CN109912683A

CN109912683A - A kind of cytotoxic molecule, conjugate and its preparation method and application

Info

Publication number: CN109912683A
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: 2019-06-21
Anticipated expiration: 2037-12-13
Also published as: CN109912683B; CN115505029A

Abstract

The present invention relates to a kind of Tubulysin derivative cytotoxic molecule, its conjugate to be formed (conjugated body), the application of conjugate synthetic method, conjugate formulations and these conjugates in terms of targeted therapy of cancer, self-immunity systems disease and infectious diseases are coupled with cell surface receptor molecule.

Description

A kind of cytotoxic molecule, conjugate and its preparation method and application

Technical field

The present invention relates to Tubulysin derivative species cytotoxic molecules, are coupled to be formed with cell surface receptor molecule Conjugate (conjugated body), conjugate synthetic method, conjugate formulations and conjugate in targeted therapy of cancer, self-immunity systems Application in terms of disease and infectious diseases.

Background technique

Targeted therapy of cancer, immune deficiency and infectious diseases etc. are the cores of accurate medical attention at present.Permitted for many years More document reports form conjugate using cell surface receptor binding molecule as drug means of delivery and cytotoxic molecule (conjugated body), carry out targeting conveying all kinds of pathogenic cells of cytotoxic molecule attack (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.).This cell surface receptor binding molecule-small-molecule drug conjugate can improve small point of classic chemotherapy The pharmacokinetics and pharmacodynamics of sub- drug, at the same reduce traditional chemical drug in human body toxic side effect (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).What is had been reported conveys work as drug The cell surface receptor binding molecule of tool has: peptide molecule (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；Temming,et al, Bioconjugate Chem.2006,17,1385-1394)；Antibody (Ducry, L. (2013) Antibody-drug conjugates,New York,Humana Press；Springer,ISBN 9781627035408；Sela et al,in Immunoconjugates 189-216(C.Vogel,ed.1987)； Ghose et al,in Targeted Drugs 1-22 (E.Goldberg,ed.1983)；Diener et al,in Antibody mediated 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 Oncology 2007,4,245-255； Singh R.et Rickson H.K.,Therapeutic Antibodies:Methods and Protocols,2009, 525,445-467)；Small molecule class such as folic acid (Kumar, R., W.S.Shin,et al.(2015)Chem Soc Rev 44(19):6670-6683；Leamon,C.P.and P.S.Low (2001),Drug Discov Today 6(1):44-51；Lu,Y.,E.Sega,et al.(2004),Adv Drug Deliv Rev 56(8):1161-1176； Reddy,et al,2009,Mol.Pharm.6,1518-25)；Single domain antibody or antibody are similar Object (Wold, E. D., J.Y.Axup, et al. (2015), Bioconjug Chem 26 (12): 2311-2314)；Prostate is special Specific membran antigene binding partner (PMSA) (Low, et al, WO 2009/026177A1)；Cobalamin and protein (Gupta, et al,Crit.Rev.Therap.Drug Carrier Syst.2008,25,347-79；Petrus,et al, Angew.Chem.Int.Ed.2009,48,1022-8)；Carbohydrate molecule (Darbre, et al, Curr.Top. Med.Chem..2008,8,1286-93；Zhang, Y., J.W.Chan, et al. (2015), J Control Release 219:355-368)；Bioactive polymer (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.)；Contain antibody or albumen point The macromolecule (Rihova, B. (1995), Folia Microbiol (Praha) 40 (4): 367-384) of son；Dendron shaped polymer (Bai,S.,C.Thomas,et al.(2006)Crit Rev Ther 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)；It is connected with nano particle (Ma, the P.and of binding partner 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)；Protease or protein (such as albumin) (Veronese, F.M.and M.Morpurgo (1999), Farmaco 54 (8): 497-516；Stehle,G.,A.Wunder, et al.(1997),Anticancer Drugs 8(9):835-844；Kratz,F.(2008),J Control Release 132(3):171-183；Fiume, L.,M.Manerba,et al.(2014)Expert Opin Drug Deliv 11(8): 1203-1217；Vhora,I., S.Patil,et al.(2015),Adv Protein Chem Struct Biol 98:1- 55)；Liposome (Medinai, et al, Curr.Phar.Des.2004,10,2981-9)；Viral capsid (Flenniken, et Al, Viruses Nanotechnol.2009,327,71-93), etc..In above-mentioned cell surface receptor binding molecule clinic at Ripe application is then antibody molecule.4 monoclonal antibody-drug conjugates of Administration of Food and Drug, the U.S. (FDA) approved at present Molecule is used for targeted therapy of cancer, this 4 conjugate drugs are Gemtuzumab ozogamicin, Brentuximab Vedotin, Trastuzumab emtansine and Inotuzumab ozogamicin.

Antibody-drug conjugates consist of three parts: antibody (often monoclonal antibody), cytotoxic molecule and connection Chain junctor between the two (Thomas, A., B.A.Teicher, et al. (2016), Lancet Oncol 17 (6): e254- e262).Respectively have unique function between three: antibody needs to combine tumor cell specific, and cytotoxic molecule needs Activity enough to tumour cell and universal, chain junctor need unique functional-stable in blood circulation, arrival tumour Cytotoxic molecule (Chari, R.V. (2008), Acc Chem Res 41 (1): 98-107), three are released effectively after cell Reasonable construction could obtain good clinical structure (Singh, S.K., D.L.Luisi, et al. (2015), Pharm Res 32 (11): 3541-3571；Hamilton, G.S. (2015), Biologicals 43 (5): 318-332).The cell of numerous species Toxin has been used to form antibody drug conjugated body (Wu, et al, Nat. with cell combination body especially antibody Biotechnol.2005,23,1137-1146.Ricart,et al,Nat.Clin.Pract.Oncol.2007,4, 245– 255).These cytotoxins include: calicheamicin derivative (Giles, et al Cancer 2003,98,2095-104； Hamann,et al,Bioconjug 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 derivative (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；United States Patent (USP) 6,441,163；6,716,821, 7,276,497,7,301,019,7,303,749,7, 368,565,7,411,063,7,851,432, and 8,163,888)； auristatins(Sutherland,et al,J Biol Chem 2006,281,10540-7；Doronina,et al, Bioconjug Chem 2006,17,114-24；United States Patent (USP) 7837995,7902338,7964566, 7964567,7851437,7994135.)；Tubulysin class (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/IB2012/053554) purple China fir class medicaments derivative (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)； United States Patent (USP) 7,276,499；7,598,290；With 7,667,054；WO 06061258), leptomycine derivative (WO 07144709), CC-1065 and the like object (Zhao, R., et al J. Med.Chem.2012,55,766-782； 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；WO 2007102069)； Amatoxin class (Moldenhauer, G., et al, J. Natl.Cancer Inst.2012,104,622-34； A.Moshnikova,et al；Biochemistry 2013,52, 1171–8；Zhao,L.,et al.,Chembiochem, 2015.16(10):1420-5；Zhou,B.,et al., Biosens Bioelectron,2015.68:189-96；WO2014/ 043403, US20150218220, EP 1661584, WO2017/046658)；A kind of Exatecan derivative (camptothecin point Son) (Nakada, T., T.Masuda, et al. (2016), Bioorg Med Chem Lett 26 (6): 1542-1545)；It cuts Junctor inhibitor (Puthenveetil, S., F.Loganzo, et al. (2016), Bioconjug Chem 27 (8): 1880- 1888)；Tall and erect nitrogen (PBD) dimer class (United States Patent (USP) 8,163,736 of benzene (simultaneously) two；8,153,627； 8,034,808；7,834, 005；7,741,319；WO2015028850)；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 (MTX), vincristine, vincaleukoblastinum, daunorubicin, Mitomycin C, melphalan and Chlorambucil derivative etc. (Wang, J., H.Xiao, et al. (2017), Trends Biotechnol 35 (5): 466-478, Puthenveetil, S., F.Loganzo, et al. (2016), Bioconjug Chem 27(8):1880-1888)。

In above-mentioned cytotoxin compounds molecule, Tubulysin class drug is strong due to its unique tumour cell The bioactivity of bioactivity, especially its cells of resistant tumors is imitated by concerned (Cohen, R., D. J.Vugts, et al. (2014),Cancer Res 74(20):5700-5710；Xiangming,X.,G.K. Friestad,et al,Mini Rev Med Chem 13(11):1572-1578；Pando,O.,S.Stark,et al. (2011),J Am Chem Soc 133 (20):7692-7695；Shibue,T.,I.Okamoto,et al. (2011),Bioorg Med Chem Lett 21(1): 431-434；Kubicek,K.,et al.(2010)Angew Chem Int Ed Engl 49(28):4809-4812； Nicolaou,K.C.,et al.(2016)J Am Chem Soc 138(5):1698-1708；Sani,M.,et al.(2017) Chemistry 23(24):5842-5850).We once reported even using Tubulysin analog construction cell-binding molecules Join object and the application (WO2014/009774 in targeted therapy；Huang, Y.Y.et al, American Society National Meeting, Med.Chem.#44,2014,Dever,CO,USA).Here we further application by The conjugate of Tubulysin class molecular configuration, and with such conjugate in targeted therapy of cancer, self-immunity systems disease and Application in terms of infectious diseases.

Summary of the invention

The present invention provides a kind of cytotoxic molecules, have structure shown in Formulas I:

Or using with structure shown in Formulas I as the pharmaceutically acceptable salt of parent, hydrate or hydrated salt；Or there is formula The optical isomer of structure shown in the polymorph or Formulas I of structure shown in I；

The R¹、R²、R³And R⁴Independently selected from H；C₁~C₈Alkyl or miscellaneous alkyl；C₂~C₈Allylic alkylation, alkynes alkyl, Heterocycle, Heterocyclylalkyl；C₃~C₈Naphthenic base, aryl, heteroaryl perfume base, aralkyl, alkyl-carbonyl；C₄~C₈Alkyl-cycloalkyl, Miscellaneous alkyl naphthenic base；

The R⁵、R⁶、R⁸、R¹⁰And R¹¹Independently selected from-H or C₁~C₄Alkyl or miscellaneous alkyl；

The R⁷Independently selected from H, R¹、-R¹⁵C (=O) X¹R¹⁶Or-R¹⁵X¹R¹⁶；

The X¹Selected from-O- ,-S ,-S-S ,-NH ,-CH₂Or-NR¹-；

The R⁹Selected from-H ,-OH ,=O ,-OR¹⁵,-OC (=O) R¹⁵,-OC (=O) NHR¹⁵,-OC (=O) R¹⁵SSR¹⁶、OP (=O) (OR¹⁵) or OR¹⁵OP (=O) (OR¹⁶)；

The R¹²Selected from-R¹⁵、-OH、-SH、-NH₂、-NH、-NHNH₂、-NH(R¹⁵)、-OR¹⁵、 -R¹⁵COR¹⁶、- R¹⁵COOR¹⁶、-R¹⁵C(O)NH₂、-R¹⁵C(O)NHR¹⁷、-SR¹⁶、R¹⁵S (=O) R¹⁶、 -R¹⁵P (=O) (OR¹⁷)₂、-R¹⁵OP (=O) (OR¹⁷)₂、-CH₂OP (=O) (OR¹⁷)₂、-R¹⁵SO₂R¹⁷、 -R¹⁵X²R¹⁶、-R¹⁵C (=O) X³；

The X²Selected from-O- ,-S- ,-NH- ,-NHNH- ,-N (R¹⁵)-、-O-R¹⁵-、-S-R¹⁵, S (=O)-R¹⁵Or- NHR¹⁵One of；

The X³Selected from-OH ,-SH ,-NH₂、-NH(R¹⁵)、-NHNH(R¹⁵)、-OR¹⁵、-S-R¹⁵Or-NR¹⁵R¹⁶In one Kind；

The R¹³And R¹⁴Independently selected from H ,-OH ,-SH ,-NH₂、-NHNH₂、-NH(R¹⁵)、 -OR¹⁵、-COX²、- COX²R¹⁶、-R¹⁷、-F、-Cl、-Br、-I、-SR¹⁶、-NR¹⁶R¹⁷,-N=NR¹⁶,-N=R¹⁶、-NO₂、-SOR¹⁶R¹⁷、-SO₂R¹⁶、- SO₃R¹⁶、-OSO₃R¹⁶、-PR¹⁶R¹⁷、-POR¹⁶R¹⁷、 -PO₂R¹⁶R¹⁷、-OP(O)(OR¹⁷)₂、-OCH₂OP(O)(OR¹⁷)₂、-OC(O) OP(O)(OR¹⁷)₂、 -PO(OR¹⁶)(OR¹⁷)、-OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、-OC(O)R¹⁷,-OC (O) NHR¹⁷；-O- (C₄-C₁₂Glucosides) ,-N- (C₄-C₁₂Glucosides)；C₁-C₈Alkyl, miscellaneous alkyl, C₂-C₈Alkenyl, alkynyl, heterocycle；C₃-C₈Carbocylic radical, Heterocyclylalkyl, miscellaneous alkyl naphthenic base, aryl, heteroarylalkyl, alkyl-carbonyl；C₄-C₈Alkyl-cycloalkyl；-NH(Aa)_1~4Or-CO (Aa)_1~4；The C₃-C₈Carbocylic radical includes naphthenic base；(Aa)_1~4For 1 to 4 identical or different natural or non-naturals Amino acid unit；

The R¹⁵、R¹⁶And R¹⁷Independently selected from C₁-C₈Alkyl, miscellaneous alkyl, C₂-C₈Alkenyl, alkynyl, heterocycle or C₃-C₈ Carbocylic radical, aryl, benzyl, alkylaryl, Heterocyclylalkyl, miscellaneous alkyl naphthenic base, heteroarylalkyl, alkyl-carbonyl, C₄-C₈Alkyl ring Alkyl or Na⁺、K⁺、Cs⁺、Li⁺、Ca²⁺、Mg⁺、 Zn²⁺、N⁺(R¹)(R²)(R³)(R⁴)、HN⁺(C₂H₅OH)₃Cationic salts；It is described C₃-C₈Carbocylic radical includes naphthenic base；

The Y¹And Y²Independently selected from N or CH；Q is 0 or 1；Work as q=0, Y³It is not present, Y⁴, Y⁵, Y⁶And Y⁷Independently Selected from CH, N, NH, O, S or N (R¹), such Y², Y⁴, Y⁵, Y⁶And Y⁷Pyrroles, furans, thiophene, thiazole, oxazole, miaow are constituted together Azoles, triazole, tetrazolium, thiadiazoles, oxadiazoles miscellaneous aromatic rings；Work as q=1, Y³, Y⁴, Y⁵, Y⁶And Y⁷Independently selected from CH or N, this Sample Y², Y³, Y⁴, Y⁵, Y⁶And Y⁷Phenyl ring, pyridine, pyrazine, pyridazine, triazine, the miscellaneous aromatic ring structure of tetrazine are constituted together.

The present invention also provides a kind of cytotoxic molecule, the group R¹、R², R²、R³Or R³、 R⁴By-CH₂Appoint Meaning connection respectively with Y¹Atomic building cyclic group or R⁵、R⁶By-CH₂It arbitrarily connects coupled carbon atom and constitutes ring Shape group or R¹¹、R¹²By-CH₂Any connection constitutes cyclic group or R with its connected carbon atom and carbonyl¹³、R¹⁴It is logical Cross-CH₂Any connection and Y⁵And Y⁶Atomic building cyclic group；

The cyclic group is selected from C₃-C₇Naphthenic base, alkyl naphthene, cycloalkanes heteroaryl perfume base, cycloalkanes miscellaneous alkyl, heterocycle, Heterocyclylalkyl, carbonyl naphthene, cycloalkanes carboxyl, cycloalkanes amide groups, cycloalkanes amido, cycloalkanes oxygen ether, cycloalkanes thioether group, cycloalkanes selenide One of base, aromatic radical, miscellaneous aromatic alkyl and heteroaryl perfume base.

The present invention also provides a kind of cytotoxic molecule, the R¹、R²、R⁷、R⁹、R¹²、R¹³And R¹⁴End also contain One can with chain junctor or the functional group being coupled with cell surface bind receptor molecule, the functional group be selected from SH, NH₂、COOH、-O-NH₂、-N₃、NHNH₂, (Py is C to-SSPy₅H₄N)、-SSAr、-SSC₆H₄NO₂、-SSC₆H₃(NO₂)(COOH)、- SC(O)R¹、 -SSC₆H₃(NO₂)₂、-C(O)NH₂、-C(O)H、-C(O)NHNH₂、-C(O)R¹-C(O)C(O)R¹、 -ArC(O) R¹、-C(O)CH₂X³、-C(O)X³、-ArCH₂X³、 The X³And X⁴Independently select From F, Cl, Br, I ,-OS (O)₂Ar、-OS(O)₂R¹、-OS(O)₂CF₃)、-OC₆F₅、 -OC₆FH₄,-OC₆F₂H₃,-OC₆H₄(NO₂)、- OC₆H₃(NO₂)₂。

The present invention also provides more than one to state the conjugate that toxicity molecule is parent, has structure shown in Formula II:

Perhaps structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound；Or The optical isomer of this structural formula of person；

The T is cell surface receptor binding molecule；The L and L ' independently is the chain junctor that can be dissociated；

--- -- represents the link key and connection site that L is connected with the atom in structure in bracket；

The integer that the n is 1~20；The integer that the m is 1~10；The integer that the m ' is 0~10；

When m ' is 0, L ' is not present；When m ' is not 0, conjugate connects cell table by two or more chain junctors Face receptor binding molecule T；

Two sites in structure shown in structure bracket shown in the other linking II of two chain junctors or this two A chain junctor connects a site in structure shown in structure bracket shown in knot II by branched chain junctor again；

The structural formula of chain the junctor L and L ' independently are-Ww- (Aa) r-Vv--；

The W is ennation, target binding molecule unit T is connected on Amino Acid Unit (Aa), if straight when without Aa V is met in succession；

The unit W include each independently one self decompose interval body, peptide unit, hydrazone bond, cystine linkage thioether bond, Ester bond or amine key；The w is 0 or 1；

The Aa is natural amino acid or non-natural amino acid unit；The integer that the r is 0 to 12, (Aa) r are 0 The identical or different Amino Acid Unit of~12 structures；The natural amino acid or non-natural amino acid unit include a peptide, two Peptide, tripeptides, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, 11 peptides or dodecapeptide unit；

The V is isolation body unit, and the isolation body unit is independently selected from H, O, NH, S, C₁-C₈Alkyl, C₂-C₈Miscellaneous alkane Base, alkylene, alkynes base；C₃-C₈Aryl, heteroaryl, heterocycle, carbocylic radical, miscellaneous alkyl naphthenic base, alkyl-carbonyl；C₄-C₈Alkane Basic ring alkyl；1~4 Amino Acid Unit or (CH₂CH₂O)r；The heterocycle includes Heterocyclylalkyl；The charcoal ring group includes ring Alkyl；(the CH₂CH₂O) the integer that the r of r is 0~12；The v is 0,1 or 2；

The cell surface receptor binding molecule T is any type of cell combination body, the structure including peptide or similar peptide: Antibody, single-chain antibody can be with the antibody fragment in conjunction with target cell, and monoclonal antibody, single monoclonal antibodies can be with mesh The monoclonal antibody fragment of cell combination is marked, chimeric antibody can resist with the chimeric antibody fragment in conjunction with target cell, functional areas Body can imitate the engineered protein of antibody, fibronectin combination with the functional areas antibody fragment in conjunction with target cell Adnectin, pre-designed ankyrin repeat protein (DARPin), lymphokine, hormone, vitamin, growth factor, colony-stimulating The factor, nutrition transmits molecule, transferrins, cell surface smaller ligand, or is connected with the albumin of cell combination body, high score Cell-binding molecules (binding peptide, albumen, antibody or cell surface smaller ligand) are contained on son or branch macromolecule, surface High molecular material, albumen, liposome, nano particle, bubble crusty pancake or (virus) micro-capsule.

Detailed description of the invention

Fig. 1 display synthesis Tubulysin derivative segment Tuv.

Fig. 2 display display synthesis Tubulysin derivative segment.

Fig. 3 display display synthesis Tubulysin derivative segment.

Fig. 4 display display synthesis Tubulysin derivative segment.

Fig. 5 shows the synthesis of Tubulysin derivative component.

Fig. 6 shows the synthesis of Tubulysin derivative component.

Fig. 7 shows the synthesis of Tubulysin derivative component Tuv and Tup.

Fig. 8 shows the synthesis of the Tup segment of Tubulysin derivative junctor containing chain.

Fig. 9 shows the synthesis of Tubulysin derivative and its conjugate.

Figure 10 shows the synthesis of Tubulysin derivative and its conjugate.

Figure 11 shows the synthesis of Tubulysin derivative and its conjugate.

Figure 12 shows the synthesis of Tubulysin derivative and its conjugate.

Figure 13 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 14 shows antibody -- the synthesis of Tubulysin derivative conjugated body.

Figure 15 shows antibody -- the synthesis of Tubulysin derivative conjugated body.

Figure 16 shows a kind of synthesis of chirality Tubulysin derivative segment.

Figure 17 shows a kind of synthesis of Tubulysin derivative segment.

Figure 18 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 19 shows a kind of synthesis of antibody-Tubulysin derivative conjugated body.

Figure 20 shows a kind of synthesis of Tubulysin derivative that can be conjugated bonding.

Figure 21 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 22 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 23 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 24 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 25 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 26 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 27 shows the synthesis of antibody-Tubulysin derivative.

Figure 28 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 29 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 30 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 31 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 32 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 33 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 34 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 35 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 36 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 37 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 38 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 39 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 40 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 41 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 42 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 43 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 44 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 45 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 46 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 47 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 48 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 49 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 50 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 51 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 52 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 53 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 54 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 55 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 56 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 57 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 58 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 59 shows the synthesis of antibody-Tubulysin derivative and its conjugated body.

Figure 60 display Her2 monoclonal antibody-Tubulysin derivative conjugated body (133,129,181,317 and 467, drug/anti- Body mole ratio DAR=3.5-4.0), N87 (gastric cancer) multicellular animal interior curative effect is acted on marketed drug T-DM1.133, 129,317,467 have good inhibition tumor efficiency.Wherein 133,129,467 curative effects are better than T-DM1.

Figure 61 shows Her2 monoclonal antibody-Tubulysin derivative conjugated body (365,377,385,412,444,474,480 With 546, drug/antibody mole ratio DAR=3.5-4.0), with marketed drug T-DM1 in N87 (gastric cancer) multicellular animal body Curative effect effect.365,377,385,412,444,474 and 480 have good inhibition tumor efficiency.Wherein 377,385,412, 444,474 and 480 curative effects are better than T-DM1.

Figure 62 display Her2 monoclonal antibody-Tubulysin derivative conjugated body (506,522,564,574,695 and 677, medicine Object/antibody mole ratio DAR=3.5-4.0), N87 (gastric cancer) multicellular animal interior curative effect is acted on marketed drug T-DM1. 506,522,564,574 and 677 have good inhibition tumor efficiency.Wherein 677 curative effects are better than T-DM1.

Relational language is explained:

Alkyl refers to linear or cyclic linear or branched aliphatic hydrocarbons containing 1 to 8 carbon atom.Branch refers to linear There are one or more low alkyl group, such as methyl on alkyl, ethyl or propyl are connected.Alkyl embodiment includes methyl, Ethyl, n-propyl, isopropyl, normal-butyl, tert-butyl, pentane base, 3- pentyl, octyl, nonyl, decyl, ring penta Alkyl, cyclohexyl, 2,2- dimethylbutyls, 2,3- dimethylbutyls, 2,2- dimethyl amyl groups, 2,3- dimethyl amyl groups, 3, 3- dimethyl amyl group, 2,3,4- tri-methyl-amyls, 3- methylhexyl, 2,2- dimethylhexanyls, 2,4- dimethylhexanyls, 2,5- Dimethylhexanyl, 3,5- dimethylhexanyls, 2,4- dimethyl amyl groups, 2- methylheptyl, 3- methylheptyl, n-heptyl, different heptyl, Normal octane base and isooctane base.C₁-C₈Alkyl both can be unsubstituted or by following but one or more not limited to the following A group replaces: C₁-C₈Alkyl, C₁-C₈Alkoxy, aryl, acyl group, acyloxy, ester group ,-C (O) NH₂,-C(O)NHR’,-C(O) N(R’)₂,-NHC(O)R’, -S(O)₂R ' ,-S (O) R ' ,-OH, halogen (- F ,-Cl ,-Br ,-I) ,-N³,-NH²,-NHR’,-N (R’)₂And-CN；Wherein R ' refers to C₁-C₈Alkyl or aryl.

C₃-C₈Carbocyclic ring refers to the saturated or unsaturated non-aromatics cyclic compound containing 3,4,5,6,7, or 8 carbon atoms.Allusion quotation The C of type₃-C₈Carbocyclic ring includes but is not limited to cyclopropyl, cyclobutyl, cyclopenta, cyclopentadienyl group, cyclohexyl, cyclohexenyl group, and 1,3- Cyclohexadienyl, Isosorbide-5-Nitrae-cyclohexadienyl, suberyl, 1,3- cycloheptadiene base, 1,3,5- cycloheptatriene base, cyclooctyl and ring are pungent Dialkylene.C₃-C₈Carbocyclic ring both can be unsubstituted, can also be taken by one or more group following but not limited to the following Generation: C₁-C₈Alkyl, C₁-C₈Alkoxy, aryl, acyl group, acyloxy, ester group ,-C (O) NH₂,-C(O)NHR’,-C(O)N(R’)₂, -NHC(O)R’,-S(O)₂R ' ,-S (O) R ' ,-OH, halogen (- F ,-Cl ,-Br ,-I) ,-N₃,-NH₂, -NHR’,-N(R’)₂And- CN；Wherein R ' is C₁-C₈Alkyl or aryl.

C₃-C₈Carbocylic radical refers to above-mentioned C₃-C₈The group that a hydrogen atom on carbocyclic ring generates replaced chemical bond.

Alkylene refers to straight chain or branched aliphatic hydrocarbons containing a carbon-carbon double bond, has 2 to 8 carbon atoms in carbochain.Alkene The embodiment of alkyl includes vinyl, acrylic, n-butene base, isobutenyl, 3- methyl-2-butene base, n-pentene base, hexene Base, heptenyl and octenyl.

Alkynes base refers to straight chain or branched aliphatic hydrocarbons containing a triple carbon-carbon bonds, has 2 to 8 carbon atoms in carbochain.Alkynes The embodiment of alkyl includes acetenyl, propinyl, positive butynyl, 2- butynyl, 3- methylbutynyl, positive pentynyl, hexin base, Heptynyl and octynyl.

Miscellaneous alkyl refers to containing 2 to 8 carbon atoms and has 1 to 4 carbon atom alkyl replaced O, S or N.

Aryl or aromatic radical refer to containing one or more ring by 3 to 14 carbon atoms, (most situations is 6 to 10 A carbon atom) composition aromatic hydrocarbon or miscellaneous aromatic hydrocarbon group.Miscellaneous aromatic hydrocarbon group, which refers to, has one or more carbon atoms (most Situation is 1,2,3 or 4 carbon atom) the generated aryl radical replaced O, N, Si, Se, P or S (preferably O, S, N) Group.Aryl or aromatic radical also refer to that the substituted aromatic hydrocarbon group of one or more hydrogen atoms, these substituent groups have: R¹³,F, Cl,Br,I, OR¹³,SR¹³,NR¹³R¹⁴, N=NR¹³, N=R¹³,NR¹³R¹⁴,NO₂,SOR¹³R¹⁴,SO₂R¹³,SO₃R¹³, OSO₃R¹³, PR¹³R¹⁴,POR¹³R¹⁴,PO₂R¹³R¹⁴,OPO₃R¹³R¹⁴Or PO₃R¹³R¹⁴.Wherein R¹³And R¹⁴Respectively H, alkyl, alkylene, alkynes Alkyl, miscellaneous alkyl, aryl, aralkyl, carbonyl or salt that can be medicinal.

Halogen atom refers to fluorine, chlorine, bromine, iodine atom, preferably fluorine and chlorine.

Heterocycle refers to the fragrance containing 2 to 8 carbon atoms, has 1 to 4 carbon atom by miscellaneous member on the ring of nonaro-maticity ring or heterocycle Replaced element.These miscellaneous elements are O, N, S, Se and P, preferably O, N and S.Available heterocycle also can be found in " The Handbook Of Chemistry and Physics ", the 78th edition, CRC Press, 1997-1998, the page 225 to 226.Suitable is non-miscellaneous Aryl includes but is not limited to epoxy group, azirane base, thiirane base, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, epoxy Ethyl group, tetrahydrofuran base, dioxolanyl, THP trtrahydropyranyl, dioxane base, piperidyl, piperazinyl, morpholinyl, Pyranose, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydro sulphur pyranose, dithianyl, thiomorpholine base, two Hydrogen pyranose, THP trtrahydropyranyl, tetrahydro pyridyl, dihydropyridine base, tetrahydro-pyrimidine base, dihydro sulphur pyranose, azepan Base and the condensed ring of they and phenyl generation.

Heteroaryl refers to the miscellaneous monocycle of armaticity containing 3 to 14 (preferably 5 to 10) atoms, two rings or multiring structure.Example Such as pyrrole radicals, pyridyl group, pyrazolyl, thienyl, pyrimidine radicals, pyrazinyl, tetrazole base, indyl, quinolyl, purine radicals, miaow Oxazolyl, thienyl, thiazolyl, benzothiazolyl, furyl, benzofuranyl, 1,2,4- thiadiazolyl group, isothiazolyl, three nitrogen Oxazolyl, tetrazole base, isoquinolyl, benzothienyl, isobenzofuran-base, pyrazolyl, carbazyl, benzimidazolyl, different evil The condensed ring that oxazolyl, N- oxy picolinate base and they and phenyl generate.

Alkyl, naphthenic base, alkylene, alkynes base, aryl, the terms such as heteroaryl and heterocycle also refer to be lost by corresponding hydrocarbon The alkylidene that two hydrogen atoms generate, ring alkylidene, alkenylene, alkynylene, arlydene, heteroarylidene and miscellaneous sub- ring etc..

" aralkyl " refer to the alkyl diradical of a non-annularity a hydrogen atom connection carbon atom, usually one The carbon atom of a terminal or sp3 hydridization is replaced by an aryl.Typical aryl alkyl includes benzyl, 2- vinylbenzene base, 2- benzene Vinyl, menaphthyl, 2- naphthalene ethyl group, 2- naphthalene vinyl, naphthalene benzyl, 2- naphthalene vinylbenzene base etc..

" heteroarylalkyl " refers to that a hydrogen atom of non-ring-like alkyl diradical is linked on a carbon atom, usually one The carbon atom of terminal or sp3 hydridization is replaced by a heteroaryl.Heteroarylalkyl is represented such as 2- benzimidazole methyl, 2- furans second Base.

" hydroxyl protection group " refers to, methyl oxygen methyl ether (MOM), 2- methyl oxygen ethyl oxygen methyl ether (2-MOEOM), Tetrahydropyranyl ethers, benzyl oxide, to methyl oxygen benzyl oxide, trimethyl silicon ether, triethyl group silicon ether, triisopropyl silicon ether, fert-butyidimethylsilyl Silicon ether, trityl group silicon ether, ethyl acetate, the ethyl acetate containing substituent group, benzoic ether, benzyl formate, chloracetic acid ester, Methoxyacetic acid ester, phenoxyacetic acid ester, pivalate (pivaloate), adamantate, trimethylbenzoic acid ester (mesitoate), methanesulfonate ester and toluene fulfonate.

Amino acid can be natural and/or non-natural amino acid, preferably alpha amino acid.Natural amino acid is by base Because of the layout of coding, they are alanine, arginine, asparagine, asparatate, cysteine, glutamic acid, glutamy Amine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, Tyrosine, tryptophan and valine.Unnatural amino acid is form derived from Argine Monohydrochloride.Such as hydroxyproline, wool sulphur Propylhomoserin；, 2- aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (neurotransmission molecule), ornithine, citrulling, β-the third ammonia Sour (3- amidos propionic acid), gamma carboxyglutamate, selenocysteine is (at present in many non-eucaryotes and most of true Core biology in, rather than be directly made of DNA encoding), pyrrolysine (only being found in some archeobacterias and bacterium), N-formylmethionine (it is bacterium, mitochondria and the initial amino acid of chloroplast protein), 5HTP, L- dihydroxy Phenylalanine, triiodo thryonine, L-DOPA (DOPA) and O- phosphoserine.The art of amino acid Language further includes amino acid analogue and approximation.Analog H having the same₂N(R)CHCO₂The structure of H natural amino acid, but R Group is not found in natural amino acid.Such as analog includes class serine, glycoleucine, methionine sulfoxide and methionine methyl Sulfonium salt.Preferably, amino acid has the structure different from the conventional chemical structure of alpha amino acid, but its function is similar.Term " unnatural amino acid " with the solid shape of " D ", natural amino acid with " L " solid shape.In the present patent application using 1~ When 12 amino acid, amino acid sequence is preferably by the identification of protease and cracking.Many identifications and cutting amino acid sequence are It is known (such as Matayoshi et al.Science 247:954 (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), Also containing with cited documents in its Literature), in particular, amino acid sequence is selected as Val-Cit, Ala-Val, Ala-Lys, Gly-Lys,Ala-Ala,Val-Val,Val-Ala-Val,Lys-Lys,Ala-Asn-Val, Val-Leu-Lys,Cit-Cit, The molecule of Val-Lys, Asp-Lys, Glu-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.

" peptide " is by the amino of an amino acid and the carboxyl of another amino acid by two or more amino acid with peptide Key (i.e. amido bond) is combined into.Two amino acid are known as dipeptides with the compound that peptide bond is connected；Three amino acid are with peptide bond phase Compound even is known as tripeptides, and so on, 30 amino acid are known as three decapeptides with the compound that peptide bond is connected.Completely with day The peptide that right alpha amino acid is constituted is native peptides (native protein).Contain one or more unnatural amino acids or amino acid analogue Peptide be non-natural peptide (class peptide compounds).The peptide of two or more amino acid is a peptide unit.

" glucosides " is that glycosyl is rolled into a ball to the molecule that another group is keyed to by its anomer carbon by its glycosyl bond. Glucosides can pass through O- (O-glycosides), N- (osamine), S- (thio glycoside) or C- (C- glucosides) glucosides key connection.Its core is Empirical equation is C_m(H₂O)_n(it is < 36 that wherein m, which can be different from n, m and n), glucosides herein includes glucose (grape Sugar), fructose (levulose) allose, altrose, mannose, gulose, idose, galactolipin, support Lip river sugar, galactosamine, Aminoglucose, sialic acid, N-acetyl-glucosamine, sulfo group quinolone (6- deoxidation -6- sulfo group-D- glucopyranose), ribose are Arabic Sugar, xylose, haemolysis sugar, sorbierite, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, gossypose, glucose Aldehydic acid (glucosiduronic acid) and stachyose.Glucosides can be D pattern or L-type formula, 5 atom cycloalkyl furanose forms, 6 atom rings Shape pyranose form or acyclic form, the alpha-isomer (- OH of the different carbon of end group below the carbon atom of Haworth projection) or β- The isomers (- OH of the anomer carbon above Haworth projection plane).Common glucosides is monosaccharide herein for it, disaccharides, Polyalcohol or oligosaccharides (containing 3-6 sugar unit).

" antibody " specifically covers complete monoclonal antibody used herein of being used in broadest sense, polyclonal Antibody, specific antibody, multi-specificity antibody (for example, bispecific antibody and antibody fragment) have ideal bioactivity, The link of the necessary amount of the drug binding site of antibody fragment.A kind of primary form of antibody is a tetramer, by two Identical immunoglobulin chain pair, it is each pair of to have a light chain and heavy chain.In each pair, light chain and heavy chain variable region (VL and VH) It is collectively responsible for and antigen binding.Light chain and heavy chain variable region are interrupted by three hypervariable region of framework region, and also referred to as " complementation determines Area " or " service ".Continuous region may be generally acknowledged, with immune system interaction.A kind of antibody can be any class Type (such as IgG, IgE, IgM, IgD and IgA), major class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Antibody can be obtained from any suitable species.In some aspects, antibody is the origin of people or mouse.Antibody can be people's, Humanization or chimeric.

Term " specific bond " and " specific bond " mean that antibody or antibody derivatives can be in a manner of high selectivities Its corresponding target antigen combines, rather than with many other antigen bindings.Under normal conditions, antibody or antibody have at least about 1x10^-7The compatibility derivative of M combines.Preferably 1x10^-8M to 10^-9M、 10^-10M、10^-11M or 10^-12M is combined.Predetermined antigens Affinity be at least twice be greater than combine heterogenetic antigen compatibility (such as bovine serum albumin(BSA), casein).

" medicinal " or " can be medicinal " refers to that corresponding compound or compound composition do not generate nocuousness on animal or human body , hypersensitive or other adverse reactions.

Pharmaceutically acceptable auxiliary material includes all carriers, diluent, auxiliary agent or binder, such as preservative, antioxygen Agent, filler, disintegrating agent, wetting agent, emulsifier, suspending agent, solvent, decentralized medium, coating, antibacterial agent, antifungal agent are isotonic With absorb delayer etc..In field of medicaments, it is very common do that these auxiliary materials are added in active drug ingedient Method.It can be said that unless auxiliary material and medicine activity component and incompatible, are added auxiliary material in drug ingedient and are not without reason. To be having obtained as a result, active helper component can also be added into drug ingedient.

In the present invention, can medicinal salt refer to the salt derivative of the compounds of this invention.By modification appropriate, this hair Bright compound can form corresponding hydrochlorate or alkali salt.Can medicinal salt include it is common do not have virose salt or quaternary ammonium salt, These salt can not have virose inorganic acid or organic acid to be made with the compounds of this invention and accordingly.For example, with inorganic acid packet Hydrochloric acid is included, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid etc. and organic acid include acetic acid, propionic acid, succinic acid, winestone Acid, citric acid, methanesulfonic acid, benzene sulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid And lactic acid etc. can be made into pharmaceutical salts.Other salt include tromethamine, Triaethanolamine, meglumine, epolamine Equal ammonium salts and sodium, potassium, calcium, zinc, the metal salts such as magnesium.

The present invention can be medicinal salt can be made of conventional chemical method.In general, these salt can by It is added in the free acid of the compounds of this invention or the aqueous solution of alkali or the mixed solution of organic solution or both other appropriate etc. The alkali or acid of equivalent and formed.The reaction medium of nonaqueous phase is generally ether, ethyl acetate, ethyl alcohol, isopropanol or acetonitrile.It can It can be found in " Remington ' s Pharmaceutical Sciences ", the 17th edition .Mack to be applicable in the list of salt Publishing Company, Easton, PA, 1985, the of page 1418

Term " pharmaceutically acceptable salt " refers to can pharmaceutically connecing for ligand drug conjugate or connector drug conjugate The organic or inorganic salt received.Conjugate can contain at least one amino, therefore can form acid-addition salts with amino.Nitric acid Salt, disulfate, phosphate, acid phosphate, isonicotinic acid salt, lactate, salicylate, acid citrate, tartrate, Oleate, tannate, pantothenate, tartrate, ascorbate, succinate, maleate, gentisate, fumarate, Gluconate, glucuronate, sugar lime, formates, benzoate, glutamate, mesylate, esilate, benzene sulphur Hydrochlorate, tosilate and embonate (i.e. 1,1'- di-2-ethylhexylphosphine oxide-(2 hydroxy naphthalene formates)).It is pharmaceutically acceptable Salt may include other molecule, such as acetate ion, succinate ion or other counter ion counterionsl gegenions.Counter ion counterionsl gegenions can be with It is any organic or inorganic part for making the charge stable on parent compound.In addition, pharmaceutically acceptable salt is in its structure In can have more than one charge atom.Multiple charge atoms are that the embodiment of a part of pharmaceutically acceptable salt can With multiple counter ion counterionsl gegenions.Therefore, pharmaceutically acceptable salt can have one or more charge atoms and/or one or Multiple counter ion counterionsl gegenions.

Phrase " pharmaceutically acceptable solvate " or " solvate " refer to one or more solvent molecules and ligand The association of drug conjugates or connector drug conjugate.The embodiment for forming the solvent of pharmaceutically acceptable solvate includes But it is not limited to water, isopropanol, ethyl alcohol, butanol, the tert-butyl alcohol, acetone, glycerol, DMSO, ethyl acetate, formic acid, acetic acid, triethanolamine And ethanol amine.

Hydrate (Hydrate) refers to the compound containing water.Wherein water can be connected with coordinate bond with other parts, If metal ion hydration ligand forms complex compound, it is also possible to combine with covalent bond, such as chloral hydrate.Can also refer to is The crystal or fluid molecule that certain compounds and moisture are formed under certain temperature, pressure condition.Water in hydrate is with true Existing for fixed amount, such as anhydrous sodium sulfate Na₂SO₄Hydrate group become Na₂SO₄·10H₂O.Water in hydrate has Several different combinations: it one is as ligand, is coordinated on metal ion, referred to as the coordination crystallization water；It is another then tie It closes on anion, referred to as the anion crystallization water.Water can not also be direct with cationic or anion binding and according to certain proportion It is present in crystal, certain position is occupied in lattice.The water of this combining form is known as lattice watter, typically contains 12 water Molecule.Some amorphous compounds are also aqueous, but without certain proportion.The salt of hydrate refers to the medicine formed on the basis of this hydrate Acceptable salt on.

Optical isomer is also known as enantiomter, to the palm isomeric compound, optical isomeric compound, mirror image isomerism object, enantiomter Or chiral isomer, molecule that cannot be completely overlapped with stereoisomer mirror image each other.Contain a chiral carbon containing a substance Atomic time, there are two optical isomer, they have relationships in kind and mirror image each other, therefore also referred to as enantiomer.Enantiomter With equal optical activity, but direction of rotation is on the contrary, its physics and chemical property are most probably similar.Containing there are two same alike results The molecule of carbon atom has 3 optical isomers.In molecule when containing several different chiral atoms, optical isomer Number is 2ⁿ, n is the number of different chiral atoms.Two kinds of substances of equivalent optical isomer each other uniformly mix, and optical activity is mutual It offsets, just constitutes racemic modification.

The embodiment of " patient " or " subject " include but is not limited to people, rat, mouse, cavy, monkey, pig, goat, ox, Horse, dog, cat, bird and poultry.In the exemplary embodiment, patient or subject are people.

" administration ", which refers to, introduces or is shipped for any mode of subject for drug or the transfer of other medicaments, conveying, including Oral, localized contact, intravenously, in peritonaeum, intramuscular is intralesional, and intranasally, subcutaneous or intrathecal administration is present invention contemplates that make Medicament is applied with device or instrument, this device can use transport actively or passively, and can be sustained release or quick Release delivery device.

Following abbreviation can be used herein, and there is specified definition: Boc, tert-butoxycarbonyl；BroP, three pyrrole of bromo Ka Wan Phosphonium hexafluorophosphate；CDI, 1,1'- carbonyl dimidazoles；DCC, dicyclohexylcarbodiimide；DCE, dichloroethanes；DCM, Methylene chloride；DIAD, diisopropyl azo-2-carboxylic acid；DIBAL-H, diisobutyl aluminium hydride；DIPEA, diisopropylethylamine； DEPC, dicyano diethyl phosphate；DMA, n,N-dimethylacetamide；DMAP, 4- (N, N- dimethylamino) pyridine；DMF, N,N-Dimethylformamide；DMSO, dimethyl sulfoxide；DTT, dithiothreitol (DTT)；EDC, 1- (3- dimethylaminopropyl) -3- ethyl Carbodiimide hydrochloride；ESI-MS, electrospray ionization mass spectrum；HATU, O- (7- azepine benzo triazol-1-yl)-N, N, N', N'- tetramethyl Base urea hexafluorophosphate；HOBt, I-hydroxybenzotriazole；HPLC, high pressure liquid chromatography；NHS, n-hydroxysuccinimide； MMP, 4- methyl morpholine；PAB, aminobenzyl；PBS, phosphate buffered saline (PBS) (pH 7.0~7.5)；PEG, polyethylene glycol； SEC, size exclusion chromatography method；TCEP, three (2- carboxyethyl) phosphines；TFA, trifluoroacetic acid；THF, tetrahydrofuran；Val, valine.

Specific embodiment

The X¹Selected from-O- ,-S ,-S-S ,-NH ,-CH₂Or-NR¹-；

The R¹²Selected from-R¹⁵、-OH、-SH、-NH₂、-NH、-NHNH₂、-NH(R¹⁵)、-OR¹⁵、 -R¹⁵COR¹⁶、- R¹⁵COOR¹⁶、-R¹⁵C(O)NH₂、-R¹⁵C(O)NHR¹⁷、-SR¹⁶、R¹⁵S (=O) R¹⁶、-R¹⁵P (=O) (OR¹⁷)₂、-R¹⁵OP (=O) (OR¹⁷)₂、-CH₂OP (=O) (OR¹⁷)₂、-R¹⁵SO₂R¹⁷、 -R¹⁵X²R¹⁶、-R¹⁵C (=O) X³；

The R¹³And R¹⁴Independently selected from H ,-OH ,-SH ,-NH₂、-NHNH₂、-NH(R¹⁵)、 -OR¹⁵、-COX²、- COX²R¹⁶、-R¹⁷、-F、-Cl、-Br、-I、-SR¹⁶、-NR¹⁶R¹⁷,-N=NR¹⁶,-N=R¹⁶、-NO₂、-SOR¹⁶R¹⁷、-SO₂R¹⁶、- SO₃R¹⁶、-OSO₃R¹⁶、-PR¹⁶R¹⁷、-POR¹⁶R¹⁷、 -PO₂R¹⁶R¹⁷、-OP(O)(OR¹⁷)₂、-OCH₂OP(O)(OR¹⁷)₂、-OC(O) OP(O)(OR¹⁷)₂、 -PO(OR¹⁶)(OR¹⁷)、-OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、-OC(O)R¹⁷,-OC (O) NHR¹⁷； -O- (C₄-C₁₂Glucosides) ,-N- (C₄-C₁₂Glucosides)；C₁-C₈Alkyl, miscellaneous alkyl, C₂-C₈Alkenyl, alkynyl, heterocycle；C₃-C₈Carbocylic radical, Heterocyclylalkyl, miscellaneous alkyl naphthenic base, aryl, heteroarylalkyl, alkyl-carbonyl；C₄-C₈Alkyl-cycloalkyl；-NH(Aa)_1~4Or-CO (Aa)_1~4；The C₃-C₈Carbocylic radical includes naphthenic base；(Aa)_1~4For 1 to 4 identical or different natural or non-naturals Amino acid unit；

The present invention also provides a kind of cytotoxic molecule, the R¹、R²、R⁷、R⁹、R¹²、R¹³And R¹⁴End also contain One can with chain junctor or the functional group being coupled with cell surface bind receptor molecule, the functional group be selected from SH, NH₂、COOH、-O-NH₂、-N₃、NHNH₂, (Py is C to-SSPy₅H₄N)、-SSAr、-SSC₆H₄NO₂、-SSC₆H₃(NO₂)(COOH)、- SC(O)R¹、 -SSC₆H₃(NO₂)₂、-C(O)NH₂、-C(O)H、-C(O)NHNH₂、-C(O)R¹-C(O)C(O)R¹、 -ArC(O) R¹、-C(O)CH₂X³、-C(O)X³、-ArCH₂X³、 The X³And X⁴Independently selected from F、Cl、Br、I、-OS(O)₂Ar、-OS(O)₂R¹、-OS(O)₂CF₃)、-OC₆F₅、-OC₆FH₄, -OC₆F₂H₃,-OC₆H₄(NO₂)、- OC₆H₃(NO₂)₂。

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

The R²⁰Selected from C₁-C₈Linear or branched paraffin, miscellaneous alkane, C₂-C₈Linear or branched-chain alkene or alkynes, C₃-C₈Virtue Hydrocarbon, alkylaromatic hydrocarbon, heterocyclic hydrocarbon or heteroaryl hydrocarbon, carbonyl rouge-C (O) OR¹⁷, carbonyl amine-C (O) NR¹⁷R¹⁸；

The Z²And Z³Independently selected from H ,-OH ,-NH₂、OR¹⁷、NHR¹⁷、COOH、 COOR¹⁷、C(O)SR¹⁷、C(O)R¹⁷、C (O)NHR¹⁷、C(O)NHNHR¹⁷、C(O)NH、 R¹⁸、OCH₂OP(O)(OR¹⁸)₂、OC(O)OP(O)(OR¹⁸)₂、NR¹⁷CH₂OP(O) (OR¹⁸)₂、 NR¹⁷C(O)OP(O)(OR¹⁸)₂、OPO(OR¹⁸)₂、NR¹⁷PO(OR¹⁸)₂、 OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂、OC (O)R¹⁸、NR¹⁷C(O)R¹⁸、OC(O)NHR¹⁸、 NR¹⁷C(O)NHR¹⁸、NR¹⁷C(O)OR¹⁸、NR¹⁷C(O)SR¹⁸、NR¹⁷C (=NH) NHR¹⁸、 OSO₂(OR¹⁸)、O-(C₄-C₁₂Glucosides), NR¹⁷SO₂(OR¹⁸)、NR¹⁷-(C₄-C₁₂Glucosides), C₁-C₈Alkyl, C₃-C₈Carbonyl alkane Base or heterocycle；

The R¹⁷And R¹⁸Independently selected from H, C₁~C₈Alkyl or miscellaneous alkyl；C₂~C₈Carbonyl alkyl, alkylene, alkyl carbonyl, alkynes Alkyl or heterocycle；C₃~C₈Aromatic radical, naphthenic base or carbonyl naphthene；

The R¹⁹Selected from H ,-OH ,-NH₂、-OSO₂(OR¹⁸)、-XCH₂OP(O)(OR¹⁸)₂、 -XPO(OR¹⁸)₂、-XC(O)OP (O)(OR¹⁸)₂、-XC(O)R¹⁸、-XC(O)NHR¹⁸、C₁~C₈Alkyl or carboxylic acid derivates, C₂~C₈Alkylene, alkynes base, alkane carbonyl Base or carbonyl alkyl, C₃~C₈Aromatic radical, naphthenic base or carbonyl naphthene；Or pharmaceutical salts；

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

The present invention also provides a kind of using the toxicity molecule with structure shown in Formulas I as the coupling of parent, has formula Structure shown in II:

Two chain junctors be separately connected two sites in structure shown in structure bracket shown in Formula II or this Two chain junctors connect a site in structure shown in structure bracket shown in eliminant II by branched chain junctor again；

The cell surface receptor binding molecule T is any type of cell combination body, the structure including peptide or similar peptide: Antibody, single-chain antibody can be with the antibody fragment in conjunction with target cell, and monoclonal antibody, single monoclonal antibodies can be with mesh The monoclonal antibody fragment of cell combination is marked, chimeric antibody can resist with the chimeric antibody fragment in conjunction with target cell, functional areas Body can imitate the engineered protein of antibody, fibronectin combination with the functional areas antibody fragment in conjunction with target cell Adnectin, pre-designed ankyrin repeat protein (DARPin), lymphokine, hormone, vitamin, growth factor, colony-stimulating The factor, nutrition transmits molecule, transferrins, or is connected to albumin, and macromolecule, branch macromolecule, cell combination is contained on surface Molecule high molecular material, liposome, nano particle steep binding peptide, albumen, antibody or the cell surface on crusty pancake or (virus) micro-capsule Smaller ligand.

The present invention provides a kind of using the toxicity molecule of structure shown in Formulas I as the conjugate of parent, has shown in formula III Structure:

Perhaps this structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound； Or the optical isomer of this structural formula；

Described T, L, L ', m, m ' and the definition of n are identical as Formula II.

Preferably, the conjugate is with one of structure shown in formula III -01~III-19:

The n is the integer of 1-20；The p is the integer of 0-100；

The mAb is cell-binding molecules, and the cell-binding molecules include antibody；

The definition of the Z2 and Z3 and definition noted earlier are identical；

The X¹、R¹、R²、R³It is identical to define definition noted earlier；The M₁And M₂Independently selected from H⁺、Na⁺、K⁺、Li⁺、 NH₄ ⁺、N(R¹R²R³R⁴)⁺Or NH (C₂H₅OH)₃ ⁺Pharmaceutically acceptable application salt ion.

The present invention provides a kind of using the toxicity molecule with structure shown in Formulas I as the conjugate of parent, has formula IV institute Show structure:

The definition of described T, L, m and n are identical as Formula II.

Preferably, the conjugate with structure shown in formula IV is with one in structure shown in formula IV -01~IV-11 Kind:

The n is the integer of 1-20；P is the integer of 0-100；

The Z²And Z³It is identical as definition noted earlier；

The X¹、R¹、R²And R³Define it is same as previously described,

The M₁And M₂Salt ion H independently selected from pharmaceutically acceptable application⁺、Na⁺、K⁺、Li⁺、 NH₄ ⁺、N (R¹R²R³R⁴)⁺Or NH (C₂H₅OH)₃ ⁺。

A cell bound receptor molecule-drug conjugates (conjugated body) of the invention have shown in following structural formula V:

Perhaps this structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound； Or the optical isomer of this structural formula, racemic modification, diastereoisomer or enantiomter；

Wherein R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、Y¹、 Y²、Y³、Y⁴、Y⁵、Y⁶、Y⁷, T, L, m and The definition of n is identical with structure formula (II).

R⁹For-O- ,-OR¹⁴,-OC(O)R¹⁴,-OC(O)NHR¹⁴,-OC (O) R¹⁴SSR¹⁵, or-OP (O) (OR¹⁴) O-, wherein R¹⁴And R¹⁵Respectively C₁~C₈Alkyl or miscellaneous alkyl, C₃~C₈Aryl, heteroaryl, heterocycle, carbocylic radical, naphthenic base, alkyl-cycloalk Base, Heterocyclylalkyl, miscellaneous alkyl naphthenic base, heteroarylalkyl, carbonyl alkyl or drug salts.In addition, R⁹It can also be with vacancy.

Preferably, it is described with structure conjugate shown in Formula V be with one of structure shown in Formula V -01~V-18:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹、Y⁷It is identical as definition noted earlier；

The Z²It is identical as definition noted earlier.

It is a kind of using the toxicity molecule with structure shown in Formulas I as the conjugate of parent, have Formula IV shown in structure:

The definition of described T, L, m and n are identical as Formula II.

Preferably, the conjugate with structure shown in Formula IV is with one in structure shown in Formula IV -01~VI-30 Kind:

The n is the integer of 1-20；P is the integer of 0-100；

The Z²And Z³It is as defined above

The X¹、R¹、R²、R³It defines identical as the definition in Formulas I；

The present invention provides the conjugate that a kind of toxicity molecule with structure shown in Formulas I is parent, has shown in Formula VII Structure:

The definition of described T, L, m and n are identical as with Formula II.

Preferably, the conjugate is with one of structure shown in Formula VII -01~VII-15:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹、Z³It is as defined above.

It is a kind of using the toxicity molecule with structure shown in Formulas I as the conjugate of parent, have Formula VIII shown in structure:

Described T, L, m and n are as defined above.

Preferably, the conjugate with structure shown in Formula VIII is in structure shown in Formula VII -01~VII-43 One kind:

The n is the integer of 1-20；The p is the integer of 1-100；

The Z²And Z³It is as defined above.

The present invention provides a kind of using the toxicity molecule with structure shown in Formulas I as the conjugate of parent, has Formula IX institute Show structure:

Preferably, the conjugate with structure shown in Formula IX is with one in structure shown in Formula IX -01~IX-28 Kind:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹、X²Definition it is identical as the definition in Formulas I；

The Z²And Z³With noted earlier.

Toxicity molecule of the present invention and they and cell combination body of the invention are formed by conjugated body and pass through this The subsequent example of specification, but it is not limited to these examples and Figure of description 1-60 is showed.

As specific a kind of conjugated chain junctor of the invention, the chain junctor (L) that can especially dissociate is one section and contains C, N, The chemical chain of O, S, Si or P atom.In conjugated body, the chemical chain is on one side with cell combination body (T) by being covalently keyed, separately Pass through covalent linkage with Tubulysin derivative on one side.Chain junctor can have length different in size, such as from 2 to 100 The length of atom is ok.All atoms on chain junctor can be linked together with various chemical methodes appropriate, such as With at alkene, sub- alkene, alkynes, ether, polyoxyalkylene, ester, amine, imines, polyamine, hydrazine, hydrazone, amide, urea, semicarbazides, carbazide, alkoxy Amine, urea ethane, amino acid, the connection types such as acyl-oxygen amine or hydroxamic acid.Certainly, these atoms that can be dissociated on chain junctor both may be used To be saturation, be also possible to unsaturated, can be free radical, can mutually cyclization and form the ring structure of divalent, such as Cycloalkane, cyclic ethers, cyclammonium, arlydene and heteroarylidene etc..

The chain junctor that can be dissociated refers generally to the chemical bond that can be at least dissociated in physiological conditions containing one, such as to pH Sensitivity, it is unstable to acid, it is unstable to alkali, it is oxidized easily, is easy to be metabolized, it is unstable on biochemistry, or be easy to be digested Chemical bond.It should be noted that this physiological condition that can make chemical bond rupture is not necessarily a biology or was metabolized Journey, conventional chemical process also should completely can be with, such as hydrolysis or substitution reaction.Due to endosome than cytosol pH more Low, the chemical bond that can be hydrolyzed in acid condition is a kind of selection well.Same reason, it is contemplated that the paddy in tumour cell The exchange reaction of the sweet peptide concentration of Guang up to mM grade, sulfydryl and cystine linkage is also feasible way.

The structure for dissociating chain junctor L of the invention can be expressed as :-Ww-(Aa) r-Vv-prolongs wherein-W-refers to Stretch body, w is 0 or 1, and-Aa-is Amino Acid Unit, the integer that r is 0 to 12, and-V-refers to that interval body, v are 1 or 2.

In the presence of ennation-Ww-, it play be connected to cell combination body (T) and Amino Acid Unit-Aa-or The effect of spacer-V-(in the absence of-Aa -).It may include the slider that self can be decomposed in ennation W, Polypeptide unit, hydrazone bond, cystine linkage or thioether bond.Obviously, cell combination body (T) can be to function corresponding on ennation containing one The group of bonding can be rolled into a ball.On cell combination body originally exist or by chemical mode generate attachable functional group include but Be not limited to: sulfydryl, amino, hydroxyl, carbonyl, sugared hydroxyl and carboxyl etc., best functional group are sulfydryl, carboxyl and amino. Sulfydryl can be generated by restoring the cystine linkage of cell combination body (such as antibody) intramolecular.In addition, sulfydryl can also or pass through Generation is reacted with the amino in lysine on cell combination body using 2- iminothiolane (Traut reagent) or thiolactone, or The other sulfydryl generation methods of person's application, such as first with chain junctor or thioesters modified cells combination containing cystine linkage, then distinguish By restoring or hydrolyzing generation sulfydryl.

The embodiment of some W being connected on T is seen below:

Here R²⁰, R²¹Selected from-C₁~C₉Alkylene-,-C₁~C₇Carbocylic radical-,-O- (- (C₁~C₈Alkyl)-,-Ya Fang Base-,-C₁~C₉Alkylene-arlydene-,-arlydene ,-C₁~C₉Alkylene ,-C₁~C₉Alkylene-(C₁~C₈Carbocylic radical)-,- (C₃~C₈Carbocylic radical)-C₁~C₉Alkylene-,-C₃~C₈Heterocycle-,-C₁~C₁₀Alkylene-(C₃~C₈Heterocycle)-,-(C₃~ C₈Heterocycle)-C₁~C₉Alkylene-,-(CH₂CH₂O)_k-, -(CH(CH₃)CH₂O)_kAnd-(CH₂CH₂O)_k-CH₂-；K is 1- Integer between 20；R ' and R " is respectively H or CH₃。

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

Such as form amido bond:

Wherein extend body unit contain active reactive group an E, E can on cell combination body primary amine or secondary amine it is raw At amido bond.Feasible active reaction position E includes but is not limited to hydroxysuccinimide eater (NHS and S-NHS etc.), 4- nitro Phenylester, pentafluorophenyl group ester, tetrafluoro phenylester (including S- tetrafluoro phenylester), acid anhydrides, acyl chlorides, sulfonic acid chloride, isocyanates and different Thiocyanates etc..

Here is the example connected with thioether or cystine linkage:

Wherein extend body unit and contain the sulfydryl that can participate in reaction, it can be formed between cell combination body T Thioether or cystine linkage.To form cystine linkage, the sulfydryl on cell combination body T both can be by restoring the intramolecular disulfide bond in it It generates, can also be generated by other chemical process modified cells combinations T；R²⁰, R ', R ", J, and the face as defined above T or Described in this specification.

There can also be the active group that can be reacted with aldehyde radical or ketone group on ennation.It is repaired by chemistry appropriate Decorations, such aldehyde radical or ketone group be directed into the appropriate location of cell combination body T.For example, in the sugar of cell combination body T On, by the way that aldehyde radical or ketone group can be generated with oxidant such as sodium periodate oxidation.For another example, antibody (or protein or more Peptide) amido in N-terminal amino acid can react with pyridoxime 5-phosphate (PLP) and introduce ketone group.These aldehyde radicals or ketone group (- C= O) can with such as hydrazides of the active reactive group on ennation, oxime, primary amine or secondary amine, hydrazine, thiosemicarbazones, hydrazinecarboxylate or Aryl hydrazine reaction, so that the two be linked together.

Here is the example with hydrazone, oxime or imines connection:

Wherein R²⁰And R²¹It is defined as described above, R²⁵Refer to the organic substituents or glucosides or H of an amino acid.

Ennation (can contain a slider V and an or amino acid) can be linked first with cell combination body T, so This cell combination body-ennation segment and a Tubulysin derivative conjugation are connected again afterwards, connection procedure can be slow It is carried out in bath solution.Here is that some examples for being conjugated connection in two steps in this manner (are connected to R¹⁶On cell toxicant Plain molecule (Tubulysin derivative) is omitted):

Wherein E includes but are not limited to hydroxysuccinimide eater (NHS and S-NHS etc.), 4- nitrobenzene base ester, phenyl-pentafluoride Base ester, tetrafluoro phenylester (including S- tetrafluoro phenylester), acid anhydrides, acyl chlorides, sulfonic acid chloride, isocyanates and isothiocyanates etc.；R' And R " is respectively H or CH₃；R²⁰, R¹⁶It is as defined above with Ar；R²⁶For H, F or NO₂；J is F, Cl, Br, I, tosylate group (TsO) or methanesulfonic acid ester group (MsO)；In above structureContain at least one Tubulysin derivative or other drugs Or cytotoxic molecule, structure is such as(To omit mark)；The cyclic annular two sulphur structures containing T refer in particular to cell combination Intramolecular disulfide bond.

Ennation can also first and Tubulysin derivative link, then again with cell combination body T pH for 3-10 (most It is well conjugation connection in the aqueous solution (organic solvent containing the upper limit to 50%) of pH 5.0-8.5).

Here is some examples for being conjugated connection in two steps in this manner:

Wherein E includes but are not limited to hydroxysuccinimide eater (NHS and S-NHS etc.), 4- nitrobenzene base ester, phenyl-pentafluoride Base ester, tetrafluoro phenylester (including S- tetrafluoro phenylester), acid anhydrides, acyl chlorides, sulfonic acid chloride, isocyanates and isothiocyanates etc.；R' And R " is respectively H or CH₃；R²⁰, R¹⁶It is as defined above with Ar；R²⁶For H, F or NO₂；J is F, Cl, Br, I, tosylate group (TsO) or methanesulfonic acid ester group (MsO)；In above structureContain at least one Tubulysin derivative or other drugs Structure, such as(To omit mark).

In the presence of Amino Acid Unit (-- Aa--), it connects ennation and slider, and when slider missing, it is connected Ennation and Tubulysin derivative, if ennation and slider all lack, it be directly connected to cell combination body T and Tubulysin derivative.-- (Aa) r-can be natural or non-natural amino acids, can be dipeptides, tripeptides, tetrapeptide, pentapeptide, Hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, 11 peptides or dodecapeptide unit, integer of the r between 0-12.Herein, amino acid Aminoalkyl carboxylic acid is referred broadly to, wherein alkyl can be by such as alkyl, acyl group, hydroxyalkyl, mercapto alkyl, aminoalkyl or carboxylic alkane Alkyl replaced the groups such as base.Natural and unnatural amino acid and the structure of polypeptide are in G.C. Barrett and D.T.Elmore Have well in written " Amino Acid and Peptides " (Cambridge University Press, 2004) book Explanation.In addition, amino acid also refers to beta amino acid, gamma amino acid and intramolecular contain methyl, benzyl, methylol, thiopurine methyltransferase, The long chain amino acid of carboxyl, carboxymethyl or guanidinopropyl etc..Best amino acid is spermine acid, and asparagine is sour, aspartic acid, Citrulling, cysteine, glycine, glutamic acid, leucine, lysine, glutamine, serine, ornithine, phenylalanine, Threonine, tyrosine and valine etc..

Amino Acid Unit of the invention can be included in the institute of proteolytic enzyme present in tumour enzyme by one or more enzymes It solves and releases Tubulysin derivative.Implement in explanation one, it is first protonated in vivo, then becomes protonation Tubulysin derivative.

In the presence of slider (- V-), it connects Amino Acid Unit and Tubulysin derivative, when Amino Acid Unit lacks When mistake, it connects ennation and Tubulysin derivative.When Amino Acid Unit and ennation all lack, slider connection Tubulysin derivative and cell-binding molecules T.It may include some functional groups in slider, can be mentioned with these functional groups The water solubility of high conjugated body, biology transmission, suitable kidney are removed, are taken in, absorb, bio distribution and bioavilability.Isolation Body is there are two types of fundamental type: from destruction type and non-from destruction type.It is non-from destruction type slider refer to an Amino Acid Unit from In Tubulysin derivative-chain junctor-cell combination body or Tubulysin derivative-chain junctor interruption is split and is especially digested Later, all or part of slider remains attached on Tubulysin derivative.

Here is some examples from destruction type slider:

The chain junctor that can be dissociated, the access point of Tubulysin derivative or cell combination body T；Here X, Y and Z³Respectively For NH, O or S；Here Z²For H, NH, O or S；V is 0 or 1；Q is H, OH, C₁~C₆Alkyl, (OCH₂CH₂)_n, F, Cl, Br, I, OR¹⁷,or SR¹⁷,NR¹⁷R¹⁸, N=NR¹⁷, N=R¹⁷, NR¹⁷R¹⁸,NO₂,SOR¹⁷R¹⁸,SO₂R¹⁷,SO₃R¹⁷,OSO₃R¹⁷,PR¹⁷R¹⁸, POR¹⁷R¹⁸,PO₂R¹⁷R¹⁸, OPO(OR¹⁷)(OR¹⁸⁾,OCH₂PO(OR¹⁷(OR¹⁸) or glucosides, wherein R¹⁷And R¹⁸Respectively H, C₁- C₈Alkyl, C₂-C₈Alkylene, alkynes base, miscellaneous alkyl, C₃-C₈Aryl, heterocycle, carbocylic radical, naphthenic base, Heterocyclylalkyl, heteroaryl alkane Base, alkyl carbonyl or cation salt that can be medicinal.

Here is some non-examples from destruction type slider:

*(CH₂CH₂O)_n*；

It is wherein slider, the chain junctor that can be dissociated with the atom that (*) is marked, Tubulysin derivative or cell combination The access point of body；Q be it is described above, m be 1~10；N is 1~20.

Cell combination body T can be known to any one can with can be used to treat on target cell or modified The molecule that segment is combined, is complexed or reacted.By with specific target cell combination, cell combination body is Tubulysin Derivative is transmitted to such target cell.

Cell combination body includes but is not limited to the albumen of macromolecule, such as complete antibody (polyclonal or monoclonal)； Single-chain antibody；Antibody fragment such as Fab, Fab ', F (ab ') 2, Fv [Parham, J.Immunol.131,2895-2902 (1983)], the segment that Fab expression library generates, the antibody (anti-Id) of anti-idiotype, CDR's, any energy and cancer cell Epitope, virus antigen epitope or microbial antigen epitope specific bond above-mentioned object segment；Interferon (I, II, III Type)；Polypeptide；Lymphokine such as IL-2, IL-3, IL-4, IL-6, GM-CSF, or IFN-γ；Hormone such as insulin, thyroid Hormone releasing hormone (TRH), melanocyte stimulating hormone (MSH), steroid hormone such as androgen, estrogen or melanotropin (MSH)；Growth factor and colony stimulating factor such as epidermal growth factor (EFG), granulocyte macrophage colony stimulating factor (GM-CSF), transforming growth factor (TGF) such as TGFα, TGF β, insulin and insulin-like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5,155-158 (1984)] etc.；Vaccina growth factor (VGF)；Fibroblast growth factor (FGF)；Small molecular weight protein, polypeptide, peptide and peptide hormone, such as bombesin, stomach are secreted sharp Element and gastrin releasing peptide；Platelet derived growth factor；Interleukins and cell factor such as interleukin 2 (IL-2) are white Cytokine -6 (IL-6), LIF ELISA, granulocyte macrophage colony stimulating factor (GM-CSF) and vitamin are such as Folic acid；Apoprotein and glycoprotein such as transferrins [O ' Keefe et al, J.Bio. Chem.260,932-927 (1985)]；Carbohydrate-binding protein or lipoprotein such as agglutinin；Cytotrophy transmits molecule；And micromolecular inhibitor such as prostate Specific membrane antigen (PSMA) inhibitor, small molecule tyrosine kinase inhibitors (TKI), non-peptide or other cell-binding molecules or Substance, such as bioactive polymer (Dhar, et al, Proc.Natl.Acad.Sci.2008,105,17356-61), it is tree-like Macromolecule (Lee, et al, Nat.Biotechnol.2005,23,1517-26；Almutairi,et al； Proc.Natl.Acad.Sci.2009,106,685-90), nano particle (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), liposome (Medinai, et al, Curr.Phar.Des.2004,10,2981-9), disease Malicious capsid (Flenniken, et al, Viruses Nanotechnol.2009,327,71-93), especially these macromolecules Or the molecule that particle surface energy of attachment is combined with target cell.In general, if there is suitable monoclonal antibody, monoclonal Antibody is best cell combination body.

Before with Tubulysin derivative conjugated, modified by using the crosslinking agent containing double-functional group, cell knot Zoarium can first connect specific polypeptide, albumen, drug molecule or other function molecule.These crosslinking agents containing double-functional group It can be the non-selective functional group crosslinking agent of amine-(succinimide (NHS)-phenodiazine cyclopropylene (SDA), succinimide ester- Azide), amido-thiol crosslinkers (NHS ester-maleimide, NHS ester-pyridine dimercapto, NHS ester-haloacetyl Base), sulfydryl-sugar crosslinking agent (maleimide-hydrazides, pyridine dimercapto-hydrazides), hydroxyl-thiol crosslinkers (isocyanates- Maleimide), amine-DNA crosslinking agent (NHS ester/psoralen) and amine-carboxyl crosslinking agent (carbodiimide) etc..

NHS in the method for modifying using succinimide (NHS)-phenodiazine cyclopropylene (SDA) crosslinking agent, on crosslinking agent Ester can be first with the amido reaction (being in the buffer solution of 6-9 in pH) on cell combination body skeleton, this will form one surely Fixed amido bond.Then, it is irradiated by the long wave that is 330-370nm with wavelength, phenodiazine cyclopropylene is activated and to generate Cabbeen living Property intermediate, the company of can be completed after the amido reaction on the intermediate and specific polypeptide, albumen or other function molecule It connects.The order of this two-step reaction also can change are as follows: first allow NHS ester reaction on amine and crosslinking agent on functional molecular, so It is chemically reacted under the irradiation of light (330-370nm) with cell combination body again afterwards.Succinimide (NHS)-phenodiazine cyclopropyl Alkene (SDA) crosslinking agent is also possible to that (just as wherein the having SDAD crosslinking agent existing for cystine linkage) of formula can be dissociated.

WhereinIt is cell-binding molecules skeleton,It is functional molecular.

Using in NHS ester-azide crosslinking agent method of modifying, NHS ester on crosslinking agent can first and cell Amido on combination skeleton reacts (in the buffer solution of pH6-9), this will form a stable amido bond.Then, lead to Cross Huisgen nitrine-alkynes cycloaddition reaction, specific polypeptide, protein or alkynyl and crosslinking agent on other function molecule The nitrine of the other end is reacted, and forms 1,2,3-triazoles chain junctor.In addition, the NHS ester of this crosslinking agent can also first with Amido on functional molecular reacts to form stable amido bond (being in the buffer solution of 6-9 in pH), then uses combination again On alkynyl and the crosslinking agent other end azido group carry out Huisgen nitrine-alkynes cycloaddition reaction, and formed 1,2,3- tri- Azoles chain junctor.

Above-mentioned nitrine group can also introduce the unnatural amino acid containing nitrine by antibody engineering, or be drawn using antibody engineering Enter glucosides, chemical degradation and glucoside transferase transfer introduce the glucosides containing nitrine, then carry out carrying out Huisgen nitrine-alkynes ring Addition reaction (click chemistry reaction).

Using in amido-thiol crosslinkers method of modifying, NHS ester on crosslinking agent can first with cell combination body Amido on skeleton reacts (in the buffer solution of pH=6~9), this will form a stable amido bond.Then, it is in pH Under conditions of 4.5~8.5, the horse of specific polypeptide, protein or sulfydryl and the crosslinking agent other end on other function molecule Come acid imide, pyridine dimercapto or haloacetyl reaction, and forms thioether or cystine linkage.The order of this cross-linking reaction can also According to circumstances to change.For example, the amino on functional molecular can also react first with crosslinking agent and generate amido bond, then again It is reacted with the sulfydryl on cell combination body.For another example, sulfydryl under conditions of pH is 4.5~7.0, on functional molecular It first can react to form thioether or cystine linkage with crosslinking agent, then again under conditions of pH is 6~9 and on cell combination body Amino reacts to form amido bond.

Using the mercapto in sulfydryl-sugar crosslinking agent method of modifying, under conditions of pH is 4.5~8, on cell combination body Base can first on crosslinking agent maleimide or pyridine dimercapto react and to form thioether or cystine linkage.Then, functional Carbonyl (aldehydes or ketones) on molecule is reacted with hydrazides again generates hydrazone bond.In addition to this, under conditions of pH is 4.5~8, function point Sulfydryl on son can also react to form thioether or cystine linkage with crosslinking agent first, then again on cell combination body sugar, Oxosugar or carbonyl (aldehydes or ketones) reaction generate hydrazone bond.

Using the mercapto in hydroxyl-thiol crosslinkers method of modifying, under conditions of pH is 6~8, on cell combination body Base can first on crosslinking agent maleimide or the reaction of pyridine dimercapto generate thioether or cystine linkage.Then, in pH Under conditions of 8~9, then the hydroxyl on functional molecular is allowed to react generation carbamate with the isocyanates on crosslinking agent. In addition, the sulfydryl on functional molecular can also first and crosslinking agent reaction under conditions of pH is 6~8 generates thioether or double sulphur Key.Then, then with the hydroxyl on cell combination body in the conditioned response that pH is 8~9 generate carbamate.

In the direct-type method of modifying using sulfydryl reactant cross-linker, sulfydryl reactive group on crosslinking agent can be with With on cell combination body skeleton sulfydryl or seleno reaction (in the buffer solution of pH=6~9), formed a stable sulphur Ether, selenide key or cystine linkage.Firstly, specific polypeptide, protein, antibody are (especially under conditions of pH is 4.5~8.5 IgG1, Ig/2, IgG3 or IgG4 monoclonal antibody) or disulfide group on other function molecule or pass through what genetic engineering transformation introduced Reduction reaction occurs for disulfide group, two selenos or sulphur seleno and TCEP or DTT or 2 mercapto ethanol, forms sulfydryl or seleno group. Cell-binding molecules are reacted with crosslinking agent after purification, and form thioether, selenide key or cystine linkage.In the reduction process with TCEP In, TCEP may not need in reaction system and remove, (or directly in crosslinking agent together addition system) directly is reacted with crosslinking agent, Or azido compound can be added after TCEP reduction reaction is complete, excessive TCEP is neutralized, crosslinking agent is then added and carries out occasionally Connection reaction.Such sulfydryl coupling reaction can a solely sulfydryl on coupled cell combination, can also be simultaneously to dimercapto Form the coupling reaction of bridging type

In additional embodiment, when each connector is connected with the cytotoxic molecules of two molecules in conjugation couplet, this A little conjugated bodies have general structure below:

Wherein D₁And D₂For cytotoxic molecule or drug, the Tubulysin derivative of structure formula (I) is independently selected；T is Targeting or binding partner or cell-binding molecules.X₁、X₂、X₃And X₄It is independently NH, NR¹、O、S、CH₂, Se and NHNH； R¹、R², n and L definition as described in structure formula (II).Furthermore work as D₁And D₂Any one of structure formula (I) or I-01 to I-67 In Tubulysin derivative；In addition a cytotoxic molecule or drug D can be selected from can effective treating cancer Chemical molecular, such as calicheamicin, maytansine derivative, aplysiatoxin (dolastatins), CC-1065 analog, Ah mould Element, Taxane derivative, pyrroles's Benzodiazepine (PBD) dimer, tomaymycin dimer, anthramycin dimer, Indolinobenzodiazepines dimer, imidazobenzothiadiazepines dimer, or Oxazolidinobenzodiazepines dimer, amatoxins, indolecarboxamide, actinomycin, Azaserines, bleomycins, epirubicin, eribulin, tamoxifen, idarubicin, Ali's statin (auristatins), (monomethyl Ali's statin E, MMAE, MMAF, Ali's statin PYE, Ali's statin TP, dolastatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, geldanamycins, HSP90 inhibitor, Centanamycin, methotrexates, thio-tepa, vindesines, vincristines, hemiasterlins, Nazumamides, microginins, radiosumins, streptonigtin, SN38 and its derivative or camptothecine are metabolized Product, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU-15968, The combination of the polyethylene glycol (PEG) or the above substance of siRNA or molecular weight less than 100KD, pharmaceutically acceptable salt And acid, or the derivative of the above substance.

Cell-binding molecules of the invention are preferably antibody, mainly monoclonal antibody.The production of antibody includes it in life In object, external generation process or anabolic process.The production method of the polyclonal antibody of anti-receptor peptide fragment has been many institutes It is known, such as US patent number 4,493,795 (Nestor etc.).The classical way of preparation monoclonal antibody is using specific antigen Immune mouse, isolated mouse boosting cell merge (Kohler, G with myeloma cell；Milstein,C.1975Nature 256:495-497).Detailed operating procedure is shown in antibodies-A Laboratory Manual, Harlow and Lane, Eds., cold spring harbor laboratory press, new York (1988)), the method shifting, which is included in this article, is drawn Document.It in particular, can be by the way that mouse, rat, the method for hamster or other mammals be immunized with purpose antigen To obtain special monoclonal antibody.Wherein purpose antigen includes: complete cell, and isolated antigen, complete from cell Whole virus, the intact virus of reduction and virus protein.Splenocyte is merged with myeloma cell using PEG6000.Fusion The hybridoma obtained afterwards screens it using its sensibility to HAT.The monoclonal antibody that hybridoma generates is logical It crosses it immune response occurs with specific receptor in target cell or inhibits receptor active, this works in implementing this invention.

The monoclonal hybridoma obtained after fusion can secrete the monoclonal antibody for specific antigen.Currently, invention Used in monoclonal antibody, be exactly based on and cultivate monoclonal hybridoma in culture medium full of nutrition to which enrichment is anti- Body.Condition of culture need to ensure that hybridoma has time enough to enter the antibody-secreting of generation in culture medium.Antibody will be contained Culture supernatant collect after, purified by well known technical antagonism body.Separation method includes: the affine layer of albumin A Analysis method；Anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography and molecular sieve chromatography (especially use The affinity chromatography and molecular sieve chromatography of antigen crosslinking albumin A are with relatively broad)；Centrifugal process；The precipitation method or other marks Quasi- purification process

Effective culture medium and artificial synthetic medium needed for hybridoma culture can be synthesized by technology or business way Diameter obtains.Wherein, typical artificial synthetic medium: 4.5mg/ is added in DMEM (Virol such as Dulbecco 8:396 (1959)) L glucose, 20mM glutamine, 20% fetal calf serum and defoaming agent, such as: poloxalkol

In addition to cell-fusion techniques, the cell line of building production antibody can also be by other methods, such as: directly by tumour Originality DNA transfects bone-marrow-derived lymphocyte, or oncogenic virus gene (such as: EBV is also known as HHV-4 or KSHV) is imported bone-marrow-derived lymphocyte, It is detailed in US patent number .4341761；4399121；4427783；4444887；4451570；4466917；4472500； 4491632；4493890. monoclonal antibodies can also be by anti-receptor polypeptides or prepared by polypeptide containing carboxyl terminal.It is detailed in The Proc. such as Niman Natl.Acad.Sci.USA, 80:4949-4953 (1983)；The Proc.Natl.Acad.Sci. such as Geysen USA,82:178-182(1985)；(1995) the Biochemistry such as Lei 34 (20): 6675-6688.In general, anti-receptor polypeptides Either polypeptide analog can be used alone or be crosslinked the list that immunogenic carrier prepares anti-receptor polypeptides as immunogene Clonal antibody.

As the antibody of binding molecule, there are also other mature production methods in invention.Wherein of particular concern is production The process of human antibody.Display technique of bacteriophage is obtained from human antibody library by affine screening to be resisted with known The human antibody of original specific binding.Display technique of bacteriophage itself, vector construction and library screening all have in the literature Record in detail.It is detailed in the Gene.148 such as Dente (1): 7-13 (1994)；The Biotechnol such as Little Adv.12 (3): 539-55(1994)；The Nature such as Clackson 352:264-628 (1991)；The Science such as Huse 246:1275-1281 (1989)。

The monoclonal antibody obtained with hybridoma technology by other kinds (such as: mouse, rabbit, alpaca) need to its into Row is humanization modified.Antibody by transformation can greatly reduce heterologous antibody and side reaction is immunized caused by human body.Its In, the relatively conventional method of the humanization of antibody is the complementary transplanting and remodeling for determining domain.It is detailed in: US patent number 5859205 and 6797492；Liu et al., Immunol Rev. 222:9-27 (2008)；Almagro etc., Front Biosci.1； 13:1619-33(2008)；The Mol such as Lazar Immunol.44 (8): 1986-98 (2007)；Li et al. Proc.Natl.Acad.Sci.USA.103 (10): 3557-62 (2006) above-mentioned article is incorporated herein as a reference.Entirely Human antibody can also pass through the immune transgenic mice for carrying a large amount of human immunoglobulin(HIg) light chains and heavy chain of antigen, rabbit, monkey It is prepared by other mammals such as son.By taking mouse as an example: Xenomouse (Abgenix, Inc.), HuMab-Mouse (Medarex/BMS), VelociMouse (Regeneron), is detailed in: US patent number: 6596541,6207418, 6150584,6111166,6075181,5922545,5661016,5545806,5436149 and 5569825.Human treatment's mistake Cheng Zhong, the chimeric antibody of mouse antibody variable region gene and the building of constant area of human antibody gene integration is produced in human body Immunogenicity reaction to be significantly less than mouse resist (Kipriyanov etc., Mol Biotechnol.26:39-60 (2004)； Houdebine, Curr Opin Biotechnol.13:625-9 (2002) above-mentioned article be incorporated herein as a reference this Outside, carrying out rite-directed mutagenesis to the moiety site of antibody variable region can effectively improve the affinity and specificity of antibody (Brannigan etc., Nat Rev Mol Cell Biol.3:964-70 (2002)；Adams etc., J Immunol Methods. 231:249-60(1999)).The parent of itself and the immunological effect factor can also effectively be promoted by carrying out partial replacement to antibody constant region With power to enhance cytotoxic effect, or antibody is reduced to normal organ tissue (such as the swollen tissue Fc- γ combination of liver) Affinity, to reduce toxic side effect.

There is the antibody of immunologic specificity to malignant cell antigen, the technical side of commercial sources or some maturations can be passed through Method obtains, such as: chemical synthesis or recombination and expression techniques.The encoding gene of this kind of antibody again may be by some business ways Diameter, such as GenBank database or other similar databases, published document, or conventional cloning and sequencing method are obtained.

In addition to antibody, polypeptide or albumen can be used as binding molecule similarly to combine, and block, attack or pass through it Its mode receptor corresponding with target cell surface or epitope interaction.As long as these polypeptides or albumen energy specific bond Defined epitope or its correspondence receptor, then they are not necessarily intended to belong to immunoglobulin class.These polypeptides can also pass through class (Szardenings, J Recept Signal Transduct is separated like the technology of phage displaying antibody Res.2003,23 (4): 307-49).The peptide fragment obtained from random polypeptide libraries and antibody and antibody fragment using similar. Polypeptide or protein molecular can connect the specificity to keep its antigen binding by its binding molecule with some macromoleculars or medium. These macromoleculars and medium include: albumin, polymer, liposome, nano particle or dendrimers.

In the treatment of cancer, autoimmune disease and infectious diseases, for being coupled the anti-of Tubulysin derivative Body is exemplified below (but not limited to this): 3F8 (anti-GD2 antibody), A Bafu monoclonal antibody (anti-CA-125 antibody), and Abciximab is (anti- CD41 antibody (integrin alpha-IIB), adalimumab (anti-TNF-Alpha antibodies), A De wood monoclonal antibody (anti-EpCAM antibody, CD326), Afelimomab (anti-TNF-α)；Ah husband's soil pearl (anti-CD 20 antibodies), (anti-vegf R2 is anti-by Alacizumabpegol Body), ALD518 (anti-IL-6 antibody), (alias: Campath, MabCampath, Alemtuzumab, anti-CD52 are anti-for alemtuzumab Body), Altumomab (CEA antibodie), Anatumomab (anti-TAG-72 antibody), Anrukinzumab (alias: IMA- 638, anti-IL-13 antibody), A Bozhu monoclonal antibody (anti-HLA-DR antibody), Arcitumomab (CEA antibodie), A Saizhu monoclonal antibody (anti-L-selectin (CD62L) antibody, Atlizumab (alias: Torr pearl monoclonal antibody, Actemra, RoActemra, anti-IL-6 receptor Antibody), Atorolimumab (anti-rh factor antibody), bapineuzumab (anti-β-amyloid), Bali former times (Shu Lai, antiCD25 (the α chain of IL-2 receptor) antibody, Ba Wei former times monoclonal antibody (anti-phosphatidylserine antibody), shellfish be not appropriate single for monoclonal antibody Anti- (alias: LymphoScan, anti-CD22 antibody), Baily wood monoclonal antibody (alias: Benlysta, LymphoStat-B, anti-BAFF Antibody), Benralizumab (anti-CD125 antibody), cypress replaces the wooden monoclonal antibody, and (anti-CCL11 (eosinophil chemokine -1) is anti- Body), shellfish Suo Dankang (alias: Scintimun, anti-CEA- associated antigen), (alias: Avastin resists bevacizumab VEGF-A antibody) Biciromab (alias: FibriScint, II β chain antibody of antiplasmin), Bivatuzumab is (anti- CD44v6 antibody), blinatumomab (alias: BiTE, anti-CD19 antibody), Brentuximab (CAC10, anti-CD30, TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), (alias: Ilaris, anti-IL-1 are anti-for Kang Na monoclonal antibody Body), Cantuzumab (alias: C242, anti-CanAg antibody), Capromab, (alias: removab resists catumaxomab EpCAM, anti-CD 3 antibodies), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD 4 antibodies) match trastuzumab (alias Cimzia anti-TNF-α antibody), Cetuximab (alias: Erbitux, IMC-C225, anti-EGFR-antibodies), his native pearl of west is (anti- EpCAM antibody), Cixutumumab (anti-IGF-1 antibody), clenoliximab (anti-CD 4 antibodies), Clivatuzumab is (anti- MUC1 antibody), Conatumumab (anti-TRAIL-R2 antibody), CR6261 (anti-A type influenza hemagglutinin antibody), Dacetuzumab (anti-CD 40 antibodies), daclizumab (alias: Zenapax, anti-CD25 (the α chain of IL-2 receptor) antibody), Daratumumab (anti-cd 38 (cyclisation ADP ribose hydrolase) antibody), (alias: Prolia, anti-RANKL are anti-by Di Nuosaimai Body), Detumomab (anti-B- lymphoma cell antibody), Dorlimomab Aritox, Dorlixizumab, Ecromeximab (anti-GD3 mind Warp knuckle glycosides rouge antibody), Yi Kuli monoclonal antibody (alias: Soliris, anti-C5 antibody), Edobacomab (anti-endotoxin antibody), according to certainly Lip river monoclonal antibody (alias: Panorex, MAb17-1A, anti-EpCAM antibody), efalizumab (alias: Raptiva, anti-LFA- 1 (CD11a) antibody), Yi Fengu monoclonal antibody (alias: Mycograb, anti-Hsp90 antibody), (anti-SLAMF7 is anti-by Elotuzumab Body), Yi Silimo (anti-IL-6 antibodies), Enlimomab (anti-ICAM-1 (CD54) antibody), Epitumomab (anti-episialin Antibody), epratuzumab (anti-CD22 antibody), Erlizumab (anti-ITGB2 (CD18) antibody), Ertumaxomab (alias: Rexomun, anti-HER2/neu, CD3 antibody), Yi Ruixi pearl (alias: Abegrin, anti-integrin alpha v beta 3 antibody), Ai Wei monoclonal antibody (anti-HBs antibody), Fanolesomab (alias: NeutroSpec, anti-CD15 antibody), faralimomab (anti-interferon receptor antibody), Farletuzumab (anti-1 antibody of folacin receptor), Felvizumab (anti respiratory syncytial virus Antibody), Fezakinumab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor antibody), Fontolizumab are (anti- IFN-γ antibody), such as (Antibody against the glycoprotein of rabies viruse), Fresolimumab (anti-TGF-beta antibodies) adds sharp former times to Fu Ruiwei Monoclonal antibody (Anti-CD80 McAb), Gantenerumab (anti-beta amyloid antibody), Gavilimomab (anti-CD14 7 (basigin) antibody), WAY-CMA 676 (anti-CD 33 antibody), Girentuximab (anti-9 antibody of carbonic anhydrase), Glembatumumab (alias: CR011, anti-GPNMB antibody), golimumab (alias: SIMPONI, anti-TNF-α antibody), Gomiliximab (anti-CD23 (IgE receptor) antibody), Ibalizumab (anti-CD 4 antibodies), (anti-CD20 is anti-for ibritumomab tiuxetan Body), Igovomab (alias: Indimacis-125, anti-CA-125 antibody), Imciromab (alias: Myoscint, the anti-heart Flesh anti-tropomyosin antibody), infliximab (alias: infliximab, anti-TNF-α antibody), Intetumumab (anti-CD51 Antibody), Inolimomab (anti-CD25 (IL-2 receptor alpha chain) antibody), Inotuzumab (anti-CD22 antibody), Yi Puli nurse Ma (anti-CD152 antibody), Iratumumab (AntiCD3 McAb 0 (TNFRSF8) antibody), Keliximab (anti-CD 4 antibodies), Labetuzumab (alias: CEA-Cide, CEA antibodie), Lebrikizumab (anti-il-13 antibody), Lemalesomab are (anti- NCA-90 (G-Ag) antibody), Le Demu monoclonal antibody (anti-TGF β -2 antibody), Lai Shamu monoclonal antibody (2 antibody of anti-TRAIL-R), Sharp Wei Dankang (anti-HBs antibody), lintuzumab (anti-CD 33 antibody), (anti-CD40 is anti-by Lucatumumab Body), Shandong former times monoclonal antibody (anti-CD23 (IgE receptor) antibody), Mapatumumab (1 antibody of anti-TRAIL-R), Maslimomab is (anti- T-cell receptors antibody), matuzumab (anti-EGFR-antibodies), mepolizumab (alias: Bosatria, anti-IL-5 antibody), (anti-TAG-72 is anti-by Metelimumab (anti-TGF β -1 antibody), Milatuzumab (anti-CD74 antibody), Minretumomab Body), mitumomab (alias BEC-2, anti-GD3 ganglioside antibody), (anti-rh factor is anti-by Morolimumab Body), Mo Weizhu monoclonal antibody (alias: NUMAX, anti respiratory syncitial virus antibodies), muromonab-CD3 (alias: OKT3, ORTHOCLONE, AntiCD3 McAb antibody), Nacolomab tafenatox (anti-C242 antibody), Ta Namo monoclonal antibody (anti-5T4 antibody), his pearl is single Anti- (alias: Tysabri, anti-4 antibody of integrin alpha), Nebacumab (anti-endotoxin antibody), Necitumumab (anti-EGFR Antibody), Nerelimomab (anti-TNF-α antibody), (alias: Theracim, Theraloc, anti-EGFR are anti-for Buddhist nun's trastuzumab Body), Nofetumomab, ocrelizumab (anti-CD 20 antibodies), Odulimomab (alias: Afolimomab, anti-LFA-1 (CD11A) antibody), the wooden monoclonal antibody (alias: Arzerra, anti-CD 20 antibodies), Olaratumab (anti-PDGF-R Alpha antibodies), Ma Zhu difficult to understand Monoclonal antibody (alias: Sorel, anti-IgEFc domain antibodies), Oportuzumab (anti-EpCAM antibody), Ao Gefu monoclonal antibody (alias: OvaRex, anti-CA-125 antibody), Otelixizumab (AntiCD3 McAb antibody), pa Ji former times monoclonal antibody (anti lipoteichoic acid antibody), Pa Li Pearl monoclonal antibody (alias: Synagis, Abbosynagis, anti respiratory syncitial virus antibodies), Victibix (alias: dimension gram for than, ABX-EGF, anti-EGFR antibody), Panobacumab (anti Bacillus pyocyaneu Flugge antibody), Pascolizumab (anti-IL-4 antibody), Pemtumomab (alias: Theragyn, anti-MUC1 antibody), handkerchief trastuzumab (alias: OMNITARG, 2C4, anti-HER2/neu Antibody), it trains gram pearl monoclonal antibody (anti-C5 antibody), Pintumomab (anti-gland cancer antigen-antibody), priliximab (anti-CD 4 antibodies), General bolster monoclonal antibody (anti-vimentin antibodies), PRO140 (antibodies against CCR 5), Racotumomab (alias: 1E10, anti-(N- hydroxyl N acetylneuraminic acid n (NeuGc, NGNA)-Ganglioside GM3) antibody), Rui Feiweilu (Antibody against the glycoprotein of rabies viruse), Ramucirumab (anti-vegf R2 antibody), Lucentis (alias: Lucentis, anti-vegf-A antibody), the Baku Rui Xi (anti-charcoal Deep carbuncle element, protective antigens antibody), regavirumab (the Glycoprotein B antibody of anti-cytomegalovirus), Reslizumab (anti-IL- 5 antibody), Rilotumumab (anti-HGF antibody), Rituximab (alias: Mabthera, Rituxanmab, anti-CD 20 antibodies), Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-ifn-alpha antibody), Rovelizumab (alias: LeukArrest, anti-CD11, CD18 antibody), Ruplizumab (alias: Antova, anti-CD154 (CD40L) antibody), it is husky it is appropriate not Monoclonal antibody (anti-TAG-72 antibody), Sevirumab (anti-cytomegalovirus antibody), Sibrotuzumab (anti-FAP antibody), Sifalimumab (anti-ifn-alpha antibody), Siltuximab (anti-IL-6 antibodies), Siplizumab (anti-CD2 antibody), (Smart) MI95 (anti-CD 33 antibody), Solanezumab (anti-β-amyloid), Sonepcizumab (anti-sphingosine- 1- phospho-AB), Suo Tuzhu monoclonal antibody (anti-episialin antibody), Si Tamolu (anti-myostatin antibody), sulphur Suo Dan Anti- (alias: LeukoScan, (anti-NCA-90 (G-Ag) antibody))), (Anti-α-Fetoprotein is anti-by Tacatuzumab Body), he spends pearl monoclonal antibody (anti-3 antibody of integrin alpha IIb β), his sharp pearl monoclonal antibody (anti-IgE antibodies), his Buddhist nun pearl (anti-ngf antibodies), Taplitumomab (anti-CD 19 antibodies), Tefibazumab (alias: Aurexis, resistant to aggregation factors A antibody), Ah Ti be not single It is anti-, Tenatumomab (anti-fixing arsenic roasting C antibody), for how former times monoclonal antibody (anti-CD 40 antibodies), (AntiCD3 McAb is anti-by Teplizumab Body), TGN1412 (anti-CD28 antibody), Ticilimumab (alias: Tremelimumab (anti-CTLA-4 antibody), Tigatuzumab (2 antibody of anti-TRAIL-R), TNX-650 (anti-il-13 antibody), Torr pearl monoclonal antibody (alias Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor antibody), Toralizumab (anti-CD154 (CD40L) antibody), Tosi be not single Anti- (anti-CD20 antibody), Herceptin (Trastuzumab, (anti-HER2/neu protein antibodies), Tremelimumab (anti-CTLA-4 Antibody), Tucotuzumabcelmoleukin (anti-EpCAM antibody), Tuvirumab (anti-B Hepatitis virus antibody), Urtoxazumab (anti-Escherichia coli antibody), this excellent its monoclonal antibody (alias: Stelara, anti-IL-12, IL-23 antibody), cuts down sharp former times Monoclonal antibody (anti-AOC3 (VAP-1) antibody), Vedolizumab, (anti-4 β of integrin alpha, 7 antibody), (anti-CD20 is anti-by Veltuzumab Body), vepalimomab (anti-AOC3 (VAP-1) antibody, Visilizumab (alias: Nuvion, anti-cd 3 antibodies), Vitaxin (anti-angiogenic integrin av b3 antibody), Volociximab (anti-Integrin α5 β1), Votumumab (alias: HumaSPECT, Antitumor antigens CTAA16.88 antibody), prick the appropriate wooden monoclonal antibody (alias: HUMAX-EGFR, (anti-egfr antibodies), Zanolimumab (alias: HUMAX-CD4, anti-CD 4 antibodies), Ziralimumab (anti-CD147 (fundamental immunity globulin) antibody), A Zuomo is mono- Anti- (anti-CD5 antibody), Etanercept (ENBREL), Ah method's Saite (AMEVIVE), OrenciaBenefit Nahsi is general (ARCALYST), and (anti-GD2 gangliosides are anti-by 14F7 [anti-IRP-2 (iron ion regulatory protein 2) antibody], 14G2a Body treats melanoma and solid tumor, fromnat.cancer inst.), (anti-PSMA antibody treats prostate cancer, health to J591 Medical college like this), 225.28S [anti-HMW-MAA (high molecular weight melanoma related antigen) antibody, Suo Lin Radiofarmaci SRL (Milan, ITA) treats melanoma], COL-1 (anti-CEACAM3 antibody, CGM1, (American National Cancer research institute), treat colorectal cancer and gastric cancer), CYT-356 ( Treat prostate cancer), HNK20 (OraVax Company, for treating Respiratory Syncytial Virus(RSV)), ImmuRAIT (treats non-Hodgkin lymphoma from IMMUNOMEDICS), Lym-1 (anti-HLA-DR10 antibody, BNP Paribas Peregrine medicine, be used for cancer), [anti-TNF antibodies are (also known as: tumor necrosis factor by MAK-195F Son；TNFA, tumor necrosis factor-alpha；TNFSF2), Alpert/Nore, for treating septicemia toxic shock], MEDI-500 [alias: T10B9, anti-cd 3 antibodies, TR α β (T cell receptor α/β), compound, MedImmune company are anti-for treating graft Host disease], RINGSCAN [anti-TAG72 (72 antibody of tumor-associated glycoprotein), Neoprobe group, for treat breast cancer, Colon cancer and the carcinoma of the rectum, (anti-EpCAM antibody (epithelial cell adhesion molecule), (tumour is related for anti-TACSTD1 antibody by Avicidin Ca2+ oscillations tranducin 11), anti-GA733-2 (stomach and intestine tumor GAP-associated protein GAP 2), anti-EGP-2 antibody (Glycoproteins in Epithelial 2)；Anti- KSA is anti- Body；KS1/4 antigen；M4S；Tumour antigen 17-1A；CD326, from NeoRx company for treating colon cancer, oophoroma, forefront Gland cancer and non-Hodgkin lymphoma；LymphoCide (Immunomedics company), smartID10 (Protein Design Labs), Oncolym (Techniclone company), Allomune (BioTransplant), anti-VEGF antibody (Genentech Company)；CEAcide (Immunomedics company), IMC-1C11 (ImClone) and Cetuximab (ImClone company).

Other antibody for combining antigen include (but being not limited only to this): Aminopeptidase N (CD13), Annexin A1, B7- H3 (CD276, various cancers), CA125, CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), LewisY (cancer), LewisX (cancer), Alpha-fetoprotein (cancer), CA242, P-ALP (cancer), prostate-specific antigen (prostate cancer), prostate gland acid phosphorus Sour enzyme (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, the lymthoma of non-Hodgkin lymphoma, multiple marrow Tumor), the ε (t cell lymphoma, lung cancer, breast cancer, gastric cancer, oophoroma, autoimmune disease, malignant ascite) of CD3, CD19 (B cell malignant tumour), CD20 (non-Hodgkin lymphoma), CD22 (leukaemia, lymthoma, Huppert's disease, systematicness Lupus erythematosus), CD30, CD33, CD37, CD38 (Huppert's disease), CD40 (lymthoma, Huppert's disease, white blood Disease), CD51 (metastatic melanoma, sarcoma), CD52, CD56 (Small Cell Lung Cancer cancer, oophoroma, Merkel cell cancer, and Liquid tumors, Huppert's disease), CD66e (cancer), CD70 (metastatic renal cell cancer and non-Hodgkin lymphoma), CD74 (Huppert's disease), CD79, CD80 (lymthoma) CD98 (cancer), mucoprotein (cancer), CD221 (solid tumor), CD227 (cream Gland cancer, oophoroma), CD262 (non-small cell lung cancer and other cancers), CD309 (oophoroma), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinomebryonic antigen；CEA, CD66e) (breast cancer, colorectal cancer and lung cancer), DLL3 (Δ shape- 3), DLL4 (Δ shape -4), EGFR (EGF-R ELISA, various cancers), CTLA4 (melanoma), CXCR4 (CD184, Ferroheme tumour, entity tumor), Endoglin (CD105, solid tumor), EPCAM (epithelial cell adhesion molecule, bladder cancer, Head, neck, colon, prostate non-Hodgkin lymphoma and oophoroma), ERBB2 (epidermal growth factor acceptor 2；Lung cancer, mammary gland Cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folacin receptor, oophoroma), GD2 gangliosides (cancer), G-28 (a kind of cell surface antigen glyvolipid, melanoma), idiotype GD3 (cancer), heat shock protein (cancer), HER1 (lung, gastric cancer), HER2 (breast cancer, lung cancer and oophoroma), (non-Hodgkin lymphoma, B are thin by HLA-DR10 (NHL), HLA-DRB Born of the same parents' leukaemia), human chorionic gonadotrophin (cancer), IGF1R (type-1 insulin like growth factor receptor, solid tumor, blood cancer Disease), IL-2 receptor (Interleukin-2 Receptor, T- chronic myeloid leukemia and lymthoma), IL-6R (interleukin-6 receptor, it is multiple Property myeloma, rheumatoid arthritis, Castleman disease, IL6 dependent tumors), integrin (5 β 1 of plain α v β 3, α, alpha 6 beta 4, α Ll β 3, α 5 cell attachment fac of 5 β 5, α v β, to various cancers), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE4 (cancer), anti-rotation Human Placental Ferritin Receptor (cancer), P97 (melanoma), MS4A1 (4 subfamily A member 1 of transmembrane domain, non-Hodgkin's B Cell lymphoma, leukaemia), MUC1 or MUC1-KLH (breast cancer, oophoroma, cervix cancer, bronchus and gastrointestinal cancer Disease), MUC16 (CA125) (oophoroma), CEA large intestine), GP100 (melanoma), MART1 (melanoma) MPG (melanin Tumor), MS4A1 (4 albumin A of transmembrane domain, Small Cell Lung Cancer, non-Hodgkin lymphoma), kernel, neural oncoprotein (cancer), P21 (cancer) resists (NeuGc ALPHA2-3Gal, breast cancer, the cancer of melanoma), PLAP sample testis alkaline phosphatase (oophoroma, Carcinoma of testis), PSMA (tumor of prostate), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukaemia, stomach Cancer, colorectal cancer, oophoroma) in, the transmembrane protein (cancer) of T cell, Tie (CD202b), TNFRSF10B (neoplasm necrosis Factor acceptor superfamily member 10B, cancer), TNFRSF13B (A member of the TNF receptor family 13B, multiple bone Myeloma, non-Hodgkin lymphoma and other cancers, rheumatoid arthritis and systemic loupus erythematosus), TPBG (trophoderm Glycoprotein, clear-cell carcinoma), and TRAIL-R1 (neoplasm necrosis apoptosis induction ligand receptor 1, lymthoma, non-Hodgkin lymphoma, greatly Intestinal cancer, lung cancer), VCAM-1 (CD106, melanoma), vascular endothelial growth factor, vascular endothelial growth factor-A, VEGF- 2 (CD309) various cancers).By antibody identify some other tumor associated antigens checked (Gerber etc., mAbs 1: 3,247-253 (2009)；Novellino etc., Cancer Immunol Immunother.54 (3), 187-207 (2005)； Franke etc., Cancer Biother Radiopharm. 2000,15,459-76).There are many more other antigens to be: other Different collection group antigen (such as CD1, 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, CD43, 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,CD85e,CD85f,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, 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 or CD389), APO2, ASLG659, BMPR1B (bone morphogenetic protein receptor), CRIPTO, Annexin A1, kernel, Endoglin (CD105), ROBO4, aminopeptidase N, CTLA-4, Δ-sample 3 (DLL3), Δ-sample 4 (DLL4), VEGFR-2 (CD309), CXCR49, CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6E7, EGFRvIII, EGFR, HER-2/neu, idiotype, MAGE A3, P53nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Napi3b (NAPI-3B, NPTIIb, SLC34A2, it is molten Matter carrier families 34, member 2, the phosphorus transporter 3b that II type sodium relies on), Ras mutation, gp100, p53 mutant, Proteinase3 (PR1), growth factor derived from BCR-abl, Tetratocarcinoma), EphA receptor, EphB receptor, EGFR, EGFRvIII, ETBR (Endothelin), HER2/neu, HER3, HLA-DOB (mhc class ii molecule IA antigen), integrin, IRTA2, MPF (MPF, MSLN, SMR, megacaryocyte intensifier, mesothelin), CRIPTO, PD-1, PD-L1, Sema 5b (domain FLJ10372, KIAA1445, Mm42015, SEMA5B, 5EMAG, semaphoring 5bHlog, sdema, seven blood are small Plate repetitive sequence, cytoplasmic domain), PSCA, STEAP1 (6 cross-film epithelium prostate antigens) and STEAP2 (HGNC 8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate), tyrosinase, survivin, hTERT, sarcoma translocation breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2ETS fusion), NA17, PAX3, ALK, androgen receptor, Cyclin B1, poly sialic acid, MYCN, RhoC, TRP-2, GD3, fucose gangliosides, 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, Human sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, beanpod egg It is white, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR- β, MAD-CT-2, Fos albumen related antigen 1.

Another special applications is Tubulysin derivative-cell-binding molecules conjugated body (coupling in the present invention Object) treatment of cancer can be suitable for method in different combinations.These cancers include, however it is not limited to, adrenocortical carcinoma, directly Intestinal cancer, bladder cancer, brain tumor are (adult: brain stem glioma, children, cerebellar astrocytoma, astrocytoma, ependymoma, marrow Blastoma, Supratentorial primitive neuroectodermal tumour, pineal body, pathways for vision and hypothalamic gliomas), breast cancer, class cancerous swelling Tumor, gastrointestinal tract, unknown primary carcinoma, cervical carcinoma, colon cancer, carcinoma of endometrium, the cancer of the esophagus, cholangiocarcinoma, Ewing family of tumor (PNET), the outer malignant germ cell tumors of cranium, cancer eye, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germinoma, Outside sexual gland, gestational trophoblastic tumor, head and neck neoplasm, hypopharyngeal cancer, islet-cell carcinoma, kidney (clear-cell carcinoma), laryngocarcinoma, white blood Sick (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelocytic, hairy cell), lip and carcinoma of mouth, liver cancer, (non-small cell, cellule, (AIDS is related, central nervous system, cutaneous T-cell, Hodgkin's disease, Fei Huoqi for lymthoma for lung cancer Golden disease, malignant mesothelioma, melanoma, merkel's cells cancer, the invisible squamous cervical metastatic cancer of primary, Huppert's disease With other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, bone marrow proliferative diseases, nasopharyngeal carcinoma, nerve mother is carefully Born of the same parents' tumor, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, oophoroma (epithelial cell, gonioma, low potential malignancy potential tumour), cancer of pancreas (outer secretion, islet-cell carcinoma), nasal sinus and CARCINOMA OF THE NASAL CAVITY, parathyroid carcinoma, carcinoma of penis, pheochromocytoma cancer, pituitary tumor, slurry are thin Palpebral edema tumor, prostate cancer rhabdomyosarcoma, the carcinoma of the rectum, clear-cell carcinoma (kidney), renal plevis and ureter (migratory cell), salivary gland Cancer, plug prick syndrome, and cutaneum carcinoma, cutaneum carcinoma (skin sample t cell lymphoma, Kaposi sarcoma, melanoma), carcinoma of small intestine is soft Sarcomatous tissue, gastric cancer, carcinoma of testis, thymoma (pernicious), thyroid cancer, carcinoma of urethra, uterine cancer (sarcoma), children's abnormal tumor, Carcinoma of vagina, carcinoma of vulva, the nephroblastoma.

Another special applications is Tubulysin derivative-cell-binding molecules conjugated body (conjugate) in the present invention Can in different combinations and method, the prevention and treatment suitable for autoimmune disease.Autoimmune disease includes, but unlimited In, gastric anacidity autoimmune chronic active hepatitis, acute diseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Ah Di's Sen Shi disease, Agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/ Renal tubular basement membrane ephritis, antiphospholipid syndrome, anti-synzyme syndrome, arthritis, atopy allergy, allergic dermatitis, Autoimmune alpastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune liver Inflammation, Autoimmune Inner Ear Disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral nervous disease, Autoimmune pancreatitis autoimmunity more endocrine disease I, II, type III, autoimmune progesterone dermatitis, autoimmune blood Platelet reduction property purpura, autoimmune uveitis, Balo disease/Balo concentric sclerosis, bechet's syndrome, Berger Disease, Bickerstaff brainstem encephalitis, Blau syndrome, big bud class day bud sore, castleman disease, American trypanosomiasis, slowly Fatigue immunologic function disorder syndrome, the multiple neuropathy of chronic inflammation demyelinating, the how Focal marrow of chronic recurrent Inflammation, chronic Lyme disease, Chronic Obstructive Pulmonary Disease, allergic granulomatous angiitis, cicatricial pemphigoid, abdominal disease, Cogan's syndrome, cold coagulation disease, complement C2 deficiency disease, cranial arteritis, CREST syndrome, clone disease are (a kind of Idiopathic inflammatory bowel diseases), Ke's Xing Shi disease, skin leukocytoclastic vasculitis, malignant atrophic papulosis, pain obesity Disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffusivity skin chorionitis, postmyocardial infarction syndrome, plate-like erythema wolf Sore, eczema, endometriosis, juvenile idiopathic arthritis, eosinophilic fasciitis, eosinophilic fasciitis, knot Section property erythema, essential mixed cryoglobulinemia, Evan's syndome, gradual ossificans structure of fibrous tissue is bad, Fibromyalgia, fibromyositis, fibrosing alveolitis, gastritis, stomach and intestine pemphigoid, giant cell arteritis, glomerulonephritis, lung go out Blood nephritic syndrome, Graves' disease, guillain-Barre ramitis, this encephalitis of bridge, struma lymphomatosa, hemolytic Anaemia, anaphylactoid purpura is more, gestational period bleb, suppurative hidradenitis, Hughes's syndrome (antiphospholipid antibody syndrome), low third ball Proteinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (autoimmunity Property thrombocytopenic purpura), IgA nephrosis (Berger disease), inclusion body myositis, inflammatory Demyelination, chromic fibrous bladder Inflammation, irritable bowel syndrome, juvenile idiopathic arthritis, infantile rheumatoid arthritis, mucocutaneous lymph node syndrome are blue Bert myasthenic syndrome, leukocytoclastic angiitis, lichen planus, lichen sclerosus, linear IgA is sick (LAD), amyotrophia Property lateral schlerosis, lupoid hepatitis, lupus erythematosus, Ma Jide syndrome, Meniere's syndrome, more blood vessels under microscope Inflammation, Miller fisher's syndrome, mixed connective tissue disease, morphoea, Mu breathe out Er Shi disease, and Wells syndrome is multiple Myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (devic's disease), nerve Myotonia, eye cicatricial pemphigoid, opsoclonus-myoclonic syndrome, Ao Deshi thyroiditis, palindromic rheumatism, bear Cat syndrome (the children's autoimmunity neuropsychiatric abnormalities for merging streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal is slept Moultinism hemoglobinuria, progressive hemifacial atrophy, two Cotard of Ba-spy, pars planitis, pemphigus are ordinary Type pemphigus, pernicious anaemia, periphlebitis, POEMS syndrome, nodular polyarteritis, polymyalgia rheumatica, multiple flesh Inflammation, primary biliary cirrhosis, primary sclerotic cholangitis, progressive inflammatory neuropathies become, psoriasis, psoriasis arthropathica Inflammation, gangraenosum purulence skin, pure red cell aplasia, Ross are write from memory Sen Shi encephalitis, Raynaud's disease, relapsing polychondritis, Lay Te Er syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia It is comprehensive to glue blood for disease, Schmidt syndrome, Schnitzler syndrome, sclerotitis, chorionitis, Sjogren syndrome, spondyloarthropathy Sign, Still disease, stiff man syndrome, subacute bacterial endocarditis, Susac Cotard, acute febrile neutrophilia skin Disease, sydenham chorea, sympathetic ophthalmia, high iS-One arteritis, temporal arteritis (giant cell arteritis), Tolosa Hunt syndrome Syndrome, transverse myelitis, ulcerative colitis (a kind of idiopathic inflammatory intestinal disease), undifferentiated connective tissue disease, Undeveloped ovules, vasculitis, leucoderma, Wei Genashi granulomatosis, Wilson's syndrome, prestige Scott-Ao Er Delhi surprise syndrome.

In another embodiment, for treating or preventing autoimmune disease, the antigen binding of coupling Molecule includes, but are not limited to: elastoresistance protein antibodies；The anti-epithelial cell antibody of Abys；Anti- basilar memebrane IV collagen type antibody； Antinuclear antibodies；Anti-dsDNA antibody；Anti-ssDNA antibody, anticardiolipin antibodies IgM, IgG；Anti- celiac (anti- Celiac antibody) antibody；Anti-phospholipid antibody IgK, IgG；Anti- nucleoglucoprotein antibody；Anti-mitochondrial antibody；Thyroid gland is anti- Body；Microsomal antibody, T- cell antibody；Thyroglobulin antibody, anti-- 70 antibody of chorionitis (anti-SCL-70)；People is anti- Jo antibody (anti-jo)；Anti- Autoantibodies in Pateints with Systemic L upus Erythematosus；Resist drying syndrome antibody (Anti-La/SSB)； Anti- systemic loupus erythematosus antibody；Anti-parietal cell anti-body；Histonic antibody；Anti ribonucleoprotein antibody (anti-RNP)；In Property cytoplasm of granulocytes antibody (C-ANCA)；Anti- Antineutrophil antibody (P-ANCA) around nucleus；Anti centromere antibody；Anti- core Fibrin antibody and AGBM antibody (GBM) antibody, anti-gangliosides (Anti-ganglioside) are anti- Body；Anti desmoglein 3 antibody (anti-Desmogein3)；Anti-human P62 antibody；Anti-human sp100 antibody；Resist mitochondria M2 Antibody；Rheumatoid factor antibodies；Anti- saltant type citrulling vimentin antibodies (anti-MCV)；Anti- topoisomerase enzyme antibody；It is anti- Neutrophil leucocyte endochylema (CANCA) antibody.

It in certain preferred aspects, can be with autoimmunity for being conjugated the binding molecule of coupling in the present invention Property disease relevant activated lymphocyte expression receptor or receptor complex combine.Including immunoglobulin gene superfamily at Member (such as 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 member (such as 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 30APO-3), integrin, cytokine receptor, chemokine receptors, mainly Tissue compatible protein, agglutinin (C type, S type or I type) or complement regulatory proteins.

In another embodiment, there is the useful combination of immunologic specificity to viral or bacterial antigen It is source of people or human monoclonal antibodies.Term " viral antigen " as used herein includes, but are not limited to: any can induce is immunized The viral peptide fragment of reaction, polypeptide protein is (for example, HIVgp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase Enzyme, influenza virus hemagglutinin, human T-lymphocyte virus infection regulatory factor tax, herpes virus glycoprotein (for example, gB, GC, gD and gE) and hepatitis B surface antibody).Term " bacterial antigen " as used herein including but not limited to: it is any The microorganism peptide fragment of immune response can be induced, polypeptide protein, carbohydrate, polysaccharide, (for example, bacterium, fungi is pathogenic for lipid molecular Protozoan, yeast polypeptides include, for example, lipopolysaccharides and capsular polysaccharide 5/8).It can be used for treating viral or bacterial infection Useful I type antibody include, but are not limited to: palivizumab, it is a kind of for treat the source of people of rsv infection preventing respiratory close born of the same parents Precursor virus monoclonal antibody；PRO542, it is a kind of for treating the CD4 fusion antibody of HIV infection；Ostavir, it is a kind of to treat hepatitis B Viral human antibody；PROTVIR, one kind is for treating cytomegalovirus humanized antibody IgG.sub.1, and there are also anti-grease polysaccharide (anti-LPS) antibody.

Binding molecule of the invention --- Tubulysin derivative conjugate can be used for treating infectious diseases.These Infectious disease includes, but are not limited to: acinetobacter, lumpy jawl clams, lethargus (African typanosomiasis nagana), and AIDS (obtains Property acquired immunodeficiency syndrome), amcbiasis, Anaplasmosis, anthrax, arcanobacterium haemolyticum infection, Argentinian hemorrhagic fever, ascarid Parasitosis, aspergillosis, astrovirus infection, babesiasis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis BV, Bacteroides infection, balantidiasis, Baily ascarid nematode infections, BK virus infection, black piedra, infection with Blastocystis hominis, bud Raw bacterium, Bolivian hemorrhagic fever, borrelia infection, botulismus (and baby's botulismus), Brazilian hemorrhagic fever, Brucella Disease, Bai Huoerde bacillus infection, Bu Luli ulcer, infection calicivirus (norovirus and Sapporo virus), campylobacteriosis is read Pearl bacterium infects (candidiasis, thrush), cat scratch disease, cellulitis, Chaggs'disease (American trypanosomiasis), chancroid, varicella, Chlamydia, infection involving chlamydia pneumoniae, cholera, chromoblastomycosis, liver rot, Clestridium difficile infection, coccidioides immitis Disease, colorado tick fever, common cold (acute virus nasopharyngitis；Acute rhinitis), creutzfeldt-Jacob disease, Crimean Congo hemorrhagic Heat, cryptococcosis, Cryptosporidiosis, cutaneous larva migrans, Cyclospora infection, cysticercosis, cytomegalovirus infection, Dengue Heat, Dientamoeba disease, diphtheria, diphyllobothrium, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, Ehrlichiosis, enterobiasis (retrofection), enterococcal infection, enterovirus infection, epidemic typhus, erythema infectioum (the 5th disease), exanthem subitum, Fasciolopsiasis, fatal familial insomnia, filariasis, the caused food poisoning of C.perfringens, non-parasitism amoeba infection, Fusobacterium infections, emphysematous gangrene (clostridial myonecrosis), geotrichosis, lucky Stedman-Astrid Strauss syndrome, Giardiasis, Glanders, ganthostomiasis, stranguria syndrome, granuloma inguinale, the infection of A group of streptococcus, the infection of B group of streptococcus, haemophilus influenzae Infection, hand-foot-and-mouth disease (HFMD), Hantavirus pulmonary syndrome, helicobacter pylori infections, hemolytic uremic syndrome, hemorrhagic fever renal Sick syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, Hepatitis E, herpe simplex, histoplasmosis, Hookworm infection, people's Bao Kan virus infection, people angstrom Wen Ehrlichia disease, Human granulocytic anaplasmosis, human metapneumovirus infection, Human monocyte's Ehrlichia disease, human papilloma virus infection, human parainfluenza viruses' infection, hymenolepiasis, Ai Posi Smooth-epstein-Barr virus infectious mononucleosis (single), influenza, isosporiasis, Kawasaki disease, keratitis, Jin Shi gold Family name's bacillus infection, kuru, Lassa fever, legionellosis (legion member disease), legionellosis (Pang Diyake Heat), leishmaniasis, leprosy, leptospirosis, listerellosis, Lyme disease (lime borreliosis), lymph filaria Sick (elephant hide swell), lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever, morbilli, glander-like disease (Hui Temoershi Disease), meningitis, meningococcosis, metagonimiasis, microsporidiosis, molluscum contagiosum, mumps, macula wound Cold (matlazahuatl), Eaton agent pneumonia, madura diseasemadura foot, fly-blown, neonatal ophthalmia (ophthalmia neomatorum), gram-sub- comprehensive Simulator sickness (vCJD, nvCJD), nocardiasis, onchocercosis (river blindness), paracoccidioidomycosis (Paracoccidioidomycosis), lung Fluke disease, pasteurellosis, pediculosis capillitli (head louse), pediculosis corporis (body louse), pediculosis pubis (crab louse), pelvic inflammatory disease, pertussis, the plague, Pneumococcal infection, pneumocystis carinii pneumonia, pneumonia, polio, general Salmonella infection, primary amebic meningo encephalitis, The more lesion leukoencephalopathies of progressive, psittacosis, Q heat, rabies, rat-bite fever, respiratory syncytial virus infection, rhinosporidiosis, Rhinovirus infection, rickettsial infection, rickettsia, Rift Valley fever, exanthematic typhus of Sao Paulo, rotavirus infection, rubeola, sramana Salmonella disease, SARS (SARS (Severe Acute Respiratory Syndrome)), scabies, snail fever, septicemia, dysentery (bacillary dysentery), shingles zoster (shingles zoster), smallpox (smallpox), sporotrichosis, staphylococcal food poisoning, staphy lococcus infection, nematode, syphilis, tapeworm Disease, tetanus (lockjaw), barber's itch, favus of the scalp, ringworm of the body, jock itch, the tinea manuum, tinea nigra, tinea pedis, onychomycosis, tinea versicolor bend roundworm Sick (larva migrans,ocular), toxocarasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomoniasis, trichuriasis (whip Insect infection), pulmonary tuberculosis, yatobyo, ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, disease Toxicity pneumonia, West Nile fever, white piedra (white piedra), false knot solani infection, Yersiniosis, yellow heat Disease, zygomycosis.

The binding molecule of the previously described antibody of this patent can be used for anti-pathogenic strain, include, but are not limited to: Bao Man is not Lever bacterium, actinomyces Israeli, Ge Shi actinomyces and propionic acid propionate bacillus, trypanosoma bocagei, HIV (human immunodeficiency Poison), Entamoeba histolytica, Anaplasma, bacillus anthracis, arcanobacterium haemolyticum, Junin virus, roundworm, aspergillus, starlike disease Malicious family, Babesia category, Bacillus cereus, more bacillus, Bacteroides, balantidium Coli, Baylisascaris, BK Virus, piedraia hortai, blastocystis hominis, Blastomyces dermatitidis, arenavirus, Borrelia, clostridium botulinum, Sabia japonica, Brucella, usual Burkholderia cepacia and other Burkholderia cepacia kinds, mycobacterium buruli, Caliciviridae man Race, campylobacter, usually Candida albicans and other Mycotoruloides, Bartonella, A group of streptococcus and staphylococcus, gram Family name's trypanosome, haemophilus ducreyi, varicellazoster virus (VZV), chlamydia trachomatis, chlamydia pneumoniae, comma bacillus, Pei Family name colours mould, clonorchis sinensis, Clestridium difficile, posadasis spheriforme and Coccidioides posadasii, section sieve Draw more tick fever virus, rhinovirus, coronavirus, prion creutzfeldt-Jacob disease, crimean-Congo hemorrhagic fever virus, Novel hidden ball Bacterium, Cryptosporidium, ancylostoma braziliense；Multiple parasites, ring spore, taeniasis suis, cytomegalovirus, Dengue pyreticosis Malicious (DEN-1, DEN-2, DEN-3 and DEN-4) arboviruse, dientamoeba fragilis, Bacterium diphtheriae, Diphyllobothrium, wheat field That imperial nematode, Ebola virus, Echinococcus, Ehrlichia category, pinworm, enterococcus spp, enterovirus genus, Rickettsia prowazeki, Assays for parvovirus B 19, human herpes virus-6 and Human Herpesvirus 7, fasciolopsis buski, Fasciola hepatica and fasciola gigantica, FFI prion, Filarioidea superfamily, Bacillus perfringens, Fusobacterium, C.perfringens, other fusobacteriums, geotrichum candidum, GSS prion, cercomonas intestinalis, Burkholderia mallei, gnathostoma siamense and gnathostoma hispidum, NEISSERIA GONORRHOEAE, Klebsiella granulomatis, micrococcus scarlatinae, Streptococcusagalactiae, haemophilus influenzae, enterovirus, most of Coxsackie A disease poison and Enteric virus71 type, Xin Nuowa virus, helicobacter pylori, Escherichia coli O 157: H7, bunyaviridae family, hepatitis A Virus, hepatitis type B virus, Hepatitis C Virus, Hepatitis D virus, Hepatitis E virus, herpes simplex virus type 1, merely Herpes virus type 2, histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, haemophilus influenzae, Human bocavirus, Angstrom Wen's Ehrlichia, Anaplasmataceae, human metapneumovirus, sand take Ehrlichia, human papilloma virus, human parainfluenza viruses, Hymenolepis nana and diminution coating tapeworm, Epstein-Barr virus, orthomyxoviridae family, Isosporabelli, Jin Shi Jin Shi bacillus, pneumonia gram The primary bacterium of thunder, Klebsiella ozaenas, kuru prion, Lassa virus, legionella pneumophilia, legionella pneumophilia, Li Shiman Proteromonas, Mycobacterium leprae and the more unrestrained crazy mycobacteria of type Ma, Leptospira, Listeria, Bo Shi borrelia vincentii With other Borrelias, bancroft's filaria and Wuchereria malayi, lymphocytic choriomeningitis virus (LCMV), plasmodium Belong to, Marburg virus, measles virus, glander-like disease Burkholderia, Neisseria meningitidis, Metagonimus Yokogawai, Microspora, passes Metachromia contagiosum (MCV), mumps virus, Richettsia bacillus, mycoplasma pneumoniae, various bacteria (Madura mycomycete Disease) and fungi (Madura mycomycete disease), parasitic Diptera fly maggot, chlamydia trachomatis and gonococcus, vCJD prion, starlike promise Cattell bacterium and other Nocardia species, Onchocerca caecutiens, blastomyces brasiliensis, Paragonismus westermani and other paragonimus Belong to, pasteurella, lice favus of the scalp, body louse, crab louse, Bordetella pertussis, Yersinia pestis, streptococcus pneumonia, Pneumocystis carinii, poliovirus, prevotella, naegleria fowleri, JC virus, chlamydia psittaci, Richettsia, rabies viruses, Streptobacillus moniliformis and borrelia muris, Respiratory Syncytial Virus(RSV), rhinosporidium seeberi, rhinopathy Poison, rickettsiae, dermacetor akari, Rift valley fever virus, rickettsia rickettsii, rotavirus, rubella virus, sramana Bordetella, SARS coronavirus, itch mite, Schistosoma, Shigella, varicellazoster virus, variola major Or alastrim, Sporothrix schenckii, staphylococcus, staphylococcus aureus, micrococcus scarlatinae, strongyloides intestinalis, syphilis Conveyor screw, tapeworm category, clostridium tetani, trichophyton, Trichophyton tonsurans, Trichophyton, acrothesium floccosum, Trichophyton rubrum And alpha fungus, exophiala werneckii, trichophyton, Malassezia, Toxocara canis or belascaris cati, toxoplasma, rotation Caterpillar, trichomonas vaginalis, whipworm, tubercle bacillus, francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Comma bacillus, guanarito virus, west nile virus, trichosporon beigelii, yersinia pseudotuberculosis, enterocolitis yersinia genus Bacterium, yellow fever virus, Mucoales (mucormycosis) and Entomophthorales (worm mycosis), pseudomonas aeruginosa, campylobacter fetus (arc Bacterium), Aeromonas hydrophila, Edwardsiella tarda, Yersinia, shigella, shigella flexneri, shiga in Song Bacterium, salmonella typhimurium, Treponema pertenue, Treponema carateneum, borrelia vincentii, the soft spiral of Bo Shi packet Body, Leptospira icterohemorrhagiae, Pneumocystis carinii, brucella, Brucella suis, Brucella, Mycoplasma, macula Rickettsia, Rickettsia tsutsugumushi, coating Pseudomonas；Disease fungus (aspergillus, Candida albicans, pod membrane group Knit endochylema bacterium)；Protozoan (Entamoeba histolytica, trichomonas vaginalis, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, crithidia cunninghami, leishmania brasiliensis, pneumocystis carinii pneumonia, tertian fever are former Worm, plasmodium falciparum, pernicious malaria)；Or worm (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworm)

Other antibody include, but are not limited to as binding partner of the present invention for treating viral disease: antibody acts on The antigen of pathogenic virus includes following example but is not limited to: variola virus, herpesviral, adenovirus, papovaviridae, enteron aisle Viraceae, Pironavirus section, Parvoviridae, reovirus, Retroviridae, influenza virus, parainfluenza virus, Parotitis, morbilli, Respiratory Syncytial Virus(RSV), rubeola, arboviruse, rhabdovirus, Arenaviridae, Non-A/Non-B hepatitis Virus, rhinovirus, coronavirus, rotavirus section, tumour virus is [for example, hepatitis type B virus (hepatocellular carcinoma), human milk head Shape tumor virus (cervical carcinoma, cancer of anus), kaposi sarcoma-associate herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal carcinoma, primary Base spy's lymthoma, primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40 (simian virus 40), third Hepatitis virus (liver cancer), Human T-lymphotropic virus 1 (adult T-cell leukemia/lymthoma), immune disorder causes Virus: [such as human immunodeficiency virus (AIDS)]；Central nervous system virus: [e.g., (the more lesion brains of progressive are white by JCV Matter disease), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitides are just glued Viraceae (possible) (lethargic encephalitis), RV (rabies), vesicular stomatitis-India's Tobamovirus, exanthema toxicity meningitis, Ramsay Hunter syndrome II type；Polio (infantile paralysis, Postpolio syndrome), the thermophilic T lymph of the mankind are thin 1 type of cellular virus (tropical spastic paraplegia)]；Cytomegalovirus (cytomegaloviral retinitis, HSV (herpetic cornea It is scorching))；Cardiovascular disease virus [such as Coxsackie virus (pericarditis, myocarditis)]；Respiratory system/acute nasopharyngitis virus/viral Pneumonia: [african lymphoma virus (4 type infection of herpesviral/infectious mononucleosis), cytomegalovirus；SARS Coronavirus (Severe acute respiratory syndrome) orthomyxovirus: influenza virus A/B/C (influenza/bird flu), paramyxovirus: Human parainfluenza virus' (parainfluenza), Respiratory Syncytial Virus(RSV) (human airway syncytial virus), Pneumovirinae]；Digestive disease Malicious [MUV (parotitis), cytomegalovirus (cytomegalovirus esophagitis)；Adenovirus (adenovirus infection)；Rotavirus, promise Such as virus, astrovirus, coronavirus；HBV (hepatitis B), Coxsackie virus, hepatitis A (hepatitis A virus), HCV (the third type Hepatitis virus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HGV RNA)]；Genito-urinary system The virus [e.g., BK virus, MUV (parotitis)] of system.

According to a further goal, the invention also includes above-mentioned conjugation drug ingedients to combine other feasible pharmaceutical carriers Therapeutic agent as cancer and autoimmune disease.Treating cancer of the present invention and the method for autoimmune disease include it is external, Internal or indirect in vivo.Ex vivo Application Example, including drug-treated cultured cell in vitro are killed in addition to not having table All cells other than up to the cell of target antigen；Or kill the cell for expressing undesired antigen.As treatment in vivo indirectly One example of the treatment method of method: handling candidate stem cell in vitro, kills defeated time former patient's body after illness or malignant cell It is interior.For example, clinically first passing through indirect interior therapeutic removes tumour cell in marrow or lymphocyte then defeated time former patient Come treating cancer and autoimmune disease, or before transplantation remove marrow in T cell and other lymphocytes to prevent from moving The immune Antagonistic reaction of plant.Implementation method is as follows: obtaining bone marrow cell from patient or other individuals, the present invention then is being added 37 DEG C of cultures in the serum-containing media of drug are conjugated, drug concentration range is 1pM to 0.1mM, and incubation time is 30 minutes left sides The right side to 96 hours or so.The specific concentration of drug and incubation time are determined by veteran clinician.After culture, marrow Cell passes through after being washed with serum-containing media to be injected intravenously in defeated the Huis' body.If patient is obtaining at bone marrow cell and feedback In the case where needing to receive other treatments, such as ablation chemotherapy or total body irradiation before reason, treated bone marrow cell can be with It is stored in qualified liquid nitrogen Medical Devices.

When for internal clinical application, jelly that conjugation drug of the invention will be injected with solution or after capable of being dissolved by sterile water The form of dry solid (powder needle) provides.Being conjugated mass ratio of the drug in solution or lyophilized solid (powder needle) can be 0.01%- 99%.In order to prepare stable freeze-dried powder, the preparation for being used for freeze-dried powder is added in feed liquid in one or more following auxiliary materials. Applicable auxiliary material includes: one or more polyol compounds (including sugar (reduction and nonreducing sugar), sugar alcohol and sugar-acids. Reducing sugar such as fructose, mannose, maltose, lactose, sugar, xylose, ribose, mannose, galactolipin and glucose；Irreducibility Sugar such as sucrose, trehalose, sorbose, melezitose and gossypose；Sugar alcohol is selected from mannitol, xylitol, antierythrite, maltose Alcohol, xylitol, antierythrite, threitol, sorbierite and glycerol；Saccharic acid such as gluconic acid and its salt) 0.0%- is accounted in ingredient 25%；Buffer reagent (such as citric acid, succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, amber Acid, Tris hydrochloride, phthalandione or phosphoric acid) 5mM-500mM (mass ratio 0.05%~25% after freeze-drying) is used for finally in feed liquid It is adjusted to pH 4.5~8.5；Surfactant (such as polysorbate 20,40,60,65,80 or 85；Poloxamer (poloxamer)- 407, polyethylene glycol propylene glycol, lauryl sodium sulfate (SDS), Sodium Laurylsulfate, sodium octyl glycoside and the like etc.) 0.0%-2.0% is accounted in ingredient；Antioxidant (such as ascorbic acid and/or methionine) accounts for 0.0%-5.0% in ingredient；Bullet Property/tension regulator (such as mannitol, sorbierite or NaCl) 0.0%-10%；Chelating agent such as EDTA or the EGTA content in ingredient 0.0mM- 5mM；Metal complex (such as zinc protein complex) accounts for 0.0%-1% in ingredient；；Biodegradable polymerization Object, as polyester accounts for 0.0%-1.5% in ingredient；Preservative (such as benzyl alcohol, phenol, butanol, benzylalcohol, Arrcostab such as methyl or Buddhist nun Moor golden propyl ester, catechol, resorcinol, cyclohexanol, 3- amylalcohol, metacresol, anesin, 18 carbon-based dimethyl benzyl chlorine Change ammonium etc.) 0.0%-5% is accounted in ingredient；And/or (such as arginine, lysine, histidine, ornithine are different for free amino acid Leucine, leucine, alanine, glycine, glutamic acid or aspartic acid) 0.0%-30% is accounted in ingredient.It prepares (prepackage) Injection/redissolution lyophilized injection should control this molar concentration range of its osmol 225-370mOsm/L.Injection/dilution physiology salt Osmol this molar concentration range after water should ensure that as 285-310mOsm/L.Said medicine component solution can be prepared into Prepackage injection is filling to being lyophilized into minute hand after cillin bottle, or is prepared into powder by efficient spray drying, for dispensing storage And sale.Human body generates pain when injecting injection to mitigate, and should drink and add local analgesia agent.Common analgesic and dosage: benzene Methanol (1% or so), procaine hydrochloride (0.2%~2.0%), lidocaine hydrochloride (0.2%~2.0%), the tertiary fourth of trichlorine Alcohol (0.3%~0.5%), C16H25NO2, morphine, morphine sulfate, Hydromorphone, dihydrohydroxycodeinone, Gabapentin, cyclobenzaprine, Trazodone, clonidine.

Suitable this patent conjugation drug administration method example is as follows: conjugation drug weekly or every two weeks or every 3 week, Or every 4 weeks, it is monthly primary by intravenous injection, continued for 3~52 weeks.Being conjugated drug single dose, (freeze-dried powder passes through addition Water for injection redissolves) by being dissolved in 50 to 500 milliliters of physiological saline, human seralbumin is added with can choose in physiological saline Albumen (for example, concentration human serum albumins of 0.5 to 1 milliliter of 100mg/ml).Drug dose is probably every in 50 μ g to 80mg Kg body weight is per week, is injected intravenously (per injection 10ug to 200mg/kg weight).After 1~52 week treatment end, patient It can receive the treatment of a new round again.Detailed treatment method includes administration route, excipient, diluent, drug dose, treatment Time etc. can be determined by experienced surgeon.

It includes any for cell mass being killed by internal or indirect intracorporal method choice come the example for treating disease The malignant tumour of type, autoimmune disease, graft rejection and infection (including virus, bacterium or helminth).

The amount for being conjugated drug for reaching ideal biology effect and needing, will be different because of Multiple factors, these factors Property feature including compound, the bioavilability of curative effect and conjugation drug, the type of disease, the race of patient, patient suffer from The state of disease, the approach of administration, all of these factors taken together codetermine administration time table and administration mode.

Generally, the conjugation drug in the present invention can be slow by being dissolved in physiology with 0.1 to 10% mass volume ratio Parenterai administration is used in fliud flushing.Typical drug dose range every kg body weight from 1ug to 0.1g is (or every 2 weeks or every weekly 3 weeks or 4 weeks every)；Every kg body weight is every 3 weeks or the youngster of equivalent dose from 0.01mg to 50mg for the drug dose range of recommendation Virgin dosage.The dosage of recommendation depends on multiple variables, the type including disease or dysfunction, and the whole of individual patient is good for Health state, the relative biological activity of coupling drug, the dosage form of compound, administration mode (intravenous injection, intramuscular injection, or It is other), the pharmacokinetic properties in the case where choosing administration mode, and administration speed (single injection or continuous drip) and The timetable (administration number of times within a certain period of time) of administration.

Conjugation drug of the invention can be equally administered in the form of unit dose, and " unit dose " here refers to one The drug of the dosage of patient's single administration, unit dose simply and easily can be packed and be used, and the drug of unit dose is to protect Hold physics and chemically stable activity conjugation drug itself, or the pharmaceutically acceptable mixture as introducing below.Allusion quotation One day dosage range of type every kg body weight from 0.01 to 100mg.In general, the unit dosage ranges of people weekly from 1 to 3000mg.The unit dose of recommendation is 1.0mg to 500mg, is monthly administered four times or 1.0mg to 500mg, every 3 weeks once. Pharmaceutical preparation can be made by the way that one or more pharmaceutically acceptable auxiliary materials are added in conjugation drug of the invention.This unit The drug of dosage can be used for being administered orally, for example be tablet, simple capsule or soft capsule；Or intranasal administration, such as powder Shape, nasal drop or spray；Or pass through percutaneous drug delivery, such as ointment, cream, washing lotion, gelling agent or spray or skin Skin patch.What medicament can be convenient is administered in the form of unit dose, and is made with any of pharmaceutical methods, such as Remington:The Science and Practice of Pharmacy,21th ed.；Lippincott Williams& Method described in Wilkins:Philadelphia, PA, 2005 years.

Pharmaceutical dosage form comprising the compound of the present invention includes pharmaceutical composition first choice administered orally or parentally mode.It is right In oral administered dosage form, such as tablet, pulvis, capsule, tablet (pastille) etc. may include one or more following raw materials or There are other compounds of similarity: adhesive, such as microcrystalline cellulose or tragacanth gum；Diluent, such as starch or lactose；Point Powder, such as starch and cellulose derivative；Lubricant, such as magnesium stearate；Glidant, such as silicon dioxide colloid；Sweet taste Agent, such as sucrose or saccharin；Flavoring agent, such as peppermint or gaultherolin.Capsule can be the form of hard capsule or soft capsule, Generally mixing with plasticiser by gelatin mixture selectivity, starch capsule is also such.In addition, the physical form of unit dose It can be changed by the way that a variety of different raw materials are added, for example, sugar-coat, shellac or enteric agents.Other peroral dosage forms such as syrup Or elixir can contain sweetener, preservative, pigment, colorant and flavoring agent.In addition, reactive compound can be by different Processing and formula, become rapidly dissolvable dosage form, slow release dosage form or sustained release agent, sustained release agent therein is preferable Dosage form.Tablet first choice includes lactose, cornstarch, magnesium silicate, croscarmellose sodium, polyvinyl pyrrolidone, stearic acid Magnesium, in the combined dosage form such as talcum.

The liquid preparation of parenterai administration includes sterile aqueous solution or non-aqueous solution, suspension and emulsion.Liquid preparation Adhesive, buffer, preservative, chelating agent, sweetener, flavoring agent and colorant etc. can also be contained.Nonaqueous solvents includes Ethyl alcohol, propylene glycol, polyethylene glycol, vegetable oil such as olive oil and organic lipid, such as ethyl oleate.Aqueous solvent includes The mixture of water, ethyl alcohol, buffer reagent and salt, in particular, biocompatibility, degradable lactide polymer, lactide/ Co-glycolide polymers or polyethylene glycol/polyglycerol copolymer can be used as the auxiliary material of control active medicine release.Intravenous injection In excipient may include liquid and nutritional supplement, electrolyte supplements, and the auxiliary material based on woods grignard glucose, with And similar auxiliary material.It include ethylene-vinyl acetate for the other feasible parenterai administration systems of active medicine of the invention Copolymer particle, implanted osmotic pumps and liposome.

Other feasible administration modes include inhalant, and inhalant includes dry powder doses, aerosol and water droplet agent.Inhalant can To be that can pass through nasal drop, nose containing the solution of such as polyoxyethylene -9- laurel ether, glycocholate, dexycholate or oil The form of interior colloid is administered.Sucking medicament includes Pastilles etc. such as pastille, can contain flavoring agent such as sucrose, Arabic gum with And other auxiliary materials such as glycocholate etc..Suppository is suitable for the form of unit dose, using solid such as cupu oil as carrier, can also add Enter salicylic acid.Local skin drug formulation, with plaster, emulsion, washing lotion, patch, gel, spray, headed by aerosol or finish Choosing.Vaseline, lanolin, polyethylene glycol, alcohols and their mixture can be used as pharmaceutical carriers.The dosage form of percutaneous drug delivery It can be patch, emulsion, buffer solution is dissolved or dispersed in polymer or adhesive.

Particularly, conjugation drug of the invention can be put with other known or unknown therapeutic agent, such as chemotherapeutics Therapy is penetrated, immunotherapeutic agents, autoimmune disease drug, anti-infectives or other antibody drugs conjugation drug are made jointly With reaching synergy.Cooperative drug or radiotherapy can be administered before or after conjugation drug administration of the invention or It implements.1 hour is can be before or after taking conjugation drug of the invention, 12 hours, one day, a week, two weeks, three One month, it is also possible to some months in week.

In other embodiments, conjugation drug of the invention and coordination drug are applied together.Play the medicine of synergistic effect Object includes but is not limited to:

1) chemotherapeutics: a) alkylating reagent: such as [mustargen: (and Chlorambucil, cyclophosphamide, ifosfamide, Mustargen, melphalan, trofosfamide)；Nitroso ureas: (Carmustine, lomustine)；Alkylsulfonate: (busulfan, Soviet Union Disappear peace)；Triazenes: (Dacarbazine)；Compound containing platinum: (carboplatin, cis-platinum, oxaliplatin)]；B) plant alkaloid: such as [vinca alkaloids: (vincaleukoblastinum, vincristine, eldisine, vinorelbine)；Bearing taxanes: (taxol, taxol Supreme Being)]；C) .DNA topoisomerase enzyme inhibitor: such as [table podophyllin: (9-aminocamptothecin, camptothecine, crisnatol, foot Leaf second glucoside, etoposide phosphate, Irinotecan, Teniposide, topotecan)；Mitomycin: (mitomycin C)]；D) is anti- Metabolic drug: such as { [anti-folic acid: dihydrofolate reductase inhibitor: (methotrexate (MTX), Trimetrexate)；IMP dehydrogenase inhibitor (mycophenolic acid, formamido thiazole, Ribavirin, EICAR)；Ribonucleotide reductase inhibitors (hydroxycarbamide, Deferoxamine)]； [pyrimidine analogue: uracil analogues: (5 FU 5 fluorouracil, doxifluridine, Raltitrexed (Tomudex))；Cytimidine is similar Object: (cytarabine, cytarabine, fludarabine)；Purine analogue: (imuran, purinethol, guanine)] }；E) swashs Plain class: such as { receptor antagonist: [antiestrogenic: (megestrol acetate, Raloxifene, tamoxifen)；LHRH agonist: (Ge Sherui Woods, leuprorelin acetate)；Antiandrogen: (Bicalutamide, Flutamide)]；Vitamin A acid/deltoid muscle: [vitamin D 3 analogs (CB 1093, EB 1089KH 1060, Vitamin D3, calciferol)；Photodynamic therapy: (in this regard, Phthalocyanines, PC4 remove methoxy A prime)；Cell factor: (interferon-' alpha ', interferon-γ, tumor necrosis factor (tumor necrosis factor), the protein of the mankind contain The domain TNF)] } f) kinase inhibitor, such as bibw 2992 (anti-EGFR/Erb2), Imatinib, Gefitinib, piperazine Jia Tani, rope La Feini, Dasatinib, Sutent, Tarceva, nilotinib, Lapatinib, Axitinib, pazopanib are quite received and are replaced Buddhist nun, Vande Thani, fluorine imatinib, e7080 (anti-vegf R2), Mubritinib, U.S. enlightening replace Buddhist nun, Ponatinib (ap24534), HQP1351, Ba Fei replace Buddhist nun (INNO-406), posupini (SKI-606), Sutent, and card is rich to replace Buddhist nun, irrigate benefit and replace Buddhist nun, dimension is not De Ji, Iniparib, Luso benefit replace Buddhist nun, CYT387, Axitinib, tivozanib, bevacizumab, Sorafenib, toltrazuril list It is anti-, Cetuximab, ranibizumab, Victibix, ispinesib；G) the other classes of: such as gemcitabine, epoxomicins (such as lenalidomide), bortezomib Thalidomide, lenalidomide, pomalidomide, Tuo Sheduote, zybrestat, PLX4032, sta-9090, Stimuvax,

Allovectin-7, xegeva, Provenge, Yervoy, isoprenylation inhibitor (such as Lovastatin), DOPA Amine energy neurotoxin (such as 1-methyl-4-phenylpyridinium ion), cell cycle inhibitor (such as staurosporin), D actinomycin D (such as actinomycin D, dactinomycin D), bleomycin A5 (such as bleomycin Bleornycin A2, B2, Peplomycin), anthracycline antibiosis Element (such as erythromycin, adriamycin (adriamycin), idarubicin, Epi-ADM, pirarubicin, zorubicin, mitoxantrone, MDR suppression Preparation (such as Verapamil), Ca²⁺Atpase inhibitor (such as thapsigargin), histon deacetylase (HDAC) inhibitor (Vorinostat, Romidepsin, pabishta, valproic acid, Mo Tinuo he (MGCD0103), belinostat, PCI-24781, grace replace Nuo Te, SB939, resminostat, givinostat, AR-42, a kind of, sulforaphen, Trichostatin A)；Thapsigargin is filled in and carrys out former times Cloth, glitazone, Epigallo-catechin gallate (EGCG), 5 disulfirams, salinosporamide A.

2) the anti-autoimmunity disease agent of includes, but are not limited to: cyclosporine, ciclosporin A, imuran, aminocaproic acid, bromine are hidden Pavilion, Chlorambucil, chloroquine, cyclophosphamide, glucocorticoid (such as amcinonide, betamethasone, budesonide, flunisolide, FLUTICASONE PROPIONATE, hydrocortisone, dexamethasone, fluocortolone danazol, Triamcinolone acetonide, beclomeasone propionate), dehydrogenation table Androsterone, Etanercept, hydroxychloroquine, infliximab, Meloxicam, methotrexate (MTX), mycophenolate, sirolimus, Ta Kemo Department, prednisone

3) resistant to infection disease agent includes, but are not limited to: a) aminoglycoside: amikacin, Astromicin, and celebrating is big mould Plain (Netilmicin, sisomicin, Isepamicin), hygromycin, kanamycins (amikacin, Arbekacin, amino deoxidation Kanamycins, dibekacin, tobramycin) neomycin (actiline, paromomycin, ribostamycin), Netilmicin, grand sight Mycin, streptomysin, tobramycin, verdamicin；B) amphenicols: azidamfenicol, chloramphenicol, Florfenicol, first Hyrazin；C) ansamycins: geldanamycin, herbimycin；D) Carbapenems: Biapenem, donipenem, strategic point he Training south, Imipenem/cilastatin, Meropenem, Panipenem；E) cephalo-type: carbacephem (Loracarbef), cephalo second Nitrile, Cefaclor, Cefradine, cefadroxil, Cefalonium, cefaloridine, cefoxitin or cephalosporin, cephalo ammonia Benzyl, cefaloglycin, Cefamandole, cefapirin, ceftriaxone, Cefazaflur, Cefazedone, cephazoline, cefbuperazone, head Spore card product, Cefdaloxime, Cefepime, Cefminox, Cefoxitin, Cefprozil, cefroxadine, ceftezole, cephalo furan It is pungent, Cefixime, Cefdinir cefoperon, Cefepime, cefetamet, Cefmenoxime, Cefodizime, cefonicid, head Spore piperazine ketone, ceforanide, cefotaxime, Cefotiam, Cefozopran, cefalexin, Cefpimizole, cefpiramide, cephalo Sieve, Cefpodoxime, Cefprozil, Cefquinome, Cefsulodin, cefotaxime, Cefteram, Ceftibuten, Ceftiolene, head Spore azoles oxime, cephalo, ceftriaxone, cefuroxime, cefuzonam, cephamycin (Cefoxitin, cefotetan, cefmetazole) oxygen head Spore alkene (Flomoxef, latamoxef)；F) glycopeptide: bleomycin, vancomycin (oritavancin, Te Lawan star), teicoplanin (Dalbavancin) Ramoplanin, Daptomycin；G) glycyl: such as tigecycline；H) beta-lactamase inhibitor: penam (Sulbactam, Tazobactam Sodium), carat tie up alkane (clavulanic acid)；I) LIN Kesheng: clindamycin, lincomycin；J) lipopeptid: Daptomycin, A54145, Ca-dependent antibiotic (CDA)；K) macrolides: azithromycin, cethromycin cethromycin quinoline Erythromycin, clarithromycin, Dirithromycin, erythromycin, Flurithromycin, josamycin, ketolide (Ketek, cethromycin Cethromycin cethromycin cethromycin) medecamycin, miocamycin, oleandomycin, rifamycin (rifampin, rifampin, Rifabutin, Rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramvcin, tacrolimus (FK506), vinegar bamboo peach Mycin, Ketek；L) Momocycle-β-lactam Antibiotics: aztreonam, Tigemonam；M) (oxazolidinon-5-yl-methyl)-2-thiophene-carboxamides: Linezolid；n). Penicillins: Amoxicillin, ampicillin (Pivampicillin, hetacillin, Bacampicillin, metampicillin, Talampicillin) nitrine XiLin, azlocillin, penicillin, tardocillin, phenoxy group tardocillin, clometocillin, procaine penicillin, carboxylic benzyl Penicillin (carindacillin), cloxacillin, dicloxacillin, cephaloridnum, flucloxacillin, Mecillinam (Amdinocillin Dibasic acid esters), mezlocillin, methicillin, naphthlazole, oxacillin, penamecillin, penicillin, how XiLin, penicillin, piperazine draw XiLin, phenoxypropyl penicillin potassium, sulbenicillin, temocillin, Ticarcillin；O) polypeptide: bacitracin, polymyxin e, polymyxin B； P) quinolone drugs: alafloxacin, Balofloxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin, Difloxacin, Yi Nuosha Star, Enrofloxacin, Floxin, T-3811, gatifloxacin, gemifloxacin, Grepafloxacin, trovafloxacin Kano, levofloxacin Star, Lomefloxacin, marbofloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Orbifloxacin, Ofloxacin, pefloxacin are bent Cut down Sha Xing, Grepafloxacin, sitafloxacin, Sparfloxacin, Temafloxacin, tosufloxacin, trovafloxacin；Q) rhzomorph: Pune is mould Element, Quinupristin/Dalfopristin)；R) sulfa drugs: mafenide, Prontosil, sulfacetamide, sulfalene, sulfanilamide (SN), willow Nitrogen sulfapryidine, bacteresulf, methoxybenzyl aminopyrimidine, trimethoprim-sulfamethoxazole (Compound New Nomin)；S) class Sterol antibacterials: such as Fusidic Acid；T) Tetracyclines: fortimicin, aureomycin, chlorine hydroxytetracycline, demeclocycline rely first Ring element, chloromethoxazole, metacycline, minocycline, terramycin, Fenobiotic, rolitetracycline, tetracycline, glycyl (such as tigecycline)；U) the other kinds of antibiotic of: Annonaceae, arsphenamine, bactoprenol inhibitor (bacitracin), Dadal/AR inhibitor (seromycin), dictyostatin, discodermolide, Eleutherobin, Epothilones, ethambutol, Etoposide, faropenem, Fusidic Acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, Mycolactones, NAM synthetic inhibitor (such as phosphonomycin), furantoin, taxol, plate mycin, pyrazinamide, Kui slave are general Fourth/Dalfopristin, rifampin (rifampin), Tazobactam Sodium Tinidazole, Annona lactone；

4) antiviral drug: a)/fusion inhibitor: aplaviroc, the maraviro, (grace of Wei Liweiluo, gp41 Fu Wei), PRO140, CD4 (Ibalizumab)；B) integrase inhibitor: draw for draw Wei, angstrom for draw Wei, globoidnan A； C) maturation inhibitor: Bei Wei pulls up a horse, vivecon；D) neuraminidase inhibitor: Oseltamivir, zanamivir, para rice Wei；E) nucleosides and nucleotide: Abacavir, acyclovir, adefovirdipivoxil, amdoxovir, apricitabine, Brivudine, west More good fortune Wei Clevudines, right Elvucitabine, Didanosine (DDI), Elvucitabine, emtricitabine (FTC), Entecavir, general former times Luo Wei, fluorouracil (5-FU), the fluorine-substituted 2' of 3'-, 3'- di-deoxynucleoside analog is (for example, 3'- fluoro- 2', 3'- bis- are de- Oxygen thymidine (FLT) and the fluoro- 2' of 3'-, 3'- double deoxidation (FLG) Fomivirsen, Ganciclovir, iodoxuridine, Lamivudine (3TC), L- core Glycosides (such as β-L- thymidine, β-L-2'- deoxycytidine), Penciclovir, Racivir, Ribavirin, stampidine, stavudine (d4T's), Ta Liweilin (big drawing miaow is fixed), Sebivo, tenofovir, Valaciclovir trifluorothymidine, valganciclovir, Zha Xi His shore (DDC), Zidovudine (AZT)；F) non-nucleoside: amantadine, ateviridine, capravirine, diaryl pyrimidine (etravirine, rilpivirine), Delavirdine, tadenan, emivirine, efavirenz, phosphonic acid (phosphonoformic acid), Imiquimod, interferon-' alpha ', Loviride, lodenosine, methimazol, nevirapine, NOV-205, Peg-IFN alpha-2b α, Podophyllum emodi var chinense Toxin, rifampin, Rimantadine, resiquimod (R-848), tromantadine；G) protease inhibitors: anpunave, A Zha That Wei, Bo Saipowei, Prezista, fosamprenavir, indinavir, Lopinavir, Nai Feinawei, pleconaril, Li Tuona Wei, inverase, telavi (VX-950) tipranavir；H) the other kinds of antiviral drug of: abzyme, A Biduo You, calanolides A, cerulenin, cyanobacteria-N, diaryl pyrimidine, Epigallo-catechin gallate (EGCG) (EGCG), phosphine Sodium formate, Ge Ruifusen, Ta Liweilin (big drawing miaow is fixed), hydroxycarbamide, KP-1461, Miltefosine, pleconaril, synthesis suppression Preparation, Ribavirin, seliciclib；

5) the other immunotherapy medicaments of: such as imiquimod, interferon (such as α, β), granulocyte colony stimulating factor, cell The factor, interleukins (IL-1~IL-35), antibody (such as trastuzumab, handkerchief trastuzumab, bevacizumab, western appropriate former times are single It is anti-, Victibix, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, Ipilimumab, Nivolumab, Pembrolizumab, BGB-A317, atezolizumab, avelumab, Durvalumab), protein binding drug (for example, Abraxane), a kind of antibody combination drug are selected from calicheamicin derivative, Maytansine derivative (DM1 and DM4), CC-1065 and multi-kanamycin ditch agent, effective paclitaxel derivatives, adriamycin, Ali's statin anti-mitosis medicine conjugate, such as Herceptin-DM1, Inotuzumab monoclonal antibody, brentuximab Vedotin, Glembatumumab vedotin, lorvotuzumab mertansine, Gemtuzumab ozogamicin, Trastuzumab emtansine, Inotuzumab ozogamicin, Sacituzumab govitecan, Anetumab Ravtansine, Denintuzumab mafodotin, Rovalpituzumab tesirine, Labetuzumab Govitecan, BMS-986148, AN-152LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, IMGN529, Mirvetuximab soravtansine (IMGN853), Vadastuximab talirine, milatuzumab- adriamycin, Depatuxizumab mafodotin (ABT-414), AGS-16C3F, Humax-TF-ADC, TAK-264 (MLN-0264), Milatuzumab doxorubicin, Trastuzumab deruxtecan, SGN-75 (anti-CD70), anti-CD22-MCC-DM1).

When carrying out treatment or prevention cancer or autoimmune disease or infectious disease in another specific embodiment, often Using drug combination mode.Its pharmaceutical composition, this patent comprising treatment effective dose describe logical formula (II) conjugate and pass through With other drugs such as chemotherapy, radiotherapy, immunotherapeutic agent, autoimmune disease drug, anti-infectives or other objects It is administered simultaneously or consecutive administration carries out cooperateing with effective treatment or prevention.Synergist preferably is selected from following one or more of drug: Ah Brazilian general, abiraterone acetic acid esters, albumin taxol, paracetamol/hydrocodone, No. CAS is 1420477-60-6's Scholar does not replace Buddhist nun to Acala, and adalimumab, RhIL-2, Afatinib dimaleate, Ai Le is for Buddhist nun, A Lundan Anti-, alitretinoin, No. CAS is 1018448-65-1 Love Capital trastuzumab maytansine, amphetamine or amphetamine/right phenylpropyl alcohol Amine salt-mixture, Anastrozole, anthracycline antibiotic, Aripiprazole, atazanavir, Aunar pearl monoclonal antibody, Atorvastatin, No. CAS For the A Wei monoclonal antibody of 1537032-82-8, pazopanib, No. CAS Baily department for 866323-14-0 he, Avastin, bud Salol fourth, Beaune spit monoclonal antibody, and bortezomib, bosutinib, originally appropriate former times monoclonal antibody, cloth add for Buddhist nun, budesonide, budesonide/good fortune Mo Teluo, buprenorphine, No. CAS Cabazitaxel for 183133-96-2, card, which is won, replaces Buddhist nun, capecitabine, and Carfilzomib is fitted into The cell of antigen receptor engineering T, celecoxib, match is vertical to replace Buddhist nun, and Cetuximab, cyclosporine, cinacalcet, gram azoles is for Buddhist nun, CAS Number examining than for Buddhist nun for 934660-93-2, dabigatran, dabrafenib, Dacarbazine, daclizumab, Daptomycin, No. CAS For the auspicious monoclonal antibody of moral of 945721-28-8, darbepoetin, No. CAS be 206361-99-1 darunavir, methanesulfonic acid she horse replace Buddhist nun, Dasatinib, No. CAS be 173146-27-5 denileukin -2, No. CAS be 615258-49-7 ground promise monoclonal antibody, it is double Sodium vedproate, dexamethasone, No. CAS R-lansoprazole for 138530-94-6, methylphenidate, No. CAS is 1363687-32-4's Appropriate former times monoclonal antibody is exerted on ground, and Doxycycline, Duloxetine, No. CAS moral prestige monoclonal antibody for 1428935-60-7, No. CAS is 915296- 00-3 according to Torr pearl monoclonal antibody, te Yi Feimosi, tenofovir/efavirenz replaces Buddhist nun according to promise, ezetimibe, ezetimibe/ Simvastatin, fenofibrate/fenofibrate/according to promise replace Buddhist nun, fingomode, fluticasone propionate, fluticasone/salmeterol, Fulvestrant, Gefitinib, copaxone, goserelin acetate, Yi Qu replace Buddhist nun, and Imatinib, she replaces not monoclonal antibody by cloth, replaces according to Shandong Buddhist nun, Yi Qu replace Buddhist nun, ifosfamide, and infliximab replaces Buddhist nun, insulin aspart, insulin detemir, sweet essence pancreas islet according to benefit Element, insulin lispro, interferon beta 1a, interferon beta 1b, Lapatinib, Yi Puli nurse Ma, Ipratropium Bromide/, Li Namoli is happy It cuts down for Buddhist nun, Letrozole, levothyrocine, levothyrocine, lidocaine, Linezolid, Liraglutide, No. CAS is 608137-33-3 lysine amphetamine, Memantine, methylphenidate, metoprolol, modafinil, Mometasone, No. CAS is The appropriate monoclonal antibody of resistance to former times of 906805-06-9, No. CAS linatinib for 698387-09-6, No. CAS Buddhist nun sieve for 641571-10-0 For Buddhist nun, No. CAS Ni Lapani for 1038915-60-4, No. CAS Wu Dankang that receives for 946414-94-4, difficult to understand is difficult to understand Pa pearl monoclonal antibody, Olmesartan, Olmesartan, oxaliplatin, Hydrochioro, omalizumab, -3 fatty-acid ethyl ester of omega are difficult to understand Si Tawei, No. CAS is replaced Buddhist nun, oxycodone hydrochloride, the auspicious monoclonal antibody of pa, pa pearl monoclonal antibody, pa Lip river monoclonal antibody, pa for difficult to understand this of 1421373-65-0 Azoles pa Buddhist nun, PD-1 antibody, PD-L1 antibody, Peg-IFN alpha-2b α -2a, No. CAS pyridine aldoxime methyliodide (PAM) monoclonal antibody for 1374853-91-4, Pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, Pregabalin, Propranolol, Quetiapine, thunder shellfish Azoles, 223Ra chloride are drawn, Raloxifene draws for Wei, Lei Molu monoclonal antibody, Lucentis, Rui Gefeini, Rituximab, benefit is drawn Husky class is cut down, romidepsin, rosuvastatin, Luso benefit is for Buddhist nun, and salbutamol, No. CAS is replaced for the Sa Woli of 1313725-88-0 Buddhist nun irrigates benefit and replaces Buddhist nun, and Suo Malu peptide, sevelamer, silaenafil, No. CAS is the appropriate former times monoclonal antibody of 541502-14-1 department, Xi Talie Spit of fland, sitagliptin/melbine, Solifenacin, Sony De Ji, Sorafenib, Sutent, tacrolimus is bright for Rameau pa, Temozolomide, tesirolimus, tenofovir/emtricitabine, testosterone gel, Thalidomide, Tiotropium Bromide, Toremifene are bent Beauty replace Buddhist nun, Herceptin, No. CAS be 114899-77-3 Qu Beite tincture, No. CAS for 183204-72-0 trifluorothymidine/ West is disturbed for, vitamin A acid, excellent special gram, Valsartan, Wei Lipani, Vande Thani, Wei Luofeini, No. CAS is 1257044-40-8 Outer Buddhist nun hide can Lay this, Vorinostat, A Puxibai, shingles zoster vaccine, and their analog, derivative, pharmaceutically The combination of acceptable salt, carrier, diluent or excipient or its drug molecule above and its various dosage forms.

As a further goal of the present invention, the invention further relates to the preparation process of antibody drug conjugated body therein. Conjugated body of the invention can be prepared with the well-known process in a variety of fields, for example, the Tubulysin in conjugated body of the present invention spreads out Biology can by the following method or improved following methods synthesize.For the professional person in the field, these improvement sides Method is all to be easy to method obtain from scientific and technical literature, well-known, apparent.In particular, these methods exist " Comprehensive Organic Transformations " (R.C.Larock writes, and 1999, Wiley-VCH publish, and the 2nd Version) there are many introductions in a book.

During reactions described herein, sometimes it is necessary to protect the active function groups that may participate in reaction, such as Hydroxyl, amino, imino group, sulfydryl and carboxyl, to avoid the generation of side reaction.The common application method of GPF (General Protection False functional group can With reference to written " the Greene's Protective Groups in Organic of P.G.Wuts and T.W.Greene Synthesis " (2006, Wiley-Interscience is published, and the 4th edition).Some reactions can it is appropriate containing acid or It is carried out in the solution of alkali.Acid, alkali and the solvent of this kind of reaction are not particularly limited, any as long as no detrimental effect Conventional acid, alkali and solvent can be used herein.Moreover, these reactions can carry out within a wide temperature range. But, generally speaking, the reaction temperature for being easier to operate is usually between -80 DEG C -150 DEG C (between room temperature and 100 DEG C More preferably).Time needed for reaction can equally vary widely range, and certainly, this depends on many factors, especially react The property of temperature and solvent for use.In general, for more satisfactory reaction, 3 to 20 hours reaction time compared Properly.

Operation processing after reaction can carry out according to a conventional method.For example, can by solvent from reaction system In the mode that evaporates recycle reaction product.Alternatively, if necessary, first residue can be poured into after solvent is steamed In water, then extracted with the immiscible organic solvent of water.Finally, after extractant is steamed, so that it may obtain reaction and produce Object.In addition, if there is the needs of higher purity, it can be also further purified by various common methods, such as recrystallization, sedimentation Or the method for various chromatographies.In general, the method for column chromatography and preparation thin plate chromatography is more commonly used.Of the invention Fig. 1-60 is shown in the synthesis of Tubulysin derivative and its conjugate.

In the following embodiments, cell combination body-Tubulysin derivative conjugate of the invention will be obtained further More specifically description.Certainly, the present invention is not limited to the following examples.

Experimental section:

NMR spectra is recorded on Bruker 500MHz nuclear magnetic resonance spectrometer.Chemical shift (δ) is on the basis of tetramethylsilane (0.00ppm) indicates that coupling constant (J) is as unit of hertz by hundred a ten thousandths (ppm).High resolution mass spectrum data Waters Acquity UPLC-MS (Xevo QTOF) or Agilent 6120LC-MS (Quadrapole) LC-MS instrument or upper acquisition. Amino acid and its derivative and pre-fill resin are international purchased from Merck chemical industry international corporation, Synthetech company, Peptides Limited liability company, Chembridge international corporation or Sigma-Aldrich.Partial cross-linked dose such as NHS ester/Malaysia acyl Imines (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/pyridine dimercapto (SPDP, LC-SPDP or Sulfo-LC-SPDP,SMPT, Sulfo-LC-SMPT)；NHS ester/haloacetyl (SIA, SBAP, SIAB or Sulfo-SIAB)；NHS ester/phenodiazine cyclopropylene (SDA or Sulfo-SDA, LC-SDA or Sulfo-LC-SDA, SDAD or Sulfo-SDAD)；Maleimide/hydrazides (BMPH, EMCH, MPBH, KMUH)；Pyridine dimercapto/hydrazides (PDPH)；With Isocyanates/maleimide (PMPI) is purchased from Thermo Fisher Scientific company.Trastuzumab is purchased from Genentech company and our unit's self-control.All anhydrous solvents are all commercially available, and are stored in hermetically sealed bottle under nitrogen.Institute There are other reagents or solvent all to use commercially available highest specification, is not further purified and directly uses.

The synthesis of 1 compound 2 of embodiment

Diethoxy acetonitrile (1.00kg, 7.74mol) is dissolved in methanol (6.0L) in 10-L reaction kettle, at room temperature (NH is added₄)₂S (48% aqueous solution, 1.41kg, 9.29mol).Temperature in the kettle rises to 33 DEG C then and drops back to room temperature.It is stirred overnight Afterwards, concentration of reaction solution.Ethyl acetate (5L) is added into residue, with saturation NaHCO₃Solution (4 × 1.0L) is washed, water phase second Acetoacetic ester (5 × 1.0L) back extraction.Merge organic phase, is washed with saturated salt solution (3L), after being dried, filtered later with anhydrous sodium sulfate It is concentrated under reduced pressure.Crude product is beaten with petroleum ether, vacuum filtration, is collected solid and is used petroleum ether.Petroleum is used again after filtrate concentration Ether mashing, merges and collects obtained solid, 1.1kg (87% yield) target product is always obtained, for white or bright yellow solid.¹H NMR(500MHz,CDCl₃) δ 7.81 (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=7.1 Hz, 6H).

The synthesis of 2. compound 3 of embodiment

A reflux condensing tube and a constant pressure funnel are equipped on tri- neck round-bottomed bottle of 5-L.Molecule is added wherein SieveWith the ethanol solution (3L) of sulphamide 2 (350g, 2.14mol), 3-BrPA ethyl ester is instilled in 30 minutes (purity 80%, 404mL, 2.57mol).Temperature slightly rises in during being added dropwise, and then reaction solution is heated to flowing back and be stirred 30 minutes.Insoluble matter is filtered to remove with diatomite after reaction solution is cooled to room temperature, and with ethyl acetate filter wash cake.Filtrate concentration Crude product and silica gel (1.5 kg) mixing afterwards is mixed thoroughly, chromatographs (10-20% ethyl acetate/petroleum by silicagel column (10kg) column Ether gradient elution) purifying, brown oil is obtained, is target compound (509g, 92% yield).

The synthesis of 3. compound 4 of embodiment

Acetone (3.0L) solution of acetal (300g, 1.16mol) is heated to flowing back, 4N HCl is added dropwise in 1.0 hours Solution (250mL).TLC shows starting material fully reacting.Reaction solution separates two-phase after being concentrated under reduced pressure.It is organic to be added to acetic acid second Ester dilutes (1.5L), and successively with saturation NaHCO₃Aqueous solution (1.0L), water (1.0L) and saline solution (1.0L) are washed, and are then used Anhydrous Na₂SO₄It is dry.Merge all water phases and be stripped with ethyl acetate, and uses anhydrous Na₂SO₄Dry organic phase.It filters out dry Organic phase is concentrated after drying prescription, gained crude product is beaten with petrol ether/ethyl acetate (5:1) solution, and the solid of precipitation is through vacuum mistake Filter is collected, and is washed with petrol ether/ethyl acetate (10:1) solution.Filtrate concentration column chromatographic purifying (0-15% ethyl acetate/stone Oily ether), all solids merge, and target product 40g (43% yield) are obtained, for white or bright yellow solid.¹H NMR(500MHz, CDCl₃) δ 10.08-10.06 (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 C₇H₈NO₃S [M+H]⁺Calculated value 186.01；Measured value 186.01.

The synthesis of 4. compound 10 of embodiment

In N₂It is molten to the tetrahydrofuran (1L) of (S)-tert-butyl sulfinic acid amine (100g, 0.825mol) at room temperature under protection Ti (OEt) is added in liquid₄(345mL, 1.82mol) and 3- methyl -2- butanone (81mL, 0.825mol).Reaction solution is heated, is returned Stream is cooled to room temperature after 16 hours, is subsequently poured into ice water (1L).It filters and with ethyl acetate filter wash cake.Having in separating filtrate Machine phase is concentrated under reduced pressure after anhydrous sodium sulfate is dry, and residue is evaporated under reduced pressure (15-20torr, 95 DEG C) and obtains target product 10 (141g, 90% yield) is yellow oil.1H NMR(500MHz,CDCl₃)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 C₉H₁₉NaNOS[M+Na]⁺Calculated value 212.12； Measured value 212.11.

The synthesis of 5. compound 11 of embodiment

In N₂Under protection, positive fourth is added into the tetrahydrofuran solution of diisopropylamine (264mL, 1.87mol) at -78 DEG C Base lithium solution (2.5M, 681mL, 1.70mol).Reaction is warming up to 0 DEG C in 30 minutes and then is cooled to -78 DEG C again, Xiang Qi Middle addition compound 10 (258g, 1.36mol) is simultaneously rinsed with tetrahydrofuran (50mL).ClTi (O is added dropwise after stirring 1 hourⁱPr)₃ Tetrahydrofuran (1.0L) solution of (834g, 3.17mol).Be added dropwise to complete after 1 hour be slowly dropped into again compound 4 (210g, Tetrahydrofuran (500mL) solution 1.13mol), it is 1 hour time-consuming.Acquired solution continues stirring 3 hours at -78 DEG C.TLC monitoring After reaction carries out completely, with the mixed liquor quenching reaction of acetic acid and tetrahydrofuran (volume ratio 1:1,300mL), then by reaction solution Salt water (2L) is poured into, (8 × 1L) is extracted with ethyl acetate.Organic phase water and salt washing, anhydrous Na₂SO₄It dries, filters simultaneously Concentration.Residue obtains compound 11 (298g, 74% yield) through column chromatography (DCM/ ethyl acetate/petroleum ether 2:1:2) purifying, For colorless oil.¹H-NMR (500MHz,CDCl₃) δ 8.13 (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 C₁₆H₂₆NaN₂O₄S₂[M+Na]⁺Calculated value 397.13 measured value 397.11.

The synthesis of 6. compound 7 of embodiment

Compound 11 (509g, 1.35mol) is dissolved in tetrahydrofuran (200mL), is cooled to -78 DEG C, is slowly added thereto Enter Ti (OEt)₄(570mL, 2.72mol) is stirred 1 hour after adding.Then NaBH is added portionwise in 90 minutes₄(51.3g, 1.36mol), reaction is stirred 3 hours at -78 DEG C.TLC monitoring discovery starting material still has residue.It is slowly added to EtOH Reaction solution is poured into saturated salt solution (2L, HOAc containing 250mL) after continuing stirring 1.5 hours, is warmed to room temperature by (50mL).By Diatomite filtering, separates organic phase.Washing, saturated common salt washing, the dry organic phase of anhydrous sodium sulfate, filtering are simultaneously concentrated.Residual Object purifies to obtain compound 12 (364g, 71% yield) through column chromatography (ethyl acetate/petroleum ether 1:1), is white solid.¹H NMR (500MHz,CDCl₃) 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=7.1Hz, 3H), 1.29 (s, 9H), 0.93 (d, J=7.3Hz, 3H), 0.90 (d, J=7.2Hz, 3H) .MS ESI m/z calculated value C₁₆H₂₈NaN₂O₄S₂[M+Na]⁺399.15 measured value 399.14.

The synthesis of 7. compound 13 of embodiment

The Isosorbide-5-Nitrae-two of 4N HCl is added in ethyl alcohol (590mL) solution of compound 12 (600g, 1.60mol) at 0 DEG C Six ring of oxygen (590mL) solution.Reaction is stirred 2.5 hours after being gradually increased to room temperature.The white solid of precipitation is collected by filtration and uses second Acetoacetic ester is washed.It is beaten after filtrate concentration with ethyl acetate.Merge the white solid obtained twice, total 446g (90% yield)

The synthesis of 8. compound 6 of embodiment

By NaN₃(740g, 11.4mol) is dissolved in water (2.0L) and methylene chloride (2.0L) is added afterwards and is cooled to 0 DEG C, to this Tf is added in solution₂O (700mL, 4.10mol) lasts 1.5 hours.Continue stirring 3 hours after adding at 0 DEG C.It has isolated (2 × 500mL) is extracted with dichloromethane in machine phase, water phase.Merge organic phase, with saturation NaHCO₃(3 × 1.0L) is washed.At room temperature will This dichloromethane solution is added to (L)-isoleucine (300g, 2.28mol), potassium carbonate (472g, 3.42mol), five water sulfuric acid In mixing methanol/water (1:1v/v, 6.0L) solution of copper (5.7g, 22.8 mmol).Temperature meeting in reaction system during charging Slightly increase.Mixed liquor is stirred at room temperature 16 hours, and decompression boils off solvent, and water phase is acidified to concentrated hydrochloric acid (about 280mL) PH 6-6.5 (about 280mL), then with phosphate buffer dilute (0.25M, pH 6.2,6.0L), with ethyl acetate (6 × 2.0L) wash away sulfanilic acid by-product.Water phase uses ethyl acetate (4 × 2.0L) after being acidified to pH 3 with concentrated hydrochloric acid (about 400 mL) Extraction.Merge organic phase, saturated common salt washes (2.0 L), anhydrous Na₂SO₄It dries, filters and is concentrated to give product 6 (320g, 89% production Rate), it is glassy yellow grease.¹H NMR(500MHz,CDCl₃) δ 12.01 (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).

The synthesis (method one) of 9. compound 14 of embodiment

Oxalyl chloride is added into the 100ml hexane solution of Azido-Ile-OH 6 (3.0g, 19.1mmol) at 0 DEG C (7.3mL, 86mmol) and DMF (1.5ml).It after adding, is warmed to room temperature, is concentrated after continuing stirring 2 hours, and toluene azeotropic is added It is spin-dried for stand-by.The methylene chloride suspension (90mL) of compound 13 (3.92g, 12.7mmol) is cooled to 0 DEG C, DIPEA is added Above-mentioned solution of acid chloride is added after (6.6mL, 38.1mmol).Reaction is gradually increased to room temperature and stirs 16 hours, uses saturated common salt Water (50mL) is quenched.Organic phase is separated, water phase is extracted with ethyl acetate.Merge organic phase and drying, filtering, concentration.Residue Product 14 (2.5g, 48% yield) is purified to obtain through column chromatography (0-30% ethyl acetate/petroleum ether), is white solid.¹H NMR (500MHz,CDCl₃) 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=6.4Hz, 3H), 1.01-0.94 (m, 9H) .MS ESI m/z calculated value C₁₈H₃₀N₅O₄S[M+H]⁺412.19 measured value 412.19.

The synthesis (method two) of 10. compound 14 of embodiment

Azido-Ile-OH (6,153g, 0.97mol) is dissolved in tetrahydrofuran (1.5L) and is cooled to 0 DEG C, is sequentially added NMM (214mL, 1.94mol) and isobutyl chlorocarbonate (95mL, 0.73mol).Chemical combination is added portionwise after stirring 1 hour at 0 DEG C Object 13 (150g, 0.49mmol).After 0 DEG C is stirred 30 minutes, it is gradually increased to room temperature and continues stirring 2 hours.Reaction is at 0 DEG C Under water quenching on the rocks go out and be extracted with ethyl acetate three times.Merge organic phase to be washed with 1N HCl, is saturated NaHCO₃It washes and is washed with salt, Anhydrous Na₂SO₄It is dry.Filtering and concentrating, residue obtain white solid through column chromatography (0-30% ethyl acetate/petroleum ether) purifying (140g, 70% yield).

The synthesis of 11. compound 15 of embodiment

Imidazoles successively is added into methylene chloride (50mL) solution of compound 14 (436g, 1.05mol) at 0 DEG C (94g, 1.37mmol) and chlorotriethyl silane (222mL, 1.32mol).After reaction solution lasts 1 hour and is warmed to room temperature, continue to stir It mixes 1 hour.Saturated salt solution is added to be quenched, separates organic phase, water phase is extracted with ethyl acetate.Organic phase after merging is through dry Product 15 (557.4g, 95% yield) is purified to obtain with column chromatography (15-35% ethyl acetate/petroleum ether) after dry, filtering, concentration, For colorless oil.¹H NMR(500MHz,CDCl₃) 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 (dd, J=19.1,6.8Hz, 6H) MS ESI m/z calculated value C₂₄H₄₄N₅O₄SSi[M+H]⁺ 526.28 measured value 526.28.

12. compound 16 of embodiment synthesizes (method one)

Tetrahydrofuran (50mL) solution of compound 15 (5.20g, 9.9mmol.) is cooled to -45 DEG C, is added thereto KHMDS (1M toluene solution, 23.8mL, 23.8mmol).After -45 DEG C are stirred 20 minutes, addition iodomethane (1.85mL, 29.7mmol).Reaction is gradually increased to room temperature, lasts 4.5 hours.Ethyl alcohol (10mL) quenching reaction is added, uses ethyl acetate (250mL) dilution simultaneously washes (100mL) with saturated common salt.Organic phase is separated, water phase is extracted with ethyl acetate (3 × 50ml).It closes Organic phase after and purifies to obtain product 16 with column chromatography (15-35% ethyl acetate/petroleum ether) after drying, filtering, concentration (3.33g, 63% yield) is glassy yellow grease.¹H NMR(500MHz,CDCl₃) 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=8.0,2.9 Hz, 15H), 0.92 (d, J=6.6Hz, 3H), 0.90 (d, J= 6.7Hz, 3H) .MS ESI m/z calculated value C₂₅H₄₆N₅O₄SSi[M+H]⁺540.30 measured value 540.30.

The synthesis (method two) of 13. compound 16 of embodiment

To the tetrahydrofuran (4L) of compound 15 (408g, 0.77mol) and iodomethane (145mL, 2.32mol) at 0 DEG C Sodium hydrogen (60%, 62.2g, 1.55mol) is added in solution.Resulting reaction solution is stirred overnight at 0 DEG C, fierceness is then poured into and stirs In ice water saturated ammonium chloride (5L) solution mixed.It is extracted with ethyl acetate (3 × 500mL).Organic phase after merging through drying, Product 16 (388g, 93% yield) is purified to obtain with column chromatography (15-35% ethyl acetate/petroleum ether) after filtering, concentration, is bright orange Color grease.¹H NMR(500MHz,CDCl₃) δ 8.09 (s, 1H), 4.95 (d, J=6.6Hz, 1H), 4.41 (q, J=7.1 Hz, 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=6.6 Hz, 3H), 0.96 (dt, J= 8.0,2.9Hz, 15H), 0.92 (d, J=6.6Hz, 3H), 0.90 (d, J=6.7 Hz, 3H) .MS ESI m/z calculated value C₂₅H₄₆N₅O₄SSi[M+H]⁺540.30 measured value 540.30.

The synthesis of 14. compound 22 of embodiment

Into methanol (100mL) solution of D- nipecotic acid (10.0g, 77.4mmol) be added formaldehyde (37% aqueous solution, 30.8mL, 154.8mmol) and Pd/C (10wt%, 1.0g).By the reaction solution in H₂It is stirred overnight in (1atm) environment, then It is filtered with diatomite, methanol is washed.Filtrate is concentrated to give compound 22 (10.0g, 90% yield), is white solid.

The synthesis of 15. compound 23 of embodiment

Pentafluorophenol is added into ethyl acetate (50mL) solution of D-N- methyl piperidine sour (2.65g, 18.5mmol) (3.75g, 20.4mmol) and DCC (4.21g, 20.4mmol).Reaction is stirred at room temperature 16 hours, and diatomite filtering is used 10mL ethyl acetate is washed.Filtrate does not have to be further purified, and directly uses.

The synthesis (method one) of 16. compound 30 of embodiment

Dry Pd/C (10wt%, 300mg) and azido compound 16 are added into the ethyl acetate solution of pentafluorophenyl group ester 23 (3.33g,6.61mmol).Reaction solution stirs 27 hours in hydrogen (1atm) environment, is then filtered with diatomite, ethyl acetate It washes.Filtrate is spin-dried for purifying to obtain chemical combination 30 (3.90g, 86% yield) through column chromatography (0-5% methanol/ethyl acetate).MS ESI m/z C₃₂H₅₉N₄O₅SSi[M+H]⁺Calculated value 639.39, measured value 639.39.

The synthesis of 17. compound 17 of embodiment

0.1N HCl is instilled into methanol (15mL) solution of compound 16 (1.01g, 1.87mmol) until during pH is Property.It is added the mixed solution after Pd/C (10wt%, 583mg) in H₂(1atm) environment is stirred at room temperature 16 hours.Filter off Pd/ C, methanol are washed.Filtrate is spin-dried for, and residue redissolves in ethyl acetate (50mL), uses anhydrous Na₂SO₄It is concentrated to give after drying, filtering Compound 17 (900mg, 94% yield) is light yellow oil.

The synthesis (method two) of 18. compound 30 of embodiment

Add in methylene chloride (20mL) solution under nitrogen protection to compound 17 (1.35g, 2.63mmol) in room temperature Enter D-N- methyl piperidine acid 22 (2.02g, 14.1mmol) and EDCIHCl (2.97g, 15.5 mmol).After stirring 17 hours, 15mL water is added into residue and is extracted with ethyl acetate (3 × 30mL) for concentration of reaction solution.Organic phase after merging is through nothing Aqueous sodium persulfate is dry, after filtering and concentration with column chromatography (ethyl acetate/methanol 40:1) purify compound 30 (1.12g, 67% yield), it is light yellow oil.

The synthesis of 19. compound 31 of embodiment

Compound 30 (3.90g, 6.1mmol) is dissolved in acetic acid/water/tetrahydrofuran (v/v/v 3:1:1,100mL) to mix In solution, it is stirred at room temperature 48 hours.Then it is concentrated, residue is purified through column chromatography (2:98 to 15:85 methanol/ethyl acetate) Obtain compound 31 (2.50g, two step yields 72%) .MS ESI m/z C₂₆H₄₅N₄O₅S [M+H]⁺Calculated value 525.30, measured value 525.33.

The synthesis of 20. compound 32 of embodiment

At 0 DEG C by LiOH (0.4N, 47.7mL, 19.1mmol) aqueous solution be added compound 31 (2.50g, In Isosorbide-5-Nitrae-dioxane (47.7mL) solution 4.76mmol).It is concentrated after being stirred at room temperature 2 hours.Residue is chromatographed through column (100% methylene chloride to methylene chloride/methanol/ammonium hydroxide 80:20:1) purifies to obtain compound 32 (2.36g, 99% yield), is Amorphous white solid.MS ESI m/z C₂₄H₄₁N₄O₅S[M+H]⁺Calculated value 497.27, measured value 497.28.

The synthesis of 21. compound 33 of embodiment

Acetic anhydride is instilled into pyridine (50mL) solution of compound 32 (2.36g, 4.75mmol) at 0 DEG C (2.25mL,24mmol).Reaction was gradually increased to room temperature through 2 hours, and continued stirring 24 hours.Isosorbide-5-Nitrae-dioxy six is added after concentration Ring/water (v/v 1:1,10mL) mixed liquor stirs 1 hour, to destroy the acid anhydrides that may be generated.Residue after concentration is through column layer (100% methylene chloride to methylene chloride/methanol/ammonium hydroxide 50:50:1) purifies, and obtains compound 33 (2.25g, 88% yield) for analysis For amorphous white solid.MS ESI m/z C₂₆H₄₃N₄O₆S[M+H]⁺Calculated value 539.28, measured value 539.28.

The synthesis (method one) of 22. compound 28 of embodiment

To 2- methylalanine (5.00g, 48.5mmol, 1.0eq.) and formaldehyde (37% aqueous solution, 20mL, Pd/C (10wt%, 0.5g) is added in methanol (20mL) solution 242.5mmol).Reaction solution is in H₂65 under (100 psi) environment DEG C stirring 16 hours.Diatomite filtering, methanol are washed.Filtrate is spin-dried for obtaining compound 28 (6.00g, 94%yield), solid for white Body.

The synthesis (method two) of 23. compound 28 of embodiment

By 2- methylalanine (500g, 4.85mol), formaldehyde (37% aqueous solution, 1.0L, 12.1mol) and formic acid The mixed liquor of (1.0L) is heated to reflux (80 DEG C).Stirring is cooled to room temperature after 3.0 hours, is added 6N HCl (850mL), then Concentration of reaction solution.Resulting solid is collected by filtration and is washed three times (1.0L) with ethyl acetate.Solid is dissolved in water (1.5L) to be used in combination 4N NaOH (about 1.0L) is neutralized to pH 7.It is concentrated and uses ethyl alcohol (2.0L) azeotropic to remove water.Residue is molten with methanol (2.0L) Solution, is filtered to remove NaCl solid, ethyl acetate is washed.White solid 639.2g is obtained after filtrate concentration, includes a small amount of NaCl, it can be straight It connects using without being further purified.

The synthesis of 24. compound 29 of embodiment

Into ethyl acetate (1L) solution of compound 28 (97g, 0.74mol) be added Pentafluorophenol (163g, 0.88mol) and DIC (126mL, 0.81mol).Reaction is filtered after being stirred at room temperature 24 hours with diatomite, with 10mL ethyl acetate It washes.Filtrate directly uses without further purification.

The synthesis of 25. compound 38 of embodiment

Into the ethyl acetate solution of above-mentioned pentafluorophenyl group ester 29 be added compound 16 (200g, 0.37mol) and do Pd/C (10wt%, 10g).Reaction solution stirs 27 hours in hydrogen (1atm) environment.Diatomite filtering, ethyl acetate are washed.It closes And organic phase concentration after through column chromatography (0-5% methanol/ethyl acetate) purify to obtain compound 38 (184g, 79% yield) .MS ESI m/z C₃₁H₅₈N₄O₅SSi[M+H]⁺Calculated value 627.39, measured value 627.39.

The synthesis of 26. compound 39 of embodiment

Compound 38 (200g, 0.32mmol) is dissolved in acetic acid/water/tetrahydrofuran (v/v/v 3:1:1,638 mL) to mix In solution, it is stirred at room temperature 4 days.Dry with toluene azeotropic band after reaction solution concentration, this step obtains compound 39 after repeating twice, directly For reacting in next step.MS ESI m/z C₂₅H₄₅N₄O₅S[M+H]⁺Calculated value 513.30, measured value 513.30.

The synthesis of 27. compound 40 of embodiment

At 0 DEG C by the aqueous solution of lithium hydroxide (0.4N, 600mL, 2.55mol) be added compound 39 (160g, 0.319mol, 1.0eq.) methanol (1.2L) solution in.Reaction is concentrated after being stirred at room temperature 2 hours.Through column chromatography (100% 2 Chloromethanes is to 80:20:1 methylene chloride/methanol/ammonium hydroxide) compound 40 (140 g, two step yields 91%) are amorphous white Solid.MS ESI m/z C₂₃H₄₀N₄O₅S[M+H]⁺Calculated value 485.27, measured value 485.27.

The synthesis of 28. compound 41 of embodiment

Compound 40 (143g, 0.30mol) and DMAP (0.36g, 2.95mmol) are dissolved in anhydrous tetrahydro furan (1.4L) In the mixed solution of anhydrous DMF (75mL).Be cooled to 0 DEG C, be added TEA (82.2mL, 0.59mmol) and acetic anhydride (56mL, 0.59mmol).Reaction is gradually increased to room temperature and stirs 24 hours.It is pure through column chromatography (5-50% ethanol/methylene) after concentration Change to obtain compound 41 (147g, 95% yield), is amorphous white solid.MS ESI m/z C₂₅H₄₄N₄O₆S[M+H]⁺Calculated value 527.28 measured value 527.28.

The synthesis of 29. compound 98 of embodiment

To Boc-L- methyl-P-tyrosine (670g, 2.27mol), potassium carbonate (358g, 2.5mol) and potassium iodide (38g, Benzyl bromide (283mL, 2.38mol) is slowly added in acetone (3L) solution 0.227mol).It is refluxed overnight, water is added after cooling (6L) is extracted with ethyl acetate (5 × 100L).Organic phase after merging is washed with saturated salt solution (2L), anhydrous Na₂SO₄It is dry, Filtering, through SiO after concentration₂Column chromatographs (4:1 petrol ether/ethyl acetate) and obtains white solid 98 (795g, 91% yield)¹H NMR (500MHz,CDCl₃) δ 7.43 (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 C₂₂H₂₈NO₅[M+H]⁺: calculated value 386.19 measured value 386.19.

The synthesis of 30. compound 99 of embodiment

It is slowly added dropwise at -78 DEG C into anhydrous methylene chloride (1L) solution of compound 98 (380g, 987mmol) DIBAL (1.0M hexane solution, 2.9L), lasts 3 hours.3L ethyl alcohol quenching reaction is used after adding.1N HCl to pH 4 is added dropwise. Gained mixed liquor is gradually increased to 0 DEG C, separates organic phase, and water phase is extracted with ethyl acetate (3 × 3L).Organic phase after merging is used full It is washed with salt, anhydrous sodium sulfate is dry, and is concentrated.Through petrol ether/ethyl acetate be beaten white solid 99 (263g, 75% Yield) .1H NMR (500MHz, CDCl3) δ 9.65 (s, 1H), 7.45 (d, J=7.1Hz, 2H), 7.41 (t, J=7.4 Hz, 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 C₂₁H₂₆NO₄ [M+H]⁺: calculated value 356.18, measured value 356.19.

The synthesis of 31. compound 95 of embodiment

Be added into 500mL round-bottomed bottle triphenylphosphine (100g, 381mmol) and 2 bromopropionic acid ethyl ester (100mL, 762mmol).Reaction solution is in N₂It is heated to 50 DEG C under protection, is stirred overnight.As white solid (PPh₃) all dissolution after, largely White product starts to be precipitated.The collected by suction white solid after petrol ether/ethyl acetate is beaten, obtain compound 95 (135g, 80% Yield).MS ESI m/z C₂₃H₂₄O₂P [M-Br]⁺Calculated value 363.15, measured value 363.13.

The synthesis of 32. compound 96 of embodiment

Methylene chloride (500mL) solution of compound 95 (135.42g, 305.7mmol) is added under violent stirring Into 10%NaOH (450mL) aqueous solution.Organic phase turns yellow immediately, and after stirring 30 minutes, TLC shows fully reacting.It isolates Organic phase, water phase are extracted with methylene chloride (2 × 200mL).Organic phase after merging is washed through saturated common salt, and anhydrous sodium sulfate is dry Yellow solid 96 (104g, 94% yield) is concentrated to give after dry.MS ESI m/z C₂₃H₂₄O₂P[M+H]⁺Calculated value 362.14, actual measurement Value 363.13.

The synthesis of 33. compound 100 of embodiment

Ylide is added into anhydrous methylene chloride (800mL) solution of compound 99 (81.4g, 229mmol) at room temperature Anhydrous methylene chloride (800mL) solution of 96 (2.0eq.), lasts 30 minutes.Reaction is concentrated after being stirred at room temperature overnight, and passes through SiO₂Column chromatographs (6:1 petrol ether/ethyl acetate) and obtains white solid 100 (63.4g, 63% yield) after purification¹H NMR (500MHz,CDCl₃) δ 7.45-7.41 (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 C₂₆H₃₃NaNO₅ [M+Na]⁺Calculated value 462.24, measured value 462.22.

The synthesis of 34. compound 101 of embodiment

Pd/C (1.83g, 10wt%) is added to compound 100 (30.2g, 68.9 mmol) in hydrogenation bottle In tetrahydrofuran (100mL) and methanol (300mL) mixed solution.Mixture is in 1atm H₂Shaken overnight under environment.Diatomite mistake Filter, filtrate are concentrated to give compound 101 (25.0g, theoretical yield), are colorless oil.¹H NMR(500MHz,CDCl₃)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 C₁₉H₃₁NO₅[M+H]⁺Calculated value 352.20, measured value 352.19.

The synthesis (method one) of 35. compound 102 of embodiment

Second is added into anhydrous methylene chloride (200mL) solution of compound 101 (5.96g, 35.9mmol) at room temperature Acid anhydrides (3.2mL, 33.9mmol) and nitric acid (65%-68% aqueous solution, 3.5mL, 50.79 mmol).It is stirred at room temperature 30 minutes Afterwards, TLC shows fully reacting.Reaction solution is washed with water (3 × 200mL), and water phase is stripped with methylene chloride (3 × 100mL).Merge Methylene chloride mutually washed with saturated common salt, anhydrous sodium sulfate is dried, filtered and concentrated.Through SiO₂Column chromatography (5:1 petroleum ether/ Ethyl acetate) compound 102 (4.18g, 72% yield) is purified to obtain, it is a yellow solid.¹H NMR(500MHz,CDCl₃)δ 10.49 (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=13.0,6.8 Hz, 2H), 2.59 (s, 1H), 1.88 (s, 1H), 1.37 (t, J=8.7Hz, 9H), 1.25 (dd, J=13.5,6.9 Hz, 4H), 1.16 (t, J =8.0Hz, 3H) .MS ESI m/z C₁₉H₂₈NaN₂O₇[M+Na]⁺Calculated value 419.19, measured value 419.17.

The synthesis (method two) of 36. compound 102 of embodiment

Nitrous acid uncle is added into tetrahydrofuran (150mL) solution of compound 101 (15.6g, 44.3mmol) at room temperature Butyl ester (18.3g, 177.2mol).TLC shows fully reacting, concentration of reaction solution, SiO after being stirred at room temperature 4 hours₂Column chromatographs (3:1 Petrol ether/ethyl acetate) 102 (14.3g, 81% yields) are obtained after purification, it is a yellow solid.

The synthesis of 37. compound 103 of embodiment

It is mixed at room temperature to the tetrahydrofuran (100mL) of compound 102 (15.3g, 38.6mmol) with methanol (100mL) LiOHH is added in solution₂The aqueous solution (190mL) of O (16.3g, 389mmol).Reaction is stirred at room temperature after forty minutes, adds water (400mL) dilution and with 1N KHSO₄It is adjusted to pH 3-4.After ethyl acetate (3 × 300mL) extraction, organic phase saturated common salt Washing, anhydrous Na₂SO₄It dries, filters, is concentrated to give 103 (14.4g, theoretical yields), be a yellow solid.¹H NMR(500MHz, CDCl₃) δ 10.48 (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 C₁₇H₂₃N₂O₇ [M-H]^-Calculated value 367.16, measured value 367.14.

The synthesis of 38. compound 104 of embodiment

Pd/C (2.60g, 10wt%) is added to compound 103 (26.0g, 70.6 mmol) in hydrogenation bottle In methanol (260mL) solution.Mixture is in 1atm H₂It is stirred to react under environment overnight.Diatomite filtering, filtrate are concentrated to give chemical combination Object 104 (24.0g, theoretical yield) is green oil object.

The synthesis of 39. compound 106 of embodiment

By the drying acetonitrile (1L) of the 2 bromopropionic acid tert-butyl ester (255g, 1.22mol) and triphenylphosphine (320g, 1.22mol) Solution is stirred at room temperature 18 hours.Decompression rotation goes addition toluene after acetonitrile that white solid is precipitated.Being poured out after toluene will be white Solid is dissolved in methylene chloride (1L) and is transferred to separatory funnel.10% NaOH aqueous solution (1L) is added, rocks rear organic phase quickly Turn yellow.Organic phase is separated, water phase is stripped primary with methylene chloride (1L).Merge methylene chloride phase, saturated salt solution (400mL) It washes, anhydrous Na₂SO₄It is concentrated to give ylide 106 (280g, 58%) after drying, filtering, is a yellow solid.

The synthesis of 40. compound 107 of embodiment

Into dry methylene chloride (3L) solution of compound 99 (450g, 1.27mol) be added ylide 106 (546g, 1.40mmol).It is stirred overnight at room temperature.After TLC monitors fully reacting, through column chromatography (10-50% ethyl acetate/petroleum ether) purifying It obtains compound 107 (444g, 75% yield), is a white solid.ESI m/z C₂₈H₃₈NO₅[M+H]⁺: calculated value 468.27, it is real Measured value 468.22..

The synthesis of 41. compound 108 of embodiment

Compound 107 (63g, 0.13mol) is dissolved in methanol (315mL), Pd/C is added, and (10wt%, 6.3 g), in hydrogen It is stirred overnight at room temperature under (1atm) environment.Filtrate is concentrated after Filtration of catalyst and obtains compound 108 (45.8g, 93% yield)

The synthesis of 42. compound 109 of embodiment

Nitrite tert-butyl is added into tetrahydrofuran (4L) solution of compound 108 (390g, 1.03mol) at room temperature (1.06kg, 10.3mol.).It is stirred overnight back spin and removes tetrahydrofuran, residue chromatographs (10-50% ethyl acetate/petroleum through column Ether) compound 109 (314g, 72% yield) is purified to obtain, it is a bright yellow solid.

The synthesis of 43. compound 110 of embodiment

Pd/C is added into ethyl acetate (500mL) solution of compound 109 (166g, 0.392mol) under nitrogen protection (10wt%, 16g).It is passed through hydrogen, vacuum displacement is three times.Reaction solution is stirred at room temperature 16 hours in hydrogen (1atm) environment. Diatomite filtering, is concentrated to give compound 110 (146g, 97% yield), is a yellow foamy solid.¹H NMR(400MHz, CDCl₃) δ 6.62 (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 C₂₁H₃₅N₂O₅[M+H]⁺Calculated value 394.25, measured value 395.25.

The synthesis of 44. compound 114 of embodiment

Under nitrogen protection, (S) -4- isopropyl oxazolidine -2- ketone (5.00g, 38.7mmol) is dissolved in anhydrous tetrahydro In furans (200mL), it is cooled in -78 DEG C, 30 minutes and n-BuLi (2.5M hexane solution, 17.0mL) is added dropwise into anti- It answers in bottle, is reacted 1 hour at -78 DEG C.Then propionyl chloride (4.0mL, 42.58mmol) is added dropwise, after being added dropwise, at -78 DEG C Lower reaction 1 hour, thin-layer chromatography monitors end of reaction, reaction solution is poured into saturated common salt aqueous solution (250mL), ethyl acetate It extracts (3 × 100mL), merges organic phase, washed once with 1N NaOH solution (200mL), saturated common salt aqueous solution (300mL) It washed once, anhydrous sodium sulfate dries, filters, and is spin-dried for, and obtains colorless oil with silica gel column purification (7:1 petrol ether/ethyl acetate) Shape object 6.36g, yield 89%.MS ESI m/z C₉H₁₆NO₃[M+H]⁺Calculated value 186.10, measured value 186.10.

The synthesis of 45. compound 115 of embodiment

Under nitrogen protection, compound 114 (2.00g, 11.9mmol) is dissolved in anhydrous methylene chloride (200 mL), is dropped Temperature is to 0 DEG C, by diisopropyl ethyl amine (2.3mL, 12.9mmol) and n-Bu₂BOTf (1.0 M dichloromethane solutions, 12.0mL) It is added dropwise into reaction flask, is reacted 45 minutes at 0 DEG C, then be cooled to -78 DEG C, by the two of compound 99 (4.24mL, 10.8mmol) Chloromethanes solution is added dropwise into reaction flask, reacts 1 hour at -78 DEG C, is then slowly ramped to room temperature and reacts overnight.Next day, Phosphate buffer (0.1M, pH 7.0,100mL) is added in reaction flask, liquid separation, water phase is extracted with dichloromethane (3 × 50 ML), merge organic phase, washed once with saturated common salt aqueous solution (200mL), anhydrous sodium sulfate is dry, is spin-dried for obtaining crude product.It will be thick Product are dissolved in methanol (100mL), are cooled to 0 DEG C, by H₂O₂(30% aqueous solution, 26mL) is instilled, and is reacted 3 hours at 0 DEG C, is rotated Methanol is removed, adds water (100mL), is extracted with ethyl acetate (3 × 100mL), merges organic phase, saturated common salt aqueous solution (300mL) washed once, and anhydrous sodium sulfate dries, filters, and be spin-dried for, and silica gel column purification (3:1 petrol ether/ethyl acetate) obtains white Color foaming solid 2.7g, yield 49%.¹H NMR(400MHz,CDCl₃) δ 7.36 (ddd, J=24.2,14.2,7.1 Hz, 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=8.6 Hz, 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 C₃₀H₄₁N₂O₇[M+H]⁺Calculated value 541.28, actual measurement Value 541.30.

The synthesis of 46. compound 116 of embodiment

Under nitrogen protection, compound 115 (2.50g, 4.63mmol) is dissolved in anhydrous tetrahydro furan (46mL), is added Enter two thiocarbonyl group imidazoles (2.48g, 13.89mmol), back flow reaction is overnight.Next day adds water (100mL), ethyl acetate extraction (3 × 50mL), merge organic phase, washed once with saturated common salt aqueous solution (300mL), anhydrous sodium sulfate dries, filters, it is spin-dried for, Silica gel column purification (3:1 petrol ether/ethyl acetate) obtains yellow foamy solid 2.33g, yield 77%.¹H NMR(400MHz, CDCl₃) δ 8.41 (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 C₃₄H₄₃N₄O₇S[M+H]⁺Calculated value 651.27, Measured value 651.39.

The synthesis of 47. compound 117 of embodiment

Under nitrogen protection, compound 116 (1.90g, 2.92mmol) is dissolved in dry toluene (30mL), azo is added Bis-isobutyronitrile (0.05g, 0.584mmol) and tributyl stannane (1.6mL, 5.84mmol), back flow reaction 2.5 hours, are spin-dried for, silicon Rubber column gel column purifying (5:1 petrol ether/ethyl acetate) obtains white foam solid 1.21g, yield 79%.¹H NMR(400MHz, CDCl₃) δ 7.36 (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 C₃₀H₄₁N₂O₆[M+H]⁺Calculated value 525.28, measured value 525.37.

The synthesis of 48. compound 118 of embodiment

Compound 117 (1.20g, 2.29mmol) is dissolved in tetrahydrofuran (30mL) and water (6mL), is added under ice bath LiOH (0.192g, 4.58mmol) and H₂O₂(30% aqueous solution, 1.4mL), ice bath react 3 hours, addition sodium sulfite (1.5M, 30mL), 1N KHSO is used after stirring 30 minutes under ice bath₄PH to 4 is adjusted, ethyl acetate extracts (3 × 50mL), merges organic Phase, saturated common salt aqueous solution (200mL) washed once, and anhydrous sodium sulfate dries, filters, and be spin-dried for, silica gel column purification (3:1 petroleum Ether/ethyl acetate) obtain white solid 0.78g, yield 82%.¹H NMR(400MHz,CDCl₃)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 C₂₄H₃₂NO₅[M+H]⁺Calculated value 414.22, measured value 414.21.

The synthesis of 49. compound 119 of embodiment

Compound 118 (0.77g, 1.86mmol) is dissolved in methanol (15mL), is added Pd/C (10wt%, 0.25g), Catalytic hydrogenation (1atm H₂) reaction 16 hours, filtering is spin-dried for obtaining white solid 0.58g, yield 96%.¹H NMR(400MHz, CDCl₃) δ 7.00 (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=12.2 Hz, 1H), 1.42 (s, 9H), 1.14 (d, J=6.6Hz, 3H). MS ESI m/z C₁₇H₂₆NO₅[M+H]⁺: calculated value 324.17, measured value 324.16.

The synthesis of 50. compound 120 of embodiment

Compound 119 (0.57g, 1.76mmol) is dissolved in anhydrous tetrahydro furan (10mL), nitrous acid is added dropwise under ice bath The tert-butyl ester (0.63mL, 5.28mmol) finishes and is warming up to room temperature reaction 2h, adds water (50mL), be extracted with ethyl acetate (3 × 30mL), merge organic phase, washed once with saturated common salt aqueous solution (100mL), anhydrous sodium sulfate dries, filters, and is spin-dried for, silicon Rubber column gel column purifying (2:1 petrol ether/ethyl acetate contains 0.1% acetic acid) obtains yellow solid 0.50g, yield 77%.¹H NMR (400MHz, DMSO) δ 12.00 (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 C₁₇H₂₅N₂O₇[M+H]⁺Calculated value 369.15, measured value 369.14.

The synthesis of 51. compound 121 of embodiment

Compound 120 (0.77g, 1.86mmol) is dissolved in methanol (10mL), is added Pd/C (10wt%, 0.02g), Catalytic hydrogenation (1atm H₂) reaction 1 hour, filtering is spin-dried for obtaining white solid 0.43g, yield 93%.MS ESI m/z C₁₇H₂₇N₂O₅[M+H]⁺Calculated value 339.18, measured value 339.17.

The synthesis of 52. compound 124 of embodiment

Maleic anhydride (285g, 2.76mol) is dissolved in acetic acid (1L), addition 4-Aminobutanoicacid (285g, 2.76mol, 1.0eq.), it reacts at room temperature 30 minutes, reacts 1.5 hours, be cooled to room temperature after being warming up to reflux, revolving removes solvent, uses acetic acid Ethyl ester dissolution, washing, saturated common salt washing, anhydrous sodium sulfate are dried, filtered, are spin-dried for, crude product petroleum ether and ethyl acetate weight Crystallization obtains white solid 400g, yield 80%.¹H NMR (500MHz, CDCl3) δ 6.71 (s, 2H), 3.60 (t, J= 6.7Hz, 2H), 2.38 (t, J=7.3Hz, 2H), 2.00-1.84 (m, 2H).

The synthesis (method one) of 53. compound 125 of embodiment

Compound 124 (400g, 2.18mol) is dissolved in anhydrous methylene chloride (1.5L), N- hydroxyl maleimide is added Amine (276g, 2.40mmol) and DIC (303g, 2.40mol), room temperature reaction overnight, are spin-dried for, silica gel column purification (1:2 petroleum ether/ Ethyl acetate) obtain white solid 382g, yield 63%.¹H NMR(500 MHz,CDCl₃) δ 6.74 (s, 2H), 3.67 (t, J= 6.8Hz, 2H), 2.85 (s, 4H), 2.68 (t, J=7.5 Hz, 2H), 2.13-2.03 (m, 2H).

The synthesis (method two) of 54. compound 125 of embodiment

Maleic anhydride (9.84g, 100mmol) and 4-Aminobutanoicacid (10.3g, 100mmol) are dissolved in DMA (100mL), N- hydroxy maleimide (14.4g, 125 mmol) and DCC (40.0g, 194mmol) is added in room temperature reaction 1 hour, ice bath, It finishing, is warming up to room temperature reaction 16 hours, filtrate is spin-dried for, adds water by filtering, and it is extracted with dichloromethane three times, merges organic phase, It washed once with saturated sodium bicarbonate, saturated common salt aqueous solution (100mL) washed once, and anhydrous sodium sulfate dries, filters, rotation Dry, silica gel column purification (10:1DCM/MeOH) obtains white solid 6.85g, yield 24%.

The synthesis (method one) of 55. compound 126 of embodiment

Compound 104 (13.8g, 40.8mmol) is dissolved in EtOH (100mL) and phosphate buffer (0.1M, pH 7.5,15mL) it in, is added compound 125 (12.6g, 44.9mmol).Overnight, revolving removes ethyl alcohol for room temperature reaction, adds water (100mL) is extracted with ethyl acetate (3 × 150mL), merges organic phase, and saturated common salt aqueous solution (100mL) washed once, nothing Aqueous sodium persulfate dries, filters, and is spin-dried for, and silica gel column purification (5-10% DCM/MeOH) obtains yellow foamy solid 15.3g, produces Rate 65%.¹H NMR (500MHz, MeOD) δ 7.36 (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= 6.7 Hz, 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=13.7,9.8,3.7 Hz, 1H), 1.39- 1.33(m,9H),1.30–1.24(m,3H),1.19–1.09(m,4H)。MS ESI m/z C₂₅H₃₂N₃O₈[M-H]^-Calculated value 502.23 measured value 502.22.

The synthesis of 56. compound 127 of embodiment

Compound 126 (0.20g, 0.4mmol) is dissolved in DCM (18mL) and TFA (2mL), is reacted at room temperature 2 hours, it is dense Contracting obtains compound 127, is directly used in and reacts in next step.MS ESI m/z C₂₀H₂₄N₃O₆ [M-H]^-Calculated value 402.17, it is real Measured value 402.17.

The synthesis (method two) of 57. compound 126 of embodiment

Compound 124 (60g, 328mmol) is dissolved in THF (600mL), ice bath, dropwise addition N-methylmorpholine (85.3mL, 984mmol) and isobutyl chlorocarbonate (44.6mL, 426mmol), after react 2 hours at 0 DEG C, dropwise addition compound 104 (102g, THF (400mL) solution 259mmol), finishes, and 0 DEG C is reacted 30 minutes, and water (300mL) is added, be extracted with ethyl acetate (3 × 300mL), merge organic phase, saturated common salt aqueous solution (100mL) washed once, and anhydrous sodium sulfate dries, filters, and is spin-dried for, silica gel Column purification (9-35% ethyl acetate/petroleum ether) obtains light yellow solid 104g, yield 73%.¹H NMR(400MHz,CDCl₃) δ 8.86 (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=6.0 Hz, 3H).

The synthesis of 58. compound 128 of embodiment

Compound 33 (40mg, 0.074mmol) is dissolved in ethyl acetate, addition Pentafluorophenol (27mg, 0.148mmol) is eluted, will be filtered with ethyl acetate with DCC (23mg, 0.111mmol), room temperature reaction 16 hours, silicagel pad filtering Liquid concentration, is re-dissolved in DMA (6mL), be added compound 127 (56.6mg, 0.13mmol) and DIPEA (47.4 μ L, 0.18mmol), it reacts at room temperature 24 hours, concentration prepares HPLC (C₁₈Column, 10-100% acetonitrile/water) purifying obtain white solid 43mg, yield 63%.MS ESI m/z C₄₆H₆₆N₇O₁₁S[M+H]⁺Calculated value 924.45, measured value 924.45.

The synthesis of 59. compound 147 of embodiment

(R) -4- isopropyl -2- oxazolidone (25.0g, 0.194mol) is dissolved in the anhydrous THF of 1150mL, in N₂Protection Under, -70 DEG C are cooled to, n-BuLi (85.0mL, 0.213mol) (temperature controls between -70 to -65 DEG C) is added dropwise, was added dropwise The a large amount of white solids of Cheng Zhongyou are precipitated, and are added dropwise, 1h is reacted at -70 DEG C, then propionyl chloride (20.0mL, 0.232mol) is added dropwise, It is added dropwise, 1h, thin-layer chromatography tracking, end of reaction is reacted at -70 DEG C.Reaction solution is poured into 1.2L saturated ammonium chloride solution In, ethyl acetate extracts (700mL × 1,350mL × 2), and combined ethyl acetate phase washs one with 1L 1mol/LNaOH solution Secondary, 1L water washing is primary, and 1L saturated sodium chloride solution washed once, and anhydrous sodium sulfate is dry, is spin-dried for, obtains colourless liquid.Acetic acid Ethyl ester/petroleum ether (6-10%) crosses column, obtains product 26.6g, yield 74.1%.ESI: m/z:C₉H₁₇NO₃[M+H]⁺Calculated value: 186.1 measured value 186.1.¹H NMR(400MHz, CDCl₃) δ 4.48-4.37 (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).

The synthesis of 60. compound 148 of embodiment

Compound 147 (18.4g, 99.5mmol) is dissolved in the anhydrous DCM of 200mL, dry N is added to₂The 1L of protection Three-necked bottle, ice bath are added dropwise anhydrous DIPEA (19mL, 108.6mmol), then Bu are slowly added dropwise₂BOTf DCM solution (1.0M, 100mL, 100mmol), be added dropwise, stirred 45 minutes under ice bath, be cooled to -78 DEG C, be added dropwise aldehyde (32.2g, 90.5mmol, The anhydrous DCM solution of 320mL 1.0eq) reacts 1h at -78 DEG C, is warming up to 0 DEG C, reacts under ice bath, and is slowly warmed to room temperature anti- It should stay overnight, solution is turned yellow by colourless in temperature-rise period.Thin-layer chromatography tracking, end of reaction.Reaction solution is poured into 700mL In PBS (0.1M, PH7.0) solution, liquid separation merges DCM phase, isometric saturated sodium chloride solution with 200mL DCM aqueous phase extracted It washed once, anhydrous sodium sulfate is dry, is spin-dried for obtaining 79.8g crocus grease, and the dissolution of 730mL methanol is added, and ice bath slowly drips Add 30% H of 225mL₂O₂(23eq) reacts 3h under ice bath.Add water 750mL, revolving removes most of methanol, adds water 100 ML, ethyl acetate extract (500mL × 1,150mL × 2), and combined ethyl acetate phase, saturated sodium chloride solution washed once, anhydrous Sodium sulphate is dry, is spin-dried for, ethyl acetate/petroleum ether crosses column, obtains product 28.3g white foam solid, yield: 58.7%. ESI:m/z:C₃₀H₄₁N₂O₇[M+H]⁺Calculated value: 541.3, measured value 541.3.¹H NMR(400MHz,CDCl₃)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).

The synthesis of 61. compound 149 of embodiment

Claim compound 148 (28.3g52.3mmol) and TCDI (35.1g, 157.0mmol) in 500mL N₂The drying of protection Two neck bottles in, 350mL anhydrous THF dissolution is added, back flow reaction is overnight.Thin-layer chromatography tracking, end of reaction.Saturated sodium-chloride Solution (200mL) washed once, and ethyl acetate (75mL × 2) extraction, anhydrous sodium sulfate is dried, filtered, is spin-dried for, and ethyl acetate/ Petroleum ether crosses column and obtains product 26.1g weak yellow foam shape solid, yield: 76.8%.ESI:m/z:C₃₄H₄₃N₄O₇S[M+H]⁺It calculates Value: 651.3, measured value 651.3.¹H NMR(400MHz,CDCl₃) δ 8.21 (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).

The synthesis of 62. compound 150 of embodiment

Claim the two neck bottles that compound 149 (26.0g, 40.0mmol) and AIBN (0.066g, 0.01eq) are dry in 500mL In, N₂Under protection, the dissolution of 350mL dry toluene is added, n-Bu is added dropwise₃SnH (21.5mL, 80.0mmol), back flow reaction 1h.It is thin Layer chromatography tracking, end of reaction.It is spin-dried for, ethyl acetate/petroleum ether crosses column and obtains product 6.0g white foam solid, yield: 37.3%.ESI:m/z:C₃₀H₄₁N₂O₆[M+H]⁺Calculated value: 525.3, measured value 525.3.¹H NMR(400MHz,CDCl₃)δ 7.37 (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).

The synthesis of 63. compound 151 of embodiment

Compound 150 (7.84g, 15.0mmol) is dissolved in 90mL THF and 30mL water, ice bath.By LiOHH₂O (1.57g, 37.5mmol) is dissolved in 30%H₂O₂In (11.4mL, 112.5mmol), and the solution is added drop-wise in reaction system, is had Light yellow solid is precipitated, and 3h, thin-layer chromatography tracking, end of reaction are reacted under ice bath.160mL 1.5M Na is added dropwise₂SO₃Solution, ice Bath reaction 30min, with 1N KHSO₄Solution adjusts pH to 4, and ethyl acetate extracts (200mL × 1,750mL × 2), merges acetic acid Ethyl ester phase, saturated sodium chloride solution washed once, and anhydrous sodium sulfate dries, filters, and be spin-dried for, and ethyl acetate/petroleum ether crosses column, Obtain 6.18g white solid, yield: 100%.ESI:m/z:C₂₄H₃₂NO₅[M+H]⁺Calculated value: 414.2, measured value 414.2.¹H NMR(400MHz,CDCl₃) δ 7.39 (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).

The synthesis of 64. compound 152 of embodiment

Compound 151 (6.18g, 15.0mmol) is dissolved in 50mL methanol, 0.6g Pd/C, catalytic hydrogenation, room temperature is added Overnight, thin-layer chromatography tracking, end of reaction.50mL ethyl acetate, filtering is added, ethyl acetate washing is spin-dried for, and vacuum is drained, Obtain the colorless and transparent oily object of 4.8g, yield: 99.4%. ESI:m/z:C₁₇H₂₆NO₅[M+H]⁺Calculated value: 324.2, measured value 324.2。¹H NMR(400MHz, CDCl₃) δ 6.97 (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).

The synthesis of 65. compound 153 of embodiment

Compound 152 (4.8g, 15.0mmol) is dissolved in the anhydrous THF of 75mL, ice bath, N₂Protection is lower to be added dropwise t-BuONO (18.0mL, 150mmol) is warming up to room temperature reaction 3h, thin-layer chromatography tracking, end of reaction.It is slowly added dropwise under condition of ice bath 100mL is saturated NaHCO₃Solution has a large amount of bubbles to generate, then with 1N KHSO₄Adjusting pH to 4, ethyl acetate extraction (150mL × 1,75mL × 2), saturated sodium-chloride washed once, and anhydrous sodium sulfate dries, filters, and be spin-dried for, and ethyl acetate/petroleum ether crosses column, obtain 3.6 g yield of product: 65.4%.ESI:m/z:C₁₇H₂₅N₂O₇[M+H]⁺Calculated value: 369.2, measured value 369.2.¹H NMR (400MHz, MeOD) δ 7.93 (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)。

The synthesis of 66. compound 154 of embodiment

Compound 153 (3.2g, 7.74mmol) is dissolved in 20mL methanol, 0.2g Pd/C, catalytic hydrogenation, room temperature is added React 3h, thin-layer chromatography tracking, end of reaction.20mL ethyl acetate, filtering is added, ethyl acetate washing is spin-dried for, and vacuum is taken out It is dry, 2.3g white foam solid is obtained, yield: 92.0%. ESI:m/z:C₁₇H₂₇N₂O₅[M+H]⁺Calculated value: 339.2, it is real Measured value 339.2.¹H NMR (400 MHz, MeOD) δ 6.61 (d, J=8.0Hz, 2H), 6.45 (d, J=6.3Hz, 1H), 3.72 (d, J=7.3 Hz, 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).

The synthesis of 67. compound 41a of embodiment

Compound 41 (10g, 19mmol) is dissolved in DCM, ice bath, Pentafluorophenol (7g, 38 mmol) and EDCI is added (7.2g, 38mmol) is warming up to room temperature reaction overnight, and concentration, silica gel column purification (0-60% ethyl acetate/petroleum ether) obtains nothing Amorphous solid 11g, yield 83%.MS ESI m/z C₃₁H₄₁F₅N₄O₆S [M+H]⁺Calculated value 693.27, measured value 693.27.

The synthesis of 68. compound 132 of embodiment

Compound 41a (11g, 15.9mmol) and compound 127 (12.3g, 23.8mmol) are dissolved in DMF (100mL) In, DIPEA (6.9mL, 39.7mmol) is added under ice bath, is warming up to room temperature reaction 1 hour, concentration, silica gel column purification (0-10% Ethanol/methylene) obtain unformed solid 10g, yield 69%.MS ESI m/z C₄₅H₆₅N₇O₁₁S[M+H]⁺Calculated value 912.45 measured value 912.45.

The synthesis of 69. compound 171 of embodiment

Compound 102 (1.00g, 2.52mmol) is dissolved in acetonitrile (10mL), is cooled to -25 DEG C, CCl is added₄ (2.2mL, 22.7mmol), stirring 10 minutes, addition DIPEA (0.88mL, 5.04mmol) and DMAP (0.03g, 0.252mmol), it then is added dropwise dibenzyl phosphite (0.84mL, 3.78mmol), is added dropwise, it is small to be warming up to room temperature reaction 1.5 When, use KH₂PO₄(0.5M, 50mL) is quenched, and ethyl acetate extracts (3 × 50mL), and anhydrous sodium sulfate dries, filters, and is spin-dried for, silica gel Column purification (10-50% ethyl acetate/petroleum ether) obtains colorless oil 1.60g, yield 96%.MS ESI m/z C₃₃H₄₁N₂O₁₀P[M+H]⁺Calculated value 657, measured value 657.

The synthesis of 70. compound 172 of embodiment

Compound 171 (1.60g, 2.43mmol) is dissolved in methanol (20mL), is added Pd/C (10wt%, 160mg), Catalytic hydrogenation (1atm H₂) reaction 1 hour, filtering is spin-dried for obtaining white solid 1.00g, yield 91%.MS ESI m/z C₁₉H₃₁N₂O₈P[M-H]^-Calculated value 447, measured value 447.

The synthesis of 71. compound 173 of embodiment

Compound 172 (730mg, 1.63mmol) is dissolved in methanol (10mL), addition 1N NaOH (16 mL, 16.3mmol), overnight, concentration adds water (20mL) to dilute, is acidified to pH 6 with 1N HCl for room temperature reaction, is concentrated, by residue It is beaten with methanol/ethyl acetate (80:20,5mL), filtering, collection obtains white solid 0.68g, yield 99%.MS ESI m/z C₁₇H₂₇N₂O₈P[M-H]^-Calculated value 417, measured value 417.

The synthesis of 72. compound 174 of embodiment

Compound 173 (300mg, 0.72mmol) is dissolved in ethanol/dichloromethane/water (v/v/v 5:3:1,2 mL), Compound 125 (262mg, 0.94mmol) and TEA (0.15mL, 1.08mmol) is added, is stirred overnight at room temperature, is concentrated, reverse phase color Compose column HPLC (C₁₈Column, 10-90% acetonitrile/water) purifying obtain white solid 200mg, yield 48%.MS ESI m/z C₂₅H₃₄N₃O₁₁P[M-H]^-Calculated value 582, measured value 582.

The synthesis of 73. compound 175 of embodiment

Compound 174 (140mg, 0.24mmol) is dissolved in DCM (2mL) and TFA (1mL), is reacted at room temperature 2 hours, it is dense Contracting obtains compound 175, is directly used in and reacts in next step.MS ESI m/z C₂₀H₂₇N₃O₉P[M-H]^-Calculated value 482, measured value 482。

The synthesis of 74. compound 180 of embodiment

Compound 33 (86mg, 0.16mmol, 1.0eq.) is dissolved in DCM (2mL), ice bath, Pentafluorophenol is added (44mg, 0.24mmol, 1.5eq.) and DIC (22mg, 0.175mmol, 1.1eq.) is warming up to room temperature reaction overnight, concentration, EtOAc (2mL) dissolution, filtering are concentrated to get phenyl-pentafluoride phenolic ester crude product again, are dissolved in DMA (2mL), and compound is added 175 (0.24mmol, 1.5eq.) and DIPEA (42 μ L, 0.24mmol, 1.5eq.), room temperature reaction overnight, are concentrated, reverse-phase chromatography Column HPLC (C₁₈Column, 10-90% acetonitrile/water) purifying obtain unformed solid 90mg, yield 56%.MS ESI m/z C₄₆H₆₇N₇O₁₄PS [M+H]⁺Calculated value 1004.41, measured value 1004.41.

The synthesis of 75. compound 289 of embodiment

2- (2- amino ethoxy) ethyl alcohol (21.0g, 200mmol) and potassium carbonate (83.0g, 600mmol) are mixed in second In nitrile (350mL), it is added benzyl bromide (57.0mL, 480mmol).After mixture is refluxed overnight, it is added water (1L), with acetic acid second Ester (3 × 300mL) extraction.Combined organic layer is washed with saturated salt solution (1000mL), mistake dry with anhydrous sodium sulfate Filter, is concentrated and passes through SiO₂Column chromatography (4:1 petrol ether/ethyl acetate) purifying, obtains colorless oil (50.97g, yield 89.2%). MS ESI m/z C₁₈H₂₃NO₂Na[M+Na]⁺: calculated value 309.17, measured value 309.19.

The synthesis of 76. compound 290 of embodiment

To 2- (2- (dibenzyl amino) ethyoxyl) ethyl alcohol (47.17g, 165.3mmol), tert-butyl acrylate (72.0mL, 495.9mmol) and 50% hydrogen-oxygen is added in methylene chloride (560mL) solution of tetrabutylammonium iodide (6.10g, 16.53mmol) Change sodium water solution (300mL).The mixture was stirred overnight.Separate organic layer, aqueous layer with ethyl acetate (3 × 100mL) extraction.It closes And organic layer and washed with water (3 × 300mL) and saturated salt solution (300mL), it is dried, filtered with anhydrous sodium sulfate, concentration is simultaneously Pass through SiO₂Column chromatography (7:1 petrol ether/ethyl acetate) purifying, obtains colorless oil (61.1g, 89.4% yield).MS ESI m/z C₂₅H₃₆NO₄[M+H]⁺: calculated value 414.2566, measured value 414.2384.

The synthesis of 77. compound 291 of embodiment

Hydrogenation bottle in 3- (2- (2- (2- (dibenzyl amino) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (20.00g, 48.36mmol, 1.0 equivalents) tetrahydrofuran (30mL) and methanol (60mL) solution in be added Pd/C (2.00g, 10wt%).It will (1atm) shaken overnight is concentrated filtrate, obtains colorless oil mixture by diatomite (filter aid) filtering in the hydrogen gas atmosphere Object (10.58g, 93.8% yield).MS ESI m/z C₁₁H₂₄NO₄[M+H]⁺: calculated value 234.1627, measured value 234.1810.

The synthesis of 78. compound 292 of embodiment

It is sour (0.15g, 1mmol) to (E) -3- bromopropene at 0 DEG C, DMAP (0.15g, 1.2mmol) and DCC Compound 291 (0.23g, 1mmol) is added in methylene chloride (10ml) solution of (0.21g, 1mmol).By reaction mixture liter It warms to room temperature, is stirred overnight.Crude product is concentrated and passes through SiO₂Column chromatography ethyl acetate/dichloromethane gradient elution, obtains To compound 292 (0.31g, 85% yield). ESI MS m/z C₁₄H₂₅BrNO₅[M+H]⁺: calculated value 366.08, measured value 366.08。

The synthesis of 79. compound 293 of embodiment

Compound 292 (0.31g, 0.84mmol) is dissolved in formic acid (4mL) at 0 DEG C, water (2mL) then is added. Reaction mixture is warming up to room temperature, and is stirred overnight.It is directly used in after crude product is concentrated in next step.ESI MS m/z C₁₀H₁₇BrNO₅[M+H]⁺: calculated value 310.02, measured value 310.03.

The synthesis of 80. compound 294 of embodiment

By compound 293 (0.12g, 0.39mmol), n-hydroxysuccinimide (0.067g, 0.58mmol) and EDCI (0.11g, 0.58mmol) is dissolved in methylene chloride (10mL), and mixture is stirred at room temperature overnight, and is concentrated and is passed through SiO₂Column Chromatogram purification obtains compound 294 (0.13g, 82% yield).ESI MS m/z C₁₄H₂₀BrN₂O₇[M+H]⁺: calculated value 407.04 measured value 407.04.

The synthesis of 81. compound 297 of embodiment

To DMA (3ml) solution of compound 256 (50mg, 0.066mmol) and compound 294 (60mg, 0.148mmol) Middle addition sodium dihydrogen phosphate (17.8mg, 0.15mmol).It is stirred at room temperature overnight, is concentrated and (is used by preparation HPLC purifying Acetonitrile/water gradient elution), obtain compound 297 (22.6mg, 33% yield).ESI MS m/z C₄₈H₇₃BrN₇O₁₂S[M+H ]⁺: calculated value 1052.41, measured value 1052.40.

The synthesis of 82. compound 302 of embodiment

By 3- (2- (2- amino ethoxy) ethyoxyl) propanoic acid tert-butyl ester (466mg, 2mmol) and propiolic acid (210mg, It 3mmol) is dissolved in methylene chloride (50mL), DCC (618mg, 3mmol) is added thereto.Acquired solution is stirred at room temperature It is concentrated again after 3 hours.It is purified by column chromatography (10% ethyl acetate/petroleum ether to 100% ethyl acetate), obtains compound 302 (400mg, 70%).ESI MS m/z 286.17([M+H]⁺)。

The synthesis of 83. compound 303 of embodiment

Compound 302 (200mg, 0.7mmol) is dissolved in methylene chloride (5mL), formic acid (7mL) is added thereto.By institute Solution is obtained to be stirred overnight at 38 DEG C.Vacuum distillation removes all volatile matters, obtains compound 303 (160mg).ESI MS m/z 230.11([M+H]⁺)。

The synthesis of 84. compound 304 of embodiment

N-hydroxysuccinimide (115mg, 1mmol) and EDC (192mg, 1mmol) are added to compound 303 (149mg, 0.65mmol) is in the solution in methylene chloride (15mL).It is stirred overnight at room temperature, reaction is concentrated and passes through column color Spectrometry (0% to 10% ethanol/methylene) purifying, obtains title compound (180mg, 85%).ESI MS m/z 327.11 ([M+H]⁺)。

The synthesis of 85. compound 305 of embodiment

Sodium dihydrogen phosphate (0.1M, 1.5mL) is added to compound 304 (90mg, 0.276mmol) and compound 110 In ethyl alcohol (7.5mL) solution of (109mg, 0.276mmol).Acquired solution is stirred at room temperature 24 hours.Vacuum distillation removes All volatile matters are removed, and by column chromatography (30% ethyl acetate/petroleum ether to 100% ethyl acetate) purification residues, are obtained Title compound (160mg, 96%). ESI MS m/z 606.34([M+H]⁺)

The synthesis of 86. compound 306 of embodiment

Compound 305 (40mg, 0.066mmol) is dissolved in methylene chloride (3mL), and at room temperature with trifluoroacetic acid (3mL) is stirred 2 hours.Vacuum distillation removes all volatile matters, obtains title compound (29mg, 99%).ESI MS m/z 450.23([M+H]⁺)。

The synthesis of 87. compound 307 of embodiment

Compound 306 (29mg, 0.066mmol) and compound 41a (46mg, 0.066mmol) are dissolved in DMA (3mL). Then DIPEA (10mg, 0.078mmol) is added, and is stirred at room temperature 1.5 hours.Vacuum distillation removes solvent, residue With preparation HPLC (C₁₈Column, 10-90% acetonitrile/water) purifying, obtain title compound (15mg, 24%).ESI MS m/z 958.47([M+H]⁺)。

The synthesis of 88. compound 309 of embodiment

Be added in the solution in methylene chloride (20mL) to compound 110 (424mg, 1mmol) imidazoles (408mg, 6mmol) and tertiary butyl chloride dimethylsilane (602mg, 4mmol).Acquired solution is stirred at room temperature 3 hours.Concentration reaction Liquid is simultaneously purified by column chromatography (10% to 30% ethyl acetate/petroleum ether), and compound 309 (344mg, 64% yield) is obtained.

The synthesis of 89. compound 310 of embodiment

Compound 309 (200mg, 0.37mmol) is dissolved in ethyl acetate (30mL), in room temperature hydrogen (1atm) environment Lower and Pd/C (10wt%, 100mg) is stirred 2 hours.After filtering out catalyst, all volatile matters are distilled off in filtrate decompression, are obtained Compound 310 (187mg, 99% yield).

The synthesis of 90. compound 312 of embodiment

Acetylenedicarboxylic acid (26.5mg, 0.232mmol) is dissolved in NMP (1.0mL) and is cooled to 0 DEG C, is added thereto Then DMTMM (0.18g, 0.65mmol) is added in compound 291 (0.15g, 0.557mmol).Reaction is stirred 5 at 0 DEG C Hour, it is then diluted with ethyl acetate, with water and saturated common salt water washing.Organic solution is concentrated and is purified by column chromatography (80-90% ethyl acetate/petroleum ether).The fraction of needs is concentrated and is stored in refrigerator overnight, filters out the solid of precipitation.It will Filtrate concentration, obtains light yellow oil (0.37g, > 100% yield), for reacting in next step.MS ESI m/z C₂₆H₄₅N₂O₁₀[M+H]⁺: calculated value 545.30；Measured value 545.30.

The synthesis of 91. compound 313 of embodiment

Compound 312 (0.21g) is dissolved in methylene chloride (2.5mL) and is cooled to 0 DEG C, addition trifluoroacetic acid Reaction is warming up to room temperature and stirred 45 minutes, then removes solvent and remaining TFA on the rotary evaporator by (2.5mL).Slightly Product is purified by column chromatography (0-15% ethanol/methylene), obtains colorless oil (58.7mg, two step yields 99%). MS ESI m/z C₁₈H₂₉N₂O₁₀[M+H]⁺: calculated value 433.17；Measured value 433.17.

The synthesis of 92. compound 314 of embodiment

By compound 313 (0.085g, 0.197mmol), compound 310 (0.1g, 0.197mmol) and HATU (0.112mg, 0.30mmol) mixing in DMF (6ml), is added triethylamine (55 μ L, 0.40mmol) thereto.Reaction is in room It is stirred overnight under temperature, vacuum distillation removes solvent, and residue passes through SiO₂Column chromatography purifying, obtains title product 314 (0.065mg, 36% yield).ESI MS m/z C₄₅H₇₅N₄O₁₄Si[M+H]⁺: calculated value 923.50, measured value 923.50.

The synthesis of 93. compound 315 of embodiment

Compound 314 (64.8mg, 0.070mmol) is dissolved in methylene chloride (3mL), is added TBAF (1M, 0.1mL), Reactant is stirred at room temperature 1 hour, is then concentrated and passes through SiO₂Column chromatography purifying, obtains title product 315 (34.5mg, 61% yield).ESI MS m/z C₃₉H₆₁N₄O₁₄ [M+H]⁺: calculated value 809.41, measured value 809.41.

The synthesis of 94. compound 316 of embodiment

Compound 315 (34.5mg, 0.043mmol) is dissolved in methylene chloride (1mL), trifluoroacetic acid is then added (0.5mL).Reactant is stirred at room temperature 2 hours, is re-dissolved in after concentration in DMA (4mL), pentafluorophenyl group ester is added (45mg, 0.065mmol) and DIPEA (15 μ L, 0.09mmol), reaction are concentrated after being stirred overnight at room temperature, pure by preparation HPLC Change and (use acetonitrile/water gradient elution), obtains title product 316 (22.8mg, 46% yield).ESI MS m/z C₅₆H₈₅N₈O₁₇S [M+H]⁺: calculated value 1173.57, measured value 1173.58.

The synthesis of 95. compound 334 of embodiment

4-Aminobutanoicacid (30g, 300mmol) and NaOH (24g, 600mmol) are dissolved in water (160mL), are added at 0 DEG C Tetrahydrofuran (130mL) solution of benzyl chloroformate (64.4g, 380mmol).Reaction is stirred 1 hour at 0 DEG C, room temperature 3 is small When.Vacuum distillation removes tetrahydrofuran, adjusts pH value of water solution to 3 with concentrated hydrochloric acid at 0 DEG C.It is extracted with ethyl acetate, saturation food Salt washing, anhydrous sodium sulfate is dry, is concentrated to get white solid (70.8g, 99%).ESI m/z C₁₂H₁₆NO₄[M+H]⁺: it calculates Value 238.10, measured value 238.22.

The synthesis of 96. compound 335 of embodiment

DMAP (0.8g, 6.56mmol) and DCC (17.1g, 83mmol) are added to 4- ((((benzyloxy) carbonyl) amino) Methylene chloride (100mL) solution of butyric acid (16.4g, 69.2mmol) and the tert-butyl alcohol (15.4g, 208mmol).It was stirred at room temperature Reactant is filtered and filtrate is concentrated by night.It dissolves the residue in ethyl acetate, with 1N HCl, saturated common salt water washing uses sulphur Sour sodium dries, filters, and is purified after concentration by column chromatography (10 to 50% ethyl acetate/petroleum ether), obtains compound 335 (7.5g, 37% yield).MS ESI m/z C₁₆H₂₃NO₄Na[M+Na]⁺: calculated value 316.16, measured value 316.13.

The synthesis of 97. compound 336 of embodiment

4- (((benzyloxy) carbonyl) amino) tert-butyl acetate (560mg, 1.91mmol) is dissolved in methanol (50mL), with Then Pd/C catalyst (10wt%, 100mg) mixing hydrogenates (1atm) and reacts 3 hours.Catalyst is filtered out, vacuum distillation removes All volatile matters obtain compound 336 (272mg, 90% yield).MS ESI m/z C₈H₁₈NO₂[M+H]⁺: calculated value 160.13 measured value 160.13.

The synthesis of 98. compound 338 of embodiment

By the 4-Aminobutanoicacid tert-butyl ester (477mg, 3mmol) and 2, bis- bromosuccinic acid of 3- (414mg, 1.5mmol) is dissolved in In methylene chloride (35mL), DIPEA (1.16g, 9mmol) and EDC (0.86g, 4.5mmol) are added thereto.By acquired solution It is stirred at room temperature overnight, then uses saturated common salt water washing, dried, filtered with sodium sulphate, it is (pure by column chromatography after concentration Methylene chloride is to 10% ethanol/methylene) purifying, obtain compound 338 (160mg, 22% yield).MS ESI m/z C₂₀H₃₄BrN₂O₆[M+H]⁺: calculated value 477.15, measured value 477.16.

The synthesis of 99. compound 339 of embodiment

Compound 338 (80mg, 0.168mmol) is dissolved in methylene chloride (5mL), and at 38 DEG C with formic acid (8mL) It is stirred overnight.Vacuum distillation removes all volatile matters, obtains compound 339 (61mg, 99% yield).MS ESI m/z C₁₂H₁₈BrN₂O₆[M+H]⁺: calculated value 365.03, measured value 365.05.

The synthesis of 100. compound 340 of embodiment

N-hydroxysuccinimide (60mg, 0.504mmol) and EDCI (97mg, 0.504mmol) are added to compound In methylene chloride (10mL) solution of 339 (61mg, 0.168mmol).It is stirred at room temperature overnight, reaction mixture is concentrated and is led to Column chromatography (0 to 10% ethanol/methylene) purifying is crossed, compound 340 (72mg, 77% yield) is obtained.MS ESI m/z C₂₀H₂₄BrN₄O₁₀[M+H]⁺: calculated value 559.06, measured value 559.78.

The synthesis of 101. compound 342 of embodiment

Biphosphate sodium water solution (0.1M, 1mL) is added to compound 340 (36mg, 0.065mmol) and compound In ethyl alcohol (5mL) solution of 110 (25mg, 0.063mmol).Acquired solution is stirred at room temperature overnight, is then added thereto Enter HO- (PEG)₂₄-NH₂(95mg), is stirred overnight at room temperature.Vacuum distillation removes all volatile matters, and passes through column chromatography (pure two Chloromethanes is to 10% ethanol/methylene) purification residues, obtain compound 342 (28mg, 24% yield).MS ESI m/z 1798.93([M+H]⁺)。

The synthesis of 102. compound 344 of embodiment

Compound 342 (28mg, 0.0156mmol) is dissolved in methylene chloride (2mL), is added trifluoroacetic acid (2mL), It stirs 2 hours at room temperature.Vacuum distillation removes all volatile matters, obtains compound 344 (25mg, 98% yield).MS ESI m/z 1642.82([M+H]⁺)。

The synthesis of 103. compound 346 of embodiment

Compound 344 (25mg, 0.0152mmol) and pentafluorophenyl group ester (15mg, 0.0213mmol) are dissolved in DMA In (5mL).DIPEA (10mg, 0.077mmol) is added thereto.Gained mixture is stirred at room temperature overnight, is led to after concentration Cross preparation HPLC (C₁₈Column, 10-90% acetonitrile/water) purifying, obtain compound 346 (13mg, 40% yield) .MS ESI m/z 2163.82([M+H]⁺)。

The synthesis of 104. compound 350 of embodiment

To the anhydrous tetrahydro furan of 2,2'- (ethane -1,2- diyl is bis- (oxygen)) diethanol (55.0mL, 410.75mmol) Sodium (0.1g) is added in (200mL) solution.Stir the mixture for until sodium disappear, then be added dropwise tert-butyl acrylate (20.0mL, 137.79mmol), it is stirred overnight, is then quenched at 0 DEG C with hydrochloric acid solution (20.0mL, 1N).Four are removed by rotary evaporation Hydrogen furans is added saturated salt solution (300mL), and gained mixture is extracted with ethyl acetate (3 × 100mL).Organic layer saturation Brine It (3 × 300mL), is dried, filtered and concentrated with anhydrous sodium sulfate, obtains colorless oil (30.20g, yield 79.0%) it, can be used without being further purified.MS ESI m/zC₁₃H₂₇O₆[M+H]⁺: calculated value 278.1729, measured value 278.1730。

The synthesis of 105. compound 351 of embodiment

To 3- (2- (2- (2- (2- hydroxyl-oxethyl) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (30.20g, 108.5mmol) and three are added in anhydrous methylene chloride (220mL) solution of paratoluensulfonyl chloride (41.37g, 217.0mmol) Ethamine (30.0mL, 217.0mmol).Reaction is stirred at room temperature overnight, water (3 × 300mL) and saturated salt solution are then used (300mL) washing, is dried, filtered with anhydrous sodium sulfate, is concentrated and passes through SiO₂Column chromatography (3:1 petrol ether/ethyl acetate) is pure Change to obtaining colorless oil (39.4g, yield 84.0%).MS ESI m/zC₂₀H₃₃O₈S[M+H]⁺: calculated value 433.1818, Measured value 433.2838.

The synthesis of 106. compound 352 of embodiment

To 3- (2- (2- (2- (2- methoxyl group) ethyoxyl) ethyoxyl) ethyoxyl) propanoic acid tert-butyl ester (39.4g, NaN is added in anhydrous DMF (100mL) solution 91.1mmol)₃(20.67g, 316.6mmol), is stirred at room temperature overnight.Add Enter water (500mL), is extracted with ethyl acetate (3 × 300mL).By combined organic layer water (3 × 900mL) and saturated salt solution (900mL) washing, is dried, filtered with anhydrous sodium sulfate, is concentrated and passes through SiO₂Column chromatography (5:1 petrol ether/ethyl acetate) is pure Change, obtains light yellow oil (23.8g, 85.5% yield).MS ESI m/z C₁₃H₂₅O₃N₅Na[M+Na]⁺: calculated value 326.2 measured value 326.2.

The synthesis of 107. compound 353 of embodiment

Raney's nickel (7.5g, suspend in water) is washed with water (three times) and isopropanol (three times), and with compound 101 The mixing of (5.0g, 16.5mmol) aqueous isopropanol.Mixture is stirred 16 hours under room temperature hydrogen balloon, then uses Celite pad Filtering, is washed with isopropanol.Filtrate is concentrated to and is passed through column chromatography (5-25% ethanol/methylene) purifying, is obtained faint yellow Grease (2.60g, 57% yield). MS ESI m/z C₁₃H₂₈NO₅[M+H]⁺: calculated value 279.19；Measured value 279.19.

The synthesis of 108. compound 354 of embodiment

Acetylenedicarboxylic acid (0.35g, 3.09mmol) is dissolved in NMP (10mL) and is cooled to 0 DEG C, thereto additionization It closes object 353 (2.06g, 7.43mmol), DMTMM (2.39g, 8.65mmol) then is added.It is small that the stirring 6 at 0 DEG C will be reacted When, it is then diluted with ethyl acetate, with water and saturated common salt water washing.Organic solution is concentrated and uses ethyl acetate and petroleum ether Mixed solvent mashing.Solid is filtered out, filtrate is concentrated and is purified by column chromatography (80-90% ethyl acetate/petroleum ether), is obtained Light yellow oil (2.26g, > 100% yield) directly uses without further purification.MS ESI m/z C₃₀H₅₃N₂O₁₂[M+ H]⁺: calculated value 633.35；Measured value 633.30.

The synthesis of 109. compound 355 of embodiment

Compound 354 (2.26g) is dissolved in methylene chloride (15mL) and is cooled to 0 DEG C, trifluoroacetic acid is then added Reaction is warming up to room temperature, and stirred 45 minutes by (15mL), then removes solvent and remaining TFA on the rotary evaporator.It will Crude product is purified by column chromatography (0-15% ethanol/methylene), obtains light yellow oil (1.39g, two step yields 86%).MS ESI m/zC₂₂H₃₇N₂O₁₂ [M+H]⁺: calculated value 521.23；Measured value 521.24.

The synthesis of 110. compound 358 of embodiment

In Z-L-Ala-OH (0.84g, 5mmol), methylene chloride (20mL) solution of H-Gly-OtBu (0.66g, 5mmol) Middle addition HOBt (0.68g, 5mmol), EDCI (1.44g, 7.5mmol) and DIPEA (1.7mL, 10mmol).Reaction solution is existed It is stirred overnight, is then washed with water (100mL) at room temperature, aqueous layer with ethyl acetate back extraction.By combined organic layer saturated common salt Washing, magnesium sulfate are dried, filtered and concentrated.Residue SiO₂Column purification obtains title product (0.87g, 52% yield). ESI m/zC₁₇H₂₅N₂O₅[M+H]⁺: calculated value 337.17, measured value 337.17.

The synthesis of 111. compound 359 of embodiment

Compound 358 (0.25g, 0.74mmol) is dissolved in methylene chloride (30mL), trifluoroacetic acid is added at room temperature (10mL), reaction is stirred overnight.Then reactant is concentrated, obtains compound 359, be directly used in without further purification next Step.

The synthesis of 112. compound 360 of embodiment

By compound 359 (0.20g, 0.70mmol), compound 110 (0.19g, 0.48mmol) and HATU (0.18g, It 0.48mmol) is dissolved in methylene chloride (20mL), triethylamine (134 μ L, 0.96mmol) is added thereto.By reaction solution in room It is stirred overnight, is concentrated under reduced pressure, residue SiO under temperature₂Column purification obtains title product 360 (0.30g, 95% yield).ESI m/zC₃₄H₄₉N₄O₉[M+H]⁺: calculated value 657.34, measured value 657.34.

The synthesis of 113. compound 361 of embodiment

In hydrogenation bottle, methanol (10mL) solution and Pd/C of compound 360 (0.30g, 0.46mmol) is added (0.10g, 10wt%).By mixture in 1 atmospheric pressure H₂Then lower shaken overnight is filtered by diatomite (filter aid), will Filtrate concentration, obtains compound 361 (0.21g, 87% yield), is directly used in without further purification in next step.ESI m/ zC₂₆H₄₃N₄O₇[M+H]⁺: calculated value 523.31, measured value 523.31.

The synthesis of 114. compound 362 of embodiment

To compound 361 (0.11g, 0.2mmol), the methylene chloride (10mL) of compound 355 (0.104g, 0.2mmol) HATU (0.07g, 0.2mmol) and triethylamine (55 μ L, 0.4mmol) are added in solution.Reaction mixture is stirred at room temperature Overnight, solvent, residue SiO are then removed under reduced pressure₂Column purification, obtaining title product 362, (0.046g, 23% produces Rate).ESI m/zC₄₈H₇₅N₆O₁₇[M+H]⁺: calculated value 1007.51, measured value 1007.52.

The synthesis of 115. compound 363 of embodiment

Compound 362 (0.046g, 0.045mmol) is dissolved in methylene chloride (5mL), trifluoroacetic acid is added at room temperature Reactant is concentrated after 2 hours for (5mL), stirring, obtains compound 363, is directly used in without further purification in next step.

The synthesis of 116. compound 364 of embodiment

Pentafluorophenyl group ester 41a (31.1mg, 0.045mmol) is added into DMA (4mL) solution of compound 363, then It is added DIPEA (28 μ l, 0.159mmol), is stirred overnight.Reaction solution is concentrated, crude product preparation HPLC purifies (10- 90% acetonitrile/water), obtain title product 364 (7.9mg, 13% yield).ESI MSm/zC₆₄H₉₉N₁₀O₂₀S[M+H]⁺: it calculates Value 1359.67, measured value 1359.62.

The synthesis of 117. compound 319 of embodiment

By compound 104 (0.19g, 0.48mmol), compound 313 (0.173g, 0.4mmol) and HATU (0.30g, It 0.8mmol) is dissolved in methylene chloride (50mL), triethylamine (110 μ L, 0.8mmol) then is added.By reaction mixture in room It is stirred overnight under temperature, then vacuum distillation removes solvent, residue SiO₂Column purification obtains title compound 319 (0.25g, 80% yield).ESI m/z C₃₉H₅₉N₄O₁₃ [M+H]⁺: calculated value 791.40, measured value 791.40.

The synthesis of 118. compound 320 of embodiment

Compound 319 (0.10g, 0.14mmol) is dissolved in methylene chloride (1.0mL), TFA (1.0mL) then is added. Reactant is stirred at room temperature 2 hours, compound 320 is obtained after concentration, is used for next step without further purification.

The synthesis of 119. compound 321 of embodiment

Pentafluorophenyl group ester 41a is added into DMA (1.0mL) solution of compound 320 (88.8mg, 0.14mmol) (96.9mg, 0.14mmol) and DIPEA (47.5 μ L, 0.28mmol).Reaction is concentrated after being stirred overnight, then pure with preparation HPLC Change (acetonitrile/water gradient elution), obtains title compound 321 (40mg, 25% yield).ESI m/z C₅₅H₈₃N₈O₁₆S[M+H ]⁺: calculated value 1143.56, measured value 1143.56.

The synthesis of 120. compound 386 of embodiment

HATU (39.9g, 105mmol) is added to DMF (300mL) solution of compound 334 (26.1g, 110mmol). After being stirred at room temperature 30 minutes, which is added to compound 110 (39.4g, 100mmol) and triethylamine DMF (300mL) solution of (20.2g, 200mmol).Reaction is stirred at room temperature 2 hours, is diluted with water, extracts through ethyl acetate, Organic layer is washed with saturated common salt, and sodium sulphate is dry.(20-70% ethyl acetate/petroleum is purified by silica gel column chromatography after concentration Ether), obtain white solid (45g, yield 73%).ESI m/z C₃₃H₄₈N₃O₈[M+H]⁺: calculated value 614.34, measured value 614.15。

The synthesis of 121. compound 387 of embodiment

Compound 386 (100g, 163mmol) is dissolved in methanol (500mL), addition Pd/C catalyst (10wt%, 10g), in room temperature hydrogenation (1atm H₂) overnight.After catalyst filtration is fallen, filtrate decompression concentration obtains brown foam Solid 387 (75.8g, yield 97%).¹H NMR (400 MHz, CDCl3) δ 7.11 (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 C₂₅H₄₂N₃O₆[M+H]⁺: calculated value 480.30, measured value 480.59.

The synthesis of 122. compound 388 of embodiment

By compound 387 (39mg, 0.08mmol), compound 313 (43mg, 0.1mmol) and HATU (30.4mg, It 0.08mmol) is dissolved in methylene chloride (20ml), triethylamine (22 μ L, 0.16mmol) then is added.By reaction mixture in room It is stirred overnight under temperature, then vacuum distillation removes solvent, and residue passes through SiO₂Column purification obtains title compound 388 (42mg, 60% yield).ESI m/z C₄₃H₆₆N₅O₁₄[M+H]⁺: calculated value 876.45, measured value 876.40.

The synthesis of 123. compound 389 of embodiment

Compound 388 (17mg, 0.019mmol) is dissolved in methylene chloride (1.0mL), TFA is added thereto (1.0mL).Reactant is stirred at room temperature 2 hours, is then concentrated, compound 389 is obtained, it is i.e. available without further purification In next step.

The synthesis of 124. compound 390 of embodiment

Pentafluorophenyl group ester 41a is added into DMA (1.0mL) solution of compound 389 (13.6mg, 0.019mmol) (13mg, 0.019mmol) and DIPEA (6.4 μ L, 0.038mmol).It is concentrated after reactant is stirred overnight, residue passes through system Standby HPLC (acetonitrile/water gradient elution) purifies, and obtains title compound 390 (9.9mg, 42% yield).ESI m/z C₅₉H₉₀N₉O₁₇S[M+H]⁺: calculated value 1228.61, measured value 1228.60.

The synthesis of 125. compound 392 of embodiment

By compound 110 (68mg, 0.17mmol), compound 124 (94.5mg, 0.52mmol) and HATU (162mg, It 0.425mmol) is dissolved in methylene chloride (50mL).Then triethylamine (73 μ L, 0.52mmol) is added.Reaction mixture is existed It is stirred overnight at room temperature, then vacuum distillation removes solvent, residue SiO₂Column purification obtains title compound 392 (98mg, 80% yield).ESI m/z C₃₇H₄₉N₄O₁₁[M+H]⁺: calculated value 725.33, measured value 725.34.

The synthesis of 126. compound 393 of embodiment

Compound 392 (98mg, 0.135mmol) is dissolved in methylene chloride (1.0mL), TFA is added at room temperature (1.0mL) is stirred 2 hours.Then it is concentrated, obtains compound 393, be used for next step without further purification.

The synthesis of 127. compound 394 of embodiment

Into DMA (1mL) solution of compound 393 (76.9mg, 0.135mmol) be added pentafluorophenyl group ester 41a (44mg, 0.06mmol) and DIPEA (45.8 μ L, 0.27mmol).Reaction is stirred overnight, is then concentrated, residue passes through preparation HPLC (acetonitrile/H₂O gradient elution) purifying, obtain title compound 394 (37mg, 55% yield).ESI m/z C₅₃H₇₃N₈O₁₄S[M+ H]⁺: calculated value 1077.49, measured value 1077.50.

The synthesis of 128. compound 409 of embodiment

By compound 110 (100mg, 0.25mmol), 3- (2- (2- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- Base) ethyoxyl) ethyoxyl) propionic acid (65mg, 0.25mmol) and HATU (190mg, 0.5mmol) be dissolved in methylene chloride (50ml) In.Reaction mixture is stirred at room temperature overnight after triethylamine (73 μ L, 0.5mmol) is added.Then vacuum distillation removes molten Agent, residue SiO₂Column purification obtains title compound 409 (164mg, 75% yield).ESI m/z C₄₃H₆₁N₄O₁₅[M+ H]⁺: calculated value 873.41, measured value 873.41.

The synthesis of 129. compound 410 of embodiment

Compound 409 (1634mg, 0.187mmol) is dissolved in methylene chloride (1.0mL), TFA is added at room temperature (1.0mL), stirring are concentrated after 2 hours, obtain compound 410, are used for next step without further purification.

The synthesis of 130. compound 411 of embodiment

Into DMA (1mL) solution of compound 410 (43mg, 0.06mmol) be added pentafluorophenyl group ester 41a (44mg, 0.06mmol) with DIPEA (34 μ L, 0.20mmol), it is stirred overnight.Then reaction solution is concentrated, residue passes through preparation HPLC (acetonitrile/H₂O gradient elution) purifying, obtain title compound 411 (33mg, 45% yield).ESI m/z C₅₉H₈₅N₈O₁₈S[M+ H]⁺: calculated value 1225.56, measured value 1225.55.

The synthesis of 131. compound 418 of embodiment

At 0 DEG C, N- methyl is added into EtOAc (120mL) solution of maleimide (6.35g, 65.4mmol) Quinoline (8.6mL, 78.5mmol) and methylchloroformate (6.0mL, 78.5mmol).Reaction is stirred 30 minutes at 0 DEG C, room temperature is stirred It mixes 1 hour.It filters out solid and filtrate is concentrated, filtered after dissolving the residue in methylene chloride by silicagel column, and use methylene chloride Elution, to remove color.Product is collected and is concentrated, obtained solid is beaten with 10% ethyl acetate/petroleum ether, and it is solid to obtain white Body 9.00g (89% yield).

The synthesis of 132. compound 419 of embodiment

By N-Boc- ethylenediamine (5.6mL, 35.4mmol) and saturation NaHCO₃The mixture of (60mL) is cooled to 0 DEG C, point It criticizes and compound 418 (5.00g, 32.2mmol) is added.After being stirred 30 minutes at 0 DEG C, reaction temperature is warmed to room temperature and stirs 1 Hour.Filtering collects and precipitates and washed with cold water, is then dissolved in ethyl acetate, with saturated common salt water washing, anhydrous Na₂SO₄It is dried and concentrated, obtains white solid (6.69g, 87% yield).

The synthesis of 133. compound 420 of embodiment

By compound 419 (6.00g, 25.0mmol) in reaction under high pressure pipe, the toluene (120mL) of furans (18.0mL) is molten Liquid is heated to flowing back and stirring 16 hours.Colourless solution turns yellow during the reaction.It is concentrated after reactant is cooled to room temperature, it will Gained white solid is beaten with ether, obtains compound 420 (6.5g, 84% yield).

The synthesis of 134. compound 421 of embodiment

Compound 420 (9.93g, 32.2mmol) is dissolved in Isosorbide-5-Nitrae-dioxane (15mL), dense HCl is added at room temperature Reaction solution is concentrated after 3 hours, obtained solid is collected by filtration, washs filter cake with ethyl acetate by (15mL), stirring.By solid It is dried overnight in baking oven (50 DEG C), obtains compound 421 (6.94g, 88% yield).

The synthesis of 135. compound 422 of embodiment

At -10 DEG C, phosphorus oxychloride is added into THF (10mL) solution of compound 421 (0.85g, 3.47mmol) Then triethylamine (966 μ L, 6.95mmol) is added in (162 μ L, 1.73mmol).Reactant is stirred 3 hours at -10 DEG C, so It is diluted with methylene chloride (20mL) and is filtered by diatomite afterwards, filtrate is concentrated, compound 422 is obtained, is directly used in down One step.ESI m/z C₂₀H₂₃ClN₄O₇P [M+H]⁺: calculated value 497.09, measured value 497.09.

The synthesis of 136. compound 423 of embodiment

Compound 422 (0.50g, 1.0mmol) and DIPEA (0.4mL, 2.4mmol) are dissolved in methylene chloride at 0 DEG C In (5.0mL), compound 291 (0.23g, 1.0mmol) then is added.Reaction is stirred 2.5 hours at 0 DEG C, is concentrated and leads to Cross SiO₂Column purification obtains title compound 423 (0.30g, 43%).ESI m/z C₃₁H₄₅N₅O₁₁P[M+H]⁺: calculated value 694.28 measured value 694.28.

The synthesis of 137. compound 424 of embodiment

Compound 423 (0.30g, 0.5mmol) is dissolved in methylene chloride (3mL), after TFA (3mL) is added at room temperature Stirring 2 hours, obtains compound 424 after concentration, be used for next step without further purification.

The synthesis of 138. compound 425 of embodiment

By compound 424 (40mg, 0.063mmol), compound 110 (40mg, 0.10mmol), HATU (24mg, It 0.063mmol) is dissolved in methylene chloride (5mL), triethylamine (27.8 μ L, 0.2mmol) then is added.Reaction mixture is existed It is stirred overnight at room temperature, then vacuum distillation removes solvent, residue SiO₂Column purification obtains title compound 425 (53.4mg, yield 84%).ESI m/z C₄₈H₆₉N₇O₁₅P[M+H]⁺: calculated value 1014.45, measured value 1014.45.

The synthesis of 139. compound 426 of embodiment

Compound 425 (53.4mg, 0.053mmol) is dissolved in methylene chloride (2mL), TFA (2mL) is added at room temperature It stirs 2 hours afterwards, compound 426 is obtained after concentration, be used for next step without further purification.

The synthesis of 140. compound 427 of embodiment

Pentafluorophenyl group ester 41a is added into DMA (1mL) solution of compound 426 (45.0mg, 0.053mmol) (37.0mg, 0.053mmol) and DIPEA (17 μ L, 0.1mmol).Reactant is stirred overnight and is concentrated, by preparing HPLC (acetonitrile/water gradient elution) purification residues obtain title compound 427 (26.2mg, 36% yield).ESI m/z C₆₄H₉₃N₁₁O₁₈PS[M+H]⁺: calculated value 1366.61, measured value 1366.61.

The synthesis of 141. compound 428 of embodiment

Compound 427 (8.0mg, 0.0058mmol) is dissolved in toluene (5.0mL), then heated overnight at reflux is concentrated, With preparation HPLC (acetonitrile/water gradient elution) purifying, title compound 428 (6.4mg, 90% yield) is obtained.ESI m/z C₅₆H₈₅N₁₁O₁₆PS[M+H]⁺: calculated value 1230.56, measured value 1230.56.

The synthesis of 142. compound 432 of embodiment

HO-PEG is added in sodium hydrogen (60%, 8g, 200mmol) at room temperature₉The tetrahydro of-OMe (42.8g, 100mmol) In the solution of furans (1L).After stirring 30 minutes, bromo-acetic acid tert-butyl (48.8g, 250mmol) is added, is stirred at room temperature It 1 hour, is subsequently poured into ice water, is extracted with dichloromethane, organic layer is washed with saturated common salt, and anhydrous sodium sulfate is dry.Silica gel Column chromatographic purifying (0-5% ethanol/methylene) obtains compound 156, is yellow oil shape object (32g, 59% yield).

The synthesis of 143. compound 433 of embodiment

Compound 432 (40g, 73.8mmol) is dissolved in methylene chloride (400mL), formic acid (600mL) then is added, 25 DEG C are stirred overnight.Vacuum distillation removes all volatile matters, obtains yellow oil (36g).ESI m/z C₂₁H₄₃O₁₂[M+ H]⁺: calculated value 487.27, measured value 487.24.

The synthesis of 144. compound 434 of embodiment

Compound 433 (36g, 73.8mmol) is dissolved in methylene chloride (640mL), sequentially add oxalyl chloride (100mL), DMF (52g, 0.74mmol).Obtained solution is stirred at room temperature 4 hours, vacuum distillation removes all volatile matters, obtains Yellow oil.

The synthesis of 145. compound 436 of embodiment

By Z-L-Lys-OH (41.4g, 147.6mmol), sodium carbonate (23.4g, 221.4mmol) and NaOH (5.9g, It 147.6mmol) is dissolved in water (720ml), is cooled to 0 DEG C, the four of compound 434 (37.2g, 73.8mmol) are then added Hydrogen tetrahydrofuran solution (20mL).Obtained mixture is stirred at room temperature 1 hour, and vacuum distillation removes THF, with dense under ice bath Hydrochloric acid is adjusted to pH 3.Solution is extracted with dichloromethane, and saturated common salt washing, anhydrous sodium sulfate is dry, obtains yellow oil (55g, 99% yield). ESI m/z C₃₅H₆₀N₂O₁₅[M+H]⁺: calculated value 749.40, measured value 749.39.

The synthesis of 146. compound 439 of embodiment

Sequentially add in DMF (500mL) solution of compound 436 (130g, 174mmol) at 0 DEG C triethylamine (66mL, 474mmol) with HATU (72g, 190mmol), then reaction mixture L is warmed to room temperature, is stirred 2 hours.By compound 387 DMF (500mL) solution of (75.8g, 158mmol) is added in above-mentioned solution at 0 DEG C, and it is small that reaction is stirred at room temperature 1 When.Reaction solution is poured into water (4L), is extracted with ethyl acetate (3 × 500ml), merges organic layer, is washed with saturated common salt (2L), crude product 439 (190g) is directly used in reaction in next step after sodium sulphate dries, filters concentration.ESI m/z C₆₀H₁₀₀N₅O₂₀[M +H]⁺: calculated value 1210.69, measured value 1210.69.

The synthesis of 147. compound 440 of embodiment

439 crude products (190g) that previous step is reacted are dissolved in methanol (900mL), Pd/C catalyst is added (10wt%, 19g), and (1atm H is hydrogenated in room temperature₂) reaction is overnight.Catalyst is filtered out, filtrate decompression is concentrated, uses silicagel column Purifying (0-10% ethanol/methylene) obtains a brown oil (105g, two step yields 62%).ESI m/z C₅₂H₉₅N₅O₁₈[M+H]⁺: calculated value 1077.65, measured value 1077.65.

The synthesis of 148. compound 441 of embodiment

At room temperature in EtOH (5.3L) solution of compound 440 (105g, 97.1mmol), compound 125 is added (54.4g, 194.2mmol) and 0.1N sodium dihydrogen phosphate (1.1L), reaction is stirred at room temperature overnight.Vacuum distillation removes EtOH, residual aqueous solution are poured into water (3L), are then extracted with ethyl acetate (4 × 500ml), are merged organic phase, are eaten with saturation Salt washes (2L), and sodium sulphate is dry, is concentrated, and crude product obtains a yellow with silica gel column purification (0-10% ethanol/methylene) Grease (100g, 83% yield).¹H NMR(400MHz,CDCl₃) δ 9.43 (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 C₆₀H₁₀₁N₆O₂₁[M+H]⁺: calculated value 1241.69, measured value 1241.69。

The synthesis of 149. compound 442 of embodiment

Compound 441 (79.1mg, 0.062mmol) is dissolved in methylene chloride (2mL), is added after TFA (2mL) in room temperature Stirring 2 hours, be evaporated under reduced pressure and with toluene azeotropic distillation, obtain compound 441, be directly used in next step.

The synthesis of 150. compound 443 of embodiment

Compound 441 (67mg, 0.062mmol) and compound 41a (43mg, 0.062mmol) are dissolved in DMA (4mL) In, DIPEA (43 μ l, 0.248mmol) then is added.After being stirred at room temperature 3 hours, vacuum distillation removes solvent, residue With preparation HPLC (C₁₈Column, acetonitrile/water 10-90%) purifying, obtain compound 443 (59mg, 60% yield).ESI m/z C₇₆H₁₂₅N₁₀O₂₄S[M+H]⁺: calculated value 1594.92, measured value 1594.24.

The synthesis of 151. compound 457 of embodiment

HO-PEG is added portionwise in sodium hydrogen (60%, 0.64g, 16mmol)₆The tetrahydrofuran of-OMe (2.37g, 8mmol) (25mL) solution.After 15min is stirred at room temperature, it is added bromo-acetic acid tert-butyl (3.90g, 20mmol), was stirred at room temperature Night.Reaction mixture is poured into ice water, is extracted with dichloromethane, organic phase is washed with saturated common salt, and sodium sulphate is dry, through silicon Gel column chromatography eluting (20-50% ethyl acetate/petroleum ether) obtains a colorless oil (1.47g, 45%).ESI m/z C₁₉H₃₉O₉[M+H]⁺: calculated value 411.25, measured value 411.15.

The synthesis of 152. compound 458 of embodiment

Compound 457 (1.47g, 3.60mmol) is dissolved in methylene chloride (30mL), with formic acid (50mL) at 38 DEG C It is stirred overnight.Vacuum distillation removes all volatile matters, obtains title compound (1.20g, 94% yield), is a yellow oil. ESI m/z C₁₅H₃₁O₉[M+H]⁺: calculated value 355.19, measured value 355.18.

The synthesis of 153. compound 459 of embodiment

Compound 458 (1.10g, 3.20mmol) is dissolved in methylene chloride (20mL), oxalyl chloride (4mL) is sequentially added With DMF (3 drop).Obtained solution is stirred at room temperature 4 hours, vacuum distillation removes all volatile matters, yellow oil is obtained, It is directly used in next step.

The synthesis of 154. compound 460 of embodiment

By Z-L-Lys-OH (1.80g, 6.4mmol), sodium carbonate (1g, 9.6mmol) and NaOH (0.26g, 6.4mmol) It is dissolved in water (30mL), after being cooled to 0 DEG C, tetrahydrofuran (10mL) solution of compound 459 (1.20g, 3.2mmol) is added. Gained mixture is stirred at room temperature 1 hour, and vacuum distillation removes THF, and aqueous solution uses concentrated hydrochloric acid condition pH to 3 under ice bath. It is extracted with dichloromethane, saturated common salt washing, anhydrous sodium sulfate is dry, obtains title compound (1.77g, 90%), is a brown Grease.ESI m/z C₂₉H₄₉N₂O₁₂[M+H]⁺: calculated value 617.32, measured value 617.31.

The synthesis of 155. compound 461 of embodiment

By NHS (644mg, 5.60mmol) and EDC (1.08g, 5.60mmol) be added to compound 460 (2.30g, In methylene chloride (100mL) solution 3.70mmol).After being stirred at room temperature overnight, directly on a silica gel column by reaction mixture Sample purifies (0-10% ethanol/methylene), obtains a brown oil (2.10g, yield 80%).ESI m/z C₃₃H₅₂N₃O₁₄[M+H]⁺: calculated value 714.34, measured value 714.32.

The synthesis of 156. compound 462 of embodiment

Sodium dihydrogen phosphate (0.1M, 3mL) is added to compound 461 (357mg, 0.50mmol) and compound 110 Ethyl alcohol (15mL) solution of (200mg, 0.50mmol).Acquired solution is stirred at room temperature 24 hours, vacuum distillation removes complete Portion's volatile matter, residue be purified by silica gel column chromatography (5-10% ethanol/methylene) obtain title compound (216mg, 44% Yield), it is a brown oil. ESI m/z C₅₀H₈₁N₄O₁₆[M+H]⁺: calculated value 993.56, measured value 993.57.

The synthesis of 157. compound 463 of embodiment

Compound 462 (108mg, 0.109mmol) is dissolved in methanol (5mL), addition palladium catalyst (10wt%, After 50mg), (1atm) is stirred at room temperature 3 hours under a hydrogen atmosphere, filters out catalyst, and filtrate decompression distillation obtains title compound (94mg) is a yellow oil.ESI m/z C₄₂H₇₅N₄O₁₄[M+H]⁺: calculated value 859.52, measured value 859.93.

The synthesis of 158. compound 464 of embodiment

Sodium dihydrogen phosphate (0.1M, 2mL) is added to compound 463 (94mg, 0.109mmol) and compound 125 Ethyl alcohol (10mL) solution of (61mg, 0.218mmol).Obtained solution is stirred at room temperature 24 hours, vacuum distillation removes Whole volatile matters, residue by column chromatography eluting (5-10% ethanol/methylene) a yellow oil (40mg, 36% yield).ESI m/z C₅₀H₈₂N₅O₁₇ [M+H]⁺: calculated value 1024.56, measured value 1024.98.

The synthesis of 159. compound 465 of embodiment

By compound 464 (20mg, 0.0196mmol) be dissolved in methylene chloride (3mL) and TFA (3mL) at room temperature Stirring 2 hours, vacuum distillation remove all volatile matters, obtain title compound (17mg), are a yellow oil.ESI m/z C₄₁H₆₆N₅O₁₅[M+H]⁺: calculated value 868.45, measured value 868.47.

The synthesis of 160. compound 466 of embodiment

Compound 465 (17mg, 0.0196mmol) and compound 41a (14mg, 0.0196mmol) are dissolved in DMA In (3mL).It is stirred at room temperature 3 hours after DIPEA (10 μ l, 0.0588mmol) is added, then vacuum distillation removes solvent, residual Object obtains compound 466 (15mg, yield 64%) by preparation HPLC (C18 column, acetonitrile/water 10-90%) purifying, is a yellow Grease.ESI m/z C₆₆H₁₀₆N₉O₂₀S [M+H]⁺: calculated value 1376.72, measured value 1376.72.

The synthesis of 161. compound 378 of embodiment

By compound 110 (0.30g, 0.76mmol), Z-L-Ala-OH (0.17g, 0.76mmol) and HATU (0.29g, It 0.76mmol) is dissolved in methylene chloride (20mL), triethylamine (110 μ L, 0.8mmol) is added thereto.It was stirred at room temperature At night, then vacuum distillation removes solvent, and residue passes through SiO₂Column purification, obtaining title compound 378, (0.43g, 95% produces Rate).ESI m/z C₃₂H₄₆N₃O₈[M+H]⁺: calculated value 600.32, measured value 600.32.

The synthesis of 162. compound 379 of embodiment

In hydrogenation bottle, Pd/C is added into methanol (10mL) solution of compound 378 (0.3g, 0.5mmol) (0.10g, 33wt%).By mixture in 1atm H₂Then lower shaken overnight is filtered by diatomite, filtrate is concentrated It closes object 379 (0.24g), is directly used in next step without being further purified.ESI m/z C₂₄H₄₀N₃O₆[M+H]⁺: calculated value 466.28 measured value 466.28.

The synthesis of 163. compound 487 of embodiment

Sodium dihydrogen phosphate (0.1M, 4mL) is added to compound 379 (233mg, 0.5mmol) and compound 461 Ethyl alcohol (20mL) solution of (357mg, 0.5mmol).Obtained solution is stirred at room temperature 24 hours, vacuum distillation removes complete Portion's volatile matter, residue by column chromatography (5-10% ethanol/methylene) purify, obtain a yellow oil (176mg, 33%).ESI m/z C₅₃H₈₆N₅O₁₇[M+H]⁺: calculated value 1064.59, measured value 1064.60.

The synthesis of 164. compound 488 of embodiment

Compound 487 (176mg, 0.166mmol) is dissolved in methanol (15mL), addition palladium catalyst (10%, 80mg), it is reacted 3 hours in room temperature hydrogenation (1atm), after catalyst is filtered, vacuum distillation concentration filtrate obtains title compound Object (154mg) is a yellow oil.ESI m/z C₄₅H₈₀N₅O₁₅[M+H]⁺: calculated value 930.56, measured value 930.56.

The synthesis of 165. compound 489 of embodiment

Sodium dihydrogen phosphate (0.1M, 4mL) is added to compound 488 (154mg, 0.166mmol) and compound 125 Ethyl alcohol (20mL) solution of (93mg, 0.332mmol).Obtained solution is stirred at room temperature 24 hours, vacuum distillation removes Whole volatile matters, residue purify (5-10% ethanol/methylene) by silica gel column chromatography, obtain a yellow oil (117mg, 64%).ESI m/z C₅₃H₈₇N₆O₁₈[M+H]⁺: calculated value 1095.60, measured value 1095.61.

The synthesis of 166. compound 490 of embodiment

By compound 489 (39mg, 0.0356mmol) be dissolved in methylene chloride (3mL) and TFA (3mL) at room temperature Stirring 2 hours, vacuum distillation remove whole volatile matters, obtain title compound (33mg), are a yellow oil.ESI m/z C₄₄H₇₁N₆O₁₆[M+H]⁺: calculated value 939.48, measured value 939.49.

The synthesis of 167. compound 491 of embodiment

Compound 490 (33mg, 0.0356mmol) and compound 41a (25mg, 0.0356mmol) are dissolved in DMA In (3mL), DIPEA (15mg, 0.116mmol) being added thereto, is stirred at room temperature 3 hours, vacuum distillation removes reaction dissolvent, Residue obtains compound 491 (17mg, 33%) by preparation HPLC (C18 column, acetonitrile/water 10-90%) purifying, is a yellow Grease.ESI m/z C₆₉H₁₁₁N₁₀O₂₁S[M+H]⁺: calculated value 1447.76, measured value 1448.78.

The synthesis of 168. compound 494 of embodiment

Compound 2- (dimethylamino) acetic acid (0.60g, 4.30mmol) and HATU (1.08g, 2.86mmol) are dissolved in DMF (2mL) is stirred at room temperature 1H after TEA (1mL, 7.16mmol) is added, is added Z-L-LyS-OH (0.80g, 2.86mmol) DMF (2mL) solution.2H is stirred at room temperature in reaction solution, is then concentrated under reduced pressure.Crude product preparation HPLC (C₁₈Column, acetonitrile/water It 10-90%) purifies, obtains colorless oil 494 (0.50g, 50%).MS ESI m/z C₁₈H₂₈N₃O₅[M+H]⁺Calculated value: 366.20 measured value 366.20.

The synthesis of 169. compound 495 of embodiment

Compound 494 (0.50g, 1.37mmol) is dissolved in methylene chloride (15mL), Pentafluorophenol is added thereto (0.38g, 2.05mmol) and EDCI (0.52g, 2.74mmol).It is stirred overnight at room temperature, reaction solution is filtered by diatomite, with two Chloromethanes washs filter cake.The ester for the Pentafluorophenol that filtrate is concentrated to get is dissolved in 10mL methylene chloride.By compound 387 (0.44g, 0.91mmol) and DIPEA (0.32mL, 1.82mmol) are added in above-mentioned solution, are stirred at room temperature 2 hours, so After be concentrated.With silica gel column purification crude product, ethanol/methylene gradient elution obtains compound 495 (1.02g, theoretical yield).MS ESI m/z C₄₃H₆₇N₆O₁₀[M+H]⁺Calculated value: 827.48, measured value 827.48.

The synthesis of 170. compound 496 of embodiment

Compound 495 (1.02g, 1.23mmol) is dissolved in methanol (10mL), addition palladium catalyst (10%, 100mg), in H₂(1atm) is stirred overnight at room temperature under atmosphere.Catalyst is removed by suction filtration under vacuum, concentration of reaction solution obtains yellow oil Shape object 496 (0.76g, 89%).MS ESI m/z C₃₅H₆₁N₆O₈ [M+H]⁺Calculated value: 693.45, measured value 693.45.

The synthesis of 171. compound 497 of embodiment

Compound 496 (0.25g, 0.36mmol) and compound 125 (0.15g, 0.54mmol) are dissolved in ethyl alcohol (5mL), After sodium dihydrogen phosphate (0.1M, 1mL) is added, it is stirred at room temperature overnight.It is concentrated in vacuo reaction solution, crude product is pure by column chromatography Change (5%~10% ethanol/methylene) and obtains compound 497 (0.15g, 48%).MS ESI m/z C₄₃H₆₈N₇O₁₁[M+H]⁺ Calculated value: 858.49, measured value: 858.49.

The synthesis of 172. compound 498 of embodiment

Compound 497 (0.15g, 0.175mmol) is dissolved in methylene chloride (1mL) and trifluoroacetic acid (2mL), It stirs two hours at room temperature, vacuum concentration obtains yellow oil, is dissolved in DMA (2mL), pentafluorophenyl group is added thereto Ester 41a (121.1mg, 0.175mmol) is subsequently added into DIPEA (91 μ L, 0.525mmol).Reaction is stirred at room temperature overnight, so Low pressure is concentrated afterwards, crude product preparation HPLC (C₁₈Column, acetonitrile/water 10-90%) purifying, obtain compound 498 (30.7mg, 14%). MS ESI m/z C₅₉H₉₂N₁₁O₁₄S[M+H]⁺Calculated value: 1210.65, measured value 1210.62.

The synthesis of 173. compound 501 of embodiment

H-LyS-OH (0.31g, 2.14mmol) is dissolved in ethyl alcohol (20mL), thereto be added compound 125 (1.80g, 6.42mmol) with the disodium hydrogen phosphate of 0.5M (4mL), reaction solution is stirred at room temperature overnight.After concentration, crude product preparation HPLC (C₁₈Column, acetonitrile/water 10-90%) purifying, obtain yellow oil 501 (0.26g, 26%).MS ESI m/z C₂₂H₂₉N₄O₈Meter Calculation value: 477.19, measured value 477.19.

The synthesis of 174. compound 502 of embodiment

Compound 501 (0.26g, 0.55mmol) is dissolved in methylene chloride (10mL), addition NHS (0.095g, 0.825mmol) and EDCI (0.16g, 0.825mmol).Reaction mixture is stirred overnight at room temperature, is then concentrated, and is used After water (50mL) dilution, (2 × 20mL) is extracted with ethyl acetate.Merge organic layer, Na₂SO₄It dries, filters, is concentrated to get thick production Product 502 (0.34g) are directly used in and react in next step.MS ESI m/z C₂₆H₃₂N₅O₁₀[M+H]⁺Calculated value: 574.21, measured value 574.21。

The synthesis of 175. compound 503 of embodiment

Compound 438 (0.19g, 0.4mmol) is dissolved in ethyl alcohol (30mL), addition compound 502 (0.34g, 0.6mmol) and the sodium dihydrogen phosphate of 0.1M (6mL).Reaction mixture is stirred at room temperature overnight, and is then concentrated in vacuo.Residual Object is diluted with water (100mL), and ethyl acetate extracts (2 × 40mL).Merge organic layer, Na₂SO₄It is dry, silica gel column purification (dichloro Methane/methanol is eluent gradient elution), obtain compound 503 (0.115g, 31%).MS ESI m/z C₄₇H₆₈N₇O₁₃[M+H]⁺ Calculated value: 938.48, measured value 938.49.

The synthesis of 176. compound 504 of embodiment

Compound 503 (0.115g, 0.12mmol) is dissolved in methylene chloride (1mL), the TFA of 2mL is added.Reaction mixture Low pressure is concentrated after 2h is stirred at room temperature, crude product preparation HPLC (C₁₈Column, acetonitrile/water 10-90%) purifying, obtain yellow oil Shape object 504 (0.0312g, 33%).MS ESI m/z C₂₂H₂₉N₄O₈ [M+H]⁺Calculated value: 477.19, measured value: 477.19.

The synthesis of 177. compound 505 of embodiment

Compound 504 (31.2mg, 0.04mmol) is dissolved in DMA (2mL), pentafluorophenyl group ester 41a is added thereto (27mg, 0.04mmol) is subsequently added into DIPEA (16 μ L, 0.08mmol).Reaction solution is stirred at room temperature overnight, and low pressure concentration is led to Preparative liquid chromatography (10-90% acetonitrile/water) purifying is crossed, compound 505 (11.9mg, 24%) is obtained.MS ESI m/z: C₆₃H₉₂N₁₁O₁₆S[M+H]⁺Calculated value: 1290.64, measured value: 1290.64.

The synthesis of 178. compound 508 of embodiment

3- (2- (2- (dibenzyl amino) ethyoxyl) ethyoxyl) propanoate (5g, 12.1mmol) is dissolved in two In chloromethanes (10mL), the trifluoroacetic acid of 5mL is added.Reaction mixture is stirred at room temperature 1 hour, is then concentrated.Crude product It is dissolved in methylene chloride (50ml), sequentially adds NHS (4.25g, 37mmol) and EDCI (7.10g, 37mmol).Reaction mixture It is stirred at room temperature overnight, is then concentrated, silica gel column purification (methylene chloride/methanol gradient elution) obtains yellow oil 508 (5g, 91%).MS ESI m/z C₂₅H₃₁N₂O₆[M+H]⁺Calculated value: 455.21, measured value 455.21.

The synthesis of 179. compound 509 of embodiment

Compound 110 (1g, 2.5mmol) is dissolved in ethyl alcohol (10ml), be added compound 508 (1.80g, 3.9mmol) and The sodium dihydrogen phosphate (2mL) of 0.1M, reaction overnight, is then concentrated at room temperature.Residue is diluted with water (100mL), uses acetic acid Ethyl ester extracts (3 × 50ml).Merge organic layer Na₂SO₄It dries, filters, is concentrated, silica gel column purification (methylene chloride/methanol body It is gradient elution), obtain yellow oil 509 (0.93g, 50%).MS ESI m/z C₄₂H₆₀N₃O₈[M+H]⁺Calculated value: 734.43 measured value 734.43.

The synthesis of 180. compound 510 of embodiment

By compound 509 (0.93g, 1.27mmol), ethyl acetate (20mL) and Pd/C (0.093g, 10wt%) are added Into hydrogenation bottle, it is stirred overnight at hydrogen (1atm), filter solid is then crossed by diatomite.Filtrate is concentrated to get compound 510 (0.57g, 81%), product do not have to be further purified. MS ESI m/z C₂₈H₄₈N₃O₈[M+H]⁺Calculated value: 554.34, it is real Measured value 554.34.

The synthesis of 181. compound 511 of embodiment

Compound 510 (0.25g, 0.45mmol) is dissolved in ethyl alcohol (5mL), at room temperature be added compound 502 (0.39g, 0.68mmol) with sodium dihydrogen phosphate (0.1M, 1mL), reaction mixture is stirred at room temperature overnight.Water is used after reaction solution vacuum concentration (100mL) dilution, is extracted with ethyl acetate (2 × 50mL).Merge organic layer, Na₂SO₄It dries, filters, is concentrated, silica gel column purification (ethanol/methylene gradient elution) obtains yellow oil 511 (0.076g, 17%).MS ESI m/z C₅₀H₇₄N₇O₁₅[M +H]⁺Calculated value: 1012.52, measured value: 1012.53

The synthesis of 182. compound 512 of embodiment

Compound 511 (0.076g, 75mmol) is dissolved in methylene chloride (2mL), is stirred at room temperature after the TFA of 4mL is added It mixes 1 hour, is concentrated, obtained crude product 512 is directly used in react in next step.MS ESI m/z C₄₁H₅₈N₇O₁₃[M+H]⁺Calculated value: 856.40 measured value 856.40.

The synthesis of 183. compound 513 of embodiment

Compound 512 is dissolved in DMA (2mL), be added compound 41a (33mg, 0.048mmol) and DIPEA (25 μ L, 0.144mmol).Reaction is stirred at room temperature 3 hours, is then concentrated and prepares HPLC purifying (acetonitrile/water gradient elution), obtain To yellow oil 513 (21.3mg, 32%).MS ESI m/z C₆₆H₉₈N₁₁O₁₈S[M+H]⁺Calculated value: 1364.67, measured value 1364.67。

The synthesis of 184. compound 515 of embodiment

Compound 386 (1g, 1.63mmol) is dissolved in methylene chloride (1mL), 2mL TFA, reaction mixing are added thereto Object is stirred at room temperature 1 hour, is then concentrated, and obtained crude product 515 is directly used in react in next step.MS ESI m/z C₂₄H₃₂N₃O₆[M+H]⁺Calculated value: 458.22, measured value: 458.22.

The synthesis of 185. compound 516 of embodiment synthesizes

Compound 515 is dissolved in DMF (3mL), thereto be added pentafluorophenyl group ester 41a (0.63g, 0.91mmol) and DIPEA (0.46mL, 2.73mmol).Reaction is stirred at room temperature overnight, and is then concentrated, silica gel column purification (methylene chloride/methanol Gradient elution), obtain yellow oil 516 (1.75g).MS ESI m/z C₄₉H₇₂N₇O₁₁S[M+H]⁺Calculated value: 966.49, it is real Measured value 966.49.

The synthesis of 186. compound 517 of embodiment synthesizes

By compound 516 (0.20g, 0.20mmol), methanol (15mL) and Pd/C (0.02g, 10wt%) are added to hydrogenation In bottle, pH value then is adjusted to 4 or so with 1N HCl.In H₂It is stirred overnight under (1atm), pad diatomite filtering, filtrate is concentrated to give To compound 517, it is directly used in and reacts in next step.MS ESI m/z C₄₁H₆₆N₇O₉S[M+H]⁺Calculated value: 832.46, measured value 832.46。

The synthesis of 187. compound 519 of embodiment

By compound H-Dap (Boc)-OH (1g, 4.9mmol) and saturation NaHCO₃Solution (20mL) mixing, at 0 DEG C It is added compound 418 (2.30g, 14.7mmol).Then reaction is warming up to and 1h is stirred at room temperature in 0 DEG C of stirring 1h.Hydrogen sulfate is added Potassium (1M) adjusts pH to about 6, and (2 × 50ml) is extracted with ethyl acetate in the mixture of generation.Merge organic layer, Na₂SO₄It is dry, mistake Filter, is concentrated to give compound 519 (0.42g, 30%).MS ESI m/z C₁₂H₁₅N₂O₆[M-H]^-Calculated value: 283.10, measured value 283.10。

The synthesis of 188. compound 520 of embodiment

Compound 519 (0.21g, 0.74mmol) is dissolved in ethyl acetate (10mL), then be added Pentafluorophenol (0.27g, 1.48mmol) and DCC (0.30g, 1.48mmol).Reaction mixture is stirred at room temperature overnight, and is then filtered, and is washed with EtOAC Wash filter cake.Concentration filtrate obtains Acibenzolar (0.17g, 0.37mmol), is dissolved in 1mL DMF, and compound 517 is added thereto (0.36g, 0.43mmol) and DIPEA (0.13mL, 0.74mmol), reaction mixture are stirred at room temperature 2 hours, are concentrated, system Standby HPLC purifying (acetonitrile/water of gradient), obtains compound 520 (50mg, 13%).MS ESI m/z C₅₃H₈₀N₉O₁₄S [M+ H]⁺Calculated value: 1098.55, calculate 1098.55.

The synthesis of 189. compound 521 of embodiment

Compound 520 (50mg, 0.046mmol) is dissolved in the methylene chloride of 0.5mL, be added 1mL trifluoroacetic acid after It reacts 1 hour at room temperature, be concentrated and obtain product 521 (11mg, 25% production with preparation HPLC (mobile phase acetonitrile/water) purifying Rate).MS ESI m/z C₄₈H₇₂N₉O₁₂S[M+H]⁺Calculated value: 998.49, measured value 998.49.

The synthesis of 190. compound 523 of embodiment

Compound 509 (1.00g, 1.36mmol) is dissolved in 2mL methylene chloride, and 4 mL trifluoro second are added thereto Acid adds rear react at room temperature 1 hour.It is concentrated to get compound 523.MS ESI m/z C₃₃H₄₄N₃O₆[M+H]⁺Calculated value: 578.32 measured value 578.32.

The synthesis of 191. compound 524 of embodiment

Compound 523 is dissolved in 5mL DMF, compound 41a (0.78g, 1.13mmol) and DIPEA are added thereto (0.8mL,4.52mmol).It is stirred overnight at room temperature after after adding, is obtained after concentration with silica gel column purification (mobile phase MeOH/DCM) Product 524 (1.64g, 100% yield).MS MS ESI m/z C₅₈H₈₄N₇O₁₁S[M+H]⁺Calculated value: 1086.59, measured value 1086.58。

The synthesis of 192. compound 525 of embodiment

Compound 524 (0.80g, 0.20mmol) is dissolved in 10mL methanol, thereto be added Pd/C (0.08g, 10wt%), and with 1N HCl tune pH to 4.After hydrogen is replaced 3 times, it is stirred overnight.Pad diatomite filtering, being concentrated to get of filtrate Close object 525.MS ESI m/z C₄₁H₆₆N₇O₉S[M+H]⁺Calculated value: 832.46, measured value 832.46.

The synthesis of 193. compound 527 of embodiment

H-Lys (Boc)-OH (1.00g, 3.8mmol) is dissolved in 16mL ethyl alcohol, compound 125 is added thereto (1.00g, 5.6mmol) and sodium dihydrogen phosphate (0.1M, 3mL) adds rear be stirred overnight at room temperature.Reaction solution is concentrated and uses silica gel Column purification (mobile phase MeOH/DCM) obtains product 527 (1.62 g, 100% yield).MS ESI m/z C₁₉H₃₀N₃O₇[M+H ]⁺Calculated value: 412.20, measured value 412.20.

The synthesis of 194. compound 528 of embodiment

Compound 527 (0.24g, 0.58mmol) is dissolved in 10mL ethyl acetate, addition Pentafluorophenol (0.21g, 1.17mmol) and DCC (0.24g, 1.17mmol).It is stirred overnight at room temperature after after adding, filters and filtrate is concentrated and obtain product This compound is dissolved in DMF by (32mg, 0.056mmol), thereto be added compound 525 (50mg, 0.056mmol) and DIPEA(29μL,0.168mmol).Room temperature reaction 2 hours after after adding, after concentration with prepare HPLC purify (mobile phase: acetonitrile/ Water), obtain product 528 (3mg, 4% yield).MS ESI m/z C₆₃H₉₉N₁₀O₁₇S[M+H]⁺Calculated value: 1299.68, actual measurement Value 1299.68.

The synthesis of 195. compound 529 of embodiment

Compound 528 (3mg, 0.002mmol) is dissolved in 0.5mL DCM, 1mL trifluoroacetic acid is added.Add rear chamber Temperature reaction 1 hour is concentrated and is purified (mobile phase: acetonitrile/water) with preparation HPLC, and obtaining product 529, (1.43mg, 52% produces Rate).MS ESI m/z C₅₈H₉₁N₁₀O₁₅S[M+H]⁺Calculated value: 1199.63, measured value 1199.62.

The synthesis of 196. compound 532 of embodiment

The phenyl-pentafluoride phenolic ester of compound 527 (0.11g, 0.19mmol) is dissolved in 1mL DMF, compound 517 is added (0.21g, 0.25mmol) and DIPEA (86uL, 0.5mmol).It reacts at room temperature 2 hours and is concentrated after after adding, with preparation HPLC Purifying (mobile phase: acetonitrile/water) obtains product 532 (20mg, 9% yield).MS ESI m/z calculated value C₆₀H₉₃N₁₀O₁₅S[M+ H]⁺: 1225.65, measured value 1225.66.

The synthesis of 197. compound 533 of embodiment

Compound 532 (20mg, 0.016mmol) is dissolved in 1mL methylene chloride, 2mL trifluoroacetic acid is added, is added rear Room temperature reaction 1 hour.Reaction solution be concentrated and with preparation HPLC purify (mobile phase: acetonitrile/water) obtain compound 533 (8.9mg, 18% yield).MS ESI m/z C₅₅H₈₅N₁₀O₁₃S [M+H]⁺Calculated value: 1125.59, measured value 1125.59.

The synthesis of 198. compound 536 of embodiment

Compound 3- [[tertbutyloxycarbonyl] amino]-l-Alanine (1.00g, 7.3mmol) is dissolved in 30mL ethyl alcohol, Compound 125 (2.00g, 7.3mmol) and 0.1M sodium dihydrogen phosphate (6mL) is added.It is stirred overnight at room temperature after adding, is used after concentration Silica gel column purification (mobile phase: MeOH/DCM) obtains product 536 (1.41g, 78% yield).MS ESI m/z C₁₆H₂₄N₃O₇ [M+H]⁺Calculated value: 370.15, measured value 370.15.

The synthesis of 199. compound 537 of embodiment

Compound 536 (1.41g, 3.8mmol) is dissolved in the methylene chloride of 2mL, room temperature is anti-after 5mL trifluoroacetic acid is added It answers 1 hour.Reaction solution is concentrated to get product 537.MS ESI m/z C₁₁H₁₆N₃O₅ [M+H]⁺Calculated value: 270.10, measured value 270.10。

The synthesis of 200. compound 538 of embodiment

Compound 537 is dissolved in 20mL ethyl alcohol, compound 125 (1.90g, 6.9mmol) and 0.1M phosphoric acid is then added Sodium dihydrogen (4mL), is stirred overnight at room temperature after adding.Purifying (mobile phase: acetonitrile/water) with preparation HPLC after reaction solution concentration must To product 538 (0.45g, 22% yield).MS ESI m/z C₁₉H₂₃N₄O₈[M+H]⁺Calculated value: 435.14, measured value 435.14。

The synthesis of 201. compound 539 of embodiment

Compound 538 (0.15g, 0.34mmol) and compound 387 (0.17g, 0.34mmol) are dissolved in DMF (2mL) In, HATU (0.16g, 0.41mmol) and triethylamine (95 μ L, 0.68mmol) are added thereto, it is small that 1 is reacted at room temperature after adding When.Reaction solution concentration after with prepare HPLC purify (mobile phase: acetonitrile/water) obtain product 539 (34mg, 11% yield).MS ESI m/z C₄₄H₆₂N₇O₁₃[M+H]⁺Calculated value: 896.43, measured value 896.42.

The synthesis of 202. compound 540 of embodiment

Compound 539 (34mg, 0.04mmol) is dissolved in 0.5mL methylene chloride, after 1mL trifluoroacetic acid is added, room temperature Reaction 2 hours.Compound 540 is obtained after reaction solution concentration.MS ESI m/z C₃₅H₄₆N₇O₁₁[M+H]⁺Calculated value: 740.30, it is real Measured value 740.32.

The synthesis of 203. compound 541 of embodiment

Compound 540 is dissolved in DMA (2mL), compound 41a (28mg, 0.04 mmol) and DIPEA are added thereto (21 μ L, 0.12mmol).It is stirred overnight after adding, is purified to obtain product 541 (14.4mg, 29% production with preparation HPLC after concentration Rate).MS ESI m/z C₆₀H₈₆N₁₁O₁₆S[M+H]⁺Calculated value: 1248.59, measured value 1248.60.

The synthesis of 204. compound 544 of embodiment

By compound 132 (0.300g, 0.329mmol) and N-BOC- ethylenediamine-hydrochloride (0.063g, 0.395 mmol) It is dissolved in methylene chloride (30mL), EDCI (0.189g, 0.988mmol) is added thereto at 0 DEG C, is warmed to room temperature after ten minutes, It is stirred overnight.After reaction solution is diluted with methylene chloride, washing, salt washing, sodium sulphate are dry, are concentrated and with silica gel column purification, obtain To product (0.132g, 54% yield).MS ESI m/z C₅₂H₈₀N₉O₁₂S[M+H]⁺Calculated value: 1054.6, measured value: 1054.6。

The synthesis of 205. compound 545 of embodiment

Compound 544 (0.132g, 0.125mmol) is dissolved in methylene chloride (45mL), 1.5mL trifluoroacetic acid, room is added Temperature reaction 1 hour.Purify to obtain product 545 (111 mg, 93% yield) with preparation HPLC after reaction solution concentration.MS ESI m/z C₄₇H₇₂N₉O₁₀S[M+H]⁺Calculated value: 954.5, measured value: 954.5.

The synthesis of 206. compound 548 of embodiment

By compound 132 (0.050g, 0.0549mmol) and N-Boc-1,11- diamino -3,6,9- trioxaundecane (0.024g, 0.0549mmol) is dissolved in methylene chloride (10mL), be added thereto at 0 DEG C EDCI (0.032g, 0.1647mmol), reaction after ten minutes, is warmed to room temperature, is stirred overnight.Reaction solution is washed after being diluted with methylene chloride, salt is washed, Sodium sulphate drying, filtering and concentrating, obtain product 548 (0.030g, 46% yield) with silica gel column purification.MS ESI m/z C₅₈H₉₂N₉O₁₅S[M+H]⁺Calculated value: 1186.6, measured value: 1186.6.

The synthesis of 207. compound 549 of embodiment

Compound 548 (0.030g, 0.0253mmol) is dissolved in 3mL methylene chloride, 1mL trifluoroacetic acid, room temperature is added Reaction 1 hour.Yellow oily liquid is obtained after concentration, purifies to obtain product 549 (11.7mg, 43% yield) with preparation HPLC. MS ESI m/z C₅₃H₈₄N₉O₁₃S[M+H]⁺Calculated value: 1086.6, measured value: 1086.6.

The synthesis of 208. compound 552 of embodiment

Compound diethyl triamine (28.7g, 275mmol) and DMAP (0.034g, 0.000275 mmol) are dissolved in dichloro Methane (350mL), is added Boc at 0 DEG C₂O(6.0g,0.0275mmol).It is stirred overnight at room temperature after adding, concentration of reaction solution, And product (45g, 80% yield) is obtained with silica gel column purification.MS ESI m/z C₉H₂₂N₃O₂[M+H]⁺Calculated value: 204.2, it is real Measured value: 204.2.

The synthesis of 209. compound 553 of embodiment

By compound 132 (0.060g, 0.0658mmol) and N-Boc-2,2 '-imino-diacetic ethamine (0.016 g, It 0.0790mmol) is dissolved in methylene chloride (6mL), EDCI (0.038g, 0.1974 mmol) is added at 0 DEG C, rises to room after adding Temperature is stirred overnight.Purify to obtain product 553 (48mg, 66% yield) with preparation HPLC after reaction solution concentration.MS ESI m/z C₅₄H₈₅N₁₀O₁₂S[M+H]⁺Calculated value: 1097.6, measured value: 1097.6.

The synthesis of 210. compound 554 of embodiment

Compound 553 (0.048g, 0.0437mmol) is dissolved in 3mL methylene chloride, after 1mL trifluoroacetic acid is added, room Temperature reaction 1 hour.Purify to obtain product 554 (11.1 mg, 25% yield) with preparation HPLC after reaction solution concentration.MS ESI m/ z C₄₉H₇₇N₁₀O₁₀S[M+H]⁺Calculated value: 997.5, measured value: 997.5.

The synthesis of 211. compound 558 of embodiment

By compound 132 (0.400g, 0.439mmol) and H-Lys (Boc)-O^tBu·HCl(0.135g,0.528 Mmol it) is dissolved in methylene chloride (40mL), EDCI (0.189g, 1.317mmol) is added at 0 DEG C, is warmed to room temperature, stirs after adding It mixes overnight.After reaction solution is diluted with methylene chloride, washing, salt washing, sodium sulphate is dry, filtering, is concentrated and with silica gel column purification Obtain product 558 (0.43g, 82% yield).MS ESI m/z C₆₀H₉₄N₉O₁₄S[M+H]⁺Calculated value: 1196.7, measured value: 1196.7。

The synthesis of 212. compound 559 of embodiment

Compound 558 (0.230g, 0.192mmol) is dissolved in methylene chloride (6mL), is added trifluoroacetic acid (2mL), room Temperature reaction 3 hours purifies (C with preparing HPLC after concentration₁₈Column, mobile phase A: water, Mobile phase B: acetonitrile；60 minutes from 10% B B to 80%) obtain product 559 (153mg, 76% yield).MS ESI m/z C₅₁H₇₈N₉O₁₂S[M+H]⁺Calculated value: 1040.5 measured value: 1040.5.

The synthesis of 213. compound 562 of embodiment

By two t-butoxycarbonyl amino caproic acid of compound 558 (0.200g, 0.167mmol) and (S) -2,6- (0.070g, 0.200mmol) be dissolved in methylene chloride (10mL), be added at 0 DEG C HATU (0.095g, 0.250mmol) and triethylamine (46 μ L, 0.334mmol).Reaction solution is warmed to room temperature, and is stirred overnight, wash, salt washing, sodium sulphate drying, filter after methylene chloride dilution, It is concentrated and is used silica gel column purification, obtains product 562 (0.270g, theoretical yield).MS ESI m/z C₇₆H₁₂₂N₁₁O₁₉S[M+H]⁺ Calculated value: 1524.9, measured value: 1524.9.

The synthesis of 214. compound 563 of embodiment

Compound 562 (0.27g, 0.177mmol) is dissolved in methylene chloride (6mL), is added trifluoroacetic acid (2mL), room temperature Reaction 4 hours.Purify (C with preparing HPLC after reaction solution concentration₁₈Column, mobile phase A: water, Mobile phase B: acetonitrile；60 minutes from The B of 10% B to 80%) obtain product 563 (172mg, 83% yield).MS ESI m/z C₅₇H₉₀N₁₁O₁₃S[M+H]⁺It calculates Value: 1168.6, measured value: 1168.6.

The synthesis of 215. compound 566 of embodiment

Ethylenediamine (3.0g, 0.5mmol) is dissolved in methylene chloride (500mL), 0 DEG C is cooled to, CbzCl is then added (8.53g, 0.050mol) is warmed to room temperature after adding, is stirred overnight.Reaction solution washing, salt washing, sodium sulphate is dry, filters simultaneously It is concentrated to get N- benzyloxycarbonyl group -1,2- diaminoethanes (7.0g, 94% yield) .MS ESI m/z C₁₀H₁₄N₂O₂[M+H]⁺Meter Calculation value: 195.1, measured value: 195.2.

The synthesis of 216. compound 567 of embodiment

Compound 566 (7g, 35.8mmol) and 37% formaldehyde (14mL, 0.1772mmol) are dissolved in methanol (120mL), Sodium cyanoborohydride (3.9g, 0.062mol) is added at 0 DEG C, adjusts pH to 7 with acetic acid (3mL), is warmed to room temperature, stirs after adding It mixes overnight.Reaction solution is diluted with methylene chloride (200mL), and washing, salt washing, sodium sulphate is dry, filters, is concentrated and uses silica gel Column purification obtains product yellow oil (6.4g, 80% yield).MS ESI m/z C₁₂H₁₈N₂O₂[M+H]⁺Calculated value: 224.1, measured value: 224.1.

The synthesis of 217. compound 568 of embodiment

Compound 567 (3.0g, 13.4mmol) is dissolved in methanol (100mL), then be added Pd/C (0.3g, 10wt%) with HCl (3mL), reacted 5 hours under the hydrogen of 100psi pressure after adding.React the filtering of fluid cushion diatomite, concentration Obtain product 568 (2.1g, 98% yield).¹H NMR(400MHz, D₂O) δ 3.33 (d, J=4.6Hz, 2H), 3.27 (s, 2H), 2.79(s,6H)。

The synthesis of 218. compound 569 of embodiment

Compound 103 (0.58g, 1.58mmol) and compound 568 (0.051g, 3.15mmol) are dissolved in anhydrous DMF In (10mL), HATU (0.09g, 2.37mmol) and triethylamine (0.656mL, 4.74mmol) are added at 0 DEG C.It is risen to after adding Room temperature is reacted 90 minutes.Reaction is diluted with water, and (3 × 100mL) is extracted with ethyl acetate, and merges organic phase, washing, salt water It washes, sodium sulphate is dry, filters and is concentrated to get yellow solid (0.67g, 97% yield).MS ESI m/z C₂₁H₃₅N₄O₆[M+H ]⁺Calculated value: 439.2, measured value: 439.2.

The synthesis of 219. compound 570 of embodiment

The ethyl acetate (10mL) that Pd/C (0.2g, 10wt%) is added to compound 569 (0.6g, 13.7mmol) is molten In liquid, reacted 4 hours under the Hydrogen Vapor Pressure of 100psi.Reaction fluid cushion diatomite filtering, be concentrated to get product (5.5g, 98% Yield).MS ESI m/z C₂₁H₃₇N₄O₆₄[M+H]⁺Calculated value: 409.3, measured value: 409.3.

The synthesis of 220. compound 571 of embodiment

Compound 570 (0.5g, 1.22mmol) is dissolved in 95% ethyl alcohol (10mL), 0.1M NaH is added₂PO₄(2mL) and Compound 125 (0.683g, 2.44mmol), is stirred overnight after adding.Reaction solution be concentrated and use silica gel column purification (mobile phase: MeOH/DCM product yellow oil (0.624 g, 89% yield)) is obtained.MS ESI m/z calculated value C₂₉H₄₄N₅O₇[M+H]⁺: 574.3, measured value: 574.3.

The synthesis of 221. compound 572 of embodiment

Compound 571 (0.2g, 0.349mmol) is dissolved in 6mL methylene chloride, after the trifluoroacetic acid of 2mL is added, room temperature Reaction 2 hours.Reaction solution is concentrated to get product (165mg, theoretical yield).MS ESI m/z C₂₄H₃₆N₅O₅[M+H]⁺Calculated value: 474.3, measured value: 474.3.

The synthesis of 222. compound 573 of embodiment

Compound 572 (0.165g, 0.349mmol) is dissolved in 2mL DMF, compound 41a is added at 0 DEG C DMF (3mL) solution of (0.29g, 1.047mmol), is warmed to room temperature after adding, and reacts 1 hour.Reaction solution is concentrated and uses preparation HPLC purifies (C₁₈Column, mobile phase A: water, Mobile phase B: acetonitrile；60 minutes from 10% B to 80% B) obtain product (58mg, 17% yield).MS ESI m/z C₄₉H₇₆N₉O₁₀S[M+H]⁺Calculated value: 982.5, measured value: 982.5.

The synthesis of 223. compound 576 of embodiment

2- bromo acid (3g, 17.9mmol) is dissolved in tetrahydrofuran (30mL), trimethylamine (1M is added thereto In THF, 17.9mL, 35.9mmol).It is stirred overnight after adding, ethyl acetate is added, solid is collected by filtration, obtains product 576 (4g, theoretical yield).MS ESI m/z C₇H₁₆NO₂ [M+H]⁺Calculated value: 146, measured value 146.

The synthesis of 224. compound 577 of embodiment

Compound 576 (1.55g, 6.9mmol) and Pentafluorophenol (2.5g, 13.8mmol) are dissolved in methylene chloride (20mL) In, it is added DCC (2.8g, 13.8mmol), is stirred overnight after adding.Reaction solution filters and is concentrated to get oil product 577.MS ESI m/z C₁₃H₁₅F₅NO₂[M+H]⁺Calculated value: 312, measured value 312.

The synthesis of 225. compound 578 of embodiment

Compound 16 (2g, 3.7mmol) is dissolved in ethyl acetate (20mL), after Pd/C (10wt%, 0.20g) is added, In H₂It is stirred overnight at room temperature under (1atm), product 17 (1.78g, 3.4mmol) is obtained by filtration.By compound 17 and compound 577 (6.9mmol) is dissolved in DMF (20mL), and DIPEA (1.8mL, 10.4mmol) is added at 0 DEG C, room temperature reaction is warming up to after adding 1 hour.Reaction solution be concentrated and use silica gel column purification (1:100 to 1:5MeOH/DCM), obtain product 578 (1.2g, 54% produce Rate).MS ESI m/z C₃₂H₆₁N₄O₅SSi[M+H]⁺Calculated value: 642, measured value 642.

The synthesis of 226. compound 579 of embodiment

Compound 578 (1.20g, 1.86mmol) is dissolved in AcOH/THF/H₂In O (volume ratio 3:1:1,20 mL), room temperature It is stirred overnight.Reaction solution is concentrated to get foaming solid (1.20g, 54% yield). MS ESI m/z C₂₆H₄₇N₄O₅S[M+H]⁺ Calculated value: 527, measured value 527.

The synthesis of 227. compound 580 of embodiment

Compound 579 (1.86mmol) is dissolved in Isosorbide-5-Nitrae-dioxane (10mL), 1N sodium hydroxide is added thereto (9.3mL) is reacted at room temperature 2 hours.1N hydrochloric acid tune pH to 4 is used after reaction solution concentration, is concentrated to get white solid product again 580。MS ESI m/z C₂₄H₄₃N₄O₅S[M+H]⁺Calculated value: 499, measured value 499.

The synthesis of 228. compound 581 of embodiment

Compound 580 (1.86mmol) is dissolved in pyridine (10mL), at 0 DEG C be added acetic anhydride (884 μ L, 9.36mmol), reaction is warming up to room temperature, is stirred overnight.After reaction solution is diluted with water (20mL), washed with ethyl acetate (3 × 10mL), water phase is concentrated to get yellow solid product 581.MS ESI m/z C₂₆H₄₅N₄O₆S[M+H]⁺Calculated value: 541, measured value 541。

The synthesis of 229. compound 582 of embodiment

Compound 581 (150mg, 0.277mmol) and Pentafluorophenol (76.5mg, 0.415mmol) are dissolved in methylene chloride In (2mL), after EDCI (63.7mg, 0.33mmol) is added, react at room temperature 3 hours.Reaction solution is concentrated to get yellow liquid 582. MS ESI m/z C₃₂H₄₄F₅N₄O₆S[M+H]⁺Calculated value: 707, measured value 707.

The synthesis of 230. compound 583 of embodiment

Compound 127 (50mg, 0.10mmol) and compound 582 (0.14mmol) are dissolved in DMF (2mL), at 0 DEG C Lower addition DIPEA (49 μ L, 0.28mmol), reacts at room temperature 1 hour after adding.Reaction solution is concentrated and is purified with preparation HPLC, obtains To white solid product 583 (30mg, 46% yield).MS ESI m/z C₄₆H₆₈N₇O₁₁S[M+H]⁺Calculated value: 926, measured value 926。

The synthesis of 231. compound 573 of embodiment

Thionyl chloride (10mL) suspension of glycine betaine (870mg, 7.4mmol) is heated to 70 DEG C to react 2 hours.Reaction Liquid is concentrated and uses toluene (3 × 10mL) azeotropic, obtains yellow solid 586.

The synthesis of 232. compound 587 of embodiment

Compound 17 (1.90g, 3.71mmol) is dissolved in methylene chloride (20mL), addition DIPEA (2.58mL, 14.8mmol), it is cooled to 0 DEG C, methylene chloride (20mL) solution of compound 586 is added dropwise, it is small that reaction 1 is warmed to room temperature after adding When.Reaction solution is concentrated and uses silica gel column purification (1:100 to 1:5MeOH/DCM) obtains product 587 (2.3g, theoretical yield). MS ESI m/z C₃₀H₅₇N₄O₅SSi [M+H]⁺Calculated value: 613, measured value 613.

The synthesis of 233. compound 588 of embodiment

Compound 587 (2.3g, 3.7mmol) is dissolved in AcOH/THF/H2O (volume ratio 3:1:1,40mL), it is stirred Night.After reaction solution concentration, compound 588 is obtained.MS ESI m/z C₂₄H₄₃N₄O₅S [M+H]⁺Calculated value: 499, measured value 499。

The synthesis of 234. compound 589 of embodiment

Compound 588 (3.7mmol) is dissolved in Isosorbide-5-Nitrae-dioxane (20mL), 1N sodium hydroxide is added thereto (18.5mL) is reacted at room temperature 2 hours.After reaction solution concentration, 1N hydrochloric acid tune pH to 4 is added, is concentrated to give white solid product again 589 (1.00g, 57% yields).MS ESI m/z C₂₂H₃₉N₄O₅S [M+H]⁺Calculated value: 471, measured value 471.

The synthesis of 235. compound 590 of embodiment

Compound 589 (1.00g, 2.12mmol) is dissolved in pyridine (10mL), at 0 DEG C be added acetic anhydride (1mL, 10.6mmol), reaction is warmed to room temperature, and is stirred overnight.(3 × 10mL) is washed with ethyl acetate after reaction solution 20mL water dilution, Concentration water phase obtains yellow solid product 590.MS ESI m/z C₂₄H₄₁N₄O₆S[M+H]⁺Calculated value: 513, measured value 513.

The synthesis of 236. compound 591 of embodiment

Compound 590 (70mg, 0.136mmol) and Pentafluorophenol (30mg, 0.163mmol) are dissolved in methylene chloride In (2mL), it is added DCC (33.7mg, 0.163mmol), is reacted 3 hours after adding.Yellow oily production is obtained after reaction solution concentration Object 591 is directly used in and reacts in next step.MS ESI m/z C₃₀H₄₀F₅N₄O₆S[M+H]⁺Calculated value: 679, measured value 679.

The synthesis of 237. compound 592 of embodiment

Compound 591 (0.136mmol) and compound 127 (0.11g, 0.273mmol) are dissolved in DMF (2mL), at 0 DEG C Lower addition DIPEA (71 μ L, 0.408mmol).Reaction is warmed to room temperature, and is concentrated after carrying out 1 hour, and is purified with preparation HPLC To yellow solid product (30.9mg, 25% yield). MS ESI m/z C₄₄H₆₄N₇O₁₁S[M+H]⁺Calculated value: 899, measured value 899。

The synthesis of 238. compound 603 of embodiment

By compound N '-(2- aminoethyl) ethyl -1,2- diamines (4.2g, 41mmol) is dissolved in 50mL tetrahydrofuran, and it is cold But to 0 DEG C, it is slowly added into the tetrahydrofuran of 2- (tert-Butoxycarbonyloxyimino) -2- benzene acetonitrile (20.4g, 82 mmol) (50mL) solution reacts 1 hour at 0 DEG C again after adding.After concentration of reaction solution, with silica gel column purification (0-10%MeOH/DCM), Obtain colorless oil (12g, 96% yield). HRMS(ESI)m/z C₁₄H₂₉N₃O₄[M+H]⁺Calculated value: 304.22, actual measurement Value: 304.22.

The synthesis of 239. compound 604 of embodiment

Compound 603 (5g, 16.4mmol) is dissolved in methylene chloride (50mL), after being cooled to 0 DEG C, succinic anhydride is added (1.64g, 16.4mmol) is added and is warmed to room temperature reaction 1 hour.Concentration of reaction solution, with silica gel column purification (0-10%MeOH/ DCM product (5.4g, 81% yield)) is obtained.HRMS (ESI)m/z C₁₈H₃₃N₃O₇[M+H]⁺Calculated value: 404.23, measured value: 404.23.

The synthesis of 240. compound 605 of embodiment

Compound 604 (5.4g, 13.3mmol) is dissolved in ethyl acetate (30mL), concentrated hydrochloric acid (10mL) is added at 0 DEG C, It removes ice bath and is vigorously stirred reaction 30 minutes.Vacuum distillation removes ethyl acetate and hydrochloric acid, and obtaining product, (3.69g, 100% produces Rate).HRMS(ESI)m/z C₈H₁₇N₃O₃[M+H]⁺Calculated value: 204.13, measured value: 204.13.

The synthesis of 241. compound 606 of embodiment

Compound 605 (0.5g, 1.81mmol) is dissolved in 5mL water, 2N sodium hydrate aqueous solution is added, adjusts pH to 7, it is dense It is reduced to dry, adds the saturated solution of sodium bicarbonate of 10mL, be cooled to 5 DEG C or so, N- methoxycarbonyl maleoyl- is added Imines (0.56g, 3.62mmol), reacts 2 hours at 5 DEG C after adding.With 2N HCl tune pH to 5, water phase is extracted with methylene chloride It takes (3 × 10mL), is dried, filtered after merging organic phase with sodium sulphate, is concentrated and purifies to obtain white solid with preparation HPLC (200mg, 30% yield).HRMS(ESI)m/z C₁₆H₁₇N₃O₇[M+H]⁺Calculated value: 364.11, measured value: 364.11.

The synthesis of 242. compound 608 of embodiment

Compound 606 (37mg, 0.102mmol) is dissolved in DMF (2mL), at 0 DEG C be added HATU (39mg, 0.102mmol), add be warmed to room temperature reaction 1 hour, then by above-mentioned solution be added to compound 440 (100mg, 0.093mmol) and in DMF (2mL) solution of triethylamine (25.8 μ L, 0.186mmol), react at room temperature 1 hour.DMF is fallen in concentration Afterwards, product (99mg, 75% yield) is obtained with silica gel column purification.HRMS(ESI)m/z C₆₈H₁₀₈N₈O₂₄[M+H]⁺Calculated value: 1421.75 measured value: 1421.75.

The synthesis of 243. compound 609 of embodiment

Compound 608 (50mg, 0.035mmol) is dissolved in methylene chloride (3mL), after TFA (1mL) is added, room temperature is anti- It answers 2 hours.It is concentrated to get product (44mg, 100% yield).HRMS(ESI) m/z C₅₉H₉₂N₈O₂₂[M+H]⁺Calculated value: 1265.63 measured value: 1265.63.

The synthesis of 244. compound 670 of embodiment

Compound 609 (20mg, 0.0158mmol) and compound 41a (10.9mg, 0.0158mmol) are dissolved in DMA In (2mL), DIPEA (5.5 μ L, 0.0316mmol) is added at 0 DEG C, adds and is warming up to room temperature reaction 1 hour.After DMA is fallen in concentration, Purify to obtain product (11mg, 40% yield) with preparation HPLC.HRMS(ESI)m/z C₈₄H₁₃₂N₁₂O₂₇S[M+H]⁺Calculated value: 1773.90 measured value: 1773.90.

The synthesis of 245. compound 672 of embodiment

The 4-Aminobutanoicacid tert-butyl ester (1.03g, 6.12mmol) and compound 436 (4.16g, 5.56mmol) are dissolved in DMF In (18mL), HATU (2.32g, 6.12mmol) and TEA (1.2mL, 8.34mmol) are added at 0 DEG C, adds after stirring 50 minutes Water (300mL), is extracted with ethyl acetate (250mL × 3), merges organic phase, saturated common salt washing, anhydrous sodium sulfate is dry, mistake Filter, concentration cross column purification (3% MeOH/DCM), obtain compound 672 (5.98g).[M+H]⁺Calculated value 890.51, measured value 891.09.

The synthesis of 246. compound 673 of embodiment

Compound 672 (5.98g, 6.73mmol) is dissolved in methanol (30mL), is added Pd/C (10wt%, 0.6g), Under 1atm hydrogen, it is stirred overnight at room temperature.Filtering and concentrating obtains compound 673 (4.57g).ESI:m/z: C₃₅H₇₀N₃O₁₄[M+H]⁺Meter Calculation value: 756.48, measured value 756.47.

The synthesis of 247. compound 674 of embodiment

Compound 673 (1g, 1.32mmol) is dissolved in NaHCO₃In saturated solution (20mL), compound is added under ice bath 418 (0.4g, 2.64mmol) are reacted under ice bath and are stirred 30 minutes.Then above-mentioned reaction solution is poured into equipped with 100mL acetic acid second In the separatory funnel of ester, organic phase is collected in liquid separation, then organic phase successively respectively washed once with the water of 50mL and 50mL saline solution, Merge organic phase, dried, filtered with anhydrous sodium sulfate, is concentrated, obtains compound 674 (0.8g, yield 72%).ESI:m/z: C₃₉H₇₀N₃O₁₆[M+H]⁺Calculated value: 836.47, measured value 836.47

The synthesis of 248. compound 675 of embodiment

After compound 674 (0.40g, 0.48mmol) is dissolved in DCM (4.5mL), it is added TFA (1.5mL), stirs at room temperature After mixing 1 hour, concentration is spin-dried for, and is finally placed on oil pump and is drained twice with methylene chloride band is dry, obtained residue and chemical combination Object 110 (0.35g, 0.48mmol) is dissolved in DMF (10mL), and HATU (0.36g, 0.96mmol) is added under ice bath, TEA (0.15mL, 1.44mmol), it then stirs 30 minutes.Then above-mentioned reaction solution is poured into the separatory funnel equipped with 100mL water, with acetic acid second Ester extracts (50mL × 2), merges organic phase, is dried, filtered with anhydrous sodium sulfate, is concentrated, and crosses silicagel column (0-10% MeOH/ DCM), compound 675 (21mg, 2.1%) is obtained.ESI:m/z:C₉₁H₁₅₃N₈O₃₅[M+H]⁺Calculated value: 1919.04, measured value 1919.04.

The synthesis of 249. compound 676 of embodiment

After compound 675 (21mg, 0.01mmol) is dissolved in DCM (0.5mL), it is added TFA (1mL), stirs 1 at room temperature After hour, concentration is spin-dried for, and is finally placed on oil pump and is drained twice with methylene chloride band is dry, obtained compound and compound 41a (6.93g, 0.01mmol) is dissolved in DMA (2mL), and DIPEA (17 μ L, 0.1mmol) then is added, and stirs 30 minutes at room temperature. The direct loading of above-mentioned reaction solution is crossed into preparation HPLC, eluting solvent MeCN/H₂O, it is purified obtain product 676 (10mg, 44%).ESI: m/z:C₁₀₇H₁₇₇N₁₂O₃₈S[M+H]⁺Calculated value: 2271.20, measured value 2271.20.

The synthesis of 250. compound 678 of embodiment

Under nitrogen protection, NaH (3.1g, 129.15mmol) is dissolved in DMF (50mL), di-t-butyl is added thereto DMF (30mL) solution of nitrine oxalic acid (10g, 43.05mmol), is stirred after twenty minutes at room temperature, and bromoacetic acid uncle is added under ice bath DMF (20mL) solution of butyl ester (28mL, 172.2mmol), stirring are slowly warmed to room temperature after twenty minutes, are stirred 1 hour.It will be upper It states reaction solution and pours into NH₄It in Cl (aq) (400mL), is extracted with ethyl acetate (300mL), merges organic phase, be washed with water (100mL × 3), saturated salt solution (100mL) are washed, and anhydrous magnesium sulfate dries, filters, concentration.Concentrate is washed through silica gel column purification Desolventizing is petrol ether/ethyl acetate, obtains compound 678 (16.5g, 83%).ESI:m/z: calculated value C₂₂H₄₁N₂O₈[M+H ]⁺: 461.28, measured value 461.27.

The synthesis of 251. compound 679 of embodiment

Compound 678 (10g, 21.7mmol) is dissolved in TFA (50mL), is stirred overnight at room temperature, is concentrated, and use dichloro Methane band is dry twice, and the solid being collected into finally is placed in through methylene chloride washing until solid is white by resulting solid It is drained on oil pump, obtains compound 679 (3g, 94%).ESI:m/z: C₄H₉N₂O₄[M+H]⁺Calculated value: 149.05, measured value 148.06.

The synthesis of 252. compound 680 of embodiment

Compound 679 (0.56g, 3.8mmol) is dissolved in THF (20mL), under ice bath be added chloracetyl chloride (3mL, 38mmol), it after stirring 30 minutes, is slowly warmed to room temperature and is stirred overnight.Above-mentioned reaction solution is concentrated to dryness, and does three with toluene band Time, finally compound 680 (1.16g, 100%) is drained to obtain with oil pump. ESI:m/z:C₈H₁₁Cl₂N₂O₆[M+H]⁺Calculated value: 300.99 measured value 301.00.

The synthesis of 253. compound 681 of embodiment

By compound L-alanine tert-butyl ester (2.6g, 14.35mmol), L-Z-Gly-O^tBu (3g, 14.35mmol) is molten HATU (6g, 15.78mmol) is added under DMF (15mL), ice bath, TEA (4.1mL, 28.7mmol), stirring rises to after five minutes 1h is stirred at room temperature.Above-mentioned reaction solution is poured into the separatory funnel equipped with 300mL water, ethyl acetate (100mL) extraction, collection has Machine phase, water phase are extracted with ethyl acetate (100mL), merge organic phase, then washed once with saturated salt solution (100mL), anhydrous sulphur Sour sodium dries, filters, and concentration obtains compound 681 (7.8g).ESI:m/z:C₁₇H₂₅N₂O₅[M+H]⁺Calculated value: 337.17, it is real Measured value 338.19.

The synthesis of 254. compound 682 of embodiment

It after compound 681 (3g, 8.9mmol) is dissolved in DCM (3mL), is added TFA (3mL), stirs 3h at room temperature, be concentrated, And it is dry twice with methylene chloride band, it is finally placed on oil pump and drains, obtain compound 682 (2.5g, 8.9mmol).ESI:m/z: C₁₃H₁₇N₂O₅[M+H]⁺Calculated value: 281.11, measured value 281.12.

The synthesis of 255. compound 683 of embodiment

By compound 682 (2.5g, 8.9mmol), the 3- alanine tert-butyl ester (1.6g, 8.9mmol) is dissolved in DMF HATU (3.4g, 8.9mmol) is added under ice bath in (10mL), TEA (2.4mL, 17.8mmol), and stirring is warmed to room temperature after five minutes stirs Mix 1h.Above-mentioned reaction solution to be poured into the separatory funnel equipped with 300mL water, organic phase is collected in ethyl acetate (100mL) extraction, Water phase is extracted with ethyl acetate (100mL), merges organic phase, then washed once with saturated salt solution (300mL), and anhydrous sodium sulfate is dry It is dry, it filters, concentration, crosses silicagel column (0-10%MeOH/DCM) and purify to obtain compound 683 (2.16g, 60%).ESI:m/z: C₂₀H₃₀N₃O₆[M+H]⁺Calculated value: 408.21, measured value 408.21.

The synthesis of 256. compound 684 of embodiment

Compound 683 (2.16g, 5.31mmol) is dissolved in methanol (20mL), is added Pd/C (0.2g, 10 wt%), instead Should be placed under hydrogen (1atm) stir 50 minutes, filtered through diatomite, by filtrate collect be concentrated, obtain crude product 684 (1.4g, 5.31mmol)。ESI:m/z:C₁₂H₂₄N₃O₄[M+H]⁺Calculated value: 274.17, measured value 274.17.

The synthesis of 257. compound 685 of embodiment

By compound 684 (1.4g, 5.31mmol), compound 680 (0.5g, 1.66mmol) is dissolved in DMF (30mL), ice Bath is lower to be added Brop (1.9g, 4.98mmol), and DIPEA (0.6mL, 3.32mmol), stirring is slowly warmed to room temperature after ten minutes, It is stirred overnight.Above-mentioned reaction solution is poured into the separatory funnel equipped with 300mL water, ethyl acetate (100mL) extraction is collected organic Phase, water phase are extracted with ethyl acetate (100mL), merge organic phase, and anhydrous sodium sulfate dries, filters, and are concentrated, and silicagel column (0- is crossed It 10%MeOH/DCM) purifies, obtains compound 685 (0.37g, 28%).ESI:m/z:C₃₂H₅₃Cl₂N₈O₁₂[M+H]⁺Calculated value: 811.31 measured value 811.31.

The synthesis of 258. compound 686 of embodiment

After compound 685 (0.37g, 0.46mmol) is dissolved in DCM (1mL), it is added TFA (3mL), stirs 1h at room temperature Afterwards, be spin-dried for being concentrated, and dry twice with methylene chloride band, be finally placed on oil pump and drain, by obtained compound (0.32g, 0.46mmol) be dissolved in DMF (20mL) with compound 110 (0.18g, 0.46mmol), be added under ice bath HATU (0.35g, 0.92mmol), TEA (0.13mL, 0.92mmol) is stirred 30 minutes.Above-mentioned reaction solution is poured into the leakage of the liquid separation equipped with 300mL water In bucket, organic phase is collected in ethyl acetate (100mL) extraction, and water phase is extracted with ethyl acetate (100mL), merges organic phase, is satisfied It is washed once with saline solution (100mL), anhydrous sodium sulfate dries, filters, concentration, silicagel column (0-10% MeOH/DCM) purifying is crossed, It obtains compound 686 (0.16g, 33%).ESI:m/z:C₄₅H₆₇Cl₂N₁₀O₁₅ [M+H]⁺Calculated value: 1057.41, measured value 1057.40.

The synthesis of 259. compound 687 of embodiment

After compound 686 (0.16g, 0.15mmol) is dissolved in DCM (0.5mL), it is added TFA (3mL), stirs at room temperature It after 1h, is spin-dried for being concentrated, and dry twice with methylene chloride band, is finally placed on oil pump and drains, compound and compound 41a will be obtained (0.1g, 0.15mmol) is dissolved in DMA (3mL), and DIPEA (0.1mL, 0.6mmol) then is added, and is stirred 1 hour at room temperature.It will 1mL acetonitrile is added after above-mentioned reaction solution concentration, loading crosses preparation HPLC, eluting solvent MeCN/H₂O, purifying obtain product 687 (59mg, 28%).ESI:m/z:C₆₁H₉₁Cl₂N₁₄O₁₈S[M+H]⁺Calculated value: 1409.57, measured value 1409.57.

The synthesis of 260. compound 690 of embodiment

Compound 672 (5.98g, 6.73mmol) is dissolved in methanol (30mL), is added Pd/C (0.6g, 10 wt%), 1 Under the hydrogen of a atmospheric pressure, it is stirred overnight at room temperature.After being filtered by diatomite, be concentrated filtrate, by obtained compound (4.57g, 6.05mmol is dissolved in tetrahydrofuran (60mL), be added compound 689 (1.01g, 2.42mmol) and HOBt (817g, 6.05mmol), DCC (1.25g, 6.05mmol) and DIPEA (2.1mL, 12.10mmol) are added after being cooled to 0 DEG C, gradually rises It warms to room temperature, is stirred overnight.Add ethyl acetate (400mL) to dilute, is washed with saturated sodium bicarbonate, 1N HCl is washed, saturated salt solution It washes, anhydrous sodium sulfate dries, filters, concentration, column chromatographic purifying (4%MeOH/DCM), and obtaining compound 690, (5.65g, 49% receives Rate).[M+H]⁺Calculated value 1892.06, measured value 1892.83.

The synthesis of 261. compound 691 of embodiment

Compound 690 (3.71g, 1.96mmol) is dissolved in methanol (50mL), is added Pd/C (0.40g, 10 wt%), 1 Under the hydrogen of a atmospheric pressure, it is stirred overnight at room temperature.After being filtered by diatomite, be concentrated filtrate, obtain compound 691 (1.64g, 51% yield).[M+H]⁺Calculated value 1623.98, measured value 1624.42.

The synthesis of 262. compound 692 of embodiment

Compound 691 (315mg, 0.194mmol) is dissolved in ethyl alcohol (10mL), addition compound 125 (136mg, 0.485mmol) and 0.5M Na₂HPO₄(2.5mL) is stirred at room temperature 3 days, after concentration, purifies (40%MeCN/ by preparation HPLC H₂O).It obtains compound 692 (50mg, 13% yield).[M+H]⁺Calculated value 1954.07, measured value 1955.05.

The synthesis of 263. compound 693 of embodiment

Compound 692 (50mg, 0.114mmol) is dissolved in DCM (5mL), is added TFA (5mL), is stirred overnight at room temperature. Concentration of reaction solution adds toluene azeotropic distillation, and obtained compound and compound 110 (33mg, 0.0837mmol) are dissolved in DMF In (6mL), after HATU (64mg, 0.1674mmol) and TEA (46uL, 0.0.3348mmol) is added at 0 DEG C, stir 1 hour. Add water (100mL) to dilute, extracted with ethyl acetate (100mL × 3), merge organic phase, saturated common salt washing, anhydrous sodium sulfate is done It is dry, it filters, concentration, silica gel column chromatography purifies (15%MeOH/DCM), obtains compound 693 (98mg, 53% yield).[M+H]⁺Meter Calculation value 2200.17, measured value 2200.15.

The synthesis of 264. compound 694 of embodiment

Compound 693 (98mg, 0.045mmol) is dissolved in DCM (3mL), is added TFA (6mL), is stirred at room temperature 1 hour. Concentration of reaction solution adds toluene azeotropic distillation, is dissolved in DMA by obtained compound and with compound 41a (31mg, 0.045mmol) (1mL) is added DIPEA (12 μ L, 0.068mmol), is stirred at room temperature 90 minutes.Concentration of reaction solution is purified by preparation HPLC, It obtains compound 694 (33.56mg, 30% yield).[M/2+H]⁺Calculated value 1276.66, measured value 1276.65.

The synthesis of 265. compound 696 of embodiment

Compound 689 (3.01g, 7.23mmol) is dissolved in tetrahydrofuran (30mL), H-Gly-O is added^tBu (3.03g, 18.09mmol) and HOBt (2.44g, 18.09mmol), DCC (3.73g, 18.09mmol) and DIPEA is added at 0 DEG C (6.3mL, 36.18mmol) is gradually heated to room temperature, stirs 3 days.Add ethyl acetate (200mL) to dilute, uses saturated sodium bicarbonate It washes, 1N HCl is washed, and saturated common salt washing, anhydrous sodium sulfate dries, filters, and is concentrated, is obtained compound 696 (10.17g).[M+H]⁺ Calculated value 643.29, measured value 643.29.

The synthesis of 266. compound 697 of embodiment

Compound 696 (5.38g, 8.38mmol) is dissolved in DCM (20mL), is added TFA (20mL), it is small to be stirred at room temperature 3 When.Concentration, toluene azeotropic distillation are beaten with methylene chloride, obtain white solid (1.00g, 1.89mmol), be dissolved in tetrahydro H-L-Ala-O is added in furans (20mL)^tBu (857mg, 4.72mmol) and HOBt (638g, 4.72mmol) is added at 0 DEG C DIC (735uL, 4.72mmol) and DIPEA (1.64mL, 9.43mmol), is gradually heated to 30 DEG C, is stirred overnight.Add acetic acid second Ester (100mL) dilution, is washed with saturated sodium bicarbonate, and 1N HCl is washed, and saturated common salt washing, anhydrous sodium sulfate dries, filters, dense Contracting, silica gel column chromatography purify (6%MeOH/DCM), obtain compound 697 (734mg, 50% yield).[M+H]⁺Calculated value 785.36 measured value 785.70.

The synthesis of 267. compound 698 of embodiment

Compound 697 (743mg, 0.936mmol) is dissolved in DCM (6mL), is added TFA (6mL), is stirred overnight at room temperature. After concentration, with toluene azeotropic distillation, solid chemical compound (629mg) is obtained, is dissolved in tetrahydrofuran (20mL), 4- amino fourth is added Tert-butyl acrylate (372mg, 2.340mmol) and HOBt (316g, 2.340mmol), be added at 0 DEG C DIC (365uL, 2.340mmol) with DIPEA (815uL, 9.43mmol), it is gradually heated to room temperature, is stirred overnight.Add ethyl acetate (150mL) Dilution, is washed with saturated sodium bicarbonate, and 1N HCl is washed, and saturated common salt washing, anhydrous sodium sulfate dries, filters, and is concentrated, and column chromatography is pure Change (8%MeOH/DCM), obtains compound 698 (612mg, 68% yield).[M+H]⁺Calculated value 955.47, measured value 955.75.

The synthesis of 268. compound 699 of embodiment

Compound 698 (500mg, 0.524mmol) is dissolved in DCM (6mL), is added TFA (6mL), it is small to be stirred at room temperature 2 When.After concentration, with toluene azeotropic distillation, solid chemical compound (441mg) is obtained, takes 186mg (0.17mmol, 1.0eq) and compound 110 (67mg, 0.17mmol) are dissolved in DMF (6mL), be added at 0 DEG C HATU (131mg, 0.34mmol) and TEA (94uL, 0.68mmol), it stirs 1 hour for 0 DEG C.Add water (100L) to dilute, extracted with ethyl acetate (50mL × 3), merge organic phase, with full It is washed with salt, anhydrous sodium sulfate dries, filters concentration, and silica gel column chromatography purifies (7%MeOH/DCM), obtains compound 699 (100mg)。[M+H]⁺Calculated value 1201.57, measured value 1201.53.

The synthesis of 269. compound 700 of embodiment

Compound 699 (228mg, 0.19mmol) is dissolved in methanol (30mL), is added Pd/C (30mg, 10wt%), 1 It is stirred overnight at room temperature under a atmospheric pressure hydrogen.Concentrate is dissolved in ethyl alcohol (5mL) by filtering and concentrating, and compound 110 is added 0.1M NaH is added in (50mg, 0.180mmol)₂PO₄(1.25mL), is stirred overnight at room temperature.Concentration of reaction solution obtains compound 700 (100mg)。[M+H]⁺Calculated value 1263.58, measured value 1263.52.

The synthesis of 270. compound 701 of embodiment

Compound 700 (100mg, 0.079mmol) is dissolved in DCM (2mL), is added TFA (3mL), is stirred at room temperature 90 points Clock.Concentration of reaction solution adds toluene azeotropic to rise distillation, and residue and compound 41a (55mg, 0.079mmol) are dissolved in DMA (2mL) is added DIPEA (28 μ L, 0.158mmol), 2h is stirred at room temperature.Concentration of reaction solution obtains chemical combination by preparation HPLC purifying Object 701 (1.2mg, 1% yield). [M+H]⁺Calculated value 1615.74, measured value 1615.68.

The synthesis of 271. compound 703 of embodiment

Compound 436 (2.93g, 3.92mmol) and the tert-butyl alcohol (1.87Ml, 19.59mmol) are dissolved in methylene chloride In (20mL), 0 DEG C sequentially adds DCC (970mg, 4.70mmol) and DMAP (48mg, 0.39mmol), is gradually heated to room temperature, It is stirred overnight.Filtrate is concentrated in filtering, adds ethyl acetate (50mL) to dilute, is washed with 1N HCl, and 5% sodium bicarbonate solution is washed, and satisfies It is washed with salt, anhydrous sodium sulfate dries, filters, and silica gel column chromatography purifying (7%MeOH/DCM), obtains compound 703 after concentration (2.28g, 72% yield).[M+H]⁺Calculated value 805.46, measured value 805.41.

The synthesis of 272. compound 704 of embodiment

Compound 703 (2.28g, 2.84mmol) is dissolved in methanol (80mL), is added Pd/C (0.23g, 10wt%), 1 Under a atmospheric pressure hydrogen, it is stirred overnight at room temperature.Filtering and concentrating filtrate, through column chromatographic purifying (7%MeOH/DCM), obtained chemical combination Object (1.39g, 2.08mmol) is dissolved in tetrahydrofuran (20mL), and compound 689 (346mg, 0.83mmol) and HOBt is added (281mg, 2.08mmol) DIC (324uL, 2.08mmol) and DIPEA (725uL, 4.16mmol) are added at 0 DEG C, is gradually warmed up To room temperature, it is stirred overnight.Adding ethyl acetate (100mL) to dilute, is washed with saturated sodium bicarbonate, 1N HCl is washed, saturated common salt washing, Anhydrous sodium sulfate dries, filters, and (7%MeOH/DCM) is purified by silica gel column chromatography after concentration, obtain compound 704 (673mg, 47% yield).[M+H]⁺Calculated value 1721.95, measured value 1722.59.

The synthesis of 273. compound 705 of embodiment

Compound 704 (673mg, 0.39mmol) is dissolved in DCM (6mL), is added TFA (6mL), is stirred overnight at room temperature. Concentration, toluene azeotropic distillation obtain a white solid (627mg), take 335mg (0.208mmol, 1.0eq) and compound 110 (82mg, 0.208mmol) is dissolved in DMF (6mL), be added at 0 DEG C HATU (158mg, 0.416mmol) and TEA (115uL, 0.832mmol), it stirs 2 hours for 0 DEG C.Add water (80mL) to dilute, extracted with ethyl acetate (80mL × 3), merge organic phase, satisfies It is washed with salt, anhydrous sodium sulfate dries, filters, and is concentrated, and column chromatographic purifying (6%MeOH/DCM) obtains compound 705 (318mg, 77% yield).[M+H]⁺Calculated value 1968.05, measured value 1969.32.

The synthesis of 274. compound 706 of embodiment

Compound 705 (318mg, 0.16mmol) is dissolved in methanol (20mL), is added Pd/C (30mg, 10wt%), 1 Under a atmospheric pressure hydrogen, it is stirred at room temperature 3 hours.Filtering and concentrating obtains a white solid (287mg).Take 98mg (0.0577mmol) It is dissolved in anhydrous tetrahydro furan (7mL), chloracetyl chloride (9 μ L) and DIPEA (20 μ L) is added at 0 DEG C, is stirred overnight.Concentration, must change It closes object 706 (27mg, 25% yield).[M+H]⁺Calculated value 1851.92, measured value 1852.94.

The synthesis of 275. compound 707 of embodiment

Compound 706 (27mg, 0.0146mmol) is dissolved in DCM (2mL), is added TFA (2mL), it is small to be stirred at room temperature 1 When.Concentration of reaction solution adds toluene azeotropic distillation, by obtained compound (25mg, 0.0146mmol) and compound 41a (10mg, 0.0146mmol) is dissolved in DMA (2mL), is added DIPEA (5 μ L, 0.0292mmol), is stirred at room temperature 90 minutes.Concentration is anti- Liquid is answered, preparation HPLC purifies concentrate, obtains compound 707 (10.2mg, 32% yield).[M/2+H]⁺Calculated value 1102.54, it is real Measured value 1103.27.

The synthesis of 276. compound 711 of embodiment

By compound Boc-N- methyl-L-Valine (33mg, 0.14mmol), Pentafluorophenol (39mg, 0.21mmol) and DCC (32mg, 0.154mmol) is dissolved in EtOAc (20mL).Mixture is stirred at room temperature overnight, diatomite is then passed through Filtering, filtrate is concentrated and is again dissolved in DMA (2mL), and compound 710 (52mg, 0.14mmol) and DIPEA is then added (48.5 μ L, 0.28mmol).It is concentrated after reaction mixture is stirred at room temperature overnight, and passes through preparation HPLC (C₁₈Column, 10- 100% acetonitrile/water) purifying, obtain compound 711 (40.2mg, 49%).ESI:m/z:C₂₈H₄₉N₄O₇S[M+H]⁺Calculated value: 585.32 measured value 585.32.

The synthesis of 277. compound 712 of embodiment

Compound 711 (40mg, 0.069mmol) is dissolved in pyridine (8mL), acetic anhydride then is added at 0 DEG C (20.4mg, 0.2mmol), reactant is warmed to room temperature, and is stirred at room temperature overnight.Mixture is concentrated and uses silicagel column (0-10%MeOH, DCM) purifying, obtains compound 712 (48.1mg ,~100%).ESI:m/z:C₃₀H₅₁N₄O₈S[M+H]⁺Meter Calculation value: 627.33, measured value 627.33.

The synthesis of 278. compound 715 of embodiment

Into EtOAc (10mL) solution of compound 712 (48.1mg, 0.077mmol) be added Pentafluorophenol (21.2mg, 0.115mmol) and DCC (17.4mg, 0.085mmol).Mixture is stirred at room temperature overnight, diatomite mistake is then passed through Filtrate is concentrated and is dissolved in DMA (4mL) again by filter, and compound 714 (20.7mg, 0.1mmol) and DIPEA is then added (26.8 μ L, 0.154mmol).Reaction mixture is stirred at room temperature overnight, is then concentrated and by preparation HPLC (C₁₈Column, Acetonitrile/water) purifying, obtain compound 715 (63mg ,~100%).ESI:m/z:C₄₂H₆₆N₅O₉S[M+H]⁺Calculated value: 816.45 measured value 816.45.

The synthesis of 279. compound 716 of embodiment

Compound 715 (63mg, 0.077mmol) is dissolved in DCM (1mL), TFA (3mL) then is added, will react Object is stirred at room temperature 2 hours, is then concentrated, and obtained compound (55mg, 0.077mmol) is dissolved in DMA (4mL), so Compound 125 (30.8mg, 0.11mmol) and DIPEA (27 μ L, 0.154mmol) are added afterwards.At room temperature by reaction mixture It is stirred overnight, is then concentrated and by preparation HPLC (C₁₈Column, acetonitrile/water) purifying, obtain compound 716 (28.5mg, 42%). ESI:m/z:C₄₅H₆₅N₆O₁₀S[M+H]⁺Calculated value: 881.44, measured value 881.44.

The synthesis of 280. compound 719 of embodiment

Compound 715 (63mg, 0.077mmol) is dissolved in DCM (1mL), TFA (3mL) then is added, will react Object is stirred at room temperature 2 hours, is then concentrated, and obtained compound (55mg, 0.077mmol) is dissolved in DMA (4mL), so Compound 718 (65.8mg, 0.11mmol) and DIPEA (27 μ L, 0.154mmol) are added afterwards.At room temperature by reaction mixture It is stirred overnight, is then concentrated and by preparation HPLC (C₁₈Column, acetonitrile/water) purifying, obtain compound 719 (14mg, 16%). ESI:m/z:C₅₅H₈₄N₇O₁₆S[M+H]⁺Calculated value: 1130.56, measured value 1130.57.

The synthesis of 281. compound 721 of embodiment

Compound 436 (3g, 4mmol) is dissolved in DMF (50mL), HATU (2.3g, 6mmol) is added under ice bath, TEA (1.7mL, 12mmol), reacts under ice bath to stir and is warmed to room temperature after twenty minutes, stirs 3 hours.Then above-mentioned reaction solution is slow Slowly it is added in p-aminophenyl methanol (0.99g, 8mmol), after stirring 1.5 hours at room temperature, pours into point equipped with 150mL water It in liquid funnel, is extracted with ethyl acetate (50mL × 2), merges organic phase, dried, filtered with anhydrous sodium sulfate, be concentrated, cross silicon Rubber column gel column (0-10%MeOH/DCM) purifying, obtains compound 721 (3.9g ,~100% yield).ESI:m/z: C₄₂H₆₈N₃O₁₅[M+ H]⁺Calculated value: 854.46, measured value 854.46.

The synthesis of 282. compound 722 of embodiment

Compound 721 (1.9g, 2.22mmol) is dissolved in methanol (20mL), is added Pd/C (0.19g), reaction is placed in hydrogen It after (1atm) is stirred 1.5 hours under gas, is filtered through diatomite, filtrate is collected and is concentrated, obtained crude product is dissolved in EtOH The NaH of compound 125 (0.61g, 2.2mmol) and 0.1M is then added in (100mL)₂PO₄(20mL), it is stirred at room temperature Night.Then above-mentioned reaction solution is concentrated to dryness, with methanol loading (being filtered to remove insoluble matter, take filtrate), by preparing HPLC (C₁₈ Column, acetonitrile/water) purifying, obtain compound 722 (0.3g, 19%).ESI:m/z:C₄₂H₆₉N₄O₁₆[M+H]⁺Calculated value: 885.46 measured value 885.44.

The synthesis of 283. compound 723 of embodiment

Compound 723 (0.12g, 0.14mmol) is dissolved in DMF (50mL), SOCl is added under ice bath₂(11 μL, 0.154mmol), it reacts under ice bath and stirs 1 hour.Then above-mentioned reaction solution is concentrated to dryness, obtains compound 723 (0.13g, 0.14mmol).ESI:m/z:C₄₂H₆₈ClN₄O₁₅[M+H]⁺Calculated value: 903.43, measured value 903.44.

The synthesis of 284. compound 725 of embodiment

By compound 723 (0.13g, 0.14mmol), compound 724 (0.06g, 0.07mmol) is dissolved in DMF (10mL), TBAI (16mg, 0.042mmol) and DIPEA (64 μ L, 0.35mmol) then are sequentially added, is stirred 1 hour at room temperature.So Above-mentioned reaction solution is concentrated to dryness afterwards, by preparing HPLC (C₁₈Column, acetonitrile/water) purifying, obtain compound 725 (10mg, 8.9%).ESI:m/z:C₇₉H₁₂₅N₉O₂₃S [M+H]⁺Calculated value: 1599.85, measured value 1599.82.

The synthesis of 285. compound 727 of embodiment

To the THF (20mL) of compound 15 (4.0g, 7.6mmol) and iodoethane (914mg, 22.85mmol) at 0 DEG C Sodium hydrogen (60wt%, 9.5g, 60.9mmol) is added in solution.Reaction solution holding is stirred overnight for 0 DEG C, high degree of agitation is then poured into Ice water saturated ammonium chloride (5L) solution in.It is extracted with ethyl acetate (3 × 100mL).Organic phase after merging is through drying, mistake After filter, concentration, column chromatographs (15-35% ethyl acetate/petroleum ether) purifying, obtains product 727 (1.5g, 36% yield), is bright orange Color grease.

The synthesis of 286. compound 728 of embodiment

29 (7.0mmol) and dry Pd/C are added into the ethyl acetate solution of compound 12 (1.5g, 2.8mmol) (10wt%, 80mg).Reaction solution stirs 20 hours in the hydrogen gas atmosphere (1atm).Diatomite filtering, ethyl acetate are washed.Merge Through column chromatography (0-13% ethyl acetate/petroleum ether) purifying after organic phase concentration, compound 728 (950mg, 55% yield) .. is obtained

The synthesis of 287. compound 729 of embodiment

It is mixed that compound 728 (950mg, 1.48mmol) is dissolved in acetic acid/water/tetrahydrofuran (v/v/v 3:1:1,50 mL) It closes in solution, is stirred at room temperature 3 days.Dry with toluene azeotropic band after reaction solution concentration, after this step repeats twice, residue is through column layer (0-6%MeOH/DCM) purifying is analysed, compound 729 (700mg, 90% yield) is obtained, is colourless liquid.

The synthesis of 288. compound 730 of embodiment

At 5 DEG C by the aqueous solution of LiOH (3.3N, 2mL, 6.67mmol, 5.0eq.) be added compound 26 (700mg, In methanol (5.0mL) solution 1.33mmol).It is concentrated after being stirred at room temperature 2 hours.Through column chromatography, (15%MeOH/DCM contains 0.1% NH₃.H₂O it) purifies, obtains compound 730 (533mg, 80%), be white solid.

The synthesis of 289. compound 731 of embodiment

By compound 730 (520mg, 1.03mmol) and DMAP (13mg, 0.1mmol) be dissolved in anhydrous THF (5.0mL) with In the mixed solution of anhydrous DMF (0.25mL).It is cooled to 0 DEG C, TEA (0.22 mL, 1.54mmol) and acetic anhydride is added (0.15mL,1.54mmol).Reaction solution is gradually increased to room temperature and stirs 3 hours.(5-50%MeOH/ is chromatographed through column after concentration DCM it) purifies, obtains compound 731 (553mg, 94% yield), be amorphous white solid.

The synthesis of 290. compound 732 of embodiment

By compound 731 (0.1g, 0.185mmol), DIC (42.9 μ L, 0.28mmol) and Pentafluorophenol (40mg, It 0.22mmol) is dissolved in ethyl acetate (20mL), is stirred overnight at room temperature.Then above-mentioned reaction is concentrated to dryness, is changed It closes object 732 (63.5mg, crude product).ESI:m/z:C₃₂H₄₄F₅N₄O₆S [M+H]⁺Calculated value: 707.28, measured value 707.28.

The synthesis of 291. compound 733 of embodiment

By compound 732 (63.5mg, 0.09mmol), compound 127 (80.6mg, 0.2mmol) is dissolved in DMA (2mL) In, DIPEA (63 μ L, 0.36mmol) then is added, reaction is stirred 1 hour at room temperature.By the direct loading of above-mentioned reaction solution, system Standby HPLC (MeCN/H₂O elution) purifying, obtain product 733 (8.38mg, 10%).ESI:m/z:C₄₆H₆₈N₇O₁₁S[M+H]⁺Meter Calculation value: 926.46, measured value 926.45.

The synthesis of 292. compound 735 of embodiment

By compound 732 (63.5mg, 0.09mmol), compound 442 (293mg, 0.27mmol) is dissolved in DMA (4mL), DIPEA (63 μ L, 0.36mmol) then is added, is stirred overnight at room temperature.Above-mentioned reaction solution is concentrated into after 2mL on directly Sample prepares HPLC (MeCN/H₂O elution) purifying, obtain product 735 (10.5mg, 7.3%).ESI:m/z:C₇₇H₁₂₇N₁₀O₂₄S[M +H]⁺Calculated value: 1607.87, measured value 1607.84.

The synthesis of 293. compound 737 of embodiment

Compound 673 (48.7g, 64.4mmol) and compound 125 (27.0g, 96.6mmol) are dissolved in 250mL 95% In ethyl alcohol, 50mL 0.1M NaH is added₂PO₄, overnight, revolving removes ethyl alcohol for room temperature reaction, adds water (300mL), DCM extraction (300mL × 1,150mL × 2) merge organic phase, wash (500mL × 2), and saturated common salt washes (500mL), anhydrous sodium sulfate It drying, filtering, crude product silica gel column chromatography is purified (3-4%MeOH/DCM) after concentration, 36.6g light yellow oil is obtained, yield: 61.7%.ESI:m/z:C₄₃H₇₇N₄O₁₇[M+H]⁺Calculated value: 921.5, measured value: 921.5.

The synthesis of 294. compound 738 of embodiment

Compound 737 (16.6g, 0.018mmol) is dissolved in 100mL anhydrous formic acid, is stirred overnight at room temperature, has reacted Finish.Revolving removes HCO₂H is added a large amount of DCM and is spin-dried for, and vacuum pump drains to obtain 15.6g yellow oil, yield: 100%.It will be upper It states crude product (0.018mol, 1.0eq) to be dissolved with the anhydrous DCM of 180mL, in N₂Under protection, be added EDC (8.6g, 0.045mol) and NHS (5.2g, 0.045mol), room temperature reaction are spin-dried for after 3 hours, and 50mL ethyl acetate is added, and filtering is washed with a small amount of ethyl acetate It washs, filtrate is spin-dried for, and vacuum pump drains to obtain 18.0g yellow oil, is directly used in and synthesizes in next step.ESI:m/z: C₄₃H₇₂N₅O₁₉ [M+H]⁺Calculated value: 962.5, measured value: 962.5.

The synthesis of 295. compound 739 of embodiment

Compound 121 (2.0g, 5.91mmol) and compound 737 (8.5g, 8.87mmol) are dissolved in 95% ethyl alcohol of 20mL In, it is added 2.0mL PBS (0.1M, pH7.5), overnight, revolving removes ethyl alcohol for room temperature reaction, adds 100mL ethyl acetate to dissolve, water It washes (50mL × 5), water phase is saturated with NaCl solid, is extracted with ethyl acetate (150mL × 1,75mL × 3), and organic phase, nothing are merged Aqueous sodium persulfate dries, filters, and is spin-dried for, and obtains product 3.56g, yield: 50%.ESI:m/z:C₅₆H₉₃N₆O₂₁[M+H]⁺Calculated value: 1185.6, measured value 1185.6.

The synthesis of 296. compound 740 of embodiment

Compound 739 (1.0g, 0.844mmol) is dissolved in 12mL DCM, and 4mL TFA is added, and is reacted at room temperature 1 hour, rotation Solvent is evaporated off, a large amount of DCM are added and are spin-dried for, add diethyl ether, is stirred at room temperature 30 minutes, stands, pours out ether, repeats the operation 2 times, Residue drains to obtain 0.91g yellow oil, by its (0.844mmol, 1.0eq) and compound 41a (0.70g, 0.101mmol) It is dissolved in the anhydrous DMA of 15mL, ice bath, N₂Protection is lower to be added dropwise DIPEA (0.44mL, 2.53mmol), finishes, it is anti-to be warming up to room temperature It is spin-dried for after answering 3 hours, a small amount of DCM is added to dissolve, first acid for adjusting pH to 3, direct loading silicagel column first uses ethyl acetate/petroleum ether (0%-100%, contain 0.1% formic acid) removes the lesser impurity of depolarization, then with MeOH/DCM (0%-20%, containing 0.1% formic acid) Elution, obtains 1.43g yellow foamy solid.It is dissolved in acetonitrile and water, with preparation HPLC (C₁₈Column, mobile phase A: water, stream Phase B: acetonitrile is moved, acetonitrile is raised to from 20% and 80%) purifies in 120 min, obtains 1.0g white foam solid, yield 74.6%. ESI: m/z:C₇₆H₁₂₅N₁₀O₂₄S[M+H]⁺: calculated value 1593.9, measured value 1593.9.

The synthesis of 297. compound 742 of embodiment

Compound 154 (2.0g, 5.91mmol) and compound 738 (8.5g, 8.87mmol) are dissolved in 95% second of 20mL In alcohol, it is added 2.0mL PBS (0.1M, PH7.5), overnight, revolving removes ethyl alcohol for room temperature reaction, and add 100mL ethyl acetate to dissolve, It washes (50mL × 5), water phase is saturated with NaCl solid, is extracted with ethyl acetate (150mL × 1,75mL × 3), merge organic phase, Anhydrous sodium sulfate dries, filters, and is spin-dried for, and obtains product 4.85g, yield: 69.2%.ESI:m/z:C₅₆H₉₃N₆O₂₁[M+H]⁺It calculates Value: 1185.6, measured value 1185.6.

The synthesis of 298. compound 743 of embodiment

Compound 742 (1.0g, 0.844mmol) is dissolved in 12mL DCM, 4mL TFA is added, is reacted at room temperature 1 hour, Revolving removes solvent, and a large amount of DCM are added and are spin-dried for, add diethyl ether, are stirred at room temperature 30 minutes, stands, and outwells ether, repeats the operation 2 It is secondary, residue is drained into obtain 0.90g yellow oil, by its (0.843mmol, 1.0eq) and compound 41a (0.70g, It 0.100mmol) is dissolved in the anhydrous DMA of 15mL, ice bath is added dropwise DIPEA (0.44mL, 2.53mmol), and it is small to be warming up to room temperature reaction 3 When, it is spin-dried for, a small amount of DCM is added to dissolve, first acid for adjusting pH to 3, direct loading silicagel column first uses ethyl acetate/petroleum ether (0%- 100%, contain 0.1% formic acid) the lesser impurity of depolarization is removed, then eluted with MeOH/DCM (0%-20% contains 0.1% formic acid), Obtain 1.43g yellow foamy solid.It is dissolved in acetonitrile and water, with preparation HPLC (C₁₈Column, mobile phase A: water, mobile phase B: acetonitrile, acetonitrile is raised to from 20% and 80%) purifies in 120min, obtains 1.0g white foam solid, yield 75.0%.ESI:m/ z: C₇₆H₁₂₅N₁₀O₂₄S[M+H]⁺Calculated value: 1593.9, measured value 1593.9.

Bis- (2- ethoxy) amido -4- oxy butyrate methyl esters of embodiment 299.

Dimethyl succinate (20.0g, 136.9mmol) and diethanolamine (7.20g, 68.7mmol) are dissolved in no water beetle In the mixed solution of benzene (500ml) and pyridine (50ml), flow back 28 hours at 150 DEG C.Through SiO after mixed solution is concentrated₂Column It chromatographs (from EtOAc/DCM=5%-25% to EtOAc) and obtains title compound (12.5g, 83%).ESI MS m/z+ ion Peak: C₉H₁₇NaNO₅(M+Na) calculated value 242.2, experiment value 242.4.

Bis- (2- (the methylsulphur base oxygroup) ethyl) amido -4- oxy butyrate methyl esters of embodiment 300..

Will be bis- (2- ethoxy) amido -4- oxy butyrate methyl esters (12.0g, 49.56mmol) be dissolved in anhydrous pyridine (350ml), It is added mesyl chloride (20.0g, 175.4mmol).Solution is concentrated after reaction overnight, then plus EtOAc (350ml) dilutes, then With cooling 1M NaH₂PO₄Solution (2x 300ml) washing, uses MgSO₄Dry, filter obtained after being spin-dried for crude product (18.8g, 101%).Not purified be just directly used in of the crude product is reacted in next step.ESI MS m/z+ quasi-molecular ions: C₁₁H₂₁NaNO₉S₂(M+ Na) calculated value 398.2, experiment value 398.4.

Bis- (2- (thioacetyl) ethyl) amido -4- oxy butyrate methyl esters of embodiment 301..

Will be bis- (2- (methylsulphur base oxygroup) ethyl) amido -4- oxy butyrate methyl esters (brand-new, 90% purity, 8.5g, about 20mmol) be dissolved in DMA (350ml), sequentially added at 0 DEG C triethylamine (30ml, 215mmol) and thioacetic acid (10ml, 134mmol).It is stirred overnight at room temperature, is concentrated, after EtOAc (350ml) dilution, successively use NaHCO₃Saturated solution (300ml), NaCl saturated solution (300ml) and 1M NaH₂PO₄Solution (300ml) washing.Then, organic phase Na₂SO₄Dry, filter rotation Through SiO after dry₂Column chromatographs EtOAc/hexane (10%-25%EtOAc) and obtains title compound (5.1g, 76%).ESI MS M/z+ quasi-molecular ions: C₁₃H₂₁NaNO₅S₂(M+Na) calculated value 358.1, experiment value 358.2.

Embodiment 302.4- (bis- (2- (pyridine -2- base disulfide group) ethyl) amidos) -4- oxy butyrate.

Will be bis- (2- (thioacetyl) ethyl) amido -4- oxy butyrate methyl esters (5.0g, 14.9mmol) be dissolved in THF (150ml) The aqueous solution (100ml) of NaOH (5.0g, 125mmol) is added.H is used after being stirred at room temperature 35 minutes₃PO₄It is neutralized to pH 7.Then, THF (100ml) solution of 1,2-, bis- pyridine disulfide (Aldrithiol-2,26.0g, 118 mmol) is added, and it is small to stir 4 When.Through SiO after concentration₂Column chromatography MeOH/DCM/HOAc (1:20/1%) obtains title compound (5.8g, 85.6%).ESI MS m/z+ quasi-molecular ions: C₁₈H₂₁NaN₃O₃S₄(M+Na) calculated value 478.0, experiment value 478.2.

Embodiment 303.2,5- dioxo pyrrolidin -1- base-bis- (2- (pyridine -2- base disulfide group) ethyl) amido -4- oxygen Butyrate.

4- (bis- (2- (pyridine -2- base disulfide group) ethyl) amidos) -4- oxy butyrate (5.2g, 11.5mmol) is dissolved in DMA NHS (1.6g, 13.9mmol) and EDC (5.0g, 26.1mmol) is added in (100ml).It is stirred overnight, through SiO after being spin-dried for₂Column layer Analysis (from EtOAc/DCM=5%-15% to EtOAc) obtains title compound (5.8g, 85.6%).ESI MS m/z+ ion Peak: C₂₂H₂₄NaN₄O₅S₄(M+Na) calculated value 575.1, experiment value 575.2.

Outside embodiment 304. -3,6- epoxy-Δ-tetrahydrophthalimide.

Will in toluene (200ml) solution of maleimide (10.0g, 103.0mmol) be added furans (10.0ml, 137.4mmol), then mixed solution is placed in 1L autoclave and is heated to 100 DEG C of reactions 8 hours.After being cooled to room temperature, by kettle Interior solid is rinsed with methanol, is recrystallized to give title compound in ethyl acetate and n-hexane mixed solution after concentration (16.7g, 99%).¹H NMR(CDCl₃):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+ quasi-molecular ions: C₈H₇NaNO₃(M+Na) calculated value 188.04, experiment value 188.04.

((((3aR, 4R, 7S, 7aS) -1,3- dioxy 3a, 4,7,7a- tetrahydro -1H-4,7- epoxy is different by 2- by embodiment 305.4- Indoles -3H)-yl) ethyl) (2- ((4R, 7S, 7aS) -1,3- dioxy -3a, 4,7,7a- tetrahydro -1H-4,7- epoxy iso-indoles -2 (3H)-yl) ethyl) amido) -4- oxy butyrate methyl esters.

By 4- (bis- (2- mesyl) ethoxy amine) -4- oxobutyrate (brand-new, 90% purity, 8.5g ,~ 20mmol) be dissolved in DMA (350mL), sequentially add 3,6- bridging oxygen-△-tetrahydric phthalimide (10.2g, 61.8mmol), sodium carbonate (8.0g, 75.5mmol) and sodium iodide (0.3g, 2.0mmol).Reaction mixture stirs at room temperature Overnight.After reaction solution is concentrated, add ethyl acetate (350mL), and successively with saturated sodium bicarbonate aqueous solution (300mL), saturation Saline solution (300mL) and 1M NaH₂PO₄(300mL) washing.Organic phase is dried, filtered with anhydrous sodium sulfate, filtrate concentration；Institute Product through silica gel column chromatography separation (10%~30% ethyl acetate/n-hexane) obtain after purification target compound (7.9g, 77%).ESI MS m/z+:C₂₅H₂₇NaN₃O₉(M+Na): calculated value: 536.2, experiment value: 536.4.

Embodiment 306.4- (bis- (2- (2,5- dioxy -2,5- dihydro -1H- pyrrolidines) ethamine) -4- ketobutyric acids

By 4- ((2- ((3aR, 4R, 7S, 7aS) -1,3- dioxy 3a, 4,7,7a- tetrahydro -1H-4,7- epoxy iso-indoles - 3H)-yl) ethyl) (2- ((4R, 7S, 7aS) -1,3- dioxy -3a, 4,7,7a- tetrahydro -1H-4,7- epoxy iso-indoles -2 (3H) - Base) ethyl) amido) -4- oxy butyrate methyl esters (3.0g, 5.8mmol) and trimethyl stannic hydroxide (4.8g, 26.4 mmol) be dissolved in In 1,2- dichloroethanes (150mL), flow back 8 hours at 80 DEG C.It is cooled to room temperature, reaction solution is filtered with silica gel, uses dichloro Methane/methanol rinse is to remove remaining trimethyl stannic hydroxide.Merging filtrate after reduced pressure, adds N, N- dimethylacetamide Amine and toluene flow back at 120 DEG C and are stirred overnight.It is purified through silica gel column chromatography separation (5%~10% ethanol/methylene) To target compound (1.62 g, 76%).ESI MS m/z+C₁₆H₁₇NaN₃O₉(M+Na): calculated value: 386.1；Experiment value: 386.2。

Embodiment 307.2,5- dioxypyrrole alkane -4- (bis- (2- (2,5- dioxy -2,5- dihydro -1H- pyrrolidines) ethamine) - 4- oxobutyric

By 4- (bis- (2- (2,5- dioxy -2,5- dihydro -1H- pyrrolidines) ethamine) -4- ketobutyric acids (16.0g, It 4.4mmol) is dissolved in DMA (100mL), NHS (0.76g, 6.61mmol) and EDC (1.70 g, 8.90mmol) is added.Reaction is mixed Liquid is closed to be stirred overnight at room temperature.Then reaction solution is concentrated, separates (5%~15% ethyl acetate/dichloro through silica gel column chromatography Methane) purifying obtain target compound (1.72g, 85.0%).ESI MS m/z+C₂₀H₂₀NaN₄O₉(M+Na): calculated value: 483.1；Experiment value: 483.2.

Embodiment 308.5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) pentanoate

The 5- hydroxypentanoic acid tert-butyl ester (10.0g, 57.4mmol) is dissolved in pyridine (60mL), mesyl chloride is added (8.0mL, 103.3mmol).Reaction mixture stirs 6h at room temperature.After reaction solution is concentrated, ethyl acetate is added to dilute, use is cold 1M NaH₂PO₄The washing of (pH 6) solution, anhydrous magnesium sulfate dry, filter, and mother liquor, which is concentrated under reduced pressure, to be done, and obtain mesylate.It will It is added to compound 12 (9.90g, 60.0mmol) and Na₂CO₃In DMF (80mL) solution of (8.5g, 80.1mmol).Mixing Reaction solution is stirred overnight at room temperature.After reaction solution is concentrated, add ethyl acetate, with saturated salt solution and 1M NaH₂PO₄(pH 6) solution washs, and anhydrous magnesium sulfate dries, filters, after mother liquor concentrations through silica gel chromatograph post separation (ethyl acetate/dichloromethane= It 1:12) purifies, obtains target compound (14.01g, 76%). ESI MS m/z+C₁₇H₂₃NaNO₅(M+Na): calculated value: 344.16；Experiment value: 344.16.

Embodiment 309.5- dimaleoyl imino valeric acid.

5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) pentanoate (5.0g, 15.57mmol) is dissolved in In Isosorbide-5-Nitrae-dioxane (40mL), concentrated hydrochloric acid (10mL) then is added at 4 DEG C, reaction mixture stirs 30min at room temperature. 5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) valeric acid (4.08g, 99%) is obtained after reaction solution concentration is done.It will be upper It states compound and is dissolved in back flow reaction 6h in n,N-dimethylacetamide/toluene (1:1,40mL).Reaction solution concentration after, with ethyl alcohol/ Ether/n-hexane is recrystallized to give target compound (2.76g, 90%).ESI MS m/z+C₉H₁₂NO₄(M+H): calculated value: 198.07；Experiment value: 198.07.

Embodiment 310.5- (dimaleoyl imino) caproic acid succinimide ester (DMPS linker)

5- maleic acylamino valeric acid (2.0g, 10.1mL) is dissolved in methylene chloride (20mL), NHS is then added (1.50g, 13.0mmol) and EDC (7.0g, 36.4mmol), reaction mixture is stirred overnight at room temperature.Reaction solution is concentrated After dry, target compound (2.43g, 82%) is obtained through silica gel chromatograph post separation (ethyl acetate/dichloromethane=1:10).ESI MS m/z+C₁₃H₁₄NaN₂O₆(M+Na): calculated value: 317.09；Experiment value: 317.09.

Embodiment 311.5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) valeric acid hydrazides t-butyl formate

5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) valeric acid (1.0g, 3.77mmol) is dissolved in DMF In (30mL), t-butylcarbamate (0.60g, 4.53mmol) and EDC (2.0g, 10.4mmol) is then added, reaction is mixed Liquid is closed to be stirred overnight at room temperature.It is pure through silica gel chromatographic column (ethyl acetate/dichloromethane=1:10) after reaction solution concentration is done Change obtains target compound (1.18g, 83%). ESI MS m/z+C₁₈H₂₅NaN₃O₆(M+Na): calculated value: 402.17；Experiment Value: 402.18.

Embodiment 312.5- (dimaleoyl imino) valeric acid hydrazides

By 5- (3', 6'- bridging oxygen-△-tetrahydric phthalimide) valeric acid hydrazides t-butyl formate (1.18g, It 3.11mmol) is dissolved in n,N-Dimethylformamide/toluene (1:1,20mL), back flow reaction 6h.Reaction solution is concentrated, Isosorbide-5-Nitrae-is added Then HCl (5mL, 36%) is added at 4 DEG C in dioxane (20mL), stirs 30min.Reaction solution is concentrated to dryness, second is used Alcohol/ether/n-hexane is recrystallized to give target compound (577mg, 88%).ESI MS m/z+C₉H₁₄N₃O₃(M+H): calculating Value: 212.10,；Experiment value: 212.10.

The bromo- maleimide compound 39 and 40 of embodiment 313.3'- and the bromo- maleimation of 3', 4'- bis- close Object 43 and 44

Compound 37 or 38 (~6g) are dissolved in DMF (60mL), then be added bromomaleic acid acid anhydride (1eq) or Person 2, and bis- bromo- maleic anhydride (1eq) of 3-, reaction mixture is stirred overnight.It is obtained after reaction solution is concentrated to dryness pure anti- Formula acid.It is added acetic acid (~50mL) and acetic anhydride (2~4g) into above-mentioned trans acids, reaction mixture flows back 6 at 120 DEG C~ 12h.Reaction solution is concentrated, obtains the bromo- Malaysia 3'- through silica gel chromatograph post separation (ethyl acetate/dichloromethane=1:10~1:1) Imide compound 39 and 40 obtains 3', the bromo- maleimide compound 43 and 44 (61%~87%) of 4'- bis- with method.

5- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3-) valeric acid

ESI MS m/z+C₉H₁₁BrNO₄(M+H): calculated value: 275.98；Experiment value: 275.98.

3- (2- (2- (2- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3-) ethyoxyl) ethyoxyl) ethoxy Base)-propionic acid.

ESI MS m/z+C₁₃H₁₉BrNO₇(M+H): calculated value: 380.03；Experiment value: 380.03.

5- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3,4- bis-) valeric acid

ESI MS m/z+C₉H₁₀Br₂NO₄(M+H): calculated value: 353.89；Experiment value: 353.89.

3 (2- (2- (2- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3,4- bis-) ethyoxyl) ethyoxyl) Ethyoxyl)-propionic acid

ESI MS m/z+C₁₃H₁₈Br₂NO₇(M+H): calculated value: 457.94；Experiment value: 457.94.

The n-hydroxysuccinimide etherate 41 and 42 and 3', 4'- bis- of the bromo- maleimide of embodiment 314.3'- The n-hydroxysuccinimide etherate 45 and 46. of bromo- maleimide

R=C₁~C₈alkyl or C₂H₄(OC₂H₄) n, n=1~20；X³=H or Br；41,42,X³=H；45,46,X³ =Br

By the bromo- maleimide compound 39 and 40 (1eq) of 3'- or the bromo- maleimide compound 43 of 3', 4'- bis- It is dissolved in DMA (~0.15ml) with 44, NHS (1.1eq) and EDC (2~4eq) is then added, reaction mixture is stirred overnight.It will Reaction solution concentration, obtains the bromo- maleimide of 3'- through silica gel chromatograph post separation (ethyl acetate/dichloromethane=1:20~1:5) N-hydroxysuccinimide etherate 41 and 42 and the bromo- maleimide of 3', 4'- bis- n-hydroxysuccinimide ether Compound 45 and 46 (70%~93%).

2,5- dioxo pyrrolidin -1- base 5- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3-) valerate (41)

ESI MS m/z+C₁₃H₁₃BrN₂NaO₇(M+Na): calculated value: 395.00；Experiment value: 395.00.

2,5- dioxo pyrrolidin -1- base 5- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base of 3,4- bis-) penta Acid esters (45)

ESI MS m/z+C₁₃H₁₂Br₂N₂NaO₆(M+Na): calculated value: 472.91；Experiment value: 472.91.

2,5- dioxo pyrrolidin -1- base 3- (2- (2- (2- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles 1- of 3- Base) ethyoxyl) ethyoxyl) ethyoxyl) propionic ester (42)

ESI MS m/z+C₁₇H₂₁BrN₂NaO₉(M+Na): calculated value: 499.04；Experiment value: 499.04.

(2- (2- (2- (the bromo- 2,5- dioxo -2,5- dihydro -1H- pyrroles of 3,4- bis- of 2,5- dioxo pyrrolidin -1- base 3 1- yl) ethyoxyl) ethyoxyl) ethyoxyl) propionic ester (46)

ESI MS m/z+C₁₇H₂₀Br₂N₂NaO₉(M+Na): calculated value: 576.95；Experiment value: 576.95.

Embodiment 315.4- (two sulphur of 2- pyridyl group) -4- methylvaleric acid

By 4- sulfydryl -4- methylvaleric acid (Goff, D. etc., Bioconjugate Chem.1990,1,381-386) (4.67g, 31.5mmol) is dissolved in methanol (15mL), is then added 2,2, two pyridine of-two sulphur (30.0 g, 136.2mmol) The sodium phosphate buffer (pH 7.5,70mL) of methanol solution (80mL) and 100mM, reaction solution stir 6h.Reaction solution is concentrated Afterwards, extracted with ethyl acetate/n-hexane (1:1), water layer be then transferred to pH 3 with 1M HCl, then with ethyl acetate (3 × 100mL) extract.Organic phase is merged, is dried, filtered with anhydrous sodium sulfate, is concentrated.Through silica gel chromatograph post separation (methanol/dichloro Methane/acetic acid=1:15:0.01) obtain target compound (7.05g, 87%).ESI MS m/z+C₁₁H₁₆NO₂S₂(M+H): Calculated value: 258.05；Experiment value: 258.05.

Embodiment 316.4- (two sulphur of 2- pyridine) -4- methylvaleric acid succinimide ester (SMDP linker)

4- (two sulphur of 2- pyridine) -4- methylvaleric acid (2.0g, 7.78mmol) is dissolved in methylene chloride (20mL), then NHS (1.10g, 9.56mmol) and EDC (4.0g, 20.8mmol) is added, reaction mixture is stirred overnight.Reaction solution is concentrated Afterwards, target compound (2.48g, 90%) is obtained through silica gel chromatograph post separation (ethyl acetate/dichloromethane=1:10).ESI MS m/z+C₁₅H₁₈NaN₂O₄S₂(M+Na): calculated value: 377.07；Experiment value: 377.08.

Embodiment 317.2- ((1R, 3R) -3- (tertbutyloxycarbonyl) -1- hydrogen -4- methyl amine amyl) 4-thiazolecarboxylic acid first Ester

By (3R, 5R)-tert-butyl -3- isopropyl -3- (4- (methoxycarbonyl group) thiazole) isopropyl oxazole -2- methyl formate (1.00g, 2.81mmol) is dissolved in acetonitrile (20mL) and H₂In O (2mL), it is added Mo (CO)₆(1.10g, 3.12mmol), reaction are mixed It closes liquid and stirs 16h at 70 DEG C.After reaction solution is concentrated, add ethyl acetate (50mL) and 10% aqueous citric acid solution (50mL), Add NaIO₄Until water layer becomes to clarify, it is extracted with ethyl acetate.Organic phase 10%Na₂S₂O₃Aqueous solution and saturated common salt Water washing, anhydrous sodium sulfate dry, filter, and concentration obtains nothing through silica gel chromatograph post separation (n-hexane/ethyl acetate=3:2) Color solid chemical compound (906mg, 90%).ESI MS m/z+C₁₆H₂₆N₂NaO₅S (M+Na): calculated value: 381.14；Experiment value: 381.14。

Embodiment 318.2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- amyl) thiazole-4- first Sour methyl esters.

By 2- ((1R, 3R) -3- (tertbutyloxycarbonyl) -1- hydrogen -4- methyl amine amyl) 4-thiazolecarboxylic acid methyl esters (900 Mg, 2.51mmol) it is dissolved in pyridine (15mL), it is added acetic anhydride (0.5mL, 5.29mmol), it is after being stirred overnight that reaction solution is dense Contracting, obtains colorless solid compounds (950mg, 95%) through silica gel chromatograph post separation (n-hexane/ethyl acetate=4:1).ESI MS m/z+C₁₈H₂₈N₂NaO₆S (M+Na): calculated value: 423.15；Experiment value: 423.16.

Embodiment 319.2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- amyl) thiazole-4- first Acid.

By 2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- amyl) 4-thiazolecarboxylic acid methyl esters (940mg, 2.35mmol) is dissolved in THF (15mL), and NaH (120mg, 3.0mmmol, 60%) is added at 4 DEG C, reaction mixing CH is added after liquid stirring 2h₃I (0.155mL, 2.49mmol), reaction mixture is stirred overnight.Reaction solution is concentrated, second is re-dissolved in Acetoacetic ester, filtering, is concentrated to dryness, obtains pure 2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- penta Base) 4-thiazolecarboxylic acid methyl esters (73a).Above-mentioned gained compound (73a) is dissolved in 1,2- dichloroethanes (20mL), front three is added Base stannic hydroxide (620mg, 3.43mmol), reaction mixture are stirred overnight at 80 DEG C.Reaction solution is concentrated, first is dissolved in It in alcohol/methylene chloride/acetic acid (1:5:0.01,20mL), filters, concentration, mother liquor is concentrated to get dry change after adding toluene again Close object.Then, above-mentioned gained compound is dissolved in pyridine (15mL), is added acetic anhydride (0.4mL, 4.23mmol), reaction is mixed Liquid is closed to be stirred overnight.After reaction solution is concentrated, obtained through silica gel chromatograph post separation (ethanol/methylene/acetic acid=1:10:0.01) To anhydrous solid compound (735mg, 78%).ESI MS m/z+C₁₈H₂₈N₂NaO₆S (M+Na): calculated value: 423.15；Experiment Value: 423.16.

Embodiment 320.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1,3- dioxoisoindolin) - 4- methyl amine-amyl) 4-thiazolecarboxylic acid methyl esters

By 2- ((1R, 3R) -3- (tertbutyloxycarbonyl) -1- hydrogen -4- methyl amine amyl) 4-thiazolecarboxylic acid methyl esters (850 mg, It 2.37mmol) is dissolved in THF (15mL), in -20 DEG C of addition NaH (100mg, 2.5mmol, 60%), and stirs at such a temperature 20min adds N- (3- bromopropyl) phthalimide (655 mg, 2.4mmol), and above-mentioned reaction mixture is at -20 DEG C After lower stirring 30min, it is warmed to room temperature reaction 4h.Methanol (0.5mL) quenching reaction is added into reaction solution, adds methylene chloride (60mL) is filtered, and concentration obtains 2- (1R, 3R) -3- (tert-butoxy-methyl amine) -1- (3- (1,3- dioxoisoindolin -2- Propoxyl group -4- amyl) 4-thiazolecarboxylic acid methyl esters, gained compound without purifying, can directly carry out in next step.By above-mentioned gained Compound is dissolved in THF (10mL), and NaH (170mg, 4.25mmol, 60%) then is added at room temperature, is stirred 45min, is added Enter CH₃I (0.20mL, 3.21mmol), reaction mixture is stirred overnight at room temperature.By reaction solution NaH₂PO₄(2.0M, 2mL) It is quenched, DMA (5mL) then is added, be concentrated under reduced pressure, through silica gel chromatograph post separation (ethyl acetate/dichloromethane=1:10~1:4) Obtain target compound (921mg, 69%).ESI MS m/z+C₂₈H₃₇N₃NaO₇S (M+Na): calculated value: 582.22；Experiment value: 582.22。

Embodiment 321.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1,3- dioxoisoindolin) - 4- methyl amine-amyl) 4-thiazolecarboxylic acid.

By dry 2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1,3- dioxoisoindolin) -4- Methyl amine-amyl) 4-thiazolecarboxylic acid methyl esters (910mg, 1.63mmol) is dissolved in 1,2- dichloroethanes (20mL), front three is added Base stannic hydroxide (400mg, 2.21mmol), reaction mixture are stirred overnight at 80 DEG C.Reaction solution is concentrated, through silica gel color Spectrum post separation (ethanol/methylene/acetic acid=1:10:0.01) obtains target compound (756mg, 85%).ESI MS m/z+ C₂₇H₃₇N₃O₇S (M+H): calculated value: 546.22；Experiment value: 546.22.

Embodiment 322.2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy (3- (bis--oxoisoindolines of 1,3--2- Amylamine) -4- methyl amine) -4- amyl) 4-thiazolecarboxylic acid methyl esters

By 2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- amyl) 4-thiazolecarboxylic acid methyl esters (800mg, 2.00mmol) is dissolved in THF (30mL), and NaH (150mg, 3.75mmol, 60%) is added at room temperature, stirring 45min adds N- (3- bromopropyl) phthalimide (655 mg, 2.4mmol).Reaction mixture stirs at room temperature Overnight.By reaction solution NaH₂PO₄(2.0M, 2mL) is quenched, and DMA (5mL) then is added, and is concentrated under reduced pressure, through silica gel chromatographic column point Target compound (971mg, 82%) is obtained from (ethyl acetate/dichloromethane=1:10~1:4).ESI MS m/z+ C₂₉H₃₇N₃NaO₈S (M+Na): calculated value: 610.22；Experiment value: 610.22.

Embodiment 323.2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy (3- (bis--oxoisoindolines of 1,3--2- Amylamine) -4- methyl amine) -4- amyl) 4-thiazolecarboxylic acid.

By dry 2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy (3- (bis--oxoisoindolines of 1,3--2- Amylamine) -4- methyl amine) -4- amyl) 4-thiazolecarboxylic acid methyl esters (900mg, 1.53mmol) is dissolved in 1,2- dichloroethanes (35mL) In, it is added trimethyl stannic hydroxide (400mg, 2.21mmol), reaction mixture is stirred overnight at 80 DEG C.Reaction solution is dense Contracting, products therefrom are dissolved in pyridine (20mL), add acetic anhydride (3mL), reaction mixture is stirred overnight.Reaction solution is concentrated Afterwards, target compound (755mg, 86%) is obtained through silica gel chromatograph post separation (ethanol/methylene/acetic acid=1:10:0.01). ESI MS m/z+C₂₈H₃₅N₃NaO₈S (M+Na): calculated value: 596.20；Experiment value: 596.20.

Embodiment 324. (S) -5- (4- (benzyloxy) phenyl) -4- (tertbutyloxycarbonylamino) -2- methylpentene acetoacetic ester (185)

- 78 DEG C at a temperature of, diisobutyl aluminium hydride (40ml, 40mmol, 1.0M) is slowly added to (S) -3- (4- (benzyloxy) phenyl) -2- (tertbutyloxycarbonylamino)-methyl propionate 184 (8.00g, 20.76mmol) DCM (250ml) In solution, after reaction 2 hours, MeOH (5ml) is added and is quenched.It is warmed to room temperature to reaction mixture, 1M HCl is added and is adjusted to pH= 4, liquid separation.Water layer is extracted with DCM (2x 150ml), and organic layer is washed with water, and is merged organic layer, is used anhydrous Na₂SO₄It dries, filters, It is concentrated to get the head product of aldehyde radical intermediate.The head product is dissolved in DCM, is added at room temperature by 1- (1- ethoxycarbonyl-ethyl) The synthesis of methylene chloride (80ml) solution of triphenylphosphinebromide (18.0g, 40.64mmol) and KOtBu (5.00g, 44.64mmol) Ylide solution, be concentrated after object reaction overnight to be mixed, column chromatography (ethyl acetate/n-hexane, 1:8-1:4) obtains target Product (185) (6.90g, 76%), ESI:m/z:[M+Na]+, quasi-molecular ions: C₂₆H₃₃NNaO₅, 462.22, experiment value, 462.22.

Embodiment 325. (4R) -4- (tertbutyloxycarbonylamino) -5- (4- hydroxy phenyl) -2 methyl valeric acid ethyl ester

(S) -5- (4- (benzyloxy) phenyl) -4- (tertbutyloxycarbonylamino) -2- methylpent is added in hydrogen reaction flask Olefin(e) acid ethyl ester (185) (6.70g, 15.26mmol), methanol (150ml), 10%Pd/C (0.3g) react 6 under 30psi hydrogen Hour, it is filtered with diatomite, is concentrated, target product (186) (4.61g, 86%) is recrystallized to give from ethyl alcohol/n-hexane. ESI:m/z:[M+Na]+, quasi-molecular ions: C₁₉H₂₉NNaO₅, 374.20, experiment value, 374.30.

Embodiment 326. (4R) -4- (tertbutyloxycarbonylamino) -5- (4- hydroxyl -3- nitrobenzophenone) -2 methyl valeric acid Ethyl ester

Past (4R) -4- (tertbutyloxycarbonylamino) -5- (4- hydroxy phenyl) -2 methyl valeric acid ethyl ester 4.50g, Anhydrous CH 12.81mmol)₂Cl₂Ac is added in (200ml) solution₂O (2ml, 21.16mmol) and fuming nitric aicd (0.65ml, 14.07mmol), it is stirred at room temperature 4 hours, is diluted with water (150ml), separation, water phase is extracted with ethyl acetate.Merge organic Layer, uses anhydrous Na₂SO₄It dries, filters, is concentrated, column chromatography (ethyl acetate/dichloromethane=1:10) obtains target product (4.21g, 83%).ESI:m/z:[M+Na]⁺, quasi-molecular ions: C₁₉H₂₈N₂NaO₇, 419.19, experiment value, 419.20.

Embodiment 327.4- (tertbutyloxycarbonylamino) -2- methyl -5- (3- nitro -4- phosphonato phenyl) valeric acid second Ester

(4R) -4- (tertbutyloxycarbonylamino) -5- (4- hydroxyl -3- nitrobenzophenone) -2 methyl valeric acid ethyl ester (4.00g, 10.09mmol) is dissolved in acetonitrile (70ml) and n,N-dimethylacetamide (30ml), after being cooled to 0 DEG C, is added Phosphorus oxychloride (2.00ml, 21.45mmol) is added after two minutes in N, N '-diisopropylethylamine (4.00ml, 23.00mmol).? It stirs 8 hours at room temperature, is cooled to 0 DEG C, slowly addition sodium bicarbonate (3.5g, 41.60mmol) inward is made into water (20ml) Solution, continuation are stirred overnight at 0 DEG C, are concentrated, and are purified with C-18 column (20x4cm), condition of gradient elution: 25ml/min, A: 0.5% acetic acid, B: methanol, 100%A are washed 10 minutes, then arrive 75%A and 25%B through 45 minutes.Collect target components, concentration Obtain target compound (3.89g, 81%).ESI:m/z:[M-H]^-, quasi-molecular ions: C₁₉H₂₈N₂O₁₀P, 475.16, experiment value, 475.20.

Embodiment 328. (4R) -4- (tertbutyloxycarbonylamino) -2- methyl -5- (3- nitro -4- phosphonato phenyl) Valeric acid.

Toward 4- (tertbutyloxycarbonylamino) -2- methyl -5- (3- nitro -4- phosphonato phenyl) ethyl valerate The water (60ml) that lithium hydroxide (5.0g, 208.7mmol) is added in THF (100ml) solution of (3.75g, 7.87mmol) is molten Liquid reacts 4 hours at 0 DEG C, is adjusted to pH6 with the hydrochloric acid of 4M, is concentrated, C-18 column purification, gradient elution, 25ml/min, A:0.5% Acetic acid, B: methanol, 100%A are kept for 10 minutes, then arrive 75%A and 25%B through 45 minutes.Target components are collected, are concentrated to get Target compound (2.82g, 80%).ESI:m/z:[M-H]^-, quasi-molecular ions: C₁₇H₂₄N₂O₁₀P, 447.12, experiment value, 447.20.

Embodiment 329. (4R) -5- (3- amino -4- phosphonato phenyl) -4- (tertbutyloxycarbonylamino) -2- methyl Valeric acid.

(4R) -4- (tertbutyloxycarbonylamino) -2- methyl -5- (3- nitro -4- is sequentially added into hydrogen reaction flask Phosphonato phenyl) valeric acid (2.6g, 5.80mmol), methanol (80ml), 10%Pd/C (0.2g).Under 35psi Hydrogen Vapor Pressure Reaction 6 hours.Mixture is filtered with diatomite, is concentrated to give crude product (2.18g, 90%), is directly used in without being further purified It reacts in next step.ESI:m/z:[M-H]^-, quasi-molecular ions: C₁₇H₂₆N₂O₈P, 417.15, experiment value, 417.15.

Embodiment 330. (S) -2- (tertbutyloxycarbonylamino) -3- (4- hydroxyl -3- nitrobenzophenone)-methyl propionate (196)。

Toward (S) -2- (tertbutyloxycarbonylamino) -3- (4- hydroxy phenyl)-methyl propionate (4.5g, 15.24mmol) Be added in anhydrous methylene chloride (240ml) solution acetic anhydride (4ml, 42.32mmol) and fuming nitric aicd (0.85ml, 18.40mmol).It after being stirred at room temperature 4 hours, is diluted with water (150ml), separation, water phase is extracted with ethyl acetate.It is associated with Machine layer, anhydrous sodium sulfate dry, filter, and concentration, column chromatography (ethyl acetate/dichloromethane, 1:10) obtains target product (4.30g, 83%).ESI:m/z:[M+Na]⁺, quasi-molecular ions: C₁₅H₂₀N₂NaO₇, 363.13, experiment value, 363.20.

Embodiment 331. (S) -2- (tertbutyloxycarbonylamino) -3- (3- nitro -4- phosphonato phenyl)-propionic acid first Ester

At 0 DEG C, toward (S) -2- (tertbutyloxycarbonylamino) -3- (4- hydroxyl -3- nitrobenzophenone)-methyl propionate (4.10 G, 12.05mmol) acetonitrile (90ml) solution in be added N, N '-diisopropyl ethyl amine (4.00ml, 23.00 mmol), stirring After 2 minutes, it is added phosphorus oxychloride (2.00ml, 21.45mmol), after reaction mixture is stirred at room temperature 8 hours, is cooled to 0 DEG C, The solution that sodium bicarbonate (3.5g, 41.60mmol) and water (20ml) is made into slowly is added inward, continues stirred at 0 DEG C Then night is concentrated, C-18 column (20x4cm) purifying, gradient elution, 25ml/min, A:0.5% acetic acid, B: methanol, 100%A are protected It holds 10 minutes, then arrived 75%A and 25%B through 45 minutes.Collect target components, be concentrated to get target compound (4.20g, 83%).ESI:m/z:[M-H]^-, quasi-molecular ions: C₁₅H₂₀N₂O₁₀P, 419.08, experiment value, 419.10.

Embodiment 332.3- (3- amino -4- phosphonato phenyl)-(2R) -2- (tertbutyloxycarbonylamino)-propionic acid.

By (S) -2- (tertbutyloxycarbonylamino) -3- (3- nitro -4- phosphonato phenyl)-methyl propionate after drying (4.0g, 9.52mmol) is dissolved in mixed solvent 1,2- dichloroethanes (50ml) and n,N-dimethylacetamide (60ml), past to be somebody's turn to do Trimethyl stannic hydroxide (4.00g, 22.1mmol) is added in solution.Mixture reacts 6 hours at 80 DEG C, concentration, column chromatography (water/acetonitrile 1:4) collects target components, is concentrated to give (S) -2- (tertbutyloxycarbonylamino) -3- (3- nitro -4- phosphonato Phenyl)-propionic acid.Resulting compound and DMAC N,N' dimethyl acetamide (70ml) and 10%Pd/C (0.3g) are added to hydrogen together In solid/liquid/gas reactions bottle.It is reacted 6 hours under 35psi Hydrogen Vapor Pressure.Mixture is filtered with diatomite, and concentration is recrystallized to give target Product (2.86g, 80%) reacts in next step without being further purified to be directly used in.ESI:m/z:[M-H]^-, quasi-molecular ions: C₁₄H₂₀N₂O₈P, 375.10, experiment value, 375.10.

Embodiment 333.3- (4- benzyloxy-phenyl)-(2R) -2- [(tert-butoxycarbonyl)-methylamino]-propionic acid benzyl Ester

Toward 3- (4- benzyloxy-phenyl)-(2R) -2- [(tertbutyloxycarbonylamino)-propionic acid benzyl ester (and 4.0g, 8.67 Mmol sodium hydride (430mg, 10.75mmol, 60% oil close object) is added in tetrahydrofuran (60ml) solution), it is small to be stirred at room temperature 1 Shi Hou is added iodomethane (1.82g, 12.82mmol), and mixed liquor is stirred overnight, with methanol (0.5ml) quenching reaction, concentration, column Chromatography (ethyl acetate/dichloromethane, 1:10) obtains target product (3.83g, 93%).MS ESI:m/z:[M+Na]⁺, ion Peak: C₂₉H₃₃NNaO₅, 498.24, experiment value, 498.24.

Embodiment 334. (2R) -2- [(tert-butoxycarbonyl)-methylamino] -3- (4- hydroxyl -2- nitrobenzophenone) propionic acid

3- (4- benzyloxy-phenyl)-(2R) -2- [(tert-butoxycarbonyl)-methyl ammonia is sequentially added into hydrogen reaction flask Base]-propionic acid benzyl ester (3.8g, 8.00mmol), methanol (80ml), 10%Pd/C (0.3g).It is anti-under 35psi Hydrogen Vapor Pressure It after answering 6 hours, is filtered with diatomite, is concentrated to give crude product (2R) -2- [(tert-butoxycarbonyl)-methylamino] -3- (4- hydroxyl Phenyl) propionic acid, the compound is without being further purified.- 25 DEG C at a temperature of, toward this compound anhydrous methylene chloride The dichloro of tin tetrachloride (1.5ml, 12.75 mmol) and fuming nitric aicd (0.60ml, 12.98mmol) are added in (240ml) solution Methane (40ml) solution, mixture are stirred 75 minutes at -25 DEG C, are quenched with saturated sodium bicarbonate, are adjusted to pH=3-4, water phase It being extracted with ethyl acetate, merges organic layer, anhydrous sodium sulfate dries, filters, it is concentrated, column chromatography (MeOH/DCM/HOAc 1:8: 0.01) target compound (1.98g, 73%) is obtained.ESI:m/z:[M+Na]⁺, quasi-molecular ions: C₁₅H₂₀N₂NaO₇, 363.13, it is real Test value, 363.13.

Embodiment 335. (2R) -2- [(tert-butoxycarbonyl)-methylamino] -3- (3- nitro -4- phosphonato phenyl) Propionic acid

By (2R) -2- [(tert-butoxycarbonyl)-methylamino] -3- (4- hydroxyl -2- nitrobenzophenone) propionic acid (1.98g, It 5.82mmol) being dissolved in acetonitrile (30ml) and n,N-dimethylacetamide (30ml), N is added at 0 DEG C in resulting solution, N '-diisopropylethylamine (2.00ml, 11.50mmol)), and phosphorus oxychloride is added after reacting two minutes at this temperature (1.10ml,11.79mmol).After reaction mixture is stirred at room temperature 8 hours, it is cooled to 0 DEG C, sodium bicarbonate is slowly added inward The solution that (2.0g, 23.80mmol) and water (10ml) is made into, continuation are stirred overnight at 0 DEG C, mixture concentration, C-18 column (20x4cm) purifying, gradient elution, 25ml/min, A:0.5% acetic acid, B: methanol, 100%A is kept for 10 minutes, then through 45 Minute arrives 75%A and 25%B.Target components are collected, target compound (1.96,80%) is concentrated to get.ESI: m/z:[M- H]^-, quasi-molecular ions: C₁₅H₂₀N₂O₁₀P, 419.09, experiment value, 419.09.

Embodiment 336.3- (3- amino -4- phosphonato phenyl)-(2R) -2- [(tert-butoxycarbonyl)-methylamino] Propionic acid

(2R) -2- [(tert-butoxycarbonyl)-methylamino] -3- (3- nitro -4- is sequentially added into hydrogen reaction flask Phosphonato phenyl) propionic acid (1.96g, 4.67mmol), n,N-dimethylacetamide (60ml), 10%Pd/C (0.2g).? It is reacted 6 hours under 30psi Hydrogen Vapor Pressure.The filtering of mixture diatomite, is concentrated to give crude product (1.74 g, 95%)), it is not necessarily into one Step purifying is directly used in next reaction.ESI:m/z:[M-H]-,calcd for C₁₅H₂₂N₂O₈P,389.12,Found, 389.12.

337. phenyl -2- (2R) of embodiment-tertbutyloxycarbonylamino -1- acetone.

(1S, 2R)-(+)-demethyl ephedrine (7.0g, 46.29mmol) is added to mixed liquor tetrahydrofuran (40 ml) In 1M sodium bicarbonate (100ml), 4 DEG C at a temperature of, in be slowly added into 45 minutes di-tert-butyl dicarbonate (10.15g, Tetrahydrofuran (60ml) solution 46.53mmol), after reaction mixture is stirred at room temperature 6 hours, concentration, ethyl acetate extraction It takes, anhydrous sodium sulfate dries, filters, and concentration, column chromatographs (EtOAc/Hexane 1:2), collects target components, is concentrated to give thick production Object (1S)-phenyl -2- (2R)-tertbutyloxycarbonylamino -1- propyl alcohol (10.81,93%), crude product are not necessarily to be further purified, MS ESI:m/z+: [M+Na]⁺, quasi-molecular ions: C₁₄H₂₁NaNO₃, 274.15, experiment value, 274.15.Toward the dichloromethane of this compound Dai Si-Martin reagent methylene chloride (180ml, 0.3M) solution is added in alkane (50ml) solution, after stirring one hour, toward mixing The sodium hydroxide solution (1M, 100ml) of frost is added in object, separates, organic phase is adjusted to pH=with 1M sodium dihydrogen phosphate (100ml) 6, anhydrous sodium sulfate dries, filters, and concentration, column chromatography (EtOAc/Hexane 1:5) obtains target product (9.34g, 81%in two steps)。MS ESI:m/z+: [M+Na]⁺, quasi-molecular ions: C₁₄H₁₉NaNO₃, 272.14, experiment value, 272.14.

Embodiment 338.2,5- dioxo pyrrolidin-1- base 2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- Dimethyl -2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxylic acid ethyl ester

Toward the N of compound 33 (788.1mg, 1.464mmol), N- hydroxyl is added in N '-dimethyl formamide (10ml) solution Base succinimide (202.0mg, 1.756mmol), 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (980mg, 5.104mmol), after mixture is stirred overnight, concentration, column chromatographs (EtOAc/CH₂Cl₂=1:3) obtain target chemical combination Object (762.8mg, 82%).MS ESI:m/z+: [M+Na]⁺, quasi-molecular ions: C₃₀H₄₅NaN₅O₈S, 658.30, experiment value, 658.30.

Embodiment 339. (4R) -4- (tertbutyloxycarbonylamino) -5- (3- (5- (2,5- dioxo -2,5- dihydro -1H- Pyrroles -1- base) valeryl amino) -4- (phosphonato) phenyl) -2 methyl valeric acid

Toward (4R) -5- (3- amino -4- phosphonato phenyl) -4- (tertbutyloxycarbonylamino) -2 methyl valeric acid 2,5- dioxo pyrrolidin -1- base -5- is added in N '-dimethyl formamide (7ml) solution by the N of (825.1mg, 1.973mmol) (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ethyl valerate (711mg, 2.417 mmol), N, N '-diisopropyl second Amine (0.250ml, 1.438mmol), after mixture is stirred overnight, concentration, C-18 column chromatography (4x25cm, v=15ml/min, from The 1% HOAc/25%MeOH in 45min of 100% 1%HOAc to 75%) obtain target compound (895.7mg, 76%).MS ESI:m/z-: [M-H]^-, quasi-molecular ions: C₂₆H₃₅N₃O₁₁P, 596.21, experiment value, 596.21.

Embodiment 340. (4R)-4- (tertbutyloxycarbonylamino)-5-(3- (3- (2- (2- (2- (dioxo-2 2,5-, 5- dihydro -1H- pyrroles -1- base) ethyoxyl) ethyoxyl) ethyoxyl) propionamido) -4- (phosphonato) phenyl) -2- methylpent Acid.

Toward (4R) -5- (3- amino -4- phosphonato phenyl) -4- (tertbutyloxycarbonylamino) -2 methyl valeric acid The N of (632.5mg, 1.512mmol), in N '-dimethyl formamide (7ml) solution plus 2,5- dioxo pyrrolidin -1- base -3- (2- (2- (2- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ethyoxyl) ethyoxyl) ethyoxyl) ethyl propionate (727mg, 1.826mmol), N, N '-diisopropylethylamine (0.250ml, 1.438mmol), after mixture is stirred overnight, concentration, C-18 column chromatographs (4x25cm, v=15ml/min, from the 1%HOAc/25%MeOH in of 100% 1%HOAc to 75% 45min) obtain target compound (763.2mg, 72%).MS ESI:m/z-:[M-H]^-, quasi-molecular ions: C₃₀H₄₄N₃O₁₄P, 700.25 experiment value, 700.25.

Embodiment 341. (4R)-4- (2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxamide base) -5- (3- (5- (2, 5- dioxo -2,5- dihydro -1H- pyrroles -1- base) valeryl amino) -4- (phosphonato) phenyl) -2- methylvaleric acid

Toward (4R) -4- (tertbutyloxycarbonylamino) -5- (3- (5- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- Base) valeryl amino) -4- (phosphonato) phenyl) and -2 methyl valeric acid (102mg, 0.171mmol) Isosorbide-5-Nitrae-dioxane (4ml) Concentrated hydrochloric acid (1.0ml, 37%) is added in solution, after mixture stirs 30 minutes, is concentrated to dryness to obtain slightly de- Boc product.By this Crude product is dissolved in n,N-dimethylacetamide (5ml), and 2,5- dioxo pyrrolidin -1- base 2- is sequentially added into the solution ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R)-1- methyl piperidine-2- formamido group) pentanamide Base) -4- methyl amyl) thiazole -4-carboxylic acid ethyl ester (110mg, 0.173mmol), N, N '-diisopropylethylamine (30ul, 0.172mmol), after mixture is stirred overnight, concentration, column chromatography (water/acetone containing 1%HOAc, 1:9-1:4) obtains targeted It closes object (123.2 mg, 71%).MS ESI:m/z-:[M-H]^-, quasi-molecular ions: C₄₇H₆₇N₇O₁₄PS, 1016.42, experiment value, 1016.42.

Embodiment 342. (4R)-4- (2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxamide base) -3- (3- (2- (2- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ethyoxyl) ethyoxyl) ethyoxyl) propionamido) -4- (phosphonato) Phenyl) -2 methyl valeric acid

Toward-5-(3- (3- (2- (2- (2- (2,5- dioxo-2,5- dihydro-of (4R)-4- (tertbutyloxycarbonylamino) 1H- pyrroles -1- base) ethyoxyl) ethyoxyl) ethyoxyl) propionamido) -4- (phosphonato) phenyl) -2 methyl valeric acid Concentrated hydrochloric acid (1.0ml, 37%) is added in Isosorbide-5-Nitrae-dioxane (4ml) solution of (108mg, 0.154mmol), mixture stirring 30 After minute, it is concentrated to dryness to obtain de- Boc crude product.This crude product is dissolved in n,N-dimethylacetamide (5ml), into the solution Sequentially add 2,5- dioxo pyrrolidin-1- base 2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxylic acid ethyl ester (110mg, 0.173mmol), N, N '-diisopropylethylamine (30ul, 0.172mmol), after mixture is stirred overnight, concentration, column chromatography (contains Water/acetone of 1%HOAc, 1:9-1:4) obtain target compound (131.2mg, 76%).MS ESI:m/z-:[M-H]^-, from Sub- peak: C₅₁H₇₅N₇O₁₇PS, 1120.47, experiment value, 1120.48.

Embodiment 343. (4R) -4- (tertbutyloxycarbonylamino) -2- methyl -5- (4- (phosphonato) -3- (4- (pyrrole Pyridine -2- disulfide group) butyrylamino) phenyl) valeric acid.

Toward (4R) -5- (3- amino -4- phosphonato phenyl) -4- (tertbutyloxycarbonylamino) -2 methyl valeric acid The N of (548.3mg, 1.311mmol), in N '-dimethyl formamide (10ml) solution plus succinimide base 4- (pyridine -2- two Sulfenyl) butyric acid (550.2mg, 1.687mmol) and N, N '-diisopropylethylamine (0.18ml, 1.03mmol), mixture is overnight After stirring, concentration, column chromatography (water/acetone containing 1%HOAc, 1:9-1:4) obtains target compound (660.2mg, 80%). MS ESI:m/z-:[M-H]^-, quasi-molecular ions: C₂₆H₃₅N₃O₉PS₂, 628.16, experiment value, 628.16.

Embodiment 344. (4R)-4- (2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxamide base) -2- methyl -5- (4- (phosphonato) -3- (4- (pyridine -2- disulfide group) butyrylamino) phenyl) valeric acid

4 DEG C at a temperature of, toward (4R) -4- (tertbutyloxycarbonylamino) -2- methyl -5- (4- (phosphonato) -3- (4- (pyridine -2- disulfide group) butyrylamino) phenyl) valeric acid (110.5mg, 0.175mmol) Isosorbide-5-Nitrae-dioxane (4ml) it is molten Concentrated hydrochloric acid (1.0ml, 37%) is added in liquid, after mixture stirs 30 minutes, is concentrated to dryness to obtain de- Boc crude product.This is thick Product is dissolved in n,N-dimethylacetamide (5ml), sequentially added into the solution 2,5- dioxo pyrrolidin -1- base 2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R)-1- methyl piperidine-2- formamido group) valeryl amino)- 4- methyl amyl) thiazole -4-carboxylic acid ethyl ester (110mg, 0.173mmol), N, N '-diisopropylethylamine (30ul, 0.172mmol), after mixture is stirred overnight, concentration, column chromatography (water/acetone containing 1%HOAc, 1:9-1:4) obtains targeted It closes object (129.1mg, 71%).MS ESI:m/z-:[M-H]^-, quasi-molecular ions: C₄₇H₆₇N₇O₁₂PS₃, 1048.38, experiment value, 1048.38.

Embodiment 345. (4R)-4- (2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R) -1- methyl piperidine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxamide base) -5- (3- (4- sulfydryl Amide-based small) -4- (phosphonato) phenyl) -2 methyl valeric acid.

By (4R)-4- (2- ((1R, 3R)-1- acetoxy-3-((2S, 3S)-N, 3- dimethyl-2- ((R)-1- methyl piperazine Pyridine -2- formamido group) valeryl amino) -4- methyl amyl) thiazole -4-carboxamide base) -2- methyl -5- (4- (phosphonato) -3- (4- (pyridine -2- disulfide group) butyrylamino) phenyl) valeric acid (30mg, 0.0285mmol) is added to DMAC N,N' dimethyl acetamide In (2ml) and sodium dihydrogen phosphate (0.1M, pH 7), dithiothreitol (DTT) (20mg, 0.129mmol) is added into the mixed liquor.It is mixed After closing object stirring 2 hours, concentration, column chromatographs the (H containing 1%HOAc₂O/MeCN, 1:9-1:4) obtain target compound (22mg, 85%).MS ESI:m/z-:[M-H]^-, quasi-molecular ions: C₄₂H₆₄N₆O₁₂PS₂, 939.38, experiment value, 939.38.

Embodiment 346.4- (4- brombutyl) -10- oxa- -4- aza-tricycle [5.2.1.0^ { 2,6 }] decyl- 8- alkene -3,5- Diketone.

By 10- oxa- -4- aza-tricycle [5.2.1.0^ { 2,6 }] decyl- 8- alkene -3,5- diketone (6.0g, 36.35mmol) It is added in n,N-dimethylacetamide (60ml), stirs 1 hour with sodium hydride (60% oil closes object, 1.50g, 37.50mmol) Afterwards, Isosorbide-5-Nitrae-dibromobutane (35.0g, 162.10mmol) and sodium iodide (0.50g, 3.33mmol) is added.Mixture is stirred Night is quenched with methanol (0.5ml), concentration, column chromatography (EtOAc/Hexane=1:8) obtain target compound (9.34g, 86%).MS ESI:m/z+:[M+Na]⁺, quasi-molecular ions: C₁₂H₁₄BrNaNO₃, 322.02, experiment value, 322.02.

347. methyl 2- of embodiment ((1R, 3R) -3- (tertbutyloxycarbonylamino) -1- [4'- (3 ", 6 "-bridging oxygens-tetrahydro Phthaloylamino) butoxy] -4- methyl amyl) thiazole -4-carboxylic acid's methyl esters.

By methyl 2- ((1R, 3R) -3- (tertbutyloxycarbonylamino) -1- hydroxy-4-methyl amyl) thiazole -4-carboxylic acid's first Ester (1.0g, 2.79mmol) and sodium hydride (120mg, 3.00mmol, 60% oil close object) are added to tetrahydrofuran (30ml), are stirred After 30 minutes, 4- (4- brombutyl) -10- oxa- -4- aza-tricycle [5.2.1.0^ { 2,6 }] decyl- 8- alkene -3,5- diketone is added (1.00g, 3.34mmol) and sodium iodide (50mg, 0.33mmol).Mixture is stirred overnight, and is quenched with methanol (0.5ml), dense Contracting, column chromatograph (EtOAc/CH₂Cl₂=1:10) obtain target compound (1.36g, 84%).MS ESI:m/z+:[M+Na]⁺, Quasi-molecular ions: C₂₈H₃₉NaN₃O₈S, 600.25, experiment value, 600.25.

348. methyl 2- of embodiment ((1R, 3R) -3- (N, N- tert-Butoxycarbonyl-methyl amino) -1- [4'- (3 ", 6 " - Bridging oxygen-tetrahydro phthaloylamino) butoxy] -4- methyl amyl) thiazole -4-carboxylic acid's methyl esters.

By methyl 2- ((1R, 3R) -3- (tertbutyloxycarbonylamino) -1- [4'- (3 ", 6 "-bridging oxygens-tetrahydro O-phthalic Acylamino-) butoxy] -4- methyl amyl) thiazole -4-carboxylic acid's methyl esters (1.30g, 2.25mmol) and sodium hydride (108mg, 2.70mmol, 60% oil close object) it is added in n,N-Dimethylformamide (80ml), after stirring 1 hour, iodomethane is added (460mg,3.24mmol).Mixture is stirred overnight, and concentration, column chromatographs (EtOAc/CH₂Cl₂=1:12-1:8) obtain targeted It closes object (1.01g, 76%).MS ESI:m/z+: [M+Na]⁺, quasi-molecular ions: C₂₉H₄₁NaN₃O₈S,614.26,Found,614.26.

Embodiment 349.2- ((1R, 3R) -3- (N, N- tert-Butoxycarbonyl-methyl amino) -1- [4'- (3 ", 6 "-bridging oxygens - Tetrahydro phthaloylamino) butoxy] -4- methyl amyl) thiazole -4-carboxylic acid

By methyl 2- ((1R, 3R) -3- (N, N- tert-Butoxycarbonyl-methyl amino) -1- [4'- (3 ", 6 "-after drying Bridging oxygen-tetrahydro phthaloylamino) butoxy] -4- methyl amyl) thiazole -4-carboxylic acid's methyl esters (900mg, 1.52mmol) is molten In 1,2- dichloroethanes (30ml) and toluene Mixed Solvent, it is added trimethyl stannic hydroxide (400mg, 2.21mmol).Mixing Object is stirred overnight at 100 DEG C, and concentration, column chromatographs (MeOH/CH₂Cl₂/ HOAc=1:10:0.01) obtain target compound (730mg, 94%).ESI: m/z:[M+Na]⁺, quasi-molecular ions: C₂₈H₃₉N₃NaO₈S, 600.22, experiment value, 600.22.

350. methyl 2- of embodiment ((1R, 3R)-1- acetoxy-3-(N, N- (tertbutyloxycarbonyl) (4'- (3 ", 6 "-bridges Oxygen-tetrahydro) butyl) amino) -4- methyl amyl)-thiazole -4-carboxylic acid.

By 2- ((1R, 3R)-1- acetoxy-3-(tert-butoxy-methyl amine)-4- amyl) 4-thiazolecarboxylic acid methyl esters (1.50g, 3.74mmol) and sodium hydride (180mg, 4.50mmol, 60% oil close object) are added to N, N- dimethylformamide In (80ml), after stirring 1 hour, 4- (4- brombutyl) -10- oxa- -4- aza-tricycle [5.2.1.0^ { 2,6 }] decyl- 8- is added Alkene -3,5- diketone (1.48g, 4.94mmol) and iodomethane (70mg, 0.467mmol).Mixture is stirred overnight, concentration, column layer Analyse (EtOAc/CH₂Cl₂=1:10-1:6) obtain target compound (1.60g, 69%).MS ESI:m/z+:[M+Na]⁺, ion Peak: C₃₀H₄₁NaN₃O₉S, 642.26, experiment value, 642.26.

Embodiment 351.2- ((1R, 3R)-1- acetoxy-3-(N, N- (tertbutyloxycarbonyl) (4'- maleimide fourth Base) amino) -4- methyl amyl)-thiazole -4-carboxylic acid.

By methyl 2- ((1R, 3R)-1- acetoxy-3-(N, N- (tertbutyloxycarbonyl) (4'- (3 ", the 6 "-bridges after drying Oxygen-tetrahydro) butyl) amino) -4- methyl amyl)-thiazole -4-carboxylic acid (800mg, 1.29mmol) is dissolved in 1,2- dichloroethanes In (40ml) and toluene Mixed Solvent, it is added trimethyl stannic hydroxide (400mg, 2.21 mmol).Mixture stirs at 100 DEG C It mixes overnight, concentration, column chromatographs (MeOH/CH₂Cl₂/ HOAc=1:5:0.01), component concentration is collected, it is dry.By resulting thick production Object is dissolved in pyridine (15ml), acetic anhydride (0.3ml, 3.17mmol) is added into the solution at 0 DEG C, mixture is stirred at room temperature Overnight, concentration, column chromatograph (MeOH/CH₂Cl₂/ HOAc=1:10:0.01) obtain target compound (578.4mg, 74%). ESI:m/z:[M+Na]⁺, quasi-molecular ions: C₂₉H₃₉N₃NaO₉S, 628.24, experiment value, 628.24.

Embodiment 352.1- (2- methyl -2- Oxyranyle) -2- phenylethylamine

0 DEG C at a temperature of, toward 1- (2- methyl -2- Oxyranyle) -2- phenylethyl) t-butyl carbamate (Sun, L.et al, J.Mol.Catalysis A:Chem., 2005,234 (1-2), 29-34) (300mg, 1.08 mmol's) Concentrated hydrochloric acid (37%, 2mL) is added in Isosorbide-5-Nitrae-dioxane (8mL) solution, continues stirring 1 hour at such a temperature, TLC is shown Through no starting reaction raw materials.Resulting mixed liquor is diluted with toluene (10ml), concentration is recrystallized from EtOH/Hexane Obtain the hydrochloride (201mg, 87%) of target compound. ESI:m/z:[M+H]⁺, quasi-molecular ions: C₁₁H₁₆NO, 178.12, experiment Value, 178.12.

Embodiment 353. (S) -5- phenyl -4- (tertbutyloxycarbonylamino) -2- methylpentene acetoacetic ester

- 78 DEG C at a temperature of, diisobutyl aluminium hydride (40ml, 40mmol, 1.0M) is slowly added to (S) -3- benzene Base -2- (tertbutyloxycarbonylamino)-methyl propionate (5.60g, 20.05mmol) CH₂Cl₂In (80ml) solution, reaction 45 After minute, it is added uses 1- (1- ethoxycarbonyl-ethyl) triphenylphosphinebromide (18.0g, 40.64mmol) and KOtBu at such a temperature Methylene chloride (80ml) solution of (5.00g, 44.64mmol) is formed by ylide solution, and object to be mixed reacts 1 at -78 DEG C It after hour, is stirred overnight at room temperature, then 1 liter of sodium dihydrogen phosphate is added into the mixture, be vigorously stirred, liquid separation, water It is mutually extracted with dichloromethane, merges organic layer, anhydrous sodium sulfate is dry, and concentration, column chromatographs (ethyl acetate/n-hexane=1:7- 1:5) obtain target product (5.50g, 83%), ESI:m/z:[M+Na]⁺, quasi-molecular ions: C₁₉H₂₇NNaO₄, 356.19, experiment Value, 356.20.

The general step of 354. polypeptide of embodiment condensation

By the hydrochloride salt of amine in methylene chloride or n,N-Dimethylformamide (0.2M), it is cooled to 4 DEG C, sequentially adds The amino acid (1.3eq) of tert-butoxycarbonyl protection, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (2eq), Or O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester (2eq) or tripyrrole alkane base phosphonium bromide hexafluorophosphate (2eq), I-hydroxybenzotriazole (1.5eq), n,N-diisopropylethylamine (3.5eq).Continue after reaction is slowly warmed to room temperature Reaction 15 hours, is diluted with ethyl acetate, and then successively uses 1M aqueous hydrochloric acid solution, saturated sodium bicarbonate, water and saturation chlorination Sodium water solution washing.Merge organic phase, anhydrous sodium sulfate dries, filters, and is concentrated, and column chromatographs (0%-20%MeOH:CH2Cl2) Obtain the polypeptide of tert-butoxycarbonyl protection.

Embodiment 355. takes off the general step of tert-butoxycarbonyl

The amino acid that tert-butoxycarbonyl is protected is dissolved in the dichloromethane solution containing 20% trifluoroacetic acid or salt containing 4M In Isosorbide-5-Nitrae-dioxane solution of acid, stir 30 minutes, or judged whether reaction is completed by TLC tracking.It is concentrated under reduced pressure Corresponding trifluoroacetate or hydrochloride polypeptide compound.Polypeptide containing trifluoroacetate can use the dichloromethane containing 2% hydrochloric acid Alkane/toluene solution is concentrated 3-4 times to form corresponding hydrochloride compound.

The general step of 356. Solid phase peptide synthesis of embodiment (SPPS)

The SPPS of tert-butoxycarbonyl protection uses Merrifield resin or modified PAM resin or MBHA resin. The SPPS of 9-fluorenylmethyloxycarbonyl protection uses Wang resin or 2- chlorine trityl chloride resin or HMPB, MBHA resin.Root According to the operating guidance of production of resins quotient, the loading of pre-treatment (pre-expansion) and amino-compound is carried out to resin.Uncle is used on resin The dichloromethane solution of 20% trifluoroacetic acid of amino acid of butoxy carbonyl protection or Isosorbide-5-Nitrae-dioxane of the hydrochloric acid containing 4M are molten Liquid deprotection (stirring 30 minutes), then successively uses n,N-Dimethylformamide, and methanol contains 50%N, N- diisopropyl second The methylene chloride and absolute dichloromethane of amine wash, complete for ensure to react for being related to the deprotection step of multiple unhindered aminas, The step needs to repeat before acylation once.Unhindered amina is suspended in the amino acid (3 equivalents of unhindered amina) by protecting, O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester or tripyrrole alkane base phosphonium bromide hexafluorophosphate (unhindered amina 3 equivalents) and n,N-diisopropylethylamine (5 equivalents of unhindered amina) composition solution in, after hybrid reaction 4 hours, successively use N,N-Dimethylformamide, methanol and methylene chloride washing.The reaction being acylated for being related to multiple unhindered aminas, to ensure to react It is complete, the Coupling step before deprotection need to repeat once.These steps need to be generally repeated, until needed for synthesis Peptide.

Embodiment 357. cuts the general step of peptide from Wang resin or 2- chlorine trityl chloride resin

The Wang resin and the methylene chloride containing 50% trifluoroacetic acid and tri isopropyl silane (1- of peptide will be combined 5%) it mixes, or the peptide that 2- chlorine trityl chloride resin combines is mixed with the methylene chloride containing 1% trifluoroacetic acid, mixing 2 It is filtered after hour, successively uses methylene chloride (3x30ml), methanol (3x30ml) elution, merging filtrate is concentrated, and is added after dry cold Ether, gained precipitating be de-protected peptide.

Embodiment 358. cuts the general step of peptide from Merrifield resin, modified PAM resin or MBHA resin

The resin and HF/Me of peptide will be combined₂S/anisole (10:1:1) or CH₃SO₃H/Me₂S/anisole (20:1: 1) mix (for the peptide containing cysteine, it should be with HF/anisole/Me₂S/p-thiocresol (10:1:1:0.2) Mixing), it after 2 hours, is concentrated, then diluted with trifluoroacetic acid, filters under nitrogen protection.Then methylene chloride is successively used (3x30ml) and methanol (3x30ml) elute resin, merging filtrate, and cold ether is added in concentration after dry, gained precipitating is De-protected peptide.

359. chromatogram purification of embodiment

By crude product mixtures of polypeptides by silica gel column chromatography purifying (5% to 25% methanol: dichloromethane eluent) or Reversed-phase HPLC purifying (0% to 70% aqueous methanol gradient elutes (preferably adding 1% acetic acid), and reaction terminates in 1 hour, after Compound target component collects sample after evaporation.

360. conjugated body of embodiment (conjugate) preparation

Antibody can pass through amide, thioether or disulfide bond and division inhibitor coupling as a kind of binding molecule.With containing The PBS buffer solution (pH8.0) of 50mM Boratex dilutes antibody (> 5mg/mL), is added dithiothreitol (DTT) (final concentration of 10mM), 35 DEG C processing 30 minutes, the releasable free sulfhydryl group of antibody.(1mM is added in PBS buffer solution with G-25 column gel permeation chromatography EDTA it after), is measured by Ellman reagent [5,5 '-two thiobis (2- nitrobenzoic acid)], is about coupled 8 on each antibody A sulfydryl.Antibody and Traut's reagent (2- imino group thiophene) (Jue, R., et al.Biochem.1978,17 (25): 5399- 5405) also releasable sulfydryl, or under the conditions of pH7~8, with SATP (N- succinimide-S- acetyl thio propionate) or N- The different attachment reaction such as succinimide-S- acetyl group (thio four ethanedioic acid) (SAT (PEG) 4), acts on by azanol, Formation sulfydryl (Duncan, R, et al, Anal.Biochem.1983,132,68-73, Fuji, N.et al, Chem.Pharm.Bull.1985,33,362-367).Substantially, 5~8 thiol molecules are connected on each antibody molecule.

Under the conditions of 4 DEG C, the dimethyl acetamide (DMA) (2~20% v/v) of the antibody containing free sulfhydryl group of Xiang Bingleng Middle be added (needs the dobell's solution (pH 9) of 0.5M to facilitate antibody and acetbromamide containing maleimide or acetbromamide group Alkylation) drug (drug of connection and the molar ratio of sulfydryl should be 1.2~1.5:1).After 1~2 hour, excessive addition Cysteine makes reaction terminating；Pass through the idol being concentrated after ultrafiltration, gel chromatography (G-25, buffer PBS), aseptic filtration Co-product.Pass through the absorbance measurement protein concentration and the drug number that is connected of each antibody at measurement 280nm and 252nm. Molecular exclusion HPLC can be used to measure specific gravity shared by the monomeric form of attachment, and the unbonded drug lower than 0.5% can It is used to measure with RP-HPLC.For thioether is keyed the monomer medicine to be formed, each antibody molecule can averagely be connected The small molecule of 3.2-4.2 mitotic inhibitor.

The type of jointing can be divided into dimethyl (phenyl) silicyl (DMPS), SMDP, 4- succinimido oxygen Butoxycarbonyl-methyl-α (two sulphur of 2- pyridyl group) toluene (SMPT), N- succinimide -4- (2- pyridine sulphur) valerate (SPP), N- Succinimide -4- (2- pyridine sulphur) propionic ester (SPDP), N- succinimide -4- (2- pyridine sulphur) butyrate (SPDB), amber Amber imide 4- (N- maleimidomehyl) hexamethylene -1- carboxylation ester (SMCC), n-hydroxysuccinimide-(poly- second two Alcohol) n- maleimide (SM (PEG) n) etc..Antibody (> 5mg/mL) be diluted in buffer (pH 6.5~7.5,5mM PBS, 50mM NaCl, 1mM EDTA) in, and Ligature 2 hours, and the molar ratio of chain junctor and antibody is 6~10 times or more.Instead Answer mixture that can separate by Sephadex G25 gel chromatography, the lower molecule of molecular weight can be removed.(the concentration of antibody It is measured by spectrophotometry, attachment includes pyridine thio.Extinction coefficient of the antibody at 280nm is 2067550M-1cm- 1.After the antibody of modification is handled with excessive dithiothreitol (DTT) (20 times or more equivalents), the 2- thiopyridines group of release is measured, 343, the extinction coefficient at 280nm is respectively 8080 and 5100M-1cm-1).1.2~1.5 equivalent bands are added into modification antibody There is the Tubulysin derivative molecular of mercapto groups.The reaction is being to carry out 5~18 hours at room temperature.Reaction mixture passes through Sephadex G25 gel chromatography removes the substance of not connected drug or other low molecular weights.Again by measurement 280nm and Absorbance at 252nm measures the concentration of connection product.Connection product is monomeric form, average each antibody molecule connection 3.2~4.5 drug molecules.

Be typically prepared side of the embodiment 361. containing the Tubulysin derivative molecular conjugate that can be reacted with mercapto groups Method.

In solution of 2.0 milliliter of 10 mg/ml pH value for 6.0~8.0 HER2 antibody, addition 0.70ml~ 2.0ml 100mM phosphoric acid (PBS), pH value be 6.5~8.5 buffers, TCEP (aqueous solution of 16-20 μ l, 20mM), at room temperature ~37.5 DEG C are incubated for 1~4 hour, and azido compound (azidobenzoic acid or the 2- (2- (2- hydroxyl-oxethyl) with equivalent is added Ethyoxyl) ethyoxyl azido compound) it is incubated for 1~4 hour at room temperature~37.5 DEG C, it is then added containing can be reacted with mercapto groups Tubulysin derivative molecular (the DMA solution of 28-32 μ l, 20mM).Mixture is incubated for 4~18 at room temperature~37.5 DEG C Hour, then DHAA (135 μ l, 50 millimeters) is added.After RT continuous culture overnight, mixture through G-25 column 100mM phosphoric acid, The buffer solution of 50mM NaCl pH 6~7.5 purifies to obtain conjugated body compound (75%~90% yield).Pass through liquid phase color Spectrum-mass spectrography mass spectroscopy drug/antibody ratio (DAR) is that 3.1~4.2, HPLC analysis is 94~99% monomer (Tosoh Bioscience, Tskgel G3000SW pillar, 7.8 millimeters of internal diameter, 30 centimetres of x, 0.5 ml/min, 100 minutes).

The test of 362. in vitro toxicity of embodiment

BJAB (Burkitt lymphoma cell), (people's Burkitt lymphoma is thin by BT-474 (breast cancer cell), Namalwa Born of the same parents), Ramos (people's Burkitt lymphoma cell), N87 (stomach cancer cell) and SK-OV-3 (ovarian cancer cell) are purchased from ATCC. Above-mentioned cell is all being grown in 1640 culture medium of RPMI of inactivated fetal bovine serum (FBS) containing 10%, condition of culture is 37 DEG C, In the incubator of 6%CO2.Colony formation can be used as a kind of method of toxicity test detection, referring to document (Franken, et al,Nature Protocols 1,2315-2319(2006)).The cell of test is according to 5000, every hole cell inoculation in 6 In orifice plate, the drug (mitotic inhibitor or conjugate) of 1pM~50nM gradient dilution is added, is incubated for 72 hours.It replaces old Some culture mediums, cell continue to cultivate, and have Clone formation after 7~10 days.Fixed cell, then with the (dilution of 0.2% crystal violet In 10% formalin or PBS) dyeing, count cell clone.The number of untreated cell (only plus culture medium) can pass through Clone's number for being formed in hole measures.The survival rate of cell be after treated with medicaments with formed in control group (drug is untreated) hole The ratio of clone number calculate.External activity inhibits the IC50 value of N87 (stomach cancer cell) to be listed in following table one.

Anti-tumor activity (balb/c/c nude mice, NCI-N87 transplantable tumor) in 363. body of embodiment.

In human gastric cancer N-87 cell lines Tumor xenograft model, her2 antibody-Tubulysin analog conjugated body (coupled object) (its structural formula is listed in table one) is divided into 3 batches and carries out Comparison of therapeutic experiment.First batch 129,133,181, 317,467；Second lot 365,377,385,412,444,474,480,546；Third batch 506,522,564,574,695, 677.Above-mentioned 3 batch drug is respectively with T-DM1 and physiological saline (PBS)) carry out evaluating drug effect in Mice Body.Five weeks old female N-87 cancer cell (5x 10 of the property balb/c/c nude mice (three batches totally 150 animals) under right shoulder⁷Cell/mouse) in no blood It is subcutaneously injected in clear culture medium.Tumour growth 8 days, mean size 130mm³.Then these animals are randomly divided into group (every group of 6 animals).Each group of control mice group is treated with phosphate buffered saline (PBS) (PBS).Conjugated body drug and T-DM1 difference To 6 mg/kg once daily of animals iv injection dosage.Measurements in volume three-dimensional degree every 3-4 days of tumour are primary, tumour Volume is calculated with formula tumor volume=1/2 (length x width x height).The weight of animal is also measured simultaneously.Work as satisfaction When following either condition, will kill animal: (1) loss of weight is more than 20%, and (2) gross tumor volume is greater than 2000mm³,(3) It is too seriously ill oneself to feed object and drinking-water, or (4) cutaneous necrosis.If mouse is considered as without swollen without apparent tumour Tumor.

Experimental result is painted on Figure 60,61 and 62.19 all conjugates do not cause the weight of animals to decline.Wherein it is conjugated Object 133,129,317,467,365,377,385,412,444,474,480,506,522,564,574,677 shows relatively strong Antitumor curative effect effect.T- is compared in the especially display of conjugate 133,129,467,377,385,412,444,474,480,677 The better anti-tumor activity of DM1.

The structural formula and its IC of some antibody-Tubulysin derivative conjugated bodies of table 1₅₀Value

Claims

1. a kind of cytotoxic molecule, has structure shown in Formulas I:

Or using with structure shown in Formulas I as the pharmaceutically acceptable salt of parent, hydrate or hydrated salt；Or there is Formulas I institute Show structure polymorph or Formulas I shown in structure optical isomer；

The R¹、R²、R³And R⁴Independently selected from H；C₁~C₈Alkyl or miscellaneous alkyl；C₂~C₈Allylic alkylation, alkynes alkyl, heterocycle, Heterocyclylalkyl；C₃~C₈Naphthenic base, aryl, heteroaryl perfume base, aralkyl, alkyl-carbonyl；C₄~C₈Alkyl-cycloalkyl, miscellaneous alkane Basic ring alkyl；

The X¹Selected from-O- ,-S ,-S-S ,-NH ,-CH₂Or-NR¹-；

The R⁹Selected from-H ,-OH ,=O ,-OR¹⁵,-OC (=O) R¹⁵,-OC (=O) NHR¹⁵,-OC (=O) R¹⁵SSR¹⁶, OP (=O) (OR¹⁵) or OR¹⁵OP (=O) (OR¹⁶)；

The R¹²Selected from-R¹⁵、-OH、-SH、-NH₂、-NH、-NHNH₂、-NH(R¹⁵)、-OR¹⁵、-R¹⁵COR¹⁶、-R¹⁵COOR¹⁶、- R¹⁵C(O)NH₂、-R¹⁵C(O)NHR¹⁷、-SR¹⁶、R¹⁵S (=O) R¹⁶、-R¹⁵P (=O) (OR¹⁷)₂、-R¹⁵OP (=O) (OR¹⁷)₂、- CH₂OP (=O) (OR¹⁷)₂、-R¹⁵SO₂R¹⁷、-R¹⁵X²R¹⁶、-R¹⁵C (=O) X³；

The X²Selected from-O- ,-S- ,-NH- ,-NHNH- ,-N (R¹⁵)-、-O-R¹⁵-、-S-R¹⁵, S (=O)-R¹⁵Or-NHR¹⁵In One kind；

The X³Selected from-OH ,-SH ,-NH₂、-NH(R¹⁵)、-NHNH(R¹⁵)、-OR¹⁵、-S-R¹⁵Or-NR¹⁵R¹⁶One of；

The R¹³And R¹⁴Independently selected from H ,-OH ,-SH ,-NH₂、-NHNH₂、-NH(R¹⁵)、-OR¹⁵、-COX²、-COX²R¹⁶、- R¹⁷、-F、-Cl、-Br、-I、-SR¹⁶、-NR¹⁶R¹⁷,-N=NR¹⁶,-N=R¹⁶、-NO₂、-SOR¹⁶R¹⁷、-SO₂R¹⁶、-SO₃R¹⁶、- OSO₃R¹⁶、-PR¹⁶R¹⁷、-POR¹⁶R¹⁷、-PO₂R¹⁶R¹⁷、-OP(O)(OR¹⁷)₂、-OCH₂OP(O)(OR¹⁷)₂、-OC(O)OP(O) (OR¹⁷)₂、-PO(OR¹⁶)(OR¹⁷)、-OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、-OC(O)R¹⁷,-OC (O) NHR¹⁷；-O-(C₄-C₁₂Sugar Glycosides) ,-N- (C₄-C₁₂Glucosides)；C₁-C₈Alkyl, miscellaneous alkyl, C₂-C₈Alkenyl, alkynyl, heterocycle；C₃-C₈Carbocylic radical, Heterocyclylalkyl, Miscellaneous alkyl naphthenic base, aryl, heteroarylalkyl, alkyl-carbonyl；C₄-C₈Alkyl-cycloalkyl；-NH(Aa)_1~4Or-CO (Aa)_1~4；It is described C₃-C₈Carbocylic radical includes naphthenic base；(Aa)_1~4For 1 to 4 identical or different natural or unnatural amino acid lists Position；

The R¹⁵、R¹⁶And R¹⁷Independently selected from C₁-C₈Alkyl, miscellaneous alkyl, C₂-C₈Alkenyl, alkynyl, heterocycle or C₃-C₈Carbocyclic ring Base, aryl, benzyl, alkylaryl, Heterocyclylalkyl, miscellaneous alkyl naphthenic base, heteroarylalkyl, alkyl-carbonyl, C₄-C₈Alkyl-cycloalkyl, Or Na⁺、K⁺、Cs⁺、Li⁺、Ca²⁺、Mg⁺、Zn²⁺、N⁺(R¹)(R²)(R³)(R⁴)、HN⁺(C₂H₅OH)₃Cationic salts；The C₃-C₈Carbon Ring group includes naphthenic base；

The Y¹And Y²Independently selected from N or CH；Q is 0 or 1；Work as q=0, Y³It is not present, Y⁴, Y⁵, Y⁶And Y⁷Independently selected from CH, N, NH, O, S or N (R¹), such Y², Y⁴, Y⁵, Y⁶And Y⁷Composition pyrroles, furans, thiophene, thiazole, oxazole, imidazoles together, three Azoles, tetrazolium, thiadiazoles, oxadiazoles miscellaneous aromatic rings；Work as q=1, Y³, Y⁴, Y⁵, Y⁶And Y⁷Independently selected from CH or N, such Y², Y³, Y⁴, Y⁵, Y⁶And Y⁷Phenyl ring, pyridine, pyrazine, pyridazine, triazine, the miscellaneous aromatic ring structure of tetrazine are constituted together.

2. cytotoxic molecule according to claim 1, which is characterized in that the group R¹、R², R²、R³Or R³、R⁴It is logical Cross-CH₂Any connection respectively with Y¹Atomic building cyclic group or R⁵、R⁶By-CH₂It is former arbitrarily to connect coupled carbon Son constitutes cyclic group or R¹¹、R¹²By-CH₂Any connection constitutes cyclic group with its connected carbon atom and carbonyl, or R¹³、R¹⁴By-CH₂Any connection and Y⁵And Y⁶Atomic building cyclic group；

The cyclic group is selected from C₃-C₇Naphthenic base, alkyl naphthene, cycloalkanes heteroaryl perfume base, cycloalkanes miscellaneous alkyl, heterocycle, heterocycle Alkyl, carbonyl naphthene, cycloalkanes carboxyl, cycloalkanes amide groups, cycloalkanes amido, cycloalkanes oxygen ether, cycloalkanes thioether group, cycloalkanes selenide base, One of aromatic radical, miscellaneous aromatic alkyl and heteroaryl perfume base.

3. cytotoxic molecule according to claim 1, it is characterised in that the R¹、R²、R⁷、R⁹、R¹²、R¹³And R¹⁴Not End can also be the same as chain junctor or the functional group being coupled with cell surface bind receptor molecule, the functional group containing one Selected from SH, NH₂、COOH、-O-NH₂、-N₃、NHNH₂, (Py is C to-SSPy₅H₄N)、-SSAr、-SSC₆H₄NO₂、-SSC₆H₃(NO₂) (COOH)、-SC(O)R¹、-SSC₆H₃(NO₂)₂、-C(O)NH₂、-C(O)H、-C(O)NHNH₂、-C(O)R¹、-C(O)C(O)R¹、- ArC(O)R¹、-C(O)CH₂X³、-C(O)X³、-ArCH₂X³、

The X³And X⁴Independently selected from F, Cl, Br, I ,-OS (O)₂Ar、-OS(O)₂R¹、-OS(O)₂CF₃)、

-OC₆F₅、-OC₆FH₄,-OC₆F₂H₃,-OC₆H₄(NO₂)、-OC₆H₃(NO₂)₂。

4. cytotoxic molecule according to any one of claims 1 to 3, which is characterized in that the cytotoxic molecule For with one of structure shown in Formulas I -01~I-67:

The R²⁰Selected from C₁-C₈Linear or branched paraffin, miscellaneous alkane, C₂-C₈Linear or branched-chain alkene or alkynes, C₃-C₈Aromatic hydrocarbons, alkane Base aromatic hydrocarbons, heterocyclic hydrocarbon or heteroaryl hydrocarbon, carbonyl rouge-C (O) OR¹⁷, carbonyl amine-C (O) NR¹⁷R¹⁸；

The Z²And Z³Independently selected from H ,-OH ,-NH₂、OR¹⁷、NHR¹⁷、COOH、COOR¹⁷、C(O)SR¹⁷、C(O)R¹⁷、C(O) NHR¹⁷、C(O)NHNHR¹⁷、C(O)NH、R¹⁸、OCH₂OP(O)(OR¹⁸)₂、OC(O)OP(O)(OR¹⁸)₂、NR¹⁷CH₂OP(O) (OR¹⁸)₂、NR¹⁷C(O)OP(O)(OR¹⁸)₂、OPO(OR¹⁸)₂、NR¹⁷PO(OR¹⁸)₂、OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂、OC(O) R¹⁸、NR¹⁷C(O)R¹⁸、OC(O)NHR¹⁸、NR¹⁷C(O)NHR¹⁸、NR¹⁷C(O)OR¹⁸、NR¹⁷C(O)SR¹⁸、NR¹⁷C (=NH) NHR¹⁸、 OSO₂(OR¹⁸)、O-(C₄-C₁₂Glucosides), NR¹⁷SO₂(OR¹⁸)、NR¹⁷-(C₄-C₁₂Glucosides), C₁-C₈Alkyl, C₃-C₈Carbonyl alkyl is miscellaneous Ring；

The R¹⁷And R¹⁸Independently selected from H, C₁~C₈Alkyl or miscellaneous alkyl；C₂~C₈Carbonyl alkyl, alkylene, alkyl carbonyl, alkynes base Or heterocycle；C₃~C₈Aromatic radical, naphthenic base or carbonyl naphthene；

The R¹⁹Selected from H ,-OH ,-NH₂、-OSO₂(OR¹⁸)、-XCH₂OP(O)(OR¹⁸)₂、-XPO(OR¹⁸)₂、-XC(O)OP(O) (OR¹⁸)₂、-XC(O)R¹⁸、-XC(O)NHR¹⁸、C₁~C₈Alkyl or carboxylic acid derivates, C₂~C₈Alkylene, alkynes base, alkyl carbonyl or Carbonyl alkyl, C₃~C₈Aromatic radical, naphthenic base or carbonyl naphthene；Or pharmaceutical salts；

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

5. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula II:

Perhaps structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound；Or this The optical isomer of structural formula；

When m ' is 0, L ' is not present；When m ' is not 0, conjugate by two or more chain junctor connect cell surface by Body binding molecule T；

Two sites or this two chains in structure shown in structure bracket shown in the other linking II of two chain junctors Junctor connects a site in structure shown in structure bracket shown in knot II by branched chain junctor again；

The W is ennation, target binding molecule unit T is connected on Amino Acid Unit (Aa), if directly connecting when without Aa Meet V；

The unit W includes the interval body self decomposed, a peptide unit, hydrazone bond, cystine linkage thioether bond, ester each independently Key or amine key；The w is 0 or 1；

The Aa is natural amino acid or non-natural amino acid unit；The integer that the r is 0 to 12, (Aa) r are 0~12 The identical or different Amino Acid Unit of a structure；The natural amino acid or non-natural amino acid unit include a peptide, dipeptides, three Peptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, 11 peptides or dodecapeptide unit；

The V is isolation body unit, and the isolation body unit is independently selected from H, O, NH, S, C₁-C₈Alkyl, C₂-C₈Miscellaneous alkyl, alkene Alkyl, alkynes base；C₃-C₈Aryl, heteroaryl, heterocycle, carbocylic radical, miscellaneous alkyl naphthenic base, alkyl-carbonyl；C₄-C₈Alkyl-cycloalk Base；1~4 Amino Acid Unit or (CH₂CH₂O)r；The heterocycle includes Heterocyclylalkyl；The charcoal ring group includes naphthenic base；Institute State (CH₂CH₂O) the integer that the r of r is 0~12；The v is 0,1 or 2；

The cell surface receptor binding molecule T is any type of cell combination body, the structure including peptide or similar peptide: anti- Body, single-chain antibody can be with the antibody fragment in conjunction with target cell, and monoclonal antibody, single monoclonal antibodies can be with target The monoclonal antibody fragment of cell combination, chimeric antibody can resist with the chimeric antibody fragment in conjunction with target cell, functional areas Body can imitate the engineered protein of antibody, fibronectin combination with the functional areas antibody fragment in conjunction with target cell Adnectin, pre-designed ankyrin repeat protein (DARPin), lymphokine, hormone, vitamin, growth factor, colony-stimulating The factor, nutrition transmits molecule, transferrins, cell surface smaller ligand, or is connected with the albumin of cell combination body, high score Cell-binding molecules (binding peptide, albumen, antibody or cell surface smaller ligand) are contained on son or branch macromolecule, surface High molecular material, protein, liposome, nano particle, bubble crusty pancake or (virus) micro-capsule.

6. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in formula III:

Perhaps this structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound；Or The optical isomer of this structural formula；

Described T, L, L ', m, m ' and the definition of n are identical as claim 5.

7. conjugate according to claim 6, which is characterized in that the conjugate is with formula III -01~III-19 institute Show one of structure:

The n is the integer of 1-20；The p is the integer of 0-100；

The definition of the Z2 and Z3 is identical with the definition in claim 4；

The X¹、R¹、R²、R³It defines identical as claim 1；The M₁And M₂Independently selected from H⁺、Na⁺、K⁺、Li⁺、NH₄ ⁺、N (R¹R²R³R⁴)⁺Or NH (C₂H₅OH)₃ ⁺Pharmaceutically acceptable application salt ion.

8. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in formula IV:

The definition of described T, L, m and n are identical as claim 5.

9. conjugate according to claim 8, which is characterized in that the conjugate is with shown in formula IV -01~IV-11 One of structure:

The n is the integer of 1-20；P is the integer of 0-100；

The Z²And Z³It is identical as the definition in claim 4；

The X¹、R¹、R²And R³Definition is identical as claim 1,

The M₁And M₂Salt ion H independently selected from pharmaceutically acceptable application⁺、Na⁺、K⁺、Li⁺、NH₄ ⁺、N(HR¹R²R³)⁺Or NH(C₂H₅OH)₃ ⁺。

10. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula V:

Perhaps this structural formula pharmaceutically acceptable salt hydrate or hydrated salt；Or the polymorph of this compound；Or The optical isomer of this structural formula, racemic modification, diastereoisomer or enantiomter；

The definition of described T, L, m and n are identical as claim 5.

11. conjugate according to claim 10, which is characterized in that the conjugate is with shown in Formula V -01~V-18 One of structure:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹、Y⁷It is identical as being defined in claim 1；

The Z²It is identical as being defined in claim 4.

12. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula IV:

The definition of described T, L, m and n are identical as claim 5.

13. conjugate according to claim 12, which is characterized in that the conjugate is with Formula IV -01~VI-30 institute Show one of structure:

The n is the integer of 1-20；P is the integer of 0-100；

The Z²And Z³It is identical as the definition described in claim 4；

The X¹、R¹、R²、R³It defines identical as the definition in claim 1；

14. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula VII:

The definition of described T, L, m and n are identical as with claim 5.

15. conjugate according to claim 14, which is characterized in that the conjugate is with Formula VII -01~VII-15 One of shown structure:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹Definition it is identical as the definition in claim 1, the Z³Definition it is identical as the definition in claim 4.

16. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula VIII:

The definition of described T, L, m and n are identical as claim 5.

17. conjugate according to claim 16, which is characterized in that the conjugate is with Formula VII -01~VII-43 One of shown structure:

The n is the integer of 1-20；The p is the integer of 1-100；

The Z²And Z³It is identical as the definition in claim 4.

18. a kind of toxicity molecule using with structure shown in Formulas I described in claim 1 is the conjugate of parent, feature exists In with structure shown in Formula IX:

Described T, L, L ', m, m ' and the definition of n are identical as claim 5.

19. conjugate according to claim 18, which is characterized in that the conjugate is with Formula IX -01~IX-28 institute Show one of structure:

The n is the integer of 1-20；The p is the integer of 1-100；

The X¹、X²Definition it is identical as the definition in claim 1；

The Z²And Z³It is identical as the definition in claim 4.

20. according to conjugate described in claim 5,6,8,10,12,14,16 or 18, which is characterized in that the T is a kind of thin Cellular surface binding reagents, the cell surface binding agent can be combined with target cell, and the target cell includes: tumour cell, disease Malicious infection cell, the cell of microorganism infection, parasite infected cells, immunocyte, active cell, bone marrow cell, activation T- Cell, B cell or melanocyte；Or the cell of the one or more following molecules of expression: Apo2, ASLG659, BAFF-R, bone Morphogenesis of proteins receptor BMPR1B, IGF-IR, CA125, CanAg, E16, EGFR, EphA2 receptor, ErbB2, interleukin, MUC1, MUC16, NAPI-3B, NPTIIb, SLC34A2,34 member 2 of sapiens's Solute Carrier family, II type sodium rely on phosphate cotransporter egg White 3b, VEGF, TF, MY9, anti-B4, EpCAM, FcRH2, C242, CD1, 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, CD43, 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,CD85e,CD85f,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, 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, CD389, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, Tetratocarcinoma derivative growth factor, APO2, ASLG659, bone morphogenetic protein receptor BMPR1B, CRIPTO, Annexin A1, kernel, Endoglin ROBO4, aminopeptidase N CEA, CEACAM3, CEACAM5, DLL3, DLL4, CTLA4, CXCR4, Endoglin, EPCAM, endothelin receptor ETBR, EphA receptor, EphB receptor, epidermis Growth factor receptors, EGFRvIII, Endothelin ETBR, ERBB2, FCGR1, FOLR, FCRH1, GD2 gangliosides, G-28, GD3 idiotype, HER2, HER2/neu, HER3, HLA-DOB, MHC II grades of molecule Ia antigens, HLA-DR10, HLA-DRB, Integral protein, IGF1R, IL-2 receptor, IL-6R, IRTA2, LIV-1, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MS4A1, MPF, MSLN, MPF, megacaryocyte synergistic factor, mesothelium albumen, MUC1, MUC1-KLH, MUC16, gp100, MART1, MPG, MS4A1, paranuclein, neu oncogene, P21, the paratope of anti-N- glycosyl neuraminic acid, GEDA, mesothelium albumen, NaPi2b, Napi3b, Ras mutant, gp100, p53 mutant, Proteinase3, BCR-abl, Tetratocarcinoma Derivative growth factor, EphA receptor, EphB receptor, EGFR, EGFRvIII, ETBR (Endothelin), PDL1, p97, Sema5b, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, brain signal albumen 5bHlog, sema structural domain, the 7th fibrin ferment Sensitive Protein repetitive unit, cytoplasmic domain, class PLAP testis alkaline phosphatase, PSA, PSCA, PSMA, ROBO4, TAG72, T Cell transmembrane albumen, Tie, TNFRSF10B, TNFRSF13B, TPBG, TRAIL-R1, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2, six prostate cross-film epithelium antigen 1STEAP1, STEAP2, HGNC8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate antigen, survivin, hTERT, sarcoma translocation breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, TMPRSS2ETS fusion, NA17, PAX3, ALK, androgen receptor, cell periodic protein B 1, poly sialic acid, MYCN, RhoC, TRP-2, GD3, fucose GM1, mesothelium albumen, PSCA, MAGEA1, sLe (a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, Human sperm protein 17, LCK、HMWMAA、AKAP-4、SSX2、XAGE1、B7H3、Legumain、Tie 2、VEGFR2、MAD-CT-1、FAP、PDGFR-β、 MAD-CT-2, Fos related antigen 1 and cell expresses insulin growth factor receptors, EGF-R ELISA or folic acid by Body.

21. according to conjugate described in claim 5,6,8,10,12,14,16 or 18, which is characterized in that the L has following One of structure:

The R²¹Independently selected from-C₁~C₉Alkylene-,-C₁~C₇Carbocylic radical-,-O- (- (C₁~C₈Alkyl)-,-arlydene-,- C₁~C₉Alkylene-arlydene-,-arlydene ,-C₁~C₉Alkylene ,-C₁~C₉Alkylene-(C₁~C₈Carbocylic radical)-,-(C₃~ C₈Carbocylic radical)-C₁~C₉Alkylene-,-C₃~C₈Heterocycle-,-C₁~C₁₀Alkylene-(C₃~C₈Heterocycle)-,-(C₃~C₈It is miscellaneous Ring group)-C₁~C₉Alkylene-,-(CH₂CH₂O)_k-,-(CH(CH₃)CH₂O)_kOr-(CH₂CH₂O)_k-CH₂-；

The k is the integer of 1-20；R ' and R " independently is H or CH₃。

22. according to conjugate described in claim 5,6,8,10,12,14,16 or 18, which is characterized in that include on the L There is self-degradation part, structural formula is one of following:

The atom of (*) label is additional slider or releasable connector, antimitotic reagent and/or combination The position of molecule T connection；

Described X, Y and Z³Independently selected from NH, O or S；

The Z²For H, NH, O or S；

The v is 0 or 1；

The Q is H, OH, C₁-C₆Alkyl, OCH₂CH₂)_n、F、Cl、Br、I、OR¹⁷Or SR¹⁷、NR¹⁷R¹⁸, N=NR¹⁷, N=R¹⁷、 NR¹⁷R¹⁸、NO₂、SOR¹⁷R¹⁸、SO₂R¹⁷、SO₃R¹⁷、OSO₃R¹⁷、PR¹⁷R¹、POR¹⁷R¹⁸、PO₂R¹⁷R¹⁸、OPO(OR¹⁷)(OR¹⁸) or OCH₂PO(OR¹⁷(OR¹⁸), the R¹⁷, R¹⁸Independently selected from H, C₁-C₈Alkyl, C₂-C₈Alkenyl, alkynyl, miscellaneous alkyl, C₃-C₈Virtue Base, heterocycle, miscellaneous alkyl, naphthenic base, Heterocyclylalkyl, heteroarylalkyl, alkyl carbonyl or cationic salts.

23. according to the conjugate in claim 5,6,8,10,12,14,16 or 18, which is characterized in that include non-on the L Self-degradation part, the structural formula of the non-self-degradation part are one of following: * (CH₂CH₂O)_n*、

The atom of (*) label is additional slider or releasable connector, antimitotic agent and/or The link position of binding molecule；

The m is the integer of 1-10, and n is the integer of 1-20.

24. a kind of conjugate for the cytotoxic molecule for being connected with two molecules has general structure below:

X₁、X₂、X₃And X₄It is independently NH, NR¹、O、S、CH₂, Se or NHNH；

The R¹And R²Definition it is identical as the definition in claim 1, n and L define identical as the definition in claim 5；

The D₁And D₂Independently selected from cytotoxic molecule described in any one of Claims 1 to 4, or in which one of be Selected from cytotoxic molecule described in any one of Claims 1 to 4, another is selected from can effective treating cancer Chemical molecular, the chemical molecular includes one or more of molecule and its pharmaceutically acceptable salt as described below: Calicheamicin, maytansine derivative, aplysiatoxin, CC-1065 analog, adriamycin, Taxane derivative, pyrroles's Benzodiazepine (PBD) dimer, tomamycin dimer, Anthramycin dimer, indoline Benzodiazepine dimer, imidazoles Benzodiazepine two Aggressiveness or oxazoline Benzodiazepine dimer, calicheamycin and Enediyne Antibiotic, goose cream Peptide toxin, Indolecarboxamide, D actinomycin D, azaserines, bleomycin, Epi-ADM, eribulin, tamoxifen, she Up to than star, Ali's statin, monomethyl Ali's statin E, Ali's statin MMAE, Ali's statin MMAF, Ali's statin PYE, Ali Statin TP, aplysiatoxin 2-AQ, aplysiatoxin 6-AQ, aplysiatoxin EB or aplysiatoxin EFP, mostly card meter Xing, phosphinothioylidynetrisaziridine, lattice Your moral mycin, HSP90 inhibitor, centanamycin it is holy it receive mycin, methotrexate (MTX), thio-tepa, eldisine, Changchun is new Alkali, hemiasterlins, nazumamides, microginins, radiosumins, streptonigrin, SN38 and its derivative, Camptothecine metabolite, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU-15968, siRNA or molecular weight are less than 100KD.

25. a kind of Pharmaceutical composition, which is characterized in that including claim 5,6,7,8,9,10,11,12,13,14,13,16, 17, conjugate described in 18 or 19 to be enough to generate the dosage of preferable therapeutic effect as treatment component, or aids in Pharmaceutically acceptable salt and their diluent, carrier or excipient and form treatment component.

26. pharmaceutical composition according to claim 25, which is characterized in that the composition comprising following weight percent

Claim 5,6,7,8,9,10,11,12,13,14,13,16,17,18 or 19

The polyol compound includes fructose, mannose, maltose, lactose, sugar, xylose, ribose, rhamnose, galactolipin, grape It is sugar, sucrose, trehalose, sorbose, melezitose, gossypose, mannitol, xylitol, antierythrite, maltitol, xylitol, red Moss sugar alcohol, sorbierite, glycerol, oxidizing ferment or one or more of gluconic acid and its metal salt；

The surfactant includes polysorbate, poloxamer, Triton, lauryl sodium sulfate SDS, lauryl sulfate Sodium, octyl glycoside sodium, lauryl-, myristyl-, sub- oil base (linoleyl)-or stearyl-sulfobetaines, laurel Base-, myristyl-, sub- oil base-or stearyl-sarcosine；Sub- oil base-, myristyl-or cetyl-glycine betaine；Laurel Amido propyl-, cocamidopropyl propyl amide-, sub- oleamide propyl-, myristamide propyl-, dodecanamide propyl, coconut oleoyl amine third Base (palmidopropyl)-or isostearoyl amine propyl-glycine betaine, myristamide propyl-, cocamidopropyl propyl amide-or different Stearamide propyl-dimethyl amine, sodium methyl cocoyl taurate or methyl oleoyl taurine disodium；Laurel, nutmeg, Sub- oil or stearic-sour sodium, sub- oil, nutmeg, dodecyl or cetyl betaine, methyl cocoyl sodium, dimethyl methyl ox Sodium sulfonate, cocounut oil both sexes glycine or different tristearin ethylimidonium ethosulfate, polypropylene glycol, polyethylene glycol, second The copolymer p luronics F68 of glycol, ethylene and propylene；The polysorbate is polysorbate 20, polysorbate 40, poly- mountain Pear ester 65, polyoxyethylene sorbitan monoleate, sorbimacrogol oleate 100 or polysorbate 85；The poloxamer is PLURONICS F87, polycyclic oxygen second Alkane-polypropylene oxide, poloxamer188 or polyethylene glycol propylene glycol；

The complexing agent is EDTA or EGTA；

The preservative be selected from benzylalcohol, stearyl dimethyl benzyl ammonium chloride, ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, In fourth benzylalcohol, Arrcostab such as methyl or propylben, catechol, resorcinol, cyclohexanol, 3- amylalcohol or metacresol It is a kind of；

Antioxidant is ascorbic acid and/or methionine；

The amino acid or proppant are selected from mannitol, sorbierite, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, sodium citrate, Or one of NaCl；

It is citric acid, succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, winestone that the buffer solution, which is selected from, Acid, succinic acid, Tris hydrochloride, one of phthalandione or phosphoric acid solution；

The pH value of the composition is 4.5~8.0, and the medicinal liquid osmosis of composition pressure is rubbed this mole for 225 to 370.

27. a kind of Pharmaceutical composition, which is characterized in that composition described in claim 26 is prepared into prepackage injection or filling It is lyophilized into minute hand after to cillin bottle, or powder is prepared by efficient spray drying.

28. Pharmaceutical composition described in claim 25 or 26 is with the application in analgesic, which is characterized in that described to want With composition and analgesic with using, the analgesic is the general Shandong of hydrochloric acid of the benzyl alcohol of 1wt%, 0.2wt%~2.0wt% Cacaine, the lidocaine hydrochloride of 0.2wt%~1.0wt%, 0.3wt%~0.5wt% anesin, C16H25NO2, morphine, sulphur Sour morphine, Hydromorphone, dihydrohydroxycodeinone, Gabapentin, cyclobenzaprine, Trazodone or clonidine.

29. Pharmaceutical composition described in claim 25 or 26 is treating or preventing cancer, disease of immune system and infectivity Pharmaceutical composition described in application or claim 25 or 26 in disease medicament is combined with synergist to be treated or prevented Application in cancer, disease of immune system and infectious disease medicament.

30. application according to claim 29, which is characterized in that the synergist is selected from following one or more of drugs: Orencia, abiraterone acetic acid esters, albumin taxol, paracetamol/hydrocodone, No. CAS is 1420477-60-6 Acala not scholar replace Buddhist nun, adalimumab, RhIL-2, Afatinib dimaleate, Ai Le replace Buddhist nun, A Lundan Anti-, alitretinoin, No. CAS is 1018448-65-1 Love Capital trastuzumab maytansine, amphetamine or amphetamine/right phenylpropyl alcohol Amine salt-mixture, Anastrozole, anthracycline antibiotic, Aripiprazole, atazanavir, Aunar pearl monoclonal antibody, Atorvastatin, No. CAS For the A Wei monoclonal antibody of 1537032-82-8, pazopanib, No. CAS Baily department for 866323-14-0 he, Avastin, bud Salol fourth, Beaune spit monoclonal antibody, and bortezomib, bosutinib, originally appropriate former times monoclonal antibody, cloth add for Buddhist nun, budesonide, budesonide/good fortune Mo Teluo, buprenorphine, No. CAS Cabazitaxel for 183133-96-2, card, which is won, replaces Buddhist nun, capecitabine, and Carfilzomib is fitted into The cell of antigen receptor engineering T, celecoxib, match is vertical to replace Buddhist nun, and Cetuximab, cyclosporine, cinacalcet, gram azoles is for Buddhist nun, CAS Number examining than for Buddhist nun for 934660-93-2, dabigatran, dabrafenib, Dacarbazine, daclizumab, Daptomycin, No. CAS For the auspicious monoclonal antibody of moral of 945721-28-8, darbepoetin, No. CAS be 206361-99-1 darunavir, imatinib mesylate, Dasatinib, No. CAS be 173146-27-5 denileukin -2, No. CAS be 615258-49-7 ground promise monoclonal antibody, double the third penta Sour sodium, dexamethasone, No. CAS R-lansoprazole for 138530-94-6, methylphenidate, No. CAS is exerted for the ground of 1363687-32-4 Appropriate former times monoclonal antibody, Doxycycline, Duloxetine, No. CAS moral prestige monoclonal antibody for 1428935-60-7, No. CAS is 915296-00-3's According to Torr pearl monoclonal antibody, te Yi Feimosi, tenofovir/efavirenz replaces Buddhist nun according to promise, ezetimibe, and ezetimibe/pungent cuts down him Spit of fland, fenofibrate/fenofibrate/according to promise replace Buddhist nun, fingomode, fluticasone propionate, fluticasone/salmeterol, fluorine dimension department Group, Gefitinib, copaxone, goserelin acetate, Yi Qu replace Buddhist nun, and Imatinib, she replaces not monoclonal antibody by cloth, replace Buddhist nun according to Shandong, she Song replaces Buddhist nun, ifosfamide, and infliximab replaces Buddhist nun, insulin aspart, insulin detemir, insulin glargine, bad dried meat according to benefit Insulin, interferon beta 1a, interferon beta 1b, Lapatinib, Yi Puli nurse Ma, Ipratropium Bromide/, Li Namoli, pleasure is cut down for Buddhist nun, Letrozole, levothyrocine, levothyrocine, lidocaine, Linezolid, Liraglutide, No. CAS is 608137-33- 3 lysine amphetamines, Memantine, methylphenidate, metoprolol, modafinil, Mometasone, No. CAS is 906805-06-9's Resistance to former times appropriate monoclonal antibody, No. CAS linatinib for 698387-09-6, No. CAS nilotinib for 641571-10-0, No. CAS is The Ni Lapani of 1038915-60-4, No. CAS Wu Dankang that receives for 946414-94-4, difficult to understand, pa pearl monoclonal antibody difficult to understand are difficult to understand Mei Shatan, Olmesartan, oxaliplatin, Hydrochioro, omalizumab, -3 fatty-acid ethyl ester of omega, Oseltamivir, CAS Number Buddhist nun, oxycodone hydrochloride, the auspicious monoclonal antibody of pa, pa pearl monoclonal antibody, pa Lip river monoclonal antibody, pazopanib, PD- are replaced for difficult to understand this of 1421373-65-0 1 antibody, PD-L1 antibody, Peg-IFN alpha-2b α -2a, No. CAS pyridine aldoxime methyliodide (PAM) monoclonal antibody for 1374853-91-4, pemetrexed, training Trastuzumab, pneumococcal conjugate vaccine, pomalidomide, Pregabalin, Propranolol, Quetiapine, Rabeprazole, 223Ra Chloride, Raloxifene draw for Wei, Lei Molu monoclonal antibody, Lucentis, Rui Gefeini, Rituximab, razaxaban, sieve is drawn Meter is pungent, rosuvastatin, and Luso benefit replaces Buddhist nun, salbutamol, and No. CAS Sa Woli for 1313725-88-0 replaces Buddhist nun, and fertile benefit is replaced Buddhist nun, Suo Malu peptide, sevelamer, silaenafil, No. CAS be the appropriate former times monoclonal antibody of 541502-14-1 department, sitagliptin, sitagliptin/ Melbine, Solifenacin, Sony De Ji, Sorafenib, Sutent, tacrolimus, bright for Rameau pa, Temozolomide replaces Sirolimus, tenofovir/emtricitabine, testosterone gel, Thalidomide, Tiotropium Bromide, Toremifene, Trimetinib are bent appropriate Pearl monoclonal antibody, No. CAS be 114899-77-3 Qu Beite tincture, No. CAS trifluorothymidine for 183204-72-0/for west is disturbed ties up Formic acid, excellent special gram, Valsartan, Wei Lipani, Vande Thani, Wei Luofeini, No. CAS outer Buddhist nun for 1257044-40-8 hides can Lay This, Vorinostat, A Puxibai, shingles zoster vaccine, and the like, derivative, pharmaceutically acceptable salt, carrier or tax Shape agent.