CN109912683B

CN109912683B - Cytotoxin molecule, conjugate, preparation method and application thereof

Info

Publication number: CN109912683B
Application number: CN201711344355.2A
Authority: CN
Inventors: 赵永新; 杨庆良; 黄圆圆; 赵林尧; 盖顺; 叶杭波; 杨成玉; 郭辉辉; 曹敏君; 徐怡芳; 雷俊; 郭智香; 贾军祥; 童茜茜; 蔡湘; 白露; 张秀珍; 孔橡飞; 李雯君; 杜勇
Original assignee: Hangzhou Dac Biotech Co Ltd
Current assignee: Hangzhou Dac Biotech Co Ltd
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2023-01-06
Anticipated expiration: 2037-12-13
Also published as: CN115505029A; CN109912683A

Abstract

The invention relates to a Tubulysin derivative cytotoxin molecule, a conjugate (conjugate) formed by coupling the Tubulysin derivative cytotoxin molecule with a cell surface receptor molecule, a synthetic method of the conjugate, a conjugate preparation and application of the conjugate in the aspects of targeted therapy of cancer, autoimmune system diseases and infectious diseases.

Description

Cytotoxin molecule, conjugate, preparation method and application thereof

Technical Field

The invention relates to a Tubulysin derivative cytotoxin molecule, a conjugate (conjugate) formed by coupling the Tubulysin derivative cytotoxin molecule with a cell surface receptor molecule, a conjugate synthesis method, a conjugate preparation and application of the conjugate in the aspect of targeted therapy of cancer, autoimmune system diseases and infectious diseases.

Background

Targeted treatment of cancer, immunodeficiency, infectious diseases, and the like is the core of current precision medical attention. Many documents have reported the use of cell surface receptor binding molecules as drug delivery vehicles to form conjugates with cytotoxic molecules for the targeted delivery of cytotoxic molecules to attack various pathogenic cells (Allen, T.M. and Cullis, P.R.,2004science,303 (5665), 1818-22, hu, Q.Y., F.Berti, et al (2016), chem Soc Rev 45 (6): 1691-1719). The cell surface receptor binding molecule-small molecule drug conjugate can improve the pharmacokinetics and pharmacodynamics of traditional chemotherapy small molecule drugs, and simultaneously reduce the toxic and side effects of traditional chemical drugs in human bodies (Alley, S.C., N.M. Okeley, et al. (2010) Curr Opin Chem Biol 14 (4): 529-537 kolhe, P., J.Khandare, et al. (2006) Biomaterials 27 (4): 660-669). Cell surface receptor binding molecules have been reported as drug delivery vehicles: peptide molecules (Yeh, C.Y, hsiao, J.K, et al.2016biomaterials 99,1-15, shen, Y.A., liu, C.S, et al.2015, cancer Lett.360 (1): 39-47 trimming, et al, bioconjugate chem.2006,17, 1385-1394); antibodies (Ducry, l. (2013), antibody-drug conjugates, new York, humana Press; springer, ISBN 9781627035408, in Immunoconjugates 189-216 (C. Vogel, ed.1987), ghose et al, in Targeted Drugs 1-22 (E.Goldberg, ed.1983), diene et al, in labeled delivery systems 1-23 (J.Rodwell, ed.1988), silverstein, nat.Immunol.2004,5,1211-7 fanning et al, clin.Immunol.Immunopathol.1996,79,1-14 Ricart A.D., et al, nature Clinical Practice 2007,4,245-255, singh R.Rickson H.K. Therapeutic, methods: 467, 2009; small molecule classes such as folic acid (Kumar, R., W.S.shin, et al (2015) Chem SocRev 44 (19): 6670-6683; single domain antibodies or antibody analogs (Wold, e.d., j.y.axup, et al. (2015), bioconjugate Chem 26 (12): 2311-2314); prostate specific membrane antigen binding ligand (PMSA) (Low, et al, WO 2009/026177 A1); cobalamin and proteins (Gupta, et al, crit. Rev. Therap. Drug Carrier Syst.2008,25,347-79; carbohydrate molecules (darby, et al, curr. Top. Med. Chem.2008, 8,1286-93; biologically active macromolecules (Dhar, et al, proc. Natl. Acad. Sci.2008,105,17356-61, chang, M., F. Zhang, et al (2016), J Drug Target 24 (6): 475-491.); macromolecules containing antibodies or protein molecules (Rihova, B. (1995), folia Microbiol (Praha) 40 (4): 367-384); dendritic polymers (Bai, S., C.Thomas, et al, (2006) Crit Rev the Drug Carrier Syst 23 (6): 437-495, lee, et al, nat.Biotechnol.2005,23,1517-26 Almutairi, et al, proc.Natl.Acad.Sci.2009,106, 685-90; nanoparticles having a binding ligand attached thereto (Ma, P.and R.J. Mumper (2013), nano Today 8 (3): 313-331, fernandes, E.et al, (2015), J Control Release 209, 288-307 Liong, et al, ACS Nano,2008,19,1309-12, medarova, et al, nat. Med.2007,13,372-7 Javier, et al, bioconjugate Chem.2008,19,1309-12 Sheikhpour, M.et al,2017, J.Control.Rel,253, 97-109; proteases or proteins (e.g., albumin) (Veronese, F.M.and M.Morpurgo (1999), farmaco 54 (8): 497-516; liposomes (Medinai, et al, curr. Phar. Des.2004,10, 2981-9); viral capsids (Flenniken, et al, virus Nanotechnol.2009,327, 71-93), and the like. The clinically mature applications of the above cell surface receptor-binding molecules are antibody molecules. Currently, the U.S. Food and Drug Administration (FDA) has approved 4 monoclonal antibody-drug conjugate molecules for targeted therapy of cancer, and the 4 conjugate drugs are Gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, and Inotuzumab ozogamicin.

The antibody-drug conjugate consists of three parts: antibodies (often monoclonal antibodies), cytotoxic molecules and linkers linking the two (Thomas, a., b.a. teicher, et al. (2016), lancet Oncol 17 (6): e254-e 262). The three have unique functions: antibodies need to bind specifically to tumor cells, cytotoxic molecules need to be sufficiently active and broad for tumor cells, linkers need to be uniquely functional-stable in the blood circulation, and efficiently release cytotoxic molecules upon reaching tumor cells (Chari, r.v. (2008), acc Chem Res 41 (1): 98-107), all three of which are rationally constructed to achieve good clinical structure (Singh, s.k., d.l.luisi, et al.352015, pharm Res 32 (11): 41-3571 hamilton, g.s. (2015), biogicals 43 (5): 318-332. Many kinds of cytotoxins have been used to form antibody drug conjugates with cell binding bodies, particularly antibodies (Wu, et al, nat. Biotechnol.2005,23,1137-1146.Ricart, et al, nat. Clin.practice. Oncol.2007,4, 245-255). These cytotoxins include: calicheamicin derivatives (Giles, et al Cancer 2003,98,2095-104, hamann, et al, bioconjugate Chem 2002,13,47-58, nicolaou, K.C. et al, science 1992,256,1172-1178, proc. Natl.Acad.Sci. USA.1993,90, 5881-8); maytansine derivatives (Zhao, r.et al, j.med.chem.2011,54,3606-3623, widdison, et al, j Med Chem 2006,49,4392-408, ikeda, et al, clin Cancer Res 2009,15,4028-37 xie, et al, expert Opin Biol Ther 2006,6,281-91; U.S. Pat. nos. 6,441,163, 716,821,7,276,497,7, 019,301,7,303,749,7,368,7,411,063,7,851,432, and 8,163,888; auristatins (Sutherland, et al, J Biol Chem 2006,281,10540-7, doronina, et al, bioconjug Chem 2006,17,114-24; U.S. Pat. Nos. 783778373702338, 7964566,7964567,7851437, 7994135.); tubulysins (Wipf, P., et al. Org. Lett.,2004,6,4057-60, pando, O., et al. J. Am. Chem. Soc.,2011,133,7692-5, domling, A., et al, angew Chem Int Ed Engl,2006,45,7235-9, PCT/IB 2012/053554) Taxus class drug derivatives (Miller, et al, J Med Chem 2004,47,4802-5, K.C.Nicolaou et al, J.am. Chem. Soc.117,2409-20 (1995); ojima et al, J.Med.chem.39:3889-3896 (1996); U.S. Pat. Nos. 7,276,499, 8,290 and 7,667,054 WO 06061258), leptomycin derivatives (WO 07144709), CC-1065 and its analogs (ZHao, R., et al, J.Med.chem.55, 766-782, suzawa, et al, J Control Release 2002,79,229-42, suzawa, et al, bioorg Med Chem 2000,8,2175-84, D.Boger et al, J.org.chem;66, 6654-61,2001; amatoxin type (Moldenhauer, G., et al, J.Natl.cancer Inst.2012,104,622-34, A.Moshannikova, et al biochemistry 2013,52,1171-8, ZHao, L., et al, chemiochem, 2015.16 (10): 1420-5 ZHou, B., et al, biosens bioelectrron, 2015.68; exatecan derivatives (a camptothecin molecule) (Nakada, T. Masuda, et al. (2016), bioorg Med Chem Lett 26 (6): 1542-1545); spliceosome inhibitors (Puthenvetoil, S., F. Logani, et al. (2016), bioconjugate Chem 27 (8): 1880-1888); benzo (o) diazepoxide (PBD) dimer species (U.S. patent nos. 8,163,736; adriamycin (Trail, et al, science 1993,261,212-5, saleh et al, J Clin Oncol 2000,18,2282-92, yang, H.M., and Reisfeld, R.A., proc.Natl.Acad.Sci.85,1189-93 (1988); and methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, and chlorambucil derivatives, among others (Wang, J., H.Xiao, et al. (2017), trends Biotechnol 35 (5): 466-478, the Punvetil, S., F.Loganzo, et al. (2016), bioconju Chem 27 (8): 1880-1888).

Among the above cytotoxic compound molecules, drugs of the Tubulysin class are of interest for their potent biological activity, particularly in drug-resistant tumor cells (Cohen, R., D.J.Vugts, et al (2014), cancer Res 74 (20) 5700-5710 Xiaoingming, X., G.K.Friested, et al, mini Rev Med Chem 13 (11) 1572-1578 pando, S.Stark, et al (20), J Am Chem Soc 133 (20) Shibue, T., I.Okamoto, et al (2011), bioorg Chem Lett 21 (1) 431-434, K., U.S. 7695, magic Bl.T., 2010) and integer End et al (2011), chem [ 4842 ] 4842, U.S.23, st.S.21 (1) 431-434, U.S.S. K., chek., USA [ 11 ] 24, st, st.I.I.W.: sangeEnd.M.4828 (1) 488, st, st.S.S.S.S.21, st.S.S.C.21, st.S.23, st.J. (1). We have reported the construction of cell binding molecule conjugates using Tubulysin analogues and their application in targeted therapy (WO 2014/009774, huang, y.y.et al, american Society National meeting, med.chem. #44,2014, dever, co, usa). Here we further apply for conjugates constructed from Tubulysin-type molecules and the use of such conjugates for the targeted treatment of cancer, autoimmune and infectious diseases.

Disclosure of Invention

The invention provides a cytotoxic molecule, which has a structure shown in a formula I:

Or a pharmaceutically acceptable salt, hydrate or hydrated salt having the structure shown in formula I as a parent; or a polymorph having a structure of formula I, or an optical isomer of a structure of formula I;

the R is ¹ 、R ² 、R ³ And R ⁴ Independently selected from H; c ₁ ～C ₈ Alkyl or heteroalkyl of (a); c ₂ ～C ₈ Alkenylalkyl, alkynylalkyl, heterocycle, heterocycloalkyl of (a); c ₃ ～C ₈ Cycloalkyl, aryl, heteroaryl, aralkyl, alkylcarbonyl of (a); c ₄ ～C ₈ Alkylcycloalkyl, heteroalkylcycloalkyl;

the R is ⁵ 、R ⁶ 、R ⁸ 、R ¹⁰ And R ¹¹ Independently selected from-H or C ₁ ～C ₄ Alkyl or heteroalkyl of (a);

the R is ⁷ Independently selected from H, R ¹ 、-R ¹⁵ C(＝O)X ¹ R ¹⁶ or-R ¹⁵ X ¹ R ¹⁶ ；

Said X ¹ Selected from-O-, -S-S, -NH, -CH ₂ -or-NR ¹ -；

The R is ⁹ Selected from-H, -OH, = O, -OR ¹⁵ 、-OC(＝O)R ¹⁵ 、-OC(＝O)NHR ¹⁵ 、-OC(＝O)R ¹⁵ SSR ¹⁶ 、OP(＝O)(OR ¹⁵ ) OR OR ¹⁵ OP(＝O)(OR ¹⁶ )；

Said R is ¹² Is selected from-R ¹⁵ 、-OH、-SH、-NH ₂ 、-NH、-NHNH ₂ 、-NH(R ¹⁵ )、-OR ¹⁵ 、-R ¹⁵ COR ¹⁶ 、-R ¹⁵ COOR ¹⁶ 、-R ¹⁵ C(O)NH ₂ 、-R ¹⁵ C(O)NHR ¹⁷ 、-SR ¹⁶ 、R ¹⁵ S(＝O)R ¹⁶ 、-R ¹⁵ P(＝O)(OR ¹⁷ ) ₂ 、-R ¹⁵ OP(＝O)(OR ¹⁷ ) ₂ 、-CH ₂ OP(＝O)(OR ¹⁷ ) ₂ 、-R ¹⁵ SO ₂ R ¹⁷ 、-R ¹⁵ X ² R ¹⁶ 、-R ¹⁵ C(＝O)X ³ ；

Said X is ² Selected from-O-, -S-, -NH-, -NHNH-, -N (R) ¹⁵ )-、-O-R ¹⁵ -、-S-R ¹⁵ -、S(＝O)-R ¹⁵ -or-NHR ¹⁵ -one of the above;

said X is ³ Is selected from-OH, -SH, -NH ₂ 、-NH(R ¹⁵ )、-NHNH(R ¹⁵ )、-OR ¹⁵ 、-S-R ¹⁵ or-NR ¹⁵ R ¹⁶ One of (a) and (b);

the R is ¹³ And R ¹⁴ Independently selected from H, -OH, -SH, -NH ₂ 、-NHNH ₂ 、-NH(R ¹⁵ )、-OR ¹⁵ 、-COX ² 、-COX ² R ¹⁶ 、-R ¹⁷ 、-F、-Cl、-Br、-I、-SR ¹⁶ 、-NR ¹⁶ R ¹⁷ 、-N＝NR ¹⁶ 、-N＝R ¹⁶ 、-NO ₂ 、-SOR ¹⁶ R ¹⁷ 、-SO ₂ R ¹⁶ 、-SO ₃ R ¹⁶ 、-OSO ₃ R ¹⁶ 、-PR ¹⁶ R ¹⁷ 、-POR ¹⁶ R ¹⁷ 、-PO ₂ R ¹⁶ R ¹⁷ 、-OP(O)(OR ¹⁷ ) ₂ 、-OCH ₂ OP(O)(OR ¹⁷ ) ₂ 、-OC(O)OP(O)(OR ¹⁷ ) ₂ 、-PO(OR ¹⁶ )(OR ¹⁷ )、-OP(O)(OR ¹⁷ )OP(O)(OR ¹⁷ ) ₂ 、-OC(O)R ¹⁷ ，-OC(O)NHR ¹⁷ ；-O-(C ₄ -C ₁₂ Glycoside), -N- (C) ₄ -C ₁₂ Glycosides); c ₁ -C ₈ Alkyl, heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl; c ₃ -C ₈ Carbocyclyl, heterocycloalkyl, heteroalkylcycloalkyl, aryl, heteroaralkyl, alkylcarbonyl; c ₄ -C ₈ An alkyl cycloalkyl group; -NH (Aa) _1～4 or-CO (Aa) _1～4 (ii) a Said C is ₃ -C ₈ Carbocyclyl includes cycloalkyl; the (Aa) _1～4 Is 1 to 4 identical or different natural or unnatural amino acid units;

the R is ¹⁵ 、R ¹⁶ And R ¹⁷ Independently selected from C ₁ -C ₈ Alkyl, heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl, or C ₃ -C ₈ Carbocyclic, aryl, benzyl, alkylaryl, heterocycloalkylHeteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, C ₄ -C ₈ Alkylcycloalkyl, or Na ⁺ 、K ⁺ 、Cs ⁺ 、Li ⁺ 、Ca ²⁺ 、Mg ⁺ 、Zn ²⁺ 、N ⁺ (R ¹ )(R ² )(R ³ )(R ⁴ )、HN ⁺ (C ₂ H ₅ OH) ₃ The cationic salt of (a); said C is ₃ -C ₈ Carbocyclyl includes cycloalkyl;

said Y ¹ And Y ² Independently selected from N or CH; q is 0 or 1; when q =0,y ³ Is absent, Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Independently selected from CH, N, NH, O, S, or N (R) ¹ ) Thus Y is ² ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Together form a heteroaromatic ring of pyrrole, furan, thiophene, thiazole, oxazole, imidazole, triazole, tetrazole, thiadiazole, oxadiazole; when q =1,Y ³ ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Independently selected from CH or N, such that Y ² ，Y ³ ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Together form a heteroaromatic ring structure of benzene ring, pyridine, pyrazine, pyridazine, triazine, tetrazine.

The invention also provides a cytotoxic molecule, the group R ¹ 、R ² ，R ² 、R ³ Or R ³ 、R ⁴ through-CH ₂ Optionally linked to Y respectively ¹ The atoms forming a cyclic group, or R ⁵ 、R ⁶ through-CH ₂ -optionally attached to the carbon atom to which it is attached to form a cyclic group, or R ¹¹ 、R ¹² through-CH ₂ -optionally linked to the carbon atom to which it is attached and the carbonyl group to form a cyclic group, or R ¹³ 、R ¹⁴ through-CH ₂ -to Y optionally ⁵ And Y ⁶ The atoms form a cyclic group;

the cyclic group is selected from C ₃ -C ₇ Cycloalkyl, cycloalkylalkyl, cycloalkylheteroaryl, cycloalkylheteroalkyl, heterocyclic, heterocycloalkyl, cycloalkylcarbonyl, cycloalkylcarboxyl,One of a cycloalkaneamide group, a cycloalkaneamine group, a cycloalkoxy ether group, a cycloalkanethioether group, a cycloalkaneselenoether group, an aromatic group, a heteroaromatic alkyl group, and a heteroaromatic group.

The invention also provides a cytotoxic molecule, wherein R is ¹ 、R ² 、R ⁷ 、R ⁹ 、R ¹² 、R ¹³ And R ¹⁴ The terminal end of (A) further contains a functional group capable of coupling with a linker or with a cell surface binding receptor molecule, wherein the functional group is selected from SH and NH ₂ 、COOH、-O-NH ₂ 、-N ₃ 、NHNH ₂ -SSPy (Py is C) ₅ H ₄ N)、-SSAr、-SSC ₆ H ₄ NO ₂ 、-SSC ₆ H ₃ (NO ₂ )(COOH)、-SC(O)R ¹ 、-SSC ₆ H ₃ (NO ₂ ) ₂ 、-C(O)NH ₂ 、-C(O)H、-C(O)NHNH ₂ 、-C(O)R ¹ -C(O)C(O)R ¹ 、-ArC(O)R ¹ 、-C(O)CH ₂ X ³ 、-C(O)X ³ 、-ArCH ₂ X ³ 、

Said X ³ And X ⁴ Independently selected from F, cl, br, I, -OS (O) ₂ Ar、-OS(O) ₂ R ¹ 、-OS(O) ₂ CF ₃ )、-OC ₆ F ₅ 、-OC ₆ FH ₄ ,-OC ₆ F ₂ H ₃ ,-OC ₆ H ₄ (NO ₂ )、-OC ₆ H ₃ (NO ₂ ) ₂ 。

The invention also provides a conjugate taking the toxic molecule as a parent, which has a structure shown in a formula II:

or a pharmaceutically acceptable salt, hydrate or hydrated salt of structural formula (la); or a polymorph of this compound; or an optical isomer of this formula;

the T is a cell surface receptor binding molecule; said L and L' are independently cleavable linkers;

the- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -represents a linking bond and a linking site of L and an atom on the structure in parentheses;

N is an integer of 1 to 20; m is an integer of 1 to 10; m' is an integer of 0 to 10;

when m 'is 0, L' is absent; when m' is not 0, the conjugate is linked to the cell surface receptor binding molecule T via two or more linkers;

the two linkers are respectively connected with two sites on the structure shown in the parenthesis in the structure shown in the formula II, or the two linkers are connected with one site on the structure shown in the parenthesis in the structure shown in the structure II through the linker with branched chains;

the structural formulas of the linker L and L' are independently-Ww- (Aa) r-Vv-;

w is an extender linking target binding molecule unit T to amino acid unit (Aa) and, if Aa is not present, directly to V;

each of the units W independently comprises a self-immolative spacer, a peptide unit, a hydrazone bond, a disulfide bond, an ester bond, or an amine bond; w is 0 or 1;

aa is a natural amino acid or an unnatural amino acid unit; r is an integer of 0 to 12, and (Aa) r is 0 to 12 amino acid units with the same or different structures; the natural amino acid or unnatural amino acid units include a mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, decapeptide, undecapeptide, or dodecapeptide unit;

V is an insulator unit independently selected from H, O, NH, S, C ₁ -C ₈ Alkyl radical, C ₂ -C ₈ Heteroalkyl, alkenyl, alkynyl; c ₃ -C ₈ Aryl, heteroaryl, heterocyclyl, carbocyclyl, heteroalkylcycloalkyl, alkylcarbonyl; c ₄ -C ₈ An alkyl cycloalkyl group; 1 to 4 amino acid units or (CH) ₂ CH ₂ O) r; the heterocyclic group includes a heterocycloalkyl group; the carbon ring group comprises a cycloalkyl group; said (CH) ₂ CH ₂ O) r is an integer of 0 to 12; v is 0,1 or 2;

the cell surface receptor binding molecule T is any form of cell binding body, including a peptide or peptide-like structure: an antibody, a single-chain antibody, an antibody fragment capable of binding to a target cell, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment capable of binding to a target cell, a chimeric antibody fragment capable of binding to a target cell, a domain antibody fragment capable of binding to a target cell, an antibody-like genetically engineered protein, a fibronectin-bound adnectin, a predesigned ankyrin repeat protein (DARPin), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transporting molecule, a transferrin, a cell-surface small molecule ligand, or an albumin, a macromolecule, or a dendrimer to which a cell-bound body is attached, a polymeric material, a protein, a liposome, a nanoparticle, a vesicle, or a (viral) microcapsule containing a cell-binding molecule (binding peptide, protein, antibody, or cell-surface small molecule ligand) on the surface.

Drawings

FIG. 1 shows the synthesis of Tubulysin derivative fragment Tuv.

FIG. 2 shows the synthesis of a fragment of a Tubulysin derivative.

FIG. 3 shows the synthesis of a fragment of a Tubulysin derivative.

FIG. 4 shows a schematic representation of the synthesis of a fragment of a Tubulysin derivative.

FIG. 5 shows the synthesis of the Tubulysin derivative component.

FIG. 6 shows the synthesis of the Tubulysin derivative component.

FIG. 7 shows the synthesis of Tubulysin derivative components Tuv and Tup.

FIG. 8 shows the synthesis of Tubulysin derivative linker-containing Tup fragments.

FIG. 9 shows the synthesis of Tubulysin derivatives and conjugates thereof.

FIG. 10 shows the synthesis of Tubulysin derivatives and conjugates thereof.

FIG. 11 shows the synthesis of Tubulysin derivatives and conjugates thereof.

FIG. 12 shows the synthesis of Tubulysin derivatives and conjugates thereof.

Figure 13 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 14 shows the synthesis of antibody-Tubulysin derivative conjugates.

FIG. 15 shows the synthesis of an antibody, a Tubulysin derivative conjugate.

FIG. 16 shows the synthesis of a chiral Tubulysin derivative fragment.

FIG. 17 shows the synthesis of a fragment of a Tubulysin derivative.

FIG. 18 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 19 shows the synthesis of an antibody-Tubulysin derivative conjugate.

FIG. 20 shows the synthesis of a Tubulysin derivative capable of conjugation to form a bond.

Figure 21 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 22 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 23 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 24 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 25 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 26 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 27 shows the synthesis of antibody-Tubulysin derivatives.

FIG. 28 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 29 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 30 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 31 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 32 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 33 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 34 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 35 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 36 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 37 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 38 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 39 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 40 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 41 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 42 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 43 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 44 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 45 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 46 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 47 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 48 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 49 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 50 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 51 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 52 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 53 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 54 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 55 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 56 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 57 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

Figure 58 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 59 shows the synthesis of antibody-Tubulysin derivatives and conjugates thereof.

FIG. 60 shows the therapeutic effect of Her2 mAb-Tubulysin derivative conjugates (133, 129, 181, 317 and 467, drug/antibody molar ratio DAR = 3.5-4.0), together with the marketed drug T-DM1, on N87 (gastric cancer) cell animals. 133. 129, 317 and 467 have good tumor inhibition curative effects. Among them, 133, 129 and 467 have better curative effect than T-DM1.

FIG. 61 shows the therapeutic effect of Her2 monoclonal antibody-Tubulysin derivative conjugates (365, 377, 385, 412, 444, 474, 480 and 546, with drug/antibody molar ratio DAR = 3.5-4.0), together with the marketed drug T-DM1, on N87 (gastric cancer) cell in vivo in animals. 365. 377, 385, 412, 444, 474 and 480 have good tumor inhibition efficacy. Among them, 377, 385, 412, 444, 474 and 480 have better curative effect than T-DM1.

FIG. 62 shows the therapeutic effect of Her2 mAb-Tubulysin derivative conjugates (506, 522, 564, 574, 695 and 677, drug/antibody molar ratio DAR = 3.5-4.0), together with the marketed drug T-DM1, on N87 (gastric cancer) cell animals in vivo. 506. 522, 564, 574 and 677 have good tumor-inhibiting effects. 677 the curative effect is better than T-DM1.

Interpretation of related terms:

alkyl refers to linear or cyclic straight or branched aliphatic hydrocarbons containing 1 to 8 carbon atoms. Branched means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl group. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a tert-butyl group, an n-pentyl group, a 3-pentyl group, an octyl group, a nonyl group, a decyl group, a cyclopentyl group, a cyclohexyl group, a 2, 2-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 2, 2-dimethylpentyl group, a 2, 3-dimethylpentyl group, a 3, 3-dimethylpentyl group, a 2,3, 4-trimethylpentyl group, a 3-methylhexyl group, a 2, 2-dimethylhexyl group, a 2, 4-dimethylhexyl group, a 2, 5-dimethylhexyl group, a 3, 5-dimethylhexyl group, a 2, 4-dimethylpentyl group, a 2-methylheptyl group, a 3-methylheptyl group, an n-heptyl group, an isoheptyl group, an n-octyl group and an isooctyl group. C ₁ -C ₈ Alkyl groups may be unsubstituted or substituted with one or more of the following, but are not limited to: c ₁ -C ₈ Alkyl radical, C ₁ -C ₈ Alkoxy, aryl, acyl, acyloxy, ester, -C (O) NH ₂ ,-C(O)NHR’,-C(O)N(R’) ₂ ,-NHC(O)R’,-S(O) ₂ R ', -S (O) R', -OH, halogen (-F, -Cl, -Br, -I), -N ³ ,-NH ² ,-NHR’,-N(R’) ₂ and-CN; wherein R' denotes C ₁ -C ₈ Alkyl or aryl.

C ₃ -C ₈ Carbocyclic refers to a saturated or unsaturated nonaromatic cyclic compound containing 3,4,5,6,7, or 8 carbon atoms. Typical of C ₃ -C ₈ Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. C ₃ -C ₈ The carbocycle may be unsubstituted or substituted with one or more of the following, but is not limited to: c ₁ -C ₈ Alkyl radical, C ₁ -C ₈ Alkoxy, aryl, acyl, acyloxy, ester, -C (O) NH ₂ ,-C(O)NHR’,-C(O)N(R’) ₂ ,-NHC(O)R’,-S(O) ₂ R ', -S (O) R', -OH, halogen (-F, -Cl, -Br, -I), -N ₃ ,-NH ₂ ,-NHR’,-N(R’) ₂ and-CN; wherein R' is C ₁ -C ₈ Alkyl or aryl.

C ₃ -C ₈ Carbocyclyl means C as defined above ₃ -C ₈ A group resulting from substitution of one hydrogen atom on a carbocyclic ring by a chemical bond.

Alkenyl refers to straight or branched aliphatic hydrocarbons having one carbon-carbon double bond, with 2 to 8 carbon atoms in the carbon chain. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl and octenyl.

Alkynyl refers to a straight or branched chain aliphatic hydrocarbon containing one carbon-carbon triple bond, with 2 to 8 carbon atoms in the carbon chain. Examples of alkynyls include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, hexynyl, heptynyl and octynyl.

Heteroalkyl means an alkyl group containing 2 to 8 carbon atoms and having 1 to 4 carbon atoms substituted by O, S or N.

Aryl or aryl refers to an aromatic or heteroaromatic hydrocarbon group consisting of 3 to 14 carbon atoms (in most cases 6 to 10 carbon atoms) containing one or more rings. The heteroaromatic hydrocarbon group means an aromatic hydrocarbon group produced by substituting one or more carbon atoms (in most cases, 1,2,3 or 4 carbon atoms) with O, N, si, se, P or S (preferably O, S, N). Aryl or aryl also means an aromatic hydrocarbon radical in which one or more hydrogen atoms are substituted, these substituents being: r ¹³ ,F,Cl,Br,I,OR ¹³ ,SR ¹³ ,NR ¹³ R ¹⁴ ,N＝NR ¹³ ,N＝R ¹³ ,NR ¹³ R ¹⁴ ,NO ₂ ,SOR ¹³ R ¹⁴ ,SO ₂ R ¹³ ,SO ₃ R ¹³ ,OSO ₃ R ¹³ ,PR ¹³ R ¹⁴ ,POR ¹³ R ¹⁴ ,PO ₂ R ¹³ R ¹⁴ ,OPO ₃ R ¹³ R ¹⁴ Or PO ₃ R ¹³ R ¹⁴ . Wherein R is ¹³ And R ¹⁴ Respectively H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl,aralkyl, carbonyl or a pharmaceutically acceptable salt.

Halogen atom means fluorine, chlorine, bromine, iodine atom, preferably fluorine and chlorine.

Heterocyclic means an aromatic, nonaromatic or heterocyclic ring containing from 2 to 8 carbon atoms, wherein from 1 to 4 carbon atoms are replaced by a hetero element. These hetero elements are O, N, S, se and P, preferably O, N and S. Useful heterocycles are also described in The Handbook of Chemistry and Physics, 78 th edition, CRCPress,1997-1998, pages 225 to 226. Suitable non-heteroaryl groups include, but are not limited to, epoxy, cycloazethyl, sulfoethyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, and fused rings thereof with phenyl.

Heteroaryl refers to aromatic heteromonocyclic, bicyclic or polycyclic structures containing 3 to 14, preferably 5 to 10 atoms. For example, pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2, 4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolinyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, N-oxopyridyl and fused rings thereof with phenyl.

The terms alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl also refer to alkylene, cycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, and heterocyclylene, among others, resulting from the loss of two hydrogen atoms from the corresponding hydrocarbon.

"aralkyl" refers to a carbon atom to which a hydrogen atom of an acyclic alkyl radical is attached, typically a terminal or sp3 hybridized carbon atom, is replaced by an aryl group. Typical arylalkyl groups include benzyl, 2-phenylethyl, 2-styryl, naphthylmethyl, 2-naphthylethyl, 2-naphthylvinyl, naphthylbenzyl, 2-naphthylphenylethyl and the like.

"Heteroaralkyl" means a noncyclic alkyl radical in which a hydrogen atom is linked to a carbon atom, usually a terminal or sp3 hybridized carbon atom, is replaced by a heteroaryl group. Heteroaralkyl represents, for example, 2-benzimidazolemethyl, 2-furoethyl.

"hydroxy-protecting group" means methyloxymethyl ether (MOM), 2-methyloxyethyloxymethyl ether (2-MOEOM), tetrahydropyran ether, benzyl ether, p-methyloxybenzyl ether, trimethylsilane, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilane, triphenylmethylsilicane, ethyl acetate, substituted ethyl acetate, benzoate, benzyl formate, chloroacetate, methoxyacetate, phenoxyacetate, pivalate (pivaloate), adamantanate, trimethylbenzoate (mesitate), methylsulfonate and tosylate.

The amino acids may be natural and/or unnatural amino acids, preferably alpha amino acids. Natural amino acids are the choreographed sequences encoded by genes such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Unnatural amino acids are protein amino acid-derived forms. Such as hydroxyproline, lanthionine; 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid (neurotransmitters), ornithine, citrulline, beta-alanine (3-aminopropionic acid), gamma-carboxyglutamate, selenocysteine (now found in many non-eukaryotes and most eukaryotes, but not directly encoded by DNA), pyrrolysine (found only in some archaea and bacteria), N-formylmethionine (which is the initial amino acid of bacterial, mitochondrial and chloroplast proteins), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3, 4-Dihydroxyphenylalanine (DOPA), and O-phosphoserine And (4) acid. The term amino acid also includes amino acid analogs and mimetics. The analogs have the same H ₂ N(R)CHCO ₂ H natural amino acid structure, but the R group is not found in natural amino acids. For example, analogs include serine-like, adipic acid, methionine sulfoxide, and methionine methyl sulfonium salts. Preferably, the amino-like acid has a structure that is different from the conventional chemical structure of an alpha amino acid, but functions similarly. The term "unnatural amino acid" is in the stereotypic form of "D", that of a natural amino acid is in the stereotypic form of "L". When 1 to 12 amino acids are used in the present patent application, the amino acid sequence is preferably recognized and cleaved by a protease. Many amino acid sequences are well known for recognition and cleavage (e.g., matayoshi et al science 247 (1990); dunn et al. Meth. Enzymol.241:254 (1994), seidah et al. Meth. Enzymol.244:175 (1994), thornberry, meth.enzymol.244:615 (1994), weber et al. Meth. Enzymol.244:595 (1994), smith et al. Meth. Enzymol.244:412 (1994), and Bouvier et al. Meth. Enzymol.248:614 (1995), wherein the literature contains the cited references), in particular, the amino acid sequence is selected as Val-Cit, ala-Val, ala-Lys, gly-Lys, ala-Ala, val-Val, ala-Val, lys-Lys, ala-Asn-Val, val-Leu-Val, cit-Cit, glu-Lys, asp-Lys, ala-Asn, ser, cit, lys, and Glu molecules.

"peptides" are peptides of two or more amino acids bonded by a peptide bond (i.e., an amide bond) between the amino group of one amino acid and the carboxyl group of another amino acid. Compounds in which two amino acids are linked by a peptide bond are called dipeptides; compounds in which three amino acids are linked by peptide bonds are called tripeptides, and so on, compounds in which thirty amino acids are linked by peptide bonds are called thirty-peptides. Peptides composed entirely of natural alpha amino acids are natural peptides (natural proteins). Peptides containing one or more unnatural amino acids or amino acid analogs are non-natural peptides (peptoid compounds). A peptide of two or more amino acids is a peptide unit.

"glycoside" is a molecule that, through its glycosyl bond, connects a glycosyl group to another group through its anomer carbon bond. The glycoside may be linked by an O- (O-glycoside), N- (sugar amine), S- (thioglycoside) or C- (C-glycoside) glycosidic bond. The core of the method is that the empirical formula is C _m (H ₂ O) _n (where m may be different from n, m and n are<36 Glycosides herein include glucose (glucose), fructose (levulose) allose, altrose, mannose, gulose, idose, galactose, tolose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinolone (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lysoglucose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide) and stachyose. The glycoside may be in the D or L form, in the form of a 5-atom cyclic furanose, in the form of a 6-atom cyclic pyranose, or in the acyclic form, the α -isomer (the-OH of the anomeric carbon below the carbon atom of the Haworth projection) or the β -isomer (the-OH of the anomeric carbon above the Haworth projection plane). The glycosides which are frequently used herein are monosaccharides, disaccharides, polyols or oligosaccharides (containing 3 to 6 sugar units).

"antibody", as used herein, is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, specific antibodies, multispecific antibodies (e.g., bispecific antibodies and antibody fragments) having the desired biological activity, the requisite number of linkages of the drug binding site of the antibody fragment. An antibody in its native form is a tetramer consisting of two identical immunoglobulin chain pairs, each pair having a light and heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) are collectively responsible for antigen binding. The light and heavy chain variable regions are interrupted by a framework region, a triple hypervariable region, also known as a "complementarity determining region" or "serving". The regions of constant duration may be recognized as interacting with the immune system. An antibody can be of any class (e.g., igG, igE, igM, igD, and IgA), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass. The antibody may be obtained from any suitable species. In certain aspects, the antibody is of human or murine origin. The antibody may be human, humanized or chimeric.

The terms "specific binding" and "specific binding" mean that an antibody or antibody derivative will bind to its corresponding target antigen in a highly selective manner, whereas Not to many other antigens. Typically, the antibody or antibodies have at least about 1x10 ^-7 Affinity derivatives of M bind. Preferably 1x10 ^-8 M to 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 And M is combined. The predetermined antigen has an affinity at least two times greater than the affinity of the non-specific antigen bound (e.g. bovine serum albumin, casein).

"pharmaceutically acceptable" or "pharmaceutically acceptable" means that the corresponding compound or composition of compounds does not produce deleterious, allergic, or other untoward effects in an animal or human.

Pharmaceutically acceptable excipients include all carriers, diluents, adjuvants or formers, such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like. In the field of medicine, it is common practice to add these adjuvants to active pharmaceutical ingredients. It can be said that it is not unreasonable to add adjuvants to pharmaceutical ingredients unless they are incompatible with the pharmaceutically active ingredient. Active auxiliary components may also be added to the pharmaceutical composition for good results.

In the present invention, pharmaceutically acceptable salts refer to salt derivatives of the compounds of the present invention. The compounds of the present invention may be modified appropriately to form the corresponding acid or base salts. Pharmaceutically acceptable salts include the usual non-toxic salts or quaternary ammonium salts which may be formed with the compounds of the invention and the corresponding non-toxic inorganic or organic acids. For example, pharmaceutically acceptable salts can be prepared with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like and organic acids including acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, lactic acid and the like. Other salts include ammonium salts such as tromethamine, aminotriethanol, meglumine, pyrroleethanol and the like, and metal salts such as sodium, potassium, calcium, zinc, magnesium and the like.

The pharmaceutically acceptable salts of the present invention may be prepared by conventional chemical methods. In general, these salts can be formed by adding other suitable equivalents of base or acid to an aqueous or organic solution or a mixture of both of the free acid or base of the compounds of this invention. The reaction medium for the non-aqueous phase is generally diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of suitable salts is available in Remington's Pharmaceutical Sciences, 17 th edition Mack Publishing Company, easton, PA,1985, page 1418.

The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a ligand drug conjugate or linker drug conjugate. The conjugate may contain at least one amino group, and thus may form an acid addition salt with an amino group. Nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1' -methylenebis- (2-hydroxynaphthoate)). Pharmaceutically acceptable salts may include additional molecules such as acetate, succinate, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, pharmaceutically acceptable salts may have more than one charged atom in their structure. Embodiments in which the plurality of charged atoms are part of a pharmaceutically acceptable salt can have a plurality of counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

The phrase "pharmaceutically acceptable solvate" or "solvate" refers to the association of one or more solvent molecules and a ligand drug conjugate or linker drug conjugate. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, butanol, tert-butanol, acetone, glycerol, DMSO, ethyl acetate, formic acid, acetic acid, triethanolamine, and ethanolamine.

Hydrate (Hydrate) refers to a compound containing water. Wherein the water can be coordinately bound to another moiety, such as a metal ion hydrated ligand to form a complex, or can be covalently bound, such as chloral hydrate. It can also refer to the crystal or liquid molecule formed by some compounds and water under certain temperature and pressure. The water in the hydrate being present in a determined amount, e.g. anhydrous sodium sulphate Na ₂ SO ₄ Has a composition of Na ₂ SO ₄ ·10H ₂ And O. There are several different ways of binding water in hydrates: one is used as a ligand, coordinated on metal ions, called coordinated crystal water; the other is bound to an anion, called anionic crystal water. Water may exist in a certain proportion in the crystal without being directly bonded to the cation or anion, and may occupy a certain portion in the crystal lattice. This bound form of water, called lattice water, typically contains 12 water molecules. Some of the crystalline compounds also contain water, but in no proportion. The hydrate salt refers to a pharmaceutically acceptable salt formed on the basis of the hydrate.

Optical isomers are also known as enantiomers, chiral, optical, mirror, enantiomeric or chiral isomers, and molecules that do not overlap completely with each other's stereoisomeric mirror images. When a substance contains a chiral carbon atom, there are two optical isomers which are in physical and mirror image relationship with each other and are therefore also called enantiomers. Enantiomers have equal optical power but opposite rotation directions and their physical and chemical properties are most likely similar. Molecules containing two carbon atoms of the same nature, have 3 optical isomers. When the molecule contains several different chiral atoms, the number of optical isomers is 2 ⁿ And n is the number of different chiral atoms. The two substances which are equal in amount and are optical isomers are uniformly mixed, and the optical rotations are counteracted with each other, so that the raceme is formed.

Examples of "patients" or "subjects" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In an exemplary embodiment, the patient or subject is a human.

"administration" refers to any means of transferring, delivering, introducing or otherwise delivering a drug or other agent to a subject, including oral, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration the invention also contemplates the use of devices or apparatus for administering the agent, such devices may utilize active or passive transport, and may be slow release or rapid release delivery devices.

The following abbreviations may be used herein, with the specified definitions: boc, tert-butoxycarbonyl; broP, bromotripyrrolidinophosphonium hexafluorophosphate; CDI, 1' -carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethyl dicyanophosphate; DMA, N-dimethylacetamide; DMAP,4- (N, N-dimethylamino) pyridine; DMF, N-dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDC,1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris (2-carboxyethyl) phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; val, valine.

Detailed Description

the R is ¹ 、R ² 、R ³ And R ⁴ Independently selected from H; c ₁ ～C ₈ Alkyl or heteroalkyl of (a); c ₂ ～C ₈ Alkenylalkyl, alkynylalkyl, heterocyclic, heterocycloalkyl of (a); c ₃ ～C ₈ Cycloalkyl, aryl, heteroaryl, aralkyl, alkylcarbonyl of (a); c ₄ ～C ₈ Alkylcycloalkyl, heteroalkylcycloalkyl;

Said X is ¹ Selected from-O-, -S-S, -NH, -CH ₂ -or-NR ¹ -；

Said R is ⁹ Selected from-H, -OH, = O, -OR ¹⁵ 、-OC(＝O)R ¹⁵ 、-OC(＝O)NHR ¹⁵ 、-OC(＝O)R ¹⁵ SSR ¹⁶ 、OP(＝O)(OR ¹⁵ ) OR OR ¹⁵ OP(＝O)(OR ¹⁶ )；

said X is ³ Selected from-OH, -SH, -NH ₂ 、-NH(R ¹⁵ )、-NHNH(R ¹⁵ )、-OR ¹⁵ 、-S-R ¹⁵ or-NR ¹⁵ R ¹⁶ One of (1);

the R is ¹⁵ 、R ¹⁶ And R ¹⁷ Independently selected from C ₁ -C ₈ Alkyl, heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl, or C ₃ -C ₈ Carbocyclyl, aryl, benzyl, alkylaryl, heterocycloalkyl, heteroalkylCycloalkyl, heteroaralkyl, alkylcarbonyl, C ₄ -C ₈ Alkylcycloalkyl, or Na ⁺ 、K ⁺ 、Cs ⁺ 、Li ⁺ 、Ca ²⁺ 、Mg ⁺ 、Zn ²⁺ 、N ⁺ (R ¹ )(R ² )(R ³ )(R ⁴ )、HN ⁺ (C ₂ H ₅ OH) ₃ The cationic salt of (a); said C is ₃ -C ₈ Carbocyclyl includes cycloalkyl;

said Y is ¹ And Y ² Independently selected from N or CH; q is 0 or 1; when q =0,Y ³ Is absent, Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Independently selected from CH, N, NH, O, S, or N (R) ¹ ) Thus Y is ² ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Together form a heteroaromatic ring of pyrrole, furan, thiophene, thiazole, oxazole, imidazole, triazole, tetrazole, thiadiazole, oxadiazole; when q =1,Y ³ ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Independently selected from CH or N, such that Y ² ，Y ³ ，Y ⁴ ，Y ⁵ ，Y ⁶ And Y ⁷ Together form a heteroaromatic ring structure of benzene ring, pyridine, pyrazine, pyridazine, triazine, tetrazine.

The invention also provides a cytotoxic molecule, the group R ¹ 、R ² ，R ² 、R ³ Or R ³ 、R ⁴ through-CH ₂ Optionally linked to Y respectively ¹ The atoms forming a cyclic group, or R ⁵ 、R ⁶ through-CH ₂ -optionally attached to the carbon atom to which it is attached to form a cyclic group, or R ¹¹ 、R ¹² through-CH ₂ Optionally linked to the carbon atom to which it is attached and to the carbonyl group to form a cyclic group, or R ¹³ 、R ¹⁴ through-CH ₂ -to Y optionally ⁵ And Y ⁶ The atoms form a cyclic group;

the cyclic group is selected from C ₃ -C ₇ Cycloalkyl, cycloalkylalkyl, cycloalkylheteroaryl, cycloalkylheteroalkyl, heterocyclic, heterocycloalkyl, cycloalkylcarbonyl, cycloalkylcarboxyl, cycloalkanoylOne of an amine group, a cycloalkaneamine group, a cycloalkoxy ether group, a cycloalkanethioether group, a cycloalkaneselenoether group, an aromatic group, a heteroaromatic alkyl group, and a heteroaromatic group.

The invention also provides a cytotoxic molecule, wherein R is ¹ 、R ² 、R ⁷ 、R ⁹ 、R ¹² 、R ¹³ And R ¹⁴ The terminal of (a) further contains a functional group capable of coupling with a linker or with a cell surface binding receptor molecule, the functional group being selected from SH, NH ₂ 、COOH、-O-NH ₂ 、-N ₃ 、NHNH ₂ -SSPy (Py is C) ₅ H ₄ N)、-SSAr、-SSC ₆ H ₄ NO ₂ 、-SSC ₆ H ₃ (NO ₂ )(COOH)、-SC(O)R ¹ 、-SSC ₆ H ₃ (NO ₂ ) ₂ 、-C(O)NH ₂ 、-C(O)H、-C(O)NHNH ₂ 、-C(O)R ¹ -C(O)C(O)R ¹ 、-ArC(O)R ¹ 、-C(O)CH ₂ X ³ 、-C(O)X ³ 、-ArCH ₂ X ³ 、

Preferably, the structure of the cytotoxic molecule is specified as follows:

said R is ²⁰ Is selected from C ₁ -C ₈ Linear or branched alkanes, hetero-alkanes, C ₂ -C ₈ Linear or branched alkenes or alkynes, C ₃ -C ₈ Aromatic, alkylaromatic, heterocyclic OR heteroaromatic hydrocarbons, carbonyl esters-C (O) OR ¹⁷ carbonylamine-C (O) NR ¹⁷ R ¹⁸ ；

Z is ² And Z ³ Independently selected from H, -OH, -NH ₂ 、OR ¹⁷ 、NHR ¹⁷ 、COOH、COOR ¹⁷ 、C(O)SR ¹⁷ 、C(O)R ¹⁷ 、C(O)NHR ¹⁷ 、C(O)NHNHR ¹⁷ 、C(O)NH、R ¹⁸ 、OCH ₂ OP(O)(OR ¹⁸ ) ₂ 、OC(O)OP(O)(OR ¹⁸ ) ₂ 、NR ¹⁷ CH ₂ OP(O)(OR ¹⁸ ) ₂ 、NR ¹⁷ C(O)OP(O)(OR ¹⁸ ) ₂ 、OPO(OR ¹⁸ ) ₂ 、NR ¹⁷ PO(OR ¹⁸ ) ₂ 、OP(O)(OR ¹⁸ )OP(O)(OR ¹⁸ ) ₂ 、OC(O)R ¹⁸ 、NR ¹⁷ C(O)R ¹⁸ 、OC(O)NHR ¹⁸ 、NR ¹⁷ C(O)NHR ¹⁸ 、NR ¹⁷ C(O)OR ¹⁸ 、NR ¹⁷ C(O)SR ¹⁸ 、NR ¹⁷ C(＝NH)NHR ¹⁸ 、OSO ₂ (OR ¹⁸ )、O-(C ₄ -C ₁₂ Glycoside), NR) ¹⁷ SO ₂ (OR ¹⁸ )、NR ¹⁷ -(C ₄ -C ₁₂ Glycoside), C ₁ -C ₈ Alkyl radical, C ₃ -C ₈ Carboxyalkyl or heterocycle;

Said R is ¹⁷ And R ¹⁸ Independently selected from H, C ₁ ～C ₈ Alkyl or heteroalkyl; c ₂ ～C ₈ A carbonylalkyl, alkenyl, alkylcarbonyl, alkynyl or heterocyclic group; c ₃ ～C ₈ Aryl, cycloalkyl or cycloalkylcarbonyl;

the R is ¹⁹ Selected from H, -OH, -NH ₂ 、-OSO ₂ (OR ¹⁸ )、-XCH ₂ OP(O)(OR ¹⁸ ) ₂ 、-XPO(OR ¹⁸ ) ₂ 、-XC(O)OP(O)(OR ¹⁸ ) ₂ 、-XC(O)R ¹⁸ 、-XC(O)NHR ¹⁸ 、C ₁ ～C ₈ Alkyl or carboxylic acid derivatives, C ₂ ～C ₈ Alkylene, alkynyl, alkylcarbonyl or carbonylalkyl, C ₃ ～C ₈ Aryl, cycloalkyl or cycloalkylcarbonyl; or a pharmaceutically acceptable salt;

the X is selected from-O-, -S-, or-NH-.

The invention also provides a coupling taking the toxic molecule with the structure shown in the formula I as a parent, which has the structure shown in the formula II:

or a pharmaceutically acceptable salt, hydrate or hydrated salt of structural formula (la); or a polymorph of such a compound; or an optical isomer of this formula;

The two linkers are respectively connected with two sites on the structure shown in the parenthesis in the structure shown in the formula II, or the two linkers are connected with one site on the structure shown in the parenthesis in the structure shown in the formula II through the linker with a branched chain;

each of said units W independently comprises an autodegradable spacer, a peptide unit, a hydrazone linkage, a disulfide linkage, an ester linkage, or an amine linkage; w is 0 or 1;

aa is a natural amino acid or an unnatural amino acid unit; r is an integer of 0 to 12, and (Aa) r is 0 to 12 amino acid units with the same or different structures; the natural amino acid or unnatural amino acid units include a mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, decapeptide-, undecapeptide, or dodecapeptide unit;

the cell surface receptor binding molecule T is any form of cell binding body, including a peptide or peptide-like structure: an antibody, a single-chain antibody, an antibody fragment capable of binding to a target cell, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment capable of binding to a target cell, a chimeric antibody fragment capable of binding to a target cell, a domain antibody fragment capable of binding to a target cell, an antibody-like engineered protein, a fibronectin-bound adnectin, a predesigned ankyrin repeat protein (DARPin), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transporting molecule, a transferrin, or a binding peptide, a protein, an antibody or a cell surface small molecule ligand attached to albumin, a macromolecule, a liposome, a nanoparticle, a pounang or a (viral) microcapsule.

The invention provides a conjugate taking a toxic molecule with a structure shown in a formula I as a parent, which has a structure shown in a formula III:

or a pharmaceutically acceptable salt, hydrate or hydrated salt of this formula; or a polymorph of such a compound; or an optical isomer of this formula;

the definitions of T, L ', m' and n are the same as those of formula II.

Preferably, the conjugate is one of the structures shown in formulas III-01 to III-19:

n is an integer of 1-20; p is an integer of 0 to 100;

the mAb is a cell-binding molecule comprising an antibody;

the definitions of Z2 and Z3 are the same as those described above;

said X ¹ 、R ¹ 、R ² 、R ³ Definitions the foregoing definitions are the same; said M ₁ And M ₂ Independently selected from H ⁺ 、Na ⁺ 、K ⁺ 、Li ⁺ 、NH ₄ ⁺ 、N(R ¹ R ² R ³ R ⁴ ) ⁺ Or NH (C) ₂ H ₅ OH) ₃ ⁺ Pharmaceutically acceptable salts of (a).

The invention provides a conjugate taking a toxic molecule with a structure shown in a formula I as a parent, which has a structure shown in a formula IV:

the definition of T, L, m and n is the same as that of formula II.

Preferably, the conjugate with the structure shown in the formula IV is one of the structures shown in the formulae IV-01 to IV-11:

N is an integer of 1-20; p is an integer of 0 to 100;

the mAb is a cell-binding molecule comprising an antibody;

z is ² And Z ³ As defined above;

said X is ¹ 、R ¹ 、R ² And R ³ The definition is the same as that described above,

said M ₁ And M ₂ Independently selected from salt ions H for pharmaceutically acceptable use ⁺ 、Na ⁺ 、K ⁺ 、Li ⁺ 、NH ₄ ⁺ 、N(R ¹ R ² R ³ R ⁴ ) ⁺ Or NH (C) ₂ H ₅ OH) ₃ ⁺ 。

One cell-binding receptor molecule-drug conjugate (conjugate) of the present invention has the following structural formula V:

or a pharmaceutically acceptable salt, hydrate or hydrated salt of this formula; or a polymorph of such a compound; or an optical isomer, racemate, diastereomer or enantiomer of this formula;

wherein R is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ 、R ¹⁰ 、R ¹¹ 、R ¹² 、R ¹³ 、R ¹⁴ 、Y ¹ 、Y ² 、Y ³ 、Y ⁴ 、Y ⁵ 、Y ⁶ 、Y ⁷ T, L, m and n are as defined for formula (II).

R ⁹ is-O-, -OR ¹⁴ ,-OC(O)R ¹⁴ ,-OC(O)NHR ¹⁴ ，-OC(O)R ¹⁴ SSR ¹⁵ OR-OP (O) (OR) ¹⁴ ) O-in which R ¹⁴ And R ¹⁵ Are respectively C ₁ ～C ₈ Alkyl or heteroalkyl, C ₃ ～C ₈ Aryl, heteroaryl, heterocyclyl, carbocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, carbonylalkyl, or a pharmaceutical salt. In addition, R ⁹ Or may be absent.

Preferably, the conjugate with the structure shown in the formula V is one of the structures shown in the formulas V-01 to V-18:

n is an integer of 1-20; p is an integer of 1 to 100;

The mAb is a cell-binding molecule comprising an antibody;

said X is ¹ 、Y ⁷ The same as defined above;

z is ² As defined above.

A conjugate comprising a toxic molecule having the structure of formula I as the parent, having the structure of formula VI:

or a pharmaceutically acceptable salt, hydrate or hydrated salt of this formula; or a polymorph of such a compound; or an optical isomer, racemate, diastereoisomer or enantiomer of this formula;

the definition of T, L, m and n is the same as that of formula II.

Preferably, the conjugate with the structure shown in the formula VI is one of the structures shown in the formulas VI-01 to VI-30:

n is an integer of 1-20; p is an integer of 0 to 100;

the mAb is a cell-binding molecule comprising an antibody;

z is ² And Z ³ Definition in the same way as before

Said X ¹ 、R ¹ 、R ² 、R ³ The definition is the same as that in formula I;

the M is ₁ And M ₂ Independently selected from pharmaceutically acceptable salt ion H ⁺ 、Na ⁺ 、K ⁺ 、Li ⁺ 、NH ₄ ⁺ 、N(R ¹ R ² R ³ R ⁴ ) ⁺ Or NH (C) ₂ H ₅ OH) ₃ ⁺ 。

The invention provides a conjugate taking a toxic molecule with a structure shown in a formula I as a parent, which has a structure shown in a formula VII:

The definitions of T, L, m and n are the same as those of the same formula II.

Preferably, the conjugate is one of structures shown in formulas VII-01 to VII-15:

n is an integer of 1-20; p is an integer of 1 to 100;

the mAb is a cell-binding molecule comprising an antibody;

said X ¹ 、Z ³ The definition of (1) is as before.

A conjugate comprising a toxic molecule having the structure of formula I as a parent, having the structure of formula VIII:

the definition of T, L, m and n is the same as that of the previous definition.

Preferably, the conjugate having the structure shown in the formula VIII is one of the structures shown in the formulas VII-01 to VII-43:

n is an integer of 1-20; p is an integer of 1 to 100;

the mAb is a cell-binding molecule comprising an antibody;

z is ² And Z ³ The definition of (2) is as before.

The invention provides a conjugate taking a toxic molecule with a structure shown in a formula I as a parent, which has a structure shown in a formula IX:

The definitions of T, L ', m' and n are the same as those of formula II.

Preferably, the conjugate having a structure represented by formula IX is one of structures represented by formulae IX-01 to IX-28:

n is an integer of 1-20; p is an integer of 1 to 100;

the mAb is a cell-binding molecule comprising an antibody;

said X is ¹ 、X ² Is as defined in formula I;

z is ² And Z ³ As described above.

The toxic molecules of the present invention and their conjugates with the cell binding agents of the present invention are shown by way of example, but not limitation, following the present specification, and in FIGS. 1-60 of the present specification.

As a specific conjugated linker of the invention, in particular the cleavable linker (L) is a chemical chain comprising C, N, O, S, si or P atoms. In the conjugate, the chemical chain is covalently linked to the cell conjugate (T) on one side and to the Tubulysin derivative on the other side. The linker may be of varying lengths, for example from 2 to 100 atoms in length. All of the atoms of the linker may be attached by any suitable chemical means, such as by an alkylene, alkenylene, alkyne, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, dihydrazocarbonyl, alkoxyamine, urethane, amino acid, acyloxyamine or hydroxamic acid. Of course, the atoms of these dissociable linkages may be saturated or unsaturated, may be free radicals, and may form rings with each other to form divalent ring structures, such as cycloalkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes, and the like.

A cleavable linker generally refers to a linker that contains at least one chemical bond that can be cleaved under physiological conditions, such as a pH sensitive, acid labile, base labile, easily oxidized, easily metabolized, biochemically labile, or easily enzymatically cleaved chemical bond. It should be noted that the physiological condition that is capable of breaking a chemical bond is not necessarily a biological or metabolic process, and that conventional chemical processes should be entirely possible, such as hydrolysis or substitution reactions. Since endosomes have a lower pH than the cytosol, chemical bonds that can be hydrolyzed under acidic conditions are a good choice. For the same reason, it is also feasible to exchange thiol groups for disulfide bonds, considering that the glutathione concentration in tumor cells is as high as millimolar.

The structure of the cleavable linker L of the invention may be expressed as: -Ww- (Aa) r-Vv-, wherein-W-denotes an extender, W is 0 or 1, -Aa-is an amino acid unit, r is an integer from 0 to 12, -V-denotes a spacer, V is 1 or 2.

When an extension-Ww-is present, it serves to link the cell-binding entity (T) to the amino acid unit-Aa-or spacer-V- (when-Aa-is absent). The extender W may contain a spacer capable of autolysis, a polypeptide unit, a hydrazone bond, a disulfide bond or a thioether bond. It is apparent that the cell binding agent (T) contains a group capable of bonding to a corresponding functional group on the extension. Ligatable functional groups that are originally present or chemically generated on the cell conjugate include, but are not limited to: mercapto, amino, hydroxy, carbonyl, hydroxyl of sugar, and carboxyl, etc., and the most preferred functional groups are mercapto, carboxyl and amino. Sulfhydryl groups may be generated by reducing disulfide bonds within a cell binding body (e.g., an antibody) molecule. Alternatively, sulfhydryl groups may be generated by reacting either 2-iminothiolane (Traut's reagent) or thiolactones with amino groups in lysines on the cell conjugate, or by other methods of sulfhydryl generation, such as first modifying the cell conjugate with a disulfide-containing linker or thioester, and then generating sulfhydryl groups by reduction or hydrolysis, respectively.

Some examples of W attached to T are given below:

where R is ²⁰ ，R ²¹ Is selected from-C ₁ ～C ₉ Alkylene-, -C ₁ ～C ₇ Carbocyclyl-, -O- (- (C) ₁ ～C ₈ Alkyl) -, -arylene-, -C ₁ ～C ₉ Alkylene-arylene-, -arylene, -C ₁ ～C ₉ Alkylene, -C ₁ ～C ₉ Alkylene- (C) ₁ ～C ₈ Carbocyclyl) - (C) ₃ ～C ₈ Carbocyclyl) -C ₁ ～C ₉ Alkylene-, -C ₃ ～C ₈ Heterocyclyl-, - (C) ₁ ～C ₁₀ Alkylene- (C) ₃ ～C ₈ Heterocyclyl) -, - (C) ₃ ～C ₈ Heterocyclyl) -C ₁ ～C ₉ Alkylene-, - (CH) ₂ CH ₂ O) _k -,-(CH(CH ₃ )CH ₂ O) _k -and- (CH) ₂ CH ₂ O) _k -CH ₂ -; k is an integer between 1 and 20; r 'and R' are each H or CH ₃ 。

The aforementioned covalent linkage of W and T can be achieved by various chemical reactions.

For example, formation of amide bonds:

wherein the extender unit contains an active reactive group E which can form an amide bond with a primary or secondary amine on the cell conjugate. Possible reactive sites E include, but are not limited to, hydroxysuccinimide esters (NHS and S-NHS, etc.), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters (including S-tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, isothiocyanates, and the like.

The following are examples of linkages with thioether or disulfide bonds:

wherein the extender unit comprises a thiol group capable of participating in a reaction which forms a thioether or disulfide bond with the cell binding entity T. In order to form disulfide bonds, sulfhydryl groups on the cell coalition T can be generated by reducing intramolecular disulfide bonds in the cell coalition T, and can also be generated by modifying the cell coalition T through other chemical processes; r is ²⁰ R', R ", J, and T are as defined above or in the specification.

The extension may also have a reactive group capable of reacting with an aldehyde or ketone group. Such aldehyde or ketone groups may be introduced into the appropriate position of the cell-bound body T by appropriate chemical modification. For example, aldehyde or ketone groups may be formed on the cell-bound carbohydrate by oxidation with an oxidizing agent such as sodium periodate. For another example, an amine group on the N-terminal amino acid of an antibody (or a protein or polypeptide) can react with pyridoxal 5-phosphate (PLP) to introduce a keto group. These aldehyde or keto groups (-C = O) can react with reactive groups on the extender such as hydrazides, oximes, primary or secondary amines, hydrazines, thiosemicarbazones, hydrazine carboxylates or arylhydrazines, linking the two together.

The following are examples of linkages with hydrazones, oximes or imines:

wherein R is ²⁰ And R ²¹ As defined above, R ²⁵ Refers to an organic substitution of an amino acid, or a glycoside or H.

The extension (which may comprise a spacer V and/or an amino acid) may be linked to the cell binding body T prior to binding the cell to the bodyThe fusion-extension fragment is conjugated to a Tubulysin derivative, and the ligation may be performed in aqueous buffer. The following are some examples of conjugated linkages in two steps in this manner (the linkage is at R) ¹⁶ The cytotoxic molecule (Tubulysin derivative) above was omitted):

wherein E includes, but is not limited to, hydroxysuccinimide ester (NHS and S-NHS, etc.), 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester (including S-tetrafluorophenyl ester), acid anhydride, acid chloride, sulfonyl chloride, isocyanate, isothiocyanate, etc.; r 'and R' are each H or CH ₃ ；R ²⁰ ，R ¹⁶ And Ar is as defined above; r is ²⁶ Is H, F or NO ₂ (ii) a J is F, cl, br, I, tosylate (TsO) or mesylate (MsO); in the above structure

Comprises at least one Tubulysin derivative or other drug or cytotoxic molecule, and is structured as

(

To omit the flag); the T-containing cyclic disulfide structure is specific for a cell-binding intramolecular disulfide bond.

The extension may be linked to the Tubulysin derivative, and then conjugated to the cell conjugate T in an aqueous solution (which may contain up to 50% organic solvent) at a pH of 3-10 (preferably pH 5.0-8.5).

The following are some examples of conjugation in two steps in this manner:

wherein E includes, but is not limited to, hydroxysuccinimide ester (NHS and S-NHS, etc.), 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester (including S-tetrafluorophenyl ester), acid anhydride, acid chloride, sulfonyl chloride, isocyanate, isothiocyanate, etc.; r 'and R' are each H or CH ₃ ；R ²⁰ ，R ¹⁶ And Ar are as defined above; r ²⁶ Is H, F or NO ₂ (ii) a J is F, cl, br, I, tosylate (TsO) or mesylate (MsO); in the above structure

Containing at least one Tubulysin derivative or other pharmaceutical structure, e.g.

(

To omit the flag).

When the amino acid unit (- -Aa- -) is present, it links the extender and the spacer, when the spacer is absent, it links the extender and the Tubulysin derivative, and if both the extender and the spacer are absent, it directly links the cell conjugate T and the Tubulysin derivative. - - (Aa) r- -may be a natural or unnatural amino acid, and may be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit, and r is an integer of 0 to 12. Herein, amino acids generally refer to aminoalkyl carboxylic acids, wherein the alkyl group may be an alkyl group substituted with a group such as an alkyl, acyl, hydroxyalkyl, mercaptoalkyl, aminoalkyl or carboxyalkyl group. The structure of natural and unnatural Amino acids and polypeptides is well described in the book "Amino acids and Peptides" (Cambridge University Press, 2004), by G.C. Barrett and D.T.Elmore. In addition, the amino acids also mean beta amino acids, gamma amino acids, and long-chain amino acids having a methyl group, a benzyl group, a hydroxymethyl group, a mercaptomethyl group, a carboxyl group, a carboxymethyl group, a guanidinopropyl group, or the like in the molecule. The most preferable amino acids are arginine, asparagine, aspartic acid, citrulline, cysteine, glycine, glutamic acid, leucine, lysine, glutamine, serine, ornithine, phenylalanine, threonine, tyrosine, valine and the like.

The amino acid unit of the invention may be enzymatically cleaved by one or more enzymes, including proteolytic enzymes present in the tumour, to release the Tubulysin derivative. In one embodiment, it is protonated in vivo and then becomes a protonated Tubulysin derivative.

When the spacer (-V-) is present, it links the amino acid unit and the Tubulysin derivative, and when the amino acid unit is deleted, it links the extender and the Tubulysin derivative. When both the amino acid unit and the extender are missing, the spacer links the Tubulysin derivative and the cell-binding molecule T. The spacer may contain functional groups that can be used to enhance the water solubility, biological transport, suitable renal clearance, uptake, absorption, biodistribution, and bioavailability of the conjugate. There are two basic types of separators: self-destructive and non-self-destructive. The non-self-destruction type spacer means that after an amino acid unit is cleaved from the Tubulysin derivative-linker-cell conjugate or the Tubulysin derivative-linker, particularly by enzymatic hydrolysis, part or all of the spacer remains attached to the Tubulysin derivative.

The following are some examples of self-destructing separators:

An access point for a cleavable linker, tubulysin derivative or cell conjugate T; where X, Y and Z ³ Are each NH, O or S; where Z is ² Is H, NH, O or S; v is 0 or 1; q is H, OH, C ₁ ～C ₆ Alkyl (OCH) ₂ CH ₂ ) _n ，F,Cl,Br,I,OR ¹⁷ ,or SR ¹⁷ ,NR ¹⁷ R ¹⁸ ,N＝NR ¹⁷ ,N＝R ¹⁷ ,NR ¹⁷ R ¹⁸ ,NO ₂ ,SOR ¹⁷ R ¹⁸ ,SO ₂ R ¹⁷ ,SO ₃ R ¹⁷ ,OSO ₃ R ¹⁷ ,PR ¹⁷ R ¹⁸ ,POR ¹⁷ R ¹⁸ ,PO ₂ R ¹⁷ R ¹⁸ ,OPO(OR ¹⁷ )(OR ¹⁸⁾ ,OCH ₂ PO(OR ¹⁷ (OR ¹⁸ ) Or a glycoside, wherein R ¹⁷ And R ¹⁸ Are respectively H, C ₁ -C ₈ Alkyl radical, C ₂ -C ₈ Alkylene, alkynylene, heteroalkyl, C ₃ -C ₈ Aryl, heterocyclyl, carbocyclyl, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl or a pharmaceutically acceptable salt of a cationic ion.

The following are some examples of non-self-destructing insulation:

*(CH ₂ CH ₂ O) _n *；

wherein the atom marked with (—) is an access point for a spacer, a cleavable linker, a Tubulysin derivative or a cell conjugate; q is as described above, and m is 1 to 10; n is 1 to 20.

The cell binding agent T may be any molecule known to bind, complex or react with a fragment of the target cell that may be used therapeutically or otherwise modified. The cell binding agent delivers the Tubulysin derivative to a specific target cell by binding to such target cell.

Cell binding bodies include, but are not limited to, large molecular weight proteins such as whole antibodies (polyclonal or monoclonal); a single chain antibody; antibody fragments such as Fab, fab ', F (ab ') 2, fv [ Parham, J.Immunol.131,2895-2902 (1983) ], fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-Id), CDR's, any fragment thereof capable of specifically binding to an epitope of a cancer cell, a viral epitope or a microbial epitope; interferons (type I, II, III); a polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL-6, GM-CSF, or IFN- γ; hormones such as insulin, thyroid stimulating hormone releasing hormone (TRH), melanocyte Stimulating Hormone (MSH), steroid hormones such as androgen, estrogen, or Melanocyte Stimulating Hormone (MSH); growth factors and colony stimulating factors such as epidermal growth factor (EFG), granulocyte macrophage colony stimulating factor (GM-CSF), transforming Growth Factors (TGF) such as TGF α, TGF β, insulin and insulin-like growth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [ Burgess, immunology Today,5,155-158 (1984) ], and the like; vaccine Growth Factor (VGF); fibroblast Growth Factor (FGF); small molecular weight proteins, polypeptides, peptides and peptide hormones, such as bombesin, gastrin and gastrin releasing peptide; platelet-derived growth factor; interleukins and cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte macrophage colony stimulating factor (GM-CSF), and vitamins such as folic acid; apoproteins and glycoproteins such as transferrin [ O' Keefe et al, J.Bio. Chem.260,932-927 (1985) ]; carbohydrate binding proteins or lipoproteins such as lectins; a cellular nutrient-transport molecule; and small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors, small molecule Tyrosine Kinase Inhibitors (TKI), non-peptides or other cell binding molecules or substances, such as bioactive macromolecules (Dhar, et al, proc.natl.acad.sci.2008,105, 17356-61), dendrimers (Lee, et al, nat. Biotechnol.2005,23,1517-26 almutairi, et al proc.natl.acad.sci.2009,106, 685-90), nanoparticles (Liong, et al, ACS Nano,2008,19,1309-12 medarova, et al, nat. Med.2007,13,372-7 javier, et al, bioconjugate chem.2008,19, 1309-12), liposomes (lininal, et al, curr.phar.10, 2981-9, 76-81, 92-9, virus, rhenek.327, 71-71, or these are attached to a surface binding molecule, especially to a target molecule, capsid, for the target molecule. Generally, monoclonal antibodies are the best cell binding agents if appropriate.

The cell binding body may be first attached to a specific polypeptide, protein, drug molecule or other functional molecule by modification with a bifunctional group containing cross-linking agent prior to conjugation with the Tubulysin derivative. These dual-functional group containing crosslinkers can be amine-nonselective functional group crosslinkers (succinimide (NHS) -diazocyclopropene (SDA), succinimide ester-azide), amine-sulfhydryl crosslinkers (NHS ester-maleimide, NHS ester-pyridinedimercapto, NHS ester-haloacetyl), sulfhydryl-sugar crosslinkers (maleimide-hydrazide, pyridinedimercapto-hydrazide), hydroxyl-sulfhydryl crosslinkers (isocyanate-maleimide), amine-DNA crosslinkers (NHS ester/psoralen), and amine-carboxyl crosslinkers (carbodiimide), among others.

In the modification method using succinimide (NHS) -cyclopropene (SDA) crosslinker, the NHS ester on the crosslinker can first react with the amine group on the cell-bound backbone (in a buffer solution at pH 6-9), which will form a stable amide bond. The diazocyclopropene is then activated by irradiation with long waves at wavelengths of 330-370nm and produces a carbene reactive intermediate which reacts with amine groups on specific polypeptides, proteins or other functional molecules to complete the linkage. The order of these two reactions can also be changed: the amine on the functional molecule and the NHS ester on the cross-linking agent are reacted first and then chemically reacted with the cell conjugate under irradiation of light (330-370 nm). Succinimide (NHS) -diazacyclopropene (SDA) crosslinkers can also be cleavable (like SDAD crosslinkers where disulfide bonds are present).

Wherein

Is a framework of cell-binding molecules,

are functional molecules.

In the modification method using the NHS ester-azide crosslinker, the NHS ester on the crosslinker can first react with the amine groups on the cytoskeleton of the cell conjugate (in a buffer solution at pH 6-9), which will form a stable amide bond. Then, by Huisgen azide-alkyne cycloaddition, the alkyne group on a particular polypeptide, protein or other functional molecule reacts with the azide at the other end of the crosslinker and forms a 1,2, 3-triazole linker. Alternatively, the NHS ester of the crosslinker can be reacted with the amine group on the functional molecule to form a stable amide bond (in a buffer solution with pH 6-9), and then the alkynyl group on the conjugate can be reacted with the azide group at the other end of the crosslinker to form a 1,2, 3-triazole linker by Huisgen azide-alkyne cycloaddition.

The azide group can also be introduced into an azide-containing unnatural amino acid through antibody engineering, or introduced into an azide-containing glycoside through antibody engineering, chemical degradation and glycosidase transfer, and then subjected to a Huisgen azide-alkyne cycloaddition reaction (click chemistry reaction).

In the modification method using an amine-thiol crosslinker, the NHS ester on the crosslinker can first react with the amine groups on the cell-bound assembly backbone (in a buffer solution with pH = 6-9), which will form a stable amide bond. Then, under the condition of pH value of 4.5-8.5, the sulfhydryl on the specific polypeptide, protein or other functional molecule reacts with maleimide, pyridine dithiol or halogenated acetyl at the other end of the cross-linking agent, and thioether or disulfide bond is formed. The order of this crosslinking reaction may also be changed as the case may be. For example, the amino group on the functional molecule can be reacted first with a cross-linking agent to form an amide bond, and then reacted with the thiol group on the cell conjugate. For another example, at a pH of 4.5 to 7.0, the thiol group on the functional molecule may first react with the cross-linking agent to form a thioether or disulfide bond, and then react with the amino group on the cell conjugate at a pH of 6 to 9 to form an amide bond.

In the modification method using a thiol-sugar crosslinker, the thiol group on the cell conjugate may first react with the maleimide or pyridinedithiol group on the crosslinker to form a thioether or disulfide bond at a pH of 4.5 to 8. Then, the carbonyl group (aldehyde or ketone) on the functional molecule is reacted with hydrazide to generate hydrazone bond. Alternatively, at a pH of 4.5 to 8, the thiol group on the functional molecule may be reacted with a crosslinking agent to form a thioether or disulfide bond, and then reacted with a sugar, oxidized sugar, or carbonyl (aldehyde or ketone) group on the cell-bound body to form a hydrazone bond.

In the modification method using the hydroxy-mercapto crosslinking agent, under the condition that the pH is 6-8, the mercapto group on the cell-bound body can firstly react with maleimide or pyridine dithiol on the crosslinking agent to generate thioether or disulfide bond. Then, under the condition of pH 8-9, the hydroxyl on the functional molecule and the isocyanate on the cross-linking agent react to generate carbamate. Alternatively, the thiol group on the functional molecule may be reacted with a crosslinking agent at a pH of 6 to 8 to form a thioether or disulfide bond. Then, the compound reacts with hydroxyl groups on the cell-bound body at a pH of 8 to 9 to form carbamate.

In a direct modification method using a thiol-reactive cross-linker, the thiol-reactive group on the cross-linker can react with the thiol or seleno group on the cytoskeleton (in a buffer solution with pH = 6-9) to form a stable thioether, seleno-ether or disulfide bond. Firstly, under the condition of pH value of 4.5-8.5, the disulfide group on the specific polypeptide, protein, antibody (especially IgG1, ig/2, igG3 or IgG4 monoclonal antibody) or other functional molecule or the disulfide group, diselenide group or sulfoselenide group introduced by genetic engineering and TCEP or DTT or 2-mercaptoethanol produce reduction reaction to form sulfhydryl group or selenide group. The cell-binding molecule is purified and then reacted with a cross-linking agent to form a thioether, selenoether, or dithio-linkage. During the reduction with TCEP, the TCEP can be directly reacted with the crosslinking agent (or directly added to the system together with the crosslinking agent) without being removed from the reaction system, or an azide compound can be added after the reduction of the TCEP is completed to neutralize the excess TCEP, and then the crosslinking agent is added to carry out the coupling reaction. The sulfhydryl coupling reaction can be singly coupled with a sulfhydryl on a cell combination body, and can also form a bridging type coupling reaction on dimercapto simultaneously

In another embodiment, when two cytotoxic molecules are attached to each linker of the conjugate, the conjugates have the following general structural formula:

wherein D ₁ And D ₂ Is a cytotoxic molecule or a drug, and independently selects a Tubulysin derivative with a structural formula (I); t is a targeting or binding ligand, or a cell binding molecule. X ₁ 、X ₂ 、X ₃ And X ₄ Independently NH, NR ¹ 、O、S、CH ₂ Se and NHNH; r ¹ 、R ² N and L are as defined in formula (II). Furthermore when D is ₁ And D ₂ A Tubulysin derivative of any one of structural formulae (I) or I-01 to I-67; an additional cytotoxic molecule or drug D may be selected from chemical molecules that are effective in the treatment of cancer, such as calicheamicin, maytansine derivatives, dolastatins, CC-1065 analogs, doxorubicin, taxane derivatives, benzodiazepine (PBD) dimers, tomaymycin dimers, antrramycin dimers, indolizinone dimers, indolizodiazepine dimers, imidazobenzothiazepine dimers, or oxolidinobenzodiazepine dimers, amatoxins, indolocaxamides, actinomycin, azaserins, bleomycin, epirubicin, eribulin, tamoxifen, idarubicin, auristatin (auristatins), (monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, dolastatins 2-AQ,6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, geldanamycins, HSP90 inhibitors, centanamycin, methotrexates, thiotepa, vindesines, vincristines, hemiasterins, azumamides, microginins, radiosumins, streptonins, SN38 and its derivatives, or camptothecin metabolites, alternactins, microscalers, theonelamides, peroxidamics, PNU-15968, siRNA, or polyethylene glycol (PEG) having a molecular weight of less than 100KD, or a combination of the above, pharmaceutically acceptable salts and acids, or derivatives thereof.

The cell binding molecules of the invention are preferably antibodies, mainly monoclonal antibodies. The production of the antibody includes the production process or the combination process in vivo or in vitro. Methods for producing polyclonal antibodies against receptor peptides are well known, for example, in U.S. Pat. No. 4,493,795 (Nestor et al). The classical method for preparing monoclonal antibodies is to immunize mice with specific antigens and to fuse the isolated mouse splenocytes with myeloma cells (Kohler, G; milstein, C.1975Nature 256. The detailed procedures are described in antibodies-A Laboratory Manual, harlow and Lane, eds., cold spring harbor Laboratory press, new York (1988)), which is incorporated herein by reference. In particular, specific monoclonal antibodies can be obtained by immunizing a mouse, rat, hamster, or other mammal with the antigen of interest. Wherein the antigen of interest comprises: intact cells, antigens isolated from cells, intact viruses, attenuated intact viruses, and viral proteins. Spleen cells were fused with myeloma cells using PEG 6000. The hybridomas obtained after the fusion are screened by using their sensitivity to HAT. Monoclonal antibodies produced by hybridoma cells play a role in practicing the invention by either immunoreacting with a particular target cell receptor or by inhibiting receptor activity.

The monoclonal hybridoma cells obtained after fusion can secrete monoclonal antibodies aiming at specific antigens. The monoclonal antibodies used in the present invention are enriched by culturing monoclonal hybridoma cells in a nutrient-rich medium. The culture conditions are such that the hybridoma cells have sufficient time to secrete the produced antibody into the culture medium. After the antibody-containing culture supernatant is collected, the antibody is purified by a well-known technique. The separation method comprises the following steps: protein a affinity chromatography; anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography (especially affinity chromatography and molecular sieve chromatography using antigen-crosslinked protein A); centrifuging; precipitation or other standard purification methods

Effective media as well as artificially synthesized media for hybridoma culture can be either technically synthesized or commercially available. Among these, typical synthetic media: DMEM (Dulbecco et al Virol 8 (1959)) was supplemented with 4.5mg/L glucose, 20mM glutamine, 20% fetal bovine serum and antifoam, as follows: polyoxyethylene polyoxypropylene copolymer

In addition to cell fusion techniques, antibody-producing cell lines can be constructed by other methods, such as: direct transfection of B lymphocytes with tumorigenic DNA or introduction of oncogenic viral genes (e.g., EBV, also known as HHV-4 or KSHV) into B lymphocytes, see, e.g., U.S. Pat. No. 4341761;4399121;4427783;4444887;4451570;4466917;4472500;4491632;4493890. monoclonal antibodies may also be prepared by anti-receptor polypeptides or polypeptides containing a carboxy terminus. See Niman et al Proc.Natl.Acad.Sci.USA, 80; geysen et al Proc.Natl.Acad.Sci.USA, 82; lei et al Biochemistry 34 (20): 6675-6688 (1995). In general, anti-receptor polypeptides or polypeptide analogs can be used alone or cross-linked to immunogenic carriers to produce monoclonal antibodies against receptor polypeptides as immunogens.

Antibodies of the invention as binding molecules have other well-established methods of production. Of particular interest are processes for the production of fully human antibodies. Phage display technology obtains fully human antibodies that specifically bind to a known antigen from a fully human antibody library by affinity screening. Phage display technology itself, vector construction and library screening are well documented in the literature. See, dente et al Gene.148 (1): 7-13 (1994); little et al Biotechnol adv.12 (3): 539-55 (1994); clackson et al Nature352:264-628 (1991); huse et al Science 246:1275-1281 (1989).

Monoclonal antibodies obtained from other species (e.g., mouse, rabbit, alpaca) using hybridoma technology need to be humanized. The engineered antibody can greatly reduce the immune side effects of the heterologous antibody on the human body. Among the more common methods for humanization of antibodies are the transplantation and remodeling of the CDRs. See in detail: U.S. patent nos. 5859205 and 6797492; liu et al, immunol Rev.222:9-27 (2008); almagro et al, front biosci.1; 13-1619-33 (2008); lazar et al mol Immunol.44 (8): 1986-98 (2007); li et al Proc. Natl. Acad. Sci. USA.103 (10): 3557-62 (2006) the above article is incorporated herein by reference. Fully human antibodies can also be prepared by antigen immunization of transgenic mice, rabbits, monkeys, and other mammals that carry large amounts of human immunoglobulin light and heavy chains. Taking a mouse as an example: xenomouse (Abgenix, inc.), huMab-Mouse (Metarex/BMS), velociMouse (Regeneron), see in detail: U.S. Pat. Nos. 6596541,6207418,6150584,6111166,6075181,5922545,5661016,5545806,5436149 and 5569855. In the course of human therapy, chimeric antibodies constructed by integrating the genes of the variable regions of murine antibodies with the genes of the constant regions of human antibodies produce a much lower immunogenic response in humans than murine antibodies (Kipriyanov et al, mol Biotechnol.26:39-60 (2004); houdbine, curr Opin Biotechnol.13:625-9 (2002) which is incorporated herein by reference).

Antibodies immunospecific for malignant cell antigens may be obtained commercially or by established techniques, such as: chemical synthesis or recombinant expression techniques. Genes encoding such antibodies are also available commercially, such as in the GenBank database or other similar databases, in published literature, or by routine clonal sequencing methods.

In addition to antibodies, polypeptides or proteins may also bind as binding molecules, blocking, attacking or otherwise interacting with a receptor or epitope on the surface of a target cell. These polypeptides or proteins do not necessarily belong to the immunoglobulin family, as long as they are capable of specifically binding to a particular epitope or its corresponding receptor. These polypeptides can also be isolated by techniques similar to phage display antibodies (Szardenengs, J Recept Signal Transmission Res.2003, 23 (4): 307-49). Peptide fragments obtained from random polypeptide libraries are used similarly to antibodies and antibody fragments. A polypeptide or protein molecule may retain its antigen-binding specificity by linking its binding molecule to some macromolecule or medium. These macromolecules and mediators include: albumin, polymer, liposome, nanoparticle, or dendrimer.

In the treatment of cancer, autoimmune diseases and infectious diseases, antibodies for coupling the Tubulysin derivative are exemplified by (but not limited to): 3F8 (anti-GD 2 antibody), abavacizumab (anti-CA-125 antibody), abciximab (anti-CD 41 antibody (integrin. Alpha. -IIB), adalimumab (anti-TNF. Alpha. -antibody)) Adalimumab (anti-EpCAM antibody, CD 326), aphidlimumab (anti-TNF-a); <xnotran> ( -CD20 ), alacizumabpegol ( VEGFR2 ), ALD518 ( -IL-6 ), (: campath, mabCampath, , CD52 ), ( CEA ), anatumomab ( -TAG-72 ), anrukinzumab (: IMA-638, -IL-13 ), ( -HLA-DR ), ( CEA ), ( -L- (CD 62L) , atlizumab (: , actemra, roActemra, -IL-6 ), atorolimumab ( ), bapineuzumab ( β - ), (, antiCD25 (IL-2 α ) , ( ), (: lymphoScan, -CD22 ), (: benlysta, lymphoStat-B, BAFF ), benralizumab ( CD125 ), ( CCL11 ( -1) ), (: scintimun, CEA- ), (: , VEGF-A ) (: fibriScint, Ⅱ β ), bivatuzumab ( CD44v6 ), </xnotran> blinatumomab (alias: biTE, anti-CD 19 antibody), brentuximab (CAC 10, anti-CD 30, TNFRSF8 antibody), briakinumab (anti-IL-12, IL-23 antibody), conatinumab (alias: ilaris, anti-IL-1 antibody), cantuzumab (alias: C242, anti-CanAg antibody), carromumab, cartuzumab (alias: removab, anti-EpCAM, anti-CD 3 antibody), CC49 (anti-72 antibody), cetuzumab (anti-CD 4 antibody), cytuzumab (alias Cimzia anti-TNF- α antibody), cetuximab (alias: erbitumomab, IMC-C225, anti-EGFR antibody), citacrolimus (anti-EpCAM antibody), cixuumumab (anti-IGF-1 antibody), clitumumab (anti-CD 4 antibody), clitumomab (anti-MUTUMc 1 antibody), contuzumab (alias-TRAUzumab (anti-TRAIL 622), anti-CDTAG-TNF-alpha antibody), protsunobu antibody (anti-TNF-alpha antibody), proteumab (anti-TNF-alpha antibody), clitumomab (anti-TNF-alpha antibody), anti-RANKL antibody), dimuzumab (anti-B-lymphoma cell antibody), atovazumab, dorlixizumab, ecromeximab (anti-GD 3 ganglioside antibody), eculizumab (alternative name: soliris, anti-C5 antibody), abazumab (anti-endotoxin antibody), epirubizumab (alias: panorex, MAb17-1A, anti-EpCAM antibody), efletuzumab (alternative name: raptiva, anti-LFA-1 (CD 11 a) antibody), efletuzumab (alternative name: mycograb, anti-Hsp 90 antibody), elotuzumab (anti-SLAMF 7 antibody), islamol (anti-IL-6 antibody), emlimumab (anti-ICAM-1 (CD 54) antibody), epituzumab (anti-epidialin antibody), epratuzumab (anti-CD 22 antibody), erlizumab (anti-ITGB 2 (CD 18) antibody), ertuzomamab (alias: rexomun, anti-HER 2/neu, CD3 antibody), israeli bead (alias: abegrin, anti-integrin α v β 3 antibody), esquiriimab (anti-hepatitis b surface antigen antibody), fanolesomab (alternative name: neutroSpec, anti-CD 15 antibody), farretazumab (anti-interferon receptor antibody), farlettuzumab (anti-folate receptor 1 antibody), felvizumab (anti-respiratory syncytial virus antibody), fezakinumab (anti-IL-22 antibody), figitumumab (anti-IGF-1 receptor antibody), fontolizumab (anti-IFN- γ antibody), vorivivir e (anti-rabies glycoprotein antibody), fresolimumab (anti-TGF- β antibody), galiximab (anti-CD 80 antibody), ganternumab (anti- β amyloid antibody), gavilimob (anti-CD 147 (bain) antibody), gemtuzumab (anti-CD 33 antibody), giretuximab (anti-carbonic anhydrase 9 antibody), glytemab (alias: CR011, anti-GPNMB antibody), golimumab antibody (alias: SIMPONI, anti-TNF- α antibody), gomiliximab (anti-CD 23 (IgE receptor) antibody), ibalizumab (anti-CD 4 antibody), ibritumomab (anti-CD 20 antibody), agovacizumab (alias: indinavis-125, anti-CA-125 antibody), infliximab (alias: myoscint, anti-cardiac myosin antibody), infliximab (alternative name: infliximab, anti-TNF- α antibody), intetumumab (anti-CD 51 antibody), inomumab (anti-CD 25 (IL-2 receptor α chain) antibody), inotuzumab (anti-CD 22 antibody), lypimma (anti-CD 152 antibody), iratuzumab (anti-CD 30 (TNFRSF 8) antibody), keliximab (anti-CD 4 antibody), labetuzumab (alias: CEA-Cide, anti-CEA antibody), lebrikizumab (anti-IL-13 antibody), lemalesamab (anti-NCA-90 (granulocyte-antigen) antibody), ledebaryabte Anti- (anti-TGF β -2 antibody), lexamu anti- (anti-TRAIL-R2 antibody), ribavirin (anti-hepatitis b surface antigen antibody), lintuzumab (anti-CD 33 antibody), lucidumab (anti-CD 40 antibody), luximab (anti-CD 23 (IgE receptor) antibody), mapatumumab (anti-TRAIL-R1 antibody), massecumab (anti-T-cell receptor antibody), matuzumab (anti-EGFR antibody), meperilizumab (alias: bosatria, anti-IL-5 antibodies), metelimumab (anti-TGF beta-1 antibody), milatuzumab (anti-CD 74 antibody), minretumumab (anti-TAG-72 antibody), mituzumab (alias BEC-2, anti-GD 3 ganglioside antibody), morolimumab (anti-rhesus factor antibody), movizumab (alias NUMAX, anti-respiratory syncytial virus antibody), moluomab-CD 3 (alias: otoCloKT 3, ORTHOCLONE, anti-CD 3 antibody), tanakemab (anti-C242 antibody), tanamomab (anti-5T 4 antibody), natalizumab (alias Tysabri, anti-integrin alpha 4 antibody), nelumboruzumab (anti-endotoxin antibody), necitumumab (anti-EGFR antibody), nerelumumab (anti-TNF-alpha antibody), nintensin (LFzeocin, theraFeloc, anti-alias antibody), metelimumab (anti-EGFR antibody), anti-EGFR antibody (anti-TNF-gamma-EGFR antibody), nortuzumab (anti-EGFR antibody), nonreformab (anti-EGFR antibody), anti-CD 20 antibody), olarataumab (anti-PDGF-R α antibody), omalizumab (alternative name: sorel, anti-IgEFc region antibody), oportuzumab (anti-EpCAM antibody), agovozumab (alternative name: ovaRex, anti-CA-125 antibody), otelixizumab (anti-CD 3 antibody), pargyximab (anti-lipoteichoic acid antibody), palivizumab (alias: synagis, abbosnagis, anti-respiratory syncytial virus antibodies), panitumumab (alternative name: victib, ABX-EGF, anti-EGFR antibody), panobaumab (anti-pseudomonas aeruginosa antibody), pascolizumab (anti-IL-4 antibody), pemtumumab (alternative name: theragyn, anti-MUC 1 antibody), pertuzumab (alias: OMNITARG,2C4, anti-HER 2/neu antibody), peclizumab (anti-C5 antibody), pintumomab (anti-adenocarcinoma antigen antibody), priliximab (anti-CD 4 antibody), promolizumab (anti-vimentin antibody), PRO140 (anti-CCR 5 antibody), racotumomab (alternative: 1E10, anti- (N-glycolylneuraminic acid (NeuGc, NGNA) -ganglioside GM 3) antibody), renfulu (anti-rabies glycoprotein antibody), ramucirumab (anti-VEGFR 2 antibody) <xnotran>, (: lucentis, VEGF-A ), ( , ), ( B ), reslizumab ( IL-5 ), rilotumumab ( HGF ), (: , rituxanmab, CD20 ), robatumumab ( IGF-1 ), rontalizumab ( IFN- α ), rovelizumab (: leukArrest, CD11, CD18 ), ruplizumab (: antova, CD154 (CD 40L) ), ( TAG-72 ), ( ), sibrotuzumab ( FAP ), sifalimumab ( IFN- α ), siltuximab ( IL-6 ), siplizumab ( CD2 ), (Smart) MI95 ( CD33 ), solanezumab ( β - ), sonepcizumab ( -1- ), ( episialin ), ( ), (: leukoScan, ( -NCA-90 ( ) ))), tacatuzumab ( α - ), ( α IIb β 3 ), ( IgE ), ( NGF ), </xnotran> <xnotran> Taplitumomab ( CD19 ), tefibazumab (: aurexis, A ), , tenatumomab ( C ), ( CD40 ), teplizumab ( CD3 ), TGN1412 ( CD28 ), ticilimumab (: tremelimumab ( CTLA-4 ), tigatuzumab ( TRAIL-R2 ), TNX-650 ( IL-13 ), ( Atlizumab, actemra, roActemra, ( IL-6 ), toralizumab ( CD154 (CD 40L) ), ( CD20 ), (, ( HER2/neu ), tremelimumab ( CTLA-4 ), tucotuzumabcelmoleukin ( EpCAM ), ( B ), urtoxazumab ( ), (: stelara, IL-12,IL-23 ), ( AOC3 (VAP-1) ), vedolizumab, ( α 4 β 7 ), veltuzumab ( CD20 ), ( AOC3 (VAP-1) , visilizumab (: nuvion, CD3 ), vitaxin ( </xnotran> Angiointegrin avb3 antibody), volociximab (anti-integrin α 5 β 1), volitumumab (alternative name: humamspect, anti-tumor antigen CTAA16.88 antibody), zatuzumab (alias: HUMAX-EGFR (anti-EGFR antibody), zanolimumab (alternative: HUMAX-CD4, anti-CD 4 antibody), ziralimumab (anti-CD 147 (basal immunoglobulin) antibody), azuolimumab (anti-CD 5 antibody), enazepul (ENBREL), alfaSite (AMEVIVE), albizumab (Abiradep)

Lencept (ARCALYST), 14F7[ anti-IRP-2 (IRP-2) antibody]14G2a (anti-GD 2 ganglioside antibody, for melanoma and solid tumors, from. Cancer inst.), J591 (anti-PSMA antibody, for prostate cancer, cannell medical school), 225.28S [ anti-HMW-MAA (high molecular weight melanoma associated antigen) antibody, sonlin Radiofarmaci SRL (Milan Italy) for melanoma treatment]COL-1 (anti-CEACAM 3 antibody, CGM1, (national cancer institute of America), for treatment of colorectal cancer and gastric cancer), CYT-356 (C)

For the treatment of prostate cancer), HNK20 (OraVax for the treatment of respiratory syncytial virus), immuRAIT (from immunomedicine for the treatment of non-hodgkin lymphoma), lym-1 (anti-HLA-DR 10 antibody, berofusk, for cancer), MAK-195F [ anti-TNF antibody (also known as: tumor necrosis factor; TNFA, tumor necrosis factor- α; TNFSF 2), abert/Norel, for treating septic shock ]MEDI-500[ alias: T10B9, anti-CD 3 antibody, TR α β (T cell receptor α/β), complex, medImmune for the treatment of graft versus host disease]RINGSCAN [ anti-TAG 72 (tumor-associated glycoprotein 72 antibody), neoprene group for the treatment of breast, colon and rectal cancers, avicidin (anti-EpCAM antibody (epithelial cell adhesion molecule), anti-TACTD 1 antibody (tumor-associated calcium signaling 1), anti-GA 733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 antibody (epithelial glycoprotein 2), anti-KSA antibody, KS1/4 antigen, M4S; tumorTumor antigen 17-1A; CD326 from NeoRx for the treatment of colon, ovarian, prostate and non-hodgkin lymphoma; lymphoCide (Immunodics Co., ltd.), smartID10 (Protein Design Labs), oncolom (Techniclone Co., ltd.), allomne (BioTransplant), anti-VEGF antibody (Genentech Co., ltd.); CEAcide (Immunodics), IMC-1C11 (Imclone) and cetuximab (Imclone).

Other antibodies for binding to an antigen include, but are not limited to: aminopeptidase N (CD 13), annexin A1, B7-H3 (CD 276, various cancers), CA125, CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), lewis Y (cancer), lewis X (cancer), alpha-fetoprotein (cancer), CA242, placental alkaline phosphatase (cancer), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, lymphoma other than Hodgkin's lymphoma, multiple myeloma), epsilon of CD3 (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B-cell malignancy), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30, CD33, CD37, CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia), CD51 (metastatic melanoma, sarcoma), CD52, CD56 (small cell lung cancer, ovarian cancer, merkel cell cancer, and liquid tumors, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD79, CD80 (lymphoma) CD98 (cancer), mucin (cancer), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66 e) (breast, colorectal and lung cancers), DLL3 (delta-3), DLL4 (delta-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD 184, heme tumors, solid tumors), endoglin (CD 105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head, neck, colon, prostate non-hodgkin lymphoma, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2; lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (a cell surface antigen glyvolipid, melanoma), idiotypic GD3 (cancer), heat shock protein (cancer), HER1 (lung, gastric cancer), HER2 (breast, lung and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (non-hodgkin's lymphoma, B-cell leukemia), human chorionic gonadotropin (cancer), IGF1R (insulin-like growth factor 1 receptor, solid tumors, hematological cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, castleman's disease, IL6 dependent tumors), integrins (hormone α v β 3, α 5 β 1, α 6 β 4, α ll β 3, α 5 β 5, α v β 5 cell attachment factor, for various cancers), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE4 (carcinoma), anti-transferrin receptor (carcinoma), P97 (melanoma), MS4A1 (transmembrane domain 4 subfamily a member 1, non-hodgkin B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA 125) (ovarian cancer), CEA large intestine, GP100 (melanoma), MART1 (melanoma) MPG (melanoma), MS4A1 (transmembrane domain 4 protein a, small cell lung carcinoma, non-hodgkin's lymphoma), nucleolus, neuro-oncogene product (carcinoma), P21 (carcinoma), anti- (N-glycolylneuraminic acid, breast carcinoma, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian carcinoma, testicular carcinoma), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukemia, gastric carcinoma, colorectal carcinoma, ovarian carcinoma), transmembrane protein of T-cells (carcinoma), tie (CD 202B), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, carcinoma), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, non-hodgkin's lymphoma, and other cancers, rheumatoid arthritis and systemic lupus erythematosus), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis-inducing apoptosis ligand receptor 1, lymphoma, non-hodgkin's lymphoma, large intestine cancer, lung cancer), VCAM-1 (CD 106, melanoma), vascular endothelial growth factor-a, VEGF-2 (CD 309) various cancers). Some other tumor-associated antigens recognized by antibodies have been reviewed (Gerber et al, mAbs 1. Still many other antigens are: other, different, cluster antigens (e.g., CD1a, CD1b, CD1c, CD1d, CD1E, CD2, CD3, CD3d, CD3E, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD12w, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66E, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85f, CD85E, CD85g, CD85g, CD85i, CD85j, CD85k, CD85m, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD123a, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CDw145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD156a, CD156b, CD156c, CD156d, CD157, CD158, CD158a, CD158b1, CD158b2, CD158c, CD158d, CD158E1, CD158E2, CD158f2, CD158g, CD158h, CD158i, CD158j, CD158k, CD159, CD159a, CD159b, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD167a, CD167b, <xnotran> CD168, CD169, CD170, CD171, CD172, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CDw186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CDw198, CDw199, CD200, CD201, CD202, CD202 (a, b), CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210a, CDw210b, CD211, CD212, CD213, CD213a1, CD213a2, CD214, CD215, CD216, CD217, CD218, CD218a, CD218, CD21b9, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD235a, CD235b, CD236, CD237, CD238, CD239, CD240, CD240ce, CD240d, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, CD300a, CD300b, CD300c, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD307a, CD307b, CD307c, CD307d, CD307e, CD307f, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD 389), </xnotran> APO2, ASLG659, BMPR1B (bone morphogenetic protein receptor), CRIPTO, annexin A1, nucleolus, endoglin (CD 105), ROBO4, aminopeptidase N, CTLA-4, delta-like 3 (DLL 3), delta-like 4 (DLL 4), VEGFR-2 (CD 309), CXCR49, CD 184), tie2, B7-H3, WT1, MUC1, LMP2, HPV E6E7, EGFRvIII, EGFR, HER-2/neu, idiotype, MAGE A3, P53 nonmutt, NY-ESO-1, GDH 2, CEA, melanNaA/MART 1, epiI 3B (NAI-3B, NPTIB, 34CA2, solute carrier 34, member 2, type II sodium-dependent phosphate transporter 3B), EGFP 100, P53 mutant, proteinPR 3 (PROINPR 1), tetraTAR-3B, SLIb-receptor, receptor derived from BCRtoR, EGFR, receptor III, growth factor RvhAA, EGFR, ETBR (endothelin), HER2/neu, HER3, HLA-DOB (MHC class I molecule IA antigen), integrin, IRTA2, MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin), CRIPTO, PD-1, PD-L1, sema5B (FLJ 10372, KIAA1445, mm42015, SEMA5B,5EMAG, semaphoring 5bHlog, sdema domain, seven platelet repeats, cytoplasmic domain), PSCA, STEAP1 (6 transmembrane epithelial prostate antigens), and STEAP2 (HGNC 8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate), tyrosinase, survivin, hTERT, sarcoma translocation, ephA2, PAP-IAP, AFP, TRPCAM, ETERG (TMPSSS 2 fusion gene), 17, PAX3, PAK, PBAT, MRAC-1, MYCI 2, rhoMA-C, GD3, fucosylganglioside, mesothelin, PSCA, MAGE A1, sLe (a), CYP1B1, PLAC1, GM3, BORIS, tn, globoH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, tie2, page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, fos protein-related antigen 1.

As another specific application, the conjugate of the Tubulysin derivative-cell binding molecule of the present invention may be used in various combinations and methods for cancer treatment. These cancers include, but are not limited to, adrenocortical carcinoma, rectal carcinoma, bladder carcinoma, brain tumors (adult: brain stem glioma, childhood, cerebellar astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, pineal, visual pathway and hypothalamic glioma), breast cancer, carcinoid tumors, gastrointestinal tract, unknown primary cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing family tumor (PNET), extracranial malignant germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck tumor, hypopharynx cancer, islet cell cancer, kidney cancer (renal cell carcinoma), larynx cancer, leukemia (acute lymphocytes, acute myeloid cells, chronic lymphocytes, chronic myeloid cells, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, lymphoma (aids-related, central nervous system, cutaneous T cell, hodgkin's disease, non-hodgkin's disease, malignant mesothelioma, melanoma, merkel cell carcinoma, primary occult squamous neck metastasis cancer, multiple myeloma and other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, myeloproliferative diseases, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial cells, germ cell tumors, low malignant potential tumors), pancreatic cancer (exocrine, islet cell cancer), sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary tumor, plasma cell tumors, prostate rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cells), salivary gland carcinoma, cerclage syndrome, skin cancer (dermatoid T-cell lymphoma, kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urinary tract cancer, uterine cancer (sarcoma), abnormal tumors in children, vaginal cancer, vulval cancer, and nephroblastoma.

Another particular application is that the conjugate of a Tubulysin derivative and a cell binding molecule according to the present invention may be used in various combinations and methods for the prevention and treatment of autoimmune diseases. Autoimmune diseases include, but are not limited to, achlorhydria autoimmune chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalopathy, addison's disease, globulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-glomerular basement membrane/renal tubule basement membrane nephritis, antiphospholipid syndrome, anti-synthetase syndrome, arthritis, atopic allergy, allergic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral nervous system disease, autoimmune pancreatitis, autoimmune polyendocrine I, II, type III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, baroreceptor disease/Barre sclerosis, black Berger's disease, bickerstaff encephalitis, blautaff syndrome, large bract pemphigus vulgaris, giant cold congestis hyperplasia, giant wheezoid hyperplasia, marigoid hyperplasia, marigonemal fatigue syndrome, chronic inflammatory bowel disease, chronic obstructive pulmonary sclerosis, chronic obstructive pulmonary disease, chronic glomerulonephritis, chronic inflammatory disease, chronic glomerulosclerosis, chronic obstructive pulmonary sclerosis, chronic inflammatory disease, chronic obstructive pulmonary sclerosis, chronic inflammatory bowel disease, chronic obstructive pulmonary sclerosis, chronic obstructive pulmonary disease, chronic inflammatory disease, chronic obstructive pulmonary sclerosis, chronic obstructive pulmonary disease, chronic inflammatory disorder of the type 1, diffuse cutaneous scleroderma, post-myocardial infarction syndrome, discoid lupus erythematosus, eczema, endometriosis, juvenile idiopathic arthritis, eosinophilic fasciitis, erythema nodosum, idiopathic mixed cryoglobulinemia, evens syndrome, progressive ossified fibrous tissue dysplasia, fibromyalgia, fibromyositis, fibrositis alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, graves ' disease, guillain-Barre radiculitis, hashimoto's encephalitis, lymphomatoid goiter, hemolytic anemia, hypersensitive purpura, herpes gestationis, hidradenitis suppurativa, househ syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), igA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating lesions, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, cutaneous mucosal lymph node syndrome, lambert's myasthenia syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), amyotrophic lateral sclerosis, lupus-like hepatitis, lupus erythematosus, magenis syndrome, menier's syndrome, microscopic polyangiitis, miller fisher syndrome, mixed connective tissue disease, maculopathy, mooha's disease, wels syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devick's disease), neuromuscular rigidity, cicatricial pemphigoid of the eye, ocular clonus-myoclonus syndrome, alder's thyroiditis, recurrent rheumatism, panda syndrome (childhood autoimmune neuropsychiatric abnormalities combined with streptococcal infection), neoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, progressive lateral atrophy, bart-Tech's syndrome, planular ciliary inflammation, pemphigus vulgaris, pernicious anemia, periphlebitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, rosemoson's anemia, raynaud's disease, recurrent polychondritis, laplace syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, schmidt's syndrome, schniella syndrome, scleritis, scleroderma, sjogren's syndrome, spondyloarthropathies, mucoid syndrome, still's disease, stiff person syndrome, subacute bacterial endocarditis, susac's syndrome, acute febrile skin disease, sidnom's chorea, sympathetic ophthalmia, takayasu's arteritis, temporal arteritis (giant cell arteritis), painful ophthalmoplegic syndrome, transverse myelitis, ulcerative colitis (an idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, wegener's granulomatosis, wilson's syndrome, wiskott-aldrich syndrome.

In another embodiment, the antigen binding molecules to which they are coupled for use in the treatment or prevention of autoimmune diseases include, but are not limited to: an anti-elastin antibody; abys anti-epithelial cell antibodies; anti-basement membrane type IV collagen antibodies; an antinuclear antibody; anti-double-stranded DNA antibodies; anti-single-chain DNA antibody, anti-cardiolipin antibody IgM, igG; anti-celiac (anti-celiac antibody) antibodies; anti-phospholipid antibodies IgK, igG; anti-ribonucleoprotein antibodies; anti-mitochondrial antibodies; a thyroid antibody; microsomal antibodies, T-cell antibodies; thyroglobulin antibody, anti-scleroderma-70 antibody (anti-SCL-70); human anti-Jo antibody (anti-Jo); autoantibodies against patients with systemic lupus erythematosus; anti-sjogren's syndrome antibody (Anti-La/SSB); anti-systemic lupus erythematosus antibody; anti-parietal cell antibodies; an anti-histone antibody; anti-ribonucleoprotein antibodies (anti-RNP); neutrophil cytoplasmic antibody (C-ANCA); perinuclear anti-neutrophil antibody (P-ANCA); anti-centromere antibodies; anti-nuclear fibrin antibodies, as well as Anti-glomerular basement membrane antibody (GBM), anti-ganglioside (Anti-ganglioside) antibodies; anti-desmoglein 3 antibody (anti-desmogenin 3); anti-human P62 antibody; anti-human sp100 antibodies; anti-mitochondrial M2 antibodies; antibodies to rheumatoid factor; anti-mutant citrullinated vimentin antibody (anti-MCV); an anti-topoisomerase antibody; anti-neutrophil cytoplasmic (CANCA) antibodies.

In certain preferred embodiments, the binding molecules used in the present invention for conjugate coupling can bind to a receptor or receptor complex expressed by activated lymphocytes associated with autoimmune diseases. Including immunoglobulin gene superfamily members (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD70, CD79, CD90, CD138, CD152/CTLA-4, PD-1, PD-L1 or ICOS), TNF receptor superfamily members (e.g., CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and 30 APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (type C, type S or type I) or complement regulatory proteins.

In another embodiment, a useful binding partner immunospecific for a viral or bacterial antigen is a human or human monoclonal antibody. The term "viral antigen" as used herein includes, but is not limited to: any viral peptide, polypeptide protein (e.g., HIV Nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, human T lymphocyte virus infection regulatory factor tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen) that can elicit an immune response. The term "bacterial antigen" as used herein includes, but is not limited to, any microbial peptide fragment, polypeptide protein, saccharide, polysaccharide, lipid molecule (e.g., bacteria, fungi, pathogenic protozoa, yeast polypeptides including, for example, lipopolysaccharide and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Useful type I antibodies useful for treating viral or bacterial infections include, but are not limited to: palivizumab, a human-derived anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody for use in the treatment of HIV infection; ostavir, a human antibody for the treatment of hepatitis b virus; PROTVIR, a humanized antibody IgG.sub.1 for the treatment of cytomegalovirus, and also anti-lipopolysaccharide (anti-LPS) antibodies.

The binding molecule-Tubulysin derivative conjugate can be used for treating infectious diseases. These infectious diseases include, but are not limited to: acinetobacter infection, actinomycosis, african sleeping sickness (african trypanosomiasis), aids (acquired immunodeficiency syndrome), amoebiasis, microsporosis, anthrax, cryptobacter haemolyticus infection, argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, bacteroidal infection, pauciosia, ascariasis, BK viral infection, black knot disease, human blastocyst protozoan infection, blastomycosis, vitronella hemorrhagic fever, borrelia infection, botulism (and infant botulism), brazil hemorrhagic fever, brucellosis, burkholderia infection, brucellosis, infection with caliciviruses (norovirus and saporovirus), campylosis, candida infection (candidiasis, thrush), cat scratch disease, cellulitis, chagas disease (trypanosomiasis americana), chancroid, chicken pox, chlamydia pneumoniae infection, cholera, chromomycosis, hepatisis, clostridium difficile infection, coccidioidomycosis, colorado tick fever, common cold (acute viral nasopharyngitis; acute rhinitis), creutzfeldt-Jakob disease, crimeria-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, skin larva migration disease, cyclosporidiosis, cysticercosis, cytomegalovirus infection, dengue fever, binuclear amebiasis, diphtheria, schistostostopsis, trichinosis, ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (enterobiasis), enterococcus infection, enterovirus infection, epidemic typhus, infectious erythema (fifth disease), acute eruption of the infant, fasciolopsiasis, familial insomnia, filariasis, clostridium perfringens-induced food poisoning, non-parasitic amoeba infection, clostridium infection, gas gangrene (clostridial muscular necrosis), filariasis, gistmann-straussonetia syndrome, giardiasis, meliodiosis, palatine nematode disease, gonorrhea, inguinal granuloma, group a streptococcal infection, group B streptococcal infection, haemophilus influenzae infection, hand-foot-and-mouth disease (HFMD), hantavirus lung syndrome, helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever nephrotic syndrome, hepatitis a, hepatitis B, hepatitis c, hepatitis d, hepatitis e, herpes simplex, histoplasmosis, hookworm infection, human baumanni virus infection, human ehrlichia disease, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrliosis, human papillomavirus infection, human parainfluenza virus infection, membranous thecal taeniasis, epstein-Barr virus infectious mononucleosis (singles), influenza, isosporosis, kawasaki disease, keratitis, king's disease, kuru disease, lassa fever, legionella disease (Ribes-Homeyer's Association disease), legionella disease (Pontiacre fever), leishmaniasis, leprosy, leptospirosis, lee's disease, lyme disease (Lyme disease), lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, marburg's hemorrhagic fever, melittia, moore's rhinoid (Tech's disease), meningitis, meningococcosis, postzotocystis, microsporidiosis, molluscum contagiosum, epidemic parotitis, typhus (endemic typhus), mycoplasmal pneumonia, podomyoma, myiasis, neonatal conjunctivitis (neonatal ophthalmia), creutzfeld-jakob syndrome (vCJD, nvCJD), nocardiosis, onchocerciasis (heumatosis), paracoccidioidomycosis (southern nystoloniasis), paracoccidiosis, barthospodiosis, pasteurellosis, pediculosis (head lice), pediculosis (body lice), pediculosis (pubis), pelvic inflammation, pertussis, plague, pneumococcal infection, pneumocystic pneumonia, poliomyelitis, prevotella infection, primary amoeba meningoencephalitis, progressive multifocal leukoencephalopathy, psilosis, Q fever, rabies, mouse bite fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsia, schizovorax, rocky mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, septicemia, dysentery (bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, nematodes, syphilis, taeniasis, tetanus (cleistolochia), tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea manuum, pityriasis nigra, tinea pedis, onychomycosis, tinea versicolor, toxocariasis (ocular larvae transmigration), toxoplasmosis, trichinosis, trichurism (trichuris infection), tuberculosis, tularemia, ureolytic ureaplasma infection, venezuelan equine encephalitis, venezuelan hemorrhagic fever, viral pneumonia, west nile fever, sarcoidosis albus (sarcoidosis albus), pseudotuberculosis infection, yersinia, yellow fever, zygomycosis.

The binding molecules for the antibodies previously described in this patent are useful against pathogenic strains, including, but not limited to: acinetobacter baumannii, actinomyces israeli, actinomyces goviensis and Propionibacterium propionate, trypanosoma brucei, HIV (human immunodeficiency virus), endomonas histolytica, anamorpha, bacillus anthracis, cryptococcus hemolyticus, huinivirus, ascaris, aspergillus, plantago virus family, babesia, bacillus cereus, polyporus, bacteroides, parietaria coli, belliobara, BK virus, oudenria, human blastocyst Protozoa, blastomyces dermatitidis, borrelia, clostridium botulinum, sinomenii, brucella, typically Burkholderia cepacis and other Burkholderia species, mycobacterium ulcerosa, the Caliciviridae family, campylobacter, typically Candida albicans and other Candida species, bartonella, group A streptococci and staphylococci, trypanosoma cruzi, haemophilus ducreyi, varicella Zoster Virus (VZV), chlamydia trachomatis, chlamydia pneumoniae, vibrio cholerae, periploca farinosa, clonorchis sinensis, clostridium difficile, coccidioides and Coccidioides posadasii, colorado tick fever virus, rhinovirus, coronavirus, prion Creutzfeldt-Jakob disease, crimia-Congo hemorrhagic fever virus, cryptococcus neoformans, cryptosporidium, hookerian; multiple parasites, cyclospores, taenia solium, cytomegalovirus, dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) arbovirus, fragile binuclear amoeba, corynebacterium diphtheriae, schistocephalus, meldonolong nematode, ebola virus, echinococcus, elekeri, enterococcus, enterovirus, rickettsia prowazekii, parvovirus B19, human herpesvirus type 6 and human herpesvirus type 7, fasciola brunetti, fasciola hepatica and fasciola magnus, FFI prions, filariales superfamily, perfringens, clostridium, perfringens, other clostridia, geotrichum, GSS prions, giardia intestinalis, burkholderia mallei, acanthopanax and acanthopanax, neisseria gonorrhoeae, granuloma, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, most coxsackie a and enterovirus type 71, sinovirus, helicobacter pylori, escherichia coli O157: h7, family of the bunyaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus type 1, herpes simplex virus type 2, histoplasma bacteria, ancylostoma duodenale and American aphrodisiae, haemophilus influenzae, human bocavirus, erwinia ehrlichia, anaplasmataceae, human metapneumovirus, serratia arenicola, human papillomavirus, human parainfluenza virus, micromembrana tenuis and minivan tapeworm, EB virus, orthomyxoviridae, belleville, king's bacillus, klebsiella pneumoniae, klebsiella oenzaas, kuru prion, lassa virus, legionella pneumophila, legionella pneumophiladensis, leishmania immaturus, mycobacterium leprae and Mycobacterium diffusible, leptospira, listeria, borrelia burgdorferi and other Borrelia species, filaria bambusicola and filarial worms, lymphocytic choriomeningitis virus (LCMV), plasmodium species, marburg virus, measles virus, burkholderia farinosa, neisseria meningitidis, retrograduate schistosomiasis, microsporophylum, molluscum Contagiosum Virus (MCV), mumps virus, rickettsia, mycoplasma pneumoniae, various bacteria (M. Duchensis) and fungi (M. Duchensis), parasitic Lepidotia, chlamydia trachomatis and gonococci, CJvD prion, nocardia asteroides and other Nocardia species, spiraptera, brucella brasiliensis, euglena and other paragonia species, pasteurella species, toxoplasma psylla, bemisia, pubic louse, bordetella pertussis, yersinia pestis, streptococcus pneumoniae, pneumocystis carinii, poliovirus, prevotella, frankia resistant Grignard, JC virus, chlamydia psittaci, rickettsia, rabies virus, streptococci candida and Treponema murinus, respiratory syncytial virus, coccidioides, rhinovirus, rickettsia, lerickettsia, rift valley fever virus, rickettsia, rotavirus, rubella virus, salmonella, atypical pneumonia coronavirus, scabies, hematodes, shigella, varicella zoster virus, smallpox or smallpox, trichosporon scherianum, staphylococcus aureus, streptococcus pyogenes, strongyloides faecalis, treponema pallidum, taenia, clostridium tetani, trichosporon, trichophyton floccosum, trichophyton rubrum and Trichophyton mentagrophytes, exophiala Venerigeron, trichophyton, malassezia, toxobolus or Toxocara, toxoplasma, trichinella, trichomonas vaginalis, trichuris trichuris, mycobacterium tuberculosis, francisella tularensis, ureaplasma urealyticum, venezuelan equine encephalitis virus, vibrio cholerae, guaratorulownia, west Nile virus, trichosporon albus, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever virus, mucor order (Mucor mucor disease) and entomomycetales (entomophthora), pseudomonas aeruginosa, campylobacter fetus (Vibrio), aeromonas hydrophila, edwardsiella tarda, yersinia, shigella dysenteriae, shigella sonnei, salmonella typhimurium, sphachis, treponema caratuum, borrelia perna, borrelia burgdorferi, leptospira hemorrhagic jaundice, pneumocystis carinii, brucella suis, brucella, mycoplasma, typhus, rickettsia tsutugumushi, chlamydomonas; pathogenic fungi (aspergillus, candida albicans, histoplasma capsulatum); protozoa (entamoeba histolytica, trichomonas vaginalis, trichomonas humanus, trypanomonas gamniense, trypanosoma rhodesiense, leishmania donovani, leishmania tropicalis, leishmania brasiliensis, pneumocystis pneumonia, plasmodium vivax, plasmodium falciparum, malaria falciparum); or helminth (Schistosoma japonicum, schistosoma mansoni, schistosoma Egypti and hookworm)

Other antibodies that are binding ligands of the invention are useful for treating viral diseases including, but not limited to: antibodies against antigens of pathogenic viruses include the following examples but are not limited to: smallpox virus, herpesvirus, adenovirus, papovaviridae, enteroviridae, picornaviridae, parvoviridae, reovirus, retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, arbovirus, rhabdovirus, arenaviridae, non-a/Non-B hepatitis virus, rhinovirus, coronavirus, rotaviridae, oncovirus [ e.g., hepatitis B virus (hepatocellular carcinoma), human papilloma virus (cervical carcinoma, anal carcinoma), kaposi's sarcoma-associated herpesvirus (kaposi's sarcoma), EB virus (nasopharyngeal carcinoma, burkitt's lymphoma, primary central nervous system lymphoma), MCPyV (merkel cell carcinoma), SV40 (simian virus 40), hepatitis c virus (hepatoma), human T-lymphotropic virus type 1 (adult T cell leukemia/lymphoma), immune-causing virus: [ such as human immunodeficiency virus (AIDS) ]; central nervous system virus: [ e.g., JCV (progressive multifocal leukoencephalopathy), meV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitis, orthomyxoviridae (possible) (narcolepsy), RV (rabies), vesicular stomatitis-Indian virus genus, herpesvirus meningitis, lamzis Hunter syndrome type II; polio (polio, post-polio syndrome), human T-lymphotropic virus type 1 (tropical spastic paraplegia) ]; cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)); cardiovascular disease viruses [ such as coxsackie virus (pericarditis, myocarditis) ]; respiratory system/acute nasopharyngitis virus/viral pneumonia: [ Epstein-Barr virus (herpes virus type 4 infection/infectious mononucleosis), cytomegalovirus; SARS coronavirus (severe acute respiratory syndrome) orthomyxovirus: influenza virus a/B/C (influenza/avian influenza), paramyxovirus: human parainfluenza virus (parainfluenza), respiratory syncytial virus (human respiratory syncytial virus), pneumovirus ]; digestive system virus [ MUV (mumps), cytomegalovirus (cytomegalovirus esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus; HBV (hepatitis b virus), coxsackievirus, hepatitis a (hepatitis a virus), HCV (hepatitis c virus), HDV (hepatitis d virus), HEV (hepatitis e virus), HGV (hepatitis g virus) ]; urogenital viruses [ e.g., BK virus, MUV (mumps) ].

According to a further object, the present invention also includes the above-described conjugated pharmaceutical compositions in combination with other possible pharmaceutical carriers as therapeutic agents for cancer and autoimmune diseases. The methods of the invention for treating cancer and autoimmune diseases include in vitro, in vivo or ex vivo therapy. Examples of the application of in vitro therapy include drug treatment of in vitro cultured cells to kill all cells except those not expressing the antigen of interest; or killing cells that express undesired antigens. As an example of a method of treatment for ex vivo therapy: hematopoietic stem cells are treated in vitro to kill diseased or malignant cells before being returned to the patient. For example, cancer and autoimmune diseases are treated clinically by ex vivo treatment to remove tumor cells or lymphocytes from the bone marrow and then transfused back to the original patient, or T cells and other lymphocytes from the bone marrow are removed prior to transplantation to prevent an immune antagonistic response of the graft. The implementation method comprises the following steps: bone marrow cells are harvested from a patient or other individual and cultured in a serum-containing medium to which the conjugate drug of the present invention is added at 37 ℃ in a drug concentration range of 1pM to 0.1mM for a period of about 30 minutes to about 96 hours. The specific concentration of the drug and the incubation time are determined by an experienced clinician. After the culture is finished, the bone marrow cells are washed by serum-containing culture medium and then are infused back into the human body through intravenous injection. If the patient is to receive other treatments, such as ablative chemotherapy or total body radiation, prior to the bone marrow cell harvesting and reinfusion treatment, the treated bone marrow cells can be stored in a qualified liquid nitrogen medical facility.

For in vivo clinical application, the conjugate drug of the present invention will be provided in the form of a solution or a lyophilized solid (powder for injection) that can be dissolved in sterile water and then injected. The mass ratio of the conjugate drug in the solution or the freeze-dried solid (powder injection) can be 0.01-99%. In order to prepare a stable lyophilized powder for injection, one or more of the following adjuvants are added to the feed solution for the preparation of lyophilized powder for injection. Suitable excipients include: one or more polyol compounds (including sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids, reducing sugars such as fructose, mannose, maltose, lactose, sugars, xylose, ribose, mannose, galactose and glucose, non-reducing sugars such as sucrose, trehalose, sorbitol, melezitose and raffinose, sugar alcohols selected from mannitol, xylitol, erythritol, maltitol, xylitol, erythritol, threitol, sorbitol and glycerol, sugar acids such as gluconic acid and its salts) in the formulation from 0.0% to 25%; 5mM-500mM (0.05% -25% of the mass ratio after freeze-drying) of buffer reagent (such as citric acid, succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, tris hydrochloride, phthalic acid or phosphoric acid) in the feed liquid is used for finally adjusting the pH value to 4.5-8.5; surfactants (such as polysorbate 20, 40, 60, 65, 80 or 85; poloxamer 407, polyethylene-polypropylene glycol, sodium Dodecyl Sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, and the like) are present in the formulation at 0.0% to 2.0%; antioxidant (such as ascorbic acid and/or methionine) 0.0% -5.0% of the ingredient; elasticity/tonicity modifier (such as mannitol, sorbitol or NaCl) 0.0% -10%; the content of chelating agent such as EDTA or EGTA in the ingredient is 0.0-5 mM; the metal complex (such as zinc-protein complex) accounts for 0.0-1% of the ingredients; (ii) a Biodegradable polymers, such as polyesters, are present in the formulation at 0.0% to 1.5%; preservatives (such as benzyl alcohol, phenol, butanol, benzyl alcohol, alkyl esters such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, chlorobutanol, 18 carbon based dimethyl benzyl ammonium chloride, and the like) are present in the formulation at 0.0% to 5%; and/or free amino acids (such as arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid or aspartic acid) in an amount of 0.0% to 30% of the formulation. The osmotemomolar concentration of the prepared (pre-filled) injection/reconstituted freeze-dried injection should be controlled within 225-370mOsm/L. The osmolality after the injection/dilution of physiological saline is guaranteed to be in the range of 285-310mOsm/L. The medicine component solution can be prepared into pre-packaged injection, or can be filled into a penicillin bottle and then freeze-dried into a needle, or can be prepared into powder through high-efficiency spray drying, and the powder is used for sub-packaging storage and sale. In order to relieve pain caused by injection, local analgesic should be added. The commonly used analgesic and dosage: benzyl alcohol (about 1%), procaine hydrochloride (0.2-2.0%), lidocaine hydrochloride (0.2-2.0%), chlorobutanol (0.3-0.5%), tramadol, morphine sulfate, hydromorphone, oxycodone, gabapentin, cyclobenzaprine, trazodone and clonidine.

Examples of suitable methods for administering the conjugate drugs of this patent are as follows: the conjugate drug is intravenously injected once a week, or every two weeks, or every 3 weeks, or every 4 weeks, or every month for 3 to 52 weeks. The single dose of the conjugate drug (lyophilized powder for injection reconstituted by addition of water for injection) is prepared by dissolving in 50 to 500 ml of physiological saline to which human serum albumin (e.g., 0.5 to 1 ml of 100mg/ml concentrated human serum albumin) may be optionally added. The dose of the drug is approximately 50 μ g to 80mg per kg of body weight per week, i.v. injection (10 ug to 200mg/kg of body weight per injection). After 1-52 weeks of treatment, the patient may receive a new cycle of treatment. The detailed treatment method including administration route, excipient, diluent, drug dose, treatment time and the like can be decided by a skilled surgeon.

Examples of diseases that can be treated by selective killing of cell populations by in vivo or ex vivo methods include any kind of malignancy, autoimmune disease, transplant rejection and infection (including viruses, bacteria or parasites).

The amount of conjugated drug required to achieve the desired biological effect will vary depending upon a number of factors including the nature of the compound, its therapeutic effect and bioavailability of the conjugated drug, the type of disease, the race of the patient, the diseased state of the patient, the route of administration, all of which together determine the timing and manner of administration.

In summary, the conjugate drug of the present invention can be used for parenteral administration by dissolving in a physiological buffer at 0.1 to 10% by mass volume. Typical drug doses range from 1ug to 0.1g per kilogram of body weight per week (or every 2 weeks, or every 3 weeks, or every 4 weeks); recommended dosages range from 0.01mg to 50mg per kilogram of body weight per 3 weeks or equivalent dosages for children. The recommended amount to be administered depends on a number of variables, including the type of disease or disorder, the general health status of the individual patient, the relative biological activity of the conjugate drug, the dosage form of the compound, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the selected mode of administration, as well as the rate of administration (single injection or continuous instillation) and the schedule of administration (number of administrations over a period of time).

The conjugate of the present invention can also be administered in the form of a unit dose, where "unit dose" refers to a dose administered once to a patient, and the unit dose of drug can be packaged and used simply and conveniently, and the unit dose of drug is an active conjugate drug itself which remains physically and chemically stable, or a pharmaceutically acceptable mixture as described later. Typical daily dosages range from 0.01 to 100mg per kg body weight. Generally, the unit dosage for humans ranges from 1 to 3000mg per week. Recommended unit dose is 1.0mg to 500mg, four times per month administration, or 1.0mg to 500mg once every 3 weeks. The conjugate medicine can be prepared into a medicinal preparation by adding one or more pharmaceutically acceptable auxiliary materials. The unit dose of the drug may be administered orally, such as in the form of a tablet, a simple capsule or a soft gel capsule; or intranasally, such as a powder, nasal drops, or spray; or by dermal administration, such as ointments, creams, lotions, gels or sprays or dermal patches. The agents may conveniently be administered in unit dosage form and may be prepared by any known pharmaceutical method, such as Remington The Science and Practice of Pharmacy,21th ed; the method described in Lippincott Williams & Wilkins, philadelphia, PA 2005.

Pharmaceutical dosage forms comprising the compounds of the invention include pharmaceutical compositions, preferably oral or parenteral administration. For oral administration, such as tablets, powders, capsules, tablets (lozenges) and the like may comprise one or more of the following materials or other compounds of similar nature: binders such as microcrystalline cellulose or tragacanth; diluents such as starch or lactose; dispersants such as starch and cellulose derivatives; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavour enhancer such as peppermint or methyl salicylate. The capsules may be in the form of hard or soft capsules, typically mixed optionally with a plasticizer from a gelatin mixture, as may starch capsules. In addition, the physical form of the unit dose can be varied by the addition of a variety of different materials, for example, sugar coatings, shellacs, or enteric agents. Other oral dosage forms such as syrups or elixirs may contain sweetening agents, preserving agents, colouring agents and flavouring agents. In addition, the active compounds can be formulated by different treatments and formulations into rapidly dissolving dosage forms, slow release dosage forms or sustained release agents, of which sustained release agents are preferred. Tablets are preferably formulated with a combination of lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, talc, and the like.

Liquid preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. The liquid agent may also contain a binder, a buffer, a preservative, a chelating agent, a sweetening agent, a flavoring agent, a coloring agent, and the like. Non-aqueous solvents include ethanol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic lipids such as ethyl oleate. The aqueous solvent comprises a mixture of water, ethanol, a buffering agent and a salt, in particular, a biocompatible, degradable lactide polymer, a lactide/glycolide polymer or a polyethylene glycol/polyglycerol copolymer can be used as an adjuvant for controlling the release of the active drug. Excipients for intravenous injection may include liquid and nutritional supplements, electrolyte supplements, and linoglycol-based excipients, and the like. Other possible parenteral delivery systems for the active agents of the invention include microparticles of ethylene-vinyl acetate copolymer, implantable osmotic pumps and liposomes.

Other possible modes of administration include inhalation, including dry powders, aerosols and drops. The inhalant may be a solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, deoxycholate or oil, and may be administered in the form of nasal drops, intranasal gel. The buccal preparation comprises lozenge, candy lozenge, etc., and optionally flavoring agent such as sucrose, acacia, and other adjuvants such as glycocholate, etc. Suppositories are suitable in unit dosage form, with solids such as cocoa butter as the carrier, and salicylic acid may also be added. The topical preparation for skin is preferably selected from plaster, emulsion, lotion, patch, gel, spray, aerosol or oil. Petrolatum, lanolin, polyethylene glycols, alcohols and mixtures thereof may be used as the pharmaceutical carrier. The formulations for dermal administration may be in the form of patches, emulsions, buffered solutions, dissolved or dispersed in polymers or adhesives.

In particular, the conjugate of the present invention may be used in combination with other known or unknown therapeutic agents, such as chemotherapeutic agents, radiation therapy, immunotherapy agents, autoimmune disease agents, anti-infective agents or other antibody drug conjugates, to achieve a synergistic effect. The co-drug or radiation therapy may be administered or administered before or after the administration of the conjugate drug of the invention. It may be 1 hour, 12 hours, one day, one week, two weeks, three weeks, one month, or several months before or after administration of the conjugate of the present invention.

In other embodiments, the conjugate drugs of the present invention are used with a coordinating drug. Drugs that act synergistically include, but are not limited to:

1) Chemotherapeutic agent a) alkylating agent: such as nitrogen mustard (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, cyclophosphamide chloroacetate); nitrosoureas (carmustine, lomustine); alkyl sulfonate (Busulfan, susulfan); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin) ]; b) Plant alkaloids such as [ vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine); taxane compounds (paclitaxel, taxotere) ]; c) DNA topoisomerase inhibitors such as [ epipodophyllotoxin (9-aminocamptothecin, camptothecin, clinostat, etoposide phosphate, irinotecan, teniposide, topotecan) ]; mitomycin: (mitomycin C) ]; d) An antimetabolite: such as { [ antifolate: dihydrofolate reductase inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase inhibitors (mycophenolic acid, formamidothiazole, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine) ]; [ pyrimidine analog: uracil analogs: (5-fluorouracil, doxifluridine, raltitrexed (tomude)); cytosine analogues: (cytarabine, fludarabine); purine analogues: (azathioprine, mercaptopurine, guanine) ] }; e) The hormones: such as { receptor antagonist: [ anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goserelin, leuprorelin acetate); anti-androgens: (bicalutamide, flutamide) ]; tretinoin/deltoid: [ vitamin D3 analogues (CB 1093, EB 1089KH 1060, cholecalciferol, vitamin D2); photodynamic therapy: (for this, phthalocyanine photosensitizer, PC4, deoxynivalenol); cytokines: (interferon- α, interferon- γ, tumor necrosis factor (tumor necrosis factor), human protein containing TNF domain) ] } f kinase inhibitors such as bibw 2992 (anti-EGFR/Erb 2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, pinatinib, vandetanib, flumatinib, e7080 (anti-VEGFR 2), molitinib, meilitinib, ponatinib (ap 245634), HQP1351, barfitinib (INNO-406), bosutinib (SKI-606), sunitinib, cabotinib, voritinib, virginib, inimod, ruxolitinib, CYT387, axizumab, tivab, tivozab, bevacizumab, sorafenib, cetuximab; g) Other classes: such as gemcitabine, epoxomicins (e.g., lenalidomide), bortezomib, lenalidomide, pomalidomide, tosedodt, zybrestat, PLX4032, sta-9090, stimuvax,

allovivin-7, xegeva, provenge, yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), pingyangmycin (e.g., bleomycin A2, B2, pelomycin), anthracyclines (e.g., erythromycin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., verapamil), ca ²⁺ Inhibitors of ATPase (e.g., thapsigargin), inhibitors of histone deacetylase (vorinostat, romidepsin, panobinostat, valproic acid, motinostat (MGCD 0103), belinostat, PCI-24781, entinostat, SB939, remininostat, givinostat, AR-42, one, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, 5 disulfiram, salinosporamide a.

2) Anti-autoimmune agents include, but are not limited to, cyclosporine A, azathioprine, aminocaproic acid, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, glucocorticoids (e.g., kininodes, betamethasone, budesonide, flunisolide, fluticasone propionate, hydrocortisone, dexamethasone, fluocortolone danazol, triamcinolone acetonide, beclomethasone dipropionate), dehydroepiandrosterone, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate, sirolimus, tacrolimus, prednisone

3) Anti-infectious disease agents include, but are not limited to: a) Aminoglycosides: amikacin, wuyimycin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin) neomycin (neomycin B, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylzeocin; b) Amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) Ansamycins: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) Cephalo species: cephem (chlorocepham), cephalosporanic nitrile, cefaclor, cephradine, cefadroxil, cefalonine, ceftiofur or cephalosporin, cephalexin, cefalexin, cefamandole, cefapirin, ceftriaxone, cefazedone, cefazolin, cefbuperazone, cefcapene, cefixime, cefdaxime, cefepime, cefoxitin, cefprozil, cefixime, ceftezole, cefuroxime, cefdinir cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefimidazole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefotiam, ceftizoxime, cefatrixime, ceftriaxone, cefuroxime, ceftizoxime, cephamycin (cefoxitin, cefotetan, cefmetazole) oxacephem (flomoxef, moxalactam); f) A glycopeptide: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin) ramoplanin, daptomycin; g) Glycyl: such as tigecycline; h) A beta-lactamase inhibitor: penicillane (sulbactam, tazobactam), clavulane (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) A lipopeptide: daptomycin, a54145, calcium Dependent Antibiotic (CDA); k) Macrolides: azithromycin, quinoxalin and quinoxalin, clarithromycin, dirithromycin, erythromycin, fluoromycin, josamycin, ketolides (telithromycin, quinoxalin and quinoxalin) medecamycin, meoxakamycin, oleandomycin, rifamycin (rifampin, rifabutin, rifapentin), natamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK 506), acearundomycin, tulithromycin; l) monocyclic beta-lactam antibiotics aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, hydracillin, bacampicillin, maytansillin, phthalazinocillin) azicillin, azlocillin, penicillin, benzathine, phenoxybenzathine, cloxacillin, procaine penicillin, carbenicillin (cairinillin), cloxacillin, dicloxacillin, cephalomycin, flucloxacillin, mexacillin (diazamidine penicillin diester), mezlocillin, methicillin, nafcillin, oxacillin, acemetacillin, penicillin, necillin, piperacillin, hydrocillin, sulbenicillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, polymyxin E, polymyxin B; p) quinolone drugs: alafloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, gatifloxacin, gemifloxacin, grepafloxacin, trovafloxacin carnot, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) a bacterin: pristinamycin, quinupristin/dalfopristin); r) sulfonamides: mafenide, chlordanese, sulfacetamide, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (compound sulfamethoxazole); s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, demeclocycline, lymecycline, chloromethycin, methacycline, minocycline, oxytetracycline, piperacycline, rolicycline, tetracycline, glycyl (e.g., tigecycline); other types of antibiotics: annonaceae, arsinamine, bacterpenol inhibitors (bacitracin), dadal/AR inhibitors (cycloserine), dictyostatin, discodermolide, exenatide, epothilones, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalamide, metronidazole, mupirocin, mycolactines, NAM synthesis inhibitors (such as fosfomycin), nitrofurantoin, taxol, platemycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, annosquamoside;

4) Antiviral agents a)./fusion inhibitors: aplaviroc, maraviroc, viriviroc, gp41 (enfuvir), PRO140, CD4 (Ibalizumab); b) An integrase inhibitor: latilatavir, eltiravir, globoidnan A; c) A maturation inhibitor: buvir lima, vivecon; d) Neuraminidase inhibitor: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir clavvudine, dexelvucitabine, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3 '-fluoro substituted 2',3 '-dideoxynucleoside analogs (e.g., 3' -fluoro-2 ',3' -dideoxythymidine (FLT) and 3 '-fluoro-2', 3 '-dideoxy (FLG) fomivirse, ganciclovir, idoside, lamivudine (3 TC), L-nucleosides (e.g., β -L-thymidine, β -L-2' -deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d 4T), tacivirine (DDC vavudine), telavavudine, tenofovir, valacyclovir, zivudine (azvir), zivudine (azvir, azvudine, azlactone, zivudine, azlactone: amantadine, atevirdine, capevirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, interferon alpha, lovirdine, lodenosine, tazozole, nevirapine, NOV-205, peginterferon alpha, podophyllotoxin, rifampin, rimantadine, resiquimod (R-848), acetimidadamantane; g) A protease inhibitor: amprenavir, atazanavir, borrelivir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950) tipranavir; h) Other types of antiviral drugs: abzyme, arbidol, calanolides a, cerulenin, cyanobacterial-N, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taliverine (velvamidine), hydroxyurea, KP-1461, miltefosine, pleconaril, synthesis inhibitors, ribavirin, seliciclib;

5) Other immunotherapeutic drugs: such as imiquimod, interferons (e.g., α, β), granulocyte colony stimulating factor, cytokines, interleukins (IL-1-IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ipilimumab, nivolumab, pembrolizumab, BGB-A317, atezolizumab, avelumab, durvalumab), protein-binding drugs (e.g., abraxane), an antibody-binding drug selected from the group consisting of calicheamicin derivatives, maytansine derivatives (DM 1 and DM 4), CC-1065, and duocarmycin minor groove agents, potent paclitaxel derivatives, doxorubicin, auristatin antimitotic drug conjugates, such as Trastuzumab-DM 1, inotuzumab, brentuximab vedotin, glembuzumab vedotin, lorvotuzumab mertansine, gemtuzumab ozogamicin, trastuzumab emtansine, inotuzumab ozogamicin, sactuzumab govitecan, anetuzumab ravtansine, denituzumab mafodotin, rovaltuzumab tesiline, labetuzumab visectan, BMS-986148, AN-LMB2, TP-38, VB4-845, cantuzumab stanzine, AVE9633, SAR3419, CAT-1528015 (anti-CD 22), IMGN388, IMGN529, mirvetuzumab soravtansine (IMGN 853), vadastuximab taline, milatuzumab-doxorubicin, depatuzumab mfododin (ABT-414), AGS-1693F, humax-TF-ADC, TAK-264 (MLN-0264), milatuzumab doxorubicin, trastuzumab derxtecan, SGN-75 (anti-CD 70), anti-CD 22-MCC-DM 1).

In another embodiment, the treatment or prevention of cancer, or autoimmune disease, or infectious disease is performed in a combination. Pharmaceutical compositions containing a therapeutically effective amount of the conjugates of formula (II) described herein are synergistically effective for treatment or prevention by simultaneous or sequential administration with other drugs such as chemotherapy, radiation therapy, immunotherapeutics, autoimmune disease drugs, anti-infective drugs, or others. The synergist is preferably selected from one or more of the following medicines: albizumab, abiraterone acetate, albumin paclitaxel, paracetamol/hydrocodone, aframoplantanib with CAS number 1420477-60-6, adalimumab, recombinant human interleukin-2, afatinib dimaleate, eletinib, alemtuzumab, aliretinic acid, CAS number 1018448-65-1 Erdituzumab maytansine, amphetamine or amphetamine/dextroamphetamine mixed salt, anastrozole, anthracycline, aripiprazole, atazanavir, atlizumab, atorvastatin, adermumab with CAS number 1537032-82-8, adixtinib, bellista with CAS number 866323-14-0, bevacizumab, besalutin, bornazumab, bortezomib, subleb, benzeximab, bugatinib, and budesonide, budesonide/formoterol, buprenorphine, cabazitaxel with CAS number 183133-96-2, cabozantinib, capecitabine, carfilzomib, cells of chimeric antigen receptor engineered T, celecoxib, cetuximab, cyclosporine, cinacalcet, crizotinib, cobicistinib with CAS number 934660-93-2, dabigatran, dabrafenib, dacarbazine, daclizumab, daptomycin, daclizumab with CAS number 945721-28-8, darbepoetin, darunavir with CAS number 206361-99-1, matatinib mesylate, dasatinib, dineburnib-2 with CAS number 173146-27-5, gienib with CAS number 526158-49-7, daclizumab, dipalmitylmetin, dexamethasone, dexlansoprazole with CAS number 530-94-6, methylphenidate, deutuximab with CAS number 1363687-32-4, doxycycline, duloxetine, dewezumab with CAS number 1428935-60-7, etobizumab with CAS number 915296-00-3, teefolimus, tenofovir/Efavirenzan, endotinib, ezetimibe/simvastatin, fenofibrate/Enitinib, fingolimod, fluticasone propionate, fluticasone/Salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, imatinib, esomelimumab, ebrutinib, imatinib, isocyclophosphamide, rituximab, ellitinib, insulin aspart, insulin detemir, insulin glargine, insulin lisine, interferon beta 1a, interferon beta 1b, lapatinib, lypramemium, ipratropium bromide/, linagli, lenvatinib, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, CAS number 608137-33-3-lysine amphetamine, memantine, methylphenidate, metoprolol, modafinil, mometasone, cetuximab with CAS number 906805-06-9, lenatinib with CAS number 69883-09-6, nilotinib with CAS number 641571-10-0, nilotinib with CAS number 1038915-60-4, nimapanib with CAS number 946414-94-4, ofamumab, olpazumab, olpadumab, olmesartan, oxaliplatin, hydrochlorothiazide, omalizumab, emimikamikamii-3 fatty acid ethyl ester, olwurtzitavir, oseltamike number 1423-65-0, oseltamiktisin with CAS number 1420, oxycodone hydrochloride, palizumab, parvizumab, paluzumab, palonozumab, pazopanib, PD-1 antibody, PD-L1 antibody, peginterferon alpha-2a, pembrolizumab with CAS number 1374853-91-4, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, propranolol, quetiapine, rabeprazole, radium chloride 223, raloxifene, latiravir, ramucirumab, ranibizumab, regorafenib, rituximab, rivaroxaban, romidepsin, rosuvastatin, luotetinib, albuterol, salbutamol, savatidine with CAS number 1313725-88-0, savatidine, nomotide, somatidelandin, sildenafil, CAS number 541502-14-1 cetuximab, sitagliptin/metformin, solifenacin, sonedgi, sorafenib, sunitinib, tacrolimus, temapamide, temozolomide, temsirolimus, tenofovir/emtricitabine, testosterone gel, thalidomide, tiotropium bromide, toremifene, tremetinib, trastuzumab, trasbrate tincture with CAS number 114899-77-3, trifluorothymidine/tipepin with CAS number 183204-72-0, tretinoin, esteke, valsartan, viliparib, vandetanib, vemurafenib, verofenib, xenopus, herpes zoster vaccines, and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof and various dosage forms thereof.

As a further object of the present invention, the present invention also relates to a process for preparing the antibody drug conjugate therein. The conjugates of the present invention may be prepared by a variety of methods well known in the art, for example, the Tubulysin derivatives of the conjugates of the present invention may be synthesized according to the following methods or modified methods described below. These modifications are well known and obvious methods readily available in the scientific literature to those skilled in the art. In particular, there are many descriptions in Comprehensive Organic Transformations (R.C. Larock, 1999, wiley-VCH, 2 nd edition).

During the reaction described herein, it may sometimes be necessary to protect reactive functional groups that may participate in the reaction, such as hydroxyl, amino, imino, mercapto, and carboxyl groups, to avoid side reactions. A common method of use of conventional Protective functional Groups is described in "Green's Protective Groups in Organic Synthesis", by P.G.Wuts and T.W.Greene (2006, wiley-Interscience, 4 th edition). Some reactions may be carried out in a suitable acid or base containing solution. The acid, base and solvent for such reaction are not particularly limited, and any conventional acid, base and solvent may be used herein as long as they have no adverse effect. Moreover, these reactions can be carried out over a wide range of temperatures. In general, however, the reaction temperature is generally between-80 ℃ and 150 ℃ (preferably between room temperature and 100 ℃). The time required for the reaction can likewise vary widely, depending, of course, on a number of factors, in particular the reaction temperature and the nature of the solvent used. In general, for a more desirable reaction, a reaction time of 3 to 20 hours is suitable.

The operation treatment after the completion of the reaction can be carried out by a conventional method. For example, the reaction product can be recovered by distilling off the solvent from the reaction system. Alternatively, after the solvent is distilled off, the residue may be poured into water and then extracted with a water-immiscible organic solvent, if necessary. Finally, after the extraction solvent has been distilled off, the reaction product is obtained. In addition, if higher purity is desired, it can be further purified by various conventional methods, such as recrystallization, sedimentation, or various chromatographic methods. Generally, column chromatography and preparative thin-plate chromatography are more commonly used. The synthesis of the Tubulysin derivatives and conjugates thereof of the present invention is illustrated in FIGS. 1-60.

In the following examples, the cell binding agent-Tubulysin derivative conjugate of the present invention will be further described in more detail. Of course, the present invention is not limited to the following examples.

Experimental part:

NMR spectra were recorded on a Bruker 500MHz NMR spectrometer. Chemical shifts (. Delta.) are expressed in parts per million (ppm) based on tetramethylsilane (0.00 ppm) and coupling constants (J) are expressed in Hertz. High resolution mass spectrum data obtained by a Waters acquisition UPLC-MS (Xevo QTOF) or Agilent 6120LC-MS (Quadrapole) liquid chromatograph or the like. Amino acids and their derivatives and pre-filled resins are available from Merck chemical International, synthetech, peptides International, inc., chembridge International, or Sigma-Aldrich. Partial cross-linkers such as NHS esters/maleimides (AMAS, BMPS, GMBS, MBS, SMCC, EMCS or Sulfo-EMCS, SMPB, SMPH, LC-SMCC, sulfo-KMUS, SM (PEG) 4,SM (PEG) 6,SM (PEG) 8,SM (PEG) 12,SM (PEG) 24); NHS ester/pyridinedimercapto (SPDP, LC-SPDP or Sulfo-LC-SPDP, SMPT, sulfo-LC-SMPT); NHS ester/haloacetyl (SIA, SBAP, SIAB or Sulfo-SIAB); NHS ester/cyclopropene (SDA or Sulfo-SDA, LC-SDA or Sulfo-LC-SDA, SDAD or Sulfo-SDAD); maleimide/hydrazide (BMPH, EMCH, MPBH, KMUH); pyridinedimercapto/hydrazide (PDPH); and isocyanate/maleimide (PMPI) from Thermo Fisher Scientific. Trastuzumab was purchased from Genentech and self-made by this unit. All anhydrous solvents were commercially available and stored under nitrogen in fully sealed bottles. All other reagents or solvents were used as received without further purification using the highest commercial specifications.

EXAMPLE 1 Synthesis of Compound 2

Diethoxyacetonitrile (1.00kg, 7.74mol) was dissolved in methanol (6.0L) in a 10-L reactor and (NH) was added at room temperature ₄ ) ₂ S (48% aqueous solution, 1.41kg, 9.29mol). The temperature in the kettle was raised to 33 ℃ and then returned to room temperature. After stirring overnight, the reaction was concentrated. Ethyl acetate (5L) was added to the residue, which was washed with saturated NaHCO ₃ The solution (4X 1.0L) was washed and the aqueous phase was back-extracted with ethyl acetate (5X 1.0L). The organic phases were combined, washed with saturated brine (3L), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Pulping the crude product with petroleum ether, vacuum filtering, collecting solid and washing with petroleum ether. The filtrate was concentrated and slurried with petroleum ether, and the resulting solids were combined and collected to yield a total of 1.1kg (87% yield) of the desired product as a white or bright yellow solid. ¹ H NMR(500MHz,CDCl ₃ )δ7.81(d,J＝71.1Hz,2H),5.03(s,1H),3.73(dq,J＝9.4,7.1Hz,2H),3.64(dq,J＝9.4,7.0Hz,2H),1.25(t,J＝7.1Hz,6H)。

Example 2 Synthesis of Compound 3

A reflux condenser tube and a constant pressure dropping funnel are arranged on a 5-L three-neck round bottom bottle. Adding molecular sieve into the mixture

And a solution (3L) of sulfamide 2 (350g, 2.14mol) in ethanol, and ethyl 3-bromopyruvate (purity 80%,404mL, 2.57mol) was added dropwise over 30 minutes. The internal temperature slightly rose during the dropwise addition, and then the reaction solution was heated to reflux and stirred for 30 minutes. The reaction solution was cooled to room temperature, and then, insoluble matter was removed by filtration through celite, and the cake was washed with ethyl acetate. The crude product after concentration of the filtrate was mixed with silica gel (1.5 kg) and stirred uniformly and purified by column chromatography on silica gel (10 kg) using 10-20% ethyl acetate/petroleum ether gradient elution to give a brown oil as the title compound (509g, 92% yield).

Example 3 Synthesis of Compound 4

A solution of the acetal (300g, 1.116mol) in acetone (3.0L) was heated to reflux and 4N HCl solution (250 mL) was added dropwise over 1.0 hour. TLC showed complete reaction of starting material. The reaction solution was concentrated under reduced pressure and the two phases were separated. The organic phase was diluted with ethyl acetate (1.5L) and successively with saturated NaHCO ₃ Washed with aqueous solution (1.0L), water (1.0L) and brine (1.0L), followed by anhydrous Na ₂ SO ₄ And (5) drying. All aqueous phases were combined and back-extracted with ethyl acetate and anhydrous Na ₂ SO ₄ The organic phase was dried. The drying agent is filtered off and the organic phase is concentrated, the crude product is slurried with a petroleum ether/ethyl acetate (5. The filtrate was concentrated and purified by column chromatography (0-15% ethyl acetate/petroleum ether) and all solids were combined to give the desired product 40g (43% yield) as a white or bright yellow solid. ¹ H NMR(500MHz,CDCl ₃ )δ10.08–10.06(m,1H),8.53–8.50(m,1H),4.49(q,J＝7.1Hz,2H),1.44(t,J＝7.1Hz,3H)。MS ESI m/z C ₇ H ₈ NO ₃ S[M+H] ⁺ Calculated value 186.01; found 186.01.

EXAMPLE 4 Synthesis of Compound 10

At N ₂ To a solution of (S) -tert-butylsulfinylamine (100g, 0.825mol) in tetrahydrofuran (1L) was added Ti (OEt) at room temperature under protection ₄ (345mL, 1.82mol) and 3-methyl-2-butanone (81mL, 0.825mol). The reaction solution was heated to reflux for 16 hours, then cooled to room temperature, and then poured into ice water (1L). Filter and wash the filter cake with ethyl acetate. The organic phase in the filtrate was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was distilled under reduced pressure (15-20torr, 95 ℃ C.) to obtain the objective product 10 (141g, 90% yield) as a yellow oil. 1H NMR (500MHz, CDCl) ₃ )δ2.54–2.44(m,1H),2.25(s,3H),1.17(s,9H),1.06(dd,J＝6.9,5.1Hz,6H)。MS ESI m/z C ₉ H ₁₉ NaNOS[M+Na] ⁺ Calculated value 212.12; found 212.11.

EXAMPLE 5 Synthesis of Compound 11

At N ₂ To a solution of diisopropylamine (264 mL, 1.87mol) in tetrahydrofuran was added a solution of n-butyllithium (2.5M, 681mL, 1.70mol) at-78 ℃ under protection. The reaction was warmed to 0 ℃ over 30 minutes and then re-cooled to-78 ℃ to which was added compound 10 (258g, 1.36mol) and rinsed with tetrahydrofuran (50 mL). After stirring for 1 hour, clTi (O) was added dropwise ⁱ Pr) ₃ (834g, 3.17mol) in tetrahydrofuran (1.0L). After completion of the dropwise addition for 1 hour, a solution of Compound 4 (210g, 1.13mol) in tetrahydrofuran (500 mL) was slowly added dropwise over 1 hour. The resulting solution was stirred at-78 ℃ for an additional 3 hours. After the completion of the reaction was monitored by TLC, the reaction was quenched with a mixture of acetic acid and tetrahydrofuran (1,300ml by volume), and then the reaction was poured into brine (2L) and extracted with ethyl acetate (8 × 1L). The organic phase was washed with water and brine, anhydrous Na ₂ SO ₄ Dried, filtered and concentrated. The residue was purified by column chromatography (DCM/ethyl acetate/petroleum ether 2. ¹ H-NMR(500MHz,CDCl ₃ )δ8.13(s,1H),6.63(d,J＝8.2Hz,1H),5.20–5.11(m,1H),4.43(q,J＝7.0Hz,2H),3.42–3.28(m,2H),2.89(dt,J＝13.1,6.5Hz,1H),1.42(t,J＝7.1Hz,3H),1.33(s,9H),1.25–1.22(m,6H)。MS ESI m/z C ₁₆ H ₂₆ NaN ₂ O ₄ S ₂ [M+Na] ⁺ Calculated 397.13, found 397.11.

EXAMPLE 6 Synthesis of Compound 7

Compound 11 (509g, 1.35mol) was dissolved in tetrahydrofuran (200 mL), cooled to-78 deg.C, and Ti (OEt) was slowly added thereto ₄ (570mL, 2.72mol), and after the addition, the mixture was stirred for 1 hour. NaBH was then added in portions over 90 minutes ₄ (51.3 g, 1.36mol), the reaction was stirred at-78 ℃ for 3 hours. TLC monitoring found the starting material still remained. EtOH (50 mL) was added slowly and stirring was continued for 1.5 h, after which the reaction was poured into saturated brine (2L, containing 250mL HOAc) and warmed to room temperature. The organic phase was separated by filtration through celite. Water washing, brine washing, drying the organic phase over anhydrous sodium sulfate, filtering and concentrating. The residue was purified by column chromatography (ethyl acetate/petroleum ether 1). ¹ H NMR(500MHz,CDCl ₃ ) δ 8.10 (s, 1H), 5.51 (d, J =5.8hz, 1h), 5.23-5.15 (m, 1H), 4.41 (q, J =7.0hz, 2h), 3.48-3.40 (m, 1H), 3.37 (d, J =8.3hz, 1h), 2.29 (t, J =13.0hz, 1h), 1.95-1.87 (m, 1H), 1.73-1.67 (m, 1H), 1.40 (t, J = 7.13h), 1.29 (s, 9H), 0.93 (d, J =7.1hz, 3h), 0.90 (d, J =7.2hz, 3h), MS ESI m/z calculated value C/z ₁₆ H ₂₈ NaN ₂ O ₄ S ₂ [M+Na] ⁺ 399.15, found 399.14.

EXAMPLE 7 Synthesis of Compound 13

A solution of 4N HCl in 1, 4-dioxane (590 mL) was added to a solution of compound 12 (600g, 1.60mol) in ethanol (590 mL) at 0 ℃. The reaction was gradually warmed to room temperature and stirred for 2.5 hours. The precipitated white solid was collected by filtration and washed with ethyl acetate. The filtrate was concentrated and slurried with ethyl acetate. The white solids obtained in two times were combined for a total of 446g (90% yield).

EXAMPLE 8 Synthesis of Compound 6

Adding NaN ₃ (740g, 11.4 mol) was dissolved in water (2.0L), and methylene chloride (2.0L) was added thereto, and the mixture was cooled to 0 ℃ to add Tf to the solution ₂ O (700mL, 4.10 mol), over 1.5 hours. After the addition was complete, stirring was continued for 3 hours at 0 ℃. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2X 500 mL). The organic phases were combined and washed with saturated NaHCO ₃ (3X 1.0L) washing. This dichloromethane solution was added to a solution of (L) -isoleucine (300g, 2.28mol), potassium carbonate (472g, 3.42mol), copper sulfate pentahydrate (5.7g, 22.8mmol) in mixed methanol/water (1. The temperature in the reaction system during the addition was slightly increased. The mixture was stirred at room temperature for 16 hours, the solvent was evaporated under reduced pressure, and the aqueous phase was acidified to pH 6-6.5 (about 280 mL) with concentrated hydrochloric acid (about 280 mL), diluted with phosphate buffer (0.25M, pH6.2, 6.0L), and washed with ethyl acetate (6X 2.0L) to remove the sulfonic acid amide by-product. The aqueous phase was acidified to pH 3 with concentrated hydrochloric acid (ca. 400 mL) and extracted with ethyl acetate (4X 2.0L). The combined organic phases were washed with saturated brine (2.0L) and anhydrous Na ₂ SO ₄ Dry, filter and concentrate to give product 6 (320g, 89% yield) as a bright yellow oil. ¹ H NMR(500MHz,CDCl ₃ )δ12.01(s,1H),3.82(d,J＝5.9Hz,1H),2.00(ddd,J＝10.6,8.6,5.5Hz,1H),1.54(dqd,J＝14.8,7.5,4.4Hz,1H),1.36–1.24(m,1H),1.08–0.99(m,3H),0.97–0.87(m,3H).

EXAMPLE 9 Synthesis of Compound 14 (method one)

To Azido-Ile-OH 6 (3) at 0 deg.C0g, 19.1mmol) to 100ml of n-hexane were added oxalyl chloride (7.3mL, 86mmol) and DMF (1.5 ml). After the addition, the temperature is raised to the room temperature, the mixture is stirred for 2 hours and then concentrated, and toluene is added for azeotropic spin drying for standby. A suspension (90 mL) of Compound 13 (3.92g, 12.7 mmol) in methylene chloride was cooled to 0 ℃ and DIPEA (6.6 mL,38.1 mmol) was added to the solution. The reaction was gradually warmed to room temperature and stirred for 16 hours, and quenched with saturated brine (50 mL). The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The organic phases are combined and dried, filtered and concentrated. The residue was purified by column chromatography (0-30% ethyl acetate/petroleum ether) to give 14 (2.5g, 48% yield) as a white solid. ¹ H NMR(500MHz,CDCl ₃ ) δ 8.14 (s, 1H), 6.57 (d, J =8.9hz, 1h), 4.91 (d, J =11.1hz, 1h), 4.44 (dd, J =13.2,6.3hz, 2h), 4.08-3.95 (m, 2H), 2.21 (dd, J =24.4,11.5hz, 2h), 1.90-1.79 (m, 3H), 1.42 (t, J =6.6hz, 3h), 1.37-1.27 (m, 2H), 1.11 (d, J = 6.hz, 3h), 1.01-0.94 (m, 9H). MS ESI m/z calculated value C/z ₁₈ H ₃₀ N ₅ O ₄ S[M+H] ⁺ 412.19, found 412.19.

EXAMPLE 10 Synthesis of Compound 14 (method two)

Azido-Ile-OH (6,153g, 0.97mol) was dissolved in tetrahydrofuran (1.5L) and cooled to 0 ℃ and NMM (214mL, 1.94mol) and isobutyl chloroformate (95mL, 0.73mol) were added in that order. After stirring at 0 ℃ for 1 hour, compound 13 (150g, 0.49mmol) was added portionwise. After stirring at 0 ℃ for 30 minutes, the temperature was gradually raised to room temperature and stirring was continued for 2 hours. The reaction was quenched with ice-water at 0 ℃ and extracted three times with ethyl acetate. The combined organic phases were washed with 1N HCl and saturated NaHCO ₃ Washing with salt water, anhydrous Na ₂ SO ₄ And (5) drying. Concentrated by filtration and the residue purified by column chromatography (0-30% ethyl acetate/petroleum ether) to give a white solid (140g, 70% yield).

EXAMPLE 11 Synthesis of Compound 15

To a solution of compound 14 (436 g, 1.05mol) in methylene chloride (50 mL) at 0 ℃ were added imidazole (94g, 1.37mmol) andtriethylchlorosilane (222mL, 1.32mol). After the reaction solution was warmed to room temperature over 1 hour, stirring was continued for 1 hour. The mixture was quenched by addition of saturated brine, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate/petroleum ether) to give 15 (557.4g, 95% yield) as a colourless oil. ¹ HNMR(500MHz,CDCl ₃ ) δ 8.12 (s, 1H), 6.75 (d, J =8.0hz, 1h), 5.20-5.12 (m, 1H), 4.44 (q, J =7.0hz, 2h), 4.06-3.97 (m, 1H), 3.87 (d, J =3.8hz, 1h), 2.14 (d, J =3.8hz, 1h), 2.01-1.91 (m, 3H), 1.42 (t, J =7.1hz, 3h), 1.34-1.25 (m, 2H), 1.06 (d, J =6.8hz, 3h), 1.00-0.93 (m, 18H), 0.88 (J =19, 19.1,6.8hz, 6h). ESI m/z C calculated value ₂₄ H ₄₄ N ₅ O ₄ SSi[M+H] ⁺ 526.28, found 526.28.

EXAMPLE 12 Synthesis of Compound 16 (method one)

A solution of compound 15 (5.20g, 9.9mmol.) in tetrahydrofuran (50 mL) was cooled to-45 ℃ and KHMDS (1M in toluene, 23.8mL, 23.8mmol) was added thereto. After stirring at-45 ℃ for 20 minutes, methyl iodide (1.85mL, 29.7 mmol) was added. The reaction was gradually warmed to room temperature over 4.5 hours. The reaction was quenched by the addition of ethanol (10 mL), diluted with ethyl acetate (250 mL) and washed with brine (100 mL). The organic phase was separated and the aqueous phase extracted with ethyl acetate (3X 50 ml). The combined organic phases were dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate/petroleum ether) to give product 16 (3.33g, 63% yield) as a bright yellow oil. ¹ H NMR(500MHz,CDCl ₃ ) δ 8.09 (s, 1H), 4.95 (d, J =6.6hz, 1h), 4.41 (q, J =7.1hz, 2h), 3.56 (d, J =9.5hz, 1h), 2.98 (s, 3H), 2.27-2.06 (m, 4H), 1.83-1.70 (m, 2H), 1.41 (t, J =7.2hz, 3h), 1.29 (ddd, J =8.9,6.8,1.6hz, 3h), 1.01 (d, J =6.6hz, 3h), 0.96 (dt, J =8.0,2.9hz, 15h), 0.92 (d, J = 6hz, 3h), 0.90 (calculated value of d, J =6.7hz, 3h), MS/z/m/z ₂₅ H ₄₆ N ₅ O ₄ SSi[M+H] ⁺ 540.30, found 540.30.

EXAMPLE 13 Synthesis of Compound 16 (method two)

To a solution of compound 15 (408g, 0.77mol) and iodomethane (145mL, 2.32mol) in tetrahydrofuran (4L) was added sodium hydrogen (60%, 62.2g, 1.55mol) at 0 ℃. The resulting reaction was stirred at 0 ℃ overnight and then poured into a vigorously stirred ice-water saturated ammonium chloride (5L) solution. Extract with ethyl acetate (3X 500 mL). The combined organic phases were dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate/petroleum ether) to give 16 (388g, 93% yield) as a bright yellow oil. ¹ HNMR(500MHz,CDCl ₃ ) δ 8.09 (s, 1H), 4.95 (d, J =6.6hz, 1h), 4.41 (q, J =7.1hz, 2h), 3.56 (d, J =9.5hz, 1h), 2.98 (s, 3H), 2.27-2.06 (m, 4H), 1.83-1.70 (m, 2H), 1.41 (t, J =7.2hz, 3h), 1.29 (ddd, J =8.9,6.8,1.6hz, 3h), 1.01 (d, J =6.6hz, 3h), 0.96 (dt, J =8.0,2.9hz, 15h), 0.92 (d, J = 6hz, 3h), 0.90 (calculated value of d, J =6.7hz, 3h), MS/z/m/z ₂₅ H ₄₆ N ₅ O ₄ SSi[M+H] ⁺ 540.30, found 540.30.

EXAMPLE 14 Synthesis of Compound 22

To a solution of D-pipecolic acid (10.0g, 77.4mmol) in methanol (100 mL) were added formaldehyde (37% aqueous solution, 30.8mL, 154.8mmol) and Pd/C (10 wt%,1.0 g). The reaction solution was reacted with hydrogen ₂ (1 atm.) ambient stirring overnight, then filtered through celite, washing with methanol. The filtrate was concentrated to give compound 22 (10.0 g,90% yield) as a white solid.

EXAMPLE 15 Synthesis of Compound 23

To a solution of D-N-methylpiperidine (2.65g, 18.5 mmol) in ethyl acetate (50 mL) were added pentafluorophenol (3.75g, 20.4 mmol) and DCC (4.21g, 20.4 mmol). The reaction was stirred at room temperature for 16 h, filtered through celite, and washed with 10mL ethyl acetate. The filtrate was used without further purification.

EXAMPLE 16 Synthesis of Compound 30 (method one)

To a solution of pentafluorophenyl ester 23 in ethyl acetate was added dry Pd/C (10 wt%,300 mg) and azide 16 (3.33g, 6.61mmol). The reaction mixture was stirred under an atmosphere of hydrogen (1 atm) for 27 hours, then filtered through celite, and washed with ethyl acetate. The filtrate was purified by column chromatography (0-5% methanol/ethyl acetate) to give compound 30 (3.90g, 86% yield). MS ESI m/z C ₃₂ H ₅₉ N ₄ O ₅ SSi[M+H] ⁺ Calculated 639.39, found 639.39.

EXAMPLE 17 Synthesis of Compound 17

To a solution of compound 16 (1.01g, 1.87mmol) in methanol (15 mL) was added dropwise HCl 0.1N until the pH was neutral. After addition of Pd/C (10 wt%,583 mg), the mixed solution was taken in H ₂ (1 atm) ambient was stirred at room temperature for 16 hours. The Pd/C is filtered off and washed by methanol. The filtrate was spun dry and the residue redissolved in ethyl acetate (50 mL) over anhydrous Na ₂ SO ₄ Drying, filtration and concentration gave compound 17 (900mg, 94% yield) as a pale yellow oil.

EXAMPLE 18 Synthesis of Compound 30 (method two)

To a solution of compound 17 (1.35g, 2.63mmol) in dichloromethane (20 mL) at room temperature under nitrogen blanket was added D-N-methylpiperidine 22 (2.02g, 14.1 mmol) and EDCI. HCl (2.97g, 15.5 mmol). After stirring for 17 hours, the reaction mixture was concentrated, and 15mL of water was added to the residue, followed by extraction with ethyl acetate (3X 30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography (ethyl acetate/methanol 40.

EXAMPLE 19 Synthesis of Compound 31

Compound 30 (3.90g, 6.1mmol) was dissolved in a mixed solution of acetic acid/water/tetrahydrofuran (v/v/v 3. Then concentrated and the residue purified by column chromatography (2 ₂₆ H ₄₅ N ₄ O ₅ S[M+H] ⁺ Calcd for 525.30, found 525.33.

EXAMPLE 20 Synthesis of Compound 32

An aqueous solution of LiOH (0.4N, 47.7mL, 19.1mmol) was added to a solution of compound 31 (2.50g, 4.76mmol) in 1, 4-dioxane (47.7 mL) at 0 ℃. After stirring at room temperature for 2 hours, the mixture was concentrated. The residue was purified by column chromatography (100% dichloromethane to dichloromethane/methanol/aqueous ammonia 80. MS ESI m/z C ₂₄ H ₄₁ N ₄ O ₅ S[M+H] ⁺ Calculated 497.27, found 497.28.

EXAMPLE 21 Synthesis of Compound 33

To a solution of compound 32 (2.36g, 4.75mmol) in pyridine (50 mL) was added dropwise acetic anhydride (2.25mL, 24mmol) at 0 ℃. The reaction was gradually warmed to room temperature over 2 hours and stirring was continued for 24 hours. After concentration, a 1, 4-dioxane/water (v/v 1. The concentrated residue was purified by column chromatography (100% dichloromethane to dichloromethane/methanol/ammonia 50. MS ESI m/z C ₂₆ H ₄₃ N ₄ O ₆ S[M+H] ⁺ Calculated 539.28 and found 539.28.

EXAMPLE 22 Synthesis of Compound 28 (method one)

To a solution of 2-methylalanine (5.00g, 48.5mmol, 1.0eq.) and formaldehyde (37% aqueous solution, 20mL, 242.5mmol) in methanol (20 mL) was added Pd/C (10 wt%,0.5 g). Reaction solution in H ₂ (100 psi) stirring at 65 ℃ for 16 hours. Filtered through celite and washed with methanol. The filtrate was spin-dried to give Compound 28 (6.00g, 94% yield) as a white solid.

EXAMPLE 23 Synthesis of Compound 28 (method two)

A mixture of 2-methylalanine (500g, 4.85mol), formaldehyde (37% aqueous solution, 1.0L,12.1 mol) and formic acid (1.0L) was heated to reflux (80 ℃ C.). After stirring for 3.0 hours, it was cooled to room temperature, 6N HCl (850 mL) was added, and the reaction solution was concentrated. The resulting solid was collected by filtration and washed three times with ethyl acetate (1.0L). The solid was dissolved in water (1.5L) and neutralized to pH 7 with 4N NaOH (ca. 1.0L). Concentrate and azeotrope with ethanol (2.0L) to remove water. The residue was dissolved in methanol (2.0L), filtered to remove NaCl solids and washed with ethyl acetate. The filtrate was concentrated to give 639.2g of a white solid containing a small amount of NaCl which was used without further purification.

EXAMPLE 24 Synthesis of Compound 29

To a solution of compound 28 (97g, 0.74mol) in ethyl acetate (1L) was added pentafluorophenol (163g, 0.88mol) and DIC (126mL, 0.81mol). The reaction was stirred at room temperature for 24 hours, filtered through celite, and washed with 10mL of ethyl acetate. The filtrate was used without further purification.

EXAMPLE 25 Synthesis of Compound 38

To an ethyl acetate solution of the above pentafluorophenyl ester 29 was added compound 16 (200g, 0.37mol) and dry Pd/C (10 wt%,10 g). The reaction solution was stirred in an atmosphere of hydrogen (1 atm) for 27 hours. Filtered through celite and washed with ethyl acetate. The combined organic phases were concentrated and purified by column chromatography (0-5% methanol/ethyl acetate) to give compound 38 (184g, 79% yield). MS ESI m/z C ₃₁ H ₅₈ N ₄ O ₅ SSi[M+H] ⁺ Calcd for 627.39, found 627.39.

EXAMPLE 26 Synthesis of Compound 39

Compound 38 (200g, 0.32mmol) was dissolved in a mixed solution of acetic acid/water/tetrahydrofuran (v/v/v 3. After the reaction solution was concentrated, it was azeotropically dried with toluene, and this step was repeated twice to obtain compound 39, which was used directly in the next reaction. MS ESI m/z C ₂₅ H ₄₅ N ₄ O ₅ S[M+H] ⁺ Calculated 513.30, found 513.30.

EXAMPLE 27 Synthesis of Compound 40

An aqueous solution of lithium hydroxide (0.4N, 600mL, 2.55mol) was added to a solution of Compound 39 (160g, 0.319mol, 1.0eq.) in methanol (1.2L) at 0 ℃. The reaction was stirred at room temperature for 2 hours and then concentrated. Column chromatography (100% dichloromethane to 80% dichloromethane/methanol/ammonia) gave compound 40 (140 g, 91% yield over two steps) as an amorphous white solid. MS ESI m/z C ₂₃ H ₄₀ N ₄ O ₅ S[M+H] ⁺ Calculated 485.27, found 485.27.

EXAMPLE 28 Synthesis of Compound 41

Compound 40 (143g, 0.30mol) and DMAP (0.36g, 2.95mmol) were dissolved in a mixed solution of anhydrous tetrahydrofuran (1.4L) and anhydrous DMF (75 mL). Cool to 0 deg.C and add TEA (82.2 mL, 0.59mmol) and acetic anhydride (56mL, 0.59mmol). The reaction was gradually warmed to room temperature and stirred for 24 hours. After concentration, purification by column chromatography (5-50% methanol/dichloromethane) gave compound 41 (147g, 95% yield) as an amorphous white solid. MS ESI m/z C ₂₅ H ₄₄ N ₄ O ₆ S[M+H] ⁺ Calcd for 527.28, found 527.28.

EXAMPLE 29 Synthesis of Compound 98

To a solution of Boc-L-tyrosine methyl ester (670g, 2.27mol), potassium carbonate (358g, 2.5mol), and potassium iodide (38g, 0.227mol) in acetone (3L) was slowly added benzyl bromide (283mL, 2.38mol). Reflux overnight, cool, add water (6L) and extract with ethyl acetate (5X 100L). The combined organic phases were washed with saturated brine (2L) anhydrous Na ₂ SO ₄ Drying, filtering, concentrating, and passing through SiO ₂ Column chromatography (4. ¹ H NMR(500MHz,CDCl ₃ )δ7.43(d,J＝7.0Hz,2H),7.38(t,J＝7.4Hz,2H),7.32(t,J＝7.2Hz,1H),7.04(d,J＝8.5Hz,2H),6.91(d,J＝8.6Hz,2H),5.04(s,2H),4.55(d,J＝6.9Hz,1H),3.71(s,3H),3.03(qd,J＝14.0,5.8Hz,2H),1.43(s,9H).MS ESI m/z C ₂₂ H ₂₈ NO ₅ [M+H] ⁺ 386.19 is calculated and 386.19 is measured.

EXAMPLE 30 Synthesis of Compound 99

DIBAL (1.0M in n-hexane, 2.9L) was slowly added dropwise over 3 hours at-78 ℃ to a solution of compound 98 (380g, 987 mmol) in anhydrous dichloromethane (1L). After the addition, the reaction was quenched with 3L ethanol. 1N HCl was added dropwise to pH 4. The mixture was gradually brought to 0 ℃ and the organic phase was separated and the aqueous phase extracted with ethyl acetate (3X 3L). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated. Slurried with petroleum ether/ethyl acetate to give white solid 99 (263g, 75% yield). 1HNMR (500mhz, cdcl3) δ 9.65 (s, 1H), 7.45 (d, J =7.1hz, 2h), 7.41 (t, J =7.4hz, 2h), 7.35 (t, J =7.1hz, 1h), 7.11 (d, J =8.6hz, 2h), 6.95 (d, J =8.6hz, 2h), 5.07 (s, 2H), 4.42 (dd, J =12.4,6.1hz, 1h), 3.09 (d, J =6.2hz, 2h), 1.46 (s, 9H). MS ESI m/z C ₂₁ H ₂₆ NO ₄ [M+H] ⁺ Calculated 356.18, found 356.19.

EXAMPLE 31 Synthesis of Compound 95

To a 500mL round bottom flask were added triphenylphosphine (100g, 381mmol) and ethyl 2-bromopropionate (100mL, 762mmol). Reaction solution in N ₂ The mixture was heated to 50 ℃ under protection and stirred overnight. White solid (PPh) ₃ ) After complete dissolution, a large amount of white product began to precipitate. After pulping with petroleum ether/ethyl acetate, a white solid was collected by suction filtration to give compound 95 (135g, 80% yield). MS ESI m/z C ₂₃ H ₂₄ O ₂ P[M-Br] ⁺ Calculated 363.15, found 363.13.

EXAMPLE 32 Synthesis of Compound 96

A solution of compound 95 (135.42g, 305.7 mmol) in dichloromethane (500 mL) was added under vigorous stirring to a 10% aqueous NaOH (450 mL). The organic phase turned yellow immediately and after stirring for 30 minutes TLC showed the reaction was complete. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2X 200 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give 96 as a yellow solid (104g, 94% yield). MS ESI m/z C ₂₃ H ₂₄ O ₂ P[M+H] ⁺ The value 362 is calculated.14, found 363.13.

EXAMPLE 33 Synthesis of Compound 100

To a solution of compound 99 (81.4 g, 229mmol) in dry dichloromethane (800 mL) was added a solution of ylide 96 (2.0 eq.) in dry dichloromethane (800 mL) at room temperature for 30 minutes. The reaction was stirred overnight at room temperature, concentrated, and then passed through SiO ₂ Column chromatography (6. ¹ H NMR(500MHz,CDCl ₃ )δ7.45–7.41(m,2H),7.40–7.35(m,2H),7.33(d,J＝7.2Hz,1H),7.10–7.06(m,2H),6.92–6.88(m,2H),6.50(dd,J＝8.8,1.3Hz,1H),5.04(s,2H),4.57(s,2H),4.18(q,J＝7.1Hz,2H),2.86(d,J＝8.5Hz,1H),2.72(dd,J＝13.6,6.8Hz,1H),1.71(d,J＝1.4Hz,3H),1.41(d,J＝2.2Hz,9H),1.28(td,J＝7.5,5.1Hz,4H).MS ESI m/z C ₂₆ H ₃₃ NaNO ₅ [M+Na] ⁺ Calculated 462.24, found 462.22.

EXAMPLE 34 Synthesis of Compound 101

Pd/C (1.83g, 10wt%) was added to a mixed solution of compound 100 (30.2g, 68.9mmol) in tetrahydrofuran (100 mL) and methanol (300 mL) in a hydrogenation reaction flask. The mixture is under 1atm H ₂ Shake overnight at ambient. Filtration through celite and concentration of the filtrate afforded compound 101 (25.0 g, theoretical yield) as a colorless oil. ¹ H NMR(500MHz,CDCl ₃ )δ6.99(d,J＝7.0Hz,2H),6.72(d,J＝7.6Hz,2H),4.39(s,1H),4.18–4.04(m,2H),3.82(s,1H),2.60(dd,J＝37.2,20.9Hz,4H),1.95–1.81(m,1H),1.39(s,11H),1.24(dd,J＝9.5,4.3Hz,3H),1.13(t,J＝8.9Hz,3H).MS ESI m/z C ₁₉ H ₃₁ NO ₅ [M+H] ⁺ Calculated 352.20, found 352.19.

EXAMPLE 35 Synthesis of Compound 102 (method one)

To a solution of compound 101 (5.96g, 35.9 mmol) in anhydrous dichloromethane (200 mL) was added acetic anhydride (3.2 mL,33.9 mmol) and nitric acid (65% -68% aqueous solution, 3.5mL, 50.79mmol) at room temperature. After stirring at room temperature for 30 min, TLC showed the reaction was complete. The reaction solution was washed with water (3X 200 mL), and the aqueous phase was back-extracted with dichloromethane (3X 100 mL). The combined dichloromethane phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. Through SiO ₂ Column chromatography (5. ¹ H NMR(500MHz,CDCl ₃ )δ10.49(s,1H),7.89(s,1H),7.44(d,J＝8.4Hz,1H),7.09(d,J＝8.6Hz,1H),4.32(d,J＝8.3Hz,1H),4.12(dd,J＝14.0,7.0Hz,2H),3.80(s,1H),2.76(dd,J＝13.0,6.8Hz,2H),2.59(s,1H),1.88(s,1H),1.37(t,J＝8.7Hz,9H),1.25(dd,J＝13.5,6.9Hz,4H),1.16(t,J＝8.0Hz,3H).MS ESI m/z C ₁₉ H ₂₈ NaN ₂ O ₇ [M+Na] ⁺ Calculated 419.19, found 419.17.

EXAMPLE 36 Synthesis of Compound 102 (method two)

To a solution of compound 101 (15.6 g,44.3 mmol) in tetrahydrofuran (150 mL) was added tert-butyl nitrite (18.3 g,177.2 mol) at room temperature. Stirring at room temperature for 4 hours, TLC showed complete reaction, concentrating the reaction solution, siO ₂ Column chromatography (3.

EXAMPLE 37 Synthesis of Compound 103

To a mixed solution of Compound 102 (15.3 g,38.6 mmol) in tetrahydrofuran (100 mL) and methanol (100 mL) at room temperature was added LiOH. H ₂ O (16.3g, 389mmol) in water (190 mL). After stirring the reaction at room temperature for 40 minutes, water (400 mL) was added to dilute and dilute with 1N KHSO ₄ Adjusting to pH 3-4. With acetic acid ethyl esterAfter extraction of the ester (3X 300 mL), the organic phase was washed with saturated brine, anhydrous Na ₂ SO ₄ Drying, filtration and concentration gave 103 (14.4 g, theoretical yield) as a yellow solid. ¹ H NMR(500MHz,CDCl ₃ )δ10.48(s,1H),7.98–7.88(m,1H),7.42(dd,J＝18.4,8.2Hz,1H),7.14–7.03(m,1H),4.48(d,J＝8.6Hz,1H),3.90(s,1H),2.82–2.53(m,3H),1.97–1.82(m,2H),1.42–1.27(m,10H),1.21(d,J＝6.7Hz,4H).MS ESI m/z C ₁₇ H ₂₃ N ₂ O ₇ [M-H] ^- 367.16 is calculated and 367.14 is found.

EXAMPLE 38 Synthesis of Compound 104

Pd/C (2.60g, 10 wt%) was added to a solution of compound 103 (26.0 g,70.6 mmol) in methanol (260 mL) in a hydrogenation flask. The mixture is under 1atm H ₂ The reaction was stirred overnight at ambient. Filtration through celite and concentration of the filtrate afforded compound 104 (24.0 g, theoretical yield) as a green oil.

Example 39 Synthesis of Compound 106

A solution of tert-butyl 2-bromopropionate (255g, 1.22mol) and triphenylphosphine (320g, 1.22mol) in dry acetonitrile (1L) was stirred at room temperature for 18 h. Acetonitrile was removed by rotary evaporation under reduced pressure, and toluene was added to precipitate a white solid. After pouring off the toluene, the white solid was dissolved in dichloromethane (1L) and transferred to a separatory funnel. 10% NaOH aqueous solution (1L) was added, and the organic phase turned yellow soon after shaking. The organic phase was separated and the aqueous phase was back-extracted once with dichloromethane (1L). The methylene chloride phases were combined, washed with brine (400 mL), anhydrous Na ₂ SO ₄ Dried, filtered and concentrated to give ylide 106 (280g, 58%) as a yellow solid.

EXAMPLE 40 Synthesis of Compound 107

To a solution of compound 99 (450g, 1.27mol) in dry dichloromethane (3L) was added ylide 106 (546g, 1.40mmol). Stir at room temperature overnight. After completion of the reaction monitored by TLC, it was purified by column chromatography (10-50% ethyl acetate/petroleum ether) to give compound 107 (444g, 75% yield) as a white solid. ESIm/z C ₂₈ H ₃₈ NO ₅ [M+H] ⁺ 468.27 calculated value and 468.22 measured value.

EXAMPLE 41 Synthesis of Compound 108

Compound 107 (63g, 0.13mol) was dissolved in methanol (315 mL), pd/C (10 wt%,6.3 g) was added, and the mixture was stirred overnight at room temperature under hydrogen (1 atm). The catalyst was removed by filtration and the filtrate was concentrated to give compound 108 (45.8g, 93% yield).

EXAMPLE 42 Synthesis of Compound 109

To a solution of compound 108 (390g, 1.03mol) in tetrahydrofuran (4L) was added tert-butyl nitrite (1.06kg, 10.3 mol.) at room temperature. After stirring overnight the tetrahydrofuran was removed by rotation and the residue was purified by column chromatography (10-50% ethyl acetate/petroleum ether) to give compound 109 (314g, 72% yield) as a bright yellow solid.

EXAMPLE 43 Synthesis of Compound 110

To a solution of compound 109 (166g, 0.392mol) in ethyl acetate (500 mL) was added Pd/C (10 wt%,16 g) under nitrogen. Hydrogen was introduced and vacuum replaced three times. The reaction solution was stirred at room temperature for 16 hours in a hydrogen (1 atm) atmosphere. DiatomiteFiltration and concentration gave compound 110 (146g, 97% yield) as a yellow foamy solid. ¹ H NMR(400MHz,CDCl ₃ )δ6.62(d,J＝7.9Hz,1H),6.55(s,1H),6.43(d,J＝7.3Hz,1H),4.39(dd,J＝53.0,44.2Hz,1H),3.77(s,4H),2.72–2.29(m,3H),1.83–1.58(m,1H),1.40(d,J＝7.6Hz,18H),1.24(s,1H),1.06(t,J＝5.7Hz,3H).MS ESI m/z C ₂₁ H ₃₅ N ₂ O ₅ [M+H] ⁺ Calculated 394.25, found 395.25.

EXAMPLE 44 Synthesis of Compound 114

(S) -4-isopropyloxazolidin-2-one (5.00g, 38.7 mmol) was dissolved in anhydrous tetrahydrofuran (200 mL) under nitrogen, cooled to-78 deg.C, and n-butyllithium (2.5M n-hexane solution, 17.0 mL) was added dropwise to the reaction flask over 30 minutes and reacted at-78 deg.C for 1 hour. Propionyl chloride (4.0 mL, 42.58mmol) was then added dropwise, and after completion of the addition, the reaction was allowed to react at-78 ℃ for 1 hour, and the reaction was monitored by thin layer chromatography to complete, and the reaction solution was poured into a saturated saline solution (250 mL), extracted with ethyl acetate (3 × 100 mL), and the organic phases were combined, washed once with a 1N NaOH solution (200 mL), washed once with a saturated saline solution (300 mL), dried over anhydrous sodium sulfate, filtered, spin-dried, and purified with a silica gel column (7. MS ESI m/z C ₉ H ₁₆ NO ₃ [M+H] ⁺ Calculated 186.10, found 186.10.

EXAMPLE 45 Synthesis of Compound 115

Compound 114 (2.00g, 11.9 mmol) was dissolved in dry dichloromethane (200 mL) under nitrogen, cooled to 0 deg.C, and diisopropylethylamine (2.3 mL,12.9 mmol) and n-Bu were added ₂ Adding BOTf (1.0M dichloromethane solution, 12.0 mL) dropwise into a reaction flask, reacting at 0 deg.C for 45 min, cooling to-78 deg.C, and mixingA dichloromethane solution of compound 99 (4.24mL, 10.8mmol) was dropped into a reaction flask and reacted at-78 ℃ for 1 hour, followed by slowly warming to room temperature and reacting overnight. The next day, phosphate buffer (0.1M, pH 7.0, 100mL) was added to the reaction flask, the layers were separated, the aqueous layer was extracted with dichloromethane (3X 50 mL), the organic layers were combined, washed once with saturated saline solution (200 mL), dried over anhydrous sodium sulfate, and spin-dried to give the crude product. The crude product was dissolved in methanol (100 mL), cooled to 0 deg.C and H was added ₂ O ₂ (30% aqueous solution, 26 mL) was added dropwise, reaction was carried out at 0 ℃ for 3 hours, methanol was removed by rotary evaporation, water (100 mL) was added, extraction was carried out with ethyl acetate (3X 100 mL), the organic phases were combined, washed once with a saturated saline solution (300 mL), dried over anhydrous sodium sulfate, filtered, rotary-dried, and purified with a silica gel column (3. ¹ H NMR(400MHz,CDCl ₃ )δ7.36(ddd,J＝24.2,14.2,7.1Hz,5H),7.12(d,J＝8.4Hz,2H),6.90(d,J＝8.5Hz,2H),5.02(s,2H),4.69(d,J＝9.0Hz,1H),4.45(d,J＝4.1Hz,1H),4.33(t,J＝8.4Hz,1H),4.15(d,J＝8.6Hz,1H),3.90(dd,J＝16.6,8.0Hz,1H),3.85–3.77(m,2H),2.81(d,J＝7.6Hz,2H),2.27(dd,J＝11.4,6.7Hz,1H),1.35(s,9H),0.89(dd,J＝14.3,6.9Hz,6H)。MS ESI m/z C ₃₀ H ₄₁ N ₂ O ₇ [M+H] ⁺ Calcd for 541.28, found 541.30.

EXAMPLE 46 Synthesis of Compound 116

Compound 115 (2.50g, 4.63mmol) was dissolved in anhydrous tetrahydrofuran (46 mL) under nitrogen, dithiocarbonylimidazole (2.48g, 13.89mmol) was added, and the reaction was refluxed overnight. The next day, water (100 mL) was added, ethyl acetate extracted (3 × 50 mL), and the organic phases were combined, washed once with saturated brine solution (300 mL), dried over anhydrous sodium sulfate, filtered, spun dry, and purified on silica gel column (3. ¹ H NMR(400MHz,CDCl ₃ )δ8.41(s,1H),7.67(s,1H),7.36(dt,J＝16.0,6.9Hz,6H),7.09(s,1H),7.05(d,J＝8.4Hz,2H),6.86(d,J＝8.4Hz,2H),6.32(d,J＝9.5Hz,1H),5.01(s,2H),4.56–4.43(m,2H),4.32(ddd,J＝16.2,15.6,7.8Hz,3H),4.19(d,J＝8.7Hz,1H),2.96(dd,J＝14.6,4.4Hz,1H),2.49(dd,J＝14.5,10.5Hz,1H),2.29(td,J＝13.4,6.7Hz,1H),1.73(s,1H),1.29(s,9H),0.91(dd,J＝13.9,6.9Hz,6H)。MS ESI m/z C ₃₄ H ₄₃ N ₄ O ₇ S[M+H] ⁺ Calculated 651.27, found 651.39.

EXAMPLE 47 Synthesis of Compound 117

Compound 116 (1.90g, 2.92mmol) was dissolved in anhydrous toluene (30 mL) under nitrogen blanket, azobisisobutyronitrile (0.05g, 0.584 mmol) and tributylstannane (1.6ml, 5.84mmol) were added, refluxed for 2.5 hours, spun dry, silica gel column purified (5. ¹ H NMR(400MHz,CDCl ₃ )δ7.36(ddd,J＝24.5,14.5,7.1Hz,5H),7.08(d,J＝8.5Hz,2H),6.90(d,J＝8.5Hz,2H),5.04(d,J＝5.1Hz,2H),4.48(d,J＝4.2Hz,1H),4.33(t,J＝8.4Hz,1H),4.22(d,J＝9.7Hz,1H),4.15(d,J＝8.8Hz,1H),3.81(s,2H),2.73(dd,J＝14.1,5.9Hz,1H),2.61(dd,J＝14.0,7.2Hz,1H),2.29(dq,J＝13.5,6.8Hz,1H),2.11–2.00(m,1H),1.60(dd,J＝15.2,6.2Hz,2H),1.35(s,9H),1.20(d,J＝6.9Hz,3H),0.89(dd,J＝14.0,6.9Hz,6H)。MS ESI m/z C ₃₀ H ₄₁ N ₂ O ₆ [M+H] ⁺ Calcd for 525.28, found 525.37.

EXAMPLE 48 Synthesis of Compound 118

Compound 117 (1.20g, 2.29mmol) was dissolved in tetrahydrofuran (30 mL) and water (6 mL), and LiOH (0.192g, 4.58mmol) and H were added under ice-cooling ₂ O ₂ (30% aqueous solution, 1.4 mL) was reacted in ice bath for 3 hours, sodium sulfite (1.5M, 30mL) was added, and the mixture was stirred in ice bath for 30 minutes and then 1N KHSO was added ₄ The pH was adjusted to 4, extracted with ethyl acetate (3 × 50 mL), the organic phases combined, washed once with saturated brine solution (200 mL), dried over anhydrous sodium sulfate, filtered, spun-dried, purified on silica gel column (3. ¹ H NMR(400MHz,CDCl ₃ )δ7.46–7.28(m,5H),7.07(d,J＝7.7Hz,2H),6.91(d,J＝7.8Hz,2H),5.04(s,2H),4.52(d,J＝8.5Hz,1H),3.87(d,J＝41.8Hz,1H),2.82–2.43(m,3H),1.85(t,J＝12.2Hz,1H),1.41(s,9H),1.17(d,J＝6.9Hz,3H)。MS ESI m/z C ₂₄ H ₃₂ NO ₅ [M+H] ⁺ Calculated 414.22, found 414.21.

EXAMPLE 49 Synthesis of Compound 119

Compound 118 (0.77g, 1.86mmol) was dissolved in methanol (15 mL), pd/C (10 wt%,0.25 g) was added, and catalytic hydrogenation (1 atm H) ₂ ) The reaction was allowed to proceed for 16 hours, filtered and spun dry to give 0.58g of a white solid in 96% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.00(d,J＝7.5Hz,2H),6.80(s,2H),4.51(d,J＝9.0Hz,1H),3.88(s,1H),2.66(dd,J＝65.6,22.6Hz,4H),1.88(t,J＝12.2Hz,1H),1.42(s,9H),1.14(d,J＝6.6Hz,3H)。MS ESI m/z C ₁₇ H ₂₆ NO ₅ [M+H] ⁺ Calculated 324.17 and found 324.16.

EXAMPLE 50 Synthesis of Compound 120

Compound 119 (0.57g, 1.76mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), tert-butyl nitrite (0.63ml, 5.28mmol) was added dropwise under ice bath, after warming to room temperature for 2h, water (50 mL) was added, extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, washed once with saturated brine solution (100 mL), dried over anhydrous sodium sulfate, filtered, spun dry, purified on silica gel column (2. ¹ H NMR(400MHz,DMSO)δ12.00(s,1H),10.68(s,1H),7.67(s,1H),7.34(d,J＝8.4Hz,1H),7.03(d,J＝8.4Hz,1H),6.69(d,J＝8.9Hz,1H),3.56(d,J＝3.8Hz,1H),2.67(dd,J＝13.5,5.1Hz,1H),2.41(dd,J＝13.8,6.6Hz,1H),1.78–1.65(m,1H),1.27(s,9H),1.18(s,1H),1.05(d,J＝7.1Hz,3H)。MS ESI m/z C ₁₇ H ₂₅ N ₂ O ₇ [M+H] ⁺ Calculated 369.15, found 369.14.

Example 51 Synthesis of Compound 121

Compound 120 (0.77g, 1.86mmol) was dissolved in methanol (10 mL), pd/C (10 wt%,0.02 g) was added, and catalytic hydrogenation (1 atm H) ₂ ) The reaction was allowed to react for 1 hour, filtered and spun dry to give 0.43g of a white solid in 93% yield. MS ESI m/z C ₁₇ H ₂₇ N ₂ O ₅ [M+H] ⁺ Calcd 339.18, found 339.17.

EXAMPLE 52 Synthesis of Compound 124

Dissolving maleic anhydride (285g, 2.76mol) in acetic acid (1L), adding 4-aminobutyric acid (285g, 2.76mol, 1.0eq.) to react at room temperature for 30 minutes, heating to reflux, reacting for 1.5 hours, cooling to room temperature, removing the solvent by rotary evaporation, dissolving with ethyl acetate, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, filtering, rotary drying, recrystallizing the crude product with petroleum ether and ethyl acetate to obtain 400g of white solid with the yield of 80%. ¹ H NMR(500MHz,CDCl3)δ6.71(s,2H),3.60(t,J＝6.7Hz,2H),2.38(t,J＝7.3Hz,2H),2.00–1.84(m,2H)。

EXAMPLE 53 Synthesis of Compound 125 (method one)

Compound 124 (400g, 2.18mol) was dissolved in anhydrous dichloromethane (1.5L), and N-hydroxymaleimide (276 g, 2.40mmol) and DIC (303g, 2.40mol) were added and reacted at room temperature overnight, spun dry, silica gel column purified (1. ¹ H NMR(500MHz,CDCl ₃ )δ6.74(s,2H),3.67(t,J＝6.8Hz,2H),2.85(s,4H),2.68(t,J＝7.5Hz,2H),2.13–2.03(m,2H)。

EXAMPLE 54 Synthesis of Compound 125 (method two)

Maleic anhydride (9.84g, 100mmol) and 4-aminobutyric acid (10.3 g, 100mmol) were dissolved in DMA (100 mL) and reacted at room temperature for 1 hour, ice-washed, N-hydroxymaleimide (14.4g, 125mmol) and DCC (40.0 g, 194mmol) were added, and after completion, warmed to room temperature for 16 hours, filtered, the filtrate was spin-dried, water was added, extracted three times with dichloromethane, the organic phases were combined, washed once with saturated sodium bicarbonate, washed once with saturated brine solution (100 mL), dried over anhydrous sodium sulfate, filtered, spin-dried, purified on silica gel column (10 dcm/MeOH), to give 6.85g of white solid, 24% yield.

EXAMPLE 55 Synthesis of Compound 126 (method one)

Compound 104 (13.8g, 40.8mmol) was dissolved in EtOH (100 mL) and phosphate buffer (0.1M, pH 7.5, 15mL), and compound 125 (12.6g, 44.9mmol) was added. The reaction was allowed to proceed overnight at room temperature, ethanol was removed by rotary evaporation, water (100 mL) was added, extraction was performed with ethyl acetate (3X 150 mL), the organic phases were combined, washed once with saturated brine solution (100 mL), dried over anhydrous sodium sulfate, filtered, rotary dried, and purified on silica gel column (5-10% DCM/MeOH) to give 15.3g of a yellow foamy solid in 65% yield. ¹ H NMR(500MHz,MeOD)δ7.36(d,J＝12.5Hz,1H),6.85(d,J＝8.2Hz,1H),6.79(s,2H),6.76(dd,J＝8.2,4.3Hz,1H),6.36(dd,J＝18.5,9.3Hz,1H),3.74(s,2H),3.60(t,J＝6.7Hz,3H),3.04(d,J＝9.9Hz,1H),2.91(s,1H),2.62(dd,J＝9.6,5.5Hz,2H),2.44(t,J＝7.4Hz,3H),2.07(s,1H),2.01–1.93(m,3H),1.84(ddd,J＝13.7,9.8,3.7Hz,1H),1.39–1.33(m,9H),1.30–1.24(m,3H),1.19–1.09(m,4H)。MS ESI m/z C ₂₅ H ₃₂ N ₃ O ₈ [M-H] ^- Calculated 502.23, found 502.22.

EXAMPLE 56 Synthesis of Compound 127

Compound 126 (0.20g, 0.4mmol) was dissolved in DCM (18 mL) and TFA (2 mL), reacted at room temperature for 2 hours, and concentrated to give compound 127, which was used directly in the next reaction. MS ESI m/z C ₂₀ H ₂₄ N ₃ O ₆ [M-H] ^- Calcd 402.17, found 402.17.

EXAMPLE 57 Synthesis of Compound 126 (method two)

Compound 124 (60g, 328mmol) was dissolved in THF (600 mL), and after reaction for 2 hours at 0 ℃ in ice bath, N-methylmorpholine (85.3mL, 984mmol) and isobutyl chloroformate (44.6 mL, 426mmol) were added dropwise, and after completion of the reaction at 0 ℃ a solution of compound 104 (102g, 259mmol) in THF (400 mL) was added dropwise, and the reaction was completed at 0 ℃ for 30 minutes, water (300 mL) was added, extraction was performed with ethyl acetate (3X 300 mL), the organic phases were combined, washed once with a saturated saline solution (100 mL), dried over anhydrous sodium sulfate, filtered, spun-dried, and purified with a silica gel column (9-35% ethyl acetate/petroleum ether) to obtain 104g of a pale yellow solid in 73% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.86(s,1H),8.40(d,J＝17.3Hz,1H),6.87(s,3H),6.70(s,2H),4.53–4.16(m,0H),3.79(s,0H),3.62(t,J＝6.1Hz,1H),2.63(s,1H),2.40(t,J＝6.9Hz,1H),2.12–1.88(m,4H),1.84–1.64(m,0H),1.38(t,J＝9.6Hz,6H),1.06(t,J＝6.0Hz,3H)。

EXAMPLE 58 Synthesis of Compound 128

Compound 33 (40mg, 0.074mmol) was dissolved in ethyl acetate, pentafluorophenol (27mg, 0.148mmol) and DCC (23mg, 0.111mmol) were added, the reaction was carried out at room temperature for 16 hours, a silica gel pad was filtered, eluted with ethyl acetate, the filtrate was concentrated, and then dissolved in DMA (6 mL)) To this solution were added compound 127 (56.6 mg, 0.13mmol) and DIPEA (47.4. Mu.L, 0.18 mmol), reacted at room temperature for 24 hours, concentrated, and subjected to preparative HPLC (C) ₁₈ Column, 10-100% acetonitrile/water) to yield 43mg of a white solid in 63% yield. MS ESI m/zC ₄₆ H ₆₆ N ₇ O ₁₁ S[M+H] ⁺ Calculated 924.45 and found 924.45.

EXAMPLE 59 Synthesis of Compound 147

(R) -4-isopropyl-2-oxazolidinone (25.0 g, 0.194mol) was dissolved in 1150mL anhydrous THF in N ₂ Under protection, the temperature is reduced to-70 ℃, n-BuLi (85.0mL and 0.213mol) is dripped (the temperature is controlled to be between-70 ℃ and-65 ℃), a large amount of white solid is separated out in the dripping process, the reaction is carried out for 1 hour at-70 ℃, propionyl chloride (20.0mL and 0.232mol) is dripped, the reaction is carried out for 1 hour at-70 ℃, thin layer chromatography tracking is carried out, and the reaction is finished. The reaction solution was poured into 1.2L of saturated ammonium chloride solution, extracted with ethyl acetate (700 mL. Times.1, 350 mL. Times.2), and the ethyl acetate phases were combined, washed once with 1L of 1mol/L NaOH solution, once with 1L of water, once with 1L of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and spin-dried to give a colorless liquid. The ethyl acetate/petroleum ether (6-10%) was passed through the column to give 26.6g of product in 74.1% yield. ESI m/z C ₉ H ₁₇ NO ₃ [M+H] ⁺ Calculated 186.1, found 186.1. ¹ H NMR(400MHz,CDCl ₃ )δ4.48–4.37(m,1H),4.27(t,J＝8.7Hz,1H),4.21(dd,J＝9.1,3.1Hz,1H),3.04–2.82(m,2H),2.45–2.30(m,1H),1.17(t,J＝7.4Hz,3H),0.90(dd,J＝17.1,7.0Hz,6H)。

EXAMPLE 60 Synthesis of Compound 148

Compound 147 (18.4g, 99.5mmol) was dissolved in 200mL of anhydrous DCM and added to dry N ₂ Protected 1L three-necked bottle, ice bath, dropwise adding withoutDIPEA (19mL, 108.6mmol) as water, and Bu is slowly dropped ₂ BOTf in DCM (1.0M, 100mL, 100mmol) was added dropwise, stirred for 45 min under ice bath, cooled to-78 deg.C, added dropwise with 320mL of anhydrous DCM solution of aldehyde (32.2g, 90.5mmol,1.0 eq) and reacted at-78 deg.C for 1h, heated to 0 deg.C, reacted under ice bath, and slowly heated to room temperature for overnight reaction, during which the solution changed from colorless to yellow. And (5) tracking by thin-layer chromatography, and finishing the reaction. Pouring the reaction solution into 700mL PBS (0.1M, PH7.0), separating, extracting the aqueous phase with 200mL DCM, combining the DCM phases, washing once with an equal volume of saturated sodium chloride solution, drying with anhydrous sodium sulfate, spin-drying to obtain 79.8g of orange oily substance, adding 730mL methanol to dissolve, cooling in ice bath, and slowly adding 225mL of 30H ₂ O ₂ (23 eq), and the reaction was carried out for 3h under ice bath. 750mL of water is added, most of the methanol is removed by rotary evaporation, 100mL of water is added, ethyl acetate is extracted (500 mL × 1, 150mL × 2), the ethyl acetate phases are combined, the mixture is washed once with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, dried by rotary evaporation, and passed through a column with ethyl acetate/petroleum ether to obtain 28.3g of a product as a white foamy solid with a yield: 58.7 percent. ESI m/z C ₃₀ H ₄₁ N ₂ O ₇ [M+H] ⁺ Calcd for 541.3, found 541.3. ¹ H NMR(400MHz,CDCl ₃ )δ7.49–7.26(m,3H),7.17(t,J＝10.7Hz,1H),6.93(d,J＝7.0Hz,2H),5.06(s,1H),4.28(dd,J＝44.4,36.4Hz,2H),4.04–3.52(m,1H),3.11–2.73(m,1H),2.35(s,1H),1.41(t,J＝16.3Hz,9H),0.91(dd,J＝15.6,6.4Hz,5H)。

EXAMPLE 61 Synthesis of Compound 149

Compound 148 (28.3g 52.3mmol) and TCDI (35.1g, 157.0mmol) were weighed into 500mL of N ₂ In a protected dry two-necked flask, 350mL of anhydrous THF was added to dissolve the mixture, and the reaction was refluxed overnight. And (5) tracking by thin-layer chromatography and finishing the reaction. Saturated sodium chloride solution (200 mL) was washed once, extracted with ethyl acetate (75 mL. Times.2), dried over anhydrous sodium sulfate, filtered, spun dry, and the product was purified by column chromatography over ethyl acetate/petroleum ether to give 26.1g of a pale yellow foamy solid, yield: 76.8 percent. ES (ES)I:m/z:C ₃₄ H ₄₃ N ₄ O ₇ S[M+H] ⁺ Calculated value 651.3, found value 651.3. ¹ H NMR(400MHz,CDCl ₃ )δ8.21(s,1H),7.43(d,J＝11.8Hz,1H),7.42–7.28(m,5H),7.06(d,J＝8.3Hz,2H),7.01(s,1H),6.80(d,J＝8.3Hz,2H),6.17(dd,J＝8.5,2.9Hz,1H),4.96(s,2H),4.42–4.04(m,5H),2.83(dd,J＝14.2,6.2Hz,1H),2.69(dd,J＝14.2,7.1Hz,1H),2.32(dd,J＝6.8,4.2Hz,1H),1.37(s,9H),1.30(d,J＝6.9Hz,3H),0.87(dd,J＝9.9,7.0Hz,6H)。

EXAMPLE 62 Synthesis of Compound 150

Compound 149 (26.0g, 40.0mmol) and AIBN (0.066g, 0.01eq) were weighed into a 500mL dry two-necked flask, N ₂ Under protection, 350mL of anhydrous toluene is added for dissolution, and n-Bu is dropwise added ₃ SnH (21.5mL, 80.0 mmol), reflux reaction for 1h. And (5) tracking by thin-layer chromatography, and finishing the reaction. Spin-dry, ethyl acetate/petroleum ether column to give 6.0g of product as a white foamy solid, yield: 37.3 percent. ESI m/z C ₃₀ H ₄₁ N ₂ O ₆ [M+H] ⁺ Calcd for 525.3, found 525.3. ¹ H NMR(400MHz,CDCl ₃ )δ7.37(ddd,J＝25.1,15.1,7.1Hz,5H),7.08(d,J＝7.9Hz,2H),6.89(d,J＝8.4Hz,2H),5.03(s,2H),4.61(d,J＝8.4Hz,1H),4.40(s,1H),4.32–4.08(m,2H),3.91–3.66(m,2H),2.83(d,J＝8.4Hz,1H),2.60(t,J＝10.1Hz,1H),2.33(s,1H),1.71(s,1H),1.41(s,9H),1.15(d,J＝6.5Hz,3H),0.87(dd,J＝17.0,7.0Hz,6H)。

EXAMPLE 63 Synthesis of Compound 151

Compound 150 (7.84g, 15.0 mmol) was dissolved in 90mL THF and 30mL water and ice-cooled. Reacting LiOH & H ₂ O (1.57g, 37.5 mmol) in 30% H ₂ O ₂ (11.4 mL,112.5 mmol), and the solution was added dropwise to the reaction system so that a pale yellow solid was presentAnd (4) separating out a body, reacting for 3 hours in ice bath, and tracking by thin layer chromatography to finish the reaction. 160mL of 1.5M Na was added dropwise ₂ SO ₃ The solution is reacted for 30min in ice bath and 1NKHSO is used ₄ The solution was adjusted to pH 4, extracted with ethyl acetate (200 mL × 1, 750mL × 2), the ethyl acetate phases combined, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, spun dry, and passed through a column of ethyl acetate/petroleum ether to give 6.18g of a white solid, yield: 100 percent. ESI m/z C ₂₄ H ₃₂ NO ₅ [M+H] ⁺ Calculated 414.2, found 414.2. ¹ H NMR(400MHz,CDCl ₃ )δ7.39(ddd,J＝24.5,15.0,7.2Hz,5H),7.11(d,J＝7.8Hz,2H),6.93(d,J＝8.3Hz,2H),5.06(s,2H),4.44(t,J＝8.3Hz,1H),3.83(d,J＝69.4Hz,1H),2.85–2.61(m,2H),2.61–2.40(m,1H),1.99–1.70(m,1H),1.39(d,J＝26.1Hz,9H),1.19(s,3H)。

EXAMPLE 64 Synthesis of Compound 152

Compound 151 (6.18g, 15.0mmol) was dissolved in 50mL of methanol, and 0.6g of Pd/C was added thereto, and the reaction was completed by catalytic hydrogenation overnight at room temperature and thin layer chromatography. 50mL ethyl acetate was added, filtered, washed with ethyl acetate, spun dry, and vacuum dried to give 4.8g of a colorless clear oil, yield: 99.4 percent. ESI m/z C ₁₇ H ₂₆ NO ₅ [M+H] ⁺ Calculated 324.2, found 324.2. ¹ H NMR(400MHz,CDCl ₃ )δ6.97(d,J＝6.5Hz,2H),6.74(d,J＝8.2Hz,2H),3.93–3.66(m,1H),2.58(tdd,J＝19.5,12.9,7.4Hz,3H),1.75(ddd,J＝20.1,16.3,7.7Hz,1H),1.37(d,J＝21.5Hz,9H),1.11(d,J＝7.0Hz,3H)。

EXAMPLE 65 Synthesis of Compound 153

Compound 152 (4.8g, 15.0mmol) was dissolved in 75mL anhydrous THF in an ice bath, N ₂ t-BuONO (18.0 mL,150 mmol), heating to room temperature for reaction for 3h, and tracking by thin layer chromatography to finish the reaction. Slowly dropwise adding 100mL of saturated NaHCO under ice bath condition ₃ The solution, with a large amount of bubbles, is treated with 1N KHSO ₄ The pH was adjusted to 4, ethyl acetate extracted (150 mL. Times.1, 75 mL. Times.2), washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, spun dry, and the product was passed through a column with ethyl acetate/petroleum ether to give 3.6g yield: 65.4 percent. ESI m/z C ₁₇ H ₂₅ N ₂ O ₇ [M+H] ⁺ Calcd for 369.2, found 369.2. ¹ HNMR(400MHz,MeOD)δ7.93(d,J＝2.0Hz,1H),7.48(dd,J＝8.6,2.1Hz,1H),7.06(d,J＝8.5Hz,1H),3.83–3.71(m,1H),2.82(dd,J＝13.6,5.0Hz,1H),2.66–2.41(m,2H),1.84(ddd,J＝14.0,10.6,5.6Hz,1H),1.65–1.51(m,1H),1.28(d,J＝24.9Hz,9H),1.15(d,J＝7.0Hz,3H)。

EXAMPLE 66 Synthesis of Compound 154

Dissolving compound 153 (3.2g, 7.74mmol) in 20mL of methanol, adding 0.2g of Pd/C, carrying out catalytic hydrogenation, reacting for 3h at room temperature, and carrying out thin layer chromatography tracking to finish the reaction. 20mL of ethyl acetate was added, filtered, washed with ethyl acetate, spun dry, and vacuum dried to give 2.3g of a white foamy solid, yield: 92.0 percent. ESI m/z C ₁₇ H ₂₇ N ₂ O ₅ [M+H] ⁺ Calculated value is 339.2, found value is 339.2. ¹ H NMR(400MHz,MeOD)δ6.61(d,J＝8.0Hz,2H),6.45(d,J＝6.3Hz,1H),3.72(d,J＝7.3Hz,1H),2.68–2.34(m,3H),1.81–1.66(m,1H),1.56–1.45(m,1H),1.36(d,J＝29.0Hz,9H),1.08(d,J＝6.9Hz,3H)。

Example 67 Synthesis of Compound 41a

Compound 41 (10g, 19mmol) was dissolved in DCM, iced, pentafluorophenol (7g, 38mmol) and EDCI (7.2g, 38mmol) were added, the mixture was allowed to react overnight at room temperature,concentration and purification on silica gel (0-60% ethyl acetate/petroleum ether) gave 11g of amorphous solid in 83% yield. MS ESI m/z C ₃₁ H ₄₁ F ₅ N ₄ O ₆ S[M+H] ⁺ Calculated 693.27, found 693.27.

EXAMPLE 68 Synthesis of Compound 132

Compound 41a (11g, 15.9mmol) and compound 127 (12.3g, 23.8mmol) were dissolved in DMF (100 mL), DIPEA (6.9mL, 39.7mmol) was added under ice bath, warmed to room temperature for 1 hour, concentrated, and purified with silica gel column (0-10% methanol/dichloromethane) to give 10g of an amorphous solid in 69% yield. MS ESI m/z C ₄₅ H ₆₅ N ₇ O ₁₁ S[M+H] ⁺ Calcd for 912.45, found 912.45.

EXAMPLE 69 Synthesis of Compound 171

Compound 102 (1.00g, 2.52mmol) was dissolved in acetonitrile (10 mL), cooled to-25 deg.C and CCl was added ₄ (2.2mL, 22.7 mmol), stirring for 10 min, adding DIPEA (0.88mL, 5.04mmol) and DMAP (0.03g, 0.252mmol), adding dibenzyl phosphite (0.84mL, 3.78mmol), adding dropwise, heating to room temperature for 1.5 hr, reacting with KH ₂ PO ₄ (0.5M, 50mL), extraction with ethyl acetate (3X 50 mL), drying over anhydrous sodium sulfate, filtration, spin-drying, purification on silica gel (10-50% ethyl acetate/petroleum ether) afforded 1.60g of a colorless oil in 96% yield. MS ESI m/z C ₃₃ H ₄₁ N ₂ O ₁₀ P[M+H] ⁺ Calculated value 657, found value 657.

EXAMPLE 70 Synthesis of Compound 172

Compound 171 (1.60g, 2.43mmol) was dissolved in methanol (20 mL), pd/C (10 wt%,160 mg) was added, and catalytic hydrogenation (1 atm H) ₂ ) The reaction was allowed to react for 1 hour, filtered and spin dried to give 1.00g of a white solid in 91% yield. MS ESI m/z C ₁₉ H ₃₁ N ₂ O ₈ P[M-H] ^- Calculated 447, found 447.

EXAMPLE 71 Synthesis of Compound 173

Compound 172 (730mg, 1.63mmol) was dissolved in methanol (10 mL), 1N NaOH (1695l, 16.3mmol) was added, the reaction was allowed to proceed overnight at room temperature, concentrated, diluted with water (20 mL), acidified to pH 6 with 1N HCl, concentrated, the residue was slurried with methanol/ethyl acetate (80, 20, 5ml), filtered, collected to give 0.68g of a white solid in 99% yield. MS ESI m/z C ₁₇ H ₂₇ N ₂ O ₈ P[M-H] ^- Calculated value 417, found value 417.

EXAMPLE 72 Synthesis of Compound 174

Compound 173 (300mg, 0.72mmol) was dissolved in ethanol/dichloromethane/water (v/v/

v

5, 1, 2ml), compound 125 (262mg, 0.94mmol) and TEA (0.15ml, 1.08mmol) were added, stirred at room temperature overnight, concentrated, reverse phase chromatography column HPLC (C ₁₈ Column, 10-90% acetonitrile/water) to yield 200mg of white solid in 48% yield. MS ESI m/z C ₂₅ H ₃₄ N ₃ O ₁₁ P[M-H] ^- Calculated 582, found 582.

EXAMPLE 73 Synthesis of Compound 175

Compound 174 (140mg, 0.24mmol) was dissolved in DCM (2 mL) and TFA (1 mL) at room temperatureAfter 2 hours of reaction, it was concentrated to give 175, which was used directly in the next reaction. MS ESI m/z C ₂₀ H ₂₇ N ₃ O ₉ P[M-H] ^- Calculated 482, found 482.

EXAMPLE 74 Synthesis of Compound 180

Dissolving compound 33 (86mg, 0.16mmol, 1.0eq.) in DCM (2 mL), ice-cooling, adding pentafluorophenol (44mg, 0.24mmol, 1.5eq.) and DIC (22mg, 0.175mmol, 1.1eq.), heating to room temperature for overnight reaction, concentrating, dissolving EtOAc (2 mL), filtering, concentrating again to obtain crude pentafluorophenol ester, dissolving it in DMA (2 mL), adding compound 175 (0.24mmol, 1.5eq.) and DIPEA (42 uL, 0.24mmol, 1.5eq.), reacting at room temperature overnight, concentrating, reverse phase chromatography column HPLC (C) ₁₈ Column, 10-90% acetonitrile/water) to give an amorphous solid 90mg, 56% yield. MS ESI m/z C ₄₆ H ₆₇ N ₇ O ₁₄ PS[M+H] ⁺ Calculated 1004.41, found 1004.41.

EXAMPLE 75 Synthesis of Compound 289

2- (2-Aminoethoxy) ethanol (21.0 g, 200mmol) and potassium carbonate (83.0 g, 600mmol) were mixed in acetonitrile (350 mL) and benzyl bromide (57.0 mL,480 mmol) was added. After the mixture was refluxed overnight, water (1L) was added and extracted with ethyl acetate (3X 300 mL). The combined organic layers were washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, filtered, concentrated and passed through SiO ₂ Column chromatography (4. MS ESI m/z C ₁₈ H ₂₃ NO ₂ Na[M+Na] ⁺ : calculated 309.17, found 309.19.

EXAMPLE 76 Synthesis of Compound 290

To a solution of 2- (2- (dibenzylamino) ethoxy) ethanol (47.17g, 165.3mmol), tert-butyl acrylate (72.0mL, 495.9mmol) and tetrabutylammonium iodide (6.10g, 16.53mmol) in dichloromethane (560 mL) was added a 50% aqueous solution of sodium hydroxide (300 mL). The mixture was stirred overnight. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3X 100 mL). The organic layers were combined and washed with water (3X 300 mL) and saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated and passed through SiO ₂ Column chromatography (7. MS ESI m/z C ₂₅ H ₃₆ NO ₄ [M+H] ⁺ : calculated 414.2566, found 414.2384.

Example 77 Synthesis of Compound 291

To a solution of tert-butyl 3- (2- (2- (2- (dibenzylamino) ethoxy) propionate (20.00g, 48.36mmol,1.0 eq) in tetrahydrofuran (30 mL) and methanol (60 mL) in a hydrogenation flask was added Pd/C (2.00g, 10wt%). The mixture was shaken overnight under hydrogen (1 atm), filtered through celite (filter aid), and the filtrate was concentrated to give a colorless oil (10.58g, 93.8% yield). MS ESI m/zC ₁₁ H ₂₄ NO ₄ [M+H] ⁺ : calculated 234.1627 and found 234.1810.

EXAMPLE 78 Synthesis of Compound 292

To a solution of (E) -3-bromoacrylic acid (0.15g, 1mmol), DMAP (0.15g, 1.2mmol) and DCC (0.21g, 1mmol) in dichloromethane (10 ml) at 0 ℃ was added compound 291 (0.23g, 1mmol). The reaction mixture was warmed to room temperature and stirred overnight. The crude product is concentrated and passed through SiO ₂ Column chromatography eluting with an ethyl acetate/dichloromethane gradient afforded compound 292 (0.31g, 85% yield). ESI MS m/z C ₁₄ H ₂₅ BrNO ₅ [M+H] ⁺ : calculated 366.08, found 366.08.

EXAMPLE 79 Synthesis of Compound 293

Compound 292 (0.31g, 0.84mmol) was dissolved in formic acid (4 mL) at 0 deg.C, followed by addition of water (2 mL). The reaction mixture was warmed to room temperature and stirred overnight. The crude product was concentrated and used directly in the next step. ESI MS m/z C ₁₀ H ₁₇ BrNO ₅ [M+H] ⁺ : calcd 310.02, found 310.03.

EXAMPLE 80 Synthesis of Compound 294

Compound 293 (0.12g, 0.39mmol), N-hydroxysuccinimide (0.067g, 0.58mmol) and EDCI (0.11g, 0.58mmol) are dissolved in dichloromethane (10 mL) and the mixture is stirred at room temperature overnight, concentrated and passed through SiO ₂ Purification by column chromatography gave compound 294 (0.13g, 82% yield). ESIMS m/z C ₁₄ H ₂₀ BrN ₂ O ₇ [M+H] ⁺ : calculated 407.04, found 407.04.

EXAMPLE 81 Synthesis of Compound 297

To a solution of compound 256 (50mg, 0.066 mmol) and compound 294 (60mg, 0.148mmol) in DMA (3 ml) was added sodium dihydrogenphosphate (17.8mg, 0.15mmol). Stirring overnight at room temperature, concentration and purification by preparative HPLC (elution with an acetonitrile/water gradient) to give the compoundSubstance 297 (22.6mg, 33% yield). ESI MS m/z C ₄₈ H ₇₃ BrN ₇ O ₁₂ S[M+H] ⁺ : calculated 1052.41 and found 1052.40.

EXAMPLE 82 Synthesis of Compound 302

Tert-butyl 3- (2- (2-aminoethoxy) ethoxy) propionate (466mg, 2mmol) and propiolic acid (210mg, 3mmol) were dissolved in dichloromethane (50 mL), to which DCC (618mg, 3mmol) was added. The resulting solution was stirred at room temperature for 3 hours and then concentrated. Purification by column chromatography (10% ethyl acetate/petroleum ether to 100% ethyl acetate) gave compound 302 (400mg, 70%). ESIMS M/z 286.17 ([ M + H ] ] ⁺ )。

EXAMPLE 83 Synthesis of Compound 303

Compound 302 (200mg, 0.7 mmol) was dissolved in dichloromethane (5 mL), to which was added formic acid (7 mL). The resulting solution was stirred at 38 ℃ overnight. All volatiles were removed by distillation under the reduced pressure to give compound 303 (160 mg). ESI MS M/z 230.11 ([ M + H ]] ⁺ )。

EXAMPLE 84 Synthesis of Compound 304

N-hydroxysuccinimide (115mg, 1mmol) and EDC (192mg, 1mmol) were added to a solution of compound 303 (149mg, 0.65mmol) in dichloromethane (15 mL). Stir at room temperature overnight, concentrate the reaction and purify by column chromatography (0% to 10% methanol/dichloromethane) to give the title compound (180mg, 85%). ESI MS M/z 327.11 ([ M + H)] ⁺ )。

EXAMPLE 85 Synthesis of Compound 305

Sodium dihydrogenphosphate (0.1M, 1.5 mL) was added to a solution of compound 304 (90mg, 0.276mmol) and compound 110 (109mg, 0.276mmol) in ethanol (7.5 mL). The resulting solution was stirred at room temperature for 24 hours. All volatiles were removed by distillation under the reduced pressure, and the residue was purified by column chromatography (30% ethyl acetate/petroleum ether to 100% ethyl acetate) to give the title compound (160mg, 96%). ESI MS M/z 606.34 ([ M + H ]] ⁺ )

EXAMPLE 86 Synthesis of Compound 306

Compound 305 (40mg, 0.066 mmol) was dissolved in dichloromethane (3 mL) and stirred with trifluoroacetic acid (3 mL) at room temperature for 2 hours. All volatiles were removed by distillation under the reduced pressure to give the title compound (29mg, 99%). ESI MS M/z 450.23 ([ M + H ] ] ⁺ )。

EXAMPLE 87 Synthesis of Compound 307

Compound 306 (29mg, 0.066 mmol) and compound 41a (46mg, 0.066 mmol) were dissolved in DMA (3 mL). DIPEA (10mg, 0.078mmol) was then added thereto, and the mixture was stirred at room temperature for 1.5 hours. The solvent was removed by distillation under the reduced pressure, and the residue was subjected to preparative HPLC (C) ₁₈ Column, 10-90% acetonitrile/water) to yield the title compound (15mg, 24%). ESI MS M/z 958.47 ([ M + H)] ⁺ )。

EXAMPLE 88 Synthesis of Compound 309

To a solution of compound 110 (424mg, 1mmol) in dichloromethane (20 mL) was added imidazole (408mg, 6mmol) and tert-butylchlorodimethylsilane (602mg, 4mmol). The resulting solution was stirred at room temperature for 3 hours. The reaction was concentrated and purified by column chromatography (10% to 30% ethyl acetate/petroleum ether) to give compound 309 (344mg, 64% yield).

EXAMPLE 89 Synthesis of Compound 310

Compound 309 (200mg, 0.37mmol) was dissolved in ethyl acetate (30 mL) and stirred with Pd/C (10 wt%,100 mg) under hydrogen (1 atm) atmosphere at room temperature for 2 hours. After filtering off the catalyst, the filtrate was distilled under reduced pressure to remove all volatiles to give compound 310 (187mg, 99% yield).

EXAMPLE 90 Synthesis of Compound 312

Acetylene dicarboxylic acid (26.5mg, 0.232mmol) was dissolved in NMP (1.0 mL) and cooled to 0 ℃ to which was added compound 291 (0.15g, 0.557mmol) followed by DMTMM (0.18g, 0.65mmol). The reaction was stirred at 0 ℃ for 5 hours, then diluted with ethyl acetate and washed with water and saturated brine. The organic solution was concentrated and purified by column chromatography (80-90% ethyl acetate/petroleum ether). The desired fractions were concentrated and stored in a refrigerator overnight, and the precipitated solid was filtered off. The filtrate was concentrated to give a pale yellow oil (0.37 g, >100% yield) was used for the next reaction. MS ESI m/z C ₂₆ H ₄₅ N ₂ O ₁₀ [M+H] ⁺ : calculated value 545.30; found 545.30.

EXAMPLE 91 Synthesis of Compound 313

Will combine withMaterial 312 (0.21 g) was dissolved in dichloromethane (2.5 mL) and cooled to 0 ℃, trifluoroacetic acid (2.5 mL) was added, the reaction was warmed to room temperature and stirred for 45 minutes, then the solvent and residual TFA were removed on a rotary evaporator. The crude product was purified by column chromatography (0-15% methanol in dichloromethane) to give a colorless oil (58.7 mg, 99% yield over two steps). MS ESI m/z C ₁₈ H ₂₉ N ₂ O ₁₀ [M+H] ⁺ : calculated 433.17; found 433.17.

EXAMPLE 92 Synthesis of Compound 314

Compound 313 (0.085g, 0.197mmol), compound 310 (0.1g, 0.197mmol) and HATU (0.112mg, 0.30mmol) were mixed in DMF (6 ml), to which was added triethylamine (55. Mu.L, 0.40 mmol). The reaction was stirred at room temperature overnight, the solvent was distilled off under reduced pressure, and the residue was passed through SiO ₂ Purification by column chromatography gave the title product 314 (0.065mg, 36% yield). ESI MS m/z C ₄₅ H ₇₅ N ₄ O ₁₄ Si[M+H] ⁺ : calculated value 923.50, found value 923.50.

EXAMPLE 93 Synthesis of Compound 315

Compound 314 (64.8mg, 0.070mmol) was dissolved in dichloromethane (3 mL), TBAF (1M, 0.1mL) was added, the reaction was stirred at room temperature for 1 h, then concentrated and passed through SiO ₂ Purification by column chromatography gave the title product 315 (34.5mg, 61% yield). ESI MS m/z C ₃₉ H ₆₁ N ₄ O ₁₄ [M+H] ⁺ : calculated 809.41, found 809.41.

EXAMPLE 94 Synthesis of Compound 316

Compound 315 (34.5mg, 0.043mmol) was dissolved in dichloromethane (1 mL), followed by addition of trifluoroacetic acid (0.5 mL). The reaction was stirred at room temperature for 2 h, concentrated and redissolved in DMA (4 mL), pentafluorophenyl ester (45mg, 0.065 mmol) and DIPEA (15 μ L,0.09 mmol) were added, the reaction stirred at room temperature overnight and concentrated, and purified by preparative HPLC (eluting with an acetonitrile/water gradient) to afford the title product 316 (22.8mg, 46% yield). ESI MS m/z C ₅₆ H ₈₅ N ₈ O ₁₇ S[M+H] ⁺ : calculated 1173.57, found 1173.58.

Example 95 Synthesis of Compound 334

4-aminobutyric acid (30g, 300mmol) and NaOH (24g, 600mmol) were dissolved in water (160 mL) and a solution of benzyl chloroformate (64.4 g, 380mmol) in tetrahydrofuran (130 mL) was added at 0 ℃. The reaction was stirred at 0 ℃ for 1 hour, room temperature for 3 hours. The tetrahydrofuran was distilled off under reduced pressure and the pH of the aqueous solution was adjusted to 3 at 0 ℃ with concentrated hydrochloric acid. Extraction with ethyl acetate, washing with saturated brine, drying over anhydrous sodium sulfate, and concentration gave a white solid (70.8g, 99%). ESI m/z C ₁₂ H ₁₆ NO ₄ [M+H] ⁺ Calculated 238.10 and found 238.22.

EXAMPLE 96 Synthesis of Compound 335

DMAP (0.8g, 6.56mmol) and DCC (17.1g, 83mmol) were added to a solution of 4- ((((benzyloxy) carbonyl) amino) butyric acid (16.4g, 69.2mmol) and t-butanol (15.4g, 208mmol) in dichloromethane (100 mL), stirred overnight at room temperature, the reaction was filtered and the filtrate was concentrated the residue was dissolved in ethyl acetate, washed with 1N HCl, saturated brine, dried over sodium sulfate, filtered, concentrated and purified by column chromatography (10 to 50% ethyl acetate/petroleum ether) to give a compoundSubstance 335 (7.5g, 37% yield). MS ESI m/z C ₁₆ H ₂₃ NO ₄ Na[M+Na] ⁺ : calculated 316.16, found 316.13.

EXAMPLE 97 Synthesis of Compound 336

Tert-butyl 4- (((benzyloxy) carbonyl) amino) butyrate (560mg, 1.91mmol) was dissolved in methanol (50 mL), mixed with a Pd/C catalyst (10 wt%,100 mg), and then reacted by hydrogenation (1 atm) for 3 hours. The catalyst was filtered off and all volatiles were distilled off under reduced pressure to give compound 336 (272mg, 90% yield). MS ESI m/z C ₈ H ₁₈ NO ₂ [M+H] ⁺ : calculated 160.13, found 160.13.

EXAMPLE 98 Synthesis of Compound 338

Tert-butyl 4-aminobutyrate (477mg, 3mmol) and 2, 3-dibromosuccinic acid (414mg, 1.5mmol) were dissolved in dichloromethane (35 mL), to which DIPEA (1.16g, 9mmol) and EDC (0.86g, 4.5mmol) were added. The resulting solution was stirred at room temperature overnight, then washed with saturated brine, dried over sodium sulfate, filtered, concentrated and purified by column chromatography (pure dichloromethane to 10% methanol/dichloromethane) to give compound 338 (160mg, 22% yield). MS ESI m/z C ₂₀ H ₃₄ BrN ₂ O ₆ [M+H] ⁺ : calculated 477.15 and found 477.16.

EXAMPLE 99 Synthesis of Compound 339

Compound 338 (80mg, 0.168mmol) was dissolved in dichloromethane (5 mL) and stirred with formic acid (8 mL) overnight at 38 ℃. Distilling under reduced pressure to removeVolatiles were present to give compound 339 (61mg, 99% yield). MS ESI m/z C ₁₂ H ₁₈ BrN ₂ O ₆ [M+H] ⁺ : calcd for 365.03, found 365.05.

Example 100 Synthesis of Compound 340

N-hydroxysuccinimide (60mg, 0.504mmol) and EDCI (97mg, 0.504mmol) were added to a solution of compound 339 (61mg, 0.168mmol) in dichloromethane (10 mL). Stirring overnight at room temperature, the reaction mixture was concentrated and purified by column chromatography (0 to 10% methanol/dichloromethane) to give compound 340 (72mg, 77% yield). MS ESI m/z C ₂₀ H ₂₄ BrN ₄ O ₁₀ [M+H] ⁺ : calculated 559.06, found 559.78.

Example 101 Synthesis of Compound 342

Aqueous sodium dihydrogen phosphate (0.1M, 1mL) was added to a solution of compound 340 (36mg, 0.065 mmol) and compound 110 (25mg, 0.063mmol) in ethanol (5 mL). The resulting solution was stirred at room temperature overnight, and then HO- (PEG) was added thereto ₂₄ -NH ₂ (95 mg), and the mixture was stirred at room temperature overnight. All volatiles were removed by distillation under the reduced pressure and the residue was purified by column chromatography (pure dichloromethane to 10% methanol/dichloromethane) to give compound 342 (28mg, 24% yield). MS ESI M/z 1798.93 ([ M + H ] ] ⁺ )。

EXAMPLE 102 Synthesis of Compound 344

Compound 342 (28mg, 0.0156mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added thereto, and the mixture was cooled to room temperatureStirred for 2 hours. All volatiles were removed by distillation under the reduced pressure to give compound 344 (25mg, 98% yield). MS ESI M/z 1642.82 ([ M + H)] ⁺ )。

EXAMPLE 103 Synthesis of Compound 346

Compound 344 (25mg, 0.0152mmol) and pentafluorophenyl ester (15mg, 0.0213mmol) were dissolved in DMA (5 mL). DIPEA (10mg, 0.077mmol) was added thereto. The resulting mixture was stirred at room temperature overnight, concentrated and subjected to preparative HPLC (C) ₁₈ Column, 10-90% acetonitrile/water) to give compound 346 (13mg, 40% yield.) MS ESI M/z 2163.82 ([ M + H)] ⁺ )。

Example 104 Synthesis of Compound 350

To a solution of 2,2' - (ethane-1, 2-diylbis (oxy)) diethanol (55.0 mL, 410.75mmol) in anhydrous tetrahydrofuran (200 mL) was added sodium (0.1 g). The mixture was stirred until sodium disappeared, then tert-butyl acrylate (20.0 mL, 137.79mmol) was added dropwise, stirred overnight, and then quenched with hydrochloric acid solution (20.0 mL, 1N) at 0 ℃. Tetrahydrofuran was removed by rotary evaporation, saturated brine (300 mL) was added, and the resulting mixture was extracted with ethyl acetate (3X 100 mL). The organic layer was washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a colorless oil (30.20 g, yield 79.0%) which was used without further purification. MS ESI m/zC ₁₃ H ₂₇ O ₆ [M+H] ⁺ : calculated 278.1729, found 278.1730.

EXAMPLE 105 Synthesis of Compound 351

To 3- (2- (2- (2))To a solution of tert-butyl- (2-hydroxyethoxy) ethoxy) propionate (30.20g, 108.5mmol) and p-toluenesulfonyl chloride (41.37g, 217.0 mmol) in anhydrous dichloromethane (220 mL) was added triethylamine (30.0mL, 217.0 mmol). The reaction was stirred at room temperature overnight, then washed with water (3X 300 mL) and saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated and passed through SiO ₂ Column chromatography (3. MS ESI m/zC ₂₀ H ₃₃ O ₈ S[M+H] ⁺ : calcd 433.1818 found 433.2838.

Example 106 Synthesis of Compound 352

To a solution of tert-butyl 3- (2- (2- (2- (2-methoxy) ethoxy) propionate (39.4g, 91.1mmol) in anhydrous DMF (100 mL) was added NaN ₃ (20.67g, 316.6 mmol) and stirred at room temperature overnight. Water (500 mL) was added and the mixture was extracted with ethyl acetate (3X 300 mL). The combined organic layers were washed with water (3X 900 mL) and saturated brine (900 mL), dried over anhydrous sodium sulfate, filtered, concentrated and passed through SiO ₂ Column chromatography (5. MS ESI m/z C ₁₃ H ₂₅ O ₃ N ₅ Na[M+Na] ⁺ : calculated 326.2 and found 326.2.

Example 107 Synthesis of Compound 353

Raney nickel (7.5 g, suspended in water) was washed with water (three times) and isopropanol (three times) and mixed with a solution of compound 101 (5.0 g,16.5 mmol) in isopropanol. The mixture was stirred under a hydrogen balloon at room temperature for 16 hours, then filtered through a pad of celite, washing with isopropanol. The filtrate was concentrated and purified by column chromatography (5-25% methanol/dichloromethane),a pale yellow oil was obtained (2.60g, 57% yield). MS ESI m/z C ₁₃ H ₂₈ NO ₅ [M+H] ⁺ : calculated value 279.19; found 279.19.

Example 108 Synthesis of Compound 354

Acetylene dicarboxylic acid (0.35g, 3.09mmol) was dissolved in NMP (10 mL) and cooled to 0 deg.C, to which was added compound 353 (2.06g, 7.43mmol) followed by DMTMM (2.39g, 8.65mmol). The reaction was stirred at 0 ℃ for 6 hours, then diluted with ethyl acetate and washed with water and saturated brine. The organic solution was concentrated and slurried with a mixed solvent of ethyl acetate and petroleum ether. The solid was filtered off, the filtrate was concentrated and purified by column chromatography (80-90% ethyl acetate/petroleum ether) to give a pale yellow oil (2.26 g,>100% yield) was used without further purification. MS ESI m/z C ₃₀ H ₅₃ N ₂ O ₁₂ [M+H] ⁺ : calculated 633.35; found 633.30.

EXAMPLE 109 Synthesis of Compound 355

Compound 354 (2.26 g) was dissolved in dichloromethane (15 mL) and cooled to 0 ℃, then trifluoroacetic acid (15 mL) was added, the reaction was warmed to room temperature and stirred for 45 minutes, then the solvent and residual TFA were removed on a rotary evaporator. The crude product was purified by column chromatography (0-15% methanol in dichloromethane) to give a light yellow oil (1.39 g, 86% over two steps). MS ESI m/zC ₂₂ H ₃₇ N ₂ O ₁₂ [M+H] ⁺ : calculated 521.23; found 521.24.

EXAMPLE 110 Synthesis of Compound 358

To a solution of Z-L-Ala-OH (0.84g, 5 mmol), H-Gly-OtBu (0.66g, 5 mmol) in methylene chloride (20 mL) were added HOBt (0.68g, 5 mmol), EDCI (1.44g, 7.5 mmol), and DIPEA (1.7 mL, 10mmol). The reaction was stirred at room temperature overnight, then washed with water (100 mL), and the aqueous layer was back-extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over magnesium sulfate, filtered and concentrated. SiO for residue ₂ Column purification gave the title product (0.87g, 52% yield). ESIm/zC ₁₇ H ₂₅ N ₂ O ₅ [M+H] ⁺ : calculated 337.17, found 337.17.

EXAMPLE 111 Synthesis of Compound 359

Compound 358 (0.25g, 0.74mmol) was dissolved in dichloromethane (30 mL), trifluoroacetic acid (10 mL) was added at room temperature, and the reaction was stirred overnight. The reaction was then concentrated to give compound 359, which was used in the next step without further purification.

EXAMPLE 112 Synthesis of Compound 360

Compound 359 (0.20g, 0.70mmol), compound 110 (0.19g, 0.48mmol) and HATU (0.18g, 0.48mmol) were dissolved in dichloromethane (20 mL), to which triethylamine (134. Mu.L, 0.96 mmol) was added. The reaction mixture was stirred at room temperature overnight, concentrated under reduced pressure, and the residue was washed with SiO ₂ Column purification gave the title product 360 (0.30g, 95% yield). ESI m/zC ₃₄ H ₄₉ N ₄ O ₉ [M+H] ⁺ : calculated 657.34 and found 657.34.

EXAMPLE 113 Synthesis of Compound 361

In a hydrogenation reaction vessel, a solution of compound 360 (0.30g, 0.46mmol) in methanol (10 mL) and Pd/C (0.10g, 10wt%) were added. The mixture is brought to 1 atmosphere H ₂ After shaking overnight, the filtrate was concentrated by filtration through celite (filter aid) to give 361 (0.21g, 87% yield) which was used in the next step without further purification. ESI m/zC ₂₆ H ₄₃ N ₄ O ₇ [M+H] ⁺ : calcd 523.31, found 523.31.

EXAMPLE 114 Synthesis of Compound 362

To a solution of compound 361 (0.11g, 0.2mmol), compound 355 (0.104g, 0.2mmol) in dichloromethane (10 mL) were added HATU (0.07g, 0.2mmol), and triethylamine (55. Mu.L, 0.4 mmol). The reaction mixture was stirred at room temperature overnight, then the solvent was removed under reduced pressure and the residue was taken up in SiO ₂ Column purification gave the title product 362 (0.046 g,23% yield). ESI m/zC ₄₈ H ₇₅ N ₆ O ₁₇ [M+H] ⁺ : calculated 1007.51 and found 1007.52.

EXAMPLE 115 Synthesis of Compound 363

Compound 362 (0.046 g, 0.045mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added at room temperature, and after stirring for 2 hours the reaction was concentrated to give compound 363, which was used in the next step without further purification.

Example 116 Synthesis of Compound 364

To a DMA (4 mL) solution of compound 363 was added pentafluorophenyl ester 41a (31.1mg, 0.045mmol) followed by DIPEA (28. Mu.l, 0.159 mmol) and stirred overnight. The reaction solution was concentrated and the crude product was purified by preparative HPLC (10-90% acetonitrile/water) to give the title product 364 (7.9 mg,13% yield). ESI MSm/zC ₆₄ H ₉₉ N ₁₀ O ₂₀ S[M+H] ⁺ : calculated 1359.67, found 1359.62.

EXAMPLE 117 Synthesis of Compound 319

Compound 104 (0.19g, 0.48mmol), compound 313 (0.173g, 0.4mmol) and HATU (0.30g, 0.8mmol) were dissolved in dichloromethane (50 mL) and triethylamine (110. Mu.L, 0.8 mmol) was added. The reaction mixture was stirred at room temperature overnight, then the solvent was distilled off under reduced pressure, and the residue was taken up with SiO ₂ Column purification gave the title compound 319 (0.25g, 80% yield). ESI m/z C ₃₉ H ₅₉ N ₄ O ₁₃ [M+H] ⁺ Calculated 791.40, found 791.40.

EXAMPLE 118 Synthesis of Compound 320

Compound 319 (0.10g, 0.14mmol) was dissolved in methylene chloride (1.0 mL), followed by addition of TFA (1.0 mL). The reaction was stirred at room temperature for 2 hours and after concentration gave compound 320, which was used in the next step without further purification.

EXAMPLE 119 Synthesis of Compound 321

To a solution of compound 320 (88.8mg, 0.14mmol) in DMA (1.0 mL) were added pentafluorophenyl ester 41a (96.9mg, 0.14mmol) and DIPEA (47.5. Mu.L, 0.28 mmol). The reaction was stirred overnight and concentrated then purified by preparative HPLC (acetonitrile/water gradient elution) to give the title compound 321 (40mg, 25% yield). ESI m/z C ₅₅ H ₈₃ N ₈ O ₁₆ S[M+H] ⁺ Calculated 1143.56 and found 1143.56.

EXAMPLE 120 Synthesis of Compound 386

HATU (39.9g, 105mmol) was added to a solution of compound 334 (26.1g, 110mmol) in DMF (300 mL). After stirring at room temperature for 30 min, the reaction mixture was added to a solution of compound 110 (39.4 g, 100mmol) and triethylamine (20.2g, 200mmol) in DMF (300 mL). The reaction was stirred at room temperature for 2 hours, diluted with water, extracted with ethyl acetate, the organic layer washed with saturated brine and dried over sodium sulfate. After concentration, purification by column chromatography on silica gel (20-70% ethyl acetate/petroleum ether) gave a white solid (45 g, 73% yield). ESI m/z C ₃₃ H ₄₈ N ₃ O ₈ [M+H] ⁺ Calculated 614.34, found 614.15.

Example 121 Synthesis of Compound 387

Compound 386 (100g, 163mmol) is dissolved in methanol (500 mL), pd/C catalyst (10 wt%,10 g) is added, and hydrogenation reaction (1 atm H) is carried out at room temperature ₂ ) Overnight. After filtration of the catalyst, the filtrate was concentrated under reduced pressure to give 387 (75.8 g, 97% yield) as a brown foamy solid. ¹ H NMR(400MHz,CDCl3)δ7.11(s,1H),6.83(d,J＝10.3Hz,2H),5.04–4.52(m,6H),3.90–3.56(m,1H),2.81(d,J＝5.3Hz,2H),2.63(dd,J＝12.5,6.1Hz,2H),2.54-2.26(dd,J＝14.0,7.6Hz,4H),1.94-1.64(m,3H),1.44–1.36(m,18H),1.08(d,J＝6.9Hz,3H)。ESI m/z C ₂₅ H ₄₂ N ₃ O ₆ [M+H] ⁺ Calculated 480.30 and found 480.59.

EXAMPLE 122 Synthesis of Compound 388

Compound 387 (39mg, 0.08mmol), compound 313 (43mg, 0.1mmol) and HATU (30.4 mg, 0.08mmol) were dissolved in dichloromethane (20 ml) and triethylamine (22. Mu.L, 0.16 mmol) was added. The reaction mixture was stirred at room temperature overnight, then the solvent was distilled off under reduced pressure and the residue was passed through SiO ₂ Column purification afforded the title compound 388 (42mg, 60% yield). ESI m/zC ₄₃ H ₆₆ N ₅ O ₁₄ [M+H] ⁺ Calculated 876.45, found 876.40.

Example 123 Synthesis of Compound 389

Compound 388 (17mg, 0.019mmol) was dissolved in methylene chloride (1.0 mL), to which TFA (1.0 mL) was added. The reaction was stirred at room temperature for 2 hours, then concentrated to give compound 389, which was used in the next step without further purification.

EXAMPLE 124 Synthesis of Compound 390

To a solution of compound 389 (13.6mg, 0.019mmol) in DMA (1.0 mL) were added pentafluorophenyl ester 41a (13mg, 0.019mmol) and DIPEA (6.4. Mu.L, 0.038 mmol). The reaction was stirred overnight and concentrated, and the residue was purified by preparative HPLC (acetonitrile/water gradient elution) to give the title compound 390 (9.9 mg,42% yield). ESI m/z C ₅₉ H ₉₀ N ₉ O ₁₇ S[M+H] ⁺ Calculated 1228.61, found 1228.60.

EXAMPLE 125 Synthesis of Compound 392

Compound 110 (68mg, 0.17mmol), compound 124 (94.5mg, 0.52mmol) and HATU (162mg, 0.425mmol) were dissolved in dichloromethane (50 mL). Triethylamine (73. Mu.L, 0.52 mmol) was then added. The reaction mixture was stirred at room temperature overnight, then the solvent was distilled off under reduced pressure, and the residue was taken up with SiO ₂ Column purification gave the title compound 392 (98mg, 80% yield). ESI m/zC ₃₇ H ₄₉ N ₄ O ₁₁ [M+H] ⁺ Calculated 725.33, found 725.34.

EXAMPLE 126 Synthesis of Compound 393

Compound 392 (98mg, 0.135mmol) was dissolved in dichloromethane (1.0 mL), TFA (1.0 mL) was added at room temperature, and stirred for 2 hours. Then concentrated to give compound 393 which was used in the next step without further purification.

EXAMPLE 127 Synthesis of Compound 394

To a solution of compound 393 (76.9mg, 0.135mmol) in DMA (1 mL) was added pentafluorophenyl ester 41a (44mg, 0.06mmol) and DIPEA (45.8. Mu.L, 0.27 mmol). The reaction was stirred overnight, then concentrated and the residue was purified by preparative HPLC (acetonitrile/H) ₂ O gradient elution) to afford the title compound 394 (37mg, 55% yield). ESI m/z C ₅₃ H ₇₃ N ₈ O ₁₄ S[M+H] ⁺ Calculated 1077.49 and found 1077.50.

EXAMPLE 128 Synthesis of Compound 409

Compound 110 (100mg, 0.25mmol), 3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionic acid (65mg, 0.25mmol) and HATU (190mg, 0.5 mmol) were dissolved in dichloromethane (50 ml). Triethylamine (73. Mu.L, 0.5 mmol) was added and the reaction mixture was stirred at room temperature overnight. Then distilling off the solvent under reduced pressure, and using SiO as the residue ₂ Column purification afforded the title compound 409 (164mg, 75% yield). ESI m/z C ₄₃ H ₆₁ N ₄ O ₁₅ [M+H] ⁺ Calculated 873.41, found 873.41.

EXAMPLE 129 Synthesis of Compound 410

Compound 409 (1634 mg, 0.187mmol) was dissolved in dichloromethane (1.0 mL), TFA (1.0 mL) was added at room temperature, stirred for 2 hours and concentrated to give compound 410, which was used in the next step without further purification.

EXAMPLE 130 Synthesis of Compound 411

To a solution of compound 410 (43mg, 0.06mmol) in DMA (1 mL) were added pentafluorophenyl ester 41a (44mg, 0.06mmol) and DIPEA (34. Mu.L, 0.20 mmol), and stirred overnight. The reaction was then concentrated and the residue was purified by preparative HPLC (acetonitrile/H) ₂ O gradient elution) to afford the title compound 411 (33mg, 45% yield). ESI m/z C ₅₉ H ₈₅ N ₈ O ₁₈ S[M+H] ⁺ Calculated 1225.56, found 1225.55.

EXAMPLE 131 Synthesis of Compound 418

To a solution of maleimide (6.35g, 65.4 mmol) in EtOAc (120 mL) was added N-methylmorpholine (8.6mL, 78.5 mmol) and methyl chloroformate (6.0mL, 78.5 mmol) at 0 ℃. The reaction was stirred at 0 ℃ for 30 minutes and at room temperature for 1 hour. The solid was filtered off and the filtrate was concentrated, the residue was dissolved in dichloromethane and filtered through a silica gel column and eluted with dichloromethane to remove the color. The product was collected and concentrated and the resulting solid was slurried with 10% ethyl acetate/petroleum ether to give 9.00g of a white solid (89% yield).

EXAMPLE 132 Synthesis of Compound 419

N-Boc-ethylenediamine (5.6 mL,35.4 mmol) and saturated NaHCO ₃ (60 mL) the mixture was cooled to 0 ℃ and compound 418 (5.00g, 32.2mmol) was added portionwise. After stirring at 0 ℃ for 30 minutes, the reaction temperature was raised to room temperature and stirred for 1 hour. Filtering, collecting precipitate, washing with cold water, dissolving in ethyl acetate, washing with saturated saline solution, and removing anhydrous Na ₂ SO ₄ Dried and concentrated to give a white solid (6.69g, 87% yield).

EXAMPLE 133 Synthesis of Compound 420

A solution of compound 419 (6.00g, 25.0 mmol), furan (18.0 mL) in toluene (120 mL) was heated to reflux in a high pressure reaction tube and stirred for 16 h. The colorless solution turned yellow during the reaction. The reaction was cooled to room temperature and concentrated, and the resulting white solid was slurried with diethyl ether to give compound 420 (6.5g, 84% yield).

EXAMPLE 134 Synthesis of Compound 421

Compound 420 (9.93g, 32.2mmol) was dissolved in 1, 4-dioxane (15 mL), concentrated HCl (15 mL) was added at room temperature, the reaction solution was stirred for 3 hours, the resulting solid was concentrated, collected by filtration, and the filter cake was washed with ethyl acetate. The solid was dried in an oven (50 ℃) overnight to give compound 421 (6.94g, 88% yield).

EXAMPLE 135 Synthesis of Compound 422

To a solution of compound 421 (0.85g, 3.47mmol) in THF (10 mL) at-10 deg.C was added phosphorus oxychloride (162. Mu.L, 1.73 mmol) followed by triethylamine (966. Mu.L, 6.95 mmol). The reaction was stirred at-10 ℃ for 3 h, then diluted with dichloromethane (20 mL) and filtered through celite, and the filtrate was concentrated to give compound 422, which was used directly in the next step. ESI m/z C ₂₀ H ₂₃ ClN ₄ O ₇ P[M+H] ⁺ Calculated 497.09, found 497.09.

EXAMPLE 136 Synthesis of Compound 423

Compound 422 (0.50g, 1.0mmol) and DIPEA (0.4mL, 2.4mmol) were dissolved in dichloromethane (5.0 mL) at 0 ℃ and then compound 291 (0.23g, 1.0mmol) was added. The reaction was stirred at 0 ℃ for 2.5 hours, concentrated and passed through SiO ₂ Column purification gave the title compound 423 (0.30g, 43%). ESI m/z C ₃₁ H ₄₅ N ₅ O ₁₁ P[M+H] ⁺ 694.28 for calculated value and 694.28 for actual value.

EXAMPLE 137 Synthesis of Compound 424

Compound 423 (0.30g, 0.5 mmol) was dissolved in dichloromethane (3 mL), added TFA (3 mL) at room temperature and stirred for 2 h to give compound 424 after concentration, which was used in the next step without further purification.

EXAMPLE 138 Synthesis of Compound 425

Compound 424 (40mg, 0.063mmol), compound 110 (40mg, 0.10mmol), HATU (24mg, 0.063mmol) were dissolved in dichloromethane (5 mL), followed by addition of triethylamine (27.8. Mu.L, 0.2 mmol). The reaction mixture was stirred at room temperature overnight, the solvent was then distilled off under reduced pressure, and the residue was washed with SiO ₂ Purification by column gave the title compound 425 (53.4 mg, 84% yield). ESI m/z C ₄₈ H ₆₉ N ₇ O ₁₅ P[M+H] ⁺ 1014.45 for calculated value and 1014.45 for found value.

EXAMPLE 139 Synthesis of Compound 426

Compound 425 (53.4mg, 0.053mmol) was dissolved in dichloromethane (2 mL), added TFA (2 mL) at room temperature and stirred for 2 h to give compound 426 after concentration, which was used in the next step without further purification.

EXAMPLE 140 Synthesis of Compound 427

To a solution of compound 426 (45.0mg, 0.053 mmol) in DMA (1 mL) were added pentafluorophenyl ester 41a (37.0mg, 0.053 mmol) and DIPEA (17. Mu.L, 0.1 mmol). The reaction was stirred overnight and concentrated and the residue was purified by preparative HPLC (acetonitrile/water gradient elution) to give the title compound 427 (26.2mg, 36% yield). ESI m/z C ₆₄ H ₉₃ N ₁₁ O ₁₈ PS[M+H] ⁺ Calculated 1366.61 and found 1366.61.

EXAMPLE 141 Synthesis of Compound 428

Compound 427 (8.0mg, 0.0058mmol) was dissolved in toluene (5.0 mL), heated under reflux overnight, then concentrated, and purified by preparative HPLC (acetonitrile/water gradient elution) to give the title compound 428 (6.4mg, 90% yield). ESI m/z C ₅₆ H ₈₅ N ₁₁ O ₁₆ PS[M+H] ⁺ Calculated 1230.56 and found 1230.56.

Example 142 Synthesis of Compound 432

Sodium hydrogen (60%, 8g, 200mmol) was added to HO-PEG at room temperature ₉ OMe (42.8g, 100mmol) in tetrahydrofuran (1L). After stirring for 30 minutes, t-butyl bromoacetate (48.8g, 250mmol) was added thereto, and the mixture was stirred at room temperature for 1 hour, then poured into ice water, extracted with dichloromethane, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Purification by column chromatography on silica gel (0-5% methanol in dichloromethane) gave compound 156 as a yellow oil (32g, 59% yield).

Example 143 Synthesis of Compound 433

Compound 432 (40g, 73.8 mmol) was dissolved in methylene chloride (400 mL), followed by addition of formic acid (600 mL) and stirring at 25 ℃ overnight. All volatiles were distilled off under reduced pressure to give a yellow oil (36 g). ESI m/z C ₂₁ H ₄₃ O ₁₂ [M+H] ⁺ Calculated 487.27 and found 487.24.

EXAMPLE 144 Synthesis of Compound 434

Compound 433 (36g, 73.8mmol) was dissolved in dichloromethane (640 mL), and oxalyl chloride (100 mL) and DMF (52g, 0.74mmol) were added in this order. The resulting solution was stirred at room temperature for 4 hours, and all volatiles were removed by distillation under reduced pressure to give a yellow oil.

EXAMPLE 145 Synthesis of Compound 436

Z-L-Lys-OH (41.4g, 147.6 mmol), sodium carbonate (23.4g, 221.4mmol) and NaOH (5.9g, 147.6 mmol) were dissolved in water (720 mL), cooled to 0 ℃ and then a solution of compound 434 (37.2g, 73.8mmol) in tetrahydrofuran (20 mL) was added. The resulting mixture was stirred at room temperature for 1 hour, THF was removed by distillation under the reduced pressure, and the pH was adjusted to 3 with concentrated hydrochloric acid in an ice bath. The solution was extracted with dichloromethane, washed with saturated brine and dried over anhydrous sodium sulfate to give a yellow oil (55g, 99% yield). ESI m/z C ₃₅ H ₆₀ N ₂ O ₁₅ [M+H] ⁺ Calculated 749.40 and found 749.39.

EXAMPLE 146 Synthesis of Compound 439

Triethylamine (66mL, 474mmol) and HATU (72g, 190mmol) were added successively to a solution of compound 436 (130g, 174mmol) in DMF (500 mL) at 0 deg.C, and the reaction mixture L was warmed to room temperature and stirred for 2 hours. A solution of compound 387 (75.8g, 158mmol) in DMF (500 mL) was added to the above solution at 0 ℃ and the reaction was stirred at room temperature for 1 hour. The reaction mixture was poured into water (4L), extracted with ethyl acetate (3X 500 ml), the organic layers were combined, washed with saturated brine (2L), dried over sodium sulfate, filtered and concentrated to give crude 439 (190 g) which was used directly in the next reaction. ESI m/z C ₆₀ H ₁₀₀ N ₅ O ₂₀ [M+H] ⁺ Calculated 1210.69, found 1210.69.

EXAMPLE 147 Synthesis of Compound 440

The crude 439 (190 g) obtained in the previous step was dissolved in methanol (900 mL), added with Pd/C catalyst (10 wt%,19 g), and hydrogenated at room temperature (1 atm H) ₂ ) The reaction was allowed to proceed overnight. The catalyst was filtered off, the filtrate was concentrated under reduced pressure and purified by silica gel column (0-10% methanol/dichloromethane) to give a brown oil (105 g, 62% over two steps). ESI m/z C ₅₂ H ₉₅ N ₅ O ₁₈ [M+H] ⁺ Calculated 1077.65 and found 1077.65.

EXAMPLE 148 Synthesis of Compound 441

To a solution of compound 440 (105g, 97.1 mmol) in EtOH (5.3L) was added compound 125 (54.4 g,194.2 mmol) and 0.1N sodium dihydrogen phosphate solution (1.1L) at room temperature, and the reaction was stirred at room temperature overnight. EtOH was distilled off under reduced pressure, the residual aqueous solution was poured into water (3L) and then extracted with ethyl acetate (4X 500 mL), the organic phases were combined, washed with saturated brine (2L), dried over sodium sulfate, concentrated and the crude product was purified on a silica gel column (0-10% methanol/dichloromethane) to give a yellow oil (100g, 83% yield). ¹ H NMR(400MHz,CDCl ₃ )δ9.43(s,1H),7.35(s,1H),7.23(t,J＝5.1Hz,1H),7.01(d,J＝4.5Hz,2H),6.89(s,2H),6.70(s,2H),4.56–4.45(m,1H),4.30(t,J＝9.7Hz,1H),3.97(s,2H),3.86-3.74(m,1H),3.66–3.63(m,36H),3.58–3.52(m,5H),3.38(s,3H),3.33–3.19(m,3H),2.47(d,J＝6.2Hz,4H),2.23(dd,J＝11.6,6.1Hz,2H),1.91(dtd,J＝26.8,13.6,6.5Hz,7H),1.71(d,J＝7.7Hz,2H),1.56–1.49(m,2H),1.42(s,9H),1.39(s,9H),1.10(d,J＝6.5Hz,3H)。ESI m/z C ₆₀ H ₁₀₁ N ₆ O ₂₁ [M+H] ⁺ Calculated value 1241.69, trueFound 1241.69.

EXAMPLE 149 Synthesis of Compound 442

Compound 441 (79.1mg, 0.062mmol) was dissolved in dichloromethane (2 mL), added with TFA (2 mL), stirred at room temperature for 2 hours, distilled under reduced pressure and azeotropically distilled with toluene to give compound 441, which was used directly in the next step.

EXAMPLE 150 Synthesis of Compound 443

Compound 441 (67mg, 0.062mmol) and compound 41a (43mg, 0.062mmol) were dissolved in DMA (4 mL) and DIPEA (43. Mu.l, 0.248 mmol) was added. After stirring at room temperature for 3 hours, the solvent was distilled off under reduced pressure and the residue was subjected to preparative HPLC (C) ₁₈ Column, acetonitrile/water 10-90%) to give compound 443 (59mg, 60% yield). ESI m/z C ₇₆ H ₁₂₅ N ₁₀ O ₂₄ S[M+H] ⁺ Calculated 1594.92 and found 1594.24.

EXAMPLE 151 Synthesis of Compound 457

Sodium hydride (60%, 0.64g, 169mol) was added portionwise to HO-PEG ₆ -OMe (2.37g, 8 mmol) in tetrahydrofuran (25 mL). After stirring at room temperature for 15min, tert-butyl bromoacetate (3.90g, 20mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was poured into ice water, extracted with dichloromethane, the organic phase washed with saturated brine, dried over sodium sulfate and purified by silica gel column chromatography (20-50% ethyl acetate/petroleum ether) to give a colorless oil (1.47g, 45%). ESI m/z C ₁₉ H ₃₉ O ₉ [M+H] ⁺ Calculated 411.25 and found 411.15.

EXAMPLE 152 Synthesis of Compound 458

Compound 457 (1.47g, 3.60mmol) is dissolved in dichloromethane (30 mL) and stirred with formic acid (50 mL) at 38 deg.C overnight. All volatiles were removed by distillation under the reduced pressure to give the title compound (1.20g, 94% yield) as a yellow oil. ESI m/z C ₁₅ H ₃₁ O ₉ [M+H] ⁺ Calculated 355.19, found 355.18.

EXAMPLE 153 Synthesis of Compound 459

Compound 458 (1.10g, 3.20mmol) was dissolved in methylene chloride (20 mL), and oxalyl chloride (4 mL) and DMF (3 drops) were added in this order. The resulting solution was stirred at room temperature for 4 hours and all volatiles were removed by distillation under reduced pressure to give a yellow oil which was used directly in the next step.

EXAMPLE 154 Synthesis of Compound 460

Z-L-Lys-OH (1.80g, 6.4 mmol), sodium carbonate (1g, 9.6 mmol) and NaOH (0.26g, 6.4 mmol) were dissolved in water (30 mL), cooled to 0 ℃ and then a solution of Compound 459 (1.20g, 3.2mmol) in tetrahydrofuran (10 mL) was added. The resulting mixture was stirred at room temperature for 1 hour, THF was removed by distillation under the reduced pressure, and the aqueous solution was brought to pH 3 with concentrated hydrochloric acid while cooling on ice. Extraction with dichloromethane, washing with saturated brine and drying over anhydrous sodium sulfate afforded the title compound (1.77g, 90%) as a brown oil. ESI m/zC ₂₉ H ₄₉ N ₂ O ₁₂ [M+H] ⁺ Calculated 617.32, found 617.31.

EXAMPLE 155 Synthesis of Compound 461

NHS (644 mg, 5.60mmol) and EDC (1.08g, 5.60mmol) were added to a solution of compound 460 (2.30g, 3.70mmol) in dichloromethane (100 mL). After stirring overnight at room temperature, the reaction mixture was directly loaded onto a silica gel column and purified (0-10% methanol/dichloromethane) to give a brown oil (2.10 g, 80% yield). ESI m/z C ₃₃ H ₅₂ N ₃ O ₁₄ [M+H] ⁺ Calculated 714.34 and found 714.32.

EXAMPLE 156 Synthesis of Compound 462

Sodium dihydrogen phosphate (0.1m, 3 mL) was added to a solution of compound 461 (357mg, 0.50mmol) and compound 110 (200mg, 0.50mmol) in ethanol (15 mL). The resulting solution was stirred at room temperature for 24 hours, all volatiles were removed by distillation under the reduced pressure, and the residue was purified by silica gel column chromatography (5-10% methanol/dichloromethane) to give the title compound (216mg, 44% yield) as a brown oil. ESI m/z C ₅₀ H ₈₁ N ₄ O ₁₆ [M+H] ⁺ Calculated 993.56, found 993.57.

EXAMPLE 157 Synthesis of Compound 463

Compound 462 (108mg, 0.109mmol) was dissolved in methanol (5 mL), a palladium catalyst (10 wt%, 50 mg) was added thereto, and the mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere (1 atm), the catalyst was filtered off, and the filtrate was distilled under reduced pressure to give the title compound (94 mg) as a yellow oil. ESI m/zC ₄₂ H ₇₅ N ₄ O ₁₄ [M+H] ⁺ Calculated 859.52, found 859.93.

Example 158 Synthesis of Compound 464

Sodium dihydrogenphosphate (0.1M, 2mL) was added to a solution of compound 463 (94mg, 0.109mmol) and compound 125 (61mg, 0.218mmol) in ethanol (10 mL). The resulting solution was stirred at room temperature for 24 hours, all volatiles were removed by distillation under reduced pressure, and the residue was purified by column chromatography (5-10% methanol in dichloromethane) to give a yellow oil (40mg, 36% yield). ESI m/z C ₅₀ H ₈₂ N ₅ O ₁₇ [M+H] ⁺ Calculated 1024.56 and found 1024.98.

EXAMPLE 159 Synthesis of Compound 465

Compound 464 (20mg, 0.0196mmol) was dissolved in dichloromethane (3 mL), and TFA (3 mL) was stirred at room temperature for 2 hours, and all volatiles were removed by distillation under the reduced pressure to give the title compound (17 mg) as a yellow oil. ESI m/z C ₄₁ H ₆₆ N ₅ O ₁₅ [M+H] ⁺ Calculated 868.45 and found 868.47.

EXAMPLE 160 Synthesis of Compound 466

Compound 465 (17mg, 0.0196mmol) and compound 41a (14mg, 0.0196mmol) were dissolved in DMA (3 mL). DIPEA (10. Mu.l, 0.0588 mmol) was added and stirred at room temperature for 3 hours, then the solvent was distilled off under reduced pressure, and the residue was purified by preparative HPLC (C18 column, acetonitrile/water 10-90%) to give 466 (15 mg, yield 64%) as a yellow oil. ESI m/z C ₆₆ H ₁₀₆ N ₉ O ₂₀ S[M+H] ⁺ Calculated 1376.72, found 1376.72.

Example 161 Synthesis of Compound 378

Compound 110 (0.30g, 0.76mmol), Z-L-Ala-OH (0.17g, 0.76mmol) and HATU (0.29g, 0.76mmol) were dissolved in dichloromethane (20 mL) to which triethylamine (110. Mu.L, 0.8 mmol) was added. Stirring overnight at room temperature, then distilling off the solvent under reduced pressure and passing the residue through SiO ₂ Column purification afforded the title compound 378 (0.43g, 95% yield). ESI m/z C ₃₂ H ₄₆ N ₃ O ₈ [M+H] ⁺ Calculated 600.32, found 600.32.

Example 162 Synthesis of Compound 379

In a hydrogenation reaction vessel, pd/C (0.10g, 33wt%) was added to a solution of compound 378 (0.3g, 0.5mmol) in methanol (10 mL). The mixture is heated at 1atm H ₂ After shaking overnight, the filtrate was filtered through celite and the filtrate was concentrated to give compound 379 (0.24 g) which was used in the next step without further purification. ESI m/z C ₂₄ H ₄₀ N ₃ O ₆ [M+H] ⁺ 466.28 calculated and 466.28 found.

Example 163 Synthesis of Compound 487

Sodium dihydrogen phosphate (0.1m, 4 mL) was added to a solution of compound 379 (233mg, 0.5mmol) and compound 461 (357mg, 0.5mmol) in ethanol (20 mL). The resulting solution was stirred at room temperature for 24 hours, all volatiles were removed by distillation under the reduced pressure, and the residue was purified by column chromatography (5-10% methanol in dichloromethane) to give a yellow oil (176mg, 33%). ESI m/z C ₅₃ H ₈₆ N ₅ O ₁₇ [M+H] ⁺ Calculated 1064.59 and found 1064.60.

EXAMPLE 164 Synthesis of Compound 488

Compound 487 (176mg, 0.166mmol) was dissolved in methanol (15 mL), and a palladium catalyst (10%, 80 mg) was added to the solution, which was hydrogenated at room temperature (1 atm) for 3 hours, after which the catalyst was filtered off, and the filtrate was concentrated by distillation under reduced pressure to give the title compound (154 mg) as a yellow oil. ESI m/zC ₄₅ H ₈₀ N ₅ O ₁₅ [M+H] ⁺ Calculated 930.56 and found 930.56.

EXAMPLE 165 Synthesis of Compound 489

Sodium dihydrogen phosphate (0.1M, 4 mL) was added to a solution of compound 488 (154mg, 0.166mmol) and compound 125 (93mg, 0.332mmol) in ethanol (20 mL). The resulting solution was stirred at room temperature for 24 hours, all volatiles were removed by distillation under the reduced pressure, and the residue was purified by column chromatography on silica gel (5-10% methanol/dichloromethane) to give a yellow oil (117mg, 64%). ESI m/zC ₅₃ H ₈₇ N ₆ O ₁₈ [M+H] ⁺ Calculated 1095.60 and found 1095.61.

EXAMPLE 166 Synthesis of Compound 490

Compound 489 (39mg, 0.0356mmol) was dissolved in dichloromethane (3 mL) and TFA (3 mL) was stirred at room temperature for 2 hours, and all volatiles were removed by distillation under the reduced pressure to give the title compound (33 mg) as a yellow oil. ESI m/z C ₄₄ H ₇₁ N ₆ O ₁₆ [M+H] ⁺ Calculated 939.48 and found 939.49.

EXAMPLE 167 Synthesis of Compound 491

Compound 490 (33mg, 0.0356mmol) and compound 41a (25mg, 0.0356mmol) were dissolved in DMA (3 mL), DIPEA (15mg, 0.116mmol) was added thereto, stirred at room temperature for 3 hours, the reaction solvent was removed by distillation under reduced pressure, and the residue was purified by preparative HPLC (C18 column, acetonitrile/water 10-90%) to give compound 491 (17mg, 33%) as a yellow oil. ESI m/zC ₆₉ H ₁₁₁ N ₁₀ O ₂₁ S[M+H] ⁺ Calculated 1447.76 and found 1448.78.

EXAMPLE 168 Synthesis of Compound 494

The compound 2- (dimethylamino) acetic acid (0.60g, 4.30mmol) and HATU (1.08g, 2.86mmol) were dissolved in DMF (2 mL), TEA (1mL, 7.16mmol) was added and 1H stirred at room temperature, and a solution of Z-L-LyS-OH (0.80g, 2.86mmol) in DMF (2 mL) was added. The reaction solution was stirred at room temperature for 2H, and then concentrated under reduced pressure. The crude product is purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water 10-90%) to give 494 (0.50g, 50%) as a colorless oil. MS ESI m/z C ₁₈ H ₂₈ N ₃ O ₅ [M+H] ⁺ Calculated values: 366.20, found 366.20.

Example 169 Synthesis of Compound 495

Compound 494 (0.50g, 1.37mmol) was dissolved in methylene chloride (15 mL), and pentafluorophenol (0.38g, 2.05mmol) and EDCI (0.52g, 2.74mmol) were added thereto. After stirring overnight at room temperature, the reaction was filtered through celite and the filter cake was washed with dichloromethane. Concentrating the filtrate to obtain pentafluorophenol ester, and dissolving the pentafluorophenol ester in the filtrate 10mL of methylene chloride. Compound 387 (0.44g, 0.91mmol) and DIPEA (0.32mL, 1.82mmol) were added to the above solution, stirred at room temperature for 2 hours, and then concentrated. The crude product was purified on a silica gel column eluting with a methanol/dichloromethane gradient to give compound 495 (1.02 g, theoretical yield). MS ESI m/zC ₄₃ H ₆₇ N ₆ O ₁₀ [M+H] ⁺ Calculated values: 827.48, found 827.48.

Example 170 Synthesis of Compound 496

Compound 495 (1.02g, 1.23mmol) was dissolved in methanol (10 mL), palladium catalyst (10%, 100 mg) was added, and the solution was taken up in H ₂ Stirred under atmosphere (1 atm) at room temperature overnight. The catalyst was removed by vacuum filtration and the reaction was concentrated to give 496 as a yellow oil (0.76g, 89%). MS ESI m/z C ₃₅ H ₆₁ N ₆ O ₈ [M+H] ⁺ Calculated values: 693.45, found 693.45.

EXAMPLE 171 Synthesis of Compound 497

Compound 496 (0.25g, 0.36mmol) and compound 125 (0.15g, 0.54mmol) were dissolved in ethanol (5 mL), and sodium dihydrogenphosphate (0.1M, 1mL) was added, followed by stirring at room temperature overnight. The reaction was concentrated in vacuo and the crude product was purified by column chromatography (5% to 10% methanol in dichloromethane) to afford compound 497 (0.15g, 48%). MS ESI m/z C ₄₃ H ₆₈ N ₇ O ₁₁ [M+H] ⁺ Calculated values are: 858.49, found: 858.49.

EXAMPLE 172 Synthesis of Compound 498

Compound 497 (0.15g, 0.175mmol) was dissolved The solution was stirred in dichloromethane (1 mL) and trifluoroacetic acid (2 mL) at room temperature for two hours and concentrated in vacuo to give a yellow oil which was dissolved in DMA (2 mL) to which pentafluorophenyl ester 41a (121.1mg, 0.175mmol) was added followed by DIPEA (91. Mu.L, 0.525 mmol). The reaction was stirred at room temperature overnight, then concentrated under reduced pressure and the crude product was purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water 10-90%) to afford compound 498 (30.7mg, 14%). MS ESI m/z C ₅₉ H ₉₂ N ₁₁ O ₁₄ S[M+H] ⁺ Calculated values: 1210.65, found 1210.62.

EXAMPLE 173 Synthesis of Compound 501

H-LyS-OH (0.31g, 2.14mmol) was dissolved in ethanol (20 mL), compound 125 (1.80g, 6.42mmol) and 0.5M disodium hydrogenphosphate (4 mL) were added thereto, and the reaction solution was stirred at room temperature overnight. After concentration, the crude product is purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water 10-90%) to give 501 as a yellow oil (0.26g, 26%). MS ESI m/z C ₂₂ H ₂₉ N ₄ O ₈ Calculated values: 477.19, found 477.19.

EXAMPLE 174 Synthesis of Compound 502

Compound 501 (0.26g, 0.55mmol) was dissolved in dichloromethane (10 mL), and NHS (0.095g, 0.825mmol) and EDCI (0.16g, 0.825mmol) were added. The reaction mixture was stirred at room temperature overnight, then concentrated, diluted with water (50 mL), and extracted with ethyl acetate (2X 20 mL). The organic layers were combined and Na ₂ SO ₄ Drying, filtration and concentration gave crude 502 (0.34 g) which was used directly in the next reaction. MS ESI m/z C ₂₆ H ₃₂ N ₅ O ₁₀ [M+H] ⁺ Calculated values: 574.21, found 574.21.

Example 175 Synthesis of Compound 503

Compound 438 (0.19g, 0.4mmol) was dissolved in ethanol (30 mL), and compound 502 (0.34g, 0.6mmol) and 0.1M sodium dihydrogenphosphate (6 mL) were added. The reaction mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was diluted with water (100 mL) and extracted with ethyl acetate (2X 40 mL). The organic layers were combined, na ₂ SO ₄ Drying and purification on a silica gel column (gradient dichloromethane/methanol) afforded compound 503 (0.115g, 31%). MS ESI m/z C ₄₇ H ₆₈ N ₇ O ₁₃ [M+H] ⁺ Calculated values: 938.48, found 938.49.

EXAMPLE 176 Synthesis of Compound 504

Compound 503 (0.115g, 0.12mmol) was dissolved in dichloromethane (1 mL) and 2mL of TFA was added. The reaction mixture is stirred at room temperature for 2h and concentrated under reduced pressure and the crude product is purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water 10-90%) to give yellow oil 504 (0.0312g, 33%). MS ESI m/z C ₂₂ H ₂₉ N ₄ O ₈ [M+H] ⁺ Calculated values: 477.19, found: 477.19.

EXAMPLE 177 Synthesis of Compound 505

Compound 504 (31.2 mg, 0.04mmol) was dissolved in DMA (2 mL), to which pentafluorophenyl ester 41a (27mg, 0.04mmol) was added, followed by DIPEA (16. Mu.L, 0.08 mmol). The reaction was stirred at room temperature overnight, concentrated under reduced pressure, and purified by preparative liquid chromatography (10-90% acetonitrile/water) to give compound 505 (11.9mg, 24%). MS ESI m/z: c ₆₃ H ₉₂ N ₁₁ O ₁₆ S[M+H] ⁺ Calculated values: 1290.64, found: 1290.64.

EXAMPLE 178 Synthesis of Compound 508

Tert-butyl 3- (2- (2- (dibenzylamino) ethoxy) propionate (5g, 12.1mmol) was dissolved in dichloromethane (10 mL) and 5mL of trifluoroacetic acid was added. The reaction mixture was stirred at room temperature for 1 hour, then concentrated. The crude product was dissolved in dichloromethane (50 ml) and NHS (4.25g, 37mmol) and EDCI (7.10g, 37mmol) were added successively. The reaction mixture was stirred at rt overnight, then concentrated and purified on silica gel (dichloromethane/methanol gradient elution) to afford 508 as a yellow oil (5g, 91%). MS ESI m/z C ₂₅ H ₃₁ N ₂ O ₆ [M+H] ⁺ Calculated values: 455.21, found 455.21.

EXAMPLE 179 Synthesis of Compound 509

Compound 110 (1g, 2.5 mmol) was dissolved in ethanol (10 mL), and compound 508 (1.80g, 3.9mmol) and 0.1M sodium dihydrogenphosphate (2 mL) were added, reacted overnight at room temperature, and then concentrated. The residue was diluted with water (100 mL) and extracted with ethyl acetate (3X 50 mL). Combine the organic layers with Na ₂ SO ₄ Drying, filtration, concentration and purification on silica gel (gradient elution with dichloromethane/methanol system) gave 509 (0.93g, 50%) as a yellow oil. MS ESI m/z C ₄₂ H ₆₀ N ₃ O ₈ [M+H] ⁺ Calculated values are: 734.43, found 734.43.

EXAMPLE 180 Synthesis of Compound 510

Will combine withThe substance 509 (0.93g, 1.27mmol), ethyl acetate (20 mL) and Pd/C (0.093g, 10wt%) were added to a hydrogenation bottle, stirred overnight under hydrogen (1 atm), and then the solid was filtered through celite. The filtrate was concentrated to give compound 510 (0.57g, 81%) without further purification. MS ESI m/z C ₂₈ H ₄₈ N ₃ O ₈ [M+H] ⁺ Calculated values: 554.34, found 554.34.

Example 181 Synthesis of Compound 511

Compound 510 (0.25g, 0.45mmol) was dissolved in ethanol (5 mL), compound 502 (0.39g, 0.68mmol) and sodium dihydrogenphosphate (0.1M, 1mL) were added at room temperature, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo, diluted with water (100 mL) and extracted with ethyl acetate (2X 50 mL). The organic layers were combined and Na ₂ SO ₄ Drying, filtration, concentration and purification on silica gel (methanol/dichloromethane gradient elution) gave 511 as a yellow oil (0.076g, 17%). MS ESI m/z C ₅₀ H ₇₄ N ₇ O ₁₅ [M+H] ⁺ Calculated values are: 1012.52, found: 1012.53

Example 182 Synthesis of Compound 512

Compound 511 (0.076 g, 75mmol) was dissolved in dichloromethane (2 mL), 4mL of TFA was added and the mixture was stirred at room temperature for 1 hour, concentrated to give crude 512 which was used directly in the next reaction. MS ESI m/z C ₄₁ H ₅₈ N ₇ O ₁₃ [M+H] ⁺ Calculated values: 856.40, found 856.40.

EXAMPLE 183 Synthesis of Compound 513

Compound 512 was dissolved in DMA (2 mL) and compound 41a (33mg, 0.048 mmol) and DIPEA (25. Mu.L, 0.144 mmol) were added. The reaction was stirred at room temperature for 3 hours, then concentrated and purified by preparative HPLC (acetonitrile/water gradient elution) to give 513 as a yellow oil (21.3 mg, 32%). MS ESI m/z C ₆₆ H ₉₈ N ₁₁ O ₁₈ S[M+H] ⁺ Calculated values: 1364.67, found 1364.67.

EXAMPLE 184 Synthesis of Compound 515

Compound 386 (1g, 1.63mmol) was dissolved in dichloromethane (1 mL), 2mL TFA was added to the reaction mixture, the reaction mixture was stirred at room temperature for 1 hour, then concentrated to give crude 515 which was used directly in the next reaction. MS ESI m/z C ₂₄ H ₃₂ N ₃ O ₆ [M+H] ⁺ Calculated values: 458.22, found: 458.22.

EXAMPLE 185 Synthesis of Compound 516

Compound 515 was dissolved in DMF (3 mL), and pentafluorophenyl ester 41a (0.63g, 0.91mmol) and DIPEA (0.46mL, 2.73mmol) were added thereto. The reaction was stirred at room temperature overnight, then concentrated and purified on a silica gel column (dichloromethane/methanol gradient elution) to give 516 (1.75 g) as a yellow oil. MS ESI m/z C ₄₉ H ₇₂ N ₇ O ₁₁ S[M+H] ⁺ Calculated values: 966.49, found 966.49.

EXAMPLE 186 Synthesis of Compound 517

Compound 516 (0.20g, 0.20mmol), methanol (15 mL) and Pd/C (0.02g, 10wt%) were added Into a hydrogenation flask, and then the pH was adjusted to around 4 with 1N HCl. At H ₂ (1 atm) overnight, filtered through celite, and the filtrate was concentrated to give compound 517, which was used directly in the next reaction. MS ESI m/z C ₄₁ H ₆₆ N ₇ O ₉ S[M+H] ⁺ Calculated values are: 832.46, found 832.46.

Example 187 Synthesis of Compound 519

The compound H-Dap (Boc) -OH (1g, 4.9mmol) and saturated NaHCO ₃ The solutions (20 mL) were mixed and compound 418 (2.30g, 14.7 mmol) was added at 0 ℃. The reaction was stirred at 0 ℃ for 1h, then warmed to room temperature and stirred for 1h. Potassium hydrogen sulfate (1M) was added to adjust the pH to about 6, and the resultant mixture was extracted with ethyl acetate (2X 50 ml). The organic layers were combined and Na ₂ SO ₄ Drying, filtration and concentration gave compound 519 (0.42g, 30%). MS ESI m/z C ₁₂ H ₁₅ N ₂ O ₆ [M-H] ^- Calculated values are: 283.10, found 283.10.

EXAMPLE 188 Synthesis of Compound 520

Compound 519 (0.21g, 0.74mmol) was dissolved in ethyl acetate (10 mL), followed by addition of pentafluorophenol (0.27g, 1.48mmol) and DCC (0.30g, 1.48mmol). The reaction mixture was stirred at room temperature overnight, then filtered and the filter cake was washed with EtOAC. The filtrate was concentrated to give the activated ester (0.17g, 0.37mmol), dissolved in 1mL of DMF, to which was added compound 517 (0.36g, 0.43mmol) and DIPEA (0.13mL, 0.74mmol), the reaction mixture was stirred at room temperature for 2 hours, concentrated and purified by preparative HPLC (gradient acetonitrile/water) to give compound 520 (50mg, 13%). MS ESI m/z C ₅₃ H ₈₀ N ₉ O ₁₄ S[M+H] ⁺ Calculated values: 1098.55, and 1098.55.

EXAMPLE 189 Synthesis of Compound 521

Compound 520 (50mg, 0.046 mmol) was dissolved in 0.5mL of dichloromethane, added 1mL of trifluoroacetic acid and reacted at room temperature for 1 hour, concentrated and purified by preparative HPLC (mobile phase acetonitrile/water) to give product 521 (11mg, 25% yield). MS ESI m/z C ₄₈ H ₇₂ N ₉ O ₁₂ S[M+H] ⁺ Calculated value of 998.49, found 998.49.

EXAMPLE 190 Synthesis of Compound 523

Compound 509 (1.00g, 1.36mmol) was dissolved in 2mL of dichloromethane, and 4mL of trifluoroacetic acid was added thereto, followed by reaction at room temperature for 1 hour after completion of the addition. Concentration gave compound 523.MS ESI m/zC ₃₃ H ₄₄ N ₃ O ₆ [M+H] ⁺ Calculated 578.32, found 578.32.

EXAMPLE 191 Synthesis of Compound 524

Compound 523 was dissolved in 5mL of DMF, and compound 41a (0.78g, 1.13mmol) and DIPEA (0.8mL, 4.52mmol) were added thereto. After addition was complete, the mixture was stirred at room temperature overnight, concentrated and purified by silica gel column (mobile phase MeOH/DCM) to provide product 524 (1.64g, 100% yield). MS MS ESI m/z C ₅₈ H ₈₄ N ₇ O ₁₁ S[M+H] ⁺ Calculated value 1086.59 and found value 1086.58.

EXAMPLE 192 Synthesis of Compound 525

Compound 524 (0.80g, 0.20mmol) was dissolved in 10mL of methanol, to which Pd/C (0.08g, 10wt%) was added, and the pH was adjusted to 4 with 1N HCl. After 3 times replacement with hydrogen, the mixture was stirred overnight. Celite was filtered and the filtrate was concentrated to give compound 525.MS ESI m/z C ₄₁ H ₆₆ N ₇ O ₉ S[M+H] ⁺ Calculated 832.46, found 832.46.

Example 193 Synthesis of Compound 527

H-Lys (Boc) -OH (1.00g, 3.8mmol) was dissolved in 16mL of ethanol, and thereto were added compound 125 (1.00g, 5.6mmol) and sodium dihydrogenphosphate (0.1M, 3mL), and after the addition, the mixture was stirred at room temperature overnight. The reaction was concentrated and purified on silica gel (mobile phase MeOH/DCM) to give product 527 (1.62g, 100% yield). MS ESI m/z C ₁₉ H ₃₀ N ₃ O ₇ [M+H] ⁺ Calcd 412.20, found 412.20.

EXAMPLE 194 Synthesis of Compound 528

Compound 527 (0.24g, 0.58mmol) was dissolved in 10mL ethyl acetate, and pentafluorophenol (0.21g, 1.17mmol) and DCC (0.24g, 1.17mmol) were added. After the addition was completed, the mixture was stirred at room temperature overnight, filtered and the filtrate was concentrated to give a product (32mg, 0.056 mmol), which was dissolved in DMF, to which was added compound 525 (50mg, 0.056 mmol) and DIPEA (29. Mu.L, 0.168 mmol). After addition was complete, the reaction was allowed to react at room temperature for 2 hours, concentrated and purified by preparative HPLC (mobile phase: acetonitrile/water) to give product 528 (3mg, 4% yield). MS ESI m/z C ₆₃ H ₉₉ N ₁₀ O ₁₇ S[M+H] ⁺ Calculated value of 1299.68, found 1299.68.

EXAMPLE 195 Synthesis of Compound 529

Compound 528 (3mg, 0.002mmol) was dissolved in 0.5mL DCM and 1mL trifluoroacetic acid was added. After addition was complete the reaction was allowed to react at room temperature for 1 hour, concentrated and purified by preparative HPLC (mobile phase: acetonitrile/water) to give the product 529 (1.43mg, 52% yield). MS ESI m/z C ₅₈ H ₉₁ N ₁₀ O ₁₅ S[M+H] ⁺ Calculated 1199.63, found 1199.62.

EXAMPLE 196 Synthesis of Compound 532

Pentafluorophenol ester of compound 527 (0.11g, 0.19mmol) was dissolved in 1mL DMF, and compound 517 (0.21g, 0.25mmol) and DIPEA (86uL, 0.5mmol) were added. After the addition was complete, the reaction was allowed to react at room temperature for 2 hours and concentrated, and purified by preparative HPLC (mobile phase: acetonitrile/water) to give product 532 (20mg, 9% yield). MS ESI m/z calculation C ₆₀ H ₉₃ N ₁₀ O ₁₅ S[M+H] ⁺ 1225.65, found 1225.66.

EXAMPLE 197 Synthesis of Compound 533

Compound 532 (20mg, 0.016mmol) was dissolved in 1mL of methylene chloride, and 2mL of trifluoroacetic acid was added thereto, followed by reaction at room temperature for 1 hour. The reaction was concentrated and purified by preparative HPLC (mobile phase: acetonitrile/water) to give compound 533 (8.9 mg,18% yield). MS ESI m/z C ₅₅ H ₈₅ N ₁₀ O ₁₃ S[M+H] ⁺ Calculated value of 1125.59, found value of 1125.59.

Example 198 Synthesis of Compound 536

Reacting compound 3- [ [ tert-butoxycarbonyl group]Amino group]L-alanine (1.00g, 7.3mmol) was dissolved in 30mL of ethanol, and compound 125 (2.00g, 7.3mmol) and 0.1M sodium dihydrogen phosphate (6 mL) were added. After addition was complete, the mixture was stirred overnight at room temperature, concentrated and purified by silica gel column (mobile phase: meOH/DCM) to give the product 536 (1.41g, 78% yield). MS ESI m/z C ₁₆ H ₂₄ N ₃ O ₇ [M+H] ⁺ Calculation 370.15, found 370.15.

Example 199 Synthesis of Compound 537

Compound 536 (1.41g, 3.8mmol) was dissolved in 2mL of methylene chloride, and reacted at room temperature for 1 hour after the addition of 5mL of trifluoroacetic acid. The reaction solution was concentrated to give a product 537.MS ESI m/z C ₁₁ H ₁₆ N ₃ O ₅ [M+H] ⁺ Calculated 270.10, found 270.10.

EXAMPLE 200 Synthesis of Compound 538

Compound 537 is dissolved in 20mL of ethanol, followed by addition of compound 125 (1.90g, 6.9mmol) and 0.1M sodium dihydrogen phosphate (4 mL), followed by stirring at room temperature overnight. The reaction was concentrated and purified by preparative HPLC (mobile phase: acetonitrile/water) to give the product 538 (0.45g, 22% yield). MS ESI m/z C ₁₉ H ₂₃ N ₄ O ₈ [M+H] ⁺ Calculated 435.14 and found 435.14.

EXAMPLE 201 Synthesis of Compound 539

Compound 538 (0.15g, 0.34mmol) and compound 387 (0.17g, 0.34mmol) were dissolved in DMF (2 mL), HATU (0.16g, 0.41mmol) and triethylamine (95. Mu.L, 0.68 mmol) were added thereto, and after completion of the addition, the reaction was carried out at room temperatureIt should be 1 hour. The reaction was concentrated and purified by preparative HPLC (mobile phase: acetonitrile/water) to give product 539 (34mg, 11% yield). MS ESI m/z C ₄₄ H ₆₂ N ₇ O ₁₃ [M+H] ⁺ Calcd for 896.43, found 896.42.

EXAMPLE 202 Synthesis of Compound 540

Compound 539 (34mg, 0.04mmol) was dissolved in 0.5mL of dichloromethane, and after 1mL of trifluoroacetic acid was added, it was reacted at room temperature for 2 hours. The reaction mixture was concentrated to obtain compound 540.MS ESI m/zC ₃₅ H ₄₆ N ₇ O ₁₁ [M+H] ⁺ Calculated 740.30, found 740.32.

Example 203 Synthesis of Compound 541

Compound 540 was dissolved in DMA (2 mL), to which was added compound 41a (28mg, 0.04mmol) and DIPEA (21. Mu.L, 0.12 mmol). After addition was complete, stirring was overnight, and after concentration purification by preparative HPLC, product 541 (14.4 mg,29% yield) was obtained. MS ESI m/z C ₆₀ H ₈₆ N ₁₁ O ₁₆ S[M+H] ⁺ Calculated value 1248.59 and found value 1248.60.

EXAMPLE 204 Synthesis of Compound 544

Compound 132 (0.300g, 0.329mmol) and N-BOC-ethylenediamine hydrochloride (0.063g, 0.395mmol) were dissolved in methylene chloride (30 mL), EDCI (0.189g, 0.988mmol) was added thereto at 0 ℃ and after 10 minutes, the mixture was warmed to room temperature and stirred overnight. The reaction was diluted with dichloromethane, washed with water, brine, dried over sodium sulfate, concentrated and purified with silica gel column to give the product (0.132g, 54% yield). MS ESI m-z C ₅₂ H ₈₀ N ₉ O ₁₂ S[M+H] ⁺ Calculated value 1054.6, found 1054.6.

EXAMPLE 205 Synthesis of Compound 545

Compound 544 (0.132g, 0.125mmol) was dissolved in dichloromethane (45 mL), and 1.5mL of trifluoroacetic acid was added to react at room temperature for 1 hour. The reaction was concentrated and purified by preparative HPLC to give 545 (111mg, 93% yield). MS ESI m/z C ₄₇ H ₇₂ N ₉ O ₁₀ S[M+H] ⁺ 954.5 for calcd value, 954.5 for found value.

EXAMPLE 206 Synthesis of Compound 548

Compound 132 (0.050g, 0.0549mmol) and N-Boc-1, 11-diamino-3, 6, 9-trioxaundecane (0.024g, 0.0549mmol) were dissolved in dichloromethane (10 mL), EDCI (0.032g, 0.1647 mmol) was added thereto at 0 deg.C, and after 10 minutes of reaction, it was allowed to warm to room temperature and stirred overnight. The reaction solution was diluted with dichloromethane, washed with water, washed with brine, dried over sodium sulfate, filtered, concentrated and purified by silica gel column to give product 548 (0.030g, 46% yield). MS ESI m/z C ₅₈ H ₉₂ N ₉ O ₁₅ S[M+H] ⁺ Calculated value 1186.6, found 1186.6.

EXAMPLE 207 Synthesis of Compound 549

Compound 548 (0.030g, 0.0253mmol) was dissolved in 3mL of dichloromethane, and 1mL of trifluoroacetic acid was added to react at room temperature for 1 hour. Concentration gave a yellow oily liquid which was purified by preparative HPLC to give product 549 (11.7mg, 43% yield). MS ESI m/z C ₅₃ H ₈₄ N ₉ O ₁₃ S[M+H] ⁺ Calculated value is 1086.6, found value is 1086.6.

Example 208 Synthesis of Compound 552

The compounds diethyltriamine (28.7g, 275mmol) and DMAP (0.034g, 0.000275mmol) were dissolved in dichloromethane (350 mL) and Boc was added at 0 deg.C ₂ O (6.0 g, 0.0275mmol). After the addition was complete, the reaction was stirred at room temperature overnight, concentrated, and purified with silica gel column to give the product (45g, 80% yield). MS ESI m/z C ₉ H ₂₂ N ₃ O ₂ [M+H] ⁺ The calculated value is 204.2, and the measured value is 204.2.

EXAMPLE 209 Synthesis of Compound 553

Compound 132 (0.060g, 0.0658mmol) and N-Boc-2,2' -iminodiethylamine (0.016g, 0.0790mmol) were dissolved in dichloromethane (6 mL), EDCI (0.038g, 0.1974mmol) was added at 0 ℃ and after completion of the addition, the mixture was warmed to room temperature and stirred overnight. The reaction was concentrated and purified by preparative HPLC to give the product 553 (48mg, 66% yield). MS ESI m/z C ₅₄ H ₈₅ N ₁₀ O ₁₂ S[M+H] ⁺ Calculated value of 1097.6, found 1097.6.

EXAMPLE 210 Synthesis of Compound 554

Compound 553 (0.048 g, 0.0437mmol) was dissolved in 3mL of dichloromethane, and after 1mL of trifluoroacetic acid was added, the reaction was carried out at room temperature for 1 hour. The reaction was concentrated and purified by preparative HPLC to give 554 (11.1mg, 25% yield). MS ESI m/z C ₄₉ H ₇₇ N ₁₀ O ₁₀ S[M+H] ⁺ The calculated value is 997.5, and the actual value is 997.5.

EXAMPLE 211 Synthesis of Compound 558

Compound 132 (0.400g, 0.439mmol) and H-Lys (Boc) -O ^t Bu.HCl (0.135g, 0.528mmol) was dissolved in methylene chloride (40 mL), EDCI (0.189g, 1.317mmol) was added at 0 ℃ and after completion of the addition, the mixture was warmed to room temperature and stirred overnight. The reaction was diluted with dichloromethane, washed with water, brine, dried over sodium sulfate, filtered, concentrated and purified over silica gel column to give 558 (0.43g, 82% yield). MS ESI m/z C ₆₀ H ₉₄ N ₉ O ₁₄ S[M+H] ⁺ Calculated 1196.7 and found 1196.7.

Example 212 Synthesis of Compound 559

Compound 558 (0.230g, 0.192mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (2 mL) was added, the reaction was allowed to react at room temperature for 3 hours, concentrated, and purified by preparative HPLC (C) ₁₈ Column, mobile phase a: water, mobile phase B: acetonitrile; from 10% B to 80% B) over 60 min yielded 559 (153mg, 76% yield). MS ESI m/z C ₅₁ H ₇₈ N ₉ O ₁₂ S[M+H] ⁺ The calculated value was 1040.5, and the found value was 1040.5.

Example 213 Synthesis of Compound 562

Compound 558 (0.200g, 0.167mmol) and (S) -2, 6-di-tert-butoxycarbonylaminocaproic acid (0.070g, 0.200mmol) were dissolved in dichloromethane (10 mL), and HATU (0.095g, 0.250mmol) and triethylamine (46. Mu.L, 0.334 mmol) were added at 0 ℃. Heating the reaction solution to room temperature, stirring overnight, diluting with dichloromethane, washing with water, washing with brine, drying with sodium sulfate, filtering, concentrating, and purifying with silica gel column to obtainProduct 562 (0.270 g, theoretical yield). MS ESI m/z C ₇₆ H ₁₂₂ N ₁₁ O ₁₉ S[M+H] ⁺ Calculated value is 1524.9, found value is 1524.9.

EXAMPLE 214 Synthesis of Compound 563

Compound 562 (0.27g, 0.177mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (2 mL) was added and the reaction was carried out at room temperature for 4 hours. The reaction mixture was concentrated and purified by preparative HPLC (C) ₁₈ Column, mobile phase a: water, mobile phase B: acetonitrile; from 10% B to 80% B) over 60 min to give product 563 (172mg, 83% yield). MS ESI m/z C ₅₇ H ₉₀ N ₁₁ O ₁₃ S[M+H] ⁺ Calculated value 1168.6, found 1168.6.

EXAMPLE 215 Synthesis of Compound 566

Ethylenediamine (3.0 g,0.5 mmol) was dissolved in dichloromethane (500 mL), cooled to 0 deg.C, then CbzCl (8.53g, 0.050mol) was added, after which it was warmed to room temperature and stirred overnight. The reaction mixture was washed with water, brine, dried over sodium sulfate, filtered and concentrated to give N-benzyloxycarbonyl-1, 2-diaminoethane (7.0 g,94% yield). MS ESI m/z C ₁₀ H ₁₄ N ₂ O ₂ [M+H] ⁺ Calculated value 195.1, found value 195.2.

EXAMPLE 216 Synthesis of Compound 567

Compound 566 (7g, 35.8mmol) and 37% formaldehyde (14mL, 0.1772mmol) were dissolved in methanol (120 mL), sodium cyanoborohydride (3.9g, 0.062mol) was added at 0 deg.C, the pH was adjusted to 7 with acetic acid (3 mL), the mixture was allowed to warm to room temperature and stirred overnight. Dichloro for reaction liquidMethane (200 mL) was diluted, washed with water, brine, dried over sodium sulfate, filtered, concentrated and purified on silica gel column to give the product as a yellow oil (6.4 g,80% yield). MS ESI m/z C ₁₂ H ₁₈ N ₂ O ₂ [M+H] ⁺ Calculated 224.1 and found 224.1.

EXAMPLE 217 Synthesis of Compound 568

Compound 567 (3.0g, 13.4mmol) was dissolved in methanol (100 mL), and then Pd/C (0.3g, 10wt%) and HCl (3 mL) were added, followed by reaction under hydrogen at a pressure of 100psi for 5 hours after completion of the addition. The reaction was filtered through celite and concentrated to give 568 (2.1g, 98% yield). ¹ H NMR(400MHz,D ₂ O)δ3.33(d,J＝4.6Hz,2H),3.27(s,2H),2.79(s,6H)。

EXAMPLE 218 Synthesis of Compound 569

Compound 103 (0.58g, 1.58mmol) and compound 568 (0.051g, 3.15mmol) were dissolved in anhydrous DMF (10 mL) and HATU (0.09g, 2.37mmol) and triethylamine (0.656 mL, 4.74mmol) were added at 0 ℃. After the addition, the temperature was raised to room temperature and the reaction was carried out for 90 minutes. The reaction was diluted with water and extracted with ethyl acetate (3 × 100 mL), and the organic phases were combined, washed with water, brine, dried over sodium sulfate, filtered and concentrated to give a yellow solid (0.67g, 97% yield). MS ESI m/z C ₂₁ H ₃₅ N ₄ O ₆ [M+H] ⁺ Calculated value was 439.2, found value was 439.2.

Example 219 Synthesis of Compound 570

Pd/C (0.2g, 10wt%) was added to a solution of compound 569 (0.6g, 13.7mmol) in ethyl acetate (10 mL) at 100psiUnder hydrogen pressure for 4 hours. The reaction mixture was filtered through celite and concentrated to give the product (5.5g, 98% yield). MS ESI m/z C ₂₁ H ₃₇ N ₄ O ₆₄ [M+H] ⁺ The calculated value is 409.3, and the measured value is 409.3.

EXAMPLE 220 Synthesis of Compound 571

Compound 570 (0.5g, 1.22mmol) was dissolved in 95% ethanol (10 mL), and 0.1MNaH was added ₂ PO ₄ (2 mL) and compound 125 (0.683g, 2.44mmol), stirring overnight after the addition. The reaction was concentrated and purified with silica gel column (mobile phase: meOH/DCM) to give the product as a yellow oil (0.624g, 89% yield). MS ESI m/z calculation C ₂₉ H ₄₄ N ₅ O ₇ [M+H] ⁺ 574.3, found 574.3.

EXAMPLE 221 Synthesis of Compound 572

Compound 571 (0.2g, 0.349mmol) was dissolved in 6mL of dichloromethane, and after 2mL of trifluoroacetic acid was added, the reaction was carried out at room temperature for 2 hours. The reaction was concentrated to give the product (165 mg, theoretical yield). MS ESIm/z C ₂₄ H ₃₆ N ₅ O ₅ [M+H] ⁺ The calculated value is 474.3, and the measured value is 474.3.

EXAMPLE 222 Synthesis of Compound 573

Compound 572 (0.165g, 0.349mmol) was dissolved in 2mL of DMF, and a solution of compound 41a (0.29g, 1.047mmol) in DMF (3 mL) was added at 0 deg.C, and the mixture was warmed to room temperature and reacted for 1 hour. The reaction was concentrated and purified by preparative HPLC (C) ₁₈ Column, mobile phase a: water, mobile phase B: acetonitrile; 60 minutes from 10% of B to80% of B) gave the product (58mg, 17% yield). MS ESI m/z C ₄₉ H ₇₆ N ₉ O ₁₀ S[M+H] ⁺ The calculated value was 982.5, and the found value was 982.5.

EXAMPLE 223 Synthesis of Compound 576

2-Bromobutyric acid (3g, 17.9 mmol) was dissolved in tetrahydrofuran (30 mL), to which was added trimethylamine (1M in THF,17.9mL,35.9 mmol). After the addition was complete, stirring overnight, ethyl acetate was added and the solid collected by filtration to give the product 576 (4 g, theoretical yield). MS ESI m/z C ₇ H ₁₆ NO ₂ [M+H] ⁺ Calculated value 146, found value 146.

Example 224 Synthesis of Compound 577

Compound 576 (1.55g, 6.9mmol) and pentafluorophenol (2.5g, 13.8mmol) were dissolved in dichloromethane (20 mL), DCC (2.8g, 13.8mmol) was added, and after the addition, stirring was carried out overnight. The reaction was filtered and concentrated to give product 577 as an oil. MS ESI m/z C ₁₃ H ₁₅ F ₅ NO ₂ [M+H] ⁺ Calculated 312, found 312.

Example 225 Synthesis of Compound 578

Compound 16 (2g, 3.7 mmol) was dissolved in ethyl acetate (20 mL), pd/C (10 wt%,0.20 g) was added, and the mixture was purified by column chromatography on hydrogen ₂ (1 atm) was stirred at room temperature overnight and filtered to give product 17 (1.78g, 3.4 mmol). Compound 17 and compound 577 (6.9 mmol) were dissolved in DMF (20 mL), DIPEA (1.8mL, 10.4mmol) was added at 0 ℃ and after completion of the addition, the reaction was warmed to room temperature for 1 hour. The reaction was concentrated and purified with a silica gel column (1,product 578 was obtained (1.2g, 54% yield). MS ESIm/z C ₃₂ H ₆₁ N ₄ O ₅ SSi[M+H] ⁺ Calculated 642, found 642.

Example 226 Synthesis of Compound 579

Compound 578 (1.20g, 1.86mmol) was dissolved in AcOH/THF/H ₂ O (volume ratio 3. The reaction was concentrated to give a foamy solid (1.20g, 54% yield). MS ESI m/z C ₂₆ H ₄₇ N ₄ O ₅ S[M+H] ⁺ Calculated 527, found 527.

EXAMPLE 227 Synthesis of Compound 580

Compound 579 (1.86 mmol) was dissolved in 1, 4-dioxane (10 mL), to which 1N sodium hydroxide (9.3 mL) was added, and reacted at room temperature for 2 hours. The reaction solution was concentrated, adjusted to pH 4 with 1N hydrochloric acid, and concentrated again to give product 580 as a white solid. MS ESI m/z C ₂₄ H ₄₃ N ₄ O ₅ S[M+H] ⁺ Calculated value of 499 and found value of 499.

Example 228 Synthesis of Compound 581

Compound 580 (1.86 mmol) was dissolved in pyridine (10 mL), acetic anhydride (884. Mu.L, 9.36 mmol) was added at 0 ℃ and the reaction warmed to room temperature and stirred overnight. The reaction was diluted with water (20 mL), washed with ethyl acetate (3X 10 mL), and the aqueous phase was concentrated to give the product 581 as a yellow solid. MS ESI m/z C ₂₆ H ₄₅ N ₄ O ₆ S[M+H] ⁺ Calcd for 541, found for 541.

EXAMPLE 229 Synthesis of Compound 582

Compound 581 (150mg, 0.277mmol) and pentafluorophenol (76.5mg, 0.415mmol) were dissolved in dichloromethane (2 mL), EDCI (63.7mg, 0.33mmol) was added, and the reaction was carried out at room temperature for 3 hours. The reaction was concentrated to give yellow liquid 582.MS ESI m/z C ₃₂ H ₄₄ F ₅ N ₄ O ₆ S[M+H] ⁺ A calculated value of 707 and an observed value of 707.

EXAMPLE 230 Synthesis of Compound 583

Compound 127 (50mg, 0.10 mmol) and compound 582 (0.14 mmol) were dissolved in DMF (2 mL), and DIPEA (49. Mu.L, 0.28 mmol) was added at 0 ℃ and reacted at room temperature for 1 hour. The reaction was concentrated and purified by preparative HPLC to give 583 (30mg, 46% yield) as a white solid. MS ESI m/z C ₄₆ H ₆₈ N ₇ O ₁₁ S[M+H] ⁺ Calculated value: 926, found value: 926.

EXAMPLE 231 Synthesis of Compound 573

A suspension of betaine (870mg, 7.4mmol) in thionyl chloride (10 mL) was heated to 70 ℃ to react for 2 hours. The reaction was concentrated and azeotroped with toluene (3X 10 mL) to give 586 as a yellow solid.

Example 232 Synthesis of Compound 587

Compound 17 (1.90g, 3.71mmol) was dissolved in dichloromethane (20 mL), DIPEA (2.58mL, 14.8mmol) was added, the temperature was decreased to 0 ℃ and dropwise addedCompound 586 in dichloromethane (20 mL) was allowed to warm to room temperature for 1 hour after the addition was complete. The reaction was concentrated and purified with a silica gel column (1. MS ESI m/z C ₃₀ H ₅₇ N ₄ O ₅ SSi[M+H] ⁺ Calcd for 613, found for 613.

Example 233 Synthesis of Compound 588

Compound 587 (2.3g, 3.7mmol) was dissolved in AcOH/THF/H2O (volume ratio 3. The reaction mixture was concentrated to obtain compound 588.MS ESI m/z C ₂₄ H ₄₃ N ₄ O ₅ S[M+H] ⁺ Calculated value of 499 and found value of 499.

Example 234 Synthesis of Compound 589

Compound 588 (3.7 mmol) was dissolved in 1, 4-dioxane (20 mL), to which was added 1N sodium hydroxide (18.5 mL), and reacted at room temperature for 2 hours. After the reaction solution was concentrated, 1N hydrochloric acid was added to adjust pH to 4, and the mixture was concentrated again to obtain 589 (1.00g, 57% yield) as a white solid. MS ESI m/z C ₂₂ H ₃₉ N ₄ O ₅ S[M+H] ⁺ Calculated value 471, found value 471.

Example 235 Synthesis of Compound 590

Compound 589 (1.00g, 2.12mmol) was dissolved in pyridine (10 mL), acetic anhydride (1mL, 10.6mmol) was added at 0 ℃ and the reaction was warmed to room temperature and stirred overnight. The reaction was diluted with 20mL of water, washed with ethyl acetate (3X 10 mL), and the aqueous phase was concentrated to give the product 590 as a yellow solid. MS ESI m/z C ₂₄ H ₄₁ N ₄ O ₆ S[M+H] ⁺ Calculated 513 and found 513.

EXAMPLE 236 Synthesis of Compound 591

Compound 590 (70mg, 0.136mmol) and pentafluorophenol (30mg, 0.163mmol) were dissolved in dichloromethane (2 mL), DCC (33.7mg, 0.163mmol) was added, and the reaction was allowed to proceed for 3 hours after the addition. The reaction was concentrated to give product 591 as a yellow oil which was used directly in the next reaction. MS ESI m/z C ₃₀ H ₄₀ F ₅ N ₄ O ₆ S[M+H] ⁺ Calculated value 679, found value 679.

EXAMPLE 237 Synthesis of Compound 592

Compound 591 (0.136 mmol) and compound 127 (0.11g, 0.273mmol) were dissolved in DMF (2 mL) and DIPEA (71. Mu.L, 0.408 mmol) was added at 0 ℃. The reaction was allowed to warm to room temperature, concentrated after 1 hour and purified by preparative HPLC to give the product as a yellow solid (30.9mg, 25% yield). MS ESI m/z C ₄₄ H ₆₄ N ₇ O ₁₁ S[M+H] ⁺ Calculated value 899, found value 899.

EXAMPLE 238 Synthesis of Compound 603

The compound N' - (2-aminoethyl) ethyl-1, 2-diamine (4.2 g, 41mmol) was dissolved in 50mL of tetrahydrofuran, cooled to 0 deg.C, and a solution of 2- (tert-butoxycarbonyloxyimino) -2-phenylacetonitrile (20.4 g, 82mmol) in tetrahydrofuran (50 mL) was slowly added, after which the reaction was continued at 0 deg.C for 1 hour. After concentrating the reaction solution, it was purified by silica gel column (0-10% MeOH/DCM), giving a colorless oil (12g, 96% yield). HRMS (ESI) m/z C ₁₄ H ₂₉ N ₃ O ₄ [M+H] ⁺ Calculated value 304.22, found value 304.22.

Example 239 Synthesis of Compound 604

Compound 603 (5g, 16.4 mmol) was dissolved in dichloromethane (50 mL), cooled to 0 ℃ and succinic anhydride (1.64g, 16.4 mmol) was added, and the reaction was allowed to warm to room temperature for 1 hour. The reaction was concentrated and purified by silica gel column (0-10% MeOH/DCM) to give the product (5.4 g,81% yield). HRMS (ESI) m/z C ₁₈ H ₃₃ N ₃ O ₇ [M+H] ⁺ Calculated value of 404.23 and found value of 404.23.

EXAMPLE 240 Synthesis of Compound 605

Compound 604 (5.4 g,13.3 mmol) was dissolved in ethyl acetate (30 mL), concentrated hydrochloric acid (10 mL) was added at 0 deg.C, and the reaction stirred vigorously for 30 minutes with the ice bath removed. The ethyl acetate and hydrochloric acid were distilled off under reduced pressure to give the product (3.69g, 100% yield). HRMS (ESI) m/z C ₈ H ₁₇ N ₃ O ₃ [M+H] ⁺ The calculated value is 204.13, and the measured value is 204.13.

EXAMPLE 241 Synthesis of Compound 606

Dissolving compound 605 (0.5g, 1.81mmol) in 5mL of water, adding 2N aqueous sodium hydroxide solution, adjusting pH to 7, concentrating to dryness, adding 10mL of saturated sodium bicarbonate solution, cooling to about 5 ℃, adding N-methoxycarbonylmaleimide (0.56g, 3.62mmol), and reacting at 5 ℃ for 2 hours after the addition. The pH was adjusted to 5 with 2N HCl, the aqueous phase was extracted with dichloromethane (3X 10 mL), the organic phases were combined and dried over sodium sulfate, filtered, concentrated and purified by preparative HPLC to give a white solid (200mg, 30% yield). HRMS (ESI) m/z C ₁₆ H ₁₇ N ₃ O ₇ [M+H] ⁺ The calculated value is 364.11, and the measured value is 364.11.

EXAMPLE 242 Synthesis of Compound 608

Compound 606 (37mg, 0.102mmol) was dissolved in DMF (2 mL), HATU (39mg, 0.102mmol) was added at 0 ℃ and allowed to warm to room temperature for 1 hour, and the above solution was added to a solution of compound 440 (100mg, 0.093 mmol) and triethylamine (25.8. Mu.L, 0.186 mmol) in DMF (2 mL) and allowed to react at room temperature for 1 hour. After concentration to remove DMF, purification on silica gel column gave the product (99mg, 75% yield). HRMS (ESI) m/z C ₆₈ H ₁₀₈ N ₈ O ₂₄ [M+H] ⁺ Calculated value 1421.75, found 1421.75.

EXAMPLE 243 Synthesis of Compound 609

Compound 608 (50mg, 0.035mmol) was dissolved in methylene chloride (3 mL), added with TFA (1 mL), and reacted at room temperature for 2 hours. Concentration gave the product (44mg, 100% yield). HRMS (ESI) m/z C ₅₉ H ₉₂ N ₈ O ₂₂ [M+H] ⁺ Calculated 1265.63 and found 1265.63.

EXAMPLE 244 Synthesis of Compound 670

Compound 609 (20mg, 0.0158mmol) and compound 41a (10.9mg, 0.0158mmol) were dissolved in DMA (2 mL), DIPEA (5.5. Mu.L, 0.0316 mmol) was added at 0 ℃ and the reaction was allowed to warm to room temperature for 1 hour. After concentration to remove DMA, purification by preparative HPLC afforded the product (11mg, 40% yield). HRMS (ESI) m/z C ₈₄ H ₁₃₂ N ₁₂ O ₂₇ S[M+H] ⁺ Calculated value of 1773.90 and found value of 1773.90.

Example 245 Synthesis of Compound 672

Tert-butyl 4-aminobutyrate (1.03g, 6.12mmol) and compound 436 (4.16g, 5.56mmol) were dissolved in DMF (18 mL), HATU (2.32g, 6.12mmol) and TEA (1.2mL, 8.34mmol) were added at 0 deg.C, after stirring for 50 minutes, water (300 mL) was added, extraction was performed with ethyl acetate (250 mL. Times.3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column-purified (3. Mu. MeOH/DCM) to give compound 672 (5.98 g). [ M + H ]] ⁺ Calcd for 890.51, found 891.09.

EXAMPLE 246 Synthesis of Compound 673

Compound 672 (5.98g, 6.73mmol) was dissolved in methanol (30 mL), pd/C (10 wt%,0.6 g) was added, and the mixture was stirred under 1atm of hydrogen at room temperature overnight. Filtration and concentration gave 673 (4.57 g). ESI m/z C ₃₅ H ₇₀ N ₃ O ₁₄ [M+H] ⁺ Calculated value of 756.48, found 756.47.

EXAMPLE 247 Synthesis of Compound 674

Compound 673 (1g, 1.32mmol) is dissolved in NaHCO ₃ To a saturated solution (20 mL), compound 418 (0.4 g, 2.64mmol) was added under ice-cooling, and the reaction was stirred for 30 minutes under ice-cooling. The reaction mixture was poured into a separatory funnel containing 100mL of ethyl acetate, the organic phase was collected by separation, washed once with 50mL of water and 50mL of brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 674 (0.8 g, 72% yield). ESI m/z C ₃₉ H ₇₀ N ₃ O ₁₆ [M+H] ⁺ 836.47, found 836.47

EXAMPLE 248 Synthesis of Compound 675

After compound 674 (0.40g, 0.48mmol) was dissolved in DCM (4.5 mL), TFA (1.5 mL) was added, after stirring at room temperature for 1 h, concentrated to dryness and taken up twice with dichloromethane, finally dried on an oil pump, the residue obtained was taken up with compound 110 (0.35g, 0.48mmol) in DMF (10 mL), HATU (0.36g, 0.96mmol), TEA (0.15mL, 1.44mmol) was added under ice bath and stirred for 30 min. The reaction mixture was poured into a separatory funnel containing 100mL of water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and passed through a silica gel column (0-10% MeOH/DCM) to give compound 675 (21mg, 2.1%). ESI m/z C ₉₁ H ₁₅₃ N ₈ O ₃₅ [M+H] ⁺ Calculated 1919.04 and found 1919.04.

Example 249. Synthesis of Compound 676

After compound 675 (21mg, 0.01mmol) was dissolved in DCM (0.5 mL), TFA (1 mL) was added, and after stirring for 1 hour at RT, concentration spin-dried and taken up twice with dichloromethane, and finally placed on an oil pump to be sucked dry, the resulting compound was dissolved with compound 41a (6.93g, 0.01mmol) in DMA (2 mL), followed by DIPEA (17. Mu.L, 0.1 mmol) and stirred for 30 minutes at RT. Directly loading the reaction solution to preparative HPLC, wherein the elution solvent is MeCN/H ₂ O, purified to give product 676 (10mg, 44%). ESI m/z C ₁₀₇ H ₁₇₇ N ₁₂ O ₃₈ S[M+H] ⁺ Calculated 2271.20, found 2271.20.

Example 250 Synthesis of Compound 678

NaH (3.1g, 129.15mmol) was dissolved in DMF (50 mL) under nitrogen protection, a solution of di-t-butyl azidooxalic acid (10g, 43.05mmol) in DMF (30 mL) was added thereto, and after stirring at room temperature for 20 minutes, a solution of t-butyl bromoacetate (28mL, 172.2mmol) in DMF (20 mL) was added under ice-bath, and after stirring for 20 minutes, the mixture was gradually warmed to room temperature, and stirred for 1 hour. Pouring the reaction solution into NH ₄ Cl (aq) (400 mL), extracted with ethyl acetate (300 mL), the combined organic phases were washed with water (100 mL. Times.3), saturated brine (100 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated. The concentrated solution was purified by silica gel column eluting with petroleum ether/ethyl acetate to obtain compound 678 (16.5g, 83%). ESI m/z calculated value C ₂₂ H ₄₁ N ₂ O ₈ [M+H] ⁺ 461.28, found 461.27.

EXAMPLE 251 Synthesis of Compound 679

Compound 678 (10g, 21.7mmol) was dissolved in TFA (50 mL), stirred at room temperature overnight, concentrated, and taken up twice with dichloromethane, the resulting solid was washed with dichloromethane until the solid became white, and finally the collected solid was put on an oil pump and pumped to dryness to give compound 679 (3g, 94%). ESI m/z C ₄ H ₉ N ₂ O ₄ [M+H] ⁺ Calculated 149.05, found 148.06.

Example 252 Synthesis of Compound 680

Compound 679 (0.56g, 3.8 mmol) was dissolved in THF (20 mL), chloroacetyl chloride (3 mL, 38mmol) was added under ice bath, and after stirring for 30 minutes, it was slowly warmed to room temperature and stirred overnight. Concentrating the reaction solution to dryness, and drying with toluene for three timesFinally, the mixture was pumped to dryness using an oil pump to obtain 680 (1.16g, 100%). ESI m/z C ₈ H ₁₁ Cl ₂ N ₂ O ₆ [M+H] ⁺ Calculated value 300.99, found value 301.00.

EXAMPLE 253 Synthesis of Compound 681

The compound L-alanine tert-butyl ester (2.6 g, 14.35mmol), L-Z-Gly-O ^t Bu (3 g, 14.35mmol) was dissolved in DMF (15 mL), HATU (6 g, 15.78mmol) and TEA (4.1 mL,28.7 mmol) were added under ice-bath, stirred for 5 min and then warmed to room temperature and stirred for 1h. The reaction mixture was poured into a separatory funnel containing 300mL of water, extracted with ethyl acetate (100 mL), the organic phases were collected, the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 681 (7.8 g). ESI m/z C ₁₇ H ₂₅ N ₂ O ₅ [M+H] ⁺ Calculated 337.17, found 338.19.

EXAMPLE 254 Synthesis of Compound 682

After compound 681 (3g, 8.9mmol) was dissolved in DCM (3 mL), TFA (3 mL) was added, stirred at RT for 3h, concentrated, and taken up twice with dichloromethane, and finally placed on an oil pump to drain, yielding compound 682 (2.5g, 8.9mmol). ESI m/z C ₁₃ H ₁₇ N ₂ O ₅ [M+H] ⁺ Calculated value 281.11, found value 281.12.

EXAMPLE 255 Synthesis of Compound 683

Compound 682 (2.5g, 8.9mmol), tert-butyl 3-aminopropionate (1.6g, 8.9mmol) were dissolved in DMF (10 mL) and ice-cooledHATU (3.4 g, 8.9mmol) and TEA (2.4 mL, 17.8mmol) were added thereto, and after stirring for 5 minutes, the mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was poured into a separatory funnel containing 300mL of water, ethyl acetate (100 mL) was extracted, the organic phases were collected, the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, washed once with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified over a silica gel column (0-10% MeOH/DCM) to give compound 683 (2.1lg, 60%). ESI m/z C ₂₀ H ₃₀ N ₃ O ₆ [M+H] ⁺ Calculated value 408.21, found value 408.21.

EXAMPLE 256 Synthesis of Compound 684

Compound 683 (2.16g, 5.31mmol) was dissolved in methanol (20 mL), pd/C (0.2g, 10wt%) was added, the reaction was stirred under hydrogen (1 atm) for 50 minutes, filtered through celite, and the filtrate was collected and concentrated to give crude 684 (1.4g, 5.31mmol). ESI m/z C ₁₂ H ₂₄ N ₃ O ₄ [M+H] ⁺ Calculated value of 274.17, found value of 274.17.

EXAMPLE 257 Synthesis of Compound 685

684 (1.4g, 5.31mmol) and 680 (0.5g, 1.66mmol) were dissolved in DMF (30 mL), and Brop (1.9g, 4.98mmol) and DIPEA (0.6mL, 3.32mmol) were added under ice-bath, stirred for 10 min, then allowed to warm to room temperature and stirred overnight. The above reaction solution was poured into a separatory funnel containing 300mL of water, ethyl acetate (100 mL) was extracted, the organic phases were collected, the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a silica gel column (0-10% MeOH/DCM) to give compound 685 (0.37g, 28%). ESI m/z C ₃₂ H ₅₃ Cl ₂ N ₈ O ₁₂ [M+H] ⁺ Calculated value 811.31, found value 811.31.

EXAMPLE 258 Synthesis of Compound 686

After compound 685 (0.37g, 0.46mmol) was dissolved in DCM (1 mL), TFA (3 mL) was added, after stirring for 1h at RT, spin-dried and concentrated, taken up twice with dichloromethane and finally placed on an oil pump to be dried, the resulting compound (0.328 g, 0.46mmol) and compound 110 (0.18g, 0.46mmol) were dissolved in DMF (20 mL), HATU (0.35g, 0.92mmol), TEA (0.13mL, 0.92mmol) were added under ice bath and stirred for 30 min. The reaction mixture was poured into a separatory funnel containing 300mL of water, extracted with ethyl acetate (100 mL), the organic phases were collected, the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified over a silica gel column (0-10% MeOH/DCM) to afford compound 686 (0.1lg, 33%). ESI m/z C ₄₅ H ₆₇ Cl ₂ N ₁₀ O ₁₅ [M+H] ⁺ Calculated 1057.41, found 1057.40.

Example 259 Synthesis of Compound 687

After compound 686 (0.16g, 0.15mmol) was dissolved in DCM (0.5 mL), TFA (3 mL) was added and after stirring for 1h at rt, spin-dried to concentrate and taken up twice with dichloromethane and finally placed on an oil pump to drain, the resulting compound was dissolved with compound 41a (0.1g, 0.15mmol) in DMA (3 mL) followed by DIPEA (0.1ml, 0.6mmol) and stirred at rt for 1 h. Concentrating the reaction solution, adding 1mL acetonitrile, loading, performing preparative HPLC, and eluting with MeCN/H ₂ Purification afforded product 687 (59mg, 28%). ESI m/z C ₆₁ H ₉₁ Cl ₂ N ₁₄ O ₁₈ S[M+H] ⁺ Calculated value of 1409.57 and found 1409.57.

EXAMPLE 260 Synthesis of Compound 690

Compound 672 (5.98g, 6.73mmol) was dissolved in methanol (30 mL), pd/C (0.6 g, 10wt%) was added, and the mixture was stirred at room temperature overnight under 1 atm of hydrogen. After filtration through celite, the filtrate was concentrated, the resulting compound (4.57g, 6.05mmol) was dissolved in tetrahydrofuran (60 mL), compound 689 (1.01g, 2.42mmol) and HOBt (817g, 6.05mmol) were added, DCC (1.25g, 6.05mmol) and DIPEA (2.1mL, 12.10mmol) were added after cooling to 0 ℃, temperature was gradually raised to room temperature, stirring was made overnight, ethyl acetate (400 mL) was added for dilution, washing with saturated sodium bicarbonate, washing with 1N HCl, washing with saturated brine, drying over anhydrous sodium sulfate, filtration, concentration, column chromatography (4 MeOH/DCM) was performed to obtain compound 690 (5.65g, 49% yield) [ M + H ])] ⁺ Calculated 1892.06 and found 1892.83.

EXAMPLE 261 Synthesis of Compound 691

Compound 690 (3.71g, 1.96mmol) was dissolved in methanol (50 mL), pd/C (0.40g, 10 wt%) was added, and the mixture was stirred at room temperature overnight under 1 atm of hydrogen. After filtration through celite, the filtrate was concentrated to give compound 691 (1.64g, 51% yield). [ M + H ]] ⁺ Calcd for 1623.98, found 1624.42.

EXAMPLE 262 Synthesis of Compound 692

Compound 691 (315mg, 0.194mmol) was dissolved in ethanol (10 mL), and compound 125 (136mg, 0.485mmol) and 0.5M Na were added ₂ HPO ₄ (2.5 mL), stirred at room temperature for 3 days, concentrated and purified by preparative HPLC (40% MeCN/H) ₂ O). Compound 692 (50mg, 13% yield) was obtained. [ M + H ]] ⁺ Calculated 1954.07, found 1955.05.

Example 263 Synthesis of Compound 693

Compound 692 (50mg, 0.114mmol) was dissolved in DCM (5 mL), TFA (5 mL) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated, subjected to azeotropic distillation with toluene, and the obtained compound and compound 110 (33mg, 0.0837mmol) were dissolved in DMF (6 mL), and after HATU (64mg, 0.1674mmol) and TEA (46uL, 0.0.3348mmol) were added at 0 ℃ and the mixture was stirred for 1 hour. Diluting with water (100 mL), extracting with ethyl acetate (100 mL. Times.3), mixing the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography (15% MeOH/DCM) to give compound 693 (98mg, 53% yield). [ M + H ]] ⁺ Calculated 2200.17 and found 2200.15.

EXAMPLE 264 Synthesis of Compound 694

Compound 693 (98mg, 0.045mmol) was dissolved in DCM (3 mL), and TFA (6 mL) was added and stirred at room temperature for 1 hour. The reaction mixture was concentrated, and subjected to azeotropic distillation with toluene, and the resulting compound and compound 41a (31mg, 0.045mmol) were dissolved in DMA (1 mL), and DIPEA (12. Mu.L, 0.068 mmol) was added thereto, followed by stirring at room temperature for 90 minutes. The reaction was concentrated and purified by preparative HPLC to give compound 694 (33.56mg, 30% yield). [ M/2+ H ] ⁺ Calculated 1276.66, found 1276.65.

EXAMPLE 265 Synthesis of Compound 696

689 (3.01g, 7.23mmol) was dissolved in tetrahydrofuran (30 mL) and H-Gly-O was added ^t Bu (3.03g, 18.09mmol) and HOBt (2.44g, 18.09mmol) were added at 0 ℃ to DCC (3.73g, 18.09mmol) and DIPEA (6.3 mL, 36.18mmol), and the mixture was gradually warmed to room temperature and stirred for 3 days. Dilute with ethyl acetate (200 mL), wash with saturated sodium bicarbonate, 1Washed with N HCl, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 696 (10.17 g). [ M + H ]] ⁺ Calculated 643.29, found 643.29.

EXAMPLE 266 Synthesis of Compound 697

Compound 696 (5.38g, 8.38mmol) was dissolved in DCM (20 mL), and TFA (20 mL) was added and stirred at room temperature for 3 hours. Concentrating, azeotropic distilling with toluene, pulping with dichloromethane to obtain white solid (1.00g, 1.89mmol), dissolving in tetrahydrofuran (20 mL), adding H-L-Ala-O ^t Bu (857mg, 4.72mmol) and HOBt (638g, 4.72mmol) were added DIC (735uL, 4.72mmol) and DIPEA (1.64mL, 9.43mmol) at 0 ℃ and the temperature was gradually raised to 30 ℃ and stirred overnight. Diluting with ethyl acetate (100 mL), washing with saturated sodium bicarbonate, 1N HCl, saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography (6% MeOH/DCM) to give compound 697 (734mg, 50% yield). [ M + H ] ] ⁺ Calcd for 785.36, found 785.70.

EXAMPLE 267 Synthesis of Compound 698

Compound 697 (743mg, 0.936mmol) was dissolved in DCM (6 mL), and TFA (6 mL) was added and stirred at room temperature overnight. After concentration, the resulting solution was subjected to azeotropic distillation with toluene to give a solid compound (629 mg), which was dissolved in tetrahydrofuran (20 mL), tert-butyl 4-aminobutyrate (372mg, 2.340mmol) and HOBt (316g, 2.340mmol) were added thereto, DIC (365uL, 2.340mmol) and DIPEA (815uL, 9.43mmol) were added thereto at 0 ℃ and the mixture was gradually warmed to room temperature and stirred overnight. Diluting with ethyl acetate (150 mL), washing with saturated sodium bicarbonate, 1N HCl, saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by column chromatography (8% MeOH/DCM) to give compound 698 (612mg, 68% yield). [ M + H ]] ⁺ Calculated 955.47, found 955.75.

EXAMPLE 268 Synthesis of Compound 699

Compound 698 (500mg, 0.524mmol) was dissolved in DCM (6 mL), TFA (6 mL) was added, and stirring was carried out at room temperature for 2 hours. After concentration, the mixture was subjected to azeotropic distillation with toluene to obtain a solid compound (441 mg), 186mg (0.17mmol, 1.0eq) and compound (67mg, 0.17mmol) of the compound 110 were dissolved in DMF (6 mL), HATU (131mg, 0.34mmol) and TEA (94uL, 0.68mmol) were added at 0 ℃ and the mixture was stirred at 0 ℃ for 1 hour. Diluting with water (100L), extracting with ethyl acetate (50 mL. Times.3), mixing the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography (7% MeOH/DCM), to obtain compound 699 (100 mg). [ M + H ] ] ⁺ Calcd 1201.57, found 1201.53.

EXAMPLE 269 Synthesis of Compound 700

Compound 699 (228mg, 0.19mmol) was dissolved in methanol (30 mL), and Pd/C (30mg, 10wt%) was added, followed by stirring overnight at room temperature under 1 atm of hydrogen. Concentrating by filtration, dissolving the concentrate in ethanol (5 mL), adding compound 110 (50mg, 0.180mmol), adding 0.1M NaH ₂ PO ₄ (1.25 mL), stir at room temperature overnight. The reaction mixture was concentrated to give Compound 700 (100 mg). [ M + H ]] ⁺ Calculated 1263.58, found 1263.52.

EXAMPLE 270 Synthesis of Compound 701

Compound 700 (100mg, 0.079mmol) was dissolved in DCM (2 mL), TFA (3 mL) was added, and the mixture was stirred at room temperature for 90 min. The reaction mixture was concentrated, subjected to azeotropic distillation with toluene, and the residue and compound 41a (55mg, 0.079mmol) were dissolved in DMA (2 mL), followed by addition of DIPEA (28. Mu.L, 0.158 mmol) and stirring at room temperature for 2 hours. Concentrating the reaction solution by preparingPurification by HPLC gave compound 701 (1.2mg, 1% yield). [ M + H ]] ⁺ Calculated 1615.74 and found 1615.68.

Example 271 Synthesis of Compound 703

Compound 436 (2.93g, 3.92mmol) and t-butanol (1.87Ml, 19.59mmol) were dissolved in methylene chloride (20 mL), DCC (970mg, 4.70mmol) and DMAP (48mg, 0.39mmol) were added in this order at 0 ℃, gradually warmed to room temperature, and stirred overnight. Filtration, concentration of the filtrate, dilution with ethyl acetate (50 mL), washing with 1N HCl, 5% sodium bicarbonate solution, saturated brine washing, drying over anhydrous sodium sulfate, filtration, concentration and purification by silica gel column chromatography (7% MeOH/DCM) gave compound 703 (2.28g, 72% yield). [ M + H ] ] ⁺ Calculated 805.46, found 805.41.

EXAMPLE 272 Synthesis of Compound 704

Compound 703 (2.28g, 2.84mmol) was dissolved in methanol (80 mL), pd/C (0.23g, 10 wt%) was added, and the mixture was stirred under 1 atm of hydrogen at room temperature overnight. The filtrate was concentrated by filtration, purified by column chromatography (7% MeOH/DCM), and the resulting compound (1.39g, 2.08mmol) was dissolved in tetrahydrofuran (20 mL), and compound 689 (346mg, 0.83mmol) and HOBt (281mg, 2.08mmol) were added, DIC (324uL, 2.08mmol) and DIPEA (725uL, 4.16mmol) were added at 0 ℃ and gradually warmed to room temperature and stirred overnight. After dilution with ethyl acetate (100 mL), washing with saturated sodium bicarbonate, 1N HCl, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (7% MeOH/DCM), compound 704 (673mg, 47% yield) was obtained. [ M + H ]] ⁺ Calculated 1721.95, found 1722.59.

EXAMPLE 273 Synthesis of Compound 705

Compound 704 (673mg, 0.39mmol) was dissolved in DCM (6 mL), and TFA (6 mL) was added and stirred at room temperature overnight. Concentration and azeotropic distillation with toluene gave a white solid (627 mg), 335mg (0.208mmol, 1.0eq) and compound 110 (82mg, 0.208mmol) were dissolved in DMF (6 mL), HATU (158mg, 0.416 mmol) and TEA (115uL, 0.832mmol) were added at 0 ℃ and stirred at 0 ℃ for 2 hours. Diluting with water (80 mL), extracting with ethyl acetate (80 mL. Times.3), combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by column chromatography (6% MeOH/DCM) to give compound 705 (318mg, 77% yield). [ M + H ] ] ⁺ Calculated 1968.05, found 1969.32.

EXAMPLE 274 Synthesis of Compound 706

Compound 705 (318mg, 0.16mmol) was dissolved in methanol (20 mL), and Pd/C (30mg, 10wt%) was added, and the mixture was stirred at room temperature for 3 hours under hydrogen gas at 1 atm. Concentrate by filtration to give a white solid (287 mg). 98mg (0.0577 mmol) was dissolved in anhydrous tetrahydrofuran (7 mL), and chloroacetyl chloride (9. Mu.L) and DIPEA (20. Mu.L) were added at 0 ℃ and stirred overnight. Concentration gave compound 706 (27mg, 25% yield). [ M + H ]] ⁺ Calcd for 1851.92, found 1852.94.

EXAMPLE 275 Synthesis of Compound 707

Compound 706 (27mg, 0.0146mmol) was dissolved in DCM (2 mL), and TFA (2 mL) was added and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated, and subjected to azeotropic distillation with toluene to dissolve the obtained compound (25mg, 0.0146mmol) and compound 41a (10mg, 0.0146mmol) in DMA (2 mL), and DIPEA (5. Mu.L, 0.0292 mmol) was added thereto and stirred at room temperature for 90 minutes. The reaction mixture was concentrated, and the concentrate was purified by preparative HPLC to obtain Compound 707 (10.2mg, 32% yield). [ M/2+ H] ⁺ Calculated 1102.54, found 1103.27.

Example 276 Synthesis of Compound 711

The compounds Boc-N-methyl-L-valine (33mg, 0.14mmol), pentafluorophenol (39mg, 0.21mmol) and DCC (32mg, 0.154mmol) were dissolved in EtOAc (20 mL). The mixture was stirred at room temperature overnight, then filtered through celite, the filtrate was concentrated and redissolved in DMA (2 mL), then compound 710 (52mg, 0.14mmol) and DIPEA (48.5. Mu.L, 0.28 mmol) were added. The reaction mixture was stirred at room temperature overnight, concentrated and purified by preparative HPLC (C) ₁₈ Column, 10-100% acetonitrile/water) to give compound 711 (40.2mg, 49%). ESI m/z C ₂₈ H ₄₉ N ₄ O ₇ S[M+H] ⁺ Calcd for 585.32, found 585.32.

EXAMPLE 277 Synthesis of Compound 712

Compound 711 (40mg, 0.069mmol) was dissolved in pyridine (8 mL), acetic anhydride (20.4 mg, 0.2mmol) was added at 0 deg.C, the reaction was warmed to room temperature, and stirred at room temperature overnight. The mixture was concentrated and purified with silica gel column (0-10% meoh, DCM) to give compound 712 (48.1 mg, 100%). ESI m/z C ₃₀ H ₅₁ N ₄ O ₈ S[M+H] ⁺ The calculated value is 627.33, and the measured value is 627.33.

EXAMPLE 278 Synthesis of Compound 715

To a solution of compound 712 (48.1mg, 0.077mmol) in EtOAc (10 mL) was added pentafluorophenol (21.2mg, 0.115mmol) and DCC (17.4mg, 0.085mmol). The mixture was stirred at room temperature overnight, then filtered through celite, and the filtrate was concentratedAnd again dissolved in DMA (4 mL) before addition of compound 714 (20.7 mg,0.1 mmol) and DIPEA (26.8 μ L,0.154 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water) to afford compound 715 (63 mg,. About.100%). ESI m/z C ₄₂ H ₆₆ N ₅ O ₉ S[M+H] ⁺ Calculated 816.45, found 816.45.

EXAMPLE 279 Synthesis of Compound 716

Compound 715 (63mg, 0.077mmol) was dissolved in DCM (1 mL), followed by addition of TFA (3 mL), the reaction was stirred at room temperature for 2 hours, then concentrated, and the resulting compound (55mg, 0.077mmol) was dissolved in DMA (4 mL), followed by addition of compound 125 (30.8mg, 0.11mmol) and DIPEA (27 μ L,0.154 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water) to yield compound 716 (28.5mg, 42%). ESI m/z C ₄₅ H ₆₅ N ₆ O ₁₀ S[M+H] ⁺ Calculated value 881.44, found value 881.44.

EXAMPLE 280 Synthesis of Compound 719

Compound 715 (63mg, 0.077mmol) was dissolved in DCM (1 mL), followed by addition of TFA (3 mL), the reaction was stirred at room temperature for 2 hours, then concentrated, and the resulting compound (55mg, 0.077mmol) was dissolved in DMA (4 mL), followed by addition of compound 718 (65.8mg, 0.11mmol) and DIPEA (27 μ L,0.154 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water) to yield compound 719 (14mg, 16%). ESI m/z C ₅₅ H ₈₄ N ₇ O ₁₆ S[M+H] ⁺ Calcd for 1130.56, found 1130.57.

Example 281 Synthesis of Compound 721

Compound 436 (3g, 4 mmol) was dissolved in DMF (50 mL), HATU (2.3g, 6 mmol) and TEA (1.7mL, 12mmol) were added under ice bath, and after stirring for 20 minutes under ice bath, the mixture was warmed to room temperature and stirred for 3 hours. The reaction was then slowly added to p-aminobenzyl alcohol (0.99g, 8mmol), stirred at room temperature for 1.5 hours, poured into a separatory funnel containing 150mL of water, extracted with ethyl acetate (50 mL. Times.2), the organic phases combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified over a silica gel column (0-10% MeOH/DCM) to give compound 721 (3.9 g, 100% yield). ESI m/z C ₄₂ H ₆₈ N ₃ O ₁₅ [M+H] ⁺ 854.46 is calculated and 854.46 is found.

EXAMPLE 282 Synthesis of Compound 722

Compound 721 (1.9g, 2.22mmol) was dissolved in methanol (20 mL), pd/C (0.19 g) was added, the reaction was stirred under hydrogen (1 atm) for 1.5 h, filtered through celite, the filtrate was collected and concentrated, the resulting crude product was dissolved in EtOH (100 mL), followed by addition of compound 125 (0.61g, 2.2mmol) and 0.1M NaH ₂ PO ₄ (20 mL) was stirred at room temperature overnight. Then, the reaction mixture was concentrated to dryness, and the mixture was subjected to preparative HPLC (C) using methanol as a sample (insoluble matter was removed by filtration and the filtrate was collected) ₁₈ Column, acetonitrile/water) to give compound 722 (0.3g, 19%). ESI m/z C ₄₂ H ₆₉ N ₄ O ₁₆ [M+H] ⁺ Calcd for 885.46, found 885.44.

Example 283 Synthesis of Compound 723

Compound 723 (0.12g, 0.14mmol) was dissolved in DMF (50 mL) and SOCl was added under ice bath ₂ (11. Mu.L, 0.154 mmol), the reaction was stirred under an ice bath for 1 hour. The reaction solution was then concentrated to dryness to give compound 723 (0.13g, 0.14mmol). ESI m/z C ₄₂ H ₆₈ ClN ₄ O ₁₅ [M+H] ⁺ Calculated value 903.43, found 903.44.

EXAMPLE 284 Synthesis of Compound 725

Compound 723 (0.13g, 0.14mmol), compound 724 (0.06g, 0.07mmol) was dissolved in DMF (10 mL), followed by the addition of TBAI (16mg, 0.042mmol) and DIPEA (64. Mu.L, 0.35 mmol) in that order, and stirred at room temperature for 1 hour. The reaction mixture was then concentrated to dryness and purified by preparative HPLC (C) ₁₈ Column, acetonitrile/water) to yield compound 725 (10mg, 8.9%). ESI m/z C ₇₉ H ₁₂₅ N ₉ O ₂₃ S[M+H] ⁺ Calculated value 1599.85, found 1599.82.

EXAMPLE 285 Synthesis of Compound 727

To a solution of compound 15 (4.0 g,7.6 mmol) and iodoethane (914mg, 22.85mmol) in THF (20 mL) at 0 deg.C was added sodium hydrogen (60 wt%,9.5g, 60.9mmol). The reaction was stirred overnight at 0 ℃ and then poured into a vigorously stirred solution of ice-water saturated ammonium chloride (5L). Extracted with ethyl acetate (3X 100 mL). The combined organic phases were dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate/petroleum ether) to give 727 (1.5 g,36% yield) as a bright yellow oil.

EXAMPLE 286 Synthesis of Compound 728

To a solution of compound 12 (1.5g, 2.8mmol) in ethyl acetate were added 29 (7.0 mmol) and dry Pd/C (10 wt%,80 mg). The reaction solution was stirred in a hydrogen atmosphere (1 atm) for 20 hours. Filtered through celite and washed with ethyl acetate. The combined organic phases were concentrated and purified by column chromatography (0-13% ethyl acetate/petroleum ether) to give compound 728 (950mg, 55% yield).

Example 287 Synthesis of Compound 729

Compound 728 (950mg, 1.48mmol) was dissolved in a mixed solution of acetic acid/water/tetrahydrofuran (v/v/v 3. The reaction was concentrated and azeotropically taken dry with toluene, this step was repeated twice and the residue was purified by column chromatography (0-6% MeOH/DCM) to give compound 729 (700mg, 90% yield) as a colorless liquid.

EXAMPLE 288 Synthesis of Compound 730

An aqueous solution of LiOH (3.3N, 2mL,6.67mmol,5.0 eq.) was added to a solution of compound 26 (700mg, 1.33mmol) in methanol (5.0 mL) at 5 ℃. After stirring at room temperature for 2 hours, the mixture was concentrated. By column chromatography (15% MeOH/DCM 0.1% NH) ₃ .H ₂ O) to afford compound 730 (533mg, 80%) as a white solid.

EXAMPLE 289 Synthesis of Compound 731

Compound 730 (520mg, 1.03mmol) and DMAP (13mg, 0.1mmol) were dissolved in a mixed solution of anhydrous THF (5.0 mL) and anhydrous DMF (0.25 mL). Cooled to 0 deg.C and TEA (0.22mL, 1.54mmol) and acetic anhydride (0.15mL, 1.54mmol) were added. The reaction solution was gradually warmed to room temperature and stirred for 3 hours. After concentration, purification by column chromatography (5-50% MeOH/DCM) gave compound 731 (553mg, 94% yield) as an amorphous white solid.

EXAMPLE 290 Synthesis of Compound 732

Compound 731 (0.1g, 0.185mmol), DIC (42.9. Mu.L, 0.28 mmol) and pentafluorophenol (40mg, 0.22mmol) were dissolved in ethyl acetate (20 mL) and stirred at room temperature overnight. The reaction was then concentrated to dryness to give compound 732 (63.5 mg, crude product). ESI m/z C ₃₂ H ₄₄ F ₅ N ₄ O ₆ S[M+H] ⁺ The calculated value is 707.28, and the measured value is 707.28.

Example 291 Synthesis of Compound 733

Compound 732 (63.5mg, 0.09mmol), compound 127 (80.6mg, 0.2mmol) was dissolved in DMA (2 mL), followed by the addition of DIPEA (63. Mu.L, 0.36 mmol), and the reaction was stirred at room temperature for 1 hour. The reaction mixture was directly applied to preparative HPLC (MeCN/H) ₂ O elution) to yield 733 (8.38mg, 10%) as product. ESI m/z C ₄₆ H ₆₈ N ₇ O ₁₁ S[M+H] ⁺ Calculated value of 926.46, found value of 926.45.

EXAMPLE 292 Synthesis of Compound 735

Compound 732 (63.5mg, 0.09mmol), compound 442 (293mg, 0.27mmol) was dissolved in DMA (4 mL), followed by the addition of DIPEA (63. Mu.L, 0.36 mmol) and stirring at room temperature overnight. The reaction mixture was concentrated to 2mL and directly subjected to HPLC (MeCN/H) ₂ O elution) to afford 735 (10.5mg, 7.3%). ESI m/z C ₇₇ H ₁₂₇ N ₁₀ O ₂₄ S[M+H] ⁺ Calculated 1607.87, found 1607.84.

EXAMPLE 293 Synthesis of Compound 737

Compound 673 (48.7g, 64.4mmol) and compound 125 (27.0g, 96.6mmol) were dissolved in 250mL of 95% ethanol, and 50mL of 0.1M NaH was added ₂ PO ₄ The reaction was allowed to proceed overnight at room temperature, ethanol was removed by rotary evaporation, water (300 mL) was added, DCM was extracted (300 mL. Times.1, 150 mL. Times.2), the organic phases were combined, washed with water (500 mL. Times.2), washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and the crude product was purified by column chromatography over silica gel (3-4% MeOH/DCM) to give 36.6g of a pale yellow oil, yield: 61.7 percent. ESI m/z C ₄₃ H ₇₇ N ₄ O ₁₇ [M+H] ⁺ The calculated value is 921.5, and the measured value is 921.5.

EXAMPLE 294 Synthesis of Compound 738

Compound 737 (16.6g, 0.018mmol) was dissolved in 100mL of anhydrous formic acid and stirred at room temperature overnight to complete the reaction. Removal of HCO by rotary evaporation ₂ H, add a large amount of DCM to spin dry and pump dry on a vacuum pump to give 15.6g of a yellow oil, yield: 100 percent. The crude product (0.018mol, 1.0eq) was dissolved in 180mL of anhydrous DCM and dissolved in N ₂ EDC (8.6 g, 0.045mol) and NHS (5.2 g, 0.045mol) were added under protection, reacted at room temperature for 3 hours and then spun dry, 50mL ethyl acetate was added, filtered, washed with a small amount of ethyl acetate, the filtrate spun dry, and pumped dry by a vacuum pump to give 18.0g yellow oil, which was used directly in the next synthesis. ESI m/z C ₄₃ H ₇₂ N ₅ O ₁₉ [M+H] ⁺ Calculated value 962.5, found 962.5.

Example 295 Synthesis of Compound 739

Compound 121 (2.0g, 5.91mmol) and compound 737 (8.5g, 8.87mmol) were dissolved in 20mL of 95% ethanol, 2.0mL of PBS (0.1m, ph 7.5) was added, reaction was performed at room temperature overnight, ethanol was removed by rotary evaporation, dissolution was performed with 100mL of ethyl acetate, washing (50 mL × 5), the aqueous phase was saturated with NaCl solid, extraction was performed with ethyl acetate (150 mL × 1, 75mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary-dried to obtain 3.56g of a product, yield: 50 percent. ESI m/z C ₅₆ H ₉₃ N ₆ O ₂₁ [M+H] ⁺ Calculated value 1185.6, found 1185.6.

EXAMPLE 296 Synthesis of Compound 740

Compound 739 (1.0g, 0.844mmol) was dissolved in 12mL DCM, 4mL TFA was added, the reaction was carried out at room temperature for 1 hour, the solvent was removed by rotary evaporation, a large amount of DCM was added and the reaction mixture was dried, diethyl ether was added, stirred at room temperature for 30 minutes, left to stand, the diethyl ether was poured out, the operation was repeated 2 times, the residue was drained to give 0.91g of a yellow oil, which was dissolved (0.844mmol, 1.0eq) and Compound 41a (0.70g, 0.101mmol) in 15mL anhydrous DMA, ice bath, N. ₂ Under protection, DIPEA (0.44mL, 2.53mmol) is added dropwise, after the reaction is finished, the reaction is heated to room temperature for 3 hours, then the reaction is dried in a spinning mode, a small amount of DCM is added for dissolution, the pH value is adjusted to 3 by formic acid, the reaction product is directly loaded on a silica gel column, impurities with smaller polarity are removed by ethyl acetate/petroleum ether (0% -100% containing 0.1% of formic acid), and then MeOH/DCM (0% -20% containing 0.1% of formic acid) is used for elution, so that 1.43g of yellow foamy solid is obtained. It was dissolved in acetonitrile and water and subjected to preparative HPLC (C) ₁₈ Column, mobile phase a water, mobile phase B acetonitrile, acetonitrile rising from 20% to 80% within 120 min) to give 1.0g of white foamy solid in 74.6% yield. ESI m/z C ₇₆ H ₁₂₅ N ₁₀ O ₂₄ S[M+H] ⁺ Calculated 1593.9 and found 1593.9.

Example 297 Synthesis of Compound 742

Compound 154 (2.0g, 5.91mmol) and compound 738 (8.5g, 8.87mmol) were dissolved in 20mL of 95% ethanol, 2.0mL of PBS (0.1m, ph7.5) was added, the reaction was performed at room temperature overnight, ethanol was removed by rotary evaporation, and 100mL of ethyl acetate was added for dissolution, water (50 mL × 5) was washed, the aqueous phase was saturated with NaCl solid, extracted with ethyl acetate (150 mL × 1, 75mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by rotary drying to obtain 4.85g of a product, yield: 69.2 percent. ESI m/z C ₅₆ H ₉₃ N ₆ O ₂₁ [M+H] ⁺ Calculated value 1185.6, found 1185.6.

EXAMPLE 298 Synthesis of Compound 743

Compound 742 (1.0g, 0.844 mmol) was dissolved in 12mL DCM, 4mL TFA was added, the reaction was carried out at room temperature for 1 hour, the solvent was removed by rotary evaporation, a large amount of DCM was added and dried by rotary evaporation, diethyl ether was added, stirring was carried out at room temperature for 30 minutes, the mixture was left to stand, the diethyl ether was decanted off, the operation was repeated 2 times, the residue was drained to give 0.90g yellow oil, which (0.843 mmol,1.0 eq) and compound 41a (0.70g, 0.100mmol) were dissolved in 15mL anhydrous DMA, ice-cooled, DIPEA (0.44ml, 2.53mmol) was added dropwise, the reaction was warmed to room temperature for 3 hours, dried by rotary evaporation, a small amount of DCM was added and the pH was adjusted to 3 with formic acid, the silica gel column was directly loaded, the less polar impurities were first removed with ethyl acetate/petroleum ether (0% -100% with 0.1% formic acid) and then MeOH/DCM (0.1% formic acid) was eluted to give 1 g yellow foamy solid, 1 g yellow solid. It was dissolved in acetonitrile and water and subjected to preparative HPLC (C) ₁₈ Column, mobile phase a water, mobile phase B acetonitrile, acetonitrile rising from 20% to 80% within 120 min) to give 1.0g of white foamy solid with a yield of 75.0%. ESI m/z C ₇₆ H ₁₂₅ N ₁₀ O ₂₄ S[M+H] ⁺ Calculated value 1593.9, found 1593.9.

Example 299 methyl bis (2-hydroxyethyl) amino-4-oxobutanoate.

Dimethyl succinate (20.0g, 136.9mmol) and dihydroxyethylamine (7.20g, 68.7mmol) were dissolved in a mixed solution of anhydrous toluene (500 ml) and pyridine (50 ml), and the mixture was refluxed at 150 ℃ for 28 hours. Concentrating the mixed solution and then passing through SiO ₂ Column chromatography (EtOAc/DCM =5% -25% to EtOAc) afforded the title compound (12.5g, 83%). ESI MS m/z + ion peak: c ₉ H ₁₇ NaNO ₅ Calculated (M + Na) 242.2, experimental 242.4.

Example 300 bis (2- (methylsulfonyloxy) ethyl) amino-4-oxobutanoic acid methyl ester.

Methyl bis (2-hydroxyethyl) amino-4-oxobutanoate (12.0 g, 49.56mmol) was dissolved in anhydrous pyridine (350 ml), and methanesulfonyl chloride (20.0 g,175.4 mmol) was added. After overnight reaction the solution was concentrated and then diluted with EtOAc (350 mL) and cooled 1M NaH ₂ PO ₄ The solution (2X 300ml) was washed with MgSO ₄ After drying, filtration and spin-drying, a crude product (18.8g, 101%) was obtained. The crude product was used directly in the next reaction without purification. ESI MS m/z + ion peak: c ₁₁ H ₂₁ NaNO ₉ S ₂ Calculated (M + Na) 398.2, experimental 398.4.

Example 301 bis (2- (thioacetyl) ethyl) amino-4-oxobutanoic acid methyl ester.

Methyl bis (2- (methylsulfonyloxy) ethyl) amino-4-oxobutanoate (freshly prepared, 90% pure, 8.5g, ca. 20 mmol) was dissolved in DMA (350 ml) and triethylamine (30ml, 215mmol) and thioacetic acid (10ml, 134mmol) were added in that order at 0 ℃. Stirred at room temperature overnight, concentrated, diluted with EtOAc (350 mL) and then sequentially with NaHCO ₃ Saturated solution (300 ml), saturated NaCl solution (300 ml) and 1M NaH ₂ PO ₄ The solution (300 ml) was washed. Then, the organic phase was treated with Na ₂ SO ₄ Drying, filtering, spin-drying, and passing through SiO ₂ Column chromatography of EtOAc/hexane (10% -25% EtOAc) afforded the title compound (5.1g, 76%). ESI MS m/z + ion peak: c ₁₃ H ₂₁ NaNO ₅ S ₂ Calculated (M + Na) 358.1, experimental 358.2.

Example 302.4- (bis (2- (pyridin-2-yldithio) ethyl) amino) -4-oxobutanoic acid.

Methyl bis (2- (thioacetyl) ethyl) amino-4-oxobutanoate (5.0 g,14.9 mmol) was dissolved in THF (150 ml) and aqueous NaOH (5.0 g, 125mmol) solution (100 ml) was added. After stirring for 35 minutes at room temperature, the mixture is washed with H ₃ PO ₄ Neutralized to pH 7. Then, a solution of 1, 2-dipyridyl disulfide (Aldrithiol-2, 26.0g, 118mmol) in THF (100 ml) was added, and stirred for 4 hours. Concentrating, and treating with SiO ₂ Column chromatography MeOH/DCM/HOAc (1. ESI MS m/z + ion peak: c ₁₈ H ₂₁ NaN ₃ O ₃ S ₄ Calculated (M + Na) is 478.0 and experimental value is 478.2.

Example 303.2, 5-dioxopyrrolidin-1-yl-bis (2- (pyridin-2-yl-dithio) ethyl) amino-4-oxobutyrate.

4- (bis (2- (pyridin-2-yldithio) ethyl) amino) -4-oxobutanoic acid (5.2 g,11.5 mmol) was dissolved in DMA (100 ml), NHS (1.6 g,13.9 mmol) and EDC (5.0 g,26.1 mmol) were added. Stirring overnight, spin-drying, and passing through SiO ₂ Column chromatography (EtOAc/DCM =5% -15% to EtOAc) afforded the title compound (5.8g, 85.6%). ESI MS m/z + ion peak: c ₂₂ H ₂₄ NaN ₄ O ₅ S ₄ Calcd for (M + Na) 575.1, experimental 575.2.

Example 304 Exo-3, 6-epoxy- Δ -tetrahydrophthalimide.

Furan (10.0 ml,137.4 mmol) was added to a solution of maleimide (10.0 g,103.0 mmol) in toluene (200 ml), and the mixed solution was placed in a 1L autoclave and heated to 100 ℃ for reaction for 8 hours. After cooling to room temperature, the in-pot solid was washed with methanol, concentrated, and recrystallized from a mixed solution of ethyl acetate and n-hexane to give the title compound (16.7 g, 99%). ¹ H NMR(CDCl ₃ ) 11.12 (s, 1H) (NH), 6.68-6.64 (m, 2H), 5.18-5.13 (m, 2H), 2.97-2.92 (m, 2H). MS m/z + ion peaks: c ₈ H ₇ NaNO ₃ Calculated (M + Na) 188.04, experimental 188.04.

Example 305.4- ((2- ((3aR, 4R,7S, 7aS) -1, 3-dioxo-3a, 4,7, 7a-tetrahydro-1H-4, 7-oxisoindol-3H) -yl) ethyl) (2- ((4R, 7S, 7aS) -1, 3-dioxo-3a, 4,7, 7a-tetrahydro-1H-4, 7-oxisoindol-2 (3H) -yl) ethyl) amino) -4-oxobutyric acid methyl ester.

Methyl 4- (bis (2-methanesulfonyl) ethoxyamine) -4-oxobutanoate (freshly made, 90% pure, 8.5g,. About.20 mmol) was dissolved in DMA (350 mL) and 3, 6-oxo-delta-tetrahydrophthalimide (10.2g, 61.8 mmol), sodium carbonate (8.0g, 75.5mmol) and sodium iodide (0.3g, 2.0mmol) were added in that order. The reaction mixture was stirred at room temperature overnight. After the reaction mixture was concentrated, ethyl acetate (350 mL) was added, and the mixture was washed with saturated aqueous sodium bicarbonate (300 mL), saturated brine (300 mL) and 1M NaH in this order ₂ PO ₄ (300 mL) washing. Drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating the filtrate; the product was purified by silica gel column chromatography (10% -30% ethyl acetate/n-hexane) to give the target compound (7.9 g, 77%). ESI MS m/z +: C ₂₅ H ₂₇ NaN ₃ O ₉ (M + Na): calculated values are: 536.2, experimental value: 536.4.

example 306.4- (bis (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrolidine) ethylamine) -4-oxobutanoic acid

Methyl 4- ((2- ((3aR, 4R,7S, 7aS) -1, 3-

dioxo

3a,4,7, 7a-tetrahydro-1H-4, 7-oxisoindol-3H) -yl) ethyl) (2- ((4R, 7S, 7aS) -1, 3-dioxo-3a, 4,7, 7a-tetrahydro-1H-4, 7-oxisoindol-2 (3H) -yl) ethyl) amino) -4-oxobutanoate (3.0g, 5.8mmol) and trimethyltin hydroxide (4.8g, 26.4mmol) were dissolved in 1, 2-dichloroethane (150 mL) and refluxed at 80 ℃ for 8 hours. After cooling to room temperature, the reaction solution was filtered through silica gel and rinsed with dichloromethane/methanol to remove residual trimethyltin hydroxide. The filtrates were combined, concentrated under reduced pressure, added with N, N-dimethylacetamide and toluene, and stirred under reflux at 120 ℃ overnight. Purification by silica gel column chromatography (5% to 10% methanol/dichloromethane) gave the title compound (1.62g, 76%). ESI MS m/z + C ₁₆ H ₁₇ NaN ₃ O ₉ (M + Na): calculated values: 386.1 of the total weight of the steel; experimental values: 386.2.

Example 307.2, 5-Dioxopyrrolidine-4- (bis (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrolidine) ethylamine) -4-oxobutanoic acid ethyl ester.

4- (bis (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrolidine) ethylamine) -4-oxobutanoic acid (16.0g, 4.4mmol) was dissolved in DMA (100 mL), NHS (0.76g, 6.61mmol) and EDC (1.70g, 8.90mmol) were added, the reaction mixture was stirred overnight at room temperature, then the reaction was concentrated and purified by silica gel column chromatography (5% -15% ethyl acetate/dichloromethane) to give the title compound (1.72g, 85.0%). ESI MS m/z + C ₂₀ H ₂₀ NaN ₄ O ₉ (M + Na): calculated values are: 483.1; experimental values: 483.2.

example 308.5- (3 ',6' -oxo- Δ -tetrahydrophthalimide) pentanoic acid tert-butyl ester

Tert-butyl 5-hydroxypentanoate (10.0 g,57.4 mmol) was dissolved in pyridine (60 mL), and methanesulfonyl chloride (8.0 mL, 103.3mmol) was added. The reaction mixture was stirred at room temperature for 6h. After concentrating the reaction mixture, diluting with ethyl acetate and diluting with cold 1M NaH ₂ PO ₄ (pH 6) washing the solution, drying the solution by anhydrous magnesium sulfate, filtering the solution, and concentrating the mother solution to dryness under reduced pressure to obtain the mesylate. This was added to compound 12 (9.90g, 60.0 mmol) and Na ₂ CO ₃ (8.5g, 80.1mmol) in DMF (80 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, ethyl acetate was added thereto, and the mixture was washed with saturated brine and 1M NaH ₂ PO ₄ The solution was washed (pH 6), dried over anhydrous magnesium sulfate, filtered, and the mother liquor was concentrated and purified by silica gel chromatography (ethyl acetate/dichloromethane =1 = 12) to obtain the objective compound (14.01g, 76%). ESI MS m/z + C ₁₇ H ₂₃ NaNO ₅ (M + Na): calculated values: 344.16; experimental values: 344.16.

example 309.5-Maleimidopentanoic acid.

Tert-butyl 5- (3 ',6' -oxo-. DELTA. -tetrahydrophthalimide) valerate (5.0g, 15.57mmol) was dissolved in 1, 4-dioxane (40 mL), concentrated hydrochloric acid (10 mL) was added at 4 ℃ and the reaction mixture was stirred at room temperature for 30min. The reaction mixture was concentrated to dryness to give 5- (3 ',6' -oxo- Δ -tetrahydrophthalimide) pentanoic acid (4.08g, 99%). The above compound was dissolved in N, N-dimethylacetamide/toluene (1, 40 mL) and reacted under reflux for 6h. After the reaction solution was concentrated, it was recrystallized from ethanol/ether/n-hexane to obtain the objective compound (2.76g, 90%). ESI MS m/z + C ₉ H ₁₂ NO ₄ (M + H): calculated values: 198.07; experimental values: 198.07.

example 310.5- (Maleimido) hexanoic acid succinimidyl ester (DMPS linker)

5-Maleamidopentanoic acid (2.0 g, 10.10 mL) was dissolved in dichloromethane (20 mL), then NHS (1.50g, 13.0 mmol) and EDC (7.0 g,36.4 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction solution was concentrated to dryness, and then subjected to silica gel chromatography (ethyl acetate/dichloromethane =1 = 10) to obtain the target compound (2.43g, 82%). ESI MS m/z + C ₁₃ H ₁₄ NaN ₂ O ₆ (M + Na): calculated values: 317.09; experimental values: 317.09.

example 311.5- (3 ',6' -endoxy-. DELTA. -tetrahydrophthalimide) pentanoic acid hydrazide tert-butyl ester formate.

5- (3 ',6' -oxo-. DELTA. -tetrahydrophthalimide) pentanoic acid (1.0g, 3.77mmol) was dissolved in DMF (30 mL), followed by addition of tert-butylcarbamate (0.60g, 4.53mmol) and EDC (2.0g, 10.4 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction solution was concentrated to dryness, and then purified by a silica gel column (ethyl acetate/dichloromethane = 1. ESI MS m/z + C ₁₈ H ₂₅ NaN ₃ O ₆ (M + Na): calculated values are: 402.17; experimental values: 402.18.

example 312.5- (Maleimido) pentanoic acid hydrazide.

Tert-butyl 5- (3 ',6' -oxo- Δ -tetrahydrophthalimide) pentanoate hydrazide carboxylate (1.18g, 3.11mmol) was dissolved in N, N-dimethylformamide/toluene (1, 20 mL) and reacted for 6h at reflux. The reaction was concentrated, 1, 4-dioxane (20 mL) was added, then HCl (5mL, 36%) was added at 4 ℃ and stirred for 30min. Concentrating the reaction solution to dryness, and recrystallizing with ethanol/diethyl ether/n-hexaneThe objective compound (577mg, 88%) was obtained. ESI MS m/z + C ₉ H ₁₄ N ₃ O ₃ (M + H): calculated values: 212.10,; experimental values: 212.10.

Example 313.3' -bromo-

maleimide compounds

39 and 40, and 3',4' -dibromo-

maleimide compounds

43 and 44

Compound 37 or 38 (6 g) was dissolved in DMF (60 mL) and then bromomaleic anhydride (1 eq) or 2, 3-dibromo-maleic anhydride (1 eq) was added and the reaction mixture was stirred overnight. The reaction solution was concentrated to dryness to obtain the pure trans acid. Acetic acid (50 mL) and acetic anhydride (2-4 g) were added to the trans acid and the reaction mixture was refluxed at 120 ℃ for 6-12 h. The reaction solution was concentrated and subjected to silica gel column separation (ethyl acetate/dichloromethane =1:10 to 1) to obtain 3' -bromo-

maleimide compounds

39 and 40, and 3',4' -dibromo-maleimide compounds 43 and 44 (61% to 87%) in the same manner.

5- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoic acid

ESI MS m/z+C ₉ H ₁₁ BrNO ₄ (M + H): calculated values are: 275.98; experimental values: 275.98.

3- (2- (2- (2- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) -propionic acid.

ESI MS m/z+C ₁₃ H ₁₉ BrNO ₇ (M + H): calculated values are: 380.03; experimental values: 380.03.

5- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoic acid

ESI MS m/z+C ₉ H ₁₀ Br ₂ NO ₄ (M + H): calculated values: 353.89; experimental values: 353.89.

3 (2- (2- (2- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) -propionic acid

ESI MS m/z+C ₁₃ H ₁₈ Br ₂ NO ₇ (M + H): calculated values: 457.94; experimental values: 457.94.

example 314.N-hydroxysuccinimide etherates 41 and 42 of 3' -bromo-maleimide and N-hydroxysuccinimide etherates 45 and 46 of 3',4' -dibromo-maleimide.

R＝C ₁ ～C ₈ alkyl or C ₂ H ₄ (OC ₂ H ₄ )n,n＝1～20；X ³ ＝H or Br；41,42,X ³ ＝H；45,46,X ³ ＝Br

3' -bromo-maleimide compounds 39 and 40 (1 eq), or 3',4' -dibromo-

maleimide compounds

43 and 44 were dissolved in DMA (. About.0.15 ml), and NHS (1.1 eq) and EDC (2-4 eq) were added and the reaction mixture was stirred overnight. The reaction solution was concentrated and subjected to silica gel column separation (ethyl acetate/dichloromethane =1:20 to 1: 5) to obtain N-hydroxysuccinimide etherates 41 and 42 of 3' -bromo-maleimide and N-hydroxysuccinimide etherates 45 and 46 (70% to 93%) of 3',4' -dibromo-maleimide.

2, 5-dioxopyrrolidin-1-yl 5- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) valerate (41)

ESI MS m/z+C ₁₃ H ₁₃ BrN ₂ NaO ₇ (M + Na): calculated values are: 395.00; experimental values: 395.00.

2, 5-dioxopyrrolidin-1-yl 5- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) valerate (45)

ESI MS m/z+C ₁₃ H ₁₂ Br ₂ N ₂ NaO ₆ (M + Na): calculated values: 472.91; experimental values: 472.91.

2, 5-dioxopyrrolidin-1-yl 3- (2- (2- (2- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propanoate (42)

ESI MS m/z+C ₁₇ H ₂₁ BrN ₂ NaO ₉ (M + Na): calculated values: 499.04; experimental values: 499.04.

2, 5-dioxopyrrolidin-1-yl 3 (2- (2- (2- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propanoate (46)

ESI MS m/z+C ₁₇ H ₂₀ Br ₂ N ₂ NaO ₉ (M + Na): calculated values: 576.95; experimental values: 576.95.

example 315.4- (2-pyridyldithio) -4-methylpentanoic acid

4-mercapto-4-methylpentanoic acid (Goff, D. Et al, bioconjugate chem.1990,1, 381-386) (4.67g, 31.5 mmol) was dissolved in methanol (15 mL), and then a methanol solution (80 mL) of 2, -dipyridyl disulfide (30.0g, 136.2 mmol) and 100mM sodium phosphate buffer (pH 7.5, 70 mL) were added, and the reaction was stirred for 6 hours. After the reaction solution was concentrated, it was extracted with ethyl acetate/n-hexane (1. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Separation by silica gel chromatography (methanol/dichloromethane/acetic acid = 1. ESI MS m/z + C ₁₁ H ₁₆ NO ₂ S ₂ (M + H): calculated values are: 258.05; experimental values: 258.05.

example 316.4- (2-Pyridinedithio) -4-Methylpentanoic acid succinimidyl ester (SMDP linker)

4- (2-Pyridyldithio) -4-methylpentanoic acid (2.0 g, 7.78mmol) was dissolved in dichloromethane (20 mL), NHS (1.10 g, 9.56mmol) and EDC (4.0 g, 20.8mmol) were then added, and the reaction mixture was stirred overnight. After the reaction solution was concentrated, the reaction solution was separated by silica gel chromatography (ethyl acetate/dichloromethane =1 = 10) to obtain the objective compound (2.48g, 90%). ESI MS m/z + C ₁₅ H ₁₈ NaN ₂ O ₄ S ₂ (M + Na): calculated values: 377.07; experimental values: 377.08.

example 317.2- ((1R, 3R) -3- (tert-butoxycarbonyl) -1-hydro-4-methylaminopentyl) thiazole-4-carboxylic acid methyl ester.

(3R, 5R) -tert-butyl-3-isopropyl-3- (4- (methoxycarbonyl) thiazole) isopropyloxazole-2-carboxylic acid methyl ester (1.00g, 2.81mmol) was dissolved in acetonitrile (20 mL) and H ₂ To O (2 mL), mo (CO) was added ₆ (1.10g, 3.12mmol), and the reaction mixture was stirred at 70 ℃ for 16 hours. After concentrating the reaction mixture, ethyl acetate (50 mL) and 10% citric acid aqueous solution (50 mL) were added, and NaIO was added thereto ₄ Until the aqueous layer became clear, it was extracted with ethyl acetate. The organic phase is 10% Na ₂ S ₂ O ₃ The aqueous solution and saturated brine were washed, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by a silica gel column chromatography (n-hexane/ethyl acetate = 3) to obtain a compound (906 mg, 90%) as a colorless solid. ESI MS m/z + C ₁₆ H ₂₆ N ₂ NaO ₅ S (M + Na): calculated values: 381.14; experimental values: 381.14.

example 318.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylic acid methyl ester.

Methyl 2- ((1r, 3r) -3- (tert-butoxycarbonyl) -1-hydro-4-methylaminopentyl) thiazole-4-carboxylate (900mg, 2.51mmol) was dissolved in pyridine (15 mL), acetic anhydride (0.5ml, 5.29mmol) was added, and after stirring overnight, the reaction was concentrated and separated by a silica gel chromatography column (n-hexane/ethyl acetate = 4) to obtain a colorless solid compound (950 mg, 95%). ESI MS m/z + C ₁₈ H ₂₈ N ₂ NaO ₆ S (M + Na): calculated values: 423.15; experimental values: 423.16.

example 319.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylic acid.

Methyl 2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylate (940mg, 2.35mmol) was dissolved in THF (15 mL), naH (120mg, 3.0mmmol, 60%) was added at 4 ℃ to the reaction mixture, and after stirring for 2 hours, CH was added ₃ I (0.155mL, 2.49mmol) and the reaction mixture was stirred overnight. Concentrating the reaction solution, dissolving in ethanolEthyl acetate, filtered and concentrated to dryness to give pure methyl 2- ((1r, 3r) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylate (73 a). The compound (73 a) obtained above was dissolved in 1, 2-dichloroethane (20 mL), trimethyltin hydroxide (620mg, 3.43mmol) was added, and the reaction mixture was stirred at 80 ℃ overnight. The reaction solution was concentrated, dissolved in methanol/dichloromethane/acetic acid (1, 5, 20ml), filtered, concentrated, and the mother liquor was added with toluene and concentrated again to obtain a dried compound. Then, the compound obtained above was dissolved in pyridine (15 mL), acetic anhydride (0.4 mL, 4.23mmol) was added, and the reaction mixture was stirred overnight. After the reaction liquid was concentrated, the reaction liquid was separated by a silica gel column (methanol/dichloromethane/acetic acid = 1. ESI MS m/z + C ₁₈ H ₂₈ N ₂ NaO ₆ S (M + Na): calculated values: 423.15; experimental values: 423.16.

example 320.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1, 3-dioxoisoindoline) -4-methylamine-pentyl) thiazole-4-carboxylic acid methyl ester.

Methyl 2- ((1R, 3R) -3- (tert-butoxycarbonyl) -1-hydro-4-methylaminopentyl) thiazole-4-carboxylate (850mg, 2.37mmol) was dissolved in THF (15 mL), naH (100mg, 2.5mmol, 60%) was added at-20 ℃ and stirred at that temperature for 20min, and N- (3-bromopropyl) phthalimide (655mg, 2.4mmol) was further added, and the above reaction mixture was stirred at-20 ℃ for 30min and then warmed to room temperature to react for 4h. The reaction was quenched by addition of methanol (0.5 mL), dichloromethane (60 mL) was added, filtered, and concentrated to afford methyl 2- (1R, 3R) -3- (tert-butoxy-methylamine) -1- (3- (1, 3-dioxoisoindoline-2-propoxy-4-pentyl) thiazole-4-carboxylate, which was used directly in the next step without purification by dissolving the above-obtained compound in THF (10 mL), followed by addition of NaH (170mg, 4.25mmol, 60%) at room temperature, stirring for 45min, addition of CH ₃ I (0.20mL, 3.21mmol), and the reaction mixture was stirred at room temperature overnight. The reaction solution is added with NaH ₂ PO ₄ (210m, 2ml), then DMA (5 mL) was added, concentrated under reduced pressure, and separated by silica gel chromatography (ethyl acetate/dichloromethane = 1. ESI MS m/z + C ₂₈ H ₃₇ N ₃ NaO ₇ S (M + Na): calculated values: 582.22; experimental values: 582.22.

example 321.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1, 3-dioxoisoindoline) -4-methylamine-pentyl) thiazole-4-carboxylic acid.

Dried methyl 2- ((1r, 3r) -3- (tert-butoxy (methyl) amine) -1- (3- (1, 3-dioxoisoindoline) -4-methylamine-pentyl) thiazole-4-carboxylate (910mg, 1.63mmol) was dissolved in 1, 2-dichloroethane (20 mL), trimethyltin hydroxide (400mg, 2.21mmol) was added, the reaction mixture was stirred at 80 ℃ overnight the reaction was concentrated, and the target compound (756mg, 85%) was obtained by silica gel chromatography (methanol/dichloromethane/acetic acid =1 ₂₇ H ₃₇ N ₃ O ₇ S (M + H): calculated values: 546.22; experimental values: 546.22.

example 322.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy (3- (1, 3-di-oxoisoindoline-2-pentylamine) -4-methylamine) -4-pentyl) thiazole-4-carboxylic acid methyl ester.

Methyl 2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylate (800mg, 2.00mmol) was dissolved in THF (30 mL), naH (150mg, 3.75mmol, 60%) was added at room temperature, stirring was performed for 45min, and N- (3-bromopropyl) phthalimide (655mg, 2.4 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction solution is added with NaH ₂ PO ₄ After quenching (2.0 m, 2ml), DMA (5 mL) was added, concentrated under reduced pressure, and separated by silica gel chromatography (ethyl acetate/dichloromethane =1Compound (971mg, 82%). ESI MS m/z + C ₂₉ H ₃₇ N ₃ NaO ₈ S (M + Na): calculated values: 610.22; experimental values: 610.22.

example 323.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy (3- (1, 3-di-oxoisoindoline-2-pentylamine) -4-methylamine) -4-pentyl) thiazole-4-carboxylic acid.

Dried methyl 2- ((1r, 3r) -1-acetoxy-3- (tert-butoxy (3- (1, 3-di-oxoisoindoline-2-pentylamine) -4-methylamine) -4-pentyl) thiazole-4-carboxylate (900mg, 1.53mmol) was dissolved in 1, 2-dichloroethane (35 mL), trimethyltin hydroxide (400mg, 2.21mmol) was added and the reaction mixture was stirred overnight at 80 ℃ ₂₈ H ₃₅ N ₃ NaO ₈ S (M + Na): calculated values: 596.20; experimental values: 596.20.

EXAMPLE 324 Ethyl (S) -5- (4- (benzyloxy) phenyl) -4- (tert-butoxycarbonylamino) -2-methylpentenoate (185)

Diisobutylaluminum hydride (40ml, 40mmol, 1.0M) was slowly added to a solution of (S) -3- (4- (benzyloxy) phenyl) -2- (tert-butoxycarbonylamino) -propionic acid methyl ester 184 (8.00g, 20.76mmol) in DCM (250 ml) at-78 deg.C, reacted for 2 hours, and quenched with MeOH (5 ml). After the reaction mixture was warmed to room temperature, 1M HCl was added to adjust pH =4, and the layers were separated. The aqueous layer was extracted with DCM (2X 150ml), the organic layer was washed with water, the organic layers were combined and washed with anhydrous Na ₂ SO ₄ Drying, filtering and concentrating to obtain the primary product of the aldehyde group intermediate. The crude product was dissolved in DCM and added with 1 at RTYlide solution synthesized from a solution of- (1-ethoxycarbonylethyl) triphenyl phosphine bromide (18.0 g, 40.64mmol) and KOtBu (5.00g, 44.64mmol) in dichloromethane (80 ml), after the mixture was reacted overnight, concentrated and column chromatographed (ethyl acetate/n-hexane, 1, 8-1]+, ion peak: c ₂₆ H ₃₃ NNaO ₅ 462.22, experimental value, 462.22.

EXAMPLE 325 Ethyl (4R) -4- (tert-butoxycarbonylamino) -5- (4-hydroxyphenyl) -2-methylpentanoate.

Ethyl (S) -5- (4- (benzyloxy) phenyl) -4- (tert-butoxycarbonylamino) -2-methylpentenoate (185) (6.70g, 15.26mmol), methanol (150 ml), 10% Pd/C (0.3 g) was charged to a hydrogen reaction flask, reacted under 30psi of hydrogen for 6 hours, filtered through celite, concentrated, and recrystallized from ethanol/n-hexane to give the desired product (186) (4.61g, 86%). ESI M/z [ M + Na ] ]+, ion peak: c ₁₉ H ₂₉ NNaO ₅ 374.20, experimental value, 374.30.

EXAMPLE 326 Ethyl (4R) -4- (tert-butoxycarbonylamino) -5- (4-hydroxy-3-nitrophenyl) -2-methylpentanoate.

To 4.50g, 12.81mmol) of ethyl (4R) -4- (tert-butoxycarbonylamino) -5- (4-hydroxyphenyl) -2-methylpentanoate in anhydrous CH ₂ Cl ₂ (200 ml) adding Ac to the solution ₂ O (2ml, 21.16mmol) and fuming nitric acid (0.65ml, 14.07mmol) were stirred at room temperature for 4 hours, diluted with water (150 ml), separated, and the aqueous phase extracted with ethyl acetate. The organic layers were combined and washed with anhydrous Na ₂ SO ₄ Drying, filtration, concentration, column chromatography (ethyl acetate/dichloromethane =1 = 10) afforded the desired product (4.21g, 83%). ESI M/z [ M + Na ]] ⁺ Ion peak: c ₁₉ H ₂₈ N ₂ NaO ₇ 419.19, experimental value, 419.20.

Example 327.4- (tert-butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) pentanoic acid ethyl ester.

Ethyl (4R) -4- (tert-butoxycarbonylamino) -5- (4-hydroxy-3-nitrophenyl) -2-methylpentanoate (4.00g, 10.09mmol) was dissolved in acetonitrile (70 ml) and N, N-dimethylacetamide (30 ml), cooled to 0 ℃ and then N, N' -diisopropylethylamine (4.00ml, 23.00mmol) was added and after two minutes phosphorus oxychloride (2.00ml, 21.45mmol) was added. Stirring at room temperature for 8 hours, cooling to 0 deg.C, slowly adding a solution of sodium bicarbonate (3.5g, 41.60mmol) and water (20 ml), continuing stirring at 0 deg.C overnight, concentrating, purifying with a C-18 column (20X 4 cm), gradient elution conditions of 25ml/min, A:0.5% acetic acid, B: methanol, 100% A for 10 minutes, and then 45 minutes to 75% A and 25% B. The objective fraction was collected and concentrated to give the objective compound (3.89g, 81%). ESI M/z [ M-H ] ] ^- Ion peak: c ₁₉ H ₂₈ N ₂ O ₁₀ P,475.16, experimental value, 475.20.

EXAMPLE 328 (4R) -4- (tert-butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) pentanoic acid.

To a solution of ethyl 4- (tert-butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) valerate (3.75g, 7.87mmol) in THF (100 ml) was added a solution of lithium hydroxide (5.0 g,208.7 mmol) in water (60 ml), reacted at 0 ℃ for 4 hours, adjusted to pH6 with 4M hydrochloric acid, concentrated, purified by C-18 column, gradient eluted, 25ml/min, A:0.5% acetic acid, B: methanol, 100% A was held for 10 minutes, then 45 minutes to 75% A and 25% B. The fractions of interest were collected and concentrated to give the title compound (2.82g, 80%). ESI M/z [ M-H ]] ^- Ion peak: c ₁₇ H ₂₄ N ₂ O ₁₀ P,447.12, experimental value, 447.20.

EXAMPLE 329 (4R) -5- (3-amino-4-phosphonoxyphenyl) -4- (tert-butoxycarbonylamino) -2-methylpentanoic acid.

To a hydrogen reaction flask were added (4R) -4- (tert-butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) pentanoic acid (2.6 g, 5.80mmol), methanol (80 ml), 10% by weight Pd/C (0.2 g) in this order. The reaction was carried out under 35psi of hydrogen pressure for 6 hours. The mixture was filtered through celite and concentrated to give the crude product (2.18g, 90%) which was used in the next reaction without further purification. ESI M/z [ M-H ] ] ^- Ion peak: c ₁₇ H ₂₆ N ₂ O ₈ P,417.15, experimental value, 417.15.

EXAMPLE 330 (S) -methyl 2- (tert-butoxycarbonylamino) -3- (4-hydroxy-3-nitrophenyl) -propionate (196).

To a solution of (S) -methyl 2- (tert-butoxycarbonylamino) -3- (4-hydroxyphenyl) -propionate (4.5g, 15.24mmol) in dry dichloromethane (240 ml) were added acetic anhydride (4 ml, 42.32mmol) and fuming nitric acid (0.85ml, 18.40mmol). After stirring at room temperature for 4 hours, it was diluted with water (150 ml), separated and the aqueous phase extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed (ethyl acetate/dichloromethane, 1. ESI M/z [ M + Na ]] ⁺ Ion peak: c ₁₅ H ₂₀ N ₂ NaO ₇ 363.13, experimental value, 363.20.

EXAMPLE 331 (S) -methyl 2- (tert-butoxycarbonylamino) -3- (3-nitro-4-phosphonoxyphenyl) -propionate.

At 0 ℃ to (S) -2- (tert-butoxy)N, N' -diisopropylethylamine (4.00ml, 23.00mmol) was added to an acetonitrile (90 ml) solution of carbonylamino) -3- (4-hydroxy-3-nitrophenyl) -propionic acid methyl ester (4.10g, 12.05mmol), after stirring for 2 minutes, phosphorus oxychloride (2.00ml, 21.45mmol) was added, the reaction mixture was stirred at room temperature for 8 hours, cooled to 0 ℃, a solution of sodium bicarbonate (3.5g, 41.60mmol) and water (20 ml) was slowly added thereto, stirring was continued at 0 ℃ overnight, and then concentrated, purified by a C-18 column (20 x4 cm), gradient eluted, 25ml/min, A:0.5% acetic acid, B: methanol, 100A held for 10 minutes, and then subjected to 45 minutes to 75% A and 25B. The objective fraction was collected and concentrated to give the objective compound (4.20g, 83%). ESI M/z [ M-H ] ] ^- Ion peak: c ₁₅ H ₂₀ N ₂ O ₁₀ P,419.08, experimental value, 419.10.

Example 332.3- (3-amino-4-phosphonooxyphenyl) - (2R) -2- (tert-butoxycarbonylamino) -propionic acid.

The dried (S) -methyl 2- (tert-butoxycarbonylamino) -3- (3-nitro-4-phosphonoxyphenyl) -propionate (4.0g, 9.52mmol) was dissolved in a mixed solvent of 1, 2-dichloroethane (50 ml) and N, N-dimethylacetamide (60 ml), and trimethyltin hydroxide (4.00g, 22.1mmol) was added to the solution. The mixture was reacted at 80 ℃ for 6 hours, concentrated, subjected to column chromatography (water/acetonitrile 1 4), and the target component was collected and concentrated to give (S) -2- (tert-butoxycarbonylamino) -3- (3-nitro-4-phosphonoxyphenyl) -propionic acid. The resulting compound was charged into a hydrogen reaction flask together with N, N-dimethylacetamide (70 ml) and 10% Pd/C (0.3 g). The reaction was carried out under 35psi of hydrogen pressure for 6 hours. The mixture was filtered through celite, concentrated, and recrystallized to give the desired product (2.86g, 80%) which was used in the next reaction without further purification. ESI M/z [ M-H ]] ^- Ion peak: c ₁₄ H ₂₀ N ₂ O ₈ P,375.10, experimental value, 375.10.

Example 333.3- (4-benzyloxyphenyl) - (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -propionic acid benzyl ester.

To a solution of benzyl 3- (4-benzyloxyphenyl) - (2R) -2- [ (tert-butoxycarbonylamino) -propionate (4.0g, 8.67mmol) in tetrahydrofuran (60 ml) was added sodium hydride (430mg, 10.75mmol,60% oil), and after stirring at room temperature for 1 hour, iodomethane (1.82g, 12.82mmol) was added, the mixture was stirred overnight, the reaction was quenched with methanol (0.5 ml), concentrated, and column-chromatographed (ethyl acetate/dichloromethane, 1. MS ESI M/z [ M + Na ] ] ⁺ Ion peak: c ₂₉ H ₃₃ NNaO ₅ 498.24, experimental value 498.24.

EXAMPLE 334 (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -3- (4-hydroxy-2-nitrophenyl) propionic acid.

To a hydrogen reaction flask was added 3- (4-benzyloxyphenyl) - (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] in that order]Benzyl propionate (3.8g, 8.00mmol), methanol (80 ml), 10% Pd/C (0.3 g). After reacting under 35psi of hydrogen for 6 hours, the reaction mixture was filtered through Celite and concentrated to give a crude product (2R) -2- [ (tert-butoxycarbonyl) -methylamino]-3- (4-hydroxyphenyl) propionic acid, which compound was not further purified. To a solution of this compound in anhydrous dichloromethane (240 ml) at-25 ℃ was added a solution of tin tetrachloride (1.5ml, 12.75mmol) and fuming nitric acid (0.60ml, 12.98mmol) in dichloromethane (40 ml), the mixture was stirred at-25 ℃ for 75 minutes, quenched with saturated sodium bicarbonate, adjusted to pH =3-4, the aqueous phase was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, column chromatographed (MeOH/DCM/HOAc 1 8. ESI M/z [ M + Na ]] ⁺ Ion peak: c ₁₅ H ₂₀ N ₂ NaO ₇ 363.13, experimental value, 363.13.

EXAMPLE 335 (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -3- (3-nitro-4-phosphonoxyphenyl) propionic acid.

Reacting (2R) -2- [ (tert-butoxycarbonyl) -methylamino]-3- (4-hydroxy-2-nitrophenyl) propionic acid (1.98g, 5.82mmol) was dissolved in acetonitrile (30 ml) and N, N-dimethylacetamide (30 ml), and the resulting solution was added N, N' -diisopropylethylamine (2.00ml, 11.50mmol) at 0 ℃ and after reacting for two minutes at that temperature, phosphorus oxychloride (1.10ml, 11.79mmol) was added. After the reaction mixture was stirred at room temperature for 8 hours, it was cooled to 0 ℃ and a solution of sodium bicarbonate (2.0 g, 23.80mmol) and water (10 ml) was slowly added thereto, and the mixture was further stirred at 0 ℃ overnight, the mixture was concentrated, purified by a C-18 column (20X 4 cm), gradient eluted at 25ml/min, A:0.5% acetic acid, B: methanol, 100% A was held for 10 minutes, and then 45 minutes to 75% A and 25% B were added. The target fractions were collected and concentrated to give the target compound (1.96, 80%). ESI M/z [ M-H ]] ^- Ion peak: c ₁₅ H ₂₀ N ₂ O ₁₀ P,419.09, experimental value, 419.09.

Example 336.3- (3-amino-4-phosphonoxyphenyl) - (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] propionic acid.

To a hydrogen reaction flask were added (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] sequentially]-3- (3-nitro-4-phosphonooxyphenyl) propionic acid (1.96g, 4.67mmol), N, N-dimethylacetamide (60 ml), 10% by weight Pd/C (0.2 g). The reaction was carried out under 30psi of hydrogen for 6 hours. The mixture was filtered through celite and concentrated to give the crude product (1.74g, 95%) which was used in the next reaction without further purification. ESI M/z [ M-H ] ]-,calcd forC ₁₅ H ₂₂ N ₂ O ₈ P,389.12,Found,389.12.

Example 337 phenyl-2- (2R) -tert-butoxycarbonylamino-1-propanone.

(1S, 2R) - (+) -demethylephedrine (7.0g, 46.29mmol) was added to a mixture of tetrahydrofuran (40 ml) and 1M sodium hydrogencarbonate (100 ml), a tetrahydrofuran (60 ml) solution of di-tert-butyl dicarbonate (10.15g, 46.53mmol) was slowly added over 45 minutes at a temperature of 4 ℃, the reaction mixture was stirred at room temperature for 6 hours, concentrated, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, subjected to column chromatography (EtOAc/Hexane 1] ⁺ Ion peak: c ₁₄ H ₂₁ NaNO ₃ 274.15, experimental value, 274.15. To a solution of this compound in dichloromethane (50 ml) was added a solution of dess-martin reagent in dichloromethane (180ml, 0.3m), stirred for one hour, frozen sodium hydroxide solution (1m, 100ml) was added to the mixture, separated, the organic phase was adjusted to pH =6 with 1M monobasic sodium phosphate (100 ml), dried over anhydrous sodium sulfate, filtered, concentrated, column chromatographed (EtOAc/Hexane 1) to give the desired product (9.34g, 81 in two steps. MS ESI M/z + [ M + Na ] ] ⁺ Ion peak: c ₁₄ H ₁₉ NaNO ₃ 272.14, experimental value, 272.14.

Example 338.2, 5-dioxopyrrolidin-1-yl 2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) valerylamino) -4-methylpentyl) thiazole-4-carboxylic acid ethyl ester.

Add N-hydroxysuccinimide (202.0 mg, 1.756mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (980mg, 5.104mmol) to a solution of Compound 33 (788.1mg, 1.464mmol) in N, N' -dimethylformamide (10 ml), stir the mixture overnight, concentrate, and perform column chromatography (EtOAc/CH) ₂ Cl ₂ = 1). MS ESI M/z + [ M + Na ]] ⁺ Ion peak: c ₃₀ H ₄₅ NaN ₅ O ₈ S,658.30, experimental value, 658.30.

EXAMPLE 339- (4R) -4- (tert-Butoxycarbonylamino) -5- (3- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid.

To a solution of (4R) -5- (3-amino-4-phosphonooxyphenyl) -4- (tert-butoxycarbonylamino) -2-methylpentanoic acid (825.1mg, 1.973mmol) in N, N '-dimethylformamide (7 ml) was added ethyl 2, 5-dioxopyrrolidin-1-yl-5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) valerate (711mg, 2.417mmol), N, N' -diisopropylethylamine (0.250ml, 1.438mmol), and after stirring the mixture overnight, the mixture was concentrated, and C-18 column chromatography (4x25cm, v =15ml/min, from 100% 1 HOAc to 75% 1 HOAc/25 MeOH in 45min) gave the target compound (895.7mg, 76%). MS ESI M/z- [ M-H ] ] ^- Ion peak: c ₂₆ H ₃₅ N ₃ O ₁₁ P,596.21, experimental value, 596.21.

EXAMPLE 340 (4R) -4- (tert-butoxycarbonylamino) -5- (3- (3- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid.

To a solution of (4R) -5- (3-amino-4-phosphonoxyphenyl) -4- (tert-butoxycarbonylamino) -2-methylpentanoic acid (632.5mg, 1.512mmol) in N, N '-dimethylformamide (7 ml) was added ethyl 2, 5-dioxopyrrolidin-1-yl-3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionate (727mg, 1.826 mmol), N, N' -diisopropylethylamine (0.250ml, 1.438mmol), and the mixture was stirred overnight, concentrated, and C-18 (4x25cm, v =, 15ml/min, column chromatography from 100% 1 HOAc to 75% 1 HOAc/25 MeOH in 4576min) yielded the target compound (3.2mg, 72%).MS ESI:m/z-:[M-H] ^- Ion peak: c ₃₀ H ₄₄ N ₃ O ₁₄ P,700.25, experimental value, 700.25.

EXAMPLE 341 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid.

To a solution of (4R) -4- (tert-butoxycarbonylamino) -5- (3- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (102mg, 0.171mmol) in 1, 4-dioxane (4 ml) was added concentrated hydrochloric acid (1.0 ml, 37%) and the mixture was stirred for 30 minutes and concentrated to dryness to give a crude Boc product. This crude product was dissolved in N, N-dimethylacetamide (5 ml), and to this solution was added ethyl 2, 5-dioxopyrrolidin-1-yl 2- ((1r, 3r) -1-acetoxy-3- ((2s, 3s) -N, 3-dimethyl-2- ((R) -1-methylpiperidin-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxylate (110mg, 0.173mmol), N' -diisopropylethylamine (30ul, 0.172mmol) in this order, and after the mixture was stirred overnight, it was concentrated, column-chromatographed (water/acetone containing 1 hoac, 1. MS ESI M/z- [ M-H ]] ^- Ion peak: c ₄₇ H ₆₇ N ₇ O ₁₄ PS,1016.42, experimental value, 1016.42.

EXAMPLE 342 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -3- (3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid.

To a solution of (4R) -4- (tert-butoxycarbonylamino) -5- (3- (3- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (108mg, 0.154mmol) in 1, 4-dioxane (4 ml) was added concentrated hydrochloric acid (1.0 ml, 37%) and the mixture was stirred for 30 minutes and then concentrated to dryness to give the crude Boc-removed product. This crude product was dissolved in N, N-dimethylacetamide (5 ml), to which was added ethyl 2, 5-dioxopyrrolidin-1-yl 2- ((1r, 3r) -1-acetoxy-3- ((2s, 3s) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) valeramido) -4-methylpentyl) thiazole-4-carboxylate (110mg, 0.173mmol), N' -diisopropylethylamine (30ul, 0.172mmol) in that order, and after stirring the mixture overnight, concentration, column chromatography (water/acetone containing 1 hoac, 1. MS ESI M/z- [ M-H ]] ^- Ion peak: c ₅₁ H ₇₅ N ₇ O ₁₇ PS,1120.47, experimental value, 1120.48.

EXAMPLE 343 (4R) -4- (tert-butoxycarbonylamino) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridin-2-dithio) butanamido) phenyl) pentanoic acid.

To a solution of (4R) -5- (3-amino-4-phosphonooxyphenyl) -4- (tert-butoxycarbonylamino) -2-methylpentanoic acid (548.3mg, 1.311mmol) in N, N '-dimethylformamide (10 ml) were added succinimidyl 4- (pyridine-2-dithio) butyric acid (550.2mg, 1.687 mmol) and N, N' -diisopropylethylamine (0.18ml, 1.03mmol), and after overnight stirring the mixture, concentration and column chromatography (water/acetone containing 1 hoac, 1. MS ESI M/z- [ M-H ] ] ^- Ion peak: c ₂₆ H ₃₅ N ₃ O ₉ PS ₂ 628.16, experimental value, 628.16.

EXAMPLE 344 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridine-2-dithio) butyrylamino) phenyl) pentanoic acid

To a solution of (4R) -4- (tert-butoxycarbonylamino) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridin-2-dithio) butyrylamino) phenyl) pentanoic acid (110.5 mg, 0.175mmol) in 1, 4-dioxane (4 ml) was added concentrated hydrochloric acid (1.0 ml, 37%) at a temperature of 4 ℃, and the mixture was stirred for 30 minutes and then concentrated to dryness to give a crude Boc-removed product. This crude product was dissolved in N, N-dimethylacetamide (5 ml), to which was added ethyl 2, 5-dioxopyrrolidin-1-yl 2- ((1r, 3r) -1-acetoxy-3- ((2s, 3s) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) valeramido) -4-methylpentyl) thiazole-4-carboxylate (110mg, 0.173mmol), N' -diisopropylethylamine (30ul, 0.172mmol) in that order, and after stirring the mixture overnight, concentration, column chromatography (water/acetone containing 1 hoac, 1. MS ESI M/z- [ M-H ]] ^- Ion peak: c ₄₇ H ₆₇ N ₇ O ₁₂ PS ₃ 1048.38, experimental value, 1048.38.

EXAMPLE 345 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) valerylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (4-mercaptobutyrylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid.

(4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) valerylamino) -4-methylpentyl) thiazole-4-carboxamido) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridine-2-dithio) butyrylamino) phenyl) pentanoic acid (30mg, 0.0285mmol) was added to N, N-dimethylacetamide (2 ml) and sodium dihydrogen phosphate (0.1M, pH 7), and dithiothreitol (20mg, 0.129mmol) was added to the mixture. Stirring the mixture for 2 hours, concentrating, and performing column chromatography (with H containing 1% HOAc) ₂ O/MeCN, 1. MS ESI M/z- [ M-H ]] ^- Ion peak: c ₄₂ H ₆₄ N ₆ O ₁₂ PS ₂ 939.38, experimental value 939.38.

Example 346.4- (4-bromobutyl) -10-oxa-4-azatricyclo [5.2.1.0 {2,6} ] dec-8-ene-3, 5-dione.

Subjecting 10-oxa-4-azatricyclo [5.2.1.0^ {2,6}]Dec-8-ene-3, 5-dione (6.0g, 36.35mmol) and sodium hydride (60% oil, 1.50g, 37.50mmol) were added to N, N-dimethylacetamide (60 ml), and after stirring for 1 hour, 1, 4-dibromobutane (35.0g, 162.10mmol) and sodium iodide (0.50g, 3.33mmol) were added. The mixture was stirred overnight, quenched with methanol (0.5 ml), concentrated, and column chromatographed (EtOAc/Hexane = 1) to give the title compound (9.34g, 86%). MS ESI M/z + [ M + Na ] ] ⁺ Ion peak: c ₁₂ H ₁₄ BrNaNO ₃ 322.02, experimental value, 322.02.

Example 347 methyl 2- ((1R, 3R) -3- (tert-butoxycarbonylamino) -1- [4' - (3 ",6" -endoxy-tetrahydrophthalimido) butoxy ] -4-methylpentyl) thiazole-4-carboxylate.

Methyl 2- ((1R, 3R) -3- (tert-butoxycarbonylamino) -1-hydroxy-4-methylpentyl) thiazole-4-carboxylate (1.0g, 2.79mmol) and sodium hydride (120mg, 3.00mmol,60% oil) were added to tetrahydrofuran (30 ml), and after stirring for 30 minutes, 4- (4-bromobutyl) -10-oxa-4-azatricyclo [5.2.1.0^ 2,6 }was added]Dec-8-ene-3, 5-dione (1.00g, 3.34mmol) and sodium iodide (50mg, 0.33mmol). The mixture was stirred overnight, quenched with methanol (0.5 ml), concentrated, and column chromatographed (EtOAc/CH) ₂ Cl ₂ = 1. MS ESI M/z + [ M + Na ]] ⁺ Ion peak: c ₂₈ H ₃₉ NaN ₃ O ₈ S,600.25, experimental value, 600.25.

Example 348 methyl 2- ((1R, 3R) -3- (N, N-tert-butoxycarbonylmethylamino) -1- [4' - (3 ",6" -endoxy-tetrahydrophthalimido) butoxy ] -4-methylpentyl) thiazole-4-carboxylate.

Methyl 2- ((1R, 3R) -3- (tert-butoxycarbonylamino) -1- [4' - (3 ', 6 ' -endoxy-tetrahydrophthalimido) butoxy]Methyl (4-methylpentyl) thiazole-4-carboxylate (1.30g, 2.25mmol) and sodium hydride (108mg, 2.70mmol,60% oil) were added to N, N-dimethylformamide (80 ml), and after stirring for 1 hour, iodomethane (460mg, 3.24mmol) was added. The mixture was stirred overnight, concentrated, and column chromatographed (EtOAc/CH) ₂ Cl ₂ 12-1). MS ESI M/z + [ M + Na ]] ⁺ Ion peak: c ₂₉ H ₄₁ NaN ₃ O ₈ S,614.26,Found,614.26.

Example 349.2- ((1R, 3R) -3- (N, N-tert-butoxycarbonylmethylamino) -1- [4' - (3 ",6" -endoxy-tetrahydrophthaloylamino) butoxy ] -4-methylpentyl) thiazole-4-carboxylic acid.

The dried methyl 2- ((1R, 3R) -3- (N, N-tert-butoxycarbonylmethylamino) -1- [4' - (3 ', 6 ' -endoxy-tetrahydrophthalimido) butoxy]Methyl (900mg, 1.52mmol) of (E) -4-methylpentyl) thiazole-4-carboxylate was dissolved in a mixed solvent of 1, 2-dichloroethane (30 ml) and toluene, and trimethyltin hydroxide (400mg, 2.21mmol) was added thereto. The mixture is stirred at 100 ℃ overnight, concentrated and subjected to column chromatography (MeOH/CH) ₂ Cl ₂ /HOAc = 1. ESI M/z [ M + Na ]] ⁺ Ion peak: c ₂₈ H ₃₉ N ₃ NaO ₈ S,600.22, experimental value, 600.22.

Example 350 methyl 2- ((1R, 3R) -1-acetoxy-3- (N, N- (tert-butoxycarbonyl) (4' - (3 ",6" -endoxy-tetrahydro) butyl) amino) -4-methylpentyl) -thiazole-4-carboxylic acid.

2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylic acid methyl ester (1.50g, 3.74mmol) and sodium hydride (180mg, 4.50mmol,60% oil) were added to N, N-dimethylformamide (80 ml), and after stirring for 1 hour, 4- (4-bromobutyl) -10-oxa-4-azatricyclo [5.2.1.0^ {2,6} was added ]Dec-8-ene-3, 5-dione (1.48g, 4.94mmol) and iodomethane (70mg, 0.467mmol). The mixture was stirred overnight, concentrated, and column chromatographed (EtOAc/CH) ₂ Cl ₂ 10-1). MS ESI M/z + [ M + Na ]] ⁺ Ion peak: c ₃₀ H ₄₁ NaN ₃ O ₉ S,642.26, experimental value, 642.26.

Example 351.2- ((1R, 3R) -1-acetoxy-3- (N, N- (tert-butoxycarbonyl) (4' -maleimidobutyl) amino) -4-methylpentyl) -thiazole-4-carboxylic acid.

The dried methyl 2- ((1R, 3R) -1-acetoxy-3- (N, N- (tert-butoxycarbonyl) (4' - (3 ",6" -endoxy-tetrahydro) butyl) amino) -4-methylpentyl) -thiazole-4-carboxylic acid (800mg, 1.29mmol) was dissolved in a mixed solvent of 1, 2-dichloroethane (40 ml) and toluene, and trimethyltin hydroxide (400mg, 2.21mmol) was added. The mixture was stirred at 100 ℃ overnight, concentrated and subjected to column chromatography (MeOH/CH) ₂ Cl ₂ /HOAc = 1), the fractions were collected, concentrated and dried. The resulting crude product was dissolved in pyridine (15 ml), acetic anhydride (0.3 ml, 3.17mmol) was added to the solution at 0 deg.C, the mixture was stirred at room temperature overnight, concentrated, and subjected to column chromatography (MeOH/CH) ₂ Cl ₂ HOAc =1, 10.01) to yield the target compound (578.4 mg, 74%). ESI M/z [ M + Na ]] ⁺ Ion peak: c ₂₉ H ₃₉ N ₃ NaO ₉ S,628.24, experimental value, 628.24.

Example 352.1- (2-methyl-2-oxiranyl) -2-phenylethylamine

To a solution of tert-butyl 1- (2-methyl-2-oxiranyl) -2-phenylethyl) carbamate (Sun, L.et al, J.mol.Catalysis A: chem.,2005,234 (1-2), 29-34) (300mg, 1.08mmol) in 1, 4-dioxane (8 mL) was added concentrated hydrochloric acid (37%, 2 mL) at a temperature of 0 deg.C, at which temperature stirring was continued for 1 hour, TLC showing no starting reaction material. The resulting mixture was diluted with toluene (10 ml), concentrated, and recrystallized from EtOH/Hexane to give the hydrochloride salt of the objective compound (201mg, 87%). ESI M/z [ M + H ]] ⁺ Ion peak: c ₁₁ H ₁₆ NO,178.12, experimental value, 178.12.

Example 353 Ethyl (S) -5-phenyl-4- (tert-butoxycarbonylamino) -2-methylpentenoate.

Diisobutylaluminum hydride (40ml, 40mmol, 1.0M) was slowly added to (S) -3-phenyl-2- (tert-butoxycarbonylamino) -propionic acid methyl ester (5.60g, 20.05mmol) in CH at-78 deg.C ₂ Cl ₂ To a solution (80 ml), after 45 minutes of reaction, a ylide solution formed of a solution of 1- (1-ethoxycarbonylethyl) triphenyl phosphonium bromide (18.0g, 40.64mmol) and KOtBu (5.00g, 44.64mmol) in dichloromethane (80 ml) was added at that temperature, and after the mixture was reacted at-78 ℃ for 1 hour, stirring was performed at room temperature overnight, to the mixture was further added 1 liter of a sodium dihydrogen phosphate solution, followed by vigorous stirring, liquid separation, extraction of the aqueous phase with dichloromethane, combination of organic layers, drying over anhydrous sodium sulfate, concentration, column chromatography (ethyl acetate/n-hexane =1 ] ⁺ Ion peak: c ₁₉ H ₂₇ NNaO ₄ ,356.19Experimental value, 356.20.

Example 354 general procedure for polypeptide condensation

The hydrochloride salt of the amine is dissolved in dichloromethane or N, N-dimethylformamide (0.2M), cooled to 4 ℃ and the tert-butoxycarbonyl protected amino acid (1.3 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2 eq), or O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate (2 eq), or tripyrrolidinylphosphonium bromide hexafluorophosphate (2 eq), 1-hydroxybenzotriazole (1.5 eq), N, N-diisopropylethylamine (3.5 eq) are added in sequence. The reaction was slowly warmed to room temperature and continued for 15 hours, diluted with ethyl acetate, and then washed successively with 1M aqueous hydrochloric acid, saturated sodium bicarbonate, water and saturated aqueous sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (0% to 20% meoh.

EXAMPLE 355 general procedure for the removal of tert-butoxycarbonyl

The tert-butoxycarbonyl protected amino acid was dissolved in a 20% trifluoroacetic acid in dichloromethane or 4M hydrochloric acid in 1, 4-dioxane and stirred for 30 minutes or followed by TLC to determine completion of the reaction. Concentrating under reduced pressure to obtain corresponding polypeptide compound of trifluoroacetate or hydrochloride. The polypeptide containing trifluoroacetate salt can be concentrated 3-4 times with a dichloromethane/toluene solution containing 2% hydrochloric acid to form the corresponding hydrochloride compound.

Example 356 general procedure for Solid Phase Peptide Synthesis (SPPS)

The tert-butoxycarbonyl protected SPPS uses Merrifield resin or modified PAM resin or MBHA resin. The 9-fluorenylmethoxycarbonyl protected SPPS used was Wang resin or 2-chlorotrityl chloride resin, or HMPB, MBHA resin. The resin was pre-treated (pre-swollen) and loaded with amino compound according to the resin manufacturer's instructions. The amino acid protected with t-butoxycarbonyl group on the resin was deprotected with 20% trifluoroacetic acid in dichloromethane or 4M hydrochloric acid in 1, 4-dioxane (stirred for 30 minutes) and washed sequentially with N, N-dimethylformamide, methanol, 50% N, N-diisopropylethylamine in dichloromethane and pure dichloromethane, and this step was repeated one more time before acylation for the deprotection step involving several free amines to ensure completion of the reaction. The free amine was suspended in a solution consisting of protected amino acid (3 equivalents of free amine), O-benzotriazol-N, N' -tetramethyluronium tetrafluoroborate or trispyrrolidinylphosphonium bromide hexafluorophosphate (3 equivalents of free amine) and N, N-diisopropylethylamine (5 equivalents of free amine), mixed for 4 hours and washed sequentially with N, N-dimethylformamide, methanol and dichloromethane. For reactions involving multiple free amine acylations, the coupling step before deprotection needs to be repeated once more to ensure completion of the reaction. These steps are generally repeated until the desired peptide is synthesized.

EXAMPLE 357 general procedure for cleaving peptides from Wang resin or 2-Chlorotriphenylmethyl chloride resin

The peptide-bound Wang resin was mixed with 50% trifluoroacetic acid in dichloromethane and triisopropylsilane (1-5%), or 2-chlorotrityl chloride resin-bound peptide was mixed with 1% trifluoroacetic acid in dichloromethane, mixed for 2 hours, filtered, eluted sequentially with dichloromethane (3 × 30 ml) and methanol (3 × 30 ml), the filtrates were combined, concentrated, dried and cold ether was added, and the resulting precipitate was the deprotected peptide.

EXAMPLE 358 general procedure for cleaving peptides from Merrifield resin, modified PAM resin or MBHA resin

Combining the peptide-bound resin with HF/Me ₂ S/anisole (10 ₃ SO ₃ H/Me ₂ S/anisole (20 ₂ S/p-thiocresol (10. Then eluting the resin with dichloromethane (3 × 30 ml) and methanol (3 × 30 ml) in sequence, combining the filtrates, concentrating, drying, and adding cold ether to obtain precipitate as the deprotected peptide.

Example 359 chromatographic purification

The crude polypeptide mixture is purified by column chromatography on silica gel (5% to 25% methanol in dichloromethane) or by reverse phase HPLC (gradient elution from 0% to 70% methanol in water (preferably with the addition of 1% acetic acid), the reaction is completed within 1 hour, the target fractions are mixed and the sample is collected after evaporation.

Example 360 preparation of conjugates

Antibodies, as a class of binding molecules, can be conjugated to the cleavage inhibitors via amide, thioether, or disulfide bonds. The antibody (> 5 mg/mL) was diluted with 50mM sodium borate in PBS buffer (pH 8.0), dithiothreitol (10 mM final concentration) was added, and the antibody released free thiol groups by treatment at 35 ℃ for 30 minutes. Approximately 8 thiol groups were coupled to each antibody as determined by Ellman's reagent [5,5' -dithiobis (2-nitrobenzoic acid) ] after gel filtration chromatography on a G-25 column (1 mM EDTA in PBS buffer). The antibody can also release a thiol group with Traut' S reagent (2-iminothiophene) (Jue, R., et al. Biochem.1978,17 (25): 5399-5405), or can react with a different linker such as SATP (N-succinimide-S-acetylthiopropionate) or N-succinimide-S-acetyl (thiotetra-acetic acid) (SAT (PEG) 4) at pH 7-8, followed by hydroxylamine to form a thiol group (Duncan, R, et al, anal. Biochem.1983,132,68-73, fuji, N.et al, chem. Pharm. Bull.1985,33, 362-367). Basically, 5 to 8 thiol molecules are attached to each antibody molecule.

Drugs containing maleimide or bromoacetamide groups (requiring 0.5M sodium borate solution (pH 9) to promote alkylation of antibody with bromoacetamide) were added to ice-cold Dimethylacetamide (DMA) containing free thiol group (2-20% v/v) at 4 ℃ (the molar ratio of attached drug to thiol group should be 1.2-1.5. After 1-2 hours, adding excessive cysteine to terminate the reaction; the concentrated coupling product is obtained by ultrafiltration, gel chromatography (G-25, buffer PBS) and sterile filtration. The protein concentration and the number of drugs attached to each antibody were determined by measuring the absorbance at 280nm and 252 nm. Size exclusion HPLC can be used to determine the specific gravity of the monomeric form of the linker, while less than 0.5% of unbound drug can be determined using RP-HPLC. For monomeric drugs formed by thioether linkages, an average of 3.2-4.2 small molecules of mitotic inhibitor will be attached per antibody molecule.

The linker may be classified into dimethyl (phenyl) silyl (DMPS), SMDP, 4-succinimidyloxycarbonyl-methyl-alpha (2-pyridyldithio) toluene (SMPT), N-succinimidyl-4- (2-pyridylthio) valerate (SPP), N-succinimidyl-4- (2-pyridylthio) propionate (SPDP), N-succinimidyl-4- (2-pyridylthio) butyrate (SPDB), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide- (polyethylene glycol) N-maleimide (SM (PEG) N), and the like. The antibody (> 5 mg/mL) was diluted in a buffer (pH 6.5-7.5, 5mM PBS,50mM NaCl, and 1mM EDTA) and reacted with the linker for 2 hours, and the molar ratio of the linker to the antibody was 6-to 10-fold or more. The reaction mixture can be separated by Sephadex G25 gel chromatography, and the lower molecular weight molecules can be removed. (the concentration of antibody is determined spectrophotometrically, the linker contains a pyridylthio group the extinction coefficient of the antibody at 280nm is 2067550M-1cm-1. The modified antibody, after treatment with excess dithiothreitol (20 fold or more equivalent), releases 2-thiopyridine groups are determined, with extinction coefficients at 343 nm and 280nm of 8080 and 5100M-1cm-1, respectively). Adding 1.2-1.5 equivalents of Tubulysin derivative molecules with sulfhydryl groups into the modified antibody. The reaction is carried out at room temperature for 5 to 18 hours. The reaction mixture was subjected to Sephadex G25 gel chromatography to remove the unbound drug or other low molecular weight substances. The concentration of the ligation product was then determined by measuring the absorbance at 280nm and 252 nm. The linking product is in a monomer form, and on average, 3.2 to 4.5 drug molecules are linked to each antibody molecule.

Example 361 general procedure for the preparation of conjugates containing Tubulysin derivative molecules reactive with sulfhydryl groups.

To 2.0ml of a 10 mg/ml solution of the HER2 antibody having a pH of 6.0 to 8.0, 0.70ml to 2.0ml of 100mM phosphoric acid (PBS), a pH of 6.5 to 8.5 buffer, TCEP (16-20. Mu.l, 20mM in water) was added, and incubated at room temperature to 37.5 ℃ for 1 to 4 hours, and then the same amount of an azide compound (azidobenzoic acid, or 2- (2- (2-hydroxyethoxy) ethoxy azide) was added and incubated at room temperature to 37.5 ℃ for 1 to 4 hours, followed by addition of a Tubulysin derivative molecule (28-32. Mu.l, 20mM DMA solution) containing a molecule capable of reacting with a thiol group. The mixture was incubated at room temperature to 37.5 ℃ for 4 to 18 hours and then DHAA (135. Mu.l, 50 mm) was added. After continuous culture at RT overnight, the mixture was purified over a G-25 column using 100mM phosphoric acid, 50mM NaCl, pH 6-7.5 buffer to give the conjugate compound (75% -90% yield). The drug/antibody ratio (DAR) was determined by liquid chromatography-mass spectrometry to be 3.1-4.2, with an HPLC analysis of 94-99% monomer (Tosoh Bioscience, tskgel G3000SW column, internal diameter 7.8 mm. Times.30 cm, 0.5 ml/min, 100 min).

Example 362 in vitro toxicity test

BJAB (Burkitt lymphoma cells), BT-474 (breast cancer cells), namalwa (human Burkitt lymphoma cells), ramos (human Burkitt lymphoma cells), N87 (gastric cancer cells) and SK-OV-3 (ovarian cancer cells) were purchased from ATCC. The cells were grown in RPMI 1640 medium containing 10% inactivated Fetal Bovine Serum (FBS) under 37 ℃ in 6% CO2 incubator. Colony formation assays can be used as a method for toxicity assay detection, see literature (Franken, et al, nature Protocols 1,2315-2319 (2006)). The test cells were seeded into 6-well plates at 5000 cells per well, and a gradient dilution of the drug (mitotic inhibitor or conjugate) ranging from 1pM to 50nM was added and incubated for 72 hours. The old culture medium is replaced, the cells are continuously cultured, and the clone is formed after 7 to 10 days. Cells were fixed, then stained with 0.2% crystal violet (diluted in 10% formalin or PBS) and cell clones counted. The number of untreated cells (medium only) can be determined by the number of clones formed in the well. Cell viability was calculated as the ratio of the number of colonies formed in wells treated with drug to those in control (drug-untreated). The IC50 values for inhibition of N87 (gastric cancer cells) by in vitro activity are given in Table one below.

Example 363 in vivo antitumor Activity (balb/c/c nude mice, NCI-N87 transplantation tumor).

In the xenograft model of human gastric cancer N-87 cell line tumor, her2 antibody-Tubulysin analog conjugate (conjugate) with the structural formula shown in table one was divided into 3 batches for efficacy comparison experiments.

First lot

129, 133, 181, 317, 467;

second lot

365, 377, 385, 412, 444, 474, 480, 546;

third batches

506, 522, 564, 574, 695, 677. The 3 batches of the medicineThe compounds were evaluated for in vivo efficacy in mice with T-DM1 and physiological saline (PBS)), respectively. Female balb/c/c nude mice five weeks old (three batches total 150 animals) have N-87 cancer cells under the right shoulder (5x 10) ⁷ Cells/mouse) were injected subcutaneously in serum-free medium. The tumors grew for 8 days with an average size of 130mm ³ . The animals were then randomly divided into groups (6 animals per group). Each group of mouse controls was treated with Phosphate Buffered Saline (PBS). The conjugate drug and T-DM1 were each administered to animals in a single intravenous dose of 6 mg/kg. Tumor volume was measured every 3-4 days in three dimensions, and tumor volume was calculated with the formula tumor volume =1/2 (length x width x height). The weight of the animals was also measured. The animals are killed when either of (1) the loss of body weight is more than 20% and (2) the tumor volume is greater than 2000mm ³ (3) too ill to feed and drink on its own, or (4) skin necrosis. If there is no significant tumor, the mice are considered tumor-free.

The results of the experiment are depicted in figures 60, 61 and 62. All 19 conjugates did not cause weight loss in the animals. Wherein the conjugate 133, 129, 317, 467, 365, 377, 385, 412, 444, 474, 480, 506, 522, 564, 574 and 677 shows stronger antitumor effect. In particular, conjugates 133, 129, 467, 377, 385, 412, 444, 474, 480, 677 showed better antitumor activity than T-DM 1.

TABLE 1 structural formulae of some antibody-Tubulysin derivative conjugates and their ICs ₅₀ Value of

Claims

1. A conjugate selected from the group consisting of:

wherein

n =1 to 8; the mAb is Her 2;

or a pharmaceutically acceptable salt thereof.

2. The conjugate of claim 1, selected from the group consisting of:

wherein

n =1 to 8; the mAb is Her 2;

or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising a conjugate as claimed in claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a diluent or excipient.

4. The pharmaceutical composition according to claim 3, having the following composition in weight percent:

the polyol compound is selected from one or more of fructose, mannose, maltose, lactose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, maltitol, xylitol, erythritol, sorbitol, glycerol or gluconic acid and metal salts thereof;

The surfactant is selected from polysorbate, poloxamer, triton, sodium dodecyl sulfate SDS, sodium lauryl sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, lauramidopropyl-, cocamidopropyl- (or palmitoamidopropyl) -betaine, myristamidopropyl-, cocamidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl taurate or disodium methyl oleoyl taurate; sodium lauryl, myristyl, linoleum or stearyl-ate, linoleum, myristyl, lauryl or cetyl betaine, sodium methyl cocoate, sodium dimethyl methyl taurate, cocoamphoglycine or isostearyl ethylenemidium ethosulfate, copolymer Pluronics F68 of polypropylene glycol, polyethylene glycol, ethylene and propylene; the polysorbate is polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 or polysorbate 85; the poloxamer is poloxamer 188, polyethylene oxide-polypropylene oxide, poloxamer 407 or polyethylene glycol-polypropylene glycol;

The complexing agent is EDTA or EGTA;

the preservative is selected from one of benzyl alcohol, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride, benzethonium chloride, butylbenzyl alcohol, methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol or m-cresol;

the antioxidant is ascorbic acid and/or methionine;

the amino acid or the propping agent is selected from one of mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, sodium citrate or NaCl;

the buffer solution is one of citric acid, succinic acid, acetic acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, tris hydrochloride, phthalic acid or phosphoric acid solution;

the pH value of the composition is 4.5-8.0, and the osmotic pressure of the liquid medicine of the composition is 225-370 Moscomo.

5. The pharmaceutical composition of claim 4, wherein the composition is prepared as a pre-filled injection, or filled into a vial and lyophilized into a needle, or prepared as a powder by high performance spray drying.

6. Use of a conjugate of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

7. A product comprising the pharmaceutical composition of any one of claims 3-5 in combination with an analgesic, wherein the analgesic is 1% benzyl alcohol, 0.2% to 2.0% procaine hydrochloride, 0.2% to 1.0% lidocaine hydrochloride, 0.3% to 0.5% chlorobutanol, tramadol, morphine sulfate, hydromorphone, oxycodone, gabapentin, cyclobenzaprine, trazodone, or clonidine.

8. Use of a pharmaceutical composition according to any one of claims 3-5 in the manufacture of a medicament for the treatment of cancer.

9. Use of a conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 3 to 5, in combination with a synergist in the preparation of a medicament for the treatment of cancer, wherein the synergist is selected from one or more of the following drugs: abiraplet, abiraterone acetate, albumin paclitaxel, paracetamol/hydrocodone, abiramate with CAS number 1420477-60-6, adamantil, recombinant human interleukin-2, afatinib dimaleate, elletinib, alemtuzumab, aliretinic acid, CAS number 1018448-65-1 Edotuzumab maytansine, amphetamine or amphetamine/dextroamphetamine mixed salt, anastrozole, anthracycline, aripiprazole, atazanavir, attributuzumab, atorvastatin, advacizumab with CAS number 1537032-82-8, abxitinib, bellistat with CAS number 866323-14-0, bevacizumab, besalutin, borassultap, bortezomib, botezomib, botuzumab, bugatitinib, boletinib, boletamib, bentuzumab, bugatitinib, budesonide, budesonide/formoterol, buprenorphine, cabazitaxel with CAS number 183133-96-2, cabozantinib, capecitabine, carfilzomib, cells of chimeric antigen receptor engineered T, celecoxib, ceritinib, cetuximab, cyclosporine, cinacalcet, crizotinib, cobicistinib with CAS number 934660-93-2, dabigatran, dabrafenib, dacarbazine, daclizumab, daptomycin, dermazumab with CAS number 945721-28-8, darbepoetin, darunavir with CAS number 206361-99-1, imatinib mesylate, dasatinib, disoproxil with CAS number 173146-27-5, dinebin-2 with CAS number 6158-49-7, dipalmitylmetin, dexamethasone, right lansoprazole with CAS number 530-94-6, methylphenidate, denouuximab with CAS number 1363687-32-4, doxycycline, duloxetine, dewemumab with CAS number 1428935-60-7, etorizumab with CAS number 915296-00-3, tenofovir/Efavirenz, etoritinib, ezetimibe/simvastatin, fenofibrate/Eratib, fingolimod, fluticasone propionate, fluticasone/Salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, imatinib, imatinib, imatinumab, esprutib, ibrutinib, ifosfamide, infinizumab, eritinib, asparanthin, insulin glargine, insulin lispro, interferon beta 1a, interferon beta 1b, and lapatinib, yipriomam, ipratropium bromide, linaglili, lenvatinib, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, CAS number 608137-33-3 lysine amphetamine, memantine, methylphenidate, metoprolol, modafinil, mometasone, cetuximab with CAS number 906805-06-9, lenatinib with CAS number 69887-09-6, nilotinib with CAS number 157641-10-0, nilapanib with CAS number 1038915-60-4, nirapanib with CAS number 946414-94-4, nivolumab, olmazumab, olmesartan, oxaliplatin, hydrochlorothiazide, omalizumab, ethyl omega-3 fatty acid ester, oseltamivir with CAS number 1373-65-0, oxpocitinib hydrochloride, oxycodone, ritorizanol hydrochloride, palizumab, palomab, pazopanib, PD-1 antibody, PD-L1 antibody, pevim interferon alpha-2a, pemetrexed, petuzumab, pneumococcal conjugate vaccine, pomaduramine, pregabalin, pranolol, quetiapine, rabeprazole, radium 223 chloride, raloxifen, laticavir, ramopuzumab, ranibizumab, rigefinitib, rituximab, rivaroxaban, romidexin, rosuvastatin, lusolinib, salbutamol, wollipirtinib with a CAS number of 1313725-88-0, wolitinib, somalide, sevelim, sildenafil, CAS number of 541502-14-1 Tuoximab, sitagliptin, sitagliptin/metformin, solifenacin, sonedgi, sorafenib, sunitinib, tacrolimus, temozolomide, temsirolimus, tenofovir/emtricitabine, testosterone gel, thalidomide, tiotropium bromide, toremifene, tremetinib, trastuzumab No. 114899-77-3 trasbrate tincture No. 183204-72-0 trifluorothymidine/teipidexine, tretinoin, eutex, valsartan, viliparib, vandetanib, vemurafenib, alloflacci no having CAS No. 1257044-40-8, vorinostat, aprepirubicin, herpes zoster vaccine, and pharmaceutically acceptable salts or excipients thereof.