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

Branched linker for protein drug conjugates

the application is a divisional application of an invention patent application with the application date of 2012, 2 and 23, the Chinese patent application number of 201280010479.6 (international application number of PCT/EP2012/053039) and the invention name of a branched chain connector for protein drug conjugates.

Technical Field

The present invention relates to a method for linking a protein and a drug to a protein drug conjugate, wherein the drug is linked to the protein by a specific branched linker comprising a peptide chain and derived from ortho hydroxy para amino benzyl alcohol, wherein the peptide chain is linked to a phenyl ring by a para amino group, the drug is linked to the phenyl ring by a benzylic alcohol moiety, and the protein is linked to the phenyl ring by an ortho-hydroxy group; the invention further relates to processes for preparing said protein-drug-conjugates via various intermediates, to pharmaceutical uses of such protein-drug conjugates, such as methods of controlling the growth of undesirable cells, to pharmaceutical compositions comprising such protein-drug conjugates, and to intermediates for preparing said protein-drug conjugates.

Background

Most drugs used in chemotherapy have serious side effects that limit their efficacy and use. Linking such payloads (i.e. pharmaceutically active compounds such as drugs) to targeting agents, particularly monoclonal antibodies, can provide novel Antibody Drug Conjugates (ADCs), for example for use in cancer therapy. Tissue specificity is usually controlled by monoclonal antibody (mAb) components, while drugs provide therapeutic effects. The efficiency and tolerability of ADCs depends on the interaction between the target antigen, drug potency and coupling technique. In particular, the chemical nature of the linker strongly affects the specificity and safety of the ADC.

Instead of chemically unstable linkers with limited stability under physiological extracellular conditions, such as hydrazone and disulfide based linkers, linkers that are stable under physiological extracellular conditions, in particular linkers with high plasma stability, are desirable for improving the applicability of the treatment, since the drug should only be released inside the cell, which is targeted by the protein to which it is attached (target), and not outside the cell.

Non-split couplings have the disadvantage that: for payload release, ADC internalization is required after complete hydrolysis of the polypeptide backbone of the mAb, and when ADC internalization is poor, a decrease in potency may be encountered. Therefore, ADCs carrying non-cleaved linkers are highly dependent on the biology of the target cell. Furthermore, when the payload is linked to the last amino acid of the mAb after degradation of the mAb, not all payloads retain their biological activity.

In order to allow the ADC to provide a payload not only to tumor cells, but also to adjacent antigen-negative cells (i.e. a bystander effect), the released payload must readily diffuse through the hydrophobic cell membrane, but this is not the case when an ADC with a non-cleavable linker releases its payload in the form of an amino acid, which carries a drug with the amino acid in a zwitterionic state (i.e. with a positively charged ammonium and a negatively charged carboxylate).

Thus, it would be desirable to have a linker that exhibits high plasma stability and releases a drug without the need to chemically modify the drug after its release.

Furthermore, it would be desirable to have a protein drug conjugate that exhibits reduced agglomeration or aggregation that would impair its performance.

A drug ligand conjugate is disclosed in EP 624377, wherein the linker comprises a linear peptide.

Fanny Guzman et al, Electronic Journal of Biotechnology, 2007, 10, 279-314; yoshio Okada, Current Organic Chemistry, 2001, 5, 1-43; US 6897289B and the textbook Houben-Weyl Synthesis of Peptides and Peptidomimetics (Methods in organic chemistry), "Murray Goodman et al, Thieme Publishing Group, 2001, in particular Volumes E22a and E22B; protecting groups, the formation of peptide bonds, general and specific methods for synthesizing polypeptides and proteins, and analytical techniques for determining the structure and composition of peptides are disclosed.

Known linkers comprising linear peptide chains still show deficiencies. Linkers and protein drug conjugates based on such linkers that exhibit improved performance are needed. Surprisingly, certain branched linkages derived from ortho hydroxy para amino benzyl alcohols exhibit desirable properties.

Disclosure of Invention

In the following text, the following abbreviations are used if not specifically stated:

DCC N, N' -dicyclohexylcarbodiimide

EDC N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide

Fmoc 9-fluorenylmethyloxycarbonyl

Boc tert-butoxycarbonyl

Boc₂O-di-tert-butyl dicarbonate

Cit citrulline

NBS N-bromosuccinimide

NHS Compound of formula (HOSu), N-hydroxysuccinimide

NIS N-iodosuccinimide

-OTs tosylate

-OMs methanesulfonate

-OTf triflate

PBS phosphate buffered saline

Red-Al sodium bis (2-methoxyethoxy) aluminum hydride

TCEP tris (2-carboxyethyl) phosphine hydrochloride

Tos or Ts tosyl or p-tosyl

TsCl tosyl chloride or p-toluenesulfonyl chloride

Z or Cbz benzyloxycarbonyl

The subject of the invention is a Method (MI) for linking a ligand LI to a drug DR,

LI is selected from: amino acid LI-AA, monoclonal or polyclonal antibody LI-Ab, antibody fragment LI-AbFrag, protein LI-Prot and peptide LI-Pep;

DR is a pharmaceutically active drug;

characterized in that the coupling body LIN is used for covalently connecting LI and DR;

LIN comprises the linking group CG 2;

CG2 is derived from o-hydroxy p-aminobenzyl alcohol and is a linking group of formula (CG 2-1);

(xvii) denotes a connection site for connecting LI;

(x) represents a connection site for connecting DR;

(xv) represents a connection site to a linear peptide, said peptide having from 2 to 8 amino acid residues;

(4) represents the para-amino group of the ortho-hydroxy para-aminobenzyl alcohol from which CG2 is derived.

A further subject of the invention is a process (MI), as defined in the present description, and all its preferred embodiments,

wherein LI and DR are covalently linked in the form of a compound of formula (I);

CG2 is as defined in the specification, and has all its preferred embodiments;

CG1 is a linking group selected from the group consisting of a linking group of formula (CG1-I), a linking group of formula (CG1-II), a linking group of formula (CG1-III), and a linking group of formula (CG 1-IV);

m30 and m32 are identical or different and independently of one another are 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;

in said formula CG 1(×) represents the bond between T1 and CG1,

covalently linked LI forms the ligand residue LIRes in the compound of formula (i), which is covalently linked to CG1 via T1;

LI is as defined in the specification, also has all its preferred embodiments, and is a compound of formula (LIRes-T1-H);

LIRes-T1-H (LIRes-H)

the LIRes is selected from: amino acid residues LIRes-AA, mono-or polyclonal antibody residues LIRes-Ab, antibody fragment residues LIRes-AbFrag, protein residues LIRes-Prot and peptide residues LIRes-Pep;

LI has a structure selected from SH, OH or NH₂The functional group in formula (I) forming said T1, said T1 being bonded to CG1 through said bond (±);

t1 is-S-, -O-, or-NH-;

n1 is 0 or 1;

SG is a spacer group selected from the group consisting of a spacer group of formula (SG-II) and a spacer group of formula (SG-III);

m1 and m2 are the same or different and are independently of each other 0 or 1;

m10, m11 and m12 are the same or different and are independently of each other 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;

provided that m2 and m10 are not simultaneously 0;

provided that m1, m11 and m12 are not simultaneously 0;

SGPEG is a linking group of formula (SGPEG-I);

m20 is 1,2, 3, 4, 5 or 6;

n2 is 0 or 1;

t4 is-O-;

CG3 is selected from a linker of formula (CG3-I) and a linker of formula (CG 3-II);

r5 and R6 are the same or different and are independently of each other C_1-4An alkyl group;

for n1 being 1, said (×) in said formula CG1 and said formula SG represents the bond between CG1 and SG, said (×) in said formula SG and said formula CG2 represents the bond between SG and CG 2; if CG1 is a linker of formula (CG1-I), then the nitrogen atom represented by (×) in SG forms a ring-internal nitrogen atom, such that the hydrogen atom of said nitrogen atom is replaced by a ring-internal bond;

said (×) in said formula CG1 and said (×) in said formula CG2 represent said bond between CG1 and CG2 for n1 being 0;

with the proviso that if n1 is 0, then CG1 is not a linking group of formula (CG 1-I);

for n2 being 1, said (×) in said formula CG2 and said formula CG3 represents a bond connecting CG2 and CG3 to T4; and said (×) in formula CG3 represents a bond between CG3 and T2;

for n2 being 0, said (×) of said formula CG2 represents the bond between CG2 and T2;

said (. times. times.) in said formula CG2 represents CG2 and AAⁿ⁴A bond between;

the covalently linked DR forms a drug residue DRRes in the compound of formula (I) which is covalently linked to CG2 via T2;

DR is as defined in the specification, has all its preferred embodiments, and is a compound of formula (DRRes-T2-H);

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

DRRes is a drug residue derived from DR;

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

t2 is-N (R4) -, -O-or-S-;

r4 is H or C_1-4An alkyl group;

n3 is 2, 3, 4, 5, 6, 7 or 8;

n4 is an integer from 1 to n 3;

AAⁿ⁴is an amino acid residue, n4 is an index of said amino acid residue, and (AA)ⁿ⁴)_n3Is AA having n3 amino acid residuesⁿ⁴And n4 represents the amino acid residue AA in the peptide starting from CG2ⁿ⁴Position of a single amino acid in said peptideThe residues being linked to one another by peptide bonds, AA¹Is the first amino acid residue in the chain and is linked to CG2 by a bond (. times.¹A bond between the carboxylic acid group of (a) and the amino group represented by (4) of CG2, and AAⁿ³Is the last amino acid residue in the chain, a single AAⁿ⁴Are the same or different independently of one another;

(3) represents AAⁿ³The N-terminal amino group of (1);

r1 and R2 are the same or different and are independently selected from hydrogen, C_1-4Alkyl, C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-R3 and PGN;

r3 is C_1-4An alkyl group;

m4 is 0 or 1;

m5 is 0 or 1;

PGN is a protecting group;

GRPEG is a linking group of formula (GRPEG-I);

m21 is 1,2, 3, 4, 5 or 6.

LI, DR and CG2 are as defined in the specification, with all its preferred embodiments as well.

Preferably, LIN is a compound of formula (LIN).

Preferably, the drug DR is selected from cytotoxic agents, other chemotherapeutic agents and anti-metastatic agents.

Preferably, the other chemotherapeutic agent and the anti-metastatic agent are selected from tyrosine kinase inhibitors and Rac1 inhibitors.

Preferably, the tyrosine kinase inhibitor is selected from the group of Active Pharmaceutical Ingredients (API) consisting of Imatinib (Imatinib), lapatinib (lapatinib), sunitinib (sunitinib), nilotinib (nilotiib), dasatinib (dasatiib).

Preferably, the Rac inhibitor is NSC 23766.

Preferred cytotoxic agents are those used to treat cancer.

Preferred classes of cytotoxic agents include, for example, enzyme inhibitors such as anthracyclines, bleomycin, cytotoxic nucleosides, dihydrofolate reductase inhibitors, differentiation inducers, DNA lytic agents, DNA intercalators, enediynes (diynenes), mitomycins (mitomycins), podophyllotoxins (podophyllotoxins), pteridine (pteridine) family drugs, paclitaxel (taxols), thymidylate synthase (thymidylate synthase) inhibitors, topoisomerase (topoisomerase) inhibitors, and vinblastine (vinca) drugs.

Preferred useful members of the various classes of cytotoxic agents are selected from: n8-acetylspermidine, actinomycin, 9-aminocamptothecin, aminopterin, serpentin (anguidine), anthracycline, auristatin, bleomycin, calicheamicin (calicheamicin), camptothecin (lactone or lactone in ring-opened form), carminomycin, CC-1065, clofarabine (clofarabine), 1- (2-chloroethyl) -1, 2-dimethanesulfonyl hydrazide, cumenoindol-4-one (CBI), cytarabine, cytosine arabinoside (cytosine arabinoside), daunomycin, dichloromethotrexate, N- (5, 5-diacetoxypentyl) doxorubicin, 1, 8-dihydroxy-bicyclo [7.3.1] trideca-4-9-diene-2, 6-diyn-13-one, difluoronucleoside, doxorubicin, Duocarmycin (duocarmycin), epirubicin (epirubicin), esperamicin (esperamicin), etoposide, 5-fluorouracil, irinotecan, leudosideine, vinpocetine, maytansine, melphalan, 6-mercaptopurine, methopterin (methopterin), methotrexate, mitomycin A, mitomycin C, morpholine-doxorubicin, nemorubicin (nemorubicin), podophyllotoxin and podophyllotoxin derivatives such as etoposide or phosphate, tretinoin, saponin, telithromycin (tallymycin), vinblastine, vincristine, vindesine, taxanes such as paclitaxel (Taxol) or paclitaxel (paclitaxel), Taxotere (Taxotere) or docetaxel, and Taxotere and derivatives thereof.

More preferred cytotoxic drugs are selected from the group consisting of anthracyclines, auristatins, calicheamicin (calicheamicin), cumyl indol-4-one (CBI), doxorubicin, duocarmycin, maytansine, mitomycin C, paclitaxel and its analogs and derivatives of these substances.

Preferably, R4 is H.

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

Preferably, n2 is 1, T2 is-NH-, -O-, or-S-, and is linked to CG3 by a bond (×); more preferably, T2 is-NH-or-S-.

Doxorubicin has CAS number 23214-92-8 and is a compound of formula (DOXO).

The compound of formula (DOXO) may also be used in the form of its hydrochloride salt.

Doxorubicin can be linked to either CG3 or CG2, respectively, through one of its functional groups, as represented by one of the functional groups represented by (D1), (D2), (D3), and (D4) used in formula (DOXO).

(d1) The functional groups represented by (D2), (D3) and (D4) function as T2 in formula (I).

Preferably, doxorubicin is linked to CG3 via the amino group represented by (d1) in formula (DOXO) and via said bond (×).

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

Maytansine can be linked to CG3 or CG2, respectively, via an-OH represented by (m2) in formula (MAYT).

or-C (O) -CH represented by (m1) in the formula (MAYT)₃Group exchange of acyl group with nucleophilic group-SH, -NH-in turn correspondingly attached to CG3 or CG2₂or-OH.

The nucleophilic group of the-OH or acyl group represented by (m2) then acts as T2 as in formula (I).

A preferred taxane is paclitaxel having CAS number 33069-62-4 and formula (TAXO).

Paclitaxel may be linked to CG3 or CG2, respectively, by one of-OH represented by (t1), (t2) and (t3) in formula (TAXO). the-OH then acts as T2 in formula (I).

In a preferred embodiment, n2 is 0, T2 is-O-, and is linked to CG2 by a bond (×) and DRRes is derived from DR, which is a compound of formula (TAXO).

In another preferred embodiment, n2 is 1, T2 is-O-and is linked to CG3 by a bond (. + -.) and CG3 is a linking group of formula (CG3-II) and DRRes is derived from DR which is a compound of formula (TAXO), one possible paclitaxel intermediate of this embodiment is a compound of formula (TAXO-T1-1).

Camptothecin is (S) - (+) -camptothecin having CAS number 7689-03-4 and is a compound of formula (CAMPTO).

Camptothecin can be linked to CG3 or CG2, respectively, via the functional group represented in formula (CAMPTO) (c 1).

The functional group represented by (c1) then functions as T2 as in formula (I).

In a preferred embodiment, n2 is 0, T2 is-O-, and is linked to CG2 by a bond (. + -), DRRes is derived from DR, which is a compound of formula (CAMPTO).

In another preferred embodiment, n2 is 1, T2 is-O-, and is linked to CG3 by a bond (. + -.) and CG3 is a linking group of formula (CG3-II), DRRes is derived from DR which is a compound of formula (CAMPTO).

In a preferred embodiment, CG1 is a linking group of formula (CG1-I), (CG1-III) or (CG1-IV), and the sulphur atom of the side chain of the Cys residue of LIRes is T1 and is linked to CG1 by a bond (#).

In another preferred embodiment, CG1 is a linking group of formula (CG1-II) and T1 is-N-or-O-of LIRes and is linked to CG1 by a bond (#). The amino or hydroxyl group of such a LIRes attached to CG1 is preferably the N-terminal amino group of LIRes or the amino or hydroxyl group of the side chain of an amino acid residue of LIRes. Preferably, if T1 is an amino group attached to the side chain of an amino acid residue of LIRes of CG1, then said amino acid residue of LIRes is preferably Lys; if T1 is a hydroxyl group attached to the side chain of an amino acid residue of the LIRes of CG1, then the amino acid residue of the LIRes is preferably Tyr, Ser or Thr.

Preferably, LI is selected from: a mono-or polyclonal antibody LI-Ab, an antibody fragment LI-AbFrag, a protein LI-Prot and a peptide LI-Pep; and

the LIRes is selected from: mono-or polyclonal antibody residues LIRes-Ab, antibody fragment residues LIRes-AbFrag, protein residues LIRes-Prot, and peptide residues LIRes-Pep.

If the LIRes is LIRes-AA, then the LIRes is preferably an alpha-amino acid residue.

The LIRes may preferably be linked to CG1 through one of two possible functional groups of the LIRes: via an N-terminal amino group or via a functional group of a side chain of the LIRes, provided that the LIRes has such a side chain with a functional group. Such a functional group linking LIRes to CG1 is T1. If the LIRes is attached via a functional group of the side chain of the LIRes, the side chain is preferably the side chain of a Cys, Lys, Tyr, Ser or Thr residue of the LIRes.

If the LIRes is LIRes-AA, the remaining functional groups of the LIAARes-AA not attached to CG1 may be protected by protecting groups commonly used in peptide chemistry, e.g., the non-attached amino group may carry acetic acid and the non-attached carboxyl group may be protected by C_1-4The alcohol is esterified and the non-linking functional group of the side chain may carry side chain protecting groups commonly used in peptide chemistry.

More preferably, the LIRes-AA is an alpha amino acid residue having a side chain with a functional group and being linked to CG1 through the functional group, even more preferably the LIRes-AA is derived from Cys, Lys, Tyr, Ser or Thr.

Preferably, if LIRes is attached via the side chain of a Cys residue of LIRes, T1 is formed via the sulphur atom of the side chain of said Cys residue and is preferably attached to CG1 via a bond (±), CG1 being a linking group of formula (CG1-I), (CG1-III) or (CG 1-IV); or

If the LIRes is attached via the side chain of a Lys, Tyr, Ser or Thr residue of the LIRes, T1 is formed via the nitrogen or oxygen atom of the side chain of said Lys, Tyr, Ser or Thr residue and is attached to CG1 via a bond (—), preferably CG1 is a linker of formula (CG 1-II).

The LIRes-Pep is derived from a cell penetrating peptide.

The LIRes-AB and LIRes-ABFrag are preferably derived from antibodies and antibody fragments used in the treatment of disease, preferably cancer.

Preferably, m30 and m32 are the same or different and are independently of each other 2, 3, 4, 5 or 6.

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

Preferably, when CG1 is a linking group of formula (CG1-II), m32 is 2.

Preferably, n1 is 1.

Preferably, m1 and m2 are 0 or 1.

Preferably, m10, m11 and m12 are the same or different and are independently of each other 0, 1,2, 3, 4, 5 or 6.

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

More preferably still, the first and second liquid crystal compositions are,

m10 is 6 and m2 is 0; or

m2 is 1, m10 is 0 and m20 is 1;

m1 is 1, m11 is 1, m12 is 1 and m20 is 3; or

m1 is 0, m11 is 3, and m12 is 0.

Preferably, R5 and R6 are CH₃。

In a particular embodiment, CG1 is a linker of formula (CG1-I), SG is a spacer of formula (SG-II), m10 is 6 and m2 is 0.

In another specific embodiment, CG1 is a linking group of formula (CG1-II), m32 is 2; or a linking group of formula (CG 1-III); or a linking group of formula (CG1-IV), m30 is 2; and SG is a spacer of formula (SG-II), m10 is 0, m2 is 1 and m20 is 1.

In another embodiment, CG1 is a linking group of formula (CG1-II), m32 is 2; or a linking group of formula (CG 1-III); or a linking group of formula (CG1-IV), m30 is 2; and SG is a spacer of formula (SG-III), m1 is 1, m11 is 1, m12 is 1 and m20 is 3; or SG is a spacer of formula (SG-III), m1 is 0, m11 is 3, and m12 is 0.

Preferably, n3 is 2, 3, 4, 5 or 6; more preferably 2, 3 or 4; more preferably, n3 is 2 or 3.

Preferably, AAⁿ⁴Is α amino acid residues.

More preferably, AAⁿ⁴Selected from: alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, an acetyl or formyl protected lysine side chain, arginine side chain protected (preferably with tosyl or nitro) arginine, histidine, ornithine, side chain protected (preferably with acetyl or formyl) ornithine, and citrulline.

Even more preferably, AAⁿ⁴Is alanine, glycine, phenylalanine, valine, lysine, leucine, tryptophan, arginine, side chain-protected arginine or citrulline, in particular alanine, glycine, phenylalanine, valine, lysine or citrulline.

If AAⁿ⁴Having a side chain with a functional group, the functional group may be protected by a protecting group that is commonly used to protect functional groups of amino acid side chains.

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

(AAⁿ⁴)_n3Examples of peptides of peptide chains are 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-Cit and D-Phe-L-Phe-Lys; any lysine side chains are optionally protected, preferably with acetyl.

Particularly preferably, n3 is 2 or 3, and AAⁿ⁴Is alanine, glycine, valine or citrulline;

more particularly, it is preferred that the first and second,

n3 is 2, AA¹Is citrulline, and AA²Is valine or alanine;

n3 is 3, AA¹Is citrulline, AA²Is valine and AA³Is glycine.

Preferably, m4 is 1.

Preferably, R3 is methyl.

Preferably, R1 and R2 are the same or different and are independently selected from: hydrogen, methyl, C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-CH₃And a PGN.

Preferably, m21 is 2, 3 or 4.

Preferably, PGNs are protecting groups commonly used in peptide chemistry to protect the N-terminus of a peptide or to protect the alpha-amino group of an alpha amino acid used as a building block in peptide synthesis.

More preferably, the PGN is selected from: boc, Fmoc and Z.

Even more preferably, R1 is hydrogen, methyl, acetyl or C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-CH₃M4 is 1 and m21 is 3, and R2 is hydrogen or methyl.

In particular, it is possible to use, for example,

r1 is acetyl, R2 is hydrogen; or

R1 and R2 are methyl; or

R1 is C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-CH₃M5 is 0, m4 is 1, and m21 is 3 and R2 is hydrogen; or

R1 is C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-CH₃M5 is 1, m4 is 1, and m21 is 2 and R2 is hydrogen.

A further subject of the invention is a Method (MI);

wherein the compound of formula (I) is prepared in step (MI);

step (MI) comprises reacting (MI), wherein the compound of formula (II) is reacted with a compound of formula (LIRes-T1-H);

CG1M is a linking group selected from the group consisting of a linking group of formula (CG1M-I), a linking group of formula (CG1M-II), a linking group of formula (CG1M-III), and a linking group of formula (CG 1M-IV);

x1 is Cl, Br or I;

SG、n1、n4、n3、AAⁿ⁴(3), (R1), (R2), (T4), (CG 3), (n 2), (T2), (DRRes), (m 30) and (m 32) are as defined in the specification, with all its preferred embodiments;

CG2 is as defined in the specification, and has all its preferred embodiments.

Preferably, X1 is Cl or Br, more preferably X1 is Br.

Preferably, CG1M is a linking group of formula (CG 1M-IV).

More preferably, CG1M is a linking group of formula (CG1M-IV) and m30 is 2.

In a particular embodiment, CG1M is a linking group of formula (CG1M-I), SG is a spacer of formula (SG-II), m10 is 6 and m2 is 0.

In another embodiment, CG1M is a linking group of formula (CG1M-II) and m32 is 2, or a linking group of formula (CG1M-iii) or a linking group of formula (CG1M-IV) and m30 is 2, and SG is a spacer of formula (SG-II), m10 is 0, m2 is 1, m20 is 1.

In another embodiment, CG1M is a linking group of formula (CG1-II) and m32 is 2, or a linking group of formula (CG1-III), or a linking group of formula (CG1M-IV) and m30 is 2, and SG is a spacer of formula (SG-III) and m1 is 1, m11 is 1, m12 is 1 and m20 is 3 or SG is a spacer of formula (SG-III) and m1 is 0, m11 is 3, and m1 is 0.

Preferably, the reaction (MI) has a reaction temperature of from 0 to 150 ℃, more preferably 5 to 50 ℃, more preferably 10 to 40 ℃.

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

The reaction (MI) is generally carried out in a solvent (MI).

Preferably, the solvent (MI) is selected from the group consisting of water, N-dimethylacetamide, N-dimethylformamide, N-dimethylsulfoxide and mixtures thereof.

In the case of water, the water may comprise a buffer (MI), preferably a buffer (MI) conventionally used in protein chemistry, more preferably a buffer (MI) from a buffer substance selected from: acetic acid, citric acid, Dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), glycine, histidine, phosphoric acid (including phosphate buffered saline, PBS), polysorbate 20, polysorbate 80, sucrose, sodium chloride, succinic acid, trehalose, tris (hydroxymethyl) aminomethane, mixtures thereof, and salts thereof.

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

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

Preferably, in reaction (MI), 1 to 100 molar equivalents, more preferably 2 to 20 molar equivalents, even more preferably 3 to 10 molar equivalents of compound of formula (II) are used, based on the number of moles of compound of formula (LIRes-T1-H).

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

TCEP is preferably used when LI is the mono-or polyclonal antibody LI-Ab or the antibody fragment LI-AbFrag.

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

After reaction (MI), the compound of formula (I) can be isolated by standard methods such as washing, extraction, filtration, concentration, drying, etc. The compounds of formula (i) may be purified before or after isolation, preferably by chromatography or crystallisation from a suitable solvent.

Alternatively, the compound of formula (I) may be purified by standard methods such as filtration, ultrafiltration, diafiltration and chromatography, and may be stored or further used as a solvent.

A further subject of the invention is a process (MII) for preparing compounds of the formula (II), as defined in the description, with all its preferred embodiments,

wherein,

if n2 is 1 and CG3 is a linking group of formula (CG3-I), then Method (MII) comprises step (MIIa) and step (MIIb);

if n2 is 1 and CG3 is a linking group of formula (CG3-II), then Method (MII) comprises said step (MIIa), step (MIic), step (MIId) and step (MIIe);

if n2 is 0 and CG1M is a linking group of formula (CG1M-IV), then process (MII) comprises one step, step (MII0-IV), or two steps, step (MII0-I-IVa) and step (MII 0-I-IVb);

if n2 is 0 and CG1M is a linking group of formula (CG1M-III), then Method (MII) comprises the steps of (MII 0-III);

if n2 is 0 and CG1M is a linking group of formula (CG1M-II), then process (MII) comprises two steps, step (MII0-IIa) and step (MII0-IIb), or one step, step (MII 0-IIc);

if n2 is 0 and CG1M is a linking group of formula (CG1M-I), then process (MII) comprises one step, step (MII0-I), or two steps, step (MII0-I-IVa) and step (MII 0-I-IVb);

step (MIIa) comprises a reaction (MIIa) in which a compound of formula (III) is reacted with a compound of formula (II-I);

n2 in formula (III) is as defined in the specification, and has all its preferred embodiments;

the compound of formula (II-I) is selected from the group consisting of compounds of formula (II-1), 1' -carbonyldiimidazole, 4-nitrophenylchloroformate, phosgene, diphosgene, triphosgene and mixtures thereof;

step (MIIb) comprises a reaction (MIIb), wherein the reaction product from the reaction (MIIa) is reacted with a compound of formula (DRRes-T2-H);

step (MIic) comprises a reaction (MIic), wherein the reaction product from said reaction (MIIa) is reacted with a compound of formula (CG3M-II) to provide a compound of formula (IIc);

in formula (IIc) n2 is as defined in claim 2;

step (MIId) comprises a reaction (MIId) wherein a compound of formula (IIc) prepared in said reaction (MIic) is reacted with said compound of formula (II-I);

step (MIIe) comprises a reaction (MIIe), wherein the reaction product from said reaction (MIId) is reacted with a compound of formula (DRRes-T2-H);

step (MII0-I-IVa) comprises a reaction (MII0-I-IVa), wherein a compound of formula (III) is reacted with compound (II0-I-IVa) to provide a compound of formula (III 0-I-IVa);

n2 is 0 in formula (III 0-I-IVa);

compound (II0-I-IVa) is selected from the group consisting of p-toluenesulfonyl chloride, p-toluenesulphonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride and mixtures thereof;

x2 is selected from the group consisting of-OTs, -OMs and-OTf;

step (MII0-I-IVb) comprises a reaction (MII0-I-IVb), wherein the compound of formula (III0-I-IVa) prepared in the reaction (MII0-I-IVa) is reacted with a compound of formula (DRRes-T2-H);

for the case where CG1M is a linking group of formula (CG1M-IV), in step (MIII-IV); or

For the case where CG1M is a linking group of formula (CG1M-III), in step (MIII-III); or

For the case where CG1M is a linking group of formula (CG1M-II), in two steps, in steps (MIII-IIa) and (MIII-IIb), or in one step, in steps (MIII-IIc); or

For the case where CG1M is a linking group of formula (CG1M-I), in step (MIII-I); the preparation of the compound of formula (III),

step (MIII-IV) comprises the reaction (MIII-IV) of a compound of formula (IV) with a compound of formula (CG1MR-IV),

in formula (IV) n2 is as defined in the specification, with all its preferred embodiments;

m30 is as defined in the specification, with all its preferred embodiments;

r20 is a residue of formula (R20-1);

step (MIII-III) comprises the reaction (MIII-III) of a compound of formula (IV) with a compound of formula (CG1 MR-III);

x1 is as defined in the specification, with all its preferred embodiments;

step (MIII-IIa) comprises reacting (MIII-IIa) the compound of formula (IV) with a compound of formula (CG1MR-IIa) to provide a compound of formula (IV-IIa);

m32 is as defined in the specification, with all its preferred embodiments;

step (MIII-IIb) comprises the reaction (MIII-IIb) of the compound (IV-IIa) prepared in step (MIII-IIa) with a compound of formula (HOSu);

step (MIII-IIc) comprises the reaction (MIII-IIc) of said compound of formula (IV) with a compound of formula (CG1 MR-IIc);

m32 is as defined in the specification, with all its preferred embodiments;

step (MIII-I) comprises the reaction of a compound of formula (IV) with a compound of formula (MA) (MIII-I);

the compound of formula (IV) is prepared in step (MIV);

step (MIV) comprises reaction (MIV), which is a reduction reaction with a compound of formula (V) of compound (IV);

compound (IV) is selected from: NaBH₄、BH₃DIBAL-H, sodium bis (2-methoxyethoxy) aluminum hydride, and mixtures thereof;

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

the compound of formula (V) is prepared in step (MVb);

step (MVb) comprises a reaction (MVb) wherein R30 is cleaved from the compound of formula (Va) with HCl;

r30 is linked to SG by the bond represented by (×) in the formula SG and is Boc;

step (MII0-IV) comprises the reaction of a compound of formula (III0) with a compound of formula (CG1MR-IV) (MII 0-IV);

SG, CG2, n1, AA, n4, n3, (3), R1, R2, T2 and DRRes are as defined in the specification, with all their preferred embodiments, and in formula (III0) n2 is 0;

step (MII0-III) comprises the reaction of a compound of formula (III0) with a compound of formula (CG1MR-III) (MII 0-III);

step (MII0-IIa) comprises reaction (MII0-IIa) of the compound of formula (III0) with a compound of formula (CG1MR-IIa) to provide a compound of formula (III 0-IIa);

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

step (MII0-IIb) comprises the reaction of the compound of formula (III0-IIa) prepared in step (MII0-IIa) with the compound of formula (HOSu) (MII 0-IIb);

step (MII0-IIc) comprises the reaction of the compound of formula (III0) with a compound of formula (CG1MR-IIc) (MII 0-IIc);

step (MII0-I) comprises the reaction of a compound of formula (III0) with a compound of formula (MA) (MII 0-I);

the compound of formula (III0) was prepared in step (MIII0),

step (MIII0) comprises a reaction (MIII0) wherein R30 is cleaved from the compound of formula (IV0) with HCl;

n2 is 0 in formula (IV 0);

the compound of formula (IV0) was prepared in step (MIV0a) and step (MIV0b),

step (MIV0a) comprises reacting (MIV0a), wherein a compound of formula (V0) is reacted with a compound of formula (RIV0a) to provide a compound of formula (IV0 a);

r30, SG, CG2, n1, AA, n4, n3, (3), R1 and R2 are as defined in the specification, with all their preferred embodiments;

compound (RIV0a) is selected from: p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride, trifluoromethanesulfonic anhydride, SOCl₂、(COCl)₂、POCl₃、PCl₃、PCl₅、POBr₃、PBr₃、PBr₅N-bromosuccinimide, N-iodosuccinimide, HCl, HBr, HI, and mixtures thereof;

x3 is selected from the group consisting of-OTs, -OMs, -OTf, -Cl, -Br, and-I;

step (MIV0b) comprises a reaction (MIV0b) in which the compound of formula (IV0a) prepared in reaction (MIV0) is reacted with a compound of formula (DRRes-T2-H);

the compound of formula (V0) is prepared in step (MV0),

step (MV0) comprises reaction (MV0), reaction (MV0) is a reduction reaction with compound (IV) of formula (Va);

CG1M and X1 are as defined herein, with all its preferred embodiments;

SG、n1、n4、n3、AAⁿ⁴(3), R1, R2, T4, CG3, n2, T2, DRRes, compounds of formula (DRRes-T2-H), m30, m32, R5 and R6 are as defined in the specification, with all its preferred embodiments;

CG2 is as defined herein and has all its preferred embodiments.

The compounds (II-I) and the compounds of the formula (CG3M-II) are well known compounds and can be prepared according to well known methods, which are usually even commercially available.

Reaction (mia) and reaction (MIID) are similar reactions and may be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

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

The reaction (MIIa) and the reaction (MIId) are generally carried out in a solvent (MIIa).

Preferably, the solvent (MIIa) is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF and mixtures thereof.

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

Preferably, in reaction (MIIa) and in reaction (MIId) 0.5 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, more preferably 1 to 2 molar equivalents of compound (II-I) are used, based on the molar amount of compound of formula (III).

The reaction (MIIa) and the reaction (MIId) may be carried out in the presence of a base (MIIa).

Preferably, the base (MIIa) is selected from the group consisting of potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures thereof.

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

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

After reaction (MIIa) and reaction (MIId), the reaction products of reaction (MIIa) and reaction (MIId) may be isolated by standard methods such as washing, extraction, filtration, concentration, drying, and the like. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

The crude reaction mixture from reaction (MIIa) and reaction (MIId) may also be used directly in reaction (MIIb) or reaction (MIIEe).

More preferably, reaction (MIIa) and reaction (MIIb) and reaction (MIId) and reaction (MIIe) are all in the same solvent and are carried out continuously in one pot.

Reaction (MIIb) and reaction (MIIEe) are similar reactions and may be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

The reaction (MIIb) and the reaction (MIIEe) are generally carried out in a solvent (MIIb).

Preferably, the solvent (MIIb) is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and mixtures thereof.

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

Preferably, in reaction (MIIb) and reaction (MIIEe), 0.2 to 10 molar equivalents, more preferably 0.5 to 5 molar equivalents, more preferably 0.8 to 2 molar equivalents of the compound of formula (DRRes-T2-H) are used, based on the molar amount of reaction product of reaction (MIIa) or reaction product of reaction (MIId).

Preferably, the reaction (MIIb) and the reaction (MIIEe) are carried out under an inert atmosphere.

The reactions (MII0-I-IVb) and (MIV0b) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

The reactions (MII0-I-IVb) and (MIV0b) are usually carried out in a solvent (MIV0 b).

Preferably, the solvent (MIV0b) is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF and mixtures thereof.

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

Preferably, in reaction (MII0-I-IVb) and reaction (MIV0b) 0.2 to 10 molar equivalents, more preferably 0.5 to 5 molar equivalents, more preferably 0.8 to 2 molar equivalents of the compound of formula (DRRes-T2-H) are used, which are based correspondingly on the molar amount of the compound of formula (III0-I-IVb) or the compound of formula (IV0 a).

Preferably, the reactions (MII0-I-IVb) and (MIV0b) are carried out under an inert atmosphere.

The reaction (MIIC) is generally carried out in a solvent (MIIC).

Preferably, the solvent (MIic) is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and mixtures thereof.

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

Preferably, in reaction (MIIc) 0.2 to 20 molar equivalents, more preferably 0.5 to 10 molar equivalents, more preferably 0.8 to 5 molar equivalents of compound of formula (CG3M-II) are used, based on the molar amount of reaction product of reaction (MIIa).

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

After the reaction (MIIC), the reaction product of the reaction (MIIC) can be isolated by standard methods such as washing, extraction, filtration, concentration, drying, etc. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

After reaction (MIIb), reaction (MIIe), reaction (MII0-I-IVb) and reaction (MIV0b), the compound of formula (II) or the compound of formula (IV0), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration, drying, and the like. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

Preferably, CG1M is a linking group of formula (CG 1M-IV);

more preferably, CG1M is a linking group of formula (CG1M-IV) and m30 is 2.

The compounds of formula (V) and of formula (III0) can be used in the reaction (MIV), in the reaction (MII0-I), in the reaction (MII0-IIa), in the reaction (MII0-III) and in the reaction (MII0-IV) in the non-protonated form or in the protonated form as salts.

The reactions (MIII-IV) and (MII0-IV) are similar reactions and may be performed under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

The reaction (MIII-IV) and the reaction (MII0-IV) are usually carried out in a solvent (MIII-IV).

Preferably, the solvent (MIII-IV) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

Preferably, in reaction (MIII-IV) and in reaction (MII0-IV) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents of compound (CG1MR-IV) are used, said molar equivalents corresponding to the molar amount of compound of formula (IV) or formula (III 0).

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

The reaction (MIII-IV) and the reaction (MII0-IV) are usually carried out in the presence of a base (MIII-IV).

Preferably, the base (MIII-IV) is selected from: sodium carbonate, potassium carbonate, diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures thereof.

Preferably, in reaction (MIII-IV) and reaction (MII0-IV) 0.5 to 50 molar equivalents, more preferably from 1 to 20 molar equivalents, even more preferably 2 to 10 molar equivalents of base (MIII-IV) are used, based on the molar amount of the compound of formula (IV) or formula (III0), respectively.

After reaction (MIII-IV) and reaction (MII0-IV), the compound of formula (III) or the compound of formula (II), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration, drying, etc. The compound of formula (III) or the compound of formula (II) may be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

The reactions (MIII-III) and (MII0-III) are similar reactions and may be performed under similar reaction parameter ranges as defined in the present specification, with individual reaction parameters for each of the two reactions being selected independently of each other.

The reaction (MIII-III) and the reaction (MII0-III) are generally carried out in a solvent (MIII-III).

Preferably, the solvent (MIII-III) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

Preferably, in reaction (MIII-III) and in reaction (MII0-III) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of compound (CG1MR-III) are used, based on the molar amount of compound of formula (IV) or compound of formula (III 0).

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

After reaction (MIII-III) and reaction (MII0-III), the compound of formula (III) or the compound of formula (II), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration, drying, etc. The compound of formula (III) or the compound of formula (II) may be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

The reactions (MIII-IIa) and (MII0-IIa) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

The reaction (MIII-IIa) and the reaction (MIII-IIa) are generally carried out in a solvent (MIII-IIa).

Preferably, the solvent (MIII-IIa) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

Preferably, in reaction (MIII-IIa) and reaction (MII0-IIa) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of compound (CG1MR-IIa) are used, based on the molar amount of compound of formula (IV) or compound of formula (III 0).

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

After reaction (MIII-IIa) and reaction (MII0-IIa), the compound of formula (IV-IIa) or the compound of formula (III0-IIa), respectively, can be isolated by standard methods such as washing, extraction, filtration, concentration, drying, and the like. The compound of formula (IV-IIa) or the compound of formula (III0-IIa) can be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

The reactions (MIII-IIb) and (MII0-IIa) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other. .

Preferably, the reactions (MIII-IIb) and (MII0-IIb) are carried out in the presence of a Compound (COUPADD).

Compounds (coupasdd) are coupling additives routinely used in peptide chemistry for coupling amino acids into peptides through the formation of amide bonds. Preferably, the compound (coupasdd) is selected from DCC, EDC and mixtures thereof.

Preferably, in the reactions (MIII-IIb) and (MII0-IIb) from 1 to 20 molar equivalents, more preferably from 1 to 10 molar equivalents, even more preferably from 1 to 5 molar equivalents of the Compound (COUPADD) are used, said molar equivalents corresponding to the molar amount of the compound of the formula (IV-IIa) or the compound of the formula (III 0-IIa).

The reactions (MIII-IIb) and (MII0-IIb) are generally carried out in a solvent (MIII-IIb).

Preferably, the solvent (MIII-IIb) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF and mixtures thereof.

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

Preferably, in the reactions (MIII-IIb) and (MII0-IIb) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of the compound of formula (HOSu) are used, based on the molar amount of the compound of formula (IV-IIa) or of formula (III0-IIa), respectively.

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

After reactions (MIII-IIb) and (MII0-IIb), the compound of formula (III) or the compound of formula (II), respectively, can be isolated by standard methods such as washing, extraction, filtration, concentration and drying. The compound of formula (III) or the compound of formula (II) may be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

The reactions (MIII-IIc) and (MII0-IIc) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the specification, with individual reaction parameters for each of the two reactions being selected independently of each other.

The reactions (MIII-IIc) and (MII0-IIc) are generally carried out in a solvent (MIII-IIc).

Preferably, the solvent (MIII-IIc) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

Preferably, in the reactions (MIII-IIc) and in the reactions (MII0-IIc) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of compound (CG1MR-IIa) are used, based on the molar amount of compound of formula (IV) or compound of formula (III0), respectively.

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

The reactions (MIII-IIc) and (MII0-IIc) are generally carried out in the presence of a base (MIII-IIc).

Preferably, the base (MIII-IIc) is selected from: potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures thereof.

Preferably, in reactions (MIII-IIc) and (MII0-IIc) 0.5 to 50 molar equivalents, more preferably 1 to 20 molar equivalents, even more preferably 2 to 10 molar equivalents of base (MIII-IIc) are used, based on the molar amount of compound of formula (IV) or compound of formula (III0), respectively.

After reactions (MIII-IIc) and (MII0-IIc), the compound of formula (III) or the compound of formula (II), respectively, can be isolated by standard methods such as washing, extraction, filtration, concentration and drying. The compound of formula (III) or the compound of formula (II) may be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

Reaction (MIII-I) and reaction (MII0-I) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

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

The reaction (MIII-I) and the reaction (MII0-I) are usually carried out in a solvent (MIII-I).

Preferably, the solvent (MIII-I) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF and mixtures thereof.

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

Preferably, in reaction (MIII-I) and reaction (MII0-I) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents of the compound of formula (MA) are used, which correspond to the molar amount of the compound of formula (IV) or the compound of formula (III 0).

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

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

Preferably, from 1 to 20 molar equivalents, more preferably from 1 to 10 molar equivalents, even more preferably from 1 to 5 molar equivalents of compound (coupasdd), based in each case on the molar amount of compound of formula (IV) or compound of formula (III0), are used in reaction (MIII-I) and reaction (MII 0-I).

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

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

Preferably, in reaction (MIII-I) and reaction (MII0-I) 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of the compound of formula (HOSu) are used, which molar equivalents are based in each case on the molar amount of the compound of formula (IV) or the compound of formula (III 0).

After reaction (MIII-I) and reaction (MII0-I), the compound of formula (III) or the compound of formula (II), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration and drying. The compound of formula (III) or the compound of formula (II) may be purified before or after isolation, respectively, preferably by chromatography or crystallization from a suitable solvent.

Reaction (MIV) and reaction (MV0) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

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

The reaction (MIV) and the reaction (MV0) are usually carried out in a solvent (MIV).

Preferably, the solvent (MIV) is selected from: water, methanol, ethanol, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol, and a mixture thereof.

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

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

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

The compound (IV) used in the reaction (MIV) and the compound (IV) used in the reaction (MV0) may be the same or different independently from each other.

Preferably, in reaction (MIV) and reaction (MV0), 1 to 50 molar equivalents, more preferably 1 to 20 molar equivalents, even more preferably 2 to 10 molar equivalents of compound (IV) are used, which molar equivalents are based in each case on the molar amount of compound of formula (V) or compound of formula (Va).

Reaction (MIV) and reaction (MV0) may be carried out in the presence of a salt (MIV), which may be selected from lithium chloride, calcium chloride, aluminum chloride, zinc chloride and mixtures thereof.

Preferably, if salt (MIV) is used in reaction (MIV) and reaction (MV0), 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1.5 to 5 molar equivalents of salt (MIV) are used, which are based in each case on the molar amount of compound of formula (V) or compound of formula (Va).

After reaction (MIV) and reaction (MV0), the compound of formula (IV) or the compound of formula (V0), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration and drying. The compound of formula (IV) or the compound of formula (V0), respectively, may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

The reactions (MII0-IVa) and (MIV0a) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the present specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

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

The reactions (MII0-I-IVa) and (MIV0a) are usually carried out in a solvent (MIV0 a).

Preferably, the solvent (MIV0a) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

Preferably, from 1 to 20 molar equivalents, more preferably from 1 to 10 molar equivalents, even more preferably from 1 to 5 molar equivalents of compound (II0-I-IVa) or of compound (RIV0a), based on the molar amount of compound of formula (V0), are used in reaction (MII0-I-IVa) and reaction (MIV0a), respectively.

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

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

Preferably, from 1 to 20 molar equivalents, more preferably from 1 to 10 molar equivalents, even more preferably from 1 to 5 molar equivalents of the compound of formula (HOSu) are used in the reaction (MII0-I-IVa) and in the reaction (MIV0a), said molar equivalents corresponding to the molar amount of compound (II0-I-IVa) or compound (RIV0 a).

After reaction (MII0-I-IVa) and reaction (MIV0a), the compound of formula (III0-I-IVa) or the compound of formula (IV0a), respectively, can be isolated by standard methods such as washing, extraction, filtration, concentration and drying. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

A further subject of the invention is a process (MVa) for the preparation of compounds of formula (Va), as defined in the description, with all its preferred embodiments;

the method (MVa) comprises a step (MVa);

step (MVa) comprises a reaction (MVa) wherein a compound of formula (VI) is reacted with a compound of formula (SGM);

r31 is linked to formula SG by said bond represented by (×) in formula SG and is-OTs, -OMs, -OTf, -Br, -Cl or-I;

r30 is as defined in the specification, and also includes all preferred embodiments thereof;

SG、n4、n3、AAⁿ⁴(3), (R1), (R2) are as defined in the specification, and all preferred embodiments thereof are also included.

Preferably, process (MII) comprises as a further step (MVa) the preparation of the compound of formula (Va).

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

Preferably, the reaction time of the reaction (MVa) is from 1 minute to 168 hours, more preferably from 1 to 144 hours, more preferably from 12 to 120 hours.

The reaction (MVa) is usually carried out in a solvent (MVa).

Preferably, the solvent (MVa) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, and a mixture thereof.

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

Preferably, in the reaction (MVa), 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1.5 to 5 molar equivalents of compound (SGM) are used, based on the molar amount of compound (VI).

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

The reaction (MVa) is usually carried out in the presence of a base (MVa).

Preferably, the base (MVa) is selected from: potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, pyridine, 4-dimethylaminopyridine and mixtures thereof.

Preferably, in the reaction (MVa), 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, more preferably 1.5 to 5 molar equivalents of base (MVa) are used, based on the molar amount of the compound of formula (VI).

After reaction (MVa), the compound of formula (Va) can be isolated by standard methods such as washing, extraction, filtration, concentration and drying. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

Reaction (MVb) and reaction (MIII0) are similar reactions and can be carried out under similar reaction parameter ranges as defined in the specification, the individual reaction parameters of each of the two reactions being selected independently of each other.

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

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

The reaction (MVb) and the reaction (MIII0) are generally carried out in a solvent (MVb).

Preferably, the solvent (MVb) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

Preferably, the amount of solvent (MVb) is 1 to 500 times, more preferably 5 to 50 times, even more preferably 10 to 30 times the weight of the compound of formula (Va) or the compound of formula (IV 0).

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

After reaction (MVb) and reaction (MIII0), the compound of formula (V) or the compound of formula (III0), respectively, may be isolated by standard methods such as washing, extraction, filtration, concentration and drying. They may be isolated in the protic or aprotic form of their salts.

Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

Another subject of the present invention is a process for the preparation of the compounds of formula (VI), as defined in the present description, also with all its preferred embodiments;

the Method (MVI) comprises a step (MVIa) and optionally a step (MVIb);

said n3AA in step (MVIa)ⁿ⁴(ii) is attached sequentially to the compound of formula (VII-1) by a peptide coupling reaction and then to the corresponding product of the peptide coupling reaction as described previously;

step (MVIb) includes reaction (MVIb) in which AA represented by (3) in formula (VI)ⁿ³Reacting said N-terminal amino group of (a) with the compound NTermProt;

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

PGNPrec is AA represented by (3) in the formula (VI)ⁿ³An agent for introducing PGN to the N-terminal amino group of (a);

GRPEG, m4, R3 and PGN have the same definitions as above, and also all of their preferred embodiments;

n4、n3、AAⁿ⁴and (3) as defined in the specification, and all of its preferred embodiments.

Preferably, process (MII) comprises as further steps a step (MVa) in which the compound of formula (Va) is prepared and a step (MVIa) and optionally a step (MVIb) in which the compound of formula (VI) is prepared.

The compound of the formula (VII-1) is a known compound and can be produced by a known method.

For simplicity, in this context, AAⁿ⁴Both the amino acid residues used for covalent attachment (e.g. in formula (II)) and the amino acids used in the method (MVIa).

If AAⁿ⁴Having a side chain with a functional group that can be protected by a protecting group commonly used to protect amino acid side chain functional groups.

Preferably NTermProt is Ac₂O。

Preferably, PGNPrec is Boc₂O, FmocCl or CbzCl.

The method (MVIa) is carried out by using methods, parameters and reagents which are customary in peptide synthesis and are well known to the person skilled in the art. The references cited above provide the necessary information.

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

The reaction (MVIb) is generally carried out in a solvent (MVIb).

Preferably, the solvent (MVIb) is selected from: n, N-dimethylformamide, N-dimethylacetamide, N-dimethylsulfoxide, acetonitrile, acetone, 1, 4-dioxane, THF, water, methanol, ethanol and a mixture thereof.

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

More preferably, the process (MVIa) and the reaction (MVIb) are carried out continuously in the same solvent.

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

Preferably, in this process (MVIa), 1 to 20 molar equivalents, more preferably 1 to 10 molar equivalents, even more preferably 1 to 5 molar equivalents of NTermProt are used, based on the molar amount of the compound of formula (VII-1).

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

Following process (MVIa) or reaction (MVIb), the reaction product of process (MVIa) or reaction (MVIb), each of which is the corresponding compound of formula (VI), may be isolated by standard methods such as washing, extraction, filtration, concentration, and drying. Any compound may be purified before or after isolation, preferably by chromatography or crystallization from a suitable solvent.

The compounds of formula (SGM) are well known compounds and can be prepared according to well known methods.

Preferably, the compound of formula (SGM) is selected from the group consisting of compound SGM-II and compound SGM-III.

Preferably, the compound SGM-II is prepared by first reacting a compound of formula (HSGH-II) with Boc₂O, and then with a compound (SGM-II-R31).

The compound (SGM-II-R31) is selected from: p-toluenesulfonyl chloride, p-toluenesulphonic anhydride, methanesulfonyl chloride, methanesulfonic anhydride, trifluoromethanesulfonyl chloride and trifluoromethanesulfonic anhydride, SOCl₂、(COCl)₂、POCl₃、PCl₃、PCl₅、POBr₃、PBr₃、PBr₅N-bromosuccinimide, N-iodosuccinimide, HCl, HBr, HI, and mixtures thereof.

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

Preferably, the compound SGM-III is prepared by first reacting a compound of formula (HSGH-III) with Boc₂O and then reacting with a compound of formula (HSGHReac-1).

The compound of formula (HSGH-II) is preferably a compound of formula (HSGH-II-1) or a compound of formula (HSGH-II-2).

The compound SGM-II is preferably a compound of formula (SGM-II-1) or a compound of formula (SGM-II-2).

The compound of formula (HSGH-III) is preferably a compound of formula (HSGH-III-1) or a compound of formula (HSGH-III-2).

The compound SGM-III is preferably a compound of formula (SGM-III-1) or a compound of formula (SGM-III-2).

The compounds of formula (HSGH-II) and (HSGH-III) are well known compounds, can be prepared according to well known methods, and are often commercially available.

The compounds of formula (CG1MR-IV), the compounds of formula (CG1MR-III), the compounds of formula (CG1MR-IIa), the compounds of formula (HOSu) and the compounds of formula (COUPADD) are well known compounds, can be prepared by well known methods, and are commonly commercially available.

Preferably, the compound of formula (CG1MR-IV) is prepared by reacting a compound of formula (CG1MR-IV-OH) with a compound of formula (HOSu);

the compound of formula (HOSu) is as defined in the specification;

preferably, the compound of formula (CG1MR-IV-OH) is prepared by reacting a compound of formula (MA) with a compound of formula (AC);

the compounds of formula (MA) and m30 are as defined in the specification, with all its preferred embodiments also.

Any of the reactions defined above may be carried out under similar reaction conditions, with the reaction parameters being selected independently of each other for each of these reactions:

with respect to the pressure: any of the reactions defined above may be carried out under vacuum, at atmospheric pressure or even at pressures, for example, up to 10 bar, preferably they are carried out at atmospheric pressure;

with respect to the temperature: preferably, the reaction temperature of any of the above defined reactions is from-20 to 100 ℃, more preferably from 0 to 75 ℃, even more preferably from 10 to 50 ℃;

with respect to the reaction time: the reaction time of any of the above defined reactions is from 1 minute to 168 hours, more preferably from 0.5 to 24 hours, even more preferably from 1 to 12 hours;

with the proviso that no further description is given for any of the reactions defined above.

In particular, the compounds of formula (I) are selected from: a compound of formula (10), a compound of formula (11), a compound of formula (12-101), a compound of formula (13), a compound of formula (14), a compound of formula (15-102), and a compound of formula (16);

monoclonal anti-interleukin-1 beta antibodies produced in mice

Monoclonal anti-interleukin-1 beta antibodies produced in mice

Wherein doxorubicin is a compound of formula (DOXO) which is linked through an amino group represented by (d1) in formulae (10), (11), (12-101), (13), (14), (15-102) and (16) and in formula (DOXO), respectively.

In particular, the compound of formula (II) is selected from: a compound of formula (20), a compound of formula (21), a compound of formula (22), a compound of formula (23), a compound of formula (24), a compound of formula (25), a compound of formula (26), a compound of formula (20-CAMPTO), a compound of formula (21-CAMPTO), a compound of formula (22-CAMPTO), a compound of formula (23-CAMPTO) and a compound of formula (21-TAXO-t 1-1);

wherein,

doxorubicin is a compound of formula (DOXO) linked through an amino group represented by (d1) in formulae (20), (21), (22), (23), (24), (25), and (26), respectively, and in formula (DOXO);

camptothecin is a compound of formula (CAMPTO) linked through a hydroxyl group represented by (c1) in formulae (20-CAMPTO), (21-CAMPTO), (22-CAMPTO), (23-CAMPTO) and in formula (CAMPTO), respectively;

taxo-t1-1 is a compound of formula (TAXO) which is linked through a hydroxyl group represented by (t1) in formula (21-TAXO-t1-1), formula (TAXO-t1-1) and in formula (TAXO).

In particular, the compound of formula (IIc) is a compound of formula (20 c).

In particular, the compound of formula (III) is selected from: a compound of formula (30), a compound of formula (31), a compound of formula (32), a compound of formula (33), a compound of formula (34), a compound of formula (35) and a compound of formula (36).

In particular, the compound of formula (III0) is a compound of formula (300);

wherein camptothecin is a compound of formula (camp to) linked through the hydroxy group represented by (c1) in formula (300) and in formula (camp to), respectively.

In particular, the compounds of formula (III0-I-IVa) are compounds of formula (320).

In particular, the compound of formula (IV) is selected from: a compound of formula (40), a compound of formula (41), a compound of formula (42), a compound of formula (43), a compound of formula (44), a compound of formula (45) and a compound of formula (46).

In particular, the compound of formula (IV0) is a compound of formula (400);

wherein,

camptothecin is a compound of formula (camp to) linked through the hydroxy group represented by (c1) in formula (400) and in formula (camp to), respectively.

In particular, the compound of formula (IV0a) is a compound of formula (400 a).

In particular, the compound of formula (V) is selected from: a compound of formula (50), a compound of formula (50-1), a compound of formula (51-1), a compound of formula (52-1), a compound of formula (53-1), a compound of formula (54-1), a compound of formula (54-2), a compound of formula (54-3), a compound of formula (55-1), a compound of formula (56) and a compound of formula (56-1).

In particular, the compound of formula (V0) is a compound of formula (500).

In particular, the compound of formula (VI) is selected from: the compound of the formula (6), the compound of the formula (6-1), the compound of the formula (6-2), the compound of the formula (6-3), the compound of the formula (6-4), the compound of the formula (6b-1), the compound of the formula (6b-2), the compound of the formula (6b-3), the compound of the formula (6b-4), the compound of the formula (6c) and the compound of the formula (6-5).

A further subject of the invention is a compound selected from: compounds of formula (I), compounds of formula (II), compounds of formula (IIc), compounds of formula (III), compounds of formula (IV-IIa), compounds of formula (V), compounds of formula (Va), compounds of formula (VI), compounds of formula (III0-IIa), compounds of formula (III0), compounds of formula (IV0), compounds of formula (IV0a), compounds of formula (V0), and compounds of formula (III 0-I-IVa); these compounds are as defined in the specification, with all of their preferred embodiments.

Further subject of the present invention are compounds selected from the group consisting of compounds of formula (10), compounds of formula (11), compounds of formula (12-101), compounds of formula (13), compounds of formula (14), compounds of formula (15-102), compounds of formula (16), compounds of formula (20c), compounds of formula (20), compounds of formula (21), compounds of formula (22), compounds of formula (23), compounds of formula (24), compounds of formula (25), compounds of formula (26), compounds of formula (20-CAMPTO), compounds of formula (21-CAMPTO), compounds of formula (22-CAMPTO), compounds of formula (23-CAMPTO), compounds of formula (21-TAXO-t1-1), compounds of formula (30), compounds of formula (31), compounds of formula (32), compounds of formula (33), A compound of formula (34), a compound of formula (35), a compound of formula (36), a compound of formula (300), a compound of formula (320), a compound of formula (40), a compound of formula (41), a compound of formula (42), a compound of formula (43), a compound of formula (44), a compound of formula (45), a compound of formula (46), a compound of formula (400a), a compound of formula (50-1), a compound of formula (51-1), a compound of formula (52-1), a compound of formula (53-1), a compound of formula (54-1), a compound of formula (54-2), a compound of formula (54-3), a compound of formula (55-1), a compound of formula (40), a compound of formula (41), a compound of formula (56), a compound of formula (56-1), a compound of formula (500), a compound of formula (6-1), a compound of formula (6-2), a compound of formula (6-3), a compound of formula (6-4), a compound of formula (6b-1), a compound of formula (6b-2), a compound of formula (6b-3), a compound of formula (6b-4), a compound of formula (6c) and a compound of formula (6-5); these compounds are as defined in the specification.

Another subject of the present invention is the use of a compound of formula (I) as defined in the present description, and as defined in all their preferred embodiments, for the preparation of a pharmaceutical composition or a medicament.

A further subject of the present invention is a pharmaceutical composition or medicament, wherein said pharmaceutical composition and medicament comprise a compound of formula (I) as defined in the present description, and also in all their preferred embodiments.

A further subject of the present invention is a compound of formula (I), a pharmaceutical composition or a medicament for its use in the treatment of a disease or a disorder, preferably in the treatment of cancer, wherein said pharmaceutical composition and said medicament comprise a compound of formula (I), said compound of formula (I) being as defined in the present description and also as defined in all its preferred embodiments.

The compounds of formula (II) can be readily covalently linked to a ligand LI. Surprisingly, the protein drug conjugates according to the present invention comprising a linking group CG2, CG2 being derived from o-hydroxy-p-aminobenzyl alcohol and comprising a linear peptide residue, in particular the compounds of formula (I), show enhanced plasma stability and release the drug without chemically modifying the drug, and furthermore they show good water solubility and low aggregation.

Detailed Description

Abbreviations

DCM dichloromethane

DIBAL-H diisobutylaluminum hydride

DIPEA N, N-diisopropylethylamine

DMA N, N-dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF N, N-dimethylformamide

EDTA ethylene diamine tetraacetic acid

EEDQ 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline

ESI-MS electrospray ionization mass spectrometry

EtOAc ethyl acetate

HIC hydrophobic interaction chromatography

MeCN acetonitrile

NAP-25 column GE Healthcare NAP-25 column is Sephadex^TMG-25 DNA grade prepackaged disposable column and operation only requires gravity

NMR nuclear magnetic resonance

PE Petroleum Ether

Retention factor of Rf in TLC

RP-HPLC reversed phase HPLC

RT Room temperature

SAFC Sigma Aldrich Fine Chemicals

SEC-HPLC size exclusion chromatography HPLC

TBTU 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethylammonium tetrafluoroborate

THF tetrahydrofuran

TFA trifluoroacetic acid

TLC thin layer chromatography

Raw material

The compound doxorubicin of formula (DOXO), available as the hydrochloride salt from the scientific and technological development company, Mitsuo, Beijing

Daunomycin (daunorubicin) is commercially available as the hydrochloride salt from Aldrich

The PBS used in the PBS experiment had the following composition, KH₂PO₄144mg/L, NaCl 9000mg/L and Na₂HPO₄:795mg/L

Example 1

Concentrated sulfuric acid (30.0 ml) was added dropwise to a mixture of p-aminosalicylic acid (15.0 g) and methanol (113.0 ml) at 0 ℃. The resulting mixture was heated to reflux and stirred for 2 hours to form a homogeneous solution. The reaction mixture was then cooled to room temperature. Water (360 ml) was added followed by solid sodium bicarbonate until pH 7. The resulting mixture was filtered, the wet cake was washed with water (3 times 80 ml each) and dried under vacuum at 55 ℃ to give 14.7 g of the compound of formula (VII-1) as a solid (yield 89%).

¹H NMR(400MHz，CDCl₃20 ℃ C. 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)+

Example 2

To a mixture of H-Cit-OH (40.0 g, 1.0 eq) and sodium carbonate (50.0 g, 2.0 eq) in water (300 ml) and THF (150 ml) was added dropwise a solution of di-tert-butyl dicarbonate (60.0 g, 1.2 eq) in THF (100 ml) over 1 hour. The resulting mixture was stirred at RT overnight. After that, the suspension was washed (2 times with 150 ml each time) with PE (polyethylene) and the mixture was concentrated under vacuum to about 300 ml. The mixture was acidified to pH 2 with 4.0M aqueous potassium hydrogen sulfate and then extracted with EtOAc (5 times 150 ml each). The organic phases were combined and washed with saturated brine (100 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness to give 52.0 g of Boc-Cit-OH as a white solid (yield 83%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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)+

Example 3

EEDQ (72.0 g, 1.5 eq) and Boc-Cit-OH (53.0 g, 1.0 eq) prepared according to example 2 were added to THF (400 mL). To this mixture was then added the compound of formula (VII-1) (49.0 g, 1.5 eq) prepared according to example 1. Will obtainThe mixture was stirred at room temperature for 14 hours. The reaction mixture was then diluted with water (500 ml) and extracted with EtOAc (3 times 250 ml each). The organic phases were combined and washed with 1.0M aqueous citric acid (2 times 150 ml each time) and saturated brine (150 ml). Then, using anhydrous Na₂SO₄The organic phase was dried, then concentrated to dryness and the crude product was purified by silica gel column chromatography (eluent PE: EtOAc ═ 4:1 to 2:1 to 1: 1(v/v) to pure EtOAc) and isolated to give 62.5 g of the compound of formula (6-4) as a yellow solid (yield 76%).

Analysis by silica gel TLC: EtOAc was eluent (Rf ═ 0.3, UV 254).

¹H NMR(400MHz，CDCl₃20 ℃)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)+

Example 4

The compound of formula (6-4) (62.0 g, 1 equivalent) prepared according to example 3 was suspended in a solution of 15% (w/w) hydrochloric acid in 1, 4-dioxane (100 ml), and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated in vacuo to give 51.6 g of the compound of formula (6-3) as the HCl salt as a white solid (yield 98%).

Example 5

The compound of formula (6-3) (75.6 g, 1.0 eq) as the HCl salt prepared in example 4, Boc-L-Val (43.0 g, 1.0 eq) and TBTU (135.5 g, 2.0 eq) were dissolved in DMF (250 ml). Then, DIPEA (71.2 g, 2.6 equivalents) was added. The resulting solution was stirred at room temperature for 17 hours. Then will be reversedThe mixture was diluted with water (750 ml), extracted with EtOAc (5X 200 ml) and the organic phases combined and washed with 1.0M NaHCO₃The aqueous solution was washed (3 times with 300 ml each) and then with saturated brine (150 ml). The organic phase was collected and concentrated to dryness, then the crude product was purified by chromatography on silica gel (eluent DCM: methanol MeOH ═ 20:1 to 10:1 to 7: 1(v/v) and isolated to give 62.0 g of compound of formula (6-2) as a solid (yield 57%).

Analysis by silica gel TLC: DCM: MeOH ═ 6:1(V/V) as eluent (Rf ═ 0.4, UV254)

¹H NMR(400MHz，DMSO-d₆20 ℃ 0.84(6H, dd, J1 ═ 17.2Hz, J2 ═ 6.8Hz), 1.39(9H, s), 1.42 to 1.51(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 ═ 2.0), 7.73(1H, d, J ═ 8.8Hz), 7.34H, 1H, d, 8.8H, 1H, d, 8H, 1.08 Hz, 1.8H, 1H, d, 8Hz, 1.8H, 1.

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

Example 6

The compound of formula (6-2) (62.0 g, 1 equivalent) prepared according to example 5 was suspended in a solution of 15% (w/w) hydrochloric acid in 1, 4-dioxane (200 ml), and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated in vacuo to give 52.4 g of the compound of formula (6-1) as an HCl salt as a white solid (yield 97%).

Example 7

The compound of formula (6-1) as an HCl salt prepared according to example 6 (52.4 g, 1.0 eq), acetic anhydride (60.0 g, 5.0 eq), pyridine (100.0 g, 11.0 eq), and methanol (150 ml) were mixed and stirred at room temperature for 7 days. The suspension was filtered and the resulting wet cake was washed with MeOH (4 times 200 ml each) and then dried under vacuum to give 32.9 g of the compound of formula (6) as a white solid (yield 62%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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, J, 8.8Hz), 7.41(1H, d, J ═ 8.8Hz), 7.8H, 8H, 1.8Hz, 1.8H, 8H, 1.8Hz, 1H, 8H, 1.6Hz, 7.8H, 8H, 1.8Hz, J, 1.8 Hz.

ESI-MS:466.3(M+H)+，931.3(2M+H)+

Example 8

The compound of formula (MA) (5.5 g, 1.0 eq) and β -alanine (5.0 g, 1.0 eq) were stirred in DMF (30.0 ml) under nitrogen for 2 hours. The mixture was then cooled to 0 ℃. The compound of formula (HOSu) (8.0 g, 1.3 eq.) and DCC (24.0 g, 2.0 eq.) were added. The reaction mixture was then warmed to room temperature and stirred at room temperature overnight. The reaction mixture was then filtered, the resulting wet cake was washed with DMF (40.0 ml), the organic phases were combined, then diluted with water (120 ml) and extracted with DCM (3 times 50 ml each). The organic phases were combined, washed with water (50 ml), then 5% (w/w) aqueous sodium bicarbonate (50 ml), then saturated brine (50 ml). The organic phase was dried over anhydrous sodium sulfate and then concentrated until a solid began to precipitate. Then, PE (20 ml) was added to the mixture, and the resulting mixture was stirred at room temperature for 10 minutes. The mixture was then filtered and the wet cake was washed with PE (20 ml) and then dried under vacuum at 40 ℃ overnight to give 4.0 g of the compound of formula (CG1MR-IV-1) as a white solid (yield 27%).

¹H NMR(400MHz，CDCl₃，20℃)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)+

Example 9

To a mixture of the compound of formula (HSGH-II-1) (10.0 g, 1.0 eq), methanol (50 mL) and Et3N (10.7 g, 1.1 eq) was added Boc dropwise₂O in MeOH (22.8 g, 1.1 eq Boc₂O in 50 ml of methanol). The resulting mixture was then stirred at room temperature for 15 hours and then dried under vacuum to give 20.0 g of the compound of formula (Boc-HSGH-II-1) as a pale yellow oil (quantitative yield).

¹H NMR(400MHz，CDCl₃20 ℃ 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).

Example 10

DMAP (0.366 g, 0.1 eq) was added to a mixture of the compound of formula (Boc-HSGH-II-1) (6.17 g, 1 eq), pyridine (2.86 g, 1.2 eq) and DCM (30 ml) prepared according to example 9 at 0 ℃. A mixture of tosyl chloride (6.31 g, 1.1 eq) and DCM (45ml) was added dropwise. The resulting mixture was stirred at room temperature for 8 days. The resulting mixture was then poured into DCM (100 ml), the resulting mixture was washed with water (1 time, with 100 ml), and the organic phase was dried over anhydrous sodium sulfate. The resulting solution was further concentrated and purified by silica gel chromatography (PE: EtOAc: 6:1 to PE: EtOAc: 1 (v/v)) to give 8.1 g of the compound of formula (SGM-II-1) as a colorless oil (yield 75%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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, J, 8.8Hz), 7.41(1H, d, J ═ 8.8Hz), 7.8H, 8H, 1.8Hz, 1.8H, 8H, 1.8Hz, 1H, 8H, 1.6Hz, 7.8H, 8H, 1.8Hz, J, 1.8 Hz.

Example 11

A compound of formula (6) prepared according to example 7 (3.00 g, 1 equivalent), a compound of formula (SGM-II-1) prepared according to example 10 (4.65 g, 2 equivalents), K₂CO₃A mixture of (1.82 g, 2eq) and anhydrous DMF (30 ml) was heated to 50 ℃ and stirred under nitrogen for 7 days. The reaction mixture was then concentrated to dryness under vacuum. Methanol (30 ml) was added to the residue, and the resulting mixture was stirred for 10 minutes, followed by filtration. The wet cake was washed with methanol (3 times 10 ml each). The organic phases were combined and then concentrated. The residue was purified by silica gel column chromatography (DCM: methanol ═ 7:1 (v/v)) to give 3.40 g of the compound of formula (50-1) as a pale yellow solid (yield 81%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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)+

Example 12

A mixture of the compound of formula (50-1) (500 mg) prepared according to example 11, and a solution of 10% (w/w) HCl in 1, 4-dioxane (10 ml) was stirred at room temperature for 15 hours. The solvent was removed in vacuo to give 431 mg of the compound of formula (50) as the HCl salt as a yellowish solid (quantitative yield).

ESI-MS:553.5(M+H)+，1105.2(2M+H)+

Example 13

To a mixture of compound of formula (50) as the HCl salt prepared according to example 12 (1.10 g, 1.0 eq) and anhydrous THF (20 ml) at-30 ℃ was added a solution of DIBAL-H in hexane (1M, 11.9ml, 6.0 eq). The resulting mixture was then heated to 0 ℃ and stirred under nitrogen for 15 hours. Methanol (2.0 ml) was then added. Saturated aqueous sodium potassium tartrate (10 ml) was then added to the mixture and the mixture was stirred at room temperature for 30 minutes. The resulting mixture was evaporated to dryness to give a white residue which was washed with methanol (5 times 10 ml each). The combined organic phases were concentrated and purified by silica gel column chromatography (DCM: MeOH: Et3N ═ 65: 33: 2 (v/v)) to give 0.65g of compound of formula (40) as a white solid (yield 72%).

¹H NMR(400MHz，DMSO-d₆60 ℃ C. 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)+

Example 14

To a mixture of the compound of formula (40) (500.2 mg, 1.0 eq) prepared according to example 13, the compound of formula (CG1MR-IV-1) (281.0 mg, 1.1 eq) prepared according to example 8 and DMF (9.5 ml) was added DIPEA (140.3 mg, 1.1 eq) at room temperature. The resulting mixture was stirred at room temperature for 17 hours. DMF was then removed under vacuum to give a pale yellow residue. The residue was then mixed with acetone (10 ml) and stirred at room temperature for 18 hours. The mixture was filtered and the wet cake was washed with acetone (2 times 5ml each) and then dried under vacuum to give 515.0 mg of the compound of formula (30) as a pale yellow solid (yield 80%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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, J36 ═ 11.2 Hz), 7.7H, 7.7H, 7.8Hz), 7.7H, 7.7H, 7.6H, 7H, 7.6H, 7.8H, 7H, 7.7H, 7H, 7.7.7.8H, 7H, 7.7H, 7.8H, 7H, 7.7.7.7.7H, 7H, 7.8H, 7H, 7.8H, 7H, 7.7H, 7H, 7.7.7H, 7H, 7.7.7, 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).

Example 15

A mixture of the compound of formula (30) (400 mg, 1.0 eq), DIPEA (231.0 mg, 3.0 eq), 4 angstrom molecular sieve (800 mg) and anhydrous DMF (8.0 ml) prepared according to example 14 was stirred for 5 min. Then, the compound of formula (II-1) (361.2 mg, 2 equivalents) was added. The resulting mixture was stirred at room temperature for 3 hours. The compound of formula (DOXO) (342.8 mg, 1.0 eq) was then added as a HCl salt and the mixture was stirred for 4 hours. MeCN (40.0 ml) was then added. A precipitate formed and was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 2 times 5ml each). The filtrates were combined and dried in vacuo at 45 ℃ to give a dark red residue. The residue was washed with MeCN (2 times 10 ml each time), then the residue was dissolved in a mixture of acetone and water (20:1 (v/v)) and purified by preparative silica gel TLC (DCM: MeOH ═ 5: 1(v/v), Rf ═ 0.15). The product was extracted from silica gel with a mixture of acetone and water (20: 1(v/v), 6 times 20 ml each) and the combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (5.0 ml), and the mixture was stirred at room temperature for 5 hours, and then filtered. The filter cake was mixed with acetonitrile (5.0 ml) and the mixture was stirred at room temperature for 3 hours. The mixture was then filtered. The filter cake was dried under vacuum at room temperature to give 48.0 mg of the compound of formula (20) as a red solid (yield 7%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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 ═ 7.68, 3.41(2H, t, J ═ 5.6Hz), 3.45 to 3.46 (4H, m), 3.54(2H, t, J ═ 7.68, 3.4.4H, 4.6H, t, 3.6H, 4.6, 4H, 4.6Hz), 3.6H, 4H, 4.6, 4H, 3.6, 4H, 6, 4H, 3.9H, 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)+

Example 16

To a mixture of a compound of formula (HSGH-III-1) (110.16 g, 5 eq.) and dioxane (400 mL) was added Boc dropwise at 0 deg.C₂A mixture of O (22.10 g, 1 eq) in dioxane (200 mL). The resulting mixture was then warmed to room temperature and then stirred for 20 hours. The solvent was then removed under vacuum. The resulting residue was added to water (300 ml) and the resulting mixture was extracted with DCM (2 times 300 ml each time). The organic layer was dried over anhydrous sodium sulfate and then evaporated to dryness. The residue was purified by silica gel column chromatography (DCM: ethyl acetate: 1(v/v) followed by DCM: MeOH: Et 3N: 89: 9: 2 (v/v)) to give 17.7 g of the compound of formula (Boc-HSGH-III-1) as a light yellow oil (yield 55%).

1H NMR(400MHz，CDCl3，20℃)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)。

Example 17

A compound of formula (Boc-HSGH-III-1) (5.00 g, 1.0 eq), K, prepared according to example 18, was added at 0 deg.C over 1 hour₂CO₃A mixture of (4.30 g, 2.0 eq) and DCM (40 ml) was added dropwise a mixture of compound of formula (HSGHReac-1) (2.17 g, 1.2 eq) in DCM (20 ml). The resulting mixture was warmed to room temperature and stirred for 20 hours. The solid was then filtered and the filter cake was washed with DCM (2X 5ml each). The filtrates were combined and evaporated to dryness. The residue was then purified by silica gel column chromatography (eluent: EtOAc: PE ═ 3: 1) to give 5.6 g of the compound of formula (SGM-III-1) as a pale yellow oil (yield 90%).

1H NMR(400MHz，CDCl3，20℃)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).

Example 18

A compound of formula (6) prepared according to example 7 (5.80 g, 1.0 eq.), K₂CO₃(5.18 g, 2.0 equiv.), the compound of formula (SGM-III-1) (9.95 g, 2.0 equiv.) prepared according to preparation 17 and DMF (45ml) were combined. The resulting mixture was then heated to 50 ℃ and stirred under nitrogen for 7 days. The resulting reaction mixture was evaporated to dryness under vacuum. Methanol (40 ml) was added to the residue, and the resulting mixture was stirred for 10 minutes, and then filtered. The wet cake was washed with methanol (3 times 10 ml each). The organic filtrates were collected and combined and then evaporated. The residue was purified by silica gel column chromatography (DCM: MeOH ═ 7:1 (v/v)) to give 7.20 g of the compound of formula (51-1) as a pale yellow solid (yield 70%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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 (6H, dd), 4.8Hz), J3532.8 HzTo 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)+

Example 19

A solution of the compound of formula (51-1) prepared according to example 18 (1.00 g) and 15% (v/v) hydrochloric acid in 1, 4-dioxane (10 ml) was mixed. The mixture was stirred at room temperature for 3 hours. The solvent was removed in vacuo to give 0.99 g of the compound of formula (51) as the HCl salt as a yellowish solid (quantitative yield).

ESI-MS:726.6(M+H)+

Example 20

A mixture of compound of formula (51) as the HCl salt prepared according to example 19 (6.08 g, 1 eq), water (100 ml), calcium chloride (1.86 g, 2.0 eq), and sodium borohydride (1.27 g, 4.0 eq) was stirred at room temperature. Additional sodium borohydride (1.27 g, 4.0 eq. after a total of 15 hours of stirring; 1.27 g, 4.0 eq. after a total of 20 hours of stirring; 1.27 g, 4.0 eq. after a total of 24 hours of stirring) was added in portions. After stirring for a total of 36 hours, methanol (30 ml) was added to the mixture. The reaction mixture was then filtered and the wet cake was washed with methanol (3 times with 10 ml each). The liquid phases were collected, combined and then evaporated to dryness. The residue was purified by silica gel column chromatography (eluent DCM: MeOH: Et3N ═ 65: 33: 2 (v/v)) to give 2.70 g of compound of formula (41) as a light yellow solid (yield 48%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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 ═ 5.7.8H, 7.8H, 8H, s).

ESI-MS:698.7(M+H)+

Example 21

The compound of formula (41) (2.78 g, 1.0 eq) prepared according to example 20, the compound of formula (CG1MR-IV-1) (1.18 g, 1.1 eq) prepared according to example 8, and DMF (30 ml) were mixed at room temperature. DIPEA (0.58 g, 1.1 eq) was then added. The resulting mixture was stirred at room temperature for 16 hours. DMF was then removed under vacuum to give a pale yellow residue. The residue was then mixed with acetone (30 ml) and the mixture was stirred at room temperature for 5 hours. The mixture was then filtered and the wet cake was washed with acetone (2 times 15ml each) and then dried under vacuum to give 2.55 g of compound of formula (31) as a pale yellow solid (yield 75%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 ═ 6.8), 4.35 to 4.40(1H, t, J ═ 7.2H, 5H, 5.5H, 7.5H, 5H, 5.5H, 5, 7.5H, 5H, 5H, 5H, d, J ═ 7.6Hz), 9.92(1H, s).

ESI-MS:831.6(M-OH)+，849.4(M+H)+

Example 22

A mixture of the compound of formula (31) (500.4 mg, 1.0 eq), DIPEA (305.7 mg, 4.0 eq), 4 angstrom molecular sieve (500.5 mg) and anhydrous DMF (10.0 ml) prepared according to example 21 was stirred for 5 min. Then, the compound of formula (II-1) (271.1 mg, 1.5 eq) was added. The resulting mixture was stirred at room temperature for 5 hours. Then, the compound of formula (DOXO) as HCl salt (342.8 mg, 1.0 eq) was added and the resulting mixture was stirred for 3.5 hours. MeCN (50.0 ml) was then added. A precipitate formed, was filtered, and washed with MeCN and DMF (5: 1(v/v), 3 times 10 ml each). The filtrates were combined and dried under vacuum at 45 ℃ to give a dark red residue. The residue was dissolved in a mixture of DCM and MeOH (7: 1 (vol/vol)) and purified by preparative silica gel TLC (DCM: MeOH ═ 7:1 (vol/vol), Rf ═ 0.15). The product was extracted from silica gel with a mixture of acetone and water (20: 1(v/v), 5 times 50 ml each), and the combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (30 ml), and the mixture was stirred at room temperature for 18 hours, then filtered. The filter cake was mixed with acetonitrile (10 ml) and the mixture was stirred at room temperature for 3 hours. The mixture was then filtered. The filter cake was dried under vacuum at room temperature to give 100.3 mg of the compound of formula (21) as a red solid (yield 12%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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 ═ 7.2), 3.68 to 3.76(1H, 3.99, 3.19, 4H, 4.19, 4H, 4.33, 4Hz), 4H, 4.19 to 3.4 Hz, 4H, m, 4Hz, 4H, 4.9 Hz, 4H, m, 3.9, 3.2H, m, 3.9, 3.41(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, 13.04), 14H (1 s), 14.6 Hz).

ESI-MS:1417.8(M+H)+

Example 23

The compound of formula (HSGH-III-2) (84.9 g, 5.0 equiv.) and CHCl were added over a period of 2 hours at room temperature₃(500 ml) of the mixture to which Boc was added dropwise₂O (50.0 g, 1.0 eq.) in CHCl₃(200 ml). The resulting solution was stirred at room temperature for 16 hours. The resulting suspension was filtered and the wet cake was washed with DCM (50 ml). The filtrates were combined and evaporated to dryness to give a colorless oil. The oil was then dissolved in DCM (200 ml), washed with water (300 ml) and dried over anhydrous sodium sulfate. The resulting organic phase was then evaporated to dryness and the product was purified by silica gel column chromatography (eluent DCM: MeOH: 20:1 to 5:1 (v/v)) to give 13.0 g of the compound of formula (Boc-HSGH-III-2) (yield 33%).

¹H NMR(400MHz，CDCl₃20 ℃ 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).

Example 24

To a compound of formula (Boc-HSGH-III-2) (11.3 g), K, prepared according to example 23 was added over 40 minutes at 0 deg.C₂CO₃(18.0 g) and DCM (200 ml) of the mixture was added dropwise a mixture of the compound of formula (HSGHReac-1) and DCM (50 ml). The resulting mixture was then warmed to room temperature and stirred for 2 hours. Then, an aqueous citric acid solution (% (by weight) based on the weight of the solution, 180 ml) was added. The organic phase was separated, washed with saturated brine (100 ml), dried over anhydrous sodium sulfate and then evaporated to dryness. The residue was then purified by silica gel column chromatography (eluent PE: EtOAc 2:1 to 1:2 (v/v)) to give 13.9 g of compound of formula (SGM-III-2) as a white solid (yield 89%).

¹H NMR(400MHz，CDCl₃20 ℃ 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).

Example 25

A compound of formula (6) prepared according to example 7 (5.51 g, 1.0 equivalent), a compound of formula (SGM-III-2) prepared according to example 24 (6.02 g, 2.0 equivalents), K₂CO₃A mixture of (3.31 g, 2.0 eq), and anhydrous DMF (30 ml) was heated to 50 ℃ and stirred under a nitrogen atmosphere for 7 days. The reaction mixture was then concentrated to dryness under vacuum. MeOH (40 ml) was added to the residue, and the resulting mixture was stirred for 10 minutes, then filtered. The wet cake was washed with methanol (3 times with 10 ml each). The filtrates were collected, combined and then concentrated. The residue was purified by silica gel column chromatography (eluent DCM: MeOH ═ 7:1 (v/v)) to give 3.8 g of the compound of formula (52-1) as a pale yellow solid (yield 46%).

¹H NMR(400MHz，DMSO-d₆0.88(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)+

Example 26

A mixture of the compound of formula (52-1) (3.40 g) prepared according to example 25, and a solution of 10% (w/w) hydrochloric acid in 1, 4-dioxane (50 ml) was stirred at room temperature for 15 hours. The reaction mixture was then concentrated in vacuo to afford 3.20 g of compound of formula (52) as the HCl salt as a white solid (quantitative yield). The crude mixture was used directly in the next step.

Example 27

A mixture of the compound of formula (52) (3.00 g, 1 eq) prepared according to example 26 as the HCl salt, water (60 ml), calcium chloride (1.09 g, 2.0 eq), and sodium borohydride (0.75 g, 4.0 eq) was stirred at room temperature. Additional sodium borohydride (0.75 g, 4.0 eq. after 15 hours of stirring; 0.74 g, 4.0 eq. after 19 hours of stirring; 0.74 g, 4.0 eq. after 23 hours of stirring) was added in portions. After stirring for a total of 40 hours, methanol (15ml) was added to the mixture. The reaction mixture was then filtered and the wet cake was washed with methanol (3 times 10 ml each). The filtrates were collected, combined and then evaporated to dryness. The residue was purified by silica gel column chromatography (eluent DCM: MeOH: Et3N ═ 65: 33: 2 (v/v)) to give 1.05 g of the compound of formula (42) as a pale yellow solid (yield 39%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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)+

Example 28

The compound of formula (42) (940.4 mg, 1.0 equivalent) prepared according to example 27, the compound of formula (CG1MR-IV-1) (501.2 mg, 1.1 equivalent) prepared according to example 8, and DMF (10 ml) were mixed. Then, DIPEA (247.0 mg, 1.1 eq) was added at room temperature. The resulting mixture was stirred at room temperature for 4 hours. DMF was then removed under vacuum to give a pale yellow residue. The residue was then mixed with acetone (20 ml) and the resulting mixture was stirred at room temperature for 2 hours. The mixture was then filtered and the wet cake was washed with acetone (3 times 5ml each) and then dried under vacuum to give 950.0 mg of the compound of formula (32) as a pale yellow solid (yield 79%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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, 6.92 ═ 7H, 7.5, 7.5.5H, 7H, 7.5.5, 7.5H, 7H, 7.5H, 7H, 8, 5H, 5.5H, 7H, 5H, 1H, 5.5H, 7.5H, 1H, 5H, 8, 1H, 5H, 1H, 5H.

Example 29

A mixture of compound of formula (32) (402.5 mg, 1.0 eq) prepared according to example 28, 4 angstrom molecular sieve (800.0 mg), anhydrous DMF (8.0 ml) and compound of formula (II-1) (347.3 mg, 2.0 eq) was stirred for 5 minutes. Then, DIPEA (297.7 mg, 4.0 eq) was added. The resulting mixture was stirred at room temperature for 5 hours. Then, the compound of formula (DOXO) as HCl salt (332.2 mg, 1.0 eq) was added and the mixture was stirred for 4 hours. MeCN (40.0 ml) was then added. A precipitate formed, which was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 2 times 5ml each). The filtrates were combined and dried under vacuum at 45 ℃ to give a dark red residue. The residue was dissolved in a mixture of DCM and MeOH (10:1 (v/v)) and purified by preparative silica gel thin layer chromatography (DCM: MeOH ═ 7: 1(v/v), Rf ═ 0.15). The product was extracted from silica gel with a mixture of acetone and water (20: 1(v/v), 5 times 50 ml each time), and the combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (20 ml), and the mixture was stirred at room temperature for 2 hours, then filtered. The filter cake was dried under vacuum at room temperature to give 74.0 mg of the compound of formula (22) as a red solid (yield 10%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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, 38H, 4.38H, 4.2H, 5H, 5 brs), 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)+

Example 30

To a compound of formula (HSGH-II-2) (21.24 g, 1.0 eq) and Et in MeOH (100 mL) at 0 deg.C over 1h₃Boc was added dropwise to a mixture of N (21.03 g, 1.1 eq.)₂A solution of O (43.83 g, 1.1 eq) in MeOH (50 ml). The mixture was then warmed to room temperature and stirred for 24 hours. The resulting mixture was then evaporated to dryness under vacuum to give 43.05 g of the compound of formula (Boc-HSGH-II-2) as a yellow oil (quantitative yield).

¹H NMR(400MHz，CDCl₃20 ℃ 1.30 to 1.43(4H, m), 1.45(9H, s), 1.48 to 1.61(4H, m), 1.70(1H, brs), 3.12(2H, q, J ═ 6.8Hz), 3.64(2H, t, J ═ 6.8Hz), 4.57(1H, brs).

Example 31

To a mixture of the compound of formula (Boc-HSGH-II-2) (20.02 g, 1.0 eq), pyridine (8.02 g, 1.1 eq) and dichloromethane (50 ml) prepared according to example 30 at 0 deg.C was added T dropwise over 2 hours_SA mixture of Cl (19.33 g, 1.1 eq) in dichloromethane (75 mL). The resulting mixture was stirred at room temperature for 7 days. The solution was then evaporated to dryness. The residue was washed with a mixture of solvents (PE: ethyl acetate 6:1(v/v), 4 times 100 ml each) and filtered. The filtrates were combined and then evaporated to dryness. The crude product was further purified by silica gel column chromatography (PE: ethyl acetate 6:1 (v/v)) to give 14.20 g of the compound of formula (SGM-II-2) as a white solid (yield 41%).

¹H NMR(400MHz，CDCl₃20 ℃ 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).

Example 32

A compound of formula (6) prepared according to example 7 (9.31 g, 1.0 equivalent), a compound of formula (SGM-II-2) prepared according to example 31 (14.90 g, 2.0 equivalents), K₂CO₃A mixture of (5.65 g, 2.0 eq) and anhydrous DMF (60 ml) was heated to 50 ℃ and stirred under a nitrogen atmosphere for 9 days. The reaction mixture was evaporated to dryness. Methanol (40 ml) was added to the residue and the resulting mixture was stirred for 10 minutes, then filtered and the filter cake was further washed with MeOH (3 times with 15ml each). The filtrates were combined and evaporated to dryness. The residue was purified by silica gel column chromatography to give 6.35 g of the compound of formula (53-1) as a yellowish solid (yield 48%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝12.0Hz，J₂6.8Hz), 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, J ═ 5.24 (1H, dd, J ═ 5.8Hz), and the like₁＝8.6Hz，J₂1.8Hz), 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)⁺

Example 33

A solution of the compound of formula (53-1) (8.10 g) prepared according to example 32 and 10% (w/w) HCl in 1, 4-dioxane (50 ml) was mixed. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed in vacuo to give 9.00 g of compound of formula (53) as the HCl salt as a white solid (quantitative yield).

Example 34

To a mixture of compound of formula (53) as the HCl salt prepared according to example 33 (4.01 g, 1.0 eq) and anhydrous THF (40 ml) at-30 ℃ was added a solution of DIBAL-H in hexane (1M, 51.1 ml, 8.1 eq). The resulting mixture was then heated to 0 ℃ and stirred under a nitrogen atmosphere for 2.5 hours, then allowed to warm to room temperature naturally and stirred for 16 hours. The mixture was then cooled to 0 ℃. Methanol (10 ml) was added. Saturated aqueous sodium potassium tartrate (100 ml) was then added to the mixture and the mixture was stirred at room temperature for 1 hour. The resulting mixture was evaporated to dryness to give a white residue which was mixed with methanol (60 ml) and stirred for 1 hour. The resulting suspension was filtered and the filter cake was further washed with methanol (3 times 30 ml each). The combined filtrates were concentrated and purified by silica gel column chromatography (DCM: methanol: Et)₃N-66: 32: 2 (v/v)) to yield 1.81 g of the compound of formula (43) as a white solid (yield 53%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝10.4Hz，J₂6.8Hz), 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-6.8 Hz), 2.91 to 3.05(2H, m), 3.92(2H, t, J-6.4 Hz), 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-5.6 Hz), 7.14 to 7.17(1H, m), 7.25(1H, d, J-8.0 Hz), 7.32(1H, d, J-2.0 Hz), 7.94(1H, d, J-6 Hz), J-1H, d, J-8.0 Hz), 7.32(1H, d, J-1H, m), J-6H, J-8.0 Hz), 1H, J-d, J-1H, m, 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)⁺

Example 35

A mixture of the compound of formula (43) (1.13 g, 1.0 eq) prepared according to example 34, maleic anhydride (207.3 mg, 1.1 eq), and DMF (10 ml) was stirred at room temperature for 20 hours. To the resulting solution was added the compound of formula (HOSu) (225.1 mg, 1.0 eq) and EDC (in its monohydrochloride form) (745.0 mg, 2.0 eq). The resulting mixture was further stirred at room temperature for 4 days. The mixture was evaporated to dryness and then mixed with acetone (30 ml). The resulting mixture was stirred at room temperature for 20 hours, and then filtered. The filter cake was washed with acetone (3 times 15ml each time) and then dried under vacuum to give 1.10 g of the compound of formula (33) as a pale yellow solid (yield 90%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝10.2Hz，J₂6.8Hz), 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, J ═ 6H, 7.87H, 1H, m), and 7.87(1H, m).

ESI-MS:599.6(M-OH^-)⁺，639.8(M+Na)⁺

Example 36

A mixture of the compound of formula (33) (50.2 mg, 1.0 eq.) prepared according to example 35, 4 angstrom molecular sieve (100.0 mg), anhydrous DMF (1.0 ml) and the compound of formula (II-1) (37.0 mg, 1.6 eq.) was stirred at room temperature for 5 minutes. DIPEA (46.5 mg, 4.4 eq) was then added. The resulting mixture was stirred at room temperature for 3.5 hours. Then, the compound of formula (DOXO) as HCl salt (37.8 mg, 0.86 eq) was added and the mixture was then stirred for 4.5 hours. Then MeCN (5.0 ml) was added. A precipitate formed and was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 2 times with 1 ml each). The filtrates were combined and dried under vacuum at 45 ℃ to give a dark residue. The residue was dissolved in a mixture of DCM and MeOH (10:1, v/v, 3ml) and purified by preparative silica gel TLC (DCM: MeOH ═ 7: 1(v/v), Rf ═ 0.35). The product was extracted from silica gel by a mixture of acetone and water (20:1, v/v, 6 times with 3ml each). The combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was mixed with acetonitrile (5ml), and the mixture was stirred at room temperature for 2 hours, followed by filtration. The filter cake was washed with acetonitrile (2 times 1 ml each time) and then dried under vacuum at 25 ℃ to give 9.0 mg of the compound of formula (23) as a red solid (yield 9%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.84(6H，dd，J₁＝10.2Hz，J₂6.8Hz), 1.13(3H, d, J6.4 Hz), 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, J6.8 Hz), 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, J5, 4.6H, 6H, 5H, 6H, 6.6H, 5H, 6H, 6.5H, 6H, 5H, 6, 5H, 5, 6, 5H, 6H, 5H, 6H, 5H, 6H, 5H, 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)⁺

Example 37

A mixture of Z-L-Val (1.47 g, 0.95 eq), anhydrous DMF (15ml), TBTU (2.97 g, 1.5 eq) and DIPEA (2.00 g, 2.5 eq) was stirred at room temperature for 15 min. To the resulting mixture was added the compound of formula (6-3) (2.23 g, 1.0 eq) as the HCl salt prepared according to example 4. The mixture was further stirred at room temperature for 14 hours and then evaporated to dryness at 45 ℃. Water (150 ml) was then added and the resulting mixture was extracted with EtOAc (3 times 200 ml each). The combined organic phases were washed with 250 ml of saturated brine and then dried over anhydrous sodium sulfate. The resulting solution was concentrated and then purified by silica gel column chromatography (PE: EA ═ 1:2 to DCM: MeOH ═ 10:1 (v/v)) to give 2.92 g of the compound of formula (6-5) as a white solid (yield 85%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.87(6H，dd，J₁＝17.6Hz，J₂6.8Hz), 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, J ═ 1H, m)₁＝8.8Hz，J₂2.0Hz), 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)⁺

Example 38

A compound of formula (6-5) (20.00 g, 1.0 eq.) prepared according to example 37, K, was mixed₂CO₃(9.91 g, 2.0 eq.) and anhydrous DMF (80 mL). The resulting mixture was heated to 50 ℃ and stirred for 0.5 hour. The compound of formula (SGM-II-1) (25.79 g, 2.0 equivalents) prepared according to example 10 was added to the reaction mixture in 4 portions over 2 hours. The reaction mixture was stirred at 50 ℃ for 3 days and then evaporated to dryness at 45 ℃. The residue was washed with MeOH (4 times 20 ml each) and the filtrates combined and then concentrated to dryness. The residue was further purified by silica gel column chromatography (DCM: MeOH ═ 20:1, vol/vol) to give 22.17 g of the compound of formula (54-1) as a white solid (yield 83%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.87(6H，dd，J₁＝17.6Hz，J₂6.8Hz), 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, J ═ 5.4Hz)₁＝8.6Hz，J₂1.56Hz), 7.30 to 7.36(6H, m), 7.48(1H, s), 7.69(1H, d, J-8.6 Hz), 8.15(1H, d, J-7.2 Hz), 10.28(1H, s).

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

Example 39

A mixture of the compound of formula (54-1) (4.80 g, 1.0 eq), prepared according to example 38, methanol (200 ml) and Pd/C (5%, 0.24 g) was degassed three times and then stirred under an atmosphere of hydrogen (6 bar) at 30 ℃ for 18 hours. The resulting mixture was filtered and the filtrate was concentrated to dryness in vacuo to give 3.96 g of the compound of formula (54-2) as a white solid (quantitative yield).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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,j6.0 Hz), 3.75 to 3.77(5H, m), 4.09(2H, t, J4.8 Hz), 4.47(1H, s), 5.45(2H, s), 6.07(1H, t, J5.6 Hz), 6.72(1H, t, J5.2 Hz), 7.24(1H, dd, J)₁＝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)⁺

Example 40

A mixture of N, N-dimethylglycine (0.48 g, 1.0 eq), DMF (15ml) and TBTU (3.01 g, 2.0 eq) was cooled to 0 ℃. The resulting mixture was then stirred for 15 minutes, followed by addition of the compound of formula (54-2) (2.86 g, 1.0 eq) prepared according to example 39. The mixture was then allowed to warm to room temperature and stirred for a further 18 hours. The resulting mixture was then evaporated to dryness. The resulting residue was purified by silica gel column chromatography (DCM: MeOH ═ 7:1 (v/v)) to give 2.78 g of the compound of formula (54-3) as a white solid (yield 85%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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 ═ 5.8), 6.73(1H, t, J ═ 6.73, 7H, 7, J ═ 6.7H, ddh, 1, 7, ddh, 5.7 Hz), 1H, 5.7, J ═ 4.7, ddh, m₁＝8.6Hz，J₂1.8Hz), 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)⁺

EXAMPLE 41

A mixture of the compound of formula (54-3) prepared according to example 40 and a solution of 10% (w/w) HCl in 1, 4-dioxane (50 ml) was stirred at room temperature for 18 hours. The solvent was removed in vacuo to give 5.60 g of compound of formula (54) as the dihydrochloride salt as a white solid (quantitative yield).

ESI-MS：596.4(M+H)⁺，1191.3(2M+H)⁺

Example 42

To a mixture of compound of formula (54) as the dihydrochloride salt prepared according to example 41 (1.00 g, 1.0 eq) and anhydrous THF (15ml) at 0 ℃ was added a solution of DIBAL-H in hexane (1M, 10 ml, 6.7 eq). The resulting mixture was further stirred at 0 ℃ for 1.5 hours. Methanol (10 ml) was then added dropwise. Then, saturated aqueous sodium potassium tartrate solution (30 ml) was added and stirred at room temperature for 1 hour. The resulting mixture was evaporated to dryness. The residue obtained is washed with methanol (3 times 10 ml each). The combined filtrates were concentrated and then purified by silica gel column chromatography (DCM: MeOH: Et)₃N65: 33: 2 (v/v)) to yield 0.43 g of the compound of formula (44) as a white solid (yield 51%).

¹H NMR(400MHz，DMSO-d₆60 deg.c 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), 6H, 1.02 (1H, 7, 15H, ddh, J ═ 6Hz), 1.15H, ddh, 5, J ═ 5.6Hz, 1H, 5, 15H, 15, ddh, m, 1H, 5, J ═ 5,5₁＝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)⁺

Example 43

To a mixture of the compound of formula (44) (1.10 g, 1.0 eq) prepared according to example 42, the compound of formula (CG1MR-IV-1) (0.57 g, 1.1 eq) prepared according to example 8, and DMF (4.0 ml) was added DIPEA (0.28 g, 1.1 eq) at room temperature. The resulting mixture was stirred at room temperature for 18 hours. DMF was then removed under vacuum to give a pale yellow residue, which was then mixed with acetone (20 ml) and stirred at room temperature for 18 h. The mixture was filtered and the filter cake was washed with acetone (2 times 5ml each time) and then dried under vacuum to give 1.11 g of the compound of formula (34) (yield 80%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 4.35 (1H, 4.35, 4H, 7.35, 7.5H, 7.8Hz), 7.7H, 7.5H, 7.7H, 7H, 7.5H, 7, 7.5H, 7.7.5H, 7, 7.5H, 7H, 7.8H, 7.7, 7H, 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)⁺

Example 44

A mixture of compound of formula (II-1) (169.4 mg, 2.0 equiv.), anhydrous DMF (4.0 ml) and 4 angstrom molecular sieves (400.0 mg) was stirred at room temperature for 15 minutes. To the resulting mixture was added DIPEA (107.9 mg, 3.0 equivalents) and the compound of formula (34) prepared according to example 43 (200.0 mg, 1.0 equivalent). The resulting mixture was stirred for an additional 2.5 hours. The compound of formula (DOXO) as HCl salt (161.8 mg, 1.0 eq) was then added and stirred for a further 2 hours. Then, acetonitrile (20 ml) was added. A precipitate formed, which was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 3 times with 3ml each time). The filtrates were combined and dried under vacuum at 48 ℃. The resulting residue was dissolved in a mixture of DCM and MeOH (5: 1(v/v), 5ml) and purified by preparative silica gel TLC (DCM: MeOH ═ 4: 1(v/v), Rf ═ 0.5). The product was extracted from silica gel by a mixture of acetone and water (7: 1(v/v), 6 times with 10 ml each). The combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (5ml), stirred at room temperature for 2 hours and then filtered. The wet cake was dried under vacuum at room temperature to give 7.0 mg of the compound of formula (24) as a red solid (yield 2%)

¹H NMR(400MHz，DMSO-d₆20 ℃)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 ═ 7.69, 3.4H, 3.69 (3.4H, m), 3.4H, 4 m), 3.9H, 4H, 3.6H, 4H, 5.6Hz, 3.5H, 3.45 (4H, 4H, 5H, 4H, 5H, 4H, 3.6, 5H, 3.9, 4H, m, 3.9, 4, 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(2H4.8Hz)，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)⁺

Example 45

A mixture of the compound of formula (VII-1) (26.10 g, 1.5 eq), prepared according to example 1 (THF (150 mL), Boc-L-Lys (Ac) -OH (30.00 g, 1.0 eq), and EEDQ (51.45 g, 2.0 eq) was stirred at room temperature for 24 hours. The mixture was then evaporated to dryness. The residue was purified by silica gel column chromatography (PE: EtOAc ═ 2: 1(v/v) to pure EtOAc) to give 26.00 g of compound of formula (6b-4) as a light yellow solid (yield 57%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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)⁺

Example 46

The compound of formula (6b-4) (26.00 g) prepared according to example 45 was suspended in a solution of 10% (w/w) HCl in 1, 4-dioxane (150 ml) and the resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was then concentrated in vacuo to give 22.05 g of the compound of formula (6b-3) as the HCl salt as a yellow solid (quantitative yield).

ESI-MS：339.2(M+H)+

Example 47

A mixture of the compound of formula (6b-3) prepared according to example 46 as the HCl salt (25.21 g, 1.0 eq), Boc-L-Val-OH (13.25 g, 1.0 eq), TBTU (23.66 g, 1.1 eq, 18 h), DIPEA (19.05 g, 2.2eq) and DMF (100 ml) was stirred at room temperature for 18 h. The resulting mixture was evaporated to dryness and then purified by silica gel chromatography (DCM: MeOH ═ 10:1 to 7:1 (v/v)) to give 30.80 g of the compound of formula (6b-2) as a pale yellow solid (yield 90%).

¹H NMR(400MHz，CDCl₃20 ℃ C.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, J ═ 8.0Hz)₁＝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)⁺

Example 48

The compound of formula (6b-2) (30.80 g) prepared according to example 47 was suspended in a solution of 10% (w/w) HCl in 1, 4-dioxane (150 ml) and the resulting mixture was stirred at room temperature for 23 hours. The reaction mixture was then concentrated in vacuo to give 25.90 g of the compound of formula (6b-1) as the HCl salt as a white solid (96% yield).

ESI-MS：409.0(M+H)⁺

Example 49

A mixture of the compound of formula (6b-1) prepared according to example 48 as the hydrochloride salt (15.32 g, 1.0 eq), methanol (100 ml), pyridine (9.0 ml, 3.2 eq) and acetic anhydride (14.0 ml, 4.3 eq) was stirred at room temperature for 60 hours. The resulting mixture was evaporated to dryness under vacuum. The residue was purified by silica gel column chromatography (DCM: MeOH ═ 15: 1 to 7:1 (v/v)) to give 12.51 g of the compound of formula (6b) as a pale yellow solid (yield 80%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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)⁺

Example 50

A compound of formula (6b) prepared according to example 49 (6.73 g, 1.0 equivalent), a compound of formula (SGM-II-1) prepared according to example 10 (11.50 g, 2.1 equivalents), K₂CO₃A mixture of (4.40 g, 2.1 eq) and anhydrous DMF (60 ml) was heated to 0 ℃ and stirred under a nitrogen atmosphere for 8 days. The resulting mixture was evaporated to dryness and the residue was washed with MeOH (3 times 15ml each). The filtrates were combined and evaporated to dryness. The residue was purified by silica gel column chromatography (DCM: MeOH ═ 15: 1 to 7:1 (v/v)) to give 7.64 g of the compound of formula (55-1) as a white solid (yield 81%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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), 4.2 ℃, (2H, d, J ═ 7.2Hz), 1.25 to 1.40(13H, m), 1.59 to 1.74(2H, m), 1.78(3H, s)4 to 4.34(2H, m), 6.74(1H, t, J ═ 5.4Hz), 7.28(1H, dd, J)₁＝8.6Hz，J₂1.6Hz), 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)⁺

Example 51

The compound of formula (55-1) (7.60 g) prepared according to example 50 was suspended in a solution of 10% (w/w) hydrochloric acid in 1, 4-dioxane (50 ml), and the resulting mixture was stirred at room temperature for 5 hours. The reaction mixture was then concentrated in vacuo to afford 6.72 g of compound of formula (55) as the HCl salt as a white solid (yield 98%).

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

Example 52

To a mixture of compound of formula (55) as the HCl salt prepared according to example 51 (6.00 g, 1.0 eq) and anhydrous THF (50 ml) at 0 ℃ was added a solution of DIBAL-H in hexane (1M, 63.0 ml, 6.0 eq). The resulting mixture was then heated to 0 ℃ and stirred under a nitrogen atmosphere for 6 hours. Methanol (10 ml) was then added. Thereafter, a saturated aqueous solution of sodium potassium tartrate (150 ml) was added to the mixture, and the mixture was stirred at room temperature for 15 hours. The resulting mixture was evaporated to dryness, yielding a white residue, which was further washed with methanol (3 times 50 ml each). The filtrates were combined, concentrated and purified by silica gel column chromatography (DCM: methanol: Et)₃N-75: 25: 2 (v/v)) to yield 3.27 g of the compound of formula (45) as a white solid (yield 61%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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 ═ 4.8.8, 8.8 (1H, 8.8, 8H, 8, 8.8, 1H, 8H, 8, 1H, 8H, 8, 1H.

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

Example 53

To a mixture of the compound of formula (45) prepared according to example 52 (1.20 g, 1.0 eq), the compound of formula (CG1MR-IV-1) prepared according to example 8 (0.69 g, 1.1 eq), and DMF (25ml) was added DIPEA (0.34 g, 1.1 eq) at room temperature. The resulting mixture was stirred at room temperature for 2 hours. DMF was then removed under vacuum to give a pale yellow residue, which was then mixed with acetone (50 ml) and stirred at room temperature for 1 hour. The mixture was filtered and the filter cake was washed with acetone (2 times 10 ml each time) and then dried under vacuum to give 1.12 g of the compound of formula (35) (yield 72%).

¹H NMR(400MHz，DMSO-d₆20 deg.c 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 ═ 5.6), 4.86(1H, t, 6, J ═ 4.7, 7.7, 7, 7.7H, 7.7, 7, 7.7H, 7, 7.7, 7H, 7.8Hz), 1H, 7.7.7H, 7, 7.7H, 7, 7.8Hz, 7H, 7.7H, 7H, 7.7.8, 7H, 7.8, 7H, 7.8Hz, j ═ 6.8Hz), 9.79(1H,s).

ESI-MS:661.5(M+H)⁺，643.6(M-OH^-)⁺

example 54

A mixture of compound of formula (35) (400.0 mg, 1.0 eq), prepared according to example 53, 4 angstrom molecular sieve (800.0 mg), anhydrous DMF (4.0 ml) and compound of formula (II-1) (373.0 mg, 2.0 eq) was stirred at room temperature for 5 minutes. DIPEA (313.5 mg, 4.0 equiv.) was then added. The resulting mixture was stirred at room temperature for 5 hours. Then, the compound of formula (DOXO) as HCl salt (328.0 mg, 0.9 eq) was added and the mixture was stirred for 4 hours. Then, acetonitrile (20 ml) was added. A precipitate formed, which was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 2 times with 10 ml each time). The filtrates were combined and dried in vacuo at 45 ℃ to give a dark residue. The residue was dissolved in a mixture of DCM and MeOH (7: 1, vol/vol, 5ml) and purified by preparative silica gel thin layer chromatography (DCM: MeOH ═ 7:1 (vol/vol), Rf ═ 0.15). The product was extracted from silica gel from a mixture of acetone and water (20:1, v/v, 10 times 20 ml each). The combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (10 ml), and the mixture was stirred at room temperature for 0.5 hour, and then filtered. The filter cake was washed with acetonitrile (5ml) and then dried under vacuum at room temperature to give 100.7 mg of the compound of formula (25) as a red solid (yield 13%).

¹H NMR(400MHz，DMSO-d₆20 ℃)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, 3.03H, 4 s), 4.22H, 4 brs), 4.18H, 14.18H, 4H, m, 3.18, 1.2H, m, 3.2H, m, 3.6Hz, 3.1H, 1H, 3.2H, 14(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)⁺

Example 55

The compound of formula (6-1) prepared according to example 6 as the HCl salt (22.81 g, 1.0 eq), 2- [2- (2-methoxyethoxy) ethoxy ] acetic acid (8.00 g, 0.9 eq) purchased from Aldrich, TBTU (24.00 g, 1.5 eq) and DIPEA (16.00 g) were dissolved in DMF (100 ml). The resulting solution was stirred at room temperature for 20 hours. The reaction mixture was concentrated to dryness, then the residue was purified by silica gel column chromatography (eluent DCM: MeOH: 10:1 to 7:1 (v/v)) to give 12.02 g of the compound of formula (6c) as a pale yellow solid (yield 41%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝24.4Hz，J₂6.8Hz), 1.37(9H, s), 1.39 to 1.52(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, 8J ═ 8H, 7.79(1H, 8J ═ 8Hz), 7.7.8H, 8J ═ 8H, 8Hz, 7.7.7, 8H, J ═ 8Hz, 1H, 8H, 1H, 8, J ═ 8, 1H, 8, H, J ═ 8, 1H, 8, H, J ═ 8, H.

ESI-MS:584.7(M+H)⁺，1167.2(2M+H)⁺

Example 56

A compound of formula (6c) prepared according to example 55 (8.01 g, 1.0 eq), a compound of formula (SGM-III-2) prepared according to example 24 (6.89 g, 2.0 eq), K₂CO₃A mixture of (3.80 g, 2.0 eq) and anhydrous DMF (60 ml) was heated to 50 ℃ and stirred under a nitrogen atmosphere for 7 days. The reaction mixture was then concentrated to dryness under vacuum. Dichloromethane (15ml) was added to the residue and the resulting mixture was purified by silica gel column chromatography (eluent: DCM: MeOH ═ 7:1 (v/v)) to give 5.52 g of the compound of formula (56-1) as a yellowish solid (yield 50%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝24.4Hz，J₂6.8Hz), 1.37(9H, s), 1.39 to 1.52(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, 8J ═ 8H, 7.79(1H, 8J ═ 8Hz), 7.7.8H, 8J ═ 8H, 8Hz, 7.7.7, 8H, J ═ 8Hz, 1H, 8H, 1H, 8, J ═ 8, 1H, 8, H, J ═ 8, 1H, 8, H, J ═ 8, H.

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

Example 57

The compound of formula (56-1) prepared according to example 56 was suspended in a solution of 10% (w/w) HCl in 1, 4-dioxane (60 ml). The resulting mixture was stirred at room temperature for 8 hours, then concentrated to give 4.99 g of the compound of formula (56) as a white solid (quantitative yield).

ESI-MS:698.4(M+H)⁺，1394.6(2M+H)⁺

Example 58

A mixture of the compound of formula (56) as the HCl salt prepared in example 57 (4.91 g, 1.0 eq), water (45ml), calcium chloride (1.49 g, 2.0 eq), and sodium borohydride (1.02 g, 4.0 eq) was stirred at room temperature. An additional amount of sodium borohydride (1.02 g, 4.0 eq. after a total of 2.5 hours of stirring; 1.01 g, 4.0 eq. after a total of 4 hours of stirring; 1.03 g, 4.0 eq. after a total of 6 hours of stirring) was added in portions. After stirring for a total of 22 hours, methanol (20 ml) was added to the mixture. The reaction mixture was then filtered and the wet cake was washed with methanol (3 times 15ml each). The filtrates were collected, combined and then evaporated to dryness. The residue was purified by column chromatography on silica gel (eluent DCM: MeOH: Et)₃N80: 20: 2.5 (v/v)) to yield 1.12 g of the compound of formula (46) as a pale yellow solid (yield 25%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.86(6H，dd，J₁＝22.8Hz，J₂6.8Hz), 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, 8, J ═ 8, 8H, t, J ═ 5.6Hz), 7.22 to 7.27(3H, m), 7.48(1H, J ═ 8, 8H, 8, J ═ 1H, 10, 1.05, H, m), 1H, J ═ 5.6Hz, 1H, m).

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

Example 59

To a mixture of the compound of formula (46) prepared according to example 58 (250.7 mg, 1.0 eq), the compound of formula (CG1MR-IV-1) prepared according to example 8 (103.1 g, 1.1 eq), and DMF (4ml) was added DIPEA (53.6 mg, 1.1 eq) at room temperature. The resulting mixture was stirred at room temperature for 1 hour. Then, DMF was removed under vacuum to give a pale yellow residue, which was then mixed with acetone (5ml) and stirred at room temperature for 1 hour. The mixture was filtered and the filter cake was washed with acetone (3 times with 3ml each) and then dried under vacuum to give 253.7 g of the compound of formula (36) (83% yield).

¹H NMR(400MHz，DMSO-d₆，20℃)0.85(6H，dd，J₁＝23.0Hz，J₂6.8Hz), 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, 6.01(1H, s), 7.8H, 7.5H, 7.5.5H, 7J ═ 8H, 7.5.5.5H, 7H, 7.8, 7H, 7.5.5H, 7J ═ 8H, 7.9 (1H, 8H, 7H, 7.9, 7H, 7J ═ 8, 7.9, 7H, J ═ 8, 7H, 7, 7.9 (1H, 7H, s).

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

Example 60

A mixture of compound of formula (36) (201.3 mg, 1.0 eq), prepared according to example 59 (400.0 mg), 4 angstrom molecular sieve, anhydrous DMF (4.0 ml) and compound of formula (II-1) (146.9 mg, 2.0 eq) was stirred at room temperature for 10 minutes. DIPEA (105.7 mg, 3.3 equiv.) was then added. The resulting mixture was stirred at room temperature for 4.5 hours. Then, the compound of formula (DOXO) as HCl salt (142.1 mg, 1.0 eq) was added and the mixture was stirred for 2.5 hours. Then MeCN (20 ml) was added. A precipitate formed, which was filtered and washed with a mixture of MeCN and DMF (5: 1(v/v), 4 times with 4ml each time). The filtrates were combined and dried in vacuo at 45 ℃ to give a dark residue. The residue was dissolved in a mixture of DCM and methanol (7: 1, vol/vol, 3ml) and purified by preparative silica gel thin layer chromatography (DCM: methanol 7:1 (vol/vol), Rf 0.3). The product was extracted from silica gel from a mixture of acetone and water (10:1, v/v, 6 times 10 ml each). The combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (5ml), and the mixture was stirred at room temperature for 2 hours, followed by filtration. The filter cake was washed with acetonitrile (2 times 2 ml each time) and then dried under vacuum at room temperature to give 33.2 mg of the compound of formula (26) as a red solid (yield 10%).

¹H NMR(400MHz，DMSO-d₆，20℃)0.84(6H，dd，J₁＝23.2Hz，J₂6.8Hz), 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, s), 4.5H, 5H, 5H, 5H, 5H, 5, 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)⁺

Example 61

A mixture of compound of formula (TAXO) (200.0 mg, 1.0 eq.), 4A molecular sieve (100.0 mg), anhydrous DCM (4.0 ml) and compound of formula (II-1) (146.9 mg, 2.0 eq.) was stirred at 0 ℃ for 10 min. Pyridine (28.1 mg, 1.5 eq) was then added to the mixture. The resulting mixture was stirred at 0 ℃ under nitrogen for an additional 41 hours. N, N' -dimethyl-1, 2-ethylenediamine (102.0 mg, 5.0 equivalents) was then added to the mixture at 0 ℃ under nitrogen. After stirring for 2 hours, the mixture was filtered. The filter cake was washed with dichloromethane (1 ml). The filtrates were combined and then evaporated to dryness to give a yellow residue. The residue was further purified by preparative silica gel thin layer chromatography (DCM: methanol ═ 7:1 (v/v)) in isolated yield of 96% to give 220.0 mg of the compound of formula (TAXO-t1-1) as a light yellow solid.

ESI-MS:968.5(M+Na)⁺

Example 62

A mixture of the compound of formula (31) (252.0 mg, 1.2 equivalents) prepared according to example 21, the compound of formula (II-1) (136.0 mg, 1.8 equivalents), 4A molecular sieve (100.0 mg) and anhydrous DMF (1 ml) was stirred at room temperature for 10 minutes. DIPEA (80.1 mg, 2.5 equivalents) was then added. The resulting mixture was stirred at room temperature for 2 hours. Then, the compound of formula (XXXX) prepared according to example 61 is added. The resulting mixture was stirred for 2 hours. Filtering the obtained suspension; the filter cake was further washed with DCM (3 times 10 ml each). The filtrates were combined and then evaporated to dryness at 35 ℃. The resulting residue was mixed with DCM (30 ml) and stirred for 0.5 h. The resulting suspension was filtered and the filter cake was further washed with DCM (2 times 5ml each time). The filtrate was dried under vacuum at room temperature. The crude product was dissolved in CH₂Cl₂MeOH in mixed solvent (8:1 (v/v)) and further purified by preparative silica gel thin layer chromatography (DCM: MeOH ═ 8:1 (v/v)). The product was extracted from the gel by a mixture of THF-water (20: 1(v/v), 6 times with 30 ml each) and dried under vacuum to give the crude product as a white solid. The crude product was then reacted with Et₂O (5ml) was mixed, stirred at 25 ℃ for 0.5 hour, and then filtered. With Et₂The filter cake was O-washed (3 times with 5ml each) and then dried under vacuum at room temperatureDrying, isolated in 21% yield, gave 100.0 mg of the compound of formula (21-TAXO-t1-1) as a white solid.

ESI-MS:1842.1(M+H)⁺，1864.5(M+Na)⁺

Comparative example 1

To a mixture of Boc-Cit-OH (1.00 g, 1.0 eq.), EEDQ (1.35 g, 1.5 eq.) and THF (15mL) prepared according to example 2 was added methyl p-aminobenzoate (0.82 g, 1.5 eq.). The resulting mixture was stirred at room temperature for 14 hours. Then, the solvent was removed under vacuum and the residue was purified by silica gel column chromatography (eluent: PE: EtOAc: 6:1(v/v) followed by DCM: MeOH: 10:1 (v/v)) to give 1.2 g of the compound of formula (Comp-6-4) as a white solid (yield 81%).

Silica gel TLC analysis: eluent EtOAc (Rf ═ 0.35, UV254)

¹H NMR(400MHz，CDCl₃20 ℃ 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

A mixture of the compound of the formula (Comp-6-4) (1.66 g) prepared according to comparative example 1 and a solution of 15% w/w HC1 in 1, 4-dioxane (10 ml) was stirred at room temperature for 3 hours. The reaction mixture was then concentrated in vacuo to give 1.38g of the compound of formula (Comp-6-3) as the HCl salt as a white solid (yield 98%).

Comparative example 3

To a mixture of compound of formula (Comp-6-3) as HCl salt (14.50 g, 1.0 eq), Boc-L-Val (10.05 g, 1.1 eq), TBTU (27.00 g, 2.0 eq) and DMF (80 ml) prepared according to comparative example 2 was added DIPEA (16.31 g, 3.0 eq). The resulting mixture was stirred at room temperature for 16 hours. The mixture was then diluted with water (120 ml) and then extracted with EtOAc (5 times 100 ml each), the organic phases were combined and evaporated to dryness, and the resulting residue was purified by silica gel column chromatography (eluent: DCM: MeOH: 20:1 to 10:1 to 7:1 (v/v)) to give 12.9 g of the compound of formula (Comp-6-2) as a solid (yield 60%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 ═ 7.6, 10.6 Hz), 7.73 to 7.76(2H, m).

Comparative example 4

A mixture of the compound of formula (Comp-6-2) (12.3 g) prepared according to comparative example 3 and a solution of 10% (w/w) hydrochloric acid in dioxane (60 ml) was stirred at room temperature for 3 hours. The mixture was then concentrated in vacuo to give 10.9 g of compound of formula (Comp-6-1) as HCl salt as a white solid (quantitative yield).

Comparative example 5

To a mixture of compound of formula (Comp-6-1) as HCl salt (10.20 g, 1.0 eq) prepared according to comparative example 4 and anhydrous THF (150 ml) was added a solution of DIBAL-H in hexane (1M, 120.0 ml, 6.2 eq) at-20 ℃. The mixture was warmed to room temperature and stirred for 14 hours. MeOH (20 ml) was then added followed by saturated sodium potassium tartrate solution (180 ml), and the mixture was stirred at room temperature for 30 minutes. The resulting mixture was evaporated to dryness to give a white residue, which was washed with MeOH (5 times 50 ml each time). The eluates were combined, concentrated and purified by silica gel column chromatography (eluent DCM: MeOH 10:1 to 7:1 to 5:1 (v/v)) to give 5.10 g of the compound of formula (Comp-10) as a white solid (yield 58%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 ═ 5.8, 11.11H, s).

ESI-MS:380.4(M+H)+，759.2(2M+H)+

Comparative example 6

To a compound of formula (Comp-10) (1.02 g, 1.0 eq.), a compound of formula (CG1MR-IV-2) (0.92 g, 1.1 eq.) prepared according to comparative example 5,

And DMF (15ml) was added DIPEA (0.39 g, 1.1 eq) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. DMF was then removed under vacuum to give a pale yellow residue. The residue was then mixed with acetone (20 ml), and the mixture was stirred at room temperature for 5 hours. The mixture was filtered and the wet cake was washed with acetone (2 times 10 ml each) and then dried under vacuum to give 1.05 g of compound of formula (Comp-11) as a pale yellow solid (yield 67%).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 ═ 5.6), 7.00(2H, s), 7.23.7 (7H, 7.81, J ═ 8H, 7.8Hz), 7.8H, J ═ 8H, 8Hz, J ═ 4.8Hz, 7.8H, 8Hz, J ═ 8H, 8Hz, 7.8H, J ═ 8Hz, 8H, 8, J ═ 8H, 8H, 8, J ═ 8H, J ═ 8, 8H, 8, J.

Comparative example 7

A mixture of compound of formula (Comp-11) (350.0 mg, 1.0 equiv) prepared according to comparative example 6, DIPEA (307.0 mg, 3.9 equiv), 500.0 mg of 4A molecular sieves and anhydrous DMF (5.0 mL) was stirred for 25 minutes. Then, the compound of formula (II-1) (280.0 mg, 1.5 equiv) was added. The resulting mixture was stirred at room temperature for 3 hours. Then, the compound of formula (DOXO) as HCl salt (368.8 mg, 1.0 eq) was added and the mixture was stirred for 12 hours. Then MeCN (25.0 ml) was added to the reaction mixture. A precipitate formed, which was filtered and washed with a mixture of MeCN and DMF (5:1 (vol/vol), 3 times with 5ml each). The filtrates were combined and dried under vacuum at 45 ℃ to give a dark red residue. The residue was dissolved in a mixture of DCM and methanol (7: 1 (v/v)) and purified by preparative silica gel TLC (DCM: MeOH ═ 7: 1(v/v) and Rf ═ 0.15). The product was extracted from silica gel from a mixture of acetone and water (20: 1(v/v), 10 times with 50 ml each time), and the combined extracts were dried under vacuum to give the crude product as a red solid. The crude product was then mixed with acetonitrile (20 ml), and the mixture was stirred at room temperature for 18 hours, then filtered. The filter cake was mixed with acetonitrile (10 ml) and the mixture was stirred at room temperature for 3 hours. The mixture was then filtered. The filter cake was dried under vacuum at room temperature to give 61.0 mg of compound of formula (Comp-12) as a red solid (yield 8.7%);

in formula (Comp-12), doxorubicin (doxorubicin) is a compound of formula (DOXO) linked through an amino group represented by (d1) in formula (Comp-12) and in formula (DOXO).

¹H NMR(400MHz，DMSO-d₆20 ℃ 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 (4H, 5H, 5.5H, 5, 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)+

Example 100

General procedure description for the preparation of the compounds of formula (I) given in table 1:

to a 10mM aqueous solution of N-acetyl-cysteine (2500. mu.l, 5 equivalents) was added a 10mM solution of the corresponding compound of formula (II) in N, N-dimethylacetamide (500. mu.l). The pH was adjusted to 7.5 with 0.3M sodium hydrogen phosphate solution and the reaction mixture was stirred at 20 ℃ for 2 hours. The resulting solution of the corresponding compound of formula (I) was used/tested without further purification.

Details are listed in Table (1)

TABLE 1
			Examples	A compound of formula (II)	A compound of formula (I)
100-1	A compound of formula (Comp-12) prepared according to comparative example 7	A compound of formula (Comp-13)
			100-2	A compound of formula (23) prepared according to example 36	A compound of formula (13)
100-3	A compound of formula (20) prepared according to example 15	A compound of formula (10)
			100-4	A compound of formula (21) prepared according to example 22	A compound of formula (11)
100-5	A compound of formula (22) prepared according to example 29	A compound of formula (12)
			100-6	A compound of formula (25) prepared according to example 54	A compound of formula (15)
100-7	A compound of formula (26) prepared according to example 60	A compound of formula (16)
			100-8	A compound of formula (24) prepared according to example 44	A compound of formula (14)

Method RP-HPLC

RP-HPLC analysis of a solution of the corresponding compound of formula (I) prepared according to example 100 was accomplished using the following parameters:

luna 5U C18250 × 4.6mm column (purchased from Phenomenex corporation), solvent a: 0.1% (v/v) TFA in water, solvent B: 0.1% (v/v) TFA in acetonitrile, 100% solvent a for 10 minutes, a gradient from 0 to 70% solvent B over the next 70 minutes, and to 100%, 1 ml/min over the next 3 minutes, detected at 254 nm.

Retention time RT ═

The values given in Table 2

TABLE 2	RT
		A compound of formula (DOXO)	44.9 minutes
A compound of formula (Comp-13)	54.5 minutes
		A compound of formula (13)	55.1 minutes
A compound of formula (10)	51.2 minutes
		A compound of formula (11)	51.7 minutes

A compound of formula (12)	50.7 minutes
		A compound of formula (15)	50.6 minutes
A compound of formula (16)	52.2 minutes
		A compound of formula (14)	48.3 minutes

Solubility test

Solutions of the corresponding compound of formula (I) prepared according to example 100 (15, 48 and 96 microliters, respectively) were mixed with water (135, 102 and 54 microliters, respectively) to give a total of 150 microliters of the corresponding three diluted solutions. The DMA concentrations of these three dilutions were 1.5%, 5.0% and 10.0%, respectively, volume percent, based on the total volume of water in each solution. These dilutions were stirred at 20 ℃ for 1 hour and then analyzed by method RP-HPLC.

The solubility of the compound of formula (I) was evaluated by comparing the peak areas (with respect to the most soluble conjugate, i.e. the compound of formula (11), whose peak areas are set to 100%, expressed in table 3(× ref)), with the values shown in table 3:

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

The higher solubility of the compound of formula (i), in particular at the more relevant low DMA concentrations, compared to the benchmark linked compound of formula (Comp-13) offers two advantages:

1) higher yields in the synthesis of compounds of formula (I) due to reduced polymerization

2) Excellent pharmacokinetics

Cathepsin B release from compounds of formula (DOXO)

Bovine spleen cathepsin B (SAFC C6286-10UN, 10 units) was dissolved in 1 ml of acetate buffer (25mM acetate and 1mM EDTA) at pH 5.0 to provide a stock solution of cathepsin B.

This cathepsin B stock solution (16. mu.l) was mixed with 30mM dithiothreitol and 15mM EDTA aqueous solution (32. mu.l), and the resulting solution was left to stand without stirring at 20 ℃ for 15 minutes. Then 25mM acetate and 1mM EDTA in water (1175. mu.l), the corresponding compound of formula (I) in solution (142. mu.l, prepared according to example 100), N-dimethylacetamide (53.7. mu.l), and 10mM daunomycin in water (157.7. mu.l) according to retention time as an internal standard for the partition peak were added. The resulting solution was incubated at 37 ℃ for 2 days. Undiluted aliquots (100 microliters) were periodically removed and analyzed by method RP-HPLC as given in table 4. The relative percentage of release of the compound of formula (DOXO) over the course of the experiment relative to the corresponding compound of formula (I) is given in table 4:

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

All compounds showed drug release in the presence of cathepsin B.

Stability in human serum

To human serum (SAFC H4522, 950. mu.l) were added a solution of the corresponding compound of formula (I) (950. mu.l, prepared according to example 100) and an aqueous solution of 10mM daunomycin as an internal standard (100. mu.l). The resulting solution was incubated at 37 ℃ for 7 days. Aliquots (100. mu.L) were periodically removed, diluted with 0 to 5 ℃ methanol (400. mu.L), filtered and the resulting filtrate analyzed by method RP-HPLC as listed in Table 4. The released compound of formula (DOXO) was quantified relative to daunomycin (internal standard) and the values are given in table 5:

TABLE 5

0 hour

4 hours

8 hours

24 hours

48 hours

72 hours

A compound of formula (13)

<0.1％

<0.1％

<0.1％

4.4％

6.3％

6.9％

A compound of formula (10)

<0.1％

<0.1％

<0.1％

2.4％

4.3％

4.3％

A compound of formula (11)

<0.1％

<0.1％

<0.1％

<0.1％

<0.1％

<0.1％

A compound of formula (12)

<0.1％

<0.1％

<0.1％

<0.1％

<0.1％

<0.1％

A compound of formula (15)

<0.1％

<0.1％

0.2％

3.2％

5.2％

6.0％

A compound of formula (16)

<0.1％

1.3％

1.2％

1.9％

3.2％

1.6％

A compound of formula (14)

<0.1％

<0.1％

<0.1％

1.1％

2.5％

2.6％

All branched linkages were demonstrated to have good stability.

Example 101

The compounds of formula (12-101) derived from the compound of formula (22) and monoclonal anti-interleukin-1 β antibody are prepared as follows:

monoclonal anti-interleukin-1 β antibody (5 mg, Sigma cat # I3642) produced in mice was mixed in PBS (8.0 ml).

To an aliquot (3ml) was added a 1.0mM aqueous solution of tris (2-carboxyethyl) phosphine hydrochloride (25. mu.l, 2.0 equiv.) and the mixture was stirred for 90 minutes at 20 ℃. A solution of 1.0mM of the compound of formula (22) prepared according to example 29 in N, N-dimethylacetamide (64.4. mu.l, 5.15 equivalents) was added, and the resulting mixture was further stirred at 20 ℃ for 30 minutes. 1.0mM of N-acetyl-cysteine (64.4. mu.l, 5.15 equivalents) in water was added and the resulting mixture was stirred at 20 ℃ for an additional 36 minutes to yield a so-called conjugate mixture. The NAP-25 column was rinsed with PBS (25ml), loaded with conjugate mix (2.5 ml), and eluted with PBS (5.0 ml). Fractions were collected and those containing proteins were pooled. The pooled protein solutions comprising the compounds of formulae (12-101) were analyzed by method SEC-HPLC (results are listed in Table 6) and method HIC using monoclonal anti-interleukin-1 β antibody suspended in PBS.

Example 102

The compounds of formula (15-102) derived from the compound of formula (25) and monoclonal anti-interleukin-1 β antibody are prepared as follows:

monoclonal anti-interleukin-1 β antibody produced in mice (5 mg, Sigma cat # I3642, as used in example 101) was mixed in PBS (8.0 ml).

To an aliquot (3ml) was added a 1.0mM aqueous solution of tris (2-carboxyethyl) phosphine hydrochloride (25. mu.l, 2.0 equiv.) and the mixture was stirred for 90 minutes at 20 ℃. A solution of 1.0mM of the compound of formula (25) prepared according to example 54 in N, N-dimethylacetamide (64.4. mu.l, 5.15 equivalents) was added, and the resulting mixture was further stirred at 20 ℃ for 30 minutes. 1.0mM of N-acetyl-cysteine (64.4. mu.l, 5.15 equivalents) in water was added and the resulting mixture was stirred at 20 ℃ for a further 42 minutes to yield a so-called conjugate mixture. The NAP-25 column was rinsed with PBS (25ml), loaded with conjugate mix (2.5 ml), and eluted with PBS (5.0 ml). Fractions were collected and those containing proteins were pooled. The pooled protein solutions comprising the compounds of formulae (15-102) were analyzed by method SEC-HPLC (results are listed in Table 6) and method HIC using monoclonal anti-interleukin-1 β antibody suspended in PBS.

Method SEC-HPLC

SEC-HPLC analysis of the corresponding pooled protein solutions was done using the following parameters:

TSK G3000SWXL 300 × 7.8mm column (average pore diameter 250 angstroms, average particle size 5 microns, silica-based column available from Tosoh Bioscience corporation), eluent: 10% (v/v) isopropanol 0.2M potassium phosphate buffer, 0.5 ml/min, detected at 280 nm.

Retention time RT ═

The peak values and their retention times of the corresponding compounds of formula (I) were determined by comparing the unmodified monoclonal anti-interleukin-1 beta antibody, which was used as the culture medium in examples 101 and 102.

HMW ═ high molecular weight fraction, unassigned

LMW is low molecular weight fraction and is not distributed

Method HIC

HIC analysis of the corresponding pooled protein solutions was done using the following parameters:

TSK-gelbutyl-NPR 4.6mm x 35mm column (NPR refers to a non-porous resin, a matrix material of polymethacrylate with an average particle size of 2.5 microns, available from Tosoh biosciences), solvent a: 50mM sodium phosphate buffer, solvent B: 25% (v/v) isopropanol in 50mM sodium phosphate buffer, solvent gradient from 100% solvent A to 100% solvent B over 12 minutes, 0.8 ml/min, detected at 280 nm.

The resulting chromatogram is shown in fig. 1.

The symbols used in FIG. 1 illustrate:

continuous line- -monoclonal anti-interleukin-1 beta antibody used in examples 101 and 102

Dash-line-compound of formula (12-101) prepared according to example 101

Dotted line ■ ■ ■ ■ ■ ■ ■ Compounds of formula (15-102) prepared according to example 102

AU absorption unit

Claims (2)

1. A compound of the formula (VI),

wherein

n3 is 1;

n4 is 1;

AAⁿ⁴is an amino acid residue, n4 is an index of the amino acid residue, n3 is the number of the amino acid residues, and (AA)¹)₁Represents an amino acid residue AA¹；

(3) Represents the N-terminal amino group of the amino acid residue;

r1 and R2 are the same or different and are independently selected from hydrogen, C_1-4Alkyl, C (O) - (CH)₂-O-)_m5-(GRPEG)_m4-R3 and PGN;

r3 is C_1-4An alkyl group;

m4 is 0 or 1;

m5 is 0 or 1;

PGN is a protecting group;

GRPEG is a linking group of formula (GRPEG-I);

m21 is 1,2, 3, 4, 5 or 6.

2. The compound of formula (VI) according to claim 1, wherein the compound of formula (VI) is selected from the group consisting of a compound of formula (6-3), a compound of formula (6-4), a compound of formula (6b-3), and a compound of formula (6 b-4):