WO2022253051A1 - 多肽偶联药物及其应用 - Google Patents

多肽偶联药物及其应用 Download PDF

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WO2022253051A1
WO2022253051A1 PCT/CN2022/094740 CN2022094740W WO2022253051A1 WO 2022253051 A1 WO2022253051 A1 WO 2022253051A1 CN 2022094740 W CN2022094740 W CN 2022094740W WO 2022253051 A1 WO2022253051 A1 WO 2022253051A1
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trp
pro
cys
dasp
harg
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French (fr)
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李慧宁
夏建华
江志赶
贺海鹰
陈曙辉
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南京明德新药研发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a polypeptide-coupled drug and its application, in particular to the compound represented by formula (I) and its pharmaceutically acceptable salt.
  • Nectin-4 (poliovirus receptor like 4, PVRL4, connexin-4) is an emerging tumor-related target in recent years, which belongs to the nectin protein family, mainly has four subtypes of nectin 1-4, and nectin-like molecules ( Necl) together constitute immunoglobulin-like cell adhesion molecules, which play an important role in the formation and maintenance of intercellular adhesion and tight junctions.
  • Nectin 1, 2, and 3 widely exist in normal tissues of the human body, and nectin-4 is mainly highly expressed in embryos and placenta, and its expression level is greatly reduced in adults.
  • nectin-4 is overexpressed in a variety of tumors, such as bladder cancer, urothelial cancer, breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, esophageal cancer, etc., making it a potential target for the treatment of related cancers.
  • tumors such as bladder cancer, urothelial cancer, breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, esophageal cancer, etc.
  • Enfortumab vedotin a biological antibody conjugate drug developed for this target, has been approved for marketing in the United States in 2019.
  • As the only marketed drug for this target its main indication is metastatic urothelial carcinoma. Clinical effectiveness studies for multiple indications are underway. Therefore, the development of chemotherapeutic drugs targeting nectin-4 has broad application prospects.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • SEQ consists of a peptide ligand and a linker to which the peptide ligand is covalently bonded;
  • the peptide ligand contains two loop sequences separated by three amino acid residues, the first of which is Pro-1Nal-dAsp and the second is Met-hArg-Asp-Trp-Ser- Thr-Pro-Hyp-Trp;
  • the amino acid residues are Xi, Xii and Xiii, said Xi, Xii and Xiii are independently selected from Cys, hCys, ⁇ Cys, Pen, Dap and N-methyl-Dap, and Xi, Xii and Xiii are not all Cys;
  • the above-mentioned peptide ligand is selected from: H-Xi-Pro-1Nal-dAsp-Xii-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Xiii-NH 2 , Wherein, Xi, Xii and Xiii are as defined in the present invention.
  • the above-mentioned peptide ligands are selected from sequences 1-7:
  • H-hCys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys- NH2 (SEQ ID NO: 7), other variables are as defined in the present invention.
  • Xi, Xii and Xiii are as defined in the present invention.
  • the present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
  • Trp-Cys-NH 2 Trp-Cys-NH 2 , TATA,
  • the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating solid tumors overexpressing Nectin-4.
  • the compound of the present invention has a strong binding effect with Nectin-4, and the mouse subcutaneous tumor model in vivo shows a strong tumor-inhibiting effect.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
  • Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure (such as the alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, formazine Thionine sulfoxide, methionine methylsulfonium.
  • Such analogs can have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics are chemical compounds whose structure differs from the general chemical structure of amino acids, but which function similarly to naturally occurring amino acids.
  • amino acid sequences of the present invention contain the standard one-letter or three-letter codes for the twenty natural amino acids.
  • treating includes inhibiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.
  • the compounds of the invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomers or “geometric isomers” arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
  • diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
  • the terms “enriched in an isomer”, “enriched in an isomer”, “enriched in an enantiomer” or “enantiomerically enriched” refer to one of the isomers or enantiomers
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the terms “isomer excess” or “enantiomeric excess” refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • linking group listed does not indicate its linking direction
  • its linking direction is arbitrary, for example,
  • the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
  • any one or more sites of the group can be linked to other groups through chemical bonds.
  • the connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
  • the chemical bonds that the site connects with other groups can use straight solid line bonds Straight dotted key or tilde express.
  • the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted line bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups.
  • the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • SXRD single crystal X-ray diffraction
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the solvent used in the present invention is commercially available.
  • the present invention adopts the following abbreviations: eq. stands for equivalent, equivalent; SPPS stands for polypeptide solid-phase synthesis; TFA stands for trifluoroacetic acid; DIEA stands for diisopropylethylamine; DMF stands for N,N-dimethyl Formamide; HATU stands for 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC stands for 1-(3-dimethylaminopropyl )-3-ethylcarbodiimide hydrochloride; NHS stands for N-hydroxysuccinimide; TIS stands for triisopropylsilane; DTT stands for DL-1,4-dithiothreitol; MMAE stands for mono Methyl auristatin E, the specific structure is: PABC representative Cit stands for L-citrulline; Val stands for L-valine; Glutaryl stands for ⁇ -A
  • polypeptides were synthesized using standard step-by-step synthetic methods.
  • step 2 Repeat step 2 to step 5, adding raw materials 2-26 each time to condense the next amino acid. See Table 1 for details.
  • polypeptide was settled with ice isopropyl ether, and centrifuged with a centrifuge (3min at 3000rpm).
  • Peptide 1 (0.7g, 0.25mmol) was dissolved in 50% CH 3 CN/H 2 O (1L), and TATA (95.9mg, 0.38mmol) was slowly added to the stirring solution at room temperature, and the reaction mixture was After stirring for 30 minutes, the pH was adjusted to 8 with NH4HCO3 , and the reaction was continued to stir at room temperature for 12 hours. LCMS showed that the reaction was complete, the stirring was stopped, and the TFA salt of Peptide 2 was obtained by purification by reverse phase preparation (A: 0.075% TFA aqueous solution, B: CH 3 CN).
  • the acetates of the other three peptide-conjugated drugs PDC_2-7 were obtained by referring to the synthesis of the acetates of PDC_1 (respectively replace the raw materials with the corresponding Peptides in the table below), see Table 4.
  • Test example 1 The binding ability test of compound of the present invention and Nectin 4 protein
  • the affinity between the analyte and the target protein Nectin 4 was detected by SPR method.
  • CM5 chip (Cat#BR-1005-30, GE Healthcare)
  • Nectin 4 (Cat#1006-72, GE Healthcare)
  • the amino coupling method was used, that is, the target protein Nectin 4 was directly immobilized on the CM5 chip by using Biacore 8K, and then the analyte was used as the analyte, and the buffer solution (10mM PBS, pH7.4, 137mM NaCl, 2.7mM KCl , 5% DMSO, 0.05% P20) dilute the analyte to the required concentration gradient, and perform multi-cycle kinetic detection. Each cycle is injected for 180 seconds, dissociated for 180 seconds, and then the next cycle is performed to obtain the target Affinity kinetic analysis data of protein Nectin 4 and analytes. The final data were analyzed by Kinetics fitting with a 1:1 model using Biacore Insight Evaluation Software (V 2.0.15.12933).
  • Preparation buffer 10mM PBS, pH7.4, 137mM NaCl, 2.7mM KCl, 5% DMSO, 0.05% P20.
  • CM5 chip activation activate with 400mM EDC and 100mM NHS at a flow rate of 10 ⁇ L/min for 420 seconds.
  • Target protein coupling Dilute the target protein to 10 ⁇ g/mL with 10 mM sodium acetate (pH 4.5), and couple at a flow rate of 10 ⁇ L/min for 284 s.
  • the experiment used the 1#, 2# and 3# channels on the chip, and the coupling results were 1639.9RU, 1747.8RU and 1702.2RU respectively.
  • CM5 chip blocking block with 1M ethanolamine at a flow rate of 10 ⁇ L/min for 420 seconds.
  • Analyte concentration use running buffer to dilute the analyte. The test substance was serially diluted 2 times from 100nM to 0.78nM.
  • each concentration of the working solution of the analyte is a cycle, with a flow rate of 30 ⁇ L/min for 180 seconds of binding and 180 seconds of dissociation.
  • the last cycle is a 5% DMSO solvent calibration cycle.
  • the listed value is the average value of multiple measurements.
  • the compound of the present invention has strong binding ability to Human Nectin-4 protein.
  • test example 2 In vivo pharmacodynamics and PK study of test example 2 on human lung cancer NCI-H292 cell subcutaneous xenograft tumor BALB/c nude mouse model
  • NCI-H292 cells Human lung cancer NCI-H292 cells (ATCC, Manassas, Virginia, Cat. No.: CRL-1848) were cultured in vitro as a single layer.
  • the culture conditions were RPMI 1640 medium plus 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, cultured in a 5% CO 2 incubator at 37°C. Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • mice BALB/c nude mice, female, 6-8 weeks old, weighing 17-21 grams. Provided by Beijing Weitong Lihua Company.
  • Tumor inoculation 0.1 mL (1 ⁇ 10 7 cells) of NCI-H292 cells were subcutaneously inoculated on the right back of each mouse.
  • IV intravenous injection
  • the dosage is: 3mg/kg.
  • Tweight and Cweight represent the tumor weights of the administration group and the vehicle control group, respectively.
  • T test was used for comparison between two groups.
  • One-way ANOVA was used for comparison among three or more groups. If there is a significant difference in F values, multiple comparisons should be performed after ANOVA analysis. All data analyzes were performed with SPSS 17.0. P ⁇ 0.05 was considered to have a significant difference.
  • the compound of the present invention exhibits significant tumor inhibitory effect in the subcutaneous xenograft tumor BALB/c nude mouse model of human lung cancer NCI-H292 cells.
  • test example 3 In vivo pharmacodynamic study of test example 3 on human breast cancer MDA-MB-468 cell subcutaneous xenograft tumor BALB/c nude mouse model
  • Human breast cancer MDA-MB-468 cells (ATCC, Manassas, Virginia, Cat. No.: HTB-132) were cultured in vitro as a monolayer, and the culture conditions were L-15 medium with 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin, cultured in 37°C, 0% CO2 incubator. Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • mice BALB/c nude mice, female, 6-8 weeks old, weighing 18-22 grams. Provided by Beijing Weitong Lihua Company.
  • Tumor inoculation 0.2 mL (1 ⁇ 10 7 cells) of MDA-MB-468 cells (plus matrigel, volume ratio 1:1) were subcutaneously inoculated on the right back of each mouse.
  • IV intravenous injection
  • the dosage is: 3mg/kg.
  • the compound of the present invention exhibits significant tumor inhibitory effect in the subcutaneous xenograft tumor BALB/c nude mouse model of human breast cancer MDA-MB-468 cells.

Abstract

一种多肽偶联药物及其应用，如式(I)所示化合物及其药学上可接受的盐，MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-SEQ(I)。

Description

多肽偶联药物及其应用
本申请主张如下优先权：
CN2021106108895，申请日：2021年6月1日；
CN2021108381694，申请日：2021年7月23日；
CN202111222422X，申请日：2021年10月20日。
技术领域
本发明涉及一种多肽偶联药物及其应用，具体涉及式(I)所示化合物及其药学上可接受的盐。
背景技术
2020年全球新增癌症病例约1930万例，死亡病例约1000万例，其中中国癌症死亡人数约300万，位居全球第一。肿瘤对传统治疗方式的耐药性以及对现有治疗方式的不敏感等原因，使得部分癌症患者尤其是晚期癌症患者的治疗手段有限且治疗效果并不理想。因此研究具有新靶点、新机制和新结构的肿瘤治疗药物始终是肿瘤治疗领域亟待解决的问题。
Nectin-4(poliovirus receptor like 4，PVRL4，结合素-4)是近年来新兴的肿瘤相关靶点，其隶属于nectin蛋白家族，主要有nectin 1-4四种亚型，与nectin-like分子(Necl)共同构成免疫球蛋白样细胞粘附分子，对细胞间粘附和紧密连接的形成和保持具有重要作用。Nectin 1，2，3广泛存在于人体正常组织中，nectin-4主要在胚胎和胎盘中高表达，在成年人体内表达量大幅度下降。研究表明，nectin-4在多种肿瘤中过度表达，如膀胱癌、尿路上皮癌、乳腺癌、三阴性乳腺癌、肺癌、胃癌、食道癌等，使其成为治疗相关癌症的潜在靶点。目前，针对该靶点研发的生物类抗体偶联药物Enfortumab vedotin已于2019年在美国批准上市，作为该靶点的唯一上市药物，其主要适应症被为转移型尿路上皮癌，同时另有多个适应症的临床有效性研究正在开展。因此开发以nectin-4为靶点的化学治疗药物具有广泛的应用前景。
发明内容
本发明提供了式(I)所示化合物或其药学上可接受的盐，
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-SEQ
(I)
其中，
SEQ由肽配体和连接子组成，所述肽配体以共价键形式与连接子相连；
肽配体包含被三个氨基酸残基隔开的两个环序列，所述两个环序列中的第一个为Pro-1Nal-dAsp，第二个为Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp；
氨基酸残基为Xi、Xii和Xiii，所述Xi、Xii和Xiii分别独立地选自Cys、hCys、βCys、Pen、Dap和N-methyl-Dap，且Xi、Xii和Xiii不全是Cys；
连接子选自
Figure PCTCN2022094740-appb-000001
在本发明的一些方案中，上述肽配体选自：H-Xi-Pro-1Nal-dAsp-Xii-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Xiii-NH 2，其中，Xi、Xii和Xiii如本发明所定义。
在本发明的一些方案中，上述肽配体选自序列1-7：
H-hCys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列1)、
H-Pen-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列2)、
H-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列3)、
H-Cys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Pen-NH 2(序列4)、
H-Cys-Pro-1Nal-dAsp-hCys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列5)、
H-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-hCys-NH 2(序列6)和
H-hCys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列7)，其他变量如本发明所定义。
在本发明的一些方案中，上述SEQ选自式(S-a)所示结构，
Figure PCTCN2022094740-appb-000002
其中，Xi、Xii和Xiii如本发明所定义。
本发明还有一些方案由上述变量任意组合而来。
本发明还提供了下式所示化合物或其药学上可接受的盐，
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-hCys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Pen-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Pen-NH 2，TATA、
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-hCys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-
Trp-Cys-NH 2，TATA、
MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-hCys-NH 2，TATA
和MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-hCys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA。
本发明还提供了上述化合物或其药学上可接受的盐在制备治疗Nectin-4过表达的实体瘤的药物中的应用。
技术效果
本发明化合物与Nectin-4具有很强的结合作用，体内小鼠皮下瘤模型表现出强抑瘤效果。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计 量的适当的碱或酸反应来制备。
“氨基酸”是指天然存在的和合成的氨基酸，以及起到与天然存在的氨基酸类似的作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如，羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构(例如与氢、羧基基团、氨基基团和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这样的类似物可以具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指其结构不同于一般的氨基酸化学结构，但起到与天然存在的氨基酸相似的作用的化学化合物。
本发明的氨基酸序列含有二十种天然氨基酸的标准单字母或三字母代码。
术语“治疗”包括抑制、减缓、停止或逆转现有症状或病患的进展或严重程度。
除非另有说明，术语“异构体”意在包括几何异构体、顺反异构体、立体异构体、对映异构体、旋光异构体、非对映异构体和互变异构体。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2022094740-appb-000003
和楔形虚线键
Figure PCTCN2022094740-appb-000004
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2022094740-appb-000005
和直形虚线键
Figure PCTCN2022094740-appb-000006
表示立体中心的相对构型，用波浪线
Figure PCTCN2022094740-appb-000007
表示楔形实线键
Figure PCTCN2022094740-appb-000008
或楔形虚线键
Figure PCTCN2022094740-appb-000009
或用波浪线
Figure PCTCN2022094740-appb-000010
表示直形实线键
Figure PCTCN2022094740-appb-000011
或直形虚线键
Figure PCTCN2022094740-appb-000012
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或 者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2022094740-appb-000013
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2022094740-appb-000014
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2022094740-appb-000015
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2022094740-appb-000016
直形虚线键
Figure PCTCN2022094740-appb-000017
或波浪线
Figure PCTCN2022094740-appb-000018
表示。例如-OCH 3中的直形 实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2022094740-appb-000019
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2022094740-appb-000020
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2022094740-appb-000021
软件命名，市售化合物采用供应商目录名称。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：eq.代表当量、等量；SPPS代表多肽固相合成法；TFA代表三氟乙酸；DIEA代表二异丙基乙基胺；DMF代表N，N-二甲基甲酰胺；HATU代表2-(7-氮杂苯并三氮唑)-N，N，N′，N′-四甲基脲六氟磷酸酯；EDC代表1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；NHS代表N-羟基琥珀酰亚胺；TIS代表三异丙基硅烷；DTT代表DL-1，4-二硫代苏糖醇；MMAE代表单甲基奥瑞他汀E，具体结构为：
Figure PCTCN2022094740-appb-000022
PABC代表
Figure PCTCN2022094740-appb-000023
Cit代表L-瓜氨酸；Val代表L-缬氨酸；Glutaryl代表
Figure PCTCN2022094740-appb-000024
β-Ala代表
Figure PCTCN2022094740-appb-000025
Sar代表
Figure PCTCN2022094740-appb-000026
Sar10代表
Figure PCTCN2022094740-appb-000027
Cys代表L-半胱氨酸；hCys代表
Figure PCTCN2022094740-appb-000028
βCys代表
Figure PCTCN2022094740-appb-000029
Pen代表
Figure PCTCN2022094740-appb-000030
N-methyl-Dap代表
Figure PCTCN2022094740-appb-000031
1Nal代表1-萘丙氨酸；hArg代表L-高精氨酸；Hyp代表L-羟脯氨酸；Trp代表L-色氨酸；Pro代表L-脯氨酸；Thr代表L-苏氨酸；Ser代表L-丝氨酸；Asp代表L-天门冬氨酸；dAsp代表D-天门冬氨酸；Fmoc代表9-芴甲氧羰基；Boc代表叔丁氧基羰基(Boc)；Trt代表三苯甲基；Pbf代表2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基；PBS代表磷酸盐缓冲液。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情 况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
Figure PCTCN2022094740-appb-000032
合成路线：
Figure PCTCN2022094740-appb-000033
Figure PCTCN2022094740-appb-000034
Figure PCTCN2022094740-appb-000035
步骤1：Peptide 1的TFA盐的合成
1.1多肽的偶联
该多肽使用标准的逐步合成方法合成。
1)将DMF加入到含有Rink amide MBHA树脂(0.5mmol，1.56g，sub：0.32mmol/g)的容器中并且让树脂溶胀2小时。
2)抽干，然后用DMF冲洗三次，每次氮气鼓动30秒。
3)加入20％哌啶/DMF，然后反应30min。缩合反应用显色反应进行检测，反应后用DMF冲洗树脂3～5次。
4)抽干，然后用DMF冲洗五次，每次氮气鼓动30秒。
5)加入下表原料1溶液，充氮30秒，然后加入缩合试剂，N 2鼓吹反应将近1小时。
6)重复步骤2至步骤5，每次顺序加入原料2-26缩合下一个氨基酸。详见表1。
表1加料顺序
# 原料 偶联试剂
1 Fmoc-Cys(Trt)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
2 Fmoc-Trp(Boc)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
3 Fmoc-HyP(tBu)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
4 Fmoc-Pro-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
5 Fmoc-Thr(tBu)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
6 Fmoc-Ser(tBu)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
7 Fmoc-Trp(Boc)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
8 Fmoc-Asp(OtBu)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
9 Fmoc-hArg(Pbf)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
10 Fmoc-Met-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
11 Fmoc-Cys(Trt)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
12 Fmoc-dAsp(OtBu)-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
13 Fmoc-1Nal-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
14 Fmoc-Pro-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
15 Fmoc-hCys(Trt)-OH(2.0eq.) HATU(1.90eq.)、DIEA(4.0eq.)
16 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
17 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
18 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
19 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
20 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
21 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
22 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
23 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
24 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
25 Fmoc-Sar-OH(5.0eq.) HATU(4.75eq.)、DIEA(10.0eq.)
26 Fmoc-β-Ala-OH(3.0eq.) HATU(2.85eq.)、DIEA(6.0eq.)
其他直链多肽的合成方法与Peptide 1的TFA盐的合成方法相同。
1.2多肽切割和纯化
1)向含有侧链保护的多肽的烧瓶中加入切割缓冲溶液(90％TFA/2.5％TIS/2.5％H 2O/5.0％DTT)，置于室温下搅拌2小时。
2)用冰的异丙醚将该多肽沉降出来，并用离心机(3min at 3000rpm)离心分离。
3)用异丙醚再洗两次。
4)干燥得Peptide 1的TFA盐。
步骤2：Peptide 2的TFA盐的合成
Peptide 1(0.7g，0.25mmol)溶解在50％CH 3CN/H 2O(1L)中，在室温下向搅拌着的溶液中缓慢加入TATA(95.9mg，0.38mmol)，反应混合物在室温下搅拌30分钟，然后用NH 4HCO 3调节pH值到8，反应继续在室温搅拌12小时。LCMS显示反应完全，停止搅拌，通过反相制备(A：0.075％TFA水溶液，B：CH 3CN)进行纯化得到Peptide 2的TFA盐。
其他多肽的合成方法与Peptide 2的合成方法相似。
表2多肽序列表
Figure PCTCN2022094740-appb-000036
步骤3：INT_1的TFA盐的合成
将化合物1-1(200.0mg，178.0μmol)溶于DMF(5mL)中，在0℃下加入DIEA(31.0μL，178.0μmol)，搅拌10mins，同时将化合物1-2(290.4mg，890.1μmol)用另外一个反应瓶，也溶于DMF(5mL)，在0℃下搅拌10mins，然后在0℃下将化合物1-1的反应液滴加到搅拌着的化合物1-2的反应液中，此反应液在0℃下搅拌30mins，将反应液过滤除去不溶的残渣，滤液直接用反相制备进行(TFA体系)纯化，得到化合物INT_1的TFA盐。
步骤4：PDC_1的乙酸盐的合成
将Peptide 2(50.0mg，16.8μmol)溶于DMF(0.3mL)中，然后加入DIEA(11.7μL，67.3μmol)，在室温下搅拌10mins。接着将化合物INT_1(22.5mg，16.8μmol)溶于DMF(0.5mL)中，滴加到上述Peptide 2的反应液中，然后在室温下搅拌1小时，将反应液过滤除去不溶的残渣，滤液直接用反相制备(TFA体系)纯化，然后再通过制备转化成AcOH盐，得到PDC_1的乙酸盐。纯化条件见表3。
表3纯化条件
Figure PCTCN2022094740-appb-000037
其他三个多肽偶联药物PDC_2-7的乙酸盐参考PDC_1的乙酸盐的合成(分别将原料替换为下表中的对应Peptide)得到，见表4。
表4 PDC结构信息
Figure PCTCN2022094740-appb-000038
Figure PCTCN2022094740-appb-000039
生物测试数据
测试例1本发明化合物与Nectin 4蛋白的结合能力测试
1.实验目的
用SPR法检测待测物与靶蛋白Nectin 4的亲和力。
2.材料和仪器
·Biacore 8K(GE Healthcare)
·96-well Plate(Cat#650101，greiner bio-one)
·CM5芯片(Cat#BR-1005-30，GE Healthcare)
·Amine Coupling Kit(Cat#BR-1000-50，GE Healthcare)
EDC
NHS
1M乙醇胺
·10mM醋酸钠pH4.5(Cat#BR-1003-50，GE Healthcare)
·DMSO(Cat#D4540，Sigma)
·P20(Cat#BR-1000-54，GE Healthcare)
·PBS(Cat#BR-1006-72，GE Healthcare)
·Nectin 4(Cat#1006-72，GE Healthcare)
3.实验方案
本实验采用氨基偶联法，即利用Biacore 8K将靶蛋白Nectin 4直接固定在CM5芯片上，然后将待测物作为分析物，用缓冲液(10mM PBS，pH7.4，137mM NaCl，2.7mM KCl，5％DMSO，0.05％P20)将待测物稀释到所需的浓度梯度，进行多循环动力学检测，每个循环进样180秒，解离180秒，然后再进行下一循环，获得靶蛋白Nectin 4与待测物的亲和力动力学分析数据。最终的数据用Biacore Insight Evaluation Software(V 2.0.15.12933)按1∶1模型进行Kinetics拟合分析。
4.实验方法及流程
1)配制缓冲液：10mM PBS，pH7.4，137mM NaCl，2.7mM KCl，5％DMSO，0.05％P20。
2)CM5芯片活化：用400mM EDC和100mM NHS以10μL/min的流速活化420秒。
3)靶蛋白偶联：用10mM醋酸钠(pH 4.5)将靶蛋白稀释到10μg/mL，以10μL/min的流速偶联284s。实验使用了芯片上的1#，2#和3#通道，偶联结果分别为1639.9RU，1747.8RU和1702.2RU。
4)CM5芯片封闭：用1M乙醇胺以10μL/min的流速封闭420秒。
5)分析物浓度：使用running buffer稀释待测物。待测物从100nM开始2倍梯度稀释至0.78nM。
6)进样分析：待测物工作液每个浓度为一个循环，以30μL/min的流速结合180秒，解离180秒。最后一个循环为5％DMSO溶剂校正循环。
7)所有结果按1∶1模型进行kinetics拟合分析。
5.实验结果
选取5个有效浓度的实验数据对本发明化合物用Biacore Insight Evaluation Software(V 2.0.15.12933)按1∶1模型进行Kinetics拟合分析，结果见表5：
表5本发明化合物与Human Nectin-4 SPR结合结果
化合物 Human Nectin-4 SPR k D，nM(测试次数)
PDC_1的乙酸盐 40.0(2)
PDC_3的乙酸盐 3.0(3)
PDC_4的乙酸盐 246.5(2)
PDC_5的乙酸盐 80.7
PDC_6的乙酸盐 9.9(2)
PDC_7的乙酸盐 3.3
若测试次数大于1则所列数值为多次测量的平均值。
结论：本发明化合物与Human Nectin-4蛋白具有强结合能力。
测试例2对人肺癌NCI-H292细胞皮下异种移植肿瘤BALB/c裸小鼠模型的体内药效学和PK研究
实验目的：评价本发明化合物在人肺癌NCI-H292细胞皮下异种移植肿瘤模型上的体内药效。
细胞培养：人肺癌NCI-H292细胞(ATCC，马纳萨斯，弗吉尼亚州，货号：CRL-1848)体外单层培养，培养条件为RPMI 1640培养基中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃ 5％CO 2孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
动物：BALB/c裸小鼠，雌性，6-8周龄，体重17-21克。由北京维通利华公司提供。
肿瘤接种：将0.1mL(1×10 7个)NCI-H292细胞皮下接种于每只小鼠的右后背。
药物配制：化合物以25mM L-Histidine(pH＝7)10％sucrose为溶媒配成均一溶液。用于IV(静注)组给药。
给药剂量为：3mg/kg。
实验指标：实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[(1-(某处理组给药结束时平均瘤体积-该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]×100％。
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV ＝V t/V 0，其中V 0是分组给药时(即d 0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
在实验结束后将检测肿瘤重量，并计算T/Cweight百分比，Tweight和Cweight分别表示给药组和溶媒对照组的瘤重。
数据分析：T检验用于两组间比较。三组或多组间比较用one-way ANOVA。如果F值有显著性差异，应在ANOVA分析之后再进行多重比较。用SPSS 17.0进行所有数据分析。p＜0.05认为有显著性差异。
结论：本发明化合物在人肺癌NCI-H292细胞皮下异种移植肿瘤BALB/c裸小鼠模型中展现出显著的抑瘤效果。
测试例3对人乳腺癌MDA-MB-468细胞皮下异种移植肿瘤BALB/c裸小鼠模型的体内药效学研究
实验目的：评价受试药BT8009和衍生物在人乳腺癌MDA-MB-468细胞皮下异种移植肿瘤模型上的体内药效。
细胞培养：人乳腺癌MDA-MB-468细胞(ATCC，马纳萨斯，弗吉尼亚州，货号：HTB-132)体外单层培养，培养条件为L-15培养基中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃，0％CO 2孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
动物：BALB/c裸小鼠，雌性，6-8周龄，体重18-22克。由北京维通利华公司提供。
肿瘤接种：将0.2mL(1×10 7个)MDA-MB-468细胞(加基质胶，体积比为1∶1)皮下接种于每只小鼠的右后背。
药物配制：化合物以25mM L-Histidine(pH＝7)10％sucrose为溶媒配成均一溶液。用于IV(静注)组给药。
给药剂量为：3mg/kg。
结论：本发明化合物在人乳腺癌MDA-MB-468细胞皮下异种移植肿瘤BALB/c裸小鼠模型中展现出显著的抑瘤效果。

Claims (7)

  1. 式(I)所示化合物或其药学上可接受的盐，
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-SEQ
    (I)
    其中，
    SEQ由肽配体和连接子组成，所述肽配体以共价键形式与连接子相连；
    肽配体包含被三个氨基酸残基隔开的两个环序列，所述两个环序列中的第一个为Pro-1Nal-dAsp，第二个为Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp；
    氨基酸残基为Xi、Xii和Xiii，所述Xi、Xii和Xiii分别独立地选自Cys、hCys、βCys、Pen、Dap和N-methyl-Dap，且Xi、Xii和Xiii不全是Cys；
    连接子选自
    Figure PCTCN2022094740-appb-100001
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，肽配体选自：
    H-Xi-Pro-1Nal-dAsp-Xii-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Xiii-NH 2，
    其中，Xi、Xii和Xiii如权利要求1所定义。
  3. 根据权利要求2所述化合物或其药学上可接受的盐，其中，肽配体选自序列1-7：
    H-hCys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列1)、
    H-Pen-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列2)、
    H-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列3)、
    H-Cys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Pen-NH 2(序列4)、
    H-Cys-Pro-1Nal-dAsp-hCys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列5)、
    H-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-hCys-NH 2(序列6)和
    H-hCys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2(序列7)。
  4. 根据权利要求1所述化合物或其药学上可接受的盐，其中，SEQ选自式(S-a)所示结构，
    Figure PCTCN2022094740-appb-100002
    其中，Xi、Xii和Xiii如权利要求1所定义。
  5. 根据权利要求1所述化合物或其药学上可接受的盐，其中，化合物选自式(I-1)所示结构，
    Figure PCTCN2022094740-appb-100003
    其中，Xi、Xii和Xiii如权利要求1所定义。
  6. 下列化合物或其药学上可接受的盐，
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-hCys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Pen-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Cys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Pen-NH 2，TATA、
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-hCys-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA、
    MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-Cys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp- Trp-hCys-NH 2，TATA
    和MMAE-PABC-Cit-Val-Glutaryl-β-Ala-Sar10-hCys-Pro-1Nal-dAsp-Pen-Met-hArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-NH 2，TATA。
  7. 权利要求1～6任意一项所述的化合物或其药学上可接受的盐在制备治疗Nectin-4过表达的实体瘤的药物中的应用。
PCT/CN2022/094740 2021-06-01 2022-05-24 多肽偶联药物及其应用 WO2022253051A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067487A1 (en) * 2008-12-12 2010-06-17 Oncotherapy Science, Inc. Nectin-4 for target genes of cancer therapy and diagnosis
CN110099682A (zh) * 2016-11-14 2019-08-06 杭州多禧生物科技有限公司 偶联连接体，含有此连接体的细胞结合分子-药物偶联物及其制备和应用
US20190307836A1 (en) * 2018-04-04 2019-10-10 Bicycletx Limited Heterotandem bicyclic peptide complexes
CN112601539A (zh) * 2018-06-22 2021-04-02 拜斯科技术开发有限公司 特异于结合素-4的双环肽配体
WO2021064428A1 (en) * 2019-10-03 2021-04-08 Bicycletx Limited Heterotandem bicyclic peptide complexes
WO2021074622A1 (en) * 2019-10-15 2021-04-22 Bicycletx Limited Bicyclic peptide ligand drug conjugates

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067487A1 (en) * 2008-12-12 2010-06-17 Oncotherapy Science, Inc. Nectin-4 for target genes of cancer therapy and diagnosis
CN110099682A (zh) * 2016-11-14 2019-08-06 杭州多禧生物科技有限公司 偶联连接体，含有此连接体的细胞结合分子-药物偶联物及其制备和应用
US20190307836A1 (en) * 2018-04-04 2019-10-10 Bicycletx Limited Heterotandem bicyclic peptide complexes
CN112601539A (zh) * 2018-06-22 2021-04-02 拜斯科技术开发有限公司 特异于结合素-4的双环肽配体
WO2021064428A1 (en) * 2019-10-03 2021-04-08 Bicycletx Limited Heterotandem bicyclic peptide complexes
WO2021074622A1 (en) * 2019-10-15 2021-04-22 Bicycletx Limited Bicyclic peptide ligand drug conjugates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WONG JEFFREY L., ROSENBERG JONATHAN E.: "Targeting nectin-4 by antibody-drug conjugates for the treatment of urothelial carcinoma", EXPERT OPINION ON BIOLOGICAL THERAPY, INFORMA HEALTHCARE, vol. 21, no. 7, 3 July 2021 (2021-07-03), pages 863 - 873, XP093010380, ISSN: 1471-2598, DOI: 10.1080/14712598.2021.1929168 *

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