TWI750194B - Egfr antibody-cytotoxic drug conjugates and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to EGFR antibody-cytotoxic drug conjugates and pharmaceutical use thereof. In particular, the present invention relates to an EGFR antibody variant-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate compound thereof, and pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt or solvate compound thereof, and use thereof as anti-cancer agents, especially in the preparation of a medicament for the treatment of EGFR-mediated disease or symptom.

Description

EGFR antibody-drug conjugate and its application in medicine

The present invention relates to a novel structure of antibody-drug conjugates. Specifically, the present invention relates to an EGFR antibody variant-cytotoxic drug conjugate, a pharmaceutical composition comprising the conjugate, and the application of the conjugate or the pharmaceutical composition in medicine.

Epidermal growth factor receptor (EGFR) is a huge transmembrane glycoprotein with a molecular weight of about 170KDa, which belongs to a member of the ErbB receptor family. The EGFR receptor itself is a tyrosine kinase. When combined with ligands such as EGF, TNF-a, etc., it can form a dimer and activate downstream signals (such as MAPK, PI3K, Stat, etc.) by transmitting phosphorylation. Thereby maintaining cell growth and promoting cell division and proliferation. Due to the conservation of ErbB family receptors, EGFR can also form heterodimers with other family proteins (such as Her2, Her3, Her4), thereby regulating cell growth more broadly.

Gene mutation or overexpression of EGFR is commonly found in a variety of epidermal cell-derived malignancies, such as head and neck cancer, colorectal cancer, lung cancer, pancreatic cancer, skin cancer, etc., and is considered to be the driver gene leading to these cancers. Therefore, the research and development of anticancer drugs targeting EGFR has always been a hot spot in the medical field. Currently So far, small molecule tyrosine kinase inhibitors of EGFR (such as Gefitinib, Erlotinib) and EGFR monoclonal antibodies (such as Cetuximab, Panitumumab) (Panitumumab), Nimotuzumab (Nimotuzumab, EP0699755, EP0712863), etc. have all been listed, and have achieved significant clinical effects. However, these EGFR-targeted drugs are not perfect, and they are only suitable for small patient groups. For example, small molecule inhibitors are usually only effective for lung cancer patients with specific EGFR mutations, and after a period of use, drug resistance often occurs; EGFR monoclonal antibodies are usually only for downstream KRAS, BRAF, PIK3CA and other genes without mutations. Colorectal cancer, head and neck cancer and other patient groups are more effective. Not only that, these drugs also partially inhibit the normal physiological function of EGFR in ordinary cells, which is often accompanied by target-related side effects such as rash and diarrhea. Therefore, whether we can find a new type of anticancer drug that can more effectively target EGFR to overcome the drug resistance caused by the existing EGFR targeted therapy, expand the indications, and reduce the side effects related to normal tissue targets is a difficult problem that we urgently need to overcome. . In addition, the stability of the antibody and the drug activity are also very important. It has been reported in the literature that the stability of the antibody or ADCC and CDC activities can be improved by the modification of the antibody constant region and framework region, such as CN1237076C, EP2203180 and so on.

Antibody-Drug Conjugate or ADC, commonly known as "bio-missile", is a kind of using the specificity of monoclonal antibody to accumulate ADC molecules in the tumor microenvironment, and mediated by antibody-tumor antigen Endocytosis is a revolutionary technology for the targeted delivery of toxin molecules into the interior of cancer cells to kill cancer cells. Compared with traditional chemotherapy, the amount of toxin molecules used by ADC is greatly reduced, and more toxin molecules are accumulated in the lesions, which have side effects on normal tissues. In addition, ADC mainly uses toxin molecules to directly bind to microtubule bundles to inhibit the division and proliferation of cancer cells, which is different from the mechanism of action of monoclonal antibodies by inhibiting receptor signaling pathways. Therefore, downstream signaling proteins Whether there is a genetic mutation will not have much impact on the effect of ADC on inhibiting cancer cells, so theoretically, ADC can have a wider range of indications than traditional therapy, and the efficacy is more powerful. . References to ADCs include WO2007008603, WO2013173393, WO2005081711, WO2013173391, WO2013173392, WO2013173393, WO2012010287 and so on.

The inventor's previous application PCT/CN2016/072129 relates to a new class of toxin molecules and conjugates, but does not disclose specific EGFR antibody-related conjugates.

There is still a need to find antibody conjugates with significant drug efficacy and less toxic and side effects; at the same time, reducing the frequency of administration, improving compliance, and reducing treatment costs while achieving the same drug efficacy is also an important problem to be solved in the art.

In order to improve the coupling effect of ligands, especially antibodies and drugs, the present invention provides an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof:

Wherein: L ₁ , L ₂ are linker units; y is 1-8, preferably 2-5; Ab is an anti-EGFR antibody or an antigen-binding fragment thereof, and the anti-EGFR antibody or an antigen-binding fragment thereof comprises the following CDR regions: LCDR1: RSSQNIVHSNGNTYLD SEQ ID NO: 7; LCDR2: KVSNRFS SEQ ID NO: 8; LCDR3: FQYSHVPWT SEQ ID NO: 9; HCDR1: NYYIY SEQ ID NO: 10; HCDR2: GINPTSGGSNFNEKFKT SEQ ID NO: 11; HCDR3: QGLWFDSDGRGFDF; SEQ ID NO: 12; the Ab has three site mutations of M258Y/S260T/T262E (YTE) on the heavy chain.

In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-EGFR antibody or antigen-binding fragment thereof is human Antibody or fragment thereof.

In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein the humanized antibody heavy chain variable region is further The heavy chain FR region comprising human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human IgG1 heavy chain FR region, and/or the light chain variable region of a humanized antibody further comprises a human kappa chain Or the light chain FR region of a human kappa chain variant, or the light chain FR region of a human lambda chain or a human lambda chain variant.

In a preferred embodiment of the present invention, a general formula (I) Antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain constant region of the humanized antibody comprises human IgG1 or a variant thereof, a human IgG2 or a variant thereof, a human IgG3 or Its variant or the constant region of human IgG4 or its variant, preferably comprising the constant region of human IgG1 or its variant, and/or the light chain of the humanized antibody further comprises human κ chain or human κ The light chain constant region of a chain variant, or the light chain constant region of a human lambda chain or a human lambda chain variant.

SEQ ID NO：3；重鏈

SEQ ID NO：4。 In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, wherein the anti-EGFR antibody comprises: a light chain

SEQ ID NO: 3; Heavy chain

SEQ ID NO:4.

其中X₁選自氫原子、鹵素、羥基、氰基、烷基、烷氧基和環烷基；X₂選自烷基、環烷基和雜環基；m為0-5，較佳為1-3；S為硫原子。 In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein L _{2 is} as represented by the following general formula (L ₂ ) Show:

Wherein X _{1 is} selected from hydrogen atom, halogen, hydroxyl, cyano group, alkyl group, alkoxy group and cycloalkyl group; X _{2 is} selected from alkyl group, cycloalkyl group and heterocyclic group; m is 0-5, preferably 1-3; S is a sulfur atom.

In a preferred embodiment of the present invention, a compound _{represented by the general formula (L 2} ) preferably has the following structure:

其中X₃為烷基，該的烷基視需要進一步被鹵素、羥基、氰基、烷基的取代基所取代；n為0-5，較佳為1-3。 In a preferred embodiment of the present invention, a compound represented by general formula (I) or a pharmaceutically acceptable salt or solvent compound thereof, wherein L1 is a compound represented by the _{following general formula (L 1 ):}

Wherein X ₃ is an alkyl group, and the alkyl group is further substituted by halogen, hydroxyl, cyano and alkyl substituents as needed; n is 0-5, preferably 1-3.

In a preferred embodiment of the present invention, a compound _{represented by the general formula (L 1} ) preferably has the following structure:

Wherein X ₃ is an alkyl group, and the alkyl group is further substituted by halogen, hydroxyl, cyano and alkyl substituents as needed; n is 0-5, preferably 1-3.

其中，Ab、L₂、y如通式(I)中所定義。 In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof is selected from antibodies represented by general formula (II) - a drug conjugate or a pharmaceutically acceptable salt or solvate thereof:

Wherein, Ab, L ₂ and y are as defined in the general formula (I).

其中，Ab、L₁、y如通式(I)中所定義。 In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof is selected from antibodies represented by general formula (III) - a drug conjugate or a pharmaceutically acceptable salt or solvate thereof:

Wherein, Ab, L ₁ , and y are as defined in the general formula (I).

其中，mAb002包括胺基酸序列如SEQ ID NO：3所示的輕鏈，和如SEQ ID NO：4所示的重鏈，y如通式(I)中所定義。 In a preferred embodiment of the present invention, an antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof has the following general structure:

Wherein, mAb002 includes a light chain whose amino acid sequence is shown in SEQ ID NO: 3, and a heavy chain whose amino acid sequence is shown in SEQ ID NO: 4, and y is as defined in general formula (I).

Another aspect of the present invention relates to an EGFR antibody, which can be used to prepare the antibody-drug conjugate represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof, comprising a sequence such as SEQ ID NO: 7. The LCDR1, LCDR2, LCDR3 regions shown in SEQ ID NO: 8, SEQ ID NO: 9 and their variants, and as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and their variants The HCDR1, HCDR2, and HCDR3 regions shown are characterized by three site mutations of M258Y/S260T/T262E (YTE) on the heavy chain.

In a preferred embodiment of the present invention, an EGFR antibody is provided, which can be used to prepare the antibody-drug conjugate represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof, the EGFR The antibody includes a light chain whose amino acid sequence is set forth in SEQ ID NO:3, and a heavy chain whose amino acid sequence is set forth in SEQ ID NO:4.

Another aspect of the present invention relates to a pharmaceutical composition comprising the antibody-drug conjugate represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable compounds excipients, diluents or carriers.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention, in the preparation of a drug for the treatment of cancer Preferably, the cancer is a cancer with high expression of EGFR, a cancer with mutations in the EGFR polypeptide, and a cancer with mutations in the downstream genes of the EGFR signaling pathway; more preferably, gastric cancer, pancreatic cancer, liver cancer, breast cancer, Lung cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer, or head and neck cancer; preferably lung, colon, rectal, colorectal, or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same as described above, which is used in the preparation of a medicine for the treatment of mammalian cancer. Preferably, the cancer is a cancer with high expression of EGFR, a cancer with mutations in the EGFR polypeptide, and a cancer with mutations in the downstream genes of the EGFR signaling pathway; more preferably, gastric cancer, pancreatic cancer, liver cancer, breast cancer, Lung cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, tuberculosis Rectal or head and neck cancer; preferably lung, colon, rectal, colorectal, or head and neck cancer.

The present invention further provides a method for treating cancer in a mammal, the method comprising administering to the mammal an effective amount of the antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; the cancer is preferably a cancer that highly expresses EGFR, a cancer with mutations in the EGFR polypeptide, and a cancer with mutations in genes downstream of the EGFR signaling pathway; more Preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer; Rectal, colorectal, or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention, in the preparation of a drug for the treatment of cancer Preferably, the cancer is a cancer with a mutation in an EGFR polypeptide, and the EGFR mutant polypeptide comprises at least one EGFR mutation selected from the group consisting of: L858R, L858R/T790M, L858R/T790M/C797S, Del19, Del19/T790M , Del19/T790M/C797S, T790M, G719X, L861Q, S768I, exon (Exon) 18 indel/E709X, exon 19 insertion (insertion), exon 20 insertion, A763_Y764insFQEA, exon 18-25 repeat (duplication) and EGFR-RAD51 rearrangement; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer with mutations in EGFR polypeptide cancer, rectal cancer, colorectal cancer, or head and neck cancer; most preferably lung, colon, rectal, Colorectal or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same as described above, which is used in the preparation of a medicine for the treatment of mammalian cancer. Preferably, the cancer is a cancer with a mutation in an EGFR polypeptide, and the EGFR mutant polypeptide comprises at least one EGFR mutation selected from the group consisting of: L858R, L858R/T790M, L858R/T790M/C797S, Del19, Del19/T790M , Del19/T790M/C797S, T790M, G719X, L861Q, S768I, exon 18 indel/E709X, exon 19 insertion, exon 20 insertion, A763_Y764insFQEA, exon 18-25 duplication and EGFR-RAD51 rearrangement ; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer with mutations in the EGFR polypeptide; The best are lung, colon, rectal, colorectal, or head and neck cancer.

The present invention further provides a method for treating cancer in a mammal, the method comprising administering to the mammal an effective amount of the antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; preferably, the cancer is a cancer with a mutation in an EGFR polypeptide, and the EGFR mutant polypeptide comprises at least one EGFR mutation selected from the group consisting of: L858R, L858R /T790M, L858R/T790M/C797S, Del19, Del19/T790M, Del19/T790M/C797S, T790M, G719X, L861Q, S768I, exon 18 indel/E709X, exon 19 insertion, exon 20 insertion, A763_Y764insFQEA , exon 18-25 repeats and EGFR-RAD51 Rearranged; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer, or head and neck cancer with mutations in the EGFR polypeptide Cancer; preferably lung, colon, rectal, colorectal, or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention, which is used in the preparation of a drug for the treatment of cancer. Preferably, the cancer is a cancer with mutations in the downstream genes of the EGFR signaling pathway, and the downstream genes at least include one selected from the following genes Ras, B-Raf, PI3K, wherein the downstream gene mutations at least include selected from the following Mutations of: K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110 γ E1021K; preferably downstream of EGFR signaling Gastric, pancreatic, liver, breast, lung, bowel, kidney, melanoma, non-small cell lung, colon, rectal, colorectal, or head and neck cancers with genetic mutations; preferably lung, colon cancer, rectal cancer, colorectal cancer, or head and neck cancer.

The present invention further provides an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising it as described above, which is used in the preparation of an anti-cancer drug for the treatment of mammalian cancer. Use of the drug; preferably, the cancer is a cancer with mutations in the downstream genes of the EGFR signaling pathway, and the downstream genes at least include one selected from the following genes Ras, B-Raf, PI3K, wherein the downstream gene mutations at least include selected from the following Mutations of: K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110 γ E1021K; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, Colorectal or head and neck cancer; preferably lung, colon, rectal, colorectal, or head and neck cancer.

The present invention further provides a method for treating cancer in a mammal, the method comprising administering to the mammal an effective amount of the antibody-drug conjugate represented by general formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; wherein the mammal is preferably a human; preferably, the cancer is a cancer with mutations in the downstream genes of the EGFR signaling pathway, and the downstream genes at least comprise one selected from the following genes Ras, B-Raf , PI3K, wherein the downstream gene mutation at least comprises a mutation selected from the group consisting of: K-Ras G12V, K-Ras G13D, N-Ras Q61K, H-Ras G12S; B-Raf V600E, B-Raf G468A; PIK3CA H1047R, PIK3CB E633K and p110 γ E1021K; more preferably gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, rectal cancer, Colorectal or head and neck cancer; preferably lung, colon, rectal, colorectal, or head and neck cancer.

[Detailed description of the invention]

Unless otherwise defined, all technical and scientific terms used herein are the same as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. In describing and claiming the present invention, the following terms are used in accordance with the following definitions.

When a trade name is used in the present invention, the applicant intends to include the trade name The formulation of the product, the generic and active drug portions of the trade name product.

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms An alkyl group of carbon atoms, preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl , 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl- 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl , 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethyhexyl Methylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl , 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4 -Ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3 -Ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof, etc. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, th Dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl , n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2, 2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3- Dimethylbutyl, etc. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from the group consisting of alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, ring Alkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, keto.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 20 carbon atoms. 10 carbon atoms, preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2) heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio.

The term "bond" refers to a covalent bond represented by "-".

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

The term "pharmaceutically acceptable salts" refers to the salts of the antibody-drug conjugates of the present invention, such salts are safe and effective when used in mammals, and have due biological activity, the antibodies of the present invention -Drug conjugates contain at least one amine group and thus can form salts with acids, pharmaceutically acceptable Non-limiting examples of salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, Pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, Ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

The term "solvate" refers to the formation of a pharmaceutically acceptable solvate of the antibody-drug conjugates of the invention with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, acetic acid ethyl ester.

The term antibody-drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxin via a stable chemical linker compound.

The term "antigen or receptor" refers to presence on a target cell for a ligand to recognize and bind to the target cell. Cell surface antigens or receptors expressed on target cells and/or tissues in proliferative diseases such as cancer are preferred in the present invention.

"Antibody" as used herein refers to any form of antibody that exhibits the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, full-length antibodies, antibody-binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies (eg, bispecific antibodies).

The term "full-length antibody" refers to an immunoglobulin molecule (eg, IgM) comprising 4 polypeptide chains, ie, 2 heavy chains and 2 light chains, which are cross-linked to each other by disulfide bonds to form multimers. Each heavy chain contains a heavy chain variable region (referred to as VH) and Heavy chain constant region, the heavy chain constant region contains 3 domains: CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated as VL) and a light chain constant region, and the light chain constant region includes one structural domain (CL1). The VH and VL regions can be further divided into hypervariable regions, termed complementarity determining regions (CDRs), with more conserved domains interspersed between them, called framework regions (FRs).

The term "antigen-binding fragment or derivative" includes any naturally-occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that can specifically bind to an antigen to form a complex; usually At least a portion of the antigen-binding or variable region (eg, one or more CDRs) of the present antibody retains at least some of the binding specificity of the parent antibody. "Antibody-binding fragments or derivatives" may be derived from antibodies, for example, by suitable standard techniques including proteolytic or recombinant genetic engineering techniques (including manipulation and expression of DNA expressing antibody variable and partial constant regions) against The full length of the body is modified. "Antigen-binding fragments or derivatives" include, but are not limited to: (i) Fab fragments; (ii) F(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) ); (vi) a dAb fragment; and (vii) a minimal recognition unit (eg, a separate complementarity determining region (CDR)) that mimics the amino acid residues of the hypervariable region of an antibody. Other engineered molecules such as diabodies, tribodies, tetrabodies, and minibodies are also within the scope of "antigen-binding fragments or derivatives."

The term "EGFR" according to the invention especially refers to human epidermal growth factor receptor 1, also known as ErbB-1 or HER1. EGFR is a receptor tyrosine kinase comprising an extracellular ligand-binding domain, a membrane-spanning domain and an intracellular kinase domain. Binds to its ligands such as epidermal growth factor (EGF) and transforming Following growth factor alpha (TGF alpha), EGFR forms homodimers or heterodimers with other ErbB receptors, and its kinase function is activated, resulting in the autophosphorylation of several tyrosines in the intracellular domain change. Anti-EGFR antibodies are antibodies that specifically bind to EGFR. In certain embodiments, anti-EGFR antibodies can interfere with or inhibit the activation of EGFR, eg, by preventing ligand binding and/or receptor dimerization. Activation of EGFR can transmit signals to downstream effectors, including PI3-K, RAS-RAF-MAPK P44/P42 and protein kinase C signaling pathways, and finally transmit signals to the nucleus to regulate cell proliferation.

The antibody of the present invention is preferably a specific antibody against cell surface antigens on target cells, and a non-limiting example is Nimotuzumab (trade name Taixinsheng), an epidermal growth factor receptor (EGFR) as the target of the monoclonal antibody drug, can be used for the treatment of malignant tumor humanized monoclonal antibody. EGFR is overexpressed in a variety of solid tumors, such as head and neck cancer, lung cancer, and colorectal cancer.

The term "Ras gene" refers to the human Ras gene family, which includes three functional genes, namely H-ras, K-ras and N-ras genes (Adjei, 2001, J. Nat. Cancer Instit. 93: 1062 -1073), all contain 1 5' non-coding exon and 4 coding exons, the relative molecular quality of the encoded products is G protein monomer of 21 000, these proteins have purine triphosphate binding sites, It plays an important role in many various intracellular signaling pathways.

The term "Raf gene" refers to the Raf family, which includes three cellular homologs, referred to as A-raf, B-raf and C-raf (also referred to as raf-1). This gene encodes a highly conserved serine-threonine-specific protein kinase, which is one of the effectors of Ras and is involved in the transduction of signals regulating cell proliferation. plays an important regulatory role in the induction process. Among them, B-Raf has the complete amino acid sequence shown in Gene Accession No. NP_004324.

The term "PI3K gene" refers to the phosphoinositide-3-kinase gene (phosphoinositide-3-kinase, referred to as PI3K, also known as phosphoinositide-4,5-bisphosphate 3-kinase), which belongs to the proto-oncogene, In the present invention, "PI3K" includes all members of the PI3K family including class IA (eg PI3K alpha, beta and delta ), class IB (eg PI3K gamma), class II (eg PI3KC2 alpha, beta and gamma) and class III (eg Vps34 yeast homolog).

The term "cytotoxic drug" refers to a chemical molecule that has a strong ability to disrupt normal growth within tumor cells. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, when killing tumor cells, they can also lead to apoptosis of normal cells, resulting in serious side effects.

The term "linker unit" in the present invention is L1 and L2, and refers to a chemical structural fragment or bond that is covalently linked to an antibody at one end and a cytotoxic drug at the other end.

The term "drug loading" refers to the average amount of cytotoxic drug loaded on each ligand in the molecule, and can also be expressed as the ratio of drug to antibody, and the drug loading can range from each ligand (Pc) 1-8 cytotoxic drugs are linked, in the embodiment of the present invention, the drug loading is represented as y, and each ADC molecule after the coupling reaction can be characterized by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC. average number of drugs.

In the present invention, y may be limited by the number of attachment sites. this invention In one embodiment, the cytotoxic drug is coupled to the N-terminal amine group of the ligand and/or the ε-amine group of the lysine residue through a linker unit, generally, it can be coupled to the antibody in a coupling reaction. The number of drug molecules will be less than the theoretical maximum.

The loading of the antibody cytotoxic drug conjugate can be controlled by the following non-limiting methods, including: (1) controlling the molar ratio of the linking reagent and the mAb, (2) controlling the reaction time and temperature, (3) selecting different Reaction reagents.

The term "carrier" used for the drug of the present invention refers to a system capable of changing the way the drug enters the human body and its distribution in the body, controlling the release rate of the drug, and delivering the drug to the targeted organs. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure. Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.

The term "excipient" refers to an addition other than the main drug in a pharmaceutical preparation, and may also be called an adjuvant. Such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in ointments and creams of semi-solid preparations; preservatives, antioxidants, flavoring agents, fragrances, and cosolvents in liquid preparations , emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can be called excipients.

The term "diluent" is also known as filler, its main purpose is to increase the weight and volume. The addition of the diluent not only ensures a certain volume, but also reduces the dosage deviation of the main components and improves the compression molding of the drug. When the medicine in the tablet contains oily components, it is necessary to add an absorbent to absorb the oily substances, so as to keep the "dry" state, so as to facilitate the tableting. Such as starch, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

Pharmaceutical compositions may be in the form of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oily phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then processed into a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the patient by local bolus injection. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous drug delivery device can be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. In addition, sterile fixed oils are conveniently employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can also be used in the preparation of injectables.

Figure 1 shows the mean pharmacokinetics of the animals in the two dose groups after a single intravenous injection of 5 mg/kg of nimotuzumab (mAb001) and the IgG1-YTE variant of nimotuzumab (mAb002) in cynomolgus monkeys Parametric graph.

Figure 2 is a graph comparing the in vivo pharmacodynamics of ADC-9 of the present invention relative to ADC-8 and naked anti-mAb002 in nude mouse lung cancer xenograft HCC827 model.

The present invention is further described below in conjunction with the examples, but these examples do not limit the scope of the present invention.

The experimental methods that do not specify specific conditions in the examples of the present invention generally follow conventional conditions or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). NMR shifts ([delta]) are given in units of ^{10<"6> (ppm).} NMR was measured by Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO- d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), and the internal standard was four Methylsilane (TMS).

The MS was measured with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

The determination of HPLC used an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6mm column).

Chiral HPLC analysis was determined using LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.).

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for TLC separation and purification products is 0.4mm ~0.5mm.

Column chromatography generally uses Yantai Huanghai silica gel 200~300 mesh silica gel as the carrier.

Chiral preparative column chromatography used Prep Star SD-1 (Varian Instruments Inc.) or SFC-multigram (Berger Instruments Inc.).

The average inhibition rate and IC ₅₀ value of kinases were measured with NovoStar microplate reader (BMG, Germany).

The known starting materials of the present invention can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co.KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals and other companies.

There is no special description in the examples, and the reactions can all be carried out in an argon atmosphere or a nitrogen atmosphere.

Argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.

Hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L.

The pressurized hydrogenation reaction uses Parr 3916EKX hydrogenation apparatus and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenation apparatus.

The hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.

The microwave reaction used a CEM Discover-S 908860 microwave reactor.

There is no special description in the examples, and the solution refers to an aqueous solution.

There is no special description in the examples, and the temperature of the reaction is room temperature, which is 20°C to 30°C.

The preparation of pH=6.5 PBS buffer in the embodiment: take KH ₂ PO ₄ 8.5g, K ₂ HPO ₄ .3H ₂ O 8.56g, NaCl 5.85g, EDTA 1.5g are placed in a bottle, set the volume to 2L, ultrasonically Make it all dissolved and shake well.

Preparation of acetic acid/sodium acetate buffer with pH=4.5 in the example: take 9g of anhydrous sodium acetate and put it in a bottle, add purified water, make up to 2L, shake well, add 4.9mL of sodium acetate, and shake well.

Preparation of phosphate buffer (pH=7.0) in the example: 0.2 M NaH ₂ PO ₄ 39 mL was added to 0.2 M Na ₂ HPO ₄ 61 mL and shaken to obtain a 0.2 M pH=7 buffer.

The monitoring of the reaction progress in the embodiment adopts thin layer chromatography (TLC), and the systems of the developing solvent used in the reaction are: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether And ethyl acetate system, D: acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound.

The eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purifying the compound include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: dichloromethane and In the acetone system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Some of the compounds of the present invention were characterized by Q-TOF LC/MS. Q-TOF LC/MS used an Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm , 2.1×75 mm column).

The known starting materials of the present invention can be synthesized by adopting or according to methods known in the art. The experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions, or in accordance with conditions suggested by raw material or commodity manufacturers. . Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1. Construction and expression of mAb001 antibody

The mAb001 antibody, Nimotuzumab (EGFR antibody), can specifically bind to the EGFR target. The antibody can be prepared according to conventional methods: for example, after constructing the vector, it can be transfected into eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), and purified and expressed.

SEQ ID NO：1；mAb001的重鏈胺基酸序列：

SEQ ID NO：2。其CDR區序列如下：LCDR1：RSSQNIVHSNGNTYLD SEQ ID NO：7；LCDR2：KVSNRFS SEQ ID NO：8；LCDR3：FQYSHVPWT SEQ ID NO：9；HCDR1：NYYIY SEQ ID NO：10；HCDR2：GINPTSGGSNFNEKFKT SEQ ID NO：11；HCDR3：QGLWFDSDGRGFDF SEQ ID NO：12。 The sequence is as follows: Light chain amino acid sequence of mAb001:

SEQ ID NO: 1; Heavy chain amino acid sequence of mAb001:

SEQ ID NO:2. Its CDR region sequence is as follows: LCDR1: RSSQNIVHSNGNTYLD SEQ ID NO: 7; LCDR2: KVSNRFS SEQ ID NO: 8; LCDR3: FQYSHVPWT SEQ ID NO: 9; HCDR1: NYYIY SEQ ID NO: 10; HCDR2: GINPTSGGSNFNEKFKT SEQ ID NO: 11 ;HCDR3:QGLWFDSDGRGFDF SEQ ID NO:12.

Example 2. Construction and expression of mAb002 antibody

The mAb002 antibody is a Nimotuzumab modified IgG1-YTE variant obtained by site mutation of Nimotuzumab.

SEQ ID NO：3；mAb002重鏈胺基酸序列：

SEQ ID NO：4。注：雙底線即代表YTE突變位點。在實施例中設計的輕重鏈DNA序列如下：mAb002輕鏈DNA序列：

SEQ ID NO：5；注：底線部分為信號肽mAb002重鏈DNA序列：

SEQ ID NO：6。注：底線部分為信號肽 Design primers PCR to build antibody VH/VK gene fragment, and then carry out homologous recombination with expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct antibody full-length expression vector VH-CH1-FC-pHr /VK-CL-pHr. The original form of the plasmid is IgG1, and the IgG1-YTE antibody form is obtained by point mutation, namely M258Y/S260T/T262E (YTE) three site mutations. The final mAb002 antibody sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. After the plasmid has been verified by sequencing, it is extracted by methods well known in the art and transiently expressed in 293 cells to obtain the culture supernatant containing the target antibody protein for separation and purification. Antibody sequence of mAb002: mAb002 light chain amino acid sequence:

SEQ ID NO: 3; mAb002 heavy chain amino acid sequence:

SEQ ID NO:4. Note: The double bottom line represents the YTE mutation site. The light and heavy chain DNA sequences designed in the examples are as follows: mAb002 light chain DNA sequence:

SEQ ID NO: 5; Note: The bottom line is the signal peptide mAb002 heavy chain DNA sequence:

SEQ ID NO:6. Note: The bottom line is the signal peptide

mAb002 antibody purification analysis:

The above cell culture supernatant was centrifuged at high speed to remove impurities and then applied to Protein A column affinity chromatography. The column was flushed with PBS until the A280 reading dropped to baseline. The target protein was eluted with 100 mM sodium acetate pH 3.0 and neutralized with 1 M Tris-HCl. After the eluted samples were properly concentrated, they were further purified by molecular sieves using a PBS-equilibrated gel chromatography column Superdex200 (GE), and the absorption peak samples of antibody monomers were collected together. The sample can be concentrated or buffer exchanged by ultrafiltration methods well known in the art to obtain a final sample of suitable concentration.

Example 3 ( S )-2-((2 R ,3 R )-3-(( S )-1-((3 R ,4 R ,5 S )-4-(( S ) -N ,3-dimethyl -2-(( S )-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanyl)pyrrolidin-2-yl) -3-Methoxy-2-methylpropionamide)-3-phenylpropionic acid

The compound of this example was prepared by the method disclosed in the patent application "WO2005081711".

MS m/z(ESI): 732.8[M+1]

¹ H NMR (400MHz, DMSO) δ 7.30-7.19 (m, 5H), 4.71-4.69 (m, 2H), 4.16-4.15 (m, 1H), 4.07-4.06 (m, 1H), 3.86-3.84 (m ,1H),3.70-3.66(m,3H),3.52-3.48(m,1H),3.41-3.40(m,1H),3.34(s,4H),3.28(s,1H),3.27-3.26(m ,1H),3.22(s,2H),3.13(s,1H),295-2.88(m,2H),2.67-2.65(m,3H),2.47-2.45(m,2H),2.33-2.31(m ,1H),2.19-2.18(m,2H),2.08-2.05(m,1H),1.89-1.86(m,2H),1.78-1.76(m,2H),1.57-1.52(m,2H),1.43 -1.38(m,2H),1.30-1.261(m,1H),1.21-1.19(dd,2H),1.15-1.11(m,3H),1.07-0.97(m,13H),0.88-0.85(m, 3H).

Example 4 ( S )-2-((2 R ,3 R )-3-(( S )-1-((3 R ,4 R ,5 S )-4-(( S )-2-(( S )- 2-(6-(2,5-Dioxo-2,5-dihydro- 1H -pyrrol-1-yl) -N -methylhexanamide)-3-methylbutanamide) -N , 3-Dimethylbutanamide)-3-methoxy-5-methylheptanyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamide)-3-benzene propionic acid

The compound of this example was prepared by the method disclosed in the patent application "WO2005081711".

MS m/z(ESI): 925.8[M+1]

¹ H NMR (400MHz, DMSO) δ 8.54-8.51 (m, 1H), 8.35-8.34 (m, 1H), 8.13-8.11 (m, 1H), 7.22-7.19 (m, 5H), 7.00-6.99 (m ,2H),4.73-4.68(m,1H),4.62-4.58(m,2H),4.45-4.40(m,1H),3.98-3.94(m,2H),3.74-3.72(m,1H),3.62 -3.59(m,1H),3.45-3.43(m,1H),3.38-3.35(m,2H),3.27-3.26(m,1H),3.23(s,1H),3.18-3.14(m,4H) ,3.05-3.03(m,2H),2.96-2.94(m,1H),2.90-2.89(m,1H),2.83-2.81(m,3H),2.35-2.28(m,2H),2.24-2.19( m, 2H), 2.00-1.98 (m, 2H), 1.78-1.72 (m, 2H), 1.51-1.43 (m, 5H), 1.25-1.21 (m, 11H), 0.92-0.70 (m, 18H).

Example 5 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((3 R ,4 S ,5 S )-4-(( S )- N ,3-dimethyl-2-(( S )-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl )-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid

first step (1 S ,3 S ,5 S )-3-((1 R ,2 R )-1-hydroxy-2-methyl-3-((4 R ,5 S )-4-methyl-2-carbonyl -5-Phenyloxazol-3-yl)-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid tert-butyl ester 5c

The starting material (4 R, 5 S) -4- methyl-5-phenyl-propan-acyl oxazolidinone 5b (1.96g, 9.26mmol, using well-known methods "Journal of the American Chemical Society, 2003 , 125(50), 15512-15520 "prepared) was dissolved in 25 mL of dichloromethane, and the temperature was lowered to 0 ℃ under an argon atmosphere. The reaction solution was added dropwise triethylamine (1.49 mL, 10.93 mmol) at 0 °C, and then dibutylboron trifluoromethanesulfonate (9.7 mL, 9.72 mmol) was added dropwise, and the mixture was stirred at 0 °C for 50 minutes. the reaction mixture was cooled to -75 ℃, was added (1 S, 3 S, 5 S) -3- methyl acyl-2-azabicyclo [3.1.0] hexane-2-carboxylic acid tert-butyl ester 5a ( 2.16g, 9.26mmol, prepared by the method disclosed in the patent application "US20100249190") dissolved in 7mL of dichloromethane solution, stirred at -75°C for 1.5 hours, at 0°C for 2 hours, and stirred at room temperature for 1 hour . After the reaction, a mixture of 36 mL of phosphate buffer (pH=7.0) and methanol (V/V=1:3) was added. A mixture of 36 mL of methanol and hydrogen peroxide (30%) (V/V=2:1) was added at 0°C, and the mixture was stirred at room temperature for 1 hour. Concentrate under reduced pressure to remove the organic phase, add a small amount of water, extract with ether (50 mL×3), wash with 5% sodium bicarbonate solution and saturated sodium chloride solution (150 mL) successively, dry with anhydrous sodium sulfate, filter, and reduce the pressure of the filtrate Concentration and purification of the residue by silica gel column chromatography with eluent system B afforded the title product 5c (2.4 g, white foamy solid) in 58.5% yield.

MS m/z(ESI): 345.1[M-100+1]

second step (1 S ,3 S ,5 S )-3-((1 R ,2 R )-1-methoxy-2-methyl-3-((4 R ,5 S )-4-methyl-2 -Carbonyl-5-phenyloxazol-3-yl)-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid tert-butyl ester 5d

The raw material 5c (1.4 g, 3.15 mmol) was dissolved in 20 mL of dichloromethane, 1.4 g of crushed molecular sieves were added, and 1,8-bisdimethylaminonaphthalene (1.75 g, 8.19 mmol) was added at 0 °C under an argon atmosphere. ), trimethyloxonium tetrafluoroborate (1.16 g, 7.87 mmol), the reaction was protected from light and stirred at room temperature for 40 hours. After the reaction, filtered, the filter cake was washed with dichloromethane, the filtrate was washed with saturated ammonium chloride solution (50mL×4) to remove excess 1,8-bisdimethylaminonaphthalene, and then saturated sodium chloride solution (120mL) Washed, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 5d (400 mg, white solid) in 27.8% yield.

MS m/z (ESI): 459.4 [M+1].

third step (2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-3-yl)- 3-Methoxy-2-methylpropionic acid 5e

The raw material 5d (400 mg, 0.87 mmol) was dissolved in 24 mL of tetrahydrofuran, cooled to 0 °C under argon atmosphere, 30% hydrogen peroxide (0.34 mL/0.38 g, 3.31 mmol) was slowly added dropwise, and lithium hydroxide monohydrate (62 mg) was added dropwise. ,, 1.48 mmol), the reaction system was reacted at room temperature for 20 hours. After the reaction, solid sodium sulfite (440 mg, 3.48 mmol) was added to the reaction solution, stirred at room temperature for 1 hour, 10 mL of water was added, the organic phase was concentrated under reduced pressure, and the obtained residue was extracted with dichloromethane (40 mL×2). The aqueous phase was added dropwise with 2 N hydrochloric acid under an ice bath until the pH of the reaction solution was 3~4, extracted with ethyl acetate (25 mL×3), and the ethyl acetate layer was washed with water (50 mL) and saturated sodium chloride solution (50 mL) in turn. , dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title product 5e (230 mg, colorless liquid) in a yield of 88.0%.

MS m/z (ESI): 200.1 [M-100+1].

the fourth step (S)-Third-butyl 2-amino-3-(2-fluorophenyl)propanoate 5g

The raw material (( S )-2-amino-3-(2-fluorophenyl)propionic acid 5f (400 mg, 2.18 mmol) was prepared by a known method "Advanced Synthesis & Catalysis , 2012, 354(17), 3327-3332 "prepared) was dissolved in 10 mL of tert-butyl acetate, added perchloric acid (300 mg (70%), 3.3 mmol), and stirred at room temperature for 16 hours. After the reaction was completed, 6 mL of water was added, and the organic phase was separated with Saturated sodium bicarbonate solution (5 mL) was washed. The aqueous phase was adjusted to pH=8 with saturated sodium bicarbonate solution, extracted with dichloromethane (5 mL×3), and the organic phases were combined, followed by water (3 mL), saturated sodium chloride solution ( 5mL) was washed, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude title product 5g (390mg, yellow oil). The product was directly subjected to the next reaction without purification.

the fifth step (1 S ,3 S ,5 S )-3-((1 R ,2 R )-3-((( S )-1-(tert-butoxy)-3-(2-fluorophenyl)- 1-Carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid tributyl ester 5h

The raw material 5e (100 mg, 0.334 mmol) was dissolved in 6 mL of a mixed solvent of dichloromethane and dimethylformamide (V/V=5:1), and 5 g (80 mg, 0.334 mmol) of the crude reactant was added. Then N , N -diisopropylethylamine (0.29mL, 1.67mmol) and 2-(7-azobenzotriazole) -N , N , N ', N' -tetramethylureahexanol were added Fluorophosphate (152.3 mg, 0.40 mmol). The reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction, 10 mL of water was added to stir, and the layers were separated. The dichloromethane layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 5h (173 mg, colorless liquid) in 99.5% yield.

MS m/z (ESI): 521.2 [M+1].

Step 6 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-azabicyclo[3.1.0]hexane-3-yl)-3-methyl Oxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid tert-butyl ester 5i

The raw material 5h (173 mg, 0.33 mmol) was dissolved in 2 mL of dioxane, 5.6 M hydrogen chloride solution in dioxane (0.21 mL, 1.16 mmol) was added, under argon atmosphere, stirred at room temperature for 1 hour, and placed in a 0°C refrigerator within 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, diluted with 5 mL of dichloromethane, and 10 mL of saturated sodium bicarbonate solution was added, followed by stirring for 10 minutes. The system layers were separated, and the aqueous layer was extracted with dichloromethane (5 mL×3). The dichloromethane layers were combined, washed with saturated sodium chloride solution (10 mL), and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave the crude title product 5i (77 mg, yellow liquid), which was directly subjected to the next reaction without purification.

MS m/z (ESI): 421.2 [M+1].

Step 7 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((5 S ,8 S ,11 S ,12 R )-11-(( S) - sec-butyl) -1- (9 H - fluoren-9-yl) -5,8-diisopropyl-12-methoxy-4,10-dimethyl -3,6,9 -Tricarbonyl-2-oxo-4,7,10-triazatetradecyl-14-acyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy tert-butyl-2-methylpropionamide)-3-(2-fluorophenyl)propanoate 5k

The crude product 5i (77mg, 0.183mmol), ( 5 S, 8 S, 11 S, 12 R) -11 - ((S) - sec-butyl) -1- (9 H - fluoren-9-yl) -5,8-Diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxa-4,7,10-triazatetradecane -14-Carboxylic acid 5j (116.8 mg, 0.183 mmol, prepared by the method disclosed in the patent application "WO 2013072813") was dissolved in 6 mL of dichloromethane and mixed with dimethylformamide (V/V=5:1) In the solvent, add N,N -diisopropylethylamine (0.16mL, 0.915mmol) and 2-(7-azobenzotriazole) -N , N , N' , N' -tetramethyl Urea hexafluorophosphate (84 mg, 0.22 mmol). The reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 mL of water was added to stir, and the layers were separated. The dichloromethane layer was washed with saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with eluent system B to give the title product 5k (190.5 mg, yellow sticky substance) in 100% yield.

MS m/z (ESI): 1040.6 [M+1].

Step 8 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((3 R ,4 S ,5 S )-4-(( S )- N ,3-dimethyl-2-(( S )-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl )-3-(2-fluorophenyl)propionic acid tert-butyl ester 51

The starting material 5k (190.5 mg, 0.183 mmol) was dissolved in 1.5 mL of dichloromethane and 2 mL of diethylamine was added. The reaction system was stirred at room temperature for 3 hours under an argon atmosphere. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude title product 51 (150 mg, yellow viscous substance), which was directly subjected to the next reaction without purification.

MS m/z (ESI): 818.5 [M+1].

Step 9 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((3 R ,4 S ,5 S )-4-(( S )- N ,3-dimethyl-2-(( S )-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl )-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid 5

The crude product 51 (150 mg, 0.183 mmol) was dissolved in 1 mL of dioxane, 3 mL of a 5.6 M hydrogen chloride solution in dioxane was added, and the mixture was stirred at room temperature for 12 hours under an argon atmosphere. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the solvent was swirled with diethyl ether. The obtained residue was purified by high performance liquid chromatography to obtain the title product 5 (28 mg, white powder solid) in a yield of 20%.

MS m/z(ESI): 762.7[M+1]

¹ H NMR (400 MHz, CD ₃ OD): δ 7.38-7.18 (m, 2H), 7.13-7.01 (m, 2H), 4.80-4.67 (m, 2H), 4.30-4.15 (m, 1H), 4.13- 4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H),3.42-3.11(m,9H),3.06-2.95(m,1H),2.70-2.58(m,4H ),2.28-2.01(m,4H),1.88-1.70(m,3H),1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65 (m, 1H).

Example 6 ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((3 R ,4 S ,5 S )-4-(( S )- 2-(( S )-2-(6-(2,5-Dicarbonyl-2,5-dihydro- 1H -pyrrol-1-yl) -N -methylhexanoamide)-3-methyl butanamide) -N ,3-dimethylbutanamide)-3-methoxy-5-methylheptanyl)-2-azabicyclo[3.1.0]hexane-3-yl)- 3-Methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid

first step 6-(2,5-Dicarbonyl-2,5-dihydro- 1H -pyrrol-1-yl)hexanoyl chloride 6b

In the raw material 6-(2,5-dicarbonyl-2,5-dihydro- 1H -pyrrol-1-yl)hexanoic acid 6a (1.5g, 7.10mmol, using a well-known method " Journal of Medcinal Chemistry , 2013, 56(24), 9955-9968 "prepared"), drop a drop of N , N -dimethylformamide, under argon atmosphere, after cooling in a dry ice bath, slowly drop 15mL of oxalic chloride, vigorously stir during dropwise addition , and the reaction was completed at room temperature for 1 hour. After the reaction, the reaction solution was concentrated under reduced pressure, the resulting residue was dissolved in dichloromethane, and concentrated under reduced pressure to obtain the crude title product 6b, which was directly subjected to the next reaction without purification.

second step ( S )-2-((2 R ,3 R )-3-((1 S ,3 S ,5 S )-2-((3 R ,4 S ,5 S )-4-(( S )- 2-(( S )-2-(6-(2,5-Dicarbonyl-2,5-dihydro- 1H -pyrrol-1-yl) -N -methylhexanoamide)-3-methyl butanamide) -N ,3-dimethylbutanamide)-3-methoxy-5-methylheptanyl)-2-azabicyclo[3.1.0]hexane-3-yl)- 3-Methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid 6

The raw material 5 (25 mg, 0.033 mmol) was dissolved in 3 mL of dichloromethane, N,N-diisopropylethylamine (0.029 mL, 0.164 mmol) was added, and the reaction system was added dropwise under an argon atmosphere under an ice bath. A solution of 6b (11.3 mg, 0.049 mmol) in dichloromethane was reacted at room temperature for 3 hours. After the reaction, 5 mL of water was added, stirred for 20 minutes, and the layers were separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography to obtain the title product 6 (7 mg, yellow viscous substance) , the yield is 22.4%.

MS m/z(ESI): 955.4[M+1]

¹ H NMR (400 MHz, CD ₃ OD): δ 7.36-7.30 (m, 1H), 7.29-7.21 (m, 1H), 7.17-7.02 (m, 2H), 6.83-6.79 (m, 2H), 4.81- 4.71(m, 2H), 4.69-4.55(m, 2H), 4.25-4.15(m, 1H), 4.13-4.04(m, 1H), 3.96-3.85(m, 2H), 3.70-3.61(m, 1H ), 3.55-3.46(m, 3H), 3.40-3.21(m, 4H), 3.18-3.10(m, 2H), 3.07-2.96(m, 4H), 2.67-2.56(m, 2H), 2.54-2.34 (m,3H), 2.29-2.17(m,2H), 2.10-1.99(m,1H), 1.89-1.57(m,7H), 1.52-1.28(m,6H), 1.21-1.11(m,4H) ,1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).

Example 7 Preparation of ADC-7

first step

The thioacetic acid S - (3- propyl-carbonyl) acetate (1.61mg, 12.2 μ mol), was dissolved in 3.0mL acetonitrile standby; to pH = 4.3 containing the mAb001 antibody acetic acid / sodium acetate buffer (10.22 mg /ml,30mL,2.04mmol) was added the above preformed thioacetic acid S - acetonitrile (3-oxopropyl) ester, sodium cyanoborohydride followed by dropwise addition of 1.2mL (49.86mg, 793 μ mol) of an aqueous solution , and shake the reaction at 25 °C for 2 hours. The reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5) to remove unreacted S- (3-carbonylpropyl) thioacetate and cyanoborohydrogenation sodium, and then concentrated to a concentration of about 10 mg/ml to obtain a PBS buffer solution (about 35 mL) of the title product 7a, which was directly carried out to the next reaction.

second step

About 0.4 mL of 2.0 M hydroxylamine hydrochloride solution was added to the PBS buffer solution (35.0 mL) of 7a. After the addition was completed, it was placed on a water bath shaker, and the reaction was shaken at 25° C. for 30 minutes to stop the reaction. The reaction solution was purified by Sephadex G25 gel column to obtain the title product mAb001-propanethiol 7b in PBS buffer (concentration 5.38 mg/ml, 55 mL).

third step

Compound 6 (4.32mg, 4.52 μ mol) was dissolved in 1.1mL acetonitrile was added propanethiol mAb001- PBS buffer solution 7b (5.38mg / mL, 11mL) and placed in a water bath shaker and shaken at 25 deg.] C The reaction was stopped after 4 hours of reaction.

The reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05 M PBS solution with pH 6.5) to obtain the crude title product 7 in PBS buffer (2.92 mg/mL, 20 mL), which was further concentrated by centrifugation to about 5.5 mL, and then desalted and purified by Sephadex G25 gel column (elution phase: 0.05 M PBS solution with pH 6.5) to obtain the PBS buffer solution of the title product 7 (4.25 mg/mL, 11.6 mL) , stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150186.98(M _Ab +0D), 151374.09(M _Ab +1D), 152287.22(M _Ab +2D), 153353.26(M _Ab +3D), 154501.80(M _Ab +4D), 155575.57 (M _Ab +5D).

Average: y=2.2.

Example 8 Preparation of ADC-8

first step

The thioacetic acid S - (3- propyl-carbonyl) acetate (0.7mg, 5.3 μ mol), was dissolved in 0.9mL acetonitrile standby; to pH = 4.3 containing the mAb002 antibody acetic acid / sodium acetate buffer (10.13mg /ml,9.0mL,0.95mmol) was added the above preformed thioacetic acid S - acetonitrile (3-oxopropyl) ester, followed by dropwise addition of 1.0mL of sodium cyanoborohydride (14.1mg, 224 μ mol) of The aqueous solution was shaken at 25°C for 2 hours. After the reaction, it was desalted and purified by Sephadex G25 gel column (elution phase: 0.05 M PBS solution with pH 6.5) to obtain the title product 8a solution, which was concentrated to about 10 mg/ml and then directly proceeded to the next reaction.

second step

0.3 mL of 2.0 M hydroxylamine hydrochloride solution was added to the 8a solution (10.0 mL), and the reaction solution was shaken at 25°C for 30 minutes, and the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05 M with pH 6.5). PBS solution) to obtain the title product mAb002-propanethiol 8b solution (concentration 6.2 mg/ml, 14 mL).

third step

The starting material 4 (1.1mg, 1.2 μ mol) was dissolved in 0.3mL acetonitrile was added a solution of 8b (6.2mg / mL, 3.0mL) in shaking at 25 deg.] C for 4 hours, the reaction was treated with Sephadex G25 gel tube Column desalting purification (elution phase: pH 6.5 containing 0.05 M PBS solution), filtered through a 0.2 μm filter under sterile conditions to obtain the title structural formula 8 in PBS buffer (3.5 mg/mL, 4.8 mL) ), stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150332.9 (M _Ab + OD), 1514911.2 (M _Ab + 1D), 152374.3 (M _Ab + 2D), 153530.1 (M _Ab + 3D), 154450.7 (M _Ab + 4D).

Mean: y=2.0.

Example 9 Preparation of ADC-9

The starting material 6 (1.1mg, 1.2 μ mol) was dissolved in 0.3mL acetonitrile was added a solution of 8b (6.2mg / mL, 3.0mL) in shaking at 25 deg.] C for 4 hours, the reaction was treated with Sephadex G25 gel tube Column desalting purification (elution phase: pH 6.5 containing 0.05 M PBS solution), filtered through a 0.2 μm filter under sterile conditions to obtain the title structural formula product 9 in PBS buffer (3.4 mg/mL, 4.7 mL ), stored frozen at 4°C.

Q-TOF LC/MS: Characteristic peaks: 150336.6 (M _Ab + OD), 151530.5 (M _Ab + 1D), 152436.1 (M _Ab + 2D), 153625.1 (M _Ab + 3D), 154607.5 (M _Ab + 4D).

Mean: y=2.0.

Biological evaluation Test Example 1: HCC827 Cell Proliferation Experiment

1. Purpose of the test:

The inhibitory effect of the samples of the present invention on the proliferation of HCC827 cells was detected.

2. Test materials:

Sample of the invention: ADC-9

Poplar reference drug: ADC-8

HCC827 cells: Cell Bank of Chinese Academy of Sciences, Cat. No. #TCHu153; CCK-8: Cell Counting Kit-8, purchased from Dojindo, Cat. No.: CK04; FBS: Fetal Bovine Serum, purchased from Gibco, Cat. No. 10099-141; RPMI1640: purchased from Hyclone , Article number: SH30809.01B; VICTOR 3 multi-function microplate reader (PerkinElmer company).

3. Test method:

1. In a 96-well plate, 100 μl of RPMI1640 medium containing 5000 HCC827 cells in 10% FBS was added to each well, and the culture plate was placed in a 37° C., 5% CO ₂ incubator for 16 h.

2. The samples were serially diluted three-fold with RPMI1640 medium containing 10% FBS, a total of 10 points was diluted starting dilution of 10 μ g / ml.

3. The HCC827 cell culture plate laid on the previous day was taken out from the incubator, the supernatant medium was discarded, and 100 μl /well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and a control well without any drug was set at the same time. At 37 ℃, 5% CO ₂ conditions of continuous culture cells.

4.72 hours later, add 10 μl of CCK-8 solution to each well for color development, put it into a 37° C., 5% CO ₂ incubator to incubate for color development for 2 hours, read OD ₄₅₀ on a microplate reader, and use Graphpad Prism 5 software IC ₅₀ was obtained by the post-processing.

4. Test results:

The biological activity of the sample of the present invention obtained by the above analysis, the calculated IC ₅₀ values are listed in Table 1:

Conclusion: The preferred sample of the present invention has obvious proliferation inhibitory activity on HCC827 cells.

Test Example 2: Lovo Cell Proliferation Experiment

1. Purpose of the test:

The inhibitory effect of the samples of the present invention on the proliferation of Lovo cells was detected.

2. Test materials:

Sample of the invention: ADC-9

Positive control drug: ADC-8

Lovo cells: Cell Bank of Chinese Academy of Sciences, Cat. No. #TCHu82; CCK-8: Cell Counting Kit-8, purchased from Dojindo, Cat. No.: CK04; FBS: Fetal Bovine Serum, purchased from Gibco, Cat. No. 10099-141; DMEM/F12: purchased In Hyclone, product number: SH30023.01; VICTOR 3 multifunctional microplate reader (PerkinElmer).

3. Test method:

1. In a 96-well plate, add 100 μl of DMEM/F12 medium containing 4000 Lovo cells in 10% FBS to each well, and place the culture plate in a 37°C, 5% CO ₂ incubator for 16 hours.

2. The samples containing 10% FBS in DMEM / F12 medium threefold dilution gradient were diluted with 10 points, the initial dilution of 100 μ g / ml.

3. The Lovo cell culture plate laid on the previous day was taken out from the incubator, the supernatant medium was discarded, and 100 μl /well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and a control well without any drug was set at the same time. At 37 ℃, 5% CO ₂ conditions of continuous culture cells.

4.72 hours later, add 10 μl of CCK-8 solution to each well for color development, put it into a 37° C., 5% CO ₂ incubator to incubate for color development for 2 hours, read OD ₄₅₀ on a microplate reader, and use Graphpad Prism 5 software IC ₅₀ was obtained by the post-processing.

4. Test results:

The biological activity of the sample of the present invention obtained by the above analysis, the calculated IC ₅₀ values are listed in Table 2:

Conclusion: The preferred sample of the present invention has obvious proliferation inhibitory activity on Lovo cells.

Test Example 3: HCC827-Del19/T790M/C797S(DTC) Cell Proliferation Experiment

1. Purpose of the test:

The inhibitory effect of the samples of the present invention on the proliferation of HCC827-DTC cells was detected.

2. Test materials:

Sample of the invention: ADC-9

Control drug: mAb002, AZD-9291

HCC827 cells: Chinese Academy of Sciences Cell Bank, Cat.#TCHu153; HCC827-DTC cells: pCDH-EGFR Del19/T790M/C797S(DTC), △R8.9 and VSVG transfected in 293T cells (ATCC, CRL-3216), 48 hours Then, the virus supernatant was collected, filtered with a 0.45 μm membrane filter to discard cell debris, centrifuged at 50,000 g for 2 hours to resuspend, and the virus was concentrated 10 times. HCC827 cells plated the previous day of virus added to the concentrate, while adding 8 μ g / ml polybrene, replaced with fresh media after 24 hours, 48 hours after the addition of 2 μ g / ml puromycin were screened to obtain cells HCC827-DTC.

CCK-8: Cell Counting Kit-8, purchased from Dojindo, Item No.: CK04; FBS: Fetal Bovine Serum, available from Gibco, item number: 10099-141; RPMI1640: available from Hyclone, item number: SH30809.01B; VICTOR 3 multifunctional microplate reader (PerkinElmer).

3. Test method:

1.96-well plate, 100 μ l added to each well containing 5000 cells HCC827-DTC 10% FBS RPMI1640 medium (puromycin 2 μ g / ml) , plates were placed in 37 ℃, 5% CO ₂ incubator 16h .

2. The samples were serially diluted with RPMI1640 medium containing 10% FBS, and diluted 10 points in total. The initial dilution concentration of antibody and ADC was 10ug/ml (66.7nM), and 3-fold dilution was performed. AZD-9291 was initially diluted at a concentration of 2500 nM and was diluted 4-fold.

3. The HCC827-DTC cell culture plate laid on the previous day was taken out from the incubator, the supernatant medium was discarded, and 100 μl /well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and a control well without any drug was set at the same time. At 37 ℃, 5% CO ₂ conditions of continuous culture cells.

4.72 hours later, add 10 μl of CCK-8 solution to each well for color development, put it into a 37° C., 5% CO ₂ incubator to incubate for color development for 2 hours, read OD ₄₅₀ on a microplate reader, and use Graphpad Prism 5 software IC ₅₀ was obtained by the post-processing.

4. Test results:

The biological activity of the samples of the present invention was obtained from the above analysis, and the calculated IC _{50 values are listed in Table 3 below: Table 3. IC 50 of the} samples of the present invention for inhibition of proliferation of HCC827-DTC cells

Conclusion: After expressing EGFR-DTC gene, non-small cell lung cancer HCC827 cells have obvious drug resistance to AZD-9291; naked anti-mAb002 has only partial inhibitory effect on the cells. But the sample ADC-9 of the present invention has a strong proliferation inhibitory activity against the drug-resistant strain HCC827-DTC.

Test Example 4: H1975-L858R/T790M/C797S(LTC) Cell Proliferation Experiment

1. Purpose of the test:

The inhibitory effect of the samples of the present invention on the proliferation of H1975-LTC cells was detected.

2. Test materials:

Sample of the invention: ADC-9

Control drug: mAb002, AZD-9291

H1975 cells: Chinese Academy of Sciences Cell Bank, Cat. No. #TCHu193;

H1975-LTC cells: 293T cells (ATCC, CRL-3216) were transfected with pCDH-EGFR L858R/T790M/C797S (LTC), △R8.9 and VSVG, and the virus supernatant was collected 48 hours later, and filtered with a 0.45 μm filter to discard Cell debris was removed, centrifuged at 50,000g for 2 hours to resuspend, and the virus was concentrated 10 times. H1975 cells were plated the day before the virus added to the concentrate, while adding 8 μ g / ml polybrene, replaced with fresh media after 24 hours, 48 hours after the addition of 2 μ g / ml puromycin for screening, H1975-LTC cells.

CCK-8: Cell Counting Kit-8, available from Dojindo, item number: CK04; FBS: Fetal Bovine Serum, available from Gibco, item number: 10099-141; RPMI1640: available from Hyclone, item number: SH30809.01B; VICTOR 3 multifunctional Microplate reader (PerkinElmer).

3. Test method:

1.96-well plate, 100μl added to each well containing 10% FBS 5000 th H1975-LTC cells RPMI1640 medium (puromycin 2 μ g / ml) , plates were placed in 37 ℃, 5% CO ₂ incubator 16h.

2. The samples were serially diluted with RPMI1640 medium containing 10% FBS, and diluted 10 points in total. The initial dilution concentration of antibody and ADC was 10ug/ml (66.7nM), and 3-fold dilution was performed. The initial dilution concentration of AZD-9291 was 2500nM, and 4-fold dilution was performed.

3. The H1975-LTC cell culture plate laid on the previous day was taken out from the incubator, the supernatant medium was discarded, and 100 μl /well of the medium containing the diluted sample was added. Two duplicate wells were set for each concentration, and a control well without any drug was set at the same time. At 37 ℃, 5% CO ₂ conditions of continuous culture cells.

4.72 hours later, add 10 μl of CCK-8 solution to each well for color development, put it into a 37° C., 5% CO ₂ incubator to incubate for color development for 2 hours, read OD ₄₅₀ on a microplate reader, and use Graphpad Prism 5 software IC ₅₀ was obtained by the post-processing.

4. Test results:

The biological activity of the sample of the present invention obtained by the above analysis, the calculated IC ₅₀ values are listed in Table 4:

Conclusion: Non-small cell lung cancer H1975 cells have obvious drug resistance to AZD-9291 after expressing EGFR-LTC gene; naked anti-mAb002 has almost no inhibitory effect on the cells. But the sample ADC-9 of the present invention has a strong proliferation inhibitory activity against the drug-resistant strain H1975-LTC.

Test Example 5: Pharmacokinetic test of EGFR antibody in cynomolgus monkeys

1. Purpose of the test:

The pharmacokinetic changes of mAb002 relative to nimotuzumab (mAb001) in cynomolgus monkeys were compared.

2. Test materials:

Sample of the invention: mAb002 (IgG1-YTE variant of Nimotuzumab)

Positive control drug: mAb001, Nimotuzumab injection, purchased from Baintai Biotechnology, Cat. No. 0120131240

PBS: purchased from Sangon Biotechnology, product number PD0100

BSA: Available from Amresco, Cat. No. 0332

Proclin300: Available from Supelco, Cat. No. 48912-U

EGFR: purchased from Sino Biological, Cat. No. 10001-H08H

Anti-Human IgG(Fc)-peroxidase: purchased from Sigma, Cat. No. A0170

Human IgG: purchased from R&D; Cat. No. 1-001-A

Microplate reader: purchased from Thermo Scientific, model Multiskan FC, catalog number EQP-LI-030

3. Test method:

ELISA is a commonly used method for the detection of pharmacokinetics in biological serum samples. In this study, the ELISA method was used to detect the concentration of the antibody to be tested in the serum of cynomolgus monkeys.

Two groups of cynomolgus monkeys (3 in each group) were injected intravenously with 5 mg/kg mAb001 and mAb002 antibodies by constant flow pump respectively, before administration (0h) and 5min (±10s), 2h (±2min) after the end of administration ), 4h(±5min), 8h(±5min), 1d(±10min), 2d(±10min), 3d(±10min), 5d(±10min), 7d(±10min), 10d(±10min), 13d(±10min), 17d(±10min), 21d(±10min), and 28d(±10min) about 1 mL of whole blood was collected from the saphenous vein of the lower extremity, and the blood samples were allowed to stand at room temperature for 1h~2h until fully coagulated. Serum was obtained by centrifugation at 4000g for 5 minutes.

EGFR-coated microplates were used to capture the antibody to be tested in diluted serum samples. The detection antibody was HRP-labeled goat anti-human IgG antibody (against the Fc segment). HRP catalyzes TMB substrate to produce a soluble blue compound. The hydrochloric acid solution terminates the reaction, and the absorbance value is detected at the working wavelength of 450nm and the reference wavelength of 620nm. The OD value is positively correlated with the concentration of the antibody to be tested in the sample. Using SoftMax Pro v5.4.1 software, draw a standard curve for the logarithm value (X axis) and OD value (Y axis) of the antibody concentration to be tested, and fit a four-parameter Logistic model, using the weighting method of 1/y to obtain to the curve parameters. The plasma concentration of the unknown sample was calculated by regressing its OD value on the standard curve.

4. Experimental results:

After a single intravenous injection of 5 mg/kg of mAb001 and mAb002 in cynomolgus monkeys, the average pharmacokinetic parameters of the animals in the two dose groups are shown in Table 5 and Figure 1 below.

_{The average C max of} mAb002 group and mAb001 group are basically the same, indicating that the amount of drug entering the animal is basically the same, but the pharmacokinetic characteristics of mAb002 group and mAb001 group in cynomolgus monkeys are significantly different, which is expressed as AUC ( _0-28d ) High, long terminal elimination half-life, low clearance rate, and long average retention time, indicating that the mutated antibody mAb002 (the IgG1-YTE variant of nimotuzumab) has significantly improved monkey drug metabolism properties.

Test Example 6: EGFR-ADC in vivo efficacy experiment

1. Purpose of the test:

Compare the in vivo efficacy of ADC-9 of the present invention relative to ADC-8 and naked anti-mAb002 in nude mouse lung cancer xenograft HCC827 model.

2. Test materials:

Sample of the invention: ADC-9 ADC-8 mAb002

HCC827 cells: Cell Bank of Chinese Academy of Sciences, Cat. No. #TCHu153; Animals: Nude mice, SPF, 16-20g, ♀, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

3. Test method:

1 Nude mice were acclimated to the laboratory environment for three days and were randomly divided into 5 groups with 9 mice in each group.

2 Tumor cell transplantation

Nude mice were inoculated subcutaneously with HCC827 cells (5×10 ⁶ +50% matrigel/mouse) in the right flank. On the 7th day after inoculation, the tumor grew to 202.07±6.22mm3 (d1) and the administration started.

3 Dosage and method of administration

The route of administration was intraperitoneal injection (ip), once a week, 3 times in total. The specific dosing schedule is shown in Table 6.

4. Determination of transplanted tumor volume and body weight of nude mice

The tumor volume was measured twice a week, the body weight was weighed and the data were recorded.

5 Statistics

Using Excel statistical software: mean value is calculated by avg; SD value is calculated by STDEV; SEM value is calculated by STDEV/SQRT; P value of difference between groups Calculated in TTEST.

The formula for calculating tumor volume (V) is: V=1/2×L _long ×L _short ²

Relative volume (RTV)=V _T /V ₀

Tumor inhibition rate (%)=(C _RTV -T _RTV )/C _RTV (%)

where V ₀ and V _T are the tumor volumes at the beginning and end of the experiment, respectively. C _RTV and T _RTV are the relative tumor volumes of the blank control group (Blank) and the experimental group at the end of the experiment, respectively.

4. Experimental results:

The tumor inhibitory effect is shown in Table 6 and Figure 2 below

The results of this experiment showed that mAb002 (an IgG1-YTE variant of Nimotuzumab, see Example 2), ADC-8 (see Example 8) and ADC-9 (see Example 9) were effective on mouse lung cancer xenografts HCC827 has a significant tumor suppressor effect. That At the same dose (0.1mg/dose) or lower dose (0.03mg/dose), ADC-9 showed more obvious tumor inhibition effect than naked anti-mAb002 (0.1mg/dose), indicating that ADC-9 phase Compared with the naked antibody in terms of efficacy. In addition, when different ADCs were compared at the same dose (0.03 mg/dose), ADC-9 showed better efficacy than ADC-8, and the tumor inhibition rates at 34 days of administration were 65.33% vs. 48.96%, respectively. , indicating that the ADC with toxin compound 6 (former compound 5) showed stronger tumor-inhibitory activity than the ADC with toxin compound 4 (MC-MMAF, whose original form was compound 3).

<110> Jiangsu Hengrui Pharmaceutical Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.

<120> EGFR antibody-drug conjugate and its application in medicine

<130> 770048CPCT-360160

<160> 12

<170> PatentIn version 3.3

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<220>

<223> Light chain amino acid sequence of mAb001

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<223> HCDR3 sequence of mAb001

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Since the graph of this case is experimental data, it is not a representative graph of this case. Therefore, there is no designated representative map in this case.

Claims (13)

An antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt thereof,

Wherein: L ₁ and L ₂ are linker units; y is 1 to 8; Ab is an anti-EGFR antibody comprising a light chain whose amino acid sequence is shown in SEQ ID NO: 3, and an amino acid sequence such as that shown in SEQ ID NO: 3. The heavy chain shown in SEQ ID NO:4. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to item 1 of the claimed scope, wherein y is 2 to 5. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof _{as described in item 1 or 2 of the patent application scope, wherein L 2 is represented by the} following general formula (L ₂ ):

wherein X _{1 is} selected from a hydrogen atom, a C _1-6 alkyl group and a C _1-6 alkoxy group; X ₂ is a C _1-6 alkyl group; m is 0 to 5; and S is a sulfur atom. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to item 3 of the claimed scope, wherein m is 1 to 3. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to any one of items 1 to 4 of the patent application scope, wherein L ₁ is the following general formula (L ₁ ) Compounds shown:

wherein X ₃ is C _1-6 alkyl; n is 0 to 5. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to item 5 of the patent application scope, wherein n is 1 to 3. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof as described in item 1 of the patent application scope is selected from the antibody-drug conjugate represented by the general formula (II) or a pharmaceutically acceptable salt thereof,

Wherein, Ab, L ₂ , and y are as defined in item 1 of the scope of the application. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof as described in item 1 of the patent application scope is selected from the antibody-drug conjugate represented by the general formula (III) or a pharmaceutically acceptable salt thereof,

Wherein, Ab, L ₁ , and y are as defined in item 1 of the scope of the application. The antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to any one of items 1 to 8 of the scope of the application, which is a compound represented by the following formula:

Wherein, mAb002 includes a light chain whose amino acid sequence is shown in SEQ ID NO: 3, and a heavy chain whose amino acid sequence is shown in SEQ ID NO: 4, and y is as defined in item 1 of the patent application scope. A pharmaceutical composition comprising the antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt thereof as described in item 1 of the scope of application, and one or more pharmaceutically acceptable excipients agent, diluent or carrier. A use of the antibody-drug conjugate represented by the general formula (I) described in item 1 of the claim scope or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in item 10 of the claim scope, which uses in the preparation of drugs for the treatment of cancer. The use according to item 11 of the scope of application, wherein the cancer is gastric cancer, pancreatic cancer, liver cancer, breast cancer, lung cancer, intestinal cancer, kidney cancer, melanoma tumor, non-small cell lung cancer, colon cancer, rectal cancer, colorectal cancer, or head and neck cancer. The use according to claim 11, wherein the cancer is lung cancer, colon cancer, rectal cancer, colorectal cancer or head and neck cancer.

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