CN104906592B - Side chain connected body for pharmaceutical grade protein conjugate - Google Patents

Abstract

The present invention relates to for connecting protein and connection medicine to the method for pharmaceutical grade protein conjugate, wherein described medicine is connected to protein by specific side chain connected body, the side chain connected body is comprising peptide chain and derived from adjacent hydroxyl to aminobenzyl alcohol, wherein, the peptide chain to amino by being connected to benzyl ring, the medicine is attached partially on phenyl ring by benzyl alcohol, and the protein is connected on phenyl ring by adjacent oh group；The invention further relates to the method for the pharmaceutical grade protein conjugate is prepared by various intermediates, it is related to the pharmaceutical use of such pharmaceutical grade protein conjugate, such as control the growth of undesirable cell, it is related to the pharmaceutical composition for including such pharmaceutical grade protein conjugate, and is related to the intermediate for preparing the pharmaceutical grade protein conjugate.

Description

Side chain connected body for pharmaceutical grade protein conjugate

It is on 2 23rd, 2012 the applying date that the application, which is, Chinese Patent Application No. is 201280010479.6 (international applications Number be PCT/EP2012/053039), the patent of invention of entitled the side chain connected body of pharmaceutical grade protein conjugate " be used for " The divisional application of application.

Technical field

The present invention relates to for connecting protein and connection medicine to the method for pharmaceutical grade protein conjugate, wherein the medicine Thing is connected to protein by specific side chain connected body, and the side chain connected body is comprising peptide chain and derived from adjacent hydroxyl to amino Benzylalcohol, wherein, the peptide chain to amino by being connected to benzyl ring, and the medicine is attached partially on phenyl ring by benzyl alcohol, The protein is connected on phenyl ring by o- oh group；It is described the invention further relates to be prepared by various intermediates The method of protein-drug-conjugate, is related to the pharmaceutical use of such pharmaceutical grade protein conjugate, such as control undesirable cell Growth method, be related to the pharmaceutical composition for including such pharmaceutical grade protein conjugate, and be related to and prepare the albumen The intermediate of matter drug conjugates.

Background technology

Most of medicines for chemotherapy have serious side effect, and which has limited their curative effect and purposes.Will be such Payload (payload) (i.e. the compound of pharmaceutical activity, such as medicine) is connected to targeting agent (particularly monoclonal antibody) energy New antibody drug conjugates (ADC) are provided, such as treatment of cancer.The usual receptor 3 monoclonal antibody (mAb) of tissue specificity Composition Control, while medicine provides therapeutic effect.ADC efficiency and tolerance is dependent on target antigen, drug titers and coupling skill Interaction between art.Especially, the chemical property of connected body consumingly influences ADC specificity and security.

There is the connected body of the unstable chemcial property of limited stability under conditions of substituting outside physiological cells, be such as based on The connected body of hydrazone and disulphide, outside physiological cells under conditions of stable connected body, particularly with high plasma stability Connected body, for improve treatment applicability be desirably because medicine should only discharge in the cell, this is thin Born of the same parents target (target) by protein, and wherein the medicine is connected to the protein, and not extracellular.

The connected body of non-cracking has the disadvantage in that：For payload release, needed after the complete hydrolysis of mAb polypeptide backbone ADC internalizations (internalization) are wanted, and when ADC internalizations are poor, are likely encountered drug effect reduction.Therefore, carry non- The ADC of the connected body of cracking is highly dependent on the biology of target cell.In addition, when payload is connected to after mAb degraded During mAb last amino acid, and the payload of not all retains its bioactivity.

In order to allow ADC not only to tumour cell, and adjacent antigen negative cells offer payload (is looked on Person's effect), the payload of release must readily diffuse through hydrophobic cell membrane, but when the connected body with non-cracking ADC is when to discharge its payload in the form of amino acid, situation is really not so, and the payload, which carries to have, is in amphion The medicine of the amino acid (i.e. with positively charged ammonium and negatively charged carboxylate) of state.

Therefore, have such connected body, it shows high plasma stability, and discharge medicine and the insoluble drug release it Afterwards without by its chemical modification, this is desirably.

In addition, desirably, with pharmaceutical grade protein conjugate, it shows the agglomeration or aggregation of reduction, agglomeration or Aggregation can damage its performance.

A kind of Drug Ligand conjugate is disclosed in EP 624377, wherein, the connected body includes linear peptides.

Fanny Guzman etc., Electronic Journal of Biotechnology, 2007,10,279-314； Yoshio Okada, Current Organic Chemistry, 2001,5,1-43；US 6897289B and textbook Houben- Weyl Synthesis of Peptides and Peptidomimetics(Methods in Organic Chemistry) ", Murray Goodman etc., Thieme Publishing Group, particularly 2001, Volumes E22a And E22b；Blocking group is disclosed, the formation of peptide bond describes the general and specific side of synthesis polypeptide and protein in detail Method, and for the analytical technology for the structure and composition for determining peptide.

The known connected body including linear peptide chain still shows deficiency.Display carries high performance connected body and based on so The pharmaceutical grade protein conjugate of connected body be in need., it is surprising that derived from spy of the adjacent hydroxyl to aminobenzyl alcohol Determine side chain connected body and show required performance.

The content of the invention

In following text, if without special declaration, using following abbreviations：

DCC N, N'- dicyclohexylcarbodiimides

EDC N- (3- dimethylaminopropyls)-N'- ethyl carbodiimides

Fmoc 9-fluorenylmethyloxycarbonyls

Boc tert-butoxycarbonyls

Boc₂O di-tert-butyl dicarbonates

Cit citrulling

NBS N- bromo-succinimides

NHS formulas (HOSu) compound, n-hydroxysuccinimide

NIS N- N-iodosuccinimides

- OTs toluene fulfonates

- OMs mesylates

- OTf fluoroform sulphonates

PBS phosphate buffered saline (PBS)s

Red-Al bis- (2- methoxy ethoxies) sodium aluminum hydride

TCEP tri- (2- carboxyethyls) phosphonium salt hydrochlorate

Tos or Ts tosyls or ptoluene-sulfonyl

TsCl toluene sulfochlorides or paratoluensulfonyl chloride

Z or Cbz benzyloxycarbonyl groups

Subject of the present invention is a kind of method (MI) for linking ligand LI and medicine DR,

LI is selected from：Amino acid LI-AA, single or multiple clonal antibody LI-Ab, antibody fragment LI-AbFrag, Protein L I- Prot and peptide LI-Pep；

DR is a kind of active medicine pharmaceutically；

It is characterised by that connected body LIN is used to be covalently attached LI and DR；

LIN includes linking group CG2；

CG2 derives from adjacent hydroxyl to aminobenzyl alcohol, and is formula (CG2-1) linking group；

(* * *) represents the connecting portion for connecting LI；

(* * * *) represents the connecting portion for connecting DR；

(* * * * * *) represents the connecting portion of connection linear peptides, and the peptide has 2 to 8 amino acid residues；

(4) represent CG2 from its derivative described adjacent hydroxyl to aminobenzyl alcohol to amino group.

Further subject matter of the present invention is a kind of method (MI), and method (MI) as defined in the description, and owns Its preferred embodiment,

Wherein, LI and DR is covalently attached in the form of formula (I) compound；

CG2 as defined in the description, and has its all preferred embodiment；

CG1 is linking group, and it is selected from formula (CG1-I) linking group, (CG1-II) linking group, (CG1-III) even Connect group and formula (CG1-IV) linking group；

M30 and m32 are same or different, and are 1,2,3,4,5,6,7,8,9 or 10 independently of one another；

(*) in the formula CG1 represents the key between T1 and CG1,

The LI of covalent attachment forms ligand residue LIRes in formula (I) compound, and LIRes is covalently attached to by T1 CG1；

LI is as defined in the description, it may have its all preferred embodiment, and is that formula (LIRes-T1-H) is changed Compound；

LIRes-T1-H (LIRes-H)

LIRes is selected from：Amino acid residue LIRes-AA, single or multiple clonal antibody residue LIRes-Ab, antibody fragment residue LIRes-AbFrag, residue of protein LIRes-Prot and peptide residue LIRes-Pep；

LI, which has, is selected from SH, OH or NH₂In functional group, the functional group forms the T1 in the formula (I), and the T1 passes through The key (*) is bonded to CG1；

T1 is-S- ,-O- or-NH-；

N1 is 0 or 1；

SG is the spacer group being selected from formula (SG-II) spacer group and formula (SG-III) spacer group；

M1 and m2 are same or different, and are 0 or 1 independently of one another；

M10, m11 and m12 are same or different, and are 0,1,2,3,4,5,6,7,8,9 or 10 independently of one another；

Its condition is 0 when being m2 different with m10；

Its condition is 0 when being m1, m11 different with m12；

SGPEG is formula (SGPEG-I) linking group；

M20 is 1,2,3,4,5 or 6；

N2 is 0 or 1；

T4 is-O-；

CG3 is selected from formula (CG3-I) linking group and formula (CG3-II) linking group；

R5 and R6 are same or different, and are C independently of one another_1-4Alkyl；

It is 1 for n1, (* *) described in the formula CG1 and the formula SG represents the key between CG1 and SG, described (the * * *) in formula SG and the formula CG2 represents the key between SG and CG2；If CG1 is formula (CG1-I) linking group, that Nitrogen-atoms in the nitrogen-atoms formation ring that (* *) in SG is represented is used in, so that the hydrogen atom of the nitrogen-atoms is taken by inner ring key Generation；

It is 0 for n1, (* *) described in the formula CG1 and (* * *) described in the molecular formula CG2 is represented The key between CG1 and CG2；

Its condition is, if n1 is 0, then CG1 is not formula (CG1-I) linking group；

It is 1 for n2, (* * * *) described in the formula CG2 and the formula CG3 represents CG2 and CG3 being connected to T4 Key；And (* * * * *) described in formula CG3 represents the key between CG3 and T2；

It is 0 for n2, (the * * * *) in the formula CG2 represents the key between CG2 and T2；

(the * * * * * *) in the formula CG2 represents CG2 and AAⁿ⁴Between key；

The DR of covalent attachment forms drug residue DRRes in formula (I) compound, and the DRRes is covalently attached by T2 To CG2；

DR with its all preferred embodiment, and is formula (DRRes-T2-H) chemical combination as defined in the description Thing；

H-T2-DRRes (DRRes-T2-H)

DRRes is the drug residue derived from DR；

DR has the functional group selected from-N (R4) H ,-OH or-SH, and the functional group forms the T2 in formula (I)；

T2 be-N (R4)-,-O- or-S-；

R4 is H or C_1-4Alkyl；

N3 is 2,3,4,5,6,7 or 8；

N4 is 1 to n3 integer；

AAⁿ⁴It is amino acid residue, n4 is the index of the amino acid residue, and (AAⁿ⁴)_n3It is residual with n3 amino acid Base AAⁿ⁴Linear peptides, and n4 represented since the amino acid residue AA in the peptide CG2ⁿ⁴Position, in the peptide Middle single amino acids residue is connected to each other by peptide bond, AA¹It is the first amino acid residue in the chain, and passes through key (* * * * * *) is connected to CG2, and the key (* * * * * *) is AA¹Hydroxy-acid group and the amino group that is represented with CG2 (4) Middle key, and AAⁿ³It is the last amino acid residue in the chain, single AAⁿ⁴It is identical or different independently of one another；

(3) AA is representedⁿ³N- terminal amino groups；

R1 and R2 are same or different, and are independently from each other hydrogen, C_1-4Alkyl, C (O)-(CH₂-O-)_m5- (GRPEG)_m4- R3 and PGN；

R3 is C_1-4Alkyl；

M4 is 0 or 1；

M5 is 0 or 1；

PGN is blocking group；

GRPEG is formula (GRPEG-I) linking group；

M21 is 1,2,3,4,5 or 6.

LI, DR and CG2 are as defined in the description, it may have its all preferred embodiment.

Preferably, LIN is formula (LIN) compound.

Preferably, medicine DR is selected from cytotoxic agent, other chemotherapeutics and anti-transfer agent.

Preferably, other chemotherapeutics and anti-transfer agent are selected from tyrosine kinase inhibitor and Rac1 inhibitor.

Preferably, tyrosine kinase inhibitor be selected from by Imatinib (Imatinib), Lapatinib (Lapatimib), Sutent (Sunitimib), nilotinib (Nilotimib), the active pharmaceutical ingredient of Dasatinib (Dasatimib) composition (API) group.

Preferably, Rac inhibitor is NSC 23766.

It is preferred that cytotoxic agent be cytotoxic agent for treating cancer.

The preferred classes of cytotoxic agent include, for example, enzyme inhibitor, such as anthracene nucleus medicament, bleomycin, cytotoxicity Nucleosides, dihydrofolate reductase inhibitor, differentiating inducer, DNA decomposition agents, DNA intercalators, enediyne (diynenes), silk Rimocidin (mitomycins), podophyllotoxin (podophyllotoxins), pteridine (pteridine) race medicine, taxol (taxols), thymidylate synthetase (thymidylate synthase) inhibitor, topoisomerase (topoisomerase) suppression Preparation, and vinblastine (vinca) medicine.

The preferred useful member of various types of other cytotoxic agent is selected from：N8- acetylspermidines, D actinomycin D, 9- ammonia Base camptothecine, aminopterin-induced syndrome, anguidin (anguidine), anthracene nucleus medicament, auristatin, bleomycin, card Ritchie are mould Plain (calicheamycin), camptothecine (lactone or the lactone of open loop form), carminomycin, CC-1065, clofarabine (clofaribine), 1- (2- chloroethyls)-1,2-dimethanesulfonyl hydrazides, ring propyl benzene diindyl-4- ketone (CBI), arabinose born of the same parents Glycosides, cytarabin (cytosine arabinoside), daunomycin, dichioromethotrexate, n- (5,5- diethyls Acyloxy amyl group) Doxorubicin, the carbon -4-9- diene -2 of 1,8- dihydroxy-bicyclic [7.3.1] 13,6- diine -13- ketone, difluoro Nucleosides, Doxorubicin, times carcinomycin (duocarmycin), epirubicin (epirubicin), Ai Sipeila mycins (esperamicin), etoposide, 5 FU 5 fluorouracil, Irinotecan, leurosideine, leurosine (leurosine), maytansine, melphalan, Ismipur, methopterin (methopterin), methopterin, Mitomycin A, Mitomycin C, morpholine-Doxorubicin, Nemorubicin (nemorubicin), podophyllotoxin and podophyllotoxin derivative, such as according to Support pool glycosides or etoposide phosphate, vitamin A acid, saponin, its Citropten (tallysomycin), vinblastine, vincristine, length Fields for spring sowing are pungent, taxane, such as taxol (Taxol) or taxol (paclitaxel), taxotere (Taxotere) or polyenoid Japanese yew Alcohol and taxotere retinoic acid and their homologue and derivative.

Preferred cytotoxic drug, selected from anthracene nucleus medicament, auristatin, Calicheamicin (calicheamycin), ring propyl benzene diindyl -4- ketone (CBI), Doxorubicin, duocarmycin, maytansine, mitomycin C, The derivative of taxol and its homologue and these materials.

Preferably, R4 is H.

Preferably, T1 is-NH- or-S-.

Preferably, n2 is that 1, T2 is-NH- ,-O- or-S-, and is connected by key (* * * * *) with CG3；It is further preferred that T2 It is-NH- or-S-.

Doxorubicin has CAS 23214-92-8, and is formula (DOXO) compound.

Formula (DOXO) compound can also be used in the form of its hydrochloride salt.

Can be accordingly connected to by one in its functional group in CG3 or CG2, functional group one of Doxorubicin such as passes through An expression in the functional group that (d1), (d2), (D3) and (D4) in formula (DOXO) is represented.

(d1), T2 of the functional group that (d2), (D3) and (D4) is represented such as in formula (I) works.

Preferably, Doxorubicin by with formula (DOXO) (d1) represent amino and by the key (* * * * *) with CG3 connections.

Maytansine has CAS 35846-53-8 and formula (MAYT).

Maytansine can accordingly be connected to CG3 or CG2 by the-OH represented of (m2) in formula (MAYT).

Or-C (O)-CH that (m1) in formula (MAYT) is represented₃Base exchange acyl group, the acyl group has again corresponding connection To CG3 or CG2 nucleophilic group-SH ,-NH₂Or-OH.

(m2)-the OH or the nucleophilic group of acyl group represented is then as the T2 in formula (I) works.

It is preferred that taxane be the taxol with CAS 33069-62-4 and formula (TAXO).

One in the-OH that taxol can be represented by (t1) in formula (TAXO), (t2) and (t3) is accordingly connected to CG3 or CG2.- the OH is then as the T2 in formula (I) works.

In one preferred embodiment, n2 is that 0, T2 is-O-, and is connected by key (* * * *) with CG2, DRRes from DR derives, and DR is formula (TAXO) compound.

In another preferred embodiment, n2 is that 1, T2 is-O- and be connected by key (* * * * *) with CG3 that CG3 is formula (CG3-II) linking group, and DRRes, from DR derivatives, DR is formula (TAXO) compound, a kind of possible purple of present embodiment China fir alcohol intermediate is formula (TAXO-t1-1) compound.

Camptothecine is (S)-(+)-camptothecine, and it has CAS 7689-03-4, and is formula (CAMPTO) compound.

Camptothecine can accordingly be connected to CG3 or CG2 by the functional group represented in (c1) in formula (CAMPTO).

With (c1) functional group represented and then as the T2 in formula (I) works.

In one preferred embodiment, n2 is that 0, T2 is-O-, and is connected by key (* * * *) with CG2, DRRes from DR derives, and DR is formula (CAMPTO) compound.

In another preferred embodiment, n2 is that 1, T2 is-O-, and is connected by key (* * * * *) with CG3, and CG3 is formula (CG3-II) linking group, DRRes derives from DR, and DR is formula (CAMPTO) compound.

In one preferred embodiment, CG1 is formula (CG1-I), (CG1-III) or (CG1-IV) linking group, and The sulphur atom of the side chain of LIRes Cys residues is T1, and is connected to CG1 by key (*).

In another preferred embodiment, CG1 is formula (CG1-II) linking group, and T1 is LIRes-N- or-O- And CG1 is connected to by key (*).The amino or oh group for being connected to CG1 this LIRes are preferably LIRes N- ends The amino or oh group of the side chain of amino group or LIRes amino acid residue.Preferably, if T1 is attached to CG1's The amino group of the side chain of LIRes amino acid residue, then the amino acid residue of the LIRes is preferably Lys；If T1 is It is connected to the oh group of the side chain of CG1 LIRes amino acid residue, then the amino acid residue of the LIRes is preferably Tyr, Ser or Thr.

Preferably, LI is selected from：Single or multiple clonal antibody LI-Ab, antibody fragment LI-AbFrag, Protein L I-Prot and peptide LI-Pep；And

LIRes is selected from：Single or multiple clonal antibody residue LIRes-Ab, antibody fragment residue LIRes-AbFrag, protein Residue LIRes-Prot and peptide residue LIRes-Pep.

If LIRes is LIRes-AA, then LIRes is preferably a-amino acid residue.

One in two kinds of possible functional groups that LIRes passes through LIRes can be preferably connected to CG1：Pass through N- ends The functional group of Amino End Group group or the side chain for passing through LIRes, on condition that LIRes has such side chain with functional group.Connection LIRes and CG1 this functional group is T1.If the functional group's connection for the side chain that LIRes passes through LIRes, then the side chain The side chain of preferably LIRes Cys, Lys, Tyr, Ser or Thr residue.

If LIRes is LIRes-AA, then being not connected to CG1 LIAARes-AA remaining functional group can pass through The blocking group commonly used in chemistry of peptides is protected, for example, disconnected amino can carry acetic acid, and disconnected carboxyl can be with Use C_1-4Alcohol is esterified, and the disconnected functional group of side chain can carry the side chain protecting group commonly used in chemistry of peptides.

It is highly preferred that LIRes-AA is the alpha amino acid residue with side chain, the side chain has functional group and by the official It can roll into a ball and be connected with CG1, even further preferably, LIRes-AA is derived from Cys, Lys, Tyr, Ser or Thr.

Preferably, if the side chain connection for the Cys residues that LIRes passes through LIRes, then T1 passes through the Cys residues The sulphur atom of side chain is formed, and is preferably connected by key (*) with CG1, and CG1 is formula (CG1-I), (CG1-III) or (CG1-IV) Linking group；Or

If Lys, Tyr, Ser or Thr residue that LIRes passes through LIRes side chain connection, then T1 by the Lys, The nitrogen or oxygen atom of the side chain of Tyr, Ser or Thr residue are formed, and are connected by key (*) with CG1, and preferably CG1 is formula (CG1- II) linking group.

LIRes-Pep is derived from cell-penetrating peptides.

LIRes-AB and LIRes-ABFrag are preferably derived from disease treatment, the antibody preferably used in treatment of cancer and Antibody fragment.

Preferably, m30 and m32 are identical or different, and are 2,3,4,5 or 6 independently of one another.

Preferably, when CG1 is formula (CG1-IV) linking group, m30 is 2.

Preferably, when CG1 is formula (CG1-II) linking group, m 32 is 2.

Preferably, n1 is 1.

Preferably, m1 and m2 are 0 or 1.

Preferably, m10, m11 and m12 are identical or different, and are 0,1,2,3,4,5 or 6 independently of one another.

Preferably, m20 is 1,2,3 or 4.

It is highly preferred that

M10 is that 6 and m2 is 0；Or

M2 is that 1, m10 is that 0 and m20 is 1；

M1 is that 1, m11 is that 1, m12 is that 1 and m20 is 3；Or

M1 is that 0, m11 is that 3, m12 is 0.

Preferably, R5 and R6 are CH₃。

In specific embodiment, CG1 is formula (CG1-I) linking group, and SG is formula (SG-II) spacer group, and m10 is 6 and m2 is 0.

In another specific embodiment, CG1 is formula (CG1-II) linking group, and m32 is 2；Or formula (CG1-III) is even Connect group；Or formula (CG1-IV) linking group, m30 is 2；And SG is formula (SG-II) spacer group, m10 be 0, m2 be 1 and M20 is 1.

In another embodiment, CG1 is formula (CG1-II) linking group, and m32 is 2；Or formula (CG1-III) connection Group；Or formula (CG1-IV) linking group, m30 is 2；And SG is formula (SG-III) spacer group, m1 is that 1, m11 is 1, m12 It is 3 for 1 and m20；Or SG is formula (SG-III) spacer group, m1 is that 0, m11 is 3, and m12 is 0.

Preferably, n3 is 2,3,4,5 or 6；More preferably 2,3 or 4；It is highly preferred that n3 is 2 or 3.

Preferably, AAⁿ⁴It is alpha amino acid residue.

It is highly preferred that AAⁿ⁴It is selected from：Alanine, valine, leucine, isoleucine, methionine, phenylalanine, color ammonia The lysine side-chain of acid, proline, lysine, acetyl group or formoxyl protection, arginine, side chain, which is protected, (preferably uses toluene Sulfonyl or nitro protection) arginine, histidine, ornithine, side chain protect (preferably with acetyl group or formoxyl protection) Ornithine, and citrulling.

Even further preferably, AAⁿ⁴It is alanine, glycine, phenylalanine, valine, lysine, leucine, tryptophan, Arginine, the protected arginine of side chain or citrulling, particularly alanine, glycine, phenylalanine, valine, lysine Or citrulling.

If AAⁿ⁴With the side chain with functional group, then the functional group can be generally used for protected amino acid side chain The protection group of functional group is protected.

In the case of lysine, side chain is most preferably protected with acetyl group or formoxyl.

(AAⁿ⁴)_n3The example of the peptide of peptide chain is Phe-Lys, Val-Lys, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly- Phe-Lys、Ala-Lys、Val-Cit、Phe-Cit、Leu-Cit、Trp-Cit、Phe-Ala、Gly-Phe-Leu-Gly、Ala- Leu-Ala-Leu, Phe-N9-tosyl-Arg and Phe-N9-Nitro-Arg, preferably Phe-Lys, Val-Lys, Val-Cit and D-Phe-L-Phe-Lys；Any lysine side-chain is optionally protected, and preferably uses acetyl group.

It is particularly preferred that n3 is 2 or 3, and AAⁿ⁴It is alanine, glycine, valine or citrulling；

More particularly,

N3 is 2, AA¹It is citrulling, and AA²It is valine or alanine；

N3 is 3, AA¹It is citrulling, AA²It is valine and AA³It is glycine.

Preferably, m4 is 1.

Preferably, R3 is methyl.

Preferably, R1 and R2 are identical or different, and are independently from each other：Hydrogen, methyl, C (O)-(CH₂-O- )_m5-(GRPEG)_m4-CH₃And PGN.

Preferably, m21 is 2,3 or 4.

Preferably, PGN is to be the N- ends of protection peptide in chemistry of peptides or is that protection is used as construction block in peptide symthesis The alpha-amido of the alpha amino acid of (building block) and conventional blocking group.

It is highly preferred that PGN is selected from：Boc, Fmoc and Z.

Even further preferably, R1 is hydrogen, methyl, acetyl group or C (O)-(CH₂-O-)_m5-(GRPEG)_m4-CH₃, m4 is 1 He M21 is 3, and R2 is hydrogen or methyl.

Especially,

R1 is acetyl group, and R 2 is hydrogen；Or

R1 and R2 are methyl；Or

R1 is C (O)-(CH₂-O-)_m5-(GRPEG)_m4-CH₃, m5 is that 0, m4 is 1, and m 21 is that 3 and R2 is hydrogen；Or

R1 is C (O)-(CH₂-O-)_m5-(GRPEG)_m4-CH₃, m5 is that 1, m4 is 1, and m 21 is that 2 and R2 is hydrogen.

The further subject matter of the present invention is a kind of method (MI)；

Wherein formula (I) compound is prepared in step (MI)；

Step (MI) includes reaction (MI), and wherein formula (II) compound is reacted with formula (LIRes-T1-H) compound；

CG1M is to be selected from formula (CG1M-I) linking group, formula (CG1M-II) linking group, formula (CG1M-III) linking group With the linking group in formula (CG1M-IV) linking group；

X1 is Cl, Br or I；

SG、n1、n4、n3、AAⁿ⁴, (3), R1, R2, T4, CG3, n2, T2, DRRes, m30 and m32 such as institute in this manual Definition, and with it is all its preferred embodiment；

CG2 as defined in this manual, and with it is all its preferred embodiment.

Preferably, X1 is Cl or Br, and more preferably X 1 is Br.

Preferably, CG1M is formula (CG1M-IV) linking group.

It is highly preferred that CG1M is formula (CG1M-IV) linking group, and m30 is 2.

In certain embodiments, CG1M is formula (CG1M-I) linking group, and SG is formula (SG-II) spacer group, and m10 is 6 and m2 is 0.

In another embodiment, CG1M is formula (CG1M-II) linking group and m32 is 2, or formula (CG1M- III) Linking group or formula (CG1M-IV) linking group and m30 is 2, and SG is formula (SG-II) spacer group, and m10 is that 0, m2 is 1, M20 is 1.

In another embodiment, CG1M is formula (CG1-II) linking group and m32 is 2, or formula (CG1-III) is even Connect group, or formula (CG1M-IV) linking group and m30 is 2, and SG is formula (SG-III) spacer group and m1 is that 1, m11 is 1, M12 is that 1 and m20 is that 3 or SG is formula (SG-III) spacer group and m1 is that 0, m11 is 3, and m1 is 0.

Preferably, the reaction temperature of the reaction (MI) be from 0 to 150 DEG C, more preferably 5 to 50 DEG C, more preferably 10 to 40℃。

Preferably, the reaction time of the reaction (MI) is more preferably 10 minutes to 24 hours from 1 minute to 168 hours, Even more preferably from 15 minutes to 3 hours.

It is typically to be carried out in solvent (MI) to react (MI).

Preferably, solvent (MI) is selected from water, DMA, DMF, N, N- dimethyl sulfoxides With their mixture.

In the case of water, the water may include buffer solution (MI), preferred buffer solution (MI) be in protein chemistry often The buffer solution used is advised, preferred buffer solution (MI) is come since the buffer substance chosen in following substances：Acetic acid, lemon Acid, dithiothreitol (DTT) (DTT), ethylenediamine tetra-acetic acid (EDTA), glycine, histidine, phosphoric acid (including phosphoric acid buffer physiology salt Water, PBS), polysorbate 20, polyoxyethylene sorbitan monoleate, sucrose, sodium chloride, butanedioic acid, trehalose, three (methylol) aminomethanes, it Mixture and their salt.

The salt of the buffer substance is preferably sodium salt, sylvite or hydrochloride.

Preferably, the amount of the solvent (MI) is 5 to 10000 times of weight of compound (LIRes-T1-H), more preferably For from 10 to 5000 times, more preferably 50 to 500 times.

Preferably, in reaction (MI), using 1 to 100 molar equivalent, more preferably 2 to 20 molar equivalents, even more preferably Formula (II) compound of 3 to 10 molar equivalents, the molar equivalent is based on the molal quantity of formula (LIRes-T1-H) compound.

Reaction (MI) can be carried out in the presence of TCEP.

When LI is single or multiple clonal antibody LI-Ab or antibody fragment LI-AbFrag, TCEP is preferably used.

Preferably, in reaction (MI), 0.5 to 20 molar equivalent, more preferably 1 to 10 molar equivalent, more preferably 1.5 are used To the TCEP of 5 molar equivalents, the molar equivalent is based on the molal quantity of formula (LIRes-T1-H) compound.

After (MI) is reacted, formula (I) can be isolated by the standard method such as such as washing, extraction, filtering, concentration, dry Compound.Formula (I) compound can be purified before separation or afterwards, preferably by chromatographing or crystallizing from appropriate solvent.

Alternatively, formula (I) compound can be purified by standard methods such as such as filtering, ultrafiltration, diafiltration and chromatographies, and It can be stored or further be used as solvent.

The further subject matter of the present invention is a kind of method (MII) for being used to prepare formula (II) compound, formula (II) chemical combination Thing as defined in this manual, and with its all preferred embodiment,

Wherein,

If it is formula (CG3-I) linking group that n2, which is 1 and CG3, method (MII) includes step (MIIa) and step (MIIb)；

If it is formula (CG3-II) linking group that n2, which is 1 and CG3, method (MII) includes the step (MIIa), step (MIIc), step (MIId) and step (MIIe)；

If it is formula (CG1M-IV) linking group that n2, which is 0 and CG1M, method (MII) includes a step, step , or two steps, step (MII0-I-IVa) and step (MII0-I-IVb) (MII0-IV)；

If it is formula (CG1M-III) linking group that n2, which is 0 and CG1M, method (MII) includes step (MII0-III)；

If it is formula (CG1M-II) linking group that n2, which is 0 and CG1M, method (MII) includes two steps, step (MII0-IIa) and step (MII0-IIb), or a step, step (MII0-IIc)；

If it is formula (CG1M-I) linking group that n2, which is 0 and CG1M, method (MII) includes a step, step , or two steps, step (MII0-I-IVa) and step (MII0-I-IVb) (MII0-I)；

Step (MIIa) includes reaction (MIIa), wherein, formula (III) compound is reacted with formula (II-I) compound；

N2 in formula (III) as defined in the description, and has its all preferred embodiment；

Formula (II-I) compound is selected from formula (II-1) compound, 1,1'- N,N'-carbonyldiimidazoles, 4- chloroformate nitrophenyl esters, light Gas, surpalite, triphosgene and its mixture；

Step (MIIb) includes reaction (MIIb), wherein, reaction product and formula (DRRes- from the reaction (MIIa) T2-H) compound is reacted；

Step (MIIc) includes reaction (MIIc), wherein, reaction product and formula (CG3M- from the reaction (MIIa) II) compound reacts to provide formula (IIc) compound；

N2 is as defined in claim 2 in formula (IIc)；

Step (MIId) include reaction (MIId), wherein, it is described reaction (MIIc) in prepare formula (IIc) compound with Formula (II-I) the compound reaction；

Step (MIIe) includes reaction (MIIe), wherein, reaction product and formula (DRRes- from the reaction (MIId) T2-H) compound is reacted；

Step (MII0-I-IVa) includes reaction (MII0-I-IVa), wherein, formula (III) compound and compound (II0- I-IVa) react to provide formula (III0-I-IVa) compound；

N2 is 0 in formula (III0-I-IVa)；

Compound (II0-I-IVa) is selected from paratoluensulfonyl chloride, p-toluenesulfonic anhydride, mesyl chloride, methanesulfonic acid acid anhydride, trifluoro Mesyl chloride and trifluoromethanesulfanhydride anhydride and their mixture；

X2 is selected from-OTs ,-OMs and-OTf；

Step (MII0-I-IVb) includes reaction (MII0-I-IVb), wherein, prepared in reaction (MII0-I-IVa) Formula (III0-I-IVa) compound is reacted with formula (DRRes-T2-H) compound；

When CG1M is formula (CG1M-IV) linking group, in step (MIII-IV)；Or

When CG1M is formula (CG1M-III) linking group, in step (MIII-III)；Or

When CG1M is formula (CG1M-II) linking group, in two steps, in step (MIII-IIa) and step Suddenly in (MIII-IIb), or in one step, in step (MIII-IIc)；Or

When CG1M is formula (CG1M-I) linking group, in step (MIII-I)；Prepare formula (III) chemical combination Thing,

Step (MIII-IV) includes the reaction (MIII-IV) of formula (IV) compound and formula (CG1MR-IV) compound,

In formula (IV) n2 as defined in the description, and with its all preferred embodiment；

M30 as defined in the description, and has its all preferred embodiment；

R20 is formula (R20-1) residue；

Step (MIII-III) includes the reaction (MIII-III) of formula (IV) compound and formula (CG1MR-III) compound；

X1 as defined in the description, also with its all preferred embodiment；

Step (MIII-IIa) includes the reaction (MIII- of the formula (IV) compound and formula (CG1MR-IIa) compound IIa) to provide formula (IV-IIa) compound；

M32 as defined in the description, also with its all preferred embodiment；

Step (MIII-IIb) includes (IV-IIa) compound prepared in step (MIII-IIa) and formula (HOSu) chemical combination The reaction (MIII-IIb) of thing；

Step (MIII-IIc) includes the reaction (MIII- of the formula (IV) compound and formula (CG1MR-IIc) compound IIc)；

M32 as defined in the description, also with its all preferred embodiment；

Step (MIII-I) includes the reaction (MIII-I) of formula (IV) compound and formula (MA) compound；

Formula (IV) compound is prepared in step (MIV)；

Step (MIV) includes reaction (MIV), and reaction (MIV) is anti-with the reduction of formula (V) compound of compound (IV) Should；

Compound (IV) is selected from：NaBH₄、BH₃, DIBAL-H, two (2- methoxy ethoxies) sodium aluminum hydrides and theirs is mixed Compound；

SG, n1, AA, n4, n3, (3), R1 and R2 as defined above, also with they all preferred embodiments；

Formula (V) compound is prepared in step (MVb)；

Step (MVb) includes reaction (MVb), wherein, R30 is cracked with HCl from formula (Va) compound；

R30 is connected to SG by using the keys represented of (the * *) in the formula SG and is Boc；

Step (MII0-IV) includes the reaction (MII0-IV) of formula (III0) compound and formula (CG1MR-IV) compound；

SG, CG2, n1, AA, n4, n3, (3), R1, R2, T2 and DRRes as defined in the description, also with it is all it Preferred embodiment, and n2 is 0 in formula (III0)；

Step (MII0-III) includes the reaction (MII0- of formula (III0) compound and formula (CG1MR-III) compound III)；

Step (MII0-IIa) includes the reaction (MII0- of the formula (III0) compound and formula (CG1MR-IIa) compound IIa) to provide formula (III0-IIa) compound；

SG, CG2, n1, AA, n4, n3, (3), R1, R2, T2 and DRRes and m32 as defined herein, also with institute There is their preferred embodiment, and n2 is 0 in formula (III0-IIa)；

Step (MII0-IIb) is included in formula (III0-IIa) compound and formula prepared in step (MII0-IIa) (HOSu) reaction (MII0-IIb) of compound；

Step (MII0-IIc) includes the reaction (MII0- of the formula (III0) compound and formula (CG1MR-IIc) compound IIc)；

Step (MII0-I) includes the reaction (MII0-I) of formula (III0) compound and formula (MA) compound；

Formula (III0) compound is prepared in step (MIII0),

Step (MIII0) includes reaction (MIII0), wherein, R30 is cracked with HCl from formula (IV0) compound；

N2 is 0 in formula (IV0)；

Formula (IV0) compound is prepared in step (MIV0a) and step (MIV0b),

Step (MIV0a) includes reaction (MIV0a), and wherein formula (V0) compound reacts to provide with (RIV0a) compound Formula (IV0a) compound；

R30, SG, CG2, n1, AA, n4, n3, (3), R1 and R2 as defined in the description, also with it is all they Preferred embodiment；

Compound (RIV0a) is selected from：Paratoluensulfonyl chloride, p-toluenesulfonic anhydride, mesyl chloride, methanesulfonic acid acid anhydride, fluoroform Sulfonic acid chloride, trifluoromethanesulfanhydride anhydride, SOCl₂、(COCl)₂、POCl₃、PCl₃、PCl₅、POBr₃、PBr₃、PBr₅, N- bromos succinyl it is sub- Amine, N-iodosuccinimide, HCl, HBr, HI and their mixture；

X3 is selected from-OTs ,-OMs ,-OTf ,-Cl ,-Br and-I；

Step (MIV0b) include reaction (MIV0b), wherein, reaction (MIV0) in prepare formula (IV0a) compound with Formula (DRRes-T2-H) compound is reacted；

Formula (V0) compound is prepared in step (MV0),

Step (MV0) includes reaction (MV0), and reaction (MV0) is anti-with the reduction of formula (Va) compound of compound (IV) Should；

CG1M and X1 as defined herein, also with its all preferred embodiment；

SG、n1、n4、n3、AAⁿ⁴, (3), R1, R2, T4, CG3, n2, T2, DRRes, formula (DRRes-T2-H) compound, M30, m32, R5 and R6 as defined in the description, also with its all preferred embodiment；

CG2 as defined herein, also with its all preferred embodiment.

Compound (II-I) and formula (CG3M-II) compound are known compounds, and can be according to known method system Standby, they are typically even available commercially.

React (MIIA) and reaction (MIID) be similar reaction, it is possible to as defined in the description it is similar instead Answer and carried out under parameter area, each independent response parameter in described two reactions is selected independently of one another.

Preferably, compound (II-I) is formula (II-1) compound.

It is typically to be carried out in solvent (MIIa) to react (MIIa) and reaction (MIId).

Preferably, solvent (MIIa) be selected from DMF, DMA, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF and their mixture.

Preferably, the amount of solvent (MIIa) is 1 to 500 times of the weight of formula (III) compound, more preferably 5 to 50 times, very To more preferably from 10 to 30 times.

Preferably, in reaction (MIIa) and in reaction (MIId), rubbed using 0.5 to 20 molar equivalent, more preferably 1 to 10 That equivalent, (II-I) compound of more preferably 1 to 2 molar equivalent, molar equivalent is based on the mole of formula (III) compound.

Reaction (MIIa) and reaction (MIId) can be carried out in the presence of alkali (MIIa).

Preferably, the alkali (MIIa) is selected from potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4- diformazans Base aminopyridine and their mixture.

Preferably, in reaction (MIIa) and reaction (MIId), 0.5 to 50 molar equivalent, more preferably 1 to 20 mole are used The alkali (MIIa) of equivalent, even more preferably 2 to 10 molar equivalents, molar equivalent is based on the mole of formula (III) compound.

Preferably, reaction (MIIa) and reaction (MIId) are carried out under an inert atmosphere.

React after (MIIa) and reaction (MIId), the reaction product of reaction (MIIa) and reaction (MIId) can be by such as Washing, extraction, filtering, concentration, the standard method such as dry and separate.Any compound can be purified before separation or afterwards, It is preferred that being purified by chromatographing or crystallizing from appropriate solvent.

The crude product mixture for coming autoreaction (MIIa) and reaction (MIId) can also be directly in reaction (MIIb) or reaction (MIIEe) use.

It is further preferred that reaction (MIIa) and reaction (MIIb) and reaction (MIId) and reaction (MIIe) are all identical In solvent, and it is carried out continuously with one pot.

It is similar reaction to react (MIIb) and react (MIIEe), it is possible to similar as defined in the description Carried out under response parameter scope, each independent response parameter in described two reactions is selected independently of one another.

(MIIb) and reaction (MIIEe) are typically reacted in solvent (MIIb).

Preferably, solvent (MIIb) be selected from DMF, DMA, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIIb) respectively be react (MIIa) reaction product weight or reaction (MIId) it is anti- Answer the weight of product 1 to 500 times, more preferably 5 to 50 times, even more preferably 10 to 30 times.

Preferably, in reaction (MIIb) and reaction (MIIEe), rubbed using 0.2 to 10 molar equivalent, more preferably 0.5 to 5 That equivalent, formula (DRRes-T2-H) compound of more preferably 0.8 to 2 molar equivalent, molar equivalent is based on reaction (MIIa) The mole of reaction product or the reaction product of reaction (MIId).

Preferably, (MIIb) and reaction (MIIEe) are reacted under an inert atmosphere.

It is similar reaction to react (MII0-I-IVb) and reaction (MIV0b), it is possible to as defined in the description Similar response parameter scope under carry out, each independent response parameter in described two reactions is selected independently of one another.

It is typically to be carried out in solvent (MIV0b) to react (MII0-I-IVb) and reaction (MIV0b).

Preferably, solvent (MIV0b) be selected from DMF, DMA, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF and their mixture.

Preferably, the amount of solvent (MIV0b) is the weight or formula (IV0a) chemical combination of formula (III0-I-IVb) compound respectively 1 to 500 times of the weight of thing, more preferably more preferably 5 to 50 times, 10 to 30 times.

Preferably, in reaction (MII0-I-IVb) and reacting (MIV0b), using 0.2 to 10 molar equivalent, more preferably Formula (DRRes-T2-H) compound of 0.5 to 5 molar equivalent, more preferably 0.8 to 2 molar equivalent, the corresponding ground of the molar equivalent In formula (III0-I-IVb) compound or the mole of formula (IV0a) compound.

Preferably, (MII0-I-IVb) and reaction (MIV0b) are reacted under an inert atmosphere.

It is typically to be carried out in solvent (MIIc) to react (MIIc).

Preferably, solvent (MIIc) be selected from DMF, DMA, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIIc) is preferably 1 to 500 times of weight of the reaction product for reacting (MIIa), more excellent Select 5 to 50 times, even more preferably 10 to 30 times.

Preferably, it is more excellent using 0.2 to 20 molar equivalent, more preferably 0.5 to 10 molar equivalent in reaction (MIIc) Formula (CG3M-II) compound of 0.8 to 5 molar equivalent is selected, the molar equivalent is based on mole of the reaction product of reaction (MIIa) Amount.

Preferably, reaction (MIIc) is carried out under an inert atmosphere.

After reaction (MIIc), the reaction product of the reaction (MIIc) can by such as washing, extracting, filtering, concentrate, The standard method separation such as dry.Any compound can be purified before separation or afterwards, preferably by from appropriate solvent Chromatography or crystallization are purified.

After reaction (MIIb), reaction (MIIe), reaction (MII0-I-IVb) and reaction (MIV0b), formula (II) chemical combination Thing or formula (IV0) compound difference can pass through the standard method separation such as such as washing, extraction, filtering, concentration, dry.Anyization Compound can be purified before separation or afterwards, be preferably purified by chromatographing or crystallizing from appropriate solvent.

Preferably, CG1M is formula (CG1M-IV) linking group；

It is highly preferred that CG1M is formula (CG1M-IV) linking group, and m30 is 2.

Formula (V) compound and formula (III0) compound can be used in instead using unprotonated forms or protonated form as salt Answer (MIV), reaction (MII0-I), reaction (MII0-IIa), reaction (MII0-III) and react in (MII0-IV).

It is similar reaction to react (MIII-IV) and react (MII0-IV), it is possible to as defined in the description Carried out under similar response parameter scope, each independent response parameter in described two reactions is selected independently of one another.

It is typically to be carried out in solvent (MIII-IV) to react (MIII-IV) and reaction (MII0-IV).

Preferably, solvent (MIII-IV) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans are sub- Sulfone, acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIII-IV) is 1 to 500 times of the weight of formula (IV) compound, more preferably 5 to 50 times, More preferably 10 to 30 times.

Preferably, in the reaction in (MIII-IV) and reaction (MII0-IV), using 1 to 20 molar equivalent, more preferably 1 to The compound (CG1MR-IV) of 10 molar equivalents, more preferably 1 to 5 molar equivalent, the molar equivalent accordingly based on formula (IV) or The mole of formula (III0) compound.

Preferably, reaction (MIII-IV) and reaction (MII0-IV) are carried out under an inert atmosphere.

Reaction (MIII-IV) and reaction (MII0-IV) are typically what is carried out in the presence of alkali (MIII-IV).

Preferably, the alkali (MIII-IV) is selected from：Sodium carbonate, potassium carbonate, diisopropylethylamine, triethylamine, pyridine, 4- Dimethyl aminopyridine and their mixture.

Preferably, in reaction (MIII-IV) and reaction (MII0-IV), using 0.5 to 50 molar equivalent, more preferably from 1 To 20 molar equivalents, the alkali (MIII-IV) of even more preferably 2 to 10 molar equivalents, the molar equivalent is accordingly based on formula (IV) or formula (III0) compound mole.

After reacting (MIII-IV) and reaction (MII0-IV), formula (III) compound or formula (II) compound are distinguished can be with Pass through the standard method separation such as such as washing, extraction, filtering, concentration, dry.Formula (III) compound or formula (II) compound can divide It is not purified, is preferably purified by chromatographing or crystallizing from appropriate solvent before separation or afterwards.

It is similar reaction to react (MIII-III) and reaction (MII0-III), it is possible to as defined in the description Similar response parameter scope under carry out, each independent response parameter in described two reactions is selected independently of one another.

It is typically to be carried out in solvent (MIII-III) to react (MIII-III) and reaction (MII0-III).

Preferably, solvent (MIII-III) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans Sulfoxide, acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIII-III) is 1 to 500 times of the weight of formula (IV) compound, more preferably 5 to 50 Times, more preferably 10 to 30 times.

Preferably, in reaction (MIII-III) and in reaction (MII0-III), 1 to 20 molar equivalent, more preferably 1 are used To 10 molar equivalents, the compound (CG1MR-III) of even more preferably 1 to 5 molar equivalent, the molar equivalent is based on formula (IV) The mole of compound or formula (III0) compound.

Preferably, reaction (MIII-III) and reaction (MII0-III) are carried out under an inert atmosphere.

React after (MIII-III) and reaction (MII0-III), formula (III) compound or formula (II) compound respectively can To pass through the standard method separation such as such as washing, extraction, filtering, concentration, dry.Formula (III) compound or formula (II) compound can It is purified, is preferably purified by chromatographing or crystallizing from appropriate solvent before separation or afterwards respectively.

It is similar reaction to react (MIII-IIa) and reaction (MII0-IIa), it is possible to as defined in the description Similar response parameter scope under carry out, each independent response parameter in described two reactions is selected independently of one another.

It is typically to be carried out in solvent (MIII-IIa) to react (MIII-IIa) and reaction (MIII-IIa).

Preferably, solvent (MIII-IIa) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- bis- First sulfoxide, acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of the solvent (MIII-IIa) is 1 to 500 times of the weight of formula (IV) compound, more preferably 5 to 50 times, even more preferably 10 to 30 times.

Preferably, in reaction (MIII-IIa) and reacting (MII0-IIa), using 1 to 20 molar equivalent, more preferably 1 to The compound (CG1MR-IIa) of 10 molar equivalents, even more preferably 1 to 5 molar equivalent, the molar equivalent is changed based on formula (IV) The mole of compound or formula (III0) compound.

Preferably, reaction (MIII-IIa) and reaction (MII0-IIa) are carried out under an inert atmosphere.

After reaction (MIII-IIa) and reaction (MII0-IIa), formula (IV-IIa) compound or formula (III0-IIa) chemical combination Thing difference can pass through the standard method separation such as such as washing, extraction, filtering, concentration, dry.Formula (IV-IIa) compound or formula (III0-IIa) compound can be purified before separation or afterwards respectively, preferably by chromatographing or crystallizing from appropriate solvent It is purified.

It is similar reaction to react (MIII-IIb) and reaction (MII0-IIa), it is possible to as defined in the description Similar response parameter scope under carry out, each independent response parameter in described two reactions is selected independently of one another..

Preferably, reaction (MIII-IIb) and reaction (MII0-IIb) are carried out in the presence of compound (COUPADD).

Compound (COUPADD) is conventional use of coupling additive in chemistry of peptides, and the chemistry of peptides is used to pass through acid amides The formation of key makes amino acid couplings react into peptide.Preferably, compound (COUPADD) is selected from DCC, EDC and their mixture.

Preferably, in reaction (MIII-IIb) and reacting (MII0-IIb), using 1 to 20 molar equivalent, more preferably 1 to The compound (COUPADD) of 10 molar equivalents, even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on formula (IV- IIa) the mole of compound or formula (III0-IIa) compound.

It is typically to be carried out in solvent (MIII-IIb) to react (MIII-IIb) and reaction (MII0-IIb).

Preferably, solvent (MIII-IIb) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans Sulfoxide, acetonitrile, acetone, 1,4- dioxanes, THF and their mixture.

Preferably, the amount of the solvent (MIII-IIb) is 1 to 500 times of the weight of formula (IV-IIa) compound, more excellent Select 5 to 50 times, more preferably 10 to 30 times.

Preferably, in reaction (MIII-IIb) and reacting (MII0-IIb), using 1 to 20 molar equivalent, more preferably 1 to Formula (HOSu) compound of 10 molar equivalents, even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on formula (IV-IIa) mole of compound or formula (III0-IIa).

Preferably, reaction (MIII-IIb) and reaction (MII0-IIb) are carried out under an inert atmosphere.

React after (MIII-IIb) and reaction (MII0-IIb), formula (III) compound or formula (II) compound respectively can With by such as wash, extract, filtering, concentration and dry etc. standard method separate.Formula (III) compound or formula (II) compound can It is purified, is preferably purified by chromatographing or crystallizing from appropriate solvent before separation or afterwards respectively.

It is similar reaction to react (MIII-IIc) and reaction (MII0-IIc), it is possible to as defined in the description Similar response parameter scope under carry out, each independent response parameter in described two reactions is selected independently of one another.

(MIII-IIc) and reaction (MII0-IIc) is reacted generally to carry out in solvent (MIII-IIc).

Preferably, solvent (MIII-IIc) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans Sulfoxide, acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIII-IIc) is 1 to 500 times of the weight of formula (IV) compound, more preferably 5 to 50 Times, even more preferably 10 to 30 times.

Preferably, in reaction (MIII-IIc) and in reaction (MII0-IIc), 1 to 20 molar equivalent, more preferably 1 are used To 10 molar equivalents, the compound (CG1MR-IIa) of even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on The mole of formula (IV) compound or formula (III0) compound.

Preferably, reaction (MIII-IIc) and reaction (MII0-IIc) are carried out under an inert atmosphere.

It is typically to be carried out in the presence of alkali (MIII-IIc) to react (MIII-IIc) and reaction (MII0-IIc).

Preferably, the alkali (MIII-IIc) is selected from：Potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4- Dimethyl aminopyridine and their mixture.

Preferably, in reaction (MIII-IIc) and reaction (MII0-IIc), 0.5 to 50 molar equivalent, more preferably 1 are used To 20 molar equivalents, the alkali (MIII-IIc) of even more preferably 2 to 10 molar equivalents, the molar equivalent is accordingly based on formula (IV) mole of compound or formula (III0) compound.

After reaction (MIII-IIc) and reaction (MII0-IIc), formula (III) compound or formula (II) compound difference Can by such as wash, extract, filtering, concentration and dry etc. standard method separate.Formula (III) compound or formula (II) compound It can before separation or afterwards be purified, be preferably purified by chromatographing or crystallizing from appropriate solvent respectively.

It is similar reaction to react (MIII-I) and reaction (MII0-I), it is possible in phase as defined in the description As carry out under response parameter scope, each independent response parameter in described two reactions is selected independently of one another.

Preferably, the reaction time of reaction (MIII-I) and reaction (MII0-I) is 1 minute to 168 hours, more preferably 2 To 144 hours, even more preferably 12 to 120 hours.

It is typically to be carried out in solvent (MIII-I) to react (MIII-I) and reaction (MII0-I).

Preferably, solvent (MIII-I) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans are sub- Sulfone, acetonitrile, acetone, 1,4- dioxanes, THF and their mixture.

Preferably, the amount of solvent (MIII-I) be respectively the weight of formula (IV) compound or formula (III0) compound 1 to 500 times, even more preferably more preferably 5 to 50 times, 10 to 30 times.

Preferably, in reaction (MIII-I) and reaction (MII0-I), rubbed using 1 to 20 molar equivalent, more preferably 1 to 10 That equivalent, formula (MA) compound of more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on formula (IV) compound or formula (III0) mole of compound.

Preferably, reaction (MIII-I) and reaction (MII0-I) are carried out under an inert atmosphere.

Preferably, reaction (MIII-I) and reaction (MII0-I) are carried out in the presence of compound (COUPADD).

Preferably, 1 to 20 molar equivalent is used in reaction (MIII-I) and reaction (MII0-I), more preferably 1 to 10 rubs That equivalent, the compound (COUPADD) of even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on formula (IV) chemical combination The mole of thing or formula (III0) compound.

Preferably, reaction (MIII-I) and reaction (MII0-I) are carried out in the presence of formula (HOSu) compound.

Preferably, reaction (MIII-I) and (MII0-I) is reacted in compound (COUPADD) and formula (HOSu) compound In the presence of carry out.

Preferably, in reaction (MIII-I) and reaction (MII0-I), rubbed using 1 to 20 molar equivalent, more preferably 1 to 10 That equivalent, formula (HOSu) compound of even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on formula (IV) chemical combination The mole of thing or formula (III0) compound.

React after (MIII-I) and reaction (MII0-I), formula (III) compound or formula (II) compound can lead to respectively Cross and such as wash, extract, filter, concentrate and dry standard method separation.Formula (III) compound or formula (II) compound can be distinguished It is purified, is preferably purified by chromatographing or crystallizing from appropriate solvent before separation or afterwards.

It is similar reaction to react (MIV) and react (MV0), it is possible to similar anti-as defined in the description Answer and carried out under parameter area, each single response parameter in described two reactions is selected independently of one another.

Preferably, the reaction time of reaction (MIV) and reaction (MV0) be from 1 minute to 168 hours, more preferably 1 to 120 hours, even more preferably 6 to 48 hours.

It is typically to be carried out in solvent (MIV) to react (MIV) and reaction (MV0).

Preferably, solvent (MIV) is selected from：Water, methanol, ethanol, N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- dimethyl sulfoxide, acetonitrile, acetone, Isosorbide-5-Nitrae-dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIV) is 1 to 500 times of the weight of formula (V) or formula (Va) compound, more preferably 5 respectively To 50 times, even more preferably 10 to 30 times.

Preferably, reaction (MIV) and reaction (MV0) are carried out under an inert atmosphere.

Preferably, compound (IV) is selected from sodium borohydride, DIBAL-H and their mixture.

The compound (IV) used in reaction (MIV) and the compound (IV) used in reaction (MV0) can be each other It is independently identical or different.

Preferably, in reaction (MIV) and reaction (MV0), using 1 to 50 molar equivalent, more preferably 1 to 20 mole is worked as Amount, the compound (IV) of even more preferably 2 to 10 molar equivalents, the molar equivalent is accordingly based on formula (V) compound or formula (Va) mole of compound.

Reaction (MIV) and reaction (MV0) can be carried out in the presence of salt (MIV), salt (MIV) can be selected from lithium chloride, Calcium chloride, aluminium chloride, zinc chloride and their mixture.

Preferably, if using salt (MIV) in reaction (MIV) and reaction (MV0), then use 1 to 20 molar equivalent, The salt (MIV) of more preferably 1 to 10 molar equivalent, even more preferably 1.5 to 5 molar equivalents, the molar equivalent is accordingly based on formula (V) mole of compound or formula (Va) compound.

React after (MIV) and reaction (MV0), formula (IV) compound or formula (V0) compound respectively can be by such as washing The standard method separation such as wash, extract, filter, concentrate and dry.Formula (IV) compound or formula (V0) compound can be respectively in separation Before or after be purified, preferably by from appropriate solvent chromatograph or crystallize be purified.

It is similar reaction to react (MII0-IVa) and react (MIV0a), it is possible to as defined in the description Carried out under similar response parameter scope, each independent response parameter in described two reactions is selected independently of one another.

Preferably, the reaction time of reaction (MII0-I-IVa) and reaction (MIV0a) is 1 minute to 168 hours, more preferably For 2 to 144 hours, even more preferably 12 to 120 hours.

It is typically to be carried out in solvent (MIV0a) to react (MII0-I-IVa) and reaction (MIV0a).

Preferably, solvent (MIV0a) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- diformazans are sub- Sulfone, acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MIV0a) is 1 to 500 times of the weight of formula (V0) compound, more preferably 5 to 50 times, very To more preferably 10 to 30 times.

Preferably, in reaction (MII0-I-IVa) and reaction (MIV0a), respectively using 1 to 20 molar equivalent, more preferably Compound (II0-I-IVa) or the compound (RIV0a) of 1 to 10 molar equivalent, even more preferably 1 to 5 molar equivalent, it is described Molar equivalent is based on the mole of formula (V0) compound.

Preferably, reaction (MII0-I-IVa) and reaction (MIV0a) are carried out under an inert atmosphere.

Preferably, reaction (MII0-I-IVa) and reaction (MIV0a) are carried out in the presence of formula (HOSu) compound.

Preferably, 1 to 20 molar equivalent, more preferably 1 to 10 are used in reaction (MII0-I-IVa) and reaction (MIV0a) Formula (HOSu) compound of molar equivalent, even more preferably 1 to 5 molar equivalent, the molar equivalent is accordingly based on compound (II0-I-IVa) or compound (RIV0a) mole.

After reaction (MII0-I-IVa) and reaction (MIV0a), formula (III0-I-IVa) compound or formula (IV0a) chemical combination Thing respectively can by such as wash, extract, filtering, concentration and dry etc. standard method separation.Any compound can be in separation It is preceding or be purified afterwards, preferably it is purified by chromatographing or crystallizing from appropriate solvent.

The further subject matter of the present invention is a kind of method (MVa) for formula (Va) compound, formula (Va) chemical combination Thing has its all preferred embodiment as defined in this manual；

Method (MVa) includes step (MVa)；

Step (MVa) includes reaction (MVa), and wherein formula (VI) compound is reacted with formula (SGM) compound；

R31 by being connected to formula SG with (* * *) described key represented in formula SG, and for-OTs ,-OMs ,-OTf ,-Br ,- Cl or-I；

R30 as defined in the description, in addition to its all preferred embodiment；

SG、n4、n3、AAⁿ⁴, (3), R1, R2 as defined in the description, in addition to its all preferred embodiment.

Preferably, method (MII) is included as (MVa) the step of further step, the formula in the step (MVa) (Va) compound.

Preferably, the reaction temperature of reaction (MVa) is more preferably 20 to 100 DEG C from 0 to 150 DEG C, even more preferably 30 to 60 DEG C.

Preferably, reaction (MVa) reaction time be from 1 minute to 168 hours, it is more preferably 1 to 144 hour, more excellent Elect as 12 to 120 hours.

It is typically to be carried out in solvent (MVa) to react (MVa).

Preferably, solvent (MVa) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- dimethyl sulfoxides, Acetonitrile, acetone, Isosorbide-5-Nitrae-dioxanes, THF, and their mixture.

Preferably, the amount of solvent (MVa) is 1 to 500 times of the weight of formula (VI) compound, more preferably 5 to 50 times, more excellent Select 10 to 30 times.

Preferably, in reaction (MVa), using 1 to 20 molar equivalent, more preferably 1 to 10 molar equivalent, even more preferably The compound (SGM) of 1.5 to 5 molar equivalents, the molar equivalent is based on the mole of compound (VI).

Preferably, reaction (MVa) is carried out under an inert atmosphere.

(MVa) is reacted to carry out generally in the presence of alkali (MVa).

Preferably, the alkali (MVa) is selected from：Potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4- diformazans Base aminopyridine and their mixture.

Preferably, in reaction (MVa), 1 to 20 molar equivalent, more preferably 1 to 10 molar equivalent, more preferably 1.5 are used To the alkali (MVa) of 5 molar equivalents, the molar equivalent is based on the mole of formula (VI) compound.

After reacting (MVa), formula (Va) compound can by such as washing, extracting, filtering, concentration and the standard side such as dry Method is separated.Any compound can be purified before separation or afterwards, preferably by the way that quilt is chromatographed or crystallized from appropriate solvent Purifying.

It is similar reaction to react (MVb) and react (MIII0), it is possible to similar as defined in the description Carried out under response parameter scope, each independent response parameter in described two reactions is selected independently of one another.

Preferably, in reaction (MVb) and reaction (MIII0), 1 to 500 molar equivalent, more preferably 5 to 100 moles are used The HCl of equivalent, even more preferably 10 to 50 molar equivalents, the molar equivalent is based respectively on formula (Va) compound or formula (IV0) The mole of compound.

Preferably, the reaction time of reaction (MVb) and reaction (MIII0) be from 1 minute to 168 hours, more preferably 1 to 48 hours, even more preferably from 2 to 24 hours.

(MVb) and reaction (MIII0) is reacted generally to carry out in solvent (MVb).

Preferably, solvent (MVb) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of solvent (MVb) is 1 to 500 times of the weight of formula (Va) compound or formula (IV0) compound, more It is preferred that 5 to 50 times, even more preferably 10 to 30 times.

Preferably, reaction (MVb) and reaction (MIII0) are carried out under an inert atmosphere.

React after (MVb) and reaction (MIII0), formula (V) compound or formula (III0) compound respectively can be by such as Washing, extraction, filtering, concentration and dry etc. standard method separation.They can be with the proton form of their salt or non-proton Form is separated.

Any compound can be purified before separation or afterwards, preferably by the way that quilt is chromatographed or crystallized from appropriate solvent Purifying.

During another theme of the present invention is method for preparing formula (VI) compound, formula (VI) compound such as this specification Defined, it may have its all preferred embodiment；

Method (MVI) includes step (MVIa) and optional step (MVIb)；

The n3AA described in step (MVIa)ⁿ⁴By peptide coupling reaction be continuously attached to formula (VII-1) compound and It is then attached to the corresponding product of peptide coupling reaction as the aforementioned；

Step (MVIb) includes reaction (MVIb), wherein, the AA represented with (3) in formula (VI)ⁿ³N- ends ammonia Base group reacts with compound N TermProt；

NTermProt is selected from C_1-4Alkyl iodide, C_1-4Alkyl bromide, Cl-C (O)-(GRPEG)_m4-R3、R3-C(O)-O-C(O)- R3 and PGNPrec；

PGNPrec is in the AA represented with (3) in formula (VI)ⁿ³The N- terminal amino groups on introduce PGN examination Agent；

GRPEG, m4, R3 and PGN are defined with as above identical, it may have its all preferred embodiment；

n4、n3、AAⁿ⁴(3) as defined in the description, it may have its all preferred embodiment.

Preferably, method (MII) is included as (MVa) the step of further step, and step (MVIa) and optionally Step (MVIb), formula (Va) compound in step (MVa) is prepared in step (MVIa) and optional step (MVIb) Formula (VI) compound.

Formula (VII-1) compound is known compound, and can be prepared by known method.

For the sake of simplicity, herein, AAⁿ⁴Both the amino acid residue of covalent attachment had been used in, (such as in formula (II)), The amino acid used in method (MVIa) is used in again.

If AAⁿ⁴With the side chain with functional group, the functional group can be generally used for protected amino acid side chain functionalities Protection group protection.

Preferably, NTermProt is Ac₂O。

Preferably, PGNPrec is Boc₂O, FmocCl or CbzCl.

Method (MVIa) by using in peptide symthesis commonly use and be that those skilled in the art are known method, ginseng Number and reagent are carried out.Bibliography cited above is submitted necessary information.

Preferably, the reaction time of reaction (MVIb) is from 1 minute to 168 hours, more preferably 1 to 48 hour, even More preferably from 2 to 24 hours.

It is typically to be carried out in solvent (MVIb) to react (MVIb).

Preferably, solvent (MVIb) is selected from：N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, N, N- dimethyl sulfoxides, Acetonitrile, acetone, 1,4- dioxanes, THF, water, methanol, ethanol and their mixture.

Preferably, the amount of the solvent (MVIb) is 1 to 500 times of the weight of formula (VII -1) compound, more preferably 2 to 50 Times, even more preferably 5 to 20 times.

It is highly preferred that method (MVIa) and reaction (MVIb) are continuously carried out in same solvent.

More preferably, method (MVIa) and reaction (MVIb) are continuously carried out in same solvent and in one pot.

Preferably, in this method (MVIa), using 1 to 20 molar equivalent, more preferably 1 to 10 molar equivalent, even more It is preferred that the NTermProt of 1 to 5 molar equivalent, the molar equivalent is based on the mole of formula (VII-1) compound.

Preferably, reaction (MVIb) is carried out under an inert atmosphere.

After method (MVIa) or reaction (MVIb), method (MVIa) or the reaction product for reacting (MVIb), wherein each Corresponding formula (VI) compound, can by such as wash, extract, filtering, concentration and dry etc. standard method separate.Anyization Compound can be purified before separation or afterwards, be preferably purified by chromatographing or crystallizing from appropriate solvent.

Formula (SGM) compound is known compound, can be prepared according to known method.

Preferably, formula (SGM) compound is selected from compound SGM-II and compound SGM-III.

Preferably, compound SGM-II by by formula (HSGH-II) compound first with Boc₂O reacts, then with chemical combination Thing (SGM-II-R31) reacts to prepare.

Compound (SGM-II-R31) is selected from：Paratoluensulfonyl chloride, p-toluenesulfonic anhydride, mesyl chloride, methanesulfonic acid acid anhydride, three Fluorine mesyl chloride and trifluoromethanesulfanhydride anhydride, SOCl₂、(COCl)₂、POCl₃、PCl₃、PCl₅、POBr₃、PBr₃、PBr₅, N- bromo fourths Imidodicarbonic diamide, N-iodosuccinimide, HCl, HBr, HI and their mixture.

Preferably, compound (SGM-II-R31) is TsCl.

Preferably, compound SGM-III be by by formula (HSGH-III) compound first with Boc₂O reacts, Ran Houyu Formula (HSGHReac-1) compound is reacted to prepare.

Formula (HSGH-II) compound is preferably formula (HSGH-II-1) compound or formula (HSGH-II-2) compound.

Compound SGM-II is preferably formula (SGM-II-1) compound or formula (SGM-II-2) compound.

Formula (HSGH-III) compound is preferably formula (HSGH-III-1) compound or formula (HSGH-III-2) compound.

Compound SGM-III is preferably formula (SGM-III-1) compound or formula (SGM-III-2) compound.

Formula (HSGH-II) compound and formula (HSGH-III) compound are known compounds, can be according to known side Prepared by method, and often commercially available.

Formula (CG1MR-IV) compound, formula (CG1MR- III) compound, formula (CG1MR-IIa) compound, formula (HOSu) are changed Compound and formula (COUPADD) compound are known compounds, can be prepared by known method, and often commercially available.

Preferably, formula (CG1MR-IV) compound is by by formula (CG1MR-IV-OH) compound and formula (HOSu) compound React to prepare；

Formula (HOSu) compound is as defined in the description；

Preferably, formula (CG1MR-IV-OH) compound is made by the way that formula (MA) compound is reacted with formula (AC) compound It is standby；

Formula (MA) compound and m30 are as defined in the description, it may have its all preferred embodiment.

Any reaction defined above can be carried out under similar reaction condition, in these reactions it is each each other Response parameter is selected independently：

On pressure：The reaction of any of above definition can be carried out under vacuo, had for example under atmospheric pressure or even Carried out up under the pressure of 10 bars, preferably they are carried out under atmospheric pressure；

On temperature：Preferably, the reaction temperature of the reaction of any of above definition be -20 to 100 DEG C, more preferably 0 to 75 DEG C, even more preferably 10 to 50 DEG C；

On the reaction time：The reaction time of the reaction of any of above definition is 1 minute to 168 hours, more preferably 0.5 To 24 hours, even more preferably 1 to 12 hour；

With the proviso that, for the reaction of any of above definition, without other explanation.

Especially, formula (I) compound is selected from：Formula (10) compound, formula (11) compound, formula (12) compound, formula (12- 101) compound, formula (13) compound, formula (14) compound, formula (15) compound, formula (15-102) compound and formula (16) are changed Compound；

The anti-Interleukin -1β antibody of monoclonal produced in mouse

The anti-Interleukin -1β antibody of monoclonal produced in mouse

Wherein, Doxorubicin is formula (DOXO) compound, its by respectively formula (10), (11), (12), (12-101), (13) the amino connection that, (d1) in (14), (15), (15-102) and (16) and in formula (DOXO) is represented.

Especially, formula (II) compound is selected from：Formula (20) compound, formula (21) compound, formula (22) compound, formula (23) Compound, formula (24) compound, formula (25) compound, formula (26) compound, formula (20-CAMPTO) compound, formula (21- CAMPTO) compound, formula (22-CAMPTO) compound, formula (23-CAMPTO) and formula (21-TAXO-t1-1) compound；

Wherein,

Doxorubicin is formula (DOXO) compound, and it passes through respectively in formula (20), (21), (22), (23), (24), (25) (26) the amino connection that (d1) in and in formula (DOXO) is represented；

Camptothecine is formula (CAMPTO) compound, and it passes through respectively in formula (20-CAMPTO), (21-CAMPTO), (22- CAMPTO the hydroxyl connection that), (c1) in (23-CAMPTO) and in formula (CAMPTO) is represented；

Taxo-t1-1 is formula (TAXO) compound, its by formula (21-TAXO-t1-1), formula (TAXO-t1-1) simultaneously And the hydroxyl connection that (t1) in formula (TAXO) is represented.

Especially, formula (IIc) compound is formula (20c) compound.

Especially, formula (III) compound is selected from：Formula (30) compound, formula (31) compound, formula (32) compound, formula (33) compound, formula (34) compound, formula (35) compound and formula (36) compound.

Especially, formula (III0) compound is formula (300) compound；

Wherein, camptothecine is formula (CAMPTO) compound, and it is by using correspondingly in formula (300) and in formula (CAMPTO) the hydroxyl connection that (c1) in is represented.

Especially, formula (a of III0-I- IV) compound is formula (320) compound.

Especially, formula (IV) compound is selected from：Formula (40) compound, formula (41) compound, formula (42) compound, formula (43) Compound, formula (44) compound, formula (45) compound and formula (46) compound.

Especially, formula (IV0) compound is formula (400) compound；

Wherein,

Camptothecine is formula (CAMPTO) compound, and it is by using correspondingly in formula (400) and in formula (CAMPTO) (c1) represent hydroxyl connection.

Especially, formula (IV0a) compound is formula (400a) compound.

Especially, formula (V) compound is selected from：Formula (50) compound, formula (50-1) compound, formula (51) compound, formula (51-1) compound, formula (52) compound, formula (52-1) compound, formula (53) compound, formula (53-1) compound, formula (54) are changed Compound, formula (54-1) compound, formula (54-2) compound, formula (54-3) compound, formula (55) compound, formula (55-1) chemical combination Thing, formula (56) compound and formula (56-1) compound.

Especially, formula (V0) compound is formula (500) compound.

Especially, formula (VI) compound is selected from：Formula (6) compound, formula (6-1) compound, formula (6-2) compound, formula (6- 3) compound, formula (6-4) compound, formula (6b) compound, formula (6b-1) compound, formula (6b-2) compound, formula (6b-3) are changed Compound, formula (6b-4) compound, formula (6c) compound and formula (6-5) compound.

The further subject matter of the present invention is compound, and it is selected from：Formula (I) compound, formula (II) compound, formula (IIc) Compound, formula (III) compound, formula (IV) compound, formula (IV-IIa) compound, formula (V) compound, formula (Va) compound, Formula (VI) compound, formula (III0-IIa) compound, formula (III0) compound, formula (IV0) compound, formula (IV0a) compound, Formula (V0) compound and formula (III0-I-IVa) compound；These compounds as defined in the description, and with it is all its Preferred embodiment.

The further subject matter of the present invention is compound, and it is selected from formula (10) compound, formula (11) compound, formula (12) and changed Compound, formula (12-101) compound, formula (13) compound, formula (14) compound, formula (15) compound, formula (15-102) chemical combination Thing, formula (16) compound, formula (20c) compound, formula (20) compound, formula (21) compound, formula (22) compound, formula (23) are changed Compound, formula (24) compound, formula (25) compound, formula (26) compound, formula (20-CAMPTO) compound, formula (21-CAMPTO) Compound, formula (22-CAMPTO) compound, formula (23-CAMPTO) compound, formula (21-TAXO-t1-1) compound, formula (30) Compound, formula (31) compound, formula (32) compound, formula (33) compound, formula (34) compound, formula (35) compound, formula (36) compound, formula (300) compound, formula (320) compound, formula (40) compound, formula (41) compound, formula (42) chemical combination Thing, formula (43) compound, formula (44) compound, formula (45) compound, formula (46) compound, formula (400) compound, formula (400a) Compound, formula (50) compound, formula (50-1) compound, formula (51) compound, formula (51-1) compound, formula (52) compound, Formula (52-1) compound, formula (53) compound, formula (53-1) compound, formula (54) compound, formula (54-1) compound, formula (54- 2) compound, formula (54-3) compound, formula (55) compound, formula (55-1) compound, formula (56) compound, formula (56-1) chemical combination Thing, formula (500) compound, formula (6) compound, formula (6-1) compound, formula (6-2) compound, formula (6-3) compound, formula (6- 4) compound, formula (6b) compound, formula (6b-1) compound, formula (6b-2) compound, formula (6b-3) compound, formula (6b-4) are changed Compound, formula (6c) compound and formula (6-5) compound；These compounds are as defined in the description.

Another theme of the present invention is formula (I) compound as the purposes for preparing pharmaceutical composition or medicine, the formula (I) Compound as defined in the description, and as defined in they all preferred embodiments.

The further subject matter of the present invention is a kind of pharmaceutical composition or medicine, wherein described pharmaceutical composition and medicine bag Include formula (I) compound, formula (I) compound as defined in the description, also as in they all preferred embodiments Defined.

The further subject matter of the present invention is formula (I) compound, pharmaceutical composition or medicine, and it is in treatment disease or disease In purposes, the purposes preferably in treating cancer, wherein described pharmaceutical composition and the medicine include formula (I) compound, Formula (I) compound as defined in the description, also as defined in its all preferred embodiment.

Formula (II) compound can easily be covalently attached to ligand LI.It is especially surprising that of the present invention Protein drug conjugate includes linking group CG2, CG2 and comes from o- hydroxyl-p- aminobenzyl alcohol, and the protein drug conjugate bag Include linear peptide residue, particularly formula (I) compound and show enhanced plasma stability, and discharge medicine without allowing medicine Learn and be modified, moreover, they show good water-soluble and low reunion.

Embodiment

Abbreviation

DCM dichloromethane

DIBAL-H diisobutyl aluminium hydrides

DIPEA N, N- diisopropylethylamine

DMA DMAC N,N' dimethyl acetamides

DMAP 4-dimethylaminopyridines

DMF N,N-dimethylformamides

EDTA ethylenediamine tetra-acetic acids

EEDQ 2- ethyoxyl -1- carbethoxyl group -1,2- EEDQs

ESI-MS electrospray ionization mass spectrometries

EtOAc ethyl acetate

HIC hydrophobic interaction chromatographies

MeCN acetonitriles

NAP-25 post GE Healthcare NAP-25 posts are to use Sephadex^TMDNA grades of G-25 It is pre-packaged disposable with post and operation only needs to gravity

NMR nuclear magnetic resonance

PE petroleum ethers

Retention factors of the Rf in TLC

RP-HPLC reversed-phase HPLCs

RT room temperatures

SAFC Sigma Aldrich fine chemicals

SEC-HPLC SECs HPLC

TBTU 2- (1H- BTA -1- bases) -1,1,3,3- tetrafluoro boric acid tetramethyl-ammonium

THF tetrahydrofurans

TFA trifluoroacetic acids

TLC thin-layered chromatography

Raw material

Formula (DOXO) compound Doxorubicin, as hydrochloride can from Beijing large medicine company development in science and technology it is limited Company buys

Daunomycin (daunorubicin) is as hydrochloride commercially available from Aldrich

PBS used has following component, KH in PBS experiments₂PO₄:144mg/L, NaCl: 9000mg/L and Na₂HPO₄:795mg/L

Embodiment 1

The concentrated sulfuric acid is added dropwise in 0 DEG C of mixture to p- aminosalicylic acid (15.0 grams) and methanol (113.0 milliliters) (30.0 milliliters).Obtained mixture is heated to reflux and stirred 2 hours, to form uniform solution.Then reaction is mixed Thing is cooled to room temperature.Water (360 milliliters) is added, solid sodium bicarbonate is subsequently added into until pH value is 7.By gained mixture mistake Filter, is washed with water (3 times, every time with 80 milliliters) wet cake, and is dried in vacuo at 55 DEG C, obtains 14.7 grams of the formula for solid (VII-1) compound (yield 89%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 3.90 (1H, s), 4.12 (2H, brs), 6.16 to 6.19 (2H, m), 7.62 to 7.65 (1H, m)

ESI-MS:168.0(M+H)+

Embodiment 2

To H-Cit-OH (40.0 grams, 1.0 equivalents) and sodium carbonate (50.0 grams, 2.0 equivalents) in water (300 milliliters) and THF Di-tert-butyl dicarbonate (60.0 grams, 1.2 equivalents) was added dropwise in THF in mixture in (150 milliliters) in 1 hour Solution in (100 milliliters).Obtained mixture is stirred overnight under RT.Afterwards, washed with PE (polyethylene) (2 times, every time With 150 milliliters) suspension, mixture is then concentrated under vacuum to about 300 milliliters.Will be mixed with 4.0M aqueous potassium hydrogen sulfates It is 2 that compound, which is acidified to pH, is then extracted with EtOAc (5 times, every time with 150 milliliters).Merge organic phase, and use saturated brine (100 milliliters) washings, with anhydrous sodium sulfate drying and are filtered.Filtrate is evaporated to drying, 52.0 grams of Boc-Cit-OH is obtained, It is white solid (yield 83%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 1.38 (9H, s), 1.36 to 1.43 (2H, m), 1.47 to 1.69 (2H, M), 2.93 (2H, q, J=6.3Hz), 3.82 to 3.87 (1H, m), 5.36 (2H, brs), 5.92 (1H, t, J=5.6Hz), 7.07 (1H, d, J=8.0Hz), 12.44 (1H, brs)

ESI-MS:275.8 (M+H)+, 550.8 (2M+H)+

Embodiment 3

The Boc-Cit-OH (53.0 grams, 1.0 equivalents) prepared by EEDQ (72.0 grams, 1.5 equivalents) and according to embodiment 2 adds Enter in THF (400 milliliters).Then added into this mixture according to embodiment 1 prepare formula (VII-1) compound (49.0 grams, 1.5 equivalents).Obtained mixture is stirred at room temperature 14 hours.Then by reactant mixture (500 milliliters) dilutions of water, Extracted with EtOAc (3 times, every time with 250 milliliters).Merge organic phase, and washed with 1.0M aqueous citric acid solution (2 times, every time With 150 milliliters) and saturated brine (150 milliliters) wash.Afterwards, anhydrous Na is used₂SO₄Organic phase is dried, is then concentrated to dryness, Crude product purifies (eluant, eluent PE by silica gel column chromatography：EtOAc=4:1 to 2:1 to 1:1 (volume/volume) extremely pure EtOAc) And separate, 62.5 grams formula (6-4) compound is obtained, it is yellow solid (yield 76%).

Pass through silica-gel TLC analysis：EtOAc is eluent (Rf=0.3, UV254).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.40 (9H, s), 1.57 to 1.83 (4H, m), 3.08 to 3.16 (2H, M), 3.90 (3H, s), 4.43 (1H, s), 5.26 (2H, s), 5.85 (2H, s), 7.11 (1H, d, J=8.8Hz), 7.28 (1H, S), 7.68 (1H, d, J=8.8Hz), 9.74 (1H, s), 10.82 (1H, brs)

ESI-MS:325.2(M-tBuOCO+2H)+

Embodiment 4

Formula (6-4) compound (62.0 grams, 1 equivalent) prepared according to embodiment 3 is suspended at 15% (w/w) In solution of the hydrochloric acid in Isosorbide-5-Nitrae-dioxanes (100 milliliters), the mixture of generation is stirred at room temperature 1 hour.Then will be anti- Answer mixture to be concentrated under vacuum, obtain formula (6-3) compound of 51.6 grams of HCl salt form, it is white solid (yield 98%).

Embodiment 5

By formula (6-3) compound (75.6 grams, 1.0 equivalents) according to the HCl salt form prepared in embodiment 4, Boc-L- Val (43.0 grams, 1.0 equivalents) and TBTU (135.5 grams, 2.0 equivalents) are dissolved in DMF (250 milliliters).Then, DIPEA is added (71.2 grams, 2.6 equivalents).Obtained solution is stirred at room temperature 17 hours.Then by reactant mixture with water (750 milliliters) Dilution, is extracted (5 times, every time 200 milliliters) with EtOAc, is merged organic phase, is used 1.0M NaHCO₃Aqueous solution washing (3 times, every time With 300 milliliters), then washed with saturated brine (150 milliliters).Collect organic phase and be concentrated to dryness, then use crude product Silica gel chromatography (eluent DCM：Methanol MeOH=20：1 to 10：1 to 7：1 (volume/volume) is simultaneously separated, and obtains 62.0 Gram formula (6-2) compound (yield 57%) for solid.

Pass through silica-gel TLC analysis：DCM：MeOH=6:1 (V/V) is used as eluant, eluent (Rf=0.4, UV254)

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.84 (6H, dd, J1=17.2Hz, J2=6.8Hz), 1.39 (9H, 1.42 to 1.51 s), (2H, m), 1.55 to 1.73 (2H, m), 1.93 to 1.98 (1H, m), 2.90 to 3.08 (2H, m), 3.82 to 3.84 (1H, m), 3.88 (3H, s), 4.39 to 4.45 (1H, m), 5.43 (2H, s), 6.01 (1H, t, J=5.8Hz), 6.72 (1H, d, J=8.8Hz), 7.09 (1H, dd, J1=8.8Hz, J2=2.0Hz), 7.73 (1H, d, J=8.8Hz), 8.08 (1H, D, J=7.2Hz), 10.34 (1H, s), 10.62 (1H, brs).

ESI-MS:524.3 (M+H)+, 424.3 (M-tBuOCO+2H)+

Embodiment 6

Formula (6-2) compound (62.0 grams, 1 equivalent) prepared according to embodiment 5 is suspended at 15% (w/w) In solution of the hydrochloric acid in Isosorbide-5-Nitrae-dioxanes (200 milliliters), the mixture of generation is stirred at room temperature 1 hour.Then will be anti- Answer mixture to be concentrated under vacuum, obtain formula (6-1) compound of 52.4 grams of HCl salt form, it is white solid (yield 97%).

Embodiment 7

By formula (6-1) compound (52.4 grams, 1.0 equivalents) of the HCl salt form prepared according to embodiment 6, acetic anhydride (60.0 grams, 5.0 equivalents), (150 milliliters) mixing of pyridine (100.0 grams, 11.0 equivalents) and methanol, and be stirred at room temperature 7 days. Suspension is filtered, the wet cake of gained is washed into (4 times, every time 200 milliliters) with MeOH, is then dried under vacuum, obtains 32.9 grams of formula (6) compounds, it is white solid (yield 62%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=12.4Hz, J2=6.8Hz), 1.35 to 1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to 2.00 (1H, m), 2.93 to 3.06 (2H, m), 3.87 (3H, s), 4.20 (1H, t, J=7.6Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J=5.2Hz), 7.11 (1H, dd, J1=8.8Hz, J2=1.6Hz), 7.41 (1H, d, J=1.6Hz), 7.73 (1H, d, J=8.8Hz), 7.89 (1H, d, J=8.4Hz), 8.21 (1H, d, J=7.2Hz), 10.24 (1H, s), 10.62 (1H, s)

ESI-MS:466.3 (M+H)+, 931.3 (2M+H)+

Embodiment 8

Under nitrogen atmosphere by formula (MA) compound (5.5 grams, 1.0 equivalents) and Beta-alanine (5.0 grams, 1.0 equivalents) in DMF Stirring 2 hours in (30.0 milliliters).Then mixture is cooled to 0 DEG C.Addition formula (HOSu) compound (8.0 grams, 1.3 equivalents) With DCC (24.0 grams, 2.0 equivalents).Then reactant mixture is warmed to room temperature, and be stirred at room temperature overnight.Then will be anti- Answer mixture to filter, the wet cake of gained is washed with DMF (40.0 milliliters), merge organic phase, it is then dilute with water (120 milliliters) Release and use (3 times, every time 50 milliliters) extractions of DCM.Merge organic phase, washed with water (50 milliliters), then with 5% (weight/weight Amount) the washing of (50 milliliters) of sodium bicarbonate aqueous solution, then washed with saturated brine (50 milliliters).By organic phase anhydrous slufuric acid Sodium is dried, and is then concentrated, until solid starts precipitation.Then, PE (20 milliliters) is added in mixture, and it is mixed by what is obtained Compound is stirred at room temperature 10 minutes.Then mixture is filtered, wet cake is washed with PE (20 milliliters), is then done under vacuo It is dry, at 40 DEG C overnight, 4.0 grams formula (CG1MR-IV-1) compound is obtained, it is white solid (yield 27%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) and δ 2.84 (4H, s), 3.04 (2H, t, J=7.0Hz), 3.95 (2H, t, J= 7.0Hz), 6.75 (2H, s)

ESI-MS:267.2 (M+H)+, 289.4 (M+Na)+

Embodiment 9

To formula (HSGH-II-1) compound (10.0 grams, 1.0 equivalents), methanol (50 milliliters) and Et3N, (10.7 grams, 1.1 work as Amount) mixture Boc is added dropwise₂O is in MeOH (22.8 grams, 1.1 equivalent Boc₂O is in 50 ml methanols) in solution.Then Gained mixture is stirred at room temperature 15 hours, is then dried under vacuum, 20.0 grams of formula (Boc-HSGH-II-1) is obtained Compound, it is light yellow oil (quantitative yield).

¹H NMR (400MHz, CDCl₃, 20 DEG C) and δ 1.44 (9H, s), 2.73 (1H, brs), 3.32 (2H, q, J=5.2Hz), 3.54 to 3.58 (4H, m), 3.72 to 3.74 (2H, m), 5.15 (1H, brs).

Embodiment 10

At 0 DEG C to formula (Boc-HSGH-II-1) compound (6.17 grams, 1 equivalent) prepared according to embodiment 9, pyridine DMAP (0.366 gram, 0.1 equivalent) is added in (2.86 grams, 1.2 equivalents) and DCM (30 milliliters) mixture.Toluene is added dropwise The mixture of sulfonic acid chloride (6.31 grams, 1.1 equivalents) and DCM (45 milliliters).Obtained mixture is stirred at room temperature 8 days.So Gained mixture is poured into DCM (100 milliliters) afterwards, (1 time, with 100 milliliters) is washed with water in gained mixture, then will be organic Mutually use anhydrous sodium sulfate drying.Obtained solution is further concentrated, and passes through Silica gel chromatography (PE:EtOAc=6:1 To PE:EtOAc=1:1 (volume/volume)), 8.1 grams formula (SGM-II-1) compound is obtained, it is colorless oil (yield 75%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=12.4Hz, J2=6.8Hz), 1.35 to 1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to 2.00 (1H, m), 2.93 to 3.06 (2H, m), 3.87 (3H, s), 4.20 (1H, t, J=7.6Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J=5.2Hz), 7.11 (1H, dd, J1=8.8Hz, J2=1.6Hz), 7.41 (1H, d, J=1.6Hz), 7.73 (1H, d, J=8.8Hz), 7.89 (1H, d, J=8.4Hz), 8.21 (1H, d, J=7.2Hz), 10.24 (1H, s), 10.62 (1H, s).

Embodiment 11

By formula (6) compound (3.00 grams, 1 equivalent) prepared according to embodiment 7, the formula prepared according to embodiment 10 (SGM-II-1) compound (4.65 grams, 2 equivalents), K₂CO₃(1.82 grams, 2 equivalents) and the mixture of dry DMF (30 milliliters) add Heat is stirred 7 days to 50 DEG C, and under nitrogen atmosphere.Then reactant mixture is concentrated under vacuum to drying.Added to residue Methanol (30 milliliters), gained mixture is stirred 10 minutes, then filtered.Wet cake is washed to (3 times, every time 10 milli with methanol Rise).Merge organic phase, then concentrate.Residue purifies (DCM by silica gel column chromatography：Methanol=7:1 (volume/volume)), 3.40 grams formula (50-1) compound is obtained, is light yellow solid (yield 81%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=12.8Hz, J2=6.8Hz), 1.38 to 1.51 (2H, m), 1.56 to 1.80 (2H, m), 1.89 (3H, s), 1.92 to 2.01 (1H, m), 2.92 to 3.05 (2H, m), 3.07 To 3.12 (2H, m), 3.50 (2H, t, J=6.0Hz), 3.75 to 3.77 (5H, m), 4.08 to 4.10 (2H, m), 4.20 (1H, Dd, J1=8.4Hz, J2=6.8Hz), 4.34 to 4.40 (1H, m), 5.42 (2H, s), 5.99 (1H, t, J=5.8Hz), 6.74 (1H, t, J=5.6Hz), 7.26 (1H, dd, J1=8.4Hz, J2=1.6Hz), 7.49 (1H, d, J=2.0Hz), 7.68 (1H, D, J=8.8Hz), 7.91 (1H, d, J=8.4Hz), 8.19 (1H, d, J=7.6Hz), 10.20 (1H, s).

ESI-MS:653.4 (M+H)+, 675.5 (M+Na)+, 553.5 (M-tBuOCO+2H)+

Embodiment 12

Formula (50-1) compound (500 milligrams) prepared according to embodiment 11, and the HCl of 10% (w/w) are existed The mixture of solution in 1,4- dioxanes (10 milliliters) is stirred at room temperature 15 hours.Solvent is removed under vacuum, 431 millis are obtained Gram HCl salt form formula (50) compound, its be slightly yellow solid (quantitative yield).

ESI-MS:553.5 (M+H)+, 1105.2 (2M+H)+

Embodiment 13

To formula (50) compound (1.10 grams, 1.0 equivalents) of the HCl salt form prepared according to embodiment 12 at -30 DEG C With the solution (1M, 11.9ml, 6.0 equivalent) of addition DIBAL-H in hexane in anhydrous THF (20 milliliters) mixture.Then Obtained mixture is heated to 0 DEG C, and stirred 15 hours under nitrogen atmosphere.Then methanol (2.0 milliliters) is added.Then will be full It is added to aqueous sodium potassium tartrate (10 milliliters) in mixture, and the mixture is stirred at room temperature 30 minutes.By institute The mixture obtained is evaporated to drying, and to produce the residue of white, (5 times, the every time 10 milliliters) residue is washed with methanol.It is dense Be condensed and organic phase, and purify with silica gel column chromatography (DCM：MeOH：Et3N=65：33：2 (volume/volumes)), obtain 0.65g formula (40) compound, it is white solid (yield 72%).

¹H NMR (400MHz, DMSO-d₆, 60 DEG C) and δ 0.86 (6H, t, J=6.6Hz), 1.37 to 1.53 (2H, m), 1.63 To 1.79 (2H, m), 1.92 (3H, s), 1.99 to 2.07 (1H, m), 2.70 (2H, brs), 2.99 to 3.02 (2H, m), 3.47 (2H, t, J=5.6Hz), 4.07 (2H, t, J=5.8Hz), 4.18 (1H, t, J=7.6Hz), 4.34 to 4.41 (1H, m), 5.25 (2H, s), 5.90 (1H, brs), 7.16 (1H, d, J=8.0Hz), 7.26 (2H, d, J=8.0Hz), 7.32 (1H, s), 7.76 (1H, d, J=7.6Hz), 7.91 (1H, d, J=4.4Hz), 9.69 (1H, s)

ESI-MS:525.6(M+H)+

Embodiment 14

At room temperature to formula (40) compound (500.2 milligrams, 1.0 equivalents) prepared according to embodiment 13, according to implementation Added in formula (CG1MR-IV-1) compound (281.0 milligrams, 1.1 equivalents) and DMF (9.5 milliliters) mixture prepared by example 8 DIPEA (140.3 milligrams, 1.1 equivalents).Obtained mixture is stirred at room temperature 17 hours.Then, remove under vacuo DMF, obtains light yellow residue.Then residue is mixed with acetone (10 milliliters), and be stirred at room temperature 18 hours.Will be mixed Compound is filtered, and (2 times, every time with 5 milliliters) wet cake is washed with acetone, is then dried under vacuum, obtains 515.0 milligrams of formula (30) compound, it is light yellow solid (yield 80%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=11.2Hz, J2=6.8Hz), 1.32 to 1.48 (2H, m), 1.55 to 1.76 (2H, m), 1.89 (3H, s), 1.94 to 2.02 (1H, m), 2.33 (2H, t, J=7.2Hz), 2.93 to 3.04 (2H, m), 3.16 to 3.20 (2H, q, J=5.6Hz), 3.46 (2H, t, J=5.8Hz), 3.60 (2H, t, J= 7.8Hz), 3.73 (2H, t, J=4.6Hz), 4.04 (2H, t, J=4.6Hz), 4.18 (1H, dd, J1=8.4Hz, J2= 6.8Hz), 4.34 to 4.39 (1H, m), 4.44 (2H, s), 4.88 (1H, brs), 5.42 (2H, s), 6.00 (1H, t, J= 5.4Hz), 7.00 (2H, s), 7.16 (1H, dd, J1=8.4Hz, J2=2.0Hz), 7.26 (1H, d, J=8.4Hz), 7.32 (1H, d, J=1.6Hz), 7.92 (1H, d, J=8.4Hz), 8.05 (1H, t, J=5.6Hz), 8.12 (1H, d, J=7.6Hz), 9.88 (1H, s).

Embodiment 15

By formula (30) compound (400 milligrams, 1.0 equivalents) prepared according to embodiment 14, DIPEA (231.0 milligrams, 3.0 Equivalent), the mixture of 4 angstroms of molecular sieves (800 milligrams) and dry DMF (8.0 milliliters) is stirred 5 minutes.Then, formula (II-1) is added Compound (361.2 milligrams, 2 equivalents).Obtained mixture is stirred at room temperature 3 hours.Then the formula of HCl salt form is added (DOXO) compound (342.8 milligrams, 1.0 equivalents), and the mixture is stirred 4 hours.Then MeCN (40.0 milliliters) is added. Precipitation is formed, and is filtered, with MeCN and DMF (5：1 (volume/volume), 2 times, every time with 5 milliliters) mixture wash.Merge Filtrate, and in 45 DEG C of vacuum drying, obtain dark red residue.Residue is washed into (2 times, every time with 10 milliliters) with MeCN, Then residue is dissolved in the mixture (20 of acetone and water：1 (volume/volume)) in, and purified by preparative silica gel tlc (DCM：MeOH=5：1 (volume/volume), Rf=0.15).By product by acetone and water mixture (20：1 (volume/volume), 6 times, every time 20 milliliters) from silica gel extraction, the extract of merging is dried under vacuum, the crude product for red solid is obtained. Then crude product is mixed with acetonitrile (5.0 milliliters), mixture is stirred at room temperature 5 hours, then filtered.By filter cake and second (5.0 milliliters) mixing of nitrile, mixture is stirred at room temperature 3 hours.Then mixture is filtered.At room temperature by filter cake vacuum Dry, obtain 48.0 milligrams formula (20) compound, it is red solid (yield 7%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.85 (6H, dd, J1=11.2Hz, J2=6.8Hz), 1.13 (3H, D, J=6.4Hz), 1.34 to 1.47 (3H, m), 1.57 to 1.72 (2H, m), 1.82 to 1.90 (1H, m), 1.90 (3H, s), 1.93 to 1.98 (1H, m), 2.08 to 2.22 (2H, m), 2.29 (2H, t, J=7.2Hz), 2.91 to 3.01 (4H, m), 3.11 to 3.16 (2H, m), 3.41 (2H, t, J=5.6Hz), 3.45 to 3.46 (1H, m), 3.54 (2H, t, J=7.2Hz), 3.68 to 3.74 (3H, m), 3.98 (3H, s), 4.03 (2H, t, J=4.0Hz), 4.17 (2H, t, J=7.6Hz), 4.32 to 4.38 (1H, M), 4.58 (2H, d, J=5.2Hz), 4.69 (2H, d, J=5.6Hz), 4.85 to 4.89 (2H, m), 4.94 (1H, brs), 5.22 (1H, brs), 5.40 (2H, s), 5.46 (1H, brs), 5.98 (1H, t, J=4.8Hz), 6.80 (1H, d, J=8.0Hz), 6.95 (2H, s), 7.12 (1H, d, J=8.4Hz), 7.19 (1H, d, J=8.4Hz), 7.36 (1H, s), 7.64 (1H, brs), 7.89 to 7.91 (3H, m), 7.99 (1H, t, J=5.2Hz), 8.10 (1H, d, J=7.2Hz), 9.92 (1H, s), 13.26 (1H, s), 14.02 (1H, s).

ESI-MS:1245.5(M+H)+

Embodiment 16

In the mixing of 0 DEG C of downdraft mode (HSGH-III-1) compound (110.16 grams, 5 equivalents) and dioxanes (400 milliliters) Boc is added dropwise in thing₂Mixtures of the O (22.10 grams, 1 equivalent) in dioxanes (200 milliliters).Then gained mixture is added Then heat stir 20 hours to room temperature.Then solvent is removed under vacuo.Obtained residue is added in water (300 milliliters), Gained mixture is extracted (2 times, every time with 300 milliliters) with DCM.By organic layer anhydrous sodium sulfate drying, then it is evaporated to Dry.Residue purifies (DCM by silica gel column chromatography：Ethyl acetate=1：1 (volume/volume), then uses DCM：MeOH： Et3N=89：9：2 (volume/volumes)), 17.7 grams formula (Boc-HSGH-III-1) compound is obtained, it is pale yellowish oil Thing (yield 55%).

1H NMR (400MHz, CDCl3,20 DEG C) δ 1.44 (9H, s), 1.64 (2H, s), 1.72-1.78 (4H, m), 2.81 (2H, t, J=6.6Hz), 3.23 (2H, q, J=6.0Hz), 3.55-3.62 (8H, m), 3.64-3.66 (4H, m), 5.14 (1H, brs)。

Embodiment 17

At 0 DEG C in 1 hour to prepared according to embodiment 18 formula (Boc-HSGH-III-1) compound (5.00 grams, 1.0 equivalents), K₂CO₃Formula (HSGHReac-1) chemical combination is added dropwise in (4.30 grams, 2.0 equivalents) and DCM (40 milliliters) mixture Mixture of the thing (2.17 grams, 1.2 equivalents) in DCM (20 milliliters).Obtained mixture is warmed to room temperature and to stir 20 small When.Then solid is filtered, filter cake is washed with DCM (2 times, every time with 5ml).Merging filtrate is simultaneously evaporated to drying.Then will be residual Thing is stayed to purify (eluant, eluent with silica gel column chromatography：EtOAc：PE=3：1) 5.6 grams formula (SGM-III-1) compound, is obtained, It is light yellow oil (yield 90%).

1H NMR (400MHz, CDCl3,20 DEG C) δ 1.44 (9H, s), 1.64 (2H, s), 1.73-1.86 (4H, m), 3.23 (2H, q, J=6.0Hz), 3.44 (2H, q, J=6.0Hz), 3.54 (2H, t, J=6.0Hz), 3.58-3.66 (10H, m), 4.04 (2H, s), 4.98 (1H, brs), 7.31 (1H, brs)

Embodiment 18

By formula (6) compound (5.80 grams, 1.0 equivalents) prepared according to embodiment 7, K₂CO₃(5.18 grams, 2.0 equivalents), Formula (SGM-III-1) compound (9.95 grams, 2.0 equivalents) and DMF (45ml) mixing prepared according to preparation example 17.Then incite somebody to action To mixture be heated to 50 DEG C, and stir 7 days under nitrogen atmosphere.Obtained reactant mixture is evaporated to dryness under vacuo It is dry.Methanol (40 milliliters) is added into residue, gained mixture is stirred 10 minutes, then filtered.By wet cake methanol Wash (3 times, every time 10 milliliters).Organic filtrate is collected and merged, is then evaporated.Residue is purified by silica gel column chromatography (DCM：MeOH=7:1 (volume/volume)), 7.20 grams of formula (51-1) compounds are obtained, it is light yellow solid (yield 70%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=13.6Hz, J2=6.8Hz), 1.32 to 1.49 (2H, m), 1.37 (9H, s), 1.55 to 1.78 (6H, m), 1.89 (3H, s), 1.94 to 1.99 (1H, m), 2.93 to 3.07 (4H, m), 3.26 (2H, q, J=6.8Hz), 3.37 (2H, t, J=6.4Hz), 3.43 to 3.52 (10H, m), 3.81 (3H, s), 4.20 (1H, dd, J1=8.4Hz, J2=6.8Hz), 4.35 to 4.41 (1H, m), 4.53 (2H, s), 5.44 (2H, s), 6.03 (1H, t, J=5.6Hz), 6.74 (1H, t, J=5.6Hz), 7.36 (1H, dd, J1=8.4Hz, J2=2.0Hz), 7.41 (1H, D, J=2.0Hz), 7.80 (1H, d, J=8.8Hz), 7.90 (1H, d, J=8.4Hz), 8.06 (1H, t, J=5.6Hz), 8.21 (1H, d, J=7.2Hz), 10.32 (1H, s).

ESI-MS:726.6 (M-tBuOCO+2H)+, 826.3 (M+H)+, 848.5 (M+Na)+

Embodiment 19

By the hydrochloric acid of formula (51-1) compound (1.00 grams) prepared according to embodiment 18 and 15% (volume/volume) 1, Solution mixing in 4- dioxanes (10 milliliters).Mixture is stirred at room temperature 3 hours.Solvent is removed under vacuum, is obtained Formula (51) compound of 0.99 gram of HCl salt form, it is slightly yellow solid (quantitative yield).

ESI-MS:726.6(M+H)+

Embodiment 20

By formula (51) compound (6.08 grams, 1 equivalent) of the HCl salt form prepared according to embodiment 19, water (100 millis Rise), the mixture of calcium chloride (1.86 grams, 2.0 equivalents) and sodium borohydride (1.27 grams, 4.0 equivalents) is stirred at room temperature.Point Criticize and add other sodium borohydride (1.27 grams, 4.0 equivalents, after the stirring of totally 15 hours；1.27 grams, 4.0 equivalents, warp Cross after the stirring of totally 20 hours；1.27 grams, 4.0 equivalents, after the stirring of totally 24 hours).By totally 36 hours After stirring, methanol (30 milliliters) is added into mixture.Then reactant mixture is filtered, washed with methanol (3 times, use every time 10 milliliters) wet cake.By liquid-phase collection, merge and be then evaporated to drying.Residue is purified by silica gel column chromatography (eluent DCM：MeOH：Et3N=65：33：2 (volume/volumes)), 2.70 grams formula (41) compound is obtained, it is light yellow Solid (yield 48%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=10.8Hz, J2=6.8Hz), 1.31 to 1.49 (2H, m), 1.55 to 1.82 (6H, m), 1.90 (3H, s), 1.93 to 2.02 (1H, m), 2.82 (2H, t, J=7.4Hz), 2.98 to 3.05 (2H, m), 3.15 to 3.20 (2H, q, J=6.8Hz), 3.34 to 3.51 (12H, m), 4.16 to 4.20 (1H, M), 4.34 to 4.41 (1H, m), 4.46 (2H, s), 4.50 (2H, s), 5.47 (2H, s), 6.13 (1H, t, J=5.6Hz), 7.25 (2H, s), 7.26 (1H, s), 7.95 (1H, d, J=8.8Hz), 8.10 (1H, t, J=5.6Hz), 8.14 (1H, d, J= 7.6Hz), 10.01 (1H, s).

ESI-MS:698.7(M+H)+

Embodiment 21

The formula prepared by formula (41) compound (2.78 grams, 1.0 equivalents) prepared according to embodiment 20, according to embodiment 8 (CG1MR-IV-1) compound (1.18 grams, 1.1 equivalents) and DMF (30 milliliters) are mixed at room temperature.Then DIPEA is added (0.58 gram, 1.1 equivalents).Obtained mixture is stirred at room temperature 16 hours.Then DMF is removed under vacuo, obtains shallow Yellow residue.Then residue is mixed with acetone (30 milliliters), and the mixture is stirred at room temperature 5 hours.Then Mixture is filtered, wet cake washs (2 times, every time with 15 milliliters) with acetone, is then dried under vacuum, obtains 2.55 grams Formula (31) compound, it is light yellow solid (yield 75%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=11.2Hz, J2=6.8Hz), 1.32 to 1.48 (2H, m), 1.55 to 1.73 (2H, m), 1.89 (3H, s), 1.93 to 2.01 (1H, m), 2.31 (2H, t, J=7.2Hz), 2.90 to 3.11 (4H, m), 3.17 (2H, q, J=6.4Hz), 3.36 (2H, t, J=6.4Hz), 3.44 to 3.60 (8H, m), 3.60 (2H, t, J=7.2Hz), 4.19 (1H, dd, J1=8.4Hz, J2=6.8Hz), 4.35 to 4.40 (1H, m), 4.46 (2H, S), 4.51 (2H, s), 5.08 (1H, brs), 5.42 (2H, s), 6.00 (1H, t, J=5.6Hz), 7.00 (2H, s), 7.22 (1H, S), 7.25 (1H, s), 7.89 to 7.91 (1H, m), 8.04 (1H, t, J=5.6Hz), 8.10 (1H, d, J=7.6Hz), 9.92 (1H, s)

ESI-MS:831.6 (M-OH)+, 849.4 (M+H)+

Embodiment 22

By formula (31) compound (500.4 milligrams, 1.0 equivalents) prepared according to embodiment 21, DIPEA (305.7 milligrams, 4.0 equivalents), the mixture of 4 angstroms of molecular sieves (500.5 milligrams) and dry DMF (10.0 milliliters) stirs 5 minutes.Then, formula is added (II-1) compound (271.1 milligrams, 1.5 equivalents).Obtained mixture is stirred at room temperature 5 hours.Then, HCl is added Formula (DOXO) compound (342.8 milligrams, 1.0 equivalents) of salt form, gained mixture is stirred 3.5 hours.Then add MeCN (50.0 milliliters).Form precipitation, filtering, and with MeCN and DMF (5：1 (volume/volume), 3 times, every time 10 milliliters) wash Wash.Filtrate is merged, is dried in vacuo at 45 DEG C, obtains dark red residue.Residue is dissolved in DCM and MeOH (7：1 (volume/volume)) mixture in, and pass through preparative silica gel tlc (DCM：MeOH=7:1 (volume/volume), Rf=0.15) Purifying.With the mixture (20 of acetone and water：1 (volume/volume), 5 times, every time with 50 milliliters) extract product from silica gel, will The extract of merging is dried under vacuum, and obtains the crude product for red solid.Then (30 milliliters) of crude product and acetonitrile are mixed Close, mixture is stirred at room temperature 18 hours, then filtered.Filter cake is mixed with acetonitrile (10 milliliters), and stirred at room temperature Mix the mixture 3 hours.Then mixture is filtered.At room temperature, filter cake is dried under vacuum, obtains 100.3 milligrams Formula (21) compound, it is red solid (yield 12%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.85 (6H, dd, J1=11.8Hz, J2=6.8Hz), 1.13 (3H, D, J=6.4Hz), 1.34 to 1.68 (9H, m), 1.82 to 1.90 (1H, m), 1.90 (3H, s), 1.93 to 2.00 (1H, m), 2.13 to 2.24 (2H, m), 2.30 (2H, t, J=7.2Hz), 2.91 to 3.04 (6H, m), 3.10 to 3.15 (2H, m), 3.29 (2H, t, J=6.0Hz), 3.37 to 3.49 (11H, m), 3.59 (2H, t, J=7.2Hz), 3.68 to 3.76 (1H, m), 3.99 (3H, s), 4.13 to 4.19 (2H, m), 4.33 to 4.41 (3H, m), 4.58 (2H, d, J=5.6Hz), 4.71 (1H, d, J= 5.2Hz), 4.86 (1H, t, J=5.8Hz), 4.95 (1H, brs), 5.02 (2H, s), 5.23 (1H, brs), 5.41 (2H, s), 5.46 (1H, brs), 5.99 (1H, t, J=4.6Hz), 6.87 (1H, d, J=7.2Hz), 7.00 (2H, s), 7.21 to 7.25 (3H, m), 7.66 (1H, t, J=4.6Hz), 8.11 (1H, d, J=7.2Hz), 9.97 (1H, s), 13.28 (1H, s), 14.04 (1H, s)

ESI-MS:1417.8(M+H)+

Embodiment 23

2 hours endo form (HSGH-III-2) compounds (84.9 grams, 5.0 equivalents) and CHCl at room temperature₃(500 milliliters) Mixture in Boc is added dropwise₂O (50.0 grams, 1.0 equivalents) is in CHCl₃Mixture in (200 milliliters).To at room temperature The solution arrived is stirred 16 hours.Gained suspension is filtered, and wet cake is washed with DCM (50 milliliters).Filtrate is merged, evaporated To drying, colorless oil is obtained.Then the grease is dissolved in DCM (200 milliliters), is washed, be used in combination with water (300 milliliters) Anhydrous sodium sulfate drying.Then the organic phase of gained is evaporated to drying, product purifies (eluent DCM with silica gel column chromatography： MeOH=20：1 to 5：1 (volume/volume)), obtain 13.0 grams formula (Boc-HSGH-III-2) compound (yield 33%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) and δ 1.32 (9H, s), 1.46-1.53 (4H, m), 2.64 (2H, t, J= 6.4Hz), 3.06 to 3.09 (2H, m), 5.22 (1H, brs)

Embodiment 24

To formula (Boc-HSGH-III-2) compound (11.3 prepared according to embodiment 23 in 40 minutes at 0 DEG C Gram), K₂CO₃Formula (HSGHReac-1) compound and DCM (50 is added dropwise in (18.0 grams) and DCM (200 milliliters) mixture Milliliter) mixture.Then gained mixture is heated to room temperature and stirred 2 hours.Then, aqueous citric acid solution (% is added (weight), the weight based on solution, 180 milliliters).Organic phase is separated, is washed with saturated brine (100 milliliters), uses anhydrous slufuric acid Sodium is dried, and is then evaporated to drying.Then residue purifies (eluant, eluent PE by silica gel column chromatography：EtOAc 2：1 to 1：1 To 1：2 (volume/volumes)), 13.9 grams formula (SGM-III-2) compound is obtained, it is white solid (yield 89%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.46 (9H, s), 1.68 to 1.71 (2H, m), 3.20 (2H, q, J= 6.2Hz), 3.39 (2H, q, J=6.4Hz), 4.07 (2H, s), 4.87 (1H, brs), 7.17 (1H, brs)

Embodiment 25

The formula prepared by formula (6) compound (5.51 grams, 1.0 equivalents) prepared according to embodiment 7, according to embodiment 24 (SGM-III-2) compound (6.02 grams, 2.0 equivalents), K₂CO₃(3.31 grams, 2.0 equivalents) and dry DMF (30 milliliters) it is mixed Compound is heated to 50 DEG C, and stirs 7 days in a nitrogen atmosphere.Then reactant mixture is concentrated under vacuum to drying.Will MeOH (40 milliliters) is added in residue, and gained mixture is stirred 10 minutes, then filtered.Wet cake is washed with methanol (3 times, every time with 10 milliliters).Filtrate is collected, merges, then concentrates.Residue passes through silica gel column chromatography (eluent DCM： MeOH=7:1 (volume/volume)) purify, 3.8 grams formula (52-1) compound is obtained, it is light yellow solid (yield 46%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.88 (6H, dd, J1=9.6Hz, J2=6.8Hz), 1.32 to 1.50 (2H, m), 1.52 to 1.82 (4H, m), 1.90 (3H, s), 1.92 to 1.97 (1H, m), 2.91 to 3.05 (4H, m), 3.17 to 3.22 (2H, m), 3.81 (3H, s), 4.13 (1H, t, J=7.6Hz), 4.33 to 4.39 (1H, m), 5.42 (2H, s), 5.98 (1H, t, J=5.8Hz), 6.80 (1H, t, J=5.4Hz), 7.45 (1H, dd, J1=8.6Hz, J2=1.8Hz), 7.50 (1H, D, J=1.6Hz), 7.80 (1H, d, J=8.6Hz), 8.02 to 8.05 (2H, m), 8.50 (1H, d, J=11.6Hz), 10.14 (1H, s)

ESI-MS:580.5 (M-tBuOCO+2H)+, 680.4 (M+H)+, 1359.0 (2M+H)+

Embodiment 26

At room temperature by formula (52-1) compound (3.40 grams) prepared according to embodiment 25, and 10% (w/w) The mixture of solution of the hydrochloric acid in 1,4- dioxanes (50 milliliters) stir 15 hours.Then by reactant mixture under vacuo Concentration, obtains formula (52) compound of 3.20 grams of HCl salt forms, and it is white solid (quantitative yield).It is straight in the next step Connect and use the crude mixture.

Embodiment 27

By formula (52) compound (3.00 grams, 1 equivalent) of the HCl salt form prepared according to embodiment 26, water (60 milliliters), The mixture of calcium chloride (1.09 grams, 2.0 equivalents) and sodium borohydride (0.75 gram, 4.0 equivalents) is stirred at room temperature.It is added portionwise Extra sodium borohydride (0.75 gram, 4.0 equivalents, after the stirring of totally 15 hours；0.74 gram, 4.0 equivalents, by totally 19 After the stirring of hour；0.74 gram, 4.0 equivalents, after the stirring of totally 23 hours).After the stirring of totally 40 hours, to mixing Methanol (15 milliliters) is added in thing.Then reactant mixture is filtered, wet cake is washed (3 times, every time with 10 milliliters) with methanol. Filtrate is collected, merges, is then evaporated to drying.Residue purifies (eluent DCM by silica gel column chromatography：MeOH：Et3N =65：33：2 (volume/volumes)), 1.05 grams formula (42) compound is obtained, it is light yellow solid (yield 39%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=11.0Hz, J2=6.8Hz), 1.30 to 1.46 (2H, m), 1.48 to 1.73 (4H, m), 1.89 (3H, s), 1.93 to 2.01 (1H, m), 2.56 (1H, t, J=6.8Hz), 2.91 to 3.06 (4H, m), 3.13 to 3.22 (2H, m), 4.16 to 4.20 (1H, m), 4.34 to 4.39 (1H, m), 4.46 (2H, S), 4.50 (2H, s), 5.45 (2H, s), 6.06 (1H, t, J=5.6Hz), 7.22 to 7.27 (3H, m), 7.94 (1H, d, J= 8.4Hz), 8.16 to 8.19 (2H, m), 9.96 (1H, s)

ESI-MS:534.2 (M-OH)+, 552.2 (M+H)+

Embodiment 28

Prepared by formula (42) compound (940.4 milligrams, 1.0 equivalents) prepared according to embodiment 27, according to embodiment 8 (10 milliliters) mixing of formula (CG1MR-IV-1) compound (501.2 milligrams, 1.1 equivalents) and DMF.Then, add at room temperature DIPEA (247.0 milligrams, 1.1 equivalents).Obtained mixture is stirred at room temperature 4 hours.Then DMF is removed under vacuo, Obtain light yellow residue.Then residue is mixed with acetone (20 milliliters), and obtained mixture is stirred at room temperature 2 Hour.Then mixture is filtered, wet cake washs (3 times, every time with 5 milliliters) with acetone, is then dried under vacuum, obtains 950.0 milligrams formula (32) compound, it is light yellow solid (yield 79%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.86 (6H, dd, J1=11.2Hz, J2=6.8Hz), 1.34 to 1.46 (2H, m), 1.47 to 1.73 (4H, m), 1.89 (3H, s), 1.94 to 2.01 (1H, m), 2.32 (2H, t, J=7.6Hz), 2.92 to 3.05 (4H, m), 3.10 to 3.15 (2H, m), 3.60 (2H, t, J=7.2Hz), 4.17 to 4.20 (1H, m), 4.34 to 4.42 (1H, m), 4.46 (2H, s), 4.52 (2H, s), 5.08 (1H, brs), 5.42 (2H, s), 5.99 (1H, t, J=5.6Hz), 7.00 (2H, s), 7.21 (1H, s), 7.25 (2H, s), 7.90 (1H, d, J=8.4Hz), 7.95 (1H, t, J=5.6Hz), 8.05 (1H, t, J=5.6Hz), 8.11 (1H, d, J=7.6Hz), 9.92 (1H, s).

Embodiment 29

By formula (32) compound (402.5 milligrams, 1.0 equivalents) prepared according to embodiment 28,4 angstroms of molecular sieve (800.0 millis Gram), the mixture of dry DMF (8.0 milliliters) and formula (II-1) compound (347.3 milligrams, 2.0 equivalents) stirs 5 minutes.So Afterwards, DIPEA (297.7 milligrams, 4.0 equivalents) is added.Obtained mixture is stirred at room temperature 5 hours.Then, HCl is added Formula (DOXO) compound (332.2 milligrams, 1.0 equivalents) of salt form, is then stirred the mixture for 4 hours.Then MeCN is added (40.0 milliliters).Precipitation is formed, is filtered and with MeCN and DMF (5：1 (volume/volume), 2 times, every time with 5 milliliters) mixture Washing.Merging filtrate, is dried in vacuo at 45 DEG C, obtains dark red residue.Residue is dissolved in DCM and MeOH (10：1 (body Product/volume)) mixture, and pass through preparative silica gel thin-layer chromatography purify (DCM：MeOH=7:In 1 (volume/volume), Rf=0.15).With the mixture (20 of acetone and water：1 (volume/volume), 5 times, every time with 50 milliliters) extract the production from silica gel Thing, the extract of merging is dried under vacuum, and obtains the crude product for red solid.Then by crude product and acetonitrile (20 millis Rise) mixing, mixture is stirred at room temperature 2 hours, then filtered.Filter cake is dried under vacuum at room temperature, obtained 74.0 milligrams formula (22) compound, it is red solid (yield 10%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.84 (6H, dd, J1=10.8Hz, J2=6.8Hz), 1.13 (3H, D, J=6.4Hz), 1.30 to 1.49 (5H, m), 1.56 to 1.69 (2H, m), 1.81 to 1.88 (4H, m), 1.93 to 1.98 (1H, M), 2.13 to 2.25 (2H, m), 2.28 (2H, t, J=7.2Hz), 2.92 to 3.07 (8H, m), 3.45 (1H, brs), 3.56 (2H, t, J=7.2Hz), 3.73 (1H, brs), 3.99 (3H, s), 4.15 to 4.19 (2H, m), 4.35 to 4.38 (1H, m), 4.41 (2H, s), 4.58 (2H, d, J=5.8Hz), 4.71 (1H, d, J=5.6Hz), 4.85 (1H, t, J=6.0Hz), 4.94 (1H, brs), 5.03 (2H, s), 5.22 (1H, brs), 5.42 (2H, s), 5.47 (1H, brs), 6.02 (1H, t, J=4.6Hz), 6.87 (1H, d, J=8.0Hz), 6.97 (2H, s), 7.22 to 7.25 (3H, m), 7.65 (1H, t, J=4.8Hz), 7.88 to 7.90 (5H, m), 8.11 (1H, d, J=7.6Hz), 9.98 (1H, s), 13.27 (1H, s), 14.02 (1H, s)

ESI-MS:1271.9 (M+H)+, 1294.4 (M+Na)+

Embodiment 30

At 0 DEG C in 1 hour endo form (HSGH-II-2) compound (21.24 grams, 1.0 equivalents) and at MeOH (100 milliliters) In Et₃Boc is added dropwise in N (21.03 grams, 1.1 equivalents) mixture₂O (43.83 grams, 1.1 equivalents) is in MeOH (50 millis Rise) in solution.Then mixture is warmed to room temperature and stirred 24 hours.Then obtained mixture is evaporated under vacuo To drying, 43.05 grams formula (Boc-HSGH-II-2) compound is obtained, it is yellow oil (quantitative yield).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.30 to 1.43 (4H, m), 1.45 (9H, s), 1.48 to 1.61 (4H, M), (1H, the brs) of 1.70 (1H, brs), 3.12 (2H, q, J=6.8Hz), 3.64 (2H, t, J=6.8Hz), 4.57

Embodiment 31

At 0 DEG C to formula (Boc-HSGH-II-2) compound (20.02 grams, 1.0 equivalents), pyrrole prepared according to embodiment 30 T was added dropwise in 2 hours in the mixture of pyridine (8.02 grams, 1.1 equivalents) and dichloromethane (50 milliliters)_SCl (19.33 grams, 1.1 equivalents) mixture in dichloromethane (75mL).Obtained mixture is stirred at room temperature 7 days.Then solution is steamed It is sent to drying.The mixture of residue and solvent is washed into (PE：Ethyl acetate=6:1 (volume/volume), 4 times, every time with 100 Milliliter), and filter.Merging filtrate, is then evaporated to drying.Further by the column chromatography eluting (PE of crude product purified by silica gel：Acetic acid Ethyl ester=6:1 (volume/volume)), 14.20 grams of formula (SGM-II-2) compounds are obtained, it is white solid (yield 41%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.22 to 1.37 (4H, m), 1.39 to 1.47 (11H, ms), 1.62 to 1.68 (2H, m), 2.46 (3H, s), 3.07 (2H, t, J=6.6Hz), 4.02 (2H, t, J=6.4Hz), 4.52 (1H, brs), 7.36 (2H, d, J=8.0Hz), 7.78 to 7.82 (2H, m)

Embodiment 32

The formula prepared by formula (6) compound (9.31 grams, 1.0 equivalents) prepared according to embodiment 7, according to embodiment 31 (SGM-II-2) compound (14.90 grams, 2.0 equivalents), K₂CO₃The mixing of (5.65 grams, 2.0 equivalents) and dry DMF (60 milliliters) Thing is heated to 50 DEG C, and stirs 9 days in a nitrogen atmosphere.Reactant mixture is evaporated to drying.Methanol is added to the residue (40 milliliters), and obtained mixture is stirred 10 minutes, then filter, further wash filter cake with MeOH (3 times, every time With 15 milliliters).Merging filtrate, is evaporated to drying.Residue is purified by silica gel column chromatography, 6.35 grams of formula (53- is obtained 1) compound, it is slightly yellow solid (yield 48%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=12.0Hz, J₂=6.8Hz), 1.24 to 1.33 (2H, m), 1.35 to 1.48 (15H, m), 1.56 to 1.76 (4H, m), 1.89 (3H, s), 1.92 to 2.01 (1H, m), 2.89 to 3.05 (4H, m), 3.75 (3H, s), 3.97 (2H, t, J=6.2Hz), 4.17 to 4.21 (1H, m), 4.34 to 4.39 (1H, m), 5.41 (2H, s), 5.99 (1H, t, J=5.8Hz), 6.77 (1H, t, J=5.2Hz), 7.24 (1H, dd, J₁= 8.6Hz, J₂=1.8Hz), 7.47 to 7.48 (1H, m), 7.67 (1H, d, J=8.6Hz), 7.90 (1H, d, J=8.6Hz), 8.18 (1H, d, J=7.4Hz), 10.17 (1H, s)

ESI-MS:565.0(M-^tBuOCO+2H)⁺, 664.8 (M+H)⁺, 1329.1 (2M+H)⁺

Embodiment 33

By the HCl of formula (53-1) compound (8.10 grams) prepared according to embodiment 32 and 10% (w/w) 1, Solution in 4- dioxanes (50 milliliters) is mixed.Mixture is stirred at room temperature 1.5 hours.Solvent is removed under vacuum, Formula (53) compound of 9.00 grams of HCl salt forms is obtained, it is white solid (quantitative yield).

Embodiment 34

To formula (53) compound (4.01 grams, 1.0 equivalents) of the HCl salt form prepared according to embodiment 33 at -30 DEG C With solution of the addition DIBAL-H in hexane (1M, 51.1 milliliters, 8.1 equivalents) in anhydrous THF (40 milliliters) mixture.So Obtained mixture is heated to 0 DEG C afterwards, and stirred 2.5 hours in a nitrogen atmosphere, then warms to room temperature and stirs naturally Mix 16 hours.Then the mixture is cooled to 0 DEG C.Add methanol (10 milliliters).Then by saturation potassium sodium tartrate (100 millis Rising) aqueous solution is added in mixture, the mixture is stirred at room temperature 1 hour.The mixture of gained is evaporated to drying, The residue of white is produced, the white residue is mixed with methanol (60 milliliters), and is stirred 1 hour.Filtering gained suspension, (3 times, every time with 30 milliliters) filter cake is further washed with methanol.The filtrate of merging is concentrated and purified by silica gel column chromatography (DCM：Methanol：Et₃N=66：32：2 (volume/volumes)), 1.81 grams formula (43) compound is obtained, it is that white solid (is received Rate 53%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=10.4Hz, J₂=6.8Hz), 1.31 to 1.46 (8H, m), 1.55 to 1.74 (4H, m), 1.90 (3H, s), 1.94 to 2.02 (1H, m), 2.60 (2H, t, J=6.8Hz), 2.91 to 3.05 (2H, m), 3.92 (2H, t, J=6.4Hz), 4.16 to 4.20 (1H, m), 4.34 to 4.39 (1H, m), 4.44 (2H, s), 5.43 (2H, s), 6.07 (1H, t, J=5.6Hz), 7.14 to 7.17 (1H, m), 7.25 (1H, d, J=8.0Hz), 7.32 (1H, d, J=2.0Hz), 7.94 (1H, d, J=8.4Hz), 8.13 (1H, d, J=7.6Hz), 9.90 (1H, s)

ESI-MS:519.9(M-OH^-)⁺, 538.0 (M+H)⁺, 1129.5 (2M+H)⁺

Embodiment 35

By formula (43) compound (1.13 grams, 1.0 equivalents) prepared according to embodiment 34, maleic anhydride (207.3 milligrams, 1.1 equivalents) and DMF (10 milliliters) mixture be stirred at room temperature 20 hours.Formula (HOSu) is added into the solution of gained to change Compound (225.1 milligrams, 1.0 equivalents) and EDC (presence in the form of its single hydrochloride) (745.0 milligrams, 2.0 equivalents).Will Obtained mixture is further stirred at room temperature 4 days.Mixture is evaporated to drying, then mixed with acetone (30 milliliters). Obtained mixture is stirred at room temperature 20 hours, then filtered.Filter cake is washed into (3 times, every time with 15 milliliters) with acetone, so After be dried under vacuum, obtain 1.10 grams formula (33) compound, its be light yellow solid (yield 90%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=10.2Hz, J₂=6.8Hz), 1.26 to 1.55 (8H, m), 1.59 to 1.78 (4H, m), 1.90 (3H, s), 1.93 to 2.02 (1H, m), 2.95 to 3.06 (2H, m), 3.41 (2H, t, J=7.0Hz), 3.89 (2H, t, J=6.2Hz), 4.16 to 4.20 (1H, m), 4.34 to 4.40 (1H, m), 4.43 (2H, s), 5.42 (2H, brs), 6.05 (1H, brs), 7.00 (2H, s), 7.15 to 7.18 (1H, m), 7.24 to 7.29 (2H, M), 7.93 (1H, d, J=8.4Hz), 8.11 (1H, d, J=7.6Hz), 9.87 (1H, s)

ESI-MS:599.6(M-OH^-)⁺, 639.8 (M+Na)⁺

Embodiment 36

By formula (33) compound (50.2 milligrams, 1.0 equivalents) prepared according to embodiment 35,4 angstroms of molecular sieve (100.0 millis Gram), the mixture of dry DMF (1.0 milliliters) and formula (II-1) compound (37.0 milligrams, 1.6 equivalents) be stirred at room temperature 5 points Clock.Then DIPEA (46.5 milligrams, 4.4 equivalents) is added.Obtained mixture is stirred at room temperature 3.5 hours.Then, plus Enter formula (DOXO) compound (37.8 milligrams, 0.86 equivalent) of HCl salt form, and then stir the mixture for 4.5 hours.So Afterwards, MeCN (5.0 milliliters) is added.Precipitation is formed, and is filtered, and with MeCN and DMF mixture (5:1 (volume/volume), divides 2 It is secondary, every time with 1 milliliter) wash.Merging filtrate, is dried in vacuo at 45 DEG C, obtains dark residue.Residue is dissolved in DCM and MeOH mixture (10:1, volume/volume, 3 milliliters) in, and (DCM is purified by preparative silica gel tlc：MeOH= 7:1 (volume/volume), Rf=0.35).Pass through the mixture (20 of acetone and water:1, volume/volume, 6 times, every time with 3 milliliters) Product is extracted from silica gel.The extract of merging is dried under vacuum, the crude product for red solid is obtained.By crude product Mixed with acetonitrile (5ml), mixture is stirred at room temperature 2 hours, then filtered.Filter cake washed with acetonitrile (2 times, every time with 1 Milliliter), then it is dried in vacuo at 25 DEG C, obtains 9.0 milligrams formula (23) compound, it is red solid (yield 9%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.84 (6H, dd, J₁=10.2Hz, J₂=6.8Hz), 1.13 (3H, D, J=6.4Hz), 1.19 to 1.23 (2H, m), 1.35 to 1.50 (7H, m), 1.56 to 1.68 (4H, m), 1.81 to 1.86 (1H, M), 1.88 (3H, s), 1.91 to 2.01 (1H, m), 2.11 to 2.23 (2H, m), 2.91 to 3.01 (4H, m), 3.32 (2H, t, J =6.8Hz), 3.45 to 3.47 (1H, m), 3.69 to 3.74 (1H, m), 3.98 (3H, s), 4.15 to 4.19 (2H, m), 4.32 to 4.37 (1H, m), 4.58 (2H, d, J=5.8Hz), 4.66 (2H, d, J=6.8Hz), 4.82 to 4.93 (4H, m), 5.23 (1H, Brs), 5.39 (2H, s), 5.46 (1H, brs), 5.96 (1H, t, J=5.6Hz), 6.74 (1H, d, J=8.0Hz), 6.93 (2H, S), 7.10 to 7.18 (2H, m), 7.31 (1H, brs), 7.62 to 7.65 (1H, m), 7.89 to 7.90 (3H, m), 8.09 (1H, d, J=7.4Hz), 9.88 (1H, s), 13.26 (1H, s), 14.02 (1H, s)

ESI-MS:1185.8(M+H)⁺, 1208.4 (M+Na)⁺

Embodiment 37

By Z-L-Val (1.47 grams, 0.95 equivalent), dry DMF (15 milliliters), TBTU (2.97 grams, 1.5 equivalents) and DIPEA (2.00 grams, 2.5 equivalents) mixture is stirred at room temperature 15 minutes.Added into the mixture of gained according to implementation Formula (6-3) compound (2.23 grams, 1.0 equivalents) of HCl salt form prepared by example 4.Mixture is further stirred at room temperature 14 hours, then it is evaporated to drying at 45 DEG C.Then water (150 milliliters) is added, gained mixture is extracted with EtOAc (3 times, Every time with 200 milliliters).The organic phase merged is washed with 250 milliliters of saturated brines, anhydrous sodium sulfate drying is then used.It will obtain Solution concentration, then purify (PE with silica gel column chromatography：EA=1:2 to DCM：MeOH=10:1 (volume/volume)), obtain 2.92 grams formula (6-5) compound, it is white solid (yield 85%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.87 (6H, dd, J₁=17.6Hz, J₂=6.8Hz), 1.36 to 1.50 (2H, m), 1.58 to 1.74 (2H, m), 1.94 to 2.02 (1H, m), 2.91 to 3.08 (2H, m), 3.87 (3H, s), 3.90 To 3.96 (1H, m), 4.37 to 4.43 (1H, m), 5.04 (2H, s), 5.43 (2H, s), 5.99 (1H, t, J=5.6Hz), 7.10 (1H, dd, J₁=8.8Hz, J₂=2.0Hz), 7.30 to 7.37 (6H, m), 7.41 (1H, d, J=2.0Hz), 7.74 (1H, d, J= 8.8Hz), 8.19 (1H, d, J=7.2Hz), 10.32 (1H, s), 10.62 (1H, s)

ESI-MS:557.6(M+H)⁺, 579.9 (M+Na)⁺, 1114.8 (2M+H)⁺, 1136.5 (2M+Na)⁺

Embodiment 38

Mix formula (6-5) compound (20.00 grams, 1.0 equivalents) prepared according to embodiment 37, K₂CO₃(9.91 grams, 2.0 Equivalent) and dry DMF (80 milliliters).Obtained mixture is heated to 50 DEG C, and stirred 0.5 hour.Divided 4 parts in 2 hours Formula (SGM-II-1) compound (25.79 grams, 2.0 equivalents) prepared according to embodiment 10 is added in reactant mixture.Will Reactant mixture is stirred 3 days at 50 DEG C, is then evaporated to drying at 45 DEG C.(4 times, every time with 20 milliliters) are washed with MeOH residual Then excess, merging filtrate is concentrated to dryness.Residue is further purified into (DCM by silica gel column chromatography：MeOH=20： 1, volume/volume), 22.17 grams formula (54-1) compound is obtained, it is white solid (yield 83%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.87 (6H, dd, J₁=17.6Hz, J₂=6.8Hz), 1.36 to 1.49 (11H, m), 1.59 to 1.73 (2H, m), 1.96 to 2.03 (1H, m), 2.92 to 3.14 (4H, m), 3.50 (2H, t, J= 6.0Hz), 3.74 to 3.76 (5H, m), 3.93 to 3.97 (1H, m), 4.08 (2H, t, J=4.6Hz), 4.39 to 4.44 (1H, M), 5.05 (2H, s), 5.42 (2H, s), 5.98 (1H, t, J=5.6Hz), 6.73 (1H, t, J=5.4Hz), 7.25 (1H, dd, J₁=8.6Hz, J₂=1.56Hz), 7.30 to 7.36 (6H, m), 7.48 (1H, s), 7.69 (1H, d, J=8.6Hz), 8.15 (1H, D, J=7.2Hz), 10.28 (1H, s)

ESI-MS:745.0(M+H)⁺, 1488.8 (2M+H)⁺, 645.3 (M-^tBuOCO+2H)⁺

Embodiment 39

By formula (54-1) compound (4.80 grams, 1.0 equivalents), methanol (200 milliliters) and the Pd/ that are prepared according to embodiment 38 C (5%, 0.24 gram) mixture deaerates three times, is then stirred 18 hours at 30 DEG C under hydrogen atmosphere (6 bar).By gained Mixture is filtered, and filtrate is concentrated under vacuum into drying, 3.96 grams formula (54-2) compound is obtained, it is white solid (quantitative yield).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.81 (3H, d, J=6.8Hz), 0.90 (3H, d, J=6.8Hz), 1.36 to 1.49 (11H, m), 1.58 to 1.78 (2H, m), 1.91 to 1.99 (1H, m), 2.92 to 3.12 (5H, m), 3.50 (2H, T, J=6.0Hz), 3.75 to 3.77 (5H, m), 4.09 (2H, t, J=4.8Hz), 4.47 (1H, s), 5.45 (2H, s), 6.07 (1H, t, J=5.6Hz), 6.72 (1H, t, J=5.2Hz), 7.24 (1H, dd, J₁=8.6Hz, J₂=1.6Hz), 7.51 (1H, d, J=1.4Hz), 7.69 (1H, d, J=8.6Hz), 8.24 (1H, d, J=8.6Hz), 10.42 (1H, s)

ESI-MS:611.8(M+H)⁺, 1221.4 (2M+Na)⁺, 512.0 (M-^tBuOCO+2H)⁺

Embodiment 40

By DMG (0.48 gram, 1.0 equivalents), DMF (15 milliliters) and TBTU (3.01 grams, 2.0 equivalents) Mixture be cooled to 0 DEG C.Then obtained mixture is stirred 15 minutes, is subsequently added into the formula prepared according to embodiment 39 (54-2) compound (2.86 grams, 1.0 equivalents).Then mixture is warmed to room temperature naturally, further stirring 18 hours.Then will The mixture of gained is evaporated to drying.The residue of gained purifies (DCM by silica gel column chromatography：MeOH=7:1 (volume/body Product)), 2.78 grams formula (54-3) compound is obtained, it is white solid (yield 85%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.82 (3H, d, J=6.8Hz), 0.89 (3H, d, J=6.8Hz), 1.36 to 1.49 (11H, m), 1.56 to 1.75 (sH, m), 1.98 to 2.06 (1H, m), 2.33 (6H, s), 2.92 to 3.04 (2H, M), 3.06 to 3.11 (4H, m), 3.50 (2H, t, J=6.0Hz), 3.74 to 3.77 (5H, m), 4.09 (2H, t, J=4.6Hz), 4.30 to 4.34 (1H, m), 4.36 to 4.41 (1H, m), 5.43 (2H, s), 6.01 (1H, t, J=5.8Hz), 6.73 (1H, t, J =5.2Hz), 7.25 (1H, dd, J₁=8.6Hz, J₂=1.8Hz), 7.28 to 7.32 (1H, m), 7.35 to 7.39 (1H, m), 7.46 (1H, d, J=1.6Hz), 7.55 (1H, d, J=8.4Hz), 7.68 (1H, d, J=8.6Hz), 7.82 (1H, d, J= 9.0Hz), 7.85 to 7.87 (1H, m), 8.38 (1H, d, J=7.2Hz), 10.30 (1H, s)

ESI-MS:696.2(M+H)⁺, 1391.1 (2M+H)⁺, 596.3 (M-^tBuOCO+2H)⁺

Embodiment 41

By the HCl of formula (54-3) compound prepared according to embodiment 40 and 10% (w/w) in 1,4- dioxanes The mixture of solution in (50 milliliters) is stirred at room temperature 18 hours.Solvent is removed under vacuum, 5.60 grams of dihydrochlorides are obtained Formula (54) compound of form, it is white solid (quantitative yield).

ESI-MS：596.4(M+H)⁺, 1191.3 (2M+H)⁺

Embodiment 42

0 DEG C of formula (54) compound (1.00 grams, 1.0 equivalents) to the dihydrochloride form prepared according to embodiment 41 with Solution of the DIBAL-H in hexane (1M, 10 milliliters, 6.7 equivalents) is added in the mixture of anhydrous THF (15 milliliters).It will obtain Mixture 0 DEG C further stirring 1.5 hours.Then methanol (10 milliliters) is added dropwise.Then saturation potassium tartrate is added Sodium water solution (30 milliliters), and be stirred at room temperature 1 hour.The mixture of gained is evaporated to drying.Gained residue is used Methanol washs (3 times, every time 10 milliliters).The filtrate of merging is concentrated, then purifies (DCM with silica gel column chromatography：MeOH： Et₃N=65：33：2 (volume/volumes)), 0.43 gram formula (44) compound is obtained, it is white solid (yield 51%).

¹H NMR (400MHz, DMSO-d₆, 60 DEG C) and δ 0.82 (3H, d, J=6.8Hz), 0.87 (3H, d, J=6.8Hz), 1.32 to 1.50 (2H, m), 1.55 to 1.74 (2H, m), 1.97 to 2.06 (1H, m), 2.23 (6H, s), 2.73 (2H, t, J= 5.6Hz), 2.91 (2H, brs), 2.93 to 3.06 (2H, m), 3.50 (2H, t, J=5.6Hz), 3.75 (2H, t, J=4.6Hz), 4.05 to 4.07 (2H, m), 4.29 to 4.33 (1H, m), 4.35 to 4.40 (1H, m), 4.45 (2H, s), 5.41 (2H, s), 6.02 (1H, t, J=5.6Hz), 7.15 (1H, dd, J₁=8.4Hz, J₂=1.6Hz), 7.26 (1H, d, J=8.0Hz), 7.31 (1H, d, J=1.6Hz), 7.63 (1H, d, J=9.2Hz), 8.31 (1H, d, J=7.4Hz), 9.97 (1H, s)

ESI-MS:550.3((M-OH^-)⁺, 568.2 (M+H)⁺, 1134.7 (2M+H)⁺

Embodiment 43

At room temperature to formula (44) compound (1.10 grams, 1.0 equivalents) prepared according to embodiment 42, according to embodiment 8 DIPEA is added in formula (CG1MR-IV-1) compound (0.57 gram, 1.1 equivalents) of preparation and DMF (4.0 milliliters) mixture (0.28 gram, 1.1 equivalents).Obtained mixture is stirred at room temperature 18 hours.Then, DMF is removed under vacuo, obtains shallow Yellow residue, is then mixed, and be stirred at room temperature 18 hours with acetone (20 milliliters).Mixture is filtered, washed with acetone (2 times, every time with 5ml) filter cake is washed, is then dried under vacuum, 1.11 grams formula (34) compound (yield 80%) is obtained.

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.82 (3H, d, J=6.8Hz), 0.88 (3H, d, J=6.8Hz), 1.34 to 1.46 (2H, m), 1.55 to 1.73 (2H, m), 1.97 to 2.05 (1H, m), 2.25 (6H, s), 2.34 (2H, t, J= 7.0Hz), 2.95 to 3.06 (4H, m), 3.17 to 3.21 (2H, m), 3.46 (2H, t, J=5.8Hz), 3.60 (2H, t, J= 7.2Hz), 3.73 (2H, t, J=4.4Hz), 4.03 (2H, t, J=4.4Hz), 4.29 to 4.33 (1H, m), 4.35 to 4.40 (1H, m), 4.45 (2H, s), 4.86 (1H, brs), 5.41 (2H, s), 5.99 (1H, t, J=5.6Hz), 7.00 (2H, s), 7.14 To 7.16 (1H, m), 7.26 to 7.30 (2H, m), 7.67 (1H, d, J=9.2Hz), 8.03 (1H, t, J=5.2Hz), 8.30 (1H, d, J=7.4Hz), 9.95 (1H, s)

ESI-MS:701.3(M-OH^-)⁺, 719.2 (M+H)⁺, 1436.7 (2M+H)⁺

Embodiment 44

By formula (II-1) compound (169.4 milligrams, 2.0 equivalents), dry DMF (4.0 milliliters) and 4 angstroms of molecular sieves (400.0 Milligram) mixture be stirred at room temperature 15 minutes.DIPEA (107.9 milligrams, 3.0 equivalents) is added into the mixture of gained With formula (34) compound (200.0 milligrams, 1.0 equivalents) prepared according to embodiment 43.Obtained mixture is stirred for 2.5 small When.Then formula (DOXO) compound (161.8 milligrams, 1.0 equivalents) of HCl salt form is added, and is further stirred 2 hours.So Afterwards, acetonitrile (20 milliliters) is added.Form precipitation, filtering, and with MeCN and DMF mixture (5：1 (volume/volume), 3 times, often It is secondary with 3ml) wash.Filtrate is merged, and is dried in vacuo at 48 DEG C.The residue of gained is dissolved in the mixed of DCM and MeOH Compound (5：1 (volume/volume), 5 milliliters), and (DCM is purified by preparative silica gel tlc：MeOH=4:1 (volume/volume), Rf=0.5).By the mixture (7 of acetone and water：1 (volume/volume), 6 times, every time with 10 milliliters) extract product from silica gel. The extract of merging is dried under vacuum, the crude product for red solid is obtained.Then crude product and acetonitrile (5ml) are mixed Close, be stirred at room temperature 2 hours, then filter.Wet cake is dried in vacuo at room temperature, the formula (24) for obtaining 7.0 milligrams is changed Compound, it is red solid (yield 2%)

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.80 (3H, d, J=6.8Hz), 0.86 (3H, d, J=6.6Hz), 1.13 (3H, d, J=6.4Hz), 1.32 to 1.52 (3H, m), 1.57 to 1.69 (2H, m), 1.82 to 1.88 (1H, m), 1.95 to 2.04 (1H, m), 2.10 to 2.18 (2H, m), 2.24 (6H, s), 2.29 (2H, t, J=7.4Hz), 2.92 to 3.02 (6H, m), 3.12 to 3.16 (2H, m), 3.41 (2H, t, J=5.6Hz), 3.45 (1H, brs), 3.55 (2H, t, J=7.2Hz), 3.69 to 3.74 (3H, m), 3.99 (3H, s), 4.03 (2H, brs), 4.13 to 4.17 (1H, m), 4.28 to 4.31 (1H, m), 4.33 to 4.38 (1H, m), 4.58 (2H, d, J=5.2Hz), 4.69 (1H, d, J=5.6Hz), 4.86 (1H, t, J=6.0Hz), 4.90 (1H, s), 4.95 (1H, t, J=4.4Hz), 5.23 (1H, brs), 5.40 (2H, s), 5.47 (1H, s), 6.00 (1H, t, J= 5.6Hz), 6.79 (1H, d, J=8.0Hz), 6.95 (2H, s), 7.12 (1H, d, J=8.2Hz), 7.19 (1H, d, J= 8.2Hz), 7.34 (1H, s), 7.64 to 7.68 (2H, m), 7.91 (2H, d, J=4.8Hz), 7.98 (1H, t, J=5.2Hz), 8.30 (1H, d, J=7.2Hz), 10.02 (1H, s), 13.27 (1H, s), 14.02 (1H, s)

ESI-MS:1288.3(M+H)⁺

Embodiment 45

By formula (VII-1) compound (26.10 grams, 1.5 equivalents) prepared according to embodiment 1, THF (150 milliliters), Boc- It is small that L-Lys (Ac)-OH (30.00 grams, 1.0 equivalents) and EEDQ (51.45 grams, 2.0 equivalents) mixture are stirred at room temperature 24 When.Then mixture is evaporated to drying.Residue is purified into (PE by silica gel column chromatography：EtOAc=2：1 (volume/body Product) to pure EtOAc), 26.00 grams formula (6b-4) compound is obtained, it is light yellow solid (yield 57%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 1.21 to 1.34 (4H, m), 1.38 (9H, s), 1.58 to 1.66 (2H, M), 1.77 (3H, s), 3.00 to 3.04 (2H, m), 3.87 (3H, s), 3.99 to 4.07 (1H, m), 7.08 to 7.11 (2H, m), 7.40 (1H, d, J=2.0Hz), 7.74 (1H, d, J=8.8Hz), 7.78 to 7.81 (1H, m), 10.20 (1H, s), 10.62 (1H, s)

ESI-MS:437.9(M+H)⁺, 460.3 (M+Na)⁺, 875.1 (2M+H)⁺, 896.9 (2M+Na)⁺, 338.1 (M-^tBuOCO+2H)⁺

Embodiment 46

Formula (6b-4) compound (26.00 grams) prepared according to embodiment 45 is suspended to the HCl of 10% (w/w) In solution in Isosorbide-5-Nitrae-dioxanes (150 milliliters), gained mixture is stirred at room temperature 3 hours.Then reaction is mixed Thing is concentrated under vacuum, and obtains formula (6b-3) compound of 22.05 grams of HCl salt forms, and it is yellow solid (quantitative yield).

ESI-MS：339.2(M+H)+

Embodiment 47

By formula (6b-3) compound (25.21 grams, 1.0 equivalents) of the HCl salt form prepared according to embodiment 46, Boc-L- Val-OH (13.25 grams, 1.0 equivalents), TBTU (23.66 grams, 1.1 equivalents 18 hours), DIPEA (19.05 grams, 2.2eq) and DMF The mixture of (100 milliliters) is stirred at room temperature 18 hours.The mixture of gained is evaporated to drying, silica gel chromatography is then used Purify (DCM：MeOH=10：1 to 7：1 (volume/volume)), 30.80 grams formula (6b-2) compound is obtained, it is light yellow solid Body (yield 90%).

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.39 to 1.46 (14H, m), 1.49 to 1.56 (2H, m), 1.74 to 1.83 (1H, m), 1.95 to 2.02 (4H, m), 3.17 to 3.30 (2H, m), 3.92 (3H, s), 4.17 to 4.22 (1H, m), 4.54 To 4.60 (1H, m), 5.53 (1H, brs), 6.32 (1H, brs), 7.15 (1H, d, J=8.0Hz), 7.31 (1H, brs), 7.38 (1H, s), 7.72 (1H, dd, J₁=8.8Hz, J₂=1.0Hz), 9.38 (1H, brs), 10.80 (1H, s)

ESI-MS:509.3(M+H)⁺, 1017.0 (2M+H)⁺, 1038.8 (2M+Na)⁺, 409.5 (M-^tBuOCO+2H)⁺

Embodiment 48

Formula (6b-2) compound (30.80 grams) prepared according to embodiment 47 is suspended in the HCl of 10% (w/w) In the solution of Isosorbide-5-Nitrae-dioxanes (150 milliliters), gained mixture is stirred at room temperature 23 hours.Then by reactant mixture It is concentrated under vacuum, obtains formula (6b-1) compound of 25.90 grams of HCl salt forms, it is white solid (yield 96%).

ESI-MS：409.0(M+H)⁺

Embodiment 49

By formula (6b-1) compound (15.32 grams, 1.0 equivalents) of the hydrochloride form prepared according to embodiment 48, methanol The mixture of (100 milliliters), pyridine (9.0 milliliters, 3.2 equivalents) and acetic anhydride (14.0 milliliters, 4.3 equivalents) is stirred at room temperature 60 hours.Obtained mixture is evaporated to drying under vacuo.Residue purifies (DCM by silica gel column chromatography：MeOH= 15：1 to 7：1 (volume/volume)), 12.51 grams formula (6b) compound is obtained, it is light yellow solid (yield 80%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 1.21 (3H, d, J=7.0Hz), 1.29 to 1.38 (4H, m), 1.61 To 1.74 (2H, m), 1.78 (3H, s), 1.86 (3H, s), 2.99 to 3.04 (2H, m), 3.87 (3H, s), 4.27 to 4.34 (2H, M), 7.14 (1H, d, J=8.8Hz), 7.42 (1H, brs), 7.73 (1H, d, J=8.6Hz), 7.81 (1H, t, J=5.2Hz), 8.09 (1H, d, J=7.0Hz), 10.18 (1H, s), 10.62 (1H, s)

ESI-MS:451.3(M+H)⁺, 473.6 (M+Na)⁺, 900.9 (2M+H)⁺, 923.1 (2M+Na)⁺

Embodiment 50

The formula prepared by formula (6b) compound (6.73 grams, 1.0 equivalents) prepared according to embodiment 49, according to embodiment 10 (SGM-II-1) compound (11.50 grams, 2.1 equivalents), K₂CO₃The mixing of (4.40 grams, 2.1 equivalents) and dry DMF (60 milliliters) Thing is heated to 0 DEG C, and stirs 8 days in a nitrogen atmosphere.The mixture of gained is evaporated to drying, residue washs (3 with MeOH It is secondary, every time with 15 milliliters).Merging filtrate, and it is evaporated to drying.Residue is purified into (DCM by silica gel column chromatography：MeOH =15：1 to 7：1 (volume/volume)), 7.64 grams formula (55-1) compound is obtained, it is white solid (yield 81%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 1.21 (3H, d, J=7.2Hz), 1.25 to 1.40 (13H, m), 1.59 To 1.74 (2H, m), 1.78 (3H, s), 1.87 (3H, s), 2.99 to 3.04 (2H, m), 3.08 to 3.12 (2H, m), 3.50 (2H, T, J=6.0Hz), 3.75 to 3.77 (5H, m), 4.09 (2H, t, J=4.6Hz), 4.24 to 4.34 (2H, m), 6.74 (1H, t, J =5.4Hz), 7.28 (1H, dd, J₁=8.6Hz, J₂=1.6Hz), 7.51 (1H, d, J=1.6Hz), 7.69 (1H, d, J= 8.6Hz), 7.81 (1H, t, J=5.2Hz), 8.08 to 8.13 (2H, m), 10.14 (1H, s)

ESI-MS:638.2(M+H)⁺, 660.4 (M+Na)⁺, 1275.0 (2M+H)⁺, 538.4 (M-^tBuOCO+2H)⁺

Embodiment 51

Formula (55-1) compound (7.60 grams) prepared according to embodiment 50 is suspended to the hydrochloric acid of 10% (w/w) In solution in Isosorbide-5-Nitrae-dioxanes (50 milliliters), gained mixture is stirred at room temperature 5 hours.Then by reactant mixture It is concentrated under vacuum, obtains formula (55) compound of 6.72 grams of HCl salt forms, it is white solid (yield 98%).

ESI-MS:538.5(M+H)⁺, 1074.8 (2M+H)⁺, 1097.0 (2M+Na)⁺

Embodiment 52

In 0 DEG C of formula (55) compound (6.00 grams, 1.0 equivalents) and nothing to the HCl salt form prepared according to embodiment 51 Solution of the DIBAL-H in hexane (1M, 63.0 milliliters, 6.0 equivalents) is added in water THF (50 milliliters) mixture.Then will Obtained mixture is heated to 0 DEG C, and stirs 6 hours in a nitrogen atmosphere.Then methanol (10 milliliters) is added.Afterwards, will be full It is added to potassium sodium tartrate (150 milliliters) aqueous solution in mixture, and the mixture is stirred at room temperature 15 hours.By institute Mixture be evaporated to drying, produce white residue, the residue is further washed with methanol (3 times, every time with 50 in the least Rise).Merging filtrate, is then concentrated, and (DCM is purified by silica gel column chromatography：Methanol：Et₃N=75：25：2 (volumes/body Product)), 3.27 grams of formula (45) compounds are obtained, it is white solid (yield 61%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 1.22 (3H, d, J=4.4Hz), 1.27 to 1.41 (4H, m), 1.62 To 1.78 (2H, m), 1.79 (3H, s), 1.88 (3H, s), 2.68 (2H, t, J=5.6Hz), 2.99 to 3.03 (2H, m), 3.46 (2H, t, J=5.6Hz), 3.73 (2H, t, J=4.4Hz), 4.04 (2H, t, J=4.4Hz), 4.21 to 4.38 (2H, m), 4.45 (2H, s), 5.36 (1H, s), 7.22 to 7.28 (2H, m), 7.40 (1H, s), 8.00 (1H, t, J=5.4Hz), 8.16 (1H, d, J =8.0Hz), 8.27 (1H, d, J=6.8Hz), 10.05 (1H, s)

ESI-MS:510.5(M+H)⁺, 1019.6 (2M+H)⁺, 492.6 (M-OH^-)⁺

Embodiment 53

At room temperature to formula (45) compound (1.20 grams, 1.0 equivalents) prepared according to embodiment 52, according to embodiment 8 DIPEA is added in formula (CG1MR-IV-1) compound (0.69 gram, 1.1 equivalents) and DMF (25ml) mixture of preparation (0.34 gram, 1.1 equivalents).Obtained mixture is stirred at room temperature 2 hours.Then, DMF is removed under vacuo, obtains shallow Yellow residue, then mixes the residue with acetone (50 milliliters), and be stirred at room temperature 1 hour.Mixture is filtered, Filter cake is washed with acetone (2 times, every time with 10 milliliters), is then dried under vacuum, formula (35) compound for obtaining 1.12 grams (is received Rate 72%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 1.20 (3H, d, J=7.2Hz), 1.24 to 1.40 (4H, m), 1.58 To 1.75 (2H, m), 1.78 (3H, s), 1.87 (3H, s), 2.33 (2H, t, J=7.2Hz), 2.98 to 3.03 (2H, m), 3.16 To 3.21 (2H, m), 3.46 (2H, t, J=5.8Hz), 3.60 (2H, t, J=7.2Hz), 3.72 to 3.75 (2H, m), 4.04 (2H, t, J=4.4Hz), 4.24 to 4.36 (2H, m), 4.44 (2H, d, J=5.6Hz), 4.86 (1H, t, J=5.6Hz), 7.00 (2H, s), 7.16 to 7.18 (1H, m), 7.26 to 7.28 (1H, m), 7.34 (1H, d, J=1.4Hz), 7.80 (1H, t, J= 5.4Hz), 7.99 (1H, d, J=8.0Hz), 8.03 (1H, t, J=5.4Hz), 8.10 (1H, d, J=6.8Hz), 9.79 (1H, s).

ESI-MS:661.5(M+H)⁺, 643.6 (M-OH^-)⁺

Embodiment 54

By formula (35) compound (400.0 milligrams, 1.0 equivalents) prepared according to embodiment 53,4 angstroms of molecular sieve (800.0 millis Gram), the mixture of dry DMF (4.0 milliliters) and formula (II-1) compound (373.0 milligrams, 2.0 equivalents) be stirred at room temperature 5 Minute.Then DIPEA (313.5 milligrams, 4.0 equivalents) is added.Obtained mixture is stirred at room temperature 5 hours.Then, plus Enter formula (DOXO) compound (328.0 milligrams, 0.9 equivalent) of HCl salt form, then stir the mixture for 4 hours.Then, plus Enter acetonitrile (20 milliliters).Precipitation, filtering are formed, and (5 are washed with MeCN and DMF mixture：1 (volume/volume), 2 times, often It is secondary with 10 milliliters).Merging filtrate, is dried in vacuo at 45 DEG C, obtains dark residue.Residue is dissolved in DCM and MeOH Mixture (7：1, volume/volume, 5ml) in, and (DCM is purified by preparative silica gel thin-layer chromatography：MeOH=7:1 (body Product/volume), Rf=0.15).By the mixture (20 of acetone and water:1, volume/volume, 10 times, every time 20 milliliters) by product from Extracted in silica gel.The extract of merging is dried under vacuum, the crude product for red solid is obtained.Then crude product and acetonitrile (10 milliliters) mixing, mixture is stirred at room temperature 0.5 hour, then filtered.Filter cake is washed with acetonitrile (5ml), Ran Hou It is dried in vacuo at room temperature, obtains 100.7 milligrams formula (25) compound, it is red solid (yield 13%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 1.13 (3H, d, J=6.4Hz), 1.20 (3H, d, J=7.2Hz), 1.24 to 1.38 (4H, m), 1.48 to 1.51 (1H, m), 1.59 to 1.73 (2H, m), 1.77 (3H, s), 1.82 to 1.86 (4H, M), 2.12 to 2.23 (2H, m), 2.29 (2H, t, J=7.2Hz), 2.89 to 3.02 (4H, m), 3.11 to 3.15 (2H, m), 3.41 (2H, t, J=5.6Hz), 3.46 (1H, brs), 3.54 (2H, t, J=7.2Hz), 3.68 to 3.76 (3H, m), 3.98 (3H, s), 4.03 (2H, brs), 4.14 to 4.18 (1H, m), 4.22 to 4.34 (2H, m), 4.58 (2H, d, J=5.2Hz), 4.70 (1H, d, J=5.8Hz), 4.85 to 4.92 (4H, m), 5.22 (1H, brs), 5.46 (1H, s), 6.81 (1H, d, J= 8.0Hz), 6.95 (2H, s), 7.14 to 7.20 (2H, m), 7.39 (1H, s), 7.61 to 7.64 (1H, m), 7.82 (1H, t, J= 5.2Hz), 7.88 to 7.91 (2H, m), 7.98 to 8.03 (2H, m), 8.12 (1H, d, J=6.8Hz), 9.89 (1H, s), 13.24 (1H, s), 14.00 (1H, s)

ESI-MS:1252.1(M+Na)⁺

Embodiment 55

By formula (6-1) compound (22.81 grams, 1.0 equivalents) of the HCl salt form prepared according to embodiment 6, it is purchased from (24.00 grams, 1.5 work as by Aldrich 2- [2- (2- methoxy ethoxies) ethyoxyl] acetic acid (8.00 grams, 0.9 equivalent), TBTU Amount) and DIPEA (16.00 grams) be dissolved in DMF (100 milliliters).Obtained solution is stirred at room temperature 20 hours.Will reaction Mixture is concentrated to dryness, and residue then purified into (eluent DCM with silica gel column chromatography：MeOH=10：1 to 7：1 (body Product/volume)), 12.02 grams formula (6c) compound is obtained, it is light yellow solid (yield 41%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=24.4Hz, J₂=6.8Hz), 1.37 (9H, 1.39 to 1.52 s), (2H, m), 1.56 to 1.75 (4H, m), 1.97 to 2.06 (1H, m), 2.91 to 3.07 (4H, m), 3.19 to 3.22 (2H, m), 3.42 to 3.47 (2H, m), 3.52 to 3.64 (6H, m), 3.81 (3H, s), 3.95 (2H, brs), 4.30 to 4.34 (1H, m), 4.36 to 4.41 (1H, m), 4.53 (2H, s), 5.43 (2H, s), 6.01 (1H, t, J=5.8Hz), 6.78 (1H, t, J=5.2Hz), 7.34 to 7.40 (2H, m), 7.45 (1H, d, J=8.8Hz), 7.79 (1H, d, J=8.4Hz), 8.04 (1H, t, J=5.6Hz), 8.38 (1H, d, J=7.2Hz), 10.36 (1H, s)

ESI-MS:584.7(M+H)⁺, 1167.2 (2M+H)⁺

Embodiment 56

The formula prepared by formula (6c) compound (8.01 grams, 1.0 equivalents) prepared according to embodiment 55, according to embodiment 24 (SGM-III-2) compound (6.89 grams, 2.0 equivalents), K₂CO₃The mixing of (3.80 grams, 2.0 equivalents) and dry DMF (60 milliliters) Thing is heated to 50 DEG C, and stirs 7 days in a nitrogen atmosphere.Then reactant mixture is concentrated under vacuum to drying.By dichloro Methane (15ml) is added in residue, and obtained mixture purifies (eluant, eluent with silica gel column chromatography：DCM：MeOH=7:1 (volume/volume)), 5.52 grams formula (56-1) compound is obtained, it is slightly yellow solid (yield 50%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=24.4Hz, J₂=6.8Hz), 1.37 (9H, 1.39 to 1.52 s), (2H, m), 1.56 to 1.75 (4H, m), 1.97 to 2.06 (1H, m), 2.91 to 3.07 (4H, m), 3.19 to 3.22 (2H, m), 3.42 to 3.47 (2H, m), 3.52 to 3.64 (6H, m), 3.81 (3H, s), 3.95 (2H, brs), 4.30 to 4.34 (1H, m), 4.36 to 4.41 (1H, m), 4.53 (2H, s), 5.43 (2H, s), 6.01 (1H, t, J=5.8Hz), 6.78 (1H, t, J=5.2Hz), 7.34 to 7.40 (2H, m), 7.45 (1H, d, J=8.8Hz), 7.79 (1H, d, J=8.4Hz), 8.04 (1H, t, J=5.6Hz), 8.38 (1H, d, J=7.2Hz), 10.36 (1H, s)

ESI-MS:698.7(M-^tBuOCO+2H)⁺, 798.3 (M+H)⁺, 820.7 (M+Na)⁺, 1595.7 (2M+H)⁺

Embodiment 57

Formula (56-1) compound prepared according to embodiment 56 is suspended at the HCl of 10% (w/w) in 1,4- bis- In solution in oxane (60 milliliters).Obtained mixture is stirred at room temperature 8 hours, then concentrated, 4.99 grams are obtained Formula (56) compound, it is white solid (quantitative yield).

ESI-MS:698.4(M+H)⁺, 1394.6 (2M+H)⁺

Embodiment 58

By formula (56) compound (4.91 grams, 1.0 equivalents) according to the HCl salt form prepared in embodiment 57, water (45 millis Rise), the mixture of calcium chloride (1.49 grams, 2.0 equivalents) and sodium borohydride (1.02 grams, 4.0 equivalents) is stirred at room temperature.In batches Add sodium borohydride (1.02 grams, 4.0 equivalents, after the stirring of totally 2.5 hours of additional quantity；1.01 grams, 4.0 equivalents pass through After the stirring of totally 4 hours；1.03 grams, 4.0 equivalents, after the stirring of totally 6 hours).After stirring 22 hours altogether, into mixture Add methanol (20 milliliters).Then reactant mixture is filtered, wet cake is washed (3 times, every time with 15 milliliters) with methanol.Collect Filtrate, merges, is then evaporated to drying.Residue purifies (eluent DCM by silica gel column chromatography：MeOH：Et₃N=80： 20：2.5 (volume/volumes)), 1.12 grams formula (46) compound is obtained, it is light yellow solid (yield 25%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.86 (6H, dd, J₁=22.8Hz, J₂=6.8Hz), 1.32 to 1.48 (4H, m), 1.51 to 1.73 (4H, m), 1.96 to 2.06 (1H, m), 2.58 (2H, t, J=6.8Hz), 2.91 to 3.06 (2H, m), 3.16 to 3.21 (2H, m), 3.22 (3H, s), 3.52 to 3.64 (8H, m), 3.95 (2H, brs), 4.29 to 4.33 (1H, m), 4.36 to 4.41 (2H, m), 4.46 (2H, s), 4.51 (2H, s), 5.44 (2H, s), 6.10 (1H, t, J=5.6Hz), 7.22 to 7.27 (3H, m), 7.48 (1H, d, J=8.8Hz), 8.19 (1H, t, J=5.4Hz), 8.34 (1H, d, J=7.6Hz), 10.05 (1H, s)

ESI-MS:652.4(M-OH^-)⁺, 670.4 (M+H)⁺, 1338.7 (2M+H)⁺

Embodiment 59

Prepared to formula (46) compound (250.7 milligrams, 1.0 equivalents) prepared according to embodiment 58, according to embodiment 8 Formula (CG1MR-IV-1) compound (103.1 grams, 1.1 equivalents) and DMF (4ml) mixture add DIPEA at room temperature (53.6 milligrams, 1.1 equivalents).Obtained mixture is stirred at room temperature 1 hour.Then, DMF is removed under vacuo, is obtained Light yellow residue, then the residue is had and is mixed with acetone (5ml), and be stirred at room temperature 1 hour.By mixture mistake Filter, filter cake washs (3 times, every time with 3 milliliters) with acetone, is then dried under vacuum, obtains 253.7 grams formula (36) compound (yield 83%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.85 (6H, dd, J₁=23.0Hz, J₂=6.8Hz), 1.36 to 1.54 (4H, m), 1.58 to 1.72 (2H, m), 1.97 to 2.05 (1H, m), 2.32 (2H, t, J=7.2Hz), 2.92 to 3.05 (4H, m), 3.08 to 3.13 (2H, m), 3.22 (3H, s), 3.44 (2H, t, J=4.8Hz), 3.52 to 3.62 (8H, m), 3.95 (2H, s), 4.29 to 4.33 (1H, m), 4.35 to 4.40 (1H, m), 4.46 (2H, s), 4.52 (2H, d, J=5.2Hz), 5.08 (1H, t, J=5.6Hz), 5.42 (2H, s), 6.01 (1H, t, J=5.2Hz), 6.99 (2H, s), 7.20 to 7.27 (3H, m), 7.46 (1H, d, J=8.8Hz), 7.95 (1H, t, J=5.4Hz), 8.05 (1H, t, J=5.8Hz), 8.31 (1H, d, J= 7.6Hz), 10.00 (1H, s)

ESI-MS:803.4(M-OH^-)⁺, 821.1 (M+H)⁺, 1641.1 (2M+H)⁺

Embodiment 60

By formula (36) compound (201.3 milligrams, 1.0 equivalents) prepared according to embodiment 59,4 angstroms of molecular sieve (400.0 millis Gram), the mixture of dry DMF (4.0 milliliters) and formula (II-1) compound (146.9 milligrams, 2.0 equivalents) be stirred at room temperature 10 Minute.Then DIPEA (105.7 milligrams, 3.3 equivalents) is added.Obtained mixture is stirred at room temperature 4.5 hours.Then, Formula (DOXO) compound (142.1 milligrams, 1.0 equivalents) of HCl salt form is added, is then stirred the mixture for 2.5 hours.So Afterwards, MeCN (20 milliliters) is added.Precipitation, filtering are formed, and (5 are washed with MeCN and DMF mixture：1 (volume/volume), 4 It is secondary, every time with 4ml).Merging filtrate, is dried in vacuo at 45 DEG C, obtains dark residue.Residue is dissolved in DCM and first The mixture (7 of alcohol：1, volume/volume, 3 milliliters) in, and (DCM is purified by preparative silica gel thin-layer chromatography：Methanol=7: 1 (volume/volume), Rf=0.3).By the mixture (10 of acetone and water:1, volume/volume, 6 times, every time 10 milliliters) by product Extracted from silica gel.The extract of merging is dried under vacuum, the crude product for red solid is obtained.Then by the crude product Mixed with acetonitrile (5ml), mixture is stirred at room temperature 2 hours, then filtered.Filter cake with acetonitrile (2 times, every time with 2 in the least Rise) washing, then it is dried in vacuo at room temperature, obtains 33.2 milligrams formula (26) compound, it is red solid (yield 10%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.84 (6H, dd, J₁=23.2Hz, J₂=6.8Hz), 1.13 (3H, D, J=6.4Hz), 1.30 to 1.70 (7H, m), 1.82 to 1.87 (4H, m), 1.97 to 2.04 (1H, m), 2.08 to 2.22 (2H, M), 2.28 (2H, t, J=7.2Hz), 2.91 to 3.09 (8H, m), 3.22 (3H, s), 3.42 to 3.46 (3H, m), 3.52 to 3.61 (8H, m), 3.70 to 3.76 (1H, m), 3.95 (2H, s), 3.99 (3H, s), 4.13 to 4.18 (2H, m), 4.28 to 4.32 (1H, m), 4.34 to 4.41 (3H, m), 4.58 (2H, d, J=5.6Hz), 4.70 (1H, d, J=5.2Hz), 4.85 (1H, t, J= 5.8Hz), 4.94 (1H, brs), 5.03 (2H, s), 5.22 (1H, brs), 5.41 (2H, s), 5.46 (1H, brs), 5.99 (1H, T, J=5.4Hz), 6.86 (1H, d, J=8.0Hz), 6.97 (2H, s), 7.19-7.24 (3H, m), 7.44 (1H, d, J= 8.8Hz), 7.65 (1H, t, J=4.8Hz), 7.85 to 7.91 (4H, m), 8.30 (1H, d, J=7.2Hz), 10.04 (1H, s), 13.26 (1H, s), 14.02 (1H, s)

ESI-MS:1390.2(M+H)⁺

Embodiment 61

By formula (TAXO) compound (200.0 milligrams, 1.0 equivalents), 4 angstroms of molecular sieves (100.0 milligrams), anhydrous DCM (4.0 Milliliter) and formula (II-1) compound (146.9 milligrams, 2.0 equivalents) mixture 0 DEG C stirring 10 minutes.Then to mixture Middle addition pyridine (28.1 milligrams, 1.5 equivalents).At 0 DEG C under a nitrogen, gained mixture is stirred for 41 hours.Then at 0 DEG C N, N'- dimethyl -1,2- ethylenediamine (102.0 milligrams, 5.0 equivalents) are added into mixture under a nitrogen.After stirring 2 hours, Mixture is filtered.Filter cake is washed with dichloromethane (1 milliliter).Merging filtrate, is then evaporated to drying, obtains yellow residual Thing.Residue is further purified into (DCM by preparative silica gel thin-layer chromatography：Methanol=7:1 (volume/volume)), with 96% separation yield, obtains 220.0 milligrams formula (TAXO-t1-1) compound, it is light yellow solid.

ESI-MS:968.5(M+Na)⁺

Embodiment 62

By formula (31) compound (252.0 milligrams, 1.2 equivalents), formula (II-1) compound for being prepared according to embodiment 21 The mixture of (136.0 milligrams, 1.8 equivalents), 4 angstroms of molecular sieves (100.0 milligrams) and dry DMF (1 milliliter) is stirred at room temperature 10 minutes.Then DIPEA (80.1 milligrams, 2.5 equivalents) is added.Obtained mixture is stirred at room temperature 2 hours.Then, Add formula (XXXX) compound prepared according to embodiment 61.Obtained mixture is stirred 2 hours.By gained suspension mistake Filter；(3 times, every time with 10 milliliters) filter cake is further washed with DCM.Merging filtrate, is then evaporated to drying at 35 DEG C.By gained Residue is mixed with DCM (30 milliliters), and is stirred 0.5 hour.Gained suspension is filtered, filter cake is further washed with DCM (2 times, every time with 5 milliliters).At room temperature, filtrate is dried under vacuum.Crude product is dissolved in CH₂Cl₂- MeOH mixing is molten Agent (8:1 (volume/volume)) in, and further purify (DCM with preparative silica gel thin-layer chromatography：MeOH=8:1 (volume/body Product)).Pass through THF- water (20:1 (volume/volume), 6 times, every time with 30 milliliters) mixture by the product from gel extraction, It is dried under vacuum, obtains the crude product for white solid.Then by crude product and Et₂(5 milliliters) mixing of O, are stirred at 25 DEG C Mix 0.5 hour, then filter.Use Et₂O washings (3 times, every time with 5 milliliters) filter cake, is then dried in vacuo at room temperature, with 21% separation yield, obtains 100.0 milligrams formula (21-TAXO-t1-1) compound, it is white solid.

ESI-MS:1842.1(M+H)⁺, 1864.5 (M+Na)⁺

Comparative example 1

To the Boc-Cit-OH (1.00 grams, 1.0 equivalents) prepared according to embodiment 2, EEDQ (1.35 grams, 1.5 equivalents) and Para-Aminobenzoic methyl esters (0.82 gram, 1.5 equivalents) is added in THF (15 milliliters) mixture.By obtained mixture in room The lower stirring of temperature 14 hours.Then, solvent is removed under vacuo, and residue is purified into (eluant, eluent by silica gel column chromatography： PE：EtOAc=6:1 (volume/volume), then DCM：MeOH=10：1 (volume/volume)), obtain 1.2 grams of formula (Comp-6- 4) compound, it is white solid (yield 81%).

Silica-gel TLC analysis：Eluent EtOAc (Rf=0.35, UV254)

¹H NMR (400MHz, CDCl₃, 20 DEG C) δ 1.28 to 1.45 (11H, m), 1.55 to 1.65 (2H, m), 2.91 to 3.08 (2H, m), 3.83 (3H, s), 4.09 to 4.14 (1H, m), 5.43 (2H, s), 5.99 (1H, t, J=5.6Hz), 7.10 (1H, d, J=7.6Hz), 7.75 to 7.77 (2H, m), 7.91 to 7.93 (2H, m), 10.32 (1H, s)

ESI-MS:309.3 (M-tBuOCO+2H)+, 409.2 (M+H)+, 817.1 (2M+H)+

Comparative example 2

The HC1 of formula (Comp-6-4) compound (1.66 grams) prepared according to comparative example 1 and 15% (w/w) is existed The mixture of solution in 1,4- dioxanes (10 milliliters) is stirred at room temperature 3 hours.Then by reactant mixture under vacuo Concentration, obtains formula (Comp-6-3) compound of 1.38g HCl salt form, and it is white solid (yield 98%).

Comparative example 3

To formula (Comp-6-3) compound (14.50 grams, 1.0 equivalents) of the HCl salt form prepared according to comparative example 2, Added in Boc-L-Val (10.05 grams, 1.1 equivalents), TBTU (27.00 grams, 2.0 equivalents) and DMF (80 milliliters) mixture DIPEA (16.31 grams, 3.0 equivalents).Obtained mixture is stirred at room temperature 16 hours.Then by mixture water (120 Milliliter) dilution, (5 times, every time with 100 milliliters) are then extracted with EtOAc, merges organic phase and is evaporated to drying, gained is residual Excess purifies (eluant, eluent by silica gel column chromatography：DCM：MeOH=20：1 to 10：1 to 7：1 (volume/volume)), obtain 12.9 grams formula (Comp-6-2) compound, it is solid (yield 60%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.85 (6H, dd, J1=17.4Hz, J2=6.6Hz), 1.39 (9H, S), 1.43 to 1.50 (2H, m), 1.57 to 1.76 (2H, m), 1.91 to 1.97 (1H, m), 2.91 to 3.09 (2H, m), 3.83 (3H, s), 3.85 to 3.88 (1H, m), 4.43 to 4.48 (1H, m), 5.43 (2H, s), 5.99 (1H, t, J=5.8Hz), 6.72 (1H, d, J=8.8Hz), 7.73 to 7.76 (2H, m), 7.90 to 7.94 (2H, m), 8.07 (1H, d, J=7.6Hz), 10.39 (1H, s)

Comparative example 4

By the hydrochloric acid of formula (Comp-6-2) compound (12.3 grams) prepared according to comparative example 3 and 10% (w/w) The mixture of solution in dioxanes (60 milliliters) is stirred at room temperature 3 hours.Then mixture is concentrated under vacuum, obtained To formula (Comp-6-1) compound of 10.9 grams of HCl salt forms, it is white solid (quantitative yield).

Comparative example 5

In -20 DEG C of formula (Comp-6-1) compounds to the HCl salt form prepared according to comparative example 4, (10.20 grams, 1.0 work as Amount) and anhydrous THF (150 milliliters) mixture in addition DIBAL-H hexane solution (1M, 120.0 milliliters, 6.2 equivalents).Will Mixture is warmed to room temperature and stirred 14 hours.Then MeOH (20 milliliters) is added, saturation potassium sodium tartrate solution is subsequently added into (180 milliliters), and the mixture is stirred at room temperature 30 minutes.The mixture of gained is evaporated to drying, white is obtained residual Excess, is washed (5 times, every time with 50 milliliters) with MeOH.Eluent is merged, concentrated, (elution is purified by silica gel column chromatography Liquid DCM：MeOH=10:1 to 7:1 to 5:1 (volume/volume)), 5.10 grams formula (Comp-10) compound is obtained, it is white Solid (yield 58%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.89 (6H, dd, J1=24.0Hz, J2=6.8Hz), 1.33 to 1.50 (2H, m), 1.56 to 1.77 (2H, m), 1.96 to 2.04 (1H, m), 2.92 to 3.07 (2H, m), 3.30 (1H, d, J= 5.2Hz), 4.12 (2H, brs), 4.43 (2H, s), 4.47 to 4.52 (1H, m), 5.12 (1H, brs), 5.50 (2H, s), 6.10 (1H, t, J=5.6Hz), 7.24 (2H, d, J=8.8Hz), 7.54 to 7.57 (2H, m), 8.37 (1H, d, J=5.8Hz), 10.11 (1H, s)

ESI-MS:380.4 (M+H)+, 759.2 (2M+H)+

Comparative example 6

Change to formula (Comp-10) compound (1.02 grams, 1.0 equivalents), formula (CG1MR-IV-2) for being prepared according to comparative example 5 Compound (0.92 gram, 1.1 equivalents),

With DIPEA (0.39 gram, 1.1 equivalents) is added in DMF (15 milliliters) mixture at room temperature.By obtained mixing Thing is stirred at room temperature 16 hours.Then, DMF is removed under vacuo, obtains light yellow residue.Then by residue and acetone (20 milliliters) mixing, then mixture is stirred at room temperature 5 hours.Mixture is filtered, wet cake is washed (2 with acetone It is secondary, every time with 10 milliliters), then it is dried under vacuum, obtains 1.05 grams formula (Comp-11) compound, it is light yellow solid Body (yield 67%).

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) δ 0.84 (6H, dd, J1=12.2Hz, J2=6.6Hz), 1.17 to 1.31 (2H, m), 1.37 to 1.75 (8H, m), 1.95 to 2.00 (1H, m), 2.11 to 2.21 (2H, m), 2.92 to 3.06 (2H, M), 3.38 (2H, t, J=6.8Hz), 4.18 to 4.22 (1H, m), 4.38 to 4.39 (1H, m), 4.43 (2H, d, J=4.8Hz), 5.09 (1H, t, J=5.2Hz), 5.41 (2H, s), 5.98 (1H, t, J=5.6Hz), 7.00 (2H, s), 7.23 (2H, d, J= 8.0Hz), 7.55 (2H, d, J=8.2Hz), 7.81 (2H, d, J=8.4Hz), 8.06 (1H, d, J=7.2Hz), 9.90 (1H, s).

Comparative example 7

By formula (Comp-11) compound (350.0 milligrams, 1.0 equivalents) prepared according to comparative example 6, DIPEA (307.0 millis Gram, 3.9 equivalents), the mixture of 500.0 milligrams of 4 angstroms of molecular sieves and dry DMF (5.0 milliliters) stirs 25 minutes.Then, plus Enter formula (II-1) compound (280.0 milligrams, 1.5 equivalents).Obtained mixture is stirred at room temperature 3 hours.Then, add Formula (DOXO) compound (368.8 milligrams, 1.0 equivalents) of HCl salt form, is then stirred the mixture for 12 hours.Then, will MeCN (25.0 milliliters) is added in reactant mixture.Precipitation, filtering are formed, and (5 are washed with MeCN and DMF mixture：1 (volume/volume), 3 times, every time with 5 milliliters).Filtrate is merged, is dried under vacuum at 45 DEG C, kermesinus is obtained remaining Thing.Residue is dissolved in DCM and methanol (7：1 (volume/volume)) mixture, pass through preparative silica gel tlc purify (DCM： MeOH=7:1 (volume/volume), Rf=0.15).By the mixture (20 of acetone and water：1 (volume/volume), 10 times, is used every time 50 milliliters) product is extracted from silica gel, the extract of merging is dried under vacuum, the crude product for red solid is obtained. Then crude product is mixed with acetonitrile (20 milliliters), mixture is stirred at room temperature 18 hours, then filtered.By filter cake and second (10 milliliters) mixing of nitrile, are stirred at room temperature the mixture 3 hours.Then mixture is filtered.At room temperature by filter cake true Sky descends drying, obtains 61.0 milligrams formula (Comp-12) compound, and it is red solid (yield 8.7%)；

In formula (Comp-12), Doxorubicin (doxorubicin) is formula (DOXO) compound, and it passes through in formula (Comp-12) the amino connection that (d1) in and in formula (DOXO) is represented.

¹H NMR (400MHz, DMSO-d₆, 20 DEG C) and δ 0.85 (6H, dd, J1=12.0Hz, J2=6.8Hz), 1.12 (3H, D, J=6.4Hz), 1.16 to 1.22 (2H, m), 1.31 to 1.50 (7H, m), 1.54 to 1.71 (2H, m), 1.82 to 1.88 (1H, M), 1.91 to 2.01 (1H, m), 2.10 to 2.22 (4H, m), 2.89 to 3.03 (4H, m), 3.38 (2H, t, J=6.8Hz), 3.44 (1H, m), 3.71 to 3.75 (1H, m), 3.99 (3H, s), 4.16 to 4.20 (2H, m), 4.33 to 4.39 (1H, m), 4.58 (2H, d, J=5.6Hz), 4.71 (1H, d, J=5.6Hz), 4.86 (1H, t, J=6.0Hz), 4.89 (2H, s), 4.95 (1H, Brs), 5.22 (1H, d, J=2.8Hz), 5.40 (2H, s), 5.47 (1H, s), 5.99 (1H, t, J=4.8Hz), 6.84 (1H, d, J=8.0Hz), 7.00 (2H, s), 7.24 (2H, d, J=8.4Hz), 7.55 (2H, d, J=8.4Hz), 7.66 (1H, t, J= 4.8Hz), 7.80 (1H, d, J=8.4Hz), 7.92 (2H, d, J=4.8Hz), 8.06 (1H, d, J=7.6Hz), 9.97 (1H, S), 13.28 (1H, s), 14.04 (1H, s)

ESI-MS:1141.7(M+H)+

Embodiment 100

The usual method explanation of the preparation of formula (I) compound being given in Table 1：

The corresponding formulas of 10mM (II) are added into 10mM N- acetyl-aqueous cystein solution (2500 microlitres, 5 equivalents) to change Solution of the compound in DMAC N,N' dimethyl acetamide (500 microlitres).PH value is adjusted to 7.5 with 0.3M dibastic sodium phosphate solution, Reactant mixture is stirred 2 hours at 20 DEG C.The solution of produced corresponding formula (I) compound need not be further purified i.e. / test can be used.

Details are listed in table (1)

Table 1
			Embodiment	Formula (II) compound	Formula (I) compound
100-1	Formula (Comp-12) compound prepared according to comparing embodiment 7	Formula (Comp-13) compound
			100-2	Formula (23) compound prepared according to embodiment 36	Formula (13) compound
100-3	Formula (20) compound prepared according to embodiment 15	Formula (10) compound
			100-4	Formula (21) compound prepared according to embodiment 22	Formula (11) compound
100-5	Formula (22) compound prepared according to embodiment 29	Formula (12) compound
			100-6	Formula (25) compound prepared according to embodiment 54	Formula (15) compound
100-7	Formula (26) compound prepared according to embodiment 60	Formula (16) compound
			100-8	Formula (24) compound prepared according to embodiment 44	Formula (14) compound

Method RP-HPLC

RP-HPLC points of the solution of corresponding formula (I) compound for completing to be prepared according to embodiment 100 using following parameter Analysis：

Luna 5U C18250 × 4.6mm posts (from the purchase of Phenomenex companies), solvent orange 2 A：0.1% (volume/volume) TFA in water, solvent B：The TFA of 0.1% (volume/volume) is in acetonitrile, and 100% solvent orange 2 A continues 10 minutes, following Solvent B of 70 minutes inside gradients from 0 to 70%, and to 100%, 1 ml/min in ensuing 3 minutes, in 254nm Place's detection.

RT=retention times

The value being given in Table 2

Table 2	RT
		Formula (DOXO) compound	44.9 minutes
Formula (Comp-13) compound	54.5 minutes
		Formula (13) compound	55.1 minutes
Formula (10) compound	51.2 minutes
		Formula (11) compound	51.7 minutes

Formula (12) compound	50.7 minutes
		Formula (15) compound	50.6 minutes
Formula (16) compound	52.2 minutes
		Formula (14) compound	48.3 minutes

Dissolubility is tested

The solution (respectively 15,48 and 96 microlitres) that corresponding formula (I) compound will be prepared according to embodiment 100 (divides with water Other 135,102 and 54 microlitres) mixing, to obtain totally 150 microlitres of corresponding three kinds of dilute solutions.Water in each solution Cumulative volume, the DMA concentration of these three dilutions is respectively 1.5%, 5.0% and 10.0%, and the percentage is percent by volume.Will These dilutions are stirred 1 hour at 20 DEG C, are then analyzed by method RP-HPLC.

By comparing peak area, (relative to most diffluent conjugate, i.e. formula (11) compound, its peak area is arranged to 100%, represented with (the * ref) in table 3) solubility of formula (I) compound is estimated, value is as shown in table 3：

Table 3	1.5%DMA	5.0%DMA	10.0%DMA
				Formula (Comp-13) compound	8%	41%	77%
Formula (13) compound	17%	53%	82%
				Formula (10) compound	15%	47%	84%
Formula (11) compound	19%	58%	100% (* ref)
				Formula (12) compound	16%	49%	85%
Formula (15) compound	16%	45%	77%
				Formula (16) compound	16%	47%	80%
Formula (14) compound	12%	36%	66%

With reference connection formula (Comp-13) compound phase ratio, formula (I) compound is especially in more relevant low DMA concentration In the case of higher solubility provide two advantages：

1) due to reducing polymerization, the higher yields when synthesizing formula (I) compound

2) excellent pharmacokinetics

Cathepsin B's release of formula (DOXO) compound

Niu Pi cathepsin Bs (SAFC C6286-10UN, 10 units) are dissolved in the acetate that 1 ml of ph is 5.0 Buffer solution (25mM acetate and 1mM EDTA) is to provide cathepsin B's stoste.

By the EDTA of cathepsin B's stoste (16 microlitres) and the 30mM dithiothreitol (DTT) and 15mM aqueous solution (32 microlitres) mixing, resulting solution is stood do not stir lasting 15 minutes at 20 DEG C.Then 25mM acetate and 1mM is added The EDTA aqueous solution (1175 microlitres), the solution (142 microlitres, prepared according to embodiment 100) of corresponding formula (I) compound, DMAC N,N' dimethyl acetamide (53.7 microlitres) and according to retention time as internal standard be used for distribute peak 10mM daunomycin water Solution (157.7 microlitres).Resulting solution is cultivated 2 days at 37 DEG C.As provided in table 4, periodically taking out and passing through method RP- HPLC analyzes undiluted aliquot (100 microlitres).In experimentation, relative to corresponding formula (I) compound, formula (DOXO) relative percentage of compound release is given in Table 4：

Table 4	0h	4h	8h	24h	48h
						Formula (13) compound	<0.1%	18.0%	24.4%	32.2%	33.8%
Formula (10) compound	<0.1%	15.8%	23.5%	35.5%	41.4%
						Formula (11) compound	<0.1%	6.4%	8.7%	13.5%	15.4%
Formula (12) compound	<0.1%	3.0%	3.8%	5.3%	5.0%
						Formula (15) compound	<0.1%	1.3%	1.6%	2.3%	4.1%
Formula (16) compound	<0.1%	0.6%	1.0%	2.0%	2.2%
						Formula (14) compound	<0.1%	<0.1%	1.0%	1.7%	2.2%

All compounds show insoluble drug release in the presence of cathepsin B.

Stability in human serum

Solution (950 microlitres, root of corresponding formula (I) compound are added into human serum (SAFC H4522,950 microlitres) Prepared according to embodiment 100) and 10mM daunomycin the aqueous solution as internal standard (100 microlitres).By resulting solution at 37 DEG C it is warm Educate 7 days.Table 4 is such as listed in, aliquot (100 microlitres) is periodically taken out, with (400 microlitres) dilutions of 0 to 5 DEG C of methanol, filtered, will Obtained filtrate is analyzed by method RP-HPLC.Formula (DOXO) compound of release is carried out relative to daunomycin (internal standard) Quantitative, value is given in Table 5：

Table 5

0 hour

4 hours

8 hours

24 hours

48 hours

72 hours

Formula (13) compound

<0.1%

<0.1%

<0.1%

4.4%

6.3%

6.9%

Formula (10) compound

<0.1%

<0.1%

<0.1%

2.4%

4.3%

4.3%

Formula (11) compound

<0.1%

<0.1%

<0.1%

<0.1%

<0.1%

<0.1%

Formula (12) compound

<0.1%

<0.1%

<0.1%

<0.1%

<0.1%

<0.1%

Formula (15) compound

<0.1%

<0.1%

0.2%

3.2%

5.2%

6.0%

Formula (16) compound

<0.1%

1.3%

1.2%

1.9%

3.2%

1.6%

Formula (14) compound

<0.1%

<0.1%

<0.1%

1.1%

2.5%

2.6%

All side chain connected bodies are proved to good stability.

Embodiment 101

The preparation method of formula (12-101) compound derived from formula (22) compound and the anti-Interleukin -1β antibody of monoclonal It is as follows：

The anti-Interleukin -1β antibody of the monoclonal produced in mouse (5 milligrams, Sigma article No. I3642) is blended in PBS In (8.0 milliliters).

1.0mM three (2- carboxy ethyls) phosphonium salt hydrochlorates (25 microlitres, 2.0 equivalents) are added into equal portions solution (3ml) The aqueous solution, and the mixture is stirred 90 minutes at 20 DEG C.Add the 1.0mM prepared according to embodiment 29 formula (22) chemical combination Solution of the thing in DMA (64.4 microlitres, 5.15 equivalents), and further by gained mixture at 20 DEG C Stirring 30 minutes.The aqueous solution of 1.0mM N- acetyl-cysteine (64.4 microlitres, 5.15 equivalents) is added, and gained is mixed Thing is stirred for 36 minutes at 20 DEG C, to produce so-called conjugate mixture.NAP-25 posts are rinsed with PBS (25ml), and filling is even Join thing mixture (2.5 milliliters), and eluted with (5.0 milliliters) of PBS.Part is collected, collects those parts for containing protein.With The anti-Interleukin -1β antibody of monoclonal being suspended in PBS is analyzed by method SEC-HPLC (result is listed in table 6) and method HIC Include the protein solution collected of formula (12-101) compound.

Embodiment 102

The preparation method of formula (15-102) compound derived from formula (25) compound and the anti-Interleukin -1β antibody of monoclonal It is as follows：

By the anti-Interleukin -1β antibody of the monoclonal produced in mouse (5 milligrams, Sigma article No. I3642, such as embodiment It is used in 101) it is blended in PBS (8.0 milliliters).

1.0mM three (2- carboxy ethyls) phosphonium salt hydrochlorates (25 microlitres, 2.0 equivalents) are added into equal portions solution (3ml) The aqueous solution, and the mixture is stirred 90 minutes at 20 DEG C.Add the 1.0mM prepared according to embodiment 54 formula (25) chemical combination Solution of the thing in DMA (64.4 microlitres, 5.15 equivalents), and further by gained mixture at 20 DEG C Stirring 30 minutes.The aqueous solution of 1.0mM N- acetyl-cysteine (64.4 microlitres, 5.15 equivalents) is added, and gained is mixed Thing is stirred for 42 minutes at 20 DEG C, to produce so-called conjugate mixture.NAP-25 posts are rinsed with PBS (25ml), and filling is even Join thing mixture (2.5 milliliters), and eluted with (5.0 milliliters) of PBS.Part is collected, collects those parts for containing protein.With The anti-Interleukin -1β antibody of monoclonal being suspended in PBS is analyzed by method SEC-HPLC (result is listed in table 6) and method HIC Include the protein solution collected of formula (15-102) compound.

Method SEC-HPLC

The SEC-HPLC analyses for the protein solution for completing to be collected accordingly using following parameter：

(average pore size is 250 angstroms to TSK 300 × 7.8mm of G3000SWXL posts, and average particle size particle size is 5 microns, is purchased from The post based on silica of Tosoh Bioscience companies), eluant, eluent：The 0.2M phosphorus of 10% (volume/volume) isopropanol Sour potassium buffer solution, 0.5 ml/min is detected at 280nm.

RT=retention times

Determined by the relatively unmodified anti-Interleukin -1β antibody of monoclonal corresponding formula (I) compound peak value and And its retention time, the anti-Interleukin -1β antibody of unmodified monoclonal is as the culture medium in embodiment 101 and 102.

HMW=high molecular weight moieties, it is unallocated

LMW=low molecular weight parts, it is unallocated

Method HIC

The HIC analyses for the protein solution for completing to be collected accordingly using following parameter：

(NPR refers to Non-porous resin to TSK- 4.6 millimeters × 35mm of gel butyl-NPR posts, with 2.5 microns of average grain diameter Polymethacrylates matrix material, purchased from Tosoh Bioscience companies), solvent orange 2 A：50mM sodium phosphate buffers, Solvent B：25% (volume/volume) isopropanol is molten from 100% in 12 minutes internal solvent gradients in 50mM sodium phosphate buffer Agent A to 100% solvent B, 0.8 ml/min is detected at 280nm.

Fig. 1 shows resulting chromatogram.

Symbol description used in Fig. 1：

The continuous lines ----anti-Interleukin -1β antibody of monoclonal used in embodiment 101 and 102

Formula (12-101) compound that dash line ---is prepared according to embodiment 101

Formula (15-102) compound that dotted line ■ ■ ■ ■ ■ ■ ■ are prepared according to embodiment 102

AU absorbance unitses

Claims (2)

1. a kind of compound of formula (VI),

Wherein

N3 is 1；

N4 is 1；

AAⁿ⁴It is amino acid residue, n4 is the index of the amino acid residue, and n3 is the quantity of the amino acid residue, and (AA¹)₁Represent an amino acid residue AA¹；

(3) the N- terminal amino groups of the amino acid residue are represented；

R1 and R2 are same or different, and are independently from each other hydrogen, C_1-4Alkyl, C (O)-(CH₂-O-)_m5- (GRPEG)_m4- R3 and PGN；

R3 is C_1-4Alkyl；

M4 is 0 or 1；

M5 is 0 or 1；

PGN is blocking group；

GRPEG is formula (GRPEG-I) linking group；

M21 is 1,2,3,4,5 or 6.

2. formula (VI) compound according to claim 1, wherein the formula (VI) compound be selected from formula (6-3) compound, Formula (6-4) compound, formula (6b-3) compound and formula (6b-4) compound：