WO2022199237A1 - Modified amino acids and application thereof in adc - Google Patents

Modified amino acids and application thereof in adc Download PDF

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WO2022199237A1
WO2022199237A1 PCT/CN2022/072943 CN2022072943W WO2022199237A1 WO 2022199237 A1 WO2022199237 A1 WO 2022199237A1 CN 2022072943 W CN2022072943 W CN 2022072943W WO 2022199237 A1 WO2022199237 A1 WO 2022199237A1
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antibody
group
compound
amino acid
mmol
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PCT/CN2022/072943
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French (fr)
Chinese (zh)
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郭茂君
李辉
李海泓
许喆
王威威
赵磊
刘海东
戚文科
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联宁（苏州）生物制药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/093—Polyol derivatives esterified at least twice by phosphoric acid groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of medicine, in particular to antibody drug conjugates (ADC), and more particularly to modified amino acids and their application in ADC.
  • ADC antibody drug conjugates
  • Antibody-drug conjugate is to link a biologically active small molecule drug to a monoclonal antibody through a chemical link, and the monoclonal antibody acts as a carrier to target and transport the small molecule drug to the target cells.
  • ADC drug structure is relatively complex, and there are great differences between different ADC drug designs. Even for different drugs of the same target, the differences in toxicity are obvious due to the differences in recognition sites, attachment sites, linkers and small molecules to which they are attached.
  • a first aspect herein provides a phosphorylcholine group-modified amino acid of Formula I below:
  • R 1 is a phosphorylcholine group
  • N represents the nitrogen atom on the amino acid molecule
  • R 2 is H, C1-C4 alkyl or -L 1 -phosphorylcholine group
  • Each L 1 is independently a linking group
  • R3 represents the remainder of the amino acid molecule other than the indicated N atom, which is optionally protected by a protecting group.
  • a second aspect herein provides a phosphorylcholine-modified peptide, the peptide comprising at least one phosphorylcholine group, the phosphorylcholine group having a relationship with one or more amino acids contained in the peptide
  • the N atoms and/or oxygen atoms are covalently linked via a linking group.
  • the third aspect of the present invention provides a linker compound having the structure shown in the following formula II:
  • R 4 is a group that can react with a sulfhydryl group in a cysteine residue in a protein or a free amino group on a lysine residue to attach Formula II to the protein;
  • L 2 is a linking group
  • P is the amino acid of any one of claims 1-3 or the peptide of any one of claims 4-8 ; the amino acid or peptide is covalently linked to L2 and R5 ;
  • R5 is:
  • R 6 is H, halogen, C1-10 alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl, 6-14-membered aryl, aralkyl
  • R 9 is H, -C(O)-N(R 7 )-(CH 2 ) n -NHR 8 or nitro-substituted phenoxy
  • R 7 is H or C1-6 alkyl
  • R 8 is H or C1-6 alkyl
  • n is an integer of 1-6.
  • R 4 , L 2 , P and R 5 are as described in any of the embodiments herein; D represents a group obtained by removing one H atom from the drug molecule, which is covalently linked to R 5 ; preferably, the drug molecule is A carbonate bond (-OCO-) or a carbamate (-OCNH-) is formed with R 5 to connect with R 5 .
  • R 4 , L 2 , P and R 5 are as described in any of the embodiments herein, D is as described in any of the embodiments herein, and A is an antibody or an antigen-binding fragment thereof; wherein, the antibody or antigen thereof The binding fragment is covalently linked to R through the sulfhydryl group it contains.
  • the sixth aspect herein also provides the use of the amino acids, peptides, linker compounds and drugs described in any of the embodiments herein in the preparation of ADCs, or in improving the solubility of antibodies, or in the preparation of antibodies for improving the solubility of antibodies. application in formulations.
  • a seventh aspect of the present invention also provides a composition comprising the amino acid, peptide, linker compound or drug covalently linked to the linker compound according to any of the embodiments herein.
  • the composition may contain other suitable excipients, such as solvents and the like.
  • Figure 1 HIC-HPLC profile of LA003.
  • FIG. 1 HIC-HPLC profile of LA004.
  • FIG. 3 HIC-HPLC profile of LA005.
  • FIG. 4 HIC-HPLC profile of LA006.
  • Figure 7 HIC-HPLC comparison of LA003, LA004, LA005 and LA006.
  • the following conclusions can be drawn from the figure: the four ADCs with DAR values of 4, the ADC with two phosphatidylcholines in the small molecule part is more hydrophilic, and the alkyl form of phosphatidylcholine is more hydrophilic It is more potent than the amide form of phosphatidylcholine.
  • Figure 8 Cytotoxic activity of phosphorylcholine-modified HER2 ADCs against human SK-BR-3, HCC1954 and MDA-MB-468 breast cancer cell lines.
  • A Phosphorylcholine-containing modification of the Lys side chain in the Val-Lys dipeptide linker;
  • B Direct modification of Gly or Ser in the Gly/Ser-Val-Cit tripeptide linker;
  • C The side chain of Lys and directly linked to Ser in the Ser-Val-Lys tripeptide linker,
  • D the side chain of Lys and/or directly linked to Ser within a Val-Lys or Ser-Val-Lys linker.
  • Anti-HER2 ADC data points represent mean normalized cell viability ⁇ SEM of at least three replicates. Results of at least two independent experiments.
  • FIG. 9 HIC profiles of LA003, LA005 and control Her2-McVCPABMMAE.
  • a, b and c represent HIC profiles of control, LA003 and LA005, respectively.
  • the present invention aims to provide a new modified amino acid or modified peptide, and the ADC prepared with such modified amino acid or modified peptide has significantly improved solubility, while still retaining the desired biological activity or pharmacological effect .
  • the present invention uses phosphorylcholine to modify amino acids to construct modified amino acids or modified peptides.
  • reaction and purification can be carried out using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention.
  • the techniques and methods described above can generally be carried out according to conventional methods well known in the art from the descriptions in the various general and more specific documents cited and discussed in this specification.
  • groups and their substituents can be selected by those skilled in the art to provide stable moieties and compounds.
  • substituents When substituents are described by conventional chemical formulae written from left to right, the substituents also include the chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH2O- is equivalent to -OCH2- .
  • C1-6 alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms.
  • the total number of carbon atoms in the simplified notation does not include carbons that may be present in the substituents of the group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • Hydroxyalkyl refers to an alkyl group as defined below substituted with hydroxy (-OH).
  • Niro refers to -NO2 .
  • Cyano refers to -CN.
  • Amino refers to -NH2 .
  • Carboxyl refers to -COOH.
  • alkyl refers to a fully saturated straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having, for example, 1 to 10 ( Preferably from 1 to 8, more preferably from 1 to 6) carbon atoms and is attached to the rest of the molecule by a single bond.
  • Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2 - Dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl and decyl, etc.
  • the alkyl group is a C1-C4 alkyl group.
  • alkenyl means consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, having, for example, 2 to 10 (preferably 2 to 8, More preferably a straight or branched hydrocarbon chain group of 2 to 6, more preferably 2 to 4) carbon atoms and linked to the rest of the molecule by a single bond.
  • Alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-1,4-dienyl, and the like.
  • alkynyl means consisting only of carbon atoms and hydrogen atoms, containing at least one triple bond, having, for example, 2 to 10 (preferably 2 to 8, More preferably a straight or branched hydrocarbon chain group of 2 to 6, more preferably 2 to 4) carbon atoms and linked to the rest of the molecule by a single bond.
  • Alkynyl groups include, but are not limited to, ethynyl, propynyl, and the like.
  • cycloalkyl refers to a stable non-aromatic monocyclic hydrocarbon group consisting only of carbon and hydrogen atoms.
  • the number of ring carbon atoms of the cycloalkyl group is usually 3 to 8.
  • Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • heterocyclyl means from 2 to 14 carbon atoms (eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 carbon atoms) and a stable 5- to 10-membered non-aromatic cyclic group consisting of 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heterocyclyl group may be a monocyclic or bicyclic ring system.
  • Heterocyclyl groups can be partially or fully saturated.
  • a heterocyclyl group can be attached to the rest of the molecule via a carbon atom or a heteroatom and through a single bond.
  • heterocyclyl groups include, but are not limited to: azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, pyranyl, tetrahydro Pyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolane, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinazinyl, thiazolidinyl , isothiazolidinyl, isoxazolidinyl, indoline, octahydroindolyl, octahydroisoindolyl and pyrazolidine, etc.
  • aryl means having 6 to 14 carbon atoms (preferably having 6 to 10 carbon atoms, such as 6, 7, 8, 9 or 10 carbon atoms) carbon atoms) of the conjugated hydrocarbon ring system group.
  • Aryl groups can be monocyclic or bicyclic ring systems. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.
  • aralkyl refers to an alkyl group as defined above substituted with an aryl group as defined above.
  • heteroaryl means a ring having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, a 5- to 16-membered, preferably 5-10 membered, conjugated ring system group.
  • a heteroaryl group can be a monocyclic, bicyclic, tricyclic or multicyclic ring system.
  • heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, Benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indazinyl, isoindolyl, indazolyl, isoindazolyl , purinyl, quinolinyl, isoquinolinyl, diazanaphthyl, naphthyridinyl, quinoxalinyl, pteridyl, carbazolyl, carboline, phenanthridine, phenanthroline, acridine base, phena
  • groups may be optionally substituted.
  • “Optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes both instances where the event or circumstance occurs and instances where it does not.
  • “optionally substituted aryl” means that the aryl group is substituted or unsubstituted, and the description includes both substituted and unsubstituted aryl groups.
  • substituents described in the claims and specification sections of the present invention include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloepoxy, haloalkenyl, haloalkyne , cyano, cycloalkyl-O-, nitro, amino, optionally substituted amino, alkoxy, cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted One or more of cycloalkyl and optionally substituted heterocyclyl.
  • substituents include, but are not limited to, C1-C6 alkyl, haloalkyl, cycloalkyl, amino, C1-C6 alkyl substituted amino, halogen, cyano, C3-C8 cycloalkyl-O -, one or more of aryl and heteroaryl.
  • the number of substituents may be 1-5, such as 1-3, depending on the structure of the substituted group.
  • phenyl may be substituted with 1-3 substituents selected from C1-C6 alkoxy, C3-C8 cycloalkyl-O-, halogen and amino.
  • intermediate compound functional groups may need to be protected by suitable protecting groups.
  • suitable protecting groups include trialkylsilyl or diarylalkylsilyl groups (eg tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl) , tetrahydropyranyl, benzyl, etc.
  • Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like.
  • Suitable thiol protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, and the like.
  • Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.
  • Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, T.W. and P.G.M. Wuts, Protective Groups in Organi Synthesis, (1999), 4th Ed., Wiley.
  • the protecting group of the present application is a protecting group for an amino group, such as Boc (tert-butoxycarbonyl).
  • the protecting group can also be a polymeric resin.
  • amino acids or amino acid molecules are used interchangeably and have meanings well known in the art.
  • the amino acid is a natural amino acid.
  • amino acids synthesized by biosynthesis are collectively referred to as "natural amino acids”.
  • amino acid molecules include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, Cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, pyrrole Lysine and Ornithine.
  • the application provides modified amino acids in which one nitrogen atom is replaced by a group R 2 and a group R 1 -L 1 -.
  • the modified amino acid of the present application can be the compound shown in the following formula I:
  • R 1 is a phosphorylcholine group
  • N represents the nitrogen atom on the amino acid molecule
  • R 2 is H, C1-C4 alkyl or -L 1 -phosphorylcholine group
  • Each L 1 is independently a linking group
  • R3 represents the remainder of the amino acid molecule other than the indicated N atom, which remainder is optionally protected by a protecting group.
  • the phosphorylcholine group is shown in the following formula:
  • the wavy line indicates the position where the phosphorylcholine group is linked to L 1 .
  • each L 1 can be independently represented as -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; L b is absent, or is a carbonyl group, an ester group (-COO-), -O- SO2- or -NH- SO2- . Preferably, L b is absent or carbonyl. Preferably, L 1 is covalently linked to said N through L b . In some embodiments, L b and the indicated N form an amide group or a sulfonamido group. In preferred embodiments, each L 1 is independently C1-C6 alkylene or C1-C6 alkylenecarbonyl.
  • the amino acid molecule described in R is selected from: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine , tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenium half Cystine, pyrrolysine and ornithine.
  • the amino acid molecule is selected from: lysine, ornithine, valine, tyrosine, glutamine, alanine, arginine, asparagine, leucine, isoleucine , threonine, serine, phenylalanine, valine and glycine. More preferably, the amino acid molecule is selected from the group consisting of: valine, ornithine, lysine, serine, glycine and citrulline. More preferably, the amino acid molecule is selected from the group consisting of: ornithine, lysine, serine and glycine.
  • the N is not a ring nitrogen atom on an aryl group, a heteroaryl group or a heterocyclic group in the amino acid molecule. In some embodiments, the N is not a nitrogen atom used to form peptide bonds in amino acid molecules.
  • amino acids have free amino groups, and the free amino groups refer to amino groups that do not normally form peptide bonds with other amino acids.
  • Amino acids with free amino groups include lysine, arginine, glutamine and asparagine.
  • the free amino group can be protected, such as protection with Boc
  • the groups are covalently linked to form a "tert-butoxycarbonyl-NH-" group.
  • the modified amino acid is selected from the compounds shown in LN001 to LN010 and LN012 to LN025.
  • the present application provides a phosphorylcholine-modified peptide, the peptide includes at least one phosphorylcholine group, and the phosphorylcholine group is combined with one or more phosphorylcholine groups contained in the peptide.
  • the N and/or oxygen atoms of the amino acid are covalently linked via a linking group. It will be understood that the amino acid residues in the peptide are linked by peptide bonds.
  • the phosphorylcholine gene can be connected to the N that forms a peptide bond, or it can be connected to the N that does not form a peptide bond; preferably, it is not connected to the ring nitrogen of an aryl, heteroaryl or heterocyclic group of an amino acid residue atom.
  • the peptide described in this application has 2-10 amino acid residues, more preferably 2-5 amino acid residues, that is, the peptide is a dipeptide, tripeptide, tetrapeptide or pentapeptide.
  • the peptide is a peptide that can be hydrolyzed by enzymes in a pathological environment.
  • the enzyme may be a proteolytic enzyme, protease or peptidase. More preferably, the enzyme is selected from the group consisting of cysteine proteases, asparagine proteases, aspartic proteases, glutamic proteases, threonine proteases, gelatinases, metalloproteases and asparagine peptide lyases one or more of.
  • the peptide can be hydrolyzed by one in a pathological environment, or by multiple enzymes simultaneously.
  • the pathological environment can be various environments known in the art where treatment is desired, for example, a pathological environment where tumor cells exist, such as cancer tissue, or a site of inflammation, or a site of infection, or other tissues to be treated.
  • the enzyme is selected from asparagine endopeptidase (Legumain).
  • the amino acid residues contained in the peptide may be various amino acid residues known in the art, and the amino acid residues may be any one or more amino acid residues described in the amino acid molecule defined by R3 above. It will be appreciated that the selection of the number and type of amino acid residues in a peptide can be determined by the enzymes present in the environment in which the peptide will be used. For example, if the pathological environment contains asparagine endopeptidase, preferably, the peptide is one that can be recognized and hydrolyzed/cleaved by asparagine endopeptidase to release the therapeutic agent. In some embodiments, the amino acid residues in the peptide are selected from one or more of valine, ornithine, lysine, serine, glycine, and citrulline.
  • the linking group may be L 1 as described in any of the embodiments herein.
  • the peptide contains at least one phosphorylcholine group-modified amino acid as described in any of the embodiments herein.
  • at least one amino acid residue modified by a phosphorylcholine group in the peptide is selected from the group consisting of compounds represented by LN001 to LN010 and LN012 to LN025.
  • the phosphorylcholine group-modified amino acid residue in the peptide is selected from one or more of ornithine, lysine, serine and glycine.
  • the amino acid residues in the peptide modified with phosphorylcholine groups are derived from one or more of the following molecules: LN001-LN010, LN022 and LN025.
  • the "derivatization" refers to the removal of one H on the amino group and the H on the carboxyl group of the molecule to form peptide bonds, respectively, with other amino acid residues in the peptide. It will be understood that when the amino acid residue derived from the molecule is located at the N- or C-terminus of the peptide, then the amino acid residue is a monovalent group, otherwise it is a divalent group.
  • the peptide is Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Val-Cit, Gly-Gly-Gly-Val- Lys or Gly-Gly-Val-Lys.
  • one or more of ornithine, lysine, serine and glycine in these peptides is modified with a phosphorylcholine group.
  • the peptide is shown as LN011.
  • the phosphorylcholine-modified peptide is selected from the group consisting of: Val-Orn(N-PC), Val-Lys(N-PC), Val-Lys(N-PC2), Val-Lys(N -PC-PC), Val-Lys(N-Me-PC), Gly(N-PC)-Val-Lys(N-PC), Ser(O-PC)-Val-Lys(N-PC), Ser (O-PC)-Val-Cit, Gly(N-PC)-Val-Cit, Gly(N-PC)-Lys(N-PC), Gly-Gly-Gly-Val-Lys(N-PC) and Gly-Gly-Val-Lys (N-PC):
  • R 4 is a group that can react with the S in the cysteine residue in the protein, or the amino group on the -lysine, thereby connecting the formula II to the protein;
  • L 2 is a linking group
  • P is the modified amino acid or modified peptide of any of the embodiments herein ; the modified amino acid or modified peptide is covalently linked to L and R ;
  • R5 is:
  • R 6 is H, halogen, C1-10 alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl, 6-14-membered aryl, aralkyl, 5-10-membered heteroaryl or 4-10-membered heterocyclyl;
  • R 9 is H, -C(O)-N(R 7 )-(CH 2 ) m -NHR 8 or nitro-substituted phenoxycarbonyl;
  • R 7 is H or C1-6 alkyl
  • R 8 is H or C1-6 alkyl
  • n is an integer of 1-6.
  • R4 is selected from:
  • the wavy line represents the position where R 4 is connected to L 2 ; F 5 represents 5 fluorine atoms.
  • L 2 is -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; L b is absent, or It is a carbonyl group, an ester group (-COO-), -O-SO 2 - or -NH-SO 2 -.
  • L b is absent or carbonyl.
  • L2 is covalently linked to said P through Lb.
  • an amide or sulfonamido group is formed between L b and P.
  • each L 2 is independently C1-C6 alkylene or C1-C6 alkylenecarbonyl.
  • the R 4 -L 2 and P and by the group selected from the group consisting of amide (-CO-NH-), ester (-COO-), -O-SO 2 -NH- and -NH -SO 2 -NH- is covalently linked.
  • R 4 -L 2 and P are covalently linked through an amide group.
  • connection between R 4 and L 2 can be made through La .
  • between R 4 and L 2 can be selected from carbon-carbon single bond, amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO
  • the connection mode of 2 -NH- is covalent connection, preferably through carbon-carbon single bond or amide group.
  • the P and R 5 are selected from the group consisting of amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO 2 -NH - is covalently attached, preferably via an amide group.
  • R 5 is:
  • R 6 is H or C1-C6 alkyl.
  • R 7 is H or C1-C4 alkyl.
  • R 8 is H or C1-C4 alkyl.
  • n is an integer of 1-4.
  • R 4 (C1-C6 alkylene)-C(O)-[NH-PC(O)]-R 5 .
  • the compound of formula II such as compound 1-8, 1-9, 2-6, 2-7, 3-5, 3-6, 4-4, 4-5, 5- 3, 5-4, 6-3, 6-4, 7-4, 7-5, 8-3, 8-4, 15-4, 15-5, 16-3, 16-4, 23-2 or 23-3.
  • linker compounds of the present application can be used to modify drug molecules of interest. Also provide a medicine covalently linked with the linker compound described in the application, which has the structure shown in the following formula III:
  • R 4 , L 2 , P and R 5 are as defined in any of the preceding embodiments; D represents a group obtained by removing one H atom from the drug molecule.
  • the drug molecule is attached to R5 by forming a carbonate bond (-OCO-) or a carbamate ( -OCNH- ) with R5.
  • the drug molecule is selected from the group consisting of: MMAE, Duo-5, DXD, ixatecan, camptothecin, 10-hydroxycamptothecin, topotecan, fluuridine, desoxy Floxuridine, Cytarabine, Etoposide, Fludarabine, Capecitabine, Vincristine, Epothilone B, Paclitaxel, Docetaxel, Daunorubicin, Epirubicin, A Ammopterin, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin.
  • These drugs are generally linked to formula II through the formation of a carbonate bond (-OCO-) or a carbamate (-OCNH-) with R5 in formula II through a hydroxy or amino group.
  • the position on the drug molecule to which the linker compound is attached should not affect the biological activity of the drug molecule.
  • a position far from its active center can be selected to be covalently linked to the linker compound.
  • Another consideration is the ease with which the linker compound can react with the drug molecule.
  • the linking position can be selected under the premise that it is convenient to link the linker compound to the drug molecule without affecting the biological activity of the linked drug molecule, which can be implemented and tested by those skilled in the art according to well-known techniques in the art.
  • the biological activity of linker compound-modified drug molecules Preferably, the covalently linked drug molecule of the linker compound described herein retains at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% more biological academic activity.
  • the drugs are shown as LD001 to LD0025.
  • the present application provides an ADC whose structural formula is shown in the following formula IV:
  • R 4 , L 2 , P, R 5 and D are as defined in any of the preceding embodiments, A is an antibody or an antigen-binding fragment thereof, and n is an integer of 1-8.
  • antibody has the meaning well known in the art and includes any form of antibody having the desired biological activity, such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies, etc.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • “Full-length antibody” refers to an immunoglobulin molecule comprising at least four peptide chains in nature: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • an "antigen-binding fragment" of an antibody includes a fragment or derivative of an antibody, typically including at least a fragment of the antigen-binding or variable region (eg, one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody .
  • antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments Sexual antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • a “single-chain Fv” or “scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Domain antibodies are immunofunctional fragments of immunoglobulins that contain only heavy or light chain variable regions. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.
  • a “bivalent antibody” contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • “Diabodies” refer to small antibody fragments with two antigen-binding sites comprising a heavy chain variable domain (VL) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites.
  • the antibody is linked to R4 through the sulfhydryl group of cysteine or the free amino group of lysine contained in the antibody.
  • the antibody can be various types of antibodies or antigen-binding fragments thereof known in the art with the desired biological activity.
  • the antibody or functional fragment thereof can be selected from: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody , anti-CTLA-4 antibody, anti-TNF ⁇ antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, or anti-ICOS Antibody, Anti-CD25 Antibody, Anti-CD30 Antibody, Anti-CD3 Antibody, Anti-CD22 Antibody, Anti-CCR4 Antibody, Anti-CD38 Antibody, Anti-CD52 Antibody, Anti-Complement C5 Antibody, Anti-RSV F Protein, Anti- -GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib/Illa antibody, anti-ICO
  • the number of -R 4 -L 2 -PR 5 -D moieties conjugated with the antibody can be 1-8, and the number is generally related to the number of disulfide bonds in the antibody, and is also related to the coupling reaction. group related. Those skilled in the art can easily determine the number of n according to conditions such as the amino acid sequence of the antibody and the groups involved in the coupling reaction. In some embodiments, n is an integer from 2-6. In some embodiments, n is 4.
  • the ADC is selected from any one or more of LA001 to LA025.
  • the phosphorylcholine group-modified amino acids, phosphorylcholine-modified peptides, linker compounds, drugs covalently linked to linker compounds, and ADCs of the present application can be prepared with reference to the methods described in the examples of the present application. Corresponding preparation methods are exemplarily described below.
  • Reductive amination can be carried out by the amino group of a suitably protected amino acid and an aldehyde containing a phosphorylcholine group (such as LN027), or by an amino group on the side chain of an amino acid and a carboxylic acid containing a phosphorylcholine group (LN028)
  • An amide is formed, or an amino acid modified with a phosphorylcholine group is prepared by forming an amide from a carboxyl group on the amino acid side chain and an amino group containing a phosphorylcholine group (LN029).
  • phosphorylcholine-modified peptides and phosphorylcholine-modified linker compounds are usually prepared by combining a suitably protected amino acid with p-aminobenzyl alcohol.
  • PAB undergoes a condensation reaction to yield the p-aminobenzyl alcohol (PAB) amide of the appropriately protected amino acid, which is then deprotected for conventional polypeptide synthesis.
  • L 2 and R 5 in formula II and R 4 and D in formula III as required.
  • the antibody can be reduced first, then the reduced antibody can be mixed with the drug covalently linked to the linker compound represented by the formula III of the present invention, and after standing at room temperature for a sufficient time, the ADC of the present invention can be separated and purified.
  • Methods for reducing antibodies are well known in the art.
  • the purpose of reduction includes reducing disulfide bonds in antibodies to sulfhydryl groups.
  • Suitable reducing agents include TCEP and the like. Different reducing agents and buffer systems can be selected according to different antibody species.
  • compositions uses and methods of treatment and prevention of diseases
  • the present application provides a pharmaceutical composition comprising an effective amount of the ADC described in any of the embodiments of the present application and a pharmaceutically acceptable carrier.
  • the present application also provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the ADC of the present application or a pharmaceutical composition thereof.
  • Also provided are the phosphorylcholine-modified amino acids, phosphorylcholine-modified peptides, linker compounds and drugs covalently linked to the linker compounds described in this application in the preparation of ADCs, and the methods described in any of the embodiments herein.
  • prevention and “prophylaxis” include reducing the likelihood of the occurrence or exacerbation of a disease or disorder in a patient; the terms also include: preventing the occurrence of a disease or disorder in mammals, especially when such mammals are susceptible to When you have the disease or condition, but have not been diagnosed with the disease or condition.
  • Treatment and other similar synonyms include the following meanings: (i) inhibiting a disease or disorder, ie, arresting its progression; (ii) alleviating a disease or disorder, ie, causing regression of the state of the disease or disorder; or (iii) alleviating the Symptoms caused by a disease or condition.
  • administration refers to a method capable of delivering a compound or composition to a desired site for biological action.
  • Administration methods well known in the art can be used in the present invention. These methods include, but are not limited to, the oral route, the duodenal route, parenteral injection (including intrapulmonary, intranasal, intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), Topical and rectal administration.
  • an effective amount includes a therapeutically effective amount and a prophylactically effective amount, which means that the ADC of the present application, when administered to a subject alone or in combination with other therapeutic agents, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the The amount of development of a disease or condition.
  • a therapeutically effective amount also refers to an amount of ADC sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as weight, age and sex, the duration of the treatment, the co-administered treatment (if any), and the specific formulation used .
  • a pharmaceutically acceptable carrier refers to an ingredient other than the active ingredient in a pharmaceutical formulation or composition that is not toxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavors, surfactants, wetting agents, Dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.
  • the pharmaceutical compositions of the present application can be formulated using pharmaceutically acceptable carriers well known in the art.
  • the pharmaceutical composition of the present application can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the intended purpose.
  • it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranically, intranasally or externally.
  • the dose of the drug may depend on the patient's age, health and weight, concurrent treatments, and the frequency of treatments, among others.
  • the pharmaceutical compositions of the present application can be administered to any subject in need thereof, such as mammals, especially humans.
  • the pharmaceutical composition of the present application can be used to treat or prevent the corresponding disease.
  • the drug is an anticancer drug
  • the pharmaceutical composition of the present application can be used to treat cancers that can be treated by the anticancer drug, including but not limited to bladder, brain, breast, cervix, colon-rectum, esophagus, kidney , Cancer in liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and blood, etc.
  • cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, Ovarian, testicular and blood cancers.
  • the present application also provides a method for improving the solubility of an ADC, especially a method for improving the water solubility of the ADC, the method comprising modifying a peptide chain as a linker in the ADC drug with a phosphorylcholine group A step of. More specifically, the method includes the step of preparing an ADC using the amino acid or peptide or linker compound of any of the embodiments of the present invention.
  • the preparation method varies according to the different antibodies, amino acids, peptides, linker compounds used, but can be implemented with reference to the preparation schemes disclosed herein.
  • the present application also provides the amino acids, peptides, linker compounds and drugs covalently linked to the linker compounds described in any of the embodiments of the present application in improving the solubility (especially water solubility) of antibodies. applications, or in the preparation of formulations for improving the solubility (especially water-solubility) of antibodies, or in the preparation of ADCs with improved solubility (especially water-solubility).
  • the starting materials used in the following examples can be purchased from chemical vendors such as Aldrich, TCI, Alfa Aesar, Bidder, Anegi, etc., or can be synthesized by known methods.
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • Boc-lysine (LN001-1, 10g, 41mmol) and tert-butyl acetate (100ml) were added to a 250ml round-bottomed flask, and after stirring evenly, perchloric acid (8.2g, 82mmol) was slowly added dropwise. After the reaction was dissolved, the mixture was stirred at room temperature overnight. Ethyl acetate was added, the pH was adjusted to about 7 with saturated sodium bicarbonate, the organic phase was separated, the aqueous phase was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a colorless oily liquid LN001-2, 3.0g. ESI-MS (m/z); 303.41.
  • LN028 (450mg, 2.0mmol) was added to a 10ml round-bottomed flask, and then 5ml of DMF was added. After stirring, HATU (760mg, 2mmol) and DIPEA (516mg, 4mmol) were added. After the reaction mixture was stirred for 30min, the compound was added. LN001-2 (300mg, 1mmol), after the reaction for 2h, LCMS monitoring after the reaction was completed, the reaction mixture was directly subjected to medium pressure reverse-phase purification (acetonitrile/water), lyophilized to obtain a colorless solid LN001-3, 180mg.ESI -MS(m/z); 526.56
  • Embodiment 2 the synthesis of compound LN002:
  • LN001-2 1.0 g, 3.3 mmol
  • LN027 (0.25 g, 1.1 mmol
  • methanol (30 ml)
  • sodium acetoxyborohydride 1.0 g, 4.7 mmol
  • LCMS was used to monitor the reaction. /water), concentrated and lyophilized to give LN002-2 as a colorless solid, 420 mg, ESI-MS (m/z): 512.44.
  • Embodiment three the synthesis of LN003:
  • LN001-2 1.0 g, 3.3 mmol
  • LN027 1.5 g, 6.6 mmol
  • methanol 30 ml
  • sodium acetoxyborohydride 2.1 g, 9.9 mmol
  • LCMS LCMS was used to monitor the reaction.
  • the mixture was concentrated to 2 ml of the reaction solution, and purified by medium pressure reverse phase (acetonitrile). /water), concentrated and lyophilized to give LN003-2 as a colorless solid, 350 mg
  • Embodiment four the synthesis of LN004
  • Embodiment five the synthesis of LN005
  • Step 4 Add compound LN011-4 (40 mg) and 1 ml of trifluoroacetic acid to a 5 ml round-bottomed flask, react at room temperature for one hour and then concentrate to obtain a crude product, dissolve the crude product in water, wash with ethyl acetate, and separate the water phase , lyophilized to obtain LN011, 25 mg. LC-MS; 469.33.
  • step 1 The compound LN012-2 (200 mg, 0.39 mmol) obtained in step 1 was dissolved in TFA (trifluoroacetic acid) and reacted at room temperature for 35 minutes. After spin-drying, add water and lyophilize to obtain 120 mg of product (light yellow solid) LN012, ESI-MS (m/z): 356.2.
  • TFA trifluoroacetic acid
  • Embodiment 8 the synthesis of compound LN013:
  • valine (2.0g) and tert-butyl acetate (20ml) to a 250ml round-bottomed flask, stir evenly, and then slowly add perchloric acid (1.2g) dropwise. After the reaction is dissolved, stir at room temperature overnight. Ethyl acetate was added, the pH was adjusted to about 7 with saturated sodium bicarbonate, the organic phase was separated, the aqueous phase was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a colorless oily liquid valine tertiary Butyl ester 400mg, ESI-MS (m/z); 174.14
  • step 3 The product LN013-2 obtained in step 3 was added to a 25ml reaction flask, 3ml of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 30 minutes. After spin-drying, add water and freeze-dried to obtain 150mg product LN013 (light yellow solid), ESI-MS (m/z): 327.2
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • Glycerophosphorylcholine (100g, 389mmol) was dissolved in 1.5 liters of water, cooled with an ice-water bath, sodium periodate (124.7g, 583mmol) was added in batches, the temperature was controlled not to exceed 20 degrees, and the reaction was controlled in LC-MS , after the completion of the reaction, concentrate under reduced pressure, add methanol to make slurry for 1-2 hours, filter to remove solids, concentrate the organic phase, continue to add ethanol, filter to remove solids, concentrate the organic phase, and dry under an oil pump to obtain compound LN027, 65g , ESI-MS(m/z): 226.1
  • Embodiment 12 the synthesis of compound LD001
  • Embodiment 13 Synthesis of compound LD002
  • Step 1 Add compound 2-1 (52 g, 10.0 mmol) into the reaction flask, add 1000 mL of dichloromethane, slowly add fluorene methoxycarbonyl chloride (Fmoc-Cl) (28 g, 10.8 mmol), and dropwise add triethylamine ( 13.5g, 13.4mmol), react at room temperature for 12-20 hours, add MTBE to the reaction solution, beat and stir for 1-2 hours, filter, and dry the solid under reduced pressure to obtain the target compound 2-2, 72g.
  • Fmoc-Cl fluorene methoxycarbonyl chloride
  • Step 2 Take the compound 2-2 (2.1g, 2.8mmol) obtained in step 1, add 30ml of dichloromethane and 10ml of dichloroacetic acid into the reaction flask, stir at room temperature for 90 minutes, and control the reaction by LC-MS. After the reaction is completed, Methyl tert-butyl ether was added to precipitate a solid, and after stirring for 1 hour, compound 2-3 was obtained by filtration as a pale yellow solid, 1.3 g, ESI-MS (m/z): 474.3 [M+H] + .
  • Step 3 Take LN-028 (0.5g, 2.1mmol), add it to the reaction flask, add 5ml DMF, HATU (0.76g, 2.1mmol), DIPEA (0.81g, 6.3mmol), react for half an hour, add the compound The DMF solution of 2-3 was continued to react for 30 minutes, and the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 2-4, 0.9 g, ESI-MS (m/z): 697.3 [M+H] + .
  • Step 4 Add the compound 2-4 (0.9g, 1.3mmol) obtained in step 3 into a 25ml single-neck reaction flask, add 10ml of DMF, add 2ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , and vacuum-dried under oil pump to obtain compound 2-5, which was directly used in the next step without purification, ESI-MS (m/z): 475.2 [M+H] + .
  • Step 5 Add 10 ml of DMF to the crude product of compound 2-5 obtained in step 4, add 0.5 g (1.62 mmol) of Mc-OSu, react at room temperature overnight, prepare and purify at medium pressure to obtain 0.85 g of compound 2-6, ESI - MS (m/z): 668.3 [M+H] + .
  • Step 6 Take 0.4g (0.60mmol) of compound 2-6 and 5ml of DMF into the reaction flask, then add 0.3g (1.0mmol) of bis(p-nitrobenzene) carbonate and 0.26g (2.0mmol) of DIPEA, room temperature The reaction was carried out for 2 hours. After the reaction was completed, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 2-7, 0.35 g, ESI-MS (m/z): 833.3 [M+H] + .
  • Step 7 Take 0.2g (0.24mmol) of compound 2-7 into a 25ml reaction flask, add 5ml of DMF, (0.1g, 0.74mmol) HOBT, 0.2g (0.28mmol) MMAE and 0.2g (1.56mmol) DIPEA , reacted at room temperature for 1 hour, the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD002 0.25g, ESI-MS (m/z): 1411.8[M+H] + .
  • Step 1 Take 50g (62.9mmol) of compound 3-1 into the reaction flask, add 500ml of dichloromethane and 170ml of dichloroacetic acid, react at room temperature for 1.5 hours, pour the reaction solution into ice water, stir vigorously for 0.5 hours, and precipitate out The white solid was filtered to obtain compound 3-2, which was dried under vacuum to obtain 31 g of the crude product of compound 3-2, which was directly used in the next reaction without purification.
  • Step 2 Take compound LN-028 (14.0g, 58.1mmol), put it into a reaction flask, add 100ml of DMF, HATU (22.1g, 58.1mmol), DIPEA (22.5g, 174.3mmol), react for one hour, add 10.0 g (17.5 mmol) DMF solution of compound 3-2, continued to react for 30 minutes, the obtained reaction solution was directly purified by medium pressure preparation to obtain 7.9 g of compound 3-3, ESI-MS (m/z): 796.4 [M+ H] +
  • Step 3 Take the compound 3-3 (7.9g, 9.9mmol) obtained in step 2 and add it to a 250ml single-neck reaction flask, add 100ml of DMF, add 20ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , DMF was removed under oil pump to obtain compound 3-4, which was directly used in the next step without purification, ESI-MS (m/z): 574.3 [M+H] + .
  • Step 4 Add 100 ml of DMF to the crude compound 3-4, add Mc-OSu (4.5 g, 14.6), react at room temperature overnight, prepare and purify at medium pressure to obtain 4.2 g of compound 3-5, ESI-MS (m/ z): 767.4 [M+H] + .
  • Step 5 Take compound 3-5 (4.0g, 5.2mmol), add 50ml of DMF to the reaction flask, then add bis(p-nitrobenzene)carbonate (3.5g, 11.5mmol) and DIPEA (3.5g, 27mmol) , and reacted at room temperature for 2 hours. After the reaction, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 3-6, 3.6g, ESI-MS (m/z): 932.4[M+H] + .
  • Step 6 Take compound 3-6 (0.2g, 0.21mmol) into a 25ml reaction flask, add 5ml DMF, 0.1g (0.7mmol) HOBT, 0.2g (0.28mmol) MMAE and 0.2g (1.55mmol) DIPEA , reacted at room temperature for 1 hour, and the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD003, 0.23 g, ESI-MS (m/z): 1510.9 [M+H] + .
  • Embodiment fifteen the synthesis of compound LD004
  • Step 1 Take 5.0 g (8.7 mmol) of compound 3-2 and compound LN027 (3.2 g, 14.1 mmol) into the reaction flask, add 150 ml of methanol, stir at room temperature for 2 hours, add 1.0 eq of STAB, and continue the reaction for 2 more After 1 hour, 1.0 eq of STAB was added, and after continuing the reaction for 2 hours, 0.5 eq of STAB was added, and the reaction was continued for 1 hour. After concentration, an appropriate amount of purified water was added, and the compound 4-1 was prepared and purified by medium pressure to obtain compound 4-1, 3.6 g, ESI - MS (m/z): 782.4 [M+H] + .
  • Step 2 Take 1.2 g (1.5 mmol) of compound 4-1 into the reaction flask, add 30 ml of methanol, dropwise add 0.1 ml of aqueous formaldehyde solution, add 2.5 eq of STAB in batches, react for 0.5 hours, and concentrate to about 5 ml, An appropriate amount of purified water was added, and the compound 4-2 was prepared and purified under medium pressure to obtain compound 4-2, 1.1 g, ESI-MS (m/z): 796.4 [M+H] + .
  • Step 3 Take the compound 4-2 (1.1g, 1.4mmol) obtained in step 2 and add it to a 50ml single-neck reaction flask, add 10ml of DMF, add 2ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , the DMF was removed under the oil pump to obtain the crude product of compound 4-3, which was directly used in the next step without purification, ESI-MS (m/z): 574.3 [M+H] + .
  • Step 4 Add 10 ml of DMF to the crude compound 4-3 (0.8 g) obtained in step 3, add Mc-OSu (0.4 g, 1.3 mmol), react overnight at room temperature, and prepare and purify at medium pressure to obtain 0.52 g of compound 4 -4, ESI-MS (m/z): 767.4 [M+H] + .
  • Step 5 Take 0.5g (0.65mmol) of compound 4-4, add 5ml of DMF into the reaction flask, then add 0.30g (1.0mmol) and 0.3g (2.3mmol) DIPEA, and react at room temperature for 2 hours. After the reaction is completed, The obtained reaction solution was directly purified by medium pressure preparation to obtain compound 4-5, 0.31 g, ESI-MS (m/z): 932.4 [M+H] + .
  • Step 6 Take 0.2g (0.21mmol) of compound 4-5 into a 5ml reaction flask, add 2ml DMF, 80mg (0.59mmol) HOBT, 0.2g (0.28) MMAE and 0.2g (1.55mmol) DIPEA, react at room temperature After 1 hour, the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD004 0.22 g, ESI-MS (m/z): 1510.9 [M+H] + .
  • Embodiment seventeen the synthesis of LD006
  • Embodiment 18 Synthesis of compound LD007
  • Embodiment 35 the synthesis of compound LD024
  • Step 1 Take compound 3-3 (4.14g, 5.2mmol), add 50ml of DMF to the reaction flask, then add bis(p-nitrobenzene)carbonate (3.5g, 11.5mmol) and DIPEA (3.5g, 27mmol) , and reacted at room temperature for 2 hours. After the reaction, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 24-1, 3.4 g, ESI-MS (m/z): 961.4 [M+H] + .
  • Embodiment thirty-eight the synthesis of compound LD027
  • n 4.
  • Herceptin antibody (anti-Her-2, 10 mg/mL), its buffer is 50 mM PB, 5 mM EDTA, pH 7.2, add 10 mM TCEP (18 microliters) solution and mix well, place at room temperature for 2 hours, add two 30 microliters of methyl sulfoxide was added to the above solution, and then slowly added 5mM LD001 in dimethyl sulfoxide (93 microliters), mixed well, allowed to stand at room temperature for 2 hours, and finally replaced the buffer with a G-25 gel column As a buffer of pH 6.5, a product of LD001 coupled with Herceptin antibody was obtained, named LA001, and its structure is shown below.
  • LD001 was replaced with LD002 to obtain a product of LD002 coupled with the Herceptin antibody, named LA002, the structure of which is shown below.
  • Embodiment 41 is a diagrammatic representation of Embodiment 41.
  • LA003 Herceptin antibody
  • the HIC-HPLC chart of compound LA003 is shown in Figure 1 .
  • LA004 Herceptin antibody
  • the HIC-HPLC profile of LA004 is shown in Figure 2.
  • LA005 Herceptin antibody
  • LA006 Herceptin antibody
  • the HIC-HPLC chart of LA006 is shown in FIG. 4
  • the SEC-HPLC chart is shown in FIG. 5 . According to the SEC retention time and peak area ratio, it can be confirmed that the main conjugated product still maintains the intact structure of the antibody.
  • LA007 Herceptin antibody
  • LA008 Herceptin antibody
  • Herceptin antibody (anti-Her-2, 10 mg/mL), its buffer is 50 mM PB, 5 mM EDTA, pH 7.2, add 10 mM TCEP (67 microliters) solution and mix well, stand at room temperature for 2 hours, and then use desalting
  • the G-25 gel column was used to replace the buffer with a buffer of pH 6.5 to obtain a product conjugated with LD009 and Herceptin antibody, named LA009, whose structure is shown below:
  • LA010 Herceptin antibody
  • LD009 was replaced with LD0011 to obtain a product conjugated with LD011 and Herceptin antibody, named LA011, the structure of which is shown below.
  • LD009 was replaced with LD012 to obtain a product conjugated between LD012 and Herceptin antibody, named LA012, the structure of which is shown below.
  • LD009 was replaced with LD013 to obtain a product of LD013 coupled with Herceptin antibody, named LA013, the structure of which is shown below.
  • LD009 was replaced with LD014 to obtain a product of LD014 coupled with Herceptin antibody, named LA014, the structure of which is shown below.
  • LD001 was replaced with LD015 to obtain a product conjugated between LD015 and Herceptin antibody, named LA015, the structure of which is shown below.
  • LD001 was replaced with LD016 to obtain a product conjugated between LD016 and Herceptin antibody, named LA016, the structure of which is shown below.
  • LA017 Herceptin antibody
  • LD009 was replaced with LD018 to obtain a product of LD018 coupled with the Herceptin antibody, named LA018, the structure of which is shown below.
  • LA019 a product of LD019 coupled with Herceptin antibody, named LA019, the structure of which is shown below.
  • LD001 was replaced with LD020 to obtain a product of LD020 coupled with Herceptin antibody, named LA020, whose structure is shown below.
  • LD009 was replaced with LD021 to obtain a product of LD021 coupled with Herceptin antibody, named LA021, the structure of which is shown below.
  • LD001 was replaced with LD022 to obtain a product conjugated between LD022 and Herceptin antibody, named LA022, the structure of which is shown below.
  • LD001 was replaced with LD023 to obtain a product conjugated with LD023 and Herceptin antibody, named LA023, the structure of which is shown below.
  • LD001 was replaced with LD024 to obtain a product conjugated between LD024 and Herceptin antibody, named LA024, the structure of which is shown below.
  • LD001 was replaced with LD025 to obtain a product of LD025 coupled with Herceptin antibody, named LA025, whose structure is shown below.
  • LD001 was replaced with LD025 to obtain a product of LD025 coupled with Herceptin antibody, named LA026, the structure of which is shown below.
  • LD001 was replaced with LD025 to obtain a product conjugated with LD025 and Herceptin antibody, named LA027, the structure of which is shown below.
  • the cell lines used in the activity assay were purchased from the American Type Culture Collection (ATCC; Manassas, VA), culture medium (Gibco ThermoFisher; Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning; Corning, NY, USA) and 1X penicillin-streptomycin (Corning), and routinely in RPMI-1640 (HCC1954 and SK-BR-3) or DMEM:F-12 (MDA-MB-468), maintained at 37°C Incubate in a humidified environment under 5% CO 2 .
  • Tumor cells were harvested by non-enzymatic dissociation with Cell Stripper Dissociation Reagent (Corning), seeded into 384-well flat-bottom white-walled plates (875 cells per well in 12.5 ⁇ L of complete medium), and maintained at 37°C for 2- Cells were allowed to adhere for 4 hours. Cells were then treated with 12.5 [mu]L of test reagents at a final concentration of 2X (serial dilution) and incubated at 37[deg.]C for 120 hours. According to the instrument manufacturer's usage protocol, use Cell viability assay (Promega; Madison, WI, USA) to determine inhibition of cancer cell growth. Luminescence was measured using a Tecan Spark multimode microplate reader (Tecan Group Ltd.; Mandorf, Switzerland).
  • EC50 median inhibitory maximal effective concentration
  • ADCs antibody drug conjugates
  • phosphorylcholine functional groups were introduced: (1) the Lys side chain of the Val-Lys dipeptide, (2) directly linked to Gly or Ser within the Gly/Ser-Val-Cit/Lys tripeptide.
  • Figure 8(A) and Table 1 below between the four series 1 MMAE payload ADCs containing phosphorylcholine-modified Lys side chains (LA003-LA006), no targeting of HER2-positive HCC1954 and SK was observed - Significant differences in activity of BR-3 cells.
  • MDA-MB-468 cells (Fig. 8, B; Table 1). As expected, LA015 and LA016 showed higher targeting specificity than MMAE alone in HCC1954 and SK-BR-3, but not in MDA-MB-468 cells.
  • the solubility of VL(PC)-PAB in water is >100mg/ml
  • the solubility of MC-VL(PC)-PAB in water is >33mg/ml
  • the solubility of VC-PAB in 1000ml of water is ⁇ 0.1mg/ml
  • MC - The solubility of VC-PAB in water is ⁇ 0.1 mg/ml.
  • the water solubility of the phosphorylcholine-modified dipeptide linkers VL(PC)-PAB and MC-VL(PC)-PAB is higher than that of conventional VC-PAB and MC-PAB, respectively.
  • VC-PAB is at least 1000 times and 330 times larger.

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Abstract

Provided are modified amino acids and an application thereof in ADC. The modified amino acids are phosphorylcholine group-modified amino acids represented by R1-L1-N(R2)-R3, wherein R1, L1, N, R2 and R3 are as defined herein. Further provided are peptides containing the modified amino acids. ADCs made by using such modified amino acids or modified peptides have significantly improved solubility.

Description

修饰的氨基酸及其在ADC中的应用Modified amino acids and their applications in ADCs 技术领域technical field
本发明涉及药物领域，具体涉及抗体药物偶联物(ADC)，更具体涉及修饰的氨基酸及其在ADC中的应用。The present invention relates to the field of medicine, in particular to antibody drug conjugates (ADC), and more particularly to modified amino acids and their application in ADC.
背景技术Background technique
抗体药物偶联物(antibody-drug conjugate，ADC)是通过一个化学链接将具有生物活性的小分子药物连接到单抗上，单抗作为载体将小分子药物靶向运输到目标细胞中。Antibody-drug conjugate (ADC) is to link a biologically active small molecule drug to a monoclonal antibody through a chemical link, and the monoclonal antibody acts as a carrier to target and transport the small molecule drug to the target cells.
ADC药物结构较为复杂，且不同ADC药物设计之间存在较大的差异。即使同一靶点的不同药物，由于识别位点、连接位点、连接子及所连接小分子的不同，其毒性的差异显而易见。ADC drug structure is relatively complex, and there are great differences between different ADC drug designs. Even for different drugs of the same target, the differences in toxicity are obvious due to the differences in recognition sites, attachment sites, linkers and small molecules to which they are attached.
发明内容SUMMARY OF THE INVENTION
本文第一方面提供下式I所示的磷酰胆碱基团修饰的氨基酸：A first aspect herein provides a phosphorylcholine group-modified amino acid of Formula I below:
R 1-L 1-N(R 2)-R 3   (I) R 1 -L 1 -N(R 2 )-R 3 (I)
式I中：In formula I:
R 1为磷酰胆碱基团； R 1 is a phosphorylcholine group;
N代表氨基酸分子上的氮原子；N represents the nitrogen atom on the amino acid molecule;
R 2为H、C1-C4烷基或-L 1-磷酰胆碱基团； R 2 is H, C1-C4 alkyl or -L 1 -phosphorylcholine group;
各L 1独立为连接基团； Each L 1 is independently a linking group;
R 3代表氨基酸分子上除所示N原子外的其余部分，所述其余部分任选地被保护基团保护。 R3 represents the remainder of the amino acid molecule other than the indicated N atom, which is optionally protected by a protecting group.
本文第二方面提供一种磷酰胆碱修饰的肽，所述肽至少包括一个磷酰胆碱基团，所述磷酰胆碱基团与所述肽所含的1个或多个氨基酸的N原子和/或氧 原子经由连接基团共价连接。A second aspect herein provides a phosphorylcholine-modified peptide, the peptide comprising at least one phosphorylcholine group, the phosphorylcholine group having a relationship with one or more amino acids contained in the peptide The N atoms and/or oxygen atoms are covalently linked via a linking group.
本发明第三方面提供具有下式II所示结构的连接子化合物：The third aspect of the present invention provides a linker compound having the structure shown in the following formula II:
R 4-L 2-P-R 5   (II) R 4 -L 2 -PR 5 (II)
式中：where:
R 4为可与蛋白中的半胱氨酸残基中的巯基或赖氨酸残基上的游离氨基发生反应、从而将式II连接到蛋白的基团； R 4 is a group that can react with a sulfhydryl group in a cysteine residue in a protein or a free amino group on a lysine residue to attach Formula II to the protein;
L 2为连接基团； L 2 is a linking group;
P为权利要求1-3中任一项所述的氨基酸或权利要求4-8中任一项所述的肽；所述氨基酸或肽与L 2和R 5共价连接； P is the amino acid of any one of claims 1-3 or the peptide of any one of claims 4-8 ; the amino acid or peptide is covalently linked to L2 and R5 ;
R 5为： R5 is:
Figure PCTCN2022072943-appb-000001
Figure PCTCN2022072943-appb-000001
其中，R 6为H、卤素、C1-10烷基、C3-C8环烷基、C2-C8烯基、C2-C8炔基、C3-C8环烯基、6-14元芳基、芳烷基、5-10元杂芳基或4-10元杂环基；R 9为H、-C(O)-N(R 7)-(CH 2) n-NHR 8或硝基取代的苯氧基羰基；R 7为H或C1-6烷基；R 8为H或C1-6烷基；和n为1-6的整数。 Wherein, R 6 is H, halogen, C1-10 alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl, 6-14-membered aryl, aralkyl R 9 is H, -C(O)-N(R 7 )-(CH 2 ) n -NHR 8 or nitro-substituted phenoxy R 7 is H or C1-6 alkyl; R 8 is H or C1-6 alkyl; and n is an integer of 1-6.
本文第四方面提供下式III所示的与本发明任一实施方案所述的连接子化合物共价连接的药物：A fourth aspect herein provides a drug covalently linked to the linker compound described in any of the embodiments of the present invention represented by the following formula III:
R 4-L 2-P-R 5-D    (III) R 4 -L 2 -PR 5 -D (III)
式中：R 4、L 2、P和R 5如本文任一实施方案所述；D表示药物分子去除了一个H原子后获得的基团，与R 5共价连接；优选地，药物分子通过与R 5形成碳酸酯键(-OCO-)或氨基甲酸酯(-OCNH-)而与R 5相连。 In the formula: R 4 , L 2 , P and R 5 are as described in any of the embodiments herein; D represents a group obtained by removing one H atom from the drug molecule, which is covalently linked to R 5 ; preferably, the drug molecule is A carbonate bond (-OCO-) or a carbamate (-OCNH-) is formed with R 5 to connect with R 5 .
本文第五方面提供一种ADC，其结构式如下式IV所示：The fifth aspect of this paper provides an ADC, the structural formula of which is shown in the following formula IV:
A-R 4-L 2-P-R 5-D    (IV) AR 4 -L 2 -PR 5 -D (IV)
式中，R 4、L 2、P和R 5如本文任一实施方案所述，D如如本文任一实施方案所述，A为抗体或其抗原结合片段；其中，所述抗体或其抗原结合片段通过其所含有的巯基与R 4共价连接。 In the formula, R 4 , L 2 , P and R 5 are as described in any of the embodiments herein, D is as described in any of the embodiments herein, and A is an antibody or an antigen-binding fragment thereof; wherein, the antibody or antigen thereof The binding fragment is covalently linked to R through the sulfhydryl group it contains.
本文第六方面还提供本文任一实施方案所述的氨基酸、肽、连接子化合物以及药物在制备ADC中的应用，或在改善抗体溶解性中的应用，或者在制备用于改善抗体溶解性的制剂中的应用。The sixth aspect herein also provides the use of the amino acids, peptides, linker compounds and drugs described in any of the embodiments herein in the preparation of ADCs, or in improving the solubility of antibodies, or in the preparation of antibodies for improving the solubility of antibodies. application in formulations.
本发明第七方面还提供一种组合物，其含有本文任一实施方案所述的氨基酸、肽、连接子化合物或与连接子化合物共价连接的药物。组合物中可含有其它合适的辅料，如溶剂等。A seventh aspect of the present invention also provides a composition comprising the amino acid, peptide, linker compound or drug covalently linked to the linker compound according to any of the embodiments herein. The composition may contain other suitable excipients, such as solvents and the like.
本文各方面的详细内容如下文所详述。Details of various aspects of this document are set forth in detail below.
附图说明Description of drawings
图1：LA003的HIC-HPLC图。Figure 1: HIC-HPLC profile of LA003.
图2：LA004的HIC-HPLC图。Figure 2: HIC-HPLC profile of LA004.
图3：LA005的HIC-HPLC图。Figure 3: HIC-HPLC profile of LA005.
图4：LA006的HIC-HPLC图。Figure 4: HIC-HPLC profile of LA006.
图5：LA006的SEC-HPLC图。Figure 5: SEC-HPLC profile of LA006.
图6：LA009的HIC-HPLC图。Figure 6: HIC-HPLC profile of LA009.
图7：LA003、LA004、LA005和LA006的HIC-HPLC对比图。从图中可以得出以下结论：DAR值均为4的四个ADC，小分子部分带有两个磷脂酰胆碱的ADC的亲水性更强，烷基形式的磷脂酰胆碱的亲水性强于酰胺形式的磷脂酰胆碱。Figure 7: HIC-HPLC comparison of LA003, LA004, LA005 and LA006. The following conclusions can be drawn from the figure: the four ADCs with DAR values of 4, the ADC with two phosphatidylcholines in the small molecule part is more hydrophilic, and the alkyl form of phosphatidylcholine is more hydrophilic It is more potent than the amide form of phosphatidylcholine.
图8：含磷酰胆碱修饰的HER2 ADC对人SK-BR-3，HCC1954和MDA-MB-468乳腺癌细胞系的细胞毒活性。(A)Val-Lys二肽连接子内的Lys侧链进行含磷酰胆碱修饰；(B)对Gly/Ser-Val-Cit三肽连接子内的Gly或Ser进行直接修饰；(C)Lys的侧链，并直接连接到Ser-Val-Lys三肽连接子中的Ser，(D)Lys的侧链和/或在Val-Lys或Ser-Val-Lys连接子内直接与Ser连接。抗HER2 ADC数据点代表至少三次重复的平均归一化细胞生存力±SEM。至少两个独立实验的结果。Figure 8: Cytotoxic activity of phosphorylcholine-modified HER2 ADCs against human SK-BR-3, HCC1954 and MDA-MB-468 breast cancer cell lines. (A) Phosphorylcholine-containing modification of the Lys side chain in the Val-Lys dipeptide linker; (B) Direct modification of Gly or Ser in the Gly/Ser-Val-Cit tripeptide linker; (C) The side chain of Lys and directly linked to Ser in the Ser-Val-Lys tripeptide linker, (D) the side chain of Lys and/or directly linked to Ser within a Val-Lys or Ser-Val-Lys linker. Anti-HER2 ADC data points represent mean normalized cell viability ± SEM of at least three replicates. Results of at least two independent experiments.
图9：LA003、LA005和对照Her2-McVCPABMMAE的HIC图谱。a、b和c分别表示对照、LA003和LA005的HIC图谱。Figure 9: HIC profiles of LA003, LA005 and control Her2-McVCPABMMAE. a, b and c represent HIC profiles of control, LA003 and LA005, respectively.
具体实施方式Detailed ways
本发明旨在提供一种新的修饰的氨基酸或修饰的肽，用这类修饰的氨基酸或修饰的肽制得的ADC具有明显提高的溶解度，同时还保留了所需的生物学活性或药效。具体而言，本发明使用磷酰胆碱来修饰氨基酸，构建修饰的氨基酸或修饰的肽。The present invention aims to provide a new modified amino acid or modified peptide, and the ADC prepared with such modified amino acid or modified peptide has significantly improved solubility, while still retaining the desired biological activity or pharmacological effect . Specifically, the present invention uses phosphorylcholine to modify amino acids to construct modified amino acids or modified peptides.
术语the term
除非另有定义，否则本文所有科技术语具有的涵义与权利要求主题所属领域技术人员通常理解的涵义相同。除非另有说明，本文全文引用的所有专利、专利申请、公开材料通过引用方式整体并入本文。Unless otherwise defined, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, publications cited throughout this document are incorporated by reference in their entirety unless otherwise indicated.
应理解，上述简述和下文的详述为示例性且仅用于解释，而不对本发明主题作任何限制。在本申请中，除非另有具体说明，否则使用单数时也包括复数。必须注意，除非文中另有清楚的说明，否则在本说明书和权利要求书中所用的单数形式包括所指事物的复数形式。还应注意，除非另有说明，否则所用“或”、“或者”表示“和/或”。此外，所用术语“包括”以及其它形式，例如“包含”、“含”和“含有”并非限制性，其可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”之义。It is to be understood that both the foregoing brief description and the following detailed description are exemplary and explanatory only and do not limit the subject matter of the invention in any way. In this application, the use of the singular also includes the plural unless specifically stated otherwise. It must be noted that the singular forms used in this specification and the claims include the plural forms of referents unless the context clearly dictates otherwise. It should also be noted that the use of "or" and "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "comprising" and other forms such as "comprising", "including" and "containing" is not limiting, and it can be open, semi-closed and closed. In other words, the term also includes the meaning of "consisting essentially of," or "consisting of."
可在参考文献(包括Carey and Sundberg"ADVANCED ORGANIC CHEMISTRY 4TH ED."Vols.A(2000)and B(2001),Plenum Press,New York)中找到对标准化学术语的定义。除非另有说明，否则采用本领域技术范围内的常规方法，如质谱、NMR、IR和UV/VIS光谱法和药理学方法。除非提出具体定义，否则本文在分析化学、有机合成化学以及药物和药物化学的有关描述中采用的术语是本领域已知的。可在化学合成、化学分析、药物制备、制剂和递送，以及对患者的治疗中使用标准技术。例如，可利用厂商对试剂盒的使用说明，或者按照本领域公知的方式或本发明的说明来实施反应和进行纯化。通常可根据本说明书中引用和讨论的多个概要性和较具体的文献中的描述，按照本领域熟知的常规方法实施上述技术和方法。在本说明书中，可由本领域技术 人员选择基团及其取代基以提供稳定的结构部分和化合物。Definitions of standard chemical terms can be found in references including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise stated, conventional methods within the skill in the art, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy and pharmacological methods are employed. Unless specific definitions are presented, the terms employed herein in the related descriptions of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients. For example, the reaction and purification can be carried out using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention. The techniques and methods described above can generally be carried out according to conventional methods well known in the art from the descriptions in the various general and more specific documents cited and discussed in this specification. In this specification, groups and their substituents can be selected by those skilled in the art to provide stable moieties and compounds.
当通过从左向右书写的常规化学式描述取代基时，该取代基也同样包括从右向左书写结构式时所得到的在化学上等同的取代基。举例而言，-CH 2O-等同于-OCH 2-。 When substituents are described by conventional chemical formulae written from left to right, the substituents also include the chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH2O- is equivalent to -OCH2- .
在本文中定义的某些化学基团前面通过简化符号来表示该基团中存在的碳原子总数。例如，C1-6烷基是指具有总共1至6个碳原子的如下文所定义的烷基。简化符号中的碳原子总数不包括可能存在于所述基团的取代基中的碳。Certain chemical groups defined herein are preceded by abbreviated symbols to indicate the total number of carbon atoms present in the group. For example, C1-6 alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the simplified notation does not include carbons that may be present in the substituents of the group.
除前述以外，当用于本申请的说明书及权利要求书中时，除非另外特别指明，否则以下术语具有如下所示的含义。In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings shown below unless specifically indicated otherwise.
在本申请中，术语“卤素”是指氟、氯、溴或碘。In this application, the term "halogen" refers to fluorine, chlorine, bromine or iodine.
“羟基”是指-OH基团。"Hydroxy" refers to the -OH group.
“羟基烷基”是指被羟基(-OH)取代的如下文所定义的烷基。"Hydroxyalkyl" refers to an alkyl group as defined below substituted with hydroxy (-OH).
“羰基”是指-C(＝O)-基团。"Carbonyl" refers to a -C(=O)- group.
“硝基”是指-NO 2。 "Nitro" refers to -NO2 .
“氰基”是指-CN。"Cyano" refers to -CN.
“氨基”是指-NH 2。 "Amino" refers to -NH2 .
“羧基”是指-COOH。"Carboxyl" refers to -COOH.
在本申请中，作为基团或是其它基团的一部分，“烷基”指完全饱和的直链或支链的烃链基，仅由碳原子和氢原子组成、具有例如1至10个(优选为1至8个，更优选为1至6个)碳原子，且通过单键与分子的其余部分连接。烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、2-甲基丁基、2,2-二甲基丙基、正己基、庚基、2-甲基己基、3-甲基己基、辛基、壬基和癸基等。在一些实施方案中，烷基是C1-C4烷基。In this application, as a group or as part of another group, "alkyl" refers to a fully saturated straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having, for example, 1 to 10 ( Preferably from 1 to 8, more preferably from 1 to 6) carbon atoms and is attached to the rest of the molecule by a single bond. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2 - Dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl and decyl, etc. In some embodiments, the alkyl group is a C1-C4 alkyl group.
在本申请中，作为基团或是其它基团的一部分，“烯基”指仅由碳原子和氢原子组成、含有至少一个双键、具有例如2至10个(优选为2至8个，更优选为2至6个，更优选2至4个)碳原子且通过单键与分子的其余部分连接的直链或支链的烃链基团。烯基包括但不限于乙烯基、丙烯基、烯丙基、丁-1-烯基、丁-2-烯基、戊-1-烯基、戊-1,4-二烯基等。In this application, as a group or as part of another group, "alkenyl" means consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, having, for example, 2 to 10 (preferably 2 to 8, More preferably a straight or branched hydrocarbon chain group of 2 to 6, more preferably 2 to 4) carbon atoms and linked to the rest of the molecule by a single bond. Alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-1,4-dienyl, and the like.
在本申请中，作为基团或是其它基团的一部分，“炔基”指仅由碳原子和氢原子组成、含有至少一个三键、具有例如2至10个(优选为2至8个，更优选为2至6个，更优选2至4个)碳原子且通过单键与分子的其余部分连接的直链或支链的烃链基团。炔基包括但不限于乙炔基、丙炔基等。In this application, as a group or as part of another group, "alkynyl" means consisting only of carbon atoms and hydrogen atoms, containing at least one triple bond, having, for example, 2 to 10 (preferably 2 to 8, More preferably a straight or branched hydrocarbon chain group of 2 to 6, more preferably 2 to 4) carbon atoms and linked to the rest of the molecule by a single bond. Alkynyl groups include, but are not limited to, ethynyl, propynyl, and the like.
在本申请中，作为基团或是其它基团的一部分，“环烷基”指仅由碳原子和氢原子组成的稳定的非芳香族单环烃基。环烷基的环碳原子数通常为3-8个。环烷基的实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基和环辛基。As used herein, as a group or as part of another group, "cycloalkyl" refers to a stable non-aromatic monocyclic hydrocarbon group consisting only of carbon and hydrogen atoms. The number of ring carbon atoms of the cycloalkyl group is usually 3 to 8. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
在本申请中，作为基团或是其它基团的一部分，“杂环基”指由2至14个碳原子(例如2、3、4、5、6、7、8、9、10、11、12、13或14个碳原子)以及1至6个选自氮、氧和硫的杂原子组成的稳定的5元至10元非芳香族环状基团。除非本说明书中另外特别指明，否则杂环基可以为单环或双环的环体系。杂环基可为部分或完全饱和。杂环基可以经由碳原子或者杂原子并通过单键与分子其余部分连接。杂环基的实例包括但不限于：氮杂环丁烷基、吡咯烷基、吗啉基、哌嗪基、高哌嗪基、哌啶基、硫代吗啉基、吡喃基、四氢吡喃基、噻喃基、四氢呋喃基、噁嗪基、二氧环戊基、四氢异喹啉基、十氢异喹啉基、咪唑啉基、咪唑烷基、喹嗪基、噻唑烷基、异噻唑烷基、异噁唑烷基、二氢吲哚基、八氢吲哚基、八氢异吲哚基和吡唑烷基等。As used herein, as a group or as part of another group, "heterocyclyl" means from 2 to 14 carbon atoms ( eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 carbon atoms) and a stable 5- to 10-membered non-aromatic cyclic group consisting of 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless specifically stated otherwise in this specification, a heterocyclyl group may be a monocyclic or bicyclic ring system. Heterocyclyl groups can be partially or fully saturated. A heterocyclyl group can be attached to the rest of the molecule via a carbon atom or a heteroatom and through a single bond. Examples of heterocyclyl groups include, but are not limited to: azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, pyranyl, tetrahydro Pyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolane, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinazinyl, thiazolidinyl , isothiazolidinyl, isoxazolidinyl, indoline, octahydroindolyl, octahydroisoindolyl and pyrazolidine, etc.
在本申请中，作为基团或是其它基团的一部分，“芳基”意指具有6至14个碳原子(优选具有6至10个碳原子，例如6、7、8、9或10个碳原子)的共轭烃环体系基团。芳基可以为单环或双环的环体系。芳基的实例包括但不限于苯基和萘基等。In this application, as a group or as part of another group, "aryl" means having 6 to 14 carbon atoms (preferably having 6 to 10 carbon atoms, such as 6, 7, 8, 9 or 10 carbon atoms) carbon atoms) of the conjugated hydrocarbon ring system group. Aryl groups can be monocyclic or bicyclic ring systems. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.
在本申请中，“芳烷基”是指被上文所定义的芳基所取代的上文所定义的烷基。In this application, "aralkyl" refers to an alkyl group as defined above substituted with an aryl group as defined above.
在本申请中，作为基团或是其它基团的一部分，“杂芳基”意指环内具有1至15个碳原子(优选具有1至10个碳原子，例如1、2、3、4、5、6、7、8、9或10个碳原子)和1至6个选自氮、氧和硫的杂原子的5元至16元、优选5-10元共轭环系基团。除非本说明书中另外特别指明，否则杂芳基可为单环、 双环、三环或更多环的环体系。杂芳基的实例包括但不限于噻吩基、咪唑基、吡唑基、噻唑基、噁唑基、噁二唑基、异噁唑基、吡啶基、嘧啶基、吡嗪基、哒嗪基、苯并咪唑基、苯并吡唑基、吲哚基、呋喃基、吡咯基、***基、四唑基、三嗪基、吲嗪基、异吲哚基、吲唑基、异吲唑基、嘌呤基、喹啉基、异喹啉基、二氮萘基、萘啶基、喹噁啉基、蝶啶基、咔唑基、咔啉基、菲啶基、菲咯啉基、吖啶基、吩嗪基、异噻唑基、苯并噻唑基、吡咯并吡啶基、苯并吡咯基、苯并噻吩基、噁***基、噌啉基、喹唑啉基、苯硫基、中氮茚基、邻二氮杂菲基、异噁唑基、吩噁嗪基、吩噻嗪基等。In this application, as a group or part of another group, "heteroaryl" means a ring having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, a 5- to 16-membered, preferably 5-10 membered, conjugated ring system group. Unless specifically stated otherwise in this specification, a heteroaryl group can be a monocyclic, bicyclic, tricyclic or multicyclic ring system. Examples of heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, Benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indazinyl, isoindolyl, indazolyl, isoindazolyl , purinyl, quinolinyl, isoquinolinyl, diazanaphthyl, naphthyridinyl, quinoxalinyl, pteridyl, carbazolyl, carboline, phenanthridine, phenanthroline, acridine base, phenazinyl, isothiazolyl, benzothiazolyl, pyrrolopyridyl, benzopyrrolyl, benzothienyl, oxtriazolyl, cinnoline, quinazolinyl, phenylthio, middle nitrogen Indenyl, o-phenanthrenyl, isoxazolyl, phenoxazinyl, phenothiazinyl, etc.
在本申请中，基团可任选地被取代。“任选地”表示随后描述的事件或状况可能发生也可能不发生，且该描述同时包括该事件或状况发生和不发生的情况。例如，“任选取代的芳基”表示芳基被取代或未被取代，且该描述同时包括被取代的芳基与未被取代的芳基。本发明权利要求书和说明书部分所述的“任选”的取代基包括但不限于烷基、烯基、炔基、卤素、卤代烷基、卤代环氧基、卤代烯基、卤代炔基、氰基、环烷基-O-、硝基、氨基、任选取代的氨基、烷氧基、环烷基、任选取代的芳基、任选取代的杂芳基、任选取代的环烃基和任选取代的杂环基中的一个或多个。在优选的实施方案中，取代基包括但不限于C1-C6烷基、卤代烷基、环烷基、氨基、C1-C6烷基取代的氨基、卤素、氰基、C3-C8环烷基-O-、芳基和杂芳基中的一个或多个。在本申请中，当被取代时，视被取代的基团的结构，取代基的数量可以是1-5个，如1-3个。例如，苯基可被1-3个选自C1-C6烷氧基、C3-C8环烷基-O-、卤素和氨基的取代基取代。In this application, groups may be optionally substituted. "Optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted and unsubstituted aryl groups. The "optional" substituents described in the claims and specification sections of the present invention include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloepoxy, haloalkenyl, haloalkyne , cyano, cycloalkyl-O-, nitro, amino, optionally substituted amino, alkoxy, cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted One or more of cycloalkyl and optionally substituted heterocyclyl. In preferred embodiments, substituents include, but are not limited to, C1-C6 alkyl, haloalkyl, cycloalkyl, amino, C1-C6 alkyl substituted amino, halogen, cyano, C3-C8 cycloalkyl-O -, one or more of aryl and heteroaryl. In the present application, when substituted, the number of substituents may be 1-5, such as 1-3, depending on the structure of the substituted group. For example, phenyl may be substituted with 1-3 substituents selected from C1-C6 alkoxy, C3-C8 cycloalkyl-O-, halogen and amino.
本领域技术人员还应当理解，在下文所述的方法中，中间体化合物官能团可能需要由适当的保护基保护。这样的官能团包括羟基、氨基、巯基及羧酸。合适的羟基保护基包括三烷基甲硅烷基或二芳基烷基甲硅烷基(例如叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基或三甲基甲硅烷基)、四氢吡喃基、苄基等。合适的氨基、脒基及胍基的保护基包括叔丁氧羰基、苄氧羰基等。合适的巯基保护基包括-C(O)-R”(其中R”为烷基、芳基或芳烷基)、对甲氧基苄基、三苯甲基等。合适的羧基保护基包括烷基、芳基或芳烷基酯类。It will also be understood by those skilled in the art that in the methods described below, intermediate compound functional groups may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto, and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl groups (eg tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl) , tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable thiol protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, and the like. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.
保护基可根据本领域技术人员已知的和如本文所述的标准技术来引入和除去。保护基的使用详述于Greene,T.W.与P.G.M.Wuts,Protective Groups in Organi Synthesis,(1999),4th Ed.,Wiley中。在一些实施方案中，本申请的保护基团为氨基的保护基团，如Boc(叔丁氧羰基)。保护基还可为聚合物树脂。Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, T.W. and P.G.M. Wuts, Protective Groups in Organi Synthesis, (1999), 4th Ed., Wiley. In some embodiments, the protecting group of the present application is a protecting group for an amino group, such as Boc (tert-butoxycarbonyl). The protecting group can also be a polymeric resin.
磷酰胆碱基团修饰的氨基酸Amino acids modified with phosphorylcholine groups
本文中，氨基酸或氨基酸分子可互换使用，其具有本领域周知含义。在一些实施方案中，氨基酸为天然氨基酸。本文中，由生物合成的氨基酸统称为“天然氨基酸”。氨基酸分子的例子包括但不限于本领域周知的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、硒半胱氨酸、吡咯赖氨酸和鸟氨酸。Herein, amino acids or amino acid molecules are used interchangeably and have meanings well known in the art. In some embodiments, the amino acid is a natural amino acid. Herein, amino acids synthesized by biosynthesis are collectively referred to as "natural amino acids". Examples of amino acid molecules include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, Cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, pyrrole Lysine and Ornithine.
本申请提供修饰的氨基酸，其一个氮原子被基团R 2和基团R 1-L 1-取代。本申请的修饰的氨基酸可为下式I所示的化合物： The application provides modified amino acids in which one nitrogen atom is replaced by a group R 2 and a group R 1 -L 1 -. The modified amino acid of the present application can be the compound shown in the following formula I:
R 1-L 1-N(R 2)-R 3   (I) R 1 -L 1 -N(R 2 )-R 3 (I)
式I中：In formula I:
R 1为磷酰胆碱基团； R 1 is a phosphorylcholine group;
N代表氨基酸分子上的氮原子；N represents the nitrogen atom on the amino acid molecule;
R 2为H、C1-C4烷基或-L 1-磷酰胆碱基团； R 2 is H, C1-C4 alkyl or -L 1 -phosphorylcholine group;
各L 1独立为连接基团； Each L 1 is independently a linking group;
R 3代表氨基酸分子上除所示N原子外的其余部分，该其余部分任选地被保护基团保护。 R3 represents the remainder of the amino acid molecule other than the indicated N atom, which remainder is optionally protected by a protecting group.
式I中，优选地，所述磷酰胆碱基团如下式所示：In formula I, preferably, the phosphorylcholine group is shown in the following formula:
Figure PCTCN2022072943-appb-000002
Figure PCTCN2022072943-appb-000002
式中，波浪线表示该磷酰胆碱基团与L 1连接的位置。 In the formula, the wavy line indicates the position where the phosphorylcholine group is linked to L 1 .
式I中，优选地，所述各L 1可独立表示为-L a-L b-，其中L a选自C1-C6亚 烷基、C2-C6亚烯基或C2-C6亚炔基；L b不存在，或者为羰基、酯基(-COO-)、-O-SO 2-或-NH-SO 2-。优选地，L b不存在或为羰基。优选地，L 1通过L b与所述N共价连接。在一些实施方案中，L b和所示的N形成酰胺基或磺酰氨基。在优选的实施方案中，各L 1独立为C1-C6亚烷基或C1-C6亚烷基羰基。 In formula I, preferably, each L 1 can be independently represented as -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; L b is absent, or is a carbonyl group, an ester group (-COO-), -O- SO2- or -NH- SO2- . Preferably, L b is absent or carbonyl. Preferably, L 1 is covalently linked to said N through L b . In some embodiments, L b and the indicated N form an amide group or a sulfonamido group. In preferred embodiments, each L 1 is independently C1-C6 alkylene or C1-C6 alkylenecarbonyl.
式I中，优选地，R 3所述的氨基酸分子选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、硒半胱氨酸、吡咯赖氨酸和鸟氨酸。优选地，所述氨基酸分子选自：赖氨酸、鸟氨酸、缬氨酸、酪氨酸、谷氨酰胺、丙氨酸、精氨酸、天冬酰胺、亮氨酸、异亮氨酸、苏氨酸、丝氨酸、苯丙氨酸、缬氨酸和甘氨酸。更优选地，所述氨基酸分子选自：缬氨酸、鸟氨酸、赖氨酸、丝氨酸、甘氨酸和瓜氨酸。更优选地，所述氨基酸分子选自：鸟氨酸、赖氨酸、丝氨酸和甘氨酸。 In formula I, preferably, the amino acid molecule described in R is selected from: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine , tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenium half Cystine, pyrrolysine and ornithine. Preferably, the amino acid molecule is selected from: lysine, ornithine, valine, tyrosine, glutamine, alanine, arginine, asparagine, leucine, isoleucine , threonine, serine, phenylalanine, valine and glycine. More preferably, the amino acid molecule is selected from the group consisting of: valine, ornithine, lysine, serine, glycine and citrulline. More preferably, the amino acid molecule is selected from the group consisting of: ornithine, lysine, serine and glycine.
式I中，优选地，所述N不是氨基酸分子中的芳基、杂芳基或杂环基上的环氮原子。在一些实施方案中，所述N不是氨基酸分子中用来形成肽键的氮原子。In formula I, preferably, the N is not a ring nitrogen atom on an aryl group, a heteroaryl group or a heterocyclic group in the amino acid molecule. In some embodiments, the N is not a nitrogen atom used to form peptide bonds in amino acid molecules.
本领域周知，部分氨基酸存在游离的氨基，所述游离氨基指通常不与其它氨基酸形成肽键的氨基。具有游离氨基的氨基酸包括赖氨酸、精氨酸、谷氨酰胺和天冬酰胺。在本发明提供的磷酰胆碱基团修饰的氨基酸中，当该氨基酸为赖氨酸、精氨酸、谷氨酰胺和天冬酰胺时，所述游离的氨基可被保护，如与Boc保护基团共价连接，形成“叔丁氧基羰基-NH-”基团。It is well known in the art that some amino acids have free amino groups, and the free amino groups refer to amino groups that do not normally form peptide bonds with other amino acids. Amino acids with free amino groups include lysine, arginine, glutamine and asparagine. In the amino acid modified with phosphorylcholine group provided by the present invention, when the amino acid is lysine, arginine, glutamine and asparagine, the free amino group can be protected, such as protection with Boc The groups are covalently linked to form a "tert-butoxycarbonyl-NH-" group.
在一个或多个实施方案中，所述修饰的氨基酸选自LN001至LN010和LN012至LN025所示的化合物。In one or more embodiments, the modified amino acid is selected from the compounds shown in LN001 to LN010 and LN012 to LN025.
磷酰胆碱修饰的肽Phosphorylcholine-modified peptides
另一方面，本申请提供一种磷酰胆碱修饰的肽，所述肽至少包括一个磷酰胆碱基团，所述磷酰胆碱基团与所述肽所含的1个或多个氨基酸的N原子和/或氧原子经由连接基团共价连接。应理解，所述肽中的氨基酸残基之间通过肽键连接。所述磷酰胆碱基因可连接至形成肽键的N上，也可连接指未形成肽键 的N上；优选不连接于氨基酸残基的芳基、杂芳基或杂环基的环氮原子。In another aspect, the present application provides a phosphorylcholine-modified peptide, the peptide includes at least one phosphorylcholine group, and the phosphorylcholine group is combined with one or more phosphorylcholine groups contained in the peptide. The N and/or oxygen atoms of the amino acid are covalently linked via a linking group. It will be understood that the amino acid residues in the peptide are linked by peptide bonds. The phosphorylcholine gene can be connected to the N that forms a peptide bond, or it can be connected to the N that does not form a peptide bond; preferably, it is not connected to the ring nitrogen of an aryl, heteroaryl or heterocyclic group of an amino acid residue atom.
优选地，本申请所述的肽具有2-10个氨基酸残基，更优选具有2-5个氨基酸残基，即所述肽为二肽、三肽、四肽或五肽。Preferably, the peptide described in this application has 2-10 amino acid residues, more preferably 2-5 amino acid residues, that is, the peptide is a dipeptide, tripeptide, tetrapeptide or pentapeptide.
优选地，所述肽为可被病理环境中的酶水解的肽。优选地，所述酶可为蛋白水解酶、蛋白酶或肽酶。更优选地，所述酶选自：半胱氨酸蛋白酶、天冬酰胺蛋白酶、天冬氨酸蛋白酶、谷氨酸蛋白酶、苏氨酸蛋白酶、明胶酶、金属蛋白酶和天冬酰胺肽裂解酶中的一种或多种。优选地，所述肽可被病理环境中的一种水解，或被多种酶同时水解。所述病理环境可以是本领域周知的各种期望进行治疗的环境，例如肿瘤细胞所存在的病理环境，如癌组织，或者为炎症部位，或者为感染部位，或者为需治疗的其它组织。在优选的实施方案中，所述酶选自天冬酰胺内肽酶(Legumain)。Preferably, the peptide is a peptide that can be hydrolyzed by enzymes in a pathological environment. Preferably, the enzyme may be a proteolytic enzyme, protease or peptidase. More preferably, the enzyme is selected from the group consisting of cysteine proteases, asparagine proteases, aspartic proteases, glutamic proteases, threonine proteases, gelatinases, metalloproteases and asparagine peptide lyases one or more of. Preferably, the peptide can be hydrolyzed by one in a pathological environment, or by multiple enzymes simultaneously. The pathological environment can be various environments known in the art where treatment is desired, for example, a pathological environment where tumor cells exist, such as cancer tissue, or a site of inflammation, or a site of infection, or other tissues to be treated. In a preferred embodiment, the enzyme is selected from asparagine endopeptidase (Legumain).
所述肽所含的氨基酸残基可以是本领域周知的各种氨基酸残基，所述氨基酸残基可以是前文R 3定义的氨基酸分子中所述的任意一种或多种氨基酸残基。应理解，肽中氨基酸残基的数量及种类的选择可根据该肽将被使用的环境中所存在的酶决定。例如，若病理环境中含有天冬酰胺内肽酶，则优选地，该肽为可被天冬酰胺内肽酶识别并水解/切割以释放出治疗剂的肽。在一些实施方案中，所述肽中的氨基酸残基选自：缬氨酸、鸟氨酸、赖氨酸、丝氨酸、甘氨酸和瓜氨酸中的一种或多种。 The amino acid residues contained in the peptide may be various amino acid residues known in the art, and the amino acid residues may be any one or more amino acid residues described in the amino acid molecule defined by R3 above. It will be appreciated that the selection of the number and type of amino acid residues in a peptide can be determined by the enzymes present in the environment in which the peptide will be used. For example, if the pathological environment contains asparagine endopeptidase, preferably, the peptide is one that can be recognized and hydrolyzed/cleaved by asparagine endopeptidase to release the therapeutic agent. In some embodiments, the amino acid residues in the peptide are selected from one or more of valine, ornithine, lysine, serine, glycine, and citrulline.
所述连接基团可以为本文任一实施方案所述的L 1。 The linking group may be L 1 as described in any of the embodiments herein.
在一个或多个实施方案中，所述肽含有至少一个本文任一实施方案所述的磷酰胆碱基团修饰的氨基酸。优选地，所述肽中的至少一个被磷酰胆碱基团修饰的氨基酸残基选自：LN001至LN010以及LN012至LN025所示的化合物。In one or more embodiments, the peptide contains at least one phosphorylcholine group-modified amino acid as described in any of the embodiments herein. Preferably, at least one amino acid residue modified by a phosphorylcholine group in the peptide is selected from the group consisting of compounds represented by LN001 to LN010 and LN012 to LN025.
在优选的实施方案中，所述肽中磷酰胆碱基团修饰的氨基酸残基选自：鸟氨酸、赖氨酸、丝氨酸和甘氨酸中的一个或多个。在进一步优选的实施方案中，所述肽中被磷酰胆碱基团修饰的氨基酸残基衍生自以下分子中的一种或多种：LN001-LN010、LN022和LN025。所述“衍生”指所述分子的氨基上的一个H以及羧基上的H被去除，以分别与肽中的其它氨基酸残基形成肽键。应理解，当衍生自所述分子的氨基酸残基位于肽的N端或C端时，则该氨基酸残基为 单价基团，否则为二价基团。In a preferred embodiment, the phosphorylcholine group-modified amino acid residue in the peptide is selected from one or more of ornithine, lysine, serine and glycine. In a further preferred embodiment, the amino acid residues in the peptide modified with phosphorylcholine groups are derived from one or more of the following molecules: LN001-LN010, LN022 and LN025. The "derivatization" refers to the removal of one H on the amino group and the H on the carboxyl group of the molecule to form peptide bonds, respectively, with other amino acid residues in the peptide. It will be understood that when the amino acid residue derived from the molecule is located at the N- or C-terminus of the peptide, then the amino acid residue is a monovalent group, otherwise it is a divalent group.
在一些实施方案中，所述肽为Val-Orn、Val-Lys、Ser-Val-Lys、Gly-Val-Lys、Ser-Val-Cit、Gly-Val-Cit、Gly-Gly-Gly-Val-Lys或Gly-Gly-Val-Lys。优选地，这些肽中的鸟氨酸、赖氨酸、丝氨酸和甘氨酸中的一个或多个被磷酰胆碱基团修饰。在一些实施方案中，所述肽如LN011所示。In some embodiments, the peptide is Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Val-Cit, Gly-Gly-Gly-Val- Lys or Gly-Gly-Val-Lys. Preferably, one or more of ornithine, lysine, serine and glycine in these peptides is modified with a phosphorylcholine group. In some embodiments, the peptide is shown as LN011.
在一些实施方案中，所述磷酰胆碱修饰的肽选自：Val-Orn(N-PC)、Val-Lys(N-PC)、Val-Lys(N-PC2)、Val-Lys(N-PC-PC)、Val-Lys(N-Me-PC)、Gly(N-PC)-Val-Lys(N-PC)、Ser(O-PC)-Val-Lys(N-PC)、Ser(O-PC)-Val-Cit、Gly(N-PC)-Val-Cit、Gly(N-PC)-Lys(N-PC)、Gly-Gly-Gly-Val-Lys(N-PC)和Gly-Gly-Val-Lys(N-PC)：In some embodiments, the phosphorylcholine-modified peptide is selected from the group consisting of: Val-Orn(N-PC), Val-Lys(N-PC), Val-Lys(N-PC2), Val-Lys(N -PC-PC), Val-Lys(N-Me-PC), Gly(N-PC)-Val-Lys(N-PC), Ser(O-PC)-Val-Lys(N-PC), Ser (O-PC)-Val-Cit, Gly(N-PC)-Val-Cit, Gly(N-PC)-Lys(N-PC), Gly-Gly-Gly-Val-Lys(N-PC) and Gly-Gly-Val-Lys (N-PC):
Figure PCTCN2022072943-appb-000003
Figure PCTCN2022072943-appb-000003
Figure PCTCN2022072943-appb-000004
Figure PCTCN2022072943-appb-000004
连接子化合物linker compound
本申请提供下式II所示的连接子化合物：The application provides a linker compound represented by the following formula II:
R 4-L 2-P-R 5    (II) R 4 -L 2 -PR 5 (II)
式中：where:
R 4为可与蛋白中的半胱氨酸残基中的S，或-赖氨酸上的氨基发生反应，从而将式II连接到蛋白的基团； R 4 is a group that can react with the S in the cysteine residue in the protein, or the amino group on the -lysine, thereby connecting the formula II to the protein;
L 2为连接基团； L 2 is a linking group;
P为本文任一实施方案所述的修饰的氨基酸或修饰的肽；所述修饰的氨基 酸或修饰的肽与L 2和R 5共价连接； P is the modified amino acid or modified peptide of any of the embodiments herein ; the modified amino acid or modified peptide is covalently linked to L and R ;
R 5为： R5 is:
Figure PCTCN2022072943-appb-000005
Figure PCTCN2022072943-appb-000005
R 6为H、卤素、C1-10烷基、C3-C8环烷基、C2-C8烯基、C2-C8炔基、C3-C8环烯基、6-14元芳基、芳烷基、5-10元杂芳基或4-10元杂环基； R 6 is H, halogen, C1-10 alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl, 6-14-membered aryl, aralkyl, 5-10-membered heteroaryl or 4-10-membered heterocyclyl;
R 9为H、-C(O)-N(R 7)-(CH 2) m-NHR 8或硝基取代的苯氧基羰基； R 9 is H, -C(O)-N(R 7 )-(CH 2 ) m -NHR 8 or nitro-substituted phenoxycarbonyl;
R 7为H或C1-6烷基； R 7 is H or C1-6 alkyl;
R 8为H或C1-6烷基；和 R 8 is H or C1-6 alkyl; and
m为1-6的整数。m is an integer of 1-6.
在一个或多个实施方案中，R 4选自： In one or more embodiments, R4 is selected from:
Figure PCTCN2022072943-appb-000006
Figure PCTCN2022072943-appb-000006
其中，波浪线表示R 4与L 2连接的位置；F 5表示5个氟原子。 Among them, the wavy line represents the position where R 4 is connected to L 2 ; F 5 represents 5 fluorine atoms.
式II中，优选地，L 2为-L a-L b-，其中L a选自C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基；L b不存在，或者为羰基、酯基(-COO-)、-O-SO 2-或-NH-SO 2-。优选地，L b不存在或为羰基。优选地，L 2通过L b与所述P共价连接。在一些实施方案中，L b和P间形成酰胺基或磺酰氨基。在优选的实施方案中，各L 2独立为C1-C6亚烷基或C1-C6亚烷基羰基。 In formula II, preferably, L 2 is -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; L b is absent, or It is a carbonyl group, an ester group (-COO-), -O-SO 2 - or -NH-SO 2 -. Preferably, L b is absent or carbonyl. Preferably, L2 is covalently linked to said P through Lb. In some embodiments, an amide or sulfonamido group is formed between L b and P. In preferred embodiments, each L 2 is independently C1-C6 alkylene or C1-C6 alkylenecarbonyl.
因此，在一些实施方案中，所述R 4-L 2与P以及通过选自酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接。优选地，R 4-L 2与P通过酰胺基共价连接。 Thus, in some embodiments, the R 4 -L 2 and P and by the group selected from the group consisting of amide (-CO-NH-), ester (-COO-), -O-SO 2 -NH- and -NH -SO 2 -NH- is covalently linked. Preferably, R 4 -L 2 and P are covalently linked through an amide group.
R 4与L 2之间可通过L a进行连接。优选地，R 4与L 2之间可通过选自碳碳单键、酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接，优选通过碳碳单键或酰胺基进行共价连接。 The connection between R 4 and L 2 can be made through La . Preferably, between R 4 and L 2 can be selected from carbon-carbon single bond, amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO The connection mode of 2 -NH- is covalent connection, preferably through carbon-carbon single bond or amide group.
优选地，式II中，所述P与R 5通过选自酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接，优选通过酰胺基共价连接。 Preferably, in formula II, the P and R 5 are selected from the group consisting of amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO 2 -NH - is covalently attached, preferably via an amide group.
优选地，式II中，R 5为： Preferably, in formula II, R 5 is:
Figure PCTCN2022072943-appb-000007
Figure PCTCN2022072943-appb-000007
优选地，式II中，R 6为H或C1-C6烷基。 Preferably, in formula II, R 6 is H or C1-C6 alkyl.
优选地，式II中，R 7为H或C1-C4烷基。 Preferably, in formula II, R 7 is H or C1-C4 alkyl.
优选地，式II中，R 8为H或C1-C4烷基。 Preferably, in formula II, R 8 is H or C1-C4 alkyl.
优选地，式II中，m为1-4的整数。Preferably, in formula II, m is an integer of 1-4.
在优选的实施方案中，所述式II的结构表示为：In a preferred embodiment, the structure of formula II is represented as:
R 4-(C1-C6亚烷基)-C(O)-[NH-P-C(O)]-R 5。 R 4 -(C1-C6 alkylene)-C(O)-[NH-PC(O)]-R 5 .
在一个或多个实施方案中，所述式II化合物如化合物1-8、1-9、2-6、2-7、3-5、3-6、4-4、4-5、5-3、5-4、6-3、6-4、7-4、7-5、8-3、8-4、15-4、15-5、16-3、16-4、23-2或23-3所示。In one or more embodiments, the compound of formula II such as compound 1-8, 1-9, 2-6, 2-7, 3-5, 3-6, 4-4, 4-5, 5- 3, 5-4, 6-3, 6-4, 7-4, 7-5, 8-3, 8-4, 15-4, 15-5, 16-3, 16-4, 23-2 or 23-3.
连接子化合物共价连接的药物Linker Compounds Covalently Linked Drugs
本申请的连接子化合物可用于修饰感兴趣的药物分子。还提供一种与本申请所述的连接子化合物共价连接的药物，其具有下式III所示结构：The linker compounds of the present application can be used to modify drug molecules of interest. Also provide a medicine covalently linked with the linker compound described in the application, which has the structure shown in the following formula III:
R 4-L 2-P-R 5-D    (III) R 4 -L 2 -PR 5 -D (III)
式中：R 4、L 2、P和R 5如前文任一实施方案所定义；D表示药物分子去除了一个H原子后获得的基团。 In the formula: R 4 , L 2 , P and R 5 are as defined in any of the preceding embodiments; D represents a group obtained by removing one H atom from the drug molecule.
在一个或多个实施方案中，所述药物分子通过与R 5形成碳酸酯键(-OCO-)或氨基甲酸酯(-OCNH-)而与R 5相连。 In one or more embodiments, the drug molecule is attached to R5 by forming a carbonate bond (-OCO-) or a carbamate ( -OCNH- ) with R5.
在一个或多个实施方案中，所述药物分子选自：MMAE、Duo-5、DXD、依沙替康、喜树碱、10-羟基喜树碱、拓扑替康、氟脲苷、去氧氟尿苷、阿糖胞苷、依托泊苷、氟达拉滨、卡培他滨、长春新碱、埃坡霉素B、紫杉醇、多烯紫杉醇、柔红霉素、表阿霉素、甲氨蝶呤、吉西他滨、美法仑、尼莫司汀、米托蒽醌、阿霉素和丝裂霉素。In one or more embodiments, the drug molecule is selected from the group consisting of: MMAE, Duo-5, DXD, ixatecan, camptothecin, 10-hydroxycamptothecin, topotecan, fluuridine, desoxy Floxuridine, Cytarabine, Etoposide, Fludarabine, Capecitabine, Vincristine, Epothilone B, Paclitaxel, Docetaxel, Daunorubicin, Epirubicin, A Ammopterin, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin.
这些药物一般通过通过羟基或氨基与式II中的R 5形成碳酸酯键(-OCO-)或氨基甲酸酯(-OCNH-)而与式II连接。 These drugs are generally linked to formula II through the formation of a carbonate bond (-OCO-) or a carbamate (-OCNH-) with R5 in formula II through a hydroxy or amino group.
通常，连接子化合物所连接的药物分子上的位置应不影响该药物分子的生物学活性。可根据药物分子的结构功能特性，选择远离其活性中心的位置与连接子化合物共价连接。另外可以考虑的是连接子化合物与药物分子发生反应的便利性。通常，可在方便将连接子化合物连接到药物分子、同时又不影响连接后的药物分子的生物学活性的前提下选择连接的位置，这可由本领域技术人员根据本领域公知技术实施并测试所得的经连接子化合物修饰的药物分子的生物学活性。优选地，本文所述的连接子化合物共价连接的药物分子与未连接的药物分子相比，保留了至少70％、优选至少80％、更优选至少90％、更优选至少95％以上的生物学活性。Generally, the position on the drug molecule to which the linker compound is attached should not affect the biological activity of the drug molecule. According to the structural and functional properties of the drug molecule, a position far from its active center can be selected to be covalently linked to the linker compound. Another consideration is the ease with which the linker compound can react with the drug molecule. Generally, the linking position can be selected under the premise that it is convenient to link the linker compound to the drug molecule without affecting the biological activity of the linked drug molecule, which can be implemented and tested by those skilled in the art according to well-known techniques in the art. The biological activity of linker compound-modified drug molecules. Preferably, the covalently linked drug molecule of the linker compound described herein retains at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% more biological academic activity.
优选地，所述药物如LD001到LD0025所示。Preferably, the drugs are shown as LD001 to LD0025.
ADCADC
再一方面，本申请提供一种ADC，其结构式如下式IV所示：In another aspect, the present application provides an ADC whose structural formula is shown in the following formula IV:
A-(R 4-L 2-P-R 5-D) n   (IV) A-(R 4 -L 2 -PR 5 -D) n (IV)
式中，R 4、L 2、P、R 5和D如前文任一实施方案所定义，A为抗体或其抗原结合片段，n为1-8的整数。 In the formula, R 4 , L 2 , P, R 5 and D are as defined in any of the preceding embodiments, A is an antibody or an antigen-binding fragment thereof, and n is an integer of 1-8.
本文中，“抗体”具有本领域周知含义，包括具有所需生物活性的任何形式的抗体，如单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体等。As used herein, "antibody" has the meaning well known in the art and includes any form of antibody having the desired biological activity, such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies, etc.
“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。"Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
“全长抗体”指在天然存在时包含至少四条肽链的免疫球蛋白分子：两条重(H)链和两条轻(L)链通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(Clq))的结合。"Full-length antibody" refers to an immunoglobulin molecule comprising at least four peptide chains in nature: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs). Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
抗体的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。 术语“结合化合物”是指抗体及其结合片段两者。An "antigen-binding fragment" of an antibody includes a fragment or derivative of an antibody, typically including at least a fragment of the antigen-binding or variable region (eg, one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody . Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments Sexual antibodies. A binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of an antibody). The term "binding compound" refers to both antibodies and binding fragments thereof.
“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。A "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. "Domain antibodies" are immunofunctional fragments of immunoglobulins that contain only heavy or light chain variable regions. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens. A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific. "Diabodies" refer to small antibody fragments with two antigen-binding sites comprising a heavy chain variable domain (VL) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites.
优选地，式IV中，抗体通过该抗体所含的半胱氨酸的巯基或赖氨酸的游离氨基与R 4相连。 Preferably, in formula IV , the antibody is linked to R4 through the sulfhydryl group of cysteine or the free amino group of lysine contained in the antibody.
优选地，式IV中，抗体可以是本领域周知的各类具有所需生物学活性的抗体或其抗原结合片段。例如，抗体或其功能性片段可选自：抗-Her2抗体，抗-EGFR抗体，抗-VEGFR抗体，抗-CD20抗体，抗-CD33抗体，抗-PD-L1抗体，抗-PD-1抗体，抗-CTLA-4抗体，抗-TNFα抗体，抗-CD28抗体，抗-4-1BB抗体，抗-OX40抗体，抗-GITR抗体，抗-CD27抗体，抗b-CD40抗体，或抗-ICOS抗体，抗-CD25抗体，抗-CD30抗体，抗-CD3抗体，抗-CD22抗体，抗-CCR4抗体，抗-CD38抗体，抗-CD52抗体，抗-补体C5抗体，抗RSV的F蛋白，抗-GD2抗体，抗-GITR抗体，抗-糖蛋白受体lib/Illa抗体，抗-ICOS抗体，抗-IL2R抗体，抗-LAG3抗体，抗-Integrinα4抗体，抗-lgE抗体，抗-PDGFRa抗体，抗-RANKL抗体，抗-SLAMF7抗体，抗-LTIGIT抗体，抗-TIM-3抗体，抗-VEGFR2抗体，抗-VISTA抗体，抗C-Met抗体，抗BCMA抗体，抗Claudin 18抗体、抗Nectin-4抗体，抗CD79b抗体，和抗Trop 2抗体。Preferably, in formula IV, the antibody can be various types of antibodies or antigen-binding fragments thereof known in the art with the desired biological activity. For example, the antibody or functional fragment thereof can be selected from: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody , anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, or anti-ICOS Antibody, Anti-CD25 Antibody, Anti-CD30 Antibody, Anti-CD3 Antibody, Anti-CD22 Antibody, Anti-CCR4 Antibody, Anti-CD38 Antibody, Anti-CD52 Antibody, Anti-Complement C5 Antibody, Anti-RSV F Protein, Anti- -GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib/Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-Integrin α4 antibody, anti-lgE antibody, anti-PDGFRa antibody, Anti-RANKL Antibody, Anti-SLAMF7 Antibody, Anti-LTIGIT Antibody, Anti-TIM-3 Antibody, Anti-VEGFR2 Antibody, Anti-VISTA Antibody, Anti-C-Met Antibody, Anti-BCMA Antibody, Anti-Claudin 18 Antibody, Anti-Nectin- 4 antibody, anti-CD79b antibody, and anti-Trop 2 antibody.
式IV中，与抗体辍合的-R 4-L 2-P-R 5-D部分的数量可以为1-8个，该数量一般与抗体中二硫键的数量有关，也与参与偶联反应的基团有关。本领域技术 人员可根据抗体的氨基酸序列、参与偶联反应的基团等条件，容易确定n的数量。在一些实施方案中，n为2-6的整数。在一些实施方案中，n为4。 In formula IV, the number of -R 4 -L 2 -PR 5 -D moieties conjugated with the antibody can be 1-8, and the number is generally related to the number of disulfide bonds in the antibody, and is also related to the coupling reaction. group related. Those skilled in the art can easily determine the number of n according to conditions such as the amino acid sequence of the antibody and the groups involved in the coupling reaction. In some embodiments, n is an integer from 2-6. In some embodiments, n is 4.
在优选的实施方案中，所述ADC选自LA001至LA025中的任意一个或多个。In a preferred embodiment, the ADC is selected from any one or more of LA001 to LA025.
制备方法Preparation
可参照本申请实施例所述的方法制备本申请的磷酰胆碱基团修饰的氨基酸、磷酰胆碱修饰的肽、连接子化合物、连接子化合物共价连接的药物以及ADC。下文示例性地描述相应的制备方法。The phosphorylcholine group-modified amino acids, phosphorylcholine-modified peptides, linker compounds, drugs covalently linked to linker compounds, and ADCs of the present application can be prepared with reference to the methods described in the examples of the present application. Corresponding preparation methods are exemplarily described below.
磷酰胆碱基团修饰的氨基酸Amino acids modified with phosphorylcholine groups
可通过将合适保护的氨基酸的氨基和含磷酰胆碱基团的醛(如LN027)进行还原胺化，或由氨基酸侧链上的氨基和含磷酰胆碱基团的羧酸(LN028)形成酰胺，或由氨基酸侧链上的羧基和含磷酰胆碱基团的氨基(LN029)成酰胺制备而得到磷酰胆碱基团修饰的氨基酸。Reductive amination can be carried out by the amino group of a suitably protected amino acid and an aldehyde containing a phosphorylcholine group (such as LN027), or by an amino group on the side chain of an amino acid and a carboxylic acid containing a phosphorylcholine group (LN028) An amide is formed, or an amino acid modified with a phosphorylcholine group is prepared by forming an amide from a carboxyl group on the amino acid side chain and an amino group containing a phosphorylcholine group (LN029).
磷酰胆碱修饰的肽和磷酰胆碱修饰的连接子化合物的制备Preparation of Phosphorylcholine-Modified Peptides and Phosphorylcholine-Modified Linker Compounds
为避免多肽缩合对氨基苄醇(PAB)时产生消漩异构体，磷酰胆碱修饰的肽和磷酰胆碱修饰的连接子化合物的制备通常采用将合适保护的氨基酸和对氨基苄醇(PAB)进行缩合反应，生成合适保护的氨基酸的对氨基苄醇(PAB)酰胺，然后脱保护，进行常规的多肽合成。并按需要引入式II中L 2和R 5的以及式III中的R 4和D。 To avoid the generation of racemic isomers when the polypeptide is condensed with p-aminobenzyl alcohol (PAB), phosphorylcholine-modified peptides and phosphorylcholine-modified linker compounds are usually prepared by combining a suitably protected amino acid with p-aminobenzyl alcohol. (PAB) undergoes a condensation reaction to yield the p-aminobenzyl alcohol (PAB) amide of the appropriately protected amino acid, which is then deprotected for conventional polypeptide synthesis. And introduce L 2 and R 5 in formula II and R 4 and D in formula III as required.
ADC的制备Preparation of ADCs
可使先还原抗体，然后混合还原的抗体与本发明的式III所示的连接子化合物共价连接的药物，室温下静置足够时间后，分离纯化得到本发明的ADC。还原抗体的方法为本领域所周知。还原的目的包括使抗体中的二硫键还原成巯基。合适的还原剂包括TCEP等。可根据不同的抗体种类选自不同的还原剂以及缓冲体系。The antibody can be reduced first, then the reduced antibody can be mixed with the drug covalently linked to the linker compound represented by the formula III of the present invention, and after standing at room temperature for a sufficient time, the ADC of the present invention can be separated and purified. Methods for reducing antibodies are well known in the art. The purpose of reduction includes reducing disulfide bonds in antibodies to sulfhydryl groups. Suitable reducing agents include TCEP and the like. Different reducing agents and buffer systems can be selected according to different antibody species.
药物组合物、用途和疾病的治疗和预防方法Pharmaceutical compositions, uses and methods of treatment and prevention of diseases
本申请提供一种药物组合物，其含有有效量的本申请任一实施方案所述的ADC和药学上可接受的载体。本申请还提供疾病的治疗或预防方法，包括给予需要的对象治疗有效量或预防有效量的本申请的ADC或其药物组合物。还提供的是本申请所述磷酰胆碱修饰的氨基酸、磷酰胆碱修饰的肽、连接子化合物以及连接子化合物共价连接的药物在制备ADC中的应用，以及本文任一实施方案所述的ADC在制备治疗或预防疾病的药物中的应用。The present application provides a pharmaceutical composition comprising an effective amount of the ADC described in any of the embodiments of the present application and a pharmaceutically acceptable carrier. The present application also provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the ADC of the present application or a pharmaceutical composition thereof. Also provided are the phosphorylcholine-modified amino acids, phosphorylcholine-modified peptides, linker compounds and drugs covalently linked to the linker compounds described in this application in the preparation of ADCs, and the methods described in any of the embodiments herein. Application of the ADC in the preparation of medicines for treating or preventing diseases.
本文中，“预防”和“预防”包括使病患减少疾病或病症的发生或恶化的可能性；该术语也包括：预防疾病或病症在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病或病症，但尚未被诊断为已患有该疾病或病症时。“治疗”和其它类似的同义词包括以下含义：(i)抑制疾病或病症，即遏制其发展；(ii)缓解疾病或病症，即，使该疾病或病症的状态消退；或者(iii)减轻该疾病或病症所造成的症状。As used herein, "prevention" and "prophylaxis" include reducing the likelihood of the occurrence or exacerbation of a disease or disorder in a patient; the terms also include: preventing the occurrence of a disease or disorder in mammals, especially when such mammals are susceptible to When you have the disease or condition, but have not been diagnosed with the disease or condition. "Treat" and other similar synonyms include the following meanings: (i) inhibiting a disease or disorder, ie, arresting its progression; (ii) alleviating a disease or disorder, ie, causing regression of the state of the disease or disorder; or (iii) alleviating the Symptoms caused by a disease or condition.
本文中，“给予”指能够将化合物或组合物递送到进行生物作用的所需位点的方法。本领域周知的给药方法均可用于本发明。这些方法包括但不限于口服途径、经十二指肠途径、胃肠外注射(包括肺内、鼻内、鞘内、静脉内、皮下、腹膜内、肌内、动脉内注射或输注)、局部给药和经直肠给药。本领域技术人员熟知可用于本文所述化合物和方法的施用技术，例如在Goodman and Gilman,The Pharmacological Basis of Therapeutics,current ed.；Pergamon；and Remington’s,Pharmaceutical Sciences(current edition),Mack Publishing Co.,Easton,Pa中讨论的那些。As used herein, "administration" refers to a method capable of delivering a compound or composition to a desired site for biological action. Administration methods well known in the art can be used in the present invention. These methods include, but are not limited to, the oral route, the duodenal route, parenteral injection (including intrapulmonary, intranasal, intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), Topical and rectal administration. Those skilled in the art are familiar with administration techniques useful for the compounds and methods described herein, for example in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Those discussed in Easton, Pa.
本文中，有效量包括治疗有效量和预防有效量，是指本申请的ADC当单独或与其它治疗药物组合给予受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效量还指足以导致症状改善的ADC的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。具体有效量取决于各种因素，例如待治疗的具体疾病，患者的身体状况，如体重，年龄和性别，治疗的持续时间，共同给药的治疗(如果有的话)，以及使用的具体配方。Herein, an effective amount includes a therapeutically effective amount and a prophylactically effective amount, which means that the ADC of the present application, when administered to a subject alone or in combination with other therapeutic agents, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the The amount of development of a disease or condition. A therapeutically effective amount also refers to an amount of ADC sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as weight, age and sex, the duration of the treatment, the co-administered treatment (if any), and the specific formulation used .
药学上可接受的载体指药物制剂或组合物中除活性成分以外的对受试者 无毒的成分。药学上可接受的载体包括但不限于佐剂、载体、赋形剂、助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。可采用本领域周知的药学上可接受的载体来配制本申请的药物组合物。A pharmaceutically acceptable carrier refers to an ingredient other than the active ingredient in a pharmaceutical formulation or composition that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavors, surfactants, wetting agents, Dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. The pharmaceutical compositions of the present application can be formulated using pharmaceutically acceptable carriers well known in the art.
本申请的药物组合物可以配制成各种合适的剂型，包括但不限于片剂，胶囊，注射剂等，并且可以通过任何合适的途径给药以达到预期目的。例如，它可以肠胃外，皮下，静脉内，肌肉内，腹膜内，透皮，口服，鞘内，颅内，鼻内或外给药。药物的剂量可取决于患者的年龄，健康状况和体重，并行进行的治疗，以及治疗的频率等。本申请的药物组合物可以施用于任何有此需要的受试者，例如哺乳动物，尤其是人类。The pharmaceutical composition of the present application can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the intended purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranically, intranasally or externally. The dose of the drug may depend on the patient's age, health and weight, concurrent treatments, and the frequency of treatments, among others. The pharmaceutical compositions of the present application can be administered to any subject in need thereof, such as mammals, especially humans.
根据ADC中的抗体或其抗原结合片段以及药物，本申请的药物组合物可用于治疗或预防相应的疾病。例如，当所述药物是抗癌药时，本申请的药物组合物可用于治疗该抗癌药可治疗的癌症，包括但不限于膀胱，脑，乳腺，子宫颈，结肠-直肠，食道，肾，肝，肺，鼻咽，胰腺，***，皮肤，胃，子宫，卵巢，睾丸和血液中的癌症等。具体而言，癌症包括膀胱癌，脑癌，乳腺癌，***，结直肠癌，食道癌，肾癌，肝癌，肺癌，鼻咽癌，胰腺癌，***癌，皮肤癌，胃癌，子宫癌，卵巢癌，睾丸癌和血癌。According to the antibody or antigen-binding fragment thereof and the drug in the ADC, the pharmaceutical composition of the present application can be used to treat or prevent the corresponding disease. For example, when the drug is an anticancer drug, the pharmaceutical composition of the present application can be used to treat cancers that can be treated by the anticancer drug, including but not limited to bladder, brain, breast, cervix, colon-rectum, esophagus, kidney , Cancer in liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and blood, etc. Specifically, cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, Ovarian, testicular and blood cancers.
在一些实施方案中，本申请还提供一种改善ADC溶解性的方法，尤其是改善ADC的水溶性的方法，所述方法包括用磷酰胆碱基团修饰ADC药物中作为连接子的肽链的步骤。更具体而言，所述方法包括使用本发明任一实施方案所述的氨基酸或肽或连接子化合物来制备ADC的步骤。所述制备方法根据所使用的不同的抗体、氨基酸、肽、连接子化合物而不同，但可参照本文所公开的制备方案实施。在其它一些实施方案中，本申请还提供本申请任一实施方案所述的氨基酸、肽、连接子化合物以及与连接子化合物共价连接的药物在改善抗体溶解性(尤其是水溶性)中的应用，或在制备用于改善抗体溶解性(尤其是水溶性)的制剂中的应用，或在制备溶解性(尤其是水溶性)改善的ADC中的应用。In some embodiments, the present application also provides a method for improving the solubility of an ADC, especially a method for improving the water solubility of the ADC, the method comprising modifying a peptide chain as a linker in the ADC drug with a phosphorylcholine group A step of. More specifically, the method includes the step of preparing an ADC using the amino acid or peptide or linker compound of any of the embodiments of the present invention. The preparation method varies according to the different antibodies, amino acids, peptides, linker compounds used, but can be implemented with reference to the preparation schemes disclosed herein. In some other embodiments, the present application also provides the amino acids, peptides, linker compounds and drugs covalently linked to the linker compounds described in any of the embodiments of the present application in improving the solubility (especially water solubility) of antibodies. applications, or in the preparation of formulations for improving the solubility (especially water-solubility) of antibodies, or in the preparation of ADCs with improved solubility (especially water-solubility).
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
下述实施例中所用的起始物可由化学品销售商如Aldrich、TCI、Alfa Aesar、毕得、安耐吉等处购得，或者可通过已知的方法来合成。The starting materials used in the following examples can be purchased from chemical vendors such as Aldrich, TCI, Alfa Aesar, Bidder, Anegi, etc., or can be synthesized by known methods.
缩写对照表：Abbreviation comparison table:
Figure PCTCN2022072943-appb-000008
Figure PCTCN2022072943-appb-000008
分离纯化方法：Separation and purification method:
中压制备方法：Medium pressure preparation method:
色谱柱Spherical C18,40-75μm，100Chromatographic column Spherical C18, 40-75μm, 100
流动相A：水(0.05％TFA)；流动相B：乙腈Mobile Phase A: Water (0.05% TFA); Mobile Phase B: Acetonitrile
时间[min]time [min] 流动相A[％]Mobile phase A[%] 流动相B[％]Mobile phase B [%] 流速[mL/min]Flow rate [mL/min]
0.000.00 90.090.0 10.010.0 50.050.0
3.003.00 90.090.0 10.010.0 50.050.0
35.035.0 10.010.0 90.090.0 50.050.0
38.038.0 10.010.0 90.090.0 50.050.0
39.039.0 90.090.0 10.010.0 50.050.0
40.040.0 90.090.0 10.010.0 50.050.0
高压制备方法：High pressure preparation method:
高压制备：High pressure preparation:
色谱柱Gemini 5μm NX-C18
Figure PCTCN2022072943-appb-000009
150*50mm Column Gemini 5μm NX-C18
Figure PCTCN2022072943-appb-000009
150*50mm
流动相A：水(0.05％TFA)；流动相B：乙腈Mobile Phase A: Water (0.05% TFA); Mobile Phase B: Acetonitrile
时间[min]time [min] 流动相A[％]Mobile phase A[%] 流动相B[％]Mobile phase B [%] 流速[mL/min]Flow rate [mL/min]
0.000.00 90.090.0 10.010.0 50.050.0
6.006.00 70.070.0 30.030.0 50.050.0
25.0025.00 35.035.0 65.065.0 50.050.0
2727 10.010.0 90.090.0 50.050.0
29.029.0 10.010.0 90.090.0 50.050.0
29.129.1 90.090.0 10.010.0 50.050.0
31.031.0 90.090.0 10.010.0 50.050.0
疏水作用色谱法(HIC)谱图分析所用材料和试剂以及分析方法如下：The materials, reagents and analytical methods used in hydrophobic interaction chromatography (HIC) spectral analysis are as follows:
Figure PCTCN2022072943-appb-000010
Figure PCTCN2022072943-appb-000010
Figure PCTCN2022072943-appb-000011
Figure PCTCN2022072943-appb-000011
尺寸排阻色谱分析(SEC)所用材料和试剂以及分析方法如下：The materials and reagents and analytical methods used in size exclusion chromatography (SEC) are as follows:
色谱柱Column TSKgel G3000SW XL 5μm*7.8mm*300mm TSKgel G3000SW XL 5μm*7.8mm*300mm
流动相mobile phase 100mM PBS+100mM NaCl+15％IPA pH7.0100mM PBS+100mM NaCl+15%IPA pH7.0
进样量Injection volume 10μL10μL
检测波长Detection wavelength 280nm 280nm
柱温column temperature 30℃30℃
流速flow rate 0.5ml/min0.5ml/min
洗脱方式Elution method 等度isocratic
分析时间Analysis time 30min30min
一、磷酰胆碱修饰的氨基酸的合成1. Synthesis of phosphorylcholine-modified amino acids
实施例一：LN001的合成Example 1: Synthesis of LN001
Figure PCTCN2022072943-appb-000012
Figure PCTCN2022072943-appb-000012
步骤一：LN001-2的合成：Step 1: Synthesis of LN001-2:
于250ml的圆底烧瓶中加入Boc-赖氨酸(LN001-1,10g，41mmol)，乙酸叔丁脂(100ml)，搅拌均匀后，再缓慢滴加高氯酸(8.2g,82mmol)，待反应溶清后，常温搅拌过夜。加入乙酸乙酯，用饱和碳酸氢钠调节pH至7左右，分出有机相，水相用乙酸乙酯萃取，合并有机层，用无水硫酸钠干燥，浓缩得到无色油状液体LN001-2，3.0g。ESI-MS(m/z)；303.41。Boc-lysine (LN001-1, 10g, 41mmol) and tert-butyl acetate (100ml) were added to a 250ml round-bottomed flask, and after stirring evenly, perchloric acid (8.2g, 82mmol) was slowly added dropwise. After the reaction was dissolved, the mixture was stirred at room temperature overnight. Ethyl acetate was added, the pH was adjusted to about 7 with saturated sodium bicarbonate, the organic phase was separated, the aqueous phase was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a colorless oily liquid LN001-2, 3.0g. ESI-MS (m/z); 303.41.
步骤二：LN001-3的合成：Step 2: Synthesis of LN001-3:
取化合物LN028(450mg，2.0mmol)加入10ml的圆底烧瓶中，再加入5ml的DMF,搅拌均匀后，加入HATU(760mg，2mmol)，DIPEA(516mg，4mmol)反应混合液搅拌30min以后，加入化合物LN001-2(300mg，1mmol)，反应2h后，LCMS监控反应完全后，将反应混合液直接进行中压反相纯化(乙腈/水)，冻干后得到无色固体LN001-3，180mg.ESI-MS(m/z)；526.56The compound LN028 (450mg, 2.0mmol) was added to a 10ml round-bottomed flask, and then 5ml of DMF was added. After stirring, HATU (760mg, 2mmol) and DIPEA (516mg, 4mmol) were added. After the reaction mixture was stirred for 30min, the compound was added. LN001-2 (300mg, 1mmol), after the reaction for 2h, LCMS monitoring after the reaction was completed, the reaction mixture was directly subjected to medium pressure reverse-phase purification (acetonitrile/water), lyophilized to obtain a colorless solid LN001-3, 180mg.ESI -MS(m/z); 526.56
步骤三：化合物LN001的合成：Step 3: Synthesis of compound LN001:
于5ml圆底烧瓶中加入上步骤得到的化合物LN001-3(50mg)和1ml的三氟乙酸，常温反应一小时后浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水相，冻干得到绿色油状物LN001，35mg。ESI-MS(m/z)：370.32.HNMR(400MHz，D 2O)：4.23-4.18(m,4H),3.94(t,1H),3.52(t,2H),3.14(t,2H),3.06(s,9H),1.87-1.78(m,2H),1.48-1.43(m,2H),1.37-1.27(m,2H). The compound LN001-3 (50 mg) obtained in the above step and 1 ml of trifluoroacetic acid were added to a 5 ml round-bottomed flask, reacted at room temperature for one hour and concentrated to obtain a crude product. The crude product was dissolved in water, washed with ethyl acetate, and the water was separated. phase, lyophilized to give LN001 as a green oil, 35 mg. ESI-MS (m/z): 370.32. HNMR (400 MHz, D 2 O): 4.23-4.18 (m, 4H), 3.94 (t, 1H), 3.52 (t, 2H), 3.14 (t, 2H), 3.06(s, 9H), 1.87-1.78(m, 2H), 1.48-1.43(m, 2H), 1.37-1.27(m, 2H).
实施例二：化合物LN002的合成：Embodiment 2: the synthesis of compound LN002:
Figure PCTCN2022072943-appb-000013
Figure PCTCN2022072943-appb-000013
步骤一：(LN002-2)的合成：Step 1: Synthesis of (LN002-2):
于50ml圆底烧瓶中加入LN001-2(1.0g，3.3mmol)，LN027(0.25g，1.1mmol)和甲醇(30ml)。搅拌均匀后，常温条件下分批加入乙酰氧基硼氢化钠(1.0g，4.7mmol),反应2h后，LCMS监控反应，原料消耗完全后，浓缩至2ml反应液，中压反相纯化(乙腈/水)，浓缩后冻干，得到无色固体LN002-2，420mg,ESI-MS(m/z)：512.44。In a 50 ml round bottom flask were added LN001-2 (1.0 g, 3.3 mmol), LN027 (0.25 g, 1.1 mmol) and methanol (30 ml). After stirring evenly, sodium acetoxyborohydride (1.0 g, 4.7 mmol) was added in batches under normal temperature conditions. After 2 h of reaction, LCMS was used to monitor the reaction. /water), concentrated and lyophilized to give LN002-2 as a colorless solid, 420 mg, ESI-MS (m/z): 512.44.
步骤二：LN002的合成：Step 2: Synthesis of LN002:
于5ml圆底烧瓶中加入化合物LN002-2(50mg)和1ml的三氟乙酸，常温反应一小时后浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水 相，冻干得到绿色油状物LN002(24mg)。ESI-MS(m/z)：356.32.HNMR(400MHz，D2O)：4.20-4.18(m,2H),4.06-4.05(m,1H),4.01-3.96(m,2H),3.21-3.16(m,2H),3.07(s,9H),3.0-2.96(m,2H)1.92-1.80(m,2H),1.71-1.59(m,2H),1.43-1.37(m,2H).Compound LN002-2 (50mg) and 1ml of trifluoroacetic acid were added to a 5ml round-bottomed flask, reacted at room temperature for one hour and concentrated to obtain a crude product. After the crude product was dissolved in water, washed with ethyl acetate, the aqueous phase was separated and lyophilized. LN002 (24 mg) was obtained as a green oil. ESI-MS(m/z): 356.32.HNMR(400MHz, D2O): 4.20-4.18(m, 2H), 4.06-4.05(m, 1H), 4.01-3.96(m, 2H), 3.21-3.16(m ,2H),3.07(s,9H),3.0-2.96(m,2H)1.92-1.80(m,2H),1.71-1.59(m,2H),1.43-1.37(m,2H).
实施例三：LN003的合成：Embodiment three: the synthesis of LN003:
Figure PCTCN2022072943-appb-000014
Figure PCTCN2022072943-appb-000014
步骤一：LN003-2的合成：Step 1: Synthesis of LN003-2:
于50ml圆底烧瓶中加入LN001-2(1.0g，3.3mmol)，LN027(1.5g，6.6mmol)和甲醇(30ml)。搅拌均匀后，常温条件下分批加入乙酰氧基硼氢化钠(2.1g，9.9mmol),反应2h后，LCMS监控反应，原料消耗完全后，浓缩至2ml反应液，中压反相纯化(乙腈/水)，浓缩后冻干，得到无色固体LN003-2，350mg,ESI-MS(m/z)：721.74。In a 50 ml round bottom flask were added LN001-2 (1.0 g, 3.3 mmol), LN027 (1.5 g, 6.6 mmol) and methanol (30 ml). After stirring evenly, sodium acetoxyborohydride (2.1 g, 9.9 mmol) was added in batches under normal temperature conditions. After 2 h of reaction, LCMS was used to monitor the reaction. After the raw materials were completely consumed, the mixture was concentrated to 2 ml of the reaction solution, and purified by medium pressure reverse phase (acetonitrile). /water), concentrated and lyophilized to give LN003-2 as a colorless solid, 350 mg, ESI-MS (m/z): 721.74.
步骤二：LN003的合成：Step 2: Synthesis of LN003:
于5ml圆底烧瓶中加入化合物LN003-2(50mg)和1ml的三氟乙酸，常温反应一小时后浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水相，冻干得到LN003，32mg。ESI-MS(m/z)：565.49.HNMR(400MHz，D2O)：4.30-4.28(m,4H),4.24-4.19(m,3H),4.12-4.05(m,3H),3.64-3.62(m,4H),3.55-3.54(m,3H),3.34-3.28(m,2H),3.17(s,18H),2.04-1.92(m,2H),1.83-1.71(m,2H),1.55-1.45(m,2H).Compound LN003-2 (50 mg) and 1 ml of trifluoroacetic acid were added to a 5 ml round-bottomed flask, reacted at room temperature for one hour and concentrated to obtain a crude product. After the crude product was dissolved in water, washed with ethyl acetate, the aqueous phase was separated and lyophilized. LN003 was obtained, 32 mg. ESI-MS(m/z): 565.49.HNMR(400MHz, D2O): 4.30-4.28(m,4H), 4.24-4.19(m,3H), 4.12-4.05(m,3H), 3.64-3.62(m ,4H),3.55-3.54(m,3H),3.34-3.28(m,2H),3.17(s,18H),2.04-1.92(m,2H),1.83-1.71(m,2H),1.55-1.45 (m,2H).
实施例四：LN004的合成Embodiment four: the synthesis of LN004
Figure PCTCN2022072943-appb-000015
Figure PCTCN2022072943-appb-000015
步骤一：LN004-2的合成：Step 1: Synthesis of LN004-2:
于10ml圆底烧瓶中加入化合物LN002-2(200mg，0.39mmol)，甲醛水溶液(0.5ml)和甲醇(5ml)。搅拌均匀后，常温条件下分批加入乙酰氧基硼氢化钠(249mg，1.2mmol),反应2h后，LCMS监控反应，原料消耗完全后，浓缩至2ml反应液，中压反相纯化(乙腈/水)，浓缩后冻干，得到绿色油状物LN004-2，110mg。ESI-MS(m/z)：526.49。Compound LN002-2 (200 mg, 0.39 mmol), aqueous formaldehyde (0.5 ml) and methanol (5 ml) were added to a 10 ml round bottom flask. After stirring, sodium acetoxyborohydride (249 mg, 1.2 mmol) was added in batches under normal temperature conditions. After 2 h of reaction, LCMS was used to monitor the reaction. water), concentrated and lyophilized to give LN004-2 as a green oil, 110 mg. ESI-MS (m/z): 526.49.
步骤二：LN004的合成：Step 2: Synthesis of LN004:
于5ml圆底烧瓶中加入50mg化合物LN004-2和1ml的三氟乙酸，常温反应一小时后浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水相，冻冻干燥得到LN004，18mg。ESI-MS(m/z)：370.32.HNMR(400MHz，D 2O)：4.18-4.16(m,2H),4.06-4.05(m,1H),4.0-3.95(m,2H),3.21-3.16(m,2H),3.07(s,9H),3.0-2.96(m,2H),2.88(s,3H),1.92-1.80(m,2H),1.71-1.59(m,2H),1.43-1.37(m,2H). 50mg of compound LN004-2 and 1ml of trifluoroacetic acid were added to a 5ml round-bottomed flask, reacted at room temperature for one hour and concentrated to obtain a crude product. After the crude product was dissolved in water, washed with ethyl acetate, the aqueous phase was separated, and freeze-dried to obtain LN004, 18 mg. ESI-MS (m/z): 370.32. HNMR (400 MHz, D 2 O): 4.18-4.16 (m, 2H), 4.06-4.05 (m, 1H), 4.0-3.95 (m, 2H), 3.21-3.16 (m,2H),3.07(s,9H),3.0-2.96(m,2H),2.88(s,3H),1.92-1.80(m,2H),1.71-1.59(m,2H),1.43-1.37 (m,2H).
实施例五：LN005的合成Embodiment five: the synthesis of LN005
Figure PCTCN2022072943-appb-000016
Figure PCTCN2022072943-appb-000016
步骤一：LN005-2的合成：Step 1: Synthesis of LN005-2:
取LN028(264mg，1.18mmol)加入10ml的圆底烧瓶中，再加入5ml的DMF，搅拌均匀后，加入HATU(448mg，1.18mmol)，DIPEA(304mg，2.36mmol)反应混合液搅拌30min以后，加入化合物LN002-2(300mg，0.59mmol)，反应2h后，LCMS监控反应完全后，将反应混合液直接进行中压反相纯化(乙腈/水)，冻干后得到无色固体LN005-2，145mg，ESI-MS(m/z)； 735.65。Take LN028 (264mg, 1.18mmol) and add it to a 10ml round-bottomed flask, then add 5ml of DMF, stir evenly, add HATU (448mg, 1.18mmol), DIPEA (304mg, 2.36mmol) After the reaction mixture is stirred for 30min, add Compound LN002-2 (300 mg, 0.59 mmol) was reacted for 2 h, and after LCMS monitoring was completed, the reaction mixture was directly subjected to medium-pressure reverse-phase purification (acetonitrile/water), and lyophilized to obtain colorless solid LN005-2, 145 mg , ESI-MS (m/z); 735.65.
步骤二：LN005的合成：Step 2: Synthesis of LN005:
于5ml圆底烧瓶中加入60mg化合物LN005-2和1ml的三氟乙酸，常温反应一小时后，LCMS监控反应进程，浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水相，冻干得到LN005,18mg。，ESI-MS(m/z)：579.48。 1HNMR(400MHz，D 2O)：4.72-4.57(m,2H),4.25-4.24(m,1H),4.17-4.11(m,2H),4.02-3.93(m,4H),3.59-3.49(m,4H),3.42-3.39(m,2H),3.35-3.23(m,2H),3.12-3.09(m,18H),1.96-1.84(m,2H),1.73-1.54(m,2H),1.42-1.34(m,2H). 60mg of compound LN005-2 and 1ml of trifluoroacetic acid were added to a 5ml round-bottomed flask. After one hour of reaction at room temperature, the reaction progress was monitored by LCMS, and the crude product was concentrated to obtain the crude product. After the crude product was dissolved in water, washed with ethyl acetate, and the water was separated. phase, lyophilized to give LN005, 18 mg. , ESI-MS (m/z): 579.48. 1 H NMR (400 MHz, D 2 O): 4.72-4.57 (m, 2H), 4.25-4.24 (m, 1H), 4.17-4.11 (m, 2H), 4.02-3.93 (m, 4H), 3.59-3.49 ( m, 4H), 3.42-3.39(m, 2H), 3.35-3.23(m, 2H), 3.12-3.09(m, 18H), 1.96-1.84(m, 2H), 1.73-1.54(m, 2H), 1.42-1.34(m,2H).
采用类似的方法可合成以下分子：Using a similar approach, the following molecules can be synthesized:
Figure PCTCN2022072943-appb-000017
Figure PCTCN2022072943-appb-000017
实施例六：化合物LN011的合成Example 6: Synthesis of compound LN011
Figure PCTCN2022072943-appb-000018
Figure PCTCN2022072943-appb-000018
步骤一：化合物LN011-2的合成Step 1: Synthesis of compound LN011-2
于50ml圆底烧瓶中加入叔丁基N6-((苄氧基)羰基)-L-赖氨酸(1.3g，6.0mmol)，HATU(2.7g，7.2mmol)，DIPEA(2.3g，18mmol)和DCM(15ml)。反应20min后，常温条件下加入LN011-1(2.0g，6mmol)，反应2h后，TLC监控反应，原料消耗完全后，反应液加入20ml的DCM，用水洗后，干燥浓缩，经硅胶柱纯化(PE/EA＝20/1-1/1)，得到白色固体LN011-2共1.8g。ESI-MS(m/z)：536.62。In a 50ml round-bottomed flask was added tert-butyl N6-((benzyloxy)carbonyl)-L-lysine (1.3g, 6.0mmol), HATU (2.7g, 7.2mmol), DIPEA (2.3g, 18mmol) and DCM (15ml). After 20 min of reaction, LN011-1 (2.0 g, 6 mmol) was added at room temperature, and after 2 h of reaction, TLC was used to monitor the reaction. After the raw materials were completely consumed, 20 ml of DCM was added to the reaction solution, washed with water, dried and concentrated, and purified by silica gel column ( PE/EA=20/1-1/1), a total of 1.8 g of white solid LN011-2 was obtained. ESI-MS (m/z): 536.62.
步骤二：Step 2:
于50ml圆底烧瓶中加入化合物LN011-2(1.8g，3.4mmol)，Pd/C(360mg)和甲醇(10ml)，抽换气后，在氢气氛围下，常温反应2h。LCMS监控反应完全后，过滤浓缩，得到白色固体LN011-3，1.4g。ESI-MS(m/z)：402.51Compound LN011-2 (1.8g, 3.4mmol), Pd/C (360mg) and methanol (10ml) were added to a 50ml round-bottomed flask, and after ventilation, the reaction was carried out at room temperature for 2h under a hydrogen atmosphere. After monitoring the reaction by LCMS, it was filtered and concentrated to obtain LN011-3 as a white solid, 1.4 g. ESI-MS(m/z): 402.51
步骤三：Step 3:
取LN028(450mg，2.0mmol)加入10ml的圆底烧瓶中，再加入5ml的DMF,搅拌均匀后，加入HATU(760mg，2mmol)，DIPEA(516mg，4mmol)反应混合液搅拌30min以后，加入化合物LN011-3(400mg，1mmol)，反应2h后，LCMS监控反应完全后，将反应混合液直接进行中压反相纯化(乙腈/ 水)，冻干后得到无色固体LN011-4(120mg).ESI-MS(m/z)：625.65。Take LN028 (450mg, 2.0mmol) into a 10ml round-bottomed flask, then add 5ml of DMF, stir well, add HATU (760mg, 2mmol), DIPEA (516mg, 4mmol) After the reaction mixture is stirred for 30min, the compound LN011 is added -3 (400mg, 1mmol), after the reaction for 2h, after LCMS monitoring the reaction was complete, the reaction mixture was directly subjected to medium-pressure reverse-phase purification (acetonitrile/water), and lyophilized to obtain a colorless solid LN011-4 (120mg).ESI - MS (m/z): 625.65.
步骤四：于5ml圆底烧瓶中加入化合物LN011-4(40mg)和1ml的三氟乙酸，常温反应一小时后浓缩得到粗品，将粗品溶于水后，用乙酸乙酯洗涤后，分出水相，冻干得到LN011，25mg。LC-MS；469.33.HNMR(400MHz，D 2O)：4.34-4.24(m,4H),3.78(d,1H),3.66-3.59(m,2H),3.22-3.19(m,2H),3.14(s,9H),3.12-3.10(m,1H),2.22-2.13(m,2H),1.87-1.71(m,2H),1.53-1.47(m,2H),1.36-1.33(m,2H),0.99-0.96(m,6H). Step 4: Add compound LN011-4 (40 mg) and 1 ml of trifluoroacetic acid to a 5 ml round-bottomed flask, react at room temperature for one hour and then concentrate to obtain a crude product, dissolve the crude product in water, wash with ethyl acetate, and separate the water phase , lyophilized to obtain LN011, 25 mg. LC-MS; 469.33. HNMR (400 MHz, D2O ): 4.34-4.24 (m, 4H), 3.78 (d, 1H), 3.66-3.59 (m, 2H), 3.22-3.19 (m, 2H), 3.14 (s,9H),3.12-3.10(m,1H),2.22-2.13(m,2H),1.87-1.71(m,2H),1.53-1.47(m,2H),1.36-1.33(m,2H) ,0.99-0.96(m,6H).
实施例七：化合物LN012的合成Example 7: Synthesis of compound LN012
Figure PCTCN2022072943-appb-000019
Figure PCTCN2022072943-appb-000019
步骤一：LN012-2的合成Step 1: Synthesis of LN012-2
将LN012-1(0.4g，1.4mmol)溶于10mL DMF中，加入0.4g(1.67mmol)化合物LN029，加入HATU(0.59g，1.54mmol)和DIPEA(0.45g，3.5mmol)，室温反应1个小时，中压制备纯化得目标产物LN012-2，0.43g，ESI-MS(m/z)：512.3。Dissolve LN012-1 (0.4 g, 1.4 mmol) in 10 mL of DMF, add 0.4 g (1.67 mmol) of compound LN029, add HATU (0.59 g, 1.54 mmol) and DIPEA (0.45 g, 3.5 mmol), and react for 1 at room temperature hours, the target product LN012-2, 0.43 g, ESI-MS (m/z): 512.3 was obtained by medium pressure preparation and purification.
核磁：δ(400MHz，D 2O)4.23-4.48(m,3H),3.97-4.08(m,2H),3.52-3.78(m,4H),3.28-3.42(m,1H),3.22(s,9H),3.13(s,1.5H),2.92-3.03(m,2.5H),1.48(s,6H),1.43-1.47(m,6H),1.24(s,6H). NMR: δ(400MHz, D 2 O) 4.23-4.48(m, 3H), 3.97-4.08(m, 2H), 3.52-3.78(m, 4H), 3.28-3.42(m, 1H), 3.22(s, 9H), 3.13(s, 1.5H), 2.92-3.03(m, 2.5H), 1.48(s, 6H), 1.43-1.47(m, 6H), 1.24(s, 6H).
步骤二：化合物LN012的合成Step 2: Synthesis of compound LN012
将步骤一得到的化合物LN012-2(200mg,0.39mmol)溶于TFA(三氟乙酸)中，常温反应三十五分钟。旋干后加水冻干，得120mg产品(淡黄色固体)LN012，ESI-MS(m/z)：356.2。The compound LN012-2 (200 mg, 0.39 mmol) obtained in step 1 was dissolved in TFA (trifluoroacetic acid) and reacted at room temperature for 35 minutes. After spin-drying, add water and lyophilize to obtain 120 mg of product (light yellow solid) LN012, ESI-MS (m/z): 356.2.
氢谱：δ(400MHz，D 2O)4.34-4.39(m,1H),4.23-4.32(m,2H),3.98-4.08(m,2H),3.59-3.68(m,4H),3.23-3.41(m,2H),3.21(s,9H),3.11(s,1.5H),2.95(s,1.5H). Hydrogen spectrum: δ(400MHz, D 2 O) 4.34-4.39(m, 1H), 4.23-4.32(m, 2H), 3.98-4.08(m, 2H), 3.59-3.68(m, 4H), 3.23-3.41 (m, 2H), 3.21(s, 9H), 3.11(s, 1.5H), 2.95(s, 1.5H).
实施例八：化合物LN013的合成：Embodiment 8: the synthesis of compound LN013:
Figure PCTCN2022072943-appb-000020
Figure PCTCN2022072943-appb-000020
步骤一：step one:
于250ml的圆底烧瓶中加入缬氨酸(2.0g)，乙酸叔丁脂(20ml)，搅拌均匀后，再缓慢滴加高氯酸(1.2g)，待反应溶清后，常温搅拌过夜。加入乙酸乙酯，用饱和碳酸氢钠调节pH至7左右，分出有机相，水相用乙酸乙酯萃取，合并有机层，用无水硫酸钠干燥，浓缩得到无色油状液体缬氨酸叔丁酯400mg，ESI-MS(m/z)；174.14Add valine (2.0g) and tert-butyl acetate (20ml) to a 250ml round-bottomed flask, stir evenly, and then slowly add perchloric acid (1.2g) dropwise. After the reaction is dissolved, stir at room temperature overnight. Ethyl acetate was added, the pH was adjusted to about 7 with saturated sodium bicarbonate, the organic phase was separated, the aqueous phase was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a colorless oily liquid valine tertiary Butyl ester 400mg, ESI-MS (m/z); 174.14
步骤二：Step 2:
于50ml圆底烧瓶中加入步骤一的产物缬氨酸叔丁酯400mg，化合物LN027 600mg和甲醇(30ml)。搅拌均匀后，常温条件下分批加入乙酰氧基硼氢化钠2.1g,反应2h后，LCMS监控反应，原料消耗完全后，浓缩反应液得到类白色固体LN013-1 3.0g,ESI-MS(m/z)：383.2In a 50ml round-bottomed flask, 400mg of valine tert-butyl ester, the product of step 1, 600mg of compound LN027 and methanol (30ml) were added. After stirring, 2.1 g of sodium acetoxyborohydride was added in batches under normal temperature conditions. After 2 h of reaction, LCMS monitored the reaction. After the raw materials were completely consumed, the reaction solution was concentrated to obtain 3.0 g of off-white solid LN013-1. ESI-MS (m /z): 383.2
步骤三：Step 3:
于50ml圆底烧瓶中加入步骤二的产物LN013-1 3.0g粗品，加入碳酸钾水溶液20ml，1.2gBoc酸酐的甲醇溶液，慢慢加入反应液中，调节pH值在8.0-10.0之间常温反应，反应1h后，LCMS监控反应，原料消耗完全后，浓缩反应液，经中压制备纯化得到产物LN013-2，230mg,ESI-MS(m/z)：483.3In a 50ml round-bottomed flask, add 3.0g crude product of the product LN013-1 of step 2, add 20ml of potassium carbonate aqueous solution, the methanol solution of 1.2g Boc acid anhydride, slowly add it into the reaction solution, adjust the pH value to react at room temperature between 8.0-10.0, After the reaction for 1 h, LCMS monitored the reaction. After the raw materials were completely consumed, the reaction solution was concentrated and purified by medium pressure preparation to obtain the product LN013-2, 230 mg, ESI-MS (m/z): 483.3
步骤四：Step 4:
将步骤三得到的产物LN013-2加入25ml反应瓶中，加入三氟乙酸3mL，常温反应30分钟。旋干后加水冻干，得150mg产品LN013(淡黄色固体)，ESI-MS(m/z)：327.2The product LN013-2 obtained in step 3 was added to a 25ml reaction flask, 3ml of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 30 minutes. After spin-drying, add water and freeze-dried to obtain 150mg product LN013 (light yellow solid), ESI-MS (m/z): 327.2
采用类似的方法可合成以下分子：Using a similar approach, the following molecules can be synthesized:
Figure PCTCN2022072943-appb-000021
Figure PCTCN2022072943-appb-000021
Figure PCTCN2022072943-appb-000022
Figure PCTCN2022072943-appb-000022
Figure PCTCN2022072943-appb-000023
Figure PCTCN2022072943-appb-000023
Figure PCTCN2022072943-appb-000024
Figure PCTCN2022072943-appb-000024
实施例九：Embodiment 9:
A：化合物LN027的合成A: Synthesis of compound LN027
Figure PCTCN2022072943-appb-000025
Figure PCTCN2022072943-appb-000025
将甘油磷酰胆碱(100g，389mmol)溶于1.5升水中，用冰水浴降温，分批次加入高碘酸钠(124.7g，583mmol)，控制温度不超过20度，LC-MS中控反应，反应完毕后，减压条件下浓缩，加入甲醇打浆1-2小时，过滤除去固体，有机相浓缩，继续加入乙醇，过滤除去固体，将有机相浓缩后，油泵下干燥，得到化合物LN027，65g，ESI-MS(m/z)：226.1Glycerophosphorylcholine (100g, 389mmol) was dissolved in 1.5 liters of water, cooled with an ice-water bath, sodium periodate (124.7g, 583mmol) was added in batches, the temperature was controlled not to exceed 20 degrees, and the reaction was controlled in LC-MS , after the completion of the reaction, concentrate under reduced pressure, add methanol to make slurry for 1-2 hours, filter to remove solids, concentrate the organic phase, continue to add ethanol, filter to remove solids, concentrate the organic phase, and dry under an oil pump to obtain compound LN027, 65g , ESI-MS(m/z): 226.1
B：化合物LN028的合成B: Synthesis of compound LN028
Figure PCTCN2022072943-appb-000026
Figure PCTCN2022072943-appb-000026
将甘油磷酰胆碱(31g，121mmol)溶于350毫升水中，加入150毫升乙腈，加入0.4g三氯化钌，分批次加入高碘酸钠(110g，514mmol)，控制温度不超过50度，LC-MS中控反应，反应完毕后，减压条件下浓缩，加入甲醇打浆1-2小时，过滤除去固体，有机相浓缩，继续加入醇，过滤除去固体，将有 机相浓缩后，油泵下干燥，得到化合物LN028，19g，ESI-MS(m/z)：242.1Dissolve glycerophosphorylcholine (31g, 121mmol) in 350ml of water, add 150ml of acetonitrile, add 0.4g of ruthenium trichloride, add sodium periodate (110g, 514mmol) in batches, and control the temperature not to exceed 50 degrees , LC-MS control the reaction, after the reaction is completed, concentrate under reduced pressure, add methanol to make a slurry for 1-2 hours, filter to remove the solid, concentrate the organic phase, continue to add alcohol, filter to remove the solid, after the organic phase is concentrated, under the oil pump Dry to give compound LN028, 19 g, ESI-MS (m/z): 242.1
实施例十：化合物LN029的合成Example 10: Synthesis of compound LN029
Figure PCTCN2022072943-appb-000027
Figure PCTCN2022072943-appb-000027
于100ml圆底烧瓶中加入LN027(1.5g，6.6mmol)，甲胺盐酸盐(440mg，6.6mmol)和甲醇(60ml)。室温搅拌4个小时，分批加入乙酰氧基硼氢化钠(4.2g，19.8mmol),反应2h后，LC-MS中控反应，反应结束后，浓缩，中压反相制备纯化，冷冻干燥得目标化合物，1.2g,ESI-MS(m/z)：241.1。Into a 100 ml round bottom flask were added LN027 (1.5 g, 6.6 mmol), methylamine hydrochloride (440 mg, 6.6 mmol) and methanol (60 ml). Stir at room temperature for 4 hours, add sodium acetoxyborohydride (4.2 g, 19.8 mmol) in batches, react for 2 hours, control the reaction by LC-MS, after the reaction is completed, concentrate, prepare and purify by medium pressure reverse phase, and freeze-dry to obtain The title compound, 1.2 g, ESI-MS (m/z): 241.1.
实施例十一：化合物LN030的合成Example 11: Synthesis of compound LN030
Figure PCTCN2022072943-appb-000028
Figure PCTCN2022072943-appb-000028
步骤一：化合物LN030-2的合成Step 1: Synthesis of compound LN030-2
将化合物LN030-1(1.2g，3.5mmol)加入反应瓶中，加入10mL DMF，缓慢加入芴甲氧羰酰氯(Fmoc-Cl)(1.3g，5.0mmol)，滴加DIPEA(1.1g，8.4mmol)，室温反应12-20小时，中压制备纯化，冷冻干燥得化合物LN030-2，1.5g，ESI-MS(m/z)：563.2。507.2。The compound LN030-1 (1.2 g, 3.5 mmol) was added to the reaction flask, 10 mL of DMF was added, fluorene methoxycarbonyl chloride (Fmoc-Cl) (1.3 g, 5.0 mmol) was slowly added, and DIPEA (1.1 g, 8.4 mmol) was added dropwise. ), react at room temperature for 12-20 hours, prepare and purify at medium pressure, and freeze-dry to obtain compound LN030-2, 1.5 g, ESI-MS (m/z): 563.2. 507.2.
步骤二：化合物LN030的合成Step 2: Synthesis of compound LN030
将化合物LN030-2(1.5g，2.7mmol)加入反应瓶中，加入10mL三氟乙酸，室温反应4.0小时，LC-MS中控，反应结束后，中压制备纯化，冷冻干燥得化合物LN030，1.1g，ESI-MS(m/z)：507.2。The compound LN030-2 (1.5 g, 2.7 mmol) was added to the reaction flask, 10 mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 4.0 hours, controlled by LC-MS. g, ESI-MS (m/z): 507.2.
二、包含细胞生物活性分子和连接体的化合物的合成2. Synthesis of Compounds Containing Cellular Bioactive Molecules and Linkers
实施例十二：化合物LD001的合成Embodiment 12: the synthesis of compound LD001
Figure PCTCN2022072943-appb-000029
Figure PCTCN2022072943-appb-000029
步骤一：化合物1-2的合成：Step 1: Synthesis of Compound 1-2:
将化合物1-1(5.0g,11.00mmol)加入到DCM(50mL)中，依次加入对 氨基苄醇(2.71g，22.00mmol)及EEDQ(5.44g，22.00mmol)。常温反应过夜。浓缩抽滤，固体以少量DCM洗涤，干燥后得3.8g产品。ESI-MS(m/z)：560.3Compound 1-1 (5.0 g, 11.00 mmol) was added to DCM (50 mL), followed by p-aminobenzyl alcohol (2.71 g, 22.00 mmol) and EEDQ (5.44 g, 22.00 mmol). The reaction was carried out at room temperature overnight. Concentrated and filtered, the solid was washed with a small amount of DCM, and dried to obtain 3.8 g of product. ESI-MS(m/z): 560.3
核磁：δ(400MHz，DMSO-d6)9.95(s,1H),7.82-7.89(m,2H),7.67-7.73(m,1H),7.56-7.60(m,1H),7.47-7.55(m,2H),7.37-7.44(m,2H),7.27-7.34(m,2H),7.19-7.25(m,2H),6.76(t,J＝5.4Hz,1H),5.26(s,1H),4.42(s,2H),4.02-4.30(m,4H),2.87-2.98(m,2H),1.54-1.70(m,2H),1.37-1.52(m,2H),1.34(s,9H).NMR: δ(400MHz, DMSO-d6) 9.95(s, 1H), 7.82-7.89(m, 2H), 7.67-7.73(m, 1H), 7.56-7.60(m, 1H), 7.47-7.55(m, 2H), 7.37-7.44(m, 2H), 7.27-7.34(m, 2H), 7.19-7.25(m, 2H), 6.76(t, J=5.4Hz, 1H), 5.26(s, 1H), 4.42 (s,2H),4.02-4.30(m,4H),2.87-2.98(m,2H),1.54-1.70(m,2H),1.37-1.52(m,2H),1.34(s,9H).
步骤二：化合物1-3的合成Step 2: Synthesis of Compounds 1-3
将化合物1-2(3.7g,6.61mmol)溶于甲醇中，加入盐酸，常温反应半小时。旋干后中压制备(32％乙腈/水)得到1.9g产品(白色固体)，ESI-MS(m/z)：460.2。Compound 1-2 (3.7 g, 6.61 mmol) was dissolved in methanol, hydrochloric acid was added, and the reaction was carried out at room temperature for half an hour. After spin drying, medium pressure preparation (32% acetonitrile/water) yielded 1.9 g of product (white solid), ESI-MS (m/z): 460.2.
核磁：δ(400MHz，DMSO-d6)10.23(s,1H),7.86-7.95(m,4H),7.68-7.76(m,2H),7.55-7.62(m,2H),7.38-7.45(m,2H),7.28-7.37(m,2H),7.20-7.26(m,2H),4.43(s,2H),4.15-4.34(m,4H),2.72-2.86(m,2H),1.75-1.88(m,1H),1.55-1.73(m,3H).NMR: δ(400MHz, DMSO-d6) 10.23(s, 1H), 7.86-7.95(m, 4H), 7.68-7.76(m, 2H), 7.55-7.62(m, 2H), 7.38-7.45(m, 2H), 7.28-7.37(m, 2H), 7.20-7.26(m, 2H), 4.43(s, 2H), 4.15-4.34(m, 4H), 2.72-2.86(m, 2H), 1.75-1.88( m,1H),1.55-1.73(m,3H).
步骤三：化合物1-4的合成：Step 3: Synthesis of Compounds 1-4:
将化合物LN028(1.3g，5.4mmol)溶于DMF中，加入DIPEA(2.1g，16.2mmol)及HATU(2.3g，6.0mmol)，反应半小时后加入化合物1-3(1.9g，4.1mmol)。常温反应半小时。中压制备得到化合物1-4，2.1g，ESI-MS(m/z)：683.3。Compound LN028 (1.3 g, 5.4 mmol) was dissolved in DMF, DIPEA (2.1 g, 16.2 mmol) and HATU (2.3 g, 6.0 mmol) were added, and compound 1-3 (1.9 g, 4.1 mmol) was added after the reaction for half an hour . React at room temperature for half an hour. Medium pressure preparation gave compound 1-4, 2.1 g, ESI-MS (m/z): 683.3.
步骤四：化合物1-5的合成：Step 4: Synthesis of Compounds 1-5:
将化合物1-4(2.0g，2.9mmol)溶于20毫升DMF中，加入5毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到产品1.3g，ESI-MS(m/z)：461.2。Compound 1-4 (2.0 g, 2.9 mmol) was dissolved in 20 mL of DMF and 5 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized by adding water to obtain 1.3 g of product, ESI-MS (m/z): 461.2.
步骤五：化合物1-6的合成：Step 5: Synthesis of Compounds 1-6:
将化合物1-5(1.2g，2.6mmol)及Fmoc-Val-OH(880mg，2.6mmol)溶于15毫升DMF中，加入HATU(1.1g，2.86mmol)及DIPEA(1.0g，7.8mmol)。常温反应十分钟。中压制备(35％乙腈)得到产品1.5g，ESI-MS(m/z)：782.4。Compound 1-5 (1.2 g, 2.6 mmol) and Fmoc-Val-OH (880 mg, 2.6 mmol) were dissolved in 15 mL of DMF, HATU (1.1 g, 2.86 mmol) and DIPEA (1.0 g, 7.8 mmol) were added. React at room temperature for ten minutes. Medium pressure preparation (35% acetonitrile) gave product 1.5 g, ESI-MS (m/z): 782.4.
步骤六：化合物1-7的合成：Step 6: Synthesis of Compounds 1-7:
将化合物1-6(1.2g，1.5mmol)溶于20毫升DMF中，加入5毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到产品0.8g，ESI-MS(m/z)：560.3。Compound 1-6 (1.2 g, 1.5 mmol) was dissolved in 20 mL of DMF and 5 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized by adding water to obtain 0.8 g of product, ESI-MS (m/z): 560.3.
步骤七：化合物1-8的合成：Step 7: Synthesis of Compounds 1-8:
将化合物1-7(0.8g，1.4mmol)溶于10毫升DMF中，加入Mc-OSu(462mg，1.5mmol)。常温反应过夜。中压制备纯化，冷冻干燥得产品0.9g，ESI-MS(m/z)：753.4。Compound 1-7 (0.8 g, 1.4 mmol) was dissolved in 10 mL of DMF and Mc-OSu (462 mg, 1.5 mmol) was added. The reaction was carried out at room temperature overnight. Preparation and purification under medium pressure, freeze-drying to obtain 0.9 g of product, ESI-MS (m/z): 753.4.
步骤八：化合物1-9的合成：Step 8: Synthesis of Compounds 1-9:
将化合物1-8(0.5g，0.66mmol)溶于10毫升DMF中，加入二(对硝基苯)碳酸酯(0.4，1.32mmol)及DIPEA(258mg，2.0mmol)。常温反应两小时。中压制备纯化，冷冻干燥得化合物1-9 0.5g，ESI-MS(m/z)：918.4。Compound 1-8 (0.5 g, 0.66 mmol) was dissolved in 10 mL of DMF, and bis(p-nitrobenzene)carbonate (0.4, 1.32 mmol) and DIPEA (258 mg, 2.0 mmol) were added. React at room temperature for two hours. Preparation and purification under medium pressure, freeze-drying to obtain compound 1-9 0.5 g, ESI-MS (m/z): 918.4.
核磁：δ(400MHz，DMSO-d6)10.31(s,1H),8.28-8.32(m,2H),8.10-8.20(m,2H),7.93(d,J＝8.8Hz,1H),7.66-7.72(m,2H),7.54-7.59(m,2H),7.37-7.43(m,2H),7.00(s,2H),5.24(s,2H),4.31-4.39(m,1H),4.10-4.22(m,5H),3.55-3.59(m,2H),3.34-3.39(m,2H),3.02-3.22(m,12H),2.08-2.24(m,2H),1.94-2.03(m,1H),1.75-1.86(m,1H),1.42-1.55(m,6H),1.12-1.28(m,3H),0.85(dd,J＝6.8,10.4Hz,6H).NMR: δ(400MHz, DMSO-d6) 10.31(s, 1H), 8.28-8.32(m, 2H), 8.10-8.20(m, 2H), 7.93(d, J=8.8Hz, 1H), 7.66-7.72 (m,2H),7.54-7.59(m,2H),7.37-7.43(m,2H),7.00(s,2H),5.24(s,2H),4.31-4.39(m,1H),4.10-4.22 (m,5H),3.55-3.59(m,2H),3.34-3.39(m,2H),3.02-3.22(m,12H),2.08-2.24(m,2H),1.94-2.03(m,1H) ,1.75-1.86(m,1H),1.42-1.55(m,6H),1.12-1.28(m,3H),0.85(dd,J=6.8,10.4Hz,6H).
步骤九：化合物LD001的合成Step 9: Synthesis of compound LD001
将化合物1-9(0.5g，0.54mmol)溶于5毫升DMF中，依次加入MMAE(390mg，0.54mmol)、HOBT(75mg，0.54mmol)及DIPEA(209mg，1.6mmol)。常温反应四小时。中压制备纯化，冷冻干燥得化合物LD001 0.6g，ESI-MS(m/z)：1496.8。Compound 1-9 (0.5 g, 0.54 mmol) was dissolved in 5 mL of DMF, MMAE (390 mg, 0.54 mmol), HOBT (75 mg, 0.54 mmol) and DIPEA (209 mg, 1.6 mmol) were added sequentially. The reaction was carried out at room temperature for four hours. Preparation and purification under medium pressure, freeze-drying to obtain 0.6 g of compound LD001, ESI-MS (m/z): 1496.8.
实施例十三：化合物LD002的合成Embodiment 13: Synthesis of compound LD002
Figure PCTCN2022072943-appb-000030
Figure PCTCN2022072943-appb-000030
步骤一：将化合物2-1(52g，10.0mmol)加入反应瓶中，加入二氯甲烷1000mL，缓慢加入芴甲氧羰酰氯(Fmoc-Cl)(28g，10.8mmol)，滴加三乙胺(13.5g，13.4mmol)，室温反应12-20小时，往反应液中加入MTBE，打浆搅拌1-2小时，过滤，固体在减压条件下干燥得目标化合物2-2，72g。Step 1: Add compound 2-1 (52 g, 10.0 mmol) into the reaction flask, add 1000 mL of dichloromethane, slowly add fluorene methoxycarbonyl chloride (Fmoc-Cl) (28 g, 10.8 mmol), and dropwise add triethylamine ( 13.5g, 13.4mmol), react at room temperature for 12-20 hours, add MTBE to the reaction solution, beat and stir for 1-2 hours, filter, and dry the solid under reduced pressure to obtain the target compound 2-2, 72g.
步骤二：取步骤一得到的化合物2-2(2.1g，2.8mmol)，30ml二氯甲烷和10ml二氯乙酸加入反应瓶中，室温搅拌90分钟，LC-MS中控反应，反应结束后，加入甲基叔丁基醚，析出固体，继续搅拌1个小时后，过滤得到化合物2-3，淡黄色固体，1.3g，ESI-MS(m/z)：474.3[M+H] +。 Step 2: Take the compound 2-2 (2.1g, 2.8mmol) obtained in step 1, add 30ml of dichloromethane and 10ml of dichloroacetic acid into the reaction flask, stir at room temperature for 90 minutes, and control the reaction by LC-MS. After the reaction is completed, Methyl tert-butyl ether was added to precipitate a solid, and after stirring for 1 hour, compound 2-3 was obtained by filtration as a pale yellow solid, 1.3 g, ESI-MS (m/z): 474.3 [M+H] + .
步骤三：取LN-028(0.5g，2.1mmol)，加入反应瓶中，加入5ml DMF， HATU(0.76g，2.1mmol),DIPEA(0.81g，6.3mmol)，反应半个小时后，加入化合物2-3的DMF溶液，继续反应30分钟，所得反应液直接用中压制备进行纯化，得化合物2-4，0.9g，ESI-MS(m/z)：697.3[M+H] +。 Step 3: Take LN-028 (0.5g, 2.1mmol), add it to the reaction flask, add 5ml DMF, HATU (0.76g, 2.1mmol), DIPEA (0.81g, 6.3mmol), react for half an hour, add the compound The DMF solution of 2-3 was continued to react for 30 minutes, and the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 2-4, 0.9 g, ESI-MS (m/z): 697.3 [M+H] + .
步骤四：将步骤三得到的化合物2-4(0.9g，1.3mmol)加入到25ml单口反应瓶中，加入10毫升DMF，溶清后加入2毫升二乙胺，室温反应30分钟，减压蒸馏，在油泵下真空干燥得到化合物2-5，不用纯化直接用于下一步骤，ESI-MS(m/z)：475.2[M+H] +。 Step 4: Add the compound 2-4 (0.9g, 1.3mmol) obtained in step 3 into a 25ml single-neck reaction flask, add 10ml of DMF, add 2ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , and vacuum-dried under oil pump to obtain compound 2-5, which was directly used in the next step without purification, ESI-MS (m/z): 475.2 [M+H] + .
步骤五：将10毫升DMF加入到步骤四得到的化合物2-5的粗品中，加入0.5g(1.62mmol)Mc-OSu，室温反应过夜，用中压制备纯化得到0.85g化合物2-6，ESI-MS(m/z)：668.3[M+H] +。 Step 5: Add 10 ml of DMF to the crude product of compound 2-5 obtained in step 4, add 0.5 g (1.62 mmol) of Mc-OSu, react at room temperature overnight, prepare and purify at medium pressure to obtain 0.85 g of compound 2-6, ESI - MS (m/z): 668.3 [M+H] + .
步骤六：取0.4g(0.60mmol)化合物2-6和5毫升DMF加入反应瓶中，再加入0.3g(1.0mmol)二(对硝基苯)碳酸酯和0.26g(2.0mmol)DIPEA，室温反应2小时，反应结束后，所得反应液直接用中压制备进行纯化，得化合物2-7，0.35g，ESI-MS(m/z)：833.3[M+H] +。 Step 6: Take 0.4g (0.60mmol) of compound 2-6 and 5ml of DMF into the reaction flask, then add 0.3g (1.0mmol) of bis(p-nitrobenzene) carbonate and 0.26g (2.0mmol) of DIPEA, room temperature The reaction was carried out for 2 hours. After the reaction was completed, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 2-7, 0.35 g, ESI-MS (m/z): 833.3 [M+H] + .
步骤七：取0.2g(0.24mmol)化合物2-7加入25毫升反应瓶中，加入5毫升DMF、(0.1g，0.74mmol)HOBT、0.2g(0.28mmol)MMAE和0.2g(1.56mmol)DIPEA，室温反应1小时，所得反应液直接用高压制备进行纯化，得化合物LD002 0.25g，ESI-MS(m/z)：1411.8[M+H] +。 Step 7: Take 0.2g (0.24mmol) of compound 2-7 into a 25ml reaction flask, add 5ml of DMF, (0.1g, 0.74mmol) HOBT, 0.2g (0.28mmol) MMAE and 0.2g (1.56mmol) DIPEA , reacted at room temperature for 1 hour, the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD002 0.25g, ESI-MS (m/z): 1411.8[M+H] + .
实施例十四：化合物LD003的合成Example 14: Synthesis of compound LD003
Figure PCTCN2022072943-appb-000031
Figure PCTCN2022072943-appb-000031
步骤一：取50g(62.9mmol)化合物3-1加入反应瓶中，加入500毫升二氯甲烷和170毫升二氯乙酸，室温反应1.5小时，将反应液倒入冰水中，剧烈搅拌0.5小时，析出白色固体，过滤得到化合物3-2，真空下干燥得化合物3-2的粗品31g，不用纯化直接用于下一步反应。Step 1: Take 50g (62.9mmol) of compound 3-1 into the reaction flask, add 500ml of dichloromethane and 170ml of dichloroacetic acid, react at room temperature for 1.5 hours, pour the reaction solution into ice water, stir vigorously for 0.5 hours, and precipitate out The white solid was filtered to obtain compound 3-2, which was dried under vacuum to obtain 31 g of the crude product of compound 3-2, which was directly used in the next reaction without purification.
步骤二：取化合物LN-028(14.0g，58.1mmol)，加入反应瓶中，加入100毫升DMF，HATU(22.1g,58.1mmol),DIPEA(22.5g,174.3mmol)，反应一个小时，加入10.0g(17.5mmol)化合物3-2的DMF溶液，继续反应30分钟，所得反应液直接用中压制备进行纯化，得到7.9g化合物3-3，ESI-MS(m/z)：796.4[M+H] + Step 2: Take compound LN-028 (14.0g, 58.1mmol), put it into a reaction flask, add 100ml of DMF, HATU (22.1g, 58.1mmol), DIPEA (22.5g, 174.3mmol), react for one hour, add 10.0 g (17.5 mmol) DMF solution of compound 3-2, continued to react for 30 minutes, the obtained reaction solution was directly purified by medium pressure preparation to obtain 7.9 g of compound 3-3, ESI-MS (m/z): 796.4 [M+ H] +
步骤三：取步骤二得到的化合物3-3(7.9g，9.9mmol)加入到250ml单口 反应瓶中，加入100毫升DMF，溶清后加入20毫升二乙胺，室温反应30分钟，减压蒸馏，在油泵下除去DMF得到化合物3-4，不用纯化直接用于下一步骤，ESI-MS(m/z)：574.3[M+H] +。 Step 3: Take the compound 3-3 (7.9g, 9.9mmol) obtained in step 2 and add it to a 250ml single-neck reaction flask, add 100ml of DMF, add 20ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , DMF was removed under oil pump to obtain compound 3-4, which was directly used in the next step without purification, ESI-MS (m/z): 574.3 [M+H] + .
步骤四：将100毫升DMF加入到化合物3-4粗品中，加入Mc-OSu(4.5g，14.6)，室温反应过夜，用中压制备纯化得到4.2g化合物3-5，ESI-MS(m/z)：767.4[M+H] +。 Step 4: Add 100 ml of DMF to the crude compound 3-4, add Mc-OSu (4.5 g, 14.6), react at room temperature overnight, prepare and purify at medium pressure to obtain 4.2 g of compound 3-5, ESI-MS (m/ z): 767.4 [M+H] + .
步骤五：取化合物3-5(4.0g，5.2mmol)，50毫升DMF加入反应瓶中，再加入二(对硝基苯)碳酸酯(3.5g，11.5mmol)和DIPEA(3.5g，27mmol)，室温反应2小时，反应结束后，所得反应液直接用中压制备进行纯化，得化合物3-6，3.6g，ESI-MS(m/z)：932.4[M+H] +。 Step 5: Take compound 3-5 (4.0g, 5.2mmol), add 50ml of DMF to the reaction flask, then add bis(p-nitrobenzene)carbonate (3.5g, 11.5mmol) and DIPEA (3.5g, 27mmol) , and reacted at room temperature for 2 hours. After the reaction, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 3-6, 3.6g, ESI-MS (m/z): 932.4[M+H] + .
步骤六：取化合物3-6(0.2g，0.21mmol)加入25毫升反应瓶中，加入5毫升DMF、0.1g(0.7mmol)HOBT、0.2g(0.28mmol)MMAE和0.2g(1.55mmol)DIPEA，室温反应1小时，所得反应液直接用高压制备进行纯化，得化合物LD003，0.23g，ESI-MS(m/z)：1510.9[M+H] +。 Step 6: Take compound 3-6 (0.2g, 0.21mmol) into a 25ml reaction flask, add 5ml DMF, 0.1g (0.7mmol) HOBT, 0.2g (0.28mmol) MMAE and 0.2g (1.55mmol) DIPEA , reacted at room temperature for 1 hour, and the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD003, 0.23 g, ESI-MS (m/z): 1510.9 [M+H] + .
实施例十五：化合物LD004的合成Embodiment fifteen: the synthesis of compound LD004
Figure PCTCN2022072943-appb-000032
Figure PCTCN2022072943-appb-000032
Figure PCTCN2022072943-appb-000033
Figure PCTCN2022072943-appb-000033
步骤一：取5.0g(8.7mmol)化合物3-2和化合物LN027(3.2g，14.1mmol)加入反应瓶中，加入150毫升甲醇，室温搅拌2个小时，加入1.0eq的STAB，继续反应2个小时后，加入1.0eq的STAB，继续反应2个小时后，加入0.5eq的STAB，继续反应1个小时，浓缩后，加入适量纯化水，中压制备纯化得到化合物4-1，3.6g，ESI-MS(m/z)：782.4[M+H] +。 Step 1: Take 5.0 g (8.7 mmol) of compound 3-2 and compound LN027 (3.2 g, 14.1 mmol) into the reaction flask, add 150 ml of methanol, stir at room temperature for 2 hours, add 1.0 eq of STAB, and continue the reaction for 2 more After 1 hour, 1.0 eq of STAB was added, and after continuing the reaction for 2 hours, 0.5 eq of STAB was added, and the reaction was continued for 1 hour. After concentration, an appropriate amount of purified water was added, and the compound 4-1 was prepared and purified by medium pressure to obtain compound 4-1, 3.6 g, ESI - MS (m/z): 782.4 [M+H] + .
步骤二：取1.2g(1.5mmol)化合物4-1加入反应瓶中，加入30毫升甲醇，滴加0.1毫升甲醛水溶液，分批加入2.5eq的STAB，反应0.5小时后，浓缩至约5毫升，加入适量纯化水，中压制备纯化得到化合物4-2，1.1g，ESI-MS(m/z)：796.4[M+H] +。 Step 2: Take 1.2 g (1.5 mmol) of compound 4-1 into the reaction flask, add 30 ml of methanol, dropwise add 0.1 ml of aqueous formaldehyde solution, add 2.5 eq of STAB in batches, react for 0.5 hours, and concentrate to about 5 ml, An appropriate amount of purified water was added, and the compound 4-2 was prepared and purified under medium pressure to obtain compound 4-2, 1.1 g, ESI-MS (m/z): 796.4 [M+H] + .
步骤三：取步骤二得到的化合物4-2(1.1g,1.4mmol)加入到50ml单口反应瓶中，加入10毫升DMF，溶清后加入2毫升二乙胺，室温反应30分钟，减压蒸馏，在油泵下将DMF除去得到化合物4-3的粗品，不用纯化直接用于下一步骤，ESI-MS(m/z)：574.3[M+H] +。 Step 3: Take the compound 4-2 (1.1g, 1.4mmol) obtained in step 2 and add it to a 50ml single-neck reaction flask, add 10ml of DMF, add 2ml of diethylamine after dissolving, react at room temperature for 30 minutes, and distill under reduced pressure , the DMF was removed under the oil pump to obtain the crude product of compound 4-3, which was directly used in the next step without purification, ESI-MS (m/z): 574.3 [M+H] + .
步骤四：将10毫升DMF加入到步骤三得到的化合物4-3粗品(0.8g)中，加入Mc-OSu(0.4g，1.3mmol)，室温反应过夜，用中压制备纯化得到0.52g化合物4-4，ESI-MS(m/z)：767.4[M+H] +。 Step 4: Add 10 ml of DMF to the crude compound 4-3 (0.8 g) obtained in step 3, add Mc-OSu (0.4 g, 1.3 mmol), react overnight at room temperature, and prepare and purify at medium pressure to obtain 0.52 g of compound 4 -4, ESI-MS (m/z): 767.4 [M+H] + .
步骤五：取0.5g(0.65mmol)化合物4-4，5毫升DMF加入反应瓶中，再加入0.30g DNPC(1.0mmol)和0.3g(2.3mmol)DIPEA，室温反应2小时，反应 结束后，所得反应液直接用中压制备进行纯化，得化合物4-5，0.31g，ESI-MS(m/z)：932.4[M+H] +。 Step 5: Take 0.5g (0.65mmol) of compound 4-4, add 5ml of DMF into the reaction flask, then add 0.30g (1.0mmol) and 0.3g (2.3mmol) DIPEA, and react at room temperature for 2 hours. After the reaction is completed, The obtained reaction solution was directly purified by medium pressure preparation to obtain compound 4-5, 0.31 g, ESI-MS (m/z): 932.4 [M+H] + .
步骤六：取0.2g(0.21mmol)化合物4-5加入5毫升反应瓶中，加入2毫升DMF、80mg(0.59mmol)HOBT、0.2g(0.28)MMAE和0.2g(1.55mmol)DIPEA，室温反应1小时，所得反应液直接用高压制备进行纯化，得化合物LD004 0.22g，ESI-MS(m/z)：1510.9[M+H] +。 Step 6: Take 0.2g (0.21mmol) of compound 4-5 into a 5ml reaction flask, add 2ml DMF, 80mg (0.59mmol) HOBT, 0.2g (0.28) MMAE and 0.2g (1.55mmol) DIPEA, react at room temperature After 1 hour, the obtained reaction solution was directly purified by high pressure preparation to obtain compound LD004 0.22 g, ESI-MS (m/z): 1510.9 [M+H] + .
实施例十六：化合物LD005的合成Example 16: Synthesis of compound LD005
Figure PCTCN2022072943-appb-000034
Figure PCTCN2022072943-appb-000034
步骤一：(化合物5-1)的合成Step 1: Synthesis of (Compound 5-1)
将化合物3-2(3.0g，5.24mmol)溶于甲醇(40mL)，加入化合物LN027(2.95g，13.10mmol)。反应液常温反应十分钟后，分批次加入三乙酰氧基硼氢化钠(3.33g，15.71mmol)，待加入完毕后常温反应三十分钟。反应液抽滤后中压制备(41％乙腈/水)得到3.2g产品(黄色油状物)。ESI-MS(m/z)：991.5Compound 3-2 (3.0 g, 5.24 mmol) was dissolved in methanol (40 mL), and compound LN027 (2.95 g, 13.10 mmol) was added. After the reaction solution was reacted at room temperature for 10 minutes, sodium triacetoxyborohydride (3.33 g, 15.71 mmol) was added in batches, and after the addition was completed, the reaction was performed at room temperature for 30 minutes. After suction filtration of the reaction solution, it was prepared under medium pressure (41% acetonitrile/water) to obtain 3.2 g of product (yellow oil). ESI-MS(m/z): 991.5
步骤二：化合物5-2的合成Step 2: Synthesis of Compound 5-2
将化合物5-1(3.1g，3.13mmol)溶于DMF(20mL)中，加入二乙胺(10mL)。 常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到产品5-2。ESI-MS(m/z)：769.4Compound 5-1 (3.1 g, 3.13 mmol) was dissolved in DMF (20 mL), and diethylamine (10 mL) was added. React at room temperature for twenty minutes. Rotary evaporation to remove diethylamine, add water and freeze-dried to obtain product 5-2. ESI-MS (m/z): 769.4
步骤三：化合物5-3的合成Step 3: Synthesis of Compound 5-3
将化合物5-2(500mg，0.65mmol)溶于DMF(10mL)中，加入Mc-OSu(401mg，1.30mmol)。常温搅拌过夜。中压制备(35％乙腈/水)得到260mg化合物5-3(黄色固体)。ESI-MS(m/z)：962.63Compound 5-2 (500 mg, 0.65 mmol) was dissolved in DMF (10 mL), and Mc-OSu (401 mg, 1.30 mmol) was added. Stir overnight at room temperature. Medium pressure preparation (35% acetonitrile/water) yielded 260 mg of compound 5-3 (yellow solid). ESI-MS(m/z): 962.63
步骤四：化合物5-4的合成Step 4: Synthesis of Compounds 5-4
将化合物5-3(50mg，0.05mmol)溶于DMF(1mL)中，加入DNPC(30mg，0.10mmol)及DIPEA(26mg，0.20mmol)。常温反应30分钟后，LCMS中控反应，中压制备纯化，冷冻干燥后得到化合物5-4，47mg，ESI-MS(m/z)：1127.5Compound 5-3 (50 mg, 0.05 mmol) was dissolved in DMF (1 mL), DNPC (30 mg, 0.10 mmol) and DIPEA (26 mg, 0.20 mmol) were added. After 30 minutes of reaction at room temperature, the reaction was controlled by LCMS, prepared and purified by medium pressure, and lyophilized to obtain compound 5-4, 47 mg, ESI-MS (m/z): 1127.5
步骤五：化合物LD005的合成Step 5: Synthesis of compound LD005
向化合物5-4粗品的DMF溶液中加入MMAE(42mg，0.06mmol)及HOBT(7mg，0.05mmol)，加入DIPEA调节pH至9～11，常温反应过夜。高压制备得12mg产品(白色固体)。ESI-MS(m/z)：1705.6.To the DMF solution of the crude product of compound 5-4, MMAE (42 mg, 0.06 mmol) and HOBT (7 mg, 0.05 mmol) were added, and DIPEA was added to adjust the pH to 9-11, and the reaction was carried out at room temperature overnight. 12 mg of product (white solid) were prepared under high pressure. ESI-MS(m/z): 1705.6.
实施例十七：LD006的合成Embodiment seventeen: the synthesis of LD006
Figure PCTCN2022072943-appb-000035
Figure PCTCN2022072943-appb-000035
步骤一：化合物6-1的合成Step 1: Synthesis of Compound 6-1
将(0.2g，0.83mmol)化合物LN028溶于DMF中，加入DIPEA(0.3g，2，4mmol)及HATU(0.32g，0.83mmol)，反应半小时后加入化合物4-1(0.4g，0.5mmol)。常温反应半小时。中压制备(28％乙腈/水)得到化合物6-1，ESI-MS(m/z)：1005.52Compound LN028 (0.2 g, 0.83 mmol) was dissolved in DMF, DIPEA (0.3 g, 2, 4 mmol) and HATU (0.32 g, 0.83 mmol) were added, and compound 4-1 (0.4 g, 0.5 mmol) was added after the reaction for half an hour. ). React at room temperature for half an hour. Medium pressure preparation (28% acetonitrile/water) gave compound 6-1, ESI-MS (m/z): 1005.52
步骤二：化合物6-2的合成Step 2: Synthesis of Compound 6-2
将化合物6-1(1.0g，1.00mmol)溶于DMF(20mL)中，加二乙胺(10mL)。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到600mg化合物6-2。ESI-MS(m/z)：783.40Compound 6-1 (1.0 g, 1.00 mmol) was dissolved in DMF (20 mL), and diethylamine (10 mL) was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized with water to obtain 600 mg of compound 6-2. ESI-MS(m/z): 783.40
步骤三：化合物6-3的合成Step 3: Synthesis of Compound 6-3
将化合物6-2(500mg，0.64mmol)溶于DMF(10mL)中，加入Mc-OSu(401mg，1.30mmol)。常温搅拌过夜。中压制备(30％乙腈/水)得到160mg化合物6-3(白色固体)。ESI-MS(m/z)：976.43Compound 6-2 (500 mg, 0.64 mmol) was dissolved in DMF (10 mL), and Mc-OSu (401 mg, 1.30 mmol) was added. Stir overnight at room temperature. Medium pressure preparation (30% acetonitrile/water) yielded 160 mg of compound 6-3 (white solid). ESI-MS(m/z): 976.43
步骤四：化合物6-4的合成Step 4: Synthesis of Compound 6-4
将化合物6-3(600mg，0.61mmol)溶于DMF(10mL)中，加入DNPC (374mg，1.23mmol)及DIPEA(159mg，1.23mmol)。常温反应30分钟。中压制备(32％乙腈/水)得560mg化合物6-4(黄色固体)。ESI-MS(m/z)：1141.4Compound 6-3 (600 mg, 0.61 mmol) was dissolved in DMF (10 mL), DNPC (374 mg, 1.23 mmol) and DIPEA (159 mg, 1.23 mmol) were added. React at room temperature for 30 minutes. Medium pressure preparation (32% acetonitrile/water) yielded 560 mg of compound 6-4 (yellow solid). ESI-MS(m/z): 1141.4
步骤五：化合物LD006的合成Step 5: Synthesis of compound LD006
将化合物6-4(0.2g，0.18mmol)溶于DMF中，加入MMAE(0.13g，0.18mmol)和HOBT(25mg，0.19mmol)，加入DIPEA(0.1g，0.78mmol)，常温反应过夜。高压制备得化合物LD1006(白色固体)。ESI-MS(m/z)：1720.8Compound 6-4 (0.2 g, 0.18 mmol) was dissolved in DMF, MMAE (0.13 g, 0.18 mmol) and HOBT (25 mg, 0.19 mmol) were added, DIPEA (0.1 g, 0.78 mmol) was added, and the reaction was carried out at room temperature overnight. The compound LD1006 (white solid) was prepared under high pressure. ESI-MS(m/z): 1720.8
实施例十八：化合物LD007的合成Embodiment 18: Synthesis of compound LD007
Figure PCTCN2022072943-appb-000036
Figure PCTCN2022072943-appb-000036
步骤一：化合物7-2的合成：Step 1: Synthesis of Compound 7-2:
将化合物3-4(0.5g，1.31mmol)及化合物7-1(WO2017/30973；(2017))(0.64g，1.31mmol)溶于DMF中，加入HATU(0.55g。1.44mmol)及DIPEA(0.5g，3.9mmol)。常温反应60分钟。中压制备(35％乙腈)得到化合物7-2，0.61g，ESI-MS(m/z)：1048.5。Compound 3-4 (0.5 g, 1.31 mmol) and compound 7-1 (WO2017/30973; (2017)) (0.64 g, 1.31 mmol) were dissolved in DMF, HATU (0.55 g. 1.44 mmol) and DIPEA ( 0.5 g, 3.9 mmol). The reaction was carried out at room temperature for 60 minutes. Medium pressure preparation (35% acetonitrile) gave compound 7-2, 0.61 g, ESI-MS (m/z): 1048.5.
步骤二：化合物7-3的合成：Step 2: Synthesis of Compound 7-3:
将化合物7-2(0.6g，0.71mmol)溶于10毫升DMF中，加入2毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物7-3，0.39g，ESI-MS(m/z)：826.4。Compound 7-2 (0.6 g, 0.71 mmol) was dissolved in 10 mL of DMF, and 2 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized with water to obtain compound 7-3, 0.39 g, ESI-MS (m/z): 826.4.
步骤三：化合物7-4的合成：Step 3: Synthesis of Compound 7-4:
将化合物7-3(0.35g，0.55mmol)溶于DMF中，加入Mc-OSu(0.17g，0.55mmol)。常温反应过夜。中压制备(43％乙腈/水)得到化合物7-4，0.36g，ESI-MS(m/z)：1019.5。Compound 7-3 (0.35 g, 0.55 mmol) was dissolved in DMF and Mc-OSu (0.17 g, 0.55 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (43% acetonitrile/water) gave compound 7-4, 0.36 g, ESI-MS (m/z): 1019.5.
步骤四：化合物7-5的合成：Step 4: Synthesis of Compounds 7-5:
将化合物7-4(0.35g，0.42mmol)溶于DMF中，加入DNPC(0.26g，0.85mmol)及DIPEA(0.17g，1.3mmol)。常温反应7小时。中压制备(55％乙腈/水)得到化合物7-5，0.27g，ESI-MS(m/z)：1184.5。Compound 7-4 (0.35 g, 0.42 mmol) was dissolved in DMF, and DNPC (0.26 g, 0.85 mmol) and DIPEA (0.17 g, 1.3 mmol) were added. The reaction was carried out at room temperature for 7 hours. Medium pressure preparation (55% acetonitrile/water) gave compound 7-5, 0.27 g, ESI-MS (m/z): 1184.5.
步骤五：化合物LD007的合成：Step 5: Synthesis of compound LD007:
将化合物7-5(0.25g，0.25mmol)溶于DMF中，依次加入MMAE(0.18g，0.25mmol)、HOBT(35mg，0.25mmol)及DIPEA(97mg，0.75mmol)。常温反应5小时。中压制备(43％乙腈/水)得到化合物LD007，ESI-MS(m/z)：1763.0。Compound 7-5 (0.25 g, 0.25 mmol) was dissolved in DMF, MMAE (0.18 g, 0.25 mmol), HOBT (35 mg, 0.25 mmol) and DIPEA (97 mg, 0.75 mmol) were added sequentially. The reaction was carried out at room temperature for 5 hours. Medium pressure preparation (43% acetonitrile/water) gave compound LD007, ESI-MS (m/z): 1763.0.
实施例十九：化合物LD008的合成Example 19: Synthesis of compound LD008
Figure PCTCN2022072943-appb-000037
Figure PCTCN2022072943-appb-000037
步骤一：化合物8-1的合成：Step 1: Synthesis of Compound 8-1:
将化合物3-4(0.7g，1.2mmol)及化合物LN030(0.95g，1.9mmol)溶于10毫升DMF中，加入HATU(800mg，2.1mmol)及DIPEA(1.0g，7.8mmol)。常温反应60分钟。中压制备(34％乙腈/水)得到化合物8-1，0.67g，ESI-MS(m/z)：1062.5。Compound 3-4 (0.7 g, 1.2 mmol) and compound LN030 (0.95 g, 1.9 mmol) were dissolved in 10 mL of DMF, and HATU (800 mg, 2.1 mmol) and DIPEA (1.0 g, 7.8 mmol) were added. The reaction was carried out at room temperature for 60 minutes. Medium pressure preparation (34% acetonitrile/water) gave compound 8-1, 0.67 g, ESI-MS (m/z): 1062.5.
步骤二：化合物8-2的合成：Step 2: Synthesis of Compound 8-2:
将化合物8-1(0.65g，0.61mmol)溶于5毫升DMF中，加入1毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物8-2，0.43g，ESI-MS(m/z)：840.4。Compound 8-1 (0.65 g, 0.61 mmol) was dissolved in 5 mL of DMF, and 1 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized with water to obtain compound 8-2, 0.43 g, ESI-MS (m/z): 840.4.
步骤三：化合物8-3的合成：Step 3: Synthesis of Compound 8-3:
将化合物8-2(0.4g，0.48mmol)溶于DMF中，加入Mc-OSu(0.2g，0.65mmol)。常温反应过夜。中压制备(43％乙腈/水)得到化合物8-3，0.33g，ESI-MS(m/z)：1033.5。Compound 8-2 (0.4 g, 0.48 mmol) was dissolved in DMF and Mc-OSu (0.2 g, 0.65 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (43% acetonitrile/water) gave compound 8-3, 0.33 g, ESI-MS (m/z): 1033.5.
步骤四：化合物8-4的合成：Step 4: Synthesis of Compound 8-4:
将化合物8-3(0.30g，0.29mmol)溶于DMF中，加入DNPC(192mg， 0.63mmol)及DIPEA(163mg，1.26mmol)。常温反应7小时。中压制备(55％乙腈/水)得到化合物8-4，0.27g，ESI-MS(m/z)：1198.5。Compound 8-3 (0.30 g, 0.29 mmol) was dissolved in DMF, and DNPC (192 mg, 0.63 mmol) and DIPEA (163 mg, 1.26 mmol) were added. The reaction was carried out at room temperature for 7 hours. Medium pressure preparation (55% acetonitrile/water) gave compound 8-4, 0.27 g, ESI-MS (m/z): 1198.5.
步骤五：化合物LD008的合成：Step 5: Synthesis of compound LD008:
将化合物8-4(0.2g，0.17mmol)溶于DMF中，依次加入MMAE(143mg，0.20mmol)、HOBT(30mg，0.22mmol)及DIPEA(90mg，0.70mmol)。常温反应5小时。中压制备(45％乙腈/水)得到LD008，135mg，ESI-MS(m/z)：1777.0。Compound 8-4 (0.2 g, 0.17 mmol) was dissolved in DMF, MMAE (143 mg, 0.20 mmol), HOBT (30 mg, 0.22 mmol) and DIPEA (90 mg, 0.70 mmol) were added sequentially. The reaction was carried out at room temperature for 5 hours. Medium pressure preparation (45% acetonitrile/water) gave LD008, 135 mg, ESI-MS (m/z): 1777.0.
实施例二十：化合物LD009的合成Example 20: Synthesis of compound LD009
Figure PCTCN2022072943-appb-000038
Figure PCTCN2022072943-appb-000038
将0.21g(0.23mmol)化合物3-6溶于DMF中，加入化合物9-1(CN 111499685 A)(0.28g，0.45mmol)和HOBT(35.2mg，0.26mmol)，加入DIPEA调节pH至9～10，常温反应过夜。高压制备得化合物LD009(白色固体)，0.16g。ESI-MS(m/z)：1299.6Dissolve 0.21 g (0.23 mmol) of compound 3-6 in DMF, add compound 9-1 (CN 111499685 A) (0.28 g, 0.45 mmol) and HOBT (35.2 mg, 0.26 mmol), add DIPEA to adjust the pH to 9～ 10. React at room temperature overnight. The compound LD009 (white solid) was prepared under high pressure, 0.16 g. ESI-MS(m/z): 1299.6
实施例二十一：化合物LD010的合成Example 21: Synthesis of compound LD010
Figure PCTCN2022072943-appb-000039
Figure PCTCN2022072943-appb-000039
将0.25g(0.22mmol)化合物3-6溶于DMF中，加入0.12g(0.23mmol)化合物9-1(CN 111499685 A)和HOBT(35.2mg，0.26mmol)，加入DIPEA调节pH至9～10，常温反应过夜。高压制备得化合物LD010(白色固体)，0.12g。ESI-MS(m/z)：1494.7Dissolve 0.25g (0.22mmol) of compound 3-6 in DMF, add 0.12g (0.23mmol) of compound 9-1 (CN 111499685 A) and HOBT (35.2mg, 0.26mmol), add DIPEA to adjust the pH to 9-10 , and reacted at room temperature overnight. The compound LD010 (white solid) was prepared under high pressure, 0.12 g. ESI-MS(m/z): 1494.7
实施例二十二：化合物LD011的合成Example 22: Synthesis of compound LD011
Figure PCTCN2022072943-appb-000040
Figure PCTCN2022072943-appb-000040
将0.21g(0.23mmol)化合物3-6溶于DMF中，加入0.11g(0.24mmol)exatecan和HOBT(35.2mg，0.26mmol)，加入DIPEA调节pH至9～10，常温反应过夜。高压制备得化合物LD011(白色固体)，0.14g。ESI-MS(m/z)：1299.60.21 g (0.23 mmol) of compound 3-6 was dissolved in DMF, 0.11 g (0.24 mmol) of exatecan and HOBT (35.2 mg, 0.26 mmol) were added, DIPEA was added to adjust the pH to 9-10, and the reaction was carried out at room temperature overnight. The compound LD011 (white solid) was prepared under high pressure, 0.14 g. ESI-MS(m/z): 1299.6
实施例二十三：化合物LD012的合成Example 23: Synthesis of compound LD012
Figure PCTCN2022072943-appb-000041
Figure PCTCN2022072943-appb-000041
将0.25g(0.22mmol)化合物5-4溶于DMF中，加入0.11g(0.24mmol)exatecan和HOBT(35.2mg，0.26mmol)，加入DIPEA调节pH至9～10，常温反应过夜。高压制备得化合物LD012(白色固体)，0.13g。ESI-MS(m/z)：1423.60.25 g (0.22 mmol) of compound 5-4 was dissolved in DMF, 0.11 g (0.24 mmol) of exatecan and HOBT (35.2 mg, 0.26 mmol) were added, DIPEA was added to adjust the pH to 9-10, and the reaction was carried out at room temperature overnight. The compound LD012 (white solid) was prepared under high pressure, 0.13 g. ESI-MS(m/z): 1423.6
实施例二十四：化合物LD013的合成Example 24: Synthesis of compound LD013
Figure PCTCN2022072943-appb-000042
Figure PCTCN2022072943-appb-000042
将0.25g(0.21mmol)化合物3-6溶于DMF中，加入0.11g(0.24mmol)exatecan和HOBT(35.2mg，0.26mmol)，加入DIPEA调节pH至9～10，常温 反应过夜。高压制备得化合物LD013(白色固体)，0.13g。ESI-MS(m/z)：1480.6。0.25 g (0.21 mmol) of compound 3-6 was dissolved in DMF, 0.11 g (0.24 mmol) of exatecan and HOBT (35.2 mg, 0.26 mmol) were added, DIPEA was added to adjust the pH to 9-10, and the reaction was carried out at room temperature overnight. The compound LD013 (white solid) was prepared under high pressure, 0.13 g. ESI-MS (m/z): 1480.6.
实施例二十五：化合物LD014的合成Example 25: Synthesis of compound LD014
Figure PCTCN2022072943-appb-000043
Figure PCTCN2022072943-appb-000043
将化合物8-4(80mg，0.068mmol)溶于干燥的DMF中，依次加入exatecan(44mg，0.10mmol)、HOBT(15mg，0.11mmol)及DIPEA(40mg，0.31mmol)。常温反应八小时。中压制备(41％乙腈/水)得到产品，56mg，ESI-MS(m/z)：1494.6。Compound 8-4 (80 mg, 0.068 mmol) was dissolved in dry DMF and exatecan (44 mg, 0.10 mmol), HOBT (15 mg, 0.11 mmol) and DIPEA (40 mg, 0.31 mmol) were added sequentially. The reaction was carried out at room temperature for eight hours. Medium pressure preparation (41% acetonitrile/water) gave the product, 56 mg, ESI-MS (m/z): 1494.6.
实施例二十六：化合物LD015的合成Example 26: Synthesis of compound LD015
Figure PCTCN2022072943-appb-000044
Figure PCTCN2022072943-appb-000044
步骤一：化合物15-2的合成：Step 1: Synthesis of Compound 15-2:
将化合物15-1(0.5g，1.31mmol)及化合物7-1(WO2017/30973；(2017))(0.64g，1.31mmol)溶于DMF中，加入HATU(0.55g。1.44mmol)及DIPEA(0.5g，3.9mmol)。常温反应60分钟。中压制备(35％乙腈)得到化合物15-2，0.61g，ESI-MS(m/z)：854.4。Compound 15-1 (0.5 g, 1.31 mmol) and compound 7-1 (WO2017/30973; (2017)) (0.64 g, 1.31 mmol) were dissolved in DMF, HATU (0.55 g. 1.44 mmol) and DIPEA ( 0.5 g, 3.9 mmol). The reaction was carried out at room temperature for 60 minutes. Medium pressure preparation (35% acetonitrile) gave compound 15-2, 0.61 g, ESI-MS (m/z): 854.4.
步骤二：化合物15-3的合成：Step 2: Synthesis of Compound 15-3:
将化合物15-2(0.6g，0.71mmol)溶于10毫升DMF中，加入2毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物15-3，0.39g，ESI-MS(m/z)：632.3。Compound 15-2 (0.6 g, 0.71 mmol) was dissolved in 10 mL of DMF and 2 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized with water to obtain compound 15-3, 0.39 g, ESI-MS (m/z): 632.3.
步骤三：化合物15-4的合成：Step 3: Synthesis of Compound 15-4:
将化合物15-3(0.35g，0.55mmol)溶于DMF中，加入Mc-OSu(0.17g，0.55mmol)。常温反应过夜。中压制备(43％乙腈/水)得到化合物15-4，0.36g， ESI-MS(m/z)：825.4。Compound 15-3 (0.35 g, 0.55 mmol) was dissolved in DMF and Mc-OSu (0.17 g, 0.55 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (43% acetonitrile/water) gave compound 15-4, 0.36 g, ESI-MS (m/z): 825.4.
步骤四：化合物15-5的合成：Step 4: Synthesis of Compound 15-5:
将化合物15-4(0.35g，0.42mmol)溶于DMF中，加入DNPC(0.26g，0.85mmol)及DIPEA(0.17g，1.3mmol)。常温反应7小时。中压制备(55％乙腈/水)得到化合物15-5，0.27g，ESI-MS(m/z)：990.4。Compound 15-4 (0.35 g, 0.42 mmol) was dissolved in DMF, and DNPC (0.26 g, 0.85 mmol) and DIPEA (0.17 g, 1.3 mmol) were added. The reaction was carried out at room temperature for 7 hours. Medium pressure preparation (55% acetonitrile/water) gave compound 15-5, 0.27 g, ESI-MS (m/z): 990.4.
步骤五：化合物LD015的合成：Step 5: Synthesis of compound LD015:
将化合物15-5(0.25g，0.25mmol)溶于DMF中，依次加入MMAE(0.18g，0.25mmol)、HOBT(35mg，0.25mmol)及DIPEA(97mg，0.75mmol)。常温反应5小时。中压制备(43％乙腈/水)得到化合物LD015，ESI-MS(m/z)：1568.9。Compound 15-5 (0.25 g, 0.25 mmol) was dissolved in DMF, MMAE (0.18 g, 0.25 mmol), HOBT (35 mg, 0.25 mmol) and DIPEA (97 mg, 0.75 mmol) were added sequentially. The reaction was carried out at room temperature for 5 hours. Medium pressure preparation (43% acetonitrile/water) gave compound LD015, ESI-MS (m/z): 1568.9.
实施例二十七：化合物LD016的合成Example 27: Synthesis of compound LD016
Figure PCTCN2022072943-appb-000045
Figure PCTCN2022072943-appb-000045
Figure PCTCN2022072943-appb-000046
Figure PCTCN2022072943-appb-000046
步骤一：化合物16-1的合成：Step 1: Synthesis of Compound 16-1:
将化合物3-4(0.7g，0.18mmol)及化合物LN030(0.95g，0.19mmol)溶于10毫升DMF中，加入HATU(80mg，0.21mmol)及DIPEA(100mg，0.78mmol)。常温反应60分钟。中压制备(34％乙腈/水)得到化合物16-1，0.67g，ESI-MS(m/z)：1062.5。Compound 3-4 (0.7 g, 0.18 mmol) and compound LN030 (0.95 g, 0.19 mmol) were dissolved in 10 mL of DMF, HATU (80 mg, 0.21 mmol) and DIPEA (100 mg, 0.78 mmol) were added. The reaction was carried out at room temperature for 60 minutes. Medium pressure preparation (34% acetonitrile/water) gave compound 16-1, 0.67 g, ESI-MS (m/z): 1062.5.
步骤二：化合物16-2的合成：Step 2: Synthesis of Compound 16-2:
将化合物16-1(0.65g，0.75mmol)溶于5毫升DMF中，加入1毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物16-2，0.43g，ESI-MS(m/z)：840.4。Compound 16-1 (0.65 g, 0.75 mmol) was dissolved in 5 mL of DMF, and 1 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary-evaporated to remove diethylamine, and lyophilized with water to obtain compound 16-2, 0.43 g, ESI-MS (m/z): 840.4.
步骤三：化合物16-3的合成：Step 3: Synthesis of Compound 16-3:
将化合物16-2(0.4g，0.62mmol)溶于DMF中，加入Mc-OSu(0.2g，0.65mmol)。常温反应过夜。中压制备(43％乙腈/水)得到化合物16-3，0.37g，ESI-MS(m/z)：1033.5。Compound 16-2 (0.4 g, 0.62 mmol) was dissolved in DMF and Mc-OSu (0.2 g, 0.65 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (43% acetonitrile/water) gave compound 16-3, 0.37 g, ESI-MS (m/z): 1033.5.
步骤四：化合物16-4的合成：Step 4: Synthesis of Compound 16-4:
将化合物16-3(0.35g，0.42mmol)溶于DMF中，加入DNPC(192mg，0.63mmol)及DIPEA(163mg，1.26mmol)。常温反应7小时。中压制备(55％乙腈/水)得到化合物16-4，0.31g，ESI-MS(m/z)：1198.5。Compound 16-3 (0.35 g, 0.42 mmol) was dissolved in DMF, and DNPC (192 mg, 0.63 mmol) and DIPEA (163 mg, 1.26 mmol) were added. The reaction was carried out at room temperature for 7 hours. Medium pressure preparation (55% acetonitrile/water) afforded compound 16-4, 0.31 g, ESI-MS (m/z): 1198.5.
步骤五：化合物LD016的合成：Step 5: Synthesis of compound LD016:
将化合物16-4(0.2g，0.20mmol)溶于DMF中，依次加入MMAE(143mg，0.20mmol)、HOBT(30mg，0.22mmol)及DIPEA(90mg，0.70mmol)。常温反应5小时。中压制备(45％乙腈/水)得到LD016，135mg，ESI-MS(m/z)：1777.0。Compound 16-4 (0.2 g, 0.20 mmol) was dissolved in DMF, MMAE (143 mg, 0.20 mmol), HOBT (30 mg, 0.22 mmol) and DIPEA (90 mg, 0.70 mmol) were added sequentially. The reaction was carried out at room temperature for 5 hours. Medium pressure preparation (45% acetonitrile/water) gave LD016, 135 mg, ESI-MS (m/z): 1777.0.
实施例二十八：化合物LD017的合成Example 28: Synthesis of compound LD017
Figure PCTCN2022072943-appb-000047
Figure PCTCN2022072943-appb-000047
步骤一：化合物17-1的合成：Step 1: Synthesis of Compound 17-1:
将化合物3-3(1.1g，1.38mmol)溶于DMF(10ml)中，加入二乙胺(2ml)。常温反应四小时。中压制备(30％乙腈/水)得到产品17-1(0.84g)，ESI-MS(m/z)：573.3。Compound 3-3 (1.1 g, 1.38 mmol) was dissolved in DMF (10 ml), and diethylamine (2 ml) was added. The reaction was carried out at room temperature for four hours. Medium pressure preparation (30% acetonitrile/water) gave product 17-1 (0.84 g), ESI-MS (m/z): 573.3.
步骤二：化合物17-3的合成：Step 2: Synthesis of Compound 17-3:
将化合物17-1(840mg，0.59mmol)和17-2(505mg，0.71mmol)溶于DMF(5ml)中，加入HATU(314mg，0.83mmol)和DIPEA(152mg，1.08mmol)。常温反应2小时。中压制备(50％乙腈/水)得到产品17-3(960mg)，ESI-MS(m/z)：1264.6。Compounds 17-1 (840 mg, 0.59 mmol) and 17-2 (505 mg, 0.71 mmol) were dissolved in DMF (5 ml), HATU (314 mg, 0.83 mmol) and DIPEA (152 mg, 1.08 mmol) were added. The reaction was carried out at room temperature for 2 hours. Medium pressure preparation (50% acetonitrile/water) gave product 17-3 (960 mg), ESI-MS (m/z): 1264.6.
步骤三：化合物17-4的合成：Step 3: Synthesis of Compound 17-4:
将化合物17-3(850mg)溶于5ml DMF中，加入DNPC(408mg)及DIPEA(9mg)。常温反应18小时。中压制备(40％乙腈/水)得到750mg产品，ESI-MS(m/z)：1429.6。Compound 17-3 (850 mg) was dissolved in 5 ml of DMF, and DNPC (408 mg) and DIPEA (9 mg) were added. The reaction was carried out at room temperature for 18 hours. Medium pressure preparation (40% acetonitrile/water) gave 750 mg of product, ESI-MS (m/z): 1429.6.
步骤四：化合物17-5的合成：Step 4: Synthesis of Compound 17-5:
将化合物17-4(170mg)溶于2ml DMF中，依次加入MMAE(140mg)、HOBT(16mg)及DIPEA(50mg)。常温反应16小时。中压制备(42％乙腈/水)得到120mg产品，ESI-MS(m/z)：2008.0。Compound 17-4 (170 mg) was dissolved in 2 ml of DMF, and MMAE (140 mg), HOBT (16 mg) and DIPEA (50 mg) were added sequentially. The reaction was carried out at room temperature for 16 hours. Medium pressure preparation (42% acetonitrile/water) gave 120 mg of product, ESI-MS (m/z): 2008.0.
步骤五：化合物17-6的合成：Step 5: Synthesis of Compound 17-6:
将化合物17-5(110mg)溶于2ml DMF中，加入二乙胺(0.2ml)。常温反应2小时。中压制备(30％乙腈/水)得到30mg产品，ESI-MS(m/z)：1563.9。Compound 17-5 (110 mg) was dissolved in 2 ml of DMF and diethylamine (0.2 ml) was added. The reaction was carried out at room temperature for 2 hours. Medium pressure preparation (30% acetonitrile/water) gave 30 mg of product, ESI-MS (m/z): 1563.9.
步骤六：化合物LD017的合成：Step 6: Synthesis of compound LD017:
将化合物17-6(30mg)溶于1ml DMF中，加入DBBK(10mg)。常温反应5分钟。高压制备(60％乙腈/水)得到27mg产品，ESI-MS(m/z)：1769.7。Compound 17-6 (30 mg) was dissolved in 1 ml of DMF and DBBK (10 mg) was added. React at room temperature for 5 minutes. High pressure preparation (60% acetonitrile/water) gave 27 mg of product, ESI-MS (m/z): 1769.7.
实施例二十九：化合物LD018的合成Example 29: Synthesis of compound LD018
Figure PCTCN2022072943-appb-000048
Figure PCTCN2022072943-appb-000048
将0.17g(0.18mmol)化合物3-6溶于干燥的2ml DMF中，依次加入HOBT(27.5mg，0.18mmol)、化合物19-6(97.3mg，0.16mmol)，用DIPEA调节碱性至pH值为9～10。常温反应1小时。中压制备(41％乙腈/水)得到化合物LD018 110mg，ESI-MS(m/z)：1400.6。Dissolve 0.17 g (0.18 mmol) of compound 3-6 in dry 2 ml of DMF, add HOBT (27.5 mg, 0.18 mmol), and compound 19-6 (97.3 mg, 0.16 mmol) in turn, adjust the basicity to pH value with DIPEA 9 to 10. The reaction was carried out at room temperature for 1 hour. Medium pressure preparation (41% acetonitrile/water) gave compound LD018 110 mg, ESI-MS (m/z): 1400.6.
实施例三十：化合物LD019的合成：Example 30: Synthesis of compound LD019:
Figure PCTCN2022072943-appb-000049
Figure PCTCN2022072943-appb-000049
Figure PCTCN2022072943-appb-000050
Figure PCTCN2022072943-appb-000050
步骤一：化合物19-2的合成：Step 1: Synthesis of compound 19-2:
将化合物19-1(200mg)溶于2ml DMA中，加入乙醇酸(42mg)、DIPEA(149mg)和HATU(184mg)。常温反应5分钟。中压制备(40％乙腈/水)得到180mg化合物19-2，ESI-MS(m/z)：493.2。Compound 19-1 (200 mg) was dissolved in 2 ml of DMA and glycolic acid (42 mg), DIPEA (149 mg) and HATU (184 mg) were added. React at room temperature for 5 minutes. Medium pressure preparation (40% acetonitrile/water) gave 180 mg of compound 19-2, ESI-MS (m/z): 493.2.
步骤二：化合物19-3的合成：Step 2: Synthesis of Compound 19-3:
将化合物19-2(180mg)溶于4ml THF中，加入PNC(100mg)、DIPEA(52mg)。常温反应四小时。中压制备(40％乙腈/水)得到200mg化合物19-3，ESI-MS(m/z)：658.2。Compound 19-2 (180 mg) was dissolved in 4 ml of THF, and PNC (100 mg) and DIPEA (52 mg) were added. The reaction was carried out at room temperature for four hours. Medium pressure preparation (40% acetonitrile/water) gave 200 mg of compound 19-3, ESI-MS (m/z): 658.2.
步骤三：化合物19-5的合成：Step 3: Synthesis of Compound 19-5:
将化合物19-3(200mg)和19-4(85mg)溶于4ml DMF中，加入HOBT(41mg)、DIPEA(77mg)。常温反应18小时。中压制备(40％乙腈/水)得到150mg化合物19-5，ESI-MS(m/z)：707.3。Compounds 19-3 (200 mg) and 19-4 (85 mg) were dissolved in 4 ml of DMF, and HOBT (41 mg), DIPEA (77 mg) were added. The reaction was carried out at room temperature for 18 hours. Medium pressure preparation (40% acetonitrile/water) gave 150 mg of compound 19-5, ESI-MS (m/z): 707.3.
步骤四：化合物19-6的合成：Step 4: Synthesis of Compound 19-6:
将化合物19-5(150mg)溶于3ml DCM中，加入1ml TFA。常温反应2小时。中压制备(30％乙腈/水)得到140mg化合物19-6，MS：607.3。Compound 19-5 (150 mg) was dissolved in 3 ml of DCM and 1 ml of TFA was added. The reaction was carried out at room temperature for 2 hours. Medium pressure preparation (30% acetonitrile/water) gave 140 mg of compound 19-6, MS: 607.3.
步骤五：化合物19-7的合成：Step 5: Synthesis of Compound 19-7:
将化合物17-4(170mg)溶于2ml DMF中，依次加入19-6(140mg)、HOBT(16mg)及DIPEA(50mg)。常温反应16小时。中压制备(42％乙腈/水)得到110mg化合物19-7，ESI-MS(m/z)：1897.8。Compound 17-4 (170 mg) was dissolved in 2 ml of DMF, and 19-6 (140 mg), HOBT (16 mg) and DIPEA (50 mg) were added sequentially. The reaction was carried out at room temperature for 16 hours. Medium pressure preparation (42% acetonitrile/water) gave 110 mg of compound 19-7, ESI-MS (m/z): 1897.8.
步骤六：化合物19-8的合成：Step 6: Synthesis of Compound 19-8:
将化合物19-7(110mg)溶于2ml DMF中，加入二乙胺(0.2ml)。常温反应2小时。中压制备(30％乙腈)得到30mg化合物19-8，ESI-MS(m/z)：1453.6。Compound 19-7 (110 mg) was dissolved in 2 ml of DMF, and diethylamine (0.2 ml) was added. The reaction was carried out at room temperature for 2 hours. Medium pressure preparation (30% acetonitrile) gave 30 mg of compound 19-8, ESI-MS (m/z): 1453.6.
步骤七：化合物LD019的合成：Step 7: Synthesis of compound LD019:
将化合物19-8(30mg)溶于1ml DMF中，加入DBBK(10mg)。常温反应5分钟。高压制备(60％乙腈/水)得到22mg化合物LD019，ESI-MS(m/z)：1659.5。Compound 19-8 (30 mg) was dissolved in 1 ml of DMF and DBBK (10 mg) was added. React at room temperature for 5 minutes. High pressure preparation (60% acetonitrile/water) gave 22 mg of compound LD019, ESI-MS (m/z): 1659.5.
实施例三十一：化合物LD020的合成：Example 31: Synthesis of compound LD020:
Figure PCTCN2022072943-appb-000051
Figure PCTCN2022072943-appb-000051
Figure PCTCN2022072943-appb-000052
Figure PCTCN2022072943-appb-000052
步骤一：化合物20-2的合成：Step 1: Synthesis of Compound 20-2:
将化合物17-4(170mg)溶于2ml DMF中，依次加入20-1(120mg)、HOBT(16mg)及DIPEA(50mg)。常温反应16小时。中压制备(42％乙腈/水)得到110mg产品，ESI-MS(m/z)：2062.1。Compound 17-4 (170 mg) was dissolved in 2 ml of DMF, and 20-1 (120 mg), HOBT (16 mg) and DIPEA (50 mg) were added sequentially. The reaction was carried out at room temperature for 16 hours. Medium pressure preparation (42% acetonitrile/water) gave 110 mg of product, ESI-MS (m/z): 2062.1.
步骤二：化合物20-3的合成：Step 2: Synthesis of Compound 20-3:
将化合物20-2(110mg)溶于2ml DMF中，加入DEA(0.2ml)。常温反应2小时。中压制备(30％乙腈)得到50mg产品，ESI-MS(m/z)：1617.9。Compound 20-2 (110 mg) was dissolved in 2 ml of DMF and DEA (0.2 ml) was added. The reaction was carried out at room temperature for 2 hours. Medium pressure preparation (30% acetonitrile) gave 50 mg of product, ESI-MS (m/z): 1617.9.
步骤三：化合物LD020的合成：Step 3: Synthesis of compound LD020:
将化合物20-3(50mg)溶于1ml DMF中，加入DBBK(15mg)。常温反应5分钟。高压制备(60％乙腈)得到35mg化合物LD020，ESI-MS(m/z)：1823.8。Compound 20-3 (50 mg) was dissolved in 1 ml of DMF and DBBK (15 mg) was added. React at room temperature for 5 minutes. High pressure preparation (60% acetonitrile) gave 35 mg of compound LD020, ESI-MS (m/z): 1823.8.
实施例三十二：化合物LD021的合成Example 32: Synthesis of compound LD021
Figure PCTCN2022072943-appb-000053
Figure PCTCN2022072943-appb-000053
步骤一：化合物21-2的合成：Step 1: Synthesis of Compound 21-2:
将化合物21-1(2g，4.1mmol)溶于干燥的DCM中，氮气保护下，依次加入三光气(0.49g，1.64mmol)和DMAP(1.5g，12.3mmol)，反应15min后得到化合物21-2的粗品。Compound 21-1 (2 g, 4.1 mmol) was dissolved in dry DCM, and under nitrogen protection, triphosgene (0.49 g, 1.64 mmol) and DMAP (1.5 g, 12.3 mmol) were added successively, and compound 21- was obtained after reaction for 15 min. 2 of the crude product.
步骤二：化合物21-3的合成：Step 2: Synthesis of Compound 21-3:
将化合物3-5(1.0g，1.3mmol)溶于干燥的DMF中，然后加入化合物21-2的反应液，反应30min后，减压浓缩，出去DCM后，中压反相(60％乙腈/水)纯化，冻干得到化合物21-3，0.6g，ESI-MS(m/z)：1285.6。Compound 3-5 (1.0 g, 1.3 mmol) was dissolved in dry DMF, then the reaction solution of compound 21-2 was added, reacted for 30 min, concentrated under reduced pressure, removed DCM, reversed-phase (60% acetonitrile/ water), and lyophilized to give compound 21-3, 0.6 g, ESI-MS (m/z): 1285.6.
步骤三：化合物LN021的合成：Step 3: Synthesis of compound LN021:
将化合物21-3(0.5g，0.39mmol)溶于10mL乙腈中，然后加入3mL TFA,常温反应7h，真空浓缩后，高压制备纯化(50％乙腈/水)，冻干后得到化合物LN021，341mg，ESI-MS(m/z)：1185.5。Compound 21-3 (0.5 g, 0.39 mmol) was dissolved in 10 mL of acetonitrile, then 3 mL of TFA was added, reacted at room temperature for 7 h, concentrated in vacuo, purified by high pressure preparation (50% acetonitrile/water), and lyophilized to obtain compound LN021, 341 mg , ESI-MS (m/z): 1185.5.
实施例三十三：化合物LD022的合成Example 33: Synthesis of compound LD022
Figure PCTCN2022072943-appb-000054
Figure PCTCN2022072943-appb-000054
将0.17g(0.18mmol)化合物3-6溶于干燥的2ml DMF中，依次加入HOBT(27.5mg，0.18mmol)、22-1(CN 107857798 A)(124mg，0.16mmol)，用DIPEA调节碱性至pH值为9～11。常温反应1小时。中压制备(41％乙腈/水)得到产品53mg，ESI-MS(m/z)：1400.6。Dissolve 0.17g (0.18mmol) of compound 3-6 in dry 2ml DMF, add HOBT (27.5mg, 0.18mmol), 22-1 (CN 107857798 A) (124mg, 0.16mmol) successively, adjust the basicity with DIPEA to pH 9-11. The reaction was carried out at room temperature for 1 hour. Medium pressure preparation (41% acetonitrile/water) gave 53 mg of product, ESI-MS (m/z): 1400.6.
实施例三十四：化合物LD023的合成Example 34: Synthesis of compound LD023
Figure PCTCN2022072943-appb-000055
Figure PCTCN2022072943-appb-000055
Figure PCTCN2022072943-appb-000056
Figure PCTCN2022072943-appb-000056
步骤一：化合物23-2的合成：Step 1: Synthesis of Compound 23-2:
将化合物23-1(150mg，0.31mmol)及1-5(145mg，0.31mmol)溶于5毫升DMF中，加入HATU(153mg，0.40mmol)及DIPEA(120mg，0.93mmol)。常温反应15分钟后。中压制备(36％乙腈/水)得到化合物23-2，207mg，ESI-MS(m/z)：934.5。Compounds 23-1 (150 mg, 0.31 mmol) and 1-5 (145 mg, 0.31 mmol) were dissolved in 5 mL of DMF, HATU (153 mg, 0.40 mmol) and DIPEA (120 mg, 0.93 mmol) were added. After 15 minutes of reaction at room temperature. Medium pressure preparation (36% acetonitrile/water) afforded compound 23-2, 207 mg, ESI-MS (m/z): 934.5.
步骤二：化合物23-3的合成：Step 2: Synthesis of Compound 23-3:
将化合物23-2(200mg，0.21mmol)溶于2毫升DMF中，加入DNPC(100mg，0.35mmol)及DIPEA(85mg，0.66mmol)。常温反应8小时后。中压制备(52％乙腈/水)得到化合物23-3，157mg，ESI-MS(m/z)：1099.5。Compound 23-2 (200 mg, 0.21 mmol) was dissolved in 2 mL of DMF, and DNPC (100 mg, 0.35 mmol) and DIPEA (85 mg, 0.66 mmol) were added. After 8 hours of reaction at room temperature. Medium pressure preparation (52% acetonitrile/water) gave compound 23-3, 157 mg, ESI-MS (m/z): 1099.5.
步骤三：化合物LD023的合成：Step 3: Synthesis of compound LD023:
将化合物23-3(150mg，0.14mmol)溶于2毫升DMF中，依次加入MMAE(98mg，0.14mmol)、HOBT(18mg，0.14mmol)及DIPEA(54mg，0.42mmol)。常温反应四小时。中压制备(43％乙腈/水)得到产品，78mg，ESI-MS(m/z)：1677.9。Compound 23-3 (150 mg, 0.14 mmol) was dissolved in 2 mL of DMF, MMAE (98 mg, 0.14 mmol), HOBT (18 mg, 0.14 mmol) and DIPEA (54 mg, 0.42 mmol) were added sequentially. The reaction was carried out at room temperature for four hours. Medium pressure preparation (43% acetonitrile/water) gave the product, 78 mg, ESI-MS (m/z): 1677.9.
实施例三十五：化合物LD024的合成Embodiment 35: the synthesis of compound LD024
Figure PCTCN2022072943-appb-000057
Figure PCTCN2022072943-appb-000057
步骤一：取化合物3-3(4.14g，5.2mmol)，50毫升DMF加入反应瓶中， 再加入二(对硝基苯)碳酸酯(3.5g，11.5mmol)和DIPEA(3.5g，27mmol)，室温反应2小时，反应结束后，所得反应液直接用中压制备进行纯化，得化合物24-1，3.4g，ESI-MS(m/z)：961.4[M+H] +。 Step 1: Take compound 3-3 (4.14g, 5.2mmol), add 50ml of DMF to the reaction flask, then add bis(p-nitrobenzene)carbonate (3.5g, 11.5mmol) and DIPEA (3.5g, 27mmol) , and reacted at room temperature for 2 hours. After the reaction, the obtained reaction solution was directly purified by medium pressure preparation to obtain compound 24-1, 3.4 g, ESI-MS (m/z): 961.4 [M+H] + .
步骤二：化合物24-2的合成：Step 2: Synthesis of Compound 24-2:
取化合物24-1(2.0g，2.1mmol)加入250毫升反应瓶中，加入50毫升DMF、1.0g(7mmol)HOBT、2.0g(2.8mmol)MMAE和2.0g(15.5mmol)DIPEA，室温反应1小时，所得反应液直接用高压制备进行纯化，得化合物24-2，2.3g，ESI-MS(m/z)：1539.9[M+H] +。 Take compound 24-1 (2.0g, 2.1mmol) into a 250ml reaction flask, add 50ml DMF, 1.0g (7mmol) HOBT, 2.0g (2.8mmol) MMAE and 2.0g (15.5mmol) DIPEA, and react at room temperature for 1 After hours, the obtained reaction solution was directly purified by high pressure preparation to obtain compound 24-2, 2.3 g, ESI-MS (m/z): 1539.9 [M+H] + .
步骤三：化合物24-3的合成：Step 3: Synthesis of Compound 24-3:
将化合物24-2(2.12g，1.38mmol)溶于DMF(10ml)中，加入二乙胺(2ml)。常温反应四小时。中压制备(30％乙腈/水)得到产品24-3(1.6g)，ESI-MS(m/z)：1317.9。Compound 24-2 (2.12 g, 1.38 mmol) was dissolved in DMF (10 ml), and diethylamine (2 ml) was added. The reaction was carried out at room temperature for four hours. Medium pressure preparation (30% acetonitrile/water) gave product 24-3 (1.6 g), ESI-MS (m/z): 1317.9.
步骤四：化合物24-5的合成：Step 4: Synthesis of Compound 24-5:
将化合物24-4(100mg，0.25mmol)及24-3(90mg，0.068mmol)溶于DMF中，加入HATU(114mg，0.3mmol)及DIPEA(81mg，0.625mmol)。常温反应十分钟。中压制备(43％乙腈/水)得到化合物24-5，81mg，ESI-MS(m/z)：1711.0。Compounds 24-4 (100 mg, 0.25 mmol) and 24-3 (90 mg, 0.068 mmol) were dissolved in DMF, and HATU (114 mg, 0.3 mmol) and DIPEA (81 mg, 0.625 mmol) were added. React at room temperature for ten minutes. Medium pressure preparation (43% acetonitrile/water) afforded compound 24-5, 81 mg, ESI-MS (m/z): 1711.0.
步骤五：化合物24-6的合成：Step 5: Synthesis of Compound 24-6:
将化合物24-5(75mg，0.44mmol)溶于2毫升DMF中，加入0.2毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物24-6，ESI-MS(m/z)：53mg，1488.9。Compound 24-5 (75 mg, 0.44 mmol) was dissolved in 2 mL of DMF and 0.2 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary evaporated to remove diethylamine, and lyophilized with water to give compound 24-6, ESI-MS (m/z): 53 mg, 1488.9.
步骤六：化合物LD024的合成：Step 6: Synthesis of compound LD024:
将化合物24-6(50mg，0.34mmol)溶于2毫升DMF中，加入Mc-OSu(16mg，0.05mmol)。常温反应过夜。中压制备(41％乙腈/水)得到化合物LD024，31mg，ESI-MS(m/z)：1682.0。Compound 24-6 (50 mg, 0.34 mmol) was dissolved in 2 mL of DMF and Mc-OSu (16 mg, 0.05 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (41% acetonitrile/water) afforded compound LD024, 31 mg, ESI-MS (m/z): 1682.0.
实施例三十六：化合物LD025的合成Example 36: Synthesis of compound LD025
Figure PCTCN2022072943-appb-000058
Figure PCTCN2022072943-appb-000058
步骤一：化合物25-2的合成：Step 1: Synthesis of Compound 25-2:
将化合物25-1(80mg，0.23mmol)及24-3(90mg，0.068mmol)溶于DMF中，加入HATU(114mg，0.3mmol)及DIPEA(81mg，0.625mmol)。常温反应十分钟。中压制备(40％乙腈/水)得到化合物25-2，71mg，ESI-MS(m/z)：1653.9。Compounds 25-1 (80 mg, 0.23 mmol) and 24-3 (90 mg, 0.068 mmol) were dissolved in DMF, and HATU (114 mg, 0.3 mmol) and DIPEA (81 mg, 0.625 mmol) were added. React at room temperature for ten minutes. Medium pressure preparation (40% acetonitrile/water) afforded compound 25-2, 71 mg, ESI-MS (m/z): 1653.9.
步骤二：化合物25-3的合成：Step 2: Synthesis of Compound 25-3:
将化合物25-2(65mg，0.40mmol)溶于2毫升DMF中，加入0.2毫升二乙胺。常温反应二十分钟。旋蒸以除去二乙胺，加水冻干后得到化合物25-3， 47mg，ESI-MS(m/z)：1431.9。Compound 25-2 (65 mg, 0.40 mmol) was dissolved in 2 mL of DMF and 0.2 mL of diethylamine was added. React at room temperature for twenty minutes. Rotary evaporated to remove diethylamine, and lyophilized with water to obtain compound 25-3, 47 mg, ESI-MS (m/z): 1431.9.
步骤三：化合物LD025的合成：Step 3: Synthesis of compound LD025:
将化合物25-3(40mg，0.028mmol)溶于DMF中，加入Mc-OSu(13mg，0.042mmol)。常温反应过夜。中压制备(38％乙腈/水)得到化合物LD025 21mg，ESI-MS(m/z)：1624.9。Compound 25-3 (40 mg, 0.028 mmol) was dissolved in DMF and Mc-OSu (13 mg, 0.042 mmol) was added. The reaction was carried out at room temperature overnight. Medium pressure preparation (38% acetonitrile/water) gave compound LD025 21 mg, ESI-MS (m/z): 1624.9.
实施例三十七：化合物LD026的合成Example 37: Synthesis of compound LD026
Figure PCTCN2022072943-appb-000059
Figure PCTCN2022072943-appb-000059
将化合物26-1(300mg，0.2mmol)加入3毫升二氯甲烷中，加入1.2毫升的二氯乙酸，室温反应3小时，LC-MS中控，反应结束后，浓缩，中压制备纯化后，冷冻干燥得化合物LD026，213mg，ESI-MS(m/z)：1287.8。Compound 26-1 (300 mg, 0.2 mmol) was added to 3 mL of dichloromethane, 1.2 mL of dichloroacetic acid was added, and the reaction was carried out at room temperature for 3 hours, controlled by LC-MS, after the reaction was completed, concentrated, and purified by medium pressure preparation, Freeze-dried to obtain compound LD026, 213 mg, ESI-MS (m/z): 1287.8.
实施例三十八：化合物LD027的合成Embodiment thirty-eight: the synthesis of compound LD027
Figure PCTCN2022072943-appb-000060
Figure PCTCN2022072943-appb-000060
步骤一：化合物27-2的合成Step 1: Synthesis of Compound 27-2
将化合物27-1(500mg，1.32mmol)溶于DMF(10mL)中，加入Mc-OSu(401mg，1.30mmol)。常温搅拌过夜。中压制备(35％乙腈/水)得到615mg化合物27-2，ESI-MS(m/z)：572.4。Compound 27-1 (500 mg, 1.32 mmol) was dissolved in DMF (10 mL), and Mc-OSu (401 mg, 1.30 mmol) was added. Stir overnight at room temperature. Medium pressure preparation (35% acetonitrile/water) gave 615 mg of compound 27-2, ESI-MS (m/z): 572.4.
步骤二：化合物27-3的合成Step 2: Synthesis of Compound 27-3
将化合物27-2(600mg，1.0mmol)溶于DMF(10mL)中，加入DNPC(300mg，1.0mmol)及DIPEA(130mg，1.0mmol)。常温搅拌过夜。中压制备纯化，冷冻干燥，得到570mg化合物27-3，ESI-MS(m/z)：737.4。Compound 27-2 (600 mg, 1.0 mmol) was dissolved in DMF (10 mL), and DNPC (300 mg, 1.0 mmol) and DIPEA (130 mg, 1.0 mmol) were added. Stir overnight at room temperature. Purification by medium pressure preparation and lyophilization to give 570 mg of compound 27-3, ESI-MS (m/z): 737.4.
步骤三：化合物LD027的合成Step 3: Synthesis of compound LD027
将化合物27-3(200mg，0.27mmol)溶于2毫升DMF中，加入MMAE(194mg，0.27mmol)及DIPEA(70mg，0.54mmol)。常温搅拌过夜。中压制备纯化，冷冻干燥，得到230mg化合物LD027，ESI-MS(m/z)：1315.8。Compound 27-3 (200 mg, 0.27 mmol) was dissolved in 2 mL of DMF, MMAE (194 mg, 0.27 mmol) and DIPEA (70 mg, 0.54 mmol) were added. Stir overnight at room temperature. Purification by medium pressure preparation and lyophilization to obtain 230 mg of compound LD027, ESI-MS (m/z): 1315.8.
三、包含细胞生物活性分子和连接体的化合物与抗体的偶联3. Conjugation of Compounds Containing Cell Bioactive Molecules and Linkers and Antibodies
下述实施例制备得到的LA001-LA027化合物中，n为4。In the LA001-LA027 compounds prepared in the following examples, n is 4.
实施例三十九：Embodiment thirty-ninth:
取1毫升Herceptin抗体(抗Her-2，10mg/mL)，其缓冲液为50mM PB，5mM EDTA，pH7.2，加入10mM的TCEP(18微升)溶液混匀，室温放置2小时，加入二甲亚砜30微升至上述溶液中，然后缓慢加入5mM的LD001的二甲亚砜溶液(93微升)，混匀，室温静置2小时，最后采用G-25凝胶柱将缓冲液置换为pH6.5的缓冲液，得到LD001与Herceptin抗体偶联的产物，命名为LA001，其结构如下所示。Take 1 ml of Herceptin antibody (anti-Her-2, 10 mg/mL), its buffer is 50 mM PB, 5 mM EDTA, pH 7.2, add 10 mM TCEP (18 microliters) solution and mix well, place at room temperature for 2 hours, add two 30 microliters of methyl sulfoxide was added to the above solution, and then slowly added 5mM LD001 in dimethyl sulfoxide (93 microliters), mixed well, allowed to stand at room temperature for 2 hours, and finally replaced the buffer with a G-25 gel column As a buffer of pH 6.5, a product of LD001 coupled with Herceptin antibody was obtained, named LA001, and its structure is shown below.
Figure PCTCN2022072943-appb-000061
Figure PCTCN2022072943-appb-000061
实施例四十：Example forty:
采用与实施例三十九类似的方法，将LD001替换为LD002，得到LD002与Herceptin抗体偶联的产物，命名为LA002，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD002 to obtain a product of LD002 coupled with the Herceptin antibody, named LA002, the structure of which is shown below.
Figure PCTCN2022072943-appb-000062
Figure PCTCN2022072943-appb-000062
实施例四十一：Embodiment 41:
采用与实施例三十九类似的方法，将LD001替换为LD003，得到LD003与Herceptin抗体偶联的产物，命名为LA003，其结构如下所示：Using a method similar to that of Example 39, LD001 was replaced with LD003 to obtain a product coupled with LD003 and Herceptin antibody, named LA003, whose structure is as follows:
Figure PCTCN2022072943-appb-000063
Figure PCTCN2022072943-appb-000063
化合物LA003的HIC-HPLC图如图1所示。The HIC-HPLC chart of compound LA003 is shown in Figure 1 .
实施例四十二：Embodiment forty-two:
采用与实施例三十九类似的方法，将LD001替换为LD004，得到LD004与Herceptin抗体偶联的产物，命名为LA004，其结构如下所示：Using a method similar to Example 39, LD001 was replaced with LD004 to obtain a product coupled with LD004 and Herceptin antibody, named LA004, and its structure is as follows:
Figure PCTCN2022072943-appb-000064
Figure PCTCN2022072943-appb-000064
LA004的HIC-HPLC图如图2所示。The HIC-HPLC profile of LA004 is shown in Figure 2.
实施例四十三：Embodiment forty-three:
采用与实施例三十九类似的方法，将LD001替换为LD005，得到LD005与Herceptin抗体偶联的产物，命名为LA005，其结构如下所示：Using a method similar to that of Example 39, LD001 was replaced with LD005 to obtain a product coupled with LD005 and Herceptin antibody, named LA005, whose structure is as follows:
Figure PCTCN2022072943-appb-000065
Figure PCTCN2022072943-appb-000065
LA005的HIC-HPLC图如图3所示。The HIC-HPLC profile of LA005 is shown in Figure 3.
实施例四十四：Embodiment forty-four:
采用与实施例三十九类似的方法，将LD001替换为LD006，得到LD006与Herceptin抗体偶联的产物，命名为LA006，其结构如下所示：Using a method similar to that of Example 39, LD001 was replaced with LD006 to obtain a product coupled with LD006 and Herceptin antibody, named LA006, whose structure is as follows:
Figure PCTCN2022072943-appb-000066
Figure PCTCN2022072943-appb-000066
LA006的HIC-HPLC图如图4所示，SEC-HPLC图如图5所示。根据SEC保留时间及峰面积比例，可以确认主要偶联产物仍然保持抗体的完整结构。The HIC-HPLC chart of LA006 is shown in FIG. 4 , and the SEC-HPLC chart is shown in FIG. 5 . According to the SEC retention time and peak area ratio, it can be confirmed that the main conjugated product still maintains the intact structure of the antibody.
实施例四十五：Embodiment forty-five:
采用与实施例三十九类似的方法，将LD001替换为LD007，得到LD007与Herceptin抗体偶联的产物，命名为LA007，其结构如下所示：Using a method similar to that of Example 39, LD001 was replaced with LD007 to obtain a product coupled with LD007 and Herceptin antibody, named LA007, whose structure is as follows:
Figure PCTCN2022072943-appb-000067
Figure PCTCN2022072943-appb-000067
实施例四十六：Embodiment forty-six:
采用与实施例三十九类似的方法，将LD001替换为LD008，得到LD008与Herceptin抗体偶联的产物，命名为LA008，其结构如下所示：Using a method similar to that of Example 39, LD001 was replaced with LD008 to obtain a product coupled with LD008 and Herceptin antibody, named LA008, whose structure is as follows:
Figure PCTCN2022072943-appb-000068
Figure PCTCN2022072943-appb-000068
实施例四十七：Embodiment forty-seven:
取1毫升Herceptin抗体(抗Her-2，10mg/mL)，其缓冲液为50mM PB，5mM EDTA，pH7.2，加入10mM的TCEP(67微升)溶液混匀，室温放置2小时，用脱盐柱将还原后的抗体分离，缓冲液为50mM PB，5mM EDTA，浓缩至1毫升，然后缓慢加入5mM的LD009的二甲亚砜溶液(160微升)，混匀，室温静置2小时，最后采用G-25凝胶柱将缓冲液置换为pH6.5的缓冲液，得到LD009与Herceptin抗体偶联的产物，命名为LA009，其结构如下所示：Take 1 ml of Herceptin antibody (anti-Her-2, 10 mg/mL), its buffer is 50 mM PB, 5 mM EDTA, pH 7.2, add 10 mM TCEP (67 microliters) solution and mix well, stand at room temperature for 2 hours, and then use desalting The column separated the reduced antibody, the buffer was 50mM PB, 5mM EDTA, concentrated to 1 ml, then slowly added 5mM LD009 in dimethyl sulfoxide (160 μl), mixed well, and allowed to stand at room temperature for 2 hours. The G-25 gel column was used to replace the buffer with a buffer of pH 6.5 to obtain a product conjugated with LD009 and Herceptin antibody, named LA009, whose structure is shown below:
Figure PCTCN2022072943-appb-000069
Figure PCTCN2022072943-appb-000069
LA009的HIC-HPLC图如图6所示。The HIC-HPLC profile of LA009 is shown in Figure 6.
实施例四十八：Embodiment forty-eight:
采用与实施例四十七类似的方法，将LD009替换为LD0010，得到LD010与Herceptin抗体偶联的产物，命名为LA010，其结构如下所示：Using a method similar to Example 47, LD009 was replaced with LD0010 to obtain a product coupled with LD010 and Herceptin antibody, named LA010, whose structure is as follows:
Figure PCTCN2022072943-appb-000070
Figure PCTCN2022072943-appb-000070
实施例四十九：Embodiment forty-ninth:
采用与实施例四十七类似的方法，将LD009替换为LD0011，得到LD011与Herceptin抗体偶联的产物，命名为LA011，其结构如下所示。Using a method similar to that in Example 47, LD009 was replaced with LD0011 to obtain a product conjugated with LD011 and Herceptin antibody, named LA011, the structure of which is shown below.
Figure PCTCN2022072943-appb-000071
Figure PCTCN2022072943-appb-000071
实施例五十：Example 50:
采用与实施例四十七类似的方法，将LD009替换为LD012，得到LD012与Herceptin抗体偶联的产物，命名为LA012，其结构如下所示。Using a method similar to that in Example 47, LD009 was replaced with LD012 to obtain a product conjugated between LD012 and Herceptin antibody, named LA012, the structure of which is shown below.
Figure PCTCN2022072943-appb-000072
Figure PCTCN2022072943-appb-000072
实施例五十一：Embodiment fifty-one:
采用与实施例四十七类似的方法，将LD009替换为LD013，得到LD013与Herceptin抗体偶联的产物，命名为LA013，其结构如下所示。Using a method similar to that in Example 47, LD009 was replaced with LD013 to obtain a product of LD013 coupled with Herceptin antibody, named LA013, the structure of which is shown below.
Figure PCTCN2022072943-appb-000073
Figure PCTCN2022072943-appb-000073
实施例五十二：Embodiment fifty-two:
采用与实施例四十七类似的方法，将LD009替换为LD014，得到LD014与Herceptin抗体偶联的产物，命名为LA014，其结构如下所示。Using a method similar to Example 47, LD009 was replaced with LD014 to obtain a product of LD014 coupled with Herceptin antibody, named LA014, the structure of which is shown below.
Figure PCTCN2022072943-appb-000074
Figure PCTCN2022072943-appb-000074
实施例五十三：Embodiment fifty-three:
采用与实施例三十九类似的方法，将LD001替换为LD015，得到LD015与Herceptin抗体偶联的产物，命名为LA015，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD015 to obtain a product conjugated between LD015 and Herceptin antibody, named LA015, the structure of which is shown below.
Figure PCTCN2022072943-appb-000075
Figure PCTCN2022072943-appb-000075
实施例五十四：Embodiment fifty-four:
采用与实施例三十九类似的方法，将LD001替换为LD016，得到LD016与Herceptin抗体偶联的产物，命名为LA016，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD016 to obtain a product conjugated between LD016 and Herceptin antibody, named LA016, the structure of which is shown below.
Figure PCTCN2022072943-appb-000076
Figure PCTCN2022072943-appb-000076
实施例五十五：Embodiment fifty-five:
采用与实施例三十九类似的方法，将LD001替换为LD017，得到LD017与Herceptin抗体偶联的产物，命名为LA017，其结构如下所示。Using a method similar to Example 39, LD001 was replaced with LD017 to obtain a product of LD017 coupled with Herceptin antibody, named LA017, the structure of which is shown below.
Figure PCTCN2022072943-appb-000077
Figure PCTCN2022072943-appb-000077
实施例五十六：Embodiment fifty-six:
采用与实施例四十七类似的方法，将LD009替换为LD018，得到LD018与Herceptin抗体偶联的产物，命名为LA018，其结构如下所示。Using a method similar to Example 47, LD009 was replaced with LD018 to obtain a product of LD018 coupled with the Herceptin antibody, named LA018, the structure of which is shown below.
Figure PCTCN2022072943-appb-000078
Figure PCTCN2022072943-appb-000078
实施例五十七：Embodiment fifty-seven:
采用与实施例四十七类似的方法，将LD009替换为LD019，得到LD019与Herceptin抗体偶联的产物，命名为LA019，其结构如下所示。Using a method similar to Example 47, LD009 was replaced with LD019 to obtain a product of LD019 coupled with Herceptin antibody, named LA019, the structure of which is shown below.
Figure PCTCN2022072943-appb-000079
Figure PCTCN2022072943-appb-000079
实施例五十八：Embodiment fifty-eight:
采用与实施例三十九类似的方法，将LD001替换为LD020，得到LD020与Herceptin抗体偶联的产物，命名为LA020，其结构如下所示。Using a method similar to Example 39, LD001 was replaced with LD020 to obtain a product of LD020 coupled with Herceptin antibody, named LA020, whose structure is shown below.
Figure PCTCN2022072943-appb-000080
Figure PCTCN2022072943-appb-000080
实施例五十九：Embodiment fifty-ninth:
采用与实施例四十七类似的方法，将LD009替换为LD021，得到LD021与Herceptin抗体偶联的产物，命名为LA021，其结构如下所示。Using a method similar to Example 47, LD009 was replaced with LD021 to obtain a product of LD021 coupled with Herceptin antibody, named LA021, the structure of which is shown below.
Figure PCTCN2022072943-appb-000081
Figure PCTCN2022072943-appb-000081
实施例六十：Example sixty:
采用与实施例三十九类似的方法，将LD001替换为LD022，得到LD022与Herceptin抗体偶联的产物，命名为LA022，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD022 to obtain a product conjugated between LD022 and Herceptin antibody, named LA022, the structure of which is shown below.
Figure PCTCN2022072943-appb-000082
Figure PCTCN2022072943-appb-000082
实施例六十一：Embodiment sixty-one:
采用与实施例三十九类似的方法，将LD001替换为LD023，得到LD023与Herceptin抗体偶联的产物，命名为LA023，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD023 to obtain a product conjugated with LD023 and Herceptin antibody, named LA023, the structure of which is shown below.
Figure PCTCN2022072943-appb-000083
Figure PCTCN2022072943-appb-000083
实施例六十二：Embodiment sixty-two:
采用与实施例三十九类似的方法，将LD001替换为LD024，得到LD024与Herceptin抗体偶联的产物，命名为LA024，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD024 to obtain a product conjugated between LD024 and Herceptin antibody, named LA024, the structure of which is shown below.
Figure PCTCN2022072943-appb-000084
Figure PCTCN2022072943-appb-000084
实施例六十三：Embodiment sixty-three:
采用与实施例三十九类似的方法，将LD001替换为LD025，得到LD025与Herceptin抗体偶联的产物，命名为LA025，其结构如下所示。Using a method similar to Example 39, LD001 was replaced with LD025 to obtain a product of LD025 coupled with Herceptin antibody, named LA025, whose structure is shown below.
Figure PCTCN2022072943-appb-000085
Figure PCTCN2022072943-appb-000085
实施例六十四：Embodiment sixty-four:
采用与实施例三十九类似的方法，将LD001替换为LD025，得到LD025与Herceptin抗体偶联的产物，命名为LA026，其结构如下所示。Using a method similar to Example 39, LD001 was replaced with LD025 to obtain a product of LD025 coupled with Herceptin antibody, named LA026, the structure of which is shown below.
Figure PCTCN2022072943-appb-000086
Figure PCTCN2022072943-appb-000086
实施例六十五：Embodiment sixty-five:
采用与实施例三十九类似的方法，将LD001替换为LD025，得到LD025与Herceptin抗体偶联的产物，命名为LA027，其结构如下所示。Using a method similar to that in Example 39, LD001 was replaced with LD025 to obtain a product conjugated with LD025 and Herceptin antibody, named LA027, the structure of which is shown below.
Figure PCTCN2022072943-appb-000087
Figure PCTCN2022072943-appb-000087
四、细胞活性测试4. Cell Viability Test
试剂reagent
10mM的毒素小分子和毒素连接子样品的溶液使用100％DMSO制备。抗HER2抗体药物偶联物(ADC)的所有样品均以PBS形式提供。10 mM solutions of toxin small molecule and toxin linker samples were prepared using 100% DMSO. All samples of anti-HER2 antibody drug conjugates (ADCs) were provided in PBS.
细胞培养cell culture
活性测试中使用的细胞系购自美国Type Culture Collection(ATCC；Manassas，VA)，培养基(Gibco ThermoFisher；Waltham,美国马萨诸塞州)，加有10％热灭活的胎牛血清(FBS；Corning；美国纽约州Corning)和1X青霉素-链霉素(Corning)，并按常规在RPMI-1640(HCC1954和SK-BR-3)或DMEM：F-12(MDA-MB-468)，保持在37℃下5％的CO 2加湿环境中培养。 The cell lines used in the activity assay were purchased from the American Type Culture Collection (ATCC; Manassas, VA), culture medium (Gibco ThermoFisher; Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning; Corning, NY, USA) and 1X penicillin-streptomycin (Corning), and routinely in RPMI-1640 (HCC1954 and SK-BR-3) or DMEM:F-12 (MDA-MB-468), maintained at 37°C Incubate in a humidified environment under 5% CO 2 .
细胞活力测定Cell viability assay
通过用Cell Stripper Dissociation Reagent(Corning)进行非酶解分离来收获肿瘤细胞，将其接种到384孔平底白壁板中(在12.5μL全培养基中每孔875细胞)，并在37℃保持2-4小时让细胞黏附。然后将细胞用12.5μL最终浓度为2X(连续稀释)的测试试剂处理，将其在37℃下孵育120小时。根据仪器制造商的使用方案，使用
Figure PCTCN2022072943-appb-000088
细胞生存力测定法(Promega；美国威斯康星州麦迪逊)，确定对癌细胞生长的抑制作用。使用Tecan Spark多模式酶标仪(Tecan Group Ltd.；瑞士曼多夫)测量发光。 Tumor cells were harvested by non-enzymatic dissociation with Cell Stripper Dissociation Reagent (Corning), seeded into 384-well flat-bottom white-walled plates (875 cells per well in 12.5 μL of complete medium), and maintained at 37°C for 2- Cells were allowed to adhere for 4 hours. Cells were then treated with 12.5 [mu]L of test reagents at a final concentration of 2X (serial dilution) and incubated at 37[deg.]C for 120 hours. According to the instrument manufacturer's usage protocol, use
Figure PCTCN2022072943-appb-000088
Cell viability assay (Promega; Madison, WI, USA) to determine inhibition of cancer cell growth. Luminescence was measured using a Tecan Spark multimode microplate reader (Tecan Group Ltd.; Mandorf, Switzerland).
数据分析data analysis
使用Microsoft Excel(美国华盛顿州雷德蒙德)用未加测试样品处理的对照将数据归一化，并使用GraphPad Prism软件(版本8；美国加利福尼亚州拉霍亚)进行分析。使用适合于4参数对数方程的非线性回归分析，从剂量响应曲线得出半数抑制最大有效浓度(EC50)。ADC的治疗窗口定义为抗原阴性和抗原阳性细胞系之间EC50的比率。Data were normalized to controls untreated with test samples using Microsoft Excel (Redmond, WA, USA) and analyzed using GraphPad Prism software (version 8; La Jolla, CA, USA). The median inhibitory maximal effective concentration (EC50) was derived from the dose-response curve using nonlinear regression analysis fitted to a 4-parameter logarithmic equation. The therapeutic window of ADC was defined as the ratio of EC50 between antigen-negative and antigen-positive cell lines.
结果result
含磷酰胆碱链接有效载荷对ADC活性的影响Effect of phosphorylcholine-containing link payload on ADC activity
通过与HER2靶向抗体偶联形成抗体药物偶联物(ADC)，测试了含磷酰胆碱连接子的ADC对细胞毒素活性的影响。在ADC系列中，磷酰胆碱功能基被引入：(1)Val-Lys二肽的Lys侧链，(2)直接和Gly/Ser-Val-Cit/Lys三肽内的Gly或Ser相连。如图8(A)和下表1所示，在系列1四个含磷酰胆碱修饰的Lys侧链(LA003-LA006)的MMAE有效载荷ADC之间，未观察到针对HER2阳性HCC1954和SK-BR-3细胞的明显活性差异。通过比较，这些含磷酰胆碱修饰的Val-Lys二肽接头ADC的活性比未修饰的Val-Lys接头ADC对照(LA026)略强，但活性却比Val-N-二甲基Lys修饰对照(LA027)小。然而，值得注意的是，在HER2阴性的MDA-MB-468乳腺癌细胞中，LA004的效能
Figure PCTCN2022072943-appb-000089
高于非磷酰胆碱修饰修饰的ADC对照，而LA003、LA005和LA006与对照组相比均显示出明显降低的活性。观察到LA003和LA006的治疗窗口明显增加。 The effect of phosphorylcholine linker-containing ADCs on cytotoxic activity was tested by coupling with HER2-targeting antibodies to form antibody drug conjugates (ADCs). In the ADC series, phosphorylcholine functional groups were introduced: (1) the Lys side chain of the Val-Lys dipeptide, (2) directly linked to Gly or Ser within the Gly/Ser-Val-Cit/Lys tripeptide. As shown in Figure 8(A) and Table 1 below, between the four series 1 MMAE payload ADCs containing phosphorylcholine-modified Lys side chains (LA003-LA006), no targeting of HER2-positive HCC1954 and SK was observed - Significant differences in activity of BR-3 cells. By comparison, these phosphorylcholine-modified Val-Lys dipeptide linker ADCs were slightly more active than the unmodified Val-Lys linker ADC control (LA026), but were more active than the Val-N-dimethyl Lys modification control (LA027) Small. Notably, however, in HER2-negative MDA-MB-468 breast cancer cells, the potency of LA004
Figure PCTCN2022072943-appb-000089
higher than that of the non-phosphorylcholine-modified ADC control, while LA003, LA005, and LA006 all showed significantly reduced activity compared to the control. A marked increase in the therapeutic window was observed for LA003 and LA006.
对于系列2中包含MMAE有效载荷的含磷酰胆碱修饰的ADC，在两个抗-HER2ADC中评估了Ser-Val-Cit/Gly-Val-Cit三肽中Ser(O-PC)或Gly(N-PC)的含磷酰胆碱修饰的偶联物LA015和LA016。结果显示在HER2阳性HCC1954和SK-BR-3及HER2阴性MDA-MD-468中，Gly(N-PC,LA016)修饰的效力始终比Ser(O-PC,LA015)高1.3-2.5倍。MDA-MB-468细胞(图8，B；表1)。如预期的那样，LA015和LA016在HCC1954和SK-BR-3中显示出比单独的MMAE更高的靶向特异性，但在MDA-MB-468细胞中却没有。在具有含磷酰胆碱修饰的三肽的ADC和与将磷酰胆碱修饰的组成连接至Val-Lys二肽接头的Lys侧链的ADC之间(LA003-LA006)，几乎没有观察到活性差异。The Ser-Val-Cit/Gly-Val-Cit tripeptide in Ser(O-PC) or Gly( N-PC) containing phosphorylcholine-modified conjugates LA015 and LA016. The results showed that in HER2-positive HCC1954 and SK-BR-3 and HER2-negative MDA-MD-468, the efficacy of Gly(N-PC, LA016) modification was consistently 1.3-2.5 times higher than that of Ser(O-PC, LA015). MDA-MB-468 cells (Fig. 8, B; Table 1). As expected, LA015 and LA016 showed higher targeting specificity than MMAE alone in HCC1954 and SK-BR-3, but not in MDA-MB-468 cells. Little activity was observed between ADCs with phosphorylcholine-modified tripeptides and ADCs with phosphorylcholine-modified compositions attached to the Lys side chain of the Val-Lys dipeptide linker (LA003-LA006) difference.
接下来，在系列3中将含磷酰胆碱修饰连接到Lys的侧链和/或直接连接到二肽或三肽抗HER2-MMAE或DXd ADC的Ser上的效果与未经含磷酰胆碱修饰修饰的ADC对照进行了比较。与带有马来酰亚胺偶联的(McVCMMAE,LN360-66-1)或C-Lock的(C-Lock-VCMMAE,LN360-66-3)的抗HER2 ADC相比，在LA007的Ser-Val-Lys接头添加两个含磷酰胆碱修饰基团不会影响活性。尽管所有三个ADC都显示出与HER2阳性SK-BR-3(EC50 0.01至0.03nM) 和HCC1954(EC50 0.03至0.05nM)和HER2阴性MDA-MB-468(EC50>20nM)相当的细胞毒活性，包含两个PC组的LA007的治疗窗口最大(图8，C；表1)。Next, in series 3, the effect of phosphorylcholine-containing modification attached to the side chain of Lys and/or directly to Ser of a dipeptide or tripeptide anti-HER2-MMAE or DXd ADC was comparable to that without phosphorylcholine. Base-modified ADC controls were compared. Ser- The addition of two phosphorylcholine-containing modification groups to the Val-Lys linker did not affect activity. Although all three ADCs showed comparable cytotoxic activity to HER2-positive SK-BR-3 (EC50 0.01 to 0.03 nM) and HCC1954 (EC50 0.03 to 0.05 nM) and HER2-negative MDA-MB-468 (EC50 > 20 nM) , the LA007 containing both PC groups had the largest therapeutic window (Fig. 8, C; Table 1).
最后，在系列4中，针对带有Gly-Gly-Phe-Gly-DXd药物接头LN360-64-1的对照ADC评估了带有含磷酰胆碱修饰的Val-Lys(LA011)或Ser(O-PC)-Val-Lys(LA013)连接子的抗HER2-DXd ADC。与SK-BR-3细胞中的(LN360-64-1)(EC50为0.04568nM)相比，LA011(EC50为0.07640nM)和LA013(EC50为0.1824nM)的偶联物导致活性适度降低2-4倍，LA011在HCC1954细胞中显示出比LN360-64-1高13倍的活性(图8，D；表1)。LA011和LA013的HER2靶向特异性也保留。Finally, in series 4, Val-Lys (LA011) or Ser (O -PC)-Val-Lys(LA013) linker anti-HER2-DXd ADC. Conjugates of LA011 (EC50 of 0.07640 nM) and LA013 (EC50 of 0.1824 nM) resulted in modest reductions in activity compared to (LN360-64-1) (EC50 of 0.04568 nM) in SK-BR-3 cells 2- 4-fold, LA011 showed 13-fold higher activity than LN360-64-1 in HCC1954 cells (Fig. 8, D; Table 1). The HER2 targeting specificity of LA011 and LA013 was also retained.
LN360-66-1LN360-66-1
Figure PCTCN2022072943-appb-000090
Figure PCTCN2022072943-appb-000090
LN360-66-3LN360-66-3
Figure PCTCN2022072943-appb-000091
Figure PCTCN2022072943-appb-000091
LN360-64-1LN360-64-1
Figure PCTCN2022072943-appb-000092
Figure PCTCN2022072943-appb-000092
表1：含磷酰胆碱修饰的抗HER2 ADC对人乳腺癌细胞的效力Table 1: Efficacy of phosphorylcholine-modified anti-HER2 ADCs on human breast cancer cells
Figure PCTCN2022072943-appb-000093
Figure PCTCN2022072943-appb-000093
Figure PCTCN2022072943-appb-000094
Figure PCTCN2022072943-appb-000094
五、溶解度实验5. Solubility test
仪器：Mettler天平XPE105Instrument: Mettler balance XPE105
供试品名称：Test article name:
VL(PC)-PAB(3-4)：VL(PC)-PAB(3-4):
Figure PCTCN2022072943-appb-000095
Figure PCTCN2022072943-appb-000095
MC-VL(PC)-PAB(3-5)：MC-VL(PC)-PAB(3-5):
Figure PCTCN2022072943-appb-000096
Figure PCTCN2022072943-appb-000096
VC-PAB：VC-PAB:
Figure PCTCN2022072943-appb-000097
Figure PCTCN2022072943-appb-000097
MC-VC-PAB：MC-VC-PAB:
Figure PCTCN2022072943-appb-000098
Figure PCTCN2022072943-appb-000098
实验步骤Experimental procedure
分别称取100mg的MC-VL(PC)-PAB(3-5)、100mg的VL(PC)-PAB(3-4)、100mg的VC-PAB、100mg的MC-VC-PAB放置于不同的10ml顶空瓶中，按下表2的顺序加入相应体积的超纯水，每隔5分钟强力振摇30秒钟，观察30分钟内的溶解情况，如无目视可见的溶质颗粒时，即视为完全溶解。Weigh 100mg of MC-VL(PC)-PAB(3-5), 100mg of VL(PC)-PAB(3-4), 100mg of VC-PAB, and 100mg of MC-VC-PAB and place them in different In a 10ml headspace bottle, add the corresponding volume of ultrapure water in the order in Table 2, shake vigorously for 30 seconds every 5 minutes, and observe the dissolution within 30 minutes. If there are no visible solute particles, that is, considered completely dissolved.
表2Table 2
序号serial number 加入溶剂体积Add solvent volume 备注Remark
11 0.1ml0.1ml   
22 0.9ml0.9ml   
33 2ml2ml   
44 7ml 7ml   
55 100ml100ml 将4转移至100ml量瓶中，加超纯水至刻度线Transfer 4 to a 100ml volumetric flask and add ultrapure water to the mark
66 1000ml 1000ml 将5转移至1000ml量瓶中，加超纯水至刻度线Transfer 5 to a 1000ml volumetric flask and add ultrapure water to the mark
实验结果Experimental results
结果显示，100mg的VL(PC)-PAB在1ml超纯水中完全溶解，100mg的MC-VL(PC)-PAB在3ml超纯水中完全溶解，100mg的VC-PAB在1000ml超纯水中有目视可见的溶质颗粒，100mg的MC-VC-PAB在1000ml超纯水中有目视可见的溶质颗粒。The results showed that 100mg of VL(PC)-PAB was completely dissolved in 1ml of ultrapure water, 100mg of MC-VL(PC)-PAB was completely dissolved in 3ml of ultrapure water, and 100mg of VC-PAB was completely dissolved in 1000ml of ultrapure water There are visually visible solute particles, and 100 mg of MC-VC-PAB in 1000 ml of ultrapure water has visually visible solute particles.
因此，VL(PC)-PAB在水中的溶解度>100mg/ml，MC-VL(PC)-PAB在水中的溶解度>33mg/ml，VC-PAB在1000ml在水中的溶解度<0.1mg/ml，MC-VC-PAB在水中的溶解度<0.1mg/ml。按完全溶解和未完全溶解来定性比较，含磷酰胆碱修饰的二肽连接子VL(PC)-PAB和MC-VL(PC)-PAB的水溶性分别比常规的VC-PAB和MC-VC-PAB至少大1000倍和330倍。Therefore, the solubility of VL(PC)-PAB in water is >100mg/ml, the solubility of MC-VL(PC)-PAB in water is >33mg/ml, the solubility of VC-PAB in 1000ml of water is <0.1mg/ml, MC - The solubility of VC-PAB in water is <0.1 mg/ml. According to the qualitative comparison of complete dissolution and incomplete dissolution, the water solubility of the phosphorylcholine-modified dipeptide linkers VL(PC)-PAB and MC-VL(PC)-PAB is higher than that of conventional VC-PAB and MC-PAB, respectively. VC-PAB is at least 1000 times and 330 times larger.
此外，采用HIC法检测LA003、LA005和对照Her2-McVCPABMMAE的水溶性。结果如图9所示。In addition, the water solubility of LA003, LA005 and control Her2-McVCPABMMAE was detected by HIC method. The results are shown in Figure 9.

Claims (20)

  1. 下式I所示的磷酰胆碱基团修饰的氨基酸：A phosphorylcholine group-modified amino acid shown in the following formula I:
    R 1-L 1-N(R 2)-R 3  (I) R 1 -L 1 -N(R 2 )-R 3 (I)
    式I中：In formula I:
    R 1为磷酰胆碱基团； R 1 is a phosphorylcholine group;
    N代表氨基酸分子上的氮原子；N represents the nitrogen atom on the amino acid molecule;
    R 2为H、C1-C4烷基或-L 1-磷酰胆碱基团； R 2 is H, C1-C4 alkyl or -L 1 -phosphorylcholine group;
    各L 1独立为连接基团； Each L 1 is independently a linking group;
    R 3代表氨基酸分子上除所示N原子外的其余部分，所述其余部分任选地被保护基团保护。 R3 represents the remainder of the amino acid molecule other than the indicated N atom, which is optionally protected by a protecting group.
  2. 如权利要求1所述的氨基酸，其特征在于，The amino acid of claim 1, wherein
    所述磷酰胆碱基团如下式所示：The phosphorylcholine group is represented by the following formula:
    Figure PCTCN2022072943-appb-100001
    Figure PCTCN2022072943-appb-100001
    式中，波浪线表示该磷酰胆碱基团与L 1连接的位置；和/或 In the formula, the wavy line indicates the position where the phosphorylcholine group is attached to L 1 ; and/or
    所述各L 1独立表示为-L a-L b-，其中L a选自C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基；L b不存在，或者为羰基、酯基(-COO-)、-O-SO 2-或-NH-SO 2-；优选地，L b不存在或为羰基；优选地，L 1通过L b与所述N共价连接；优选地，L b和所示的N形成酰胺基或磺酰氨基；优选地，各L 1独立为C1-C6亚烷基或C1-C6亚烷基羰基；和/或 Said each L 1 is independently represented as -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; L b does not exist, or is carbonyl , ester group (-COO-), -O-SO 2 - or -NH-SO 2 -; preferably, L b does not exist or is a carbonyl group; preferably, L 1 is covalently linked to the N through L b ; Preferably, L b and indicated N form an amide group or a sulfonamido group; preferably, each L 1 is independently a C1-C6 alkylene group or a C1-C6 alkylene carbonyl group; and/or
    R 3所述的氨基酸分子选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸、硒半胱氨酸、吡咯赖氨酸和鸟氨酸；优选地，所述氨基酸分子选自：赖氨酸、鸟氨酸、缬氨酸、酪氨酸、谷氨酰胺、丙氨酸、精氨酸、天冬酰胺、亮氨酸、异亮氨酸、苏氨酸、丝氨酸、苯丙氨酸、缬氨酸和甘氨酸；更优选地，所述氨 基酸分子选自：缬氨酸、鸟氨酸、赖氨酸、丝氨酸、甘氨酸和瓜氨酸。更优选地，所述氨基酸分子选自：鸟氨酸、赖氨酸、丝氨酸和甘氨酸；和/或 The amino acid molecule described in R3 is selected from: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine Amino acid, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, pyrrolysine acid and ornithine; preferably, the amino acid molecule is selected from: lysine, ornithine, valine, tyrosine, glutamine, alanine, arginine, asparagine, leucine acid, isoleucine, threonine, serine, phenylalanine, valine and glycine; more preferably, the amino acid molecule is selected from: valine, ornithine, lysine, serine, glycine and citrulline. More preferably, the amino acid molecule is selected from the group consisting of: ornithine, lysine, serine and glycine; and/or
    所述N不是氨基酸分子中的芳基、杂芳基或杂环基上的环氮原子，或所述N不是氨基酸分子中用来形成肽键的氮原子。The N is not a ring nitrogen atom on an aryl, heteroaryl or heterocyclyl group in an amino acid molecule, or the N is not a nitrogen atom in an amino acid molecule used to form peptide bonds.
  3. 如权利要求1所述的氨基酸，其特征在于，所述修饰的氨基酸选自LN001至LN010以及LN012至LN025中任一所示的化合物。The amino acid of claim 1, wherein the modified amino acid is selected from the compounds shown in any one of LN001 to LN010 and LN012 to LN025.
  4. 一种磷酰胆碱修饰的肽，其特征在于，所述肽至少包括一个磷酰胆碱基团，所述磷酰胆碱基团与所述肽所含的1个或多个氨基酸的N原子和/或氧原子经由连接基团共价连接；优选地，本申请所述的肽具有2-10个氨基酸残基，更优选具有2-5个氨基酸残基；优选地，所述磷酰胆碱基团和连接基团如权利要求2所定义。A phosphorylcholine-modified peptide, characterized in that the peptide comprises at least one phosphorylcholine group, the phosphorylcholine group and the N of one or more amino acids contained in the peptide Atoms and/or oxygen atoms are covalently linked via a linking group; preferably, the peptides described in this application have 2-10 amino acid residues, more preferably 2-5 amino acid residues; preferably, the phosphoryl The choline group and the linking group are as defined in claim 2.
  5. 如权利要求4所述的肽，其特征在于，所述肽为可被病理环境中的酶水解的肽；优选地，所述酶可为蛋白水解酶、蛋白酶或肽酶。更优选地，所述酶选自：半胱氨酸蛋白酶、天冬酰胺蛋白酶、天冬氨酸蛋白酶、谷氨酸蛋白酶、苏氨酸蛋白酶、明胶酶、金属蛋白酶和天冬酰胺肽裂解酶中的一种或多种；更有效的，所述酶为天冬酰胺内肽酶。The peptide of claim 4, wherein the peptide is a peptide that can be hydrolyzed by an enzyme in a pathological environment; preferably, the enzyme can be a proteolytic enzyme, a protease, or a peptidase. More preferably, the enzyme is selected from the group consisting of cysteine proteases, asparagine proteases, aspartic proteases, glutamic proteases, threonine proteases, gelatinases, metalloproteases and asparagine peptide lyases one or more of; more effectively, the enzyme is asparagine endopeptidase.
  6. 如权利要求4或5所述的肽，其特征在于：The peptide of claim 4 or 5, characterized in that:
    所述肽中的氨基酸残基选自：缬氨酸、鸟氨酸、赖氨酸、丝氨酸、甘氨酸和瓜氨酸中的一种或多种；和/或The amino acid residues in the peptide are selected from: one or more of valine, ornithine, lysine, serine, glycine and citrulline; and/or
    所述肽中磷酰胆碱基团修饰的氨基酸残基选自：鸟氨酸、赖氨酸、丝氨酸和甘氨酸中的一个或多个；和/或The amino acid residue modified by the phosphorylcholine group in the peptide is selected from: one or more of ornithine, lysine, serine and glycine; and/or
    所述肽中的至少一个被磷酰胆碱基团修饰的氨基酸残基为权利要求1-3中任一项所述的磷酰胆碱基团修饰的氨基酸。At least one amino acid residue modified with a phosphorylcholine group in the peptide is the phosphorylcholine group-modified amino acid of any one of claims 1-3.
  7. 如权利要求4所述的肽，其特征在于，所述肽选自Val-Orn、Val-Lys、Ser-Val-Lys、Gly-Val-Lys、Ser-Val-Cit、Gly-Val-Cit、Gly-Gly-Gly-Val-Lys和Gly-Gly-Val-Lys中的一种或多种；选地，所述肽中的鸟氨酸、赖氨酸、丝氨酸和甘氨酸中的一个或多个被磷酰胆碱基团修饰。The peptide of claim 4, wherein the peptide is selected from the group consisting of Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Val-Cit, One or more of Gly-Gly-Gly-Val-Lys and Gly-Gly-Val-Lys; optionally, one or more of ornithine, lysine, serine and glycine in the peptide Modified with phosphorylcholine groups.
  8. 如权利要求4所述的肽，其特征在于，所述肽选自下组中的一个或多 个：The peptide of claim 4, wherein the peptide is selected from one or more of the group consisting of:
    Figure PCTCN2022072943-appb-100002
    Figure PCTCN2022072943-appb-100002
    Figure PCTCN2022072943-appb-100003
    Figure PCTCN2022072943-appb-100003
  9. 具有下式II所示结构的连接子化合物：A linker compound having the structure shown in the following formula II:
    R 4-L 2-P-R 5  (II) R 4 -L 2 -PR 5 (II)
    式中：where:
    R 4为可与蛋白中的半胱氨酸残基中的巯基或赖氨酸残基上的游离氨基发生反应、从而将式II连接到蛋白的基团； R 4 is a group that can react with a sulfhydryl group in a cysteine residue in a protein or a free amino group on a lysine residue to attach Formula II to the protein;
    L 2为连接基团； L 2 is a linking group;
    P为权利要求1-3中任一项所述的氨基酸或权利要求4-8中任一项所述的肽；所述氨基酸或肽与L 2和R 5共价连接； P is the amino acid of any one of claims 1-3 or the peptide of any one of claims 4-8 ; the amino acid or peptide is covalently linked to L2 and R5 ;
    R 5为： R5 is:
    Figure PCTCN2022072943-appb-100004
    Figure PCTCN2022072943-appb-100004
    其中，R 6为H、卤素、C1-10烷基、C3-C8环烷基、C2-C8烯基、C2-C8炔基、C3-C8环烯基、6-14元芳基、芳烷基、5-10元杂芳基或4-10元杂环基；R 9为H、-C(O)-N(R 7)-(CH 2) n-NHR 8或硝基取代的苯氧基羰基；R 7为H或C1-6烷基；R 8为H或C1-6烷基；和n为1-6的整数。 Wherein, R 6 is H, halogen, C1-10 alkyl, C3-C8 cycloalkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl, 6-14-membered aryl, aralkyl R 9 is H, -C(O)-N(R 7 )-(CH 2 ) n -NHR 8 or nitro-substituted phenoxy R 7 is H or C1-6 alkyl; R 8 is H or C1-6 alkyl; and n is an integer of 1-6.
  10. 如权利要求9所述的连接子化合物，其特征在于，R 4选自： The linker compound of claim 9, wherein R 4 is selected from:
    Figure PCTCN2022072943-appb-100005
    Figure PCTCN2022072943-appb-100005
    Figure PCTCN2022072943-appb-100006
    Figure PCTCN2022072943-appb-100006
    其中，波浪线表示R 4与L 2连接的位置；F 5表示5个氟原子。 Among them, the wavy line represents the position where R 4 is connected to L 2 ; F 5 represents 5 fluorine atoms.
  11. 如权利要求9或10所述的连接子化合物，其特征在于，L 2为-L a-L b-，其中L a选自C1-C6亚烷基、C2-C6亚烯基或C2-C6亚炔基；L b不存在，或者为羰基、酯基(-COO-)、-O-SO 2-或-NH-SO 2-；优选地，L b不存在或为羰基；优选地，L 2通过L b与所述P共价连接；优选地，L b和P间形成酰胺基或磺酰氨基；优选地，各L 2独立为C1-C6亚烷基或C1-C6亚烷基羰基。 The linker compound according to claim 9 or 10, wherein L 2 is -L a -L b -, wherein L a is selected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 Alkynylene; L b is absent, or is carbonyl, ester group (-COO-), -O-SO 2 - or -NH-SO 2 -; preferably, L b is absent or carbonyl; preferably, L 2 is covalently linked to the P through L b ; preferably, an amide group or a sulfonamido group is formed between L b and P; preferably, each L 2 is independently a C1-C6 alkylene group or a C1-C6 alkylene carbonyl group .
  12. 如权利要求9或10所述的连接子化合物，其特征在于，The linker compound of claim 9 or 10, wherein,
    所述R 4-L 2与P以及通过选自酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接；优选地，R 4-L 2与P通过酰胺基共价连接；和/或 The R 4 -L 2 is linked to P and through a group selected from the group consisting of amide (-CO-NH-), ester (-COO-), -O-SO 2 -NH- and -NH-SO 2 -NH- is covalently attached; preferably, R 4 -L 2 is covalently attached to P through an amide group; and/or
    R 4与L 2之间通过选自碳碳单键、酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接，优选通过碳碳单键进行共价连接；和/或 Between R 4 and L 2 is selected from carbon-carbon single bond, amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO 2 -NH- The connection method is covalently connected, preferably through a carbon-carbon single bond; and/or
    所述P与R 5通过选自酰胺基(-CO-NH-)、酯基(-COO-)、-O-SO 2-NH-和-NH-SO 2-NH-的连接方式共价连接，优选通过酰胺基共价连接。 The P and R 5 are covalently linked through a linking mode selected from the group consisting of amide group (-CO-NH-), ester group (-COO-), -O-SO 2 -NH- and -NH-SO 2 -NH- , preferably covalently linked via an amide group.
  13. 如权利要求9-12中任一项所述的连接子化合物，其特征在于，The linker compound of any one of claims 9-12, wherein
    R 5为： R5 is:
    Figure PCTCN2022072943-appb-100007
    Figure PCTCN2022072943-appb-100007
    Figure PCTCN2022072943-appb-100008
    和/或
    Figure PCTCN2022072943-appb-100008
    and / or
    R 6为H或C1-C6烷基；和/或 R 6 is H or C1-C6 alkyl; and/or
    R 7为H或C1-C4烷基；和/或 R 7 is H or C1-C4 alkyl; and/or
    R 8为H或C1-C4烷基；和/或 R 8 is H or C1-C4 alkyl; and/or
    n为1-4的整数。n is an integer of 1-4.
  14. 如权利要求9所述的连接子化合物，其特征在于，所述式II化合物如化合物1-8、1-9、2-6、2-7、3-5、3-6、4-4、4-5、5-3、5-4、6-3、6-4、7-4、7-5、8-3、8-4、15-4、15-5、16-3、16-4、23-2或23-3所示。The linker compound according to claim 9, wherein the compound of formula II is compound 1-8, 1-9, 2-6, 2-7, 3-5, 3-6, 4-4, 4-5, 5-3, 5-4, 6-3, 6-4, 7-4, 7-5, 8-3, 8-4, 15-4, 15-5, 16-3, 16- 4, 23-2 or 23-3.
  15. 下式III所示的与权利要求9-14中任一项所述的连接子化合物共价连接的药物：A drug covalently linked to the linker compound described in any one of claims 9-14 shown in the following formula III:
    R 4-L 2-P-R 5-D  (III) R 4 -L 2 -PR 5 -D (III)
    式中：R 4、L 2、P和R 5如权利要求9-14中任一项所述；D表示药物分子去除了一个H原子后获得的基团，与R 5共价连接；优选地，药物分子通过与R 5形成碳酸酯键(-OCO-)或氨基甲酸酯(-OCNH-)而与R 5相连。 In the formula: R 4 , L 2 , P and R 5 are as described in any one of claims 9-14; D represents a group obtained by removing one H atom from the drug molecule, which is covalently linked to R 5 ; preferably , the drug molecule is linked to R 5 by forming a carbonate bond (-OCO-) or a carbamate (-OCNH-) with R 5 .
  16. 如权利要求15所述的药物，其特征在于，所述药物分子选自：MMAE、Duo-5、DXD、依沙替康、喜树碱、10-羟基喜树碱、拓扑替康、氟脲苷、去氧氟尿苷、阿糖胞苷、依托泊苷、氟达拉滨、卡培他滨、长春新碱、埃坡霉素B、紫杉醇、多烯紫杉醇、柔红霉素、表阿霉素、甲氨蝶呤、吉西他滨、美法仑、尼莫司汀、米托蒽醌、阿霉素和丝裂霉素；The drug of claim 15, wherein the drug molecule is selected from the group consisting of: MMAE, Duo-5, DXD, ixatecan, camptothecin, 10-hydroxycamptothecin, topotecan, fluoxuron glycosides, deoxyfluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, daunorubicin, epirubicin Mycin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin;
    优选地，所述药物如LD001到LD0025所示。Preferably, the drugs are shown as LD001 to LD0025.
  17. 一种ADC，其结构式如下式IV所示：A kind of ADC, its structural formula is shown as following formula IV:
    A-R 4-L 2-P-R 5-D  (IV) AR 4 -L 2 -PR 5 -D (IV)
    式中，R 4、L 2、P和R 5如权利要求9-14中任一项所述，D如权利要求15或16所述，A为抗体或其抗原结合片段；其中，所述抗体或其抗原结合片段通过其所含有的巯基与R 4共价连接。 In the formula, R 4 , L 2 , P and R 5 are as described in any one of claims 9-14, D is as described in claim 15 or 16, and A is an antibody or an antigen-binding fragment thereof; wherein, the antibody or its antigen - binding fragment is covalently linked to R4 through the sulfhydryl group it contains.
  18. 如权利要求17所述的ADC，其特征在于，所述抗体选自：抗-Her2 抗体，抗-EGFR抗体，抗-VEGFR抗体，抗-CD20抗体，抗-CD33抗体，抗-PD-L1抗体，抗-PD-1抗体，抗-CTLA-4抗体，抗-TNFα抗体，抗-CD28抗体，抗-4-1BB抗体，抗-OX40抗体，抗-GITR抗体，抗-CD27抗体，抗b-CD40抗体，或抗-ICOS抗体，抗-CD25抗体，抗-CD30抗体，抗-CD3抗体，抗-CD22抗体，抗-CCR4抗体，抗-CD38抗体，抗-CD52抗体，抗-补体C5抗体，抗RSV的F蛋白，抗-GD2抗体，抗-GITR抗体，抗-糖蛋白受体lib/Illa抗体，抗-ICOS抗体，抗-IL2R抗体，抗-LAG3抗体，抗-Integrinα4抗体，抗-lgE抗体，抗-PDGFRa抗体，抗-RANKL抗体，抗-SLAMF7抗体，抗-LTIGIT抗体，抗-TIM-3抗体，抗-VEGFR2抗体，抗-VISTA抗体，抗C-Met抗体，抗BCMA抗体，抗Claudin 18抗体、抗Nectin-4抗体，抗CD79b抗体，和抗Trop 2抗体。The ADC of claim 17, wherein the antibody is selected from the group consisting of: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, and anti-PD-L1 antibody , Anti-PD-1 Antibody, Anti-CTLA-4 Antibody, Anti-TNFα Antibody, Anti-CD28 Antibody, Anti-4-1BB Antibody, Anti-OX40 Antibody, Anti-GITR Antibody, Anti-CD27 Antibody, Anti-b- Anti-CD40 Antibody, or Anti-ICOS Antibody, Anti-CD25 Antibody, Anti-CD30 Antibody, Anti-CD3 Antibody, Anti-CD22 Antibody, Anti-CCR4 Antibody, Anti-CD38 Antibody, Anti-CD52 Antibody, Anti-Complement C5 Antibody, Anti-RSV F protein, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib/Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-Integrinα4 antibody, anti-lgE Antibody, Anti-PDGFRa Antibody, Anti-RANKL Antibody, Anti-SLAMF7 Antibody, Anti-LTIGIT Antibody, Anti-TIM-3 Antibody, Anti-VEGFR2 Antibody, Anti-VISTA Antibody, Anti-C-Met Antibody, Anti-BCMA Antibody, Anti- Claudin 18 antibody, anti-Nectin-4 antibody, anti-CD79b antibody, and anti-Trop 2 antibody.
  19. 如权利要求17所述的ADC，其特征在于，所述ADC选自LA001至LA025中的任意一个或多个。The ADC of claim 17, wherein the ADC is selected from any one or more of LA001 to LA025.
  20. 权利要求1-3中任一项所述的氨基酸、权利要求4-8中任一项所述的肽、权利要求9-14中任一项所述的连接子化合物以及权利要求15-16中任一项所述的药物在制备ADC中的应用，或在改善抗体溶解性中的应用，或者在制备用于改善抗体溶解性的制剂中的应用。The amino acid of any one of claims 1-3, the peptide of any one of claims 4-8, the linker compound of any one of claims 9-14, and the linker compound of any one of claims 15-16 Use of any one of the medicaments in preparing ADC, or in improving antibody solubility, or in preparing a preparation for improving antibody solubility.
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