WO2022199237A1 - Acides aminés modifiés et leur utilisation dans un adc - Google Patents

Acides aminés modifiés et leur utilisation dans un 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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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

L'invention concerne des acides aminés modifiés et leur utilisation dans un ADC. Les acides aminés modifiés sont des acides aminés modifiés par un groupe phosphorylcholine représentés par R1-L1-N(R2)-R3, dans laquelle R1, L1, N, R2 et R3 sont tels que définis dans la description. L'invention concerne en outre des peptides contenant les acides aminés modifiés. Les ADC fabriqués à l'aide de tels acides aminés modifiés ou les peptides modifiés présentent une solubilité améliorée de manière significative.
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