CN109517039B

CN109517039B - Peptide compound, application thereof and composition containing peptide compound

Info

Publication number: CN109517039B
Application number: CN201811100581.0A
Authority: CN
Inventors: 武芸; 胡永韩; 胥诗华; 潘国龙
Original assignee: Shanghua Pharmaceutical Technology Jiangxi Co ltd
Current assignee: Shanghua Pharmaceutical Technology (Jiangxi) Co.,Ltd.
Priority date: 2017-09-20
Filing date: 2018-09-20
Publication date: 2021-03-19
Anticipated expiration: 2038-09-20
Also published as: CN109517039A; CN113150068B; CN113150068A

Abstract

The invention discloses a peptide compound, application thereof and a composition containing the same. The invention provides a peptide compound shown as a formula III, pharmaceutically acceptable salt, solvate, prodrug, tautomer or crystal form thereof, and the compound improves the activity and stability of polypeptide on the basis of LY 2510924.

Description

Peptide compound, application thereof and composition containing peptide compound

Technical Field

The invention relates to a peptide compound, application thereof and a composition containing the same.

Background

The chemokine receptor CXCR4(chemokine receptor-4) belongs to the chemokine family and is a G-protein coupled 7-transmembrane receptor protein (GPCR). CXCR4 is expressed or overexpressed in a variety of tumor cell lines and tissues, including breast, prostate, lung, ovary, colon, pancreas, kidney, and brain, as well as non-hodgkin's lymphoma and chronic lymphocytic leukemia. Stromal-derived factor (SDF-1), also known as CXCL12, is the only ligand for this receptor. CXCL12 can be specifically combined with the N end of CXCR4, and can start a downstream signal path to form a CXCL12/CXCR4 biological axis after the extracellular interaction with the No. 2 of CXCR4, and plays an important role in maintaining embryonic development, mediating immune and inflammatory responses, regulating hematopoiesis, HIV infection, inducing angiogenesis, tumor invasion and metastasis and other physiological and pathological processes. (Tamamura et al Mini-Reviews in Medicinal Chemistry,2006,6,989-

The CXCR4 receptor, initially focused on HIV infection as a drug target, was recently discovered to play an important role in tumor pathology as well. CXCR4 is currently expressed in 23 different types of tumors including breast cancer, lung cancer, Acute Myeloid Leukemia (AML), melanoma, prostate cancer, pancreatic cancer, ovarian cancer, renal cancer, colorectal cancer, non-hodgkin lymphoma (NHL), multiple myeloma, glioblastoma multiforme, and the like. The CXCR4 receptor is a chemokine receptor which is most commonly expressed by tumor cells, is closely related to the proliferation, invasion, metastasis and prognosis of the tumor cells, and is expected to become a new hotspot of tumor gene therapy research aiming at CXCR4 targeted therapy.

AMD3100 (trade name Plerixafor octahydrochloride) is the first approved small molecule CXCR4 antagonist drug on the market (2008), more precisely a CXCR4 α antagonist and an allosteric CXCR7 receptor agonist. AMD3100 is marketed as a rare drug for stem cell mobilization and transplantation in adult tumor patients with multiple myeloma and non-hodgkin lymphoma, which receive stem cell bone marrow transplantation. The AMD3100 can rapidly and effectively mobilize bone marrow hematopoietic stem cells and mesenchymal stem cells, inhibit the growth and metastasis of tumor cells, block the binding sites of HIV-1 infected T lymphocytes, and inhibit the progression of certain inflammatory and immune diseases.

CTCE-9908 is a polypeptide antagonist of the CXCR4 receptor and is marketed as an orphan drug for the treatment of osteosarcoma. Huang et al reported that CTCE-9908 is effective in inhibiting the growth and metastasis of breast cancer (Journal of scientific Research,2009,155, 231-. Can induce mitogenic mutations in ovarian cancer cells and can elicit a superimposed cytotoxic effect when used in combination with paclitaxel. CTCE-9908 also exhibits significant anti-angiogenic activity, a neovascular system essential to prevent tumor tissue growth.

LY2510924 is a lactam cyclic peptide CXCR4 antagonist against non-hodgkin lymphoma, acute myelogenous leukemia, small cell lung cancer, colon cancer, kidney cancer, etc. Shows remarkable anti-tumor activity. Phase 1 clinical studies of LY2510924 in combination with Durvalumab to treat solid tumors are ongoing. While clinical study II (NCT01439568) of LY2510924 in combination with carboplatin and etoposide for the treatment of a wide range of small cell lung cancers has been concluded.

Compared with most of organic small molecular drugs, the polypeptide drug has the outstanding characteristics of high biological activity, small dosage, low toxic and side effects, amino acid as a final product of metabolism and the like. Compared with macromolecular protein or antibody drug, the compound has smaller molecular weight, similar protein activity, more obvious function, chemical synthesis, high product purity, controllable quality, little immunogenicity of small peptide and very good drug development prospect. The research and development of polypeptide drugs become an international emerging high-tech field of biology and have great market potential.

Disclosure of Invention

The invention provides a peptide compound, application thereof and a composition containing the same, aiming at improving the activity and stability of polypeptide on the basis of LY 2510924.

The invention provides a peptide compound shown as a formula III, pharmaceutically acceptable salt thereof, solvate thereof, prodrug thereof, tautomer thereof or crystal form thereof:

wherein the content of the first and second substances,

is in S configuration, R configuration or a mixture thereof;

n is 0 or 1;

Z¹is composed of

When Z is¹Is composed of

When Z is¹The carbon atom is in S configuration;

Z²is composed of

When Z is²Is composed of

When Z is²The carbon atom is in S configuration;

Z³is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

Z⁶Is H or C₁～C₄Alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and also for example isopropyl);

Z⁷is H, C₁～C₄Alkyl (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, also e.g. isopropyl), C₃～C₆Cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, again e.g. cyclohexyl), or

Wherein R is²Is H or C₁～C₄Alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and also for example methyl);

Z⁵is-C (═ O) -NH-<Its left end and Z⁴Is connected with, or at its left end with

Connection of>、-C(＝S)-NH-<Its left end and Z⁴Is connected or/and with

Connection of>、-CH₂-NR¹-<Its left end and Z⁴Is connected, wherein R¹Is H or benzyl>A 3-7 membered heterocycloalkylene group having 1-4 heteroatoms (S) of one or more of N, O and S (the heteroatoms may include at least N, or only N; the heteroatoms may be one or more of N, N and S)1 or 2, also 1; the number of the elements of the heterocycloalkylene can be 5 or 6, and can also be 5; the "hetero atom is one or more of N, O and S, the hetero atom number is 1-4, and the 3-7 membered heterocycloalkylene group" may be "hetero atom at least contains N, the hetero atom number is 1 or 2,5 or 6 membered heterocycloalkylene group", may also be "hetero atom is N, the hetero atom number is 1, 5-6 membered heterocycloalkylene group", and may also be tetrahydropyrrolylene or tetrahydropyridinylene group "; said tetrahydropyrrolyl group may be

<Its left end and Z⁴Connection of>And can also be

<Its left end and Z⁴Connection of>(ii) a The heteroatom is one or more of N, O and S, the heteroatom number is 1-4, and the Z is neutralized in 3-7-membered heterocycloalkylene⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the heteroatom is one or more of N, O and S, the heteroatom number is 1-4, and the 3-7 membered heterocycloalkylene is neutralized

The connecting atoms can be C atoms or N atoms, and can also be N atoms; when the heterocycloalkylene group is a five-membered ring, "it is bonded to Z⁴And "its connection point with

The connecting points of (A) can be ortho-position or meta-position, and can be ortho-position; when the heterocycloalkylene group is a six-membered ring, "it is bonded to Z⁴And "its connection point with

The connecting point of (A) is one or more of N, O and S, and the hetero atom number is 1-4, 5-7 membered heterocycloalkenylene group"(said" oxo "means-CH on the ring)₂-substituted by-C (═ O) -; the number of said "oxo" may be one or more<E.g. 2,3 or 4>May be one; the heteroatom may contain at least N, and may be N only; the number of the hetero atoms can be 1 or 2, and can also be 2; the number of the elements of the heterocycloalkylene group can be 5 or 6, or 5; the "heterocycloalkylene group having 1 to 4 hetero atoms and 5 to 7 hetero atoms" may be "heterocycloalkylene group having 1 or 2 hetero atoms or 5 or 6 hetero atoms and containing at least N, or" heterocycloalkylene group having 2 hetero atoms or 5 to 6 hetero atoms ", or" heterocycloalkylene group having 2 hetero atoms or 2, 3-dihydroimidazolyl group or more "or 2, 3-dihydroimidazolyl group or more

<Its left end and Z⁴Connection of>(ii) a The oxo or non-oxo heteroatom is one or more of N, O and S, the heteroatom number is 1-4, 5-7 membered heterocyclylene radical⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the oxo or non-oxo heteroatom is one or more of N, O and S, the heteroatom number is 1-4, 5-7 membered heterocyclylene

The atom to which the linking atom is linked may be a C atom or a N atom, and may also be a N atom; when the heterocycloalkenylene is a five-membered ring, "it is bonded to Z⁴And "its connection point with

The connecting points of (A) can be in ortho-position or meta-position with each other, and can also be in meta-position with each other; when the heterocycloalkylene group is a six-membered ring, "it is bonded to Z⁴And "its connection point with

The connection points of (A) can be in ortho, meta or para position and can be in meta position; said "oxo heteroatomN, O and S, the 5-7 membered heterocycloalkenylene group having 1 to 4 hetero atoms may be

) Or, the heteroatom is one or more of N, O and S, the number of heteroatoms is 1-3, 5-6 heteroarylene "(the heteroatom may at least contain N, and may be only N; the number of the hetero atoms can be 2 or 3, and can also be 3; the "heteroarylene group with 1-3, 5 or 6 members and 1-3 or 6 members in heteroatom number" may be "a heteroarylene group with 2 or 3,5 or 6 members in heteroatom number" or "a heteroarylene group with 1, 5 or 6 members in heteroatom number" with at least N; the "hetero atom at least comprises N, the number of hetero atoms is 2 or 3, and 5-6 membered heteroarylene" can be "hetero atom is N, the number of hetero atoms is 2 or 3, and 5 membered heteroarylene", and can also be pyrazolyl, imidazolyl or 1,2, 3-triazolyl; the pyrazolylene radical can be

<Its left end and Z⁴Connection of>(ii) a The imidazolyl group may be

<Its left end and Z⁴Connection of>(ii) a The 1,2, 3-triazolylene group may be

<Its left end and Z⁴Connection of>(ii) a The "heteroarylene group containing at least N as a heteroatom and having 1, 5 or 6 membered heteroatoms" may be "a heteroarylene group containing N as a heteroatom and 1, 5 membered heteroatoms", and may be a pyrrolylene group; the hetero atom is one or more of N, O and S, the hetero atom number is 1-3, and 5-6-membered heteroarylene is subjected to neutralization with Z⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the 'hetero atom is one or more of N, O and S, the number of hetero atoms is 1-3, and 5-6 membered heteroarylene'Neutralization of

The connecting atoms can be C atoms or N atoms, and can also be N atoms; when the heteroarylene group is a five-membered ring, "it is bonded to Z⁴And "its connection point with

The connecting points of (A) can be in ortho-position or meta-position with each other, and can also be in meta-position with each other; when the heteroarylene group is a six-membered ring, "it is bonded to Z⁴And "its connection point with

The connecting points of (A) may be ortho-, meta-or para-position to each other, or meta-position to each other);

but which is not the following compound:

in one embodiment, certain substituents of compound III are as defined below, and substituents not mentioned are as defined in any of the above embodiments:

and n is 0.

z is¹、Z²、Z³And Z⁴At least one (e.g. 1,2 or 3) of which is an N atom and the remainder are C atoms (e.g. C atoms)

)。

z is¹、Z²、Z³And Z⁴Only one of them is a N atom and the others are C atoms.

z is¹、Z²、Z³And Z⁴In, only Z¹Is a N atom, and the remainder are C atoms.

z is¹、Z²、Z³And Z⁴In, only Z²Is a N atom, and the remainder are C atoms.

z is¹、Z²、Z³And Z⁴In, only Z³Is a N atom, and the remainder are C atoms.

z is¹、Z²、Z³And Z⁴In, only Z⁴Is a N atom, and the remainder are C atoms.

z is⁵is-C (═ O) -NH-, -C (═ S) -NH-or-CH₂-NR¹-。

z is⁵is-C (═ O) -NH-, having the left end thereof in combination with Z⁴Connecting; preferably Z⁴Is a C atom.

In one embodiment, certain substituents of compound I are as defined below, and substituents not mentioned are as defined in any of the above embodiments:

z is⁵is-C (═ S) -NH-<Its left end and Z⁴Connecting; preferably Z⁴Is a C atom>。

z is⁵is-CH₂-NR¹-<Its left end and Z⁴Connecting; preferably Z⁴Is a C atom>。

z is⁵The compound is 'one or more of N, O heteroatoms and S, 1-4, 3-7 membered heterocycloalkylene', oxo or non-oxo 'the heteroatoms are one or more of N, O and S, 1-4, 5-7 membered heterocycloalkenylene', or 'one or more of N, O and S, the heteroatoms are 1-3, 5-6 membered heteroarylene'.

z is⁵The heterocyclic group is' heterocyclic alkylidene with 1-4, 3-7 members of hetero atoms which is one or more of N, O and S.

z is⁵"hetero atoms" oxo or non-oxo are N, O and SOne or more of (1-4) heterocyclic atoms, and (5-7) membered heterocycloalkenylene.

z is⁵The heteroaryl group is a heteroarylene group with 1-3 hetero atoms and 5-6 hetero atoms, wherein the hetero atoms are one or more of N, O and S.

z is⁶Is H.

z is⁷Is H.

z is⁷Is C₃～C₆A cycloalkyl group.

z is⁷Is composed of

n is 0 or 1;

Z¹is composed of

When Z is¹

When, the carbon atom is in S configuration;

Z²is composed of

When Z is²Is composed of

When Z is²The carbon atom is in S configuration;

Z³is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

Z⁵is-C (═ O) -NH-, -C (═ S) -NH-, -CH₂-NR¹-, "hetero atom is one or more of N, O and S, hetero atom is 1-4, 3-7 membered heterocycloalkylene", or "hetero atom is one or more of N, O and S, hetero atom is 1-3, 5-6 membered heteroarylene";

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

n is 0 or 1;

Z¹is composed of

When Z is¹

When, the carbon atom is in S configuration;

Z²is composed of

The carbon atom is in S configuration;

Z³is composed of

The carbon atom is in S configuration;

Z⁴is composed of

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

In one embodiment, certain substituents of compound III are as defined below, and substituents not mentioned are as defined in any of the above embodiments: n is 0 or 1;

Z¹、Z²、Z³and Z⁴At least one (e.g., 1,2, or 3) of them is an N atom, and the others are C atoms; preferably Z⁴Is an N atom;

Z⁵is-C (═ O) -NH-, -C (═ S) -NH-, -CH₂-NR¹-, "hetero atom is one or more of N, O and S, hetero atom is 1-4, 3-7 membered heterocycloalkylene", or "hetero atom is one or more of N, O and S, hetero atom is 1-3, 5-6 membered heteroarylene"; preferably-C (═ O) -NH-<Its left end and Z⁴Connection of>；

Z⁶Is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹、Z²、Z³and Z⁴Are all C atoms;

Z⁵is 'hetero atom is N, O or one or more of S, hetero atom is 1-3, 5-6 membered heteroarylene group';

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹、Z²、Z³and Z⁴Are all C atoms;

Z⁵is 'heterocyclic alkylidene with one or more of hetero atoms of N, O and S, 1-4 hetero atoms and 3-7 members';

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹、Z²、Z³and Z⁴Are all C atoms;

"hetero atom is N, O and one or more of S, hetero atom number is 1-3, 5-6 membered heteroarylene group";

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹、Z²、Z³and Z⁴Are all C atoms;

Z⁵is-CH₂-NR¹-；

Z⁶Is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹、Z²、Z³and Z⁴Are all C atoms;

Z⁵is-C (═ O) -NH-or-C (═ S) -NH-; the left end of-C (═ O) -NH-or-C (═ S) -NH-is linked with

Connecting;

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹and Z²Are all made of

Z³And Z⁴At least one of which is an N atom;

Z⁶Is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

Preferably C₁～C₄An alkyl group.

Z¹is composed of

When Z is¹Is composed of

When Z is²The carbon atom is in S configuration;

Z²is composed of

Z³Is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁵is-C (═ O) -NH-, having the left end thereof in combination with Z⁴Connecting;

Z⁶is H or C₁～C₄An alkyl group;

Z⁷is H, C₁～C₄Alkyl radical, C₃～C₆Cycloalkyl radicals or

In one embodiment, certain substituents of compound III are as defined below, and substituents not mentioned are as defined in any of the above embodiments: the compound III is a compound shown as a formula I:

wherein Z is²Is composed of

When Z is²Is composed of

When Z is²The carbon atom is in S configuration;

Z³is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

Z⁵is-C (═ O) -NH-<Its left end and Z⁴Connection of>The "hetero atom is one or more of N, O and S, the hetero atom number is 1-4, 3-7 membered heterocycloalkylene" (the hetero atom may at least contain N, or only N; the hetero atom number may be 1 or 2, or also may be 1 or 2)1, the number of the active ingredients is 1; the number of the elements of the heterocycloalkylene can be 5 or 6, and can also be 5; the "hetero atom is one or more of N, O and S, the hetero atom number is 1-4, and the 3-7 membered heterocycloalkylene group" may be "hetero atom at least contains N, the hetero atom number is 1 or 2,5 or 6 membered heterocycloalkylene group", may also be "hetero atom is N, the hetero atom number is 1, 5-6 membered heterocycloalkylene group", and may also be tetrahydropyrrolylene or tetrahydropyridinylene group "; said tetrahydropyrrolyl group may be

<Its left end and Z⁴Connection of>And can also be

<Its left end and Z⁴Connection of>(ii) a The heteroatom is one or more of N, O and S, the heteroatom number is 1-4, and the Z is neutralized in 3-7-membered heterocycloalkylene⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the heteroatom is one or more of N, O and S, the heteroatom number is 1-4, and the atom connected with the chiral carbon atom in the 3-7-membered heterocycloalkylene can be a C atom or an N atom and can also be an N atom; when the heterocycloalkylene group is a five-membered ring, "it is bonded to Z⁴The "connecting point to the chiral carbon atom" may be either ortho-or meta-or ortho-to each other; when the heterocycloalkylene group is a six-membered ring, "it is bonded to Z⁴And the "connecting point with chiral carbon atom" may be ortho, meta or para to each other, and may be ortho to each other), oxo or non-oxo "hetero atom is one or more of N, O and S, the number of hetero atoms is 1-4, 5-7 membered heterocycloalkylene group" (the "oxo" refers to-CH on the ring)₂-substituted by-C (═ O) -; the number of said "oxo" may be one or more<E.g. 2,3 or 4>May be one; the heteroatom may contain at least N, and may be N only; the number of the hetero atoms can be 1 or 2, and can also be 2; the number of the elements of the heterocycloalkylene group can be 5 or 6, or 5; said "hetero atomN, O and S, wherein the hetero atom number is 1-4, 5-7 membered heterocycloalkylene group can be a "hetero atom at least containing N, hetero atom number is 1 or 2,5 or 6 membered heterocycloalkylene group", can be a "hetero atom is N, hetero atom number is 2, 5-6 membered heterocycloalkylene group", can also be 2, 3-dihydroimidazolyl group, and can further be 2, 3-dihydroimidazolyl group

<Its left end and Z⁴Connection of>(ii) a The oxo or non-oxo heteroatom is one or more of N, O and S, the heteroatom number is 1-4, 5-7 membered heterocyclylene radical⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the heteroatom which is oxo or non-oxo is one or more of N, O and S, the heteroatom number is 1-4, and the atom which is connected with the chiral carbon atom in the 5-7-membered heterocycloalkenylene can be a C atom or an N atom and can also be an N atom; when the heterocycloalkenylene is a five-membered ring, "it is bonded to Z⁴The "connecting point to the chiral carbon atom" and the "connecting point to the chiral carbon atom" may be either ortho-or meta-positions to each other, or meta-positions to each other; when the heterocycloalkylene group is a six-membered ring, "it is bonded to Z⁴The "connecting point to the chiral carbon atom" and "the connecting point to the chiral carbon atom" may be either ortho, meta or para, or meta; the oxo-heterocyclic-ene-containing group may be one or more of N, O and S, and the hetero atom number is 1-4, 5-7

) Or, the heteroatom is one or more of N, O and S, the number of heteroatoms is 1-3, 5-6 heteroarylene "(the heteroatom may at least contain N, and may be only N; the number of the hetero atoms can be 2 or 3, and can also be 3; the "heteroarylene group with 1-3, 5 or 6 members and 1-3 or 6 members in heteroatom number" may be "a heteroarylene group with 2 or 3,5 or 6 members in heteroatom number" or "a heteroarylene group with 1, 5 or 6 members in heteroatom number" with at least N; the "hetero atom" ofThe heteroarylene group having 2 or 3 hetero atoms and 5 to 6 members may be a heteroarylene group having 2 or 3 hetero atoms and 5 members, and may be a pyrazolylene group, an imidazolylene group or a 1,2, 3-triazolylene group; the pyrazolylene radical can be

<Its left end and Z⁴Connection of>(ii) a The imidazolyl group may be

<Its left end and Z⁴Connection of>(ii) a The 1,2, 3-triazolylene group may be

<Its left end and Z⁴Connection of>(ii) a The "heteroarylene group containing at least N as a heteroatom and having 1, 5 or 6 membered heteroatoms" may be "a heteroarylene group containing N as a heteroatom and 1, 5 membered heteroatoms", and may be a pyrrolylene group; the hetero atom is one or more of N, O and S, the hetero atom number is 1-3, and 5-6-membered heteroarylene is subjected to neutralization with Z⁴The atoms to which they are bonded may be C atoms or N atoms, and may also be C atoms; the heteroatom is one or more of N, O and S, the number of the heteroatoms is 1-3, and the atom connected with the chiral carbon atom in the 5-6-membered heteroarylene group can be a C atom or an N atom and can also be an N atom; when the heteroarylene group is a five-membered ring, "it is bonded to Z⁴The "connecting point to the chiral carbon atom" and the "connecting point to the chiral carbon atom" may be either ortho-or meta-positions to each other, or meta-positions to each other; when the heteroarylene group is a six-membered ring, "it is bonded to Z⁴And "the point of attachment to a chiral carbon atom" may be ortho, meta, or para to each other, and may be meta to each other);

but which is not the following compound:

z is²、Z³And Z⁴Are all C atoms.

z is²、Z³And Z⁴At least one (e.g., 1,2, or 3) of which is an N atom, and the remainder are C atoms.

z is²、Z³And Z⁴Only one of them is a N atom and the others are C atoms.

z is²、Z³And Z⁴In, only Z²Is a N atom, and the remainder are C atoms.

z is²、Z³And Z⁴In, only Z³Is a N atom, and the remainder are C atoms.

z is²、Z³And Z⁴In, only Z⁴Is a N atom, and the remainder are C atoms.

Z⁵is-C (═ O) -NH-.

Z⁵the compound is 'one or more of N, O heteroatoms and S, 1-4, 3-7 membered heterocycloalkylene', oxo or non-oxo 'the heteroatoms are one or more of N, O and S, 1-4, 5-7 membered heterocycloalkenylene', or 'one or more of N, O and S, the heteroatoms are 1-3, 5-6 membered heteroarylene'.

Z⁵the heterocyclic group is' heterocyclic alkylidene with 1-4, 3-7 members of hetero atoms which is one or more of N, O and S.

Z⁵the 'heteroatom is one or more of N, O and S, and the number of the heteroatoms is 1-4, 5-7-membered heterocyclylene' which is oxo or non-oxo.

Z⁵the heteroaryl group is a heteroarylene group with 1-3 hetero atoms and 5-6 hetero atoms, wherein the hetero atoms are one or more of N, O and S.

z is²、Z³And Z⁴Are all made ofA C atom;

z is²、Z³And Z⁴Are all C atoms;

z is²、Z³And Z⁴At least one (e.g., 1,2, or 3) of them is an N atom, and the others are C atoms;

Z⁵is-C (═ O) -NH-<Its left end and Z⁴Connection of>。

z is²、Z³And Z⁴Only one of them is N atom, and the others are C atoms;

Z⁵is-C (═ O) -NH-<Its left end and Z⁴Connection of>。

z is²、Z³And Z⁴Only one of them is N atom, and the others are C atoms;

Z⁵is one or more of N, O heteroatoms and S, the number of the heteroatoms is 1-4, 3-7A meta heterocycloalkylene group ".

z is²、Z³And Z⁴Only one of them is N atom, and the others are C atoms;

Z²is composed of

Z³Is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

Z⁵Is "heteroatom is one or more of N, O and S, heteroatomThe compound is a 3-7 membered heterocycloalkylene group with a sub-number of 1-4, wherein the oxo or non-oxo 'heteroatom is one or more of N, O and S, and the heteroatom number is 1-4, 5-7 membered heterocycloalkylene group', or the 'heteroatom is one or more of N, O and S, and the heteroatom number is 1-3, and 5-6 membered heteroarylene group'.

Z²is composed of

Z³Is composed of

When Z is³Is composed of

When Z is³The carbon atom is in S configuration;

Z⁴is composed of

The compound I can be any one of the following compounds:

the invention also provides application of the compound I, pharmaceutically acceptable salts, solvates, prodrugs, tautomers or crystal forms thereof in preparation of medicaments for treating and/or preventing CXCR4 related diseases.

The "CXCR 4 related disease" may be an immune disease, an inflammatory disease, pulmonary fibrosis, HIV infection or cancer.

The inflammatory disease may be rheumatoid arthritis.

The cancer may be breast cancer, pancreatic cancer, melanoma, prostate cancer, renal cancer, neuroblastoma, non-hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, osteosarcoma or leukemia. The leukemia may be acute myelogenous leukemia. The lung cancer can be small cell lung cancer.

The invention also provides application of the compound I, pharmaceutically acceptable salts, solvates, prodrugs, tautomers or crystal forms thereof in preparation of CXCR4 antagonists.

The invention also provides a pharmaceutical composition, which comprises the compound I, pharmaceutically acceptable salts thereof, solvates thereof, prodrugs thereof, tautomers thereof or crystal forms thereof, and pharmaceutic adjuvants.

The dosage of the compound I, the pharmaceutically acceptable salt thereof, the solvate thereof, the prodrug thereof, the tautomer thereof or the crystal form thereof is therapeutically effective amount.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be formulated for administration in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention may also be in a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations which can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts. Exemplary acid salts include, but are not limited to: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1-1-methylene-bis (2-hydroxy-3-naphthoate)). The compounds used in the present invention may form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, bismuth, and diethanolamine salts. For reviews of pharmaceutically acceptable Salts, see Handbook of Pharmaceutical Salts, Properties, Selection, and Use (P.Heinrich Stahl and Camile G.Wermuth, ed., Wiley-VCH, 2002).

The term "crystalline form" refers to one or more crystal structures formed by the arrangement of molecules in lattice space that differ when crystallized.

The term "solvate" is a crystalline form that contains, in addition to the active molecule, one or more solvent molecules incorporated into the crystal structure. Solvates may include stoichiometric or non-stoichiometric amounts of solvent, and the solvent molecules in the solvent may be present in ordered or non-ordered arrangements. Solvates containing non-stoichiometric amounts of solvent molecules may result from solvates that have lost at least a portion (but not all) of the solvent molecules. In a particular embodiment, a solvate is a hydrate, meaning that a crystalline form of the compound may include water molecules.

The term "prodrug" refers to a derivative of a compound that contains a biologically reactive functional group such that, under biological conditions (in vitro or in vivo), the biologically reactive functional group can be cleaved or otherwise reacted from the compound to provide the compound. Typically, prodrugs are inactive, or at least less active than the compound itself, such that the compound does not exert its activity until cleaved from a biologically reactive functional group. The bioreactive functional group can be hydrolyzed or oxidized under biological conditions to provide the compound. For example, the prodrug may comprise a biohydrolyzable group, examples of which include, but are not limited to, biohydrolyzable phosphates, biohydrolyzable esters, biohydrolyzable amides, biohydrolyzable carbonates, biohydrolyzable carbamates, and biohydrolyzable ureides. For a review of Prodrugs see, e.g., J.Rautio et al, Nature Reviews Drug Discovery (2008)7, 255-.

The positive progress effects of the invention are as follows: the peptide compound of the invention has better stability and better activity.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 WY-2

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4- (isopropylamino) butyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2,4,9,12,15,18, 21-octaazacycloeicosanoic-5-carboxamide

Preparation of Fmoc-2Nal-azaGly- [ D-Glu (OAll) ] -OH (WY-2-I):

(5S,11R) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -5- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

Step 1.(R) -5-allyl-1-tert-butyl-2- (tert-butoxycarbonylamino) glutaric acid diester (WY-2-B)

(R) -5-tert-butoxy-4- (tert-butoxycarbonylamino) -5-oxopentanoic acid (2g,6.59mmol) and 3-bromo-1-propene (1.6g,13.19mmol) were dissolved in DMF (30mL), sodium carbonate (1.4g,13.19mmol) was slowly added, and after stirring at room temperature for 4 hours, water (100mL) was added, followed by extraction with ethyl acetate (100 mL. times.3). The organic phases were combined, washed with water (50mL × 4), dried over anhydrous sodium sulfate, filtered, concentrated and the resulting crude product was isolated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 9/1) to give the title compound (2.1g, 92.80%) as a colorless oily liquid. LCMS (ESI) [ M +23 ]]⁺＝367.9.

Step 2 (R) -5-allyl-1-tert-butyl-2-aminoglutaric diester (WY-2-C)

(R) -5-allyl-1-tert-butyl-2- (tert-butoxycarbonylamino) glutaric acid diester (2.1g,6.11mmol) was placed in a 250mL one-necked flask, and then hydrogen chloride-ethyl acetate solution (2M,90mL) was slowly added. The reaction solution was stirred at room temperature for 1 hour. Then adding diethyl ether (400mL) for dilution, concentrating to obtain 100mL volume, adding diethyl ether (400mL) and rotary evaporating to obtain 100mL volume, and repeating the stepsThree post-spin-drying times afforded the title compound (1.5g, 87.88%) as a yellow oily liquid. This compound was used directly in the next reaction. LCMS (ESI) [ M + H ]]⁺＝244.0.

Step 3 (R) -5-allyl-1-tert-butyl-2-isocyanatoglutaric diester (WY-2-D)

A solution of triphosgene (1.125g,3.79mmol) in dry dichloromethane (10mL) was cooled to 0 deg.C and a solution of (R) -5-allyl-1-tert-butyl-2-aminoglutaric diester hydrochloride (1.842g,7.58mmol) in dry dichloromethane (50mL) was slowly added dropwise to the solution and the mixture was stirred at 0 deg.C for 1 hour. The reaction mixture was slowly quenched dropwise with triethylamine (2.301g, 22.74mmol), and the mixture was stirred at room temperature for 20 minutes to give a solution containing the title compound (about 7.58mmol), which was used directly in the next reaction. LC-MS has no ion current and obvious ultraviolet absorption of the product.

Step 4 (S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionyl) hydrazinocarbonate (WY-2-F)

(S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionic acid (2.5g,5.72mmol), tert-butoxycarbonylhydrazine (756mg, 5.72mmol), HATU (2.829g, 7.44mmol), HOBT (1.159g, 8.58mmol) was dissolved in dichloromethane (100mL), and DIPEA (2.218g, 17.16mmol) was slowly added. The reaction solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated, and the resulting residue was purified by flash chromatography (silica gel column, eluent: ethyl acetate/petroleum ether ═ 1/1) to give the title compound (3g, 95.08%) as a white solid. LCMS (ESI) [ M + H-100 ]]⁺＝452.1.

Step 5 (S) - (9-hydro-fluoren-9-yl) methyl-1-hydrazino-3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbamate (WY-2-G)

(S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propanoyl) hydrazinocarbonate (3g,5.44mmol) was dissolved in dichloromethane (50mL), a hydrogen chloride-dioxane solution (4N,50mL) was slowly added, and the reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated to give the title compound (2.4g, 97.72%) as a white solid. The resulting solid was used directly in the next reaction. LCMS (ESI) [ M + H ]]⁺＝452.1.

Step 6 (R) -5-allyl-1-tert-butyl-2- (2- ((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionyl) hydrazinocarbonylamino) glutaric acid diester (WY-2-H)

Hydrochloride (2400mg,5.32mmol) of (S) - (9-hydro-fluoren-9-yl) methyl-1-hydrazino-3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbonate and a solution containing (R) -5-allyl-1-tert-butyl-2-isocyanatoglutaric diester (about 7.58mmol, i.e. the solution of the title compound from step 3) were dissolved in dichloromethane (25mL), the reaction was stirred at room temperature for 20 minutes, and triethylamine (1.613g, 15.94mmol) was slowly added to the reaction mixture and stirred at room temperature for 16 hours. The reaction mixture was concentrated, and the residue was added to water (100mL) and extracted with ethyl acetate (100 mL. times.3). The organic layers were combined, washed with water (50mL × 3), the organic phase was spin dried and isolated and purified by silica gel column chromatography to give the title compound (1.5g, 39.12%) as a white solid. LCMS (ESI) [ M + H-56 ]]⁺＝665.0.

Step 7 (5S,11R) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -5- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid (WY-2-I)

(R) -5-allyl-1-tert-butyl-2- (2- ((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propanoyl) hydrazinocarbonylamino) glutaric acid diester (1.400g,1.94mmol) was dissolved in dichloromethane (50mL) and trifluoroacetic acid (50mL) was added slowly and the reaction stirred at room temperature for 2 hours. The reaction solution was spun dry and dichloromethane (50 mL. times.3) was added and spun dry three times to remove excess trifluoroacetic acid, and the residue was isolated and purified by reverse phase column chromatography (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (650mg, 50.41%) as a white solid. LCMS (ESI) [ M + H-]⁺＝665.0.¹H NMR(400MHz,DMSO-d₆)δ12.73(s,1H),9.94(s,1H),8.07(s,1H),7.83(q,J＝22.8Hz,7H),7.36-7.62(m,,8H),7.30(t,J＝3.52Hz,1H),7.19(t,J＝9Hz,1H),6.56(d,J＝7.6Hz,1H),5.86-5.93(m,1H),5.28(d,J＝16.0Hz,1H),5.18(d,J＝8.8Hz,1H),4.35-4.54(m,3H),4.08-4.17(m,4H),3.23(d,J＝9.6Hz,1H),2.99(t,J＝11.2Hz,1H).2.39(t,J＝6.4Hz,2H),2.01-2.05(m,1H),1.80-1.86(m,1H).

B solid phase Synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (225mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc-2Nal-azaGly- [ D-Glu (OAll) was added]-OH (483mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF and DIPEA (193mg,1.5mmol) was added and the reaction was allowed to proceed at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give 2Nal-azaGly- [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added Fmoc-D-Arg (Pbf) -OH (648mg,1mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF, followed by DIPEA (193mg,1.5mmol), and reacted at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, methylene chloride, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence, and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] ] -2Nal-azaGly- (D-Glu) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:82/18-72/28 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, the mixture was purified using Sunfire, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 7.8mg of a white solid.

Mass spectrum [ M +2H]/2⁺:596.0

HPLC elution time: 11.03 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Linear concentration gradient elution: eluent a/B-95/5-35/65, eluent a was 0.05% TFA in water, eluent B was 0.05% TFA in acetonitrile (20 min)

Flow rate: 1.0 mL/min

Example 2 WY-3

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-NMe-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4- (isopropylamino) butyl) -1-methyl-22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Preparation of Fmoc-2Nal-NMe-azaGly- [ D-Glu (OAll) ] -OH (WY-3-I):

(5S,11R) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -7-methyl-5- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

Step 1.(R) -5-allyl-1-tert-butyl-2- (tert-butoxycarbonylamino) glutaric acid diester (WY-3-B)

(R) -5-tert-butoxy-4- (tert-butoxycarbonylamino) -5-oxopentanoic acid (2g,6.59mmol) and 3-bromo-1-propene (1.6g,13.19mmol) were dissolved in DMF (30mL), sodium carbonate (1.4g,13.19mmol) was slowly added, and after stirring at room temperature for 4 hours, water (100mL) was added, followed by extraction with ethyl acetate (100 mL. times.3). The organic phases were combined, washed with water (50mL × 4), dried over anhydrous sodium sulfate, filtered, and spin-dried, and the resulting crude product was isolated and purified by flash chromatography (silica gel column, petroleum ether/ethyl acetate ═ 9/1) to give the title compound (2.1g, 92.80%) as a colorless oily liquid. LCMS (ESI) [ M +23 ]]⁺＝367.9.

Step 2 (R) -5-allyl-1-tert-butyl-2-aminoglutaric diester (WY-3-C)

(R) -5-allyl-1-tert-butyl-2- (tert-butoxycarbonylamino) glutaric acid diester (2.1g,6.11mmol) was placed in a 250mL one-necked flask, and then hydrogen chloride-ethyl acetate solution (2M,90mL) was slowly added. The reaction solution was stirred at room temperature for 1 hour. Then adding 400The solution was diluted with mL of diethyl ether, rotary evaporated to a volume of 100mL, and 400mL of diethyl ether was added, and rotary evaporated to a volume of 100mL, which was repeated three times and then rotary dried to give the title compound (1.5g, 87.88%) as a yellow oily liquid, which was used directly in the next reaction. LCMS (ESI) [ M + H ]]⁺＝244.0.

Step 3 (S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionyl) -2-methylhydrazinocarbonate (WY-3-E)

(S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionic acid (3.5g,8mmol), tert-butoxycarbonylhydrazine (1.171g, 8mmol), HATU (3.955g, 10.4mmol), HOBT (1.622g,12mmol) was dissolved in dichloromethane (150mL) and diisopropylethylamine (3.120g, 24mmol) was slowly added. The reaction solution was stirred at room temperature for 4 hours, then, it was spin-dried, and the residue was added with water (50mL) and extracted with ethyl acetate (50 mL. times.3). The organic layers were combined, washed with water (50mL × 3), the organic phase was spin dried and purified by flash chromatography (silica gel column, eluent: ethyl acetate/petroleum ether ═ 3/2) to afford the title compound (4g, 88.40%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝565.8.

Step 4 (S) - (9-hydro-fluoren-9-yl) methyl-1- (1-methylhydrazino) -3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbamate (WY-3-F)

(S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionyl) -2-methylhydrazinocarbonate (4g,7.07mmol) was dissolved in methylene chloride (100mL), and a hydrogen chloride-dioxane solution (4N,100mL) was slowly added, and the reaction solution was stirred at room temperature for 5 hours. The reaction mixture was concentrated and dried by spinning, ethyl acetate (20mL) and petroleum ether (80mL) were added, slurried and stirred for 30 minutes, filtered, washed three times with 60mL of a mixed petroleum ether/ethyl acetate solution (v: v ═ 4/1), the filter cake was collected and dried by spinning to give the title compound (3g, 91.16%) as a white solid, which was used directly in the next reaction. LCMS (ESI) [ M + H ]]⁺＝465.7.

Step 5 (S) - (9H-fluoren-9-yl) methyl-1- (isocyanato (methyl) amino) -3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbamate (WY-3-G)

A solution of triphosgene (673mg,2.27mmol) in dry dichloromethane (20mL) was cooled toA solution of the hydrochloride salt of (S) - (9-hydro-fluoren-9-yl) methyl-1- (1-methylhydrazino) -3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbamate (2.29g,4.54mmol) in anhydrous dichloromethane (30mL) was slowly added dropwise to the above solution at 0 deg.C, and the resulting mixture was stirred at 0 deg.C for 3 hours. The reaction mixture was slowly quenched dropwise with triethylamine (1.377g, 13.608mmol), and the mixture was stirred at room temperature for 20 minutes to give a solution containing the title compound (about 4.54mmol), which was used directly in the next reaction. LCMS (ESI) [ M + H ]]⁺＝491.8.

Step 6 (R) -5-allyl-1-tert-butyl-2- (2- ((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionyl) -2-methylhydrazinocarbonylamino) glutaric acid diester (WY-3-H)

The hydrochloride of (R) -5-allyl-1-tert-butyl-2-aminoglutaric diester (1.267G,4.54mmol) and (S) - (9-hydro-fluoren-9-yl) methyl-1- (isocyanato (methyl) amino) -3- (naphthalen-2-yl) -1-oxopropyl-2-yl-carbamate (WY-3-G) (about 4.54mol, i.e., the solution of the title compound from step 5) were dissolved in dichloromethane (50mL), the reaction was stirred at room temperature for 20 minutes, and then triethylamine (1.377G, 13.61mmol) was slowly added to the reaction, and the reaction was stirred at room temperature for 16 hours. The reaction mixture was concentrated, and the residue was added to water (100mL) and extracted with ethyl acetate (100 mL. times.3). The organic layers were combined, washed with water (50mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was isolated and purified by silica gel column chromatography to give the title compound (1.6g, 48%) as a white solid. LCMS (ESI) [ M +23 ]]⁺＝757.0.

Step 7 (5S,11R) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -7-methyl-5- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid (WY-3-I)

(R) -5-allyl-1-tert-butyl-2- (2- ((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propanoyl) -2-methylhydrazinocarbonylamino) glutaric acid diester (1550mg,2.11mmol) was dissolved in dichloromethane (50mL) and trifluoroacetic acid (50mL) was added slowly and the reaction stirred at room temperature for 2 h. The reaction was spun dry and dichloromethane (50 mL. times.3) was added and spun dry three times to remove excess trifluoroethyl etherThe mixture was separated and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile/water system containing 0.1% trifluoroacetic acid) to give the title compound (780mg, 54.47%) as a white solid. LCMS (ESI) [ M + H-]⁺＝678.7。¹H NMR(400MHz,DMSO-d₆)δ12.73(s,1H),8.76(s,1H),7.72-7.86(m,7H),7.60(q,J＝15.2Hz,2H),7.20-7.46(m,,7H),7.15(d,J＝7.2Hz,1H),5.78(s,1H),5.19(t,J＝17.6Hz,2H),4.87(s,1H),4.21-4.40(m,3H),4.07(s,3H),3.16(s,1H),3.08(s,3H),2.78(s,1H),2.41(s,2H).1.97(d,J＝24.8Hz,2H).

B solid phase Synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (225mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20mL was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -RinkAmide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc-2Nal-NMe-azaGly- [ D-Glu (OAll) was added]A solution of-OH (400mg,0.75mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was then added DIPEA (193mg,1.5mmol) and reacted at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give 2Nal-NMe-azaGly- [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added Fmoc-D-Arg (Pbf) -OH (648mg,1mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF, followed by DIPEA (193mg,1.5mmol), and reacted at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2Nal-NMe-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe, etc. are introduced in sequence, and the resulting resin is treated with DMF, methylene chloride, methanol, methylThe tert-butyl ether is washed in sequence and then drained to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-NMe-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 80mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal-NMe-azaGly- (D-Glu) ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:84/16-74/26 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, the mixture was purified using Sunfire, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 3.7mg of a white solid.

Mass spectrum [ M +2H]/2⁺:603.0

HPLC elution time: 10.73 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 3 WY-4

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-Amtp]]-Lys(iPr)-NH₂

(2R,7S,10S,13S,16R,19S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -7-benzyl-16- (3-guanidinopropyl) -10- (4-hydroxybenzyl) -13- (4- (isopropylamino) butyl) -19- (naphthalen-2-ylmethyl) -5,8,11,14,17, 20-hexaoxo-1, 6,9,12,15,18,21,24, 25-nonaazabicyclo [21.2.1] hexacosane 23(26), 24-diene-2-carboxamide

Amtp＝(R)-2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)pentanedioic acid

Preparation of Fmoc-2 Nal-Ampp-OH (WY-4-G):

(R) -2- (4- (((S) -2- (((9 hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionamido) methyl) -1 hydro-1, 2, 3-triazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid:

step 1.(R) -4-azido-5-tert-butoxy-5-oxobutanoic acid (WY-4-B)

(R) -4-amino-5-tert-butoxy-5-oxobutanoic acid (1g,4.9mmol), 1H-imidazole-1-sulfonylazide hydrochloride (1.55g,7.4mmol), copper sulfate pentahydrate (246mg,1mmol) and potassium carbonate (2.72g,19.7mmol) were added to methanol (400mL), and the resulting mixture was stirred at room temperature overnight. The solvent was then removed under reduced pressure and the pH adjusted to 2 with 0.5N hydrochloric acid. The obtained mixture is treated with acetic acidExtraction with ethyl ester (3X 200 mL). The organic layers were combined, washed with saturated brine (200mL), dried over sodium sulfate, and filtered. The filtrate was concentrated to give the title mixture (1.2g, 89%) as a colorless oil. LCMS (ESI) [ M + Na ]]⁺＝251.9.

Step 2.(S) - (9 hydro-fluoren-9-yl) methyl-3- (naphthalen-2-yl) -1-oxo-1- (prop-2-ynylamino) propyl-2-yl-carbamate (WY-4-D)

(S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionic acid (3g,6.9mmol), propynylamine (0.76g,13.7mmol), HATU (2.87g,7.6mmol) and DIPEA (1.77g,13.7mmol) were added to dichloromethane (100mL), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then filtered, and the filtrate was washed with saturated brine (20mL), dried over sodium sulfate, and filtered. The filtrate was concentrated to give the title mixture (2.3g, 71%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝475.2.

Step 3 (R) -4- (4- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carboxamido) -3- (naphthalen-2-yl) propionamido) methyl) -1-hydro-1, 2, 3-triazol-1-yl) -5-tert-butoxy-5-oxopropanoic acid (WY-4-E)

(S) - (9-hydro-fluoren-9-yl) methyl-3- (naphthalen-2-yl) -1-oxo-1- (propyl-2-alkynylamino) propyl-2-yl-carbamate (2.69g,5.7mmol), (R) -4-azido-5-tert-butoxy-5-oxopentanoic acid (1.3g,5.7mmol), copper sulfate pentahydrate (284mg,1.1mmol) and sodium ascorbate (450mg,2.3mmol) were added to dimethyl sulfoxide (60mL) and water (10mL), and the resulting mixture was stirred overnight at room temperature. The mixture was adjusted to pH 2 with 0.5N aqueous hydrochloric acid and extracted with ethyl acetate (500 mL). The ethyl acetate layer was then washed with saturated brine (5X 150mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and the crude product obtained was purified by column chromatography over silica gel (eluent: dichloromethane/methanol-20/1). After purification the title compound (2.1g, 53%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝704.0.¹H NMR(400MHz,DMSO-d₆)δ12.32(s,1H)；8.64(s,1H)；7.95(s,1H)；7.86-7.74(m,7H)；7.59(q,J＝7.1Hz,2H)；7.05-7.45(m,3H)；7.37(q,J＝8Hz,2H)；7.24(t,J＝7.4Hz,1H)；7.15(t,J＝7.4Hz,1H)；5.37(q,J＝5.1Hz,1H)；4.39-4.34(m,3H)；4.13-4.00(m,3H)；3.16(dd,J₁＝13.4Hz,J₂＝4Hz,1H)；3.01-2.95(m,1H)；2.42-2.05(m,4H)；1.38(s,9H).

Step 4.(R) -5-allyl-1-tert-butyl-2- (4- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionamido) methyl) -1-hydro-1, 2, 3-triazol-1-yl) glutaric acid diester (WY-4-F)

(R) -4- (4- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionamido) methyl) -1-hydro-1, 2, 3-triazol-1-yl) -5-tert-butoxy-5-oxopentanoic acid (1.3g,1.8mmol), 3-bromopropyl-1-ene (448mg,3.7mmol) and sodium carbonate (588mg,5.5mmol) were added to DMF (30mL) and the resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate (400mL), and washed with water (3X 100mL) and saturated brine (2X 100mL), respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate 2/3). The title compound (1.3g, 95%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝744.4.

Step 5.(R) -2- (4- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionamido) methyl) -1-hydro-1, 2, 3-triazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-4-G)

(R) -5-allyl-1-tert-butyl-2- (4- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-2-yl) propionamido) methyl) -1-hydro-1, 2, 3-triazol-1-yl) glutaric acid diester (1.28g,1.7mmol) was added to dichloromethane (20mL) and trifluoroacetic acid (20mL), and the resulting mixture was stirred at room temperature for 6 hours. The mixture was concentrated to give the title compound (1.15g, 97%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝688.0.¹H NMR(400MHz,DMSO-d₆)δ12.85(s,1H)；8.63(t,J＝5.0Hz,1H)；7.96(s,1H)；7.86-7.73(m,7H)；7.60(q,J＝6.9Hz,2H)；7.51-7.45(m,3H)；7.40-7.34(m,2H)；7.24(t,J＝7.4Hz,1H)；7.15(t,J＝7.4Hz,1H)；5.90-5.83(m,1H)；8.42-8.38(m,1H)；5.28-5.16(m,2H)；4.51-4.50(m,2H)；4.40-4.35(m,3H)；4.13-4.08(m,3H)；3.19-3.15(m,1H)；3.01-2.95(m,1H)；2.51-2.46(m,1H)；2.37-2.26(m,2H)；2.22-2.15(m,1H).

B solid phase Synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (225mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF 6:10:84 solution 20mL was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and a solution of Fmoc-2 Nal-Ampp-OH (400mg,0.75mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL of DMF was added followed by DIPEA (193mg,1.5mmol) and the reaction was carried out at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give 2 Nal-Ampp-Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added Fmoc-D-Arg (Pbf) -OH (648mg,1mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF, followed by DIPEA (193mg,1.5mmol), and reacted at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2 Nal-Ampp-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, methylene chloride, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2 Nal-Ampp-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 80mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence, and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2 Nal-http ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:76/24-72/28 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, the mixture was purified using Sunfire, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 7.6mg of a white solid.

Mass spectrum [ M +2H]/2⁺:607.5

HPLC elution time: 10.66 minutes

Column: SunAire C18, 4.6X 150mm,3.5um

Linear concentration gradient elution: eluent a/B95/5-35/65, eluent a was 0.01% TFA in water, eluent B was 0.01% TFA in acetonitrile (20 min)

Flow rate: 1.0 mL/min

Example 4 WY-6

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-aza2Nal-Gly-(D-Glu)]]-Lys(iPr)-NH₂

(8R,13S,16S,19S,22R) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -13-benzyl-22- (3-guanidinopropyl) -16- (4-hydroxybenzyl) -19- (4- (isopropylamino) butyl) -2- (naphthalen-2-ylmethyl) -3,6,11,14,17,20, 23-heptaoxo-1, 2,4-,7,12,15,18, 21-octaazacycloeicosatrine-8-carboxamide

Preparation of Fmoc- [ D-Arg (Pbf) ] -aza2Nal-Gly-OH (WY-6-F):

(R) -1- (9H-fluoren-9-yl) -8- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-5- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid.

Step 1. tert-butyl-2- (naphthalen-2-ylmethyl) hydrazinocarbonate (WY-6-B).

2-Naphthalenecarboxaldehyde (5g,32mmol) and tert-butylhydrazinocarbonate (4.23g,3mmol) were added to ethanol (200mL) and the mixture was heated under reflux for 6 hours. The mixture was concentrated and dissolved in tetrahydrofuran (200mL), and palladium hydroxide-carbon (20%, 2g) was added, followed by connection to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 16 hours. The reaction solution was filtered, the filtrate was concentrated, and the obtained crude product was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate-5/1) to obtain the title compound (6.9g, 79%) as a white solid. LCMS (ESI) [ M + Na ]]⁺＝295.0.

Step 2. tert-butyl-2- (2- (benzyloxy) -2-oxoethylcarbamoyl) -2- (naphthalen-2-ylmethyl) hydrazinocarbonate (WY-6-C)

A solution of triphosgene (2.46g,8.3mmol) in dichloromethane (50mL) was cooled to 0 deg.C and to the above solution was added a solution of benzyl-2-aminoacetate hydrochloride (3.34g,16.5mmol) and triethylamine (1.11g,11mmol) in dichloromethane (60mL) and the resulting mixture was stirred at 0 deg.C for 2 h. Triethylamine (7.24g,71.7mmol) was then added to the mixture, and the mixture was stirred at 0 ℃ for 30 minutes. Then, addTert-butyl-2- (naphthalen-2-ylmethyl) hydrazinocarbonate (3g,11mmol), and the resulting mixture was stirred at room temperature overnight. To the mixture was added saturated sodium bicarbonate (200mL) and extracted with dichloromethane (3X 200 mL). The organic layers were combined, washed with saturated brine (200mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate 3/1) to obtain the title compound (4.7g, 92%) as a white solid. LCMS (ESI) [ M + Na ]]⁺＝486.1.¹H NMR(400MHz,DMSO-d₆)δ9.7(s,1H)；7.90-7.84(m,3H)；7.74(s,1H)；7.52-7.42(m,3H)；6.92(s,1H)；4.67(s,2H)；6.38(s,2H)；1.42(s,9H)；1.34(s,9H).

Step 3, benzyl-2- (1- (naphthalen-2-ylmethyl) hydrazinocarbonylamino) acetate (WY-6-D).

A mixture of tert-butyl-2- (2- (benzyloxy) -2-oxoethylcarbamoyl) -2- (naphthalen-2-ylmethyl) hydrazinocarbonate (2.5g,5.4mmol) dissolved in hexafluoroisopropanol (30mL) was microwaved to 100 ℃ and reacted for 1 hour. The resulting mixture was concentrated to give the crude title compound (2g, 91%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝364.1.

Step 4.(R) -benzyl-1- (9 hydro-fluoren-9-yl) -8- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-5- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (WY-6-E).

A solution of benzyl-2- (1- (naphthalen-2-ylmethyl) hydrazinocarbonylamino) acetate (1.9g,5.2mmol), Fmoc-D-Arg (Pbf) -OH (5.09g,7.5mmol) and pyridine (5mL) in dichloromethane (40mL) was cooled to 0 deg.C, phosphorus oxychloride (1.6g,10.5mmol) was added to the solution, and the resulting reaction was stirred at 0 deg.C for 2 hours. The resulting mixture was concentrated, and the residue was purified by column chromatography on silica gel (eluent: dichloromethane/ethyl acetate 2/3). The title compound (3.3g, 63%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝994.4.

Step 5, Fmoc- [ D-Arg (Pbf) ] -aza2Nal-Gly-OH (WY-6-F)

(R) -1- (9-hydro-fluoren-9-yl) -8- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-5- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

(R) -benzyl-1- (9H-fluoren-9-yl) -8- (naphthalen-2-ylmethyl) -3,6, 9-trioxo-5- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (3.3g,3.3mmol) and palladium on carbon (10%, 1g) were added to methanol (100mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 6 hours. The reaction solution was filtered, the filtrate was concentrated, and the obtained crude product was purified by silica gel column chromatography (eluent: dichloromethane/methanol-10/1). The title compound (1.7g, 57%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝904.3.¹H NMR(400MHz,DMSO-d₆)δ10.24(s,1H)；7.90-7.65(m,10H)；7.46-7.30(m,8H)；7.15-6.90(m,1H)；6.72-6.57(m,2H)；4.31-4.17(m,3H)；3.92-3.88(m,1H)；3.62-3.50(m,2H)；2.97-2.86(m,4H)；2.51-2.49(m,2H)；2.49(s,3H)；2.42(s,3H)；1.99(s,3H)；1.68 1.52(m,2H)；1.38-1.34(m,8H).

B. Solid phase synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (255mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc- [ D-Glu (OAll) was added]-OH 410mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In thatFmoc- [ D-Arg (Pbf) is added into the resin]-aza2Nal-Gly-OH (677mg,1mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give Fmoc- [ D-Arg (Pbf)]-aza2Nal-Gly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, methylene chloride, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-aza2Nal-Gly-OH-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence, and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -aza2Nal-Gly- (D-Glu) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (1)0 min), flow rate 25 mL/min, eluent A/B78/22-74/26, using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 27.9mg of a white solid.

Mass spectrum [ M +2H]/2⁺:596.1

HPLC elution time: 12.89 minutes

Elution conditions: c

Column: xbridge Peptide BEH column C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 5.WY-7

[cyclo side[Phe-Tyr-azaLys(iPr)-(D-Arg)-2Nal-Gly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,8S,14R,19S,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -19-benzyl-5- (3-guanidinopropyl) -22- (4-hydroxybenzyl) -2- (4- (isopropylamino) butyl) -8- (naphthalen-2-ylmethyl) -3,6,9,12,17,20, 23-heptaoxo-1, 2,4,7,10,13,18, 21-octaazacycloeicosatriane-14-carboxamide

Preparation of Fmoc-Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf) ] -OH (WY-7-M):

(5S,11R) -5- (4-tert-Butoxybenzyl) -8- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-11- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid.

Step 1.(R) -benzyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) pentanoate (WY-7-B).

Fmoc-D-Arg (Pbf) -OH (5g,7.7mmol), benzyl bromide (1.58g,9.3mmol) and sodium carbonate (1.64g,15.4mmol) were added to DMF (50mL) and the resulting mixture was stirred at room temperature for 16 h. To the mixture was added ethyl acetate (300mL), which was then washed with saturated brine (5X 100mL), and the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained crude product was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate-2/1) to give the title compound (5.6g, 98%) as a colorless oil. LCMS (ESI) [ M + H ]]⁺＝739.3.¹H NMR(400MHz,DMSO-d₆)δ7.89(d,J＝7.6Hz,2H)；7.84(d,J＝8.0Hz,1H)；7.70(d,J＝7.2Hz,2H)；7.41(t,J＝7.4Hz,2H)；7.35-7.30(m,8H)；6.66(s,1H)；6.43(s,1H)；5.09(s,2H)；4.32-4.26(m,2H)；4.21(t,J＝6.8Hz,1H)；4.10-4.06(m,1H)；3.06-2.99(m,2H)；2.92(s,2H)；2.48(s,3H)；2.42(s,3H)；1.99(s,3H)；1.75-1.70(m,1H)；1.62-1.57(m,1H)；1.44(s,2H)；1.38(s,6H).

Step 2.(R) -benzyl-2-amino-5- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) pentanoate (WY-7-C).

(R) -benzyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) pentanoate (5.6g,7.6mmol) was added to dichloromethane (50mL) and diethylamine (50mL), and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol-10/1) to give the title compound (3.8g, 97%) as a brown oil. LCMS (ESI) [ M + H ]]⁺＝517.3.

Step 3.4- (isopropylamino) -butan-1-ol (WY-7-E).

4-Aminobutan-1-ol (10g,112mmol), acetone (9.78g,169mmol) and acetic acid (0.5mL) were added to methanol (100mL) and the resulting mixture was allowed to stand at room temperatureStirred for 3 hours. To the reaction mixture was added sodium cyanoborohydride (10.6g,169mmol), and the mixture was stirred at room temperature for 6 hours. The resulting mixture was concentrated to give the crude title compound (25g, 80%) as a colorless oil, which was used directly in the next step without purification. LCMS (ESI) [ M + H ]]⁺＝132.1.

Step 4. tert-butyl-4-hydroxybutyl (isopropyl) carbamate (WY-7-F).

4- (isopropylamino) butan-1-ol (14.5g,110.7mmol), di-tert-butyl dicarbonate (36.2g,166mmol) and triethylamine (33.5g,332mmol) were added to dichloromethane (1000mL), and the resulting mixture was stirred at room temperature overnight. The resultant mixture was concentrated and purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate 4/1) to give the title compound (20.5g, 80%) as a colorless oil. LCMS (ESI) [ M + Na ]]⁺＝254.1.

Step 5. tert-butyl-isopropyl- (4-oxobutyl) carbamate (WY-7-G).

A dry three-necked flask was charged with dry dichloromethane (40mL), cooled to-78 ℃ using a dry ice-acetone bath, and then oxalyl chloride (4.12g,32mmol) and dimethyl sulfoxide (5.06g,65mmol) were added dropwise in that order. The reaction was kept at-78 deg.C and stirred for 1 hour, then a solution of tert-butyl-4-hydroxybutyl (isopropyl) carbamate (5g,22mmol) in dry dichloromethane (20mL) was added dropwise. The reaction mixture was further reacted at-78 ℃ for 1.5 hours, and then triethylamine (15.3g,152mmol) was added thereto, and the reaction mixture was warmed to room temperature and stirred for 30 minutes. Water (100mL) was added to the reaction mixture, which was then extracted with methylene chloride (3X 200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude title compound (5.3g, 92%) as a colorless oil. The crude product was used in the next step without purification. LCMS (ESI) [ M + Na ]]⁺＝252.0.

And step 6, benzyl-2- (4- (tert-butyloxycarbonyl (isopropyl) amino) butyl) hydrazinocarbonate (WY-7-H).

Tert-butyl-isopropyl (4-oxobutyl) carbamate (5g,21.8mmol), benzylhydrazinocarbonate (7.25g,43.7mmol) and acetic acid (1mL) were added to methanol (50mL) and the resulting mixture was stirred at room temperature for 16 h. Adding cyanoborohydride into the reaction liquidSodium (2.75g,43.7mmol) and stirring at room temperature was continued for 1 hour. The resultant mixture was concentrated and purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate-3/1) to give the title compound (5.7g, 69%) as a colorless oil. LCMS (ESI) [ M + H ]]⁺＝380.3.

Step 7.(R) -benzyl-5- (benzyloxycarbonylamino) -10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-carboxylate (WY-7-I).

A solution of triphosgene (1.1g,3.7mmol) in dichloromethane (40mL) was cooled to 0 deg.C, to which was added dropwise a solution of (R) -benzyl-2-amino-5- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) valerate (3.8g,7.4mmol) in dichloromethane (20mL), and the reaction was stirred at 0 deg.C for 1.5 hours. Then benzyl-2- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) hydrazinocarbonate (4.19g,11mmol) was added and the reaction was stirred at room temperature for 2 hours. Water (200mL) was added to the reaction mixture, which was then extracted with methylene chloride (3X 200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate-1/1). The title compound (4.76g, 70%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝922.5.

Step 8.(R) -5-amino-10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-oxocarboxylic acid (WY-7-J).

(R) -benzyl-5- (benzyloxycarbonylamino) -10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-carboxylate (4.5g,4.9mmol) and palladium-carbon (10%, 1.5g) were added to tetrahydrofuran (100mL) and then a hydrogen generator was attached. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 3 hours. The reaction was filtered and the filtrate was concentrated to give the crude title compound (3.4g, 99%) as a white solid which was used in the next step without purification. LCMS (ESI) [ M + H ]]⁺＝698.4.

Step 9.(5R) -methyl-amino-10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-carboxylic acid ester (WY-7-K).

(triethylsilane) diazomethane (2.0M in n-hexane, 2.95mL,5.9mol) was slowly added to a solution of (R) -5-amino-10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-oxocarboxylic acid (3.4g,4.9mmol) in dichloromethane (40mL) and methanol (8mL) at 0 ℃. The reaction solution was stirred at 0 ℃ for 1 hour. Then, acetic acid (1mL) was added to the reaction mixture, and the mixture was warmed to room temperature and stirred for 15 minutes. The resulting mixture was concentrated and purified by reverse phase column chromatography (C18 column, eluent: 0-75% acetonitrile/water, 10mM ammonium bicarbonate in water). The title compound (3.3g, 95%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝712.3.¹H NMR(400MHz,DMSO-d₆)δ6.87(d,J＝8.4Hz,1H)；6.70(s,1H)；6.40(s,2H)；4.46(s,2H)；4.13-4.06(m,1H)；3.60(s,3H)；3.36-3.31(m,2H)；3.05-2.99(m,4H)；2.96(s,2H)；2.47(s,3H)；2.42(s,3H)；2.01(s,3H)；1.69-1.52(m,2H)；1.48-1.23(m,22H)；1.05(d,J＝6.0Hz,6H).

Step 10.(5S,11R) -methyl-5- (4-tert-butoxybenzyl) -8- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-11- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (WY-7-L).

(5R) -methyl-5-amino-10-isopropyl-13, 13-dimethyl-4, 11-dioxo-2- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -12-oxa-3, 5, 10-triazatetradecane-1-carboxylate (3.3g,4.6mmol), Fmoc-Tyr (C: (R) (3.3g,4.6 mmol))^tA solution of Bu) -OH (2.77g,6.0mmol) and pyridine (5mL) in dichloromethane (60mL) was cooled to 0 deg.C, phosphorus oxychloride (1.42g,9.3mmol) was added to the solution, and the reaction was stirred at 0 deg.C for 1.5 h. The reaction was concentrated and purified by reverse phase column chromatography (C18 column, eluent:0-90% acetonitrile/water, 0.01% trifluoroacetic acid in water). The title compound (4g, 75%) was obtained as a white solid. LCMS (ESI) [ M + H ]]⁺＝1153.6.

Step 11.Fmoc-Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf) ] -OH (WY-7-M).

(5S,11R) -5- (4-tert-Butoxybenzyl) -8- (4- (tert-Butoxycarbonylamino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-11- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

(5S,11R) -methyl-5- (4-tert-Butoxybenzyl) -8- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-11- (3- (3- (2,2,4,6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-ylsulfonyl) guanidino) propyl) -2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (4.0g,3.5mmol) and lithium hydroxide monohydrate (583mg,13.9mmol) were added to water (50mL) and tetrahydrofuran (50mL), the resulting mixture was stirred at room temperature for 2 hours, and then tetrahydrofuran was removed under reduced pressure. The reaction solution was adjusted to pH 4 with 0.5N hydrochloric acid, 1, 4-dioxane (50mL) was added, and (9H-fluoren-9-yl) methyl-2, 5-dioxopyrrolidine-1-carboxylate (1.4g,4.2mmol) and sodium carbonate (736mg,6.9mmol) were added. The reaction was stirred overnight at room temperature. The reaction mixture was adjusted to pH 3-4 with 0.5N hydrochloric acid and extracted with ethyl acetate (3X 300 mL). The organic layers were combined, washed with saturated brine (200mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained crude product was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate 1/10) to obtain the title compound (2.8g, 71%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝1139.6.¹H NMR(400MHz,DMSO-d₆)δ12.38(s,1H)；10.12(s,1H)；7.98(s,1H)；7.88(d,J＝7.6Hz,2H)；7.64(q,J＝10.8Hz,2H)；7.41(t,J＝7.4Hz,2H)；7.31(t,J＝7.4Hz,2H)；7.16(d,J＝7.6Hz,2H)；6.85(d,J＝8.0Hz,2H)；6.67(s,1H)；6.44(s,2H)；4.23-3.99(m,7H)；3.02-2.84(m,9H)；2.46(s,3H)；2.40(s,3H)；1.96(s,3H)；1.77-1.71(m,1H)；1.60-0.97(m,38H).

B. Solid phase synthesis

2.5g of a commercially available Rink Amide MBHA resin was added(0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (255mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc- [ D-Glu (OAll) was added]-OH 410mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, two amino acids, Gly and 2Nal, are introduced sequentially to give 2Nal-Gly- [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. Fmoc-Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf) was added to the resin]-OH (853mg,0.75mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-Gly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, Phe amino acid was introduced and the resulting resin was washed with DMF, dichloromethane, methanol, methyl tert-butyl ether in sequence and then dried by suction to give Fmoc-Phe-Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-Gly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally the cyclized resin cyclo side [ Phe-Tyr (tBu) -azaLys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal-Gly- [ D-Glu (OAll)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:78/22-68/32 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xtimate C18,10 μm,

column (20 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 31.0mg of a white solid.

Mass spectrum [ M +2H]/2⁺:596.0

HPLC elution time: 12.39 minutes

Elution conditions: c

Column: xbridge Peptide BEH column C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 6: WY-8

[cyclo side[Phe-azaTyr-Lys(iPr)-(D-Arg)-2Nal-Gly-(D-Glu)]]-Lys(iPr)-NH₂

(5S,8R,11S,17R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -22-benzyl-8- (3-guanidinopropyl) -2- (4-hydroxybenzyl) -5- (4- (isopropylamino) butyl) -11- (naphthalen-2-ylmethyl) -3,6,9,12,15,20, 23-heptaoxo-1, 2,4-,7,10,13,16, 21-octaazacycloeicosatrine-17-carboxamide

Preparation of Fmoc-Phe-azaTyr (tBu) -Lys (iPr, Boc) -OH (WY-8-J)

(5S,11S) -5-benzyl-8- (4-tert-Butoxybenzyl) -11- (4- (tert-Butoxycarbonylamino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

Step 1 (S) -benzyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -6- (tert-butoxycarbonyl (isopropyl) amino) -hexanoate (WY-8-B):

(S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -6- (tert-butoxycarbonyl (isopropyl) amino) hexanoic acid (5g,9.79mmol), benzyl bromide (2.512g,14.685mmol) were dissolved in DMF (100mL) and sodium carbonate (2.075g,19.58mmol) was slowly added. The reaction mixture was stirred at room temperature for 2 hours, water (100mL) was added, and the mixture was extracted with ethyl acetate (100 mL. times.3). The organic phases were combined, washed with water (100mL × 4), dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue, which was purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 7/3) to give the title compound (4.6g, 78.22%) as a colorless oily liquid. LCMS (ESI) [ M-100+ H]⁺＝501.1.

Step 2 (S) -benzyl-2-amino-6- (tert-butoxycarbonyl (isopropyl) amino) -hexanoate ester (WY-8-C)

Reacting (S) -benzyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -6- (tert-butyl) carbonylOxycarbonyl (isopropyl) amino) -hexanoate (4.6g,7.66mmol) was dissolved in dichloromethane (30mL) and diethylamine (30mL) was added slowly. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the obtained residue was roughly purified by a silica gel column (eluent: dichloromethane/methanol 10/1) and then purified by reverse phase preparative purification (C18 column, eluent: ammonium bicarbonate/acetonitrile/water) to obtain the title compound (1.7g, 58.64%) as a colorless oily liquid. LCMS (ESI) [ M + H ]]⁺＝379.1.

Step 3 (S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3-phenylpropionyl) hydrazinocarbonic acid ester (WY-8-E)

(((9-hydro-fluoren-9-yl) methoxy) carbonyl) -L-phenylalanine (3.77g,9.73mmol), tert-butoxycarbonylhydrazine (1.286g, 9.73mmol), HATU (4.810g, 12.65mmol) and HOBt (1.972g,14.60mmol) were dissolved in dichloromethane (200mL), diisopropylethylamine (6.288g, 48.65mmol) was slowly added, and the reaction mixture was stirred at room temperature for 3 hours. The resulting mixture was spun dry, water (100mL) was added, and extraction was performed with ethyl acetate (100 mL. times.3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and spin-dried. The residue was slurried with ethyl acetate (30mL) and petroleum ether (150mL) and filtered to give the title compound (4.15g, 84.94%) as a white solid. LCMS (ESI) [ M +23 ]]⁺＝524.0.

Step 4 (S) - (9-hydro-fluoren-9-yl) methyl-1-hydrazino-1-oxo-3-phenylpropyl-2-ylcarbamate (WY-8-F)

Compound (S) -tert-butyl-2- (2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3-phenylpropionyl) hydrazinocarbonic acid ester (4.15g,8.27mmol) was dissolved in methylene chloride (50mL), a solution of hydrogen chloride-1, 4-dioxane (4N,50mL) was slowly added, and the reaction solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated to dryness, ethyl acetate (20mL) and petroleum ether (80mL) were added, slurried and stirred for 30 minutes, filtered, the filter cake was washed three times with 60mL (petroleum ether/ethyl acetate ═ 4/1), the filter cake was collected and dried to give the title compound (3.17g, 87.52%) as a white solid. LCMS (ESI) [ M + H-36 ]]⁺＝402.0.

Step 5 (S) - (9-hydro-fluoren-9-yl) methyl-1- (2- (4-tert-butoxybenzyl) hydrazino) -1-oxo-3-phenylpropyl-2-ylcarbamate (WY-8-G)

The hydrochloride (3.17g,7.25mmol) of (S) - (9-hydro-fluoren-9-yl) methyl-1-hydrazino-1-oxo-3-phenylpropyl-2-ylcarbamate and p-tert-butoxybenzaldehyde (1.292g, 7.25mmol) were dissolved in tetrahydrofuran (100mL), concentrated hydrochloric acid (1mL) was added, and the mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (1.367g, 21.75mmol) was then slowly added to the reaction solution, and the reaction solution was stirred at room temperature for 3 hours. Water (20mL) was added to the reaction solution, the reaction solution was spin-dried, water (50mL) was added to the residue, extraction was performed with ethyl acetate (50 mL. times.3), and the organic layers were combined and washed with water (50 mL. times.3). The organic layer was spin dried and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 3/1) to afford the title compound (2.38g, 58.10%). LCMS (ESI) [ M + H ]]⁺＝564.3.

Step 6 benzyl- (S) -6- ((tert-Butoxycarbonyl) (isopropyl) amino) -2-isocyanatohexanoate (WY-8-H)

A solution of triphosgene (1.333g, 4.49mmol) in anhydrous dichloromethane (40mL) was cooled to 0 deg.C, and then a solution of (S) -benzyl-2-amino-6- (tert-butoxycarbonyl (isopropyl) amino) hexanoate (1.700g,4.49mmol) in anhydrous dichloromethane (60mL) was slowly added to the solution at 0 deg.C. The reaction was stirred at room temperature for 2 hours, then triethylamine (4.089g, 40.41mmol) was added slowly, and the reaction was stirred at room temperature for twenty minutes to give a solution containing the title compound (about 4.49mmol) which was used directly in the next reaction. LCMS (ESI) [ M +23 ]]⁺＝427.0。

Step 7 (5S,11S) -benzyl-5-benzyl-8- (4-tert-butoxybenzyl) -11- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (WY-8-I)

To a solution of benzyl- (S) -6- ((tert-butoxycarbonyl) (isopropyl) amino) -2-isocyanatohexanoate (about 4.49mmol, i.e. the solution of the WY-8-H compound obtained in step 6) was slowly added a solution of (S) - (9 hydro-fluoren-9-yl) methyl-1- (2- (4-tert-butoxybenzyl) hydrazino) -1-oxo-3-phenylprop-2-ylcarbamate (2.38g, 4.23mmol) in dichloromethane (15mL) and the reaction was stirred at room temperature for 2 hours and then concentrated to dryness. The residue was added to ethyl acetate (100mL), washed with water (50 mL. times.3), and the organic layer was washedThe layers were dried by spinning and purified by flash chromatography (silica gel column, eluent: ethyl acetate/petroleum ether-1/3) to give the title compound (2.2g, 53.72%) as a colorless liquid. LCMS (ESI) [ M-100+ H]⁺＝868.1。

Step 8 Fmoc-Phe-azaTyr (tBu) -Lys (iPr, Boc) -OH (WY-8-J)

(5S,11S) -5-benzyl-8- (4-tert-Butoxybenzyl) -11- (4- (tert-Butoxycarbonylamino) butyl) -1- (9H-fluoren-9-yl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-oxocarboxylic acid

(5S,11S) -benzyl-5-benzyl-8- (4-tert-butoxybenzyl) -11- (4- (tert-butoxycarbonyl (isopropyl) amino) butyl) -1- (9-hydro-fluoren-9-yl) -3,6, 9-trioxo-2-oxa-4, 7,8, 10-tetraazadodecane-12-carboxylate (2g,2.07mmol) and palladium on carbon (10%, 100mg) were added to tetrahydrofuran (60mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 4 hours. The reaction was filtered, the filtrate was concentrated, and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (680mg, 37.42%) as a white solid. LCMS (ESI) [ M-100+ H]⁺＝778.1。¹H NMR(400MHz,DMSO-d₆)δ10.38(s,1H),7.88(d,J＝7.2Hz,3H),7.64(q,J＝12Hz,2H),7.18-7.42(m,10H),7.01(d,J＝8Hz,2H),6.84(d,J＝8Hz,2H),6.26(d,J＝7.2Hz,1H),4.15-4.25(m,7H),2.84(s,5H),1.51-1.86(m,2H),1.33(s,11H),1.24(s,11H),0.98-1.05(m,6H).

B. Solid phase synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (255mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMFAddition of Fmoc- [ D-Glu (OAll)]-OH 410mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Gly, 2Nal, D-Arg (Pbf) and the like are introduced in sequence to obtain [ D-Arg (Pbf)]-2Nal-Gly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

To the resin was added Fmoc-Phe-azaTyr (tBu) -Lys (iPr, Boc) -OH (650mg,0.75mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF, followed by DIPEA (193mg,1.5mmol) and reaction at room temperature for 1.5 hours; the obtained resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally Fmoc-Phe-azaTyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal-Gly- [ D-Glu (OAll)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-aza Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal-Gly- [ D-Glu (OAll)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:78/22-73/27 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xtimate C18,10 μm,

column (20 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 24.2mg of a white solid.

Mass spectrum [ M +2H]/2⁺:596.0

HPLC elution time: 11.50 minutes

Elution conditions: c

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 7: WY-9

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-Pro-(D-Glu)]]-Lys(iPr)-NH₂

(3R,8S,11S,14S,17R,20S,25AS) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -8-benzyl-17- (3-guanidinopropyl) -11- (4-hydroxybenzyl) -14- (4- (isopropylamino) butyl) -20- (naphthalen-2-ylmethyl) -1,6,9,12,15,18, 21-heptaoxoeicosahydro-1H-pyrrolo [2,1-C ] [1,4,7,10,13,16,19] heptaazacycloeicosatrine-3-carboxamide

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twiceDo this. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (255mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (193mg,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc- [ D-Glu (OAll) was added]A solution of-OH (410mg,0.75mmol), HATU (570mg,1.5mmol) and HOAt (203mg,1.5mmol) in 20mL DMF was reacted with DIPEA (193mg,1.5mmol) at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Pro,2Nal, D-Arg (Pbf), Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then dried to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-Pro-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence, and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal-Pro- (D-Glu) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (15 min) at a flow rate of 25 mL/min, eluent A/B:77/23-69/31 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 28.4mg of a white solid.

Mass spectrum [ M +2H]/2⁺:615.7

HPLC elution time: 13.46 minutes

Elution conditions: c

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 8: WY-10-a

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-(R-2-AMPP acid)]]-Lys(iPr)-NH₂

(4S,7R,10S,13S,16S,21R,25aS) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -16-benzyl-7- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -10- (4- (isopropylamino) butyl) -4- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo- ((R) -1H-pyrrolo [1,2-a ]) -1,2,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Preparation of (R) -2- ((S) -2- (9-fluorenyl) methoxy) carbonylamino) methyl) pyrrolidin-1-yl) -5- (allyloxy-5-oxopentanoic acid (WY-10-a-E, Fmoc-Allyl-R-2-AMPP acid):

step 1.(R) -5-benzyl-1-tert-butyl-2- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (WY-10-a-A)

(S) -5-benzyl-1-tert-butyl-2-bromoglutaric diester (4.28g,12mmol), (S) -tert-butylpyrrolidine-2-methylcarbamate (2g,10mmol) and sodium carbonate (2.12g,20mmol) were dissolved in DMF (30mL) and N-methylpyrrolidone (10mL), and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (500mL) and washed with water (150 mL. times.5). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate-15/1) to give the title compound (1.7g, 36%) as a colorless oil. MS (ESI) M/z 477.5[ M + H ]]⁺.

Step 2.(R) -5-tert-butoxy-4- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) -5-oxopentanoic acid (WY-10-a-B)

Bis (R) -5-benzyl-1-tert-butyl-2- ((S) -2 ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutarate (1.7g,3.57mmol) and palladium-carbon (10%, 500mg) were added to methanol (50mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 2 hours. The reaction was filtered and the filtrate was concentrated to give the crude title compound (1.4g, 100%) as a colorless oil, which was used in the next step without purification. MS (ESI) M/z 387.1[ M + H ]]⁺.

Step 3 (R) -5-allyl-1-tert-butyl-2- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (WY-10-a-C)

(R) -5-tert-butoxy-4- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) -5-oxopentanoic acid (1.4g,3.6mmol) and 3-bromo-1-propene (1.32g,10.9 mmol)l) was dissolved in dimethylformamide (40mL), sodium carbonate (770mg,7.3mmol) was slowly added, and after stirring at room temperature for 4 hours, water (100mL) was added, followed by extraction with ethyl acetate (100 mL. times.3). The organic phases were combined, washed with water (100mL × 4), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude title compound (1.5g, 97%) as a colorless oil which was used in the next step without purification. MS (ESI): M/z 427.4[ M + H]⁺.

Step 4.(R) -5- (allyloxy) -2- ((S) -2- (aminomethyl) pyrrolidin-1-yl) -5-oxopentanoic acid trifluoroacetic acid hydrochloride (WY-10-a-D)

(R) -5-allyl-1-tert-butyl-2- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (1.5g,3.5mmol) was dissolved in dichloromethane (20mL), then trifluoroacetic acid (20mL) was slowly added and the reaction was stirred at room temperature for 6 hours. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess trifluoroacetic acid to give the crude title compound (1.28g, 85%) as a colorless oil, which was used in the next step without purification. MS (ESI) M/z 271.1[ M + H ]]⁺.

Step 5.(R) -2- ((S) -2- (((9-fluorenyl) methoxy) carbonylamino) methyl) pyrrolidin-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-10-a-E)

(R) -5- (allyloxy) -2- ((S) -2- (aminomethyl) pyrrolidin-1-yl) -5-oxopentanoic acid trifluoroacetic acid hydrochloride (1.28g,3.5mmol), 9-fluorenylmethyl-N-succinimidyl carbonate (1.29g,3.8mmol) and sodium carbonate (1.11g,10.5mmol) were dissolved in water (50mL) and dioxane (50mL) and stirred at room temperature overnight. The reaction mixture was adjusted to pH 3 to 4 with 0.5mol/L hydrochloric acid, extracted with ethyl acetate (200 mL. times.3), and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (1.4g, 82%) as a white solid. MS (ESI) M/z 493.5[ M + H ]]⁺.

¹H NMR(400MHz,DMSO-d₆)δ12.08(s,1H)；7.89(d,J＝7.6Hz,2H)；7.69(d,J＝7.6Hz,2H)；7.41(t,J＝7.4Hz,2H)；7.35-7.30(m,3H)；5.96-5.86(m,1H)；5.31-5.18(m,2H)；4.54(d,J＝5.2Hz,2H)；4.36-4.19(m,3H)；3.34(t,J＝7.2Hz,1H)；3.20-3.18(m,1H)；3.04-2.99(m,1H)；2.92-2.89(m,1H)；2.80-2.72(m,2H)；2.47-2.39(m,2H)；2.03-1.83(m,2H)；1.75-1.60(m,4H).

B solid phase Synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (225mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (0.26mL,1.5mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and a solution of (WY-10-a-E, Fmoc-Allyl-R-2-AMPP acid) (687mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF was added followed by DIPEA (0.26mL,1.5mmol) and reacted at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, this procedure was repeated twice, and the resin was washed with DMF to give (R) -2-AMPP (Allyl) -Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added Fmoc-2Nal-OH (437mg, 1mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL DMF followed by DIPEA (0.26mL,1.5mmol) and reacted at room temperature for 1.5 h; the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give 2Nal- [ (R) -2-AMPP (Allyl)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as D-Arg (Pdf), Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-[(R)-2-AMPP(Allyl)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added followed by DIPEA (0.26mL,1.5mmol) and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [ (R) -2-AMPP ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:79/21-75/25 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, the mixture was purified using Sunfire, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 25.2mg of a white solid.

Mass spectrum [ M +2H]/2⁺:608.6

HPLC elution time: 10.96 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 9: WY-10-b

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-(S-2-AMPP acid)]]-Lys(iPr)-NH₂

(4S,7R,10S,13S,16S,21R,25aS) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -16-benzyl-7- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -10- (4- (isopropylamino) butyl) -4- (naphthalen-2-ylmethyl) -3,6,9,12,15,18,20, 23-heptaoxo- ((S) -1-hydro-pyrrolo [1,2-a ]) -1,2,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Preparation of (S) -2- ((S) -2- (9-fluorenyl) methoxy) carbonylamino) methyl) pyrrolidin-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-10-b-E, Fmoc-Allyl- (S) -2-AMPP acid):

step 1.(S) -5-benzyl-1-tert-butyl-2- ((S) -2 ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (WY-10-b-A)

(R) -5-benzyl-1-tert-butyl-2-bromoglutaric diester (1.3g,3.6mmol), (S) -tert-butylpyrrolidine-2-methylcarbamate (1.09g,5.5mmol) and potassium carbonate (1.01g,7.63mmol) were dissolved in acetonitrile (10mL) and N-methylpyrrolidone (10 mL). The reaction solution was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (300mL) and washed with water (150 mL. times.5). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate-15/1) to give the title compound (1.11g, 64%) as a colorless oil. MS (ESI) M/z 477.1[ M + H ]]⁺.

Step 2 (S) -5-tert-butoxy-4- ((S) -2- ((tert-butoxycarbonylamino) methylpyrrolidin-1-yl) -5-oxopentanoic acid (WY-10-B-B)

Reacting (S) -5-benzyl-1-tert-butylButyl-2- ((S) -2 ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (1.9g,4mmol) and palladium-carbon (10%, 600mg) were added to methanol (60mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 2 hours. The reaction was filtered and the filtrate was concentrated to give the crude title compound (1.5g, 97%) as a colorless oil which was used in the next step without purification. MS (ESI) M/z 387.2[ M + H ]]⁺.

Step 3 (S) -5-allyl-1-tert-butyl-2- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (WY-10-b-C)

(S) -5-tert-butoxy-4- ((S) -2- ((tert-butoxycarbonylamino) methylpyrrolidin-1-yl) -5-oxopentanoic acid (1.5g,3.9mmol) and 3-bromo-1-propene (1.41g,11.7mmol) were dissolved in dimethylformamide (40mL), sodium carbonate (823mg,7.8mmol) was slowly added, after stirring at room temperature for 4 hours, water (100mL) was added, extraction was performed with ethyl acetate (150 mL. times.3.) the organic phases were combined, washed with water (100 mL. times.4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude title compound (1.5g, 90%) as a colorless oil, which was used in the next step without purification]⁺.

Step 4 (S) -5- (allyloxy) -2- ((S) -2- (aminomethyl) pyrrolidin-1-yl) -5-oxopentanoic acid trifluoroacetic acid hydrochloride (WY-10-b-D)

(S) -5-allyl-1-tert-butyl-2- ((S) -2- ((tert-butoxycarbonylamino) methyl) pyrrolidin-1-yl) glutaric acid diester (1.5g,3.5mmol) was dissolved in dichloromethane (20mL), trifluoroacetic acid (20mL) was slowly added, and the reaction was stirred at room temperature for 6 hours. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess trifluoroacetic acid to give the crude title compound (1.4g, 95%) as a colorless oil, which was used in the next step without purification. MS (ESI) M/z 271.0[ M + H ]]⁺.

Step 5 (S) -2- ((S) -2- (9-fluorenyl) methoxy) carbonylamino) methyl) pyrrolidin-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-10-b-E)

(S) -5- (allyloxy) -2- ((S) -2- (aminomethyl) pyrrolidin-1-yl) -5-oxopentanoic acid trifluoroacetic acid hydrochloride (1)3g,3.5mmol), 9-fluorenylmethyl-N-succinimidyl carbonate (1.31g,3.9mmol) and sodium carbonate (751mg,7.1mmol) were dissolved in water (50mL) and dioxane (50mL) and stirred at room temperature overnight. The reaction mixture was adjusted to pH 3 to 4 with 0.5mol/L hydrochloric acid, extracted with ethyl acetate (200 mL. times.3), and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (1.1g, 65%) as a white solid. MS (ESI) M/z 493.0[ M + H ]]⁺.

¹H NMR(400MHz,DMSO-d₆)δ12.37(s,1H)；7.89(d,J＝7.6Hz,2H)；7.68(d,J＝7.6Hz,2H)；7.41(t,J＝7.6Hz,2H)；7.34-7.30(m,2H)；7.16(t,J＝5.6Hz,1H)；5.92-5.83(m,1H)；5.29-5.15(m,2H)；4.50(d,J＝5.6Hz,2H)；4.33-4.19(m,3H)；3.44-3.40(m,1H)；3.40-2.99(m,2H)；2.80-2.77(m,2H)；2.67-2.61(m,1H)；2.42-2.35(m,2H)；1.95-1.50(m,6H).

B solid phase Synthesis

Proceeding from 2.5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 9B. Except that (WY-10-b-E, Fmoc-Allyl- (S) -2-AMPP acid) (687mg,0.75mmol) was used in place of (WY-10-b-E, Fmoc-Allyl- (S) -2-AMPP acid) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) - ] -2Nal- [ (S) -2-AMPP (Allyl) - ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ (S) -2-AMPP (Allyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min using eluent A/B79/21-75/25, use: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, the mixture was purified using Sunfire, C18,10 μm,

Mass spectrum [ M +2H]/2⁺:608.6

HPLC elution time: 11.04 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 10: WY-13

[cyclo side[azaPhe-Tyr-Lys(iPr)-(D-Arg)-2Nal-Gly-(D-Glu)]-Lys(iPr)-NH₂

(5R,8S,11S,14S,20R) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -2-benzyl-11- (3-guanidinopropyl) -5- (4-hydroxybenzyl) -8- (4- (isopropylamino) butyl) -14- (naphthalen-2-ylmethyl) -3,6,9,12,15,18, 23-heptaoxo-1, 2,4,7,10,13,16, 19-octaazacycloeicosatrine-20-carboxamide

Preparation of (R) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (2- ((S) -1- (allyloxy) -3- (4-tert-butoxyphenyl) -1-oxopropyl-2-carbamoyl) -2-benzylhydrazino) -5-oxopentanoic acid (WY-13-I)

Step 1.(S) -allyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (4-tert-butoxyphenyl) propanoate (WY-13-A)

Fmoc-O-tert-butyl-L-tyrosine (5g,10.89mmol) and 3-bromo-1-propene (1.8mL,20.8mmol) were dissolved in DMF (40mL), sodium carbonate (2.3g,21.7mmol) was slowly added, and after stirring at room temperature for 2 hours, water (200mL) was added and extraction was performed with ethyl acetate (250 mL. times.3). The organic phases were combined, washed with water (100mL × 4), dried over anhydrous sodium sulfate, filtered and the resulting crude product was isolated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate ═ 6/1) to give the title compound (3.99g, 73%) as a colorless oily liquid. MS (ESI) M/z 500.2[ M + H ]]⁺.

Step 2.(S) -allyl-2-amino-3- (4-tert-butoxyphenyl) propionate (WY-13-B)

(S) -allyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (4-tert-butoxyphenyl) propanoate (3.99g,8mmol) was added to dichloromethane (50mL) and diethylamine (50mL), and the resulting mixture was stirred at room temperature for 3 hours. The mixture was concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 2/1 to pure ethyl acetate) to give the title compound (1.87g, 84%) as a colorless oil. MS (ESI) M/z 278.1[ M + H ]]⁺.

Step 3 (R) -5-tert-butyl-1-methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) glutaric acid diester (WY-13-C)

(trimethylsilyl) diazomethane (2.0M in N-hexane, 14.1mL,28.2mol) was slowly added to a solution of N- (((9-hydro-fluoren-9-yl) methoxy) carbonyl) -D-glutamic acid-5-tert-butyl ester (10g,23.5mmol) dissolved in dichloromethane (200mL) and methanol (40mL) at 0 ℃. The reaction solution was stirred at 0 ℃ for 1.5 hours. Then, acetic acid (2mL) was added to the reaction mixture, and the mixture was warmed to room temperature and stirred for 30 minutes. The resulting mixture was concentrated and the crude product was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate-4/1) to give the title compound (9.6g, 93%) as a colorless oil. MS (ESI) M/z 440.2[ M + H ]]⁺.

Step 4.(R) -4- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-methoxy-5-oxopentanoic acid (WY-13-D)

(R) -5-tert-butyl-1-methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) glutaric acid diester (9.6g,21.9mmol) was dissolved in dichloromethane (100mL) thenThereafter, trifluoroacetic acid (100mL) was added slowly, and the reaction mixture was stirred at room temperature for 3 hours. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess trifluoroacetic acid to give the crude title compound (8.4g, 99%) as a brown oil which was used in the next step without purification. 384.2[ M + H ] M/z]⁺.

Step 5 (R) -benzyl-2- (4- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-methoxy-5-oxopentanoyl) hydrazinoformate (WY-13-E)

(R) -4- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-methoxy-5-oxopentanoic acid (4.16g,25.1mmol), benzyloxycarbonylhydrazine (4.16g,25.1mmol) and HATU (9.52g,25.1mmol) were dissolved in dichloromethane (200mL), triethylamine (6.33g,62.7mmol) was slowly added, and the reaction mixture was stirred at room temperature for 2 hours. The resulting mixture was spun dry and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (10.5g, 95%) as a white solid. MS (ESI) M/z 532.3[ M + H ]]⁺.

(R) -methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-hydrazino-5-oxopentanoate (WY-13-F)

(R) -benzyl-2- (4- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-methoxy-5-oxopentanoyl) hydrazinoformate (10.4g,19.6mmol) and palladium-on-carbon (10%, 3g) were added to methanol (250mL) and acetic acid (10mL), and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 1 hour. The reaction was filtered and the filtrate was concentrated to give the crude title compound (7.5g, 96%) as a white solid, which was used in the next step without purification. MS (ESI) 398.2[ M + H ]]⁺.

Step 7.(R) -methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (2-benzylhydrazino) -5-oxopentanoate (WY-13-G)

(R) -methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5-hydrazino-5-oxopentanoate (7.4g,18.6mmol) and benzaldehyde (1.98g,18.6mmol) were dissolved in methanol (200mL), formic acid (5mL) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and then dissolved in tetrahydrofuran (200mL), palladium hydroxide-carbon (20%, 2.5g) was added, and the mixture was addedAnd a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature overnight. The reaction was filtered and the filtrate was concentrated to give the crude product, which was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (7.3g, 80%) as a white solid. MS (ESI) M/z 488.2[ M + H ]]⁺.

Step 8 (5R,13S) -allyl-10-benzyl-13- (4-tert-butoxybenzyl) -1- (9H-fluoren-9-yl) -5- (methoxycarbonyl) -3,8, 11-trioxo-2-oxa-4, 9,10, 12-tetraazatetradecane-14-carboxylic acid ester (WY-13-H)

A solution of triphosgene (997mg,3.4mmol) in methylene chloride (40mL) was cooled to 0 ℃ and a solution of (S) -allyl-2-amino-3- (4-tert-butoxyphenyl) propionate (1.86g,6.7mmol) in methylene chloride (40mL) was added dropwise to the solution, and the reaction mixture was stirred at 0 ℃ for 1 hour. Triethylamine (2.03g,20.1mmol) was added thereto, and the reaction mixture was stirred at 0 ℃ for 0.5 hour. Then (R) -methyl-2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (2-benzylhydrazino) -5-oxopentanoate (3.27g,6.7mmol) was added and the reaction stirred at room temperature for 4 h. Water (200mL) was added to the reaction mixture, which was then extracted with methylene chloride (3X 200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 1/1). The title compound (4.8g, 90%) was obtained as a white solid. MS (ESI) M/z 791.3[ M + H ]]⁺.

Step 9.(R) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -5- (2- ((S) -1- (allyloxy) -3- (4-tert-butoxyphenyl) -1-oxopropyl-2-carbamoyl) -2-benzylhydrazino) -5-oxopentanoic acid (WY-13-I)

(5R,13S) -allyl-10-benzyl-13- (4-tert-butoxybenzyl) -1- (9H-fluoren-9-yl) -5- (methoxycarbonyl) -3,8, 11-trioxo-2-oxa-4, 9,10, 12-tetraazatetradecane-14-carboxylate (4.7g,5.9mmol) and sodium carbonate (1.26g,11.9mmol) were dissolved in water (100mL) and tetrahydrofuran (100mL), and the reaction was stirred at room temperature overnight. The reaction mixture was adjusted to pH 3 to 4 with acetic acid, extracted with ethyl acetate (300 mL. times.3), and washed with saturated brine (200 mL). The organic phase is separated off, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue is purified by Prep-HPLC (C18 column, eluent: trifluoroethyl fluorideAcid/acetonitrile/water) to afford the title compound (1.9g, 41%) as a white solid. MS (ESI) M/z 777.5[ M + H ]]⁺.

¹H NMR(400MHz,DMSO-d₆)δ12.72(s,1H)；9.83(s,1H)；7.89(d,J＝7.6Hz,2H)；7.71(d,J＝7.6Hz,3H)；7.41(t,J＝7.4Hz,2H)；7.33-7.17(m,7H)；7.10(d,J＝8.4Hz,2H)；6.83(d,J＝8.0Hz,3H)；5.84-5.78(m,1H)；5.25(d,J＝17.2Hz,1H)；5.16(d,J＝10.4Hz,1H)；4.52-4.17(m,8H)；4.03-3.98(m,1H)；2.97(d,J＝7.2Hz,2H)；2.20(t,J＝7.2Hz,2H)；2.08-2.03(m,1H)；1.79-1.76(m,1H)；1.25(s,9H).

B. Solid phase synthesis

2.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (255mg,0.5mmol), HATU (568mg,1.5mmol) and HOAt (202mg,1.5mmol) in 20mL of DMF was added followed by DIPEA (0.26mL,1.2mmol) and reacted at room temperature for 40 min. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF-6: 10:84 solution 20ml was reacted for one hour, and the resin was washed with DMF to give Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc- [ D-Glu (Azape-Tyr (OtBu)) (COOAllyl) was added]A solution of-OH (WY-13-I,410mg,0.75mmol), HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL DMF was reacted with DIPEA (0.26mL,1.5mmol) at room temperature for 1.5 h. The resin was treated with 20mL of 20% piperidine/DMF for 20 minutes to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give [ D-Glu (Azape-Tyr (OtBu) (COOAllyl))]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Gly, 2Nal, D-Arg (Pbf), Lys (iPr, Boc) and the like are introduced in sequence to obtain Fmoc-Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-Gly-[D-Glu(AzaPhe-Tyr(OtBu)(COOAllyl)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin, palladium tetratriphenylphosphine (1.16g,1mmol) and 1, 3-dimethylbarbituric acid (390mg,2.5mmol) were added to 40mL of anhydrous dichloromethane, and the reaction mixture was reacted at room temperature under argon atmosphere for 3 hours. After the reaction was completed, the resin was washed with a DMF solution of sodium diethyldithiocarbamate trihydrate (2g/400mL,50mL), a DMF solution of DIPEA (2mL/400mL,50mL) and a DMF solution of HOBT (5.4g/400mL,50mL) in that order, and the resin was washed with the above three solutions again for 5 times in the same order. The resin was treated with 20mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. After the resin was washed with DMF, a solution of HBTU (568mg,1.5mmol) and HOBt (202mg,1.5mmol) in 20mL of DMF was added, followed by DIPEA (193mg,1.5mmol), and the reaction was carried out at room temperature for 1.5 hours. The resin is washed by DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction, and finally the cyclized resin [ cyclo side [ azaPhe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal-Gly- (D-Glu) ] -Lys (iPr, Boc) -Rink Amide MBHA resin is obtained.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:83/17-73/27 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xtimate C18,10 μm,

column (20 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 10.8mg of a white solid.

Mass spectrum [ M +2H]/2⁺:596.2

HPLC elution time: 11.38 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 11: WY-15

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-(4-AMIP acid)]]-Lys(iPr)-NH₂

(7S,10S,13S,16R,19S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -7-benzyl-16- (3-guanidinopropyl) -10- (4-hydroxybenzyl) -13- (4- (isopropylamino) butyl) -19- (naphthalen-2-ylmethyl) -5,8,11,14,17, 20-heptaoxo-1, 6,9,12,15,18,21, 24-octaazacyclo [21,2.2] hexocosa-23 (26), 24-diene-2-carboxamide

Preparation of 2- (4- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-imidazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-15-I):

step 1.N- ((1H-imidazol-4-yl) methyl) -1-phenylmethanamine (WY-15-A)

4-Formylimidazole (10g,104.2mmol), benzylamine (22.3g,208.3mmol) and acetic acid (2mL) were added to methanol (100mL) and tetrahydrofuran (100mL), and the resulting mixture was stirred at room temperature for 5 hours. To the above reaction solution was added sodium cyanoborohydride (13.1g,208.3mmol), and the mixture was further stirred at room temperature overnight. Water (200mL) was added, and the mixture was extracted with ethyl acetate (500 mL. times.5). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude title compound (24g, 62%) as a brown oil which was used in the next step without purification. MS (ESI) 188.1[ M + H ] M/z]⁺.

Step 2. tert-butyl-4- ((benzyl (tert-butoxycarbonyl) amino) methyl) -1 h-imidazole-1-carboxylate (WY-15-B)

N- ((1H-imidazol-4-yl) methyl) -1-phenylmethylamine (11.5g,61.5mmol), di-tert-butyldicarbonate (40.2g,184.5mmol) and triethylamine (23.8g,307.5mmol) were dissolved in dichloromethane (400mL), and the reaction was stirred at room temperature for 3 hours. The reaction solution is concentrated, and then,purification by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 5/1) gave the title compound (17.6g, 74%) as a brown oil. MS (ESI) M/z 388.4[ M + H ]]⁺.

Step 3. tert-butyl- (1H-imidazol-4-yl) methyl (benzyl) carbamate (WY-15-C)

Tert-butyl-4- ((benzyl (tert-butoxycarbonyl) amino) methyl) -1 h-imidazole-1-carboxylate (21.4g,55.3mmol) and sodium hydroxide (3.32g,82.9mmol) were dissolved in methanol (70mL) and water (20mL), and the reaction was stirred at room temperature for 1 h. The reaction was concentrated and purified by Prep-HPLC (C18 column, eluent: ammonium bicarbonate/acetonitrile/water) to give the title compound (15.6g, 98%) as a brown oil. MS (ESI) M/z 288.2[ M + H ]]⁺.

Step 4.5-benzyl-1-tert-butyl-2- (4- ((benzyl (tert-butoxycarbonyl) amino) methyl) -1 h-imidazol-1-yl) glutarate diester (WY-15-D)

(S) -5-benzyl-1-tert-butyl-2-bromoglutaric diester (7.6g,21.3mmol), tert-butyl- (1H-imidazol-4-yl) methyl (benzyl) carbamate (6.1g,21.3mmol) and potassium carbonate (5.9g,42.6mmol) were dissolved in N-methylpyrrolidone (80 mL). The reaction solution was stirred at room temperature for 60 hours. The reaction mixture was diluted with ethyl acetate (800mL) and washed with water (150 mL. times.5). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-5/2) to give the title compound (7g, 58%) as a brown oil. MS (ESI) M/z 564.1[ M + H ]]⁺.

Step 5.5-benzyl-1-tert-butyl-2- (4- ((benzylamino) methyl) -1H-imidazol-1-yl) glutarate diester hydrochloride (WY-15-E)

5-benzyl-1-tert-butyl-2- (4- ((benzyl (tert-butoxycarbonyl) amino) methyl) -1 h-imidazol-1-yl) glutarate diester (5.2g,9.2mmol) was dissolved in 2mol/L ethyl acetate hydrochloride solution (150mL), and the reaction solution was stirred at room temperature for 50 minutes. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess hydrochloric acid to give the crude title compound (4.7g, 95%) as a brown oil, which was used in the next step without purification. MS (ESI) M/z 464.4[ M + H ]]⁺.

Step 6.4- (4- (aminomethyl) -1 h-imidazol-1-yl) -5-tert-butoxy-5-oxopentanoic acid hydrochloride (WY-15-F)

5-benzyl-1-tert-butyl-2- (4- ((benzylamino) methyl) -1 hydro-imidazol-1-yl) glutarate diester hydrochloride (4.7g,9.4mmol) and palladium-carbon (10%, 1.8g) were added to methanol (100mL) and acetic acid (6mL), and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature overnight. The reaction was filtered and the filtrate was concentrated to give the crude title compound (3g, 75%) as a yellow oil which was used in the next step without purification. MS (ESI) M/z 284.4[ M + H ]]⁺.

Step 7.4- (4- ((((9 hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1 hydro-imidazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (WY-15-G)

4- (4- (aminomethyl) -1 h-imidazol-1-yl) -5-tert-butoxy-5-oxopentanoic acid hydrochloride (3g,9.4mmol), 9-fluorenylmethyl-N-succinimidyl carbonate (3.16g,9.4mmol) and sodium carbonate (1.99g,18.8mmol) were dissolved in water (30mL) and dioxane (60mL) and stirred at room temperature overnight. The reaction mixture was adjusted to pH 3 to 4 with 0.5mol/L hydrochloric acid, extracted with ethyl acetate (300 mL. times.3), and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by flash chromatography (silica gel column, eluent: dichloromethane/methanol-10/1) to give the title compound (2.9g, 62%) as a white solid. MS (ESI) 506.1[ M + H ]]⁺.

Step 8.5-allyl-1-tert-butyl-2- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-imidazol-1-yl) glutarate diester (WY-15-H)

4- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-imidazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (2.9g,5.8mmol) and 3-bromo-1-propene (1.4g,11.6mmol) were dissolved in dimethylformamide (50mL), sodium carbonate (1.22g,11.6mmol) was slowly added, stirred overnight at room temperature, water (100mL) was added, and extracted with ethyl acetate (150 mL. times.3). The organic phases are combined, washed with water (100 mL. times.4), dried over anhydrous sodium sulfate, filtered, the filtrate is concentrated to give the crude product, which is purified by flash chromatography (silica gel column, eluent: stone)Oil ether/ethyl acetate 1/1) to give the title compound (1.9g, 61%) as a brown oil. MS (ESI) M/z 546.2[ M + H ]]⁺.

Step 9.2- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-imidazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-15-I)

5-allyl-1-tert-butyl-2- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-imidazol-1-yl) glutarate diester (1.9g,3.5mmol) was dissolved in dichloromethane (60mL), trifluoroacetic acid (60mL) was slowly added, and the reaction was stirred at room temperature for 6 hours. The reaction solution was spun dry and dichloromethane (50 mL. times.3) was added and spun dry three times to remove excess trifluoroacetic acid to give the crude product, which was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (1.07g, 63%) as a white solid. MS (ESI) M/z 490.0[ M + H ]]⁺.

¹H NMR(400MHz,DMSO-d₆)δ13.47(s,1H)；7.89(d,J＝7.6Hz,2H)；7.73-7.65(m,4H)；7.41(t,J＝7.2Hz,2H)；7.32(t,J＝7.4Hz,2H)；7.01(s,1H)；5.93-5.84(m,1H)；5.29-5.18(m,2H)；4.97-4.93(m,1H)；4.52(d,J＝5.2Hz,2H)；4.30-4.20(m,3H)；4.10(d,J＝5.6Hz,2H)；2.40-2.08(m,4H).

B. Solid phase synthesis

Proceeding from 2.5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 9B. Except that (WY-15-I, Fmoc-Allyl- (S) -2-AMIP acid) (366mg,0.75mmol) was used in place of (WY-10-a-E, Fmoc-Allyl- (R) -2-AMPP acid) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ (S) -2-AMIP (Allyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [ (S) -2-AMIP ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. Mixing filtrates, adding into the filtrateEther (200mL) was added and the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant removed, and the solid washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:81/19-71/29 using: eluent A was 0.1% TFA in water, and eluent B was 0.1% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 75mg of a white solid.

Mass spectrum [ M +2H]/2⁺:607.0

HPLC elution time: 11.23 and 11.29 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 12: WY-16

Cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-AMPyP]-Lys(iPr)-NH₂

(7S,10S,13S,16R,19S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -7-benzyl-16- (3-guanidinopropyl) -10- (4-hydroxybenzyl) -13- (4- (isopropylamino) butyl) -19- (naphthalen-2-ylmethyl) -5,8,11,14,17, 20-heptaoxo-1, 6,9,12,15,18,21, 25-octaazacyclo [21,2.1] hexocosa-23 (26), 24-diene-2-carboxamide

Preparation of 2- (4- ((((9 hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1 hydro-pyrazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid (Fmoc- (WY-16-F):

step 1.N- ((1H-pyrazol-4-yl) methyl) -N-benzyl-1-phenylmethylamine (WY-16-A)

4-formylpyrazole (6.6g,68.7mmol), benzylamine (11.03g,103.1mmol) and acetic acid (10mL) were added to methanol (200mL), and the resulting mixture was stirred at room temperature overnight. Sodium cyanoborohydride (8.66g,137.5mmol) was then added slowly and stirring continued at room temperature for 2 h. Benzaldehyde (36.43g,343.7mmol) was added thereto, and the mixture was stirred at room temperature for 2 hours. Finally, another portion of sodium cyanoborohydride (8.66g,137.5mmol) was added slowly and stirring continued at room temperature for 2 hours. Water (300mL) was added, and the mixture was extracted with ethyl acetate (300 mL. times.3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude product which was purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 1/1) to give the title compound (14.2g, 74%) as a brown oil. MS (ESI) M/z 278.2[ M + H ]]⁺.

Step 2.5-benzyl-1-tert-butyl-2- (4- ((dibenzylamino) methyl) -1H-pyrazol-1-yl) glutaric acid diester (WY-16-B)

(S) -5-benzyl-1-tert-butyl-2-bromoglutaric diester (10g,28mmol), N- ((1H-pyrazol-4-yl) methyl) -N-benzyl-1-phenylmethanamine (7.76g,28mmol) and potassium carbonate (7.73g,56mmol) were dissolved in N-methylpyrrolidone (100 mL). The reaction solution was stirred at 45 ℃ for two days. The reaction mixture was diluted with ethyl acetate (700mL) and washed with saturated brine (200 mL. times.5). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-9/1) to give the title compound (7.4g, 48%) as a yellow oil. MS (ESI) M/z 554.3[ M + H ]]⁺.

Step 3.4- (4- (aminomethyl) -1H-pyrazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (WY-16-C)

5-benzyl-1-tert-butyl-2- (4- ((dibenzylamino) methyl) -1 hydro-pyrazol-1-yl) glutaric acid diester (7.3g,13.2mmol) and palladium-carbon (10%, 3g) were added to methanol (200mL) and acetic acid (10mL), and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at 45 ℃ for 5 hours. Reaction ofThe solution was filtered and the filtrate was concentrated to give the crude title compound (3.7g, 99%) as a colorless oil which was used in the next step without purification. MS (ESI) 284.1[ M + H ]: M/z]⁺.

Step 4.4- (4- ((((9 hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1 hydro-pyrazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (WY-16-D)

4- (4- (aminomethyl) -1-hydro-pyrazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (3.7g,13.1mmol), 9-fluorenylmethyl-N-succinimidyl carbonate (4.41g,13.1mmol) and sodium carbonate (2.77g,26.1mmol) were dissolved in water (150mL) and dioxane (150mL) and stirred at room temperature overnight. The reaction mixture was adjusted to pH 3 to 4 with 0.5mol/L hydrochloric acid, extracted with ethyl acetate (300 mL. times.3), and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 1/3) to give the title compound (6.3g, 95%) as a colorless oil. MS (ESI) 506.1[ M + H ]]⁺.

Step 5.5-allyl-1-tert-butyl-2- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-pyrazol-1-yl) glutaric acid diester (WY-16-E)

4- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-pyrazol-1-yl) -5-tert-butyl-5-oxopentanoic acid (6.2g,12.3mmol) and 3-bromo-1-propene (2.23g,18.4mmol) were dissolved in dimethylformamide (120mL), sodium carbonate (2.6g,24.6mmol) was slowly added, stirred overnight at room temperature, water (200mL) was added, and extracted with ethyl acetate (200 mL. times.3). The organic phases were combined, washed with water (100mL × 4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product, which was purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate ═ 3/1) to give the title compound (6.2g, 92.5%) as a colorless oil. MS (ESI) M/z 546.2[ M + H ]]⁺.

Step 6.2- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-pyrazol-1-yl) -5- (allyloxy) -5-oxopentanoic acid (WY-16-F)

Di-tert-butyl-5-allyl-1- (4- ((((9-hydro-fluoren-9-yl) methoxy) carbonylamino) methyl) -1-hydro-pyrazol-1-yl) glutarateThe ester (6.15g,11.3mmol) was dissolved in dichloromethane (120mL), then trifluoroacetic acid (120mL) was added slowly and the reaction stirred at room temperature for 6 h. The reaction solution was spun dry and dichloromethane (50 mL. times.3) was added and spun dry three times to remove excess trifluoroacetic acid to give the crude product, which was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (4.5g, 82%) as a white solid. MS (ESI) M/z 490.1[ M + H ]]⁺.

¹H NMR(400MHz,DMSO-d₆)δ13.13(s,1H)；7.89(d,J＝7.2Hz,2H)；7.70-7.64(m,4H)；7.41(t,J＝7.4Hz,2H)；7.37(s,1H)；7.32(t,J＝7.4Hz,2H)；5.93-5.84(m,1H)；5.29-5.18(m,2H)；5.03-4.99(m,1H)；4.52(d,J＝5.6Hz,2H)；4.32(d,J＝7.2Hz,2H)；4.22(t,J＝6.6Hz,1H)；4.05(d,J＝5.6Hz,2H)；2.37-2.22(m,3H)；2.16-2.10(m,1H).

B. Solid-phase synthesis:

proceeding from 2.5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 9B. Except that (WY-16-F, Fmoc-4-AMPyP acid) (368mg,0.75mmol) was used in place of (WY-10-a-E, Fmoc-Allyl- (R) -2-AMPP acid) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) - ] -2Nal- [4-AMPyP (Allyl) - ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used for 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [4-AMPyP ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:77/23-67/33 using: eluent A was 0.1% TFA in water, and eluent B was 0.1% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 87.5mg of a white solid.

Mass spectrum [ M +2H]/2⁺:607.1

HPLC elution time: 12.11 and 12.34 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 13: WY-23

Cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-ψ(NHCO)Gly-Glu]-Lys(iPr)-NH₂

(5S,8R,11S,14R,17S,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -17-benzyl-8- (3-guanidinopropyl) -14- (4-hydroxybenzyl) -11- (4- (isopropylamino) butyl) -5- (naphthalen-2-ylmethyl) -4,7,10,13,16,19, 23-heptaoxo-1, 3,6,9,12,15, 18-octaazacycloeicosatriene-22-carboxamide

Preparation of (5S) -11- (3- (allyloxy) -3-oxopropyl) -1- (9-hydro-fluoren-9-yl) -5- (2-naphthylmethyl) -3,6, 10-trioxo-2-oxa-4, 7, 9-triazadecane-12-oic acid (WY-23-H):

step 1.(9 hydro-fluoren-9-yl) methyl (S) - (1- ((2-amino-2-oxoethyl) amino) -3- (2-naphthyl) -1-oxopropyl-2-yl) carbamate (WY-23-A)

Fmoc-L-3- (2-naphthyl) -alanine (10g,22.9mmol), glycinamide hydrochloride (2.78g,25.2mmol), HATU (9.6g,25.2mmol) were dissolved in dichloro-methaneTo methane (400mL), triethylamine (5.8g,57.2mmol) was added slowly, and the reaction mixture was stirred at room temperature overnight. The reaction was filtered, the solid washed with dichloromethane (200mL) and dried to afford the title compound (11g, 97%) as a white solid. MS (ESI) M/z 494.1[ M + H ]]⁺.

Step 2 ((9-hydro-fluoren-9-yl) methyl (S) -1- ((aminomethyl) amino) -3- (2-naphthyl) -1-oxopropyl-2-yl) carbamate hydrochloride (WY-23-B)

(9H-fluoren-9-yl) methyl (S) - (1- ((2-amino-2-oxoethyl) amino) -3- (2-naphthyl) -1-oxopropyl-2-yl) carbamate (11g,22.3mmol) was dissolved in tetrahydrofuran (200mL) and water (50mL), and [ bis (trifluoroacetyloxy) iodonium [ bis (trifluoroacetoxy) was added slowly]Benzene (19.2g,44.6mmol) was added and the reaction was stirred at room temperature overnight. The reaction was spin dried to give the crude product, which was purified by Prep-HPLC (C18 column, eluent: hydrochloric acid/acetonitrile/water) to give the title compound (1.34g, 12%) as a yellow solid. MS (ESI) M/z 488.1[ M + Na ]]⁺.

Step 3.2- (3-benzyloxy-3-oxopropyl) -malonic acid di-tert-butyl ester (WY-23-C)

Di-tert-butyl malonate (20g,92.6mmol) was dissolved in anhydrous tetrahydrofuran (500mL), sodium tert-butoxide (10.7g,111.1mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. Benzyl 3-bromopropionate (22.5g,92.6mmol) was then added and stirred at room temperature for two hours. Water (300mL) was added, and the mixture was extracted with ethyl acetate (300 mL. times.3). The organic phases were combined, washed with saturated brine (200), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product, which was isolated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-30/1) to give the title compound (26.6g, 76%) as a colorless oil. MS (ESI) M/z 401.1[ M + Na ]]⁺.

Step 4.4- (tert-Butoxycarbonyl) -5-tert-butoxy-5-oxopentanoic acid (WY-23-D)

Di-tert-butyl 2- (3-benzyloxy-3-oxopropyl) -malonate (10g,26.5mmol) and palladium-carbon (10%, 2g) were added to methanol (100mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 4 hours. The reaction was filtered and the filtrate was concentrated to give the crude title compound (7.4g, 97%) It was a colorless oil and was used in the next step without purification. MS (ESI) M/z 311.1[ M + Na ]]⁺.

Step 5.2- (3-allyloxy-3-oxopropyl) -malonic acid di-tert-butyl ester (WY-23-E)

4- (tert-Butoxycarbonyl) -5-tert-butoxy-5-oxopentanoic acid (7.4g,25.7mmol), 3-bromo-1-propene (4.7g,38.5mmol) and sodium carbonate (5.4g,51.4mmol) were dissolved in dimethylformamide (100mL), stirred at room temperature for 4 hours and then diluted with ethyl acetate (700 mL). Washed with saturated brine (150mL × 5), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a crude product, which was then subjected to separation and purification by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate ═ 20/1) to give the title compound (26.6g, 76%) as a colorless oil. MS (ESI) M/z 351.1[ M + Na ]]⁺.

Step 6.2- (tert-Butoxycarbonyl) -5- (allyloxy) -5-oxopentanoic acid (WY-23-F)

Di-tert-butyl 2- (3-allyloxy-3-oxopropyl) -malonate (6.3g,19.2mmol) was dissolved in 4mol/L dioxane hydrochloride solution (200mL), and the reaction mixture was stirred at room temperature for 6 hours. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess hydrochloric acid to give the crude title compound (4.7g, 95%) as a colorless oil, which was used in the next step without purification. MS (ESI) M/z 295.0[ M + H ]]⁺.

Step 7.5-allyl-1-tert-butyl-2- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (2-naphthyl) propionamido) methylcarbamoyl) glutarate diester (WY-23-G)

2- (tert-Butoxycarbonyl) -5- (allyloxy) -5-oxopentanoic acid (1.41g,5.2mmol), (9-hydro-fluoren-9-yl) methyl (S) -1- ((aminomethyl) amino) -3- (2-naphthyl) -1-oxopropyl-2-yl) carbamate hydrochloride (1.3g,2.6mmol) and HATU (1.08g,2.9mmol) were dissolved in dichloromethane (50mL), triethylamine (0.79g,7.8mmol) was slowly added, and the reaction mixture was stirred at room temperature for 6 hours. The resulting mixture was spun dry and the residue was purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 3/1) to give the title compound (1.1g, 59%) as a yellow solid. MS (ESI) 720.3[ M + H ] M/z]⁺.

Step 8.(5S) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -5- (2-naphthylmethyl) -3,6, 10-trioxo-2-oxa-4, 7, 9-triazadecane-12-oic acid (WY-23-H)

5-allyl-1-tert-butyl-2- (((S) -2- (((9-hydro-fluoren-9-yl) methoxy) carbonylamino) -3- (2-naphthyl) propionamido) methylcarbamoyl) glutaric acid diester (1.1g,1.53mmol) was dissolved in dichloromethane (20mL), trifluoroacetic acid (20mL) was slowly added, and the reaction was stirred at room temperature for 4 hours. The reaction solution was spun dry and dichloromethane (50mL × 3) was added and spun dry three times to remove excess trifluoroacetic acid to give crude product, which was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (780mg, 78%) as yellow solid ms (esi) M/z 664.3[ M + H/water]⁺.

¹H NMR(400MHz,DMSO-d₆)δ13.57(s,1H)；8.88-8.81(m,2H)；7.90-7.79(m,6H)；7.70(t,J＝8.8Hz,1H)；7.59(d,J＝7.2Hz,1H)；7.55-7.53(m,2H)；7.49-7.43(m,2H)；7.40-7.33(m,2H)；7.24(t,J＝7.2Hz,1H)；7.12(t,J＝7.4Hz,1H)；5.88-5.76(m,1H)；5.25-5.08(m,2H)；4.52-4.42(m,4H)；4.38-4.33(m,1H)；4.10-4.04(m,3H)；3.29(t,J＝7.2Hz,1H)；3.14-3.09(m,1H)；2.91(t,J＝12.2Hz,1H)；2.30(t,J＝7.8Hz,2H)；2.00-1.89(m,2H).

B. Solid phase synthesis

Proceeding from 2.5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 9B. Except that (WY-23-H, Fmoc- ψ (NHCO) Gly-Glu (OAllyl) -OH) (498mg,0.75mmol) was used in place of (WY-10-a-E, Fmoc-Allyl- (R) -2-AMPyP acid) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ psi (NHCO) Gly-Glu (OAllyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used for 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [ psi (NHCO) Gly-Glu ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 40mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TI was addedS/H₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (200mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (10 min) at a flow rate of 25 mL/min, eluent A/B:77/23-67/33 using: eluent A was 0.1% TFA in water, and eluent B was 0.1% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 10.1mg of a white solid.

Mass spectrum [ M +2H]/2⁺:595.7

HPLC elution time: 11.98 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 14: WY-24

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-2- (4-aminobutyl) -10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4-butyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Solid-phase synthesis:

0.5g of commercially available Rink Amide MBHA resin (0.4mmol/g) was swollen in DMF and the resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and Fmoc-Lys (Boc) -O was addedA solution of H (100mg,0.2mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL DMF was reacted with DIPEA (0.2mL,1.2mmol) at room temperature for 2H. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF 6:10:84 solution 10ml reaction for one hour, resin with DMF washing, get Fmoc-Lys (Boc) -Rink Amide MBHA resin. The resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc-2Nal-azaGly- [ D-Glu (OAll) was added]-OH (400mg,0.6mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL DMF and DIPEA (0.2mL,1.2mmol) was added and the reaction was allowed to proceed at room temperature for 1 h. The resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give 2Nal-azaGly- [ D-Glu (OAll)]-Lys (Boc) -Rink Amide MBHA resin. To the resin was added a solution of Fmoc-D-Arg (Pbf) -OH (390mg,0.6mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL of DMF followed by DIPEA (0.2mL,1.2mmol) and reacted at room temperature for 2 hours; the resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, methylene chloride, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (Boc) -Rink Amide MBHA resin.

The Ally and Fmoc protecting groups were removed as used in example 1B and the ring was closed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal-azaGly- (D-Glu) ] ] -Lys (Boc) -Rink Amide MBHA resin.

The dried resin was added to 10mL of TFA/TIS/H₂O (95/2.5/2.5) solution, the mixture was stirred for 2 hours, the resin was removed by filtration, and 2mL of TFA/TIS/H was used₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (60mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. To obtainThe precipitate obtained was dissolved in DMF and then subjected to a linear concentration gradient elution (20 min) at a flow rate of 25 mL/min, eluent A/B:78/22-68/32 using: eluent A is 0.1% TFA aqueous solution, eluent B is acetonitrile, on the preparative HPLC, Xbridge Peptide BEH C1810 μm is used,

column (19 mm. times.250 mm). The fractions containing the product were collected and lyophilized to give 10.0mg of a white solid.

Mass spectrum: 1148.6[ M + H ] +,574.8[ M +2H ] +/2,383.5[ M +3H ] +/3.

HPLC elution time: 14.78 minutes

Column: waters Xbridge C184.6X 250mm 5 μm

Linear concentration gradient elution: eluent a/B-85/15-55/45, eluent a was 0.1% TFA in water, eluent B was 0.05% TFA in acetonitrile (30 min)

Flow rate: 1.0 mL/min

Example 15: WY-25

[cyclo side[Phe-Tyr-Lys-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl-10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4-aminobutyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2,4,9,12,15,18, 21-octaazacycloeicosatrine-5-carboxamide

Solid-phase synthesis:

0.5g of commercially available Rink Amide MBHA resin (0.4mmol/g) was swollen in DMF and the resin was treated with 8mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF, and a solution of Fmoc-Lys (iPr, Boc) -OH (104mg,0.2mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL of DMF was added, followed by DIPEA (0.2mL,1.2mmol) and reaction at room temperature for 2 hours. The resin was washed with DMF and excess Ac₂O/DIPEA/DMF 6:10:84 solution 10ml reaction for one hour, resin with DMF washing, get Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. The resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc-2Nal-azaGly- [ D-Glu (OAll) was added]-OH (400mg,0.6mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL DMF and DIPEA (0.2mL,1.2mmol) was added and the reaction was allowed to proceed at room temperature for 1 h. The resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give 2Nal-azaGly- [ D-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added a solution of Fmoc-D-Arg (Pbf) -OH (390mg,0.6mmol), HBTU (228mg,0.6mmol) and HOBt (81mg,0.6mmol) in 10mL of DMF followed by DIPEA (0.2mL,1.2mmol) and reacted at room temperature for 2 hours; the resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, dichloromethane, methanol and methyl tert-butyl ether in sequence and then is dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (Boc) - [ D-Arg (Pbf)]-2Nal-azaGly-[D-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The Ally and Fmoc protecting groups were removed as used in example 1B and the ring was closed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (Boc) - [ D-Arg (Pbf) ] -2Nal-azaGly- (D-Glu) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 20mL of TFA/TIS/H₂O (95/2.5/2.5) solution, the mixture was stirred for 2 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was used₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (100mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (20 min) at a flow rate of 25 mL/min using eluent A/B: 77/23-67/33: eluent A0.1% TFA water solution, eluentAcetonitrile, prepared by using Xbridge Peptide BEH C1810 mu m on preparative HPLC,

column (19 mm. times.250 mm). The fractions containing the product were collected and lyophilized to give 26.42mg of a white solid.

Mass spectrum: 1148.6[ M + H]⁺,574.8[M+2H]⁺/2,383.6[M+3H]⁺/3.

HPLC elution time: 12.53 minutes

Column: welch Ultimate LB-C18, 4.6X250mm 5um

Linear concentration gradient elution: eluent a/B-80/20-50/50, eluent a was 0.1% TFA in water, eluent B was acetonitrile (30 min)

Flow rate: 1.0 mL/min

Example 16: WY-26

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-azaGly-Glu]]-Lys(iPr)-NH₂

(5S,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl-10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4- (isopropylamino) butyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2-,4,9,12,15,18, 21-octaazacycloeicosanoic-5-carboxamide

Solid-phase synthesis:

0.5g of commercially available Rink Amide MBHA resin (0.432mmol/g) was swollen in DMF and the resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc and this procedure was repeated twice. The resulting resin was washed with DMF and a solution of Fmoc-Lys (iPr, Boc) -OH (120mg,0.23mmol), HBTU (84mg,0.22mmol) and HOBt (33mg,0.23mmol) in 7mL DMF was added followed by DIPEA ((0.26mL,1.5mmol) and the reaction was carried out at room temperature for 2h₂O/DIPEA/DMF 6:10:84 solution 10ml reaction for one hour, resin with DMF washing, get Fmoc-Lys (iPr, Boc) -Rink Amide MBHA resin. TheThe resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice. The resin was washed with DMF and Fmoc-2Nal-azaGly- [ L-Glu (OAll) was added]-OH (464mg,0.7mmol, synthesized essentially as WY-2-I), HBTU (268mg,0.7mmol) and HOBt (98mg,0.73mmol) in 7mL DMF followed by DIPEA (0.2mL,1.2mmol) and reaction at room temperature for 1 h. The resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, this was repeated twice, and the resin was washed with DMF to give 2Nal-azaGly- [ L-Glu (OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. To the resin was added a solution of Fmoc-D-Arg (Pbf) -OH (454mg,0.7mmol), HBTU (268mg,0.7mmol) and HOBt (98mg,0.73mmol) in 7mL of DMF followed by DIPEA (0.26mL,1.5mmol) and reacted at room temperature for 2 hours; the resin was treated with 10mL of 20% piperidine/DMF for 20 min to remove Fmoc, and this procedure was repeated twice; the resin was washed with DMF to give [ D-Arg (Pbf)]-2Nal-azaGly-[L-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin. In a similar manner, amino acids such as Lys (iPr, Boc), Tyr (tBu), Phe and the like are introduced in sequence, and the obtained resin is washed with DMF, methylene chloride, methanol and methyl tert-butyl ether in sequence and then dried by suction to finally obtain Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)]-2Nal-azaGly-[L-Glu(OAll)]-Lys (iPr, Boc) -Rink Amide MBHA resin.

The Ally and Fmoc protecting groups were removed as used in example 1B and the ring was closed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal-azaGly- (L-Glu) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 10mL of TFA/TIS/H₂O (95/2.5/2.5) solution, the mixture was stirred for 2 hours, the resin was removed by filtration, and 2.5mL of TFA/TIS/H was used₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (50mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (20 min) at a flow rate of 25 mL/min using eluent A/B: 82/18-72/28: eluent A was 0.1% TFA in water, eluent B was acetonitrile, and Xbridge Peptide BEH C18 was used for preparative HPLC,10μm,

Column (19 mm. times.250 mm). The fractions containing the product were collected and lyophilized to give 10.15mg of a white solid.

Mass spectrum: 1190.7[ M + H]+,595.9[M+2H]⁺/2,397.6[M+3H]⁺/3

HPLC elution time: 12.11 minutes

Column: welch Ultimate LB-C18, 4.6X250mm, 5 μm

Linear concentration gradient elution: eluent a/B-80/20-50/50, eluent a was 0.1% TFA in water, eluent B was acetonitrile (25 min)

Flow rate: 1.0 mL/min

Example 17: WY-27

cyclo side[Phe-Tyr-Lys(cyc)-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl-10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (4- (cyclohexylamino) butyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2-,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Preparation of Fmoc-Lys (cychexa, Boc) -OH:

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (tert-butoxycarbonyl (cyclohexyl) amino) hexanoic acid

Step 1.(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (cyclohexylamino) hexanoic acid

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6-amino-hexanoic acid (2g,4.94mmol) was dissolved in methanol (50mL) and a solution of cyclohexanone (1.45g,14.82mmol) in methanol (2mL), acetic acid (0.1mL) and sodium cyanoborohydride (1.24g,19.76mmol) were added under nitrogen. After stirring at room temperature for 16 h, water (20mL) was added and concentrated, water (30mL) was added again to give a suspension, which was filtered, concentrated and dried to give the crude title compound (1.8g,3.99mmol) as a white solid, which was used directly in the next reaction.

Step 2 (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (tert-butoxycarbonyl (cyclohexyl) amino) hexanoic acid

(S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (cyclohexylamino) hexanoic acid (1.6g,3.55mmol) was dissolved in dichloromethane (40mL) and Boc was added₂O (1.16g,5.33mmol) and triethylamine (1.08g,10.65 mmol). The reaction was carried out at room temperature for 16 hours. The reaction solution was directly concentrated, and added to water (40mL), extracted with ethyl acetate (50mL × 3), dried and concentrated, and passed through a silica gel column (dichloromethane/methanol ═ 0-10%) to obtain the title compound (0.8g, yield: 36.36%) as a white solid.

B. Solid-phase synthesis:

proceeding in analogy to the solid phase method of example 14B, starting from 500mg of commercially available Rink Amide MBHA resin (0.432 mmol/g). Except that [ Fmoc-Lys (cychexa, Boc) -OH ] (315mg,0.75mmol) was used in place of Fmoc-Lys (Boc) -OH for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (cychexa, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ D-Glu (Allyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (cytohexa, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ D-Glu (Allyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 10mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 2mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (50mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (20 min) at a flow rate of 25 mL/min using eluent A/B: 80/20-70/30: eluent A was 0.1% TFA in water and eluent B was acetonitrile, and on preparative HPLC, the mixture was purified using Xbridge Peptide BEH C18,10 μm,

column (19 mm. times.250 mm). The fractions containing the product were collected and lyophilized to give 18.7mg of a white solid.

Mass spectrum 1230.7[ M + H]⁺,615.9[M+2H]⁺/2

HPLC elution time: 13.64 minutes

Column: welch Ultimate LB-C18, 4.6X250mm, 5 μm

Flow rate: 1.0 mL/min

Example 18: WY-28

[cycloside[Phe-Tyr-Arg(Me)-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(5R,10S,13S,16S,19R,22S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl-10-benzyl-19- (3-guanidinopropyl) -13- (4-hydroxybenzyl) -16- (3- (3-methylguanidino) propyl) -22- (naphthalen-2-ylmethyl) -3,8,11,14,17,20, 23-heptaoxo-1, 2,4,9,12,15,18, 21-octaazacycloeicosatriene-5-carboxamide

Solid-phase synthesis:

proceeding in analogy to the solid phase method of example 14B, starting from 500mg of commercially available Rink Amide MBHA resin (0.432 mmol/g). Except that [ Fmoc-Arg (Me, Pbf) -OH ] (315mg,0.75mmol) was used in place of Fmoc-Lys (Boc) -OH for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Arg (Me, Pbf) - [ D-Arg (Pbf) - ] -2Nal- [ D-Glu (Allyl) - ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Arg (Me, Pbf) - [ D-Arg (Pbf)) ] -2Nal- [ D-Glu (Allyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 10mL of TFA/TIS/H₂O (95/2.5/2.5) solution, the mixture was stirred for 2 hoursWhen the resin was removed by filtration, 2mL of TFA/TIS/H was used₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (50mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (20 min) at a flow rate of 25 mL/min using eluent A/B: 81/19-71/29: eluent A was 0.1% TFA in water and eluent B was acetonitrile, and on preparative HPLC, the mixture was purified using Xbridge Peptide BEH C18,10 μm,

column (19 mm. times.250 mm). The fractions containing the product were collected and lyophilized to give 31.2mg of a white solid.

Mass spectrum 1190.7[ M + H]⁺,595.8[M+2H]⁺/2,397.6[M+3H]⁺/3

HPLC elution time: 15.01 minutes

Column: waters Xbridge C18, 4.6X250mm, 5 μm

Linear concentration gradient elution: eluent a/B-85/15-55/45, eluent a was 0.1% TFA in water, eluent B was acetonitrile (25 min)

Flow rate: 1.0 mL/min

Example 19: WY-30

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-ψ(CH2NBn)G-(D-Glu)]]-Lys(iPr)-NH₂

(2S,5S,8S,11R,14S,20R) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -2, 19-dibenzyl-11- (3-guanidinopropyl) -5- (4-hydroxybenzyl) -8- (4- (hydroxybenzyl) -14- (naphthalen-2-ylmethyl) -3,6,9,12,15, 23-hexa-oxo-1, 4,7,10,13,16, 19-heptaazacycloeicosatrine-20-carboxamide

A: preparation of (R) -2- ((2- (((9-fluorenyl) methoxy) carbonylamino) ethyl (benzyl) amino) -5- (allyloxy) -5-oxopentanoic acid (WY-11-B)

Step 1.(R) -5- (allyloxy) -2-amino-5-oxopentanoic acid trifluoroacetic acid hydrochloride (WY-11-A)

(R) -5-allyl-1-tert-butyl-2- (tert-butoxycarbonylamino) glutaric acid diester (4.9g,14.3mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (50mL) was slowly added to stir the reaction at room temperature for 6 hours. The reaction was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess trifluoroacetic acid to give the crude title compound (5.3g, 89%) as a colorless oil, which was used in the next step without purification. MS (ESI) 188.2[ M + H ] M/z]⁺。

Step 2.(R) -2- ((2- (((9-fluorenyl) methoxy) carbonylamino) ethyl (benzyl) amino) -5- (allyloxy) -5-oxopentanoic acid (WY-11-B)

(R) -5- (allyloxy) -2-amino-5-oxopentanoic acid trifluoroacetic acid hydrochloride (5.3g,17.6mmol), 2- (((9-fluorenyl) methoxy) carbonylamino) acetaldehyde (4.95g,17.6mmol) and acetic acid (8mL) were added to methanol (200mL), and the resulting mixture was stirred at room temperature overnight. Sodium cyanoborohydride (2.21g,35.2mmol) was then added slowly and stirring continued at room temperature for 2 h. Benzaldehyde (5.6g,52.8mmol) was added thereto, and the mixture was stirred at room temperature for 4 hours. Finally, another portion of sodium cyanoborohydride (2.21g,35.2mmol) was added slowly and stirring continued at room temperature for 2 hours. The reaction mixture was adjusted to pH 3 to 4 with 0.5mol/L hydrochloric acid, extracted with ethyl acetate (200 mL. times.3), and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (870mg, 9.1%) as a white solid. MS (ESI) M/z 543.2[ M + H ]]⁺。

¹H NMR(400MHz,DMSO-d₆)δ12.50(s,1H)；7.89(d,J＝7.6Hz,2H)；7.68(dd,J₁＝2.0Hz,J₂＝7.6Hz；2H)；7.41(t,J＝7.4Hz,2H)；7.34-7.19(m,8H)；5.87-5.78(m,1H)；5.25-5.14(m,2H)；4.41-4.39(m,2H)；4.29-4.18(m,3H)；3.84(d,J＝14.0Hz,1H)；3.60(d,J＝14.0Hz,1H)；3.18-3.15(m,1H)；3.04-2.99(m,2H)；2.76-2.68(m,1H)；2.56-2.53(m,1H)；2.37-2.32(m,2H)；1.88-1.69(m,2H).

B. Solid-phase synthesis:

proceeding from 5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 9B. Except that [ Fmoc-psi (CH2NBn) Gly- [ D-Glu (OAlly) ] (813mg,1.5mmol) was used in place of (WY-10-b-E, Fmoc-Allyl- (S) -2-AMPPacid) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2 Nal-psi (CH2NBn) Gly- [ D-Glu (OAlly)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2 Nal-psi (CH2NBn) Gly- (D-GluOAlly) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 75mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (600mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (15 min) at a flow rate of 25 mL/min, eluent A/B:77/23-69/32 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 161.5mg of a white solid.

Mass spectrum [ M +2H]/2⁺:633.6

HPLC elution time: 13.61 minutes

Elution conditions: c

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 20: WY-11

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-2Nal-azaGly-(D-Glu)]]-Lys(iPr)-NH₂

(2S,5S,8S,11R,14S,20R) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -2-benzyl-11- (3-guanidinopropyl) -5- (4-hydroxybenzyl) -8- (4- (isopropylamino) butyl) -14- (naphthalen-2-ylmethyl) -3,6,9,12,15, 23-hexa-oxo-1, 4,7,10,13,16,19-7 azacycloeicosatrine-20-carboxamide

B. Solid-phase synthesis:

WY-30 and palladium-carbon (10%, 50mg) were added to methanol (50mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 16 hours. The reaction was filtered, the filtrate was concentrated, and then subjected to linear concentration gradient elution (15 min) at a flow rate of 25 mL/min and eluent A/B:77/23-69/33, using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide, C18,10 μm,

column (19 mm. times.250 mm). Fractions containing the product were collected and lyophilized to give 10.3mg of a white solid.

Mass spectrum [ M +2H]/2⁺:588.6

HPLC elution time: 10.91 minutes

Elution conditions: c

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 21: WY-36

[cyclo side[Tyr-Lys(iPr)-(D-Arg)-2Nal-ψ(CH2NBn)Gly-(D-Glu)]]-Lys(iPr)-NH₂

(2S,5S,8R,11S,17R) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -16-benzyl-8- (3-guanidinopropyl) -2- (4-hydroxybenzyl) -5- (4- (isopropylamino) -11- (naphthalen-2-ylmethyl) -3,6,9,12, 20-pentoxazole-1, 4,7,10,13, 16-heptaazacycloeicosatrine-17-carboxamide

Solid-phase synthesis:

starting from 5g of Rink Amide MBHA resin (0.432mmol/g), the procedure was carried out in analogy to the solid phase method of preparation 19B to give Fmoc-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ ψ (CH2NBn) Gly- [ D-Glu (OAlly)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used in 19B and ring-closed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [ psi (CH2NBn) Gly- (D-GluOAlly) ] ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

The dried resin was added to 75mL of TFA/TIS/H₂O (95/3/2) solution, the mixture was stirred for 2.5 hours, the resin was removed by filtration, and 5mL of TFA/TIS/H was added₂The O (95/3/2) solution washes the resin. The filtrates were combined, ether (600mL) was added to the filtrate, the resulting mixture was centrifuged at 3000 rpm for 1 min, the supernatant was removed, and the solid was washed 2 times with ether and drained. The resulting precipitate was dissolved in DMF and then subjected to linear concentration gradient elution (15 min) at a flow rate of 25 mL/min, eluent A/B:77/23-69/31 using: eluent A was 0.05% TFA in water, and eluent B was 0.05% TFA in acetonitrile. On preparative HPLC, Xbridge BEH peptide, C18,10 μm,

Mass spectrum [ M +2H]/2⁺:560.0

HPLC elution time: 12.10 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

Example 22: WY-37

[cyclo side[Phe-Tyr-Lys(iPr)-(D-Arg)-ψ(NHCS)Gly-Glu]]-Lys(iPr)-NH₂

(5S,8R,11S,14S,17S) -N- ((S) -1-amino-6- (isopropylamino) -1-oxohex-2-yl) -17-benzyl-8- (3-guanidinopropyl) -14- (4-hydroxybenzyl) -11- (4- (isopropylaminobutyl) -5- (naphthalen-2-ylmethyl) -4,7,10,13,16, 19-hexa-oxo-23-thioxo-1, 3,6,9,12,15, 18-heptaazacycloeicosatrine-22-carboxamide

Preparation of (5S) -11- (3- (allyloxy) -3-oxopropyl) -1- (9-hydro-fluoren-9-yl) -5- (2-naphthylmethyl) -3,6, -dioxo-10-thioxo-2-oxa-4, 7, 9-triazadecane-12-oic acid (WY-37-K)

Step 1. N-benzyloxycarbonyl glycinamide (WY-37-A)

Sodium carbonate (9.6g,90.5mmol) and glycinamide hydrochloride (10g,90.5mmol) were dissolved in water (100mL) and dioxane (100mL), benzyl chloroformate (15.43g,90.5mmol) was added slowly at 0 deg.C, and the reaction was stirred at room temperature for 16 h. The reaction was filtered, the solid washed with water (400mL) and dried to give the title compound (12g, 64%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝209.1。

Step 2. N-benzyloxycarbonylaminomethylamine hydrochloride (WY-37-B)

N-benzyloxycarbonyl glycinamide (24g,115mmol) was dissolved in dichloromethane (1L) and water (20mL) and [ bis (trifluoroacetyloxy) iodide was added]Benzene (54.6g,127mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. Ethyl acetate hydrochloride solution (2mol/L,150mL) was then added, the reaction was filtered, the solid was washed with n-hexane (100mL), and the solid was dried to give the title compound (20g, 80%) as a white solid. LCMS (ESI) [ M + H ]]⁺＝181.1。

Step 3.2- (3-benzyloxy-3-oxopropyl) -malonic acid methyl ester tert-butyl ester (WY-37-C)

Methyl tert-butyl malonate (25g,144mmol) was dissolved in anhydrous tetrahydrofuran (500mL), sodium tert-butoxide (13.4g,144mmol) was added, and the reaction solution was stirred at room temperature for half an hour. Benzyl 3-bromopropionate (34.9g,144mmol) was then added and stirred at room temperature for two hours. Water (300mL) was added, and the mixture was extracted with ethyl acetate (300 mL. times.3). The organic phases were combined, washed with saturated brine (200), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product, which was isolated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-20/1) to give the title compound (33g, 69%) as a colorless oil. MS (ESI) 359.1[ M + Na ] M/z]⁺。

Step 4.4- (methoxycarbonyl) -5-tert-butoxy-5-oxopentanoic acid (WY-37-D)

Methyl 2- (3-benzyloxy-3-oxopropyl) -malonate tert-butyl ester (16.5g,49.1mmol) and palladium-carbon (10%, 5g) were added to methanol (400mL) and the mixture was connected to a hydrogen generator. The air in the system was replaced with hydrogen, and the mixture was stirred at room temperature for 4 hours. The reaction was filtered and the filtrate was concentrated to give the crude title compound (12g, 99%) as a colorless oil, which was used in the next step without purification. MS (ESI) M/z 269.2[ M + Na ]]⁺。

Step 5.2- (3-allyloxy-3-oxopropyl) -malonic acid methyl ester tert-butyl ester (WY-37-E)

4- (methoxycarbonyl) -5-tert-butoxy-5-oxopentanoic acid (24g,97.6mmol), 3-bromo-1-propene (23.6g,195.1mmol) and sodium carbonate (10.3g,97.6mmol) were dissolved in dimethylformamide (200mL), and after stirring at room temperature for 5 hours, ethyl acetate (700mL) was added to dilute the solution. Washing with saturated brine (200 mL. times.5) and washing with brineDried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to give the crude product, which was then purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-20/1) to give the title compound (26g, 93%) as a colorless oil. MS (ESI) M/z 309.2[ M + Na ]]⁺。

Step 6.2- (methoxycarbonyl) -5- (allyloxy) -5-oxopentanoic acid (WY-37-F)

Methyl 2- (3-allyloxy-3-oxopropyl) -malonate tert-butyl ester (20g,69.9mmol) was dissolved in dichloromethane (50mL), then trifluoroacetic acid (100mL) was added slowly and the reaction stirred at room temperature for 7 hours. The reaction solution was spun dry and dichloromethane (50mL × 3) was added to spin dry three times to remove excess trifluoroacetic acid to give a crude product, which was then subjected to separation and purification by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate ═ 2/1) to give the title compound (13.5g, 84%) as a colorless oil. MS (ESI) M/z 231.2[ M + H ]]⁺。

Step 7.5-allyl-1-methyl-2- ((((((benzyloxy) carbonyl) amino) methyl) carbamoyl) glutarate diester (WY-37-G)

2- (methoxycarbonyl) -5- (allyloxy) -5-oxopentanoic acid (10g,43.5mmol), N-benzyloxycarbonylaminomethylamine hydrochloride (14.1g,65.2mmol) and HATU (19.8g,52.2mmol) were dissolved in dichloromethane (500mL), triethylamine (13.2g,130.4mmol) was slowly added, and the reaction mixture was stirred at room temperature overnight. Water (500mL) was added and the mixture was extracted with methylene chloride (600 mL. times.3). The organic phases were combined, washed with saturated brine (400), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product, which was isolated and purified by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate-2/1) to give the title compound (14.5g, 85%) as a colorless oil. MS (ESI) M/z 393.1[ M + H ]]⁺。

Step 8.5-allyl-1-methyl-2- ((((((benzyloxy) carbonyl) amino) methyl) aminosulfamoyl) glutarate diester (WY-37-H)

5-allyl-1-methyl-2- (((((benzyloxy) carbonyl) amino) methyl) carbamoyl) glutaric acid diester (14.5g,37mmol), and Lawson's reagent (14.9g,37mmol) were dissolved in toluene (400mL), and the reaction was stirred at 90 ℃ for 16 hours. The resulting mixture was spin-dried and the residue was usedPurification by flash chromatography (silica gel column, eluent: petroleum ether/ethyl acetate 3/1) gave the title compound (13.5g, 84%) as a yellow liquid. MS (ESI) M/z 409.2[ M + H ]]⁺.

Step 9.5-allyl-1-methyl-2- ((aminomethyl) aminosulfanyl) glutaric acid diester hydrobromide hydrochloride (WY-37-I)

5-allyl-1-methyl-2- (((((benzyloxy) carbonyl) amino) methyl) aminosulfamoyl) glutaric acid diester (14g,34.3mmol) was dissolved in 33% hydrobromic acid in acetic acid (50mL), and the reaction was stirred at room temperature for 20 minutes. The reaction was concentrated to give the crude title compound (11g, 92%) as a yellow material, which was used in the next step without purification. MS (ESI) M/z 297.1[ M + Na ]]⁺。

Step 10.5-allyl-1-methyl-2- ((((S) -2- (((((9-hydro-fluoren-9-yl) methoxy) carbonyl) amino) -3- (2-naphthyl) propionamido) methyl) aminosulfanyl) glutaryl ester (WY-37-J)

N-Fmoc-L-3- (2-naphthyl) -alanine (13.5g,31.1mmol), 5-allyl-1-methyl-2- ((aminomethyl) aminosulfanyl) glutarate diester hydrobromide hydrochloride (11g,31.1mmol), HATU (14.17g,37.3mmol) were dissolved in dichloromethane (500mL), triethylamine (7.85g,77.7mmol) was slowly added, and the reaction was stirred at room temperature for 5 hours. Water (350mL) was added and the mixture was extracted with methylene chloride (500 mL. times.3). The organic phases were combined, washed with saturated brine (400), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude product, which was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (4.06g, 19%) as a white solid. MS (ESI) M/z 694.2[ M + H ]]⁺.

Step 11.(5S) -11- (3- (allyloxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -5- (2-naphthylmethyl) -3,6, -dioxo-10-thioxo-2-oxa-4, 7, 9-triazodecane-12-oic acid (WY-37-K)

5-allyl-1-methyl-2- ((((S) -2- (((((9-hydro-fluoren-9-yl) methoxy) carbonyl) amino) -3- (2-naphthyl) propionamido) methyl) aminosulfanyl) glutaryl ester (4g,5.8mmol) and sodium carbonate (2.45g,23.1mmol) were dissolved in water (50mL) and tetrahydrofuran (50mL) and the reaction was stirred at room temperature for three days. 0.5mol/L for reaction liquidThe pH was adjusted to 3 to 4 with hydrochloric acid, and the mixture was extracted with ethyl acetate (300 mL. times.3) and washed with saturated brine (200 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by Prep-HPLC (C18 column, eluent: trifluoroacetic acid/acetonitrile/water) to give the title compound (0.7g, 18%) as a white solid. MS (ESI) M/z 680.1[ M + H ]]⁺.1H NMR(400MHz,DMSO-d₆)δ12.53(s,1H)；10.74(s,1H)；8.95-8.89(m,1H)；7.85-7.81(m,6H)；7.75-7.72(m,1H)；7.60-7.52(m,3H)；7.49-7.43(m,2H)；7.40-7.33(m,2H)；7.24(t,J＝7.4Hz,1H)；7.13(t,J＝7.4Hz,1H)；5.90-5.75(m,1H)；5.26-5.10(m,2H)；4.97-4.78(m,2H)；4.50-4.40(m,3H)；4.11-4.06(m,3H)；3.73(t,J＝7.2Hz,1H)；3.21-3.16(m,1H)；2.94(t,J＝12.2Hz,1H)；2.25(t,J＝7.8Hz,2H)；2.16-2.07(m,2H).

B. Solid phase synthesis

Proceeding from 2.5g of Rink Amide MBHA resin (0.432mmol/g), the procedure is analogous to the solid phase procedure of example 13B. Except that (WY-23-H, Fmoc- ψ (NHCO) Gly-Glu (OAlly) -OH) (498mg,0.75mmol) was used in place of (WY-37-K, Fmoc- ψ (NHCS) Gly-Glu (OAlly) -OH) for solid phase condensation. Finally obtaining Fmoc-Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf)) ] -2Nal- [ psi (NHCS) Gly-Glu (OAllyl)) ] -Lys (iPr, Boc) -Rink Amide MBHA resin. And the Ally and Fmoc protecting groups were removed by the method used for 9B, and ring closure was performed with HBTU/HOBt/DIEA. Finally obtaining the cyclized resin [ cyclo side [ Phe-Tyr (tBu) -Lys (iPr, Boc) - [ D-Arg (Pbf) ] -2Nal- [ psi (NHCS) Gly-Glu ] -Lys (iPr, Boc) -Rink Amide MBHA resin.

Mass spectrum [ M +2H]/2⁺:603.5

HPLC elution time: 13.31 minutes

Column: XBridge Peptide BEH C18, 4.6X 150mm,3.5 μm

Flow rate: 1.0 mL/min

The MS data for the polypeptides prepared in the above examples are shown in Table 1.

Effect example 1:

a-1 purpose of the experiment:

detecting the antagonistic activity of the compound on the CXCR4 chemokine receptor.

Test unit: shanghai national drug screening center

A-2. test drug and positive control drug

A-2.1 test drugs

The characteristics are as follows: powder of

The storage method comprises the following steps: storing in dark place under refrigeration

The dosage concentration: 10mM, 1mM, 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10nM, water.

Working concentration: 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM, 100pM, water.

A-2.2 Positive control drugs:

A-2.2.1T140(CXCR4 antagonist)

Name: t140

The storage method comprises the following steps: keeping in dark place at-80 deg.C

The dosage concentration: 1mM, 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM, water.

Working concentration: 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM, 100pM, 10pM, water.

A-2.2.2SDF-1(CXCR4 agonist)

Name: SDF-1

The dosage concentration: 1 μ M.

Working concentration: 10 nM.

A-3. reagents and apparatus:

a-3.1 Main reagents: f12 medium (GIBCO);

dimethyl sulfoxide (sigma)

FLUO-4,AM PACKAGED FOR(invitrogen)

A-3.2 Main instruments: flexstation-3(Molecular Devices)

Group, dose setting:

a-4.1 dose setting basis:

according to the test IC₅₀Setting a test concentration gradient of the compound and setting a plurality of holes.

A-4.2 dose settings and groups:

all the test substances are provided with 8 concentrations, and each concentration is provided with 3 multiple holes.

A-5. Experimental principles and methods:

the experimental principle is as follows:

by establishing a cell line which co-transfers a target receptor and the G alpha 16, the activation of the receptor can cause the activation of the G alpha 16 protein, and further activates phospholipase C (PLC) to generate IP3 and DAG, IP3 can be combined with an IP3 receptor on an endoplasmic reticulum and mitochondria in a cell, thereby causing the release of intracellular calcium. Therefore, measurement of changes in intracellular calcium can be used as a means for detecting the activation state of a target receptor. Fluo-4/AM is a calcium fluorescent probe indicator used for measuring calcium ions, is used as a nonpolar fat-soluble compound, and after entering cells, under the action of cell lipolytic enzyme, an AM group is dissociated to release Fluo-4; since Fluo-4 is a polar molecule and does not readily pass through a lipid bilayer membrane, it can retain Fluo-4 in the cell for a long period of time. The level of activation of the G.alpha.protein can ultimately be reflected by measuring the intensity of the fluorescence that is excited. If the compound to be screened is capable of agonizing the target receptor, the calcium flux response can be greatly increased. Conversely, if the compound to be screened is capable of antagonizing the target receptor, the calcium flux response may be greatly reduced.

The experimental steps are as follows:

i. cells stably expressing the target receptor/G.alpha.16 were seeded in 96-well plates and cultured overnight.

The culture medium in the wells seeded with cells was aspirated, 40. mu.l/well of freshly prepared dye was added, and incubation was carried out in an incubator at 37 ℃ for 40 minutes.

The drug to be tested was diluted with calcium buffer and mixed well.

Antagonistic mode:

iv, the dye is aspirated and discarded, and after washing once with freshly prepared calcium buffer, 50 μ l of calcium buffer in which the drug to be tested is dissolved is replaced.

v. detection with Flexstation II instrument, starting at 15 seconds 25. mu.l of calcium buffer with known agonist dissolved is automatically added by the instrument and the fluorescence at 525nm is finally read.

A-6, data processing and statistical analysis:

antagonistic mode

The cell Response (% Response) at each concentration of each sample was calculated by the following equation.

L_SampleIndicating the value of the detection signal, L, of the sample to be measured_BlankValue of detection signal, L, indicating complete inhibition of positive antagonist_AgonistRepresents the detection signal value after positive agonist stimulates the DMSO group.

IC₅₀Values were calculated by GraphPad Prism. To give IC of the corresponding compound₅₀As shown in table 1.

Effect example 2:

b-1 purpose of the test:

the inhibitory activity of the compounds on cell migration was examined.

B-2. test drug and positive control drug

B-2.1 test drugs

The characteristics are as follows: powder of

The dosage concentration: 10mM, 1mM, 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10nM and 1pM

Working concentration: 100. mu.M, 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM, 100pM and 10pM

B-2.2 Positive control drugs:

b-2.2.1WY-1-1(CXCR4 antagonist, LY2510924)

Name: LY2510924

B-3. reagents and apparatus:

b-3.1 Main reagents:

● Cytoselect TM 96-well CELL migration kit, (CELL BIOLABS, cat # CBA-105-5)

● Cytoselect TM 96-well CELL migration kit, (CELL BIOLABS, cat # CBA-106-5)

● human SDF-alpha, (PEPROTECH, cat # 300-28A-10)

● F12K Medium, (GE hyclone, cat # SH30526.01)

● McCoys 5A Med Mod medium, (invitrogen, cat # 16600082)

● fetal bovine serum FBS, (France origin, cat # FB-1280-500)

● double resistance to penicillin (invitrogen, cat No. 15070063)

B-3.2 Main instruments: flexstation-3(Molecular Devices)

B-4. Experimental methods:

the experimental steps are as follows:

i. cells were made 5.0X 10 with serum-free medium (RMPI1640+ 0.3% BSA +10mM HEPES)⁶cells/ml cell suspension, CXCR4 antagonist was added directly to the cell suspension.

mu.L of serum-free medium containing 10ng/ml BDF-alpha (RMPI1640+ 0.3% BSA +10mM HEPES) was added to the 96-well cell migration plate external chamber.

iii.96 well transfer plates the inner chamber was carefully replaced in the outer chamber to ensure that no air bubbles were formed between the membrane of the inner chamber and the culture medium.

iv, mixing the cell suspension evenly, and adding 90ul of the cell suspension into the inner chamber.

v.96 well cell culture plates at 37 ℃ in 5% CO₂Was cultured in an incubator for 3 hours.

Before the end of the cell incubation, 150ul of pre-warmed cell separation medium was added to a 96-well cell collection plate provided in the kit.

After 3 hours of incubation, the medium in the chamber of the cell culture plate was blotted, the chamber was transferred to the cell collection plate containing 150ul of cell separation medium and incubated at 37 ℃ for 30 minutes.

Gently shaking the inner chamber several times in the cell collection fluid to detach the cells that have migrated to the outside of the inner chamber from the membrane.

ix, 75ul of medium was removed from the external culture chamber and mixed with 75ul of the cell harvest described above and placed in a clean 96-well plate.

And x, diluting the staining solution with 4x lysis solution according to the ratio of 1:75 to prepare working solution, and adding 50ul of staining working solution into the cell collection mixed solution. After mixing well, the mixture was left at room temperature for 20 minutes.

And xi, taking 150ul of the mixed solution, and transferring the mixed solution to a 96-well plate which can be used for fluorescence detection.

Reading values at excitation 480nm and emission 520 nm.

B-5, processing experimental data:

setting Positive and negative controls in the experiment

Positive control: the outer chamber of the transfer plate contains 10ng/ml BDF-alpha, and the inner chamber of the transfer plate contains no compound

Negative control: no 10ng/ml BDF-alpha in the outer chamber of the transfer plate

Using the fluorescence values read as Y-axis, the compound concentrations as X-axis, using Graphpad Prism6 as a curve relating fluorescence values to compound concentrations, and calculating the IC of each compound from the curve using software₅₀As shown in table 1.

TABLE 1

NA ═ inactive

As can be seen from Table 1, the compound of the invention shows better activity in cell migration inhibition, the whole activity is below 125nM, wherein the activity of WY-2, WY-4, WY-7, WY-10-a, WY-10-b, WY-11, WY-15-16, WY-23, WY-30 and WY-36 is basically equivalent to that of the control compound LY2510924, while the activity of the compounds WY-2, WY-6, WY-13 and WY-24-27 is obviously better (<25nM) than that of the control compound LY2510924, especially the activity of WY-6 and WY-24-27 is below 15 nM.

Claims

1. A peptide compound of formula III, a pharmaceutically acceptable salt thereof:

the peptide compound shown in the formula III is characterized in that the peptide compound shown in the formula III is any one of the following compounds:

2. use of a peptide compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prevention of a CXCR4 related disease or CXCR4 antagonist.

3. The use of claim 2, wherein said "CXCR 4 related disease" is an immune disease, an inflammatory disease, pulmonary fibrosis, HIV infection or cancer.

4. The use according to claim 3, wherein when said "CXCR 4-related disease" is an inflammatory disease, said inflammatory disease is rheumatoid arthritis;

and/or, when the "CXCR 4 related disease" is cancer, the cancer is breast cancer, pancreatic cancer, melanoma, prostate cancer, renal cancer, neuroblastoma, non-hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, glioblastoma multiforme, osteosarcoma, or leukemia.

5. A pharmaceutical composition comprising a peptide compound as claimed in claim 1, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.