Formula (I) and Formula (II),

[0111] In some embodiments of the compounds of Formula (I), the Linker comprises a covalently bound warhead. In some embodiments, the covalently bound warhead is -OSO2R S In some embodiments, R S is halogen. In some embodiments, R S is F.

[0112] In some embodiments of the compounds of Formula (I), the Linker moiety is *-(CH2) p -L1-L2-L3-L4-L5-(CH2) e -L6-(CH2) r - wherein the * end is attached to P; each of L1, L2, L4, and L5 is independently selected from a direct bond, -CO-, -NH-, -CONH-, or -NHCO-; L3 is wherein X1 is CH or N; L6 is selected from -S-, -O-, -NH-, or -N(alkyl)-; and each of p, a, b, c, e, and r is independently an integer from 0 to 6. In some embodiments, L3 is selected from In some embodiments, R S is halogen. In some embodiments, R S is F.

[0113] In some embodiments of the compounds of Formula (I), the Linker moiety is wherein the * end is attached to P. In some embodiments, P is

[0114] In some embodiments of the compounds of Formula (I), Xaa1 is a Cys derivative residue having the structure:

[0115] wherein R 1 is alkyl-L 1 -L 2 -; each of L 1 and L 2 is independently selected from a direct bond, -CO-, -NH-, -CONH-, -NHCO-, or -SO2-; the * end indicates the point of attachment of Xaa1 to Xaa2.

[0116] In some embodiments, L 1 is selected from a direct bond, -NH-, or -NHCO-, L 2 is -CO-.​

[0117] In some embodiments, R 1 is C1-C6alkyl-CO-, C1-C6alkyl-NHCO- or C1-C6alkyl-NHCOCO-.

[0118] In some embodiments, Xaazis a Cys residue having the structure: wherein R2is -OR or -NR2, R is H or alkyl, and the * end indicates the point of attachment of Xaazto Xaaz-1.

[0119] In some embodiments, z is an integer from 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, or 6-7. In some embodiments, z is 8. In some embodiments, z is 7. In some embodiments, z is 6.

[0120] In some embodiments, z is 7, Xaa2and Xaa3are Pro, Xaa4is Thr, Xaa5is Gln, and Xaa6is Phe.

[0121] In some embodiments, the present disclosure provides a compound selected from the group consisting of:

[0122] In some embodiments, the present disclosure provides a compound selected from the group consisting of:

[0123] Chelate

[0124] In yet another aspect, the present disclosure provides a chelate comprising a compound of the present disclosure, a tautomer, stereoisomer, or solvate thereof, or a pharmaceutically acceptable salt thereof, and a radionuclide.

[0125] In some embodiments, the radionuclide is selected from the group consisting of: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99 mTc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153Sm, 166 Ho, 86 Y, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th and 199 Ag.

[0126] In some embodiments, the chelates of the present disclosure can be inhibitors of fibroblast activation protein. Accordingly, the present disclosure provides the use of the chelates as inhibitors of fibroblast activation protein.

[0127] Pharmaceutical compositions and kits

[0128] The present disclosure also provides pharmaceutical compositions comprising the chelates of the present disclosure and a pharmaceutically acceptable carrier.

[0129] The present disclosure also provides pharmaceutical compositions for use in: (a) a method of treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body; or (b) a method for the treatment or prevention of a subject having or at risk of having a disease or disorder; or (c) a method for directing surgery on a subject having or at risk of having a disease or disorder; or (d) a method for diagnosing a disease or disorder, the method practiced on the human or animal body and involving nuclear medicine imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT); or (e) a method for targeted delivery of a therapeutic or diagnostic agent to a subject having or at risk of having a disease or disorder, wherein in each of the foregoing (b)-(e), the disease or disorder is characterized by overexpression of fibroblast activation protein (FAP), e.g., the disease or disorder is independently selected from a cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, or a scarring disease. In some embodiments, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular cancer, clear cell kidney cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymic carcinoma, glioma, glioblastoma, astrocytoma, cervical cancer, or prostate cancer.

[0130] Acceptable carriers for therapeutic use are well-known in the medical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier can be selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions can comprise, as

[0131] Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0132] Depending on the different delivery systems, there can be different composition / formulation requirements. For example, the pharmaceutical composition can be formulated for administration using a mini-pump or by a mucosal route, for example as a nasal spray or aerosol for inhalation or as an ingestible solution, or parenterally, wherein the composition is formulated in an injectable form for delivery by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the pharmaceutical composition / formulation can be designed for administration by a variety of routes.

[0133] If the agent is to be administered transmucosally through the gastrointestinal mucosa, it should be able to remain stable during transit through the gastrointestinal tract; for example, it should resist proteolytic degradation, be stable at acidic pH and resist the detergent action of bile.

[0134] Where appropriate, the pharmaceutical composition can be administered by inhalation; in the form of a suppository; topically in the form of a lotion, solution, cream, salve, or powder; by use of a skin patch; orally in the form of tablets containing excipients such as starch or lactose, or capsules or ovules either alone or in combination with excipients, or elixirs, solutions or suspensions containing flavouring or colouring agents; or the pharmaceutical composition can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the pharmaceutical composition is best used in the form of a sterile aqueous solution, which can contain other substances, for example enough salts or monosaccharides to make the solution isotonic with the blood. For buccal or sublingual administration the pharmaceutical composition can be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0135] Routes for administration (delivery) can include, but are not limited to, one or more of oral (for example in the form of tablets, capsules or ingestible solutions), topical, transmucosal (for example in the form of nasal sprays or aerosols for inhalation), nasal, parenteral (for example by injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, trans-ocular (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

[0136] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. For any particular patient, specific dosage levels and frequency of administration will vary depending on a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health condition, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition being treated, and the individual undergoing therapy.

[0137] The formulations can be packaged in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind previously described. Exemplary unit-dose formulations contain a daily dose or unit daily sub-dose of an active ingredient.

[0138] In yet another aspect, the present disclosure provides a kit comprising a chelate or pharmaceutical composition of the present disclosure, and instructions for diagnosing or treating a disease.

[0139] Treatment

[0140] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof. The method can comprise administering to a subject in need thereof a chelate or pharmaceutical composition of the present disclosure. The method can provide therapeutic and / or prophylactic benefit to a subject in need thereof, including administering a chelate or pharmaceutical composition of the present disclosure.

[0141] In some embodiments, the methods of the present disclosure are used to treat a disease or disorder characterized by overexpression of fibroblast-activated protein (FAP). In some embodiments, the disease or disorder is independently selected from a cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, or a keloid disease. In some embodiments, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular cancer, clear cell kidney cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic carcinoma, glioma, glioblastoma, astrocytoma, cervical cancer, or prostate cancer.

[0142] In addition to the treatment methods described above, the chelates or pharmaceutical compositions described herein can be used in diagnostic methods practiced in a subject in need thereof, the methods comprising administering to a subject in need thereof a diagnostically effective amount of a chelate or pharmaceutical composition as described herein. Methods of the present disclosure for diagnosing a disease or disorder include, but are not limited to, diagnostic methods practiced on a subject in need thereof and / or involving nuclear medicine imaging techniques (e.g., positron emission tomography (PET) or single photon emission computed tomography (SPECT)), methods for targeted delivery of a diagnostic agent to a subject having a disease or disorder or a subject at risk of having a disease or disorder, and the like.

[0143] The application is further illustrated by the following examples. Unless otherwise defined, the terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For convenience, some of the compounds involved in the preparation of the application are referred to by number.

[0144] Compounds are named according to the nomenclature rules agreed upon by various chemical publications, or using software nomenclature, and commercially available compounds are named using the vendor catalog name.

[0145] Solvents used in the present application are commercially available.

[0146] The following abbreviations are used in the present application: eq represents equivalent, equivalents; TFA represents trifluoroacetic acid; DIEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide; HATU represents O-(7-azabenzotriazol-l-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate; HBTU represents benzotriazol-N,N,N,N-tetramethyluronium hexafluorophosphate; EDC represents l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS represents N-hydroxysuccinimide; Fmoc represents 9-fluorenylmethoxycarbonyl; Boc represents tert-butoxycarbonyl; Trt represents trityl; tBu represents tert-butyl; PBS represents phosphate buffered saline; 2-CTC Resin represents 2-chloro-tritylchloride.

[0147] Examples

[0148] Reagents and models used

[0149] The starting materials of the examples are commercially available and / or can be prepared in a variety of ways well known to one skilled in the art of organic synthesis. The skilled artisan in the art of organic synthesis will choose appropriate reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of the experiment, and work-up) in the following synthetic methods. The skilled artisan in the art of organic synthesis will appreciate that the functional groups present on various portions of the molecule should be compatible with the reagents and reactions proposed.

[0150] All reagents, compounds used in the synthesis are commercially available in China (excluding Hong Kong, Macau and Taiwan) through general commercial channels.

[0151] Synthetic route:

[0152] Reference Example 1: Preparation method of intermediate compound B1

[0153] Step 1: Synthesis of B1-2

[0154] ​To a solution of compound B1-1 (7.00 g, 20.75 mmol, 1.00 eq) in tetrahydrofuran (90 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (6.32 g, 41.49 mmol, 6.25 mL, 2.00 eq) and 4-(acetylamino)phenyl] imidodisulfide (7.17 g, 22.82 mmol, 1.10 eq) at 25 °C, and the reaction mixture was stirred at 25 °C for 20 min. After the reaction was completed, it was diluted with ethyl acetate (300 mL) and washed with saturated brine (60 mL x 3). It was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 0 / 1-1 / 1, volume ratio) to give compound B1-2. MS-ESI m / z: 420.2 [M+H] + . 1 H NMR (400 MHz, MeOD) δ: 7.44-7.40 (m, 2H), 7.39-7.35 (m, 2H), 4.26 (br dd, J = 6.3, 8.7 Hz, 1H), 3.12 (dd, J = 6.1, 13.8 Hz, 1H), 2.95 (dd, J = 9.1, 13.8 Hz, 1H), 1.41 (s, 9H), 1.39 (s, 9H). 19 F NMR (376 MHz, MeOD) δ: 35.20 (s, 1F).

[0155] Step 2: Synthesis of trifluoroacetate salt of B1-3

[0156] To a solution of compound B1-2 (5.00 g, 11.92 mmol, 1.00 eq) in dichloromethane (50 mL) was added trifluoroacetic acid (25 mL) at 25 °C, and the reaction mixture was stirred at 25 °C for 40 min. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give the trifluoroacetate salt of compound B1-3, which was used directly in the next step. MS-ESI m / z: 263.9 [M+H] + .

[0157] Step 3: Synthesis of B1

[0158] To a solution of compound B1-3 (10.00 g, 11.66 mmol, 1.00 eq, 44% purity, trifluoroacetate salt) in dioxane (100 mL) was added 9-fluorenylmethyl N-succinimidyl carbonate (3.93 g, 11.66 mmol, 1.00 eq) at 25 °C. The reaction was adjusted to pH = 8.5 with sodium bicarbonate solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, it was diluted with ethyl acetate (300 mL), and the aqueous phase was adjusted to pH = 5 with hydrochloric acid solution (1 M). The organic phases were combined, washed with saturated brine (25 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 0 / 1-1 / 9, volume ratio) to obtain compound B1. MS-ESI m / z: 484.0 [M-H] - . 1 H NMR (400 MHz, MeOD) δ: 7.77 (d, J = 7.5 Hz, 2H), 7.61-7.55 (m, 2H), 7.40-7.34 (m, 4H), 7.32-7.25 (m, 4H), 4.44 (dd, J = 4.7, 9.6 Hz, 1H), 4.33-4.21 (m, 2H), 4.15-4.11 (m, 1H), 3.26 (dd, J = 4.6, 13.9 Hz, 1H), 2.99 (dd, J = 9.7, 13.9 Hz, 1H). 19 F NMR (376 MHz, MeOD) δ: 35.35 (s, 1F).

[0159] Reference Example 2: Preparation method of intermediate compound B2

[0160] Step 1: Synthesis of B2-2

[0161] To a solution of N,N'-sulfonyldiimidazole (7.40 g, 37.33 mmol, 3.00 eq) in trifluoroacetic acid (24 mL) was added potassium fluoride (5.78 g, 99.54 mmol, 8.00 eq) at 25 °C, and the reaction mixture was stirred at 25 °C for 18 h. Then, in another reaction device, compound B2-1 (3.50 g, 12.44 mmol, 1.00 eq), triethylamine (5.20 mL, 37.33 mmol, 3.00 eq) were added to dichloromethane (50 mL), and the reaction mixture was stirred at 25 °C for 18 h. The two reaction devices were connected. After the reaction was completed, the reaction was concentrated under reduced pressure to obtain compound B2-2. MS-ESI m / z: 263.8 [M-Boc+H] + .

[0162] Step 2: Synthesis of trifluoroacetate salt of B2-3

[0163] To a solution of compound B2-2 (5.90 g, 11.37 mmol, 1.00 eq, 70% purity) in dichloromethane (20 mL) was added trifluoroacetic acid (20 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 2 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give the trifluoroacetate salt of compound B2-3, which was used directly in the next step. MS-ESI m / z: 263.8 [M+H] + .

[0164] Step 3: Synthesis of B2

[0165] To a solution of compound B2-3 (6.00 g, 11.13 mmol, 1.00 eq, trifluoroacetate salt, 70% purity) in dioxane (100 mL) was added 9-fluorenylmethyl-N-succinimidyl carbonate (3.76 g, 11.13 mmol, 1.00 eq) at 25 °C. The reaction solution was adjusted to pH = 8.5 with aqueous sodium bicarbonate solution, and the reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution was diluted with ethyl acetate (100 mL) and acidified with dilute hydrochloric acid solution (1 M) to adjust to pH = 5. The organic phase was combined, washed with saturated brine (60 mL x 2 x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by reverse phase high performance liquid chromatography (column: Biotage; 40 g Agela, C18, 20-35 μm, mobile phase: 0-60% acetonitrile / water (0.1% trifluoroacetic acid); flow rate: 35 mL / min) to give compound B2. MS-ESI m / z: 508.0 [M+Na] + . 1 H NMR (400 MHz, MeOD) δ: 7.78 (d, J = 7.5 Hz, 2H), 7.62-7.54 (m, 2H), 7.49-7.42 (m, 1H), 7.41-7.33 (m, 4H), 7.32-7.26 (m, 3H), 4.50-4.42 (m, 1H), 4.35-4.20 (m, 2H), 4.19-4.11 (m, 1H), 3.30-3.27 (m, 1H), 2.96 (br s, 1H). 19 F NMR (376 MHz, MeOD) δ: -77.54 (s, 1F).

[0166] Reference Example 3: Preparation method of intermediate compound B3

[0167] Step 1: Synthesis of B3-2

[0168] Into a 500 mL three-necked flask, was placed compound B3-1 (5.00 g, 19.13 mmol, 1.00 eq), triphenylphosphine (6.27 g, 23.92 mmol, 1.25 eq), imidazole (1.69 g, 24.87 mmol, 1.30 eq) and dichloromethane (150.0 mL). The reaction mixture was cooled to 0 °C, and iodine (6.31 g, 24.87 mmol, 5.01 mL, 1.30 eq) was added in three portions with 10 minutes interval under stirring. Finally, compound B3-1 (5.00 g, 19.13 mmol, 1.00 eq) was dissolved in 50.0 mL dichloromethane and added dropwise into the reaction mixture. The reaction mixture was stirred at 0 °C for 1 hour, and then stirred at 25 °C for 1 hour. After the reaction was completed, the reaction was quenched with 10.0 mL methanol. The reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 0 / 1-1 / 9, volume ratio) to give compound B3-2. MS-ESI m / z: 215.8 [M+H-Boc-t-Bu] + . 1 H NMR (400 MHz, CDC13) δ: 5.39-5.28 (m, 1H), 4.35 (td, J = 3.5, 7.2 Hz, 1H), 3.57 (br d, J = 3.3 Hz, 2H), 1.51 (s, 9H), 1.46 (s, 9H).

[0169] Step 2: Synthesis of B3-3

[0170] To a solution of zinc powder (2.80 g, 42.79 mmol, 3.53 eq) in N,N-dimethylformamide (50.0 mL) was added iodine (769.20 mg, 3.03 mmol, 0.25 eq) at 25 °C under nitrogen protection. The reaction mixture was stirred at 25 °C for 10 min under nitrogen protection. Then to the reaction mixture was added compound B3-2 (4.50 g, 12.12 mmol, 1.00 eq) and iodine (769.20 mg, 3.03 mmol, 0.25 eq) at 25 °C under nitrogen protection. The reaction mixture was stirred at 25 °C for 30 min under nitrogen protection. Then to the reaction mixture was added a mixture of 3-phenylmethoxy-5-bromopyridine (3.20 g, 12.12 mmol, 1.00 eq), tris(dibenzylideneacetone)dipalladium (1.11 g, 1.21 mmol, 0.10 eq), 2-dicylohexylphosphino-2,6-dimethoxybiphenyl (1.99 g, 4.85 mmol, 0.40 eq) in N,N-dimethylformamide (20.0 mL) at 50 °C under nitrogen protection. The reaction mixture was stirred at 50 °C for 16 h under nitrogen protection. After completion of the reaction, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (200.0 mL), filtered over celite, and the filtrate was concentrated under reduced pressure. The resulting crude was purified by column chromatography on silica gel (eluent: ethyl acetate / petroleum ether = 0 / 1-1 / 1, volume ratio) to give compound B3-3. MS-ESI m / z: 429.6 [M+H] + . 1 H NMR (400 MHz, CDCl3) d: 8.37-8.29 (m, 1H), 8.11 (br s, 1H), 7.43-7.37 (m, 5H), 7.35-7.32 (m, 1H), 5.08 (s, 3H), 4.50-4.38 (m, 1H), 3.16-3.00 (m, 2H), 1.40 (d, J = 2.9 Hz, 18H).

[0171] Step 3: Synthesis of B3-4

[0172] A fixed bed (named FLR1, volume 5 mL) was packed with granular catalyst 5% palladium on alumina (WXC1035, 3.30 g), and a hydrogen back pressure regulator was adjusted to 1.0 MPa with a hydrogen flow rate of 30 mL / min. Then a solution of compound B3-3 (2.80 g, 6.53 mmol, 1.00 eq) in methanol (30.0 mL) was pumped into the fixed bed (FLR1, SS, fixed bed, 6.350 (1 / 4) mm, 50 °C) with pump No. 1 (S1, P1, 0.3 mL / min). After the reaction was completed, the reaction mixture was collected from the output end of the reactor. The reaction mixture was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 0 / 1-1 / 9, volume ratio) to obtain compound B3-4. ESI m / z: 339.2 [M+H] + . 1 H NMR (400 MHz, MeOD_d4) δ: 7.98-7.93 (m, 1H), 7.89 (s, 1H), 7.14 (br d, J = 1.2 Hz, 1H), 4.26-4.18 (m, 1H), 3.07-2.99 (m, 1H), 2.92-2.84 (m, 1H), 1.41 (br d, J = 10.0 Hz, 18H).

[0173] Step 4: Synthesis of B3-5

[0174] To a solution of N,N'-sulfonyldiimidazole (5.62 g, 28.37 mmol, 6.00 eq) in trifluoroacetic acid (10.0 mL) was slowly added potassium fluoride (4.40 g, 75.65 mmol, 16.00 eq) at 0 °C, which slowly generated white gas. Meanwhile, in another reaction device, a solution of compound B3-4 (1.60 g, 4.73 mmol, 1.00 eq) in dichloromethane (16.0 mL) was added with triethylamine (1.97 mL, 14.18 mmol, 3.00 eq). The two reaction devices were connected, and the reaction mixture was stirred at 25 °C for 16 hours. After the reaction was completed, the reaction liquid was concentrated under reduced pressure to obtain compound B3-5, which was directly used in the next step. MS-ESI m / z: 421.4 [M+H] + .

[0175] Step 5: Synthesis of trifluoroacetate salt of B3-6

[0176] To a solution of compound B3-5 (3.50 g, 4.08 mmol, 1.00 eq, 49% purity) in dichloromethane (20.0 mL) was added trifluoroacetic acid (20.0 mL) at 15 °C. The reaction mixture was stirred at 15 °C for 8 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give trifluoroacetate salt of compound B3-6, which was used directly in the next step. MS-ESI m / z: 265.0 [M+H] + .

[0177] Step 2: Synthesis of B3

[0178] To a solution of trifluoroacetate salt of compound B3-6 (1.50 g, 3.97 mmol, 1.00 eq) in dioxane (15.0 mL) was added 9-fluorenylmethyl-N-succinimidyl carbonate (1.34 g, 3.97 mmol, 1.00 eq) at 15 °C. The reaction solution was adjusted to pH = 8.5 with aqueous sodium bicarbonate solution, and the reaction mixture was stirred at 15 °C for 2 h. After the reaction was completed, the reaction solution was diluted with ethyl acetate (100.0 mL) and acidified with dilute hydrochloric acid solution (1 M) to adjust to pH = 5. The organic phase was combined, washed with saturated brine (60.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 0 / 1-1 / 9, volume ratio) and then by high performance liquid chromatography preparation (column: 53-YMC Actus Triart C18 150 x 30 mm, 5 μm; mobile phase: [water (0.1% trifluoroacetic acid) - acetonitrile]; acetonitrile: 38% - 68%, 11 min) to give compound B3. MS-ESI m / z: 487.2 [M+H] + . 1 H NMR (400 MHz, MeOD_d4) δ: 8.62-8.56 (m, 2H), 7.91 (br s, 1H), 7.78 (d, J = 7.5 Hz, 2H), 7.62-7.56 (m, 2H), 7.40-7.35 (m, 2H), 7.31-7.26 (m, 2H), 4.51 (br dd, J = 4.7, 9.6 Hz, 1H), 4.33-4.23 (m, 2H), 4.18-4.12 (m, 1H), 3.38 (br dd, J = 4.6, 14.1 Hz, 1H), 3.08 (br dd, J = 10.0, 14.0 Hz, 1H).

[0179] Reference Example 4: Preparation method of intermediate compound B4

[0180] Step 1: Synthesis of B4-2

[0181] To a solution of compound B4-1 (2.00 g, 10.30 mmol, 1.00 eq) in tetrahydrofuran (20.0 mL) was added [4-(acetylamino)phenyl] imino- dithiodifluoride (3.88 g, 12.36 mmol, 1.20 eq) and 1,8-diazabicyclo[5.4.0]undec-7- ene (2.04 g, 13.39 mmol, 2.02 mL, 1.30 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 10 min. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-4 / 1, volume ratio) to obtain compound B4-2. 1 H NMR (400 MHz, CDCl3) δ: 8.06 (td, J = 1.4, 7.4 Hz, 1H), 7.94 (s, 1H), 7.58-7.51 (m, 2H), 1.61 (s, 9H). 19 F NMR (376 MHz, CDCl3) δ: 38.20 (s, 1F).

[0182] Step 2: Synthesis of B4-3

[0183] To a solution of compound B4-2 (2.23 g, 8.07 mmol, 1.00 eq) in dichloromethane (10.0 mL) was added trifluoroacetic acid (10.0 mL) at 25 °C. The reaction mixture was stirred at 25 °C for 20 min. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product of compound B4-3, which was used directly in the next step.

[0184] Step 3: Synthesis of B4-4

[0185] To a solution of compound B4-3 (1.17 g, 5.31 mmol, 1.00 eq) in tetrahydrofuran (20.0 mL) was added N,N-dicyclohexylcarbodiimide (1.32 g, 6.38 mmol, 1.29 mL, 1.20 eq) and N-hydroxysuccinimide (672.73 mg, 5.85 mmol, 1.10 eq) at 20 °C. The reaction mixture was stirred at 20 °C for 1 h. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product of compound B4-4.

[0186] Step 4: Synthesis of B4-5

[0187] To a solution of compound B4-4 (1.69 g, 5.33 mmol, 1.00 eq) in tetrahydrofuran (15.0 mL) was added a solution of Boc-L-lysine (1.57 g, 6.39 mmol, 1.20 eq) and triethylamine (1.48 mL, 10.65 mmol, 2.00 eq) in N,N-dimethylformamide (15.0 mL) at 20 °C, and the reaction mixture was continued to stir at 20 °C for 16 h. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the crude compound B4-5. MS-ESI m / z: 449.3 [M+H] + .

[0188] Step 5: Synthesis of B4

[0189] To a solution of compound B4-5 (2.39 g, 5.33 mmol, 1.00 eq) and N-hydroxysuccinimide (920.01 mg, 7.99 mmol, 1.50 eq) in tetrahydrofuran (30.0 mL) was added N,N-dicyclohexylcarbodiimide (1.65 g, 7.99 mmol, 1.62 mL, 1.50 eq) at 20 °C, and the reaction mixture was stirred at 20 °C for 1 h. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-1 / 1, by volume) to give compound B4. MS-ESI m / z: 446.0 [M-Boc+H] + . 1 H NMR (400 MHz, CDCl3) d: 7.92 (d, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.59-7.51 (m, 1H), 7.50-7.44 (m, 1H), 6.95-6.75 (m, 1H), 5.18 (br d, J = 6.7 Hz, 1H), 4.73 (br s, 1H), 3.65-3.40 (m, 2H), 2.85 (br s, 4H), 1.97 (br d, J = 6.9 Hz, 2H), 1.78-1.67 (m, 2H), 1.63-1.54 (m, 2H), 1.49 (s, 9H). 19 F NMR (376 MHz, CDCl3) d: 38.23 (s, 1F).

[0190] Reference Example 5: Preparation method of intermediate compound B5

[0191] Step 1: Synthesis of B5-2

[0192] To a solution of 1,1-sulfonyldiimidazole (8.55 g, 43.13 mmol, 3.00 eq) in trifluoroacetic acid (30.0 mL) was added potassium fluoride (6.68 g, 115.02 mmol, 8.00 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. Then compound B5-1 (2.00 g, 14.38 mmol, 1.00 eq), triethylamine (6.00 mL, 43.13 mmol, 3.00 eq) was added to dichloromethane (50.0 mL) in another reaction apparatus, and the reaction mixture was stirred at 25 °C for 16 h. The two reaction apparatuses were connected, and after the reaction was completed, the reaction solution was adjusted to pH = 5 with 1M dilute hydrochloric acid, and the reaction solution was filtered, the filtrate was added with dichloromethane (30.0 mL x 2), the organic phase was washed with saturated brine 4.5 mL (1.5 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was combined with another batch and slurried with petroleum ether / ethyl acetate (1 / 1, 30.0 mL) to give compound B5-2. MS-ESI m / z: 219.9 [M-H] - . 1 H NMR (400 MHz, DMSO-d6) δ: 9.18 (d, J = 1.5 Hz, 1H), 9.15 (d, J = 2.7 Hz, 1H), 8.54 (s, 1H).

[0193] Step 2: Synthesis of B5-3

[0194] To a solution of compound B5-2 (3.30 g, 14.92 mmol, 1.00 eq) in tetrahydrofuran (30.0 mL) was added N-hydroxysuccinimide (2.06 g, 17.91 mmol, 1.20 eq) and N,N-dicyclohexylcarbodiimide (3.69 g, 17.91 mmol, 3.62 mL, 1.20 eq) at 20 °C. The reaction mixture was stirred at 20 °C for 16 h. Then (S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid (3.66 g, 17.91 mmol, 1.20 eq) and triethylamine (4.15 mL, 29.84 mmol, 2.00 eq) in N,N-dimethylformamide (15.0 mL) was added to the reaction solution, and the reaction mixture was stirred at 20 °C for 16 h. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound B5-3, which was used directly in the next step. MS-ESI m / z: 408.0 [M+H] + .

[0195] Step 3: Synthesis of B5

[0196] To a solution of compound B5-3 (4.00 g, 9.82 mmol, 1.00 eq), N-hydroxysuccinimide (1.70 g, 14.73 mmol, 1.50 eq) in tetrahydrofuran (50.0 mL) was added N,N-dicyclohexylcarbodiimide (3.04 g, 14.73 mmol, 2.98 mL, 1.50 eq) at 20 °C. The reaction mixture was stirred at 20 °C for 30 min. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 0 / 1-1 / 1, volume ratio) to obtain compound B5. MS-ESI m / z: 505.1 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ: 9.16 (s, 1H), 8.81 (d, J = 2.4 Hz, 1H), 8.26 (br s, 1H), 7.84 (br d, J = 3.7 Hz, 1H), 5.85 (br d, J = 1.5 Hz, 1H), 4.92 (br s, 1H), 4.44-4.29 (m, 1H), 3.93-3.79 (m, 1H), 2.92 (br s, 4H), 1.42 (s, 9H). 19 F NMR (376 MHz, CDCl3) δ: 39.37 (s, 1F).

[0197] Example 1: Preparation method of compound BR001

[0198] Step 1: Synthesis of trifluoroacetate salt of BR001-2

[0199] 1.1 Synthesis of polypeptide

[0200] The polypeptide was synthesized using standard step-by-step synthesis method.

[0201] 1) Resin loading: To a solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin substitution degree: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropyl ethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.50 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0202] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0203] 3) Condensation: A solution of compound B1 (1.05 mmol, 1.50 eq), N,N- diisopropylethylamine (2.10 mmol, 3.00 eq) and benzotriazol-1-yl-oxy- tris-(dimethylamino)-phosphonium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and stirred under nitrogen at 25 °C for 1 hour. The resin was then washed with N,N- dimethylformamide (20.0 mL*5) and filtered.

[0204] 4) Steps 2 to 3 were repeated and the next amino acid was added in sequence to the starting material 3-8 for condensation. See Table 1 for details.

[0205] Table 1 Charge sequence

[0206] 1.2 Polypeptide cleavage and purification

[0207] 1) After completion of condensation, the resin was washed with N,N- dimethylformamide (20.0 mL*5), then washed with methanol (20.0 mL*3) and dried under vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the side chain protected peptide resin and stirred at 25 °C for 1.5 hours.

[0208] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (100.0 mL) and filtered to collect the filter cake, which was washed with isopropyl ether (100.0 mL*2) and dried under vacuum for 2 hours to obtain the crude trifluoroacetate salt of compound BR001-2 (620.00 mg).

[0209] Step 2: Synthesis of the trifluoroacetate salt of BR001-3

[0210] 1) The crude trifluoroacetate salt of compound BR001-2 (620.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (223.00 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (300.0 mL) and 1 M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10. The reaction mixture was stirred at 25 °C for 1 hour. Then mercaptoethylamine (72.00 mg, 1.50 eq) was added to the reaction mixture and stirring was continued for 1 hour.

[0211] 2) The pH of the reaction mixture was adjusted to 6-7 by adding 1 M aqueous hydrochloric acid solution and directly lyophilized to obtain the crude compound BR001-3 (700.00 mg).

[0212] 3) The crude compound BR001-3 was purified by preparative high performance liquid chromatography (column: Gemini, 5 μm, C18, C18, 10 μm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 27% - 57% acetonitrile, 50 min; retention time: 25 min) to give the trifluoroacetate salt of compound BR001-3. Purification conditions are shown in Table 2.

[0213] Table 2 Purification conditions

[0214] Step 3: Synthesis of the trifluoroacetate salt of BR001

[0215] Compound BR001-3 (40.00 mg, 1.00 eq, trifluoroacetate salt) and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (25.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (3.0 mL) at 25 °C, then N,N- diisopropylethylamine (21.70 mg, 5.00 eq) was added, and the reaction solution was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Gemin, 5 μm, C18, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 7 min; 22% - 52% acetonitrile, 60 min; retention time: 30.5 min) to give the trifluoroacetate salt of the target compound BR001. HRMS-ESI m / z: 1568.5958 [M+H] + .

[0216] Example 2: Preparation method of compound BR002

[0217] Step 1: Synthesis of the trifluoroacetate salt of BR002-1

[0218] 1.1 Synthesis of polypeptides

[0219] The polypeptides were synthesized using standard stepwise synthesis methods.

[0220] 1) Resin loading: To a solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin loading: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropyl- ethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled nitrogen for 2 hours, then 1.50 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0221] 2) Deprotection: 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0222] 3) Condensation: A solution of compound B2 (1.05 mmol, 1.50 eq), N,N-diisopropyl- ethylamine (2.10 mmol, 3.00 eq) and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and nitrogen was bubbled at 25 °C for 1 hour, then the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0223] 4) Steps 2 to 3 were repeated and the next amino acid was added in sequence according to the raw materials 3~8. See Table 3 for details.

[0224] Table 3 Charge sequence

[0225] 1.2 Polypeptide cleavage and purification

[0226] 1) After completion of condensation, the resin was washed with N,N-dimethylformamide (20.0 mL*5), then with methanol (20.0 mL*3) and dried under vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the resin of the side chain protected peptide, and stirred at 25 °C for 1.5 hours.

[0227] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (100.0 mL), filtered and the filter cake was washed with isopropyl ether (100.0 mL*2) and dried under vacuum for 2 hours to obtain the crude trifluoroacetate salt of compound BR002-1 (600.00 mg).

[0228] Step 2: Synthesis of trifluoroacetate salt of BR002-2

[0229] 1) Dissolve the crude trifluoroacetate salt of compound BR002-1 (600.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (216.00 mg, 1.00 eq) in 50% acetonitrile / water (300.0 mL), dropwise add 1 M aqueous cesium carbonate solution to adjust pH to 9-10, the reaction mixture is reacted at 25 °C for 1 hour. Then add mercaptoethylamine (70.00 mg, 1.50 eq) to the reaction solution and continue stirring for 1 hour.

[0230] 2) Add 1 M aqueous hydrochloric acid solution to the reaction solution to adjust pH to 6-7, directly freeze-dried to obtain the crude product of compound BR002-2 (706.00 mg).

[0231] 3) The crude product of compound BR002-2 above is purified by high performance liquid chromatography preparation (chromatographic column: Gemini, 5 μm, C18, C18, 10 μm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 37% - 57% acetonitrile, 50 minutes; retention time: 15 minutes) to obtain the trifluoroacetate salt of compound BR002-2. The purification conditions are shown in Table 4.

[0232] Table 4 Purification conditions

[0233] Step 3: Synthesis of trifluoroacetate salt of BR002

[0234] Dissolve compound BR002-2 (45.00 mg, 1.00 eq, trifluoroacetate salt) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (29.00 mg, 1.50 eq) in N,N-dimethylformamide (3.0 mL) at 25 °C, then add N,N-diisopropylethylamine (24.00 mg, 5.00 eq), the reaction solution is stirred at 25 °C for 1 hour. After the reaction is completed, the reaction solution is purified by high performance liquid chromatography (chromatographic column: Gemin, 5 μm, C18, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 6% - 6% acetonitrile, 13 minutes; 24% - 39% acetonitrile, 25 minutes; retention time: 36 minutes) to obtain the trifluoroacetate salt of the target compound BR002. HRMS-ESI m / z: 1568.6488 [M+H] + .

[0235] Example 3: Preparation method of compound BR003

[0236] Step 1: Synthesis of trifluoroacetate salt of BR003-1

[0237] 1.1 Synthesis of polypeptide

[0238] The polypeptide was synthesized using standard stepwise synthesis methods.

[0239] 1) Resin loading: To a solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin substitution: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropyl ethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.50 mL of methanol was added and the nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0240] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0241] 3) Condensation: A solution of compound B1 (1.05 mmol, 1.50 eq), N,N-diisopropyl ethylamine (2.10 mmol, 3.00 eq) and benzotriazol-N,N,N,N-tetramethyluronium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin, and nitrogen was bubbled at 25°C for 1 hour, then the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0242] 4) Steps 2 to 3 were repeated, and the next amino acid was added in sequence according to the order of raw materials 3-7. See Table 5 for details.

[0243] Table 5 Order of feeding

[0244] 5) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0245] 6) Condensation: A solution of di(p-nitrophenyl) carbonate (3.50 mmol, 5.00 eq) and N,N-diisopropyl ethylamine (7.00 mmol, 10.00 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin, and nitrogen was bubbled at 25°C for 1 hour, then the resin was washed with N,N-dimethylformamide (20.0 mL*5), methanol (20.0 mL*3) and methyl tert-butyl ether (15.0 mL*3) in sequence, and dried under vacuum.

[0246] 7) Condensation: n-Butylamine (2.10 mmol, 3.00 eq) and N-hydroxy-7-azabenzotriazole (1.40 mmol, 2.00 eq) in pyridine (8.0 mL) were added to the resin and stirred at 25 °C for 12 hours under nitrogen. The resin was then washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0247] 1.2 Polypeptide cleavage and purification

[0248] 1) After completion of condensation, the resin was washed with N,N-dimethylformamide (20.0 mL*5) and then with methanol (20.0 mL*3) and dried under vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the resin of the side chain protected peptide and stirred at 25 °C for 1.5 hours.

[0249] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (100.0 mL), filtered and the filter cake was washed with isopropyl ether (100.0 mL*2) and dried under vacuum for 2 hours to obtain the crude trifluoroacetate salt of compound BR003-1 (400.00 mg).

[0250] Step 2: Synthesis of trifluoroacetate salt of BR003-2

[0251] 1) The crude trifluoroacetate salt of compound BR003-1 (400.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (143.00 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (200.0 mL) and 1 M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10. The reaction mixture was stirred at 25 °C for 1 hour. Then mercaptoethylamine (46.00 mg, 1.50 eq) was added to the reaction and stirring was continued for 1 hour.

[0252] 2) The pH of the reaction was adjusted to 6-7 by adding 1 M aqueous hydrochloric acid solution and lyophilized directly to obtain the crude compound BR003-2 (450.00 mg).

[0253] 3) The crude compound BR003-2 described above was purified by preparative high performance liquid chromatography (column: Gemini, 5 pm, C18, C18, 10 pm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 25% - 55% acetonitrile, 50 minutes; retention time: 25 minutes) to obtain the trifluoroacetate salt of compound BR003-2. The purification conditions are shown in Table 6.

[0254] Table 6 Purification conditions

[0255] Step 3: Synthesis of trifluoroacetate salt of BR003

[0256] Compound BR003-2 (40.00 mg, 1.00 eq, trifluoroacetate salt) and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (25.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (3.0 mL) at 25 °C, then N,N- diisopropylethylamine (21.70 mg, 5.00 eq) was added, and the reaction solution was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (chromatography column: Gemin, 5 μm, C18, mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 7 minutes; 18% - 48% acetonitrile, 60 minutes; retention time: 34 minutes) to obtain the trifluoroacetate salt of the target compound BR003. HRMS-ESI m / z: 1569.5907 [M+H] + .

[0257] Example 4: Preparation method of compound BR004

[0258] Step 1: Synthesis of trifluoroacetate salt of BR004-1

[0259] 1.1 Synthesis of polypeptide

[0260] The polypeptide was synthesized using standard step-by-step synthesis methods.

[0261] 1) Resin loading: A solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin substitution degree: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N- diisopropylethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.50 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0262] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N- dimethylformamide (20.0 mL*5) and filtered.

[0263] 3) Condensation: A solution of compound B2 (1.05 mmol, 1.50 eq), N,N- diisopropylethylamine (2.10 mmol, 3.00 eq) and benzotriazol-1- yloxytris-(dimethylamino)-phosphonium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and stirred under nitrogen at 25 °C for 1 h. The resin was then washed with N,N- dimethylformamide (20.0 mL*5) and filtered.

[0264] 4) Steps 2 to 3 were repeated and the next amino acid was added in sequence to the raw material 3-7 condensation. See Table 7 for details.

[0265] Table 7 Charge sequence

[0266] 5) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, followed by nitrogen sparging (30 s*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0267] 6) Condensation: A solution of di(p-nitrophenyl)carbonate (3.50 mmol, 5.00 eq) and N,N-diisopropylethylamine (7.00 mmol, 10.00 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and stirred under nitrogen at 25 °C for 1 h. The resin was then washed with N,N-dimethylformamide (20.0 mL*5), methanol (20.0 mL*3) and methyl tert-butyl ether (15.0 mL*3) in sequence and dried under vacuum.

[0268] 7) Condensation: A solution of n-butylamine (2.10 mmol, 3.00 eq) and N-hydroxy-7-azabenzotriazole (1.40 mmol, 2.00 eq) in pyridine (8.0 mL) was added to the resin and stirred under nitrogen at 25 °C for 12 h. The resin was then washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0269] 1.2 Polypeptide cleavage and purification

[0270] 1) After completion of the condensation, the resin was washed with N,N- dimethylformamide (20.0 mL*5), followed by methanol (20.0 mL*3) and dried under vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the resin of the side chain protected peptide and stirred at 25 °C for 1.5 h.

[0271] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (100.0 mL) and filtered to collect the filter cake, which was washed with isopropyl ether (100.0 mL*3) and dried under vacuum.

[0272] mL*2) washed the filter cake, and vacuum dried for 2 hours to obtain the crude trifluoroacetate salt of compound BR004-1 (370.00 mg).

[0273] Step 2: Synthesis of the trifluoroacetate salt of BR004-2

[0274] 1) The crude trifluoroacetate salt of compound BR004-1 (370.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (133.00 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (200.0 mL), 1M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10, and the reaction mixture was reacted at 25°C for 1 hour. Then mercaptoethylamine (43.00 mg, 1.50 eq) was added to the reaction solution and stirring was continued for 1 hour.

[0275] 2) 1M aqueous hydrochloric acid solution was added to the reaction solution to adjust the pH to 6-7, and direct lyophilization was performed to obtain the crude compound BR004-2 (440.00 mg).

[0276] 3) The crude compound BR004-2 described above was purified by preparative high performance liquid chromatography (column: Gemini, 5 μm, C18, C18, 10 μm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 22% - 52% acetonitrile, 50 minutes; retention time: 29.5 minutes) to obtain the trifluoroacetate salt of compound BR004-2. The purification conditions are shown in Table 8.

[0277] Table 8 Purification conditions

[0278] Step 3: Synthesis of the trifluoroacetate salt of BR004

[0279] Compound BR004-2 (20.00 mg, 1.00 eq, trifluoroacetate salt) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (15.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (1.5 mL) at 25°C, then N,N-diisopropylethylamine (13.00 mg, 5.00 eq) was added, and the reaction solution was stirred at 25°C for 1 hour. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography (column: Gemin, 5 μm, C18, C18, 10 μm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 10 min; 22% - 52% acetonitrile, 50 min; retention time: 35 min) purification to give the trifluoroacetate salt of the target compound BR004. HRMS-ESI m / z: 1569.6105 [M+H] + .

[0280] Example 5: Method for the preparation of compound BR005

[0281] Step 1: Synthesis of the trifluoroacetate salt of BR005-1

[0282] 1.1 Synthesis of the polypeptide

[0283] The polypeptide was synthesized using standard stepwise synthesis methods.

[0284] 1) Resin loading: To a solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin loading: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropylethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled nitrogen for 2 hours, then 1.50 mL of methanol was added and the bubbling of nitrogen was continued for 30 minutes. The reaction mixture was filtered to give the loaded resin.

[0285] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds*2). The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0286] 3) Condensation: A solution of compound B1 (1.05 mmol, 1.50 eq), N,N- diisopropylethylamine (2.10 mmol, 3.00 eq) and benzotriazol- N,N,N,N-tetramethyluronium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and nitrogen was bubbled at 25 °C for 1 hour, then the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0287] 4) Steps 2 to 3 were repeated and the next amino acid was added in order according to the sequence of the starting materials 3~8. See Table 9 for details.

[0288] Table 9 Order of addition of materials

[0289] 1.2 Cleavage and purification of the polypeptide

[0290] 1) After completion of condensation, the resin was washed with N,N- dimethylformamide (20.0 mL*5), then washed with methanol (20.0 mL*3), and dried in vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the side chain protected peptide resin and stirred at 25 °C for 1.5 hours.

[0291] 2) The polypeptide was precipitated by the addition of ice-cold isopropyl ether (100.0 mL), filtered and the filter cake was washed with isopropyl ether (100.0 mL*2) and dried in vacuum for 2 hours to give the crude trifluoroacetate salt of compound BR005-1 (470.00 mg).

[0292] Step 2: Synthesis of the trifluoroacetate salt of BR005-2

[0293] 1) The crude trifluoroacetate salt of compound BR005-1 (470.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (166.00 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (250.0 mL) and 1 M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10. The reaction mixture was stirred at 25 °C for 1 hour. Then mercaptoethylamine (54.00 mg, 1.50 eq) was added to the reaction and stirring was continued for 1 hour.

[0294] 2) To the reaction was added 1 M aqueous hydrochloric acid solution to adjust the pH to 6-7 and lyophilized directly to give the crude compound BR005-2 (550.00 mg).

[0295] 3) The crude compound BR005-2 above was purified by preparative high performance liquid chromatography (column: Gemini, 5 μm, C18, C18, 10 μm, mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 20% - 50% acetonitrile, 50 minutes; retention time: 32 minutes) to give the trifluoroacetate salt of compound BR005-2. The purification conditions are shown in Table 10.

[0296] Table 10 Purification conditions

[0297] Step 3: Synthesis of the trifluoroacetate salt of BR005

[0298] Compound BR005-2 (50.00 mg, 1.00 eq, trifluoroacetate salt) and l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid 1-(2,5-dioxo-l-pyrrolidinyl) ester (31.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (3.0 mL) at 25 °C, then N,N-diisopropylethylamine (26.00 mg, 5.00 eq) was added, and the reaction solution was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (chromatography column: Gemin, 5 μm, C18, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 7 minutes; 20% - 50% acetonitrile, 60 minutes; retention time: 30.5 minutes) to obtain the trifluoroacetate salt of the target compound BR005. HRMS-ESI m / z: 1583.6013 [M+H] + .

[0299] Example 6: Preparation method of compound BR006

[0300] Step 1: Synthesis of trifluoroacetate salt of BR006-1

[0301] 1.1 Synthesis of polypeptide

[0302] The polypeptide was synthesized using standard step-by-step synthesis methods.

[0303] 1) Resin loading: A solution of compound BR001-1 (0.70 mmol, 1.00 eq, resin substitution degree: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropylethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.50 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0304] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, then nitrogen was bubbled (30 seconds * 2). The resin was washed with N,N-dimethylformamide (20.0 mL * 5) and filtered.

[0305] 3) Condensation: a solution of compound B2 (1.05 mmol, 1.50 eq), N,N- diisopropylethylamine (2.10 mmol, 3.00 eq) and benzotriazol-1-yl-oxy- tris-(dimethylamino)-phosphonium hexafluorophosphate (1.00 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and stirred under nitrogen at 25 °C for 1 hour, then the resin was washed with N,N- dimethylformamide (20.0 mL*5) and filtered.

[0306] 4) Steps 2 to 3 were repeated and the next amino acid was added in sequence to condense with raw materials 3~8. See Table 11 for details.

[0307] Table 11 Charge sequence

[0308] 1.2 Polypeptide cleavage and purification

[0309] 1) After completion of condensation, the resin was washed with N,N- dimethylformamide (20.0 mL*5), then washed with methanol (20.0 mL*3) and dried in vacuum. 20.0 mL of cleavage buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the resin of side chain protected peptide, and stirred at 25 °C for 1.5 hours.

[0310] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (100.0 mL), filtered and the filter cake was washed with isopropyl ether (100.0 mL*2) and dried in vacuum for 2 hours to obtain the crude trifluoroacetate salt of compound BR006-1 (470.00 mg).

[0311] Step 2: Synthesis of the trifluoroacetate salt of BR006-2

[0312] 1) The crude trifluoroacetate salt of compound BR006-1 (470.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (166.00 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (250.0 mL), and 1M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10. The reaction mixture was reacted at 25 °C for 1 hour. Then mercaptoethylamine (54.00 mg, 1.50 eq) was added to the reaction solution and stirring was continued for 1 hour.

[0313] 2) 1M aqueous hydrochloric acid solution was added to the reaction solution to adjust the pH to 6-7, and directly freeze-dried to obtain the crude compound BR006-2 (560.00 mg).

[0314] 3) The above crude compound BR006-2 was prepared by high performance liquid chromatography (column: Gemini, 5 μm, C18, 250*50 mm, 10 nm, 40 mL / min, 220 nm, 10-50% acetonitrile / water, 30 min), and the eluate was collected and freeze-dried to obtain the trifluoroacetate salt of compound BR006-2 (100.00 mg). C18, 10 pm, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 22% - 42% acetonitrile, 50 min; retention time: 25 min) purification to give trifluoroacetate salt of compound BR006-2. See Table 12 for purification conditions.

[0315] Table 12 Purification conditions

[0316] Step 3: Synthesis of trifluoroacetate salt of BR006

[0317] Compound BR006-2 (50.00 mg, 1.00 eq, trifluoroacetate salt) and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (31.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (3.0 mL) at 25 °C, then N,N- diisopropylethylamine (26.00 mg, 5.00 eq) was added, and the reaction solution was stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Gemin, 5 pm, C18, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 5 min; 16% - 46% acetonitrile, 50 min; retention time: 34 min) purification to give trifluoroacetate salt of target compound BR006. HRMS-ESI m / z: 1583.5936 [M+H] + .

[0318] Example 7: Preparation method of compound BR007

[0319] Step 1: Synthesis of BR007-2

[0320] The polypeptide was synthesized using standard Fmoc chemistry.

[0321] 1) Resin loading: To a solution of compound BR001-1 (3.00 mmol, 1.00 eq, resin substitution degree: 0.50 mmol / g) and Fmoc-Gln(Trt)-OH (1.00 eq), N,N- diisopropylethylamine (4.00 eq) in dichloromethane (80.0 mL) was bubbled with nitrogen for 2 h, then 6.0 mL of methanol was added and nitrogen bubbling was continued for 30 min. The reaction mixture was filtered to obtain the loaded resin.

[0322] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (100.0 mL) was added, then nitrogen was bubbled for 15 min. The resin was washed with N,N- dimethylformamide (100.0 mL*5) and filtered.

[0323] 3) Condensation: Fmoc-Thr(tBu)-OH (9.00 mmol, 3.00 eq), N,N- diisopropylethylamine (18.00 mmol, 6.00 eq) and benzotriazol- N,N,N,N-tetramethyluronium hexafluorophosphate (8.55 mmol, 2.85 eq) in N,N-dimethylformamide (50.0 mL) were added to the resin and stirred under nitrogen at 25 °C for 1 hour. The resin was then washed with N,N-dimethylformamide (100.0 mL*5) and filtered.

[0324] 4) Steps 2 to 3 were repeated and the next amino acid was added in sequence. See Table 13 for details.

[0325] Table 13 Charge sequence

[0326] 5) Deprotection: N,N-dimethylformamide (100.0 mL) containing 20% piperidine was added to the resin and stirred under nitrogen for 15 minutes. The resin was then washed with N,N-dimethylformamide (100.0 mL*5) and filtered.

[0327] 6) Condensation: Bis(p-nitrophenyl)carbonate (15.00 mmol, 5.00 eq) and N,N- diisopropylethylamine (30.00 mmol, 10.00 eq) in N,N-dimethylformamide (50.0 mL) were added to the resin and stirred under nitrogen at 25 °C for 1 hour. The resin was then washed with 100.0 mL N,N-dimethylformamide 5 times, 50.0 mL methanol 3 times and finally 15.0 mL methyl tert-butyl ether 3 times.

[0328] 7) Condensation: n-Butylamine (18.00 mmol, 6.00 eq) and 1-hydroxy-7- azabenzotriazole (12.00 mmol, 4.00 eq) in pyridine (60.0 mL) were added to the resin and stirred under nitrogen at 25 °C for 12 hours.

[0329] 8) The resin was washed with 100.0 mL N,N-dimethylformamide 5 times, 20.0 mL methanol 3 times and filtered, and vacuum dried. 100.0 mL cleavage solution (20% hexafluoroisopropanol / dichloromethane) was added to the flask containing the side chain fully protected peptide resin, stirred at 25 °C for 15 minutes, filtered and the filtrate collected, the operation repeated twice, and the filtrate vacuum dried for 1 hour to give the side chain protected peptide which was dissolved in 50% acetonitrile / water and then lyophilized to give the crude compound BR007-1 (1.20 g).

[0330] Step 2: Synthesis of trifluoroacetate salt of BR007-6

[0331] The polypeptide was synthesized using standard Fmoc chemistry.

[0332] 1) Resin loading: To a solution of compound BR001-1 (0.50 mmol, 1.00 eq, resin loading: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropyl ethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.0 mL of methanol was added and the nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0333] 2) Deprotection: 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, followed by nitrogen bubbling for 15 minutes. The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0334] 3) Condensation: A solution of compound B3 (0.75 mmol, 1.50 eq), N,N-diisopropyl ethylamine (1.50 mmol, 3.00 eq) and benzotriazol-N,N,N,N-tetramethyluronium hexafluorophosphate (0.713 mmol, 1.42 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and nitrogen was bubbled at 25 °C for 1 hour. The resin was then washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0335] 4) Deprotection: 10% 1,8-diazabicyclo[5.4.0]undec-7-ene in N,N-dimethylformamide (20.0 mL) was added to the resin and nitrogen was bubbled (30 seconds*2), then the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0336] 5) Condensation: A solution of compound BR007-1 (1.50 mmol, 3.00 eq), N,N-diisopropyl ethylamine (3.00 mmol, 6.00 eq) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.43 mmol, 2.85 eq) in N,N-dimethylformamide (8.0 mL) was added to the resin and nitrogen was bubbled at 25 °C for 1 hour, the resin was washed with 20.0 mL of N,N-dimethylformamide five times in turn.

[0337] Polypeptide cleavage and purification:

[0338] 1) After completion of condensation, the resin was washed with 20.0 mL of N,N- dimethylformamide for 5 times, and finally washed with 20.0 mL of methanol for 3 times, and dried in vacuum. 15.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the side chain fully protected peptide resin, and stirred at 25°C for 1 hour.

[0339] 2) The polypeptide was precipitated by adding cold isopropyl ether (100.0 mL), filtered and the filter cake was collected. The filter cake was washed with 100.0 mL of isopropyl ether for 2 times, and dried in vacuum for 2 hours to obtain the crude compound BR007-2 (205.00 mg).

[0340] 3) The crude compound BR007-2 (205.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (73.50 mg, 1.00 eq) were dissolved in 50% acetonitrile / water (100.0 mL), and then 1M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10, and the reaction mixture was reacted at 25°C for 1 hour. After completion of the reaction, mercaptoethylamine (35.00 mg, 1.50 eq) was added to the reaction solution and stirred for 1 hour.

[0341] 4) 1M aqueous hydrochloric acid solution was added to the reaction solution to adjust the pH to 6-7, and directly lyophilized to obtain the crude compound BR007-3 (220.00 mg).

[0342] 5) The crude compound BR007-3 above was purified by high performance liquid chromatography preparation (chromatography column: Gemini, 5 μm, C18, + luna, C18, 10 μm, Mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]; gradient: 15%-45% acetonitrile, 60 minutes; retention time: 37 minutes) to obtain the trifluoroacetate salt of compound BR007-3. The purification conditions are shown in Table 14.

[0343] Table 14 Purification conditions

[0344] Step 3: Synthesis of the trifluoroacetate salt of BR007

[0345] The trifluoroacetate salt of compound BR007-3 (12.50 mg, 1.00 eq) and l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid 1-(2,5-dioxo-l-pyrrolidinyl) ester (8.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (1.0 mL) at 25 °C, and then N,N-diisopropylethylamine (6.81 mg, 5.00 eq) was added. The reaction solution was stirred at 25-40 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Gemin, 5 μm, C18, Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 0% - 0% acetonitrile, 5.5 minutes; 14% - 34% acetonitrile, 35 minutes; retention time: 39 minutes) to give the trifluoroacetate salt of the target compound BR007. HRMS-ESI m / z: 1570.6674 [M+H] + .

[0346] Example 8: Preparation method of compound BR008

[0347] Step 1: Synthesis of BR008-2

[0348] The polypeptide was synthesized using standard Fmoc chemistry.

[0349] 1) Resin loading: A solution of compound BR001-1 (0.50 mmol, 1.00 eq, resin substitution degree: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropylethylamine (4.00 eq) in dichloromethane (10.0 mL) was bubbled with nitrogen for 2 hours, then 1.00 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to give the loaded resin.

[0350] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) was added, and then nitrogen was bubbled for 15 minutes. The resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0351] 3) Condensation: A solution of Fmoc-Phe-OH (1.50 mmol, 3.00 eq), N,N-diisopropylethylamine (3.00 mmol, 6.00 eq) and benzotriazol-N,N,N,N-tetramethyluronium hexafluorophosphate (1.43 mmol, 2.85 eq) in N,N-dimethylformamide (10.0 mL) was added to the resin, and nitrogen was bubbled at 25 °C for 1 hour, then the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered.

[0352] 4) Repeat steps 2 to 3, and add raw materials 3-8 to condense the next amino acid in order. See Table 15 for details.

[0353] Table 15 Charge order

[0354] Polypeptide cleavage and purification:

[0355] 1) After completion of condensation, wash the resin with N,N-dimethylformamide (20.0 mL*5), and then wash the resin with methanol (15.0 mL*3), and dry under vacuum. Add 15.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) to the flask containing the side chain fully protected peptide resin, and stir at 25°C for 1 hour.

[0356] 2) Add cold isopropyl ether (100.0 mL) to precipitate the polypeptide, filter and collect the filter cake. Wash the filter cake with isopropyl ether (100.0 mL*2), and dry under vacuum for 2 hours to obtain the crude compound BR008-1 (409.00 mg).

[0357] 3) Dissolve the crude compound BR008-1 (409.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (163.00 mg, 1.00 eq) in 50% acetonitrile / water (200.0 mL), then add 1M aqueous cesium carbonate solution to adjust the pH to 9-10, and react the mixture at 25°C for 1 hour. Then add mercaptoethylamine (62.00 mg) to the reaction solution and continue stirring for 1 hour.

[0358] 4) Add 1M aqueous hydrochloric acid solution to the reaction solution to adjust the pH to 6-7, and directly lyophilize to obtain the crude compound BR008-2 (428.00 mg).

[0359] 5) Purify the crude compound BR008-2 described above by high performance liquid chromatography preparation (chromatography column: Gemini, 5 μm, C18, C18, 10 μm, mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]; gradient: 18%-48% acetonitrile, 50 minutes; retention time: 30 minutes) to obtain compound BR008-2. See Table 16 for purification conditions.

[0360] Table 16 Purification conditions

[0361] Step 3: Synthesis of trifluoroacetate salt of BR008

[0362] 1) Compound BR008-2 (50.00 mg, 1.00 eq) and compound B4 (37.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (4.0 mL) at 25 °C, then N,N-diisopropyl ethylamine (5.95 mg, 1.00 eq) was added, and the reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound BR008-3. Then 9.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the polypeptide, and stirred at 25 °C for 20 minutes, and isopropyl ether was added to precipitate the polypeptide. The crude peptide was collected by centrifugation and dried under vacuum to obtain compound BR008-4.

[0363] 2) Compound BR008-4 (55.00 mg, 1.00 eq) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (27.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (2.5 mL) at 25 °C, then N,N-diisopropyl ethylamine (23.50 mg, 5.00 eq) was added, and the reaction solution was stirred at 25-40 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (chromatography column: Gemini, 5 μm, C18, 4.6 x 50 mm) to obtain the trifluoroacetate salt of the target compound BR008. HRMS-ESI m / z: 1800.7391 [M+H] Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 15% acetonitrile, 5 minutes; 25%-55% acetonitrile, 50 minutes; retention time: 34 minutes) to obtain the trifluoroacetate salt of the target compound BR008. HRMS-ESI m / z: 1800.7391 [M+H] + .

[0364] Example 9: Preparation method of compound BR009

[0365] Step 1: Synthesis of the trifluoroacetate salt of BR009

[0366] 1) Compound BR008-2 (50.00 mg, 1.00 eq) and compound B5 (37.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (4.0 mL) at 25 °C, then N,N-diisopropyl ethylamine (5.95 mg, 1.00 eq) was added, and the reaction solution was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound BR009-1. Then 9.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the polypeptide, and stirred at 25 °C for 20 minutes, and ice isopropyl ether was added to precipitate the polypeptide. The crude peptide was collected by centrifugation and vacuum dried to obtain the trifluoroacetate salt of compound BR009-2.

[0367] 2) The trifluoroacetate salt of compound BR009-2 (52.00 mg, 1.00 eq) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (29.04 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (2.5 mL) at 25 °C, then N,N-diisopropyl ethylamine (24.90 mg, 5.00 eq) was added, and the reaction solution was stirred at 25-40 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Gemini, 5 μm, C18, 150 mm x 21.1 mm, 100 A; mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 15% acetonitrile, 5 minutes; 23%-53% acetonitrile, 50 minutes; retention time: 34 minutes) to obtain the trifluoroacetate salt of the target compound BR009. HRMS-ESI m / z: 1759.6859 [M+H] . + .

[0368] Example 10: Method for preparing compound BR010

[0369] Step 1: Synthesis of BR010-2

[0370] The polypeptide was synthesized using standard Fmoc chemistry.

[0371] 1) Resin loading: To a solution of compound BR001-1 (3.00 mmol, 1.00 eq, resin substitution: 0.50 mmol / g) and Fmoc-Cys(Trt)-OH (1.00 eq), N,N-diisopropyl ethylamine (4.00 eq) in dichloromethane (60.0 mL) was bubbled with nitrogen for 2 hours. Then 6.0 mL of methanol was added and nitrogen bubbling was continued for 30 minutes. The reaction mixture was filtered to obtain the loaded resin.

[0372] 2) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (100.0 mL) was added to the resin and then nitrogen was bubbled for 15 minutes. The resin was washed with N,N-dimethylformamide (100.0 mL*5) and filtered.

[0373] 3) Condensation: A solution of Fmoc-Phe-OH (9.00 mmol, 3.00 eq), N,N- diisopropylethylamine (18.00 mmol, 6.00 eq) and benzotriazol- N,N,N,N-tetramethyluronium hexafluorophosphate (8.55 mmol, 2.85 eq) in N,N-dimethylformamide (50.0 mL) was added to the resin and nitrogen was bubbled for 1 hour at 25 °C. The resin was then washed with N,N-dimethylformamide (100.0 mL*5) and filtered.

[0374] 4) Steps 2 to 3 were repeated and the next amino acid was added in the order of raw materials 3~7. See Table 17 for details.

[0375] Table 17 Charge sequence

[0376] 5) Deprotection: A solution of 20% piperidine in N,N-dimethylformamide (100.0 mL) was added to the resin and then nitrogen was bubbled for 15 minutes. The resin was washed with N,N-dimethylformamide (100.0 mL*5) and filtered.

[0377] 6) Condensation: A solution of di(p-nitrophenyl)carbonate (15.00 mmol, 5.00 eq) and N,N-diisopropylethylamine (30.00 mmol, 10.0 eq) in N,N-dimethylformamide (50.0 mL) was added to the resin and nitrogen was bubbled for 1 hour at 25 °C. The resin was then washed with 100.0 mL of N,N-dimethylformamide 5 times, 50.0 mL of methanol 3 times, and finally 50.0 mL of methyl tert-butyl ether 3 times.

[0378] 7) Condensation: A solution of n-butylamine (18.00 mmol, 6.00 eq) and 1- hydroxy-7-azabenzotriazole (12.00 mmol, 4.00 eq) in pyridine (60.0 mL) was added to the resin and nitrogen was bubbled for 12 hours at 25 °C. The resin was then washed with N,N-dimethylformamide (100.0 mL)*5 and filtered.

[0379] Polypeptide cleavage and purification:

[0380] 1) After completion of condensation, the resin was washed with 100.0 mL of N,N- dimethylformamide for 5 times and finally washed with 50.0 mL of methanol for 3 times and dried under vacuum. 120.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the side chain fully protected peptide resin and stirred for 1 hour at 25 °C.

[0381] 2) The polypeptide was precipitated by adding ice-cold isopropyl ether (1000.0 mL), filtered and the filter cake was washed with 1000.0 mL of isopropyl ether for 2 times and dried under vacuum for 1 hour to obtain the crude compound BR010-1 (850.00 mg).

[0382] 3) The crude compound BR010-1 (850.00 mg, 1.00 eq) and 1,3,5-tris(bromomethyl)benzene (509.00 mg, 1.50 eq) were dissolved in 50% acetonitrile / water (450.0 mL) and then 1 M aqueous cesium carbonate solution was added dropwise to adjust the pH to 9-10 and the reaction mixture was reacted at 20 °C for 15 minutes. After completion of the reaction, mercaptoethylamine (161.00 mg) was added to the reaction solution and stirring was continued for 0.5 hours.

[0383] 4) 1 M aqueous hydrochloric acid solution was added to the reaction solution to adjust the pH to 6-7 and directly lyophilized to obtain the crude compound BR010-2 (1000.0 mg).

[0384] 5) The above crude compound BR010-2 was purified by high performance liquid chromatography preparation (column: Gemini, 5 μm, C18, C18, 10 μm, mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 18% - 48% acetonitrile, 50 minutes; retention time: 27 minutes) to obtain the compound BR010-2. The purification conditions are shown in Table 18.

[0385] Table 18 Purification conditions

[0386] Step 3: Synthesis of trifluoroacetate salt of BR010

[0387] 1) Compound BR010-2 (50.00 mg, 1.00 eq) and compound B4 (37.70 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (4.0 mL) at 25 °C, then N,N-diisopropyl ethylamine (5.95 mg, 1.00 eq) was added, and the reaction mixture was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound BR010-3. Then 9.0 mL of cleavage solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to the flask containing the polypeptide, and stirred at 25 °C for 20 minutes, and ice isopropyl ether was added to precipitate the polypeptide. The crude peptide was collected by centrifugation, vacuum dried to obtain compound BR010-4.

[0388] 2) Compound BR010-4 (58.00 mg, 1.00 eq) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (30.82 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (2.5 mL) at 25 °C, then N,N-diisopropyl ethylamine (26.45 mg, 5.00 eq) was added, and the reaction solution was stirred at 25-40 °C for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (chromatography column: Gemini, 5 μm, C18, 4.6 x 50 mm) to obtain the trifluoroacetate salt of the target compound BR010. HRMS-ESI m / z: 1801.7985 [M+H] Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 15% acetonitrile, 5 minutes; 23%-53% acetonitrile, 50 minutes; retention time: 37.5 minutes) to obtain the trifluoroacetate salt of the target compound BR010. HRMS-ESI m / z: 1801.7985 [M+H] + .

[0389] Example 11: Preparation method of compound BR011

[0390] Step 1: Synthesis of trifluoroacetate salt of BR011

[0391] 1) At 25°C, compound BR010-2 (50.00 mg, 1.00 eq) and compound B5 (37.00 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (4.0 mL), followed by the addition of N,N-diisopropylethylamine (5.95 mg, 1.00 eq). The reaction mixture was stirred at 25°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain compound BR011-1. Then, 9.0 mL of lysis buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% 3-mercaptopropionic acid) was added to a flask containing the peptide, and the mixture was stirred at 25°C for 20 minutes. Icy-rich isopropyl ether was added to precipitate the peptide. The crude peptide was collected by centrifugation and dried under vacuum to obtain compound BR011-2.

[0392] 2) At 25°C, compound BR011-2 (53.00 mg, 1.00 eq) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (29.59 mg, 1.50 eq) were dissolved in N,N-dimethylformamide (2.5 mL), followed by the addition of N,N-diisopropylethylamine (24.90 mg, 5.00 eq). The reaction mixture was stirred at 25-40°C for 1 hour. After the reaction was complete, the reaction mixture was analyzed by high-performance liquid chromatography (HPLC) (column: Gemini, 5 μm, C18). Mobile phase: [water (0.075% trifluoroacetic acid) - acetonitrile]; gradient: 1-1% acetonitrile, 5 min; 15-45% acetonitrile, 31 min; retention time: 31 min) Purification yielded the trifluoroacetate of the target compound BR011. HRMS-ESI m / z: 1760.6851 [M+H] + .

[0393] 177 Preparation and formulation of Lu-labeled compounds

[0394] Add approximately 185 MBq to a 2 mL centrifuge tube. 177 LuCl3 solution, 10 μL of DMSO solution of the compound (concentration 1 mg / mL), 0.17 mL of 0.22 M sodium acetate / 0.15 M ascorbic acid / 0.5% Tween 80 aqueous solution, and 20 μL of ethanol were mixed and reacted at 75 °C for 15 min, then cooled to room temperature for 5 min. The solution was diluted with 2.8 mL of 0.15 M sodium ascorbate / 0.002 M gentianic acid aqueous solution and aseptically filtered into sterile vacuum vials to obtain... 177 Lu-labeled FAPI complex injection solution.

[0395] Furthermore, this labeling and purification method, except for the substitution of individual reagents with reagents of similar function or the adjustment of the ratio, is applicable to all compounds of the present invention.

[0396] The purity of the labeled compounds was characterized by Radio-HPLC method, and the results are shown in Table 19.

[0397] Table 19 Radiochemical purity of radioactive compounds

[0398] Biological test data

[0399] Test Example 1 Test of the binding ability of the compound prepared in the embodiment of the present application to FAP (fibroblast activation protein)

[0400] 1. Purpose of the experiment

[0401] The affinity of the test substance to the target protein FAP (fibroblast activation protein) was detected by SPR method.

[0402] 2. Materials and instruments

[0403] Biacore 8K (Cytiva)

[0404] CM5 chip (Cat# 29149603, Cytiva)

[0405] Amine Coupling Kit (Cat# BR100633, Cytiva)

[0406] Acetate buffer pH 5.5 (Cat# BR100352, Cytiva)

[0407] DMSO (Cat# D4540, Sigma Aldrich)

[0408] P20 (Cat# BR-1000-54, GE Healthcare)

[0409] HBS-N buffer (Cat# BR100670, Cytiva)

[0410] Biotinylated His-Avi-FAP (Cat# FAP-H82Q6, Acrobiosystems)

[0411] Biotin (Cat# HY-101884, MedChem Express)

[0412] Neutravidin (Cat# 31000, Thermo Scientific)

[0413] 3. Experimental protocol

[0414] The biotin coupling method was used in this experiment. 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed at a ratio of 1:1, and the CM5 sensor chip was activated for 420 seconds at a flow rate of 10 μL / min. 50 μg / mL of neutral avidin was injected into the chip at a flow rate of 20 μL / min, and then 1M of ethylolamine-acetate-NaOH in the amino coupling kit (type 2) was used to block the unreacted chip for 180 seconds at a flow rate of 30 μL / min.

[0415] 20 μg / mL of FAP protein was injected into the chip Fc2 sample channel at a flow rate of 1 μL / min, and then 10 μg / mL of biotin was used to block the chip Fc1-Fc2 that did not bind the biotin-labeled protein. The buffer was injected into the chip at a flow rate of 60 μL / min, and after the baseline was stabilized, different gradient concentrations of test and control solutions were injected into different channels Fc1-Fc2 at a flow rate of 30 μL / min. The single-cycle kinetics method was used to perform 120 seconds of binding and 1200 seconds of dissociation stages and collect data.

[0416] The collected data was analyzed using the Biacore Insight Evaluation software version 4.0 using the double reference method. The obtained curve was fitted using a 1:1 binding model to calculate the dissociation constant (K D ).

[0417] 4. Experimental methods and procedures

[0418] 1) Prepare the buffer: use 10x HBS-N as the mother liquor to add Tween 20 and DMSO to prepare a 1x HBS-N, 0.0048% (w / v) Tween 20 and 0.1% (v / v) DMSO, pH 7.2-7.4 buffer solution.

[0419] 2) CM5 chip activation: mix 75 mg / mL EDC and 11.5 mg / mL NHS in the amino coupling kit (type 2) at a ratio of 1:1, and use the mixture to activate the CM5 sensor chip at a flow rate of 10 μL / min for 420 seconds.

[0420] 3) Target protein coupling: FAP protein was captured on the CM5 chip coupled with NeutrAvidin protein amine at a concentration of 20 μg / mL, and reached a fixed level of about 10-12 kRU.

[0421] 4) CM5 chip blocking: use 10 μg / mL of biotin to block the chip Fc1-Fc2 that did not bind the biotin-labeled protein.

[0422] 5) Analyte concentration: Dilute the analyte with running buffer (1X HBS-N, 0.0048% (w / v) Tween 20 and 0.1% (v / v) DMSO, pH 7.2-7.4).

[0423] 6) Injection analysis: Inject the test sample and control sample solutions of different gradient concentrations into the Fc1-Fc2 of different channels at a flow rate of 30 μL / min, using a single-cycle kinetic method, and collect data for a binding phase of 120 seconds and a dissociation phase of 1200 seconds.

[0424] 7) All results are subjected to kinetics fitting analysis according to a 1:1 binding model.

[0425] 5. Experimental results

[0426] The experimental data of five effective concentrations are selected, and the compound prepared in the embodiment of the application is subjected to kinetics fitting analysis according to a 1:1 binding model by using Biacore Insight Evaluation software version 4.0, and the results are shown in Table 20.

[0427] Table 20: SPR binding results of the compound of the application with Human FAP

[0428] Conclusion: The compound prepared in the embodiment of the application has strong binding capacity with Human FAP protein.

[0429] Test Example 2: Enzyme activity test of the compound prepared in the embodiment of the application on FAP (fibroblast activation protein)

[0430] 1. Experimental purpose

[0431] The activity of the compound prepared in the embodiment of the application in the target protein FAP (fibroblast activation protein) is tested.

[0432] 2. Experimental operation

[0433] The test compound was dissolved into 10 mM stock solution with DMSO, 10 μL of 2x human recombinant FAP protein solution (Yiqiao China 10464-H07H) was added into the well containing the test compound, centrifuged at 1000 rpm (revolutions per minute) for 1 minute, after incubation at 37℃ for 5 minutes, 10 μL of GP-AMC solution (MedChem Express HY-137834) was added into each well to make the final concentration 100 μM, and the enzymatic reaction was started. The reaction was quantified by dynamic reading, using Biotek Synergy2 microplate reader, recording the fluorescence signal at excitation wavelength 370 nm and emission wavelength 440 nm for 30 minutes continuously at 37℃, and the IC50value of the test compound was calculated based on the test results 50 .

[0434] 3. Experimental results

[0435] The results of the test of some of the compounds prepared in the embodiments of the present application are shown in Table 21.

[0436] Table 21. Activity inhibition of Human FAP by the compounds of the present application

[0437] Conclusion: The compounds prepared in the embodiments of the present application show good activity inhibition on the target protein Human FAP.

[0438] Test Example 3 177 Biodistribution experiment of Lu-labeled compounds in HT1080-FAP tumor-bearing mice

[0439] 1. Purpose of the experiment

[0440] Evaluation 177 The in vivo tissue distribution characteristics of Lu-labeled compounds in HT1080-FAP model mice after single intravenous injection.

[0441] 2. Experimental instruments and reagents

[0442] Main instruments:

[0443] Main reagents:

[0444] 3. Experimental method

[0445] 1) HT1080-FAP cells were inoculated into NU / NU mice (female, about 16-20 g, about 5-6 weeks, 5x10 6 HT1080-FAP cells were subcutaneously inoculated on the right shoulder of the animals), and HT1080-FAP model mice were constructed;

[0446] 2) Randomly grouped HT1080-FAP model mice (tumor volume >100 mm 3 ) for study 177 Tissue distribution characteristics of Lu-labeled compounds. HT1080-FAP model mice were given a single intravenous injection of 3.7 MBq (100 μCi) per mouse 177 Lu-labeled compounds, and the animals were sacrificed at 4 h, 24 h and 72 h after administration (n = 9), respectively. The whole blood, heart, liver, spleen, lung, kidney, bone, muscle and tumor of the animals were collected, and the total radioactivity of the samples was detected using a γ counter.

[0447] 4. Experimental results

[0448] Part of the present application 177 The tissue distribution results of Lu-labeled compounds are shown in Tables 22, 23 and 24.

[0449] Table 22 177 Tissue distribution results of Lu-labeled compounds in HT1080-FAP tumor-bearing mice

[0450] Table 23 177 Tissue distribution results of Lu-labeled compounds in HT1080-FAP tumor-bearing mice

[0451] Table 24 177 Tissue distribution results of Lu-labeled compounds in HT1080-FAP tumor-bearing mice

[0452] Conclusion: 177 The Lu-labeled compounds exhibited high uptake and targeting to tumors in FAP-positive HT1080 model mice, and the uptake of other normal tissues was low, and the tissue selectivity was good.

[0453] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods described herein can be performed in any sequence unless otherwise specified. Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0454] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the application. Indeed, various modifications of the examples, in addition to those shown and described herein, will become apparent to those skilled in the art, and the generic principles defined herein can be applied to other examples and equivalents without the use of the inventive faculty. The examples are intended only to further illustrate the application and are not intended to limit the scope of the application. Accordingly, the application is not limited to that precisely as shown and described.

Claims

1. A compound of Formula (I’), a tautomer, stereoisomer, solvate thereof, or a pharmaceutically acceptable salt thereof: wherein: Ring W is a cyclic peptide moiety comprising 6 to 22 residues of amino acids or amino acid derivatives; P is a chelating moiety that can chelate a radionuclide; Linker is a linker moiety covalently linking Ring W to P; at least one of said ring W and Linker comprises a covalent binding warhead selected from -SO2R S , -OSO2R S , -N(R)SO2R S , -S(=N)O(R)(R S ), -OP(O)(R S )N(R)2, -N(R)COCH=CH2, or -N(R)COC≡CCH3; R S is halogen; and R is H or alkyl.

2. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 1, wherein the Ring W comprises a covalently bound warhead.

3. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 1, wherein the Linker comprises a covalently bound warhead.

4. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 1, wherein the compound is of Formula (I): ###0002### (I) ​ wherein: z is an integer from 6 to 22; Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa18, Xaa19, Xaa20, Xaa21, and Xaa22are each independently an amino acid residue selected from Cys, Pro, Thr, Gin, Phe, Tyr, Asn, Gly, Glu, Ala, Val, Leu, He, Trp, Asp, His, Lys, Met, Arg, Ser, Sec, Pyl, and derivatives thereof; Y is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; and m and n are each independently an integer from 0 to 6.

5. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-4, wherein Y is C4-C7 cycloalkylene, 5- to 10-membered heterocycloalkylene, C6-C8 arylene, or 5- to 10-membered heteroarylene.

6. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 5, wherein Y is cyclohexyl, phenyl, thienyl, furanyl, imidazolyl, pyrrolidinyl, pyranyl, pyridyl, piperazinyl, piperidinyl, pyrimidinyl, pyridazinyl, quinolinyl, or naphthrydinyl.

7. The compound, tautomer, stereoisomer, or solvate of claim 6, or a pharmaceutically acceptable salt thereof, wherein Y is is preferably 8. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-7, wherein P is selected from the group consisting of:

9. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-8, wherein P is 10. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 4-9, wherein at least one of Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa18, Xaa19, Xaa20, Xaa21, and Xaa22comprises a covalently bound warhead.

11. The compound, tautomer, stereoisomer, or solvate of claim 10, or a pharmaceutically acceptable salt thereof, wherein the covalently bound warhead is -OSO2R S .

12. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 10 or 11, wherein at least one of Xaa2, Xaa3,..., Xaaz-2, and Xaaz-i is an amino acid residue having the structure: wherein X1is CH or N.

13. The compound, tautomer, stereoisomer, or solvate of claim 12, or a pharmaceutically acceptable salt thereof, wherein selected from 14. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 12 or 13, wherein Xaaz-i has the structure 15. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-15, wherein R S is halogen, preferably F.

16. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 4-15, wherein Xaal is a Cys derivative residue having the structure: wherein R 1 is alkyl-L 1 -L 2 -; L 1 and L 2 are each independently selected from a direct bond, -O-, -CO-, -NH-, -CONH-, -NHCO- or -SO2-; the * end indicates the point of attachment of Xaai to Xaa2.

17. The compound, tautomer, stereoisomer, or solvate of claim 16, or a pharmaceutically acceptable salt thereof, wherein L 1 is selected from a direct bond, -NH-, or -NHCO-, L 2 is -CO-.

18. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 16 or 17, wherein R 1 is Ci-C6alkyl-CO-, Ci-C6alkyl-NHCO- or Ci-C6alkyl-NHCOCO-.

19. The compound, tautomer, stereoisomer, or solvate of any one of claims 4-18, or a pharmaceutically acceptable salt thereof, wherein Xaaz is a Cys residue having the structure: wherein R 2 is -OR or -N(R)2, R is H or alkyl, and the * end indicates the point of attachment of Xaazto Xaaz-1.

20. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 4-18, wherein z is 7.

21. The compound, tautomer, stereoisomer, or solvate of claim 20, or a pharmaceutically acceptable salt thereof, wherein the compound is of Formula (II):

22. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 21, wherein R 1 is C3-C5alkyl-CO-, C3-C5alkyl-NHCO- or C3-C5alkyl-NHCOCO-, and R 2 is -OH or -NH2.

23. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 21 or 22, wherein Xaa2and Xaa3are Pro, Xaa4is Thr, and Xaa5is Gin.

24. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 10-23, wherein Linker is *-(CH2) p -L1-L2-L3-L4-L5-(CH2) e -L6-(CH2) r - wherein the * end is attached to P; L1, L2, L4, and L5are each independently selected from a direct bond, -O-, -CO-, -NH-, -CONH-, or -NHCO-; L3is alkylene; L6is selected from -S-, -O-, -NH-, or -N(alkyl)-; and L1, L2, L4, and L5are each independently selected from a direct bond, -O-, -CO-, -NH-, -CONH-, or -NHCO-; L3is alkylene; L6is selected from -S-, -O-, -NH-, or -N(alkyl)-; and each of p, e and r is independently an integer from 0 to 6.

25. The compound, tautomer, stereoisomer, or solvate of claim 24, or a pharmaceutically acceptable salt thereof, wherein Linker is 26. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 10-25, wherein is 27. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 4-9, wherein the Linker comprises a covalently bound warhead.

28. The compound, tautomer, stereoisomer, or solvate of claim 27, or a pharmaceutically acceptable salt thereof, wherein the covalently bound warhead is -OSO2R S .

29. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 28, wherein the linker moiety is *-(CH2) p -L1-L2-L3-L4-L5-(CH2) e -L6-(CH2) r - wherein the * end is attached to P; L1, L2, L4and L5are each independently selected from a direct bond, -CO-, -NH-, -CONH- or -NHCO-; L3 is wherein X1is CH or N; L6is selected from -S-, -O-, -NH- or -N(alkyl)-; and each of p, a, b, c, e and r is independently an integer from 0 to 6.

30. The compound, tautomer, stereoisomer, or solvate of Formula (I), or a pharmaceutically acceptable salt thereof, of claim 29, wherein L3is selected from:

31. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 29 or 30, wherein R S is halogen, preferably F.

32. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 29-31, wherein the Linker moiety is: wherein the * end is attached to P.

33. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 29-32, wherein P is 34. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 29-33, wherein Xaa1is a Cys derivative residue having the structure: wherein R 1 is alkyl-L 1 -L 2 -; L 1 and L 2 are each independently selected from a direct bond, -CO-, -NH-, -CONH-, -NHCO- or -SO2-; * end indicates the site of attachment of Xaa1to Xaa2.

35. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 34, wherein L 1 is selected from a direct bond, -NH-, or -NHCO-, L 2 is -CO-.

36. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of claim 34 or 35, wherein R 1 is Ci-C6alkyl-CO-, Ci-C6alkyl-NHCO- or Ci-C6alkyl-NHCOCO-.

37. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 29-36, wherein Xaaz is a Cys residue having the structure: wherein R2is -OR or -NR2, R is H or alkyl, and * end indicates the site of attachment of Xaazto Xaaz-i.

38. The compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 29-37, wherein z is 7, Xaa2and Xaa3are Pro, Xaa4is Thr, Xaa5is Gin, and Xaa6is Phe.

39. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-38, wherein the compound is selected from the group consisting of:

40. The compound, tautomer, stereoisomer, or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1-39, wherein the compound is selected from the group consisting of:

41. A chelate comprising the compound, tautomer, stereoisomer or solvate thereof, or pharmaceutically acceptable salt thereof, of any one of claims 1 to 40, and a radionuclide.

42. The chelate according to claim 41, wherein the radionuclide is selected from the group consisting of: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99 mTc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th and 199 Ag.

43. Use of the chelate of claim 41 or 42 as an inhibitor of fibroblast activation protein.

44. A pharmaceutical composition comprising the chelate of claim 41 or 42, and a pharmaceutically acceptable carrier.

45. Use of the chelate of claim 41 or 42 or the pharmaceutical composition of claim 39 in the manufacture of a medicament for the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in a subject.

46. The use of claim 45, wherein the disease characterized by overexpression of fibroblast activation protein (FAP) is selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling or a scarring disease, preferably wherein the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular cancer, clear cell kidney cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymic carcinoma, glioma, glioblastoma, astrocytoma, cervical cancer or prostate cancer.

47. A kit comprising or consisting of the chelate of claim 41 or 42 or the pharmaceutical composition of claim 44, and instructions for the diagnosis or treatment of a disease.

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