CN110090308B

CN110090308B - Method for preparing conjugate

Info

Publication number: CN110090308B
Application number: CN201910061779.0A
Authority: CN
Inventors: 宋帅; 田强; 唐祖建; 邓汉文; 汪静; 刘登念; 胡瑞斌; 肖亮; 薛彤彤; 蔡家强; 王利春; 王晶翼
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2018-01-30
Filing date: 2019-01-23
Publication date: 2023-03-24
Anticipated expiration: 2039-01-23
Also published as: WO2019149116A1; CN111447939B; CN111447939A; CN110090308A

Description

Method for preparing conjugate

Technical Field

The invention belongs to the technical field of medicines. In particular, the present invention relates to a method for preparing a targeted drug-bioactive molecule conjugate, products prepared by said method and the use of the products in the prevention and/or treatment of proliferative diseases associated with abnormal cellular activity, including but not limited to cancer diseases.

Background

Chemotherapy was once the standard therapy for cancer, but highly lethal bioactive molecules can miskill normal cells, causing serious side effects. The targeting antitumor drug has both targeting property and antitumor activity, and has become a hotspot in the current tumor research field. Since the 20 th century, the development of biomacromolecule drugs (such as therapeutic antibodies or antibody fragments) and targeting small molecule ligands for antitumor drugs and tumor targeting therapies has made breakthrough progress. However, although the biomacromolecule drug has strong targeting property, the treatment effect on solid tumors is limited; the bioactive molecules have high killing effect on cancer cells, but often lack targeting property, and often injure normal cells by mistake, thereby causing serious toxic and side effects.

In recent years, it has been discovered that therapeutic antibodies can be linked to biologically active molecules to form antibody-drug conjugates (ADCs). ADC combines the targeting effect of the antibody and the high activity of bioactive molecules to form a biological missile. The antibody directs ADC binding to the target cell, which is then internalized by the cell, releasing the drug to treat the disease. Because the antibody has specificity and targeting property on the tumor cell related target, the application value of the antibody is reflected in the aspect of treatment, and the antibody also becomes an ideal carrier for targeted delivery of the drug, so that the side effect of the drug is reduced. The design principle of Small Molecule Drug Conjugate (SMDC) and antibody-drug conjugate (ADC) is the same, i.e. bioactive molecule is coupled with some small molecule ligands which can selectively bind to tumor cell surface receptors by chemical method, thereby improving the targeting of effector molecule to tumor cells. The chemical structure of SMDCs is almost identical to ADCs, but small molecule ligands are used instead of antibodies. At present, no SMDC is on the market.

Currently, there are four types of ADCs on the market: mylotarg (Gemtuzumab Oxogamicin, gemtuzumab oxazole Mi Xing), adcetris (Brentuximab Vedotin, CD30 monoclonal antibody-MMAE), kadcyla (Trastuzumab Emtansine, trastuzumab-maytansine alkaloid) and Besponsa (Inotuzumab Ozogamicin, CD22 monoclonal antibody-calicheamicin).

Typically, ADC drugs consist of an antibody, a biologically active molecule, and a linker. The biologically active molecule is covalently coupled to the antibody through a linker. The antibody (such as a monoclonal antibody) can specifically recognize a specific target on the surface of the cancer cell, and further can guide the ADC to the surface of the cancer cell, so that the ADC enters the cancer cell through an endocytosis effect. Bioactive molecules can be released in cells, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

Lysine is the most common linking site in antibodies, and the epsilon-amino group can react with the activated carboxyl group of the linking group to form an amide bond, which is the most common coupling technique at present. With the progress of research, a technology for realizing site-directed coupling has appeared, namely, pentafluorophenol is used for activating carboxyl of a connecting group, and then the carboxyl and the lysine epsilon-amino in an antibody form an amido bond so as to complete the coupling of a bioactive molecule and the antibody. However, the coupling technology has some defects, and due to the fact that the pentafluorophenol ester obtained by carboxyl activation has high reaction activity and poor chemical stability, and even cannot stably exist in certain specific structures, the coupling reaction is difficult to carry out, and further the ADC coupling efficiency is extremely low, and even cannot be obtained.

Disclosure of Invention

The quaternary ammonium salt or the nitroxide structure has good physicochemical properties (such as hydrophilicity), so that the pharmacokinetic properties of the ADC are greatly improved, and the hydrophilicity of the ADC with the quaternary ammonium salt or the nitroxide structure is improved, so that the crosslinking of an antibody part is reduced, and the toxicity of the ADC drug is reduced. In addition, in the preparation process of ADC drugs, an activating group is required to be used for activating the terminal carboxyl in a connecting group, and because pentafluorophenol has a good leaving property, the pentafluorophenol is used for activating the carboxyl at present. However, in the process of preparing the ADC having the quaternary ammonium salt or the oxynitride structure, it is found that the pentafluorophenol ester having the quaternary ammonium salt or the oxynitride structure has too high reaction activity to stably exist, so that the coupling reaction cannot be performed, and the yield of the ADC obtained is extremely low. Through intensive research, a novel method for coupling the target drug and the bioactive molecule is surprisingly found, namely, the stability of activated carboxyl in a connecting group is greatly improved by utilizing a novel carboxyl activation mode, the problem that stable pentafluorophenol ester cannot be obtained by using a coupling technology of pentafluorophenol activated carboxyl is successfully overcome, and the coupling of the bioactive molecule and the target drug is realized at a higher coupling ratio. The coupling method can be widely applied to the synthesis of the targeted drug-bioactive molecule conjugate.

A first aspect of the invention provides a process for the preparation of a conjugate of general formula (II),

the method comprises reacting a compound of formula (I) with a compound containing one or more amino groups (-NH) ₂ ) The target-oriented drug coupling of (1),

wherein:

t is selected from the following structures:

L ₁ is composed of

The group being linked to T via one of the two positions marked 1 or 2 and to L via the other position ₂ Connecting; preferably, the group is linked to T via the 1-labelled position and L via the 2-labelled position ₂ Connecting;

L ₂ is a group selected from:

the radical being bound to L via one of the two positions marked by 1 or 2 ₁ Is connected to L via another position ₃ Connecting; preferably, the position of the group marked by 1 is linked to L ₁ Is connected and passes the position marked by 2 and L ₃ Connecting; more preferably, L ₂ Is->

The position of the group marked by 1 and L ₁ Is connected and passes the position marked by 2 and L ₃ Connecting;

L ₃ selected from optionally substituted by one or more R _i Substituted of the following groups:

the radical being bound to R via one of the two positions marked by 1 or 2 ₁ Is connected to L via another position ₂ Connecting; preferably, the position of the group marked by 1 is linked to R ₁ Is connected and passes the position marked by 2 and L ₂ Connecting;

wherein:

e is a counterion, preferably a halide anion, and more preferably a chloride, bromide or iodide;

R _q each occurrence of which is independently selected from C _1-6 Alkyl radical, C _2-6 Alkenyl radical, C _2-6 Alkynyl and C _3-8 CycloalkanesA base;

R _p each occurrence of which is independently selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl radical, C _1-6 Alkoxy and-C _1-2 Alkyl-cyano, preferably-CH ₂ CN and-CH ₂ CH ₂ CN；

Beta is an integer of 0, 1 or 2; and is

γ is independently at each occurrence an integer of 1,2, or 3;

R _i selected from H (hydrogen), D (deuterium), halogen, = O, CF ₃ 、CN、CH ₂ CN、C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _2-10 Alkenyl radical, C _3-8 Cycloalkyl radical, C _2-10 Alkynyl, COOH and SO ₃ H, preferably, R _i Selected from H (hydrogen), D (deuterium), = O, CN, CH ₂ CN, methyl and CF ₃ ；

R ₁ Selected from optionally substituted by one or more R _b Substituted of the following groups: c _1-6 Alkylene radical, C _3-10 Cycloalkylene radical, C _6-10 Arylene and 5-10 membered heteroarylene, wherein R _b Is H (hydrogen), D (deuterium), halogen, cyano, nitro, trifluoromethyl, COOH or SO ₃ H；

Z is an oxygen atom or a sulfur atom, and preferably an oxygen atom;

R ₂ selected from optionally substituted by one or more R _a Substituted of the following groups: c _1-6 Alkyl radicals and

and R is _a Is fluorine or nitro, with the proviso that when R is _a When it is fluorine, R ₂ Phenyl which is not perfluorinated;

a is a group obtained after alpha amino groups are removed from a targeted drug, and the targeted drug is selected from trastuzumab, pertuzumab or Sacituzumab;

m, n, and r are each independently at each occurrence an integer of 0, 1,2,3, 4,5, 6, 7, 8, 9, or 10; preferably, m, n are each independently selected from 0, 1,2,3, and r is selected from 0, 1, 2; more preferably, m, n are 1, and r is 0; and is

α is selected from 1,2,3 or 4, preferably α is selected from 1 or 2.

The second aspect of the present invention provides a conjugate of the general formula (II), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, which is prepared by the above-mentioned method,

wherein each group is as defined above.

A third aspect of the present invention provides a process for preparing a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, which comprises reacting a compound of formula (I-0) with R ₂ -ZH reaction to give the compound of formula (I),

wherein each group is as defined above.

A fourth aspect of the invention provides a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,

wherein each group is as defined above.

A fifth aspect of the invention provides a pharmaceutical composition comprising a conjugate of general formula (II) according to the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or solvate thereof, and one or more pharmaceutically acceptable carriers, and optionally further comprising one or more other anti-cancer drugs such as chemotherapeutic agents and/or antibodies. The pharmaceutical composition is preferably a solid formulation, a semi-solid formulation, a liquid formulation or a gaseous formulation.

A sixth aspect of the invention provides a pharmaceutical formulation comprising a conjugate of general formula (II) according to the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, or a pharmaceutical composition according to the fifth aspect of the invention.

A seventh aspect of the present invention provides the use of a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof for the manufacture of a medicament for the prevention or treatment of a cancer disease.

An eighth aspect of the present invention provides a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, for use in the prevention or treatment of a cancer disease.

A ninth aspect of the present invention provides a method for preventing or treating a cancer disease, the method comprising administering to a subject in need thereof an effective amount of a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof.

Definition of terms

In the present invention, unless otherwise specified, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present invention, the term "conjugate" refers to a substance obtained by linking a bioactive molecule to a targeted drug. In some embodiments of the invention, the bioactive molecule is linked to the targeting drug via a linker. The linker is capable of cleavage in a specific environment (e.g., an intracellular low pH environment) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive molecule from the targeted drug. In some embodiments of the invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the invention, the bioactive molecule is directly attached to the targeted drug via a covalent bond that is capable of breaking under a particular environment or action, thereby separating the bioactive molecule from the targeted drug.

A "small molecule" is defined herein as a small molecule drug having biological activity. In certain embodiments, the small molecule has a molecular weight of no greater than 2000Da, such as no greater than 1500Da, 1000Da, or 500Da.

In the present invention, the term "linker" refers to a structural fragment that links a biologically active molecule to a targeted drug.

In the present invention, the term "targeted drug" refers to a drug capable of specifically binding to a target (or a portion of a target) on the surface of a cell. The conjugate can be delivered to a specific cell population by the interaction of the targeting drug moiety with the target.

In the present invention, when the targeted drug is an antibody, the conjugate may be referred to as an "antibody-drug conjugate". In the present invention, "antibody-drug conjugate" and "immunoconjugate" can be used interchangeably.

In the present invention, the term "antibody" is to be interpreted in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, so long as they possess the desired biological activity. Herein, "antibody" and "immunoglobulin" may be used interchangeably.

In an embodiment of the invention, the targeted drug is a monoclonal antibody against Her2, such as trastuzumab, pertuzumab; or the targeted drug is an anti-Trop-2 monoclonal antibody, such as, for example, sacituzumab.

In some embodiments of the invention, the targeted drug is trastuzumab or pertuzumab. Trastuzumab is an anti-Her 2 monoclonal antibody whose amino acid sequence is known to those skilled in the art, and whose schematic sequence can be found, for example, in CN103319599. Trastuzumab heavy chain terminal Lys is easily deleted, but this deletion does not affect biological activity, see Dick, l.w. et al, biotechnol.bioeng., 100. Exemplary heavy and light chain sequences of pertuzumab are set forth in SEQ ID nos. 16 and 15 of US 7560111.

In some embodiments of the invention, the targeted drug anti-Trop-2 antibody is hRS7 (i.e., sacituzumab of the invention) described in US 2012/0237518.

In the present invention, erbB2 and Her2 are used interchangeably, both of which refer to the native sequence of the human Her2 protein (Genebank accession number X03363, see, e.g., semba et al, 1985, PNAS,82 6497-6501; and Yamamoto et al, 1986, nature, 319) and functional derivatives thereof, e.g., amino acid sequence variants. ErbB2 denotes the gene encoding human Her2 and neu denotes the gene encoding rat p185 neu. In some embodiments, a compound or conjugate of the invention is capable of inhibiting or killing a cell that expresses an ErbB2 receptor, such as a breast cancer cell, ovarian cancer cell, gastric cancer cell, endometrial cancer cell, salivary gland cancer cell, lung cancer cell, kidney cancer cell, colon cancer cell, thyroid cancer cell, pancreatic cancer cell, bladder cancer cell, or liver cancer cell.

In the present invention, trop-2 or Trop2 refers to human trophoblast cell-surface antigens-2 (also known as tactd 2, M1S1, GA733-1, EGP-1), which are cell surface receptors expressed by many human tumor (e.g., breast, colorectal, lung, pancreatic, ovarian, prostate, or cervical cancer) cells. In some embodiments, the compound or conjugate of the invention is capable of inhibiting or killing a cell that expresses a TROP2 receptor, such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, or cervical cancer.

In the present invention, the term "C _1-6 Alkyl "denotes straight or branched alkyl having 1 to 6 carbon atoms, including for example" C _1-4 Alkyl group "," C _1-3 Alkyl "and the like, specific examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropylN-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, and 1,2-dimethylpropyl, and the like. The term "C _1-6 Alkylene "is a corresponding divalent radical, including, for example," C _1-4 Alkylene group "," C _1-3 Alkylene "and the like, specific examples include, but are not limited to: methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like.

In the present invention, the term "C _2-10 The "alkenyl group" means a straight-chain, branched or cyclic alkenyl group having 2 to 10 carbon atoms and containing at least one double bond, and includes, for example, "C _2-6 Alkenyl group "," C _2-4 Alkenyl groups "and the like. Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, and 1,4-cyclohexadienyl and the like. The term "C _2-10 Alkenylene "is a corresponding divalent radical, including, for example," C _2-6 Alkenylene group and C _2-4 Alkenylene "and the like. Examples thereof include, but are not limited to: vinylene, propenylene, butenylene, pentenylene, hexenylene, cyclopentenylene, cyclohexenylene, and the like.

In the present invention, the term "C _2-10 Alkynyl "refers to a straight or branched chain alkynyl group containing at least one triple bond and having 2 to 10 carbon atoms, including, for example," C _2-6 Alkynyl group "," C _2-4 Alkynyl "and the like. Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl and the like. The term "C _2-10 Alkynylene "is a corresponding divalent radical, including, for example," C _2-6 Alkynylene group and C _2-4 Alkynylene "and the like. Examples include, but are not limited to: ethynylene, propynyl, butynyl, pentynyl, hexynyl and the like。

In the present invention, the term "halogen" includes fluorine, chlorine, bromine and iodine.

In the present invention, the term "3-to 10-membered cycloalkyl" or "C _3-10 Cycloalkyl "refers to saturated cyclic alkyl groups containing 3 to 10 carbon atoms, including, for example," C _3-8 Cycloalkyl group "," C _3-6 Cycloalkyl group "," C _4-6 Cycloalkyl group "," C _5-7 Cycloalkyl radicals "or" C _5-6 Cycloalkyl groups "and the like. Specific examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, and the like. The term "3-10 membered cycloalkylene" or "C _3-10 Cycloalkylene "is a corresponding divalent radical, including, for example," C _3-8 Cycloalkylene group "," C _3-6 Cycloalkylene group "," C _4-6 Cycloalkylene group "," C _5-7 Cycloalkylene "or" C _5-6 Cycloalkylene "and the like. Specific examples include, but are not limited to: cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, and the like.

In the present invention, the term "3-8 membered heterocyclic group" means a cyclic group containing 3 to 8 ring atoms, at least one of which is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. "3-8 membered heterocyclic group" includes, for example, "3-8 membered nitrogen-containing heterocyclic group", "3-8 membered oxygen-containing heterocyclic group", "3-6 membered oxygen-containing heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered nitrogen-containing heterocyclic group", preferably includes, but is not limited to, oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and homopiperazinyl groups and the like. The term "3-to 8-membered heterocyclylene" is a corresponding divalent group, and includes, for example, "3-to 8-membered nitrogen-containing heterocyclylene", "3-to 8-membered oxygen-containing heterocyclylene", "3-to 6-membered oxygen-containing heterocyclylene", "4-to 7-membered heterocyclylene", "4-to 6-membered heterocyclylene", "5-to 7-membered heterocyclylene", "5-to 6-membered nitrogen-containing heterocyclylene", and preferably includes, but is not limited to, oxiranylene, tetrahydrofurylene, piperidylene, piperazinylene, tetrahydropyrylene, and homopiperazinylene, and the like.

In the present invention, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromatic character, such as C _6-10 Aryl radical, C _5-8 Aryl, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, and the like. The term "arylene" is a corresponding divalent radical, e.g. C _6-10 Arylene radical, C _5-8 Arylene groups, and the like. Specific examples include, but are not limited to, phenylene, naphthylene, anthracenylene, phenanthrenylene, and the like.

In the present invention, the term "heteroaryl" refers to a cyclic group having aromaticity, wherein at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. Specific examples include, but are not limited to, 5-10 membered heteroaryl, 5-10 membered nitrogen-containing heteroaryl, 6-10 membered oxygen-containing heteroaryl, 6-8 membered nitrogen-containing heteroaryl, and 5-8 membered oxygen-containing heteroaryl, and the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-6272 zxft 5272-oxazinyl, 4H-3445-oxazinyl, 6H-3272-oxazolyl, 4H-3424 zzft 497984-357923-triazinyl, 354235-triazinyl, 354284-567923-triazinyl, 35-5623-triazinyl, 35-5660-triazinyl, and the like. The term "heteroarylene" is a corresponding divalent group, and specific examples include, but are not limited to, nitrogen-containing heteroarylene, 5-10 membered nitrogen-containing heteroarylene, 5-6 membered nitrogen-containing heteroarylene, 6-10 membered oxygen-containing heteroarylene, 6-8 membered nitrogen-containing heteroarylene, and 5-8 membered oxygen-containing heteroarylene, and the like, such as furanylene, thienyl, pyrrolylene, thiazolyl, oxazolylene, imidazolyl, pyrazolyl, triazolylene, pyridyl, pyrimidinylene, oxazinylene, pyridazinylene, pyrazinylene, triazinylene, and tetrazinylene.

In the present invention, the term "counter ion" refers to an ion accompanying an ionic substance to maintain electrical neutrality, for example, in sodium chloride, the sodium cation is the counter ion of the chloride anion, and vice versa.

Optionally, the hydrogen in the groups referred to in the present invention may be substituted by deuterium.

The groups mentioned in the invention are obtained by replacing 1,2 or 3 hydrogen atoms in the compound/conjugate corresponding to the group with other atoms, and the number of the replaced hydrogen atoms can be determined according to the valence number formed by the group in the compound or conjugate. For example, an alkyl group is a group obtained by substituting one hydrogen atom in an alkane, an alkylene group is a group obtained by substituting two hydrogen atoms in an alkane, a methyl group and an ethyl group are groups obtained by substituting one hydrogen atom in methane and ethane, respectively, and a methylene group and an ethylene group are groups obtained by substituting two hydrogen atoms in methane and ethane, respectively.

As used herein, the term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the current circumstances is not exceeded and that the substitution results in a stable compound/conjugate. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds/conjugates.

If a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced individually and/or together with an independently selected optional substituent. If the nitrogen of a substituent is described as being optionally substituted with one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogen is present) may each be replaced with an independently selected optional substituent.

If a substituent is described as being "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2,3, 4,5 or 10, under reasonable conditions.

As used herein, unless otherwise indicated, the point of attachment of a substituent may be from any suitable position of the substituent.

When a bond of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

As used herein, the term "solvate" refers to a substance formed by association of a compound/conjugate with a solvent molecule. The solvent may be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.) or the like. For example, the compounds/conjugates of the invention may form ethanolates with ethanol.

In the present invention, the term "hydrate" refers to the substance formed by association of a compound/conjugate with a water molecule.

In embodiments of the invention, if a chiral carbon is present in the compound/conjugate, the invention includes isomers formed based on any stereoconfiguration of the chiral carbon, including, for example, racemates or any mirror image isomers. Moreover, the present invention includes all other stereoisomers that may be present. That is, the compounds/conjugates of the present invention include all enantiomers, diastereomers, cis-trans isomers, racemates, and the like.

Solid lines may be used herein

Real wedge shaped->

Or a virtual wedge>

Chemical bonds of the compounds/conjugates of the invention are depicted. The use of a solid line to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomers shown are present. When present in a racemic mixture, solid and dotted wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds/conjugates of the present invention are intended to exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformers, atropisomers, and mixtures thereof. The compounds/conjugates of the invention may exhibit more than one type of isomerization and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

The present invention encompasses all possible crystalline forms or polymorphs of the compounds/conjugates of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

Pharmaceutically acceptable salts of the compounds/conjugates of the present invention include acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, glucoheptonate, gluconate, orotate, palmitate and other similar salts.

Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts, and other similar salts.

For a review of suitable Salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: properties, selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds/conjugates of the present invention are known to those skilled in the art.

Also included within the scope of the present invention are metabolites of the compounds/conjugates of the invention, i.e., substances formed in vivo upon administration of the compounds/conjugates of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound/conjugate. Accordingly, the present invention includes metabolites of the compounds/conjugates of the present invention, including compounds/conjugates made by the process of contacting the compounds/conjugates of the present invention with a mammal for a time sufficient to produce a metabolite thereof.

Preparation method

In some embodiments, the invention provides a method of preparing a conjugate of formula (II),

wherein:

t is selected from the following structures:

L ₁ is composed of

The group being linked to T via one of the two positions marked 1 or 2 and to L via the other position ₂ Connecting; preferably, said group is labelled by 1Is connected to T and is marked by a position of 2 and L ₂ Connecting; />

L ₂ Is a group selected from:

the radical being bound to L via one of the two positions marked by 1 or 2 ₁ Is connected to L via another position ₃ Connecting; preferably, the position of the group marked by 1 is linked to L ₁ Is connected and passes through the 2 marked position and L ₃ Connecting; more preferably, L ₂ Is->

The position of the group marked by 1 and L ₁ Is connected and passes the position marked by 2 and L ₃ And (4) connecting.

wherein:

R _q each at each occurrence is independently selected from C _1-6 Alkyl radical, C _2-6 Alkenyl radical, C _2-6 Alkynyl and C _3-8 A cycloalkyl group;

R _p each occurrence of which is independently selected from C _1-6 Alkyl radical, C _3-6 Cycloalkyl, C _1-6 Alkoxy and-C _1-2 Alkyl-cyano, preferably-CH ₂ CN and-CH ₂ CH ₂ CN；

Beta is an integer of 0, 1 or 2; and is

γ is independently at each occurrence an integer of 1,2, or 3;

Z is an oxygen atom or a sulfur atom, and is preferably an oxygen atom;

R ₂ selected from the group consisting of optionally substituted by one or more R _a Substituted of the following groups: c _1-6 Alkyl radicals and

R _a is fluorine or nitro, with the proviso that when R is _a When it is fluorine, R ₂ Phenyl which is not perfluorinated;

α is selected from 1,2,3 or 4, preferably α is selected from 1 or 2.

In a preferred embodiment, L ₃ Selected from the group consisting of optionally substituted by one or more R _i Substituted of the following groups:

the radical being bound to R via one of the two positions marked by 1 or 2 ₁ Is connected and passes another position with L ₂ Connecting; preferably, the position of the group marked by 1 is linked to R ₁ Is connected and passes the position marked by 2 and L ₂ Connecting;

wherein:

e is a counterion, preferably a halide anion, and more preferably a chloride, bromide, or iodide;

R _q each occurrence of which is independently selected from C _1-3 Alkyl radical, C _2-4 Alkenyl radical, C _2-4 Alkynyl and C _3-6 A cycloalkyl group;

R _i selected from H (hydrogen), D (deuterium), CN, CH ₂ CN, methyl and CF ₃ ；

More preferably, L ₃ Selected from:

one of the two positions of said group marked by 1 or 2 is linked to R ₁ Is connected to L via another position ₂ Connecting; preferably, the position of the group marked by 1 is linked to R ₁ Is connected and passes the position marked by 2 and L ₂ Connecting;

wherein:

Further preferably, L ₃ Selected from:

wherein:

in a preferred embodiment of the process according to the invention,

selected from:

the position of the group marked by 1 and L ₁ Attached and linked to the carbonyl through the 2-labeled position;

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

selected from the group consisting of:

the position of the group marked by 1 and L ₁ Attached and linked to the carbonyl group via the 2-labeled position.

In a preferred embodiment, R ₂ Is phenyl substituted by four fluorine atoms, phenyl substituted by three fluorine atoms, phenyl substituted by two fluorine atoms, phenyl substituted by one fluorine atom, phenyl substituted by nitro, C substituted by six fluorine atoms _3-6 Alkyl, five fluorine atom substituted C _2-6 Alkyl, tetraC substituted by fluorine atoms _2-6 Alkyl or C substituted by three fluorine atoms _1-6 An alkyl group.

More preferably, R ₂ Selected from:

further preferably, R ₂ Is 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl or 2,6-difluorophenyl.

In a preferred embodiment of the process according to the invention,

selected from:

preferably, the first and second electrodes are formed of a metal,

selected from:

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

selected from:

it is further preferred that the first and second liquid crystal compositions,

selected from the group consisting of:

in a preferred embodiment, the method comprises mixing the targeting agent with a compound of general formula (I).

Preferably, the molar ratio of the targeting drug to the compound of the general formula (I) is 1 (1-20).

Preferably, the method comprises mixing a solution comprising the targeted drug with the compound of formula (I).

Preferably, the process is carried out in water or an organic solvent.

Preferably, the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol or ethanol), and any combination thereof.

Preferably, the method further comprises purification by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography and affinity chromatography.

In some embodiments, the present invention provides a conjugate of formula (II), a pharmaceutically acceptable salt, stereoisomer, or metabolite thereof, or a solvate thereof,

wherein each group is as defined above.

Preferably, the conjugate of formula (II) is prepared by the above method.

Preferably, the conjugate of formula (II) is an antibody-drug conjugate.

Preferably, the antibody-drug conjugate is:

wherein α is an integer of 1,2,3 or 4; and A is a group obtained by removing alpha amino groups from trastuzumab, pertuzumab or Sacituzumab.

Preferably, the antibody-drug conjugate is:

wherein A1 is a group obtained after removing 2 amino groups from trastuzumab.

Preferably, the antibody-drug conjugate is:

wherein A2 is a group obtained by removing 2 amino groups from pertuzumab.

Preferably, the antibody-drug conjugate is:

wherein A3 is a group obtained by removing 2 amino groups from Sacituzumab.

Preferably, the antibody-drug conjugate is:

wherein A1' is a group obtained by removing 1 amino group from trastuzumab.

Preferably, the antibody-drug conjugate is:

wherein A2' is a group obtained by removing 1 amino group from pertuzumab.

Preferably, the antibody-drug conjugate is:

wherein A3' is the group obtained after removing 1 amino group in Sacituzumab.

In some embodiments, the present invention provides a method of preparing a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer, or metabolite thereof, or a solvate thereof, comprising reacting a compound of formula (I-0) with R ₂ -ZH reaction to give the compound of the general formula (I),

wherein each group is as defined above; and is provided with

The reaction is preferably carried out in water or an organic solvent.

Preferably, the reaction is carried out in the presence of a base (including organic bases (such as triethylamine, DIPEA, pyridine, NMM or DMAP) or inorganic bases (such as NaH, naOH, na) ₂ CO ₃ 、NaHCO ₃ 、K ₂ CO ₃ ) And a condensing agent (e.g., HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, pyAOP, or PyBOP).

In some embodiments, the present invention provides a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer, or metabolite thereof, or a solvate thereof,

wherein each group is as defined above.

In a preferred embodiment, the compounds of formula (I) are prepared by the methods described above.

In a preferred embodiment, the compound of formula (I) has a structure of formula (Ia),

preferably, the compound is selected from:

pharmaceutical compositions, pharmaceutical formulations and methods of treatment

In some embodiments, the present invention provides a pharmaceutical composition comprising a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or solvate thereof and one or more pharmaceutically acceptable carriers, and optionally further comprising one or more other anti-cancer agents such as chemotherapeutic agents and/or antibodies. The pharmaceutical composition is preferably a solid formulation, a semi-solid formulation, a liquid formulation or a gaseous formulation.

In some embodiments, the present invention provides a pharmaceutical formulation comprising a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, or a pharmaceutical composition according to the present invention.

In some embodiments, the present invention provides the use of a conjugate of general formula (II) according to the present invention, or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof, for the manufacture of a medicament for the prevention or treatment of a cancer disease.

In some embodiments, the present invention provides a conjugate of general formula (II) according to the present invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof for use in the prevention or treatment of a cancer disease.

In some embodiments, the present invention provides a method for preventing or treating a cancer disease, the method comprising administering to a subject in need thereof an effective amount of a conjugate prepared by the method of the present invention or a pharmaceutically acceptable salt, stereoisomer, or metabolite thereof or a solvate thereof.

In some embodiments, the cancer disease is selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small-cell lung cancer or non-small cell lung cancer), squamous cell cancer, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, salivary gland cancer, colon cancer, colorectal cancer, liver cancer, kidney cancer, solid tumors, non-hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme or glioma or sarcoma), prostate cancer, and thyroid cancer.

By "pharmaceutically acceptable carrier" in the context of the present invention is meant a diluent, adjuvant, excipient, or vehicle that is administered together with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that may be employed in the pharmaceutical compositions or formulations of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition or pharmaceutical formulation is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical composition or pharmaceutical formulation of the invention may act systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

For these administration routes, the pharmaceutical composition or pharmaceutical preparation of the present invention can be administered in a suitable dosage form.

Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

The term "effective amount" as used herein refers to an amount of conjugate that will alleviate one or more symptoms of the condition being treated to some extent after administration.

The dosing regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering the composition or supervising the administration of the composition.

The amount of the conjugate of the invention administered will depend on the severity of the individual, disorder or condition being treated, the rate of administration, the disposition of the conjugate, and the judgment of the prescribing physician. Generally, an effective dose is from about 0.0001 to about 50mg per kg body weight per day, e.g., from about 0.01 to about 10 mg/kg/day (single or divided administration). For a 70kg person, this may amount to about 0.007 mg/day to about 3500 mg/day, for example about 0.7 mg/day to about 700 mg/day. In some cases, dosage levels not higher than the lower limit of the aforesaid range may be sufficient, while in other cases still larger doses may be employed without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.

The amount or amount of the conjugate of the invention in a pharmaceutical composition or pharmaceutical preparation may be from about 0.01mg to about 1000mg, suitably 0.1 to 500mg, preferably 0.5 to 300mg, more preferably 1 to 150mg, especially 1 to 50mg, for example 1.5mg, 2mg, 4mg, 10mg, 25mg etc.

As used herein, unless otherwise specified, the term "treating" or "treatment" means reversing, alleviating, inhibiting the progression of, or preventing such a disorder or condition, or one or more symptoms of such a disorder or condition, to which such term applies.

As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Advantageous effects of the invention

The invention utilizes a new carboxyl activation mode, greatly improves the stability of the intermediate obtained after the carboxyl in the connecting group is activated, successfully overcomes the difficult problem that the pentafluorophenol ester intermediate is unstable in the presence of a quaternary ammonium salt or a nitrogen oxide structure in the coupling technology, and simultaneously realizes the coupling of bioactive molecules and targeted drugs with higher coupling ratio (for example, about 33 percent). The coupling method can be widely applied to the synthesis of the targeted drug-bioactive molecule conjugate.

Drawings

FIG. 1 is a Hydrophobic Interaction Chromatography (HIC) analysis of naked antibody.

FIG. 2 is a HIC analysis pattern of Compound 2 conjugated to an antibody.

FIG. 3 is a HIC analysis pattern of Compound 7 conjugated to an antibody.

FIG. 4 is a HIC analysis pattern of Compound 8 conjugated to an antibody.

FIG. 5 is a HIC analysis pattern of Compound 9 conjugated to an antibody.

FIG. 6 is a HIC analysis pattern of Compound 10 conjugated to an antibody.

FIG. 7 is a HIC analysis pattern of Compound 11 conjugated to an antibody.

FIG. 8 is a liquid chromatography-mass spectrometry (LCMS) spectrum of Compound 2 after conjugation with antibody.

Figure 9 is a LCMS spectrum of compound 7 after conjugation with antibody.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

The structures of the compounds/conjugates described in the following examples were determined by nuclear magnetic resonance ( ¹ H NMR) or Mass Spectrometry (MS).

Nuclear magnetic resonance ( ¹ H NMR) was performed using a Bruker 400MHz nuclear magnetic resonance apparatus; the solvent was determined to be deuterated methanol (CD) ₃ OD), deuterated chloroform (CDCl) ₃ ) Or hexadeutero dimethyl sulfoxide (DMSO-d) ₆ ) (ii) a The internal standard substance is Tetramethylsilane (TMS).

The abbreviations used in the Nuclear Magnetic Resonance (NMR) spectra in the examples have the following meanings:

s: singlet (singlet), d doublet (doublet), t triplet (triplet), q quartet (quatet), dd doublet (doublet), qd quartet (doublet), ddd doublet (doublet), ddt doublet, dddd doublet (doublet), m multiplet (multiplt), br broad (broad), J coupling constant, hz, DMSO-d ₆ Hexadeutero dimethyl sulfoxide.

All delta values are expressed in ppm.

Mass Spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Synthesis of compounds comprising biologically active molecules and linkers

Example 1

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methyl-4- ((8978 zft 8978-tetrafluorophenoxy) carbonyl) piperidin-1-ium iodide (Compound 1)

The method comprises the following steps:

synthesis of ethyl 1- (4- (tert-butoxy) -4-oxobutyl) piperidine-4-carboxylate (Compound 1-2)

Compound 1-1 (1.0g, 6.4mmol) was dissolved in N, N-dimethylformamide (10 mL) at room temperature, and tert-butyl 4-bromobutyrate (1.7g, 7.6mmol), potassium carbonate (1.77g, 12.8mmol) and potassium iodide (0.53g, 3.2mmol) were added to the reaction mixture. After the addition, the mixture was stirred at room temperature for 5 hours, and the reaction mixture was poured into water, extracted with ethyl acetate (20 mL × 3), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered to remove a drying agent, and evaporated under reduced pressure to remove the solvent, and purified with silica gel (petroleum ether/ethyl acetate = 3/2) to obtain 1.5g of the objective compound. ESI-MS (m/z) 300.2[ 2 ], [ M ] +H] ⁺ 。

Step two:

synthesis of 1- (4- (tert-butoxy) -4-oxobutyl) -4- (ethoxycarbonyl) -1-methylpiperidin-1-ium iodide (Compound 1-3)

Compound 1-2 (500mg, 1.7mmol) and iodomethane (2413mg, 17.0mmol) were dissolved in dichloromethane (10 mL) at room temperature, and the reaction solution was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure to give the title compound (530 mg). ESI-MS (m/z) 314.2[ M/z ]] ⁺ 。

Step three:

synthesis of 1- (3-carboxypropyl) -4- (ethoxycarbonyl) -1-methylpiperidin-1-ium iodide (Compound 1-4)

Compound 1-3 (530mg, 1.7 mmol) was dissolved in dichloromethane (3 mL) at room temperature, and trifluoroacetic acid (5 mL) was added to the reaction solution and the reaction solution was stirred at room temperature for 2h. The solvent was distilled off under reduced pressure to give the title compound (500 mg). ESI-MS (m/z) 258.2[ M ] 2] ⁺ 。

Step four:

synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- (ethoxycarbonyl) -1-methylpiperidin-1-ium iodide (Compound 1-5)

(S) -2- ((S) -2-amino-3-methylbutanamide) -N- (4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamide) -N, 3-dimethylbutanamide) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropanamide) methyl) phenyl) -5-ureidopentanamide (300mg, 0.27mmol) and compound 1-4 (127mg, 0.33mmol) N, N-dimethylformamide (3 mL) were dissolved at room temperature, cooled to 0 ℃ and N, N-diisopropylethylamine (105mg, 0.81mmol) and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (281mg, 0.54mmol) were added successively and the reaction was stirred for 3H at room temperature. Preparative liquid chromatography gave 200mg of the title compound. ESI-MS (m/z): 1346.2[ M ], [ M] ⁺ 。

Step five:

synthesis of 4-carboxy-1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-penta-ureido-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 1-6)

Compound 1-5 (200mg, 0.15mmol) was dissolved in a mixed solvent of tetrahydrofuran (5 mL) and water (5 mL), and lithium hydroxide (36mg, 1.5mmol) was added thereto, followed by stirring at room temperature for 2.0 hours. Hydrochloric acid solution (0.5 mol/L) was added to adjust pH =4, and the solvent was distilled off under reduced pressure to purify by preparative liquid chromatography to obtain 150mg of the objective compound. ESI-MS (m/z) 1318.2[ M ], [ M] ⁺ 。

Step six:

synthesis of Compound 1

Dissolving the compounds 1-6 (50mg, 0.04mmol) in N, N-dimethylformamide (2 mL), under nitrogen protection, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (11mg, 0.06mmol) and 4-dimethylaminopyridine (15mg, 0.12mmol) to the reaction solution, cooling to 0 ℃, adding 2,3,5,6-tetrafluorophenol (67mg, 0.4mmol) thereto, heating to room temperature, and reacting overnight. Purification by preparative liquid chromatography to give the titleCompound 5.6mg. ESI-MS (m/z) 1466.2[ 2 ] M] ⁺ 。

Example 2

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- ((2,4,6-trifluorophenoxy) carbonyl) piperidine-1-oxide (Compound 2)

The method comprises the following steps:

synthesis of 1- (4- (tert-butoxy) -4-oxobutyl) -4- (ethoxycarbonyl) piperidine-1-oxide (Compound 2-2)

Compound 1-2 (700mg, 2.34mmol) was dissolved in dichloromethane (10 mL) at room temperature, and m-chloroperoxybenzoic acid (808mg, 4.68mmol) was added under nitrogen and stirred at room temperature overnight. The starting material reaction was monitored by hplc-ms for complete reaction, the reaction was quenched with saturated aqueous sodium bicarbonate, extracted with dichloromethane (50 mL × 3), washed with saturated aqueous sodium bicarbonate (100 mL × 2), the combined organic phases were washed with water, dried over anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was evaporated under reduced pressure to give the title compound as a yellow oily liquid 700mg. ESI-MS (m/z) 316.2[ 2 ], [ M + H ]] ⁺ 。

Step two:

synthesis of 1- (3-carboxypropyl) -4- (ethoxycarbonyl) piperidine-1-oxide (Compound 2-3)

Using a similar procedure as described in step three of example 1, compound 2-2 was used instead of compound 1-3 to give 500mg of the title compound. ESI-MS (m/z) 260.2[ M ], [ M] ⁺ 。

Step three:

synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrol-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- (ethoxycarbonyl) piperidine-1-oxide (Compound 2-4)

Using a similar procedure as described in step four of example 1, compound 2-3 was used instead of compound 1-4 to give 15mg of the title compound. ESI-MS (m/z) 674.5[ 2 ] M/2+1] ⁺ 。

Step four:

synthesis of 4-carboxylic acid-1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamide) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrol-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) piperidine-1-oxide (Compound 2-5)

Using a similar procedure as described in step five of example 1, compound 2-4 was substituted for compound 1-5 to give the title compound 15mg. ESI-MS (m/z) 660.5[ M/8978 ] zxft 8978] ⁺ 。

Step five:

synthesis of Compound 2

Using a similar procedure as described in step six of example 1, compound 2-5 was used instead of compound 1-6, and 2,4,6-trifluorophenol was used instead of 2,3,5,6-tetrafluorophenol, to give the title compound 2.0mg. ESI-MS (m/z) 725.6[ M/2+1] ⁺ 。

Example 3

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methyl-4- ((8978 zft 8978-trifluorophenoxy) carbonyl) piperidin-1-ium iodide (Compound 3)

Using a similar procedure as described in step six of example 1, substituting 2,4,6-trifluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained at 21mg. ESI-MS (m/z) 1448.2[ M ]] ⁺ 。

Example 4

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-allopentyl-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methyl-4- ((8978 zft 8978-pentafluoropropoxy) carbonyl) piperidin-1-ium iodide (Compound 4)

Using a similar procedure as described in step six of example 1, substituting 2,2,3,3,3-pentafluoropropan-1-ol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in 21mg. ESI-MS (m/z) 1450.2[ M ], [ m] ⁺ 。

Example 5

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- ((3-fluorophenoxy) carbonyl) -1-methylpiperidin-1-ium iodide (Compound 5)

Example 1 procedure six institute was usedSimilar procedure as described, substituting 3-fluorophenol for 2,3,5,6-tetrafluorophenol gave the title compound 5mg. ESI-MS (m/z) 1412.2[ M ] M] ⁺ 。

Example 6

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- ((4-fluorophenoxy) carbonyl) -1-methylpiperidin-1-ium iodide (Compound 6)

Using a similar procedure as described in example 1, step six, substituting 4-fluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in the amount of 4mg. ESI-MS (m/z) 1412.2[ M ] M] ⁺ 。

Example 7

Synthesis of 4- ((2,3-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 7)

Using a similar procedure as described in step six of example 1, substituting 2,3-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in the amount of 5mg. ESI-MS (m/z) 1430.2[ M ], [ M] ⁺ 。

Example 8

Synthesis of 4- ((2,5-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 8)

Using a similar procedure as described in step six of example 1, substituting 2,5-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in 6mg. ESI-MS (m/z) 1430.2[ 2 ] M] ⁺ 。

Example 9

Synthesis of 4- ((2,6-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 9)

Using a similar procedure as described in step six of example 1, substituting 2,6-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in 6mg. ESI-MS (m/z) 1430.2[ M ], [ M] ⁺ 。

Example 10

Synthesis of 4- ((3,4-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 10)

Using a similar procedure as described in step six of example 1, substituting 3,4-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in the amount of 5mg. ESI-MS (m/z) 1430.2[ M ], [ M] ⁺ 。

Example 11

Synthesis of 4- ((2,3-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) quinuclidin-1-ium bromide (Compound 11)

The method comprises the following steps:

synthesis of 1- (4- (tert-butoxy) -4-oxobutyl) -4- (ethoxycarbonyl) quinuclidin-1-ium bromide (Compound 11-2)

Compound 11-1 (500mg, 2.7 mmol) was dissolved in methylene chloride (5 mL) at room temperature, and tert-butyl 4-bromobutyrate (602mg, 2.7 mmol) was added to the reaction mixture. After the addition was completed, the mixture was stirred at room temperature overnight, and the solvent was distilled off under reduced pressure to obtain the title compound 1100mg as a pale yellow solid which was used in the next reaction without purification. ESI-MS (m/z) 326.2[ 2 ], [ M + H ]] ⁺ 。

Step two:

synthesis of 1- (3-carboxypropyl) -4- (ethoxycarbonyl) quinuclidin-1-ium bromide (Compound 11-3)

Compound 11-2 (200mg, 0.44mmol) was dissolved in dioxane hydrochloride at room temperatureTo the solution (4M, 3mL), the reaction solution was stirred for 2h at room temperature. The solvent was distilled off under reduced pressure, and recrystallized from ether to yield the title compound (150 mg). ESI-MS (m/z) 270.2[ 2 ] M] ⁺ 。

Step three:

synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- (ethoxycarbonyl) quinuclidin-1-ium bromide (Compound 11-4)

(S) -2- ((S) -2-amino-3-methylbutyrylamino) -N- (4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutyrylamino) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) -5-ureidopentanamide (300mg, 0.27mmol) and compound 11-3 (131mg, 0.33mmol) were dissolved in N, N-dimethylformamide (3 mL) at room temperature, cooled to 0 ℃ and N, N-diisopropylethylamine (105mg, 0.81mmol) and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (281mg, 0.54mmol) were added successively and the reaction stirred at room temperature for 3H. Preparative liquid phase purification gave 210mg of the title compound. ESI-MS (m/z) 1358.2[ M ], [ M] ⁺ 。

Step four:

synthesis of 4-carboxy-1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamide) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) quinuclidin-1-ium bromide (Compound 11-5)

Compound 11-4 (210mg, 0.14mmol) was dissolved in a mixed solvent of tetrahydrofuran (5 mL) and water (5 mL), and lithium hydroxide (36mg, 1.5mmol) was added thereto, followed by stirring at room temperature for 2.0 hours. Adding hydrochloric acid for dissolvingThe solution (0.5 mol/L) was adjusted to pH =3 to 4, and a part of the solvent was distilled off under reduced pressure to prepare a liquid phase, which was purified to obtain 140mg of the objective compound. ESI-MS (m/z) 1330.2[ M ], [ m] ⁺ 。

Step five:

synthesis of Compound 11

Compound 11-5 (58mg, 0.04mmol) was dissolved in N, N-dimethylformamide (2 mL), protected with nitrogen, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (11mg, 0.06mmol) and 4-dimethylaminopyridine (15mg, 0.12mmol) were added to the reaction solution, cooled to 0 deg.C, 2,3-difluorophenol (52mg, 0.4mmol) was added thereto, warmed to room temperature, and reacted overnight. Purification by preparative liquid phase gave the title compound 4mg. ESI-MS (m/z) 1442.2[ M ], [ M] ⁺ 。

Example 12

Synthesis of 4- ((2,3-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1,4-dimethylpiperidin-1-ium iodide (Compound 12)

The method comprises the following steps:

synthesis of ethyl 1- (4- (tert-butoxy) -4-oxobutyl) -4-methylpiperidine-4-carboxylate (Compound 12-2)

Using a similar procedure as described in step one of example 1, compound 12-1 was substituted for compound 1-1 to give 130mg of the title compound. ESI-MS (m/z) 314.2[ 2 ], [ M + H ]] ⁺ 。

Step two:

synthesis of 1- (4- (tert-butoxy) -4-oxobutyl) -4- (ethoxycarbonyl) -1,4-dimethylpiperidin-1-ium iodide (Compound 12-3)

Similar to that described in step two of example 1 was usedOperation, compound 12-2 instead of compound 1-2, gave the title compound, which was used in the next reaction without purification. ESI-MS (m/z) 328.2[ M ], [ m/z ]] ⁺ 。

Step three:

synthesis of 1- (3-carboxypropyl) -4- (ethoxycarbonyl) -1,4-dimethylpiperidin-1-ium iodide (Compound 12-4)

Using a similar procedure as described in step three of example 1, compound 12-3 was substituted for compound 1-3 to give 100mg of the title compound. ESI-MS (m/z) 270.2[ 2 ] M] ⁺ 。

Step four:

synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -4- (ethoxycarbonyl) -1,4-dimethylpiperidin-1-ium iodide (Compound 12-5)

Using a similar procedure as described in step four of example 1, compound 12-4 was substituted for compound 1-4 to give the title compound 66mg. ESI-MS (m/z): 1360.2[ M ], [ 2 ]] ⁺ 。

Step five:

synthesis of 4-carboxy-1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-pentaureido-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1,4-dimethylpiperidin-1-ium iodide (Compound 12-6)

Using a similar procedure as described in step five of example 1, compound 12-5 was substituted for compound 1-5 to give the title compound 50mg. ESI-MS (m/z) 1332.2[ M ], [ M] ⁺ 。

Step six:

synthesis of Compound 12

By usingSimilar operation as described in example 1, step six, substituting compounds 12-6 for compounds 1-6 and 2,3-difluorophenol for 2,3,5,6-tetrafluorophenol gave the title compound 5mg. ESI-MS (m/z) 1444.2[ M ]] ⁺ 。

Example 13

Synthesis of 4- ((2,4-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 14)

Using a similar procedure as described in step six of example 1, substituting 2,4-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in the amount of 5mg. ESI-MS (m/z) 1430.2[ M ], [ M] ⁺ 。

Example 14

Synthesis of 4- ((3,5-difluorophenoxy) carbonyl) -1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutylamino) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methylpiperidin-1-ium iodide (Compound 15)

Using a similar procedure as described in step six of example 1, substituting 3,5-difluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained in the amount of 5mg. ESI-MS (m/z) 1430.2[ M ], [ M] ⁺ 。

Example 15

Synthesis of 1- (4- (((S) -1- (((S) -1- ((4- (((S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamido) -3-phenylpropanamido) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -4-oxobutyl) -1-methyl-4- ((8978 zft 8978-trifluorophenoxy) carbonyl) piperidin-1-ium iodide (Compound 20)

Using a similar procedure as described in step six of example 1, substituting 3,4,5-trifluorophenol for 2,3,5,6-tetrafluorophenol, the title compound was obtained. ESI-MS (m/z) 1448.2[ M ], [ M] ⁺ 。

Preparation of conjugates

Example 16 conjugation of Compound 2 with trastuzumab

To 1mL of a trastuzumab solution having a pH of 7.5 and a concentration of 18mg/ml, a 6-fold amount of compound 2 dissolved in N, N-Dimethylacetamide (DMA) was added under stirring, and the coupling reaction was performed for 4 hours at room temperature with exclusion of light. The components with different peak time are enriched by HIC-HPLC to obtain specific conjugates with drug/antibody ratio (DAR) of 1 and 2, which are named as 2-H1-1 and 2-H1-2 respectively.

Example 17 conjugation of Compound 7 with trastuzumab

Using a coupling procedure similar to that of example 16, compound 2 was replaced by compound 7 and the fractions with different peak times were enriched by HIC-HPLC to give specific conjugates with DAR values of 1 and 2, designated 1-H1-1 and 1-H1-2, respectively.

Example 18 conjugation of Compound 8 with trastuzumab

Using a coupling procedure similar to that of example 16, compound 2 was replaced with compound 8 and the fractions with different peak times were enriched by HIC-HPLC to give specific conjugates with DAR values of 1 and 2, designated 1-H1-1 and 1-H1-2, respectively.

Example 19 conjugation of Compound 9 with trastuzumab

Using a coupling procedure similar to that of example 16, compound 2 was replaced with compound 9 and the fractions with different peak times were enriched by HIC-HPLC to give specific conjugates with DAR values of 1 and 2, designated 1-H1-1 and 1-H1-2, respectively.

Example 20 conjugation of Compound 10 with trastuzumab

Using a coupling procedure similar to that of example 16, compound 2 was replaced with compound 10 and the fractions with different peak times were enriched by HIC-HPLC to give specific conjugates with DAR values of 1 and 2, designated 1-H1-1 and 1-H1-2, respectively.

Example 21 conjugation of Compound 11 with trastuzumab

Using a coupling procedure similar to that of example 16, compound 2 was replaced with compound 11 and the fractions with different peak times were enriched by HIC-HPLC to give specific conjugates with DAR values of 1 and 2, designated 11-H1-1 and 11-H1-2, respectively.

Example 22 analysis of conjugates Using Hydrophobic Interaction Chromatography (HIC)

The reaction was monitored by HIC-HPLC and the conjugate was tested for HIC.

HIC conditions were as follows:

liquid chromatography column: TOSOH TSKgel Butyl-NPR,4.6x100mm

Mobile phase A:1.5M ammonium sulfate

Mobile phase B:25mM Na ₂ HPO ₄ pH 7.0, 25% isopropanol

Flow rate: 0.5ml/min

Detection wavelength: 280nm

Column temperature: 30 deg.C

Temperature of the sample chamber: 8 deg.C

Elution conditions:

time (minutes)	0	3	25	30	30.1	35
							Mobile phase A (% by volume)	70	70	55	15	70	70
Mobile phase B (% by volume)	30	30	45	85	30	30

HIC-HPLC analysis was performed on samples of the naked antibody and compounds 2, 7, 8, 9, 10 and 11 conjugated to the antibody, and the spectra were shown in FIGS. 1 to 7. As can be seen from FIG. 1, the retention time of the naked antibody is about 8.00 min. As can be seen from the overall analysis of FIGS. 2 to 7, the amount of residual naked antibody in the coupled system is small, and a plurality of conjugate peaks appear after the naked antibody peaks, which proves that the bioactive molecule and the antibody are efficiently coupled.

EXAMPLE 23 determination of molecular weight

LCMS molecular weight analysis was performed on the coupled samples.

The measurement conditions were as follows:

liquid chromatography column: ACQUITU

Protein BEH C4 1.7μm 2.1mm x 100mm

A mobile phase A:0.1% Formic Acid (FA)/98% ₂ O/2% Acetonitrile (ACN)

And (3) mobile phase B:0.1% of FA/2%H ₂ O/98％ACN

Flow rate: 0.25ml/min

Temperature of the sample chamber: 8 deg.C

Time (minutes)	1	7	8	9	13
						Mobile phase a (% by volume)	90	20	20	90	90
Mobile phase B (% by volume)	10	80	80	10	10

Mass spectrum: triple TOF 5600

GS1 60；GS2 60；CUR30；TEM600；ISVF5000；DP300；CE10m/z 600-5000

The results are as follows: the theoretical molecular weights calculated after coupling compound 2 and compound 7 to the antibody were as follows:

sugar type	mAb	DAR1	DAR2	DAR3	DAR4
						G0F/G0F	148057.8	149359.5	150661.1	151962.8	153264.5
G0F/G1F	148220.0	149521.6	150823.3	152124.9	153426.6
						G0F/G2F	148382.1	149683.8	150985.4	152287.1	153588.7

In the table, mAb represents an antibody, DAR1 represents a conjugate comprising one biologically active molecule and one antibody, DAR2 represents a conjugate comprising two biologically active molecules and one antibody, DAR3 represents a conjugate comprising three biologically active molecules and one antibody, DAR4 represents a conjugate comprising four biologically active molecules and one antibody; the glycoform represents a sugar chain structure on both heavy chains, G0F represents fucosylated galactose-free, G1F represents fucosylated monogalactose, and G2F represents fucosylated digalactose.

LCMS molecular weight analysis was performed on samples of compound 2 and compound 7 after conjugation with the antibody, and the spectra obtained are shown in fig. 8 and fig. 9. As can be seen from FIGS. 8 and 9, both Compound 2 and Compound 7 were successfully conjugated to the antibody, and both DAR1 and DAR2 were produced as the conjugation products.

Example 24 coupling efficiency test

The coupling efficiency was calculated from the results of the analysis of the conjugate by Hydrophobic Interaction Chromatography (HIC), and the coupling efficiency of compounds 2, 7, 8, 9, 10 and 11 having a novel carboxyl activating group to the antibody was compared with that of a control compound using a coupling technique in which pentafluorophenol activates the carboxyl group to the antibody, and the results are shown in Table 1. Where coupling efficiency refers to the molar percentage of conjugates with the corresponding DAR value over the whole (total conjugates and naked antibody).

The structure of the control compound used in the coupling technique using pentafluorophenol to activate the carboxyl group is as follows:

TABLE 1 comparison of coupling efficiencies

As can be seen from Table 1, conjugates comprising a biologically active molecule and one or two antibodies can be obtained using the compounds of the present invention having a novel carboxyl activating group. The compound with the new carboxyl activating group obviously improves the coupling efficiency of the bioactive molecules and the antibody by more than 20 times, preferably more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times or more than 80 times, and successfully overcomes the problem that the bioactive molecules are difficult to couple with the antibody when the connecting group contains a quaternary ammonium salt or a nitric oxide structure.

Biological experiments

Example 25 detection of the inhibitory Effect of antibody-drug conjugates on in vitro cellular Activity

Tumor cells HCC1954 were first cultured in RPMI1640+10% FBS. HCC1954 is a Her2 positive cell and has endocytosis to a conjugate (e.g., an anti-Her 2 antibody trastuzumab-drug conjugate). The conjugates (starting at 1. Mu.g/mL, 2-fold diluted, 10 concentration gradients diluted) were diluted with the corresponding assay medium (containing 2% FBS), the tumor cells were digested by conventional methods using pancreatin, the tumor cells were collected, and resuspended with the corresponding assay medium (containing 2% FBS). The diluted conjugate was added to a 96-well plate, and the resuspended cells were added to the corresponding wells containing the conjugate (10000 cells/well), and incubated for 3 days. Then, 20. Mu.L of CCK8 reagent (Donnell chemical Co., ltd.) was added to each well, and the reaction was carried out for 1.5 hours, and the inhibition of cell proliferation by the antibody-drug conjugate was evaluated by detecting the activity of dehydrogenase in vivo using a microplate reader reading at 450nm (manufacturer: molecular Devices, model: spectraMax M2).

The results of the experiment are shown in table 2.

TABLE 2

ADC name	EC ₅₀ (ng/mL)
		1-H1-2	13.48±0.45

EC of conjugate 1-H1-2 of the invention ₅₀ 13.48. + -. 0.45ng/mL. EC of marketed Kadcyla (trastuzumab-maytansine alkaloid T-DM1, i.e., trastuzumab-DM 1 conjugate) ₅₀ 43ng/mL (see Howard A. Burris III et al, clinical Breast Cancer, vol.11, no.5,275-82). It can be seen that the conjugates of the invention have a stronger inhibitory activity on cell proliferation than Kadcyla.

While the invention has been illustrated by the foregoing specific embodiments, it should be understood that it is not to be construed as being limited thereby. The present invention covers the general aspects previously disclosed, and various modifications or changes in detail thereof may be made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

Claims

1. A process for the preparation of a conjugate of formula (II),

the method comprises reacting a compound of formula (I) with a compound containing one or more amino groups (-NH) ₂ ) The target-oriented drug coupling of (2),

wherein:

t is the following structure:

L ₁ is composed of

The group is linked to T via the 1-labelled position and L via the 2-labelled position ₂ Connecting;

L ₂ is composed of

The radical being bound to L via one of the two positions marked by 1 or 2 ₁ Is connected to L via another position ₃ Connecting;

L ₃ selected from:

the radical being bound to R via one of the two positions marked by 1 or 2 ₁ Is connected to L via another position ₂ Connecting;

wherein:

e is a counterion;

R ₁ selected from optionally substituted by one or more R _b Substituted of the following groups: c _1-6 Alkylene radical, C _3-10 Cycloalkylene radical, C _6-10 Arylene and 5-10 membered heteroarylene,wherein R is _b Is H (hydrogen), D (deuterium), halogen, cyano, nitro, trifluoromethyl, COOH or SO ₃ H；

Z is an oxygen atom;

R ₂ selected from the group consisting of _a Substituted by

And R is _a Is fluorine, and R ₂ Phenyl which is not perfluorinated;

a is a group obtained after alpha amino groups are removed from a targeted drug, and the targeted drug is trastuzumab;

m and n are 1, and r is 0; and is

Alpha is selected from 1 or 2.

2. The method of claim 1, wherein E is a halide anion.

3. The method of claim 2, wherein E is chloride, bromide, or iodide.

4. The method of claim 1, wherein L ₃ Position marked by 1 and R ₁ Is connected and passes the position marked by 2 and L ₂ And (4) connecting.

5. The method of claim 1, wherein

Selected from:

6. The method of claim 1, wherein R ₂ Is benzene substituted by four fluorine atomsA phenyl group substituted by three fluorine atoms, a phenyl group substituted by two fluorine atoms or a phenyl group substituted by one fluorine atom.

7. The method of claim 6, wherein R ₂ Selected from:

8. the method of claim 7, wherein R ₂ Is 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl or 2,6-difluorophenyl.

9. The method of claim 1, wherein

Selected from:

10. the method of claim 9, wherein

Selected from:

11. the method of claim 10, wherein

Selected from:

12. the method of claim 1, wherein the compound of formula (I) is selected from:

13. the method of any one of claims 1-12, comprising mixing a targeted drug with the compound of formula (I).

14. The method of claim 13, wherein the molar ratio of the targeting drug to the compound of formula (I) is 1 (1-20).

15. The method of claim 13, wherein the method comprises mixing a solution comprising a targeted drug with a compound of formula (I).

16. The process of claim 13, wherein the process is carried out in water or an organic solvent.

17. The method of claim 16, wherein the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons, halogenated hydrocarbons, ethers, nitriles, alcohols, and any combination thereof.

18. The process of claim 17 wherein the saturated hydrocarbon is cyclohexane or hexane;

and/or

The halogenated hydrocarbon is dichloromethane, chloroform or 1,2-dichloroethane;

and/or

The ether is tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxyethane;

and/or

The nitrile is acetonitrile;

and/or

The alcohol is methanol or ethanol.

19. The method of claim 13, wherein the method further comprises purification by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, and affinity chromatography.

20. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof,

wherein the conjugate is:

wherein A1 is a group obtained by removing 2 amino groups from trastuzumab.

21. A process for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, which comprises reacting a compound of formula (I-0) with R ₂ -ZH reaction to give the compound of formula (I),

wherein each group is as defined in any one of claims 1 to 13.

22. A compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein each group is as defined in any one of claims 1 to 13.

23. A compound according to claim 22, or a pharmaceutically acceptable salt thereof, prepared by a process according to claim 21.

24. The compound of claim 22, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the structure of formula (Ia),

25. the compound of claim 22, or a pharmaceutically acceptable salt thereof, selected from:

26. a pharmaceutical composition comprising a conjugate prepared by the process of any one of claims 1-19, the conjugate of claim 20, or a pharmaceutically acceptable salt of the conjugate, and one or more pharmaceutically acceptable carriers.

27. A pharmaceutical formulation comprising a conjugate prepared by the process of any one of claims 1 to 19, the conjugate or a pharmaceutically acceptable salt of the conjugate of claim 20, or the pharmaceutical composition of claim 26.

28. Use of a conjugate prepared by the method of any one of claims 1-19, the conjugate or a pharmaceutically acceptable salt of the conjugate of claim 20, the pharmaceutical composition of claim 26, or the pharmaceutical formulation of claim 27 in the manufacture of a medicament for the prevention or treatment of a HER 2-associated cancer disease.

29. The use of claim 28, wherein:

the cancer disease is selected from esophageal cancer, lung cancer, squamous cell cancer, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, salivary gland cancer, colon cancer, colorectal cancer, liver cancer, kidney cancer, prostate cancer and thyroid cancer.

30. The use of claim 29, wherein:

the esophageal cancer is esophageal adenocarcinoma or esophageal squamous cell carcinoma;

and/or

The lung cancer is small cell lung cancer or non-small cell lung cancer.