Preparation methods for camptothecin derivative and intermediate thereof

Abstract

The present invention provides preparation methods for a camptothecin derivative and an intermediate thereof, in particular to a preparation method for the compound of formula (A), and a synthetic intermediate compound of the compound of formula (A) and a preparation method therefor.

Description

Process for the preparation of camptothecin derivatives and intermediates thereof

[0001] This application claims priority to Chinese application 202410635925.7 filed on May 21, 2024, which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to a process for the preparation of a camptothecin derivative, in particular to a process for the preparation of a compound of formula (A), and an intermediate compound in the synthesis of the compound of formula (A) and a process for the preparation thereof. BACKGROUND

[0003] The concept of antibody-drug conjugate (ADC) or ADC drug has a long history. As early as 1913, Professor Paul Ehrlich, a Nobel Prize winner, first proposed the concept of "magic bullet", that is, to install cytotoxic drugs on specific monoclonal antibodies to achieve targeted killing of tumor cells. The ADC drug that has been marketed is based on this theory, which connects cytotoxic drugs to monoclonal antibodies, so that monoclonal antibodies act as carriers to transport small molecule cytotoxic drugs to target tumor cells in a targeted manner.

[0004] ADC drug is a combination of antibody and small molecule cytotoxic drug through a specific linker. The main components include antibody, linker and small molecule cytotoxic drug. After entering the blood, the antibody component of the ADC drug can recognize the target and bind to tumor cells with high expression of cell surface antigens. When the ADC-antigen complex enters the tumor cell through endocytosis, the cytotoxic load (drug) is released under the degradation of lysosomes, which destroys DNA or inhibits tumor cell division, thereby killing tumor cells.

[0005] The synthesis of ADC drug is usually to synthesize the structure of "linker-drug molecule" first, and then couple the linker with the antibody to finally obtain the ADC drug. Many methods for preparing linker-drug molecules have low yield, which leads to high cost and is not conducive to industrial production.

[0006] Therefore, it is of great significance to develop a synthetic method with high yield and economic efficiency. SUMMARY

[0007] The present inventors have found a method suitable for industrial production of the compound of formula (A), which can produce the compound of formula (A) with high yield.

[0008] Specifically, the present application relates to a process for preparing a compound of formula (A):

[0009] comprising the step of:

[0010] reacting a compound of formula (B) with a compound of formula (C) to form a compound of formula (A):

[0011] or, reacting the amino acid residue A'4 of a compound of formula (D) with the amino acid residue A'5 of a compound of formula (E) to form a peptide bond to form a compound of formula (A):

[0012] wherein the variables are as defined herein.

[0013] In one aspect, the present application relates to a compound of formula (B):

[0014] wherein the variables are as defined herein.

[0015] In another aspect, the present application relates to a compound of formula (E):

[0016] wherein the variables are as defined herein.

[0017] In another aspect, the present application relates to a compound of formula (G):

[0018] wherein the variables are as defined herein.

[0019] In another aspect, the present application relates to a compound of formula (D):

[0020] T-A1-A2-A3-A'4

[0021] D

[0022] wherein the variables are as defined herein.

[0023] In another aspect, the present application relates to a compound of formula (C7):

[0024] wherein R D5 is as defined herein.

[0025] In another aspect, the present application relates to a compound of formula (C1):

[0026] wherein R D5 is as defined herein.

[0027] In another aspect, the present application relates to a method of making a compound of formula (B) comprising the step of:

[0028] reacting a compound of formula (F) with a compound of formula (G) to form a compound of formula (B):

[0029] or, reacting a compound of formula (H) with a compound of formula (I) to form a compound of formula (B):

[0030] wherein the variables are as defined herein.

[0031] In another aspect, the present application relates to a method of preparing a compound of formula (C3-a):

[0032] reacting a compound of formula (C1-a) with a compound of formula (C2) to form a compound of formula (C3-a):

[0033] wherein the variables are as defined herein.

[0034] In another aspect, the present application relates to a method of preparing a compound of formula (C4-a):

[0035] deprotecting a compound of formula (C4-a) to form a compound of formula (C5-a) and subjecting the compound of formula (C5-a) to an amino protection reaction to form a compound of formula (C6-a):

[0036] wherein the variables are as defined herein.

[0037] In another aspect, the present application relates to a method of preparing a compound of formula (C6-a):

[0038] deprotecting a compound of formula (C4-a) to form a compound of formula (C5-a) and subjecting the compound of formula (C5-a) to an amino protection reaction to form a compound of formula (C6-a):

[0039] wherein the variables are as defined herein.

[0040] In another aspect, the present application relates to a method of preparing a compound of formula (C-a):

[0041] wherein the variables are as defined herein.

[0042] In another aspect, the present application relates to a method of preparing a compound of formula (E):

[0043] reacting a compound of formula (E1) with a compound of formula (C) to form a compound of formula (E’), and deprotecting the compound of formula (E’) to form a compound of formula (E):

[0044] wherein the variables are as defined herein.

[0045] In another aspect, the present application relates to a method of preparing a compound of formula (F3):

[0046] reacting a compound of formula (Fl) with a compound of formula (F2) to form a compound of formula (F3):

[0047] wherein the variables are as defined herein.

[0048] In another aspect, the present application relates to a method of preparing a compound of formula (F):

[0049] deprotecting a compound of formula (F3) and then performing a carboxyl protection reaction to form a compound of formula (F):

[0050] wherein the variables are as defined herein.

[0051] In another aspect, the present application relates to a method of preparing a compound of formula (G):

[0052] reacting a carboxyl group of an amino acid residue A'4 of a compound of formula (Gl) with a compound of formula (G2) to form a compound of formula (G3), and then deprotecting a compound of formula (G3) to form a compound of formula (G):

[0053] wherein A'4 is as defined herein and the amino terminus is attached to A3 and the carboxyl group is involved in the reaction;

[0054] A5 is as defined herein and the carboxyl terminus is attached to L3;

[0055] the remaining variables are as defined herein.

[0056] In another aspect, the present application relates to a method of preparing a compound of formula (D):

[0057] reacting a compound of formula (F) with a compound of formula (J) to form a compound of formula (D):

[0058] wherein the variables are as defined herein.

[0059] In another aspect, the present application relates to a method of preparing a compound of formula (C7):

[0060] reacting a compound of formula (C8) with an oxidizing agent to form a compound of formula (C7):

[0061] wherein the variables are as defined herein.

[0062] In another aspect, the present application relates to a method of preparing a compound of formula (Cl):

[0063] deprotecting the compound of formula (C7) to form a compound of formula (Cl):

[0064] wherein R D5 as defined herein.

[0065] In another aspect, the present application relates to a method of preparing an antibody drug conjugate (ADC), comprising conjugating a compound of formula (A) to an antibody; said compound of formula (A) being as defined herein. DETAILED DESCRIPTION

[0066] DEFINITIONS

[0067] CHEMICAL DEFINITIONS

[0068] The definitions of specific functional groups and chemical terms are described in more detail below.

[0069] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0070] "C 1-10 "alkyl" refers to a straight or branched chain saturated hydrocarbon radical having from 1 to 10 carbon atoms. In some embodiments, C 1-9 alkyl, C 1-8 alkyl, C 1-6 alkyl, C 1-4 alkyl and C 1-3 alkyl are preferred. Examples of C 1-6 alkyl include: methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5) and n-hexyl (C6). The term "C 1-6"Alkyl" also includes heteroalkyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0071] "C 2-10 "Alkenyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-6 Alkenyl groups are preferred. C 2-6 Examples of alkenyl groups include: ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 "Alkenyl" also includes heteroalkenyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkenyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0072] "C 2-10 "Akynyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-6 Akynyl groups are preferred. C 2-6 Examples of akynyl groups include, but are not limited to: ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 "Akynyl" also includes heteroakynyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Akynyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0073] "Halo" or "halogen" refers to fluoro (F), chloro (Cl), bromo (Br), and iodo (I).

[0074] Thus, "C 1-6 "Haloalkyl" refers to "C 1-6"alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 haloalkyl is particularly preferred, more preferably C 1-2 haloalkyl. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCI3, -CH2CI, -CHCI2, 2,2,2-trifluoro-l,l-dimethyl-ethyl, and the like. A haloalkyl group can be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0075] "C 1-6 "alkoxy" means an -OR group where R is an alkyl group as defined above. In some embodiments, C 1-6 alkyl. C 1-4 alkoxy is preferred.

[0076] "C 1-10 "alkylene" means a divalent group formed by removing an additional hydrogen from a C 1-10 alkyl group and can be substituted or unsubstituted. In some embodiments, C 1-8 alkylene, C 1-6 alkylene, C 1-4 alkylene, and C 1-2 alkylene is preferred. Unsubstituted alkylene groups include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, for example, alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- 2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0077] "C 2-10 "alkenylene" means a divalent group formed by removing an additional hydrogen from a C 2-10 alkenyl group and can be substituted or unsubstituted. In some embodiments, C 2-8alkenylene, C 4-6 alkenylene, C 1-4 alkenylene, C 2-4 Alkenylene is preferred.

[0078] "C 2-10 alkynylene" refers to a divalent radical formed by the removal of two hydrogens from a C 2-10 alkynyl group, and can be substituted or unsubstituted. In some embodiments, C 2-8 alkynylene, C 4-6 alkynylene, C 1-4 alkynylene, C 2-4 Alkenylene is preferred.

[0079] "C 3-10 "Cycloalkyl" refers to a non-aromatic hydrocarbon radical of from 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 5-7 cycloalkyl, C 3-7 cycloalkyl and C 3-5 Cycloalkyl is particularly preferred, more preferably C 5-6 cycloalkyl. Cycloalkyl also includes ring systems in which the above cycloalkyl ring is fused with one or more aryl or heteroaryl rings, where the point of attachment is to the cycloalkyl ring, and in such cases the number of carbons continues to designate the number of carbons in the cycloalkyl ring system. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. Cycloalkyl groups can be optionally substituted with one or more substituents, for example, with one to five substituents, one to three substituents, or one substituent.

[0080] "3-10 membered heterocyclyl" refers to a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 4-9 membered heterocyclyl groups are preferred, which are 4- to 9-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 5-8 membered heterocyclyl groups are preferred, which are 5- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl groups are preferred, which are 3- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; 3-7 membered heterocyclyl groups are preferred, which are 3- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 2 ring heteroatoms; 4-7 membered heterocyclyl groups are preferred, which are 4- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 4-6 membered heterocyclyl groups are preferred, which are 4- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 3-5 membered heterocyclyl groups are preferred, which are 3- to 5-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; and 5-6 membered heterocyclyl groups are preferred, which are 5- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the cycloalkyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, dioxanyl.Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, hexahydrotriazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0081] "C 6-10 Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. 10 Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment being on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0082] "5-10 membered heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-9 membered heteroaryl is preferred, which is a 5-9 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Heteroaryl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0083] "C 3-10 "Cycloalkylene", "3-10 membered heterocyclylene", "C 6-10 "Arylene" and "5-10 membered heteroarylene" refer to divalent groups derived from "C3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" are defined as previously described. 3-10 cycloalkyl", "3-10 membered heterocyclyl", "C 6-10 aryl" and "5-10 membered heteroaryl" are defined as previously described.

[0084] "Carbonyl" whether used alone or as part of a larger moiety, e.g., amino carbonyl, oxalyl, represents -C(O)-.

[0085] "Oxo" represents =O.

[0086] "Thio" represents =S.

[0087] Alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, heterocyclyl, aryl and heteroaryl groups defined herein are optionally substituted.

[0088] Exemplary substituents on carbon atoms include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa, -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa)2, -BR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0089] or two geminal hydrogens on a carbon atom are replaced with a group =0, =S, =NN(R bb )2, =NNR bb C(=0)R aa , =NNR bb C(=0)OR aa , =NNR bb S(=0)2R aa , =NR bb , or =NOR cc ;

[0090] each R aa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0091] each R bb is independently selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=0)R aa , -C(=0)N(R cc )2, -C02R aa , -S02R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -S02N(R cc )2, -S02R cc , -S02OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=0)SR cc , -C(=S)SR cc , -P(=0)2R aa , -P(=0)(R aa)2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0092] R cc each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0093] R dd each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(Rff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0094] R ee each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0095] R ff each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0096] R gg each is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1-6 alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocyclyl, C5-C 10 heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0097] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SRcc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups attached to a nitrogen atom are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd , R aa , R bb , R cc , and R dd are as described above.

[0098] Other Definitions

[0099] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody drug conjugate” includes a plurality of antibody drug conjugates and reference to “an antibody drug conjugate” in some embodiments includes a plurality of antibody drug conjugates, and so forth.

[0100] The term “comprising” and variations thereof as used herein are intended to encompass the presence of stated elements or steps and / or groups of elements or steps, but do not preclude the presence or addition of one or more other elements or steps or groups of elements or steps, unless otherwise indicated or unless it would be clear to an ordinary skilled person that the presence of such elements, steps, or groups of elements or steps is precluded.

[0101] These antigen binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as the intact antibodies.

[0102] As used herein, unless otherwise indicated, a bivalent structure can be attached to the remainder of the compound in either direction, left to right or right to left. In one embodiment, the bivalent structure is preferably attached to the remainder of the compound in the left to right orientation.

[0103] The term "amino acid residue" means to include any natural or synthetic amino acid residue, not limited to amino acid residues in the group consisting of the 20 naturally occurring amino acids, wherein residue refers to the moiety remaining after removal of water from an amino acid linked by a peptide bond. The 20 naturally occurring amino acid residues are selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

[0104] "Protecting groups" include "amino protecting groups," "hydroxyl protecting groups," and "carboxyl protecting groups," which are used to protect certain functional groups, such as amino, hydroxyl, and carboxyl groups, from undesirable reactions. The selection of suitable protecting groups for particular functional groups, as well as suitable conditions for protection and deprotection, are well known in the art. For example, many protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0105] Common amino protecting groups include alkoxycarbonyl-type protecting groups, acyl-type protecting groups, or alkyl-type protecting groups. In some embodiments, alkoxycarbonyl-type protecting groups are preferred. In some embodiments, acyl-type protecting groups are preferred.

[0106] Alkoxycarbonyl-type protecting groups include, but are not limited to, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl, or ethoxycarbonyl. Preferably, in some embodiments, Fmoc is preferred. In some embodiments, Cbz is preferred. In some embodiments, Boc is preferred.

[0107] Acyl-type protecting groups include, but are not limited to, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), formyl, acetyl (Ac), or benzoyl (Bz). In some embodiments, Ac is preferred.

[0108] Alkyl-based protecting groups include, but are not limited to, trityl (Trt), 2,4- dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB), benzyl (Bn), or (trimethylsilyl)ethoxymethyl (Sem).

[0109] Common hydroxyl protecting groups include silyl ether-based protecting groups, alkyl ether-based protecting groups, or alkyl methyl ether-based protecting groups.

[0110] Silyl ether-based protecting groups are represented by the structure -SiR1R2R3, where R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl. For example, silyl ether-based protecting groups include, but are not limited to, trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS). In some embodiments, TES is preferred.

[0111] Alkyl ether-based protecting groups include, but are not limited to, C 1-10 alkyl (e.g., methyl, ethyl, or t-butyl), benzyl, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMB), or trityl.

[0112] Alkyl methyl ether-based protecting groups include, but are not limited to, methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBOM), methylthiomethyl (MTM), 2-tetrahydropyranyl, or 2-tetrahydrofuranyl.

[0113] Common carboxyl protecting groups include, but are not limited to, C 1-10 alkyl (preferably C 1-6 alkyl, e.g., methyl, ethyl, or t-butyl), methoxymethyl (MOM), benzyl, p-methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2-methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl (Allyl), 2-tetrahydropyranyl, 2-tetrahydrofuranyl, or -SiR1R2R3(where R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl). In some embodiments, benzyl, p-methoxybenzyl (PMB), or 2,4-dimethoxybenzyl (DMB) are preferred. In some embodiments, is preferred. In some embodiments, C 1-10 alkyl (preferably C 1-6Preferred are alkyl groups, such as methyl, ethyl or tert-butyl, preferably tert-butyl.

[0114] The term "antibody conjugate" generally refers to an antibody linked to another chemical moiety. The chemical moiety can be a cytotoxic drug, an immunostimulatory molecule, and a detectable label. The drug can be, for example, a microtubulin inhibitor, an antibiotic, a DNA synthesis inhibitor, a topoisomerase inhibitor, an RNA polymerase II inhibitor, and an RNA spliceosome inhibitor. The terms "antibody-drug conjugate", "antibody conjugate", and "ADC" can be used interchangeably.

[0115] The terms "alcohol" or "alcoholic compound" are used interchangeably and are to be understood in their broadest sense to mean a compound having a hydroxyl group. "Alkyl alcohol" refers to a compound formed when an alkane is substituted with one or more hydroxyl groups.

[0116] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition for formulation and does not substantially toxicize the recipient thereof, and / or such carrier or excipient is approved or can be used in a pharmaceutical composition for parenteral administration to humans.

[0117] As used herein, the terms "treat," "treatment," "therapy," and the like, refer to the application of a pharmaceutical agent or performance of a procedure for the purpose of effecting an outcome. These outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease (i.e., including an individual which can have a disease which can be related to or caused by the primary disease) but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter such as reduction of symptoms; alleviation of disease symptoms or making the disease condition more tolerable to the patient; slowing in rate of decline or degrading; or amelioration of any disease condition resulting from the disease. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including results of a physician examination. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0118] As used herein, the term "effective amount" means an amount of a pharmaceutical agent administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0119] As used herein, the term "subject" refers to any mammalian subject in whom diagnosis, treatment, or therapy is desired. "Mammalian" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, etc.

[0120] "stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0121] "chiral" is a molecule which has the property of not being superimposable on its mirror image; whereas "achiral" refers to a molecule which is superimposable on its mirror image.

[0122] "enantiomers" refer to two isomers of a compound which are nonsuperimposable mirror images of one another.

[0123] "diastereomers" refer to two or more stereoisomers of a compound which are not mirror images of one another and which have different physical properties.

[0124] The stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0125] Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of the rotation of plane-polarized light by the compound; (-) or 1 indicating that the compound is levorotatory. A compound, which is ( + ) or d is dextrorotatory. A specific stereoisomer is an enantiomer; a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate when there is no stereo selectivity or stereospecificity in a chemical reaction or process.

[0126] Any asymmetric atom (e.g., carbon, etc.) of a compound disclosed herein can exist in a racemic or enantiomeric enriched form, e.g., in the (R)-, (S)-, or (R,S)-configurational form. In certain embodiments, each asymmetric atom has at least a 50% enantiomeric excess in the (R)- or (S)- configuration, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess, or at least a 99% enantiomeric excess.

[0127] Depending on the choice of starting materials and methods, the compounds of the present application can be present in the form of one or more of possible isomers, such as racemates and mixtures of diastereomers, depending on the number of asymmetric carbon atoms. The optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If a compound contains a double bond, the substituents can be in the E or the Z configuration; if the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent can have the cis- or trans-configuration.

[0128] Any mixture of stereoisomers can be separated into their individual components by conventional techniques, such as HPLC or fractional crystallization. If desired, a specific stereoisomer can be derivatized into a different stereoisomer of the same or a different compound.

[0129] Any resulting end products or intermediates of racemates can be resolved into the optical antipodes by methods well known to those skilled in the art, such as, for example, by separation of the resulting diastereomeric salts thereof. The racemic products can also be separated by chiral chromatography, such as, for example, high performance liquid chromatography (HPLC) using a chiral adsorbent. In particular, the enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0130] The present application includes tautomers, which are isomers of functional groups resulting from the movement of a certain atom in a molecule between two positions. Compounds exist in different tautomeric forms, one said compound is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

[0131] The compounds of the present application can include one or more asymmetric centers and can thus occur as individual enantiomers, diastereomers, or as mixtures of stereoisomers, including racemates. The isolation of the individual enantiomers can be achieved by chiral chromatography or by the formation of a chiral salt. The preferred isomers are separated by chiral high pressure liquid chromatography (HPLC) and chiral salt formation and crystallization. Alternatively, the preferred isomers can be synthesized by asymmetric synthesis.

[0132] The skilled artisan will appreciate that organic compounds can form complexes with solvents in which they are reacted or precipitated from solution or crystallized from solvent. These complexes are known as "solvates". When the solvent is water, the solvate is known as a "hydrate". The present application encompasses all solvates of the compounds of the present application.

[0133] The present application also includes isotopically-labelled compounds (isotopic variants) which are identical to those recited in Formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be suitably replaced include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. The present application includes compounds of the present application, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms. Certain isotopically-labelled compounds of the present application, for example those into which radioactive isotopes such as 3 H and 14 C) are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can offer therapeutic advantages resulting from greater metabolic stability, for example, increased half-life or reduced dosage requirements in vivo, and can therefore be preferred in some circumstances. Isotopically-labeled compounds of formula (I) of the present application and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and / or in the examples and preparations described below by substituting a readily available isotopically-labeled reagent for a non-isotopically labeled reagent.

[0134] As described herein, the compounds of the present application can optionally be substituted with one or more substituents, such as described above for the Compounds of the Formulae, or as in the specific examples in the Examples, and classes of compounds encompassed by this application.

[0135] "Pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio.

[0136] As used herein, "pharmaceutically acceptable salts" means organic and inorganic salts of the compounds of the application. Pharmaceutically acceptable salts are well known in the art, e.g., S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable nontoxic acid addition salts include those derived from inorganic acids, such as hydrochloric, hydrobromic, phosphoric, sulfuric, perchloric, and organic acids, such as acetic, oxalic, maleic, tartaric, citric, succinic, malonic, and other similar acids. Salts prepared from bases include those derived from ammonia, N+(C1-C4alkyl)4, and the like. Also contemplated are quaternary ammonium salts of any group containing N in the compound. Water or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed by the addition of inorganic or organic acids to a free amine group of the compound, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, nitric, C1-8sulfonic, and aromatic sulfonic acids.

[0137] Certain embodiments of the application are now described in greater detail by reference to the following examples and the accompanying structural and chemical formulas. The application is intended to encompass all alternatives, modifications and equivalents of the described embodiments, which include all of the features and benefits described herein, and which include those now known or later developed. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The application is intended to encompass all such equivalents. The application is not limited to the specific embodiments described herein, but includes any and all modifications and equivalents thereof within the scope of the claims.

[0138] It is further recognized that certain of the present features can be described in the context of a number of separate embodiments, but can also be provided in combination in a single embodiment. Conversely, various features of the present application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications identified are incorporated herein by reference in their entirety.

[0140] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications identified are incorporated herein by reference in their entirety.

[0141] Abbreviations DCM: dichloromethane DCE: dichloroethane THF: tetrahydrofuran DMF: N,N-dimethylformamide DMA: N,N-dimethylacetamide DMSO: dimethylsulfoxide NMP: N-methylpyrrolidone TEA: triethylamine DIPEA: N,N-diisopropylethylamine NMM: N-methylmorpholine DMAP: 4-dimethylaminopyridine DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DABCO: 1,4-diazabicyclo[2.2.2]octane HATU: 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU: benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate HCTU: 6-chlorobenzotriazol-l,1,3,3-tetramethyluronium hexafluorophosphate BOP: benzotriazol-l-yl-octadecarbodiimide (Carter's reagent) PyBOP: 1H-benzotriazol-l-yloxytrispyrrolidinophosphonium hexafluorophosphate DIC: N,N'-diisopropylcarbodiimide DCC: N,N'-dicyclohexylcarbodiimide EDC: l-ethyl-(3-dimethylaminopropyl)carbodiimide HOBt: 1-hydroxybenzotriazole HOAt: N-hydroxy-7-azabenzotriazole

[0142] Methods of preparation

[0143] The methods of the application can be performed using the methods disclosed herein and routine modifications thereof, which will be apparent in light of the disclosure herein and methods well known in the art. In addition to those teachings herein, routine and well-known synthetic methods can be used. The synthesis of typical compounds described herein (e.g., compounds of Formula (A) and compounds of Formula (B)) can be accomplished as described in the following examples.

[0144] Typical embodiments of compounds according to the application can be synthesized using the general reaction schemes described below. As will be apparent to one of ordinary skill in the art in light of the description herein, the general schemes can be altered by substituting other materials having similar structures for the starting materials to produce correspondingly different products. Given a desired product in which substituents are defined, the desired starting materials can often be identified by inspection. The starting materials are often obtained from commercial sources or synthesized using published methods. To synthesize a compound of the embodiments disclosed, inspection of the structure of the compound to be synthesized will provide the identification of each substituent. In light of the examples herein, the properties of the desired starting materials will often become apparent through a simple process of elimination.

[0145] The compounds of the present disclosure can be prepared from readily available starting materials using, for example, the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimal reaction conditions can vary depending on the particular reactants or solvent used, but such conditions can be determined by a person skilled in the art by routine optimisation procedures.

[0146] In addition, the compounds of the present disclosure can contain one or more chiral centers. Thus, if desired, the compounds can be prepared, or isolated, as pure stereoisomers, that is, as individual enantiomers or diastereomers, or as an enantiomeric or stereoisomerically enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present disclosure unless it is otherwise indicated. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of the compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0147] The starting materials for the following reactions are generally known compounds, or can be prepared by known methods or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Shanghai T&b Chemical Technology Co., Ltd. (Shanghai, China), Sanland Chemical Technology (Shanghai) Co., Ltd. (Shanghai, China), Shanghai Hupu Chemical Technology Co., Ltd. (Shanghai, China), Shanghai Jinlu Pharmaceutical Technology Co., Ltd. (Shanghai, China), Anhui Deshengjia Biological Medicine Co., Ltd. (Anhui, China), Tianjin Famosi Pharmaceutical Technology Co., Ltd. (Tianjin, China), Hunan Hezhong Pharmaceutical Technology Co., Ltd. (Hunan, China). Others can be prepared by following the procedures described in standard reference texts or obvious modifications thereof, such as Fieser and Fieser’s Reagents for Organic Synthesis (John Wiley and Sons, 1991), Rodd’s Chemistry of Carbon Compounds (Elsevier Science Publishers, 1989), Organic Reactions (John Wiley and Sons, 1991), March’s Advanced Organic Chemistry (John Wiley and Sons, 5th Edition, 2001), and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0148] In each of the exemplary schemes, it can be advantageous to separate the reaction products from one another and / or from the starting materials. The desired product of each step or series of steps is isolated and / or purified (hereinafter isolated) to the desired degree of homogeneity using techniques generally known in the art. Typically, such isolation will involve multiphase extraction, crystallization from a solvent or mixture of solvents, distillation, sublimation, or chromatography. Chromatography can involve any number of methods including, for example: reverse and normal phase chromatography; size exclusion chromatography; ion exchange chromatography; high, medium, and low pressure liquid chromatography and equipment; small scale analytical chromatography; simulated moving bed (SMB) and preparative thin or thick layer chromatography, and small scale thin layer and flash chromatography techniques.

[0149] Another class of separation methods involves treating the mixture with a reagent that selectively binds to or separates the product, unreacted starting material, reaction by-products, etc. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent can be an acid (in the case of basic materials), a base (in the case of acidic materials), a binding reagent such as an antibody, a binding protein, a selective chelator such as a crown ether, a liquid / liquid example extraction reagent (LIX), etc.

[0150] The selection of the appropriate separation method depends on the nature of the materials involved. For example, boiling points and molecular weights in distillation and sublimation, the presence or absence of polar functional groups in chromatography, the stability of the material in acidic and basic media in multiphase extraction, etc. The skilled artisan will apply the technique most likely to achieve the desired separation.

[0151] A single stereoisomer, such as an enantiomer, which is substantially free of its stereoisomer, can be obtained by resolution of a racemic mixture, for example, by the formation of diastereomeric salts using, for example, an optically active resolving agent (Sterechemistry of Carbon Compounds, (1962), E. L. Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3) 283-302). A racemic mixture of a chiral compound of the present application can be separated and resolved into the pure stereoisomers by any suitable method, including: (1) forming ionizable diastereomeric salts with a chiral compound and separating the diastereomeric salts by fractional crystallization or other methods; (2) forming diastereomeric compounds with a chiral derivatizing reagent, separating the diastereomeric compounds by chromatography or other methods, and converting the separated stereoisomers to the pure stereoisomers; and (3) resolution of a racemic mixture under a chiral condition to produce a first eluant which is substantially enriched in one enantiomer and a second eluant which is substantially enriched in the other enantiomer.

[0152] As described above, the present application provides, in some embodiments, a method of preparing a compound of formula (A):

[0153] comprising the steps of:

[0154] reacting a compound of formula (B) with a compound of formula (C) to form a compound of formula (A):

[0155] or, reacting the amino acid residue A'4 of a compound of formula (D) with the amino acid residue A'5 of a compound of formula (E) to form a peptide bond to form a compound of formula (A):

[0156] wherein,

[0157] *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0158] T is T 1a -L 1b -L 1c -L 1d -L 1e -;

[0159] T 1a selected from

[0160] LG is a leaving group; preferably, LG is wherein R is selected from C 1-6 alkyl, C 1-6 haloalkyl, -OR a , -NR a R b , C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl, R being optionally substituted with m R';

[0161] m = 1, 2, 3, 4 or 5;

[0162] each R' is independently selected from halogen, -NO2, -CN, -NR a R b , -NR a C(O)R b , C 1-6 alkyl, C 1-6 haloalkyl, -O-C 1-6 alkyl or phenyl;

[0163] R a , R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; or Ra R b and the N atom to which they are attached forms a 3-10 membered heterocyclyl or 5-10 membered heteroaryl;

[0164] L 1b is selected from C 1-10 alkylene, C 2-10 alkenylene, or C 2-10 alkynylene, wherein each CH2in said C 1-10 alkylene, C 2-10 alkenylene, and C 2-10 alkynylene is optionally substituted with 1, 2, or 3 R x ;

[0165] each R x is independently selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl; or, two R x on any identical or different carbon atom can be connected to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene;

[0166] L 1c is selected from a bond, -C(O)-, -C(O)NH-, or -NHC(O)-;

[0167] L 1d is selected from a bond, C 1-8 alkylene, or -(CH2CH2O) n -C 1-4 alkylene-; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl, or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0168] n and w are independently selected from 1, 2, 3, 4, or 5;

[0169] L 1e is selected from -C(O)-, -NHC(O)-, or -C(O)-NHC(O)-;

[0170] L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, having the structure -A1-A2-A3-A4-A5-;

[0171] A1, A2, A3, A4, and A5are independently selected from a bond, an optionally substituted natural amino acid divalent residue, or an optionally substituted non-natural amino acid divalent residue, wherein at least two are not a bond;

[0172] A'4 and A'5 are independently an optionally substituted natural amino acid monovalent residue or an optionally substituted non-natural amino acid monovalent residue;

[0173] said divalent residue or monovalent residue of each amino acid is optionally substituted with 1, 2, or 3 R y substituents;

[0174] each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy;

[0175] L3 is an optional substituted or unsubstituted spacer, for example -NH2-CH2-,

[0176] L3 is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy;

[0177] L D2 is selected from -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0178] q is 0, 1, 2, 3, or 4, preferably 1, 2, or 3;

[0179] R D1 and R D2 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0180] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7Cycloalkylene or 3-7-membered heterocyclic groups, preferably C-shaped 3-5 Cycloalkylene;

[0181] R D5 Selected from halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl, 5-10 heteroaryl, -C 1-6 Alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10-membered heterocyclic group, -C 1-6 Alkylene-C 6-10 Aryl or -C 1-6 Alkyl-5-10 heteroaryl groups, preferably -C 1-6 Alkylene-C 3-10 Cycloalkyl.

[0182] T

[0183] In one implementation, T is T 1a -L 1b -L 1c -L 1d -L 1e -

[0184] T 1a

[0185] In one implementation, T 1a for For example In another implementation, T 1a for In another implementation, T 1a for In another implementation, T 1a for

[0186] In a specific implementation plan, T 1a for

[0187] In one implementation, LG is a leaving group, for example For example

[0188] In one specific implementation scheme, R is selected from C. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OR a -NR a R b C 3-10cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; in another specific embodiment, R is selected from the group consisting of C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, 3-7 membered heterocyclyl, phenyl or 5-6 membered heteroaryl; in another specific embodiment, R is selected from the group consisting of C 1-6 alkyl or C 1-6 haloalkyl; in another specific embodiment, R is selected from the group consisting of methyl, p-nitrophenyl, p-nitrile phenyl, p-tolyl, p-trifluoromethylphenyl, p-chlorophenyl, preferably methyl.

[0189] In one specific embodiment, R is optionally substituted with m R'; in another specific embodiment, R is unsubstituted.

[0190] In one specific embodiment, m = 1, 2, 3, 4 or 5.

[0191] In one specific embodiment, each R' is independently selected from the group consisting of halogen, -NO2, -CN, -NR a R b , -NR a C(O)R b , C 1-6 alkyl, C 1-6 haloalkyl, -O-C 1-6 alkyl or phenyl; in another specific embodiment, each R' is independently selected from the group consisting of halogen, -NO2, -CN, C 1-6 alkyl or C 1-6 haloalkyl.

[0192] In one specific embodiment, R a , R b are independently selected from the group consisting of H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; in another specific embodiment, R a , R b are independently selected from the group consisting of H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, 3-7 membered heterocyclyl, phenyl or 5-6 membered heteroaryl; in another specific embodiment, R a , R b are independently selected from the group consisting of H, C 1-6 alkyl or C 1-6 haloalkyl.

[0193] In one embodiment, R a , R b and the N atom to which they are attached form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl; in another embodiment, R a , R b and the N atom to which they are attached form a 3-7 membered heterocyclyl or 5-6 membered heteroaryl.

[0194] L 1b

[0195] In one embodiment, L 1b is C 1-10 alkylene; in another embodiment, L 1b is C 1-6 alkylene; in another embodiment, L 1b is C 2-10 alkenylene; in another embodiment, L 1b is C 2-6 alkenylene; in another embodiment, L 1b is C 2-10 alkynylene; in another embodiment, L 1b is C 2-6 alkenylene; in another embodiment, L 1b is C 2-6 alkynylene; in another embodiment, each CH2in the foregoing C 1-10 alkylene, C 1-6 alkylene, C 2-10 alkenylene, C 2-6 alkenylene, C 2-10 alkynylene and C 2-6 alkynylene is unsubstituted; in another embodiment, each CH2in the foregoing C 1-10 alkylene, C 2-10 alkenylene and C 2-10 alkynylene is optionally substituted with 1, 2, or 3 (preferably 1 or 2) R x In one embodiment, L 1b is

[0196] In one embodiment, each R x is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; in another more specific embodiment, each R x is independently selected from H, halogen or C 1-6 alkyl; in another more specific embodiment, two R x on any identical or different carbon atom can join to form a C3-10 Cycloalkylene or 3-10 membered heterocyclyl, preferably C 3-7 Cycloalkylene or 3-7 membered heterocyclyl, more preferably C 3-5 Cycloalkylene, for example cyclopropyl.

[0197] L 1c

[0198] In one embodiment, L 1c is a bond; in another embodiment, L 1c is -C(O)-; in another embodiment, L 1c is -C(O)NH-; in another embodiment, L 1c is -NHC(O)-.

[0199] In one particular embodiment, L 1c is selected from a bond, -C(O)-, -C(O)NH- or -NHC(O)-; in another particular embodiment, L 1c is a bond or -C(O)NH-.

[0200] L 1d

[0201] In one embodiment, L 1d is a bond; in another embodiment, L 1d is C 1-8 Alkylene; in another embodiment, L 1d is C 1-6 Alkylene, preferably C 1-4 Alkylene; in another embodiment, L 1d is -(CH2CH2O) n -C 1-4 Alkylene-; in another embodiment, L 1d is C 1-8 Alkylene or C 1-6 Alkylene, said C 1-8 Alkylene or C 1-6 Alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 Alkyl; in another embodiment, L 1d is C 1-8 Alkylene or C 1-6 Alkylene, said C 1-8 Alkylene or C 1-6 Alkylene is optionally substituted with -C(O)NH-(CH2CH2O) w -C 1-4 Alkyl.

[0202] In a particular embodiment, L 1d is selected from a chemical bond or C 1-6 alkylene; in another particular embodiment, L 1d is selected from a chemical bond or C 1-4 alkylene; in another particular embodiment, L 1d is not a chemical bond.

[0203] In a particular embodiment, n is selected from 1, 2, 3, 4 or 5.

[0204] In a particular embodiment, w is selected from 1, 2, 3, 4 or 5, preferably from 2, 3 or 4, preferably 3.

[0205] L 1e

[0206] In a particular embodiment, L 1e is -C(O)-; in another embodiment, L 1e is -NHC(O)-; in another embodiment, L 1e is -C(O)-NHC(O)-.

[0207] L2

[0208] In one embodiment, L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, having the structure -A1-A2-A3-A4-A5-.

[0209] In a particular embodiment, L2 is selected from -gly-gly-, -gly-gly-gly-, -gly-gly-gly-gly-, -val-gly-gly-, -val-cit-gly-, -val-gln-gly-, -val-glu-gly-, -phe-lys-gly-, -leu-lys-gly-, -gly-val-lys-gly-, -val-lys-gly-gly-, -val-lys-gly-, -val-lys-ala-, -val-lys-leu-, -leu-leu-gly-, -gly-gly-phe-gly-, -gly-gly-phe-gly-gly-, -val-gly-, -val-cit- or val-lys-β-ala-, preferably -gly-gly-phe-gly-.

[0210] In one specific embodiment, the amino terminus of the peptide chain of the peptide group L2 is attached to T and the carboxyl terminus is attached to L3; in another specific embodiment, the carboxyl terminus of the peptide chain of the peptide group L2 is attached to T and the amino terminus is attached to L3.

[0211] In one specific embodiment, A1, A2, A3, A4, and A5 are independently selected from a chemical bond, an optionally substituted natural amino acid bivalent residue, or an optionally substituted unnatural amino acid bivalent residue, wherein at least two are not a chemical bond; in another specific embodiment, A1, A2, A3, A4, and A5 are independently selected from a chemical bond, a bivalent residue of gly, val, cit, gln, glu, phe, lys, leu, or ala, wherein at least two are not a chemical bond; in another specific embodiment, A2 is a chemical bond.

[0212] In one specific embodiment, A'4 and A'5 are independently an optionally substituted natural amino acid monovalent residue or an optionally substituted unnatural amino acid monovalent residue; in another specific embodiment, A'4 and A'5 are independently selected from a monovalent residue of gly, val, cit, gln, glu, phe, lys, leu, or ala; in another specific embodiment, A'4 is -phe; in another specific embodiment, -A1-A2-A3-A'4 is -gly-gly-phe; in another specific embodiment, A'5 is -gly.

[0213] In one specific embodiment, the amino terminus of A'4 is attached to A3 and the carboxyl is reacted with the amino group of A'5 to form a peptide bond; in another specific embodiment, the carboxyl terminus of A'4 is attached to A3 and the amino group is reacted with the carboxyl of A'5 to form a peptide bond.

[0214] In one embodiment, the amino acid is selected from glycine, phenylalanine, alanine, valine, citrulline, or lysine.

[0215] In one embodiment, the bivalent residue or monovalent residue of each amino acid is unsubstituted; in another embodiment, the bivalent residue or monovalent residue of each amino acid is optionally substituted with 1, 2, or 3 R y groups.

[0216] In one specific embodiment, each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy; in another specific embodiment, each R y is independently selected from H, halogen, C1-6 alkyl or C 1-6 haloalkyl.

[0217] L3

[0218] In one embodiment, L3 is an optional spacer, which can or can not be substituted; in a particular embodiment, L3 is -NH-CH2-(AM); in another particular embodiment, L3 is In a particular embodiment, L3 is

[0219] In a particular embodiment, L3 is unsubstituted; in another particular embodiment, L3 is optionally substituted with 1, 2, or 3 R z substituted.

[0220] In a particular embodiment, each R z is independently selected from H, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy; in another particular embodiment, each R z is independently selected from H, halo, or C 1-6 alkyl.

[0221] L D2

[0222] In one embodiment, L D2 is -NH-; in another embodiment, L D2 is -O-; in another embodiment, L D2 is -C(O)-; in another embodiment, L D2 is -NHC(O)-; in another embodiment, L D2 is -C(O)NH-.

[0223] In a particular embodiment, L D2 is selected from -NH- or -O-.

[0224] *1, *2, *3, *4, and q

[0225] In one embodiment, *1 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in one embodiment, *1 is the (S) configuration; in another embodiment, *1 is the (R) configuration.

[0226] In one embodiment, *2 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in one embodiment, *2 is in the (S) configuration; in another embodiment, *2 is in the (R) configuration.

[0227] In one embodiment, *3 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in one embodiment, *3 is in the (S) configuration; in another embodiment, *3 is in the (R) configuration.

[0228] In one embodiment, *4 is a chiral center selected from the (S) or (R) absolute configuration, or a mixture thereof; in one embodiment, *4 is in the (S) configuration; in another embodiment, *4 is in the (R) configuration.

[0229] In one embodiment, q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3, preferably 1.

[0230] R D1 , R D2 , R D3 , R D4 and R D5

[0231] In one embodiment, R D1 is H; in another embodiment, R D1 is halogen; in another embodiment, R D1 is C 1-6 alkyl, for example Me; in another embodiment, R D1 is C 1-6 haloalkyl; in another embodiment, R D1 is C 1-6 alkoxy.

[0232] In one particular embodiment, R D1 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl.

[0233] In one embodiment, R D2 is H; in another embodiment, R D2 is halogen, for example F; in another embodiment, R D2 is C 1-6 alkyl; in another embodiment, R D2 is C 1-6 haloalkyl; in another embodiment, R D2 is C 1-6 alkoxy.

[0234] In a particular embodiment, R D2 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl.

[0235] In a particular embodiment, R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl; in another particular embodiment, R D1 , R D2 and the carbon atom to which they are attached together form a 5-6 membered heterocyclyl.

[0236] In a particular embodiment, R D3 is H; in another particular embodiment, R D3 is halogen; in another particular embodiment, R D3 is C 1-6 alkyl; in another particular embodiment, R D3 is C 1-6 haloalkyl; in another particular embodiment, R D3 is C 1-6 alkoxy.

[0237] In a particular embodiment, R D4 is H; in another particular embodiment, R D4 is halogen; in another particular embodiment, R D4 is C 1-6 alkyl; in another particular embodiment, R D4 is C 1-6 haloalkyl; in another particular embodiment, R D4 is C 1-6 alkoxy.

[0238] In a particular embodiment, R D3 and R D4 are not simultaneously H.

[0239] In a particular embodiment, each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; in another particular embodiment, R D3 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably H or C 1-6 alkyl (e.g. methyl).

[0240] In a particular embodiment, R D3 , RD4 and the carbon atom to which they are attached form C 3-7 cycloalkylene or 3-7 membered heterocyclylene; in another embodiment, R D3 , R D4 and the carbon atom to which they are attached form C 3-5 cycloalkylene.

[0241] in one embodiment, R D5 is halogen; in another embodiment, R D5 is C 1-6 alkyl; in another embodiment, R D5 is C 1-6 haloalkyl; in another embodiment, R D5 is C 3-10 cycloalkyl; in another embodiment, R D5 is 3-10 membered heterocyclyl; in another embodiment, R D5 is C 6-10 aryl; in another embodiment, R D5 is 5-10 membered heteroaryl; in another embodiment, R D5 is -C 1-6 alkylene-C 3-10 cycloalkyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl, for example -CH2-cyclopropyl; in another embodiment, R D5 is -C 1-6 alkylene-3-10 membered heterocyclyl, preferably -C 1-4 alkylene-3-5 membered heterocyclyl; in another embodiment, R D5 is -C 1-6 alkylene-C 6-10 aryl; in another embodiment, R D5 is -C 1-6 alkylene-5-10 membered heteroaryl.

[0242] in one particular embodiment, R D5 is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl, or -C 1-6 alkylene-5-10 membered heteroaryl; in another particular embodiment, R D5 is selected from -C1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl.

[0243] Any of the technical solutions in any of the above specific embodiments or any combination thereof can be combined with any of the technical solutions in other specific embodiments or any combination thereof. For example, any of the technical solutions of T or any combination thereof can be combined with any of the technical solutions of y, L1, L2, L3, T 1a , L 1b , L 1c , L 1d , L 1e , L D2 , R D1 , R D2 , R D3 , R D4 and R D5 , or any combination thereof. The present application is intended to include all combinations of these technical solutions, which are not listed one by one due to the length of the article.

[0244] In one embodiment, the present application provides the following method for preparing a compound of formula (A):

[0245] reacting a compound of formula (B) with a compound of formula (C) to form a compound of formula (A):

[0246] In another embodiment, the present application provides the following method for preparing a compound of formula (A):

[0247] reacting an amino acid residue A'4 of a compound of formula (D) with an amino acid residue A'5 of a compound of formula (E) to form a peptide bond to form a compound of formula (A):

[0248] In some embodiments, in formula (1) or formula (2), *1 and *2 are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0249] T is T 1a -L 1b -L 1c -L 1d -L 1e -;

[0250] T 1a is selected from

[0251] LG is a leaving group; preferably, LG is wherein R is selected from C 1-6 alkyl, C 1-6haloalkyl, -OR a , -NR a R b , C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl, R is optionally substituted by m R’;

[0252] m = 1, 2, 3, 4 or 5;

[0253] each R’ is independently selected from halogen, -NO2, -CN, -NR a R b , -NR a C(O)R b , C 1-6 alkyl, C 1-6 haloalkyl, -O-C 1-6 alkyl or phenyl;

[0254] R a , R b are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; or R a , R b and the N atom to which they are attached form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl;

[0255] L 1b is selected from C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein each CH2in said C 1-10 alkylene, C 2-10 alkenylene and C 2-10 alkynylene is optionally substituted with 1, 2 or 3 R x ;

[0256] each R x is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, two R x on any identical or different carbon atom can be connected to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene;

[0257] L 1c is selected from a bond, -C(O)-, -C(O)NH- or -NHC(O)-;

[0258] L 1da chemical bond, C 1-8 alkylene or -(CH2CH2O) n -C 1-4 alkylene; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0259] n and w are independently selected from 1, 2, 3, 4, or 5;

[0260] L 1e selected from -C(O)-, -NHC(O)-, or -C(O)-NHC(O)-;

[0261] L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or non-natural amino acid residues, having the structure -A1-A2-A3-A4-A5-;

[0262] A1, A2, A3, A4, and A5 are independently selected from a chemical bond, an optionally substituted natural amino acid divalent residue, or an optionally substituted non-natural amino acid divalent residue, wherein at least two are not a chemical bond;

[0263] A'4 and A'5 are independently an optionally substituted natural amino acid monovalent residue or an optionally substituted non-natural amino acid monovalent residue;

[0264] each divalent residue or monovalent residue of said amino acid is optionally substituted with 1, 2, or 3 R y ;

[0265] each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy;

[0266] L3 is an optional substituted or unsubstituted spacer, such as -NH2-CH2-,

[0267] L3 is optionally substituted with 1, 2, or 3 R z , each R z is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy;

[0268] L D2 is selected from -NH-, -O-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0269] q is 0, 1, 2, 3, or 4, preferably 1, 2, or 3;

[0270] R D1 and R D2 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached together form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl;

[0271] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached together form a C 3-7 cycloalkylene or 3-7 membered heterocyclylene, preferably C 3-5 cycloalkylene;

[0272] R D5 is selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl, or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl.

[0273] In some embodiments, in reaction (1) or reaction (2), *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably, *1 is in (S) configuration;

[0274] T is T 1a -L 1b -L 1c -L 1d- L 1e -;

[0275] T 1a is preferably for example

[0276] LG is wherein R is selected from C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, 3-7 membered heterocyclyl, phenyl or 5-6 membered heteroaryl, preferably from C 1-6 alkyl or C 1-6 haloalkyl, R being optionally substituted with m R'; preferably R is selected from methyl, p-nitrophenyl, p-nitrile phenyl, p-tolyl, p-trifluoromethylphenyl, p-chlorophenyl, preferably methyl;

[0277] m = 1, 2, 3, 4 or 5;

[0278] each R' is independently selected from halogen, -NO2, -CN, C 1-6 alkyl or C 1-6 haloalkyl;

[0279] L 1b is selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, preferably C 2-6 alkynylene, wherein each CH2in said C 1-6 alkylene, C 2-6 alkenylene and C 2-6 alkynylene is optionally substituted with 1 or 2 R x ; preferably L 1b is

[0280] each R x is independently selected from H, halogen or C 1-6 alkyl; or two R x on any identical or different carbon atom can be linked to form a C 3-7 cycloalkylene or 3-7 membered heterocyclylene, preferably C 3-7 cycloalkylene, preferably C 3-5 cycloalkylene, more preferably cyclopropylene;

[0281] L 1c is selected from a chemical bond, -C(O)-, -C(O)NH- or -NHC(O)-, preferably a chemical bond or -C(O)NH-;

[0282] L 1dselected from a chemical bond or C 1-6 alkylene, preferably a chemical bond or C 1-4 alkylene; said C 1-6 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl, preferably optionally substituted with -C(O)NH-(CH2CH2O) w -C 1-4 alkyl;

[0283] w is selected from 2, 3 or 4, preferably 3;

[0284] L 1e selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably -C(O)-;

[0285] L2is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural or unnatural amino acid residues, having the structure -A1-A2-A3-A4-A5-;

[0286] A1, A2, A3, A4and A5are independently selected from a chemical bond, a divalent residue of gly, val, cit, gln, glu, phe, lys, leu or ala, wherein at least two are not a chemical bond; preferably A2is a chemical bond; preferably L2is selected from -gly-gly-, -gly-gly-gly-, -gly-gly-gly-gly-, -val-gly-gly-, -val-cit-gly-, -val-gln-gly-, -val-glu-gly-, -phe-lys-gly-, -leu-lys-gly-, -gly-val-lys-gly-, -val-lys-gly-gly-, -val-lys-gly-, -val-lys-ala-, -val-lys-leu-, -leu-leu-gly-, -gly-gly-phe-gly-, -gly-gly-phe-gly-gly-, -val-gly-, -val-cit- or val-lys-β-ala-, preferably -gly-gly-phe-gly-;

[0287] A'4and A'5are independently selected from monovalent residues of gly, val, cit, gin, glu, phe, lys, leu or ala; preferably A'4is -phe; preferably in formula (2), -A1-A2-A3-A'4is -gly-gly-phe; preferably A'5is -gly;

[0288] each amino acid of said divalent residue or monovalent residue is optionally substituted with 1, 2 or 3 R y ;

[0289] each R y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0290] L3is -NH2-CH2-;

[0291] L3is optionally substituted with 1, 2 or 3 R z , each R z is independently selected from H, halogen or C 1-6 alkyl;

[0292] L D2 is selected from -NH- or -O-, preferably -O-;

[0293] q is 1, 2 or 3, preferably 1 ;

[0294] R D1 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me;

[0295] R D2 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, for example F;

[0296] each R D3 and R D4 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; preferably R D3 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably H or C 1-6 alkyl (for example methyl); preferably R D4 is H;

[0297] R D5 is selected from -C 1-4alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3-5 membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl, for example -CH2-cyclopropyl.

[0298] In some embodiments, the compound of formula (A) is selected from:

[0299] wherein *1, *2, *3 and *4 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof;

[0300] Preferably, *3 is in (S) configuration; preferably, *4 is in (S) configuration;

[0301] R D3 as defined herein.

[0302] In some embodiments, the compound of formula (A) is selected from:

[0303] In some embodiments, in the reaction of formula (1), the method is carried out in the presence of a condensing agent; preferably, the condensing agent is an onium salt condensing agent, for example HATU, HBTU, HCTU, BOP or PyBOP, preferably HATU.

[0304] In some embodiments, the molar amount of the condensing agent is about 1-5 times, preferably about 1-3 times, preferably about 1-2 times, for example about 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.24, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times the molar amount of the compound of formula (B).

[0305] In some embodiments, the method is carried out in the presence of a base. Preferably, the base is selected from organic and inorganic bases, preferably organic bases.

[0306] In some embodiments, the organic base is selected from alkali and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases.

[0307] In some embodiments, the organic amine base is selected from TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine; preferably, the organic amine base is selected from DIPEA and TEA, preferably DIPEA.

[0308] In some embodiments, wherein the alkali and alkaline earth metal alkoxides are selected from NaOMe, KOMe, NaOEt, KOEt, NaOtBu, and KOtBu.

[0309] In some embodiments, wherein the inorganic base is selected from an alkali and alkaline earth metal carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydroxide, or hydride, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, and NaH.

[0310] In some embodiments, wherein the molar amount of the base is about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, for example about 1, 1.5, 2, 2.5, 3, 3.03, 3.06, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.93, 4, 4.5, 5 times the molar amount of the compound of formula (B).

[0311] In some embodiments, wherein the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, THF, DMF, DMA, DMSO, NMP, benzene, toluene, xylene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably selected from DMF, DMA, DMSO, NMP, toluene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, the solvent is selected from DMF and DMA, preferably DMF.

[0312] In some embodiments, wherein the molar amount of the compound of formula (C) is about 0.5-5 times, preferably about 0.5-2 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (B).

[0313] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0314] In one embodiment, the present application also provides the following method for preparing a compound of formula (B):

[0315] reacting a compound of formula (F) with a compound of formula (G) to form a compound of formula (B):

[0316] In another embodiment, the present application also provides the following method for preparing a compound of formula (B):

[0317] reacting a compound of formula (H) with a compound of formula (I) to form a compound of formula (B):

[0318] In some embodiments, in trans (3) or formula (4), PG1is a carboxyl protecting group, for example

[0319] NH2-L’2- is a monovalent radical of the amino terminus of the peptide chain of the peptide radical L2when not attached to T;

[0320] In formula (3), T is T 1a -L 1b -L 1c -L 1d -C(O)-;

[0321] In formula (4), T is T 1a -L 1b -C(O)NH-L 1d -L 1e -;

[0322] the remaining variables are as defined herein.

[0323] In some embodiments, L in formula (4) is not a chemical bond. 1d

[0324] In some embodiments, wherein in the reaction of formula (3) or formula (4), the method is carried out in the presence of a base. Preferably, wherein the base is selected from the group consisting of an organic base and an inorganic base.

[0325] In some embodiments, wherein the base is an inorganic base.

[0326] In some embodiments, wherein the inorganic base is selected from the group consisting of carbonates, bicarbonates, phosphates, hydrogenophosphates, dihydrogenophosphates, hydroxides or hydrides of alkali and alkaline earth metals, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, or NaH; preferably from carbonates or bicarbonates of alkali and alkaline earth metals, for example, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, or KHCO3; preferably from Na2CO3and K2CO3, preferably Na2CO3.

[0327] ​In some embodiments, wherein the molar amount of the base is about 0.5-5 times, preferably about 0.5-3 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5 or 3 times the molar amount of the compound of formula (G) or the compound of formula (F).

[0328] In some embodiments, wherein the molar amount of the base is about 0.5-5 times, preferably about 0.5-3 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5 or 3 times the molar amount of the compound of formula (H) or the compound of formula (I).

[0329] In some embodiments, wherein the base is an organic base; preferably an alkali and alkaline earth metal alkoxide and an organic amine base, preferably an organic amine base.

[0330] In some embodiments, wherein the organic amine base is selected from the group consisting of TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine; preferably the organic amine base is selected from the group consisting of DIPEA and TEA, preferably DIPEA.

[0331] In some embodiments, wherein the alkali and alkaline earth metal alkoxide is selected from the group consisting of NaOMe, KOMe, NaOEt, KOEt, NaOtBu and KOtBu.

[0332] In some embodiments, wherein the molar amount of the base is about 1 -10 times, preferably about 1 -8 times, preferably about 1 -5 times, for example about 1, 1.5, 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.98, 4, 4.5, 5 times the molar amount of the compound of formula (G) or the compound of formula (F).

[0333] In some embodiments, wherein the method is performed in the presence of a condensing agent; preferably the condensing agent is an onium salt condensing agent, for example HATU, HBTU, HCTU, BOP or PyBOP, preferably HATU.

[0334] In some embodiments, wherein the molar amount of the condensing agent is about 1 -5 times, preferably about 1 -3 times, preferably about 1 -2 times, for example about 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times the molar amount of the compound of formula (G) or the compound of formula (F).

[0335] In some embodiments, the process is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, isopropyl acetate, n-hexane, n-heptane, petroleum ether, acetone, acetonitrile, toluene, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, DMF, DMA, DMSO, NMP, methanol, ethanol, isobutanol, tert-butanol, isopropanol, n-propanol, n-pentanol, and iso-pentanol; preferably, the solvent is selected from DCM, DCE, acetonitrile, tetrahydrofuran, DMF, DMA, and NMP; preferably, the solvent is acetonitrile.

[0336] In some embodiments, the molar amount of one of the compound of formula (G) and the compound of formula (F) is about 0.5-5 times, preferably about 0.5-2 times, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the other.

[0337] In some embodiments, the molar amount of one of the compound of formula (H) and the compound of formula (I) is about 0.5-5 times, preferably about 0.5-2 times, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the other.

[0338] In some embodiments, the process is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0339] In one embodiment, the present application also provides the following process for preparing a compound of formula (G):

[0340] reacting the carboxyl group of the amino acid residue A'4 of a compound of formula (G1) with a compound of formula (G2) to form a compound of formula (G3), and deprotecting the compound of formula (G3) to form a compound of formula (G):

[0341] wherein,

[0342] A'4 is as defined herein, and the amino terminus is attached to A3, and the carboxyl group participates in the reaction;

[0343] A5 is as defined herein, and the carboxyl terminus is attached to L3;

[0344] PG2and PG3are independently an amino protecting group; preferably the amino protecting group is selected from the group consisting of alkoxycarbonyl protecting groups, acyl protecting groups or alkyl protecting groups; preferably an alkoxycarbonyl protecting group, such as carbobenzyloxy (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl; preferably PG2is Cbz; preferably PG3is Fmoc;

[0345] PG4is a carboxyl protecting group, such as C 1-10 alkyl, preferably C 1-6 alkyl, such as methyl, ethyl or tert-butyl), methoxymethyl (MOM), benzyl, p- methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2- methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl (Allyl), 2-tetrahydropyranyl, 2-tetrahydrofuranyl or -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; preferably selected from benzyl, p-methoxybenzyl (PMB) or 2,4-dimethoxybenzyl (DMB); preferably benzyl;

[0346] L2is as defined herein and the carboxyl end is attached to L3;

[0347] NH2-L’2- is a monovalent group when the amino end of the peptide chain of the peptide group L2is not attached to T;

[0348] each of the remaining variables is as defined herein.

[0349] In some embodiments, wherein the reaction to generate formula (G3) is carried out in the presence of a base. Preferably, wherein the base is selected from the group consisting of organic bases and inorganic bases, preferably organic bases.

[0350] In some embodiments, wherein the organic base is selected from the group consisting of alkali and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases.

[0351] In some embodiments, wherein the organic amine base is selected from the group consisting of TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine; preferably the organic amine base is selected from the group consisting of DBU and DABCO, preferably DBU.

[0352] In some embodiments, wherein the inorganic base is selected from a carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydroxide or hydride of an alkali metal and an alkaline earth metal, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, or NaH.

[0353] In some embodiments, wherein the molar amount of the base is about 0.1-5 times, preferably about 0.1-3 times, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, or 3 times the molar amount of the compound of formula (G2).

[0354] In some embodiments, wherein the reaction to generate the compound of formula (G3) is carried out in the presence of a condensing agent. Preferably, the condensing agent is a carbodiimide-based condensing agent, for example, DCC, DIC, or EDC, preferably EDC.

[0355] In some embodiments, wherein the molar amount of the condensing agent is about 1-5 times, preferably about 1-3 times, preferably about 1-2 times, for example about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (G2).

[0356] In some embodiments, wherein the reaction to generate the compound of formula (G3) is carried out in the presence of a condensing activator; preferably, the condensing activator is selected from DMAP, HOBt, and HOAt, preferably HOBt.

[0357] In some embodiments, wherein the molar amount of the condensing activator is about 1-5 times, preferably about 1-3 times, preferably about 1-2 times, for example about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (G2).

[0358] In some embodiments, wherein the reaction to generate the compound of formula (G3) is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, THF, DMF, DMA, DMSO, NMP, benzene, toluene, xylene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably selected from DMF, DMA, DMSO, NMP, toluene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, the solvent is selected from DMF and DMA, preferably DMF.

[0359] In some embodiments, the molar amount of the compound of formula (G1) is about 0.5-5 times, preferably about 0.5-2 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.05, 1.07, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (G2).

[0360] In some embodiments, the reaction to form formula (G) is conducted in the presence of a catalyst; preferably, the catalyst is a palladium catalyst, preferably palladium on carbon, for example 0.5% palladium on carbon, 5% palladium on carbon, or 10% palladium on carbon.

[0361] In some embodiments, the molar amount of the catalyst is about 1%-50%, preferably about 1%-25%, preferably about 1%-15%, for example about 1%, 2%, 3%, 4%, 5%, 5.4%, 6%, 7%, 8%, 8.8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the molar amount of the compound of formula (G2).

[0362] In some embodiments, the reaction to form formula (G) is conducted in an atmosphere of hydrogen.

[0363] In some embodiments, the reaction to form formula (G) is conducted in the presence of a solvent; preferably, the solvent is selected from the group consisting of an alcoholic solvent, an ethereal solvent, and water; preferably, the solvent is a mixed solvent of an alcoholic solvent, an ethereal solvent, and water; preferably, the volume ratio of the alcoholic solvent:the ethereal solvent:water is (0.5-5):(0.5-5):(0.5-5), preferably (0.5-2):(0.5-2):(0.5-2), preferably (0.5-1.5):(0.5-1.5):(0.5-1.5), preferably 1:1:1.

[0364] In some embodiments, the alcoholic solvent is selected from the group consisting of methanol, ethanol, isobutyl alcohol, tert-butyl alcohol, isopropyl alcohol, n-propyl alcohol, n-pentyl alcohol, and iso-pentyl alcohol, preferably methanol.

[0365] In some embodiments, the ethereal solvent is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane, preferably tetrahydrofuran.

[0366] In some embodiments, the reaction to form formula (G3) or the reaction to form formula (G) is conducted at a temperature of about 0°C to about 80°C; preferably, the temperature is about 0°C to about 50°C; preferably, the temperature is about 0°C to room temperature; preferably, the temperature is about 25°C.

[0367] In some embodiments, the reaction to form Formula (G) is conducted under an atmosphere of hydrogen.

[0368] In one embodiment, the present application also provides the following method for preparing a compound of Formula (F3):

[0369] reacting a compound of Formula (F1) with a compound of Formula (F2) to form a compound of Formula (F3):

[0370] wherein PG8is a carboxyl protecting group, for example, C 1-10 alkyl (preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl), methoxymethyl (MOM), benzyl, p- methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2- methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl, 2-tetrahydropyranyl, 2- tetrahydrofuranyl or -SiR1R2R3wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; preferably C 1-10 alkyl (preferably C 1-6 alkyl), for example methyl, ethyl or tert-butyl, preferably tert-butyl;

[0371] L 1c is -C(O)NH-;

[0372] the remaining variables are as defined herein.

[0373] In some embodiments, wherein the method is conducted in the presence of a solvent, the solvent is selected from DCM, DCE, THF, benzene, toluene, xylene, DMF, DMA and NMP; preferably selected from DCM, DCE, THF, benzene, toluene and xylene; preferably selected from DCM and DCE, more preferably DCM.

[0374] In some embodiments, wherein the method is conducted in the presence of a condensing agent; preferably, the condensing agent is an onium salt condensing agent, for example HATU, HBTU, HCTU, BOP or PyBOP, preferably HATU.

[0375] In some embodiments, wherein the molar amount of the condensing agent is about 1-5 times, preferably about 1-3 times, preferably about 1-2 times, for example about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times, of the molar amount of the compound of Formula (F1).

[0376] In some embodiments, wherein the molar amount of the compound of formula (F2) is about 0.5-5 times, preferably about 0.5-2 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times the molar amount of the compound of formula (F1).

[0377] In some embodiments, wherein the method is carried out in the presence of a base.

[0378] In some embodiments, wherein the base is selected from inorganic bases and organic bases, preferably organic bases.

[0379] In some embodiments, wherein the inorganic base is selected from carbonates, bicarbonates, phosphates, hydrogenophosphates, dihydrogenophosphates, hydroxides or hydrides of alkali metals and alkaline earth metals, for example LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4 or NaH.

[0380] In some embodiments, wherein the organic base is selected from alkoxides of alkali metals and alkaline earth metals and organic amine bases.

[0381] In some embodiments, wherein the organic amine base is selected from triethylamine, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine, preferably DIPEA.

[0382] In some embodiments, wherein the alkoxides of alkali metals and alkaline earth metals are selected from NaOMe, KOMe, NaOEt, KOEt, NaOtBu and KOtBu.

[0383] In some embodiments, wherein the molar amount of the base is about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, for example about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (F1).

[0384] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably the temperature is about 0 °C to about 50 °C; preferably the temperature is about 0 °C to room temperature; preferably the temperature is about 25 °C.

[0385] In one embodiment, the present application provides a method of preparing a compound of formula (F):

[0386] deprotecting the compound of formula (F3) and subjecting it to a carboxyl protection reaction to form a compound of formula (F):

[0387] wherein the variables are as defined herein.

[0388] In some embodiments, the reaction to generate the compound of formula (F4) is carried out in the presence of an acid; preferably, the acid is selected from HC1, H2S04, p-toluenesulfonic acid, benzenesulfonic acid, formic acid, acetic acid, and trifluoroacetic acid; preferably selected from acetic acid and trifluoroacetic acid, preferably trifluoroacetic acid.

[0389] In some embodiments, the reaction to generate the compound of formula (F4) is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, ethanol, isopropanol, and water; preferably selected from DCM and DCE, more preferably DCM.

[0390] In some embodiments, the volume ratio of the solvent:trifluoroacetic acid is about 1:(0.1-20), preferably about 1:(0.5-10), for example about 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0391] In one embodiment, the reaction to generate the compound of formula (F) is carried out in the presence of a condensing agent; preferably, the condensing agent is a carbodiimide-based condensing agent, for example DCC, DIC, or EDC, preferably EDC.

[0392] In some embodiments, the molar amount of the condensing agent is about 1-5 times, preferably about 1-3 times, for example about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3 times the molar amount of the compound of formula (F4).

[0393] In some embodiments, the molar amount of the carboxyl protecting agent in the reaction to generate the compound of formula (F) is about 1-5 times, preferably about 1-3 times, for example about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3 times the molar amount of the compound of formula (F4).

[0394] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, isopropyl acetate, n-hexane, n-heptane, petroleum ether, acetone, acetonitrile, toluene, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, DMF, DMA, and DMSO; preferably selected from DCM and DCE, more preferably DCM.

[0395] In some embodiments, the reaction to generate Formula (F4) or Formula (F) is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0396] In one embodiment, the present application provides a compound of Formula (B):

[0397] wherein each variable is as defined herein.

[0398] In some embodiments, the compound of Formula (B) is selected from:

[0399] wherein *2, *3 and *4 are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably, *3 is (S) configuration; preferably, *4 is (S) configuration;

[0400] R D3 as defined herein.

[0401] In some embodiments, the compound of Formula (B) is selected from:

[0402] In one embodiment, the present application provides a compound of Formula (G):

[0403] wherein each variable is as defined herein.

[0404] In some embodiments, the compound of Formula (G) is of Formula (G-a):

[0405] wherein *2 and *3 are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably, *3 is (S) configuration;

[0406] R D3 as defined herein.

[0407] In some embodiments, the compound of Formula (G) is selected from:

[0408] In one embodiment, the present application provides a compound of Formula (C):

[0409] wherein each variable is as defined herein.

[0410] In some embodiments, the compound of formula (C) is selected from:

[0411] preferably preferably

[0412] In one embodiment, the present application provides a compound of formula (C1):

[0413] wherein R D5 as defined herein.

[0414] In some embodiments, the compound of formula (C1) is selected from:

[0415] In one embodiment, the present application provides the following method for preparing a compound of formula (C3-a):

[0416] reacting a compound of formula (C1-a) with a compound of formula (C2) to form a compound of formula (C3-a):

[0417] wherein PG5is an amino protecting group, preferably an acyl-type protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), formyl, acetyl (Ac) and benzoyl (Bz), preferably Ac;

[0418] the remaining variables are as defined herein;

[0419] The method is carried out in the presence of a solvent, which is a mixed solvent of a benzene-based solvent and a phenol-based solvent.

[0420] In some embodiments, the benzene-based solvent is selected from benzene, toluene and xylene, preferably toluene.

[0421] In some embodiments, the phenol-based solvent is selected from phenol, o-cresol, m-cresol and p-cresol, preferably from phenol and o-cresol, preferably o-cresol.

[0422] In some embodiments, the volume ratio of benzene-based solvent:phenol-based solvent is 30-1:1, preferably 25-5:1, preferably 20-10:1, such as 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1.

[0423] In some embodiments, the method is carried out in the presence of a catalyst.

[0424] In some embodiments, the catalyst is an acid catalyst, preferably selected from pyridinium p-toluenesulfonate, p-toluenesulfonic acid (TsOH), camphorsulfonic acid ((-)-CSA), methanesulfonic acid (MsOH), and trifluoromethanesulfonic acid (TfOH), preferably selected from pyridinium p-toluenesulfonate, p-toluenesulfonic acid, and camphorsulfonic acid, preferably is pyridinium p-toluenesulfonate.

[0425] In some embodiments, the molar amount of the catalyst is about 0.1-5 times, preferably about 0.1-2 times, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (C1-a).

[0426] In some embodiments, the molar amount of the compound of formula (C2) is about 0.5-5 times, preferably about 0.5-2 times, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.07, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the molar amount of the compound of formula (C1-a).

[0427] In some embodiments, the method is carried out at a temperature of about 0 °C to about 180 °C; preferably, the temperature is about 50 °C to about 180 °C; preferably, the temperature is about 100 °C to about 150 °C; preferably, the temperature is about 110 °C to about 150 °C; preferably, the temperature is about 135 °C.

[0428] In some embodiments, the method is carried out in a nitrogen atmosphere.

[0429] In one embodiment, the present application provides the following method for preparing a compound of formula (C4-a):

[0430] subjecting a compound of formula (C3-a) to a hydroxyl protection reaction to form a compound of formula (C4-a):

[0431] PG6is a hydroxyl protecting group, preferably selected from a silyl protecting group, an alkyl ether protecting group, or an alkyl methyl ether protecting group; preferably is a silyl protecting group, for example -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; for example trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS), preferably TES;

[0432] the remaining variables are as defined herein.

[0433] In some embodiments, the process is carried out in the presence of a base. Preferably, the base is selected from the group consisting of organic bases and inorganic bases, preferably organic bases.

[0434] In some embodiments, the organic base is selected from the group consisting of alkali and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases.

[0435] In some embodiments, the organic amine base is selected from the group consisting of TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine; preferably, the organic amine base is selected from the group consisting of imidazole, pyridine and 2,6-dimethylpyridine, preferably imidazole.

[0436] In some embodiments, the inorganic base is selected from the group consisting of alkali and alkaline earth metal carbonates, bicarbonates, phosphates, hydrogenophosphates, dihydrogenophosphates, hydroxides or hydrides, for example LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4or NaH.

[0437] In some embodiments, the base is used in a molar amount of about 1-30 times, preferably about 1-20 times, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times the molar amount of the compound of formula (C3-a).

[0438] In some embodiments, the process is carried out in the presence of a catalyst; preferably, the catalyst is 4-dimethylaminopyridine.

[0439] In some embodiments, the catalyst is used in a molar amount of about 0.1-5 times, preferably about 0.1-2 times, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times the molar amount of the compound of formula (C3-a).

[0440] In some embodiments, the hydroxyl protecting agent is used in a molar amount of about 1-30 times, preferably about 1-20 times, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times the molar amount of the compound of formula (C3-a).

[0441] In some embodiments, the process is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, THF, DMF, DMA, DMSO, NMP, benzene, toluene, xylene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, from DMF, DMA, DMSO, NMP, toluene, 1,4-dioxane, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, the solvent is selected from DMF and DMA, preferably DMF.

[0442] In some embodiments, the process is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0443] In some embodiments, the process is carried out under a nitrogen atmosphere. In one embodiment, the present application provides the following process for preparing a compound of formula (C6-a):

[0444] deprotecting a compound of formula (C4-a) to form a compound of formula (C5-a), followed by an amino protection reaction to form a compound of formula (C6-a):

[0445] wherein PG7is an amino protecting group, preferably an alkoxycarbonyl protecting group, such as benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl, or ethoxycarbonyl; preferably Boc;

[0446] the remaining variables are as defined herein.

[0447] In some embodiments, the deprotection reaction is carried out in the presence of a base. Preferably, the base is selected from organic and inorganic bases, preferably organic bases.

[0448] In some embodiments, the organic base is selected from alkali and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases.

[0449] In some embodiments, the organic amine base is selected from TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine, and 2,2,6,6-tetramethylguanidine; preferably, the organic amine base is selected from imidazole, pyridine, and 2,6-dimethylpyridine, preferably 2,6-dimethylpyridine.

[0450] In some embodiments, wherein the inorganic base is selected from a carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydroxide or hydride of an alkali metal and an alkaline earth metal, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, or NaH.

[0451] In some embodiments, wherein the molar amount of the base is about 1-30 times, preferably about 1-20 times, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times the molar amount of the compound of formula (C4-a).

[0452] In some embodiments, wherein the deprotection reaction is carried out in the presence of an acidic compound; preferably, the acidic compound is oxalyl chloride.

[0453] In some embodiments, wherein the molar amount of the acidic compound is about 1-20 times, preferably about 1-10 times, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the molar amount of the compound of formula (C4-a).

[0454] In some embodiments, wherein the deprotection reaction is carried out in the presence of an alcoholic compound; preferably, the alcoholic compound is selected from C 1-12 alkyl alcohol compounds, preferably C 1-6 alkyl alcohol compounds, for example, ethylene glycol or glycerol, preferably ethylene glycol.

[0455] In some embodiments, wherein the deprotection reaction is carried out in the presence of a solvent; preferably, the solvent is an ether solvent; preferably, the ether solvent is selected from tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, ethylene glycol monomethyl ether, and ethylene glycol dimethyl ether; preferably, selected from tetrahydrofuran, 2-methyltetrahydrofuran, and 1,4-dioxane, preferably tetrahydrofuran.

[0456] In some embodiments, wherein the deprotection reaction is carried out at a temperature of about -20 °C to about 30 °C; preferably, the temperature is about -20 °C to about 10 °C; preferably, the temperature is about -10 °C to about 10 °C; preferably, the temperature is about 0 °C.

[0457] In some embodiments, wherein the reaction is carried out at a temperature of about -20 °C to about 100 °C when an alcoholic compound is involved in the reaction; preferably, the temperature is about 0 °C to about 80 °C; preferably, the temperature is about 20 °C to about 60 °C; preferably, the temperature is about 40 °C.

[0458] In some embodiments, the deprotection reaction is carried out in an atmosphere of nitrogen.

[0459] In some embodiments, the amino protection reaction is carried out in the presence of a base. Preferably, the base is selected from the group consisting of organic bases and inorganic bases, preferably organic bases.

[0460] In some embodiments, the organic base is selected from the group consisting of alkali and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases.

[0461] In some embodiments, the organic amine base is selected from the group consisting of TEA, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine; preferably, the organic amine base is selected from the group consisting of TEA, DIPEA and NMM, preferably TEA.

[0462] In some embodiments, the base is used in a molar amount of about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, for example about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (C5-a).

[0463] In some embodiments, the amino protection agent is used in a molar amount of about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, for example about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (F4).

[0464] In some embodiments, the amino protection reaction is carried out in the presence of a solvent; preferably, the solvent is selected from the group consisting of DCM, DCE, ethyl acetate, methyl acetate, isopropyl acetate, n-hexane, n-heptane, petroleum ether, acetone, acetonitrile, toluene, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, DMF, DMA and DMSO; preferably, the solvent is selected from the group consisting of DCM and DCE, more preferably DCM.

[0465] In some embodiments, the amino protection reaction is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0466] In some embodiments, the method further comprises the step of:

[0467] deprotecting the compound of formula (C6-a) to form a compound of formula (C-a): In some embodiments, the method further comprises the step of:

[0468] wherein the variables are as defined herein.

[0469] In some embodiments, the method is carried out in the presence of an acid; preferably the acid is selected from HC1, H2S04, p-toluenesulfonic acid, benzene sulfonic acid, formic acid, acetic acid and trifluoroacetic acid; preferably from acetic acid and trifluoroacetic acid, preferably trifluoroacetic acid.

[0470] In some embodiments, the method is carried out in the presence of a solvent; preferably the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, ethanol, isopropanol and water; preferably from DCM and DCE, more preferably DCM.

[0471] In some embodiments, the volume ratio of solvent: trifluoroacetic acid is about 1 : (0.1 -20), preferably about 1 : (0.5-10), for example about 1 :0.5, 1 :1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9 or 1 :10.

[0472] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably the temperature is about 0 °C to about 50 °C; preferably the temperature is about 0 °C to room temperature; preferably the temperature is about 25 °C.

[0473] In one embodiment, the present application provides a compound of formula (C7):

[0474] wherein R D5 as defined herein.

[0475] In some embodiments, the compound of formula (C7) is selected from:

[0476] In one embodiment, the present application provides the following method for preparing a compound of formula (C7):

[0477] reacting a compound of formula (C8) with an oxidizing agent to form a compound of formula (C7):

[0478] wherein PG9is a carboxyl protecting group, for example C 1-10 alkyl, preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl, preferably ethyl;

[0479] R4is selected from C 1-9 alkyl, preferably C 1-5 alkyl, preferably C 1-3 alkyl, for example methyl;

[0480] R D5 as defined herein.

[0481] In some embodiments, wherein the oxidizing agent is selected from the group consisting of oxygen, sodium dichromate, potassium dichromate, and potassium permanganate; preferably, the oxidizing agent is oxygen. In some embodiments, the method is carried out in an oxygen atmosphere.

[0482] In some embodiments, wherein the method is carried out in the presence of a base; preferably, wherein the base is selected from the group consisting of an organic base and an inorganic base, preferably an inorganic base.

[0483] In some embodiments, wherein the inorganic base is selected from the group consisting of a carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydroxide, or hydride of an alkali metal and an alkaline earth metal, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, or NaH; preferably, a carbonate or bicarbonate of an alkali metal and an alkaline earth metal, for example, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, or KHCO3; preferably, selected from the group consisting of Na2CO3and K2CO3, preferably K2CO3.

[0484] In some embodiments, wherein the base is used in a molar amount of about 0.5-5 times, preferably about 0.5-3 times, for example, about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, or 3 times, of the molar amount of the compound of formula (C8).

[0485] In some embodiments, wherein the method is carried out in the presence of a solvent; preferably, the solvent is an alcoholic solvent; preferably, the alcoholic solvent is selected from the group consisting of methanol, ethanol, isobutanol, tert-butanol, isopropanol, n-propanol, n-pentanol, and iso-pentanol, preferably methanol.

[0486] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0487] In one embodiment, the present application also provides the following method for preparing a compound of formula (C8):

[0488] heating a compound of formula (C9) to form a compound of formula (C8):

[0489] wherein each variable is as defined herein.

[0490] In some embodiments, the temperature of the heating is about 40 °C to about 100 °C; preferably, the temperature is about 60 °C to about 90 °C; preferably, the temperature is about 75 °C.

[0491] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, THF, DMF, DMA, DMSO, NMP, benzene, toluene, xylene, 1,4-dioxane, ethylene glycol monomethyl ether and ethylene glycol dimethyl ether, preferably selected from benzene, toluene and xylene, preferably toluene.

[0492] In one embodiment, the present application also provides the following method for preparing a compound of formula (C9):

[0493] reacting a compound of formula (C10) with nitrous acid and / or a nitrite salt to form a compound of formula (C9):

[0494] wherein each variable is as defined herein.

[0495] In some embodiments, the nitrite salt is selected from sodium nitrite, potassium nitrite and ammonium nitrite, preferably sodium nitrite.

[0496] In some embodiments, the molar amount of the nitrous acid and / or the nitrite salt is about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, for example about 1, 1.5, 2, 2.5, 3, 3.33, 3.5, 4, 4.5, 5 times of the molar amount of the compound of formula (C10).

[0497] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from an acidic solvent; preferably, the solvent is selected from a mixed solvent of R4-C(O)OH and R4-C(O)OC(O)-R4; preferably, the volume ratio of R4-C(O)OH:R4-C(O)OC(O)-R4 in the mixed solvent is about 1:(0.1-20), preferably about 1:(0.5-10), for example about 1:0.5, 1:1, 1:2, 1:3, 1:3.04, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0498] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0499] In some embodiments, the method is carried out in a nitrogen atmosphere.

[0500] In one embodiment, the present application provides the following method for preparing a compound of formula (C10):

[0501] reacting a compound of formula (C11) with a compound R4-C(O)OC(O)-R4 to form a compound of formula (C10):

[0502] wherein each variable is as defined herein.

[0503] In some embodiments, the method is carried out in the presence of a catalyst; preferably, the catalyst is selected from transition metal catalysts, preferably a nickel catalyst, such as Raney nickel, nickel acetylacetonate, nickel acetate, nickel chloride, nickel bromide or nickel dichlorobis(triphenylphosphine); preferably, the catalyst is Raney nickel.

[0504] In some embodiments, the catalyst is used in a molar amount of about 0.1-10 times, preferably about 0.5-5 times, such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.28, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (C11).

[0505] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from acidic solvents; preferably, the solvent is selected from a mixture of R4-C(O)OH and R4-C(O)OC(O)-R4; preferably, the volume ratio of R4-C(O)OH:R4-C(O)OC(O)-R4 in the mixture is about 1:(0.1-20), preferably about 1:(0.5-10), such as about 1:0.5, 1:1, 1:2, 1:3, 1:3.04, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0506] In some embodiments, the method is carried out at a temperature of about 30°C to about 100°C; preferably, the temperature is about 50°C to about 80°C; preferably, the temperature is about 65°C.

[0507] In some embodiments, the method is carried out in an atmosphere of hydrogen.

[0508] In one embodiment, the present application also provides the following method for preparing a compound of formula (C11):

[0509] reacting a compound of formula (C12) with a compound R4-C(O)OC(O)-R4 to form a compound of formula (C11): D5 -X to form a compound of formula (C11):

[0510] wherein X is selected from halogen, such as F, CI, Br or I, preferably Br;

[0511] the remaining variables are as defined herein.

[0512] In some embodiments, the method is carried out in the presence of a base; preferably, wherein the base is selected from an organic base and an inorganic base, preferably an inorganic base.

[0513] In some embodiments, wherein the inorganic base is selected from a carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydroxide or hydride of an alkali metal and an alkaline earth metal, such as LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4, or NaH; preferably from a carbonate or bicarbonate of an alkali metal and an alkaline earth metal, such as Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, or KHCO3; preferably from Na2CO3and K2CO3, preferably K2CO3.

[0514] In some embodiments, wherein the molar amount of the base is about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (C12).

[0515] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, isopropyl acetate, n-hexane, n-heptane, petroleum ether, acetone, acetonitrile, toluene, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, DMF, DMA, DMSO, NMP, methanol, ethanol, isobutanol, tert-butanol, isopropanol, n-propanol, n-pentanol, and iso-pentanol; preferably, the solvent is selected from DCM, DCE, acetonitrile, tetrahydrofuran, DMF, DMA, and NMP; preferably, the solvent is acetonitrile.

[0516] In some embodiments, wherein the compound R D5 the molar amount of X is about 1-10 times, preferably about 1-8 times, preferably about 1-5 times, such as about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 times the molar amount of the compound of formula (C12).

[0517] In some embodiments, the method is carried out at a temperature of about 40 °C to about 120 °C; preferably, the temperature is about 60 °C to about 100 °C; preferably, the temperature is about 80 °C.

[0518] In one embodiment, the present application also provides the following method for preparing a compound of formula (C1):

[0519] deprotecting a compound of formula (C7) to form a compound of formula (C1):

[0520] wherein R D5 as defined herein.

[0521] In some embodiments, the method is carried out in the presence of an acid; preferably, the acid is selected from HC1, H2SO4, p-toluenesulfonic acid, benzene sulfonic acid, formic acid, acetic acid and trifluoroacetic acid; preferably selected from acetic acid and trifluoroacetic acid, preferably trifluoroacetic acid.

[0522] In some embodiments, the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, ethyl acetate, methyl acetate, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, ethanol, isopropanol and water; preferably selected from methanol, ethanol, isopropanol and water, preferably water.

[0523] In some embodiments, the volume ratio of the solvent:trifluoroacetic acid is about 1:(0.1-20), preferably about 1:(0.5-10), for example about 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0524] In some embodiments, the method is carried out at a temperature of about 0 °C to about 80 °C; preferably, the temperature is about 0 °C to about 50 °C; preferably, the temperature is about 0 °C to room temperature; preferably, the temperature is about 25 °C.

[0525] In some embodiments, the compound of formula (C7) is selected from:

[0526] In one embodiment, the reaction to form a compound of formula (C1) is deprotecting a compound of formula (C7-a) to form a compound of formula (C1-a):

[0527] wherein R D5 as defined herein.

[0528] In one embodiment, the present application provides the following method for preparing a compound of formula (D):

[0529] reacting a compound of formula (F) with a compound of formula (J) to form a compound of formula (D):

[0530] A'1is an optionally substituted natural amino acid monovalent residue or an optionally substituted non-natural amino acid monovalent residue, preferably a monovalent residue selected from gly, val, cit, gin, glu, phe, lys, leu, or ala, preferably a monovalent residue of gly, optionally substituted with 1, 2, or 3 R y substituted;

[0531] each R y is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy; preferably selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl;

[0532] A'1carboxy terminus is attached to A2, and the amino group is involved in the reaction;

[0533] the other variables are as defined herein.

[0534] In one embodiment, the present application provides a compound of formula (D):

[0535] T-A1-A2-A3-A'4

[0536] D

[0537] wherein the variables are as defined herein.

[0538] In some embodiments, the compound of formula (D) is selected from:

[0539] In one embodiment, the present application provides the following method of making a compound of formula (E):

[0540] reacting a compound of formula (E1) with a compound of formula (C) to form a compound of formula (E'), and deprotecting the compound of formula (E') to form a compound of formula (E):

[0541] wherein A5and PG3are as defined herein; the remaining variables are as defined herein.

[0542] In one embodiment, the present application provides a compound of formula (E):

[0543] wherein the variables are as defined in any one of the present application.

[0544] In some embodiments, the compound of formula (E) is selected from:

[0545] In one embodiment, the present application provides a method for preparing an antibody drug conjugate (ADC), comprising conjugating a compound of formula (A) to an antibody; the compound of formula (A) is as defined herein. BRIEF DESCRIPTION OF DRAWINGS

[0546] Figure 1 is the test result of the growth inhibition of JIMT-1 mouse subcutaneous xenograft model in Test Example 1;

[0547] Figure 2 is the summary result of the body weight change of the experimental mice in Test Example 1;

[0548] Figure 3 is the test result of the growth inhibition of NCI-N87 xenograft in nude mice in Test Example 2;

[0549] Figure 4 is the summary result of the body weight change of the experimental mice in Test Example 2.

[0550] EXAMPLE

[0551] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The chemical shift δ is given in 10-6 (ppm) units. The NMR determination is carried out using a Bruker nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0552] The LCMS determination is carried out using an Agilent 1260 Infinity II (ESI) mass spectrometer, a Waters UPLC H Class plus (ESI), or a Shimadzu LCMS-2020 (ESI).

[0553] The high-performance liquid chromatography (HPLC) analysis uses an Agilent 1260 or a Shimadzu LC-20AD.

[0554] The preparative high-performance liquid chromatography (pre-HPLC) uses a GILSON GX-281 or an Agilent 1260 Infinity II preparative liquid.

[0555] The chiral preparation uses critical fluid chromatography (SFC), and the instrument uses a Shimadzu LC-30Adsf or a Shimadzu LC-20AD.

[0556] Thin layer chromatography silica gel plate uses GF254 acrylate adhesive silica gel plate of Anhui Liangchen Silicon Source Material Co., Ltd., and the silica gel plate used in thin layer chromatography has a specification of 0.2 mm silica gel plate, and the specification of the silica gel plate used in thin layer chromatography separation and purification of a product is 0.5 mm silica gel plate.

[0557] Column chromatography generally uses 200-300 mesh silica gel of Anhui Liangchen Silicon Source Material Co., Ltd. as a carrier.

[0558] The determination of the average inhibition rate of the kinase and the IC50 value is performed by using a SpectraMax i3X enzyme marker (MD Company, USA).

[0559] Known starting materials of the present disclosure can be synthesized according to methods known in the art or purchased from companies such as Bid pharmaceutical, Leyan, Shaoyuan Chemical Technology, and Anning Chemicals.

[0560] In the following examples, unless otherwise specified, the reactions are carried out under an argon atmosphere or a nitrogen atmosphere.

[0561] The argon atmosphere or the nitrogen atmosphere refers to that a reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.

[0562] The hydrogen atmosphere refers to that a reaction bottle is connected to a hydrogen balloon with a volume of about 1 L.

[0563] The hydrogenation reaction is generally performed by repeatedly operating 3 times of vacuumizing and filling hydrogen.

[0564] The oxygen atmosphere refers to that a reaction bottle is connected to an oxygen balloon with a volume of about 1 L.

[0565] In the following examples, unless otherwise specified, the solution refers to an aqueous solution, and the reaction temperature is room temperature, which is 20-30°C.

[0566] Example 1: Preparation of compounds 1-A & 1-B

[0567] (S)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1-A

[0568] (R)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H-spiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione 1-B

[0569] Step one: synthesis of 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid ethyl ester (1c)

[0570] To a mixture of 1a (2 kg, 12.6 mol) and triethyl orthoformate (2.81 kg, 19.0 mol) in ethanol (1 L) was added p-toluenesulfonic acid monohydrate (48.1 g, 253 mmol) and stirred at 25 °C for 5 hours, then a solution of 1b (1.06 kg, 12.6 mol) in acetonitrile (12 L) and potassium carbonate (1.92 kg, 13.9 mol) were added. The resulting mixture was stirred at 85 °C for 12 hours. After the reaction solution was cooled to 15 °C, diluted with 15 L of water, then slowly added concentrated hydrochloric acid to adjust pH to 3, at this time a large amount of solid precipitated, kept stirring at 15-20 °C for 1 hour. The solid was collected by filtration, washed with water (1 L*3), dried in a hot air oven at 60 °C for 12 hours to obtain compound 1c (1.80 kg, 69.2% yield) as a yellow solid.

[0571] MS m / z (ESI): 207.0 [M+1].

[0572] Step two: synthesis of 6-cyano-1-hydroxy-7-methyl-5-oxo-3,5-dihydro-1H-indolizine-2- carboxylic acid methyl ester (1e)

[0573] To a solution of 1c (800 g, 3.94 mol) in dimethyl sulfoxide (8 L) was added potassium carbonate (532 g, 3.94 mol) and 1d (1.64 g, 19.0 mol). The mixture was stirred at 75 °C for 12 hours. After the reaction solution was cooled to 20 °C, diluted with 10 L of water, then the pH was adjusted to 2-3 with concentrated hydrochloric acid, at this time a large amount of solid precipitated. The solid was collected by filtration, washed with water (800 mL*3), then dried in a hot air oven at 60 °C for 12 hours to obtain compound 1e (624 g, 64.1% yield) as a yellow solid.

[0574] MS m / z (ESI): 247.0 [M+1].

[0575] Step three: synthesis of 7-methyl-1,5-dioxo-1,2,3,5-tetrahydro-1H-indolizine-6-carbonitrile (1f)

[0576] To a solution of 1e (600 g, 386 mmol) in acetic acid (4 L) was added concentrated hydrochloric acid (4 L), the resulting mixture was stirred at 100 °C for 4 hours. Then the reaction solution was cooled to 15 °C, concentrated under reduced pressure to obtain compound 1f (414 g, 83.9% yield) as a black-brown solid.

[0577] MS m / z (ESI): 188.9 [M+1].

[0578] Step four: synthesis of 7-methyl-5-oxo-2,3-dihydro-5H-spiro[oxindole-l,2'- [l,3]dioxolane]-6-carbonitrile (1g)

[0579] To a solution of 1f (372 g, 1.74 mol) in dichloromethane (1 L) was added trimethylsilyl chloride (850 g, 7.83 mol) and ethylene glycol (324 g, 5.22 mol). The resulting mixture was stirred at 35 °C for 6 hours, then the reaction solution was cooled to 15 °C and concentrated in vacuo to give compound 1g (332 g, 81.5% yield) as a brown solid.

[0580] MS m / z (ESI): 233.0 [M+1].

[0581] Step five: synthesis of ethyl 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[oxindole-l,2'- [l,3]dioxolane]-7-yl)acetate (1h)

[0582] To a mixture of 1g (100 g, 427 mmol) and sodium ethoxide (116 g, 1.71 mol) in tetrahydrofuran (1 L) was added dropwise diethyl carbonate (177 g, 1.50 mol) at 15 °C under nitrogen atmosphere, the resulting mixture was warmed to 60 °C and stirred for 12 hours. The reaction solution was cooled to below 20 °C and slowly poured into 4 mol / L aqueous hydrochloric acid solution (2.2 L). The resulting solution was then concentrated in vacuo to remove tetrahydrofuran therein, at this time a large amount of solid was precipitated, which was filtered, and the filter cake was washed with water (300 mL). The filter cake was collected and added to methanol (2 L), then stirred and slurried at 25 °C for 12 hours, and the solid was collected by filtration, washed with methanol (200 mL), and then dried in a hot air oven at 40 °C for 2 hours to give compound 1h (92.4 g, 68.1% yield) as a brown solid.

[0583] MS m / z (ESI): 304.9 [M+1].

[0584] Step six: synthesis of ethyl 2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[oxindole-l,2'- [l,3]dioxolane]-7-yl)-3-cyclopropylpropanoate (1j)

[0585] To a solution of 1h (100 g, 311 mmol) in acetonitrile (1 L) was added potassium carbonate (86.1 g, 623 mmol) and 1i (84.1 g, 623 mmol). The resulting mixture was stirred at 80 °C for 12 h. The reaction solution was cooled to below 25 °C, then filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was diluted with dichloromethane (2 L), washed with saturated brine (1 L), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting solid was added to petroleum ether / ethyl acetate (3 / 1, v / v, 1 L), stirred and slurried at 25 °C for 12 h, the solid was collected by filtration, and then dried in a hot air oven at 40 °C for 2 h to give compound 1j (109.3 g, 91.6% yield) as a brown solid.

[0586] MS m / z (ESI): 359.0 [M+1].

[0587] Step Seven: Synthesis of 2-(6-(acetylamino methyl)-5-oxo-2,3-dihydro-5H-spiro[oxindole-1,2'- [1,3]dioxolane]-7-yl)-3-cyclopropylpropanoic acid ethyl ester (1k)

[0588] To a solution of 1j (100 g, 274 mmol) in acetic acid (230 mL) and acetic anhydride (700 mL) was added Raney nickel (30.0 g, 350 mmol) at 25 °C under nitrogen atmosphere. The resulting suspension was degassed and purged with hydrogen gas for 3 times, then the mixture was stirred at 65 °C under hydrogen gas (2.0 MPa) for 12 h. The reaction solution was cooled to below 25 °C, then filtered, and the filtrate was concentrated under reduced pressure. The resulting solid was added to methyl tert-butyl ether (2 L), stirred and slurried at 25 °C for 3 h, the solid was collected by filtration, and then dried in a hot air oven at 40 °C for 2 h to give compound 1k (94.1 g, 83.4% yield) as a yellow solid.

[0589] MS m / z (ESI): 405.2 [M+1].

[0590] Step Eight: Synthesis of 3-cyclopropyl-2-(6-((N-nitrosoacetamido)methyl)-5-oxo-2,3-dihydro-5H- spiro[oxindole-1,2'-[1,3]dioxolane]-7-yl)propanoic acid ethyl ester (1l)

[0591] To a mixture of 1k (100 g, 244 mmol) in acetic acid (230 mL) and acetic anhydride (700 mL) was added sodium nitrite (56.1 g, 813 mmol) portionwise slowly at 0 °C under nitrogen atmosphere. The resulting mixture was stirred at 25 °C for 3 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 1l (111 g, crude) as a yellow oil.

[0592] MS m / z (ESI): 434.1 [M+1].

[0593] Step Nine: Synthesis of 2-(6-(acetyloxymethyl)-5-oxo-2,3-dihydro-5H-spiro[oxindole- 1,2'-[1,3]dioxolane]-7-yl)-3-cyclopropylpropanoic acid ethyl ester (1m)

[0594] A mixture of 1 1 (111 g, crude) in toluene (1 L) was stirred at 75 °C for 3 hours. After the reaction solution was cooled to below 25 °C, it was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (Si02, dichloromethane / methanol = 1 / 0-20 / 1, v / v) to give compound 1m (86.5 g, 86.3% yield) as a yellow solid.

[0595] MS m / z (ESI): 406.0 [M+1].

[0596] Step Ten: Synthesis of 4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10H- spiro[pyrano[3,4-f]oxindole-6,2'-[1,3]dioxolane]-3,10-dione (1)

[0597] To a mixture of 1m (32.0 g, 77.7 mmol) in methanol (420 mL) was added potassium carbonate (12.9 g, 93.2 mmol) at 25 °C under oxygen atmosphere. The resulting mixture was stirred at 25 °C under oxygen atmosphere for 2 hours. The reaction solution was adjusted to pH to 3 with 1 mol / L aqueous hydrochloric acid solution, and then extracted with dichloromethane (500 mL x 2). The combined organic phase was washed with saturated brine (400 mL x 2), and then the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting solid was added into methyl tert-butyl ether / dichloromethane (1 / 1, 200 mL), stirred and slurried at 25 °C for 1 hour, the solid was collected by filtration, and then dried in a hot air oven at 40 °C for 1 hour to give compound 1 (21.2 g, 80.7% yield) as a white solid.

[0598] MS m / z (ESI): 333.9 [M+1].

[0599] 1H NMR (400 MHz, DMSO-d6) δ 6.51 (s, 1H), 6.42 (s, 1H), 5.22-5.31 (m, 2H), 4.08-4.14 (m, 4H), 3.97 (t, J = 6.8 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 1.64-1.75 (m, 2H), 0.69-0.73 (m, 1H), 0.31-0.35 (m, 2H), 0.00-0.01 (m, 1H), -0.10--0.08 (m, 1H).

[0600] Step eleven: Synthesis of (S)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8- tetrahydro-3H,10Hspiro[pyrano[3,4-f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione (1-A) & (R)-4-(cyclopropylmethyl)-4-hydroxy-1,4,7,8-tetrahydro-3H,10Hspiro[pyrano[3,4- f]indolizine-6,2'-[1,3]dioxolane]-3,10-dione (1-B)

[0601] Compound 1 (252 g, 756 mmol) was purified by preparative SFC (column: DAICEL CHIRALCEL OX (250 mm*50 mm, 10 pm); mobile phase: [A: CO2-CAN, B: MeOH (0.1% NH3-H2O)]; B%: 45%, isocratic elution mode) to give Compound 1-A (117 g, 41.5% yield) as a white solid and Compound 1-B (86.7 g, 30.7% yield) as a white solid.

[0602] Single configuration Compound 1-A

[0603] SFC analysis: Retention time 1.392 min. (Chromatographic column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-Carbon dioxide, B-Methanol (0.05% diethylamine), gradient elution: B%: 5-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0604] MS m / z (ESI): 334.0 [M+1].

[0605] Single configuration Compound 1-B

[0606] SFC analysis: Retention time 1.762 min. (Chromatographic column: Chiralpak AS-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A - Carbon dioxide, B - Methanol (0.05% Diethylamine), Gradient elution: B%: 5% - 40%, Flow rate: 3 mL / min, Instrument: Shimadzu LC-30ADsf).

[0607] MS m / z (ESI): 334.0 [M+1].

[0608] Example 2: Preparation of compound 2

[0609] (1S,9S)-1 -amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-10,13-dione trifluoroacetic acid salt 2

[0610] Step one: Synthesis of (S)-4-(cyclopropylmethyl)-4-hydroxy-7,8-dihydro-1H- pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2a)

[0611] Dissolve 1-A (20.0 g, 56.41 mmol) in a mixture solution of trifluoroacetic acid / water (3 / 1, v / v, 80 mL) and stir at 25 °C for 3 hours. Concentrate the reaction solution under reduced pressure, stir the obtained residue with methyl tert-butyl ether (200 mL) at 55 °C for 2 hours, then cool to room temperature, filter, collect the solid and dry to give compound 2a (16.2 g, 95% yield) as a white solid.

[0612] MS m / z (ESI): 290.1 [M+1].

[0613] Step two: Synthesis of N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1 -yl)acetamide (2c)

[0614] To a solution of 2a (15.0 g, 51.85 mmol), 2b (13.9 g, 55.54 mmol) and pyridinium p-toluenesulfonate (3.9 g, 15.52 mmol) in a mixture of toluene / o-cresol (15 / 1, v / v, 375 mL) was added, the resulting mixture was stirred at 135 °C under nitrogen atmosphere for 24 h. After the reaction mixture was cooled to room temperature, it was filtered, the filter cake was washed with cold toluene (100 mL), the solid was collected and dried to give compound 2c (22.2 g, 95% purity, 81% yield) as a brown solid.

[0615] MS m / z (ESI): 504.3 [M+1].

[0616] 1 H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.37 (s, 1H), 6.59 (s, 1H), 5.63 - 5.49 (m, 1H), 5.49 - 5.33 (m, 2H), 5.31 - 5.11 (m, 2H), 3.26 - 3.10 (m, 2H), 2.40 (s, 3H), 2.17 - 2.10 (m, 2H), 1.91 (s, 3H), 1.86 - 1.71 (m, 2H), 0.86 - 0.76 (m, 1H), 0.43 - 0.24 (m, 2H), 0.11 - 0.02 (m, 1H), -0.04 - -0.14 (m, 1H).

[0617] Step Three: Synthesis of N-((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-4-methyl-10,13- dioxo-9-((triethylsilyl)oxy)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)acetamide (2d)

[0618] Compound 2c (37.5 g, 72.32 mmol), imidazole (59.0 g, 867.76 mmol) and 4-dimethylaminopyridine (8.82 g, 72.32 mmol) were dissolved in N,N-dimethylformamide (600 mL), and triethylchlorosilane (109 g, 723.16 mmol) was slowly added dropwise at 25 °C under a nitrogen atmosphere. After the addition was completed, the reaction solution was stirred at 25 °C for 16 hours. The reaction solution was slowly poured into water (4 L), and then extracted with ethyl acetate (3 L x 2). The combined organic phase was washed with saturated brine (2 L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (mobile phase: A-dichloromethane, B-methanol; isocratic elution: B%: 4%), to obtain compound 2d (37.1 g, 80% yield) as a brownish foamy solid.

[0619] MS m / z (ESI): 618.4 [M+1].

[0620] Step four: Synthesis of (1S,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-4-methyl-9- ((triethylsilyl)oxy)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-10,13-dione (2e)

[0621] Compound 2d (15.0 g, 24.07 mmol) was dissolved in dry tetrahydrofuran (375 mL), and 2,6-dimethylpyridine (25.8 g, 240.68 mmol) was added. The reaction system was cooled to below 0 °C under a nitrogen atmosphere, and oxalyl chloride (15.3 g, 120.34 mmol) was slowly added dropwise, and stirred at 0 °C for 30 minutes. Then, dry ethylene glycol (29.8 g, 481.36 mmol) was slowly added, and the reaction solution was warmed to 40 °C and stirred for 30 minutes. After removing part of the tetrahydrofuran by concentrating under reduced pressure, water (300 mL) was added to the reaction solution, and extracted with dichloromethane / isopropanol (10 / 1, v / v, 300 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude compound 2e (14.2 g) was obtained as a brownish oil.

[0622] MS m / z (ESI): 576.4 [M+1].

[0623] Step five: Synthesis of tert-butyl ((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-4-methyl- 10, 13-dioxo-9-((triethylsilyl)oxy)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (2f)

[0624] Dissolve 2e (14.2 g, crude) in dichloromethane (300 mL), add triethylamine (7.36 g, 72.20 mmol) and di-tert-butyl dicarbonate (15.7 g, 72.20 mmol) successively, and stir the mixture at 25 °C for 16 hours. Wash the reaction solution with 10% aqueous citric acid (150 mL) and saturated brine (100 mL) successively, dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the obtained residue by silica gel column chromatography (mobile phase: A - petroleum ether, B - ethyl acetate; gradient elution: B%: 10% - 50%), to obtain compound 2f (14.1 g, 85% yield) in the form of a white solid.

[0625] MS m / z (ESI): 676.5 [M+1].

[0626] Step six: Synthesis of (1S,9S)-1-amino-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-10,13-dione (2)

[0627] Dissolve 2f (14.1 g, 24.19 mmol) in dichloromethane (70 mL), and add trifluoroacetic acid (70 mL). Stir the mixture at 25 °C for 16 hours. Concentrate the reaction solution under reduced pressure, add the obtained residue into acetonitrile / water (10 / 1, v / v, 1 L), stir and slurry at 60 °C under nitrogen atmosphere for 16 hours, then cool to room temperature naturally, filter, collect the solid, and dry, to obtain compound 2 (9.11 g, 99.1% purity, 64% yield) in the form of a white solid.

[0628] MS m / z (ESI): 462.2 [M+1].

[0629] 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 2H), 7.88 (d, J = 10.8 Hz, 1H), 7.41 (s, 1H), 6.63 (s, 1H), 5.71 (d, J = 19.3 Hz, 1H), 5.51 - 5.35 (m, 3H), 5.11 (s, 1H), 3.37 - 3.23 (m, 2H), 3.20 - 3.05 (m, 1H), 2.42 (s, 3H), 2.28 - 2.12 (m, 1H), 1.92 - 1.70 (m, 2H), 0.90 - 0.74 (m, 1H), 0.42 - 0.24 (m, 2H), 0.11 - 0.02 (m, 1H), -0.03 - -0.13 (m, 1H).

[0630] Example 3: Preparation of compound 3

[0631] (2R,10S)-16-amino-10-benzyl-2-methyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14- tetraazahexadecanoic acid 3

[0632] Step one: Synthesis of (benzyloxy)carbonylglycyl-L-phenylalanine tert-butyl ester (3c)

[0633] Step one: Synthesis of (benzyloxy)carbonylglycyl-L-phenylalanine tert-butyl ester (3c)

[0634] MS m / z (ESI): 414.2 [M+1-56]

[0635] Step two: Synthesis of (benzyloxy)carbonylglycylglycyl-L-phenylalanine (3d)

[0636] To a solution of 3c (7.81 g, 16.61 mmol) in dichloromethane (40 mL) was added trifluoroacetic acid (40 mL) and the reaction was stirred at room temperature for 1 h. The reaction was concentrated under reduced pressure and the resulting residue was taken up in methyl tert-butyl ether (100 mL) and stirred at 60 °C under a nitrogen atmosphere for 16 h. The mixture was allowed to cool to room temperature and filtered to collect the solid which was dried to give compound 3d (5.53 g, 80% yield) as a white solid.

[0637] MS m / z (ESI): 414.2 [M+1]

[0638] Step Three: Synthesis of methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (3f)

[0639] To a solution of 3e (15.0 g, 42.32 mmol) in tetrahydrofuran (225 mL) and acetic acid (45 mL) was added lead tetraacetate (30.1 g, 67.73 mmol) and the resulting mixture was stirred at reflux for 2 h. After cooling to room temperature, the insoluble material was filtered off and the filter cake was washed with ethyl acetate (75 mL). The washings were combined with the filtrate and the resulting solution was washed with 20 (w / v) % aqueous trisodium citrate dihydrate (75 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was taken up in acetonitrile (200 mL) and stirred at 60 °C under a nitrogen atmosphere for 16 h. The mixture was allowed to cool to room temperature and filtered to collect the solid which was dried to give compound 3f (10.4 g, 67% yield) as a white solid.

[0640] MS m / z (ESI): 369.2 [M+1]

[0641] Step Four: Synthesis of (benzyl (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazoundecan-11-oate (3h)

[0642] To a solution of 3f (10.4 g, 28.23 mmol) in ethyleneglycol dimethyl ether (150 mL) was added 3g (10.2 g, 56.46 mmol), and the resulting mixture was cooled to 0-5 °C. To it was added sodium hydroxide aqueous solution (10 mol / L, 2.77 mL, 27.66 mmol), and the resulting mixture was kept stirring at 0-5 °C for 1 hour. To the reaction was added 10 (w / v) % ammonium chloride aqueous solution (200 mL), then extracted with ethyl acetate (150 mL x 2), the combined organic phase was washed with saturated brine (150 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 30%-80%), to give compound 3h (8.91 g, 64% yield) as a white solid.

[0643] MS m / z (ESI): 489.1 [M+1]

[0644] Step five: Synthesis of benzyl (11S,19R)-11-benzyl-19-methyl-3,6,9,12,15-pentaoxo-1- phenyl-2,18-dioxa-4,7,10,13,16-pentaazahenicosan-19-yl acetate (3i)

[0645] To a solution of 3h (8.91 g, 18.24 mmol) in N,N-dimethylformamide (100 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (1.39 g, 9.12 mmol), the reaction was stirred at 25 °C for 1 hour. Then 3d (7.92 g, 19.15 mmol), 1-hydroxybenzotriazole (2.96 g, 21.89 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (3.85 g, 20.06 mmol) were added successively, the resulting mixture was stirred at 25 °C for 2 hours. The reaction was slowly poured into water (600 mL), then extracted with ethyl acetate (400 mL x 2), the combined organic phase was washed with saturated brine (400 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was added into n-heptane / ethyl acetate (1 / 1, 360 mL), stirred and slurried at 70 °C under nitrogen atmosphere for 16 hours, then filtered after natural cooling to room temperature, the solid was collected and oven-dried, to give compound 3i (10.6 g, 87% yield) as a white solid.

[0646] MS m / z (ESI): 662.4 [M+1]

[0647] Step six: synthesis of (2R, 10S)-16-amino-10-benzyl-2-methyl-6,9,12,15- tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (3)

[0648] To a solution of 3i (10.6 g, 16.02 mmol) and 10% palladium on carbon (1.5 g) in a mixture of methanol / tetrahydrofuran / water (1 / 1 / 1, 180 mL) was purged with hydrogen three times and stirred under hydrogen atmosphere at 25 °C for 6 h. The reaction mixture was filtered, the filter cake was rinsed with methanol (60 mL), and the filtrate and wash were combined and concentrated under reduced pressure. The residue was purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 6%) to give compound 3 (6.21 g, 88% yield) as a white solid.

[0649] MS m / z (ESI): 348.1 [M+1]

[0650] 1 H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.71 - 8.59 (m, 2H), 8.40 - 8.29 (m, 1H), 7.33 - 7.12 (m, 5H), 4.74 - 4.65 (m, 1H), 4.55 - 4.48 (m, 1H), 4.47 - 4.38 (m, 1H), 3.88 - 3.76 (m, 3H), 3.71 - 3.64 (m, 1H), 3.51 - 3.40 (m, 3H), 3.11 - 3.04 (m, 1H), 2.90 - 2.82 (m, 1H), 1.18 (d, J = 6.8 Hz, 3H).

[0651] Example 4: Preparation of compound 4

[0652] (13S,24S,32R)-24-benzyl-32-methyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoylamino)-12,16,19,22,25,28-hexaoxo-2,5,8,31-tetraoxa-11,17,20,23,26,29-hexaazatricopper-33-oic acid 4

[0653] Scheme One:

[0654] Step one: synthesis of (S)-tert-butyl 13-((benzyloxy)carbonyl)amino)-12-oxo-2,5,8- trioxa-11-azahexadecan-16-oate (4c)

[0655] To a solution of 4a (10.0 g, 29.64 mmol) and 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.4 g, 32.61 mmol) in dichloromethane (300 mL) was added N,N-diisopropylethylamine (11.5 g, 88.92 mmol) followed by 4b (5.08 g, 31.12 mmol) and the reaction mixture was stirred at 25 °C for 15 h. The reaction mixture was washed with water (150 mL) and the organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-dichloromethane, B-methanol; isocratic elution: B%: 5%) to give compound 4c (11.5 g, 80% yield) as a yellow oil.

[0656] MS m / z (ESI): 483.3 [M+1].

[0657] Step two: Synthesis of (S)-tert-butyl 13-amino-12-oxo-2,5,8-trioxa-11- azahexadecanoate (4d)

[0658] To a solution of 4c (9.60 g, 19.89 mmol) and 10% palladium on carbon (1.70 g) in methanol (150 mL) was added and the reaction mixture was stirred under hydrogen atmosphere at 25 °C for 2 h. The reaction mixture was filtered and the filter cake was washed with methanol (30 mL) and the filtrate and washings were combined and concentrated under reduced pressure. The crude compound 4d (7.05 g) was obtained as a colorless oil.

[0659] MS m / z (ESI): 483.3 [M+1].

[0660] Step three: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)hexyl-5- ynoate (4g)

[0661] To a solution of 4e (10 g, 48.76 mmol), 4f (11.4 g, 58.51 mmol), triethylamine (49.3 g, 487.61), cuprous iodide (928 mg, 4.88 mmol) and [1,1'- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.78 g, 2.44 mmol) in 1,4- dioxane (300 mL) was added and the reaction mixture was stirred at 100 °C under nitrogen atmosphere for 3 h. The reaction mixture was cooled to room temperature and filtered and the filter cake was washed with ethyl acetate (100 mL) and the filtrate and washings were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 10%-40%) to give compound 4g (8.76 g, 71% yield) as a colorless oil.

[0662] MS m / z (ESI): 251.0 [M+1].

[0663] Step four: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)hexyl-5-yne acid (4h)

[0664] A mixture of 4g (8.70mg, 42.42mmol) in tetrahydrofuran / water (3 / 1, v / v, 80mL) was added lithium hydroxide monohydrate (7.12g, 169.69mmol) and the resulting mixture was stirred at 25°C for 2 hours.

[0665] The pH of the reaction solution was adjusted to about 3 by slowly adding 1 mol / L aqueous hydrochloric acid, at which time a large amount of white solid was precipitated, filtered, the filter cake was washed with water (150mL), and the filter cake was collected and dried. Compound 4h (7.21g, 71% yield) was obtained as a white solid.

[0666] MS m / z (ESI): 237.0 [M+1].

[0667] Step five: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-yne acid (4i)

[0668] To a mixture of 4h (7.21g, 30.51mmol) in tetrahydrofuran / water (1 / 1, 400mL) was added potassium peroxymonosulfate (52.8g, 152.57mmol) and the resulting mixture was stirred at 45°C for 3 hours. Water (200mL) was added to the reaction solution, which was then extracted with dichloromethane / isopropanol (10 / 1, v / v, 300mL x 3), and the combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Compound 4i (7.72g, 93% yield) was obtained as a white solid.

[0669] MS m / z (ESI): 269.0 [M+1].

[0670] Step six: Synthesis of (S)-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5- amido)-12-oxo-2,5,8-trioxa-11-azahexadecan-16-oic acid tert-butyl ester (4j)

[0671] Step 1 : Synthesis of 2-(6-bromohexyl)-5-oxopyrrolidine-l-carboxylic acid tert-butyl ester (4a)

[0672] MS m / z (ESI): 543.3 [M + 1 - 56]

[0673] Step 7: Synthesis of (S)-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5- carbonylamido)-12-oxo-2,5,8-trioxa-11-azahexadecan-16-oic acid (4k)

[0674] Step 7: Synthesis of (S)-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5- carbonylamido)-12-oxo-2,5,8-trioxa-11-azahexadecan-16-oic acid (4k)

[0675] MS m / z (ESI): 543.3 [M + 1 - 56]

[0676] Step 8: Synthesis of 2,5-dioxopyrrolidin-l-yl (S)-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoylamido)-12-oxo-2,5,8-trioxa-11- azahexadecan-16-oate (4l)

[0677] Step 7: Synthesis of (S)-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5- carbonylamido)-12-oxo-2,5,8-trioxa-11-azahexadecan-16-oic acid (4k)

[0678] MS m / z (ESI): 640.4 [M+1]

[0679] Step nine: Synthesis of (13S,24S,32R)-24-benzyl-32-methyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoylamido)-12,16,19,22,25,28- hexaoxo-2,5,8,31-tetraoxa-11,17,20,23,26,29-hexazatriazacyclohexadecane-33- acid (4)

[0680] To a solution of 3 (397 mg, 0.91 mmol) and sodium carbonate (96.1 mg, 0.91 mmol) in water (5 mL), 4l (580 mg, crude) was dissolved in acetonitrile (3 mL) and added slowly drop wise to the reaction. After the addition was complete, the resulting mixture was stirred at 25 °C for 1 h. The reaction was directly purified by Prep-HPLC (column: Whattman-C18 20x150mm, 10um, mobile phase: A- water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 25%) to give compound 4 (625 mg, 71% yield) as a white solid.

[0681] MS m / z (ESI): 960.6 [M-1]

[0682] 1 H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 2H), 8.54 (t, J = 6.7 Hz, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.18 - 7.88 (m, 5H), 7.34 - 7.10 (m, 5H), 4.68 - 4.43 (m, 3H), 4.30 - 4.18 (m, 1H), 4.10 - 4.01 (m, 1H), 3.81 - 3.54 (m, 6H), 3.54 - 3.45 (m, 6H), 3.44 - 3.38 (m, 7H), 3.26 - 3.10 (m, 5H), 3.08 - 2.99 (m, 1H), 2.86 - 2.73 (m, 1H), 2.55 (t, J = 7.1 Hz, 2H), 2.39 - 2.27 (m, 2H), 2.21 - 2.09 (m, 2H), 1.93 - 1.62 (m, 4H), 1.24 (d, J = 6.9 Hz, 3H).

[0683] Scheme Two:

[0684] Step one: Synthesis of Glycylglycyl-L-phenylalanine tert-butyl ester (4m)

[0685] To a solution of 3c (730 mg, 1.51 mmol) and 10% palladium on carbon (80 mg) in methanol (15 mL) was purged with hydrogen gas three times and stirred under hydrogen atmosphere at 25 °C for 1 h. The reaction was filtered, the filter cake was washed with methanol (10 mL), and the combined filtrate and washings were concentrated under reduced pressure. The crude compound 4m (520 mg) was obtained as a colorless oil.

[0686] MS m / z (ESI): 336.2 [M+1].

[0687] Step two: Synthesis of (S)-13-((benzyloxy)carbonyl)amino)-12-oxo-2,5,8-trioxa- 11-azahexadecan-16-oic acid (4n)

[0688] To a solution of 4c (600 mg, 1.23 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL), and the reaction was stirred at room temperature for 2 h. The reaction was concentrated under reduced pressure, and the resulting residue was added to methyl tert-butyl ether (30 mL) and stirred at 60 °C under nitrogen atmosphere for 2 h. The reaction was filtered after it was allowed to cool to room temperature naturally, and the solid was collected and dried to give compound 4n (471 mg, 86% yield) as a white solid.

[0689] MS m / z (ESI): 427.3 [M+1]

[0690] Step three: Synthesis of tert-butyl ((S)-13-((benzyloxy)carbonyl)amino)-12-oxo- 2,5,8-trioxa-11-azahexadecan-16-yl)glycylglycyl-L-phenylalaninate (4o)

[0691] To a solution of 4n (457 mg, 1.07 mmol) and 2-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (448 mg, 1.18 mmol) in dichloromethane (15 mL) was added N,N-diisopropylethylamine (693 mg, 5.36 mmol) followed by 4m (395 mg, 1.18 mmol) portionwise, and the reaction was stirred at 25 °C for 4 h. The reaction was washed with water (50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (mobile phase: A-dichloromethane, B-methanol; isocratic elution: B%: 5%) to give compound 4o (586 mg, 75% yield) as a yellow oil.

[0692] MS m / z (ESI): 744.4 [M+1]

[0693] Step 4: Synthesis of ((S)-13-((benzyloxy)carbonyl)amino)-12-oxo-2,5,8-trioxa-11-azahexadecane-16-yl)glycylglycyl-L-phenylalanine (4p)

[0694] 400 mg (0.53 mmol) of compound 4o was dissolved in 5 mL of dichloromethane, followed by the addition of 5 mL of trifluoroacetic acid. The mixture was stirred at room temperature for 2 hours after the addition was complete. The reaction solution was concentrated under reduced pressure, and the resulting residue was added to 30 mL of methyl tert-butyl ether. The mixture was stirred and slurried at 60 °C under a nitrogen atmosphere for 2 hours. After naturally cooling to room temperature, the mixture was filtered, and the solid was collected and dried to give compound 4p (338 mg, 88% yield) as a pale yellow solid.

[0695] MS m / z (ESI): 688.2 [M-1]

[0696] Step 5: Synthesis of benzyl(13S,24S,32R)-24-benzyl-13-((benzyloxy)carbonyl)amino)-32-methyl-12,16,19,22,25,28-hexaoxo-2,5,8,31-tetraoxa-11,17,20,23,26,29-hexaazatriazacyclobutane-33-ester (4q)

[0697] To a solution of 150 mg (0.31 mmol) in N,N-dimethylformamide (5 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (23.4 mg, 0.15 mmol) was added, and the mixture was stirred at 25 °C for 1 hour. Then, 4p (219 mg, 0.33 mmol), 1-hydroxybenzotriazole (50.2 mg, 0.37 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (65.5 mg, 0.34 mmol) were added sequentially, and the resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was slowly poured into water (30 mL), then extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: Huapu-C18 20×150mm, 10um; mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 20%-90%), to give compound 4q (209 mg, 74% yield) as a white solid.

[0698] MS m / z (ESI): 936.5 [M+1]

[0699] Step six: Synthesis of ((13S,24S,32R)-13-amino-24-benzyl 32-methyl-12,16,19,22,25,28-hexaoxo- 2,5,8,31-tetraoxo-11,17,20,23,26,29-hexaazatricyclohexanexyl-33-acid (4r)

[0700] To a solution of 4q (200 mg, 0.21 mmol) and 10% palladium on carbon (30 mg) in a mixture of methanol / tetrahydrofuran / water (1 / 1 / 1, v / v / v, 6 mL) was bubbled with hydrogen gas for three times and stirred at 25 °C for 2 h under hydrogen atmosphere. The reaction mixture was filtered, the filter cake was washed with methanol (10 mL), and the filtrate and the wash were combined and concentrated under reduced pressure. The residue was purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 4%) to give compound 4r (113 mg, 75% yield) as a white solid.

[0701] MS m / z (ESI): 712.4 [M+1]

[0702] Step seven: Synthesis of 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoate (4s)

[0703] To a solution of 4i (100 mg, 0.37 mmol) in dichloromethane (4 mL) was added N-hydroxysuccinimide (63.8 mg, 0.55 mmol) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (99.4 mg, 0.51 mmol) successively under nitrogen atmosphere, and the resulting mixture was stirred at room temperature for 2 h. To the reaction mixture was added dichloromethane (20 mL), followed by washing with water (10 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 20%-80%) to give compound 4s (112 mg, yield: 82%) as a white solid.

[0704] MS m / z (ESI): 366.1 [M+1].

[0705] Step eight: Synthesis of (13S,24S,32R)-24-benzyl-32-methyl-13-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hexyl-5-ynoylamino)-12,16,19,22,25,28-hexaoxo-2,5,8,31-tetraoxo-11,17,20,23,26,29- hexaazatricyclohexanexyl-33-acid (4)

[0706] Compound 4 (99.3 mg, 72% yield) was obtained as white solid by Prep- HPLC purification (column: Whp-C18 20x150mm, 10um, mobile phase: A- water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 25%) of the reaction mixture obtained by dissolving 4r (100 mg, 0.14 mmol) and sodium carbonate (15.1 mg, 0.14 mmol) in water (1 mL) and then slowly adding a solution of 4s (52.2 mg, 0.14 mmol) in acetonitrile (1 mL), stirring the resulting mixture at 25 °C for 0.5 h.

[0707] MS m / z (ESI): 960.6 [M-1]

[0708] 1 H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 2H), 8.54 (t, J = 6.7 Hz, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.18 - 7.88 (m, 5H), 7.34 - 7.10 (m, 5H), 4.68 - 4.43 (m, 3H), 4.30 - 4.18 (m, 1H), 4.10 - 4.01 (m, 1H), 3.81 - 3.54 (m, 6H), 3.54 - 3.45 (m, 6H), 3.44 - 3.38 (m, 7H), 3.26 - 3.10 (m, 5H), 3.08 - 2.99 (m, 1H), 2.86 - 2.73 (m, 1H), 2.55 (t, J = 7.1 Hz, 2H), 2.39 - 2.27 (m, 2H), 2.21 - 2.09 (m, 2H), 1.93 - 1.62 (m, 4H), 1.24 (d, J = 6.9 Hz, 3H).

[0709] Example 5: Preparation of compound 5

[0710] (13S,24S)-24-benzyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoyl- amino)-12,16,19,22,25,28-hexaoxa-2,5,8,31-tetraoxa-11,17,20,23,26,29-hexaazatriazacyclonane- 33-oic acid 5

[0711] Step one: Synthesis of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazoundecan-11-oic acid benzyl ester (5b)

[0712] To a solution of 3f (17.5 g, 47.50 mmol) and 5a (15.8 g, 95.01 mmol) in ethyleneglycol dimethyl ether (260 mL) was added sodium hydroxide aqueous solution (10 mol / L, 4.51 ml, 45.10 mmol) dropwise at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 0.5 h. Then 10(w / v)% ammonium chloride aqueous solution (400 mL) was added to the reaction mixture, which was extracted with ethyl acetate (300 mL x 2). The combined organic phase was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: petroleum ether (A) and ethyl acetate (B), gradient elution: B%: 20%-70%) to give compound 5b (17.8 g, 78% yield) as a white solid.

[0713] MS m / z (ESI): 475.2 [M+1]

[0714] Step two: synthesis of (S)-11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazadocosa- 20-enoic acid benzyl ester (5c)

[0715] To a solution of 5b (8.00 g, 16.86 mmol) in N,N-dimethylformamide (100 mL) was added 1,8-diazabicyclo(5,4,0)-7-undecene (1.28 g, 8.43 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 1 h. Then 3d (7.32 g, 17.70 mmol), 1-hydroxybenzotriazole (2.73 g, 20.23 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.56 g, 18.55 mmol) were added successively. The reaction mixture was stirred at 25 °C for 3 h. The reaction mixture was poured into water (800 mL) and extracted with ethyl acetate (600 mL x 2). The combined organic phase was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was dissolved in n-heptane / ethyl acetate (1 / 1, v / v, 300 mL) and stirred at 70 °C for 16 h under nitrogen atmosphere. The mixture was allowed to cool to room temperature naturally and then filtered. The solid was collected and dried to give compound 5c (9.15 g, 83% yield) as a white solid.

[0716] MS m / z (ESI): 648.4 [M+1]

[0717] Step three: synthesis of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (5d)

[0718] To a solution of 5c (4.50 g, 8.76 mmol) in a mixture of methanol / tetrahydrofuran / water (1 / 1 / 1, v / v / v, 80 mL), 10% palladium on carbon (500 mg) was added, then the reaction system was replaced with hydrogen three times and stirred under hydrogen atmosphere at 25 °C for 4 h. The reaction was filtered, the filter cake was rinsed with methanol (30 mL), the filtrate was concentrated under reduced pressure, and the resulting residue was purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 6%) to give compound 5d (2.71 g, 73% yield) as a white solid.

[0719] MS m / z (ESI): 424.1 [M+1]

[0720] Step four: Synthesis of (13S,24S)-24-benzyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoylamido)-12,16,19,22,25,28-hexaoxa-2,5,8,31-tetraoxa-11,17,20,23,26,29-hexaazatriazolidin-33- oic acid (5)

[0721] To a solution of 5d (629 mg, 1.49 mmol) and sodium carbonate (157 mg, 1.49 mmol) in water (8 mL), a solution of 41 (950 mg, crude) in acetonitrile (6 mL) was added dropwise slowly, and the resulting mixture was stirred at 25 °C for 1 h. The reaction was directly purified by Prep-HPLC (column: Whart-C18 20x150mm, 10um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 24%) to give compound 5 (1.15 g, 80% yield) as a white solid.

[0722] MS m / z (ESI): 946.5 [M-1]

[0723] Example 6: Preparation of compound 6

[0724] (9S,17R)-9-benzyl-17-methyl-1-(1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1- yl)cyclopropyl)-1,4,7,10,13-pentaoxa-16-oxa-2,5,8,11,14-pentaazaoctadecan-18-oic acid 6

[0725] Step one: Synthesis of tert-butyl 1-(3-oxopropyl)cyclopropane-1-carboxylate (6b)

[0726] To a solution of 6a (3.10 g, 17.01 mmol), silver nitrite (131 mg, 0.85 mol), bis(benzonitrile)palladium(II) chloride (655 mg, 1.70 mmol) and copper(II) chloride dihydrate (262 mg, 1.70 mmol) in a mixture solvent of tert-butyl alcohol / nitromethane (15 / 1, v / v, 50 mL) was added, and the reaction was replaced with oxygen for three times, and stirred at 25 °C for 12 h under oxygen atmosphere. The reaction was filtered, the filter cake was rinsed with ethyl acetate (30 mL), and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 0%-20%) to give compound 6b (1.55 g, 46% yield) as a colorless sticky substance.

[0727] Step two: synthesis of tert-butyl 1-(but-3-yn-1-yl)cyclopropane-1-carboxylate (6c)

[0728] To a solution of 6b (1.55 g, 7.82 mmol) in methanol (50 mL) was added potassium carbonate (2.16 g, 15.64 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (1.80 g, 9.38 mmol) successively, and stirred at 25 °C for 12 h. Water (50 mL) was added to the reaction, and the organic phase was extracted with ethyl acetate (40 mL x 2), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 0%-15%) to give compound 6c (1.13 g, 74% yield) as a colorless sticky substance.

[0729] Step three: synthesis of tert-butyl 1-(4-(2-(methylthio)pyrimidin-5-yl)but-3-yn-1-yl)cyclopropane-1-carboxylate (6d)

[0730] To a solution of 6c (1.15 g, 5.92 mmol), 5-bromo-2-methylthiopyrimidine (1.21 g, 5.92 mmol), triethylamine (5.99 g, 59.20 mmol), cuprous iodide (112 mg, 0.59 mmol) and bis(triphenylphosphine)palladium(II) chloride (216 mg, 0.30 mmol) in 1,4-dioxane (30 mL) was added, and the reaction was replaced with nitrogen for three times, and stirred at 100 °C for 3 h under nitrogen atmosphere. The reaction was filtered, the filter cake was rinsed with ethyl acetate (40 mL), and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 10%-50%) to give compound 6d (1.43 g, 73% yield) as a colorless sticky substance.

[0731] MS m / z (ESI): 319.6 [M+1].

[0732] Step four: Synthesis of tert-butyl 1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1- yl)cyclopropane-1-carboxylate (6e)

[0733] 6d (1.43 g, 4.49 mmol) was dissolved in 20 mL of dichloromethane, then m-chloroperoxybenzoic acid (2.28 g, 13.21 mmol, purity 85.0%) was added, stirred at 25 °C for 2 hours. The reaction was filtered, the filter cake was rinsed with dichloromethane (30 mL), the filtrate was concentrated under reduced pressure, the obtained residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 20%-70%), to give compound 6e (1.40 g, 87% yield) as a colorless viscous substance.

[0734] MS m / z (ESI): 351.1 [M+1].

[0735] Step five: Synthesis of 1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1-yl)cyclopropane-1- carboxylic acid (6f)

[0736] 6e (1.40 g, 4.00 mmol) was dissolved in dichloromethane (15 mL), then trifluoroacetic acid (5 mL) was added, stirred at 25 °C for 1 hour. The reaction was concentrated under reduced pressure, the obtained residue was stirred with methanol (15 mL) at 60 °C for 3 hours, then after natural cooling to room temperature, it was filtered, the filter cake was washed with cold methanol (6 mL), the filter cake was collected and dried. Compound 6f (885 mg, 75% yield) was obtained as a light yellow solid.

[0737] MS m / z (ESI): 295.2 [M+1].

[0738] Step six: Synthesis of (9S,17R)-9-benzyl-17-methyl-1-(1-(4-(2-(methylsulfonyl)pyrimidin-5- yl)but-3-yn-1-yl)cyclopropyl)-1,4,7,10,13-pentaoxo-16-oxa-2,5,8,11,14-pentaazaoctadecan-18- oic acid (6)

[0739] To a solution of 6f (200 mg, 0.68 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (271 mg, 0.71 mmol) and N,N- diisopropylethylamine (351 mg, 2.72 mmol) in acetonitrile (3 mL) was added at 25 °C and stirred for 15 min. Then the above reaction solution was slowly added to a solution of 3 (327 mg, 0.75 mmol) in water (3 mL), after addition, the reaction was kept at 25 °C for 0.5 h. The reaction solution was directly purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 5%-60%) to give compound 6 (310 mg, 64% yield) as a white solid.

[0740] MS m / z (ESI): 712.4 [M-1].

[0741] 1 H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 2H), 8.54 (t, J = 6.7 Hz, 1H), 8.30 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.02 - 7.94 (m, 1H), 7.88 (t, J = 5.8 Hz, 1H), 7.32 - 7.11 (m, 5H), 4.66 - 4.43 (m, 3H), 4.10 - 3.99 (m, 1H), 3.81 - 3.54 (m, 6H), 3.41 (s, 3H), 3.11 - 3.00 (m, 1H), 2.84 - 2.74 (m, 1H), 2.72 - 2.64 (m, 2H), 1.90 (t, J = 7.8 Hz, 2H), 1.23 (d, J = 6.9 Hz, 3H), 1.08 - 0.96 (m, 2H), 0.78 - 0.68 (m, 2H).

[0742] Example 7: Preparation of compound 7

[0743] (9S,17S)-9-benzyl-17-methyl-1-(1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1- yl)cyclopropyl)-1,4,7,10,13-pentaoxo-16-oxa-2,5,8,11,14-pentaazaoctadecan-18-oic acid 7

[0744] Step one: Synthesis of (benzyl (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7- diazoundecan-11-ate (7b)

[0745] To a solution of 3f (5.20 g, 14.12 mmol) in ethyleneglycol dimethyl ether (75 mL) was added 7a (5.10 g, 28.23 mmol), and the resulting mixture was cooled to 0-5 °C. To it was added aqueous sodium hydroxide solution (10 mol / L, 1.39 mL, 13.90 mmol), and the resulting mixture was kept stirring at 0-5 °C for 0.5 h. To the reaction was added 10 (w / v) % aqueous ammonium chloride solution (100 mL), followed by extraction with ethyl acetate (75 mL x 2), the combined organic phase was washed with saturated brine (75 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 30%-80%), to give compound 7b (5.85 g, 84% yield) as a yellow sticky solid.

[0746] MS m / z (ESI): 489.2 [M+1].

[0747] Step two: Synthesis of benzyl (11S, 19S)-11-benzyl-19-methyl-3, 6, 9, 12, 15-pentaoxo-1- phenyl-2, 18-dioxa-4, 7, 10, 13, 16-pentaazahexacosan-25-oate (7c)

[0748] To a solution of 7b (5.85 g, 11.97 mmol) in N, N-dimethylformamide (60 mL) was added 1, 8-diazabicyclo[5.4.0]undec-7-ene (912 mg, 5.98 mmol), and the resulting mixture was stirred at 25 °C for 1 h. Then 3d (5.21 g, 12.57 mmol), 1- hydroxybenzotriazole (1.94 g, 14.37 mmol) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide (2.53 g, 13.17 mmol) were added successively, and the resulting mixture was stirred at 25 °C for 2 h. The reaction was slowly poured into water (300 mL), followed by extraction with ethyl acetate (200 mL x 2), the combined organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was added into n-heptane / ethyl acetate (1 / 1, v / v, 200 mL), and the resulting mixture was stirred at 70 °C under nitrogen atmosphere for 16 h, then filtered after natural cooling to room temperature, the solid was collected and oven-dried, to give compound 7c (6.13 g, 77% yield) as a white solid.

[0749] MS m / z (ESI): 662.1 [M+1].

[0750] Step three: synthesis of (2S, 10S)-16-amino-10-benzyl-2-methyl-6,9,12,15- tetraoxo-3-oxo-5,8,11,14-tetraazahexadecanoic acid (7d)

[0751] To a solution of 7c (6.13 g, 9.26 mmol) in methanol / tetrahydrofuran / water (1 / 1 / 1, v / v / v, 100 mL) was added 10% palladium on carbon (800 mg), the resulting mixture was purged with hydrogen three times and stirred under hydrogen atmosphere at 25 °C for 4 h. The reaction was filtered, the filter cake was rinsed with methanol (40 mL), and the filtrate and wash were combined and concentrated under reduced pressure. The resulting residue was purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 6%) to give compound 7d (3.21 g, 79% yield) as a white solid.

[0752] MS m / z (ESI): 438.2 [M+1].

[0753] 1 H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.69 - 8.58 (m, 2H), 8.54 - 8.45 (m, 1H), 7.33 - 7.13 (m, 5H), 4.66 - 4.52 (m, 2H), 4.47 - 4.36 (m, 1H), 3.94 - 3.76 (m, 3H), 3.72 - 3.61 (m, 1H), 3.55 - 3.43 (m, 3H), 3.11 - 3.01 (m, 1H), 2.87 - 2.75 (m, 1H), 1.18 (d, J = 6.8 Hz, 3H).

[0754] Step four: synthesis of (9S, 17S)-9-benzyl-17-methyl-1-(1-(4-(2- (methylsulfonyl)pyrimidin-5-yl)but-3-yn-1-yl)cyclopropyl)-1,4,7,10,13- pentaoxo-16-oxo-2,5,8,11,14-pentaazaoctadecan-18-oic acid (7)

[0755] To a solution of 6f (300 mg, 1.02 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (406 mg, 1.06 mmol) and N,N- diisopropylethylamine (526 mg, 4.08 mmol) in acetonitrile (4 mL) was added at 25 °C and stirred for 15 min. Then the above reaction solution was slowly added to a solution of 7d (490 mg, 1.13 mmol) in water (4 mL) at 25 °C and stirred for 0.5 h. The reaction solution was directly purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 5%-60%) to give compound 7 (485 mg, 66% yield) as a white solid.

[0756] MS m / z (ESI): 712.1 [M-1].

[0757] 1 H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 2H), 8.55 (t, J = 6.7 Hz, 1H), 8.30 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.88 (t, J = 5.8 Hz, 1H), 7.30 - 7.10 (m, 5H), 4.65 - 4.46 (m, 3H), 4.12 - 3.99 (m, 1H), 3.80 - 3.53 (m, 6H), 3.41 (s, 3H), 3.13 - 2.99 (m, 1H), 2.84 - 2.74 (m, 1H), 2.72 - 2.61 (m, 2H), 1.90 (t, J = 7.8 Hz, 2H), 1.24 (d, J = 6.9 Hz, 3H), 1.09 - 0.94 (m, 2H), 0.81 - 0.67 (m, 2H).

[0758] Preparation of compound 8 of Example 8

[0759] (13S,24S,32S-24-benzyl-32-methyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5- ynylamino)-12,16,19,22,25,28-hexaoxo-2,5,8,31-tetraoxo-11,17,20,23,26,29-hexaazatricyclo triptorelin 8

[0760] Compound 8 was obtained as white solid (795 mg, 72% yield) by dissolving 7d (500 mg, 1.14 mmol) and sodium carbonate (121 mg, 1.14 mmol) in water (6 mL) and then slowly adding a solution of 4l (730 mg, crude) in acetonitrile (4 mL). The resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was directly purified by Prep-HPLC (column: Whiptom-C18 20x150mm,10um, mobile phase: A-water(0.1%formic acid), B-acetonitrile, isocratic elution, B%: 25%).

[0761] MS m / z (ESI): 960.6 [M-1].

[0762] 1 H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 2H), 8.55 (t, J = 6.7 Hz, 1H), 8.32 (t, J = 5.8 Hz, 1H), 8.15 - 7.93 (m, 5H), 7.30 - 7.12 (m, 5H), 4.68 - 4.44 (m, 3H), 4.30 - 4.15 (m, 1H), 4.11 - 3.97 (m, 1H), 3.83 - 3.53 (m, 6H), 3.52 - 3.46 (m, 6H), 3.44 - 3.37 (m, 7H), 3.26 - 3.12 (m, 5H), 3.10 - 2.99 (m, 1H), 2.84 - 2.74 (m, 1H), 2.55 (t, J = 7.1 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 2.15 (t, J = 7.9 Hz, 2H), 1.93 - 1.63 (m, 4H), 1.24 (d, J = 6.9 Hz, 3H).

[0763] Example 9: Preparation of compound 9

[0764] (S)-9-benzyl-1-(1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1-yl)cyclopropyl)- 1,4,7,10,13-pentaoxo-16-oxa-2,5,8,11,14-pentaazaoctadecan-18-oic acid 9

[0765] To a solution of 6f (340 mg, 1.16 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (448 mg, 1.18 mmol) and N,N- diisopropylethylamine (597 mg, 4.62 mmol) in acetonitrile (5 mL) was stirred at 25 °C for 15 min. Then the above reaction solution was added slowly into a solution of 5d (538 mg, 1.27 mmol) in water (5 mL) at 25 °C for 0.5 h. The reaction solution was directly purified by C18 flash reverse phase silica gel column (mobile phase: A-water (0.1% formic acid), B-acetonitrile, gradient elution, B%: 5%-60%) to give compound 9 (703 mg, 86% yield) as a white solid.

[0766] MS m / z (ESI): 698.3 [M-1].

[0767] Example 10: Preparation of compound 10

[0768] ((S)-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoylamido)-12-oxo- 2,5,8-trioxa-11-azahexadecan-16-yl)glycylglycyl-L-phenylalanine 10

[0769] Example 11: Preparation of compound 11

[0770] (1-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-1-yl)cyclopropane-1- carbonyl)glycylglycyl-L-phenylalanine 11

[0771] Example 12: Preparation of compound 12

[0772] (R)-2-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-(cyclopropylmethyl)-5- fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propanamide 12

[0773] Example 13: Preparation of compound 13

[0774] (S)-2-((2-aminoacetamido)methyloxy)-N-((lS,9S)-9-(cyclopropylmethyl)-5- fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)propanamide 13

[0775] Example 14: Preparation of Compound 14

[0776] 2-amino-N-((2-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[ 1,2-b]quinolin- 1 -yl)amino)-2-oxoethoxy)methyl)acetamide 14

[0777] Example 15: Preparation of Compound I

[0778] (S)-N 5 -((2R,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadecan-16-yl)-N 1 -((2R,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2,3,9,10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadecan-16-yl)-N

[0779] Scheme One:

[0780] To a solution of 4 (694 mg, 0.72 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (288 mg, 0.76 mmol), N,N- diisopropylethylamine (326 mg, 2.52 mmol) and trifluoroacetate salt of 2 (415 mg, 0.72 mmol) in N,N-dimethylformamide (10 mL) was stirred at 25 °C for 30 min. The reaction mixture was added to dichloromethane (50 mL) and washed with water (20 mL), the organic phase was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: Whp-C18 20x150mm,10um, mobile phase: A-water(0.1%formic acid), B-acetonitrile, isocratic elution, B%: 39%), to get compound I (883 mg, 81% yield) as yellow solid.

[0781] Scheme II:

[0782] MS m / z (ESI): 1405.7 [M+1].

[0783] 1H NMR (400MHz, CD3OD) δ8.95–8.88(m,2H),7.68–7.61(m,2H),7.29–7.21(m,2H),7.20–7 .12(m,3H),5.68–5.64(m,1H),5.60–5.54(m,1H),5.42–5.33(m,2H),5.16–5.10(m,1H ),4.82–4.79(m,1H),4.70–4.66(m,1H),4.43–4.35(m,2H),4.26–4.20(m,1H),3.90–3 .80(m,4H),3.78–3.73(m,1H),3.71–3.66(m,1H),3.63–3.54(m,6H),3.54–3.46(m,5H) ,3.43–3.35(m,4H),3.35–3.33(m,3H),3.27–3.22(m,1H),3.21–3.16(m,1H),3.15–3. 12(m,1H),3.07–3.02(m,1H),2.92–2.86(m,1H),2.59–2.52(m,2H),2.47–2.38(m,5H) ,2.36–2.29(m,3H),2.11–2.04(m,1H),1.99–1.87(m,4H),1.85–1.79(m,1H),1.60–1. 51(m,3H),0.92–0.85(m,1H),0.49–0.39(m,2H),0.15–0.08(m,1H),0.06–0.00(m,1H).

[0784] Example 16: Preparation of Compound II

[0785] (S)-N 5 -((S)-10-benzyl-1-(((1S,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4′:6,7]indo[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-N 1 -(2-(2-methoxyethoxy)ethoxyethyl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)glutaramide II

[0786] Option 1:

[0787] To a solution of 5 (132 mg, 0.14 mmol), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (58.1 mg, 0.15 mmol), N,N- diisopropylethylamine (53.9 mg, 0.42 mmol) and trifluoroacetate salt of 2 (80.0 mg, 0.14 mmol) in N,N-dimethylformamide (4 mL) was stirred at 25 °C for 15 min. The reaction mixture was added to dichloromethane (50 mL) and washed with water (20 mL), and the organic phase was concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC (column: Whattman-C18 20x150mm, 10um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 38%) to give compound II (156 mg, 79% yield) as a white solid.

[0788] Scheme II:

[0789] MS m / z (ESI): 1391.4 [M+1].

[0790] 1 H NMR (400 MHz, CD3OD) δ 8.97 - 8.87 (m, 2H), 7.68 - 7.58 (m, 2H), 7.29 - 7.13 (m, 5H), 5.76 - 5.69 (m, 1H), 5.61 - 5.53 (m, 1H), 5.38 - 5.27 (m, 2H), 5.16 - 5.06 (m, 1H), 4.84 - 4.79 (m, 2H), 4.68 - 4.63 (m, 1H), 4.40 - 4.32 (m, 2H), 4.27 - 4.21 (m, 1H), 4.18 - 4.13 (m, 1H), 3.94 - 3.81 (m, 4H), 3.69 - 3.64 (m, 2H), 3.62 - 3.59 (m, 1H), 3.59 - 3.58 (m, 3H), 3.54 - 3.47 (m, 4H), 3.43 - 3.36 (m, 2H), 3.36 - 3.34 (m, 3H), 3.33 - 3.32 (m, 2H), 3.28 - 3.25 (m, 2H), 3.21 - 3.17 (m, 1H), 3.00 - 2.96 (m, 1H), 2.95 - 2.90 (m, 1H), 2.58 - 2.53 (m, 2H), 2.50 - 2.31 (m, 9H), 2.28 - 2.23 (m, 1H), 2.11 - 2.04 (m, 1H), 1.96 - 1.85 (m, 4H), 1.81 - 1.74 (m, 1H), 0.89 - 0.82 (m, 1H), 0.44 - 0.35 (m, 2H), 0.10 - 0.04 (m, 1H), 0.01 - -0.05 (m, 1H).

[0791] Example 17: Preparation of compound III

[0792] N-((2R,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadec-16-yl)-l-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-l-yl)cyclopropane- 1-carboxamide III

[0793] Scheme I:

[0794] To a solution of 6 (124 mg, 0.17 mmol), 2-(7-azabenzotriazol-l-yl)-l,1,3,3- tetramethyluronium hexafluorophosphate (79.3 mg, 0.21 mmol), N,N- diisopropylethylamine (67.4 mg, 0.52 mmol) and trifluoroacetate salt of 2 (100 mg, 0.17 mmol) in N,N-dimethylformamide (4 mL) was stirred at 25 °C for 15 min. The reaction mixture was added to dichloromethane (50 mL) and washed with water (20 mL), the organic phase was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: Whp-C18 20x150mm, 10um, mobile phase: A-water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 41%) to give compound III (153 mg, 75% yield) as a white solid.

[0795] Scheme II:

[0796] MS m / z (ESI): 1157.2 [M+l].

[0797] 1H NMR (400 MHz, DMSO-d6) δ 9.12 - 9.00 (m, 2H), 8.68 - 8.60 (m, 1H), 8.60 - 8.52 (m, 1H), 8.32 - 8.26 (m, 1H), 8.15 - 8.08 (m, 1H), 8.01 - 7.92 (m, 1H), 7.90 - 7.84 (m, 1H), 7.81 - 7.74 (m, 1H), 7.36 (s, 1H), 7.27 - 7.10 (m, 5H), 6.61 - 6.56 (m, 1H), 5.63 - 5.56 (m, 1H), 5.46 - 5.35 (m, 2H), 5.25 - 5.07 (m, 2H), 4.74 - 4.66 (m, 1H), 4.58 - 4.50 (m, 1H), 4.50 - 4.43 (m, 1H), 4.17 - 4.08 (m, 1H), 3.79 - 3.53 (m, 6H), 3.44 - 3.37 (m, 3H), 3.18 - 3.05 (m, 2H), 3.04 - 2.96 (m, 1H), 2.78 - 2.63 (m, 3H), 2.39 - 2.35 (m, 3H), 2.23 - 2.13 (m, 2H), 1.94 - 1.84 (m, 2H), 1.83 - 1.71 (m, 2H), 1.48 - 1.36 (m, 3H), 1.06 - 0.95 (m, 2H), 0.86 - 0.77 (m, 1H), 0.75 - 0.66 (m, 2H), 0.39 - 0.24 (m, 2H), 0.09 - 0.01 (m, 1H), -0.04 - -0.13 (m, 1H).

[0798] Example 18: Preparation of compound IV

[0799] N-((2S,10S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadec-16-yl)-l-(4-(2-(methylsulfonyl)pyrimidin-5-yl)but-3-yn-l-yl)cyclopropane- 1-carboxamide IV

[0800] Scheme One:

[0801] To a solution of 7 (124 mg, 0.17 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (79.3 mg, 0.21 mmol), N,N- diisopropylethylamine (67.4 mg, 0.52 mmol) and trifluoroacetate salt of 2 (100 mg, 0.17 mmol) in N,N-dimethylformamide (4 mL) was stirred at 25 °C for 15 min. The reaction mixture was added to dichloromethane (50 mL) and washed with water (20 mL), the organic phase was concentrated under reduced pressure. The resulting residue was purified by Prep-HPLC (column: Whp-C18 20x150mm,10um, mobile phase: A-water(0.1%formic acid), B-acetonitrile, isocratic elution, B%: 41%), to give compound IV (154 mg, 76% yield) as a white solid.

[0802] Scheme II:

[0803] MS m / z (ESI): 1157.8 [M+1].

[0804] 1 H NMR (400 MHz, CD3OD) δ 8.89 - 8.80 (m, 2H), 7.66 - 7.58 (m, 2H), 7.26 - 7.19 (m, 2H), 7.19 - 7.08 (m, 3H), 5.71 - 5.65 (m, 1H), 5.62 - 5.54 (m, 1H), 5.50 - 5.42 (m, 1H), 5.33 - 5.25 (m, 1H), 5.22 - 5.15 (m, 1H), 4.78 - 4.71 (m, 2H), 4.33 - 4.25 (m, 2H), 3.91 - 3.72 (m, 4H), 3.61 - 3.45 (m, 3H), 3.36 - 3.33 (m, 3H), 3.22 - 3.12 (m, 1H), 2.94 - 2.84 (m, 2H), 2.73 - 2.64 (m, 2H), 2.46 - 2.37 (m, 3H), 2.37 - 2.28 (m, 1H), 2.26 - 2.18 (m, 1H), 2.01 - 1.90 (m, 2H), 1.89 - 1.81 (m, 1H), 1.76 - 1.69 (m, 1H), 1.51 - 1.42 (m, 3H), 1.17 - 1.12 (m, 2H), 0.94 - 0.83 (m, 3H), 0.45 - 0.31 (m, 2H), 0.06 - -0.08 (m, 2H).

[0805] Example 19: Preparation of compound V

[0806] (S)-N 5- ((2S, 10S)-10-benzyl-l-(((lS, 9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4- methyl-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-l-yl)amino)-2-methyl-l, 6, 9, 12, 15-pentaoxo-3-oxa-5, 8, 11, 14- tetraazahexadec-16-yl)-N 1 - (2-(2-methoxyethoxy)ethoxyethyl)-2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)pentanediamide V

[0807] Scheme One:

[0808] To a solution of 8 (293 mg, 0.30 mmol), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (127 mg, 0.33 mmol), N,N- diisopropylethylamine (118 mg, 0.91 mmol) and trifluoroacetate salt of 2 (175 mg, 0.30 mmol) in N,N-dimethylformamide (6 mL) was stirred at 25 °C for 30 min. The reaction mixture was diluted with dichloromethane (50 mL) and washed with water (20 mL). The organic phase was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: Whattman-C18 20 x 150 mm, 10 um, mobile phase: A- water (0.1% formic acid), B-acetonitrile, isocratic elution, B%: 39%) to give compound V (295 mg, 68% yield) as a light yellow solid.

[0809] Scheme Two:

[0810] MS m / z (ESI): 1405.9 [M+l].

[0811] 1H NMR (400 MHz, DMSO-d6) δ 9.14 - 9.08 (m, 2H), 8.70 - 8.64 (m, 1H), 8.61 - 8.54 (m, 1H), 8.37 - 8.29 (m, 1H), 8.14 - 7.95 (m, 5H), 7.80 - 7.75 (m, 1H), 7.40 - 7.36 (m, 1H), 7.27 - 7.13 (m, 5H), 6.63 - 6.57 (m, 1H), 5.58 - 5.50 (m, 1H), 5.47 - 5.35 (m, 2H), 5.31 - 5.15 (m, 2H), 4.77 - 4.68 (m, 1H), 4.62 - 4.54 (m, 1H), 4.51 - 4.41 (m, 1H), 4.26 - 4.18 (m, 1H), 4.16 - 4.08 (m, 1H), 3.77 - 3.70 (m, 1H), 3.69 - 3.64 (m, 3H), 3.62 - 3.54 (m, 1H), 3.51 - 3.45 (m, 6H), 3.42 - 3.36 (m, 7H), 3.34 - 3.34 (m, 2H), 3.26 - 3.09 (m, 7H), 3.04 - 2.95 (m, 1H), 2.79 - 2.69 (m, 1H), 2.57 - 2.51 (m, 2H), 2.41 - 2.34 (m, 3H), 2.34 - 2.26 (m, 2H), 2.18 - 2.11 (m, 3H), 1.90 - 1.66 (m, 6H), 1.35 - 1.27 (m, 3H), 0.86 - 0.75 (m, 1H), 0.40 - 0.25 (m, 2H), 0.09 - 0.02 (m, 1H), -0.04 - -0.13 (m, 1H).

[0812] Example 20: Preparation of compound VI

[0813] N-((S)-10-benzyl-l-(((lS,9S)-9-(cyclopropylmethyl)-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l- yl)amino)-l,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecan-16-yl)-l-(4-(2- (methylsulfonyl)pyrimidin-5-yl)but-3-yn-l-yl)cyclopropane-l-carboxamide VI

[0814] Scheme One:

[0815] To a solution of 9 (207 mg, 0.30 mmol), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (124 mg, 0.32 mmol), N,N- diisopropylethylamine (153 mg, 1.18 mmol) and trifluoroacetate salt of 2 (170 mg, 0.30 mmol) in N,N-dimethylformamide (5 mL) was stirred at 25 °C for 15 min. The reaction mixture was added to dichloromethane (50 mL) and washed with water (20 mL), the organic phase was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: Whp-C18 20x150mm,10um, mobile phase: A-water(0.1%formic acid), B-acetonitrile, isocratic elution, B%: 41%), to get compound VI (260 mg, 76% yield) as white solid.

[0816] Scheme II:

[0817] MS m / z (ESI): 1143.5 [M+l].

[0818] 1 H NMR (400 MHz, DMSO-d6) δ 9.11 - 9.00 (m, 2H), 8.67 - 8.59 (m, 1H), 8.55 - 8.48 (m, 1H), 8.34 - 8.28 (m, 1H), 8.17 - 8.11 (m, 1H), 8.00 - 7.94 (m, 1H), 7.90 - 7.83 (m, 1H), 7.81 - 7.75 (m, 1H), 7.41 - 7.35 (m, 1H), 7.28 - 7.11 (m, 5H), 6.60 - 6.55 (m, 1H), 5.63 - 5.56 (m, 1H), 5.47 - 5.35 (m, 2H), 5.25 - 5.17 (m, 2H), 4.70 - 4.59 (m, 2H), 4.51 - 4.43 (m, 1H), 4.07 - 3.98 (m, 2H), 3.79 - 3.53 (m, 6H), 3.44 - 3.36 (m, 3H), 3.20 - 3.13 (m, 2H), 3.05 - 2.99 (m, 1H), 2.81 - 2.73 (m, 1H), 2.71 - 2.62 (m, 2H), 2.40 - 2.36 (m, 3H), 2.22 - 2.13 (m, 2H), 1.92 - 1.85 (m, 2H), 1.85 - 1.79 (m, 1H), 1.78 - 1.71 (m, 1H), 1.04 - 0.95 (m, 2H), 0.83 - 0.76 (m, 1H), 0.73 - 0.65 (m, 2H), 0.37 - 0.25 (m, 2H), 0.09 - 0.01 (m, 1H), -0.05 - -0.12 (m, 1H).

[0819] Example 21: ADC conjugate preparation

[0820] The "linker-drug molecules" prepared by the method of the present application can be used to prepare ADC drugs, the effects of which can be seen in the examples and test examples disclosed in international application WO2024109840A1, which is incorporated herein by reference in its entirety.

[0821] Method for preparing antibody conjugates

[0822] Method A: The antibody was replaced into 50 mM PBS / 1.0 mM EDTA buffer (pH 7.4 adjusted by sodium hydroxide solution) using an ultrafiltration tube with a molecular weight cut-off of 50 kD, 5-10 equivalents of 10 mM TCEP aqueous solution were added, and the mixture was shaken at 25°C for 3 hours. The linker-drug conjugate was dissolved in DMSO, 10-20 equivalents of the linker-drug conjugate were taken therefrom, and added dropwise to the reduced antibody solution, which was shaken to mix uniformly, and the mixture was shaken at 25°C for 2 hours. After the reaction was completed, 40 equivalents of 100 mM NAC aqueous solution were added, and the mixture was shaken at 25°C for 20 minutes to terminate the linker reaction. Excess small molecules were removed using an ultrafiltration tube with a molecular weight cut-off of 50 kD or a Sephadex G-25 desalting column, and the antibody-drug conjugate was replaced into 50 mM PBS buffer (pH 6.0). The sample was filtered using a 0.22 μm filter membrane, and the antibody-drug conjugate was obtained and stored in a refrigerator at 4°C. The DAR value of the conjugate was determined using reverse phase high performance liquid chromatography or mass spectrometry.

[0823] Method B: The antibody was replaced into 50 mM PBS / 1.0 mM EDTA buffer (pH 6.5 adjusted by sodium hydroxide solution) using an ultrafiltration tube with a molecular weight cut-off of 50 kD, 5-10 equivalents of 10 mM TCEP aqueous solution were added, and the mixture was shaken at 25°C for 3 hours. The linker-drug conjugate was dissolved in DMSO, 10-20 equivalents of the linker-drug conjugate were taken therefrom, and added dropwise to the reduced antibody solution, which was shaken to mix uniformly, and the mixture was shaken at 25°C for 2 hours. After the reaction was completed, 40 equivalents of 100 mM NAC aqueous solution were added, and the mixture was shaken at 25°C for 20 minutes to terminate the linker reaction. Excess small molecules were removed using an ultrafiltration tube with a molecular weight cut-off of 50 kD or a Sephadex G-25 desalting column, and the antibody-drug conjugate was replaced into 50 mM PBS buffer (pH 6.0). The sample was filtered using a 0.22 μm filter membrane, and the antibody-drug conjugate was obtained and stored in a refrigerator at 4°C. The DAR value of the conjugate was determined using reverse phase high performance liquid chromatography or mass spectrometry.

[0824] ADC-I

[0825] Prepared by the above described method A, starting from antibody Trastuzumab and compound I. The conjugate ADC-I was obtained in PBS buffer, stored at 4°C.

[0826] Mass spectrometry calculated average drug loading: y = 6.93.

[0827] ADC-II

[0828] Prepared by the above described method A, starting from antibody Trastuzumab and compound II. The conjugate ADC-II was obtained in PBS buffer, stored at 4°C.

[0829] Mass spectrometry calculated average drug loading: y = 8.02.

[0830] ADC-III

[0831] Prepared by the above described method A, starting from antibody Trastuzumab and compound III. The conjugate ADC-III was obtained in PBS buffer, stored at 4°C.

[0832] Mass spectrometry calculated average drug loading: y = 8.02.

[0833] ADC-IV

[0834] Prepared by the above described method A, starting from antibody Trastuzumab and compound IV. The conjugate ADC-IV was obtained in PBS buffer, stored at 4°C.

[0835] Mass spectrometry calculated average drug loading: y = 8.00.

[0836] Reference 1

[0837] Prepared by the above described method B, starting from antibody Trastuzumab and N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l- yl)amino)-l,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo- 2,5-dihydro-lH-pyrrol-l-yl)hexanamide. The conjugate mixture FADC-Reference 1 was obtained in PBS buffer, stored at 4°C, as Example 4-83.

[0838] Mass spectrometry calculated average drug loading: y = 8.00.

[0839] Test Example 1: Evaluation of the efficacy of an antibody drug conjugate in JIMT-1 tumor-bearing mice

[0840] 1. Test purposes:

[0841] The purpose of this experiment is to detect the efficacy of Her2 antibody ADC drug on human breast cancer JIMT-1 tumor-bearing nude mice.

[0842] 2. Test drugs and materials:

[0843] (1) Test drug: antibody drug conjugate: 3 mg / kg, 10 mg / kg.

[0844] (2) Experimental animals: Balb / c-nude female mice, 6-8 weeks old, weighing 20-22 g (Jisujiaokang), raised in a sterile animal room at 25°C, with 12h day-night alternation, free feeding and drinking.

[0845] 3. Experimental method:

[0846] (1) JIMT-1 mouse subcutaneous tumor model

[0847] Inoculation method: Take the logarithmic growth phase of JIMT-1 cells (DSMZ, ACC 589v), 300g, centrifuge for 7min. Count after washing with pre-cooled PBS twice, adjust the cell density to 1e8 / mL, mix with Matrigel thoroughly (1:1), divide into sterile 2mL EP tubes, and place on ice for use. Use 1mL sterile syringe to subcutaneously inoculate 100μL per mouse, and pay attention not to miss the liquid.

[0848] (2) Drug administration and tumor volume detection

[0849] When the tumor volume reaches 100-300mm 3 (around the 4th day after tumor inoculation), select tumor-bearing mice with similar tumor volumes for grouping and drug administration, 6 per group. Adopt tail vein administration, 2 times on day 0 and day 7. Use a vernier caliper to measure the tumor volume every 2-3 days, take the length (a) and width (b) of the tumor, and the volume = a x b x b / 2. Draw the tumor growth curve (tumor volume does not exceed 3000mm 3 , when it reaches 3000mm 3 , the mice are sacrificed), and detect the body weight change of the mice at the same time.

[0850] (3) Data analysis:

[0851] All experimental results are expressed as mean ± standard error, and the tumor volumes of each group are statistically analyzed using Graphpad Prism 7.0 software. For tumor volume, one-way ANOVA and Dunnett's test are used for statistical analysis to evaluate the differences between groups for the solvent control group and all treatment groups, and p<0.05 is considered to have significant difference.

[0852] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0853] T / C(%)=(T-T0) / (C-C0)×100, wherein T, C are the tumor volumes of the experimental group and the control group at the end of the experiment; T0, C0 are the tumor volumes at the beginning of the experiment.

[0854] Tumor inhibition rate (TGI) (%) = 100-T / C (%).

[0855] When the tumor regresses, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100, if the tumor is smaller than the initial volume, i.e. T<T0 or C<C0, it is defined as partial regression of tumor (PR); if the tumor completely disappears, it is defined as complete regression of tumor (CR).

[0856] The average tumor volume change, relative tumor growth rate and tumor inhibition rate of the above test are summarized in Figure 1 and Table 1, which shows that the detected antibody drug conjugate can effectively inhibit the growth of JIMT-1 transplanted tumor in nude mice, and the tumor inhibition effect on the same batch of tumor cells is better than that of the reference 1 at the same dose.

[0857] The body weight changes of the experimental mice in the above test are summarized in Figure 2, and the results show that the body weight of the animals in each group is normal during the administration, indicating that the mice have good tolerance to the detected antibody drug conjugate.

[0858] Table 1:

[0859] Test Example 2: Efficacy evaluation of antibody drug conjugate in NCI-N87 transplanted tumor mice

[0860] 1. Test purpose:

[0861] The purpose of this experiment is to detect the efficacy of Her2 antibody ADC drug on human gastric cancer cell NCI-N87 transplanted tumor in nude mice.

[0862] 2. Test drugs and materials:

[0863] (1) Test drug: antibody drug conjugate: 2 mg / kg.

[0864] (2) Experimental animals: Balb / c-nude female mice, 6-8 weeks old, weighing 20-22 g (Jisujiaokang), were raised at 25°C in a 12-hour alternating light-dark sterile animal room, with free access to food and water.

[0865] 3. Experimental method:

[0866] (1) NCI-N87 mouse subcutaneous xenograft model

[0867] Inoculation method: Take NCI-N87 cells (Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 3111C0001CCC000481) in the logarithmic growth phase, 300 g, centrifuge for 7 min. Count after washing with pre-cooled PBS twice, adjust the cell density to 6e7 / mL, a total of 10 mL, mix well in PBS, divide into sterile 1.5 mL EP tubes, and place on ice for use. Use 1 mL sterile syringe to subcutaneously inoculate 100 μL per mouse, and pay attention not to miss the liquid.

[0868] (2) Drug administration and tumor volume detection

[0869] When the transplanted tumor volume reaches 100-300 mm 3 (8 days after tumor inoculation), select tumor-bearing mice with similar tumor volumes for grouping and drug administration, 5 mice per group. Tail vein administration was used, and administration was performed once on day 0. Use a vernier caliper to measure the tumor volume every 2-3 days, take the length (a) and width (b) of the tumor, and the volume = a x b x b / 2. Draw the tumor growth curve (tumor volume does not exceed 3000 mm 3 , and when the tumor volume reaches 3000 mm 3 , the mice are sacrificed), and detect the body weight change of the mice at the same time.

[0870] (3) Data analysis:

[0871] All experimental results are expressed as mean ± standard error, and the tumor volumes of each group are statistically analyzed using Graphpad Prism 7.0 software. For tumor volume, one-way ANOVA and Dunnett's test were used for statistical analysis to assess differences between groups, and p<0.05 was considered to have significant differences.

[0872] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0873] T / C (%) = (T-T0) / (C-C0) x 100, where T, C are the tumor volumes of the experimental group and the control group at the end of the experiment; T0, C0 are the tumor volumes at the beginning of the experiment.

[0874] Tumor inhibition rate (TGI) (%) = 100 - T / C (%).

[0875] When the tumor appears to regress, the tumor inhibition rate (TGI) (%) = 100 - (T - T0) / T0 x 100, if the tumor is reduced than the initial volume, i.e. T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor is completely disappeared, it is defined as complete tumor regression (CR).

[0876] The results of the average tumor volume change, relative tumor growth rate and tumor inhibition rate of the above test are summarized in Figure 3 and Table 2, which shows that the tested antibody drug conjugate can effectively inhibit the growth of NCI-N87 transplanted tumor in nude mice.

[0877] The body weight change of the experimental mice in the above test is summarized in Figure 4, which shows that the body weight of the animals in each group is normal during the administration, suggesting that the mice are well tolerated to the tested antibody drug conjugate.

[0878] Table 2:

[0879] Although the embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

A method of preparing a compound of formula (A): which comprises the steps of: reacting a compound of formula (B) with a compound of formula (C) to form a compound of formula (A): Alternatively, the amino acid residue A'4 of the compound of formula (D) is reacted with the amino acid residue A'5 of the compound of formula (E) to form a peptide bond to form the compound of formula (A): wherein *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; T is T 1a - L 1b - L 1c - L 1d - L 1e - T 1a selected from LG is a leaving group; preferably, LG is wherein R is selected from C 1-6 alkyl, C 1-6 haloalkyl, -OR a , -NR a R b , C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl, R is optionally substituted by m R'; m = 1, 2, 3, 4 or 5; each R' is independently selected from halogen, -NO2, -CN, -NR a R b , -NR a C(O)R b , C 1-6 alkyl, C 1-6 haloalkyl, -O-C 1-6 alkyl or phenyl; R a , R b is independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl; or R a , R b and the N atom to which they are attached form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl; L 1b selected from C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene, wherein each CH2in said C 1-10 alkylene, C 2-10 alkenylene and C 2-10 alkynylene is optionally substituted with 1, 2 or 3 R x ; each R x is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; or, two R x may be joined to form a C 3-10 cycloalkylene or 3-10 membered heterocyclylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-; L 1d selected from a chemical bond, C 1-8 alkylene or -(CH2CH2O) n -C 1-4 alkylene; said C 1-8 alkylene is optionally substituted with -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl; n and w are independently selected from 1, 2, 3, 4 or 5; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-; L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or unnatural amino acid residues, having the structure -A1-A2-A3-A4-A5-; A1, A2, A3, A4 and A5 are independently selected from a chemical bond, a divalent residue of an optionally substituted natural amino acid or a divalent residue of an optionally substituted unnatural amino acid, wherein at least two are not a chemical bond; A'4 and A'5 are independently a monovalent residue of an optionally substituted natural amino acid or a monovalent residue of an optionally substituted unnatural amino acid; each amino acid bivalent residue or monovalent residue is optionally substituted with 1, 2, or 3 R y substituted; each R is independently selected from H, halogen, C y independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; L3is an optional substituted or unsubstituted spacer, for example -NH2-CH2-, L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or C 1-6 haloalkoxy; L D2 is selected from -NH-, -0-, -C(O)-, -NHC(O)-, or -C(0)NH-; q is 0, 1, 2, 3 or 4, preferably 1, 2 or 3; R D1 and R D2 are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D1 , R D2 and the carbon atom to which they are attached form a C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably 5-6 membered heterocyclyl; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; or, R D3 , R D4 and the carbon atom to which they are attached form a C 3-7 cycloalkylene or 3-7 membered heterocyclyl, preferably a C 3-5 cycloalkylene; R D5 selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -C 1-6 alkylene-C 3-10 cycloalkyl, -C 1-6 alkylene-3-10 membered heterocyclyl, -C 1-6 alkylene-C 6-10 aryl or -C 1-6 alkylene-5-10 membered heteroaryl, preferably -C 1-6 alkylene-C 3-10 cycloalkyl. The method of claim 1, wherein *1 and *2 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably *1 is in (S) configuration; T is T 1a - L 1b - L 1c - L 1d - L 1e - T 1a for Preferably For example LG is wherein R is selected from C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, 3-7 membered heterocyclyl, phenyl or 5-6 membered heteroaryl, preferably selected from C 1-6 alkyl or C 1-6 haloalkyl, R is optionally substituted with m R'; preferably R is selected from methyl, p-nitrophenyl, p-nitrile phenyl, p-tolyl, p-trifluoromethylphenyl, p-chlorophenyl, preferably methyl; m = 1, 2, 3, 4 or 5; each R' is independently selected from halogen, -N02, -CN, C 1-6 alkyl or C 1-6 haloalkyl; L 1b selected from C 1-6 alkylene, C 2-6 alkenylene or C 2-6 alkynylene, preferably C 2-6 alkynylene, wherein each CH2in said C 1-6 alkylene, C 2-6 alkenylene and C 2-6 alkynylene is optionally substituted with 1 or 2 R x ; preferably L 1b is each R is independently selected from H, halogen, or C x is independently selected from H, halogen, or C 1-6 alkyl; or, two R groups on any identical or different carbon atom can be joined to form a C x may be joined to form a C 3-7 cycloalkylene or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkylene, preferably C 3-5 cycloalkylene, more preferably cyclopropylene; L 1c is selected from a chemical bond, -C(O)-, -C(O)NH-, or -NHC(O)-, preferably a chemical bond or -C(O)NH-; L 1d is selected from a chemical bond or C 1-6 alkylene, preferably a chemical bond or C 1-4 alkylene; said C 1-6 alkylene is optionally substituted by -NHC(O)-(CH2CH2O) w -C 1-4 alkyl or -C(O)NH-(CH2CH2O) w -C 1-4 alkyl, preferably optionally substituted by -C(O)NH-(CH2CH2O) w -C 1-4 alkyl; w is selected from 2, 3 or 4, preferably 3; L 1e is selected from -C(O)-, -NHC(O)- or -C(O)-NHC(O)-, preferably -C(O)-; L2 is selected from a divalent peptidyl group comprising 2 to 5 optionally substituted natural amino acid residues or unnatural amino acid residues, having the structure -A1-A2-A3-A4-A5-; A1, A2, A3, A4 and A5 are independently selected from a chemical bond, a divalent residue of gly, val, cit, gln, glu, phe, lys, leu or ala, wherein at least two are not a chemical bond; preferably A2 is a chemical bond; preferably L2 is selected from -gly-gly-, -gly-gly-gly-, -gly-gly-gly-gly-, -val-gly-gly-, -val-cit-gly-, -val-gln-gly-, -val-glu-gly-, -phe-lys-gly-, -leu-lys-gly-, -gly-val-lys-gly-, -val-lys-gly-gly-, -val-lys-gly-, -val-lys-ala-, -val-lys-leu-, -leu-leu-gly-, -gly-gly-phe-gly-, -gly-gly-phe-gly-gly-, -val-gly-, -val-cit- or val-lys-β-ala-, preferably -gly-gly-phe-gly-; A'4 and A'5 are independently selected from a monovalent residue of gly, val, cit, gln, glu, phe, lys, leu or ala; preferably A'4 is -phe; preferably in formula (2), -A1-A2-A3-A'4 is -gly-gly-phe; preferably A'5 is -gly; each amino acid bivalent residue or monovalent residue is optionally substituted with 1, 2, or 3 R y substituents; each R is independently selected from H, halogen, C y is independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; L3 is -NH2-CH2-; L3is optionally substituted with 1, 2, or 3 R z substituents, each R z is independently selected from H, halo, or C 1-6 alkyl; L D2 is selected from -NH- or -O-, preferably -O-; q is 1, 2 or 3, preferably 1; R D1 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably C 1-6 alkyl, for example Me; R D2 selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably halogen, e.g. F; each R D3 and R D4 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxy; preferably, R D3 is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl, preferably H or C 1-6 alkyl (e.g. methyl); preferably, R D4 is H; R D5 selected from -C 1-4 alkylene-C 3-5 cycloalkyl or -C 1-4 alkylene-3- to 5-membered heterocyclyl, preferably -C 1-4 alkylene-C 3-5 cycloalkyl, for example -CH2-cyclopropyl. The method of claim 1, wherein, The compound of formula (A) is selected from: wherein *1, *2, *3 and *4 are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably *3 is in (S) configuration; preferably *4 is in (S) configuration; R D3 as defined in any one of claims 1-3; Preferably, The compound of formula (A) is selected from: The method of any one of claims 1-3, further comprising the step of: reacting a compound of formula (F) with a compound of formula (G) to form a compound of formula (B): or, reacting a compound of formula (H) with a compound of formula (I) to form a compound of formula (B): wherein, PG1is a carboxyl protecting group, for example NH2-L'2- is a monovalent group when the amino terminus of the peptide chain of the peptide group L2 is not attached to T; In formula (3), T is T 1a -L 1b -L 1c -L 1d -C(O)-; In formula (4), T is T 1a -L 1b -C(O)NH-L 1d -L 1e -; the remaining variables are as defined in any one of claims 1 to 3; preferably L in formula (4) 1d is not a chemical bond; Preferably, further comprising the step of: carboxyl group of the amino acid residue A'4 of the compound of formula (G1) is reacted with a compound of formula (G2) to form a compound of formula (G3), which is then deprotected to form a compound of formula (G): wherein A'4 is as defined in any one of claims 1-3, and the amino terminus is attached to A3, the carboxyl group participates in the reaction; A5 is as defined in any one of claims 1-3, and the carboxyl terminus is attached to L3; PG2 and PG3 are independently an amino protecting group; preferably the amino protecting group is selected from the group consisting of alkoxycarbonyl protecting groups, acyl protecting groups or alkyl protecting groups; preferably alkoxycarbonyl protecting groups, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl; preferably PG2 is Cbz; preferably PG3 is Fmoc; PG4 is a carboxyl protecting group, for example, C 1-10 alkyl (preferably C 1-6 alkyl, for example, methyl, ethyl, or tert-butyl), methoxymethyl (MOM), benzyl, p- methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2- methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl, 2-tetrahydropyranyl, 2-tetrahydrofuranyl or -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; preferably selected from benzyl, p-methoxybenzyl (PMB) or 2,4-dimethoxybenzyl (DMB); preferably benzyl; L2 is as defined in any one of claims 1-3, and the carboxyl terminus is attached to L3; NH2-L'2- is a monovalent group when the amino terminus of the peptide chain of the peptide group L2 is not attached to T; the remaining variables are as defined in any one of claims 1-3; Preferably, further comprising the step of: reacting a compound of formula (F1) with a compound of formula (F2) to form a compound of formula (F3): wherein PG8is a carboxyl protecting group, for example C 1-10 alkyl (preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl), methoxymethyl (MOM), benzyl, p- methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2- methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl, 2-tetrahydropyranyl, 2- tetrahydrofuranyl or -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; preferably C 1-10 alkyl (preferably C 1-6 alkyl), for example methyl, ethyl or tert-butyl, preferably tert-butyl; L 1c -C(O)NH-; the remaining variables are as defined in any one of claims 1-3; Preferably, the method is performed in the presence of a solvent; preferably the solvent is selected from the group consisting of DCM, DCE, THF, benzene, toluene, xylene, DMF, DMA and NMP; preferably from the group consisting of DCM, DCE, THF, benzene, toluene and xylene; preferably from the group consisting of DCM and DCE, more preferably DCM; Preferably, the method is performed in the presence of a condensing agent; preferably the condensing agent is an onium salt condensing agent, such as HATU, HBTU, HCTU, BOP or PyBOP, preferably HATU; Preferably, the method is performed in the presence of a base; preferably the base is selected from the group consisting of inorganic bases and organic bases, preferably organic bases; Preferably, the inorganic base is selected from the group consisting of carbonates, bicarbonates, phosphates, hydrogenophosphates, dihydrogenophosphates, hydroxides or hydrides of alkali metals and alkaline earth metals, for example LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4 or NaH; Preferably, the organic base is selected from the group consisting of alkali metal and alkaline earth metal alkoxides and organic amine bases, preferably organic amine bases; Preferably, the organic amine base is selected from the group consisting of triethylamine, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine, preferably DIPEA; Preferably, the alkali and alkaline earth metal alkoxides are selected from the group consisting of NaOMe, KOMe, NaOEt, KOEt, NaOtBu and KOtBu. The method of any one of claims 1-4, wherein, The compound of formula (B) is selected from: wherein *2, *3 and *4 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably *3 is in (S) configuration; preferably *4 is in (S) configuration; R D3 as defined in any one of claims 1-3; Preferably, The compound of formula (B) is selected from: The method of claim 4 or 5, wherein, The compound of formula (G) is represented by the structure of formula (G-a): wherein *2 and *3 are chiral centers independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably *3 is in (S) configuration; R D3 as defined in any one of claims 1-3; Preferably, The compound of formula (G) is selected from: The method of any one of claims 1-6, wherein, the compound of formula (C) is selected from the group consisting of: preferably Preferably The method according to any one of claims 1 to 7, further comprising the step of: reacting a compound of formula (C1-a) with a compound of formula (C2) to form a compound of formula (C3-a): wherein, PG5 is an amino protecting group, preferably an acyl-type protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), formyl, acetyl (Ac) and benzoyl (Bz), preferably Ac; the remaining variables are as defined in any one of claims 1 to 3; The method is carried out in the presence of a solvent, which is a mixed solvent of a benzene-based solvent and a phenol-based solvent; Preferably, the benzene-based solvent is selected from the group consisting of benzene, toluene and xylene, preferably toluene; Preferably, the phenol-based solvent is selected from the group consisting of phenol, o-cresol, m-cresol and p-cresol, preferably from phenol and o-cresol, preferably o-cresol; Preferably, the volume ratio of benzene-based solvent:phenol-based solvent is 30-1 :1, preferably 25-5:1, preferably 20-10:1, such as 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11 :1 or 10:1 ; Preferably, the method is carried out in the presence of a catalyst; Preferably, the catalyst is an acid catalyst, preferably selected from the group consisting of pyridinium p-toluenesulfonate, p-toluenesulfonic acid (TsOH), camphorsulfonic acid ((-)-CSA), methanesulfonic acid (MsOH) and trifluoromethanesulfonic acid (TfOH), preferably selected from the group consisting of pyridinium p-toluenesulfonate, p-toluenesulfonic acid and camphorsulfonic acid, preferably pyridinium p-toluenesulfonate; Preferably, the method further comprises the step of: subjecting a compound of formula (C3-a) to a hydroxyl protection reaction to form a compound of formula (C4-a): PG6is a hydroxyl protecting group, preferably selected from silyl ether protecting groups, alkyl ether protecting groups or alkyl methyl ether protecting groups; preferably a silyl ether protecting group, such as -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; such as trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS) or t-butyldiphenylsilyl (TBDPS), preferably TES; the remaining variables are as defined in any one of claims 1 to 3; Preferably, the method further comprises the step of: deprotecting a compound of formula (C4-a) to form a compound of formula (C5-a), followed by an amino protection reaction to form a compound of formula (C6-a): wherein PG7 is an amino protecting group, preferably an alkoxycarbonyl-type protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl; preferably Boc; the remaining variables are as defined in any one of claims 1 to 3; Preferably, the method further comprises the step of: deprotecting a compound of formula (C6-a) to form a compound of formula (C-a): wherein the variables are as defined in any one of claims 1 to 3. The method according to any one of claims 1 to 8, further comprising the step of: reacting a compound of formula (C8) with an oxidizing agent to form a compound of formula (C7): wherein, PG9is a carboxyl protecting group, for example C 1-10 alkyl, preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl, preferably ethyl; R4is selected from C 1-9 alkyl, preferably C 1-5 alkyl, preferably C 1-3 alkyl, for example methyl; R D5 as defined in any one of claims 1-3; Preferably, the method further comprises the step of: heating a compound of formula (C9) to form a compound of formula (C8): wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C10) with nitrous acid and / or a nitrite salt to form a compound of formula (C9): wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C11) with a compound R4-C(O)OC(O)-R4 to form a compound of formula (C10): wherein the variables are as defined above; Preferably, the method further comprises the step of: wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C12) with a compound of formula (R) D5 to form a compound of formula (C11): wherein X is selected from halogen, such as F, Cl, Br or I, preferably Br; the remaining variables are as defined above; Preferably, the method further comprises the step of: deprotecting the compound of formula (C7) to form a compound of formula (Cl): wherein R D5 as defined in any one of claims 1-3; Preferably, said compound of formula (C7) is selected from: Preferably, the reaction to form the compound of formula (C1) is deprotection of a compound of formula (C7-a) to form a compound of formula (C1-a): wherein R D5 as defined in any one of claims 1-3. The method according to any one of claims 1 to 3, further comprising the step of: reacting a compound of formula (F) with a compound of formula (J) to form a compound of formula (D): wherein A'1is an optionally substituted natural amino acid monovalent residue or an optionally substituted non-natural amino acid monovalent residue, preferably a monovalent residue selected from gly, val, cit, gln, glu, phe, lys, leu or ala, preferably a monovalent residue of gly, which is optionally substituted with 1, 2 or 3 R y substituted; each R is independently selected from H, halogen, C y halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; preferably from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; A'1carboxyl end is connected to A2, and the amino group participates in the reaction; the remaining variables are as defined in any one of claims 1 to 3. The method of any one of claims 1-3 and 10, wherein, The compound of formula (D) is selected from: The method according to any one of claims 1 to 3 and 10 to 11, further comprising the step of: reacting a compound of formula (E1) with a compound of formula (C) to form a compound of formula (E'), and deprotecting the compound of formula (E') to form a compound of formula (E): wherein, A5and PG3are as defined in claim 4; the remaining variables are as defined in any one of claims 1 to 3. The method of any one of claims 1-3 and 10-12, wherein, said compound of formula (E) is selected from the group consisting of: Compounds of formula (B): wherein the variables are as defined in any one of claims 1 to 3; Preferably, the compound of formula (B) is selected from: wherein *2, *3and *4are chiral centers, independently selected from (S) or (R) absolute configuration, or a mixture thereof; preferably *3is in (S) configuration; preferably *4is in (S) configuration; R D3 as defined in any one of claims 1-3; Preferably, The compound of formula (B) is selected from: Compounds of formula (E): wherein the variables are as defined in any one of claims 1 to 3; Preferably, the compound of formula (E) is selected from: a compound of formula (D): T-A1-A2-A3-A'4 D wherein the variables are as defined in any one of claims 1 to 3; Preferably, the compound of formula (D) is selected from: Compounds of formula (C7): wherein R D5 as defined in any one of claims 1-3; Preferably, said compound of formula (C7) is selected from: Compounds of formula (C1): wherein R D5 as defined in any one of claims 1-3; Preferably, the compound of formula (C1) is selected from: A method for preparing a compound of formula (B), comprising the step of: reacting a compound of formula (F) with a compound of formula (G) to form a compound of formula (B): or, reacting a compound of formula (H) with a compound of formula (I) to form a compound of formula (B): wherein, PG1is a carboxyl protecting group, for example NH2-L'2- is a monovalent group when the amino end of the peptide chain of the peptide group L2is not connected to T; In formula (3), T is T 1a -L 1b -L 1c -L 1d -C(O)-; In formula (4), T is T 1a -L 1b -C(O)NH-L 1d -L 1e -; the remaining variables are as defined in any one of claims 1 to 3; preferably L in formula (4) 1d is not a chemical bond. A method for preparing a compound of formula (C3-a): reacting a compound of formula (C1-a) with a compound of formula (C2) to form a compound of formula (C3-a): wherein PG5is an amino protecting group, preferably an acyl-type protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), formyl, acetyl (Ac) and benzoyl (Bz), preferably Ac; the remaining variables are as defined in any one of claims 1 to 3. The method is carried out in the presence of a solvent, which is a mixed solvent of a benzene-based solvent and a phenol-based solvent; Preferably, the benzene-based solvent is selected from benzene, toluene and xylene, preferably toluene; Preferably, the phenol-based solvent is selected from phenol, o-cresol, m-cresol and p-cresol, preferably selected from phenol and o-cresol, preferably o-cresol; Preferably, the volume ratio of the benzene-based solvent:phenol-based solvent is 30-1:1, preferably 25-5:1, preferably 20-10:1, such as 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1; Preferably, the method is carried out in the presence of a catalyst; Preferably, the catalyst is an acid catalyst, preferably selected from pyridinium p-toluenesulfonate, p-toluenesulfonic acid (TsOH), camphorsulfonic acid ((-)-CSA), methanesulfonic acid (MsOH) and trifluoromethanesulfonic acid (TfOH), preferably selected from pyridinium p-toluenesulfonate, p-toluenesulfonic acid and camphorsulfonic acid, preferably pyridinium p-toluenesulfonate. A method for preparing a compound of formula (E): reacting a compound of formula (E1) with a compound of formula (C) to form a compound of formula (E'), and deprotecting the compound of formula (E') to form a compound of formula (E): wherein A5and PG3are as defined in claim 4; the remaining variables are as defined in any one of claims 1 to 3. A method for preparing a compound of formula (F3): reacting a compound of formula (F1) with a compound of formula (F2) to form a compound of formula (F3): wherein PG8 is a carboxyl protecting group, for example C 1-10 alkyl (preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl), methoxymethyl (MOM), benzyl, p- methoxybenzyl (PMB), 2,4-dimethoxybenzyl (DMB), benzyloxymethyl, 2- methoxyethoxymethyl, benzhydryl, pentafluorophenyl, allyl, 2-tetrahydropyranyl, 2- tetrahydrofuranyl or -SiR1R2R3, wherein R1, R2, R3are independently selected from C 1-10 alkyl or C 6-10 aryl, preferably selected from C 1-6 alkyl and phenyl; preferably C 1-10 alkyl (preferably C 1-6 alkyl), for example methyl, ethyl or tert-butyl, preferably tert-butyl; L 1c -C(O)NH-; the remaining variables are as defined in any one of claims 1 to 3; the remaining variables are as defined in any one of claims 1 to 3. Preferably, the method is carried out in the presence of a solvent; preferably, the solvent is selected from DCM, DCE, THF, benzene, toluene, xylene, DMF, DMA and NMP; preferably from DCM, DCE, THF, benzene, toluene and xylene; preferably from DCM and DCE, more preferably DCM; Preferably, the method is carried out in the presence of a condensing agent; preferably, the condensing agent is an onium salt condensing agent, such as HATU, HBTU, HCTU, BOP or PyBOP, preferably HATU; Preferably, the method is carried out in the presence of a base; preferably, the base is selected from inorganic bases and organic bases, preferably organic bases; Preferably, the inorganic base is selected from carbonates, bicarbonates, phosphates, hydrogenophosphates, dihydrogenophosphates, hydroxides or hydrides of alkali and alkaline earth metals, for example, LiOH, NaOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, NaHCO3, KHCO3, Na3PO4, K3PO4, K2HPO4, KH2PO4or NaH; Preferably, the organic base is selected from alkoxides of alkali and alkaline earth metals and organic amine bases, preferably organic amine bases; Preferably, the organic amine base is selected from triethylamine, DIPEA, NMM, DBU, DABCO, DMAP, imidazole, pyridine, 2,6-dimethylpyridine and 2,2,6,6-tetramethylguanidine, preferably DIPEA; Preferably, the alkoxides of alkali and alkaline earth metals are selected from NaOMe, KOMe, NaOEt, KOEt, NaOtBu and KOtBu. A method of preparing a compound of formula (C7) comprising the step of: reacting a compound of formula (C8) with an oxidizing agent to form a compound of formula (C7): wherein PG9 is a carboxyl protecting group, for example C 1-10 alkyl, preferably C 1-6 alkyl, for example methyl, ethyl or tert-butyl, preferably ethyl; R4is selected from C 1-9 alkyl, preferably C 1-5 alkyl, preferably C 1-3 alkyl, for example methyl; R D5 as defined in any one of claims 1-3; Preferably, the method further comprises the step of: heating a compound of formula (C9) to form a compound of formula (C8): wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C10) with nitrous acid and / or a nitrite salt to form a compound of formula (C9): wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C11) with a compound R4-C(O)OC(O)-R4 to form a compound of formula (C10): wherein the variables are as defined above; Preferably, the method further comprises the step of: reacting a compound of formula (C12) D5 with a compound of formula (C13) to form a compound of formula (C14): wherein X is selected from halogen, for example F, CI, Br or I, preferably Br; the remaining variables are as defined above. A method of preparing a compound of formula (C1): deprotecting the compound of formula (C7) to form a compound of formula (Cl): wherein R D5 as defined in any one of claims 1-3; Preferably, said compound of formula (C7) is selected from: Preferably, the reaction to form the compound of formula (C1) is deprotection of a compound of formula (C7-a) to form a compound of formula (C1-a): wherein R D5 as defined in any one of claims 1-3. A method of preparing an antibody drug conjugate (ADC), wherein, comprising coupling a compound of formula (A) to an antibody; the compound of formula (A) being as defined in any one of claims 1-3.

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